Datasets:

griffin

/

ChemSum

like 12

Photoredox-mediated hydroalkylation and hydroarylation of functionalized olefins for DNA-encoded library synthesis

DNA-encoded library (DEL) technology features a time-and cost-effective interrogation format for the discovery of therapeutic candidates in the pharmaceutical industry. To develop DEL platforms, the implementation of water-compatible transformations that facilitate the incorporation of multifunctional building blocks (BBs) with high C(sp 3 ) carbon counts is integral for success. In this report, a decarboxylative-based hydroalkylation of DNA-conjugated trifluoromethyl-substituted alkenes enabled by single-electron transfer (SET) and subsequent hydrogen atom termination through electron donoracceptor (EDA) complex activation is detailed. In a further photoredox-catalyzed hydroarylation protocol, the coupling of functionalized, electronically unbiased olefins is achieved under air and within minutes of blue light irradiation through the intermediacy of reactive (hetero)aryl radical species with full retention of the DNA tag integrity. Notably, these processes operate under mild reaction conditions, furnishing complex structural scaffolds with a high density of pendant functional groups.

photoredox-mediated_hydroalkylation_and_hydroarylation_of_functionalized_olefins_for_dna-encoded_lib

Introduction<!>Discussion<!>DNA compatibility with EDA complex photoactivation<!>DNA compatibility with aryl radical intermediates<!>Conclusions

<p>The identication of specic binding molecules remains a central theme in drug discovery efforts in academic and industrial laboratories. 1 The global pharmaceutical industry invests over $186 billion annually on research and development to meet the ever-increasing demands for safe and improved therapeutics. 2 In recent years, DNA-encoded library (DEL) technology (Fig. 1) has emerged as a novel interrogation platform to accelerate the advancement of small-molecule modulators of biomolecular targets. 3 Conceptualized by Brenner and Lerner in 1992, 4 DEL platforms confer unprecedented capabilities to overlap the versatility of chemical synthesis with the powerful features of genetic coding, allowing simultaneous testing of combinatorial libraries of exceptional magnitude (>10 6 to 10 12 discrete members). 3 Through "split and pool" synthetic cycles, diverse BBs are encoded by a unique DNA tag functioning as a molecular identier. 3 Following synthesis, DEL libraries are incubated with immobilized target proteins, aer which non-binders are washed away. The chemical structures of the potent ligands are decoded using polymerase chain reaction (PCR) amplication and posterior next-generation DNA sequencing. 3 In this vein, DELs feature a more effective and inexpensive discovery format ($0.0002 per library member) compared with conventional high-throughput screening (HTS) methods ($1000 per library member). 5 To be successful, on-DNA chemistries are required to incorporate BBs from readily available chemicals bearing multifunctional handles for further diversication under mild, dilute, and aqueous conditions. 6 In light of these considerations, the development of reliable transformations that operate through novel reactivity modes and employ an abundant, diverse set of starting materials would expedite progress in this eld.</p><p>As part of a program centered on the development of catalytic tools to yield novel structural scaffolds, we recently reported the synthesis of gem-diuoroalkenes, 7,8 carbonyl mimics that display in vivo resistance toward metabolic processes, 9 through photoinduced radical/polar crossover deuorinative alkylation. 7,8,10 As a complementary approach to build chemical diversity, we became interested in pushing the limits of photochemical paradigms to access benzylically tri-uoromethylated compounds, bioactive structural motifs in medicinal settings (Scheme 1A). 11 Specically, uorine incorporation is a powerful strategy invoked by the pharmaceutical and agrochemical industries to alter a molecule's chemical, physical, and biological properties, such as its pK a , dipole moment, and molecular conformation. 12,13 As a consequence of these factors, uorinated scaffolds are prevalent in more than 25% of marketed drugs. 11c As an important representative, the triuoromethyl (-CF 3 ) group renders increased metabolic stability, lipophilicity, and binding selectivity when embedded in therapeutic candidates. 11, 14 Typically, the triuoromethyl group can be installed through nucleophilic, electrophilic, or radical routes. 15 Although these strategies undoubtedly expand chemical space, these protocols remain elusive in the context of late-stage functionalization and the incorporation of sensitive functional groups in complex environments under DEL-like conditions. An underexplored opportunity to achieve C(sp) 3 triuoromethylation is the direct hydroalkylation of triuoromethyl-substituted olens. Specically, the carbofunctionalization of these electrophilic unsaturated systems occurs readily at room temperature with exquisite functional group compatibility, 16 thus rendering the incorporation of pharmaceutically relevant cores and complex alkyl fragments feasible in a library setting. However, given the established propensity of triuoromethyl-substituted alkenes to undergo intramolecular E1cB-type uoride elimination in metalcatalyzed cross-couplings that proceed through the intermediacy of a-CF 3 -metal species 17 via the nucleophilic addition of organometallic reagents 18,19 or in the presence of traditional photoredox catalysts irrespective of the nature of the radical precursor (Scheme 1B), 16,19a,20 hydrofunctionalization 21 efforts remain challenging. In particular, the hydroalkylation of triuoromethyl-substituted alkenes using unactivated alkyl counterparts presents a formidable, yet potentially powerful scenario to rapidly access unprecedented benzylically tri-uoromethylated building blocks from commodity radical progenitors with a high content of C(sp 3 ) carbons.</p><p>To address this challenge and unlock access to benzylically triuoromethylated motifs from commodity chemicals in DEL synthesis, we report a decarboxylative-based, radical-mediated hydroalkylation of DNA-tagged triuoromethyl-substituted alkenes enabled by the merger of electron donor-acceptor (EDA) complex photoactivation 22,22j,22k and hydrogen atom transfer (HAT) chemistry (Scheme 2). 23 Under blue light irradiation, a commercially available electron donor, Hantzsch ester (HE, diethyl 1,4-dihydro-2,6-dimethyl-3,5pyridinedicarboxylate), functions as a strong photoreductant to induce C(sp 3 ) radical generation from commercially available carboxylic acid derivatives. 24 As part of its dual role, HE subsequently serves as a suitable hydrogen atom donor, impeding the formation of anionic intermediates upon radical addition as well as circumventing the necessity for alkylmetal complexes, species intrinsically primed to undergo b-F elimination in reactions with triuoromethyl-substituted alkenes. 17e,20, 25 In this vein, the utility of this EDA paradigm is partially driven by its ability to deliver complex triuoromethyl-substituted hydrofunctionalized products with high C(sp 3 ) carbon counts selectively under mild and open-air conditions.</p><p>As a complement to the hydroalkylation protocol, a radicalmediated intermolecular hydroarylation of electronically unbiased olens was developed (Scheme 2). 26 Because alkenes are plentiful, structurally diverse, and versatile commodity feedstocks, readily available from petrochemical and renewable resources, they are ideal precursors for C-C bond formation in DELs, and the strategy developed is based on photoinduced reductive activation of DNA-conjugated (het)aryl halides to deliver reactive (het)aryl radical species that can be harnessed in useful synthetic operations followed by hydrogen atom termination. 23</p><!><p>Recently, synthetic processes driven by EDA complex photochemistry have gained considerable traction, including protocols resulting in borylation, sulfonylation, and thioetherication. 22,27 To harness the synthetic potential of EDA complex photoactivation toward DEL platforms, we examined the feasibility of the proposed decarboxylative hydroalkylation using on-DNA triuoromethyl-substituted alkene 1A and unactivated primary redox-active ester (RAE) 2a as model substrates (Table 1). Under blue Kessil irradiation (l max ¼ 456 nm), efficient conversion to the desired benzylic triuoromethylsubstituted product 3a was observed using 50 equivalents of the radical precursor under ambient reaction conditions within minutes of illumination. In contrast to radical-mediated alkylation promoted by metal reductants 17e,28 or external photoredox catalysts, 29 this open-to-air EDA paradigm provides an exceedingly low barrier to practical implementation in highthroughput settings and circumvents side reactivity stemming from singlet oxygen generation through triplet-energy transfer. 29 To examine the inuence of the dihydropyridine (DHP) backbone on the efficacy of this photochemical manifold, the reaction was conducted with four different DHP derivatives to gain a deeper understanding of their dual reactivity prole in EDA complex photoactivation and HAT catalysis. The C4substituted DHP (HE A, entry 7) thus exhibited no reactivity under the reaction conditions, whereas cyano substitution at C3 and C5 of the DHP (HE B, entry 8) led to diminished product formation. As expected, 4,4 0 -dimethyl HE C (entry 9) failed to promote the reaction, presumably because of competitive back electron transfer (BET) 22a that restores the ground-state EDA complex from its radical ion pair in the absence of a probable photooxidative aromatization event. Notably, commercially available and bench-stable HE 30 displayed optimal performance (84% yield, entry 1), accommodating water as a co-solvent and high dilution conditions (0.3-0.6 mM), with only trace amounts of the corresponding gem-diuoroalkene detected. Using UV/vis absorption studies, a bathochromic shi of the reaction mixture in 8 : 1 DMSO/H 2 O (0.6 mM) was observed, with a wavelength band tailing to 500 nm (see ESI †). This is indicative of the formation of a new molecular aggregate between the electron-decient aliphatic RAEs and the electron-rich HE. Using Job's method 31 of continuous variation, we determined a molar donor-acceptor ratio of 1 : 1 for the colored EDA complex (see ESI †). Further spectrophotometric analysis at 450 nm revealed an association constant (K EDA ) of 1.2 M À1 of HE with 1-methylcyclohexane-N-hydroxyphthalimide ester using the Benesi-Hildebrand method, 32 highlighting a plausible association event of charge-transfer complexes prior to SET events under the conditions of the reaction. Finally, control experiments validated the necessity of all reaction parameters for effective C(sp 3 )-C(sp 3 ) bond formation.</p><p>Next, we examined the scope of redox-active carboxylate derivatives using on-DNA triuoromethylated alkene 1A (Scheme 3). In general, a broad palette of primary aliphatic systems that lack any radical stabilizing factors exhibited excellent reactivity. The method further benets from broad functional group tolerance, facilitating the introduction of bifunctional handles including ketones (3a, 3f, 3q), aryl halides (3c, 3e, 3n), a terminal alkyne (3d), esters (3j, 3o), substituted alkenes (3k, 3l, 3m, 3o), free alcohols (3o, 3p), as well as medicinally-relevant heteroaromatic scaffolds (3a, 3n). In addition, Boc-and Fmoc-protected amines served as competent substrates. This is crucial in DEL settings, where library members should ideally bear multifunctional BBs that allow subsequent derivatization. The scope was further extended to the modication of biologically active molecules displaying a high density of pendant functional groups, including the herbicide 2,4-dichlorophenylacetic acid (3e), long-chain fatty acids (3k-3m), the anti-inammatory agent indomethacin (3n), mycophenolic acid (3o), as well as various steroids (3p, 3q). In particular, this method provides a clear advantage in terms of scope over previously reported on-DNA photoinduced decarboxylative alkylation protocols, which are largely limited to aheteroatom-stabilized radicals, 7,33 or exclusively restricted to secondary and tertiary radicals. 22k,34 More specically, complementary decarboxylative methods employing zinc nanopowder as a reductant under strictly deoxygenated conditions fail to incorporate primary systems on DNA, 28 presumably because of the higher reduction potentials associated with the radical precursor. Most importantly, these methods largely proceed through anionic intermediates, where in the case of the triuoromethyl-substituted olens, there is a predominant propensity for intramolecular E1cB-type uoride elimination 16a,19a,20 to afford the corresponding gem-diuoroalkenes (rather than triuoromethyl-substituted alkanes) via radical/ polar crossover pathways. 10 In a similar manner, secondary and tertiary radical architectures are harnessed effectively to afford functionalized synthetic frameworks, including scaffolds derived from proteinogenic and non-proteinogenic amino acids (3u-3y), a glycoside (3z), and lipid-lowering agent gembrozil (3za) (Scheme 3). The reaction conditions proved general for both acyclic and cyclic carboxylate derivatives, including bridged bicyclics (3zf-3zh), as well as strained ring systems, such as cyclobutanes (3s, 3t) and a cyclopropane (3zc). Notably, triuoromethylsubstituted bicyclo[1.1.1]pentane (BCP) product 3zg was obtained in good yield. These BCP derivatives serve as bioisosteres for arenes, internal alkynes, and tert-butyl groups in medicinal chemistry settings. 35 With respect to the scope of triuoromethyl-substituted alkenes, a diverse array of DNA headpieces (DNA-HPs) led to the desired benzylic triuoromethyl-substituted products without compromising yields (Scheme 4). In general, both electron-withdrawing and electron-donating groups are well tolerated under the developed conditions. Substitution at the para-, meta-, and ortho-positions of the HPs' aryl moieties was explored, whereby efficient decarboxylative photocoupling took place. Furthermore, comparable reactivity was observed for unactivated primary, secondary, tertiary, as well as stabilized benzylic-, a-oxy-, and a-amino radical species, further underscoring the versatility of this photochemical EDA paradigm.</p><p>The commercial availability and structural diversity of carboxylic acids render them particularly attractive for use as multifunctional BBs in DEL libraries. To validate the modularity of this approach further, we developed a telescoped, one-pot photoinduced decarboxylative alkylation protocol through in situ formation of aliphatic RAEs with N-hydroxyphthalimide tetramethyluronium hexauorophosphate (HITU), 28 a benchstable solid that can be readily prepared on kilogram scale (Scheme 5). This reagent features great versatility in reaction scope, accommodating a wide array of functional groups including ketone (3f), carbamate (3zi), aryl chloride (3zl), and Boc-protected amines (3zn, 3zo). Using 24-well plates, microdosing of the carboxylic acid, DIPEA, and HITU in DMSO is accomplished under air, followed by 3 h of activation time. The in situ formed RAEs can then be treated directly with a solution of HE and the corresponding DNA headpiece, reaching synthetically useful yields aer 10 min of illumination (Scheme 5, workow). Notably, this HITU-mediated alkylation performs equally well using unactivated-and a-heteroatom-stabilized radical progenitors, presenting a direct route toward C-C bond formation through oxidative quenching modes, an underexplored challenge in DEL-based environments. 28 As an extension of the hydroalkylation chemistry, an on-DNA multicomponent reaction (MCR) was In recent years, MCRs 36 have emerged as a powerful tool to furnish novel scaffolds with inherent molecular complexity from abundant feedstocks. Through sequential bond formation, MCRs enable the sampling of uncharted chemical space to accelerate drug discovery efforts. 36 An underexplored realm in DEL synthesis is the development of olen dicarbofunctionalization reactions. 3a Specically, alkenes serve as versatile BBs that possess functional group-rich handles for derivatization. However, in addition to chemo-and regio-selectivity concerns associated with DEL reactions that rely on high loadings of reagents, these processes are further complicated by the generation of undesired two-component coupling products. Keeping these considerations in mind and taking advantage of the electronically distinct nature of triuoromethyl-substituted alkenes, a polarity-reversing radical cascade/ triuoromethylation of olens has been developed through EDA complex photoactivation between HE and Umemoto's reagent, 22a a commercially available triuoromethylating agent (Scheme 6).</p><p>In particular, this open-to-air charge-transfer manifold harnesses electrophilic triuoromethyl radicals for subsequent addition to electron-neutral or electron-rich alkenes, abundant yet currently underexplored partners in photoinduced DEL synthesis. 3a, 37 The resulting nucleophilic, open-shell radical intermediates may then engage in chemoselective coupling with on-DNA triuoromethyl-substituted alkenes. As part of its dual role, the HE also functions as a hydrogen atom donor to furnish bis-triuoromethylated products of signicance in medicinal settings. 11 Remarkably, the scope of the olen partner proved general, tolerating diverse functional groups including a free alcohol (11a) and unprotected glycoside 11d. In this vein, we anticipate this mode of catalysis will help inform the design and implementation of unique synthetic disconnections toward complex, bioactive targets in DELs.</p><p>Having developed suitable conditions for the hydroalkylation of unsaturated DEL platforms, attention was turned toward hydroarylation transformations. Recently, research efforts have validated Ni/photoredox dual manifolds in DEL platforms using carboxylic acids, 7,33b,33e 1,4-dihydropyridines (DHPs), 7 a-silylamines, 8 and alkyl bromides 8,38 as radical precursors. Given our long-standing interest in the design of complex (hetero)aryl scaffolds with high C(sp 3 ) carbon counts, 39 we sought to expand reactivity in DEL synthesis through intermolecular radicalmediated hydroarylation of functionalized olens to generate alkylarenes (Scheme 7). To develop a complementary approach toward C(sp 3 )-C(sp 2 ) bond formation, we reasoned that singleelectron reduction of DNA-bound, halogenated aryl subunits 26 would grant access to reactive (het)aryl radical species in a regioselective fashion. Subsequent addition to alkenes followed by hydrogen atom termination would deliver unprecedented structures from readily available substrates. Inspired by pioneering work by Beckwith 40 and related, precedented milestones, 26 we hypothesized that photoinduced electron transfer from highly reducing transition-metal-based complexes would enable this strategy under mild reaction conditions. However, because of the high redox potentials associated with organohalides and the propensity of aryl radicals to undergo reduction through rapid HAT, 26a the adaptation of this mechanistic proposal in DEL environments posed challenges. Importantly, aryl radicals have been shown to induce DNA strand damage, 41 underscoring the requirement for a regulated generation of these high-energy intermediates and the necessity for wellorchestrated addition reactions. To achieve chemo-and regioselectivity, the following criteria was considered: (i) the rate of (het)aryl radical addition to unsaturated systems must be competitive with C-X bond reduction stemming from undesired HAT pathways. (ii) The rate of hydrogen atom abstraction by the resulting alkyl radical must be competitive with its addition to another equivalent of the alkene. (iii) The rate of single-electron reduction of the aryl halide should take place preferentially over that of the alkyl radical intermediate. Specically, the choice of both photocatalyst and hydrogen atom donor inuences product distributions. We determined that 300 equiv. of the olenic substrate and a 1 : 200 photocatalyst-to-HAT reagent ratio was optimal for reactivity. Toward this end, the combination of fac-Ir(ppy) 3 and HE enabled the construction of alkylated arenes under air within minutes of blue light irradiation. Control experiments demonstrated that all reaction components are necessary for aryl radical generation.</p><p>With optimized conditions established, we surveyed DNAtagged (het)aryl halides with norbornene as the alkyl source (Scheme 7). Aryl iodide 13A bearing a chloride substituent afforded the desired product with the electrophilic crosscoupling handle intact, delivering linchpins for further functionalization. Electron-neutral iodobenzene 13B as well as derivatives bearing electron-donating groups (13C) or electronwithdrawing groups (13G) served as excellent substrates. Further extension to less activated aryl bromides was also Scheme 6 On-DNA multicomponent trifluoromethylation promoted by photoactive EDA complex activation. Note: several diastereomers are expected to form under these conditions. possible (13D). Notably, electrophilic pyridyl radicals were employed as coupling partners, giving rise to functionalized heteroaromatics (19a, 19j-19n). Finally, in addition to the strained bicyclic norbornene, a broad array of functionalized alkenes was examined. In general, unactivated alkenes bearing unprotected alcohols (15b, 15c, 19j), an ester (19k), ketones (15i, 19l), and an epoxide were all accommodated. In addition, this photochemical paradigm was extended to the modication of activated styrene derivatives (15e, 15g) in synthetically useful yields. Even benzylically triuoromethylated product 15g could be used as a substrate to afford product with complete retention of the bromide handle, presumably because of the high loading of alkene precursor compared to the photoredox catalyst, precluding an overreduction event of the halide. From the standpoint of DEL synthesis, which benets from minimal reagent input (e.g., 10-25 nmol of HP per transformation), such equivalencies can be leveraged to achieve selectivity and unique reactivity trends that are otherwise untenable in traditional small molecule synthesis. In particular, these halogenated alkenes can further grow DEL libraries through transition metal-catalyzed cross-coupling efforts.</p><!><p>Because the integrity of the DNA barcode is essential to a successful protein target selection, mock ligations and qPCR amplications were performed to evaluate the ability of the RAE hydroalkylation conditions to be used in an actual library production. A model DNA conjugate composed of a representative headpiece ligated to a 4-cycle tag and equipped with a 2base 5 0 overhang was subjected to the standard hydroalkylation conditions. This model DNA conjugate was also subjected to control reactions where either Hantzsch ester or light was omitted. Post reactions, these DNA conjugates were further elongated by ligation to install the necessary PCR primers and quantied by qPCR. There was no signicant difference in qPCR amplication across the various experiments, suggesting full DNA integrity (see ESI †). These ndings further underscore the utility of EDA paradigms as a general blueprint toward selective on-DNA alkylation under open-to-air conditions.</p><!><p>Mindful of well-established precedent of DNA strand damage in the presence of reactive aryl radical species, 41 the hydroarylation conditions were studied to evaluate the resulting DNA integrity. Again, a model DNA conjugate composed of a representative headpiece ligated to a 4-cycle tag and equipped with a 2-base 5 0 overhang was reacted using the standard conditions. This model DNA conjugate was also subjected to control reactions where either Hantzsch ester, photocatalyst, or light was omitted. Post reactions, these DNA conjugates were further elongated by ligation to install the necessary PCR primers and quantied by qPCR. There was no signicant difference in qPCR amplication across the various experiments, suggesting full DNA integrity (see ESI †). These results emphasize the mild nature of the developed photoredox paradigm, whereby the formation of reactive aryl radical intermediates in a regulated fashion facilitates productive on-DNA alkylation.</p><!><p>In summary, this report demonstrates a mechanistically-driven proof-of-concept for the implementation of charge-transfer complex activation as an enabling technology to introduce diverse C(sp 3 )-hybridized architectures from commodity chemicals in DEL platforms (including unactivated primary, secondary, tertiary, as well as stabilized benzylic, a-alkoxy, and a-amino systems). Specically, this EDA paradigm was utilized to achieve the selective decarboxylative-based hydroalkylation of triuoromethyl-substituted alkenes through radical/HAT crossover to access complex benzylic triuoromethylated scaffolds, unlocking a complementary reactivity outcome to established carbodeuorinative protocols mediated by an external photoredox catalyst. Furthermore, a general intermolecular hydroarylation protocol of electronically unbiased olens through selective C-X bond activation in DNA-tagged organohalides is reported. Remarkably, this photochemical paradigm delivers reactive (hetero)aryl radical species in a regulated fashion without compromising the DNA integrity. Notably, these open-to-air processes are chemoselective, operate under mild and dilute reaction conditions, and are completed within minutes, rendering them suitable for late-stage functionalization and high-throughput settings in the pharmaceutical industry. We anticipate these ndings will expedite drug discovery research and provoke further development in radicalmediated DEL synthesis.</p>

Royal Society of Chemistry (RSC)

Transferrin and H-ferritin involvement in brain iron acquisition during postnatal development: impact of sex and genotype

Iron delivery to the developing brain is essential for energy and metabolic support needed for processes such as myelination and neuronal development. Iron deficiency, especially in the developing brain, can result in a number of long-term neurological deficits that persist into adulthood. There is considerable debate that excess access to iron during development may result in iron overload in the brain and subsequently predispose individuals to age-related neurodegenerative diseases. There is a significant gap in knowledge regarding how the brain acquires iron during development and how biological variables such as development, genetics and sex impact brain iron status. In this study, we used a mouse model expressing a mutant form of the iron homeostatic regulator protein HFE, (Hfe H63D), the most common gene variant in Caucasians, to determine impact of the mutation on brain iron uptake. Iron uptake was assessed by using 59Fe bound to either transferrin or H-ferritin as the iron carrier proteins. We demonstrate that at postnatal day 22, mutant mice brains take up greater amounts of iron compared to wildtype. Moreover, we introduce H-ferritin as a key protein in brain iron transport during development and identify a sex and genotype effect demonstrating female mutant mice take up more iron by transferrin while male mutant mice take up more iron from H-ferritin at PND22. Furthermore, we begin to elucidate the mechanism for uptake using immunohistochemistry to profile the regional distribution and temporal expression of transferrin receptor and Tim-2, the latter is the receptor for H-ferritin. These data demonstrate that sex and genotype have significant effects on iron uptake and that regional receptor expression may play a large role in the uptake patterns during development.

transferrin_and_h-ferritin_involvement_in_brain_iron_acquisition_during_postnatal_development:_impac

Introduction:<!>H-ferritin Preparation<!>Protein Radiolabeling<!>Genotyping<!>Uptake Studies<!>Specific Activity<!>Immunohistochemistry<!>Western Blot<!>Statistical Analysis<!>Iron Uptake into the Developing Brain<!>Developmental Expression of Iron Related Proteins<!>Distribution of Iron Related Proteins During Development<!>Tim-2<!>Transferrin Receptor<!>H-ferritin<!>Discussion:

<p>Iron is a crucial micronutrient to the brain and is required for such fundamental processes as DNA synthesis, myelination, and cellular metabolism (MacKenzie et al. 2008; Black 2003). The developing brain not only requires a substantial amount of iron but it also must be delivered in a timely manner to establish time sensitive connections within the brain. In two studies of adult neuronal connectivity, Algarin and colleagues demonstrated altered brain connectivity patterns in former iron deficient anemia individuals, suggesting long-lasting effects in adult brain function as a result of iron deficiency in adolescence (Algarín et al. 2003; Algarin et al. 2017). Iron deficiency during development often results in long-term measurable phenotypes such as poor cognitive function, motor deficits, increased hearing impairment, and other neurological disorders (Connor et al. 2011; Connor et al. 2003; Dunham et al. 2017). Decreased iron levels may also lead to depressed energy production and metabolism (Zimmermann and Hurrell 2007). Elevated levels of iron can cause increased oxidative stress through Fenton Chemistry. Thus, understanding the regulation of iron uptake into the brain and the dysregulation that may occur with disease is critical to healthy brain function. Studies of iron uptake into the brain have mainly focused on the adult brain; iron uptake into the developing brain is a heretofore underexplored area of study that may hold important therapeutic strategies for iron supplementation and intervention to treat brain iron deficiency.</p><p>A common genetic mutation that results in iron accumulation in numerous tissues including in the brain is the H63D mutation of the human iron homeostatic regulator protein (HFE) which is now under investigation in a number of neurodegenerative conditions as a disease modifier (Liu et al. 2011; Nandar et al. 2013; Nandar et al. 2014). The H63D variant of the HFE gene is the most common genetic variation in Caucasians and appears in 1 in 200 people (Girouard et al. 2002). Wild-type HFE functions by limiting transferrin (Tf) interaction with transferrin receptor 1 (TfR1) at the cell membrane, while the mutant H63D form fails to limit Tf binding to TfR1 (Nandar and Connor 2011). The lack of interaction between the HFE mutant protein and TfR1 results in increased uptake of transferrin-bound iron into the cell. Previous studies by our laboratory have demonstrated the presence of HFE at the blood-brain barrier (BBB) (Duck and Connor 2016), demonstrating a role for HFE in regulating iron uptake both into the brain as well as the endothelial cells that comprise the BBB. Although the existing clinical and biological paradigm is that individuals with hemochromatosis are protected from brain iron overload by the BBB, recent MRI studies have found they contain higher brain iron loads (Nandar and Connor 2011). We have recently demonstrated that adult H67D (mouse homolog to human H63D) mutant mice have increased total brain iron at three months of age, but no difference in rate of brain iron uptake (Duck et al. 2018). These key findings are suggestive evidence for a critical set point in the adult brain that limits or at least highly regulates the rate of iron uptake into the brain. To build upon this previous study, we aim to examine iron uptake in the developing brain. The H67D mouse is an exciting model to interrogate the mechanisms and regulation of iron uptake into the brain.</p><p>Classical studies of iron delivery to the brain have mainly focused on the ability of Tf to deliver iron both during development as well as in the adult brain (Fishman et al. 1987; Taylor and Morgan 1990). However, we have recently demonstrated the ability for H-ferritin (FtH1), to cross the BBB (Chiou et al. 2018c; Chiou et al. 2018b) and provide a substantial amount of iron into the brain (Fisher et al. 2007). This is a significant departure from the established literature that posited FtH1 as simply an iron storage protein (Arosio et al. 2009). Furthermore, our previous studies have demonstrated that the receptor for FtH1 is the T-cell immunoglobulin and mucin domain 1 (Tim-1) in humans (Chiou et al. 2018a) and Tim-2 in rodents (Todorich et al. 2008; Han et al. 2011; Chen et al. 2005). In this study we have chosen to focus on FtH1 rather than L-ferritin, as we previously demonstrated minimal L-ferritin transport in a model of the BBB (Fisher et al. 2007). Furthermore, the only known receptors for L-ferritin are not present on the BBB, thus L-ferritin is not considered as a likely source of iron for the developing brain (Chiou and Connor 2018).</p><p>The goal of this study was to develop models that may impact brain iron uptake during development. The two variables under investigation were sex and genotype. Specifically, we determined if the presence of the H67D gene variant altered brain iron uptake during development. Moreover, given the evidence for FtH1 uptake in our cell culture models (Chiou et al. 2018a; Chiou et al. 2018c) and adult mice (Fisher et al. 2007), we compared FtH1 iron delivery with Tf during development. We hypothesized that the H67D genotype and sex influences the relative levels of uptake of iron during development similar to that seen in our previous study in adults (Duck et al. 2018; Wade et al. 2019). We have observed differences in adult iron uptake, and have determined in this study that these differences appear first during development. In addition to measuring uptake as a function of development, we also assessed the developmental expression of TfR, FtH1, and Tim-2 in order to begin to address and characterize the potential mechanisms for the differential uptake observed.</p><!><p>H-ferritin was prepared according to previously published methods (Todorich et al. 2008; Chiou et al. 2018a). Briefly, FtH1 with a poly-His tag was subcloned into a pET30a(+) plasmid, to be cloned into BL21 Escherichia coli. Isopropyl-β-D-thio-galactoside (IPTG, ThermoFisher #15529019) was used to induce expression. Subsequently, bacteria were lysed and FtH1 protein collected via nickel column according to the manufacturer's instructions (GE Healthcare Bio-Sciences #17526801). Protein concentration was determined via bicinchoninic assay (BCA, Pierce #23225) prior to use. Custom-made materials will be shared upon reasonable request.</p><!><p>We used our previously published methods for labeling Tf and FtH1 with 59Fe (Chiou et al. 2018c; Duck et al. 2017). Briefly, 59Fe (Perkin Elmer #NEZ037500) was complexed with 1 mM nitriloacetic acid (NTA, Sigma #N0253), 0.5 M sodium bicarbonate (NaHCO3), and 6.17 mM ferric chloride (FeCl3) at a ratio of 100 µL NTA: 6.7 µL FeCl3: 23.3 µL NaHCO3: 50 µCi 59FeCl3 to create the 59Fe-NTA complex, adjusted for cases where greater or less than 50 µCi was necessary. The 59Fe-NTA complex was incubated with iron poor transferrin (apo-Tf, Sigma #T1147) or FtH1 for 30 minutes to allow for iron loading. Free iron was subsequently separated from solution using G-50 Sephadex QuickSpin columns (Sigma #11273973001) according to the manufacturer's instructions.</p><!><p>Wild-type or H67D knock-in mice (Jackson Laboratory, Stock #: 023025, RRID:Hfetm1.1Jrco) were genotyped prior to uptake studies as described in our previous publications (Nandar et al. 2013; Nandar et al. 2014; Duck et al. 2018). Briefly, DNA was extracted from tail clips using a DNeasy blood and tissue kit (Qiagen). PCR was performed using forward primer (5' AGG ACT CAC TCT CTG GCA GCA GGA GGT AAC CA 3') and reverse primer (5' TTT CTT TTA CAA AGC TAT ATC CCC AGG GT 3'). PCR conditions used were: 94°C for 15 min followed by 39 cycles of 94°C for 45 sec, 58°C for 45 sec, 72°C for 90 sec, further followed by 72°C for 10 minutes. Amplified DNA was further digested using the BspHI restriction enzyme for 2 hours at 37°C and then separated by 2% agarose gel electrophoresis.</p><!><p>Uptake studies were performed as previously described, with mice sample size determined based on previous study results (Duck et al. 2018). Briefly, 5 wild-type males, 5 wild-type females, 5 H67D/H67D males, and 5 H67D/H67D females at postnatal day 7, 14, or 22 received a single intraperitoneal injection of 3.4 mg/kg body weight 59Fe-Tf or 59Fe-FtH1 (60 total mice per injection condition) (Fig. 1). 24 hours after injection, mice were anesthetized using a ketamine/xylazine cocktail (100 mg per kg body weight/10 mg per kg body weight), blood was drawn via cardiac puncture and mice were transcardially perfused with 0.1M phosphate-buffered saline (PBS, pH 7.4). Plasma was separated from whole blood fractions by centrifugation at 2000 x g for 15 minutes. Whole brains and liver were collected and weighed immediately, and radioactivity in liver, brain, and 50 µL plasma measured on a Beckman Gamma 4000 (Beckman Coulter). Blank tube values were subtracted from final counts to control for background counts. All procedures were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals, formally approved by the Pennsylvania State University College of Medicine International Animal Care and Use Committee under Protocol #45975, and reported in accordance with Animal Research: Reporting In Vivo Experiments. Mice are given food and water ad libitum with a maximum number of mice per cage of 5 in Cage type T II (Velaz). Mice were randomized by taking equal numbers of mice from each litter for injections, assigned by computer based randomization (Excel, RAND() function) wherein mice from each group (M vs. F, WT vs. H67D) were assigned to a list for injection of either 59Fe-Tf or 59Fe-Hft. RAND() assigns a random number between 0 and 1 to an animal and these animals were then sorted into equal groups of 5 using the =ROUNDUP(RANK(Animal, Range)/Group size, 0) function. Following this, the Sort function was used to group the mice into their respective groups prior to injection. Person assigning subjects to groups was blinded to genotype and injection protein. No exclusion criteria was pre-determined and no animals died.</p><!><p>Specific activity was calculated from counts per minute (CPM) and reported as moles of iron per gram of tissue. Calculations were as follows: 10,000 CPM at a counting efficiency of 80% results in 12,500 disintegrations per minute (DPM). As stated previously, mice were injected with 3.4 mg/kg of either 59Fe-Tf or 59Fe-FtH1. Specific activity for 59Fe-Tf injection as DPM per mol: Tf at PND7 = 7.81*1013, PND14 = 4.56*1013, and PND22 = 3.15*1013. Specific activity for 59Fe-FtH1 injection as DPM per mol: FtH1 at PND7 = 4.42*1014, PND14 = 2.58*1014, and PND22 = 1.78*1014. To obtain moles iron per gram tissue, DPM per gram tissue obtained from the radioactivity measurements was divided by DPM/mol Tf or DPM/mol FtH1. Subsequently, DPM/mol protein was multiplied by either 2 or 121 for Tf and FtH1 respectively. This correction factor was applied because each Tf molecule can hold 2 iron atoms, while we have previously determined that 30 minutes of loading FtH1 with iron results in 121 atoms of iron within the core (Chiou et al. 2018b).</p><!><p>Mice at postnatal day 7, 14, or 22 were sacrificed and transcardially perfused first with Ringer's solution followed by 4% paraformaldehyde. Brains were isolated and moved into a 4% paraformaldehyde solution overnight. Subsequently, brains were transferred to a 0.1 M PBS solution until one day prior to paraffin embedding, when they were immersed in 70% ethanol. Brains were embedded in paraffin, sagittal sections were cut at 5 µm thickness, and sections mounted on standard microscopy slides. Immunohistochemistry was performed as previously described (Snyder et al. 2009). The slides were deparaffinized in xylene and rehydrated in an ethanol gradient followed by a dH2O rinse. Antigen retrieval was performed with 10 mM citrate buffer (pH = 6.0). The slides were blocked in a 3% H2O2/methanol solution for 20 minutes, rinsed with 1X PBS, and blocked in a 2% milk/PBS solution for 1 hour. The slides were then incubated in primary antibody overnight at 4°C using anti-Tim-2 (10 µg/mL, R&D Systems, MAB1885, RRID:AB_2201833), anti-TfR (2 µg/mL, Thermo Fisher Scientific, 13–6800, RRID:AB_2533029), anti-CC1 (1:100, Calbiochem, OP80, RRID:AB_2057371), anti-Iba1 (1:200, Wako, 019–19741, RRID:AB_839504), anti-GFAP (1:1000, Agilent, Z0334, RRID:AB_10013382) or anti-FtH1 (1 µg/mL, Cell Signaling, 4393) primary antibodies. The following day, the slides were washed with PBS and secondary antibodies (1:200) were applied using an ABC Vectastain kit (Vector Labs, RRID:AB_2336827) in accordance with manufacturer's protocol or using species-specific AlexaFluor488 and AlexaFluor555 secondary antibodies (Life Technologies). The slides treated with ABC Vectastain secondary antibodies were visualized with diaminobenzidine (DAB) application using standard procedure. DAB staining was intensified via the addition of nickel chloride to the DAB solution. Following DAB staining or fluorescent secondary antibody incubation, slides were mounted, coverslipped, and allowed to dry overnight. Fluorescent slides were mounted with Prolong Diamond Antifade Mountant with DAPI (ThermoFisher #P36962). All slides were imaged using Aperio AT2 Leica slide scanner with a 40X objective. Images were viewed using Aperio ImageScope software.</p><!><p>Western blots were performed as previously described (Chiou et al. 2018a). Briefly, 20 µg protein solubilized in RIPA buffer (Sigma #89900) was quantified by bicinchoninic assay prior to boiling for 10 minutes, loading onto a denaturing 4–20% gradient SDS-PAGE gel (BioRad #4561094), and subsequent transfer to nitrocellulose membrane. Membranes were blocked in a 5% milk solution made in Tris-buffered saline and Tween-20 (TBS-T, pH 7.6). Blots were probed for Tim-2 (2 µg/mL, R&D Systems, RRID:AB_2201833), TfR (1 µg/mL, Thermo Fisher Scientific, RRID:AB_2533029), FtH1 (1:500, Cell Signaling, 4393), or β-actin (1:3000, Sigma-Aldrich, A178, RRID:AB_476692) overnight at 4°C in a 5% milk/TBS-T solution. Corresponding species-specific secondary antibody conjugated to HRP was used (1:5000, GE Amersham), using ECL reagents (Perkin Elmer) to visualize bands on an Amersham Imager 600 (GE Amersham) with densitometry analysis using ImageJ. The sum total of all bands was used in the quantification, because the additional bands are thought to be dimers of the proteins (near exact molecular weight matches). Shown are representative western blots. Western blots were repeated 3 times for each timepoint and each band represents homogenates pooled from 3 male and 3 female mice.</p><!><p>Statistical analyses were performed using GraphPad Prism 4 Software (Graphpad Software Inc.). Data from biological replicates were averaged and are expressed as the mean ± standard deviation (SD). Specific numbers of replicates are found in each figure legend. Data were tested for normality using the Shapiro-Wilk test; data were normally distributed thus parametric statistics were used. Two-way ANOVA with Bonferroni post-hoc analysis or unpaired t-tests were used to evaluate for statistical significance where appropriate. A p-value <0.05 was considered significant. ROUT test with a Q=1% was performed in Prism for all data, but no animals or data were excluded.</p><!><p>To measure the uptake of iron into the brain, we loaded either Tf or FtH1 with 59Fe and injected the proteins intraperitoneally. In our first study, we examined the levels of iron uptake into the developing brain by both 59Fe-Tf and 59Fe-FtH1 (Fig. 2). Uptake of Tf peaked at PND14 and decreased at PND22. Overall, 59Fe-FtH1 uptake was significantly higher, with a peak uptake for wild-type mice at PND14 and a peak uptake for H67D mice at PND22 (Fig. 2A). There was a genotype and sex effect at PND22 for both Tf and FtH1: H67D mice had a significantly higher amount of uptake than wild-type mice at PND22, however, the increased iron uptake was from Tf in the female H67D mice (Fig. 2B) whereas in the male H67D mice there was significantly increased uptake of 59Fe-FtH1 (Fig. 2C).</p><p>We also measured the amount of 59Fe retained by the liver. At PND14, wild-type mice take up a significantly greater amount of 59Fe-FtH1 into their livers compared to the H67D mice (Fig. 3A). Furthermore, at PND22, H67D mice took up a greater amount of 59Fe-Tf into their livers compared to wild-type mice. At PND7, we demonstrate that both male and female wild-type mice livers take up significantly more 59Fe-Tf than their H67D counterparts (Fig. 3B). There was also a sex and genotype effect of FtH1 delivery of iron; male wild-type take up more 59Fe-FtH1 than male H67D mice into their livers on PND7, whereas female H67D mice take up more iron into their livers than wild-type mice on PND22 (Fig. 3C). While there were no significant differences in plasma levels of 59Fe-Tf, 59Fe-FtH1 levels in plasma at PND7 was significantly higher in H67D mice whereas at PND22, wild-type mice had more 59Fe-FtH1 in plasma than the H67D mice (Fig. 4A). There were no sex differences in the plasma accumulation levels of iron for either FtH1 or Tf (Fig. 4B, C).</p><!><p>To examine the mechanism of uptake of Tf and FtH1, we performed a characterization of the expression profiles of the receptors. For this, we used a separate group of mice that did not have any injections and collected whole brains at PND7, 14, and 22. To begin, we used western blot analyses on whole brain lysates to probe for levels of Tim-2, the receptor for FtH1 in mice (Todorich et al. 2008; Chen et al. 2005) (Fig. 5A), and TfR (Fig. 5B). We also probed for FtH1 as a surrogate for the amount of iron stored (Fig. 5C), with β-Actin used as a loading control (Fig. 5D). At PND7, there was significantly more TfR and FtH1 in the H67D mouse brains compared to wild-type mice but no difference in Tim-2 expression (Fig. 5E). At PND14 there were no significant differences between wild-type and mutant mice in any of the proteins measured. At PND22, Tim-2 was significantly increased in the H67D mouse brains compared to wild-type (Fig. 5E). Throughout development in both genotypes, Tim-2 expression was highest at PND7 and 14 and decreased at PND22. Wild-type TfR expression peaked at PND14 whereas the inverse was true in H67D mice – PND14 had the lowest amount of observed TfR. FtH1 expression remained consistent throughout development.</p><p>Next, we examined the cellular distribution of Tim-2 in vivo. Though we have previously published evidence for the presence of Tim-2 on mouse oligodendrocytes which is able to mediate FtH1 uptake (Todorich et al. 2008), we wanted to determine if other cell types may use Tim-2 as a receptor for FtH1 during development. Consistent with our previous results (Todorich et al. 2008), we found robust Tim-2 staining on CC1-positive cells, denoting mature oligodendrocytes (Fig. 6A). We did not observe any Iba1-positive cells that were positive for Tim-2 (Fig. 6B, white arrows). We did observe that astrocytes robustly stain for Tim-2 (Fig. 6C). Throughout all the stains, we observed strong staining for Tim-2 in cells that were negative for CC1, Iba1, and GFAP, suggesting neuronal staining.</p><!><p>In the following study, we examined the regional distribution and expression of Tim-2 (Fig. 7), TfR (Fig. 8), and FtH1 (Fig. 9) in the brain during development.</p><!><p>At PND7, 14, and 22, Tim-2 staining was present throughout the cortex in both wild-type and H67D mice (Fig. 7A). Though we observed very sparse staining in cortical layer 1, immunostaining in cortical layers 2, 3, 4, 5 and 6 was robust (data not shown). Of note is the localization of Tim-2 to the borders of the cell body, especially in the large pyramidal neurons of layer 4 and 5. Consistent with our previous studies (Todorich et al. 2008), there is robust Tim-2 staining in oligodendrocytes of the corpus callosum arranged in a tram-track orientation at PND7 (Fig. 7B). By PND14 and PND22, we observed similar clear staining of the corpus callosum. Lastly, we observed Tim-2 staining at the microvasculature throughout the brain at PND7 (Fig. 7C), which persisted throughout development (data not shown). We also observed robust staining in the Purkinje cells of the cerebellum, hippocampus, thalamus, and substantia nigra (data not shown). At all timepoints we measured, we observed no regional differences in Tim-2 staining between wild-type and H67D mice.</p><!><p>TfR staining at PND7 in the corpus callosum was very light in both wild-type and H67D mice (Fig. 8A). Similarly, staining for TfR at PND14 was also very lightl, and this staining was decreased at PND22. At PND7, we observed staining for TfR in the microvasculture (Fig. 8B) which persisted at PND14 and PND22 (data not shown), consistent with previous literature demonstrating TfR at the level of the BBB (Simpson et al. 2015). We also observed extremely dense staining in all cortical layers, the thalamus, hippocampus, substantia nigra, and cerebellum throughout development (data not shown). At all timepoints we measured, we observed no regional differences in TfR staining between wild-type and H67D mice.</p><!><p>In stark contrast to Tim-2 and TfR expression, FtH1 staining, used here as a surrogate marker for intracellular iron, was relatively sparse except in specific regions. Throughout the developmental timepoints we examined, FtH1 was consistently highest in the corpus callosum (Fig. 9A) and was present in the microvasculature (Fig. 9B). At PND22 in the corpus callosum, staining is very heavy for FtH1, especially in what appear to be mature oligodendrocytes. Similar to Tim-2 and TfR, FtH1 staining was observed heavily at PND7 which persisted throughout development (data not shown). We also examined the cortex, thalamus, hippocampus, substantia nigra, and cerebellum for FtH1 immunostaining, finding that FtH1 was heavily localized to cortex layer 4 and the white matter throughout the brain (data not shown). However no regional differences between wild-type and H67D mice were observed.</p><!><p>The aim of this study was to examine the uptake of FtH1 bound iron as a novel iron delivery system to the developing brain. Furthermore, we determined if sex or genotype could alter the uptake of Tf or the newly discovered FtH1 delivery system. The results of this study have demonstrated that FtH1 can deliver a significantly higher amount of labelled iron during development than transferrin. These data further demonstrate that brain iron acquisition from both transport proteins are modified by age, sex, and genotype. While it has previously been shown that in general, liver and plasma iron levels tend to correspond in normal humans and HFE hemochromatosis patients (Olynyk et al. 2008; Graham et al. 2010), our single time point data demonstrate that after injection of extra iron via different carriers, we find a significant difference in the handling of this iron by H67D and wild-type mice.</p><p>For Tf uptake, the transport of iron appears highly regulated (Simpson et al. 2015), increasing at PND14 and decreasing thereafter. The decrease in brain iron uptake occurs even in the presence of a gene variant that is associated with increased iron uptake by all organs in the body including the brain (Nandar et al. 2013). For FtH1 uptake, the transport of iron into the brain does not decrease after PND14 and in the H67D mouse, FtH1 uptake continues to increase with age. There is also a sex difference as males with the H67D variant show an increase, rather than a decrease, in FtH1 uptake between PND7 and PND22. Similarly, female H67D mice take up more Tf at PND22 than male H67D mice. These results clearly demonstrate the regulation of uptake of iron from FtH1 is distinct from Tf and furthermore that regulatory mechanisms between males and females may have significant differences. Our data demonstrate that gross levels of TfR in the brain do not necessarily follow the same temporal pattern of brain iron uptake, pointing towards a delay in and possibly more regional regulation of uptake at the BBB as we have proposed (Chiou et al. 2018c). We have previously published that FtH1 and Tf transport across the BBB share a common regulatory mechanism mediated by apo-Tf and holo-Tf (Chiou et al. 2018c), suggesting that this axis may be of critical significance during development. We have also begun to examine the mechanism of uptake into the brain by profiling the different cell types of the brain and various brain regions for the receptors for Tf and FtH1 during development. By tracking these receptors during development, we have demonstrated that the uptake mechanism for iron delivered via Tf and FtH1 is present. Our data showing both Tf and FtH1 delivery to the brain during development are consistent with the demonstration that both of these proteins are involved in brain iron delivery in the adult (Duck et al. 2018).</p><p>This study shows that the temporal pattern of delivery of Tf and FtH1 to the developing brain is similar. There is peak uptake of Tf at PND14, with a decrease at PND22. Our data for Tf-mediated uptake of iron are consistent with previous studies that demonstrate a similar uptake pattern during development (Moos and Morgan 2002). Though the general decline in uptake occurs in both wild-type and H67D mice, female H67D mice take up significantly higher amounts of 59Fe-Tf than the other groups. In contrast to Tf, FtH1 uptake does not decline after PND14 in either the wild-type or H67D mouse. Furthermore, male H67D mice significantly increase the amount of 59Fe-FtH1 that they take up at PND22 compared to wild-type. The different patterns of uptake for Tf and FtH1 delivered iron suggest that Tf could be providing iron more acutely to aid in the a growth spurt of the brain that accompanies peak myelination (Hulet et al. 2002) whereas FtH1 provides iron at a steadier pace for maintence of required levels for normal brain function. For example, oligodendrocytes are the highest iron staining cells in the brain (Benkovic and Connor 1993; Connor et al. 1995) and the ability of these cells to take up a substantial amount of iron during development is necessary for proper myelination; inadequate iron delivery to oligodendrocytes frequently results in hypomyelination (Todorich et al. 2009). We have reported that FtH1 is the major source of iron for mature oligodendrocytes (Todorich et al. 2011). This study provides further evidence that FtH1 plays a significant role in delivery of iron to various organs throughout the body (Blight and Morgan 1983; Kim et al. 2013; Fisher et al. 2007; Sibille et al. 1989). While we have previously demonstrated the ability of FtH1 to deliver iron to the adult rat brain (Fisher et al. 2007), this study is the first to demonstrate FtH1 delivery during development in a mouse. We have previously shown that the FtH1 in our preparation results in at least 10 times as much iron loaded as transferrin/mole (Chiou et al. 2018b). Conceptually, FtH1 could contain 4500 atoms of iron per mole or 2000 times more iron than transferrin (Arosio et al. 2017). The source of FtH1 that may be taken up in the brain has yet to be elucidated but studies have shown FtH1 is released by multiple cell types (Chiou and Connor 2018).</p><p>In this study, we performed immunostaining for intracellular FtH1 to understand the regional expression of FtH1 in the brain during development as a surrogate marker for cellular iron status (Fig. 9). It was noted that FtH1 staining becomes more punctate and region-specific during development, suggesting that there is a higher utilization of iron in specific regions of the brain during development. This suggests future studies on regional brain delivery via FtH1 are warranted. The data supported previous reports that oligodendrocytes and glial cells of the white matter tracts are the cells that accumulate the most iron (Connor et al. 1992; Connor and Menzies 1996) and FtH1 (Schonberg et al. 2012; Hulet et al. 1999a; Hulet et al. 2002). Here, we expand those findings and demonstrate that specific neurons in the hippocampus and pyramidal neurons in the cortex stain for FtH1 as well. That FtH1 staining in oligodendrocytes is highest during peak myelination strongly coincides with the necessity of iron for the myelination process and supports the reports that chronic iron deficiency frequently leads to hypomyelination in both rodent (Ortiz et al. 2004) and human models (Roncagliolo et al. 1998; Algarín et al. 2003). Consistent with these results, we confirmed the presence of Tim-2 for iron acquisition via extracellular FtH1 on oligodendrocytes (Fig. 6A).</p><p>We also demonstrate that there is a sex effect on iron uptake, a phenomena we have described previously in the adult H67D mouse (Duck et al. 2018); female H67D mice accumulate more iron in their brain than male H67D mice (Duck et al. 2018). At the earlier time points the pattern of iron uptake from either FtH1 or Tf was similar between the different sexes and genotypes. However, at PND22, there is increased Tf-iron uptake in the H67D female mice compared to the other groups, whereas FtH1-mediated iron uptake is greater in the H67D males compared to the other groups. These uptake data may account for reports of a higher volume of total brain iron in males (Hahn et al. 2009) compared to females, with the caveat that our differences were only seen in the mutant mice. We have also previously published that the adult H67D mouse brain has a higher overall iron load (Nandar et al. 2013; Duck et al. 2018), suggesting that this imbalance in iron uptake is established at PND22 and persists into adulthood. Overall, this is the first study to reveal a sex and genotype effect on brain iron acquisition during development. The mechanism regulating these uptake differences, which is occurring prior to hormonal influences associated with puberty will be explored in future studies.</p><p>Our data further support the concept that iron uptake is tightly regulated and suggests the potential for a set-point for iron delivery that is established during development and maintained throughout adulthood. We propose there is a critical window during which the brain is growing that is amenable to relatively high levels of iron uptake. This concept is evident in studies on nutritionally iron deficient animals where iron supplementation started at PND4 can correct brain iron deficiency yet supplementation at PND21 was not able to correct early iron losses (Felt et al. 2006; Beard et al. 2006; Unger et al. 2012). Our data suggest that once significant brain growth has plateaued, there are regulatory molecules such as apo-Tf and holo-Tf (Chiou et al. 2018c) from the brain signaling to the endothelial cells of the BBB regarding brain iron status. The observation that the H67D mutation and sex both affect iron uptake by Tf and FtH1 demonstrates that multiple factors exist in tandem to influence the regulation of iron uptake. Importantly, we demonstrate that the H67D mutation may result in an aberrant set-point, as there is higher uptake of 59Fe-Tf and 59Fe-FtH1 at PND22 than wild-type. This point is further illustrated in the adult brain, as brain iron in the H67D adult mouse brain is significantly higher than wild-type yet iron accumulation in the brain 24 hours after injection with 59Fe-Tf does not differ from wild-type (Duck et al. 2018). This implies that the H67D mouse brain has a higher set-point for iron uptake than the wild-type, and this persists into adulthood. Furthermore, it has been shown that H67D mutant mice have altered brain iron handling proteins such as TfR, H- and L-ferritin, and Tim-2 (Nandar et al. 2013), suggesting that in these mice the altered brain iron profile is a result of altered regulation.</p><p>The data from this study could have significant potential implications in the clinical treatment of brain iron deficiency during development. The data herein demonstrates the concept that in the mouse there may be a specific time during normal development that iron supplementation may be most effective, as Tf uptake decreases after PND14 while FtH1 uptake levels off at PND14. This is significant, as it is still controversial regarding at what age iron supplementation should be started in the treatment of iron deficiency, or if iron supplementation in an early age would lead to adult disease phenotypes. For example, there are studies demonstrating that in mice, early exposure to iron overload during a critical period of development (PND10–17) may result in higher rates of a Parkinsonian disease phenotype (Billings et al. 2016). Similarly, Kaur et al. demonstrate that increased neonatal iron intake results in adult Parkinsonian phenotypes (Kaur et al. 2007). Furthermore, in β-thalassemia patients, iron supplementation in children leads to impaired neurocognitive function (Elalfy et al. 2017). The ability to override the set point and normal regulatory mechanisms for brain iron uptake in these models could be due to significant inflammation and subsequent compromise in BBB integrity that would be associated with infusion of intravenous iron or gavage of carbonyl iron molecules (Varatharaj and Galea 2017; Chiou and Connor 2018). While needing further studies to fully explore this concept, these results may impart specific directions for clinicians to follow in the treatment of developmental iron deficiency.</p><p>An unexpected finding in this study was the identification of Tim-2 on neuronal cells. We have previously identified Tim-2 as the receptor for FtH1 on oligodendrocytes (Todorich et al. 2008); indeed, FtH1 can replace Tf as the obligate source of iron for oligodendrocytes (Todorich et al. 2011). The presence of Tim-2 on neurons may then represent a novel iron uptake mechanism in neurons. Previous literature has identified neuronal acquisition of iron through a Tf-mediated pathway (Leitner and Connor 2012) or through voltage-gated calcium channels (Lopin et al. 2012), but the presence of Tim-2 may suggest FtH1 uptake in neuronal cells as well. An alternative explanation for the high abundance of Tim-2 on neurons may lie in the other function of Tim-2 as a receptor for Sema4A (Kumanogoh et al. 2002). During neural development, the semaphorin family acts as negative regulators of neural migration, causing growth cone collapse (Yukawa et al. 2005). It has also been shown that Sema4A, a semaphorin family member, can also bind to Tim-2 (Kumanogoh et al. 2002). Other binding partners to Sema4A, namely Plexin D1 (Toyofuku et al. 2007), Plexin B2 (Ito et al. 2015), and Neuropilin-1 (Delgoffe et al. 2013), have been demonstrated to facilitate axon guidance during neural development. While we have previously shown Sema4A to be cytotoxic to oligodendrocytes (Chiou et al. 2018a; Chiou et al. 2019), it is possible that the presence of Tim-2 on neurons may lead to axonal guidance either through a pro-apoptotic mechanism or through the classical Rho/vascular endothelial growth factor (VEGF) signaling pathway (Zhou et al. 2008). A second explanation may lie in the possibility for Tim-2 to non-covalently dimerize and form a separate binding site that has potentially different effects on the cell (Santiago et al. 2007). This dimerization effect may be specific to oligodendrocytes or neurons. A different function for Tim-2, and perhaps altered ability to bind to FtH1 would explain the differences between the immunostaining data for Tim-2 and a ferritin binding study performed by Hulet et al. that demonstrated ferritin binding localizes specifically to the white matter tracts and not the gray matter regions (Hulet et al. 2002; Hulet et al. 1999b).</p><p>In addition to neurons, robust Tim-2 staining was found in astrocytes and brain microvasculature throughout the development time periods interrogated (Fig. 6C). For the microvasculature, the presence of Tim-2 in endothelial cells of the BBB demonstrates a mechanism for uptake of FtH1 into the brain parenchyma. We have previously demonstrated that FtH1 can be taken up and directly cross the endothelial cells and that the human receptor for FtH1 on the BBB is Tim-1 (Chiou et al. 2018c). The abundance of Tim-2 in the BBB is further consistent with previous literature documenting FtH1 transport in vivo (Fisher et al. 2007). For astrocytes, like neurons, the presence of Tim-2 may indicate a novel iron uptake mechanism. It has been documented that astrocytes can obtain iron through a Tf-mediated mechanism in vitro (Qian et al. 1999), but the presence of Tim-2 on astrocytes may represent an alternative method for iron uptake through FtH1. Interestingly, we found that microglia, the resident immune cells of the brain, do not express Tim-2 (Fig. 6B). Previous studies have determined that CXCR4, a microglial chemokine receptor, can bind and interact with FtH1 (Li et al. 2006) suggesting that microglia may have an alternative mechanism to take up` FtH1.</p><p>In this study, while we have focused primarily on iron uptake into the brain and transport across the BBB, it is important to also consider the effects of efflux from the BBB and the brain itself. Iron efflux is typically handled by ferroportin, the only known free iron exporter, and hepcidin, a hormone that causes the degradation of ferroportin. We have previously demonstrated that hepcidin significantly impacts iron transport across the BBB in a cell culture model (Chiou et al. 2018c) and it is likely there is a similar mechanism in vivo as well. It has previously been shown that HFE plays a role as an upstream regulator of hepcidin (Pantopoulos 2008), while studies have also shown that hepcidin expression decreases in iron loaded Hfe-knockout mice (Bridle et al. 2003). In the developing brain, there is little currently known about the interplay between hepcidin levels and how a mutation in HFE would impact its' expression and subsequently, iron uptake. Previous studies have shown that hepcidin is indeed present throughout the brain in the adult rodent (Zechel et al. 2006; Raha-Chowdhury et al. 2015), but a comprehensive analysis of hepcidin expression during development has yet to be performed. This is clearly an opportunity for additional study to interrogate another mechanism revolving around the ferroportin-hepcidin axis that may contribute to the observed increased brain iron uptake in the H67D mice.</p><p>Overall, these data provide strong evidence for a sex and genotype effect of iron uptake into the brain during development and begin to elucidate the mechanism for differential iron uptake. Importantly, this study provides developmental snapshots of Tim-2, TfR, and FtH1 expression in critical regions of the brain, demonstrating a temporal aspect for receptor expression. Moreover, the identification of Tim-2 on neurons and astrocytes represents a heretofore unexplored area of research that has strong implications for FtH1 uptake and Sema4A-mediated axonal guidance.</p>

PubMed Author Manuscript

Success of Montreal Protocol Demonstrated by Comparing High-Quality UV Measurements with “World Avoided” Calculations from Two Chemistry-Climate Models

the Montreal protocol on Substances that Deplete the ozone Layer has been hailed as the most successful environmental treaty ever (https://www.unenvironment.org/news-and-stories/story/ montreal-protocol-triumph-treaty). Yet, although our main concern about ozone depletion is the subsequent increase in harmful solar UV radiation at the earth's surface, no studies to date have demonstrated its effectiveness in that regard. Here we use long-term UV Index (UVI) data derived from high-quality UV spectroradiometer measurements to demonstrate its success in curbing increases in UV radiation. Without this landmark agreement, UVi values would have increased at mid-latitude locations by approximately 20% between the early 1990s and today and would approximately quadruple at midlatitudes by 2100. In contrast, an analysis of UVI data from multiple clean-air sites shows that maximum daily UVI values have remained essentially constant over the last ~20 years in all seasons, and may even have decreased slightly in the southern hemisphere, especially in Antarctica, where effects of ozone depletion were larger. Reconstructions of the UVi from total ozone data show evidence of increasing UVI levels in the 1980s, but unfortunately, there are no high-quality UV measurements available prior to the early 1990s to confirm these increases with direct observations.Concern about ozone depletion arose primarily because of its potential to increase UV-B radiation, and the consequent effects on health and the environment 1 . Observations of unexpected springtime decrease in stratospheric ozone over Antarctica 2 -commonly referred to as the "ozone hole" -led to the rapid adoption of the Montreal Protocol on Substances that Deplete the Ozone Layer in 1987 3 . It has been shown that without this treaty and its subsequent Amendments and Adjustments, ozone holes would by now also be occurring over the North Pole, resulting in highly elevated UVI in the Arctic 4 . Even at mid-latitudes, the UVI would by now have increased markedly, and would more than double at some latitudes by the middle of the 21 st century [5][6][7] . However, due to the success of the Montreal Protocol, ozone decreases appear to have been brought under control 8 , though any effects on surface UV irradiances have not previously been confirmed by measurements.We attempt to redress this issue by using an analysis of long-term UV measurements from instruments that meet the stringent requirements of the Network for the Detection of Atmospheric Composition Change (NDACC, www.ndacc.org).

success_of_montreal_protocol_demonstrated_by_comparing_high-quality_uv_measurements_with_“world_avoi

<!>Results from Model Simulations<!>Model<!>UVi in summer.<!>Decadal trends in UVi.

<p>Observatory global irradiance measurements were available every 30 min prior to September 2010, and every 15 min after that date. At Villeneuve d' Ascq, scans are performed every 30 min.</p><p>We considered whether to use peak daily values, peak values during a specified time window (e.g., ±1 hour of local noon), or mean values over the noon period (e.g., ±0.5 hour of local noon). Variations in seasonal mean UVIs calculated from the three quantities agreed to within ±3%, which is smaller than the measurement uncertainty of ±5% 10 . By using peak values instead of mean values, cloud effects on seasonal averages are reduced and time-series derived from measurements therefore agree slightly better with our modelling results, which do not take attenuation from clouds into account. The following analysis is therefore based on seasonal means of the daily maximum UVI observed within ±1 hour of local noon (12:00 UTC at Greenwich; 00:45 UTC at Lauder, etc). Data gaps were treated as described by Bernhard 18 . In brief, single missing days were "filled in" by calculating the average of the UVI of adjacent days. Data gaps lasting for more than one day were corrected by taking climatological variations in SZA and ozone into account. If more than 30 days were missing within a 90-day period, seasonal means were excluded from further analysis.</p><p>Seasonal changes in UVI are large at mid-to high-latitudes. For latitudes poleward of 45°, UVI values in winter are less than 10% of the summer peaks. And in polar regions, the UVI reduces to zero during the polar night. Additionally, there are large day-to-day differences in UVI due to changing cloud conditions and types. To minimize these effects, we consider seasonal means of approximately 90 days (December-February, March-May, June-August, and September-November), which are also more relevant from an environmental perspective.</p><p>Measurements of UVI from the sites in Table 1 are compared with values calculated for clear skies for four different ozone scenarios:</p><p>(1) ozone values from the NIWA/BS global total-column ozone assimilation, based on measurements between 1978 and today 19 , as described at www.bodekerscientific.com. (2) ozone values projected in the World Avoided scenario from 1974 to 2065 6 , which was calculated using the GEOS-CCM (Goddard Earth Observing System -Chemistry Climate model (NASA, USA)). ( 3) ozone values projected in the World Avoided scenario from 1974 to 2100 7 which was calculated with the NIWA-UKCA (NIWA-UK Meteorological Office Climate Assessment) model. (4) ozone values projected for the World Expected scenario, as calculated with the NIWA-UKCA model. This scenario is the REF-C2 experiment described by Morgenstern et al. 20 . In this simulation, ozone-depleting substances follow the "WMO (2010) A1" scenario, which assumes compliance with the Montreal Protocol.</p><p>Differences between the two CCM models are summarized in Table 2. Details of both models are given elsewhere 16,[20][21][22][23][24] .</p><p>We note that future changes are dependent on the RCP scenario, and in particular depend on future changes in CO 2 , CH 4 , and N 2 O 25 . For example, a strong overshoot in ozone would be expected for RCP 8.5, and by the end of the 21 st century, differences between N 2 O and CH 4 scenarios may account for differences in ozone larger than 5%. For all scenarios, the clear-sky UVI at noon was interpolated from a 5D table of UVI as a function of ozone amount, solar zenith angle (SZA), altitude (pressure), aerosol optical depth, and surface albedo that had been pre-computed with the discrete-ordinate 26 implementation in the TUV radiative transfer model 27 . Subsequent corrections were made to account for seasonal variations in Earth-Sun separation. In the World Avoided scenarios (Scenarios (2) and ( 3)) and World Expected scenario (Scenario (4)), the UVI was calculated for sea level with no aerosols and low surface albedo. Although both SO 2 and NO 2 can affect UVI, they have not been included in the clear-sky calculation. With the exception of San Diego, Thessaloniki, and Melbourne, sites considered in this paper are "clean-air" sites where the effect of absorption by these trace gases can be considered negligible.</p><p>To better approximate the measurements, corresponding UVI calculations using the ozone data at each site (Scenario (1)) were computed with TUV for the altitude at each site, assuming an aerosol optical depth of 0.05 (at wavelength 0.5 µm) with a single scattering albedo of 0.90 for all sites, and a representative (annually invariant) surface albedo, ranging from 0.05 at snow-free sites, to 0.98 at the South Pole.</p><p>As shown below, the comparison between the measured UVI data and the UVI data calculated from the assimilated ozone dataset (Scenario (1)) demonstrates that year-to-year variability in the UVI can be estimated with sufficient accuracy from ozone changes. The observed good agreement gives us confidence that UVI calculated from ozone for the period prior to the 1990s, when no direct UVI measurements are available, can be used to infer changes in UVI over this period. The comparisons with the two World Avoided scenarios were included to demonstrate the divergence between what has actually happened compared with what would have happened without the Montreal Protocol, using two independent model calculations.</p><!><p>We begin by comparing projected ozone fields from the two World Avoided models (Scenarios (2) and ( 3)).</p><p>Predicted ozone values from these two models are compared for selected latitudes in Fig. 1. The two models are in general agreement in long-term behaviour, but also show significant differences over shorter time scales, especially at low latitudes. Close agreement is not expected at time scales of less than 5 years because there is a random component to the way modes of variability such as the quasi biennial oscillation (QBO) manifest themselves in the model runs. The systematic differences between the two models are within the usual range for chemistry-climate models 28 .</p><p>Modelled ozone data for Scenarios (2), ( 3) and (4) were compared with measured ozone data (Scenario (1)) and results are provided as Supplementary Data. In general, modelled and measured total ozone column amounts (TOCs) agree reasonably well. For the reference period 1978-1987 (i.e., the period between the year when ozone measurements from space became available globally and the year when the Montreal Protocol came into effect), TOCs calculated with the NIWA-UKCA model (both World Expected and World Avoided) for sites between 45°S and 45°N -which represents 71% of the globe -are on average 8 ± 4% (±1σ) higher than the measured TOCs. The maximum bias for this latitude range is 14%. TOCs calculated with the GEOS World Avoided model are slightly smaller. It overestimates the measured TOC by 2 ± 5% on average, with a maximum bias of 12%. Deviations between the modeled and measured TOCs become larger at higher latitudes, especially in the southern hemisphere. In this latitude range, differences can exceed 20% with biases from GEOS model exceeding those from the NIWA-UKCA model. These larger biases are partly a consequence of the low ozone amounts there and partly due to the difficulty to correctly model the destruction of ozone by heterogeneous chemical processes, which strongly depend on the temperature of the lower stratosphere. Furthermore, specific sites are not necessarily well represented by the zonal means used in the model. This is especially true for high-latitude southern hemisphere sites (i.e., Ushuaia and Palmer Station) in spring when the position of the ozone hole is frequently displaced toward the South American quadrant. Some of the discrepancy may also be due to interpolation errors in the ozone assimilation during the polar night, but this is irrelevant for changes in the UVI. Even though some uncertainties remain in the models, the agreement in geographic and seasonal patterns gives confidence that they can be used to project future changes. Finally, we also note that there may be errors in the model projections if factors such as changes in the Brewer-Dobson circulation are not properly parameterised.</p><p>Previous studies have also shown that the CCM models tend to overestimate ozone amounts. For example, the GEOS-CCM used previously 6 has a high-ozone bias at mid to high latitudes 29 , which would lead to UVI estimates that are too low by approximately 10% at latitude 45° S. The green lines in Fig. 1 are the mean differences between the two models (smoothed), which shows that they depend on both latitude and time.</p><p>In the longer-term, projections by the two models are in good agreement. The reasonable level of consistency between these models adds further confidence to those predictions but also illustrates some significant differences.</p><p>It is interesting to note that by the year 2100, projected ozone declines are largest at high latitudes, and smallest in the tropics. By that time, the lowest mean ozone amounts would be in polar regions, and the highest in the tropics. time series for lauder. We now compare these UVI projections from the CCM models with observed changes in UVI and with those expected from clear-sky calculations using the assimilated ozone values. Results for Lauder are shown in Fig. 3. Figure 3a displays the time series of daily ozone derived from the NIWA/BS ozone assimilation for the period since satellite measurements became available. In Fig. 3b, the peak UVI measured within one hour of local noon is compared with the UVI calculated for clear-sky conditions using the NIWA/BS ozone data. There is good agreement in the seasonal range between measured and modelled UVI. Because of the large seasonal and day-to-day variability, it is difficult to see any long-term effects (other than in the upper envelope of data) in Fig. 3b. The lower four panels of Fig. 3 redress this by considering seasonal means, with each data point representing the mean over different 3-month periods. Because of the previously discussed biases in calculated and modelled ozone, all data shown in Fig. 3c-f were normalized. The measured UVI values were normalized to the average of all values, while the calculated UVIs were normalized to the average of only those years with measurements. The normalized peak UVI changes derived from measurements (blue lines) are compared with those calculated from the assimilated ozone dataset (black lines). Also shown in these panels are the corresponding UVI changes that would have occurred according to the World Avoided calculations reported by the GEOS-CCM model (broken red lines) and the NIWA-UKCA model (broken magenta lines). UVI values in the World Expected are also shown (broken green lines). The heavier solid lines for each model run include smoothing with an approximating spline. Both World Avoided datasets and the World Expected dataset were normalized to the average of the calculated UVI values of the years 1978 to 1987; the decade that immediately preceded the signing of the Montreal Protocol. Corresponding plots for all other sites are in Supplementary Data.</p><!><p>In the period prior to 2020 shown in Fig. 3, projected changes are generally smaller with the NIWA-UKCA model than with the GEOS-CCM model. However, this is not everywhere the case. At northern mid-latitudes, the two models are in close agreement (see Supplementary Data). They are also similar at high southern latitudes during summer, while the NIWA-UKCA values are larger at high northern latitudes. Reasons for these model differences are outside the scope of the present study. Green lines indicate the smoothed difference between the two models (i.e., NIWA-UKCA minus GEOS-CCM). Plots for latitude 15° are representative of all equatorial latitudes, while those for 75° are representative for higher polar latitudes.</p><!><p>Possible changes in UV radiation during summer are most relevant from an environmental and health perspective. In Fig. 4, we compare measured, calculated, and projected UVIs for summer at all 17 sites in Table 1. The figure shows that the World Avoided trends depend greatly on latitude and that differences between the two model runs are also latitude-dependent. For example, at latitude 40°N, there is close agreement between the two models (for other seasons, see Supplementary Data).</p><p>With the exception of Hoher Sonnblick, the measured data from all sites closely match the calculated values and are much closer to the World Expected curves than the World Avoided curves. However, there is a significant divergence between the measured values and World Expected values at higher latitudes: measured and calculated UVI ratios are more or less constant over time while the World Expected curves have a broad maximum during the first decade of the 21 st century (last two panels of Fig. 4). This suggests that the impact of the large spring-time ozone depletions persists for too long into the following summer in the model. This is a well-known limitation of CCMs 28 for which the causes are not fully understood, and is an area of current research.</p><p>The measurements at Hoher Sonnblick (Panel f) show a continuation of the previously reported upward trend in summertime UVI 30 , which was attributed to changes in aerosols or cloud cover. This increase is not present in all seasons, so it is not a calibration artefact. However, there are several seasonal gaps in the data, and the year-to-year variability is also much greater at this mountainous site (see Supplementary Data), possibly due to variable snow cover.</p><p>At some other sites, the length of the time series is too short for reliable trend estimates. Only nine of the 17 sites have more than 20 years of data coverage.</p><p>Figure 4 also indicates that UVI ratios calculated from the assimilated ozone data are generally less than unity prior to the 1990s and before the start of UV measurements. The slope in the calculated data is generally consistent with the slope in the datasets of the three CCM models. These model calculations imply that considerable changes in summer UVI occurred between 1978 and 1990 (about 5% at northern mid-latitude sites, up to 10% at southern mid-latitude sites and up to 20% at the three Antarctic sites). However, direct UVI observations needed to confirm these changes do not exist.</p><p>After accounting for differences in elevation between the sites, we note that latitude-for-latitude, the mean summer UVI values tend to be larger at the southern hemisphere (SH) sites compared with the northern www.nature.com/scientificreports www.nature.com/scientificreports/ hemisphere (NH) sites, as has been predicted and observed previously [31][32][33][34] . This NH/SH difference is as expected. For example, Fig. 2 shows that at 45°S, the peak UVI value is larger than at 45°N by approximately 13%. Approximately half of this difference is due to ozone differences and half is due to Sun-Earth separation differences. The much larger NH/SH differences reported in the past [31][32][33][34] also include effects of clouds and aerosols, which were not considered in the CCM calculations. The NH/SH asymmetry is projected to be only slightly smaller by year 2100.</p><p>UVi in spring at high latitudes. In Fig. 5, we compare normalized measured, calculated, World Avoided, and World Expected UVIs in spring for the Arctic and Antarctic sites included in this study, where the largest long-term changes in UVI have been observed. Note the expanded vertical axis scale compared with that in Fig. 4. For these high-latitude sites, the "calculated" and "measured" data sets are in almost perfect agreement, particularly in Antarctica, indicating that ozone variation is the largest contributor to UVI changes. Variability of clouds and albedo is a minor contributor in comparison.</p><p>The "hump" in the World Expected data noted previously for the summer data (Fig. 4), which peaks in the early part of the 21 st century, is also present in the spring data (Fig. 5). However, in the spring period, there is also a hint of this in the measured and calculated data for the two Antarctic sites and perhaps also at Summit. Data from Barrow show more variability, as one would expect from an Arctic coastal site that is affected by changes in albedo from snow and sea ice. summer (e) and autumn (f) determined from measurements of the NDACC instruments (blue), calculated from total ozone column (black), and projected by the two World Avoided (red and magenta) and World Expected (green) CCM model runs. UVI changes were normalized as described in the text and the "UVI ratios" shown in Panels (c-f) are ratios relative to these normalizations. Note that for the season that spans two years (summer at this southern hemisphere site), the year label refers to the year at the start of the season. For example, the summer of Dec 2017 to Feb 2018 is plotted at 2017.</p><!><p>Due to the less complete data coverage early in the period, our statistical analysis is restricted to the period since 1996. This period also excludes effects from the eruption of Mt Pinatubo, which led to significant effects on ozone and UV at some locations [35][36][37][38] . The period since 1996 also includes approximately two complete 11-year solar cycles, which cause variations in ozone of ±2% 39,40 that would lead in turn to a modulation of similar relative magnitude, but of opposite sign, in the UVI. Even for the period since 1996, measured data are unfortunately incomplete at some sites, and only a small number of sites have full data coverage through to the end of 2018 (see Table 1 www.nature.com/scientificreports www.nature.com/scientificreports/ (BSI). Their time series from Ushuaia and San Diego are also restricted to the period before 2010 and 2008 respectively. There was also a gap from 2003 to 2008 in Melbourne data. Of the 17 sites, only three (Lauder, Palmer, and Thessaloniki) have near-complete data coverage for the full period from 1996 to 2018. However, six others (Barrow, Hoher Sonnblick, Boulder, Mauna Loa, Arrival Heights and South Pole) have only a few years of missing data over that period. Greatest confidence should be placed on data from these nine stations.</p><p>There are other possible forcing mechanisms that drive interannual variability in UVI. These include variability due to the QBO or the El Nino Southern Oscillation (ENSO). However, over time scales as long as 22 years, any such effects are small, and have not been considered in the statistical analysis. In these seasonal averages, where each data point is separated from the previous value by a period of 9 months, any statistical auto-correlation effects should also be small. In the trend analysis, we have assumed that any long-term changes over the period 1996 to 2018 could be represented by a linear change, which is a reasonable assumption (e.g., see Fig. 4). Results of the statistical analysis are shown in Fig. 6. The results are not particularly sensitive to the start year, resulting in a similar picture (not shown) if the start-date for the analysis is changed by 1 or 2 years.</p><p>Note that the error bars shown for the trend estimates in Fig. 6 are 2-σ uncertainties of the regression model, which also include small random uncertainties in the measurements that propagate to the 90-day averages analysed here. However, systematic errors due to potential long-term drifts in the measurements (e.g., due to the transfer of irradiance scales between calibration standards) are not included.</p><p>Figure 6 shows the following:</p><p>1. The two World Avoided Simulations give similar trends: With the exception of Palmer Station in winter, there is good agreement between the trends derived from the two World Avoided simulations. (At Palmer Station, the sun is only 2° above the horizon (SZA min = 88°) at the winter solstice, so peak UVI values during this period are small.) The calculated UVI is also sensitive to small modelling and sampling differences (see Supplementary Data). Also, as shown in Fig. 1 (lower left panel), there are significant differences in the seasonal variability between the two model projections in this latitude region and period. 2. UV Trends are large in the World Avoided Simulations: In the World Avoided (without the Montreal Protocol), UVI levels would have increased over the last 22 years by approximately 50% per decade at high southern latitudes in spring, and by 30% per decade in summer. Increases of up to 20% per decade would have been seen at northern high latitudes. At midlatitudes, the increases would have been approximately 5-10% per decade. Such changes would have been clearly detectable in the measurement data. 3. Measurements Differ from the World Avoided Simulations: In the spring and summer, the observed trends for all nine sites with good data coverage (solid symbols in Fig. 6) are significantly different from the World Avoided trends at all mid-and high-latitudes in the southern hemisphere, and at high latitudes in the northern hemisphere. At northern mid-latitudes, differences between measured trends and trends from the World Avoided simulations are significant in summer at all sites except Hoher Sonnblick, but in other seasons the results are more mixed. 4. Measurements follow the World Expected Simulations: For all sites, trends calculated from the measurements are consistent with the World Expected scenario, though uncertainties are large at Hoher Sonnblick, and other sites (e.g., San Diego, Saint Denis and Obs. Haute-Provence), where there are only a few years of observation. While variability in UVI is close to that predicted by changes in ozone at the southern hemisphere sites, the situation is more complex at northern mid-latitude sites, such as Hoher Sonnblick, where the effects of changes in aerosols and clouds are more important in some seasons. www.nature.com/scientificreports www.nature.com/scientificreports/ confirmed, the downward trend at Arrival Heights would remain statistically significant. At the South Pole and Arrival Heights, both measured and calculated UVIs appear to have decreased during spring (the period most affected by the ozone hole), however, trends calculated from these changes are not yet statistically significant. At Lauder, small downward trends in measured UVI are observed for summer and autumn, which are on the verge of being statistically significant. These trends are generally consistent with trends derived from the NIWA/BS ozone dataset, although throughout the southern hemisphere, trends are systematically more negative for measurements than for calculations for all seasons. Where data are available in the early 1990s, most sites show evidence of increasing UVI in the earliest part of the record when ozone was declining. However, the short length of the data record prior to 2000, and possible interference from the eruption of Mt Pinatubo, hinder our ability to ascribe changes in UVI to changes in ozone.</p><p>In most cases, we find that calculated and measured trends have been small and are in most cases not significantly different from zero. With the exception of one site (Hoher Sonnblick), they also agree with each other to within their error bars, and with trends calculated with the World Expected model.</p><p>At clean-air sites in the southern hemisphere, the UVI has followed the World Expected scenario within the limits of the measurement uncertainty. Differences between measurements and the two World Avoided models are already highly significant in the Arctic, and at southern hemisphere sites, especially in Antarctica. The situation is more complex at mid-latitudes in the northern hemisphere, where the effects of changes in aerosol and clouds mask effects of ozone.</p><p>For winter and autumn, changes observed at mid-latitude sites are mixed. Measurements show a tendency towards decreasing UVI in the southern hemisphere and increasing UVI in the northern hemisphere. However, few of the observed long-term changes since 1996 are statistically significant.</p><p>A statistically significant decrease in measured summertime UVI has been observed at the Antarctic site Arrival Heights, and the reduction in UVI at Lauder is close to being statistically significant at the 95% level. However, at both sites, trends calculated from ozone remain close to zero, showing that the reductions in UVI must be due to other factors (e.g., changes in cloud, aerosol, or surface albedo). Measurements during the last few years at Lauder also show signs of decreases in UVI, which also cannot be attributed to ozone changes.</p><p>The Montreal Protocol has been effective in curbing increases in harmful UVI. Without the Montreal Protocol, UVI values at northern and southern latitudes <50° would by now be 10 to 20% larger in all seasons compared to UVIs observed during the early 1990s. These changes would have had grave consequences for public health and would have led to increases in skin cancer occurrences 41 . For latitudes >50°S, UVI values would have increased over the same period between 25% (Ushuaia in summer and autumn) to more than 100% (South Pole in spring and summer).</p><p>UVI values in the future remain uncertain because of: a. possible volcanic eruptions, which could temporarily exacerbate ozone depletion as long as chlorine levels remain elevated; b. interactions with other aspects of climate change, such as changes in clouds and aerosols; c. slowly varying natural modes of variability 42 , such as the Interdecadal Pacific Oscillation 43 , which may affect cloud cover but are not included in the study; d. effects of increasing greenhouse gases, which will lead to stratospheric cooling and changes in dynamics that may subsequently cause ozone to increase above levels observed in 1980 outside polar regions; e. non-compliance to the Montreal Protocol, such as found in a recent study 44 .</p><p>The reduced number of operational sites measuring UV irradiance is concerning. The value of time series data increases with the length of the record, as shown by the smaller error bars for the longer-term measurement sites in Fig. 6. Unfortunately, there are only a few high-quality NDACC sites with data from the early 1990s that are still operational. It is important that they continue to be supported in case of unexpected future changes.</p>

Scientific Reports - Nature

Characterization of Oligodeoxynucleotides and Modifications by 193 nm Photodissociation and Electron Photodetachment Dissociation

Ultraviolet photodissociation (UVPD) at 193 nm is compared to collision induced dissociation (CID) for sequencing and determination of modifications of multi-deprotonated 6 \xe2\x80\x93 20-mer oligodeoxynucleotides. UVPD at 193 nm causes efficient charge reduction of the deprotonated oligodeoxynucleotides via electron detachment, in addition to extensive backbone cleavages to yield sequence ions of relatively low abundance, including w, x, y, z, a, a-B, b, c, and d ions. Although internal ions populate UVPD spectra, base loss ions from the precursor are absent. Subsequent CID of the charge-reduced oligodeoxynucleotides formed upon electron detachment, in a net process called electron photodetachment dissociation (EPD), results in abundant sequence ions in terms of w, z, a, a-B and d products, with a marked decrease in the abundance of precursor base loss ions and internal fragments. Complete sequencing was possible for virtually all oligodeoxynucleotides studied. EPD of three modified oligodeoxynucleotides, a methylated oligodeoxynucleotide, a phosphorothioate-modified oligodeoxynucleotide, and an ethylated-oligodeoxynucleotide, resulted in specific and extensive backbone cleavages, specifically, w, z, a, a-B and d products, which allowed the modification site(s) to be pinpointed to a more specific location than by conventional CID.

characterization_of_oligodeoxynucleotides_and_modifications_by_193_nm_photodissociation_and_electron

Introduction<!>Chemicals<!>Mass Spectrometry<!>Results and Discussion<!>UVPD, EPD, CID, IRMPD product ions<!>Variation of laser pulse energy for UVPD<!>Base dependence on electron detachment efficiency<!>Influence of oligodeoxynucleotide length and charge state on CID, UVPD, and EPD<!>EPD for studying modified oligodeoxynucleotides<!>Conclusions

<p>Sequencing biopolymers such as nucleic acids and proteins and determining their structural modifications remains one of the most important applications of tandem mass spectrometry. Collision induced dissociation (CID) is by far the most widely used activation method for nucleic acids.1 During CID, the fragmentation of deprotonated oligodeoxynucleotides is initiated by loss of a neutral or charged base, followed by subsequent backbone fragmentation leading to complementary w and a - B ions (Scheme 1).2 Infrared multiphoton dissociation (IRMPD) has also been successful for activation of nucleic acids due to the high absorptivity of the phosphodiester backbone at 10.6 um and results in fragmentation patterns similar to those of CID.3 The high abundances of non-informative base loss and internal fragments that complicate spectra as well as the limited diversity of products are oft-cited disadvantages of CID and IRMPD.</p><p>Interest in alternative ultraviolet photon-based and electron-based ion dissociation techniques for tandem mass spectrometry of oligodeoxynucleotides continues to grow, and include ultraviolet photodissociation (UVPD) at 193 nm,4 electron photodetachment dissociation at 260 nm (EPD),5–6 electron detachment dissociation (EDD),7–13 electron capture dissociation (ECD),14–15 and electron transfer dissociation (ETD).16 Pioneering work by McLafferty et al. used 193 nm photons to irradiate multiply charged dT30 ions, causing electron photodetachment in addition to formation of w and a ions (which have the same masses as d and z ions in the sequence dT30).4 More recently, Gabelica et al. explored the electron photodetachment of single strand oligodeoxynucleotide anions and duplexes at 250–285 nm.5–6 The minimal fragmentation of oligodeoxynucleotides observed upon laser irradiation at 260 nm was almost exclusively supplanted by electron photodetachment, rendering UVPD at 260 nm inefficient for sequencing oligodeoxynucleotides.5 CID of the charge reduced radical ions arising from electron photodetachment predominantly yielded w, d, a, and z ion series compared to the w and a-B ions observed upon CID.5</p><p>EDD is promoted by interaction of an oligonucleotide with >10 eV electrons, thus generating both radical and non-radical products. Hakansson et al has investigated EDD of deoxyoligonucleotides.7–13 Upon EDD, both w/d and radical a/z series of fragment ions were observed for oligodeoxynucleotide sequences for which those ions were not distinguishable, and one radical z ion was observed for the sequence dGCATGC.7 The limited fragmentation that was observed upon EDD of longer oligodeoxynucleotides was attributed to residual secondary structure that prevented product ions from separating.7,10 Recently, EDD has been performed on oligoribonucleotides by Taucher and Breuker, with EDD resulting in the formation of abundant noncomplementary even electron w and d ions.13 A mechanism was suggested whereby the radical z ions formed upon EDD undergo facile dissociation into even electron w ion.13</p><p>ECD and ETD entail activation of oligodeoxynucleotide cations, not anions, and lead to radical cation products via electron attachment. Subsequent dissociation follows different pathways that give rise to many types of product ions.14–16 For example, upon ECD, d radical cations, a/z ions, and c/x ions were observed.15 ETD of DNA cations generated very low abundance backbone fragment ions and instead predominantly caused charge reduction (i.e. electron attachment without dissociation).16 CID of the resulting charge-reduced species produced w, a, z, and d ions, with a marked decrease in the abundance of precursor base loss ions and internal fragments compared to CID.16 Although our previous work has shown efficient cation formation of DNA oligonucleotides, it should be noted that generation of positively charged oligodeoxynucleotides is generally perceived to be less efficient than the generation of the corresponding anions due to the acidic nature of the phosphate backbone under most experimental conditions, making ECD and ETD less popular options for characterization of DNA.14</p><p>With respect to characterization of DNA modifications, tandem mass spectrometry techniques offer a promising approach for the rapid and sensitive detection of modifications based on reconstitution of the original nucleic acid sequence from characteristic fragment ions.17 Sequencing by CID and IRMPD has been used to locate modified nucleobases,18–29 and modified deoxyribose and phosphate moieties in DNA,30–32 in addition to characterization of the extremely varied modifications of RNA.33–38 Even some 3D structural aspects of DNA and RNA can be obtained by combining the use of chemical probes with tandem mass spectrometry.39–43 Furthermore, the factors governing fragmentation mechanisms of modified oligonucleotides44–48 by CID have been explored.</p><p>In the present study, we explore the fragmentation patterns of negatively charged oligodeoxynucleotides, both single strands and modified single strands, using UVPD and EPD at 193 nm. In all cases, UVPD at 193 nm causes efficient electron detachment from the multiply charged oligodeoxynucleotide anions, producing charge reduced oligodeoxynucleotide anions in addition to an impressive variety of low abundance backbone ions. Subsequent CID of the charge-reduced oligodeoxynucleotide radical ions results in backbone fragmentation which is more extensive than that produced by CID of the corresponding even-electron species but less complicated than that promoted by UVPD alone. EPD of three modified oligodeoxynucleotides resulted in specific and extensive backbone cleavages which allowed the modification site(s) to be pinpointed more readily than by conventional CID.</p><!><p>The following oligodeoxynucleotides were obtained from Integrated DNA Technologies (Coralville, IA) on the 1.0 umole scale and used without further purification: 5'- AAAAAA-3', 5'- CCCCCC-3', 5'- GGGGGG-3', 5'- TTTTTT-3', 5'- TGGCCA-3', 5'- ATGACTCG-3', 5'- GTATGACTCGCA-3', 5'- TCGTATGACTCGCAAG-3', 5'- CATCGTATGACTCGCAAGTG-3'. In addition, the following sequences were obtained with modifications: 5' – TGCATGCAAG – 3' (in which the bold cytosine is methylated at the carbon 5 position) and 5' - TAGCTAGTCsGAC – 3' (which contains one phosphorothioate bond in which a sulfur atom is substituted for a non-bridging oxygen in the phosphate backbone at the "s" position) (Table 1). Oligodeoxynucleotide single strand concentrations were determined spectrophotometrically by Beer's Law using the extinction coefficients provided by the manufacturer. For ESI-MS analysis, the solution was diluted to 10 µM of oligodeoxynucleotide in 20 mM ammonium acetate solution. For the N-ethyl-N-nitrosourea (ENU) reaction, 10 µL of 5.0 M ENU in methanol was added to 40 µL of a 50 mM solution of 5'- GTATGACTCGCA-3' in 100 mM ammonium acetate. The incubate was heated to 57 °C for one hour, diluted to a DNA concentration of 10 µM and immediately analyzed by ESI-MS.</p><!><p>Oligodeoxynucleotide samples were directly electrosprayed into a Finnigan LTQ mass spectrometer (Thermo Electron Corp., San Jose, CA). A Harvard syringe pump (Holliston, MA) at a flow rate of 3 µL/min was used. The ESI source was operated in the negative ion mode with an electrospray voltage of 3.5 kV and a heated capillary temperature of 90 °C. To assist in desolvation, nitrogen sheath and auxiliary gas were applied at 40 and 20 arbitrary units, respectively. Spectra were acquired by summing 20 scans. For the collisionally induced dissociation (CID) experiments, collisional activation voltages were applied at a level sufficient to reduce the isolated precursor ion to ~10–20% of its original abundance. The default activation time of 30 ms was used in all CID experiments with a qz value of 0.25.</p><p>The LTQ mass spectrometer was modified for UVPD in a manner described previously.49 UVPD was performed using a Coherent Excistar XS 500 ArF excimer laser (Munich, Germany), with a repetition rate of 500 Hz, and a pulse width of 5 ns. A laser gas mixture containing inert gases and a small amount of fluorine (<1% composition in mixture) was used as the active laser medium inside the laser tube. As the number of laser pulses increased, the concentration of the premix laser gas in the laser tube decreased, resulting in a decrease in laser energy per pulse. In order to maintain the same pulse energy throughout all experiments (except for the energy variable experiments), the high voltage electrical discharge was automatically varied so that the energy per pulse was 6.0 mJ. Most UVPD experiments were performed with one laser pulse, during which the total activation period was the lowest default value of the LTQ mass spectrometer, 0.03 ms. Multiple laser pulse experiments were also performed, in which the laser was pulsed every 2.0 ms at the maximum repetition rate of the laser (500 Hz). The back flange of the vacuum manifold of the instrument was modified with a CF viewport flange with a VUV grade CaF2 window with an anti-reflective 193 nm coating to allow the transmission of 193-nm radiation. The unfocused laser beam was aligned on axis with the linear ion trap such that the beam passed through a 2-mm aperture of the exit lens to irradiate the ion cloud. Upon exiting the laser aperture, the pulse per energy was ~ 6.0 mJ; the energy per pulse near the back flange of the instrument was ~ 5.0 mJ per pulse prior to the 2-mm aperture of the exit lens. The q-value of the precursor was set at 0.1 to reduce the low-mass cutoff value. The pressure in the analyzer region was nominally 9.0 × 10−6 Torr, and no changes to the He bath gas pressure were made.</p><p>For EPD experiments, ultraviolet photodissociation was performed for 1 pulse (LTQ default activation period of 0.03 msec), and a collisional activation voltage was subsequently applied at a level required to reduce the isolated precursor ion to ~10 – 20% of its original abundance. The default activation time of 30 ms was used in all EPD experiments, and the qz value was set to 0.25. The isolation width was set to 5 m/z for all MS/MS steps.</p><!><p>For the present study, the fragmentation patterns of a series of oligodeoxynucleotides obtained by UVPD and EPD are compared to those obtained by CID and IRMPD. The types and relative abundances of diagnostic sequence ions are evaluated, and the benefits of EPD for characterization of modified oligodeoxynucleotides are demonstrated.</p><!><p>For the single strand d(GTATGACTCGCA), the UVPD spectrum of the 4- charge state, the EPD spectrum of the odd electron species (3-•) produced by electron photodetachment from the 4- charge state, and the CID and IRMPD spectra of the 4- charge state are displayed in Figure 1. UVPD of [GTATGACTCGCA-4H]4- results in an extensive series of product ions arising from various backbone cleavages as well as the abundant charge-reduced product assigned as 3-• and 2-••, as shown in Figure 1a. Due to the complexity of the spectra, not all product ions are labeled in the UVPD (Figure 1a) and EPD (Figure 1b) spectra. Although of low abundance, a complete series of w ions and a near complete series of d ions is observed. Moreover, a few a, a-B, b, c, x, y, and z ions are observed. Internal ions are numerous but base loss ions are low in abundance. This array of fragmentation pathways is generally characteristic of the UVPD mass spectra obtained for all other deprotonated oligodeoxynucleotides. Electron photodetachment upon UVPD has been observed previously for oligodeoxynucleotide anions at 260 nm,5–6 and the mass spectrum in Figure 1a indicates a similar process is operative upon UVPD at 193 nm, yielding abundant charge reduced products.</p><p>The sequence ions produced by UVPD in Figure 1a are numerous, albeit at rather low abundance. To circumvent this shortcoming and to promote more abundant fragmentation, the abundant charge-reduced ion produced upon UVPD (e.g., [(GTATGACTCGCA-3H)3-• in Figure 1a) was subsequently subjected to collisional activation (shown in Figure 1b), and this two stage activation process was termed by Gabelica and coworkers as electron photodetachment dissociation (EPD, i.e. UVPD → CID).5–6 A near complete series of w and d ions (except for the 5' terminal w1 and d1 ions due to the low mass cut-off associated with CID) are formed as well as numerous a, a-B and z ions.</p><p>For comparison, the CID spectrum of (GTATGACTCGCA-4H)4- is shown in Figure 1c. CID results in production of several w ions, a - B ions, and internal ions, the latter of which often complicate spectral interpretation. Internal ions, the most abundant of which are labeled with an asterisk, are the result of sequential fragmentation leading to products that contain neither the 5'- nor 3'- terminus. Simple base loss results in the most abundant product ions in the CID mass spectrum (Figure 1c).</p><p>IRMPD is another photon-based dissociation technique that is an alternative to CID.50 The non-resonant process of ion activation by IR absorption results in rapid conversion of the uninformative base loss ions, ones that often dominate CID mass spectra acquired in quadrupole ion traps, into a - B and w sequence ions without the need for sequential stages of ion activation.3 The IRMPD spectrum of (GTATGACTCGCA-4H)4- is shown in Figure 1d. IRMPD results in production of several w ions, a - B ions, and internal ions, similar to CID, but the unwanted base loss ions are minimized by secondary dissociation into a-B and w ions. The w, z, and d series are notably absent from the CID and IRMPD spectra.</p><!><p>Energy-variable UVPD experiments were also conducted to reveal the impact of photon flux on the secondary dissociation of the charge reduced product ions. Gabelica et al measured the electron detachment yield of dG63- at 260 nm and found that the first electron photodetachment is a one-photon process, while the second electron photodetachment was a multi-photon process.5–6</p><p>To establish the one-photon or multi-photon character of electron photodetachment when using 193 nm photons, [TGGCCA]3- was exposed to one laser pulse while the laser energy per pulse was increased. Supplemental Figure 1 shows the resulting energy-variable UVPD results for the peak areas of the singly (◊) and doubly (□) charge-reduced product ions, in addition to a w52- ion (Δ). The increase in abundance of the singly charged-reduced product ion follows a linear trend (R2 > 0.99) with respect to laser pulse energy, showing that the first electron detachment is a one-photon process. In contrast, the abundance of the doubly charge-reduced and w52- product ions exhibit a nonlinear trend, indicating that the reaction is a multi-photon process. A quadratic equation fit to the data for the doubly charge-reduced and w52- product ions yields a value of R2 greater than 0.99. The combined pulse-variable and energy-variable data confirm that the dissociation of DNA oligodeoxynucleotides at 193 nm is dependent on total photon flux.</p><!><p>Oligodeoxynucleotides have been shown previously to exhibit sequence-dependent dissociation patterns attributed to the nature of the nucleobases by the ultraviolet photon-based and electron-based activation techniques, including EPD at 260 nm5–6 and EDD.7–12 In order to evaluate the potential impact of the nucleobases on the fragmentation pathways of oligodeoxynucleotides upon irradiation at 193 nm, UVPD experiments were undertaken for four six-mers, dC6, dA6, dG6, and dT6, in the 2- and 3- charge states. After exposure to a single UV pulse, each 6-mer undergoes electron photodetachment as well as fragmentation into primarily w or d sequence ions (UVPD spectra not shown). The electron detachment efficiencies for each 6-mer are summarized in Figure 2.</p><p>The percentage of charge-reduced species was determined by summing the abundances of all charge reduced ions and radical sequence ions and dividing by the summed abundances of all ions. Figure 2 shows that the electron detachment efficiency upon one laser pulse at 193 nm follows the trend dA6 > dG6 > dC6 > dT6. The propensity for electron detachment was also monitored as a function of the number of laser pulses (1 to 15) (data not shown). Based on the much slower decay of the precursor ion abundance, the rate of dissociation of the thymine strand is significantly slower than for the other strands. The precursor ion is completely dissociated after 6 pulses for [dA6]3-, [dG6]3-, and [dC6]3-, whereas 13 pulses are necessary to completely dissociate [dT6]3-. In addition, secondary dissociation of the charge reduced species for [dT6]3- also occur much slower compared to the other 6-mer strands.</p><p>The trend in electron detachment observed by EPD at 193 nm (dA6 > dG6 > dC6 > dT6) is different from EPD at 260 nm (dG6 > dA6 > dC6 > dT6), 6 different from EDD (dG6 > dT6 > dC6 > dA6)12 and also different from electron thermal autodetachment (dT7 > dC7 > dA7).51 Guanine has the lowest ionization potential and thus would be expected to undergo the most facile electron detachment. For individual nucleobases, the order of ionization potentials is G < A < C < T.52 Molar extinction coefficients, which are directly related to the absorptivity of the molecule, are not available for 193 nm, but at 260 nm follow the trend A > G > C > T. The base dependence of electron detachment at 193 nm may depend on both the ionization potentials of the individual bases and their photoabsorption efficiencies.</p><!><p>UVPD, EPD, and CID were undertaken for several oligodeoxynucleotides of varying length and sequence in order to determine the sequence coverage obtained as a function of activation method and the charge state of the precursor. A comparison of the CID, UVPD, and EPD results for the series of oligodeoxynucleotides is summarized in Figure 3. The total numbers of diagnostic sequence ions (a - B and w for CID; a, a - B, d, w, z, and other ions (including b, c, x, y) for UVPD; a, a - B, d, w and z for EPD) are displayed in bar graph form for all observed charge states for each oligodeoxynucleotide. As the oligodeoxynucleotide increases in length and the number of possible backbone cleavage site increases, the overall number of product ions increases, as expected. In most cases, the number of product ions formed upon CID does not vary significantly with charge state. In contrast, the total number of ions produced by both UVPD and EPD appear to be much more dependent on charge state, with the intermediate charge states generally leading to the greatest array of product ions for each DNA strand. The total number of product ions from EPD are generally comparable or greater than that from UVPD, even though fewer different types of ions are produced (i.e. no b, c, x or y ions upon EPD).</p><p>A detailed comparison of the distributions of product ions generated upon CID, UVPD, and EPD as a function of charge state is shown in Supplementary Figure 2 for ss12. The abundances of each type of sequence ion (categorized as w; a - B; base loss from the intact single strand (-B); d; a; z; collective other ions including b, c, x, and y; and charge-reduced ions) were tallied and normalized to 100% for each precursor charge state. Figure 3 and Supplemental Figure 2 illustrate the striking differences in the number and distributions of product ions arising from each of the activation methods, thus affording significant analytical flexibility depending on the targeted objective, as demonstrated in the next section for characterization of modified oligodeoxynucleotides.</p><!><p>As described above, EPD results in a more diverse array of product ions than CID and greater abundances of diagnostic sequence ions than UVPD, and thus the potential of EPD for characterizing modified nucleic acids was explored. Elucidation of modified nucleic acids by MS/MS methods generally poses a greater challenge because the ability to pinpoint the specific site(s) of modification depends on generating a comprehensive array of site-specific fragment ions. In particular, EPD was applied for the characterization of a methylated oligodeoxynucleotide, a phosphorothioate-modified oligodeoxynucleotide, and an ethylated-oligodeoxynucleotide.</p><p>Most cytosine residues at CpG sites are physiologically methylated in mammalian genomes.53 Although 5-methylcytosine does not alter coding information, its presence plays a number of important biological roles, such as repression of gene transcription and the maintenance of gene integrity.54 DNA methylation can also physiologically occur with adenine,55 and guanine as well is susceptible to methylation by certain methylating agents.56 The oligodeoxynucleotide Mss10 (see sequence in Table 1) was characterized by EPD and CID as a means to pinpoint the cytosine methylation site. The representative spectra and resulting backbone cleavages observed upon both EPD and CID are summarized in Figure 4. Those product ions that retain the methyl group are readily identified based on their characteristic mass shifts in the MS/MS spectra and are highlighted with crescents. Interpretation of the MS/MS spectra confirms that there is only one modification located at the seventh residue. In the CID mass spectrum, the pair of 3'-w4 and 5'-a8-AH ions are methylated, indicating the methyl group resides on the oligodeoxynucleotide somewhere between those two complementary products, a region including the cytosine nucleobase, the ribose at that residue, and the two adjacent phosphate groups. For EPD, the 3'-z4 and 5'-a7 ions are methylated, pinpointing the methylation to an even more specific location, either the cytosine nucleobase or the adjacent ribose. Comparison of the 5'-a7 methylated and the 5'-a7-C unmethylated further confirms the site of modification.</p><p>Coupled with the knowledge that cytosine nucleobases are more often methylated than other regions in a DNA oligodeoxynucleotide, CID and EPD provide similar levels of structural information for this example. In order to evaluate the ability of EPD to specifically pinpoint a modification in another location, we characterized the phosphorothioate-modified oligodeoxynucleotide, PSss12, by EPD and CID (Figure 5). For phosphorothioate (PS) modified oligodeoxynucleotides, a sulfur atom is substitute for one non-bridging oxygen in the phosphate backbone of an oligodeoxynucleotide, which renders the internucleotide linkage resistant to nuclease degradation.57,58 The summarized cleavages for both EPD and CID (Figure 5c) (with the characteristic mass shift due to the sulfur atom indicated by stars) indicate that there is only one modification, and it is located at the ninth residue. For CID, the w4 and a10-AH ions are modified, indicating the modification resides somewhere on the oligodeoxynucleotide where those two products overlap, which includes the cytosine nucleobase, the ribose at that residue, the two adjacent phosphate groups, and the ribose at the tenth residue. For EPD, the z4 and d9 ions are modified, pinpointing the modification to a more specific location: a region comprising the cytosine nucleobase, the ribose at that residue, and the 3' adjacent phosphate group. In addition, taking into account that the a9 ion is never detected with the modification, it can be assumed that the modification must reside on the phosphate backbone. In this case, EPD outperforms CID.</p><p>As a final example, N-ethyl-N-nitrosourea (ENU) is a highly potent alkylating agent which acts by transferring an ethyl moiety to nucleic acids. Much discrepancy exists regarding the site of alkylation, with some studies finding nucleobases are the target, including guanine,59–64 thymine,59, 61, 64–67 and adenine.63–65 Other studies have indicated that the major site of adduction occurs at the oxygen of the phosphate backbone.68–70 While it is likely that reaction conditions influence the extent of alkylation and the specific alkylation sites, we were interested in evaluating the use of EPD as a means to pinpoint the ethylation sites. Oligodeoxynucleotide ss12 was reacted with ENU and subsequently characterized by EPD and CID (Figure 6). The summarized cleavages upon EPD and CID of ENU-modified ss12 are shown in Figure 6c. According to the established nomenclature for oligonucleotide dissociation, the four possible cleavages along the phosphodiester chain are indicated by the lower case letters a,b,c, and d for fragments containing the 5'-OH group and w,x,y, and z for fragments containing the 3'-OH group. The numerical subscripts indicate the number of bases from the respective termini. The products formed by EPD (w, d, a, z ions) result from cleavages that occur both 5' and 3' to the phosphate group, yielding some products that consist of the nucleobase, ribose, and the phosphate at the newly terminal residue (w and d ions) in addition to the rest of the oligonucleotide, with other products formed containing only the newly terminal nucleobase and the ribose (a and z ions) with the remainder of the oligonucleotide. These complementary sets of differential products allow the ethylation sites to be pinpointed on the phosphate group or the nucleobase/ribose moiety. For ethylation of ss12, EPD allows the modification to be pinpointed to the phosphate, specifically due to the a3 and ethylated d3 ions, and the ethylated w3 and z3 ions. The limited backbone cleavages by CID do not provide sufficient information to identify the ethylation site. In addition, we noted that as the length of the product ion increased (regardless of ion type), the abundances of the modified ions increased relative to analogous unmodified ions (data not shown). The nearly linear increase in percent modification confirms the lack of sequence specificity of the ENU modification.</p><!><p>UVPD of DNA oligodeoxynucleotides at 193 nm promotes extensive charge reduction, in addition to an impressive array of w, x, y, z, a, b, c, d, and a-B ions. CID of the charge reduced ions formed upon UVPD, termed EPD, results in far greater abundances of informative sequence ions, including w, z, a, d, and occasionally a-B ions. Although the total number of different types of ions produced by EPD is lower than produced upon UVPD, the overall numbers of products produced is similar or greater, meaning that each series of sequence ions (e.g. w, z, a, d) is more extensive. The efficient and characteristic fragmentation promoted by EPD proves to be more effective than that induced by CID for pinpointing the location of modifications of oligodeoxynucleotides, such as phosphorothioate substitutions and ethylation.</p>

PubMed Author Manuscript

Catalytic Upgrading of Lignocellulosic Biomass Sugars Toward Biofuel 5-Ethoxymethylfurfural

The conversion of biomass into high-value chemicals through biorefineries is a requirement for sustainable development. Lignocellulosic biomass (LCB) contains polysaccharides and aromatic polymers and is one of the important raw materials for biorefineries. Hexose and pentose sugars can be obtained from LCB by effective pretreatment methods, and further converted into high-value chemicals and biofuels, such as 5-hydroxymethylfurfural (HMF), levulinic acid (LA), γ-valerolactone (GVL), ethyl levulinate (EL), and 5-ethoxymethylfurfural (EMF). Among these biofuels, EMF has a high cetane number and superior oxidation stability. This mini-review summarizes the mechanism of several important processes of EMF production from LCB-derived sugars and the research progress of acid catalysts used in this reaction in recent years. The influence of the properties and structures of mono- and bi-functional acid catalysts on the selectivity of EMF from glucose were discussed, and the effect of reaction conditions on the yield of EMF was also introduced.

catalytic_upgrading_of_lignocellulosic_biomass_sugars_toward_biofuel_5-ethoxymethylfurfural

Introduction<!>The Synthesis Routes<!><!>The Synthesis Routes<!>The Synthesis Mechanism<!>The Factors Impacting the Yield of EMF From LCB-Derived Sugars<!><!>Monofunctional Acid Catalysts<!>Bifunctional Acid Catalysts<!>Reaction Conditions<!>Conclusion<!>Perspectives<!>Author Contributions<!>Funding<!>Conflict of Interest<!>Publisher’s Note

<p>Extensive use of fossil fuels has caused energy depletion and serious environmental problems (e.g., greenhouse effect and acid rain). It is urgent to develop green renewable energy to replace fossil fuels for a better living environment (Li et al., 2017; Li et al., 2020; Pan et al., 2020). Lignocellulosic biomass (LCB) is a typical renewable energy with an annual global output of approximately 12 billion tons (Abraham et al., 2020). It is mainly composed of a layer of firm lignin-wrapped cellulose and hemicellulose components (Bhatia et al., 2020). Among them, cellulose is a biopolymer linking massive glucose units via β-1,4-glycosidic bonds, accounting for 38–50 wt% of LCB (Somerville et al., 2010). Thus, a large amount of glucose can be obtained by hydrolyzing cellulose. There were many researchers focused on the conversion of glucose to high value-added chemicals. Through various catalytic reactions such as dehydration, hydrogenation, hydrolysis, alcoholysis, and etherification, glucose can be turned into high value-added fuels and fine chemicals [e.g., 5-hydroxymethylfurfural (HMF), 5-ethoxymethylfurfural (EMF), levulinic acid (LA), and ethyl levulinate (EL)] (Rackemann and Doherty, 2011; Yang et al., 2012; Climent et al., 2014; Yang et al., 2019).</p><p>Furan derivatives like furfural, furfuryl alcohol, HMF, EMF, and 2,5-dimethylfuran have shown great potential in the formation of fine chemicals and alternative fossil fuels (Tong et al., 2010; Liu et al., 2021). Among these furan derivatives, EMF has the advantages for instance a higher boiling point (235°C), superior energy density (30.3 MJ/L) compare with ethanol (23.5 MJ/L), and low flash point (ca. 110°C) (Corma et al., 2007). Therefore, it has been considered one of the excellent choices of fuel additives in the future (Li et al., 2016). When 17 wt% EMF was used as an additive that mixes with fuel in a fuel engine, the engine could run stably and release fewer harmful particles and sulfides (with a 16% reduction in soot) (Mascal and Nikitin, 2008). In addition, EMF has also be used as a reaction substrate for the synthesis of various industrially significant chemicals, such as 5-ethoxymethylfurfuryl alcohol, 2,5-diethoxymethylfuran, and cyclopentenone (Ras et al., 2009; Ras et al., 2010; Bredihhin et al., 2016).</p><p>Generally, EMF can be transformed from HMF and ketose (e.g., fructose, inulin, and sucrose) with a satisfactory yield (ca. 70–90%) (Bredihhin et al., 2013; Dai et al., 2019; Hafizi et al., 2020). Yet, the industrial-scale production of EMF was limited by these high-priced feedstocks. For example, the price of HMF and fructose in Sigma-Aldrich is 12,634 and 205 EUR per kilogram, respectively. However, glucose has a lower price (88 EUR per kilogram in Sigma-Aldrich), which is reasonable to convert glucose into EMF (187 EUR per gram in Sigma-Aldrich). Moreover, the large amount of glucose can be obtained from cheap LCB, which is also a choice for economic and environmental development. At present, relevant reviews have summarized the use of various types of catalysts to convert different raw materials into EMF (Chen B. et al., 2020; Yu et al., 2021). But almost no review focused on the mechanism of EMF synthesis from glucose to EMF. Hereby, this mini-review introduces the paths and mechanisms of producing EMF from LCB derivatives, with a focus on challenges of the conversion of glucose to EMF. The aim is to provide a feasibility method for maximizing the conversion of LCB into EMF.</p><!><p>EMF can be obtained from glucose or cellulose via multi-step chemical conversion (Zheng et al., 2021). There are three paths to synthesize EMF from glucose (Figure 1). The mainstream Path I uses glucose as the starting material, which is isomerized to produce fructose, then HMF is obtained through fructose dehydration (-3H2O), and finally, HMF is etherified to EMF (Chen et al., 2019). The most important step in this path is the isomerization of glucose, which usually requires the participation of Lewis acid (Lew et al., 2012). There are two other secondary paths with ethyl-D-fructofuranoside (EDFF) as an intermediate transit. Path Ⅱ is that fructose reacts with ethanol in acidic solution to form EDFF, which is then dehydrated (-3H2O) to produce EMF (Zhang et al., 2018). Path Ⅲ is glucose and ethanol to generate ethyl-D-glucopyranoside (EDGP) in an acid medium, then isomerized to EDFF, finally dehydrated (-3H2O) to obtain EMF (Zheng et al., 2021). Currently, most EMF is obtained through Path I for the following reasons:</p><!><p>The conversion paths of LCB to EMF. (A) Cellulose hydrolysis by Brønsted acid, (B) glucose isomerization by Lewis acid, (C) the conversion of fructose to EMF.</p><!><p>(ⅰ) Compare Path I and Path Ⅱ. The difference is that fructose is more likely to be converted into HMF (Path I) or EDFF (Path Ⅱ). It has been found that fructose was inclined to be dehydrated to form HMF (Path I) rather than etherified with ethanol to form EDFF (Path Ⅱ) when Brønsted acid is present (Xiang et al., 2017).</p><p>(ⅱ) Compare Path I and Path Ⅲ. Glucose is usually isomerized to fructose when Brønsted acid and Lewis acid are present at the same time (He et al., 2022). When there is only Brønsted acid in the system, although the DFT calculation results show that the highest energy barriers required for Path I (17.7 kcal/mol) and Ⅱ (20.8 kcal/mol) are similar, the thermodynamic reaction is more favorable for Path I (Wang et al., 2021). And the intermediate EDGP in Path Ⅲ is difficult to continue further conversion.</p><!><p>The conversion of cellulose to EMF requires multiple reaction processes, namely cascade reactions. A detailed description of the synthesis mechanism of each step in Path I is shown in Figure 1.</p><p>Cellulose has a condensed structure (Figure 1), and is a high molecular polymer connected by β-1,4-glycosidic bonds and axial hydrogen bonds between numerous glucose monomers (Shrotri et al., 2018). Therefore, the hydrolysis of cellulose in the first step of Path I is a major obstacle that needs to be overcome. Many studies have shown that Brønsted acid can destroy the β-1,4-glycosidic bonds of cellulose (Zeng and Pan, 2020). As shown in Figure 1A, firstly, the oxygen atom of the β-1,4-glycosidic bond is attacked by the proton of the Brønsted acid site. Then the C-O bond between the two glucose molecules is broken for releasing glucose and glucose-ion intermediate. Finally, the hydroxyl group from water binds to the exposed carbon of glucose-ion intermediate to form glucose. And the free protons from water participate in the next hydrolysis reaction.</p><p>The second step in Path I, the isomerization of glucose into fructose, is the most important step in determining the yield of EMF. Many studies have indicated that glucose transforms into fructose via Lewis acid sites (Li et al., 2014; Rajabbeigi et al., 2014). As shown in Figure 1B, the C1-O5 bond of glucose is broken by Lewis acid and forms a linear glucose molecule. The oxygen atoms of C1 and C2 on linear glucose coordinate with the Lewis acid center. Subsequently, the hydrogen on C2 is transferred to C1, which realizes the aldehyde-ketone conversion between C1 and C2 to form linear fructose. Finally, the oxygen of C2 is linked with C5 to form a fructose molecule by C-C bond.</p><p>The third step is that fructose generates HMF by dehydration of three H2O molecules under acidic conditions. Firstly, the hydroxyl group on C2 is protonated to release the first H2O, and C=C is formed between C1 and C2. Then, the hydroxyl group on C3 is protonated to release the second H2O. Meanwhile, the C=C bond between C1 and C2 is broken, the aldehyde group is formed at the C1, and C=C is formed between C2 and C3. Finally, the hydroxyl group on C4 is protonated to release the third H2O, and C=C is formed between C4 and C5 to get HMF. After that, HMF is etherified to EMF with ethanol existence (Figure 1C).</p><!><p>Many LCB-derived sugars and compounds have been used to convert into EMF, such as cellulose, cellobiose, and glucose. The EMF yield from these substrates has displayed the order of glucose > cellobiose > cellulose > LCB (Li et al., 2016; Guo et al., 2018). In general, only moderate or low EMF yields can be obtained from these raw materials which are due to the different number of reaction steps. For example, HMF as a feedstock (high EMF yield) just needs one step, but cellulose (low EMF yield) needs four steps. Meanwhile, the lengthy chemical reaction process increased more by-products or humins (Zheng et al., 2021). Therefore, many studies were devoted to developing more efficient catalytic systems, which can obtain more satisfactory EMF yields from glucose or glucose-based carbohydrates (Guo et al., 2017; Guo et al., 2018; Karnjanakom et al., 2020; He et al., 2022). Some catalysts and reaction conditions for obtaining EMF from LCB-derived sugars were summarized in Table 1.</p><!><p>EMF from LCB-derived sugars via different catalysts and reaction systems.</p><!><p>Currently, many monofunctional (Brønsted or Lewis) acid catalysts are designed to catalyze the synthesis of EMF from glucose, including H2SO4 (Xu et al., 2017), metal salts (Liu et al., 2013), SO3H-based catalyst (Liu and Zhang, 2013), and ionic liquid (Guo et al., 2017). From the perspective of the synthesis routes, theoretically, when only Brønsted acid exists, using glucose as substrates hardly produces EMF. Yet, many studies had found that in the presence of Brønsted acid, a spot of EMF could be detected using glucose (7.46% yield), cellobiose (19.8% yield), and cellulose (3.05% yield) as raw materials (De et al., 2012; Guo et al., 2017; Xu et al., 2017). One possible reason for this is that glucose formed a bit intermediate 3-deoxyglucosone in Brønsted acid, which is then dehydrated to form HMF, and finally etherified to EMF (Jadhav et al., 2011; Jadhav et al., 2012). In addition, Brønsted acid and protonated ethanol ([C2H5OH2]+) can open the ring of glucose to form intermediate 1,2-enediol then isomerizes to fructose, which makes it possible to produce EMF in the next step (Guo et al., 2017; Wang et al., 2021). When there is only Lewis acid in the system, a moderate EMF yield (10–40%) can be obtained from glucose (Dutta et al., 2012; Liu et al., 2013; Tan et al., 2017). In the presence of a single Lewis acid, the possible reason for the failure to obtain high EMF yield is that the Lewis acid cannot provide H+, resulting in the low [C2H5OH2]+ concentration in the system which limits the fructose dehydration and subsequent etherification steps. Meanwhile, the EMF yields obtained by catalyzing glucose with different types of metal salts were quite different. Such as metal chlorides AlCl3 and CrCl3 could obtain 11.2 and 15.2% EMF yields, respectively. However, with metal sulfates Al2(SO4)3, CuSO4, Fe2(SO4)3, and Cr2(SO4)3 as catalysts, the reaction system hardly detected EMF, but more EDGP (ca. 80% yield) was detected (Yu et al., 2017). Thus, the metal chloride is more conducive to the isomerization of glucose, while the metal sulfate is more inclined to promote the etherification of glucose. Overall, the monofunctional acid catalysts cannot obtain satisfactory EMF yield from glucose. Whereas, the developed bifunctional acid catalysts with Brønsted-Lewis acids can obtain high EMF yield from glucose.</p><!><p>Generally, zeolite molecular sieve catalysts contain Brønsted acid species Al-O(H)-Si (framework four-coordinate aluminum), and Lewis acid species Al- (framework three-coordinate aluminum) can be obtained after high-temperature dealumination (Xin et al., 2019). For example, ultra-stable Y zeolite (USY) and β zeolite (H-β) after high-temperature dealumination were used to catalyze the synthesis of EMF from glucose and obtained 39.5 and 41% EMF yields, respectively (Li et al., 2016; Zheng et al., 2021). In addition, zeolite can also be modified to obtain better EMF yield. Introducing Lewis acid species Sn- and Al- into zeolite to obtain MFI-Sn/Al (Bai et al., 2018) or simultaneously introduce H4 [Si(W3O10)4] and SnCl4 (Brønsted-Lewis acids) into zeolite to obtain SBA-15 (Srinivasa Rao et al., 2020). These catalysts could obtain EMF with medium yield from glucose. A soft template HIPE was utilized to support the sulfonic acid group and Cr3+ to synthesize BFC-3 catalyst, which could be used to catalyze glucose and cellobiose and obtain 48.1 and 37.1% EMF yields, respectively (Chen et al., 2020a). Furthermore, glycerol and glucose were sulfonated into carbon spheres, then introduced into Zn- to prepare Zn-SO3H-GR-carbon (Karnjanakom and Maneechakr, 2019a) and Zn-S-C (Karnjanakom et al., 2020). Both of them can obtain amazing EMF yields from glucose (86.3 and 80.9%).</p><p>Bifunctional acid catalysts have great differences in catalytic performance. Using the same Brønsted acid and different Lewis acids to prepare various catalysts, the yields of EMF obtained from glucose were different. For example, the sulfonated carbon (SC) was doped with different metal species (Zn-, Al-, and Ni-), which exhibits different catalytic performances (Karnjanakom and Maneechakr, 2019b). When EMF was selectively produced from glucose, Zn-SC, Al-SC, and Ni-SC provided yields of 85.1, 84.4, and 32.8%, respectively. The reason for the difference is that the acidity provided by specie Ni- is lower than Zn- and Al-. A recent study also confirmed that the type of Lewis acids affects the yield of EMF (He et al., 2022). Meanwhile, this study found that the performance of the catalyst was also affected by the number of metal species in it. Specifically, the more types of metals contained in the catalyst, the better the catalytic efficiency. In addition, choosing a suitable ratio of Brønsted/Lewis acid can improve the selectivity of EMF (Srinivasa Rao et al., 2020). For the ratio of strong/weak acid, when the weak acid accounts for more, it is harmful to glucose isomerization, fructose dehydration, and HMF etherification, resulting in lower EMF selectivity. On the contrary, when more strong acids are present in the system, the generated EMF can be turned into EL by a ring-opening reaction or converted into humins (He et al., 2022). Therefore, the ratio of Brønsted/Lewis and strong/weak acids in the bifunctional acid catalysts are also critical for obtaining EMF from glucose.</p><!><p>The selectivity of EMF is also affected by the reaction conditions, such as temperature, time, ultrasound, and co-solvent.</p><p>Obtaining EMF from glucose usually requires a higher temperature and longer reaction time (Srinivasa Rao et al., 2020; Wang et al., 2021). However, continuously increasing the reaction temperature and time leads to the decrease in the yield of EMF, which is due to the unstable EMF and easily converted to EL under high temperature and long time (Zheng et al., 2021). During the conversion of glucose to EMF, water may be produced due to dehydration and etherification, which makes the hydrolysis of HMF into LA inevitable in this system (Wang et al., 2021). Since the polar co-solvent limits the conversion of HMF to LA (Morales et al., 2017), such as dimethyl sulfoxide (DMSO), tetrahydrofuran (THF), and GVL, many studies add co-solvents to this system, which significantly inhibited the production of EL (Yu et al., 2018). The amount of co-solvent also affects the yield of EMF. With the increase of co-solvent ratio, the yield of EMF first increased and then decreased, while the yield of EL continued to decrease and HMF continued to increase (He et al., 2022). The increase of EMF can be attributed to the inhibition of the conversion of EMF to EL. Then adding too much co-solvent can reduce the amount of EMF, which is attributed to the decrease of ethanol content in the system to limit the etherification of HMF into EMF (Chen et al., 2020b). Besides, several studies have shown that ultrasonic assistance can form cavitation bubbles in the system and promote bond breakage, which can promote the reaction to a certain extent (Karnjanakom and Maneechakr, 2019b). The ultrasound assistance can greatly reduce the requirement of temperature and time from glucose to high yield EMF, such as 98°C for 47 min obtained 80.9% yield (Karnjanakom et al., 2020) and 106°C for 72 min obtained 86.3% yield (Karnjanakom and Maneechakr, 2019a). Therefore, EMF can be generated rapidly under mild conditions.</p><!><p>The richness, versatility, and accessibility of LCB are the reasons for its advantages in the field of sustainable energy conversion. The mechanisms and technologies of EMF production from LCB-derived sugars in recent years were reviewed. These studies aim to develop more efficient catalysts and reaction systems to increase the yield of EMF.</p><p>Glucose as a typical LCB-derived sugar is used to synthesize EMF. It is mainly through path I (Figure 1) to synthesize EMF. In general, it shows low EMF yield when used monofunctional acid catalysts. The key to this problem is attributed to the glucose isomerization step (corresponding to Lewis acid) and low concentration of [C2H5OH2]+ (corresponding to Brønsted acid). Yet, the developed bifunctional (Brønsted-Lewis) acid catalysts can effectively solve this problem, which can obtain satisfactory EMF yields from glucose. Meanwhile, the species of Lewis acids, ratio of Brønsted/Lewis acids, and ratio of strong/weak acids in the bifunctional acid catalysts have decisive effects on EMF yield. In addition, the optimization of reaction conditions has also made efforts in EMF yield. The suitable time, temperature, and a certain concentration of co-solvent can provide upside space for the selectivity of EMF.</p><!><p>Although there are some technological breakthroughs in obtaining high EMF yield from glucose, high yield EMF has not been found directly from LCB. However, studies based on glucose can provide feasible strategies for direct conversion of LCB to obtain high EMF in the future. Firstly, the bifunctional acid solid catalysts were given priority in the choice of catalysts, and the catalysts can adjust the type and quantity of acid. Secondly, it is also crucial to select appropriate co-solvents and reaction conditions. Although the ultrasound-assisted method showed excellent effects, it is not suitable for large-scale industries. Therefore, it is of great significance to develop an efficient catalyst strategy to convert LCB into EMF under mild conditions.</p><!><p>XL and DY jointly conceived the article, discussed the outline. DY wrote the manuscript. XL and HL have made preliminary revisions to the manuscript. CL and XL coordinated the entire content of the manuscript and made detailed revisions.</p><!><p>This work was financially supported by the scientific research funds of Guiyang University (GYU-KY-(2022)), the Guizhou Provincial Key Laboratory for Rare Animal and Economic Insects of the Mountainous Region ((2018)5102), and the National Natural Science Foundation of China (22065004).</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p><!><p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, orclaim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>

PubMed Open Access

Live Cell Surface Labeling with Fluorescent Ag Nanocluster Conjugates\xe2\x80\xa0

DNA-encapsulated silver clusters are readily conjugated to proteins and serve as alternatives to organic dyes and semiconductor quantum dots. Stable and bright on the bulk and single molecule levels, Ag nanocluster fluorescence is readily observed when staining live cell surfaces. Being significantly brighter and more photostable than organics and much smaller than quantum dots with a single point of attachment, these nanomaterials offer promising new approaches for bulk and single molecule biolabeling.

live_cell_surface_labeling_with_fluorescent_ag_nanocluster_conjugates\xe2\x80\xa0

INTRODUCTION<!>MATERIALS AND METHODS<!>Conjugation of avidin-DNA<!>Staining with Avidin-C24-Ag<!>Conjugation of anti-HS ab C24<!>RESULTS AND DISCUSSION

<p>Fluorescent probes can be utilized for highly site-specific labeling to study both bulk and single molecule dynamics. Unfortunately while various methods can be used to mark particular biological processes via either genetic encoding with green fluorescent protein or by incorporating exogenous fluorophores, standard dyes suffer from fast photobleaching and low emission rates (1, 2). Combined with fluorescence intermittency (3) and O2 sensitivity (4), these issues severely limit single molecule experiments (5, 6). One approach to improve sensitivity through increasing fluorophore brightness has been to employ semiconductor quantum dots (QDs) as contrast agents (7). QDs are usually prepared in nonpolar organic solvents, however, and require further modification to improve their solubility in aqueous systems. While there have been numerous applications of QDs in cellular imaging (8-11), the large particle size upon functionalization (∼20nm), tendency to aggregate, and potential toxicity prevent them from being versatile cellular labeling agents (12). The emergence of fluorescent silver clusters that possess advantages of both small size and good brightness opens a complementary path toward biological labeling (13-15). In contrast to QDs, silver clusters protected with short peptides can diffuse through cell membranes into live cells, giving bright cell staining (16). Unfortunately, the fluorescence quantum yield (ΦF) of these initial species was quite low (∼3%), and studies to improve the ΦF are underway. Alternatively, DNA also shows strong affinity for silver (17, 18), but with much higher ΦF's. Recently, we have produced spectrally pure silver clusters encapsulated in ssDNA, ranging from blue to near IR emission, exhibiting up to 40% fluorescence quantum yields while maintaining small size (19). Herein, we demonstrate the applicability of bright DNA-encapsulated silver clusters to biological imaging in a more specific way through cell surface labeling.</p><!><p>Materials and cell culture information are reported in accompanying Supplementary Material.</p><!><p>The disulfide protected 24mer cytosine (C24, 50 μM) was deprotected with tris(2-carboxyethyl)phosphine (1 mM) at room temperature in phosphate buffered EDTA (PBE, phosphate 100 mM, sodium chloride 137 mM, potassium chloride 2.5 mM, EDTA 5 mM) in a 1000 MWCO dialysis tube (Spectrum Laboratories). The dialysis tube was then suspended in PBE overnight at 4 °C, followed by the addition of avidin (EZ-Link®Maleimide Activated NeutrAvidin™ Protein, Pierce, 270 μM). The mixture was kept at 4 °C for another 8 hrs and the protein was concentrated and washed with deionized water 5 times to remove free DNA in a 10,000 MWCO centrifugal ultrafiltration vial (Vivascience, Stonehouse, UK).</p><!><p>Cells seeded on coverslips in 12-well-plates were fixed with pre-cooled methanol for 10 min, washed with phosphate buffered saline (PBS, pH 7.9) three times, and some were incubated with biotinylation agent (800 μM in PBS) for 15 min, before being washed with PBS five times. Both the non-biotinylated and the biotinylated cells were incubated with Avidin-C24-Ag (5 μm) for 20 min, then washed with PBS four times and mounted onto slides for microscope. Live cells seeded on coverslips in 6-well-plates were incubated with Sulfo-NHS-LC-Biotin (80 μM) in PBS-Mg (Na2HPO4 20 mM, NaCl 150 mM, MgCl2 5 mM, pH 7.9) at 4 °C for 15 min to maintain maximum cell viability. The biotinylated cells were washed with ice-cooled PBS-Mg and incubated with Avidin-C24-Ag (1 μM) at 4 °C for 10 min, washed with PBS-Mg and mounted onto thick glass slides (Erie Scientific, Portsmouth, NH, USA) with a depression filled with DMEM (Dulbecco's Modified Eagle's Medium) to keep the cells alive.</p><!><p>(a) Anti-Heparin/Heparan Sulfate (anti-HS, clone T320.11, Millipore, MAB204, 0.1 mL) was added with Sulfo-SMCC(0.6 mg, Sigma) dissolved in 0.06 mL PBS (pH 7.2, 10 mM) and stored at 4 °C for 2 hrs, and then purified over a Sephadex G100 column with nitrogen-degassed PBS buffer as eluant. (b) 9 mg TCEP and 1600 nmol thiolated C24 DNA (IDT) were mixed in 0.6 mL borate buffer (pH8.5) and stored at 4 °C for 4 hrs, and then purified over a Sephadex G50 column with nitrogen-degassed PBS buffer as eluant. (c) The above two products were mixed in the presence of an extra 2 mg of TCEP and then reacted at 4 °C for 48 hrs, followed by concentration with 5k mwco membrane centrifugal tubes (Vivaspin) and purified over a Sephadex G100 column. Anti-HS ab C24-Ag was prepared in a manner identical to nanocluster formation in free ss-DNA(17, 18), but using the Anti-HS ab C24 conjugate as the scaffold. Briefly, protein-C24 conjugate and silver nitrate were mixed at a molar ratio of two bases of DNA to one silver nitrate and reduced with aqueous sodium borohydride.</p><!><p>Modified proteins, either genetically or chemically, are widely applied for specific staining, yet for long term studies or those involving low copy numbers in the presence of high background, available labels limit experimental observations. The ssDNA scaffold affords a single point of attachment, while simultaneously stabilizing the few-atom, strongly emissive nanoclusters. Since the DNA chain can be easily modified with a thiol group, this thiolated DNA is conveniently covalently conjugated to any protein activated with maleimide. Capitalizing on the strong biotin-avidin interaction (20), an avidin-C24 conjugate was prepared through maleimide coupling (EZ-Link®Maleimide Activated NeutrAvidin™ Protein, Pierce, 50 μM) to disulfide-protected 24mer oligocytosine (5′-/5ThioMC6-D/CCCCCCCCCCCCCCCCCCCCCCCC-3′, Integrated DNA Technologies (IDT), Coralville, IA, USA, abbreviated as C24, 50 μM), after deprotection with tris(2-carboxyethyl)phosphine (TCEP) (21). Labeling was intentionally kept low to ensure <1 label/avidin tetramer. Creation of avidin-C24 conjugate was confirmed with MALDI-TOF mass spectrometry (Figure 1, inset). The avidin-C24 conjugate was then mixed with silver nitrate and chemically reduced as reported in unconjugated DNA to prepare fluorescent silver clusters (Avidin-C24-Ag) (14). The silver clusters show similar photophysics as those protected with C24 alone (C24-Ag), i.e., a major emissive species with emission maximum at 634 nm and an excitation spectrum centered at 580 nm is observed (Figure 1) with a fluorescence lifetime of 2.86 +/- 0.01 ns. Without conjugated ssDNA, we see no emission from avidin when subjected to identical cluster creation conditions. The photophysical similarity of silver clusters protected with avidin-C24 conjugate or C24 alone further indicates that the silver ions are predominantly bound to DNA even in the presence of covalently linked protein, promising wide applicability of silver nanocluster biolabeling. As observed for free DNA-encapsulated nanoclusters, those conjugated to avidin exhibit excellent photostability and brightness (∼20,000 counts/s/molecule excited at 568-nm CW laser with an excitation intensity of 2 kW/cm2, Figure 2), in line with the regular bright, stable, essentially non-blinking ssDNA-encapsulated silver clusters (22). Single molecule intensity autocorrelations yield a fast decay (∼12 μs) characteristic of the very short-lived dark state of silver nanocluster emitters (22), indicating the independence of nanocluster photophysics upon protein conjugation. Detected single molecule emission rates easily exceed 40,000 counts/sec with a further 2-fold excitation intensity increase.</p><p>The avidin-biotin interaction has been widely used for molecular targeting and supramolecular assembly (23, 24). Biotin can be conjugated to targets via chemical reactions or specifically targeted using biotin ligase (25). Here sulfosuccinimidyl-6-(biotinamido) hexanoate (EZ-Link®Sulfo-NHS-LC-Biotin, Pierce) was used to react with exposed primary amines (lysine residues) to biotinylate cellular proteins. This reaction was first tested on methanol fixed NIH 3T3 cells. As shown in Figure 3, the fixed cells exhibit quite different fluorescence images, in which the biotinylated cells (Figure 3a) show much higher fluorescence intensity than the non-biotinylated cells (Figure 3b) when both are stained with Avidin-C24-Ag (5 μM). The application of Sulfo-NHS-LC-Biotin to fixed cells (800 μM) was too harsh for live cells, therefore the concentration was lowered to 80 μM to maintain cell viability. Biotinylated live cells loaded with Avidin-C24-Ag were examined at room temperature shortly after preparation, as shown in Figure 3c. The silver clusters were bound to the cell surface yielding diffuse labeling with some bright aggregates, possibly resulting from endocytic uptake. However, the non-biotinylated live cells showed only weak emission, indicative of autofluorescence alone.</p><p>Although the cell surface was readily stained with Avidin-C24-Ag under these conditions, the somewhat indiscriminate biotinylation agent was still rather toxic to NIH 3T3. The affinity to and permeability of NIH 3T3 cells to DNA are low, resulting in poor staining of cells by C24-Ag alone (supplementary information). Targeting membrane-bound components enables effective cell surface labeling and uptake via receptor mediated endocytosis (26). Heparin sulfate (HS) is a linear, polysulfated, and highly negatively-charged polysaccharide attached to the cell surface and extracellular matrix proteins (27). Cellular uptake of a wide variety of extracellular ligands such as fibroblast growth factor and cell penetrating peptides are facilitated through initial electrostatic interaction between the negatively-charged HS and positively-charged ligands, therefore HS proteoglycan is recruited as a plasma membrane carrier (28). To better maintain cell viability, we conjugated C24 ssDNA to a heparin sulfate antibody and labeled with emissive Ag nanoclusters. In this paper, cells incubated with anti-HS-C24-Ag (4 μM) at 4 °C for 15 min showed staining of only the cell surface (Fig 4a - 4c), with no internalization. While most labeling was diffuse, some aggregation on the cell surface was observed, resulting in a few bright spots. Longer incubation time and higher antibody concentration induces denser and more even surface staining. Silver clusters were quickly internalized when the above cells were incubated at 37°C and emission is concentrated in the nuclei (Fig 4d - 4f). Likely, the antibody silver cluster conjugate first bonds to cell surface HS followed by endocytosis (29), as further indicated by the lack of observed fluorescence from cells incubated with C24-Ag only at either 4°C or 37°C. The internalization of silver clusters illustrates that silver clusters can be applied not only to label cell surface proteins to investigate extracellular dynamics, but also potentially as a reporter of endocytic uptake and vesicular transport.</p><p>In summary, the oligoDNA protected silver clusters are readily covalently conjugated to proteins such as avidin, and primary antibodies without significant interference of either biological function or nanocluster photophysics. As silver cluster label size is significantly less than 10% of labeled protein molecular weight, and overall isolated label size is quite small (19), proteins are likely minimally perturbed by cluster-based labeling. Employing either avidin-biotin or antibody/antigen interactions, these silver clusters can stain the cell surface and be internalized, possibly enabling studies of transmembrane transport or drug delivery. Because silver nanocluster-based labels simultaneously exhibit small size, excellent photostability, and bright emission, they offer new opportunities in high sensitivity biolabeling.</p>

PubMed Author Manuscript

A three-dimensional tetraphenylethylene-based fluorescence covalent organic framework for molecular recognition

The development of highly-sensitive recognition of hazardous chemicals, such as volatile organic compounds (VOCs) and polycyclic aromatic hydrocarbons (PAHs), is of significant importance because of their widespread social concerns related to environment and human health. Here, we report a three-dimensional (3D) covalent organic framework (COF, termed JUC-555) bearing tetraphenylethylene (TPE) side chains as an aggregation-induced emission (AIE) fluorescence probe for sensitive molecular recognition. Due to the rotational restriction of TPE rotors in highly interpenetrated framework after inclusion of dimethylformamide (DMF), JUC-555 shows impressive AIE-based strong fluorescence. Meanwhile, owing to the large pore size (11.4 Å) and suitable intermolecular distance of aligned TPE (7.2 Å) in JUC-555, the obtained material demonstrates an excellent performance in the molecular recognition of hazardous chemicals, e.g., nitroaromatic explosives, PAHs, and even thiophene compounds, via a fluorescent quenching mechanism. The quenching constant (K SV ) is two orders of magnitude better than those of other fluorescence-based porous materials reported to date. This research thus opens 3D functionalized COFs as a promising identification tool for environmentally hazardous substances.

a_three-dimensional_tetraphenylethylene-based_fluorescence_covalent_organic_framework_for_molecular_

Introduction<!>AIE characteristics of JUC-555<!>Fluorescence quenching mechanism<!>Outlook<!>Online content

<p>The demand for sensing environmentally and biologically important molecules has attracted wide attention in exploiting fluorescent probes. 1,2 During the detecting process, molecular motions of a movable fluorescent probe can be transformed by the fixing analyte or micro-environment, thereby leading to significant alterations in visual signals. [3][4][5] Since the pioneer work of Aggregation-Induced Emission (AIE) phenomenon in 2001 by Tang group, 6 AIE molecular rotors play an essential role in the behavior of fluorescence emission. 7 AIE-based fluorescent molecules are non-emissive in their monomers, but turn into highly emissive in molecular aggregates due to the restriction of intramolecular motions. 2,8 Recently, tetraphenylethylene (TPE) and its derivatives, as one of the most important AIE luminogens, have been widely reported, which can act as AIE-active fluorescent probes for chemical sensors. [9][10][11] In particular, TPE-based molecules have been demonstrated to promote exciton migration and enhance luminescence activity in porous materials, e.g., supramolecular coordination complexes, 12-14 porous polymers, [15][16][17][18] and metal−organic frameworks (MOFs). [19][20][21][22] Covalent organic frameworks (COFs) are a class of charming crystalline porous materials, which are constructed from organic building blocks linked by covalent bonds. [23][24][25][26][27][28] Over the past decade, COFs has driven considerable research efforts in various application fields including gas adsorption and separation, [29][30][31] heterogeneous catalysis, [32][33][34][35] organic electronics, [36][37][38] and many others. [39][40][41][42] Interestingly, some studies have also proved the combination of COFs and TPE-based AIE molecular rotors to enhance the fluorescence intensity. 6,7, 43 For example, Jiang group has reported highly emissive boronate-linked COFs with TPE monomers, and showed the sensitive fluorescence in the presence of ammonia vapor. 44 Wang group has obtained an AIEgen-based COF by introducing TPE rotors, which emits yellow fluorescence upon excitation with a photoluminescence quantum yield of 20%. 45 Recently, Zhao and co-workers have realized that the COF nanosheets could display signal amplification effect in biomolecular recognition of amino acids and small pharmaceutical molecules (Ldopa). 46 Despite the above processes, TPE-based COFs for molecular recognition is limited; in particular, highly-sensitive recognition of hazardous chemicals by TPE-based COFs has yet to be reported.</p><p>Here, we report a 3D TPE-based COF (termed JUC-555) as an AIE fluorescence probe for highly-sensitive molecular recognition. Different from all previously reported structures, where TPE function as the linkers in the skeletons, JUC-555 features dangling TPE in the pores. When the pores were filled with a suitable solvent such as DMF, the rotational restriction of TPE rotors in 10-fold interpenetrated diamondoid (dia) framework induced by confinement endows JUC-555 with exceptional AIE-based fluorescent (the on-state). In addition, owing to the large pore size (11.4 Å) and suitable intermolecular distance of TPE (7.2 Å) in JUC-555, the obtained material indicates an impressive performance in the special recognition of hazardous chemicals, e.g., nitroaromatic explosives, PAHs, and even thiophene compounds, via a fluorescent quenching mechanism (the off-state). The difference between the on-state and the off-state is exceptional with quenching constant (K SV ) approaching close to 10 9 . To the best of our knowledge, these results showed two orders of magnitude higher sensitivity to those of fluorescence-based porous materials reported to date, such as Eu-MOF, 47 NUS-25 nanosheet et al., 48 demonstrating 3D functionalized COFs as a promising identification tool for environmentally and biologically important analytes. 1a). To construct a 3D architecture, we further choose a typical tetrahedral organic linker, tetra(4-aminophenyl)methane (TAPM, Fig. 1a) as a 4-connected building unit. Based on the Schiff-base chemistry, the condensation of BFTP and TAPM results in a novel 3D COF (JUC-555, Fig. 1a). Since the BFTP as a linear building unit shares the same length (~16.9 Å) with that of 4,7-bis(4-formylbenzyl)-1H-benzimidazole (BFBZ) from LZU-79, the newly synthesized COF is expected to have 10-fold interpenetrated dia network (Fig. 1b-d). 49 The structure of JUC-555 (Fig. 1c) was determined by powder X-ray diffraction (PXRD, Supplementary Fig. S2) combined with structural simulations (Supplementary Fig. S3). After a geometrical energy minimization by using the Materials Studio 7.0 software package 30 based on the 10-fold interpenetrated dia net (Fig. 1d) with disordered TPE side chains, the unit cell parameters of JUC-555 were obtained (Fig. 1b, a S1) nearly equivalent to the predictions with good agreement factors (Rp = 3.39% and ωRp = 4.30%). Some peaks after 2 ˃ 15° were strong due to disordered TPE molecules in the channel or dynamic effect of 3D framework. 50,51 It should be noted that a similar structure with 10-fold interpenetrated dia net (LZU-79) has been proved by its single crystal, and the PXRD pattern of JUC-555 was well consistent with that from LZU-79 (Supplementary Fig. S4). 49 According to these results, JUC-555 showed a microporous framework with a diameter of about 11.4 Å (Fig. 1c), which has been demonstrated by the nitrogen (N 2 ) adsorption-desorption isotherm at 77 K (Supplementary Figs. S5-7). Aligned TPE side chains in the pores of JUC-555 after optimization displayed a maximum layer to layer distance of 7.2 Å and a minimum layer to layer distance of 3.7 Å (Fig. 1e). Furthermore, JUC-555 was stable in various organic solvents and water (Supplementary Fig. S8), and was thermally stable up to 400 °C under nitrogen according to the thermogravimetric analysis (Supplementary Fig. S9). FT-IR and solid state 13 C NMR shown in Supplementary Figs. S10 and S11, respectively, also confirmed the successful transformation of aldehyde and amine groups to the C=N bonds in JUC-555.</p><!><p>Different from previous reports where TPEs were integrated as the linkers of the crosslinked skeletons, our TPE units in BFTP are designed as the side chains, which endows TPE rotors with more flexibility. In addition, upon inclusion of molecules of different sizes, the degree of molecular congestion in the pores can be tuned, which offers an ability to tune the photoluminescence of TPEs due to the AIE effect. As shown in Supplementary Fig. S12, the ultraviolet−visible (UV−vis) spectrum of the BFTP monomer exhibited an absorption band at 352 nm, whereas that of JUC-555 exhibited a peak centered at 339 nm. Upon excitation, the solid samples of the BFTP compound emitted blue luminescence with peak maxima at 466 nm and JUC-555 emitted brilliant blue luminescence with peak maxima at 482 nm. JUC-555 is yellow as a solid, and shows a greenish yellow color under UV irradiation of 365 nm. As shown in Supplementary Fig. S13, the emission color of BFTP monomer and JUC-555 show CIE coordinates of (0.16, 0.20) in the blue region and (0.21, 0.35) in cyan color, respectively. To have a better understanding of the dynamic behavior of BFTP rotor in JUC-555, we explored the AIE fluorescent properties of BFTP monomer and JUC-555 by using the mixed solvent of THF and water with different water fractions (f w ). As shown in Supplementary Fig. S14, the BFTP monomer exhibited a typical AIE characteristic. When the water component of the mixed solvent was increased to 60%, the fluorescence emission showed a sudden enhancement, and the highest fluorescence intensity was obtained at f w = 90%, which is 14.5-fold higher than that in pure THF solution (f w = 0%). Such AIE characteristic can be attributed to the rotational restriction of the phenyl rings (AIE molecular rotor) in the aggregated state in poor solvents. However, this fluorescence enhancement is much weaker for JUC-555 (a merely 1.35fold increase at f w = 90% as shown in Supplementary Fig. S15) because of the lack of rotational restriction of AIE molecular rotors in the channel. We also measured the fluorescence of JUC-555 in different organic solvents (Supplementary Fig. S16). It's quite amazing that JUC-555 showed exceptionally stronger luminescence in DMF than in other organic solvents. Photoluminescence quantum yield (PLQY) measurements also confirmed that JUC-555 in DMF showed much higher QY than those in other solvents (Supplementary Fig. S17). A quantitative comparison of the PLQY between the monomer and JUC-555 under different conditions was also investigated (Fig. 2a). BFTP monomer showed a moderate quantum yield of 8% as a solid and a very low quantum yield of 0.2% in DMF solution. When water content in DMF was raised to 90%, a quantum yield of 6% was achieved due to AIE effects of the monomer. As for JUC-555, the solid showed only a low QY of 0.6% with a lifetime of 2.187 ns (Supplementary Fig. S18), but a high QY of 13.2% was achieved in DMF solution. Dynamic Light Scattering (DLS) results show that the mean particle size of JUC-555 in DMF is around 1 μm (Supplementary Fig. S19). Such JUC-555 in DMF solution was very stable under ambient conditions (Supplementary Fig. S20). Comparing to the emission spectra of JUC-555 in other solvents, a strong red-shift to 550 nm in DMF along with an over 20-fold luminescence were observed (Supplementary Fig. S16). Such enhancement could also be explained by AIE effects due to the inclusion and molecular recognition of DMF in the pores. The conformation of DMF molecules in COF channels was simulated by Materials Studio 7.0. As shown in the optimized geometry in Fig. 2b, DMF molecules were perfectly aligned in the channels with a comfortable packing coefficient of 27.96% and the intermolecular π-π distance is 3.4 Å. Compared with that of 3.7 Å in empty JUC-555, a much tighter packing of TPE phenyl is expected. The O•••H distance of 2.2 Å and O•••H-N angle of 154.9 o shown in Fig. 2C indicate intermolecular H-bonding between the imidazole moiety of JUC-555 and DMF solvent molecules, which is consistant with the observation in the IR spectra (Supplementary Fig. S21) where the N-H vibration in imidazole moiety of JUC-555 and the C=O stretching in included DMF shifted to lower frequency and the peak being broadened when compared that that of the monomer and DMF, respectively. Such intermolecular H-bonding and the increased molecular crowding induced by inclusion of DMF in the pores rigidify the molecular conformation and impede the intramolecular motions, hence endowing JUC-555 in DMF with an over 20-fold luminescence increase. 52 Based on the strong luminescence feature of JUC-555 in DMF, we then tested it as fluorescent sensors. Firstly, we focused on the detection of explosives, nitroaromatic compounds, which are recognized as one of the major classes of dangerous as well as their highly explosive nature. For efficient nitro explosive detection, it requires high sensitivity and unveils quick response even at low concentrations. When JUC-555 was treated with nitrobenzene (5.0 × 10 -8 M), the fluorescence quenching processes were obvious (Fig. 3a). The quenching constant (K SV ) was calculated to be 7.57 × 10 8 M −1 (Fig. 3b), which shows two orders of magnitude higher quenching constant value for nitrobenzene than all previously reported porous material to our best knowledge (Fig. 3c, Table S8). Similar quenching by other nitro aromatic compounds (Supplementary Fig. S22-26) were also experiential by JUC-555 with similar K SV constants as summarized in Table S2. The K SV of 1,2-dinitrobenzene, 1,3-dinitrobenzene and 1,4-dinitrobenzene are 7.59 × 10 8 M -1 , 5.81 × 10 8 M -1 and 5.14 × 10 8 M -1 respectively (Fig. 3b). Large Ksv value means a higher sensitivity and stronger interactions between nitro explosives and JUC-555. A blue-shift of 10~15 nm was observed in the quenching process by nitroexplosive. In addition, the detection limit of JUC-555 for nitrobenzene, 1,2-dinitrobenzene, 1,3dinitrobenzene and 1,4-dinitrobenzene is able to reach 0.1673 nM (20.60 ppt), 0.1685 nM (28.32 ppt), 0.1773 nM (29.81 ppt) and 0.1786 nM (30.03 ppt) respectively (Table S3). S4) are higher than the those of previously reported porous material sensors. For example, when JUC-555 was treated with acenaphthylene (5.0 × 10 -5 M), the fluorescence quenching processes were shown in Supplementary Fig. S37. The quenching constant (K SV ) representing the binding affinity was calculated to be 5.35 × 10 5 M −1 (Supplementary Fig. S27, Table S5) and it is more than two orders of magnitude higher than that of NUS-25, which was reported as a chemical sensor for the specific detection of acenaphthylene. Similar quenching phenomenon of other PAHs was also experiential by JUC-555 with similar K SV constants. The K SV for benzene, naphthalene, p-xylene, toluene, o-xylene, m-xylene, fluorene, anthracene, phenanthrene, acenaphthylene, pyrene and triphenylene are 2.56 x 10 6 M -1 , 2.15 x 10 6 M -1 , 1.62 x 10 6 M -1 , 1.52x 10 6 M -1 , 1.41 x 10 6 M -1 , 1.41 x 10 6 M -1 , 8.28 x 10 5 M -1 , 8.10 x 10 5 M -1 , 7.39 x 10 5 M -1 , 7.00 x 10 5 M -1 , 5.04 x 10 5 M -1 , and 3.62 x 10 5 M -1 , respectively (Supplementary Figs. S28-40). A blue-shift of 15 nm was observed in the quenching process by benzene, and blue-shifts of 5 nm were observed in the quenching processes by naphthalene, fluorene, anthracene, acenaphthylene, and pyrene. In addition, the detection limit of JUC-555 for benzene, naphthalene, fluorene, anthracene, phenanthrene, acenaphthylene, pyrene and triphenylene are able to reach 200. 42 S6. The percentages of fluorescence quenching caused by PAHs range from 78.7% for benzene to 39.2% for triphenylene as shown in Supplementary Fig. S41. Sulfur in gasoline is a considerable source of sulfur oxide emissions, which have been one of the major causes of environmental pollution formed during the combustion of sulfur-containing fuels. Since sulfur is present in gasoline in forms of different thiophene derivatives, it is very important to develop a sensitive way to detect such sulfur containing compounds in gasoline. We find JUC-555 is extraordinary sensitive for thiophene derivative recognition. Fluorescence quenching was observed when JUC-555 was exposed to various benzothiophene and dibenzothiophenes (Supplementary Figs. S42-45). The quenching constant (K SV ) for thianaphthene, dibenzothiophene, and 4,6-dimethyldibenzothiophene was calculated to be 8.06 × 10 5 M −1 , 5.74 × 10 5 M -1 , and 5.92 × 10 5 M -1 (Fig. 4). A blue-shift of 5~10 nm was observed in the quenching process. In addition, the detection limit of JUC-555 for thianaphthene, dibenzothiophene, and 4,6dimethyldibenzothiophene are able to reach 94.488 nM (12.680 ppb), 133.499 nM (24.599 ppb), and 146.823 nM (31.171 ppb), respectively, as summarized in Table S7. It should be noted that the correlation between the the Ksv and packing coefficient follows a normal Guassian distribution (Fig. 4b). µ = 0.3253, sigma = 0.0653 for PAHs, and µ = 0.3253, sigma = 0.0653 for S-PAHs were fitted. Such a nice fitting indicates the inclusion is mainly determined by supramolecular recognition mechanism with the exception of benzne, anthracene and triphenylene where the first two were too slim, while the last was two bulky in size.</p><!><p>The high sensitivity of JUC-555 toward hazardous molecules prompted us to conduct a detailed study on the quenching mechanism. We selected three examples of the harmful substances, nitrobenzene, fluorene and thianaphthene, to explain the mechanism. First of all, no overlap between the absorption of the analyte and emission of JUC-555 (Supplementary Fig. S46) indicates that the quenching is not a resonance energy transfer (RET) mechanism. The blue shifts of 0~15 nm in fluorescence spectra (Tables S4-7) suggest a mechanism of photoinduced electron transfer (PET) during the fluorescence quenching process. We also conducted timeresolved photoluminescence (TRPL) measurements to provide more evidences regarding the quenching processes caused by hazardous molecules. The TRPL decay curves in Supplementary Figs. S47 and S48 show that the lifetimes (τ 0 ) of JUC-555 in DMF decrease gradually from 3.733 ns to 3.477 ns upon 0 to 50 μL of nitrobenzene titration (5 × 10 -8 M in DMF), indicating the electron transfer from JUC-555 to nitrobenzene. We also performed structural optimization of JUC-555 upon inclusion of nitrobenzene using Materials Studio 7.0 to elucidate the reason for the extreme high Ksv for nitrobenzene. The optimized conformation of nitrobenzene in the channels of JUC-555 was simulated. As shown in Fig. 5, It is clear that nitrobenzene molecules (4.29 Å × 6.10 Å) fit well into the pores of JUC-555 (5.7 Å × 5.7 Å) with a comfortable packing coefficient of 26.6% and perfectly aligned insertions of nitrobenzenes between the layers of JUC-555 (layer to layer distance of 7.2 Å) can be observed. There are strong C-H-π interactions between the TPE units and nitrobenzene molecules. We also studied the quenching dynamic of JUC-55 by PAHs with different sizes. As shown in Supplementary Figs. S53 and S54, it's evident that the fluorescence quenching process is size dependent with quenching constants of 0.02239, 0.00418 and 0.00241 for benzene, naphthalene and triphenylene, respectively. As shown in Supplementary Fig. S55, the measurement Ksv for nitrobenzene is still in the range of experimental errors after 5 cycles, indicating the fluorescence sensing of nitrobenzene is also very robust. DFT calculations of the energy levels of JUC-555 and nitrobenzene derivatives (Supplementary Fig. S56) show that the lowest unoccupied molecular orbital (LUMO) of JUC-555 fragment (-2.47 eV, Supplementary Fig. S57) is higher than those of the nitroaromatic compounds tested (-2.50 eV for nitrobenzene, -3.11 eV for o-dinitrobenzene, -3.19 eV for m-dinitrobenzene and -3.55 eV for pdinitrobenzene, respectively), which support a photoinduced electron transfer mechanism of the fluorescence quenching of JUC-555.</p><!><p>In conclusion, we have designed and synthesized a novel 3D COF (JUC-555) where TPE-based AIEgens were integrated as side chains in the pores. The synergy between the confinement effect of ordered channels and the AIE effect of dangling TPEs render JUC-555 with strong molecular recognition capability and fluorescence sensing ability. Depending on what types of molecules to be included into the pores, the fluorescence of JUC-555 can be switched between the on-state when rotation restriction enhances AIE effects and the off-state when photoinduced electron transfer induces fluorescence quenching. These phenomena were exploited to identify hazardous chemical molecules such as nitroaromatic explosives, PAHs, and thiophene-based PAHs with much higher sensitivity than those of most previously reported porous materials. This research thus starts a new path to identify dangerous molecules in environment through the combined effects of molecular recognition and fluorescence quenching. Continuing work on chemical sensing of metal ions using JUC-555 is in progress. 2D AIE-based COFs with similar design principle will be pursued in due course.</p><!><p>Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/ (60 µg mL -1 ) at intervals of 10 min. Fluorescence spectra were recorded after the addition of aromatic nitro compounds, PAHs, and S-PAHs solutions. Excitation wavelength of 339 nm was used. Fluorescence quenching was analyzed using the Stern−Völmer equations derived for 1:1 complex to determine the binding mode:</p><p>The quenching percentage was estimated using the formula (I 0 -I)/I 0 × 100%, where I 0 is the original maximum peak intensity and I is the maximum peak intensity after exposure to aromatic nitro compounds, PAHs, and S-PAHs solutions.</p><p>Computational methods for HOMO-LUMO energy calculations. To quantitatively evaluate the interactions between JUC-555 and nitro explosives molecules, the electronic properties of a JUC-555 fragment were calculated using DFT. Initially, the structure of JUC-555 was optimized by Forcite using Materials Studio 7.0 to remove geometric distortions. Then, a JUC-555 fragment was used in DFT calculations. The nitro explosives molecules and the JUC-555 fragment were optimized using the M06 functional with 6-31G(d) basis set. The HOMO and LUMO energy levels of the JUC-555 fragment and nitro explosives molecules were calculated using the M06 functional with 6-311G** basis set. All the DFT calculated were perfromed using Gaussian 16.</p><p>GCMC simulation. These simulations were performed with the Sorption module of Materials Studio 7.0. All GCMC simulations included a 4,000,000-cycle equilibration period followed by a 4,000,000-cycle production</p>

ChemRxiv

Towards Density Functional Approximations from Coupled Cluster Correlation Energy Densities

Semi-)local density functional approximations (DFAs) are the workhorse electronic structure methods in condensed matter theory and surface science. The correlation energy density c (r) (a spatial function that yields the correlation energy E c upon integration) is central to defining such DFAs. Unlike E c , c (r) is not uniquely defined, however. Indeed, there are infinitely many functions that integrate to the correct E c for a given electron density ρ. The challenge for constructing useful DFAs is thus to find a suitable connection between c (r) and ρ. Herein, we present a new such approach by deriving c (r) directly from the coupledcluster (CC) energy expression. The corresponding energy densities are analyzed for prototypical two-electron systems. To explore their usefulness for designing DFAs, we construct a semilocal functional to approximate the numerical CC correlation energy densities. Importantly, the energy densities are not simply used as reference data, but guide the choice of the functional form, leading to a remarkably simple and accurate correlation functional for the Helium isoelectronic series.

towards_density_functional_approximations_from_coupled_cluster_correlation_energy_densities

I. INTRODUCTION<!>II. THEORY<!>A. Exchange and Correlation in WFT and DFT<!>B. Correlation Energy Densities from WFT<!>C. CC Correlation Energy Densities<!>III. RESULTS<!>IV. CONCLUSIONS

<p>There is no doubt that density functional theory (DFT) has had an unrivalled impact on computational chemistry and physics [1][2][3][4] This is because modern realizations of DFT (density functional approximations, DFAs) tend to offer the best compromise between accuracy and computational cost for most applications [5][6][7][8] This is especially true for semilocal DFAs, where E xc only depends on properties of the electron density, such as the local density and its gradient. Such methods are sometimes referred to as "pure" density functionals, as opposed to, e.g., hybrid fuctionals which are based on a generalized Kohn-Sham scheme. 9 Indeed, the early adoption of semilocal DFAs in the quantum chemistry community can be largely attributed to the remarkable accuracy with which, e.g., the semilocal BLYP 10,11 functional describes energy differences in molecules at a much lower cost than post-Hartree Fock methods such as second-order Møller-Plesset perturbation theory (MP2). 12 Even though BLYP and other popular semilocal functionals based on the generalized gradient approximation (GGA) were developed in the 1980-90s, they are still widely used. More recent functionals like those of the ωB97 and Minnesota families (both based on Becke's 1997 power-series approximation) are also commonly applied in chemistry, although mostly in their hybrid variants [13][14][15] Similarly, in the solid-state community, the ubiquotous semilocal PBE 16 functional is still the most frequent choice. Here, more recent alternatives, like the constraint-based SCAN 17 functional of Perdew and co-workers and the Bayesian (m)BEEF 18,19 methods are a) Electronic mail: johannes.margraf@ch.tum.de also gaining traction.</p><p>Of course, there have been highly significant developments beyond semilocal methods. Most prominently, the already mentioned hybrid functionals (e.g. B3LYP or PBE0) complement semilocal DFA exchange with 'exact' Hartree-Fock exchange. 20,21 This makes the functional depend on the occupied Kohn-Sham (KS) orbitals, and not just on the electron density. Particularly in their more recent range-separated variant, these methods are able to extend the applicability of DFT into areas where "pure" DFAs have difficulties, e.g. charge-transfer states or reaction barrier heights. [22][23][24] In (gas-phase) molecular chemistry, these methods have become the de facto standard, whereas they are still too computationally demanding for routine application to condensed matter or nanosized systems. The higher computational demand of hybrids is a direct consequence of the fact that the exchange energy now depends on the occupied KS orbitals, and not just on the total electron density. This is even more critical for correlation functionals beyond the semilocal approximation, which depend on the unoccupied (virtual) KS orbitals as well. Such 'higher-rung' functionals are typically based on the random-phase approximation (RPA) or second-order perturbation theory (double-hybrid functionals). [25][26][27][28][29][30] This strongly improves their thermochemical accuracy, and allows for the description of van-der-Waals interactions. The virtual orbital dependence of these methods translates to a quite unfavourable formal scaling with the basis-set size (typically O(N 5 ) or worse, compared to O(N 3 ) for GGAs). This is further aggravated by the fact that they additionally require larger (correlation consistent) basis sets, though this deficiency is less critical for the more recent range-separated correlation approaches. 31,32 Such DFAs are consequently not really comparable with 'lower-rung' GGAs, in terms of applicability. Instead, they compete with wavefunction methods such as MP2 or CC.</p><p>Improving correlation functionals without resorting to virtual orbitals is therefore an exciting prospect and the focus of this work. To this end, we adhere to a purist approach to DFT. In general, the exchange-correlation energy is only dependent on the electron density ρ, and can be determined via numerical integration of a spatial function:</p><p>Here, xc [ρ](r) is the exchange-correlation energy density. The notation xc [ρ](r) implies that the energy density is both a spatial function (i.e. it has a single scalar value at a given point in space) and a functional of the electron density. In the most general case, the exchangecorrelation energy density on a given point r depends on the electron density at all other points. Semilocal approximations like the GGA use a more convenient formulation, where xc (r) only depends on local quantities like the local electron density ρ(r) or its gradient ∇ρ(r). Furthermore, the exchange and correlation components are usually treated separately, leading to expressions for x [ρ](r) and c [ρ](r). We will focus on the latter.</p><p>Within this paradigm, there are two classic approaches to designing DFAs. On one hand, there is the constraintbased philosophy championed by Perdew, Burke, Levy and others. [33][34][35] Here, exact conditions for the DFA are derived from theoretical considerations of model densities such as the homogeneous electron gas or spherical two-electron densities. 36,37 On the other hand, the property-based approach postulates a parametric form for the exchange-correlation energy density, which is then fitted to accurate reference properties of real molecular or condensed phase systems (often based on higher level calculations). [18][19][20][38][39][40][41] In this contribution, we follow a new route to constructing "pure" DFAs, namely by deriving a correlation energy density from ab initio coupled cluster (CC) wavefunctions. This can be thought of as an intermediate strategy between the constraint and property-based philosophies. On one hand, the DFA is constructed to reproduce high quality benchmark calculations, as in the property-based approach. On the other hand, it is not based on a predefined fit function. Instead, the functional form emerges naturally from the shape of the correlation energy densities of meaningful model systems, as in the constraint-based approach.</p><p>This paper is organized as follows: In the theory section, we discuss the meaning of the exchange and correlation energies in DFT and WFT and motivate why we expect the CC correlation energy density ( CC c ) to be a useful model for a correlation functional. Then the formalism for computing CC c is presented. In the results section, we analyze the properties of CC c for prototypi-cal two-electron systems. The usefulness of these energy densities is then illustrated by constructing an accurate DFA to the CC correlation energy of the He isoelectronic series.</p><!><p>We denote occupied molecular orbitals (MOs, φ(r)) with the indices i, j, k . . ., virtual MOs by a, b, c . . . and general MOs by p, q, r . . .. All calculations are performed in a one-electron basis of atom-centered, normalized basis-functions χ µ (r), with indices µ, ν, σ . . .. Following common practice in the CC community, the basisfunctions are referred to as atomic orbitals (AOs).</p><p>For clarity, it should be noted that the term "exchangecorrelation energy density" is often used in the literature for the correlation energy per particle. The exchangecorrelation energy per volume (as used in this paper) is in that case often referred to as the exchange-correlation kernel. The latter can be converted into the former by dividing through the electron density.</p><!><p>The concepts of exchange and correlation are fundamental to both WFT and DFT. In WFT methods, the correlation energy E c is defined with respect to the Hartree-Fock (HF) energy, and simply describes the difference between HF and the exact non-relativistic energy (i.e. the full configuration interaction limit) in a given basis. 42 Meanwhile, the exchange energy E x emerges naturally from the HF formalism, due to the antisymmetry of the wavefunction. 43 In DFT, exchange and correlation in principle describe the same physical phenomena, but the energies are not referenced to HF. Instead, the KS equations use the variational principle to obtain (given the exact functional) the exact density. 2 Accordingly, the exact exchange and correlation energies are referenced to that density, and not to the HF one. One would thus not expect the WFT and DFT E xc to be numerically identical unless the HF density is exact, which is only true in some special cases like the homogeneous electron gas and for one-electron systems like the hydrogen atom. From a DFT perspective, the WFT correlation energy thus contains implicit corrections to the classical and exchange energies, which otherwise carry some error due to the approximate HF density.</p><p>To understand these differences in detail, it is helpful to consider the individual components to the DFT and CC total energies. In DFT, all energy contributions are written as functionals of the exact ground state density ρ 0 :</p><p>with the non-interacting kinetic energy functional T s and the contributions of the external (U ) and the Hartree (J) potentials. Equivalently, these terms can be expressed as functionals of the occupied KS orbitals {φ KS i }, which is particularly useful for the kinetic energy.</p><p>In CC, similar components are computed in terms of the HF orbitals {φ HF p }:</p><p>Here, K[{φ HF i }] is the HF exchange energy. Given the exact exchange-correlation functional and full CC expansion, both expressions lead to the same energy (E DFT tot = E CC tot ). It is therefore tempting to equate the last term in the DFT expression with the last two terms of the CC formula leading to:</p><p>However, this is an approximation because i |φ HF i | 2 does not yield the exact ground-state density. Accordingly, for E CC tot to be exact, E CC c must also contain corrections to all other terms in the energy expression:</p><p>where</p><p>, and so on. When constructing a correlation functional based on CC reference data, we are essentially hoping for a high accuracy of eq. ( 4). In particular that,</p><p>and</p><p>Indeed, these conditions are related, since the exact DFT exchange can be computed analogously to the HF case, but using {φ KS i } instead of {φ HF i }, leading to</p><p>The difference between the WFT and DFT correlation energies thus boils down to the difference between {φ KS i } and {φ HF i }. While the exact KS orbitals are generally not available (because a general expression for the exact E xc [ρ 0 ] is unknown), it has been observed that Brueckner theory offers an excellent approximation to {φ KS i }. 44,45 Very briefly, the idea behind the Brueckner CC approach is to rotate the HF orbitals in such a way that the T 1 contribution to the correlation energy vanishes. This is equivalent to introducing a (non-local) correlation potential into the HF equations. 46 If the chosen CC expansion is exact (see below), the total energies of the canonical and Brueckner CC methods are identical. However, the individual components on the r.h.s. of eq. ( 3) change. Specifically, the sum of the first four terms (the reference energy) becomes less negative, while the last term (the correlation energy) becomes more negative by the same amount.</p><p>In the following we apply the CC singles and doubles expansion (CCSD) to two electron systems. We can use canonical and Brueckner CC calculations to numerically estimate the accuracy of eq. ( 4) for this case. For the He atom, the correlation energy difference between a canonical CCSD and Brueckner CCD calculation is 2.6 × 10 −5 E h (see the Supporting Information, table S1). Consequently, the approximation made in eq. ( 4) is very good in this particular case.</p><p>In a more general vein, it can be noted that HF electron densities are often surprisingly good. Indeed they are often better than self-consistent GGA densities as observed by Bartlett, Burke and others. [47][48][49] Note, however, that the above discussion is no longer valid if semilocal exchange functionals are used (in particular for molecular systems). Semilocal correlation functionals cannot describe the type of static (left-right) correlation that is evident, e.g. when dissociating the hydrogen molecule in a spin-restricted calculation. As was observed by Handy and others, this contribution is instead emulated by GGA exchange functionals. 50 The case is different for atomic systems, however. Many classic GGA functionals are based on the approximate equivalence of exchange and correlation in DFT and WFT for atoms. For example, Becke's 1988 exchange functional was fitted to HF exchange energies of atoms, and the Lee-Yang-Parr (LYP) correlation functional is derived from the Colle-Salvetti formula, which expresses the WFT correlation energy of the Helium atom in terms of the corresponding HF density matrix. 10,11 Even functionals which are not based on WFT at all (such as the already mentioned SCAN functional and the "nearly correct asymptotic property" NCAP functional) show reasonably good numerical agreement with the WFT based exchange and correlation energies of noble gas atoms. 17,51 It has also been found empirically that WFT and DFT correlation energies are compatible, as reflected in the success of double hybrid functionals, which describe E c as a linear combination of GGA and MP2 correlation. 25</p><!><p>The connection between WFT and DFT has long been the subject of intensive research. Most prominently, such efforts have been directed at the exchange-correlation potential, V xc . [52][53][54][55][56][57][58] These studies have underscored the limitations of most semi-local approximations to V xc , particu- larly those that are the functional derivatives of common DFAs 59,60 . Such ab initio potentials are also essential components of some of the higher-rung DFAs methods mentioned above. [61][62][63] Knowledge of V xc does not provide a route to the corresponding functional E xc , however. The latter requires an expression for the exchange correlation energy density xc (r), as given in eq. ( 1). Unfortunately, an inherent difficulty with defining xc (r) is that it is not unique. In principle, the only condition is that integrating this function over all space yields the exchange-correlation energy. Adding any function that integrates to zero to an ansatz for xc (r) therefore yields equally valid energy densities that may look completely different (see Fig. 1). 64 In this sense, xc (r) is arbitrary. However, not all possible energy densities are mappable to the electron density in an efficient way. A systematic way for defining xc (r) for different systems from ab initio calculation allows exploring this mapping, and therefore represents a promising starting point for designing new DFAs.</p><p>One strategy to this end is relating xc (r) to the exchange-correlation hole potential. 56,65 This offers a systematic route to calculating xc (r), given that the one-and two-particle density matrices are known. This has, e.g., been done for configuration interaction wavefunctions with singe and double excitations (CISD). 56 More recently, Vyboishchikov used modified "local" twoelectron integrals to calculate the correlation energy density c (r) at the MP2 and CISD level. 66 These functions were used to construct a simple local correlation functional for spherically confined atoms.</p><!><p>In the following we introduce a new method to calculate an c (r) from first principles, namely one that integrates to the CC correlation energy. The approach has several advantages: (1) By virtue of being CC-based, it is automatically size-extensive (unlike truncated CI). ( 2) Only integrals and amplitudes that are available in any standard CC code are required. (3) The c (r) obtained in this manner is by construction topologically similar to the electron density, making it amenable to semilocal approximations (see below).</p><p>In CC, the ground-state wavefunction Ψ CC is defined with respect to a reference determinant ψ 0 as: 67</p><p>By truncating T at double (N=2), triple (N=3), or quadruple (N=4) excitations one obtains specific CC methods, abbreviated as CCSD, CCSDT, and CCSDTQ respectively. [67][68][69] An important feature of these methods is that they are exact for systems with a number of electrons smaller or equal to the highest excitation level (i.e. CCSD is exact for two-electron systems).</p><p>Irrespective of the truncation, the CC correlation energy only depends on the single and double amplitudes (t a i and t ab ij ), while higher than double excitations contribute to the energy indirectly, by coupling with T 1 and T 2 . The correlation energy is calculated as:</p><p>) with τ ab ij = t ab ij + 1 2 t a i t b j , and the antisymmetrized twoelectron integrals in MO basis defined as</p><p>These integrals are obtained from the corresponding AO integrals and the MO coefficients which define ψ 0 , formally via:</p><p>We are now looking to transform the coupled cluster correlation energy into a form resembling the DFT expression:</p><p>We start from the AO-CC approach of Ayala and Scuseria, which is based on an MO to AO transformation of the T-amplitudes: 70</p><p>Given these AO amplitudes, the correlation energy can be calculated as:</p><p>We now partition the energy into atomic or AO contributions, using:</p><p>Because the AO basis-functions are normalized, the CC correlation energy can now be written as an integral over space:</p><p>This defines the CC correlation energy density as:</p><p>As noted above, CC c (r) is topologically similar to the electron density, in the sense that it is a linear combination of atomic densities. As shown in Fig. 1, the shape of the correlation energy density is in principle arbitrary. However, an energy density that is similar to the electron density can be much more easily approximated by a (semi-)local approach.</p><p>Using eqs. ( 15), ( 16), ( 18) and ( 20), CC c (r) can be calculated for any system, as long as a standard calculation is possible. In the following some exemplary calculations for atomic two-electron systems are performed at the CCSD level, using a custom Python program interfaced with the Psi4 program package. 71,72 Calculations for two-electron ions were performed with a modified uncontracted cc-pV5Z basis set for Helium, where the scaling factor of the orbital exponents was optimized individually for each ion (abbreviated u-5Z). 73 In all other DFT calculations, the pcseg-3 basis set of Jensen is used. 74 Additional CCSD calculations on He-Zn were performed with the core-polarized cc-pwCV(5+d)Z basis. 75 DFT correlation energies are calculated by numerical quadrature on Lebedev-Treutler (75,302) grids. 76 All DFT calculations (also for PBE) are performed nonself-consistently using HF densities with the same code.</p><!><p>As model systems, we calculate CC c (r) for the twoelectron ions from H − to Ne 8+ (see Fig. 2). In all cases, the correlation energy density decays in an approximately exponential fashion as a function of the distance from the nucleus, with the individual curves being highly system dependent. Specifically, CC c (r) decays slowly for the very diffuse H − ion and quickly for Ne 8+ . It is furthermore notable that the correlation energy density for He is quite similar to the one obtained by Vyboishchikov's 'local 2e-integral' approach, despite the different mathematical ansatz. 66 In the supporting information we also include the respective plots for the PBE and LDA correlation functionals (Figs. S1 and S2). While both energy densities are qualitatively similar to Fig. 2, there are important differences. In the LDA case, the functions decay at approximately the same rate as CC c (r), but they are less curved and display larger values at the nuclear cusp. In contrast, the PBE curves overall decay more quickly and display a more complex shape, with a fast initial decay close to the nucleus followed by a slower asymptotic decay.</p><p>From a DFT perspective, the more interesting dependence is between CC c (r) and ρ (Fig. 3). As the atomic electron densities are monotonically decaying, there is a unique mapping between the two for each ion. Specifically, | CC c (r)| increases approximately parabolically with ρ. Unsurprisingly, the curves are again somewhat system dependent, however. This simply means that a LDA-like correlation functional cannot represent CC c (r) exactly for all systems.</p><p>If it is to be useful for defining DFAs, it should at least be approximately possible to effectively map CC c (r) to ρ, however. Furthermore, this mapping should ideally only use readily available local features of the electron density, such as ρ(r) or the reduced density gradient s = |∇ρ(r)| 2(3π 2 ) 1/3 ρ(r) 4/3 . To explore whether this is possible in the presented formalism, we construct a simple GGA functional to approximate CC c (r). To this end, only datapoints with s < 5 were taken into account, following the observation of Burke, Perdew and coworkers that the energetically relevant range is 0 < s < 3. 77 As can be seen in Fig. 4, a simple linear fit allows an accurate description of all datapoints with s < 0.1 (i.e. those with approximately "homogeneous electron gas"-like conditions). This is reminiscent of the Wigner functional, 78,79 which is linear in ρ to leading order, but allows some more flexibility in the low density regime:</p><p>where c 1 and c 2 are coefficients to be defined. Eq. ( 21) forms the local baseline functional for our GGA (with c 1 = −0.0468 and c 2 = 0.023).</p><p>As shown in Fig. 5, the residual error of W c [ρ(r)] is strongly dependent on the reduced gradient s. The largest errors are found in the regime between 0 < s < 2. For the full GGA functional, we now choose the enhancement-factor ansatz:</p><p>Plotting CC c / W c vs. s, gives insight into the numerical distribution of an ideal enhancement factor (Fig. 6). Interestingly, all ions from He to Ne 8+ approximately fall on a curve, whereas the H − datapoints deviate significantly. This reflects the well-known inability of GGAs to adequately describe atomic anions. 80 Specifically, semilocal DFAs only attach a fractional electron to an atom in a complete basis-set due to the self-interaction error. 81,82 This is an inherent limitation of the GGA functional form, not of the CC reference calculations. 83 We therefore exclude H − when fitting parameters, though it is retained in the analysis, for comparison. The distribution of the numerical enhancement factor in Fig. 6 suggests that F (s) should have a sigmoidal form with the asymptotic behaviour:</p><p>We therefore base F (s) on the "complementary" logistic function:</p><p>with coefficients c 3−5 .</p><p>Combining equations ( 21), ( 22) and ( 25), the final functional, which we call ccDF, thus has the simple 5parameter form:</p><p>One could optimize these parameters to directly reproduce the numerical F (s) as closely as possible. However, this strategy is not optimal, as F (s) only enters the energy expression as a scaling factor for W c [ρ(r)]. Consequently, it has little effect on the total energy, whenever W c [ρ(r)] is small. A more promising approach is therefore to use total correlation energies (E c ) as reference data.</p><p>A least-squares fit of the GGA parameters to the correlation energies of He to Ne 8+ yields: The resulting enhancement factor is a good fit to the numerical F (s) (solid line in Fig. 6), and the ccDF functional accurately reproduces the CCSD correlation energies of He to Ne 8+ (Fig. 7). This figure also includes the PBE correlation energies. Unsurprisingly, ccDF more closely reproduces the CCSD correlation energies than PBE, given that it was fitted to this data. It is, however, notable that this functional achieves very high total accuracies of 10 −3 E h or better (except for H − , see above), given its simple functional form. More importantly, both functionals display the correct qualitative behaviour: As Z increases, the correlation energy converges to a constant value.</p><p>As discussed in the Theory section, exact numerical agreement between DFT and WFT correlation energies should generally not be expected. Neither is it necessary for chemical applications. For example, both MP2 and PBE correlation energies will often deviate from more accurate CC values by 10% or more, yet both methods are still quite accurate in terms of energy differences. In fact, even the CCSD/u-5Z values we used for fitting ccDF are only converged to within several milli-Hartree, since the complete basis-set limit for absolute correlation energies of isolated atoms is notoriously difficult to reach. 84 Still, a useful DFA should reproduce the qualitative behaviour of accurate WFT reference values.</p><p>Having established the accuracy of ccDF for twoelectron systems, the question arises whether this functional form can also be applied in the many-electron case. To this end, we computed the correlation energies for the closed-shell neutral atoms from He to Kr (table 1), for which highly accurate reference energies are available. 84,85 Here, ccDF and PBE show qualitatively different behaviour. For He and Be, both functionals recover >90% of the correlation energy. For all other systems, PBE continues to recover 85-100% of the correlation energy while the ccDF values range from 60-70%.</p><p>This behaviour can readily be explained by considering the spin-polarized form of the Wigner functional, upon which ccDF is based:</p><p>Here, ρ α and ρ β are the up and down-spin densities, respectively. By construction, this functional only describes correlation between electrons of opposite spin (i.e., the correlation energy for fully spin-polarized systems is zero). Though this is not widely appreciated, the LYP functional actually suffers from the same problem, since the first term in its expansion is exactly eq. ( 27). [87][88][89][90] Obviously, closed shell two-electron systems like He only display opposite spin correlation. Similarly, Be possesses filled 1s and 2s orbitals, so that there is only weak core-valence correlation between same-spin electrons, and the bulk of the correlation energy is of opposite-spin nature. ccDF describes these systems quite accurately.</p><p>For all other systems, ccDF underestimates the total correlation energy by about one third, presumably due to the missing same-spin contribution. Importantly, this is in good agreement with the relative contribution of same-spin correlation for general many-electron systems, as estimated by Grimme and Head-Gordon in the construction of the spin-component-scaled (SCS) and scaled-opposite-spin (SOS) MP2 methods. 91,92 For instance, SOS-MP2 simply scales the opposite-spin correlation energy by 1.3 to approximate the full correlation energy.</p><p>To further corroborate this interpretation, we turn to the spin component decomposition of the CC energy, which allows computing the opposite-spin contribution to the CC correlation energy as: 93</p><p>As shown in Fig. 8, E ccDF c indeed correlates with E CCSD c,OS quite well. This indicates that the physics of opposite-spin correlation are essentially transferable between two-and many-electron system. However, neither this transferability nor the GGA approximation should be expected to be perfect. Future work will therefore focus on developing general correlation functionals based on CC. We consider the presented results to be very encouraging for this endeavour.</p><!><p>In this paper, we have introduced a new approach to calculating CC correlation energy densities CC c (r) for atomic systems. These densities are derived from an AObased formulation of CC and exactly integrate to the respective CC correlation energy. The properties of CC c (r) were discussed for a range of atomic two-electron systems.</p><p>As these energy densities are by construction topologically similar to the electron density, they are well suited to be approximated by DFAs. As a proof-of-principle, a CCSD based GGA functional was constructed for the He isoelectronic series. By analysis of the numerical CCSD functional, we find that a remarkably simple enhancement-factor formula can be fitted to yield highly accurate correlation energies for these systems. Despite only being fitted to two-electron systems, we find that the ccDF functional also provides reasonable estimates of the opposite-spin correlation energies of many-electron atoms. This indicates that CC c (r) provides a robust physical basis for the design of DFAs, and that the He isoelectronics form an interesting set of model densities.</p><p>However, it should be emphasised that the proposed GGA functional is mainly intended as a proof-ofprinciple, and should not be applied to general systems as is. Most importantly, it should at least be augmented with a corresponding same-spin functional. 88 Furthermore, the proposed form of CC c (r) is only one possible choice.</p><p>An expression based on the one-and two-particle density matrices may in fact be preferable, as it would allow using the "gold-standard" CCSD(T) method as reference, which includes perturbative triple contributions. In contrast, our current approach can only be used with full coupled cluster methods (CCSD,CCSDT,CCSDTQ, etc.), of which all but CCSD display prohibitive computational scaling for all but the simplest systems. Moving beyond CCSD is a prerequisite to obtain a good description of electron correlation from systems with more than two electrons.</p><p>Importantly, the present framework is general enough to be applied to more complex functional forms (e.g. truly non-local functionals), and this will be the subject of future work. An especially promising route lies in the use of CC energy densities to train "machine-learned" functionals. 94 The fact that CC c (r) can guide the design of a simple and accurate functional form like the GGA indicates that it contains the necessary information to this end.</p>

ChemRxiv

Antimicrobial photodynamic therapy in the colon: delivering a light punch to the guts?

A paper in this issue of Photochemistry and Photobiology by Cassidy et al describes the use of a sophisticated drug delivery vehicle prepared by the hot melt extrusion process to deliver photosensitizers to the colon. The smart vehicle protects its cargo through the acidic environment of the stomach but releases the active photosensitizers in the higher pH and anaerobic environment of the colon. The goal is to use photodynamic therapy (PDT) to destroy pathogenic microorganisms that can cause disease when they grow out of control in the colon. Since the colon is an environment with a low oxygen concentration the investigators also used tetrachlorodecaoxide, an oxygen donor to boost the available oxygen concentration. The paper reports results with Enterococcus faecalis and Bacteroides fragilis but the real medical problem demanding to be solved is Clostridium difficile that can cause intractable drug-resistant infections after antibiotic use. There still remain barriers to implementing this strategy in vivo, including light delivery to the upper colon, oxygen availability and optimizing the selectivity of photosensitizers for bacteria over colon epithelial cells. Nevertheless this highly innovative paper lays the ground for the study of an entirely new and significant application for antimicrobial PDT.

antimicrobial_photodynamic_therapy_in_the_colon:_delivering_a_light_punch_to_the_guts?

Introduction<!>Novel application for antimicrobial PDT<!>Smart Drug Delivery Vehicle<!>Caveats<!>Conclusions

<p>Photodynamic therapy (PDT) was discovered over one hundred years ago by the ability of certain dyes when combined with visible light in the presence of oxygen to kill various microorganisms. However PDT has not been much developed as a treatment approach for infections until recent times, but rather has been studied as a treatment for cancer, skin diseases and choroidal neovascularization. The recent alarming rise in drug resistance not only amongst bacteria but also involving fungi, parasites, viruses and almost all classes of pathogens has acted to change these priorities. The interest in antimicrobial PDT for infections is starting to rapidly increase although the actual clinical applications so far remain very limited. In most of these clinical applications the photosensitizer is topically applied to the infection as for instance rubbed on the skin for acne, injected into the dental pocket for periodontitis, or applied to the surface of a non-healing ulcer.</p><p>The application of photosensitizers to deeper-seated, semi-localized infections clearly requires more sophisticated drug delivery methods; such as a delivery vehicle that is engineered to only release its cargo at the target site.</p><!><p>The current submission from Cassidy et al. [1] provides a model for the treatment of intestinal infection using several known photosensitizers and a methacrylate polymer delivery system to great effect against known colonic pathogens.</p><p>The alimentary tract relies on bacterial flora for efficient food breakdown, transit and elimination. A wide variety of bacteria – from Gram-positive aerobes to Gram-negative anaerobes is – present, normally in a synergistic relationship with the host. Thus when the microflora is damaged to any significant degree, for example under the action of oral antibiotics, gastric/enteric disorders usually ensue. Such disorders are due to the overgrowth of recalcitrant bacteria in areas of the GI tract previously colonized by antibiotic-susceptible species. Often in hospital patients, however, and particularly in the elderly or those being treated for multidrug-resistant bacterial infections, such is the damage to the microbial ecosystem that both morbidity and mortality rates increase. In such cases, the offending organism is usually the Gram-positive, microaerophilic bacterium Clostridium difficile [2], or the yeast Candida albicans [3].</p><p>It will be understood, of course, that there are antimicrobial agents available for both of these pathogens. For example, the glycopeptide antibiotic vancomycin and the nitroimidazole agent metronidazole are recommended for the treatment of C. difficile infection. However, as noted by Cassidy et al., the efficacy of these drugs is often variable [4]. In addition, other intestinal bacteria, such as Enterococcus spp. are a considerable cause for concern, and obviously more so where vancomycin resistance is involved.</p><p>The difficulty inherent in the application of conventional antimicrobial agents to colonic infection/superinfection surely constitutes a prima facie case for the application of the photodynamic approach. As Cassidy et al. [1] have shown, it is possible to realize high levels of bacterial kill against colonic bacteria using established photosensitizers/ALA. In addition, it is established that bacterial resistance to conventional chemotherapy does not affect susceptibility to photodynamic antimicrobial chemotherapy (PACT) – indeed, this has been reported for vancomycin-resistant Enterococcus faecalis [5].</p><p>It should also be remembered that PACT offers other advantages, particularly from the aspect of conventional drug conservation. While vancomycin is normally kept in reserve for the treatment of serious drug-resistant infection, typically with methicillin-resistant Staphylococcus aureus (MRSA), as noted it is currently a front-line treatment for C. difficile infection and this must surely impact on the wider development of resistance to vancomycin [6]. Indeed, infection with C. difficile has been correlated with vancomycin-resistant enterococcal (VRE) infection [7].</p><p>Obviously, hospital-acquired infection rates can only be decreased by breaking the infection chain, and significant falls will require increases in environmental hygiene control as well as more effective disease therapy. However, the photodynamic approach as described is antimicrobial, rather than specifically antibacterial or antifungal. Thus while specific conventional therapy of the colon using vancomycin etc. may be effective against C. difficile, by definition it would not affect VRE and could provide no check against nascent infection by Candida spp.</p><!><p>The approach taken by Cassidy et al [1] details a "smart" delivery vehicle for the potential aPDT of multi-drug resistant colon infections. It is an oral formulation prepared using the hot melt extrusion process. During the journey of the vehicle to its destination in the colon, the vehicle can "smartly" protect its PS cargo when passing through the stomach. When it reaches the colon, the vehicle releases its cargo in a time dependent manner. In this procedure, the pH value of the environment serves as the "traffic signal" for the PS release. The pH value of 1, which is the value of an acid environment (stomach), is the "withholding signal"; while the pH value of >7, the value of the anaerobic environment in the gut is the "release signal". With such a smart delivery vehicle that can recognize the "release signal", PS would be released in a controlled manner. Recognizing the low availability of oxygen in the anaerobic environment in the colon these investigators incorporated an oxygen delivery vehicle in their formulation. Tetrachlorodecaoxide (TCDO) is a chlorite derivative that is used in wound dressings and can even be injected intravenously so it should not have unacceptable toxicity in the colon. Nonetheless, this study is so far still at the stage of in vitro testing and considerable further work would be needed to advance the concept to clinical application.</p><!><p>There remain formidable potential obstacles to carrying out aPDT in the colon. How would the light be delivered? Although fiber optic devices with cylindrical diffusing tips exist that could in principle be advanced up the colon to the infected areas, it is not clear how much of the colon would need to be illuminated. Can the actual areas of infection be identified? Another serious potential problem concerns the availability of sufficient oxygen concentration in the colon. Although these investigators showed that the addition of TCDO was able to potentiate PACT-mediated bacterial killing (especially in the case of Bacteroides fragilis), oxygen limitation may still be problematic. In the clinical application it is possible that the light delivery fiber could be engineered to simultaneously deliver oxygen at the same time as the inactivating light. A third problem lies in the question of whether the photosensitizers would need to be specifically designed to bind to the bacteria rather than the colon tissue and cells, upon release from their smart vehicle. Numerous studies have reported sophisticated antimicrobial PS whose ability to selectively bind to bacteria rather than host mammalian cells has been optimized. Examples of these constructs are the polycationic conjugate between polyethylenimine and chlorin(e6) [8], specially constructed nanoparticles conjugated to PS [9] and a bacteriophage-Sn(ce6) conjugate that recognizes specific receptors on bacteria [10].</p><!><p>Even with all these caveats this innovative proposal to carry out colon-specific delivery of PS coupled with local illumination could destroy drug-resistant disease-causing bacteria and cause less damage to the commensal flora in other parts of the alimentary canal.</p>

PubMed Author Manuscript

Hydrogen Bond Networks Near Supported Lipid Bilayers from Vibrational Sum Frequency Generation Experiments and Atomistic Simulations

We report vibrational sum frequency generation (SFG) spectra in which the C-H stretches of lipid alkyl tails in fully hydrogenated single-and dual-component supported lipid bilayers are detected along with the O-H stretching continuum above the bilayer. As the salt concentration is increased from ~10 µM to 0.1 M, the SFG intensities in the O-H stretching region decrease by a factor of 2, consistent with significant absorptive-dispersive mixing between χ (2) and χ (3) contributions to the SFG signal generation process from charged interfaces.A method for estimating the surface potential from the second-order spectral lineshapes (in the OH stretching region) is presented and discussed in the context of choosing truly zero-potential reference states. Aided by atomistic simulations, we find that the strength and orientation distribution of the hydrogen bonds over the purely zwitterionic bilayers are largely invariant between sub-micromolar and hundreds of millimolar concentrations. However, specific

hydrogen_bond_networks_near_supported_lipid_bilayers_from_vibrational_sum_frequency_generation_exper

<!>I. Introduction.<!>III. Results and Discussion.<!>B. Dual-Component Supported Lipid Bilayers Formed from Zwitterionic and Negatively<!>IV. Conclusion. In conclusion

<p>interactions between water molecules and lipid headgroups are observed upon replacing phosphocholine (PC) lipids with negatively charged phosphoglycerol (PG) lipids, which coincides with SFG signal intensity reductions in the 3100 cm -1 to 3200 cm -1 frequency region.</p><p>The atomistic simulations show that this outcome is consistent with a small, albeit statistically significant, decrease in the number of water molecules adjacent to both the lipid phosphate and choline moieties per unit area, supporting the SFG observations. Ultimately, the ability to probe hydrogen-bond networks over lipid bilayers holds the promise of opening paths for understanding, controlling, and predicting specific and non-specific interactions between membranes and ions, small molecules, peptides, polycations, proteins, and coated and uncoated nanomaterials.</p><!><p>The structure of water over lipid membranes is of interest for a variety of reasons that are rooted in fundamental scientific interest and connect all the way to biological function and technological applications. [1][2][3][4][5][6] Specific questions pertain to whether there exist populations of interfacial water molecules that can undergo hydrogen-bond (H-bond) interactions with certain membrane constituents that can be strengthened or weakened with variations in ionic strength, or, as indicated by molecular dynamics simulations, 2 whether some population of water molecules exists that may interact specifically with certain lipid headgroups over others.</p><p>While interface-specific vibrational spectroscopic approaches, particularly those that are based on sum frequency generation (SFG), are in principle well suited for probing water near membranes, this method has been largely limited to probing lipid monolayers 1,[7][8][9][10][11][12][13][14][15][16][17][18][19] chemically asymmetric bilayers, [20][21][22] or the use of D 2 O as opposed to H 2 O. [23][24][25] Indeed, the use of SFG spectroscopy for probing fully hydrogenated lipid bilayers is now just emerging. Part of the reason for this relatively new application of vibrational SFG spectroscopy to probe chemically unmodified lipid bilayers is rooted in the symmetry-breaking requirement of the method, 26 which has limited its use largely to asymmetric bilayers consisting of a deuterated and a hydrogenated leaflet, or lipid monolayers, as stated above. SFG signals generated by asymmetric membranes (deuterated leaflet on one side and hydrogenated leaflet on the other side, or aliphatic lipid tail on one side and polar headgroup on the other) are strong enough to be detectable using lowrepetition rate, low peak power laser systems most commonly used in the field. Two studies known to us also report SFG spectra of unlabeled symmetric lipid bilayers, demonstrating their low signal yields when compared to labeled bilayers. [27][28] Our recent work [29][30][31] has shown, in the C-H stretching region, that commercially available broadband optical parametric amplifier laser systems running at modest (kHz) repetition rates can overcome these limitations, with reasonably high signal-to-noise ratios obtained in just a few minutes of spectral acquisition time.</p><p>Here, we report how to apply this approach to probe the C-H stretches of the alkyl tails in fully hydrogenated single-and dual-component supported lipid bilayers (SLBs) along with the O-H stretching continuum of the H-bond network system in the electrical double layer above them. The approach probes lipid tail order and disorder while also informing on changes in the H-bond network strength that result from changes in the bulk ionic strength up 100 mM NaCl. Moreover, by varying the lipid bilayer composition from 100% zwitterionic lipid to an 8:2 mixture of zwitterionic and negatively charged lipids, we identify specific H-bond interactions between water molecules and the lipid headgroup choline moieties that manifest themselves in spectral intensity changes in the 3100 cm -1 to 3200 cm -1 range. II. Methods.</p><p>A. Bilayer Preparation. 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2dimyristoyl-sn-glycero-3-phospho-(1-rac-glycerol) (DMPG) were purchased from Avanti Polar Lipids and used without further purification. Lipid bilayers from small unilamellar vesicles of pure DMPC, lipid mixtures containing 90 mol% DMPC and 10 mol% DMPG, and 80 mol% DMPC and 20 mol% DMPG were prepared by the vesicle fusion method, as described earlier, 29, 31-34 on 3 mm thick calcium fluoride windows (ISP Optics, CF-W-25-3). Prior to use, the calcium fluoride window was sonicated in HPLC-grade methanol (Fisher Scientific) for 30 min, rinsed with ultrapure water (18.2 Ω•cm resistivity; Millipore), and dried with N 2. The window was then plasma cleaned (Harrick Plasma Cleaner, 18W) for 10 min.</p><p>Experiments were carried out at room temperature (21 ± 2 o C). All SLBs were formed at 0.01 M Tris buffer and 0.1 M NaCl in the presence of 0.005 M CaCl 2 •2H 2 O at pH 7.40 ± 0.03. 33 Following bilayer formation, SLBs were rinsed with Ca-free buffer to remove excess vesicles.</p><p>The spectra were recorded at two different ionic strengths. Before the preparation of aqueous solutions, Millipore water was left overnight to equilibrate with atmospheric CO 2 . The solution pH was measured for each salt concentration and the pH was adjusted to 7.4 with minimal NaOH and HCl before the solutions were flowed across the interface resulting in ionic strengths of ~10 µM and 0.1 M for the Millipore solution and NaCl solution, respectively. B. Vibrational Sum Frequency Generation Spectroscopy. Details of our SFG approach and experimental setup for probing condensed matter interfaces in the C-H stretching region have been reported previously. [33][34][35][36][37][38] Here, we adapt this approach to extend our spectral range into the O-H stretching region, as described in detail in the Supporting Information (see part numbers of the optical elements in Supplementary Figure S1). Briefly, 90% of the output from a Ti:Sapphire amplifier laser system (Spectra Physics Solstice, 3 mJ/pulse, 795 nm pulses, 1 kHz repetition rate, 120 femtosecond pulse duration) pumps a travelling-wave optical parametric amplifier to generate a broadband tunable IR beam tuned to the C-H and O-H regions (2800-3600 cm -1 ), while the remaining portion is sent down the visible upconverter beam line, where it is attenuated using a variable density filter and spectrally narrowed using an etalon. The IR and visible beams are focused to a ~30 µm beam waist at the interface, where they overlap at the CaF 2 /water interface at 38° and 30° from the surface normal, respectively. The beams approach the interface from the CaF 2 side and the SFG signal is detected in reflection. The resultant SFG signal is dispersed on to a spectrograph (Acton SP-2558) and liquid nitrogen-cooled CCD camera. During the SFG experiments, the IR line was purged with dry house N 2 to avoid water absorption bands that appear in this stretching region. All SFG spectra were collected using the near total internal reflection geometry and the ssp polarization combination (s-polarized SFG, s-polarized 800 nm light, p-polarized IR light). All SFG spectra were recorded in triplicates and normalized to the ppp-polarized SFG response obtained from a gold window. To cover the full spectral range of interest, multiple spectra are collected at different IR center wavelengths before being combined into a single spectrum. Further details regarding spectral acquisition and analysis procedures are provided in the Supporting Information (See Figure S2).</p><p>C. FRAP Measurements. Two-dimensional diffusion coefficients, which can serve as a metric for bilayer quality, were estimated using fluorescence recovery after photobleaching (FRAP).</p><p>FRAP measurements and sample preparation were carried out in a manner consistent with our previous approach. 29 For these experiments, vesicles composed of DMPC or a 9:1 mixture of DMPC/DMPG lipids were doped with 0.1 mol% TopFluor PC (Avanti Polar Lipids, 810281).</p><p>After forming the SLB as described in Section IIA, the cell was flushed with 20 mL of 0.1 M NaCl, 0.01 M Tris buffer (pH 7.4). In a second set of experiments, the flow cell was flushed with 20 mL of pH-adjusted Millipore water with no added salt. For SLBs formed from a 9:1 mixture of DMPC/DMPG lipids, we find diffusion coefficients on the order of 0.5 ± 0.2 µm 2 /s (13 replicates over two samples) after rinsing with 0.1 M NaCl, 0.01 M Tris buffer, which is consistent with our previously reported two-dimensional diffusion coefficients 29 and indicates that a well-formed bilayer is produced from the abovementioned method. [39][40][41] Upon rinsing with pH-adjusted Millipore water with no added salt, we find that the diffusion coefficient for SLBs formed from 9:1 mixtures of DMPC/DMPG lipids are on the order of 0.03 ± 0.01 µm 2 /s ( 6replicates over 1 sample). The diffusion coefficient for SLBs formed from pure DMPC lipids on calcium fluoride is 0.4 ± 0.2 µm 2 /s (6 replicates over two samples) after rinsing with 0.1 M NaCl, 0.01 M Tris buffer. After rinsing with pH adjusted Millipore water, we find a diffusion coefficient of 0.07 ± 0.02 µm 2 /s (4 replicates over two samples). For SLBs formed from 8:2 mixtures of DMPC/DMPG lipids, the diffusion coefficient on calcium fluoride is 0.07 ± 0.04 µm 2 /s (6 replicates over one sample) after rinsing with 0.1 M NaCl, 0.01 M Tris buffer.</p><p>Representative traces, along with a detailed procedure used in these experiments, are provided in the Supporting Information (see Figure S3). These results indicate the bilayers transition between the gel and fluid phases, irrespective of the nature of the underlying substrates (CaF 2 vs fused silica). D. Computational Methods. Molecular dynamics (MD) simulations for investigating the structure of the H-bond network near the lipid-water interface were performed using the CHARMM-GUI 42 input generator to set up the DMPC and 9:1 DMPC/DMPG systems. Each system contains a 10 x 10 nm 2 lipid bilayer. For pure DMPC, systems were set up with 0.15 M NaCl or no salt added, both with a hydration level (i.e., water:lipid ratio) of 53. The 9:1 DMPC/DMPG system was set up with 0.15 M NaCl and a hydration level of 65. We performed equilibration and production runs with CHARMM-GUI generated input files using the NAMD 43 package. The CHARMM36 [44][45] force field was applied for the lipid, water, and ions. The Particle-Mesh-Ewald 46 (PME) method was used for the electrostatic interactions with a realspace cutoff of 1.2 nm. Force switching with a cutoff of 1.2 nm was applied to the van der Waals interactions. The PME grid size was set to 108, 108, and 100 for the X, Y, and Z dimensions in the DMPC simulations, and to 108, 108, and 120 for the 9:1 DMPC/DMPG simulations.</p><p>RATTLE 47 was applied to constrain all bonds involving hydrogen atoms in length. Langevin dynamics were applied for constant pressure and temperature control. A Nose-Hoover Langevin piston [48][49] was applied with constant ratio on the X-Y plane and a target pressure of 1 atm. The target temperature was set to be 303.15 K with a damping coefficient of 1.0 ps -1 . For the DMPC systems, the production run lasted for 30 ns with a 2 fs time step; for the 9:1 DMPC/DMPG systems, the production was run for 70 ns. Any unspecified details, including the equilibration process before production runs (see Table S2 for details), are consistent with the standard CHARMM-GUI protocol which have been shown to provide area per lipid and other essential properties of lipid bilayers in good agreement with experiments. 42,[50][51]</p><!><p>A. Single-Component Zwitterionic Supported Lipid Bilayers. Figure 1A shows the ssppolarized SFG response from the pure DMPC bilayer without added salt. At this low ionic strength (~10 µM), we find clear spectral signatures from the C-H oscillators of the alkyl tails, 29, 31, 34 as well as broad contributions from the O-H stretches of the water molecules. The non-zero signals are due to the fact that the molecular environment above and below the bilayer is not fully symmetric, as would be expected for a suspended bilayer. Instead, symmetry breaking occurs due to the presence of the aqueous phase on one side and the solid support on the other.</p><p>The frequencies corresponding to the signal peaks in the C-H stretching region shown in Figure 1A are comparable to the ones we observe for supported lipid bilayers formed on fused silica substrates (see Supporting Information Figure S4) 29,31,34 The two broad features in the O-H stretching continuum located at ~3200 cm -1 and ~3400 cm -1 are associated with bandwidths (full width at half maximum) of about 200 cm -1 . The peak positions are within 50 cm -1 of what has been reported for water spectra obtained from symmetric bilayers prepared from negatively charged lipids on CaF 2 . 28 The difference is attributed to the fact that our current experiments use bilayers formed from purely zwitterionic lipids.</p><p>Replacing the H 2 O phase with D 2 O while maintaining low ionic strength, shown in Supporting Information Figure S5, leads to the C-H oscillators retaining their frequencies while the O-H stretching continuum is entirely absent. This experiment indicates that 1) there are no exogenous photon sources contributing to the SFG response from the bilayer under water (H 2 O), and 2) that H 2 O that may be possibly trapped between the bilayer and the substrate is readily exchanged or associated with too little SFG intensity to be detectable by our method. Control experiments assessing the possible role that CaF 2 dissolution could have on the spectra [52][53] (see Supporting Information Figure S6) show that the presence of the bilayer eliminates any flowdependent changes in the SFG signal intensity produced by the interfacial water molecules.</p><p>The O-H stretching continuum can be viewed as a display of the various O-H … O distances sampled in the water network probed by the SFG spectrometer. As shown, for instance, by Lawrence and Skinner, 54 frequencies around 3200 cm -1 correspond to O-H stretches associated with water molecules in tighter H-bond networks, where distances between the donor hydrogen and acceptor oxygen atoms (H … O) are as short as 1.6 Å or less. Towards 3400 cm -1 , the spectrum samples water molecules in a considerably looser H-bond network, having H … Odistances as long as 2.1 Å or so. Towards 3550 cm -1 , H … O distances can be as long as 2.4 Å or more. At the very end of the spectrum, near 3700 cm -1 , would be the O-H stretch of non-Hbonded water molecules, those that "straddle the interface". 55 Such signals are not identified within our signal-to-noise ratio, even though they have been reported to be present in Langmuir monolayers prepared from DPPC lipids. 56 Figure 1B shows the SFG spectrum from the supported lipid bilayer in comparison with that of two other aqueous CaF 2 interfaces, namely that of bare CaF 2 in contact with ~10 µM ionic strength water adjusted to pH 7.4, as well as bare CaF 2 in contact with water vapor in He flow adjusted to 80% relative humidity (see Supporting Information Figure S7). The SFG response from the bare CaF 2 /water interface is in reasonable agreement with published data. 53,[57][58] We find that the peak positions from the bilayer/water interface is blue-shifted by around 25 cm -1 when compared to those obtained from the bare CaF 2 /water interface. Additionally, the SFG spectrum from the CaF 2 /water vapor interface exhibits a blue-shifted SFG spectrum when compared to the bilayer/water or CaF 2 /water interfaces, consistent with the expectation that its hydrogen-bonding environment is looser than in the case of bulk water in contact with the solids. [59][60] Upon increasing the ionic strength in the bulk aqueous phase, the sodium and chloride ions can modify the H-bond network of water molecules in the bulk in ways that are the subject of much past and ongoing scientific attention and discussion. [61][62] NaCl, whose anion and cation fall right in the middle of the familiar Hofmeister series, are not necessarily expected to modify the H-bond network over lipid bilayers at the relatively modest concentrations (0.1 M) employed here. Moreover, ion-specific interactions with the lipids used in our work are unlikely to be strong under the conditions of our experiments. Indeed, Figure 2 shows that the spectral changes we observe in response to changes in the ionic strength are largely uniform over the entire frequency region probed in our experiment (1000 cm -1 ). Between 3000 cm -1 and 3600 cm -1 , the ratio of the SFG spectral intensities at low (~10 µM) and high (0.1 M) ionic strength is computed to vary only slightly, from 1.7 at 3000 cm -1 to 2.3 at 3600 cm -1 and back to 2.0 at 3700 cm -1 (average of 2.1 ± 0.2 over all frequencies). We find this slight frequency dependence of the SFG intensity ratio to be indicative of a minor influence that the relatively modest salt concentrations used here even under what we term "high salt" have on the various contributors to the H-bond network. This interpretation is borne out in molecular dynamics simulations as well, which are described next.</p><p>To further explore the molecular details near the bilayer/water interface, we performed MD simulations for a DMPC lipid bilayer with and without 0.15 M NaCl salt. We focus here on the analysis of the interfacial water structure, specifically the orientation of interfacial water molecules and the O•••O distance of neighboring water molecules. The water orientation is characterized with the dipole angle, θ, which is defined as the angle between the dipole vector of water and the membrane normal pointing towards the bulk. The distribution of water orientations is analyzed as a function of distance from the membrane-water interface, i.e., we plot the two-dimensional distribution 63 (Figure 3):</p><p>in which 𝜃 is the dipole angle defined above, z is the normal distance of the water oxygen from the bilayer center, 𝜌(𝑧) is the number density of water, and 𝑠𝑖𝑛𝜃 is the angular Jacobian factor.</p><p>The distribution shown in Figure 3 is normalized to that of the bulk value. According to the mass density distribution (see Figure S8), the lipid-water interface is identified at z ~ 20 Å. As shown in Figure 3 (left column), in all cases studied, the water orientation distribution shifts towards smaller dipole angles near the interface, while the opposite shift is observed for the small amount of water molecules that penetrate below the lipid/water interface to interact with the lipid glycerol groups (for a snapshot, see Figure S9). The distribution approaches the bulk value at ~8-10 Å away from the lipid-water interface.</p><p>Nevertheless, the distribution of water orientation angles remains broad even at the interface, which is likely due to the dynamic nature of the lipid headgroup (see Figure S10). As a result, no statistically significant difference is observed between the two DMPC cases studied, suggesting that the impact of salt on the water orientation at the interface is subtle compared to the effect of thermal fluctuations. Regarding the distributions of the nearest O•••O distances among water molecules, which reports on the hydrogen bonding strength, our results in Figure 3 (right column) suggest again that the impact of salt on the distance dependent orientation distributions of the water molecules is small for the salt concentrations investigated.</p><p>Rather than being due to changes in the H-bonding network, we find that the SFG signal intensity reductions that coincide with raising the salt concentration from 10 µM to 0.1 M are consistent with absorptive-dispersive mixing between χ (2) and χ (3) contributions to the SFG signal generation process from charged interfaces, according to [64][65][66][67][68][69][70]</p><p>Here, the first two terms are the non-resonant and resonant 2 nd -order susceptibility and the 3 rd term is given by the inverse Debye screening length, 𝜅, the inverse of the coherence length of the SFG process, ∆𝑘 ! , and the interfacial potential, Φ(0), multiplied by the 3 nd -order susceptibility. [64][65]71 We recently showed 66-68 that for an exponential distance dependence of 𝛷(z), the χ (3) phase angle, ϕ, equals 𝑎𝑟𝑐𝑡𝑎𝑛 ∆𝑘 ! 𝜅 . Using "primitive ion" models, 72 such as Gouy-Chapman theory, we estimate at the low (resp. high) salt concentration investigated here that 𝜅 is 1 x 10 7 (resp. 1 x 10 9 ) m -1 . For our experimental geometry, ∆𝑘 ! of 2.4 x 10 7 m -1 and invariant with salt concentration. The resulting phase angle is shown in Figure 4A. At high salt concentration, eqn.</p><p>2 becomes simply additive, 73-74 75 whereas constructive and destructive interference occurs when the phase angle deviates from zero.</p><p>In the absence of phase-resolved measurements, which are proving to be considerably challenging at buried liquid-solid interfaces such as the ones studied here, it is difficult to quantitatively examine the interfacial potential, even if one uses the 3 rd order (𝜒 !"#$ ! ) term recently reported by Wen et al. 64 that should be quite universally applicable for aqueous interfaces. Moreover, it is perhaps not possible to prepare, in an experiment, a truly "zero potential" reference state: even the fully protonated reference state of a carboxylic acid monolayer, commonly used as a reference state in surface potential measurements, 64,[76][77] is subject to dipolar potentials. In the absence of 1) phase resolved data and 2) a true zero potential -and/or zero charge density -reference state, quantitative knowledge of the interfacial potential at two different solution or bilayer conditions from which a difference in surface potential, i.e.</p><p>ΔΦ, can be calculated is difficult to obtain, though methods to acquire this knowledge remain a topic of keen interest to us that we will discuss in forthcoming work.</p><p>For now, we offer the following method for estimating surface potential changes from the second-order spectral lineshapes (in the OH stretching region): an examination of Equation 2reveals that even if, as suggested by the MD simulations discussed above, the H-bond network close to the interface remains invariant or nearly invariant (implying a constant 𝜒 !"#$ ! ) upon changes in ionic strength, changes in the SFG signal intensity can still arise from the potential-dependent χ (3) term. These changes take the form of a complex multiple of the 𝜒 !"#$ ! term, which is given mainly by the 3 rd order optical properties of bulk water. Unfortunately, given the difficulties discussed above, our lack of phase-resolved measurements and our lack of access to a reference state of true Φ(0)=0, precludes us from comprehensively accounting for the phaseangle dependent χ (2) /χ (3) mixing, and thus quantitatively determining the interfacial potential from the SFG spectra reported here. Yet, surprisingly good qualitative agreement is obtained between the difference of the measured intensity spectra for the low and high salt conditions from Figure 2 and the calculated 𝜒 !"#$ ! intensity spectrum derived from the real and imaginary data reported by Wen et al. (see Figure 4). This agreement supports our conclusion that the spectral changes are not indicative of large changes in the H-bonded network of water molecules but rather result from the χ (3) -potential dependent term. Moreover, as shown Supplementary Information equations S1-S5, under conditions where the SFG responses are dominated by the χ (3) term, i.e.</p><p>χ (3) Φ >> χ (2) , an estimate of the difference in surface potential, ΔΦ, can be readily provided if the magnitude of the SFG intensity difference, ΔI SFG , observed for conditions of varying ionic strength, bulk solution pH, analyte concentration, or surface composition, is known (see Supporting Information Eqn S5).</p><!><p>Charged Lipids. Motivated by recent reports that the major contribution in the 3000 cm -1 to 3200 cm -1 frequency region originates from polarized water molecules that bridge phosphate and choline in the zwitterionic lipid headgroup (n. b.: that work focused on lipid monolayer/water interfaces as opposed to lipid bilayer/water interfaces, which are probed in the present study), 2 we proceeded to add negatively charged lipids to the zwitterionic system studied. Mixing in negatively charged lipids, such as DMPG, is then expected to reduce the population of polarized water molecules that interact specifically with the zwitterionic PC headgroup.</p><p>Figure 5 shows that this response is indeed observed. At 0.1 M NaCl, the three systems we surveyed (100% zwitterionic DMPC, 9:1 DMPC/DMPG, and 8:2 DMPC/DMPG) showed no significant changes in the 3400 cm -1 frequency region. Yet, as the percentage of negatively charged lipids increases, the SFG spectral intensity in the 3200 cm -1 region decreases, indicating the theoretical result obtained for lipid monolayer/water interfaces may also hold for lipid bilayer/water interfaces. Triplicate measurements are shown in the Supporting Information (see Figure S11).</p><p>Results from our MD simulation for 9:1 DMPC/DMPG with 0.15 M NaCl (Figure 3, bottom row) reveal similar trends when compared to the pure DMPC case, suggesting that the impact of a small amount (10%) of anionic lipids on the structure and orientation of water at the interface is minor, in the background of thermal fluctuations. Yet, computing the number of water molecules adjacent to lipid phosphate, choline, and those close to both phosphate and choline (see Figure S12 for the relevant radial distribution functions), in a manner consistent with the analysis by Morita and coworkers, 2 we find that mixing in DMPG lipids leads to a small, albeit statistically significant, decrease in the number of water molecule adjacent to both the lipid phosphate and choline moieties per area, as shown in Table 1. These computational results support the observations that the SFG signal intensities seen in the experimental spectra between 3100 cm -1 to 3200 cm -1 are due to local water molecules that specifically interact with the phosphate and choline moieties of the DMPC lipids. 2 As shown in Figure S13, these water molecules are also subject to a fairly broad molecular orientation distributions (with the second moment of the dipole angle θ in the range of 34-38°) due to thermal fluctuations at the lipid/water interface. As the salt concentration is increased from ~10 µM to 0.1 M, the SFG intensities in the O-H stretching region decrease by a factor of 2. This observed salt concentration-dependent change in the SFG signal intensity is consistent with significant absorptive-dispersive mixing between χ (2) and χ (3) contributions to the SFG signal generation process from charged interfaces.</p><!><p>Surprisingly good qualitative agreement is obtained between the difference of the measured intensity spectra for the low and high salt conditions from Figure 2 and the calculated 𝜒 !"#$ ! intensity spectrum derived from the real and imaginary data reported by Wen et al. (Figure 4).</p><p>This agreement supports our conclusion that the spectral changes are not indicative of large changes in the H-bonded network of water molecules but rather result from the χ (3)potential dependent term. As shown in the TOC graphic, at low (resp. high) salt concentration, the surface potential is high (resp. low), thus modulating the SFG response according to the functional form that gives rise to the χ (3) phase angle, 𝜑. Moreover, our analysis provides a method for estimating the difference in surface potential, ΔΦ, from the magnitude of the SFG intensity difference, ΔI SFG , observed for conditions of varying ionic strength, bulk solution pH, analyte concentration, or surface composition, is known (see Supporting Information Eqn S5).</p><p>The TOC graphic indicates that specific interactions between water molecules and lipid headgroups are observed as well: Replacement of PC lipids with negatively charged PG lipids coincides with SFG signal intensity reductions in the 3100 cm -1 to 3200 cm -1 frequency region.</p><p>Our atomistic simulations show that this outcome is consistent with a small, albeit statistically significant, decrease in the number of water molecules adjacent to both the lipid phosphate and choline moieties per unit area, supporting the SFG observations. This result further supports recent molecular dynamics simulations indicating that the major contribution in the 3000 cm -1 to 3200 cm -1 frequency region originates from polarized water molecules that bridge phosphate and choline in the zwitterionic lipid headgroup. 2 Ultimately, the ability to probe H-bond networks over lipid bilayers holds the promise of opening paths for understanding, controlling, and predicting specific and non-specific interactions membranes with solutes such as ions 34 and small molecules such as peptides, 70 or larger species such as polycations, 30,38 and coated and uncoated nanomaterials. 31,33,[78][79][80][81][82][83][84] The lines represent the data that have been binned by over nine points in x and y between 3000 cm -1 and 3600 cm -1 .</p>

ChemRxiv

Controlled Chemoenzymatic Synthesis of Heparan Sulfate Oligosaccharides

A chemoenzymatic approach has been developed for the preparation of diverse libraries of heparan sulfate (HS) oligosaccharides. It employs chemically synthesized oligosaccharides having a chemical entity at a GlcN residue, which in unanticipated manners influences the site of modification by NST, C5-Epi/2-OST and 6-OST1/6-OST3, thus resulting in oligosaccharides differing in N/O-sulfation and epimerization pattern. The enzymatic transformations defined fine substrate requirements of NST, C5-Epi, 2-OST, and 6-OST.

controlled_chemoenzymatic_synthesis_of_heparan_sulfate_oligosaccharides

<p>Heparan sulfates (HSs) are highly sulfated linear polysaccharides that occur on the cell surface and in the extracellular matrix of all animal cells.[1] The interaction between HSs and proteins is critical for many biological processes, and it has been suggested that by regulating the expression of the HS biosynthetic enzymes, cells can create unique epitopes for the recruitment of specific HS-binding proteins.[2]</p><p>The biosynthesis of HS occurs in the Golgi where a protein-linked heparosan polymer is assembled and composed of alternating N-acetylglucosamine (GlcNAc) and glucuronic acid (GlcA) moieties.[3] Discrete regions of the polymer are then modified by N-deacetylase/N-sulfotransferases (HSNdAc/NST), C5 epimerases (C5-Epi), uronosyl 2-O-sulfotransferase (2-OST), glucosaminyl 6-O-sulfotransferases (6-OST), and 3-O-sulfotransferases (3-OST). These modifications are often incomplete, thus creating considerable structural diversity.</p><p>Although it is widely accepted that HS is an information-rich polymer, ligand requirements for only a few proteins have been determined.[4] This deficiency can be addressed by preparing large collections of well-defined HS oligosaccharides for structure–activity relationship studies. Several laboratories have reported elegant chemical syntheses of HS oligosaccharides,[5] however these approaches are mainly focused on one-compound at the time of preparation. To prepare more diverse libraries of HS oligosaccharides, we have introduced a modular synthetic approach whereby a set of disaccharides, which resemble the differently sulfated disaccharides found in HSs, are used for oligosaccharide synthesis.[6] The end game, which involves the selective introduction of O- and N-sulfates, requires a relatively large number of steps, thus hampering the preparation of large collections of compounds. Enzyme-mediated synthesis of HS oligosaccharides requires significantly fewer steps, but because of the promiscuity of the biosynthetic enzymes, it is difficult to control the site of modification.[7] This deficiency is being addressed by genetically engineering sulfotransferases with alternate substrate specificities,[8] but despite these new enzymes, mainly highly sulfated compounds with repetitive IdoA-GlcN moieties are obtained.</p><p>Herein we describe a chemoenzymatic approach that addresses deficiencies associated with chemical and enzymatic HS synthesis. The premise of the approach is the chemical synthesis of heparosan oligosaccharides in which a given site is modified by a chemical entity that blocks specific enzymatic transformations. In a proof of principle study, we found that methylation of the C6 hydroxy group of a glucosamine residue of a heparosan oligosaccharide (e.g. compounds 2 and 3 in Figure 1) is tolerated by many HS biosynthetic enzymes. Obviously, the methyl ether prevented sulfation of the methylated hydroxy group. Surprisingly, it also blocked epimerization of the GlcA moiety at the +1 site, whereas GlcA at the −1 site could be readily transformed into IdoA. Furthermore, the compounds 1–3 could be chemically desulfated, and resulfation with NSTresulted in di-N-sulfated compounds in which the reducing GlcN moiety is unmodified. It was also found that a GlcNS moiety at the reducing end of a hexasaccharide cannot be modified by 6-OST. These observations offer unique possibilities to control the sites of sulfation and epimerization.</p><p>The targeted hexasaccharides 1–3 were assembled from the disaccharide building blocks 4, 5, and 6 (Scheme 1).[6] The disaccharide 5 is modified by a levulinoyl ester (Lev) at C6 of GlcN, which at a late stage of the synthesis can be removed for introduction of a methyl ether. We opted for this strategy because previous studies had shown that an ester at C6 of GlcN, in compounds such as in 5 and 6, is critical for high α-anomeric selectivity.[6a] Triflic acid catalyzed glycosylations of the glycosyl donors 5 and 6 with the acceptor 4 gave the tetrasaccharides 7 and 8, respectively, which were treated with triethylamine to remove the Fmoc-protecting group. The resulting glycosyl acceptors 9 and 10 were coupled with 6 and 5, respectively, to afford the corresponding hexasaccharides 11 and 12. The Lev ester of 11 and 12 was removed by treatment with hydrazine acetate and the corresponding alcohols 13 and 14 were methylated with TMSiCHN2/HBF4 to afford 15 and 16, respectively. The latter compounds were treated with LiOH/H2O2 to remove the base-sensitive protecting groups, with subsequent reduction of the azido functions using PMe3 in THF/H2O to give 18 and 19. In addition, 13 was subjected to base treatment and reduction with PMe3 to provide the unmethylated hexasaccharide 17. The amines of 17, 18, and 19 were sulfated with pyridinium sulfate, which was followed by catalytic hydrogenation over Pd(OH)2/C to give the hexasaccharides 20, 21, and 22, respectively. The anomeric aminopentyl moieties of 20–22 were protected as a carboxybenzyl (Cbz) moieties by reaction with benzyl chloroformate to give the corresponding fully N-sulfated target compounds 1–3.</p><p>Next, attention was focused on the chemoenzymatic diversification of 1–3. The compounds 23–25, having GlcNH2 moieties, were prepared by hydrolysis of the sulfonamides of 1–3 by conversion of the N-sulfates into pyridinium salts followed by solvolysis in a mixture of DMSO and H2O (Scheme 2).[9] Treatment of these compounds with NST in the presence of 3′-phosphoadenosine-5′-phosphosulfate (PAPS) gave the di-N-sulfated derivatives 26–28. Mass spectrometry and two-dimensional NMR experiments by COSY and HSQC confirmed the identity of the compounds, and specifically, C2 of the reducing-end GlcN (A-residue, Figure 2) was substantially shifted up-field compared to that of GlcNS residues of the C and E residues. These results highlight that NST is not affected by methylation of a C6 hydroxy group in GlcN, and furthermore a GlcA residue at the +1 site of GlcN is critical for N-sulfation. The compounds 26–28 were chemically N-acetylated with N-acetyl succinimide (AcOSu) in PBS buffer to give 29–31, respectively, after purification by BioRad P4 size exclusion column chromatography.</p><p>C5-Epi is an enzyme that establishes an equilibrium between GlcA and IdoA for residues within sequences containing GlcNS-GlcA-GlcNS/Ac.[10] By performing the epimerization in the presence of 2-OST, this transformation can be driven to completion because an IdoA2S residue is not recognized by the epimerase, and thus the reverse reaction will not take place.[7b] Thus, tri-N-sulfated compounds 1 and 2, which have two GlcA moieties flanked by GlcNS, were treated with PAPS and C5-Epi/2-OST to give compounds 32 and 33, respectively in which only one GlcA had been epimerized (Scheme 3). H1 and HSQC spectra of 33 demonstrated a significant chemical shift change of H1E (δ=5.48 to 5.18 ppm), thus indicating the epimerization had occurred at the central GlcA moiety (Figure 3). Furthermore, 2DNOESY experiments showed crosstalk between H1E and H4D/H3D, thereby unambiguously confirming the structure of the product. These results highlight that GlcA at the +2 position is critical for enzyme recognition. Interestingly, the presence of a 6-O-methyl ether of GlcN at the +1 site of GlcA, as in 2, does not interfere with the epimerization and 2-O-sulfation. In contrast, methylation of GlcN at the −1 site, as in 3, resulted in a complete block of epimerization and 2-O-sulfation. As expected, treatment of 29 and 30 with PAPS and C5-Epi/2-OST gave 34 and 35, respectively, while 31 remained unmodified because of the presence of a 6-OMe at the −1 site of GlcA (Scheme 3).</p><p>Next, sulfation of the oligosaccharides by 6-OST, which are enzymes that require N-sulfation of the targeted GlcN moiety, was explored.[11] Treatment of 1 with a mixture of 6- OST1/6-OST3 in the presence of PAPS gave the di-6-O-sulfated derivative 36 (Scheme 4). Interestingly, the GlcNS at the reducing end had remained unmodified as confirmed by diagnostic fragments such as B4, Y2, B5, Y1, C4, C5, and 2,4A6 in the MS2 spectrum and a 2D TOCSY NMR experiments (see Figures S6 and S7). These results indicate that these enzymes require, in addition to GlcNS, a GlcA residue at the +1 site. This observation is in agreement with a recently reported cocrystal structure of 6-OST3 with an octosaccharide and it showed that the majority of the interactions were made with the acceptor GlcNS and the adjacent disaccharide GlcA-GlcNS on the reducing side.[12] Next, 3, 31, 2, and 33 were subjected to 6-OSTand PAPS, and as expected the derivatives 37–40, having a single 6-O-sulfate at different positions, were readily obtained.</p><p>In summary, enzymatic modifications of three chemically synthesized hexasaccharides, with or without a 6-O-methyl ether on a GlcN residue, with NST, C5-Epi/2-OST, and 6-OST1/6-OST3 provided a library of 21 hexasaccharides that differ in N/O-sulfation and epimerization pattern. It is known that consecutive GlcA moieties that are flanked by GlcNS residues are randomly modified by C5-Epi and even in the presence of 2-OST, only a mixture of partially epimerized products is obtained.[13] Therefore, current enzymatic approaches can only provide HS sequences with repetitive IdoA moieties,[7c] and therapeutically valuable targets with discrete epimerization patterns are out of reach.[13] Our discovery that a C6 masking group at GlcN allows epimerization of only one of the neighboring GlcA residues, is laying the foundation for development of next-generation chemoenzymatic synthesis of HS oligosaccharides with control over epimerization and sulfation. In this respect, it is known that the enzyme PmHS2 can employ UDP-GlcNAc derivatives in which C6 of GlcNAc is modified and thus can provide heparanose oligosaccharides with modified GlcN residues.[14] In this study, a methyl ether was chosen to mask C6 of a GlcN residue. Future studies will focus on the identification of chemical modifications which can be removed to achieve even greater synthetic flexibility.</p>

PubMed Author Manuscript

A Chemical Approach for the Detection of Protein Sulfinylation

Protein sulfinic acids are formed by the reaction of reactive oxygen species with protein thiols. Sulfinic acid formation has long been considered an irreversible state of oxidation and is associated with high cellular oxidative stress. Increasing evidence, however, indicates that cysteine is oxidized to sulfinic acid in cells to a greater extent, and is more controlled, than first thought. The discovery of sulfiredoxin has demonstrated that cysteine sulfinic acid can be reversed, pointing to a vast array of potential implications for redox biology. Identification of the site of protein sulfinylation is crucial in clarifying the physiological and pathological effects of post-translational modifications. Currently, the only methods for detection of sulfinic acids involve mass spectroscopy and the use of specific antibodies. However, these methodologies are not suitable for proteomic studies. Herein, we report the first probe for detection of protein sulfinylation, NO-Bio, which combines a C-nitroso warhead for rapid labelling of sulfinic acid with a biotin handle. Based on this new tool, we developed a selective two-step approach. In the first, a sulfhydryl-reactive compound is introduced to selectively block free cysteine residues. Thereafter the sample is treated with NO-Bio to label sulfinic acids. This new technology represents a rapid, selective and general technology for sulfinic acid detection in biological samples. As proof of our concept, we also evaluated protein sulfinylation levels in various human lung tumour tissue lysates. Our preliminary results suggest that cancer tissues generally have higher levels of sulfinylation in comparison to matched normal tissues. A new ability to monitor protein sulfinylation directly should greatly expand the impact of sulfinic acid as a post-translational modification.

a_chemical_approach_for_the_detection_of_protein_sulfinylation

INTRODUCTION<!>RESULT AND DISCUSSION<!>Using SNL for labeling protein sulfinic acids<!>Selective block of free cysteine residues<!>Design and synthesis of NO-Bio<!>Development of a chemical approach for protein sulfinic acid detection<!>NO-Bio detects sulfinic acid-modified proteins in cell lysate<!>Protein sulfinylation levels in human lung cancer<!>CONCLUSIONS<!>METHODS<!>General reactivity of recombinant proteins toward NO-Ph (Figure 3)<!>General SDS-PAGE and Western blot procedures<!>NO-Bio labeling of DJ-1 (Figure 4)<!>Cell Culture<!>General procedure for lysate preparation<!>Labeling of protein SO2H in lysate using NO-Bio (Figure 5)

<p>Reactive oxidant species derived from oxygen or nitrogen (RNOS) were originally notorious for indiscriminately oxidizing various cellular components and for promoting aging and a broad range of pathologies. By contrast, research in the last two decades has shown that low levels of RNOS regulate basic cellular processes including growth, differentiation, and cell migration.1,2 Protein-thiols (SH) are the main target of RNOS-dependent signaling.3 The fine oxidation of specific cysteine (Cys) residues has emerged as a molecular switch for the modulation of protein function and is similar in effect to enzyme-assisted post-translational modifications (PTMs).4 In addition to the well-known disulfide, a variety of products may result from oxidation of thiols, but the most important are sulfenic acids (SOH), sulfinic acids (SO2H), and sulfonic acids (SO3H).5 The development of redox-probes for monitoring RSOH has unequivocally revealed that protein sulfenylation modulates protein activity directly or through the formation of disulfide bonds.6 Persistent lack of efficient tools for tracking SO2H, however, has confined this PTM to a minor role. Since common cellular reductants do not reduce Cys-SO2H, protein sulfinylation was long considered merely a marker of oxidative stress, though mounting evidence indicates that hyperoxidation to SO2H is a more controlled event than previously thought. In fact, increasing number of proteins have been shown to be regulated by selective sulfinylation, including matrilysin, nitrile hydratase, and the Parkinson's disease protein, DJ-1.7 The best characterized example of modulation of protein activity via sulfinylation, however, occurs in the Peroxiredoxin (Prx) family. Over-oxidation of the catalytic Cys leads to deactivation of peroxidase activity and the formation of high-molecular-weight aggregates, which exhibit molecular chaperone activity.8,9 Prx inactivation is then reversed by Sulfiredoxin (Srx), an ATP-dependent protein that specifically reduces Cys-SO2H in Prxs.10 Furthermore, it has been shown that transient sulfinylation of Prx represents a universal marker for circadian rhythms along all three domains of life.11 The discovery of Srx suggests a more fundamental role for Cys-SO2H, which may constitute an additional layer of redox regulation.12 Finally, in addition to cysteine oxidation by ROS, an enzyme-mediated oxidation has recently emerged. Several plant cysteine oxidases have been identified that can selectively oxidize the penultimate cysteine of transcription factors to SO2H and thereby control the life span of these proteins.13 Accordingly, sulfinylation of specific Cys residue has drawn wide attention as a novel PTM responsible for regulation of protein function. Studies of the role of Cys-SO2H, however, have been hampered by the technically challenging nature of selective assays for such oxoforms, and mass spectroscopy remains the main tool for monitoring this PTM.14 Although SO2H shows higher stability than SOH, mass analyses may introduce a high percentage of artifacts. Moreover, the fact that persulfide modification has the same nominal mass shift of 32 Da raises additional concerns. Antibodies able to detect hyperoxidized forms of specific proteins are known15 but, even without taking in account lack of specificity, are unsuited to global profiling studies.</p><!><p>We strongly believe that only the development of chemical probes capable of selectively trapping SO2H will allow a clear elucidation of the role of protein sulfinylation. In this connection, we recently developed chemoselective Sulfinic acid Nitroso Ligation (SNL).16 The addition of SO2H to C-nitroso compounds has been known for more than a century; however, the resulting adduct is base-labile (Figure S1). In order to trap this unstable species, we have incorporated an electrophilic center (Figure 1) in the ortho-position of a nitroso-benzene derivative (1). The transient oxyanion (2) reacts with the ester by intramolecular trans-esterification to form a stable benzisoxazolone (3). Basing our work upon this idea, we have synthesized a class of C-nitroso compounds that show fast reactivity with low molecular weight SO2H. These reagents do not react with other biologically relevant nucleophiles aside from thiols, which, however, do not form stable adducts (Figure S2).</p><!><p>Encouraged by these results, we employed SNL to develop chemical probes for detection of protein sulfinylation. First of all, we explored the ability of NO-Ph (Figure 2), the C-nitroso derivative that has shown the best reactivity, to modify SO2H within the double mutant (C64,82S) of the thiol peroxidase Gpx3 from yeast.17 In the presence of H2O2, Gpx3 forms an intramolecular disulfide bond through sulfenylation of catalytic C36, followed by condensation with the resolving C82. Mutation of C82 to serine stabilizes transient Cys36-SOH, allowing its further controlled oxidation to SO2H (see Supporting Information).</p><p>Incubation of NO-Ph with C64,82S Gpx3-SO2H (22772 Da) yields the expected sulfonamide adduct (22949 Da) as confirmed by ESI-LC/MS analysis (Figure 3A). Our preliminary experiments with small molecules have shown that thiols react with C-nitroso compounds to yield an unstable sulfenamide adduct, which is cleaved by reaction with a second thiol (Figure S2). In order to confirm these results with protein-SH, we treated fully reduced C64,82S Gpx3 (22740 Da) with NO-Ph, followed by incubation with DTT. Surprisingly, ESI-LC/MS revealed the formation of a stable adduct with a mass of 22935 Da (Figure 3B). Alkylation of the Cys residue with N-ethylmaleimide (NEM), conversely, prevented adduct formation (Figure 3C). Even considering that DTT was unable to cleave the sulfenamide formed by the addition of NO-Ph to Cys36, the detected mass increase (Δm = 195) does not correspond to the expected adduct (Δm = 177). Generally, addition of thiol to C-nitroso aryl compound yields an unstable semimercaptale, which can react with a second thiol molecule or undergo rearrangement to form a more stable sulfinamide (Figure S3).18 Spontaneous rearrangement occurs via dissociation of a hydroxyl anion and formation of a cationic nitrenium ion intermediate, which is later hydrolyzed by water. Following the same pathway, the addition of NO-Ph to C64,82S Gpx3-SH would form a sulfinamide adduct with a mass increase of 195 Da, which corresponds exactly to our observations. Acidic environment usually favors semimercaptale rearrangement. With NO-Ph, however, once the benzisoxazolone is generated, the rearrangement appears to occur even at neutral pH, probably because the carboxylate group is much more prone to dissociate from the nitrogen atom than is the hydroxyl anion (Figure S4). The formation of the rearranged sulfinamide would also explain why the adduct was not reduced by DTT. Since sulfinamide formation was never observed with low molecular weight thiols,16 we wondered why the rearrangement occurred with protein-SH. We speculated that, in such cases, the attack of a second thiol molecule on the transient sulfenamide is generally faster than the rearrangement of the latter. Once the sulfenamide is formed, however, the attack of a second Cys-SH would be precluded with the use of C64,82S Gpx3-SH because of steric hindrance. To verify this hypothesis, we tested the reactivity of NO-Ph toward C64S Gpx3, which has both redox-active and resolving cysteines. As expected, the incubation of C64S Gpx3 with NO-Ph exclusively promoted the formation of the internal disulfide (Figure 3D). The presence of the resolving Cys, which can easily interact with the sulfenamide adduct formed with catalytic Cys, prevents the rearrangement of the latter. This result suggests that the formation of stable sulfinamide and disulfide reflects competitive reaction pathways influenced by kinetic factors (Figure S5). As additional proof, we incubated 2-methyl 2-propanethiol with an excess of NO-Ph. In this case, we speculated that, because the interaction of two molecules of thiols would be more hampered for steric hindrance, sulfenamide rearrangement would be facilitated. As anticipated, LC-MS analyses showed formation of the expected sulfinamide adduct (Figure S6).</p><!><p>Sulfenamide rearrangement apparently limits the use of SNL for protein sulfinylation detection. As the experiment with NEM suggests, however, protection of the free cysteines can be employed to prevent formation of non-reducible adducts with hindered thiols. In fact, many chemical methods for detection of specific thiol modifications (e.g., S-nitrosylation) involve selective blocking of reduced thiols.19 The success of these assays relies on the selectivity of the thiol-blocking step and the reagent's efficiency in fully protecting free thiols without cross-reacting with SO2H. We evaluated the reactivity of several thiol-blocking reagents toward C64,82S Gpx3-SO2H. When we used a large excess of common alkylating agents such as NEM or iodoacetamide (IAM), ESI-LC/MS analyses detected small but significant amounts of alkylated sulfinic acid (Figures S7A and S7B). Although this result may seem unexpected, the reaction of low molecular weight SO2H with Michael acceptors and a-halo carbonyl compounds has been reported.20 Conversely, sulfhydryl reactive compounds that promote mixed-disulfide formations, such as 2,2′-dipyridyl disulfide (DPS) and S-methyl methanethiosulfonate (MMTS), showed no cross-reactivity toward SO2H (Figures S7C and S7D). Next, we examined whether protection of free Cys residues as disulfides was sufficient to prevent cross-reactivity with NO-Ph. After C64S,82S Gpx3-SH was pre-incubated with DPS (Figure S8A) or MMTS (Figure S8B), the excess of thiol-blocking reagents was removed and the protein was incubated with NO-Ph. ESI-LC/MS analyses confirmed that both DPS and MMTS efficiently blocked formation of the sulfinamide adduct with the reduced protein.</p><!><p>Having established that SNL can be efficiently employed for labeling protein SO2H, we designed and synthesized NO-Bio (Figure 2). The new chemical probe combines the C-nitroso warhead (blue) with a biotin handle (violet), which allows detection of protein sulfinylation in biological samples. The synthesis of NO-Bio (Figure S9), which is described in detail in the Supporting Information, involved the coupling of commercially available Biotin-PEG4-NHS with a diamino-linker, N-tert-Butoxycarbonyl-1,6-hexanediamine. The protected amino group was then cleaved by TFA treatment, and the generated primary amine was coupled with the N-succimidyl ester of NO-Ph to yield NO-Bio, which was purified by reverse-phase HPLC.</p><!><p>As Figure 4A shows, protein sulfinylation could be selectively detected by a two-step method. In the first, a sulfhydryl-reactive compound (DPS or MMTS) is introduced to selectively block free Cys residues. Thereafter the sample is treated with the biotin-tag probe, NO-Bio, to label sulfinic acids. We tested this approach using recombinant DJ-1 as model. The Parkinson's associate protein has a conserved Cys residue, C106, which is extremely sensitive to oxidative stress and tends to form a stable SO2H. Many studies demonstrate that DJ-1 protects cells against oxidative stress-mediated apoptosis through the formation of C106-SO2H.21,22 In addition, DJ-1 contains two other free Cys residues, C46 and C53, which are not redox-active. Though C53 is not modified by ROS, it is still very reactive toward electrophiles.23 Accordingly, DJ-1 represents an excellent model for testing the selectivity of our strategy. Reduced or oxidized WT DJ-1 was incubated with DPS, following by treatment with NO-Bio. As shown in Figure 4B, mass analysis clearly confirmed the selective modification of the solely oxidized DJ-1. Interestingly, DPS promoted the formation of an internal disulfide between C46 and C53. We speculated that DPS would first react with the highly solvent-exposed C53 to yield a mixed-disulfide. Later, the relatively more deeply buried C46 would attack the active disulfide with consequent generation of an internal disulfide bond (Figure S10). Selective protection of the free Cys can be achieved using MMTS as well (Figure S11). However, our results indicate that MMTS reacts with thiols at a relatively slower rate than does DPS. In fact, small amounts of Cys-SO2H were detected even in the reduced sample, which indicates that C106 was partially oxidized during thiol blocking. Although addition of EDTA in the buffer prevented this unwanted oxidation, we opted to use the more efficient DPS in all subsequent experiments. The biotin handle of the probe allows visualization of the labeled proteins by streptavidin blotting. Therefore, the selectivity of NO-Bio labeling was also confirmed by Western blot analysis. Reduced or oxidized DJ-1 was treated with DPS, followed by incubation with NO-Bio. The reactions were then subjected to SDS-PAGE and analyzed by streptavidin blotting. Treatment of oxidized DJ-1 with NO-Bio afforded selective protein labeling, while DJ-1 was not detected at all by streptavidin blotting in the absence of the oxidant, demonstrating the specificity of our chemoselective approach (Figure S12). NO-Bio showed also higher sensitivity in comparison to a commercially available antibody against hyperoxidized DJ-1 (Figure S13), allowing detection of sulfinylated DJ-1 at relative low concentrations.</p><p>DJ-1 possesses a highly conserved G18 residue, which facilitates the ionization of C106, reduces its pKa, and helps stabilize C106-SO2H. Small changes in this position can drastically influence the oxidative properties of C106.24 For example, the E18D DJ-1 mutant has a lower propensity to form SO2H, but the structurally similar E18N mutant shows an increased oxidation propensity thanks to a strong stabilization of C106-SO2H. We evaluated the sensitivity of NO-Bio, probing the different oxidation propensities of various DJ-1 mutants including C106S DJ-1, which does not contain a redox-active cysteine. Each DJ-1 variant (WT, E18N, E18D and C106S) was exposed to H2O2, treated with DPS, and finally incubated with NO-Bio. Western blot analysis was consistent with expected results (Figure 4c). E18N DJ-1 showed a higher fraction of sulfinylation, even in the absence of H2O2. The sulfinylation level of the E18D mutant exposed to oxidative stress, in contrast, was almost negligible. It is worth noting that C106S DJ-1 treated with H2O2 was not detected by streptavidin blotting, confirming that only C106 is able to form SO2H under relatively mild oxidation conditions.</p><!><p>Having established the specificity and sensitivity of our two-step approach in homogenous protein solutions, we next investigated whether NO-Bio could detect protein-SO2H in a complex, unfractionated cell lysate. To this end, we tested our optimized chemistry in a whole human cervical cancer (HeLa) cell extract, which was obtained by lysing the cells in modified RIPA buffer containing Catalase and DTT. The reducing lysis buffer prevents further oxidation of SO2H and maintains free Cys in the reduced form, avoiding overestimation of protein sulfinylation. Figure 5A shows an HRP-streptavidin western blot, which indicates that robust levels of protein SO2H can be detected under normal conditions. To demonstrate that DPS efficiently trapped all free thiols and therefore that the streptavidin blot revealed only sulfinylated proteins, we employed iodoacetyl-PEG2-biotin (IAM-Bio). The biotinylated reagent is able to alkylate thiols such as free Cys residues. Pre-treatment of the sample with DPS completely abrogated the IAM-Bio-dependent signal (Figure 5A, Lane 3), which indirectly proved that NO-Bio reacted only with protein SO2H. Next, we determined whether our chemical approach could detect increases in protein sulfinylation in human cell culture. HeLa cells were incubated with increasing amounts of H2O2 for 15 minutes (this time point was chosen after a preliminary time-dependent experiment – Figure S14A), lysed, and then labeled as described above. Western blot analysis showed that the level of protein sulfinylation was increased by H2O2 in a dose-dependent fashion (Figure S14B). Taken together, these results confirm that SO2H is stable enough to be successfully detected in cell lysates and does not require in vivo labeling.</p><!><p>In order to show that our method can be applied to more complex biological questions, we performed comparative sulfinic acid profiling in human lung tumor tissue. For these experiments, protein sulfinylation was characterized by western blot analysis of whole-cell lysates. Our results showed a highly variable presence of SO2H among the three patient tumor tissue samples (Figure 5B). All three tumor tissue (papillary adenocarcinoma, adenocarcinoma, and adeno-squamous cell carcinoma) exhibited significant increase in the extent of SO2H modifications vs. matched normal tissue. Although the number of samples was too small to draw broad conclusions, these initial observations suggest that elevated levels of SO2H could be used as a cancer marker.</p><!><p>In summary, sulfinic acid nitroso ligation allows quick conversion of sulfinic acids into sulfonamide adducts. We designed and synthesized a nitroso-based probe, NO-Bio, which can be used to label protein-SO2H. In model protein sulfinic acids, this compound yields stable products. We developed a two-step chemical approach that selectively labels protein sulfinic acid in vitro without cross-reactivity with thiols. Furthermore, NO-Bio was able to detect global increases in protein SO2H modification under oxidizing cellular conditions or in cancer cell lines. To the best of our knowledge, this is the first chemical approach that allows selective protein-sulfinylation detection, making NO-Bio a valuable new tool for monitoring changes in cysteine oxidation and should find a wide variety of applications for the study of biological processes. In addition, the biotin tag provides an opportunity for the enrichment and proteomic analysis of oxidized proteins. These studies are currently underway and will be reported in due course.</p><!><p>For protein expression and purification, generation of protein sulfinic acids, synthesis of NO-Bio, screening of the thiol-blocking reagent and mass spectrometry, please see the Supporting Information. Human cancer lung tissue lysates were purchased from Protein Biotechnologies Inc.</p><!><p>25 μM of recombinant protein (C64,82S Gpx3-SO2H, C64,82S Gpx3-SH, C64,82S Gpx3-SNEM or C64S Gpx3) was incubated at room temperature in the presence of NO-Ph (500 μM) in 100 mM PBS pH 7.4. After 1 hour, the reaction was quenched by passage through one Micro Bio-Spin P-30 column pre-equilibrated with ammonium bicarbonate (50 mM, pH 8.0) for analysis by ESI-LC/MS.</p><!><p>Protein samples were resolved by SDS-PAGE using Mini-Protean TGX 4–15% Tris-Glycine gels (BioRad) and transferred to a polyvinylidene difluoride (PVDF) membrane (BioRad). After transfer, the PVDF membrane was blocked with 5% BSA in TBST for 1 hour at room temperature. The membrane was washed with TBST (3X) and immunoblotting was performed with the following primary and secondary antibodies at the indicated dilutions: HRP-streptavidin (GE Healthcare, 1:80000), Actin (Santa Cruz Biotechnology, 1:1000), PARK7/DJ-1 (Abcam, 1:1000), oxidized PARK7/DJ-1 (Abcam, 1:1000), rabbit anti-goat IgG-HRP (Invitrogen, 1:2000 – 1:50000), and rabbit anti-mouse IgG-HRP (Invitrogen, 1:20000 – 1:50000). The PVDF membranes were washed with TBST (3X) and developed with ECL Plus chemiluminescence (Pierce) and imaged by film.</p><!><p>Each DJ-1 form (WT, E18N, E18D and C106S) was buffer exchanged using Micro Bio-Spin P-30 column pre-equilibrated with 50 mM HEPES, 100 mM KCl pH 7.4. Three samples of each DJ-1 form (25 μM) were then treated with 5 equivalents of H2O2 or H2O (control samples) in ice for 30 minutes. The reactions were quenched adding 2 mM of DTT. Each sample was incubated for 30 min at room temperature and then passed through one Micro Bio-Spin P-30 column. 40 equivalents of DPS were then added at room temperature. After 1 hour, each sample was quenched by passage through one Micro Bio-Spin P-30 column pre-equilibrated with 100 mM PBS pH 7.4. 10 equivalents of NO-Bio or DMSO (control samples) were then added. After 1 hour, the reactions were quenched adding non-reducing 2x Laemmli buffer. The resulting samples were subjected to SDS-PAGE and Western blot analyses as described above. Equal protein loading was verified by α-DJ1 antibody (Abcam).</p><!><p>HeLa cell line was obtained from American Type Culture Collection. HeLa cell line were grown in DMEM media supplemented 10% FBS (Invitrogen), 1% penicillin-streptomycin (Invitrogen), 1% of glutagro (Corning) and 1× non-essential amino acids (Invitrogen) at 37 °C in a humidified atmosphere of 5% CO2. For H2O2 stimulation, HeLa cells were plated in a 6-well plate. Once the cells reached 90% of confluence, they were washed with PBS. Cell were exposed to increasing concentration of H2O2 (0.2, 1, 2 mM) for 15 min, and then washed with PBS (3×).</p><!><p>Cell were harvested in modified RIPA buffer [50 mM triethanolamine, pH 7.4, 150 NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 5 mM DTT, 200 U/mL catalase (sigma) and 1x EDTA-free complete mini protease inhibitors (Roche)]. After 20 min incubation on ice with frequent mixing, unlysed cell fragments were removed by centrifugation at 14,000 g at 4 C for 20 min. Protein concentration was measured by BCA assay.</p><!><p>Cell lysate (1 mg/mL) was buffer exchanged using Micro Bio-Spin P-6 column pre-equilibrated with 100 mM PBS, 100 mM NaCl pH 7.4. Free thiols were trapped by incubation with 2 mM of DPS (in DMSO). Final reaction volumes were 0.1 mL, and the samples were incubated for 30 min at r.t. Reactions were quenched by passage through one Micro Bio-Spin P-30 column pre-equilibrated with 100 mM PBS pH 7.4. Protein sulfinylation was assayed adding 250 μM of NO-Ph. After 30 min, reactions were quenched adding non-reducing 2x Laemmli buffer. Three additional controls included untreated lysate, lysate incubated with 250 μM of iodoacetyl-LC-biotin (Thermo) and lysate treated with DPS following by incubation with 250 μM of iodoacetyl-LC-biotin. The samples treated with iodoacetyl-LC-biotin were incubated for 30 min at r.t in the dark. Reactions were quenched via the addition of non-reducing 2x Laemmli buffer. The resulting samples were subjected to SDS-PAGE and Western blot analyses as described above. Equal protein loading was verified by α-actin antibody.</p>

PubMed Author Manuscript

Correlation between optical activity and the helical molecular orbitals of allene and cumulenes

Helical frontier molecular orbitals (MOs) appear in disubstituted allenes and even-n cumulenes. Chiral molecules are optically active, but while these molecules are single-handed chiral, π-orbitals of both helicities are present. Here we computationally examine whether the optical activity of chiral cumulenes is controlled by the axial chirality or the helicity of the electronic structure. We exploit hyperconjugation with alkyl, silaalkyl, and germaalkyl substituents to adjust the MO helicity without altering the axial chirality. For the same axial chirality, we observe an inversion of the helical MOs contribution to the electronic transitions and a change of sign in the electronic circular dichroism and optical rotation dispersion spectra. While the magnitude of the chiroptical response also increases, it is similar to that of chiral cumulenes without helical π-orbitals. Overall, Helical π-orbitals correlate with the big chiroptical response in cumulenes, but are not a prerequisite for it.

correlation_between_optical_activity_and_the_helical_molecular_orbitals_of_allene_and_cumulenes

<!>Supporting Information

<p>Molecules that exhibit structural chirality are optically active. Recently, Hendon et al. 1 found that linear p-conjugated molecules with perpendicular end-groups such as allene and certain functionalised polyynes can have helical molecular orbitals (MOs) when their symmetry is reduced and the molecules become chiral. [1][2][3][4][5][6][7] For instance, in [4]cumulene, 4 being the number of double bonds, the frontier p-MOs come in degenerate pairs and look like those of butadiene as shown in Figure 1. A 1,5-disubstitution of [4]cumulene reduces the symmetry from D2d to C2. The C2 symmetry causes the rectilinear p-MOs (px and py) to mix, thus breaking the degeneracy and forming pairs of helical p-MOs. 6 The frontier MOs are near-degenerate pairs of helical MOs; one of each helicity (pP and pM of P-and M-helicity). Consequently, while the molecule is one of two enantiomers, here the R enantiomer, the electronic structure contains both helicities simultaneously. Given 1,5-disubstituted [4]cumulenes are chiral, they will be optically active, and the R-S enantiomers will have opposite chiroptical response. As can be inferred by parity, the helicities of the MOs will also be opposite between the S and R enantiomers. The relation between helical electronic structure and chiroptical response has been contemplated. Caricato analyzed MO contributions to the rotary strength of S-1,3-dimethylallene (2,3-pentadiene, structurally analogous to dimethyl[4]cumulene in Figure 1), and demonstrated that the major contributions to the specific rotation originate from electronic transitions between a few helical p-orbitals. 8 Substituted allenic systems have been found to have big chiroptical responses. [9][10][11][12][13][14][15][16] More recently, Ozcelik et al. synthesized and measured the chiroptical response of conformationally locked cyclic butadiyne systems with distinct helical electronic structure. 17 They found the chiroptical response to be among the largest measured, but could not attribute this specifically to the appearance of helical MOs in their molecules. 17 Considering the previous theoretical and experimental results that have been presented, 8,17 we find it imperative to examine the relation between the chiroptical response and helical MOs. In this letter, we exploit computationally (see Supporting Information part A for details) [18][19][20] the hyperconjugation of substituents into the helical p-system, as a strategy to provide systematic control of helicity and energetic splitting of the otherwise near-degenerate MOs of 1,5-disubstituted [4]cumulenes. These molecules enable the separation of orbital helicity from the axial chirality of the molecule (S or R), and we probe how changes of helicity in the electronic structure affects the chiroptical properties with time-dependent DFT (see Supporting Information part A for details). [20][21][22][23] We examine the optical transitions, and compute conformation-resolved electronic circular dichroism (ECD) and optical rotation dispersion (ORD) spectra of disubstituted [4]cumulenes. We focus on [4]cumulenes, but the findings apply to allenes as even-n cumulenes belong to the allene family. 1,24 The test systems are selected with the aim of conceptual understanding, although we note that syntheses of allenes with permethylated silicon substituents and [4]cumulenes have been reported. [25][26][27][28][29][30][31][32] Hyperconjugation and Helical p-systems. Methyl substituents break the symmetry and the frontier MOs are explicitly non-degenerate, however, the HOMO and HOMO-1 are split by less than 1 meV. 6 This near-degeneracy of each MO pair (HOMO-1 and HOMO, LUMO and LUMO+1, etc.) seem to be retained with larger methyl-like substituents such as trimethylsilyl (Figure S1). To break the near-degeneracy, strategies utilizing donation from lone-pairs into the helical psystem have been explored. 33,34 Inspired by these approaches, we explore a strategy that let us control the splitting of the helical MO pairs and control the MO helicity. In an ethyl-substituted</p><p>[4]cumulene (3,4-heptadiene) there is conformational freedom around the C-C-C-C dihedral angle, as illustrated in Figure 2a. We examine the conformations of Rdiethyl[4]cumulene and find local minima to have C-C-C-C dihedral angles at approximately 0° and ±120°. At ±120° the ethyl groups form separate points of chirality, as mirroring a positive dihedral angle gives the negative. We focus on the diastereomers with both dihedral angles in +120° or -120° configuration, and label them (-)R(-) and (+)R(+), indicating these have R axial chirality but opposite ethyl configuration.</p><p>Shown in Figure 2b, the helicity of the frontier MOs of diethyl[4]cumulene changes with the ethyl configuration. The order of the helical MO changes, i.e., the helicities of the MOs of (-)R(-) is opposite to those of (+)R(+). While the HOMO of (-)R(-) is a P-helix (pP), that of (+)R(+) is an M-helix (pM). As if the electronic structure is being mirrored; however, (-)R(-) and (+)R(+) are not enantiomers but diastereomers because, for example, mirroring (-)R(-) gives (+)S(+). With ethyl substituents the HOMO and HOMO-1 are split by a slightly bigger 0.012 eV, while the splitting in the (+)R(-) conformations is similar to that of the methyl-substituted [4]cumulene (see Supporting Information part B). The saturated ethyl substituent interacts with the helical p-system through hyperconjugation. We estimate the magnitude of this electronic interaction (hyperconjugation energy) with a bond separation equation as described in Supporting Information part C. 35,36 The hyperconjugation of silicon and germanium into a carbon psystem is stronger because their s-system energetically match better with the p-system. [37][38][39] This is evident by changing ethyl to silaethyl, providing more than twice the hyperconjugation energy at 0.40 eV (9.3 kcal/mol) compared to ethyl at 0.19 eV (4.4 kcal/mol). As illustrated in Figure 3a, The conformations of the bis-silaethyl system are the same as with ethyl substituents and provide the same control of the MO helicity. However, with silaethyl substituents the splitting between the HOMO-1 and HOMO is more substantial at 0.11 eV. We assess a range of such silicon and germanium-based ethyl-like substituents in Figure 3b, as well methyl-like substituents for control, and plot their MO splitting as a function of the hyperconjugation energy. There is a near-linear correlation for ethyl-like substituents, and permethylated disilyl provides an MO splitting of just over 0.3 eV (7 kcal/mol). The MO splitting is systematically changed with ethyl-like substituents, and the helicities are controlled by their conformation.</p><p>Optical Activity. The electronic transitions of [4]cumulene are analogous to those of allene; for a thorough discussion of the spectral assignment of substituted allenes, we refer to the extensive experimental and theoretical work by Rauk et al., 40 Runge and co-workers, 41, 42 and earlier work. 43,44 In [4]cumulene, only the p®p* transition of B2 symmetry is electric dipole allowed, corresponding to a transition dipole along the cumulenic axis. In agreement with prior work, 41 all four p®p* transitions are superpositions (configuration interaction) of px®px* and py®py* and only S0®S4 is electric dipole allowed based on symmetry considerations (see Supporting Information part D). When the symmetry of the molecule is reduced to C2 by substituents, the first three transitions are not symmetry forbidden, however, their oscillator strengths remain negligible in all cases studied here. The S0®S4 transition of [4]cumulene and its dimethyl, diethyl, and bis-silaethyl substituted counterparts are listed in Table 1. The transitions are almost unchanged by the substituents, having similar oscillator strengths and excitation energies. However, we note one important difference between the (-)R(-) and (+)R(+) diastereomers of bissilaethyl[4]cumulene (bottom of Table 1). As the helical MO pairs (pM and pP, pM* and pP*) split energetically, the weighting in the configuration interaction changes. In (-)R(-) the pM®pM* excitation has the bigger coefficient. This is reversed in (+)R(+) where pP®pP* has the bigger coefficient. The helicities that contribute to S0®S4 transition are thus systematically controlled by the change of conformation.</p><p>We simulate the conformation-resolved UV-Vis and ECD spectra for [4]cumulene, and its dimethyl, diethyl, and bissilaethyl counterparts. The UV-Vis spectra of these these molecules are shown in Figure 4a and 4b for reference, and are similar with a dominant p®p* transition between 200 and 250 nm (cf. Table 1). In Figure 4c, the ECD spectra of R-1,5dimethyl[4]cumulene and the (+)R(-) conformation of 1,5diethyl[4]cumulene are shown. The dominant p®p* transition has negative De. The ECD spectra of the (+)R(-) conformation of the diethyl-substituted system is very similar to that of the dimethyl-substituted system. This indicates that the effect of the ethyl substituents cancels out when they are in the mixed (+)R(-) configuration. 5a, along with that of (1S,4S)-norbonenone for reference as a small molecule with high specific rotation. 45 In agreement with work on 1,3-dimethylallene by Crawford and co-workers, and others, 46-49 these R-enantiomers of [4]cumulene have relatively small negative specific rotation. For the ORD spectra of the (-)R(-) and (+)R(+) conformations of the diethyl and bis-silaethyl substituted [4]cumulenes shown in Figure 5b, two things are notably different. The magnitude of the ORD response is stronger compared to the dimethylsubstituted case, and the sign of the specific rotation is opposite for the (-)R(-) and (+)R(+) conformations. The same effect is present in 1,3-diethyl-substituted allene (Figure S10). As in the ECD spectra, the sign of the chiroptical response correlates with the substituent configuration and the associated change of MO helicity.</p><p>The magnitudes of the specific rotation in (-)R(-) and (+)R(+) conformations of diethyl[4]cumulene reach a similar magnitude to that of norbornenone, and for the bis-silaethyl substituted cases the magnitude is almost twice that of norbonenone. This suggests that with increasing splitting of the helical frontier MOs (HOMO-1 and HOMO), the specific rotation also increases. In Figure 5c and 5d, we plot the specific rotation at 436 nm and 589 nm against the splitting of the HOMO and HOMO-1 for the substituents listed in Figure 3b. While there is some correlation, for systems with increasing MO splitting and big specific rotation the data is more spread. The big specific rotation of norbonenone has been attributed to the electronic coupling between its two separate pchromophores. 8,23,47,[50][51][52] The effect of splitting the degenerate px and py systems into helical MOs is possibly somewhat analogous. While the data suggests a connection between the optical rotation and the helical frontier MOs (and their splitting), it also indicates that it is not the only important factor. This is especially the case for the larger substituents that provide some of the biggest MO splittings in Figure 5c and 5d. Similarly, the correlation with the magnitude and sign of the ECD response also becomes less clear (see Supporting Information part E). With increasingly large substituents, the MOs, and thus the electronic transitions, will have less pcharacter as the conjugated part of the molecule is relatively small. Furthermore, strong hyperconjugation and dispersion interaction between the bulky saturated substituents distort the structures from the idealized ones illustrated in Figure 2a. We compute the vertical change of electron density, Dr, for the studied [4]cumulenes in Supporting Information part D. Chiral features appear in Dr of the S0®S4 transition of (-)R(-) and (+)R(+). However, helicity is not apparent in Dr, possibly because the transitions are superpositions of excitations occurring from one helical MO to another, cf. Table 1. Although helicity does not manifest itself in the density, the chiral features of Dr are opposite for of (-)R(-) and (+)R(+).</p><p>The data presented here, and in other recent work, 8,17 suggest that a large chiroptical response may be intrinsically connected to helical p-orbitals. [3]cumulenes are structurally similar to [4]cumulenes, but have co-planar end-groups and therefore cannot achieve helical MOs in the ground-state. 6,32 In Figure 6, we examine the ORD spectra of disubstituted [3]cumulenes and compare them with those of the equivalently disubstituted [4]cumulenes. Instead of axial chirality there is E-Z isomerism in the [3]cumulenes, and consequently only the substituents provide chirality. An overview of the structure, frontier MOs, and electronic transitions are included in Supporting Information part F. Shown in Figure 6, the specific rotation of the (-)Z(-) and (-)E(-) conformations of the diethyl and bissilaethyl substituted [3]cumulenes has the same sign as (-)R(-) of the [4]cumulenes. With ethyl substituents, (-)Z(-) matches the magnitude of (-)R(-), and with silaethyl substituents the [3]cumulene-response is slighter stronger than that of the [4]cumulene. Clearly, both chiral cumulenes with and without helical MOs can achieve high specific rotation. In summary, we have developed a hyperconjugation-based strategy that enables systematic increase of the splitting of the helical HOMO and HOMO-1 in allene and even-n cumulenes. The frontier p-MOs change helicity depending on the substituent conformation, and the helicity is thus chemically controlled without changing the axial chirality of the molecule. The sign and magnitude of De in conformation-resolved ECD spectra, and the specific rotation in ORD spectra, change with the helicity of the frontier MOs of [4]cumulenes. There is an apparent correlation between p-orbital helicity with sign and magnitude of the chiroptical response. However, the response is of similar magnitude in chiral cumulenes without helical MOs, and we conclude that helical MOs are not a prerequisite for big chiroptical response in substituted allenes and cumulenes.</p><!><p>Computational Details. Frontier MOs of [4]cumulenes and Allene. Hyperconjugation Energy. Electronic Transitions. Spectra. Electronic structure and transitions of [3]cumulene.</p><p>The Supporting Information is available free of charge on the ACS Publications website.</p>

ChemRxiv

Scope and Limitations of 3‐Iodo‐Kdo Fluoride‐Based Glycosylation Chemistry using N‐Acetyl Glucosamine Acceptors†

AbstractThe ketosidic linkage of 3‐deoxy‐d‐manno‐octulosonic acid (Kdo) to lipid A constitutes a general structural feature of the bacterial lipopolysaccharide core. Glycosylation reactions of Kdo donors, however, are challenging due to the absence of a directing group at C‐3 and elimination reactions resulting in low yields and anomeric selectivities of the glycosides. While 3‐iodo‐Kdo fluoride donors showed excellent glycosyl donor properties for the assembly of Kdo oligomers, glycosylation of N‐acetyl‐glucosamine derivatives was not straightforward. Specifically, oxazoline formation of a β‐anomeric methyl glycoside, as well as iodonium ion transfer to an allylic aglycon was found. In addition, dehalogenation of the directing group by hydrogen atom transfer proved to be incompatible with free hydroxyl groups next to benzyl groups. In contrast, glycosylation of a suitably protected methyl 2‐acetamido‐2‐deoxy‐α‐d‐glucopyranoside derivative and subsequent deiodination proceeded in excellent yields and α‐specificity, and allowed for subsequent 4‐O‐phosphorylation. This way, the disaccharides α‐Kdo‐(2→6)‐α‐GlcNAcOMe and α‐Kdo‐(2→6)‐α‐GlcNAcOMe‐4‐phosphate were obtained in good overall yields.

scope_and_limitations_of_3‐iodo‐kdo_fluoride‐based_glycosylation_chemistry_using_n‐acetyl_glucosamin

<!>Introduction<!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!>Conclusions<!>Chemistry<!>

<p>B. Pokorny, P. Kosma, ChemistryOpen 2015, 4, 722.</p><p>This article is part of the Virtual Special Issue "Carbohydrates in the 21st Century: Synthesis and Applications".</p><!><p>The eight‐carbon sugar 3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid (Kdo) is a biomedically important constituent of bacterial polysaccharides occurring in capsular polysaccharides (CPS), O‐antigens, and the core region of lipopolysaccharides (LPS). Whereas Kdo has been found in both anomeric configurations in CPS, the LPS of many Gram‐negative bacteria harbors a structurally conserved α‐(2→4)‐linked Kdo‐disaccharide which connects the endotoxically active lipid A part to the core region and the O‐antigenic polymer.1 Lipid A is composed of a bisphosphorylated β‐(1→6)‐linked N‐/O‐acylated glucosamine disaccharide which plays a decisive role in the immune response of host cells when infected by Gram‐negative bacteria.2 Kdo is present in an acid‐labile α‐ketosidic linkage to position 6 of the distal glucosamine unit. Whereas the acylated, bisphosphorylated diglucosamine unit provides the main interactions in the complex with Toll‐like receptor 4/myelodifferentiation factor MD‐2, the Kdo and adjacent heptose and outer core sugars provide additional binding epitopes for receptor interactions and recognition by antibodies and lectins.3</p><p>Thus, the chemical synthesis of relevant Kdo‐lipid A fragments constitutes an important target and has successfully been pursued by several groups.4 Coupling of Kdo donors, however, is far from trivial, and an elaborate optimization of protecting and anomeric leaving groups for each glycosylation step is often crucial for good results.5, 6 Inherent challenges in glycosidation reactions of 3‐deoxy‐2‐ulosonic acid glycosyl donors pertain to the absence of a stereodirecting group at C‐3, resulting in low anomeric selectivities. Furthermore, competing elimination reactions are common and are promoted by the deactivating C‐1 ester group. In our previous work we have presented a convenient approach towards the α‐specific and regioselective synthesis of Chlamydia‐related Kdo oligomers using 3‐iodo‐Kdo fluoride donor 1 for the coupling step followed by deiodination of the stereodirecting auxiliary group.7 Gratifyingly, the elimination reaction could also be largely suppressed, and the protocol was successfully expanded to include the formation of the sterically demanding α‐(2→5)‐linkage of Kdo units.8 In continuation of these applications, we have set out to investigate the suitability of donor 1 for the glycosylation of glucosamine acceptors, specifically addressing regioselective glycosylation at position 6 with the option for subsequent phosphorylation at position 4 in order to generate the common phosphoester substitution at the distal glucosamine unit of lipid A.2, 9</p><!><p>As glycosyl acceptors, N‐acetyl‐protected monosaccharide derivatives were selected, since glycosylation of the primary alcohol of N‐acetyl‐, N‐acyl‐, N‐Cbz‐, and N‐Troc‐protected glucosamine derivatives with various Kdo donors gave comparable yields and anomeric selectivities.10 In a first trial experiment, the easily accessible N‐acetyl‐β‐d‐glucosamine methyl glycoside derivative 2 11 was coupled with the previously described peracetylated 3‐iodo‐Kdo fluoride donor 1 7 under BF3 .Et2O‐promotion in dichloromethane (Scheme 1). Provided, that a regioselective reaction at position 6 would be achieved, the 4‐OH group should remain accessible for subsequent phosphate introduction, thus minimizing protecting group manipulations.</p><!><p>Reagents and conditions: a) BF3⋅Et2O, 3 Å molecular sieves, CH2Cl2, 0 °C→rt, 1.5 h, 3: 32 %, 4: 11 %, 5: 3 %.</p><!><p>Disappointingly, disaccharide 3 was isolated in poor yield (32 %). As expected, only traces of elimination product 6 12 were found and formation of the corresponding β‐linked Kdo‐GlcNAc disaccharide was not observed. As the main disaccharide side products, furanose 4 (11 %) and pyranose 5 oxazolines (3 %) were identified. The structure of the furanose ring form of 4 was proven by an heteronuclear multiple bond correlation (HMBC) between H‐1 and C‐4. When donor 1 was reacted with allyl glycoside 7 (see Supporting Information) under similar glycosylation conditions, a complex mixture of degradation products was obtained. Interestingly, a pronounced elimination of donor 1 giving glycal ester 6 was observed on thin‐layer chromatography (TLC).</p><p>In a preliminary experiment, the same behavior was also noted for the N‐phthalimido‐protected β‐allyl glycosyl acceptor 8 (see Supporting Info). This outcome prompted us to investigate the reaction mechanism in more detail, since this high propensity of 3‐iodo fluoride donor 1 towards elimination had previously not been observed.7 We hypothesized that fast and irreversible migration of an intermediary iodonium ion13, 14 to the allylic double bond leads to formation of the glycal ester 6 and outcompetes glycoside formation (Scheme 2).</p><!><p>Crossover experiments of intermediary iodonium ion in the presence of different olefins and ratios of obtained product mixtures.</p><!><p>We expected that a transfer of iodonium ion generated from the activated donor to the allyl aglycon could be rationalized by the distinct difference in electron density of the allyl aglycon when compared to the rather electron‐deficient α,β‐unsaturated ester 6. To investigate this hypothesis, the competitive behavior of different olefins in a coupling reaction of donor 1 with 2‐propanol in dichloromethane under BF3 .Et2O‐promotion was screened (for experimental detail see Supporting Information). The reaction was performed and directly monitored in an NMR tube. The consumption of the respective olefin was determined by comparing representative 1H NMR signals before and after donor activation in relation to the residual solvent peak. The decrease of olefin matched the amount of glycal 6 formed in the mixture. Immediate and intensive discoloration of the solutions upon activation indicated the instability of the resulting alkyl halide structures under the applied conditions that prevented identification of the iodo species by NMR. In fact the unsubstituted olefins 1‐octene and cyclohexene suppressed formation of the known 2‐propyl glycoside 9 7 and donor 1 degraded completely to glycal 6 within a few seconds. Next we used allyl methyl ether resembling the aglycon of acceptors 7 and 8. Parallel formation of glycoside 9 and glycal 6 (final composition of the mixture was based on relative 1H NMR integral values: 9:6=1:1.2) over a period of 1 h indicated that both pathways (Scheme 2) are operative. In contrast, in the presence of α,β‐unsaturated methyl crotonate, glycoside 9 was obtained without any formation of elimination product 6. The observation that this electrophilic double bond does not inhibit the glycosylation reaction was in agreement with our previous result that donor 1 readily glycosylates glycal ester acceptors.7 In summary, electronic rather than steric effects seem to dictate the halonium migration—a trend that is conclusively reflected by the different results obtained for the two terminal olefins 1‐octene and allyl methyl ether. This may further explain why 2‐iodo‐2‐deoxy sugar donors, that—in comparison to ulosonic acids like Kdo—lack a deactivating C‐1 ester group next to the anomeric center, are not affected by a competitive iodonium‐ion migration in the presence of allyl groups.15 Thus, the allyl group turned out to be an unsuitable protecting or aglyconic group for the 3‐iodo‐Kdo fluoride donor 1 under these conditions.</p><p>Next, the α‐anomeric methyl glycoside 10 (see Supporting Information) was subjected to glycosylation (Scheme 3). In contrast to the outcome of the reaction with β‐anomeric glycoside 2, the resulting disaccharide 11 was not susceptible to oxazoline formation under the applied conditions. The coupling reaction with sparingly soluble acceptor 10 provided disaccharide 11 in moderate yield (∼55 %) together with an inseparable impurity (∼5 % based on the 1H NMR data). First, we believed in the presence of the 4‐O‐regioisomer as only one m/z value was observed for the mixture. However, the expected low‐field‐shifted 13C NMR signal of C‐4 and high‐field‐shifted signal of C‐6, respectively, were not detected. Instead, signals of C‐3 and the benzylic carbon were shifted to lower field (see Supporting Information) in comparison to the major compound. For further analysis, 11 was submitted to the previously optimized dehalogenation procedure capitalizing on a hydrogen atom transfer reaction using lauroyl peroxide in cyclohexane (Scheme 3).16 Free hydroxyl groups were usually tolerated by this method in previous experiments, although decreased solubility of only partially protected substrates required prolonged reaction times.7 Notably, the free 4‐hydroxyl group vicinal to the benzyl group led to a significantly decreased yield. Among different side products, an acid labile 3,4‐O‐benzylidene protected disaccharide could be identified. Similar observations have been described by the Bols group, who later capitalized on this benzylidene formation and its ensuing cleavage as a regioselective deprotection method for benzyl groups vicinal to free OH groups.17 In agreement with their observation that free hydroxyl groups were essential for this reaction type17a—involving a radical process17b—benzyl groups did not interfere with the hydrogen atom transfer reaction when using fully protected carbohydrate moieties. Further on, the disaccharide 11 containing the unknown entity was O‐acetylated (12) prior to dehalogenation that provided disaccharide 13 in 75 % yield. Neither of these steps allowed separation of the minor unit by high‐performance liquid chromatography (HPLC). Again, the only significant differences were seen for the low‐field NMR shifts of C‐3 and the benzylic carbon of the minor species. Thus, we surmised the presence of two stable rotamers in the disaccharide product.</p><!><p>Reagents and conditions: a) 1, BF3⋅Et2O, 3 Å molecular sieves, CH2Cl2, 0 °C→rt, 1 h, 55 %; b) Ac2O, 4‐(N,N‐dimethylamino)pyridine, pyridine, rt, 5 h, 59 %; c) lauroyl peroxide, cyclohexane/1,2‐dichloroethane (8:1), reflux, 2 h, 75 %.</p><!><p>This was eventually proven by a method proposed by Ley's group wherein 1 D‐nuclear Overhauser effect (NOE)‐difference spectra are used to distinguish between equilibrating rotamers and nonequilibrating isomers.18 By selective excitation of the NH signal of the minor entity, a parallel attenuation of the distant and heavily overlapped NH of the major species was observed (Figure 1). This phenomenon only occurs for the equilibrating rotamers but not for chemically distinct isomers.</p><!><p>Evidence for the presence of two rotamers in disaccharide 13; top: 1D NOE‐difference spectrum after selective pulse at 5.64 ppm (NH signal of minor compound); bottom: expansion plot of 1H NMR of 13 showing the NH signals.</p><!><p>To increase the limited solubility of the acceptor and to avoid problems at the dehalogenation step, the protecting group pattern of the GlcNAc acceptor was revised and the 3‐O‐benzoyl‐4‐O‐benzyl‐protected acceptor 14 (see Supporting Information) was coupled with 3‐iodo fluoride donor 1 following the general protocol (Scheme 4). This way, disaccharide 15 was obtained in a high yield (89 %) as the α‐anomer only,19 and was dehalogenated (87 % of 16) by hydrogen atom transfer.20 Debenzylation by catalytic hydrogenation (Pd/C, H2) afforded 4‐OH disaccharide 17 (96 %). Subsequent phosphorylation capitalizing on the two‐step procedure using dibenzyl N,N‐diisopropylphosphoramidite/1H‐tetrazole followed by oxidation with meta‐chloroperoxybenzoic acid (mCPBA) yielded 4‐O‐phosphotriester 18 (70 %).21</p><!><p>Reagents and conditions: a) 1, BF3⋅Et2O, 3 Å molecular sieves, CH2Cl2, 0 °C→rt, 1 h, 89 %; b) lauroyl peroxide, cyclohexane/1,2‐dichloroethane (8:1), reflux, 2 h, 87 %; c) Pd/C (10 %), H2, MeOH, rt., 3 h, 96 %; d) dibenzyl N,N‐diisopropylphosphoramidite, 1H‐tetrazole, 4 Å molecular sieves, CH2Cl2, 0 °C, 70 min, then mCPBA, 0 °C, 1 h, 70 %; e) NaOMe, MeOH, 3 h, then aq. NaOH, 0 °C, 5 h, 97 %; f) Pd/C (10 %), H2, MeOH, rt, 30 min, then NaOMe, MeOH, rt, 17 h, then aq NaOH, 0 °C, 5 h, 99 %.</p><!><p>Compound 17 was subjected to transesterification with methanol under Zemplén conditions (the formed methyl benzoate had to be removed by repeated extraction between water and n‐hexane), followed by saponification of the methyl ester to give disaccharide 19 in 97 % yield. For global deprotection of 18, the phosphotriester was first debenzylated affording the free phosphate, which was no longer prone to migration/hydrolysis under basic conditions. Deprotection of the acyl groups and ester hydrolysis furnished the deblocked disaccharide 20 as sodium salt in near theoretical yield.</p><!><p>3‐Iodo‐Kdo fluoride donor 1 was suitable for stereo‐ and regioselective glycosylation of a 6‐OH GlcNAc acceptor, but high yields relied on an appropriate protecting group pattern. The acceptor had to be designed as the α‐OMe glycoside to avoid oxazoline formation. Ensuing dehalogenation by hydrogen atom transfer reaction did not tolerate free hydroxyl groups in close proximity to the benzyl protecting group. Furthermore, the 3‐iodo group was incompatible with the presence of nucleophilic olefins due to putative iodonium ion migration resulting in glycal ester 6. Within these boundary conditions, however, the α‐specific glycosylation, dehalogenation, and global deprotection proceeded in high overall yields.</p><!><p>General: All purchased chemicals were used without further purification unless stated otherwise. Solvents (CH3CN, CH2Cl2, cyclohexane, 1,2‐dichloroethane, N,N‐dimethylformamide) were dried over activated 4 Å molecular sieves. Dry MeOH (secco solv) was purchased from Merck. Cation exchange resin DOWEX 50 H+ was regenerated by consecutive washing with HCl (3 m), water, and dry MeOH. The promoter BF3⋅Et2O was used as a solution in diethyl ether (≥46 % according to the manufacturer). Concentration of organic solutions was performed under reduced pressure<40 °C. Optical rotations were measured with a PerkinElmer 243 B Polarimeter (Waltham, USA). [α]D 20 values are given in units of 10−1 deg cm2 g−1. Thin layer chromatography was performed on Merck precoated plates: generally on 5×10 cm, layer thickness 0.25 mm, Silica Gel 60F254; alternatively on HP‐TLC plates with 2.5 cm concentration zone (Merck). Spots were detected by dipping reagent (anisaldehyde‐H2SO4). For column chromatography, silica gel (0.040–0.063 mm) was used. HP‐column chromatography was performed on a prepacked column (YMC‐Pack SIL‐06, 0.005 mm, 250×10 mm). Size exclusion chromatography for desalting was performed on prepacked PD‐10 columns (GE Healthcare, Sephadex G‐25 m). NMR spectra were recorded with a Bruker Avance III 600 instrument (600.22 MHz for 1H, 150.93 MHz for 13C and 242.97 MHz for 31P) using standard Bruker NMR software (Rheinstetten, Germany). 1H spectra were referenced to 7.26 (CDCl3) and 0.00 (D2O, external calibration to 2,2‐dimethyl‐2‐silapentane‐5‐sulfonic acid) ppm unless stated otherwise. 13C spectra were referenced to 77.00 (CDCl3) and 67.40 (D2O, external calibration to 1,4‐dioxane) ppm. 31P spectra in D2O were referenced to external ortho‐phosphoric acid (0.00 ppm). Electrospray ionization mass spectrometry (ESI‐MS) data were obtained on a Waters Micromass Q‐TOF Ultima Global instrument (Santa Clara, USA) or on a Thermo Scientific Exactive Plus Orbitrap instrument (Waltham, USA).</p><p>Methyl (4,5,7,8‐tetra‐O‐acetyl‐3‐deoxy‐3‐iodo‐d‐glycero‐α‐d‐talo‐oct‐2‐ulopyranosyl)onate‐(2→6)‐methyl 2‐acetamido‐3‐O‐benzoyl‐4‐O‐benzyl‐2‐deoxy‐α‐d‐glucopyranoside (15): A suspension of 3‐iodo‐Kdo donor 1 (76.6 mg, 0.140 mmol) and acceptor 14 (50.0 mg, 0.116 mmol) in dry CH2Cl2 (4.0 mL) containing ground 3 Å molecular sieves (200 mg) was stirred for 1 h at ambient temperature. The cooled mixture (0 °C) was treated with BF3⋅Et2O (44 μL, 0.349 mmol) and kept at ambient temperature for 1 h. After dilution with CH2Cl2, the organic phase was washed successively with satd. NaHCO3 (1×5 mL), sodium thiosulfate (5 w %) (1×5 mL), and brine (1×5 mL). The solution was dried over MgSO4, filtered, and concentrated, and the crude product was purified by chromatography (toluene/EtOAc 1:2) affording disaccharide 15 as a colorless oil (99.2 mg, 89 %): R f=0.31 (toluene/EtOAc 1:2, HP‐TLC); [α]D 20=+68.2 (c=1.28 in CHCl3); 1H NMR (CDCl3): δ=8.03–8.00 (m, 2 H, Ar), 7.58–7.55 (m, 1 H, Ar), 7.46–7.42 (m, 2 H, Ar), 7.25–7.08 (m, 5 H, Ar), 5.82 (d, 1 H, J NH,2 9.5 Hz, NH), 5.55 (dd, 1 H, J 3,2 10.8, J 3,4 9.1 Hz, H‐3), 5.38–5.36 (m, 1 H, H‐5′), 5.34 (ddd, 1 H, J 7′,6' 9.5, J 7′,8′b 4.1, J 7′,8′a 2.6 Hz, H‐7′), 5.05 (dd, 1 H, J 4′,3' 4.7, J 4′,5' 3.7 Hz, H‐4′), 4.72 (d, 1 H, J 1,2 3.5 Hz, H‐1), 4.67 (dd, 1 H, J 8′a,8′b 12.3 Hz, H‐8′a), 4.64 (d, 1 H, J 11.2 Hz, CHHPh), 4.51 (d, 1 H, H‐3′), 4.44 (d, 1 H, J 11.2 Hz, CHHPh), 4.36 (ddd, 1 H, H‐2), 4.32 (dd, 1 H, J 6′,5' 1.8 Hz, H‐6′), 4.16 (dd, 1 H, H‐8′b), 3.93–3.89 (m, 1 H, H‐5), 3.74 (s, 3 H, CO2CH3), 3.61–3.52 (m, 3 H, H‐4, H‐6a, H‐6b), 3.42 (s, 3 H, OCH3), 2.11, 2.05, 2.03, 1.99, and 1.83 ppm (5 s, each 3 H, COCH3); 13C NMR (CDCl3): δ=170.3, 170.1, 170.0, 169.5, and 169.3 (5 s, 5C, COCH3), 166.8 (s, 1C, COPh), 165.6 (s, C‐1′), 137.0 (s, 1C, Ar), 133.4 (d, 1C, Ar), 129.7 (d, 2C, Ar), 129.3 (s, 1C, Ar), 128.5 (d, 2C, Ar), 128.4 (d, 2C, Ar), 128.0 (d, 1C, Ar), 127.9 (d, 2C, Ar), 101.4 (s, C‐2′), 98.0 (d, C‐1), 76.3 (d, C‐4), 74.9 (t, CH2Ph), 74.2 (d, C‐3), 69.8 (d, C‐5), 68.3 (d, C‐6′), 67.7 (d, C‐7′), 65.2 (d, C‐4′), 65.1 (t, C‐6), 63.4 (d, C‐5′), 61.8 (t, C‐8′), 55.1 (q, OCH3), 52.8 (q, CO2 CH3), 52.3 (d, C‐2), 23.1 (q, COCH3), 21.8, 20.9, 20.8, 20.7, and 20.6 ppm (4 q, 1 d, 5C, 4×COCH3, C‐3′); HRMS (ESI‐TOF) m/z [M+Na]+ calcd for C40H48INO18Na+: 980.1808, found: 980.1808.</p><p>Methyl (4,5,7,8‐tetra‐O‐acetyl‐3‐deoxy‐α‐d‐manno‐oct‐2‐ulopyranos‐yl)onate‐(2→6)‐methyl 2‐acetamido‐3‐O‐benzoyl‐4‐O‐benzyl‐2‐deoxy‐α‐d‐glucopyranoside (16): Disaccharide 15 (99.0 mg, 0.103 mmol) was dissolved in dry cyclohexane (4.0 mL) and dry 1,2‐dichloroethane (0.5 mL) and the solution was degassed with argon. After heating to reflux for 15 min, lauroyl peroxide (14.4 mg, 0.036 mmol) was added and the solution was held at reflux for 2 h. The mixture was concentrated in vacuo, and the residue was subjected to chromatography (n‐hexane/EtOAc 1:9), yielding dehalogenated compound 16 as a colorless oil (75.0 mg, 87 %); R f=0.36 (CH2Cl2/EtOAc 1:1, HP‐TLC); [α]D 20=+82.2 (c=0.66 in CHCl3); 1H NMR (CDCl3): δ=8.02–8.00 (m, 2 H, Ar), 7.58–7.54 (m, 1 H, Ar), 7.46–7.42 (m, 2 H, Ar), 7.22–7.12 (m, 5 H, Ar), 5.81 (d, 1 H, J NH,2 9.5 Hz, NH), 5.55 (dd, 1 H, J 3,2 10.8, J 3,4 9.0 Hz, H‐3), 5.37–5.33 (m, 2 H, H‐4′, H‐5′), 5.21 (ddd, 1 H, J 7′,6' 9.6, J 7′,8′b 4.6, J 7′,8′a 2.5 Hz, H‐7′), 4.72 (d, 1 H, J 1,2 3.6 Hz, H‐1), 4.65 (d, 1 H, J 11.0 Hz, CHHPh), 4.59 (dd, 1 H, J 8′a,8′b 12.3 Hz, H‐8′a), 4.49 (d, 1 H, J 11.0 Hz, CHHPh), 4.37 (ddd, 1 H, H‐2), 4.18 (dd, 1 H, J 6′,5' 1.1 Hz, H‐6′), 4.10 (dd, 1 H, H‐8′b), 3.92–3.88 (m, 1 H, H‐5), 3.76 (dd, 1 H, J 6a,6b 10.6, J 6a,5 1.7 Hz, H‐6a), 3.72 (s, 3 H, CO2CH3), 3.61 (app t, 1 H, J 4,5 ∼9.7 Hz, H‐4), 3.57 (dd, 1 H, J 6b,5 7.2 Hz, H‐6b), 3.43 (s, 3 H, OCH3), 2.24–2.20 (m, 1 H, H‐3′eq), 2.10 (app t, 1 H, J 3′ax,3′eq ∼J 3′ax,4' 12.3 Hz, H‐3′ax), 2.08, 2.023, 2.020, 1.98, and 1.83 ppm (5 s, each 3 H, COCH3); 13C NMR: δ=170.5, 170.4, 170.0, 169.9, 169.7, 167.2 and 166.9 (7 s, 7C, 5×COCH3, COPh, C‐1′), 137.2 (s, 1C, Ar), 133.4 (d, 1C, Ar), 129.8 (d, 2C, Ar), 129.4 (s, 1C, Ar), 128.5 (d, 2C, Ar), 128.4 (d, 2C, Ar), 127.91 (d, 1C, Ar), 127.87 (d, 2C, Ar), 98.5 (s, C‐2′), 98.1 (d, C‐1), 76.6 (d, C‐4), 74.9 (t, CH2Ph), 74.3 (d, C‐3), 69.9 (d, C‐5), 68.6 (d, C‐6′), 67.7 (d, C‐7′), 66.3 (d, C‐4′), 64.5 (d, C‐5′), 63.2 (t, C‐6), 62.2 (t, C‐8′), 55.2 (q, OCH3), 52.6 (q, CO2 CH3), 52.4 (d, C‐2), 31.8 (t, C‐3′), 23.1, 20.8, 20.73, 20.69, and 20.6 ppm (5 q, 5C, COCH3); HRMS (ESI‐TOF) m/z [M+Na]+ calcd for C40H49NO18Na+: 854.2842, found: 854.2847.</p><p>Methyl (4,5,7,8‐tetra‐O‐acetyl‐3‐deoxy‐α‐d‐manno‐oct‐2‐ulopyranosyl)onate‐(2→6)‐methyl 2‐acetamido‐3‐O‐benzoyl‐2‐deoxy‐α‐d‐glucopyranoside (17): Compound 16 (70.0 mg, 0.084 mmol) was dissolved in dry MeOH (3.0 mL). The atmosphere was exchanged to argon by alternating evacuation and flushing with argon. Pd/C (10 %, 3 mg) was added followed by successive exchange of atmosphere to argon and hydrogen. After hydrogenation for 3 h, the mixture was filtered via a syringe filter, rinsed with MeOH (3×2 mL), and the filtrate was concentrated providing compound 17 as a colorless oil (60.0 mg, 96 %), which was used without purification: R f=0.43 (EtOAc); [α]D 20=+99.3 (c=0.78 in CHCl3); 1H NMR (CDCl3): δ=8.05–8.03 (m, 2 H, Ar), 7.60–7.56 (m, 1 H, Ar), 7.47–7.43 (m, 2 H, Ar), 5.78 (d, 1 H, J NH,2 9.8 Hz, NH), 5.37–5.32 (m, 2 H, H‐4′, H‐5′), 5.24 (dd, 1 H, J 3,2 10.7, J 3,4 9.2 Hz, H‐3), 5.23 (ddd, 1 H, J 7′,6' 9.3, J 7′,8′b 4.8, J 7′,8′a 2.6 Hz, H‐7′), 4.75 (d, 1 H, J 1,2 3.5 Hz, H‐1), 4.59 (dd, 1 H, J 8′a,8′b 12.1 Hz, H‐8′a), 4.45 (ddd, 1 H, H‐2), 4.28 (dd, 1 H, J 6′,5' 1.3 Hz, H‐6′), 4.15 (dd, 1 H, H‐8′b), 3.89–3.84 (m, 1 H, H‐5), 3.82 (s, 3 H, CO2CH3), 3.79–3.73 (m, 3 H, H‐4, H‐6a, H‐6b), 3.45 (s, 3 H, OCH3), 3.10 (d, 1 H, J 3.8 Hz, OH), 2.21–2.17 (m, 1 H, H‐3′eq), 2.12 (app t, 1 H, J 3′ax,3′eq ∼J 3′ax,4' 12.5 Hz, H‐3′ax), 2.085, 2.077, 2.00, 1.97, and 1.86 ppm (5 s, each 3 H, COCH3); 13C NMR (CDCl3): δ=170.6, 170.4, 169.91, 169.89, 169.8, 168.2, and 167.6 (7 s, 7C, 5×COCH3, COPh, C‐1′), 133.6 (d, 1C, Ar), 130.1 (d, 2C, Ar), 129.1 (s, 1C, Ar), 128.5 (d, 2C, Ar), 98.7 (s, C‐2′), 98.3 (d, C‐1), 75.5 (d, C‐3), 70.4 (d, C‐5), 70.0 (d, C‐4), 68.7 (d, C‐6′), 67.8 (d, C‐7′), 66.3 (d, C‐4′), 64.5 (d, C‐5′), 63.5 (t, C‐6), 62.3 (t, C‐8′), 55.3 (q, OCH3), 52.9 (q, CO2 CH3), 51.4 (d, C‐2), 31.9 (t, C‐3′), 23.2, 20.8, 20.73, 20.68, and 20.6 ppm (5 q, 5C, COCH3); HRMS (ESI‐TOF) m/z [M+Na]+ calcd for C33H43NO18Na+: 764.2372, found: 764.2372.</p><p>Methyl (4,5,7,8‐tetra‐O‐acetyl‐3‐deoxy‐α‐d‐manno‐oct‐2‐ulopyranosyl)onate‐(2→6)‐methyl 2‐acetamido‐3‐O‐benzoyl‐2‐deoxy‐4‐O‐(di‐benzylphosphoryl)‐α‐d‐glucopyranoside (18): A solution of compound 17 (19.5 mg, 0.026 mmol) in dry CH2Cl2 (1.0 mL) was degassed with argon. Under argon atmosphere 1 H‐tetrazole (5.5 mg, 0.079 mmol) was added followed by ground 4 Å molecular sieves (50 mg). After 30 min the suspension was cooled to 0 °C and treated dropwise with dibenzyl N,N‐diisopropylphosphoramidite (17.3 μL, 0.053 mmol, in 3 portions within 70 min). mCPBA (70 w %, 9.7 mg, 0.039 mmol) was added at 0 °C, and the mixture was stirred for 1 h. The mixture was diluted with CH2Cl2 and satd. NaHCO3, the aqueous phase was extracted with CH2Cl2 (3×5mL), and the combined organic layers were dried over MgSO4. Filtration, concentration of the organic phase, and chromatography of the residue (toluene/EtOAc 1:4) provided phosphorylated compound 18 with a minor impurity which was separated by HP‐column chromatography (n‐hexane/EtOAc 1:2→1:4) affording 18 as a colorless oil (18.3 mg, 70 %): R f=0.41 (n‐hexane/EtOAc 1:4); [α]D 20=+64.8 (c=0.78 in CHCl3); 1H NMR (CDCl3, ref. to 0.00, TMS): δ=8.03–8.01 (m, 2 H, Ar), 7.54 −7.51 (m, 1 H, Ar), 7.39–7.35 (m, 2 H, Ar), 7.29–7.13 (m, 8 H, Ar), 6.91–6.89 (m, 2 H, Ar), 5.79 (d, 1 H, J NH,2 9.4 Hz, NH), 5.56 (dd, 1 H, J 3,2 10.9, J 3,4 9.1 Hz, H‐3), 5.41 (ddd, 1 H, J 4′,3′ax 12.2, J 4′,3′eq 5.1, J 4′,5' 3.1 Hz, H‐4′), 5.37–5.35 (m, 1 H, H‐5′), 5.21 (ddd, 1 H, J 7′,6' 9.2, J 7′,8′b 5.0, J 7′,8′a 2.4 Hz, H‐7′), 4.89 (dd, 1 H, 2 J 11.8, J H,P 7.2 Hz, CHHPh), 4.78 (dd, 1 H, 2 J 12.2, J H,P 7.9 Hz, CHHPh), 4.77 (d, 1 H, J 1,2 3.8 Hz, H‐1), 4.74 (dd, 1 H, 2 J 11.7, J H,P 7.5 Hz, CHHPh), 4.67 (dd, 1 H, J 8′a,8′b 12.2 Hz, H‐8′a), 4.56 (dd, 1 H, 2 J 11.8, J H,P 10.0 Hz, CHHPh), 4.51 (app q, 1 H, J 4,5 ∼J 4,P 9.3 Hz, H‐4), 4.41 (ddd, 1 H, H‐2), 4.34 (dd, 1 H, J 6′,5' 1.4 Hz, H‐6′), 4.09 (dd, 1 H, H‐8′b), 4.05–4.00 (m, 2 H, H‐5, H‐6a), 3.74 (dd, 1 H, J 6b,6a 11.1, J 6b,5 7.9 Hz, H‐6b), 3.68 (s, 3 H, CO2CH3), 3.48 (s, 3 H, OCH3), 2.22–2.18 (m, 1 H, H‐3′eq), 2.12 (app t, 1 H, J 3′ax,3′eq ∼12.5 Hz, H‐3′ax), 2.091, 2.086, 2.01, 1.96, and 1.84 ppm (5 s, each 3 H, COCH3); 13C NMR (CDCl3): δ=170.5, 170.4, 169.9, 169.83, 169.80, 167.2, and 166.9 (7 s, 7C, 5×COCH3, COPh, C‐1′), 135.5 (ds, 1C, J 7.2 Hz, Ar), 135.2 (ds, 1C, J 6.5 Hz, Ar), 133.4 and 130.0 (2 d, 3C, Ar), 129.1 (s, 1C, Ar), 128.44, 128.39, 128.32, 128.30, 127.69, and 127.66 (6 d, 12C, Ar), 98.5 (s, C‐2′), 97.8 (d, C‐1), 73.8 (dd, J 5.9 Hz, C‐4), 72.1 (dd, J 2.0 Hz, C‐3), 69.53 (d, C‐5), 69.47 (dt, J 5.8 Hz, CH2Ph), 69.4 (dt, J 5.9 Hz, CH2Ph), 68.8 (d, C‐6′), 68.1 (d, C‐7′), 66.3 (d, C‐4′), 64.7 (d, C‐5′), 62.7 (t, C‐6), 62.1 (t, C‐8′), 55.4 (q, OCH3), 52.6 (q, CO2 CH3), 52.2 (d, C‐2), 31.9 (t, C‐3′), 23.1, 20.8, 20.73, and 20.66 ppm (4 s, 5C, COCH3); 31P NMR (CDCl3): δ=−2.01 ppm; HRMS (ESI‐TOF) m/z [M+Na]+ calcd for C47H56NO21PNa+: 1024.2975, found: 1024.2976.</p><p>Sodium (3‐deoxy‐α‐d‐manno‐oct‐2‐ulopyranosyl)onate‐(2→6)‐methyl 2‐acetamido‐2‐deoxy‐α‐d‐glucopyranoside (19): A solution of 17 (8.2 mg, 0.011 mmol) in dry MeOH (1 mL) was treated with sodium methoxide (0.1 m in MeOH, 22 μL, 2.2 μmol) at ambient temperature for 3 h. Ion‐exchange resin DOWEX 50 (H+‐form) was added until the mixture reacted neutral. The resin was filtered off and the filtrate was concentrated in vacuo. To remove methyl benzoate, water (3 mL) and n‐hexane (3 mL) were added to the residual solid, the mixture was ultrasonicated for 10 s, and the organic layer was removed. Fresh n‐hexane (3 mL) was added and the procedure was repeated. After two more extractions, the aqueous phase was concentrated in vacuo, and the residue was stirred in aq NaOH (0.01 m, 3.0 mL) at 0 °C for 3 h. Additional aq NaOH (0.1 m, 0.5 mL) was added, and after 2 h at 0 °C the mixture was neutralized with DOWEX 50 (H+‐form). The resin was filtered off, washed with water (3×1 mL), and the filtrate was freeze‐dried. The crude product was desalted on a PD10 SEC column (H2O). Freeze‐drying of pooled fractions yielded sodium salt 19 as a colorless amorphous solid (5.1 mg, 97 %): [α]D 20=+102.8 (c=0.26 in H2O); 1H NMR (D2O, pD 7.4): δ=4.71 (d, 1 H, J 1,2 3.7 Hz, H‐1, overlapped by residual solvent peak), 4.04 (ddd, 1 H, J 4′,3′ax 12.1, J 4′,3′eq 5.0, J 4′,5' 2.9 Hz, H‐4′), 3.99–3.97 (m, 1 H, H‐5′), 3.92–3.87 (m, 3 H, H‐2, H‐7′, H‐8′a), 3.74 (ddd, 1 H, J 5,4 10.1, J 5,6a 4.5, J 5,6b 3.4 Hz, H‐5), 3.68–3.54 (m, 5 H, H‐3, H‐6a, H‐6b, H‐6′, H‐8′b), 3.44 (dd, 1 H, J 4,3 9.4 Hz, H‐4), 3.34 (s, 3 H, OCH3), 2.05 (dd, 1 H, J 3′eq,3′ax 13.1 Hz, H‐3′eq), 1.99 (s, 3 H, COCH3), and 1.76 ppm (app t, 1 H, H‐3′ax); 13C NMR (D2O, pD 7.4): δ=175.8 and 175.3 (2 s, 2C, C‐1′, COCH3), 100.7 (s, C‐2′), 98.8 (d, C‐1), 72.3 and 72.2 (2 d, 2C, C‐3, C‐6′), 71.2 (d, C‐4), 71.0 (d, C‐5), 70.3 (d, C‐7′), 67.1 and 66.9 (2 d, 2C, C‐4′, C‐5′), 64.2 (t, C‐8′), 62.7 (t, C‐6), 56.0 (q, OCH3), 54.4 (d, C‐2), 34.9 (t, C‐3′), and 22.7 ppm (q, COCH3); HRMS (ESI‐TOF) m/z [M−H]− calcd for C17H28NO13 −: 454.1555, found: 454.1564.</p><p>3‐Deoxy‐α‐d‐manno‐oct‐2‐ulopyranosylonic acid‐(2→6)‐methyl 2‐acetamido‐2‐deoxy‐4‐phosphono‐α‐d‐glucopyranoside sodium salt (20): Phosphotriester 18 (11.0 mg, 0.011 mmol) was dissolved in dry MeOH (2.0 mL). The atmosphere was exchanged to argon by alternating evacuation and flushing with argon. Pd/C (10 %, 1 mg) was added followed by successive exchange of atmosphere to argon and hydrogen. After hydrogenation for 30 min the mixture was filtered via a syringe filter into a flask containing sodium methoxide (0.1 m in MeOH, 110 μL, 0.011 mmol) in dry MeOH (2.0 mL). After rinsing the filter with MeOH (3×1 mL), the filtrate was neutralized by further addition of sodium methoxide (0.1 m in MeOH). The solvent was removed until a volume of 3 mL of MeOH remained: The pH was adjusted to 8 with sodium methoxide, and the mixture was stirred for 3 h at rt. After increasing the pH to 9, stirring was continued for 14 h. Excessive base was neutralized by adding DOWEX 50 (H+‐form) resin. The resin was filtered off, rinsed with MeOH, and the filtrate was concentrated in vacuo. Removal of methyl benzoate, ester saponification, and desalting were performed as described for compound 19, providing phosphate 20 as a colorless amorphous solid (6.3 mg, 99 %): [α]D 20=+91.6 (c=0.63 in H2O); 1H NMR (D2O, pD 7.4): δ=4.71 (d, 1 H, J 1,2 3.5 Hz, H‐1, overlapped by residual solvent peak), 4.06 (ddd, 1 H, J 4′,3′ax 12.0, J 4′,3′eq 5.2, J 4′,5' 3.0 Hz, H‐4′), 3.99–3.98 (m, 1 H, H‐5′), 3.92–3.76 (m, 7 H, H‐2, H‐3, H‐4, H‐5, H‐6′, H‐7′, H‐8′a), 3.70–3.63 (m, 2 H, H‐6a, H‐8′b), 3.57–3.53 (m, 1 H, H‐6b), 3.34 (s, 3 H, OCH3), 2.03 (dd, 1 H, J 3′eq,3′ax 13.1 Hz, H‐3′eq), 1.98 (s, 3 H, COCH3), and 1.74 ppm (app t, 1 H, H‐3′ax); 13C NMR (D2O, pD 7.4): δ=176.0 and 175.1 (2 s, 2C, C‐1′, COCH3), 100.6 (s, C‐2′), 98.1 (d, C‐1), 74.3 (dd, J 5.2 Hz, C‐4), 72.2 (d, 2C, C‐3, C‐6′), 70.5 (d, C‐7′), 70.3 (dd, J 8.7 Hz, C‐5), 67.2 (d, C‐5′), 66.9 (d, C‐4′), 64.2 (t, C‐8′), 63.2 (t, C‐6), 55.9 (q, OCH3), 54.1 (d, C‐2), 35.0 (t, C‐3′), and 22.7 ppm (q, COCH3); 31P NMR (D2O, pD 7.4): δ=−3.30 ppm; HRMS (ESI‐TOF) m/z [M−H]− calcd for C17H29NO16P−: 534.1219, found: 534.1229.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supplementary</p><p>Click here for additional data file.</p>

PubMed Open Access

Limits of the quantum cognition hypothesis: 31 P singlet order lifetimes of pyrophosphate from experiment and simulation Authors

A proposal of quantum cognition advances the hypothesis that quantum entanglement between 31 P nuclei could serve as a means of information storage in the brain. Testing this hypothesis requires an understanding of how long-lived these quantum effects may be. We used NMR spectroscopy and molecular dynamics simulations to study the mechanisms that limit these quantum processes in 18 O-enriched molecules of pyrophosphate, the simplest biomolecule that can sustain quantum-entangled 31 P nuclear spin singlet states. We confirmed that chemical shift anisotropy limits the singlet magnetization order lifetimes in high magnetic fields, and we discovered that rapid rotation of the phosphate groups limits the lifetime in low magnetic fields. These findings represent an important starting point in studying whether quantum cognition can be a true biological phenomenon.

limits_of_the_quantum_cognition_hypothesis:_31_p_singlet_order_lifetimes_of_pyrophosphate_from_exper

Introduction<!>Synthesis and NMR characterization of unsymmetrically 18 O-labeled pyrophosphate<!>NMR field-cycling relaxation measurements of uPPi<!>Molecular dynamics simulation and ab initio calculation of relaxation rate curves<!>Discussion<!>Materials and Methods<!>Field-dependent NMR spectroscopy<!>Simulation methods

<p>It has been proposed that 31 P nuclear spin entanglement may play a role in physiology and biological information storage and transmission (1, 2). Although the notion of quantum processes involving entangled nuclear spin states may appear far-fetched, the hypothesis has not been easy to directly prove or refute. The quantum cognition proposal involves the existence of so-called Posner clusters with the stoichiometric formula Ca9(PO4)6, which through an interplay between rotational and nuclear spin states may exhibit symmetryconstrained quantized states labeled by what was called 'pseudospin'. Such clusters have yet to be detected experimentally. Smaller fragments, such as pyrophosphate, have also been considered as potential actors for 31 P nuclear singlet order (SO) modulation of reaction rates, in particular enzymatic ones (1). In this work, we therefore set out to determine the underlying limitations that would have to be considered in support of such claims, in particular regarding the lifetime of 31 P SO, represented by an exponential decay time constant TS, describing the quantum entanglement memory loss.</p><p>Nuclear SO in 1 H and 13 C spin pairs has been observed to have very long TS values compared to the spin-lattice relaxation time constant T1 in a variety of compounds, in some cases one to two orders of magnitude higher (3)(4)(5)(6)(7).Very recently, SO between 31 P nuclei has been observed and characterized in large diphosphate compounds (8,9). For the 31 P spins in the compounds studied, however, singlet relaxation has been found to be much more rapid than spin-lattice relaxation, with a major reason being the anticorrelation between the chemical shift anisotropy (CSA) tensors of the two spins (8).</p><p>Two aspects of this prior work motivated us to examine 31 P-spin SO further. The compounds used previously were particularly bulky and contained large asymmetries between the two spins (either transient or constant). We therefore sought to study the small, highly symmetric molecule pyrophosphate, modified to have slight asymmetry, thereby enabling access to SO. Since the main mechanism in prior work on substituted phosphates appeared to be due to CSA, we wished to perform field-dependent studies. We present here Zeeman and SO relaxation studies over a large field range (2 𝜇T to 9.4 T) to investigate the major relaxation mechanisms as a function of magnetic field, and to determine, in particular, the underlying low-field limit to the SO relaxation time. We further identify the mechanistic contributions to SO relaxation by molecular dynamics (MD) and ab initio computation.</p><!><p>One challenge in the study of SO in the pyrophosphate (PPi) molecule is the lack of inequivalence (either chemical or magnetic), which is needed for creating and reading out SO of the 31 P spins. To overcome this challenge, we unsymmetrically labeled PPi with the 18 O isotope. The increased mass of the 18 O nuclei relative to the abundant 16 O isotope was expected to induce a small chemical shift difference between the neighboring 31 P nuclei, sufficiently large to allow creation and read-out of SO. This strategy was used previously for pairs of 13 C nuclei (10). The tetrasodium salt of the unsymmetrically labelled 18 O-PPi (uPPi) was synthesized as described in the Materials and Methods section below and prepared in D2O under highly alkaline conditions to avoid potentially interfering effects due to proton exchange, which can accelerate SO relaxation (11). An excess of potassium cations, relative to the sodium cations from the tetrasodium salt, was found to promote longer SO lifetimes than if sodium ions alone were present. Similar results were obtained by adding ethylenediaminetetraacetate (EDTA) instead (Fig. S1, Supplementary Materials). The NMR properties of the synthesized uPPi 31 P spin system were extracted from a 31 P pulse-acquire spectrum acquired at 9.4 T by multiplet simulation and fitting using the Spinach MATLAB package (http://spindynamics.org/group/) (12). Fig. 1 displays the fitting results. The unsymmetrical isotopic labelling of the uPPi induces a slight chemical shift difference ∆𝛿PP between the two 31 P nuclei of 0.0663 ppm, or 10.7 Hz at 9.4 T. The 31 P nuclei share a homonuclear J-coupling of magnitude 2 JPP = 21.5 Hz. Thus, the uPPi 31 P spin system is in a strongly coupled regime at 9.4 T. Singlet-triplet mixing can occur at high fields, but this mechanism of SO decay is eliminated when the sample is moved to lower fields. Additional peaks are observed which likely stem from partial labelling of the molecule. We could not fully identify these, but products with partial labelling should not affect the results, since the triplet-singlet transfer is tailored to a particular chemical shift / coupling combination. The isotope composition should not affect relaxation rates due to the small differences in mass. The 31 P R1 values of the unlabeled PPi and the 18 O-labeled uPPi were measured at 9.4 T as 0.107 s -1 and 0.102 s -1 , respectively, with identical solution conditions (pD 14.4, 25 °C).</p><!><p>We then performed field-dependent measurements of both SO relaxation and spin-lattice relaxation, in order to compare and contrast known relaxation mechanisms. We chose to utilize the spin-lock induced crossing (SLIC) pulse sequence (13) for preparing and reading out SO for NMR spectroscopic relaxation measurements. The SLIC pulse sequence used for field-dependent measurements of RS (= 1/TS) is displayed in Fig. 2. Optimization of the power and duration of the SLIC spin-lock pulse confirmed the spin system parameters determined via spectral fitting: the optimal pulse amplitude and duration corresponded with 2 JPP of 20.3 Hz and a ∆𝛿PP of 12.3 Hz (Fig. S2, Supplementary Materials). We performed 31 P relaxation measurements on ~300 mL of the uPPi solution in a 5 mm NMR tube using a 9.4 T Bruker NMR spectrometer equipped with a home-built field shuttling system, to transport the sample rapidly between regions of different magnetic field. The shuttling system included a shielded region above the magnet and therefore enabled access to magnetic field strengths as low as 2 µT. An inversion-recovery sequence was used with the same sample shuttling setup in order to measure R1 (= 1/T1) Conversions between detectable magnetization and singlet order were performed at 9.4 T, and the sample was shuttled to low field for the incremented relaxation delay, then shuttled back for detection. A T00 filter prior to singlet readout was used with two-step phase cycling on the first 90° pulse and receiver to remove undesired coherence pathways.</p><p>The relaxation measurements are shown in Fig. 3. Generally, the R1 and RS values tracked each other, with R1 experiencing a slight increase in the 2 µT to 200 mT range. RS also tended to be smaller than R1 in the high-field regime, above 4.5 T. Both R1 and RS approached a constant relaxation rate offset of approximately 0.018 s -1 at the lowest field values measured. The measured relaxation trends with magnetic field were well approximated using MD simulations and ab initio calculation (Fig. 3, dashed lines), as described below.</p><!><p>In order to study the CSA tensors in uPPi and their contributions to longitudinal and SO relaxation, MD simulations were performed using Gaussian 16 and Amber20 (14) software, as described in the Materials and Methods section. Briefly, the uPPi electronic structure was modeled in Gaussian, then energy minimization was performed over 5000 steps, followed by 20,000 steps of 1 fs to reach the desired temperature and pressure (300 K, 1 bar), and the final production run was performed with an isothermal/isobaric ensemble (NPT, 300 K, 1 bar) with 10 7 steps. The CSA tensors at each 31 P nucleus were then calculated ab initio in Gaussian from 100 randomly selected conformations. Fig. 4 shows average and multiplesnapshot representations of the symmetric portion of the CSA tensors experienced by the 31 P nuclei. As is seen here, the principal component appears almost completely aligned with the bond between phosphorus and the bridging oxygen. Because the -PO3 groups experience fast intramolecular rotation about the bridging P-O bond (see Fig. 4B), the CSA tensors were averaged across the 100 conformations, following molecular alignment along the P-P vector. A more detailed justification for this averaging procedure can be found in the Materials and Methods section. The difference between the average tensors at each 31 P nucleus was computed, and the average and difference tensors were separated into their symmetric and antisymmetric components. The (Frobenius) norms of the tensor components are summarized in Table 1 and were used to calculate the CSA contributions to R1 and RS using the expressions</p><p>In the equations above, 𝜔 % is the Larmor frequency, ‖𝜎‖ & and ‖∆𝜎‖ & indicate the Frobenius norms of the average and difference tensors, respectively, and 𝜏 ! and 𝜏 ' are the first-and second-rank correlation times, respectively, where 𝜏 ! = 3𝜏 ' assuming isotropic motion. The second-rank correlation time was determined to be 48.6 ps, based upon MD simulation following adjustment using the NMR-measured PPi diffusion coefficient, as described in the Materials and Methods section. It is seen that CSA accounts for the major relaxation effect at high magnetic fields. The symmetric CSA component (Fig. 5, solid lines) contributes the most to R1 and RS at high field strengths, whereas the antisymmetric contribution (Fig. 5, dotted lines) is relatively small for both but much larger for RS than it is for R1. Other smaller yet significant relaxation contributions, largely field-independent, are discussed further below. The spin-rotation contribution to R1 was calculated as follows: From MD simulations, the correlation function ω(0)ω(𝑡) for the angular rotation frequency of the -PO3 entity about the bridging P-O bond of PPi was calculated. An exponential fit was performed to this function, which yielded 𝜔(0) ' and the correlation time 𝜏 -. These values were determined as 3.1 rad 2 ps -2 and 0.0255 ps, respectively. Gaussian 16 was used to compute the spinrotation tensor for 31 P in PPi at the B3LYP/aug-cc-pVTZ level, which produced the value for 𝐶 ∥ /2π = 4.424 kHz, for rotation around the bridging P-O vector, and roughly two equivalent values for the perpendicular rotation 𝐶 / /2π = 1.095 kHz. The spin-rotation tensors are visualized in Fig. S3 in the Supplementary Materials, which indicates that the major component of this tensor also points along the bridging P-O bond similar to the CSA tensor. Given that the motion perpendicular to the P-O bond can be assumed to be very small by comparison (see Fig. 4B, showing the superposition of conformers obtained from MD trajectories), we neglect this portion and calculate the spin-rotation relaxation rate constant by the expression</p><p>where 𝐼 || = 1.758×10 -45 kg m 2 is the moment of inertia for the -PO3 entity with respect to the bridging P-O axis. This expression can be derived by combining Eq. ( 22) from McClung (15) with Eq. (4.83) from Spiess (16). The spin-rotation relaxation rate constant then becomes R1,SR = 0.0113 s -1 . The rate is essentially independent of the magnetic field due to the extremely short correlation time for the angular frequency correlation function.</p><p>Spin-rotation is also expected to affect the relaxation of SO in uPPi. We made the following considerations: were the spin-rotation field fluctuations produced by each rotating -PO3 group fully uncorrelated, we would predict RS,SR to be twice as large as R1,SR. However, in this case RS would be larger than R1 at low field strengths, whereas experimentally we observed similar low-field values of R1 and RS. We therefore determined the correlation coefficient 𝛼 for the spin-rotation interaction at each 31 P spin following the discussion about correlated mechanisms of Tayler et al (17), in particular Eq.s (1) and (2). From these considerations, once can obtain 𝑅 ,,,1 /𝑅 !,,1 = 2(1 − 𝛼), and when using the experimental values for 𝑅 ,,,1 and 𝑅 !,,1 we obtain the correlation coefficient 𝛼 = 0.5. Modeling the spinrotation contribution to RS in this manner produced an excellent fit to the experimental data (Fig. 3, dashed line). Other known relaxation contributions to R1 and RS are described below. MD simulations following the procedure of Kharkov et al (18) gave the contribution of intermolecular dipolar relaxation between 31 P and 2 D solvent spins as 5.14×10 -3 s -1 . The 31 P-31 P dipolar relaxation contribution, relevant only for R1, was determined to be 1.60×10 -3 s -1 . The correlation times for these processes range from 20-40 ps, and therefore their contributions are likewise almost completely independent of the magnetic field. The singlettriplet leakage (STL) contribution to SO relaxation cannot easily be determined in closed form, since it depends on the specifics of the relaxation mechanism. This effect was therefore estimated using the Spinach NMR simulation package in MATLAB (12), by simulating SO relaxation with and without the chemical shift difference included and calculating the difference. The contribution is field-dependent but relatively minor, as seen in Fig. 5. Finally, the 1 H-31 P dipolar relaxation contribution arising from the added KOH was estimated from the 2 D-31 P contribution as 0.00025 s -1 , which is negligible compared to other relaxation contributions.</p><!><p>The quantum cognition proposal advanced by Fisher (1) involves multiple components requiring independent validation. One of the most prominent challenges of the proposal in its current form involves assessing whether quantum entanglement can survive for an appreciable duration in a "wet" biological environment, even though some level of protection from the environment may be provided by symmetry in a Posner cluster (1). Our approach to studying entangled spin order in unsymmetrically labeled pyrophosphate represents an important initial step in studying what phenomena most strongly limit the lifetime of quantum entanglement between 31 P nuclei.</p><p>Our R1 and RS measurements show that uPPi high-field relaxation is dominated by the CSA mechanism, similar to the case in other reported diphosphates (8,9). In contrast to previous studies, the RS values observed in the high field regime are slightly lower compared with R1, which correspond well with the symmetric CSA tensor norm being somewhat lower for the difference tensor (Table 1). The norm of the antisymmetric component, however, is significantly larger for the difference tensors than for the individual tensors, with the result being a larger antisymmetric CSA contribution to RS. Still, the antisymmetric contribution to RS is smaller than one fifth of the symmetric contribution.</p><p>Importantly, we observed that towards low fields, a constant offset in relaxation rate constants is approached for the experimentally measured values of both R1 and RS. The offset at the lowest field, 2 𝜇T, was found to be approximately 0.018 s -1 for both. The same trend and a similar, albeit slightly higher R1 and RS offset were observed from measurements on a 30 mM uPPi sample with 10 mM EDTA added (Fig. S4, Supplementary Materials). We believe this constant contribution at the lowest field to be primarily comprised of spinrotation relaxation, as shown in Fig. 5. Furthermore, at very low field strengths (2 µT to 100 mT), R1 showed a peculiar increase in the rate that was consistently observed across different sample formulations (Fig. S4A). This effect is not understood at this time.</p><p>The largest values of the T1 and TS time constants appear to be approximately 65 s for uPPi under our experimental setup (in the low field range). We note, however, that many of the experimental conditions used for our relaxation measurements are different from those that would be encountered in a biological system. First, our experiments were performed at a relatively high pD, to limit deuteron exchange, whereas faster exchange at physiological pD values would be expected to reduce the TS and possibly T1 relaxation times (11). In addition, the nature of the counterion played a role in the relaxation measurements, and the longest T1 times were observed with an excess of K + ions relative to the Na + ions from the synthesized uPPi tetrasodium salt. It is worth noting that the intracellular K + concentration tends to be approximately fourfold higher than Na + , whereas Na + is much more abundant in the extracellular space (19). This finding may suggest that the intracellular environment is more conducive to long-lived quantum entanglement, at least for free pyrophosphate. Furthermore, D2O was used as a solvent rather than H2O. We note that if H2O were used as a solvent, this limit would be significantly smaller. We measured an increase in R1 of 0.028 s -1 at 9.4 T when we replaced D2O with 90% H2O plus 10% D2O. Assuming this increase to be field-independent, we would therefore expect a T1 and TS maximum of approximately 26 s if we were to repeat the field-dependent measurements with this solvent. Finally, certain paramagnetic species are abundant within cells and tissues and can contribute to relaxation. Comparison of rates observed in degassed and non-degassed samples, however, showed approximately the same rate constants in the low field region, suggesting that the effect of paramagnetic relaxation due to oxygen is low (Fig. S4, Supplementary Materials). Other paramagnetic impurities were considered, but careful and extensive cleaning of glassware with KOH/iPrOH and HCl did not produce significant changes. Examination of relaxation in the presence of EDTA (to potentially capture paramagnetic impurities) likewise did not show significant changes in the observed rate constants (Fig. S4, Supplementary Materials).</p><p>In summary, we report measurements of 31 P SO decay in isotope labeling-induced unsymmetric PPi over a wide range of field strengths. We demonstrate that CSA dominates both R1 and RS relaxation at high fields but diminishes at low fields, and that the two rates have similar values from 2 µT to 9.4 T. We observe that both R1 and RS approach a constant value at low field strengths, and that this relaxation appears to be primarily explained by spin-rotation relaxation, with minor (but non-negligible) contributions from intermolecular 31 P-2 D dipolar coupling and intramolecular 31 P-31 P dipolar coupling. The magnitude of the spin-rotation relaxation contribution in this molecular system was an unexpected discovery.</p><p>Our main experimental finding is that the relaxation rates for 31 P longitudinal magnetization and for 31 P nuclear SO are similar for pyrophosphate in solution, with multiple mechanisms contributing to both relaxation processes. Both relaxation times are of the order of 1 minute in low magnetic field under our experimental conditions, and they decrease rapidly as the magnetic field is increased. In low magnetic fields the 31 P singlet lifetime of pyrophosphate is possibly long enough to sustain the hypothesis that such entangled spin pairs might play a role in quantum cognition (1, 2). As far as the authors know, there is no evidence that cognition is significantly disturbed by high magnetic fields, as would be anticipated from the experimental results described here.</p><!><p>Unsymmetrically 18 O-labeled pyrophosphate synthesis and formulation</p><p>The synthesis of 18 O/ 16 O unsymmetrical pyrophosphate tetrasodium salt 6, henceforth referred to as uPPi, is shown in Fig. 6. Light sensitive silver phosphate salt 1 was prepared from 18 O phosphoric acid by a simple precipitation method (20). Subsequent benzylation in the presence of excess benzyl chloride provided the triester 2 in 75% yield (21). Heating triester 2 in the presence of one equivalent of sodium iodide in acetone accomplished selective mono-deprotection (21), and the resulting dibenzyl phosphate sodium salt 3 was converted to the tetrabenzyl 18 O/ 16 O pyrophosphate 4 by reaction with dibenzyl phosphoryl chloride ( 16 O, obtained by the chlorination of dibenzyl phosphite with NCS in benzene and used directly (22)) in the presence of triethylamine (23). Global debenzylation of the tetrabenzyl pyrophosphate using hydrogen over Pd required prolonged reaction times and was inefficient due to accompanying partial hydrolysis to the orthophosphate. Ultimately, a two-step procedure via the dibenzyl pyrophosphate disodium salt 5 was optimised, with the remaining two benzyl groups removed by hydrogenolysis over Pd in the presence of sodium bicarbonate in 5 hours. This six-step sequence afforded the regioselectively O 18 /O 16 labelled pyrophosphate tetrasodium salt 6 as a white crystalline solid. Isotopic incorporation was confirmed by mass spectrometry to be 96% 18 O4, 96% 18 O3. For NMR experiments, the tetrabasic sodium uPPi was formulated as a 30 mM solution in deuterium oxide plus 10 equivalents of potassium hydroxide, in order to minimize proton exchange, which can accelerate singlet relaxation (11), and minimize interactions with sodium ions in solution, which our results seem to indicate also accelerates relaxation of both longitudinal and spin order (Fig. S1, Supplementary Materials). The final concentrations of Na + and K + counterions were 120 mM and 300 mM, respectively. The pD of the solution was expected to be about 14.4, based upon room-temperature pH electrode measurements of a sample prepared identically but with unlabelled tetrabasic sodium pyrophosphate. The NMR tubes used with the samples were carefully cleaned to avoid any paramagnetic impurities by immersing in a KOH/iPrOH bath overnight followed by HCl immersion overnight, rinsing several times with acetone, and drying with argon gas. More details on sample preparation can be found in the Supplementary Materials.</p><!><p>All field-dependent NMR measurements were performed at the University of Southampton. For measurements of R1 via inversion-recovery, the uPPi 31 P populations were inverted with a 180° pulse, the sample was shuttled to a region with the desired magnetic field strength, and then the sample was returned to the bore for excitation with a 90° pulse followed by acquisition. SO was prepared with a SLIC spin-lock pulse at 9.4 T within the bore, the sample was shuttled to a region above the magnet for singlet relaxation at the desired field strength, and then returned to the magnet bore for singlet order readout via SLIC. The sample shuttling speed to and from the low field for all measurements was about 1 m/s, and the shuttling time (one-way) was no greater than 1 second. The sensitivity of singlet-triplet conversion due to transmitter offset during SLIC was mitigated by turning off the temperature regulation within the NMR scanner, in order to minimize the change in temperature between the bore and the shuttling region above the magnet. The probe temperature within the bore was measured to be about 22 °C with the temperature regulation off, and the temperature during sample shuttling was not expected to vary more than ±5 °C from the probe temperature.</p><!><p>MD simulations in Amber20 were performed with the following modifications: PPi was parametrized using ESP charges obtained from Gaussian 16 with B3LYP/6-31G(d), the polyphosphate parameters described by Meagher et al (24), with the missing parameters provided by the GAFF2 force field. Minimization was performed in 5000 steps, Timesteps were 1 fs throughout, and the final isothermal/isobaric ensemble (NPT, 300 K, 1 bar) production run contained 10 7 steps. The simulation was performed at 300 K. 100 snapshots were selected randomly to perform ab initio calculations of CSA tensors with the B3LYP/aug-cc-pVTZ combination and the GIAO method. Fig. S5 in the Supplementary Materials shows the individual tensor norms and eigenvalues of the tensor components for all conformers. To calculate the average CSA tensors across all selected conformations, the molecules were aligned along the P-P vector (i.e. along the x coordinate) with the bridging P-O vector pointing upwards in the x-z plane, as shown in Fig. 4B. The CSA tensors were rotated into this frame and averaged. For the R1 calculation, the Frobenius norms were taken of the symmetric and antisymmetric components of the average tensors. For the RS calculation, the Frobenius norm was calculated for the difference between the average tensors of each 31 P. Tensor visualizations were generated using the Ovaloid function from SpinDynamica v3.6 (25) in Mathematica, as described previously (26,27), and displaying with the MoleculePlot3D function.</p><p>The CSA tensor averaging procedure described above is strictly valid only in the limit where the internal motion is much faster than the overall tumbling rate. We justify its use as follows: from the MD trajectories the root mean square (rms) angular frequency of the -PO3 rotation around the bridging P-O bond is determined as 1.76 rad/ps. From this value, we can calculate the root-mean square rotation of -PO3 within the reorientation correlation time period determined above (48.6 ps) as 13.6π. We therefore can assume that the -PO3 rotation is much faster than the molecular reorientation, so that averaging the tensors for the two 31 P spins prior to taking the differences between them is the correct approach.</p>

ChemRxiv

Noninvasive Imaging and Quantification of Radiotherapy-Induced PD-L1 Upregulation with 89Zr\xe2\x80\x93Df\xe2\x80\x93Atezolizumab

Immune checkpoint expression is highly dynamic, and combination treatments including radiotherapy can particularly modulate this expression. PET imaging using 89Zr\xe2\x80\x93Df\xe2\x80\x93atezolizumab can provide insight into the levels of PD-L1 variation following radiotherapy treatments. In vitro screening was used to monitor PD-L1 expression by lung cancer cells following radiotherapy. Mice bearing PD-L1+ (H460) or PD-L1\xe2\x88\x92 (A549) tumors were subjected to various external beam radiotherapy regimens and then imaged using 89Zr\xe2\x80\x93Df\xe2\x80\x93atezolizumab PET. ROI analysis and ex vivo biodistribution studies were employed to quantify tracer accumulations. H460 cells were found to have PD-L1 expression at baseline, and this expression increased following daily radiotherapy of 5 fractions of 2 Gy. PD-L1 expression could not be induced on A549 cells, regardless of radiotherapy regimen. The increase in PD-L1 expression in H460 tumors following fractionated radiotherapy could be imaged in vivo using 89Zr\xe2\x80\x93Df\xe2\x80\x93atezolizumab, with statistically significant higher tracer accumulation noted in fractionated H460 tumors over that in all other H460 or A549 groups after 72 h postinjection of the tracer. Significant accumulation of the tracer was also noted in other PD-L1+ organs, including the spleen and lymph nodes. Ex vivo staining of tumor tissues verified that tumor cells as well as tumor-infiltrating immune cells were responsible for increased PD-L1 expression after radiotherapy in tumor tissues. Overall, PD-L1 expression can be modulated with radiotherapy interventions, and 89Zr\xe2\x80\x93Df\xe2\x80\x93atezolizumab is able to noninvasively monitor these changes in preclinical models.

noninvasive_imaging_and_quantification_of_radiotherapy-induced_pd-l1_upregulation_with_89zr\xe2\x80\

INTRODUCTION<!>In Vitro PD-L1 Expression Analysis.<!>PET Imaging Visualizes PD-L1 Expression Changes.<!>Ex Vivo Verification of in Vivo Trends.<!>DISCUSSION<!>Cell Culture.<!>In Vitro PD-L1 Expression Studies.<!>Animal Models.<!>Radiation Treatments.<!>PET Tracer Preparation.<!>PET Imaging and Biodistribution Studies.<!>Ex Vivo Verification.<!>Statistical Analysis.

<p>Although immune checkpoint treatments have shown promising efficacy, the problems of resistance and relapse often require their combination with other treatment options.1 In particular, combinations of immunotherapy and radiotherapy have enabled systematic treatment of many cancers.2,3 Although synergistic effects have been noted with this combination, the mechanisms and dynamic processes involved are still largely a mystery.</p><p>The programmed death protein 1 (PD-1) pathway, in particular, has been implicated as important in the synergy of radiotherapy and immunotherapy.4,5 Consistent with the inflammation that results from radiotherapy, programmed death protein ligand 1 (PD-L1) is upregulated on irradiated tumor tissues and, if left unchecked, has been shown to contribute to radiotherapy resistance. Blockade of the PD-1/PD-L1 pathway in combination with radiotherapy can reduce the presence of tumor-infiltrating myeloid-derived suppressor cells in order to maintain T-cell activity,4,6 and such trends have been demonstrated in a wide variety of cancer types.7,8 Because the majority of cancer patients receive some form of radiotherapy, a greater understanding of synergistic therapies is greatly warranted, in order to increase the proportion of patients receiving curative treatments.</p><p>Currently, PD-L1 status is determined through biopsy and immunohistochemistry analysis; however, it is becoming increasingly clear that immune checkpoint targets are highly dynamic, and single time-point biopsies cannot provide adequate information on their expression throughout a treatment regimen. Therefore, techniques such as molecular imaging are increasingly being applied to provide real-time, longitudinal information about the expression of these targets,9 complementing existing immunohistochemical techniques. Recent clinical studies have verified the potential of PD-L1 PET imaging in cancer patients, finding correlations with patient outcomes and tracer accumulation levels.10,11 Enabling visualization of these molecules' expression and their changes with different therapies will therefore certainly provide scientific insight into the mechanisms of synergy but also may help guide more rational treatment decisions for cancer patients. We herein therefore developed a PD-L1-targeting positron emission tomography (PET) tracer, reactive to both human and murine PD-L1, and demonstrated that we can image clinically relevant changes in tumor PD-L1 expression following radiotherapy, even in the presence of high uptake in lymphatic organs.</p><!><p>Screening of H460 and A549 lung cancer cells revealed notable expression of PD-L1 at baseline by H460 cells that was absent in the other line (Figure 1). Therefore, H460 cells formed the basis for the majority of these studies, and A549 cells served as a negative control. Following irradiation of H460 cells in vitro, Western blot analysis revealed upregulated PD-L1 expression in the 2 Gy × 5 Fx group (Figure 2). An over 4-fold increase in the PD-L1/β-actin ratio was observed at 24 h after completion of this fractionated regimen. Similar levels of PD-L1 were measured in the 5 Gy × 1 Fx group and the control, indicating that fractionated radiotherapy was more effective at inducing PD-L1. These findings were mimicked in flow cytometry analyses as well, with a shift toward higher PD-L1 staining noted after fractionated radiotherapy.</p><!><p>Following completion of the respective radiotherapy regimens, 24 h later, mice were administered 89Zr–Df–atezolizumab through tail vein injection. Serial PET scans were then conducted to visualize the distribution of PD-L1 expressing tissues.</p><p>Several trends were evident following analysis of H460-bearing mouse images. Most notably, the PD-L1 tracer accumulated to a very high level in the spleen (18–19%ID/g at 96 h) and lymph nodes (8–12%ID/g at 96 h) of all tumor-bearing mice, to a similar extent regardless of the radiotherapy treatment arm (Figures 3 and S2, n = 4–5). This enabled clear visualization of the entire lymph node network with high contrast, especially at later time points. The uptake of the tracer in all other normal organs was similar across all groups and below 10%ID/g at 96 h.</p><p>Given the high, specific uptake of the tracer in the lymphatic organs, the absolute amount of tracer binding in H460 tumor tissues was low. However, significant differences in the tumor accumulation were seen following the different treatment schedules. In nonirradiated mice, tumor uptake peaked at 24 h postinjection, at 2.10 ± 0.52%ID/g. In contrast, in the 5 Gy × 1 Fx group, the accumulation was 2.44 ± 1.18%ID/g at the same time point, and tumor uptake of 4.44 ± 1.52%ID/g was measured for the 2 Gy × 5 Fx mice. At the 24 h scan, this corresponded to a statistically significant higher tracer accumulation in 2 Gy × 5 Fx mice as compared to the nonirradiated group (p < 0.05, n = 4–5). Although the highest absolute uptake in the tumors was observed at the 24 h scan, peak tumor-to-muscle ratios (TMRs) were calculated at the final imaging time point, 96 h. Mice receiving the 2 Gy × 5 Fx regimen had the highest TMRs at 3.59 ± 1.53, compared to the 5 Gy × 1 Fx group at 2.59 ± 1.48 and nonirradiated mice at 2.33 ± 0.81.</p><p>As a negative control, mice bearing A549 tumors were also employed to determine the accumulation level of atezolizumab in tumor tissues not resulting from tumor cell expression of PD-L1 (Figures 4 and 5). This control was determined to be most appropriate, because many traditional control experiments (blocking, nonspecific IgG) were not applicable to this model (see Discussion section). From PET ROI analysis, nonirradiated A549 xenografts displayed the highest uptake of 89Zr–Df–atezolizumab at 12 h postinjection, at 2.38 ± 0.67% ID/g. When irradiated with 5 Gy in a single fraction, the peak A549 tumor accumulation was noted at 24 h, peaking at 2.10 ± 0.97%ID/g. In mice receiving fractionated radiotherapy, a peak tumor uptake of 1.64 ± 0.65%ID/g was calculated at 24 h postinjection as well. This provides support to a PD-L1-mediated mechanism for the increase in uptake of 89Zr–Df–atezolizumab in H460 tumors, because the accumulation at the same time point (24 h) in these tumors after fractionated radiotherapy was 4.44 ± 1.53%ID/g, a statistically significant difference (p < 0.02, n = 4–5). In fact, the A549 tumor uptake in both irradiated and nonirradiated mice was nearly identical to that of nonirradiated H460 xenografts at all time points, indicating that this ~2%ID/g can be attributed to nonspecific accumulation in tumor regions, likely because of the enhanced permeability and retention effect. No notable difference was noted for other organs and tissues between the two groups of tumor-bearing mice, as seen in the gamma counting biodistribution studies.</p><!><p>Ex vivo biodistribution studies verified the trends found through analysis of PET images of 89Zr–Df–atezolizumab (Figure 5d). The highest uptake of the tracer was noted in the spleen (33–38%ID/g) in all groups, followed by the lymph nodes (13–25%ID/g). Notably, the uptake in these tissues measured by gamma counting was higher than that measured through PET ROI analysis, likely because of partial volume effects in the ROI measurements. Low levels of accumulation were also noted in the liver (7–8%ID/g), as this is the clearance organ for antibody-based tracers. H460 tumor uptake was significantly higher in the group receiving 2 Gy × 5 Fx, at 3.38 ± 0.66%ID/g, compared to 2.18 ± 0.80 and 1.51 ± 0.61%ID/g for the 5 Gy × 1 Fx and nonirradiated groups, respectively (p < 0.05, n = 4–5). Similar tumor accumulations were noted for all of the A549 groups: 1.42 ± 0.53%ID/g for nonirradiated, 2.10 ± 0.79%ID/g for 5 Gy × 1 Fx, and 1.69 ± 0.76%ID/g for 2 Gy × 5 Fx.</p><p>Radioactivity in irradiated H460 tumors was statistically higher, as expected; however, consistently higher accumulation of the tracer was also noted in the bones of mice receiving the fractionated radiotherapy treatment, regardless of tumor type, as measured by both PET ROI analysis and ex vivo biodistribution (Figure S5). The largest differences were noted in those bones and joints that were within the radiotherapy field (i.e., the hips and coccyx regions), indicating a possible PD-L1-mediated mechanism, rather than instability of the tracer. The bone uptake values calculated from gamma counting and PET ROI analysis were well-matched, signaling minimal impact of the partial volume effect on these measurements. No other clear trends were noted in the normal tissue distribution of 89Zr–Df–atezolizumab with regard to radiotherapy regimen.</p><p>Tissues of significant tracer uptake were also excised at the time of necropsy, including the spleen and tumors (Figures 6, 7, S3, and S4). Immunofluorescent staining of these tissues verified the expression of PD-L1 and therefore specific binding of 89Zr–Df–atezolizumab. Notably, the intensity of PD-L1 staining was higher in the lymphoid organs compared to H460 tumors but also more heterogeneous. PD-L1 appeared to be expressed by a subpopulation of the cells in the spleen, with much colocalization with CD45 expression correlating with immune cell expression of PD-L1. In nonirradiated tumor tissues, some infiltrating myeloid cells were visualized and correlated with selected PD-L1 expression. Following irradiation, an increase in CD45+ cells was observed, especially around the periphery of the tumor tissues (Figures S4 and 6). However, PD-L1 staining was observed in tumor tissues that also did not overlay with CD45 or F4/80, indicating tumor cell expression. Additionally, the morphology of CD45+ cells was clearly different than that of tumor cells themselves, with most CD45+ cells presenting as circular, whereas tumor cells were more elongated and abnormal shapes. The PD-L1 staining in irradiated tumors was more uniform than that observed in nonirradiated tissues. A549 tumors did not reveal any notable PD-L1 staining, regardless of radiotherapy treatment.</p><!><p>Although the value of PD-L1 as a prognostic or predictive marker is still under debate,12,13 the importance of monitoring changing biomarker expressions throughout treatment is well-recognized.14,15 Imaging of T-cell-related targets, such as PD-1, has been widely explored preclinically, and initial clinical studies are underway.9 Recent clinical studies have additionally indicated that PD-L1 imaging may be an excellent predictor of patient response to immune checkpoint blockade, even more so than the clinical standard of biopsy analysis.10 Additionally, PD-L1 is widely known to be upregulated in tumor tissues following radiotherapy interventions, likely resulting from the inflammation caused by radiotherapy.16 Treatment with radiotherapy alone, as a result, often does not lead to complete tumor regression because of these immunosuppressive effects. However, combination of blockade of the PD-1/PD-L1 axis with local radiotherapy has led to impressive primary and secondary tumor responses.4,6,17 Not all tumors exhibit this enhancement of PD-L1 expression following radiotherapy, however, and the accompanying window of opportunity for immunotherapy intervention. Identifying patients with PD-L1+ biomarker status in both primary and metastatic tumor sites may help allot them to the proper combination treatment of radiotherapy and immune checkpoint blockade.</p><p>We have therefore demonstrated that noninvasive PET imaging can monitor the dynamic expression of PD-L1 in naïve preclinical subjects as well as following radiation treatment. Monitoring changes in PD-L1 expression in real-time has important implications clinically, especially for the optimization of synergistic therapy regimens.1,4,5 PD-L1 PET therefore not only has value as a diagnostic agent for simple tumor detection but also as a means of monitoring tumor response and allocating patients to proper therapies.</p><p>A number of studies have investigated tumor detection through molecular imaging based upon PD-L1 expression, often using murine models.18–25 The present study varies from these previous ones, particularly those noninvasively monitoring PD-L1 dynamics, on a few key points. First, we utilized an anti-human PD-L1 antibody that also cross-reacts with murine PD-L1. This means that these results should be more easily clinically translated than certain other studies that use entirely murine systems, and we also have representative off-target binding of the tracer to lymphatic organs. This off-target binding will certainly be seen in patients and thus needs to be considered in preclinical studies, which in some cases use human-specific antibodies (which therefore do not bind to the analogous murine molecules). Second, our positive cell line herein (H460) only expresses native levels of PD-L1, rather than being engineered to express high, unnatural levels of the protein—a strategy that has been employed in some past studies. Although this expression level is low compared to lymphatic organs' PD-L1 levels (spleen and lymph nodes), we are still able to visualize the tumor burden by PET after therapeutic intervention. Finally, many other studies, both preclinical and clinical, employ a predosing strategy to minimize this off-target lymphatic uptake by administering excess cold anti-PD-L1 antibody either before the tracer injection or as a coinjection.26 Even within the present study, PD-L1-specific uptake of the tracer was found to be highly dependent on the administered protein doses. Although this technique does provide higher tumor uptake and contrast, such predosing has the possibility of inducing a pharmacologic response, which is not desired for an imaging tracer. We only administered the radiolabeled protein, at a low per-mouse level, in order to avoid the chance of a pharmacologic response, even if it is not altogether common. Indeed, administering the lowest amount of protein to a patient, while achieving sufficient imaging signal, is ideal.</p><p>As aforementioned, PD-L1 imaging is more complicated than imaging other traditional tumor markers. Because PD-L1 is so widely expressed, not only is the imaging signal in the tumor reduced but also the traditionally used control experiments cannot be applied. For instance, because administration of a blocking dose would saturate the PD-L1 found in the spleen and lymph nodes, this technique would be expected to actually result in a higher tracer accumulation in the tumor and would not prove specificity for the cancer cells as in traditional studies. Additionally, another control technique is the use of a nonspecific, isotype-matched antibody. However, a nonspecific antibody would not bind to the PD-L1 that is ubiquitous throughout the body and may actually accumulate once again to a higher level in the tumor tissue. We have tested a control 89Zr–Df–IgG in nude mice bearing A549 tumors in other studies, for example, and achieved 5.05 ± 1.70%ID/g tumor uptake at 120 h postinjection.27 This is higher than any tumor uptake observed in these studies; therefore, these controls would not be helpful. For this reason, we employed the A549 xenografts as a control, because PD-L1 expression cannot be induced on them with the radiotherapy regimens employed here. This would provide a measure of the accumulation of atezolizumab in tumor tissues that do not express PD-L1 while maintaining the same background uptake in lymphoid organs. Additionally, this provides a verification that, for instance, disruption of blood vessel architecture by radiotherapy is not to blame for increased tumor accumulation of 89Zr–Df–atezolizumab.</p><p>The absolute uptake of 89Zr–Df–atezolizumab in the H460 tumor tissues was low (1–5%ID/g), even in the 2 Gy × 5 Fx group, when compared to that of cancer PET tracers targeted to markers other than PD-L1. As partially mentioned before, this is likely due to a few factors. Most importantly, the expression of PD-L1 is not limited to the tumor tissue, and several other tissues (including the spleen, lymph nodes, and brown fat) represent sinks for the tracer. Additionally, the number of cells expressing PD-L1, or even the number of copies of PD-L1 per cell, is lower than that of other commonly used imaging targets. Notably, the cutoff for PD-L1 positivity in a tumor is often that 1–5% of the total cells express the target,28 compared to, for example, the definitions of Her-2 positivity (>10% of cells).29 Even though the absolute uptake is low and there is notable off-target binding, high tumor-to-muscle ratios allowed clear visualization of irradiated tumors, especially at later time points. This is a well-recognized benefit of 89Zr-labeled antibodies—the long radioactive half-life allows for high-contrast imaging after the antibody has cleared from nonspecific binding.</p><p>The results obtained herein may not be generally applicable to all cancer types, models, and treatments, however. The expression of PD-L1 is expected to change in different cell lines and with different treatment regimens; therefore, future studies should fully explore these options. The potential of PD-L1 PET to monitor changes in normal tissue PD-L1 expression and the implications of these changes for the system-wide immune state would be an additional interesting future avenue for exploration. The present study, in particular, showed interesting changes in the bone uptake of 89Zr–Df–atezolizumab after the various radiotherapy interventions, which merits further mechanistic investigation.</p><p>The dynamic nature of immune checkpoint molecules is beginning to be realized. There is therefore a great need to longitudinally monitor this expression in cancer patients receiving any number of treatments. We have herein demonstrated that PET of tumor PD-L1 expression using 89Zr–Df–atezolizumab is able to monitor changes in PD-L1 expression following various radiotherapy regimens. Such techniques may find application clinically for monitoring patient responses and determining proper therapies, giving clinicians another tool in their personalized medicine arsenal.</p><!><p>H460 and A549 cells were purchased from the American Type Culture Collection and maintained in Roswell Park Memorial Institute-1640 or Dulbecco's Modified Eagle's medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin in a 37 °C humidified incubator with 5% CO2. For all studies, cells were utilized at 60–70% confluence.</p><!><p>To monitor changes in PD-L1 protein levels and expression, several in vitro studies were performed. To measure protein concentrations, Western blot analysis was employed. Cells were plated into T25 flasks and supplemented with 10 mL of media each (corresponding to approximately 2–3 mm of media above the cell layer). For the first study, protein was simply extracted from H460 and A549 cells at 60% confluence to explore baseline PD-L1 levels.</p><p>The cells were then subjected to one of three radiotherapy regimens: 5 Fx of 2 Gy each; 1 Fx of 5 Gy; or no radiation (but still the sham procedure of being removed from the incubator, etc.). These treatments were administered using an XRAD320 biological irradiator (Precision X-ray), and fractionated treatments were administered 24 ± 1 h apart. Protein was then extracted from the cells at the completion of their respective schedules at 24 h after completion. Cells were lysed using radioimmunoprecipitation assay buffer (Boston Bio-Products) supplemented with 1:100 Halt Inhibitor and EDTA (ThermoFisher Scientific). Supernatant protein concentration was measured using the NanoDrop One (ThermoFisher Scientific). Western blotting was performed using standard procedures,30 with the following reagents: Chameleon Duo ladder protein marker (LI-COR Biosciences), anti-hB7-H1 antibody (R&D Systems), anti-β-actin antibody (Novus Biologicals), donkey anti-goat IRDye 800CW, and donkey anti-mouse IRDye 680RD (LI-COR Biosciences). The final prepared membrane was scanned using a LI-COR Odyssey infrared imaging system (LI-COR Biosciences).</p><p>Verification of tracer binding was performed using flow cytometry. The binding properties of both atezolizumab and Df–atezolizumab to irradiated H460 cells were analyzed at 48 h after the fractionated irradiation. Preparation of cells for flow cytometry and analysis were conducted using standard procedures.30 Samples were run on a MacsQuant cytometer (Miltenyi Biotec) and analyzed with FlowJo V10 (FlowJo LLC).</p><!><p>All animal studies were conducted under an approved protocol by the Institutional Animal Care and Use Committee. Lung cancer xenograft models were generated by inoculating 4–6 week-old female athymic nude mice (Envigo) with a 1:1 mixture of H460 or A549 cells in Matrigel Matrix Basement Membrane (Corning) subcutaneously in the lower flank. When tumors reached 5–8 mm in diameter, mice were used for subsequent studies.</p><!><p>Similar to cell studies, three radiotherapy regimens were administered to H460- or A549-bearing mice (n = 4–5 per group) using the XRAD320 irradiator: 5 daily fractions of 2 Gy (2 Gy × 5 Fx), one fraction of 5 Gy (5 Gy tme 1 Fx), or no radiation. Prior to irradiation, mice were placed and taped in the prone position in lead body shields (Figure S1) to minimize radiation exposure to organs other than the tumor. Mice were irradiated one at a time, and the X-ray field was collimated to leave ~0.5 cm margins on each side of the unshielded area of the mouse.</p><!><p>Atezolizumab (Genentech Oncology) was obtained in its clinically available IV-injectable form and run through a PD-10 column (GE Healthcare) with phosphate-buffered saline mobile phase to remove any stabilizers. The antibody was then conjugated with desferrioxamine (Df, Macrocyclics) using previously reported methods in preparation for radiolabeling with 89Zr.31,32 89Zr (t1/2: 78.4 h) was produced through proton irradiation of yttrium foils.33 For development of the radiolabeled tracer, Df–atezolizumab was mixed with 89Zr–oxalate at a ratio of 50 μg of protein to 37 MBq of radionuclide and incubated for 1 h at 37 °C. PD-10 columns were then used to purify the reactants and products and formulate the final tracer into phosphate-buffered saline for injection.</p><!><p>One day after completion of the respective radiotherapy regimens, mice were intravenously injected with 4–9 megabecquerels (5–12 μg) of 89Zr–Df–atezolizumab. PET scans were acquired using an Inveon microPET/CT scanner (Siemens) at 1, 6, 12, 24, 48, 72, and 96 h postinjection, with 20 000 000 counts per mouse obtained at each time point. OSEM/3DMAP reconstructions were employed. After the 96 h scan, mice were euthanized through CO2 asphyxiation, and major organs were removed, wet-weighed, and counted using an automated gamma counter (PerkinElmer). Quantitative data from these studies are presented as percent of injected dose per gram of tissue (%ID/g), mean ± standard deviation.</p><!><p>Organs of significant tracer uptake, including the spleen, lymph nodes, and tumors, were excised from mice, embedded in TissueTek Optimal Cutting Temperature Compound (Sakura), sliced, and mounted for immunofluorescent analysis. Using the human anti-PD-L1 antibody atezolizumab (because mouse and human PD-L1 share structural similarities), rat anti-mouse F4/80 (for macrophages), and mouse anti-mouse CD45 (hematopoietic cells) primary antibodies, the tissues were stained to determine which cells express PD-L1. Secondary antibodies were used to complete the staining using standard procedures:34 donkey anti-rat Cy3, donkey anti-human DyLight650, and goat anti-mouse AlexaFluor488. Slides were then mounted with DAPI-containing hard mount (Vector Laboratories) and cover-slipped. Confocal imaging was performed using a Nikon A1RS microscope (Nikon Corporation).</p><!><p>For the statistical analysis in this study (PET imaging and biodistribution), with five mice per group, a Student's t test, which can detect a difference of 1.68 standard deviations with 80% power at 5% significance level was utilized (p < 0.05, two-sided).</p>

PubMed Author Manuscript

Impacts of simulated erosion and soil amendments on greenhouse gas fluxes and maize yield in Miamian soil of central Ohio

Erosion-induced topsoil loss is a threat to sustainable productivity. Topsoil removal from, or added to, the existing surface is an efficient technique to simulate on-site soil erosion and deposition. A 15-year simulated erosion was conducted at Waterman Farm of Ohio State University to assess impacts of topsoil depth on greenhouse gas (GHG) emissions and maize yield. Three topsoil treatments were investigated: 20 cm topsoil removal, 20 cm topsoil addition, and undisturbed control. Results show that the average global warming potential (GWP) (Mg CO 2 Eq ha −1 growing season −1 ) from the topsoil removal plot (18.07) exhibited roughly the same value as that from the undisturbed control plot (18.11), but declined evidently from the topsoil addition plot (10.58). Maize yield decreased by 51% at the topsoil removal plot, while increased by 47% at the topsoil addition plot, when compared with the undisturbed control (7.45 Mg ha −1 ). The average GWP of erosion-deposition process was 21% lower than that of the undisturbed control, but that greenhouse gas intensity (GHGI) was 22% higher due to lower yields from the topsoil removal plot. Organic manure application enhanced GWP by 15%, and promoted maize yield by 18%, but brought a small reduction GHGI (3%) against the N-fertilizer application.

impacts_of_simulated_erosion_and_soil_amendments_on_greenhouse_gas_fluxes_and_maize_yield_in_miamian

<!>Results<!>Discussion<!>Methods

<p>In agricultural ecosystem, sustainable food production and mitigation of greenhouse gas (GHG) emissions have been concerned by agricultural or environmental scientists, especially under future climate conditions. Accelerated erosion is one of the most prevalent forms of soil degradation in the world 1,2 , which poses major threat to food security 3 and a significant impact on GHG emissions 4 . Erosion translocate sediment and soil organic C laterally across landscapes (0.5-0.6 Pg C year −1 ) 5 , potentially causing approximately 0.8-1.2 Gt C year −1 emissions into the atmosphere, while burying 0.4-0.6 Gt C year −1 by deposition processes 2 . Although erosion-induced GHG emissions are dominated by CO 2 , the fluxes of CH 4 and N 2 O are also considerable 6 . IPCC (Intergovernmental Panel on Climate Change) (2007) stated 7 that the emissions of N 2 O and CH 4 have global warming potentials (GWPs) of 310 and 21 times that of CO 2 , which have not yet been adequately investigated. Soil erosion comprises three stages: detachment, transport/redistribution, and deposition 8 . The first two stages, detachment and transportation, lead to increased mineralization and emission of CO 2 . However, the prevalence of anaerobic conditions at the depositional stage reduces the emission of CO 2 and leads to flux of CH 4 and N 2 O 9 . Up to now, there is no systematically assessment on grain yield and GHG emissions under soil erosion-deposition events.</p><p>In fact, it is difficult to detect the decline of productivity that results from erosion directly, because the productivity reduction caused by erosion often occurs so slowly that it may not be recognized until crop production is no longer economically viable 10 . Moreover, improved technology often masks productivity decline caused by erosion, leading to increased rather than decreased yields 4,10 . Various indirect methods (e.g., the comparative-plot method, transect method, and desurfacing experiments) have been carried out extensively in the study of erosion-productivity relationships 10 . The simplest method is to artificially remove topsoil (which is also referred to as desurfacing experiments 10,11 ). Previous studies have reported that the yield reduction rate was faster after the top 40 cm soil was eroded, but became slower if the deeper soil was lost 12 . Moreover, desurfacing approach can also help eliminate the inherent variability of topsoil depth and landscape position 13 .</p><p>Restoration of degraded soils is a high priority in global scale 4 . When soil carbon pool in degraded cropland increased by one ton, crop yield would increase by 20-40 kg ha −1 for wheat, 10-20 kg ha −1 for maize, and 0.5-1 kg ha −1 for cowpeas 2 . Substantial studies have reported the restored productivity of de-surfaced soils by amending with fertilizer or manure [14][15][16] .</p><p>In this study, we investigated the impacts of simulated soil erosion-deposition (after 15 years of establishment) on greenhouse gas (GHG; CO 2 , N 2 O, and CH 4 ) emissions and maize yield, via topsoil depth (TSD) removal and addition 8,17 treatments during the growing season, under N-fertilizer and organic manure amendments with no-till management.</p><!><p>Soil temperature, moisture and GHG emissions. Figure 1 displays the diurnal air temperature and precipitation distribution in 2012; the inset shows the cumulative monthly precipitation and mean monthly temperature in 2012 (OARDC). Mean air temperature showed an increase from April (11.55 °C) to July (26.53 °C), followed by a decrease thereafter. Monthly cumulative precipitation varied between 3.69 and 7.96 cm from April to September, with the highest precipitation in September and lowest precipitation in August.</p><p>Figure 2 shows the variation of soil temperature and soil moisture content at 0-10 cm impacted by simulated erosion under N-fertilizer and organic manure application during the growing season in 2012. Like air temperature, soil temperature showed an increase from April to July, followed by a decrease thereafter. During the growing season, mean soil temperatures for the three TSD treatments were 25.65, 24.75, and 24.92 °C respectively for top soil removal, undisturbed control, and topsoil addition. Obviously higher soil temperature trend was observed for topsoil removal under N-fertilizer application. While, mean soil moisture content for topsoil removal was significantly higher than other two TSD treatments (P = 0.0372) at 10 cm depth.</p><p>Figure 3 shows the effects of simulated erosion on CO 2 fluxes under N-fertilizer and organic manure application during the growing season in 2012. All three TSD treatments were persistent CO 2 sources both for N-fertilizer and organic manure application during the study. Soil CO 2 fluxes differed among seasons, which stirred by fertilizer application and reached the maxima at the peak of air temperature and corn growth 18 . Under N-fertilizer application, CO 2 fluxes in all three TSD treatments were below 4 g C m −2 d −1 from April 6 th to June 8 th , 2012. CO 2 fluxes increased sharply from July to August, and observed the maximum fluxes (g C m −2 d −1 ) on August 7 th (9.22), July 3 rd (8.98) and July 20 th (4.55) respectively for topsoil removal, undisturbed control and topsoil addition. For organic manure application, peak CO 2 fluxes (g C m −2 d −1 ) appeared on July 3 rd , which were 10.4, 15.54 and 8.94 respectively for topsoil removal, undisturbed control and topsoil addition. For N-fertilizer application, there was a 19% increase in the topsoil removal treatment and a 67% decrease in the topsoil addition treatment compared with undisturbed control (Table 1). For soil receiving organic manure, no significant difference observed among the three TSD treatments (Table 1). Average cumulative CO 2 emissions for the entire growing season significantly differed among the three TSD treatments (P = 0.0003) (Table 2).</p><p>Figure 3 shows the effects of simulated erosion on N 2 O fluxes under N-fertilizer and organic manure application during the growing season in 2012. Three TSD treatments were also persistent sources of N 2 O fluxes both for receiving N-fertilizer and organic manure during the study. Soil N 2 O fluxes also displayed a small increase on May 10 th after the fertilizer application, and a peak flux coincided with the peak air and soil temperatures in July. The results indicate that the CO 2 and N 2 O fluxes were stimulated by fertilizer application and soil temperature rise. N 2 O fluxes fluctuated below 3 mg N m −2 d −1 from April to June, and rose rapidly from June 8 th , got the maxima fluxes on July 3 rd both under N-fertilizer application (7.35, 6.59 and 3.90 mg N m −2 d −1 respectively for topsoil removal, undisturbed control and topsoil addition) and organic manure application (8.02, 6.70 and 4.71 mg N m −2 d −1 respectively for topsoil removal, undisturbed control and topsoil addition), then declined to the original level (<3 mg N m −2 d −1 ) thereafter until September (Fig. 3). Our result agree with the statement that the N 2 O emission often characterized by a short time of very high flux rates that make up a substantial part of the total annual loss 19 . Under N-fertilizer application, cumulative N 2 O emission decreased by 19% and 42% respectively for topsoil removal and topsoil addition treatments, compared with the undisturbed control (Table 1). For organic manure application, cumulative N 2 O emission increased by 16% for topsoil removal compared with the undisturbed control, and no significant difference was observed between the undisturbed control and topsoil addition (Table 1). Average cumulative N 2 O emissions for the entire growing season significantly differed among the three TSD treatments (P < 0.0001) (Table 2).</p><p>Figure 3 shows the effects of simulated erosion on CH 4 fluxes under N-fertilizer and organic manure application during the growing season in 2012. Under N-fertilizer application, CH 4 fluxes in the three TSD treatments were generally low (i.e., averaged <2 mg C m −2 d −1 ) and broadly taken up during the growing season. While, under organic manure application, CH 4 fluxes fluctuated widely, and a peak positive CH 4 flux (5.68 mg C m −2 d −1 ) observed on July 3 rd for topsoil removal. For N-fertilizer application, all three TSD treatments were net CH 4 sinks from the atmosphere during the growing season (Table 1). Cumulative CH 4 uptake decreased for both topsoil removal (by 49%) and topsoil addition (by 67%) compared with the undisturbed control (−1.78 kg C ha −1 growing season −1 ) (Table 1). Under organic manure application, topsoil removal was net CH 4 source, and two other TSD treatments were net CH 4 sinks during the growing season, cumulative CH 4 emission increased by 174% for topsoil removal compared with the undisturbed control (−0.77 kg C ha −1 growing season −1 ) (Table 1). Average cumulative CH 4 emissions significantly differed among the three TSD treatments (p < 0.0001) for the entire growing season (Table 2).</p><p>Soil temperatures were negatively correlated with soil moisture contents both under N-fertilizer application (P = 0.0232, 0.0397 and 0.0046 respectively for topsoil removal, undisturbed control and topsoil addition), and organic manure application (P = 0.0350 for topsoil addition). Soil temperature was positively correlated with CO 2 fluxes (P = 0.0787) and N 2 O fluxes (P = 0.0918) from the topsoil removed plot under N-fertilizer application. Similar and much stronger correlations were also found between soil temperature and CO 2 fluxes (P = 0.0138 and 0.0453 respectively for topsoil removal and undisturbed control) as well with N 2 O fluxes (P = 0.0194 and 0.0615 respectively for topsoil removal and undisturbed control) under organic manure application. CO 2 fluxes were positively correlated with N 2 O flux both under N-fertilizer (P = 0.0073, 0.0002 and 0.0024 respectively for topsoil removal, topsoil addition and undisturbed control) and organic manure application P = 0.0025 and 0.0023 respectively for topsoil removal and topsoil addition; P < 0.0001 for undisturbed control). The CH 4 fluxes were negatively correlated with soil temperature both under N-fertilizer application (P = 0.0466 for undisturbed, P = 0.0255 for topsoil addition) and organic manure application (P = 0.0734 for topsoil addition).</p><p>Soil bulk density and SOC, total N content. Figure 4 shows the soil bulk density affected by simulated soil erosion under N-fertilizer and organic manure application. Soil bulk density was higher for topsoil removal and lower for topsoil addition at every soil layer depth from 0-40 cm, significant difference were observed among the three TSD treatments both under N-fertilizer (at 20 and 40 cm soil layer depth), and organic manure (20 cm soil layer depth) application. Soil bulk density for soil receiving organic manure was lower than soil with N-fertilizer application at every soil layer depth from 0-40 cm, with significant difference observed at 0-10 cm soil layer depth. Eq Mg grain yield growing season −1 ) = GWP/grain yield Figure 5 shows the SOC and total N content affected by simulated erosion under N-fertilizer and organic manure application. Significant differences of SOC and total N at the top soil layers displayed inverse patterns from that at the lower layer. At the soil layer of 0-10 and 10-20 cm, the SOC and total N were in the order of undisturbed control > topsoil addition > topsoil removal, while in reverse order of topsoil addition > undisturbed control > topsoil removal at 30-40 cm soil layer depth. For topsoil addition, the migrated topsoil might lose part of C from decomposition due to soil disturbance 20,21 ; meanwhile, the former topsoil buried under the plough depth was preserved from decomposition and mineralization [22][23][24] . Soil receiving organic manure had higher SOC and total N content than soil with N-fertilizer application at every soil layer depth from 0-40 cm.</p><p>Soil GWP, GHGI and maize yield. The GWP (Table 1) increased by 16% for topsoil removal and decreased by 64% for topsoil addition compared with the undisturbed control (17.36 Mg CO 2 Eq ha −1 growing season −1 ) under N-fertilizer application; and GWP decreased by 14% and 21% respectively for topsoil removal and topsoil addition compared with the undisturbed control (19.01 Mg CO 2 Eq ha −1 growing season −1 ) under organic manure application. The mean GWP for topsoil removal and topsoil addition decreased by 24% under N-fertilizer application, and decreased by 18% under organic manure application, compared with the undisturbed control.</p><p>For N-fertilizer application, the GHGI (Table 1) increased by 113% for topsoil removal and decreased by 71% for topsoil addition, compared with the undisturbed control (2.40 Mg CO 2 Eq Mg −1 grain yield growing season −1 ); the average GHGI of the topsoil removal and topsoil addition was 21% higher than that of the undisturbed control. Under organic manure application, the GHGI increased by 101% for topsoil removal and decreased by 53% for topsoil addition, compared with the undisturbed control (2.48 Mg CO 2 Eq Mg −1 grain yield growing season −1 ); the average GHGI of the topsoil removal and topsoil addition was 24% higher than that of the undisturbed control (Table 1).</p><p>Figure 6 shows the grain yield and above ground residue affected by simulated erosion under N-fertilizer and organic manure application in 2012. The grain yield significantly decreased (by 45% under N-fertilizer, by 57% under organic manure) for topsoil removal, while increased (by 24% under N-fertilizer, by 69% under organic manure) for topsoil addition compared with the undisturbed control (7.23 Mg ha −1 under N-fertilizer, 7.67 Mg ha −1 under organic manure). The average grain yield of topsoil removal and topsoil addition decreased by 10% under N-fertilizer application, and increased by 6% under organic manure application, compared with the undisturbed control (7.23 Mg ha −1 under N-fertilizer, 7.67 Mg ha −1 under organic manure). The above ground residue decreased (by 28% under N-fertilizer, by 54% under organic manure) for topsoil removal, and increased (by 23% under N-fertilizer, by 41% under organic manure) for topsoil addition, compared with the undisturbed control (6.31 Mg ha −1 under N-fertilizer, 9.46 Mg ha −1 under organic manure. The average above ground residue of topsoil removal and topsoil addition decreased by 2% under N-fertilizer application, and decreased by 7% under organic manure application compared with the undisturbed control (6.31 Mg ha −1 under N-fertilizer, 9.46 Mg ha −1 under organic manure). Significant difference were observed among three TSD treatments both for grain yield (P < 0.001) and above ground residue (P = 0.0256) (Table 2).</p><!><p>Topsoil remove and addition can not only significantly affect the greenhouse gases emissions, but also changes the crop yield (Table 1 and Fig. 6), which indicated that the erosion-deposition process can significantly alter the global warming effects, soil nutrient status and soil productivity during the erosion events.</p><p>The average cumulative CO 2 emission in our study was 3.89 Mg C ha −1 growing season −1 ; the value fell into the range of seasonal CO 2 emission reported by several global studies 20,[25][26][27] . In the present study, cumulative CO 2 emission significantly increased following 20 cm topsoil removal and decreased following 20 cm topsoil addition throughout the maize growing season compared with the undisturbed soil under N-fertilizer application (Table 1). The difference of cumulative CO 2 emissions among three TSD treatments could be explained by the soil temperature variation that caused by aboveground coverage shade 28 . Soil temperature was the primary drive to CO 2 flux 21,[28][29][30][31] . The enhanced cumulative CO 2 emission at the eroded site may primarily result from its higher soil temperature of 25.9 °C (from 17.5 to 33.9 °C) with less aboveground coverage shade. The reduced cumulative CO 2 emission at the depositional site probably due to its lower soil temperature of 24.7 °C (from 15.5 to 32.2 °C) owing to its dense above ground coverage shade (Figs 2 and 6). In addition, the reduced cumulative CO 2 emissions at depositional site probably also caused by their lower substrate availability (e.g., SOC) 28 in surface soil (Table 1 and Fig. 5). While, under organic manure application, no significant difference observed among the three TSD treatments for cumulative CO 2 emissions (Table 1), which probably due to their similar average soil temperatures (respectively 22.6, 22.5 and 22.5 °C) (Fig. 2). It was worthwhile to note that the cumulative CO 2 emission for the organic manure applied plot (4.16 Mg C ha −1 growing season −1 ) was much greater than that from the plot receiving N-fertilizer (3.62 Mg C ha −1 growing season −1 ) (P = 0.0345). This was probably due to the greater carbon substrate (SOC) under organic manure application 20 (Fig. 5). Soil moisture content did not respond to the cumulative CO 2 emission in our study, probably because the soil seldom underwent prolonged drought 28 . Similar results also reported by Sheng et al. (2010). Smith et al. reported that the release of CO 2 by aerobic respiration is primarily driven by soil temperature, but becomes moisture-dependent as soil dries out 32 .</p><p>Among the three TSD treatments, undisturbed plot exhibited the largest cumulative N 2 O emission under N-fertilizer application. This can possibly attribute to the greater SOC and total N content in 0-20 cm soil layer depth (Table 2 and Fig. 5), as N 2 O emission depended on SOC and total nitrogen contents, bulk density, clay fraction and soil moisture content that regulates N 2 O production via microbial de-nitrification as well as nitrification 33 . The second largest cumulative N 2 O emission was at the eroded site under N-fertilizer, which might due to its higher soil temperature 32 and greater soil bulk density (Table 2, Figs 2 and 4). The results are in good line with previous studies conducted with an intensively farmed organic soil in North Central Ohio, which reported that N 2 O flux was positively related to soil temperature and CO 2 flux 34 . The least cumulative N 2 O emission at the depositional site may be determined by its lower soil temperature and lower SOC, total N content (Table 2, Figs 2 and 5). In addition, the eroded site displayed the highest cumulative N 2 O emission under organic manure application, probably ascribed to the anaerobic conditions 35 in soil pore space caused by higher soil moisture content and greater soil bulk density (Figs 2 and 4). The result is in agreement with a previous report that N 2 O emissions as a result of denitrification occurred in compaction treatment 36 . Flessa et al., reported that total N 2 O emission from the potato field during the growing season was 2.0 (kg N ha −1 growing season −1 ) in 1998 19 , and Kumar et al., resulted 2.89 (kg N ha −1 year −1 ) of N 2 O emission from corn-corn cropping rotation in an Alfisol of Ohio in 2012 26 , which were similar to our results of average cumulative N 2 O emission (2.9 kg N ha −1 growing season −1 ) in corn field in 2012.</p><p>In this study, cumulative CH 4 emission was positive at the eroded site under organic manure application, which was probably due to the hypoxic conditions 35 that may have been caused by the relatively greater soil moisture (Fig. 2). Cumulative CH 4 emissions from other TSD treatments were negative, which indicated the net CH 4 sink during the growing season (Table 1). The low values and net uptake of CH 4 reported in our study were consistent with those reported for cultivated soils 25,37 In our study, the average GWP at the eroded site (18.07 Mg CO 2 Eq ha −1 growing season −1 ) generated roughly the same value with the undisturbed control (18.11 Mg CO 2 Eq ha −1 growing season −1 ), but declined dramatically at the depositional site (10.58 Mg CO 2 Eq ha −1 growing season −1 ). The cumulative GHG emission at the eroded site might be stimulated via the higher soil temperature caused by its less aboveground coverage shade, but limited by its lower available C substrate. The cumulative GHG emission at the depositional site declined because of its lower soil temperature and lower available C substrate in surface. In addition, the cumulative GHG emission may also be influenced by soil bulk density and soil moisture content. The results displayed that eroded site can neither play a net sink nor net source of greenhouse gases emission, while depositional site can be a net sink of GHG emission.</p><p>It is critical to consider the overall (net) effect of the erosion and deposition processes in comparison with undisturbed soil (the control). Therefore, in this paper, we compared important parameters for undisturbed control with the average values observed for the combination of the topsoil removal and topsoil addition conditions. The average value of GWP of eroded and depositional site was 21% lower than that of the undisturbed control, indicated the erosion-deposition process could be net sink of GHG emission.</p><p>Average GWP for soil receiving organic manure was increased by 15%, compared with that with N-fertilizer application. This might be due to the greater SOC and total N content and the higher soil moisture content after organic manure application (Figs 2 and 5).</p><p>Given the limited accessibility to the experimental field, gas fluxes were only collected once every 2 weeks. There might be other peaks in GHG fluxes that were not captured during our sampling regime. More frequent sampling intervals are highly recommended in the further study.</p><p>In our study, the average maize yield significantly decreased by 51% at eroded site, and significantly increased by 47% at depositional site compared with the undisturbed control (7.45 Mg ha −1 ) (P < 0.0001), which was coincide with maize yields on two Alfisols in central Ohio 38 , and consistent with the finding that the corn yield declined by nearly half (46%) on removal of 20 cm of the topsoil in an eroded farmland of Chinese Mollisols 16 . Crop yield usually adversely affected by the impedance of root growth, water and nutrient deficits, high bulk density, penetrometer resistance, and low field moisture capacity under erosion 10,11,16 . On the one hand, topsoil removal significantly declined the SOC and total N content, increased the soil bulk density, and further reduced the maize yield at the eroded site. On the other hand, the topsoil addition buried the former topsoil under plough depth, preserved SOC from decomposition and mineralization [22][23][24]39 , promoted deep root growth, and consequently resulted to greater crop productivity (Figs 4, 5 and 6). This result agree with the findings that the impacts of erosion on agricultural land are usually negative for eroded sites and may be positive for depositional sites 40,41 .</p><p>The average value of maize yield of eroded and depositional site declined by 2% compared with the undisturbed control (7.45 Mg ha −1 ), which indicate the erosion-deposition process resulted the equivalent production with the uneroded site.</p><p>Maize yield at the organic manure applied plot (7.97 Mg ha −1 ) increased by 18% compared with that from the plot receiving N-fertilizer (6.74 Mg ha −1 ). This was probably caused by the improved SOC, total N content and soil moisture content with organic manure application (Figs 2 and 5).</p><p>The average value of GHGI was 4.96, 2.43 and 0.96 Mg CO 2 Eq Mg −1 grain yield growing season −1 , respectively for eroded site, undisturbed control and depositional site. The eroded site enhanced the GHGI because of its lower grain yield, while the depositional site declined the GHGI due to its lower GWP and higher grain yield. The average GHGI of erosion-depositional site increased by 22% compared to the undisturbed control. The average GHGI for soil receiving organic manure exhibited a small reduction of 3% compared with that of N-fertilizer application.</p><p>In summary, our results displayed that eroded site can neither play a net sink nor net source of greenhouse gases emission, while depositional site can be a net sink of GHG emission. Eroded site significantly reduced maize yield, while depositional site significantly enhanced the maize yield. The erosion-deposition process declined the GWP, not changed maize yield, and increased the GHGI. Soil with organic manure application enhanced GWP, improved maize yield and slightly reduced GHGI compared with soil receiving N-fertilizer.</p><p>It was worthwhile to note that our results were merely concluded based on the assumption that the area of erosion equals the area of deposition. However, in natural field, eros ion tends to be dissipated over much wider area, while eroded materials often end in areas not suitable for crop growth (river beds, estuaries, etc.) or water bodies (wetlands, reservoirs, ocean). The weight of GHG emissions from eroded area could have been much larger, and depositional zone may have led to additional CH 4 emissions. This calls for systematic investigation in the future study.</p><!><p>Study area. The study was conducted in an on-going long-term experiment at Waterman Farm of the Ohio State University, Columbus, OH, USA (N + 40° 1′ 5.52″ E −83° 2′ 29.72″). The experiment was initiated in 1997 on the Crosby soil series (deep, fine, mixed, active, mesic, Aeric Epiaqualf). The deep soil developed on nearly level topography (0% to 2% slopes) is of silt loam texture, poorly drained and derived from glacial till. The mean annual rainfall is 1016 mm and the mean annual air temperature is 11 °C 8 .</p><p>The experiments were designed in a split-plot arrangement with completely randomized blocks. Three TSD levels were carried out as main plots and two amendment types as subplots. The 18 × 9 m main plots were subdivided into 6 × 4.5 m subplots, with three replications for each treatment combination. The main plots were separated by 2.7 m long border strips 8,38 . Three TSD levels created once at the beginning of the experiment to simulated soil erosion-deposition process, which were: (1) topsoil removal (eroded site) created by physically remove of 20 cm topsoil with a landscape loader; (2) undisturbed control (uneroded site); and (3) topsoil addition (depositional site) achieved by deposit of 20 cm topsoil on soil surface. Two amendments were applied in this study: N-fertilizer and organic manure. For the plots receiving N-fertilizer, 150 kg N ha −1 urea-ammonium nitrate (28% N) was side-banded on the soil surface at the 3 rd to 4 th leaf stage of corn growth. For the plots receiving organic manure, dry matter compost (20 Mg ha −1 ) was uniformly top dressed during April each year. CO 2 , N 2 O and CH 4 flux measurements. Soil-air samples for the assessment of CO 2 , N 2 O and CH 4 fluxes were collected using the static chamber method 42 . Gas chambers were made of polyvinyl chloride (PVC) pipes of 15 cm diameter and 30 cm length. The top lid was made of a PVC cap, and the lower end was trimmed to be inserted into the soil. A machine-trimmed PVC trough was coupled around the outer ring of the pipe, approximately 5 cm from the top. The PVC cap was equipped with a sampling port and a rubber septum on the top, and the cap bottom could be fitted into the trough when the cap was in place 42,43 .</p><p>The chambers were inserted 10 cm deep into the ground at each sampling point, with three replications for each treatment. Chambers were installed 1 month before gas sampling, and the chambers remained in place with the cap opened during the entire growing season, except for temporary removal during seeding or fertilizer application. Chambers were reinstalled in the same place immediately after the fertilizer and seeding operations were completed 34 .</p><p>When sampling, closed the chamber lid, taken approximately 10 cm 3 soil-air samples from each chamber headspace at 0 and 30 minutes, and transferred it to crimp sealed pre-evacuated 10 ml vials fitted with butyl rubber septa. The vials were evacuated to a pressure of −172 kPa and prepared 1 day before sampling. Soil-air samples were obtained between 11 AM and 2 PM when fluxes were expected to be maximal 43 biweekly during the entire growing season. Three replications were taken for each treatment.</p><p>The CO 2 and CH 4 in soil-air samples were analyzed using a GC-2014 gas chromatograph (GC; Shimadzu, Kyoto, Japan) equipped with a thermal conductivity detector for CO 2 , and a flame ionization detector for CH 4 . N 2 O was analyzed on a GC fitted with a 63 Ni electron capture detector 34 .</p><p>Soil temperature and moisture measurements. Soil samples for measurements of soil temperature and soil moisture content were collected biweekly in conjunction with soil-air sampling 18 . Soil temperatures at 10 cm soil depth were monitored by using a digital thermometer near each chamber simultaneously with gas sampling. Gravimetric soil moisture content was also determined by collecting soil samples close to the chambers at 0-10 cm depth.</p><p>Analysis of soil properties. Bulk and intact core samples were obtained separately in June 2012 to measure soil properties. Intact core soil samples for bulk density analysis were collected at 0-40 cm depth (10 cm intervals) using a manually-driven core sampler with diameter and height both 5 cm. Gravimetric soil moisture content (SMC) was measured by drying a portion of trimmed core samples at 105 °C for 24 h 19 . Wet bulk density was computed as the ratio of soil wet weight to core volume, and soil bulk density was calculated from the wet bulk density and soil moisture content, ρ b = ρ b ′/(1 + w), where ρ b is soil bulk density, ρ b ′ is soil wet bulk density, and w is the gravimetric moisture content. Total porosity was calculated from the equation 44 : f = 1 − (ρ b ′/ρ s ), where ρ s is the soil particle density and is estimated at 2.65 g cm −3 . Bulk soil samples were air-dried at room temperature, ground with a wooden hammer, and sieved through a 2 mm sieve before physical and chemical analysis 38 . Soil total C and N contents were analyzed by the dry combustion method using a vario Max CN analyzer (Elementar, Hanau, Germany) 18 . The SOC was assumed to be equal to the total C as inorganic C concentration was negligible with the soil pH was below 7 38 .</p><p>Crop yield. Corn (Zea mays L.) was grown from about mid-May to October in 2012 without any major disturbances, and no tillage operation was performed. Corn plants from the center two rows of each plot were hand harvested. Crop residue after the harvest was left on the soil surface.</p><p>Corn ears were separated from the stover and weighed. Corn ears were shelled, and grains were weighed separately from other parts of the ear after air drying. Subsamples of grain were weighed and then oven-dried at 60 °C for 48 h to determine the water content 38 . Grain yields are reported in Mg ha -1 at 12% moisture content.</p><p>Data calculations and statistical analysis. Daily gas fluxes (q) (in units of g CO 2 -C m −2 d −1 or mg N 2 O − N m −2 d −1 or mg CH 4 − C m −2 d −1 ) were computed using Eq. (1) 34,42 : The greenhouse gas intensity (GHGI) was calculated by dividing GWP by crop yield using Eq. ( 4) 46 :</p><p>= − GHGI GWP/grain yield(Mg CO Eq Mg grain yield growing season ) (4)</p><p>Statistical analysis was performed using the analysis of variance (ANOVA) procedure available in SAS 8.01 for Windows (1999-2000, SAS Institute Inc., Cary, NC, USA). Mean and interactive effects of treatments were separated using the F-protected least significant difference test. The probability level (P) chosen to designate significance was ≤0.05. Correlation and regression analyses were performed on selected variables at P ≤ 0.1 using the same package.</p>

Scientific Reports - Nature

Isomeric anthracene diimide polymers

N-type semiconducting polymers are attractive for organic electronics, but desirable electron-deficient units for synthesizing such polymers are still lacking. As a cousin of rylene diimides such as naphthalene diimide (NDI) and perylene diimide (PDI), anthracene diimide (ADI) is a promising candidate; its polymers, however, have not been achieved yet because of synthetic challenges for its polymerizable monomers.Herein, we present ingenious synthesis of two dibromide ADI monomers with dibromination at differently symmetrical positions of the ADI core, which are further employed to construct ADI polymers.More interestingly, the two obtained ADI polymers possess the same main-chain and alkyl-chain structures but different backbone conformations owing to varied linking positions between repeating units. This feature enables their different optoelectronic properties and film-state packing behavior. The ADI polymers offer first examples of conjugated polymer conformational isomers and are highly promising as a new class of n-type semiconductors for various organic electronics applications.

isomeric_anthracene_diimide_polymers

Introduction<!>Results and discussion<!>Conclusions<!>Conflicts of interest

<p>N-type organic semiconductors, in particular conjugated polymers, are very crucial for optoelectronic devices, but their development lags far behind that of p-type counterparts due to the lack of electron-decient building blocks. Among a handful of electron-decient units, [1][2][3][4][5][6][7][8][9][10][11][12][13] six-membered tetracarboxylic aromatic diimides, typically naphthalene diimide (NDI) and perylene diimide (PDI), attract enormous attention because of their high electron affinity and mobility. A plenty of NDI-and PDI-based polymers have been developed as n-type semiconductors for various organic electronics applications. 3,[14][15][16] The aromatic diimides can be categorized into rylene diimides and acene diimides according to the p-conjugation core. 17 The NDI and PDI are just representatives of rylene diimides having extended conjugations along the normal axis of the NDI scaffold (Fig. 1). More derivatives e.g. terrylene and quaterrylene diimides were investigated by Müllen, [18][19][20] Langhals 21 and Adachi 22 et al. However, they are not suitable for constructing polymers because of high steric hindrance at the longitudinal direction which is adverse to the charge transport and molecular crystallinity, as claimed for PDI polymers. 23 This issue is absent in another class of aromatic diimides, namely acene diimides with conjugation expansion along the equatorial axis of the NDI, yet their polymers cannot be achieved so far owing to synthetic difficulties. Wang et al. 24 and Yamada et al. 25 have performed pioneering studies on the synthesis of acene diimides such as anthracene-, tetracene-, pentacene-and hexacene-diimides. Among them, the anthracene diimide (ADI) with one ring extension relative to the NDI should possess similar electron affinity and potentially provide interesting optoelectronic properties. 26 Moreover, its polymers have been theoretically predicted to present excellent n-type characteristics for organic electronics. 27 However, access to the ADI unit and especially to its polymers is rather challenging. Although Wudl et al. 28 and Yamada et al. 25 separately reported short alkyl chain-substituted ADIs synthesized from different routes, ADI-based polymers were never materialized since synthetic chemists were plagued by challenges in synthesizing polymerizable monomers of ADI. Long alkyl chains attached to amide N atoms are, on the one hand, indispensable for ensuring the solubility of resultant polymers; on the other hand, functionalized groups on the anthracene such as halogens, boronic esters, or tin salts (bromines usually preferred) are needed for the cross-coupling polymerization. Unfortunately, precursor compounds with long alkyl chains are not easily accessible, and symmetrical dibromination of ADI obviously suffers from great complexity owing to the presence of several pairs of substitutable sites (2,6-, 3,7-, and 4,8-positions, Fig. 1). Establishing new synthetic protocols to tackle these issues is thus of critical signicance.</p><p>Herein, we report the synthesis and properties of two 2octyldodecyl-substituted dibromide ADI monomers and their derived polymers. The two monomers are dibrominated at 2,6and 3,7-positions (2,6-2Br-ADI and 3,7-2Br-ADI, Fig. 1), respectively, by different strategies, making the two obtained polymers (PADI-2,6-2T and PADI-3,7-2T) possess isomeric backbone conformations. The two polymer conformers, thereby, present noticeably different molecular congurations, optoelectronic properties, as well as packing and oriented behavior in the lm state. To the best of our knowledge, this is the rst report on ADI-based polymers with backbone isomerism showing potential as a new class of promising n-type semiconducting materials.</p><!><p>The two monomers are synthesized with pre-and postbromination approaches, respectively. The 3,7-2Br-ADI is synthesized by a post-bromination route (Scheme 1a), where the 3,7-dibromination is conducted aer obtaining the 2octyldodecyl-substituted ADI (6a). The ADI 6 is acquired via a Lewis acid-mediated reaction between acyl chloride 3 and alkyl isocyanate 5 (for their synthesis see the ESI †). This approach was initially reported by Yamada et al. using bismuth triate as a Lewis acid; 25 however, the procedure in the literature did not work for our reaction. By replacing the bismuth triate with a classic Lewis acid of AlCl 3 and swapping the addition sequence of Lewis acid and isocyanate, the reaction is successfully realized to give the ADI 6. When the substituent is 2-octyldodecyl, 6a is produced with a low yield (<10%, two steps from 2 to 6a). Instead of branched alkyl chains, linear n-hexyl and n-dodecyl are employed to synthesize ADI 6b and 6c with improved yields.</p><p>It is worthy of note that the yield of 6c with longer alkyl chains exceeds 50%, higher than that ($20%) of 6b. Based on these observations and the fact of adding AlCl 3 prior to isocyanate, we propose a mechanism of Lewis acid-mediated successive two-step Friedel-Cras-type reaction (Scheme 2). First, the AlCl 3 coordinates with the acyl chloride 3 to generate an electrophile carbocation 12. Then, with the addition of isocyanate, nucleophilic attack to 12, namely the intermolecular Friedel-Cras-type amidation, occurs to form intermediate 13, which undergoes the intramolecular Friedel-Cras acylation to offer the double-cyclized ADI. For the rst-step Friedel-Cras reaction, the nucleophilic attack can be considerably impacted by the alkyl chain of isocyanate. Therefore, the steric hindrance of branched 2-octyldodecyl results in a low yield of 6a, and the yields of linear alkyl chain-substituted 6b and 6c are much higher. Meanwhile, long linear alkyl chains can provide better solubility of intermediates 13 and 14, allowing for efficient second-step intramolecular Friedel-Cras acylation, by which the 6c containing the n-dodecyl is synthesized in a higher yield than the n-hexyl-substituted 6b. The proposed mechanism suggests that the ADI and its various derivatives could be obtained in a desirable yield via tuning side chains. In the present work, in order to ensure the solubility of the resulting polymers and to compare with classic NDI and PDI analogs, the 2octyldodecyl-substituted 6a is used to prepare its dibromide monomer. Aer careful optimization (Table S1 †), it is selectively dibrominated at 3,7-positions with a brominating agent, i.e. 5,5dimethyl-1,3-dibromohydantoin (DBH), in conc. H 2 SO 4 at a mild temperature of 65 C to produce the 3,7-2Br-ADI. On the other hand, the 2,6-2Br-ADI is synthesized by a pre-bromination route (Scheme 1b), where the 2,6-dibromination prior to annulation of diimides is performed for the starting material (1) to get tetrabromide anthracene 7 that follows similar acidation, acylation, and Friedel-Cras-type reactions to yield the target monomer. The molecular structures of two isomeric monomers are unambiguously conrmed by 1 H NMR spectroscopy (Fig. 2) and high-resolution mass spectrometry (see the ESI †).</p><p>Indicated by density functional theory (DFT) calculations (Fig. 3a-c), the ADI skeleton is highly coplanar and the 3,7dibromination does not vary such a molecular conguration. Interestingly, the 2,6-dibromination causes a small distortion of the ADI plane, most likely owing to the steric hindrance between the adjacent bromine and the carbonyl group. UV-Vis absorption spectra (Fig. 4a) display that the absorption bands of the two monomers locate in between those of dibromide NDI and PDI. Notably, relative to the absorption of the ADI 6a (Fig. S1a †), the 3,7-2Br-ADI exhibits an evident red shi, while the 2,6-2Br-ADI shows a blue shi. Such observations can be ascribed to the effect of different extensions of frontier molecular orbitals (Fig. S2 †), induced by bromine substitutions at different positions. These results suggest that dibromination positions impact on the molecular geometry, the frontier Scheme 2 Proposed mechanism of the Lewis acid-mediated twostep Friedel-Crafts-type reaction.</p><p>Fig. 2 1 H NMR spectra of ADI 6a and two dibrominated isomers. The two monomers were then copolymerized with distannyl bithiophene to synthesize two polymers, PADI-3,7-2T and PADI-2,6-2T (Scheme 1c). Both polymers exhibit good solubility in chloroform, toluene, chlorobenzene, etc. at room temperature. Their molecular weights/polydispersity index determined by GPC are 17.8 kDa/2.8 and 14.2 kDa/2.3 (Table 1). Thermogravimetric analysis reveals their excellent thermal stability and the differential scanning calorimetry plot shows no detectable thermal transitions (Fig. S3 †). The DFT simulations demonstrate that the backbone conformations of the two polymers vary profoundly. The PADI-3,7-2T shows a rigid and planar polymer backbone, with small dihedral angles between ADI and bithiophene units (Fig. 3d), whereas the PADI-2,6-2T main chains are more distorted (Fig. 3e). In view of their identical polymer structures, the isomeric backbone conformations are bound to stem from the different linking positions between repeating units and can induce diversity in various properties of the two polymers. In toluene solution, the absorption maximum (l max , 740 nm) of PADI-3,7-2T manifests a bathochromic shi of 40 nm compared with that (700 nm) of PADI-2,6-2T (Fig. 4b, c). In the lm state, the PADI-3,7-2T presents a similar absorption spectrum to that in solution (Fig. 4b), while the PADI-2,6-2T exhibits a red-shied one compared to its solution absorption (Fig. 4c). These results reect that both polymers with the same repeating units but different linkage modes afford varied optical properties and aggregation behavior. The LUMO/HOMO levels (Fig. S5 †) measured from CV are À4.02/À5.43 and À4.06/ À5.44 eV for PADI-3,7-2T and PADI-2,6-2T, respectively. The deep LUMO levels similar to those of NDI and PDI polymers suggest strong electron affinity of ADI polymers with potential as n-type semiconductors. 23,29 The effect of the different backbone linkage modes on molecular packing and orientation in lms was investigated by grazing incidence X-ray diffraction (GIXD). Both pristine lms show a (010) diffraction ring (Fig. 5), indicating their mixed face-on and edge-on orientation; however, the PADI-3,7-2T lm prefers the face-on one. This packing structure indicates the coexistence of parallel and vertical charge transportation channels in its lms. 30 Aer thermal annealing (TA), the peak intensities become signicantly stronger. It is clear that the PADI-2,6-2T lm manifests distinct (h00) lamellar diffraction peaks (q z ¼ h  0.27 A À1 , h ¼ 1-4) with large crystal coherence lengths (CCLs) (Table S2 †) in the out-of-plane direction, signifying a high degree of molecular ordering. In contrast, no signals implying ordered lamellar stacking are found for the PADI-3,7-2T lm. Since the two polymers have the same backbone structures and alkyl chains, their difference in lamellar stacking can be attributed to isomeric backbone conformationinduced disparity of side-chain ordering.</p><!><p>In conclusion, we have presented the synthesis of two dibromide 2-octyldodecyl-substituted ADI with elaborate pre-and post-bromination methods, allowing for the exploration of ADI polymers. The dibromination at different positions of the ADI unit endows two monomers with variable coplanarity which further leads to isomeric backbone conformations of the resultant polymers even though they have the same molecular structures. With the unique conformational isomerism, the PADI-2,6-2T and PADI-3,7-2T exhibit distinct optical properties and packing behavior. The advent of ADI polymers with deep LUMO levels enriches the family of aromatic diimide polymers, which are promising for electron-carrying optoelectronic devices.</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

Conformational Heterogeneity of Bax Helix 9 Dimer for Apoptotic Pore Formation

Helix α9 of Bax protein can dimerize in the mitochondrial outer membrane (MOM) and lead to apoptotic pores. However, it remains unclear how different conformations of the dimer contribute to the pore formation on the molecular level. Thus we have investigated various conformational states of the α9 dimer in a MOM model -using computer simulations supplemented with site-specific mutagenesis and crosslinking of the α9 helices. Our data not only confirmed the critical membrane environment for the α9 stability and dimerization, but also revealed the distinct lipid-binding preference of the dimer in different conformational states. In our proposed pathway, a crucial iso-parallel dimer that mediates the conformational transition was discovered computationally and validated experimentally. The corroborating evidence from simulations and experiments suggests that, helix α9 assists Bax activation via the dimer heterogeneity and interactions with specific MOM lipids, which eventually facilitate proteolipidic pore formation in apoptosis regulation.As a proapoptotic protein from the Bcl-2 family, Bax is a crucial executioner in the mitochondrial pathway of apoptosis 1-3 . In stressed cells, Bax from the cytosol is activated by the BH3-only proteins (such as Bim and Bid) and inserts into the mitochondrial outer membrane (MOM) [4][5][6][7][8][9] . Through multi-step conformational changes, Bax forms dimers and further oligomerizes into membrane permeabilizing pores [8][9][10][11] which subsequently release apoptotic mitochondrial proteins like cytochrome c and SMAC to the cytosol and lead to cell death. Nonetheless, the conformational transition of Bax after activation, involving the dimerization in the cytosolic region and/or the MOM, has not been fully understood. Upon activation, a symmetric dimer conformation with reciprocal binding of the BH3 region of one Bax to the groove of another Bax was first observed by X-ray crystallography using a truncated Bax protein 8 , and recently confirmed by crosslinking of intact Bax protein in the mitochondria 12 . While Bax dimerization has been confirmed in certain cytosolic domains, the MOM-anchoring domain -in particular, the helix α 9 (residue 169-189) -is found to form dimer interfaces that expand the oligomeric pore in the MOM 10,12,13 . In the soluble Bax, helix α 9 is buried in a hydrophobic groove formed by helices α 2, α 3, α 4, and α 5 11 . However, studies using Förster resonance energy transfer (FRET) and electron spin resonance (ESR) observed significant rearrangements of helix α 9 indicating its exposure to the cytosol before membrane binding 10,14 . In the activated Bax, helix α 9 functions as a membrane anchor to target Bax to the MOM, because its deletion could prevent Bax translocation to the mitochondria 15 . Consistently, mutations of Ser184 in helix α 9 could either abolish or enhance the Bax translocation 15 . While prior studies pointed out that helix α 9 would be a key to understand Bax activation and apoptosis regulation, its particular roles and relevant molecular mechanisms have not been fully understood.Recently we have discovered two distinct conformational states of the α 9 dimer in the MOM. The first state, termed the intersected α 9 dimer (Fig. 1A1), was based on the disulfide linkages of four cysteine mutants: I175C, G179C, A183C and I187C 12 . In this state, two α 9 helices intersect though the G 179 xxxA 183 motif with a right-handed crossing angle of ~40°1 6 . The other state is referred to as the parallel α 9 dimer (Fig. 1B1) with a right-handed crossing angle below 15°, according to the disulfide linkages of other four cysteine mutants: Q171C, A178C, T182C and L185C 12 . Although α 9 dimerization was believed to facilitate the lipidic pore 12 , the actual

conformational_heterogeneity_of_bax_helix_9_dimer_for_apoptotic_pore_formation

<!>Results<!>Discussion<!>Methods and Models

<p>roles of these two dimeric states and their connections to Bax activation remain elusive or controversial. A number of key problems on the molecular level need to be studied. First, despite other mitochondrial targeting signals in Bax, helix α 9 by itself is sufficient to target the mitochondria 17 . However, what drives helix α 9 into the MOM, and how is helix α 9 stabilized in the membrane is currently unknown. Moreover, mitochondrial specific lipids are known to actively influence the structures and functions of the Bcl-2 family proteins including Bax 18 . It has been suggested that insertion of helix α 9 into the MOM and Bax pore formation are regulated by the membrane curvature and lipid compositions 19 . Then how does helix α 9 interact with various lipids in the MOM, especially the mitochondrion-specific ones? What is the significance of these interactions for helix α 9 dimerization and mitochondrial poration? In an attempt to gain molecular insight, we herein present the first systematic study of molecular dynamics (MD) simulations for the helix α 9 dimer in a model lipid bilayer, in tandem with experimental investigations of the helix α 9 dimer interface between intact Bax proteins activated in the native MOM.</p><p>While MD simulations have been applied to study the stability of helix α 9 bound to the Bax groove in the aqueous solution [20][21][22] , a large-scale simulation study of helix α 9 with atomic details in the membrane has never been reported. Also, it is required to further investigate the roles of Bax helix α 9 in apoptosis with a detailed membrane model to mimic the MOM lipid composition. Therefore, in this work we focus on the helix α 9 dimers in a MOM-mimicking lipid bilayer, using conventional all-atom MD and free-energy simulations for both qualitative and quantitative analyses. Simulations starting from the "static" intersected and parallel dimer models 12 were performed to understand the conformational dynamics, while in return, experiments with full-length Bax variants in native mitochondria were designed to validate the predictions from the simulations. Such synergy between simulations and experiments enable us to access structural and dynamic details of helix α 9 at various spatial and temporal scales. Building on good agreements between simulations and experiments, our work shines light on the molecular mechanism of Bax dimerization via helix α 9 interactions in the MOM, which has never been fully described before. In addition, our study establishes a unique approach for further investigations of the apoptotic MOM permeabilization induced by Bax and regulated by other Bcl-2 family proteins.</p><!><p>The membrane environment is critical to stabilize the helix α9 dimers. We have compared the stabilities of the intersected and parallel α 9 dimers in a MOM-mimicking lipid bilayer versus an aqueous solution. Within the 200-ns MD simulations, both the intersected and parallel α 9 dimers appear stable in the membrane (Fig. 1), as shown by the small fluctuation around 1 Å of the backbone root-mean-square deviation (RMSD). Consistently, all the cross-linkable data 12 were reproduced by C β -C β ' distances along time evolution The second replicas reproduced the qualitative results (Supplementary Fig. 2).</p><p>(Supplementary Fig. 1). In contrast, in solution the α 9 dimers became disordered during the simulations (A2 and B2 of Fig. 1). Compared to the starting conformations, the backbone RMSDs of both dimers in the solution dramatically increase in the first 50 ns and then fluctuate around 5 Å toward the end of the simulations (Fig. 1C). The helicity of the intersected dimer decreases from 87 to 54% due to melting in the terminal regions, giving rise (see Supplementary Fig. 3) to a separation greater than 27 Å for I187-I187′ pair in the C-termini. Similarly, the helicity of the parallel dimer drops from 87 to 70%, increasing Q171-Q171′ and T182-T182′ separations to 13 and 11 Å, respectively. Additionally, rotation of the individual α 9 helices in the parallel dimer was also observed in the solution, which increased the crossing angle between two monomers by over 10°. Likewise for a single α 9 monomer in the solution, a dramatic loss of helicity was observed in our simulations.</p><p>Our MD simulations demonstrate that the membrane environment is essential to maintain the helicity of α 9, because α 9 remains helical in the membrane but becomes disordered in the solution. Combined with the previous experimental finding of helix α 9 being partially buried in a groove of the solvated Bax monomer 11 , we suggest a possible conformational transition of helix α 9 after Bax activation upon binding to a BH3 peptide or protein that displaces α 9 from the groove 5,8 . It is likely that an order-to-disorder transition occurs after helix α 9 becomes unbound to the groove of Bax, and a disorder-to-order transition follows when helix α 9 starts to insert into the MOM. α 9 adopts disordered conformations due to the exposure of its hydrophobic core to the cytosol or even membrane's charged periphery, while the membrane plays a key role in recovering the peptide helicity by providing a hydrophobic environment around α 9's hydrophobic core and charged/polar periphery around α 9's charged/ polar termini. In fact, many peptides such as melittin 23,24 are known to undergo similar disorder-to-order transitions when inserted to membranes. Therefore, the order (in groove)-to-disorder (in cytosol)-to-order (in membrane) transition may be a general pathway for α 9 targeting and insertion to the MOM during Bax activation.</p><p>The intersected and parallel α9 dimers display distinct preferences toward anionic lipids. To understand how the membrane stabilizes the α 9 dimers, we have investigated the lipid distributions and dimer-lipid interactions. First, we analyzed (Fig. 2) the average anionic lipid distributions and diffusion coefficients in three 10-ns windows during the last 30 ns, and obtained converged results (Supplementary Fig. 4 and Supplementary Table 2) with clear lipid preference near each α 9 dimer. Despite the fact that neutral lipids account for ~75% of total lipids in the MOM, the anionic ones such as PS, PI, and CL have been known to facilitate Bax activation, insertion, and pore formation 25,26 . Starting with a membrane model that had a random distribution of neutral and anionic lipids, the anionic lipids were enriched around each α 9 dimer in the end. An area with a radial cutoff of 20 Å from the dimer centroid contains 24-26% of anionic lipids and 18-21% of neutral lipids on average (given its bulky tails, a CL counts as twice the area of other lipids).</p><p>Within the radial cutoff of 20 Å from an α 9 dimer centroid, we calculated the stoichiometry of CL, PS, and PI, which is 2.0 ± 0.1, 2.7 ± 0.5, and 2.7 ± 0.5 per intersected dimer, and 3.7 ± 0.5, 1.0 ± 0.1, and 2.0 ± 0.1 per parallel dimer, respectively. Such evident discrepancy suggests a preference of the intersected dimer to PS and PI (that carry one negative charge under the neutral pH), in contrast to a preference of the parallel dimer to CL (that carries two negative charges). Second, we examined the protein-lipid interacting sites identified from the converged lipid distributions and found a structural basis for the lipid preference. Contacts between the α 9 dimer and the headgroups of PS, PI, and CL are found to involve the side chain, backbone, or the terminal groups (-COO − and -NH 3+ ) of amino acids (Fig. 3). Around the intersected dimer, PS and PI are able to form multiple polar contacts with each α 9 monomer: for example, PI contacts with W170 and Q171, and PS interacts with T169′ and W170′ (Fig. 3A1). However, CL only forms occasional, transient contacts (duration below 1 ns, Supplementary Fig. 5) with residues like K189′ (Fig. 3A2), which is insufficient to keep CL bound to α 9. This explains why more PS and PI lipids than CL dwell near the intersected dimer in our simulations. Around the parallel α 9 dimer, CL owing to its wide headgroup (~10 Å) is able to span across a large region and form multiple long-lasting contacts with both monomers. However, since more terminal residues participate in the interfacial interaction between two monomers (Fig. 3B1), in the parallel dimer there are fewer residues available for binding PI and PS (Fig. 3B2). As a result, the presence of PI and PS in the vicinity of the parallel dimer is reduced. Therefore, it is the availability of the residues exclusive from the dimer interface and the distinct lipid head-group together that determine the intersected dimer's preference to PS and PI, and the parallel dimer's preference to CL.</p><p>Our observation, which the α 9 dimer in different conformational states preferentially binds anionic lipids, is in line with a previous study showing that the α 9-membrane interaction is influenced by the presence of anionic lipids 27 . Further, while previous studies mainly focus on the role of CL and other anionic lipids on the activation of the BH3-only protein Bid or of Bax by a truncated Bid (tBid) 26,28 , our study describes a detailed picture of the α 9 dimerization that occurs downstream of Bax activation to link the BH3-in-groove Bax dimers into higher-order oligomers. Presumably anionic lipids play manifold roles in such processes. On one hand, they recruit a caspase-cleaved Bid (cBid) to the membrane and then activate tBid that in turn recruits and activates Bax 26 . On the other hand, our findings suggest that α 9 forms different dimeric states that interact with selective anionic lipids. Accordingly, Bax traps more anionic lipids into a microdomain via α 9 dimers (Supplementary Fig. 5), which may destabilize the membrane by concentrating the negative charges, or by increasing the membrane curvature (as PS generates positive curvature and CL leads to very negative curvature of the membrane 26 ). This membrane destabilization likely leads to collapse of the bilayer and formation of proteolipidic pores. Moreover, it is found that the binding affinity of cBid to the membrane without CL is similar to that to the membrane with CL as long as the total negative charge is maintained (e.g. by replacing CL with PS) 26 . However, the tBid conformational changes required for activation of Bax are impaired in the membranes that lack CL. Since the active tBid is enriched in the CL-rich domain where Bax can be recruited, the active Bax may first form the parallel dimer in the CL-rich domain and then convert into the intersected one that retains more PI and PS. In summary, the Rare PS lipids get close enough to the parallel dimer to form polar contacts (Supplementary Figs 5 and 6). α 9 dimerization may enhance the local Bax concentration to the membrane domains enriched with negatively charged CL, PS and PI, which will eventually stress the membrane and promote proteolipidic pore formation.</p><p>The helix α9 dimer switches between multiple conformational states in the MOM. According to our conventional MD simulations, the α 9 dimers in the intersected and parallel states are stable in the MOM, but interact differently with anionic lipids, and their interconversion is likely related to the Bax activation. To further investigate the conformational transition pathway of the α 9 dimer, we employed metadynamics simulations to calculate the free-energy landscape of the α 9 dimer in the membrane, regarding two collective variables: the angles of rotation (α , β ) of backbone around the principal axis of the two helical monomers. The rotation angles in the starting intersected dimer are set to zero degree. The rotation directions are chosen along the smallest rotation angle to transit. We did not constrain the monomer-monomer separation or the crossing angle, and thus they were allowed to relax when the monomers rotated along the directions shown in Fig. 4. To ensure convergence, we performed two simulations to model the transition: one for the path from the intersected to the parallel dimer while the other for the reverse path. Consistent results have been obtained from these simulations, which confirm the convergence of our free-energy calculations (Supplementary Fig. 7).</p><p>We then extracted the shortest transit pathway and identified the representative states (Fig. 4 and Supplementary Fig. 8), including four major low-energy states (I, II, IV, and VI) and two high-energy transition states (III and V). States I (α = 0°, β = 0°) and VI (α = ~70°, β = ~90°) correspond to the intersected and parallel dimer states respectively, which fit the crosslinking data from our previous study 12 . Our results suggest that the transition pathway from the intersected conformation toward the parallel one consists of three major steps (Fig. 4B). (i) The α and β rotation angles in the dimer increases to cause a slight increase in the monomer-monomer distance and a decrease in the crossing angle, which enables a transition from state I to II (α = ~30°, β = ~20°). (ii) State II converts into state IV (α = ~60°, β = ~40°), where the helix-helix crossing angle gets below 15° and T172 and T186 in one monomer approaches their counterparts in the other monomer at the dimer interface (Fig. 5A, hereafter the state IV is referred to as the iso-parallel state). There is an energy barrier of 3.1 kcal/mol (state III) when I175 and I175′ are very close. (iii) The monomers in iso-parallel state continue to rotate, resulting in state VI with Q171, A178, T182 and L185 in one monomer closer to their counterparts in the other monomer. Given the transition barrier as high as 6.2 kcal/mol (state V), this step is likely to be rate-limiting in the entire pathway.</p><p>Our metadynamics simulations not only are consistent with prior crosslinking data 12 and conventional MD simulations on the stability of the α 9 dimer (Fig. 1C), but also provide valuable quantitative, mechanistic evidence that is not readily available via other approaches. First of all, the free-energy map (Fig. 4A) and the potential transition pathway (Fig. 4B) indicate multiple conformational states of the α 9 dimer in the MOM -which is more complex than a simple two-state model. The energy map indicates the conformational diversity of the dimer in the MOM with the intermediate states (including the blue areas not labeled in Fig. 4A) with lower free energies that are more stable than the others with higher free energies. Although the shortest transition pathway between the intersected and parallel states is presented in Fig. 4B, we are also aware of other pathways to connect the multiple dimer states, which can likely promote the conformational heterogeneity. Next, the intersected dimer is found 3.5 kcal/mol lower than the parallel one in free energy. Thus, in the MOM there could be slightly more intersected dimers, since it is thermodynamically more stable. However, the intersected and parallel α 9 dimers are likely to coexist, provided an overall barrier of just 12 kcal/mol between them. In addition, we have found the iso-parallel dimer interface as an intermediate state with a small crossing angle (~15 degree) and a moderate monomer-monomer distance. Because of the small energy difference between the iso-parallel (IV) and parallel (VI) states, it is likely that the iso-parallel state is also stable or at least metastable in the membrane. Supportively, the iso-parallel α 9 dimer still resembles its initial model from the metadynamics simulation after a 282-ns conventional MD simulation, with a final backbone RMSD as low as 2.2 Å. Therefore, combining our conventional and metadynamics simulations, we are able to support the previous experimental work 12 and further predict a new dimeric state.</p><p>Recently, three compounds targeting at S184 in helix α 9 have been suggested as Bax agonists for potential treatments against the lung cancer 29 . The discovery of multiple conformational states proposes the helix α 9 dimer as a potential therapeutic target to treat diseases associated with excessive Bax activation or inhibition. To further our understanding towards this goal, we carried out experiments of the helix dimer in intact Bax proteins to validate the iso-parallel state and to show the possibility to perturb selective states.</p><p>The simulation-predicted iso-parallel α9 dimer was validated in the mitochondria. To determine whether the iso-parallel α 9 dimer interface is actually formed by the active Bax proteins in the MOM, we made two single-cysteine Bax mutants T172C and T186C. The cysteine pairs that replace the T172-T172′ and T186-T186′ pairs are within disulfide-linkable distance according to the iso-parallel dimer model (Fig. 5A), but not in the disulfide-linkable distance of the intersected and parallel dimer models. We synthesized the [ 35 S] methionine-labeled mutant proteins in vitro, activated them by a BH3 peptide from Bax (BH3) or cBid, and targeted them to the mitochondria lacking endogenous Bax and Bak as before 12 . We then oxidized the resultant mitochondria to induce disulfide linkage between each mutant. We used non-reducing SDS-PAGE and phosphor-imaging to detect the radioactive Bax monomer and the potential disulfide-linked dimer. As expected from the iso-parallel α 9 dimer model, both single-cysteine Bax mutants formed disulfide-linked homodimers at the mitochondria (Fig. 5B,C). Of note, these in vitro synthesized single-cysteine Bax mutants could release cytochrome c from the Bax and Bak deficient mitochondria in a tBid-dependent manner (data not shown). Therefore, the iso-parallel α 9 dimer detected by the crosslinking is formed by the active Bax protein.</p><p>The α9 dimer in different states can be selectively perturbed by mutations. Consistent evidence from both simulations and experiments has shown that the G179I and T182I mutations generate steric clashes in the interfaces to disrupt the intersected and parallel α 9 dimers, respectively 12 (Supplementary Figs 9 and 10). Likewise, as indicated by the simulations, A183I disrupts the iso-parallel α 9 dimer and increases the distance of the T186-T186′ pair beyond a cross-linkable range (Fig. 5A).</p><p>To verify the simulation-predicted effect of the A183I mutation on the iso-parallel and other α 9 dimers, we made the A183I mutation into the T186C or A178C mutant that was used to detect the iso-parallel or parallel dimer in crosslinking experiments, respectively. In accordance to the MD simulations (Fig. 5A), the disulfide crosslinking of the T186C mutant was inhibited by the A183I mutation (Fig. 5C, comparing lane 6 to 4), providing further support to the iso-parallel dimer model. Unexpected from the parallel dimer model but expected from the predicted iso-parallel to parallel transition path, the A183I mutation also inhibited the parallel α 9 dimerization detected by the disulfide crosslinking of the A178C mutant (Fig. 5D, comparing lane 4 to 2). Also, we found that the contacts of the iso-parallel dimer to anionic lipids are less than they are around the intersected dimer (Supplementary Fig. 11). Taking the simulation and experimental evidence together, the iso-parallel α 9 dimer represents a conformational state formed during the Bax oligomerization, which intermediates the intersected and parallel states, and facilitate their interconversion.</p><!><p>Connecting the evidence from simulations and experiments, we propose a possible molecular mechanism for the dynamic conformation and interaction of Bax helix α 9 to induce apoptotic pores in the MOM (Fig. 6). The cytosolic Bax protein is equilibrated between a fully folded state with the helix α 9 buried in the hydrophobic groove (as shown by the NMR structure) 11 and a partially unfolded state with α 9 released from the groove. Interactions The N-terminus and helix α 1 are shown as the green surface, while helices α 2 to α 5 in sky blue and helices α 6 to α 8 in deep blue, and helix α 9 is represented by yellow ribbon. After displaced by tBid from the groove formed by helices α 2 to α 5, helix α 9 loses helicity in the cytosol but then folds back within the MOM. The formation of the intersected and parallel α 9 dimers attracts different anionic lipids to the vicinity as indicated, which may increase membrane tension and curvature, thereby promoting proteolipidic pore formation to release cytochrome c. with a BH3-only protein such as tBid may shift the equilibrium to the partially unfolded state due to displacement of helix α 9 from the hydrophobic groove to an aqueous environment. Such an order-disorder transition drives α 9 to the MOM, where α 9 folds back to the helical conformation in a disorder-order transition with its hydrophobic core stabilized by the lipid tail region and the polar/charged termini stabilized by the lipid headgroup region.</p><p>During the initial activation, Bax is likely targeted to a CL-rich domain in the MOM (usually near the outer-inner membrane contact site) where the activator tBid is likely located 33,28 . Through specific interactions, helix α 9 traps more anionic lipids that are wandered by. To remain stable in the membrane, helix α 9 likely first forms the parallel dimer that preferentially binds CL. Then the parallel dimer relaxes into the intersected dimer that is more stable and able to bind other anionic lipids like PS and PI. The parallel to intersected conformational transition can go through the intermediate iso-parallel state. The dynamics of the α 9 dimer conformation may result in the dynamics of anionic lipid distribution, which destabilizes the lipid bilayer to induce a proteolipidic pore that is lined by both Bax oligomers and lipid headgroups, and able to releases cytochrome c and other intermembrane space proteins to initiate apoptosis.</p><p>In general, we have combined simulations and experiments to study the detailed conformations and dynamics of the Bax helix α 9 dimers in the MOM, a difficult task with any single approach alone. Corroborating evidence is obtained in the conformational stability of the α 9 dimer and mutants, the dimer-lipid interacting patterns, and the free-energy landscape to reflect the α 9 dynamics -suggesting a detailed mechanism of helix α 9 in the Bax-induced membrane poration, which may be facilitated by the interactions between multiple dimer conformational states and anionic lipids in the MOM. Our combined computational/experimental strategy will be useful to further explore the molecular mechanisms of the other membrane embedded/interacting helices in Bax activation and action, or even the mechanisms of other Bcl-2 family members in the MOM.</p><!><p>Models. The intersected and parallel α 9 dimer models were generated in our previous study 12 : the intersected model was predicted ab initio by CATM program 16 ; the parallel model was obtained through a systematic search of the dimer conformational space directed by the disulfide-crosslinking data. These two models fit the disulfide-crosslinking data from the single-cysteine Bax mutants illustrated in Fig. 1.</p><p>We used the membrane builder and MD simulator of CHARMM-GUI 30 to generate the protein-membrane and protein-solvent systems. The lipid bilayer consist of 46.5% L-α -phosphatidylcholine (PC), 28.4% L-α -phosphatidylethanolamine (PE), 8.9% L-α -phosphatidylinositol (PI), 8.9% L-α -phosphatidylserine (PS) and 7.3% cardiolipin (CL) in mole fraction 25 . All systems were solvated with explicit water in the TIP3P model in the periodic box. Counterions Na + and Cl − were added to keep the system charge neutral.</p><p>Simulation setup. Summary of all the MD simulations is provided in Supplementary Table 1. Most simulations have two replicas, of which the longer one lasts 100-200 ns and a shorter one 50-150 ns. The NAMD package 31 with CHARMM36 force field has been used 32 . Both equilibration and production runs were performed in the NPT ensemble (310 K, 1 bar, Nose-Hoover coupling scheme) with a time step of 2 fs. The particle mesh Ewald (PME) technique was used for the electrostatic calculations. The van der Waals and short-range electrostatics were cut off at 12.0 Å with switch at 10.0 Å. The metadynamics simulations 33,34 were carried out in NAMD to study the transitions between the intersected and parallel α 9 dimers, with the assumption that the transition between two conformations is reversible. The sampling bias was applied to two collective variables -the angles of rotation (α , β ) of the backbone atoms around the principle axis of each monomeric helix.</p>

Scientific Reports - Nature

Functional paper-based materials for diagnostics

Functional papers are the subject of extensive research efforts and have already become an irreplaceable part of our modern society. Among other issues, they enable fast and inexpensive detection of a plethora of analytes and simplify laboratory work, for example in medical tests. This article focuses on the molecular and structural fundamentals of paper and the possibilities of functionalization, commercially available assays and their production, as well as on current and future challenges in research in this field.Graphic abstract

functional_paper-based_materials_for_diagnostics

<!>Introduction<!>History, manufacture, and current use<!><!>History, manufacture, and current use<!><!>History, manufacture, and current use<!><!>Cellulose functionalization<!><!>Cellulose functionalization<!><!>Biofunctionalization of paper<!><!>General remarks<!><!>Paper test strips<!><!>Paper test strips<!><!>Lateral flow assays<!><!>Lateral flow assays<!><!>Current and future trends in µPAD research<!><!>Current and future trends in µPAD research<!><!>Advanced assay designs<!><!>Sensing techniques<!><!>Sensing techniques<!><!>Sensing techniques<!><!>Conclusion/summary<!>

<p>Open Access funding enabled and organized by Projekt DEAL.</p><!><p>Paper-based sensors are highly attractive not only for researchers. Being very low in cost, easy-to-use, and able to give, in the simplest case, a yes/no answer on questions arising in everyday life, paper-based analytical devices have found their way into all of our households [1].</p><p>A prototype of a paper-based urine test to assess the presence of glucose and protein was described by George Oliver, a physician from England, in 1883. However, the first paper dipstick aimed at specific testing for glucose, named Clinistix, was marketed in 1956 by Ames (later Bayer). Intensive research and development efforts have been made since then. Nowadays, it is even possible to test for up to ten biomarkers from just a few drops of urine within a minute using a single multitest dipstick [2].</p><p>The same decade when the first commercial dipstick test appeared on the market also provided the onset for today's commonly known lateral flow assay format, another important type of paper-based analytical device. Only 30 years later, in the 1980s, was this test strip available, e.g., as a convenient home pregnancy test, having marked a real revolution by that time. Since then, over 500 patents have been filed on the development of this technology [3].</p><p>Together with recently established diverse types of microfluidic paper-based analytical devices (µPADs), functional papers provide a broad methodological basis addressing a vast variety of analytical questions [4].</p><p>General interest in paper-based sensors is unbroken. The global biosensor market will probably grow by more than 70% by 2022, compared to 2015 [5]. Improvement of existing and design and implementation of new, low-cost, and user-friendly point-of-care devices are being cited as the main drivers of this development. Obviously, paper-based technologies have the potential to meet most of the ASSURED guidelines filed by the World Health Organization (WHO) for diagnostics in resource-constrained settings. According to the WHO, such tests must be Affordable, Sensitive, Specific, User-friendly, Rapid and robust, Equipment-free, and Deliverable [6].</p><p>In this respect, cellulose and its derivatives feature several promising properties and offer some evident benefits. Present in practically all plants, this natural product is abundantly available and therefore inexpensive [7]. It is environmentally sustainable, highly bioavailable, biodegradable, and biocompatible. Cellulose is insoluble in the majority of common organic solvents, in particular in water, and is able to transport water-soluble substances just by capillary forces, without further employment of an external power source. Moreover, substances may be stored inside the paper pores [8]. Finally, paper is chemically and thermally stable, easy to handle and store, and safely disposable or even recyclable, if desired [8, 9]. Taken together, these properties caused a still growing interest in paper-based analytical devices [9].</p><p>Regardless of the number of interesting demonstrations reported to date, a number of challenges still exist. In particular, if paper samples are used as a materials source for medical diagnostic tools. There are already numerous valuable review articles available on the newest developments on such applications. However, stepping one step further towards any successful transfer of such demonstrations into market-ready devices requires a more in-depth look at the paper material and its structure–property relationship itself. The latter progresses from looking at essential building elements of paper, the paper lignocellulosic fiber, to papermaking technology, including ways of controlling the porous inner structure, the ability to functionalize paper fibers in a tailored fashion, and finally to integrate such highly complex and functional biogenic materials into paper-based sensor devices. In order to address these points, this article will start out with looking at the historical, yet innovative papermaking process, followed by looking in detail at the chemical and geometrical structure of fibers being used in papermaking. Then we will focus attention on the chemical properties of cellulose, as the major building block of paper fibers, as well as its different opportunities and challenges for modifications as a basis for the further design of paper-based sensors. Finally, will focus on commercially available paper-based sensors, especially their setups and techniques. Here, the latest developments and their potential will be presented and discussed, as well as remaining challenges, requirements, and future directions.</p><!><p>For almost 2000 years, paper has been defined as a porous, flat, non-woven, layered material made of mostly natural fibers. Commonly, such a paper sheet is made through dewatering of a fiber suspension on a sieve and subsequent drying under compression and thermal heating of the resulting fiber fleece. This fiber fleece is composed of many statistically and randomly aligned individual fibers, constituting a highly porous organic material [10–14].</p><p>Historically, the invention of paper goes back to the year 105 ad. The Minister of the Chinese Imperial Court Cai Lun used old textiles and mulberry bast to make paper as a writing material, which is nowadays known as the birth of paper. With the takeover of Turkmenistan by the Arabs, paper finally reached Europe via Arabia around 760. The oldest existing European paper document dates back to 1102, and the first paper mill that started producing paper in Germany was in Nuremberg in 1390. As a result of the invention of letterpress printing by Johannes Gutenberg in 1435, the demand for paper experienced a strong increase [11–13, 15].</p><!><p>Shares of the individual paper grades in the total annual paper production in Germany in 2018.</p><p>[17]</p><!><p>At a first glance, paper seems to be a simple and scientifically less challenging, historic material. Up to now, research on paper has therefore mainly focused on the processing and the properties of the material with respect to its standard applications, progressing from packaging material to print media and hygiene products. The necessity of meeting our needs in the future, to a large extent from renewable resources, calls for further use of paper as a biogenic, recyclable material, beyond its classic fields of application. Paper can be functionalized in a variety of ways. This feature allows the production of functional paper-based materials for sensor technology, microfluidics, and a variety of other high-tech applications. With respect to synthesis and analysis, it is a tremendous challenge to capture the complexity of the material over all length and time scales. However, the latter is perhaps the most important key to tailor the geometric structure and chemical composition of functional paper.</p><!><p>Properties of commercially available Whatman® filter papers [21, 22]</p><!><p>The basic building blocks of paper are single lignocellulosic fibers of natural origin or of recycled fibers, so-called secondary fibers. Natural fibers are obtained directly from plant raw materials by pulping. Pulping describes the process of disintegration of the raw material wood into fibers and can either be carried out mechanically or chemically. Mechanical pulping separates fibers from woodchips by applying strong mechanical shear forces. Among other things, a rotating millstone is used for this purpose, which presses and shears the wood surface, thereby separating out individual fibers. Paper fibers obtained from this pulping process still contain a larger amount of lignin as well as hemicelluloses. The latter can affect the behavior of such fibers in contact with water, as well as the chemical composition of the surface of such fibers which can be very different from that of cellulose itself [23, 24]. Chemical pulping, on the other hand, dissolves lignin and other components of the interfiber matrix material, thus separating the individual fibers, which then typically contain only low amounts of the aromatic and nonpolar compound lignin from the rest of the wood-based materials. The pulping process, the raw material, and its origin therefore all have a strong influence on the fiber properties. For example, fibers still containing nonpolar lignin or other hemicellulosic material may absorb water and swell to a different degree than those fibers that are composed of pure cellulosic polymers. The latter may be of great importance in the design of paper-based sensors that are in contact or transporting aqueous analyte-containing fluids.</p><p>Worldwide, 90% of the raw material source is wood. Depending on the wood source, the obtained fibers moreover differ in length and stiffness, which will also impact the final mechanical properties of the resulting paper, and as such its use in different applications. In addition, pulp from jute, flax, hemp, sisal, grasses, and cotton is also used in paper production [12, 13, 25]. In particular, paper produced from cotton and cotton linters fibers is predominantly used for the design of functional papers in biomedical or other highly specialized applications. The latter is due to their high purity, related to a high cellulose content in the fibers, which far exceeds 95% [24–27].</p><!><p>Supramolecular structure of cellulose fibers.</p><p>Reproduced with permission from [29], electron microscopy pictures with permission from [30]</p><p>Chemical structure of anhydroglucose (AGU), cellobiose, and the non-reducing and reducing end groups of a cellulose polymer chain</p><p>Section of the supramolecular structure of native cellulose. Intermolecular hydrogen bonds are indicated in blue and intramolecular hydrogen bonds in red.</p><p>Reproduced with permission from [29, 32]</p><p>Fringed fibril model of the supramolecular structure of cellulose, as described by Haerle in 1958. An exemplary crystalline region is indicated in green and an exemplary amorphous region in yellow.</p><p>Reproduced with permission from [28]. Copyright (1998) Wiley-VCH GmbH</p><p>Sodium periodate oxidation of cellulose leading to 2,3-dialdehyde cellulose</p><!><p>In general, cellulose offers numerous possibilities for functionalization owing to the large amount of addressable hydroxyl groups. Indeed, each AGU has two secondary hydroxyl groups at C2 and C3 and one primary hydroxyl group at C6. These three hydroxyl groups per AGU exhibit different reactivities, depending on the type of reaction, the reaction solvent and environment, as well as on other factors.</p><p>We have already pointed out that cellulose is insoluble in most common solvents; nevertheless, some special solvent systems are capable of dissolving the macromolecule. Thus, two different types of cellulose functionalization reactions are generally possible, homogeneous, single-phase reactions in the dissolved state or heterogeneous, two-phase reactions on intact cellulose fibers. Homogeneous reactions lead to a highly uniform cellulose functionalization and distribution of the substituents along the cellulose polymer chains. In heterogeneous reactions, on the other hand, mostly just the amorphous and swellable cellulose parts are converted. In general, homogeneous functionalization under disruption of the supramolecular cellulose structure, combined with a change in the chemical structure, usually leads to products that can be hardly compared to the cellulosic starting material. In the following, the most common and important cellulose functionalization reactions are explained in detail.</p><p>Chemical cellulose functionalization is primarily based on the so-called polymer analogue reactions on hydroxyl groups. A polymer analogue reaction is principally defined as a chemical reaction on a polymer with preservation of the polymer backbone. In case of cellulose, three hydroxyl groups, namely one primary at the C6 position and two secondary ones at C2 and C3, are available. As mentioned above, these hydroxyls are all involved in the strong hydrogen bond network of cellulose. In general, the OH groups at C2 and C6 have multiple reaction possibilities compared to the hydroxyl group at C3, as they are statistically less involved in the hydrogen bond network, which in turn leads to higher chemical reactivity. The primary hydroxyl group on C6 is also more reactive than the secondary hydroxyl group on C2 for the same reason. Additionally, the steric accessibility of the hydroxyl groups also has an influence on functionalization reactions. In two-phase reactions, solely the amorphous regions of cellulose are accessible, whereas in homogenous ones nearly all hydroxyl groups are reactive. To enhance the accessibility of hydroxyl groups in a two-phase reaction, different chemical and mechanical treatments can be conducted, e.g., amplification of fiber fibrillation by fiber beating, or the increased swelling of fibers in sodium hydroxide (NaOH) [32, 37]. However, it is obvious that the maximum degree of substitution (DS), which describes the number of chemically converted hydroxyl groups per AGU, cannot exceed three.</p><p>Functionalization reactions on cellulose have been the focus of some scientific research for many years [18, 38–40]. Therefore, a wide range of reactions have been reported to date. In the scope of this review, functional papers for diagnostic and sensory applications will be discussed in more detail; hence, strategies for cellulose functionalization, which are important in this particular field of application, will be addressed in the following.</p><!><p>Scheme of typical polymer-analogue functionalization reactions on hydroxyl side groups</p><!><p>Cellulose ethers are obtained according to the commonly known Williams ether synthesis, by converting the hydroxyl groups of cellulose with alkyl halides in the presence of strong bases. A prominent example of a widely used cellulose ether derivative is carboxymethyl cellulose (CMC). CMC is produced by etherification of the cellulose hydroxyls with monochloroacetic acid in the presence of NaOH, and CMCs are utilized, for instance, in the textile industry as a coating for yarns or as binding and thickening agents in the food industry [28, 37]. Concerning functional paper applications, CMCs offer the advantage of exposing free carboxyl groups that can be easily used for subsequent immobilization reactions [42].</p><!><p>Reaction scheme of TEMPO-mediated cellulose oxidation.</p><p>Reproduced with permission from [45]. Copyright (2006) Elsevier</p><p>Schematic of the four different immobilization methods for biomolecules on surfaces. From top left to bottom right: non-site-specific, noncovalent immobilization by physical adsorption; non-site-specific, covalent immobilization by covalent binding; site-specific, noncovalent immobilization by bioaffinity coupling; and site-specific, covalent binding by bioorthogonal coupling. Note, the dashed lines between the green antibodies and the surface (upper and lower left schemes) represent weak physical interactions, whereas the solid lines (upper and lower left schemes) represent covalent strong interactions between the surface and the antibody, respectively.</p><p>Reproduced with permission from [49]. Copyright (2013) American Chemical Society</p><!><p>Among the four immobilization methods, biomolecule immobilization on paper by physical adsorption is the most straightforward and frequently used technique. Thereby, the immobilization is conducted by simply dipping paper into a biomolecule-containing solution, or coating and printing this solution onto paper with a subsequent washing step. Generally, no further pre-/post-treatments procedures are necessary. Hence, coupling is assured by hydrogen bonds, electrostatic/ionic interactions, van der Waals forces, or hydrophobic interactions between the paper substrate and the biomolecule. The main advantage of this immobilization technique is the simple coupling procedure, whereas the disadvantage is the relatively weak and non-site-specific binding [49–52].</p><!><p>Functional groups available in proteins and corresponding surface functionalities required for coupling [37]</p><p>Chemical nature of covalent bonds between model substrates (gray) and biomolecules (green).</p><p>Reproduced with the permission from the Royal Society of Chemistry from [37]. Copyright (2013) American Chemical Society</p><p>Bioaffinity-based immobilization of a biomolecule (green) an a model substrate (gray) using avidin (blue) and biotin (yellow)</p><p>Bioorthogonal biomolecule (green) immobilization via copper-catalyzed azide–alkyne cycloaddition (CuAAC) on a model substrate (gray)</p><!><p>Analytical devices which are commercially available as of today and which consist in part of cellulosic material rely on two different types of assays, paper test strips and lateral flow assays (LFAs) [59]. Test strips, also called dipsticks, are used for a broad range of applications, from environmental, food and beverage probes to diagnostics. Commonly, they are used to determine or prove pH values, the presence of heavy metals, glucose levels, or peroxidase activity [60]. LFAs, on the other hand, are used in diagnostics, environmental or food safety control. The mixtures to be assessed can be very complex. Besides urine or saliva, even whole blood can be tested [61]. The most prominent LFA is the home-use pregnancy test. Other important applications are, e.g., the detection of pesticides or pathogens in food [60] or a test against anti-SARS-CoV-2 antibodies in human blood samples [62]. The success of paper-based analytical devices on the market is based on the simplicity of the execution and their readout, which can be performed by an untrained user often by naked eye detection.</p><!><p>From left to right. A dipstick is taken from the package. The test is performed. The strip is compared to a reference depicted on the package.</p><p>Reproduced with permission from [65]. Copyright Merck KGaA Darmstadt</p><!><p>Test strips are user-friendly, inexpensive, and deliver quick results. However, despite all the ease in use of the dipstick assay, they exhibit certain drawbacks. Color can intensify and thereby falsify the test result when incubation times are exceeded. Thus, precise timing is required to obtain semiquantitative results. Additionally, the interpretation of a color is always subjective, so that the result obtained depends on the tester. Besides, the sensitivity and limit of detection are defined by the pore volume of the paper patch, as it limits the applicable sample volume [63].</p><!><p>Glucose test strip composition for a bottom readout. Figure created with biorender.com. Protein structures from PDB (GOD: 1GPE; POD: 1HCH).</p><p>Reproduced from [66]</p><!><p>Glucose is a very well-known representative to be tested in a dipstick format. It is commonly part of a combined urinalysis dipstick to determine the glucose content in urine for diabetes mellitus screening. But they are frequently used in the food and beverages industry, where the knowledge of glucose levels in raw materials and final products is important [67].</p><!><p>1. Conversion of glucose to hydrogen peroxide and gluconic acid by glucose oxidase. 2. The colorless compound o-dianisidine is converted to the oxidized state by peroxidase in the presence of hydrogen peroxide. A red dye is formed by acidification</p><p>Summary of commercially available paper test strips [68]</p><!><p>Lateral flow assays (LFA) can be subclassified into lateral flow immunoassays (LFIA) and nucleic acid lateral flow assays (NALFA). LFIAs are protein-based assays relying on antibody–antigen interactions. NALFAs are based on the hybridization of two complementary DNA or RNA strands, making use of a different interaction mode. In the following, the setup and operation mode for the LFIA will be described, as this is the predominant test setup used, but the overall logic principles are similar in both classes.</p><!><p>Setup of a lateral flow assay for the detection of one analyte</p><!><p>By capillary forces the sample migrates through the strip by passing through the conjugate pad. This component of the strip holds the detector particles. These are target-specific molecules, like antibodies, that are conjugated to fluorescent or colored particles. The so-called label is required to meet certain terms, some of them being stability in dried state and in solution under various conditions, susceptibility for detection over a large and useful dynamic range, low nonspecific binding, and commercial availability at low cost. The most widely used label are gold nanoparticles, which have an intense purple color, a high stability both in dried and liquid environments, and no need of instrumentation for visualization. Latex microspheres very versatile systems as they can be tagged with a variety of detector reagents, like color or fluorescent dyes, or magnetic and paramagnetic components. Besides, the conjugate pad contains carbohydrates, which act as preservative and resolubilization agents for the conjugate, to ensure a consistent release between individual test strips.</p><p>When the analyte is bound to the label, they both migrate to the detection zone on the porous membrane, where specific biological components, like antigens or antibodies, are immobilized in lines to interact with the conjugate-bound analyte and the conjugate itself. The membrane is considered as the most critical element of the LFA and in the majority of cases is made of nitrocellulose. Advantages and disadvantages of this polymeric material, which is essentially not a "paper", have been outlined above. Important parameters of the membrane are the capillary forces, the ease of binding or immobilizing proteins, and the flow time. The flow time is the time required for the liquid to travel through and completely fill the strip. This is crucial as it defines the time for interaction of the analyte–conjugate and the biomolecules at the test and control line. The test line is responsible for the analyte recognition, while the control line indicates the proper liquid flow throughout the membrane.</p><p>The liquid finally flows to the end of the membrane and there into the absorbent pad. This last compartment of the test strip maintains the capillary flow, and prevents backflow by wicking excess fluid. Therefore, it also allows the use of larger sample volumes, which increases the test sensitivity. The readout of either appearing or disappearing lines is performed with the naked eye or a dedicated reader [61].</p><p>The assay can be performed in two formats, as a direct, so-called sandwich assay or as a competitive one. The decision of which of the formats can be used depends on the size of the antigen. The direct assay is suitable for all larger analytes/proteins with more than one epitope, meaning a cluster of amino acids, accessible for the specific antibodies, at the same time. The specificity of the antibody–antigen interaction arises from the compatibility of the surfaces of the two species [61, 69]. In a sandwich assay, the antibodies on the label and the test line are complementary and can bind simultaneously to the antigen. The appearance of the test line indicates a positive result.</p><!><p>Formats of a LFIA: competitive format (top); sandwich format (bottom).</p><p>Adapted from [70]. Figure created with biorender.com</p><p>First µPAD for simultaneous glucose (red color change) and protein (blue color change) detection in urine.</p><p>Reproduced with permission from [71]. Copyright (2007) Wiley-VCH</p><p>Schematic representation of µPAD fabrication by laser cutting (top), photoresist application (middle), and wax printing (bottom)</p><p>Analyte and sample loss due to either adsorptive effects on the cellulose fibers before the test line, evaporation during fluid transport through the device, or insufficient binding kinetics due to a non-optimized capillary flow times.</p><p>The detection limit of µPADS with classical readout, e.g., colorimetric sensing, is often insufficient for low abundant analytes because of their restricted sensitivity.</p><!><p>LFA tests can have a variety of functionality and signal intensity because of differing paper or (bio-)molecule quality or through varying environmental conditions [73, 79, 90]. As a result of these still unsolved challenges, as of today, µPADs yield only qualitative signals and a quantitative readout is not trivial without any further equipment in the peripheries. One of the most challenging parameters to control in µPADs is the fluid flow through the device. The fluid velocity is an important factor that especially influences the reaction time (kinetics) between the analyte in the fluid front and the functional detector molecule on the paper surface, thus the sensitivity and limit of detection (LOD) of the µPAD are strongly influenced by the capillary flow times (CFT). There are several ways of controlling the CFT, which progress from adjusting the porous structure of the paper itself to inclusion of dissolvable barriers (viscosity modulators) in the channels, or a geometric control of the channel volume. Methods have been outlined in detail in the literature in various recent articles [80, 83, 84, 86, 88, 90–96].</p><!><p>a Fluid transport through µPADs with different channel geometries. The fluid front is initially at the same level for all µPADs (up to the time of image A). For an expanding channel, the fluid front slows down in the broader segment compared to if it had continued in the same initial channel width (image B). In graph C, the fluid front distance is plotted vs. the square root of time for the channels A to D. Reproduced with permission from [94]. Copyright (2011) Springer Nature b Dissolvable trehalose barrier in the right fork slows down the fluid flow, compared to the left fork with unhindered fluid flow. Reproduced from [95] with permission from the Royal Society of Chemistry. Copyright (2010) Royal Society of Chemistry c Operation principle of a dissolvable bridge: the bridge material dissolves to a permanent shut-off state after passing a well-defined volume of fluid at t3. Adapted with permission from [96]. Copyright (2013) American Chemical Society d Design of a simple two-reservoir (A1, A2) µPAD with mechanical switches (S1, S2) that control the fluid flow into the reaction zone (B)</p><p>Adapted with permission from [97]. Copyright (2008) American Chemical Society</p><!><p>Finally, CFT can be controlled well by adjusting the porous structure of the paper material itself. With respect to the latter, papermaking technologies are in place to vary, e.g., the pore sizes within the paper sheet. The latter directly affects capillary forces and thereby controls the CFT over a wide range [80].</p><!><p>a Schematic principle of fluid flow in a 3D µPAD made by hydrophilic/hydrophobic stacked paper layers. Reproduced with permission from [73]. Copyright (2018) Elsevier b 3D µPAD that distributes four different sample reservoirs into an array of 64 detection zones (A; 10 µL sample volume per reservoir) and 1024 detection zones (B, 100 µL sample volume per reservoir). Adapted with permission from [98]. Copyright (2008) National Academy of Sciences USA c Scheme of the fabrication principle for a one-paper 3D µPAD based on double-sided wax printing. Reproduced from [99] with permission from the Royal Society of Chemistry. Copyright (2015) Royal Society of Chemistry d Composition of a µPDA with switchable filter unit that is incorporated by mechanical pulling of the filter strip. Reproduced with permission from [97]. Copyright (2008) American Chemical Society e Scheme of "one paper sheet 3D µPAD", prepared by origami technique.</p><p>Reproduced with permission from [101]. Copyright (2011) American Chemical Society</p><!><p>A further trend in the field is the ongoing simplification of its production. In this context, the work of Liu et al. should be outlined; they described the production of 3D µPADs from a single sheet of paper with folding by hand, a procedure which is called as the origami technique. The device is made from patterned paper that is obtained by a single-step photolithographic modification. They showed a two-analyte assay of glucose and protein in urine on a single origami-fabricated paper 3D µPAD [101].</p><!><p>LEGO-like modular assembly for setting up a user-defined lateral-flow immunoassay.</p><p>Reproduced with permission from [102]. Copyright (2018) Wiley-VCH</p><p>a Procedure for the light-induced immobilization of benzophenone-containing polymers on paper fibers by using a photolithography mask. b Reaction mechanism of light-induced crosslinking of benzophenone moieties. c Schematic of the microfluidic paper-based glucose sensor. The glucose test solution is transported through the sensor by capillary forces only, where it is oxidized to gluconolactone, with hydrogen peroxide as a co-product, as it passes through the glucose oxidase (GOx) patch. Subsequently, hydrogen peroxide oxidizes the non-fluorescent red Amplex dye to red-fluorescent resorufin in the peroxidase (POx) patch. d Grayscale fluorescence microscopy values of GOx-modified paper (black) and glucose oxidase in solution (red; solution was dropped onto unmodified paper substrates for examination) plotted against glucose concentration.</p><p>All figures are reproduced with permission from [85]</p><!><p>The readout of new paper-based analytical devices can be divided in two classes and is based on either an optical or an electrochemical detection system. All of the systems mentioned above rely on optical detection methods, which are among the most inexpensive, simple, and universal methods available [103]. Depending on the nature of the optical signal, the readout can be as easy and cheap as naked eye detection for colorimetric methods [104, 105]. As already mentioned, different lightning conditions or varying visual perception of people can influence the outcome. Therefore, the use of color charts or detectors, among them scanners, cameras, including those of smartphones, can be suitable for the improvement of the readout. None of those devices need special skills to be operated. In particular, smartphone cameras are a promising tool, considering that 67% of the world's population own these devices, rampant with growing ownership, particularly in the developing world [75]. Images taken of a µPAD could be analyzed by user-optimized software or sent for analysis by a specialist making use of telemedicine [105, 106]. Digital cameras can be used with automatic white balance, which should improve the image quality. But one has to respect that digital cameras and smartphone cameras cannot focus objects which are too close to the lens, whereas office scanners capture images independent of external light sources, ensuring consistent color intensity. The generation of high resolution images, with the µPAD always in focus, is another advantage of scanners which are also widespread devices [105]. Picture analysis can be carried out with standard image analysis or using customized software [75].</p><!><p>The left image shows a reference card with space in the middle to place the dipstick. The right image shows the dipstick placed in the middle of the reference card and the use of the corresponding smartphone app.</p><p>Reproduced with permission from [108]. Copyright Merck KGaA, Darmstadt</p><!><p>Besides, more complex devices are used for optical readout. Photoelectric meters or transmittance colorimeters were built that are made of readily available components out of an electronics supply catalogue. Spectrophotometers, fluorometers, microplate readers, photomultiplier tubes, and gel documentation systems are useful, but more sophisticated and rather impractical for point-of-care testing [75].</p><p>A major disadvantage of optical sensors is their susceptibility to light, insoluble compounds, and dust. Challenges of this kind can be conquered by the second class of sensor systems, electrochemical detection systems [109]. Those can of course be found in laboratories. However, field measurements and detection can also be carried out with homemade systems, consisting of off-the-shelf units, which are low cost and rather simple. The systems can be designed to be mobile and powered by batteries or by the network, which is especially interesting for their usage in developing countries [110]. The type of measurement, such as voltammetric, amperometric, potentiometric, or conductivity-based, can be adapted to suit the assay and analytes to be tested [75].</p><!><p>Origami µPAD for adenosine sensing. Paper was wax-printed on the left side, to define an inlet, two hydrophilic channels, and the hourglass-shaped reaction area. There is a thin connection between the two half-cells that acts as a salt bridge. On the right side of the same paper, screen printing was used to define a hydrophilic inlet pattern. Also two carbon electrodes were printed on the right side, which are positioned above the reaction area, when the paper is folded in the middle. The paper is laminated after folding with cutouts to enable a connection of the electrodes and sample application. The reagents were preloaded in the hourglass-shaped pattern. DNA-labeled microbeads are deposited in the hydrophilic channels. An aptamer is loaded in one of the channels. When the sample is applied to the device, DNA-labeled glucose oxidase (GOx) is released downstream when the aptamer binds its target adenosine. The other channel serves as control, where no aptamer is present. The freed GOx catalyzes the oxidation of glucose and thereby the formation of [Fe(CN)6]4−. The resulting voltage between the two half-cells due to the difference in concentration of both ionic species can be measured with the help of a digital mutlimeter and a capacitor.</p><p>Adapted with permission from [111]. Copyright (2012) Wiley-VCH</p><!><p>To build the device, a foldable piece of paper was wax-printed on one side, to define an inlet, two hydrophilic channels, and the reaction area having the shape of an hourglass. The thin connection between the two half-cells acts as a salt bridge. On the opposite side, screen printing was used to define a hydrophilic inlet pattern and two carbon electrodes, which are positioned above the reaction area, when the paper is folded. The reagents, glucose and [Fe(CN)6]3−, were preloaded in the hourglass-shaped pattern. In both hydrophilic channels, DNA-labeled microbeads were deposited. In one channel an aptamer was loaded that binds the incoming target adenosine by releasing DNA-labeled glucose oxidase (GOx) downstream. The other channel serves as control, as no aptamer is present. The freed GOx catalyzes the oxidation of glucose and thereby the formation of [Fe(CN)6]4−. The resulting voltage between the two half-cells due to the difference in concentration of both ionic species can be measured as described above.</p><!><p>A test strip for complex fluids was developed for the use in combination with a commercial glucometer by Nie et al. [94]. Chromatography paper was modified using wax and screen printing. Reagents were stored in the bottom of the strip. When the sample is applied the compounds migrate through the patterned test strip, allowing the electrical readout with a commercial glucometer.</p><p>Reproduced from [109] with permission from the Royal Society of Chemistry. Copyright (2010) Royal Society of Chemistry</p><!><p>This review summarizes the basic steps to generate functional papers made of cellulose, the world's most abundant raw material source, beginning with the fundamental chemical properties of cellulose, the possible functionalization strategies, to commercially available functional papers, and the current state of the art of functional papers at the research level. The review also includes a short introduction to papermaking and its history. Thanks to intensive research work, biochemical sensors can be produced that are able to detect specific target analytes in biological samples on paper-based devices. By tuning the fluid flow, the µPAD sensitivity can be enhanced and the detection limit can be lowered. Depending on the assay and fabrication design, they can range from very simple, inexpensive, and being produced in high counts to very sophisticated assay designs with handmade devices. Although already a large number of demonstrations exist to date, there are still major challenges ahead until these types of biosensors will enable medical analysis, address even personalized medical point-of-care issues, and may be used even in remote areas in a broad applicational fashion. With respect to these challenges, answers will to a large extent be given once we start looking more deeply into understanding the highly complex material paper itself. Any successful design of a functional high-tech paper in medical analysis in the future requires a fundamental understanding of the impact of paper's intrinsic parameters (fiber source, fiber pretreatment, paper porosity, mechanical paper properties, etc.) along different length scales on functionalization, capillary transport, and colorimetric/electrochemical readout with paper-based analytical devices. Glucose test strips and pregnancy tests are most likely just the beginning of the research success with a bright future for these sustainable paper-based point-of-care diagnostic devices.</p><!><p>Publisher's Note</p><p>Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.</p><p>Laura M. Hillscher and Valentina J. Liebich contributed equally to this work.</p>

PubMed Open Access

Single Particle Assays to Determine Heterogeneities within Fluid Catalytic Cracking Catalysts

AbstractFluid catalytic cracking (FCC) is an important process in oil refinery industry to produce gasoline and propylene. Due to harsh reaction conditions, FCC catalysts are subject to deactivation through for example, metal accumulation and zeolite framework collapse. Here, we perform a screening of the influence of metal poisons on the acidity and accessibility of an industrial FCC catalyst material using laboratory‐based single particle characterization that is, μ‐XRF and fluorescence microscopy in combination with probe molecules. These methods have been performed on density‐separated FCC catalyst fractions, allowing to determine interparticle heterogeneities in the catalyst under study. It was found that with increasing catalyst density and metal content, the acidity and accessibility of the catalyst particles decreased, while their distribution narrowed with catalyst age. For example, particles containing high Ni level possessed very low acidity and were hardly accessible by a Nile Blue dye. Single catalyst particle mapping identifies minority species like the presence of a phosphated zeolite ZSM‐5‐containing FCC additive for selective propylene formation, catalyst particles without any zeolite phase and catalyst particles, which act as a trap for SOx.

single_particle_assays_to_determine_heterogeneities_within_fluid_catalytic_cracking_catalysts

<!>Introduction<!><!>Introduction<!>Results and Discussion<!><!>Bulk catalyst characterization trends<!>Particle distributions within density‐separated catalyst<!><!>Particle distributions within density‐separated catalyst<!><!>Particle distributions within density‐separated catalyst<!>Correlations between metal type and content and catalyst particle acidity and accessibility<!><!>Correlations between metal type and content and catalyst particle acidity and accessibility<!><!>Minority species<!><!>Conclusions<!>Experimental Section<!>Conflict of interest<!>

<p>A.-E. Nieuwelink, M. E. Z. Velthoen, Y. C. M. Nederstigt, K. L. Jagtenberg, F. Meirer, B. M. Weckhuysen, Chem. Eur. J. 2020, 26, 8546.</p><!><p>The main chemical process to produce gasoline and propylene is the cracking of vacuum gas oil (VGO) and heavy gas oil (HGO) with the use of a solid catalyst material. This multicomponent and hierarchically structured catalyst consists of spray‐dried porous spheres with an average diameter of 50–150 μm and contains next to zeolite also a clay and binder material, such as silica and alumina. The catalyst design is such that the mixture of hot catalyst and crude oil behaves like a liquid; hence the name fluid catalytic cracking (FCC).1, 2 The main active sites that catalyze the cracking of VGO are the Brønsted acid sites in the zeolite material, but the large crude oil molecules are pre‐cracked in the matrix of the FCC particle, prior to entering the crystalline micropores of the zeolite. Commonly employed zeolites in the FCC process include the synthetic ultra‐stable zeolite Y (ultra‐stable Y or US‐Y, FAU framework structure) and, to a lesser extent, zeolite ZSM‐5 with the MFI framework structure. Due to the smaller micropore structure of zeolite ZSM‐5 in comparison with that of zeolite US‐Y (i.e., 5.6 vs. 7.4 Å), zeolite ZSM‐5 increases the FCC catalyst selectivity towards propylene. They are added to the FCC catalyst inventory as separate FCC catalyst particles, and are visually not distinguishable from normal FCC catalyst particles containing zeolite US‐Y.3, 4</p><p>During the FCC process, VGO and HGO are cracked by the hot FCC catalyst in the riser reactor, after which the formed products are separated from the catalyst. Remaining carbonaceous species on the catalyst material block the active sites, preventing further cracking activity. The spent catalyst is, therefore, subjected to a regeneration procedure through high‐temperature calcination, making the FCC particles available for a consecutive cycle of crude oil cracking. The FCC particles can, however, also deactivate irreversibly due to the accumulation of metals originating from the crude oil feedstock, with Ni, Fe and V being the most notorious ones, resulting in a reduced porosity or metal promoted side reactions. Also, the harsh steaming conditions during catalyst regeneration induce dealumination of the embedded zeolite material, thereby decreasing the amount of available Brønsted acid sites. To preserve the overall activity in a reactor, part of the spent catalyst material is constantly being replaced with fresh FCC catalyst particles, leading to an equilibrium in the reactor with a mixture of FCC catalyst particles with different ages and degrees of deactivation. This mixture is called the equilibrium catalyst, further denoted as ECAT.5, 6</p><p>The previously mentioned deactivation processes, involving metal accumulation and zeolite dealumination, all occur simultaneously during the FCC process. Most studies, however, address only one of these processes, making use of lab‐deactivated FCC catalyst samples. Separating these two effects makes their investigation easier: the influence of dealumination due to steaming or the accumulation of metals coming from the oil feedstock on the catalytic performance of the FCC catalyst have been thoroughly studied on both the single particle and the bulk level.7, 8, 9 Furthermore, as mentioned before, due to the mix of fresh and deactivated FCC particles, the actual ECAT has a large intrinsic interparticle heterogeneity. This complicates the study of these industrially used catalyst particles even more.10</p><p>Our group has performed extensive work on the characterization of ECAT and FCC particles. Buurmans et al. used several staining techniques to visualize the acidity of ECAT and lab deactivated single FCC particles.11, 12 These staining techniques have been combined with for example TEM to correlate the acidity with structural features in one FCC particle.10 In later work by Ristanovic et al., extensive fluorescence microscopy work revealed more details on the activity of single zeolite domains by performing single molecule tracking.13 Kerssens et al. performed staining experiments on bigger samples of a few milligrams, to visualize the acidity of both fresh FCC and ECAT particles.14 For the accumulation of metals, Ruiz‐Martínez et al. revealed a core–shell deactivation due the accumulation of metals and coke using non‐invasive 2D micro X‐ray fluorescence (μ‐XRF). With X‐ray nano‐tomography, Meirer et al. studied the distribution of deactivating metals in more detail. They performed a density separation to classify three categories (low, medium and high metal loaded particles) and found that Fe forms a shell of reduced porosity in the outer 2 μm layer of the particle while Ni and V are distributed more throughout the whole particle.15 Most recently, the role of Ni in the deactivation of ECAT was studied with single particle X‐ray fluorescence‐diffraction‐absorption tomography by Gambino et al.16 Buurmans et al., however, already argued that a disadvantage of single particle characterization methods is the difficulty to obtain statistically relevant data.8 For example, due to the use of expensive and time‐consuming synchrotron related techniques,17 the amount of particles that can be analyzed is limited. Although the previously mentioned studies provided useful insights through extensive characterization of ECAT catalysts, they mainly focus on one deactivation process at the time with limited amounts of particles.</p><p>Recently, Solsona et al. did an individual assessment of multiple FCC particles via microfluidic sorting, based on the particles' magnetic moment. The sorted samples obtained were too small to perform bulk characterization techniques, but it was possible to analyze a significant number of particles on the single particle level.18 For this analysis, after sorting the FCC particles, all collected fractions were analyzed with μ‐XRF mapping and fluorescence microscopy using staining molecules to characterize the fractions' metal content, acidity and accessibility.</p><p>Here, we continue this line of single particle diagnostics research by presenting a study about the influence of different deactivation processes on the activity of an ECAT. In particular, we report a characterization method, using laboratory‐based techniques, that bridges the gap between single particle and bulk analyses, as depicted in Scheme 1. We use the same set of characterization techniques as reported by Solsona et al. and try to further correlate the single particle mapping data on ECAT fractions sorted by a classical density separation. We have previously reported such a correlation to reveal interparticle heterogeneities in polymerization catalysts.19 We use these correlations to identify relationships as well as special FCC catalyst particles.</p><!><p>Schematic of the research approach. An FCC ECAT was density separated into fractions and analyzed for its acidity, accessibility and metal content. Bulk characterization techniques revealed trends as a function of density. Micro‐spectroscopic methods were employed for large scale single particle mapping. This screening allowed for determining interparticle distributions within each fraction as well as establishing single particle correlations.</p><!><p>A calcined ECAT was sorted into six fractions, using density gradient separation with different diiodomethane/acetone mixtures.20 The increasing density per fraction is attributed to the accumulation of metals like Fe, Ni and V and, thus, to the catalytic age of a particle. It is generally accepted that the activity of an FCC particle decreases with increasing metal loading.21 By sorting this ECAT on density, the interparticle heterogeneity within each age fraction is reduced to facilitate a more precise characterization. Bulk characterization was established with traditional methods such as inductively coupled plasma‐optical emission spectroscopy (ICP‐OES), temperature programmed desorption with NH3 (NH3‐TPD) and N2 physisorption. In this work, the term "bulk technique" is used to address techniques that analyze a relatively large sample of ECAT particles and give an ensemble average as outcome. Each density separated ECAT fraction was, subsequently, characterized with multiple micro‐spectroscopic techniques to visualize the interparticle heterogeneity within each fraction. Specifically, fluorescence micro‐spectroscopy and subsequent μ‐XRF measurements were performed on a set of the same particles to obtain correlated information on many single FCC ECAT particles. Correlations between acidity, accessibility and metals accumulation could be made.</p><p>From this comprehensive approach, conclusions on three different levels could be drawn, as indicated in Scheme 1. First, bulk trends regarding the acidity, accessibility and metal accumulation as a function of catalyst age were established. Second, within each density separated fraction, large scale single particle mapping revealed the level of heterogeneity within these catalyst fractions. Finally, this particle screening resulted in detailed correlations regarding the catalyst properties and highlighted the presence of minority species that cannot be detected in neither bulk nor single particle analysis. The key of this presented methodology relies on single particle analysis with statistical relevance.</p><!><p>A regenerated ECAT from a commercial FCC unit was sorted in six fractions using a density gradient separation protocol, which was based on a sink‐float method in different acetone/diiodomethane (DIIM) mixtures. More details can be found in the Supporting Information. Figure 1 a and Table S2 demonstrate that most FCC catalyst particles end up in the fractions with the lowest density. More specifically, more than 60 % of the ECAT particles has a skeletal density below 2.47 g cm−3. In comparison, depending on the amount of zeolite material present and the source of binder material and clay, skeletal densities between 2.4 and 2.8 g cm−3 have been reported for fresh FCC catalyst particles.21−23</p><!><p>a) An FCC ECAT was density separated using a sink‐float method in different acetone/diiodomethane (DIIM) mixtures. The percentage of particles collected per fraction decreases with increasing density. Bulk characterization methods showed trends in metal accumulation, acidity and accessibility. ICP‐OES data (b) shows an increasing metal content. Quality control samples indicating the accuracy of the measurements give Fe=98 %, Ni=102 %, V=101 %, Ca=98 %. NH3‐TPD results (c) show a decreasing acidity and the pore size distribution as determined with N2 physisorption (d) shows a decrease in the amount of mesopores with increasing density. Fraction 6 does not follow these trends; this interesting outlier will be discussed in detail in the main text.</p><!><p>Bulk characterization techniques, in this work referred to as techniques that were applied to a large sample of ECAT particles and including NH3‐TPD, N2‐physisorption, and ICP‐OES, revealed the bulk trends as function of density in acidity, accessibility and metal accumulation, respectively. Together, these bulk analyses provide useful insights into the level of catalyst deactivation as a function of catalytic age. In correspondence with previous studies, the metal content of FCC particles is correlated with density, while acidity and accessibility are negatively correlated with density. Since we are investigating an industrially used and regenerated ECAT, all particles already have been used in at least one cracking cycle and could, theoretically, already be considerably deactivated. Therefore, we expect a relatively low overall acidity and accessibility in the particles.</p><p>Figure 1 b indicates increasing levels of Ca, Fe, and V with increasing density, suggesting longer residence times in the FCC reactor. Also, the overall level of Ni is relatively low in this specific FCC ECAT.</p><p>Three striking observations can be made from these bulk trends. First, as seen in Figure 1 c, the decrease in acidity appears most significant going from fraction F1 to F2 and from F2 to F3, while fractions F3 to F5 have similar acidities. CO FT‐IR spectroscopy (Figure S4) indeed confirms that most Brønsted acid sites are removed going from fraction F1 to F2 and indicate that strong Lewis acid sites even only exist in fraction F1. Also, mesopores of around 6 nm (Figure 1 d) and the micropore volume (Figure S2c) decrease significantly from fraction F1 to F4. This is a clear indication of the destruction of zeolite domains, the only microporous material in an FCC particle, during the aging of the particle. Furthermore, the mesopores of 20–30 nm as observed in fraction F1 (Figure S2b), disappear completely upon ageing. This is ascribed to the accumulation of metals in the matrix of the particles, blocking the accessibility for oil molecules.</p><p>Second, both fractions F5 and F6 appear to lack a presence of zeolite domains, as both fractions demonstrate a low level of La compared to fractions F1–4 (see ICP‐OES results in Figure S1). The La level can be used as a marker for zeolite US‐Y domains, since this rare earth metal is used as stabilizing agent during the synthesis of this zeolite.24, 25 Therefore, fraction F5 and F6 contain non‐FCC particles without zeolite domains. Considering only fractions comprising actual FCC particles (fraction F1 to F4), their La levels are fairly similar for fractions F1 to F3, which is an indication for a comparable amount of zeolite domains in these fractions, and slowly decreases to fraction F4. From literature, it is not expected that La will leave the particles upon zeolite framework collapse.25 The absence of zeolite domains in fractions F5 and F6 is further rationalized by the deviating pore size distribution as indicated in Figure 1 d. These two observations indicate that employing density gradient separation allows for filtering out most non‐FCC particles from the ECAT batch.</p><p>Third, the composition of fraction F6, the heaviest fraction, does not follow the trends set by the first five fractions. The Mg and V levels are particularly high in this most dense FCC fraction, while as shown in Figure S1 the level of Ti, a marker for the clay component of a typical FCC particles' matrix26 is very low. This suggests that fraction F6 mainly contains Mg‐based traps for vanadium or SOx,3, 27, 28 which we were able to separate from the FCC catalyst particles using density separation. These particles possess a low porosity, but have a relatively high Lewis acidity, most probably due to the presence of acidic vanadium oxide.</p><!><p>The bulk characterization techniques as discussed in Figure 1 provide overall trends and insights in the investigated ECAT material. Single‐particle mapping, however, provides a more in‐depth analysis of the interparticle heterogeneity of the investigated FCC samples. These results give distributions of the acidity, accessibility and metal accumulation in every fraction. For every fraction, a minimum of 90 particles was analyzed. The exact amounts are given in Table S3.</p><p>Figure 2 a shows histograms of the fluorescence intensity per particle after the Brønsted acid catalyzed oligomerization of 4‐methoxystyrene. This fluorescence intensity is a measure for the number of available acid sites. With this staining method, using a styrene derivative as probe molecule, the amount of available acid sites can be visualized, as was shown before by Buurmans et al.8, 11, 12 In line with our previously shown bulk results in Figure 1 c, the acidity decreases with density. For an assessment of the particles' accessibility, staining experiments were performed using Nile Blue A, a large conjugated fluorescent molecule that can enter the macro‐ and mesoporous pore structure of an FCC particle.8 Similar to the acidity measurements in Figure 2 a, the fluorescence intensity from Nile Blue reflects the particle accessibility. The distributions in Figure 2 b indicate that also the average accessibility decreases with increasing density.</p><!><p>Single particle mapping revealing interparticle distributions within each density separated ECAT fraction. For each particle in field of view, the fluorescence of styrene oligomerization reaction products indicates the level of acidity (a) and the fluorescence of Nile Blue demonstrates the accessibility (b). Each scale bar equals to 200 μm.</p><!><p>Strikingly, the trends in the acidity distributions show an almost bimodal behavior. Within the fractions F1–F3, the distributions of acidity demonstrate a large spread and thus significant interparticle heterogeneities. Fractions F4–F6, however, show more narrow distributions with low fluorescent intensities. We ascribe the heterogeneity in the lighter fractions to a convolution of a natural density spread and ageing. Due to the spray drying process, we expect a density gradient already in the fresh catalyst. Particles that are, therefore, denser from the start, can end up in a fraction together with aged particles. Fraction F4 only shows non‐fluorescent particles, suggesting the absence of acid sites and the blockage of pores. The decrease in porosity is less pronounced than the drop in acidity. The large spread in the accessibility of particles in Fraction F4 suggests that the deactivation of Fraction F4 is due to a combination of pore blocking by Fe, as reported by Meirer et al. and the absence of acid sites.21, 29</p><p>A different explanation for the non‐fluorescent particles in all fractions, relies on the presence of other type of particles, such as additives or spray‐dried particles without zeolites. The absence of Brønsted acid sites can be a result of synthesis or due to severe dealumination. Bulk analysis showed that such particles are typically found in fractions F5 and F6. Regardless of the origin, these so‐called non‐FCC particles are not as porous as the active FCC particles and do not contain Brønsted acid sites to perform the oligomerization of 4‐methoxystyrene. Due to a low metal loading, however, they can also end up in the lightest fractions. Although they show low fluorescence intensities regarding acidity and accessibility, they are properly sorted into the correct age fraction.</p><p>Finally, μ‐XRF experiments provided the XRF intensity of different metals per particle. In Figure 3, the distributions of Fe, V and Ni as a function of density are shown. The average μ‐XRF values per element and fraction follow the trends as previously observed with bulk analysis (ICP‐OES). Figure S5 presents the average μ‐XRF values for the other most abundant metals as indicated with ICP‐OES. It was found that for each metal, the average μ‐XRF intensity per density separated fraction follows the trend from bulk analysis. This means that the single particle mapping method provides an accurate representation of the bulk material, indicating that we have studied a statistically relevant number of particles. The main advantage of this μ‐XRF mapping over bulk methods like ICP‐OES, however, is that interparticle heterogeneity is revealed.</p><!><p>Single particle mapping revealing interparticle distributions within each density separated ECAT fraction. For each particle in field of view, the XRF intensity represents the content of Fe (a), V (b) and Ni (c). Each scale bar equals to 200 μm.</p><!><p>The Fe distributions appear narrower compared to styrene and Nile Blue in Figure 2. Also, the differences between fractions are less pronounced and more difficult to visualize. Therefore, to show clear trends and capture outliers with either very low or high Fe contents, we need more particles as compared to the acidity and accessibility studies. It is interesting to see the broad V distribution of fraction F6, while the other fractions contain only limited amounts of V. The distributions of Ni are narrow and at relatively low intensities and indicate an overall low amount of Ni in this ECAT. All μ‐XRF images per fraction are of the same region and can therefore be directly overlaid.</p><!><p>By performing μ‐XRF and fluorescence microscopy measurements at identical positions, single particle mapping can also provide correlations between the acidic properties and the metal content of particles. These correlations allow for detecting the presence of minority species, provide additional information about the FCC ECAT, or reveal new trends that were not revealed with bulk analyses.</p><p>In FCC deactivation research, the Ni content of a particle is often used as indication for the age of a catalyst: Ni is accumulated during the cracking process but is not present in the fresh catalyst (such as Fe in the clay component of the FCC catalyst). Furthermore, Ni inside an FCC particle is not very mobile. Therefore, the more Ni an ECAT particle contains, the longer it has been cycling through the reactor and the more Fe and V it contains.30 Consequently, it is expected that these particles show a low acidity and accessibility and thus a low fluorescence of Nile Blue (NB) and 4‐methoxystyrene.</p><p>Correlation plots show the relation between fluorescence, Fe and Ni, where each measured particle is represented with one data point. These plots originate from the overlay of the corresponding μ‐XRF maps and the resulting single particle intensities. A detailed explanation of the data analysis can be found in Figure S6. The overlaying maps of acidity and Ni in Figure 4 a demonstrate an anti‐correlation between acidity and Ni content in each fraction. Closer inspection reveals that particles with a high Ni content always show low fluorescence, and therefore low acidity. We can therefore conclude that indeed, Ni acts as a marker for catalytic age of an ECAT. It must be noted that a low level of acidity is indeed linked to a higher degree of deactivation and not to an absence of zeolite domains, as indicated in Figure S7. Due to the overall low levels of Ni present in this ECAT, bulk characterization techniques were not sufficiently accurate to detect this new trend. The correlation of Ni with NB staining (Figure 4 b) is less pronounced, but also here particles with a high Ni content show low accessibility. The correlation between Fe and acidity or accessibility is not as straightforward. We expect a relation where acidity and accessibility decrease with increasing Fe level, due to pore blocking. However, the overlay images in Figure 4 show particles with a high Fe content that still show 4‐methoxystyrene (left) or Nile Blue (right) fluorescence. In these particles, Fe is most likely deposited as clusters. These Fe clusters were already observed in the study of Solsona et al., in which it was demonstrated that particles with high Fe levels are not necessarily heavily deactivated.18</p><!><p>The correlation plots of Fe (green in image) and Ni (blue in image) with fluorescence of 4‐methoxystyrene (a) and Nile blue (b) with corresponding example images show the relation between deactivating metals and acidity/accessibility. Pearson correlations of these plots are −0.014, 0.0011, −0.11 and 0.049, indicating there is no linear correlation found in all plots.</p><!><p>The different correlations between Fe and Ni with acidity and accessibility, lead to the question how Fe and Ni relate to each other. Figure 5 a shows the Ni–Fe correlation plots as a result of overlaying the single particle XRF maps. The correlation appears to be a fan‐shaped plot, indicating that particles with high Ni concentrations generally also have elevated Fe levels. The reverse correlation, on the other hand, does not uphold, meaning that particles with a high Fe concentration do not necessarily have a high Ni content as well. The hotspot of Fe that is observed in the upper right corner of Figure 5 b, is allocated to an FCC particle with a dark spot on the optical image in Figure 5 c. This particle has, thus, a cluster with high concentrations of Fe, but overall, does not have a high skeletal density, as it was sorted in fraction F2. Interestingly, whereas bulk characterization demonstrated that the Fe and Ni contents gradually increase with increasing density, single particle mapping now indicates a significant heterogeneous interparticle distribution.</p><!><p>The correlation plot of the XRF maps of Ni and Fe (a, Pearson correlation: 0.26). Every data point represents an individual particle with Pearson correlation coefficients of 0.54, 0.16, −0.12, −0.18 for F1 to F4 respectively. The overlay images (b) of fraction 2 are shown as an example. The second example (c) shows a Fe outlier with a very high XRF intensity, attributed to a cluster. The threshold of the overview Fe image is chosen as such to display the overall image correct; the outlier is therefore saturated. See Figure S9 (Supporting Information) for details.</p><!><p>Our presented single particle mapping method provides the possibility to determine the composition of many single particles within each fraction. This allows for the detection of outliers and minority species. As was stated before, FCC particles can contain both ZSM‐5 and US‐Y as active cracking components. Like La serves as a marker for zeolite US‐Y, the presence of ZSM‐5 in a particle is indicated by the P content. The ICP‐OES measurements in Figure 1 b did not detect a significant amount or clear trend in the P content per fraction. On the single particle level, however, we observe the presence of ZSM‐5 containing particles in fractions F1 and F2. Figure 6 shows these particles in blue. Interestingly, as shown in Figure 7, these particles contain low levels of Fe and Ni. Second, the Mg based traps for V/SOx are more clearly detected with XRF mapping. Detailed correlation plots in Figure S8 show the direct link of Mg with V. The red indicated particles (Mg) in Figure 6 demonstrate that minor amounts of these traps were collected in fractions F1 to F5, but the majority is collected in fraction F6. Particles not displaying one of the pure distinct colors red, blue, or green, do not contain (enough) zeolite domains (green, blue) or Mg (red) and are indicated as grey in the pie charts. It is important to note that these thresholds are chosen manually; a detailed description can be found in the Supporting Information. From the pie charts, once more the large intrinsic heterogeneity of an FCC ECAT is emphasized. Most striking from these results is the heterogeneity of the La values found for all particles. The large spread in zeolite content is most probably due to the used spray drying technique and again highlights the importance of a method that can perform diagnostics on many single particles: bulk methods cannot give enough information on a sample as complex as an FCC ECAT material. This characterization method can be extended to other systems with a large inter‐particle heterogeneous catalysts, such as olefin polymerization catalysts or metal (oxide) impregnated support oxides.19, 31</p><!><p>Overlay of XRF maps Mg, La and P for fraction F1–F6 and relative percentages of all three. Particles with an XRF intensity for La between 200 and 600 are indicated as light green in the pie charts. More details of this analysis can be found in Figure S9. The blue colour in the overlay image of fraction F5 is below the set threshold and therefore not ascribed to ZSM‐5 domains, but to other additives. The pie charts represent the percentages of the number of particles with Mg, La or P as main content.</p><p>(a) An overlay of Ni (green) and Fe (red) in fraction F1 shows that the zeolite ZSM‐5‐containing catalyst particles (within white circles) generally contain below average levels of Fe and/or Ni. In (b) the ZSM‐5 particles are indicated red (P) while USY particles are indicated green (La).</p><!><p>We have shown that correlating information from multiple techniques per particle, provides valuable insights in the heterogeneity of a density separated FCC ECAT. Furthermore, the possibility to measure multiple particles adds statistical value to the data obtained. Where bulk measurements can give insight in the trends between the six density separated FCC ECAT fractions under study, the diagnostics of single particles can show trends even within each fraction.</p><p>Extensive characterization revealed clear trends in the acidity, accessibility and metal content of the density separated fractions: from fractions F1 to F4 the separation was according to catalytic age. We conclude this from the accumulation of deactivating metals, a decrease in acidity and accessibility. However, we have shown a large spread in acidity and accessibility for these fractions as a result of a convolution of natural density and ageing. Fractions F5 and F6 are mainly composed of additives without zeolites or Mg‐based traps for V or SOx.</p><p>The large intrinsic inter‐particle heterogeneity of each density separated fraction was visualized by overlaying XRF maps of Fe and Ni with fluorescence maps. The degree of deactivation was linked to the Ni content on a single particle level, in a sense that particles with a high Ni content do not show high acidity. Furthermore, correlations of the Mg, La and P levels revealed the presence of ZSM‐5 containing particles in the lightest fractions, while the V/SOx‐traps were collected with increasing amounts in the heavier fractions.</p><!><p>A detailed experimental approach of the described work can be found in section S1 of the Supporting Information (SI). A regenerated ECAT sample, originating from a commercial FCC unit, was calcined at 600 °C for 5 h to remove residual coke species. Subsequently, density gradient separation, as described by Dyrkacz et al.,32 was used to sort the ECAT catalyst particles in six fractions with increasing density. The density separated catalyst fractions have been characterised by Temperature Programmed Desorption (TPD) of NH3 using a Micromeritics ASAP2920 equipped with a TCD detector, Fourier Transform‐Infrared Spectroscopy (FT‐IR) spectroscopy in transmission mode and CO as probe molecule using a PerkinElmer 2000 instrument, N2‐physisorption using a Micromeritics TriStar apparatus and Inductively Coupled Plasma–Optical Emission Spectroscopy (ICP‐OES) measurements using a Spectro Arcos instrument. For the single catalyst FCC particle studies, micro‐X‐ray Fluorescence (μ‐XRF) studies were performed with an Orbis PC SDD instrument with a Rh tube as X‐ray source, while the fluorescence microscopy studies in combination with probe molecules were done using a Nikon upright A1 confocal fluorescence microscope equipped with a 488 nm excitation solid‐state laser source. The probe molecules were 4‐methoxystyrene (Sigma Aldrich, 97 %) and Nile Blue A (Acros Organic, pure). Data correlations were made using an in‐house MATLAB script to register fluorescence and μ‐XRF maps with an optical image for every density separated catalyst fraction.</p><!><p>The authors declare no conflict of interest.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supplementary</p><p>Click here for additional data file.</p>

PubMed Open Access

The Protective Role of D-Glucose Against 1-Methyl-4- Phenylpyridinium Ion (MPP+): Induced Mitochondrial Dysfunction in C6 Astroglial Cells

Impaired mitochondrial function in glial and neuronal cells in the substantia nigra is one of the most likely causes of Parkinson\xe2\x80\x99s disease. In this study, we investigated the protective role of glucose on early key events associated with MPP+-induced changes in rat C6 astroglial cells. Studies were carried out to examine alterations in mitochondrial respiratory status, membrane potential, glutathione levels, and cell cycle phase inhibition at 48 h in 2 and 10 mM glucose in media. The results obtained suggest that MPP+ caused significant cell death in 2 mM glucose with LC50 0.14 \xc2\xb1 0.005 mM, while 10 mM glucose showed highly significant protection against MPP+ toxicity with LC50 0.835 \xc2\xb1 0.03 mM. This protection was not observed with cocaine, demonstrating its compound specificity. MPP+ in 2 mM glucose decreased significantly mitochondrial respiration, membrane potential and glutathione levels in a dose dependent manner, while 10 mM glucose significantly restored them. MPP+ in 2 mM glucose arrested the cells at G0/G1 and G2/M phases, demonstrating its dual inhibitory effects. However, in 10 mM glucose, MPP+ caused G0/G1 arrest only. In summary, the results suggest that loss of cell viability in 2 mM glucose group with MPP+ treatments was due to mitochondrial dysfunction caused by multilevel mechanism, involving significant decrease in mitochondrial respiration, membrane potential, glutathione levels, and dual arrest of cell phases, while 10 mM glucose rescued astroglial cells from MPP+ toxicity by significant maintenance of these factors.

the_protective_role_of_d-glucose_against_1-methyl-4-_phenylpyridinium_ion_(mpp+):_induced_mitochondr

Introduction<!>Materials<!>C6 Astroglial Cell Culture<!>Preparation of Experimental Medium<!>Cell Doubling Period<!>Treatments with MPP+<!>Treatments with Cocaine<!>Effect of MPP+ on Mitochondrial Respiratory Activity<!>Assessment of Mitochondrial Membrane Potential<!>Estimation of Cellular Glutathione Levels<!>Cell Cycle Progression<!>Statistical Analysis<!>Excess Glucose does not Increase Cell Proliferation Rate<!>Excess Glucose Specifically Protects Glial Cells Against MPP+ Toxicity<!>MPP+ Decreases Mitochondrial Respiratory Function<!>MPP+ Decreases Mitochondrial Membrane Potential<!>MPP+ Decreases Total Cellular Glutathione Levels<!>Dual Cell Cycle Arrest by MPP+<!>Discussion<!>

<p>One of the neuropathological hallmarks of Parkinson's disease (PD) is the selective and progressive degeneration of the dopaminergic neurons in the substantia nigra of the brain [1]. Despite several years of extensive research, the exact molecular events in PD are not yet totally understood. Even though many theories are proposed for the causes of PD, none is proven. There are several drugs available to alleviate the symptoms of this disease, but unfortunately, there is no cure available. Post-mortem examinations of PD patients suggest that the impaired mitochondrial function in the neurons of substantia nigra is the most likely cause of the disease, possibly due to oxidative stress [2] or inflammation [3, 4]. Since mitochondria are the main organelles producing reactive oxygen species (ROS), they are highly vulnerable for oxidative damage, leading to their dysfunction and inhibition of ATP synthesis. The mitochondrial impairment could also lead to an unusual glucose oxidation, which is observed in certain central nervous system associated diseases namely, schizophrenia and Alzheimer's disease [5].</p><p>The environmental neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) was shown to produce several biochemical, pathological and clinical features of PD in several animal models by inhibiting complex I of the electron transport chain (ETC.) in the mitochondria. It was demonstrated that MPTP itself was not toxic to any brain cells. Upon the in vivo administration of MPTP, which is a lipophilic, it crosses the blood brain barrier with ease. Monoamine oxidase type B (MOAB) converts MPTP into 1-methyl-4-phenyl-1, 2-dihydroxypyridinium ion (MPDP+) [6, 7], which is spontaneously oxidized to toxic metabolite MPP+ (1-methyl-4-phenylpyridinium). Interestingly, this enzyme (MOAB) is absent in dopaminergic neurons [8] but exists mainly in glial cells and serotonergic neurons [9, 10]. Since astroglial cells are densely localized in substantia nigra par compacta [11], it is obvious that MPTP is converted first into the toxic MPP+ metabolite in these cells, causing damages to the mitochondria. It was demonstrated that the release of MPP+ into the extracellular spaces due to lysis of glial cells enables accumulation of these ions in nearby dopaminergic neurons via the plasma membrane dopamine transporters. MPP+ has been shown to inhibit complex I of mitochondrial ETC. selectively in these cells [12, 13].</p><p>Although the toxic nature of MPP+ against various cell cultures was well-demonstrated earlier [14–16], the mechanism of MPP+ toxicity was not investigated thoroughly so far in glial cells. Since glial cells represent a significant portion of central nervous system in maintaining the health and viability of neurons, the effect of MPP+ and the significant role of glucose against MPP+ toxicity would help in understanding MPP+ mechanism of toxicity. Glucose is the preferred energy source in the CNS and its availability can influence several vital cellular processes.</p><p>In the present investigation, we have employed rat C6 astroglial (glioma) cell line to examine the role of MPP+ on the onset of early events associated with PD, such as changes in mitochondrial respiratory status, mitochondrial membrane potential and cellular glutathione levels in 2 and 10 mM glucose. In addition, rate of cell proliferation and compound specific protection by high level of glucose were also determined. Finally, cell cycle analysis was performed to assess for the role of glucose on MPP+ effect on the different phase of cell cycle. In these studies, 2 mM glucose in the culture media represents the physiological concentration in the extra cellular fluid of brain in wake up animals [17], and thus may bring a step closer to in vivo situation. On the other hand, 10 mM glucose represents lower range hyperglycemia [18] under in vivo conditions and may help for better speculation of the early key events associated with PD.</p><!><p>Supplies include RPMI 1640 medium, Dulbecco's Modified Eagle Medium (DMEM) fetal bovine serum (FBS), penicillin/streptomycin, amphotericin B, phosphate-buffered saline (PBS), and L-glutamine were purchased from Media Tech, Inc., (Herndon, VA, US). Crystal violet, D-glucose, 5,5-dithiobis-2-nitrobenzoic acid (DTNB), EDTA, L-glutaraldehyde, trypan blue, MPP+, rhodamine—123, nicotinamide adenosine dinucleotide phosphate (NADPH) and 5-sulfosalicylic acid were supplied by Sigma Chemical Company (St. Louis, MO, US). CellTiter 96 Aqueous One Solution Reagent kit was purchased from Promega (Madison, WI, US).</p><!><p>The central nervous system derived rat C6 astroglial (glioma) cell line was purchased from American Type Culture Collection (Rockville, MD, US) and maintained as an adherent monolayer culture in RPMI 1640 1X medium (Cat. No. 10-040-CV, Media Tech, Inc., Herndon, VA, US) with phenol red, containing 11.1 mM glucose, 2 mM L-glutamine, and supplemented with 10% (v/v) FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B. Cells were grown in a humidified atmosphere containing 5% CO2 in air at 37 °C in an incubator and sub-cultured twice a week. For cytotoxic studies, the culture was harvested by treating with 0.05% EDTA in PBS for 2 min or less. Cell counts and cell viability were assessed immediately by using 0.4% trypan blue stain on a haemocytometer under a light microscope. Dye stained cells (blue) were counted as dead cells, while dye excluded cells were counted as viable cells. The actual cell numbers were determined by multiplying diluted times compared with initial cell numbers. When cell viability exceeded more than 90%, cells were diluted in complete RPMI 1640 medium and seeded in culture plates or dishes for the experiments.</p><!><p>The experimental medium was prepared with DMEM powder (Cat. No. 90-113-PB) in distilled water, supplemented with phenol red (5 μg/ml or 0.5% v/v), sodium bicarbonate (44 mM), 2 or 10 mM D-glucose, 2 mM L-glutamine, 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 0.25 μg/ml amphotericin B. The medium was filtered and used in the experimental studies.</p><!><p>Cells were plated in 96-well microtiter plates at a starting density of 5 × 103 cells per well in DMEM containing 2 or 10 mM glucose supplemented with 10% FBS. The number of cells at various time points was evaluated by crystal violet as described earlier [19]. The culture plates were read at 540 nm in a plate reader (Bio-Tek Instruments Inc, Wincoski, VT). The doubling period in hours was determined as reported earlier [20] from the average absorbance values (n = 24) of cells growing at exponential phase.</p><!><p>The cells were seeded at a starting density of 2 × 104 cells per well in a total volume of 196 μl of complete RMPI 1640 growth medium with 10% FBS. The cells were allowed to adhere overnight in the incubator. Then the medium was replaced completely with DMEM with phenol red containing 10% FBS either with 2 or 10 mM D-glucose. Stocks and working stocks of MPP+ were prepared always fresh in PBS and used in the studies. The cells (typically 60–70% confluent) were treated with MPP+ at different concentrations in a final volume of 4 μl under sterile conditions. MPP+ was added in increasing concentrations (0.1, 0.2 and 0.3 mM). In all studies, cells in the medium alone or cells in the medium containing equal volume of PBS served as controls. Controls and the treated samples were always present in the same culture plates. These plates were incubated for 48 h continuously without further renewal of growth medium in a 5% CO2 in air at 37 °C with the plates capped in the normal fashion. All studies were repeated at least twice (n = 16). At the end of incubation, the cytotoxicity of MPP+ was evaluated by dye uptake assay using crystal violet [19]. The plates were read at 540 nm in a plate reader.</p><!><p>The role of glucose concentration on cocaine- induced toxicity on glial cell was performed in the presence of 2 or 10 mM glucose in complete DMEM. Cocaine was tested at six different concentrations (2–7 mM) for 24 h as per earlier report [20]. These studies were repeated twice (n = 12). Cytotoxicity was evaluated by dye uptake assay using crystal violet [19] and the plates were read at 540 nm in a plate reader.</p><!><p>Mitochondrial respiratory activity was measured according to Denizot and Lang [21]. In brief, glial cells were exposed to various concentrations of MPP+ (0.1, 0.2 and 0.3 mM) in 2 or 10 mM glucose for 48 h. Three hours prior to end of incubation, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethonyphenol)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) was added per well. The titer plates were read in a plate reader at 490 nm. These studies were repeated at least twice (n = 6).</p><!><p>MPP+ treatments were performed at 0.1, 0.2 and 0.3 mM in 2 or 10 mM glucose for 48 h. In brief, at the end of incubation, cells were fixed with 100 μl of 0.25% aqueous glutaraldehyde, containing rhodamine—123 to yield a final concentration of 1 μM for 30 min at room temperature as described earlier [20]. The plates were read with excitation filter set at 485 nm and emission filter at 538 nm on a microplate Fluorometer model 7620, Version 5.02, Cambridge Technology, Inc., (Watertown, MA, US). These studies were repeated at least twice (n = 8).</p><!><p>Total cellular glutathione was assayed according to Smith et al. [22]. In brief, after treating with MPP+ at 0.1, 0.2 and 0.3 mM in 2 or 10 mM glucose for 48 h, the cells were deproteinized with 2% 5-sulfosalicylic acid (10 μl/well) for 30 min at 37 °C, followed by addition of 90 μl of reaction mixture containing 0.416 mM sodium EDTA, 0.416 mM NADPH, 0.835 mM DTNB and 0.083 mM sodium phosphate buffer, pH 7.5, and 0.216 units of glutathione reductase. The plates were incubated at 37°C for 30 min. The absorbance was measured at 412 nm on a plate reader. These studies were repeated at least twice (n = 12).</p><!><p>Treatments with MPP+ (0.1, 0.15 and 0.25 mM) were performed for 48 h. Then the cells were collected and fixed in 95% ice-cold ethanol for at least 24 h at 4°C. The next day, the tubes were centrifuged at 2,600 rpm for 7 min and fixed in 100 μl chilled ethanol. Cells were stained and analyzed as described elsewhere [23]. The proportion of cells in each stage was performed within 2 h by using FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). In each sample, 10,000 individual events from the gated subpopulation were analyzed separately. Cell Quest Software was used for acquisition and analysis of the data, and the percentage of cells in each phase was determined by using ModFit 3.0 Software (Verity Software House, Topsham, ME, US). The studies were repeated at least twice.</p><!><p>In this study, "n" represents the final number of wells per each treatment. The absorbance values were converted to percentage with respective to the control (100%) and the experimental results were presented as mean ± SEM (standard error mean) in the plots. The data were analyzed for significance by one-way analysis of variance (ANOVA) and then compared by Bonferroni's multiple comparison tests, using GraphPad Prism Software, Version 3.00 (San Diego, CA, US). The test values of P<0.05 and P<0.01 were considered significant and highly significant, respectively. The LC50 or EC50 values, representing the millimolar concentration of MPP+ needed to show 50% response on glial cells was determined from the graphs where both curves crossed [24].</p><!><p>The data on cell growth studies for 2 and 10 mM glucose groups (Fig. 1) was used to determine the cell doubling periods. It was found that the difference in the absorbance values between 2 and 10 mM glucose groups shown in this figure at any time point was insignificant (P > 0.05). In comparison to zero hour control (100%), the number of cells in 2 mM glucose was increased by 178, 386, and 832% after 24, 48, and 72 h, respectively (Fig. 1). This increase was almost the same as those cells in 10 mM glucose (178, 384, and 858% at 24, 48, and 72 h, respectively). These growth rates clearly suggest that cells in both glucose groups proliferated linearly with time (P<0.001) with an initial lag of about 24 h after seeding of the cells (Fig. 1). The average doubling period (±SEM) of cells in 2 and 10 mM glucose groups was 22.53 ± 0.9 and 22.63 ± 0.39 h, respectively. The data clearly suggest that fivefold difference in the glucose concentration in the medium had no significant role on the rate of cell proliferation.</p><!><p>Our subsequent studies were focused on MPP+ toxicity to glial cells, and the protection by glucose. For this purpose, the MPP+ concentrations (0.1, 0.2 and 0.3 mM) were selected based on non-linear regression analysis method (Fig. 2a). The correlation coefficient, R2, indicating the linearity was 0.9944. Ideally, an R2 value of 1.0 indicates that all data points are on the straight line, while zero R2 value indicates lack of such linearity. The R2 value obtained from our data clearly indicates that there was more than 99% linear relationship between MPP+ concentration and the cell viability. It was observed that MPP+ caused a progressive and statistically significant (P<0.01) cell death in 2 mM glucose in comparison to control after 48 h with an average LC50 of 0.14 ± 0.005 mM. This value was much lower than a previous report [0.5 mM, 25] presumably due to differences in glucose concentrations or incubation periods or serum concentrations in media or other factors, but was closer to another study [160 μM, 26]. Interestingly, 10 mM glucose significantly (P<0.01) increased the percentage of cell viability at all MPP+ treatments in comparison to the treatments in 2 mM glucose (Fig. 2b). The average LC50 in 10 mM glucose was 0.835 ± 0.03 mM MPP+. This increase of about sixfold higher value clearly demonstrates the protective role of glucose on cells.</p><p>We next designed an experiment to determine if 10 mM glucose protection is limited to MPP+-induced toxicity alone or extended to any other toxic compounds. For this purpose, studies were performed with cocaine, a widely abused drug that causes toxicity to various cells. Six different concentrations of cocaine were tested (2–7 mM) on glial cells for 24 h in the presence of 2 or 10 mM glucose in DMEM with 10% FBS. The selection of cocaine concentrations in this study was based on our previous report [20]. The results (Fig. 2c) revealed that 10 mM glucose did not protect the cells significantly at any cocaine concentrations (P>0.05). Instead, a similar significant dose dependent cytotoxicity was observed in both 2 and 10 mM glucose groups (P<0.01) at all concentrations of cocaine. The LC50 of cocaine in both 2 and 10 mM glucose was 4.4 ± 0.02 mM and 4.6 ± 0.02 mM, respectively. These values were significantly closer to earlier 24 h studies with cocaine 4.3 mM, [20, 27]. Since no protection was observed with 10 mM glucose at 24 h exposure, further increase in the incubation period to 48 h would only enhance cocaine toxicity to cells. Thus, studies were not repeated with cocaine. From these results, it is obvious that 10 mM glucose protection is compound specific.</p><!><p>Exposure of glial cells to MPP+ at increasing concentrations (0.1, 0.2 and 0.3 mM) for 48 h caused significant (P<0.01) decrease in mitochondrial respiration in 2 mM glucose group (Fig. 3). This was evidenced in terms of progressive decrease in the amount of formazan produced in MPP+ treated cells in 2 mM glucose. The average EC50 of MPP+, where 50% loss of mitochondrial respiratory activity in 2 mM glucose observed was found to be 0.174 ± 0.01 mM. Interestingly, the amount of formazan produced in MPP+ treated cells in 10 mM glucose group was significantly (P<0.01) higher in comparison to 2 mM glucose group. These observations reflect the increased state of mitochondrial respiratory activity of glial cells in 10 mM glucose. The average EC50 of MPP+ in 10 mM glucose was found to be 0.299 ± 0.06 mM. The results clearly suggest the ability of 10 mM glucose for restoring the respiratory status in MPP+ treated cells.</p><!><p>Glial cells were treated with MPP+ at 0.1, 0.2 and 0.3 mM for 48 h in the presence of 2 or 10 mM glucose in DMEM containing 10% FBS and then stained with 1 μM rhodamine 123. It is clear from Fig. 4 that MPP+ decreased significantly (P<0.05) the mitochondrial membrane potential of glial cells in 2 mM glucose in a dose dependent manner, which is consistent from earlier reports [28]. In 2 mM glucose group, the average EC50 of MPP+ was found to 0.26 ± 0.02 mM. In comparison to MPP+ treatments in 2 mM glucose, a dose dependent significant (P<0.05) increase in mitochondrial membrane potential was observed (Fig. 4) in 10 mM groups. The average EC50 in the case of 10 mM glucose was found to 0.428 ± 0.02 mM. The increased membrane potential by excess glucose was consistent with earlier studies [25, 29] albeit no quantified effective dose values were reported.</p><!><p>It was observed that MPP+ treatments at 0.1, 0.2 and 0.3 mM for 48 h decreased the total glutathione levels significantly (P<0.05) in cells of 2 mM glucose group (Fig. 5) with the EC50 of 0.36 mM. On the other hand, the total glutathione levels in 10 mM glucose group restored significantly (P<0.05) at all MPP+ treatments in comparison to MPP+ treatments in 2 mM glucose group. The EC50 in this case was>1 mM. Since we used glutathione reductase in the assay, the total glutathione measurements in our study represent both oxidized (GSSG) and reduced form (GSH) of glutathione. The data on MPP+ treatments in 10 mM glucose again confirms the protective role of 10 mM glucose.</p><!><p>The effect of MPP+ at 0.1, 0.15 and 0.25 mM in 2 and 10 mM glucose was studied on different phases of glial cell cycle for 48 h and analyzed by flow cytometry. It was observed that in 2 mM glucose, MPP+ caused a dual cell cycle inhibition. For instance, MPP+ treatment in the presence of 2 mM glucose caused a dose dependent cell cycle arrest both at G0/G1 and G2/M phases (Fig. 6a). These phase arrests were significant (P<0.05) at 0.15 and 0.25 mM MPP+. In 10 mM glucose, MPP+ caused significant arrest (P<0.05) only at G0/G1 phase, but no G2/M phase arrest was observed (Fig. 6b). In 2 and 10 mM glucose, the number of cells in S-phase was decreased significantly. While the dual inhibition by MPP+ in 2 mM glucose was not reported earlier, its G0/G1 inhibition in 10 mM glucose was consistent with previous studies [30]. The results clearly show the dual inhibitory nature of MPP+ in glial cells in 2 mM glucose.</p><!><p>The high sensitivity of cells to MPP+ in low levels of glucose (1 or 2 mM) was in agreement with previous reports, where low glucose was shown to potentiate MPP+ induced toxicity [31, 32]. In our study, the decreased cell viability due to MPP+ treatments in 2 mM glucose group does not appear to be an additive affect of both MPP+ toxicity and insufficient availability of glucose to cells. If insufficiency of glucose was the cause, then cells in both 2 and 10 mM glucose control groups would not have shown similar absorbance values as a function of the time (Fig. 1). Since cells in 2 or 10 mM glucose exhibited almost the same absorbance values at any given time point up to 72 h in our growth curve studies (Fig. 1), it is clear that the cells in 2 mM glucose were as much viable as those in 10 mM glucose group prior to MPP+ treatments, and thus the observed cell death during treatments was solely attributed to MPP+ toxicity.</p><p>Based on the cell doubling results, we used 48 h incubation, where cells were proliferating actively, as an end point in all our studies. Consistent with earlier reports on different cell lines [13, 25, 29]; we also observed that 10 mM glucose significantly protected the glial cells against MPP+ toxicity (Fig. 2b). In our study, this protection was reflected in terms of having about 6 times higher LC50 value in 10 mM glucose (0.835 ± 0.03 mM) in comparison to 2 mM glucose (0.14 ± 0.005 mM).</p><p>Various studies demonstrated that MPP+ inhibits NADH-ubiquinone oxidoreductase (complex I) of ETC. in the mitochondria [13, 33]. The inhibition at complex I results in very low or no ATP production in the mitochondria, depending on the potency of inhibition by the compounds, such as MPP+ [34]. In addition, inhibition at complex I may also generate more ROS [35], which affects the rate of mitochondrial respiration and its membrane potential. Because of several disadvantages associated with Clark oxygen electrode measurements [36–39], we preferred measuring the mitochondrial respiration by evaluating succinate dehydrogenase activity directly in cells as per the established method [21]. Since this enzyme is located on the inner membrane of mitochondria, measuring its activity may provide direct evidence on the general respiratory status of mitochondria in terms of their ability to reduce the tetrazolium compound of MTS to formazan in cells. We observed that the amount of formazan produced due to MPP+ treatment in 2 mM glucose was significantly lower than the control (Fig. 3). This indicates that MPP+ treatment significantly inhibited the mitochondrial respiration in 2 mM glucose group.</p><p>Under such circumstances, we next sought to know the state of mitochondrial membrane potential of MPP+ treated cells in 2 mM glucose group. For this purpose, we used rhodamine—123 fluorescent dye. Since this dye is selectively taken up by mitochondria [40], the amount of rhodamine —123 present in the mitochondria is directly proportional to its membrane potential. We observed that MPP+ treatment resulted significant loss of mitochondrial membrane potential in 2 mM glucose group (Fig. 4). It was further observed that MPP+ treatment decreased the total glutathione content significantly in 2 mM glucose cells, while 10 mM glucose significantly restored and almost reached (94%) to the level of control at 0.3 mM MPP+ (Fig. 5). While the decreased glutathione level was known to be associated with neurodegeneration, this is the first report to observe the glucose dependent regulation of glutathione levels by MPP+.</p><p>Significant decrease and restoration of mitochondrial respiratory status, its membrane potential and glutathione levels in MPP+ treated glial cells in 2 and 10 mM glucose respectively may indicate that MPP+ inhibition at complex I of ETC. was reversible type. This speculation was consistent with earlier reports [41, 42], where reversal of MPP+ inhibition was shown in different cell systems. The mechanism of restoration of the mitochondrial membrane potential by 10 mM glucose was not the objective of this investigation. It may be possible that high glucose in our studies relieved the MPP+ inhibition at complex I site, which resulted in increased cell viability. Further studies, however, are required to corroborate this speculation.</p><p>The data obtained from cell cycle study indicate that high level of glucose was associated with the lack of G2/M cell arrest, indicating the increase in cell mitosis and con- firm the cell viability data (Fig. 2b). The obtained results indicate that glucose protection against MPP+ and the lack of G2/M cell cycle arrest might be related to the mitochondrial protection offered by glucose. Cell protection by 10 mM glucose observed in this study seems to be compound specific based on the observation that 10 mM glucose did not offer similar protection against cocaine treated glial cells (Fig. 2c). This was evidenced by exhibiting closer LC50 values of cocaine in both 2 mM glucose (4.4 mM) and 10 mM glucose (4.6 mM). These values were also found closer to the LC50 values of earlier studies [4.3 mM, 20, 27]. Recent studies from our group indicated that cocaine interaction decreases the mitochondrial membrane potential in glial cells [20]. This observation reminds a similar trend of decrease in mitochondrial membrane potential by MPP+ in glial cells observed in the present study. The ability of 10 mM glucose to protect glial cells against MPP+ toxicity (Fig. 2b) and its inability to protect cocaine treated cells under the same conditions (Fig. 2c) may indicate that the mode of action of both MPP+ and cocaine at mitochondria was different in glial cells. This speculation is further supported from the results of our previous cell cycle analyses with cocaine [20]. For instance, our earlier studies with cocaine for 48 h under similar conditions as of the present study, resulted significant glial cell cycle arrest at G2/M phase. On the other hand, MPP+ treatment for the same period of time in the present study led to significant G0/G1 and G2/M arrests in 2 mM glucose group and G0/G1 arrest in 10 mM glucose cells (Fig. 6a, b). These observations clearly suggest that even though both MPP+ and cocaine interact with mitochondria and decrease membrane potential, their mode of action for cytotoxicity were different. Based on the differences in LC50 values or EC50 values or cell cycle arrest of MPP+ and cocaine, it appears that the MPP+ toxicity is specific rather than non-specific.</p><p>The data obtained in this investigation demonstrate the role of glucose in MPP+ induced changes on glial cell viability. While earlier reports [25] concluded that the increased glial cell viability in 10 mM glucose with MPP+ treatment was exclusively due to anaerobic glycolysis, here, based on the results of our additional studies, we found that the increased cell viability in 10 mM glucose with MPP+ treatments was not associated with a single mechanism but multi level mechanisms such as significant restoration of mitochondrial respiratory activity, its membrane potential, and increased total glutathione content. One of our findings was consistent with earlier studies on PC12 cells [29], where glucose protection against MPP+ toxicity was clearly demonstrated due to maintenance of mitochondrial membrane potential.</p><p>The data also indicate that MPTP toxicity to neurons might be related more to glial cells damage rather than the ability of glial cells to metabolize MPTP and release of the toxic metabolite, MPP+. Damaged and injured glial cells can produce toxic cytokines [43] to neurons and play important role in the inflammatory process in nervous system [43]. Inflammation may be associated with the neuropathology of PD as evidenced by the excessive glial activation and increased levels of the pro-inflammatory cytokines, tumor necrosis factor-alpha and interleukin-1beta in the substantia nigra of patients with PD [44]. Since anti-inflammatory drugs and microglial activation inhibitors decrease susceptibility to PD, it is likely that their use may emerge as a therapy in PD [44].</p><p>It was concluded that from this study that high levels of glucose were protective against MPP+-induced changes on glial cell viability, alterations in mitochondrial general respiratory status, mitochondrial membrane potential, total glutathione levels and dual cell cycle phase arrest. The data also indicate that MPTP toxicity to neurons is more likely related to glial cell activation rather than the ability of glial cells to release the toxin MPP+.</p><!><p>Growth curves of glial cells. Cells with starting density of 5 × 103 per well in 96-well plates (n = 4, and each value is the average absorbance of 6 wells, so final n at each time point = 24) with complete DMEM containing 2 or 10 mM glucose and 10% FBS were incubated for designated time points. The cells were stained with crystal violet and the absorbance at 540 nm was read in a plate reader. The absorbance values at any time point were so close to each other that error bars were not seen clearly on the plot. * Significant when compared to the respective zero hour controls (P<0.001). # There was no significant difference between the 2 and 10 mM glucose (P>0.05) groups</p><p>a Concentration-effect curve. Glial cells were treated with MPP+ (0.1, 0.2 and 0.3 mM) in 2 mM glucose for 48 h. The absorbance for viability was measured, and a graph was plotted between log of MPP+ concentrations and the mean absorbance values by non-linear regression analysis method. b Effect of MPP+ on glial cell viability in 2 and 10 mM glucose after 48 h. * MPP+ treatments had significant effect when compared to the respective controls. # MPP+ treatments had significant effect on cell viability when 2 and 10 mM glucose groups were compared. Values represent the average of three independent studies (P<0.001, n = 16). c Lack of 10 mM glucose protection in glial cells treated with cocaine after 24 h. * Treatments had significant effects when compared to the respective controls (P<0.05, n = 12). # There was no significant difference between 2 and 10 mM glucose groups (P>0.05, n = 12)</p><p>Effect of MPP+ on mitochondrial respiratory status (MRS) in glial cells. Cells were grown in DMEM with 2 or 10 mM glucose containing 10% FBS, and were treated with various concentrations of MPP+ for 48 h. Values represent the average of two independent studies (n = 6). * MPP+ had significant effect on MRS in comparison to the control containing 2 or 10 mM glucose (P<0.05, n = 6 One-way ANOVA, Bonferroni's multiple comparison test). # Significant difference between MPP+ treatment groups with 2 and 10 mM glucose (P<0.001, n = 6, One-way ANOVA, Bonferroni's multiple comparison test)</p><p>Effect of MPP+ on mitochondrial membrane potential in glial cells. Cells were grown in DMEM with 2 or 10 mM glucose containing 10% FBS, and were treated with various concentrations of MPP+ for 48 h. Values represent the average of two independent studies (n = 8). * Significant effect of MPP+ in comparison to control groups containing 2 or 10 mM glucose (P<0.05, n = 8, One-way ANOVA, Bonferroni's multiple comparison test). # Significant difference between MPP+ treatment groups with 2 and 10 mM glucose in media (P<0.001, n = 8, One-way ANOVA, Bonferroni's multiple comparison test)</p><p>Effect of MPP+ on GSH levels in glial cells. Cells were grown in DMEM with 2 or 10 mM glucose containing 10% FBS, and were treated with various concentrations of MPP+ for 48 h. Values represent the average of two independent studies (n = 12). * Significant in comparison to control containing 2 or 10 mM glucose in media (P<0.05, n = 12 One-way ANOVA, Bonferroni's multiple comparison test). # Significant difference between MPP+ treatment groups with 2 and 10 mM glucose in media (P<0.001, n = 12, One-way ANOVA, Bonferroni's multiple comparison test)</p><p>Effect of MPP+ on glial cell cycle. Cells were grown in 100 × 15 mm culture dishes in DMEM with 2 mM (a) or 10 mM (b) glucose containing 10% FBS, and were treated with various concentrations of MPP+ for 48 h. Cells were stained by propidium iodide staining solution for 1 h in dark and analyzed by flow cytometry. Data were presented as mean ± SEM (n = 2, * P<0.05, significant in comparison to control, One-way ANOVA, Bonferroni's multiple comparison test)</p>

PubMed Author Manuscript

Multidimensional persistence in biomolecular data

Persistent homology has emerged as a popular technique for the topological simplification of big data, including biomolecular data. Multidimensional persistence bears considerable promise to bridge the gap between geometry and topology. However, its practical and robust construction has been a challenge. We introduce two families of multidimensional persistence, namely pseudo-multidimensional persistence and multiscale multidimensional persistence. The former is generated via the repeated applications of persistent homology filtration to high dimensional data, such as results from molecular dynamics or partial differential equations. The latter is constructed via isotropic and anisotropic scales that create new simiplicial complexes and associated topological spaces. The utility, robustness and efficiency of the proposed topological methods are demonstrated via protein folding, protein flexibility analysis, the topological denoising of cryo-electron microscopy data, and the scale dependence of nano particles. Topological transition between partial folded and unfolded proteins has been observed in multidimensional persistence. The separation between noise topological signatures and molecular topological fingerprints is achieved by the Laplace-Beltrami flow. The multiscale multidimensional persistent homology reveals relative local features in Betti-0 invariants and the relatively global characteristics of Betti-1 and Betti-2 invariants.

multidimensional_persistence_in_biomolecular_data

1 Introduction<!>2 Multidimensional persistence in the point cloud data of protein folding<!>2.1 Protein folding/unfolding processes<!>2.2 All-atom and coarse-grained representations<!>2.3 Multidimensional persistence in protein folding process<!>3 Multidimensional persistence in biological matrices<!>3.1 Protein flexibility prediction<!>Flexibility rigidity index<!>Elastic network model<!>3.2 Persistent homology analysis of optimal cutoff distance<!>3.3 Persistent homology analysis of the FRI scale<!>4 Multidimensional persistence in volumetric data<!>4.1 Multidimensional topological fingerprints and topological denoising<!>Laplace-Beltrami flow<!>Topological fingerprint identification<!>4.2 Multiscale multidimensional persistence<!>Multiscale 2D persistence<!>Multiscale high-dimensional persistence<!>5 Conclusion

<p>The rapid progress in science and technology has led to the explosion in biomolecular data. The past decade has witnessed a rapid growth in gene sequencing. Vast sequence databases are readily available for entire genomes of many bacteria, archaea and eukaryotes. The human genome decoding that originally took 10 years to process can be achieved in a few days nowadays. The Protein Data Bank (PDB) updates new structures on a daily basis and has accumulated more than one hundred thousand tertiary structures. The availability of these structural data enables the comparative study of evolutionary processes, gene-sequence based protein homology modeling of protein structures and the decryption of the structure-function relationship. The abundant protein sequence and structural information makes it possible to build up unprecedentedly comprehensive and accurate theoretical models. One of ultimate goals is to predict protein functions from known protein sequences and structures, which remains a fabulous challenge.</p><p>Fundamental laws of physics described in quantum mechanics (QM), molecular mechanism (MM), continuum mechanics, statistical mechanics, thermodynamics, etc. underpin most physical models of biomolecular systems. QM methods are indispensable for chemical reactions, enzymatic processes and protein degradations.1, 2 MM approaches are able to elucidate the conformational landscapes of proteins.3 However, both QM and MM involve an excessively large number of degrees of freedom and their application to real-time large-scale protein dynamics becomes prohibitively expensive. For instance, current computer simulations of protein folding take many months to come up with a very poor copy of what Nature administers perfectly within a tiny fraction of a second. One way to reduce the number of degrees of freedom is to employ time-independent approaches, such as normal mode analysis (NMA),4–7 flexibility-rigidity index (FRI)8, 9 and elastic network model (ENM),10 including Gaussian network model (GNM)11–13 and anisotropic network model (ANM).14 Another way is to incorporate continuum descriptions in atomistic representation to construct multiscale models for large biological systems.1, 2, 15–19 Implicit solvent models are some of the most popular approaches for solvation analysis.20–29 Recently, differential geometry based multiscale models have been proposed for biomolecular structure, solvation, and transport.30–33 The other way is to combine several atomic particles into one or a few pseudo atoms or beads in coarse-grained (CG) models.34–37 This approach is efficient for biomolecular processes occurring at slow time scales and involving large length scales.</p><p>All of the aforementioned theoretical models share a common feature: they are geometry based approaches38–40 and depend on geometric modeling methodologies.41 Technically, these approaches utilize geometric information, namely, atomic coordinates, angles, distances, areas40, 42, 43 and sometimes curvatures44–46 as well as physical information, such as charges and their locations or distributions, for the mathematical modeling of biomolecular systems. Indeed, there is an increased importance in geometric modeling for biochemistry,39 biophysics47, 48 and bioengineering.49, 50 Nevertheless, geometry based models are typically computationally expensive and become intractable for biomolecular processes such as protein folding, signal transduction, transcription and translation. Such a failure is often associated with massive data acquisition, curation, storage, search, sharing, transfer, analysis and visualization. The challenge originated from geometric modeling call for game-changing strategies, revolutionary theories and innovative methodologies.</p><p>Topological simplification offers an entirely different strategy for big data analysis. Topology deals with the connectivity of different components in a space and is able to classify independent entities, rings and higher dimensional holes within the space. Topology captures geometric properties that are independent of metrics or coordinates. Indeed, for many biological problems, including the opening or closing of ion channels, the association or disassociation of ligands, and the assembly or disassembly of proteins, it is the qualitative topology, rather than the quantitative geometry that determines physical and biological functions. Therefore, there is a topology-function relationship in many biological processes51 such that topology is of major concern.</p><p>In contrast to geometric tools which are frequently inundated with too much structural information to be computationally practical, Topological approaches often incur too much reduction of the geometric information. Indeed, a coffee mug is topologically equivalent to a doughnut. Therefore, topology is rarely used for quantitative modeling. Persistent homology is a new branch of topology that is able to bridge the gap between traditional geometry and topology and provide a potentially revolutionary approach to complex biomolecular systems. Unlike computational homology which gives rise to truly metric free or coordinate free representations, persistent homology is able to embed additionally geometric information into topological invariants via a filtration process so that "birth" and "death" of isolated components, circles, rings, loops, voids or cavities at all geometric scales can be measured.52–54 As such, the filtration process create a multiscale representation of important topological features. Mathematically these topological features are described by simplicial complexes, i.e., topological spaces constructed by points, line segments, triangles, and their higher-dimensional counterparts. The basic concept of persistent homology was introduced by Frosini and Landi55 and Robins,56 independently. The first realization was due to Edelsbrunner et al.52 The concept was genearlized by Zomorodian and Carlsson.53 Many efficient computational algorithms have been proposed in the past decade.57–61 Many methods have been developed for the geometric representation and visualization of topological invariants computed from persistent homology. Among them, the barcode representation62 utilizes various horizontal line segments or bars to describe the "birth" and "death" of homology generators over the filtration process. Additionally, persistent diagram representation directly displays topological connectivity in the filtration process. The availability of efficient persistent homology tools63, 64 has led to applications in a diverse fields, including image analysis,65–68 image retrieval,69 chaotic dynamics,70, 71 complex network,72, 73 sensor network,74 data analysis,75–79 computer vision,67 shape recognition,80 computational biology,51, 81–83 and nano particles.84, 85</p><p>The most successful applications of persistent homology have been limited to topological characterization identification and analysis (CIA). Indeed, there is little persistent homology based physical or mathematical modeling and quantitative prediction in the literature. Recently, we have introduced persistent homology as unique means for the quantitative modeling and prediction of nano particles, proteins and other biomolecules.51, 84 Molecular topological fingerprint (MTF), a recently introduced concept,51 is utilized not only for the CIA, but also for revealing topology-function relationships in protein folding and protein flexibility. Persistent homology is found to provide excellent prediction of stability and curvature energies for hundreds of nano particles.84, 85 More recently, we have proposed a systematical variational framework to construct objective-oriented persistent homology (OPH),85 which is able to proactively extract desirable topological traits from complex data. An example realization of the OPH is achieved via differential geometry and Laplace-Beltrami flow.85 Most recently, we have developed persistent homology based topological denoising method for noise removal in volumetric data from cryo-electron microscopy (cryo-EM).86 We have shown that persistent homology provides a powerful tool for solving ill-posed inverse problems in cryo-EM structure determination.86</p><p>However, one dimensional (1D) persistent homology has its inherent limitations. It is suitable for relatively simple systems described by one or a few parameters. The emergence of complexity in self-organizing biological systems frequently requires more comprehensive topological descriptions. Therefore, multidimensional persistent homology, or multidimensional persistence, becomes valuable for biological systems as well as many other complex systems. In principle, multidimensional persistence should be able to seamlessly bridge geometry and topology. Although multidimensional persistence bears great promise, its construction is non-trivial and elusive to the scientific community.87 A major obstacle is that, theoretically, it has been proved there is no complete discrete representation for multidimensional persistent module analogous to one dimensional situation.87 State differently, the persistent barcodes or persistent diagram representation is only available in one dimension filtration, no counterparts can be found in higher dimensions. Therefore, in higher dimensional filtration, incomplete discrete invariants that are computable, compact while still maintain important persistent information, are being considered.87 Among them, a well-recognized one is persistent Betti numbers (PBNs),52 which simply displays the histogram of Betti numbers over the filtration parameter. The PBN is also known as rank invariant87 and size functions (0th homology).55 A major merit of the PBN representation is its equivalent to the persistent barcodes in one dimension, which means that this special invariant is complete in 1D filtration. Also, it has been proved that PBN is stable in the constraint of certain marching distance.88 A few mathematical algorithms have been proposed.88–90 Multi-filtration has been used in pattern recognition or shape comparison.55, 91, 92 Computationally, the realization of robust multidimensional persistent homology remains a challenge as algorithms proposed have to be topologically feasible, computationally efficient and practically useful.</p><p>The objective of this work is to introduce two classes of multidimensional persistence for biomolecular data. One class of multidimensional persistence is generated by repeated applications of 1D persistent homology to high-dimensional data, such as those from protein folding, molecular dynamics, geometric partial differential equations (PDEs), varied signal to noise ratios (SNRs), etc. The resulting high-dimensional persistent homology is a pseudo-multidimensional persistence. Another class of multidimensional persistence is created from a family of new simplicial complexes associated an isotropic scale or anisotropic scales. In general, scales behave in the same manner as wavelet scales do. They can focus on the certain features of the interest and/or defocus on undesirable characteristics. As a consequence, the proposed scale based isotropic and anisotropic filtrations give rise to new multiscale multidimensional persistence. We demonstrate the application of the proposed multidimensional persistence to a number of biomolecular and/or molecular systems, including protein flexibility analysis, protein folding characterization, topological denoising, noise removal from cryo-EM data, and analysis of fullerene molecules. Our multidimensional filtrations are carried out on three types of data formats, namely, point cloud data, matrix data and volumetric data. Therefore, the proposed methods can be easily applied to problems in other disciplines that involve similar data formats.</p><p>Our algorithm for multidimensional persistence is robust and straightforward. In a two-dimensional (2D) filtration, we fix one of the filtration parameters and perform the filtration on the second parameter to obtain PBNs. Then we systematically change the fixed parameter to sweep over its whole range, and stack all the PBNs together. This idea can be directly applied to three dimensional (3D) and higher dimensional filtrations. Essentially, we just repeat the 1D filtration over and over until the full ranges of other parameters are sampled. The PBNs are then glued together. This multidimensional persistent homology method can be applied to any other high dimensional data. In this work, point cloud data and matrix data are analyzed by using the JavaPlex.63 Volumetric data are processed with the Perseus.64</p><p>The rest of this paper is organized as follows. In Section 2, we explore the multidimensional persistence in point cloud data for protein folding. We model the protein unfolding process by an all-atom steer molecular dynamics (SMD). We consider both an all-atom representation and a coarse-grained representation to analyze the SMD data. From our multifiltration analysis, it is found that PBNs associated with local hexagonal and pentagonal ring structures in protein residues are preserved during the unfolding process while those due to global rings and cavities diminish. Coarse-grained representation is able to directly capture the dramatic topological transition during the unfolding process. In Section 3, we investigate the multidimensional persistence in matrix data. The GNM Kirchhoff (or connectivity) matrix and FRI correlation matrix are analyzed by multidimensional persistent homology. The present approach is able to predict the optimal cutoff distance of the GNM and the optimal scale of the FRI algorithm for protein flexibility analysis. Section 4 is devoted to the multidimensional persistence in volumetric data. We analyze the multidimensional topological fingerprints of Gaussian noise and demonstrate the multidimensional topological denoising of synthetic data and cryo-EM data in conjugation with the Laplace-Beltrami flow method. Finally, we construct multiscale 2D and 3D persistent homology methods to analyze the intrinsic topological patterns of protein 2YGD and fullerene C60 molecule. This paper ends with a conclusion.</p><!><p>In this section, we reveal multidimensional persistence in point cloud data associated with protein folding process. It is commonly believed that after the translation from mRNA, unfolded polypeptide or random coil folds into a unique 3D structure which defines the protein function.93 However, protein folding does not always lead to a unique 3D structure. Aggregated or misfolded proteins are often associated with sporadic neurodegenerative diseases, such as mad cow disease, Alzheimer's disease and Parkinson's disease. Currently, there is no efficient means to characterize disordered proteins or disordered aggregation, which is crucial to the understanding of the molecular mechanism of degenerative disease. In this section, we show that multidimensional persistence provides an efficient tool to characterize and visualize the orderliness of protein folding.</p><!><p>The SMD is commonly used to generate elongated protein configurations from its nature state.94–96 Our goal is to examine the associated changes in the protein topological invariants induced by SMD. There are three approaches to achieve SMD: high temperature, constant force pulling, and constant velocity pulling.94–96 Both implicit and explicit molecular dynamics can be used for SMD simulations. The mechanical properties of protein FN-III10 has been utilized to carefully design and valid SMD. Appropriate treatment of solvent environment in the implicit SMD is crucial. Typically, a large box which can hold the stretched protein is required, although the computational cost is relatively high.97 In our study, a popular SMD simulation tool NAMD is employed to generate the partially folded and unfolded protein conformations. The procedure consists of two steps: the relaxation of the given structure and unfolding simulation with constant velocity pulling. In the first step, the protein structure is downloaded from the Protein Data Bank (PDB), which is the major reservoir for protein structures with atomic details. Then, the structure is prepared through the standard procedure, including adding missed hydrogen atoms, before it is solvated with a water box which has an extra 5Å layer, comparing with the initial minimal box that barely hold the protein structure.98 The standard minimization and equilibration processes are carried out. We employ a total of 5000 time steps of equilibration iterations with the periodic boundary condition after 10000 time steps of initial energy minimization. In our simulations, we use a time increment of 2 femtoseconds (fs). We set SMDk=7. The results are recorded after each 50 time steps, i.e., one frame for each 0.1 picosecond (ps). We accumulate a total of 1000 frames or protein configurations, which are employed for our persistent homology filtration.</p><!><p>Persistent homology analysis of proteins can be carried out either in an all-atom representation or in CG representations.51 For the all-atom representation, various types of atoms, including O, C, N, S, P, etc., are all included and regarded as equally important in our computation. We deliberately ignore the Hydrogen atoms in our structure during the filtration analysis, as we found that they tend to contaminate our local protein fingerprints. The all-atom representation gives an atomic description of a given protein frame or configuration and is widely used in molecular dynamic simulation. In contrast, CG representations describe the protein molecule with the reduced number of degrees of freedom and are able to highlight important protein structure features. CG representations can be constructed in many ways. A standard coarse-grained representation of proteins used in our earlier topological analysis is to represent each amino acid by the corresponding Cα atom.51 CG representations are efficient for describing large proteins and protein complexes and significantly reduce the cost of calculating topological invariants.51</p><p>Figure 1 demonstrates the persistence information for the all-atom representation and the Cα coarse-grained representation of 1UBQ relaxation structure (i.e., the initial structure for the unfolding process). Figures 1a and b are topological invariants from the all-atom representation without hydrogen atoms. In Fig. 1a, it can be observed that β1 and β2 barcodes are clearly divided into two unconnected regions: local region (from 1.6 to 2.7 Å for β1 and from 2.4Å to 2.7Å for β2) and global region (from 2.85Å to 6.7Å for β1 and from 3.5Å to 6.7Å for β2). Local region appears first during the filtration process and it is directly related to the hexagonal ring (HR) and pentagonal ring (PR) structures from the residues. As indicated in the zoomed-in regions enclosed by dotted red rectangles, there are 7 local β1 bars and 3 β2 bars, which are topological fingerprints for phenylalanine (one HR), tyrosine (one HR), tryptophan (one HR and one PR), proline (one PR), and histidine (one PR). In Fig. 2a, we have 3 hexagonal rings (red color) corresponding to 3 local β1 bars and 3 local β2 bars. It is well known that hexagonal structures produce β2 invariants in the Vietoris–CRips complex based filtration.51 The other 4 local β1 bars are from pentagonal structures (blue color). Figures 2 c and d are results from the coarse-grained representation. It can be seen that there is barely any β2 information for the initial structure. As protein unfolds, almost no cavities or holes are detected. Therefore, we only consider β0 and β1 invariants in the coarse-grained model.</p><!><p>In our protein folding analysis, we extract 1000 configurations over the unfolding process. For each configuration, we carry out the point cloud filtration, i.e., systematically increasing the radius of ball associated with each atom, and come up with three 1D PBN graphs for β0, β1, and β2. We then stack 1000 PBN graphs of the same type, say all β0 graphs, together. In this way, the final result can be stored in a 2D matrix with the row number indicating the filtration radius, the column number indicating the configuration, and the elements are PBN values. Figures 2 a, b and c demonstrate the unfolding of protein 1UBQ in the all atom representation without hydrogen atoms and the corresponding 2D persistence diagrams. In these subfigures, we highlight residual pentagonal rings and hexagonal rings in blue and red, respectively. These ring structures correspond to the local topological invariants as indicated in Fig. 1a. Figures 2 d, e and f depict 2D persistent homology analysis of the protein unfolding process. Because all the bond lengths are around 1.5 to 2.0 Å and do not change during the unfolding process, the 2D β0 persistence shown in Fig. 2 d is relatively simple and consistent with the top panel in Fig. 1b. The 2D β1 persistence shown in Fig. 2 e is very interesting. The local rings occurred from 1.6 to 2.7 Å are due to pentagonal and hexagonal structures in the residues and are persistent over the unfolding process. However, the numbers of β1 invariants for global rings in the region from 2.85 to 6.7 Å vary dramatically during the unfolding process. Essentially, the SMD induced elongation of the polypeptide structure reduces the number of rings. Finally, the behavior of the β2 invariants in Fig. 2 f is quite similar to that of the β1. The local β2 invariants occurred from 2.4 to 2.7 Å induced by the hexagonal structures51 remain unchanged during the unfolding process, while the number of global β2 invariants occurred from 3.5 to 6.7 Å rapidly decreases during the unfolding process. Especially, at 750th configuration and beyond, the number of β2 invariants in global region has plummeted. The related PBNs for β2 drop to zero abruptly, indicates that the protein has become completely unfolded. Indeed, there is an obvious topological transition in multidimensional β1 persistence around 750th configuration as shown in Fig. 2 e. The global β1 PBNs are dramatically reduced and their distribution regions are significantly narrowed for all configurations beyond 75 picosecond simulations, which is an evidence for solely intraresidue β1 rings.</p><p>Having analyzed the multidimensional persistence in protein folding via the all-atom representation, it is interesting to further explore the same process and data set in the coarse-grained representation. Figure 3 illustrates our results. In Fig. 3 a, protein 1UBQ is plotted with all atoms except for hydrogen atoms. We use different colors to label different types of residues. The same structure is illustrated by the Cα based CG representation in Fig. 3 b. An advantage of the CG model is that it simplifies topological relations by ignoring intraresidue topological invariants, while emphasizing interresidue topological features. Figures 3 c and d respectively depict 2D β0 and β1 invariants of the protein unfolding process. Compared with the all-atom results in Figs. 2 d and e, there are some unique properties. First, the CG analysis only emphasizes the global topological relations among residues and their evolution during the protein unfolding. Additionally, the 2D β0 profile of the all-atom representation was a strict invariant over the time evolution as shown in Fig. 2 d, while that of CG model in Fig. 3 c varies obviously during the SMD simulation. The standard mean distance between two adjacent Cα atoms is about 3.8 Å, which can be enlarged under the pulling force of the SMD. The deviation from the mean residue distance indicates the strength of the pulling force. Finally, Fig. 3 d displays a clear topological transition from a partially folded state to a completely unfolded state at 75 picoseconds or 750th configuration.</p><p>As demonstrated in our earlier work,51 one can establish a quantitative model based on the PBNs of β1 to predict the relative folding energy and stability. The β1 PBNs computed from the present CG representation are particularly suitable for this purpose. A similar quantitative model can be established to describe the orderliness of disordered proteins.51 In Figure 4, we demonstrate the prediction of bond and total energies using β0 and β1 accumulated bar lengths, respectively. Basically, the PBNs for each individual configuration are added to deliver the accumulated bar lengths, which are then used to fit with the simulated results in a total of 1000 frames. It can be seen that the accumulated bar lengths of β0 give a nice prediction of the bond energy, and the accumulated bar lengths of β0 capture the essential properties of the total energy. For these two fittings, Pearson's correlation coefficients are 0.924 and 0.990, respectively. It can been seen these topological measurements capture the essential properties of the bond and total energies, and thus can be used to characterize the unfolding process.</p><p>In summary, multidimensional persistent homology analysis provides a wealth of information about protein folding and/or unfolding process including the number of atoms or residues, the numbers of hexagonal rings and pentagonal rings in the protein, bond lengths or residue distances, the strength of applied pulling force, the orderliness of disordered proteins, the relative folding energies, and topological translation from partially folded states to completely unfolded states. Therefore, multidimensional topological persistence is a powerful new tool for describing protein dynamics, protein folding and protein-protein interaction.</p><!><p>Having illustrated the construction of multidimensional topological persistence in point cloud data, we further demonstrate the development of multidimensional topological analysis of matrix data. To this end, we consider biomolecular matrices associated flexibility analysis. The proposed method can be similarly applied to other biological matrices.</p><!><p>Geometry, electrostatics, and flexibility are some of the most important properties for a protein that determine its functions. The role of protein geometry and electrostatics has been extensively studied in the literature. However, the importance of protein flexibility is often overlooked. An interesting argument is that it is the protein flexibility, not disorder, that is intrinsic to molecular recognition.99 Protein flexibility can be defined as its ability to deform from the equilibrium state under external force. The external stimuli are omnipresent either in the cellular environment and in the lattice condition. In response, protein spontaneous fluctuations orchestrate with the Brownian dynamics in living cells or lattice dynamics in solid with its degree of fluctuations determined by both the strength of external stimuli and protein flexibility. It has been shown that the Gaussian network model (GNM) and the flexibility-rigidity index (FRI) are some of most successful methods for protein flexibility analysis.8, 9 However, the performance of these methods depends on their parameters, namely, the cutoff distance of the GNM and the characteristic distance or the scale of the FRI. In this work, we develop matrix based multidimensional persistent homology methods to examine the optimal scale of FRI and optimal cutoff distance of the GNM. Brief descriptions are given to both methods to facilitate our persistent homology analysis.</p><!><p>The FRI have been proposed as a matrix diagonalization free method for the flexibility analysis of biomolecules.8, 9 The computational complexity of the fast FRI constructed by using the cell lists algorithm is of O(N), with N being the number of particles.9 In FRI, protein topological connectivity is measured by a correlation matrix. Consider a protein with N particles with coordinates given by {rj|rj ∈ ℝ3, j = 1, 2, ⋯, N}. We denote ‖ri – rj‖ the Euclidean distance between ith particle and the jth particle. For the ith particle, its correlation matrix element with the jth particles is given by Φ(‖ri – rj‖; σj), where σj is the scale depending on the particle type. The correlation matrix element is a real-valued monotonically decreasing function satisfying (1)Φ(‖ri−rj‖;σj)=1as‖ri−rj‖→0 (2)Φ(‖ri−rj‖;σj)=0as‖ri−rj‖→∞. The Delta sequences of the positive type discussed in an earlier work100 are suitable choices. For example, one can select generalized exponential functions (3)Φ(‖ri−rj‖;σj)=e−(‖ri−rj‖/σj)κ,κ>0 and generalized Lorentz functions (4)Φ(‖ri−rj‖;σj)=11+(‖ri−rj‖/σj)υ,υ>0. We have defined the atomic rigidity index μi for the ith particle as8 (5)μi=∑jNwjΦ(‖ri−rj‖;σj),∀i=1,2,⋯,N. where wj is a particle type dependent weight function. The the atomic rigidity index has a straight forward physical interpretation, i.e., a strong connectivity leads to a high rigidity.</p><p>We also defined the atomic flexibility index as the inverse of the atomic rigidity index, (6)fi=1μi,∀i=1,2,⋯,N. The atomic flexibility indices {fi} are used to predict experimental B-factors or Debye-Waller factors via a linear regression.8 The FRI theory has been intensively validated by a set of 365 proteins.8, 9 It outperforms the GNM in terms of accuracy and efficiency.8</p><p>When we only consider one type of particles, say Cα atoms in a protein, we can set wj = 1. Additionally, it is convenient to set σj = σ for Cα based CG model. We use σ as a scale parameter in our multidimensional persistent homology analysis, which leads to a 2D persistent homology.</p><!><p>The normal mode analysis (NMA)4–7 is a well developed technique and is constructed based on the matrix diagonalization of MD force field. It can be employed to study, understand and characterize the mechanical aspects of the long-time scale dynamics. The computational complexity for the matrix diagonalization is typically of O(N3), where N is the number of matrix rows or particles. Elastic network model (ENM)10 simplifies the MD force field by considering only the elastic interactions between nearby pairs of atoms. The Gaussian network model (GNM)11–13 makes a further simplification by using the coarse-grained representation of a macromolecule. This coarse-grained representation ensures the computational efficiency. Yang et al.101 have demonstrated that the GNM is about one order more efficient than most other matrix diagonalization based approaches. In fact, GNM is more accurate than the NMA.9 It should be noticed that the GNM models can be further improved by the incorporation of information from crystalline structure, residual types, and co-factors.</p><p>The performance of GNM depends on its cutoff distance parameter, which allows only the nearby neighbor atoms within the cutoff distance to be considered in the elastic Hamiltonian. In this work, we construct multidimensional persistent homology based on the cutoff distance in the GNM. We further analyze the parameter dependence of the GNM by our 2D persistence.</p><!><p>Protein elastic network models, including the GNM, usually employ the coarse-grained representation and do not distinguish between different residues. Let us denote N the total number of Cα atoms in a protein, and ‖ri – rj‖ the distance between ith and jth Cα atoms. To analyze the topological properties of protein elastic networks, we have introduced a new distance matrix D = {Dij |i = 1, 2, ⋯, N; j = 1, 2, ⋯, N}51 (7)Dij={‖ri−rj‖,‖ri−rj‖≤rc;d∞,‖ri−rj‖>rc, where d∞ is a sufficiently large value which is much larger than the final filtration size and rc is a given cutoff distance. Here d∞ is designed to ensure that atoms beyond the cutoff distance rc do not form any high order simplicial complex during the filtration process. Therefore, the resulting persistent homology shares the same topological connectivity with elastic network models. By systematically increasing the cutoff distance rc, one can analyze the topological connectivity and performance of the GNM. Additionally, the cutoff distance (rc) in Eq. (7) is also employed as the filtration parameter in our 2D persistent homology analysis of the GNM.</p><p>The performance of the GNM for the B-factor prediction and the multidimensional persistent homology analysis of protein 1PZ4 are plotted in Fig. 5. In Fig. 5 a, we compare the experimental B-factors and those predicted by the GNM with a cutoff distance 6.6 Å. The Pearson correlation coefficient for the prediction is 0.89. The GNM provides very good predictions except for the first three residues and the high flexibility around the 42nd residue. Figure 5 b shows the relation between correlation coefficient and cutoff distance. It can be seen that the largest correlation coefficients are obtained in the region when cutoff distance is in the range of 6Å to 9Å. Figures 5 c and d illustrate 2D β0 and β1 persistence, respectively. The x-axises are the cutoff distance rc in filtration matrix (7), which is the major filtration parameter. The y-axises are the cutoff distance rc in the GNM Kirchhoff matrix. The resulting β0 and β1 PBNs in the matrix representation have unique patterns which are highly symmetric along the diagonal lines. This symmetry, to a large extent, is duo to the way of forming the GNM Kirchhoff matrix. The 2D β0 persistence has an obvious interpretation in terms of 113 residues. Interestingly, patterns in Fig. 5 d can be employed to explain the behavior of the correlation coefficients under different cutoff distances. To this end, we roughly divide Fig. 5 d into four regions according to the cutoff distance, i.e., (0Å, 4.5Å), (4.5Å, 5.8Å), (5.8Å, 9Å) and (9Å, 12Å). In the first region, the network is not well constructed. As the distance between two Cα atoms is around 3.8Å, there is only a cluster of isolated atoms when cutoff distance is smaller than 4.5 Å. Therefore, the corresponding GNMs do not give any reasonable prediction. In the second region, network structures begin to form. The number of 1D ring structures within these networks increases dramatically. It reaches its maximum when cutoff is about 5Å, and then drops quickly. This behavior means that many local small-sized loops are developed. The corresponding GNMs can capture certain local properties, however, they neglect the global networks and are unable to grab the essential characteristics of the protein. As a consequence, the correlation coefficients are quite poor. In the third region, constructed networks incorporate more and more large-sized loops or rings and the corresponding GNMs improve predictions. In the last region, local rings disappear while global rings are included in the network models. It is natural to assume that only when the constructed network includes all essential topological invariants that the corresponding GNM delivers the best prediction. However, this assumption turns out to be incorrect. As indicated in Fig. 5 b, the largest correlation coefficient is actually in the third region. The best cutoff distances are around 7Å to 9Å. This happens because in the GNM, equal weights are assigned to all elastic springs once spring lengths are within the cutoff distance. Thus, there is no discrimination between local and global ring structures.</p><!><p>Unlike GNM which utilizes a cutoff distance, the FRI theory employs a scale or characteristic distance σ in its correlation kernel. The scale has a similar function as the scale in wavelet theory, and thus it emphasizes the contribution from the given scale. The FRI scale has a direct impact in the accuracy of protein B-factor prediction. Similar to the optimal cutoff distance in the GNM, the best FRI scale varies from protein to protein, although an optimal value can be found based on a statistical average over hundreds of proteins.8, 9 In the present work, we use the scale as an additional variable to construct multidimensional persistent homology.</p><p>In our recent work, we have introduced a FRI based filtration method to convert the point cloud data into matrix data.51 In this approach, we construct a new filtration matrix M = {Mij |i = 1, 2, ⋯, N; j = 1, 2, ⋯, N} (8)Mij={1−Φ(‖ri−rj‖;σ),i≠j,0,i=j, where 0 ≤ Φ(‖ri – rj‖; σ) ≤ 1 is defined in Eqs. (1) and (2). To avoid any confusion, we simply use the exponential kernel with parameter κ = 2 in the present work.</p><p>The performance of the FRI B-factor prediction and the multidimensional persistence of protein 2MCM are illustrated in Fig. 6. The filtration matrices are constructed as Mij=1.0−e−(‖ri−rj‖σ)2. The comparison of experimental B-factors and predicted B-factors with the scale σ = 9.2Å is given in Fig. 6 a. The Pearson correlation coefficient is 0.81 for the prediction. Figure 6 b, shows the relation between the correlation coefficient and the scale. It is seen that the largest correlation coefficients are obtained when the scale is in the range of 5Å to 15Å. Figures 6 c and d demonstrate respectively β0 and β1 2D persistence. Unlike the GNM results shown in Fig. 5 where different cutoff distances lead to dramatic changes in network structures, the FRI connectivity shown in Fig. 6 c increases gradually as σ increases. For all σ > 3Å, the maximal β1 values can reach 40 as shown in Fig. 6 d. However, in the region of 5Å< σ <15Å, 1D rings are established over a wide range of the matrix values, which implies a wide range of distances. The balance of the global and local rings gives rise to excellent FRI B-factor predictions.</p><p>In fact, a persistent homology based quantitative model can be established in terms of accumulated bar length.51 Essentially, if all the PBNs are added up at each scale, the accumulated PBNs give rise a good prediction of the optimal scale range. State differently, the plot of the accumulated PBNs versus the scale will have a similar shape as the curve in Fig. 6 b.</p><!><p>Volumetric data are widely available in science and engineering. In biology, density information, such as the experimental data from cryo-EM,86, 102 geometric flow based molecular hypersurface31, 33, 42, 85 and electrostatic potential,44, 103 are typically described in volumetric form. These volumetric data can be filtrated directly in terms of isovalues in persistent homology analysis. Basically, the locations of the same density value form an isosurface. The discrete Morse theory can then be used to generate cell complexes. Additionally, we have developed techniques51 to convert point cloud data from X-ray crystallography into the volumetric form by using the rigidity function or density in our FRI algorithm.86 Specifically, the atomic rigidity index μi in Eq. (5) can be generalized to a position (r) dependent rigidity function or density8, 9 (9)μ(r)=∑j=1Nwj(r)Φ(‖r−rj‖;σj). Volumetric multidimensional persistence can be constructed in many different ways. Because wj and σj are 2N independent variables, it is feasible to construct 2N + 1-dimensional persistence for an N-atom biomolecule. Here the additional dimension is due to the filtration over the density μ(r). If we set wj = 1 and σj = σ, we can construct genuine 2D persistence by filtration over two independent variables, i.e., σ and density.</p><p>In this work, we also demonstrate the construction of pseudo-multidimensional persistence. Since noise and denoising are two important issues in volumetric data, we develop methods for pseudo-multidimensional topological representation of noise and pseudo-multidimensional topological denoising.</p><!><p>To analyze the topological signature of noise, we make a case study on Gaussian noise, which is perhaps the most commonly occurred noise. The Gaussian white noise is a set of random events satisfying the normal distribution (10)n(t)=An2πσne−(t−μn)22σn2, where An, μn and σn are the amplitude, mean value and standard deviation of the noise, respectively. The strength of Gaussian white noise can be characterized by the signal to noise ratio (SNR) defined as SNR = μs / σn, where μs is the mean value of signal. We generate noise polluted volumetric data by adding different levels of Gaussian white noise to the original data.</p><p>We employ fullerene C20 as an example to illustrate the multidimensional topological fingerprints of noise. The rigidity density of C20 is given by (11)μ(r)=∑j=120e−2‖r−rj‖. The noisy data and multifiltration results are demonstrated in Fig. 7. We plot the noisy data of C20 with three SNRs, 1, 10 and 100 in Figs. 7 a1–a3. The persistent barcodes of C20 have 20 β0 bars, 11 β1 bars and one β2 bar. Figures 7 b1–b3 are respectively 2D β0, β1 and β2 persistent homology. In these figures, the vertical axises are the SNR values, which are varied over the range of 1.0 to 100.0. The horizontal axises represent the density isovalues (i.e., the main filtration parameter). In these cases, the designed filtration goes from the highest density value around 2.0 to the lowest density about −1.0. The negative values are introduced by the Gaussian noise. The resulting PBNs are plotted in the natural logarithm scale as indicated by the color bars.</p><p>First of all, the topological fingerprints of C20 stand out in Figs. 7 b1–b3 and demonstrate some invariant features as the SNR increases. In Figure 7 b1, the rectangle-like region is due to the twenty isolated parts in C20. Similarly, the rectangle-like region in Figs. 7 b2 and b3 represents the 12 rings and the central void of the C20 structure. These rectangle patterns are the intrinsic topological fingerprints of C20. In Figs. 7 b1–b3, noise topological signatures dominate the counts of Betti numbers, particularly when the SNR is smaller than 30. For example, β2 spectrum near the density value of 0.4 is essentially indistinguishable from noise induced cavities.</p><p>We have recently proposed topological denoising as a new strategy for topology-controlled noise reduction of synthetic, natural and experimental data.86 Our essential idea is to couple noise reduction with persistent homology analysis. Since persistent topology is extremely sensitive to the noise, the strength of noise signature can be monitored by persistent homology in a denoising process. As a result, one can make optimal decisions on number of deniosing iterations. It was found that contrary to popular belief, noise can have very long lifetimes in the barcode representation,86 while short lived features are part of molecular topological fingerprints.51 In the present work, we introduce 2D topological denoising methods. To this end, we present a brief review of the Laplace-Beltrami flow based denoising approach.</p><!><p>One of efficient approaches for noise reduction in signals, images and data is geometric analysis, which combines differential geometry and differential equations. The resulting geometric PDEs have become very popular in applied mathematics and computer science in the past two decades.104–106 Wei introduced some of the first families of high-order geometric PDEs for image analysis107 (12)∂u(r,t)∂t=−∑q∇·jq+e(u(r,t),|∇u(r,t)|,t),q=0,1,2,⋯ where the nonlinear hyperux term jq is given by (13)jq=−dq(u(r,t),|∇u(r,t)|,t)∇∇2qu(r,t),q=0,1,2,⋯ where r ∈ ℝn, ∇=∂∂r, u(r, t) is the processed signal, image or data, dq(u(r, t), |∇u(r, t)|, t) are edge or gradient sensitive diffusion coefficients and e(u(r, t), |∇u(r, t)|, t) is a nonlinear operator. Denote X(r) the original noise data and set the initial input u(r, 0) = X(r). There are many ways to choose hyperdiffusion coefficients dq(u, |∇u|, t) in Eq. (13). For example, one can use the exponential form (14)dq(u(r,t),|∇u(r,t)|,t)=dq0exp[−|∇u|κσqκ],κ>0, where dq0 is chosen as a constant with value depended on the noise level, and σ0 and σ1 are local statistical variance of u and ∇u (15)σq2(r)=|∇qu−∇qu¯|2¯(q=0,1). Here the notation Y(r)¯ represents the local average of Y (r) centered at position r. The existence and uniqueness of high-order geometric PDEs were investigated in the literature.108–111 Recently, we have proposed differential geometry based objective oriented persistent homology to enhance or preserve desirable traits in the original data during the filtration process and then automatically detect or extract the corresponding topological features from the data.85 From the point of view of signal processing, the above high order geometric PDEs are designed as low-pass filters. Geometric PDE based high-pass filters was pioneered by Wei and Jia by coupling two nonlinear geometric PDEs.112 Recently, this approach has been generalized to a new formalism, the PDE transform, for signal, image and data analysis.40, 113–115</p><p>Apart from their application to images,107, 116, 117 high order geometric PDEs have also been modified for macromolecular surface formation and evolution,43 (16)∂S∂t=(−1)qg(|∇∇2qS|)∇·(∇(∇2qS)g(|∇∇2qS|))+P(S,|∇S|), where S is the hypersurface function, g(|∇∇2qS|) = 1+|∇∇2qS|2 is the generalized Gram determinant and P is a generalized potential term. When q = 0 and P = 0, a Laplace-Beltrami equation is obtained,42 (17)∂S∂t=|∇S|∇·(∇S|∇S|). We employe this Laplace-Beltrami equation for the noise removal in this work.</p><p>Computationally, the finite different method is used to discretize the Laplace-Beltrami equation in 3D. Suitable time interval δt and grid spacing h are required to ensure the stability and accuracy. To avoid confusion and control the noise reduction process systematically, we simply ignore the voxel spacing in different data sets and employ a set of unified parameters of δt = 5.0E – 6 and h = 0.01 in our computation. The intensity of noise reduction is then described by the duration of time integration or the number of iterations of Eq. (17).</p><!><p>From Figures 7 b1–b3, it can been seen that, with the increase of SNR, the intrinsic topological properties emerge and persist. Persistent patterns can be seen in the PBN representation. It is interesting to know whether the topological persistence of the signal is a feature in the denoising process.</p><p>Figures 7 c1–c3 depict the topological invariants of contaminated fullerene C20 over the Laplace-Beltrami flow based denoising process. The fullerene C20 rigidity density is generated by using Eq. (11). The noise is added according to Eq. (10) with the SNR of 1.0. The Laplace Beltrami equation (17) is solved with time stepping δt = 5.0E – 6 and spatial spacing h = 0.01. Figures 7 c1–c3 illustrate respectively the β0, β1 and β2 persistent homology analysis of the denoising process. The filtration goes from density 2.0 to −1.0 (the negative values are due to the added noise). A total of 200 denoising iterations is applied to the noisy data. The PBNs are plotted in the natural logarithm scale. It can be seen that after about 40 denoising iterations, the noise intensity has been reduced dramatically. Indeed, the intrinsic topological features of C20 emerge and persist. It appears that the bandwidths of C20 PBNs reduce during the denoising process. However, such a bandwidth reduction is due to the fact that there is a dramatic density reduction during the denoising precess, particularly at the early stage of the denoising. In fact, the accumulated Betti numbers of C20 do not change and stay stable. It should be noted the color bar denotes the natural logarithm of PBNs values added by 1. The comparison between Figures 7 b1–b3 and c1–c3 demonstrates clearly the noise reduction effect in various iteration steps. It provides a criteria to distinguish between the intrinsic topological properties and noise in denoising process.</p><p>Having demonstrated the construction of 2D persistence for topological denoising, we further apply this new technique for the analysis of noisy cryo-EM data of a microtubule (EMD 1129).102 Figures 8 a, b, and c are surfaces extracted from denoising data with the numbers of iterations of 1, 100 and 200, respectively. A common isovalues of 15.0 used to extract surfaces in these plots. It is seen that the denoising process reduces not only the noise, but also the density, which leads to the shift in the topological distribution. Figures 8 d, e and f are respectively the 2D β0, β1 and β2 persistence. The filtrations in horizontal axises go from density 45 to 0. In Figures 8 d, e and f, vertical axises are the numbers of iterations. A total of 300 iterations is employed for integrating Eq. (10) with time stepping δt = 2.0E − 6 and spatial spacing h = 0.01. Color bar values represent the natural logarithm of PBNs. It can be seen that after about 100 denoising iterations, the noise intensity has been dramatically reduced. Persistent behavior can be observed in β0, β1 and β2. This persistent behavior is a manifest of the intrinsic topological features of the micortubule structure.</p><!><p>In this section, we demonstrate the construction of multiscale multidimensional persistent homology. To this end, we consider protein 2YGD in our multiscale 2D persistence analysis. The fullerene C60, whose topological properties have been analyzed in our earlier work,51 is used as an example to illustrate our multiscale high-dimensional persistence.</p><!><p>We generate volumetric density data of protein 2YGD by using the exponential kernel function (18)μ(r)=∑j=1Nwje−‖r−rj‖σ, where the resolution σ is utilized as a multiscale parameter and will be varied from 0.7Å to 14.7Å. Weight wj is chosen as the atomic number of the jth atom. We linearly rescale the density value to region [0,1] using expression μ(r)s=μ(r)μmax. Here μ(r)s is the rescaled density value. Here μmax is the largest density value in the original data. For each given scale, we carry out the density value based filtration of protein 2YGD. Our results are depicted in Fig. 9. The structure of protein 2YGD is plotted in Fig. 9 a.</p><p>The structure of protein 2YGD exhibits dramatically different scales ranging from atom, residue, secondary-structure, domain to entire protein. Figure 9 illustrates the topological representation of this multiscale structure. Generally speaking, we can roughly divide results of β0, β1 and β2 into three parts according to the resolution parameter σ. The first part is when σ is smaller than 3 Å. In this region, the topological properties related to the local structures, i.e., atoms or intra-residues, are well captured. The second part is the region when σ is larger than 3 Å and smaller than 7 Å. With the increase of the resolution value, local structures gradually disappear, more global type of structures, i.e., inter-residual and domain, begins to emerge. The rest region belongs to the third part, in which, only the global backbone structure of the protein 2YGD is captured. We can seen that the PBNs in this region are comparably consistent. In β0, we have 4 individual components corresponding to the four major domains in the protein. In β1, the PBNs are majorly 9 and 4, representing the 6 large ring and 4 small ring pattern in the structure. Finally the PBN is 1 for β2, this captures the central void in the protein.</p><!><p>Having demonstrated the construction of 2D topological persistence in a number of ways, we pursue to the development of 3D persistence. Obviously, there are a variety of ways that one can construct 3D or multidimensional persistent homology. For example, 3D persistent homology can be generated by the combination of scale, time and the matrix filtration, the combination of scale, time and density filtration, and the combination of scale, SNR and density filtration. In the present work, we illustrate 3D persistent homology by using anisotropic scales or anisotropic filtrations, which give rise to truly multidimensional simplicial complexes and truly multidimensional persistent homology. For simplicity, we take fullerene C60 as an example to illustrate our approach.</p><p>We define the density of the fullerene C60 by a multiscale function, (19)μ(r)=∑j=1601.01.0+(x−xjσjx)2+(y−yjσjy)2+(z−zjσjz)2, where (xj, yj, zj) are the atomic coordinates of C60 molecule and σjx,σjy and σjz are 180 independent scales. Obviously, each of these scales can vary independently. Therefore, together with the density, these scales are able to deliver 181-dimensional filtrations. However, the visualization of such a high-dimensional persistent homology will be a problem, not to mention its physical meaning. To reduce the dimensionality, we set σjx=σx,σjy=σy and σjz=σz, which leads to four-dimensional (4D) persistent homology. To further reduce the dimensionality, we set σx = σy to end up with 3D persistence.</p><p>Unlike the isotropic filtration created by an isotropic scale, the anisotropic filtration creates a family of distorted "molecules" for topological analysis. For the highly symmetry C60 molecule, these distorted versions are not very physical by themselves. However, C60 is a good choice for illustrating and analyzing our methodology, because any distortion is due to the method. On the other hand, the method itself is meaningful due to the fact that most molecules are not symmetric and have anisotropic shapes or anisotropic thermal fluctuations. Figure 10 depicts anisotropic C60 molecules generated by different combinations of σx = σy and σz according to Eq. (19). Figures 10 a and b are obtained with σx = σy = 0.2 Å and σz = 0.5 Å at the isovalue of 0.4. There is an elongation along the z axis. Figures 10 c and d are generated with σx = σy = 0.5 Å and σz = 0.2 Å at the isovalue of 1.0. In this case, there is an obvious compression in the z-direction.</p><p>Topologically, the anisotropic filtration systematically creates a family of truly multidimensional simplicial complexes which would be difficult to imagine otherwise in the 3D space. Figure 11 illustrates the multiscale 3D persistent homology of C60 molecule. The molecular structure is presented in Fig. 11 a with σx = σy = σz = 0.5 Å at the isovalue of 1.5. For the 2D persistent homology, the variation of PBNs over two axises can be represented by different color schemes. However, the visualization of PBNs in 3D is not trivial. Figures 11 b, c and d are respectively multiscale 3D β0, β1 and β2 persistence. Here the x-axises represent the density value (i.e., the main filtration parameter). The y-axises denote σz and the z-axises are for σx = σy. The distributions of two PBNs, β0 = 4 and β0 = 50 are plotted with blue dots and red dots respectively in Fig. 11 b. It is seen that PBNs of β0 are mainly distributed at small fix and σz scales. In Fig. 11 c, we depict the distributions of β1 = 3 and β1 = 20 with blue dots and red dots, respectively. As the scales increase, the PBNs of β1 first increase then decay. Finally, the distributions of β2 = 1 and β2 = 2 are illustrated with blue dots and red dots, respectively in Fig. 11 d. As the cavity of C60 is relatively global, the values of β2 = 1 is seen to locate at relatively large scales.</p><!><p>Recently, persistent homology, a new branch of topology, has gained considerable popularity for computational application in big data simplification. It generates a one-parameter family of topological spaces via filtration such that topological invariants can be measured at a variety of geometric scales. As a result, persistent homology is able to bridge the gap between geometry and topology. However, one-dimensional (1D) persistent homology has its limitation to represent high dimensional complex data. Multidimensional persistence, a generalization of 1D persistent homology to a multidimensional one, provides a new promise for big data analysis. Nevertheless, the realization and construction of robust multidimensional persistence have been a challenge.</p><p>In this work, we introduce two types of multidimensional persistence. The first type is called pseudo-multidimensional persistence, which is generated by the repeated applications of 1D persistent homology to high-dimensional data, such as results from molecular dynamics simulation, partial differential equations (PDEs), molecular surface evolution, video data sets, etc. The other type of multidimensional persistence is constructed by appropriate multifiltration processes. Specifically, cutoff distance and scale are introduced as new filtration variables to create multifiltration and multidimensional persistence. The scale of flexibility-rigidity index (FRI)8, 9 behaves in the same manner as the wavelet scale. It serves as an independent filtration variable and controls the formation of simplicial complexes and the corresponding topological spaces. As a result, the FRI scale creates truly multiscale multidimensional persistent homology, in conjugation with the matrix value variable or the density variable. We have developed genuine two-dimensional (2D) persistent homology. By using anisotropic scales, in which the scale in each spatial direction can vary independently, we can construct four dimensional (4D) persistent homology. A protocol is prescribed for the construction of arbitrarily high dimensional persistence. Concrete numerical example is given to three-dimensional (3D) persistence.</p><p>We have demonstrated the utility, established the robustness and explored the efficiency of the proposed multidimensional persistence by its applications to a wide range of biomolecular systems. First, we have constructed pseudo-multidimensional persistence for the protein unfolding process. It is shown that local topological features such as pentagonal and hexagonal rings in the amino acid residues are preserved during the unfolding process, whereas global topological invariants diminish over the unfolding process. Topological transition from folded or partially folded proteins to unfolded proteins can be clearly identified in the 2D persistence. We show that the β0 persistence also provides an indication of the strength of applied pulling forces in the steer molecular dynamics. Additionally, we have analyzed the optimal cutoff distance of the Gaussian network model (GNM) and the optimal scale of the FRI theory by using 2D persistence. We have revealed the relationship between the topological connectivity in terms of Betti numbers and the performance of the GNM and the FRI for the prediction of protein Debye-Waller factors. Moreover, we have utilized 2D persistence to illustrate the topological signature of Gaussian noise. The efficiency of Laplace-Beltrami flow based topological denoising is studied by the present 2D persistence. We show that the topological invariants of C20, especially β2, persist during the denoising process, whereas the topological invariants of noising diminish during the denoising process. Similar results are also observed for the topological denoising of cryo-electron microscopy (cryo-EM) data. Finally, we have employed multiscale multidimensional persistence to investigate the topological behavior of protein 2YGD. We reveal its multiscale structure properties in the our 2D persistence. We also consider the C60 over anisotropic scale variations. This study unveils that β0 invariants are intrinsically local, while β1 and β2 invariants are relatively global.</p><p>Multidimensional persistence techniques have been developed for three types of data formats, i.e., point cloud data, matrix data and volumetric data. We have also illustrated conversion of point cloud data to matrix and volumetric data via the FRI theory. Therefore, the proposed methodology can be directly applied to other biomolecular systems, biological networks, and diverse other disciplines.</p>

PubMed Author Manuscript

Fabrication of highly ordered Sn nanowires in anodic Aluminum Oxide templates by using AC electrochemical method

Sn and its nanostructures are one of the promising candidates to replace graphite in the anode of Lithium-ion batteries due to their higher capacity. One of the challenges, which limited the usage of Sn anodes for the Lithium-ion batteries, is Tin's high volumetric strain and its low cyclability. On the other hand, nanostructures show lower volume change during charge/discharge and as a result could address the cyclability issues. In this research, an alternating current (AC) electrochemical method is developed in order to facilitate the industrial scale production of Sn nanowires. The developed electrodeposition technique shows reliable controllability over chemical composition and crystalline structure of Sn nanowires. Also, the order structure of nanowires could be adjusted more accurately in comparison to conventional fabrication techniques. As a result, the Sn nanowires as well as Aluminum Oxide templates synthesized by using the developed electrochemical method are examined due to their morphology, chemical composition, and their crystalline structure in order to develop a practical relation between electrochemical composition of the solution and materials properties of Sn nanowires. The results show that the proposed electrodeposition method maintains a highly-ordered morphology as well as industrially acceptable controllability over crystalline structure of nanowires, which could be used to optimize the procedure for industrial applications due to low cost and simple experimental setup.

fabrication_of_highly_ordered_sn_nanowires_in_anodic_aluminum_oxide_templates_by_using_ac_electroche

Introduction<!>Two step anodizing<!>AC nanowire electrodeposition<!>Materials characterization<!>Aluminum Oxide morphology<!>Impedance spectroscopy of Aluminum Oxide template's barrier layer<!>Sn nanowires<!>Conclusions

<p>Exceptional mechanical properties of nanowires (e.g. low volumetric strain) in comparison to thin films make them suitable for micro-electronic device applications, where cyclability and volume changes are great concerns which may lead to mechanical failure and decrease the efficiency of the devices (e.g. Lithium-ion batteries) [1][2][3][4][5][6][7][8][9][10][11]. Tin (Sn) based anodes are thoroughly studied in order to increase the Li-ion batteries' capacities . Despite of large volumetric strains of Sn based anodes, during charge/discharge, which may lead to mechanical failure of anodes and reduce the cyclability of Lithium-ion batteries [48][49][50], alloying the anode material (i.e. Sn) with mechanically stable elements (e.g. Ni, Co, Cu, graphene, etc.) method is developed in order to achieve promising cycling performance and utilizing the higher capacity of Tin (Sn) simultaneously [12-14, 17, 23, 26]. Furthermore, nanostructured version of these Tin based alloys (e.g. nanowires, nanoparticles, etc.) could stabilize the volumetric strains more effectively, because of their small volume changes in comparison to thin films [51]. Although, synthesizing the nanostructures in large volume for industrial applications, due to their expensive and time consuming production methods such as: surfactant based techniques or self-assembling of 0D nanostructures [52][53][54], limited their application for Lithium-ion batteries' anodes productions. On the other hand, novel fabrication techniques such as electrodeposition methods (e.g. direct or alternating current techniques) have shown more accurate controllability over materials morphology and chemical composition of nanostructures in comparison to conventional production techniques as well as their easier scalability for industrial applications [55][56][57]. Electrodeposition of metallic alloys in the ordered self-assembled templates (e.g. Al 2 O 3 , TiO 2 , porous polycarbonate, etc.) is developed primarily to control the morphology of nanostructures [58][59][60][61][62]. One of the main challenges, which limited the applicability of electrochemical method to fabricate highly-ordered nanowires, is that conventional direct current (DC) electrodeposition methods are suffering from practical difficulties related to producing a conductive self-assembled structure due to low conductivity of templates' materials [63][64][65][66][67][68][69][70][71][72][73][74][75][76]. On the other hand, alternating current (AC) electrochemical deposition method is well designed to overcome the technical difficulties, related to removing the barrier layer of the anodic Aluminum Oxide (AAO) and coating this self-assembled template with conductive materials [77]. As a result, the AC electrodeposition technique has a potential to pave the way for the industrial scale production of nanostructures with a highly controlled morphology as well as their chemical structure [59]. In this research, AC electrodeposition technique has been deployed to fabricate highly-ordered Sn nanowires in Aluminum Oxide templates. Furthermore, a novel room temperature two-steps anodization technique, which is used to produce a highly-ordered template, as well as its morphological properties will be investigated in details. Finally, morphological properties as well as chemical structure of fabricated Sn nanowires will be studied in order to examine their crystalline structures as well as controllability of nanowires' chemical composition. All the experiments in this research are done at room temperature, which paves the way for its industrial scale generalizations due to technical difficulties related to costly temperature controlling systems [78].</p><!><p>Before anodizing of Aluminum samples (Merck KGaA, 99.95%, 0.3 mm thickness -annealed), electrodes are electropolished (A = 1cm 2 ) in HClO 4 60 wt. % solution. In fact, the electropolishing voltage and temperature are fixed at 2 V and room temperature (25 • C) respectively. Also, the electropolishing time is optimized at 5min in order to achieve a highly smooth surface without primary amorphous Aluminum Oxide. In both the first and second steps of anodization procedure, the electrodes are anodized in C 2 H 2 O 4 0.3M solution. Furthermore, anodization time, voltage, and temperature are fixed at 2h, 40 V and room temperature (25 • C) respectively for first and second steps of this anodizing procedure. Fabricated porous Aluminum Oxides after the first step of anodization are etched in a solution of 0.6M H 3 PO 4 85 wt. % -0.2M H 2 CrO 4 . The etching temperature and time are fixed at 60 • C and 30min respectively. The main challenge, in order to use Aluminum Oxide templates for electrodeposition purposes directly, is how to reduce the electrical resistance of Al 2 O 3 barrier layer, which prevents the electrical current flow through the thick insulative layer at the bottom of the pores (i.e. barrier layer) [79][80][81][82][83]. In this research, the barrier layer thinning (BLT) procedure (i.e. reducing the second step anodization voltage gradually) is used to reduce the electrical impedance of the Aluminum Oxide layer at the bottom of the pores. In fact, in this BLT procedure, anodization voltage at the second step is decreased step-wise with a rate of 2 V min until reaching 30 V. After that, the voltage will be decreased with a rate of 1 V min to reach 6 V. Finally, the samples will be held in the anodization solution at the 6 V electrical potential for 10 minutes before removing them from anodization solution in order remove barrier layer as much as possible and increase the electrical conductivity of self-assembled templates. Additionally, the electrical impedance of the electrodes, before and after BLT procedure, are examined by using impedance spectroscopy.</p><!><p>The AC electrochemical deposition technique is employed to reduce Sn 2+ ions in the pores of self-assembled Aluminum Oxide template. In all the experiments of this section, pH as well as Boric acid (H 3 BO 3 ) concentration, root mean square (RMS) voltage, and AC signal's frequency are fixed at 1, 0.5M, 10 V, and 200 Hz respectively. Also, the Tin Sulfate (SnSO 4 ) concentration is changed from 0.1M to 0.5M with step size of 0.1M, in order to examine the effect of concentration on crystalline structure as well as directional growth of Sn nanowires. As a result, chemical composition as well as crystalline structure of deposited nanowires are examined by using energy dispersive spectroscopy (EDS) and X-ray diffraction respectively.</p><!><p>Samples' characterization, which was used to investigate morphology of self-assembled templates as well as Sn nanowires, was done with a field emission scanning electron microscope (FE-SEM) Quanta 3D FEG (FEI, Phillips, The Netherlands). In order to examine the pore sizes as well as morphology (i.e. order structure) of nanowires, ImageJ [84] image processing software is used to obtain quantitative information on the average diameter of the self-assembled templates' pores' diameter and length, as well as diameter of the electrodeposited Sn nanowires. The crystalline structure of the Sn nanowire alloy was analyzed by X-ray diffraction using a Rigaku Ultima IV diffractometer with Co K radiation and operating parameters of 40 mA and 40 kV with a scanning speed of 1 • per minute and step size of 0.02 • . Finally, the impedance spectroscopy of the anodized samples were done by using a MultiPalmSens4 potentiostat in order to compare the electrical resistance of anodizied samples before and after barrier layer thinning procedure.</p><!><p>The porous morphology of two steps anodized Aluminum Oxide is shown in fig. 1. According to fig. 1(a), which is analyzed by using image processing techniques, it could be understood that the average pore size of this self-assembled template is 60 nm. Furthermore, the order structure of this porous medium is analyzed by using the fast Fourier transform (FFT) technique in order to examine the spatial structure of pores and their deviation from honeycomb structure. As a result, according to fig. 1(b), the FFT result of this AAO microstructure shows 6 strong bright dots, which indicates that a perfect honeycomb structure is achieved after the second step of anodization. Additionally, in order to examine the aspect ratio of self-assembled pores of AAO, a cross-sectional FE-SEM microscopy is done to estimate the thickness of Aluminum Oxide after the second step of anodization (cref. fig. 2). As shown in fig. 2(a), the thickness of AAO template is about 33 µm. As a result, the aspect ratio of pores, which is defined as the ratio of thickness over diameter, could be estimated as 550. Also, this aspect ratio will be increased for nanowires after AAO dissolution because of nanowires' radial shrinkage due to compressive residual stresses [85,86]. The wall thickness of pores in self-assembled AAO template is estimated as 50 nm, which is shown in fig. 2(b). This highly ordered structure after the second step of anodization is achieved because: the quantum dots are created on the electrode's surface after etching step, which could facilitate the directional growth of AAO as well as controlling of its diameter more precisely [87].</p><!><p>Impedance spectroscopy is done in order to examine the electrical resistance of barrier layer before and after the thinning procedure. Additionally, the impedance of barrier layer directly could be related to its thickness as [88,89]:</p><p>Where Z is the electrical impedance, j = √ −1 is the imaginary unit, ω is the frequency, a is frequency scattering factor, C bl is the barrier layer capacity, d bl is the barrier layer thickness, r is the relative electrical permittivity, 0 is the electrical permittivity of vacuum, and S is the surface area of the sample. The impedance magnitude versus frequency and its real part versus imaginary part (Nyquist plot) for before and after the barrier layer thinning procedure are shown in fig. 3(a) and fig. 3(b) respectively. The equations 1 and 2 are used to fit them into the Nyquist plots (cref. fig. 3) and as a result, the obtained values for barrier layer thicknesses before and after BLT procedure are 20nm and 5nm respectively. This calculation shows that the BLT procedure reduced the barrier layer thickness 4 times smaller, which could increase its conductivity and facilitate the AC electrochemical deposition step. Additionally, the electrical circuits equivalence of the Nyquist plots are extracted due to the fitted parameters which are shown in fig. 4. According to these electrical circuits, it could be understood that the second resistance/capacitance pair remained constant before and after BLT procedure. However, the electrical resistance of first resistance/capacitance pair is reduced by three orders of magnitude, which shows the electrical resistance is reduced after BLT procedure significantly (cref. fig. 4).</p><!><p>FE-SEM microscopy technique is used to investigate the morphology of Sn nanowires after dissolution of AAO template in 1M NaOH solution. In fig. 5, Sn nanowires are shown in two different resolutions, which show their long-range order structure (cref. fig. 5(a)) as well as the diameter of the nanowires (cref. fig. 5(b), 33 nm). As a result, according to fig. 5(b), it could be understood that the aspect ratio of the nanowires are increased by a factor of 2, which could increase their surface to volume ratio as well as their chemical reactivity for practical applications. Also, the EDS and X-ray diffraction techniques are used to examine the chemical composition and crystalline structure of Sn nanowires respectively (cref. fig. 6). According to fig. 6(a), there are some residual Al 2 O 3 due to presence of Aluminum and Oxygen peaks. These residual Aluminum Oxide could be eliminated by increasing the AAO template dissolution time. Furthermore, fig. 6(b) shows that Sn nanowire preferential growth direction could be controlled by changing Tin Sulfate concentration in the electrodeposition solution. In fact, solution's chemistry could be used as a controlling variable to optimize crystalline structure of Sn nanowires. Due to fig. 6(b), it could be observated that intensity peaks of direction (200) is maximized for concentration 0.5M. It means at 0.5M SnSO 4 concentration, the (200) direction is the most thermodynamically unstable direction in comparison to (211) direction [90], which will lead to preferential growth along the (200) direction. In fact, at the 0.5M SnSO 4 concentration the activity of the Sn 2+ ions are maximized and as a result of that it shows preferential growth. This preferential growth observed in X-ray diffraction pattern of Sn nanowires indicates that crystalline structure of Tin based nanostructures could be optimized by changing SnSO 4 concentration in the context of the proposed AC electrochemical method. Additionally, this oriented crystalline structure of Sn nanowires in (200) direction could maximize their efficiency as an anode for Lithium-ion batteries because of their maximum chemical activity as well as enhancing their cyclability due to minimized volumetric strain [91][92][93][94][95].</p><!><p>In this research, an AC electrochemical deposition technique is developed, which could be easily used to fabricate metallic nanowires with highly-ordered structure and reasonable controllability over the chemical composition as well as the crystalline structure. Additionally, this technique could facilitate the large-scale production of nanostructures due to its room temperature operating environment. As a result, this technique could be generalized to develop industrial scale coating facilities, which could be used in Lithium-ion battery production as well as other industries, such as: biomedical applications [96][97][98][99][100], oil and gas extraction plants [101][102][103][104], and nanoparticles technologies [105]. Finally, these Sn nanowires' production technique should be optimized by using the proposed electrochemical synthesizing procedure, and be examined in assembled Lithium-ion batteries to accurately measure their capacity as well as efficiency in order to achieve higher cyclability.</p>

ChemRxiv

The Class A Carbapenemases BKC-1 and GPC-1 Both Originate from the Bacterial Genus Shinella

Comparative genomics identified the environmental bacterial genus Shinella as the most likely origin of the class A carbapenemases BKC-1 and GPC-1. Available sequences and PCR analyses of additional Shinella species revealed homologous β-lactamases showing up to 85.4% and 93.3% amino acid identity to both enzymes, respectively. The genes conferred resistance to β-lactams once expressed in Escherichia coli. blaBKC-1 likely evolved from a putative ancestral Shinella gene with higher homology through duplication of a gene fragment.

the_class_a_carbapenemases_bkc-1_and_gpc-1_both_originate_from_the_bacterial_genus_shinella

INTRODUCTION<!><!>INTRODUCTION<!><!>INTRODUCTION<!>Data availability.<!>

<p>The high potential of Gram-negative bacteria to acquire exogenous DNA through horizontal gene transfer has allowed clinically relevant bacteria to acquire resistance toward many antibiotics (1, 2). The acquisition of carbapenemase genes in Enterobacteriaceae, Pseudomonas aeruginosa, and Acinetobacter baumannii represents one of the most important threats, compromising the use of the entire β-lactam family.</p><p>Recently, two novel carbapenemase genes, blaBKC-1 and blaGPC-1, were characterized (3, 4). The genes code for weak class A carbapenemases sporadically identified in Klebsiella pneumoniae and P. aeruginosa isolates, respectively. The two enzymes, BKC-1 and GPC-1, share 77% amino acid identity, but their exact origins remain unknown. The aim of this study was to investigate the origin of both blaBKC-1 and blaGPC-1.</p><p>All bacterial genomes and plasmids (n = 610,187, downloaded March 2020) available in GenBank were searched for the blaGPC-1 and blaBKC-1-like genes, using DIAMOND v0.9.24.125 at a 70% identity cutoff (5). The blaGPC-1/BKC-1-like genes were identified in 19 assemblies and plasmids. In addition to the presence of blaBKC-1 in the originally reported plasmid from K. pneumoniae, the most similar sequences were found in two Shinella zoogloeoides chromosomes (81.7% and 85.4% amino acid [aa] identity, but if the duplication of part of the gene sequence is considered, the identity is up to 90.2%; see Discussion). The blaGPC-1-like genes were found in 14 different Shinella spp. genomes (S. granuli, S. kummerowiae, S. curvata, Shinella spp.; 80.1 to 93.3% aa identity), and two genomes whose global average nucleotide identity (gANI) analysis showed they are likely to be related to Shinella species and may have been misnamed (Sinorhizobium sp. RAC02 [84.9% aa identity] and uncharacterized Rhizobiaceae bacterium UBA3138 [78.9% aa identity]). Analysis of the two genes' genetic environments in K. pneumoniae and P. aeruginosa showed the previously demonstrated association with both insertion sequences and plasmid-specific genes (Fig. 1). On the contrary, no insertion sequence or other genes indicating mobility could be associated with the homologues of blaGPC-1/BKC-1 found in Shinella spp. The blaGPC-1 and the blaBKC-1 homologs found in different Shinella spp. were located on the same chromosomal locus as indicated by strong synteny (Fig. 1). Sequence dissimilarities of 7% to ≥20% across this locus between species indicate a long-standing association with the Shinella genus (Fig. 1). Moreover, analysis of the sequences immediately up- and downstream of blaGPC-1 and blaBKC-1 showed high homologies with the corresponding loci in Shinella. The upstream and downstream regions of the blaGPC-1 gene shared 85% (44/52) and 86% (48/56) nucleotide identity with S. granuli DD12, while for the blaBKC-1 gene, the corresponding identities to S. zoogloeoides DSM287 were 80% (12/15) and 87% (87/100), respectively.</p><!><p>Comparative analysis of GPC-1/BKC-1-like loci. Striped arrows denote GPC-1/BKC-1-like genes, dark spotted arrows symbolize transposition associated genes such as IS, and light spotted arrows denote other genes associated with mobility. Light gray areas between graphs symbolize sequence alignment. Nucleotide alignment identities between GPC-1/BKC-1-like loci top to bottom: P. aeruginosa to S. spp JR1-6: 89% to 90%; S. spp JR1-6 to S. granuli, 70% to 93%; S. granuli to S. kummerowiae, 84% to 88%; S. kummerowiae to S. zoogloeoides, 84% to 86%; S. zoogloeoides to K. pneumoniae, 87%. Protein name abbreviations: Gdpd, putative glycerophosphoryl diester phosphodiesterase; Pd, phosphodiesterase; Nsps, norspermidine sensor; RNAtr, putative RNA-binding transcriptional regulator; Tr, ArsR family transcriptional regulator; Gly, glyoxalase/bleomycin resistance protein/dihydroxybiphenyl dioxygenase; Tp, l,d-transpeptidase catalytic domain protein; RecO, DNA repair protein RecO; Padp, phenylactetic acid degradation protein; DUF389, DUF389-containing protein; Pmp, predicted membrane protein; Era, GTPase Era; Phd, putative HD superfamily hydrolase. Nucleotide sequence accessions top to bottom: MN628598, SHMI01000003.1, SLVX01000002.1, WUMK01000006.1, WUML01000002.1, and KP689347.</p><!><p>The GC content of the blaBKC/GPC-like genes from all Shinella isolates and their mobile counterparts ranged from 65.3% to 69.7%. This overlaps with that of the larger (±10,000 bp) genetic contexts in Shinella (63.8% to 66.6%) but not with that of clinical species carrying blaBKC/GPC genes (59.3% to 60.5%).</p><p>Altogether, this indicates that the two resistance genes share an ancestor gene that have evolved separately into a more blaBKC-1-like gene in S. zoogloeoides and a more blaGPC-1-like gene in, e.g., S. granuli. It is therefore highly plausible that that blaGPC-1 and blaBKC-1 were mobilized from different Shinella spp.</p><p>To evaluate the phenotype provided by the blaBKC/GPC-like gene and to find other variants and potential closer homologs, four different Shinella species were recovered from the public bacterial collection bank of the Culture Collection of the University of Gothenburg (CCUG), being S. granuli 56487, S. kummerowiae 56777, S. zoogloeoides 35204, and S. fusca 55808 (6–8). PCR experiments using degenerate primers were performed, and amplified β-lactamase genes (named blaGPG, blaGPK, blaGPZ, and blaGPF, respectively) were cloned into E. coli TOP10 using the pCR2-TOPO cloning kit (Thermo Fisher) and tested for the resistance phenotype using broth microdilution. A CarbaNP test (9) confirmed the ability of the GPC/BKC-like expressing clones to hydrolyze imipenem. All clones displayed a resistance profile against β-lactams, including amino- and ureidopenicillins, first- and second-generation cephalosporins and a low level of resistance against third- and fourth-generation cephalosporins, monobactam, and carbapenems. All clones remained susceptible to the cephamycin cefoxitin. In addition, the use of clavulanic acid or avibactam restored a complete susceptibility against amoxicillin or ceftazidime, respectively, a characteristic shared by class A β-lactamases (Table 1). This phenotype is in accordance with those reported for BKC-1 and GPC-1 (3, 4). Protein alignments using SeaView Software (Prabi, Doua, France) showed that GPC-1 was most closely related to the GPC-like proteins from Shinella sp. strian DD12 (93.3%) and S. granuli (92.6%), whereas BKC-1 shared the highest amino acid identity with S. zoogloeoides (85.4%) (see Fig. S1 in the supplemental material). All variants possessed the typical conserved serine/threonine kinase motifs and the motif involved in the Ω-loop formation of class A β-lactamases (10) (Fig. 2). Deeper alignment analysis showed that BKC-1 displayed a duplication of 16 amino acids, being the repetition of the protein segment from Ala12 to Ser27. Therefore, a putative ancestral protein was designed in silico and named BKC-b (Fig. 2). Aligning the BKC-b sequence with GPZ increased the amino acid identity up to 90.2% compared to 85.4% without the duplication (Fig. S1). Production of BKC-b in E. coli TOP10 showed a weaker resistance profile while having an increased activity against cefoxitin. The use of the I-TASSER (11) in silico tool predicted the tridimensional structures of both BKC-b and BKC-1 and showed that the duplication of the protein segment in BKC-1 modified the ligand binding site of the enzyme and probably led to the increased spectrum of activity observed in BKC-1 compared to that in BKC-b but the loss of its activity against cephamycins.</p><!><p>MICs of the clones expressing the different BKC and GPC variants</p><p>AMX, amoxicillin; AMC, amoxicillin-clavulanic acid; PIP, piperacillin; CEF, cephalothin; FOX, cefoxitin; CXM, cefuroxime; CTX, cefotaxime; CAZ, ceftazidime; CZA, ceftazidime-avibactam; FEP, cefepime; ATM, aztreonam; IPM, imipenem; MEM, meropenem; ERT, ertapenem.</p><p>Amino acid sequence comparison between the different BKC/GPC-like enzymes. A dash represents an amino acid that is common among all variants, a slash represents a gap. The underlined sequence represents the duplication of the peptide Ala12 to Ser27 in BKC-1. Bolded sequences implicated the conserved motifs present in class A β-lactamases: 70STFK, 130SDN, 234KTG involved in the catalytic activity and 166EPxLN, involved in the Ω-loop (ABL numbering).</p><!><p>Here, we provide evidence that the genes blaGPC-1 and blaBKC-1 were most likely mobilized from members of bacterial genus Shinella into clinical species. This conclusion is based on the presence of a conserved locus containing a blaGPC/BKC-like gene in all investigated Shinella species, the lack of associated mobile genetic elements, and high amino acid and nucleotide identities to the clinical counterparts, but not so high that we could assign with confidence the exact origin species. However, it is highly plausible that the origins of blaGPC-1 and blaBKC-1 are Shinella species closely related to S. granuli and S. zoogloeoides, respectively. The resistance phenotype provided by the blaGPC/BKC-like genes is in line with mobilization and transfer driven by antibiotic exposure. The Shinella genus includes mesophilic, aerobic Gram-negative species mainly recovered from environmental samples. For instance, the studied S. granuli and S. zoogloeoides isolates were recovered from sludge in China, while the S. kummerowiae and S. fusca isolates were recovered from root nodules and domestic compost in Korea and Portugal, respectively (6–8). The presence of a natural and functional β-lactamase gene in this genus could be explained by the presence of β-lactam-producing microorganisms sharing the same niche (12). Additionally, we show that the BKC-1 protein presented a duplication of its Ala12-Ser27 segment, likely from a putative ancestral protein BKC-b. Hence, the blaBKC-1 gene may have evolved from blaBKC-b, likely under a selective pressure from β-lactams, eventually resulting in a more efficient enzyme. This mutation led, on the other hand, to the reduction of its activity against cefoxitin.</p><p>Emergence of new resistance genes, especially genes providing resistance to antibiotics of last resort, such as carbapenems, represents a major clinical threat. After initial emergence, they are likely to remain undetected and spread silently in the human microbiota for some time. When detected, they are often already widespread (13). Understanding the origin and mobilization history of as many and diverse clinically important resistance genes as possible could enable us to manage risks for future emergence events in a better way. The data presented here provides one additional piece in this large puzzle.</p><!><p>The nucleotide sequence of the carbapenemases genes blaGPG, blaGPK, blaGPZ, blaGPF, and blaBKC-b were submitted to GenBank with the following accession numbers, respectively: MT661611, MT661612, MT661613, MT661614, and MT661610.</p><!><p>Supplemental material is available online only.</p>

PubMed Open Access

Water Dynamics in Salt Solutions Studied with Ultrafast 2D IR Vibrational Echo Spectroscopy

Conspectus Water is ubiquitous in nature, but it exists as pure water infrequently. From the ocean to biology, water molecules interact with a wide variety of dissolved species. Many of these species are charged. In the ocean, water interacts with dissolved salts. In biological systems, water interacts with dissolved salts as well as with charged amino acids, the zwitterionic head groups of membranes, and other biological groups that carry charges. Water plays a central role in vast number of chemical processes because of its dynamic hydrogen bond network. A water molecule can form up to four hydrogen bonds in an approximately tetrahedral arrangement. These hydrogen bonds are continually being broken and new bonds are being formed on a picosecond time scale. The ability of water\xe2\x80\x99s hydrogen bond network to rapidly reconfigure enables water to accommodate and facilitate chemical processes. Therefore, the influence of charged species on water hydrogen bond dynamics is important. Recent advances in ultrafast coherent infrared spectroscopy have greatly expanded our understanding of water dynamics. Two dimensional infrared (2D IR) vibrational echo spectroscopy is providing new observables that yield direct information on the fast dynamics of molecules in their ground electronic state under thermal equilibrium conditions. 2D IR vibrational echoes are akin to 2D NMR but operate on time scales that are many orders of magnitude shorter. In a 2D IR vibrational echo experiment (see Conspectus figure), three IR pulses are tuned to the vibrational frequency of interest, which in this case is the frequency of the hydroxyl stretching mode of water. The first two pulses \xe2\x80\x9clabel\xe2\x80\x9d the initial molecular structures by their vibrational frequencies. The system evolves between pulses two and three, and the third pulse stimulates the emission of the vibrational echo pulse, which is the signal. The vibrational echo pulse is heterodyne detected by combining it with another pulse, the local oscillator. Heterodyne detection provides phase and amplitude information, which are both necessary to perform the two Fourier transforms that take the data from the time domain to a two dimensional frequency domain spectrum. The time dependence of a series of 2D IR vibrational echo spectra provides direct information on system dynamics. Here we use two types of 2D IR vibrational echo experiments to examine the influence that charged species have on water hydrogen bond dynamics. Solutions of NaBr and NaBF4 are studied. The NaBr solutions are studied as a function of concentration using vibrational echo measurements of spectral diffusion and polarization selective IR pump-probe measurements of orientational relaxation. Both types of measurements show the slowing of hydrogen bond network structural evolution with increasing salt concentration. NaBF4 is studied using vibrational echo chemical exchange spectroscopy. In these experiments it is possible to directly observe the chemical exchange of water molecules switching their hydrogen bond partners between BF4\xe2\x88\x92 and other water molecules. The results demonstrate that water interacting with ions has slower hydrogen bond dynamics than pure water, but the slowing is a factor of three or four rather than orders of magnitude.

water_dynamics_in_salt_solutions_studied_with_ultrafast_2d_ir_vibrational_echo_spectroscopy

I. Introduction<!>A. Chemical Exchange Spectroscopy Measurements of Water \xe2\x80\x94 NaBF4<!>B. Spectral Diffusion Measurements of Water \xe2\x80\x94 NaBr<!>III. Discussion

<p>The hydrogen bond network of pure water, which is responsible for water's unique properties, undergoes rapid structural evolution.1 A question of fundamental importance is how do the dynamics of water in the immediate vicinity of an ion or ionic group differ from those of pure water? Water dynamics in ion hydration shells play a significant role in the nature of systems such as proteins and micelles and in processes such as ion transport through transmembrane proteins.</p><p>In pure water the hydrogen bond network is constantly evolving with a range of time scales from tens of femtoseconds to picoseconds.2-6 Hydrogen bonds are continually forming and breaking through concerted hydrogen bond rearrangements.7 These dynamical processes can be observed on the timescales they occur using ultrafast infrared spectroscopy. Measurements of spectral diffusion using ultrafast two dimensional infrared (2D IR) vibrational echo spectroscopy5,8,9 as well as other ultrafast IR techniques4,10 have determined the multiple time scales for the hydrogen bond dynamics. The slowest time component of the FFCF (1.7 ps) is associated with the randomization of the hydrogen bond network through concerted hydrogen bond rearrangements. The orientational relaxation time of pure water (2.6 ps)4,6 is also assigned to concerted hydrogen bond rearrangement via jump reorientation.7</p><p>Ionic hydration and the dynamics of water in salt solutions have been studied extensively by NMR,11 Neutron diffraction,12 computer simulations,13 and infrared spectroscopy.9,14,15 There is information about the structure of ionic solvation shells from diffraction experiments. However, unraveling the dynamics of the solvation shell and the back and forth exchange of waters hydrogen bonded to ions and to water molecules is an ongoing experimental problem.</p><p>Here, two types of ultrafast 2D IR vibrational echo experiments as well as polarization selective IR pump-probe experiments are used to study water hydrogen bond dynamics in concentrated salt solutions. The two 2D IR vibrational echo observables are chemical exchange16,17 and spectral diffusion.5,8,9 The application of these two types of vibrational echo experiments is dictated by the IR absorption spectrum of the hydroxyl stretch of water. The OD hydroxyl stretch of dilute HOD in H2O is studied rather than pure H2O or D2O to eliminate vibrational excitation transfer.18,19 Vibrational excitation transfer interferes with orientational relaxation measurements, and it will artificially contribute to both chemical exchange and spectral diffusion. MD simulations have shown that dilute HOD in H2O does not change the behavior of water and that observations of the OD hydroxyl stretch report on the dynamics and local environment of water.20</p><p>Figure 1A shows background subtracted IR absorption spectra of the OD stretch of HOD in H2O salt solutions for NaBF417 for several salt concentrations. The numbers, n, are the number of water molecules per salt, e.g., n = 30 means that there are 30 water molecules for one NaBF4. As the concentration of the salt increases, a peak develops on the high frequency side of the water spectrum. This peak arises from ODs hydrogen bonded to the BF4− anions.17 In addition, the main water peak shifts to the blue with increasing concentration but essentially maintains its shape. The presence of distinct peaks for ODs hydrogen bonded to other water molecules and to BF4− anions makes it possible to perform the chemical exchange spectroscopy (CES) experiments, 17 in which hydrogen bond switching between anions and water oxygens is manifested by the growth of off-diagonal peaks in the 2D IR vibrational echo spectra. The CES experiments were performed on the highest concentration sample, 5.5 M with n = 7.</p><p>The NaBr spectra (Figure 1B) do not show two distinct peaks at any salt concentration. The spectra are broad21 and the peak position is increasingly blue-shifted as the NaBr concentration increases. For pure water the broad spectrum is associated with different numbers and a wide range of strengths of the hydrogen bonds.21 For pure water, subensembles of water molecules with more and/or stronger hydrogen bonds have red-shifted absorptions, while subensembles with fewer and/or weaker hydrogen bonds are blue-shifted. In the type of systems being considered here, in which the hydroxyl groups are likely to be interacting with ions, the blue-shift should not be interpreted as an indication of weakening of the hydrogen bond structure in an aqueous solutions, but rather as an overall change in the nature of the local water environments. Because the NaBr spectra do not show distinct peaks, CES cannot be employed. Instead, measurements of spectral diffusion (change in the 2D IR vibrational echo line shapes) can be used to examine the hydrogen bond dynamics.9</p><!><p>Chemical exchange occurs when two species in equilibrium interconvert without changing the overall number of either species. 2D IR vibrational echo chemical exchange spectroscopy has been developed recently16,22-25 and applied to the formation and dissociation dynamics of organic solute-solvent complexes,16,22,26 the rate of isomerization around a carbon-carbon single bond,25 the switching between well defined protein structural substates,27 and hydrogen bond dynamics in NaBF4 solutions17 discussed here.</p><p>Chemical exchange between two species has a well-defined effect on the 2D IR vibrational echo spectrum.16 At times short compared to the exchange time, the peaks corresponding to the two species appear as two positive going bands on the diagonal of the 2D spectrum. A 2D contour plot spectrum like this is shown in the Conspectus figure (also see Figure 3A below). The diagonal is the dashed line. Positive going peaks are red and negative going peaks are blue. The horizontal axis is ωτ, corresponding to the frequencies that the vibrational oscillators interact with when the first pulse in the three pulse sequence is applied. The vertical axis is ωm. It corresponds to the frequencies of oscillators' interacting with the third pulse, which gives rise to the vibrational echo pulse at the same frequencies. So ωm corresponds to the frequencies contained in the vibrational echo wave packet. The time between the first and second pulses is τ, and the time between the second and third pulses is Tw. The vibrational echo pulse is emitted at time ≤τ after the third pulse. The vibrational echo is detected through a monochromator using an MCT array detector, which measures thirty two frequencies at once (see Conspectus figure). Frequency resolving the combined echo-local oscillator wave packet performs one of the two Fourier transforms necessary to obtain a 2D spectrum. The detection of the many wavelengths in the vibrational echo pulse with the monochromator gives the ωm axis. This axis reads out the final frequencies of all of the species.</p><p>The first two pulses in the sequence label the initial species. As τ is scanned, the echo pulse moves temporally relative to the fixed local oscillator pulse, producing a temporal interferogram at each ωm where there is signal. Numerical Fourier transformation of these temporal interferograms, one at each ωm, gives the ωτ axis. The ωτ axis reports the initial frequencies of the species, and the ωm axis reports the final frequencies of the species after the system has evolved for time, Tw.</p><p>In the Conspectus figure (also see Figure 3A below), the high frequency peak on the diagonal arises from ODs hydrogen bonded to BF4− anions. The lower frequency peak on the diagonal comes from ODs hydrogen bonded to water oxygens. In addition, there are two negative going off-diagonal bands (blue in the figure) that arise from vibrational echo emission at the v = 1 to v = 2 (1-2) transition frequencies of each diagonal peak.28,29 The 1-2 peaks are shifted to lower frequency along the vibrational echo emission (ωm) axis of the 2D spectrum by the anharmonicities of the OD stretch of the two species.</p><p>It is useful to discuss the ideal CES case, in which the anharmonic shifts are so large that when projected onto the ωm axis the off-diagonal 1-2 peaks do not overlap with the 0-1 peaks.30 (Note that this is not the case for the salt solution spectrum shown in the Conspectus figure and Figure 3 below.) In the ideal case, we can consider only the 0-1 peaks. At short Tw, there are two peaks on the diagonal, call them A and B (see Figure 2A). The system is in equilibrium, so A's are constantly turning into B's, and B's are turning into A's. At longer Tw, some A's have turned into B's and some B's into A's (see Figure 2B). For those A's that turn into B's, the initial frequency on both the ωτ and ωm axes is ωA but the final frequency on ωm is ωB, producing an off-diagonal peak at (ωA,ωB). For those B's that turn into A's, a different off-diagonal peak is produced at (ωB,ωA). The time dependence of the growth of these off-diagonal peaks and the decay of the diagonal peaks yields the chemical exchange times.16 Figure 2C shows both the 0-1 and 1-2 portions of spectrum at long Tw. There are equivalent chemical exchange peaks in both portions.16</p><p>The water- BF4− chemical exchange system behaves in the same manner except that there is overlap between chemical exchange peaks and other peaks in the 2D spectrum. The overlapping peaks produce a more complex appearing spectrum as the chemical exchange peaks grow. The overlap of the peaks is taken into account in the data analysis.17</p><p>In the following, hydroxyl-water (hw) signifies hydroxyls hydrogen bonded to oxygen atoms of water molecules. Hydroxyl-anion (ha) signifies hydroxyls hydrogen bonded to BF4− anions. The first two pulses label the ha's and hw's. As time increases, additional peaks grow in due to hydrogen bond rearrangements that cause ha's to change into hw's and vice versa. The exchange dynamics are extracted from the time dependent growth of these off-diagonal peaks when the effects of the vibrational lifetimes and orientational relaxation of the two species are included in the chemical exchange kinetic model analysis.16,17 The vibrational lifetimes and orientational relaxation times were measured with polarization selective IR pump-probe experiments.17</p><p>Figure 3 displays the 2D IR chemical exchange spectrum at 200 fs (A) and Tw = 4 ps (B). The growth of the chemical exchange peaks at 4 ps is clear from the change in the spectrum compared to the Tw = 200 fs spectrum. In B, only the higher frequency region of the 2D spectrum is presented, because this region of the spectrum shows the influence of the growth of the off-diagonal peaks most clearly. Because the lifetime of hw is considerably shorter than ha (see below), the hw peak has decreased in amplitude relative to the ha peak when compared to the 200 fs spectrum. Chemical exchange will cause four additional off-diagonal peaks to grow in, two of which are positive going, arising from the 0-1 transitions, and two that are negative going and come from the 1-2 transitions. In Figure 3 the most evident chemical exchange peak is the 0-1 hw→ha peak, labeled A; this peak does not overlap with any other peak. The spectrum contains two other exchange peaks. The 1-2 hw→ha peak is labeled B. The 1-2 hw→ha peak eats away a strip from the diagonal 0-1 hw band because it is negative, relatively narrow along the ωm axis, and extended along the ωτ axis. This is seen very clearly by comparing the hw peak in Figure 3B to its short time counterpart in Figure 3A. The last exchange peak in Figure 3 is the 0-1 ha→hw peak, labeled C. It is positive going and is manifested as a reduction in the bottom portion of the negative going 1-2 ha peak. The shapes of the diagonal and off-diagonal CES peaks have been explicated theoretically and experimentally.23,30 The important point is that we know exactly where all the peaks are. The overlap of the peaks is handled in the data analysis.17</p><p>A series of 2D spectra over a range of Tw was collected and analyzed. The chemical exchange times can be obtained from the peak volumes as a function of Tw.16,23 The time dependence of the 0-1 and corresponding 1-2 peaks are the same.16,23 Because the system is in equilibrium, the ha→hw and hw→ha chemical exchange peaks grow in with the same time dependence.16,17,23 Therefore, all of the dynamics are reflected in the subset of peaks, the diagonal 0-1 ha peak, the diagonal 0-1 hw peak, and the off-diagonal 0-1 chemical exchange hw→ha peak.17 The volumes associated with these peaks are plotted in Figure 4A.</p><p>To obtain the chemical exchange rate, the data in Figure 4A are fit with a model that includes the vibrational population decay rates and the orientational relaxation rates of hw and ha.16,17,23 Polarization selective pump-probe experiments were used to determine the apparent lifetime. The data are shown in Figure 4B. Using the measured values as an initial guess, the data in Figures 4A and B were simultaneously fit to a system of coupled differential equations.23 In the fitting, the only adjustable parameters are the two vibrational lifetimes and the exchange rate for the process, ha → hw. The simultaneous fits give an exchange rate, vibrational lifetimes, and orientational relaxation times that are internally consistent with the data measured by 2D IR CES and polarization selective pump-probe spectroscopy.</p><p>The fits to the exchange model are shown as the solid curves in Figures 4A and B. The quality of the fits in both panels is excellent. In Figure 4A, the fit reproduces the time dependence of the diagonal and chemical exchange peaks. The exchange time for ha → hw is Taw = 7 ± 1 ps. The exchange time for hw → ha, Twa, is related to Taw by the equilibrium constant.17 Using R = 0.317 gives Twa = 24 ps. This value will be dependent on the concentration of salt because hw → ha can only occur if a water molecule is very close to an anion. Therefore, the exchange dynamics are better characterized by the time constant, Taw = 7 ps, which may be relatively insensitive to salt concentration.</p><p>Figure 4B shows the vibrational population relaxation data (symbols) and the exchange model (solid curves). The process of exchange modifies the vibrational population decays such that the experimentally measured decays do not provide the true lifetimes. Initially, the data are fit with single exponential decays to extract a first guess for the lifetimes. The hw decay is substantially faster than the ha decay. Because the system is in equilibrium, at t = 0, equal population is exchanged between the two species. However, as time proceeds, the hw population decays more than the ha population, and the exchange process will serve to bolster the population of hw, making the apparent lifetime longer than the true lifetime. The converse is true for ha. Note that the number of molecules undergoing exchange is always constant, but the fraction of excited molecules of each species depends on the vibrational lifetime of that species. The shapes of the population decay curves, particularly the ha curve, are substantially influenced by the chemical exchange.</p><p>Simultaneously fitting the 2D IR CES data and the pump probe data yields the true values for the lifetimes of τhw = 2.2 ± 0.1 ps and τha = 9.4 ± 1 ps. More important is that the fits using the chemical exchange model reproduce the functional form and time dependence of all of the curves in Figures 4A and B. Measurements of the orientational anisotropy decay following procedures described previously6 yields values of τrha = 5.0 ps and τrhw = 4.1 ps where τrha and τrhw are the long time orientational correlation times for ha and hw, respectively. Because the orientational time constants are close in value, the measured values are not significantly modified by the chemical exchange.</p><p>The time constants for orientational motion are faster than the time constant for chemical exchange. The ha→hw exchange time is 7 ± 1 ps. The orientational relaxation times and the chemical exchange times are not directly comparable. Nonetheless, it is reasonable to expect that orientational relaxation will be faster than chemical exchange. In pure water, orientational relaxation is modeled as jump reorientation. Here, the jumps can be from ha to hw and hw to ha, as well as ha to ha, and hw to hw. The first two types of jumps produce both chemical exchange and orientational relaxation while the last two produce only orientational relaxation. Thus, there are more pathways contributing to orientational relaxation than to chemical exchange, in accord with the observation of faster reorientation. The orientational relaxation times are less than a factor of two slower than the orientational relaxation time of pure water, τr = 2.6 ps. Thus, the chemical exchange time and the orientational relaxation times show that dynamics of water in this concentrated salt solution are not tremendously slower than in pure water.</p><!><p>The CES method depends on having two resolvable peaks (see Figure 1A) so that the growth of off-diagonal chemical exchange peaks can be observed. The spectra of NaBr solutions (Figure 1B) display a single OD stretching band because the hw and ha peaks overlap to such a great extent that they are not resolved. Therefore, spectral diffusion is used to investigate the water-ion dynamics rather than chemical exchange.</p><p>Spectral diffusion in pure water was briefly mentioned in the Introduction. The OD hydroxyl stretch frequency is very sensitive to the hydrogen bond configuration. Stronger hydrogen bonds and more hydrogen bonds shift the frequency to the red (low energy). Weaker and fewer hydrogen bonds shift the frequency to the blue. As the hydrogen bond structure evolves, the frequency of the OD stretch changes. The time dependent evolution of the frequency is called spectral diffusion. Tracking the time dependence of the frequency provides information on the time dependence of the hydrogen bond structural rearrangement.5,8 Experiments and simulations of spectral diffusion have shown that there are a variety of time scales for hydrogen bond dynamics in pure water.5,8 The very fast dynamics (tens to hundreds of femtoseconds) are associated with very local motions of the hydrogen bonds. The slowest component of the dynamics (1.7 ps) corresponds to the complete randomization of the network structure through concerted hydrogen bond rearrangements.</p><p>Spectral diffusion can be understood using the ideas introduced to discuss chemical exchange. For pure water, rather than two resolved peaks on the diagonal, imagine a continuous overlapping set of peaks along the diagonal. At short time before significant dynamics have occurred, the 2D spectrum will be elongated along the diagonal because of the many overlapping peaks. As the hydrogen bond structure changes, off-diagonal peaks will grow in. However, instead of two peaks at two specific frequencies in the 0-1 region, there will be a continuum of peaks spanning all accessible frequencies. These "off-diagonal" peaks overlap, and their effect is to change the shape of the 2D spectrum. As Tw increases, the spectrum broadens, and goes from being elongated along the diagonal to increasingly symmetrical. The change in shape can be analyzed using a variety of methods to give the frequency-frequency correlation function. Basically, the FFCF describes the time evolution of an initial frequency ω(0) to other frequencies ω(t) at later time t, which is directly related to an initial local hydrogen bonding structure experience by an OD at t = 0 evolving to other structures at time t.</p><p>In the NaBr solutions, in addition to spectral diffusion, there is also chemical exchange, in which OD hydroxyls bound to water oxygens (hw) and OD hydroxyls bound to Br- (ha) switch. The overall dynamics are illustrated schematically in Figure 5. The left well represents hw and the right well represents ha. Based on the experiments on NaBF4 we anticipate that the switching between ha and hw will occur on the many picosecond time scale. On a much shorter time scale, spectral diffusion will cause sampling of frequencies without switching between species. Because the ha and hw peaks overlap strongly, spectral diffusion in each of the wells shown in Figure 5 will sample a large range of frequencies. The diagram has the two types of species separated with distinct regions of spectral diffusion. While this is the case for NaBF4, it is not the physical situation for NaBr. In the NaBr solutions, the ranges of spectral diffusion strongly overlap. However, without chemical exchange interconverting ha's and hw's, full spectral diffusion, that is sampling of all structures and therefore all frequencies, cannot occur.</p><p>To analyze the Tw dependence of the 2D lineshapes, we employ the Center Line Slope (CLS) method, which is an accurate approach for obtaining the FFCF.31,32 Figure 6 shows 2D IR vibrational echo spectra for the 6 M (n = 8) NaBr solution at three Tws, 200 fs, 1 ps, and 9 ps. The dashed line on the top panel is the diagonal. The positive going peaks (red) on the diagonal are from the OD stretch 0-1 transition and the off-diagonal negative going peaks (blue) are from the 1-2 transition. We will focus on the 0-1 transition peaks. At 200 fs, the 0-1 band is elongated along the diagonal. By 1 ps, the band is less elongated, and by 9 ps, the band is virtually symmetrical. At sufficiently long time, the 0-1 band will become round in the absence of the 1-2 band. However, the negative 1-2 band overlaps the bottom of the positive 0-1, flattening it out. The blue lines are the center lines. They are calculated as has been described previously.31,32</p><p>The basic idea of the CLS method is that a line can be constructed from the data at each Tw that has a slope which is directly related to the shape of the spectrum. In the analysis used here, the FFCF is related to the inverse of the slope. At short time, the center line slope approaches 1, so the inverse is 1. At long time, the center line is vertical (infinite slope, see Figure 6 bottom panel), so the inverse is 0. The FFCF can be calculated from the Tw dependence of the inverse of the CLS.31</p><p>Figure 7 displays the inverse of the CLS, C(Tw), for pure water and three NaBr solutions.9,33 It is immediately clear that as the NaBr concentration increases the water dynamics slow. Extensive simulations of pure water5,8 and comparisons to vibrational echo data3,5,8 show that the FFCF decays on multiple time scales. The slowest time scale for pure water, 1.7 ps, arises from the complete randomization of the hydrogen bond network. We find that FFCFs for the NaBr solutions also decay with multiple time scales with the faster components being similar to those found for pure water.9 The slowest component of the FFCF shows the most dramatic change. In analogy to pure water, we assume that this component reflects the final complete randomization of the hydrogen bond network. However, in NaBr solutions, there will be hydrogen bonds between water molecules as well as hydrogen bonding of water to ions. The slowest FFCF component for pure water and the 1.5 M, 3 M, and 6 M NaBr solutions are 1.7 ±0.5 ps, 2.6 ±0.5 ps, 3.5 ±0.5 ps, and 4.8 ±0.6 ps, respectively. For the highest concentration there are only eight water molecules per NaBr (n = 8), which is insufficient to complete even a single solvation shell for the cation and anion. Therefore, all of the molecules will be interacting directly with ions. Even for this sample, the hydrogen bond randomization is only approximately a factor of three slower than occurs in pure water.</p><p>Figure 8 displays decays of the orientational anisotropy, r(t), of the OD hydroxyl for pure water and the three NaBr concentrations.9,34 r(t) is the second Legendre polynomial orientational correlation function divided by 2.5. The data describe the randomization of the direction of the OD bond vector. As with the vibrational echo spectral diffusion, the orientational relaxation slows as the NaBr concentration increases. The slowest component of r(t) for pure water and the 1.5 M, 3 M, and 6 M NaBr solutions are 2.6 ±0.1 ps, 3.9 ±0.3 ps, 5.1 ±0.2 ps, and 6.7 ±0.3 ps, respectively. The complete randomization of the bond vector occurs through jump reorientation rather than Gaussian orientational diffusion.7,35 The orientations of water molecules are restricted because of hydrogen bonds to other water molecules. Concerted hydrogen bond network rearrangement is required to randomize the orientation. The switching of hydrogen bonds between partners causes jumps in the orientation of ∼60°. Therefore, the slowest component of the orientational relaxation is closely related to the slowest component of the spectral diffusion. Both require hydrogen bond network randomization.</p><p>In the n = 8 NaBr solution, the slowest component of the orientational relaxation is almost three times longer than in pure water. In the model we are using, both the slowest component of the spectral diffusion and the complete randomization of the OD orientation require a global reorganization of the hydrogen bond structure. The FFCF and the orientational correlation function (r(t)) cannot be directly compared because they are different correlation functions. It is interesting to compare the effect of NaBr concentration on the slowest components of the FFCF and r(t). This can be done by taking the ratios, Ri, of the time constants of the slowest components for NaBr solutions with respect to the value for pure water. For the FFCF, RFFCF = 1.5, 2.1, and 2.8 going from low to high concentration. For r(t), Ror = 1.5, 2.0, and 2.6. Within experimental error, the two sets of ratios are identical, which supports the picture that the slowest components of both experimental observables are related to the same global hydrogen bond rearrangement.</p><p>Previously, two-color IR pump-probe experiments were performed by pumping the OH stretch of HOD in D2O salt solutions (6 M NaCl, NaBr, or NaI) near the center of the OH stretching band and measuring vibrational population relaxation at different frequencies.15 The results were interpreted as spectral diffusion and were analyzed in terms of a correlation time constants τc . The correlation times were reported to be 20 to 50 times longer than those of pure water. Both the direct chemical exchange measurements on NaBF4 and the vibrational echo spectral diffusion measurements on NaBr discussed here demonstrate that water dynamics are not slowed as much as suggested by the earlier pump-probe experiments.</p><!><p>In both the NaBF4 and NaBr solutions, the hydrogen bond rearrangement dynamics can be divided into four subsets based on the initial and final hydrogen bonding partner of the OD hydroxyl, which is the species under observation in all of the experiments. The four processes are: hw→hw, ha→ha, hw→ha, and ha→hw. In the NaBF4 chemical exchange experiments, we observe only the hw→ha and ha→hw processes, and therefore we obtain the time for switching from a water hydroxyl bound to an anion to a hydroxyl bound to a water oxygen, and vice versa. We found that the time for the ha→hw process is 7 ps. In the NaBr solutions, all four processes contribute to spectral diffusion and orientational relaxation. As the NaBr concentration goes up, the water concentration goes down, and the number of hw→hw processes decreases. For the lower NaBr concentrations, the number of ha→ha processes is probably negligible because the anions are separated. For the highest NaBr concentration, there are 8 water molecules with 16 water oxygen accepting sites per NaBr. Br- will accept ∼6 hydroxyls to solvate it in dilute solution. Therefore, even at the highest concentration, the number water accepting sites substantially out number the Br- accepting sites. So, it is likely that the ha→ha process is not substantial even at this high concentration. If this is the case, then increasing the NaBr concentration increases the importance of the chemical exchange processes ha→hw and hw→ha relative to the other two processes. Based on the results of the NaBF4 experiments, we can reasonably assume that the chemical exchange processes are slower than the hw→hw process, which in bulk water gives a spectral diffusion time of 1.7 ps and an orientational relaxation time of 2.6 ps.</p><p>In light of these ideas, the NaBr concentration dependent data can be viewed as follows. The identical concentration dependence of the ratios RFFCF and Ror show that the spectral diffusion observable and the orientational relaxation observable are measuring different aspects of the same phenomena. In contrast to the direct chemical exchange measurements, both of these observables have contributions from the chemical exchange pathways, ha→hw and hw→ha, and the non-chemical exchange pathways, hw→hw and ha→ha. As the concentration of NaBr increases, the chemical exchange pathways will become increasingly important relative to the non-chemical exchange pathways. The observed slowing of the dynamics with increasing NaBr concentration (see Figures 7 and 8) shows that the chemical exchange pathways are slower than the non-chemical exchange pathways. Even at the highest NaBr concentration (6 M, n = 8), there will still be non-chemical exchange pathways participating in the spectral diffusion and orientational relaxation dynamics. However, at all of the concentrations used in the NaBr experiments, the hw→hw process will not be the same as it is in bulk. The slowest components of the spectral diffusion and the orientational relaxation involve the randomization of the hydrogen bond network, which requires concerted motions of a number of water molecules.7,36 The OD hydroxyl under observation can switch from one water oxygen to another, but the other waters participating in the process may be associated with ions. Therefore, the slowing of the dynamics with increased NaBr concentration is not necessarily a superposition of a decreasing contribution of normal bulk water dynamics and an increasing contribution of slower chemical exchange dynamics. Because of the concerted multi-water nature of the dynamics that randomize the hydrogen bond structure, the hw→hw process is also likely to become slower as the NaBr concentration increases. At the highest concentration, the hw→hw process may not be able to occur without chemical exchange participating as part of the concerted process. Therefore, the dynamics measured at the highest concentration are likely to be dominated by and close to the chemical exchange dynamics.</p><p>The take home message is that the interaction of water with ions slows hydrogen bond network dynamics. However, even at very high salt concentrations, the lengthening of the time scale for a water to switch between being hydrogen bonded to an ion and being hydrogen bonded to another water molecule is slower by a factor of three or four, not orders of magnitude. The results present here indicate that water interacting with other types of charged species, for example, a charged amino acid at the surface of a protein, will result in a longer time to reorganize the local hydrogen bond network than pure water, but the increase in time scale should be significantly less than an order of magnitude.</p>

PubMed Author Manuscript

Selective ring-opening metathesis polymerization (ROMP) of cyclobutenes. Unsymmetrical ladderphane containing polycyclobutene and polynorbornene strands

At 0 °C in THF in the presence of Grubbs first generation catalyst, cyclobutene derivatives undergo ROMP readily, whereas norbornene derivatives remain intact. When the substrate contains both cyclobutene and norbornene moieties, the conditions using THF as the solvent at 0 °C offer a useful protocol for the selective ROMP of cyclobutene to give norbornene-appended polycyclobutene. Unsymmetrical ladderphane having polycyclobutene and polynorbornene as two strands is obtained by further ROMP of the norbornene appended polycyclobutene in the presence of Grubbs first generation catalyst in DCM at ambient temperature. Methanolysis of this unsymmetrical ladderphane gives polycyclobutene methyl ester and insoluble polynorbornene-amide-alcohol. The latter is converted into the corresponding soluble acetate. Both polymers are well characterized by spectroscopic means. No norbornene moiety is found to be incorporated into polycyclobutene strand at all. The double bonds in the polycyclobutene strand are mainly in cis configuration (ca 70%), whereas the E/Z ratio for polynorbornene strand is 8:1.

selective_ring-opening_metathesis_polymerization_(romp)_of_cyclobutenes._unsymmetrical_ladderphane_c

<!>Introduction<!><!>A comparison of the reactivity of cyclobutene versus norbornene derivatives 4 and 5 in ROMP catalyzed by Grubbs I catalyst (6)<!><!>Synthesis of monomer 9<!><!>Synthesis of unsymmetrical ladderphane 8 by sequential ROMPs catalyzed by 6<!><!>Synthesis of unsymmetrical ladderphane 8 by sequential ROMPs catalyzed by 6<!><!>Synthesis of unsymmetrical ladderphane 8 by sequential ROMPs catalyzed by 6<!>Methanolysis of unsymmetrical ladderphane 8<!><!>Methanolysis of unsymmetrical ladderphane 8<!>Conclusion<!>General<!>General procedure for kinetic measurements<!>

<p>This article is part of the thematic issue "Progress in metathesis chemistry III". In memory of the late Professor Teruaki Mukaiyama.</p><!><p>Ring-opening metathesis polymerizations (ROMP) of strained cycloalkenes offer a powerful arsenal for the synthesis of polymers having a variety of fascinating properties [1–3]. To illustrate this, polynorbornenes and polycyclobutenes are readily obtained from the corresponding monomeric norbornene and cyclobutene derivatives under various conditions. Symmetrical DNA-like double stranded ladderphanes are conveniently synthesized from bisnorbornene [4–15] or from biscyclobutene [16] linked with a range of different rigid linkers. When a flexible linker is used, bisnorbornene derivatives undergo cascade metathetical cyclopolymerization giving the corresponding polynorbornenes with hammock-like pendants [17–18]. Unsymmetrical polynorbornene-based ladderphane is obtained by a replication protocol from a single stranded polynorbornene [19–20]. Alternatively, sequential polymerization of a monomer containing a norbornene moiety and other polymerizable group furnishes an unsymmetrical ladderphane having two structurally different polymeric backbones [21–22]. It seems to be not easy if both strands are arisen from different strained rings by ROMP. It is known that norbornenes having different substituents would have different reaction rates in ROMP [23]. These discrepancies in reactivity have been used for sequence control in polymer synthesis [24]. Since the first living ROMP methods for cyclobutenes were reported in 1992 [25], cyclobutene-containing block copolymers are well documented [26–34]. Alternating cyclobutene–cyclohexene copolymers have been synthesized by ROMP of the corresponding monomers [31–33]. However, to the best of our knowledge, selective ROMPs between cyclobutene and norbornene have not been reported.</p><p>The strain energies for norbornene and cyclobutene are 25 and 31 kcal/mol, respectively [35]. It is therefore envisaged that cyclobutene would react faster than norbornene under certain ROMP conditions. As such, when monomer 1 containing a cyclobutene moiety and a norbornene moiety connected by a bridge are subjected to ROMP, it would be feasible that the cyclobutene moiety would react preferentially giving the corresponding norbornene-appended polycyclobutene 2. After all cyclobutene moieties have been consumed and quenched, further ROMP of 2 under different conditions would afford unsymmetrical double-stranded ladderphane 3 having both polycyclobutene and polynorbornene as two polymeric frameworks (Scheme 1). We have tested this viewpoint and now wish to report sequential ROMP of monomers containing both cyclobutene and norbornene moieties tethered by a linker.</p><!><p>Strategy for sequential ROMP of 1 to yield 3.</p><!><p>In the beginning of this study, we have examined the first order reaction kinetics of ROMPs of 4 and of 5 in the presence of 10 mol per cent of Grubbs first generation catalyst (6) [36] in DCM at 10 °C [37]. The rate constants for the reactions of 4 and 5 were 1.3 × 10−3 and 5.1 × 10−4 s−1, respectively. On the other hand, when the reaction was carried out in THF-d8 at 273 K, the second order rate constant for 4 was 2.1 × 10−3 M−1s−1, whereas norbornene derivative 5 was inert under these conditions. The details are described in the Experimental section and Supporting Information File 1 (Figures S1, S2 and S8–S10).</p><p>It has been suggested that the metathesis reaction may involve a fourteen-electron ruthenium species as the active catalyst [38–40]. This active species might be stabilized when the reaction is carried out in polar solvent having weak coordination ability such as THF [41–43]. As mentioned above, the difference in reactivity between the ROMP of 4 and 5 in THF at 0 °C would offer useful conditions to selectively react with 4 in the presence of 5. Thus, a mixture of an equal molar of 4 and 5 was treated with 10 mol % of 6 in THF-d8 at 0 °C. Only 4 was consumed to give the corresponding polymer 7, whereas 5 remained intact (Scheme 2). This promising observation prompted us to pursue the synthesis of unsymmetrical double-stranded ladderphane 8 by sequential ROMPs of 9 (Scheme 3).</p><!><p>ROMP of 4 and 5 in THF at 0 °C in the presence of 10 mol % of 6.</p><p>Retrosynthesis of 8 from 9.</p><!><p>4-Aminobutanol (11) was used to link norbornene and cyclobutene moieties via amide and ester groups. The use of such a linker is because the ester group could be selectively hydrolyzed in the presence of amides. This selectivity will be helpful for the structural elucidation of polymer 8. Thus, 10b was allowed to react with 11 to afford amide-alcohol 12 in 79% yield. Esterification of 12 with 13b furnished 70% yield of monomer 9 (Scheme 4).</p><!><p>Synthesis of monomer 9.</p><!><p>Polymerization of monomer 9 in the presence of 10 mol % of 6 was performed in THF at 0 °C for 4 h, followed by quenching with ethyl vinyl ether to give polymer 14 in 86% yield (Scheme 5). It is worth noting that no incorporation of the norbornene moiety into the polymeric backbone under these conditions was observed. The 1H NMR spectrum of 14 shows the olefinic proton signals at δ 5.49 and 6.12 ppm in 1:1 ratio. These signals were assigned to the absorptions of olefinic protons on the polymeric backbone and the olefinic proton of unreacted norbornene pendants, respectively. In the 13C NMR spectrum, the peak at δ 139 ppm owing to the olefinic carbon of cyclobutene shifts to δ 130 ppm due to ring opening, whereas the olefin carbon of the unreacted norbornene moiety at δ 136 ppm remained unchanged after first polymerization. These observations are consistent with the results of our preliminary studies that only the cyclobutene moiety, but not norbornene in 9, proceeds 6-catalyzed ROMP under these conditions. The degree of polymerization of 14 was estimated to be 10 based on the 1H NMR integration of relevant peaks.</p><!><p>Synthesis of 14 and 8 by selective olefin metathesis.</p><!><p>We have previously found that two norbornene derivatives connected by a flexible linker 15 may undergo cascade ring-opening–ring-closing metathesis polymerization to give single-stranded hammock-like appended polynorbornenes 17 (Scheme 6) [17–18]. The linker in 8 is flexible, and, therefore, the possibility for similar intramolecular metathesis cyclopolymerization might take place to form intermediate 16 for further transformations. However, no such reaction was observed in this study. Presumably, the 6-catalyzed metathesis reactivity of cyclobutenes would be much higher than that of norbornene derivatives. Accordingly, intermolecular metathesis reaction between two cyclobutene moieties would be favored over intramolecular ring-closing metathesis between a ruthenium carbene and the norbornene moiety.</p><!><p>Cyclopolymerization of 15 with a flexible linker.</p><!><p>Polymer 14 was treated with 10 mol % 6 in DCM at rt to give 8 in 95% yield. The 1H NMR spectrum of 8 shows that the relative intensity of the signals around δ 5.4 ppm was doubled, all signals due to olefinic protons in 9 and 14 being diminished.</p><!><p>In order to confirm the uniformity of the polymerization leading to the formation of unsymmetrical ladderphane 8, methanolysis of 8 with NaOMe in methanol at rt gave 7 and 18. Chloroform was then added and 18 was collected as a grayish precipitate in 56% yield. After filtration, the filtrate was worked up to afford 7 in 64% yield with a degree of polymerization of 10 (Mn = 2500, PDI = 1.11), in good agreement with those of 14 and 8. The 13C NMR spectrum of 7 shows two peaks at δ 40.6 and 45.4 ppm, attributed to the allylic carbons attached to a cis and a trans double bond [13], respectively, and the relative ratio of these two peaks is roughly 7:3. This result suggests that about 70% of the double bonds in 7 might adopt cis configuration. Moreover, no norbornene moiety was detected by NMR on the polymeric backbones in 7 (Scheme 7).</p><!><p>Methanolysis of unsymmetrical ladderphane 8.</p><!><p>Since 18 was insoluble in most organic solvents, acetylation of 18 with excess acetic anhydride and pyridine at 70 °C for 10 h gave the corresponding acetate 19, which had good solubility in DCM or chloroform. GPC analysis showed that the degree of polymerization of 19 (DP = 10, PDI = 1.24) was again comparable with that of the corresponding ladderphane 8, polycyclobutene 7 and 14.</p><p>The 1H NMR spectrum of 19 shows peaks at δ 5.6 and 5.3 ppm attributed to trans and cis olefinic protons, respectively, in a ratio of 8 to 1. It is well documented that 6-catalyzed ROMP of N-arylpyrrolidene appended norbornene gives polynorbornene with all double bonds in trans configuration [44–46]. The existence of both Z- and E-double bonds in the parent polycyclobutene backbone in 14 may influence the stereoselectivity of the polynorbornene strand in 7 during the course of ROMP.</p><!><p>In summary, we have demonstrated useful ROMP conditions to selectively transform cyclobutene derivatives into the corresponding polycyclobutenes in THF at 0 °C, whereas the corresponding norbornene skeleton appears to be unreactive under these conditions. This protocol has been used for the selective synthesis of unsymmetrical ladderphane having polycyclobutene in one strand and polynorbornene in the other. Further applications of this selectivity to other systems are in progress in our laboratory.</p><!><p>Unless otherwise specified, all commercially available starting materials were used without further purification. All air and moisture-sensitive reactions were carried out under an atmosphere of dry nitrogen in a glove box. All 1H and 13C NMR spectra were recorded on a Varian 400 Unity Plus NMR spectrometer using CDCl3 as solvent at ambient temperature. Chemical shifts were expressed in parts per million using residual solvent protons as internal standards (1H: chloroform: 7.26 ppm). Gel permeation chromatography (GPC) was performed on a Waters GPC instrument equipped with Waters 1515 HPLC pump using Waters 2487 absorbance detector. Polymer (approximately 0.5 mg) in THF (0.1 mL) was filtered through a 0.5-micron filter and 20 μL of the sample was injected into Shodex KF-G, Styragel HR2, Styragel HR3 and Styragel HR4 column (7.8 × 300 mm) with oven temperature at 40 °C using standard polystyrene samples (1.84 × 105 to 996 Da) for calibration. THF was used as eluent (flow rate 1.0 mL/min).</p><p>Synthesis of 12. Under N2 atomosphere, to 10a (560 mg, 2.2 mmol) in DCM (20 mL) was added oxalyl chloride (0.4 mL, 4.3 mmol) at 0 °C. The mixture was gradually warmed to rt and then stirred for 1 h. The solvent was removed in vacuo to give the crude acyl chloride 10b, to which was added DCM (15 mL), DMAP (60 mg, 0.5 mmol) and Et3N (2.0 mL, 15 mmol). 4-Amino-1-butanol (11, 178 mg, 2.0 mmol) was then added slowly at 0 °C. After stirring for 8 h at rt, the mixture was poured into H2O (50 mL) and DCM (50 mL). The organic layer was separated, washed with brine (100 mL) and dried (MgSO4). The solvent was removed in vacuo and the residue was chromatographed on silica gel (DCM/MeOH 20:1) to afford 12 (515 mg, 79%). mp 207–209 °C; IR (KBr): ν 3455, 3306, 3056, 2940, 2867, 1606, 1554, 1514, 1473, 1379, 1309, 1199, 1130, 1047, 969, 826, 768, 733, 683 cm−1; 1H NMR (400 MHz) δ 1.52 (d, J = 8.4 Hz, 1H), 1.61–1.70 (m, 6H), 2.92–2.99 (m, 4H), 2.98–2.99 (m, 2H), 3.09 (m, 2H), 3.25–3.30 (m, 2H), 3.47–3.48 (m, 2H), 3.71 (t, J = 5.6 Hz, 2H), 6.15–6.16 (m, 3H), 6.39 (d, J = 9.0 Hz, 2H), 7.61 (d, J =9.0 Hz, 2H); 13C NMR (100 MHz): δ 26.8, 30.1, 39.8, 45.6, 46.8, 50.6, 52.2, 62.3, 110.7, 120.1, 127.9, 135.3, 148.9, 167.1; HRMS (FAB, m/z): calcd for C20H26N2O2, 326.1994; found, 326.1997.</p><p>Synthesis of 9. Under N2 atomosphere, to 13a (321 mg, 1.4 mmol) in DCM (20 mL) was added oxalyl chloride (0.4 mL, 4.3 mmol) at 0 °C. The mixture was gradually warmed to rt and then stirred for 1 h. The solvent was removed in vacuo to give the crude acyl chloride 13b, to which was added DCM (15 mL), DMAP (60 mg, 0.5 mmol) and Et3N (2.0 mL, 15 mmol). Compound 12 (522 mg, 1.6 mmol) was then added slowly at 0 °C. After stirring for 8 h at rt, the mixture was poured into H2O (50 mL) and DCM (50 mL). The organic layer was separated, washed with saturated brine (100 mL) and dried (MgSO4). The solvent was removed in vacuo and the residue was chromatographed on silica gel (DCM/MeOH 20:1) to afford 9 (512 mg, 70%). mp 238–240 °C; IR (KBr): ν 3333, 3051, 2949, 2843, 1699, 1606, 1547, 1511, 1473, 1376, 1274, 1216, 1180, 1106, 1050, 963, 828, 769, 740 cm−1; 1H NMR (400 MHz) δ 1.51 (d, J = 8.2 Hz, 1H), 1.61 (d, J = 8.2 Hz, 1H), 1.72–1.85 (m, 4H), 2.92–2.98 (m, 6H), 3.07–3.09 (m, 2H), 3.25–3.29 (m, 2H), 3.49–3.56 (m, 4H), 3.65 (d, J = 10.0 Hz, 2H), 4.30 (t, J = 6.4 Hz, 2H), 6.03 (m, 1H), 6.13–6.15 (m, 4H), 6.38 (d, J = 8.6 Hz, 2H), 6.62 (d, J = 8.4 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 7.87 (d, J = 8.4 Hz, 2H); 13C NMR (100 MHz) δ 26.4, 26.6, 39.4, 45.3, 46.4, 46.5, 48.8, 50.4, 52.0, 63.7, 110.8, 111.8, 117.5, 120.4, 128.0, 130.9, 135.5, 139.1, 149.1, 152.9, 166.6, 167.1; HRMS (FAB, m/z): calcd for C33H37N3O3, 523.2835; found, 523.2839.</p><p>Synthesis of 14. Under N2 atomosphere, to a solution of 9 (84.0 mg, 0.16 mmol) in THF (10 mL) was added 6 (12.8 mg, 0.016 mmol) in THF (1 mL) at 0 °C. After stirring at 0 °C for 4 h, ethyl vinyl ether (1.0 mL) was then added and stirring was continued at 0 °C for 2 h. The mixture was concentrated and the residual solution was added to methanol. The precipitate was collected and redissolved in DCM. Reprecipitation by adding the DCM solution to methanol afforded 14 as a grayish powder (74.8 mg, 89%). IR (KBr): ν 3350, 3054, 2954, 2847, 1695, 1605, 1512, 1476, 1381, 1275, 1179, 1107, 967, 827, 768, 733, 698 cm−1; 1H NMR (400 MHz) δ 1.51–1.72 (m, 6H), 2.92–3.48 (m, 16H), 4.26 (br, 2H), 5.49 (m, 2H), 6.12 (br, 2H), 6.36 (m, 5H), 7.63 (br, 2H), 7.86 (br, 2H); degree of polymerization (DP) analysis: δ 7.86/δ 5.07 = 10, indicating a DP of 10; 13C NMR (100 MHz) δ 26.6, 39.6, 40.9, 45.5, 46.6, 50.5, 52.1, 52.9, 64.0, 110.5, 110.9, 117.0, 120.5, 128.2, 129.8, 131.3, 135.6, 149.2, 150.2, 166.8, 167.4.</p><p>Synthesis of 8. Under N2 atomosphere, to a solution of 14 (62.8 mg, 0.12 mmol) in DCM (40 mL) was added 6 (9.6 mg, 0.012 mmol) in DCM (5 mL). After stirring at rt for 4 h, ethyl vinyl ether (0.5 mL) was then added and stirring was continued for 30 min. The mixture was concentrated and the residual solution was added to methanol. The precipitate was collected and redissolved in DCM. Reprecipitation by adding the DCM solution to methanol afforded 8 as a grayish powder (59.7 mg, 95%). IR (KBr): ν 3373, 3054, 2929, 2849, 1694, 1605, 1512, 1478, 1381, 1274, 1179, 1106, 966, 827, 767, 733, 697 cm−1; 1H NMR (400 MHz) δ 1.47 (br, 1H), 1.82 (m, 5H), 2.88–3.49 (m, 16H), 4.27 (br, 2H), 5.47 (m, 4H), 6.49 (m, 5H), 7.67–7.89 (m, 4H); DP analysis: δ 4.27/δ 5.05 = 11, indicating a DP of 11. 13C NMR (100MHz) δ 26.6, 40.0, 46.1, 49.7, 53.2, 63.7, 110.6, 111.8, 116.9, 121.8, 126.0, 128.5, 131.3, 136.5, 138.7, 150.1, 166.7, 167.5.</p><p>Synthesis of 7 and 18. To a solution of 8 (52 mg, 0.1 mmol [calculated based on the molecular weight of the monomeric unit]) in DCM (20 mL) was added 30% NaOMe in methanol (6 mL). The mixture was stirred at 50 °C for 20 h and cooled to rt. The insoluble solid residue was collected and dried to give crude 18 as a grayish solid (18 mg, 56%). After filtration, the filtrate was washed with water and dried (MgSO4). The mixture was concentrated and the residual solution was added to methanol. The precipitate was collected and redissolved in DCM. Reprecipitation by adding the DCM solution to methanol afforded 7 as a grayish powder (21 mg, 64%). IR (KBr): ν 3066, 2951, 2862, 1702, 1605, 1524, 1478, 1434, 1383, 1281, 1180, 1108, 970, 828, 769, 698, 507 cm−1; 1H NMR (400 MHz) δ 3.02–3.49 (m, 6H), 3.86 (br, 3H), 5.49 (m, 2H), 6.43 (br, 2H), 7.87 (br, 2H), DP analysis by integration of peaks at δ 6.43/δ 5.06 = 10, indicating a DP of 10. 13C NMR (100 MHz) δ 40.8, 45.8, 51.6, 52.7, 110.5, 117.1, 128.4, 129.7, 131.3, 150.2, 167.2. GPC: Mn = 2500, Mw = 2800 , PDI = 1.11.</p><p>Synthesis of 19. A mixture of crude 18 (16 mg, 0.05 mmol), obtained from the above experiment, in Ac2O (0.5 mL) and pyridine (5 mL) was stirred at 70 °C for 10 h. The solvent was concentrated and the residue was dissolved in CHCl3 (15 mL) and washed first with diluted HCl (pH 3) and then with water. The organic solvent was concentrated and the residual solution was added to methanol. The precipitate was collected and redissolved in CHCl3. Reprecipitation by adding the CHCl3 solution to methanol afforded 19 as a grayish powder (12 mg, 63%). 1H NMR (400 MHz) δ 1.73 (br, 6H) 2.05 (s, 3H), 2.73–3.62 (m, 10H), 4.07 (br, 2H), 5.50 (m, 2H), 6.48 (br, 2H), 7.73 (br, 2H), DP δ 5.50/δ 5.05 = 10, indicating a DP of 10. 13C NMR (100 MHz) δ 21.1, 28.0, 39.7, 45.0, 46.5, 50.8, 64.3, 112.2, 121.9, 128.5, 131.8, 132.0, 150.5, 168.1, 171.6.</p><!><p>Monomer 4 or 5 (0.03 mmol) was dissolved in DCM-d2 or THF-d8 (0.5 mL) and was syringed into an NMR tube inside a glove-box under nitrogen atmosphere. The NMR tube was then covered with a standard tube cap and placed in the NMR spectrometer. The tube was left to equilibrate at the desired temperature and all parameters were adjusted. A solution of 6 (24 mg in 1.0 mL of the same solvent) was prepared under nitrogen atmosphere prior to the reaction. Catalyst 6 (10 mol %) was syringed into the NMR tube which was immediately put in the NMR probe again. The reaction was monitored by the decrease of the peak intensity for H-2 using the peaks for H-1 and H-1' as the internal reference (Supporting Information File 1, Figures S8–S10). The spectra were recorded every ten to twenty minutes interval depending on the reaction (Figures S8–S10). The rate constants were thus obtained (Figures S1 and S2).</p><!><p>1H and 13C NMR spectra of both monomers and polymers, as well as GPC and kinetic investigation results.</p>

PubMed Open Access

Bioenergetic Analysis of Isolated Cerebrocortical Nerve Terminals on a Microgram Scale: Spare Respiratory Capacity and Stochastic Mitochondrial Failure

Presynaptic nerve terminals (synaptosomes) require ATP for neurotransmitter exocytosis and recovery and for ionic homeostasis, and are consequently abundantly furnished with mitochondria. Presynaptic mitochondrial dysfunction is implicated in a variety of neurodegenerative disorders, although there is no precise definition of the term \xe2\x80\x98dysfunction\xe2\x80\x99. In this study we test the hypothesis that partial restriction of electron transport through Complexes I and II in synaptosomes to mimic possible defects associated with Parkinson\'s and Huntington\'s diseases respectively, sensitizes individual terminals to mitochondrial depolarization under conditions of enhanced proton current utilization, even though these stresses are within the respiratory capacity of the synaptosomes when averaged over the entire population. We combine two novel techniques, firstly using a modification of a novel plate-based respiration and glycolysis assay that requires only microgram quantities of synaptosomal protein, and secondly developing an improved method for fluorescent imaging and statistical analysis of single synaptosomes. Conditions are defined for optimal substrate supply to the in situ mitochondria within mouse cerebrocortical synaptosomes, and the energetic demands of ion cycling and action potential firing at the plasma membrane are additionally determined.

bioenergetic_analysis_of_isolated_cerebrocortical_nerve_terminals_on_a_microgram_scale:_spare_respir

<!>Reagents<!>Preparation of synaptosomes<!>Synaptosomal attachment<!>Respiration and medium acidification rates<!>Confocal microscopy<!>Analysis of mitochondrial membrane potentials in single synaptosomes<!>Statistical analysis<!>Coupling efficiency and spare respiratory capacity<!>The contribution of anaerobic glycolysis<!>Pyruvate metabolism<!>Mitochondrial coupling to plasma membrane ion fluxes<!>The bioenergetic demand of repetitive action potential firing<!>Loss of spare respiratory capacity and enhanced susceptibility to stochastic mitochondrial depolarization of synaptosomes with restricted electron transport activity<!>Methodology<!>Spare respiratory capacity<!>Synaptosomal bioenergetics<!>Loss of spare respiratory capacity and enhanced stochastic mitochondrial depolarization following restricted electron transport activity<!>Conclusions

<p>While mitochondrial dysfunction is implicated in a wide range of neurodegenerative disorders, investigations employing isolated brain mitochondria have a number of limitations. Firstly the preparations are inherently heterogeneous, since 'non-synaptic' mitochondria originate from the cell bodies of both neurons and glia. Secondly, as with all isolated mitochondrial incubations, the environment and substrate availability depart considerably from the physiological. In contrast, isolated nerve terminals (synaptosomes) largely preserve the metabolism, mitochondrial function, plasma membrane excitability, receptors, ion channels and machinery for the exocytosis and reuptake of neurotransmitters characteristic of the intact presynaptic terminal in situ (for reviews see Nicholls, 1993, 2003; Sánchez-Prieto et al., 1996) and possess the considerable advantage in contrast to primary neuronal cultures that preparations can be made from experimental animals of any age, thus allowing critical age-related changes to be monitored.</p><p>While many studies of in situ mitochondrial function have been performed with synaptosomes by this and other groups, the amount of material required for monitoring respiration (typically 0.5mg synaptosomal protein) has severely restricted the application of this technique for studies of discrete brain regions of transgenic mice. In this study we report the application of the Seahorse XF24 extra-cellular flux analyzer (Wu et al. 2007) to monitor synaptosomal respiration together with extra-cellular pH as an indication of anaerobic glycolysis. The fifty-fold decrease in the amount of material required compared to conventional respirometry, together with the ability to assay multiple samples in parallel facilitates the study of synaptosomes from specific murine brain regions. In this study we first quantify basic mitochondrial respiratory parameters, including coupling efficiency and 'spare respiratory capacity' in the presence of optimal protonophore concentrations, establishing conditions for optimal substrate supply and then determining the bioenergetic demand of ion cycling at the plasma membrane.</p><p>Recent studies with primary neuronal cultures have emphasized the importance of spare respiratory capacity for maintaining an energetic reserve in the face of oxidative stress (Vesce et al. 2005), partial Complex I inhibition (Yadava and Nicholls 2007) or in a model of 'mild-uncoupling' (Johnson-Cadwell et al. 2007). In this study we extend this to synaptosomes by in vitro restriction of Complex I and II activity to decrease spare respiratory capacity modeling putative aspects of Parkinson's and Huntington's diseases. By developing an improved technique for the functional imaging of single discrete synaptosomes it is possible to monitor stochastic changes in mitochondrial membrane potential (Δψm) between individual synaptosomes in these respiratory-restricted models. A heterogeneous response to increased energy demand, together with a time-dependent decrease in Δψm indicates that sub-populations of synaptosomes are differentially susceptible to bioenergetic failure under these conditions. The suitability of these exquisitely sensitive techniques for examining subtle bioenergetic changes in discrete brain regions of transgenic mouse models of neurodegenerative disorders is discussed. .</p><!><p>Tetramethylrhodamine methyl ester, TMRM, was from Invitrogen (Carlsbad, CA). All other reagents were from Sigma-Aldrich (St. Louis, MO).</p><!><p>Synaptosomes were isolated from CD1 mouse cerebral cortices aged from 17-21days by the method of Dunkley et al. (1986) with slight modifications. Briefly, a cortex (0.1-0.2g/brain) was rapidly removed, rinsed with ice-cold 'Sucrose Medium' (320 mM sucrose, 1 mM EDTA, 0.25 mM dithiothreitol, pH 7.4) to remove excess blood, transferred to a pre-chilled Dounce glass homogenizer containing 3 ml Sucrose Medium and homogenized gently by 8-10 up-and-down strokes. The homogenate was then centrifuged at 1000g for 10 min at 4°C. The supernatant was carefully layered on top of a discontinuous Percoll gradient (3ml layers of 3, 10 and 23% Percoll in Sucrose Medium) in a 15ml centrifuge tube, and centrifuged at 32500g for 10 min at 4°C in a JA-25.50 fixed angle rotor in a Beckman Avanti J-26 XPI centrifuge. Synaptosomes were isolated as the band between 10% and 23% Percoll. It should be noted that resealed glial membranes (gliosomes) have been isolated from a 2-6% Percoll boundary (Stigliani et al. 2006).</p><p>The synaptosomal band was diluted into 'Ionic Medium' (20 mM HEPES, 10 mM D-Glucose, 1.2 mM Na2HPO4, 1 mM MgCl2, 5 mM NaHCO3, 5 mM KCl, 140 mM NaCl, pH. 7.4) or diluted into 'Sucrose Medium'. Both were centrifuged at 15000g for 15 min at 4°C to remove Percoll. The final synaptosome pellets were resuspended in Ionic or Sucrose media respectively in prior to respirometry or confocal imaging.</p><!><p>Seahorse respirometry and confocal imaging each require that synaptosomes be robustly attached to the substrate. The polystyrene Seahorse plates and glass-bottomed 96 well plates were each coated with polyethyleneimine (1:15000 dilution from a 50% solution, Sigma-Aldrich, St. Louis) to optimize attachment. While there are a number of studies in which synaptosomes in ionic media have been allowed to sediment under unit gravity onto coverslips coated with Cell-Tak (BD Biosciences, San Jose, CA) (Nichols and Mollard, 1996; Nayak et al., 2001) or poly-D-lysine (Millán et al., 2002), the relatively high frequency of aggregates rather than discrete single synaptosomes has limited the ability to image multiple discrete terminals in a single field.</p><p>Synaptosomes aggregate when suspended in a medium with a significant ionic strength but remain discrete in a sucrose-based isolation medium with very low ionic strength. Respirometry and confocal imaging have rather different requirements, thus for the former it is important to optimize the yield of attached synaptosomes and to ensure that attachment is sufficiently firm to preclude displacement of the terminals during the rather vigorous mixing that occurs prior to each determination in the XF24 respirometer (Fig. 1b). For confocal imaging, in contrast, yield is of secondary importance, but it is preferable to optimize an even field of attached 'monmeric' synaptosomes where individual terminals can be distinguished. Preliminary experiments determined that an ionic medium gave a high yield optimal for respirometry, while the sucrose-based preparation medium yielded a uniform field of monomeric synaptosomes for imaging (Fig. 1c). The diameters of the images equilibrated with TMRM (Fig. 1d) were consistent with single discrete synaptosomes (Jones and Brearley, 1973).</p><!><p>For monitoring respiration, 'Ionic' synaptosomes (10μg protein/well unless otherwise shown) were aliquoted into 20 wells of a polyethyleneimine-coated XF24 V7 cell culture microplate (Seahorse Bioscience, North Billerica, MA). The plate was centrifuged at 3400g for 1hr at 4°C in an A-4-62 rotor in a Eppendorf 5810R centrifuge. Control experiments determined that this caused an attachment of synaptosomal aggregates that was sufficiently robust to withstand the mixing protocols of the machine. The Ionic medium was replaced with 700μl of 'Incubation Medium' (3.5mM KCl, 120mM NaCl, 1.3mM CaCl2, 0.4mM KH2PO4, 1.2mM Na2SO4, 2mM MgSO4, 15mM D-glucose, 4mg/ml BSA, 37°C). Plates were used immediately or stored on ice for not more than 3hrs. The cell culture microplate was incubated and loaded into the Seahorse XF24 extracellular flux analyzer following the manufacturer's instructions. All experiments were performed at 37°C. The XF24 utilizes a specialized multi-well plate, lowering an array of sensors to enclose a 7μl fluid volume above a layer of attached cells (or in this case synaptosomes) in 24 wells each with a total volume of approximately 700μl. During the 1-3min period when the volume is enclosed oxygen uptake and medium acidification are monitored by fluorescence probes immobilized in matrices attached to the sensor, which is subsequently raised and oscillated to ensure mixing and reoxygenation with the bulk medium prior to the next measurement or addition of reagents as appropriate. Oxygen consumption and acidification rate data points reefer to the mean rates during the measurement cycles, which consisted of a mixing time of 30s and a wait time of 2min followed by a data acquisition period of 3min (13 data points). All reagents were added at appropriate dilutions in 75μl of Incubation Medium.</p><p>The oxygen concentration in the 7μl entrapped volume was sensed by the O2-quenched fluorescence of the probe. The non-linear response of the probe was calibrated with the Stern-Volmer equation. The oxygen consumption rates were determined by using a compartment model based 'deconvolution' algorithm which compensated for oxygen diffusion phenomena occurring around the entrapped volume, and for the response time of the probe (Gerencser et al. unpublished results). Rates obtained with this algorithm were proportional to the amounts of synaptosomes attached to the plate, and were steady during basal respiration (not shown). Most respiration rates are expressed as a percentage of the basal respiration in Incubation Medium (containing 15mM glucose) except where absolute rates are reported. The rate of acidification of the medium was monitored in parallel. Results were normalized relative to the basal acidification rate of synaptosomes in glucose medium.</p><!><p>For single synaptosome confocal imaging 'Sucrose' synaptosomes (3μg protein/well) were aliquoted into a 4×3 subset of wells in a Whatman 96-well glass-bottomed multi-well plate and centrifuged in parallel with the 'Ionic' synaptosomes. Synaptosomes were subsequently loaded with 10nM tetramethylrhodamine methyl ester (TMRM) plus 1μM tetraphenylboron for 40min. Fluorescence was monitored in a Zeiss LSM510 confocal microscope with a Plan-Apochromat 40×/0.95 air objective, at 543nm excitation and >560nm emission using the 'Multi Time Lapse' module of the Zeiss LSM software. Single planes of 512×512 images at 0.088 μm/pixel resolution were recorded at ∼9 min intervals, and each image acquisition was preceded by auto focusing.</p><!><p>Time lapse image sequences were aligned to stabilize any lack of register in sequential images due to inaccuracy of the x,y-stage motors. This was done based on the transmitted light images recorded in parallel with the TMRM signal. The background was subtracted at the first percentile of the image histograms. Then a duplicate of the TMRM fluorescence image series was band pass spatial filtered at ω=0.55-2 μm/cycle (Gerencser and Nicholls 2008) to enhance details corresponding to synaptosome-sized objects and reduce noise. This was followed by binarization at Otsu's optimum threshold value (Otsu, 1979). Individual synaptosomes (sized at least 10 pixels in area and maximally 20 pixels in diameter) were located by segmentation of binarized images, and their positions were tracked throughout the time sequence. Fluorescence intensities corresponding to each segment were determined in the original, background subtracted, TMRM fluorescence images, yielding a time lapse of fluorescence intensities for each individual synaptosome. For synaptosomes that gained or lost TMRM fluorescence signal during the experiment, intensities were measured at the first or last known position. Finally the TMRM signal (F) was normalized to the mean of the two baseline data points (F0) and converted to millivolts relative to the baseline by calculating 26.7 mV × ln((F- F0)/F0+1). Therefore the negative sign denotes depolarization.</p><p>To visualize populations, histograms of changes in Δψm of single synaptosomes were calculated in Mathematica 5.2 (Wolfram Research, Champaign, IL). At t=0 the relative Δψm of each synaptosome in a field wasdefined to be zero (and was therefore omitted from the histogram). At approximately 9min intervals images of the field were taken and the change in potential from t=0 recorded. A range of +20mV (hyperpolarization0 to −50mV (depolarization) was divided into 40 bins and the percentage of synaptosomes in each bin was plotted. Image processing was carried out using custom developed image analysis software written in Pascal language (Delphi 2009; Borland, Austin, TX) and Mathematica 5.2.</p><!><p>Respiration and acidification rates are presented as the mean ±s.e.m. of at least three independent experiments with different synaptosomal preparations. Membrane potential data is shown as the mean±s.e.m. of the parameters calculated from independent synaptosomal preparations (n). Histograms reflect data pooled from all experiments performed. Significance level was determined by performing ANOVA on the complete data set with Tukey's post-hoc testing.</p><!><p>Isolated nerve terminals utilize exogenous glucose and pyruvate as substrates (Kauppinen and Nicholls, 1986b). Fig. 2 and Table 1 show mean results from multiple experiments for synaptosomes respiring in the presence of 15mM glucose, 10mM pyruvate as sole substrate and 15mM glucose supplemented with 10mM pyruvate. Coupling efficiency under these conditions is estimated by the addition of oligomycin, and spare respiratory capacity by the addition of FCCP. It should be emphasized that in all experiments the concentration of FCCP was titrated to just relieve respiratory control. Under these conditions there is predicted to be only a slight decline in Δψ (Johnson-Cadwell et al., 2007).</p><p>In order to assess the extent to which electron transport chain activity controls spare respiratory capacity it is important to consider whether substrate supply to the in situ mitochondria is optimal. Using conventional oxygen electrode assemblies, we have previously determined that pyruvate is an excellent substrate for intact synaptosomes and can further enhance the respiration in the presence of 15mM glucose (Kauppinen and Nicholls, 1986b). Fig. 2b and c show the respiratory parameters for synaptosomes respiring in the presence of 10mM pyruvate and with both pyruvate and 15mM glucose present. It is apparent that exogenous pyruvate and glucose are almost additive in enhancing spare respiratory capacity in the presence of FCCP. In addition to enhancing the basal respiration in the presence or absence of glucose it is notable that pyruvate significantly increases the proton leak conductance estimated from the oligomycin-insensitive respiration (Table 1).</p><p>Since proton leak rate is highly dependent upon Δψm (Nicholls 1974) and pyruvate produces a mean population hyperpolarization of the intra-synaptosomal mitochondria by about 6mV even in the presence of glucose (Kauppinen and Nicholls 1986b), one explanation is that the increased proton leak may be accounted for by this increased driving force. However, an alternative hypothesis is that pyruvate selectively energizes a population of synaptosomes that has impaired glucose utilization and thus possess depolarized mitochondria. In order to monitor Δψm in individual synaptosomes, fields of 'monomeric' synaptosomes (see Methods) were equilibrated with TMRM+ under non-quench conditions (Ward et al., 2000) and changes in Δψm were monitored with confocal microscopy (Fig. 3). It should be noted that strictly the fluorescence is a function of both Δψm and plasma membrane potential (Ward et al., 2000).</p><p>Single synaptosomes were tracked and the TMRM+ fluorescence intensity was monitored as a function of time (Fig. 3). The deviation of TMRM+ fluorescence intensity from the baseline of each individual synaptosome was expressed as mV change. Therefore at 0 min 100% of synaptosomes were at '0 mV' in the histograms. At later time points the population of synaptosomes was represented by the bell-shaped distributions of membrane potential change. The superimposed color-coded histograms show a time-dependent widening of the distribution (increase in the variance), emphasizing the stochastic deviation of Δψm from the value at 0 min in individual terminals.</p><p>When synaptosomes utilized glucose alone (Fig. 3A) the mean Δψm slowly decayed, by 5±1.2 mV in 38 min (Fig.3A). In contrast, this decay was insignificant (0.45±0.49 mV) in glucose medium supplemented with 10mM pyruvate (Fig.3B). When pyruvate was added to synaptosomes kept in glucose medium, mitochondria hyperpolarized by 3.8±1.3 mV by 38 min (Fig.3C) and respiration increased by 22.9±0.6% (n=4) in a parallel respirometer experiment, data not shown. The changes in potential were significantly different from each other at p<0.05 by one way ANOVA and pairwise comparison by Tukey post-hoc test. At 38min after pyruvate addition 2.6% of synaptosomes had hyperpolarized Δψm by more than 20mV, in contrast to only 0.3% of the synaptosomes in glucose medium and 0.5% in glucose plus pyruvate medium over the same period.</p><p>As shown by the images taken before (Fig. 3D) and after (Fig. 3E) pyruvate very few 'new' synaptosomes appeared in the TMRM+ image, but rather there was a brightening in general. Thus the effect of pyruvate at a single synaptosome level is to cause a significant hyperpolarization. This, combined with the slow decline in Δψm in glucose alone suggest that glycolysis is not fully stable in synaptosomes incubated at 37°C, perhaps due to a slow leakage of glycolytic intermediates. Incubating synaptosomes in glucose-only medium, or addition of pyruvate, significantly widened the histogram of the Δψm, increasing the standard deviation to 12.5±1.0 mV and 10.6±0.7 mV, respectively (both at p<0.01 significance at 38min), as compared to synaptosomes maintained throughout in glucose plus pyruvate medium (6.7±0.4 mV). This suggests that a population of synaptosomes showed a stochastic failure of glycolysis, and that this population could be rescued by the addition of pyruvate, the glycolytically less competent terminals producing stronger responses to pyruvate. For these reasons we have included 10mM pyruvate in the incubation to eliminate this factor in most of the subsequent experiments.</p><!><p>Synaptosomes display a robust Pasteur effect, enhancing glycolysis in response to an inhibition of mitochondrial ATP synthesis (Kauppinen and Nicholls, 1986b). The Seahorse respirometer can detect the resultant lactate release as an extracellular acidification. Fig. 2D shows that the rate of acidification in glucose medium increases on addition of oligomycin, consistent with an activation of anaerobic glycolysis as compensation for the inhibited mitochondrial ATP synthesis. Acidification rates in the presence of glucose plus pyruvate were similar, and in both cases the further addition of FCCP resulted in a further increase in rate. In glucose-free pyruvate medium as expected no increase is seen on addition of oligomycin, but FCCP produces an increased acidification. Since the FCCP addition is in each case associated with a large enhancement of respiration this suggests that CO2 evolution from the TCA cycle can contribute to the medium acidification detected in the medium. The slow decline in acidification rate, as well as in respiration, seen after FCCP in the presence of oligomycin is a consistent feature in these experiments and may be a consequence of the acidification of the the cytoplasm and matrix occurring under these conditions.</p><!><p>α-Cyanocinnamate is an inhibitor of the mitochondrial inner membrane pyruvate transporter (Hildyard et al., 2005). The dominant role of pyruvate as a substrate for the in situ mitochondria is shown by the extensive concentration dependent inhibition of the spare respiratory capacity (Fig. 4A). It is notable that no significant effect of the inhibitor is seen on basal or oligomycin inhibited respiration, indicating that the respiratory limitation following addition of the transport inhibitor is only apparent under conditions of maximal pyruvate utilization, i.e. pyruvate transport and pyruvate dehydrogenase have little control over the basal respiration rate. Since the activity of pyruvate dehydrogenase is inhibited by pyruvate dehydrogenase kinase it is of interest to establish whether the dehydrogenase is maximally activated under these conditions. Dichloroacetate inhibits the kinase, and hence maximizes pyruvate dehydrogenase activity (Fuller and Randle, 1984). Titration of synaptosomes with up to 1 mM dichloracetate failed to affect basal, oligomycin inhibited or FCCP stimulated respiration, and no effect on lactate extrusion (as monitored by the extra-cellular acidification rate) was detected (data not shown). This supports earlier indications that pyruvate dehydrogenase has little control over respiration in synaptosomal preparations (Kauppinen and Nicholls 1986a). Mitochondrial oxidation of glycolytic pyruvate is dependent upon the oxidation of cytoplasmic NADH. The importance of the malate/aspartate shuttle in this context (Kauppinen et al., 1987) is demonstrated by the inhibition of spare respiratory capacity in the presence of the transaminase inhibitor aminoxyacetic acid (Fig. 4B).</p><!><p>The presynaptic Na+/K+-ATPase is a major utilizer of ATP under conditions when sodium reentry is enhanced, for example following addition of veratridine to prevent inactivation of voltage activated sodium channels (Nicholls and Scott, 1980). The effect of veratridine on the respiration of the cortical synaptosomes in glucose/pyruvate medium is shown in Fig. 5A, B. An extensive enhancement of respiration is seen, accompanied by an increase in extracellular acidification (Fig 5C, D). The stimulation of respiration is oligomycin sensitive (Fig. 5B) consistent with the enhanced ATP turnover, however a slight increase in oligomycin-insensitive respiration is seen, possibly due to an increase in the rate of Ca2+ cycling across the inner membrane driven by the proton circuit as a consequence of activation of mitochondrial Na+/Ca2+ exchanger by the increased cytoplasmic Na+ (Nicholls and Scott 1980).</p><p>In glucose plus pyruvate medium the veratridine-stimulated respiration was accompanied by a reciprocal decline in FCCP-stimulated respiration both in the presence or absence of oligomycin (Fig. 5A, B). Veratridine also caused a parallel decrease in the extra-cellular acidification rate (Fig. 5C, D), indicative of a limitation in glycolysis. Consistent with this, veratridine addition in glucose-only medium led to an almost complete loss of FCCP-stimulated respiration (data not shown).</p><p>On purely bioenergetic grounds addition of the Na+/K+-ATPase inhibitor ouabain would be predicted to cause a decreased respiration by inhibiting the slow ATP hydrolysis by the pump under basal conditions. In practice, however a slight stimulation of respiration is seen on addition of the inhibitor (Fig. 6A) which was sensitive to oligomycin and thus due to increased mitochondrial ATP-turnover. Since ouabain induces plasma membrane depolarization and allows cytoplasmic Na+ to rise, it is possible that the combination of the ATP requirement for exocytosis and enhanced Na+ and Ca2+ cycling at the mitochondrial inner membrane more than compensates for the decreased ATP requirement by the Na+/K+-ATPase. In glucose plus pyruvate medium following oligomycin, the presence of 0.3mM ouabain decreased FCCP-stimulated respiration from 860±146% to 592±38% of the glucose-only basal respiration. More dramatically, ouabain almost completely abolished the spare respiratory capacity in glucose-only medium (data not shown). Thus ouabain, similarly to veratridine, inhibited glycolysis. Therefore another possible explanation is that ouabain increased respiration as compensation for the decreased contribution of glycolysis to ATP turnover.</p><!><p>Veratridine is a use-dependent inhibitor of sodium channel deactivation, and its effectiveness in this and other synaptosomal preparations indicates that the terminals undergo slow spontaneous action potential firing in low K+ medium. The potassium channel inhibitor 4-aminopyridine (4AP) greatly enhances the frequency of these spontaneous action potentials, and has been extensively employed in studies with isolated synaptosomes to induce trains of repetitive action potentials to study the mechanism and regulation of transmitter exocytosis (Tibbs et al., 1989a). 4AP should increase synaptosomal ATP demand as the terminal restores ionic homeostasis following action-potential initiated calcium and sodium entry, in addition to the ATP requirements of exocytosis and endocytosis. Concentrations of 50μM and 1mM 4AP respectively induce half-maximal and maximal glutamate exocytosis from cortical synaptosomes (Tibbs et al. 1989) and these concentrations were employed here, producing a graded respiratory response (Fig. 6B). In contrast to veratridine and ouabain, 4AP did not significantly reduce FCCP stimulated respiration (903±66% control, 819±41% in presence of 1mM 4AP).</p><!><p>Brain mitochondria prepared from rodent models of many major neurodegenerative disorders show impaired electron transport activity (reviewed in (Lin and Beal 2006). Since brain mitochondrial preparations are an inherently heterogeneous mixture originating from processes, terminals, cell bodies and glia, it is difficult to make conclusions relating the in vitro mitochondrial dysfunction to functional deficits or pathology. As a preliminary to investigating these and additional disease models, synaptosomes were titrated with low concentrations of rotenone or 3-nitropropionic acid to partially inhibit Complexes I and II respectively (Fig. 7). It is notable that spare respiratory capacity is titrated down by the inhibitors with no effect on basal or oligomycin-insensitive respiration. In other words the control that electron transport exerts over respiration is expected to be lowest in the presence of oligomycin, when proton re-entry has greatest control, Conversely, electron transport will have greatest control under conditions of maximal electron flux when proton re-entry is not restricted (Brand, 2005).</p><p>The ability to image single synaptosomes allows us to test one aspect of the 'spare respiratory capacity' concept, namely that statistical deviations in individual terminals that result in a bioenergetic deficit can result in stochastic mitochondrial depolarization within the population, and that this is exacerbated by factors that restrict maximal electron transport activity. In order to determine the viability of the synaptosomes in the face of a relatively modest increase in proton current demand, single synaptosomes were tracked and the changes of TMRM+ fluorescence intensity were recorded under control conditions or during application of rotenone or 3NPA at concentrations that partially restricted spare respiratory capacity (10 nM and 200 μM respectively, Fig. 7).</p><p>The mean Δψm was only slightly decreased compared to the untreated control by rotenone (by 7.1±1.2 mV) or 3NPA (by 6.5±1.8 mV) at 34 min after application of the inhibitors (n=10 synaptosomal preparations; p<0.05). It should be noted that the synaptosomes retain considerable spare respiratory capacity in the presence of these inhibitor concentrations (Fig. 7). However, partial electron transport inhibition results in a broadening of the spread in mitochondrial membrane potentials in the individual terminals (Fig. 8 A, C, E) signified by the greater standard deviation of Δψm (10.5±0.4 mV for rotenone and 10.5±0.8 mV for 3NPA, compared with 7.4±0.3 mV for the control at t=34 min; p<0.001; n=10.</p><p>In order to simulate an increase in energy demand, synaptosomes were treated with a very low concentration of FCCP (150 nM in the presence of 64μM BSA), which alone caused only an insignificant decrease in mean Δψm (2.7±1.5 mV at t=34 min; p>0.05). When 10nM rotenone was combined with this FCCP concentration, Δψm gradually decreased (by 13.2±1.8 mV at t=34 min, as compared to the untreated control; p<0.001; n=4), while a subset of synaptosomes was depolarized by more than 30 mV, sufficient to abolish mitochondrial ATP production. At 34min, 5.9±0.6% of the rotenone plus FCCP population passed this threshold vs. 1.1±0.2% for the control plus FCCP (p<0.05). The progressive nature of the depolarization meant that by 79min 37.9±9.3% vs. 4.5±0.3% respectively of the populations had depolarized by more than 30mV. Qualitatively similar results were obtained for 3NPA-inhibited synaptosomes (11.7±2.6 mV depolarization at t=34 min, p<0.01; n=4) for the combination of 3NPA and FCCP.</p><!><p>The synaptosome preparation has been available for almost 50 years (reviewed in Whittaker, 1993) and was subsequently refined with techniques to improve purity (Dunkley et al. 2008). Included among the more than 10,000 references exploiting the preparation are studies monitoring mitochondrial and plasma membrane potentials and respiration (e.g. Nicholls and Scott, 1980), presynaptic substrate metabolism (Erecinska et al. 1996) and oxidative stress (Tretter and Adam-Vizi 2007), glutamate release (e.g. Nicholls and Sihra, 1986) and the molecular mechanisms of vesicular trafficking (Cousin and Robinson 2000). The ability of 4AP to induce spontaneous 'action potentials' (Tibbs et al., 1989a) allowed presynaptic receptor regulation of exocytosis to be studied in great detail (Sánchez-Prieto et al., 1996). However, while synaptosomes can be prepared from animals of any age, the amount of material required to monitor respiration (typically 0.5mg protein) has limited the study of the presynaptic bioenergetics of specific brain regions of transgenic mice unless large numbers of animals are sacrificed. The present technique reduces the amount of material required to monitor respiration by a factor of 50.</p><!><p>An important aspect of aging-related changes in the brain is that loss of function is gradual, suggestive of a stochastic synaptic failure. Studies with cultured neurons exposed to energy-intensive excitotoxic stress have supported a hypothesis that neuronal cell death in this context is primarily induced by an 'energy crisis' (Nicholls et al., 2007). Thus reduction of spare respiratory capacity or maximal ATP generation by oxidative damage to in situ mitochondria (Vesce et al., 2005), Complex I restriction (Yadava and Nicholls, 2007), 'mild-uncoupling' (in an attempt to reduce oxidative stress, Johnson-Cadwell et al., 2007) or oxygen-glucose deprivation (Vesce and Nicholls, unpublished) each potentiate glutamate-induced cell death concomitant with an exhaustion of spare respiratory capacity in the neuronal cultures. The present combination of population respiration studies of spare respiratory capacity with single synaptosomal fluorescence monitoring of membrane potential allows the stochastic model of presynaptic bioenergetic failure to be tested. In particular we propose that the simple initial experiment quantifying respiration under basal conditions, in the presence of oligomycin and following carefully titrated protonophore be adopted as an initial quantitative means of assessing the rather vague concept of mitochondrial dysfunction. Basal respiration in the presence of substrate drives both proton leak and ATP synthesis. Addition of oligomycin eliminates proton re-entry via the ATP synthase and the decrease in respiration provides a minimum estimate of the proton current used to drive ATP synthesis prior to addition of the inhibitor. However, since the proton leak is voltage-dependent (Nicholls, 1974; Nobes et al., 1990) the mitochondrial hyperpolarization that accompanies ATP-synthase inhibition will enhance the proton leak and hence lead to some under-estimation of the ATP-synthesizing proton current and proportionate over-estimation of the proton leak under normal sub-maximal 'State 3½' ATP-generating conditions (i.e. intermediate between State 3 and State 4).</p><p>Damage to, and inhibition of, electron transport chain complexes is central to many neurodegenerative disorders (Beal, 2005). Neuronal mitochondria are subjected to a variable ATP demand, depending primarily on their excitation pattern, and it is critical that the electron transport chain has sufficient capacity to supply protons to the ATP synthase. We have coined the term 'spare respiratory capacity'to quantify the difference between this maximal uncontrolled respiration and the initial basal respiration (Nicholls et al., 2007) and have proposed that the maintenance of some spare respiratory capacity even under conditions of maximal physiological or pathophysiological stimulus is a major factor defining the survival of the neuron.</p><!><p>The present study has allowed us to reproduce and extend earlier findings with rat or guinea-pig cerebrocortical synaptosomes obtained with conventional oxygen electrode respirometry. The ability of exogenous pyruvate to synergize with glucose was shown earlier (Kauppinen and Nicholls, 1986a). In the present study this is shown to be due to a variable hyperpolarization (depending on the glycolytic competence of a given synaptosome) throughout the synaptosomal population. The implication is that glycolysis slowly deteriorates in synaptosomes incubated at 37°C, as shown by the progressive decrease in Δψm and that this metabolic limitation can be rescued by inclusion of exogenous pyruvate. Pyruvate does not however prevent the slow decline in maximal respiration following FCCP (see e.g. Fig. 2) suggesting that mitochondrial respiration does not continue indefinitely in a terminal whose sources of ATP (failing glycolysis and uncoupled mitochondria) are severely compromised. In an earlier study (Kauppinen and Nicholls, 1986c)a progressive failure of glycolysis and ATP depletion were reported following cyanide inhibition of respiration.</p><p>The inability of dichloracetate to further activate pyruvate dehydrognase in mouse synaptosomes, the role of the malate-aspartate shuttle in reoxidizing cytoplasmic NADH during glycolysis, and the increased respiration consequent upon Na+ channel activation by veratridine are consistent with earlier studies (Nicholls and Scott, 1980, Kauppinen and Nicholls, 1986a, Kauppinen et al., 1987). While we introduced 4AP as a means of inducing spontaneous action potentials in synaptosomes (Tibbs et al., 1989a,b) the energetic demand of exo-endocytosis and the re-establishment of ionic homeostasis has not previously been documented. It is significant that, relative to 'quiescent' synaptosomes in 3.5mM KCl medium, firing induced by 1mM 4AP at least doubles synaptosomal ATP turnover (Fig. 6).</p><p>Both in the presence and absence of oligomycin, veratridine decreases the maximal respiration achievable with FCCP (Fig. 5). Veratridine acts by preventing Na+ channel desensitization and thus increases Na+ cycling and ATP utilization by the Na+ pump, as is reflected in the increased oligomycin-sensitive respiration. At the same time it elevates cytoplasmic Na+ causing extensive efflux of glutamate and aspartate from the cytoplasm by reversal of the excitatory amino acid carrier (Seal and Amara 1999). In view of the importance of the malate/aspartate shuttle, evidenced from the inhibitory effect of AOAA (Fig. 4B), it is possible that cytoplasmic aspartate depletion may limit the activity of the shuttle and hence glycolysis. However application of exogenous aspartate did not rescue the spare respiratory capacity (data not shown).</p><p>Ouabain reduced spare respiratory capacity similarly to veratridine in glucose plus pyruvate medium. In both cases this effect was amplified in glucose-only medium, suggesting an inhibition of glycolysis. Indeed a time-dependent inhibition of glycolysis has been reported in rat brain synaptosomes in the presence of veratridine and ascribed to an inhibition of hexokinase as a result of ATP depletion (Erecinska et al. 1996). However, in contrast to veratridine, ouabain preserves synaptosomal ATP (Nicholls and Scott, 1980), therefore it is unlikely that ATP depletion fully accounts for the respiratory decline. A common feature of both veratridine and ouabain is that both trigger a sustained increase of [Na+]i paralleled by a further decrease in [K+]I, i.e. a collapse of the plasma membrane Na+-electrochemical potential. The isolated nerve terminal is deprived of physiological neurotrophic factors and there are suggestions that this may underlie a time-dependent decline in glucose and glutamate transport that can be prevented by neurotrophic factors (Guo and Mattson 2000). Finally, synaptosomes have a low internal K+ concentration as a result of the isolation procedure (Nicholls & Scott 1980). Since K+ is a necessary co-factor for pyruvate kinase (Kachmar & Boyer 1953) inhibition of this enzyme as a consequence of the loss of cytosolic K+ is an alternative explanation for the inhibition of the glycolysis.</p><!><p>There is convincing evidence associating a partial inhibition of Complex I with Parkinson's Disease (reviewed in (Beal 2005). The association of Complex II deficiency with Huntington's is more tenuous (reviewed in (Browne and Beal 2004;Orr et al. 2008). In the case of PD, degeneration of the nigro-striatal dopaminergic neurons may occur in a retrograde manner (Betarbet et al. 2006), emphasizing the relevance of investigating presynaptic striatal bioenergetics. There is a major conceptual problem in relating acute experiments to the slow progression of the human neurodegenerative disorders. We have proposed that the failure of a synapse may occur stochastically when that terminal faces an 'energy crisis', i.e. when the instantaneous ATP demand in that terminal exceeds the maximal ATP supply by the combination of oxidative phosphorylation and glycolysis (Nicholls et al. 2007). Such an event may be rare, a bioenergetic 'hundred year flood' but as mitochondrial capacity declines with age (Lenaz et al. 2006) the probability of stochastic failure will increase, contributing to the age-related component in the incidence of the neurodegenerative disorders. Spare respiratory capacity is thus a critical factor in maintaining a reserve of ATP generating capacity.</p><p>Preparations of isolated brain mitochondria are inherently heterogeneous reflecting the multiple cell types and sub-cellular localizations. In particular the expression of cyclophilin D and the associated susceptibility to the permeability transition is variable. At the single mitochondrion level isolated brain mitochondria undergo stochastic oscillations in Δψ that appear to be associated with the permeability transition since they are decreased by exogenous ADP (Vergun et al., 2003,Vergun and Reynolds 2004). Hazelton et al. (2008) have reported that cyclophilin D is preferentially expressed in GABAergic interneurons, with lower expression in synaptic rather than non-synaptic mitochondria, contrasting with an opposite conclusion on sub-cellular sensitivity from Naga et al. (2007). Conversely, mitochondria prepared from glutamatergic cerebellar granule neurons were insensitive to cyclosporine A in contrast to cortical astrocytes (Bambrick et al. 2006). Nayak et al. (1999) have reported a heterogeneous distribution of serotonin receptors on individual synaptosomes from different brain regions. Finally, sensitivity to excitotoxicity differs widely in different neuronal cultures and although much of this can be ascribed to differential NMDA receptor expression, the role of mitochondria remains unclear.</p><!><p>We have described sensitive techniques for monitoring and quantifying bioenergetic function in isolated nerve terminals acutely from a specific mouse brain region. Since synaptosomes may be prepared from animals of any age and are used acutely, they represent an ex vivo preparation that can faithfully represent the metabolism of the intact terminal in the animal before sacrifice. The approach is thus invaluable for investigating aging-related aspects of presynaptic function in a wide variety of transgenic and disease models.</p>

PubMed Author Manuscript

Efficient electrochemical synthesis of a manganese-based metal–organic framework for H₂ and CO₂ uptake

In this study Mn-DABDC (DABDC = diaminobenzenedicarboxylate, or 2,5-diaminoterephthalate) MOF was synthesised both via an electrochemical method, to make Mn-DABDC(ES), and via a conventional solvothermal approach, to make Mn-DABDC(ST). A Mn-BDC (BDC = benzenedicarboxylate) MOF was also prepared by a conventional solvothermal method for gas uptake capacity comparison. Investigation of the electrochemical synthesis parameters demonstrated that current density, electrolyte amount and reaction time were the most significant factors affecting crystal synthesis and product yield. The best conditions found for obtaining a crystalline MOF with high yield (93%) were 70 mA current, electrolyte 2.7 mmol/30 ml DMF and 2 h of reaction time. These optimized electrochemical conditions allow for a relatively fast MOF synthesis, important for reducing synthesis cost compared with conventional hydrothermal and solvothermal methods. The Mn-DABDC(ES) MOF sample was fully characterized to analyse its structure, thermal stability and surface area. The electrochemically synthesized MOF has high carbon dioxide uptake (92.4 wt% at 15 bar and 273 K) and hydrogen uptake (12.3 wt% at 80 bar and 77 K). This is the first amine-based manganese MOF synthesized electrochemically, and the method has excellent potential for reducing large-scale MOF production costs.

efficient_electrochemical_synthesis_of_a_manganese-based_metal–organic_framework_for_h₂_a

Introduction<!>Materials<!>Equipment and characterization<!>Results and discussion<!>Conclusions

<p>Coordination chemistry has evolved as a most promising route to porous materials with precisely decorated interiors to obtain specific properties for versatile applications. 1 One of the most highly studied applications of metal-organic frameworks (MOFs) is the capture of CO 2 to address global energy and environmental issues. 2,3 Metal-organic frameworks have metal coordination sites bridged by organic ligands in highly ordered networks that often afford well defined structures, high crystallinity and large surface areas that can be used in catalysis, or gas separation applications. 4,5 Over the last two decades developments in MOF synthesis have enabled MOFs to become promising candidates for carbon dioxide capture, however there is often poor carbon dioxide gas selectivity from flue gas streams. 2,6 A notable development is in the functionalization of MOF structures, including selective functional group insertion within any given framework to serve specific end functions and impart desirable properties to the MOF materials. 2,7,8 Amine sites in particular show great affinity towards carbon dioxide and are known to be highly effective for CO 2 adsorption while also being amenable to use under dry or humid conditions. 7 We have recently reported the modification of a copper-based MOF during synthesis by doping with hexamethylenetetramine, resulting in the enhancement of carbon dioxide sorption over the unmodified framework. 9 In another study we reported that amine post synthetic modification on a Mn-DOBDC framework (DOBDC = 2,5-dihydroxyterepthalate) enhances water stability and carbon dioxide uptake of the MOF. 10 A major challenge in preparing MOFs for CO 2 capture applications is still the energy intensive, tedious and laborious conventional solvothermal process for MOF synthesis. Electrochemical synthesis of MOFs was first reported by BASF in 2005, using anodic dissolution to synthesise the copperbased framework HKUST-1. 11,12 Most subsequent examples using this method have focussed on Cu and Zn frameworks, although examples exploiting Al and Fe have been reported. [13][14][15][16][17] Recent reports include Pirzadeh and co-workers, who electrochemically synthesized a Cu 3 (BTC) 2 metal-organic framework for CO 2 and CH 4 separation, 18 and, in a hybrid approach, Mitra et al. grew Cu-based MOFs onto modified thin-film electrodes to study their electrochemical properties. 19 The electrochemical MOF synthesis process has advantages over conventional MOF syntheses including the potential for shorter reaction times and lower energy consumption with a relatively simple equipment setup. 18 Perhaps the most attractive feature of electrochemical synthesis is the mild reaction conditions, since these reactions can be performed at ambient pressure and temperature. Despite these advantages, it is still an under-exploited approach, especially in the synthesis of functionalised framework materials. 20 This study demonstrates the synthesis of a new amine-functionalised Mn-DABDC MOF using electrochemical synthesis to cut synthesis costs, important for future scale-up. The prepared material was fully characterized to analyse its structure, thermal stability and surface area. For comparison, a Mn-BDC MOF that lacks amine functionalisation was also synthesized using a traditional solvothermal method to compare CO 2 and H 2 adsorption of these MOFs.</p><!><p>All the chemicals were purchased from Merck Sigma Aldrich and used as received.</p><p>Synthesis of {Mn 2 (BDC) 2 (DMF) 2 } ∞ (Mn-BDC) Mn-BDC MOF was prepared using a conventional solvothermal method reported by Huiping et al. in 2016, with slight modifications. 21 Equimolar quantities (1 : 1) of Mn (NO 3 ) 2 •6H 2 O (287 mg, 1 mmol) and terephthalic acid (160 mg, 1 mmol) were dissolved in 10 ml DMF in a 50 ml beaker. The contents were ultra-sonicated at 45 °C for 2 hours then the solution was transferred to 23 ml Teflon vials. These were each sealed in a Parr autoclave and heated in an oven at 110 °C for 24 hours to yield white crystalline material. Crystals obtained were washed thrice with DMF then thrice with THF (5 ml for each wash). The resulting crystals were dried overnight at room temperature to get 83% yield (371 mg). The sample was activated in vacuum oven at 130 °C for 12 hours before further analysis.</p><p>Electrochemical synthesis of {Mn 3 (DABDC) 3 (DMF) 4 } ∞ (Mn-DABDC(ES))</p><p>For electrochemical synthesis of Mn-DABDC, 2,5-diaminoterephthalic acid (588 mg, 3 mmol) was dissolved in 30 ml DMF. In another beaker, NaNO 3 (225 mg, 2.7 mmol) was mixed with 10 ml distilled water to serve as a conductive electrolyte for the reaction. These mixtures were combined and ultrasonicated for 1 hour at room temperature to ensure complete mixing of contents. Mn strips were prepared for reaction (10 cm long × 2 cm wide × 0.4 cm thick). Polishing was done with sandpaper (400 grit) to remove any oxide layer and washing with distilled water followed by ethanol.</p><p>The electrochemical synthesis reaction was performed by dipping these Mn strips (3 cm depth) in the reaction mixture keeping them 2 cm apart. A direct current (DC) supply was then attached to the electrodes and the current adjusted to 70 mA. As the reaction proceeded, light brown crystals were observed in the solution. The reaction was performed at ambient temperature and pressure (i.e. 20-22 °C and 1 atm). After 2 h, the product was collected, filtered and washed with DMF three times and then three times with THF (5 ml for each wash). The product obtained was dried at 60 °C in the oven for 4 hours to obtain 93% yield (1.29 g). The sample was activated in a vacuum oven at 130 °C for 12 hours before further analysis. This electrochemically synthesised material is named Mn-DABDC(ES) throughout the manuscript. Note: a series of reactions were performed to optimize time of reaction, current density and electrolyte concentration to obtain the best Mn-DABDC(ES) MOF yield; details of this series are in the ESI and Fig. S1. † Solvothermal synthesis of {Mn 3 (DABDC) 3 (DMF) 4 } ∞ (Mn-DABDC(ST))</p><p>Samples of the Mn-DABDC framework were also prepared using the conventional solvothermal method to compare synthetic outcomes with the product obtained by electrochemical synthesis. Equimolar quantities (1 : 1) of Mn (NO 3 ) 2 •6H 2 O (287 mg, 1 mmol) and 2,5-diaminoterephthalic acid (196 mg, 1 mmol) were dissolved in 10 ml DMF. After ultra-sonication at 45 °C for 2 hours the solution was transferred to 23 ml Teflon vials and sealed in a Parr autoclave and heated in an oven at 120 °C for 22 hours to yield light brown crystals. Crystals obtained were washed thrice with DMF then thrice with THF (5 ml for each wash). The resulting crystals were dried overnight at room temperature to obtain 78% yield (377 mg). The sample was activated in vacuum oven at 130 °C for 12 hours before further analysis. This solvothermally synthesised material is named Mn-DABDC(ST) throughout the manuscript.</p><!><p>Electrochemical syntheses were performed under constant current or voltage using a RIGOL DC Power supply and RIGOL millimeter DM3058E. Single crystal X-ray diffraction data for Mn-DABDC and Mn-BDC MOFs were collected on an Agilent SuperNova Dual Atlas diffractometer with Mo and Cu sources and a CCD detector. Data reduction and integration was performed using CrysAlisPro. Powder X-ray diffraction (PXRD) patterns were collected on an X'PertPro Panalytical Chiller 59 diffractometer using copper Kα (1.5406 Å) radiation. A 2θ range from 5 to 40 degrees was used to record the diffraction pattern. A SHIMADZU IR Affinitt-1S spectrometer was used to obtain IR spectra. Thermogravimetic analyses (TGA) were performed using a PerkinElmer Pyris 1 TGA equipment. The temperature was increased from 25 °C to 700 °C at a heating rate of 5 °C min −1 under a flow of air (20 ml min −1 analyses were performed using a FlashSmart NC ORG elemental analyser. CO 2 adsorption experiments were performed on a Quantachrome Isorb-HP100 volumetric type sorption analyser. Samples were degassed at 130 °C under vacuum for 12 hours and then backfilled with helium gas prior to gas sorption studies. CO 2 sorption studies were performed at two selected temperatures, 273 K and 298 K, over a pressure range of 0.5-15 bar. H 2 adsorption studies were performed at 273 K and 77 K, over a pressure range of 0.5-80 bar. N 2 adsorption studies of prepared samples were conducted to analyse surface area and pore volume using a Quantachrome Nova 2200e at 77 K at a relative pressure of P/P 0 = 0.05-1.0.</p><!><p>There are several known structures containing Mn(II) nodes and the BDC linker, the earliest being MOF-73, [21][22][23] but these were made using, and consist of, different metal : linker ratios and solvents to our material; herein we have formed a new Mn-BDC framework. Briefly, our Mn-BDC framework crystallises in a monoclinic geometry with a = 13.4484( 4 FTIR spectra of the prepared materials confirmed the presence of representative functional groups indicative of Mn-BDC and Mn-DABDC MOF formation (Fig. S2 †). Sharp peaks representative of symmetric and asymmetric stretching of carboxylates bonded to Mn are observed at 1535 cm −1 and 1367 cm −1 in the Mn-DABDC sample. 3 Both samples contain a broad band at around 3250 cm −1 , which can be attributed to O-H stretching vibrations of adsorbed atmospheric water. 24,25 In addition to the C-H stretches in both samples around The PXRD patterns of the as-synthesized Mn-BDC, Mn-DABDC(ES), Mn-DABDC(ST), and those simulated from single crystal XRD are shown in Fig. 3 and 4. PXRD patterns indicate in all cases the formation of highly crystalline material. PXRD patterns for Mn-DABDC produced from solvothermal synthesis and electrochemical synthesis indicate the same framework is synthesized with both methods, and in almost all solid products produced during the electrochemical parameter optimisation the Mn-DABDC MOF phase was formed with no apparent secondary phases (see ESI and Fig. S2 †). Exceptions to this were the presence of a peak at 25°2θ indicating unreacted crystalline DABDC linker remaining when current density was too low for efficient conversion to product (Fig. S2 pattern S1 †), and the presence of small additional peaks, most notably around 11-12°and 25-27°2θ, in the sample with the lowest quantity of electrolyte (Fig. S2 pattern S4 †). There is also good agreement between the simulated and as-synthesized (optimised synthesis) PXRD patterns, indicating that the single crystals studied are representative of the bulk samples, which in the optimised syntheses exhibit good phase purity and absence of manganese dioxide. 26 The optimised product yield of Mn-DABDC obtained by electrochemical synthesis for 2 hours at room temperature was 93%, compared with only 78% obtained from the 22 hours, 120 °C solvo-thermal method. This improvement is possibly as a result of electrochemical delivery of metal ions from the manganese electrode at a rate determined by the electrolysis, combined with ready provision of nitrate counterions from the excess present as part of the electrolyte. Indeed, the nitrate ions can be recycled during the synthesis rather than having to be supplied stoichiometrically as part of the Mn(NO 3 ) 2 salt used in the solvothermal synthesis. These differences evidently have a marked impact on the reaction kinetics and hence may affect the resulting crystal size and defect content. The SEM images of Mn-BDC and Mn-DABDC are therefore presented in Fig. S5. † SEM results show a range of particle morphologies including flat hexagonal rods stacked on each other for Mn-DABDC(ES), a mixture of hexagonal rods and flake structures for Mn-DABDC(ST), and loose laminar rod-like structures for Mn-BDC. The surface roughness of Mn-DABDC(ST) visually appears greater than that of Mn-DABDC(ES). The electrochemically-synthesised crystallites are quite clearly larger than those formed in both solvothermal syntheses, with the largest Mn-DABDC(ES) rod diameters reaching ∼8 μm in contrast to 2-3 μm for Mn-DABDC(ST) and only 1-2 μm for Mn-BDC. Both changes in morphology and size of the electrochemically synthesised framework are consistent with a different crystal growth mechanism, a feature of interest for future study beyond the scope of this present work. Thermogravimetric analysis (TGA) was performed on Mn-BDC and Mn-DABDC (Fig. S4 †). Some weight loss was observed below 100 °C for both MOFs indicating there was little surface adsorbed moisture. [27][28][29] There is a weight loss step between approx. 125-245 °C for both MOFs which we ascribe to the loss of coordinated DMF from the MOF structures. 6,30 There is prominent two-step DABDC linker degradation in the Mn-DABDC sample as the temperature increases above approximately 325 °C. No further weight losses were observed for Mn-DABDC above 560 °C, indicating residual metal oxide, while Mn-BDC MOF decomposed completely to the oxide at 425 °C, a notably lower temperature than Mn-DABDC.</p><p>CO 2 and H 2 adsorption capacities of Mn-BDC, Mn-DABDC (ES) and Mn-DABDC(ST)</p><p>The CO 2 adsorption capacity for both MOF materials was evaluated by monitoring pseudo equilibrium adsorption uptakes. Samples were first degassed at 130 °C for 12 hours. 200 mg of each sample was used for three consecutive adsorption-desorption cycles at 273 K and 298 K with adsorbate pressure ranging between 0.1 to 15 bar. The CO 2 capacities calculated at 273 K and 15 bar pressure were 11.5 mmol g −1 and 21 mmol g −1 for Mn-BDC and Mn-DABDC(ES), respectively. This trend also occurs for adsorption capacities recorded at 298 K (Fig. 5). Hydrogen uptake of Mn-DABDC(ES) MOF was 12.3 wt% at 80 bar pressure and 77 K (Fig. 6), and a pore size of 3.53 Å was calculated from gas sorption data. Moderate Q st values were calculated for both gases in Mn-DABDC(ES) (Fig. S6 †) which 7).</p><p>A comparison can also be made against the solvothermallysynthesised Mn-DABDC(ST) material. The CO 2 uptake at 55 bar of Mn-DABDC(ES) is slightly higher at both temperatures than that of Mn-DABDC(ST) (an increase of 6.3% at 273 K and 4.5% at 298 K) and markedly higher than that of Mn-BDC (an increase of 130% at 273 K and 136% at 298 K). The H 2 uptake of Mn-DABDC(ES) is similarly slightly higher than that of Mn-DABDC(ST) (an increase of 5% at 77 K and 21% at 273 K) and again markedly higher than that of Mn-BDC (an increase of 61% at 77 K and 113% at 273 K).</p><p>To put this work in a broader context, Table 1 provides a comparison of amine-based metal-organic frameworks for uptake of carbon dioxide and hydrogen. In previous studies, we reported amine-modification of Cu-BDC, a copper-based MOF, by doping the synthesis with hexamethylenetetramine (HMTA). 9 Despite a reduction in BET surface area from 708 to 590 m 2 g −1 , this modification afforded a 3-and 4-fold increase in 273 K CO 2 uptake over the unmodified Cu-BDC framework, at 1 and 14 bar respectively. We have also reported a post-synthetic modification approach, attaching ethylenediamine (EDA) to Mn-DODC, a manganese-based framework. 10 In that study, modification only reduced the BET surface area a small amount, from 1256 to 1203 m 2 g −1 , but again increased the 273 K CO 2 uptake, albeit by a smaller multiplier (see Table 1). However, it is notable in the present study that the incorporation of two primary amine groups per linker in Mn-DABDC (ES) results not only in the largest BET surface area of all three studies, 1453 m 2 g −1 , but in the highest overall CO 2 uptake of our amine-containing frameworks. At 273 K the CO 2 uptake of Mn-DABDC(ES) at 1 bar is 40.9 wt% and at 15 bar it is 92.4 wt%. These values surpass those of many related smallpore frameworks reported in the literature; some examples are given in Table 1. Given that the Q st values at zero loading for EDA-MnDOBDC and Mn-DABDC(ES) are, perhaps unsurprisingly, essentially the same (32 kJ mol −1 ) and most likely result from CO 2 binding to the primary amines in both cases, the improved performance of Mn-DABDC(ES) at higher pressure may be attributable in part to the greater surface area, and in part to the greater density of amine sites in the framework.</p><!><p>Mn-DABDC(ES) was successfully produced in good yield using electrochemical synthesis. Synthesis conditions were optimized to get a maximum product yield of 93%. Here, manganese metal cations were produced in situ using Mn electrodes, eliminating the need for the MOF-precursor metal salt as required in conventional solvothermal and hydrothermal MOF production approaches, since the counter-ions are transiently provided by the electrolyte solution and hence can be continuously recycled in the synthesis. SEM results revealed well-formed flat hexagonal rod-like crystals for Mn-DABDC(EC), larger than the rods produced for Mn-DABDC(ST) and Mn-BDC. The three MOF materials were tested for carbon dioxide and hydrogen gas uptake. Mn-DABDC(ES) demonstrated high carbon dioxide (92.4 wt% at 15 bar pressure and 273 K) and hydrogen uptake (12.3 wt% at 80 bar pressure and 77 K), a little higher than the respective CO 2 and H 2 uptake of the solvothermally synthesised Mn-DABDC(ST) material, with both outperforming the related Mn-BDC framework. These results are ascribed to the incorporation of basic amine groups into the organic ligand within the framework significantly enhancing electrostatic interactions between the framework and the guests, increasing gas sorption. The electrochemical synthesis has the following specific advantages over the traditional solvothermal synthesis: (i) the use of ambient temperature instead of 120 °C, (ii) the use of ambient pressure instead of high-pressure autoclaves, (iii) the use of mild reaction conditions that recycle the nitrate counterions, and (iv) a vastly reduced reaction time compared to the conventional solvothermal synthesis method. These advantages demonstrate a method of design and synthesis of new materials with high carbon dioxide and hydrogen uptake with the potential for cost-effective large-scale production in the future.</p><p>CCDC 1948926 † contains the supplementary crystallographic data for the Mn-DABDC MOF structure and CCDC 2027762 † contains the supplementary crystallographic data for the Mn-BDC MOF structure.</p>

Royal Society of Chemistry (RSC)

Precise Molecular Design of a Pair of New Regioisomerized Fluorophores With Opposite Fluorescent Properties

Aggregation-induced emission (AIE) has attracted much attention in the past 2 decades. To develop novel AIE-active materials, ACQ-to-AIE transformation via regioisomerization is one of the most straightforward method. However, most of the reported ACQ-to-AIE transformations are achieved by migrating bulky units. In this work, a facile conversion was realized by migrating a small pyrrolidinyl group from para- to ortho-position on the rofecoxib scaffold. As a result, a pair of new isomers named MOX2 and MOX4 exhibited AIE behavior and ACQ activity, respectively. Moreover, MOX2 also showed solvatochromic, mechanochromic, and acidochromic properties with reversible multi-stimulus behavior. Single crystal X-ray analysis of MOX2 revealed that the molecular conformation and its packing mode were responsible for the AIE emission behavior. Further investigation indicated that MOX2 showed high lipid droplets staining selectivity. Taken together, the current work not only provides a new design philosophy for achieving ACQ-to-AIE conversion by migrating a small pyrrolidinyl group but also presents a promising candidate MOX2 for potential applications such as in security ink, optical recording and biological applications.

precise_molecular_design_of_a_pair_of_new_regioisomerized_fluorophores_with_opposite_fluorescent_pro

Introduction<!><!>Materials and Instrumentation<!>Synthesis and Characterization<!>Absorption and PL Intensity in Solution<!><!>Solvatochromic Properties<!><!>ACQ and AIE Properties of MOX2 and MOX4<!><!>Crystal Analysis of MOX2<!><!>Mechanochromic Properties of MOX2<!><!>Mechanochromic Properties of MOX2<!>Sensing Properties Toward Protonic Acids<!><!>Lipid Droplets Imaging in Living Cells<!><!>Conclusion<!>Data Availability Statement<!>Author Contributions<!>Funding<!>Conflict of Interest<!>Publisher’s Note<!>Supplementary Material<!>

<p>Aggregation Induced Emission (AIE) materials have been successfully developed in different research fields since it was discovered by Tang's group back in 2001 (Luo et al., 2001; Hong et al., 2009; Hong et al., 2011; Mei et al., 2015; Arathi et al., 2016; Mallick et al., 2017; Lou and Yang, 2020). To date, a variety of AIE luminogens (AIEgens) have been reported (Lim et al., 2016; Choi et al., 2020; Li J. et al., 2020) for their inherent characteristics of high brightness in solid states by suppressing extensive π-π stackings in their packing mode (Yuan et al., 2014; Mei et al., 2015), which would otherwise result in aggregation-caused quenching (ACQ) of the fluorophores (Hong et al., 2009; Yuan et al., 2010). These AIEgens varied greatly in terms of their chemical structures, including tetraphenylethylene (TPE) (Feng H. T. et al., 2018; La et al., 2018; Wu et al., 2018), triphenylamine (TPA) (Zhang et al., 2013; Cao et al., 2014; Zhang Y. et al., 2014), cyano-substituted diarylethene (Shen et al., 2013; Zhang X. et al., 2014; Lu et al., 2015), 2,3,4,5-tetraphenylsiloles (Chen et al., 2014), silole (Chang et al., 2015), distyrylanthrancene (Niu et al., 2015; Xiong et al., 2015), and anthracene derivatives (Lu et al., 2010; Zhang et al., 2011; Li A. et al., 2019), conjugated polymers (Ravindran and Somanathan, 2017; Wang et al., 2018), and so on. Modification of the existing AIE scaffolds is the most popular strategy for constructing new AIEgens (Mei et al., 2015). In addition to this strategy, transforming ACQ molecules into AIE-active materials also provides a direct way to design highly bright AIEgens due to the rich source of ACQ (Yuan et al., 2010; Zong et al., 2016; Liu et al., 2017; Lu et al., 2019; Li Y. et al., 2020; Pratihar et al., 2020; Wang et al., 2020; Huang et al., 2021; Long et al., 2021; Zhang et al., 2021).</p><p>Traditionally, ACQ-to-AIE transformation can be achieved by integrating bulky substituents into ACQ molecules to avoid strong π-π stackings or by introducing twisted AIEgens (Dhokale et al., 2015; Gomez-Duran et al., 2015; Feng X. et al., 2018) and propeller-shaped molecules into ACQ fluorophores (Ma et al., 2016; Wang et al., 2020). However, integration of twisted AIEgens prolongs the π system thus it would probably change the already satisfactory fluorescent properties of the original ACQ molecules. Therefore, it will be highly desirable to achieve ACQ-to-AIE conversion without extending its conjugated system too much. Some progresses have been made along this line for the past decade (Sasaki et al., 2017; Li Y. et al., 2020). Among them, ACQ-to-AIE conversion was realized by Tang's group recently through a regioisomerization strategy via shifting a molecular rotor in the end position of a planar core of dithieno [2,3-a:3′,2′-c] benzo [i] phenazine (TBP) to the bay position (Li Y. et al., 2020). The same strategy was utilized by Xu's group to achieve ACQ-to-AIE conversion through changing the substituted position of a benzanthrone moiety from the meta-position to the ortho-position on the perylenetetracarboxylic diimide core (Long et al., 2021). Similarly, Prasad's group also realized ACQ-to-AIE transformation by changing a cyano group from the para-position to the meta-position on the same phenyl group (Pratihar et al., 2020). Despite the great examples above, the successful cases of the regioisomerization of small units are still rare (Pratihar et al., 2020). Therefore, it is still necessary to develop more examples of ACQ-to-AIE conversion to guide the design of novel AIEgen materials.</p><p>Herein, we report our recent work on the realization of ACQ-to-AIE transformation by migrating a small pyrrolidine group from para-to ortho-position based on the rofecoxib scaffold. The resulting compounds MOX4 with para-substitution showed ACQ effect while compound MOX2 wih ortho-substition exhibited AIE acitivity (Figure 1). Moreover, MOX2 also showed multi-stimulus fluorescent responsive properties, such as mechanochromic, acidochromic properties. In addition, its potential applications have been presented. This work is expected to provide a new guidance on designing AIE-type materials.</p><!><p>A pair of new regioisomerized fluorophores with opposite fluorescent properties.</p><!><p>All commercial reagents and solvents were used as received. Compounds p-tolylacetic acid and 2-bromo-4′-methanesulfonyl acetophenone were purchased from Shanghai Macklin Biochemical Co., Ltd. and Shanghai Bidepharm Co., Ltd., respectively. Various benzaldehydes with different substituted groups were purchased from Shanghai Shaoyuan Co., Ltd. Reactions were magnetically stirred and monitored on a TLC Silica gel 60G F254 plate from Millipore Sigma (United States). All other reagents and solvents were purchased from Sigma-Aldrich (United States) and used without further purification, unless otherwise stated. Photoluminescence (PL) spectra were recorded on a Varioskan LUX 3020-80110. Differential scanning calorimetry (DSC) were obtained on a DSC STAR system at a heating rate of 15°C/min from 40°C to 500°C under a high purity nitrogen atmosphere. Powder X-ray diffraction (PXRD) patterns were recorded on a Rigaku (D/MaX-3B) diffractometer. Single-crystal X-ray diffraction was conducted with Bruker D8 Quesr/Venture diffraction with λ = 0.77Å (MoKα). Lipid droplets (LDs) imaging was performed with a Nikon Ti-E&C2 scanning unit. 1H NMR and 13C NMR spectra were measured on a Bruker AV 600 spectrometer in appropriated deuterated chloroform solution at room temperature with the solvent residual proton signal as a standard. High resolution mass spectra (HRMS) were recorded on a GCT premier CAB048 mass spectrometer operating in MALDI-TOF mode.</p><!><p>The synthetic route of compounds is outlined in Scheme 1. The key intermediate MOX was synthesized in a one-step reaction involving cyclization of esters via 2-bromo-4′-methanesulfonylacetophenone and p-methyl phenylacetic acid in the presence of triethylamine and DBU. Finally, the target compounds MOX2 and MOX4 were prepared via Knoevenagel condensation reaction. (General procedure). Piperidine, 3 drops, was added to a mixture of 0.140 g (0.0004 mol) of intermediate (MOX) and 0.140 g of 2-(1-Pyrrolidinyl) bezaldehyde in 10 ml of methanol, and the mixture was stirred at room temperature for 12 h in dark atmosphere. The mixture was then cooled, and the precipitate was filtered off and washed with methanol on a filter. The yellow powder (MOX2, 0.1001 g) was obtained with the yield 72%. The DSC thermogram of powder MOX2 showed only one endothermic peak that corresponds to the melting point (Tm = 203.6°C) (Supplementary Figure S12). 1H NMR (600 MHz, DMSO-d 6 ) δ 8.09 (d, J = 8.3 Hz, 2H), 7.89 (d, J = 8.8 Hz, 1H), 7.72 (d, J = 8.3 Hz, 2H), 7.26 (d, J = 23.8 Hz, 3H), 7.17 (d, J = 8.1 Hz, 2H), 7.05–6.89 (m, 2H), 6.04 (s, 4H), 3.06 (s, 3H), 2.28 (s, 1H), 1.76 (s, 4H). 13C NMR (151 MHz, DMSO-d 6 ) δ 168.3, 150.3, 148.8, 146.6, 142.1, 139.2, 136.1, 131.5, 130.6, 130.5, 129.5, 129.3, 128.0, 126.5, 124.8, 123.2, 120.6, 116.8, 111.9, 52.6, 43.7, 24.9, 21.4.HR-MS (ESI): calced for C29H27NO4S: 486.1734 [(M + H)+]), found:486.1813. The red powder (MOX4, 0.1230 g) was synthesized following the general procedure with the yield 88%. The DSC thermogram of powder MOX4 showed only one endothermic peak that corresponds to the melting point (Tm = 231.3°C) (Supplementary Figure S12). 1H NMR (600 MHz, DMSO-d 6 ) δ 8.05 (d, J = 8.1 Hz, 2H), 7.67 (dd, J = 8.2, 3.9 Hz, 4H), 7.20 (d, J = 8.0 Hz, 2H), 7.13 (d, J = 8.0 Hz, 2H), 6.61 (d, J = 8.7 Hz, 1H), 5.94 (s, 1H), 3.33 (s, 3H), 2.27 (s, 4H), 1.97 (s, 4H). 13C NMR (151 MHz, DMSO-d 6 ) δ 168.4, 148.7, 148.6, 144.2, 141.9, 138.5, 136.3, 133.0, 130.8, 129.5, 129.2, 128.0, 127.0, 122.4, 120.5, 115.2, 112.5, 47.7, 43.8, 25.4, 21.3. HR-MS (ESI): calced for C29H27NO4S:486.1734 [(M + H)+], found:486.1771. The final products were characterized by 1H NMR, 13C NMR, high-resolution mass spectrometry and single-crystal X-ray diffraction. The relevant data are collected from the original spectra and listed in the Support Information (Supplementary Figures S1–S6).</p><!><p>As displayed in Figure 2A, MOX2 had two absorption bands at 350 and 430 nm, respectively. The high energy band was attributed to the π-π transition while the low energy band was probably caused by the internal charge transfer (ICT) (Pati et al., 2013; Pati et al., 2015). In contrast, MOX4 has only one maximum absorption band at 484 nm. In terms of emission, MOX2 had a maximum PL intensity at 674 nm, slightly red-shifted compared to 652 nm of MOX4 (Figure 2B). Another attractive feature of MOX2 was its large Stokes shifts (13,675 cm−1) in dimethyl sulfoxide (DMSO) as displayed in Supplementary Table S1, which may be beneficial for overcoming the self-quenching of traditional fluorophores (Li P. et al., 2019). We carried out an experiment of 50 μM concentration, this intensity was consistent with the Beer-Lambert's plot (Supplementary Figure S7). To provide insight into regioisomerization of the pyrrolidine group in compounds MOX2 and MOX4 on their photophysical properties, density functional theory (DFT) calculations at the B3LYP/6-311++G (2 d,p) level were carried out (Lee et al., 1988). The ICT was evident in comparison between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO). The energy band gap was determined to be 3.05 and 2.75 eV for MOX2 and MOX4, respectively, which was consistent with our experimental absorption data (Figure 2C).</p><!><p>Normalized absorption (A) and PL (B) spectra of MOX2 and MOX4 dyes in DMSO (50 μM). (C) The optimized structure of MOX4, MOX2, and their HOMO and LUMO at the B3LYP/6-31++G (2 d, p) level.</p><!><p>The UV-vis absorption and PL spectra of MOX2 and MOX4 were measured in different solvents, such as DMSO, dimethylformamide (DMF), chloroform, dichloromethane (DCM), ethanol (EtOH), tetrahydrofuran (THF). The photophysical data were summarized in Supplementary Table S1. As the solvent varied from chloroform to DMSO, the λ abs of MOX2, and MOX4 displayed minor changes, indicating that their ground states are hardly affected by the solvent polarity (Supplementary Figure S8). In contrast, the PL spectra of MOX2 and MOX4 exhibited different maximum wavelengths in different solvents (Figure 3). With the solvent polarity changed from chloroform to DMSO, the λ em of MOX2, and MOX4 are red-shifted of 40 and 58 nm, respectively. Furthermore, the PL intensity of MOX2 was gradually weakened with increased polarity of the solvent, indicating that the PL intensity of MOX2 was highly dependent on the polarity of solvents.</p><!><p>PL spectra of compounds MOX2 (A) and MOX4 (B) in various solvents (50 µM), such as DMSO, DMF, chloroform, DCM, EtOH, THF, respectively. Inset: photograph of MOX2 (A) and MOX4 (B) in various solvents with UV irradiation (λ = 365 nm).</p><!><p>To demonstrate the feasibility of the present strategy, the photophysical properties of the two isomers were performed to monitor the PL intensity fluctuation of the molecules in DMSO/water mixtures with various water volume fractions (f w) as shown in Figure 4. The absorption spectra as a function of the water fraction of MOX2 and MOX4 in DMSO as displayed in Supplementary Figure S9. As shown in Figure 4C, molecule MOX4 displayed strong emission in DMSO due to the presence of a large π-conjugated planar structure. As f w was increased from 0 to 100 vol%, the PL intensity was decreased gradually, and the quantum yield (QY) of MOX4 decreased significantly from DMSO (3.96%) to water (0.30%) (Table 1). This result demonstrated that compound MOX4 has typical ACQ effect. On the contrary, MOX2 exhibited week emission in DMSO (Figure 4B). In the water fraction range (f w = 0–60 vol%), the PL intensity was still weak. However, when f w reached 80 vol%, the PL intensity was increased significantly, which was ascribed to the aggregation of the compound (Figure 4D) (Liu et al., 2016; Sun et al., 2018; Gao et al., 2019). Moreover, MOX2 gave a low QY in pure DMSO (0.13%) but a high QY in pure water (2.62%) (Table 1). Therefore, MOX2 was AIE-active. The distinct difference in solid-state QY suggested a significant effect of the pyrrolidinyl position as regioisomers on the fluorescent properties.</p><!><p>(A) The photos in different solvent states of MOX2 and MOX4 at 365 nm (10 µM). (B) The MOX2 PL spectra as a function of the water fraction in DMSO. (C) The MOX4 PL spectra as a function of the water fraction in DMSO. (D) Variation in PL intensity (I/I 0 ) of MOX2 with f w (%), where I0 is the luminescence intensity in pure H2O. (E) Variation in PL intensity (I/I 0 ) of MOX4 with f w (%), where I0 is the luminescence intensity in pure DMSO.</p><p>Photoluminescence QY of MOX4 and MOX2.</p><!><p>To better understand the origin of the AIE property, the single crystal of MOX2 was obtained (CCDC 2107729) and its molecular packing was analyzed as follows. The crystal data and structure refinement for MOX2 as displayed in Supplementary Table S2. As shown in Figure 5A, the dihedral angles between A-B, A-C, and B-C planes are 80.76°, 29.94°, and 65.82°, respectively. In addition, MOX2 adopted a loose packing mode with weaker intermolecular C–H∙∙∙O (2.484 Å, 2.696 Å, 2.633 Å, 2.659 Å) interactions as shown in Figure 5B; Supplementary Figure S10. The existence of these intermolecular interactions helped rigidify the molecular conformation, thus restricting the intramolecular rotation of the phenyl group. Moreover, the non-radiative relaxation process could be largely prohibited and significant emission enhancement was observed in its solid form, yielding the remarkable AIE phenomenon. It is worth noting that such relatively weak intermolecular interactions coculd be easily destroyed upon exposure to external mechanical force, giving rise to the mechanochromic properties as studied below. The single crystal data of MOX2 was displayed in the Supporting Information (Supplementary Table S2).</p><!><p>Crystal structure of MOX2. (A) a single molecule in the crystal strucure and relevant dihedral angles; (B) a tetramer in the crystal structure.</p><!><p>Due to the AIE characteristics of MOX2, its mechanochromic luminescence (MCL) behaviors was investigated. As shown in Figure 6, the pristine MOX2 powder emitted strong yellow fluorescence with a maximum at 592 nm. The emission peak red-shifted to 660 nm after grinding with red emission under 365 nm light. The spectral shift value was 68 nm (Table 2). The emission of the ground sample was restored to its original yellow color by immersing of acetone for 2 s or heated at 50°C within 10 min (Figure 6A). Moreover, fluorescence color conversion could be repeated numerous times (Figures 6B–E) without fatigue (Chan et al., 2014). To gain insight into the MCL phenomenon, PXRD and DSC experiments were conducted on the pristine, grinding, immersing, and heating solids of MOX2 and MOX4 (Figure 7). It showed that the intensity of the diffraction peaks observed for powdered crystalline MOX2 significantly decreased upon grinding, which indicated the loss of crystallinity. The diffraction peaks of MOX4 still existed after heavily grinding. Furthermore, MOX2 exhibited greater redshift than MOX4 after grinding (Supplementary Figure S11). The transition from a crystalline structure to an amorphous state upon grinding was further confirmed by DSC experiments. In a DSC measurement of grinding MOX2 and MOX4 (Supplementary Figure S12), exothermic peaks that corresponded to the cold-crystallization transition MOX2 (T = 107.7°C) and MOX4 (T = 155.1°C) were observed followed by endothermic peaks that corresponded to the melting point of powder MOX2 (T = 203.6°C) and MOX4 (T = 231.3°C), respectively. These experimental results suggested that the transformation between crystalline structure and the amorphous state was responsible for the observed MCL behavior upon external stimuli.</p><!><p>(A) PL spectra of MOX2 powder under alternating grinding, immersing and heating; Images were taken under a 365 nm hand-held UV lamp. Change in the PL spectrum of MOX2 in grinding-immersing (B) and grinding-heating (D) circles. Repeated switching of the emission wavelength in grinding-immersing (C) and grinding-heating (E) circles.</p><p>Peak emission wavelengths (nm) of solid-state MOX2 under different conditions.</p><p>△λ = λpristine– λas-prepared.</p><p>(A) PXRD patterns of compound MOX2 in different states: pristine (black line), grinding (red line). (B) PXRD patterns of compound MOX4 in different states: pristine (black line), grinding (red line). (C) A reversible data recording device of MOX2 based on its mechanochromic phosphorescence.</p><!><p>By exploiting the excellent MCL behavior and good reversibility of MOX2, a simple rewritable phosphorescence data recording device was fabricated (Figure 7C) (Gundu et al., 2017). The specific reversible procedure is as follows. First, the original sample was put into a mortar and ground as the grinding sample. Next, it was spread on a filter paper to make a thin film, showing orange emission under UV excitation. Then, the characters "AIE" were written on the thin film with a writing brush with the acetone solvent as the "ink". Due to the change of the emission color, the yellow-emitting letters could be clearly seen. Next, the "AIE" was erased by using a cotton swab to make it completely disappear, resulting in the recovery of the original orange thin film. New letters "MCL" could be written again with the "writing brush" of the acetone solvent, which could still be removed by using a cotton swab according to the above method. This writing-erasing process can be repeated for several cycles. Based on this, MOX2 could be a potential fluorescent material for applications as repeated writing.</p><!><p>Next, the sensing properties of MOX2 was exploited because the pyrrolidine unit can be protonated. The solution of MOX2 was prepared in DCM at a concentration of 1 mM. This solution was added dropwise to a filter paper. At this point, the filter paper showed a dark red color under 365 nm illumination. Then after fumigation with trifluoroacetic acid (TFA) for 1 min, at the very beginning, the filter paper exhibited light blue under 365 nm illumination. With the passage of time, the light blue faded, and then the orange began to appear (Supplementary Figure S13). Until 480 min, the blue was completely gone. The color of the filter paper sheet returned to orange (Supplementary Figure S13) (Chen et al., 2018). Meanwhile, as shown in Figure 8A, to investigate the possible acidochromic properties of MOX2, the spectral response ability of MOX2 towards TFA in methanol solution (1.0 × 10−4 M) was also measured. When TFA was gradually added to the methanol solution of MOX2, the yellow solution by degrees converted into a light blue one (Figure 8A inset). Upon addition of TFA, the color of MOX2 in methanol solvent changed from orange to blue under 365 nm illumination, and a new blue-shifted peak formed in the PL spectrum (Figure 8A) (Cao et al., 2019; Cao et al., 2021). With the concentration of TFA increased from 0 to 21.32 mM, pronounced changes in emission suggested that MOX2 had been protonated by TFA. The ratio of PL intensity had a quadratic relationship with TFA concentration in a certain range of TFA concentrations (Figure 8B) (Cao et al., 2020). As displayed in Figure 8C, we further explored the acidochromic behavior of MOX2 for making a rewritable media by coating the solution on a filter paper. The letters were written on filter paper were stable, which could be erased only on fuming with TFA. Fumigation with ammonia could quickly return the sample to the initial color. Fumigation with TFA again can be quickly wiped off. The color would gradually return to its initial appearance after 2 h. This demonstrated that MOX2 has the potential to be used as security link.</p><!><p>(A) PL spectra of MOX2 (100 μM) in methanol solvent with different concentration (0.67-21.32 mM) of TFA. Photographs pf MOX2 (100 μM) in methanol solvent before and after acidification at 365 nm UV light showed in the inset. (B) The plot and linear fitting of PL intensity at 484 nm versus TFA (0.67-21.32 mM). (C) Photographs of filter papers coated with MOX2 (10 mM) in DCM solvent under different conditions.</p><!><p>Lipid droplets (LDs) are key organelles which are closely related to living status and death process of living cells (Kuerschner et al., 2008; Walther and Farese, 2009). Before cell imaging experiments, the potential cytotoxicity of MOX2 and MOX4 was evaluated using the MTT assay and low cytotoxicity of them was observed (Supplementary Figure S14). We first conducted co-staining experiments of LDs in living HeLa cells (Figure 9). The cells were stained with BODIPY 503 (1 × 10−5 M) and MOX2 (1 × 10–5 M) for 0.5 h, and were imaged in green channel (λ ex = 488 nm, λ em = 500–540 nm) and red channel (λ ex = 488 nm; λ em = 600–640 nm), respectively. The two channels are well overlapped with each other. This result indicated the good LDs selectivity of MOX2. When HeLa cells were incubated with MOX2 for 30 min at 37°C, strong red fluorescence signals were observed from inside cells shown in Supplementary Figure S15. This was not surprising for the far-red emitting compound MOX2, which showed a bright fluorescence image in vitro.</p><!><p>Colocalization confocal imaging of living HeLa cells stained with MOX2 and BODIPY 503: (A) bright field image; (B) imaging channel of BODIPY 503 (λ ex = 488 nm; λ em = 500–540 nm); (C) imaging channel of MOX2 (λ ex = 488 nm; λ em = 600–640 nm); (D) overlay image. Scale bar: 10 µm. (E) Red and green lines represent the fluorescence of MOX2 and BODIPY, respectively.</p><p>Synthetic route of MOX2 and MOX4. Reaction conditions: (A) Et3N, 25°C, 1 h, DBU −5°C, 1 h; (B) piperidine and 2-(1-pyrrolidinyl)benzaldehyde or 4-(1-Pyrrolidinyl)benzaldehyde.</p><!><p>In this work, ACQ-to-AIE transformation was achieved by migrating a small pyrrolidinyl group from para- to ortho-position in a new molecular system, in which the para-isomer MOX4 showed the ACQ behavior and the ortho-isomer MOX2 is AIE-active. Moreover, compound MOX2 exhibited mechanochromic properties and pressure sensing ability. PXRD and DSC revealed that the MCL mechanism was ascribed to the transformation among crystalline state and the amorphous state by grinding. Furthermore, investigation of the acidochromic behavior of MOX2 revealed that it could be further used as security link. In further biological experimental study, MOX2 could be utilized as a fluorescent probe for LDs imaging in living cells. This work provides a novel strategy to achieve the ACQ-to-AIE transformation, which will provide a new guideline for future design of new AIEgens from ACQ molecules.</p><!><p>The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.</p><!><p>ZW, RL, LC performed the fluorescence and mass spectrometry experiments, and wrote the manuscript. XZ, WL, XL, and LWC performed data collation. NC, SS, ZL, and JH reviewed manuscript drafts. ZW, RL, and LC contributed equally in this work. XC, BL, and LX conceived the entire project.</p><!><p>Financial support from the Natural Science Foundation of Fujian Province (No. 2020J06028) to LX and the NSFC (NO. 51872048) to RL, the CAS/SAFEA International Partnership Program for Creative Research Teams to XC and National Natural Science Foundation of China (Nos. 81872751 and 82104003). Liao Ning Revitalization Talents Program (No. XLYC2002115) and Key R&D Plan of Liaoning Province in 2020 (No. 2020020215-JH2/103).</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p><!><p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p><!><p>The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fchem.2021.823519/full#supplementary-material</p><!><p>Click here for additional data file.</p>

PubMed Open Access

Lifetime-based sensing of the hyaluronidase using fluorescein labeled hyaluronic acid

In this report we propose a lifetime-based sensing (LBS) for the detection of hyaluronidase (HA-ase). First, we heavily label hyaluronan macromolecules (HA) with fluorescein amine. The fluorescein labeled HA (HA-Fl) has a weak fluorescence and short fluorescence lifetime due to an efficient self-quenching. Upon the addition of HA-ase, the brightness and lifetime of the sample increase. The cleavage of an HA macromolecule reduces the energy migration between fluorescein molecules and the degree of the self-quenching. A first order of the cleavage reaction depends on the amount of the HA-ase enzyme. We describe an HA-ase sensing strategy based on the lifetime changes of the fluorescein labeled HA in the presence of HA-ase. We demonstrate that the calibration of the sensing response is the same for the average lifetime as for a single exponential decay approximation, which significantly simplifies the analysis of the sensing measurements.

lifetime-based_sensing_of_the_hyaluronidase_using_fluorescein_labeled_hyaluronic_acid

Introduction<!>Material and Methods<!>Preparation of conjugated Hyaluronan (HA-Fl Probe)<!>Fluorescence Measurements of Hyaluronan Hydrolysis<!>Lifetime Measurements of the HA-Fl Probe<!>Recovery of the brightness upon a release of the self-quenching<!>Changes to the HA-Fl fluorescence lifetimes in the presence of HA-ase<!>Lifetime-based sensing of HA-ase activity

<p>Hyaluronidases (HA-ase) are enzymes that cleave the polysaccharide, hyaluronic (HA), which is a glycosaminoglycan expressed in extracellular and pericellular matrices. In humans, five hyaluronidase genes and one hyaluronidase pseudogene has been described [1]. The hyaluronidases are endoglycosidases that predominantly catalyze hyaluronan depolymerization via cleavage of the β-N-acetyl-D-glucosaminidic bonds. In mammalian normal tissue, they are present in low concentrations; 60ng/ml in human serum [2]. It is well established that an over expression of the hyaluronidase enzymes is observed in many different cancers including prostate cancer and malignant melanomas [3, 4]. The increased activity of the hyaluronidases has been correlated with several carcinogenic cell behaviors including tissue invasion [5], resistance to apoptosis [6] and the potentiation of angiogenesis [4]. However HA-ases are also used as anticancer chemotherapeutic agents—the addition of HA-ase reduces a tumor's resistance to chemotherapy [7, 8]. HA-se can have different biological activities depending on the cancer cell type. In contrast to prostate cancer and malignant melanoma, HA-se suppresses tumorigenicity in a model of colon cancer [9].</p><p>HA, the substrate for HA-se, is a high molecular weight, linear, non-sulfated glycosaminoglycan composed of multiple subunits of D-glucuronic acid (GlcA) and N-acetylglucosamine (GlcNAc) and has the primary structure [β1→4GlcA β1→3GlcNAc]n. HA is known to exhibit diverse biological functions including: a) maintenance of tissue structural integrity, b) formation of highly hydrated matrices around individual cells, c) promotion of cellular migration including metastasis, and d) mediation of intercellular signaling. HA contributes to tumor cell behavior by: a) modulating the biomechanical properties of extracellular and pericellular matrices in which cells reside, b) forming a repetitive template for interactions with other macromolecules in the pericellular and extracellular environment and thus, contributing to the assembly, structural integrity and physiological properties of these matrices, and c) interacting with cell surface receptors through concomitant signal transduction. The digestion of fluorescently labeled HA (by HA-ase) can be used for detection of HA-ase enzyme activity. Several methods have been proposed. A simple assay for HA-ase activity using fluorescence polarization has been proposed by Murai and Kawashima [10]. However, the observed changes in polarization do not exceed 0.01 (10mP). Although polarization measurements are very precise, these changes are too small for reliable detection. Another approach involves dually labeled HA with fluorophores suitable for Forster resonance energy transfer (FRET). The cleavage of HA results in the release of FRET and change of the relative intensities of the fluorophores involved. These ratio-metric measurements offer larger signal responses in the presence of HA-ase than polarization changes but involve the dual HA labeling [11, 12].</p><p>In this manuscript we propose a simpler approach for the detection of HA-ase activity. We observed that fluorescein-labeled HA (HA-Fl) shows a very short fluorescence lifetime due to the self-quenching of fluorescein, a phenomenon known for many years. Self-quenching of fluorescein and other xantene-type dyes is one of the oldest observations in fluorescence spectroscopy and is due to resonance energy transfer between fluorescein molecules (homo FRET). This process was frequently connected to decreases in the quantum yield, lifetime and polarization of viscous solutions with high probe concentrations [13-18]. In the case of fluorescein, the Förster distance (50% probability of excitation energy transfer) for homo FRET is about 42Å [19]. Since this distance is comparable to or larger than the size of many proteins, FRET is expected to occur when a macromolecule contains more than a single fluorophore.</p><p>The digestion of HA-Fl by HA-ase enzyme releases fluorescein self-quenching, thereby increasing fluorescence brightness and lifetime. We describe here the strategy for the HA-ase detection using changes in observed lifetimes (lifetime-based sensing, LBS).</p><p>There are many advantages of LBS over intensity-based sensing methods [20, 21]. Fluorescence lifetime measurements yield absolute quantity values that are independent of the measurement platform. LBS does not depend on the excitation intensity and optical misalignments which simplifies the calibration of the sensing device. Mentioned above, LBS properties and its robustness make this approach an ideal tool for measurements of difficult-to-control "real world" samples such as physiological fluids or tissue.</p><!><p>Sodium hyaluronate from bacterial fermentation was obtained from Acros Organics (Thermo Fisher Scientific, NJ, USA). Fluorescein amine, dimethyl sulfoxide (DMSO), guanidine hydrochloride, acetaldehyde, cyclohexyl isocyanide, Sephadex G-75, and bovine testes hyaluronidase (EC 3.2.1.35, type 1-S, 451 U/mg) all were obtained from Sigma–Aldrich (Sigma-Aldrich, St. Louis, MO, USA). Dulbecco's phosphate-buffered saline (PBS) was purchased from Invitrogen Life Technologies (Invitrogen Corporation, CA, USA) and was adjusted to pH 6.0 with 0.1 N HCl after reconstitution in distilled water (dH2O). Slide-A-Lyser dialysis cassettes (10,000 molecular weight cutoff) were purchased from Pierce Chemical (Thermo Fisher Scientific).</p><!><p>Hyaluronan was covalently conjugated to fluorescein amine essentially as described [22]. Briefly, HA was dissolved to 1.25 mg/ml in dH2O. The HA solution was diluted 1:2 in DMSO, and fluorescein amine (predissolved as a DMSO stock solution) was added to a final concentration of 5 mg/ml. Acetaldehyde and cyclohexyl isocyanide were added to 0.04% (v/v), and the reaction was allowed to proceed for 16 h at 25 C. Afterward, the solution was diluted 1:14 in ethanol/guanidine HCl (50 μl of 3 M guanidine HCl per 900 μl of 100% ethanol) and the HA was allowed to precipitate overnight at −20 C. The precipitate was then dissolved in 1 ml of dH2O, followed by extensive dialysis against dH2O.</p><!><p>The HA-Fl probe (labeled hyaluronan) was incubated with a different concentration of hyaluronidase in PBS pH 6.0 at room temperature (RT). At selected time points, fluorescence emission spectra were collected using Cary Eclipse spectrofluorometer (Varian Inc., Australia). Measurements were performed in 0.4×0.4 cm quartz cells with the excitation at 480 nm and emission at 520 nm.</p><!><p>Fluorescence lifetime measurements were done using a FluoTime 200 fluorometer (PicoQuant, GmbH, Berlin, Germany). This time–resolved instrument is equipped with an ultrafast detector, a Hamamatsu R3809U-50 microchannel plate photomultiplier (MCP). For the excitation we used a 470 nm picosecond pulsed laser diode. The detection was made through a monochromator supported by a 495 nm long wave pass filter in order to eliminate a scattered excitation light. The decay data were analyzed with FluoFit, version 5.0 software (PicoQuant, GmbH). Fluorescence intensity decays were analyzed by reconvolution with the instrument response function and analyzed as a sum of experimental terms:</p><p>The intensity decays were analyzed with a multi-exponential model using FluoFit v. 5.0 software (PicoQuant, GbmH.). The data for each experiment were fitted with the multi-exponential model: (1)I(t)=∑iαiexp(−t∕τi) where τi are the decay times and αi are the pre-exponential factors (amplitudes) of the individual components (∑αi = 1). The contribution of each component to the steady state intensity is given by: (2)fi=αiτi∑jαjτj where the sum in the denominator is over all the decay times and amplitudes. The mean decay time (intensity-weighted average lifetime) is given by: (3)τ¯=∑ifiτi and the amplitude-weighted lifetime is given by: (4)〈τ〉=∑iαiτi</p><!><p>Absorption and fluorescence spectra of fluorescein-labeled HA, HA-Fl, are shown in Figure 1. A convenient blue excitation results in a green emission of the fluorescein, which can be easily filtered from the excitation light. A large spectral overlap (Figure 1, shadowed area) enables an efficient homo-transfer of the excitation energy.</p><p>Heavily labeled HA-Fl shows a relatively weak green fluorescence (Figure 2, top). Upon the addition of HA-ase, the HA macromolecule is cleaved into smaller pieces. The homo-transfer of the excitation energy and the subsequent self-quenching of fluorescein moiety are reduced which results in a stronger green fluorescence (Figure 2, bottom). The change in the fluorescence spectrum in the presence of HA-ase is shown in Figure 3. At the level of 100 U/ml HA-ase, after 30 min of incubation, the brightness of the HA-Fl solution (10 μg/ml) increases about 2.5 fold. It should be noted that, in the presence of the analyte, the observed signal increases, which is a favorable future in the sensing study.</p><!><p>In the absence of HA-ase, the lifetime of the HA-Fl solution has a very short lifetime and the fluorescence intensity decay is very heterogeneous (Figure 4, upper right). In the presence of HA-ase, the lifetimes are longer and intensity decays less heterogeneous (Figure 4). Upon the addition of 100 U/ml of HA-ase and 30 min incubation, the amplitude averaged lifetime increases more than two-fold, similar to the increase in the brightness.</p><p>We selected the 30 min incubation time after studying the time dependent traces with different amounts of HA-ase enzyme (Figure 5). As seen from this Figure, the first order of the kinetics depends strongly on the amount of the HA-ase, and the kinetics stabilize after about 30 min. The different signal responses to the different amounts of the enzyme facilitate the HA-ase detection. The lifetime changes in the presence of HA-ase are summarized in the Table 1.</p><!><p>In the case of complex intensity decays, it is not always obvious which lifetime parameters should be used to describe a desired dependence. In order to have changes in the lifetime consistent with the changes in the brightness, one should use the amplitude averaged lifetimes, <τ>, [25].</p><p>Using these (amplitude averaged lifetimes) from Table 1, we constructed a calibration curve for the sensing of HA-ase (Figure 6, left). Assuming the uncertainty of the measured lifetime is 0.02ns, the HA-ase activity can be estimated within an accuracy of 10%.</p><p>Although calculations of the average lifetimes from multi-exponential decay fittings pose no problem with today's computers, we were pleasantly surprised that the HA-ase dependence of lifetimes estimated from single exponential fits is almost identical (Figure 6, right). This could be a significant simplification for developers constructing a sensing device.</p><p>Since there are many researchers in the fluorescence field using frequency-domain instrumentation, we reconstructed time-domain lifetimes into the frequency-domain (Figure 7). At the frequency of 100 MHz the phase decreases almost 15 degrees, and the modulation is about 0.16 upon addition of 100 U/ml HA-ase. The phase and modulation data can be as well used for an efficient sensing of HA-ase.</p><p>In conclusion: we believe that the lifetime-based sensing of HA-ase presented above carries many advantages over intensity-based methods. The independence from excitation power and optical configuration are probably the most important considerations for the construction of a reliable detection device. Also, the range of observed lifetime changes is much more comfortable than the range of the polarization changes. The lifetime-based sensing of HA-ase can be done in the solution with the calibrated HA-Fl, using a titration method. However, for the surface measurements (skin cancers detection) one can prepare a water-based gel or cream containing HA-Fl and use a portable fluorometer for the detection.</p>

PubMed Author Manuscript

Stereoselectivities and Regioselectivities of (4+3) Cycloadditions Between Allenamide-Derived Chiral Oxazolidinone-Stabilized Oxyallyls and Furans: Experiment and Theory

A systematic investigation of the regioselectivities and stereoselectivities of (4+3) cycloadditions between unsymmetrical furans and a chiral oxazolidinone-substituted oxyallyl is presented. Cycloadditions were performed using an oxyallyl containing a (R)-4-phenyl-2-oxazolidinone auxiliary (2Ph), under either thermal or ZnCl2-catalyzed conditions. Reactions of 2Ph with 2-substituted furans gave syn cycloadducts selectively, while cycloadditions with 3-substituted furans gave selectively anti cycloadducts. The stereoselectivities were in favor of a single diastereoisomer (I) in all but one case (2-CO2R). Density functional theory calculations were performed to explain the selectivities. The results support a mechanism in which all cycloadducts are formed from the E isomer of the oxyallyl (in which the oxazolidinone C=O and oxyallyl oxygen are anti to each other) or the corresponding E ZnCl2 complex. The major diastereomer is derived from addition of the furan to the more crowded face of the oxyallyl. Crowded transition states are favored because they possess a stabilizing CH\xe2\x80\x93\xcf\x80 interaction between the furan and the Ph group.

stereoselectivities_and_regioselectivities_of_(4+3)_cycloadditions_between_allenamide-derived_chiral

INTRODUCTION<!>Previous Reports on Regioselectivities<!>Previous Reports on Stereoselectivities<!>RESULTS AND DISCUSSION<!>1.1 Cycloadditions in the absence of ZnCl2<!>1.2 Cycloadditions in the presence of ZnCl2<!>2. Cycloadditions of 2Ph with 3-Substituted Furans<!>3. Computational Studies<!>3.1 Cycloadditions of 2Ph with 2-Substituted Furans<!>3.2 Cycloadditions of 2Ph with 3-Substituted Furans<!>CONCLUSIONS<!>Typical Procedure for (4+3) Cycloadditions<!>THEORETICAL CALCULATIONS

<p>The (4+3) cycloadditions of oxyallyl (1) with dienes represent a valuable synthetic route to 7-membered carbocycles.1 We recently reported the cycloadditions shown in Scheme 1, where a chiral oxazolidinone-stabilized oxyallyl (2) is generated by oxidation of an allenamide, and reacts with a cyclopentadiene, pyrrole, or furan.2 This sequence provides access to a diverse range of cycloadducts having endo stereochemistry.3 Regioselective reactions with unsymmetrical furans have also been achieved. During our initial studies, we were surprised to find that the use of a 4-phenyl-2-oxazolidinone auxiliary (2, R = Ph) ("2Ph") led to unusual selectivities in reactions involving several furans. We have therefore carried out a detailed survey of the regio- and stereochemical features of the oxyallyl protocol using this auxiliary. Experimental and computational results, presented here, support a novel mode of stereoinduction for these reactions, in which the furan adds preferentially to the more crowded face of the oxyallyl. The crowded transition state is stabilized by an attractive CH–π interaction between the furan and the Ph group.</p><p> </p><p>Cycloaddition of 2Ph with furan led selectively to the cycloadduct I with a diastereomer ratio (I:II) of 82:18.2a In the presence of ZnCl2, the dr increased to ≥96:4. Originally, it was thought2 that the stereoselectivities of cycloadditions involving 2 arose through steric control, as depicted in Scheme 2. The presence of the bulky R group was thought to block one face of the oxyallyl from the approaching furan. Addition of the furan to the less hindered face of 2-Z (or its ZnCl2 chelate) would lead to the major diastereomer of the cycloadduct (I), while addition to the more hindered face would lead to the minor diastereomer (II).</p><p>Computational studies, however, revealed an altogether different mechanism.4 Computations showed that only cycloadditions involving the E isomer of 2Ph are energetically feasible (Scheme 3). The O-O repulsion is very destabilizing in conformations previously proposed (Scheme 2). This is true even in the presence of ZnCl2. Furan actually adds to the more crowded face of 2Ph-E, where a favorable CH–π interaction between the furan and the Ph group stabilizes the transition state by approximately 0.2 kcal/mol over the uncrowded TS.</p><p>We present here experimental and computational studies of the cycloadditions of 2Ph with a range of monosubstituted furans, chosen to represent all available variations of electronic character and position of substitution. The data support the anti disposition of oxygens and attractive CH–π model, and also indicate situations where the CH–π interaction can be overcome by substituents on the furan, resulting in altered selectivities.</p><!><p>Numerous regioselective cycloadditions between furans and N- or O-substituted oxyallyls have previously been reported.1 Some representative examples are shown in Scheme 4. In principle, the reactions of achiral heteroatom-substituted oxyallyls with unsymmetrical furans can yield two regioisomeric endo adducts; we denote these by the terms syn and anti, in reference to the position ultimately adopted by the substituents on the furan relative to the heteroatom.</p><p>Walters5 generated nitrogen-substituted oxyallyl intermediates from the α,α'-dihalide 3, by treatment with either Et3N in trifluoroethanol or Et3N/LiClO4 in acetonitrile. Both reacted with 2-methylfuran or 2-methoxyfuran to give predominantly anti adducts (4b). Föhlisch6 generated an oxygen-stabilized oxyallyl cation by treatment of the α-haloketone 5 with Et3N/LiClO4, and its cycloaddition with 2-methylfuran gave predominantly the anti cycloadduct 6b.7 By contrast, when Albizati8 treated the dimethylacetal 7 with a Lewis acid (TMSOTf, SnCl4, TiCl4, or BCl3), only the syn cycloadduct (6a) was obtained. In the reactions of both 3 and 5, detectable amounts of a third type of adduct resulting from the "sickle" form of the oxyallyl intermediate (rather than the "W" form) were also produced.9</p><p>We recently reported the regioselectivities of cycloadditions involving the parent achiral oxazolidinone-substituted oxyallyl (Scheme 5).10 Reactions with 2-methyl- or 2-CO2Me-furan led predominantly to syn cycloadducts, while anti adducts were obtained from 3-methyl- and 3-CO2Et-furan. Inclusion of ZnCl2 increased the yields, but had little effect on the regioselectivity.</p><!><p>Incorporation of a chiral auxiliary on the oxyallyl increases the number of possible endo cycloadducts to four. For each of the syn and anti regioisomers, there are two diastereomers, I and II, which differ according to the face of the oxyallyl intermediate to which the furan binds. Selected literature examples involving chiral nitrogen- and oxygen-substituted oxyallyls are shown in Scheme 6.</p><p>Walters generated a chiral nitrogen-stabilized oxyallyl intermediate by treatment of the α-haloketone 8 with Et3N/trifluoroethanol, and it reacted with 3-bromofuran to give selectively the anti-II adduct 9.11 Hoffmann studied the asymmetric (4+3) cycloadditions of oxygen-stabilized oxyallyl cations generated from 10 by treatment with TMSOTf; their cycloadditions with 3-substituted furans led predominantly to anti-I adducts.12 These results have recently been shown to conform to a CH–π mode of stereoinduction similar to that proposed for our oxyallyls 2.13</p><!><p>We have now investigated the asymmetric cycloadditions of 2Ph with a range of 2- and 3-substituted furans. Methyl and acyloxymethyl-substituted furans were used as electron-rich substrates,14 while ester and cyano-substituted furans were used as electron-poor substrates. The experimentally measured regio- and stereoselectivities are given in Tables 1–3.15</p><!><p>The cycloaddition of 2Ph with 2-methylfuran (Table 1) gave a 54% total yield of the endo cycloadducts 12a–d, in which the diastereomer ratio (dr) of I:II was 90:10. The regioisomer ratios were equivalent for the two diastereomers: I was obtained with a syn:anti ratio of 67:23 (12a:12b), while II was obtained with a syn:anti ratio of 70:30 (12c:12d). Stereochemical assignments were made on the basis of NMR coupling patterns and COSY spectra. The olefinic protons in both of the I isomers (12a and 12b) are shifted unusually upfield, due to anisotropic shielding by the nearby Ph ring on the oxazolidinone. Similar shielding effects were also observed with the other I isomers described below. The II isomers 12c and 12d showed no such upfield shifting. For the syn-I isomer (12a), the 1H NMR spectrum had to be collected at high temperature in order to discern the coupling patterns (Supporting Information).</p><p>In contrast, the cycloadditions of 2Ph with 2-EWG-substituted furans produced diastereomer II but no observable I. Methyl 2-furoate and allyl 2-furoate gave the syn-II isomers 13c and 13'c as the sole cycloadducts in 40–60% yield. The stereochemistry of 13c was unambiguously assigned by X-ray crystallography (Figure 1).</p><!><p>Inclusion of ZnCl2 in the reaction mixture for cycloaddition with 2-methylfuran raised the diastereomer ratio from 90:10 to 100:0 (Table 2). The syn:anti ratio increased to 80:20.</p><p>With methyl 2-furoate, the major product was still the syn-II isomer 13c, but it was now accompanied by a small amount of syn-I (13a). The structure of 13a was assigned spectroscopically; anisotropic shielding was again evident.2d Somewhat surprisingly, on going from the furoate ester to its nitrile analogue, a return in stereoselectivity back to I was observed! The syn-I isomer 14a was obtained from 2-cyanofuran with a dr of 83:17. The regioselectivity for both the ester and the nitrile was completely in favor of syn.</p><p>The regioselectivities observed here parallel those observed previously for the parent oxazolidinone-substituted oxyallyl (Scheme 5),10 although for 2-methylfuran the syn:anti ratio is lower with 2Ph than with the parent oxyallyl.</p><!><p>The cycloadditions of 2Ph with 3-substituted furans were studied under ZnCl2-catalyzed conditions. Our results are listed in Table 3. The stereochemistry of the major cycloadducts was unambiguously assigned by means of COSY and NOESY analysis and, in the cases of ethyl 3-furoate and 3-bromofuran, by X-ray single crystal structures (Figure 1). In all of the major products, the olefinic proton showed the typical anisotropic shielding by the Ph ring on the oxazolidinone. For the minor isomers, the regiochemistry could be firmly assigned as syn by COSY and NMR coupling patterns, but the stereochemistry was not determined. The syn regiochemistry ruled out the use of Ph shielding as a stereochemical diagnostic. We have tentatively assigned the II stereochemistry to the minor isomers, on the basis of our computational results reported below.</p><p>The regio- and stereoselectivities of the cycloadditions involving 3-substituted furans differ markedly from those of 2-substituted furans. All of the 3-substituted furans gave selectively the anti-I cycloadduct. For 3-methylfuran and methyl 3-furoate, the anti:syn ratio was approximately 90:10. 3-phenylfuran and 3-acetoxymethylfuran gave more modest anti:syn ratios (60:40 and 70:30, respectively), but replacement of the acetyl group in the latter by a pivaloyl group increased the anti selectivity to 100:0. 3-bromofuran also gave 100:0 anti selectivity.</p><p>The regioselectivities of these reactions mirror those of the parent oxazolidinone-substituted oxyallyl with 3-substituted furans (Scheme 5),10 both in direction and magnitude. Similar anti-I selectivity was reported by Hoffmann for the cycloadditions of 3-substituted furans with related oxygen-substituted oxyallyl cations (Scheme 6).12</p><!><p>Numerous theoretical studies of oxyallyl cations and their cycloadditions have been reported previously.5,16–18 Cramer17b,c showed that the cycloadditions can take place by various mechanisms; the highly electrophilic hydroxyallyl cation was calculated to react with furan by a two-step process commencing with electrophilic attack at C-2 of the furan. We have found4 that the less electrophilic, neutral oxyallyls 2 undergo concerted, asynchronous cycloadditions with furans.</p><p>Our earlier findings4 are summarized in Scheme 7. At the B3LYP/6-31G(d) level, only the E conformer of 2Ph was an energy minimum. Attempts to locate the Z conformer led to the isomeric cyclopropanone. ZnCl2 complexes were located for both the E and the Z conformers, but the Z complex was 6.2 kcal/mol less stable. Transition states were located for cycloadditions involving both the E and the Z conformers, but those involving the Z isomer were disfavored by ≥15.4 kcal/mol (≥5.2 kcal/mol for the ZnCl2 complexes). Electrostatic repulsion between the oxygen atoms destabilizes all of the Z structures.</p><p>The concerted transition state geometries are asynchronous such that the more advanced bonding interaction involves the more nucleophilic (CH2) carbon of the oxyallyl. This is the case also for reactions with substituted furans, shown in Scheme 5.10 On the other hand, the calculated Mulliken charges in the transition states indicate net charge transfer of 0.05–0.17e from the furan to the oxyallyl. Overall, the oxyallyls 2 are best characterized as ambiphilic toward furans. The activation barriers for reactions with either Me- or CO2Me-substituted furans (Scheme 5) are lower than those for furan itself. Methyl 2-furoate has the lowest barrier (ΔH‡ 3.2 kcal/mol lower than for furan).</p><p>To model the reactions of 2Ph with substituted furans, we calculated transition states involving addition to the E oxyallyl only. Calculations were performed at the B3LYP/6-31G(d) level (including LANL2DZ+ECP for Zn) in Gaussian 03.19 In Figure 2 are shown the four isomeric transition structures calculated for the reaction of 2Ph with 2-methylfuran. These are representative of the transition states calculated for the other furans also. Concerted, asynchronous transition states were found for all of the cycloadditions.</p><!><p>The calculated activation energies for reactions of 2Ph-E with 2-substituted furans are listed in Table 4. The experimental selectivities are also included in the table for comparison.</p><p>The calculations correctly predict the major product for each 2-substituted furan. Some differences in the minor isomers are found, which for the ZnCl2-catalyzed cycloadditions may be due to the use of excess furan and ZnCl2 in the experiment. For example, a furan molecule may coordinate to the Zn centre of 2Ph-E·ZnCl2, or ZnCl2 may coordinate to the furan that undergoes cycloaddition. These factors would lead to small differences in the proportions of the minor isomers but are unlikely to affect the overall selectivity.</p><p>The oxyallyl 2Ph is more nucleophilic at the CH2 terminus and more electrophilic at the CHN terminus. Yet, cycloadditions of 2Ph with both 2-EWG and 2-EDG-substituted furans favor the syn cycloadducts. This regioselectivity is predictable for the EWG-substituted furans, since the substituent renders the 5-position of the furan more electrophilic than the 2-position. The regioselectivity for the donor-substituted furan (2-Me-furan) is unexpected. Here electronic effects are small, and the regioselectivity instead arises from a preference for the transition state that allows the more advanced bonding interaction to be made to the less-substituted carbon (C-5) of the furan (Figure 2).</p><p>The stereoselectivities observed with 2-methylfuran and 2-cyanofuran conform to the CH–π model (Scheme 3), and have a magnitude somewhat smaller than that observed with furan itself (82:18 uncatalyzed, ≥96:4 with ZnCl2). The proton involved in the CH–π interaction is H-3 or H-4 of the furan (for the syn and anti isomers, respectively), which points roughly towards the centre of the Ph ring in the I transition states. The stabilizing role of the CH–π interaction has both electrostatic and dispersive components. To estimate the role of the latter, the dispersion energies in the transition states were calculated by means of Grimme's 2006 empirical B3LYP-D formula, which sums the dispersion energies between each pair of atoms in the structure.20 A comparison between the dispersion energies of the syn-I and syn-II transition states for each 2-substituted furan is shown in Figure 3. Dispersive stabilization is 0.02–2.9 kcal/mol stronger in the I transition states than the II transition states.</p><p>The reversal in stereoselectivity for 2-CO2Me-furan was at first suprising, but it is indeed predicted by theory. The syn-II transition state is favored over the other three TSs by 1.9 kcal/mol in the uncatalyzed reaction and by 0.6 kcal/mol in the ZnCl2-catalyzed reaction (ΔΔH‡). A top-down view of the syn-I transition state for 2-CO2Me-furan under ZnCl2-catalyzed conditions is shown in Figure 4a. A destabilizing electrostatic interaction takes place between the ester carbonyl group and the Ph π-cloud. The distance between the carbonyl oxygen and the nearby C-2 of the Ph ring (red line: 3.22 Å) is equal to the sum of the van der Waals radii for C and O. This interaction outweighs the stabilizing effect of the CH–π interaction, and the uncrowded (II) transition state is instead favored.</p><p>The calculations also correctly predict that the seemingly small change from a 2-CO2Me to a 2-CN group should induce a return to syn-I selectivity. The syn-I transition state for 2-CN-furan is shown in Figure 4b. The CN group is positioned further from the Ph ring (N–C 3.56 Å), and therefore does not present such severe electrostatic repulsion as the CO2Me group.</p><!><p>The calculated activation energies for ZnCl2-catalyzed reactions of 2Ph with 3-substituted furans are listed in Table 5, together with the experimental selectivities.</p><p>The calculations correctly predict the regio- and stereochemistry of the major product for methyl 3-furoate. For 3-methylfuran, the free energies of activation do not capture the correct stereoselectivity, but the enthalpies do predict both the regio- and stereoselectivity. As with the 2-EWG-substituted furans, the regioselectivity for 3-CO2Me-furan is under electronic control, since C-2 of the furan is more electrophilic than C-5. For 3-Me-furan, the regioselectivity is instead steric in origin, arising through avoidance of clashing between the Me group and the Ph group. This steric effect also influences the reaction of 3-CO2Me-furan: indeed, a concerted syn-I transition state could not be located, due to severe clashing.</p><p>Both 3-substituted furans favor the I stereoisomer, as do the four other 3-substituted furans in Table 3. The anti arrangement enables the CH–π interaction in the crowded TS to take place without interference from the substituent. The calculations predict that for both 3-substituted furans, the minor (syn) isomer should have the II stereochemistry, as the syn-I transition state is subject to unfavorable interaction between the Ph group and the furan 3-substituent.</p><!><p>A summary of the origins of the regio- and stereoselectivities of the (4+3) cycloadditions is given in Scheme 8. The oxyallyls 2 undergo (4+3) cycloadditions with furans through concerted transition states in most cases. Generally, bond development at the TS is most advanced at the nucleophilic (CH2) terminus of 2. The regioselectivities of (4+3) cycloadditions between 2 and furans arise through steric effects, but these are complemented by electronic effects when the sub-stituent is an electron-withdrawing group. For 2-substituted furans, syn cycloadducts are formed selectively, because this arrangement enables the stronger bonding interaction in the TS to involve the less-hindered (C-5) carbon. For 2-EWG-substituted furans, C-5 is also more electrophilic than C-2, enhancing regiocontrol. 3-Substituted furans undergo cycloaddition preferentially in the anti geometry, in order to avoid steric clashing between the 3-substituent and the Ph ring. Similar anti selectivity has even been observed when the Ph group is absent,10 indicating that the pseudoaxial group at the 4-position of the oxazolidinone has a steric influence even when it is only an H atom. For 3-EWG-substituted furans, the anti selectivity is complemented by the electronic effect of the substituent, which renders C-2 more electrophilic than C-5.</p><p>All but two of the furans studied (Tables 1–3) lead selectively to the cycloadduct I. Only for 2-CO2R-furans (R = Me, allyl) does the unexpected II isomer predominate. The CH–π model (Scheme 3) therefore appears to have broad applicability for monosubstituted furans. When the oxazolidinone Ph group is replaced by a non-aromatic group, the stabilizing CH–π interaction is lost, and the stereoselectivity reverts to favor II, as shown by our earlier studies with a 4-Bn-oxazolidinone and with Seebach et al.'s21 4-iPr-5,5-Ph2-oxazolidinone auxiliary.4 Reversals of stereoselectivity have also previously been observed to occur in hetero-Diels–Alder reactions22 and conjugate radical additions23 catalyzed by chiral Lewis acid/bis(oxazoline) complexes, when the Ph groups on the ligand were replaced by tBu groups.</p><p>Chiral oxazolidinone auxiliaries have been used to achieve a wide range of other asymmetric transformations, including Diels–Alder reactions,24 aldol condensations,25 enolate alkylation,26 hydroxylation,27 and amination,28 Michael additions,29 epoxide synthesis,30 and resolutions of α-halo carbonyl compounds.31 The results described herein for the (4+3) cycloadditions of 2Ph likely hold relevance to stereocontrol in these other oxazolidinone-directed reactions.</p><!><p>To a solution of the allenamide in CH2Cl2 (0.1 M) were added the appropriate furan (3–9 equiv) and 4Å powder molecular sieves (0.5 g). The solution was cooled to −78 °C, and ZnCl2 (1.0 M in ether, 2.0 equiv) was added. DMDO (4.0–6.0 equiv, in acetone) was then added as a chilled solution via syringe pump (at −78 °C) over 3–4 h. The reaction mixture was stirred for a further 14 h, then quenched with saturated aqueous NaHCO3, filtered through Celite, concentrated in vacuo, extracted with CH2Cl2, dried over Na2SO4, and concentrated in vacuo. The crude residue was purified via column chromatography on silica gel (gradient eluent: 10% to 75% ethyl acetate in hexane).</p><!><p>Density functional theory calculations were performed at the B3LYP/6-31G(d) level32 in Gaussian 03.19 The LANL2DZ basis set and effective core potential were used for Zn.33 Species were characterized as minima or transition states on the basis of vibrational frequency analysis and, where appropriate, IRC calculations.34 Zero-point energy and thermal corrections were derived (unscaled) from the B3LYP/6-31G(d) frequencies. Conformational searching was performed for each species in order to identify the lowest-energy conformer. Enthalpies and free energies are reported at 298.15 K, with a standard state of 1 atm.</p>

PubMed Author Manuscript

Stereoselective Ring-Opening (Co)polymerization of β-Butyrolactone and ε-Decalactone Using an Yttrium Bis(phenolate) Catalytic System

An effective route for ring-opening copolymerization of β-butyrolactone (BBL) with ε-decalactone (ε-DL) is reported. Microstructures of the block copolymers characterized by 13C NMR spectroscopy revealed syndiotactic-enriched poly(3-hydroxybutyrate) (PHB) blocks. Several di- and triblock copolymers (PDL-b-PHB and PDL-b-PHB-b-PDL, respectively) were successfully synthesized by sequential addition of the monomers using (salan)Y(III) complexes as catalysts. The results from MALDI-ToF mass spectrometry confirmed the presence of the copolymers. Moreover, thermal properties of the block copolymers were also investigated and showed that the microphase separation of PDL-b-PHB copolymers into PHB- and PDL-rich domains has an impact on the glass transition temperatures of both blocks.

stereoselective_ring-opening_(co)polymerization_of_β-butyrolactone_and_ε-decalactone_using_an_yttriu

Introduction<!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Results and Discussion<!><!>Microstructural and Statistical Analysis of the Copolymers<!><!>Microstructural and Statistical Analysis of the Copolymers<!><!>Thermal and X-Ray Analyses<!><!>Thermal and X-Ray Analyses<!><!>Thermal and X-Ray Analyses<!>Conclusion<!>Author Contributions<!>Conflict of Interest Statement

<p>Polyhydroxyalkanoates (PHAs) comprise a group of naturally occurring aliphatic polyesters produced by bacteria and other living organisms (Reddy et al., 2003; Tan et al., 2017). These biodegradable and hydrophobic materials combine the film-barrier properties of polyesters with the mechanical performances of petroleum-based polyethylene and polypropylene. Thanks to their interesting properties, these polymers are already used in packaging, automotive, hygienic, agricultural, and biomedical applications (Costa et al., 2013; Khosravi-Darani, 2015; Luef et al., 2015; Ali and Jamil, 2016; Yeo et al., 2018). The most common PHA is poly(3-hydroxybutyrate) (PHB), which is a linear, isotactic, high-molecular-weight polymer. However, this polymer has poor mechanical properties due to its brittleness and a susceptibility to thermal degradation slightly above its melting temperature. Therefore, development of a synthesis method producing PHBs with controlled molecular weight, lower melting temperature, and lower brittleness would be a solution for industrial manufacturing and practical use of ideal bio-based polymers (Li et al., 2016). One of the most effective approaches for the controlled synthesis of PHB is ring-opening polymerization (ROP) of β-butyrolactone (BBL) (Kricheldorf and Eggerstedt, 1997; Hori and Hagiwara, 1999; Rieth et al., 2002; Ajellal et al., 2010; Brulé et al., 2011, 2013; Li et al., 2016). ROP of β-butyrolactone mediated by metal systems has attracted much attention in recent years and had considerable achievements (Kricheldorf and Eggerstedt, 1997; Hori and Hagiwara, 1999; Rieth et al., 2002; Ajellal et al., 2010; Brulé et al., 2011). A major point of interest for some of these catalysts is the high degree of (stereo)control they exhibit under suitable conditions.</p><p>Various modification methods have been reported to improve the performance of PHB (Thomas, 2010; Aluthge et al., 2013; Lin et al., 2013; Olsén et al., 2013; Barouti and Guillaume, 2016). In particular, the formation of PHB-based copolymers might be a promising strategy but still remains a considerable synthetic challenge (Aluthge et al., 2013; Barouti and Guillaume, 2016). In this regard, poly(ε-decalactone) (PDL), a bio-based polymer synthesized by ROP of ε-decalactone (ε-DL), might be a good candidate to copolymerize with PHB in order to improve its mechanical properties (Lin et al., 2013; Olsén et al., 2013). Indeed, this polymer has been shown to be amorphous, with a low Tg value (i.e., −53°C) (Olsén et al., 2013). Although some initiators (co)polymerize ε-decalactone with good control, (Chuang et al., 2013; Lin et al., 2013; Olsén et al., 2013; Jasinska-Walc et al., 2014, 2015; Martello et al., 2014; Schneiderman et al., 2014, 2015; Lee et al., 2015; Zhu et al., 2015, 2018; Mannion et al., 2016; Tang et al., 2017) copolymerization of ε-DL with rac-BBL is still unknown. In this paper, we present the first example of an effective block copolymerization of BBL and DL initiated by a group 3 metal system to produce high-molecular-weight polymers with narrow molecular weight distributions.</p><!><p>In our search for new copolymerization catalysts, we focused our efforts on investigating the catalytic activity of (salan)Y(III) complexes known to be active for PHB formation (Fang et al., 2013). Therefore, we decided to study the potential of such a catalytic system in a copolymerization procedure, aiming to produce PDL-PHB block copolymers with a one-pot sequential methodology. In accordance with previous observations, the reaction of commercially available Y(OiPr)3 with the salan ligand L produces a mixture of bimetallic complexes [(L)Y(μ-OiPr)]2 (1) and (L)2Y2(μ-OiPr)(μ-OH) (2) (Scheme 1). Only volatile by-products are then removed between the yttrium alkoxide formation step and the polymerization step so that the resulting complexes are ready for polymerization without purification.</p><!><p>Synthesis of complexes 1 and 2.</p><!><p>Firstly, we investigated the homopolymerization of ε-decalactone using complexes 1 and 2. The results are summarized in Table 1. These complexes are active initiators for the controlled ROP of ε-DL under mild conditions. The corresponding polymers formed had narrow polydispersities (PDI = Mw/Mn), and GPC chromatograms of the isolated polymers are monomodal, suggesting that only one species in solution is active for the ROP of DL (Figure S1). As was already demonstrated for rac-BBL (Fang et al., 2013), we hypothesize that excess monomer cleaves the dimeric structure of both yttrium alkoxide complexes. When these species are exposed to excess DL, the monomer is coordinated and allows the formation of a mononuclear (salan)Y(OiPr)(DL) complex and a (salan)Y(OH)(DL) species, presumably inactive for polymerization.</p><!><p>Polymerization of ε-decalactone using (salan)Y(III) complexesa.</p><p>All reactions performed at 50°C.</p><p>Time was not necessarily optimized.</p><p>As determined by the integration of 1H NMR methine resonances of DL and PDL.</p><p>Mn and Mw/Mn of polymer determined by SEC-RI using polystyrene standards. Mn, calc = [DL]/[Y] × Conv. × MDL.</p><!><p>The resulting catalytic system proved to be active at 50°C in either C6D6 or toluene solutions. For [DL]/[Y] = 62.5, polymerization in C6D6 reached 92% conversion in 10 h (Table 1, Entry 2), while polymerization in toluene achieved 80% conversion within 14 h (Table 1, Entry 3). Reducing or increasing the monomer concentration resulted in no significant change of the polymerization control (Table 1, Entries 3 and 4). Although our yttrium-based system was also active at 50°C in neat DL, low catalytic activities were observed, probably due to viscosity issues (Table 1, Entry 5) (Moritz, 1989). All other polymerization reactions were therefore preferably conducted in C6D6 at [ε-DL] = 2.5 M. Interestingly, this system proved to be active in the presence of 125–625 equiv. of lactone (Table 1, Entries 6–8), resulting in a TOF of 6 h−1.</p><p>For [DL]/[Y] < 100, the polymers produced have narrow molecular weight distributions and experimental number-average molecular masses (Mn) close to the theoretical ones. However, for higher ratios, experimental Mn values do not correspond well with calculated Mn values. This mismatch possibly arises from suppression of hydrodynamic volume of PDL chains in THF or poor correlation between the polystyrene calibration of the GPC and the actual molecular weights of the PDL chains, as already reported by Olsén et al. (2013).</p><p>To confirm the mechanism, PDL was characterized by NMR spectroscopy. The results showed the polymer chain having isopropoxide and hydroxyl as end groups (Figure 1 and Figure S2), suggesting that the polymerization occurs through a coordination–insertion mechanism. The mechanism is similar to the mechanism reported in ROP of lactones or lactide catalyzed by metal alkoxide complexes (Thomas, 2010; Chuang et al., 2013; Fang et al., 2013). Matrix Assisted Laser Desorption Ionisation - Time of Flight (MALDI-ToF) mass spectrometry analysis was also performed in order to determine structure of the resulting polymer. The sample from Table 1, Entry 1, was chosen to be characterized by MALDI-ToF-MS. The spectrum reveals a repeating mass series of linear PDL having isopropoxide as an end group (OiPr[ε-DL]nH + Cs+) (Figure S3). The result is consistent with the end-group analysis by 1H NMR spectroscopy. All of the PDL samples are transparent viscous liquids under ambient temperature, indicating the amorphous nature of PDL (Lin et al., 2013; Olsén et al., 2013). Having an amorphous nature, it could be a great choice to copolymerize PDL with brittle polymers to improve the performance of the resulting copolymer. Then, we investigated the catalytic activity of our yttrium system toward diblock copolymerization of ε-DL and rac-BBL. The copolymerization was first attempted through addition of monomer mixture to a C6D6 solution of the precursor at 60°C. However, this method failed to give block copolymers, as GPC analysis showed multimodal distributions. The copolymerization was therefore carried out by one-pot sequential copolymerization of first ε-DL and then rac-BBL at room temperature. The results are summarized in Table 2. For [monomers]/[Y] < 100, the copolymerization nearly reached completion for both monomers in < 5 h in C6D6 (Table 2, Entries 1 and 2). By doubling the monomer-to-metal ratio, the resultant polymer revealed a double experimental Mn value, indicating a controlled polymerization reaction (Table 2, Entries 1 and 2). The copolymerization was then conducted in toluene with high amounts of rac-BBL (Table 2, Entries 3–7). The resulting copolymers having 5–59 mol% of ε-DL were synthesized with TOF up to 44 h−1. Interestingly, the catalytic system also proved to be active for synthesizing PDL-b-PHB-b-PDL triblock copolymers by one-pot sequential copolymerization. The copolymerization achieved ~60% conversion of ε-DL and 99% conversion of rac-BBL (Table 2, Entries 8 and 9). GPC analysis of all the copolymers showed a monomodal peak with narrow molecular weight distributions Mw/Mn (Figure S1). As observed for the homopolymerization of ε-DL, lower experimental Mn values were obtained, probably due to poor correlation between the polystyrene calibration of the GPC and the actual molecular weights. In order to determine the diffusion coefficients of two copolymers of different sizes (Table 2, Entries 1 and 7), DOSY NMR experiments were performed. For the low-molecular-weight copolymer sample (Table 2, Entry 1), we measured a diffusion coefficient of 2.32 × 10−10 m2.s−1, while the higher-molecular-weight copolymer corresponded to a diffusion coefficient of 1.88 × 10−10 m2.s−1 (Table 2, Entry 7). As the rate of diffusion is inversely related to the molecular weight/size, these results are consistent with GPC analyses (Figures S12, S13).</p><!><p>1H NMR (500 MHz, CDCl3) of PDL prepared by ROP of ε-DL with (salan)Y(III) complexes.</p><p>Copolymerization of ε-DL and BBL using (salan)Y(III) complexesa.</p><p>Polymerization of ε-DL and BBL were respectively performed at 50°C and room temperature with [DL] = 2.5 mol/L, unless otherwise stated.</p><p>[DL+BBL] = 2.5 mol/L.</p><p>DL content in copolymer.</p><p>Time was not necessarily optimized.</p><p>As determined by the integration of 1H NMR methine resonances of DL and PDL.</p><p>Mn and Mw/Mn of polymer determined by SEC-RI using polystyrene standards. Mn values were not corrected. Mn, calc = [DL]/[Y] × Conv. × MDL + [BBL]/[Y] × Conv. × MBBL.</p><!><p>In order to determine topology and end groups of the block copolymers, a diblock copolymer (PDL-b-PHB) (Table 2, Entry 1) and a triblock copolymer (PDL-b-PHB-b-PDL) (Table 2, Entry 8) were characterized by MALDI-ToF-MS (Figure 2 and Figure S4). The highest-intensity isotope distribution corresponds to linear PDL with an isopropoxide end group. Also, analysis of the minor isotope distributions confirmed the presence of the block copolymers. For instance, in Figure 2, the peak at m/z 1,980.31 corresponds to (OiPr[ε-DL]10[BBL]1H + Cs+). Similar MALDI-ToF-MS spectra showing minor series of isotope distribution of block copolymers were previously reported for poly(ε-decalactone)-b-poly(ω-pentadecalactone) copolymers (Jasinska-Walc et al., 2015). For PDL-b-PHB-b-PDL, similar isotope patterns are observed with mass higher than PDL-b-PHB (Figure S4).</p><!><p>MALDI-ToF-MS spectrum of PDL-b-PHB copolymer synthesized by ring-opening copolymerization of ε-DL and BBL with cesium trifluoroacetate as a cationizing agent.</p><!><p>Microstructural analysis of PDL-b-PHB was studied by 13C NMR spectroscopy. The resonances were assigned at the diad and triad levels. Ring-opening copolymerization of ε-DL and rac-BBL promoted by the yttrium complexes allowed the formation of syndiotactic PHB in PDL-b-PHB copolymers. By comparing 13C NMR of the block copolymers with a prior 13C NMR assignment for syndiotactic PHBs (Ajellal et al., 2009a; Fang et al., 2013), expansion of both carbonyl and methylene regions of PHB in PDL-b-PHB copolymer showed resonances that corresponded to triad sensitivities. The most intense resonances of the carbonyl and methylene regions at δ 169.32 and δ 40.79 ppm, respectively, were correlated to rr-centered triads. The lower-intensity resonances at δ 169.22 (carbonyl region) and δ 40.93 ppm (methylene region) were assigned to rm triads, and the others at δ 40.88 and δ 40.73 (methylene region) were mm and mr triads, respectively (Figure 3).</p><!><p>13C NMR spectrum (125 MHz, CDCl3) of (A) carbonyl region and (B) methylene region, (C) methyl region of PHB in PDL-b-PHB copolymers (Table 2, entry 6).</p><!><p>Notably, the resonance attributed to mm triad is almost negligible, as expected for syndiotactic-enriched polymers (Amgoune et al., 2006; Ajellal et al., 2009a,b). The methyl region shows two resonances at the diad level. The resonance at δ 19.89 ppm was correlated to r diad, and the one at δ 19.84 ppm was assigned to m diad. Therefore, probability of racemic linkages between monomer units (Pr) can be calculated (Amgoune et al., 2006; Ajellal et al., 2009a,b). As expected for syndiotactic PHBs, Pr values of PHB in PDL-b-PHB are high (up to 0.90 syndiotacticity), for all diblock copolymers with 5–25 mol% ε-DL (Table 3, Entries 2–5), although we observed slight differences in the PHB microstructures depending on the reaction conditions (e.g., the concentration of monomer) (Amgoune et al., 2006; Ajellal et al., 2009b; Kramer et al., 2009). Finally, the microstructure of PDL blocks was not determined, due to insufficient resolution of ε-DL resonances at 125.0 MHz (Figure S5).</p><!><p>Thermal data of selected syndiotactic-enriched PDL-b-PHB copolymers obtained by using (salan)Y(III) complexes. Data of a PHB homopolymer are also reported for comparison.</p><p>DL content in copolymer.</p><p>Weight % of PHB block was calculated from ε-DL mol%.</p><p>Melting and crystallization enthalpy values were calculated from the experimental data on the basis of wt% of PHB blocks (ΔH = ΔHobs/wt%PHB).</p><p>Pr is the probability of racemic linkages between monomer units and is determined by 13C{1H} NMR spectroscopy.</p><p>nd, not determined.</p><p>Value is overestimated due to possible overestimation ε-DL mol%.</p><!><p>Differential scanning calorimetry (DSC) and wide-angle x-ray diffraction (WAXD) analyses were performed to evaluate the influence of ε-DL content on the thermal and structural properties of the copolymers. The main thermal properties [i.e., glass transition temperatures (Tg), melting temperatures (Tm), and melting enthalpies (ΔHm)] of the selected PDL-b-PHB copolymers (corresponding to polymers of Table 2, Entries 3–6) are reported in Table 3. Thermal data of a PHB sample are also shown in Table 3 for comparison. All analyzed samples crystallize from melt during the DSC cooling run, and only small differences are observed for both Tm and ΔHm between first and second DSC heating runs. Second DSC heating thermograms of Entries 1–5 in Table 3 are reported in Figures S6–S11.</p><p>The detected high values of Tm (ranging from 144 to 156°C) and ΔHm (ranging from 42 to 59 J/g) of copolymers are in good agreement with those observed for other syndiotactic-enriched PHB homopolymers (0.8 < Pr < 0.9) (Ajellal et al., 2009a; Ebrahimi et al., 2016). This hypothesis was confirmed by the WAXD patterns of all copolymers (Figure 4). Positions and intensity ratios of Bragg reflections detected in the spectra correspond to those observed for the syndiotactic PHB crystalline form (Kemnitzer et al., 1993, 1995). Moreover, WAXD spectra confirm that PDL blocks are amorphous, since no other Bragg reflection, except those of the syndiotactic PHB crystal form, was detected. Both thermal (Table 3) and structural (Figure 4) data suggest that ε-DL content ranging from 5 to 25 mol% has less effect on crystallinity of PHB blocks of PDL-b-PHB copolymers. It is worth noting that even by increasing ε-DL content to 59 mol% (Table 2, Entry 7), the Tm of the PHB block remains high (i.e., 143°C). Based on these data, it appears that the PDL block, probably due to its high flexibility, has no influence on the ability of the PHB block to crystallize.</p><!><p>WAXD patterns of PHB homopolymer and PDL-b-PHB samples (entries 1–5 in Table 3) produced via ROP of rac-BBL and copolymerization of rac-BBL and DL with (salan)Y(III) complexes.</p><!><p>The observed small differences in Tm of copolymers reported in Table 3 are possibly due to small differences in stereoregularity of the BBL unit sequences rather than to the PDL block lengths (Figure S14). These differences could explain why, in the second DSC heating run, the copolymer with 25 mol% of DL units (Table 3, Entry 5) has a Tm higher than the one of the PHB homopolymer having similar Mn (Table 3, Entry 1). In addition, PDL-b-PHB copolymers exhibited two Tg values (Tg values were evaluated by second DSC heating runs and reported in Table 3), one in the range −5, +4°C, similar to the Tg of a PHB homopolymer (Ajellal et al., 2009a), and the second in the range −52, −48°C, comparable to the Tg of a PDL homopolymer (Olsén et al., 2013). This probably results in the immiscibility of PHB and PDL blocks (Olsén et al., 2013). In Figure 5, Tg of both PDL and PHB blocks of the polymers reported in Table 3 was plotted as a function of ε-DL mol% content. A roughly linear behavior for both glass transition temperatures is observed. Therefore, increasing the length of the PDL blocks increases the glass transition temperatures of the PHB blocks while slightly decreasing the ones of the PDL blocks.</p><!><p>Plot of Tg(second run) vs. ε-DL mol% content of polymers from Table 3 (entries 1–5).</p><!><p>The behavior of both glass transition temperatures does not match the typical Flory–Fox (Fox and Flory, 1950) behavior, which, for low Mn values,1 predicts the Tg increase with increasing Mn. For the PDL block, the Tg decreases from −48 to −52°C when Mn, calc increases from 1.6 to 12.5 kDa. For the PHB block, copolymers (Table 3, Entries 2–5) have Tg ranging from 4 to −5°C, while the homopolymer (Table 3, Entry 1) has both the lowest Tg (−8°C) and the highest Mn,calc (25.5 kDa). This behavior can be explained assuming that PDL-b-PHB copolymers are constituted by immiscible blocks arranged in separated microphases. Based on this assumption, the reduction of the volume fraction2 of the PHB phase (f PHB), which decreases from 0.9 to 0.55 with increasing ε-DL content from 5 to 25 mol%, corresponds to a size decrease of PHB-rich domains. Moreover, we can also assume that the PHB blocks have a decreasing mobility, as they are segregated in gradually decreasing space and surrounded by an immiscible phase. As it is generally accepted that Tg is inversely related to polymer flexibility and mobility, the observed increase of Tg of PHB can therefore be explained by the reduction of the PHB block mobility due to the microphase separation of PDL-b-PHB copolymers into PDL and PHB domains.</p><p>For the PDL block, with increasing volume fraction of the PDL phase (f PDL), which increases from 0.1 to 0.45 with increasing ε-DL content from 5 to 25 mol%, the size of PDL-rich domains, increases and the mobility of PDL blocks, segregated into increasing space, is assumed to increase. Consequently, with increasing f PDL, the PDL block mobility increases approaching the mobility of PDL homopolymer, and as a result, Tg of the PDL block approaches that of the homopolymer (i.e., −53°C) (Olsén et al., 2013).</p><!><p>We have reported for the first time the ring-opening copolymerization of ε-DL with rac-BBL catalyzed by an yttrium-based catalytic system. Di- and triblock copolymers, PDL-b-PHB and PDL-b-PHB-b-PDL, were synthesized by means of one-pot sequential polymerization of ε-DL and rac-BBL. In agreement with NMR observations, thermal and structural analyses of PDL-b-PHB copolymers suggested that the observed crystallinity is due to the syndiotactic PHB block. Our results demonstrated that the PHB block crystallizes even in the presence of long, amorphous, and highly flexible PDL blocks. Moreover, it has been also observed that the microphase separation of PDL-b-PHB copolymers into PHB- and PDL-rich domains has an impact on the glass transition temperature of both blocks, which is not in agreement with the Flory–Fox relationship. As observed for a thermoplastic elastomer, the physical behavior of these copolymers (i.e., stiffness/flexibility) can be tuned by changing the ε-DL/BBL ratio, thanks to the simultaneous presence of a highly flexible phase (PDL block) associated with a rigid crystalline phase (PHB block). Such tunable physical behavior makes these copolymers industrially relevant.</p><!><p>JK, VG, and CR performed the experiments. CT, JK, and VV wrote the manuscript with support from the co-authors. All authors analyzed the data, discussed the results, and commented on the manuscript.</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>

PubMed Open Access

Peroxiredoxins play a major role in protecting Trypanosoma cruzi against macrophage- and endogenously-derived peroxynitrite

There is increasing evidence that Trypanosoma cruzi antioxidant enzymes play a key immune evasion role by protecting the parasite against macrophage-derived reactive oxygen and nitrogen species. Using T. cruzi transformed to overexpress the peroxiredoxins TcCPX (T. cruzi cytosolic tryparedoxin peroxidase) and TcMPX (T. cruzi mitochondrial tryparedoxin peroxidase), we found that both cell lines readily detoxify cytotoxic and diffusible reactive oxygen and nitrogen species generated in vitro or released by activated macrophages. Parasites transformed to overexpress TcAPX (T. cruzi ascorbate-dependent haemoperoxidase) were also more resistant to H2O2 challenge, but unlike TcMPX and TcCPX overexpressing lines, the TcAPX overexpressing parasites were not resistant to peroxynitrite. Whereas isolated tryparedoxin peroxidases react rapidly (k = 7.2 \xc3\x97 105 M-1 \xc2\xb7 s-1) and reduce peroxynitrite to nitrite, our results demonstrate that both TcMPX and TcCPX peroxiredoxins also efficiently decompose exogenous- and endogenously-generated peroxynitrite in intact cells. The degree of protection provided by TcCPX against peroxynitrite challenge results in higher parasite proliferation rates, and is demonstrated by inhibition of intracellular redox-sensitive fluorescence probe oxidation, protein 3-nitrotyrosine and protein-DMPO (5,5-dimethylpyrroline-N-oxide) adduct formation. Additionally, peroxynitrite-mediated over-oxidation of the peroxidatic cysteine residue of peroxiredoxins was greatly decreased in TcCPX overexpressing cells. The protective effects generated by TcCPX and TcMPX after oxidant challenge were lost by mutation of the peroxidatic cysteine residue in both enzymes. We also observed that there is less peroxynitrite-dependent 3-nitrotyrosine formation in infective metacyclic trypomastigotes than in non-infective epimastigotes. Together with recent reports of up-regulation of antioxidant enzymes during metacyclogenesis, our results identify components of the antioxidant enzyme network of T. cruzi as virulence factors of emerging importance.

peroxiredoxins_play_a_major_role_in_protecting_trypanosoma_cruzi_against_macrophage-_and_endogenousl

INTRODUCTION<!>Parasites<!>Site-directed mutagenesis<!>In vitro metacyclogenesis of T. cruzi CL-Brener and transformed cells<!>Oxidant-sensitivity experiments<!>Macrophage-derived oxidants<!>Assessment of parasite viability<!>DHR (dihydrorhodamine) oxidation<!>Peroxynitrite-mediated oxidative modifications to proteins<!>Data analysis<!>Transformed T. cruzi overexpressing TcAPX, TcMPX or TcCPX show differential susceptibility to hydrogen peroxide and peroxynitrite<!>Overexpression of T. cruzi peroxiredoxins inhibits peroxynitrite-induced oxidative modifications<!>Enhanced resistance of TcMPX or TcCPX overexpressing cells against mitochondrial and cytosolic generation of peroxynitrite<!>Increased resistance of the infective metacyclic stage of T. cruzi against peroxynitrite<!>DISCUSSION

<p>Chagas disease affects 18-20 million people in Latin America and is caused by the kinetoplastida protozoan Trypanosoma cruzi [1]. This parasite undergoes extensive morphological and biochemical changes during its life cycle. Non-infective epimastigotes proliferate in the gut of the insect vector (triatomid hematophage arthropod), where they differentiate into metacyclic trypomastigotes, the infective form for the vertebrate host. Once in the dermal layers or conjunctival mucosa, the trypomastigotes invade host cells, mainly macrophages, where they transform into amastigotes, the infective replicative intracellular stage [2]. After several cycles of binary division, transformation to trypomastigotes and host-cell disruption occurs [3]. Subsequently, infective forms access the bloodstream and penetrate other nucleated cells such as myocardiocytes, smooth muscle cells and astrocytes [4].</p><p>To establish an infection, metacyclic trypomastigotes must invade macrophages and survive the highly oxidative conditions generated inside the phagosome. Several antioxidant enzymes, including a mitochondrial Fe-SOD (iron-containing superoxide dismutase), TcMPX (T. cruzi mitochondrial tryparedoxin peroxidase) and TcAPX (T. cruzi ascorbate-dependent haemoperoxidase), are upregulated during transformation of the insect-derived non-infective epimastigotes into the infective metacyclic trypomastigotes [5]. These biochemical changes may pre-adapt metacyclic forms with the capacity to detoxify reactive oxygen and nitrogen species generated by the macrophage during the T. cruzi-mammalian host-cell interactions [5].</p><p>Peroxide detoxification in trypanosomatids, including T. cruzi, relies on a sophisticated system of linked pathways in which the dithiol T(SH)2 (trypanothione; N1,N8-bisglutathionylspermidine), the flavoenzyme TR (trypanothione reductase) and the thioredoxin homologue tryparedoxin play central roles as the major donors of reducing equivalents derived from NADPH [6,7]. Five distinct peroxidases have been identified in T. cruzi which differ in their subcellular location and substrate specificity. Two of these peroxidases, TcGPX [T. cruzi GPX (glutathione-dependent peroxidase)] I and TcGPXII, have sequence similarity with non-selenium GPXs. TcGPXI is localized to the cytosol and the glycosome, whereas TcGPXII is localized to the endoplasmic reticulum. Transformation-mediated overexpression of both enzymes confers resistance against exogenous hydroperoxides [8,9]. TcCPX (T. cruzi cytosolic tryparedoxin peroxidase) and TcMPX belong to the 2-cysteine peroxiredoxin family, which have the capacity to detoxify H2O2, peroxynitrite [10] and small-chain organic hydroperoxides [11]. In the corresponding transformed cell lines, overexpressed TcCPX is localized to the cytosol and TcMPX is localized exclusively to mitochondria. Total T(SH)2-dependent peroxidase activity in these trypanosomes was 2.5- and 1.9-fold higher respectively than in wild-type parasites, with elevated levels of the peroxiredoxins conferring protection against exogenous peroxides (H2O2 and t-butyl-hydroperoxide), but no protective effects were found against the trypanocidal drugs nifurtimox and benznidazole, agents that are thought to undergo redox cycling within the cell [11]. A fifth peroxidase, TcAPX, is located in the endoplasmic reticulum. Overexpression of this enzyme resulted in a 5-fold increase in terms of activity and also conferred resistance against H2O2 challenge [12]. In addition, T. cruzi contains a repertoire of four Fe-SODs, which are located in different subcellular compartments, to detoxify O2•−. Mitochondrial Fe-SOD overexpression interferes with the mitochondrial O2•−-dependent signalling of T. cruzi programmed cell death induced by fresh human serum [13].</p><p>During phagocytosis of parasites, macrophage membrane-associated NADPH oxidase is activated, resulting in the generation of O2•−. Inside the phagosome, this is converted into H2O2, and through the action of transition metals, to •OH [14]. •NO, produced by iNOS (inducible NO synthase), can diffuse and rapidly react with O2•− in a diffusion-controlled reaction to form peroxynitrite, a strong oxidizing and cytotoxic effector molecule against T. cruzi [15]. Peroxynitrite (pKa = 6.8) is partially protonated at pH 7.4 to peroxynitrous acid, which, after homolysis, produces the one-electron oxidant •OH and •NO2. The major reactivity of peroxynitrite in vivo involves the oxidation of thiol groups [16]. Peroxynitrite also nitrates tyrosine residues in proteins by a two-step process, where the initial reaction is the oxidation of tyrosine (by one-electron oxidants) to form a tyrosyl radical which in turns adds •NO2 to yield 3-nitrotyrosine. In this context, the effectiveness of the parasite oxidative defence system at the onset of macrophage invasion is critical for successful infection. The reactivity of TcCPX against peroxynitrite has been evaluated in vitro. The enzyme catalytically reduces peroxynitrite to nitrite through a fast-reacting thiol group located at the peroxidatic cysteine residue (Cys52 and Cys81 in TcCPX and TcMPX respectively), thus acting as tryparedoxin/peroxynitrite oxidoreductases [10]. Although the capability of these peroxiredoxins to detoxify peroxynitrite is well defined in vitro, there is less information available on the in vivo relevance of tryparedoxin-dependent detoxification of macrophage-derived oxidants and the effect on the outcome of cell invasion.</p><p>In this present paper, we describe the parasite response to peroxynitrite challenge using T. cruzi epimastigotes and metacyclic trypomastigotes transformed to overexpress TcMPX, TcCPX or TcAPX. The results clearly demonstrate that both TcCPX and TcMPX act in vivo as peroxynitrite oxidoreductases, conferring major protection against this oxidant.</p><!><p>T. cruzi epimastigotes (CL-Brener, wild-type) were cultured at 28°C in BHI (brain heart infusion) medium [17]. Parasites overexpressing TcCPX, TcMPX or TcAPX were obtained as described previously [11,12]. The complete gene sequences of the enzymes were cloned into the trypanosomal vector pTEX-9E10 (Invitrogen) and a ligation was performed to insert a c-Myc-derived epitope (9E10) in frame at the 3′ end of the genes to produce the construct pTEX-enzyme-9E10 [11]. Transformed TcCPX, TcMPX and TcAPX cells were cultured in BHI medium containing 250 μg · ml-1 of geneticin (Sigma).</p><!><p>Oligonucleotide-directed in vitro mutagenesis was performed using the Stratagene QuikChange® mutagenesis kit, following the manufacturer's instructions. Briefly, amplifications were performed in a final volume of 50 μl, with pTEX-TcMPX-9E10 or pTEX-TcCPX-9E10 [8] used as the template DNA. The PCR parameters were as follows: 1 cycle of 95°C for 30 s, followed by 95°C for 30 s, 55°C for 1 min and 68°C for 6 min for 16 cycles. DpnI (10 units, Invitrogen) was then added to digest parental double-stranded DNA. The DpnI-digested PCR product (1 μl) was used to transform Escherichia coli XL1-Blue supercompetent cells. The primers used to generate each of the desired mutations were produced by MWG Biotech AG (Ebersberg, Germany) and are as follows: TcCPX C52A(F), 5′-GACTTCACCTTCGTCGCCCCCACAGAGATCTGC-3′; TcCPX C52A(R), 5′-GCAGATCTCTGTGGGGGCGACGAAGGTGAAGTC-3′; TcMPX C81A(F), 5′-GATTTTACCTTTGTGGCCCCCACAGAAATCACA-3′ and TcMPX C81A(R), 5′-TGTGATTTCTGTGGGGGCCAGAAAGGTAAAATC-3′. The relevant substitution sites, incorporating the required base changes, are underlined. Successful mutagenesis was confirmed by sequencing using a BigDye® terminator cycle sequencing kit (Applied Biosystems) and an ABI PRISM® 3730 automated sequencer (Applied Biosystems).</p><!><p>Epimastigotes were collected by centrifugation at 800 g for 10 min at 25°C and washed three times in 10 ml TAU (triatomine artificial urine) [190 mM NaCl, 17 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 8 mM sodium phosphate buffer (pH 6.0) and 0.035 % sodium bicarbonate] and resuspended at (3-5) × 108 cells · ml-1. After incubation at 28°C for 2 h, the parasites were transferred and diluted in TAU3AAG (TAU three amino acids plus glucose) medium [TAU (pH 6.0) supplemented with 10 mM l-proline, 50 mM sodium l-glutamate, 2 mM sodium l-aspartate and 10 mM glucose to (3-5) × 106 cells · ml-1, following incubation for 96 h at 28°C as described previously [18].</p><!><p>Epimastigotes (3 × 108 cells · ml-1) were incubated for 1 h at 28°C in DPBS (Dulbecco's PBS, pH 7.3; Sigma), and 0-800 μM H2O2 and peroxynitrite (synthesized from sodium nitrite and H2O2 in acidic medium using a quenched flow reactor as described previously [19]). Peroxynitrite (0-1000 μM) was added with vigorous vortex-mixing to parasite suspensions as a single or multiple doses of 100 μM, reaching the different final concentrations as indicated. O2•− was generated using the redox-cycling compound 50 μM DMNQ (2,3-dimethoxy-1-naphthoquinone; Sigma) (5 nM O2•−/1 × 108 cells per min), estimated from H2O2 formation by the p-hydroxyphenyl acetic acid/horseradish peroxidase assay [13,20]. AA (antimycin A) (5 μM; Sigma)-treated epimastigotes were used to elicit mitochondrial-derived O2•− generation [21,22]. •NO fluxes were generated by NO donors, 0-1 mM spermine NONOate (diazeniumdiolate, t½ ∼45 min; Alexis) and 0-2 mM NOC-18 (t½ ∼1080 min, Alexis), and quantified following the oxidation of oxyhaemoglobin to methaemoglobin at 577 nm (ε577 = 11 mM-1 · cm-1) [23]. Parasites were incubated in the presence of the different oxidant-generating systems for 1 h at 28°C in DPBS (pH 7.3).</p><!><p>The murine macrophage cell line J774A.1 was cultured in DMEM (Dulbecco's modified Eagle's medium; Sigma) at 37°C in a 5 % CO2 atmosphere. Production of •NO by iNOS was triggered by pre-incubating macrophages with 300-500 units · ml-1 IFNγ (interferon γ; Calbiochem) and 3 μg · ml-1 lipopolysaccharide (Sigma) for 5 h at 37°C for maximal •NO production [15]. O2•− production by NADPH oxidase in macrophages was triggered by the addition of 2 μg · ml-1 PMA (Sigma) as described previously [15]. Macrophage/T. cruzi co-culture experiments were performed at 37°C by direct contact of parasites with the macrophage monolayer, allowing cell-to-cell interactions. Cells were then harvested and assayed for [3H]thymidine (American Radiolabeled Chemicals, St Louis, MO, U.S.A.) incorporation [17] and RH (rhodamine) 123 fluorescence [15]. After iNOS induction, 2 μg · ml-1 PMA was added to macrophages 5 min before parasite addition, and co-culture experiments were performed for 90 min at 37°C.</p><!><p>After treatment, parasite viability was evaluated using the [3H]thymidine incorporation assay as described previously [17]. Briefly, following parasite exposure to the different oxidative systems, an aliquot containing 5 × 106 cells was incubated overnight at 28°C in BHI medium containing 1 μCi [3H]thymidine. When transformed parasites were used, BHI medium was supplemented with 250 μg · ml-1 of geneticin. Results are the percentage of [3H]thymidine incorporation compared with the control (no oxidant addition) sample for each cell line (wild-type and TcAPX, TcCPX or TcMPX overexpressing cells). The IC50 values for H2O2 and peroxynitrite were calculated to be the oxidant concentration that produced a 50 % inhibition in the [3H]thymidine incorporation assay compared with the control sample.</p><!><p>Parasites (1 × 109 cells · ml-1) were incubated for 30 min at 28°C in DPBS containing 50 μM DHR (Molecular Probes). After incubation, cells were centrifuged at 800 g for 10 min at 25°C and washed twice in DPBS in order to eliminate non-incorporated DHR. Detection of intracellular RH 123, the oxidation product of DHR, was performed after exposure to the different experimental conditions (oxidant and macrophage-derived oxidant treatment) using black 96-well plates and a fluorescence plate reader at 28°C (Fluostar; BMG Labtech, Offenburg, Germany), with filters set at λex = 485 nm and λem = 520 nm. In order to evaluate membrane integrity and parasite morphology after peroxynitrite addition, cells were pre-loaded for 30 min with 10 μM 6-CFDA (6-carboxyfluorescein diacetate; Molecular Probes) and washed three times in DPBS. After exposure to 250 μM peroxynitrite, parasites were examined by fluorescence microscopy (Nikon Eclipse TE-200 inverted microscope) and digital images of treated parasites were recorded.</p><!><p>Parasites were exposed to 0-1 mM peroxynitrite as described above. After treatment, cells were centrifuged at 800 g for 10 min at 25°C, resuspended in 250 μl lysis buffer [10 mM Tris/HCl (pH 8.0), 1 mM EDTA and 0.5 % Triton X-100] and incubated on ice for 15 min. Cell extracts were centrifuged at 13 000 g for 30 min at 4°C, and loading buffer [30 mM Tris/HCl (pH 6.6), 1 % SDS and 5 % (v/v) glycerol] was added to the supernatants. Proteins (50 μg) were resolved by SDS/PAGE (13 % gels), followed by Western blotting on to nitrocellulose membranes. After protein transfer, membranes were stained with Ponceau-S solution (Applichem, Darmstadt, Germany) to confirm equal protein loading. The membranes were blocked using 5 % (w/v) BSA and 0.1 % Tween 20 (Sigma) in TBS (Tris-buffered saline) [25 mM Tris/HCl (pH 7.4), 140 mM NaCl and 3 mM KCl] for 1 h at 25°C. The membranes were then probed with rabbit anti-(nitrotyrosine) serum (1:2000 dilution) [24], rabbit anti-(sulfonic peroxiredoxin) antibody (1:2000 dilution; Lab Frontier, Seoul, Korea) that recognizes cysteine sulfinic acid (Cys-SO2H) and sulfonic acid (Cys-SO3H) from human peroxiredoxin I to IV [25] and monoclonal mouse anti-c-Myc antibody (9E10, 1:300 dilution; Santa Cruz Biotechnology) for 1 h at 25°C. Immunoreactive proteins were detected using the Immun-Star™ chemiluminescence kit (Bio-Rad). Immunodetection of DMPO (5,5-dimethylpyrroline-N-oxide)-nitrone protein adducts on parasite samples was performed using a rabbit anti-(DMPO-nitrone) serum which binds to the one-electron oxidation product of the initial DMPO-nitroxyl protein spin adduct [26]. Parasites (1 × 109 cells · ml-1) were pre-incubated with DMPO (100 mM) in DPBS for 30 min at 28°C. After incubation, parasites were collected by centrifugation at 800 g for 10 min at 25°C and resuspended at a concentration of 3 × 108 cells · ml-1 in DPBS. After peroxynitrite treatment, parasite extracts were prepared as above and protein extracts (50 μg) were resolved by SDS/PAGE (12 % gels), blotted on to nitrocellulose and probed with anti-(DMPO-nitrone) serum (1:2000 dilution) as described previously [26]. Immunoreactive proteins were detected using the Immun-Star™ chemiluminescence kit (Bio-Rad).</p><!><p>All results are means ± S.D. unless otherwise stated. For comparison between two groups, the Student's t test was performed. ANOVA was performed for comparison of more than two groups. Post-hoc analysis was performed using a LSD (least significant difference) test. P < 0.05 was considered significant. All experiments were typically repeated on separate days (n ≥ 3).</p><!><p>In order to evaluate the susceptibility of the genetically transformed T. cruzi parasite lines to H2O2, cells were exposed to different concentrations of oxidant for 1 h and parasite viability was assayed. T. cruzi epimastigotes overexpressing TcAPX showed a 2-fold increase in their IC50 value compared with wild-type cells, whereas cells with elevated levels of TcMPX and TcCPX were 50 % more resistant to oxidant exposure (Table 1). Expression of the appropriate peroxidase in each transformed cell line was confirmed by the detection of the c-Myc tag epitope in parasite extracts (Figure 1).</p><p>Unlike with H2O2, TcAPX overexpression did not confer protection against peroxynitrite (Figure 2A), with an IC50 value comparable with wild-type cells (250 ± 25 and 280 ± 12 μM respectively; Table 1). In contrast, overexpression of TcMPX or TcCPX conferred resistance against peroxynitrite when added exogenously as a single dose (Table 1). The dose-response curve showed a biphasic profile for both cell lines overexpressing the peroxiredoxins (Figures 2B and 2C), suggesting there is a marked resistance at the lower concentration range tested, particularly for cells with elevated levels of TcCPX (Figure 2C). The major difference between wild-type and T. cruzi overexpressing TcCPX was observed at 400 μM peroxynitrite (90 % compared with 20 % inhibition of [3H]thymidine incorporation respectively). At higher peroxynitrite doses (≥500 μM), proliferation was inhibited in all cell lines. When these experiments were extended to investigate cellular responses to the consecutive addition of sub-lethal levels of peroxynitrite (100 μM) at 1 min intervals, parasites overexpressing TcCPX or TcMPX displayed remarkable resistance, greater than the resistance observed during the addition of a single dose (Figure 2D). Peroxiredoxins depend on a trypanothione redox cascade and, ultimately, on NADPH arising from the pentose phosphate pathway, which is stimulated after oxidant addition [13] in order to regenerate the active enzyme form. Therefore the time between each challenge allows TcCPX and TcMPX to be in the reduced-active state prior to the next oxidant assault.</p><p>Examination of cell morphology after peroxynitrite challenge (250 μM) revealed that wild-type cells lost their cellular structure, whereas parasites overexpressing TcCPX appeared to be unaffected (Figure 2E). The activity of both trypanosomal peroxiredoxins is dependent on a conserved peroxidatic cysteine residue at positions 52 and 81 in TcCPX and TcMPX respectively [10]. To investigate whether these residues play a key role in the metabolism of peroxynitrite, they were mutated to alanine residues, in order to yield inactive enzymes. The mutated enzymes were expressed at similar levels to that shown in Figure 1 for the wild-type form (results not shown). Parasites expressing TcCPX C52A and TcMPX C81A failed to confer peroxynitrite resistance, demonstrating the catalytic nature of the protection afforded by elevated levels of wild-type peroxiredoxins (inset, Figure 2C).</p><!><p>By metabolising peroxynitrite, peroxiredoxins play a key role in minimising the formation of peroxynitrite-derived radicals such as •OH, •NO2 and CO3•− [10]. To determine whether parasites overexpressing TcCPX or TcMPX have decreased levels of peroxynitrite-derived radicals, cells were pre-loaded with the redox sensitive agent DHR (which is oxidized to RH 123, a fluorescent product) and then challenged with the oxidant [15]. Overexpression of either TcMPX or TcCPX resulted in partial inhibition (∼50 %) of intracellular DHR oxidation in a dose-dependent manner compared with wild-type cells (Figure 3A). In contrast, T. cruzi overexpressing TcAPX displayed a fluorescence pattern similar to controls (results not shown).</p><p>Peroxynitrite-dependent oxidative modification of proteins was analysed by immunospin-trapping using an anti-(DMPO-nitrone) serum [27]. Intense immunostaining was observed in CL-Brener epimastigotes after peroxynitrite treatment, whereas only minimal staining was observed in TcCPX or TcMPX overexpressing cells (Figure 3B). Moreover, basal levels of modified proteins were higher in the control than in the overexpressing cells, suggesting that there are lower steady-state levels of endogenous oxidants in the overexpressing cells. The inhibition of peroxynitrite-dependent protein modifications in cells with elevated TcCPX was further confirmed by blotting for tyrosine nitration (Figure 3C).</p><p>Finally, peroxynitrite-mediated two-electron thiol oxidation processes lead to the formation of sulfenic acid, which forms an intersubunit disulfide bond at the expense of the resolving cysteine residue of the enzyme, which can then be reduced at the expense of tryparedoxin. Under conditions of excess oxidant, the peroxiredoxin catalytic thiol present in the sulfenic state can be over-oxidized to sulfinic and sulfonic acid derivatives, before reacting with the resolving cysteine residue [28,29]. Detection of sulfinic and/or sulfonic acid at the active site of TcCPX was performed immunochemically after exposure to different peroxynitrite doses [25]. In wild-type parasites exposed to peroxynitrite, over-oxidized peroxiredoxins were detected, including some staining under basal conditions. Importantly, we could not detect over-oxidation of peroxiredoxins in cells overexpressing TcCPX incubated with up to 1 mM peroxynitrite, with only minor staining at higher concentrations (2 mM) (Figure 3D).</p><!><p>To evaluate the protection conferred by TcMPX or TcCPX overexpression against intracellularly-formed peroxynitrite, the viability of parasites treated with compounds known to stimulate cytosolic (DMNQ) or mitochondrial (AA) O2•− formation in the presence of different •NO fluxes (in order to have defined ratios of both oxidants) was examined as described in the Experimental section [13,21]. For the mitochondrial O2•− generating system, AA alone was toxic to wild-type cells, as a result of intramitochondrial O2•− and/or subsequent intracellular H2O2 formation [30,31], a feature that was decreased in both TcCPX and TcMPX overexpressing trypanosomes. When AA-treated cells were also exposed to •NO fluxes (NOC-18, 0-0.25 μM •NO · min-1), overexpression of TcMPX was found to have significantly improved [3H]thymidine incorporation with respect to TcCPX and wild-type cells, indicating that TcMPX minimizes the toxic growth effects of mitochondrially-generated peroxynitrite (Figures 4A and 4B). We then assayed for the production of peroxynitrite-derived radicals in intact cells loaded with DHR. In parasites overexpressing TcMPX, and to a minor extent TcCPX (results not shown), RH 123 formation was partially inhibited, giving further support to the idea that TcMPX plays an important role in detoxifying peroxynitrite generated within mitochondria (Figure 4C). When reciprocal experiments were performed using physiologically relevant cytosolic O2•− fluxes (5 nM O2•−/108 cells · min-1), a similar resistance was observed using trypanosomes expressing elevated levels of TcCPX (see Supplementary Figure 1 at http://www.BiochemJ.org/bj/410/bj4100359add.htm).</p><p>Finally, and of most relevance, epimastigotes were exposed to activated macrophages that produced O2•−, •NO or both, in a cell-to-cell contact model as described previously [15]. Simultaneous macrophage generation of O2•− and •NO inhibited growth of wild-type and TcAPX overexpressing parasites by equivalent levels (Table 2) [15]. However, overexpression of TcMPX conferred partial protection against growth inhibition, and TcCPX overexpression completely protected cells against macrophage-derived peroxynitrite (Table 2). Consistent with these results, intracellular oxidation of DHR in TcCPX or TcMPX overexpressing cells exposed to macrophage-derived O2•− and •NO was greatly decreased compared with wild-type (results not shown).</p><!><p>A recent proteomic study reported that components of the oxidative defence system appeared to be up-regulated during metacyclogenesis [5]. Using wild-type epimastigotes and chemically-differentiated wild-type metacyclic trypomastigotes, we evaluated protein 3-nitrotyrosine formation after peroxynitrite challenge. As shown in Figure 5(A), protein nitrotyrosine in wild-type metacyclic trypomastigotes was detected at a significantly decreased level than that observed in the epimastigote stage when treated with equal amounts of peroxynitrite. These results support, from a biochemical standpoint, the previously observed increase in the levels of antioxidant enzymes during the infective stage [5]. Parasites transformed to express elevated levels of TcCPX and TcMPX were also subjected to chemical differentiation to the infective metacyclic stage. The preservation of enzyme expression was corroborated by detection of the c-Myc-tagged enzyme by Western blot analysis (Figure 5B). Peroxynitrite treatment of the metacyclic parasites overexpressing TcCPX showed inhibition of protein 3-nitrotyrosine formation (Figure 5C), and the redox-active cysteine in the peroxiredoxin displayed a decreased level of oxidation to cysteine sulfinic and/or sulfonic acid (Figure 5D), supporting the key role of this enzyme in protection against peroxynitrite.</p><!><p>Macrophages are among the first cells to be invaded by T. cruzi and, as a consequence, parasites need to cope with macrophage-derived oxidants. Since the discovery of peroxiredoxins in trypanosomatids, H2O2 and small-chain organic hydroperoxides have been assigned as the preferential biological substrates for these enzymes [11,32-34]. A fast reaction constant of TcCPX with peroxynitrite has been reported previously, with a value of 7.2 × 105 M-1 · s-1 at pH 7.4. Furthermore, in the presence of tryparedoxin, TcCPX was shown to catalytically decompose peroxynitrite [10]. Given the relevance of peroxynitrite production as an effector cytotoxic molecule against T. cruzi [15,19,35] and the in vitro kinetic considerations, we decided to evaluate the importance of TcCPX and TcMPX against peroxynitrite challenge in the intact cell.</p><p>To evaluate the protective effects of these enzymes against biologically-relevant oxidants, we conducted experiments using epimastigotes that overexpress TcCPX, TcMPX or TcAPX. Using an acute challenge of H2O2 (1 h incubation), we showed that TcAPX confers higher protection than TcCPX and TcMPX, as was demonstrated previously [11,12]. IC50 values for wild-type parasites and TcAPX overexpressing cells against peroxynitrite were not different (Table 1), suggesting that TcAPX does not play a significant role in the detoxification of exogenously-added peroxynitrite. Notably, overexpression of TcCPX or TcMPX conferred a high level of protection on epimastigotes against a broad range of peroxynitrite concentrations (0-200 μM and 0-400 μM for TcMPX and TcCPX overexpressing cells respectively) (Figures 2B and 2C). The observed protection was lost when the peroxidatic cysteine mutants of TcCPX and TcMPX were used (inset, Figure 2C). Together, our results demonstrate that peroxiredoxins efficiently detoxify peroxynitrite in cells and protect the parasites from the deleterious actions of this oxidant [36]. At higher peroxynitrite concentrations, the effect was less pronounced. This suggests that either direct inactivation of TcMPX and TcCPX by peroxynitrite occurs, owing to over-oxidation of the enzyme thiol group, or acute depletion of reducing equivalents (NADPH) occurs, ultimately derived from the pentose phosphate pathway, which would jeopardize the catalytic efficiency of the antioxidant system. Further experiments, involving the sequential addition of low concentrations of peroxynitrite, showed an increase in the total peroxynitrite concentration that the enzymes have the capacity to overcome (0-1 mM for TcMPX or TcCPX overexpressing cells; Figure 2D). This strongly suggests that TcCPX and TcMPX catalytically decompose peroxynitrite in our in vivo system. Also, these results eliminate the suggestion of inactivation of the enzymes at peroxynitrite concentrations less than 1 mM (see below).</p><p>The cytotoxic effects of peroxynitrite against T. cruzi result from direct reactions with critical parasite targets, such as essential enzymatic thiol residues, and from the actions of the induced radicals •OH, CO3•− and •NO2 [15,19,35]. We therefore explored radical-mediated damage by using probes and evaluating modifications of endogenous constituents known to react with these radicals. TcCPX or TcMPX overexpressing cells effectively decreased peroxynitrite-dependent intracellular DHR oxidation, protein radical formation (as evaluated by DMPO immuno-spin trapping) and protein 3-nitrotyrosine formation (Figures 3A-3C). The relatively decreased protection afforded by peroxiredoxin overexpression on DHR oxidation compared with the oxidation/nitration of the other tested molecular targets, as well as in cell viability, is explained by the fact that DHR readily diffuses out of the cell, and therefore some of the peroxynitrite-dependent redox chemistry on DHR occurs extracellularly. Additionally, dose-dependent over-oxidation of peroxiredoxin(s) to sulfinic and/or sulfonic acid was evident after peroxynitrite treatment of wild-type cells, indicating that basal levels of TcCPX in the epimastigote stage are not sufficient to detoxify peroxynitrite at the concentrations tested (Figure 3D). In cells with elevated TcCPX, over-oxidation of the enzyme was not detected at concentrations below 2 mM peroxynitrite, an observation which supports our suggestion that, in overexpressing cells, the effect of peroxynitrite does not result from enzyme inactivation at these concentrations.</p><p>In living systems, peroxynitrite arises from physiologically-generated •NO and O2•− fluxes at different ratios, and oxidation/nitration yields are responsive to peroxynitrite formation rates in spite of the flux ratio [37]. T. cruzi can be challenged by high levels of mammalian cell-derived •NO and of O2•− which can be formed either by activation of macrophage NADPH oxidase or by the •NO-dependent inhibition of the mitochondrial respiratory chain in the target cell [30,38]. Therefore peroxynitrite can also be formed in different cellular compartments (such as cytosol and mitochondria). In order to assess the antioxidant capabilities of TcCPX and TcMPX in their subcellular locations, we used a system which allowed site-directed generation of O2•− radicals in the presence of •NO fluxes. The inhibition in proliferation of TcCPX overexpressing and wild-type cells exposed to •NO fluxes alone were similar, while TcMPX overexpressing cells exhibited a greater level of proliferation (≃50 % compared with 20 % inhibition respectively) (Figure 4A). •NO is a known inhibitor of mitochondrial cytochrome c oxidase. This leads to an increase in mitochondrial O2•− production and consequently to peroxynitrite formation in the vicinity of mitochondria, which could explain the effect of TcMPX overexpression on parasite proliferation. When O2•− is generated in the cytosol by the redox cycling agent DMNQ, overexpression of TcCPX affords maximal protection, whereas TcMPX overexpressing cells behaved similarly to wild-type cells (results not shown). The presence of the complex III respiratory chain inhibitor AA has profound effects on parasite proliferation, with 80 % and 40 % inhibition of thymidine incorporation in wild-type and overexpressing cells respectively (Figure 4B). In the presence of •NO fluxes, enhanced levels of TcMPX sustained proliferation capabilities and inhibited peroxynitrite-dependent DHR oxidation, whereas TcCPX overexpressing cells displayed a progressive decrease in proliferation (Figures 4B and 4C). Together, these results identify the importance of the compartmentalized antioxidant defences in the parasite against localized oxidant formation.</p><p>In Leishmania chagasi, overexpression of cytosolic peroxiredoxin 1 partially improved parasite survival within naive macrophages [33]. Maximal peroxynitrite-dependent trypanocidal activity of macrophages, including human macrophages [39], is observed under iNOS induction and O2•− generation by NADPH oxidase after parasite engulfment [15]. We therefore tested the hypothesis that overexpression of TcCPX would render the parasites resistant to macrophage-derived peroxynitrite in co-culture experiments, as evaluated by proliferation and intra-parasite DHR oxidation assays. Under maximal macrophage stimulation conditions (production of both •NO and O2•−), a 50 % inhibition in parasite proliferation was found in the wild-type and TcAPX overexpressing cells, and 30 % in cells with elevated TcMPX levels. No cytotoxic effects were observed in cells overexpressing TcCPX, with the proliferation rates being the same as the untreated controls (Table 2). These results correlate well with the DHR oxidation assays, where maximal protection was observed in the TcCPX overexpressing cells (results not shown). Although levels of •NO achieved by activated human macrophages are smaller than those observed with murine macrophages, several reports have indicated that the trypanocidal activity depends on •NO production [39,40]. Finally, in order to validate our findings in the infective metacyclic stage of the parasite, we chemically transformed epimastigotes (wild-type, TcCPX or TcMPX overexpressing cells) and evaluated peroxynitrite-dependent protein oxidative modifications. In wild-type parasites, metacyclic trypomastigotes contained less protein 3-nitrotyrosine than epimastigotes, in agreement with the expected up-regulation of antioxidant defences in the infective stage (Figure 5A) [5]. We successfully differentiated parasites overexpressing TcMPX or TcCPX to the infective stage, while retaining the overexpression phenotype (Figure 5B). Overexpression of TcCPX in the infective metacyclic stage prevented peroxynitrite-dependent protein nitrotyrosine formation and over oxidation of peroxiredoxins (Figures 5C and 5D).</p><p>Peroxynitrite production inside the macrophage phagosome have been estimated to be sufficient to kill internalized trypanosomes [15,41]. In line with this, we anticipate that overexpression of TcCPX will render T. cruzi resistant to macrophage killing and therefore increase its virulence. In this present paper, we have extensively analysed the role of tryparedoxin peroxidases in T. cruzi epimastigotes and at the metacyclic stage by exposure to diverse peroxynitrite-generating systems. We provide evidence that supports the key role of parasite tryparedoxin peroxidases in defence against macrophage-derived and even endogenously produced peroxynitrite. The results we present, together with enhanced antioxidant defence observed in the infective metacyclic stage of the parasite, place the antioxidant network of T. cruzi as an emerging virulence factor.</p>

PubMed Author Manuscript

Oxygen transfer in electrophilic epoxidation probed by ¹⁷O NMR: differentiating between oxidants and role of spectator metal oxo

Peroxide compounds are used both in laboratory and industrial processes for the electrophilic epoxidation of olefins. Using NMR-spectroscopy, we investigate why certain peroxides engage in this type of reaction while others require activation by metal catalysts, e.g. methyltrioxorhenium (MTO). More precisely, an analysis of 17 O NMR chemical shift and quadrupolar coupling parameters provides insights into the relative energy of specific frontier molecular orbitals relevant for reactivity. For organic peroxides or H 2 O 2 a large deshielding is indicative of an energetically high-lying lone-pair on oxygen in combination with a low-lying s*(O-O) orbital. This feature is particularly pronounced in species that engage in electrophilic epoxidation, such as peracids or dimethyldioxirane (DMDO), and much less pronounced in unreactive peroxides such as H 2 O 2 and ROOH, which can however be activated by transition-metal catalysts. In fact, for the proposed active peroxo species in MTO-catalyzed electrophilic epoxidation with H 2 O 2 an analysis of the 17 O NMR chemical shift highlights specific p-and d-type orbital interactions between the so-called metal spectator oxo and the peroxo moieties that raise the energy of the highlying lone-pair on oxygen, thus increasing the reactivity of the peroxo species.

oxygen_transfer_in_electrophilic_epoxidation_probed_by_¹⁷o_nmr:_differentiating_between_o

Introduction<!>CSTs of non-metal-based peroxides<!>Orientation of the CSTs<!>Orbital analysis of the CSTs<!>Quadrupolar coupling parameters<!>MTO-catalyzed epoxidation<!>CST of peroxo intermediates<!>Orbital analysis of the CSTs<!>Energetic considerations in oxygen-transfer reactions<!>Conclusions<!>Epilogue<!>Conflicts of interest

<p>Electrophilic epoxidations are at the core of numerous processes, ranging from the industrial synthesis of propylene oxide to enzymatic oxygenase reactions. [1][2][3][4][5][6][7][8] In organic synthesis, this ubiquitous transformation is commonly achieved by using stoichiometric epoxidation agents such as meta-chloroperoxybenzoic acid (mCPBA), dimethyldioxirane (DMDO), or oxaziridines. 9-14 H 2 O 2 and ROOH can also be used for epoxidation but they require a catalyst. The most commonly used catalysts are (i) organorhenium trioxides (especially methyltrioxorhenium, MTO) [15][16][17][18][19][20][21] and group 6 metal dioxo compounds [22][23][24] (Fig. 1) or (ii) early transition-metal alkoxides (e.g. Ti and V), [25][26][27][28][29] that involve peroxo-species as key reaction intermediates.</p><p>While the reactivity of oxidizing agents such as mCPBA or DMDO towards olens is well established and exploited synthetically, the origin of their reactivity towards C-C double bonds has not been studied in detail. This question is particularly apparent when considering that other peroxides, such as H 2 O 2 or tBuOOH, are usually not reactive towards olens, unless combined with metal catalysts.</p><p>Recent work has shown that analysis of the 13 C NMR chemical shi tensor (CST) of metal alkyl compounds can give valuable insights into the electronic structure and the reactivity of ubiquitous reaction intermediates in organometallic chemistry and homogeneous catalysis. [30][31][32][33][34][35][36][37] Considering the large chemical shi window of 17 O nuclei (around 1200 ppm), we reasoned that analysis of the 17 O NMR chemical shi tensor of oxidants would allow for a detailed understanding of the electronic structure and associated reactivity of these molecules. In addition, the quadrupolar coupling constant of 17 O (nuclear spin I ¼ 5/2) can provide valuable information on the charge distribution around the nucleus. [38][39][40][41][42] In fact, 17 O NMR spectroscopy has been used to identify and study peroxo species as well as related compounds containing O-N bonds. [43][44][45][46] The isotropic chemical shi d iso (eqn (1)) and the three principal components (d 11 $ d 22 $ d 33 ) of the CST contain a considerable amount of information on the electronic structure of NMR active nuclei. The corresponding shielding values (s, eqn (2)), can be decomposed computationally into diamagnetic (s dia ) and paramagnetic contributions, which also include contributions from spin-orbit coupling (s para+SO , eqn (3)). While the diamagnetic contributions, which arise from a molecule's electronic ground state, lead to shielding and are usually similar for all nuclei of a given kind independent of their chemical environment, the paramagnetic contributions, which give mostly rise to deshielding, originate from magnetically induced coupling of excited states to the ground state, by action of the angular momentum operator Li , as described in a 2nd order perturbation approach in eqn (4). 47</p><p>According to eqn (4), deshielding of a nucleus is expected along the direction i, if an occupied orbital on this nucleus can be "superimposed" onto a vacant orbital on the same nucleus rotated by 90 along the axis i (Fig. 2). Since the extent of deshielding increases with a decreasing energy gap between the two orbitals, the paramagnetic contribution to shielding is most strongly affected by frontier molecular orbitals (FMOs)energetically high-lying occupied and low-lying vacant orbitals.</p><p>In this work, we make use of chemical shi to evidence specic high-lying occupied and low-lying vacant orbitals in the aforementioned oxidizing agents, thereby probing their electronic structure and connection to the observed reactivities.</p><p>As 17 O is a quadrupolar nucleus (I ¼ 5/2), the quadrupolar coupling, which typically complicates the interpretation of spectra by line broadening, holds information about the distribution of charges around the nucleus. The quadrupolar interaction is proportional to the electric eld gradient (EFG) tensor € V (eqn (5)), where e is the electron charge, Q is the quadrupolar moment of 17 O, ħ is the reduced Planck constant (ħ ¼ h/2p), Î is the nuclear spin operator and I the nuclear spin quantum number.</p><p>V is a traceless second rank tensor (V 11 + V 22 + V 33 ¼ 0) where we follow the notation |V 33 | $ |V 22 | $ |V 11 | for the three principal components. The EFG tensor can be described by two independent variablesusually the largest principal component (V 33 ) and the asymmetry parameter h Q (eqn ( 6)).</p><p>The quadrupolar coupling constant C Q arises from the interaction of the quadrupole moment of 17 O (I ¼ 5/2) with the EFG and is proportional to V 33 (eqn (7)). 38 Since the electric quadrupole moment Q of the 17 O nucleus is negative, V 33 and C Q have opposing signs.</p><p>V 33 and hence C Q are indicative of how symmetric the EFG and thus the charge distribution around the nucleus is. This has been a valuable tool to assess local symmetry around quadrupolar nuclei (e.g. 17 O, 27 Al, 45 Sc). 38,41,42,46,[48][49][50][51]</p><!><p>We calculated the chemical shi tensors (CSTs) of selected peroxides relevant to epoxidation reactions, as well as associated reduced compounds. We chose to investigate DMDO and mCPBA, two compounds showing activity towards electrophilic epoxidation, as well as H 2 O 2 and tBuOOH that do not participate in this transformation, unless activated by metal catalysts. In order to benchmark our calculations, we also experimentally determined the 17 O NMR chemical shi tensors of H 2 O 2 52 and acetone by solid-state 17 O NMR spectroscopy [53][54][55] (see ESI † for experimental details).</p><p>The measured and calculated chemical shis are given in Table 1. Generally, a good agreement between calculated and experimental data (when available) is obtained. The oxygen atoms of the unsymmetric peroxides (tBuOOH and mCPBA) are labelled (O), for the oxygen bound to a carbon atom and (OH) for the oxygen connected to the hydrogen.</p><p>A comparison of the isotropic chemical shis given in Table 1 reveals that all peroxide species (H 2 O 2 , tBuOOH, mCPBA, and DMDO) show signicantly more deshielded d iso values in comparison with H 2 O, tBuOH and mCBA, albeit less deshielded than carbonyl oxygens (e.g. mCBA (C]O) and acetone). While the differences among the various peroxides are less pronounced, the chemical shi (d iso ) of the oxygen atom which is transferred during epoxidation reactions is more deshielded for DMDO and mCPBA (OH) as compared to H 2 O 2 and tBuOOH (OH). A closer inspection of the principal components of the chemical shi tensor reveals that this is mostly due to the d 11 component of the CST which is signicantly more deshielded in DMDO and the OH-oxygen of mCPBA (636 and 540 ppm) than in H 2 O 2 and tBuOOH (364 and 386 ppm). This highly deshielded component is accompanied by a signicantly larger span U of the CST in the former compounds.</p><!><p>In order to further understand these observed trends, we investigated the orientation of the 17</p><!><p>We decided to further elucidate the origin of deshielding in the individual components of the CST in a Natural Chemical Shielding (NCS) analysis. 47,48,[60][61][62][63][64][65] This analysis allows for a decomposition of the s ii /d ii components into diamagnetic (s dia ) and paramagnetic/spin-orbit (s para+SO) contributions (eqn (3)). The paramagnetic term can then be further decomposed into contributions of the various NLMOs (bonds and lone pairs) surrounding the nucleus of interest. Due to the orbital energy difference in the denominator of eqn ( 4), the orbitals contributing most strongly to s para+SO are the frontier molecular orbitals (FMOs) of the molecule with a non-vanishing coefficient on the investigated nucleus.</p><p>Since the largest differences in the CST of the investigated peroxides originate from the s 11 /d 11 component of the CST, its orbital analysis will be further discussed (see Fig. S3 and S4 † for other components).</p><p>The NCS analysis of the s 11 component of the various compounds (Fig. 4b) reveals, that the diamagnetic contributions to this component are essentially invariant throughout the whole series of compounds. The differences in d 11 result from the paramagnetic contributions, which are mainly affected by four different Natural Localized Molecular Orbitals (NLMOs). These correspond to two lone-pairs on oxygen (denoted as LP 'p' and LP 's'), as well as the two s-bonding orbitals (denoted as s(O-R) and s(O-O)). These four NLMOs are visualized for the case of hydrogen peroxide in Fig. 4a; for the other compounds they are shown in Fig. S11-S13. † Notably, the dominant contribution to the deshielding of s 11 /d 11 originates from the LP 'p' on oxygen in all cases given in Fig. 4, with the exception of mCPBA (O) (vide infra). The observation of the large deshielding perpendicular to the O-O axis originating from this lone pair indicates the presence of a lowlying vacant orbital, oriented perpendicular to both the lonepair and the direction of s 11 /d 11 ; i.e. oriented along the O-O bond (eqn ( 4)). This vacant orbital corresponds to s*(O-O), the lowest unoccupied molecular orbital with contribution of oxygen in all of the investigated peroxides. As the extent of</p><p>b Note that the quadrupolar nature of 17 O can lead to inaccuracies in the determination of the chemical shi tensorssee ESI for a more detailed discussion of the experimental measurements. Additionally, the presence of solvents can also signicantly impact the chemical shi. The importance of this "backdonation" was further explored by investigating transition state geometries where the olen is perpendicular to the oxygen lone pair and hence co-planar with the dioxirane moiety in DMDO or the carbonyl group in mCPBA. The corresponding transition state (2nd order saddle point) energies for the epoxidation where the rotation around the reaction coordinate was restricted were found at 32.7 and 29.0 kcal mol À1 for DMDO and mCPBA, respectively. The</p><!><p>To complement the analysis of the 17 O NMR parameters we calculated the quadrupolar coupling parameters of the aforementioned peroxides. The respective 17 O C Q and h Q -values are reported in Table 2.</p><p>For all investigated peroxide oxygens, the absolute magnitude of the quadrupolar coupling constant C Q (and V 33 accordingly) is signicantly larger than for water. The orientation of the EFG tensor is similar in all of these peroxides, with the most positive component oriented along the O-O bond, and the most negative component oriented in the direction of LP 'p' (selected examples in Fig. 7a-c, see Fig. S14 † for other compounds). This orientation is hence indicative of a region of high electron density in the direction of LP 'p', and a region of depleted electron density in the direction of the O-O bond. The reversed sign of C Q observed for DMDO in contrast to other peroxides is due to the denition of V 33 , which always corresponds to the EFG tensor component with the largest absolute value (indicated by a red arrow in Fig. 7). While the EFG tensor is similar in all peroxides, the negative EFG tensor component perpendicular to the O-O bond is slightly larger for DMDO by absolute value as compared to the positive EFG tensor component along the O-O bond. For the other investigated peroxides, the situation is reversed, leading to a change in sign of V 33 and hence of C Q . The large C Q value for peroxides in combination with the specic orientation of the EFG tensor is consistent with the presence of a high-lying occupied orbital (LP 'p') oriented perpendicular to a low-lying vacant s*(O-O) orbital in these species. This observation parallels what is seen from the NCS analysis of the d 11 component of the CST.</p><!><p>As both H 2 O 2 and tBuOOH are inactive in electrophilic epoxidation but are rendered active in the presence of transitionmetal catalysts, we investigated the process of activation and the properties of the proposed active species. We chose methyltrioxorhenium (MTO) as a prototypical catalyst because of its high efficiency in olen epoxidation with H 2 O 2 (Fig. 1b), and the existence of detailed mechanistic studies (isolation of reaction intermediates, measurements of 17 O NMR parameters and computational studies). 15,[68][69][70][71] We hence calculated the chemical shi tensors of MTO (Table 3 and Fig. 8c) and of the water and pyridine adducts of the corresponding mono-and bisperoxides (Table 3). The bisperoxo pyridine adduct will be discussed in more detail; the tensors are shown in Fig. 8a and b. [17][18][19]72 The CSTs and NCS analyses for other intermediates are provided in the ESI † (Table S1, Fig. S1 and Tables S2-S4, Fig. S8-S10, † respectively) and further commented below for comparison.</p><!><p>The measured and calculated chemical shis of bis-and monoperoxo intermediates of the MTO-catalyzed olen epoxidation are given in Table 3. Both the isotropic chemical shi (d iso ) and the three principal components of the CST are signicantly more deshielded in the metal-peroxide compound by comparison with H 2 O 2 (Table 3, where cis/trans denotes the peroxo oxygen pseudo-cis/trans with respect to the methyl substituent). This observation suggests a change in the electronic structure of the peroxide oxygen atoms, which is likely connected to their increased reactivity towards olens. Note the signicantly larger deshielding found for the oxo-ligands as typically observed for metal-oxo compounds. 73,74 The CSTs of the peroxo oxygen atoms have similar orientations as shown in Fig. 8 (and Fig. S1 † for the water adducts and the monoperoxo species). For both peroxo oxygens, s 11 /d 11 and s 22 /d 22 are in the O-Re-O plane (dened by Re and the two peroxo O-atoms) whereas s 33 /d 33 is perpendicular to it. In contrast to H 2 O 2 , the most deshielded component of the metal peroxo species, d 11 , is no longer oriented perpendicularly to the O-O axis but is signicantly tilted, while remaining in the peroxo O-Re-O plane. One can also note differences of chemical shis observed for the cis and trans peroxo oxygens in the bisperoxo compounds, the former being slightly more deshielded than the latter for both d iso and d 11 . These values are not strongly affected by the apical ligand (pyridine vs. water), suggesting a similar electronic structure in all bisperoxo intermediates. For the monoperoxo species, the d iso of both peroxo oxygens are more similar, albeit slightly more deshielded than in the bisperoxo intermediates.</p><!><p>An orbital analysis reveals that the largest contribution to the paramagnetic deshielding of the s 11 /d 11 component arises from the LP 'p', as was also observed for non-metal-based peroxides (shown in Fig. 9 for the bisperoxo pyridine adduct). Note however, that a signicant contribution of the s(O-Re) bond is also observed for the metal-peroxo species. The other components and peroxo species are given in Tables S2 10). In fact, the calculated molecular orbitals suggest that the antibonding combinations of the above mentioned dand p-bonds are the LUMO and LUMO+1 of the MTO bisperoxide, respectively.</p><p>As observed for non-metal-based peroxides, the MTO-derived bisperoxide shows rather large C Q values (À15.0 MHz and À15.2 MHz for the cis-and trans-oxygen, respectively, see Table S5 and</p><p>b The monoperoxo species is proposed not to coordinate an additional water-ligand; in fact, coordination of water has little effect on the NMR parameters and the water-ligand dissociated upon optimizing the O-transfer transition state. 75 c Same data as shown in Table 1. perpendicularly to a low-lying s*(O-O) orbital. As observed for the CST, also the C Q values of the peroxo oxygen atoms are rather insensitive towards replacement of the pyridine ligand by water; the corresponding water adduct shows a C Q of À15.0 MHz on both oxygen atoms. These values differ more in the monoperoxo species, where C Q 's are equal to À17.2 and À13.0 MHz for the cis-and trans-oxygen in the pyridine adduct, respectively, consistent with a more dissymmetric structure (Table 3).</p><!><p>Transition state energy calculations were performed both for the attack of ethylene at the oxygen pseudo-cis to the methyl group and the oxygen pseudo-trans to it. The obtained geometries for the bis-peroxo pyridine adducts are shown in Fig. 11 (others are given as coordinate les in the ESI †).</p><p>The free energy barriers from the bis-peroxo pyridine adduct are 34.2 kcal mol À1 and 27.2 kcal mol À1 for transfer of the cis and trans oxygens, respectively. Notably, the more deshielded oxygen atom (pseudo-cis to the methyl ligand) is associated with a less favorable oxygen transfer step, consistent with a CST already close to what is observed in metal-oxo species, showing the connection between reactant and product. While in H 2 O 2 the dihedral angle (H-O-O-H) is calculated to be 114 with the two LP 'p' on the oxygen pointing away from each other, the lone pairs are coplanar in a peroxo metal complex, introducing again a maximized a-effect (similar to DMDO and mCPBA). In order to understand and quantify the effect of the LP 'p' backdonation into the olen p*-orbital to the transition state energy, and hence probe the importance of the a-effect, a transition state (2nd order saddle point), where the attacking olen is coplanar with the peroxo O-Re-O plane was calculated for the case of the oxygen pseudo-trans to the methyl group. The obtained free energy of activation was found to be at 31.6 kcal mol À1 and thus 4.4 kcal mol À1 higher in energy than for the perpendicular transition state. This is consistent with what has been found for the epoxidation with non-metal-based peroxides, again evidencing the importance of the backdonation of a high-lying LP 'p' on oxygenthe lled p*(O-O) orbitalinto the olen p*(C]C) orbital. Similar trends are observed for the bis-peroxo water adduct with free energy barriers of 34.3 and 27.8 kcal mol À1 , for the transfer of the cis-and trans-oxygen, respectively. For the monoperoxo intermediate, the free energy barriers for O-transfer are typically slightly higher (>29.0 kcal mol À1 ) for both oxygen atoms, at the exception of the cis-peroxo oxygen of the pyridine adduct (26.8 kcal mol À1 ). These results suggest that the bisperoxo complex is possibly the more reactive species in water, and that in the presence of pyridine both mono-and bis-peroxo complexes are reactive. 72,75 Thus pyridine may have a dual role, i.e. as a phase transfer agent and as a ligand to accelerate catalysis. 17,76 Notably, for all metal-peroxo compounds, the easier oxygen transfer is associated with the peroxo oxygen with smaller d iso and d 11 , while the oxygen atom which is not transferred displays a larger oxo-character, evidenced by the larger d iso and d 11 .</p><p>As for DMDO and mCPBA, the coplanarity of the two oxygen lone pairs LP 'p' in the MTO peroxo species maximizes the aeffect, raising the energy of the lone pairs thereby increasing their reactivity. In addition, the p-interaction of the peroxo moiety with the metal (Fig. 10) assists the oxygen transfer process in olen epoxidation, during which a fully developed p(Re]O) bond is formed.</p><p>Notably, the spectator oxo-ligand in the apical position in the bisperoxo Re complex is not innocent: this oxo-ligand interacts with the peroxo moiety via the metal d-orbitals involved in the pand d-interactions (Fig. 10). This interaction minimizes the stabilization of the peroxo LP 'p' by weakening the (stabilizing) dand p-bonds. In addition, the presence of the spectator oxoligand also provides a driving force for the formation of the metal-peroxo species in the catalytic cycle: formation of the peroxo species from MTO strengthens the bond of the apical "spectator" oxo-ligand as evidenced by a slight decrease of the Re]O bond length on going from MTO (1.69 Å) to the monoperoxo (1.68 Å) and then the bisperoxo (1.67 Å) pyridine adducts. This effect is reminiscent of the spectator oxo effect discussed for metallacyclobutane formation from metal alkylidenes during the olen metathesis reaction, as well as for the formation of metallacycle oxetane intermediates in the reaction of alkenes with metal oxo compounds. [77][78][79]</p><!><p>Overall, peroxide compounds are associated with signicantly deshielded 17 O chemical shis that indicate the presence of low-lying vacant and high-lying occupied orbitals, corresponding to the s*(O-O) and the lone pairs on oxygens, associated with p(O-O) and p*(O-O), for both metal-based and non-metalbased peroxides. These specic electronic features are particularly pronounced in peroxide species that engage in electrophilic epoxidation reactions (DMDO, mCPBA, and MTO bisperoxo), as evidenced by their remarkably large deshielding. This is due to the coplanarity of the oxygen lone-pairs in these peroxides which is induced by their strained cyclic structure or by H-bonding in the case of mCPBA. Both maximize overlaps and in ne raises the HOMO (a-effect) and increases reactivity towards electrophilic epoxidation. In metal peroxo species, this HOMO is further raised in energy by the presence of a spectator oxo-ligand in the apical position. In fact, this "spectator" oxo species participates in modulating the reactivity of peroxo intermediates in transition-metal-catalyzed oxidation processes; it is thus not surprising that such a moiety is ubiquitous in efficient epoxidation catalysts that use H 2 O 2 as a primary oxidant. The a-effect and the presence of a strained cyclic structure goes hand in hand with a weakening of the O-O bond. Both the high-lying lone pair of the lled p*(O-O) and the low-lying s*(O-O) orbital drive the observed reactivity in electrophilic epoxidation, in which these two orbitals interact with the p*(C]C) and p(C]C) orbitals of the olen, respectively. Thus, 17 O NMR chemical shi provides a powerful descriptor to pinpoint key electronic features that are decisive for reactivity in oxidation chemistry (Fig. 12).</p><!><p>The frontier orbital interactions shown in Fig. 6 highlight the following observation: while an epoxidation with DMDO or mCPBA is typically thought of as an electrophilic epoxidation, with the oxidant acting as electrophile and the olen acting as nucleophile, these molecular orbital interactions indicate that both substrates act as nucleophiles and electrophiles by exploiting the low-lying s*(O-O) orbital and the high-lying lone pair LP 'p' (O) induced by the a-effect. This is reminiscent of synergistic effects observed in transition metal chemistry, for example in olen complexes or in oxidative addition processes (Fig. 13a and b). Considering that olen epoxidation with DMDO or mCPBA is isolobal to epoxidations with oxaziridines and halogenation reactions by X 2 or NXS (X ¼ Cl, Br, I), a similar orbital picture can be anticipated for these "electrophilic" additions (Fig. 13c).</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

Thiol/disulfide homeostasis impaired in patients with primary Sjögren's syndrome

BackgroundPrimary Sjögren's syndrome (pSS) is a disease associated with the overexpression of proinflammatory cytokines, and oxidative stress is one of the factors responsible for its etiopathogenesis. This study aimed to investigate the thiol/disulphide homeostasis in pSS patients.MethodsThe study included 68 pSS patients and 69 healthy controls. Thiol/disulphide homeostasis (total thiol, native thiol, and disulphide levels) was measured using the automatic spectrophotometric method developed by Erel and Neselioglu, and the results of the 2 groups were compared.ResultsThe gender and age distributions of the pSS and control groups were similar (P = 0.988 and P = 0.065). Total thiol and native thiol levels were lower in the pSS group than in the control group (470.08 ± 33.65 µmol/L vs. 528.21 ± 44.99 µmol/L, P < 0.001, and 439.14 ± 30.67 µmol/L vs. 497.56 ± 46.70 µmol/L, P < 0.001, respectively). There were no differences in disulphide levels between groups [17.00 (range 0.70-217.0) µmol/L vs. 14.95 (range 2.10-40.10) µmol/L, P = 0.195].ConclusionsIt was concluded that the thiol/disulphide balance shifted towards disulphide in patients with pSS.

thiol/disulfide_homeostasis_impaired_in_patients_with_primary_sjögren's_syndrome

Uvod<!>Metode<!>Rezultati<!>Zaključak<!>Introduction<!>Study Population<!>Biochemical Parameters<!>Thiol/Disulphide Homeostasis<!>Statistical Analysis<!>Results<!>Clinical findings and autoimmune parameters of the patients with pSS<!>Results<!>Comparison of thiol/disulfide levels, demographic and laboratory findings of the pSS and control groups<!>Results<!>Comparison of thiol/disulfide levels of the pSS patients according to xerostomia, xerophthalmia, Schirmer’s test, minor salivary gland biopsy, and some laboratory results<!>Discussion<!>Funding<!>Conflict of interest statement<!>

<p>Primarni Sjögrenov sindrom (pSS) je bolest povezana sa prekomernom ekspresijom proinflamatornih citokina, dok je oksidativni stres jedan od faktora odgovornih za njegovu etiopatogenezu. Cilj ove studije je bio da ispita tioldisulfidnu homeostazu kod pacijenata sa pSS-om.</p><!><p>Studija je obuhvatila 68 pacijenata sa pSS-om i 69 zdravih kontrolnih pojedinaca. Homeostaza tiol-disulfida (nivoi ukupnog tiola, nativnog tiola i disulfida) je izmerena pomoću automatske spektrofotometrijske metode koju su razvili Erel i Neselioglu. Rezultati dve grupe su upoređeni.</p><!><p>Raspodela po polu i starosti pacijenata sa pSSom i pojedinaca u kontrolnoj grupi je bila slična (P = 0,988 i P = 0,065). Ukupni nivoi tiola i nativnog tiola bili su niži u grupi sa pSS-om nego u kontrolnoj grupi (470,08 ± 33,65 µmol/L naspram 528,21 ± 44,99 µmol/L, P <0,001 i 439,14 ± 30,67 µmol/L naspram 497,56 ± 46,70 µmol/L, P <0,001, respektivno). Nije bilo razlika u nivoima disulfida između grupa [17,00 (opseg 0,70-217,0) µmol/L naspram 14,95 (opseg 2,10-40,10) µmol/L, P = 0,195].</p><!><p>Zaključeno je da se ravnoteža tiol/disulfid pomera prema disulfidu kod pacijenata sa pSS-om.</p><!><p>Sjögren's syndrome (SS) is a systemic chronic inflammatory disease characterized by the lymphocytic infiltration of exocrine organs [1] [2]. It is further divided into 2, as primary and secondary SS, depending on the other accompanying autoimmune diseases. Primary SS (pSS) is an autoimmune disease characterized by lymphocytic infiltration and destruction of the salivary and tear glands, and systemic auto antibody production against SS-A/Ro and SS-B/La ribonucleoprotein particles. Although etio pathogenesis is not clearly understood, the current theory is that environmental factors trigger autoimmunity in genetically predisposed individuals. SS is associated with the overexpression of proinflammatory cytokines, including tumour necrosis factor-alpha, interleukin (IL)-7, IL-1 beta, IL-6, IL-10, IL-17, IL-18, and interferon-gamma [3]. Together with the increased inflam matory processes, the increased reactive oxygen radicals are also thought to increase the formation of immune complexes and exacerbate the destruction of exocrine glands. It has been demonstrated that the oxidative stress associated with accompanying autoimmune diseases (such as systemic lupus erythematosus, rheumatoid arthritis (RA), and systemic sclerosis) takes part in the etiopathogenesis of SS. Therefore, it is plausible that oxidative stress plays a role in the etiopathogenesis of SS.</p><p>Oxidative damage in the body is prevented by enzymatic or non-enzymatic antioxidant mechanisms [4]. Thiols are a principal member of both the enzymatic and non-enzymatic intracellular antioxidant systems. Thiols are also known as mercaptans. They are organic compounds that are formed by binding sulphur and hydrogen atoms to carbon atoms [5]. The thiol pool in the plasma consists mainly of thiols, such as albumin, proteins, low molecular weight cysteine, cysteine-glycine, glutathione, homocysteine, and gamma-glutamylcysteine [6]. When the level of free oxygen radicals' increases (i.e., increased oxidative stress), the plasma thiol/disulphide balance shifts against thiol, and disulphide levels increase [7] [8].</p><p>To the best of our knowledge, there are no studies that have investigated oxidative status via the measurement of native thiol, total thiol, and disulphide levels in patients with pSS. Therefore, it was aimed herein to evaluate the oxidative status via these parameters in patients with pSS.</p><!><p>Sixty-eight patients with pSS and 69 healthy individuals were included in the study. Patients with cardiovascular diseases, cerebrovascular diseases, kidney failure, liver failure, infections, malignancies, and individuals that used antioxidants, multivitamins, cigarettes, or alcohol were excluded from the study. SS was diagnosed according to the criteria defined by the 2016 American College of Rheumatology/European League Against Rheumatism [9].</p><p>The demographic and clinical characteristics (age, sex, xerostomia or xerophthalmia that had been present for 3 months, presence of arthritis, myositis, or vasculitis, pulmonary, renal, hematological, or hepatological involvement, presence of Raynaud's phenomenon, and Schirmer tests and salivary gland biopsy results) and laboratory results [hemogram, aspartate transaminase (AST), alanine transaminase (ALT), glucose, urea, creatinine, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), anti-Ro antibody (anti-SSA), anti-La antibody (anti-SSB), antinuclear antibody (ANA), rheumatoid factor (RF), total thiols (-SH + -S-S-), native thiols (-SH) and dynamic disulphide (-S-S-)] of the patients were recorded. The study was approved by the ethical board of the institution, and informed consent was obtained from all participants (Approval date and number:18.09.2019/26379996/96).</p><!><p>Biochemical parameters (AST, ALT, glucose, urea, and creatinine) were measured using standard laboratory methods (Cobas 501; Roche Diagnostics, Germany). The hemogram was evaluated on Sysmex XN-1000 (Sysmex Corporation, Kobe, Japan) analyzer, and serological parameters were studied using the nephelometric method (Siemens BN-11).</p><!><p>For the assessment of the thiol/disulphide homeostasis, blood samples were centrifuged at 1500 rpm for 10 min and stored at -80°C until assessment. After all of the samples had been collected, they were simultaneously evaluated for oxidative stress parameters by the same laboratory technician using the same device. Native thiol, total thiol, and disulphide levels were assessed using the novel and fully automated assay developed by Erel and Neselioglu, which is based on the reduction of dynamic disulphide bonds to free thiol groups by sodium borohydride (NaBH4) [10]. Formaldehyde was added to eliminate excess NaBH4 to avoid the extra reduction of DTNB (5,50-dithiobis-[2-nitrobenzoic acid]), and further reduction of the formed disulphide bonds after the DTNB reaction. Ellman's reagent was used to measure the total thiol content.</p><p>Next, the native thiol content was subtracted from the total thiol content, and 50% of this difference revealed the quantity of the disulphide bond. An automated clinical chemistry analyser (Cobas 501) was used to measure the amount of native thiol and disulphide. The serum thiol and disulphide values were presented as µmol/L.</p><!><p>All statistical analyses were performed with IBM SPSS Statistics 21.0 (Armonk, NY, USA) software. The Shapiro-Wilk test was used to evaluate compliance with the normal distribution. Descriptive analyses were presented as the mean ± standard deviation (SD) for normally distributed variables and median and range (min-max) for non-normally distributed variables. Categorical variables were presented as numbers and percentages. The 2 groups were compared using the Student t-test and 1-way ANOVA for parametric variables, and the Kruskal-Wallis test for non-parametric variables. P < 0.05 was considered statistically significant.</p><!><p>Of the pSS patients, 66 were female, and 2 were male, and of the controls, 67 were female, and 2 were male (P = 0.988). The mean ages of the patient and control groups were 53.71±8.17 and 51.04±8.57 years, respectively (P = 0.065). Of the pSS patients, 64.7% had Raynaud's phenomenon, 57.4% were anti-SSA-positive, and 69.1% were anti-SSB-positive. The Schirmer's test result was 2 mm in 30.9% of the patients. According to the Chisholm-Mason classification [11] [12], the salivary gland biopsy results of the pSS patients were as follows: stage 1 in 5.9%, stage 2 in 22.1%, stage 3 in 38.2%, and stage 4 in 33.8%. Of the pSS patients, 58.8% had xerostomia, and 52.9% had xerophthalmia. Data regarding the symptoms and organ involvement of the pSS patients are presented in Table 1.</p><!><p>ANA, antinuclear antibody; Anti-SSA, anti-SS-related antigen A; Anti-SSB, SS-related antigen B, RF, rheumatoid factor.</p><!><p>The ESR, CRP, and AST levels of the patient group were significantly higher and the leukocyte values were lower than in the control group (P < 0.001, P < 0.001, P < 0.001, and P= 0.009, respectively). The total thiol and native thiol levels were statistically lower in the pSS patients when compared to the controls (470.08±33.65 µmol/L vs. 528.21±44.99 µmol/L, P < 0.001, and 439.14±30.67 µmol/L vs. 497.56±46.70 µmol/L, P < 0.001, respectively). Although the disulphide levels were higher in the pSS group when compared to the control group, this difference was not statistically significant [17.00 (range 0.70-217.0) µmol/L vs. 14.95 (range 2.10-40.10) µmol/L, P = 0.195] (Table 2).</p><!><p>Note: Parameters are expressed as the means ± SD and medians [interquartile range]. P < 0.05 was considered statistically significant. AST, Aspartate transaminase; ALT, alanine transaminase; ESR, erythrocyte sedimentation rate, CRP, C-reactive protein.</p><!><p>The total thiol, native thiol, and disulphide levels were similar in patients with and without xerostomia or xerophthalmia (P > 0.05 for all of the parameters). The total thiol, native thiol, and disulphide levels were not significantly associated with the Schirmer's test or minor salivary gland biopsy results (P > 0.05 for all of the parameters). Moreover, the thiol/disulphide parameters of the anti-SSA- and anti-SSB-positive patients were similar to those of the anti-SSA- and anti-SSB-negative patients (P > 0.05 for all of the parameters) (Table 3).</p><!><p>Anti-SSA, anti-SS-related antigen A; Anti-SSB, anti-SS-related antigen B.</p><!><p>Although oxidative stress is involved in the pathophysiology of several chronic conditions, there have been a limited number of studies evaluating the relationship between SS and oxidative stress. To the best of our knowledge, the current study was the first to investigate thiol/disulphide homeostasis in pSS. In this study, it was observed that the total thiol and native thiol levels were reduced in the pSS patients when compared to the healthy controls, there was a statistically insignificant increase in the disulphide levels, and the dynamic thiol/disulphide balance shifted towards disulphide.</p><p>There is an association between proinflammatory states and oxidative stress. Moreover, studies have suggested that oxidative stress is involved in the pathogenesis of SS. A study by Kurimoto et al. [13] found that the antioxidant thioredoxin had increased in the salivary gland biopsies of SS patients when compared to the controls, and they evaluated this as a protective mechanism against oxidative stress in SS. Cejková et al. [14] measured antioxidant markers, such as superoxide dismutase, catalase, and glutathione peroxidase, in the conjunctival epithelium and showed that their levels decreased with increasing xerophthalmia. Furthermore, oxidative stress markers were found to have increased in plasma and lip biopsy samples of SS patients [15] [16].</p><p>Thiols are crucial organic compounds that contain sulfhydryl groups, which play an important role in the oxidant/antioxidant mechanism [17]. The thiol/disulphide balance is crucial to the organism. ROS increase when the balance shifts towards disulphide [18]. Native thiols are molecules containing nonreduced functional thiol groups. As a part of the antioxidant system, native thiols decrease when oxidative stress increases. Total thiol levels reflect the sum of both oxidized and non-oxidized thiols. Plasma thiol and disulphide levels can be measured both separately and combined with the novel assay developed by Erel and Neselioglu in 2014 [10].</p><p>The thiol/disulphide balance is impaired in many diseases, such as myocardial infarction, preeclampsia, polycystic kidney disease, diabetes mellitus (DM), and cancer. Disulphide levels increase in degenerative diseases, such as DM, obesity, pneumonia, and familial Mediterranean fever, and the balance shifts towards thiol in proliferative diseases, such as multiple myeloma, bladder cancer, colon cancer, and kidney cancer [10]. A recent study found that native thiol levels were high and disulphide levels were low in patients with fibromyalgia when compared to the healthy controls, and it was reported that in fibromyalgia, the thiol/disulphide balance was similar to that in benign proliferative diseases and that this was due to the proliferative pattern rather than inflammation [19]. Our hypothesis is also supported by the results of some studies that include autoimmune and inflammatory diseases. Dealing with examples of autoimmune diseases, one study compared patients with RA and healthy controls and observed that the thiol/disulphide balance shifted towards disulphide in the RA patients [20]. According to another study which compared the Graves' patients, the ratios of native thiols, total thiols, and native thiols/total thiols were lower; and the ratios of disulphide/native thiol and disulphide/total thiol were higher in the patient group comprehensively to the healthy controls [21]. The results of a study that included Celiac patients showed that total and native thiol levels in celiac patients were lower while disulphide level, disulphide/total thiol, and disulphide/native thiol ratios were higher compared to the healthy people; and also dynamic equilibrium was found shifted to disulphide side [22]. Another study that contained autoimmune gastritis patients showed altered thiol/disulphide homeostasis compared to the healthy group, and the dynamic balance was shifted through disulphide form [23]. On the other hand, to present examples of inflammatory diseases, some studies compared Psoriasis patients and healthy controls; and according to results in the patient group, disulphide, disulphide/native thiol, and disulphide/total thiol were significantly higher, but native thiol and native thiol/total thiol were significantly lower compared to healthy controls. This showed that psoriasis patients had a higher increase in oxidants compared to the healthy group [24] [25]. In a study with pregnant women with Familial Mediterranean Fever, disulphide levels were higher in patients compared to controls [26]. In another study with FMF patients, the relation of thiol/disulphide imbalance and colchicine resistance was evaluated. According to the results, native thiol and total thiol levels were lower in colchicine resistance cases compared to those not colchicine resistance ones. Also, disulphide levels were higher in colchicine resistance cases [27].</p><p>In the current study, the total thiol and native thiol levels in the pSS patients were significantly lower than in the control group, as was the case in patients with RA. This suggested that antioxidation had decreased in patients with pSS, indirectly increasing oxidation. The fact that disulphide levels did not increase as expected, despite the fact that native thiols decreased, may have been because these molecules could not be measured due to being converted into advanced oxidation products. It should be noted that the subjects herein with pSS were on a variety of drugs, including immunosuppressives and corticosteroids. Methotrexate is known to reduce oxidative stress by reducing free radicals, such as superoxide (O- 2), and reactive oxygen particles [28]. The reason that the disulphide levels did not increase in the patients herein may have been because of the antioxidation effects of the medications used by the patients, such as methotrexate. It is not known precisely how the drug combinations used by pSS patients affect the thiol/disulphide balance. Besides, the reason why the thiol/disulphide balance did not change with anti-SSA or anti-SSB positivity, salivary gland biopsy results, xerostomia, or xerophthalmia may have been ascribed to medication use as well.</p><p>This study had several limitations. First, whether the patients and controls had consumed thiol-containing nutrients was investigated only through anamnesis. Second, the pSS patients were under medical treatment, and the effects of the drugs that they used on the oxidative parameters were not evaluated. Finally, the relationship between disease duration and the oxidative parameters was not evaluated in this study.</p><p>In conclusion, the total thiol and native thiol levels decreased in patients with pSS, suggesting that antioxidation was impaired in these patients, and the oxidation/antioxidation mechanism may play a role in the pathogenesis of the disease. To fully evaluate how the thiol/disulphide mechanism is altered in pSS, prospective studies with larger samples that include recently diagnosed pSS patients, who have not yet started treatment, are needed.</p><!><p>The author(s) received no financial support for the research, authorship, and/or publication of this article.</p><!><p>All the authors declare that they have no conflict of interest in this work.</p><!><p>Conflict of Interest: The authors stated that they have no conflicts of interest regarding the publication of this article.</p>

PubMed Open Access

Exchange-Biasing in a Dinuclear Dysprosium(III) Single-Molecule Magnet with a Large Energy Barrier for Magnetization Reversal

A dichlorido-bridged dinuclear dysprosium(III) singlemolecule magnet [Dy2L2(µ-Cl)2(THF)2] has been made using a diamine-bis(phenolate) ligand, H2L. Magnetic studies show an energy barrier for magnetization reversal (Ueff) around 1000 K. Exchangebiasing effect is clearly seen in magnetic hysteresis with steps up to 4 K. Ab initio calculations exclude the possibility of pure dipolar origin of this effect leading to the conclusion that super-exchange via the chloride bridging ligands is important.

exchange-biasing_in_a_dinuclear_dysprosium(iii)_single-molecule_magnet_with_a_large_energy_barrier_f

<!>𝐻 cross = −𝐽 𝐼𝑠𝑖𝑛𝑔 /2𝑔𝛽<!>Experimental Section

<p>Individual molecules that show slow relaxation of magnetisation are known as single-molecule magnets (SMMs). 1 This field started in 1993, and SMMs have been proposed as possible media for high-density magnetic storage. 2 A key parameter to evaluate the performance of an SMM is the effective energy barrier to magnetization reversal (Ueff). Dysprosium(III) is particularly preferred as the high magnetic anisotropy arising from the 6 H15/2 state generates the highest values for Ueff when placed in strong axial crystal fields. In particular, two Dy-SMMs families have very high Ueff: the sandwich structures with biscyclopentadienyl ligands 3 and pentagonal bipyramidal complexes. 4 In the latter, the strong axial crystal field is normally defined by short coordination bonds to the ligands on the axial positions of the pentagonal bipyramid. 4,5 A feature common to many Dy-SMMs is loss of magnetization at zero field, which is attributed to the quantum tunneling of magnetization (QTM) under zero field. [3][4][5][6] Interactions between spin centers can prevent such zero-field loss of magnetization, known as exchange-bias. This was first seen 7 in a dimer of [Mn4] SMMs and has more recently been seen in Dy(III) dimers. 8 Our aim was to combine high Ueff values with exchange biasing by making a dimer of highly axial Dy(III) ions. For this strategy to work, the best molecular design would have the local anisotropy axes of the two Dy(III) sites as close as possible to being coparallel. 9 The title complexes [RE2L2(µ-Cl)2(THF)2]• toluene (RE = Dy, 1; RE = Y, 2; 5% Dy@2, 3) were prepared by deprotonation of H2L (N-(2-pyridylmethyl)-N,N-bis(2'-hydroxy-3',5'-di-tertbutylbenzyl)amine) with NaH, followed by reactions with anhydrous RECl3 in THF (see ESI for details). Since they are isomorphous as confirmed by single-crystal X-ray diffraction, only the structure of complex 1 is discussed in detail (Table S1, ESI). Compound 1 crystallizes in space group C2/c and has a two-fold rotation axis passing through the two bridging chlorides (Figures 1 and S1, ESI). The Dy(III) ion has a seven-coordinate geometry completed by two phenoxide oxygen atoms and two nitrogen atoms from one tetra-chelated L 2-, two µ2 bridging Cland one THF molecule. The two Dy-O(PhO) bond lengths are 2.152(2) and 2.168(2) Å, much shorter than the Dy-OTHF (2.412(2) Å), two Dy-N bonds (2.574(2) and 2.520(2) Å) and Dy-Cl bonds (2.7880(6) and 2.7896(6) Å) (Table S2, ESI). The local symmetry is not a regular polyhedron (as determined by Shape software, 10,11 see ESI Table S3), however the bond angle between the two short Dy-O(PhO) bonds = 149.62 (8) o and this defines the main magnetic anisotropy axis as calculated by CASSCF-SO, giving an angle between the two main anisotropy axes of ca. 68° (see below) Temperature-dependent direct-current (dc) susceptibility data of 1 were collected under 1 kOe applied field. At room temperature the χT value is 28.53 cm 3 mol -1 K, in good agreement with the expected value of 28.34 cm 3 mol -1 K for two Dy(III) ions (Figure S3, ESI). Upon cooling χT decreases slowly at first, and then more rapidly below 16 K, reaching 17.23 cm 3 mol -1 K at 2 K. The field-dependence of the magnetization reveals that the highest M value is 10.31 Nβ at 50 kOe and 2 K (Figure S4, ESI).</p><p>Alternating-current (ac) susceptibility measurements with an oscillating field of 3.5 Oe were also performed. Under zero dc field, 1 exhibits clear temperature and frequency dependence of the ac susceptibility below 53 K, showing the typical slow magnetic relaxation of SMMs (Figures S5 and S6, ESI). The relaxation time (τ) at each temperature was extracted from a simultaneous fit of χ' and χ'' using the generalized Debye model. 12 The obtained parameters are summarized in Table S4, in which α values are always less than 0.07 in the temperature range of 8-53 K, indicating a narrow distribution of relaxation times (Figure S7, ESI). The α values found were converted into experimental uncertainties in the relaxation times for each temperature using the CC-FIT2 code. 12 We observe that relaxation on the timescale of our ac susceptibility experiments is dominated by a power-law temperature dependence characteristic of a two-phonon Raman process with log[C (s -1 K -n )] = -3.8  0.2 and n = 4.3  0.2 (Figure 2, Equation 1).</p><p>At the highest temperatures there is an increase in the relaxation rate, likely indicative of a multi-phonon Orbach process with an exponential temperature dependence. Including the experimental uncertainties renders these parameters practically undefined with Ueff = 1000  1000 K and log[τo (s)] = -10  10. However, a Ueff value around 1000 K is independently supported by ab initio calculations, see below, and taking only the central relaxation rate, as has been the only approach in the literature prior to our new method, 12 a fit gives Ueff = 922  9 K, log[τo (s)] = -11.33  0.08 (Figure S8, ESI). The only higher Ueff value found for a polynuclear single-molecule magnets is in Dy2ScN@C80-Ih. 13,14 . To examine the influence of the exchange-bias, we studied the magnetic properties of compound 3, which contains a 5% concentration of Dy(III) ion doped into the isostructural diamagnetic yttrium dimer 2. At this dilution level, the paramagnetic material is dominated by isolated Dy(III) ions in the structure. 8a The dc magnetic susceptibility data and low temperature magnetisation data for 3 have very similar profiles to those of 1 (Figures S13-S15 Cycling the field between +10 and -10 kOe for 3 gives a hysteresis loop shaped like a butterfly at low temperatures (Figures 4 and S20, ESI). The loss of magnetization at zero field for the diluted sample clearly confirms that magnetic interactions between Dy(III) ions in the dimer shifts the quantum tunnel resonances away from zero field. The two steps at ca. +1000 and -300 Oe for 1 are missing after dilution (Figures 3, 4, S12 and S21), and thus are markers of the magnetic interaction between the Dy(III) ions in the dimer. To understand the local electronic structure of the Dy(III) ions, complete active space self-consistent field spin-orbit (CASSCF-SO) calculations were performed using MOLCAS 8.0. 15a Basis sets from the MOLCAS ANO-RCC library 15 were employed with the paramagnetic ion described using VTZP quality, the first coordination sphere with VDZP quality, and all other atoms with VDZ quality. To probe the single ion properties in the pure Dy compound 1, one of the two Dy(III) ions in the crystal structure was replaced with the diamagnetic Lu(III) ion, as this more closely resembles the electronic manifold of the neighboring Dy(III) ion than would Y(III) (as Lu(III) has filled 4d, 5p and 4f orbitals). As expected based on the structure with a pair of phenoxide donors at an angle of 149.6°, the crystal field stabilizes an almost pure |±15/2⟩ ground doublet. This state is well separated from the 1 st (534 K), 2 nd (868 K) and 3 rd (1053 K) excited states. While the 1 st and 2 nd excited states are fairly pure mJ = |±13/2⟩ and |±11/2⟩ functions respectively, the 3 rd excited state is only 67% |±9/2⟩ (Table S5, ESI), and thus magnetic relaxation via the Orbach process is likely to occur through this state, predicting Ueff = 1053 K (Figure S22, ESI); this is in agreement with experimental value of 1000 K.</p><p>We can calculate the dipolar interaction between the two Dy(III) sites using the g-values and relative orientation of the g-frames from CASSCF-SO (Equation 2). 9.16 Owing to the two Dy(III) sites being related by a two-fold axis of rotation, the local anisotropy axes of the ions are not co-parallel, but rather the gz axes have an angle of 68.25° between them. This gives the interaction matrix (Equation 3), which can be implemented in PHI to simulate the low temperature magnetic behavior (Equation 4). 17</p><p>This purely dipolar interaction alone does not reproduce crossings or avoided crossings along any of the three main directions (Figures S23 and S24, ESI) corresponding to the steps in the hysteresis measurements, and instead predict a step only at zero field that is inconsistent with the experimental data. Therefore, there must be a non-zero superexchange interaction via the bridging chlorides. Compound 1 is EPR silent and therefore we were unable to directly measure the exchange interactions using EPR. 9.16 For comparison, we could estimate the Ising exchange parameter considering the system as a simple Ising dimer, Equation (5), 8e,18 giving JIsing = -1.88 cm -1 where Hcross is 1018 Oe from the first derivative of the magnetization, and g equals gz = 19.87.</p><!><p>(5)</p><p>Substituting JIsing for Jxx in our simulations (because x in the molecular frame is the most magnetic direction, Figure S23) does not yield avoided crossings consistent with the QTM steps observed in the hysteresis measurements (Figure S25). 8d, 19 Combining the dipole interaction matrix with the Ising approximation (i.e. Equation 3 with Jxx replaced with JIsing) does predict an avoided crossing at ca. 1000 Oe, however, also predicts significant zero-field avoided crossing (contradicting experiment) and no evidence of the experimental feature at -300 Oe (Figure S26).</p><p>As none of these models fully explain the magnetization data, we have started with the dipole interaction matrix and added a superexchange component in order to reproduce the observed QTM steps. By addition +0.5 and -0.2 cm -1 to Jxx, and Jyy, respectively, we find Zeeman simulations that predict avoided crossings consistent with the observed magnetization steps at ca. +1000 and -300 Oe (Figure 5). However, single crystal measurements at mK temperatures would be necessary to obtain accurate measurements of the low-lying magnetic states in 1 in order to verify the exchange model proposed here. ). The magnetic field along the molecular x-axis (left), y-axis (centre) and z-axis (right). Note that there is a small avoided crossing at zero-field between the two ground states, with a gap of 4.27×10 -3 cm -1 .</p><p>The parameters used to fit the relaxation behaviour of 1 and 3 can be compared with those found for other seven-coordinate Dy-SMMs with O-donors in the axial positions. 20 For example, in regular pentagonal bipyramids 900 < Ueff < 1300 cm -1 ; log[τo (s)] = -11.63  0.57; log[C (s -1 K -n )] = -6.03  0.52; n = 4.1  1.0. Thus, while Ueff and n are very similar both τo and C are bigger in the exchange-coupled dimer (log τo = -11.33  0.08, log C = -3.41  0.06). This may also be due to the less regular coordination environment in 1. We have been able to switch off the zero-field loss of magnetisation in this high T SMM through exchangebiasing and this motivates us to improve the coupling strength while keeping the high anisotropy to construct better SMMs.</p><!><p>Experimental Details can be found in the supplementary information.</p>

ChemRxiv

Challenges in the quality assurance of elemental and isotopic analyses in the nuclear domain benefitting from high resolution ICP-OES and sector field ICP-MS

Accurate analytical data reinforces fundamentally the meaningfulness of nuclear fuel performance assessments and nuclear waste characterization. Regularly lacking matrix-matched certified reference materials, quality assurance of elemental and isotopic analysis of nuclear materials remains a challenging endeavour. In this context, this review highlights various dedicated experimental approaches envisaged at the European Commission—Joint Research Centre—Institute for Transuranium Elements to overcome this limitation, mainly focussing on the use of high resolution-inductively coupled plasma-optical emission spectrometry (HR-ICP-OES) and sector field-inductively coupled plasma-mass spectrometry (SF-ICP-MS). However, also α- and γ-spectrometry are included here to help characterise extensively the investigated actinide solutions for their actual concentration, potential impurities and isotopic purity.

challenges_in_the_quality_assurance_of_elemental_and_isotopic_analyses_in_the_nuclear_domain_benefit

Introduction<!>Analytical performance of high resolution ICP-OES<!><!>Analytical performance of high resolution ICP-OES<!><!>Nuclear forensics—uranium isotopic analysis<!>Pyrochemical treatment of spent nuclear fuel<!><!>Pyrochemical treatment of spent nuclear fuel<!><!>Pyrochemical treatment of spent nuclear fuel<!>Spent fuel analysis<!><!>Spent fuel analysis<!><!>Spent fuel analysis<!>Conclusions

<p>Over the last decades, analytical quality assurance has gained in importance in many scientific areas, including the analysis of radioactive specimens. The use of certified reference materials is an important pillar for the assessment of the quality of any acquired analytical data. Such matrix-matched certified reference materials that are employed frequently for both quality control and method validation are unfortunately not available for most investigations relevant to the nuclear domain [1]. Therefore, it would be most helpful to compare the analytical results obtained for a particular instrumental technique, e.g. inductively coupled plasma-mass spectrometry (ICP-MS), with data from another methodology whose analyte detection is based on a different physical principle, e.g. inductively coupled plasma-optical emission spectrometry (ICP-OES). Both before mentioned techniques work independently because the separation/detection of elements or more specific isotopes is based on the mass-to-charge ratio (m/z) in ICP-MS, while emission of light of element characteristic wavelengths is fundamental to ICP-OES.</p><p>Using such complementary experimental approaches reduce largely the likelihood of the occurrence of identical analytical problems related to the determination of a specific analyte leading to either a positive or negative bias of the final result. Two particular examples may highlight this important aspect: First, the presence of a large excess of 238U in the analyte solution always hampers the reliable determination of 237Np (being a direct neighbour of 238U in the mass spectrum) using ICP-MS. Depending on the employed ICP-MS instrument, the abundance sensitivity, i.e. the impact of the peak tailing of 238U on the 237Np signal, is slightly different, but leads to a positive bias of the acquired Np data at all times. For ICP-OES, in turn, suitable emission wavelengths have been identified that allow an accurate determination of 237Np in the presence of 238U [2]. Second, isobaric interferences occurring in ICP-MS, e.g. 238Pu and 238U or 241Am and 241Pu, cannot be resolved spectroscopically, even using sector field (SF-)ICP-MS [3]. Again, these analytical problems are overcome by using appropriate ICP-OES emission wavelengths for the particular analytes of interest because the above mentioned isotopes emit light at different wavelengths [3, 4].</p><p>Moreover, this cross-validation of two independent analytical procedures helps to identify potential limitations of a specific analytical method envisaged to analyse a selected element/isotope. An agreement of results obtained by at least two independent analytical methods, in turn, essentially improves the creditability of the acquired analytical data. In addition, such inter-method comparisons are carried out preferably in-house for radioactive samples basically because of the huge efforts necessary with respect to safety, security, and monetary aspects associated with a transport of such specimens to another external laboratory.</p><p>Whenever possible, the analysis of nuclear samples may be carried out directly, i.e. without the need of chemically separating off the analyte of interest from the remaining elements [2–5]. This straightforward approach is not only faster, but also results in less radiation dose originating from the sample to the laboratory personnel. The presence of a variety of fission products and minor actinides in the analyte solution, however, complicates frequently the application of a particular instrumental approach. Consequently, complementary analysis employing diverse instrumental techniques based on different physical detection principles is a key issue to ascertain the reliability of the generated analytical data, especially when "non-separated" fuel solutions, containing all fission products as well as actinides, are to be analysed.</p><p>Currently, the potential of high resolution (HR-)ICP-OES for elemental and isotopic analysis in the nuclear domain is not fully exploited, often leading to modest performance only [6–13]. Even though early work already demonstrated the successful application of this analytical technique to the nuclear field about 3 decades ago [6–9, 14], this knowledge disappeared in some way with the introduction of commercial ICP-MS instruments in the early 1990s. With the wide spread of ICP-MS, being more sensitive and providing superior performance in terms of isotopic analysis, investigations on the potential of ICP-OES for this kind of analysis largely stopped.</p><p>At the European Commission—Joint Research Centre—Institute for Transuranium Elements (EC-JRC-ITU), we aim at reconsidering this powerful analytical technique for both elemental and isotopic analysis of actinides and fission products in a substantial variety of samples within the nuclear domain. In addition, its potential to complement other well established analytical techniques such as ICP-MS, as well as α- and γ-spectrometry are highlighted in this study.</p><p>The main intention of this review is to summarize and share the experience gained during the in-house analysis of nuclear samples, thereby also raising the awareness of the importance of analytical quality assurance in the nuclear field. Using a commercial HR-ICP-OES spectrometer, sensitive emission wavelengths for potential isotopic and elemental analysis of nuclear samples were identified and inspected thoroughly. Besides, analytical procedures based on SF-ICP-MS were developed and applied subsequently to cross-validate the HR-ICP-OES results. The benefits and pitfalls of different quantification strategies applied to HR-ICP-OES and SF-ICP-MS analysis were examined carefully, complemented by α- and γ-spectrometry measurements. Specific innovative examples presented here include (1) U isotopic analysis with HR-ICP-OES, identifying depleted, natural and enriched (at various levels) abundances of 235U; (2) the accurate determination of alkaline elements, neodymium (Nd), and neptunium (Np) concentrations in nuclear specimens including samples from pyrochemical treatment of spent fuel; as well as (3) the direct elemental and isotopic analysis of americium (Am) in non-separated spent fuel solutions.</p><!><p>In contrast to conventional ICP-OES instruments equipped with a charge coupled device (CCD) detector for fast, simultaneous analysis, the sequentially working HR-ICP-OES employed in our studies benefits from a photomultiplier. Among the main differences between conventional and HR-ICP-OES is the fact that the latter consists of a superior optical path allowing an advantageous separation of individual emission wavelengths from each other. While conventional ICP-OES instruments with a CCD typically provide optical resolutions in the range of 10–20 pm (depending on the wavelength region) [13, 15], this value is well below 5 pm for most emission wavelengths of interest for HR-ICP-OES [11, 16]. The superior high optical resolution of the latter allows for measurements of peak increments of <0.5 pm and better identification of potential spectral interferences.</p><p>Normally used in its standard configuration (pneumatic nebulizer, sample uptake rate of ~1–2 ml min−1), reported ICP-OES detection limits (LODs) for the determination of selected minor actinides and fission products are in the low to mid µg kg−1 range, depending on the element considered [6–11]. While these LODs are sufficient for a number of applications in the nuclear field [10–13], instrumental performance can be improved substantially providing some distinct benefits as described in more detail below.</p><!><p>Comparison of the performance of two diverse detectors used with the identical HR-ICP-OES instrument. Old refers to the commonly employed standard photomultiplier, whereas the new detector denotes a high sensitivity multialkali photocathode providing high near-infrared sensitivity. In addition, typical wavelengths of selected elements measured frequently at EC-JRC-ITU employing ICP-OES are indicated</p><!><p>Using this advanced instrumental set-up, we showcase selected recent applications of HR-ICP-OES analysis of nuclear specimens carried out at EC-JRC-ITU and demonstrate its potential for cross-validating other well-established analytical techniques such as SF-ICP-MS (Element2, Thermo Scientific, Bremen, Germany).</p><!><p>HR-ICP-OES spectra of the certified reference material EC-NRM-199 having almost identical isotope amount fractions of 233U, 235U, and 238U. Spectra have been recorded using the optimised instrumental set-up (see text for details) at four different concentration levels. Reported concentrations refer to the total amount of U, i.e. concentrations of individual U isotopes amount to only ~1/3 of this total U concentration</p><p>Uranium isotopic analysis 235U enrichments [%] of various scrap metal samples as assessed using various instrumental techniques</p><p>aMulti collector-inductively coupled plasma-mass spectrometry</p><p>bThermal ionisation mass spectrometry</p><!><p>Among the advantages of using HR-ICP-OES for the reliable assessment of 235U enrichments is the fact that no matrix separation is required prior to analysis. As such, HR-ICP-OES can be employed as a fast screening tool, providing sufficiently accurate (<1.5 %) and precise (~1 %) U isotopic information [24]. Setting up necessary laborious chemical separation procedures for subsequent mass spectrometric measurements can benefit from such overview HR-ICP-OES analysis. Compared to most mass spectrometric techniques, no so-called "mass bias" correction is required for HR-ICP-OES analysis of U isotopes. In addition, the main assembly of the HR-ICP-OES does not get contaminated radioactively, a crucial issue that cannot be avoided using mass spectrometry. Because the difference in signal intensity of the minor (235U) and major abundant (238U) U isotopes is only about two orders of magnitude at most, no deteriorating effects limiting the intensity linearity are observed [24]. If U isotope ratios would become larger, then worsening effects such as self-absorption might hamper the reliable determination of U isotopes using HR-ICP-OES. This kind of measurement, however, is beyond the instrumental capabilities of HR-ICP-OES [24].</p><p>In addition to comparative measurements, certified reference materials such as the IRMM 184–187 series (Joint Research Centre, IRMM, Geel, Belgium) or NBS CRM U100, U500, and U850 (New Brunswick Laboratory, Argonne, IL, USA), are available for quality assurance [24]. However, it is important to note that for many nuclear applications matrix-matched certified nuclear reference materials (e.g. spent fuel) are not available [1].</p><!><p>Among several separation concepts being developed worldwide, pyrochemical treatment of spent fuel is an experimental approach aiming at minimizing the amount and radiotoxicity of both spent nuclear fuel and radioactive waste [25]. Most pyrochemical separation processes are based on electro-refining of metallic fuel dissolved in molten salts, e.g. LiCl–KCl eutectic mixtures [26]. For process optimisation, both salt mixtures and elements/isotopes deposited on the electrodes need to be analysed. Certified matrix-matched reference materials are currently not available for quality control of such analysis.</p><!><p>Cross-validation of γ-spectrometry and SF-ICP-MS measurements for the assessment of the concentration of a 237Np stock standard solution [2]</p><!><p>As there is only a few literature data available [8, 9, 27], such a well characterised 237Np standard solution can be employed to identify the corresponding most sensitive ICP-OES emission wavelengths. Subsequently those 237Np wavelengths have to be selected that are not suffering from spectral interference caused by the occurrence of other concomitant elements in the analyte solution. In the case of samples originating from experiments related to pyrochemical treatment of spent fuel, for example, the most sensitive 237Np emission line at λ = 382.92 nm cannot be employed at all because a serious spectral overlap from excess Nd, that is present is such samples, hampers the reliable determination of 237Np at this wavelength [2]. Trustworthy quantification of 237Np, however, can be carried out at the emission wavelengths λ = 410.84, 429.09 and 456.04 nm in such cases with results comparing well to SF-ICP-MS measurements carried out at m/z 237 [2]. If the investigated sample contains excess amounts of U, however, i.e. in the case of non-separated fuel solutions, both HR-ICP-OES and SF-ICP-MS analyses may suffer from spectral interferences requiring a separation of Np from the fuel matrix prior to analysis. Altogether the use of complementary instrumental techniques is highly recommended to ensure the accuracy of the obtained results, because no matrix-matched certified reference materials for the analysis of Np in spent fuel are currently available.</p><!><p>a Comparison of Na data in a suite of salt samples as determined by sector field ICP-MS and ICP-OES highlighting the high reproducibility of the ICP-OES results as well as the disagreement between both instrumental approaches. b Internally consistent Na concentration results obtained at two different emission wavelengths using HR-ICP-OES. All data from Ref. [17]. See text for details</p><!><p>The robustness and sensitivity of HR-ICP-OES analysis, in turn, offer highly reproducible results and low detection limits (down to sub-µg kg−1 levels) for Na, K, and Li [17]. Concerning quality control, the use of certified water reference materials is justified for such analysis because the actual salt samples are diluted up to ~400,000-times for HR-ICP-OES analysis [17]. As such, the concentrations of the alkali elements are at similar levels in both the employed reference materials and the analyte solutions. The experimental concentrations established for Na, K, and Li in 3 different water reference materials agreed well with the certified values underpinning the accuracy of the applied HR-ICP-OES procedures [17].</p><p>For additional quality assurance, two emission wavelengths of the same element (e.g. Na) can be used to check for internally consistent results (Fig. 3b). The validity of the graphically sound results can be further proven mathematically by the confidence levels of the regression parameters being 1.019 ± 0.020 (slope) and −5.116 ± 5.299 (intercept) (p = 0.033). This data confirms that these parameters are not significantly different from 1 and 0. If similar results are obtained for the same element at two different wavelengths at least, this fact indicates the absence of spectral interferences underpinning the quality of the obtained HR-ICP-OES data [17].</p><p>Taken together, SF-ICP-MS measurements of the alkaline elements Na, K, and li were less stable than HR-ICP-OES analysis, revealing instrumental drifts that could only be partly compensated for. The HR-ICP-OES results, however, were highly reproducible and validated through the beneficial agreement between experimental and certified concentrations of Na, K, and Li in several certified reference materials [17]. The applied HR-ICP-OES procedures proved to be reliable, robust, more straightforward and less laborious than the SF-ICP-MS approach.</p><!><p>Complementary analysis becomes especially important when dealing with spent fuel. Because of security and safety issues as well as expenses for the transport, spent fuel is normally not shipped to another laboratory for comparative analysis. Therefore, it is highly desirable to develop diverse analytical methods in-house that can be employed to cross-validate each other. Ideally, these analytical procedures can avoid laborious separation procedures, thereby also limiting the radiation dose to the analyst during sample preparation and speeding up the overall quantification step.</p><!><p>SF-ICP-MS spectrum from m/z 240 to 246 of a spent fuel sample revealing isobaric overlaps that hamper the reliable determination of Am [3]. See text for details</p><!><p>In the absence of an Am standard solution, however, calibration of the ICP-MS response is not trivial at all either. The closest element in the mass spectrum, for which a commercial standard solution can be purchased easily, is U. Calibrating the ICP-MS instrument for Am with this natural U standard solution, assumes that both elements behave very similarly during measurements though. While this assumption already gives reasonable results, the accuracy of the calibration strategy can be improved further by also taking into account the mass bias and oxide formation rate of Th and U, as described in detail elsewhere [3]. Briefly, this calibration methodology has been tested and validated through the analysis of various spent fuels [3]. The legitimacy of this experimental approach has been confirmed by the fact that the mean Am concentration for each spent fuel assessed via ICP-OES and ICP-MS differed at most by 4 % from each other [3].</p><p>Regarding ICP-OES, the above calibration strategy is not feasible at all because an Am standard solution is required for this instrumental approach. To this end, 241Am can be separated from a concentrated 241Pu solution using liquid extraction followed by extraction chromatography [4]. While the isotopic purity of the 241Am fraction can be checked via SF-ICP-MS as well as α- and γ-spectrometry, the latter allows the determination of its actual concentration [4]. With this well-characterised 241Am standard solution at hand, selective and sensitive ICP-OES Am emission lines were identified for spent fuel analysis [3]. Low detection limits (i.e. 0.07 µg kg−1 at λ = 283.226 nm) helped to increase dilution factors of actual spent fuel solutions, thereby keeping the radiation dose to the operator of the ICP-OES instrument as low as possible [3, 4]. Because 241Am and 241Pu emit light at different wavelengths, the isobaric spectral interference observed in the mass spectrum of ICP-MS, is not present in ICP-OES analysis. It is worth noting, however, that due to the complexity of the Am emission spectra and the much smaller isotopic shift of 241Am and 243Am compared to that of U isotopes, peak deconvolution strategies would be necessary to extract Am isotopic information from the HR-ICP-OES spectra. In other words, using HR-ICP-OES for Am analysis, the obtained emission signal reflects the sum of all Am isotopes, i.e. its concentration. Therefore, a direct ICP-OES determination of the Am concentration in spent fuel solutions is possible that can be used to cross-validate the results obtained via ICP-MS.</p><!><p>Representative ICP-OES calibration curves for neodymium (Nd) at the 3 emission wavelengths λ = 401.225, 410.946, and 430.367 nm highlighting the high sensitivity and performance of the upgraded instrumental set-up</p><p>Comparison of Nd concentrations (average ± SD, [µg kg−1]) in a solution of a dissolved spent fuel obtained via HR-ICP-OES analysis employing external calibration versus standard addition [5]</p><!><p>The direct determination of the Nd concentration in spent fuel via ICP-MS is complicated by the fact that its Nd isotopic composition varies distinctly from the natural one. In addition, the ICP-MS signals of some Nd isotopes are overlapped by isobaric interferences (142Nd by 142Ce, 148Nd by 148Sm, and 150Nd by 150Sm) that cannot be resolved spectroscopically, even if SF-ICP-MS is employed. Additionally, the normally small isobaric contribution of 144Ce to the signal of 144Nd also needs to be known from γ-spectrometry.</p><p>Knowing the actual Nd isotopic composition (from separate measurements, for example), the ICP-MS signal of the four Nd isotopes at m/z 143, 144, 145, and 146 provides a solid basis for the reliable determination of the Nd concentration in spent fuel. Comparison of the ICP-MS data to the results obtained by ICP-OES again serves as a valuable tool for cross-validating an analytical approach that requires careful attention to guarantee high quality analytical results [5].</p><!><p>This report highlighted some fundamental challenges related to the elemental and isotopic analysis of actinides and fission products in a variety of nuclear samples coming from different parts of the nuclear fuel cycle including nuclear forensics. The use of HR-ICP-OES and SF-ICP-MS provides accurate and precise analytical data relevant for all kind of issues related to nuclear security and safety.</p><p>Both instrumental approaches have an excellent potential for this endeavour, but their individual application may be limited by several distinct constraints in particular cases. Frequently, HR-ICP-OES and SF-ICP-MS complement each other in such difficult-to-analyse instances. While the brilliant performance of SF-ICP-MS for elemental and isotopic analysis is generally well accepted, HR-ICP-OES also serves as a powerful, yet underestimated, tool for such kind of analysis.</p><p>As it is much easier to analyse radioactive samples in-house than to ship them to another external laboratory, at least two independent analysis methods need to be available internally. In addition, it is essential to use certified reference materials for quality assurance, whenever this is possible. In the absence of such reference materials, the application of two independent analytical methods and different calibration strategies may be applied to support the quality of the acquired analytical data. Once accurate and precise analytical data are available, issues related to nuclear security and safety can be addressed accordingly.</p>

PubMed Open Access

FTIR Spectroscopic Study of the Secondary Structure of Globular Proteins in Aqueous Protic Ionic Liquids

Protein misfolding is a detrimental effect which can lead to the inactivation of enzymes, aggregation, and the formation of insoluble protein fibrils called Amyloids. Consequently, it is important to understand the mechanism of protein folding, and under which conditions it can be avoided or mitigated. Ionic liquids (ILs) have previously been shown as capable of increasing or decreasing protein stability, depending on the specific IL, IL concentration and which protein. However, a greater range of IL-proteins need to be systematically explored to enable the development of structure-property relationships. In this work, the secondary structure of four proteins, lysozyme, trypsin, β-lactoglobulin and α-amylase, were studied in aqueous solutions of 10 protic ionic liquids (PILs) with 0–50 mol% PIL present. The PILs consisted of ethyl-, ethanol-, diethanol- and triethanolammonium cations paired with nitrate, formate, acetate or glycolate anions. The secondary structure was obtained using ATR-FTIR spectroscopy. It was found that lysozyme and trypsin retained its secondary structure, consistent with a native folded state, for many of the aqueous IL solutions which contained a formate or nitrate anion at the most dilute concentrations. In contrast, α-amylase and β-lactoglobulin generally had poor stability and solubility in the IL solutions. This may be due to the isoelectric point of α-amylase and β-lactoglobulin being closer to the pH of the solvents. All four proteins were insoluble in ethyl-, ethanol- and diethanolammonium acetate, though α-amylase and trypsin retained their secondary structure in up to 20 and 30 mol% of triethanolammonium acetate, respectively. It was evident that the protein stability varied substantially depending on the protein-IL combination, and the IL concentration in water. Overall, the findings indicated that some ions and some ILs were in general better for protein solubility and stability than others, such as acetate leading to poor solubility, and EAN and EAF generally leading to better protein stability than the other PILs. This study of four proteins in 10 aqueous PILs clearly showed that there are many complexities in their interactions and no clear general trend, despite the similarities between the PIL structures. This highlights the need for more and larger studies to enable the selection and optimization of PIL solvents used with biomolecules.

ftir_spectroscopic_study_of_the_secondary_structure_of_globular_proteins_in_aqueous_protic_ionic_liq

Introduction<!><!>Experimental Methods<!>Results<!><!>Results<!><!>Results<!><!>Results<!><!>Results<!><!>Results<!><!>Results<!><!>Results<!>Discussion<!>Conclusions<!>Author Contributions<!>Conflict of Interest Statement

<p>Proteins are macromolecules made up of sequences of amino acid sub units. The folding of these amino acid chains determines the proteins three-dimensional structure and functionality. The hydrophilic and hydrophobic characteristics of amino acids are important contributors to protein shape, folding and solubility. The folding of proteins are based on their amino acid sequence, and many proteins easily unfold outside of their native environment, and then can refold, mis-fold, or aggregate (Fernández and Scott, 2003). One of the major problems in stabilizing proteins is that they can easily denature and form aggregates during the process.</p><p>Previously, it was considered that non-aqueous solvents make proteins insoluble or unstable, and have therefore not been used extensively (Rariy and Klibanov, 1997). However, while polar organic solvents are generally detrimental to proteins, studies have shown that proteins can be stable in some solutions containing polar molecules, such as glycerol, which has been shown to have a stabilizing effect on lysozyme (Rariy and Klibanov, 1997; Paciaroni et al., 2002; Cinelli et al., 2004; Gögelein et al., 2011). In addition, non-polar solvents, such as cyclohexane, can be tolerated by proteins at high concentrations (Pace et al., 2004).</p><p>Ionic liquids (ILs) have emerged as interesting solvents for biological molecules, with a broad range of polar and non-polar ILs appearing to be beneficial for protein stability (Rodrigues et al., 2014). ILs are highly tailorable solvents, which allows many solvent properties to be modified and functional groups to be included. For example, their tailorable hydrophobicity makes them potentially useful as solvents for proteins which have poor solubility in water. Due to the tailorability of ILs it is feasible that these solvents could be designed to have either a stabilizing or destabilizing effect on proteins, depending on what is needed for a specific application. This has been shown for aqueous solutions containing ILs, where depending on the IL, the protein lysozyme can be stabilized (Zhao, 2016), crystallized(Niedzialkowska et al., 2016), denatured(Ortore et al., 2009), or formed into Amyloid fibrils(Cao et al., 2004; Byrne and Angell, 2009). However, the high tailorability of ILs also means that there are many possible combinations of IL cations, anions and proteins, along with water concentrations or other additives. It is unclear whether there are particular ILs which will be beneficial for classes of proteins, or whether each protein will need the solvent optimized individually. Consequently, it is necessary to build up systematic knowledge of proteins in IL containing solvents, to enable the design of IL solvents for specific proteins.</p><p>Protic ionic liquids (PILs) have been used in this study due to the ease of modifying their structure and properties, including their polarity, cation alkyl chain length, anion nucleophilicity, hydrophobicity, pH, and ionicity. PILs are synthesized by proton transfer from a Brønsted acid to a Brønsted base with equal molar ratios (Greaves and Drummond, 2008). There are previous studies which have shown the effect of certain PILs on protein stability, with the vast majority having the PILs present in aqueous solutions. Ethylammonium nitrate (EAN) was one of the first ionic liquids trialed for potentially being capable of stabilizing proteins, as it has some similar characteristics to water, such as being able to form a hydrogen bonded network (Garlitz et al., 1999), high polarity, and the ability promote amphiphile self-assembly (Evans et al., 1982). More recent studies on hen egg white lysozyme (HEWL) in EAN and triethylammonium triflate (TEATf) solutions have shown dissolution and inhibition of amyloid fibrils formation (Byrne and Angell, 2009). However, limited work has been done to understand the secondary structural changes of proteins in PILs, which PILs are optimal, the role of the cation and anion, or what PIL-water concentrations are best.</p><p>A variety of aqueous aprotic ILs has also been explored for their ability to stabilize proteins. Similar to PILs, it appears that there is a significant variation in protein stability depending on the specific protein, ionic liquid, and water content. For example, the structural stability of cytochrome c (at 50 mM) was studied using temperature-induced FTIR spectroscopy in a few biocompatible IL solutions with 20 mg/ml of 0.5–3 M IL in water. The ILs contained dicyanamide, saccharinate, or dihydrogen phosphate (dhp) anions paired with a butylmethylpyrrolidinium cation, or choline chloride (Fujita et al., 2005). It was found that solutions containing choline dhp showed no changes in protein structure during exposure to temperatures of 110 °C, providing long term thermal stability of the protein(Fujita et al., 2005). FTIR analysis of lipase in ILs containing dicyanimide, alkylsulfate, nitrate and lactate anions paired with [BMIm][BF4] showed the secondary structure was maintained, with the conformation of the enzyme closely resembling the native one (Madeira Lau et al., 2004). There has been evidence showing that ILs can stabilize RNase at pH 7.4, with previous experiments identifying certain beneficial ions like 1-alkyl-3-methylimidazolium,N,N-dialkylpyrrolidinium and tetraalkylammonium for protein stability, whereas [PF6] and [BF4] were detrimental (Weingärtner et al., 2012).</p><p>Previously, we reported on the activity and conformation of hen egg white Lysozyme in aqueous solutions of ethylammonium nitrate (EAN), ethylammonium formate (EAF), ethanolammonium formate (EtAF), ethanolammonium nitrate (EtAN), aqueous solutions of molecular solvents, and a selection of highly concentrated salts (Wijaya et al., 2016, 2018). Of these it was observed that EAN and EtAF led to maximum lysozyme activity, comparable to conventional salts, whereas EAF and EtAN did not. Similarly, lysozyme activity was maintained in up to very highly concentrated PIL solutions for EAN and EtAF, whereas EAF and EtAN led to denaturation and aggregates at higher PIL concentrations. The maximum activity of lysozyme was lower in the molecular solvents than in EAN or EtAF solutions. Interestingly the two PILs of EAN and EtAF did not share common ions, indicating that the combination of IL ions is perhaps more important than which ions are present. It also clearly showed that comparable activities can be obtained using PILs compared to conventional salts, but without any salt solubility limit due to the miscibility of these PILs with water (Wijaya et al., 2016).</p><p>In this work we have used PILs as tuneable solvents to investigate the effect of the solvent environment on protein stability. We have investigated the secondary structure of four proteins in aqueous solutions of 10 PILs. This significantly extends upon our previous work and aims at exploring the role of individual ions and the combinations of cations and anions. The chemical structures of the 10 PILs are provided in Figure 1 and have been selected to cover short alkylammonium cations with and without hydroxyl groups, and with primary, secondary and tertiary structures. These were paired with nitrate, formate, acetate, and glycolate anions. A selection of properties of the neat ILs are provided in Table S1 of the ESI, including their molecular weight, melting point, viscosity and density. The proteins selected were lysozyme, trypsin, α-amylase, and β-lactoglobulin. The proteins were selected to provide a variety of sizes, shapes, iso electric points (pI), hydrophobicity/hydrophilicity and secondary structures. The secondary structures of the proteins in each solution were characterized using Fourier Transform Infrared Spectroscopy (FTIR).</p><!><p>Chemical structures and abbreviations of the 10 PILs used in this investigation (A) ethylammonium nitrate (EAN), (B) ethanolammonium nitrate (EtAN), (C) ethylammonium formate (EAF), (D) ethanolammonium formate (EtAF), (E) ethylammonium glycolate (EAG), (F) ethanolammonium glycolate (EtAG), (G) ethylammonium acetate (EAA) (H) ethanolammonium acetate (EtAA), (I) diethanolammonium acetate (DetAA), and (J) triethanolammonium acetate (TetAA).</p><!><p>Lysozyme from chicken egg white (EC 3.2.1.17), trypsin from bovine pancreas (EC 3.4.21.4), α-amylase from Bacillus licheniformis (EC 3.2.1.1) and β-lactoglobulin from bovine milk (EC 2329289) were obtained from Sigma Aldrich. Ethylamine (Sigma-Aldrich, 70 wt%), ethanolamine (Chem Supply, 99%), diethanolamine (Chem Supply, 98%), triethanolamine (Chem Supply, 99%), nitric acid (Chem supply, 70% w/w), acetic acid (Chem Supply, 99%), glycolic acid (Chem Supply, 99%) and formic acid (Merck, 98–100%) were used without further purification for the synthesis of the PILs.</p><p>The PILs were synthesized by slowly adding equimolar amounts of the acid to the base. The solution was continuously stirred, and the temperature maintained below 10°C using an ice bath. Small portions of methanol were added to the amine for the PILs which otherwise would form a solid during the reaction. Methanol and excess water were removed by drying under vacuum at >0.01 Torr on a rotary evaporator. Further drying was carried out using a LabconcoFreeZone® 4.5 Liter Freeze Dry System, for up to 24 h. The water content of the PILs was determined by Karl Fischer Titration, using a Mettler Toledo DL39 Karl Fischer coulometer, and the PILs all had <2 wt% water. Aqueous PIL solutions were prepared for each PIL with 5, 10, 20, 30, and 50 mol% of PIL ions present. The wt% for each of these compositions is provided in Table S2 of the ESI.</p><p>Samples of the proteins in the PIL-water solutions were prepared using 20 mg/ml of each of these four proteins in each of the 50 aqueous PIL solvents, along with water for comparison. The proteins were added to 1 ml Eppendorf tubes in a powder form, followed by addition of the solvents. The proteins were dissolved by gently vortexing for 10–30 s. Samples which did not dissolve were then further vortexed for 1–5 min and the protein state checked after 24 h.</p><p>Fourier transform infrared (FTIR) spectra were recorded using a Perkin-Elmer Frontier MID/FAR IR instrument with a diamond ATR (attenuated total reflectance) crystal. Protein concentrations of 20 mg/ml were used for all samples, and the samples were left to equilibrate for 1 h prior to measurements, with all measurements being made in the next 1 h. Each sample was characterized using 32 scans with a resolution of 2 cm−1 over the range of 400 cm−1 to 4000 cm−1. FTIR spectra of each solvent, without the protein present, were acquired under the same conditions and used for solvent subtraction.</p><!><p>Solutions of each of the 10 PILs, shown in Figure 1, were prepared with 5, 10, 20, 30, and 50 mol% of the PIL ions in water, leading to 50 individual aqueous PIL solvents (the corresponding concentrations in wt% are provided in Table S2 of the ESI). These concentrations are well-outside the concentration ranges used for conventional salts and buffers for protein stability work, and beyond the solubility of many salts in water. The protein stability of four globular proteins, lysozyme, trypsin, α-amylase and β-lactoglobulin, were investigated in each of the aqueous PIL solutions. These proteins were selected to explore a diverse range of enzymatic globular proteins. The size and isoelectric point for these four proteins is provided in Table 1.</p><!><p>Size and isoelectric point, pI, of the four proteins.</p><!><p>Visual observations were made of the resulting solutions to identify which solutions led to the 20 mg/ml proteins dissolving, forming a gel, or not fully dissolving. These observations have been provided in Tables 2–5 for lysozyme, trypsin, α-amylase, and β-lactoglobulin, respectively. The open squares represent the protein-solvent conditions where the protein did not fully dissolve, and crosses where the addition of the protein caused the solution to form a gel.</p><!><p>Summary of the physical state and secondary structure of lysozyme in the aqueous PIL solvents.</p><p>FTIR consistent with native state.</p><p>FTIR small changes.</p><p>Changes in the secondary structure.</p><p>Visually observed to not fully dissolve.</p><p>Viscous gel.</p><p>Summary of the physical state and secondary structure of α-amylase in the aqueous PIL solvents.</p><p>FTIR consistent with native state.</p><p>Changes in the secondary structure.</p><p>Visually observed to not fully dissolve.</p><p>Viscous gel.</p><p>Formed a gel after 5–10 min.</p><p>Summary of the physical state and secondary structure of trypsin in the aqueous PIL solvents.</p><p>FTIR consistent with native state.</p><p>Changes in the secondary structure.</p><p>Visually observed to not fully dissolve.</p><p>Viscous gel.</p><p>Summary of the physical state and secondary structure of β-lactoglobulin in the aqueous PIL solvents.</p><p>FTIR consistent with native state.</p><p>Changes in the secondary structure.</p><p>Visually observed to not fully dissolve.</p><p>Viscous gel.</p><p>formed a gel after 5–10 min.</p><!><p>In general, the proteins dissolved well in these aqueous PIL solutions, with the exceptions of the acetates. All four proteins did not fully dissolve in any of the EAA, EtAA, or DEtAA containing solutions. In contrast, all four proteins were soluble in the bulkier TEtAA, for TEtAA concentrations of 5, 10, and 20 mol%. For the non-acetates, α-amylase was not soluble in any of the EtAF solutions, and β-lactoglobulin was not soluble in any EAF solutions.</p><p>For a few protein-IL combinations, the proteins fully dissolved and then formed a very viscous solution, which has been referred to as a gel. The gelation of proteins is highly dependent on the solvent condition, and typically occurs when proteins partially unfold and form cross links between protein molecules. Globular proteins require higher concentration for gel formation, with interactions at low concentrations tending to occur within molecules rather than between molecules, and hence the gel network is not formed (Ziegler and Foegeding, 1990). It has been shown that some proteins like α-lactalbumin do not exhibit gelling properties because of the nature of their poly-peptide linkage (Paulsson et al., 1986). Gel formation occurs when partially unfolded proteins develop uncoiled polypeptide segments which interacts at certain points to form a three dimensional cross-linked network, and with gels forming if there is sufficient cross-linking present (Hill et al., 1998). All four proteins formed a gel in EtAN solutions immediately when the concentration of EtAN was 20 mol% or higher, as well as at 10 mol% for α-amylase in EtAN. In addition, α-amylase formed a gel in EAF and EtAG for all PIL concentrations trialed. This is consistent with what has previously been reported with gelation more likely for proteins containing more hydrophobic groups, and α-amylase was the most hydrophobic of the proteins used (Zayas, 1997). In addition, the ability of EtAN to act as a hydrogen bond donor or acceptor is likely to contribute to the gel formation, with perhaps the strong nitrate anion contributing in destabilizing the protein and exposing the hydrophobic core.</p><p>FTIR spectroscopy was used to investigate the secondary structure of the proteins in these aqueous PIL solutions. FTIR is sensitive to protein structural changes, and unlike circular dichroism it is useable for these solutions, which can be considered as extremely concentrated salt solutions. There are two broad absorption bands observed for proteins using FTIR, conventionally called amide I and amide II bands, at wavenumbers 1,700–1,600 cm−1 and 1,600-1,500 cm−1, respectively. The Amide I band is more commonly used for characterizing the secondary structure, and is due to C = O stretching vibrations of the peptide bonds, which are modulated by the secondary structure (α-helix, β-sheet, etc.). The sum of the spectral contributions from these features lead to a broad band with overlapping sub spectra, and the frequency associated with the main protein secondary structural features are provided in Table 6. The Amide I band for each of the four proteins in water is shown in Figure 2.</p><!><p>The FTIR frequency of the dominant secondary structural components of proteins contributing to the Amide I band (Creamer et al., 1983; Knubovets et al., 1999).</p><p>Amide I band from FTIR spectra for (A) lysozyme, (B) trypsin, (C) α-amylase and (D) β-lactoglobulin at 20 mg/ml in water.</p><!><p>The solvent contribution was subtracted for each sample, which is difficult due to the ILs containing features which overlap with the Amide I band. An example is shown in Figure 3 for lysozyme in 5 mol% EAN, with the FTIR spectra of the solvent, and protein in solvent, before solvent subtraction shown in Figure 3A, and the resulting spectra after subtraction in Figure 3B. Consequently, due to the solvent contributions in the wavenumber region of interest these measurements require identical solvents to be used for the solvent blank and protein containing solution, and identical volumes for both solutions. In addition, for it to be considered a good background subtraction, the baseline needed to be straight between 1,800–1,500 cm−1 and 2,000–1,750 cm−1, consistent with the literature methods (Kumosinski and Farrell, 1993; Wijaya et al., 2016).</p><!><p>FTIR spectra for (A) 5 mol% EAN in water (lower trace) and 20 mg/ml of lysozyme in this solution (upper trace), and (B) the Amide I band after solvent subtraction for the spectra in (A).</p><!><p>Subtracted FTIR spectra were obtained for all the protein-aqueous IL solutions, where the protein dissolved. These are provided in Figures S1–S25 of the ESI. Spectra were also acquired for lysozyme in a solution of EAN at 10 mol% at 1, 2, and 3 h after mixing which showed minimal variation between spectra, and these are provided in Figure S26 of the ESI. There was a significant variation in the behavior of the secondary structure for each protein-IL combination, varying between negligible change and major disruption of the secondary structure. A summary is provided in Tables 2–5 for which solvents retained the secondary structure of the protein consistent to what was seen in water (♦). For some spectra there was only minor changes to the secondary structure, and these have been denoted by an additional * next to the ♦.</p><p>The proteins in all the other PIL-water series, where they were soluble, showed evidence of changes in their secondary structures at either some, or all, PIL concentrations. It is evident from Tables 2–5 that the solvent conditions which supported the proteins in having FTIR spectra, consistent with the native state, were substantially different for the four proteins. As shown in Tables 2–5 there were only two protein-IL combinations where the protein retained its secondary structure across all concentrations of the ILs. These were lysozyme in EAN and EtAF. The amide I band for lysozyme in EAN solutions is shown in Figure 4, and it is apparent that there was little change in the secondary structure from 0 to 50 mol% of EAN. However, when the EAN concentration was 50 mol% there appeared to possibly be a small increase in the proportion of β turn present. There was good stability for lysozyme in solutions of 5 mol% of many of the PILs (EAN, EAF, EAG, EtAN, EtAF, EtAG and TEtAA), and at 10 and 20 mol% for many of these. For these solutions, the dominant peak in the FTIR spectra was consistent with the alpha helix structures being retained.</p><!><p>Amide I region of the FTIR spectra of lysozyme in aqueous solutions of 5, 10, 20, and 30 mol% of EAN.</p><!><p>The spectra for trypsin in aqueous solutions containing 5 mol% of EtAN or TEtAA are provided in Figure 5. At this lowest PIL concentration Trypsin retained its secondary structure in EtAN, shown in Figure 5, and also in EtAF and EtAG. However, for the PILs of EAG and TEtAA there were some changes to the secondary structure, consistent with a small increase in beta structure, and the spectra for Trypsin in 5 mol% TEtAA is shown in Figure 5. The secondary structure of Trypsin in 5 mol% EAN and EAF underwent significant changes, showing large proportions of beta structures.</p><!><p>Amide I region of the FTIR spectra of trypsin in aqueous solutions of 5 mol% of EtAN and TEtAA.</p><!><p>Based on the FTIR spectra, the secondary structure of amylase was altered in many of the PIL containing solvents. An example is shown in Figure 6 for amylase in aqueous EAN solvents. At 5 and 10 mol% there were some minor changes in the spectra, whereas at 20 mol% there appears to be a major loss of alpha helix structures and a large gain in beta structures. For higher PIL concentrations of 30 or 50 mol % EAN the amylase did not dissolve.</p><!><p>Amide I region of the FTIR spectra of α-amylase in aqueous solutions of 0, 5, 10, and 20 mol% of EAN.</p><!><p>It was evident that the dominant change in secondary structure, away from native structure for all of four proteins in the PIL-solutions, was an increase in the proportion of beta structures present. These beta structures then led to the aggregation and gelation of the proteins, likely through disruption to the intermolecular interactions between hydrophobic regions, leading to exposed hydrophobic groups. These exposed hydrophobic surfaces can interact between proteins, and lead to either amorphous or fibril aggregates.</p><!><p>The stability and solubility of proteins are important factors for the pharmaceutical industry (Brader et al., 2015). The stability of proteins depend on environmental properties such as pH, ionic strength, and the presence of buffers/salts. Protein stability in an ionic solution is highly complex, and depends on the protein-ion, ion-ion, ion-water, protein-water and water-water interactions. The ions can modify the local structure of water molecules, increasing or decreasing how structured it is, and hence change how entropically favorable it is for proteins to be dissolved. It was evident from this investigation that the protein stability in concentrated aqueous ILs solutions varied substantially depending on the protein-IL combination.</p><p>There have been several efforts to minimize protein aggregation, enhance stability and improve protein crystallization, by utilizing appropriate screening techniques (Kheddo et al., 2014; Liu et al., 2016; Zhou et al., 2018). Here we used visual observations and FTIR as suitable screening methods for the characterization of protein stability. These relatively fast techniques enabled the investigation of four proteins in 50 different IL-water solutions. In this work, the ion concentrations were much greater than those used for conventional protein stability solutions in buffers, with concentrations from 5 to 50 mol% of ILs. This makes it difficult to compare with much of the conventional literature using aqueous buffers. In addition, the literature discussions surrounding salting in and salting out effects are valid for low concentrations of ions, whereas at higher concentrations previous studies have indicated that most ions behave with a combination of weak electrostatic repulsion and other attractive interactions, causing proteins to aggregate (Yang et al., 2010).</p><p>The properties of the ions have a significant influence on their interactions. Consistent with previous studies, the anion in our series of ILs had a dominant effect on the protein stability. This is attributed to its hydrogen bond forming capability, nucleophilicity properties, and ability of the anion to strongly interact with the enzyme, thus leading to conformational change in the structure and activity of the enzyme (Zeuner et al., 2011).</p><p>Previously, we showed that the combination of ions plays an important role in protein stability for lysozyme in PIL solutions of EAN, EtAN, EAF, EtAF. For these PILs, the data correlated well with the "Collins law of matching water affinity" (Collins, 2004) with good protein stability occurring where both ions could be considered either kosmotropic or chaotropic, but poor if they were different (Wijaya et al., 2016). In an non-IL study it was also found that when both anions had similar kosmotropic or chaotropic behavior, proteins usually were stabilized by a kosmotropic anion and chaotropic cation and destabilized by the opposite, for IL concentrations up to 1M (Yang et al., 2010). In this study we clearly see that the combination of ions was crucial; however, they behaved differently for each protein and many trends were not consistent across all proteins. The findings from this current study showed that the trends for those four PILs with Lysozyme did not extend to the other proteins. However, it was apparent that some ILs, such as EAN and EtAF were generally more favorable, and that some ions, such as acetate, were not. This highlights the importance of larger studies for proteins in ILs which will enable more robust structure-property relationships to be developed.</p><p>The acetate ion was the least favorable ion of the anions used in this investigation. It was observed that even at low concentrations all four proteins were insoluble in ethyl-, ethanol- and diethanolammonium acetate solutions, though all were soluble in the presence of triethanolammonium acetate at lower concentrations, with lysozyme and amylase retained in a native state. Acetate ions have previously been seen to decrease the solubility of proteins, though it depends on the cations they are paired with. For example, ILs containing acetate are weak stabilizers when paired with the more hydrophobic imidazolium or phosphonium cations (Wijaya et al., 2018), while acetate paired with triethylammonium has been shown to be a strong stabilizer (Attri et al., 2011). In this study we observed that the hydroxyl version of the tertiary ammonium cation, TEtA cation, also appears to enhance protein solubility, and stability for some proteins when paired with acetate. In contrast the acetate salts of the primary and secondary cations of EtA and DEtA led to poor protein solubility. The hydration number and ion-water interactions, as well as ion-pair formation, are crucial factors for how the ion and water in the solvent will interact with proteins. These depend on the size, shape and charge density of ions. Ions can be widely classified into either kosmotropes or chaotropes according to their ability to bind to water (Manincelli et al., 2007). Acetate can be considered a kosmotropic anion, and likely to have strong interactions with multiple water molecules. Considering the very high proportion of ions in these solutions, this may result in the majority of the water present to interact with acetate anions, and therefore either having fewer, or modified water interactions with the proteins, potentially leading to the poor solubility.</p><p>The comparison of the protein stability in these IL solutions to conventional aqueous salts is difficult, particularly because of the much higher concentrations of ions used here. One common ion description is of ions leading to salting in or salting out of proteins, but this is only meaningful for ion concentrations up to 1 M (Schröder, 2017). In this study, the ion concentrations are much greater than those used for conventional protein stability solutions in buffers. In terms of salting in and salting out, all the ions will all behave the same at these higher concentrations, with the ionicity effect leading to weak electrostatic repulsion contrasted with opposing attractive interactions, decreasing protein solubility, and increasing the preference to aggregate (Yang et al., 2010).</p><p>The effect of ions on proteins can be classified for many aqueous systems by using the Hofmeister series (Timasheff, 1993; Hribar et al., 2002; Pegram and Record, 2007; Yang et al., 2010; England and Haran, 2011; Schröder, 2017). In this series, cations and anions are ordered based on their tendency to solubilize, or stabilize proteins. This series definitely does not apply to protein stability in all IL solutions, with different studies showing that IL ions can follow the Hofmeister series, follow reverse Hofmeister series, or are anomalous to the Hofmeister series (Warren et al., 1966; Yang, 2009). Similarly, in this study we have clearly seen that there is not a consistent trend in the IL ions for protein stability or solubility across all four proteins, or with the Hofmeister series. This indicates that there are more complex interactions present, and that the "observational" Hofmeister series is not sufficient to classify ILs for protein stability (Mazzini and Craig, 2018). Alternative methods have recently been discussed by Mazzini and Craig, and better alternatives involve using ion water affinity and ion sizes, and considering specific-ion effects (Mazzini and Craig, 2018).</p><p>The isoelectric point, pI, of these proteins is given in Table 1, and these proteins were selected due to the broad range of pI's. The protein stability in these ILs indicated that the pI of the proteins had an effect. Trypsin and lysozyme had higher pI's and were stable in a broader range of these IL solvent conditions. In contrast, α-amylase and β-lactoglobulin had pI's below 7 and displayed generally poorer stability and solubility in the IL solutions, forming aggregates or precipitates in many of the solvent conditions trialed. This may be due to the pI of these proteins being closer to the pH of the solvents. In aqueous solutions, the pH scale is routinely used to measure proton activity. However, the conventional pH scale is not valid in neat ionic liquids or highly concentrated ionic liquid solutions such as these.</p><!><p>This study shows that the nature of proteins and the combination of ions are all important in stabilizing proteins. The changes in the secondary structure of proteins using FTIR spectroscopic analysis, proved an efficient tool to quantify wide arrays of protein-ion combinations. Understanding protein stabilization is a vast process involving different solvent-protein properties. In this work, we identified that some of the PILs containing nitrates, formates and glycolates were able to retain the secondary structure of proteins at the lower IL proportions, with poorer protein solubility at high IL concentrations. There was a significant variation over which ILs the four different proteins were stable, with generally EAN and EtAF being good co-solvents, whereas the acetate containing PILs led to poor proteins solubility. This study highlights the need for extensive studies on protein stability in IL solutions with a broader range of ILs, proteins, and IL concentrations.</p><!><p>RA completed the experimental work and much of the analysis and writing of the paper. TG trained and assisted RA with the experimental work and data analysis. CD assisted with interpretation of the results.</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>

PubMed Open Access

Strain-Specific Peptide (SSP) Interference Reference Sample: A Genetically Encoded Quality Control for Isobaric Tagging Strategies

Isobaric tag-based sample multiplexing strategies are extensively used for global protein abundance profiling. However, such analyses are often confounded by ratio compression resulting from the co-isolation, co-fragmentation, and co-quantification of co-eluting peptides, termed \xe2\x80\x9cinterference.\xe2\x80\x9d Recent analytical strategies incorporating ion mobility and real-time database searching have helped to alleviate interference, yet further assessment is needed. Here, we present the strain-specific peptide (SSP) interference reference sample, a tandem mass tag (TMT)-pro-labeled quality control that leverages the genetic variation in the proteomes of eight phylogenetically divergent mouse strains. Typically, a peptide with a missense mutation has a different mass and retention time than the reference or native peptide. TMT reporter ion signal for the native peptide in strains that encode the mutant peptide suggests interference which can be quantified and assessed using the interference-free index (IFI). We introduce the SSP by investigating interference in three common data acquisition methods and by showcasing improvements in the IFI when using ion mobility-based gas-phase fractionation. In addition, we provide a user-friendly, online viewer to visualize the data and streamline the calculation of the IFI. The SSP will aid in developing and optimizing isobaric tag-based experiments.

strain-specific_peptide_(ssp)_interference_reference_sample:_a_genetically_encoded_quality_control_f

INTRODUCTION<!>Materials.<!>Mouse Tissue Preparation.<!>Tandem Mass Tag Labeling.<!>Liquid Chromatography and Tandem Mass Spectrometry.<!>Data Analysis.<!>Assembling the Strain-Specific Peptide (SSP) Reference Sample.<!>Using the SSP Reference Sample to Compare hrMS2, SPS-MS3, and RTS-MS3 Data Acquisition Methods.<!>SSP Reference Sample Viewer Offers a Simple Web Interface to Determine IFI Values and Visualize Individual Peptides.<!>CONCLUSIONS

<p>Isobaric labeling strategies are a predominant technique for mass spectrometry-based quantitative proteome profiling. Sample multiplexing permits increased throughput, minimizes missing values within an experiment, and improves statistical power to detect differences between samples. However, a major caveat of isobaric tag-based quantitative strategies is ratio compression due to "interference," which is a consequence of the co-isolation, co-fragmentation, and co-quantification of co-eluting peptides. Recently utilized techniques, such as high-field asymmetric-waveform ion-mobility spectrometry (FAIMS)-based gas-phase fractionation1-3 and real-time database searching (RTS),4 have reduced interference. However, the need remains to better quantify and evaluate interference to benchmark and improve emerging data acquisition strategies. To this end, we introduce the strain-specific peptide (SSP) interference reference sample. Here we leverage the absence of tandem mass tag (TMT) reporter ion signal in specific channels due to missense mutations in the protein-coding region as the premise for a novel quality control reference sample for isobaric tag-based workflows.</p><p>The basis of the SSP is the genetic variation present in a set of genetically distinct and defined inbred mouse strains, notably representing contributions from three subspecies of the house mouse (Mus musculus).5 Specifically, we use the eight founder strains of the Collaborative Cross (CC) recombinant inbred mouse panel and the related Diversity Outbred (DO) mouse population.6,7 Numerous studies have utilized these populations (founder strains, CC, and DO) to investigate complex traits, leveraging their diverse and reproducible genetic and phenotypic variation. The founder strains consist of five classical inbred strains: 129S1/SvImJ (129), A/J (AJ), C57BL/6J (B6), NOD/ShiLtJ (NOD), and NZO/H1LtJ (NZO), and three wild-derived inbred strains: CAST/EiJ (CAST), PWK/PhJ (PWK), and WSB/EiJ (WSB).5 Notably, B6 is among the most used inbred strain and thus the reference proteome of M. musculus for UniProt.8 The other founder strains possess characteristic phenotypes that make them important models for specific traits and diseases. This phenotypic diversity of the founder strains reflects thousands of mutations in the protein coding and noncoding regions of the mouse genome, providing the foundation for the SSP reference sample.</p><p>Much of the genetic variation represents single-nucleotide polymorphisms (SNPs), with differing alleles becoming fixed across the founder strains through divergent evolution and subsequent inbreeding. Some of the SNPs represent missense mutations, which are then manifested in the relative abundance of the native peptide. As such, global mass spectrometry-based protein profiling can be used to distinguish peptides that contain missense mutations. Consider the scenario of a protein that is expressed in all of the founder strains and one of the strains possessing a missense mutation. When profiling the relative peptide abundance of the native peptide across the founder strains, the strain with the missense mutation should be absent for the reporter ion signal in its corresponding TMT channel. Any TMT signal measured in the strain with the missense mutation is representative of the degree of interference, which can thus be quantified and compared across different analyses using the interference-free index (IFI).9,10</p><p>We showcase the SSP reference sample by comparing three data acquisition methods—hrMS2 (high-resolution MS2), SPS-MS3 (synchronous precursor selection-MS3),11 and RTS-MS3 (real-time database searching-MS3)4—using an Orbitrap Eclipse mass spectrometer with and without FAIMS-based gas-phase separation.1-3 We observed improvements in IFI for RTS-MS3 and FAIMS-based data acquisition methods. We have also developed a user-friendly, online tool to streamline the calculation of the IFI for the SSP reference sample. This R-Shiny application is available at: https://japaulo.shinyapps.io/ssp_app/. In all, the SSP is a valuable reference sample for developing and optimizing isobaric tag-based workflows.</p><!><p>Tandem mass tag (TMT) isobaric reagents (TMTpro) and trypsin were purchased from ThermoFisher Scientific (Waltham, MA), LysC from Wako Chemicals (Richmond, VA), and organic solvents and water were from J.T. Baker (Center Valley, PA). Unless noted otherwise, all other chemicals were from Pierce Biotechnology (Rockford, IL).</p><!><p>The murine liver tissue was provided by The Jackson Laboratory for the following strains: 129S1/SvlmJ (129), A/J (AJ), C57BL/6J (B6), CAST/EiJ (CAST), NOD/ShiLtJ (NOD), NZO/H1LtJ (NZO), PWK/PhJ (PWK), and WSB/EiJ (WSB). Liver tissue was processed for mass spectrometry analysis as described previously.12</p><!><p>TMT labeling, mass spectrometry data acquisition, and analysis were performed as described previously.13 In brief, 6 μL of TMTpro reagent (from a 20 μg/μL stock) and 14 μL of acetonitrile were added to 50 μg of peptides (dissolved in 50 μL of 200 mM EPPS, pH 8.5) and incubated for 1 h at room temperature. The reaction was then quenched with hydroxylamine to a final concentration of 0.3% (v/v). Mouse peptides were combined at a 1:1 ratio across all channels, and the pooled peptide sample was vacuum-centrifuged to near dryness and finally peptides were desalted by C18 Sep-Pak solid-phase extraction (Waters).</p><!><p>Mass spectrometry data were collected on an Orbitrap Eclipse mass spectrometer coupled to a Proxeon EASY-nLC 1200 liquid chromatograph (ThermoFisher Scientific). Peptides were separated on a 100 μm inner diameter microcapillary column that was packed with ~35 cm of Accucore150 resin (2.6 μm, 150 Å, ThermoFisher Scientific). We loaded between 0.5 and 1 μg onto the column and peptides were fractionated using a 90 min gradient of 7–27% acetonitrile in 0.125% formic acid at a flow rate of ~575 nL/min.</p><p>We collected mass spectrometric data with three different acquisition modes (hrMS2, SPS-MS3, and RTS-MS3). The data were acquired using these methods with or without FAIMS. For the hrMS2 (high-resolution MS2) method, the scan sequence initialized with an Orbitrap MS1 spectrum (resolution, 120 000; mass range, 350–1400 m/z; automatic gain control (AGC), 5 × 105; maximum injection time, 100 ms). To select MS2 precursors, a TopSpeed parameter of 3 s was used. FAIMS data were collected using 3 CVs (−40, −60, and −80 V) each as a separate 1 s TopSpeed experiment. MS2 analysis consisted of HCD (high-energy collision-induced dissociation) fragmentation with the following settings: resolution, 50 000; AGC target, 1 × 103; isolation width, 0.4 m/z; normalized collision energy (NCE), 37.5; and maximum injection time, 120 ms. For the SPS-MS3 and RTS-MS314 methods, the scan sequence began with an MS1 spectrum which was collected as in the hrMS2 method. Precursors were then selected for MS2/MS3 analysis.15 MS2 analysis consisted of CID (collision-induced dissociation) fragmentation with ion trap analysis. We used the following parameters: scan speed, turbo; NCE, 35; q-value, 0.25; AGC target, 2 × 104; maximum injection time, 120 ms; and isolation window, 0.4 m/z. MS3 precursors were fragmented by HCD and analyzed using the Orbitrap with the following parameters: resolution, 50 000; NCE, 45; maximum injection time, 150 ms; AGC, 1.5 × 105; maximum synchronous precursor selection (SPS) ions, 10; and isolation window, 1.3 m/z.</p><!><p>Spectra were first converted to mzXML using ReAdW.exe. A comet-based pipeline was used to process the acquired mass spectrometric data.16 The canonical mouse database from Uniprot (downloaded November 2019) was concatenated with one of all reversed protein sequences and used for searching. We used a 50 ppm precursor ion tolerance, while that for the product ions was set to 0.03 Da for hrMS2 and 1 Da for SPS-MS3 and RTS-MS3. TMTpro labels on lysine residues and the N-terminus (+304.207 Da), and carbamidomethylation (+57.021 Da) of cysteine residues were set as static modifications. In addition, oxidation of methionine residues (+15.995 Da) was set as a variable modification. Peptide-spectrum matches (PSMs) were adjusted to a 1% false discovery rate (FDR)17,18 and filtered using a linear discriminant analysis (LDA), as described previously.19 For LDA, we considered the following parameters: XCorr, peptide length, ΔCn, charge state, missed cleavages, and precursor mass accuracy. PSMs were then collapsed further to a final protein-level FDR of 1%. Finally, protein assembly was guided by principles of parsimony thereby generating the smallest ensemble of proteins needed for considering all observed peptides. Data were exported for further analysis in Microsoft Excel and R (v 4.0.2) using the DT, dplyr, shiny, and ggplot2 packages.</p><!><p>The strain-specific peptide (SSP) is a quality control reference sample for isobaric tag-based applications. SSP was assembled as a TMTpro16-plex of peptides from a diverse panel of eight mouse strains (the CC/DO founder strains) in duplicate. Protein was extracted from the livers of these mice and processed using the SL-TMT method,20 as illustrated in Figure 1A. SSP can be efficiently assembled, as it does not rely on heavy isotope labels, treatments/perturbations, or multiple proteome mixes. One potential caveat of the SSP is the unavailability of an appropriate mouse tissue for a given experiment. The panel of mice used here (and associated tissues) were commercially available from The Jackson Laboratory (https://www.jax.org). Milligram quantities of peptides can be readily obtained, for example from a single liver, enabling tens of thousands of analyses to be performed with an initial investment. Moreover, because the peptides were genetically encoded in the strain genomes, virtually any tissue (or a mix of tissues) with the correct genetic background can be processed to assemble the appropriate SSP reference sample.</p><p>The assembled SSP was then analyzed unfractionated over 90 min gradients. Following data acquisition, we searched against the mouse reference database consisting of the B6 proteome. Although most peptides were present across all mouse strains, strains with missense mutations within a given peptide sequence will have no TMT reporter ion signal for the native peptide if it uniquely maps to its annotated protein. As an example, we highlighted the peptide VDVVGATQ-GQAGSCSR in Figure 1B. The protein EIF4B (eukaryotic translation initiation factor 4B) was expressed in all eight founder strains. However, in AJ, a missense SNP was present in the coding region of this protein, modifying the sequence of the peptide to VDVVGATQGQTGSCSR (second alanine residue changed to a threonine). This substitution changed the mass and retention time of the peptide and precluded its quantification with respect to the native peptide in channels representing the AJ strain. Similar mutations were scattered across the strains' genomes, and measurement of TMT reporter ion signal therein reflected the presence of interference in a given run.</p><p>We calculated the interference-free index (IFI) to quantify interference (Figure 1C).9,10,21,22 We defined the IFI as the difference from 1 of the average TMT signal-to-noise value for any of the strains that should not possess the peptide of interest (due to a missense mutation) divided by the average TMT signal-to-noise value of the strains with the peptide of interest. The IFI provided a quantitative measure of interference, where "1" represented the ideal case of no interference (i.e., zero signal from the strains without the native peptide). We included further examples of peptides expected to have no TMT reporter ion signal due to SNPs specific to each founder strain and the corresponding IFI values in Figure S1A-G. The B6 strain was the reference proteome database against which we searched the data and does not have missense mutations with respect to itself, and thus a corresponding example was not presented.</p><p>Altogether, we have assembled a list of 657 560 sequence-specific peptides from which to select for calculating the IFI for a given analysis (Table S1). This list was constructed using in silico digests of all eight founder strain proteome databases with trypsin as the protease, allowing for two possible missed cleavages, and requiring a peptide length between 7 and 60 residues (Protein Digestion Simulator, omics.pnl.gov, PNNL). Although the majority of these peptides were absent in only a single strain (n = 244 822), many were absent in multiple strains. More specifically, 109 865 were absent in two strains, 59 446 were absent in three strains, 42 852 were absent in four strains, 35 249 were absent in five strains, 39 799 were absent in six strains, and 125 527 were absent in all seven nonreference strains. We used all quantified sequence-specific peptides with a TMT summed signal-to-noise greater than 200 to calculate the average IFI for each analysis.</p><!><p>We evaluated the SSP reference sample by exploring differences in interference using hrMS2, SPS-MS3, and RTS-MS3 acquisition methods (Figure 2A). We noted that hrMS2-based data were acquired by the Orbitrap at both the MS1 and MS2 stages for database matching and quantification, respectively. However, for the SPS-MS3 and RTS-MS3 strategies, MS2 data were collected in the ion trap for database matching. Fragments thereof were then analyzed at the MS3 stage in the Orbitrap for reporter ion quantification. As such, the additional ion trap scan enabled an added level of selectivity by isolating and analyzing the most abundant fragment ions for SPS-MS3 analysis. Additionally, RTS-MS3 allowed for the selection of the correct predetermined SPS ions. We designated the UniProt mouse database (based on the B6 strain) as the online search database for analyzing the SSP with RTS-MS3.</p><p>We first acquired data for evaluation of the SSP reference sample in triplicate analyses using hrMS2, SPS-MS3, and RTS-MS3 on an Orbitrap Eclipse mass spectrometer (without FAIMS). As discussed above, we calculated the IFI to determine the degree of interference (Figure 2B), where high values (approaching 1) signify reduced interference. For all of the data acquisition methods, the IFI values were consistent among strain replicates. We noted that the lowest IFI values (~0.6) were for hrMS2-acquired data. As expected, the IFI values for the MS3 data were higher, with SPS-MS3 at ~0.8 and RTS-MS3 at ~0.9. These data showed that the acquisition of MS3 data reduced interference, while RTS ensured that the correct SPS ions were always selected, further increasing quantitative accuracy.</p><p>We next performed a similar analysis of the SSP reference sample on an Orbitrap Eclipse mass spectrometer coupled to a FAIMSpro interface. Data acquisition was performed again in triplicate with the hrMS2, SPS-MS3, and RTS-MS3 strategies. FAIMS had been shown previously to reduce interference by separating ionized molecules in the gas phase based on their ion mobility.23-25 In FAIMS, a given ion traverses the electrodes at a specific DC voltage (the compensation voltage, or CV), and alternating CVs can rapidly separate different types of ions.26 We selected three commonly used CVs (−40, −60, and −80 V) for our FAIMS-based analyses.1,27 Once again, the lowest IFI values among the three data acquisition methods were those for the hrMS2-acquired data. However, the IFI values for the hrMS2 analyses were now ~0.8, ostensibly approaching that of SPS-MS3 when collected without FAIMS. Likewise, the IFI values for the MS3 methods also increased slightly, with SPS-MS3 now approaching 0.90 and RTS-MS3 approaching 0.95. Overall, our data showed additive improvements when RTS-MS3 and FAIMS were used together for data acquisition, as also shown previously.13</p><!><p>We provide a user-friendly, online viewer to visualize the data and streamline the calculation of the IFI for SSP analysis. The SSP reference sample viewer extracts strain-specific peptides from the search data results and calculates the IFI for each peptide, as well as the average IFI and associated standard deviation for each RAW data file. The input file requires columns for the search/RAW file name, peptide sequence (stripped of post-translational modifications), and 16 additional columns with TMT reporter ion values. The search results from one or more RAW files may be uploaded as a single comma-separated values (CSV) file. We provide a template file (Figure 3A) in which the user-supplied data can be inserted and uploaded using a web browser (Figure 3B). Once the data are uploaded, an R script is executed in the background and produces a downloadable table that lists the name of the search/RAW file, the average IFI, the standard deviation of the IFI, and the number of strain-specific peptides used to calculate the IFI for each RAW file in the input file (Figure 3C). The user can query an individual peptide (Figure 3D) that corresponds to a given search (Figure 3E) via dropdown menus. Once these values are selected, a bar graph of the TMT relative abundance (TMT RA) values for the selected peptide are generated. As such, the SSP reference sample viewer can simplify the assessment of interference. The viewer can be accessed at: https://japaulo.shinyapps.io/ssp_app/.</p><!><p>We introduce the strain-specific peptide (SSP) as a novel quality control reference sample for isobaric tag-based workflows. Previously, we developed the TKO (triple knock out) standard to understand and limit interference.21,22 The SSP reference sample addressed two limitations associated with the TKO standard, specifically the TKO's moderate proteome complexity and its focus on deletion strains of highly abundant proteins. The mouse reference proteome consisted of over 55 000 entries, which is approximately 10 times the size of the Saccharomyces cerevisiae proteome that comprised the TKO standard. To examine the effect of protein abundance on IFI, we estimated the iBAQ (intensity-based absolute quantification)28 values of our mouse liver proteome12 using MaxQuant.29 Plotting the iBAQ values of quantified proteins with SSP peptides showed a slight trend of better IFI values for more abundant proteins (Figure S2A-C), particularly for the most abundant proteins, and with a larger difference for hrMS2 data (Figure S2A). We noted that even for lower abundant proteins, RTS-MS3 (Figure S2C) outperforms the other sample acquisition methods. Such data could not be acquired with the TKO standard, and merit further investigation. Likewise, although we have shown previously using the TKO standard that fractionation did not affect IFI values,9 it would be interesting to explore if IFI values are affected with respect to lower abundance peptides as found in the SSP reference sample. As such, we noted how the diversity of proteins that comprise the SSP can lead to applications not possible with the various forms of the TKO standard.</p><p>Here, we showcased the SSP to assess interference by comparing three data acquisition methods (hrMS2, SPS-MS3, and RTS-MS3) on an Orbitrap Eclipse mass spectrometer. Our data showed improvements in IFI when using RTS-based data acquisition and FAIMS. Applications of the SSP reference sample extend greatly beyond what was shown here. Although we have focused on global protein profiling, the SSP can be used for targeted assay development and benchmarking. For instance, specific peptides originating from proteins across a wide abundance range can be selected for various isobaric tag-based targeted quantification strategies.4,30 In summary, the SSP reference sample is an innovative and versatile tool to further develop and continually optimize isobaric tag-based workflows.</p>

PubMed Author Manuscript

Redox-induced umpolung of transition metal carbenes

Metal carbene complexes have been at the forefront of organic and organometallic synthesis and are instrumental in guiding future sustainable chemistry efforts. While classical Fischer and Schrock type carbenes have been intensely studied, compounds that do not fall within one of these categories have attracted attention only recently. In addition, applications of carbene complexes rarely take advantage of redox processes, which could open up a new dimension for their use in practical processes. Herein, we report an umpolung of a nucleophilic palladium carbene complex, [{PC(sp 2 )P} tBu Pd(PMe 3 )] ({PC(sp 2 )P} tBu ¼ bis[2-(di-iso-propylphosphino)-4-tert-butylphenyl]methylene), realized by successive one-electron oxidations that generated a cationic carbene complex, [{PC(sp 2 )P} tBu PdI] + , via a carbene radical, [{PCc(sp 2 )P} tBu PdI]. An EPR spectroscopic study of [{PCc(sp 2 )P} tBu PdI] indicated the presence of a ligandcentered radical, also supported by the results of reactions with 9,10-dihydroanthracene and PhSSPh. The cationic carbene complex shows electrophilic behavior toward nucleophiles such as NaH, p TolNHLi, PhONa, and PMe 3 , resulting from an inversion of the electronic character of the Pd-C carbene bond in [{PC(sp 2 )P} tBu Pd(PMe 3 )]. The redox induced umpolung is reversible and unprecedented.

redox-induced_umpolung_of_transition_metal_carbenes

Introduction<!>Results and discussion<!>DFT calculations<!>Reactivity of palladium carbene radical complex<!>Reactivity of palladium cationic carbene complex<!>Conclusions<!>Experimental<!>Reduction of 4 with KC 8<!>Reaction of 5 with NaH

<p>Transition metal carbene complexes, usually classied as Fischer and Schrock type according to the polarity of the M] C carbene bond, 1 are among the most extensively studied organometallic species. 2 While the reactivity of the M]C carbene fragment is generally governed by the electronic properties of the metal center and the substituents of the carbene moiety, 3 its behavior in response to redox reactions has not been studied in detail. 4 For example, although the one-electron reduction of a Fischer-type carbene leading to a carbene radical anion, which upon a second one-electron reduction generates a dianion, is known, 5 the corresponding oxidation processes that could be applied to Schrock-type carbenes (Fig. 1) have not been reported so far. These electron transfer processes cause an umpolung of the original carbene's character, namely the inversion of the M]C carbene bond polarity, 6 and could open up new applications for these compounds in synthesis.</p><p>The development of redox chemistry of transition metal carbenes and reactivity studies of the species generated under redox conditions are still in their infancy, 7 with only a few examples reported exclusively for Fischer carbenes. 8 Interestingly, Fischer type carbenes of group 9 metals in the +2 oxidation state exhibited a remarkable radical character, their redox noninnocent behavior being considered crucial to the asymmetric cyclopropanation of olens catalyzed by Co II (porphyrins). 8d-i Pioneering work by Casey and later by Cooper et al. showed that Fischer type carbene complexes could be reduced to the corresponding radical and dianionic entities. 5 The latter reacted with CO 2 to form a malonate, indicating an umpolung from the electrophilic character of the Fischer type carbene to nucleophilic. 5b As mentioned above, the redox behavior of Schrock type carbenes has not been explored. We reasoned that palladium nucleophilic carbenes 9 represent good candidates for such a study given the higher stability of late transition metal carbene complexes when compared to that of their early metal counterparts. In addition, the combination between palladium, which can undergo two-electron processes (oxidative addition/ reductive elimination), and a redox active ligand, which can undergo sequential one electron processes, has only recently been investigated. 10 No examples in which a carbene carbon is the site of redox activity are known.</p><p>On the other hand, although it is known that palladium carbene species generated from diazo compounds show unique properties and undergo novel transformations, their isolation and characterization is still challenging due to their highly reactive nature. 11 We previously reported the nucleophilic Pd(II) carbene complexes [{PC(sp 2 )P} H Pd(PR 3 )] (1: R ¼ Me; 2: R ¼ Ph) ([PC(sp 2 )P] H ¼ bis[2-(di-iso-propylphosphino)phenyl]-methylene) 9a,12 and [{PC(sp 2 )P} tBu Pd(PMe 3 )] (3, {PC(sp 2 )P} tBu ¼ bis[2-(di-iso-propylphosphino)-4-tert-butylphenyl]methylene), 9b in which the Pd-C carbene bonds are best described as ylide-like, M + -C À type, as demonstrated by the reactivity of 1 toward MeI, HCl, MeOH and para-toluidine 9a and also by C-H activation reactions. 13 The strong nucleophilic character of 1 and 3 was also demonstrated by the rapid Lewis acid/base "quenching" reactions with B(C 6 F 5 ) 3 . 9b As indicated by DFT calculations, the HOMO of 1 is largely localized on the carbene carbon atom, therefore, an oxidation process to induce the loss of electrons might occur from that orbital. Consequently, the one-electron oxidation of 1 with 0.5 equivalents of I 2 afforded a stable radical complex, [{PCc(sp 2 )P} H PdI], which persisted in solution but dimerized in the solid state. Interestingly, the chloride and bromide congeners are monomers in both phases. 14 These results prompted us to study the oxidation of the radical species further to the corresponding cations, and therefore to realize an umpolung of a nucleophilic carbene. In order to prevent dimerization and also simplify the synthetic procedure, carbene complex 3 bearing bulky tert-butyl groups was employed instead of 1. 14 The good solubility provided by the t-Bu groups also facilitated reactivity studies. Herein, we report the successive one-electron oxidation of 3 leading to a cationic carbene complex, [{PC(sp 2 )P} tBu PdI][BAr F 4 ] (5), via a monomeric radical species [{PCc(sp 2 )P} tBu PdI] (4). Reactivity studies on both complexes are consistent with their unique electronic properties, and the umpolung of carbene complex 3 was undoubtedly demonstrated by the reactions of 5 with various nucleophiles. Notably, the electron transfer processes to generate the carbene, radical, and cationic species are reversible.</p><!><p>Synthesis and characterization of palladium radical and cationic carbene complexes Slow addition of 0.5 equivalents of I 2 to the dark-brown solution of [{PC(sp 2 )P} tBu Pd(PMe 3 )] ( 3) in THF at À35 C instantly generated a dark-green solution, from which dark-green crystals of [{PCc(sp 2 )P} tBu PdI] (4) were isolated in 97% yield (Scheme 1). Complex 4 is silent by both 1 H and 31 P{ 1 H} NMR spectroscopy. The magnetic susceptibility measurement at 298 K using the Evans method gave an effective magnetic moment m eff of 1.85 m B , thus indicating an S ¼ 1/2 ground state. The X-band EPR spectrum recorded at 298 K revealed an isotropic signal typical for a carbon centered radical complex with a g value of 2; no hyperne coupling was observed (Fig. S1 †). Complex 4 is stable toward water but highly sensitive toward oxygen. Upon exposing the dark-green solution of 4 to air, the color changed immediately to yellow. However, attempting to react 4 with a stoichiometric amount (1 or 0.5 equivalents) of pure oxygen only led to complicated mixtures of products. Interestingly, in contrast to radicals generated in situ from Fischer type carbenes, 4,5 complex 4 is thermally robust: no signicant decomposition was observed aer heating 4 in C 6 D 6 at 80 C for a week, likely a consequence of the radical delocalization over both phenyl rings and of the steric protection from the two tert-butyl groups present in 4.</p><p>Cyclic voltammetry studies of 4 performed in a THF solution (Fig. 2) showed a quasi-reversible event at E 1/2 ¼ À0.38 V, assigned to [{PC(sp 2 )P} tBu PdI] + /[{PCc(sp 2 )P} tBu PdI], and an irreversible reduction event at E ¼ À2.03 V (vs. Fc/Fc + ), assigned to the reduction to [{PC(sp 2 )P} tBu PdI] À .</p><p>Treatment of the dark-green solution of 4 with an equivalent of [Cp 2 Fe][BAr F 4 ] in diethyl ether at À35 C instantly formed a dark-red solution, and the dark-red crystalline complex [{PC(sp 2 )P} tBu PdI][BAr F 4 ] (5) was isolated in 94% yield (Scheme 1). To the best of our knowledge, only two examples of nonheteroatom-stabilized cationic Pd(II) carbene complexes are known: complex A (Fig. 3) was prepared from a cationic precursor by triate abstraction with di-p-tolyldiazomethane, 15 while compound B was synthesized by hydride abstraction from a PCHP pincer complex; 16 reactivity studies have not been reported for these species. In addition, cationic alkylidene complexes of other late transition metals are also rare. 17 Cationic Ir(III) alkylidene complexes generated either by chloride or hydride abstraction showed electrophilic reactivity toward PMe 3 and LiAlH 4 to form the phosphonium ylide and the alkyl species, respectively. 18 A cationic Pt(II) alkylidene complex synthesized by hydride abstraction also showed similar reactivity toward Lewis bases such as DMAP (4-dimethylaminopyridine). 19 It is worth mentioning that, unlike the synthesis of the previously reported cationic carbene complexes, the synthesis of 5 is based on a redox process.</p><p>Due to its ionic nature, 5 is only soluble in ethers and chlorinated solvents. The 1 H NMR spectrum of 5 in CDCl 3 indicates a C 2 symmetric species. The 31 P{ 1 H} NMR spectrum shows one sharp singlet at d ¼ 72.04 ppm, which shied to lower eld compared to that of 61.86 ppm in 3. The counter anion [BAr F 4 ] À was conrmed by singlets at d ¼ À6.62 and À65.79 ppm in the 11 B{ 1 H} and 19 F{ 1 H} NMR spectra, respectively. The characteristic signal for the carbenium carbon was observed at d ¼ 284.47 ppm as a singlet in the 13 C{ 1 H} NMR spectrum, which is signicantly shied to lower eld compared to the corresponding values of 136.06 ppm for 3, À18.3 ppm for [Cy 2 P(S)CP(S)Ph 2 ]Pd(PPh 3 ), 9d and 189.6 ppm for the cationic carbene complex [P 2 C]PdCl]X (B, Fig. 3). 16 However, this value is close to that of 313.4 ppm for [(Trpy)Pd]C(p-Tol) 2 ][BAr F 4 ] (Trpy ¼ 3,4,8,9,13,14-hexaethyl-2,15dimethyltripyrrinato, A, Fig. 3). 15 The structures of 4 and 5 were unambiguously determined by single crystal X-ray diffraction studies. As shown in Fig. 4 and 5, both complexes contain square-planar palladium centers bound to the sp 2 hybridized backbone carbons (the sum of the angles at C is 359.8 for 4 and 359.7 for 5). The Pd-C carbene distance of 2.076(3) Å in 3 contracts to 2.022(3) Å in 4 and further to 1.968(3) Å in 5. The Pd-C carbene distance of 1.968 (3) Å in 5 is comparable to that of 1.999(4) Å in [P 2 C]PdCl][PF 6 ] (B, Fig. 3). 16 Although the Pd-C carbene bond in 3 is better described as a single bond, 9a,9b,14,20 the contraction observed for 4 and 5 can be attributed to the increased bond order between the metal center and the carbene carbon, achieved by the sequential removal of electrons from the p* antibonding orbital (vide infra). This trend also indicated a change of the electronic property of this atom from an anion in 3 to a cation in 5 via the radical in 4. Therefore, the isolation of 4 and 5 is remarkable and represents the rst example of successive oxidations of a transition metal nucleophilic carbene leading to a well-dened radical and cation, with all three complexes characterized by Xray crystallography.</p><!><p>DFT calculations were performed on a model of the cationic carbene 5 (Fig. 6). In contrast to the antibonding character of HOMO observed for 3, a similar antibonding p type interaction was found for the LUMO of 5 and the SOMO of 4, resulting from the successive removal of electrons from the HOMO of 3 that is largely localized on the carbene carbon. The bonding component of this p bond was found in HOMOÀ11 for 5. Therefore, consistent with the observed contraction of the Pd-C carbene distances from 3 (2.076(3) Å) to 5 (1.968(3) Å), the frontier molecular orbital analysis also indicates an increase of the Pd- A successive addition of electrons to the LUMO of 5 should regenerate the carbene radical 4 and, ultimately, the carbene 3. Treatment of the dark-red THF solution of 5 with KC 8 (1 eq.) at room temperature immediately led to a dark-green solution of 4, as conrmed by its reaction with 9,10-dihydroanthracene in C 6 D 6 to form 6 (vide infra). In the presence of PMe 3 , the further reduction of 4 with KC 8 (1 eq.) in THF also regenerated 3. Such reversible electron transfer among the carbene species 3, 4, and 5 allowed us to tune their electronic properties by redox methods (Fig. 1). 4 Accordingly, treatment of cation 5 with 1 eq. of carbene 3 in diethyl ether immediately generated a dark green solution containing carbene radical 4 and the cationic radical complex [{PCc(sp 2 )P} tBu Pd(PMe 3 )][BAr F 4 ] (Fig. S41 †), which were conrmed by their hydrogen atom abstraction reactions with 9,10-dihydroanthracene (vide infra).</p><!><p>The carbene radical complex 4 is expected to undergo ligand centered radical-type reactions, such as hydrogen atom abstraction. 14 Despite this prediction, heating a C 6 D 6 solution of 4 with n Bu 3 SnH at 80 C for one week did not produce the hydrogen atom abstraction product, and the color of the solution remained dark green. However, 4 reacted slowly reacted with 0.5 equivalents of 9,10-dihydroanthracene under similar conditions to generate a bright yellow solution, from which the expected hydrogen abstraction product [{PC(sp 3 )HP} tBu PdI] ( 6) was isolated as an orange crystalline solid in 81% yield, aer recrystallization from n-pentane (Scheme 2). The formation of the by-product anthracene was also conrmed by NMR spectroscopy.</p><p>The 1 H NMR spectrum of 6 in C 6 D 6 showed C s symmetry as observed for its chloride analogue [{PC(sp 3 )HP} tBu PdCl]. 9b The benzylic proton of 6 was observed at d ¼ 6.41 ppm as a singlet, while the benzylic carbon was observed at d ¼ 58.35 ppm as a triplet ( 2 J PC ¼ 4.4 Hz) in the corresponding 13 C{ 1 H} NMR spectrum. Both values are slightly downeld shied compared to those of the chloride analogue (d ¼ 6.21 and 50.59 ppm for the 1 H and 13 C{ 1 H} NMR spectra, respectively). 9b In the 31 P{ 1 H} NMR spectrum, only one sharp singlet at d ¼ 51.25 ppm was observed. The crystal structure of 6 is analogous to that of [{PC(sp 3 )HP} tBu PdCl] (Fig. 7), in that both contain an sp 3 hybridized backbone carbon atom. The Pd-C backbone distance of 2.094(2) Å is also close to that of 2.078(2) Å in [{PC(sp 3 ) HP} tBu PdCl], 9b but longer than the Pd-C carbene distance of 2.022(3) Å for 4.</p><p>Coupling of the radical in 4 with a thiyl radical occurs in the reaction with diphenyl disulde (Scheme 2). Albeit slow, heating 4 with 0.5 equivalents of PhSSPh in C 6 D 6 at 80 C for 7 days formed [{PC(sp 3 )(SPh)P} tBu PdI] (7), which was isolated as a yellow waxy solid in 94% yield. The 1 H NMR spectrum of 7 in C 6 D 6 showed C s symmetry, indicating an sp 3 hybridized backbone carbon. No benzylic proton was observed and the quaternary backbone carbon showed a triplet at d ¼ 79.58 ( 2 J PC ¼ 6.1 Hz) in the 31 C{ 1 H} NMR spectrum, both facts being consistent with a C-S bond formation between the carbene radical of 4 and the [PhS]c radical. The 31 P{ 1 H} NMR spectrum showed only a sharp singlet at d ¼ 48.59 ppm. However, all attempts to crystallize 7 failed due to its high solubility in hydrocarbons. Therefore, complex 7 was treated with an equivalent of AgOTf in THF, 23 and the expected complex [{PC(sp 3 )(SPh)P} tBu Pd(OTf)] ( 8) was obtained as yellow blocks in 61% yield aer recrystallization from diethyl ether. The crystal structure of 8 was determined by X-ray diffraction (Fig. 8), showing the sp 3 hybridized backbone carbon bound to a PhS group. The phenyl ring of the PhS group is pointing somewhat toward the triate substituent with the corresponding dihedral angles between the plane of the phenyl ring and the plane dened by O, Pd, C backbone and S being 73.49 .</p><p>According to the standard redox potential of PhSSPh (À1.7 V vs. SCE, À2.1 V vs. Fc/Fc + ) 24 and the observed value for the [{PCc(sp 2 )P} tBu PdI]/[{PC(sp 2 )P} tBu PdI] + , redox couple (À0.38 V vs. Fc/Fc + ), it is less likely that the carbene radical 4 was oxidized by PhSSPh to the carbene cation [{PC(sp 2 )P} tBu PdI] + which reacted with the [PhS] À anion to form 7. Therefore, compound 7 was probably formed by direct radical coupling between the carbene radical and [PhSc] generated by the homolysis of PhSSPh.</p><p>It is also interesting to note that the carbene complex 3 can be readily oxidized by PhSSPh. Treatment of 3 with an equivalent of PhSSPh in C 6 H 6 immediately generated a bright yellow solution at room temperature, from which the yellow crystalline solid of [{PC(sp 3 )(SPh)P} tBu Pd(SPh)] (9) was isolated in quantitative yield aer recrystallization from n-pentane (Scheme 3). The 1 H NMR spectrum of 9 in C 6 D 6 also showed C s symmetry. No benzylic proton was observed and the corresponding quaternary backbone carbon showed a triplet at d ¼ 74.78 ( 2 J PC ¼ 3.2 Hz) in the corresponding 31 C{ 1 H} NMR spectrum. Only a sharp singlet at d ¼ 44.26 ppm was observed in the 31 P{ 1 H} NMR spectrum. The structure of 9 was further conrmed by X-ray diffraction studies. As shown in Fig. 9, complex 9 contains two PhS moieties, one bound to the sp 3 hybridized carbon backbone and the other to the palladium center. Unlike 8, the phenyl ring of the PhS group on the backbone carbon points away from the Pd center. When carbene 3 was treated with only 0.5 equivalents of PhSSPh in C 6 D 6 , compound 9 and unreacted 3 were observed by 1 H and 31 P NMR spectra; which upon the addition of another 0.5 equivalents of PhSSPh, the mixture led to 9. It is worth noting that a radical complex [{PCc(sp 2 )P} tBu PdSPh] (C) was not formed under these conditions, but it could be an intermediate species that generates 9 by rapid coupling with the [PhS]c radical. This intermediate, C, can be generated by an one-electron transfer from carbene 3 to PhSSPh, however, direct addition of PhSSPh across Pd + -C À carbene moiety or oxidative addition to palladium followed by the migration of the PhS À moiety to the carbene carbon to form 9, cannot be ruled out.</p><!><p>The cationic carbene complex 5 is expected to be electrophilic, therefore its reactivity toward various nucleophiles was studied. We had previously isolated complex 6 from a hydrogen atom abstraction reaction with the carbene radical 4, and thus assumed that a nucleophilic attack on the cationic carbene 5 by a H À nucleophile would also produce 6. Treatment of a dark-red solution of 5 with an equivalent of NaH in THF at room temperature gradually generated a bright yellow solution within 24 hours. Both 1 H and 31 P{ 1 H} NMR spectra showed that the 6 was formed in quantitative yield together with the by-product Na[BAr F 4 ] (Scheme 4). Since the cationic Ir(III) metallacyclic alkylidene complex was reported to react with LiAlH 4 even at low temperature, 18b the slow reaction with 5 was attributed to the poor nucleophilicity of NaH, but also to its poor solublility in THF. (Dn 1/2 z 505 Hz) ppm, respectively. Upon cooling, complex 10 showed coalescence at 278 K, and further splitting to two sharp doublets at 238 K (Fig. 12). For 11, a slightly lower temperature (268 K) was required to reach coalescence and two sharp doublets were observed at 228 K (Fig. S34 †). Both 10 and 11 showed only one sharp singlet in the corresponding 31 P{ 1 H} NMR spectra at 338 K. Since a similarly dynamic behavior was not observed for 8 and 9, in which both backbone carbons are Although the nucleophiles H À , p TolNH À , and PhO À afforded the expected products 6, 10, and 11, respectively, the reaction of 5 with PhCH 2 K did not lead to an isolable product. Also, the lithium salts MeOLi and PhOLi did not react with 5, probably due to their weaker nucleophilic character compared to that of the sodium salt PhONa.</p><p>A neutral nucleophile such as PMe 3 also reacts instantly with 5 in diethyl ether to form a bright-yellow solution; yellow crystals of [{PC(sp 3 )(PMe 3 )P} tBu PdI][BAr F 4 ] ( 12) were isolated in quantitative yield by the diffusion of n-pentane into a uorobenzene solution at room temperature. Compound 12 is only soluble in etheral and chlorinated solvents. Its 1 H NMR spectrum in CDCl 3 showed C s symmetry as was also observed for compounds 6-11. The protons of the PMe 3 group were observed at d ¼ 1.31 ppm as a doublet ( 2 J HP ¼ 11.5 Hz), which correlates with a doublet at d ¼ 13.86 ppm ( 1 J CP ¼ 54. 3 Hz) in the 31 C{ 1 H} NMR spectrum. In the 31 P{ 1 H} NMR, two sharp singlets at d ¼ 42.17 and 31.01 ppm were observed for the two i Pr 2 P phosphines and the PMe 3 on the backbone carbon, respectively. Both signals remain sharp when the temperature was lowered to 248 K, indicating a lower energy barrier for the rotation of PMe 3 group compared to those for the p TolNH and PhO groups in 10 and 11. In the 13 C{ 1 H} NMR spectrum, the backbone carbon resonance was observed at d ¼ 63.65 (dt, 1 J CP ¼ 21.5 Hz, 2 J CP ¼ 2.4 Hz) ppm, which is signicantly shied to higher eld compared to 284.47 ppm observed for 5, and close to the value of 58.35 ppm in 6. Thus, the strong donation from PMe 3 to the carbenium carbon effectively offsets the charge on this atom. The exclusive formation of 12 is consistent with its strong electrophilic character. Heating this compound at 60 C in CDCl 3 for 24 h did not lead to any decomposition. However, the cationic Ir(III) alkylidene hydride species [(C 5 Me 5 )HIr i Pr 2 P(Xyl)] [BAr F 4 ] generated by chloride abstraction was reported to react with PMe 3 to form a similar phosphonium ylide only as a kinetically favored product, which converts to the Ir(III) alkyl phosphine adduct at 45 C by migratory insertion of the hydride ligand into the alkylidene functionality. 18a The structure of 12 was also determined by X-ray diffraction studies (Fig. 13). Compound 12 contains an sp 3 hybridized backbone carbon bound to PMe 3 , with an average dihedral angle of 78.9 between the planes dened by P( 3 27 Other phosphines, such as Ph 3 P did not afford an isolable product probably because of steric crowding. A broad peak at d ¼ 72 ppm was observed in the 31 P{ 1 H} NMR spectrum of the reaction mixture of 5 and Ph 3 P, indicating that some interaction between the carbenium carbon and Ph 3 P occurred upon mixing, but a complicated mixture was formed aer heating the reaction at 60 C.</p><!><p>In conclusion, the one-electron oxidation of the palladium carbene complex [{PC(sp 2 )P} tBu Pd(PMe 3 )] (3) with I 2 generated a monomeric radical carbene complex [{PCc(sp 2 )P} tBu PdI} ( 4), which upon a second one-electron oxidation with [Cp 2 Fe][BAr F 4 ] formed a cationic carbene complex, [{PC(sp 2 )P} tBu PdI][BAr F 4 ] (5). We were able to isolate and characterize for the rst time a whole series (Fig. 1) of an anionic carbene (3), a carbene radical (4), and a cationic carbene (5). Our studies show that: (1) transition metal carbene complexes possess a rich redox chemistry that allows the tuning of the electronic properties of the carbene carbon; (2) an umpolung of the M]C carbene bond can be accomplished by successive electron transfer processes; and (3) the electron transfers among the carbene, radical, and cationic species are reversible.</p><!><p>All experiments are performed under an inert atmosphere of N 2 using standard glovebox techniques. Solvents hexanes, npentane, diethyl ether, and CH 2 Cl 2 were dried by passing through a column of activated alumina and stored in the glovebox. THF was dried over LiAlH 4 followed by vacuum transfer and stored in the glovebox. CDCl 3 was dried over 4 Å molecular sieves under N 2 , while C 6 D 6 was dried over CaH 2 followed by vacuum transfer, and stored in the glovebox. Complex 3, [Cp 2 Fe] [BAr F 4 ] and KC 8 were prepared according to literature procedures. 9b,28 p TolNHLi was prepared by deprotonation of p-toluidine with n BuLi, while PhONa was prepared from NaH and phenol. 1 H, 13 C{ 1 H}, 31 P{ 1 H}, 19 F{ 1 H} and 11 B{ 1 H} NMR spectra were recorded on a Bruker DRX 500 spectrometer. All chemical shis are reported in d (ppm) with reference to the residual solvent resonance of deuterated solvents for proton and carbon chemical shis, and to external H 3 PO 4 , BF 3 $OEt 2 , and CFCl 3 for 31 P, 11 B, and 19 F chemical shis, respectively. Magnetic moments were determined by the Evans method 29 by using a capillary containing 1,3,5-trimethoxybenzene in C 6 D 6 as a reference. EPR spectrum of compound 4 was recorded on a Bruker EMXplus EPR spectrometer with a standard X-band EMXplus resonator and an EMX premium microwave bridge. Cyclic voltammetry was performed on a Metrohm Autolab PGSTAT-128N instrument. Elemental analyses were performed on a CE-440 Elemental analyzer, or by Midwest Microlab. Gaussian 03 (revision D.02) 30 was used for all reported calculations. The B3LYP (DFT) method was used to carry out the geometry optimizations on model compounds specied in text using the LANL2DZ basis set. The validity of the true minima was checked by the absence of negative frequencies in the energy Hessian.</p><p>Synthesis of [{PCc(sp 2 )P} tBu PdI] ( 4) Iodine (11 mg, 0.043 mmol) in 1 mL of THF was slowly added to a dark-brown solution of 3 (60 mg, 0.087 mmol) in 1 mL of THF at À35 C. Upon addition, a dark-green solution was formed immediately and stirred at ambient temperature for 30 min. All volatiles were removed under reduced pressure and the darkgreen residue was extracted with n-pentane (2 Â 4 mL). Aer reducing the volume of the pentane solution to about 1 mL, the solution was stored at À35 C to give 4 as a dark-green crystalline solid; yield 62 mg (97%). Compound 4 is paramagnetic. Magnetic moment (Evans method, 298 K): m eff ¼ 1.85 m B ; EPR: g ¼ 2.0000. Anal. calcd for C 33 H 52 IP 2 Pd (744.04 g mol À1 ): C, 53.27; H, 7.04. Found: C, 53.05; H, 7.24.</p><!><p>KC 8 (2.3 mg, 0.016 mmol) was mixed with 4 (12 mg, 0.016 mmol) and PMe 3 (32 mL, 0.032 mmol, 1 M in THF) in 1 mL THF at room temperature. The mixture turned dark-brown immediately. Aer stirring the reaction mixture for about 5 min, the volatiles were removed under reduced pressure and the residue was extracted with 1 mL of n-pentane. The solution was ltered through celite. Compound 3 was obtained as dark brown crystalline solid from this n-pentane solution at À35 C. 1 H and 31 P { 1 H} NMR spectra were identical with the previously reported data. 9b Yield 9 mg (80%).</p><p>Synthesis of [{PC(sp 2 )P} tBu PdI][BAr F 4 ] (5) [Cp 2 Fe][BAr F 4 ] (83.2 mg, 0.079 mmol) in 3 mL of diethyl ether was slowly added to a dark-green solution of 4 (59 mg, 0.079 mmol) in 2 mL of diethyl ether at À35 C. Upon addition, a darkred solution was formed immediately that was stirred at room temperature for 15 min. Aer reducing the volume of the ether solution to about 1 mL, n-pentane (about 8 mL) was layered and the mixture was stored at À35 C overnight to afford compound 5 as a dark-red crystalline solid, which was washed with npentane and dried under vacuum; yield 119 mg (94%). 1 H NMR (500 MHz, CDCl 3 , 25 C): d ¼ 7.93 (td, 4 J HH ¼ 3.8 Hz, 3 J HP ¼ 1.5 Hz, 2H, ArH), 7.73 (m, 4H, ArH), 7.64 (s, 8H, ortho-Ar F H), 7.46 (s, 4H, para-Ar F H), 2.99 (m, 4H, CH(CH 3 ) 2 ), 1.39 (s, 18H, C(CH 3 ) 3 ), 1.36 (dt, 3 J HH ¼ 9.0 Hz, 3 J HP ¼ 9.5 Hz, 12H, CH(CH 3 ) 2 ), 1.26 (dt, 3 J HH ¼ 9.0 Hz, 3 J HP ¼ 8.0 Hz, 12H, CH(CH 3 ) 2 ) ppm; 13 Reduction of 5 with KC 8 KC 8 (2.6 mg, 0.018 mmol) and 5 (30 mg, 0.018 mmol) were mixed in 2 mL of THF at room temperature, which formed a dark-green solution immediately. Aer stirring the reaction mixture for about 5 min, all volatiles were removed under reduced pressure. The residue was extracted with 4 mL of npentane, ltered, and the pentane was removed under reduced pressure. The resulted green solid was mixed with 0.5 equiv. of 9,10-dihydroanthracene (2 mg, 0.010 mmol) in C 6 D 6 and heated at 80 C for 7 d to afford compound 6 (vide infra). Yield 12 mg (92%).</p><p>Synthesis of [{PC(sp 3 )HP} tBu PdI] ( 6) 9,10-dihydroanthracene (3.6 mg, 0.020 mmol) and 4 (30 mg, 0.040 mmol) were mixed in 0.6 mL of C 6 D 6 and heated at 80 C for 7 d, during which time the dark-green solution slowly turned to bright yellow. All volatiles were removed under reduced pressure and the residue was extracted with 5 mL of n-pentane.</p><p>Aer reducing the volume of the pentane solution to about 1 mL, the by-product anthracene precipitated as a white crystalline solid, which was ltered, and the solution stored at À35 C to give orange crystals. Further recrystallization from n-pentane at À35 C afforded 6 as analytically pure orange crystals; yield 24 mg (81%). 1 H NMR (500 MHz, C 6 D 6 , 25 C): d ¼ 7.46 (td, 4 J HH ¼ 4.3 Hz, 3 J HP ¼ 2.0 Hz, 2H, ArH), 7.42 (dm, 3 J HH ¼ 8.0 Hz, 2H, ArH), 7.22 (dm, 3 J HH ¼ 8.0 Hz, 2H, ArH), 6.41 (s, 1H, CH backbone ), 2.78 (m, 2H, CH(CH 3 ) 2 ), 2.60 (m, 2H, CH(CH 3 ) 2 ), 1.44 (dt, 3 J HH ¼ 7.0 Hz, 3 J HP ¼ 7.8 Hz, 6H, CH(CH 3 ) 2 ), 1.41 (dt, 3 J HH ¼ 7.5 Hz, 3 J HP ¼ 7.8 Hz, 6H, CH(CH 3 ) 2 ), 1.23 (s, 18H, C(CH 3 ) 3 ), 1.16 (dt, 3 J HH ¼ 7.5 Hz, 3 J HP ¼ 7.5 Hz, 6H, CH(CH 3 ) 2 ), 1.12 (dt, 3 J HH ¼ 7.5 Hz, 3 J HP ¼ 7. 3 Hz, 6H, CH(CH 3 ) 2 ) ppm; 13 Synthesis of [{PC(sp 3 ) (SPh)P} tBu PdOTf] (8) AgOTf (9.6 mg, 0.038 mmol) in 1 mL of THF was added to 7 (32 mg, 0.038 mmol) in 1 mL of THF at room temperature. The resulting orange slurry was stirred for 4 h, slowly turning to a pale yellow slurry. The volatiles were removed under reduced pressure and the residue was extracted with 5 mL of benzene</p><!><p>NaH (0.6 mg, 0.024 mmol) and 5 (32 mg, 0.020 mmol) were mixed in 1 mL of THF at room temperature. The dark-red mixture was allowed to stir for 24 h, during which time a yellow solution was slowly formed. Removal of volatiles under reduced pressure led to a yellow solid. The 1 H and 31 P{ 1 H} NMR of the crude yellow solid in C 6 D 6 showed clean conversion to 6 and NaBAr F 4 .</p><p>Synthesis of [{PC(sp 3 )(NH p Tol)P} tBu PdI} (10)</p><p>p TolNHLi (3.3 mg, 0.029 mmol) in 1 mL of THF was slowly added to a dark-red solution of 5 (46 mg, 0.029 mmol) in 1 mL of THF at À35 C. The resulting brownish-green solution was then stirred at room temperature for 15 min. The volatiles were removed under reduced pressure and the residue was extracted with 5 mL of benzene and ltered. Removal of volatiles under reduced pressure gave a greenish-yellow residue, which was extracted with n-pentane (8 mL), ltered, and the volume of the pentane solution reduced to about 1 mL. Storing this solution at À35 C gave compound 10 as yellow crystals; yield 12 mg (49%).</p>

Royal Society of Chemistry (RSC)

Palladium and Platinum 2,4-cis-amino Azetidine and Related Complexes

Seven N,N'-palladium(II) chloride complexes, one N,N'-palladium(II) acetate complex of 2,4-cis-azetidines where prepared and analyzed by single crystal XRD. Two platinum(II) chloride N,N'-complexes of 2,4-cis-azetidines where prepared and analyzed by single crystal XRD. Computational analysis and determination of the %Vbur was examined conducted. A CNN' metallocyclic complex was prepared by oxidative addition of palladium(0) to an ortho bromo 2,4-cis-disubstituted azetidine and its crystal structure displays a slightly pyramidalized metal-ligand orientation.

palladium_and_platinum_2,4-cis-amino_azetidine_and_related_complexes

Introduction<!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!><!>Results and discussion<!>Conclusions<!>Author contributions<!>Conflict of interest statement

<p>Complexes of group 10 metals coordinated by N,N'-ligands have found wide use from therapeutics (Graham et al., 2004) through to catalysis (Fossey et al., 2005; Saito and Fu, 2007; Binder et al., 2012; Martínez-Olid et al., 2014; Álvarez-Casao et al., 2015). In our research, through studying the synthesis and cyclisation of homoallylamine derivatives (Fossey et al.; Rixe et al., 1996; Jamieson and Lippard, 1999; Feula et al., 2010, 2013; Feula and Fossey, 2013; Hama Salih et al., 2015), the potential for use of the cis-aminoazetidine products as ligands emerged, and their use in asymmetric copper-catalyzed Henry reactions was developed and reported elsewhere (Yoshizawa et al., 2018a,b,c). Group 10 complexes of azetidine derivatives have been reported previously (Voureka et al., 1996; Keller et al., 2005; Lee et al., 2008, 2009a,b; Choi et al., 2011), and during our aforementioned studies various attempts to form and isolate racemic group 10 metal complexes have been made. Successful attempts to prepare crystals of metal complexes suitable for single crystal X-ray diffraction structure determination are reported herein, along with crystal structures of unexpected by-products, a complex formed from a pyrrolidine ligand (an isomer of the azetidines of primary interest) as well as an observation pertaining to diastereoisomer differentiation upon complexation and sample preparation under slightly different conditions. In most cases small amounts of (or even just single) crystals were obtained sometimes precluding full characterisation, however in some cases elemental analyses are reported. The structural observations facilitated by surveying the obtained crystal structures have the potential to influence catalysis design. Thus, whilst this report primarily serves as a crystal structure report the interpretation and understanding garnered is gathered here with a view to informing studies across organic and inorganic synthesis.</p><!><p>Our previously documented interest in the synthesis of 2,4-cis-disubstituted azetidines (Fossey et al.; Feula et al., 2010, 2013; Feula and Fossey, 2013; Hama Salih et al., 2015), and our recent use of the same family of azetidines as chiral ligands in asymmetric copper-catalyzed Henry reactions afforded us the opportunity to probe the complexation of these ligands and related compounds with various metal sources, in this report examples leading to samples suitable for analysis by X-ray single crystal diffraction studies are collected and compared. A previously revealed platinum complex (4a) is re-reported here since it is compared directly with a diastereomeric complex (4b) of the same ligand, and the racemic ligands used in this study have all been previously reported in the aforementioned publications arising from this programme of research1.</p><p>Treatment of a series of amino-substituted azetidines with various palladium(II) chloride derivatives afforded crystals suitable for analysis by single crystal XRD analysis in seven cases (Scheme 1). Racemic ligands 1a to g were converted to the corresponding square planar palladium chloride complexes 2a to g, by combination of various palladium(II) chloride sources and ligands in methanol and heating at reflux until ligand had been consumed by TLC analysis, see Figure 1 (and Supplementary Material) for representations of the palladium chloride complexes thus obtained. Treatment of ligand 1a with palladium(II) acetate in dichloromethane afforded mono-nuclear palladium complex 3, bearing a coordinated water molecule and displaying hydrogen bonding between the coordinated water molecule and the two acetate anions (coordinated to palladium and bridging ligand NH…H2O respectively), see Figure 2, for a representation of the crystal structure obtained for compound 3.</p><!><p>General protocol for the preparation of identified palladium(II) chloride (2a–g) and acetate (3) complexes of 2,4-cis-disubstituted azetidine N,N'-ligands.</p><p>Representations of one molecule from the unit cells of the single crystal XRD structures of racemic complexes 2a-g. Elipsoids plot at 50% probability, and rendered using Ortep-III for Windows and PovRay v3.7. For selected bond lengths, angles and torsions see ESI.</p><p>Representation of one molecule from the unit cell of the single crystal XRD structures of racemic complex 3. Elipsoids plot at 50% probability, and rendered using Ortep-III for Windows and PovRay v3.7.</p><!><p>Reactions were routinely conducted under anhydrous, oxygen-free conditions, but materials obtained from reactions were handled in air and no special precautions to exclude moisture or oxygen were taken. Crystals of palladium(II) chloride complexes (Figure 1) suitable for single crystal XRD analysis were typically obtained by crystallization from acetonitrile or acetonitrile/diethyl ether mixtures, pre-purification by flash chromatography or filtration through silica in dichloromethane/methanol mobile phase was sometimes advantageous. The XRD structures typically displayed single diastereoisomers of products as racemic mixtures. The figures of the main text show one molecule from the unit cell, displayed such that the stereochemistry of those drawn in the main text match permitting ready comparison of steric effects among the ligands compared.</p><p>Single crystals of complex 3 (Figure 2) were obtained by slow diffusion of hexane directly into the dichloromethane solvated reaction mixture.</p><p>The platinum(II) chloride complex 4a, formed by heating ligand 1a at reflux in methanol with potassium tetrachloroplatinate, was previously reported by us. We were intrigued when we later repeated the protocol, with ethanol as solvent, instead of methanol, and extended the reaction time somewhat (from 16 to 72 h), to find an alternative diastereoisomer, racemic 4b, was formed, differing only by the coordinational chirality of the amine-part's nitrogen, both complexes are discussed in this report (Scheme 2). Gagné and co-workers previous discussed persistent nitrogen chirality in square planar diamine complexes of palladium(II) in their seminal work on the topic (Pelz and Gagné, 2003; Pelz et al., 2004), and their work should be consulted for discussion about complexes displaying chirality only as a result of nitrogen's stereogenicity as a result of coordination to a metal.</p><!><p>The protocol used to obtain crystals suitable for single crystal XRD analysis of N-diastereomeric racemic platinum(II) chloride complexes 4a and 4b.</p><!><p>Crystallization from acetonitrile/diethyl ether or slow evaporation of an acetonitrile solution led to the isolation of crystals suitable for single crystal XRD analysis of 4a and 4b respectively, Figure 3.</p><!><p>Representation of one molecule from each unit cell of racemic 4a (left) and 4b (right). The diastereoisomers drawn differ by inversion of the amine-centered nitrogen stereogenicity. The two diastereoisomers depicted are 4a N(azet)RN(amin)R (left) and 4b N(azet)RN(amin)R (right). Elipsoids plot at 50% probability, and rendered using Ortep-III for Windows and PovRay v3.7. For selected bond lengths, angles and torsions see ESI.</p><!><p>Noting that platinum complex 4a was formed under reflux in methanol (boiling point 64.7 °C) and the N-diastereomeric complex 4b at reflux in ethanol (boiling point 78.4 °C) and over a longer time, it was reasoned that 4a may be a kinetic product whilst 4b the thermodynamic. If this were the case, there may be implications for the future use of such complexes in catalysis. Computational analysis revealed this assumption to be incorrect, importantly demonstrating that XRD analysis, especially in the case of this report where, in some cases, very few crystals were isolated from the bulk. Nevertheless, the computational analysis does demonstrate the constrained nature of the cis-azetidine architecture to be important in determining the underlying steric parameters around the coordination environment. The square planar N-diastereomeric complexes 4a and 4b were studied with density functional theory in order to establish the intrinsic thermodynamic preference. Calculations employed the M06-2X functional with the 6-31G* basis set for all atoms apart from platinum atom which was treated with the LANL double zeta basis set and pseudopotential. Optimized geometries were subject to single point energy evaluations to evaluate the effects of solvation. Three conformations of the N-benzyl sidechain were studied, and energies of the lowest energy conformation reported. These calculations revealed that the lowest energy structure is that of 4a. In these calculations, the preferred conformation of the sidechain is an extended one, Figure 4 (left). The alternative conformation [Figure 4 (center)], resembling that observed in the crystal structures is close in energy (0.8 kcal/mol) and so is readily accessible. The preferred conformation for 4b is 1.6 kcal/mol higher in energy than the lowest energy conformation of 4a, and its geometry as shown in Figure 4 (right) resembles the crystal structure of 4b in Figure 3. The energy difference between 4a and 4b narrows slightly (by 0.01 kcal/mol) on changing from methanol solvation to ethanol. The origin of the preference for 4a is likely the close approach required by the N-benzyl group to the hydrogen at the 2-position of the azetidine; this rigid system does not permit this close contact to be avoided. A table comparing and contrasting both crystallographically and computationally determined (minima) selected bond lengths, angles and torsions of 4a and 4b is included in the Supplementary Material. Good agreement between these data confirms the computed coordination environment matches that experimentally observed.</p><!><p>Calculated structural minima for 4a (left) and 4b (right), 4b being 1.6 kcal/mol higher in relative free energy then 4a. A crystal-structure-like accessible (0.8 kcal/mol higher in relative free energy) minima for 4a depicted in the center.</p><!><p>This computational analysis reveals that at equilibrium a mixture of 4a and 4b would consist mostly of 4a. Yet in our slightly divergent preparation and perhaps more importantly slow crystallization conditions is was possible to favor crystalline deposition of the minor component (diffusion of diethyl ether into, vs. slow evaporation of acetonitrile).</p><p>Closer inspection of the wider sphere about the metal atoms in the crystal structures shows that compound 4b places the azetidine ring N-benzyl group closer to the platinum, potentially due to the space created by placing the exocyclic N-benzyl group on the opposite face of the square plane. This may be judged by a shortest aryl-H…Pt distance of 2.8806 (3) Å for 4b (Figure 5) whereas aryl-H…Pt distances in 4a are ≥3.00 Å (Hambley, 1998).</p><!><p>Representation of a molecule of 4b showing a 2.8806 (3) Å distance between and aryl-H (H17) and the platinum(II) center (Pt1).</p><!><p>Whilst computationally reasoned to be primarily due to solid state, crystallization condition-dependent, phenomena we were interested to probe the consequences of the nitrogen atom stereogenicity upon the asymmetric, or more precisely the differing topological, properties of the cleft described by ligand 1a in complexes 4a and 4b, as such the buried volume (%Vbur) described by the ligand geometries was calculated. Inspired by the work of Nolan (Clavier and Nolan, 2010), Cavallo (Poater et al., 2009) and their co-workers the SambVca 2.0 tool was employed (Falivene et al., 2016), topological maps generated and their features compared2, to describe the steric constraints imposed about the exchangeable (halide) sites. PDB files containing one molecule of the single crystal X-ray diffraction-determined unit cell were uploaded to the SambVca 2.0 web-portal. The x axis being described as parallel with the N-N orientation of the square plane about the metal; the y axis lies perpendicular to the square plane of the metals' coordination; and the z axis bisects the both the two nitrogens and the two chlorides. The chlorides were removed in the web-portal and the %Vbur calculation run under otherwise default settings.</p><p>Viewed from the front (down the z-axis as depicted in Figure 6) the diastereoisomeric complexes show similar steric constraints on the left upper and lower (NW and SW) quadrants. As expected the right side (Easterly) differs markedly between the diastereomeric complexes 4a and 4b, essentially leaving the NE and SE quadrants sterically unencumbered respectively. Thus, demonstrating the significance of nitrogen's coordinational chirality in defining the asymmetric environment a chiral ligand describes around a metal. In principle it may be reasoned that were the formed diastereomeric complexes stable and isolated as single enantiomers, that the same sense of carbon-centered stereogenicity could deliver opposite enantiomers of products in catalysis as a result of deviation in nitrogen-centered stereogenicity, a phenomenon warranting future further investigation. In order to permit direct comparison, steric maps of the percentage buried volume of all complexes of this report, generated by the SambVca 2.0 online tool, are included in the Supporting Information of this manuscript.</p><!><p>Burried volume analysis of ligand geometries in complexes 4a and 4b, conducted using online tool SambVca 2.0. (i-iv) Various orientations of the considered structures; (v) SambVca-generated %Vbur map ; and (vi) %Vbur and quadrantwise contributions.</p><!><p>Having previously accessed ortho bromo ligand 1i and having previously investigated oxidative addition of 10 metals to form NCN pincer complexes (Fossey and Richards, 2004; Fossey et al., 2007), it was reasoned that ligand 1i may form a metallocyclic complex upon reaction with palladium(0), Scheme 3. To our delight a small amount of a CNN' metallocyclic complex (5) was identified, see Figure 7 for a representation of the crystal structure.</p><!><p>Oxidative addition of palladium(0) to 1i to generate a palladium(II) CNN' metallocyclic complex.</p><p>Representation of one molecule from the unit cell of the single crystal XRD structure of racemic complex 5. Elipsoids plot at 50% probability, and rendered using Ortep-III for Windows and PovRay v3.7.</p><!><p>A unique opportunity provided by the 2,4-cis-disubstituted azetidine geometry is that a tridentate complexation of palladium is possible and concave ligand architecture is able to asymmetrically envelop the metal, and distort the metal coordination geometry away from an ideal square plane. The trans bond angles C-Pd-N and N-Pd-Br deviate from 180°, being 160.8° and 173.8° respectively, leading to slight pyramidalisation of the palladium(II) center (Figure 7).</p><p>When a salt of an amino azetidine ligand (6) was exposed to sodium tetrachloropalladate and heated to reflux in methanol or ethanol ring-opened complexes (7a and b) were obtained, Scheme 4. Very few crystals were formed precluding analysis other than by single crystal XRD analysis alongside intractable material and it is not yet possible to say if all the ligand was consumed in this manner (Figure 8). But since prolonged heating of free-base ligands in methanol or ethanol with a metal source did not furnish any detectable ring-opened material it was reasoned that the small amount of crystals obtained resulted from acid promoted ring opening followed by coordination to palladium rather than the complex being unstable to the reaction conditions. This could be the subject of further future study, especially if the accessed ring opened complexes offer advantages in catalysis or other applications. The %Vbur of complexes 7a and b are also calculated, and details are provided in the Supporting Information to allow for comparison amongst the complexes detailed in this manuscript.</p><!><p>Methyl and ethyl ether complexes 7a and 7b resulting from azetidine ring opening of salt 6 and complexation to palladium(II) identified by single crystal XRD investigation.</p><p>Representation of one molecule from the unit cells of the single crystal XRD structures of racemic complex 7a and 7b. Elipsoids plot at 50% probability, and rendered using Ortep-III for Windows and PovRay v3.7.</p><!><p>The crystal structures of racemic complexes 7a and 7b display typical NN' binding modes in these palladium(II) chloride complexes, the stereogenic center on the carbon backbone imposes a trans stereochemical relationship upon the coordinationally chiral neighboring nitrogen atom (Fossey et al., 2005, 2008). The bulky groups point away from each other, and there is no evidence, in the solid state, of ancillary ether oxygen coordination. The crystals analyzed are single diastereoisomer racemates, meaning the ring opening step appears to have proceeded with high stereochemical fidelity, i.e., with clean inversion upon O-attack on the aryl substitute azetidinium ring stereogenic carbon (Gaertner, 1968; Leonard and Durand, 1968).</p><p>The synthesis of 2,4-cis-disubstituted azetidines by an iodocyclisation protocol used in the synthesis of azetidines 1a-g can give rise to pyrrolidine by-products (see previous reports for details). In the case of the synthesis of complex 2d a minor pyrrolidine impurity led to the formation and initial isolation of a complex derived from 2,4-cis-disubstituted pyrrolidine 8.</p><p>Whilst only a single crystal of the formed complex 9 was isolated and analyzed Scheme 5, as the minor component of the mixture, the complex itself displays some noteworthy features (Figure 9). Firstly, that even as a minor component, complex 9 readily formed a prominent crystal bodes well for the future targeted synthesis and isolation of it and its analogs. Secondly the cis relative stereochemistry across the 2,4-positions confers a geometry similar to, but subtly different from, azetidine analogs (compare 2d with 9). Importantly, in our previous work we showed how we can access both diastereoisomers of 8-like pyrrolidines and a future planned programme of study will look in detail at the potential of 2,4-cis-pyrrolidines as scaffolds for catalyst construction (Feula et al., 2010, 2013).</p><!><p>The formation of complex 9. Namely through complexation of a minor impurity (8), leading to a crystalline material, one crystal, of a prominent crystal upon attempted crystallization, alter leading to 2d after removal of the obtain crystal of 9.</p><p>Representation of one molecule from the unit cell of the single crystal XRD structure of racemic complex 9. Elipsoids plot at 50% probability, and rendered using Ortep-III for Windows and PovRay v3.7.</p><!><p>Since ligands 1d and 8 are accessible form the same source, depending on divergent synthesis conditions (Feula et al., 2013), complexes 2d and 9 were compared using the SambVca 2.0 online tool, Figure 10.</p><!><p>Burried volume analysis of ligand geometries in complexes 2d and 9, conducted using online tool SambVca 2.0. (i-iv) Various orientations of the considered structures; (v) SambVca-generated %Vbur map ; and (vi) %Vbur and quadrantwise contributions.</p><!><p>Under the same inspection protocols both the azetidine (2d) and pyrrolidine (9) complexes offer a similar level of steric constraint about their respective palladium(II) metal centers. However, the subtle impact of employing isomeric 4- and 5-membered heterocyclic ligands (1d and 8 respectively) can be witnessed in the buried volume differences between western and eastern hemispheres (as drawn). The average difference between the (western) left vs. (eastern) right increases from 13 to 21% across 2d and 9 respectively. Thus, were single enantiomer asymmetric catalyst to be developed it may be expected that 9 offers greater steric discrimination across the described x axis. Whilst beyond the scope of this crystal structure report the displayed steric divergence between these isomeric ligands warrants further attention in an onward programme of research.</p><!><p>Seven N,N'-palladium(II) chloride complexes, one N,N'-palladium(II) acetate complex of 2,4-cis-azetidines where prepared and analyzed by single crystal XRD. The racemic ligands adopted a single diastereoismer form upon coordination to palladium the same chirality at nitrogen. In the palladium(II) acetate complex a coordinated water molecule and H-bonding acetates formed the identified complex. Two platinum(II) chloride N,N'-complexes of 2,4-cis-azetidines where prepared and analyzed by single crystal XRD, and two diastereoisomers were generated upon amine coordination to platinum (under different preparation conditions). Computational analysis revealed which diastereoisomer was more stable and provided a rationale for why this is the case, and the %Vbur described by the diastereomeric coordination geometry was examined. A CNN' metallocyclic complex was prepared by oxidative addition of palladium(0) to an ortho bromo 2,4-cis-disubstituted azetidine and its crystal structure displays a slightly pyramidalized metal-ligand orientation. Ligand salts were not suitable for the synthesis of azetidine complexes, instead leading to N,N' complexation of stereospecifically ring-opened congeners. A minor, pyrrolidine, impurity in an azetidine ligand sample led, initially, to the formation of a highly crystalline complex, identified by single crystal XRD, as well as (later) from the same sample the expected azetidine complex. The isomeric azetidine and pyrrolidine complexes, characterized by single crystal XRD, were studied using the SambVca 2.0 online tool and their steric parameters contrasted revealing the potential for both azetidine and pyrrolidine ligands in future catalytic applications3.</p><!><p>AF developed the ligand synthesis protocol (reported elsewhere) and synthesized some cis-aminoazetidines and prepared XRD-quality crystals of the first N,N'-platinum and -palladium complexes of them, the first organometallic CNN'-palladium azetidine complex was also prepared, AF co-wrote sections of the manuscript; JF is the lead and corresponding author who conceived and led the project, JF conceived the project and co-wrote the manuscript; AL: calculated the free energies of the diastereomeric platinum complexes and wrote the aspects of the manuscript pertaining to that aspect; LM recorded, analyzed and interpreted some of the X-ray crystallography data of this report and has oversight of all of the X-ray crystallography data herein. LM conducted training of AY, in order for AY to carry out XRD analysis, LM co-wrote aspects of the report pertaining to XRD data collection and analysis; AY synthesized some cis-aminoazetidines and prepared XRD-quality crystals of N,N'-platinum and -palladium complexes of them. AY obtained the crystal of the cis-pyrrolidine palladium complex and probed the solvent-mediated ring opening of an azetidine salt during a palladium complexation protocol. AY prepared ligands and complexes, analyzed and interpreted some of the XRD data in this report and co-wrote aspects of the report. All authors made critical contributions to the report, gave significant input into the report writing process and analysis of data herein.</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p>

PubMed Open Access

Cell-level Comparisons between Literature and Industry for Lithium-Sulfur, Lithium-Ion, Lithium-Oxygen, and other Next-Generation Batteries

A set of 212 lithium-sulfur, lithium-ion, lithium-oxygen, and other next-generation experimental battery articles was reviewed, and 15 articles provided at least one cell-level performance metric and/or a complete set of information to calculate it. This subset was compared against 27 commercial technologies in key metrics across various energy storage applications using an Excel database tool. While many high cell-level specific energy batteries are reported in the literature, there is a lack of demonstrated high cell-level specific power batteries for any chemistry. Li-S applications are comparable to Li-ion in terms of specific power and generally have higher specific energy, but also exhibit lower cycle life. In future work and as more articles include cell-level information, this tool can be expanded to include thousands of data points, enabling development of new models via machine learning and other methods to predict relationships between battery structure and performance. Additionally, the tool would become more useful for industry experts to effectively search and sort through the vast amount of knowledge accessible in research journals.

cell-level_comparisons_between_literature_and_industry_for_lithium-sulfur,_lithium-ion,_lithium-oxyg

Introduction<!>Visual Database via Radar Chart<!>Tool Features<!>Assessment of Next-Generation Literature<!>Ragone and Radar Data<!>Conclusion

<p>Battery technologies have helped to better the standard of living of millions of people through renewable energy [1][2][3][4], electric vehicle [1,[5][6][7][8][9][10], consumer electronic [11][12][13], medical [14][15][16][17], unmanned aerial vehicle (UAV) [18,19], military [3,[20][21][22] and other applications. Batteries will continue transforming these sectors as systematic improvements are made in a range of key areas including specific energy [1,3,[8][9][10]23], specific power [1,[23][24][25], affordability (i.e. low-cost) [1,[26][27][28][29][30][31][32][33][34][35][36][37], cycle life [1,24,[38][39][40][41][42][43][44], amples of keywords include "lithium-sulfur", "lithium-air", "silicon anode", "flow battery", "specific energy", "specific power", and "cell-level."</p><p>From a master list of articles, those that were experimental in nature were manually sorted from those that were theoretical, computational, or a review article, resulting in the 212 experimental articles considered for review. The experimental articles were manually evaluated to determine whether they reported or included enough information to calculate a cell-level metric. If sufficient information was included, experimental details from these articles were extracted and used to calculate cell-level metrics.</p><p>Due to the differences in type of information reported and cell-design, each article required an individualized approach to make cell-level calculations (see for example, Table ??). It was also noted whether a paper with cell-level information made a claim to commercial potential of the battery and if a celllevel metric or set of metrics from the article was comparable to or exceeded that of a commercialized state-of-the-art solution.</p><!><p>Cell-level performance of characteristic commercial technologies from the high-impact applications highlighted in this work were obtained from online datasheets, retail websites, and personal communications with companies. Experimental battery articles are compared with commercial solutions.</p><!><p>The tool offers versatile features to visually display the database information using sorting and filtering. A demonstration of the various features is provided in the supporting information. Best-in-Class sorting is a feature useful for identifying leaders in particular categories and can be helpful in evaluating the potential of ones own work in comparison with commercial technologies. In addition, it may provide insight for those looking to find available options or compare their products against competition.</p><p>When evaluating whether or not a technology meets the needs of a particular application, custom filtering can help in comparing the oftentimes multiple minimum requirements involved. Data from multiple categories that is below a particular value can be excluded, resulting in a condensed and more complexly sorted list. There are 15 articles that report sufficient information to determine cell performance criteria such as specific energy and specific power. Within these 15 articles, some were even competitive with state-of-the-art commercial technologies in that a metric or combination of metrics was comparable with state-of-the-art technologies (e.g. high specific energy, high affordability).</p><p>Because not all criteria are essential to know for each application, for simplicity, criteria can be manually added or removed. When the above two techniques (Best-in-Class Sorting and Custom Filtering) do not properly refine the data, technologies can similarly be manually included or excluded.</p><!><p>This research reviews 212 experimental battery articles up until 2017 with a focus towards Li-S articles (Figure 1). Other types of batteries considered include lithium-air, silicon anode, lithium-ion, and flow batteries. Ones that either include enough information to calculate or directly report cell-level performance are shown in Table 1. The first set [27,46,[56][57][58][59][60] represent articles that cite manufacturing potential, include a complete set of component information to make cell-level calculations, and do not directly report these metrics. The second set [61][62][63][64] consists of those that directly report a metric at the cell-level and do not include a complete set of component level information to calculate cell-level metrics. The third set consists of articles that include a complete set of component level information and report a cell-level metric [65][66][67]. There is a trade-off between specific energy and specific power, as well as a lack of representation above 100 W/kg for specific power in the literature. Arrows highlight which batteries are primary batteries (i.e. cycle life of 1). In the case of EV and RE applications, the two batteries highlighted are used in both applications. For the first set, cell-level performance was calculated from component-level information. The component-level information from each paper typically required an individualized approach to calculate cell-level performance due to cell configurations (e.g. coin cell vs. flow cell) and reported information (e.g. areal loading thickness vs. molarities) differing between papers.</p><p>For the second set, some component-level information was not present that would be necessary to calculate a cell-level performance metric. This made it infeasible to make reasonable cost predictions.</p><p>The percentage of experimental articles that contain some type of cell-level information and the importance of this type of information is consistent with observations by various groups [70][71][72][73]. Including both comprehensive component-level information and cell-level metrics provides an opportunity for industry to easily make evaluations and comparisons of cutting-edge battery research and helps research groups narrow down key system limitations [31,73]. This can help researchers compare potential for impact and high-priority gaps in knowledge. Quantities of materials such as electrolyte and lithium metal are often left unmeasured or unreported [73]. By tracking and reporting these material types and quantities (e.g. "100 µL 1M LiTFSI in DME/DIOX (1:1) used as electrolyte" or "50 mg, 2 cm 2 dia. lithium metal foil used as anode"), failure mechanisms may be easier to elucidate because the performance of many next-generation chemistries change drastically based on parameters [61] such as excess electrolyte or lithium [73]. With an increase in reported information, these trends could be analyzed in broader ways through methods such as machine learning [70]. Because processing methods and characterization parameters can vary drastically and the number of parameters are often extensive, many articles with such information are essential for reliable insights [70].</p><p>This perspective is not meant to detract from the importance of understanding fundamental chemistries [9,51] and cross-component and system-level challenges [3] of next-generation energy storage technology. An interdisciplinary approach that balances basic studies of chemistry systems and battery components with a knowledge of practical energy storage application requirements is required for successful implementation of next-generation technologies.</p><p>Due to lack of reported information and the subjective nature of storage characteristics and safety categories, for the purposes of this study, qualitative rankings are assigned based on chemical composition and material choice. For example, literature batteries that contain additives such as LiNO 3 [71] are given Table 2: Summary of experimental data from industry. Six performance metrics -Specific energy (SE), specific power (SP), number of cycles to 80% capacity, affordability (energy affordability and lifetime energy affordability), safety, storage characteristics, and energy density (ED) -are given of characteristic technologies from commercial, start-up, and literature batteries. Safety and storage characteristics are assigned qualitative rankings based on the chemistry and the constituents of the cell/system. Commercial affordability data was obtained through online sources (see "Ref" column), whereas literature affordability data was estimated from raw material costs and complexity of processing. * BPS stands for Battery Power Solutions. lower safety rankings.</p><!><p>Data is presented detailing Ragone plot comparisons within literature (Figure 2), among literature, start-ups, and commercial technologies (Figure 3), among various chemistries(Figure 4), and corresponding radar charts.</p><p>In literature, very high specific energies can be achieved (Figure 2); however, many fall short of specific power values obtained in industry (Figure 3). Interestingly, when considering all secondary literature batteries (i.e. non-primary batteries), there is not a significant trade-off between specific energy and specific power; rather, a generally increasing trend is observed. This could be related to the amount of excess materials in the battery and the battery's ability to function well with an optimized amount of excess material or to a trade-off not seen in metrics other than cell-level specific energy and specific power.</p><p>There are no start-up affordability data points due to lack of available information. This is to be expected, since estimating cost from components would require proprietary information from the, and the batteries are usually only available as evaluation cells or through private contracts. Li-S, Li-ion, and Li-O2 batteries each have examples of high specific energy (Figure 4). Several</p><p>Li-S and Li-ion data points are near each other, with Li-S generally exhibiting higher cell-level specific energy. One of the major differences is that the cycle life of the Li-ion batteries is generally higher than the Li-S batteries (Table 2).</p><p>The differences in shapes of the radar charts (Figures 5 and 6) illustrate that maximizing a single criteria does not necessarily produce the same kinds batteries. This supports the notion that multiple battery requirements change for a given application. Literature data is again noticeably lacking in terms of cell-level specific power.</p><!><p>A framework is provided to systematically integrate and compare experimental battery research data via a visual database tool aimed at helping to bridge the gap between science and industry. Cell-level data had limited availability in the literature. Reporting such information enables calculations of critical battery performance metrics including cell-level specific energy, cell-level specific power, and cell-level affordability estimates. Ragone and radar charts are used to compare technologies and identify trends across literature and industry.</p><p>While great strides have been made towards higher specific energy batteries, this study reveals that high specific power batteries are lacking in the literature. Cycle life information is typically available in most experimental secondary battery publications; however, metrics such as energy retention (alternatively, self-discharge) are often not reported. Safety is more difficult to quantify but can be done to some extent by reporting the aforementioned information and evaluating the energy, power, and materials.</p><p>Safety can be more rigorously evaluated through shock, short-circuit, and puncture tests. Additionally, and as application-specific focuses arise, research groups may consider studying and reporting other metrics such as temperature performance, energy efficiency, volumetric energy and power density, fast charge characteristics, and voltage stability.</p><p>Ragone and radar charts supplied in the visual database tool are useful for planning and comparison purposes in both science and industry. The readers are encouraged to use these tools to inform their studies of high-impact battery chemistries. The visual database tool and relevant data are provided as an attachment for the reader.</p><p>Funding: This work was supported through a BYU Office of Research and Creativities (ORCA)</p><p>Grant.</p>

ChemRxiv

Click Chemistry and Radiochemistry: The First 10 Years

The advent of click chemistry has had a profound influence on almost all branches of chemical science. This is particularly true of radiochemistry and the synthesis of agents for positron emission tomography (PET), single photon emission computed tomography (SPECT), and targeted radiotherapy. The selectivity, ease, rapidity, and modularity of click ligations make them nearly ideally suited for the construction of radiotracers, a process that often involves working with biomolecules in aqueous conditions with inexorably decaying radioisotopes. In the following pages, our goal is to provide a broad overview of the first 10 years of research at the intersection of click chemistry and radiochemistry. The discussion will focus on four areas that we believe underscore the critical advantages provided by click chemistry: (i) the use of prosthetic groups for radiolabeling reactions, (ii) the creation of coordination scaffolds for radiometals, (iii) the site-specific radiolabeling of proteins and peptides, and (iv) the development of strategies for in vivo pretargeting. Particular emphasis will be placed on the four most prevalent click reactions\xe2\x80\x94the Cu-catalyzed azide-alkyne cycloaddition (CuAAC), the strainpromoted azide-alkyne cycloaddition (SPAAC), the inverse electron demand Diels-Alder reaction (IEDDA), and the Staudinger ligation\xe2\x80\x94although less well-known click ligations will be discussed as well. Ultimately, it is our hope that this review will not only serve to educate readers but will also act as a springboard, inspiring synthetic chemists and radiochemists alike to harness click chemistry in even more innovative and ambitious ways as we embark upon the second decade of this fruitful collaboration.

click_chemistry_and_radiochemistry:_the_first_10_years

INTRODUCTION<!><!>INTRODUCTION<!>RADIOLABELING WITH PROSTHETIC GROUPS<!>CREATING COORDINATION SCAFFOLDS<!>SITE-SPECIFIC BIOCONJUGATION<!>IN VIVO PRETARGETING<!>EMERGING APPLICATIONS<!>CONCLUSIONS AND FUTURE DIRECTIONS

<p>A decade and a half have passed since Kolb, Finn, and Sharpless published the landmark review that introduced the concept of click chemistry.1 In the intervening years, the influence of click chemistry has grown by leaps and bounds. To wit, the number of publications with "click chemistry" in the title has grown from 6 in 2003 to 252 in 2009 to 2014 in 2015!2</p><!><p>"The reaction must be modular, wide in scope, give very high yields, generate only inoffensive byproducts that can be removed by non-chromatographic methods, and be stereospecific (but not necessarily enantioselective). The required process characteristics include simple reaction conditions (ideally, the process should be insensitive to oxygen and water), readily availably starting materials and reagents, the use of no solvent or a solvent that is benign (such as water) or easily removed, and simple product isolation."</p><!><p>A handful of reactions that satisfy (or, at the very least, come close to satisfying) these criteria have been uncovered, including nucleophilic ring opening reactions with epoxides, aziridines, and aziridinium ions; the formation of ureas, oximes, and hydrazones via nonaldol carbonyl chemistry; and oxidative and Michael additions to carbon–carbon double bonds.3 Yet one particularly powerful reaction has emerged as the canonical click ligation and has proven remarkably useful in myriad applications: the copper-catalyzed [3 + 2] cycloaddition between an azide and a terminal alkyne (Figure 1A).4,5 More recently, Bertozzi and others have pioneered a subset of click reactions that boast an additional boundary condition: bioorthogonality.6–9 Bioorthogonal click ligations satisfy all of the requirements of standard click reactions but are also inert within biological systems. Not surprisingly, these reactions are hard to come by, yet a handful (most notably the Staudinger ligation, the strain-promoted azide–alkyne cycloaddition reaction, and the inverse electron demand Diels–Alder cycloaddition) have been developed and proven powerful in the hands of chemical biologists, biochemists, and biomedical scientists (Figure 1B–D).7,10–16</p><p>Click chemistry has had a paradigm-shifting influence on a wide range of chemical fields, from drug development17,18 and polymer chemistry19,20 to chemical biology21 and nanoscience.22 However, it is hard to imagine a field that has more to gain from harnessing click chemistry than radiochemistry. The principal reason for this lies in what makes radiochemistry unique: the inexorable physical decay of radioisotopes during synthesis. As a result, radiolabeling reactions—and especially radiolabeling reactions using short-lived isotopes such as 11C (t1/2 ≈ 20 min) and 68Ga (t1/2≈68 min)—must be rapid and efficient tomaximize yield as well as selective and clean to eliminate time-sapping purification steps. Furthermore, the widespread use of biomolecules as targeting vectors has also placed a premium on bioconjugation reactions that are both selective and unencumbered by water. Finally, the proliferation of an ever-growing list of prosthetic groups and radiometal chelators has made modularity a critical feature of radiosynthetic protocols as well. Remarkably, all of these traits can be found in click chemistry ligations.</p><p>In light of these benefits, it is somewhat surprising that the first publications describing radiopharmaceuticals synthesized using click chemistry came rather late: a 2006 work from Mindt et al. describing the use of click chemistry to create coordination scaffolds for 99mTc and a 2007 report from Wuest and co-workers detailing the use of the CuAAC reaction to create an 18F-labeled variant of neurotensin(8–13).23 Yet in the years since this somewhat belated start, work at the nexus of these two fields has expanded dramatically.24–27 This growth means that an exhaustive review covering every instance in which click chemistry has been applied to nuclear imaging would almost certainly be an exhausting read. Instead, in the pages that follow, it is our goal to highlight the most interesting, exciting, and useful points of intersection between click chemistry and nuclear medicine. More specifically, we will focus on the use of click chemistry for (i) radiolabeling reactions with prosthetic groups, (ii) the creation of novel chelation architectures, (iii) site-specific bioconjugation, and (iv) in vivo pretargeting. Taken together, we believe that these four areas underscore how the rapidity, efficiency, selectivity, modularity, and bioorthogonality of click chemistry have empowered radiochemists to create innovative agents for imaging and therapy. Ultimately, we sincerely hope that this review not only informs the reader about research at the intersection of chemistry and radiochemistry but also inspires new and seasoned researchers alike to apply this remarkably useful chemical technique to the development radiopharmaceuticals.</p><!><p>One of the first reported, and still most extensively employed, applications of click chemistry to radiochemistry lies in the use of "clickable" prosthetic groups for radiolabeling. The everincreasing use of imaging agents based on biomolecular vectors has put a premium on radiosynthesis strategies that are both mild and selective. Put simply, peptides, proteins, and antibodies should be radiolabeled under aqueous conditions at room temperature to ensure that their structural integrity is preserved, yet critically, many radiolabeling reactions require elevated temperatures, nonaqueous solvents, or (at the very least) pH conditions outside of the physiological norm. This is especially true for 18F-radiofluorination reactions, which often require organic solvents and high temperatures.</p><p>Radiolabeled prosthetic groups provide an efficient way to circumvent these issues. Prosthetic groups are radiolabeled reactive small molecules that can be appended to biomolecules under benign conditions. Until recently, the vast majority of prosthetic groups have relied upon reactions with natural amino acids (most notably, couplings between N-hydroxysuccinimidyl (NHS) esters and lysines and Michael additions between maleimides and cysteines).28–30 Yet prosthetic groups of this ilk present a number of problems. Most concerning is the complete loss of regiochemical control during the labeling of a peptide or protein containing more than one lysine or cysteine. This, of course, can only be remedied by yield-sapping separations or the addition of time-consuming protection and deprotection steps.31 On top of this, both NHS esters and their isothiocyanate cousins are unstable under aqueous conditions, and maleimide–thiol linkages are prone to reversible substitution reactions in vivo.32</p><p>In response to these limitations, radiochemists have increasingly turned to "clickable" prosthetic groups. Not surprisingly, the canonical CuAAC ligation leads the pack. In this regard, the relative age of the reaction certainly plays a role. Yet another critical advantage of the CuAAC ligation is that its "footprint" — a 1,2,3-triazole ring — is unlikely to perturb the structure or activity of the vector: the heterocycle is both relatively small and a rigid stereoisomer of an amide linkage. At this junction, we would be remiss if we did not mention the CuAAC reaction's lesser-known cousin: the ruthenium-catalyzed azide–alkyne cycloaddition (RuAAC).33 The RuAAC reaction produces 1,5-disubstituted 1,2,3-triazoles as opposed to the 1,4-disubstituted 1,2,3-triazoles created by the Cu-catalyzed cycloaddition. Even though it is regarded as a "click reaction", the RuAAC ligation requires organic solvents, elevated temperatures, and inert gas atmosphere. Furthermore, the 1,5-disubstituted 1,2,3-triazoles produced by the reaction are—unlike 1,4-disubstituted 1,2,3-triazoles—metabolically active and can be degraded via enzymatic N3 oxidation to produce highly reactive and potentially toxic metabolites.34 Given both of these issues, it is not surprising that, to the best of our knowledge, the RuAAC reaction has not been applied to the synthesis of radiopharmaceuticals.</p><p>Moving back to the topic at hand, an extensive body of work has emerged on the design, synthesis, and optimization of radiolabeled CuAAC-ready building blocks. Much, although not all, of this work has focused on 18F.35–38 Indeed, a variety of radiosynthetic methods have been employed to create azide- and alkyne-bearing 18F-labeled prosthetic groups (Figure 2A).37,39,40 These tools and the CuAAC reaction have been harnessed with great success in the radiolabeling of a wide variety of vectors, including phosphonium ions,41 peptides,42–50 oligonucleotides,39,47 and proteins.27,47 This application of the CuAAC reaction is not without its flaws, however. These stem primarily from the two reagents needed to facilitate the cycloaddition: Cu(I/II) cations and a sacrificial reductant. The latter, most often ascorbic acid, can inadvertently reduce particularly fragile peptides and proteins.27 The Cu cations can be even more of a problem. Peptides and proteins (specifically serine, histidine, and arginine residues) can coordinate Cu2+ ions, resulting in structural and functional alterations to the peptide.51 For example, Pretze et al. observed the accidental formation of Cu–peptide complexes following the CuAAC-mediated ligation of an 18F-labeled, alkyne-containing prosthetic group to an azide-bearing SNEW peptide.45 The coordination of the oxidative Cu(I) species can also lead to dramatic alterations to the chelating amino acid residues, as demonstrated very recently.52 These issues are compounded even further for radiometal-containing constructs. In these cases, not only can the chelator capture the copper catalyst and prevent the reaction from happening, but residual Cu2+ ions can also outcompete the far less abundant radiometal cations for coordination by the chelator.53 On top of these coordination-related concerns, the presence of Cu+ can also increase the likelihood of undesired side reactions such as Glaser couplings or the formation of copper-acetylides.45,54,55 Some of these issues can be ameliorated through the use of Cu+-stabilizing chelators such as THPTA or N-heterocyclic carbene complexes of Cu+; however, these reagents can create their own set of complications.56–58</p><p>In light of the limitations of the CuAAC ligation, researchers have turned to a handful of "second generation" click reactions that are both bioorthogonal and catalyst-free. The most obvious place to start is the strain-promoted azide–alkyne cycloaddition (SPAAC). The SPAAC reaction is an azide–alkyne cycloaddition in which ring strain built into a cyclic alkyne—often a dibenzocyclooctyne (DBCO)—drives the reaction and eliminates the need for a catalyst.59,60 Campbell-Verduyn et al. were among the first to use this approach for radiochemistry, creating a series of 18F-labeled bombesin derivatives via the reaction of a DBCO-modified peptide with an array of 18F-bearing, azide-containing prosthetic groups.61 Following a similar strategy, another laboratory modified a series of ανβ3-targeting RGD peptides with DBCO and radiolabeled them using an [18F]fluoro–PEG4–azide prosthetic group.50,62 In a creative twist, the authors scavenged excess unlabeled peptide using an azide-grafted resin, allowing them to achieve specific activities of up to 62.5GBq/μmol. Critically, all of the 18F-labeled peptides bore biological affinity comparable to their unlabeled cousins and were shown to be effective for the visualization ανβ3-expressing U87MG xenografts (Figure 3). Of course, radiolabeling via the SPAAC reaction goes both ways: several laboratories have created 18F-labeled cyclooctynes for the radiofluorination of azide-modified small molecules, sugars, and peptides (Figure 2B).63–65</p><p>The SPAAC reaction has also been used for radioiodinations and radiometalations. Choi et al., for example, used a DBCO-bearing cRGD peptide and a prosthetic group composed of a PEG4–azide moiety grafted to an 125I-labeled pyridine to create an 125I-labeled cRGD.66 Evans et al. labeled an azide-modified DOTA with 68Ga for the radiometalation of several DBCO-modified peptides.53 Likewise, the Anderson group has conjugated DIBO-bearing copper chelators to an azide-modified cetuximab antibody and an azide-bearing somatostatin analogue.67,68</p><p>Despite its utility, the SPAAC ligation has one critical limitation: its dibenzocyclooctatriazole "footprint". The work of Hausner and co-workers provides a particularly useful cautionary example.69 Here, the authors radiolabeled an azide-modified A20FMDV2-peptide using an 18F-labeled variant of DBCO. While in vitro experiments confirmed that the 18F-labeled peptide retained its affinity and specificity for ανβ6-expressing cells, in vivo imaging suggested that the bulky and hydrophobic benzocyclooctatriazole footprint introduced by the SPAAC ligation led to dramatic changes in the pharmacokinetics of the tracer and significantly impaired its uptake in ανβ6-expressing xenografts.</p><p>The inverse electron demand Diels–Alder (IEDDA) cycloaddition between tetrazine (Tz) and a dienophile, most commonly trans-cyclooctene (TCO) but also norbornene (NB), has also provided fertile ground for the development of prosthetic groups. Like the SPAAC ligation, the IEDDA reaction is bioorthogonal and proceeds without a catalyst. The principal advantage of the IEDDA ligation is its extraordinary speed (vide infra), which makes it particularly well suited for applications with short-lived radioisotopes. In 2010, the laboratories of Fox and Conti reported the first 18F-labeled TCO (Figure 2C).70 This prosthetic group was used for the rapid (t < 5 min) radiolabeling of a range of tetrazine-bearing peptides, including RGD and the GLP agonist Exendin.71–73 The 18F-labeled Exendin proved particularly promising, enabling the PET imaging of GLP-1R-positive insulinoma xenografts in mice. The same 18F–TCO was also used to great effect by Weissleder and co-workers for labeling a Tz-bearing analog of the PARP1 inhibitor AZD2281. In this work, however, the authors added a creative wrinkle: removing unlabeled AZD2281–Tz using a TCO-coated magnetic resin.74,75 Finally, a number of 18F-labeled tetrazines have also been synthesized, but the in vivo use of radiopharmaceuticals created using these moieties has thus far remained somewhat sparing.76,77</p><p>The utility of the IEDDA reaction extends beyond radiofluorination. 53 To wit, a handful of radioiodinated tetrazine constructs have been successfully developed (Figure 2C). Albu et al., for example, synthesized an 125I-labeled tetrazine and conjugated this building block to a TCO-modified anti-VEGFR2 antibody.78 Interestingly, in vivo studies using this tracer revealed an additional benefit of this approach: the 125I-labeled antibody proved to be more than 10-fold more stable to deiodination over 48 h compared to traditionally radioiodinated analogs. More recently, Choi et al. used a similar strategy for the radiolabeleling of both a cRGD peptide and human serum albumin (HSA).79 The 125I-labeled HSA displayed impressive in vivo behavior, with a deiodination rate reduced by 50-fold compared to constructs created via traditional radioiodination. In 2011, Zeglis et al. employed the IEDDA reaction to create a modular strategy for the bioconjugation of a trastuzumab–TCO immunoconjugate with Tz–desferrioxamine (for 89Zr4+) and Tz–DOTA (for 64Cu2+).80 More recently, Kumar and co-workers harnessed the IEDDA reaction to circumvent the incompatibility of antibodies with the high temperatures required to radiolabel the CB-TE2A-1C chelator with 64Cu.81 To this end, the authors modified the chelator with a norbornene moiety and grafted tetrazine onto an anti-PSMA antibody (YPSMA). After radiolabeling of the chelator-NB building block with 64Cu at 85 °C, the 64Cu–CBTE2A1C-NB synthon was attached to YPSMA–Tz under mild conditions, and the 64Cu-labeled radioimmunoconjugate was successfully deployed for the PET imaging of PSMA-expressing tumors in a murine model of prostate cancer.</p><p>Although the rapidity of the IEDDA reaction provides a marked improvement over the sluggish SPAAC ligation, it fails to solve one of the latter's major issues: a bulky, hydrophobic footprint. As we have discussed, the SPAAC reaction leaves a benzocyclooctatriazole moiety in its wake. The IEDDA ligation creates an equally large footprint: a bicyclic [6.4.0] ring system. Both structures have the potential to interfere with the biological activity and pharmacokinetics of vectors, particularly small molecules and short peptides. The traceless version of the Staudinger ligation offers an exciting alternative (Figure 4A). This ligation relies on an initial reaction between a phosphine-based moiety and an azide followed by a rearrangement that produces a simple amide linkage. Along these lines, the radiolabeling of peptides with 18F has been achieved via the reaction between (diphenylphosphanyl)methanethiol thioester-bearing peptides and an 18F-labeled azide as well as that between a radiolabeled 2-(diphenylphosphanyl)phenol ester with an azide-bearing peptide (Figure 2D).82–84 Unfortunately, however, the traceless Staudinger ligation requires high temperatures (90–130 °C) to achieve speeds that are compatible with short-lived isotopes. This undoubtedly limits its utility with fragile small molecules, peptides, and proteins; however, we are optimistic about the potential applications of this elegant transformation with longer-lived isotopes.</p><p>Finally, a handful of other, less-well-known click ligations have made sparing yet interesting appearances in the literature of prosthetic groups. In 2012, Zlatopolskiy et al. reported the formation of a reactive nitrone from 18F-fluorobenzaldehyde and phenylhydroxylamine.85 The authors showed that this 18F-labeled nitrone could undergo a [3 + 2] cycloaddition with a maleimide, resulting in quantitative conversion in less than 15 min at 80 °C (Figure 4B). It must be said, however, these reaction conditions leave much to be desired when it comes to labeling biomolecules. Later the same year, the same group probed the potential of cycloaddition reactions between nitriloxides and dipolarophiles (Figure 4C).86 An 18F-labeled nitriloxide was synthesized from 18F-p-fluorobenzaldehyde and reacted with a series of dipolarophiles, producing quantitative conversions in <10 min at 40 °C. However, these reactions were performed in alcohol, and no data was presented regarding the feasibility of this transformation under aqueous conditions. Recently, other groups have harnessed the reactivity of 2-cyanobenzothiazoles toward 1,2-aminothiols to radiolabel peptides and proteins containing N-terminal cysteines (Figure 4D).87,88 To this end, 18F-labeled 2-cyanobenzothiazoles were synthesized and appended to RGD and diRGD peptides bearing N-terminal cysteines as well as a genetically engineered variant of luciferase with a cysteine at the N-terminus. Lastly, just this year, Chiotellis et al. have explored phenyloxadiazole methylsulfones (PODS) as more stable alternatives to maleimides for conjugations with thiols (Figure 4E).89 In this work, an 18F-labeled PODS was used to radiolabel both a cysteine-bearing peptide and a cysteine-modified affibody, and the resulting constructs were used to HER2-positive tumors in a mouse model of breast cancer.</p><!><p>The use of click chemistry to create radiometal chelation architectures provides one of the best examples of the unique modularity conferred by this synthetic approach.90,91 Easily the best known of these methods, dubbed "click-to-chelate" by its inventors, was introduced in 2006 by Mindt et al. (Figure 5).92–94 This strategy employs the CuI-catalyzed azide–alkyne cycloaddition (CuAAC) reaction to attach small molecule "pro-chelators" to peptides and small molecules. However, the 1,2,3-triazole produced by the click ligation becomes far more than just a simple link between the subunits of the construct. Indeed, the heterocycle forms an integral part of a tripodal coordination scaffold capable of the rapid chelation of [M(CO)3]+ synthons, in which M can be the γ-emitting radiometal 99mTc (t1/2 = 6.01 h) or the β-emitting radiometal 188Re (t1/2 = 16.98 h). In this way, "click-to-chelate" facilitates the creation of a chelator and its subsequent radiometalation in a rapid, robust, and reproducible one-pot reaction. This is particularly important given the mercurial coordination chemistry of 99mTc.</p><p>In their initial proof-of-concept report, the authors created seven different tripodal scaffolds—including N3, N2S, and N2O ligand architectures—using a series of azide-modified small molecules. Subsequent labeling with M(CO3) [M = Re, 99mTc] synthons resulted in a series of highly stable, low-spin d6-complexes despite differences in the size, molecular charge, and hydrophilicity of the prochelator.92–95 The creation of a 99mTc-labeled variant of folate using "click-to-chelate" provides an excellent example of the approach (Figure 6). The 1,2,3-triazole ring formed in the first phase of the reaction between the azide-bearing folate construct (1) and the alkyne-modified amino acid (2) not only connects the pro-chelator to the folate vector but also serves as an essential part of the N2O coordination scaffold for the [99mTc(CO)3]+ moiety. The incubation of the chelator-bearing construct with [99mTc(CO)3(H2O)3]+ reproducibly yields 99mTc-labeled folate (3) in high yield and specific activity.92</p><p>In subsequent work, this technique was applied to peptides as well as an array of other biologically active small molecules such as sugars, nucleosides, and steroids.96–100 Fernandez et al., for example, developed a 99mTc-labeled glucose derivative as an imaging probe for glucose metabolism.97 Similarly, Struthers et al. developed an elegant one-pot "click-to-chelate" synthesis of a 99mTc-labeled thymidine analogue as a SPECT surrogate for the clinically successful proliferation marker 18F–FLT.98 Taken together, this work clearly demonstrates that 99mTc-labeled tracers created using the "click-to-chelate" methodology demonstrate in vivo behavior that is comparable, and in some cases superior, to the current "gold standard" chelators for [99mTc-(CO)3]+: Nτ-derivatized histidine and Nα-acetylated histidine. Indeed, studies using 99mTc-labeled folate revealed that the click-to-chelate approach furnished compounds in purities and radiochemical yields equal to those achieved using traditional radiolabeling techniques. Furthermore, in this work, the click-to-chelate approach did not alter biodistribution patterns or pharmacodynamic parameters such as receptor affinities and selectivities. Finally, the superiority of the click-to-chelate methodology becomes most obvious in the context of synthetically challenging molecules. In the case of the azide-modified folate construct, for example, the differences in synthetic effort and yield are striking: "click-to-chelate" furnished an 99mTc-labeled tracer in 80% overall yield in 8 steps, whereas 10 steps were required to muster approximately 1% yield with a histidine-based chelator.92</p><p>From a chemical standpoint, it is important to note that the inherent asymmetry of the CuAAC reaction means that two different 1,2,3-triazoles can be formed when linking the vector and the chelator (Figure 7).93,95 In the first, the "regular click ligand", the pro-chelator bears the alkyne moiety while the vector contains the azide group, and the N3 atom of the triazole participates in the coordination of 99mTc. In the second, the "inverse click ligand", the pro-chelator boasts the azide moiety while the vector wields the alkyne group, and the N2 atom of the triazole participates in the coordination of 99mTc. Somewhat surprisingly, the two different chelation environments display quite different behavior when radiolabeled with [99mTc(CO)3]+ and [188Re(CO)3]+, with the "inverse click ligand" offering significantly lower labeling efficiency and decreased in vivo stability.93 Although a concrete explanation for this phenomenon remains elusive, the most likely hypothesis points to the decreased electron density in the N2 position compared to the N3 site.</p><p>Before moving on, it is worth noting that a handful of other groups have also used click chemistry in the synthesis of radiometal chelators. Bailey et al., for example, used the CuAAC reaction in the synthesis H4azapa: a carboxypyridine-based chelator for 111In3+ and 177Lu3+ (Figure 8A).91 In addition, Bottorff et al. have developed a synthetic strategy to generate isoxazole ligands via click chemistry (Figure 8B).101 Yet in the end, it is undeniable that the "click-to-chelate" methodology represents the gold standard in this area. Indeed, this approach not only provides a cardinal example of the modularity and flexibility provided by click chemistry but also stands as one of the most useful and innovative developments in 99mTc chemistry of the past decade.2–4</p><!><p>The selectivity and bioorthogonality of click chemistry have also been leveraged for the site-specific modification of proteins and antibodies. This process has become ubiquitous in the synthesis of biomolecular therapeutics such as antibody-drug conjugates, and it is increasingly important in the creation of radiolabeled probes as well. Until recently, the overwhelming majority of bioconjugation methods were predicated on ligations between reactive bifunctional probes—e.g., N-hydroxysuccinimide-bearing chelators or maleimide-modified toxins—and amino acids within the biomolecule, most often lysines and cysteines. While these methods are undeniably simple, they are far from precise. Control over the location and frequency of these ligations is impossible because proteins have multiple copies of these amino acids distributed throughout their structures. As a result, these bioconjugation strategies produce constructs that are both heterogeneous and poorly defined. Furthermore, random conjugation strategies can decrease the reactivity of constructs if the cargo is inadvertently appended to the target-binding domains of the biomolecule.</p><p>In response to these issues, significant effort has been dedicated to the creation of strategies for the site-specific bioconjugation of proteins and antibodies. A wide variety of methods have been developed, including variants predicated on the selective reduction of disulfide bridges and the oxidative manipulation of the heavy chain glycans. Yet regardless of the exact strategy, a wealth of preclinical data makes the bottom line clear: site-specifically labeled proteins and antibodies are more homogeneous, better defined, and exhibit superior in vivo behavior compared to constructs synthesized using traditional, random bioconjugation techniques.102–105 A handful of the most promising site-specific bioconjugation strategies combine the selectivity of enzymatic reactions with the modularity of chemical ligations. Generally speaking, these chemoenzymatic strategies have two steps. In the first, an enzyme is used to site-specifically incorporate a substrate bearing a reactive handle into the biomolecule. Then, in the second, a cargo bearing a complementary reactive handle is appended to its partner in the biomolecule. In this context, the selectivity and bioorthogonality of click chemistry are particularly valuable, as the two handles must only react with each other and not the enzyme or biomolecule.</p><p>A recently developed strategy for the site-specific modification of the heavy chain glycans (the biantennary sugar chains attached to the CH2 domains of the FC region of antibodies) provides an excellent example of a chemoenzymatic approach that employs the SPAAC ligation. Inspired by the work of Hsieh-Wilson and Qasba, this methodology employs two enzymes and has three steps: (i) the removal of the terminal sugars of the heavy chain glycans using β-(1,4)-galactosidase; (ii) the incorporation of azide-modified galactose residues (GalNAz) into the sugar chains with a promiscuous galactosyltransferase [GalT-(Y289L)]; and (iii) the attachment of dibenzocyclooctyne (DIBO)-bearing cargoes to the azide-presenting sugars (Figure 9).106–108 Ultimately, this approach has the potential to yield highly homogeneous and well-defined immunoconjugates carrying up to four cargo molecules per antibody. In their initial report, Zeglis et al. used a DIBO-modified desferrioxamine (DFO) to create a site-specifically modified, 89Zr-labeled radioimmunoconjugate based on the PSMA-targeting antibody J591. In subsequent work, the authors demonstrated the modularity and flexibility of this approach through the development of a series of immunoconjugates for the PET, NIRF, and multimodal PET/NIRF imaging of colorectal and pancreatic cancer. Importantly, in all three cases, the in vivo performance of the site-specifically modified imaging agents was equivalent, and in some respects superior, to that of analogous constructs synthesized using traditional techniques.109,110 More recently, Geel et al. reported an interesting variation on this theme.111 In this work, EndoS, an enzyme that trims each glycans chain down to its innermost residues, is used instead of β-(1,4)-galactosidase. This change ultimately produces an immunoconjugate with two azides per antibody after treatment with GalT(Y289L) and GalNAz. To date, this strategy has only been used in conjunction with DIBOmodified chemotherapeutics, but the modularity of the SPAAC ligation could easily facilitate the adaptation of this approach to the synthesis of radiopharmaceuticals.</p><p>Shifting gears to another family of enzymes, transglutaminases catalyze the formation of isopeptide bonds between the acyl functionality of glutamine residues and primary amines. While antibodies certainly contain multiple glutamine residues, transglutaminases have been found to react exclusively with the Q295 glutamines within the CH2 domain of deglycosylated or aglycosylated IgGs. This unique reactivity has led a number of laboratories (most notably, that of Roger Schibli at ETH Zurich) to harness these enzymes for the site-specific modification of antibodies. One-step and two-step approaches have been developed (Figure 10). In the former, transglutaminase is used to directly append cadaverine-modified cargoes to the antibody. This method was used by Jeger et al. to site-specifically append two DFO chelators to a deglycosylated variant of the L1CAM-targeting antibody chCE7 for radiometalation with 64Cu, 67Ga and 89Zr.112 In an interesting twist, this group used transglutaminase to modify amutant version of the chCE7 antibody that contained two additional glutamines in place of the N297 residues, thereby creating an immunoconjugate with four DFO/mAb. More germane to the topic at hand, transglutaminase has also been used to modify proteins with azide- and cyclooctyne-modified cadeverines that can then be reacted via the SPAAC ligation with DIBO- or azide-bearing cargoes, respectively.113,114 In a very recent proof-of-concept study, Puthenveetil et al. have used this strategy to label a model antibody with both Cy5.5 and BODIPY fluorophores. 115 While this approach has not yet been applied to radioimmunoconjugates, it could easily be adapted to create a modular route for the conjugation of radiometal chelators.</p><p>The last bioconjugation technique that we will discuss relies not on post-translational modifications but, rather, on harnessing the cell's translational machinery itself. The expansion of the genetic code to enable the incorporation of unnatural and noncanonical amino acids (uAAs and ncAAs, respectively) into proteins has quickly become a vital component of the molecular biologist's toolkit. The union of this technology and click chemistry has proven particularly powerful. p-Azido-L-phenylalanine (pAzF) is one of the most commonly used uAAs, and residues bearing trans-cyclooctene, tetrazine, cyclooctyne, and norbornene groups have been incorporated into proteins as well (Figure 11A).116–119 pAzF and the SPAAC ligation have been used to create both antibody-drug conjugates and immunoglobulins modified with fluorophores. Yet until very recently, only one instance of the use of this technology to create a radiolabeled compound had been reported. In this work, Wållberg et al. developed affibodies containing selenocysteine, a natural ncAA, and exploited the unique reactivity of this residue with maleimides and iodomethane to site-specifically radiolabel the vectors with both 68Ga and 11C (Figure 11B).120 Finally, just prior to the submission of this review, Wu et al. reported the first example of a radioimmunoconjugate created using an uAA. In this case, the authors incorporated an azide-bearing lysine residue (Az–K) into the heavy chain of the anti-CD20 antibody Rituximab and subsequently used the SPAAC ligation to attach a DIBO-bearing DOTA to the azide-containing immunoconjugate. 121 The site-specifically modified antibody showed in vitro and in vivo behavior comparable with an analogous construct synthesized using traditional methods. Ultimately, we are confident that more laboratories will use genetic engineering and click chemistry for the synthesis of radiopharmaceuticals as the technology underlying the former becomes more widely accessible and less technically demanding.</p><!><p>The next area of discussion, in vivo pretargeting, places the bioorthogonality and speed of click chemistry on center stage. To provide some background, in vivo pretargeting strategies have been developed in direct response to a core limitation of radiolabeled antibodies. Immunoglobulins are extraordinarily promising vectors for nuclear medicine due to their exquisite affinity and selectivity for their molecular targets. However, because antibodies have multiday biological half-lives, they must necessarily be labeled with isotopes with multiday physical half-lives such as 124I (t1/2 ≈ 4.2 days) or 89Zr (t1/2 ≈ 3.2 days) to create effective radioimmunoconjugates.122 Unfortunately, however, this combination of lengthy circulation times and slow radioactive decay can create prohibitively high radiation dose rates to healthy organs.</p><p>Pretargeting methodologies seek to circumvent this problem by decoupling the antibody from the radioisotope and injecting the two components separately, in essence synthesizing the radioimmunoconjugate at the target tissue itself. A pair of components form the core of any pretargeting strategy: a small molecule radiolabeled hapten and an antibody capable of binding both an antigen and said hapten. The antibody is injected first and is given a number of days to accumulate at the target site and clear from the blood. After this interval, the radiolabeled hapten is administered. Because it is a small molecule, the hapten travels through the bloodstream quickly, either combining with its immunoconjugate partner or clearing from the body. This approach offers two distinct advantages over traditional immunoconjugates.123,124 First, the rapid clearance of any unreacted radioligand limits the activity concentrations in and radiation dose to healthy organs. Second, and more importantly, this strategy facilitates the use of short-lived radioisotopes—e.g., 64Cu (t1/2 = 12.7 h), 18F (t1/2 = 109 min), and 68Ga (t1/2 = 68 min)—that would normally be incompatible with antibody-based vectors. The latter trait not only produces a dosimetric benefit but also has the potential to accelerate imaging workflows. A variety of approaches to in vivo pretargeting have been attempted, including the use of streptavidin-modified antibodies and biotin-based radioligands,125–128 genetically engineered bispecific antibodies capable of binding radiometal chelate complexes,124,129–131 and antibodies and radioligands conjugated to complementary oligonucleotide strands.132–134 While all of these strategies have proven promising in the preclinical arena, their ultimate clinical implementation has been derailed somewhat by intrinsic issues such as the immunogenicity of streptavidin-modified bioconjugates.</p><p>In the end, it is not surprising that bioorthogonal click chemistry has attracted attention as a tool for in vivo pretargeting. Indeed, the selectivity, speed, and, above all, bioorthogonality of these reactions make them seem almost perfectly suited to the task. Attempts at in vivo pretargeting have been made using a variety of bioorthogonal click ligations. In 2011, for instance, Vugts et al. described the development of a pretargeting approach based on the Staudinger ligation between an azide-bearing antibody and phosphine-containing small molecule probes labeled with 68Ga, 89Zr, 177Lu, and 123I.135 In this work, the authors reported that the Staudinger ligation product could not be observed in vivo, leading to the conclusion that in vivo pretargeting with the Staudinger ligation is not possible due to the reaction's sluggish kinetics, the inherent instability of the phosphine radioligands, or a combination thereof. The SPAAC reaction also has a history of in vivo use dating back to the groundbreaking work in zebrafish performed by Carolyn Bertozzi's laboratory.136,137 Van den Bosch et al. investigated the feasibility of the SPAAC reaction for in vivo pretargeting using an 125I- and azide-bearing Rituximab immunoconjugate (125I–Rtx–N3) and 177Lu-labeled cyclooctyne radioligands.138 Unfortunately, however, dual-isotope biodistribution experiments revealed disappointing activity concentrations of 177Lu in the target tissue, suggesting that the somewhat slow reaction kinetics of the SPAAC ligation limit its application in vivo. Intriguingly, however, this position has been countered by recent work on the use of nanoparticles for SPAAC-mediated in vivo pretargeting (vide infra).139</p><p>Over the past 5 years, one of the newest additions of the click chemistry toolbox, the inverse electron demand Diels–Alder (IEDDA) reaction, has proven particularly well suited for pretargeting. 10,24,140,141 Like the Staudinger and SPAAC ligations, the IEDDA reaction is catalyst-free, clean, selective, and bioorthogonal. From a pretargeting perspective, the true advantage of the IEDDA cycloaddition is speed. The first-order rate constants for reactions between 1,2,4,5-tetrazines (Tz) and trans-cyclooctenes (TCO) hover in the range of 104–105M−1 s−1. In contrast, the rate constants for the Staudinger and SPAAC ligations are orders of magnitude slower: approximately 0.002 and 0.07M−1 s−1, respectively.142,143 Surely, this added speed could play a pivotal roll in the feasibility of click chemistry in the in vivo environment.</p><p>The vast majority of IEDDA-based pretargeting approaches employ a TCO-labeled antibody and a tetrazine-based radioligand (Figure 12A). Rossin et al. were the first to report a pretargeting strategy based on the ligation. The authors successfully employed a TCO-labeled immunoconjugate (CC49-TCO) and an 111In-labeled dipyridyltetrazine radioligand to facilitate the pretargeted SPECT imaging of TAG72-expressing colorectal cancer xenografts (Figure 12B).144 Since this initial report, this group has continued to be a pioneer in the field, producing investigations on alternative trans-cyclooctene moieties,145 tetrazine-bearing clearing agents,146 and pretargeting with antibody fragments and affibodies.147,148 In parallel to the Dutch work, Zeglis et al. have developed and optimized a 64Cu-based pretargeting approach for the PET imaging of colorectal carcinoma that produces images with excellent quality and contrast at only a fraction of the radiation dose to healthy tissue created by traditional radioimmunoconjugates (Figure 12C).108,149,150 Even more recently, Houghton,151 Meyer,152 and colleagues used 5B1-TCO, a CA19.9-targeting immunoconjugate, as well as 64Cu- and 18F-labeled tetrazines to demonstrate the feasibility of the pretargeted imaging of an antigen that is both shed and internalized. Other laboratories have contributed to the advent of IEDDA-based pretargeting as well, developing 11C-, 68Ga-, and 99mTc-labeled tetrazine radioligands153–156 as well as 18F- and tetrazine-labeled nanoparticles (Figure 13).157 Finally, Rossin et al. very recently expanded the scope of this methodology even further by harnessing the newly developed IEDDA pyridazine elimination reaction to trigger the selective in vivo cleavage of tumor-bound antibody-drug conjugates (ADCs).158 This innovative "click-to-release" approach has the potential to add a powerful new tool to the arsenal of ADC therapies.</p><p>Of course, the extremely promising preclinical results for IEDDA-based pretargeting beg the question: will it work in humans? Not surprisingly, some legitimate concerns have been leveled on this front (most notably, the dramatic increase in blood volume upon moving from mice to humans). Although no trials have yet been reported, a number of laboratories are currently working toward bringing these exciting technologies to the clinic, and the field is collectively hopeful that the speed, selectivity, and bioorthogonality of the IEDDA reaction will be up to the task.</p><!><p>Of course, the four areas we have discussed so far are not the only points of intersection between click chemistry and radiopharmaceutical science. Indeed, the past few years have played witness to the increasingly innovative use of click chemistry in the synthesis of radiopharmaceuticals. For example, click chemistry has played an important role in the advent of nanoparticles as vectors for molecular imaging.159 In this regard, the modularity, selectivity, and chemically mild nature of click chemistry have proven especially useful. Along these lines, Zeng et al. used the strain-promoted alkyne–azide cycloaddition (SPAAC) to modify the surface of azide-bearing shell-cross-linked nanoparticles with ~500 64Cu–DOTA moieties per particle, ultimately achieving specific activities of up to 975 Ci/μmol.160 Similarly, Lee et al. reported the synthesis and in vivo evaluation of 64Cu-labeled chitosan nanoparticles constructed via the SPAAC reaction between 64Cu–DOTA–DBCO prosthetic groups and azide-modified chitosan NPs (Figure 14).161 Click chemistry has also been used to enable pretargeted imaging using nanoparticulate vectors. For instance, Lee et al. have developed an SPAAC-based pretargeting strategy based on mesoporous silica nanoparticles (MSN).139 In this work, DBCO-modified mesoporous silica nanoparticles were injected into mice bearing U87MG tumors. A total of 24 h later, the same mice were injected with an 18F-labeled, azide-functionalized radioligand. This strategy successfully enabled the noninvasive visualization of tumor tissue (up to 1.4% ID/g at 2 h post-injection) with promising tumor-to-background contrast, a truly remarkable result given the somewhat sluggish kinetics of the SPAAC ligation.</p><p>Shifting gears somewhat, a number of recent reports have emerged in which click chemistry (and the triazole-forming reactions, in particular) has been harnessed to enhance the in vivo stability of peptide-based imaging agents.162,163 1,2,3-Triazoles possess two critical physicochemical similarities to the amide bonds that normally link amino acids: planarity and the ability to act as hydrogen bond acceptors.163,164 Yet unlike amide bonds, triazoles are resistant to protease or peptidase metabolism in vivo. As a result, radiolabeled peptides in which triazole linkages replace some of the traditional amide bonds offer enticing prospects for nuclear imaging. Just last year, Valverde et al.165 demonstrated the potential of this approach by synthesizing a series of triazole-containing, 177Lu-labeled peptidomimetic radiotracers that target the gastrin-releasing peptide receptor (GRPr) (Figure 15A). In vivo studies revealed a significant increase in the in vivo stability of the triazole-containing compounds, a result that is likely responsible for the observation that the amide-to-triazole substituted derivatives exhibited an approximately 2-fold increase in tumor uptake. Very recently, the same group applied this methodology to bombesin-derived peptides, aiming to create tracers with improved tumor-to-kidney activity concentration ratios.166 This work demonstrated that the triazole-containing constructs boast improved (~5-fold) serum stability without sacrificing binding affinity. In vivo biodistribution experiments in mice bearing antigen-expressing PC3 and AR42J xenografts further revealed that the backbone-modified constructs possessed superior in vivo properties (Figure 15B).166</p><p>Finally, a very recent paper from Thurber and co-workers (although admittedly one that focuses on fluorescence rather than nuclear imaging agents) describes a complementary way to use click chemistry to enhance the in vivo stability of peptides.167,168 In this work, the authors use an innovative "double-click" approach that simultaneously enables the conjugation of a fluorophore to the peptide and creates an internal cross-link that stabilizes the α-helical structure of the peptide.168 The authors were able to demonstrate that the "double-clicked" peptides exhibited improved metabolic stability compared to analogous constructs. Furthermore, in vivo studies in C57BL/6 mice revealed that the click-stabilized peptides possessed increased protease resistance and significantly enhanced bioavailability.</p><!><p>In the preceding pages, we have highlighted what we believe to be the most important and innovative advances from the first 10 years of research at the intersection of click chemistry and radiopharmaceutical chemistry. The bulk of this work has been concentrated in four areas: (i) the radiolabeling of molecules using prosthetic groups, (ii) the assembly of radiometal coordination architectures, (iii) the site-specific modification of immunoglobulins, and (iv) the creation of in vivo pretargeting strategies. Of course, in each, the details differ. Far more important, though, is that in every case, the refrain remains the same: the intrinsic selectivity and modularity of click chemistry can—and very often do—dramatically improve the construction of radiopharmaceuticals.</p><p>However, despite a wealth of preclinical data, click-based radiopharmaceuticals seem to have stalled just short of the clinic. This is somewhat surprising given the manifold advantages click chemistry offers for the construction of radiolabeled agents. For example, click chemistry could dramatically streamline the logistics of clinical probe production, as a single "clickable" prosthetic group could be used in the production of multiple radiotracers. Yet still, only a small handful of click-based radiopharmaceuticals have been the subject of clinical trials, most notably the αvβ3-targeting peptide 18F–RGD–K5 and the somatostatin receptor targeting peptide 18F–fluoroethyltriazole–Tyr3–octreotate (Figure 16).169–171 These two agents certainly represent a great start, but they must be considered just that: a start. We believe that as we move into the field's second decade, clinical translation must be the top priority. To be sure, pushing back the frontiers of basic preclinical radiochemistry will remain vital. Yet ultimately, only the clinical translation of a variety of click-based probes will demonstrate once and for all the utility of this chemical technology in nuclear medicine.</p>

PubMed Author Manuscript

Computational Modeling of Virucidal Inhibition Mechanism for Broad-Spectrum Antiviral Nanoparticles and HPV16 Capsid Segments

Solid core nanoparticles coated with sulfonated ligands that mimic heparan sulfate proteoglycans (HSPG) can exhibit virucidal activity against many viruses that utilize HSPG interactions with host cells for the initial stages of the infection. How the interactions of these nanoparticles with large capsid segments of HSPG-interacting viruses lead to their virucidal activity has been unclear. Here, we describe the interactions between sulfonated nanoparticles and segments of the human papilloma virus type 16 (HPV16) capsids using atomistic molecular dynamics simulations. The simulations demonstrate that nanoparticles primarily bind at interfaces of two HPV16 capsid proteins. After equilibration, distances and angles between capsid proteins in the capsid segments are larger for the systems in which the nanoparticles bind at interfaces of capsid proteins. Over time, the nanoparticle binding can lead to breaking of contacts between two neighboring proteins. The revealed mechanism of nanoparticles targeting the interfaces between pairs of capsid proteins can be utilized for designing new generations of virucidal materials and contribute to the development of new broad-spectrum non-toxic virucidal materials.

computational_modeling_of_virucidal_inhibition_mechanism_for_broad-spectrum_antiviral_nanoparticles_

Introduction<!>Constructing HPV16 Capsid Segments.<!>Ligand Docking on HPV Pentamer.<!>Nanoparticle Model.<!>Molecular Dynamics Simulations.<!>Distance between two pentamers.<!>Angle between two pentamers.<!>Residue Contacts.<!>Results and Discussion<!>MD Simulations of HPV16 Capsid Segments in the Presence and Absence of Nanoparticles.<!>Effects of Nanoparticle Binding on the Positions of Proteins within Capsid Segments.<!>Effects of Nanoparticle Binding on the Pentamer Interface and the Integrity of Capsid Segments.<!>Conclusion

<p>Among many infectious disease threats that humans face from microbes, viral infections are arguably the biggest pandemic threat at present. Even in the absence of pandemics, viral infections kill millions of people every year, as viruses have high rates of replication, mutation and transmissibility1. Available antiviral drugs often target specific viruses or, in some cases, members of a viral family2. Current antiviral drugs include small molecules, such as nucleoside analogues and peptidomimetics, monoclonal antibodies, proteins that are able to stimulate the immune response, such as interferon, and oligonucleotides2. Many of these drugs act intracellularly with selectivity for viral enzymes. However, since viruses mostly depend for their replication on infected host cells, the specificity of antiviral drugs for viral proteins is not ideal, often causing general intrinsic toxicity upon administration3,4. Furthermore, due to high mutation rates, most viruses develop drug resistance5–7.</p><p>When antiviral drug development is based on single virus-specific protein as a target, the discovered therapeutics lack the broad-spectrum effects and thus the capability of targeting many viruses that are phylogenetically unrelated and structurally different. Yet, there is a demand for development of broad-spectrum antiviral therapeutics that are non-toxic to hosts. One development approach is to identify substances that can favorably interact with many viruses outside of host cells and determine if these interactions can prevent the first stages of infection and the viral replication cycle. Previous efforts, based on the principle of targeting virus-cell interactions that are common to many viruses, identified many non-toxic substances that interact with a broad spectrum of viruses, such as heparin and polyanions8–12. However, most of these substances exhibit only virustatic properties where their activity depends on a reversible binding event. Binding reversibility makes these substances therapeutically ineffective: upon dilution, these materials detach from intact viral particles allowing the viruses to infect again.</p><p>Virucidal materials show large promise for use against viral infections. Virucidal molecules cause irreversible viral deactivation, which is not adversely affected by dilution13. Virucidal materials can include detergents, strong acids, polymers, and nanoparticles14–18. However, most of these materials have intrinsic cellular toxicity, since materials that can chemically damage the virus often also affect the host in which the virus replicates.</p><p>Recent studies demonstrated that non-toxic broad-spectrum virucidal materials can be developed based on the principle of mimicking the virus-host interactions common to many viruses19,20. In particular, virucidal material design can be inspired by viral attachment ligands / host cell receptors called heparan sulfate proteoglycans (HSPG), which are responsible for the initial steps of the virus replication cycle21. Many viruses, including human papillomavirus type 16 (HPV-16), human immunodeficiency virus 1 (HIV-1), herpex simplex virus 1 and 2 (HSV-1 and HSV-2), attach to HSPGs, which are expressed on the surfaces of almost all eukaryotic cell types22. Nanoparticles (NPs) coated with long and flexible sulfonated ligands mimicking HSPG were shown to effectively associate with such viruses and eventually lead to irreversible viral deformation. These non-toxic nanoparticles (NPs) show in vitro irreversible virucidal activity against HSV-2, HPV-16, RSV, Dengue and lentivirus, and are also active ex vivo in human cervicovaginal histocultures infected by HSV-2 and in vivo in mice infected with RSV19.</p><p>Previous transmission electron microscopy analyses suggested the mechanism of the virucidal activity of NPs coated with sulfonated ligands against the HPV16 virus: NPs eventually break the capsid or the virus particle19. Few materials with reported virucidal effects have been examined with computational methods19,20,23–25, to explore their mechanisms of virucidal activity with atomistic resolution. In the present study, we use large scale atomistic molecular dynamics (MD) simulations to explore this suggested mechanism on systems containing 2.4 nm gold core nanoparticles coated with HSPG-mimicking ligands and extended capsid segments of two, three and four HPV16 major late pentamer proteins (L1). Our studies expand on previous modeling of sulfonated nanoparticles interacting with single HPV16 capsid proteins19. The present simulations explore the preferred sites of interaction between sulfonated NPs and capsid segments, as well as the perturbations induced in the capsid segments by the presence of NPs.</p><!><p>The cryo-electron microscopy (cryo-EM) structure of the Human Papillomavirus Type 16 (HPV16) L1 capsid proteins, assembled into a capsid structure, shown in Figure 1a, was obtained from the RCSB protein databank (PDB: 3J6R)26. Segments of either two, three or four L1 proteins with the least empty space between them were selected and then extracted from the capsid. As L1 protein structures in the extracted segments were defined via the coordinates of the backbone atoms only, each L1 protein structure in three extracted segments was replaced by a crystal structure of HPV16 L1 protein (PDB: 5W1O)27. In all three segments, each L1 pentamer structure was docked into the cryo-EM structure-based protein density using colores tool in Situs software using a resolution of 5 Å28. Structures of resulting three HPV16 capsid segments were then further flexibly fitted into cryo-EM-extracted densities of L1 proteins using the molecular dynamics flexible fitting (MDFF) method in NAMD 2.1329–31. MDFF simulations, using the CHARMM36 force field32, were carried out for 50,000 steps with timestep of 1 fs in the constant volume and temperature conditions at 310 K in vacuum, with the scaling factor for potential, gscale (ξ), set to the value of 0.3 and with Langevin constant γLang set to 5.0 ps–1. Secondary structure, cispeptide bonds, and chirality restraints were applied on L1 pentamers to prevent the unnatural distortions during the fitting procedure.</p><p>Individual chains of L1 protein crystal structures fitted into capsid segments, based on PDB: 5W1O, were missing residues 404 to 431 and several residues at C- and N-termini. The structures of these missing residues (backbone atoms) were provided in pdbID: 3J6R, where they assumed flexible coil secondary structures. Since these missing residues form important contacts with the neighboring pentamers within capsid segments (Figure S1c), they were reconstructed in our models fully based on the structures reported in pdbID: 3J6R and covalently attached to the initial models L1 proteins (after MDFF fitting). Disulfide bonds were built between C161 and C324 residues for all the chains in L1 proteins. Final completed capsid segments were solvated with TIP3P water and neutralized with 0.15 M NaCl with solvate and ionize VMD plugins33, respectively.</p><!><p>Small sulfonated ligands (CH3CH2SO3−) were docked onto a single L1 protein and a pair of L1 proteins using the Autodock Vina software34,35. Docking was performed using the finalized structures of one and two L1 proteins described above. Sulfonated ligand structures were built using AMPAC graphical user interface. In Autodock Vina, the grid box was centered at various positions on a grid, scanning the protein surfaces with five fixed z-coordinates and changing the x and y coordinates from −40 Å to 40 Å for both, with an interval of 1 nm. Docking procedure was automated using our in-house Linux shell script. Each grid box dimension was 1 × 1 × 1 nm3 with a default spacing and exhaustiveness of 0.0375 nm and 8, respectively. The above procedure determined the locations of the ligands with the lowest docking scores on the L1 protein surfaces (Figure S1). These locations guided the placement of the nanoparticles with sulfonated ligands above the capsid segments.</p><!><p>Atomistic model of a spherical nanoparticle was prepared by cutting a face centered cubic lattice of gold (Au) atoms (Au-Au bond length 2.88 Å) into a sphere with diameter of ~2.4 nm. The gold core was ligated with a random spherical array of 40 1-octanethiol (OT) and 40 11-mercapto-1-undecanesulfonate (MUS) ligands. All ligands were built with AMPAC, and then arranged into a random spherical array with 0.3949 per Au atom surface density using our own code. The built NP was then solvated in TIP3P water and ionized with sodium (Na+) and chloride (Cl−) ions at a 0.15 M NaCl concentration using the VMD solvate and ionize plugins33. After solvation and ionization, short MD simulations were performed to obtain a structure of the equilibrated NP as an input for the NP-capsid segment simulations. NPs, described with the generalized CHARMM force field36, were minimized for 2,000 steps using NAMD2.13 software29 and equilibrated for 20 ns in the NPT ensemble with Langevin dynamics (Langevin constant γLang = 1.0 ps–1), where temperature and pressure remained constant at 310 K and 1 bar, respectively. The particle-mesh Ewald (PME) method was used to calculate the Coulomb interaction energies, with periodic boundary conditions applied in all directions. Evaluation of van der Waals and Coulombic interactions was performed every 1- and 2-timesteps, respectively. A timestep of 2 fs was used for equilibration of NPs.</p><!><p>Atomistic simulations were conducted to investigate interactions of HPV16 capsid segments alone and in the presence of a 2.4 nm-core MUS:OT NP. All the systems were described with CHARMM36 force field parameters32,36. MD simulations were performed with the NAMD2.13 package29. All the simulations were conducted with Langevin dynamics (Langevin constant γLang = 1.0 ps–1) in the NPT ensemble, where temperature and pressure remained constant at 310 K and 1 bar, respectively. The particle-mesh Ewald (PME) method was used to calculate the Coulomb interaction energies, with periodic boundary conditions applied in all directions. The evaluation of van der Waals and Coulombic interactions was performed every 1- and 2-timesteps, respectively.</p><p>In total, six systems were prepared for final MD simulation runs. They consisted of two, three and four HPV16 L1 pentamers with and without the nanoparticle. Sizes and the number of atoms in all the simulated systems are listed in Table S1. Guided by docking calculations, which identified multiple sites on L1 pentamer surfaces where the bound sulfonated ligands have the most favorable docking scores (Figure S1), three systems with nanoparticles were prepared by placing a single nanoparticle 10 Å above the interfaces of two, three, or four pentamers that contained these sites identified in docking calculations. For all systems, 10,000 steps of minimization were performed, followed by 2 ns equilibration of solvent molecules around the pentamers, which were restrained by using harmonic forces with a spring constant of 1 kcal/(mol Å2). Next, the systems were equilibrated in MD production runs. To prevent the translation of the simulated capsid segments across the unit cell boundaries, the centers-of-mass (COMs) of these segments were restrained to remain at their initial values with a force constant of 2.0 kcal/mol∙Å2. COMs of capsid segments were calculated from the coordinates and masses of the α-carbon atoms of several selected buried residues (chosen to be residue 331) for all the chains of L1 pentamers. The applied single point COM restraint still allowed full rotation, capsid disassembly, and internal conformational changes of the simulated capsid segments. Yet, rigid body rotations of the capsid segments are of no interest to the present study, but could occur spontaneously by diffusion and would result in the unwanted capsid segment self-interactions across the unit cell boundaries in the rectangular prism unit cells (Table S1). Therefore, we applied an additional restraint on L1 pentamers, that would effectively prevent capsid segments to rotate as rigid bodies, but still allow L1 pentamers to move freely with respect to each other within the capsid segments. This additional restraint was applied using a collective variable distanceZ in NAMD, which allowed the z-coordinate of pentamer COMs (the coordinate along the z-axis labeled in Figure 1b) to fluctuate freely without any restraints within a 10 Å-wide window with respect to its initial value, but prevented that z-coordinate of pentamer COMs to fall outside the defined 10 Å-wide window. The distanceZ collective variable was implemented using the potential constant of 100 kcal/ mol∙Å2. The applied restraint had no effect on pentamers / capsid segments motion in the other two dimensions.</p><!><p>To analyze distances between two L1 pentamers, we calculated distances between centers of mass (COM) of backbone atoms of individual pentamers over the duration of simulations using our scripts in VMD33.</p><!><p>To analyze tilting of two pentamers with respect to each other, we calculated angles between vectors that align with axes of the cylindrically shaped void space in pentamer centers. Directions of these vectors were calculated by approximating shapes of pentamers as cylinders and evaluating the direction of the plane passing through COM of atoms of every alternating monomer (three in total) that forms each pentamer protein.</p><!><p>To analyze interaction between neighboring pentamers and between NP and pentamers, we evaluated contacts between different protein residues and between protein residues and NP over the duration of simulations. A contact was defined to exist at a given time frame if any atom of the concerned protein residue was located within 5 Å of the other pentamer or the NP.</p><!><p>The present study examines the interactions between gold nanoparticles coated with sulfonated ligands and segments of HPV16 capsids of varying sizes. Previous modeling of these virucidal NPs interacting with a single L1 capsid protein of the HPV16 virus suggested that the initial steps of the virucidal mechanism involve NPs binding multivalently to L1 proteins via charged, polar and hydrophobic interactions19. However, the atomistic details behind the NP interactions with larger HPV16 capsid segments and the eventual distortion and breaking of the capsid, proposed to be at the core of the virucidal activity, remained unexamined and unclear. Here, we probe the effects of the NP binding on the stability of the HPV16 capsid segments. We first modeled several segments of HPV16 capsid and relaxed them in MD simulations. Then, these segments were also simulated in the presence of virucidal MUS:OT NPs. Two sets of simulations were then analyzed to determine the mechanism of virucidal activity by these nanoparticles.</p><!><p>A total of six systems, containing segments of two, three and four L1 proteins by themselves and in the presence of single MUS:OT nanoparticles, were modeled in aqueous solution and examined in atomistic MD simulations. The snapshots of these systems after between 200 ns (for two- and three-pentamer systems) and 115 ns (for four-pentamer systems) of equilibration are shown in Figures 1 and 2. During equilibration, the proteins comprising the capsid segments moved closer to each other in all the systems, and the flexible coils that make contacts between neighboring pentamers rearranged from their initial conformations. The change of the two-pentamer system from the initial to a more compact state is shown in Figure 1b. This observed change in the pentamer positions and interfacial flexible coils may indicate a need for more careful modeling of the capsid segment interfaces. However, the present study relied on modeling the interfaces based on the structural information reported in Ref26. In systems where NPs were present, pentamers also moved closer to each other, while the NPs diffused over the initial 7–9 ns from the aqueous solution towards surfaces of the capsid segments, where they remained bound for the rest of the simulations (Supplementary Movie).</p><p>The primary binding mode between NP and capsid segments, as observed in the simulations, is shown in Figures 1c and 2b,d. In all the cases, the 2.4 nm gold core NP interacts with the capsid segment at the interface of two L1 pentamers, and over time, it is observed to wedge in between two pentamers that it binds. The NP binds to the interface of two pentamers even in three- and four-pentamer capsid segments, in which there are interfaces of three pentamers within the segments (Figure 2). Over the course of the three- and four-pentamer systems simulations, the NPs shifts from three pentamer junctions to two pentamer junctions, and eventually bind to A-F pentamer junction in three-pentamer system (after ~30 ns) and B-F pentamer junction in four-pentamer system (after ~70 ns), as shown in Figure 2b,d. The binding mode of NP to the capsid segment is likely determined by the NP size: larger NPs may have different binding modes, potentially including the junctions of three pentamers.</p><p>In all the examined systems, internal stabilities of individual L1 proteins were tracked by calculating root mean square deviation (RMSD) of parts with defined secondary structures from the systems simulated without and with the NPs. As Figures S2 and S3 show, there are no significant differences in RMSDs of analogous L1 proteins in the systems simulated without and with the NPs. Therefore, the internal stabilities of L1 proteins are not compromised in the presence of NPs.</p><!><p>MD trajectories of all the systems, both without and with NPs, clearly show that L1 proteins move with respect to each other. To measure the changes in the positions of proteins within capsid segments, we tracked distances between centers of mass (COM) of the adjacent pentamer pairs and angles between the central axes of the adjacent pentamer pairs. The resulting distances and angles between adjacent proteins in two- and three-pentamer systems are shown in Figure 3, and their values averaged over the last 100 ns of trajectories are reported in Table S2. In two-pentamer systems, distances between A and F pentamers decrease over time from their initial values, both for the lone capsid segment and the capsid segment binding to the NP (Figure 3b), in agreement with the visual observations that capsid proteins move closer to each other over time. However, these distances fluctuate and decrease by different amounts in the absence and presence of NPs. Namely, the pentamers equilibrate at COM distances of ~99.6 Å and ~ 104 Å in the absence and presence of NP, respectively. When averaged over the last 100 ns, NP binding to two-pentamer segment leads to a COM distance between the adjacent pentamers that is greater by ~ 4.5 Å than the COM distance in the system without NP. Similar trends are observed when measuring the distances between only the chains of A and F pentamers that are in direct contact, A1, A5 and F1, F2 (Figure S15). The pentamers in two-pentamer systems also equilibrate at angles of ~22° and ~27° in the absence and presence of NP, respectively (Figure 3c). Overall, these pentamers have an angle that is ~ 5° larger when averaged over the last 100 ns in the presence of the NP than in its absence. The observations indicate that NP acts as a wedge between the pentamers.</p><p>The effects of the NP on protein positions are similar in two- and three-pentamer systems. The distances and the angles between A and F pentamers, averaged over the last 100 ns, are greater by ~ 6 Å and ~ 10°, respectively, in the system with the NP than in the system without the NP. In three-pentamer systems, distances between pentamers whose interfaces do not participate in NP binding are similar in systems with and without the NP (Figure S4). For the four-pentamer system, the NP has no clear effects on the segment perturbation, as shown in Figures S5–S6. These negligible effects are likely due to a combination of short simulation timescales and a larger network of interactions between the pentamers within the extended capsid that may increase the times over which the segment changes occur.</p><!><p>When the NP binds at the interface of two pentamers, it interacts with two out of five chains on each pentamer. These chains on A and F pentamers, labeled as A1, A5, F1 and F2, are highlighted in Figure 4a. Similar interactions are observed for NPs within three- and four-pentamer systems. The individual chains preserve their secondary structures while interacting with NPs (Figures S7–S9), as it is mostly the flexible loop regions of the chains, protruding from the capsid surface into the solvent, that interact with NPs (Figures 4b, S10). These loop regions and specifically some of the lysine residues on them (K278, K356, K361, K54 and K59) were found to be implicated in the recognition of heparan sulfate proteoglycans27,37, indicating that NPs coated with MUS and OT ligands can mimic HSPG cell receptors allowing for effective viral association.</p><p>As each chain is equivalent in sequence, similar in structure, but different in orientation, MUS:OT NP interacts with two unique surfaces of A1, A5, F1 and F2 chains. Figures 4b and S10 highlight the residues of these chains that interact significantly with the NP: chains F1 and A5 interact with NP via three loop regions (residues 52–62, 345–362 and 424–432), whereas chains A1 and F2 interact with NP via two loop regions (residues 172–186 and 265–286). Contacts between these loop regions and the NP are largely maintained over the simulation time, as shown in the contact maps in Figure 4c. These loop regions contain many charged and non-polar amino acids (Figure S10). For example, the loop region with residues 52–62 interacts strongly with the NP via K53, K54, and K59, as well as the polar 56–58 residues. Interactions of NP with this loop region (residues 52–62) is significant in all three examined systems indicating the importance of this region for establishing the initial NP-capsid binding through long range electrostatic interactions and preserving it later via local electrostatic and H-bond interactions. All the loops, and especially the loop with residues 172–186, also interact with the NP via its hydrophobic residues. The interactions of NP with capsid segments via charge-charge and hydrophobic interactions correlates with observations of a previous MD study of a single L1 pentamer with MUS:OT NP19. The charge-charge interactions occur between negative sulfonate groups on MUS:OT-NP and the positive HSPG-binding lysine residues of loop regions, while the hydrophobic interactions occur between non-polar alkyl chains of NP ligands and nonpolar groups on L1 protein surface. Similar pentamer-NP contacts are observed in the three-pentamer system as in the two-pentamer system (Figure S11). However, significantly fewer pentamer-NP contacts are observed in the four-pentamer system (Figure S12), indicating a need for longer simulation times in this system to capture the effects of the NP presence on the capsid segment integrity.</p><p>We next examine in Figure 5 how the contacts between neighboring pentamers evolve over time in the absence and presence of the NP within two-pentamer systems. Most of the time, the same inter-pentamer contacts are observed in the absence and presence of the NP. However, towards the end of the 240 ns trajectories, some contacts between pentamers become less frequent or are lost in the presence of the NP, such as the contacts that A5 segment makes with the F pentamer (residues 52 – 62 and amino acids surrounding the residue 449) and the contacts that F2 segment makes with the A pentamer (residues 169 – 189). Similar loss of interactions between pentamers interacting with the NP is observed in three-pentamer and four-pentamer systems (although less pronounced, as shown Figures S13 and S14). All these contact maps indicate that NPs are able to disrupt some interactions between neighboring pentamers over the simulation timescales.</p><p>Overall, our studies also show that most of the residues involved in pentamer-NP and pentamer-pentamer interactions are mutually exclusive over the course of 115 – 240 ns simulations. With longer simulation times, more significant effects of NP presence are expected on pentamer-pentamer interactions.</p><!><p>In this work, we described the interactions between virucidal MUS:OT nanoparticles with 2.4 nm gold cores and segments of HPV16 capsids using atomistic MD simulations. It was previously shown that HSPG-mimicking ligands of these NPs form multivalent interactions with L1 capsid proteins19. Here, we determine the initial effects of the MUS:OT NP presence on HPV 16 capsid segments and depict it in the scheme of Figure 6. The first step of this activity is the binding of MUS:OT NPs at interfaces of two L1 proteins forming the capsid segments. The insertion of the NP at the interface of two L1 proteins leads to distances and angles between these neighboring proteins being greater than when the NP is not present. As the time progresses, the NP presence can lead to loss of some contacts between two neighboring proteins, although our simulations captured the very initial stages of this stage of the virucidal activity. Our findings suggest that the disruption of the HPV 16 capsid by MUS:OT NPs may be starting at interfaces of two L1 capsid proteins. However, we note a possibility that our findings for two-, three-, and four-pentamer capsid segments are not necessarily translatable to the disruption of larger capsid lattice segments or the intact capsid. Further investigations of larger lattices and intact capsids are needed to reveal further insights into pathways of the NP approach and the full capsid breaking process. However, the revealed mechanism of the NP disruptions at interfaces can be experimentally tested and utilized for designing new generations of materials that can perturb and disintegrate viral capsids38 or control the integrity of other naturally occurring or engineered protein assemblies39,40.</p>

PubMed Author Manuscript

Ligand bias in receptor tyrosine kinase signaling

Ligand bias is the ability of ligands to differentially activate certain receptor signaling responses compared with others. It reflects differences in the responses of a receptor to specific ligands and has implications for the development of highly specific therapeutics. Whereas ligand bias has been studied primarily for G protein–coupled receptors (GPCRs), there are also reports of ligand bias for receptor tyrosine kinases (RTKs). However, the understanding of RTK ligand bias is lagging behind the knowledge of GPCR ligand bias. In this review, we highlight how protocols that were developed to study GPCR signaling can be used to identify and quantify RTK ligand bias. We also introduce an operational model that can provide insights into the biophysical basis of RTK activation and ligand bias. Finally, we discuss possible mechanisms underpinning RTK ligand bias. Thus, this review serves as a primer for researchers interested in investigating ligand bias in RTK signaling.

ligand_bias_in_receptor_tyrosine_kinase_signaling

<!>Quantitative differences in signaling responses and ligand bias<!>Identifying and quantifying ligand bias: A practical demonstration<!><!>Identifying and quantifying ligand bias: A practical demonstration<!><!>Identifying and quantifying ligand bias: A practical demonstration<!>GPCRs<!>RTKs<!><!>RTKs<!>Mechanisms of RTK ligand bias<!>Do biased ligands stabilize different dimeric RTK catalytic conformations?<!>Can ligand bias be due to differences in the stability of RTK dimers bound to different ligands?<!><!>Can ligand bias be due to differences in the stability of RTK dimers bound to different ligands?<!>Beyond biased ligands: Toward new biased modulators<!>Prospective<!>Data availability

<p>Edited by Alex Toker</p><p>"Ligand bias" (also known as "biased agonism" or "ligand functional selectivity") is the ability of ligands to differentially activate a subset of receptor signaling pathways (1). Research focused on G protein–coupled receptors (GPCRs) has revealed that these seven-transmembrane-helix receptors engage in biased signaling in natural, physiologic systems (2). These discoveries have transformed our understanding of GPCR signaling and have empowered the search for synthetic biased ligands that selectively target therapeutically relevant signaling pathways (2, 3, 4, 5). These new ligands can modify specific signaling responses, leading to different functional and physiological consequences as compared with natural ligands (6).</p><p>Ligand bias can lead to fundamentally different receptor signaling outcomes. A schematic illustrates different cases in which two ligands induce dimerization of a RTK and downstream signaling. The stars represent two signaling responses: response A in green and response B in red. The size of the stars represents the efficiency of a response (i.e. a combination of the potency of the ligand and the magnitude (or efficacy) of the response) (Table 1). In case i, ligand 1 induces both responses more efficiently than ligand 2, but the two responses are equally increased with ligand 1, and therefore there is no bias. In case ii, response B is more efficient for both ligands, and in addition ligand 1 induces both responses more efficiently, but there is no bias in the responses induced by the two ligands. There is bias only in case iii, where only response B is more efficiently induced by ligand 1 compared with ligand 2, and thus ligand 1 is biased toward response B. EC, extracellular region; TM, transmembrane helix; IC, intracellular region.</p><p>There have been reports that propose the existence of ligand bias for various RTK families. These include the ERBB receptor family (7, 21, 22, 23, 24, 25), the fibroblast growth factor receptor (FGFR) family (26, 27), the TRK receptor family (28, 29, 30, 31, 32, 33), the insulin receptor family (34, 35, 36, 37, 38, 39, 40, 41, 42), the platelet-derived growth factor receptor (PDGFR) family (43), the RET receptor (44), and the EPH receptor family (45, 46). It is conceivable that many, if not all, RTKs engage in biased signaling (9), but this aspect of their signaling activity has not been thoroughly investigated thus far. To accelerate progress in the study of RTK signaling bias, we can use existing tools developed for GPCRs.</p><p>A goal of this review is to highlight the concept of ligand bias in RTK signaling and to outline protocols that can be used to identify and quantify bias by analyzing dose-response curves. A second goal is to provide insights into the biophysical basis of RTK ligand bias and give an overview of mechanistic hypotheses proposed in the literature to explain ligand bias. A third goal is to encourage new research into RTK ligand bias and the mechanisms that underpin it.</p><!><p>"Ligand bias" or "biased agonism" is the ability of distinct ligands to differentially activate specific signaling responses downstream of a single receptor. A "response" is defined as an effect due to ligand binding and may include receptor phosphorylation and changes in components of downstream signaling pathways. Responses could also include effects on cell behavior that depend on complex coordination of multiple signaling pathways, such as changes in cell proliferation, metabolism, differentiation, or migration. Ligand bias can lead to fundamental differences in the signaling output of a RTK in response to different ligands (2, 5). It does not just reflect, for example, similar quantitative differences in all of the signaling responses induced by different ligands.</p><p>This is illustrated in Fig. 1 for two ligands that activate the same two signaling responses, A and B, through the same RTK. Cases i and ii show examples of quantitative differences in the responses induced by the two ligands, but without bias. In case i, ligand 1 induces more efficient responses than ligand 2, but the two responses are equally increased with ligand 1 and therefore are not biased. Ligand 1 is also more efficient in case ii, and in addition, response B is more efficient than response A for both ligands. However, the variation in the two responses is the same for both ligands, and therefore the quantitative differences shown in case ii also do not represent ligand bias. Case iii illustrates an example of ligand bias. In case iii, both ligands activate response A similarly, but ligand 1 activates response B more efficiently than ligand 2. Only in this case the two responses are differentially activated by the two ligands, and ligand 1 is biased toward response B.</p><p>The identification and quantification of bias is not trivial. Ligand bias is a property of the ligand/receptor system and its coupling to downstream signaling responses. To determine whether signaling is biased or not, at least two ligands and at least two responses need to be evaluated, as illustrated in Fig. 1. Furthermore, dose-response curves have to be acquired because parameters obtained from fitting such curves are needed for the quantification of ligand bias (1, 47, 48) (see below). In addition, as emphasized in the GPCR literature, to arrive at a correct assessment of signaling bias, it is important to eliminate potential bias due to the experimental system (such as, for example, the cellular context) and the type of measurement or assay used (1, 2, 5, 6, 48). Correctly quantified bias should not be influenced by these factors.</p><p>Properly designed experiments can involve measuring different responses in different cell lines and with different assay systems. However, a response induced by different ligands should be measured using the same assay system and in the same cell line. Thus, experimental designs that should be avoided include measuring (i) the response to a ligand in one cell line and the same response to a different ligand in a different cell line or (ii) the response to a ligand with one assay and the same response to a different ligand with a different assay. It is also important to acquire a complete dose-response curve over a broad ligand concentration range rather than using only one or several ligand concentrations.</p><!><p>This section illustrates how bias can be quantified for RTKs using protocols borrowed from the GPCR field. The data we use for this demonstration have been published previously (49). We chose two engineered peptide ligands that activate the EphA2 receptor that was endogenously expressed in the PC3 prostate cancer cell line. The two peptides, designated here YSK (βAWLAYPDSVPYSK-biotin) and YSPK (βAWLAY-PDSVPYSPK-biotin), differ only by one residue (Pro-13 in YSPK, which is not present in YSK). Both peptides induce two responses that are also induced by the natural ephrinA ligands (49). Response A involves phosphorylation of Tyr-588 in the juxtamembrane region of EphA2, which is an autophosphorylation site known to promote receptor kinase activity. Response B involves inhibition of AKT phosphorylation on Ser-473. EphA2 Tyr-588 phosphorylation and AKT Ser-473 phosphorylation were detected in cells treated for 15 min with different peptide concentrations by immunoblotting cell lysates using phosphospecific antibodies. The phosphorylation signals were quantified by measuring chemiluminescence in the immunoblots.</p><!><p>Dose-response curves and bias plots for ligand bias assessment.A, schematic illustrating the two ligand-induced EphA2 signaling responses analyzed and dose-response curves for two peptide ligands, YSPK and YSK, that activate the EphA2 RTK in PC3 prostate cancer cells. The two responses measured are autophosphorylation on tyrosine 588 (pY588, which is normalized to total EphA2 levels) and inhibition of AKT phosphorylation (pAKT). The 100 nm concentration is highlighted in the graphs to emphasize the different potency of the two ligands. B, bias plot comparing the two responses for the two ligands. The color coding for the two responses (green and red) and the two ligands (blue and orange) are the same as in Fig. 1.</p><!><p>When comparing the dose-response curves for the two ligands in Fig. 2A, we see that a lower concentration of YSPK is needed to reach the maximum EphA2 phospho-Tyr-588 (pY588) phosphorylation level, and therefore the YSPK peptide is much more potent than the YSK peptide. Much lower YSPK concentrations stimulate both EphA2 Tyr-588 phosphorylation (EC50 values are ∼40 nm for YSPK and ∼1,400 nm for YSK) and AKT inhibition (EC50 values are ∼30 nm for YSPK and ∼1,000 nm for YSK). The maximal responses (Etop) are also different. However, this does not necessarily mean that the YSK ligand is biased compared with the YSPK reference ligand (see Fig. 1, case ii). To determine whether significant ligand bias exists or not, we can create a bias plot to visualize potential bias. Furthermore, we need to calculate a bias coefficient that takes into account the relationship of EC50 and Etop for different ligands and responses.</p><p>The bias plot (Fig. 2B) is a graphical representation that shows response A (Tyr-588 phosphorylation) as a function of response B (pAKT inhibition) for each ligand (2). The bias plot is directly derived from the dose-response data, by plotting the magnitude of response A versus the magnitude of response B for each ligand concentration. The bias plot reports on the relative ability of a ligand to induce the two responses, without the need for assumptions or mathematical modeling (2). The data points for different ligands can be directly compared because they incorporate system and measurement bias in identical ways. We see that the YSK data points in Fig. 2B overlap with the YSPK data points, despite the fact that one ligand is more potent than the other. This suggests that there is no bias in the responses induced by the two ligands. Note that this analysis can be used even if response A for the two ligands is measured in one cell line and response B in a different cell line. As long as each response is measured in the same way for both ligands (2, 3, 50), differences between the two ligands in the bias plot are entirely due to ligand bias.</p><p>Quantification of bias involves the calculation of a single numerical value (a bias coefficient) for each ligand, which reports on the existence and magnitude of ligand bias (48, 51). Different types of bias coefficients are described in the GPCR literature, and readers are encouraged to familiarize themselves with them (2, 3, 5, 51, 52, 53). Here, we demonstrate how to calculate a bias coefficient known as "βlig" (48). βlig was chosen from several available mathematical formalisms to assess bias, because it can yield an accurate description of GPCR bias (53) and because it is based on simple mathematical equations. In the next section, we confirm that βlig can be used to evaluate bias for RTKs.</p><!><p>Definition of parameters</p><!><p>The ratio of the two fitted parameters, Etop/EC50, has been recognized as an important descriptor of ligand efficiency (54), and ligand bias coefficients can be calculated using these ratios for different ligands and responses. In particular, the equation to calculate βlig is as follows (48, 54), (Eq. 2)βlig=logEtop,AEC50,BEC50,AEtop,BligEtop,BEC50,AEC50,BEtop,Aref where "lig" indicates the ligand (YSK) being compared with the reference ligand "ref" (YSPK), and "A" and "B" are the two responses, Tyr-588 phosphorylation and pAKT inhibition. Thus, based on this equation, the value of βlig for the reference ligand (in our case, YSPK) is 0 (i.e. βYSPK = 0). Using Equation 2, we calculated βYSK as −0.02 ± 0.12. This value is not significantly different from βYSPK = 0, based on a one-sample t test. Thus, YSK is an unbiased ligand when compared with YSPK, despite being less potent.</p><p>Equation 2 can be applied to other published data. For instance, we used it to compare the c-Kit ligand stem cell factor and an engineered partial agonist derivative with impaired ability to promote dimerization of the c-Kit receptor (43). We used the dose-response curves for these two ligands as reported and fitted by Ho et al. (43) in Fig. 5 (B and D) of their article. In these dose-response curves, they analyzed ex vivo proliferation of hematopoietic stem and progenitor cells purified from mouse bone marrow (response A) and the release of IL-6 by mouse bone marrow–derived cultured mast cells (response B). Using Equation 2, we calculated the value of βlig for the engineered ligand as compared with WT stem cell factor as the reference ligand. This βlig value (−0.72 ± 0.16) is significantly different from 0, supporting the conclusion that the engineered ligand is biased toward cell proliferation and against IL-6 secretion (43).</p><p>This approach can be used to determine whether bias exists or not, provided that the data are well-described by Equation 1. In our experience, Equation 1 generally gives a reasonable fit to RTK dose-response curves. If it is necessary to use a Hill equation with n ≠ 1 to fit the data, then a more complex method needs to be used to calculate bias (2, 3, 53). However, it has been suggested that Equation 2 can be used even if n ≠ 1, as long as Etop > 0.35 and n > 0.5 (54).</p><!><p>In the previous section, we demonstrated that Equation 1 can be used to fit experimental dose-response curves. Whereas the approach we used is phenomenological (i.e. Equation 1 is based on the shape of experimental dose-response curves), there are mechanistic models of receptor activation. In this section, we outline a physicochemical mechanistic model of GPCR activation and show how the parameters in the phenomenological Equation 1 relate to the parameters of this mechanistic model.</p><p>Many physicochemical models of GPCR activation that vary in sophistication have been developed (55, 56, 57). The simplest, and perhaps most widely used, is the so-called "operational model" introduced by Black and Leff (58). This model is based on the experimental observation that the relationship between the concentration of activated receptors and a measured response is often hyperbolic. In other words, this model assumes that the ligand-bound receptor, RL, activates the cellular response with an effective equilibrium dissociation constant denoted as Kresp according to the equation, (Eq. 3)Response=RLEmax[RL]+Kresp</p><p>Mathematically, Kresp is the concentration of ligand-bound receptors that produces 50% of the largest possible response that can be achieved by the system (Emax) (Table 1). Data are often normalized so that Emax = 1. Physically, Kresp describes the propensity of the ligand-bound receptor to induce a response. Thus, Kresp is a measure of the efficiency of the response: the more efficient the process, the smaller the value of Kresp (Table 1).</p><p>The concentration of the ligand-bound receptor [RL] in Equation 3 depends on the concentrations of free receptor [R] and ligand [L] and on the ligand-receptor dissociation constant KL according to the following equation. (Eq. 4)RL=RLKL</p><p>The total receptor concentration [Rt] is as follows. (Eq. 5)Rt=R+RL</p><p>Therefore, substitution of Equation 5 into Equation 4 yields the following. (Eq. 6)RL=RtLKL1+LKL</p><p>Substitution of Equation 6 into Equation 3 yields the following. (Eq. 7)Response=RtLEmaxRt[L]+Kresp(KL+L)=RtLEmaxL(Rt+Kresp)+(KLKresp)</p><p>If we divide both the numerator and the denominator by Kresp, we obtain Equation 8, (Eq. 8)Response=RtKrespLEmaxRtKresp+1L+(KL)=τLEmax(τ+1)L+(KL)</p><p>Equation 8 is the "operational model," and τ is the "transducer coefficient" defined as follows. (Eq. 9)τ=RtKresp</p><p>Equation 8 can also be written as follows. (Eq. 10)Response=τ/(τ+1)LEmaxL+(KL)/(τ+1)</p><p>Equation 10 is the same as Equation 1, where the following is true. (Eq. 11)Etop=τEmax(τ+1)(Eq. 12)EC50=KL(τ+1)</p><p>Thus, the Black and Leff model provides a justification for using the simple phenomenological Equation 1 when fitting dose-response curves for GPCRs. Additional models have been developed in the GPCR field, including the "ternary complex" (55), "extended ternary complex" (56), and "cubic ternary complex" (57, 59) models. These models incorporate specific interactions of the GPCR with signaling proteins that mediate the downstream responses. Within the operational model, the effects of these interactions are incorporated into the Kresp value.</p><p>We can now ask which parameters of the Black and Leff model carry information about ligand bias. To answer this question, we substitute the expressions in Equations 11 and 12 into Equation 2 and simplify to obtain the following. (Eq. 13)βlig=logEtop,AEC50,BEC50,AEtop,BligEtop,BEC50,AEC50,BEtop,Aref=logτAKL,BKL,AτBligτBKL,AKL,BτAref</p><p>This equation shows that βlig depends on the values of the ligand-receptor dissociation constant KL and the transducer coefficient τ for both the ligand of interest and the reference ligand. Whereas it is known that ligand-binding affinity can be influenced by the identity of the downstream molecules that interact with the receptor (3), here we assume that KL does not depend on the particular receptor signaling response measured in the dose-response curves. This will be a valid assumption when the measured responses are averaged for receptors bound to multiple downstream signaling proteins, as is the case for many types of receptors in the cellular environment.</p><p>Thus, KL for response A (KL,A) is the same as KL for response B (KL,B), and Equation 13 can be simplified as follows. (Eq. 14)βlig=logτAτBligτBτAref=logRt,A/Kresp,ARt,B/Kresp,BligRt,B/Kresp,BRt,A/Kresp,Aref</p><p>The concentration of the receptors [Rt] is also independent of the type of response measured in the experiments (i.e. Rt,A=Rt,B), and thus the following is true. (Eq. 15)βlig=logKresp,BKresp,AligKresp,AKresp,Bref=log(Kresp,A)ref(Kresp,A)lig(Kresp,B)lig(Kresp,B)ref</p><p>Equation 15 shows that bias arises due to differences in Kresp, the parameter describing the coupling of the ligand-bound receptor to downstream signaling responses. It allows us to formulate mathematically the conditions resulting in bias. There is bias when the argument of the logarithm is significantly different from 1 (and βlig is significantly different from 0). In other words: (Eq. 16)(Kresp,A)lig(Kresp,A)ref≠(Kresp,B)lig(Kresp,B)refbias and (Eq. 17)(Kresp,A)lig(Kresp,A)ref=(Kresp,B)lig(Kresp,B)refnobias</p><p>Equation 15 shows that the ligand-receptor dissociation constant KL has no relevance for ligand bias. It also shows that the value of βlig is related to Kresp, in agreement with the definition of ligand bias. This provides a justification for the utility of Equation 2 in bias quantification and clarifies the physical meaning of βlig. In practice, βlig is calculated using Equation 2 and then analyzed for significance as illustrated in the previous section. Generally, Kresp cannot be calculated from Equation 3 because the concentration [RL] is unknown in most experiments.</p><!><p>In this section, we show that a mathematical formalism that is analogous to the GPCR operational model can be developed for RTKs to help us understand RTK ligand bias. The operational model of Black and Leff describes the case of one ligand binding to one GPCR. On the other hand, RTK activation generally requires receptor dimerization and sometimes oligomerization. To develop an operational model of RTK activation, we will consider a model in which ligand binding is coupled to RTK dimerization. Different models should be used for RTKs that are constitutively dimerized through disulfide bonds (which can be described using Equation 4) and for RTKs that form oligomers larger than dimers (which require more complicated equations) (60, 61). This coupling can be understood though the use of thermodynamic cycles, which show how increases in both receptor and ligand concentration drive the transition to active liganded RTK dimers via intermediate states.</p><!><p>Predictions of responses for dimeric ligands based on the RTK operational model.A, schematic and thermodynamic cycle describing VEGFA binding to VEGFR2 and VEGFR2 dimerization. B, definitions of the three principal dissociation constants. C, definition of the other dissociation constants in terms of the three principal ones. D, predicted orange curve: abundance of VEGFR2 dimers bound to VEGFA [DL] as a function of VEGFA (ligand 1) concentration. Predicted blue curve, abundance of VEGFR2 dimers [DL] bound to a hypothetical ligand 2 as a function of ligand 2 concentration. E, measured dissociation constant values for VEGFA from Ref. 60 and assigned dissociation constant values for the hypothetical ligand 2, which binds with 10-fold higher affinity than ligand 1 to the VEGFR2 dimer. F, dose-response curves predicted using Equation 24 based on the values shown in G. G, values of Kresp for responses A or B and for ligands 1 or 2 that were used to simulate the cases of bias and no bias in F. In all cases, [Rt] = 500 receptors/µm2. H, bias plots calculated from the predictions in F.</p><!><p>To develop an operational model that links ligand-receptor interactions that occur extracellularly to intracellular responses, we follow the formalism of Black and Leff and use Equation 3 but substitute the concentration of ligand-bound receptor [RL] with the concentration of ligand-bound dimer [DL] to obtain Equation 18, (Eq. 18)Response=[DL]Emax[DL]+Kresp</p><p>Kresp in Equation 18 is the concentration of liganded dimer [DL] that produces 50% of the maximum possible response. For RTKs, Kresp can be considered as an equilibrium constant that describes the efficiency of the response of interest, including autophosphorylation, interaction with a binding partner, activation of a downstream signaling molecule, or changes in cellular behavior. As with GPCRs, the more efficient the process, the smaller the value of Kresp. Emax is the largest possible response that can be observed for the system with any ligand and will be set to 1.</p><p>The concentration of active dimer [DL] is given by Equation 19, (Eq. 19)DL=[M]2[L]KL′KR where KL′ and KR are the dissociation constants defined in Fig. 3 (A and B), [M] is the concentration of receptor monomers, and [L] is the free ligand concentration. [M] is unknown, but it can be determined if the total receptor concentration [Rt] is known. (Eq. 20)Rt=ML+M+2D+2DL</p><p>Substituting the dissociation constants defined in Fig. 3 (A and B) for [ML], [D], and [DL] in Equation 20 yields a quadratic equation in terms of [M]. (Eq. 21)[Rt]=MLKL+M+2M2KR+2M2LKL′KR</p><p>Accordingly, [M] can be solved for in terms of [L], [Rt], and the dissociation constants. (Eq. 22)M=-(L+KL)KL+L+KLKL2+8KL′+[L]KL′KR[Rt]4KL′+[L]KL′KR</p><p>Equation 22 can then be substituted into Equation 19 as follows. (Eq. 23)DL=-L+KLKL+L+KLKL2+8KL′+[L]KL′KR[Rt]4KL′+[L]KL′KR2[L]KL′KR</p><p>Finally, Equation 23 can be substituted into Equation 18 to express the response as a function of the dissociation constants that govern RTK ligand binding and dimerization as follows. (Eq. 24)Response=-L+KLKL+L+KLKL2+8KL′+[L]KL′KR[Rt]4KL′+[L]KL′KR2KL′KREmax[L]-L+KLKL+L+KLKL2+8KL′+[L]KL′KR[Rt]4KL′+[L]KL′KR2[L]KL′KR+Kresp</p><p>Equation 24 for the binding of a dimeric ligand to a RTK is the analog of Equation 7 for GPCRs but is more complicated due to the coupling between ligand binding and receptor dimerization. In the case of GPCRs, we simplified Equation 7 to yield Equation 10, which is the same as Equation 1. However, we cannot similarly simplify the more complicated Equation 24. Nevertheless, Equation 24 allows us to simulate the response for any value of the equilibrium constants shown in Fig. 3A. In Fig. 3F, we show examples of simulated responses for different VEGFA ligand concentrations (orange dotted lines) using the experimentally measured values of KL, KL′, and KR (Fig. 3E) and hypothetical values of Kresp for responses A and B (Fig. 3G). Thus, we can use Equation 24 to simulate dose-response curves and determine whether Equation 1 can be used to fit these simulated dose-response curves in a meaningful way.</p><p>In a previous section, we used Equation 1 to fit experimental dose-response curves obtained for the EphA2 RTK activated by two engineered peptide ligands and Equation 2 to calculate bias coefficients and draw conclusions about the existence of bias. EphA2 is activated by the peptide ligands through a different mechanism than VEGFR2 by its ligand VEGFA because each biotinylated peptide is a monomer that bivalently interacts with two EphA2 molecules to ultimately form a complex of two peptides bound to two receptors (49). To investigate whether Equations 1 and 2 are applicable for analysis of bias in RTKs with different dimerization mechanisms, we first simulated dose-response curves for the cases of bias and no bias for two VEGFR2 ligands (Fig. 3F) and then asked whether these two cases can be correctly identified. Ligand 1 is the natural ligand, VEGFA, which we denote as the reference ligand. Ligand 2 is a hypothetical ligand that binds to the VEGFR2 monomer with the same affinity as VEGFA but binds with 10-fold higher affinity to the VEGFR2 dimer (Fig. 3, E and F). To simulate bias, we used different relative values of Kresp for the two different ligands and the two different responses, according to Equation 16 (Fig. 3G, Bias). To simulate the condition of no bias, we used the same relative values of Kresp for the two different ligands and the two different responses, according to Equation 17 (Fig. 3G, No bias). Indeed, Kresp is the only parameter in the operational model that accounts for the coupling of the RTK with signaling responses. Equations 16 and 17 are intuitive and are consistent with Fig. 1. Thus, they can be expected to be valid for both GPCRs and RTKs.</p><p>From the predicted dose-response data points, we can construct bias plots for response A versus response B for the two ligands (Fig. 3H). As expected, the curves for the two ligands are very different in the case of bias but overlap in the case of no bias. This suggests that bias plots, as developed for GPCRs, are also applicable for RTKs despite the fact that the RTK mechanism of activation is more complex.</p><p>Next, we fit the dose-response curves in Fig. 3F with Equation 1, and we determined the best fit Etop and EC50 values. Although we could not mathematically transform the complex Equation 24 into Equation 1, the fits with Equation 1 are very good (R2 > 0.9999), suggesting that Equations 1 and 24 have a very similar functional dependence on [L]. We then used Equation 2 to calculate the bias coefficients. We obtained βlig = −1.01 in the bias case and βlig = −0.01 in the no bias case. Thus, the βlig value in the case of no bias is, as expected, very close to 0. βlig is different from zero in the bias case, also as expected, suggesting that Equation 2 can successfully differentiate between the cases of RTK bias and no bias. Therefore, equations 1 and 2 can be a valuable tool in RTK research, provided that dose-response curves for at least two responses are measured.</p><p>A wealth of knowledge already exists about RTK signaling responses, and many tools are available to study them. For example, the major RTK downstream signaling pathways have been delineated, and phosphospecific antibodies are commercially available to detect phosphorylation events in the RTKs and in downstream signaling proteins. Bias plots and bias coefficients will reveal whether some of the RTK tyrosines are preferentially phosphorylated in response to a specific ligand, as compared with other ligands. These approaches can further reveal whether some pathways—such as the ERK mitogen-activated protein kinase, phophoinositide-3-kinase, protein kinase C, and signal transducer and activator of transcription pathways (10, 11, 17, 66, 67, 68)—are differentially activated by different ligands. If complex functional responses (such as cell proliferation, migration, differentiation, and metabolic responses) are measured as a function of ligand concentration, correlations may be revealed between the bias in the phosphorylation of specific tyrosines in the RTK and the bias in the activation of particular downstream signaling proteins or responses. Such correlations may suggest functional links that can be further investigated. Comparisons of different ligands using such quantitative assessments will yield fundamental knowledge on the regulation of RTK signaling responses that is not currently available.</p><p>To compare multiple ligands, it is useful to rewrite Equation 2 in the form, (Eq. 25)βlig=βlig′-βref′ where βlig′ and βref′ are defined as follows. (Eq. 26)βlig′=logEtop,AEC50,BEC50,AEtop,Blig(Eq. 27)βref′=logEtop,AEC50,BEC50,AEtop,Bref</p><p>Multiple β′ values can then be compared using one-way analysis of variance to determine whether there are significant differences in the bias exhibited by multiple different ligands.</p><!><p>In this section, we discuss molecular hypotheses that could explain how RTKs may differentially engage downstream signaling pathways depending on the bound ligand. For GPCRs, the explanation for this phenomenon could be that biased ligands stabilize distinctly different GPCR conformations that support preferential activation of a specific signaling pathway. For instance, the presence of multiple ligand-specific conformations of the β2-adrenergic receptor has been revealed using a quantitative MS approach (69). In addition, fluorine NMR experiments have shown that the cytoplasmic ends of helices VI and VII in the β2-adrenergic receptor adopt different conformations in response to G protein biased ligands and β-arrestin biased ligands (70). This view is supported by many other studies (5, 6, 71, 72, 73, 74) and is now the prevailing view in the field (75). Although dimerization can in some cases regulate GPCR signaling (75), it is the main mechanism of activation for RTKs. Thus, factors related to dimerization are particularly relevant for understanding RTK signaling bias.</p><!><p>In analogy to the ligand-dependent conformational differences documented for GPCRs, it has been hypothesized that different ligands stabilize different RTK dimeric catalytic conformations, leading to ligand-specific preferential phosphor-ylation of selected tyrosines in the intracellular region (7). There is evidence that the transmembrane helix and the juxtamembrane segment in RTK dimers can sense the identity of the bound ligand and adopt different conformations (26, 65, 76). However, it is not known whether these conformational differences are transmitted to the kinase domain. Despite many years of research, it remains controversial whether the structural information about the bound ligand propagates to the kinase domain, such that different kinase-kinase interfaces are preferentially engaged in response to different ligands. Whereas some researchers believe that this is the case, others argue that linkers in RTKs (such as the linker connecting the extracellular region and the transmembrane helix or the juxtamembrane segment linker connecting the transmembrane helix and the kinase domain) are flexible. Thus, distinct structural changes that might occur in the extracellular region in response to the binding of different ligands may not be propagated to the intracellular region (24, 77, 78). If so, the two kinase domains in the dimer interact with each other based on their physicochemical properties and, therefore, in the same way, regardless of which ligand is bound to the extracellular region.</p><p>To resolve this controversy, it will be critical to monitor the configuration of the kinase domains to determine which kinase interfaces are used when an RTK is bound to different ligands. Researchers have long hoped that the structures of full-length RTKs would provide this information. The cryogenic EM (cryoEM) structures of the full-length insulin receptor bound to insulin and of the full-length insulin-like growth factor 1 receptor (IGF1R) bound to IGF1 have been recently reported (79, 80). In both cases, however, the intracellular regions are not resolved in the cryoEM maps. Only the extracellular regions are resolved, despite the fact that full-length receptors were analyzed. This likely indicates that the kinase domains explore many configurations within the active RTK dimer, consistent with the observation that tyrosines in different parts of the intracellular region undergo autophosphorylation. Because a substrate tyrosine must come in contact with the active-site cleft in the kinase domain to become phosphorylated, the kinase domains presumably exist in multiple configurations.</p><p>X-ray crystallography studies can characterize the structural arrangements of two dimerized kinase domains that are stabilized in RTK crystals. However, configurations that may be critically important for RTK autophosphorylation or for the binding and phosphorylation of downstream proteins may remain unresolved. Because there are many published high-resolution structures of RTK kinase domains, it may be useful to examine their crystallographic interfaces. It is possible that some of them are in fact biologically relevant interfaces that mediate kinase domain interactions within the context of the full-length RTK dimer in the plasma membrane. Indeed, analysis of crystal structures has identified interfaces that place a tyrosine in close proximity to the active site of the neighboring kinase (81, 82). A strategy to evaluate whether such contacts are important for biological function is to weaken the contacts through mutagenesis and then determine whether this perturbs the function of the full-length RTK (while not perturbing the folding of the kinase domain). If the mutations have effects on RTK signaling, they likely affect biologically relevant contacts. Such mutagenesis studies are rare (83) but can be very informative. There may also be interfaces that stabilize the kinase dimer but are not directly involved in phosphorylation and signaling. In this case, the interfaces can be identified by measuring RTK interactions rather than phosphorylation or functional outcomes.</p><p>If ligand bias arises from differential engagement of distinct interfaces that involve the kinase domain, the effects of destabilizing a particular interface will be different when different ligands are bound. Experiments to examine this hypothesis are feasible, because the effects of mutations in the putative kinase interfaces on dimer stability and phosphorylation can be measured using well-established techniques. These techniques include FRET (84, 85), co-immunoimmobilization (86), and fluorescence intensity fluctuations (87) to measure dimer stability as well as immunoblotting and mass spectrometry to measure tyrosine phosphorylation.</p><p>A possible outcome of these experiments is that a particular mutation will substantially affect RTK phosphorylation and/or dimer stability only when a specific ligand is bound but will have a much smaller effect when other ligands are bound. This would indicate that this specific ligand promotes a particular kinase domain interaction involving the mutated amino acid and that this interaction is not as important when other ligands are bound. This, in turn, would indicate that the identity of the bound ligand affects the arrangement of the two intracellular regions, supporting the view that structural information that depends on the identity of the ligand can be transmitted along the length of a RTK and reach the kinase domain.</p><p>Alternatively, it is possible that engineered mutations have the same effects on autophosphorylation and dimer stability, no matter what ligand is bound. This would suggest that kinase domain interactions and the pattern of phosphorylation sites do not depend on the identity of the bound ligand. This result would support the view that structural information related to the identity of the bound ligand does not reach the kinase domain. Such an outcome would suggest that factors other than the configuration of RTK molecules in a dimer or oligomer may differentially control signaling responses and ligand bias. A factor affecting bias that has been proposed in the literature is the stability of RTK dimers bound to different ligands, which is discussed in the next section.</p><!><p>The stability of ligand-bound RTK dimers has been proposed to explain some instances of RTK ligand bias, because dimer stability can determine whether the response to a ligand is sustained or transient, which may ultimately affect biological outcomes (24, 27, 43, 88). In one example, the receptor-binding affinity of a ligand has been proposed to determine the strength and stability/durability of receptor dimerization to generate qualitatively different responses. This example involves the FGFR family, which plays a role in brain development (27, 89, 90). Two splice isoforms of the ligand FGF8 that differ by 11 amino acids (present at the N terminus of FGF8b but not FGF8a) have been shown to induce different phenotypes and gene expression patterns when electroporated in the midbrain of chicken embryos (89, 90). In particular, FGF8b induces the formation of the cerebellum by repressing the expression of the transcription factor orthodenticle homeobox 2 (Otx2). In contrast, FGF8a is not capable of repressing Otx2 and causes expansion of the optic tectum. Thus, these two FGF8 isoforms appear to act as biased ligands, because they induce different biological responses by activating the same receptor, FGFR1c (the c isoform of FGFR1, which is the major receptor for FGF8 in the developing midbrain). FGF8b binds with ∼10-fold higher affinity than FGF8a to the FGFR1c, FGFR2c, and FGFR3c extracellular domains. This effect is mediated by phenylalanine 32, a residue that is present only in FGF8b and contributes an additional hydrophobic contact with the receptor (89). Mutation of this phenylalanine to alanine weakens the FGFRc-binding affinity of FGF8b, functionally converting it into FGF8a. Thus, the binding affinity of the ligand for the receptor was proposed to be the defining factor that controls the functional outcome of receptor signaling. Furthermore, a connection was proposed between the stability of FGF8a-FGFRc or FGF8b-FGFRc dimers and the nature of the signaling, with the more stable FGF8b-FGFRc dimers transmitting stronger and more sustained intracellular signals (88).</p><p>Another study proposing that dimer stability correlates with ligand bias focuses on the epidermal growth factor receptor (EGFR), a member of the ERBB family that can be activated by multiple ligands (24). Some of them, such as EGF, bind with high affinity (apparent Kd of 0.1–1 nm). Other ligands, such as epiregulin and epigen, bind 10–100 times more weakly. These ligands appear biased, as EGF has been shown to induce cell proliferation under conditions where epiregulin and epigen induce differentiation. The different cellular responses were found to correlate with the kinetics of EGFR activation. In the MCF-7 and T47D breast cancer cell lines, tyrosine phosphorylation of EGFR was sustained upon activation with epigen and epiregulin, whereas it was transient upon activation with EGF. The activation kinetics, in turn, were found to correlate with the stability of the different ligand-bound EGFR dimers. In particular, the stable EGF-bound EGFR dimers were proposed to induce a transient response, whereas the less stable epiregulin and epigen dimers were proposed to induce a more sustained response. The opposite effects of dimer stability on FGFR and EGFR activation kinetics are not well-understood but may depend on distinct mechanisms regulating the phosphorylation of the two RTK families and/or different interplays with phosphatases regulating their dephosphorylation (91, 92, 93).</p><!><p>Model describing the binding of a monomeric ligand to an RTK and RTK dimerization.A, thermodynamic cycle. B, definitions of the four principal equilibrium dissociation constants. C, definitions of the other equilibrium constants in terms of the four principal ones.</p><!><p>EGF is a monomeric ligand that binds to a single receptor molecule, which is different from the case discussed above of dimeric VEGFA binding to VEGFR2. The thermodynamic cycle for a monomeric ligand (Fig. 4A) is more complex than the cycle for a dimeric ligand such as VEGFA (Fig. 3A) because the receptor dimers can bind either one or two monomeric ligands. As a consequence, there are three ligand-receptor dissociation constants denoted as KL,KL′, and KL″ besides the three receptor-receptor dissociation constants denoted as KR, KR′, and KR″. Four equilibrium dissociation constants are needed to define this cycle; their definitions are shown in Fig. 4B, and the links between the dissociation constants are shown in Fig. 4C.</p><p>KR is the dissociation constant for dimerization of the unliganded monomeric receptor M, and the stability of the unliganded dimer is the dimerization free energy ΔGR defined as follows, (Eq. 28)ΔGR=RTlnKR where R is the gas constant and T is the absolute temperature. Similarly, the dissociation constant KR′ describes the lateral interaction between an unliganded receptor monomer M and a liganded monomer ML, producing the dimer DL with a single ligand bound. The corresponding dimerization free energy is as follows. (Eq. 29)ΔGR′=RTlnKR′</p><p>Finally, the dissociation constant KR″ describes the lateral interaction between two liganded receptor monomers ML, producing the dimer DLL with two ligands bound. The dimerization free energy associated with this process is as follows. (Eq. 30)ΔGR″=RTlnKR″</p><p>The dimerization free energy values ΔGR, ΔGR′, and ΔGR″ can be used to define the differences in the stability of receptor dimers with different ligand occupancy. For example, the extent of dimer stabilization due to the binding of two ligands is given by the following. (Eq. 31)ΔΔGlig″=ΔGR″-ΔGR=RTlnKR″KR</p><p>This increase in stability can be quantified, because KR and KR″ can be measured in the plasma membrane of live cells using techniques such as FRET, co-immunoimmobilization, or fluorescence intensity fluctuations (84, 86, 87, 94). KR can be measured in the absence of ligand, and KR′′ can be measured at saturating ligand concentrations. At saturating ligand concentrations, all receptors (both monomers and dimers) are ligand-bound, because the ligand concentration [L] is much higher than the ligand-receptor dissociation constants KL, KL′, and KL″.</p><p>If a correlation exists between dimer stability and ligand bias, then the values of ΔΔGlig″ in Equation 31 and βlig′ in Equation 26 should be correlated. The correlation between ΔΔGlig″ and βlig′ measured for several ligands can be analyzed in a plot of βlig′ versus ΔΔGlig″, using either linear or nonlinear regression depending on the functional dependence.</p><p>It should be noted that within the context of the thermodynamic cycle–based RTK operational model, the information about bias depends entirely on the values of Kresp, which have no direct link to dimer stability. Thus, we would expect no correlation between ΔΔGlig″ and βlig′ if the operational model provides a good description of RTK activation and RTK ligand bias. It is therefore important to probe this correlation experimentally, to directly verify whether a link exists between ligand bias and receptor dimer stability.</p><p>As discussed above, FGFR and EGFR dimer stability has been correlated with the kinetics of the response, either sustained or transient (24, 88). The factors affecting the kinetics of RTK autophosphorylation and dephosphorylation are complex and are not yet completely understood. For EGFR, it has been proposed that the kinetics of phosphorylation and dephosphorylation/degradation depend mainly on the identity of the ligand, but not on the ligand and receptor concentrations (24). However, other RTKs, such as EphA2, exhibit phosphorylation kinetics that depend strongly on ligand concentration (95). Currently, it is also not known how the kinetics of RTK autophosphorylation may contribute to ligand bias, as quantified using Equation 2. To gain insights, dose-response curves could be acquired at different time points, yielding time-resolved bias plots and time-resolved bias coefficients. This approach can reveal whether ligand bias depends on time for RTKs.</p><!><p>It would be expected that not only activating ligands, but also other modulatory agents that bind to a receptor could bias downstream signaling responses by stabilizing a particular receptor structural conformation. For example, a characteristic of allosteric modulators is their ability to differentially affect distinct ligand-induced signaling responses, a phenomenon known as pathway bias (96, 97). Although this has been poorly studied so far, there are some examples of RTK inhibitors that act at allosteric sites and preferentially inhibit some of the responses induced by ligand binding, suggesting pathway-biased pharmacology (8). One of the examples is the small molecule AG1296, which targets the kinase domain of the PDGFRβ through a complex mechanism that depends on receptor activation state (98). AG1296 was shown to differentially inhibit PDGFR autophosphorylation on different tyrosines. Another example is the negative allosteric modulator SSR128129E, a small molecule that binds to the extracellular region of all FGFRs and induces a structural change that decreases fibroblast growth factor ligand efficacy (99, 100). SSR128129E was shown to inhibit tyrosine phosphorylation of an FGFR substrate (FRS2) but not another (PLCγ). Other interesting examples are matuzumab and cetuximab, two EGFR-inhibitory therapeutic antibodies with different binding sites in the EGFR extracellular region (101, 102). These antibodies can promote the formation of EGFR dimers that presumably have different configurations than the dimers induced by EGF but nevertheless can undergo autophosphorylation on at least some of the same tyrosines (102, 103). Despite inducing EGFR autophosphorylation, matuzumab and cetuximab do not activate the downstream AKT and ERK pathways (103). Similarly, artificial EGFR dimerization induced by bivalent synthetic ligands can induce autophosphorylation and recruitment of adaptor proteins, but not activation of AKT and ERK (104). Additional factors that could also bias signaling responses induced by ligands include receptor oligomerization, association with co-receptors, and subcellular trafficking (9). Thus, future studies will likely lead to a broader view of RTK biased signaling mechanisms and to the development of pathway-biased agents with novel modes of action.</p><!><p>Studies of GPCR biased signaling have revolutionized basic research and pharmacological discoveries (2, 6, 105, 106). It can be expected that an increased focus on RTK biased signaling will also transform the RTK field. It is enticing to think that studies of RTK ligand bias may reveal new biology that has been hidden from us due to a lack of quantification in our methodologies. Whereas the work is still in its early stages, it can progress rapidly if it takes advantage of the wealth of knowledge accumulated in the GPCR field and of the mathematical methods developed to identify biased agonism. Such rapid progress is highly desirable because the therapeutic implications of RTK ligand bias are far-reaching. We look forward to a new generation of smarter drugs that selectively target therapeutically relevant RTK signaling responses and have reduced side effects.</p><!><p>All data are included in the article.</p>

PubMed Open Access

Roles of Changes in Active Glutamine Transport in Brain Edema Development During Hepatic Encephalopathy: An Emerging Concept

Excessive glutamine (Gln) synthesis in ammonia-overloaded astrocytes contributes to astrocytic swelling and brain edema, the major complication of hepatic encephalopathy (HE). Much of the newly formed Gln is believed to enter mitochondria, where it is recycled to ammonia, which causes mitochondrial dysfunction (a “Trojan horse” mode of action). A portion of Gln may increase osmotic pressure in astrocytes and the interstitial space, directly and independently contributing to brain tissue swelling. Here we discuss the possibility that altered functioning of Gln transport proteins located in the cellular or mitochondrial membranes, modulates the effects of increased Gln synthesis. Accumulation of excess Gln in mitochondria involves a carrier-mediated transport which is activated by ammonia. Studies on the expression of the cell membrane N-system transporters SN1 (SNAT3) and SN2 (SNAT5), which mediate Gln efflux from astrocytes rendered HE model-dependent effects. HE lowered the expression of SN1 at the RNA and protein level in the cerebral cortex (cc) in the thioacetamide (TAA) model of HE and the effect paralleled induction of cerebral cortical edema. Neither SN1 nor SN2 expression was affected by simple hyperammonemia, which produces no cc edema. TAA-induced HE is also associated with decreased expression of mRNA coding for the system A carriers SAT1 and SAT2, which stimulate Gln influx to neurons. Taken together, changes in the expression of Gln transporters during HE appear to favor retention of Gln in astrocytes and/or the interstitial space of the brain. HE may also affect arginine (Arg)/Gln exchange across the astrocytic cell membrane due to changes in the expression of the hybrid Arg/Gln transporter y+LAT2. Gln export from brain across the blood–brain barrier may be stimulated by HE via its increased exchange with peripheral tryptophan.

roles_of_changes_in_active_glutamine_transport_in_brain_edema_development_during_hepatic_encephalopa

Gln Accumulation as a Cause of Astrocytic Swelling and Brain Edema in HE: A Brief Overview<!>Mitochondrial Gln Transport<!>Gln Transport in Astrocytes and Neurons<!>Gln Transport in the Blood–Brain Barrier-Forming Cerebral Capillary Endothelial Cells<!><!>Mitochondria<!><!>Astrocytes and Neurons<!>Transport Across the BBB<!>Conclusions and Perspectives

<p>Hepatic encephalopathy (HE) is a complex neuropsychiatric syndrome caused by liver failure where excessive accumulation of blood-derived ammonia in the brain is a primary causative factor. Acute HE or aggravation of a chronic condition due to hyperammonemic incidents is associated with brain edema, which often leads to the patients' death in consequence of increased intracranial pressure and herniation (reviewed in [1, 2]). Brain edema is primarily cytotoxic in nature and is mainly due to astrocytic swelling (AS [1]). The current view is that excessive synthesis of Gln from ammonia and glutamate catalyzed by an astrocyte-specific enzyme, glutamine synthetase (GS) plays a major role in the pathogenesis of AS. The natural history of this view has been exhaustively described in recent review articles [3, 4]. Briefly, the concept originates from the observation that Gln in cultured astrocytic increases oxidative stress in 6-diazo-5-oxo–l-norleucine (DON), histidine (His) and cyclosporine A-sensitive manner. It has also been shown that HE in experimental animals subside or became attenuated upon treatment with a GS inhibitor, methionine sulfoximine (MSO). The abnormalities in the brain corrected by MSO include, among other events, decreased oxygen consumption and large neutral amino acid imbalance [5], and, at the physiological level, decreased specific gravity and/or increased water content of the tissue, reflecting brain edema [6, 7]. At the cellular level, MSO reduces perivascular astrocytic and pericytic swelling in cerebral cortex (cc) of hyperammonemic rats [8, 9], and swelling of cultured astrocytes exposed to ammonia [10]. The toxic effects of Gln are believed to be largely due to its entry to mitochondria and subsequent intra-mitochondrial release of toxic concentrations of ammonia, which leads to mitochondrial permeability transition (mPT) and swelling [11]. This sequence of events has been derived from in vitro studies and has been summarized as the "Trojan horse" hypothesis [3]. According to this hypothesis, Gln acting as a "Trojan horse" would contribute to other actions of ammonia at different cellular targets, collectively resulting in the complex interplay of oxidative/nitrosative stress and impairment of in- and out-transport of different osmolytes, leading to intracellular osmotic imbalance [12, 13]. However, the Trojan horse hypothesis still needs to be unequivocally validated in the in vivo setting. One of the weak points of the hypothesis is related to the controversy whether glutamine is located in the inner mitochondrial membrane or in the inter-membrane space as suggested by Kvamme et al. [14]. Independently, accumulation of Gln is believed to directly contribute to the osmotic imbalance in astrocytes [15].</p><p>While part of newly accumulated Gln may passively diffuse from the locus of its synthesis, its considerable fraction is directed towards different destinations by active transport mediated by specific carriers. The present paper addresses the as yet unresolved question whether and in what degree modulation of the carriers by the pathogenic condition affects the Gln-related pathogenesis. In the following section we provide basic information about how Gln is shuttled between the cells and subcellular compartments of the brain and how it manages to egress the brain to the periphery.</p><!><p>Cerebral mitochondria possess an active, saturable transport system for Gln. The system operates at relatively high affinity and low capacity and is therefore well suited for the regulation of the entry of Gln from cytoplasm, where it is present at low milimolar concentrations [16]. Mitochondrial Gln uptake is inhibited by several neutral amino acids, of which histidine (His) appears to exert the strongest inhibitory effect [17].</p><!><p>Gln transporting proteins are present both in astrocytes and neurons, but are distributed between them asymmetrically. Astrocytes are enriched in two bi-directional system N transporters: SN1 [18] and SN2 [19]. SN1 is the best candidate to specifically mediate Gln efflux from astrocytes. SN1 has an activity optimum at physiological extracellular Gln concentration (Km ~0.4 mM) and shows independence on a substrate on the trans-side, which predisposes it to be a mediator of net efflux [18]. Furthermore, SN1 activity is positively controlled by intracellular Glu [20], and the significance of this interdependence is underscored by the fact that neuron-derived neurotransmitter Glu accounts for ~80–90 % of the substrate pool for Gln synthesis in astrocytes [21]. SN2 is less ubiquitous in the CNS than SN1 and in addition to Gln release, mediates glycine release for regulation of the N-methyl-d-aspartate (NMDA) receptor function [22]. Still, the precise role of each of the two transporters in regulating Gln efflux from astrocytes and its distribution in the different compartments of the CNS is not completely clear as yet. In neurons, Gln uses as carriers two neuronal system A transporters: SAT1 and SAT2, of which SAT2 specifically serves to replenish Glu in glutamatergic neurons [23]. Part of Gln migrates between the different cellular compartment in exchange for arginine (Arg), using the y+L system. The y+L system is represented by 4F2hc/y+LAT1 and 4F2hc/y+LAT2 transporters, which in addition to cationic amino acids accept neutral amino acids; the transport of the latter is coupled to Na+ [24]. Of these two transporters, the brain expresses only y+LAT2 [25, 26], which appears to abound in astrocytes and as such is well suited for catalyzing Gln/Arg exchange in these cells [27].</p><!><p>Available evidence suggests that Gln transport at the blood–brain barrier (BBB) is primarily mediated by system N transporters [18, 28]. Studies with luminal and abluminal plasma membrane vesicles derived from bovine brain endothelial cells disclosed that system N transporters account for ~80 % of Gln transport from brain to peripheral blood [29], and the presence of SN1 in the endothelial cells has been well documented [30]. Gln out-transport from the brain is also facilitated by system L-mediated exchange with other large neutral amino acids, mainly tryptophan (Try) [31, 32]. The Arg/Gln exchanger y+LAT2 appear to be likewise expressed in the cerebral capillary endothelial cells in culture, but much less so in situ [33].</p><!><p>Effect of the presence of 10 mM BCH, Leu and cyclo-Leu on [3H] Gln uptake in control and hyperammonemic (HA) rat cerebral cortical slices. Cerebral cortical slices of HA rat were pre-incubated for 30 min at 37 °C and the uptake was started by adding L-[3H] Gln (Perkin-Elmer) at 100 mmol/l final concentration and the incubation was continued for 4 min. The incubation was terminated by rapid vacuum filtration, followed by three washes with 2 ml with Krebs buffer maintained at 4 °C. The radioactivity on filter disks was measured in a liquid scintillation spectrometer. The control value for [3H] Gln uptake was 29.5 nmol/min/mg wet tissues. Values in each group are mean ± SD for n = 5. (*p < 0.05; Dunnet's test)</p><!><p>Convincing evidence that transport of excess of newly formed Gln to brain mitochondria is carrier-mediated has come from studies analyzing the effects of the transport competitor, His. Co-incubation with His abolished Gln-evoked mPT and swelling, and the response was well correlated with inhibition of Gln uptake [34]. His inhibited swelling of ammonia-treated astrocytes and the effect showed cooperativity with MSO [35]. His administered i.p. attenuated brain edema and other oxidative stress-related responses in the brain in rats with thioacetamide (TAA)-induced HE [36]. However, results obtained with His must be interpreted with caution do to its pleiotropic effects, including direct amelioration of HE-induced oxidative stress, as manifested by the recovery of mitochondrial glutathione [37], and, independently of the tissue status, interference with system N-mediated Gln transport at the astrocytic cell membranes [38], and references therein). An earlier observation indicated that ammonia increases Gln uptake to non-synaptic (astrocytic) mitochondria [39], which is likely to augment the mitochondrial effects of excess Gln in the setting of HE, where brain ammonia is elevated as a rule [2, 32].</p><!><p>a, b Expression of SN1, SN2 and y+LAT2 at the mRNA (a) and protein (b) level in cerebral cortex of control rats and rats with TAA-induced HE. a Relative quantification of SN1, SN2, y+LAT2 mRNA. Total RNA was isolated using TRI Reagent (Sigma-Aldrich), and reverse-transcribed using High Capacity cDNA Reverse Transcribed Kit (Life Technologies; Applied Biosystems). Probes for SN1, SN2, y+LAT2 and β-actin (Rn 01447660, Rn 00684896, Rn 01431908_m1 and Rn 00667869, respectively) were purchased from Applied Biosystems. Further details were as described in Ref. [27]. Values in each group are mean ± SD for n = 8; *p < 0.05; T test. b Quantification of SN1, SN2, y+LAT2 protein densities. The antibodies used included SN1 (Santa Cruz Biotechnology; goat polyclonal, 1:500), SN2 (Santa Cruz Biotechnology; goat polyclonal, 1:1,000), y+LAT2 (Santa Cruz Biotechnology; 1:1,000, rabbit polyclonal) and GAPDH (Sigma-Aldrich; rabbit polyclonal, 1:3,000). Representative immunoblots of SN1, SN2, y+LAT2 and GAPDH (loading control) corresponding to the immunoblots of transporters. See Ref. [27] for further experimental details. Values in each group are mean ± SD for n = 5–8. (*p < 0.05; T test)</p><p>a, b Expression of SN1 and SN2 at the mRNA (a) and protein (b) level in cerebral cortex of control rats and rats with ammonium acetate-induced HA. Values in each group are mean ± SD for n = 4</p><!><p>A variable response of the y+LAT2 carrier has been noted in the two different models. Simple hyperammonemia (HA) stimulated the expression of y+LAT2 mRNA and protein in the brain, accounting for an increased back-flux of Gln to the cells (probably astrocytes) on the expense of Arg [27]. HE in the TAA model decreased the brain y+LAT2 expression at the mRNA level, leaving the y+LAT2 protein unchanged (Fig. 2a, b). The ambiguity of the responses noted, and the impact of the observed changes on the overall tissue balance of Gln remain to be clarified.</p><!><p>The effect of HE or ammonia on the system N-mediated Gln transport in the cerebral capillary endothelial cells has not been examined as yet. The L system-dependent exchange of systemic Try with intracerebral Gln has been found increased in cerebral capillary endothelial cells treated with ammonia [31], or isolated from rats with HE [32]. While the results tend to support increased efflux of newly formed Gln from the brain to the periphery in the setting of HE, the significance of the observation cannot be appreciated before its role relative to the system N-mediated transport is unraveled. Ammonia also increased y+LAT2 expression in the cerebral capillary endothelial cell line [43], but how this increase translates into Gln/Arg exchange and whether, and in what degree, it is eventually reflected in the tissue distribution of Gln is unknown.</p><!><p>Collectively, the above presented data suggest that HE-induced alterations in the expression of the Gln transporting proteins may favor Gln trapping within the vulnerable compartments of the brain. According to the most plausible scenario, decreased expression of the astrocytic Gln transporters SN1 (SNAT3) and SN2 (SNAT5), if translated into their decreased activity (this presumption remains to be tested), will impair Gln efflux from astrocytes. This in turn will lead to increased intra-astrocytic and in consequence, intra-mitochondrial accumulation of Gln, the latter process being additionally stimulated by ammonia-induced activation of the mitochondrial Gln carrier. This sequence of mutually amplifying events would contribute to astrocytic swelling both by the cytoplasmic—osmotic mechanism, and by the mitochondrial—Trojan horse model.</p><p>A question that remains to be resolved is how to reconcile the elevation of intraastrocytic Gln content with the increase of extracellular Gln, frequently observed in HE [15, 44, 45]. One possible explanation would be that the apparent increase of extracellular Gln concentration is due to the shrinkage of extracellular space subsequent to astrocytic swelling, as earlier suggested for the increased accumulation of extracellular K+ in the brains of hyperammonemic rats [46]; in such a case, however, the increase would encompass, in a nonselective way, other amino acids as well. One other possibility would be a decreased backflux of Gln to astrocytes, due to an unfavorable concentration gradient. Clearly, further work is needed to resolve between these two possibilities.</p><p>Consequences of the decreased expression of the neuronal transporters SAT1 and SAT2 are more difficult to predict. If a portion of Gln newly derived from astrocytes indeed "omits" neurons, this may either lead to increased Gln efflux across the BBB, or contribute to its increased trapping in the extracellular space. The latter interpretation would be consistent with the earlier discussed increase of extracellular Gln often recorded in human HE patients [15] and animal HE models [44, 45].</p><p>Clearly, interpretations of the results derived so far will have to be verified by detailed investigation on how the changes expression of the different Gln-transporting moieties are translated into their functional status. While preliminary data obtained in this laboratory demonstrate that ammonia, the key primary cause of astrocytic swelling does inhibit Gln uptake in vitro (Fig. 1), the inhibition appears to primarily involve the sodium-independent L system, which does not appear compatible with the transporter expression data. However, the complexity of cellular composition of the slice and the ensuing difficulty to analyze the contribution of the different cell components to the final result, do not permit simple extrapolation of the results obtained in vitro to the in vivo conditions. Assessment of the role of alterations in Gln transport in the redistribution of Gln overload between the different compartments of the CNS will not be possible without knowledge is acquired about quantitative contribution of active transport and diffusion to Gln fluxes. To this end, Gln fluxes will have to be compared in the brains of normal rats and rats with selectively inactivated transporters (knockout animals, siRNA), using the NMR technique.</p>

PubMed Open Access

Iron complexes of tetramine ligands catalyse allylic hydroxyamination via a nitroso–ene mechanism

Iron(II) complexes of the tetradentate amines tris(2-pyridylmethyl)amine (TPA) and N,N′-bis(2-pyridylmethyl)-N,N′dimethylethane-1,2-diamine (BPMEN) are established catalysts of C-O bond formation, oxidising hydrocarbon substrates via hydroxylation, epoxidation and dihydroxylation pathways. Herein we report the capacity of these catalysts to promote C-N bond formation, via allylic amination of alkenes. The combination of N-Boc-hydroxylamine with either FeTPA (1 mol %) or FeBPMEN (10 mol %) converts cyclohexene to the allylic hydroxylamine (tert-butyl cyclohex-2-en-1-yl(hydroxy)carbamate) in moderate yields. Spectroscopic studies and trapping experiments suggest the reaction proceeds via a nitroso-ene mechanism, with involvement of a free N-Boc-nitroso intermediate. Asymmetric induction is not observed using the chiral tetramine ligand (+)-(2R,2′R)-1,1′-bis(2-pyridylmethyl)-2,2′-bipyrrolidine ((R,R′)-PDP).

iron_complexes_of_tetramine_ligands_catalyse_allylic_hydroxyamination_via_a_nitroso–ene_mechanism

Introduction<!>Synthesis of metal complexes<!>Allylic amination reactions<!>Reaction using a chiral catalyst<!>Mechanistic studies<!>Conclusion<!>Experimental General experimental<!>Hydroxyamination reactions

<p>The selective functionalization of C-H bonds is an area of considerable current research interest [1][2][3][4][5]. The development of methods for catalytic C-H amination has attracted particular attention [6][7][8][9][10][11], given the significance of C-N bonds to the structures of biologically active natural products and pharmaceuticals. In this context there has been a renewed focus on the chemistry of acylnitroso species in recent times [12][13][14][15], in particular on α-hydroxyamination of carbonyl compounds via nitrosocarbonyl aldol reactions [16][17][18][19][20][21] and allylic hydroxyami-nation of alkenes via nitroso-ene reactions [22][23][24][25][26]. Several new developments in the related hetero-Diels-Alder reaction of acylnitroso species have also been reported recently [27][28][29][30].</p><p>These methodologies generally involve in situ generation of the acylnitroso species, achieved using a variety of oxidants including vanadium- [28], manganese- [19][20][21], iron- [23,24], copper- [22,31], rhenium- [26], and rhodium- [27] based reagents. The recent resurgence of interest in the nitroso-ene reaction builds on earlier work by Sharpless, Nicolas, Jørgensen and others. Sharpless reported allylic amination of 2-methyl-2hexene with N-(p-chlorophenyl)hydroxylamine using a molybdenum complex [32], a process that was made catalytic by adding excess N-phenylhydroxylamine [33]. The combination of iron(II) phthalocyanines [34,35] or iron(II)/iron(III) chloride [36][37][38] and N-phenylhydroxylamine effect allylic amination reactions that are believed to follow a nitroso-ene mechanism. Similar reactions have been reported using copper salts and N-phenylhydroxylamine [39] or N-Boc-hydroxylamine [40,41], presumably via oxidation of the hydroxylamine to a nitroso species which then undergoes the nitroso-ene reaction.</p><p>Stemming from our interest in iron-catalysed hydrocarbon oxidation using systems inspired by the non-heme iron-dependent enzyme family [42][43][44][45][46][47], we have investigated the capacity of iron complexes of simple tetramine ligands to promote the reaction between an alkene and N-Boc-hydroxylamine. Herein we report that iron complexes of tris(2-pyridylmethyl)amine (TPA, 1) [48,49], N,N′-bis(2-pyridylmethyl)-N,N′-dimethylethane-1,2diamine (BPMEN, 2) [48,50] and (+)-(2R,2′R)-1,1′-bis(2pyridylmethyl)-2,2′-bipyrrolidine ((R,R′)-PDP, 3) [51] (Figure 1) catalyse the allylic amination of cyclohexene. Mechanistic investigations suggest the reaction proceeds via nitroso-ene reaction of the oxidised hydroxylamine and the alkene.</p><!><p>The tetramine ligands TPA (1), BPMEN (2) and (R,R′)-PDP (3) were synthesised following literature procedures [48,50,51], then combined with iron(II) triflate as previously reported to generate the complexes [Fe(TPA)(CH 3 CN) 2 ](OTf) 2 (FeTPA, 4) [52], [Fe(BPMEN)(OTf) 2 ] (FeBPMEN, 5) [48] and [Fe(R,R′-PDP)(OTf) 2 ] (Fe(R,R′)-PDP, 6) [51].</p><!><p>As an extension of our previously reported iron-catalysed allylic oxidation of cyclohexene (7) [45][46][47], we wished to explore potential C-N bond formation at this position using iron catalysis. Combining cyclohexene (7, in excess) with N-Bochydroxylamine (8) as the nitrogen source and the iron complex FeTPA (4) or FeBPMEN (5) afforded a mixture of products: the allylic hydroxylamine 9 alongside the Fenton oxidation products alcohol 10 and ketone 11 [53], and a small amount of tertbutyl carbamate (12, Scheme 1). Initial reactions under an argon or air atmosphere returned product mixtures in the ratios shown in Table 1.</p><p>Scheme 1: Allylic hydroxyamination of cyclohexene (7) using iron catalysts 4 and 5; i. 4 or 5 (10 mol %), BocNHOH (8), CH 3 CN, rt, 18 h; for yields see Table 1.</p><p>Under an argon atmosphere, the allylic hydroxylamine 9 was produced in ~10% yield with either ligand; performing the reaction open to air lifted the yield of the allylic hydroxyamination product 9 as high as 40%, but also substantially increased yields of 10 and 11 (Table 1).</p><p>Control experiments using just the metal salt or each of the ligands on their own returned trace quantities of product 9 and varying levels of Fenton-type pathways (Table S3, Supporting Information File 1), confirming that FeTPA (4) and FeBPMEN (5) are active agents in promoting allylic hydroxyamination of cyclohexene.</p><p>The effect of catalyst loading was screened under an air atmosphere, since initial results indicated that better yields of 9 are obtained under air than argon. Thus cyclohexene (0.7 mL, 7 mmol, 100 equiv) was added to a solution of catalyst 4 or 5 (1-20 mol %) and BocNHOH (70 μmol, 1 equiv) in CH 3 CN (Table S4, Supporting Information File 1). Lowering the catalyst loading of FeTPA from 10 to 5 mol % led to a small a Each reaction was performed in triplicate; data are averages of at least three runs. b Yields determined by GC using single point internal standard method (Tables S1 and S2, Supporting Information File 1). c Yields are quoted relative to the initial amount of BocNHOH ( 8), limiting reagent for the hydroxyamination reaction of interest. Formation of products 10 and 11 is not dependent on hydroxylamine 8 so the combined yields for some entries in this table are more than 100%.</p><p>increase in the yield of allylic hydroxylamine 9 with a significant decrease in the appearance of allylic oxidation products 10 and 11. The amount of FeTPA ( 4) could be further lowered to 2 and 1 mol %, bringing further small increases in the yield of 9.</p><p>However increasing loading of FeTPA (4) to 20 mol % halts the amination reaction, returning only allylic oxidation products 10 and 11. In contrast, changing the catalyst loading of FeBPMEN ( 5) up or down from 10 mol % lowers yields of 9; at 1 mol % or 20 mol % loading of catalyst 5, increased levels of 10 and 11 are observed, but at 5 mol % catalyst 5, the yields of all three oxidation products are diminished. Clearly the competing hydroxyamination and Fenton reaction pathways are sensitive to the amount of catalyst used relative to BocNHOH; optimum catalyst loadings are 1 mol % for 4 and 10 mol % for 5.</p><p>Nicholas and Kalita have reported that the addition of hydrogen peroxide can improve yields in their copper-catalysed allylic amination reactions using BocNHOH [41]. Thus the addition of hydrogen peroxide (1:1 relative to BocNHOH) to reactions with 4 or 5 was investigated. Using a 1:1:1 ratio of cyclohexene:BocNHOH:H 2 O 2 with FeTPA (4) at 1 mol %, allylic hydroxylamine 9 was formed in only 4% yield, with the allylic oxidation products 9 and 10 predominant. This is not unexpected given the propensity of hydrogen peroxide to react directly with iron complexes to produce 10 and 11 via Fentontype pathways [47,53].</p><p>We have previously observed solvent-dependent behaviour by non-heme iron complexes when mediating oxidation of cyclohexene in methanol versus acetonitrile as solvent [46,47]. Using methanol as solvent in the allylic amination reactions with FeTPA (4, 1 mol %) and cyclohexene in excess, yields of allylic hydroxylamine 9 dropped: 9 was formed in 10% yield (vs 27% in acetonitrile), while yields of allylic oxidation products 10 and 11 were also lowered, to 25% and 38% respectively (vs 54% and 36% in acetonitrile). BocNH 2 (12) was not observed. Using FeBPMEN (5, 10 mol %) in methanol, 10 and 11 were formed but target compound 9 was not observed at all. Presumably with methanol as solvent, the oxidising power of iron:ligand system is partially redirected to oxidise the solvent.</p><p>With a view to improving the synthetic potential of this reaction, the transformation was attempted at a 1:1 ratio of BocNHOH to cyclohexene. Thus BocNHOH (70 μmol), FeTPA 4 (1 mol %) and cyclohexene (70 μmol) were combined in acetonitrile and stirred for 18 hours at room temperature, open to the air. Allylic hydroxylamine 9 was formed in 6% yield; allylic oxidation products 10 and 11 were each observed in ≤1%. Reaction at 2:1 BocNHOH:cyclohexene did not significantly improve the yield of allylic amine 9 (7%). Similar results were obtained using FeBPMEN (5, 10 mol %) as catalyst, which yielded small amounts of 9 (8%) and 10 (2%) but not ketone 11. In their work using copper(I) iodide to catalyse similar reactions, Iwasa et al. conducted reactions at much higher concentrations of hydroxylamine and alkene (0.5 mmol BocNHOH and 0.75 mmol alkene in a total reaction volume of 1 mL) [40]. With this in mind, the FeBPMEN (5) reaction was repeated at 10-fold higher concentration (i.e., 1:1 BocNHOH/cyclohexene in a total reaction volume of 1 mL). Under these conditions the yield of allylic amine 9 doubled relative to the more dilute 1:1 reaction, to 17%; 10 and 11 were not observed.</p><!><p>The chiral catalyst Fe(R,R′)-PDP (6) has been used previously to promote asymmetric C-H oxidation reactions [51]. With a view to developing an asymmetric iron-catalysed allylic hydroxyamination reaction, catalyst 6 was prepared and used to effect conversion of cyclohexene 7 to hydroxylamine 9. This reaction afforded 9 in 13% yield, but only as the racemate: analysis by chiral GC (CP-Chirasil-Dex CB column) revealed two peaks with equal peak areas (t R = 8.6 and 8.8 minutes); the same peaks in the same ratio were observed using a reference sample of racemic 9.</p><p>Scheme 2: Proposed mechanism for hydroxyamination of cyclohexene (7) by FeTPA (4) and FeBPMEN ( 5): (a) iron-mediated oxidation of BocNHOH (8) by O 2 affords the nitroso-species 13, which (b) undergoes an ene reaction with the alkene substrate; (c) an alternative disproportionation reaction to convert 8 to 13 can occur without involvement of O 2 and also generates BocNH 2 (12), observed as a byproduct at low levels (Tables 1, S3 and S4, Supporting Information File 1). The mechanistic evidence gathered to date suggests the formation of a free nitroso species and 'off-metal' reaction with the alkene.</p><p>Several groups have recently reported efficient methods for the asymmetric hydroxyamination of carbonyl compounds using acylnitroso species generated in situ along with chiral oxazolines [16,17], N-oxides [19] or amines [18,20,21] as ligands or organocatalysts. In these nitrosoaldol contexts, the chiral agents induce asymmetry by virtue of their influence over the enolate reaction partner. Achieving asymmetric induction in the nitroso-ene reaction is a trickier proposition [14], although this has been demonstrated in an intramolecular context [22].</p><!><p>Previous studies of iron-promoted allylic amination reactions with N-phenylhydroxylamine, and copper-catalysed reactions with N-Boc-hydroxylamine (8) return regio-and chemoselectivity profiles that are consistent with reaction via nitroso-ene mechanisms [35,36,38,41]. Thus we hypothesised that the hydroxyamination reactions mediated by FeTPA (4) and FeBPMEN (5) follow a similar mechanism: iron-catalysed oxidation of 8 to generate the N-Boc-nitroso intermediate 13, which then participates in an ene reaction with cyclohexene (7, Scheme 2). Several experiments were conducted to test this hypothesis.</p><p>Spectroscopic experiments evince interactions between N-Bochydroxylamine (8) and FeTPA (4) in solution. An acetonitrile solution of FeTPA ( 4) is a dark red-brown colour; adding BocNHOH prompts a colour change to deep purple (λ max = 550 nm, ε ≈ 300 L mol −1 cm −1 ). Monitoring the electronic spectrum between 400 and 900 nm (Figure S1, Supporting Information File 1), this absorption peak reaches a maximum when 1 equivalent of N-Boc-hydroxylamine (8) has been added, consistent with coordination of hydroxylamine 8 to the FeTPA complex (4). A similar result is observed using 1 H NMR: titrating a solution of hydroxylamine 8 in d 3 -acetonitrile with FeTPA (4) indicates coordination of hydroxylamine 8 to the complex 4 (Figure S2, Supporting Information File 1).</p><p>Direct observation of the proposed Boc-nitroso intermediate 13 is difficult given its reactive nature. However BocNH 2 (12), the other product of the proposed disproportionation of N-Bochydroxylamine (8, Scheme 2c) is observed as a side product.</p><p>When N-Boc-hydroxylamine (8) was treated with FeTPA (4) in the absence of an alkene partner, BocNH 2 (12) was isolated in increased yield (25%). This mirrors the results of Jørgensen and Nicholas who have observed an analogous iron-catalysed reduction of N-phenylhydroxylamine to aniline, and confirms that FeTPA (4) can mediate the conversion of BocNHOH to BocNH 2 .</p><p>Nitroso species can be trapped by hetero-Diels-Alder reaction with dienes [54,55], and detection of the resulting hetero-Diels-Alder adducts used to confirm the formation of free nitroso intermediates. Thus trapping experiments were conducted using isoprene (14) to investigate the formation of a nitroso species in this reaction. First a 1:1 mixture of cycloadducts 15 and 16 was synthesised as a reference sample using Kirby's conditions for the hetero-Diels-Alder reaction (N-Boc-hydroxylamine (8), isoprene (14) and sodium periodate, Scheme 3a) [54,55]. Then isoprene (14) and N-Boc-hydroxylamine (8) were combined in acetonitrile in the presence of FeTPA (4) or FeBPMEN (5). Cycloadducts 15 and 16 were formed, along with the allylic amination product 17 in a 1:1:3 ratio (Scheme 3b); the observation of 15 and 16 in this reaction confirms that a Boc-nitroso species 13 is formed in the FeTPA/ FeBPMEN-catalysed reaction. It is interesting to note that the nitroso-ene product is not generally observed under the Kirby conditions, as the allylic hydroxyamination product 17 is unstable in the highly oxidising environment rendered by sodium periodate [56]. Observation of this product in the FeTPA-and FeBPMEN-mediated reaction of isoprene indicates the relative mildness of these conditions for nitroso formation.</p><p>Nicholas et al. have reported a similar experiment to study the iron(II,III) chloride-catalysed reaction of N-phenylhydroxylamine with 2,3-dimethyl-1,3-butadiene, in which the Diels-Alder cylcoadduct was not observed, and only the allylic amination product was formed [36,38]. Conversely Jørgensen and Johannsen report that N-phenylhydroxylamine and 1,3cyclohexadiene in the presence of iron(II) phthalocyanine do form the Diels-Alder cycloadduct [35]. Although the outcomes with the different catalysts were contrasting, only one product was observed in each case. Cenini et al. have reported that both cycloadduct and ene product are formed in the ruthenium-catalysed reaction of nitrobenzene (an alternate route to a nitrosobenzene intermediate) with isoprene [57]. As mentioned above, isoprene produces two different regioisomers in the hetero-Diels-Alder reaction with nitroso compounds, yet Cenini et al. only detected one isomer. From this observation they concluded that their Ru-catalysed amination reaction and the Diels-Alder reactions were occurring 'on metal' without generation of a free nitroso species [57].</p><p>In contrast, the FeTPA-mediated reaction generates both BocNO cycloadducts of isoprene along with the amination product. Furthermore, the reaction with the chiral system 6 renders zero asymmetric induction. Thus we conclude that the Fe-TPA/BPMEN reaction involves the nitroso-ene reaction of a free nitroso intermediate.</p><!><p>FeTPA (4) and FeBPMEN ( 5) are established catalysts for the hydroxylation, dihydroxylation and epoxidation of hydrocarbon substrates [48,[58][59][60]. In this study we have shown that they can also catalyse the allylic hydroxyamination of alkenes with N-Boc-hydroxylamine. Mechanistic investigations suggest the involvement of a free nitroso species which undergoes a nitroso-ene reaction with the alkene. The intermediacy of a free nitroso species means that asymmetric induction is not observed in reactions with the chiral catalyst Fe(R,R′)-PDP (6).</p><!><p>All commercially available reagents were used without purification unless otherwise specified. Solvents for extraction and chromatography were distilled before use. Solvents for reactions were freshly distilled immediately prior to use. Tetrahydrofuran (THF) was dried over sodium wire and benzophenone. Dichloromethane and acetonitrile were dried over calcium hydride. Acetonitrile was degassed using three freeze-thaw cycles when it was to be used in an atmosphere of argon. Methanol (MeOH) was dried over magnesium methoxide. Alkenes used in allylic amination reactions were passed through a micro-column of neutral alumina immediately before use. Water was purified using a Millipore purification system. Analytical thin-layer chromatography (TLC) was performed using preconditioned plates (Merck Kieselgel 60 F254). Developed TLC plates were viewed using a UV lamp at a wavelength of 254 nm and visualised with a ninhydrin stain. Flash column chromatography was performed on Davisil Grace Davison 40-63 μm (230-400 mesh) silica gel using distilled solvents.</p><p>Melting points were recorded on a Stanford Research Systems Optimelt automated melting system and are uncorrected. and are reported as (c mg mL −1 , solvent). Infrared spectra were recorded on a Bruker ALPHA FTIR spectrophotometer (ZnSe ATR). Gas chromatography was carried out on a Hewlett Packard 5890A and 5890 Series II Gas chromatographs with ChemStation software using HP1 (Crosslinked Methyl Silicone Gum) and CP-Chirasil-Dex CB columns, respectively. Both chromatographs were equipped with split/splitless capillary inlets and flame ionization detectors (FID). UV-vis spectra were recorded on a Varian Carey 4000 UV-vis spectrophotometer.</p><p>Synthesis of iron complexes 4, 5 and 6 and N-Boc-hydroxylamine (8) Tris(2-pyridylmethyl)amine (TPA, 1) [48] and N,N′-bis(2pyridylmethyl)-N,N′-dimethylethane-1,2-diamine (BPMEN, 2) [50] were synthesised in good yields following literature procedures (Supporting Information File 1). (+)-(2R,2′R)-1,1′-Bis(2-pyridylmethyl)-2,2′-bipyrrolidine ((R,R′)-PDP, 3) was synthesised from commercially available (R,R′)-2,2′-bipyrrolidine L-tartrate trihydrate according to the procedure reported by White and Chen [51]. Ligands 1-3 were combined with iron(II) triflate using literature protocols to generate [Fe(TPA)(CH 3 CN) 2 ](OTf) 2 (FeTPA, 4) [52], [Fe(BPMEN)(OTf) 2 ] (FeBPMEN, 5) [48] and [Fe(R,R′-PDP)(OTf) 2 ] (Fe(R,R′)-PDP, 6) (Supporting Information File 1) [51]. N-Boc-hydroxylamine (tert-butyl hydroxycarbamate, BocNHOH, 8) was prepared using a modified literature procedure (Supporting Information File 1) [61].</p><!><p>Acetonitrile was freshly distilled from calcium hydride; for reactions under argon (i.e., anaerobic conditions), the solvent was subjected to three freeze-thaw degassing cycles immediately before use. Stock solutions of iron complex (22.6 mmol L −1 ) and BocNHOH (8, 70 mmol L −1 ) in degassed acetonitrile were prepared under an atmosphere of argon. Acetonitrile (8.0 mL) was stirred under the required environment (argon or air) while iron complex stock solution (0.3 mL, 6.8 μmol) and cyclohexene (0.7 mL, 6.9 mmol) were added. Using a syringe pump, the BocNHOH stock solution (1.0 mL, 70 μmol) was added to the reaction mixture over 30 min. The reaction was stirred for 18 h after which time the solvent was removed in vacuo. The residue was dissolved in ethyl acetate and passed through a micro-column of silica to remove the iron complex. The sample was subjected to analysis by GC using n-decane as an internal standard and the single point internal standard method (Supporting Information File 1) [62,63]. Each reaction was performed in triplicate and data presented above are the average of the three runs.</p>

Beilstein

Disparate Catalytic Scaffolds for Atroposelective Cyclodehydration

Catalysts that control stereochemistry are prized tools in chemical synthesis. When an effective catalyst is found, it is often explored for other types of reactions, frequently under the auspices of different mechanisms. As successes mount, a unique catalyst scaffold may become viewed as \xe2\x80\x9cprivileged\xe2\x80\x9d. However, the mechanistic hallmarks of privileged catalysts are not easily enumerated, nor readily generalized to genuinely different classes of reactions or substrates. We explored the concept of scaffold uniqueness with two catalyst types for an unusual atropisomer-selective cyclodehydration: (a) C2-symmetric chiral phosphoric acids, and (b) phosphothreonine-embedded, peptidic phosphoric acids. Pragmatically, both catalyst scaffolds proved fertile for enantioselective/atroposelective cyclodehydrations. Mechanistic studies revealed that the determinants of often equivalent and high atroposelectivity are different for the two catalyst classes. A data-descriptive classification of these asymmetric catalysts reveals an increasingly broad set of catalyst chemotypes, operating with different mechanistic features, that creates new opportunities for broad and complementary application of catalyst scaffolds in diverse substrate space.

disparate_catalytic_scaffolds_for_atroposelective_cyclodehydration

INTRODUCTION<!>Catalyst optimizations.<!>Substrate evaluation.<!>Mechanistic experiments for P19.<!>DFT calculations for B14.<!>Comprehensive model and determinants.<!>CONCLUSIONS

<p>Chemists rarely know what type of catalyst will be well-suited to a new substrate class or reaction type a priori. The closer to precedent the desired transformation is, the more readily chemists can predict an effective catalyst scaffold.1,2 To that extent, a few principles have emerged to guide chemists in uncharted reaction territory.3 Among these, the evaluation of catalysts deemed "privileged" is a common starting point for the consideration of a catalyst chemotype.4,5 This leads to questions: How unique are privileged catalysts? To what extent are privileged catalysts exclusively matched for a number of reactions proceeding through a common mode of activation? Can a different catalyst scaffold also achieve high selectivity for the same reactions through alternative modes of action? In addition, do privileged catalysts remain effective beyond simple substrates, or can there be a different catalyst scaffold that offers complementarity when substrates become more complex? Episodic disclosures of different catalysts classes for the same transformation, on the same substrate, exist in several widely-explored asymmetric reactions such as the catalytic hydrogenation,6 aldol7 and Diels-Alder reactions8 (Scheme 1). Harnessing different catalyst designs for effective enantiocontrol is therefore possible, but direct comparative studies of the substrate preferences, and their different mechanistic underpinnings has been difficult to achieve rigorously and retrospectively, given the multiple laboratories and diverse reaction conditions often engaged. The present study confronts these questions in an experimentally and computationally driven manner through the comparative study of two distinct catalyst classes that are effective for the enantioselective and atropisomer-selective cyclodehydration of o-sub-stituted aniline derivatives to deliver optically enriched benzimidazoles,9 N-heterocycles of high interest in drug-like molecules10 as shown in Figure 1c.</p><p>The two catalyst classes hail from disparate realms of the asymmetric catalysis landscape. One catalyst class, C2-symmetric chiral phosphoric acids derived from BINOL (C2-type),11 is widely accepted as a privileged scaffold, which has been effectively utilized in an exceedingly large number of reports (Figure 1a).12 The second class incorporates peptide-based phosphoric acids, inspired by the ubiquitous biochemically relevant moiety, phosphothreonine (pThr) as shown in Figure 1a. pThr is formed by enzymatic phosphorylation, resulting in numerous biochemical signal transduction cascades.13 Interestingly, this motif is not to our knowledge known to play a catalytic role implicated in bond formation or cleavage in any enzyme. Yet, pThr-based catalysts have recently been found to be effective for asymmetric reactions such as transfer hydrogenation14 and the Baeyer-Villiger oxidation of cyclobutanones15 (Figure 1b). Critically, in each case good selectivity was found for C2-type-and peptide-based chiral phosphoric acids. In the case of Baeyer-Villiger oxidation, the pThr-type catalyst actually gives better selectivity than a canonical C2-type catalyst with a functionalized substrate.</p><p>Detailed below is the experimental documentation that both C2-type and pThr-type CPAs can catalyze a wide array of atroposelective cyclodehydrations in very high enantioselectivity. Challenging each catalyst class with a range of substrate types reveals not only intriguing strengths and weaknesses for each as a function of substrate, but perhaps even more curiously, high functional homology for other substrates. Catalyst and substrate structure-selectivity relationships (SSRs) and context dependent mechanistic studies point to subtle but a distinct set of control elements that lead to common selectivity outcomes in many cases.16</p><!><p>The context for this study is in the area of catalytic asymmetric synthesis of atropisomers, which is a relatively young subfield of asymmetric catalysis, with a variety of ap-proaches now reported,17,18 including asymmetric cross-coupling19 and dynamic kinetic resolutions.20 Few catalytic atropisomer-selective methods target medicinally relevant heterocycles, and accordingly this study began with the targeted conversion of 1a to 2a (Table 1, Eq 1). While the precedent for the atroposelective cyclodehydration was initially focused on diastereoselectivity,9 we commenced the comparative study by expanding this finding to an enantioselective method for atropisomer-selective synthesis of axially chiral benzimidazoles with both the pThr-type and C2-type of CPA. We initially examined 19 pThr-type catalysts for the atropisoselective cyclization. Catalyst P1 (with a canonical DPro-Aib sequence at the i+1 and i+2 positions)21 was evaluated to benchmark the catalyst-dependent enantioselectivity against a well-studied β-turn inducing peptide sequence resulting in product, 2a with a 43% e.e. at 74% con-version (Table 1A, entry 1). Standard variations of the peptide sequence led to significant improvements. Essentially all residues transmit stereochemical information to the reaction coordinate, whether the individual residues are locally chiral or not.22 While ex-tended commentary could be provided about the complete set, significant conclusions pointed to the critical nature of a DPro residue at i+1 (Table 1A, entry 1 versus entry 2), as well as a synergistic advantage conferred by an aminocyclopropane carboxylic acid residue (Acpc; Table 1A, entries 4, 10–19) at the i+2 position, and a branched L-configured cyclohexylglycine residue (Chg; Table 1A, entries 15–19) at the i+3 residue. An N-terminal benzoyl group was also found to be optimal, such that catalyst P19 was declared the lead catalyst, delivering 2a with a 94% e.e. at 94% conversion (Table 1A, entry 19).23</p><p>In parallel, we asked exactly the same question with C2-type CPAs. Shown in Table 1B are the results of atroposelective cyclodehydrations with 14 variously substituted common BINOL-derived CPAs. Notably, the results, from a purely enantioselectivity-centric per-spective are comparable to the results obtained with the pThr-type catalysts. For example, p-substituted catalysts B1 and B2 provide a modest level of initial selectivity at good conversion (58% e.e. and 52% e.e., respectively; Table 1B, entries 1 and 2). o-Substitution with B3, B4, and B5 leads to improvement, with up to 89% e.e. (Table 1B, entries 3–5). Apparent electronic effects also emerged, as the m-substituted catalysts B6, B7, and B8 gave a range of results (Table 1B, entries 6–8), with B9, the m,m-diphenyl substituted catalyst, giving 89% e.e. in excellent conversion (Table 1B, entry 9). Further substitution leveraged apparent combinations of various features (vide infra). Tris-substitution on the BINOL-aryl substituent gave a set of catalysts that were quite effective for the atropoisomer-selective cyclodehydration, as B10–13 all converged to give 2a with between 90% and 92% e.e. for the canonical TRIP catalyst24 (Table 1B, entries 10–13). Catalyst B14 bearing 9-anthracenyl groups afforded the highest selectivity observed, delivering 2a with 96% e.e. at full conversion (Table 1B, entry 14).</p><!><p>From these optimization studies a ques-tion arises: is the privileged C2-symmetric type catalyst B14 (96% e.e., 100% conversion) clearly better than peptidic catalyst P19 (94% e.e., 97% conversion)? Analysis of each against a panel of 22 systematically modified substrates allows a data-driven inquisition (Figure 2). Noteworthy, cyclodehydrations procced efficiently with both catalysts, while moderate conversions were observed with sterically congested o,o'-disubstituted substrates.23 We first evaluated three additional 6-substituted substrates, 2b–d (Figure 2a). Removing the remote tBu group (i.e., 1b to 2b) impacted enantioselectivity, as both P19 and B14 process this substrate with a slightly lower level of selectivity (P19, 91% e.e., B14, 88% e.e.). An electron donating group also leads to a slight drop in selectivity for both catalysts (P19, 92% e.e.; B14, 94% e.e.), while an electron withdrawing group enhances the asymmetric catalysis (P19, 97% e.e.; B14, 97% e.e.). But, most noteworthy is that P19 and B14 are all but indistinguishable with these substrates. Substitution at the 7-position, however, cre-ates an entirely different scenario. For five different substrates (1e–i), peptidic catalyst P19 appears to offer more generality, as benzimidazoles 2e–i are delivered with 89–93% e.e. for this class (Figure 2b). In contrast, C2-type catalyst B14 seems poorly suited to these substrates, as the products are obtained with 44–76% e.e. (2e–h), with the exception of 2i being isolated with 83% e.e. (cf. 89% e.e. with P19). Several other substrates also reveal the differential attributes of pThr-type and C2-type CPAs. For example, as shown in Figure 2c, substitution at the 5-position (i.e., 2j) is better tolerated with P19 (94% e.e.) than with B14 (90% e.e.). Incorporating differ-ent substitution patterns on the bottom arene of the diarylaniline moiety also reveals curious effects. Highly substituted axially chiral benzimidazoles 2k–m (Figure 2d) are all formed with excellent enantioselectivity when either P19 (93–97% e.e.) or B14 (93–96% e.e.) are used as the catalysts. Exceptions appear in the formation of 2n where B14 (95% e.e.) is slightly more effective than P19 (85% e.e.) and vice versa in the formation of 2o (P19, 89% e.e.; B14, 88% e.e.). Some striking differences also emerge with o,o′-disubstituted substrates 2p–r (Figure 2e). In these cases, low enantioselectivities are observed with both P19 and B14 as the catalyst. While B14 pro-vides a somewhat higher enantioselectivity in the cases of 2p and 2r, neither catalyst gives promising results. However, when a second R-group is added to the remote arene, as in the case 2s and 2t, quite good enantioselectivities are recorded by both catalysts. Benzannu-lated analogue (2u) is slightly better accommodated with P19 (94% e.e.) than with B14 (91% e.e.). Yet, both P19 and B14 are poor catalysts for the formation of 2v, with a basic N-atom introduced re-mote from the loci of bond formation (Figure 2i, 16% e.e. and 2% e.e., respectively). The absolute configuration of 2a was determined by X-ray crystallographic analysis after recrystallization (Figure 2j), and those of other products in Figure 2 were displayed by analogy.</p><!><p>As a synthetic method, per-haps most would agree that P19 and B14 are promising new tools for an underexplored area of enantioselective catalysis. However, what do their similarities and differences in performance tell us about their respective mode of stereoinduction? For this question, we turned to mechanistic studies. The manner in which one can in-terrogate the mechanistic details of P19 and B14 is different.</p><p>Computation, particularly at the DFT level, has emerged as a pow-erful tool for assessing the feasibility of mechanistic steps involved in catalysis and the origins of enantioinduction.25 These techniques are renowned for the interrogation of rigid C2-type catalysts like B1–14. In contrast, peptide-based catalysts like P19 present special challenges, and computational analyses of such flexible systems are far from routine.26 The complication arises from the many plausible ground state (GS) and transition state (TS) conformations, which require exhaustive sampling and may require molecular dynamic simulations for effective conformation generation, an inherently time consuming process.27 A range of experimental techniques, however, offers a complementary way to probe the effect of the catalyst structure on enantioselectivity, and in some instances are faster to complete than computations. In this regard, we performed two types of studies including, (a) evaluation of catalyst analogs, and (b) investigation of the catalyst structure by NMR techniques. For the former, truncated peptides P20–22 were prepared and evaluated to isolate the minimal determinants of selectivity and striking results were obtained (Figure 3a). To begin, phosphorylated threonine monomer, P20, shows moderate enantioselectivity favoring the enantiomeric product of 2a, suggesting that the high enantioselectivity of P19 is not dominated by the local stereoconfiguration of threonine, but ra-ther on the global conformation of the peptide.28 Elongation to the dipeptide (i.e., P21) also affords ent-2a as a major enantiomer (−42% e.e.), while the tripeptide P22 gives almost racemic mixture of 2a. High enantioselectivity was only observed with the Fmoc protected tetrapeptide, P15.</p><p>These results also imply additional non-covalent interactions be-tween the cyclohexyl group at i+3 residue and 1a and/or considerable conformational changes induced by the introduction of i+3 residue. For direct observation of the catalyst structure, 1H−1H ROESY NMR studies of P19 were performed. We observed 15 non-sequential ROE correlations as shown in Figure 3b. Contacts (red arrows) of N‒Hacpc with the bottom face of DPro and the proton of the αC-H of pThr suggest that the N‒H bond is likely located underneath the DPro residue, in proximity to potential hydrogen bond acceptors C=OThr and C=OBz. Considering ROE contacts (blue arrows) of the cyclohexyl group with the protons of C‒H of DPro and βC-H of Thr, a β-turn conformation appears supported by the hydrogen bond be-tween N–Hchg and C=OThr as shown in Figure 3c. Multiple correlations (green arrows in Figure 3b) between the dimethylamide at the C-terminus and benzylic and aromatic protons of -OBn/-NHBz of pThr are consistent with our hypothetical conformation and also in-dicate that the i+3 residue would be in proximity with the phosphoric acid. Given the steric effect of the i+3 residue as described in Table 1A, secondary interaction between the cyclohexyl group and substrate may have a beneficial influence on atroposelectivity.</p><!><p>In evaluating the mechanism of C2-type catalysts like B14, DFT and experimental results strongly suggest that cyclization is both rate and enantiodetermining.23 To probe the origins of enantioinduction, we performed geometry optimizations with ωB97XD/6–31G(d) followed by single-point calculations at the ωB97XD/6–31G(d,p) level in toluene with the polarizable continuum model, IEFPCM using optimal catalyst B14 containing an anthryl group. The calculations corroborate the magnitude (exp. 96% e.e., calc. 93% e.e.) and sense of enantioinduction (Figure 4a). The key controlling element appears to be a steric interaction be-tween the naphthyl group on the substrate and the 3,3′ substituents on the catalyst (Figure 4a). To precisely explore the contributions of the t-butyl and the naphthyl substituents on the substrate structure to the TS energies, two truncation studies were performed computationally. Firstly, the t-butyl substituent was replaced for a proton and a single-point energy was taken of the resulting structures without re-optimization.29 The energy difference (ΔΔE‡) between the competing structures increased from 2.4 to 4.0 kcal mol–1, which suggests that the t-butyl group is not a major element in the catalyst-substrate interactions. However, replacing the naphthyl group for a phenyl group led to a significant decrease in ΔΔE‡ from 2.4 to 0.8 kcal mol–1. These truncation studies are consistent with the primary determinants of enantioselectivity being the interactions between the 3,3′ substituents on the catalyst and the naphthyl substituent of the substrate. Further interrogation of the mechanistic basis for the selectivity afforded by C2-type catalysts was accomplished using statistical comparison of catalysts substituent changes to reaction enantioselectivity (expressed as G‡).30 Collected parameters for the correlations included IR vibrational frequencies and intensities, torsion angles, NBO charges and Sterimol steric descriptors (L, B1, B5). A simple model prioritizes steric effects measured though torsion and two other terms: the P=Oas stretch likely describing H-bond contacts and the cross term *B1C3, a steric correction for the inclusion of 9-anthryl, as additional selectivity discriminants.</p><!><p>Perhaps the most powerful analysis of the differences between the two catalyst classes can be achieved by using a substrate profiling technique wherein the performance of each catalyst is analyzed through correlation of substrate outcomes. Specifically, we anticipated that the subtle differences in substrate performance as a function of catalyst class could be related through overlapping and varying sensitivities to the substrate, providing insight into how these catalysts induce stereoselectivity. We hypothesized that 2a–t offered the requisite changes to both the electronic and steric environments of the substrate (Figure 5) but also incorporates sufficient overlapping features for analysis.</p><p>To define the parameter library, DFT optimizations were per-formed on these substrates at the M06–2X/def2-TZVP level of the-ory wherein NBO charges, IR vibrations and Sterimol values were collected to probe structural effects.31 A statistical model consisting of four terms was determined for the BINOL-derived phosphoric acids. The included parameters suggest that the process proceeds via a TS that minimizes steric repulsions between the large 3,3′ groups on the catalyst and the substrate (Figure 5a), which is consistent with both the DFT and catalyst correlation studies detailed above. For example, the negative coefficient with the LC7 term describes likely repulsive interactions in TSRe with the C7 substituent and the 3,3′ substituents. The terms B5C6 and the B1ortho both describe the preferred orientation of the bottom ring in the TS. Reducing the size of the substituent at C6 decreases the repulsion between the top and the bottom rings in the cyclization step. In this case, the more compact, the third TS, in which the larger substituent on the bottom aromatic ring points towards the top aromatic ring (second TS structure in Figure 5b), would be more plausible leading to decreased enantioselectivity. Complementary to the catalyst statistical model (Figure 4b and 5a), the NBOO term is likely describing H-bond contacts between the catalyst and substrate.</p><p>Intriguingly, the pThr-type catalysts counterparts do not function through an analogous model of enantioinduction, despite the same catalytic apparatus for bond formation (i.e., the CPA moiety itself). For the pThr-type catalysts, the mathematical model consists of three conserved terms. Two of these terms essentially describe the bottom ring orientation preferences, an apparent locus of critical catalyst-substrate interactions. The NBO term is likely indicative of the hydrogen bonding features of phosphoric acid with the substrate. However, most notably, the peptide system does not include an additional penalizing steric term in the model. Despite steric effects and the proximity of the i+3 residue to the phosphoric acid as observed in Table 1A and Figure 3, this catalyst class still provides an alternative mode of enantioinduction. This effect may be consistent with the enhanced generality in scope for substrate variation in this vicinity; the flexible peptide system may also be adaptive to substrate steric demands, as is evidenced in other peptide-based catalysts.26,27 Comparison of the magnitudes of the coefficient terms can provide further insight into the stereocontrolling elements responsible for enantioinduction. Intriguingly, the B5C6 term is statistically more significant for the C2-type CPAs. This suggests that due to steric repulsion between the catalyst 3,3′ substituents and the bottom aromatic ring of the substrate that a compact arrangement may be more readily favored. The proposed physical meaning behind each of the terms in the mathematical equations have been summarized in Figure 5b.</p><!><p>Although the general principles of energetic differentiation underlying asymmetric catalysis by distinct catalyst families are essentially the same, significant mechanistic differences can lead to similar selectivity outcomes. Fundamentally, destabilizing effects and stabilizing noncovalent interactions of a variety of types can all contribute to the differential energies of competing transition states within an ensemble. In the present study, steric effects from reasonably large groups on both the catalyst and substrate appear to dictate enantioselectivity for C2-type CPAs. In contrast, pThr-type CPAs appear to work through an alternative mode of enantioinduction, where conformational adaptation presumably limits repulsive interactions. These experimental and computational findings allow us to revisit some of the questions posed in our introduction. For example, it seems ever clearer that various catalyst types may be found to achieve high levels of enantioselectivity for a given transformation of interest, and that if the mode of activation is general (e.g., Lewis/Brønsted acid activation, bifunctional catalysis, etc.), application to other reactions with mechanistic similarity can follow. Yet, the redundancy of mechanistic solutions to common overall transformations may expand the concept of the privileged catalyst. In this sense, as the field evolves, it seems there may be less privilege, indeed less uniqueness of solution, as more catalyst types are discovered for a given type of enantioselectivity transformation. The expanded array of catalyst available also enables exploration of ever-expanding substrate space. Accordingly, new layers of mechanistic complexity are introduced when unusual substrate types are evaluated. Therein, situations emerge where catalyst functional equivalences may be lifted. In such situations, mechanistic complementarity among catalyst types may be particularly critical for broad spectrum success with highly diverse substrates. And of course, these same issues will likely emerge as each catalyst type is profiled in an ever-expanding list of reaction types. In this sense, a case may exist for an ever expanding, perhaps less privileged catalyst catalog, placing a premium on catalytic structural diversity and diverse catalytic mechanisms that control secondary, outer sphere interactions. This is a hallmark of enzymatic catalysis, where diversity of function on common catalyst scaffolds has emerged alongside an impressive array of catalyst specificities.32</p>

PubMed Author Manuscript

Top-Down Protein Identification/Characterization of a priori Unknown Proteins via Ion Trap Collision-Induced Dissociation and Ion/Ion Reactions in a Quadrupole/Time-of-Flight Tandem Mass Spectrometer

The identification and characterization of a priori unknown proteins from an Escherichia coli (E. coli) soluble protein lysate using ion trap collision-induced dissociation of intact protein ions followed by ion/ion reactions in a quadrupole/time-of-flight tandem mass spectrometer is illustrated. The procedure involved the submission of uninterpreted product ion spectra to a peak picking program and then to ProSightPTM for searching against an E. coli database. Examples are provided for the identification and characterization of both modified and unmodified unknown proteins with masses up to \xe2\x88\xbc28 kDa. The availability of protein intact mass along with sequence information makes possible the characterization of proteins with post translational modifications, such as disulfide linkages, as well as protein isoforms whose sequences are absent from a database, provided that a related form of the gene product is present in the database. This work demonstrates that the quadrupole/time-of-flight platform, in conjunction with ion-ion proton transfer reactions, can be adapted to obtain primary structure information from entire protein ions, rather than simply N- or C-terminal information from low mass-to-charge products, for proteins as large as several tens of kilodaltons.

top-down_protein_identification/characterization_of_a_priori_unknown_proteins_via_ion_trap_collision

1. Introduction<!>Material<!>Growth and Lysis of E. coli<!>Fractionation of Proteins from the Soluble E. coli Whole Cell Lysate by RP-HPLC<!>Mass Spectrometer<!>Database Search<!>Tandem Mass Spectrometry of Unknown Proteins from a Whole Cell E. coli Lysate Fraction<!>Protein Identification by Database Search of Un-interpreted Whole Protein MS/MS Spectra<!>Proteins without post-translational modification<!>Identification of Proteins with Post-translational Modifications or Absent from the Database<!>4. Conclusions<!>

<p>Mass spectrometry has emerged as a rapid and sensitive technique for protein identification and characterization.1, 2 Initial success with protein identification was based on an approach referred to as "peptide mass fingerprinting",3-5 which involved a single stage mass measurement of peptide components from enzymatically or chemically digested pure proteins or proteins in a simple mixture. As the complexity of the protein mixture increases, tandem mass spectrometry of the digested peptides is typically used to increase the specificity of the approach with database analysis of the product ion spectrum.6, 7 Both approaches are based on the digestion of proteins and are generally referred to as "bottom-up" approaches. To date, "bottom-up" proteomics has developed into a powerful approach for protein identification; variations thereof are widely used on a range of platforms. However, valuable information about a protein, such as its molecular weight and modification state, can be lost when the protein is subjected to complete digestion, which is a widely recognized disadvantage in the 'bottom-up" approach.</p><p>With the advent of new ionization approaches, such as electrospray ionization (ESI),8, 9 as well as advances in mass spectrometry instrumentation, intact proteins can be interrogated directly by mass spectrometry without resort to digestion. As a result, intact protein mass information can be readily obtained from various platforms, including mass spectrometers of moderate mass-to-charge upper limits due to the multiple charging phenomenon associated with ESI. The derivation of structural information from a whole protein ion, however, is more challenging than the measurement of its mass due to challenges associated with dissociation of the protein ions and the interpretation of the product ion spectra. Nevertheless, various approaches have been developed for the tandem mass spectrometry of intact protein ions. Strategies based on whole protein tandem mass spectrometry form the basis of "top-down" proteomics,10-12 the utility of which for protein identification/characterization is increasingly recognized, particularly for the characterization of protein post-translational modifications.13</p><p>In a typical "top-down" approach, protein identification is generally made via a database search of a protein database or a translated genomic database, in which an experimental product ion spectrum from dissociation of a whole protein is compared with a predicted spectrum derived from in silico dissociation of database candidate proteins, whose molecular weights are within a specified mass window of the unknown protein to be identified. The candidate protein is then ranked based on an algorithm-assigned score that depends primarily on the number of matched peaks.14, 15 The confidence level for a proposed identification via database search is affected by many factors and is heavily dependent on the information that can be drawn from a product ion spectrum. The quality of the structural information in a product ion mass spectrum is largely defined by factors such as the mass measurement accuracy of the instrument, signal-to-noise ratio (S/N) of the products in the spectrum, etc., while the quantity of the information depends both on the amount of potential information that can be initially produced in a tandem mass spectrometry experiment (typically a dissociation experiment) as well as the fraction of the total amount of information that can be extracted from such an experiment. For a polypeptide ion, for example, the quantity of information might be measured by the fraction of inter-residue cleavages observed.</p><p>The amount of structural information that can be produced in a tandem mass spectrometry experiment is determined largely by factors like the forms of the protein precursor ions (e.g. positive vs. negative, open shell vs. closed shell, protonated vs. metal cationized, etc.) and the dissociation method employed. Compared to the fragmentation of peptides, dissociation of intact proteins of large size (e.g., >200 kDa) is much more challenging.16 To date, for proteins in the tens of kilodaltons range, a variety of approaches has been used for intact protein ion dissociation that are based on either ion/electron interaction, such as electron capture dissociation (ECD)17 and electron transfer dissociation (ETD),18, 19 ion/photon interaction, such as infra-red multiple photon dissociation (IRMPD),20, 21 or ion/neutral interaction, such as ion trap collisional-induced dissociation (CID)22 and collision-induced dissociation by sustained off-resonance irradiation (SORI CID).23</p><p>Relative to the "bottom-up" approach, it is also more difficult to extract sequence information from dissociation of a whole protein ion due to the challenges in the charge state assignment of large fragment ions and the potentially severe peak overlap in the spectrum from ions of different mass but similar mass-to-charge ratio. The percentage of mass information that can be extracted from an MS/MS experiment is largely determined by the informing power24 of the approach employed, which is reflected by its ability to address the charge state ambiguity associated with a highly charged large fragment ion as well as its capability to deal with peak overlap in a spectrum. With the high resolving powers of the Fourier transform ion cyclotron resonance (FTICR) mass spectrometer and Oribtrap™,25, 26 assignment of product ion charge state can be facilitated by measuring the spacing between the isotopic peaks,27 which has greatly facilitated the exploration of whole protein tandem mass spectrometry.28 It is also noteworthy that a quadrupole/TOF platform, with its moderate resolving power, has been reported for "top-down" protein identification from a database search based on peptide sequence tags identified from the spectrum.29 However, identification of a sequence tag from a tandem mass spectrum is not always guaranteed on such a platform and full characterization of the protein is precluded by overlap of high mass and high charge products. The problems of peak overlap and charge state ambiguity can be alleviated to some extent in top-down analysis of intact proteins using MALDI TOF-TOF platforms in which singly charged precursor ions are examined.30, 31 However, the extent of sequence information available from the dissociation of large singly charged polypeptides can be limited. Alternative approaches to obtain product ion mass information without resorting to ultra-high resolving power have employed charge manipulation strategies such as ion/ion reactions to remove the charge state ambiguity by charge reducing the product ions to largely singly charged ions.32 Additional advantages from the use of ion/ion charge reduction include a reduction of peak overlap in the post-ion/ion CID spectrum by distributing the overlapped peaks over a wider mass space. A recent study24 showed that the informing power of an electrospray-based tandem mass spectrometry approach for protein mixture analysis can be significantly improved when coupled with ion/ion reactions for charge state manipulation, particularly when mass analyzers of low to moderate resolving powers are used, such as with ion traps and time-of-flight, respectively. Consistent with this result, identification of a priori unknown proteins has been successfully demonstrated on 3D ion traps coupled with ion/ion reactions.33-35 Moreover, successful "top-down" protein identification has been demonstrated via database search of low mass product ions from a linear ion trap coupled with ion/ion reactions for both protein dissociation (i.e. ETD) and product ion charge reduction (i.e. proton transfer reactions).36, 37 However, the limited mass range (<∼ m/z 2000) associated with such a platform imposed a limitation on the derivation of the sequence information from the internal region of a large protein. A QqTOF platform, with a much larger mass range and higher resolving power relative to a 3D or linear ion trap, coupled with ion/ion reactions has been predicted to provide informing power comparable to that obtained from very high resolution approaches that do not employ charge state manipulation, such as those based on Orbitrap™ and FTICR MS.24 This suggests that ion/ion reactions can enhance the utility of the QqTOF platform for top-down proteomics. In this work, we apply a QqTOF approach coupled with ion/ion reactions to the identification and characterization of components of protein mixtures derived from Escherichia coli protein lysates. The results serve to illustrate that the predicted improvements in informing power are largely realized and that a QqTOF instrument of moderate mass resolving power and mass measurement accuracy is capable of confident identification and characterization of intact protein ions of several tens of kilodaltons in mass.</p><!><p>Methanol and glacial acetic acid were purchased from Mallinckrodt (Phillipsburg, NJ). Perfluoro-1-octanol (PFO) was obtained from Sigma-Aldrich (Milwaukee, WI). Difco nutrient broth and bacto-agar were purchased from BD (Franklin Lakes, NJ), while Bacterial protease inhibitor cocktail was purchased from Sigma-Aldrich (St. Louis, MO).</p><!><p>E. coli ATCC 15224 was obtained from the American Type Culture Collection (Rockville, MD). The freeze-dried E. coli was reactivated in nutrient broth at 37 °C under aerobic growth conditions. The E. coli cells were harvested during stationary phase by centrifugation at 3000g for 10 min. About 4g of the pellets were resuspended in 10 mL of water with 1mL of protease inhibitor cocktail and then were subjected to French press under 700 psi. The lysate was then centrifuged at 16000g for 20 min, and the resulted supernatants were stored at -70 °C.</p><!><p>The supernatant was fractionated by reverse-phase HPLC on an Agilent 1100 series with a C4 column (Vydac, 4.6 × 100 mm). A linear 60-min gradient from 0 to 100% buffer B was used (buffer A: 0.1% aqueous TFA and buffer B: 60% acetonitrile/40% H2O containing 0.09% TFA). The column temperature was set at 40 °C. The absorbance was monitored at 215 nm. The fraction collected was lyophilized to dryness and then dissolved in 49:49:2 methanol/water/acetic acid solution for nano-ESI.</p><!><p>All experiments were performed on a quadrupole/TOF (Q-STAR XL, Applied BioSystems/MDS Sciex, Concord, ON, Canada) modified for ion/ion reactions.38 A home-built pulsed dual ion source39 was coupled directly to the interface of the Q-STAR instrument for the generation of ions of both polarities, which consists of two nano-ESI emitters, one for the generation of protein cations and the other for the production of reagent PFO anions. Ion/ion proton transfer reactions were implemented in the Q2 quadrupole linear ion trap (LIT) in mutual storage mode,40 in which ions of opposite polarity were stored simultaneously by superposing an auxiliary radio frequency signal (rf) (250 kHz, 500 Vpp) on the end lenses (IQ2 and IQ3) to store ions in the axial direction, while the normal operation of the oscillating quadrupole field of the Q2 quadrupole array stored ions of both polarities in the radial plane. All electronics are controlled by Daetalyst 3.14, a version of research software developed by MDS Sciex.</p><p>A typical scan function in the ion/ion reaction of product ions derived from the ion trap CID consists of the following steps: protein ion injection and isolation (100 ms), ion trap CID of the isolated ions (200 ms), anion injection and isolation (100 ms), mutual storage mode ion/ion reaction (300 ms), and mass analysis by TOF (50 ms). Specifically, a positive high voltage (∼+1.5 kV) applied to one of the emitters was initially pulsed on to generate protein cations, which were sampled and transferred into the Q2 linear ion trap (LIT) with Q1 operated in mass resolving mode to isolate the charge state of interest. Protein ions were cooled in the Q2 LIT for a short time (30 ms) with nitrogen as buffer gas at a pressure of ∼ 8 mTorr, during which time the positive high voltage was turned off. After the cation cooling step, an auxiliary dipolar AC signal corresponding to the secular frequency of the isolated ions was applied to one pair of the Q2 rods to induce ion trap CID. The product ions were cooled in the Q2 LIT for 30 ms, after which time the negative high voltage was turned on to generate the PFO reagent anions, which were subsequently introduced into the Q2 LIT with relatively low kinetic energies (∼ 8 eV) with Q1 operated in mass resolving mode to isolate the singly charged PFO dimer reagent anions (i.e., [(PFO)2-H]-) for the subsequent ion/ion reaction. During this period, the DC potentials on the IQ2 and IQ3 lenses were adjusted to a common value and set ∼ 0.5 V more positive relative to the Q2 DC offset while an auxiliary rf voltage (250 kHz, 500 Vpp) was applied to the IQ3 lens. During the subsequent mutual storage step, the negative high voltage was turned off and a common DC offset potential was set for both the Q2 rods and the Q2 containment lenses. An auxiliary rf voltage was also applied to the IQ2 and IQ3 lenses to enable the axial trapping of both polarities. After a specified mutual storage time, a positive DC potential was applied to the containment lenses to remove the residual reagent anions while the auxiliary rf voltage was terminated. In the final step, the positive ions consisting of the ion/ion reaction products and the unreacted precursor ions were released from Q2 LIT to the orthogonal reflectron TOF for mass analysis.</p><!><p>ProSight PTM Retriever41, 42 was used in its absolute mass search mode as a "top-down" database search engine searching against the annotated SWISS-PROT E. coli K12 strain database. Input data to the search engine were a product ion list selected using the Origin 6.0 program from the post-ion/ion CID spectrum. Instead of a S/N criterion, Origin 6.0 uses a search rectangle to find a peak and a minimum height percentage value to eliminate noise from consideration. The typical search rectangle set for the current study is one with a width of 0.3 and length of 0.3, and the minimum height percentage value of 3. The product ions selected were searched as average masses against the protein database with an intact protein mass window of 2,000 Da and product ion mass tolerance of 1.5 Da.</p><!><p>A simple LC separation was employed to reduce the complexity of the protein mixture in the supernatant of the E. coli whole cell lysate after centrifugation. The acquired chromatogram is shown in Figure 1, in which the four labeled peaks correspond to fractions selected for study. No attempt was made to examine each fraction. The fractions examined were chosen more-or-less randomly with the provision that at least one abundant protein greater in mass than 9 kDa was present. In all of the examples described herein, the protein subjected to characterization was a major component in the mass spectrum. One of the most abundant charge states of the protein was selected for tandem mass spectrometry. All proteins that met these criteria (i.e., mass greater than 9 kDa that yielded strong signals) provided sufficient information for protein characterization. However, in most of the fractions, the signals were dominated by species of mass lower than about 5 kDa. It is generally straightforward to perform tandem mass spectrometry on these low mass species without recourse to ion/ion reactions and they were therefore not targeted in this work. There were also many low levels signals for larger proteins but these species proved not to provide sufficient product ion signal for protein characterization. In the absence of further purification to isolate abundant low mass polypeptides from the mixtures, which tended to consume much of the charge in the mass spectrum, the fraction of proteins of mass greater than 9 kDa that can be characterized under the conditions used in this work is expected to be low. However, no attempt was made here to take measures either to improve the dynamic range or to determine the fraction of proteins in the entire protein lysate that can be characterized. This is an important area for emphasis in future work. The electrospray mass spectrum of the fraction at a retention time of 38.26 min., labeled as #1 in Fig. 1, is shown in Figure 2(a), in which one major component was apparent with a charge state distribution corresponding to +15-+10 ions and a molecular weight of 9534.25 Da. A charge state of relatively high abundance was arbitrarily selected, isolated, and subjected to ion trap CID. The CID spectrum derived from the +12 charge state of this unknown protein is shown in Figure 2(b) by use of a dipolar excitation frequency of 164.6 kHz with a voltage amplitude of 470 mVpp for 200 ms.</p><p>The resolving power of the TOF analyzer (∼8000 M/ΔMFWHM) allows confident charge state assignment of most of the ions in the low m/z region of the CID spectrum by measuring the spacing between the isotope peaks of a product ion. However, charge state assignment in the high m/z region was complicated by the overlap of peaks, as evident by the clustering of products around the precursor ion shown in Figure 2(b). Therefore, CID products shown in Figure 2(b) were subjected to ion/ion proton transfer reactions to simplify the CID spectrum. The post-ion/ion CID spectrum is shown in Figure 3, in which the majority of the product ions are reduced to singly and doubly charged ions, as indicated by the predominant +2 and +1 residual precursor ions in the spectrum. All the product ions with m/z values falling above the residual doubly charged precursor ion must be singly charged ions; therefore, the doubly charged version of these product ions can be readily identified on the basis of their m/z values and abundances relative to the residual doubly charged precursor ions, because the intensity ratio of such a doubly charged product ion relative to the residual doubly charged precursor ion is close to the intensity ratio of the singly charged version of that product over the residual singly charged precursor ion. However, no attempt was made to identify the doubly charged ions in the post-ion/ion spectrum. Rather, all the peaks in the post-ion/ion spectrum were subjected to a peak picking process using Origin 6.0 and the masses derived therefrom were based on the assumption that all ions were singly protonated. The selected peaks are labeled with red crosses in Figure 3. The automatically selected product ions with m/z less than that of the singly charged precursor ion were subjected to database searches for protein identification. While it is possible to correct for the doubly charged ions, we chose to evaluate how well the overall approach works when there is essentially no post-processing of the raw product ion spectrum. A similar procedure as described above was applied to other E. coli LC fractions of interest to provide protein intact mass and CID product ion mass information for ProSightPTM database search. In cases in which the protein mixture was significantly more complex than that of Figure 1(a), the protein ion mixture was first subjected to ion/ion proton transfer reactions to determine the masses of the protein components in the mixture. This information was used to determine which peaks to select from the pre-ion/ion protein mixture spectrum for tandem mass spectrometry.</p><!><p>In a typical "top-down" database search, candidate proteins selected from a database are fragmented in silico to generate a series of theoretical product ions, which are compared to the experimental product ion masses to determine the number of matched ions, from which a search score is assigned.14 Either the monoisotopic mass or the average mass, but not a combination of both, is normally used in such a mass comparison process in most search algorithms, including ProSightPTM. However, the product ion mass selected by the Origin program from the post-ion/ion spectrum collected using the current approach is a mixture of monoisotopic and average masses, i.e. monoisotopic masses for the low mass resolvable ions and average masses for high mass ions whose isotopes are unresolved, because the Origin program tends to select peaks with the highest abundance within a specified search rectangle. To accommodate this feature of the product ion mass data, experimental product ion masses selected by Origin program were searched in ProSighPTM as average masses with a relatively large product ion mass tolerance of 1.5 Da. An intact protein ion mass window of 2000 Da was also used in the search to accommodate a large range of possible PTMs on the protein.</p><!><p>The ProSightPTM database search of the major unknown protein component in the soluble whole cell E. coli lysate HPLC fraction #1 resulted in 1412 candidate proteins within the 2000 Da mass window of the unknown protein. Of this group, 1406 had at least one b-/y-type product ion matching one of the 116 experimental product ions selected by the Origin program from the post-ion/ion spectrum in Figure 3. All candidate proteins were ranked by probability score14 (viz., lowest scores are most highly ranked) and the three top ranked proteins are listed in Table 1, in which the highest ranked protein is DBHA_ECOLIm with a MW of 9535.0 Da. This top ranked protein has a total match of 44 ions (19 b-ions and 25 y-ions) with a probability score of 1.17×10-19. The second highest ranked protein is DBHA_ECOLI, which has the same sequence as the top ranked protein except for the absence of the N-terminal methionine residue. The DBHA_ECOLI entry has a total match of 26 ions with a probability score of 3.49×10-6 and the third highest ranked protein matched only 17 ions with an assigned probability score of 0.266. With a good match to the measured protein mass (mass difference of 0.78 Da) and such a small value for the probability score, it is very unlikely that the top ranked DBHA_ECOLIm protein is a random match. The peaks in the post-ion/ion product ion spectrum shown in Figure 3 were assigned according to the sequence of DBHA_ECOLIm with a summary of the fragmentation shown in the upper panel, which represents cleavage of 38.2% of the amide bonds. It is noteworthy that matches of b-type ions from the candidate protein DBHA_ECOLI are unexpected because the absence of an N-terminal methionine residue relative to the top-ranked protein DBHA_ECOLIm shifts all its b-ion masses by a constant value equal to the mass of the methionine residue. Therefore, the match of a b-type ion for DBHA_ECOLI most likely comes from a random match of a noise peak. A close examination of this match reveals that the matched b4 ion with neutral mass of 471.500 Da is 0.708 Da smaller than the observed mass, which corresponds to a 1499.3 ppm mass difference, which is much larger than the routine mass accuracy of ∼ 30 ppm obtained with this TOF using external calibration. This result indicates that the potential performance of the current platform was not fully exploited by the employment of a relatively large product ion mass tolerance of 1.5 Da to accommodate the limitations associated with the ProSightPTM search with only monoisotopic or average masses. It can be envisioned that if the monoisotopic mass information available for the low mass ions can be exploited in a mixed search with both monoisotopic and average product ion masses, random matches would significantly decrease, which can improve the specificity of the current approach.</p><p>The previous case illustrated the situation in which the N-terminal initiating methionine residue is retained. An example of the opposite situation is briefly described below. The procedure just described was performed with the major protein component in the LC fraction No. 2, which has a measured MW of 15407.9 Da. The post-ion/ion CID spectrum of this unknown protein is shown in Figure 4 with peaks selected by the Origin program for database search indicated with red crosses. The ProSightPTM database search resulted in a top ranked protein, HNS_ECOLI, which is 0.59 Da higher than the experimentally measured mass. This protein matched a total of 61 product ions in the spectrum with 32 b- ions and 29 y- ions, yielding a probability score of 1.29×10-28, while the proteins ranked second and third showed much fewer matches relative to the top ranked protein with probability scores of 1.09×10-7 and 0.941, respectively. The second ranked protein, which shows a moderately good score, is related to the top ranked protein in that it has the same sequence with the lone exception that it contains the N-terminal methionine residue. Based on the identification made here, the product ions were assigned and the fragmentation pattern of this protein is summarized in the upper panel of Figure 4. For the selected charge state, evidence for cleavage of roughly 33.3% of the amide bonds were cleaved via ion trap CID.</p><!><p>One of the major causes for errors in protein identification arises from incompleteness of a database. That is, they do not contain all of the possible mature gene products, including those with PTMs, mutations, etc., that may be present in the sample. However, it is often possible to characterize a protein even when it is not in the database and this is greatly facilitated if a closely related protein happens to be in the database. Several examples of situations of this type are presented that illustrate the capabilities of this approach.</p><p>The first example involves the database search of the major unknown protein component in the E. coli LC fraction #3, the post ion/ion CID spectrum of which is shown in Figure 5. Compared to the post-ion/ion CID spectrum of the protein of similar intact mass (Figure 3), this protein, with a measured intact mass of 9738.49 Da, shows significantly fewer product ions upon collisional activation. The ProSightPTM database search of the post-ion/ion CID spectrum results in a best candidate protein HDEA_ECOLIc with a probability score of 0.0111, which is slightly better than the commonly accepted score of 0.05 for positive identification at a confidence level of 95%. The proteins ranked second and third, i.e. HDEA_ECOLIcm and MINE_ECOLI, respectively, have a probability score larger than 0.05 and are most likely random matches. Although the probability score of 0.0111 for the top-ranked protein is below the commonly used significance level, the mass difference of 2.44 Da (∼ 250 ppm) between the measured mass and the predicted mass from HDEA_ECOLIcm is much larger than the mass accuracy of the current platform. Therefore, while the protein under investigation may be related to the top-ranked protein in the search, it is clearly not the entity in the database. A close examination of the post-ion/ion spectrum reveals that the top-ranked protein matches only the peaks in the low mass region of the spectrum, which are labeled in blue in Figure 5, with the exception of one peak of low abundance in the high mass region matching the b87+ ion of HDEA_ECOLIc. With matches of only low mass product ions, the discrepancy between the spectrum and the best candidate protein suggests a possible PTM in the middle of the protein. The most common protein PTM giving a mass deficiency of 2.44 Da relative to the unmodified version is the formation of a disulfide linkage within the protein molecule, which can occur in HDEA_ECOLIc between Cysteine 18 and Cysteine 66. When the formation of a disulfide bond between these two cysteine residues is considered, most of the abundant peaks in the high mass region can be successfully identified and are labeled in green in Figure 5. With the additional matches, a much better probability score of 1.85×10-5 was obtained. Except for the three charge reduced precursor ions, the three most abundant peaks in the high mass region of the post-ion/ion spectrum correspond to the singly, doubly and triply charged fragment ions of y81, which is the complementary ion of b8+, the most abundant product ion in the spectrum. The b8/y81 complementary pair arises from cleavage C-terminal to an aspartic acid residue, a favorable channel commonly observed in collisional activation of protein cations of relatively low charge states. The existence of a disulfide bond is also consistent with the fact that no cleavage was observed in the region between Cysteine 18 and Cysteine 66, which is a common phenomenon for proteins with disulfide linkages43. Moreover, the formation of a disulfide bond in the protein is also supported by the ESI spectrum of the protein (data not shown) that gave a charge distribution of only three low charge states of +9, +8, and +7, a result that may very well reflect a relatively compact conformation of the protein due to the existence of a disulfide linkage in the molecule. Collectively, this evidence suggests that a disulfide-linked version of HDEA_ECOLIc has been identified in the E. coli sample.</p><p>Another case in which the observed protein was not present in the database is illustrated by the major protein component in the LC fraction #4, which has a measured MW of 28462.14 Da. The post-ion/ion CID spectrum of its most abundant charge state, [M+31H]31+, is shown in Figure 6. A ProSightPTM database search of this spectrum gave a top-ranked protein of RBSB_ECOLIc with a probability score of 7.49×10-12. The protein ranked second, RBSB_ECOLIcm, shares the same sequence with the top-ranked protein except for the absence of the N-terminal methionine residue and was assigned a probability score of 1.77×10-4. The protein THIM_ECOLI was ranked third with a probability score of 1.7, which is most likely a random match to the spectrum.</p><p>The top-ranked protein, RBSB_ECOLIcm, gave 34 matches (13 b-type ions and 23 y-type ions) to the total 123 peaks selected by Origin program from the post-ion/ion spectrum shown in Figure 6. However, 31 out of the 34 ions matched only the low mass ion peaks in the spectrum, which are labeled in blue in the spectrum. The three matches to high mass ions (b262+, y214+, y191+) were peaks of very low abundances, which are not labeled in the spectrum. Although the top-ranked protein has an excellent probability score, it has an intact mass 12.37 Da (∼435 ppm) higher than the experimentally measured mass. This large mass discrepancy between the measured mass and the predicted mass indicates that RBSB_ECOLIc is not a correct assignment, although it is likely to be related to the unknown protein. The dominant matches of the low mass b- and y-type ions of RBSB_ECOLIc strongly suggest that the protein in the sample shares much of the same sequence with RBSB_ECOLIc in both N- and C- terminals but not in the middle region, where the 12.37 Da mass discrepancy originates. Such a difference in mass cannot be accounted for by the presence of multiple di-sulfide linkages because there are no cysteine residues in the sequence.</p><p>One way to account for such a mass discrepancy could come from a substitution of some amino acid residues in the middle region of the RBSB_ECOLIc sequence. To test this hypothesis, a sequence similarity search of protein RBSB_ECOLIc was performed using protein BLAST44 to identify any RBSB_ECOLIc protein isoforms containing amino acid changes giving such a mass deficit. The results showed that RBSB_ECOLIc shares the same sequence with 19 protein isoforms with removed signal peptides except for residue isoleucine 167, which is threonine 167 in these isoforms. The mass difference (12.037 Da) between threonine and isoleucine can account for the 12.37 Da mass difference observed between the measured mass and the mass for RBSB_ECOLIc. The 19 protein isoforms include a D-ribose periplasmic binding protein [rbsB] from E. coli O157:H7, i.e. Q8XAW6_ECO57, as well as another 18 proteins from E. coli strains other than K12. When the post ion/ion spectrum shown in Figure 6 was interpreted based on the sequence of Q8XAW6_ECO57 without the signal peptide sequence, the majority of the abundant peaks in Figure 6 can be identified that gave a total of 36 b-ions and 30 y-ions, with the new identifications being labeled in green. Twenty-one complementary b-/y-pairs were identified from the 36 b-ions and 30 y-ions (top panel in Figure 6). As a result, an excellent probability score of 2.8×10-31 was obtained when the 66 b-/y-ion identifications were considered. It is noteworthy that the identified peaks labeled in red in Figure 6 are those missed in the ProSightPTM identification because the mass determined by Origin is beyond the mass tolerance window of 1.5 Da due to the irregularity of the peak shape. From the above discussion, it is clear that the protein in the sample is not included in the E. coli K12 database used by ProSightPTM; however, with the assistance of a BLAST sequence similarity search, the high informing power and high mass accuracy of the current platform allows confident identification of the correct protein which has lost its signal peptide and also has a substituted amino acid residue relative to the top ranked protein RBSB_ECOLIc identified directly from the ProSightPTM database search.</p><!><p>With a simple LC separation of an E. coli lysate soluble protein mixture, unknown protein identification with high confidence was demonstrated on a quadrupole/TOF platform with ion/ion reaction capabilities. The high informing power of the current platform allows sequence information to be derived from entire proteins with molecular weights up to at least 28 kDa in a tandem mass spectrometry experiment by removing the product ion charge state ambiguity and reducing the peak overlap via ion/ion reactions. The specificity provided by the large amount of sequence information is high enough to allow confident identification of unknown proteins even with a relatively large product ion mass tolerance that was made necessary by the current version of the ProSightPTM search engine in the handling of mixed data of both monoisotopic masses and average mass. It can be envisioned that when the high mass accuracy associated with the monoisotopic mass in the low mass region can be exploited in the database search, an even higher specificity can be achieved from the current approach. Although the use of the Origin program for peak picking can compromise the overall performance of the approach, as just mentioned, it is desirable to have an automated means for picking the peaks for subsequent database searching. The high mass accuracy achieved from the current approach with large intact proteins allows facile determination of the existence of an unknown protein with PTMs or a protein absent from the searched database. When this accurate intact mass information is linked with protein sequence information, it is possible to identify and localize protein PTMs, such as disulfide linkages, as well as to identify proteins whose primary structures are slightly different from those of proteins in the database.</p><p>This work represents a significant improvement in top-down protein characterization using a quadrupole/TOF platform. Further improvements can be readily envisioned by adapting the search algorithm to accept both monoisotopic and average masses. Furthermore, several other means for ion manipulation may improve the specificity and overall utility of the approach. These include the use of beam-type collisional activation to access higher dissociation rate processes45, the use of electron transfer dissociation to provide complementary structural information46, 47, the use of ion parking techniques for parent ion charge state concentration and purification31, 38, 48, and the use of de-convolution algorithms to convert product spectra to zero-charge spectra. All of these measures are currently under development and evaluation for unknown protein characterization on the quadrupole/TOF platform and promise to improve further the capabilities of this type of instrument for top-down protein analysis. In addition to improvements in specificity, key areas for improvement in whole protein mixture analysis in general include matrix effects upon ionization and dynamic range. Future work will also be directed to addressing these issues.</p><!><p>RP-HPLC fractionation of the soluble proteins from the supernatant of the whole cell E. coli lysate. The labeled peaks correspond to the fractions containing unknown proteins discussed in this study.</p><p>Spectra derived from (a) ESI of the LC fraction labeled as #1 in Figure 1, and (b) ion trap CID of protein ions with m/z 795.5 shown in (a) (frequency: 164.6 kHz, amplitude: 470 mV ).</p><p>Post-ion/ion reaction MS/MS spectrum derived from the [M+12H]12+ ion, m/z 795.5, of the unknown protein in fraction #1 followed by ion/ion reactions with the PFO dimer anion. (Peaks are labeled based on the matches of the protein DBHA_ECOLIm identified from ProSightPTM database search.)</p><p>Post-ion/ion MS/MS spectrum derived from ion trap CID of the [M+15H]15+ ion of the unknown protein in fraction #2 followed by ion/ion reactions with the anions of PFO dimer. (Peaks are labeled based on the matches of the protein HNS_ECOLI identified from ProSightPTM database search.)</p><p>Post-ion/ion MS/MS spectrum derived from ion trap CID of the [M+8H]8+ ion of an unknown protein of mass 9738.49 Da in fraction #3 followed by ion/ion reactions with the anions of PFO dimer. A zoomed region is shown in the inset. (Peaks are labeled based on the sequence of the protein HDEA_ECOLIc. Product ions labeled in blue are identified from ProSightPTM database search, while ions labeled in green are identified when disulfide linkage is considered.)</p><p>Post-ion/ion MS/MS spectrum derived from ion trap CID of the [M+31H]31+ (m/z = 919.13) ion of an unknown protein in fraction #4 followed by ion/ion reactions with the anions of PFO dimer. The bottom panel shows the zoomed mass region of m/z 19000 – 23000. (Peaks are labeled based on the sequence of the protein RBSB_ECOLIc with a substitute of isoleucine 167 by threonine, which is indicated by the red box in the top panel showing the fragmentation pattern. Product ions labeled in blue are identified directly from ProSightPTM database search, while ions labeled in green are identified when the amino acid substitute is considered. The peaks labled in red are those missed by the algorithm.)</p><p>Summary of the ProSightPTM Database Search Results for Proteins from the Soluble Whole Cell Lysate HPLC Fraction of E. coli. (Candidate proteins are ranked based on the probability scores)</p>

PubMed Author Manuscript

Mechanism-Based Design of Quinoline Potassium Acyltrifluoroborates (KATs) for Rapid Amide-Forming Ligations at Physiological pH

Potassium acyltrifluoroborates (KATs) undergo chemoselective amide-forming ligations with hydroxylamines. Under aqueous, acidic conditions these ligations can proceed rapidly, with rate constants of ~20 M -1 s -1 . The requirement for lower pH to obtain the fastest rates, however, limits their use with certain biomolecules and precludes in vivo applications. By mechanistic investigations into the KAT ligation, including kinetic studies, X-ray crystallography, and DFT calculations, we have identified a key role for a proton in accelerating the ligation. We applied this knowledge to the design and synthesis of 8-quinolyl acyltrifluoroborates, a new class of KATs that ligates with hydroxylamines at pH 7.4 with rate constants >4 M -1 s -1 . We trace the enhanced rate at physiological pH to unexpectedly high basicity of the 8-quinoline-KATs, which leads to their protonation even under neutral conditions. This proton assists the formation of the key tetrahedral intermediate and activates the leaving groups on the hydroxylamine towards a concerted 1,2-BF3 shift that leads to the amide product. We demonstrate that the fast ligations at pH 7.4 can be carried out with a protein substrate at micromolar concentrations.

mechanism-based_design_of_quinoline_potassium_acyltrifluoroborates_(kats)_for_rapid_amide-forming_li

Introduction<!>Results and Discussion<!>Kinetics of KAT ligations.<!>Mechanism of KAT ligations.<!>TS-c<!>Conclusion

<p>Potassium acyltrifluoroborates (KATs) are robust, bench stable compounds that undergo amideforming ligations with O-substituted hydroxylamines under dilute, aqueous conditions. [1][2][3] KAT ligation has already found applications in protein PEGylation, hydrogel formation and modification, and post-polymerization modification. [4][5][6][7] The reaction is exceptionally fast at lower pH, with rate constants >20 M -1 s -1 at pH 2, but becomes slower as the pH is increased. 8 At pH 7, amide formation can still proceed but at rates that are not suitable for reactions at micromolar concentrations. We have previously employed 2-pyridine-derived KATs as they were superior to aryl derivatives at pH 3-6, 4 with rate constants of ~10 M -1 s -1 at pH 3.6. These improved substrates, however, showed only modest reaction rates at physiological pH, limiting their use for many desirable applications of chemoselective ligations. 9,10 In this report we document the synthesis and rapid amide-forming ligations of 8-quinoline-derived KATs, which undergo KAT ligation at pH 7.4 with second order rate constants up to 4 M -1 s -1more than 300 times faster than aryl derivatives. This report also provides detailed rate constants of various KAT derivatives at both pH 7.4 and pH 3.8. DFT calculations on the protonated and unprotonated reaction pathways provide insights into the mechanistic basis for the role of a proton in accelerating the KAT ligation. Characterization of the pyridyl-and quinolyl KATs -including titrations and X-ray crystallography -confirm the unexpectedly high basicity of the quinolyl KATs, which translates to superior reaction rates at higher pH.</p><!><p>Synthesis of KAT substrates. While initially challenging to produce, synthetic access to KATs has expanded markedly in the past few years. 2,[11][12][13][14][15][16][17][18][19] These advances have allowed us to prepare a broad range of KATs containing a diverse range of functional groups and structural features.</p><p>Based on our previous finding of superior ligation rates with pyridyl KATs, which we attributed to protonation of the pyridine nitrogen under acidic conditions, we aimed to synthesize a series of pyridyl-and quinolyl KATs that contain a basic atom near the reactive KAT group. Exploration of a number of hypotheses led to the initial finding that 8-quinolyl KATs performed better at higher pH, and we elected to systematically study this class of compounds further -along with the aryl and pyridyl KATs -with regard to the role of electron-donating (-OMe) and withdrawing (-Cl) groups incorporated onto the aromatic rings. 20,21 Quinolines are typically less basic than pyridines (pKa = 4.9 for quinoline, pKa = 5.2 for pyridine), 22 but place the basic nitrogen three, rather than two atoms away from the acylboronate carbonyl. 23 Phenyl-and pyridyl KATs were synthesized from their corresponding halides using one equivalent of n-BuLi and reagent 1 each at -78 °C in THF according to previous reports. 11 Applying these conditions to 8-quinolyl halides, however, failed to yield any product and further optimizations were needed. Screening of reaction conditions showed that by conducting the lithium-halogen exchange at -110 °C in a solvent mixture of diethyl ether and THF (4:1 v/v) and allowing the reaction mixture to stir for one hour prior to the addition of reagent 1, followed by quenching at this temperature with aqueous KF and overnight mixing to form a precipitate provided access to the desired compounds (Scheme 2). Scheme 2. All reactions were performed in an oven-dried Schlenk flask. n-BuLi was added dropwise over 30 min via syringe pump at -78 °C for phenyl-and pyridyl KATs and at -110 °C for quinolyl KATs; the reactions were quenched with 3.0 equiv 6.5 M KF(aq) solution and the cooling bath was removed, allowing the reaction mixture to warm to rt. Despite these improvements, the reaction between the 8-lithioquinolines and reagent 1 was sluggish and remaining reagent 1 formed a hydrolyzed side-product 2 that must be separated from the desired KAT. This could be accomplished by washing the crude mixtures with acetone to extract 2, as 8-quinolyl KATs, unlike most phenyl-and pyridyl KATs, do not dissolve in acetone.</p><p>The desired KAT can be isolated from the remaining crude mixture using DMF, leaving behind the residual inorganic salts.</p><!><p>With KAT substrates 3-5 in hand, we carefully evaluated the rates of their ligation with hydroxylamine 6. For the determination of rate constants for KAT ligation, we selected UV-vis spectroscopy, as KATs have unique absorption bands that can be used for accurate determination of their concentration. We established calibration curves in buffered solutions at various pH values used for kinetic studies over the concentration range of 0 -15 mM (Figure 1b). Quinolyl KAT 5c failed to dissolve above 0.25 mM in the buffers used for UV-vis measurements, therefore KAT 5d was synthesized and used instead. We selected 1:1 (v/v) mixtures of acetonitrile with aqueous buffer solutions for the measurement of the ligation kinetics to ensure solubility of all reagents. KAT ligations work well in purely aqueous conditions, but these substrates required an organic co-solvent to ensure homogeneity throughout the course of the reaction at the various pH ranges and concentrations studied. To improve solubility of the starting materials and products, we designed and prepared water-soluble hydroxylamine 6. Potassium acetate buffer was employed for ligations at pH 3.8, and potassium phosphate buffer was used for experiments at pH 7.4. The ligation reaction was initiated by mixing pre-buffered stock solutions of the KATs (15 mM) with hydroxylamine 6 (15 mM) to give an initial concentration of 7.5 mM in each reactant. UV-vis spectra were recorded over a period of 1000 s to monitor the consumption of KATs.</p><p>A representative set of UV absorption profiles over time is illustrated in Fig. 1c and shows the decay of the KAT specific band as the ligation progress. Approximately 100 time points were recorded over the 1000 seconds of measurement. The concentration of the KATs was determined by Beer's law using the determined molar absorption coefficients. Initial studies determined that the reaction is single order with respect to each reactant and the reaction was treated as an overall second order reaction with equimolar amounts of KAT and hydroxylamine. Linear regression of the reciprocal concentration values against time was used to determine the apparent second order rate constants (Figure 1d). With the uncertainty in absorbance measurement being as low as 0.001 a.u. in terms of standard deviation from blank measurements, the number of time points enabled the reliable determination of the background absorption as well as a reasonable fit to the assumed second order kinetics. Only the later portions of the data deviated slightly from ideal second order behavior. All measurements were repeated three times (marked in color) to calculate the averages and standard deviations of the rate constants. increase in rate with a methoxy substituent, and an 8.3-fold increase with a chloro substituent.</p><p>The same trend also held true within the quinolyl KAT 5 series. Electron-donating groups enhance the basicity of the nitrogen, which should lead to a higher proportion of the protonated forms. 20 This would account for the fact that methoxy-substituted pyridyl KAT 4c and quinolyl KAT 5d were the most reactive ones in their respective aryl subgroups at pH 7.4, but the slowest at pH 3.8.</p><p>At pH 3.8, all of the KATs examined participated in the KAT ligation with second order rate constants higher than 1 M -1 s -1 , but the alkoxy-substituted pyridine 4c and quinoline 5d were slower than their analogs. This was consistent with the expectation that all pyridyl-and quinolyl KATs studied would be protonated at pH 3.8 and inductive substituent effects that modulate the electrophilicity of the KAT dominated. Overall, the most reactive KAT at pH 3.8 was chloropyridyl KAT 4b. This confirms that, as expected, more electron deficient KATs are more reactive overall, but that the presence of a proton near the KAT groups greatly accelerates the reaction and becomes the determining factor at higher pH regimes. This UV-vis method was able to determine the reaction rate constants of KATs with hydroxylamine 6 by monitoring the consumption of KATs, rather than the formation of the ligation product amide.</p><p>To verify that the rate of product formation correlated with the reactivity of the KATs, and not just a difference in the formation of an initial adduct, a competition experiment was designed. KATs 4a, 4b, and 4c with relative reactivities of 3.34, 8.33 and 16.7 to the reference at pH 7.4, were selected for pairwise competition with a limited amount of hydroxylamine 7 and the final product distribution was measured by 1 H-NMR. The parallel competition outcomes were compared to modeled results based on the initial concentration of reactants and rate constants. The agreement of observed and predicted results are depicted in Figure 3 (see SI for model and parameters).</p><!><p>Our basic understanding of the mechanism of KAT ligation featured a nucleophilic addition of the hydroxylamine to the KAT carbonyl, forming a tetrahedral intermediate, followed by its breakdown (Scheme 3). At the outset of our studies, however, we had little insight into the specific bond-forming and bond-breaking processes or the role of protonation in accelerating the KAT ligation. leading to a C to N 1,2-BF3 migration. 26 1,2-migration of the tetracoordinated boron group to carbon, nitrogen, and oxygen atoms has been shown for B-MIDA (N-methyliminodiacetic acid)</p><p>acylboronates. 27 These migrations proceeded via a tetrahedral intermediate, where a leaving group at the β-position of the boronate initiated the migration (Scheme 5). The stereochemistry between the leaving group and the boronate group has also been found to effect the migratory aptitude of the boronate group. 27 The computed reaction pathways shown in Scheme 4 led to the formation of an N-BF3 amide as the primary ligation product, which was also observed in real time mass spectrometry analysis of the reaction mixture (see Supporting Information Fig. 22).</p><!><p>BF3 -Scheme 4. Computed KAT ligation reaction of KATs 4a and 5a. Reaction paths with the pyridine or quinoline being protonated were colored in brown. The vertical axes between 0 and -80 kcal/mol were truncated to accommodate the free energy of the reaction products. Sums of free energies of the hydroxylamine and (protonated) KATs were set to zero as references. Computations were performed at the def2/ma-TZVP level. 24,25 Scheme 5. Instances of 1,2-migration of boronate groups in B-MIDA acylboronates reported by Yudin. 27 [B] = B-N-Methyliminodiacetate.</p><p>In the computed ligation pathway of quinolyl KAT 5a, quinoline protonation also favored the formation of a hydrogen bond-tethered intermediate I-c. The quinolinium proton was in vicinity to three basic atoms: the quinoline nitrogen, the hemiaminal oxygen, and the carbamoyl oxygen.</p><p>Interaction between the proton and these atoms fixed the F3B-C-N-O dihedral angle (marked in orange), across the C-N bond between the KAT carbonyl and hydroxylamine nitrogen, close to an antiperiplanar 180°. This conformation would facilitate the concerted C-B bond / N-O bond cleavage towards the transition state. The activation barrier from intermediate I-c, formed from protonated KAT 5a, to TS-c, was found to be slightly higher than that of from I-b to TS-b. This corresponded to the slightly higher observed rate of 4a ligating at pH 3.8, where both KATs are mostly protonated.</p><p>Nitrogen containing heteroaryl aldehydes are known to participate in reactions of neighboring groups. [28][29][30] For example, pyridine-2-carbaldehyde and quinoline-8-carbaldehyde form hydrazones faster than their corresponding hydrocarbon variants, benzaldehydes and naphthaldehydes. Kool et al. proposed that an intramolecular proton donor could facilitate both</p><p>the formation and breakdown of the tetrahedral intermediate. 28 Our findings suggest that a similar mode of rate acceleration was operative in KAT ligations, especially for quinolyl KATs 5 -with some qualifications.</p><p>Quinolyl KATs had faster ligation rates than pyridyl KATs at neutral pH. This suggestedcounterintuitively -that quinolyl KATs were protonated to a greater extent than their pyridine counterparts, as pyridine (pKa = 5.2) is known to be more basic than quinoline (pKa = 4.9). To examine further, we performed titrations of the KATs with hydrochloric acid in aqueous acetonitrile to measure their basicities, and determined that the conjugate acid of quinolyl KAT 5a has a pKa of 5.96, while the conjugate acid of pyridyl KAT 4a has a lower pKa of 4.92.</p><p>The inversion of the inherent basicity of pyridyl-and quinolyl KATs implies that the proximal KAT group facilitates the protonation of quinoline KATs, possibly due to the formation of an internal salt resulting in a zwitterionic species that does not require the potassium gegenion. To investigate this further, we grew crystals of various quinolyl-and pyridyl KATs from either pH 7.4</p><p>buffers or dilute HCl solutions and determined their structures by X-ray diffraction. In the case of quinolyl KAT 5a, crystals grown from either dilute HCl or from pH 7.4 buffer both delivered the protonated, zwitterionic form (Figure 4a). Notably, electronic rich quinolyl KAT 5d also crystalized as the protonated form, while the chloro derivative 5b afforded unprotonated potassium salts from the same buffer. In contrast, all pyridine KAT crystals were obtained as their potassium salts. The carbonyl group for the zwitterionic species 5a is almost coplanar to the aromatic ring while KAT 5b shows the highest torsional angle between the carbonyl group and its aryl ring (Figure 4b). The computational investigations indicated the importance of carbamoyl protonation during the N-O bond cleavage during KAT ligation. This is consistent with the observation that all KATs ligate faster at lower pH and follow specific acid catalysis. 31 The phenyl KATs 3a-c showed rate constants of ~3 M -1 s -1 at pH 3.8. For KATs 4 and 5, protonation is likely to both increase their electrophilicity and accelerate the N-O bond cleavage by protonation of the carbamate, increasing the ligation rates to around 10 M -1 s -1 at the same pH. The more basic quinoline KATs 5a-d will have higher protonated fractions at neutral pH than pyridyl KATs 4, and are more likely KAT Ligations on Folded Proteins at pH 7.4. At the outset of this project, we sought to identify KAT ligation partners that formed amide bonds under physiological conditions with rate constants of at least 1 M -1 s -1 , which would make it suitable for bioconjugations at micromolar concentrations and potential in vivo applications. Although detailed applications of the quinolyl KATs to these goals are beyond the scope of this manuscript, we have confirmed the benefit of higher ligation rates of quinolyl KATs for protein modification.</p><p>According to our previously reported protocol, a sfGFP(S147C) mutant was expressed from chemically competent Escherichia coli BL21(DE3) cells in high yields and treated with the thiophilic hydroxylamine derivative. 4 A 100 μM solution of the sfGFP(S147C)-hydroxylamine adduct 8 was treated with quinolyl KAT 5d (5.0 equiv) at pH 7.4 in the same buffer system used for the kinetic measurements and was fully converted to the amide product 9 within 90 min. In contrast, the same experiment with pyridyl KAT 4a returned only unreacted protein after 90 min at pH 7.4 (Figure 5). The reaction mixture was passed through a size-exclusion column to elute the protein in MilliQ water prior to mass analysis (ESI-MS).</p><!><p>In summary, we have identified 8-quinolyl KATs as uniquely suited for rapid amide-forming ligations at pH 7.4. They exhibit second order rate constants of 1-5 M -1 s -1 at physiological pH, hundreds of times faster than previously examined KATs. The origin of the enhanced reactivity was traced -through kinetic studies, titrations, X-ray crystallography, and DFT calculations -to an unexpectedly high basicity of the quinolyl KATs and a key role for the proton in the mechanism of the KAT ligation. These findings were translated to ligation of a model protein at micromolar concentration in pH 7.4 buffer and pave the way for applications of KAT ligation on systems that require rapid ligations at physiological pH.</p>

ChemRxiv

In silico identification and docking-based drug repurposing against the main protease of SARS-CoV-2, causative agent of COVID-19

The rapidly enlarging COVID-19 pandemic caused by novel SARS-coronavirus 2 is a global public health emergency of unprecedented level. Therefore the need of a drug or vaccine that counter SARS-CoV-2 is an utmost requirement at this time. Upon infection the ssRNA genome of SARS-CoV-2 is translated into large polyprotein which further processed into different nonstructural proteins to form viral replication complex by virtue of virus specific proteases: main protease (3-CL protease) and papain protease. This indispensable function of main protease in virus replication makes this enzyme a promising target for the development of inhibitors and potential treatment therapy for novel coronavirus infection. The recently concluded α-ketoamide ligand bound X-ray crystal structure of SARS-CoV-2 M pro (PDB ID: 6Y2F) from Zhang et al.has revealed the potential inhibitor binding mechanism and the determinants responsible for involved molecular interactions. Here, we have carried out a virtual screening and molecular docking study of FDA approved drugs primarily targeted for other viral infections, to investigate their binding affinity in M pro active site. Virtual screening has identified a number of antiviral drugs, top ten of which on the basis of their bending energy score are further examined through molecular docking with M pro . Docking studies revealed that drug Lopinavir-Ritonavir, Tipranavir and Raltegravir among others binds in the active site of the protease with similar or higher affinity than the crystal bound inhibitor α-ketoamide. However, the in-vitro efficacies of the drug molecules tested in this study, further needs to be corroborated by carrying out biochemical and structural investigation. Moreover, this study advances the potential use of existing drugs to be investigated and used to contain the rapidly expanding SARS-CoV-2 infection.

in_silico_identification_and_docking-based_drug_repurposing_against_the_main_protease_of_sars-cov-2,

Introduction<!>Phylogenetic analysis of SARS-CoV-2 genome<!>Molecular docking<!>Genome sequence alignment and phylogenetic analysisof SAR-CoV-2<!>Inhibitor binding cleft of M pro<!>Docking analysis<!>Conclusion

<p>The ongoing and rapidly spreading outbreak of the Covid-19 pandemic, which is being caused by the highly contagious and pathogenic SARS-Coronavirus-2 (SARS-CoV-2), has endangered the global public health and alarmed the scientific community at an unprecedented magnitude.</p><p>The novel SARS-CoV-2 was first reported to have emerged in the live wildlife market in the Wuhan region of Hubei province, where it has caused mystic pneumonia-like respiratory illnesses in the human population of the area (1,2). Later, the virus has made its way under extensive and fast-paced human travel to other geographic locations in the world such as Japan, Australia, Southeast Asian countries, Western European countries, Middle Eastern Countries, and finally to USA, Canada, and India. According to data presented by the World Health Organization (WHO), as of March 23, 2020, the virus has infected more than 693,224 people in more than 190 countries around the world including a staggering 33,106 deaths, with a cumulative mortality rate of >4.7% (3). Despite the instantaneous, collaborative, and monumental research efforts from the scientific community around the globe, no vaccine or therapeutic intervention could be developed to cure or mitigate the Covid-19 so far. The treatment of severely ill patients has been limited to the use of prophylactic and symptomatic management. In contrast, the prevention of disease has been only effective through strong and preemptive measures from health organizations and governmental authorities such as the sought outs for practicing social distancing, maintaining respiratory hygiene, and impositions of public curfews and state-wide lockdowns.</p><p>Coronaviruses (CoVs), which belong to family Coronaviridae of viruses, constitute an essential class of pathogens for humans and other vertebrates (4). Before the current SARS-CoV-2 induced pathogenesis, only six of the CoVs were known to cause mild to severe illnesses in humans. HCoV-229E, HCoV-OC43, HCoV-NL63, and HKU1, which fall in genera alphacoronavirus, cause milder upper respiratory disease in adults, and sometimes can also cause severe infection in infants and young children. Whereas the betacoronaviruses like SARS-CoV (severe acute respiratory syndrome coronavirus; which has triggered an epidemic in China during 2002-03) and MERS-CoV (Middle East respiratory syndrome coronavirus; an etiological agent of middle East coronavirus epidemic of 2012) have potential to cause infection in lower respiratory tract along with cough & fever and triggers severe respiratory illness in humans (5,6).</p><p>The causative agent of the current outbreak, SARS-CoV-2, also belongs to betacoronavirusesand is closely related to SARS-CoV with an overall genomic sequence similarity of >79% (7).</p><p>The virion of SARS-CoV-2 is consists of crown-shaped peplomers, 80-160 nm in diameter, and consists of a ~30 kb long single-stranded RNA molecule of positive polarity with 5' cap and 3' Poly-A tail (8). The RNA genome is composed of at least six open reading frames (ORFs) of which the first ORF (ORF1a/b) makes up the 5'two-third and encodes two polypeptides pp1a and pp1ab both of which furthermore leads to the production of 16 nonstructural proteins (nsPs). Other ORFs that make up the remaining one-third of the viral genome give rise to the production of four main structural factors of the virion: Spike protein (S), Envelope protein (E), Membrane protein (M) and Nucleocapsid protein (N) (9).</p><p>The virus uses the heterotrimeric Spike (S) protein, which consists of S1 and S2 subunit, on its surface to interacts with the ACE2 (angiotensin-converting enzyme 2) cellular receptor, abundantly expressed on many cell types in human tissues (10). Upon internalization into the cell, genomic RNA is used as a template for direct translation of two polyprotein pp1a and pp1ab which encodes a number of crucial nonstructural proteins (nsPs) including two proteases; Chymotrypsin-like protease (3CL pro ) or main protease (M pro ) -nsP5 and a papain like protease (P pro ) -nsP3, both of which processes the polypeptide pp1a and pp1ab in a sequence specific manner to produce 16 different nsPs (11,12). The papain protease processes the polyprotein to generate nsP1-4 while the M pro operates at as many as 11 cleavage sites by specifically recognizing the sequence Leu-Gln*Ser-Ala-Gly (* marks the cleavage site) to generate rest of the critical nsPs including helicase, methyltransferase, and RNA dependent RNA polymerase (RdRp) all of which play a critical role in the viral infection cycle by forming a replicationtranscription complex (RTC) (13). Therefore, the main protease constitutes a major and attractive drug target to block the production of nonstructural viral components and thereby to hamper the replication event of the virus life cycle. Additionally, no human protease with similar cleavage specificity is known to rule out the possibility of cellular toxicity upon the potential inhibition of main viral protease.</p><p>In recent years drug repurposing screens have emerged as a resourceful alternative to fasten the drug development process against rapidly spreading emerging infections such as the one of SARS-CoV-2 (14,15). The approach of drug repurposing has successfully led to the discoveries of potential drug candidates against several diseases such as Ebola disease, hepatitis C virus, and zika virus infection (16)(17)(18)(19). In the present study, we have applied the approach of in silico virtual screening and protein-ligand docking of a spectrum of Food and Drug Administration (FDA) -approved antiviral drugs against SARS-CoV-2 M pro . To this end a recently elucidated X-ray crystal structure of SARS-CoV-2 M pro (PDB ID: 6Y2F) which have been shown to harbor an α-ketoamide as a potent inhibitor in the enzyme's active site, was chosen and screened for a number of FDA approved antiviral drugs to simulate the M pro -αketoamide interactions and thereby blocking the active pocket (20). The crystal structure of SARS-CoV-2 M pro in apo form (PDB ID: 6Y2E) and α-ketoamide bound form (PDB ID: 6Y2F) shows that the protein makes a crystallographic dimer composed of two monomers of identical conformations. Each protomer furthermore is made up of three domains. The interface of domain I and domain II form the active site of the protein, which is composed of a Cys 145 -His 41 dyad where α-ketoamide derivative 13b is bound (Fig. 1A). The uniquely globular domain III is linked to domain II through a linker region and deemed essential for the catalytic activity of this chymotrypsin-like protease (20,21). The α-ketoamide derivative 13b is shown bound in the active site and is stabilized by a number of interactions with the active site residues His 41 & Cys 145 and adjacent residues in substrate binding cleft such as Gly 143 and Ser 144 (20) (Fig. 1B). We have selected a number of existing drugs, most of which are reported to be used in humans for countering certain viral infections and screened them for binding in the active cleft of M pro . Our results have shown that some of the drugs occupied the active site of M pro with even increased binding affinity than that of the bound α-ketoamide 13b. Whilethe rest of the compounds has shown appreciable binding while holding most of the crucial active site determinants. We envisage that further in vitro examination of the inhibitory potential of these drugs on the catalytic activity of the main protease could lead the way to repurpose one or more of the tested FDA approved drugs in this study as a treatment therapy for SARS-CoV-2 induced disease.</p><!><p>To understand the evolutionary relationship between the previously known human coronavirus and the novel SARS-CoV-2, we have performed the phylogenetic analysis. For analysis, all the closely related and complete reference genome sequences of SARS-CoV-2 were downloaded from the NCBI GenBank database. A total of 50 genomes were considered for the study. MEGA 6.0 was used for multiple sequence alignment and construction of a phylogenetic tree and 1000 bootstrap replicates is performed using Neighbor-joining method (22).</p><!><p>The recently elucidated X-ray crystal structure coordinates of SAR-CoV-2 M pro was downloaded from RCSB PDB (PDB ID: 6Y2F), having 1.75Å resolution (20). In this structure the M pro was co-crystallized with the bound improved α-ketoamide (13b) inhibitor and multiple intermolecular interaction of ligand with the active site residues are characterized. To further identify the potent inhibitors for SAR-CoV-2 M pro among the FDA approved antiviraldrugs, we have downloaded more than 75 drug compounds from the PubChem chemical database. For molecular docking based drug repurposing, the download 3D structures of compounds and protein was prepared.</p><p>The docking study was performed by AutoDock Vina, which uses a lamrackian genetic algorithm (GA) in combination with grid based energy estimation (23), to check the docking accuracy of software we have performed re-docking to co-crystal bound ligand.The main aim of this molecular interaction study was to identify the highly interacting drug with SAR-CoV-2 protein crystal structure and to propose the drug by in-silico repurposing method. All the interaction visualization analysis studies were performed by Discovery Studio Visualizer (DS), PyMol molecular visualization tool and LIGPLOT + (24,25).</p><!><p>The sequence alignment of SAR-CoV-2 genome shows high similarity with the closely related reference genomes of other coronaviruses. The Blastn search of the complete genome of SARS-CoV-2 reveals that the most closely related virus available in GenBank is SL-CoVZXC45</p><!><p>Coronaviruses uses a chymotrypsin like protease along with papain protease to process and cleaves its long polyprotein precursor into individually functional nsPs. Multiple sequence analysis of the main protease of SARS-CoV-2 with that of SARS-CoV reveals that amino acid sequence is conserved with a sequence identity of 96% (Fig. 3). The active site residues are thoroughly conserved and makes a catalytic Cys 145 -His 41 dyad. Additionally there are substrate binding subsites positioned in the active site groove of the protease. The specific subsite residues located in the enzyme active site are named as S1', S1, S2 , S3 and S4 depending on their relative position to the cleavage site and subsites P1', P1,P2, P3 and P4 in the polyprotein. In the M pro of SARS-CoV-2 active site region, the S1' residues are contributed by Cys145, Gly143 ans Ser144 which also serve as the oxyanion hole. The S1 residue is His163 while Glu166 & Gln189 located at the S2 position. Bulky Gln189 and Pro168 makes the S4 site (20) (Fig. 4A). The main protease recognizes and bind specific residues at each subsite of the peptide substrate to determine the initiation of proteolysis and production of nsPs for the formation of replication-transcription complex.</p><!><p>The molecular docking based virtual screening of FDA approved antiviral drugs against the SARS-CoV-2 M pro revealed the strong interaction with higher docking energyand binding affinities. All the potential drugs docked with the independent conformation in the active site of protein where the co-crystal structure ligand (improved α-ketoamide 13b) bound. Molecular docking binding affinity of all the docked and analysed drugs with their binding energy ranking is shown in table S1 (Supplementary material). The molecular re-docking was also performed to check the docking accuracy of the software AutoDock Vina, and it was observed that the cocrystal bound ligand and re-docked ligand shows RMSD value of 0.51 Å, suggesting the high fidelity of docking method (Fig. 4B). In the present study, we focused on the top 10 docking results for further analysis as these drug compounds showing higher binding affinity ranging from (-10.6 to -7.9 kcal/mol). Although among the top 10 drugs,the top three drug compounds were showing binding affinity even higher than that of the improved α-ketoamide 13b compound (Fig. 5A-D) (Table 1). From the recently published studies for SARS-CoV-2 it was observed that virus binds with angiotensin-converting enzymes 2 (ACE2) receptors in the lower respiratory tracts of infected patients to gain entry into the lungs. The study reveals that SARS-CoV-2 main protease (M pro ) is the best drug target among coronaviruses (30). Interestingly, one of the most characterized and promising drug targetin against coronavirus infection is the main protease (M pro , also known as 3CL pro ) which has been co-crystallized with a bound ligand 'improved α-ketoamide 13b' in case of SARS-Cov-2 main protease (20,31). This crystal structure reveals that the α-ketoamide 13b is occupying the active site of the protein and making a number of hydrogen bonds and hydrophobic interactions with the active site residues as well as other substrate binding residues of the binding pocket. In the present study, we have screened more than 75 antiviral, anticancer, and anti-malarial drugs for the identification of potent drug molecules using drug repurposing virtual screening methods. Molecular docking studies have revealed that maximum of the screened drug compounds interact with SARS-CoV-2 M pro active pocket and also share same interacting amino acids residues. The SARS-CoV-2 bound ligand (improved-α-ketoamide)</p><p>shows strong bond in interactions with surrounding amino acids within the region of 4 Å at different subsites with His164, Glu166, Gly143, His163, Cys145, His41, and Phe140 where it forms hydrogen bonds with active site His41 and also accept hydrogen bond from the backbone amides of Gly143, Cys145 and Ser144. This protein ligand interaction reveals a strong inhibition of virus protease (Fig. 5D) (20). The screening and molecular docking of at least 75 preexisting drugs we have carried out have shown to fit in the active site of protease in independent conformation and appreciable binding energy score (Fig. 6A-D). Further,we have analyzed and repurposing the top 10 drugs which showed higher or similar binding affinity as compared to the co-crystal bound ligand of SARS-CoV-2. The top 3 drugs that are exhibiting the interaction with same amino acid residues as of the α-ketoamide with themain protease are Lopinavir-Ritonavir</p><p>showing binding affinity of (-10.6 kcal/mol) and Tipranavir (-8.7 kcal/mol), whereas</p><p>Raltegravirhas binding affinity of (-8.3 kcal/mol), which is similar to improved-α-ketomaide 13b (-8.3kcal/mol). While the rest of the drug compounds have also shown good binding energy score, as presented in table1.</p><p>The drug Lopinavir-Ritonavir is a combination product contains two medications lopinavir and ritonavir. This drug is mainly used for HIV-AIDS to control HIV infection by inhibiting the protease and help to decrease the amount of HIV in the body by promoting thefunction of body's natural immune system to work better (32,33).The enzyme SARS-CoV-2</p><p>M pro along with the papain-like proteases is essential for processing the polyproteins into various nonstructural protein by cleaving at specific sites, that are translated from the viral RNA. The interacting amino acids in the M pro enzyme active site were reported to be Leu, Gln, Ser, Ala, Glyalong with the Cys-His dyad which marks the cleavage site, similarly our in silico docking study shows that top screened drug Lopinavir-Ritonavir combination interacts with Glu166 (also form strong hydrogen bonding), Gln189, Leu167, Met165, Asp187, Met49, His41, Cys145, and Leu141(Fig. 5A). Interestingly, the binding energy score of Lopinavir-Ritonavir in protein-ligand docking was found to be even better than that of the docked α-ketoamide and the in silico inhibition constant (Ki) was obtained to be 16 nM. In silico inhibition constant (Ki), as obtained by docking is given in table 2 for top 10 drugs.</p><p>Drug tipranavir or tipranavir disodium is another nonpeptidic protease inhibitor used in combination with ritonavir to treat HIV infection (34)(35)(36). In our study, the drug shows interaction with Gln192, Met165 (both form hydrogen bonding), Gln189, Asp187, Met49, Arg188, Ser46, Cys44, Thr25, and His41 in different conformation from that of α-ketoamide inhibitor (Fig. 5B). We hypothesizes that tipranavir or its other derivatives with even improved binding affinity in combination with ritonavir could serve as the potential protease inhibitor to counter SARS-CoV-2 multiplication in cell based assay. Another drug which has shown comparable binding affinity and binding energy with that of the docked α-ketoamide in M pro active site, theraltegraviris a characterized antiretroviral medication which work by inhibiting the integrase strand transfer and is used in combination with other drugs to relieve the HIV infection (37)(38)(39). In the present study, raltegravir drug shows interaction with His164, Arg188, Gln192, Glu166 (all residues were bonded with strong hydrogen bond), Met49, Met165, Phe140, Pro168, and Leu167. The drug shows four H-bonds with nearest interacting amino acids of SARS-CoV-2 M pro enzyme, which indicates good inhibition (Fig. 5C). This drug could also be used with other combinations like raltegravir and lopinavir for the treatment of COVID-19, if found producing desirable inhibitory effect against SARS-CoV-2 protease in biochemical activity assay or cell based assays. Additionally, other drugs which were screened and docked in the substrate binding cleft of the M pro , has shown good binding energy score which is comparably similar to the original compound in the protein crystal structure. Many of these drugs such as dolutegravir, letermovir & Nelfinavir are commonly used for treating different infections ranging from HIV to cytomegalovirus by employing different mechanism of action (40)(41)(42)(43)(44)(45). The identified repurposed drug and their interaction with binding amino acids in the M pro active site have been shown in table 1.</p><p>After screening the different FDA approved drugs, the present study enabled us to understand the mode of interaction of approved antiretroviral drugs with new coronavirus SARS-CoV-2 main protease enzyme. However, we believe that all the drugs studied and screened for repurposing against COVID-19 in this study should furthermore be tested and their in vitro inhibitory potential needs to be investigated through robust biochemical proteolytic activity assays and other biophysical & structural studies.</p><!><p>From this study, we conclude that the repurposed drugs may be helpful for the treatment of novel coronavirus disease and can serve as potential drug candidates to curb the ongoing and everenlarging COVID-19 pandemic. Sinceall the drugs used in this study are of known pharmacokinetics standards and approved by FDA for human use they do not need to undergo specific long term clinical trials and therefore can fasten up the process of the therapeutics development. Our phylogenetic analysis of the available genomes of SARS-CoV-2 isolated from different sources also reveals that the virus is not showing any sign of mutation or diversification rapidly, therefore the repurposed drug combinations could be used against SARS-CoV-2 on pancommunity level.</p>

ChemRxiv

Facing Diseases Caused by Trypanosomatid Parasites: Rational Design of Pd and Pt Complexes With Bioactive Ligands

Human African Trypanosomiasis (HAT), Chagas disease or American Trypanosomiasis (CD), and leishmaniases are protozoan infections produced by trypanosomatid parasites belonging to the kinetoplastid order and they constitute an urgent global health problem. In fact, there is an urgent need of more efficient and less toxic chemotherapy for these diseases. Medicinal inorganic chemistry currently offers an attractive option for the rational design of new drugs and, in particular, antiparasitic ones. In this sense, one of the main strategies for the design of metal-based antiparasitic compounds has been the coordination of an organic ligand with known or potential biological activity, to a metal centre or an organometallic core. Classical metal coordination complexes or organometallic compounds could be designed as multifunctional agents joining, in a single molecule, different chemical species that could affect different parasitic targets. This review is focused on the rational design of palladium(II) and platinum(II) compounds with bioactive ligands as prospective drugs against trypanosomatid parasites that has been conducted by our group during the last 20 years.

facing_diseases_caused_by_trypanosomatid_parasites:_rational_design_of_pd_and_pt_complexes_with_bioa

Introduction<!><!>Introduction<!><!>Introduction<!><!>Introduction<!>Strategies for the Rational Design of Antiparasitic Metal Complexes<!>Palladium and Platinum Complexes With Bioactive Ligands<!><!>Palladium and Platinum Complexes With Bioactive Ligands<!><!>Palladium and Platinum Complexes With Bioactive Ligands<!><!>Palladium and Platinum Complexes With Bioactive Ligands<!><!>Palladium and Platinum Complexes With Bioactive Ligands<!><!>Palladium and Platinum Complexes With Bioactive Ligands<!>Ferrocenyl Derivatives<!><!>Ferrocenyl Derivatives<!><!>Ferrocenyl Derivatives<!><!>Ferrocenyl Derivatives<!>Concluding Remarks<!>Author Contributions<!>Conflict of Interest<!>Publisher’s Note

<p>Seventeen infectious diseases have been named by the World Health Organization (WHO) as Neglected Tropical Diseases (NTDs), mainly due to low pharmaceutical industry investment in drug research because of the low associated revenue. Consequently, most of the efforts related to the search of new chemotherapeutics against these diseases has come from the academy. More than 20% of the world's population lives in regions where these diseases are endemic. The impact of NTDs on the affected countries is enormous because of mortality and morbidity that causes important economic losses (World Health Organization, 2012; De Rycker et al., 2018; Chami and Bundy, 2019; World Health Organization, 2021a).</p><p>Human African Trypanosomiasis (HAT), Chagas disease or American Trypanosomiasis (CD), and leishmaniases are protozoan infections produced by trypanosomatid parasites belonging to the kinetoplastid order. These are among the most important neglected diseases and constitute an urgent health problem in developing countries. They are often co-endemic in certain regions of the world (Leishmaniasis and Chagas' disease in South America and Leishmaniasis and HAT in Africa) and they have span worldwide because of globalization caused by human migration. Despite being some of the most life-threatening infective diseases, only a poor and inadequate chemotherapy is available (De Rycker et al., 2018; Santos et al., 2020; Kourbeli et al., 2021).</p><p>American trypanosomiasis also called Chagas' disease after its discoverer, the Brazilian scientist Carlos Chagas, is endemic in Latin America where there are 7–8 million infected people, 10,000 annual deaths and 25 million people at risk of infection. In the last decades, the number of cases in non-endemic regions, like United States, Australia, Europe and Japan, has increased due to migration flows. The disease is caused by the protozoan parasite Trypanosoma cruzi (T. cruzi) that is mainly transmitted to mammalian hosts by infected blood-sucking bugs. In addition, other modes of transmission, responsible of the spreading of the disease to non-endemic regions, involve blood transfusion, organ transplant and congenital transmission. The parasite shows a complex life cycle that involves stages in the host and in the biological vector. Stages in the host show different susceptibility to drugs, which hampers the development of an effective chemotherapy (Santos et al., 2020; World Health Organization, nd).</p><p>The available chemotherapy includes drugs developed more than 50 years ago, Benznidazole and Nifurtimox (Figure 1), which proved to be toxic, require long treatments, are not effective in chronic stage of the disease, and often develop resistance.</p><!><p>Structure of Nifurtimox and Benznidazole.</p><!><p>Many natural products and synthetic inorganic and organic compounds have been successful assayed against T. cruzi and some of them entered clinical trials but finally did not reach the clinics (Paucar et al., 2016; Chatelain and Ioset, 2018; Scarim et al., 2018; Francisco et al., 2020).</p><p>Leishmaniases are a group of diseases caused by various Leishmania species. The insect vector that transmits the disease through its bite is a female phlebotomine sandfly. Leishmaniasis affect 350 million people in 98 countries, in four continents. There are three main forms of the disease: cutaneous, visceral and mucocutaneous with different levels of severity. In addition, Leishmania–HIV co-infection has been reported leading to more severe forms that are also more difficult to face (Nagle et al., 2014; Burza et al., 2018; Kwofie et al., 2020; Brindha et al., 2021; World Health Organization, 2021b). Some current drugs for the treatment of this disease are shown in Figure 2.</p><!><p>Some current drugs for the treatment of Leishmaniasis.</p><!><p>HAT mainly occurs in the sub-Saharan regions of Africa and it is transmitted through the bite of a tsetse fly. During the 20th century, the epidemics of this disease was devasting being people living in rural areas the most affected ones. Despite control efforts have reduced the number of annual cases, the relax of surveillance policies, the lack of new drugs for its treatment and the emergence of resistance to the old ones, have contributed to the reappearance of the disease. The protozoan parasite causing this disease is Trypanosoma brucei (T. brucei) and in particular, Trypanosoma brucei gambiense and Trypanosoma brucei rhodesiense subspecies. Most cases of chronic-like infections are caused by T. b. gambiense while T. b. rhodesiense causes the most serious form of the disease. In the late stage of this illness the parasites enter the central nervous system leading to profound sleep effects that give rise to the common name of HAT, sleeping sickness. A similar disease, called Nagana importantly affects cattle since antiquity but, interestingly, humans are resistant to the trypanosome species causing it. All forms of HAT are fatal if they are not properly treated. Five drugs are currently recommended: pentamidine, melarsoprol, eflornithine, suramin, and fexinidazole (Figure 3). Nifurtimox-eflornithine combination therapy is currently a first line treatment for HAT.</p><!><p>Drugs for the treatment of HAT.</p><!><p>However, most of these drugs show toxicity problems and their efficacy is variable depending on the type and stage of the disease. Although the mortality rates for HAT have decreased substantially in the last years with less than 1,000 cases found in 2019, the development of improved drugs bearing high bioavailability and low toxicity is crucial to definitively fight HAT (Nagle et al., 2014; Kourbeli et al., 2021; World Health Organization, 2021c).</p><p>Even though in the last years public-private consortia have pushed forward the drug discovery process, in general, no new drugs to treat diseases caused by kinetoplastids have been registered in more than 30 years. As previously stated, current chemotherapy against diseases caused by trypanosomatid parasites is very deficient so more research is urgently needed to discover safe and effective therapies (Nagle et al., 2014; Rao et al., 2019; Kayode et al., 2020).</p><p>During the preclinical development of new prospective drugs, it is important to know that these parasites exhibit complicated lifecycles alternating between stages in the insect vectors gut and in the mammalian host. This knowledge is relevant since it is well known that these different stages show different biological properties and, most importantly, different susceptibility to drugs. Shortly, T. cruzi is transmitted to the vertebrate host by the release of metacyclic trypomastigote form of the parasite in the feces of an infected triatome insect vector during its bite. Once inside the host, the parasite converts into the infective blood circulating trypomastigote stage that invades the tissues. Intracellularly, the amastigote form emerges that divides by binary fission and produces cell´s lysis. The released amastigotes convert into blood circulating trypomastigotes. After an insect meal they infect new insects and close the life cycle by converting into the non-infective epimastigote stage that only exists inside the gut of the insect. A similar scenario is observed in the lifecycle of T. brucei that shows four main developmental stages that occur in the infected insect and in the mammalian host, epimastigotes and procyclic forms, slender metacyclic trypomastigotes, and stumpy bloodstream proliferative metacyclic trypomastigotes, being the last one the most relevant form in the development of therapeutic agents. In contrast to other trypanosomatids, the part of the life cycle of T. brucei in the mammalian host occurs completely in the extracellular space. During the lifecycle of Leishmania sp the parasite alternates between the infective promastigote stage generated in the intestine of the insect vector and the replicative amastigote form inside the host macrophages (Brindha et al., 2021; Kourbeli et al., 2021).</p><p>Although these trypanosomatid protozoan parasites show important differences, they are transmitted by different insect vectors and are responsible for clinically different human diseases, it has been demonstrated that they show relevant similarities related to their biology. Their genomes have been sequenced in 2005 and they are available in a database for parasites of the family Trypanosomatidae (TriTrypDB, http://tritrypdb.org) (El-Sayed et al., 2005; Aslett et al., 2010). These studies showed that Leishmania spp, T. cruzi and Trypanosoma brucei have many common features, like gene conservation, genome architecture, high synteny, identical amino acid sequences in proteins and common subcellular structures like kinetoplasts. In particular, they show more than 6,100 closely related genes codifying proteins of a total of 8,000–12,000 genes. This paves the way for planning the development of broad-spectrum compounds that could be trypanosomatid-specific, affecting the three main parasites instead of affecting only one of them. This could lead to drugs that affect common targets in the different parasites showing activity against Leishmania spp, T. cruzi and T. brucei (Stuart et al., 2008; Ilari et al., 2018).</p><p>Metal-based medicines have been used since ancient times. However, the discovery of the potent anti-tumor activity of cisplatin, [PtCl2(NH3)2] determined the beginning of the modern era of Medicinal inorganic chemistry, accompanied by a huge impact of metal compounds in modern medicine. By exploiting the unique properties of metal ions, Medicinal inorganic chemistry currently offers an attractive option for the rational design of new drugs, looking for defined targets and activities, and new diagnostic and theranostic tools (Zhang and Sadler, 2017; Hanif and Hartinger, 2018; Englinger et al., 2019; Kostelnik and Orvig, 2019; Soldevila-Barreda and Metzler-Nolte, 2019; WahsnerGale et al., 2019; Wang et al., 2019; Anthony et al., 2020; Chamberlain et al., 2020; Chellan and Sadler, 2020; Imberti et al., 2020; Murray and Dyson, 2020; Cirri et al., 2021; Karges and Cohen, 2021; Lin et al., 2021; Silva et al., 2021; Yousuf et al., 2021). In the last decades, the development of metal-based drugs has proven to be a promising approach in the search for new therapeutic tools against parasitic diseases. As a result of the research performed by several academic groups, a great number of classical coordination compounds and organometallics bearing antiparasitic activity were developed (Sánchez-Delgado et al., 2004; Navarro, 2009; Navarro et al., 2010; Gambino, 2011; Gambino and Otero, 2012; Salas et al., 2013; Tahghighi, 2014; Brown and Hyland, 2015; Pessoa et al., 2015; Camarada et al., 2016; Gambino and Otero, 2018; Marcelino et al., 2018; Ravera et al., 2018; Gambino and Otero, 2019; Markwalter et al., 2019; Ong et al., 2019; Mbaba et al., 2020; Păunescu et al., 2021).</p><!><p>As previously stated, current drugs for the treatment of diseases caused by trypanosomatid parasites are characterized for their severe side effects, the need for prolonged treatments, and the emergence of resistance. In the search for new improved treatment options based on organic compounds, both phenotypic and target-based approaches have been used. In a phenotypic approach, many compounds are evaluated in vitro against the parasites searching for a hit or lead compound and, in the target-based drug design, a specific parasite target is selected, and drugs are drawn up to affect this target specifically (Haanstra and Bakker, 2015; Field et al., 2017; Gambino and Otero, 2019).</p><p>The phenotypic strategy has been the method of choice for the discovery of potential metal-based drugs and, in particular, antiparasitic ones. In fact, although metal compounds have the potentiality of interacting with selected parasitic enzymes and biomolecules, they are likely to act on different targets at the same time. In addition, they are usually transformed in vivo into active species by ligand exchange or redox reactions which sometimes makes it difficult to design them to act on specific targets (Navarro et al., 2010; Field et al., 2017; Anthony et al., 2020).</p><p>However, metal-based classical coordination complexes or organometallic compounds could be designed as multifunctional agents joining, in a single molecule, different chemical species that could affect different parasitic targets: a bioactive metal ion or metal centre and one or more antiparasitic organic compounds included as ligands. In fact, metal complexes with compounds bearing antiparasitic activity as ligands are expected to maintain the target of the bioactive compound. In addition, the presence of some bioactive metal ions (like palladium or platinum, as will be stated below) could give rise to the appearance of other targets like DNA or parasitic enzymes (Sánchez-Delgado et al., 2004; Gambino, 2011; Gambino and Otero, 2012; Gambino and Otero, 2018; Gambino and Otero, 2019).</p><p>In this sense, the peculiar chemical properties of metal complexes impart them the ability to interact with different biomolecules including proteins, enzymes, small peptides, nucleic acids, carbohydrates, among many others. To avoid unspecific toxicity, the design of metal-based compounds bearing antiparasitic activity should include specific targets like proteins or enzymes that are essential for the parasites and are not present in the host (like NADH-fumarate reductase, see below). When the mode of action of metal compounds are involved in metabolic pathways or targets that are common between parasite and mammals, specificity in the toxic action could also be achieved by taking advantage of other differences like the presence of specific organelles in the parasites or the poor mechanisms of detoxification of some toxic species as will be stated in the following sections (Gambino and Otero, 2019).</p><p>In particular, DNA is not a specific parasitic target as it is for metal-based antitumor compounds. However, parasites and cancer cells have some common features like their capacity for rapid cell division, some immune evasion and defense strategies, high rate of aerobic glycolysis with key glycolytic enzymes, need of nucleic acid biosynthesis, etc. These similarities noy only support DNA as a valid target for antiparasitic metal-based drugs but also the selection of metal centres that have proved to be useful in cancer therapy, like platinum or palladium (see below) (Williamson and Scott-Finnigan, 1975; Kinnamon et al., 1979; Fuertes et al., 2008; Dorosti et al., 2014; Sullivan et al., 2015).</p><p>In the framework of these general features, one of the main strategies for the design of metal-based antiparasitic compounds has been the coordination of an organic ligand with known or potential biological activity, to a metal centre or an organometallic core. The activity of the selected ligand could be enhanced because of metal complexation and its pharmacological properties could be improved as the stabilization of the organic drug may allow it to have a better performance in reaching and affecting the biological targets. In addition, the potential toxicity of the metal may be reduced because the organic ligand may limit the ability of the metal to interact with biomolecules that leads to toxicity. This approach could also be useful to circumvent drug resistance, as the metal complex would mask the organic drug. Finally, as stated, these metallodrugs would be capable of affecting multiple parasitic targets simultaneously (Sánchez-Delgado et al., 2004; Gambino and Otero, 2012; Ong et al., 2019; Navarro and Visbal, 2021).</p><p>For a metal compound to be able to accomplish an adequate pharmacological behavior, a very strict control of all its chemical features should be considered. The selection of the nature and oxidation state of the metal, the nature and number of coordinated ligands and the coordination geometry are essential for controlling complexes' reactivity and tuning their thermodynamic and kinetic stability. In addition, other physicochemical properties like lipophilicity, solubility or protein interaction can also be tuned by the choice of the metal ion, its oxidation state, and the inclusion of auxiliary co-ligands (Barry and Sadler, 2014; Ong et al., 2019; Czarnomysy et al., 2021; Mjos and Orvig, 2014).</p><p>This review is focused on the rational design of palladium(II) and platinum(II) compounds with bioactive ligands as prospective antiparasitic drugs that has been conducted by our group during the last 20 years. The selection of these metal ions is based on the resemblance between tumour cells and parasites as stated above. In this sense, the current treatment of cancer is based on platinum complexes, alone or in combination with other chemotherapeutic agents. In addition, most efforts related to the development of more effective and less toxic anticancer agents have been devoted to platinum compounds. On the other hand, complexes containing palladium are closely related to their platinum analogues. Therefore, Pd(II) has been selected as an alternative to Pt(II) in the search of new compounds for cancer therapy. The most significant resemblances are related to their coordination chemistry and, in particular, to the coordination number (four) and geometry (square planar) of their compounds. However, the ligand exchange kinetics of Pd(II) compounds is 104–105 times faster than that of the Pt(II) analogues. This means that Pd(II) compounds are much more reactive in solution which could lead to a higher toxicity and a different biological behaviour. However, the adequate selection of ligands could be able to stabilize Pd(II) complexes affecting their reactivity and imparting substitution inertness (Jahromi et al., 2016; Lazarević et al., 2017; Cirri et al., 2021; Czarnomysy et al., 2021; Yousuf et al., 2021).</p><!><p>In the search of ligands bearing anti T. cruzi activity, we had developed 5-nitrofuryl-containing thiosemicarbazones (HTS) maintaining the 5-nitrofuryl moiety that has proved to be the pharmacophore group of Nifurtimox (Figure 4). HTS were more active in vitro against T. cruzi than this reference drug. These compounds were designed to act on T. cruzi by the same mechanism than Nifurtimox: the generation of toxic reactive oxygen species (ROS) through the reduction of the nitro moiety followed by redox cycling. In addition, they have proved to affect the activity of trypanothione reductase parasitic specific enzyme through the nitrofuran group as well as to inhibit the main parasitic cysteine protease, cruzipain, through the thiosemicarbazone moiety (Aguirre et al., 2004; Rigol et al., 2005; Otero et al., 2008).</p><!><p>Bioactive 5-nitrofuryl-containing thiosemicarbazones (HTS) and their Pt(II) and Pd(II) complexes.</p><!><p>In order to address the effect of palladium and platinum complexation on the anti T. cruzi activity of these organic compounds, a large series of complexes with different stoichiometries and including different co-ligands was developed (Figure 4).</p><p>Thirty two analogous platinum and palladium complexes of the formula [MCl2(HTS)] and [M(TS)2] with M = Pd(II) and Pt(II) were obtained. Most of them were, in vitro, equally or more active than Nifurtimox against T. cruzi (epimastigote form, Tulahuen 2 strain). For palladium complexes, the activity of the ligands was maintained or increased as a consequence of metal complexation. Although, the obtained IC50 differences between the assayed species were quite low (IC50/5 days between 3 and 6 µM), a general trend was found: [PdCl2(HTS)] > HTS > [Pd(TS)2]. However, no similar trend was observed for platinum compounds showing, in most cases, lower activity than the palladium counterparts and the free ligands. Some platinum compounds were also tested against Dm28c epimastigotes and trypomastigotes of T. cruzi. Indeed, the infective trypomastigote form of the parasite resulted more susceptible to most of the tested Pt compounds than the epimastigote form, being these compounds more active on trypomastigotes than Nifurtimox (Otero et al., 2006; Vieites et al., 2008a; Vieites et al., 2009).</p><p>On the other hand, eight new palladium(II) and platinum(II) complexes of formula [MCl(TS)(PTA)] with PTA (1,3,5-triaza-7-phosphaadamantane) as co-ligand were obtained. PTA was included with the aim of modulating the solubility and lipophilicity of the new species. Most [MCl(TS)(PTA)] complexes showed similar activities against T. cruzi (trypomastigote form, Dm28c clone) to those of the corresponding HTS ligands and [PtCl2(HTS)] complexes. In contrast to what was observed for [MCl2(HTS)] compounds, no significant differences between palladium and platinum complexes were observed. However, for the most active compound, [MCl(TS4)(PTA)], the selectivity index (SI) was higher for the platinum complex (SI =20) than for the palladium one (SI = 10). It also should be noted that for the whole series of compounds, no correlation between the anti T. cruzi activity and the nature of substituent in the thiosemicarbazone chain was observed (Cipriani et al., 2014).</p><p>All prepared palladium and platinum complexes with the 5-nitrofuryl-containing thiosemicarbazones as bioactive ligands were supposed to show various mechanisms of action i.e., affecting the same targets or processes than the bioactive ligands or maintaining those mechanisms related to the PTA co-ligand and/or to the metal ions. Therefore, different studies were performed in order to sustain the proposed targets and to look into the potential mode of action of the metal compounds. Related to the bioactive ligands, the reduction of the nitro moiety had been demonstrated to be the first step of their mechanism of anti T. cruzi action. The nitro anion formed would be responsible of generating other toxic radical species through a redox cycling process. Therefore, the effect of metal complexation on the redox potential of the nitro moiety was studied using cyclic voltammetry. This potential slightly changed because of both palladium or platinum complexes formation (ΔE = 0.05–0.1 V) and no significant differences were observed between both metal ions. However, it should be stated that the nitro moiety of all compounds resulted more easily reducible than that of Nifurtimox, and, therefore, the capacity of generating toxic free radicals would be better for the complexes. The production of free radicals inside the parasite cells was assessed by ESR spectroscopy using 5,5-dimethyl-1-pirroline-N-oxide (DMPO) for spin trapping of radical species having short half-lives. A 10–13 lines spectral pattern was observed for all the studied complexes which is consistent with the intracellular generation of the hydroxyl radical and the nitroheterocyclic radical of the complexes. So, the mechanism of anti T. cruzi action of the HTS compounds seems to remain in the obtained complexes. However, only for [PdCl2(HTS)] and [Pd(TS)2] series, a good correlation between the concentration of the detected radicals (measured through the EPR signal intensities) and the IC50 values for the anti T. cruzi activity was observed. On the other hand, the described redox cycling processes should increase the parasite oxygen consumption. Thus, the parasite oxygen uptake in the presence of the compounds was determined. In general, complexes increased oxygen consumption which confirms that redox cycling processes are occurring inside the parasites treated with the complexes (Otero et al., 2006; Vieites et al., 2008a; Cipriani et al., 2014).</p><p>On the other hand, trypanothione reductase and cruzipain inhibition was also observed for some of the obtained metal compounds but no correlation with the anti T. cruzi activity was observed (Otero et al., 2006; Cipriani et al., 2014).</p><p>DNA was also tested as a potential target for the developed palladium and platinum compounds. The binding of [MCl2(HTS)] and [M(TS)2] compounds was studied by combining quantification of the bound metal by atomic absorption spectrometry and quantification of DNA by electronic absorption measurements. The amount of metal bound to DNA for platinum complexes was comparable to that previously reported for cytotoxic metal complexes and it was lower than the one determined for palladium complexes (Otero et al., 2006; Vieites et al., 2008a; Vieites et al., 2009).</p><p>[MCl2(HTS)] interaction with DNA was characterized by using gel electrophoresis, DNA viscosity measurements, circular dichroism (CD) and atomic force microscopy (AFM). Electrophoresis results showed that all complexes caused the loss of DNA superhelicity and modifications in the shape of plasmid DNA were observed in AFM studies. The effect on DNA was more significant for palladium complexes than for platinum analogues. In addition, CD results showed that while palladium complexes induce modifications in calf thymus DNA structure, no effect was observed for platinum ones. Finally, either Pd or Pt complexes increased the viscosity of DNA which agrees with an intercalative mode of interaction. An explanation for the observed differential intensity of the effect on DNA between palladium and platinum complexes could be the differences in exchange reactions kinetics between both metal ions (Vieites et al., 2011).</p><p>Similar results were obtained for [MCl(TS)(PTA)] complexes (Figure 4). The effect of these compounds on DNA was characterized by gel electrophoresis and ethidium bromide fluorescence experiments. Results of both experiments are in accordance with an intercalating-like mode of interaction between DNA and these compounds. However, the intensity of the effect on DNA was dependent on the nature of the metal ion. In fact, all Pd complexes showed a more significant effect on DNA than the platinum ones. In fact, some palladium compounds induce decomposition of DNA at high DNA/complex molar ratios (Cipriani et al., 2014).</p><p>It is interesting to note that no correlation between the anti T. cruzi activity and DNA binding was observed for the different series of palladium and platinum compounds. In fact, theoretical calculations performed for [MCl2(HTS)] complexes suggested that, in the cell, these complexes would not interact with DNA because they would react with the cell content before accessing to DNA. In addition, the potency and mode of interaction of both HTS and their metal complexes with T. cruzi cruzipain and trypanothione reductase enzymes was also studied using molecular docking. Results showed that the mode of action of these compounds involved multiple mechanisms and that, depending on the nature of the species, one mode of action would be predominant over others (Merlino et al., 2011).</p><p>Other thiosemicarbazone containing bioactive ligands were selected to study the effect of palladium and platinum complexation on the anti T. cruzi activity. Eight Pd(II) and Pt(II) complexes, [MCl2(HIn)] and [M(HIn)(In)]Cl with HIn = thiosemicarbazones derived from 1-indanones were obtained (Figure 5; Gómez et al., 2011).</p><!><p>Bioactive thiosemicarbazones derived from 1-indanones (HIn) and their Pt(II) and Pd(II) complexes.</p><!><p>The in vitro activity on T. cruzi (epimastigote form, Tulahuen 2 strain) and the unspecific cytotoxicity on red blood cells, were studied. All compounds showed higher activity than the corresponding free ligands with IC50 values in the low micromolar range. Most palladium compounds showed higher trypanosomicidal activity than their platinum counterparts. A quite good correlation between lipophilicity and antiproliferative activity was observed for these complexes. On one hand, lipophilicity was enhanced as a consequence of metal complexation and, in most cases, anti T. cruzi activity was also increased. In addition, being most palladium complexes more lipophilic than platinum ones, they showed higher anti T. cruzi activity. Unfortunately, obtained complexes showed low selectivity for the antiparasitic action, being less selective than the free ligands. In this case, coordination to Pd and Pt led to an increase in bioactivity but had a deleterious effect on unspecific cytotoxicity.</p><p>On the other hand, obtained complexes were tested for their antitumoral activity. Free ligands had no cytotoxic effect, but platinum and palladium complexes showed anti-leukemia properties and induced apoptosis. However, in this case no clear correlation between antitrypanosomal and antitumoral activities could be detected (Gómez et al., 2011; Santos et al., 2012).</p><p>Using the same approach, palladium and platinum compounds with pyridine-2-thiol N-oxide (2-mercaptopyridine N-oxide, Hmpo) as bioactive ligand were studied as potential antitrypanosomal agents (Figure 6). Mpo had shown a high anti T. cruzi activity against all forms of the parasite and no unspecific toxicity on mammalian cells. The antiparasitic action of mpo was related to the inhibition of NADH-fumarate reductase enzyme which is responsible for producing succinate from fumarate in the parasite. The lack of this enzyme in mammalian cells makes it a promising target for the development of antichagasic compounds. In addition, mpo, as similar amine N-oxides do, could suffer bioreduction leading to the release of radical species that are toxic for the parasite (Vieites et al., 2008b).</p><!><p>pyridine-2-thiol N-oxide (Hmpo) and their Pt(II) and Pd(II) complexes.</p><!><p>[Pd(mpo)2] and [Pt(mpo)2] compounds were synthesized and fully characterized. Both complexes showed very high in vitro growth inhibition activity of T. cruzi (epimastigote form, Tulahuen 2 strain) with IC50 values in the nanomolar range (IC50/5 days = 0.067 and 0.200 µM for palladium and platinum complexes, respectively). They were 39–115 times more active than Nifurtimox. In addition, the palladium complex showed an approximately threefold enhancement of the activity compared with the free mpo while only a low increase in the activity was observed for the platinum compound. In addition, owing to their low unspecific cytotoxicity on mammalian macrophages, the complexes showed a highly selective antiparasitic activity.</p><p>These complexes were also good candidates for a multi-target activity. In this sense, free-radical production, inhibition of the parasite-specific enzymes trypanothione reductase and NADH-fumarate reductase were studied. Additionally, studies on DNA interaction were performed.</p><p>Although radical species could be obtained electrochemically for both metal complexes, no free radical species were detected when they were incubated with the epimastigote form of T. cruzi. In addition, neither trypanothione reductase inhibition nor DNA interaction could be observed.</p><p>However, in the assayed conditions, both complexes have an inhibitory effect on NADH fumarate reductase. [Pd(mpo)2] showed the highest inhibition levels while the effect of [Pt(mpo)2] on the anzyme was similar to that of the free ligand. A similar behaviour was observed when analysing the IC50 values of these compounds which strongly suggest the involvement of this enzyme in the mode of action of the obtained complexes.</p><p>On the other hand, homology modelling combined with enzyme-cofactor docking were used to propose tertiary structures for NADH-dependent T. cruzi fumarate reductase. This model was used to explain the inhibitory effect and the binding modes of Pd- and Pt-mpo complexes. In fact, obtained theoretical inhibition constants (K i values) showed a good correlation with the experimental data. Both complexes bind to the enzyme into the cleft between domains 2 and 3, near to the nicotinamide ring of the NADH cofactor. However, [Pd(mpo)2] seems to bind closer to the cofactor which could affect the orientation of the nicotinamide ring for a proper position for catalysis, explaining the better inhibitory capacity displayed by this complex (Merlino et al., 2014).</p><p>Quinoxalines (3-aminoquinoxaline-2-carbonitrile 1,4-dioxides) are a family of compounds that had shown anti T. cruzi activity. Previous QSAR studies had shown that these compounds' activity was dependent on the electronic characteristics of the substituents as well as on the volume at the 3-amino level of the compounds. However, the low activities displayed by some derivatives could be the result of their low solubilities in the physiological media. In this sense, [Pd(quino)2] complexes were obtained in order to improve on one hand, the volume at the 3-amino level (Figure 7) and on the other hand, the bioavailability of the organic ligands. Results showed that the antitrypanosomal activity (epimastigote form, Tulahuen 2 strain) of the quinoxalines was improved upon complexation. The parent ligands having poor trypanosomicidal activity became 20- to 80-times more active upon complexation with palladium (Urquiola et al., 2009; Benítez et al., 2012).</p><!><p>Trypanocidal 3-aminoquinoxaline-2- carbonitrile 1,4-dioxides (quino) and their Pd(II) complexes.</p><!><p>In order to diminish the costs of developing new drugs, "repositioning" was used as one of the strategies for obtaining new antiparasitic compounds. Biphosphonates are examples of this approach. These compounds are the most prescribed drugs for osteoporosis and other bone diseases. Among bisphosphonates, those containing nitrogen (NBPs) have proved to inhibit farnesyl diphosphate synthase (FPPS) enzyme of the osteoclastic cells as their main mode of action. This enzyme is also present in trypanosomatid parasites. In addition, the specificity of the anti T. cruzi action of NBPs could be facilitated by the presence in parasites of specific organelles called acidocalcisomes. Acidocalcisomes are acidic structures involved in the storage and metabolism of phosphorous and calcium in parasites. Their composition is equivalent to the bone mineral so accumulation of bisphosphonates in these organelles would facilitate their antiparasitic action (Gambino and Otero, 2019). Therefore, bisphosphonates were selected as bioactive ligands for our multi-target based approach. In this sense, complexes of the formula [Pd(NBP)2(NN)] with NBP = commercial bisphosphonates (alendronate (ale) or pamidronate (pam)) and NN = 1,10 phenanthroline (phen) or 2,2′-bipyridine (bpy) were obtained (Figure 8). The selection of palladium as metal ion and the NN compounds as co-ligands points at DNA as target. In fact, besides the potential covalent interaction of Pd ion with DNA, metal complexes with these planar aromatic ligands could interact with DNA through intercalation between nucleobases. Additionally, parasitic enzymes of the mevalonate pathway (like FPPS) would also be a potential target for these compounds as they are for the NBP ligands.</p><!><p>Pamidronate (pam) and alendronate (ale) mixed-ligand Pd(II) compounds.</p><!><p>All the obtained compounds showed an increased anti-T. cruzi activity (amastigotes, CL strain) when compared to the free NBP ligands showing only slight signs of unspecific toxicity at high concentrations. In addition, Pd–NBP–phen complexes (IC50/3 days = 1.30 and 1.44 µM for ale and pam, respectively) resulted 15 times more active than the corresponding bpy analogues (IC50/3 days = 17.4 and 21.4 µM for ale and pam, respectively). However, all the complexes were able to similarly inhibit T. cruzi farnesyl diphosphate synthase and solanesyl diphosphate synthase enzymes suggesting that enzymatic inhibition would not be responsible for the observed differences in the biological activity. On the contrary, differences in the anti T. cruzi activity could be explained through the interaction of the complexes with DNA. As expected, the nature of the NN ligand determined the complexes' interaction with DNA. In fact, both Pd–NBP–phen complexes showed a much higher affinity for DNA in the fluorescent ethidium bromide displacement experiments than Pd–NBP–bpy analogues. It should be noted that, for these complexes, a good correlation between the antiparasitic and antitumor activities was observed. Additionally, the compounds were tested for their antitumoral activities. The cytotoxicity of the complexes on MG-63 osteosarcoma cells was dependent on the nature of the NBP as expected but for the A549 lung adenocarcinoma cells, Pd–NBP–phen compounds showed the highest cytotoxicities (Cipriani et al., 2020).</p><!><p>Organometallic compounds, characterized by showing at least one σ metal-carbon bond, offer a promising opportunity for the rational design of novel metal-based drugs. They show a wide structural diversity, their lability can be modulated leading to kinetically stable compounds, and they show adequate lipophilicity which favors their in vivo behavior (Allardyce et al., 2005; Hartinger and Dyson, 2009; Noffke et al., 2012; Zhang and Sadler, 2017; Chellan and Sadler, 2020).</p><p>In particular, the "sandwich type" ferrocene moiety has shown high potentiality in the development of novel organometallic drugs (Figure 9).</p><!><p>(A) ferrocene moiety; (B) ferrocifen (C) ferroquine (D) 1,1'-bis(dipheny1phosphino) ferrocene, dppf.</p><!><p>For instance, compounds including it, like ferrocifen, an analogue of the antitumoral drug tamoxifen, and ferroquine, an analogue of the antimalarial drug chloroquine, have achieved clinical or preclinical trials (Figure 9). In general, ferrocene derivatives are low cost, they are stable both in air and in solution and they are easy to derivatize. In addition, they are hardly not cytotoxic and have adequate lipophilicity. Furthermore, ferrocene derivatives show improved bioaccumulation when compared to the ionic forms of iron. In addition, ferrocenes are able to undergo one electron oxidation which could catalyze the generations of radicals in a Fenton-like manner, leading to the oxidation of macromolecules. The formation of ROS could be significant for antiparasitic therapy because trypanosomatids have a very primitive system of radical species detoxification (Biot, 2004; Dubar et al., 2008; Biot et al., 2010; Braga and Silva, 2013; Jaouen et al., 2015; Gambino and Otero, 2018).</p><p>In order to further address the therapeutic potential of palladium and platinum compounds with the selected bioactive ligands, we followed the rational design strategy previously delineated by including a ferrocene moiety in the new structures (Figure 9). Instead of coupling, as usual, the ferrocene scaffold to an organic skeleton as in ferroquine or ferrocifen, our synthetic strategy was to include the ferrocene fragment as a coligand in the platinum or palladium coordination sphere. The selected coligand, 1,1'-bis(dipheny1phosphino) ferrocene, dppf (Figure 9), acts as bidentate ligand binding to the metal center through the two phosphorus donor atoms and leaving the two extra coordination positions of the metal centre able to coordinate to the selected bioactive bidentate ligand (Figure 10). Accordingly, the twenty-four structurally related ferrocenyl compounds shown in Figure 10 were synthesized and fully characterized in the solid state and in solution and they were evaluated on trypanosomes as well as on mammalian cell models. Their effect on some selected molecular targets was studied and omic studies were performed for the most promising pyridine-2-thiolato-1-oxide (mpo) compounds. In general, the inclusion of the ferrocene moiety led to interesting effects on the biological profile of the compounds (Rodríguez Arce et al., 2015; Mosquillo et al., 2018a; Mosquillo et al., 2018b; Rivas et al., 2018; Rivas et al., 2019; Rodríguez Arce et al., 2019; Mosquillo et al., 2020; Rivas et al., 2021).</p><!><p>Pd(II) and Pt(II) dppf compounds with selected bioactive ligands L, [M(dppf)(L)](PF6).</p><!><p>At a first stage [M(dppf)(L)](PF6) compounds with L = pyridine-2-thiolato-1-oxide (mpo) as bioactive ligand were synthesized and characterized and their biological behavior compared with the previously developed classical coordination compounds [M(mpo)2] (Figure 11; Rodríguez Arce et al., 2015). Both ferrocenyl compounds showed IC50 values in the nanomolar range on T. cruzi epimastigotes (Dm28c strain) as well as low cytotoxicity on VERO epithelial cells (ATCC CCL81) as mammalian cell model, leading to good selectivity towards the parasite (Table 1). The complexes were about 10–20 times more active than the antitrypanosomal drug Nifurtimox (IC50/5 days = 6.0 μM) and two- to five-fold more active than mpo sodium salt. Moreover, epimastigotes of CL Brener strain (type VI) resulted more susceptible to the compounds than the type I Dm28c strain (Table 1). It is well known that genetic diversity of the parasite can lead to different susceptibility to drugs.</p><!><p>Structure of [M(dppf)(mpo)] (PF6) compounds, where M = Pd or Pt, mpo = pyridine-2-thiolato-1-oxide.</p><p>Activity against trypanosomes of M-dppf-L compounds.</p><p>SI = IC50 mammalian cells/IC50 parasite.</p><p>Dm28c strain epimastigotes, 24 h incubation.</p><p>CL Brener strain epimastigotes, 24 h incubation.</p><p>Dm28c strain tripomastigotes, 24 h incubation; EA.hy926 endothelial cell line: permanent human cell line derived by fusing human umbilical vein endothelial cells-HUVEC with human lung cells-A549; VERO cells, VERO kidney epithelial cells from African green monkey (ATCC CCL81); J774, J774 murine macrophages.</p><!><p>The compounds induce necrosis after 24 h of parasite incubation. Both complexes also affected the trypomastigote infection process as well as the number of amastigotes per cell. As expected, these dppf compounds showed lower unspecific cytotoxicity on mammalian cells than the [M(mpo)2] compounds (Table 1; Mosquillo et al., 2018a; Mosquillo et al., 2018b).</p><p>Molecular docking studies conducted on a model structure of the T. cruzi NADH fumarate reductase (TcFR) together with experimental in vitro studies on T. cruzi protein extracts demonstrated the inhibitory effect of the compounds on TcFR. As a consequence of palladium and platinum complexation, an increase in the inhibitory effect in respect to the free mpo was observed. Interestingly, the Pt compound had both the highest inhibition values and the highest activity against T. cruzi suggesting that TcFR could be involved in the mode of action of the compounds. Additionally, theoretical calculations confirmed that both compounds could be able to undergo oxidation at the ferrocene moiety which could aid to the biological activity. The generation of radical species inside the parasite cells by the action of M-dppf-mpo compounds was confirmed through EPR experiments (unpublished results) (Gambino and Otero, 2018).</p><p>These compounds resulted highly promising deserving further studies. The identification of molecular targets and the understanding of the mode of action of drug candidates are essential data for their clinical development. This knowledge is commonly elusive for metal-based drugs due to complicated mechanisms of action involving the interaction of metal compounds with multiple targets and biomolecules. Inorganic medicinal chemists have traditionally tried to identify and characterize, using in vitro approaches, molecular targets of a metal-based drug that mainly depend on the nature of the organic ligands and the metal center. Omic studies are relevant tools for uncovering the whole mechanism of action of metal-based drugs. Up and down regulated cellular proteins due to effects of drug and molecular targets interactions, identified and quantified by proteomics, together with cell uptake and subcellular distribution, quantified by metallomics, and changes in gene expression, determined by transcriptomics, allow to get a deeper insight into what occurs in the cells after administering a metallodrug. The knowledge achieved is a useful income for the rational design of novel metallodrugs (Wang et al., 2020).</p><p>High-throughput omic studies have proved to be powerful tools for going further into the basic biology of parasites like T. cruzi and also for the validation of drug targets (Atwood et al., 2005; Chávez et al., 2017; García-Huertas et al., 2017; Van den Kerkhof et al., 2020). Although these studies have been also used to explore organic drugs action in kinetoplastid parasites, similar studies for metal-based drugs were not reported until part of our group recently performed a high throughput omic study on T. cruzi for the two analogous Pt and Pd organometallic hit compounds [MII(dppf)(mpo)](PF6) (Mosquillo et al., 2018a; Mosquillo et al., 2018b; Mosquillo et al., 2020).</p><p>Metallomic studies showed a high Pt and Pd uptake by parasite epimastigotes. For the same dose a higher nanomolar uptake per 106 parasites was determined for Pt-dppf-mpo than for Pd-dppf-mpo. A similar pattern of metal distribution among the four analyzed macromolecules fractions (DNA, RNA, soluble proteins and insoluble proteins including membrane lipids) was found, with a preferential association to DNA. It is well known that metal complexes often could suffer reactions in biological media. Therefore, to evaluate if the M-dppf-mpo compounds are taken up intact by the parasites, the M (Pd or Pt) and Fe (present in the dppf moiety) levels were quantified in the selected parasite fractions at the same time. A 1:1 stoichiometric relationship between Fe and M in each fraction was obtained which would be consistent with the presence of the intact M-dppf-mpo species bound to the selected macromolecules (Mosquillo et al., 2018a; Mosquillo et al., 2018b). Proteomic and transcriptomic analyses allowed to identify differentially expressed transcripts and proteins in the treated parasites. The number of differentially expressed proteins was 342 for Pd-dppf-mpo and 411 for Pt-dppf-mpo. In addition, the number of soluble and insoluble proteins modified after treatment with both compounds was similar. The effect of Pd-dppf-mpo treatment showed more modulated transcripts (2,327 of 10,785 identified transcripts) than Pt-dppf-mpo treatment (201 of 10,773 identified transcripts). These results suggest that the mechanism of action for Pd-dppf-mpo is at the transcriptome level. Differentially expressed transcripts were functionally categorized which allowed to identify the cellular processes and pathways that were affected by the treatment with the compounds. Transcripts involved in DNA binding, protein metabolism, transmembrane transport (ABC transporters related with T. cruzi response and resistance to drugs, among others), oxidative defense, and the biosynthesis of ergosterol pathways were found to be modulated by the presence of the compounds. The whole set of data obtained allowed to suppose that the antitrypanosomal mechanism of action of Pd-dppf-mpo and Pt-dppf-mpo is multimodal. Interestingly, significant biological differences were assessed for both structurally analogous compounds showing the significance of the nature of the metal center on the biological behavior. In particular, metallomic studies allowed to assess that the Pt(II) compound showed higher cellular uptake than the Pd one. In addition, proteomic and transcriptomic studies showed different biological effects of both chemically analogous compounds. Both metals belong to the same group of the periodic table allowing to expect many chemical and physicochemical similarities between their compounds. Nevertheless, differences are expected based on the differential lability of the metal centers. The omic work performed gives details about the biological implications emerging of these chemical differences (Mosquillo et al., 2020).</p><p>As previously discussed, NADH-fumarate reductase, a specific parasite enzyme absent in the host, had been previously identified as a potential target for both M-dppf-mpo compounds (Mosquillo et al., 2018a). Although proteomic analysis agrees with this previous finding showing that, the amount of enzyme is modified in the treated parasites respect to untreated control parasites, omic studies unveiled, in addition, several down and up regulated proteins and a wide range of pathways affected by the compounds. Another remarkable point is that in vitro target identification should be in agreement with observed cellular uptake and subcellular localization. In the case of the M-dppf-mpo compounds, DNA was not considered for the in vitro target identification as an important biological target to be tested but metallomics showed a high accumulation of the complexes in the parasite DNA fraction. Globally, this study constitutes the first omic contribution to unravel the whole scenario of effects involved in the mechanism of action of potential metal-based drugs for the treatment of Chagas disease.</p><p>Following the promising results obtained with the M-dppf-mpo compounds and in an attempt to get broad spectrum Pd and Pt dppf compounds that could affect both T. brucei and T. cruzi parasites, four 5-nitrofuryl containing thiosemicarbazones (HTS) were coordinated as bioactive bidentate ligands to the {M-dppf} centers leading to eight new heterobimetallic [MII(TS)(dppf)](PF6) Pt(II) or Pd(II) compounds (Figure 10). IC50 values for most compounds were in the low micromolar or submicromolar range against both parasites, having the platinum compounds higher activities than the corresponding palladium ones (Table 1). Their activities were significantly higher than those of the free thiosemicarbazone ligands, showing a 3- to 24-fold increase for T. cruzi and up to 99-fold increase for T. brucei. The presence of the organometallic dppf co-ligand also seems to be responsible for a lower toxicity on mammalian cells and higher selectivity towards both parasites when compared to the free thiosemicarbazone compounds. In addition, new compounds showed higher activity and selectivity than the several groups of Pd and Pt classical coordination complexes with 5-nitrofuryl thiosemicarbazones previously described in this review.</p><p>The M-dppf-TS compounds resulted up to 26 times more active on T. cruzi than Nifurtimox (IC50/24 h = 20 μM) and up to 30 times (IC50/24 h = 15 μM) on T. brucei. These Pd and Pt compounds demonstrated to affect the redox metabolism of T. cruzi seeming to retain the mechanism of anti-T. cruzi action of the free ligands previously described in this review. However, no correlation between oxygen uptake and the generation of free oxygen radical species in the parasite, and the anti-T. cruzi activity was observed. Additionally, these compounds demonstrated to interact with DNA. Fluorescence results showed that ethidium bromide (EB) was displaced from the {DNA–EB} adduct as a result of the interaction of the Pd and Pt complexes with the biomolecule. This effect could be related to an intercalative-like mode of interaction or to the generation of DNA conformational changes causing the disruption of EB binding (Log K SV 4.3–5.0, with K SV = Stern Volmer quenching constant). Obtained K SV values could be related to a high affinity of the compounds for DNA. However, no correlation between the interaction with DNA and the biological activity was observed, discarding this biomolecule as a main target.</p><p>Zebrafish (Danio rerio) is employed as a toxicological model, among others, for evaluating in vivo toxicity in drug development. The most active and selective compound of the new series, [Pt(dppf)(TS3)] (PF6), showed no in vivo toxicity in zebrafish embryos. All embryos were alive in the 1–100 μM concentration range examined, and no apparent toxicity was observed after 48 h of treatment (Rodríguez Arce et al., 2019).</p><p>Based on the very nice results obtained for these complexes on both, T. cruzi and T. brucei, other ferrocenyl compounds, [M(dppf)(L)](PF6), with M=Pd(II) or Pt(II) and HL=tropolone (HTrop) or hinokitiol (HHino), were obtained and evaluated against the bloodstream form of T. brucei and L. infantum amastigotes (Figure 10; Rivas et al., 2018). Tropolones and their derivatives were selected as bioactive ligands because they are considered lead-like natural products. The tropolone moiety shows high possibilities for derivatization including improvements in the metal binding abilities. Tropolone, hinokitiol and their derivatives as well as the metal complexes with these compounds as ligands have shown various biological activities, for example antimicrobial one (Ononye et al., 2013; Saniewski et al., 2014; El Hachlafi et al., 2021).</p><p>The obtained complexes showed IC50/24 h values in the range 1.2–4.5 μM against T. brucei together with a significant increase of the activity against this parasite with respect to the free ligands (Table 1). In addition, obtained heterobimetallic compounds showed higher selectivity indexes towards the parasite than the free ligands. Platinum complexes were more selective than palladium ones. Moreover, coordination of the bioactive ligands to the {M-dppf} moiety also led to a slight increase of the anti-leishmanial potency. Studies performed to unravel the mechanism of action of the compounds indicated that no effect on the thiol-redox homeostasis of the parasites is produced by the complexes' action. On the other hand, fluorescence measurements of displacement of ethidium bromide from the adduct {DNA-EB} showed that DNA could be a probable, but not main, target of these compounds.</p><p>Later on, the series of structurally related Pd and Pt dppf compounds was expanded with ten new compounds that include five 8-hydroxyquinoline derivatives (8HQs) as bioactive bidentate co-ligands (Figure 10; Rivas et al., 2019; Rivas et al., 2021). These compounds showed IC50 values against bloodstream T. brucei form, in the submicromolar or micromolar range (IC50/24 h: Pt compounds 0.14–0.93 μM; Pd compounds 0.33–1.2 μM). In addition, they displayed good to very good selectivity towards the parasite (SI: Pt compounds 11–48; Pd compounds 4–102) with respect to murine macrophages (cell line J774) (Table 1). In most cases, an increase of the activity (11- to 41-fold) was observed as a consequence of coordination of the bioactive 8HQs to the {Pt-dppf} moiety. Only part of the Pd compounds were more active than the corresponding 8HQ ligands and most of the Pd compounds were less active than their Pt analogues. It should be stated that, the palladium compounds were 2- to 45-fold more potent than the drug Nifurtimox while platinum complexes resulted 16- to 107-fold more potent than the same reference drug. All the complexes interacted with DNA (Pt compounds Log K SV 3.3–3.9; Pd compounds Log K SV 3.8–4.6) but the Pd compounds show higher Log K SV values than the Pt analogues. In addition, the most active Pt ones induced reactive oxygen species (ROS) formation in tumor cells. Results suggest that the mechanism of action for these complexes against T. brucei may be mediated by interaction with DNA and additionally oxidative stress for the Pt compounds.</p><p>An exploratory pre-clinical therapeutic efficacy study was performed in an acute murine model for Human African Trypanosomiasis (HAT) for the most promising Pt compound, Pt-dppf-8HQ Cl,I, (IC50/24 h = 0.14 μM, SI = 48) using mice infected with a bioluminescent cell line of T. brucei that allows in vivo mice imaging. Although preliminary, the in vivo study showed that the assayed compound does not show acute toxicity to animals. In addition, results suggested that although the compound exerts an anti-proliferative activity that prolongs animal survival, it does not exert a curative effect (Rivas et al., 2021).</p><p>Having developed a long series of twenty-two structurally related M-dppf-L compounds with different bioactive ligands L (Figure 10) that had been evaluated against bloodstream T. brucei and with the aim of understanding and assessing the main structural parameters that determine the anti-T. brucei activity, a quantitative structure–activity relationships (QSAR) study was performed (Rivas et al., 2021). For this study, the dependent variable was the log10 of the IC50 values on T. brucei. As independent variables, different physicochemical characteristics including lipophilic, electronic, and steric/topological properties were considered (Hansch et al., 1963). Lipophilicity was indirectly determined by a reverse phase TLC method adequate for non-soluble in water compounds that led to experimental Rf and calculated RM values. As electronic parameter a signal of the 1H NMR spectra of the studied compounds was selected. Owing to the chemical variability of the studied compounds, i.e. palladium or platinum and three different families of ligands, the cyclopentadienyl-moiety was selected as common feature to determine the contribution of the compounds' electronic effect on the measured anti T. brucei activities. The displacement δ of the 1H NMR signals of the protons of the cyclopentadienyl-framework resulted indicative of the coordination and the nature of compound. Consequently, Δδ defined as the largest difference between δ of cyclopentadienyl-framework protons in the complexes and δ of cyclopentadienyl-framework protons without coordination was used as electronic descriptor. Additionally, an indicator variable, IVPd, was defined that adopts value 1 for palladium compounds or 0 for platinum complexes. According to the QSAR study, ligands with electron withdrawing substituents and with high lipophilicity and having platinum as central atom would result in complexes with increased anti T. brucei activity. Among all these descriptors, the electronic properties and the nature of the metal ion were the most relevant ones. QSAR studies are relevant to guide the rational design of further bioactive compounds. However, they are not common in inorganic medicinal chemistry due to the need of having numerous structurally related compounds to perform them (Rivas et al., 2021).</p><!><p>The development of metal-based compounds for the treatment of diseases caused by trypanosomatid parasites has evolved from rather isolated serendipitous efforts to a more rational and systematic strategy. In this sense, the development of palladium and platinum compounds described in this review constitute an example of this rational phenotypic approach. In fact, from the selection of the metal centers and the bioactive ligands to the inclusion of different co-ligands, the design was based on both chemical and biological arguments. The establishment of structure-activity relationships and the deep insight into the molecular modes of action of the metallic compounds also aided to redesign new compounds with improved pharmacological properties. In this sense, the development of structurally related series of compounds has let us perform quantitative structure activity relationship (QSAR) studies that are not common in Medicinal Inorganic Chemistry.</p><p>On the other hand, all the data previously discussed clearly show that the strategy of combining, in a single molecule, palladium or platinum with ligands bearing activity against parasites produce, in most cases, an enhancement of the activity of the ligand and/or a reduction in toxicity. In addition, it was demonstrated that this approach could lead to multifunctional compounds generating single chemical entities that can act simultaneously on multiple targets. In this sense, the omic approach, also new for metal-based antiparasitic drug development, allowed us to go further into the study of the whole mechanism of action of the prospective antiparasitic agents.</p><p>In the process of rational design of palladium and platinum complexes bearing antiparasitic activity, the selection of dppf as co-ligand deserves to be highlighted. The inclusion of a bioactive ligand in the {M-dppf} moiety gave the most promising results as most developed ferrocenyl derivatives not only showed low IC50 values in both T. cruzi and T. brucei but also excellent selectivity index values. In particular, the hypothesis of developing broad spectrum drugs that affect more than one trypanosomatid parasite, based on the discovery of common genomic features for T. cruzi, T. brucei and L. major, was verified in the case of the 5-nitrofurylthiosemicarbazone M-dppf-L complexes. These compounds showed IC50 values in the submicromolar or micromolar range on both, T. cruzi and T. brucei. Some of these compounds were good candidates for in vivo studies. Currently, our group is working on the development of delivery systems based on different nano-systems with the aim of avoiding or diminishing usual toxicity and solubility problems of metal-based drugs and to improve bioavailability.</p><!><p>DG and LO are responsible of conceptualization, original draft preparation, writing, review and editing the manuscript. Both authors have read and agreed to the published version of the manuscript.</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p><!><p>All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.</p>

PubMed Open Access

Natural and anthropogenic variations in atmospheric mercury deposition during the Holocene near Quelccaya Ice Cap, Peru

Mercury (Hg) is a toxic metal that is transported globally through the atmosphere. The emission of Hg from mineral reservoirs and subsequent recycling in surface reservoirs (i.e., soil/biomass, ocean, and atmosphere) are fundamental to the modern global Hg cycle, yet past emissions from anthropogenic and natural sources are not fully constrained. We use a sediment core from Yanacocha, a headwater lake in southeastern Peru, to study the anthropogenic and natural controls on atmospheric Hg deposition during the Holocene. From 12.3 to 3.5 ka, Hg fluxes in the record are relatively constant (mean \xc2\xb1 1\xcf\x83: 1.4 \xc2\xb1 0.6 \xce\xbcg m\xe2\x88\x922 a\xe2\x88\x921, n = 189). Past Hg deposition does not correlate with changes in regional temperature and precipitation, inferred from nearby paleoclimate records, or with most large volcanic events that occurred regionally, in the Andean Central Volcanic Zone (~300\xe2\x80\x93400 km from Yanacocha), and globally. In B.C. 1450 (3.4 ka), Hg fluxes abruptly increased and reached the Holocene-maximum flux (6.7 \xce\xbcg m\xe2\x88\x922 a\xe2\x88\x921) in B.C. 1200, concurrent with a ~100-year peak in Fe and chalcophile metals (As, Ag, Tl) and the presence of framboidal pyrite. Continuously elevated Hg fluxes from B.C. 1200\xe2\x80\x93500 suggest a protracted mining-dust source near Yanacocha that is identical in timing to documented pre-Incan cinnabar mining in central Peru. During Incan and Colonial time (A.D. 1450\xe2\x80\x931650), Hg deposition remains elevated relative to background levels but lower relative to other Hg records from sediment cores in central Peru, indicating a limited spatial extent of preindustrial Hg emissions. Hg fluxes from A.D. 1980 to 2011 (4.0 \xc2\xb1 1.0 \xce\xbcg m\xe2\x88\x922 a\xe2\x88\x921, n = 5) are 3.0 \xc2\xb1 1.5 times greater than pre-anthropogenic fluxes and are similar to modern fluxes documented in remote lakes around the world.

natural_and_anthropogenic_variations_in_atmospheric_mercury_deposition_during_the_holocene_near_quel

Introduction<!>Study Site<!>Core Collection and Processing<!>Geochemical Analyses<!>Loss on Ignition and Biogenic Silica<!>Major and Trace Metals<!>Heavy Mineral Separation and Analysis<!>Chronology<!>Flux Calculations<!>Stratigraphy and Age-depth Model<!>Holocene Sedimentology<!>Hg variability during the Holocene<!>Heavy Mineral Characterization<!>Depositional Pathway<!>Climate and Hg Deposition<!>Precipitation<!>Temperature<!>Volcanism and Hg Deposition<!>B.C. 1450\xe2\x80\x93500<!>A.D. 1480\xe2\x80\x932011<!>Modern Flux Ratio<!>Conclusions

<p>Rapidly rising anthropogenic emissions of mercury (Hg) to the atmosphere during the past decade are superimposed on a longer-term increasing trend since the industrial revolution [Streets et al., 2011]. Hg is transported globally as gaseous Hg0 (e.g. Mason et al. [1994]), deposited to the land/water surface as Hg2+, and rapidly transferred to biota as extremely toxic methyl-Hg [Harris et al., 2007], posing a great risk to human and ecosystem health. An accurate understanding of the global Hg cycle is required to assess the role of anthropogenic emissions on current and future Hg deposition. Information on the biogeochemical cycling of Hg primarily comes from reconstructions of Hg deposition over time in sedimentary archives (i.e., lake sediment, peat, and ice) and from global Hg models. A wealth of lake sediment records from around the world provide direct evidence for an average 3.5 fold increase in Hg deposition since ~A.D. 1850 [Biester et al., 2007], but very few records extend earlier in time. A recent model of global Hg cycling, forced with estimates of anthropogenic Hg emissions from B.C. 2000 to A.D. 2008 and constant natural emissions, yields a similar amount of increase (2.6 times) since A.D. 1840 but a much larger increase (7.5 times) since B.C. 2000 [Amos et al., 2013]. The apparent importance of anthropogenic emissions before ~A.D. 1850 (i.e., during preindustrial time) and the assumption of constant natural emissions require independent validation with geophysical evidence, such as Hg contained in sedimentary archives.</p><p>Natural variations in Hg emissions to the atmosphere can be caused by changes in volcanism, low-temperature volatilization, and external factors which affect exchanges between surface Hg reservoirs (soil/biomass, ocean, and atmosphere) [Fitzgerald and Lamborg, 2007]. Terrestrial volcanic Hg sources are somewhat constrained [Nriagu and Becker, 2003; Pyle and Mather, 2003], but large uncertainties remain in estimates of the inputs from submarine volcanism [Lamborg et al., 2006] and low-temperature volatilization [Gustin et al., 2000] due to limited observational data. A number of factors are thought to affect the exchange of Hg between surface reservoirs including biomass burning [Friedli et al., 2003], permafrost thaw/freeze [Rydberg et al., 2010], and oceanic evasion [Strode et al., 2007].</p><p>Use of Hg by humans began as early as B.C. 1500 in Egypt and Peru, and continued later in parts of Asia and the Roman Empire [Nriagu, 1979; Cooke et al., 2009]. This early use primarily consisted of extracting the common mineral form cinnabar (HgS) as the bright red pigment vermilion, although there are also early accounts of metal amalgamation using liquid Hg0 [Nriagu, 1979]. Anthropogenic Hg emissions increased dramatically in the late 16th century when Hg amalgamation for silver extraction was introduced to South and Central America [Nriagu, 1993]. Hg was emitted during the smelting of cinnabar to form liquid Hg0, which occurred extensively in Huancavelica, central Peru, and during the heating of silver amalgams, which occurred throughout the Andes but most notably in Potosí, Bolivia (Figure 1) [Robins and Hagan, 2012]. Estimates of preindustrial Hg emissions are based on historical records and anecdotes of past metal use coupled with assumed emission factors, and they are subject to high uncertainty [Nriagu, 1993; Streets et al., 2011]. In addition, the spatial distribution of Hg emissions from preindustrial mining remains uncertain. There is strong evidence for local deposition in highly enriched soils and sediments near mining sites [Cooke et al., 2009; Robins et al., 2012], limited evidence for regional (~200–500 km) transport [Beal et al., 2013; Cooke et al., 2013], and no evidence for an impact of preindustrial Hg emissions on a global scale [Lamborg et al., 2002].</p><p>In this study, we reconstruct atmospheric Hg deposition during the Holocene in a sediment core from a headwater lake in southeastern Peru near Quelccaya Ice Cap (QIC). Past Hg deposition is recorded reliabily in lake sediments and is not affected by diagenetic changes [e.g., Biester et al., 2007; Rydberg et al., 2008]. We use this continuous record of atmospheric Hg deposition and co-registered proxies for paleo-environmental change to: 1) assess natural variability in Hg deposition by comparing the Hg record to local and regional paleoclimate conditions and major volcanic eruptions; 2) evaluate the impact of preindustrial anthropogenic emissions on Hg deposition in the study lake by examining the Hg record during periods of known preindustrial metal use, and 3) quantify the extent of anthropogenic modification to natural Hg cycling by calculating an atmospheric deposition Hg flux ratio using modern and pre-anthropogenic Hg fluxes.</p><!><p>The study lake informally known as Yanacocha is located in the South Fork valley on the western side of Quelccaya Ice Cap (QIC) in the Cordillera Vilcanota of southeastern Peru (13.945 °S, 70.875 °W, 4910 m asl; Figure 1). Yanacocha is a tarn that occupies 0.036 km2 in a catchment of 0.11 km2. The catchment is composed of a sparsely vegetated and gently sloping colluvial apron that extends radially ~100 m from the edge of the lake, beyond which a near-vertical ~100-m high ignimbrite bedrock headwall surrounds the north, east, and south sides of the lake (Figure 1). Inflows are limited to surface runoff from the catchment, and a single outflow on the west side of the lake is active only during the wet season. During the field season in June 2011, the lake exhibited constant pH (~8), temperature (~6 °C), and conductivity (~10 μS) with depth (Figure S1, Table S1), characteristic of a holomictic lake.</p><p>Situated near the eastern edge of the Andes at 4910 m above sea level, Yanacocha likely receives most of its precipitation from easterly mid-upper troposphere flows in Austral Summer that bring moisture from the Amazon Basin (e.g. Garreaud et al. [2003]). Precipitation and atmospheric conditions at the study site have likely changed with the position of the Inter-Tropical Convergence Zone and El Niño-Southern Oscillation, with drier conditions during modern-day El Niño and wetter conditions during modern-day La Niña (e.g. Garreaud et al. [2003]).</p><p>An expanded QIC prior to ~12.8 ka (kilo-annum; defined here as thousands of years before A.D. 1950) had a terminus position ~2 km downvalley from Yanacocha, covering the lake with glacial ice [Kelly et al., 2012]. Retreat of QIC began ~12.3 ka, leaving the Yanacocha catchment by at least 11.6 ka and remaining ~3 km upvalley of Yanacocha during the Holocene [Kelly et al., 2012]. The bedrock of the headwall surrounding Yanacocha prevented inflows of QIC meltwater from entering the lake during the Holocene. Therefore any material transported to the lake occurred either by surface runoff within the relatively small catchment or atmospheric deposition.</p><p>Yanacocha is removed from major development. The nearest major population center is Cusco, located ~130 km away. Present-day land use in the vicinity of Yanacocha is limited to sparse livestock grazing. We are not aware of any mining near the margins of QIC and, although there are currently no large-scale mining operations in the region, a large silver-lead-zinc mine is in planning stages ~25 km northwest of Yanacocha. Small-scale and artisanal gold mining is prevalent in the Amazon basin ~120 km away, but this mining was shown not to be a major contributor of Hg to high-elevation lakes in southeastern Peru [Beal et al., 2013].</p><!><p>We collected a long (4-m) sediment core, YANA11, near the center of Yanacocha and at its greatest water depth (5.5 m) in June 2011. We used a Bolivian coring system from a floating platform to retrieve ~1-meter drives of sediment into polycarbonate tubes, collecting two adjacent cores offset by ~50 cm. Core tubes were capped, kept unfrozen in the field, and then shipped from Cusco to the National Lacustrine Core Facility (LacCore) at the University of Minnesota. At LacCore, we split the polycarbonate core tubes and took high resolution core images. Working halves of each core drive were shipped to Dartmouth College for subsequent analyses and archive halves are stored at the LacCore repository.</p><p>We also collected a short (40-cm) sediment core, YC1, adjacent to the YANA11 core using a gravity corer that preserves the sediment-water interface. This core was collected prior to YANA11 to avoid disturbance of the sediment-water interface. YC1 was extruded in the field at 1-cm intervals and stored in Whirlpak bags [Beal et al., 2013].</p><!><p>We sampled the YANA11 core at continuous 1-cm intervals using acid-clean polystyrene spoons. The samples from YANA11 and YC1 were freeze-dried in new polypropylene centrifuge tubes, homogenized in an agate mortar and pestle, and sub-sampled for loss on ignition (LOI), biogenic silica (BSi), major and trace metals, and heavy mineral separations.</p><!><p>We performed LOI in three stages: 110 °C overnight, 550 °C for 4 hours, and 1000 °C for 2 hours. BSi was determined at Northern Arizona University by molybdate-blue reaction and spectrophotometry following Mortlock and Froelich [1989]. Bulk density was calculated based on water content and assumed densities for the organic (1.4 g cm−3), carbonate (2.7 g cm−3), and inorganic (2.0 g cm−3) components determined by LOI.</p><!><p>We determined total Hg using a Milestone DMA-80 on ~50-mg sub-samples. One of the Standard Reference Materials (SRMs) IAEA-SL-1 (lake sediment), STSD-1 and STSD-2 (stream sediment), and NIST-1547 (peach leaves) was run every 10 samples. Measured SRM concentrations (Table S2) were within their published 95% confidence intervals. Sample replicates were run every 10 samples with typical precision (relative percent difference for n = 2, relative standard deviation for n ≥ 3) of less than 10 %. We also extracted ~200-mg sub-samples by strong acid (9:1 HNO3:HCl) in open microwave vessels at 90 °C and analyzed the leachates for metal concentrations (henceforth referred to as Mext) by quadrupole ICP-MS (Agilent 7700x), running calibration checks and blanks every 10 samples. Typical precision on replicate samples for detectable analytes was less than 10%. All concentrations are expressed as mass of metal per mass of dry sediment. In addition, total metals were measured at 0.5-cm resolution on archive core halves by ITRAX core-scanning XRF at the University of Minnesota Duluth with a dwell time of 30 seconds [Croudace et al., 2006].</p><!><p>We separated the heavy mineral fraction of selected samples by mixing ~500 mg of freeze-dried sediment with 10 ml of sodium polytungstate adjusted to a density of 2.8 g cm−3, placing the mixtures in an ultrasonic bath for 30 minutes, and centrifuging the mixtures for 90 minutes at 4500 rpm. This separation procedure accommodates a theoretical minimum cinnabar (8.1 g cm−3) particle diameter of 65 nm following the equation in Plathe et al. [2013]. We rinsed the heavy fraction by following the above ultrasonic and centrifugation steps with 10 ml of deionized H2O, repeated three times. We digested and analyzed selected heavy fraction samples for metal concentrations (henceforth referred to as Mhvy) using the same methods described above for bulk samples, while accounting for contamination by the heavy liquids with one procedural blank for every five samples. For certain non-digested samples, we dried the heavy fractions and studied them using a Scanning Electron Microscope (SEM; Hitachi TM3000) with Energy-dispersive X-ray Spectroscopy (EDS).</p><!><p>The composite record (henceforth the Yanacocha record) includes YC1 from 0–27 cm depth and YANA11 from 27–333 cm depth. We correlated the offset drives from YANA11 based on visual stratigraphy and then correlated YC1 to YANA11 using LOI550 (R = 0.90, p < 0.001; Figure S2). Age control in the Yanacocha record consists of twelve 210Pb ages from YC1 (details previously published in Beal et al. [2013]) and ten AMS 14C ages on macrofossils isolated from discrete sediment layers throughout YANA11 (Table S3). The age-depth model uses 210Pb ages from 0–12 cm, linear interpolation from 12–27 cm, Bayesian age-modeling from 27–303 cm using the program Bacon [Blaauw and Christen, 2011], and linear interpolation from 303–333 cm (Figure 2). The following non-default parameters were used in Bacon: mean accumulation rate 0.0167 cm yr−1, memory strength 20, and memory mean 0.1. All ages were calibrated in Bacon using SHCal04 [McCormac et al., 2004], except for the basal age which was calibrated using IntCal09 [Reimer et al., 2011] because its 14C age exceeds SHCal04. Linear interpolation was used from 303–333 cm because of deviations in Bacon's fit near the base of cores where accumulation rates decrease rapidly.</p><!><p>Hg fluxes were calculated as the product of concentration and dry mass sedimentation rate. Dry mass sedimentation rate (g m−2 a−1) was calculated as the product of bulk density (g cm−3) and accumulation rate (cm a−1). Because Bacon assumes unrealistic changes in accumulation rate between adjacent sections [Jacobson et al., 2012], a second order polynomial fit was used to calculate accumulation rates (Figure S3).</p><!><p>The composition of the Yanacocha record is a diatomaceous gyttja from the top of the core to a depth of 333 cm (Figure 2). Below 333 cm, the lithology is uniformly silt and clay. A macrofossil just above this abrupt transition from silt and clay to gyttja dates to 12.3 ka and likely marks the termination of meltwater input caused by recession of QIC behind the bedrock ridge surrounding Yanacocha. This basal age is older than two previously reported 14C ages (both 11.2 ka) in Yanacocha basal sediments from slightly above the transition in another core [Kelly et al., 2012]. The Yanacocha record exhibits constant sedimentation throughout the Holocene with no evidence for a hiatus in either the age-depth model or the stratigraphy.</p><!><p>Organic matter (LOI550) and BSi, proxies for productivity in the lake, each comprise between ~20 and 60 % of Yanacocha sediments and are significantly inversely correlated throughout the Holocene (Figure 3). BSi is high (~38–55 %) and LOI550 is low (~10–30 %) from 12.3 ka to 6.5 ka (Figure 4), followed by relatively low BSi (~26–38 %) and high LOI550 (~31–50 %) from 6.5 to 4.7 ka. Subsequent to 4.7 ka, BSi and LOI550 remain within their early Holocene values, except for a brief reversal from 1.1 to 0.6 ka when BSi is low and LOI550 is high. Total Ti, a proxy for total lithogenic input, is relatively high in the early Holocene from ~12.3 to 9 ka, followed by lower values from ~7 to 5 ka. Higher than average Ti persists from ~4.8 to 3 ka and then is variable from ~3 ka through the late Holocene.</p><!><p>Hg concentrations in the Yanacocha record range from a minimum of 13 μg kg−1 at 8.9 ka to a maximum of 115 μg kg−1 at 3.2 ka (Figure 4). Pre-3.5 ka Hg concentrations are relatively stable (mean ± 1σ: 32 ± 9 μg kg−1), except from ~10 to 9 ka when Hg concentrations are relatively elevated (~40–60 μg kg−1). An abrupt increase in Hg concentration occurs at 3.4 ka and reaches the Holocene maximum concentration at 3.2 ka, followed by a steady decline to pre-3.5 ka values by ~2.5 ka. Slightly elevated Hg concentrations (~45 μg kg−1) persist from 1.5 to 0.5 ka. An abrupt increase beginning in ~A.D. 1480 is followed by consistently elevated concentrations (46–75 μg kg−1) until the most recent sediment in A.D. 2011.</p><p>The record of Hg flux is largely a reflection of the record of Hg concentration, as it is the product of Hg concentration and sedimentation rate (Figure S4). Pre-3.5 ka Hg fluxes are ~1.0–1.5 μg m−2 a−1, compared to a maximum of 6.7 μg m−2 a−1 at 3.2 ka and average post-A.D. 1980 fluxes of ~4.1 μg m−2 a-1. The main deviation of Hg flux from concentration occurs from ~1.5 to 0.5 ka, concurrent with increased LOI550 (Figure 4). Because estimates of Hg flux are subject to high uncertainty, particularly in older records such as this one that are dependent upon a limited number of ages, we use Hg concentrations to determine secular changes in Hg deposition and time-averaged Hg fluxes to calculate an Hg flux ratio.</p><!><p>The composition and morphology of minerals contained in the heavy fraction of sediment (>2.8 g cm−3) provide insight into the role of sulfide minerals in Hg deposition. We analyzed fourteen heavy fraction samples for metal composition and six heavy fraction samples by SEM, with a particular focus on the period 3.3–3.2 ka that is characterized by a peak in Feext concentrations and Holocene-maximum Hg concentrations (Figure 5b). We did not identify Hg sulfides in any of the six samples analyzed by SEM, but we found abundant framboidal pyrite in one sample from 3.3 ka with diameters of 10–15 μm (Figure 5a) and Fe, S, and C spectral peaks identified by EDS. A pronounced one-sample peak in concentrations of Fehvy and Shvy at 3.2 ka (Figure 5) has a molar Fe:S ratio of 1:1.79 similar to observed framboidal pyrite and highly elevated concentrations of Ashvy, Aghvy, and Tlhvy [Large et al., 2001]. Although the Hghvy concentration is relatively elevated in this sample, the percent of Hg in the heavy fraction (% Hghvy) is not relatively elevated (Figure 5b).</p><!><p>We first test the hypothesis that atmospheric deposition is the primary source of Hg to Yanacocha by comparing Hg concentrations and sedimentology in the Yanacocha record during the entire record (12.3 to 0 ka) and just the pre-anthropogenic period (defined and used herein as 12.3 to 3.5 ka, based on previous records and historical information [Nriagu, 1979; Martínez-Cortizas et al., 1999; Cooke et al., 2009]). Previous millennial-scale Hg records in lake sediments relate changes in Hg fluxes to groundwater level [Jacobson et al., 2012], lithogenic input from weathering within the catchment [Thevenon et al., 2011], and mobilization of Hg from soils [Cannon et al., 2003]. However, the lack of correlations of Hg concentration with LOI550 and with Ti (Figure 6) show that organic matter and lithogenic input, respectively, do not have significant effects on Hg deposition in Yanacocha. The only statistically significant correlation is between Hg and LOI550 during the pre-anthropogenic period, but this correlation has a very weak effect (R2 = 0.049). The absence of increased Hg deposition when lithogenic input was relatively high during the lake's early stage (~12.3 to 11 ka; Figure 4) indicates that weathering of surrounding bedrock is not a significant source of Hg. Based on these correlations, the small catchment area, and the lack of stream inputs, we conclude that atmospheric Hg deposition is the primary source of Hg to Yanacocha sediments.</p><p>One exception to this interpretation is the brief association of increased Feext concentrations and framboidal pyrite with near-maximum Hg concentrations from 3.3 to 3.2 ka. Framboidal pyrite often contains many heavy metals including Hg (e.g. Schoonen [2004]), presumably due to the affinity that Hg has for S and the large pyrite surface area afforded by the crystallite sub-units within each framboid (e.g., Figure 5a). Chemical preservation of framboidal pyrite is not influenced by diagenesis in lake sediments [Suits and Wilkin, 1998]. Framboidal pyrite is formed either in euxinic water columns or within upper sediments, near the sediment-water interface, where anoxic conditions occur [Suits and Wilkin, 1998]. The relatively large diameters of the observed framboids (10–15 μm; Figure 5a) and low modern water sulfate concentration (238 μg L−1; Table S1) are consistent with formation within the sediment as opposed to within the water column [Wilkin et al., 1996]. Therefore, we hypothesize that framboidal pyrite was formed within Yanacocha's uppermost sediments due to external input of oxidized Fe and S, which may have sequestered Hg from the lake during the period of elevated Feext concentrations from 3.3 to 3.2 ka</p><p>Atmospheric Hg deposition to a lake can occur by oxidation of Hg0 vapor in the atmosphere to Hg2+, that then binds with particles or is dissolved in precipitation, and by atmospheric transport and deposition of Hg-bearing particles [e.g., Mason et al., 1994]. Either form of deposited Hg likely had a residence time on the order of years due to Yanacocha's small catchment size and surrounding colluvium that lacks developed soil. In the sections below, we examine the possible influences of climate (i.e., precipitation and temperature), volcanism, and anthropogenic activity on atmospheric Hg deposition to Yanacocha.</p><!><p>We investigate the possible response of atmospheric Hg deposition to past changes in regional precipitation and temperature during the pre-anthropogenic period using nearby paleoclimate records and co-registered proxy data in the Yanacocha record. Long-term monitoring in North America has found that wet deposition of Hg (Hg bound in precipitation) represents ~50–90 % of modern total atmospheric Hg deposition and can vary seasonally [Prestbo and Gay, 2009]. Temperature has been proposed as a controlling factor on Hg in sedimentary systems either directly, by affecting the thermal stability of Hg in peat [Martínez-Cortizas et al., 1999], or indirectly, through warming-driven increases in algal productivity that scavenges Hg from the lake water column (e.g. Outridge et al. [2007]).</p><!><p>Past precipitation in the central Andes is inferred primarily from lake level records and δ18O records of carbonate lake sediments and cave deposits. Lake level records from Lake Titicaca are interpreted to reflect precipitation amounts on the Altiplano (14–21 °S) and show high lake levels from ~21 to 10 ka, followed by low lake levels from ~8 to 5 ka, [Baker et al., 2001; Rowe et al., 2002]. From ~4 ka to the present-day, Lake Titicaca levels were near modern and are interpreted to reflect relatively high precipitation [Rowe et al., 2002]. Records of precipitation inferred from δ18O values of carbonate lake sediments and cave deposits in central Peru (11 °S) show a similar pattern to Lake Titicaca, although they indicate more gradually increasing precipitation throughout the Holocene [Bird et al., 2011; Kanner et al., 2013]. Precipitation-related changes in biomass burning, which cause Hg release to the air primarily as Hg0 [Friedli et al., 2003], are unlikely due to the rarity of natural fires in the Amazon Basin during the Holocene [Bush et al., 2007].</p><p>We interpret BSi in the Yanacocha record as a proxy for precipitation on the catchment, resulting from runoff delivery of Si to the lake for diatom frustule formation. An inverse correlation between BSi and LOI550 throughout the Yanacocha record (Figure 3) likely reflects the dilution of organic matter deposition with diatoms. We infer high precipitation at the lake from high BSi (low LOI550) prior to ~7 ka and lower precipitation from near minimum BSi (maximum LOI550) between ~7 and 4.5 ka (Figure 4). Continuously below average Ti between ~7 and 4.5 ka supports decreased lithogenic input from runoff during this time. Subsequent to ~4.5 ka, generally high BSi (low LOI550) and approximately average Ti suggest relatively high precipitation. The general pattern of precipitation changes interpreted from BSi in Yanacocha is similar to that inferred from Lake Titicaca [Baker et al., 2001; Rowe et al., 2002] and δ18O records in central Peru [e.g., Kanner et al., 2013]. A shift in hydrology during the appearance of framboidal pyrite at ~3.3 ka is not evident in the Yanacocha sedimentology, and regional proxy records do not show precipitation changes during this time in central Peru [Kanner et al., 2013]. Despite millennial-scale changes in precipitation inferred from BSi in the Yanacocha record, Hg concentrations and fluxes remain relatively constant during pre-anthropogenic time (Figure 4).</p><!><p>While Holocene paleotemperature proxies in the Central Andes are scarce, some paleotemperature information has been inferred from past glacier extents of QIC [Kelly et al., 2012; Thompson et al., 2013; Stroup et al., 2014]. Radiocarbon ages of in situ plants show that QIC was smaller than at present prior to ~7 ka, suggesting relatively warm conditions during this time. A subsequent advance of QIC that overran and entombed plants dating to between ~7 and 5 ka [Thompson et al., 2006, 2013; Buffen et al., 2009], during relatively dry middle Holocene conditions (see Section 5.2.1), was likely influenced by cooling. Relatively constant Hg concentrations and fluxes from ~8 to 5 ka (Figure 4) suggest that regional temperatures did not strongly influence atmospheric Hg deposition in Yanacocha. This finding is consistent with an Hg record from lake sediments in arctic Canada in which there is no relationship between Hg deposition and Holocene temperature changes [Cooke et al., 2012].</p><!><p>Volcanic eruptions with a Volcanic Explosivity Index (VEI) ≥ 6 (i.e., Plinian eruptions that inject volcanic gases into the stratosphere) are known to have occurred throughout the Holocene [Siebert and Simkin, 2002]. Hg records from peat cores in Switzerland [Roos-Barraclough et al., 2002] and ice cores in Wyoming, United States [Schuster et al., 2002] report short-lived (~100-year for peat, ~1–10-year for ice) peaks in Hg deposition, usually manifested as a greater than tripling of Hg flux, that are similar in timing to explosive volcanic eruptions in both the Northern and Southern Hemispheres. Based on the temporal resolution of the Yanacocha record (median = 26 years per sample), we would expect to find Hg peaks during times of known volcanic eruptions. However, Hg deposition in the Yanacocha record during the pre-anthropogenic period is relatively stable, and eruption-related increases in Hg deposition are not distinguishable from the noise (Figure 4). Continuous volcanic degassing and more frequent smaller eruptions may contribute significant amounts of natural Hg to the atmosphere [Pyle and Mather, 2003] but similarly cannot be distinguished in the Yanacocha record.</p><p>The Andean Central Volcanic Zone (CVZ) is located ~300–400 km from Yanacocha (Figure 1) and has hosted a number of Plinian eruptions since ~3.5 ka (Figure 7). The VEI 5 eruption of the volcano Yucamane (~3270 14C yr BP) [Siebert and Simkin, 2002] roughly overlaps in timing with the abrupt increase in Hg and Feext concentrations and framboidal pyrite appearance from ~3.3 to 3.2 ka. Deposition of volcanic sulfate and Fe to Yanacocha from this eruption may have provided adequate reactants for framboid formation within the lake's surface sediments and sequestration of Hg from the water column or volcanic ash. Further evidence for volcanism at ~3.2 ka comes from highly enriched Ashvy, Aghvy, and Tlhvy concentrations in Yanacocha sediments (Figure 5b), which, in addition to having an affinity for framboidal pyrite [Schoonen, 2004; Neumann et al., 2013], are also emitted predominantly from volcanic sources [Kellerhals et al., 2010]. If volcanism were responsible for the sharp increase in Hg concentration at ~3.3 ka, then the Hg is retained in the less dense fraction of sediment (< 2.8 g cm−3) or in nanometer-scale particles because of near-constant % Hghvy during this time (Figure 5b).</p><p>The largest eruption in the CVZ during the Holocene was the VEI 6 eruption of the volcano Huaynaputina, historically dated to February 19, A.D. 1600. Ashfall from this eruption with particle diameters of ~20 μm is registered in ice cores from QIC [Thompson et al., 1986], and lava flows on Huaynaputina have a similar Fe content (~3–6 wt %) to those on Yucamane [Mamani et al., 2008]. Hg concentrations in the Yanacocha record do not register this volcanic eruption, but instead generally decline between A.D. 1590 and 1730 (Figure 7). This finding is consistent with a lake sediment record from Southern Chile that shows relatively constant Hg fluxes within and subsequent to visible tephra layers from three separate Holocene eruptions [Hermanns and Biester, 2013]. In contrast to the peat and ice core records that show volcanic Hg peaks, the overall lack of volcanic events registered in the Yanacocha Hg record from both regional and global eruptions suggests that large volcanic events during the Holocene had negligible decadal to century-scale effects on atmospheric Hg levels.</p><!><p>An early phase of increased atmospheric Hg deposition in the Yanacocha record began at B.C. 1450 (3.4 ka), reached a maximum at B.C. 1200 (3.15 ka), and remained elevated until at least B.C. 500 (2.45 ka; Figure 7). This peak is not associated with a change in any of the other bulk analytes in the Yanacocha record except for a brief peak in Feext concentrations from B.C. 1340 to 1240 associated with the presence of framboidal pyrite and discussed above. A mining dust source of Fe and S for framboidal pyrite formation is unlikely due to the low solubility of most sulfide ore minerals. However, increased concentrations of Cuhvy, Cohvy, Nihvy, Mohvy, and Pbhvy from B.C. 1650 to 1500 (Figure 5b, Figure S5) suggest an early mining dust source to Yanacocha. These metals are commonly found together within the same sulfide deposits and can be accessible at the surface in areas affected by glaciation in Peru [Petersen, 1965]. This period of enhanced chalcophile deposition preceded the abrupt increase in Hg deposition at B.C. 1400 and is concurrent with a slight monotonic increase in Hg concentration and flux. Following the peak in Hg deposition at B.C. 1200, the endurance of elevated Hg deposition (5.0 to 6.8 μg m−2 a−1) for nearly a millennium implies a persistent local anthropogenic source of Hg to Yanacocha. Furthermore, the shapes of Hg concentration and flux peaks, characterized by onsets with abrupt increases and subsequent slow declines to background levels, are similar to pre-industrial anthropogenic peaks found in cores from the headwater lake Laguna Negrilla in Peru (Figure 7) [Cooke et al., 2013] and a saltwater lagoon in France [Elbaz-Poulichet et al., 2011]. Near-constant % Hghvy (Figure 5b) suggests that Hg from mining during this time was likely emitted either as ultrafine (< 65 nm diameter) cinnabar particles or as Hg0/Hg2+ that was subsequently bound to less dense materials.</p><p>The timing of the early phase of Hg deposition in Yanacocha is identical to pre-colonial cinnabar mining registered in the lakes LY1 and LY2 located ~10 km from Huancavelica (Figure 7) [Cooke et al., 2009]. Cooke et al. [2009] found that the Hg deposited during pre-Incan time was primarily bound as cinnabar, and neither an increase in Hg fluxes nor a distinct change in Hg isotopes was observed during pre-Incan time in a sediment core from Laguna Negrilla, located ~200 km southeast of Huancavelica (Figure 1) [Cooke et al., 2013]. This spatial limitation of Hg emissions from Huancavelica would have likely precluded the longer distance transport to Yanacocha, located ~460 km southeast of Huancavelica (Figure 1), which suggests that the early phase of Hg deposition in Yanacocha is from pre-Incan metal use near the catchment.</p><p>We hypothesize that the early phase of anthropogenic Hg deposition in Yanacocha was due to a three-part sequence of events. First, mining of a nearby polymetallic sulfide deposit provided minimal Hg contributions to Yanacocha from B.C. 1650 to 1450. Second, a combination of nearby mining emissions, potential volcanic emissions, and/or an affinity of Hg for framboidal pyrite caused the abrupt increase in Hg deposition from B.C. 1450 to 1200. Third, on-going nearby mining supplied decreasing amounts of Hg to Yanacocha from B.C. 1200 to at least B.C. 500.</p><!><p>A later phase of enhanced atmospheric Hg deposition in Yanacocha is registered from ~A.D. 1480 to 2011. Increased Hg concentrations (55–73 μg kg−1) from ~A.D. 1480 to 1640 (Figure 7) may reflect both cinnabar mining in Huancavelica, first by the Inca from ~A.D. 1450 and then by the Spanish from A.D. 1564 onward [Cooke et al., 2009], and the concurrent growth of Ag refining using Hg amalgamation throughout the Andes beginning ~A.D. 1570 [Robins and Hagan, 2012]. A simultaneous peak in Pbext concentrations from ~A.D. 1500 to 1670 (Figure 7) is similar in timing to the initial use of Pb for smelting Ag ores [Guerrero, 2012]. If Hg was co-transported with aerosol-based smelting emissions, it must either reside as cinnabar with particle diameters less than 65 nm (because % Hghvy does not change substantially [Figure 5b]) or as Hg adsorbed to less dense aerosols. Atmospheric transport of Hg from Huancavelica to Laguna Negrilla between ~A.D. 1450 and 1650 is supported by a pronounced increase in Hg fluxes (~10 fold increase, up to 82 μg m−2 a−1; Figure 7) and a shift in the mass-dependent fractionation of Hg isotopes [Cooke et al., 2013]. The relatively small increase in Hg deposition in Yanacocha compared to Laguna Negrilla suggests that Hg emissions from Huancavelica were, at least during the time of Inca control (~A.D. 1450–1564), predominantly in the solid phase and decreased in spatial extent with distance from Huancavelica.</p><p>The shift to elemental Hg production for silver mining between A.D. 1564 and 1810 likely influenced more globally distributed Hg emissions [Nriagu, 1993; Robins and Hagan, 2012]. Decreased Hg concentrations and fluxes in the Yanacocha record from ~A.D. 1650 to 1750 are followed by a general increase coincident in timing with estimated maximum Hg0 emissions in South and Central America from ~A.D. 1750 to 1810 [Nriagu, 1993]. However, increasing Hg fluxes are not evident during this period in Laguna Negrilla (Figure 7) or in two lakes ~65 km west of Yanacocha [Beal et al., 2013]. The spatially inconsistent signal of Hg fluxes in this region suggests that mining dust continued to contribute significant amounts of Hg to certain lakes and that any increase in Hg deposition due to anthropogenic Hg0 emissions was relatively negligible during the preindustrial period. A more localized distribution of preindustrial Hg emissions is consistent with new chemical modeling by Guerrero et al. [2013] that shows solid calomel (Hg2Cl2) comprised up to 90% of Hg losses from Ag refining in the Hispanic New World. Post-industrial increases in Hg deposition in the Yanacocha record were likely caused by global Hg0 emissions.</p><!><p>The extent of anthropogenic modification to natural Hg cycling is typically represented by an Hg flux ratio, which is the ratio of recent Hg fluxes to background fluxes that occurred at some earlier time (i.e., from A.D. 1800 to 1850 in most sediment records). Table 1 lists mean Hg concentrations and fluxes in the Yanacocha record for key time periods during the Holocene, weighted on the length of time each sample represents. Because of the evidence for significant pre-A.D. 1850 anthropogenic deposition in the Yanacocha record, natural Hg fluxes are likely only represented prior to 3.5 ka in this record. Whereas Hg concentrations remain remarkably constant from 12.3 to 3.5 ka, Hg fluxes gradually decrease with increasing age (Table 1). This is likely an artifact of the age-depth model. We therefore calculate a best approximation of the Hg flux ratio in the Yanacocha record as time-weighted mean post-A.D. 1980 fluxes (4.0 μg m−2 a−1) over pre-3.5 ka fluxes (1.4 μg m−2 a−1), yielding a flux ratio of 3.0 ± 1.5. This flux ratio, which accounts for total anthropogenic modification to the global Hg cycle during the Holocene, is in good agreement with sediment records that use the period A.D. 1800 to 1850 as background fluxes from two other lakes in southeastern Peru (i.e., 4.0 ± 1.0 [Beal et al., 2013]) and from lakes around the world (i.e., on average 3.5 [Biester et al., 2007]). The discrepancy between our Holocene Hg flux ratio (3.0 ± 1.5) and the modeled 7.5-fold enrichment since B.C. 2000 by Amos et al. [2013] indicates that preindustrial Hg emissions were either not as globally distributed as assumed in the model or were not as persistent in labile surface reservoirs. Revised accounting for losses of Hg0 to the atmosphere from preindustrial mining may improve the accuracy of global Hg models and help reconcile them with sedimentary records.</p><!><p>During the pre-anthropogenic period, atmospheric Hg deposition recorded in Yanacocha was relatively constant and did not vary with changes in local and regional climate. Holocene volcanic eruptions are generally not registered in the Hg record despite a number of Plinian eruptions that occurred both globally and within the Andean CVZ. An early phase of enhanced Hg deposition in Yanacocha began in B.C. 1450 (3.4 ka) likely due to a combination of nearby mining emissions and volcanic input of Fe and S that led to framboidal pyrite formation and possible Hg sequestration between ~B.C. 1340 and 1240. The endurance of this early phase of enhanced Hg deposition until B.C. 500 is coincident with known pre-Incan cinnabar mining in Huancavelica. The limited spatial distribution of Hg emissions from Huancavelica and the magnitude of Hg fluxes during this early phase, which are greater than modern fluxes, indicate a separate and nearby mining source of Hg to Yanacocha, likely from within the Cordillera Vilcanota. Increased concentrations of Hg and Pbext from ~A.D. 1480 to 1640 suggest sources of Hg to the lake first from Incan cinnabar mining and then from colonial Hg-production and Ag-refining. The agreement of the Holocene flux ratio determined from the Yanacocha record with flux ratios determined from post-industrial lake sediment records suggests that preindustrial Hg emissions either were not well distributed globally or did not have a long-lasting impact on the global atmospheric Hg burden.</p>

PubMed Author Manuscript

Contrast-Matched Small Angle X-ray Scattering from a Heavy Atom-Labeled Protein in Structure Determination: Application to a Lead-Substituted Calmodulin-Peptide Complex

The information content in one-dimensional solution X-ray scattering profiles is generally restricted to low-resolution shape and size information that, on its own, cannot lead to unique three-dimensional structures of biological macromolecules comparable to all-atom models derived from X-ray crystallography or NMR spectroscopy. Here we show that contrast-matched X-ray scattering data collected on a protein incorporating specific heavy atom labels in 65% aqueous sucrose buffer can dramatically enhance the power of conventional small and wide angle X-ray scattering (SAXS/WAXS) measurements. Under contrast-matching conditions the protein is effectively invisible and the main contribution to the X-ray scattering intensity arises from the heavy atoms, allowing direct extraction of pairwise distances between them. In combination with conventional aqueous SAXS/WAXS data, supplemented by NMR-derived residual dipolar couplings (RDCs) measured in a weakly aligning medium, we show that it is possible to position protein domains relative to one another within a precision of 1 \xc3\x85. We demonstrate this approach with respect to the determination of domain positions in a complex between calmodulin, in which the four Ca2+ ions have been substituted by Pb2+, and a target peptide from myosin light chain kinase. The uniqueness of the resulting solution is established by an exhaustive search over all models compatible with the experimental data, and could not have been achieved using aqueous SAXS and RDC data alone. Moreover, we show that the correct structural solution can be recovered using only contrast-matched SAXS and aqueous SAXS/WAXS data.

contrast-matched_small_angle_x-ray_scattering_from_a_heavy_atom-labeled_protein_in_structure_determi

<p>Small and wide angle X-ray scattering (SAXS/WAXS) in solution yield one-dimensional profiles that are determined by the pairwise distances between all atoms in a molecule.1 Because of the convoluted nature of SAXS/WAXS, it is not possible, outside of the low q-range, to directly relate features of the scattering profiles to a particular structure. Further, it is generally not feasible to derive unique three-dimensional structures from one-dimensional curves, as many models may be compatible with a given scattering profile. On the other hand, direct refinement against SAXS/WAXS data in combination with other constraints, such as those from NMR data, can be extremely powerful.2 In this communication, we investigate the utility of scattering arising from a few heavy atoms, such as Pb2+, under contrast-matched conditions at which the protein is rendered effectively invisible at low scattering angles by using a 65% aqueous sucrose solution as a solvent.3 The contrast-matched scattering profiles arise from only a small number of heavy atoms and should therefore effectively constrain the distances between them. We demonstrate the utility of contrast-matched SAXS using, as an example, a calmodulin (CaM)-myosin light chain kinase (MLCK) peptide complex in which the four coordinated Ca2+ ions have been substituted by Pb2+. We show that the combination of contrast-matched SAXS, aqueous SAXS/WAXS and NMR-derived residual dipolar couplings (RDCs)4 is sufficient to define the positions of the two domains of CaM within a precision of 1 Å, and that the contrast-matched SAXS data are critical for discriminating between several models that are fully consistent with aqueous SAXS/WAXS and RDC data.</p><p>Examination of Ca2+-loaded CaM-peptide complexes solved by NMR and crystallography reveals considerable variability in the relative positions of the N- and C-terminal domains (Fig. 1). While some of this variability can be attributed to differences in the target peptide sequences leading to distinct binding modes,5 even unique binding modes exhibit substantial structural differences. Further, none of these structures fit the X-ray scattering data within experimental error (Figs. S4 and S5 and Table S1) and only in a handful of cases are the fits of the RDC data (measured in a weakly aligning medium of phage pf16) to the whole complex comparable to those for the individual domains (Table S1).</p><p>Substitution of Ca2+ by Pb2+ does not significantly perturb the structure of CaM (as judged by crystallography,7 NMR and SAXS/WAXS), its function,8 or binding affinity for the MLCK peptide (see Supplementary). Similarly, the structure of the CaM-MLCK complex appears unperturbed by the addition of sucrose as judged by NMR (see Supplementary).</p><p>Rather than refine directly against all available SAXS/WAXS and NMR data by simulated annealing, starting from a limited set of initial coordinates as is commonly done,2 we chose to evaluate the agreement with the experimental data by exhaustively sampling all possible, stereochemically feasible, relative geometries of the two domains of CaM. The latter approach guarantees that the best-fitting solution corresponds to the global minimum of the target function and is not simply one of many possible solutions, thereby removing the issue of model degeneracy which is universally recognized as one of the main pitfalls of biomolecular SAXS.</p><p>Fits of the RDC data to the individual domains of CaM from a large array of crystal structures indicated that some of the most accurate representations of the N- (residues 1–75) and C-(residues 82–148) domains in solution were those from the 1.7 Å resolution structure of a CaM-peptide complex from CaM-dependent protein kinase I (PDB accession code 1MXE)9 with RDC R-factors of 17% (Table S1). We therefore used the N- and C-domain coordinates from the 1MXE structure throughout this study.</p><p>The rotational and translational parameters (three of each) describing the position of the two domains of CaM were systematically sampled in 2° and 1 Å steps, respectively, to generate an initial set of ~4.5x1011 geometries with a spatial resolution of ~1 Å. Structures were removed that (i) had steric clashes <2.5 Å between backbone or Cβ atoms of the two domains; (ii) exhibited an increase of >10% in the RDC R-factor (equivalent to ~10° orientational uncertainty) relative to the minimum value obtained by rigid-body minimization of the relative domain orientations; or (iii) whose radius of gyration (Rgyr) lay outside a range of 13–19 Å. (Note the experimental Rgyrobtained from the aqueous SAXS data via Guinier and P(r) analyses is 17.8±0.4 Å for the CaM-MLCK complex with Ca2+ and 18.3±0.4 Å for the Pb2+ substituted complex). For relative domain geometries that passed the latter requirements, rotation and translation of the MLCK peptide taken from the solution structure of the CaM-MLCK complex (2BBM),10 were obtained by minimization of a target function comprising the intermolecular NOE distance restraints from the 2BBM deposition and a repulsion term between the heavy atoms of the two CaM domains and MLCK to prevent atomic overlap. Additional geometries were then excluded in instances with steric clashes <2.5 Å between the heavy atoms of MLCK and the two domains of CaM or with NOE distance restraint violations >3 Å. Finally the linker (residues 76–81) was built from a PDB database of 6250 non-redundant protein chains encompassing a total of 2.2x106 residues. All contiguous 8-residue stretches that did not contain either Gly or Pro (consistent with the composition of the CaM linker) were selected. The terminal residues of these 8-residue fragments were best-fitted to the backbone atoms of Lys75 of the N-domain and Glu82 of the C-domain for each CaM domain geometry. All linkers exhibiting backbone rms differences <1 Å relative to the coordinates of Lys75 and Glu82 were retained. The backbone linker geometries that did not exhibit steric clashes <2.5 Å with the two CaM domains and the MLCK peptide were processed further, and the best-fitting six-residue backbone segment corresponding to Met76-Ser81 was decorated with the appropriate side chains using residue-specific rotamers,11 avoiding steric clashes with other atoms of the complex.</p><p>The calculated SAXS/WAXS curves for the resulting 75,000 models of the CaM-MLCK complex were best-fitted to the experimental data (recorded on beamlines 12-IDC and 12-IDB, APS) using the AXES formalism which makes use of explicit water molecules to model the solvent boundary layer.12 The scattering intensity from the Pb-substituted sample in 65% sucrose was predicted via the Debye formula from the coordinates of the Pb sites. Agreement of the aqueous SAXS/WAXS and contrast-matched SAXS data sets with structural models was evaluated using χ2 statistics.</p><p>As is clear from Fig. 2, the discriminating power of the Pb-substituted contrast-matched SAXS data is essential for selecting between the candidate solutions. Three clusters of structures fit equally well to the aqueous SAXS/WAXS data (Figs. 2A and 3A), and the RDCs (Fig. 3C) within a few percentage points of their minimum values (Table 1). These clusters, depicted in blue (Cluster I), green (Cluster II) and red (Cluster III) in Figs. 2–4 also satisfy the NOE distance restraints between CaM and the bound MLCK peptide (Fig. 2B) indicating that all three capture the placement of the MLCK peptide in the complex within experimental error. The only observable that discriminates between the three clusters is the Pb-substituted contrast-matched SAXS data, and only one cluster, namely Cluster I, satisfies the contrast-matched SAXS data within experimental error (Figs. 2A, 3B and Table 1).</p><p>A structural comparison of the three clusters is shown in Fig. 4 and the rms differences within and between the clusters is provided in Table 2. For Cluster I, the relative position of the two CaM domains is defined with a precision of 1 Å. When best-fitted to the N-domain, the backbone rms displacements of the C-domains of Clusters II and III relative to Cluster I are much larger, 1.8 and 5.4 Å, respectively, well outside the coordinate precision of Cluster I. The structural differences between the three clusters reflect systematic lengthening of the Pb-Pb distances from Cluster I to Clusters II and III (Fig. 4 and Table 1).</p><p>Given the discriminating power of the Pb-substituted contrast-matched SAXS data, can such data be directly used to accurately extract Pb-Pb distances? To assess this we carried out fits to the contrast-matched SAXS data using a Monte Carlo-generated random sampling of 4-atom geometries to represent the Pb sites within CaM (see Supplementary). We consider three cases: 4 variable inter-domain Pb-Pb distances with the two intradomain distances fixed to the values measured from the domain coordinates (11.7 Å); 5 variables comprising the 4 inter-domain Pb-Pb distances with the two intradomain distances constrained to have the same value; and 6 variables in which all four interdomain and two intradomain Pb-Pb distances are allowed to vary. The results are summarized in Table 3. For the 4-variable case, corresponding to knowledge of the atomic structures of the two CaM domains, the average interdomain Pb-Pb distances derived from the contrast-matched SAXS data have uncertainties of only ~1 Å and are in excellent agreement with the corresponding Pb-Pb distances in Cluster I (cf. Table 1). Introducing an additional variable, with the assumption that the two intradomain Pb-Pb distances are the same, results in larger uncertainties, but agreement with the Cluster I Pb-Pb distances is still excellent and within the uncertainties of the distance estimates. This case could be modeled based on the high sequence similarity of the N- and C-domains of CaM if their atomic structures were unknown. Finally, the completely unrestrained 6-variable simulation still results in reasonable agreement with the longer, interdomain Pb-Pb distances from the all-atom model but exhibits marginal agreement with the correct intradomain Pb-Pb separations. These calculations therefore indicate that up to 5 (from a total of 6) Pb-Pb distances can be extracted both precisely and accurately from a single curve of the contrast-matched SAXS data arising from 4 Pb labels. Accelerated deterioration of the uncertainties of the shorter separations is likely due to increased uncertainty at wider angles and the limited qmax of the fitted data, as the impact of the shorter Pb-Pb distances is increasingly felt at higher q values.</p><p>In light of the above results we sought to investigate whether the contrast-matched SAXS and aqueous SAXS/WAXS data alone are sufficient to arrive at the correct solution without recourse to filtering by RDCs and intermolecular NOE distance violations. Using the same grid procedure, structures were filtered by the fits to the X-ray scattering data using a cutoff of χ 2SAXS,water < 1.69 and χ2SAXS,sucrose < 0.72, the absence of steric clashes, an Rgyr of 13–19 Å, and the ability to form a linkage between the N- and C-domains. The results in Fig. 5 indicate that the cluster with the lowest normalized SAXS fitness score yields solutions with a coordinate accuracy of better than 1 Å by reference to Cluster I.</p><p>While this work capitalizes on the ability of CaM to specifically bind Pb2+ in place of Ca2+, applications of this approach can be readily extended beyond metal-binding proteins by incorporating heavy atom ions such as Pb2+ or Hg2+ into EDTA moieties conjugated via disulfide bonds to engineered surface cysteines as routinely done in NMR paramagnetic relaxation enhancement studies.13 Although the EDTA-metal moiety samples a large region of conformational space, the metal-metal separations measured by contrast-matched SAXS are simple linear averages of all the conformations present in solution and each metal atom can therefore be represented by a single average position.</p><p>Although, in principle, similar information has been obtained from neutron scattering of 240Pu,14 or X-ray scattering of DNA with attached gold nanoclusters,15 the present approach does not suffer from the lower signal-to-noise of small angle neutron scattering and complications of having to deal with a highly radioactive isotope, or the necessity to decompose the observed data into individual scattering functions from measurements on a series of samples.</p><p>Finally, the results obtained without recourse to filtering by NMR data suggest that accurate 'triangulation-driven' assembly of multi-component protein architectures, based only on a combination of aqueous and contrast-matched/heavy-atom-labeled SAXS data, is feasible providing the structures of the individual subunits are known.</p>

PubMed Author Manuscript

Nano-encapsulation Method for High Selectivity Sensing of Hydrogen Peroxide inside Live Cells

Reactive oxygen species (ROS) are ubiquitous in life and death processes of cells1, with a major role played by the most stable ROS, hydrogen peroxide (H2O2). However, the study of H2O2 in live cells has been hampered by the absence of selective probes. Described here is a novel nanoprobe (\xe2\x80\x9cnanoPEBBLE\xe2\x80\x9d) with dramatically improved H2O2 selectivity. The traditional molecular probe, 2\xe2\x80\xb2,7\xe2\x80\xb2-dichlorofluorescin (DCFH), which is also sensitive to most other ROS, was empowered with high selectivity by a nanomatrix that blocks the interference from all other ROS (hydroxyl radical, superoxide, nitric oxide, peroxynitrite, hypochlorous acid and alkylperoxyl radical), as well as from enzymes such as peroxidases. The blocking is based on the combination of multiple exclusion principles: time barrier, hydrophobic energy barrier, size barrier. However, H2O2 sensitivity is maintained down to low nM. The surface of the nanoprobe was engineered to address biological applications, and the power of this new nanoPEBBLE is demonstrated by its use on RAW264.7 murine macrophages. These nanoprobes may provide a powerful chemical detection/imaging tool for investigating biological mechanisms related to H2O2, or other species, with high spatial and temporal resolution.

nano-encapsulation_method_for_high_selectivity_sensing_of_hydrogen_peroxide_inside_live_cells

<p>H2O2 is a key player in intracellular signaling2,3, host defense and phagocytosis4,5,6,7, wound detection and healing8, as well as in the initiation of several pathophysiological cascades that terminate in cell death or adaptation9,10. H2O2 is a by-product from other ROS related reactions, such as the dismutation of the superoxide radical anion (O2−) under the mediation of superoxide dismutase. It is also a common precursor for the further generation of other, more damaging, ROS, such as hydroxyl radical (·OH), hypochlorous acid (HOCl) and peroxynitrite (ONOO−)11,12,13. H2O2 is a relatively stable ROS, amenable to measurement, and, as such, has drawn much interest. However, specific and readily deployable tools for reliable and precise physiological/intracellular measurements of H2O2 are still lacking.</p><p>Many ROS sensitive molecular probes have severe limitations, such as poor selectivity and instability, thus often leading to spontaneous oxidation (Supplementary figure 1)14,15. Further complications, such as the inability of specific probes to permeate biological membranes and the potential interference by unwanted enzymatic reactions, need to be considered for quantitative measurements under physiological conditions. DCFH (2′,7′-dichlorofluorescin) dye is highly sensitive and is one of the most widely used probes for the detection of ROS and oxidative stress in biolological systems16,17,18. For intracellular measurements, the non-fluorescent and lipophilic precursor molecule, DCFDA (2′,7′-dichlorofluorescin diacetate), is used to freely pass into the plasma membrane. The DCFDA dye then converts into the hydrophilic DCFH, inside the cell, by losing two acetate groups due to esterase activity. DCFH can readily generate the strongly fluorescent and hydrophilic product, 2′,7′-dichlorofluorescein (DCF), upon oxidation by ROS generated inside of cells. Although this dye has often been used to monitor H2O2, its poor selectivity strictly limits the utility of the measurements to systems that are relatively free from interferences. Furthermore, its pH sensitivity is a well known drawback19. Also, its use often depends on a catalytic activity of peroxidases, which can complete the transition to DCF even without ROS, and thus hurting the accuracy of detection16,17. Those limitations hamper its application in most biological situations.</p><p>Nanoparticle platforms provide a working solution that addresses many of the problems identified above. Our group has developed nanoparticle based biosensors, termed PEBBLEs (Photonic Explorers for Biomedical uses with Biologically Localized Embedding)20,21,22, for the intracellular detection of a large number of biologically important analytes. NanoPEBBLE sensors provide several important advantages over conventional sensors such as molecular probes: minimal physical/chemical invasiveness, minimal interference between probe and cell ingredients, a wider choice of probe materials, synergistic detection schemes, biological targeting, autofluorescence background suppression, and a ratiometric measurement capability23. Here we describe an added benefit: induced selectivity.</p><p>Recently, several attempts to improve H2O2 detection have been reported, based on either chemical modification of the ROS probes or nanoparticle-based approaches. Gong et al.24, Dickinson et al.25, Maeda et al.26 and Setsukinai et al.14 suggested various chemical derivatizations of DCFDA. Lee et al.27,28 developed a polymer nanoparticle based on a peroxalate chemiluminescence (POCL)29, which is a well known chemiluminence technique for H2O2 detection. Kim et al.30 and Poulson et al.31 reported nanoparticles that embed peroxidases in hydrophilic polymer matrices. Both approaches are based on the selectivity of peroxidases and their catalytic effect. In spite of these advances, some limitations still remain, such as imperfect selectivity, interferences by enzymes and by non-ROS chemicals, unverified particle biocompatibility, leaching from particles, and insufficient dynamic ranges of detection.</p><p>In contrast, the approach given below utilizes the nanoparticle as a screening device that empowers high H2O2 selectivity to an otherwise non-selective probe, DCFH. Such induced selectivity can be achieved by a delicate complimentarity of properties between the nanoparticle matrix and the embedded molecular probe (Scheme 1). In addition, this nanoprobe has other typical benefits of nanoPEBBLEs, such as biocompatibility, protection of content and targeting capability.</p><p>Ormosil nanoplatforms, which are bio-inert, relatively hydrophobic but still water-suspendable, with a 100 nm size (Supplementary figure 2), were synthesized by a previously described method32,33. The nanoparticle surface is readily modifiable by amine functionalization (resulting in about 20 mV of zeta potential, indicating a positive surface charge). DCFDA was loaded into preformed Ormosil particles by a solvent displacement technique (Supplementary figure 3)34,35, wherein hydrophobic dye molecules and particles are dissolved in a volatile organic solvent first, mixed with aqueous media and then the solvent is evaporated. The resulting nanoparticle retains the dye molecule in its inactive/lypophilic form, DCFDA. We discovered that the DCFDA dye shows H2O2 sensitivity without conversion to the activated hydrophilic intermediate (DCFH), in contrast to previous reports16–18 (Table 1 and Supplementary figure 4). The DCFDA nanoprobes (0.1 mg/mL) displayed an increase in fluorescence intensity over time, upon addition of H2O2. The fluorescence intensity value reached a plateau after very long time (supplementary figure 5). The plateau value varied monotonously with H2O2 concentration. The early-time rate of fluorescence generation also depends on H2O2 concentration, as demonstrated in Table 1. The nanoprobes detected H2O2 over a concentration range of 10 – 100 μM—a significantly wider detectable range than previous probes'27,30,31. These DCFDA nanoprobes showed negligible dye leaching, confirming that the H2O2 response was not from released dye molecules in solution (Figure 1).</p><p>The H2O2 selectivity of the DCFDA nanoprobe and the DCFDA free dye has been examined by monitoring the change in fluorescence intensity upon exposure to various ROS interferents, such as ·OH, O2−, ·NO, ONOO−, HOCl, and ROO· (alkylperoxyl radical) (Table 2). The DCFDA free dye was prepared by solubilizing DCFDA in a trace amount of DMSO and diluting it in excess aqueous medium. The tests were performed as previously reported for the DCFH dye probe14. We note that when the DCFH dye was used as a H2O2 probe, it suffered very large interferences from most ROS14. For instance, the fluorescence increase of the DCFH dye due to ·OH, ONOO− and ROO· was 40, 35 and 4 times that due to H2O2, respectively, under the same tetsing conditions as for Table 2.</p><p>The DCFDA nanoprobes, in comparison to DCFDA dye, showed a significantly enhanced selectivity towards H2O2 over other ROS. Upon exposure to these ROS, DCFDA free dye showed noticeable increases in fluorescence intensity, except for ROO·. In contrast, the DCFDA nanoprobes exhibited significantly less changes (see Table 2). In the case of ·OH interference, the slight decrease in the nanoprobe response just reflects the reduced concentration of H2O2, by the reaction to produce ·OH, suggesting negligible interference from ·OH itself. Notably, ·OH has the shortest lifetime (nsec or shorter) among all the ROS36. Our previous study on ·OH sensing nanoprobes showed that ·OH could only be detected at the surface of nanoprobes, but not inside them. This suggests the general principle that nanoparticle matrixes may filter species based on lifetime, thus achieving improved selectivity towards longer-lived ROS. In the case of the HOCl interference, the decrease in the nanoprobe's fluorescence may be because of a pH change in the testing solution, due to HCl production from HOCl19. The H2O2 response of the DCFDA nanoprobes shows a monotonic pH dependent behavior (Supplementary figure 6)</p><p>The ROS like O2−, ·NO, ONOO−, HOCl, and ROO· (alkylperoxyl radical) are moderately unstable ROS molecules, with lifetimes of msec or longer. Exclusion by short lifetime alone may not be sufficient to explain why they are not detected by the nanoprobes. Note that our previous studies have shown that singlet oxygen (1O2), with a lifetime of only about 2 μsec in aqueous media, can be successfully detected with the same Ormosil-based nanoprobes37. It is well known that organic solvents greatly stabilize 1O2, resulting in extension of lifetime38. We hypothesize that the relatively hydrophobic Ormosil matrix must offer a similarly favorable environment for lipophilic ROS, such as 1O2 and H2O2. On the other hand, it is reasonable to assume that the Ormosil matrix would behave as a hydrophobic energy barrier, reducing the stability of most other polar ROS above. In the presence of ROO·, neither the free DCFDA dye nor the nanoprobe exhibited recognizable fluorescence generation, in contrast to the DCFH dye probe14.</p><p>Another important advantage of using DCFDA encapsulated in nanoprobes is the avoidance of unwanted enzymatic interactions. The DCFDA nanoprobes showed no interference from horseradish peroxidase (HRP) but the DCFD free dyes showed significant interference (Table 2). Typically, the ROS assay using DCFH dye is performed in association with HRP16,17, for faster and more vivid detection, despite the fact that DCFH can be oxidized by HRP alone, without ROS16,17. Although this effect is not a significant drawback for general ROS detection, it can cause a very significant error in the H2O2-specific quantification. The matrix resistance to HRP interference is due to the large size of the HRP, being too large to penetrate into the Ormosil matrix. The same size exclusion may also eliminate potential confounding interactions from other enzymes or macromolecules. For example, the nanoprobes would be free from unwanted conversion of DCFDA to hydrophilic DCFH by the intracellular esterase, retaining their H2O2 selectivity.</p><p>Based on the improved H2O2 selectivity, H2O2 generation from stimulated macrophages, which are well known for oxidative bursts, could be evaluated quantitatively. The surfaces of the DCFDA nanoprobes were engineered for in vitro measurement as follows: First, the DCFDA nanoprobes were labeled with a secondary fluorophore, Alexa Fluor 568 succinimidyl ester, because otherwise the nanoprobes are invisible until oxidation (Figure 2-a). Second, a membrane penetrating peptide, cystein terminated TAT peptide (TAT-cys), was conjugated onto the surface of the nanoprobes, so as to deliver them directly to the cytosol39,40. As the DCFDA nanoprobe is pH sensitive (Supplementary figure 6), the nanoprobes must be actively delivered to the cytosol, bypassing phagocytotic uptake of macrophages, which is associated with significant acidification41.</p><p>The generation of H2O2 from RAW264.7 murine macrophage cells, stimulated by a macrophage stimulant, N-formyl-Methiony-L-Leucyl-L-Phenylalanine (fMLP)42, was monitored by DCFDA nanoprobes. Two control experiments were performed in parallel, one with the nanoprobes without TAT on the surface, so as to observe the effect of cytosolic delivery, and the other with the TAT-conjugated nanoprobes but without the addition of fMLP, so as to confirm that the response was induced by an oxidative burst. Macrophages without incubation with nanoprobes and without addition of fMLP were used to determine the autofluorescence background of the cells.</p><p>The fluorescence intensity changes of the nanoprobes were monitored over 6 hours after addition of fLMP, until the fluorescence was stabilized (Figure 2-b). The TAT conjugated nanoprobes, with stimulation, showed a fast increase of emission intensity for 1 hour, then the rate of increase slowed down until 4 hours, and stabilization occurred later on. The quantity of H2O2 detected by the nanoprobes was assessed by comparison with the final product, DCF (from Sigma-Aldrich), applying a 1:1 stoichiometry in the reaction between H2O2 and DCFDA dye molecules (overall, DCFDA+H2O2→DCF+H2O), based on a literature method15,43,44 (Supplementary figure 7). Based on the Alexa 568 fluroescence, we estimated the concentration of nanoprobes in the cells to be about 12 mg/mL. This guarantees that there were enough DCFDA nanoprobes inside each cell for a reliable measurement. The H2O2 concentration detected by the TAT conjugated nanoprobes, with fMLP stimulation, was equivalent to 18 nM. This result shows that low nM concentrations of H2O2 can indeed be successfully detected in live cells by these nanoprobes. The nanoprobes without TAT exhibited a significantly reduced response to H2O2, which may imply that the nanoprobe's sensitivity towards H2O2 was hindered by the phagocytotic acidification.</p><p>In summary, the improved H2O2 selectivity of the DCFDA Ormosil nanoprobes is an outcome of a synergistic combination of three different matrix exclusion principles: time barrier, hydrophobic energy barrier, and size barrier. The resultant high specificity is assisted further by negligible dye leaching and by a wide dynamic range, thus enhancing the accuracy and applicability of the measurements. It appears that these DFCDA nanoprobes may provide a powerful chemical detection/imaging tool for investigating biological mechanisms related to H2O2, with high spatial and temporal resolution. Similar principles and nanoplatforms may be applied to the determination of other important biochemical species.</p><p>Negligible dye leaching from DCFDA nanoprobes. Filtrate of DCFDA nanoprobes did not respond to H2O2, indicating that the reaction between DCFDA dye and H2O2 was confined to the inside of the nanoparticle matrix. Note that the change of filtrate's fluorescence values are so small that they are barely visible in the chart. This Figure validates the hypothesis of induced selectivity by nanoencapsulation.</p><p>In vitro H2O2 detection performed with DCFDA nanoprobes. a) Confocal image showing cellular uptake of DCFDA nanoprobes. The nanoprobes were incubated with RAW264.7 macrophage cells overnight. The nanoprobes were delivered to the cytosol by the TAT peptide function. The cells were stained by Calcein-AM (green colored, ex: 488 nm, em: 525 nm) and the nanoprobes were visualized by a secondary label, Alexa 568 (red colored, ex: 568 nm, em: 600 nm). b) Fluorescence increase over time due to H2O2 generation from macrophages upon stimulation with fMLP: TAT-conjugated nanoprobes (◆), control without TAT (■), and control without stimulation (▲). The DCFDA nanoprobes showed a significantly larger increase of fluorescence from stimulated cells than from the control (caused by auto-oxidation or natural H2O2 generation).</p><p>Schematic representation of induced H2O2 selectivity by encapsulating DCFDA into relatively hydrophobic Ormosil nanoparticles. The left scheme shows that DCFH (activated form) can be oxidized by a variety of interferents. The right scheme shows that a relatively hydrophobic nanoparticle matrix encapsulating DCFDA (precursor form) can filter the interferences, thus only allowing penetration of H2O2, resulting in high selectivity towards this specific analyte.</p><p>The DCFDA nanoprobes' H2O2 sensitivity vs concentration. DCFDA nanoprobes show a fluorescence generation that increases monotonically with the H2O2 concentration.</p><p>DCFDA nanoprobe resistance to interferences from other ROS and HRP. The fluorescence generation upon introduction of various ROS interferents is compared between a solubilized DCFDA free dye solution (dissolved in a trace amount of DMSO and then diluted in an excess amount of aqueous media) and the DCFDA nanoprobes solution. Note, however, that the activated free dye, DCFH, is much more sensitive to ROO· (in Supplementary figure 8-f).</p><p>Generated from Fenton reaction (Fe2+ + H2O2), ferrous chlorate (final 1 μM) and H2O2 (final 8.7 μM). Note that the fluorescence increase here results from both ·OH radical and H2O2.</p><p>Generated by ionization of KO2 in aqueous medium, (final 100 μM O2−)</p><p>From ·NO saturated stock solution, (final 20 μM ·NO)</p><p>From commercially available ONOO− solution (final 3.5 μM, from Cayman Chemicals)</p><p>Generation from ionization of NaOCl (final 3 μM −OCl)</p><p>Generated by thermolysis of 2,2-Azobis(2-amidinopropane)dihydrochloride in 37 °C (final 100 μM ROO·)</p><p>HRP was added into the solution (final 10 μM HRP).</p>

PubMed Author Manuscript

Impact of Macroporosity on Catalytic Upgrading of Fast Pyrolysis Bio‐Oil by Esterification over Silica Sulfonic Acids

AbstractFast pyrolysis bio‐oils possess unfavorable physicochemical properties and poor stability, in large part, owing to the presence of carboxylic acids, which hinders their use as biofuels. Catalytic esterification offers an atom‐ and energy‐efficient route to upgrade pyrolysis bio‐oils. Propyl sulfonic acid (PrSO3H) silicas are active for carboxylic acid esterification but suffer mass‐transport limitations for bulky substrates. The incorporation of macropores (200 nm) enhances the activity of mesoporous SBA‐15 architectures (post‐functionalized by hydrothermal saline‐promoted grafting) for the esterification of linear carboxylic acids, with the magnitude of the turnover frequency (TOF) enhancement increasing with carboxylic acid chain length from 5 % (C3) to 110 % (C12). Macroporous–mesoporous PrSO3H/SBA‐15 also provides a two‐fold TOF enhancement over its mesoporous analogue for the esterification of a real, thermal fast‐pyrolysis bio‐oil derived from woodchips. The total acid number was reduced by 57 %, as determined by GC×GC–time‐of‐flight mass spectrometry (GC×GC–ToFMS), which indicated ester and ether formation accompanying the loss of acid, phenolic, aldehyde, and ketone components.

impact_of_macroporosity_on_catalytic_upgrading_of_fast_pyrolysis_bio‐oil_by_esterification_over_sili

<!>Introduction<!>Catalyst characterization<!><!>Catalyst characterization<!>Esterification of model carboxylic acids<!><!>Esterification of thermal pyrolysis bio‐oil<!><!>Esterification of thermal pyrolysis bio‐oil<!><!>Esterification of thermal pyrolysis bio‐oil<!>Conclusions<!>Experimental Section<!>Conflict of interest<!>

<p>J. C. Manayil, A. Osatiashtiani, A. Mendoza, C. M. Parlett, M. A. Isaacs, L. J. Durndell, C. Michailof, E. Heracleous, A. Lappas, A. F. Lee, K. Wilson, ChemSusChem 2017, 10, 3506.</p><!><p>Biofuels have an important role to play in mitigating anthropogenic climate change arising from the combustion of fossil fuels.1 In the context of energy, despite significant growth in fossil fuel reserves, great uncertainties remain in the economic and environmental impact of exploitation, and crucially, approximately 65–80 % of such carbon resources cannot be burned without breaching the United Nations framework convention on climate change (UNFCC) target to keep the global temperature rise this century well below 2 °C. Biofuels will prove critical in helping many countries meet their renewable energy commitments, which for the UK are 15 % by 2020, alongside greenhouse gas (GHG) emission reductions of 34 % by 2020 and 80 % by 2050 (compared with 1990 levels). They also represent drop‐in fuels able to utilize existing pipeline and filling station distribution networks.2 Thermochemical processing of waste biomass such as lignocellulosic materials sourced from agriculture or municipal waste offers a promising route to biofuels through pyrolysis.3</p><p>Pyrolysis is a widespread approach for bio‐oil4 synthesis, in which biomass is thermally decomposed in an oxygen‐free or oxygen‐limited environment.5 The resulting crude bio‐oil is a complex mixture of acids, alcohols, furans, aldehydes, esters, ketones, sugars, and multifunctional compounds such as hydroxyacetic acid, hydroxyl‐acetaldehyde and hydroxyacetone (derived from cellulose and hemicellulose), together with 3‐hydroxy‐3‐methoxy benzaldehyde, phenols, guaiacols, and syringols derived from the lignin component.1b, 6 Pyrolysis bio‐oils thus require "upgrading" through deoxygenation and neutralization to enhance their energy density, stability, and physical properties.6a, 7 A range of catalytic upgrading methods are known,8 at least at the laboratory scale, including esterification,9 ketonization,10 hydrodeoxygenation,11 and condensation.12</p><p>Carboxylic acids comprise 5–10 wt % of pyrolysis bio‐oils,9, 13 and are largely responsible for their poor chemical stability. Hence, esterification (particularly employing bio‐derived alcohols such as methanol, ethanol, or phenols9, 14) offers an energy‐efficient and atom‐economical route to upgrading.8b, 15 Homogeneous mineral acid catalysts are historically employed for esterification, however their process disadvantages and poor (environmental) E‐factors are well‐documented; hence, strong drivers remain for the development of heterogeneous solid acid counterparts.11 Although base catalysts are widely used for the transesterification of vegetable oils (triacylglycerides) to yield biodiesel, they are unsuitable for catalytic esterification owing to neutralization/saponification.1d</p><p>Diverse solid acids have been explored for esterification, including zeolites,16 heteropolyacids,17 sulfated metal oxides,18 carbon‐based acid catalysts,19 and functionalized mesoporous silicas.20 Research on the latter indicates that mesoporous SBA‐15,21 KIT‐6,22 and PMO23 sulfonic acids, and a macroporous–mesoporous SBA‐15 (MM‐SBA‐15)20g analogue, are among the most promising owing to their tunable pore architecture strong Brønsted acidity and hydrophobicity.2a, 14, 20g, 23, 24 3‐Propylsulfonic acid (PrSO3H)/SBA‐15 has been reported as an efficient catalyst for acetic acid esterification with methanol2a, 25 and other alcohols in simulated bio‐oils,26 and the most widely used sulfonic acid in solid acid catalyzed esterification.27 Such catalysts exhibit improved water tolerance during esterification when the sulfonated silica surface is co‐functionalized with alkyl chains.2a, 5, 25b We recently reported a post‐modification hydrothermal saline‐promoted grafting (HSPG) route to introduce higher sulfonic acid loadings into mesoporous silicas than those achievable by conventional grafting methods,24a and confer stability towards leaching during the esterification of model acids.24b, 28 Hydrophobicity and catalytic reactivity, can also be enhanced through incorporating organic groups into the silica framework.24b Mesopore interconnectivity also plays a role in controlling esterification activity, with interconnectivity between the hexagonal cylindrical mesopores of PrSO3H/KIT‐6 offering superior mass transport and active site accessibility to non‐interconnected PrSO3H/SBA‐15.20g Mesopore expansion (from ≈5 to 14 nm),14 and macropore incorporation23 offer alternative approaches to enhance the esterification activity of PrSO3H/SBA‐15 for long chain fatty acid esterification.</p><p>With respect to bio‐oil upgrading through catalytic esterification, most studies have employed only model compounds owing to the complex nature of real pyrolysis bio‐oils7a and the associated analytical challenge. We previously reported the application of PrSO3H/SBA‐15 for acetic acid esterification of model bio‐oils.26, 28 Here, we report the synthesis and application of HSPG‐derived mesoporous PrSO3H/SBA‐15, and a macroporous counterpart, for the esterification of simple carboxylic acids (C3, C6, and C12), and the upgrading of thermal fast pyrolysis bio‐oil derived from woodchips.</p><!><p>The successful synthesis of an ordered mesoporous skeleton for SBA‐15 and a macroporous–mesoporous (MM) skeleton for MM‐SBA‐15 (with a mean macropore diameter of ≈200 nm, close to that of the polystyrene colloidal hard template, Figure S1 in the Supporting Information) supports was confirmed by TEM. An ordered, 2D hexagonal mesopore channel network was observed for the former, and a well‐defined interconnecting macropore‐mesopore network for the latter (Figure S2). Formation of the desired p6mm pore architecture for both SBA‐15 and MM‐SBA‐15 was confirmed by low angle X‐ray diffraction (Figure S3), which revealed reflections characteristic of hexagonally ordered mesostructures. Both supports retained hexagonal close packed pore architectures following functionalization by propylsulfonic acid in a H2O/NaCl mixture (the HSPG method). However, a shift in the diffraction peaks to higher angle was observed post‐functionalization owing to mesopore contraction.23 Mesopore generation (and retention after sulfonation) was further evidenced by N2 porosimetry, which showed type IV isotherms with H1 hysteresis loops for all materials (Figure S4). The textural properties of PrSO3H/SBA‐15 and PrSO3H/MM‐SBA‐15 are summarized in Table 1. The BET surface areas decreased after sulfonic acid grafting over both silicas owing to micropore blockage, which was apparent as a dramatic drop in the micropore area and pore volume. These changes were accompanied by a decrease in pore diameter and an increase in wall thickness, suggesting the uniform grafting of sulfonic acid groups throughout both pore networks without distortion of their unit cells. Previous studies have shown the macropores in such hierarchical frameworks are open and interconnected by bottleneck pore openings.23, 29</p><!><p>Physicochemical properties of mesoporous SBA‐15 and macroporous–mesoporous SBA‐15 and their sulfonic acid analogues.</p><p>[a] BET, [b] BJH, [c] t‐plot, [d] CHNS, [e] propylamine adsorption/TGA‐MS.</p><!><p>Diffuse reflectance infrared fourier transform spectra (DRIFTS) of the parent silicas showed bands at 700–1400 cm−1 and 3000–3800 cm−1, which were indicative of framework Si‐O‐Si and surface silanols, respectively (Figure S5).15 Additional bands appeared at approximately 2960‐2830 cm−1after sulfonation of both materials, which were attributed to CH2 vibrations of the propyl backbone, and a new CH2−Si band centered at 1360 cm−1. CHNS elemental analysis of the sulfonated silicas revealed that both contained approximately 6 wt % sulfur (Table 1), which represented a five‐fold increase over conventional toluene grafting,14, 23 in good agreement with our preliminary results using the HSPG method.24a S 2p XP spectra of both sulfonic‐acid‐functionalized materials in Figure S6 reveal two distinct S chemical environments; a low binding energy centered at 164.5 eV associated with unoxidized thiol, and a higher energy doublet arising from sulfonic acid groups centered at 168.9 eV.30 Quantitative XPS analysis (Table S1) showed that approximately 85 % of S was incorporated as sulfonic acid groups. Thermogravimetric analysis (Figure S7 b) highlighted two major weight losses; one below 100 °C, which was attributed to physisorbed water; and the second between 250–650 °C owing to propylsulfonic acid decomposition.31 The bulk S content estimated from this second loss feature was approximately 5 wt % in accordance with elemental analysis. Acid properties of both sulfonated silica were subsequently probed through pyridine and propylamine adsorption. DRIFT spectra of pyridine‐titrated materials (Figure S8) evidenced only Brønsted acid sites.26 Temperature‐programmed analysis of reactively formed propene from chemisorbed propylamine confirmed that PrSO3H/SBA‐15 and PrSO3H/MM‐SBA‐15 possessed similar acid strengths and loadings (Figure S9 and Figure S10). Therefore, the incorporation of macropores into the SBA‐15 architecture had minimal impact on silica functionalization; the propylsulfonic acid functions grafted over silica surfaces in PrSO3H/SBA‐15 and PrSO3H/MM‐SBA‐15 catalysts were chemically identical. Therefore, any differences in TOFs between the two catalysts must arise solely from diffusion phenomena. However, despite their similar acid site loadings, the surface coverage of acid sites was higher over the macroporous material (which possessed a lower surface area). Note that the higher S loadings accessible through the HSPG method offer acid loadings of approximately 1.5 mmol g−1, approximately twice those obtained through sulfonic acid grafting in toluene (0.6–0.8 mmol g−1).2a Molecular dynamics simulations and adsorption calorimetry revealed that cooperative effects between silanol and sulfonic acid functions can weaken their acidity in PrSO3H/MCM‐41 owing to hydrogen bonding and associate sulfonate reorientation.32 However, such effects only operated for low sulfonic acid loadings, and were absent on crowded surfaces such as those employed in this work; hence, cooperative effects were not expected to influence the catalytic performance.</p><!><p>The catalytic performance of mesoporous and macroporous–mesoporous sulfonic acid silicas was evaluated in the esterification of propanoic (C3), hexanoic (C6), and lauric acids (C12) with methanol to explore the influence of the macropores on the reactivity under previously optimized conditions.2a Because both catalysts possessed similar acid loadings and strength, any differences in activity must arise from their pore architecture. Both sulfonic acid catalysts were active for methylic esterification of the C3, C6, and C12 acids (Figure S11), which were 100 % selective to their corresponding methyl esters. The rate of esterification decreased with increasing alkyl chain length owing to polar and steric effects.33</p><p>The associated turnover frequencies (TOFs) for carboxylic acid esterification were similar over both catalysts for the C3 and C6 acids (Figure 1), whereas the TOF for lauric acid over the hierarchical PrSO3H/MM‐SBA‐15 was twice that observed for the purely mesoporous PrSO3H/SBA‐15 (Figure S12). This rate enhancement for the bulky lauric acid esterification could be explained in terms of improved sulfonic acid accessibility through (i) faster in‐pore diffusion of the reactant/ester product; (ii) shorter mesopore channel lengths owing to truncation by macropores; and (iii) an increased number of mesopore openings, which may boost the sulfonic acid density at mesopore entrances.23</p><!><p>TOF for esterification of various carboxylic acids over PrSO3H/SBA‐15 and PrSO3H/MM‐SBA‐15 catalysts. (Reaction conditions: 25 mg catalyst, 5 mmol acid, acid/MeOH molar ratio=1:30, 60 °C).</p><!><p>The performance of both sulfonic acid silicas was also assessed for the upgrading of a bio‐oil produced by thermal fast pyrolysis of oak woodchips at a bench‐scale, continuous fluidized bed reactor at 500 °C. Some physicochemical properties of the parent biomass feedstock are presented in Table S2, and of the crude bio‐oil in Table S3. Although the bio‐oil possessed a similar calorific value to the woodchips, the volumetric energy density of the former was significantly higher than that of the original biomass, whose density was only 600–900 kg m−3. The bio‐oil contained 23 wt % water, typical of fast pyrolysis bio‐oil,6b, 34 although the total acid number (TAN) of 61.6 mg KOH g−1 measured by the Modified D664A acid number titration method35 was relatively low.34</p><p>Figure 2 compares TOFs for total acid removal (as determined by KOH titration) through catalytic esterification with methanol, and the corresponding reaction profiles for total acid conversion (Figure 2 inset). The PrSO3H/MM‐SBA‐15 catalyst was almost three times more active in terms of TOF, and converted twice the amount of acid than the PrSO3H/SBA‐15 after 6 h. Because the pyrolysis oil contains numerous bulky compounds as described in Table 2, Table 3, and Table S4, we attributed the superior performance of the hierarchical catalyst to improved active site accessibility akin to that for lauric acid esterification. The carboxylic acid constituents of fast pyrolysis bio‐oils may drive low level (<5 %) autocatalytic esterification.36 This was consistent with a control experiment in the absence of any sulfonic acid catalyst, which revealed <8 % total acid conversion of the pyrolysis bio‐oil. Hence, autocatalysis exerted minimal impact on our results.</p><!><p>Effect of support architecture on the TOFs of sulfonic acid catalyzed bio‐oil esterification. Inset: acid conversion profiles for bio‐oil esterification using sulfonic acid catalysts. (Reaction conditions: 9.2 g bio‐oil ≈10 mmol acid, 12.1 mL MeOH (acid/MeOH molar ratio=1:30), 100 mg catalyst, 85 °C).</p><p>Compositions of crude and upgraded bio‐oils following treatment with PrSO3H/MM‐SBA‐15 catalyst.</p><p>Esters present in crude and upgraded thermal fast pyrolysis bio‐oils following treatment with PrSO3H/MM‐SBA‐15 catalyst.</p><!><p>The chemical composition of the crude and upgraded bio‐oil following catalytic treatment by PrSO3H/MM‐SBA‐15 were analyzed in detail by GC×GC–time‐of‐flight mass spectrometry (GC×GC–ToFMS), and the resulting 2D chromatograms are shown in Figure 3. For both the crude and upgraded bio‐oils, the chromatographic space was divided into six discreet molecular groups: acids and esters; aldehydes and ketones (including furanoics and cyclic carbonyls); hydrocarbons (saturated and unsaturated non‐aromatic); aromatic hydrocarbons; phenolic compounds; and sugars. Compounds that could not be identified by the library and/or did not meet the required identification criteria (as detailed in the Supporting Information) were classified as "unidentified". A more detailed classification of each molecular group and their relative chromatographic area is presented in Table 2. Almost complete loss of organic acids (from 19.7 to 0.9 %) and a significant decrease in phenolics, ketones, aldehydes, and sugars was observed following catalytic upgrading, accompanied by a significant increase in ester and alcohol components, consistent with esterification. Additional details on the removal/formation of specific phenolics, ethers, and carbonyls is presented in Table S4. Acetic acid was the major organic acid in both the crude and upgraded bio‐oils. Esters with relative areas >0.1 in the crude and upgraded bio‐oils are presented in Table 3.</p><!><p>GC×GC–ToFMS chromatogram of a) crude thermal fast pyrolysis bio‐oil and b) bio‐oil after esterification over PrSO3H/MM‐SBA‐15.</p><!><p>Methyl acetate accounted for 10.8 % of the total chromatographic area of the esterified bio‐oil, as compared to only 1.4 % of the crude bio‐oil, alongside a range of methyl and dimethyl esters from C3–C11 compounds. Identifiable ethers were mainly C3–C6 methoxy‐compounds, with 1,1,2,2‐tetramethoxyethane predominant. Considering phenolics, upgrading principally removed methoxy‐phenols, whereas cresol and catechol derivatives were recalcitrant. The increase in alcohols appeared to arise from glycolaldehyde dimethyl acetal (GDA) formation from levoglucosan.37 Previous studies have revealed that levoglucosan can be transformed in alcohol media by acid catalysts to methyl levulinate, through intermediate glycolaldehyde (GA) formation38 (which may itself form glycolaldehyde dimethyl acetal). GA and GDA were detected in the upgraded bio‐oil, supporting this proposed reaction pathway. Future work will address the recyclability of PrSO3H/MM‐SBA‐15 for the esterification of real bio‐oils, wherein we expect strong adsorption of organics that will require the development of low‐temperature regeneration protocols that avoid decomposition of the grafted sulfonate.</p><p>In summary, GC×GC–ToFMS analysis confirmed that PrSO3H/MM‐SBA‐15 was an effective catalyst for the esterification of a real thermal pyrolysis bio‐oil, significantly reducing the bio‐oil acidity through esterification of organic acids under mild reaction conditions.</p><!><p>Mesoporous and hierarchical macroporous–mesoporous (MM) propyl sulfonic acid (PrSO3H) silicas were synthesized by hydrothermal saline‐promoted grafting of the pre‐formed architectures. The textural properties of the parent silicas were unperturbed by sulfonation, which resulted in similar sulfonic acid loadings and strengths for both pore networks. The turnover frequencies for catalytic esterification of model C3–C12 carboxylic acids with methanol decreased with alkyl chain length over both materials, however the introduction of 200 nm macropores into the SBA‐15 framework doubled the activity per acid site for the bulkiest lauric acid, which was attributed to enhanced mass transport and active site access, and a higher −PrSO3H surface density. Macropore incorporation also enhanced the esterification activity for the upgrading of a real bio‐oil derived from thermal fast pyrolysis of oak woodchips; the TOF for total organic acid removal increased three‐fold relative to the mesoporous sulfonic acid silica, which was also attributed to superior in‐pore mass transport and active site accessibility. The total acid number was reduced by 57 % over a 6 h reaction at 85 °C using the hierarchical PrSO3H/MM‐SBA‐15 catalyst. GC×GC–time‐of‐flight mass spectrometry (GC×GC–ToFMS) confirmed that catalytic upgrading removed almost all organic acids, and significantly lowered the concentration of reactive, phenolic, aldehyde, and ketone components, accompanied by the formation of carboxylic acids methyl esters and ethers.</p><!><p>Full details of the catalyst synthesis, bulk and surface characterization (TEM, XRD, N2 porosimetry, DRIFTS, XPS, TGA, pyridine adsorption/DRIFTS, propylamine adsorption/TGA‐MS), and catalytic esterification and bio‐oil analysis protocols are provided in the Supporting Information.</p><!><p>The authors declare no conflict of interest.</p><!><p>As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.</p><p>Supplementary</p><p>Click here for additional data file.</p>

PubMed Open Access

Dicationic phenyl-2, 2\xe2\x80\xb2-bichalcophenes and analogues as antiprotozoal agents

A series of phenyl-2, 2\xe2\x80\xb2-bichalcophene diamidines 1a-h were synthesized from the corresponding dinitriles either via a direct reaction with LiN(TMS)2, followed by deprotection with ethanolic HCl or through the bis-O-acetoxyamidoxime followed by hydrogenation in acetic acid and EtOH over Pd-C. These diamidines show a wide range of DNA affinities as judged from their \xce\x94Tm values which are remarkably sensitive to replacement of a furan unit with a thiophene one. These differences are explained in terms of the effect of subtle changes in geometry of the diamidines on binding efficacy. Five of the eight compounds were highly active (below 6 nM IC50) in vitro against Trypanosoma brucei rhodesiense (T. b. r.) and four gave IC50values less than 7 nM against Plasmodium falciparum (P. f.). Only one of the compounds was as effective as reference compounds in the T. b. r. mouse model for the acute phase of African trypanosomiasis.

dicationic_phenyl-2,_2\xe2\x80\xb2-bichalcophenes_and_analogues_as_antiprotozoal_agents

1. Introduction<!>2.1. Chemistry<!>2.2. Biology<!>2.3. Conclusion<!>3.1.1. \xce\x94Tm Measurements<!>3.1.2. In vitro activity determination<!>3.1.3. STIB900 Acute Mouse Model of Trypanosomiasis<!>3.2. Chemistry<!>General procedure of Heck reaction (2a, b)<!>5\xe2\x80\xb2-(2-Cyanopyridin-5-yl)-2,2\xe2\x80\xb2-bifuran-5-carbonitrile (2a)<!>5\xe2\x80\xb2-(2-Cyanopyridin-5-yl)-2,2\xe2\x80\xb2-bithiophene-5-carbonitrile (2b)<!>General procedure of Suzuki coupling (2c\xe2\x80\x93f)<!>5\xe2\x80\xb2-(4-Cyanophenyl)-2,2\xe2\x80\xb2-bifuran-5-carbonitrile (2c)<!>5-[5-(4-Cyanophenyl)-furan-2-yl]-thiophene-2-carbonitrile (2d)<!>5\xe2\x80\xb2-(4-Cyanophenyl)-2,2\xe2\x80\xb2-bithiophene-5-carbonitrile (2e)<!>5-[5-(4-Cyanophenyl)-thiophen-2-yl]-furan-2-carbonitrile (2f)<!>5\xe2\x80\xb2-[(4-Cyanophenyl)ethynyl]-2,2\xe2\x80\xb2-bifuran-5-carbonitrile (2g)<!>6-(2,2\xe2\x80\xb2-Bifuran-5-yl)-nicotinonitrile (6)<!>6-(5\xe2\x80\xb2-Bromo-2,2\xe2\x80\xb2-bifuran-5-yl)-nicotinonitrile (7)<!>6-(5\xe2\x80\xb2-Cyano-2,2\xe2\x80\xb2-bifuran-5-yl)-nicotinonitrile (2h)<!>General procedure of diamidines synthesis (Method A)<!>5\xe2\x80\xb2-(2-Amidinopyridin-5-yl)-2,2\xe2\x80\xb2-bifuran-5-carboxamidine hydrochloride (1a)<!>5\xe2\x80\xb2-(2-Amidinopyridin-5-yl)-2,2\xe2\x80\xb2-bithiophene-5-carboxamidine hydrochloride (1b)<!>5\xe2\x80\xb2-(4-Amidinophenyl)-2,2\xe2\x80\xb2-bifuran-5-carboxamidine hydrochloride (1c)<!>5-[5-(4-Amidinophenyl)-furan-2-yl]-thiophene-2-carboxamidine hydrochloride (1d)<!>5\xe2\x80\xb2-(4-Amidinophenyl)-2,2\xe2\x80\xb2-bithiophene-5-carboxamidine hydrochloride (1e)<!>5-[5-(4-Amidinophenyl)-thiophen-2-yl]-furan-2-carboxamidine hydrochloride (1f)<!>5\xe2\x80\xb2-[(4-Amidinophenyl)ethynyl]-2,2\xe2\x80\xb2-bifuran-5-amidine hydrochloride (1g)<!>N-Hydroxy-5\xe2\x80\xb2-[4-(N-hydroxyamidino)-phenyl]-2,2\xe2\x80\xb2-bifuran-5-carboxamidine (8a)<!>N-Hydroxy-6-[5\xe2\x80\xb2-(N-hydroxyamidino)-2,2\xe2\x80\xb2-bifuran-5-yl]-nicotinamidine (8b)<!>5\xe2\x80\xb2-(4-Amidinophenyl)-2,2\xe2\x80\xb2-bifuran-5-carboxamidine acetate (1c)<!>5\xe2\x80\xb2-(4-Amidinophenyl)-2,2\xe2\x80\xb2-bifuran-5-carboxamidine (1c)<!>6-(5\xe2\x80\xb2-Amidino-2,2\xe2\x80\xb2-bifuran-5-yl)-nicotinamidine acetate (1h)<!>

<p>Aromatic diamidines exhibit broad-spectrum antimicrobial activity including effectiveness against the protozoan diseases caused by Trypanosoma sp and Plasmodium sp.1 The broad activity of the aromatic diamidines notwithstanding, pentamidine (I) is the only compound of this class to be used extensively in the clinic.1 Furamidine (IIa), a diamidino diphenylfuran, has been shown to be more potent and less toxic than pentamidine in murine models of trypanosomiasis.2 The oral prodrug of furamidine, 2,5-bis[4-(methoxyamidino)phenyl]furan (pafuramidine), showed promising results in Phase I and II clinical trials against both human African trypanosomiasis (HAT) and malaria.1 Unfortunately, in an additional safety study of pafuramidine paralleling the Phase III trials, liver and kidney toxicities in some volunteers were found and the development of pafuramidine was terminated.2 Many of the active aromatic diamidines for various structural classes have been shown to bind to the minor groove of DNA at AT rich sites.3–9 It has been assumed that the minor groove binding of these type of compounds leads to inhibition of one or more DNA dependant enzymes which gives rise to the anti-microbial effect.10–12 Recently, we have made compounds in which the phenyl group(s) of furamidine have been replaced with pyridyl group(s) (IIb). Several of these aza-analogues show in vivo activity which is superior to that of furamidine.13 More recently, one of these aza analogs was found to be effective in a mouse model for second stage African sleeping sickness.14 Furan units are often key structural elements in the aromatic frame work for the more effective diamidines.15–17 The furan analogue III has been found to bind to DNA in a unique stacked dimer array which has potential for development of new gene regulation molecules.18–21 Compound III has shown some activity in an immunosupressed rat model for Pneumocystis carinii pneumonia18 and in the STIB900 mouse model for acute stage African trypanosomiasis.22 The bichalcophene diamidine IV from our laboratory has been shown to recognize G-quadruplex DNA.23 More recently, the diverse modes of the interaction of IV with multi-stranded DNA structures has been reported.24</p><p>As part of a research program directed to drug discovery of antiprotozoal agents and due to the unusual DNA binding properties of III and IV we decided to prepare additional analogues of this series of bichalcophene diamidines in order to investigate structure-activity relationships (SAR) and their DNA binding profiles. We report here the synthesis of novel diamidino phenyl-bifuran derivatives and structural isosteres and their evaluation versus Trypanosoma brucei rhodesiense (T. b. r.) and Plasmodium falciparum (P. f.).</p><!><p>Our strategy for the synthesis of diamidines 1 is based upon the conversion of dinitriles either by direct reaction using lithium bis(trimethylsilyl)amide LiN(TMS)2 or through the bis-O-acetoxyamidoxime followed by hydrogenation in acetic acid. Retrosynthetic studies (Figure 2) for this strategy suggested that the triaryl structure 2 could be obtained by the application of palladium catalyzed reactions which permit the formation of C-C bond with full regioselectivity. The bichalcophenes 3 could be converted into dinitriles 2 using the Heck coupling reaction. The compounds 4 could be converted into the dinitriles 2 using a Suzuki coupling reaction. For SAR study, a two-carbon spacer could be inserted into dinitriles 2 using Sonogashira coupling27 by the reaction of 4a with trimethyl silyl acetylene derivative. Furthermore, Stille coupling is another Pd catalyzed reaction which is an alternative pathway to furnish certain analogues using compound 5.</p><p>We initiated our study by the synthesis of dinitrile 2a which was obtained from 2,2′-bifuran-5-carbonitrile (3a), prepared according to our published procedure,25 via a Heck coupling reaction with the commercially available 5-bromopyridine-2-carbonitrile as shown in Scheme 1. The dinitrile 2b was prepared employing similar procedures used for 2a using 2, 2′-bithiophene-5-carbonitrile (3b). The synthesis of dinitriles 2c–f was accomplished in two steps. The regioselective bromination of 3a–d using N-bromosuccinamide (NBS) in DMF furnished bromo derivatives 4a–d. Suzuki coupling of 4a–d with p-cyanophenylboronic acid was accomplished in the presence of Pd (PPh3)4 as a catalyst, Na2CO3 as base and toluene as solvent.</p><p>Compound 2h was obtained in three steps starting with a Stille coupling reaction of the readily available 6-(5-bromofuran-2-yl)nicotinonitrile 5 and 2-tributylstannylfuran to form the corresponding 6-(2,2′-bifuran-5-yl)-nicotinonitrile (6) (Scheme 2). Bromination of 6 with N-bromosuccinimide in DMF, furnished bromo-bifuran derivative 7 in a 58% yield. A subsequent cyanation reaction of compound 7 with Cu(I)CN in DMF gave the dinitrile 2h.</p><p>As outlined in Scheme 3, a series of diamidines 1a–h was obtained from the dinitriles 2a–h either by direct reaction using LiN(TMS)2 (cf. 1a–g) or through the bis-O-acetoxyamidoxime followed by hydrogenation in glacial acetic acid as in case of 1c,h. Thus, 5′-(2-amidinopyridin-5-yl)-2,2′-bifuran-5-carboxamidine (1a) was synthesized from the corresponding dinitrile 2a by treatment with LiN(TMS)2 followed by deprotection with ethanolic HCl(g). 6-(5′-Amidino-2, 2′-bifuran-5-yl)-nicotinamidine acetate salt (1h) was synthesized from 6-(5′-cyano-2, 2′bifuran-5-yl)-nicotinonitrile (2h), through the bis-O-acetoxyamidoxime followed by hydrogenation. The hydrochloride salts of the diamidines 1a–g were obtained by passing hydrogen chloride gas into ethanolic solutions of their free bases</p><!><p>Aromatic diamidines are well known cationic molecules that have a strong and reversible interaction with DNA. Recent work has shown that both curved and linear molecules exhibit excellent biological activity against trypanosomes.22,26 The mechanism of action for antitrypanosomal activity has been suggested to involve the inhibition of DNA dependent enzymes or direct inhibition of transcription.1,9,10</p><p>Table 1 contains the DNA binding affinities for the new bichalcophene diamidines as well as the in vitro activities for these compounds against T. b. r. and P. f. For comparative purposes, similar data for furamidine and pentamidine are included. The use of the thermal melting increase ΔTm (Tm of complex – Tm of free DNA) is a rapid and reliable method for comparing binding affinities for a large number of diverse classes of diamidines. The complexes between poly (dA-dT) and the bichalcophene diamidines give an unexpected wide range (from 4 to 18°C) of ΔTm values. The ΔTm value (8.5°C) for the bifuran 1c is significantly lower than that of both pentamidine (12.6°C) and furamidine (25°C). Due to structural similarities this analysis will focus on comparisons between furamidine and the bichalcophene analogues. The large drop in ΔTm value on replacing one of the furamidine phenyl groups with a furan ring is probably due to a combination of factors presented by 1c including a more curved structure, a shorter distance between the two amidine units and a smaller stacking surface provided by the three aryl rings. The first two differences seem likely to be the more important contributors to the reduction in ΔTm value since the more curved structure would be expected to prevent optimal fit in the groove by pushing the central portion of the molecule away from the floor of the groove and the shorter distance between the amidine units could lead to a disadvantageous offset in H-bond indexing with the minor groove base pairs. Replacement of one of the furan rings with a thiophene ring (1d or 1f) results in an increase in the ΔTm by approximately 5°C. This result is consistent with the increased affinity of other thiophene based diamidines in comparison to furan counterparts.28 There is a small increase in bond angle for C-S-C in thiophene compared to that of C-O-C in furan attributable to the differences in van der Waals radii between S and O. This small angle difference when amplified to the terminal amidine units leads to a significant difference in the positions of the amidines in the two different molecules and likely leads to the increase in binding affinities for the thiophene analogues.28 The bithiophene analog 1e gives the highest ΔTm value (18.1 °C) for the bichalcophenes studied, consistent with the previous observations. Introduction of nitrogen into the phenyl ring in this system (1a, 1b, 1h) results in a 1.4 to 3.0 °C decrease in ΔTm values compared to that of their carbocycle analogues. This type decline has been noted previously for other diamidines and has been attributed to differences in hydration of the phenyl and pyridyl ring systems.13,29–31 The bifuran 1g in which a carbon-carbon triple bond has been inserted between the furan and the phenyl rings yields the bichalcophene compound with the lowest ΔTm value of those investigated. A similar reduction in binding affinity was noted in DAPI analogues in which a carbon-carbon triple bond had been inserted.30 The origin of the decline seems likely to be associated with poor stacking interactions of the triple bond and possible changes in H-bond indexing as a result of further separation of the amidine groups.</p><p>The IC50 values for the bichalcophene diamidines against T. b. r. range from 2 nM to 97 nM. Five (1a, 1b, 1d, 1e, 1f) of the eight compounds studied give IC50 values of 6 nM or less which are all in the range of the values of pentamidine and furamidine. These compounds were quite effective against P. f. with IC50 values ranging from of 0.9 to 41 nM. Against this organism four compounds (1a 1b, 1f, 1h) gave IC50 values less than 7 nM. On comparison of the T. b. r. activity of non-pyridyl containing analogues (1c–g) the thiophene compounds (1b, 1e) are the most effective. A similar trend is also seen for the pyridyl analogues (1a, 1b, 1h); however, the difference in IC50 values for 1a and 1b is within experimental error. There is not an obvious SAR pattern for the P. f. activity. As we have noted previously, there is no direct correlation between DNA binding affinity and antiparasitic activity.1 While some DNA affinity seems essential, transport of diamidines into the parasite plays and important role.1 There does appear to be a rough correlation between DNA affinity and cytotoxicity.31 Nevertheless, the selectivity of these compounds for the parasitic organisms is generally quite high as judged from their cytotoxicity to cultured L6 rat myoblast cells shown in Table 1. The selectivity indices range from 277 to 38500.</p><p>Given the in vitro selectivity and activity of the bichalcophenes they were evaluated in the stringent T. b. r. mouse model for the acute phase of African trypanosomiasis.14 The compounds were evaluated by daily intraperitoneal dosage of 5 mg/kg (except for 1c which was tested in an earlier protocol at 20 mg/kg) for four consecutive days. The results for these studies are included in Table 1. At the low dosage of 5 mg/kg only 1f provided cures (2/4) which represents activity comparable to that of the reference compounds pentamidine and furamidine. Compound 1f merits further evaluation in other models for HAT.</p><!><p>A new series of bichalcophene diamidines has been prepared in good yields. These analogues show a wide range of DNA affinities as judged from their ΔTm values which are remarkably sensitive to replacement of a furan unit with a thiophene one. These differences are explained in terms of the effect of subtle changes in geometry of the diamidines on binding efficacy. Five of the eight compounds were highly active (below 6 nM IC50) in vitro against T. b. r. and four gave IC50values less than 7 nM against P. f. Only one of the compounds was as effective as reference compounds in the T. b. r. mouse model for the acute phase of African trypanosomiasis.</p><!><p>Thermal melting experiments were conducted with a Cary 300 spectrophotometer. Cuvettes for the experiment are mounted in a thermal block and the solution temperatures are monitored by a thermistor in the reference cuvette. Temperatures were maintained under computer control and are increased at 0.5 °C/min. The experiments were conducted in 1 cm path length quartz cuvettes in CAC 10 buffer (cacodylic acid 10 mM, EDTA 1 mM, NaCl 100 mM with NaOH added to give pH 7.0). The concentrations of DNA were determined by measuring the absorbance at 260 nm. A ratio of 0.3 mol compound per mole of DNA was used for the complex and DNA with no compound was used as a control.26, 32</p><!><p>In vitro assays with Trypanosoma b. rhodesiense STIB 900 and Plasmodium falciparum K1 strain and cell toxicity assays using cultured L6 rat myoblast cells were carried out as previously described.33</p><!><p>Experiments were performed as previously reported14 with minor modifications. Briefly, female NMRI mice were infected intraperitoneally (ip) with 2 × 104 STIB900 bloodstream forms. Groups of four mice were treated ip with the tested dications on 4 consecutive days from day 3 through 6 post infection. A control group was infected but was not treated. The tail blood of all mice was checked for parasitemia until 60 days post infection. Surviving and parasite free mice at day 60 were considered cured and then euthanized. Death of animals (including the aparasitemic mice, >60) was recorded to calculate the mean survival time in days.</p><!><p>Melting points were recorded using a Thomas-Hoover (Uni-Melt) capillary melting point apparatus and are uncorrected. TLC analysis was carried out on silica gel 60 F254 precoated aluminum sheets and detected under UV light. 1H and 13C NMR spectra were recorded employing a Varian Unity Plus 300 spectrometer and a Bruker Avance 400 MHz spectrometer, and chemical shifts (δ) are in ppm relative to TMS as internal standard. Mass spectra were recorded on a VG analytical 70-SE spectrometer. Elemental analyses were obtained from Atlantic Microlab Inc. (Norcross, GA) and are within ±0.4 of the theoretical values. The compounds reported as salts frequently analyzed correctly for fractional moles by water and/or ethanol of solvation. In each case proton NMR showed the presence of indicated solvent (s). All chemicals and solvents were purchased from Aldrich Chemical Co., Fisher Scientific or Frontier.</p><!><p>A mixture of 2,2′-bifuran-5-carbonitrile 3a or 2,2′-bithiophene-5-carbonitrile 3b (10 mmol), 5-bromopyridine-2-carbonitrile (1.82 g, 10 mmol), Pd(PPh3)4(0) (400 mg) and KOAc (5 g, 50 mmol) in dry DMF (10 mL) was heated under nitrogen at 130–135 °C for overnight. The reaction mixture then poured onto cold-water. The precipitate which formed was collected, recrystallized from DMF.</p><!><p>Yellow solid, yield 55%, mp 213–214 °C. 1H NMR (DMSO-d6); δ 7.25 (d, J = 3.6 Hz, 1H), 7.27 (d, J = 3.6 Hz, 1H), 7.59 (d, J = 3.6 Hz, 1H), 7.80 (d, J = 3.6 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 8.41 (dd, J = 8.0, 2.4 Hz, 1H), 9.22 (d, J = 2.4 Hz, 1H). 13C NMR (DMSO-d6); δ 149.9, 149.1, 146.5, 145.1, 131.7, 130.7, 129.3, 128.3, 125.6, 124.4, 117.6, 113.1, 112.1, 111.8, 108.5. MS (EI) m/e (rel.int.); 262 (M+ +1, 100), 207 (18), 179 (24), 148 (80). Anal. Calcd for C15H7N3O2: C, 68.96; H, 2.70. Found: C, 68.72.; H, 2.79.</p><!><p>Yellow solid, yield 60%, mp 257–258.5 °C. 1H NMR (DMSO-d6); δ 7.59 (d, J = 4.0 Hz, 1H), 7.72 (d, J = 4.0 Hz, 1H), 7.95 (d, J = 4.0 Hz, 1H), 8.00 (d, J = 4.0 Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 8.34 (dd, J = 8.0, 2.4 Hz, 1H), 9.14 (d, J = 2.4 Hz, 1H). 13C NMR (DMSO-d6); δ 147.6, 143.0, 140.4, 138.9, 136.3, 133.6, 132.2, 131.0, 129.3, 129.0, 128.5, 125.4, 117.5, 114.1, 107.0. MS (EI) m/e (rel.int.); 293 (M+, 10), 152 (12), 133 (15), 69 (100). Anal. Calcd for C15H7N3S2: C, 61.41; H, 2.41. Found: C, 61.54; H, 2.47.</p><!><p>To a stirred solution of 5′-bromo-2,2′-bifuran-5-carbonitrile25 4a (4.74 g, 20 mmol), and Pd(PPh3)4(0) (600 mg) in toluene (40 mL) under a nitrogen atmosphere was added 20 mL of a 1.5 M aqueous solution of Na2CO3 followed by 4-cyanophenylboronic acid (3.21 g, 22 mmol) in 5 mL of methanol. The vigorously stirred mixture was warmed to 80 °C for 16 h. The solvent was evaporated, the precipitate was partitioned between CH2Cl2 (300 mL) and aqueous solution containing 15 mL of concentrated ammonia. The organic layer was dried (Na2SO4), and then concentrated to dryness under reduced pressure.</p><!><p>Yellow solid, yield 74%, mp 194–195 °C (DMF).25 1H NMR (DMSO-d6) δ 7.17 (d, J = 3.6 Hz, 1H), 7.21 (d, J = 3.6 Hz, 1H), 7.43 (d, J = 3.6 Hz, 1H), 7.76 (d, J = 3.6 Hz, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.99 (d, J = 8.4 Hz, 2H).</p><!><p>Yellowish green solid, yield 76%, mp 187–188 °C.25 1H NMR (DMSO-d6); δ 7.22 (d, J = 3.6 Hz, 1H), 7.35 (d, J = 3.6 Hz, 1H), 7.62 (d, J = 3.9 Hz, 1H), 7.85–7.94 (m, 5H).</p><!><p>Yellow solid, yield 70%, mp 231–233 °C.25 1H NMR (DMSO-d6) δ 7.50 (d, J = 3.9 Hz, 1H), 7.59 (d, J = 3.9 Hz, 1H), 7.73 (d, J = 3.9 Hz, 1H), 7.84–7.87 (m, 4H), 7.91 (d, J = 3.9 Hz, 1H).</p><!><p>Yellow solid, yield 75%, mp 216.5–218 °C.25 1H NMR (DMSO-d6) δ 7.09 (d, J = 3.9 Hz, 1H), 7.64–7.66 (m, 2H), 7.75 (d, J = 3.9 Hz, 1H), 7.84–7.88 (m, 4H).</p><!><p>A mixture of 4-[(trimethylsilyl)ethynyl]benzonitrile (1.99 g, 10 mmol), 5′-bromo-2,2′-bifuran-5-carbonitrile (4a) (1.19 g, 5 mmol), palladium acetate (56 mg, 0.25 mmol), tri(o-tolyl)phosphine (152 mg, 0.5 mmol), Bu4NCl (1.39 g, 5 mmol), and sodium acetate (1.64 g, 20 mmol) in DMF (20 mL) was heated at 70 °C for 3 hr. The reaction mixture was poured onto water and extracted with ethyl acetate 200 mL (3X). The extract was evaporated, and the residue was chromatographed on silica gel using hexanes/EtOAc (95:5) as an eluent to furnish compound 2g as yellow solid in 25% yield, mp 172–173 °C. 1H NMR (DMSO-d6); δ 7.16 (d, J = 3.6 Hz, 1H), 7.21 (d, J = 3.6 Hz, 1H), 7.23 (d, J = 3.6 Hz, 1H), 7.76–7.79 (m, 3H), 7.93 (d, J = 8.0 Hz, 2H). 13C NMR (DMSO-d6); δ 148.7, 145.1, 136.1, 132.7, 131.9, 125.6, 125.5, 124.5, 119.8, 118.3, 111.7, 111.2, 108.6, 93.8, 82.5. MS (EI) m/e (rel.int.); 284 (M+, 10), 228 (35), 201 (40), 164 (60), 64 (100). Anal. Calcd for C18H8N2O2: C, 76.05; H, 2.83. Found: C, 75.80; H, 2.96.</p><!><p>A mixture of 6-(5-bromofuran-2-yl)nicotinonitrile13 5 (2.48 g, 10 mmol), 2-n-tributyltin furan (3.58 g, 10 mmol), and tetrakis(triphenylphosphine) palladium (300 mg) in dry dioxane (20 mL) was heated under nitrogen at 100–110 °C for 24 h. The solvent was evaporated under reduced pressure and the resulting residue was dissolved in ethyl acetate. This solution was passed through celite to remove Pd. The solution was evaporated, and the residue was chromatographed on silica gel using hexanes/EtOAc (70:30) as an eluent to furnish compound 6 as a golden yellow solid in 78% yield, mp 169–170 °C. 1H NMR (CDCl3); δ 6.52 (dd, J = 3.6, 1.8 Hz, 1H), 6.72–6.76 (m, 2H), 7.31 (d, J = 3.6 Hz, 1H), 7.49 (d, J = 1.8 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.95 (dd, J = 8.4, 2.1 Hz, 1H), 8.80 (d, J = 2.1 Hz, 1H). 13C NMR (CDCl3); δ 152.5, 151.3, 151.0, 148.7, 145.5, 142.9, 139.6, 117.6, 117.0, 114.3, 111.7, 108.1, 107.1, 106.6. Anal. Calcd for C14H8N2O2: C, 71.18; H, 3.41; N, 11.85. Found. C, 70.83; H, 3.61; N, 11.84.</p><!><p>To a solution of 6 (1.41 g, 6 mmol) in DMF (20 mL) was added portionwise N-bromosuccinimide (1.07 g, 6 mmol) with stirring. The reaction mixture was stirred overnight, then poured onto cold water. The precipitate which formed was collected, washed with water and dried to give 7 as a golden yellow solid in 58% (EtOH), mp 143–145 °C. 1H NMR (CDCl3); δ 6.44 (d, J = 3.6 Hz, 1H), 6.70 (d, J = 3.6 Hz, 1H), 6.75 (d, J = 3.6 Hz, 1H), 7.30 (d, J = 3.6 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.95 (dd, J = 8.4, 2.1 Hz, 1H), 8.80 (d, J = 2.1 Hz, 1H). 13C NMR (CDCl3); δ 152.5, 151.2, 151.1, 147.5, 147.3, 139.7, 122.8, 117.7, 117.0, 114.3, 113.5, 109.3, 108.6, 106.9. MS (EI) m/e (rel.int.); 314 (M+, 60), 285 (10), 235 (20), 207 (100), 179 (10). HRMS calcd for C14H7BrN2O2: 313.9690. Observed 313.9661.</p><!><p>A mixture of 7 (942 mg, 3 mmol) and Cu(1)CN (540 mg, 6 mmol) in dry DMF (25 mL) was refluxed for 48 h. The reaction mixture was poured onto water/ammonia and extracted with methylene chloride. The extract was washed with water and brine, dried over Na2SO4, then passed on silica gel to give compound 2h as yellow solid in 27% yield, mp 209–210.5 °C. 1H NMR (DMSO-d6); δ 7.23 (d, J = 3.6 Hz, 1H), 7.27 (d, J = 3.6 Hz, 1H), 7.52 (d, J = 3.6 Hz, 1H), 7.79 (d, J = 3.6 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 8.39 (dd, J = 8.4, 2.4 Hz, 1H), 9.03 (d, J = 2.4 Hz, 1H). Anal. Calcd for C15H7N3O2: C, 68.96; H, 2.70. Found: C, 68.81; H, 2.64.</p><!><p>The dinitrile 2a (0.87 g, 3.34 mmol), suspended in freshly distilled THF (10 mL), was treated with LiN(TMS)2 (1M solution in THF, 8 mL, 8 mmol) and the reaction was allowed to stir overnight. The reaction mixture was then cooled to 0 °C to which was added HCl saturated ethanol (40 mL) whereupon a precipitate started forming. The mixture was left to run overnight whereafter it was diluted with ether and the resultant solid was collected by filtration. The diamidine was purified by neutralization with 1N NaOH followed by filtration of the resultant solid and washing with water (3X 10 mL). Finally, the free base was stirred with ethanolic HCl overnight, diluted with ether, and the solid formed was filtered and dried to give the diamidine 1a hydrochloride salt</p><!><p>Yellow solid, yield 65%, mp >320 °C. 1H NMR (DMSO-d6); δ 7.30 (d, J = 2.4 Hz, 1H), 7.38 (d, J = 2.4 Hz, 1H), 7.67 (d, J = 2.4 Hz, 1H), 8.05 (d, J = 2.4 Hz, 1H), 8.51–8.57 (m, 2H), 9.26 (s, 2H), 9.29 (s, 1H), 9.53 (s, 2H), 9.60 (s, 2H), 9.69 (s, 2H). 13C NMR (DMSO-d6); δ 161.4, 153.3, 150.3, 148.8, 145.4, 145.0, 142.2, 140.5, 132.1, 129.1, 123.8, 120.9, 113.0, 112.4, 109.5. MS (ESI) m/e (rel.int.); 296 (M+ + 1, 100), 208 (7), 148 (52). HRMS calcd for C15H14N5O2: 296.1147. Observed: 296.1144. Anal. Calcd for C15H13N5O2-2.0HCl-1.25H2O: C, 46.10; H, 4.51; N, 17.92. Found: C, 46.01; H, 4.44; N, 17.63.</p><!><p>The same procedure described for preparation of 1a was used starting with the dinitrile 2b. Orange solid, yield 71%, mp 318–321 °C. 1H NMR (D2O/DMSO-d6); δ 7.55 (d, J = 4.0 Hz, 1H), 7.60 (d, J = 4.0 Hz, 1H), 7.82 (d, J = 4.0 Hz, 1H), 7.94 (d, J = 4.0 Hz, 1H), 8.19 (d, J = 8.4 Hz, 1H), 8.30 (dd, J = 8.4, 2.0 Hz, 1H), 9.06 (d, J = 2.0 Hz, 1H). 13C NMR (D2O/DMSO-d6); δ 161.5, 158.4, 146.6, 143.7, 142.4, 139.4, 137.0, 135.8, 134.3, 133.4, 129.2, 128.8, 127.2, 126.3, 123.9. MS (ESI) m/e (rel.int.); 328 (M+ + 1, 75), 224 (10), 203 (7), 164 (100). HRMS calcd. for C15H14N5S2: 328.0691. Observed: 328.0696. Anal. Calcd for C15H13N5S2-2.0HCl-0.5H2O-0.25EtOH: C, 44.23; H, 4.19; N, 16.64. Found: C, 44.18; H, 4.13; N, 16.61.</p><!><p>The same procedure described for preparation of 1a was used starting with the dinitrile 2c. Yellow solid, yield 73%, mp > 300 °C. 1H NMR (D2O/DMSO-d6); δ 7.16 (d, J = 3.6 Hz, 1H), 7.23 (d, J = 3.6 Hz, 1H), 7.36 (d, J = 3.6 Hz, 1H), 7.75 (d, J = 3.6 Hz, 1H), 7.85 (d, J = 7.8 Hz, 2H), 8.01 (d, J = 7.8 Hz, 2H). Anal. Calcd for C16H14N4O2-2.0HCl-1.0H2O: C, 49.88; H, 4.70; N, 14.54. Found. C, 49.92; H, 4.50; N, 14.41.</p><!><p>The same procedure described for preparation of 1a was used starting with the dinitrile 2d. Brown solid, yield 70%, mp 250–252 °C. 1H NMR (D2O/DMSO-d6); δ 7.14 (d, J = 3.6 Hz, 1H), 7.25 (d, J = 3.6 Hz, 1H), 7.58 (d, J = 3.6 Hz, 1H), 7.79 (d, J = 7.8 Hz, 2H), 7.85–7.93 (m, 3H). 13C NMR (D2O/DMSO-d6); δ 165.9, 159.2, 153.2, 148.8, 140.2, 136.1, 134.8, 129.8, 127.6, 127.5, 125.5, 125.0, 112.9, 112.7. MS (ESI) m/e (rel.int.); 311 (M+ +1, 92), 156 (100). HRMS calcd for C16H15N4OS: 311.0967. Observed: 311.0977. Anal. Calcd for C16H14N4OS-2.0HCl-1.5H2O-0.25EtOH: C, 46.97; H, 4.89; N, 13.28. Found: C, 46.82; H, 4.69; N, 13.13.</p><!><p>The same procedure described for preparation of 1a was used starting with the dinitrile 2e. Redish brown solid, yield 81%, mp 271–273 °C. 1H NMR (D2O/DMSO-d6); δ 7.49 (d, J = 3.6 Hz, 1H), 7.53 (d, J = 3.6 Hz, 1H), 7.67 (d, J = 3.6 Hz, 1H), 7.84 (s, 4H), 7.94 (d, J = 3.6 Hz, 1H). 13C NMR (D2O/DMSO-d6); δ 165.5, 158.7, 144.1, 143.3, 138.4, 136.1, 136.0, 129.8, 129.0, 128.1, 127.5, 127.4, 126.4, 126.2. MS (ESI) m/e (rel.int.); 327 (M+ +1, 100), 208 (17), 164 (90). HRMS calcd. for C16H15N4S2: 327.0738. Observed: 327.0739. Anal. Calcd for C16H14N4S2-2.0HCl-1.5H2O: C, 45.06; H, 4.49; N, 13.13. Found: C, 45.00; H, 4.38; N, 13.03.</p><!><p>The same procedure described for preparation of 1a was used starting with the dinitrile 2f. Yellow solid, yield 77%, mp 248–249.5 °C. 1H NMR (D2O/DMSO-d6); δ 7.19 (d, J = 3.6 Hz, 1H), 7.78–7.82 (m, 3H), 7.91–7.96 ((m, 4H). 13C NMR (D2O/DMSO-d6); δ 165.5, 153.8, 153.4, 143.7, 140.2, 138.5, 131.9, 129.6, 128.9, 127.4, 126.4, 126.0, 123.8, 121.8. MS (ESI) m/e (rel.int.); 311 (M+ +1, 100), 156 (87). HRMS calcd. for C16H15N4OS: 311.0967. Observed: 311.0966. Anal. Calcd for C16H14N4OS-2.0HCl-1.5H2O: C, 46.83; H, 4.67; N, 13.65. Found: C, 46.90; H, 4.66; N, 13.30.</p><!><p>The same procedure described for preparation of 1a was used starting with the dinitrile 2g. Yellow solid, yield 78%, mp >320 °C. 1H NMR (DMSO-d6); δ 7.19 (d, J = 3.6 Hz, 1H), 7.25 (d, J = 3.6 Hz, 1H), 7.32 (d, J = 3.6 Hz, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.93 (d, J = 8.4 Hz, 2H), 8.03 (d, J = 3.6 Hz, 1H), 9.26 (s, 2H), 9.40 (s, 2H), 9.58, 9.61 (2s, 4H). 13C NMR (DMSO-d6); δ 164.9, 153.2, 148.3, 145.4, 140.5, 136.2, 131.4, 128.7, 128.4, 125.9, 120.9, 119.7, 111.5, 109.6, 94.1, 81.95. MS (ESI) m/e (rel.int.); 319 (M+ + 1, 100), 160 (75). HRMS calcd for C18H15N4O2: 319.1195. Observed 319.1206. Anal. Calcd for C18H14N4O2-2.0HCl-1.25H2O: C, 52.24; H, 4.51; N, 13.54. Found: C, 52.50; H, 4.54; N, 13.23.</p><!><p>A mixture of hydroxylamine hydrochloride (695 mg, 10 mmol, 10 equiv.) in anhydrous DMSO (8 mL) was cooled to 5 °C under nitrogen and potassium t-butoxide (1.12 g, 10 mmol, 10 equiv.) was added in portions. The mixture was stirred for 30 min. This mixture was added to the dinitrile derivative 2c (260 mg, 1 mmol, 1 equiv.). The reaction mixture was stirred overnight at room temperature. The reaction mixture was then poured slowly onto ice-water. The precipitate was filtered and washed with water and then ethanol to afford 8a (free base) in 93%, mp 205–206 °C. 1H NMR (DMSO-d6); δ 5.83 (br s, 4H), 6.86–6.89 (m, 3H), 7.14 (s, 1H), 7.75 (s, 4H), 9.70 (s, 1H), 9.75 (s, 1H). 13C NMR (DMSO-d6); δ 152.3, 150.3, 146.7, 145.0, 144.9, 144.1, 132.3, 129.9, 125.8, 123.1, 109.7, 108.5, 107.2. MS (FAB) m/e (rel.int.); 327 (M++1, 40), 307 (100), 299 (60), 273 (10), 220 (30). HRMS calcd for C16H15N4O4 ms 327.1093. Observed 327.1137.</p><!><p>The same procedure described for 8a was used starting with 2h. Yield 89%, mp 248–250 °C. 1H NMR (DMSO-d6); δ 5.88 (s, 2H), 6.04 (s, 2H), 6.92–6.96 (m, 3H), 7.29 (d, J = 3.6 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 8.11 (dd, J = 8.4, 2.1 Hz, 1H), 8.88 (d, J = 2.1 Hz, 1H), 9.80 (s, 1H), 9.92 (s, 1H). 13C NMR (DMSO-d6); δ 152.2, 148.7, 147.8, 147.0, 146.6, 146.0, 144.6, 144.0, 133.6, 127.3, 117.7, 111.3, 109.7, 108.5, 107.9. MS (EI) m/e (rel.int.);; 327 (M+, 15), 311 (5), 295 (10), 278 (85), 261 (100). HRMS calcd for C15H13N5O4: 327.0967. Observed 327.0974.</p><!><p>To a solution of the diamidoxime 8a (326 mg, 1 mmol) in glacial acetic acid (10 mL) was slowly added acetic anhydride (0.35 mL). After stirring for overnight TLC indicated complete acylation of the starting material, ethanol (10 mL) and 10% palladium on carbon (80 mg) were then added. The mixture was placed on Parr hydrogenation apparatus at 50 psi for 4 h at room temperature. The mixture was filtered through hyflo and the filter pad washed with water. The filtrate was evaporated under reduced pressure and the precipitate was collected and washed with ether to give 1c acetate salt in 67% yield, mp 240–242 °C. 1H NMR (D2O/DMSO-d6); δ 1.80 (s, 2 x CH3), 7.06 (d, J = 3.6 Hz, 1H), 7.11 (d, J = 3.6 Hz, 1H), 7.36 (d, J = 3.6 Hz, 1H), 7.39 (d, J = 3.6 Hz, 1H), 7.89 (d, J = 8.4 Hz, 2H), 7.98 (d, J = 8.4 Hz, 2H). Anal. Calcd for C16H14N4O2-2.0AcOH-2.4H2O: C, 52.48; H, 5.87; N, 12.23. Found. C, 52.28; H, 5.49; N, 11.81.</p><!><p>It was prepared by dissolving the acetate salt 1c (50 mg) in water (5 mL) and by neutralization with 1N NaOH. The precipitate was filtered, dried to afford free amidine of 1c. mp 202–203.5 °C. 1H NMR (D2O/DMSO-d6); δ 6.93 (d, J = 3.6 Hz, 1H), 7.01 (d, J = 3.6 Hz, 1H), 7.11 (d, J = 3.6 Hz, 1H), 7.22 (d, J = 3.6 Hz, 1H), 7.83 (s, 4H). 13C NMR (D2O/DMSO-d6); δ 162.2, 153.9, 152.4, 147.4, 145.7, 145.0, 134.1, 131.1, 127.3, 123.1, 111.9, 109.4, 109.2, 107.7. MS (EI) m/e (rel.int.); 294 (M+, 50), 277 (100), 261 (25). HRMS calcd for C16H14N4O2: 294.1116. Observed: 294.1101.</p><!><p>The same procedure described for preparation of 1c was used starting with 8b. Yellow solid, yield 59%, mp 269–271 °C. 1H NMR (D2O/DMSO-d6); δ 7.15 (d, J = 3.6 Hz, 1H), 7.23 (d, J = 3.6 Hz, 1H), 7.47 (d, J = 3.6 Hz, 1H), 7.57 (d, J = 3.6 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H), 8.99 (s, 1H). MS (EI) m/e (rel.int.); 296 (M++1, 100), 273 (12), 239 (40). HRMS calcd for C15H14N5O2: 296.1147. Observed 296.1189. Anal. Calcd for C15H13N5O2-2.0AcOH-2.65H2O-0.5EtOH: C, 49.41; H, 6.07; N, 14.40. Found. C, 49.72; H, 5.96; N, 14.02.</p><!><p>Retrosynthetic study of Diam id i n e s</p><p>Reagents and conditions:(i) DMF, KOAc, Pd(PPh3)4, 125–135 °C; (ii) NBS, DMF; (iii) Toluene, Pd(PPh3)4, Na2CO3, 80 °C; (iv) Pd(OAc)2, NaOAc, Bu4NCl, DMF, 70 °C.</p><p>Reagents and conditions: (i) Pd(PPh3)4, 1,4-dioxane, 100–110 °C; (ii) NBS, DMF; (iii) Cu(I)CN, DMF.</p><p>Reagents and conditions: (i) a- LiN(TMS)2, THF, r.t., 12h; b- HCl(gas), dry EtOH, r.t. 12h. (ii) NH2OH.HCl/KO-t-Bu, DMSO; (iii) a- AcOH/Ac2O; b- H2/Pd-C, AcOH.</p><p>DNA affinities, in vitro anti-protozoan data and in vivo anti-trypanosomal activity of bichalcophene diamidines.</p><p>Increase in thermal melting of Poly d(A-T)n.32</p><p>Cytotoxicity was evaluated using cultured L6 rat myoblast cells using an Alamar Blue assay.33</p><p>The T.b.r.(Trypanosoma brucei rhodesiense) strain was STIB900, and the P. f. (Plasmodium falciparum) strain was K1. The values are the average of duplicate determination.33</p><p>Selectivity index for T. b. r. (SIT) expressed as the ratio: IC50(L6)/IC50(T. b. r.).</p><p>Selectivity index for P. f. (SIP) expressed as the ratio: IC50(L6)/IC50(P. f.).</p><p>Intraperitoneal administration.14</p><p>Number of mice that survive and are parasite free for 60 days.14</p><p>Average days of survival; untreated controls expired between day 7 and 10 post infection. 14</p>

PubMed Author Manuscript

Muscle-specific TGR5 overexpression improves glucose clearance in glucose-intolerant mice

TGR5, a G protein–coupled bile acid receptor, is expressed in various tissues and regulates several physiological processes. In the skeletal muscle, TGR5 activation is known to induce muscle hypertrophy; however, the effects on glucose and lipid metabolism are not well understood, despite the fact that the skeletal muscle plays a major role in energy metabolism. Here, we demonstrate that skeletal muscle–specific TGR5 transgenic (Tg) mice exhibit increased glucose utilization, without altering the expression of major genes related to glucose and lipid metabolism. Metabolite profiling analysis by capillary electrophoresis time-of-flight mass spectrometry showed that glycolytic flux was activated in the skeletal muscle of Tg mice, leading to an increase in glucose utilization. Upon long-term, high-fat diet challenge, blood glucose clearance was improved in Tg mice without an accompanying increase in insulin sensitivity in skeletal muscle and a reduction of body weight. Moreover, Tg mice showed improved age-associated glucose intolerance. These results strongly suggest that TGR5 ameliorated glucose metabolism disorder that is caused by diet-induced obesity and aging by enhancing the glucose metabolic capacity of the skeletal muscle. Our study demonstrates that TGR5 activation in the skeletal muscle is effective in improving glucose metabolism and may be beneficial in developing a novel strategy for the prevention or treatment of hyperglycemia.

muscle-specific_tgr5_overexpression_improves_glucose_clearance_in_glucose-intolerant_mice

<!>Muscle-specific TGR5 overexpression increased RER and glycolytic flux without changing the gene expression related to glucose and lipid metabolism in mice<!><!>Discussion<!><!>Antibodies<!>Animals and diets<!>Metabolic analysis<!>Serum biochemistry<!>Metabolite profiling with CE-TOF MS<!>Cell culture<!>Real-time PCR<!>Immunoblotting<!>Intracellular TG measurements<!>Statistical analysis<!>Data availability<!>Conflict of interest<!>Supporting information

<p>Edited by Qi Qun Tang</p><p>Bile acids, the primary component of bile, are released from the gallbladder after meals to promote the absorption of lipids and fat-soluble vitamins in the small intestine. Almost 95% of bile acids are reabsorbed in the ileum and transported back to the liver through the portal blood and recycled. Therefore, the concentration of blood bile acids temporarily reaches high levels in the postprandial state (1). Interestingly, blood bile acids have been reported to function as metabolic regulators by activating several bile acid receptors.</p><p>TGR5 (also known as G protein–coupled bile acid receptor 1) is a G protein–coupled receptor that exists in the plasma membrane and recognizes bile acid as its ligand (2, 3). Ligand-bound TGR5 interacts with Gαs subunit and then activates the cAMP signaling pathway. TGR5 is expressed in various tissues, such as brown adipose tissue (BAT), white adipose tissue (WAT), and intestinal L cells. In BAT and WAT, TGR5 promotes energy expenditure, causing the amelioration of obesity (4, 5, 6). In contrast, TGR5 activation in intestinal L cells enhances GLP-1 secretion and improves diabetes in mice (7, 8). TGR5 is also expressed in the skeletal muscle, and its expression is increased by exercise (9). We have previously demonstrated that TGR5 activation induces muscle cell differentiation in cultured muscle cells and muscle hypertrophy in mice (9). Because skeletal muscle–specific TGR5 overexpression increases muscle strength, TGR5 may be a feasible target for maintaining muscle function. In fact, several compounds possessing TGR5 agonistic activity, such as citrus limonoid nomilin and obacunone, have antiobesity, antidiabetic, and muscle hypertrophy effects (10, 11, 12).</p><p>As the skeletal muscle is not only central to the locomotor system but also the largest glucose-metabolizing organ, a higher muscle mass is associated with better glycemic control (13). However, a lower skeletal muscle mass is significantly associated with type 2 diabetes (14), suggesting that increasing the skeletal muscle mass is effective in improving diabetes. Therefore, it could be expected that TGR5 activation in the skeletal muscle improves glucose metabolism by inducing muscle hypertrophy, although this has not been verified till date.</p><p>In this study, we evaluated the effects of muscle TGR5 on energy metabolism using skeletal muscle–specific hTGR5 Tg mice. Interestingly, skeletal muscle–specific overexpression of TGR5 induced an increase in the respiratory exchange ratio (RER) along with the activation of glycolytic flux in the skeletal muscle. As anticipated from these results and the fact that TGR5 induces muscle hypertrophy, Tg mice exhibited better glucose clearance under long-term high-fat diet (HFD) challenge, which was independent of changes in muscle insulin sensitivity. We also observed that Tg mice showed improvement in aging-associated glucose intolerance. Altogether, our study indicates that muscle TGR5 activation contributes to improving glucose intolerance by increasing muscle mass and glucose utilization and may be beneficial in developing a novel strategy for the prevention or treatment of hyperglycemia caused by obesity and aging.</p><!><p>To evaluate the effect of muscle TGR5 on energy metabolism, we compared muscle weight and the expression of several genes related to glucose and lipid metabolism between WT and Tg mice. As we had reported previously, Tg mice exhibited significantly increased muscle weight (Fig. S1A); however, no differences were observed in mRNA expression involved in lipid and glucose metabolism (Fig. S1B). Moreover, periodic acid–Schiff staining of gastrocnemius muscle samples revealed almost identical glycogen storage between WT and Tg mice (Fig. S1C).</p><!><p>Tg mice exhibit significantly higher RER under a normal diet.A–D, RER and energy expenditure, evaluated by oxygen consumption and carbon dioxide production, were monitored for 48 h under normal diet (n = 6). E–H, average values in light and dark periods (n = 6). I, blood glucose levels in the free-feeding condition (n = 6–9). J, blood glucose levels after fasting for 18 h. Data are mean ± SE. Statistical analyses were conducted using a two-tailed unpaired Student's t test. ∗, p < 0.05. RER, respiratory exchange ratio.</p><p>TGR5 activates glycolytic flux in skeletal muscle. Comparison of major metabolites produced in glycolysis and citric acid cycle in gastrocnemius muscle (n = 5). Red indicates a significant increase and blue indicates a significant decrease in Tg mice. Data are mean ± SE. Statistical analyses were conducted using a two-tailed unpaired Student's t test. ∗, p < 0.05; ∗∗, p < 0.01. 1,3BPG, 1,3-Bisphosphoglycerate; 2PG, 2-phosphoglycerate; 3PG, 3-phosphoglycerate; DHAP, dihydroxyacetone phosphate; F1,6BP, fructose-1,6-bisphosphate; F6P, fructose 6-phosphate; G3P, glyceraldehyde 3-phosphate; G6P, glucose 6-phosphate; PEP, phosphoenolpyruvic acid.</p><p>WT and Tg mice show similar RER under HFD.A–D, RER and energy expenditure, evaluated by oxygen consumption and carbon dioxide production, were monitored for 48 h under 4 weeks of HFD. E–H, the average value in light and dark periods (n = 5). Data are mean ± S.E. Statistical analyses were conducted using a two-tailed unpaired Student's t test. HFD, high-fat diet; RER, respiratory exchange ratio.</p><p>TGR5 activation does not affect lipid metabolism.A, C2C12 myotubes infected with adenovirus expressing TGR5 or LacZ were treated with TLCA (50 μM) for 3 h. mRNA levels were determined by RT-PCR (n = 3). B, TG accumulation normalized by total protein in C2C12 myotubes expressing LacZ or TGR5 and treated with palmitate (250 μM) and TLCA (50 μM) for 18 h (n = 3). Data are mean ± S.E. Statistical analyses were conducted using one-way ANOVA (Tukey–Kramer post hoc test). ∗, p < 0.05; ∗∗, p < 0.01; n.s., not significant. TG, triacylglycerol; TLCA, taurolithocholic acid.</p><p>Muscle TGR5 preserves obesity-induced dysfunction of glucose homeostasis.A–C, body weight (A), food intake (B), and tissue weight (C) of WT mice and Tg littermates fed HFD. D, E, oral glucose tolerance test (D) and insulin tolerance test (E). The right panel shows AUC. Data are mean ± S.E. (n = 12–14). Statistical analyses were conducted using a two-tailed unpaired Student's t test. ∗, p < 0.05; ∗∗, p < 0.01. AUC, area under the curve; HFD, high-fat diet; OGTT, oral glucose tolerance test; ITT, insulin tolerance test.</p><p>Muscle TGR5 does not improve insulin sensitivity.A, B, Akt phospho- and total protein, FLAG, and β-actin or GAPDH protein were measured by Western blotting. A, C2C12 myotubes were precultured with palmitate (500 μM) and TLCA (50 μM) for 18 h and then treated with insulin (100 nM) for 30 min (B) WT and Tg littermates fed an HFD for 8 weeks were injected with saline or insulin (0.75 IU/kg body weight) for 30 min after 18 h of fasting and then the gastrocnemius muscle was isolated. Data are mean ± S.E. Statistical analyses were conducted using one-way ANOVA (Tukey–Kramer post hoc test). ∗, p < 0.05; ∗∗, p < 0.01; n.s., not significant. HFD, high-fat diet; TLCA, taurolithocholic acid.</p><p>Muscle TGR5 improves age-associated dysfunction of glucose homeostasis. A, B, body weight (A) and tissue weight (B) of 23- to 24-month-old WT and Tg mice (n = 5–6). C-E, RER, energy expenditure, and act count of old WT and Tg mice were monitored for 48 h (n = 5–6). F, intraperitoneal glucose tolerance test in elderly (23- to 24-month-old) and young (2- to 5-month-old) WT and Tg mice (n = 6–7). The right panel shows the AUC. Data are mean ± S.E. Statistical analyses were conducted using a two-tailed unpaired Student's t test or one-way ANOVA (Tukey–Kramer post hoc test). ∗, p < 0.05; ∗∗, p < 0.01. AUC, area under the curve; RER, respiratory exchange ratio.</p><!><p>Various TGR5 agonists have been developed till date, which exert antiobesity and antidiabetic effects by activating thermogenesis in WAT and BAT and promoting GLP-1 secretion from enteroendocrine L cells (4, 5, 6, 7). It has been reported that the administration of TGR5 agonists, such as INT777, betulinic acid, and nomilin, to HFD-induced obese mice decreased body weight gain and blood glucose levels (10, 11, 12). We have also have previously demonstrated that TGR5 activation in skeletal muscle induces muscle hypertrophy (9). Skeletal muscle–specific TGR5 Tg mice exhibited increased muscle mass and enhanced muscle strength at both young and old ages. In general, the skeletal muscle is a major organ that consumes glucose, and hence, an increase in skeletal muscle mass is expected to improve glucose metabolism. However, the effects of skeletal muscle TGR5 on glucose and lipid metabolism remain unclear.</p><p>We first explored the role of muscle TGR5 in energy metabolism using skeletal muscle–specific TGR5 Tg mice that exhibited a characteristic increase in muscle mass (Fig. S1A). The exhaled breath analysis under the ND condition showed an increase in RER in Tg mice compared with control WT mice, indicating that TGR5 activation in skeletal muscle promotes glucose utilization (Fig. 1, C and G). As an increase in RER was not observed upon HFD challenge, it is possible that the utilization of glucose was promoted only when there was abundant dietary glucose available in the diet (Fig. 3, C and G). In contrast, there was no significant difference in energy expenditure between WT and Tg mice under both ND and HFD conditions (Fig. 1, D, H, and 3, D and H). In fact, body weight and food intake of Tg mice were comparable with those of WT mice under HFD challenge (Fig. 5, A and B). These data indicate that TGR5 does not promote energy expenditure and has no effect in preventing obesity at least in the mouse skeletal muscle. However, in the human skeletal muscle, it has been reported that TGR5 activation increases the expression of genes involved in energy production (4). Therefore, the effect of muscle TGR5 on energy metabolism may be different between mice and humans.</p><p>In glycolysis, which is one of the core metabolic pathways for energy production, glucose is converted into pyruvate by a 10-step enzymatic reaction. PFK is the most important rate-limiting glycolytic enzyme that catalyzes the conversion of F6P and ATP into F1,6BP, and ADP. PFK is phosphorylated by PKA, thereby modifying its enzymatic activity (17). A previous study demonstrated that activation of the cAMP-PKA pathway by epinephrine increases PFK activity through the stabilization of its tetrameric conformation in rabbit skeletal muscle (18). Moreover, it has been reported that PKA lowers the inhibition against PFK activity by lactate (19). These reports clearly indicate that PKA upregulates PFK activity in the skeletal muscle. Because TGR5 is known to activate PKA by increasing the intracellular cAMP levels, it is possible that PFK is activated in the skeletal muscle of Tg mice. To explore this possibility, we conducted metabolite profiling analysis by CE-TOF MS and observed a decrease in the levels of G6P and F6P, indicating the activation of PFK in the skeletal muscle of Tg mice (Fig. 2). In addition to PFK, there are two other known rate-limiting enzymes in glycolysis; one is hexokinase, which catalyzes the phosphorylation of glucose by ATP to G6P, and the other is pyruvate kinase, which catalyzes the transphosphorylation from PEP to ADP. The significant changes in the levels of G6P and F6P as well as 3PG and 2PG may partially be due to these rate-limiting steps other than PFK activation. These results obtained from the metabolite profiling analysis clearly explain the increased RER observed in Tg mice. Glycolysis in the skeletal muscle begins with glucose uptake from the blood or intracellular glycogen. The lack of difference in glycogen levels in the skeletal muscle between WT and Tg mice strongly suggests that the increased glycolysis in Tg mice was due to the increased uptake of blood glucose (Fig. S1C). Therefore, we conclude that activation of glycolysis in Tg mice is one of the reasons for improved glucose clearance.</p><p>As the skeletal muscle is a prominent organ of glucose disposal, TGR5-induced muscle hypertrophy and enhanced glucose utilization may lead to an increase in glucose consumption and improve hyperglycemia. To test this assumption, OGTT and ITT were performed using glucose-intolerant mice challenged by long-term HFD feeding. We observed that HFD-fed Tg mice exhibited improved glucose clearance compared with control WT mice (Fig. 5, D and E). In general, glucose intolerance in obese mice is caused by insulin resistance in peripheral tissues (20). In particular, the skeletal muscle has a significant influence on glucose clearance because it plays a major role in glucose uptake during hyperinsulinemia (21). Long-term HFD feeding promotes the accumulation of TG and lipid intermediates, such as diacylglycerol and ceramide, which hamper insulin signaling in the skeletal muscle (22, 23, 24, 25). Therefore, the TG content in the skeletal muscle correlates with insulin resistance (26, 27). Interestingly, we found little difference in the expression of genes related to lipid metabolism between the skeletal muscle of WT and Tg mice (Fig. S1B). Similar results were also obtained from TGR5-activated C2C12 myotubes, and as anticipated, TGR5 activation had no influence on intramuscular TG accumulation under palmitate treatment (Fig. 4, A and B). Consequently, TGR5 activation did not improve palmitate-induced insulin resistance in C2C12 myotubes, as well as HFD-induced insulin resistance in mice (Fig. 6, A and B). Furthermore, the levels of fasting plasma glucose, TG, NEFAs, and insulin, which are indicators of insulin resistance, were not different between Tg and WT mice after HFD challenge (Fig. S3). These results are consistent with our data showing that increased glucose utilization in Tg mice occurs when glucose is abundantly ingested in the form of ND rather than HFD. Our findings suggest that the improvement of glucose clearance by muscle TGR5 is independent of enhanced insulin sensitivity but rather caused by skeletal muscle hypertrophy and increased glucose utilization.</p><p>Unlike our study results, a recent study reported that TGR5 activation in the skeletal muscle improves insulin sensitivity and glucose homeostasis in a diabetic mice model (28). In the present study, palmitate-induced insulin resistance was prevented by TGR5 ligand and it was canceled by TGR5 silencing. Although the reason for this discrepancy remains unclear, it might be due to the type of the agonist used, as various agonists can lead to a bias in downstream signaling of G protein–coupled receptors (29).</p><p>In addition to obesity, aging is an important risk factor for glucose intolerance and insulin resistance (15, 16). Therefore, we examined the effects of skeletal muscle TGR5 on glucose metabolism in aged mice and detected better glucose tolerance in aged Tg mice (Fig. 7F). In general, age-related glucose intolerance is caused by body fat accumulation (30, 31), physical inactivity (30), and skeletal muscle atrophy (32). We compared body weight, fat weight, and physical activity in elderly WT and Tg mice and found that these parameters were comparable (Fig. 7, A, B and E). In contrast, there were significant increases in skeletal muscle weight and RER in elderly Tg mice (Fig. 7, B–C). These results suggest the involvement of muscle hypertrophy and increased glucose utilization in the improvement of glucose clearance in both elderly mice and HFD-induced obese mice.</p><!><p>Schematic representation of the effect of muscle TGR5 on glucose clearance.</p><!><p>The following antibodies were used in this study: anti-Akt (#9272) and anti-phospho-AKT (thr308) (#9275) (Cell Signaling Technology); anti-FLAG (M2) and anti-β-actin (AC-15) (Sigma); anti-GAPDH (10494-1-AP) (Proteintech); and horseradish peroxidase–coupled anti-mouse IgG and anti-rabbit IgG (Jackson Immune Research).</p><!><p>Muscle-specific TGR5 transgenic mice were generated as previously described (9). In brief, 3 × FLAG hTGR5 was cloned into MCK promoter–containing plasmid, and the purified transgene was injected into C57BL/6 oocytes. Mice were housed with a 12:12-h light–dark cycle and provided free access to water and standard chow (Labo MR Stock; Nosan Corporation Bio-Department). HFD pellets with 60% energy supplied by fat were purchased from Research Diet (D12492). The number of mice used in each experiment is described in the figure legend. All animal experiments were conducted according to the guidelines of the Animal Usage Committee of the University of Tokyo.</p><!><p>O2 consumption and CO2 production in Tg and WT mice were measured using an ARCO-2000 Mass Spectrometer (ARCO system) with one mouse per chamber as previously described (36). The chambers were maintained at 21 °C ± 3 °C, with 50% ± 10% relative humidity. The physical activity was quantitated using an infrared beam sensor (NS-AS01, Neuroscience) placed approximately 11 cm above the center of the cage.</p><p>For OGTT, mice were fasted for 16 h, and glucose water was orally administered (2 mg/g body weight). For IPGTT, mice were fasted for 16 h, and glucose water was intraperitoneally administered (1 mg/g body weight). For ITT, mice were fasted for 6 h and then injected with insulin intraperitoneally (0.75 IU/kg body weight). Blood glucose level was measured using a handheld glucometer (Ascensia Breeze 2; Bayer Diagnostics).</p><!><p>Levels of serum glucose, TG, and NEFAs were determined using kits purchased from FUJIFILM Wako Pure Chemical. Serum insulin concentrations were estimated using the Mouse Insulin ELISA Kit purchased from FUJIFILM Wako Shibayagi Corporation.</p><!><p>Gastrocnemius muscles collected from WT and Tg mice were homogenized and used for the analysis of ionic metabolites. Before analysis, hydrophobic and high-molecular-weight compounds were removed by the preparative processes of liquid–liquid separation using chloroform and water and ultrafiltration using a 5-kDa cutoff filter (37). A comprehensive analysis of ionic metabolites by CE-TOF MS was performed as described previously (38).</p><!><p>C2C12 myoblasts obtained from ATCC were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin. To induce differentiation, C2C12 myoblasts were cultured in DMEM supplemented with 2% horse serum, 100 U/ml penicillin, and 100 μg/ml streptomycin for 4 to 5 days. Cells were maintained at 37 °C in 95% humidity with 5% CO2.</p><p>For TGR5 overexpression experiments, C2C12 myotubes were infected overnight with 2.5 × 106 plaque-forming units/ml adenovirus medium. Then, the cells were washed with PBS three times and incubated with fresh medium.</p><!><p>Total RNA was extracted from C2C12 myotubes or the skeletal muscle of mice using ISOGEN (NIPPON GENE), according to the manufacturer's instructions. The high-capacity cDNA reverse transcription kit (Applied Biosystems) was used to synthesize and amplify cDNA from total RNA. Quantitative real-time PCR analyses were performed using an Applied Biosystems StepOnePlus instrument. Expression was normalized to an 18S ribosomal RNA (18S). The primers used for the PCR analysis are described in Supporting information Table S1.</p><!><p>Cells and mouse skeletal muscle were lysed in radio immunoprecipitation assay buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (v/v) Triton X-100, 0.5% (w/v) deoxycholate, and 0.1% (w/v) SDS) supplemented with a protease inhibitor mixture (Nacalai Tesque) and a phosphatase inhibitor mixture (Sigma-Aldrich). The lysates were subjected to SDS-PAGE, transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA), and then probed with the antibodies indicated in the figure legends. For cell lysates of C2C12 myotubes, the β-actin protein was used as an internal control, and for mouse skeletal muscle lysates, GAPDH protein was used as a control due to the low expression of β-actin in vivo.</p><!><p>C2C12 myotubes were washed with PBS, and lipids were extracted by hexane in 2-propanol (3:2, v/v). The levels of intracellular TG were determined using the Triglyceride E-test Wako Kit (FUJIFILM Wako Pure Chemical) and normalized to the levels of total cellular protein determined using a BCA protein assay kit (Pierce), according to each manufacturer's instructions.</p><!><p>All data are presented as mean ± SE. Two-tailed unpaired Student's t tests or one-way ANOVAs (Tukey–Kramer post hoc test) were used to determine p values. Statistical significance was defined as p < 0.05.</p><!><p>All the data are in the manuscript.</p><!><p>The authors declare that they have no conflicts of interest with the contents of this article.</p><!><p>Tables and figures</p>

PubMed Open Access

Peptidyl Acyloxymethyl Ketones as Activity-Based Probes for the Main Protease of SARS-CoV-2

The global pandemic caused by SARS-CoV-2 calls for a fast development of antiviral drugs against this particular coronavirus. Chemical tools to facilitate inhibitor discovery as well as detection of target engagement by hit or lead compounds from high throughput screens are therefore in urgent need. We here report novel, selective activity-based probes that enable detection of the SARS-CoV-2 main protease. The probes are based on acyloxymethyl ketone reactive electrophiles combined with a peptide sequence including non-natural amino acids that targets the non-primed site of the main protease substrate binding cleft. They are the first activity-based probes for the main protease of coronaviruses and display target labeling within in a human proteome without background. We expect that these reagents will be useful in the drug development pipeline, not only for the current SARS-CoV-2, but also for other coronaviruses.

peptidyl_acyloxymethyl_ketones_as_activity-based_probes_for_the_main_protease_of_sars-cov-2

<!>Results and discussion<!>Conclusion

<p>Although coronaviruses that infect humans have been known since 1966, 1 2 they have gained more global attention since the turn of the millennium, with the appearance of several strains that caused more severe symptoms and were more widely spread. Two outbreaks in 2002 and 2012, which were caused by severe acute respiratory syndrome coronavirus (SARS-CoV) 3 and Middle-East respiratory syndrome coronavirus (MERS-CoV), 4 respectively, were contained regionally. In December 2019, a novel coronavirus, later termed SARS-CoV-2 appeared in the province of Wuhan, China, 5 which led to a global pandemic that is currently ongoing. The clinical manifestations range from asymptomatic to fever and severe pneumonia known as covid-19, which in some cases can lead to death. 6 The virus has caused a worldwide crisis in healthcare, economy and society, and has painfully exposed the vulnerability of modern civilization. Currently, there is an ongoing global effort to develop vaccines and drugs that can eradicate SARS-CoV-2 and contain the pandemic. Unfortunately, the immune response against the virus is not well understood, and it is unclear whether infected people build up immunity against reinfection. This emphasizes the importance for the development of drugs that inhibit coronavirus replication.</p><p>Coronaviruses -or coronaviridae -are enveloped, single stranded RNA viruses with a typical appearance of the solar corona caused by the viral spike protein that is part of the viral capsid.</p><p>The replicase gene of the virus contains two overlapping open reading frames (ORFs) that are translated in two large polyproteins. 7 These polyproteins are proteolytically processed into 16 non-structural proteins (nsp1 to nsp16) by two viral proteases. Whereas the papain-like protease (PL pro ) performs three cuts to release nsp1-3, the main protease (M pro , also called 3CL pro for 3C-like protease) cuts at 11 sites, thereby liberating the most important protein products for replication. 7 8 Therefore, the M pro has attracted major interest as a potential drug target.</p><p>M pro is a protease from the C30 family. 9 Its active site comprises a catalytic dyad formed by a cysteine and histidine residue. The substrate specificity is governed by a requirement for Gln in the P1 position, and preferences for large hydrophobic residues in P2 and small residues in P1'. 10 11 Recently, the Drag laboratory has reported a substrate library screen incorporating a large series of non-natural amino acids in a comparison of M pro from SARS-CoV and SARS-CoV-2. 12 In the past, information on substrate specificity has been instrumental in the design of active site directed M pro inhibitors -especially of peptidomimetics, 13 whereas crystallography has increased our structural understanding of the interactions between inhibitors and M pro for further optimization. 14 Since the start of the 2019 SARS-CoV-2 pandemic, several crystal structures of the M pro in complex with various inhibitors, such as peptidyl ketoamides, peptide aldehydes and peptidyl vinyl methyl esters, have been published. 15 16 17 In the past, activity-based protein profiling (ABPP) has been particularly useful for the study of proteases. 18 19 Activity-based protein profiling (ABPP) is a powerful technique to detect active enzymes in complex proteomes, such as cell lysates, whole cells or in vivo. 20 It relies on small molecules called activity-based probes (ABPs) that specifically form covalent bonds with active site residues of the target enzyme in a mechanism-based reaction. Although ABPP has been extensively applied for the characterization for the study of cancer-related enzymes 21 as well as enzymes from infectious bacteria, 22 application to viral infections has been much less investigated. 23 One of the underlying reasons may be the lack of ABPs specifically designed for viral targets, except for probes targeting the NS2B-NS3 protease of flaviviruses 24 25 and the smallpox virus protease K7L. 26 Here, we report the first ABPs for the Mpro of coronaviruses, exemplified by a probe for the Mpro of the recent SARS-CoV-2. It is based on a cysteine specific reactive electrophile and recent substrate specificity information combining natural and non-natural amino acids. We show that these probes detect the active form of the Mpro, are highly selective and can distinguish the protease within a human proteome.</p><!><p>Probe design -As a warhead for the M pro ABPs we chose the acyloxymethyl ketone (AOMK) reactive electrophile, because it is selective for cysteine proteases and compatible with solid phase peptide synthesis. 27 To guide this warhead towards the active site cysteine of the SARS-CoV-2 M pro , we incorporated a peptide element matching the substrate specificity. Because the glutamine side chain may react with the ketone moiety of the AOMK in a cyclization reaction, as reported before for glutaminyl fluoromethyl ketones, 28 we also incorporated a citrulline as possible glutamine mimic, a dimethylated Gln and a His, which both have been used as P1 residue in M pro inhibitors. 13 For the P2-P4 position, we chose Abu-Tle-Leu, which is the recently reported optimal sequence for SARS-CoV-2 M pro . 12 However, for the P1 His molecules, a Thr-Val-Cha sequence was selected, because this was reported to be a potent sequence for the biologically similar SARS 3CL Protease. 29 As a detection handle, we chose a hexynoic acid, since it allows for click chemistry-mediated introduction of various tags.</p><p>Probe synthesis -For the solid phase synthesis of the AOMK M pro ABPs, we first synthesized the necessary chloromethyl ketone (CMK) building blocks of properly protected glutamine, dimethyl glutamine (made from commercially available building block 1), citrulline and histidine, by activation of the carboxylic acid with isobutyl chloroformate, subsequent reaction with diazomethane and quenching with hydrochloric acid (Scheme 1A). The His CMK could not be made with the more standard trityl protecting group, not only because of the lability of the protecting group under acidic conditions, but also because of substitution of the chloride by the imidazole nitrogen in part of the product. Fortunately, with the more electron withdrawing Boc protecting group on the His side chain, CMK building block 3d was obtained in a satisfactory crude yield of 82%.</p><p>For the construction of the probes, we took two different solid phase approaches. The probe with a P1 Gln side chain offered the opportunity to attach it by its side chain to a solid support.</p><p>To this end, CMK building block 3a was first converted into an AOMK by reaction with dimethylbenzoic acid, before removal of the tert-butyl protecting group and coupling to the Rink amide resin (Scheme 1B). Elongation with the appropriate Fmoc-protected amino acids, capping with a hexynoic acid and cleavage from resin yielded probe 4. The compounds with a dimethyl Gln, Cit or His in the P1 position were made by loading the corresponding CMKs 3bd onto a semicarbazide resin, follwed by substitution with dimethylbenzoic acid, elongation and cleavage from the resin (Scheme 1C). Unfortunately, after cleavage from the resin and 6 concomitant cleavage of side chain protecting groups (where present), the compounds with a P1 His residue immediately hydrolyzed to hydroxymethyl ketones (HMKs) 7-8, probably assisted by the imidazole side chain (Scheme 1D). Evaluation of inhibition -Compounds 4-8, were evaluated an inhibition assay on purified SARS-CoV-2 M pro , and compared to the recently reported M pro active site inhibitor carmofur. 16 30 In short, the protease was incubated with the compound for 1 hour, after which a fluorogenic aminomethyl coumarin substrate 12 was added to measure residual activity. Compounds 4 and 6 showed clear inhibition of the protease, with probe 6 being most potent, but 5 and 8 did not show any activity (Figure 1A). Interestingly, probe 7 was still able to inhibit M pro to some degree, even without its ability to form a covalent bond with the protease, illustrating that a His is still accepted in the P1 position.</p><p>To gain insight in the binding modes of the different probes, we performed covalent docking of the probe 4 -8 in complex with Mpro using the flexible side chain method of Autodock 4.2. 31 The P1 and P2 amino acid residues of probe 4 and 6 both dock well into the corresponding S1 and S2 pockets of the enzyme (Figure 1C-E). Moreover, they display good overlap with crystal structures of various different peptidomimetic inhibitors (Figure 1D and Figure S1). However, probe 5 with Cit in the P1 position does not fit well into the active site (Figure 1F). Repeated runs of docking did not result in placement of the P1 Cit into the S1 pocket, likely due to its larger side in comparison with Gln, which explains the lack of potency of probe 5 and confirms the importance of the S1 pocket for inhibitor design. In-gel fluorescence clearly shows that M pro is covalently modified by both 4 and 6 at concentrations as low as 200 nM (Figure 2A). Biotin blot was also able to detect the covalent probe-Mpro complex, albeit with slightly lower sensitivity. Overall, probe 6 with the dimethyl-Gln in the P1 position is more sensitive than probe 4, in line with the results from the inhibition assay. This may be attributed to the possible reversible formation of a cyclic hemiamidal by reaction of the glutamine side chain with the ketone (Figure 2B).</p><p>We next investigated the capacity of the probes to label SARS-CoV-2 M pro in a complex proteome. To this end, prepared lysates of E. coli that were induced or not induced to express recombinant M pro . Because the click chemistry could potentially be problematic in more complex samples, we also synthesized a pre-clicked TAMRA version of probe 4 (probe 9; see Figure S2). Both probe 4 and 6 were able to label M pro selectively in the bacterial lysate, as the few other observed bands were caused by click chemistry background labeling as judged from a no probe control sample (Figure 2C). Labeling by probe 4 and 6 is largely prevented by the active site inhibitor carmofur and no substantial labeling was observed in the lysates of uninduced bacteria. Both results demonstrate the selectivity of the probes.</p><p>For future application in infected human cells, we wanted to show that the probes do not show crossreactivity with human proteins. As we lack the facilities in our laboratory to work with live viruses, we spiked lysates of HEK293T cells with different amounts of purified M pro and labeled with preclicked probe 9. SDS-PAGE followed by fluorescent scanning revealed that M pro was detected with a sensitivity down to 0.07% of the total proteome content. No other substantial gel bands were detected, illustrating that probe 9 specifically labels M pro within a complete human proteome.</p><!><p>In conclusion, we designed and synthesized different ABPs targeting the protease M pro of SARS-CoV-2. They are based on cysteine reactive AOMK electrophiles and peptides resembling the substrate specificity of M pro . AOMKs have not been reported before as inhibitors for M pro . We have here demonstrated that they act as covalent, active site inhibitors.</p><p>Admittedly, the P1 residues in this study are not yet optimal, as the side chain of Gln may form a cyclic hemiamidal and the GlnMe2 is generally less favorable for M pro . Hence, we anticipate that further optimization of the peptide element as well as the primed site leaving group will lead to AOMK probes and inhibitors with higher potency. Nevertheless, the here described probes are able to detect M pro in complex proteomes without background labeling of other</p>

ChemRxiv

Enhancement of Clathrate Hydrate Formation Kinetics Using Carbon-Based Material Promotion

Although hydrate-based technology has been considered as a safe and environmentally friendly approach for gas storage and transportation in recent decades, there are still inherent problems during hydrate production, such as a long induction time, slow formation kinetics, and limited hydrate storage capacity. Attempts to resolve these issues have resulted in the development of various kinetics promoters, among which carbon-based materials have become one of the most attractive owing to their unique promotion effect. Herein, results on promotion by bulk wetted carbon materials in the forms of a packed bed, carbon particles in a suspension, and nano-carbon materials in a nanofluid are collected from the published literature. Meanwhile, the promotion mechanisms and influencing factors of the carbon-based promoters are discussed. The purpose of this mini-review is to summarize recent advances and highlight the prospects and future challenges for the use of carbon-based materials in hydrate production.

enhancement_of_clathrate_hydrate_formation_kinetics_using_carbon-based_material_promotion

Introduction<!>Gas Hydrate Formation With Carbon-Based Materials<!>Gas Hydrate Formation With Bulk Carbon Materials<!><!>Gas Hydrate Formation With Bulk Carbon Materials<!>Gas Hydrate Formation With Carbon-Based Suspensions<!>Gas Hydrate Formation With Carbon-Based Nanofluid<!>Conclusion and Prospects<!><!>Author Contributions<!>Conflict of Interest<!>

<p>Natural gas hydrate, also referring to the methane hydrate, is an ice-like clathrate constituted by hydrogen-bonded water molecules and light molecules like methane that have filled in the cavities via Van der Waals force (Sloan, 1998). This solidified natural gas (SNG) has been viewed as a potential alternative for natural gas transportation and storage because of several advantages (Thomas, 2003; Javanmardi et al., 2005; Koh et al., 2011; Veluswamy et al., 2018): the high volumetric storage capacity of 160–180 v/v, much milder formation and storing conditions than CNG and LNG, e.g., at 273.15 K and 3.2 MPa for methane hydrate formation, and safe and environmentally benign manufacturing process. However, technical challenges arise in the production process, primarily the slow kinetics of hydrate formation, large amount of heat generated, and limited gas storage capacity. Hydrate formation is always accompanied by heat release, which will impede hydrate growth if the heat is not removed in time, particularly in large-scale industrialization. Moreover, the theoretical gas storage capacity is hard to achieve due to the retarded mass transfer caused by the formation of thin hydrate layers at gas–liquid interfaces (Lee et al., 2006; Aman and Koh, 2016).</p><p>A great deal of effort has been focused on developing efficient methods for overcoming the above issues. To date, the most well-studied field is the formation of methane hydrates in the presence of surfactant, among which sodium dodecyl sulfate (SDS) showed the best performance (Zhong and Rogers, 2000; Kumar et al., 2015). In a recent review article, He et al. He et al. (2019) have provided a good review of surfactant-promoted gas hydrate formation during the past three decades. Given the enormous amount of foam production in hydrate dissociation and the difficulty of recycling the surfactant, non-surfactant-based methods for improving hydrate formation have attracted growing attention over the last 10 years. A review by Veluswamy et al. (2018) documented and discussed in detail the different materials applied for methane hydrate formation, e.g., silica gel, dry water, dry gel, sand, zeolite, and hollow silica, which are used as a fixed bed for hydrate reaction. Another review conducted by Nashed et al. (2018) shed light on the nanomaterials for gas hydrate formation, where various metal-based particles, like nano-Ag, Cu, CuO, and ZnO were discussed, and it was concluded that nanoparticles not only could help to promote mass transfer but they could also contribute to heat transfer enhancement in the hydrate reaction. Additionally, some non-metal materials such as silica nanoparticles (Wang et al., 2019), graphene (Wang et al., 2017), and carbon nanotubes (Pasieka et al., 2014) exhibited excellent performance in promoting gas storage capacities and hydrate formation rate.</p><p>As carbon-based materials (e.g., activated carbon, graphite, graphene, and carbon nanotubes) have been widely employed in gas hydrate formation in recent years, this mini-review summarizes the published studies where the promotion effects of carbon-based materials on gas hydrate formation were investigated. With an attempt to draw critical conclusions after compiling this knowledge into a single article, this review provides significant guidance for developing novel methods for hydrate-based technology.</p><!><p>Porous carbon-based materials, such as active carbon, graphite, carbon nanotubes, and graphene, can realize gas adsorption due to their porosity and high specific areas when utilized for hydrogen or methane storage (Nikitin et al., 2008; Mohan et al., 2019). During research on the gas adsorption process, scientists discovered that when carbon materials were wetted by water or dispersed in water, a higher methane storage capacity was obtained via hydrate formation under certain conditions. Hence, carbon materials attracted research interest as efficient promoters for the gas hydrate formation, resulting in numerous investigations in the last 10 years. Referring to the literature concerning different kinds of carbon materials, this section is divided into three parts: the promotion of gas hydrate formation by bulk carbon materials, carbon-based suspensions, and carbon-based nanofluid, respectively.</p><!><p>Since natural gas hydrates are usually stored within porous sediments in nature, it is essential to understand the characteristics of hydrate formation in porous space. In experiments, the reactor is often filled with bulk materials with adsorbed water in the form of a packed bed for hydrate formation. The mass ratio of water to bulk materials, the material types, and the pore size are the primary factors that affect the gas hydrate formation rate and storage capacity.</p><p>The literature regarding the use of porous carbon materials (mainly referring to activated carbon) in methane hydrate formation is listed in chronological order in Table 1A. The porous material first reported as being for hydrate formation was active carbon, in an investigation by Zhou and Sun (2001). They found that wet activated carbon caused an increase in methane adsorption isotherms and enhanced gas uptake by 60% at a water ratio of 1.4. Later on, many studies proved the optimal water/carbon mass ratio to be about 1 (Perrin et al., 2003; Yan et al., 2005; Celzard and Marêché, 2006). By analysis of pore styles, Perrin et al. found that microporosity seemed to be useless for clathrate formation, while mesoporous and macroporous carbon materials were more favorable to enhancing hydrate formation. Following this work, Celzard and Marêché (2006) proved, however, that saturated pore space would slow down the hydrate formation kinetics since gas diffusion pathways became scarce when the small spaces in the pore network were filled by water. Similarly, another study showed that a 96.5% enhancement of water conversion was obtained due to the larger interstitial pore space between activated particles than between other smaller particles under 8 MPa and 4°C (Siangsai et al., 2015). Via observation of the morphology of methane hydrate formed in porous media of activated carbon, Babu et al. (2013) confirmed that the hydrates primarily nucleated on the surface of the activated carbon and that whether the hydrates further developed into stable hydrate crystals depended on the interstitial space between the activated carbon particles. As a consequence, porous activated carbons with an optimal water ratio can provide excellent interfaces that enlarge the area of gas–liquid contact for hydrate nucleation and growth, and the hydrate formation process is only accelerated by active carbons with large pore size rather than micropores.</p><!><p>List of the carbon-based materials used in methane hydrate formation.</p><p>Rw is the mass ratio of water to carbon; "-" means "not found"; SSA refers to specific surface area; P and T are the pressure and temperature respectively; the storage efficiency improvement was calculated based on a pure water system.</p><!><p>Aiming to determine the critical hydrate formation conditions, phase equilibrium estimations of gas hydrate formation in porous carbon materials have been conducted in many experimental or theoretical studies. The methane hydrate equilibrium was usually shifted to a higher pressure or lower temperature in bulk carbons compared to pure water (Najibi et al., 2008; Mingjun et al., 2010; Yang et al., 2012). For example, Liu et al. (2018) measured the methane hydrate formation or dissociation conditions in eleven porous materials, verifying that smaller pores size (below 6.2 nm) exerted a negative influence on the hydrate formation conditions due to extra capillary pressure in these pores. Taking the pore size, pore distribution, capillary pressure, and hydrate–liquid interfacial tension into consideration, some new equilibrium models were established and also supported the experimental results (Zhang et al., 2020).</p><p>The addition of a traditional promoter such as a surfactant or thermodynamic promoter into the water or offering hydrophobic/hydrophilic groups on the carbon surface have proved to be efficient ways of improving gas storage capacity and the hydrate formation rate in porous media (Casco et al., 2017; Cuadrado-Collados et al., 2020; Palodkar and Jana, 2020; Zhang et al., 2020). In the latest research, Cuadrado-Collados et al. (2020) reported the promotion effects of various additives, such as the sodium dodecyl sulfate (SDS), leucine, and tetrahydrofuran (THF) in the confined nanospace of the carbon surface, where hydrate nucleation and growth rate were both accelerated significantly. A similar work was conducted by Zhang et al. (2020), who noted that, when anionic active groups aggregated onto the surface of the porous media, the modified carbon could promote gas adsorption and enhance formation process because of micellar solubilization in the presence of SDS. By introducing oxygen-containing groups on the activated carbon, the carbons performed better after being wetted by water, as shown by the result that the methane hydrate yield was elevated to 51% for oxidation-treated carbons under the conditions of 3.3 MPa and 2°C. It was assumed that the locations of the oxygen groups on the surfaces of carbons acted as nucleation centers for water clustering, which benefited further hydrate growth (Casco et al., 2017). Herein, after functionalization or being attached to other promoters, porous carbon materials provided more efficient reaction media for hydrate formation.</p><p>There are two basic kinds of promotion mechanism for hydrate formation in wetted porous carbon materials. The generally accepted mechanism is the interface adsorption theory (Zhou and Sun, 2001; Mingjun et al., 2010; Cuadrado-Collados et al., 2018; Andres-Garcia et al., 2019). Unlike in the gas–free water system, there are many voids among and inside the carbon particles when water is absorbed in porous activated carbon, and these will provide efficient contact areas for gas and water. The hydrate formation process can then be described as: liquid water films gradually form at the surface of the carbon interface, followed by hydrate formation after gas adsorption at the water–carbon interface. This theory also points out that methane hydrates tend to form in wider pores and the intersectional spaces between particles. Another promotion mechanism is the capillary effect caused by the pores or interstitial space in the porous media. As the capillary force can enhance liquid phase migration in the pores, continuous gas–liquid contact is realized, and hydrate formation distributions are changed constantly. This promotion mechanism became more obvious when surfactant was added to the porous materials (Zhang et al., 2020). However, in this light, a minimum pore size of about 3 nm is required for methane hydrate formation considering the hydrate crystal size. Conversely, in some cases, the pore capillary force was assumed to reduce the activity of the pore-confined water that hindered hydrate formation (Liu et al., 2018).</p><!><p>Suspensions formed by carbon particles in an aqueous solution are considered another potential reaction medium for rapid hydrate formation (Takahata et al., 2010; Govindaraj et al., 2015; Yu et al., 2016, 2018). In case of severe sedimentation of hydrophobic particles in the reaction system, mechanical agitation is necessary during the hydrate formation. A carbon-based suspension is preferable to bulk materials in a fixed bed as the hydrate reaction system, since there are three distinct advantages when particles are dispersed in a liquid phase: the greater gas–liquid contact area of stirred suspensions, a more uniformly distributed hydrate crystallization process, and the feasibility of a continuous production process (Govindaraj et al., 2015).</p><p>By investigating methane hydrate formation kinetics in an activated carbon particle suspension at loadings of 0.5, 1.0, and 2.0 wt%, Govindaraj et al. elucidated that suspensions with a higher fraction of activated carbon particles had stronger promotion effects on hydrate formation kinetics (Govindaraj et al., 2015). Meanwhile, a prominent positive correlation was established between the activated carbon concentration and the hydrate gas storage capacity, where the gas storage capacity was increased by 60% in a 2.0 wt% suspension compared to a pure water system. Although in several studies, the graphite had marginal promotion effects on methane hydrates, mixtures of graphite and other promoters, e.g., a mixture of graphite and hematite or a mixture of graphite and surfactant could lead to rapid hydrate formation (Takahata et al., 2010; Yu et al., 2016). Carbon nanotubes, in particular, attracted most interest for the excellent thermal properties reported in some literature. By adding multi-walled or single-walled carbon nanotubes to pure water, the gas consumption and hydrate reaction rate during hydrate formation were dramatically improved (Park et al., 2010). A comparative study on the enhanced formation of methane hydrate by different types of CNTs indicated that a shorter nucleation stage and more rapid growth process were obtained when short nanotubes (CM-95) rather CM-100 were applied as additives as a result of the larger specific area of the shorter MWCNTs (Kim et al., 2011).</p><p>In summary, carbon particle suspensions have obvious promotion effects on gas hydrate formation. The primary reason for this is the enlarged gas–liquid contact area provided by suspended particles, which leads to a mass transfer enhancement. However, it is noted that hydrate formation must be carried out with the aid of stirring, and it thus requires extra energy consumption and the use of an agitation apparatus.</p><!><p>Nanofluid is actually a stable dispersion formed by nanoparticles dispersed homogeneously in an aqueous phase. Nanofluid is considered an excellent hydrate reaction medium based on its superior mass transfer and heat transfer properties (Li et al., 2017; Nashed et al., 2018). The behaviors of nanoparticles in nanofluid that promote hydrate formation are as follows. Firstly, the nanoparticles move like microstirrers in the liquid through Brownian motion, resulting in constant updating of the gas–liquid interface. Secondly, the nanoparticles have high specific surface areas and can thus offer plenty of nucleation sites for hydrate formation. Lastly, the continuous movement of carbon nanoparticles helps to remove the heat generated during hydrate formation. Carbon nanomaterials such as carbon nanotubes and graphene are more beneficial to heat transfer due to their intrinsic high thermal conductivity.</p><p>Nanofluid constituted by water-soluble carbon nanotubes has been verified to be an excellent promoter for gas hydrate formation (as listed in Table 1B). When an oxidized CNT nanofluid was used as the reaction system, the gas consumption was up to 4.5 times higher than in water (Park et al., 2012; Li et al., 2017). The promotion efficiency of chemically or physically treated CNT nanofluid exceeded that of pristine CNTs. For instance, acid-treated CNTs, SDS-coated CNTs and plasma-functionalized CNTs could efficiently reduce induction time, increase gas consumption, and enhance growth rate (Park et al., 2010; Pasieka et al., 2013, 2015). The promotion efficiency of the CNT-based nanofluid, however, is affected by the particle fraction, the surface functional groups, and the treatment methods. The best concentration of OCNTs for promoting the growth of methane hydrate was 0.003% in Park et al. (2010). In view of the marked reactivity of the sulfonate groups contained in SDS, some researchers have coated the CNT surfaces with SDS, long-chain polymers containing SO3-, or Reactive Red 195 molecules and then dispersed the CNTs in water for use as the reaction system. Hydrates formed in these nanofluids all exhibited gas storage capacities that were elevated to 140–150 v/v, and the hydrate reactions finished within 100 min (Song Y. et al., 2017; Song Y. M. et al., 2017; Song et al., 2019). Moreover, with the aid of a high-speed ball milling process, the obtained functionalized CNTs (such as RR195@CNTs) had excellent recycling performance in the hydrate formation process (Song et al., 2019). Due to the thermal conductivity of metal nanoparticles (nano-Ag or nano-Cu), a prepared compound nanofluid containing OCNTs grafted by metal nanoparticles had a stronger promotion effect than the one-component nanofluid, with the exception that the metal nanoparticle-grafted OCNT nanofluid was not as stable as an OCNT nanofluid (Song et al., 2018).</p><p>Since graphene has smooth surfaces and is easy to functionalize by sulfonate groups or to load with metal nanoparticles, this two-dimensional carbon material is also introduced to hydrate formation reactions. Wang et al. (2017) grafted sulfonate groups successfully to graphene by covalent bonding and used it in methane hydrate formation. The results showed that the promotion efficiency of SGO (sulfated graphene) was better than that of GO (oxidized graphene). In another work, nano-Ag coated SGO was prepared for methane hydrate formation, and a shorter hydrate formation stage was achieved compared to SGO (He and Wang, 2018).</p><p>Considering that the fraction of carbon nanoparticles in the nanofluid is far smaller, the promotion efficiency of the equivalent carbon-based material in nanofluid is superior to the materials in suspension or a packed bed. Besides, the stable carbon-based nanofluid has excellent recycling performance in repeated hydrate formation, which thus contributes to more economical hydrate production.</p><!><p>This work is devoted to the summary of hydrate formation in various carbon media of different forms: porous carbon materials in packed beds, particles in suspension, and nanoparticles in nanofluid. Figure 1 highlights the themes of this mini review. Porous carbons provide a large interface area for gas-liquid contact, and particles in suspension or nanofluids are helpful for heat and mass transfer enhancement. To sum up, carbon-based materials, either in macro or micro forms, all show unique promotion effects on gas hydrate formation. Carbon-based nanofluid is the preferable medium among these for achieving economical and efficient hydrate production. Accordingly, it is necessary to develop more economical and efficient carbon-based nanofluids via surface modifications or coupling with other promoters. Besides, a majority of current research focuses on experimental investigation, while few works have attempted molecular illustration of the gas hydrates promoted by those carbon materials. Molecular simulation or mathematical modeling to investigate the hydrate formation characteristics and hydrate growth mechanism in carbon-based materials is therefore required, and this would also be helpful for designing and propelling the application of novel carbon materials for hydrate-based technology.</p><!><p>The schematic summary of the promotion of the carbon-based materials on the gas hydrate formation.</p><!><p>Y-MS was in charge of literature collection, review, and writing. R-QL contributed to the tools and the internet search. FW and D-BZ helped write the manuscript. J-HS and LY assisted with manuscript enhancement. All authors contributed to the article and approved the submitted version.</p><!><p>The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.</p><!><p>Funding. This work was supported financially by the National Natural Science Foundation of China under the grants 51676031, 51976087, 21706269, and 21978142 and by the Natural Science Foundation of Shandong Province with grant number ZR2018PEE004.</p>

PubMed Open Access

Hi-Plex targeted sequencing is effective using DNA derived from archival dried blood spots

Many genetic epidemiology resources have collected dried blood spots (predominantly as Guthrie Cards) as an economical and efficient means of archiving sources of DNA, conferring great value to genetic screening methods that are compatible with this medium. We applied Hi-Plex to screen the breast cancer predisposition gene PALB2 in 93 Guthrie Card-derived DNA specimens previously characterised for PALB2 genetic variants via DNA derived from lymphoblastoid cell lines, whole blood and buffy coat. 92 of the 93 archival Guthrie Card-derived DNAs (99%) were processed successfully and sequenced using approximately half of a MiSeq run. From these 92, all 59 known variants were detected and no false-positive variant calls were yielded. 98.13% of amplicons (5417/5520) were represented within 15-fold of the median coverage (2786 reads) and 99.98% of amplicons (5519/5520) were represented at a depth of 10 read-pairs or greater. With Hi-Plex, we show for the first time that a high-plex amplicon based MPS system can be applied effectively to DNA prepared from dried blood spot archival specimens and, as such, dramatically increase the scopes of both method and resource.

hi-plex_targeted_sequencing_is_effective_using_dna_derived_from_archival_dried_blood_spots

Introduction<!>DNA samples<!>Mutation Screening using Hi-Plex<!>Results and Discussion<!>Conclusions<!>

<p>Dried blood spots, or Guthrie Cards specimens, provide a long-term, cost-effective and convenient alternative to freezing blood [1]. In many developed countries, they are obtained routinely from newborns to screen for metabolic disorders [2]. Dried blood spots have been collected by large epidemiological studies such as the Breast Cancer Family Registry [3] and the Melbourne Collaborative Cohort Study [4] as well as numerous others [5]. The ability to use dried blood-derived DNA in the context of such large study resources would allow researchers to make a considerable contribution to our understanding of the genetics of human diseases. However, there is evidence for DNA fragmentation over time with storage of dried blood spots [6], and this can influence the efficiency of downstream applications. Dried blood spots have been reported as a source of DNA suitable for downstream SNP genotyping via prior whole-genome amplification [7, 8] and, more recently, without the requirement for pre-amplification [9]. Archived neonatal dried blood spot samples have also been used, following whole-genome amplification, for accurate whole-genome and exome-targeted massively parallel sequencing [10]. A 'low amplicon-plexity'-based approach has been published to show that massively parallel sequencing (MPS) of dried blood spot specimens can offer a novel approach to HIV drug-resistance surveillance [11]. To our knowledge, no prior study has been published that demonstrates or validates the accuracy of 'high amplicon-plexity' targeted enrichment applied to dried blood spot-derived DNA for genetic screening via MPS.</p><p>We previously developed and reported Hi-Plex, a streamlined and cost-effective highly multiplexed PCR approach for MPS library preparation. In addition to superior cost-effectiveness and accuracy [12] and Hsu et al. (submitted manuscript), Hi-Plex confers mechanistic advantages over alternative amplicon-based targeted enrichment systems for application to fragmented DNA since it can define a small and uniform size of amplicons [13, 14] and Nguyen-Dumont et al. (submitted manuscript). In this study, we assess the performance of Hi-Plex applied to archival dried blood spot-derived DNA.</p><!><p>Our sample set comprised 93 Guthrie Card-derived DNAs from women affected by breast cancer that had been screened previously for mutations in the coding and flanking intronic regions of PALB2 via Hi-Plex and Sanger sequencing and/or high resolution melting curve analysis of lymphoblastoid cell line, whole blood or buffy coat-derived DNA [15–17]. All participants provided written informed consent for participation in the study. This study was approved by The University of Melbourne Human Research Ethics Committee.</p><p>Guthrie Card samples were provided by the Australian Breast Cancer Family Registry [3] (ABCFR, 89 specimens, including one duplicated sample) and the Kathleen Cuningham Foundation Consortium for research into Familial Breast cancer (kConFab, Melbourne, Australia, four specimens). The samples were archived between six and 21 years prior to this study (mean: 12 years, median: 10 years, standard deviation: 4 years). DNA extractions from 2 mm diameter circular punches were performed using the QIAamp® 96 DNA blood kit 4 (Qiagen, Hilden, Germany) according to the manufacturer's instructions, including a proteinase K incubation step. Quant-iT™ PicoGreen® dsDNA Assay Kit (Life Technologies) was used for quantification.</p><!><p>This Hi-Plex assay was designed to target the PALB2 and XRCC2 genes. However, genotyping aspects of this study focus on PALB2 only, as we did not have a similar test set with genotyping data for XRCC2. Sixty primer pairs targeting the protein coding and some flanking intronic and untranslated regions of PALB2 and XRCC2, dual-indexing hybrid adapter primer sets and 'Bridge' primers are described in [13], [15] and Nguyen-Dumont et al. (submitted manuscript), respectively and listed in Supplementary Table 1. Supplementary Figure 1 schematically indicates how gene-specific primers target PALB2. All oligonucleotides used in this study were manufactured to standard desalting grade by Integrated DNA Technologies (Coralville, IA, USA). 94 individual PCR reactions (93 specimens and one no-template control) were conducted in wells of a skirted PCR plate in a final volume of 25μl with 1xPhusion® HF PCR buffer (ThermoScientific, Waltham, MA, USA), 1 unit of Phusion Hot Start II High-Fidelity DNA Polymerase (ThermoScientific), 400μM dNTPs (Bioline, London, UK), approximately 1 μM gene-specific primer pool aggregate (individual gene-specific primer concentrations vary and are described in [14] – those deviating from 4nM final reaction concentration are listed in Supplementary Table 2), 1μM 'Bridge' primers F8-bridge and R5-bridge (Nguyen-Dumont et al., submitted), 2.5mM MgCl2 (ThermoScientific) and 25 ng input genomic DNA. The following steps were then applied: 98°C for 1 min, 20 cycles of [98°C for 30 sec, 50°C for 1 min, 55°C for 1 min, 60°C for 1 min, 65°C for 1 min, 70°C for 1 min], addition of 1 μM dual-indexed hybrid adapter primers, then a further four cycles of [98°C for 30 sec, 68°C for 1 min, 70°C for 1 min], followed by incubation at 68°C for 20 min. Pooled library size-selection, quantification and sequencing were performed as detailed in [15], except that only ~53% of the sequencing run was dedicated to this experiment. Briefly, equal volumes of Hi-Plex products from each specimen were pooled and 40μl of the pooled library was resolved on a single wide lane (~2 cm) by 2% (w/v) agarose TBE gel electrophoresis using HR-agarose (Life Technologies, Carlsbad, CA, USA). The ~275bp library was excised from the gel and purified using the QIAEXII gel extraction kit (Qiagen, Dusseldorf, Germany). The library was sequenced on a MiSeq instrument using the MiSeq Reagent kit v2 300 cycles (Illumina). Prior to performing the sequencing run, 3.4μl of 100μM sequencing primers TSIT_Read1, TSIT_Read2 or TSIT_i7_read (Supplementary Table 1) were added respectively to the read1, read2 and i7 primer reservoirs in the MiSeq reagent cartridge. Mapping to hg19 was performed using bowtie-2-2.1.0 [18] with default parameters except for –trim5 20 and –trim3 20. ROVER variant caller [12] was applied using a variant proportion threshold of 0.15 and minimum required variant depth of two read-pairs.</p><!><p>Of the 93 specimens, 92 (99%) were sequenced successfully. The remaining specimen conferred low polymerase fidelity during amplification and a very low yield, indicative of PCR inhibition. The failed specimen was unexceptional in that it had been archived for the median time prior to this study. It is likely that this specimen was affected by unusual handling at some point during collection, storage or DNA extraction, although the timing of collection, transport and processing to produce the Guthrie Card specimen was routine.</p><p>For the sequenced 92 specimens, using ~53% of the MiSeq run, the median coverage depth for all specimens and all amplicons was 2786 reads. Figure 1 illustrates a high degree of coverage uniformity; shown are the median coverage and median absolute deviation from the median for all specimens for each of the 60 amplicons. The median coverage for the lowest represented amplicon (419 reads) was 6.7-fold lower than the overall median (2786 reads), while the median for the highest represented amplicon (11759 reads) was 4.3-fold higher than overall. As another way of representing the data, 98.13% (5417/5520) of amplicons were represented within 15-fold of the overall median (2786 reads). 99.98% (5519/5520) of amplicons were covered at a depth of 10 read-pairs or greater. All 59 known genetic variants were detected by the ROVER variant calling software (Table 1). Furthermore, no false-positive calls were made, resulting in 100% sensitivity and 100% specificity. The uniformity of amplicon coverage across the targeted regions was high and consistent with the profile observed previously following application of Hi-Plex to matched specimens derived from other blood-based sources of DNA, including freshly cultured lymphoblastoid cell lines. This high performance is probably at least partly a consequence of Hi-Plex enabling the use of relatively small and highly uniform amplicon lengths. Other amplicon-based target enrichment systems are more constrained in this regard and would be predicted to struggle for coverage uniformity with increasing DNA fragmentation. As such, it is expected that Hi-Plex will offer performance advantages in other contexts in which DNA integrity is compromised, e.g. DNA-derived from formalin-fixed, paraffin-embedded tumour specimens or ancient DNA specimens. The accuracy and stringent artefact filtering afforded by Hi-Plex are expected to confer advantages in applications of genetic variant detection in sub-populations, such as identifying emerging drug resistance in heterogeneous tumours, for example.</p><p>If we use the MiSeq performance metrics for the two genes targeted in this study and assume a target mean coverage depth of 200 reads per specimen amplicon and factor in the lower cost per base of HiSeq2500 sequencing compared with MiSeq sequencing, we can realistically project that for large-scale screening, the cost per specimen would currently be ~65 Australian cents or ~36 British pence per specimen.</p><p>The ability to apply Hi-Plex in the context of dried blood spot material opens a wide variety of possibilities for genetic epidemiology and diagnostic applications.</p><!><p>With Hi-Plex, we show for the first time that highly multiplex amplicon-based target enrichment for MPS can produce robust and highly accurate sequence screening in the context of archival dried blood spot-derived DNA. This empowers genetic epidemiologists and diagnosticians with the ability to use this very important bioresource for a broad range of applications to address many research questions.</p><!><p>Supplementary Table 1: Oligonucleotides used in this study. For gene-specific primers, lower case sequence text relates to adapter sequence regions and upper case sequence text indicates gene-specific sequence regions. For adapter primers, upper case sequence text relates to TruSeq-based sequences, underlined sequence text relates to Nextera-dual indices and lower case relates to Ion Torrent-based sequences.</p><p>Supplementary Table 2: Adjustment factor and reaction concentration of 'over-achieving' gene-specific primers. # Forward and reverse primers were decreased by the same factor. All other gene-specific primers were used at 4 nM each for this 60-plex format experiment.</p><p>Supplementary Figure 1: Integrative Genome Viewer screenshot showing typical Hi-Plex-derived coverage for PALB2. The locations of gene-specific primers are represented by arrows for the largest exon. Vertical lines demark the bounds of separate target 'insert' sequences included in resulting amplicons.</p>

PubMed Author Manuscript

Identification and validation of the microRNA response elements in the 3\xe2\x80\x99-untranslated region of the UDP glucuronosyltransferase (UGT) 2B7 and 2B15 genes by a functional genomics approach

Posttranscriptional repression of UDP-glucuronosyltransferase (UGT) 2B7 and 2B15 expression by microRNAs (miRNAs) may be an important mechanism underlying inter-individual variability in drug glucuronidation. Furthermore, the UGT2B15 3\xe2\x80\x99-UTR contains a common SNP (rs3100) that could influence miRNA binding. The aim of this study was to identify the complete complement of miRNAs that could regulate UGT2B7 and UGT2B15 expression through binding to the reference and/or variant 3\xe2\x80\x99-UTRs. Luciferase reporter plasmids containing either the reference or variant 3\xe2\x80\x99-UTRs were screened against a 2,048 human miRNA library to identify those miRNAs that decrease luciferase activity by at least 30% when co-transfected into HEK293 cells. Six novel miRNAs (miR-1293, miR-3664\xe2\x80\x933p, miR-4317, miR 513c-3p, miR-4483, and miR-142\xe2\x80\x933p) were identified that repressed the reference UGT2B7 3\xe2\x80\x99-UTR, while twelve novel miRNAs (miR-770\xe2\x80\x935p, miR-103b, miR-3924, miR-376b-3p, miR-455\xe2\x80\x935p, miR-605, miR-624\xe2\x80\x933p, miR-4712\xe2\x80\x935p, miR-3675\xe2\x80\x933p, miR-6500\xe2\x80\x935p, miR-548as-3p, and miR-4292) repressed both the reference and rs3100 variant UGT2B15 3\xe2\x80\x99-UTR. Deletion and mutagenesis studies confirmed the binding site location of each miRNA. Although the UGT2B15 rs3100 SNP was located within the miR-376c-3p response element, there was no effect on miRNA binding. miR 142\xe2\x80\x933p, miR-3664\xe2\x80\x933p, miR-4317, miR-455\xe2\x80\x935p, miR-376c-3p, miR-770\xe2\x80\x935p, miR-3675\xe2\x80\x933p, miR-331\xe2\x80\x935p, miR-605, and miR-376b-3p transcript levels were measured by quantitative PCR and correlated with UGT2B7 and UGT2B15 enzyme activities in 27 human liver samples. A significant negative correlation (Rs = -0.53; p = 0.005) was demonstrated between hepatic miR-455\xe2\x80\x935p transcript levels and UGT2B15-mediated S-oxazepam glucuronidation activities. Thus, the UGT2B7 and UGT2B15 3\xe2\x80\x99-UTRs contain miRNA response elements for multiple miRNAs that may contribute to variable drug glucuronidation.

identification_and_validation_of_the_microrna_response_elements_in_the_3\xe2\x80\x99-untranslated_re

Introduction<!>Chemicals and reagents.<!>Cell lines and culture conditions.<!>Construction of reporter plasmids.<!>Luciferase reporter assays.<!>Quantitative Real-time PCR (qPCR).<!>Statistical analysis.<!>A genome-wide approach identifies novel miRNAs that decrease the UGT2B7 and UGT2B15 3\xe2\x80\x99-UTR-dependent luciferase reporter activity.<!>The UGT2B7 and UGT2B15 3\xe2\x80\x99-UTRs contain functional microRNA response elements (MREs) for the novel candidate miRNAs.<!>Effect of the rs3100 single nucleotide polymorphism (SNP) in the UGT2B15 3\xe2\x80\x99-UTR on miRNA binding.<!>Expression of candidate miRNAs in human liver bank samples and correlation with hepatic UGT2B7 and UGT2B15 activity.<!>Discussion

<p>The UDP-glucuronosyltransferases (UGTs) comprise a large family of conjugation enzymes capable of detoxifying a wide variety of both endogenous and exogenous substrates. The UGTs consist of 19 functional enzymes in humans that are subdivided by genetic similarity into three subfamilies, UGT1A, UGT2A, and UGT2B.The UGT2B subfamily includes 7 functional enzymes in humans (UGT2B4, 2B7, 2B10, 2B11, 2B15, 2B17, and 2B28), located on chromosome 4q13 which are expressed as individual genes each with a unique 3′-untranslated region (3'-UTR) [1]. The UGTs are critical for the efficient elimination of the majority of the top 200 prescribed drugs in the United States, ranked second only to the cytochrome P450 enzymes [2]. UGT2B7 is the most commonly listed UGT enzyme known to contribute tothe glucuronidation of the top 200 prescribed drugs [2]. Examples of clinically important drugs that are eliminated by UGT2B7-mediated glucuronidation include morphine and zidovudine (also called 3′-azido-3′-deoxythymidine or AZT) [3].Furthermore, UGT2B7 plays a major role in the glucuronidation of endogenous compounds such as steroids and retinoids [4–6]. UGT2B15 contributes to the glucuronidation of androgenic steroids and various drugs e.g. oxazepam and lorazepam [7, 8]. Interestingly the major elimination pathway of oxazepam in humans isthrough stereoselective hepatic glucuronidation mediated primarily by the UGT2B15 and UGT2B7 enzymes [8]. Specifically in human liver S-oxazepam is mainly glucuronidated by UGT2B15 while R-oxazepam is glucuronidated by both UGT2B7 and UGT1A9 [8, 9]. Furthermore, the glucuronidation of this drug by human subjects is known to be polymorphic although the molecular mechanisms responsible for this variability are only partly understood [9].</p><p>We demonstrated previously that the single nucleotide polymorphism (SNP) c.735A>G (UGT2B7*1c; rs28365062) in the UGT2B7 exon 2 explained only part of the relatively high interindividual variability in the clearance of a UGT2B7-specific probe substrate namely zidovudine, observed in HIV patients [10]. Furthermore, results from different groups showed that the SNPs located in the UGT2B7 promoter and coding region cannot explain completely the observed variability in the UGT2B7 mediated glucuronidation of various drugs [11, 12]. Previous work from our lab identified SNP D85Y (253G>T, rs1902023) in exon 1 of the UGT2B15 gene [13]. Moreover we demonstrated that the high frequency variant allele D85Y (allele frequency 47%) was a major determinant of the observed inter-individual variability ofoxazepam glucuronidation in vivo [14]. However, SNP D85Y could explain only 34% of the observed interindividual variability of hepatic oxazepam glucuronidation in vivo [14]. Consequently, other trans-acting factors could regulate UGT2B7 and UGT2B15 expression and contribute to this variability.</p><p>In recent years, microRNAs (miRNAs) have emerged as a crucial suppressive regulator of gene expression. miRNAs are a class of small non-coding RNAs that have been shown to control gene expression via imperfect base pairing to the 3' UTR of target genes [15]. Accumulating data demonstrate that miRNAs modulate the expression of drug disposition genes, including cytochrome P450 enzymes, UGTs, and transporters [16]. Recently we showed using an unbiased genome-wide screen that five novel miRNA response elements (MREs) are located in the common UGT1A 3'-UTR [17]. In addition two recent studies identified and validated the functional MREs of miR-376c and miR-331–5p to the 3'-UTRs of UGT2B15 and UGT2B17 in prostate cancer cells [18, 19]. However there is still a large gap in our knowledge regarding the role of miRNAs in the regulation of two important members of the UGT2B subfamily, namely UGT2B15 and UGT2B7. Essentially we lack knowledge of the full complement of miRNAs other than miR-376c and miR-331–5p which are involved in the direct regulation of UGT2B7 and UGT2B15. Moreover, the studies that are based on a bioinformatics scan of the target gene 3'-UTR to identify novel miRNA candidates tend to bias the results to those miRNAs that match best using available bioinformatics algorithms, which have a significant false negative discovery rate because of the imperfect binding of miRNAs to their targets.Consequently, alternate unbiased genome-wide approaches are likely needed to reveal the full complement of miRNAs regulating the UGT2B7 and UGT2B15 enzyme.</p><p>The objective of the current work was to identify the complete complement of miRNAs that could regulate UGT2B7 and UGT2B15 expression through binding to the respective 3'-UTRs. For this purpose, we employed the luciferase reporter system to perform a genome-wide screen of the UGT2B7 and UGT2B15 3'-UTRs against a miRNA mimic library representing all of the available human miRNAs sequenced so far. This was followed by identification and functional validation of the miRNA response elements in the 3'-UTRs by a combination of in silico analysis and luciferase reporter construct assays. Furthermore we determined whether a common SNPin the UGT2B15 3'-UTR (rs3100) that was previously associated with UGT2B15 mRNA allelic imbalance [14] can disrupt miRNA binding. Finally, we measured the transcript amounts of the candidate miRNAs in human liver samples to determine whether they are correlated with hepatic UGT2B7 or UGT2B15 activities.</p><!><p>The miRNA mimic library (miRIDIAN microRNA Mimic Library, v.19), the miRNA mimic negative controls (Negative controls 1 and 2), the short interfering RNA directed against the UGT2B7 and UGT2B15 3'-UTR [positive controls: (siUGT2B7), 5'- AGA AGT CCA CTG ACA GTA T-3' – (siUGT2B15) 5'- GAC CAA ATA GGA ACA GCT CCA -3'], and the transfection reagent DharmaFECT Duo were purchased from GE Dharmacon (Lafayette, CO).The pMIR-REPORT plasmid was from Life Technologies (Grand Island, NY) and the Renilla luciferase vector pRL-CMV was purchased from Promega (Madison, WI). All synthesized DNA oligos used in this study were obtained from Integrated DNA Technologies, Inc. (Coralville, IA).</p><!><p>HEK293 cells were cultured in DMEM (Life Technologies) supplemented with 10% FBS (Life Technologies). Cells were grown in a humidified chamber at 37oC and 5% CO2.</p><!><p>The pMIR-UGT2B7 and pMIR-UGT2B15 luciferase plasmids containing the entire UGT2B7 3'-UTR (hg38, chr4: 69,112,737–69,113,408) and UGT2B15 3'-UTR (hg38, chr4: 68,646,196–68,647,103; contains the rs3100 c.1761 C allele) were de novo synthesized by Blue Heron (Bothell, WA). For the generation of all deletion constructs and the pMIR-UGT2B15 rs3100 c.1761 T allele variant vector we used the KAPA HiFi Polymerase (Kapa Biosystems, Inc., Wilmington, MA) together with the pMIR-UGT2B15 luciferase plasmid and the respective set of primers (Table 1). All the triplicated constructs were generated using synthesized 5'-end phosphorylated oligonucleotides that were annealed with their complementary sequence (Table 1) and cloned into the pMIRREPORT vector at the HindIII sites directly downstream of the firefly luciferase gene.All plasmids used in this study were verified by Sanger sequencing performed at the Washington State University Genomics Core.</p><!><p>For all luciferase assays HEK293 cells were co-transfected with a firefly luciferase construct, the pRL-CMV Renilla luciferase control vector and the miRNA mimic or one of the mimic controls (negative or positive) as described previously [17].</p><!><p>Total RNA was isolated from human liver samples using Trizol (Life Technologies). Mature miRNAs were isolated from individual liver samples of our human liver bank (n=27) using the mirVana™ miRNA Isolation Kit, with phenol (Life Technologies) according to the manufacturers' instructions. The source of the liver bank tissues have been published recently [20, 21]. These de-identified liver samples (Table 2) were obtained from publicly available sources, including the National Disease Research Interchange (Philadelphia, PA) and the Liver Tissue Procurement and Distribution Service (Minneapolis, MN). The use of these tissues for this study was approved by the Washington State University Institutional Review Board. mRNA and mature miRNA concentrations were determined by qPCR using reverse transcribed total RNA. For mRNA, cDNAs were first synthesized from total RNA using TaqMan Reverse Transcription Reagents (Life Technologies). qPCR was performed using TaqMan Gene Expression Master Mix and Taqman primer-probe mixes from Life Technologies, including UGT2B15 (Hs00870076_s1) and GAPDH (Hs02758991_g1). Mature miRNAs were also quantified from total RNA after reverse transcription with miRNA-specific primers and MultiScribe Reverse Transcriptase (Life Technologies). qPCR was performed using TaqMan Universal Master Mix II, no UNG and Taqman miRNA assays from Life Technologies (Table 3). All assays were performed in triplicate. Gene expression was compared to an endogenous internal control (GAPDH for mRNA or the geometric mean of miR-1521 3p/miR-23b for miRNA as described previously [22]. There was not commercially available a set of primers and probes of the Applied Biosystems™ TaqMan™ microRNA assays for miR-4292 and miR-3924. Thus we used the GTEx database (http://www.gtexportal.org/) to determine the hepatic transcript amounts of these miRNAs. The ΔCt values for each miRNA were calculated this way for all of the samples in our human liver bank (n=27). Subsequently these values were normalized to the liver sample with the highest amount for each miRNA. These normalized values were then used to determine the correlation between the miRNA amounts in human liver samples and the hepatic activities of UGT2B7 and UGT2B15.</p><!><p>Statistical analysis was performed with a two-tailed Student's t test using SigmaPlot Version 12 (Systat Software, Inc., San Jose, CA). Correlation analysis was performed by a Spearman's rank method. A p value of less than 0.05 was considered statistically significant.</p><!><p>To identify candidate miRNAs involved in the regulation of UGT2B7 and UGT2B15 expression via direct binding to the respective 3'-UTRs, we used a commercially available miRNA mimic library (miRIDIAN microRNA Mimic Library, v.19) that contained all human miRNAs sequenced at that time (2,048 miRNAs as listed in miRBase version 19). We transiently co-transfected pMIR-UGT2B7 or the pMIR-UGT2B15 luciferase reporter plasmids and the pRL-CMV vector (Renilla transfection control) together with the miRNA mimics or the respective positive (siUGT2B7, siUGT2B15) and negative controls into HEK293 cells. This cell line is ideal for the heterologous expression of UGT2B7 and UGT2B15 since the mRNA transcript amounts of these enzymes in HEK293 cells are very low [23].</p><p>Subsequently we determined the effect of each miRNA mimic on the UGT2B7 and UGT2B15 3'-UTR-dependent luciferase activities and classified them according to the degree of decrease of the relative UGT 3'-UTR-mediated luciferase activity. We chose to study further the miRNAs that showed at least 30% decrease in the relative UGT 3'-UTR-dependent luciferase activity in the initial library screen [19 miRNAs for UGT2B7 (Table 4 and 20 miRNAs for UGT2B15 (Table 5)]. Although miR-331–5p and miR-376c-3p caused a 38% and 54% decrease respectively in UGT2B15 3'-UTR-dependent luciferase activity we did not study these miRNAs further since recent studies demonstrated that they can regulate UGT2B15 expression [18, 19]. After counter-screening with the empty pMIR-REPORT vector to exclude nonselective effects, only six miRNAs for UGT2B7 (miR-1293, miR-3664–3p, miR-4317, miR-513c-3p, miR-4483, and miR-142–3p) and twelve for UGT2B15 (miR-770–5p, miR-103b, miR-3924, miR-376b-3p, miR-455–5p, miR-605, miR-624–3p, miR-47125p, miR-3675–3p, miR-6500–5p, miR-548as-3p, and miR-4292) were found to significantly reduce the luciferase activity of cells transfected with pMIR-UGT2B7 and pMIR-UGT2B15 luciferase reporter plasmids (Fig. 1A, 1B).</p><!><p>miRanda and RNAhybrid are well-verified online bioinformatics programs that predict the location of MREs in the 3'-UTRs of target mRNAs [24–26]. Using these programs, we identified putative MREs in the UGT2B7 and UGT2B15 3'-UTRs for each of the candidate miRNAs (Fig. 2). To confirm the functionality of the candidate MREs in the 3'-UTRs we generated constructs that carried the full length UGT2B7 and UGT2B15 3'-UTRs with a deletion (Δ) of the predicted MREs cloned directly downstream of the firefly luciferase gene. We also cloned each predicted MRE sequence as a triplicate in the forward or reverse (negative control) orientation in the pMIR-REPORT firefly luciferase vector. We then transiently co-transfected the full length, deletion, triplicate constructs or the empty pMIR-REPORT vector with miRNA mimics) or the miRNA mimic negative controls into HEK293 cells and quantified the luciferase activity of each construct. As shown in Figure 3 all miRNA mimics tested significantly reduced the luciferase activity of constructs carrying the full length UGT2B7 and UGT2B15 3'-UTRs (left set of columns in each panel A-T). However, these reductions were abrogated when the respective MRE was deleted (Figure 3; second set of columns from left in each panel A-T). Moreover, cells transfected with the miRNA mimics and pMIR constructs carrying the MREs in the forward orientation showed a significant decrease in the luciferase activity compared with the control, while those with MREs in the reverse orientation showed no miRNA mimic effect (Figure 3; third and fourth set of columns from left in each panel A-T). As expected, none of the miRNA mimics affected luciferase activity of the empty pMIR-REPORT vector (Figure 3; right set of columns in each panel A-T).</p><!><p>We next sought to determine whether the common SNP rs3100 c.1761 C/T [27] present in the UGT2B15 3'-UTR had an effect on binding of any of the candidate UGT2B15-regulating miRNAs. Interestingly, in silico analysis using the bioinformatics program miRanda determined that the rs3100 SNP was located within the predicted miR-376c-3p MRE (Fig. 2, panels J, K). Furthermore, the variant c.1761T allele (Fig. 2, panel K) appeared to create an additional base-pairing site between the UGT2B15 3'-UTR and miR-376c-3p when compared with the reference c.1761C allele (Fig. 2, panel J). Consequently, lower luciferase activities would have been expected for the pMIR-UGT2B15-variant construct compared with the reference pMIR-UGT2B15. However, as shown in Figure 4 luciferase activities were reduced by all the candidate UGT2B15-regulating miRNAs (including miR-376c-3p) in both the pMIR-UGT2B15 reference and variant constructs to the same extent indicating this SNP does not affect regulation by any of these miRNAs.</p><!><p>qPCR was then used to determine which of the candidate miRNAs are normally expressed in human liver. Mean (± SD) threshold cycle (Ct) values for each miRNA were determined using RNA extracted from 27 of our human liver bank samples (Table 2). For the UGT2B7 candidate miRNAs relatively high transcript amounts (lowest Ct values) were observed only for miR-142–3p (25.5 ± 1.1). Relatively low transcript amounts were observed for miR-3664–3p (36.1 ± 1.5) and miR-4317 (36.4 ± 1.7), while miR-513c-3p, miR-1293, and miR-4483 showed no measurable expression in any of the liver samples tested (Ct value over 40).</p><p>For the UGT2B15 candidate miRNAs relatively high transcript amounts (low Ct values) were observed for miR-455–5p (27.8 ± 0.6), miR-376c-3p (29.9 ± 1.4), miR-770–5p (29.7 ± 0.3), and miR-3675–3p (29.9 ± 0.4). Relatively low transcript amounts (high Ct values) were observed for miR-331–5p (32.3 ± 0.8), miR-605 (35.7 ± 1), and miR-376b-3p (37.6 ± 1.4). We did not measure miR-3924 and miR-4292 transcripts since publicly available RNA-seq data (http://www.gtexportal.org/) indicated that both these microRNAs are not expressed in human liver. Moreover recent results from a different group demonstrated that miR-4292 is not expressed in human liver [28].</p><p>Finally, we sought to determine whether expression levels of any of the candidate miRNAs were negatively associated with glucuronidation activities selective for UGT2B7 (zidovudine glucuronidation, morphine-3-glucuronidation, and morphine-6glucuronidation) and UGT2B15 (S-oxazepam glucuronidation) when measured in the same human liver bank samples. These glucuronidation activities had been previously reported in [3, 13]). As shown in Table 6 although hepatic UGT2B7 activities were not negatively correlated with any of the miRNAs assayed, UGT2B15mediated S-oxazepam glucuronidation activity showed a statistically significant negative correlation (Rs = −0.53; p = 0.005) with miR-455–5p levels (Table 7, Fig. 5).</p><!><p>The most important novel findings of our study were the discovery and validation of 6 novel MREs for miR-1293, miR-3664–3p, miR-4317, miR-513c-3p, miR-4483, and miR-142–3p in the UGT2B7 3'-UTR, and 12 novel MREs for miR-770–5p, miR-103b, miR-3924, miR-376b-3p, miR-455–5p, miR-605, miR-624–3p, miR-4712–5p, miR-3675–3p, miR-6500–5p, miR-548as-3p, and miR-4292 in the UGT2B15 3'-UTR The location of each MRE in the UGT2B7 and UGT2B15 3'-UTRs are shown in Fig. 6 as well as the MREs for miR-376c and miR-331–5p, which have been previously characterized [18, 19].</p><p>We showed that half of the miRNAs we identified were expressed in human liver samples. More specifically 3 out of 6 candidate miRNAs for UGT2B7 and 7 out of 14 candidate miRNAs for UGT2B15 had detectable transcript amounts in human liver samples. Correlation analysis demonstrated that UGT2B7 activity did not show statistically significant correlation with any of the miRNAs while UGT2B15 activity showed a significant negative correlation with miR-455–5p hepatic levels (Fig. 5). While this suggests that miR-455–5p may contribute to inter-individual variability in hepatic UGT2B15 activity, the number of liver samples used for this analysis was limited (n=27). Thus, it will be necessary to study more human liver samples to clarify the contribution of miRNAs to the variable hepatic UGT2B7 and UGT2B15 activities in vivo.</p><p>Previous results from our laboratory demonstrated that miR-103b and miR-376b-3p have functional MREs in the common UGT1A 3'-UTR [17]. In the current work we showed that miR-103b and miR-376b-3p were also among the miRNAs with functional MREs in the UGT2B15 3'-UTR. Thus, it is plausible that a network of miRNAs contributes to the regulation of the expression of various UGT isoforms. A genome-wide approach is required to validate this hypothesis and evaluate the role of specific miRNAs in the observed interindividual variability in the expression of UGT enzymes.</p><p>UGT2B7 activity, protein, and mRNA levels were previously demonstrated to be significantly lower in livers and kidneys obtained from diabetic patients when compared matched tissues from non-diabetic patients [29]. A meta-analysis of miRNA expression profiling studies identified miR-142–3p among the top miRNAs that were significantly and consistently upregulated in blood samples of patients with type 2 diabetes [30]. Taken together these results imply that the observed decrease of hepatic and renal UGT2B7 activity and expression in diabetic patients could be due to increased miR-142–3p expression. However, a detailed analysis of the hepatic and renal UGT2B7 activity and expression and the blood and/or tissue concentrations of miR-142–3p in a large number of samples would be required to prove the correlation between UGT2B7 altered expression and the increased levels of this miRNA in diabetes.</p><p>In recent work we used the same in vitro system to perform a genome-wide screen of the miRNA mimic library with the pMIR-REPORT luciferase vector carrying the reference UGT1A 3'-UTR or the variant 3'-UTR [17]. This way we demonstrated that SNP rs10929303 C>T located in the common UGT1A 3'-UTR created a novel MRE for miR-21–3p. Reporter gene assays by another group showed that the UGT2B15 3'-UTR rs3100 c.1761C (reference) allele resulted in lower UGT2B15-dependent luciferase reporter activity compared with the (variant) c.1761T allele in liver, breast, colon, and prostate cancer cell lines [27]. Those authors suggested that this SNP might be located within a miRNA binding site that could influence UGT2B15 expression. Consequently we had hypothesized that the rs3100 c.1761T allele destroys the MRE of a miRNA that normally binds to the UGT2B15 3'-UTR rs3100 c.1761C allele. After screening for miRNAs that bind to the rs3100 c.1761C or c.1761T alleles, only a single miRNA (miR-376c-3p) was identified with a predicted MRE that included the rs3100 SNP. However, in silico analysis showed that the c.1761T allele (Fig. 2, panel K) enhances rather that reduces base-pairing to miR-376c-3p when compared with the c.1761C allele (Fig. 2, panel J). Despite this in silico prediction we were not able to experimentally demonstrate a significant difference in UGT2B15-dependent luciferase activity between the rs3100 c.1761C and c.1761T constructs when coexpressed with miR-376c-3p, or with any other miRNA demonstrated to bind to the UGT2B15 3'-UTR (Fig. 4). This lack of effect of rs3100 on miR-376c-3p binding may be because it is not located within the critical miRNA seed sequence (nucleotides 2–7). Regardless, it is still possible that rs3100 affects UGT2B15 expression through influencing binding of another (yet to be identified) miRNA, or perhaps indirectly through another mechanism.</p><p>There are several limitations to our study. We used a 30% cutoff for luciferase reporter inhibition in the initial library screen to focus our efforts on those miRNAs most likely to influence UGT2B7 and UGT2B15 expression. This threshold was sensitive enough to identify miR-376c-3p and miR-331–5p, which were the only other miRNAs with a functionally validated MRE [18, 19]. This cutoff also allowed us to minimize the significant amount of counter-screening that was needed since approximately 80% of candidate miRNAs were subsequently found to inhibit reporters lacking the UGT 3'-UTRs (i.e. false positives). However, it is possible that some of the miRNAs evaluated in our initial screen that showed less than 30% inhibition of reporter activity could also be important regulators of UGT2B7 and UGT2B15 expression. Furthermore, overexpression and knock-down experiments of these miRNAs in the appropriate cell line and in primary human hepatocytes are required to demonstrate their functional role in the regulation of UGT2B7 and UGT2B15 expression and enzyme activity.</p><p>In summary we used a genome-wide approach to identify functional MREs in the UGT2B7 and UGT2B15 3'-UTRs. The genome-wide approach we employed could be further used to identify candidate miRNAs that bind directly in the 3'-UTR of other hepatic genes of pharmacological importance.</p>

PubMed Author Manuscript

Triazoles as T2-Exchange Magnetic Resonance Imaging Contrast Agents for the Detection of Nitrilase Activity

We characterized the T2-exchange (T2ex) magnetic resonance imaging (MRI) contrast of azole protons that have large chemical shifts from the water proton resonance as a function of pH, temperature, and chemical modification. Our results showed that 1,2,4-triazoles could be tuned into excellent diamagnetic T2ex contrast agents, with an optimal exchange-based relaxivity r2ex of 0.10 s\xe2\x88\x921 mm\xe2\x88\x921 at physiological pH and B0 = 9.4 T. A fit of r2ex data to the Swift\xe2\x80\x93Connick equation indicated that imino proton exchange of triazoles is dominated by a base-catalyzed process at higher pH values and an acid-catalyzed process at lower pH. The magnitude of r2ex was also found to be heavily dependent on chemical modifications, that is, enhanced by electron-donating groups, such as amines and methyls, or by intramolecular hydrogen bonding between the imino proton and the carboxyl, and weakened by electron-withdrawing groups like bromo, cyano, and nitro. In light of these findings, we applied T2ex MRI to assess the activity of nitrilase, an enzyme catalyzing the hydrolysis of 1,2,4-triazole-3-carbonitrile to 1,2,4-triazole-3-carboxylic acid, resulting in the enhancement of R2ex. Our findings suggest that 1,2,4-triazoles have potential to provide sensitive and tunable diagnostic probes for MRI.

triazoles_as_t2-exchange_magnetic_resonance_imaging_contrast_agents_for_the_detection_of_nitrilase_a

Introduction<!>Selection of 1,2,4-triazole over other azoles for the T2ex characterization<!>T2ex effects of 1,2,4-triazole derivatives<!>The detection of nitrilase activity by T2ex MRI<!>Conclusion<!>Chemicals<!>MRI<!>NMR<!>Theory<!>Analysis of enzyme kinetics

<p>1H magnetic resonance imaging (MRI) is one of the most versatile medical imaging modalities. The sensitivity and specificity of MRI can be boosted dramatically by the use of contrast agents. Currently, T1 and T2/T2* contrast agents are most widely used, for example, gadolinium chelates and iron oxide nanoparticles, respectively, modulating the water proton longitudinal relaxation times and transverse relaxation times to generate detectable contrast.[1] Targeted molecular imaging can be achieved by combining these agents with specific moieties or functional groups.[2] Recently, chemical exchange saturation transfer (CEST)[3] and chemical exchange-sensitive spin-lock (CESL)[4] have been developed as new MRI approaches to visualize low-concentration diamagnetic molecules containing exchangeable protons. Numerous diamagnetic agents have been reported for CEST and CESL MRI detection in a broad spectrum of biomedical applications.[5] An invaluable merit of CEST/CESL is the possibility to noninvasively visualize bio-relevant compounds (diamagnetic agents), including clinically accessible ones, thus allowing their use as MRI contrast agents. This feature has the potential to greatly simplify the process for development of new imaging agents and their clinical translation. It is noted that CEST/CESL MRI requires the exchange rate (kex) of exchangeable protons to fall in the slow-to-intermediate range, that is, kex ≤ Δω where Δω is the chemical shift difference (in radians s−1) between the resonance frequencies of labile protons and water protons.[6] This condition limits the applications of CEST MRI in a broad spectrum of biological molecules whose exchangeable protons fall in the fast regime at physiological pH.</p><p>To improve the detectability of exchangeable protons whose kex is in intermediate-to-fast range, T2-exchange (T2ex) MRI has been developed. It has been known for a long time that the proton exchange of agents with bulk water can reduce water transverse (T2) relaxation times without affecting the T1.[7] The fast exchanging protons hence can be utilized to modulate water T2 relaxation times to generate T2 contrast,[8] extending MRI detection of labile protons from the slow-to-intermediate range (CEST/CESL) to the intermediate-to-fast range. At a given B0, the T2ex effect (quantified by the transverse relaxivity, r2ex) of 1 mm of exchangeable protons can be described by the Swift–Connick equation[9] [Eq. (1) and Figure S1 in the Supporting Information].</p><p>Consequently, an optimal r2ex is obtained when kex ≈ Δω. To date, the T2ex principle has been successfully extended to a few diamagnetic compounds, including glycogen,[10] iopamidol,[11] glucose,[12] and phenols.[13] The feasibility of the in vivo applications of T2ex agents has been demonstrated by a recently published T2-weighted dynamic contrast-enhanced MRI (DCE-MRI) study in which d-maltose was used as the contrast agent to characterize the permeability of tumors.[14] However, the limited Δω (typically < 6 ppm) creates a sensitivity barrier for most diamagnetic agents (r2ex < 0.1 s−1 mm−1). According to Equation (1), the magnitude of the maximum r2ex increases linearly with Δω, indicating one way to enhance the T2ex effect is to use agents whose exchangeable protons have chemical shifts far from water.</p><p>Azoles are nitrogen containing heterocyclic compounds, widely used in the development of medicinal drugs such as antifungal, anticancer, antiviral agents, and so on.[15] Their N–H protons can exhibit a large chemical shift (up to 15.3 ppm, or about 10.6 ppm from water). Additionally, azoles are often incorporated into polymers towards the fabrication of proton-exchange membranes because of their fast exchange property.[16] We thereby hypothesized that azoles could be tuned as a new class of diamagnetic T2ex agents.</p><!><p>We first chose 1,2,4-triazole (1), 1,2,3-triazole (2), and imidazole (3) as model compounds to study T2ex contrast. As evident from the NMR spectra (Figure S2 in the Supporting Information), their exchangeable imino proton, respectively resonates at 9.2, 10.3, and 7.3 ppm from the water proton frequency (at 4.7 ppm). It turned out that 1 is the best azole scaffold, showing the largest T2ex effect (Figure S3), and thus we took it for more detailed characterization. As shown in Figures 1 A–B, 1 exhibited a concentration-dependent T2 effect, with a r2ex estimated to be 0.045 s−1 mm−1 in PBS at pH 7.4 and at 37 °C. The r2ex is strongly dependent on pH above pH 7.0 (Figure 1 C). Interestingly, the pH-dependence exhibited two regional maxima, which we speculated to be due to the involvement of two catalytic mechanisms, that is, base-catalysis when pH > 6.5 with the regional maximum r2ex occurring at pH 7.0, and acid-catalysis when pH< 6.5 with the regional maximum r2ex occurring at pH 6.3. In order to determine the kex values, we measured r2ex values at three temperatures (i.e., 20, 30, and 37 °C) and fitted them to Equation (1) at each pH (Figures 1D and E), under the assumption that kex increases with temperature (Figure S4).[12] The two-phase pH dependence of kex of 1 is markedly different from other reported protons such as phenol.[13] At pH 7.0, the kex was estimated to be 27.3 ± 4.0 kHz, which is close to the Δω of 1 [i.e., 2π × 9.2 (ppm) × 400 (Hz ppm−1) = 23.1 × 103 rad s−1], enabling the highest enhancement (r2ex = 0.10 s−1 mm−1). It should be noted that our assumption that the temperature dependence of the exchangeable protons follows the Arrhenius equation is valid only for one-step exchange model and caution should be taken when the exchange model involves two steps such as those of salicylic acid.[17]</p><!><p>A library of 1,2,4-triazole analogues was then tested and their measured r2ex, Δω, and kex values are listed in Scheme 1. When electron-donating groups such as amine- (4) and methyl- (5 and 6) were introduced, the proton exchange slowed down towards the optimum (4 and 5), or behind the optimum (6). However, the substitution of electron-donating groups also reduced the Δω (7.2 ppm for 4, 8.8 ppm for 5, and 8.1 ppm for 6). The overall effect reached maximum for 6 and the r2ex was doubled at pH 7.4 (0.088 vs. 0.045 s−1 mm−1) with a kex of 15 kHz. As expected, the introduction of electron-withdrawing groups, such as bromo (7) and cyano (8) (chloro- and nitro-substituted compounds were also tested, Figure S6) dramatically increased proton exchange rates, leading to strongly weakened T2ex contrast. Interestingly, our results also showed that the introduction of carboxyl (9) was favorable and a r2ex of 0.059 s−1 mm−1 was observed. We speculated the considerably decreased kex (77 kHz) was a result of the formation of an intramolecular hydrogen bonding (or possibly intermolecular bonding when concentration is high) between the carboxyl and the exchangeable imino proton.[18]</p><p>We next characterized the T2ex effects of 9 at different pH (5.3 to 7.4) and temperatures (20, 30, and 37 °C), and studied the relationship among r2ex, pH, and kex, as shown in Figure 2. At 37 °C (Figure 2 A), the maximum r2ex (0.092 s−1 mm−1) was observed at pH 6.0, which is slightly smaller than the theoretical maximum (0.106 s−1 mm−1) calculated using the Swift–Connick equation, indicating that the proton exchange is still a bit fast compared to the shift difference with water. The theoretically maximal r2ex could be attained at pH 6.2 at 20 °C (Figure 2 B). Based on the measurements at different temperatures, we estimated the kex values at different pH by fitting the r2ex data at 20, 30, and 37 °C to the Equation (1) (Figure S7). Figures 2 C, D show the correlation between r2ex and kex. At pH > 6.0, the exchange rate increases with pH, implying the proton exchange is base-catalyzed, and at pH < 6.0, the exchange rate decreases with decreasing pH, suggesting that the proton exchange is dominated by an acid-catalyzed process. This two-phase pH dependence of kex is consistent with what we have observed for 1.</p><!><p>T2ex MRI contrast can be utilized to develop responsive agents for specific detection of biomarkers and enzymes, for example, nitric oxide[19] and tyrosinase.[13] Such an MRI detection can be achieved when a biomarker or enzyme acts on the exchangeable protons, either by enhancing (by the so-called "turn-on" mechanism) or attenuating (by the "turn-off" mechanism) the T2ex contrast. In the present study, we designed a "turn-on" mechanism for detecting nitrilase using the low r2ex compound 1,2,4-triazole-3-carbonitrile (8) as the probe and also the substrate of nitrilase, which is converted to high r2ex compound 9 by the enzyme (Figure 3 A). Nitrilases (EC 3.5.5.1) are a family of enzymes[20] that are abundant in microorganisms, especially in bacteria, filamentous fungi, yeasts, and plants, catalyzing the hydrolysis of organonitriles to related carboxylic acids under mild conditions.[21] Nitrilase-mediated biocatalysis is widely used in industry as a "green method" for the production of a number of commercial compounds, which underlines the critical importance of developing strategies to assess the activity of nitrilase for screening nitrilase-expressing organisms. Conventional assays rely on the determination of the amounts of NH3 produced equimolarly with acids using solution-based and instrument-based methods.[20] High-throughput screening strategies have been developed either by the formation of chromophores or fluorophores or by pH indicators.[22] On the basis of the large difference in T2ex between the substrate (8) and the product (9), we hypothesized that the activity of nitrilase can be monitored with MRI, and since the R2 relaxation rate is increased with the conversion, the enzymatic reaction can be temporally imaged via from hyperintense T2 states to hypointense T2 states.</p><p>To demonstrate this, we incubated 10 mm of 8 with nitrilase at a range of concentrations (0, 0.24, 0.48, 0.72, 0.96, 1.2, 1.8, and 2.4 U, U = units mL−1) for 4h at 37 °C in PBS (pH 7.2, 10 mm), followed by T2 MRI measurement (20 °C, pH 7.2). The reaction time and temperature were chosen to obtain the optimal enzymatic conversion (Figure S8). The T2 test was performed at 20 °C due to a more appreciable T2ex effect of 9 (Figure 2 B). At 20 °C and pH 7.2, the r2ex of 9 was determined to be 0.086 s−1 mm−1 vs. 0.063 s−1 mm−1 at 37 °C (Figure S9). The R2 map of Figure 3B clearly shows that the presence of nitrilase increases the R2 relaxation rate, which is proportional to the enzyme concentration (Figure 3 C, round mark), thereby allowing the direct quantification of enzyme activity and simplifying the detection. Because neither the substrate nor nitrilase produced noticeable T2ex effects (Figure S10), the net differences in R2 between the samples before and after reaction [ΔR2 = R2 (after reaction)–R2 (before reaction)] can be used to quantify 9, that is, C(9) = ΔR2/r2ex(9), in which C(9) is the final concentration of 9 (See details in Supporting Information). As a consequence, the conversion ratio can be calculated for each enzyme concentration (Figure 3 C, square mark), showing that a maximum conversion ratio of 65 % was obtained under the present circumstances. It also allowed us to estimate the kinetic parameters, KM (7.5 mm), Vmax (0.19 mm min−1), kcat (33 min−1), and kcat/KM (4.4 mm−1 min−1), using the Michaelis–Menten kinetics model, as depicted in Figure 3 D. Given that compound 8 has not been used as substrate for nitrilase before, we could not find other kinetics data for comparison. Overall, these data demonstrate that the activity of nitrilase can be assessed quantitatively by the inherent T2ex MRI contrast of triazoles.</p><p>It is worth noting that the T2ex MRI is similar to CEST in that it exploits the chemical exchange of protons of agents with surrounding water protons. CEST is more suitable for detecting agents whose proton exchange rates fall in the slow to intermediate regime, whereas T2ex is useful for detecting those with intermediate and fast exchange rates. For diamagnetic agents, the sensitivity of T2ex agents is on the same order (≈ mm) as CEST agents. In addition, since the R1 relaxation rate is characteristically not affected by exchange for diamagnetic T2ex agents, the use of a R2/R1 ratio can provide an alternative way of quantification.[19] As CEST MRI is being demonstrated in more and more biomedical applications, we expect T2ex MRI to be developed similarly, but as a unique tool complementary to CEST. For example, the T2ex strategy might be used for in vivo detection of enzymatic activity using non-toxic substrates (and non-toxic products) that can be administered at a relatively high dose to generate high local concentrations in the targeted tissues such as tumors[5k,23] (typically with long T2 times) or kidney (high local concentrations can be reached[12]). It should be noted that, as shown in Figure 2, pH has a strong effect on T2ex contrast, indicating the precise determination of the concentration of T2ex agents requires a priori knowledge of local pH. On the other hand, similar to CEST agents, the pH-dependence of T2ex agents may be utilized to measure local pH.</p><!><p>We characterized the T2ex effects of a series of triazoles by modulating proton exchange properties with respect to pH, temperature, and chemical modification. Exchange rates were studied using the Swift–Connick equation to optimize the T2ex effect. In addition, as a proof-of-concept study, we applied the T2ex differences between compounds to detect the enzymatic activity of nitrilase. This type of detection for enzyme activities by exploiting the signal difference between the substrate and the product is a common advantage characteristic of CEST MRI and T2ex MRI. Given its simplicity and compatibility with multiple analysis,[24] this triazoles-based T2ex strategy can for instance be used as a high-throughput screening method for discovering nitrilase-expressing organisms. Taken together, the T2ex MRI is a useful extension of CEST MRI for the detection of diamagnetic compounds with fast exchangeable protons, potentially enabling the label-free detection of many drugs and enzymes.</p><!><p>Imidazole, 1,2,3-triazole, 1,2,4-triazole, 3-amine-1,2,4-triazole, 3-methyl-1,2,4-triazole, 3,5-dimethyl-1,2,4-triazole, 1,2,4-triazole-3-carboxylic acid, 3-bromo-1,2,4-triazole, 3-chloro-1,2,4-triazole, 1,2,4-triazole-3-carbonitrile, and 3-nitro-1,2,4-triazole were purchased from Combi-Blocks (San Diego, CA). Nitrilase (recombinant, expressed in E. coli, 4.8 unit mg−1) was purchased from Sigma–Aldrich (St. Louis, MO).</p><!><p>Aqueous solutions of each chemical were prepared in PBS (1x), with pH adjusted by using 1 m NaOH or HCl solutions. Sample phantoms were prepared by placing the solutions into 1 mm-diameter glass capillaries, and assembled in a customized holder for MR imaging on a Bruker 9.4 T vertical scanner with a 20-mm birdcage transmit/receive coil. We acquired T2 relaxation times using a Carr-Purcell-Meiboom-Gill (CPMG) method. Briefly, a T2 preparation module was added in front of a fast spin-echo imaging readout, i.e., Rapid Acquisition with Relaxation Enhancement (RARE) pulse sequence. The T2 preparation period consisted of a series of single spin-echo elements with tCPMG = 10 ms together forming a CPMG pulse train. We used loop numbers ranging from 2 to 512, making echo times from 20 ms to 5.12 sec. The imaging parameters were: TR/TE = 25 s/4.3 ms, RARE factor = 16, a 64 × 64 acquisition matrix with a spatial resolution of 0.25 × 0.25 mm2, and a slice thickness of 2 mm. The acquisition time for each T2-weighted image was 1 min 40 s. To obtain the exchange-based relaxivity, r2ex, of each compound, the R2 = 1/T2 water proton relaxation rates of the compound solutions were measured at different concentrations, i.e., 1.25, 2.5, 5 and 10 mm, and fitted to Eq. (2).</p><p>Where R20 is the inherent relaxation rate of the water protons, R2 the relaxation rate of the solutions, and [C] the concentration of the agent.</p><!><p>1H-NMR spectra of the compounds dissolved in [D6]DMSO were collected on a Bruker Avance III 500 MHz NMR spectrometer equipped with an autosampler. The chemical shifts are reported as δ values (ppm) relative to TMS.</p><!><p>The relationship between Δω, kex, and T2ex relaxation times can be described by the Swift–Connick equation (Eq. 3),[9a,b] in which R2B is the transverse relaxation rate of the exchangeable solute proton and PB is the mole fraction of exchangeable protons. The Δω in the unit of rad s−1 is the shift difference between the water frequency and the chemical shift of the exchangeable proton, and kex is the rate of exchange.</p><p>In the case of Δω≫R2Bkex≫R2B and 1 mm of labile proton, this can be simplified to Equation (1).</p><!><p>At 37 °C in PBS (pH 7.2, 10 mm), 1.2 U of nitrilase was incubated with various concentrations of 8 ranging from 2 to 40 mm for 10 min. The reaction was halted by refrigerating the samples, after which solution pH was adjusted to 7.2 and T2 MRI was measured at 20 °C. We assume that the conversion of substrate is linearly correlated with time over this short time period. The correlation of reaction velocity with the concentration of substrate can be fitted by the Michaelis–Menten model, based on which the Michaelis constant KM and the maximum rate Vmax are obtained. The kcat was calculated from the Vmax/[enzyme], where [enzyme] was measured to be 5.8 µm.</p>

PubMed Author Manuscript

Spectrally-selective all-inorganic scattering luminophores for solar energy-harvesting clear glass windows

All-inorganic visibly-transparent energy-harvesting clear laminated glass windows are the most practical solution to boosting building-integrated photovoltaics (BIPV) energy outputs significantly while reducing cooling-and heating-related energy consumption in buildings. By incorporating luminophore materials into lamination interlayers and using spectrally-selective thin-film coatings in conjunction with CuInSe 2 solar cells, most of the visible solar radiation can be transmitted through the glass window with minimum attenuation while ultraviolet (UV) radiation is down-converted and routed together with a significant part of infrared radiation to the edges for collection by solar cells. Experimental results demonstrate a 10 cm 3 10 cm vertically-placed energy-harvesting clear glass panel of transparency exceeding 60%, invisible solar energy attenuation greater than 90% and electrical power output near 30 W p /m 2 mainly generated by infrared (IR) and UV radiations. These results open the way for the realization of large-area visibly-transparent energy-harvesting clear glass windows for BIPV systems.

spectrally-selective_all-inorganic_scattering_luminophores_for_solar_energy-harvesting_clear_glass_w

<!>Principal Results and Discussion<!>Conclusion<!>Methods

<p>T he role of renewable energy in addressing the challenges associated with implementing CO 2 emissions reduction, addressing the climate change and energy supply concerns, has been recognised globally. Photovoltaics (PV), the conversion of sunlight to electricity has been reported to be the fastest-growing technology for electricity generation 1 . One-fifth of the world's total energy consumption is delivered for civil applications, residential and commercial 2 . Developing energy-efficient buildings is of prime importance, and future building industry regulations will have energy-efficiency requirements that meet the growing demand on energy resources. Ideally, new energy technologies must be integrated into the existing and future infrastructure elements (e.g. buildings), and at the same time provide savings by reducing the energy consumption. Designing ''energy-saving and energy-producing solar windows'' provides a way of boosting building-integrated PV (BIPV) energy outputs dramatically and beyond what is possible currently, simultaneously with reducing the coolingand heating-related energy consumption.</p><p>Various glazings and coated-type windows are in use worldwide as they offer thermal comfort improvements, energy savings, aesthetic value and ultraviolet (UV) protection. With the widespread use of glass panels covering vast areas of walls and roofs in modern buildings, significant economic benefits could be achieved if these glass surfaces had energy-generation capabilities. An economically-ideal energy-saving approach is to use window glazings that offer a combination of IR-range transparency blocking and energy-harvesting (due to the ability to efficiently convert the lighting-unrelated IR and UV solar energy into usable electric energy). It is likely that windows possessing these useful properties offer substantial economic and environmental benefits and would rapidly be recognized and marketed globally. It is well-known that industrial solar and wind farms require allocation of sizeable land areas in order to generate significant power. Conventionally, the power generation capability of green-energy PV installations is limited by the available roof area, especially for tall buildings. Wallmountable BIPV modules are starting to appear on the market, however, these mainly rely on thin-film photovoltaics of limited efficiency, and also cannot provide high optical transparency or energy savings related to solar-control properties.</p><p>Solar energy contains strong IR radiation flux coming from direct, diffused, reflected and re-emitted radiation from illuminated surfaces. Therefore, by using high-efficiency photovoltaic solar cells, energy-harvesting windows can generate measurable electric power even in non-ideal illumination conditions.</p><p>The development of principally new BIPV systems capable of high transparency and energy saving simultaneous with energy harvesting will bring the future goal of achieving net zero energy consumption in buildings closer to reality. The Luminescent Solar Concentrator (LSC) technology [3][4][5] is currently being considered poten-tially suitable for engineering the PV windows of the future 6 , even though ensuring the high transparency in high-efficiency concentrators remains problematic for reasons of IR-luminophore limited availability. Organic dye-based LSCs have been employed to concentrate sunlight re-emitted at wavelength bands efficient for power generation by PV cells 4,5,7,8 . This type of concentrators commonly use dyes as luminescent organic species dispersed in optically clear polymer matrices 7,8 . However, it has been reported that when using organic dyes, the overlapping of absorption and emission bands reduces the concentration capabilities of LSC, which also contributes to their poor stability under long-term solar illumination exposure 5 . Moreover, most of LSC technologies and demonstrator samples featuring PV conversion of light concentrated by planar structures reported in the literature to date 9,10 , share the following major limitations that still prohibit product-level development of energy-harvesting windows and at the same time emphasize the need for further research in this field:</p><p>. The concentrator-area scalability of all current LSC technologies is severely limited by the effects of re-absorption that reduce the propagation path-length of luminescence emissions within lightguiding structures; . No LSC structures reported so far in the literature were designed to enhance the visible transparency simultaneously with spectrally-selective harvesting of non-visible solar radiation using inorganic-only luminophores. This is despite the fact that over 50% of solar energy reaching the ground level is distributed spectrally outside the visible range. . The best power conversion efficiency of LSC devices reported to date remains within 2-4% if using Si cells. The sizes of most recently-reported high-performing concentrator panels never exceeded 100 mm 3 100 mm 10,11 .</p><p>To the best of our knowledge, no literature sources reported on achieving these 2-4% efficiency figures in samples of significant visible-range transparency, and most luminophores used have either been not disclosed or their compositions implied a limited environmental stability [9][10][11] . Also, no spectrally-selective IR-specific photon-trapping structures suitable for use in window systems or glazing-type applications have ever been reported. Glass structure, technology and materials. A window design reflecting and redirecting the invisible solar radiation (Fig. 1(a)) exhibits LSC capability that relies on the efficient absorption of UV and IR light across as broad spectral range as is possible. This can be achieved using an active luminescent functionalized interlayer that provides photo-induced re-emission of the absorbed photons. The glass structure is integrated to act as spectrally-selective concentrator in such a way as to provide efficient transport of the re-emitted and otherwise internally deflected photons towards its edges.</p><p>Our solar concentration approach simultaneously enables electric power generation through PV cells attached to glass panel edges and maintains a maximized visible-range (400-700 nm) transparency. Concentrators of this type can be called ''hybrid'', since luminescence effects play only a partial role in energy harvesting functionality, together with multiple scattering and diffraction enabled by embedding quasi-random disordered arrays of luminophore particles inside polymer interlayers. Despite its contributions to optical losses within the concentrator structure 12 , multiple scattering effects can enhance light-trapping performance in planar-geometry structures [13][14][15] . Our approach to the development of transparent hybridtype solar concentrators relies on the experimental optimization of electric energy output generated by the solar cells attached to the structure edges, by trialing a large number of luminescent material combinations. Some of these materials are presented in Table 1 and some others cannot be disclosed due to the confidentiality agreements. Generally, these materials are capable of converting the non-visible solar energy into near-IR emissions. Different ways of distributing the luminescent powders geometrically within interlayers have been trialed, including varying the particle sizes, epoxy loading concentrations, and layer thicknesses. Since a rather complex combination of several inter-related physical mechanisms and effects acting simultaneously was expected to lead (at least statistically) towards the increased IR illumination levels at sample edges, we chose to characterize the resulting structures' performance in terms of the actual energy harvesting output observed, in both the outdoor and solar-simulator experiments. Using PV cells attached to the sample edges was in this case the best practical way of detecting and measuring the total integrated contribution from all possible light redirection pathways present within the system structures, through measuring the electric output parameters such as open-circuit voltage (V oc ) and short-circuit current (I sc ). The relative contributions of different physical effects e.g. luminescence-assisted energy conversion versus multiple scattering or multiple diffraction events on powder particles is subject to our ongoing study.</p><p>A thin film interference-coating was especially designed to achieve minimized transmission of UV and IR energy as well as fulfil durability requirements related to environmental-stability. Fig. 1(c, d) shows the modelled and measured transmission and reflection spectra of a metal-dielectric thin film, which was developed using an ebeam/thermal evaporator. The heat-mirror-film design was adjusted in terms of materials selection and number of layers deposited to ensure wide-band reflection of IR radiation and the associated ther-mal insulation performance. Multiple coatings of this type were deposited onto glass substrates following the methods described in 16 .</p><p>Functionalized luminescent interlayers. To improve the light collection efficiency of light-trapping glass structures, we adopted an approach based on incorporating inorganic luminophore mixes into a transparent epoxy-based lamination layer, thus realizing a transparent, spectrally-selective, coating-assisted all-inorganic solar concentrator.</p><p>The selection of inorganic luminophore types for developing the interlayers as well as the optimization of powder particle sizes, concentrations, and interlayer thicknesses were subject to application constraints imposed by our industry partner (Tropiglas Technologies Ltd). The principal requirements were related to ensuring a substantial visible-range sample transparency and clear, non-colorbiased glass appearance. These requirements limited the selection of potential luminophores to inorganic substances possessing photoluminescence excitation ranges within either the UV or near-IR ranges, which could be ground into fine (of the order of 1 mm) particles, and then incorporated effectively by either dissolution or ultrasoundassisted disperse suspension, into the interlayer host material (epoxy). The required transparency and visual clarity of the functionalised interlayers were the variables that primarily controlled the upper limits of powder concentrations and interlayer thicknesses used. Prior to undertaking the research activities described in this paper, we had evaluated the relative energy-collection performance of a large number of 20 3 20 3 6 mm samples of similar structure, which employed different inorganic luminophore mixes of various concentrations and layer thicknesses as well as some luminescent organic dyes (Lumogen F Red and others). After extensive experimentation with multiple high-performance commercial photoluminophore substances from a range of suppliers, we identified four inorganic compositions from three different manufacturers which, when used individually or mixed together in small concentrations (sub-1 wt% of each), led to substantial measured increases in the electric output from edge-mounted PV cells, when tested using a solar simulator beam directed at normal incidence (compared to a reference sample that did not contain luminophore materials within its epoxy-based interlayer). The beam of simulated sunlight was significantly smaller than the sample dimensions (20diam. vs 100 mm size), therefore some effective photon redirection mechanism within the glass structure (e.g. luminescence, Mie scattering promoting the wavevector deflection, diffraction events occurring on powder particles, or a combination of these) was crucial for increasing the photon flux reaching the side-mounted PV cells, as was evidenced by the significant dependency of electric output parameters on the interlayer type and composition. All samples constructed had interlayer thicknesses below 3 mm, to ensure large visible transmission and good visual clarity, despite some noticeable scattering and diffused transmission effects. Here, we report on the empiric optimisation of the pre-selected functionalised interlayer compositions in 100 3 100 3 18 mm 3 clear glass samples to maximise the electric power output in PV cells attached to structure edges to detect and convert the optical power deflected towards the edges of glass structures. The effective path length of the luminescence excitation was difficult to evaluate, since the multi-pass propagation through the luminophore-loaded interlayer assisted by the multiple coating reflections and scattering events was engineered to occur within the structures. Four different finely-ground luminophore powder types (described in Table 1) were selected (based on their performance characteristics such as excitation and emission spectral bands and quantum efficiency), and, in order to optimize the performance of the proposed energy-harvesting clear glass panel structure, various mixes were used to construct a number of concentrator prototypes of different interlayer chemistries.</p><p>The luminophore powders and their mixes were primarily selected to enable improved energy harvesting performance by providing both the UV, short-wave visible and also near-IR wavelength regions for photoluminescence excitation, and special care was also taken to place the emission wavelength regions inside the near-IR band, where the solar cells used had good responsivity, and also within the high-reflectance regions of the spectrally-selective coating. The function of (Zn, Cd)S:Cu luminophore (c) was to convert a fraction of light from (300-550) nm band into (840-1040) nm. The main feature of this material and also of luminophore d, is related to the practical absence of overlapping between the excitation and emission wavelength bands, which effectively eliminates reabsorption-related energy losses. Emissions from all four phosphor types were within the high-responsivity band of the solar cell type used.</p><p>Partial concentrations of luminophores (in wt%) were selected carefully in order to maintain the balance between optical clarity of the samples, reasonably high visible-range direct transmission, and enhancing the diffused transmission fraction together with material compatibility issues in terms of increasing scattering with increased number of powder types and concentrations used.</p><p>Several energy harvesting clear glass samples were assembled with different functionalized interlayer properties, such as luminophore compositions, concentrations and thicknesses as shown in Table 2. We carried out a large number of interlayer property adjustment experiments in order to keep the visible-range sample transparency above 40% while trialing the use of various simple, binary and ternary luminophore powder mixes and different loading concentrations to develop the most suitable interlayer material system for the routing of incident radiation towards solar cells.</p><p>Sample A of Table 2 was used as reference sample for benchmarking other samples' performance.</p><p>Sample testing methodology and energy harvesting performance characterization. We designed the concentrator and interlayer characterization experiments in order to identify the bestperforming samples (in terms of energy conversion and routing capability). The electric output parameters of samples were measured when illuminating the glass energy-collecting areas using a normally-incident, 20 collimated (and also diffused) solar simulator beam spectrally equivalent to AM1.5G irradiation (Fig. 2(a)). A reference sample which intentionally did not incorporate any of the technology features designed to assist in energy routing towards glass edges, was selected for benchmarking the performance of all other samples. We define the ''Area Collection Gain'' parameter (ACG) to characterize the relative flux-deflection capability in our concentrator samples as follows:</p><p>Parameter ACG (equation ( 1)) defines the way in which the reference sample is used and also describes the energy-routing performance differences between all samples measured through their electric output.</p><p>Figure 2(b) summarizes the results of performance testing experiments performed with all 12 concentrator samples each containing a different interlayer type. The ACG measured in each sample varied depending on factors such as the luminophore chemistries used and Phosphor a was always used after dry grinding for several hours using Fritsch Pulverisette Premium Line 7 ball mill at 700 rpm to reduce the mean particle size to the vicinity of 1-2 mm.</p><p>the concentrations of the powders, together with interlayer thickness. The goal was to search for an optimized interlayer type and properties, so as to deflect most of the non-transmitted incident optical power towards sample edges more effectively. For example, the results showed (as was somewhat expected) that the ability to deflect more radiation towards edges correlated inversely with visible transmission of samples. It is important to note that only the spectrum of direct transmission was measured, however diffuse transmission was also significant in our samples, as was evident from their visual appearance as well as from their light diffusion properties as illustrated in Fig. 3.</p><p>As an example, the functionalized interlayer of sample C was made by mixing two luminophore powders a and b the excitation bands of which overlapped significantly which, in conjunction with a relatively large scattering observed in all samples containing powder b, resulted in a relatively weak light collection performance. The optimization of light scattering intensity and the related light diffusion effects on the light collection efficiency and samples transmission was made only empirically in our experiments, since multiple factors affecting concentrator performance (from the varying solubilities of various powders in polymer materials to the final particle or agglomerate size ranges available from grinding and homogenization processes) could not be realistically predicted computationally. Sample E, on the other hand, contained three different powders which provided a combination of wide excitation bands placed in the UV, blue and also the near-IR wavelength regions, a rather large Stokes shift of luminophore c, and the IR-range emission bands within the high responsivity wavelength range of CIS cells, showed considerably improved light concentration in comparison to most other samples. Luminophore c dissolved fairly well into our epoxies, had a rather small mean particle size of about 1 mm, and did not cause significant scattering at concentrations not exceeding about 0.05 wt%. The addition of other luminophores to the same sample increased scattering and decreased direct transmission in the visible range, while increasing the diffused transmission component. As shown in Fig. 2(b), the same ACG trends were seen across all samples in our batch, when using either the collimated-beam or diffused-beam illumination geometry.</p><p>The effects of multiple scattering on weakly-absorbing luminophore powder particles distributed in a quasi-random, quasi-3D fashion inside our interlayers, on the character of light propagation through concentrator samples are rather complex and lead to an additional scattering-related loss mechanism. However, the disorder-induced light trapping also occurs, and the possible light-trapping mechanisms involving multiple scattering effects in disordered photonic systems were reported recently in [13][14][15] . Additionally, the potential role of scattering effects in assisting the light trapping functionality in LSC systems requiring only modest flux gains has been recognized as early as 1981 5 . Fig. 3 shows that beam expansion and light diffusion effects are easily observable, as well as the appearance of fringe structure within a halo of diffused light formed due to multiple scattering on powder particles.</p><p>The disorder-induced beam expansion effects and their role in assisting the light trapping in planar hybrid-type optical concentrators is subject to our ongoing study.</p><p>The evaluation of the performance of LSC-type light concentration systems is commonly related to geometric flux gain measurements as well as to the measurements of the power conversion efficiency (overall system efficiency g) 4,5,10,11 . For concentrator systems designed to provide significant spectral selectivity and thus spectrally separating the energy-harvesting wavelength bands from the enhanced-transmission bands, the definition of flux gain can be adjusted to account for the limited spectral bandwidth available for radiation routing and flux concentration. Parameters measurable directly in all PV systems include the short-circuit current (I sc ) or its density J sc , open-circuit voltage (V oc ) and/or their product, or the output power I sc * V oc *FF. Within the framework of our experimental methodology, we define another figure of merit to characterize the energy routing (flux deflection) performance of concentrator samples assisted by spectrally-selective coatings, namely the Deflection Efficiency Factor (DEF), in the following way:</p><p>First, we calculate the optical power (within the response bandwidth of PV cells used) of the normally-incident optical power, after the first coating-assisted reflection, that is</p><p>Calculations according to equation (2) with the integration limits between 300 nm and 1220 nm show that the total optical power within the bandwidth of interest (for circular incident beam of 20 diameter) after its first coating reflection (P refl , opt ) was only a small fraction (26%) of the incident power. Figure 4(a) shows the effect of our highly-transparent spectrallyselective solar-control coating on the spectral modification of the spectral power density distribution of incident light after the first reflection off the coating. We integrated the product of standard AM1.5G spectral distribution and the coating's reflectivity function numerically.</p><p>Second, we calculate the DEF as follows:</p><p>This Deflection Efficiency Factor (DEF) quantifies the optical power within the spectral responsivity band of CIS cells that has actually reached the cell surfaces, as a fraction of the optical power within the same spectral band, within the incident-beam area, available after the first reflection off the spectrally-selective coating placed at the back surface of the samples. This definition emphasizes the capability of the samples to deflect the energy available, accounting for the effects of transmission, scattering and absorption which limit the possible energy flux available for trapping or re-direction within the structure of the samples and interlayers. This way of evaluating the performance of the concentrator samples can be used for all types of PV cells, coatings and illumination conditions used, as long as the beam is centered within the glass collection area at near-normal incidence conditions. Figure 4(b) shows a summary of the relative performance of our energy harvesting clear glass samples in terms of DEF as calculated using equation (3). Not surprisingly, significant dependency of DEF on the interlayer type was observed.</p><!><p>As was expected, the radiation deflection performance trends quantified using the DEF (Fig. 4 showed a somewhat remarkable result of routing in excess of 20% of the total radiation energy reflected off its back coating towards the PV modules, considering that the input radiation was incident normally and multiple scattering and internal reflection events were required to propagate the photons incident onto the central region of glass collection area towards sample edges over a path length of several centimeters. In addition, the V oc generated by this sample (5.12 V) was approaching its saturation value (near 7.4 V for CIS modules of 100 mm length) despite not being directly illuminated by the light source. Performance comparisons were also made in outdoor illumination conditions using vertically-oriented glass concentrator samples. The performance differences between the different sample types (within this batch of 100 3 100 mm samples) were less pronounced compared to the results presented in Figs. 2 and 4, due to the increased direct (background) illumination reaching the CIS active areas, and the fact that the total area of CIS cells was as large as the glass collection area (which was in fact a design consideration for increasing the electric output of samples in lab conditions, where the background illumination of cells was quite easy to avoid), for reducing the ACG and DEF measurement errors.</p><p>Somewhat surprisingly, sample J showed the best performance in peak outdoor illumination conditions (I sc 5 60.5 mA; V oc 5 7.4 V and thus P out 5 268.6 mW). When placed vertically, all samples intercepted a total flux of about 700 W/m 2 (7 W incident power), which brings the power conversion efficiency to g 5 3.8%. We believe that this result is outstanding for a glazing system-based concentrator of 55% visible transparency employing inorganic-only luminophore combination. The cells were able to partially collect some background solar illumination in addition to collecting the concentrated light from inside the glass structure. Since approximately 50% of the air-exposed cell areas (at top and one side of sample, which accounted for 14% of the total CIS area) were essentially shadowed from direct sunlight, and the other exposed cell areas received only a fraction of incident flux due to geometry, most of the output was generated through concentration effects.</p><p>The results of interlayer optimization were used successfully to build up-scaled visibly-transparent glazing system concentrators of size 200 3 200 mm, however the detailed description of concentrator up-scaling results are beyond the scope of this article and will be reported separately. Fig. 5 shows typical I-V curves of our 100 3 100 mm and advanced 200 3 200 mm samples measured at ''average'' and peak outdoor illumination conditions, respectively.</p><p>The performance characteristics of our energy-harvesting glass samples quantified in terms of the parameters used commonly in the LSC field (such as power conversion efficiency g, optical collection probability P defined by g/ g 0 as a ratio of wall-plug device efficiency to bare-cell efficiency, and geometric gain G [11]) are summarized in Table 3. All of 100 3 100 mm 2 samples had a small geometric gain of near unity (G 5 1.02), whereas the up-scaled 200 3 200 mm 2 sample had G 5 1.87.</p><p>The wall-plug (power conversion) efficiency and photon collection probability of sample J both increased by almost 50% compared to sample B which relied on a blank (luminophore-free) epoxy interlayer and an identical structure and heat-mirror coating, due to adding an optimized concentration and mix of functional luminescent materials into its interlayer structure. Notable differences in the electric output stability versus the horizontal-angle orientation of these two samples were also seen, which were expected, because LSCtype collectors have an advantage of relative insensitivity to the incoming radiation flux direction. It is important to also note that, despite photoluminescence effects, other physical mechanisms including Mie scattering, multiple diffraction on a disordered array of powder particles and refractive-type coating-assisted geometric concentration, also contributed to energy collection at cells. The detailed analysis of relative contributions of all mechanisms leading to radiation flux re-direction and energy harvesting in visually-clear glass concentrators are subject to our ongoing studies.</p><!><p>We have proposed the use of all-inorganic spectrally-selective scattering luminophores in conjunction with spectrally-selective thin- film coatings and CuInSe 2 solar cells to realize visibly-transparent energy-harvesting clear laminated glass panels. Luminophore chemistries and concentrations have been optimized and incorporated into an optical epoxy lamination interlayer to maximize the power conversion efficiency of a 100 mm 3 100 mm vertically-placed energy-harvesting clear glass panel. Experimental results have demonstrated a transparency exceeding 55%, invisible solar energy attenuation greater than 90%, power conversion efficiency of 3.8%, a solar heat gain coefficient (SHGC) of 0.41, and a U-factor less than 1.8 W/(m 2 K).</p><!><p>Thin film coatings. Metal-dielectric coatings were manufactured by physical vapor deposition (PVD) techniques using sputtering targets and pellets of 99.99% pure materials purchased from AJA international, Inc. Chemically ultrasonically cleaned low iron borofloat glass cut-outs of size 100 3 100 3 6 mm were used as substrates. (KVE-ENT 200 hl) thermal/e-beam evaporator and (KVS-T4065) sputtering system, supplied by Korea Vacuum Tech, LTD, were used to deposit thin film layer sequences. The desired thin film characteristics were predicted using OptiLayer software package. The thin films transmission spectra were characterized using Agilent Cary 5000 UV-VIS-NIR and Beckman Coulter DU 640 B UV/Visible spectrophotometers.</p><p>Functionalized interlayer formation. The laminated glass functional interlayer composites were made with various concentrations and combinations of luminophore powders as shown in table 1 and table 2. NOA61 UV-curable epoxy (Norland, Inc.) and WTS-80203 (H.K. Wan Ta Shing, Industrial Ltd.) were used as polymer host matrices. No significant epoxy performance differences were noted when using these epoxies interchangeably.</p><p>Powders formed partially dissolved suspensions within the epoxy materials and were dispersed ultrasonically after mechanical stirring to de-agglomerate the suspended particles. Sonication of 100-150 ml epoxy volumes was performed for 5-10 minutes using Bandelin Sonopuls HD2200 fitted with 12 mm stainless steel horn, running at 10-20% of its transducer power. Interlayers were formed by pouring luminophore-loaded epoxy over glass sample surfaces manually. Layer thickness adapters were used at all sample corners during the liquid phase of the lamination process and then removed during layer solidification. Light curing system (ELC-410 Thorlabs, Inc.) was used as UV-blue light source for epoxy curing.</p><p>Solar cells integration and electrical circuits. 5 W CuInSe 2 thin film solar cell modules of size 200 3 270 mm on 2 mm -thick glass substrates (AD Solar, Inc.) were cut into strips of size 98 3 25 mm using diamond-wheel glass cutter. The number of p-n junctions integrated along the cut-out lengths had to be made identical which was confirmed by making I sc and V oc measurements at full illumination to ensure uniform electric performance characteristics. Soldering connections to tabbing wires were made using ultrasonic soldering iron (PVLED Technology, Ltd.) and SB-220 (S-Bond, Inc.) active solder compound. Clear UV-curable epoxy identical to the interlayer host material was used as adhesive to attach the solar cell modules to each of the four laminated sample sides. All four cell modules were connected electrically in parallel using blocking diodes, since significantly non-uniform illumination conditions were expected to affect the system performance during outdoor operation of samples placed vertically. Device characterization. Indoor tests were performed using a solar simulator system (ScienceTech, Inc.) as described in Principal Results and Discussion. Care was taken to ensure that all samples were tested at normal incidence conditions at a fixed (100 mm) distance from the source's output aperture, and that the AM 1.5G beam was centered onto the glass surface. Background illumination level was minimized by using curtains and removing room lighting.</p><p>Outdoor tests were performed in natural daylight conditions, and I-V characteristics were recorded manually (point by point) using 3700 Series Programmable DC Electronic Load (Array Electronic Co., Ltd.) after orienting the vertically-placed samples in the horizontal plane to obtain peak output. The ''peak'' conditions were observed typically for horizontal-plane sample orientation angles near the 45u flux incidence condition with respect to the Sun azimuth direction. 3.19% 34.71% 0.649 Geometric gain G 5 1.87; energy collection area of 400 cm 2 .</p><p>varied slightly during each of the stages. The design of thin film coating and running the deposition processes was mainly accomplished by R.A., M.V. and M.N. M.V. and M.N. designed the electrical interconnections circuitry and performed the integration of solar modules. All four authors (R.A., M.V., K.A. and M.N.) worked on selecting the luminophore chemistries suitable for use in functionalised lamination interlayers and contributed to the fine-tuning of glass panels lamination processes. All authors participated in the design of both the indoor and outdoor solar energy harvesting performance characterisation experiments. The final data analysis was accomplished by R.A. and M.V. The figures and images included in the manuscript were generated throughout the research program, and all authors contributed actively to their contents. The research progress and the results achieved at each stage were supervised by K.A. from the intial stages; M.N. and K.A. reviewed the manuscipt after it had been written by R.A. and M.V., after which all authors again contrubuted to producing the final manuscript version.</p>

Scientific Reports - Nature

Microwave-assisted synthesis of <i>N,N</i>-bis(phosphinoylmethyl)amines and <i>N,N,N</i>-tris(phosphinoylmethyl)amines bearing different substituents on the phosphorus atoms

A family of N, N-bis(phosphinoylmethyl)amines bearing different substituents on the phosphorus atoms was synthesized by the microwave-assisted and catalyst-free Kabachnik-Fields reaction of (aminomethyl)phosphine oxides with paraformaldehyde and diphenylphosphine oxide. The three-component condensation of N,N-bis(phosphinoylmethyl)amine, paraformaldehyde and a secondary phosphine oxide affording N,N,N-tris(phosphinoylmethyl)amine derivatives was also elaborated. This method is a novel approach for the synthesis of the target products.

microwave-assisted_synthesis_of_<i>n,n</i>-bis(phosphinoylmethyl)amines_and_<i>n,n,n</i>-tris(phosph

Introduction<!>Results and Discussion<!>Synthesis of N,N,Ntris(phosphinoylmethyl)amines<!>Conclusion

<p>α-Aminophosphine oxides are of considerable importance as potential precursors of α-aminophosphine ligands [1]. α-Aminophosphines play an important role in the synthesis of P(III)-transition metal complexes [2], which are often applied catalysts in homogeneous catalytic reactions [2][3][4]. In addition, a few Pt, Ru and Au complexes incorporating phosphine ligands show significant anticancer activity [5,6].</p><p>One of the most common synthetic routes to α-aminophosphine oxides is the Kabachnik-Fields (phospha-Mannich) reaction, where an amine, an oxo compound (aldehyde or ketone) and a secondary phosphine oxide react in a condensation reaction [1]. However, only a few papers deal with the synthesis of α-aminophosphine oxides. (Phenylaminomethyl)dibenzylphosphine oxide was prepared by the three-component reaction of aniline, paraformaldehyde and dibenzylphosphine oxide [7], as well as by the reaction of (hydroxymethyl)dibenzylphosphine oxide and aniline [8]. The condensation of butylamine, paraformaldehyde and di(p-tolyl)phosphine oxide to afford (butylaminomethyl)di(p-tolyl)phosphine oxide was also described [9]. A microwave (MW)-assisted, catalyst-free method was elaborated by us for the synthesis of several (aminomethyl)phosphine oxides [10,11].</p><p>As regards α-aminophosphine oxides with different P-substituents, only two different types were reported. Olszewski and co-workers synthesized chiral thiazole-substituted aminophosphine oxides 2 through the Pudovik reaction of alkylphenylphosphine oxides and the corresponding aldimine derivatives of thiazole 1 (Scheme 1) [12].</p><p>Cherkasov and his group applied the Kabachnik-Fields reaction to synthesize a P-chiral aminophosphine oxide with a 2-pyridyl substituent 3 (Scheme 2) [13].</p><p>Bis(aminophosphine oxide) derivatives were also prepared by the double Kabachnik-Fields reaction using primary amines [11,14,15], amino acids [16,17] or aminoethanol [14] as the amine component.</p><p>To the best of our knowledge, only one example can be found for a bis(α-aminophosphine oxide) containing different P-functions that was prepared by the condensation of (octylaminomethyl)dihexylphosphine oxide, paraformaldehyde and di(ptolyl)phosphine oxide in the presence of p-toluenesulfonic acid in boiling acetonitrile (Scheme 3) [12]. Furthermore, tris(α-aminophosphine oxide) derivatives have not been described in the literature up to now. In this paper, we report the efficient, catalyst-free and MW-assisted synthesis of N,N-bis(phosphinoylmethyl)amine and N,N,N-tris(phosphinoylmethyl)amine derivatives bearing different substituents on the phosphorus atoms.</p><!><p>Synthesis of N,N-bis(phosphinoylmethyl)alkylamines containing different substituents on the phosphorus atoms First, the (aminomethyl)phosphine oxide starting materials 5-7 were synthesized following our previous protocol [11]. Thus, the MW-assisted Kabachnik-Fields reaction of primary amines (butyl-, cyclohexyl-or benzylamine), paraformaldehyde and di(p-tolyl)-or dibenzylphosphine oxide was carried out in acetonitrile at 100 °C for 1 h affording the products with excellent yields (Scheme 4).</p><p>Then, (aminomethyl)diphenylphosphine oxide (9) was prepared through debenzylation of (benzylaminomethyl)diphenylphosphine oxide (8, Scheme 5). The reduction was carried out in the presence of a 10% palladium on carbon catalyst (Selcat Q), in methanol, at 75 °C for 3 h, and the (aminomethyl)diphenylphosphine oxide (9) was obtained in a yield of 47% after column chromatography. In the next step, (aminomethyl)phosphine oxides 5-7 were converted to bis(phosphinoylmethyl)amine derivatives bearing different substituents at the phosphorous atoms (Y 2 P=O) by reacting them with one equivalent of paraformaldehyde and diphenylphosphine oxide under MW conditions (Scheme 6). The three-component condensations were performed in the absence of any catalyst in acetonitrile as the solvent to over-come the heterogeneity of the reaction mixture. After an irradiation of 1 h at 100 °C, the mixed N,N-bis(phosphinoylmethyl)amines 10a,b, 11a,b and 12a,b were obtained in yields of 92-97% and their structures were confirmed by 31 P, 13 C and 1 H NMR, as well as HRMS measurements. Due to the two differently substituted phosphorous nuclei in the molecules, two signals were observed in the 31 P NMR spectra.</p><p>The valuable intermediate 9 was then utilized in the synthesis of N,N-bis(phosphinoylmethyl)amines 13a-c (Scheme 7). The condensation of (aminomethyl)diphenylphosphine oxide (9), paraformaldehyde and various secondary phosphine oxides, such as diphenyl, di(p-tolyl) or dibenzylphosphine oxide, at 100 °C for 40 min led to the corresponding N,N-bis(phosphinoylmethyl)amines containing identical (13a) or different substituents on the phosphorus atoms (13b and 13c) in excellent yields (95-97%).</p><!><p>Finally, N,N-bis(phosphinoylmethyl)amines 13a and 13b were reacted further with paraformaldehyde and a secondary phosphine oxide (diphenyl-, di(p-tolyl)-or dibenzylphosphine oxide) to afford the N,N,N-tris(phosphinoylmethyl)amine derivatives bearing identical (14) and different Y 2 P=O groups (15-17) (Scheme 8). The condensations were performed as mentioned above. The introduction of a third phosphinoylmethyl moiety into the bis-derivatives containing an NH unit (13a and 13b) required a longer reaction time (2 h) at 100 °C. In these cases, the conversion was 70-95%, and the corresponding N,N,Ntris(phosphinoylmethyl)amine derivatives 14-17 were isolated in yields of 27-77%. However, applying a higher temperature and/or longer reaction time, lead to decomposition.</p><!><p>In summary, we have developed an efficient, catalyst-free and MW-assisted method for the synthesis of N,N-bis(phosphinoylmethyl)amines and N,N,N-tris(phosphinoylmethyl)amines bearing different substituents on the phosphorus atoms by the Kabachnik-Fields reaction. This method is a novel approach for the synthesis of the target products. In all, thirteen new derivatives were isolated in high yields and fully characterized.</p>

Beilstein

A Synthesis of Alsmaphorazine B Demonstrates the Chemical Feasibility of a New Biogenetic Hypothesis

An N-oxide fragmentation/hydroxylamine oxidation/intramolecular 1,3-dipolar cycloaddition cascade efficiently converted an oxidized congener of akuammicine into the complex, hexacyclic architecture of the alsmaphorazine alkaloids. This dramatic structural change shows the chemical feasibility of our novel proposal for alsmaphorazine biogenesis. Critical to these endeavors was a marked improvement in our previously reported Zincke aldehyde cycloaddition approach to indole alkaloids, which permitted the gram-scale synthesis of akuammicine. The chemoselective oxidations of akuammicine leading up to the key rearrangement also generated several biogenetically related alkaloids of the alstolucine and alpneumine families.

a_synthesis_of_alsmaphorazine_b_demonstrates_the_chemical_feasibility_of_a_new_biogenetic_hypothesis

<p>Inspiration from presumed biogeneses has led to compelling outcomes in numerous alkaloid syntheses.1 In one historically fascinating example, Woodward patterned aspects of his landmark strychnine synthesis2 on a hypothesis that its biosynthesis might involve an arene oxidative cleavage event.3 While this attractive idea turned out to be incorrect, his thoughts about strychnine's biogenesis were likely critical to the design of the successful approach. Here, we put forth a new biogenetic hypothesis for the origins of the hexacyclic alsmaphorazine alkaloids—one that might or might not be correct—and demonstrate how this way of thinking enabled the first synthesis of alsmaphorazine B.</p><p>Alsmaphorazines A and B (1 and 2, Figure 1) feature a stereochemically dense, highly oxidized, and cage-like hexacyclic skeleton with an endocyclic N–O bond that is rarely encountered in indole alkaloids. In their report on the isolation and structural elucidation of these compounds,4 the Morita group put forth a proposed biosynthesis via akuammicine-type intermediates involving multiple stepwise oxidations and rearrangements to access the alsmaphorazines. Our analysis of the structural relationship between the almaphorazines, which were isolated from the tree Alstonia pneumatophora, and other well-known alkaloids from Alstonia led us to propose a new biogenesis of 1 and 2.</p><p>Our alternative biogenetic hypothesis also centered on the idea of performing multiple oxidations of akuammicine.5,6 Of course, 1,2-oxazolidines are retrons for application of the nitrone-alkene 1,3-dipolar cycloaddition,7 and we viewed nitrone/enone 3 (Scheme 1) as a key progenitor of the alsmaphorazine framework. This fleeting intermediate might be formed by β-elimination of a tertiary amine N-oxide and in situ hydroxylamine oxidation starting from intermediates of type 4, one diastereoisomer of which corresponds to the enantiomeric natural products alstolucine C and alpneumine C.8 The requisite C19 and N4-oxidations of akuammicine (5) to 4 appeared straightforward, and during the course of our investigations, Andrade et al. reported just these types of transformations.6i Numerous syntheses of akuammicine have been reported, with Andrade's six-step synthesis of (±)-5 currently standing as the most concise.6f The directness of our previously described Zincke aldehyde-based approach to Strychnos-type alkaloids was also attractive.9 We recognized that the alsmaphorazine challenge provided an excellent opportunity for optimization and extension of our chemistry to procure the quantities of akuammicine needed to study our hypothesis for biogenesis of the alsmaphorazines.</p><p>Our synthesis of akuammicine began with reductive amination of tryptamine with p-anisaldehyde to give secondary amine 7 (Scheme 2), which was treated with potassium glutaconate salt 8 according to modified Marazano conditions10 (in lieu of Zincke pyridinium ring opening9,11) to deliver Zincke aldehyde 9 in 93% yield from tryptamine. Notably, this improved procedure greatly simplifies the purification of the key Zincke aldehyde, requiring only trituration of the crude material. Subsequent base-mediated formal cycloaddition9 provided tetracyclic enal 10 in 89% yield. Numerous attempts were made at direct C17 oxidation; however, the free indoline proved to be a liability. In the end, several steps were needed to reliably change the C17 oxidation state and to refunctionalize N4; however, each of these reactions proved efficient even on multigram scales, and the intramolecular Heck reaction precursor 13 could be reliably accessed. This final ring closure, patterned on the work of Rawal,6e,12 MacMillan,6g and Andrade,6f,h,i was remarkably efficient. The nine-step sequence to akuammicine proceeded in nearly 40% overall yield and procured gram quantities of the racemic alkaloid for our alsmaphorazine studies.</p><p>For the next stage of our plan, we sought to perform selective oxidations of akuammicine to arrive at the ketone natural products of the alstolucine and alpneumine series (Scheme 3).13 Our efforts to engage the C19–C20 alkene with Wacker and peracid oxidations, as well as thiol-ene, oxymercuration, and hydroboration reactions, were met either with outright failure or undesired regioselectivity. Inspired by the precedent of Brown and Djerassi,14 in which stoichiometric osmium tetroxide was used to dihydroxylate akuammicine, we evaluated catalytic variants, eventually settling on the conditions developed by Fokin and Sharpless15 owing to the relative ease of extractive removal of osmium remnants. Attempts to extend this oxidation process to an alkene ketohydroxylation by employing the conditions of DuBois16 proved unsuccessful, and only starting material and minor products of dihydroxylation were observed. We made many attempts to effect a hydride shift of the intermediate diol directly to the C19 ketone with no success. We performed the oxidation of crude diol with CrO3 as prescribed by Brown and Djerassi,14 but only obtained 27% yield (two steps) of α-hydroxyketone 14. Oxidation by the Dess–Martin periodinane proved sluggish depending on the batch of diol. Careful evaluation of the reaction conditions led to the identification of t-BuOH as a critical accelerant,17 providing product 14 in 68% yield over two steps. SmI2 reduction of the α-ketol18 provided an inconsequential 1:1.5 mixture of alstolucine F/alpneumine E (15) and alstolucine B (16). The components can be separated by chromatography, providing the former in 33% yield and the latter in 61% yield; however, we typically use the crude mixture of ketones in subsequent operations. Exposure of the diastereomeric mixture of methyl ketones to DMDO at −78 °C led to quantitative conversion to the corresponding N-oxides, 17 and 18, the latter of which is the racemate of the enantiomeric natural products alstolucine C and alpneumine C. Minimal C20 epimerization was observed during this reaction, as determined by 1H NMR (1:1.4 dr).</p><p>The final and key stage in our plan was also the riskiest. We needed to initiate a base-mediated elimination or thermal Cope elimination to liberate an enone and a hydroxylamine (see 19), the latter of which needed to be oxidized in situ to a reactive nitrone for 1,3-dipolar cycloaddition. The precedent of Ciganek19 showed that it is indeed possible to execute E1cb eliminations of cyclic piperidine N-oxides with adjacent electron-accepting groups, but the hydroxylamines thus formed readily recyclize by conjugate addition upon standing. We reasoned that the intermediate hydroxylamine might be intercepted by air oxidation20 to selectively generate the desired nitrone intermediate, which would participate in the intramolecular cycloaddition. With this idea in mind, we heated the mixture of N-oxides21 in toluene in the presence of DBU under an atmosphere of air. After complete consumption of starting material, we painstakingly separated the complex mixture of cycloadduct 20, nitrone isomer 21, and amines 15 and 16 along with other unidentified side products. The structure of cycloadduct 20 was confirmed by single crystal X-ray diffraction. The formation of amines 15 and 16 might be explained by a disproportionation process22 in which the intermediate hydroxylamine 19 might shuffle oxidation states to give amines and nitrones.</p><p>On the basis of this result, we reasoned that a more strongly oxidizing atmosphere might facilitate complete oxidation to the desired nitrone and mitigate the presumed disproportionation. Indeed, conducting the same reaction under an oxygen balloon led to an isolated yield of cycloadduct 19 in 49% yield over three steps along with fully substituted nitrone 20 in 29% yield.</p><p>Only oxidation of the vinylogous carbamate, which was expected to be highly stereoselective by virtue of the cage-like ring system, separated 20 from alsmaphorazine B. On the basis of numerous studies toward Vinca alkaloids,23 we treated the cycloadduct with m-CPBA in the presence of various additives; however, only decomposition was observed. DMDO and peroxide reagents also proved ineffective. It appeared that N4 underwent competitive oxidation, and Baeyer–Villiger oxidation of the ketone might also have occurred under some conditions. Furthermore, the indolenine of alsmaphorazine B could potentially undergo oxidation, further complicating our efforts at a selective reaction. We reasoned that deprotonation of the vinylogous carbamate moiety could increase its relative reactivity and facilitate oxidation to the tertiary alcohol. Indeed, metallation of the vinylogous carbamate of 20 followed by treatment with Davis oxaziridine provided alsmaphorazine B in 82% yield. The structure of our synthetic sample was confirmed by single crystal X-ray diffraction, and the accumulated spectral data were in agreement with those reported by Morita.4</p><p>We have completed the first synthesis of alsmaphorazine B in 15 steps and 10.6% overall yield by recognizing its potential biosynthetic connection to akuammicine via oxidation-induced fragmentation and nitrone/alkene dipolar cycloaddition. To achieve this goal, we improved and extended our Zincke aldehyde methodology to facilitate the gram-scale synthesis of akuammicine. Subsequent chemoselective oxidation of the pentacyclic framework provided several alkaloids of intermediate levels of oxidation. Although our synthesis produces racemic material, we recognize that many of the intermediate alkaloids have been isolated in both enantiomeric forms,8 with representatives in each series demonstrating interesting biological activities; resolutions of synthetic 15 and 18, for example, will each yield two natural products. In our key fragmentation/oxidation/dipolar cycloaddition step, the cogeneration of the two complementary reactive functionalities for the dipolar cycloaddition is noteworthy. Our synthetic work provides a meaningful biosynthetic oxidation pathway that connects all of these Alstonia alkaloids and provides strong support for our biogenetic proposal that features a nitrone/alkene 1,3-dipolar cycloaddition, adding to the current literature of such dipolar cycloadditions in complex alkaloid biosynthesis.24,25</p><p> ASSOCIATED CONTENT </p><p> Supporting Information </p><p>Experimental procedures and characterization data for all new compounds, X-ray crystallographic structures, and information for 2 and 20 (CIF). The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/jacs.5b04686.</p><p>The authors declare no competing financial interest.</p>

PubMed Author Manuscript

Combined ionic liquid and supercritical carbon dioxide based dynamic extraction of six cannabinoids from <i>Cannabis sativa</i> L.

The potential of supercritical CO 2 and ionic liquids (ILs) as alternatives to traditional extraction of natural compounds from plant material is of increasing importance. Both techniques offer several advantages over conventional extraction methods. These two alternatives have been separately employed on numerous ocassions, however, until now, they have never been combined for the extraction of secondary metabolites from natural sources, despite properties that complement each other perfectly. Herein, we present the first application of an IL-based dynamic supercritical CO 2 extraction of six cannabinoids (CBD, CBDA, Δ 9 -THC, THCA, CBG and CBGA) from industrial hemp (Cannabis sativa L.). Various process parameters were optimized, i.e., IL-based pre-treatment time and pre-treatment temperature, as well as pressure and temperature during supercritical fluid extraction. In addition, the impact of different ILs on cannabinoid extraction yield was evaluated, namely, 1-ethyl-3-methylimidazolium acetate, choline acetate and 1-ethyl-3-methylimidazolium dimethylphosphate. This novel technique exhibits a synergistic effect that allows the solvent-free acquisition of cannabinoids from industrial hemp, avoiding further processing steps and the additional use of resources. The newly developed IL-based supercritical CO 2 extraction results in high yields of the investigated cannabinoids, thus, demonstrating an effective and reliable alternative to established extraction methods. Ultimately, the ILs can be recycled to reduce costs and to improve the sustainability of the developed extraction process. † Electronic supplementary information (ESI) available. See

combined_ionic_liquid_and_supercritical_carbon_dioxide_based_dynamic_extraction_of_six_cannabinoids_

Introduction<!>Results and discussion<!>Ratio of ionic liquid to water (Stage 2)<!>SFE extraction parameterspressure and temperature (Stage 3)<!>Type of ionic liquid<!>Plant material<!>Ionic liquid-supercritical fluid extraction<!>Solvent-based extraction<!>Ionic liquid recovering<!>Cannabinoid quantification<!>Statistical analysis<!>Addendum

<p>Cannabis sativa L. is an annual herbaceous blossoming plant that has been used throughout history in the textile industry, for recreational purposes and in medical applications. It is regarded as one of the oldest cultivated plants, and one of the most essential crops for the progress of humankind. Although native to Eastern Asia, its extensive applications led to its global spread. 1 The medicinal properties of Cannabis sativa L. can be attributed to the many bioactive compounds present in the plant, such as terpenes, polyphenols, phytosterols, tocopherols, fatty acids, and, specifically, cannabinoids, which are terpenophenolic secondary metabolites. 2,3 It is important to mention that cannabinoids are not equally distributed in the plant. They are mainly found in the trichomes and in smaller to negligible amounts in the seeds, while roots contain none. 4 Presently, over 100 cannabinoids have been identified. 5 They are primarily encountered in their carboxylated form in the plant which constitutes a structure of 22 carbon atoms. So far, cannabinoids have been categorized into 11 subclasses: (1) (−)-Δ 9 -tetrahydrocannabinol (Δ 9 -THC), (2) (−)-Δ 8 -tetrahydrocannabinol (Δ 8 -THC), ( 3) cannabidiol (CBD), ( 4) cannabigerol (CBG), ( 5) cannabichromene (CBC), ( 6) cannabinol (CBN), ( 7) cannabinodiol (CBND), ( 8) cannabicyclol (CBL), (9) cannabielsoin (CBE), (10) cannabitriol (CBT) and (11) miscellaneous. The structures of cannabinoids from hemp investigated in this study are depicted in Fig. 1. 6 In terms of the biosynthesis of cannabinoids, CBGA is the main precursor for THCA and CBDA. 7 However, under high temperatures, both acids are prone to degrade into their respective decarboxylated analogues, Δ 9 -THC and CBD. 8 Δ 9 -THC and CBD are the most abundant cannabinoids present in cannabis plants. Δ 9 -THC is well-known as a psychoactive compound, which influences the central nervous and cardiovascular systems. Contrarily, CBD is non-psychoac-tive, but is regarded as a compound of enormous medical interest, as it has demonstrated numerous health benefits. It has been reported to have anti-inflammatory, antiepileptic and anticonvulsive properties, among many others. [9][10][11] Excellent medicinal potential have been attributed to cannabinoids; thus, significant effort has been made in the past decades towards the research of the functions and mechanisms of cannabis-derived secondary metabolites in the human body.</p><p>Due to the growing medicinal interest in cannabinoids over the years, scientists have undertaken efforts in the development of extraction methods for these valuable bioactive compounds. Traditionally, Δ 9 -THC and other cannabinoids have been isolated by solvent-based extractions, with hydrocarbons and alcohols delivering the highest yields. 12,13 Soxhlet extraction (SE) is also a commonly used technique, 14,15 which is characterized by shortcomings, namely, long extraction times and high temperature that may promote thermal degradation of the target compounds. 16 Other advanced extraction techniques, such as microwaveassisted extraction (MAE) allow higher yields, shorter extraction times, less solvent and reduced energy consumption. 14,17 Nevertheless, uneven heating and/or overheating may cause thermal degradation, and thus negatively impact the extraction efficiency. 18 Alternatively, the use of ultrasound-assisted extraction (UAE) achieves high yields in short times; 19 however, the distribution of ultrasound energy lacks uniformity and over time the power decreases, which can lead to inefficient use of the ultrasound-generated energy. 20 Supercritical fluid extraction (SFE) is an innovative separation technique, which has thus far been employed for extractions of valuable constituents from over 300 plant species. 21 Carbon dioxide is a widespread choice for SFEs due to its several advantageous properties, such as low reactivity, nontoxicity, non-flammability, affordability, availability, and recyclability. Additionally, its selectivity can be adjusted by modification of pressure and temperature, while product fractionation and recovery with high purity is feasible. Nevertheless, due to its low polarity, addition of small quantities of organic solvents (co-solvents or modifiers) is necessary to access more polar compounds, thereby expanding its extraction range. 22 The selection of an appropriate co-solvent is key for achieving optimum solubility of the bioactive compounds present in the plant. 23 Supercritical carbon dioxide has previously been used to assess the solubility of individual cannabinoids, for example, Δ 9 -THC, 24 CBD 25 and CBG. 25 Moreover, several extractions of cannabinoids from different parts of the cannabis plant, for instance, leaves, trimmings, buds, flowers and threshing residues, have been performed using ethanol as a co-solvent. [26][27][28][29] Within the past years, ionic liquids have also emerged as alternative reaction media for the extraction of biomass that is regarded as a source of natural medicinally relevant complex compounds. Many different properties are attributed to ionic liquids, such as exceptional dissolution properties, high thermal stability and broad liquid range, to name a few. Furthermore, ILs display high tuneability, as the combination of different cations and anions leads to hydrophilicity or hydrophobicity and different polarity. 30 The dissolution and processing of lignocellulosic biomass is a particularly interesting application of ionic liquids (ILs), as they can directly dissolve and fractionate (ligno-)cellulose in an overall less energy intensive process. 31,32 The biomass dissolution capability of ILs is impacted by both their cation and anion, however, current publications suggest that anions have a more significant impact, since they play a role in breaking the many intermolecular hydrogen bonds. 30 Regarding the cation, imidazolium-based ILs were the most successful for the direct dissolution of cellulose, followed by pyridinium-and ammonium-based ones. 33 In addition, increasing the chain length of the cation had a negative influence on the dissolving capabilities of the ILs, as the viscosity increased, and the H-bond acidity decreased. As far as the anion is concerned, dissolving efficiency seems to be determined by the H-acceptor properties of the anion. In general, anions with weak H-bond basicity, for instance, [BF 4 ] − and [PF 6 ] − , could not successfully dissolve cellulose, while ionic liquids based on halide or acetate anions are typically the candidates of choice. 30,34 The growing research on ILs as solvents for lignocellulosic biorefinery also prompted innovations for the extraction of valuable ingredients from plant materials. 35 There are several aspects of ILs that are potentially advantageous for the extraction of highvalue compounds: apart from their unique solvent properties and potential environmental benefits, the ability of ILs to dissolve biomass can lead to a better, and higher, yielding access to valuable ingredients embedded in the biopolymers and contribute to a value-added biorefinery. 36,37 However, the recovery of natural products from ionic liquids is often more demanding than the mere extraction: many studies require extensive back-extraction with volatile solvents to actually isolate the valuable ingredients from ILs, thereby rendering the original solvent reduction less significant or even negating it altogether.</p><p>The combination of non-volatile polar ILs with volatile nonpolar scCO 2 has several advantages for extractions, as well as for catalysis. Since scCO 2 is highly soluble in ILs, but ILs cannot dissolve in scCO 2 , it can easily penetrate the IL-phase. This allows the extraction of compounds from the IL-phase into the scCO 2 phase, taken into account that the organic compound of interest is soluble in scCO 2 . Ultimately they are transported into an extraction vessel in a pure, solvent-free and solid form. 38 Furthermore, ILs in the presence of CO 2 expand their applicability, as their melting point and viscosity decrease, thus, promoting mass transportation. 39 Consequently, the combination of ionic liquids with scCO 2 has found application in several catalytic processes, such as hydroformylations, hydrogenations or carboxylations of alkenes in IL-scCO 2 biphasic reaction media. [40][41][42][43] In the IL-scCO 2 reaction systems, the reactants and products are carried by the scCO 2 and IL is used as a reaction media. 44,45 Additionally, it is demonstrated that IL-scCO 2 biphasic systems avoid cross-contamination of the extracted solute. 38,46 Until now, IL-based pre-treatment and subsequent SFE (IL-SFE) for natural products has not been described, although ideal conditions arise from the unique properties of both media. Hence, by comparing IL-scCO 2 extraction with the utilization of both applications individually or to traditional solvent-extraction, the IL-scCO 2 approach is preferable. To begin with, less additional preparation, e.g., filtration of the raw material and consequent evaporation of solvents or separation of IL from the organic solvent is required to obtain a solvent-free and solid extract (Fig. 2). Consequently, there is a lower chance of loss of product or impurities, due to less post processing steps. On the other hand, IL-SFE is performed without additional co-solvents, therefore it reduces further solvent consumption and leads to lower expenses. Ultimately, if chosen appropriately, the ionic liquid can be recovered and re-used to improve the sustainability of the extraction process.</p><p>Recently, an investigation of the extraction of cannabidiol with the aid of ILs has been published; however, isolation of cannabidiol required tedious back-extraction with organic solvents or with an aqueous AgNO 3 solution. 47 To the best of our knowledge, no data has been reported thus far regarding a combined extraction process that takes advantage of the complementing properties.</p><p>Herein, we present the first application of IL-SFE from industrial hemp of six cannabinoids (Δ 9 -THC, THCA, CBD, CBDA, CBG and CBGA). Several parameters during the IL-assisted pretreatment, such as time, temperature and dilution with H 2 O, were investigated. In addition, pressure and temperature during SFE were evaluated. Ultimately, the optimized process for 1-ethyl-3-methylimidazolium acetate ([C 2 mim][OAc]) was additionally performed with choline acetate ([Ch][OAc]) and 1-ethyl-3-methylimidazolium dimethyl phosphate ([C 2 mim][DMP]) to compare the extraction efficiency of the investigated cannabinoids. In addition, the developed extraction process is complemented by a simple ionic liquid recovering process without the usage of additional organic solvents.</p><!><p>The focus of this research was the investigation and optimization of various parameters for the extraction of CBD, CBDA, Δ 9 -THC, THCA, CBG and CBGA from partially pre-dissolved hemp in various room-temperature ILs with supercritical CO 2 . The optimization was divided into three successive stages (Scheme 1). In the first stage, the pre-treatment conditions to digest and partially dissolve hemp using [C 2 mim][OAc] before SFE were investigated. The lignocellulosic composition of hemp hurds is reported to contain 43.0% cellulose, 24.4% lignin and 29.0% hemicellulose. 48 ILs are known to dissolve a variety of carbohydrates, e.g., cellulose, by combining strongly basic anions (e.g., Cl − or OAc − ) with various cations. [49][50][51] In particular, [C 2 mim][OAc] was selected in this study as it was used to pre-treat various lignocellulosic biomasses 52 and it is known to effectively dissolve, hemicellulose 53 and lignin. 54 . Both ILs are liquid at room temperature, non-halogenated and hydrophilic. Moreover, both ILs have been reported for pre-treatment of biomass. 55,56 In addition, the positive rating of choline-based ILs in terms of toxicity and biodegradation renders them ideally suited for natural product extractions. 57,58 Pre-treatment with ionic liquid (Stage 1)</p><p>Herein, the influence of temperature and time for the partial dissolution of Cannabis sativa L. in [C 2 mim][OAc] before the scCO 2 extraction is evaluated.</p><p>Initially, the conditions to partially dissolve industrial hemp in [C 2 mim][OAc] were investigated in experiments 1-4 (Table 1).</p><p>Therefore, the pre-treatments were carried out at S1 and S2 †).</p><p>The cannabinoids CBD and CBDA are predominantly accumulated in industrial hemp compared to THC, THCA, CBG and CBGA, which are considered minor compounds.</p><p>The pre-treatment with [C 2 mim][OAc] of industrial hemp at 25 °C and 70 °C indicated comparable cannabinoid yields. Increasing the time from 15 to 60 min at 70 °C in exp. 2 led to a small decrease of roughly 5% ∑(CBD) and 8% ∑(THC). However, similar ∑(CBD), but significantly more CBD (6.58 mg g −1 ) and less CBDA (6.3 mg g −1 ) at 60 min, was yielded in exp. 2 compared with exp. 4 (15 min), which led to 5.29 mg g −1 CBD and 8.8 mg g −1 CBDA, respectively ( p < 0.05, Fig. 3, Table S2 †). It was reported that an extraction process including [C 6 mim][NTf 2 ] at 60 °C and 50 min leads to high amounts of CBD and that the IL preserves CBD, 47 which correlates with the observations herein. In addition, the decarboxylation of cannabinoids at higher temperatures for longer times has been described before. 8 The IL [C 6 mim][NTf 2 ] was not utilized in this study, as the anion [NTf 2 ] − renders it is less suit- able to dissolve cellulose compared to the basic [OAc] − or [DMP] − and similarly, the longer alkyl side chain of the cation would be disadvantageous for this purpose. 59 Ultimately, [NTf 2 ] − was not considered for the extraction process, as it is hydrophobic and not mixable with H 2 O and thus, not suitable for the IL recovering process shown in here. A total time of 15 min instead of 60 min seems to be sufficient to release the investigated cannabinoids from the plant tissue with [C 2 mim][OAc] and hence, allows a significantly shorter pre-treatment time The highest cannabinoids yields were obtained at 70 °C for 15 min in exp. 4, namely 13.6 mg g −1 ∑(CBD), 0.513 mg g −1 ∑(THC)and 0.247 mg g −1 ∑(CBG) (Table 1).</p><!><p>Optimization of temperature and time during the pre-treatment was performed with a constant ratio of 1 : 2</p><p>Here, the influence of several IL : H 2 O ratios was investigated and compared with the sole use of IL as well as pure H 2 O in the extraction vessel (Table 2 and Fig. 4).</p><p>A decrease of water in the IL : H 2 O ratio from 1 : 2 in exp. 4 to 1 : 1 in exp. 5 led to a significant reduction of ∑(CBD) as well as ∑(THC) yield (Table 2) at 20 MPa and 70 °C. However, the significantly highest yield of CBD (7.45 mg g −1 ) of all performed IL-SFE was obtained under these conditions in exp. 5 ( p < 0.05) and additionally, low yields of CBDA (1.09 mg g −1 ) and no CBGA were extracted (Fig. 4, Table S2 † 60 Therefore, H 2 O was added to the IL after the initial pre-treatment. The addition of H 2 O resulted in a reduction of the mixture's viscosity, and thus improved mass transport. 60 It is reported that the viscosity of [C 2 mim][OAc] is reduced by 50% when mixed with 10 wt% H 2 O and that the IL is less viscous at higher temperatures. 61 Lower viscosity of the IL : H 2 O mixture led to higher yields, possibly due to the higher mobility of dissolved cannabinoids and better penetration of scCO 2 . An increase in carboxylated cannabinoids was observed by adding more water (Fig. 4). Furthermore, water is the only solvent without any negative impacts on the environment. Additionally, it is reported to have low solubility in scCO 2 62 and therefore less potential con- 0.260 mg g −1 ( p < 0.05, Table 2). In particular, the use of H 2 O alone tends to yield fewer neutral cannabinoids (Fig. 4), which verifies what has previously been reported; ILs preserve neutral CBD. 47 When comparing exp. 8 with exp. 4, even though the same total quantity of liquid was added in the high-pressure vessel, significantly less yields of ∑(CBD) by 12% and ∑(THC) by 27% are observed ( p < 0.05, Table 2, Fig. 4) in the sole water-based SFE extraction. Therefore, a pre-treatment with IL to liberate the cannabinoids from the plant tissue and subsequent dilution with H 2 O positively affects the yield. Ultimately, a reference scCO 2 extraction in the absence of both IL and H 2 O in exp. 9 (no pre-treatment) yielded 10.1 mg g −1 ∑(CBD), 0.355 mg g −1 ∑(THC), 0.196 mg g −1 ∑(CBG) at 70 °C and 20 MPa (Table 2). Thus, IL-SFE with [C 2 mim][OAc] : H 2 O 1 : 3 in exp. 6 and 1 : 2 in exp. 4, led to significantly higher yields of ∑(CBD, THC, CBG) than sole SFE ( p < 0.05). It has been reported that the cannabinoid yields during SFE can be enhanced by adding EtOH as a modifier. 26,28 In preliminary studies SFE with EtOH as a modifier at different temperatures and vol% EtOH as well as various conventional ethanolic extractions were carried out with another batch of industrial hemp. High yields of the targeted cannabinoids were obtained at 35 °C, 10 MPa and 120 min dynamic extraction with 10 and 20 vol% EtOH. In comparison to the performed conventional extraction, similar ∑(THC) yields, but less ∑(CBD) and ∑(CBG) were yielded ( p < 0.05, Table S3 †). All data is presented in the ESI. †</p><p>The addition of EtOH as a co-solvent to IL-SFE would lead to the extraction of both IL and cannabinoids, thus, leading to impurities in the extract. In particular, IL-SFE does not require the use of a co-solvent to obtain cannabinoids in high yields, avoiding further solvent consumption.</p><p>Hence, the highest extraction yields were obtained with a IL : H 2 O ratio of 1 : 3 in exp. 6, which achieved 15.6 mg g −1 ∑(CBD), 0.542 mg g −1 ∑(THC) and 0.335 mg g −1 ∑(CBG) (Table 2).</p><!><p>Apart from the optimization of pre-treatment conditions and the ratio of [C 2 mim][OAc] to H 2 O, temperature and pressure during SFE were investigated (Table 3).</p><p>Initially, the pressure was reduced from 20 MPa in exp. 6 to 15 MPa in exp. 11 at 70 °C and led to a significant reduction by 17% ∑(CBD) to 13.00 mg g −1 , 16% ∑(THC) to 0.457 mg g −1 and 23% ∑(CBG) to 0.245 mg g −1 ( p < 0.05, Table 3). After further decreasing the pressure to 10 MPa in exp. 10, a significantly diminished yield of 3.66 mg g −1 ∑(CBD), 0.0885 mg g −1 ∑(THC), and 0.045 mg g −1 ∑(CBG) was observed (Table 3). Even though lower cannabinoid yields were obtained at 10 MPa and 70 °C in exp. 10, the extraction of neutral cannabinoids was favoured (Fig. 5). In literature, sole scCO 2 extractions yield neither CBD nor CBDA at 10 MPa at 70 °C for 120 min, 63 but SFE can be improved upon by adding EtOH 26 or by the pre-treatment with IL, as herein reported. In addition, the pressure was increased to 30 MPa at 70 °C in exp. 12, which resulted in comparable yields of ∑(CBD, THC, CBG) as IL-SFE at 20 MPa in exp. 6 (Table 3). It can be assumed that 20 MPa at 70 °C are sufficient to extract cannabinoids during IL-SFE.</p><p>Furthermore, the temperature was lowered to 35 °C at 20 MPa during SFE in exp. 13. This led to comparable yields of ∑(CBD) and ∑(CBG), but significantly lower ∑(THC) yields (0.493 mg g −1 ) compared to 70 °C in exp. 6 ( p < 0.05, Table 3). This corresponds to literature data, where similar yields of ∑(CBD) were extracted during SFE at 35 °C and 70 °C at 50 MPa. 63 Lower temperatures are known to reduce the viscosity of H 2 O and additionally, have been reported to decrease the viscosity of [C 2 mim][OAc]. 61 Hence, the mixture is less pene- trable for scCO 2 to extract the target cannabinoids. In comparison of exp. 13 and exp. 6, the yields of decarboxylated cannabinoids decreased significantly (CBD by 28%; Δ 9 -THC by 16%; CBG by 33%) and similar yields of THCA and CBDA, but significantly more CBGA by 23% was obtained in exp. 13 ( p < 0.05, Table S3 †).</p><p>A further decrease from 20 MPa at 35 °C in exp. 13 to 10 MPa in exp. 14 led to a slight reduction in ∑(CBD) by 8% and ∑(THC) by 5%, and significant reduction in ∑(CBG) by 23% ( p < 0.05, Table 3). Therefore, a combination of 35 °C and 10 MPa seems to affect the total cannabinoid yield negatively, but increasing the temperature to 70 °C at the same pressure further reduces the yields. Lower CBD and CBDA yields at 10 MPa at 70 °C compared with 35 °C during SFE have been described in literature. 63 Thus, 10 MPa at 70 °C during SFE seem to be unfeasible to extract cannabinoids from industrial hemp. At 20 MPa, the temperature seems to have a minor effect on the total yields of cannabinoids.</p><p>Consequently, the optimum cannabinoid yields were obtained at 20 MPa and 70 °C in exp. 6 during supercritical CO 2 extraction. 4 and Fig. 6).</p><!><p>Ionic liquid assisted SFE with [Ch][OAc] in exp. 15 yielded comparable yields of ∑(CBD) (15.4 mg g −1 ) and ∑(THC) (0.535 mg g −1 ), but significantly more ∑(CBG) (0.401 mg g −1 ) than IL-SFE with [C 2 mim][OAc] in exp. 6 ( p < 0.05, Table 4). The change of cation does affect the yields of cannabinoids, however, the role of the cation during the dissolution of lignocellulose structure is not yet fully understood. 64 On the other hand, anions, such as [OAc] − , are described to effectively support the dissolution of cellulose by forming hydrogen bonds. 34 To investigate the influence of the anion in IL-SFE of cannabinoids from industrial hemp, the imidazolium-based IL [C 2 mim][DMP] was used in exp. 16. This resulted in a significant reduction of ∑(CBD) to 11.8 mg g −1 and total THC to 0.449 mg g −1 compared with the acetate-based ILs in exp. 6 and exp. 15 ( p < 0.05, Table 4). [C 2 mim][DMP] is described as effectively dissolving biomass, but has a high viscosity, 56,65 which could affect the extraction at supercritical conditions, due to the weaker penetration of scCO 2 . Nonetheless, phosphate based and acetate based IL-SFE yielded higher amounts of ∑(CBD, THC, CBG) compared with sole supercritical CO 2 extraction without IL pre-treatment (Fig. 6).</p><p>The following mechanism can be proposed for IL-SFE. Firstly, the biomass is partially dissolved by breaking down the lignocellulose structure of the industrial hemp powder. This depends on the anion and cation of the ILs. 34,64 The cannabinoids are released from the plant tissues and the IL possibly stabilizes them. 47 Secondly, the water is added, which reduces the viscosity of the mixture 61 and lowers the solubility of the target cannabinoids. Due to the lower surface tension and higher mobility of cannabinoids, a higher mass transfer between the scCO 2 phase and the IL : H 2 O phase is generated. As reported the scCO 2 dissolves in ILs, however, neither the IL nor the H 2 O does dissolve in scCO 2 . 38,62 Finally, these synergic effects allow the scCO 2 to extract the targeted cannabinoids, due to better solubility in the supercritical phase without contaminating it with IL or H 2 O. Thus, no further organic solvents are necessary to purify the compounds from the IL phase and consequently, no additional work up is needed to obtain a solid and solvent free product (Fig. 2). Ultimately, IL-SFE was compared with reference solvent extraction (exp. 17-19). Ethanol is one of the most commonly used solvents to extract cannabinoids. 66 Herein, a conventional extraction for 2 h, at 70 °C, with EtOH in exp. 17, sufficiently extracted the investigated cannabinoids; however, employing H 2 O in exp. 19 alone under the same conditions, low yields of cannabinoids were obtained (Table 4). A control extraction in EtOH for 24 h was carried out in exp. 18 to investigate the influence of longer extraction times. Longer times at high temperatures seem to degrade carboxylated cannabinoids significantly, reducing CBDA by 52%, THCA by 65% and CBGA by 53% ( p < 0.05, Table S2 †). The decarboxylation of cannabinoic acids at high temperatures for longer times is described in literature. 8 However, it can be reported that the degradation over time does not affect the overall cannabinoid yields.</p><p>By comparing the two-hour ethanolic extraction (exp. 17) with acetate based ionic liquid-SFE, several differences can be observed. Firstly, the yields of ∑(CBD) by SFE with [C 2 mim][OAc] in exp. 6 and [Ch][OAc] in exp. 15 are slightly higher but comparable to the 2 h ethanolic extraction (Table 4). Secondly, significantly more ∑(THC) with 4). Hence, the results underline the importance of appropriately selecting the IL cation and anion, as well as the optimal extraction parameters for IL-SFE to extract cannabinoids from industrial hemp. Ultimately, [Ch][OAc] based SFE yields high amounts of the investigated cannabinoids and also provides environmental and economic benefits. Not only is [Ch][OAc] biodegradable, but it is also considered relatively cheap (88 € for 25 g), easy to synthesize, as well as less toxic compared to other ionic liquids. 57,58,67,68 Furthermore, no co-solvents are applied during IL-SFE, which avoids additional solvent consumption and consequently leads to a purer, solid extract (Fig. S2 †). Ultimately, all three ILs were purified without any additional use of organic solvents. Neither water nor significant impurities were detected by NMR spectroscopic analysis for the purified ILs (Table S4 and Hence, the type of IL is of great importance and affects the cannabinoid yield significantly. However, not only the type of IL needs to be selected carefully, also the SFE parameters. In dependence of various parameters, e.g. IL pre-treatment temperature or the ratio of IL : H 2 O during SFE, it is possible to adjust the proportion of carboxylated and decarboxylated cannabinoids in the extracts. In addition, IL-SFE allows extracting cannabinoids in highest yields and, therefore, it can be reported as a novel competitive alternative to traditional extraction techniques or supercritical fluid extraction with co-solvents. Ultimately, the ILs can be recycled without additional usage of further organic solvents to reduce costs and improve the sustainability of the process. IL-SFE offers the opportunity to extract secondary metabolites from different natural sources without volatile organic solvents and the presented process has great potential for future industrial applications.</p><!><p>The type III chemovar Futura 75 was cultivated in Austria, in the fields of Biobloom (Apetlon, Austria, 7°41′23.4″N 16°56′ 26.7″E), in September 2020. After the harvest, the plants (flowers, leaves and stems) were stored under mild conditions at 40 °C for 14 h. The samples were milled with a Fritsch Universal Pulverisette 19 mill through a 2 mm sieve (Fritsch, Oberstein, Germany). The dry matter was 94.73 ± 0.05 wt% (n = 3). A second batch of the same industrial hemp harvested in 2019 was used for the preliminary experiments, mentioned in section Results and discussion. The dry matter was 93.68 ± 0.03 wt% (n = 3). The hemp raw material was stored in the dark, at −20 °C, between experiments.</p><!><p>For pre-treatment, a high-pressure vessel of approximately 50 mL (EV-3), produced by Jasco (Jasco Corporation, Tokyo, Japan), containing one input and one output connections on the lid, was used. The batch reactor was charged with 0. The SFE setup is presented in Fig. 7. All extractions were performed with a scCO 2 device manufactured by Jasco (Jasco Corporation, Tokyo, Japan). Liquid CO 2 (>99.995% purity; with ascension pipe; Messer GmbH, Vienna, Austria) was pressurized by two CO 2 -pumps (PU-2086, Jasco Corporation, Tokyo, Japan) with cooled heads (CF40, JULABO GmbH, Seelbach, Germany). An oven (CO-2060, Jasco Corporation, Tokyo, Japan) with a heating coil was used and was thermostated to the desired temperature. The vessel containing the IL pre-treated hemp was placed on a heating mantle set to a certain temperature and a stirring rate of 500 rpm and, subsequently, connected to the supercritical carbon dioxide (scCO 2 ) device. A back-pressure regulator (BP-2080, Jasco Corporation, Tokyo, Japan), a gas/liquid separator (HC-2086-01, Jasco Corporation, Tokyo, Japan), and a product collector (SCF-Vch-Bp, Jasco Corporation, Tokyo, Japan) were used to obtain the extracts.</p><p>The conditions employed for the SFE of cannabinoids were based on literature data 63,69 and adapted for our purposes.</p><!><p>For comparison, conventional solvent extractions were performed in 30 mL Teflon screw cap vials. The hemp quantity used in each extraction was 0.2 g. Two extractions were performed in triplicate using 2 mL solvent, more precisely, H 2 O and EtOH, for 2 h at 70 °C and a third one, also in triplicate, using 10 mL EtOH for 24 h and 70 °C. 70</p><!><p>After extraction, the scCO 2 device was depressurized, the metallic extraction reactor was disconnected and brought to room temperature. The IL-water-hemp mixture (Fig. S3 †) was filtered to remove hemp particles, the water was evaporated in vacuo and the remaining ionic liquid was dried under vacuum (0.65 mbar) for 24 h. Afterwards 20 mg of purified IL (Fig. S4 †) were dissolved in chloroform-d 3 (Sigma Aldrich, St Louis, USA) and a 1 H-NMR was recorded with a 400 MHz Bruker Advanced Ultra Shield 400 spectrometer (Bruker, Billerica, USA). Spectroscopic data and NMR spectra are given in the ESI (Table S4 and Fig. S5-7 †)</p><!><p>The determination of CBDA, CBD, CBGA, CBG, THCA, Δ 9 -THC, was carried out on a High-Performance Liquid Chromatography (HPLC) in a Dionex UltiMate© RSLC System, with DAD-3000RS Photodiode Array Detector (Thermo Scientific, Germering, Germany), on a Dionex Acclaim™ RSLC 120 C18 (2.2 µm, 120 Å, 2.1 × 150 mm, Bonded Silica Products: no. 01425071, Thermo Scientific, Germering, Germany). A mobile phase flow rate of 0.2 mL min −1 was employed and the oven temperature was set to 25 °C. As a mobile phase, H 2 O with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B) were used. The following gradient was carried out: 2 min of pre-equilibration at 70% B, 6 min hold at 70% B, 6 min from 70% B to 77% B, 18 min hold at 77% B, 0.5 min from 77% B to 95% B, 1.5 min at 95% B, 0.5 min from 95% B to 70% B, and 5 min at 70% B. 71 Acetonitrile was purchased from VWR Chemicals (Radnor, PA, USA) and formic acid from Merck (Darmstadt, Germany). All solvents for HPLC were of analytical grade.</p><p>The cannabinoid standards CBD, CBDA, THCA, Δ 9 -THC, CBG and CBGA were provided by Medical Cannabinoids Research and Analysis GmbH (Brunn am Gebirge, Austria) in the course of previous joint research. A mixed cannabinoid stock solution (1 mg mL −1 ) in MeOH of the investigated cannabinoids diluted for calibration.</p><!><p>Statistical data analysis was performed with Origin 2021. Oneway ANOVA for multiple groups, followed by Tukey honestly significant difference (HSD) post hoc test at the 0.05 significance level, was carried out.</p><!><p>The authors would like to point out that the focus of this study was the extraction of cannabinoids as a class, not THC specifically. Any THC extraction is purely incidental, and bound to be negligible, given that industrial hemp was used, which in the EU must have a THC content not in excess of 0.2%.</p><p>The relevant EU law can be perused under: https://eur-lex. europa.eu/legal-content/EN/TXT/HTML/?uri=CELEX:32013R13 07&from=de.</p><p>In particular, we refer to Article 32, paragraph 6. Additionally, the authors do hold a licence to for the purposes of research, in accordance with Austrian law, available under:</p>

Royal Society of Chemistry (RSC)

REDUCTION OF PM2.5 TOXICITY ON HUMAN ALVEOLAR EPITHELIAL CELLS A549 BY TEA POLYPHENOLS

Tea-derived polyphenols have anticancer and antioxidant properties, and they can regulate oxidative stress. This study was designed to quantify both the toxic effects of fine particulate matter with aerodynamic diameter less than 2.5 \xce\xbcm (PM2.5) and determine whether tea polyphenols could provide a protective effect against PM2.5 toxicity on human alveolar epithelial A549 cells in vitro. Cytotoxic effects of the PM2.5 on A549 cells were measured by means of cell viability, the expression of caspase-3, bax/bcl-2 and C/EBP-homologous protein (CHOP), and the generation of intracellular reactive oxygen species, malondialdehyde and superoxide dismutase. The results showed that tea polyphenols ameliorated some of the adverse effects of PM2.5 on A549 cell viability and superoxide dismutase levels. In addition, tea polyphenols decreased the production of reactive oxygen species, malondialdehyde generation, and apoptosis in response to PM2.5 exposure. Therefore, our results support a role for tea polyphenols in reducing the toxicity of PM2.5, particularly with regard to targeting oxidative stress and apoptosis.

reduction_of_pm2.5_toxicity_on_human_alveolar_epithelial_cells_a549_by_tea_polyphenols

INTRODUCTION<!>Preparation of PM2.5<!>Cell Culture and Treatment<!>Cell Viability Assay<!>Measurement of Intracellular ROS level, SOD activity and MDA Levels<!>Apoptosis Detection Assay<!>Western Blot Analysis for Cleaved Caspase-3, Bax/Bcl-2 and CHOP<!>Statistical analysis<!>PM2.5 and Tea Polyphenols Cytotoxicity<!>Tea Polyphenols Reduce Oxidative Stress Induced by PM2.5 in Lung Cells<!>Tea Polyphenols Reduce Cell Apoptosis Level after PM2.5 treatment<!>DISCUSSION

<p>PM2.5 is a major environmental pollutant that has become a significant threat to human health. The World Health Organization maintains an ongoing database identifying global exposure rates for fine particulate matter (PM2.5) and their data suggests that a majority of the human population that live in cities have been exposed to PM2.5 to varying degrees (http://www.who.int/gho/phe/air_pollution_pm25_concentrations/en/). Moreover, extensive epidemiological studies have identified PM2.5 exposure as a major variable associated with increased morbidity and mortality for cardiopulmonary and respiratory diseases, such as cerebrovascular disease, stroke, heart rhythm disturbances, chronic obstructive pulmonary disease, and respiratory tract infection (Leiva et al., 2013; Bell et al., 2014; Karlsson et al., 2005; Pardo et al., 2015). One reason for the danger associated with PM2.5 exposure is based on its size; with a diameter less than 2.5 μm, these particles are able to penetrate deeply into the alveolar tissue of the respiratory tract. The alveolar duct cells and adjacent capillary beds are, therefore, vulnerable to PM2.5 and this may constitute a critical route of physiological challenge associated with the exposure risk of PM2.5 and the subsequent adverse effects for human health (Bell et al; 2010; Guo et al., 2015; Loxham et al., 2015).</p><p>Current standard practice for protecting individuals against PM2.5 exposure have largely utilized barrier or filtration approaches to try and reduce particle inhalation. Outdoor interventions primarily involve reduced exposure time and/or the use of facial masks as a primary inhalant barrier while efforts to reduce indoor PM2.5 exposure largely rely on central air conditioner and airflow systems in buildings to filter out particles (Cai and He, 2016). One study demonstrated the potential of using polyacrylonitrile transparent materials associated with windows to increase filtration efficiency for particulate matter, including PM2.5 (Liu et al., 2015). Despite these precautions, inhalation of PM2.5 is often unavoidable, particularly during cold, seasonal weather (Hu et al., 2017; Tahri et al., 2017). If the PM2.5 is inhaled, there is the potential for injury to the lung cells, and possibly the associated capillaries (Xu et al., 2017).</p><p>The hazardous effects of PM2.5 particles are largely caused by its small size, but composition of the particles may play a role as well. Indeed, composition can vary depending on source location as well as temporal and regional variation (Villar-Vidal et al., 2014; Akhtar et al., 2014; Sun et al., 2014; Liang et al., 2017). Because of their small size and high surface area/volume ratio, PM2.5 have been shown to be able to penetrate into mucous-lined epithelial cells in culture, along with much smaller ultrafine particles (Loxham et al., 2015). Moreover, the PM2.5 often contain toxic components such as polycyclic aromatic hydrocarbons (PAHs; Oh et al., 2011) and transition metals (Al, Pb, Cu, etc.; Rodríguez-Cotto et al., 2014) that increase the potential toxicity of PM2.5. One of the primary mechanisms whereby PM2.5 appears to mediate cellular toxicity is via its ability to increase oxidative stress in a cell or tissue system. Oxidative stress is one indicator of current or predictive tissue damage and oxidative stress is generally characterized by an increase in the levels of reactive oxygen species (ROS) and a concomitant reduction in the activity of superoxide dismutase (SOD; Cachon et al., 2014). Therefore, this research focuses on examining a potential treatment method, tea polyphenols, and their ability to ameliorate the toxic effects of PM2.5 exposure.</p><p>Polyphenols derived from tea extracts have been characterized for their potential health benefits in managing oxidative stress (reviewed in Mao, 2017; Yiannakopoulou, 2013; Saeed et al., 2017; Malongane et al., 2017) and for their ameliorating effects in a variety of human diseases and animal models of human diseases (Paola et al., 2005; Xie et al., 2012; Lorenz, 2013; Renno et al., 2017). The current study focuses on the antioxidant capacity of tea polyphenols to treat or prevent apoptosis caused by PM2.5 exposure in a lung culture model. Herein, we quantified changes in two toxicity metrics, oxidative stress indicators and apoptosis, and quantified the ability of tea polyphenols to ameliorate the damage induced by PM2.5 exposure in lung cells.</p><!><p>PM2.5 samples from Shijiazhuang city were collected in an area known for heavy vehicular traffic with a high exposure to diesel exhaust. PM2.5 samples were collected on glass filters (diameter = 90 mm, Whatman, USA) using a high volume sampler (average collection rate at 24 L/min, Beijing Geology Device Company, China) for 12 hours (8:00–20:00). The glass filters were preheated at 500 °C for 4 hours before sampling, and weighed on a microbalance (Mettler Toledo, USA) to measure atmospheric daily PM2.5 concentration collected. All sample filters were stored in the dark at −20 °C before further chemical and physical characterization to avoid light-based sample degradation or contamination. Unexposed filters were used as a control and were prepared in parallel using the same methods. The PM2.5 samples were extracted from sample filter strips or control strips by immersing them in methanol:deionized water (v: v=4:1) and sonicating for 60 min at room temperature. The sonicated methanol:water mixture constituted the extracted material and these samples were then incubated at 65 °C for 12 hours to dry the material. The dried samples were suspended in serum free culture medium containing 0.1% DMSO and stored at 4 °C.</p><!><p>Human lung carcinoma cell line A549 (ATCC CCL-185) was grown and maintained in Dulbecco's modified Eagle medium (DMEM, Sigma–Aldrich, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 2 mmol/L glutamine, and 100 IU/mL penicillin, 100 μg/mL streptomycin. During the whole process, growing cells were cultured at 37°C, in a humidified environment containing 5% CO2. The A549 cells were seeded onto 60 mm tissue culture dishes and allowed to adhere for 18 hours. The media was removed and replaced with either control media or media containing PM2.5 for 24 hours. After the PM2.5 was removed, tea polyphenol was added to the media for 24 hours. Tea polyphenols were purchased as powder and added at the defined concentrations indicated after solubilization in water (Shanghai Jonln Reagent Company, Shanghai, China).</p><!><p>The effect of PM2.5 and tea polyphenols on cell viability was measured using the Cell Counting Assay Kit 8 (CCK-8, Yeasen, China) according to the manufacturer's protocol. A549 cells were seeded at a density of 2×103 cells/well into 96-well flat-bottomed plates and allowed to adhere overnight (18 hours). Cells were then exposed to 0.1 – 500 μg/mL of PM2.5 for 24 hours and 0.1 – 100 μg/mL of tea polyphenols for 24 hours, in series. At the end of the assay time period, 10 μL of CCK-8 developing solution was added to each well and incubated for 2 hours at 37°C. The proportion of viable cells was determined as a function of absorption at 450 nm (Bio-Tek Microplate Reader, USA).</p><!><p>The assays to measure ROS, SOD, and MDA were carried out by reactive oxygen species assay kit, total superoxide dismutase (T-SOD) assay kit and catalase (cat) assay kit (Jian Cheng Bioengineering Institute, Nanjing, China), respectively, according to the manufacturers' protocols. The assays were set up as described for the viability assay above. At the end of the assay period, the cells were washed with phosphate buffered saline solution (PBS) and incubated with 10 μmol/L 2′,7′-dichlorofluorescin diacetate (DCFH-DA) in DMEM medium in a CO2 incubator at 37°C for 30 minutes. The DCFH-DA is de-esterified intracellularly and produces a measurable green fluorescent oxidation product in the presence of ROS. After the 30-minute incubation, the cells were washed again with PBS to remove excess DCFH-DA and 1.0 mL of PBS was added to each well. The fluorescence intensity as a reflection of intracellular ROS levels was quantified using a FACS Calibur flow cytometer (BD Biosciences, CA, USA), with an excitation wavelength set at 488 nm and an emission wavelength of 525 nm. Similar assay approaches were used to quantify SOD activity and the level of MDA product, using absorbance wavelengths set at 550 nm 532 nm, respectively, measured on a standard microplate reader (Bio-Tek, USA).</p><!><p>The levels of apoptotic cells in the different treatment conditions were determined using double labeling for Annexin V and propidium iodide (PI) (Annexin V/PI assay kit, BD bioscience, US) and detection with flow cytometry according to the manufacturer's protocol. The A549 cells plated at a density of 1 × 105 cells/well in 6-well plates and then challenged with 0.1, 1 and 10 μg/mL PM2.5 followed by tea polyphenol treatment as described previously. At the end of the assay, the cells were harvested, washed with cold PBS, and incubated in the dark with 1× Annexin V working solution containing PI (1 μg/mL final concentration) for 5 min at room temperature. The cells were exposed to binding buffer according to the manufacturer's recommended protocol (400 μL of 1× binding buffer and sample cells). The proportion of apoptotic cells was determined using CellQuest software on a flow cytometer with gates set at default sensitivity to determine single and double-labeled populations. The cell number was set at 2×104 per test.</p><!><p>The A549 cells were plated in 6-well plates and then challenged with PM2.5 followed by tea polyphenol treatment, as described previously. At the end of the treatments, A549 cells were washed once with PBS and then lysed with radioimmunoprecipitation assay (RIPA) buffer containing 1% phenylmethylsulfonylfluoride (PMSF) for 5 minutes on ice. The whole-cell lysates were centrifuged at 12,000 rpm for 5 min at 4 °C, and the supernatants were collected. Protein concentrations were determined by bicinchoninic acid assay against a standard curve (BCA/Smith Assay, Jian Cheng Bioengineering Institute, China). Equal amounts of protein (10 μg) were separated by electrophoresis on 10% sodium dodecyl sulphate- (SDS-) polyacrylamide gels and transferred onto 0.22 μm polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with 5% (w/v) non-fat milk powder in Tris-buffered saline containing 0.1% (v/v) Tween-20 (TBST) for 90 min to block nonspecific binding sites on the membrane. The PVDF membranes were then incubated overnight at 4 °C with diluted primary antibodies (caspase-3 antibody, bax antibody, bcl-2 antibody and CHOP antibody, Cell Signaling, US). After extensive washing with TBST, the membranes were incubated for 90 min at room temperature with the horseradish peroxidase conjugated anti-rabbit secondary antibodies (Cell Signaling, US). After being rewashed with TBST, the bands were developed by enhanced chemi-luminescence detection reagents kit (Beijing Solarbio Science & Technology Co., Ltd, Beijing, China).</p><!><p>The data were analyzed using a One Way ANOVA analysis (SPSS 21.0) with treatment condition as the dependent variable and the significance set at the 0.05 level. When both PM2.5 and tea polyphenol treatments were included in an assay, the results were analyzed with Two Way ANOVA, the post hoc test used was Tukey HST and the P values were provided in the figure legends and indicated on the graphs.</p><!><p>Since PM2.5 has considerable toxic impact based on the composition of the chemical components and the ability of the fine particulate matter to penetrate deep into the lung alveolar buds, we wanted to determine the adverse effects of PM2.5 on cultured human lung cells. We treated A549 human lung carcinoma cells with a range of PM2.5 doses from 0.1 to 500 μg/mL for 24 hours (Fig. 1). We observed statistically significant, concentration-dependent reduction in cell viability determined via the CCK-8 assay with the highest doses resulting in nearly 100% cell loss. Even at a concentration of 1 μg/mL PM2.5, cell viability was 77.25 ± 4.88 % relative to control cells. Based on these results we chose to use PM2.5 at a concentration of 50 μg/mL which resulted in ~50% cell viability. Our rationale for this concentration was to use a mid-point effective dose that would result in a damaged, but not a crashing cell population.</p><p>In order to investigate tea polyphenols as a potential therapeutic intervention for PM2.5-induced toxicity in lung cells, we first needed to identify a dose that would allow for an effective range without having a secondary detrimental impact on the cells. While tea polyphenols have been shown to be beneficial to human health and nutrition, some reports indicate adverse effects at high tea polyphenols concentrations in kidney and liver cells (Yang et al., 1998; Murakami, 2014; Inoue et al., 2011) with both anti-oxidant and pro-oxidant effects reported, depending on cell type and dose (reviewed in Lorenz et al., 2013). Therefore, we treated A549 cells with a range of tea polyphenol from 0.1 μg/mL to 100 μg/mL and then quantified cell viability in order to identify an optimal concentration for our assays (Fig. 2). Tea polyphenol concentrations at 20 μg/mL and higher significantly reduced cell viability with 100% cell loss observed at 80 μg/mL and 100 μg/mL. This result demonstrated that tea polyphenols have cytotoxicity at high concentrations in agreement with the results of others (Murakami, 2014; Inoue et al., 2011). Based on our cell viability assessment, we chose to use the non-toxic concentration of tea polyphenols in the range of 0.1 to 10 μg/mL for subsequent experiments.</p><!><p>Since one of the proposed mechanisms of action for PM2.5 toxicity is via oxidative stress, we anticipated that PM2.5 treatment could stimulate an oxidative stress response in the A549 lung carcinoma cells. We therefore chose to quantify levels of ROS, SOD and MDA as biological metrics of oxidative stress in A549 cells treated with PM2.5. Tea polyphenols have previously been demonstrated to reduce ROS in rat aortic endothelial cells fed on a high fat diet (Zuo et al., 2014). We postulated that a similar protective effect might occur in the A549 cells stressed with PM2.5 treatment. We quantified ROS levels in A549 cells treated with 50 μg/mL PM2.5 for 24 hours and then subsequently treated with 0.1 to 10 μg/mL of tea polyphenols (Fig. 3). PM2.5 induced an increase of ROS in comparison with the control group (shown in Fig. 3, P<0.01). Treatment with 1 μg/mL or 10 μg/mL tea polyphenols reduced ROS levels compared to A549 cells treated with PM2.5 alone (Fig. 3 P<0.01). The ROS levels in the samples treated with tea polyphenols did not reach the levels of ROS in the control, untreated population. However, obviously, ROS level of tea polyphenols treatment groups was lower than only PM2.5 treatment group, and the treatment effect of 10 μg/mL tea polyphenols was better than 1 μg/mL.</p><p>The activity levels of SOD in a healthy cell are maintained to regulate superoxide production downstream of oxygen metabolism (Griess et al., 2017). We quantified SOD activity levels in A549 cells treated as described for Figure 3 and determined that SOD activity levels were significantly decreased after exposure to PM2.5 for 24 hours (Fig. 4). However, after treatment with tea polyphenols for 24 hours, the SOD activity levels were increased at the higher doses with recovery of control levels observed at 10 μg/mL of tea polyphenols (shown in Fig. 4, P>0.05).</p><p>MDA is one of the primary byproducts of lipid oxidation and its production can be used as a biomarker of oxidative stress (Czerska et al., 2015). We quantified MDA levels in our PM2.5/tea polyphenol treatment paradigm and observed significantly elevated MDA levels in A549 cells treated with PM2.5 compared to the control group (Fig. 5, P<0.001). However, treatment with tea polyphenols after PM2.5 exposure ameliorated the MDA elevation with levels returning to ~90% of control at the 1 and 10 nmol/mg protein doses.</p><!><p>One of the hallmarks of PM2.5 toxicity includes the feature that these particles are able to directly impact cells and induce oxidative stress resulting in apoptosis. (Deng et al., 2013). We wanted to determine whether the tea polyphenols would have a protective effect against PM2.5-induced apoptosis in A549 cells. To that end, we quantified apoptosis levels in our PM2.5 toxicity lung cell model using colabeling for Annexin V/PI (Fig. 6). Based on ROS, SOD activity and MDA quantity level assay, 10 μg/mL of tea polyphenols had effective therapeutic effect as a treatment concentration. The tea polyphenols reduced the apoptotic effect of the PM2.5 significantly at each dose tested and the cell apoptosis quantified was dependent on the tea polyphenol concentration (Fig. 6). PM2.5 at 0.1, 1 and 10 μg/mL induced cell apoptosis up to 10.98 ± 1.13%, 15.76 ± 2.6 %, and 26.50 ± 2.37%, respectively. In contrast, treatment with tea polyphenols reduced the percentage of apoptotic cells to 3.55 ± 0.32%, 9.19 ± 0.68 %, and 18.25 ± 2.20%, respectively. These results support a role for the tea polyphenols in reducing the apoptosis of PM2.5-treated lung cells.</p><p>To further explore the potential mechanism for the protective effect of tea polyphenols on PM2.5-induced apoptosis, we wanted to determine protein levels of caspase-3, bax, bcl-2, and CHOP as key indicators of apoptosis (Cao et al., 2015; Liu et al., 2017). We quantified the bax/bcl-2 ratio and the caspase-3 expression levels in A549 cells treated with PM2.5 alone or PM2.5 followed by tea polyphenol treatment (Fig. 7). PM2.5 induced a significant increase in Caspase 3 and in the ratio of bax/bcl-2 compared with the control groups (P<0.05). However, with tea polyphenol treatment, the protein levels of caspase 3 (Fig. 7B) and bax decreased (P<0.05) while that of bcl-2 was increased (P<0.05) compared to the control group. As a result, the ratio of bax/bcl-2 dropped to control levels in the presence of tea polyphenols (Fig. 7C). CHOP protein levels also increased with PM2.5 treatment (Fig. 8) with CHOP levels reduced to control in the A549 cells that were also treated with tea polyphenols. Taken together our results indicate that PM2.5-induced cytotoxicity as measured by cell viability, oxidative stress assessment, and apoptosis in A549 lung cells was ameliorated by treatment with tea polyphenols.</p><!><p>PM2.5 are a major component of urban pollution and the complex chemical components and particle size of PM2.5 can directly aggravate the respiratory system and cause disease (Leiva et al., 2013; Bell et al., 2014; Karlsson et al., 2005; Pardo et al., 2015). The primary route of exposure is via PM2.5 inhalation into the lungs, resulting in tissue damage or distress that can disrupt normal lung function. Therefore, reducing or preventing inhalation, at the outset, in combination with reducing the end point toxic effects of PM2.5 are important goals with regard to human health and exposure to small particle pollutants. The tea polyphenols have been reported to be effective scavengers of reactive oxygen and nitrogen species and may help keep antioxidant enzyme activities in check in vivo (reviewed in Kim et al., 2000; Frei and Higdon, 2013; Raza and John, 2008). In the current study, we investigated the impact of tea polyphenols on alleviating the cytotoxicity of PM2.5 in a lung cell culture model. Using a variety of methods to assess cell survival and oxidative stress, we have obtained results indicating that tea polyphenols reduce the overall toxicity of PM2.5 on A549 cells.</p><p>PM2.5 exposure and increased oxidative stress have been associated in a variety of model systems, including rat cardiac cells that show increased ROS and subsequent activation of MAP kinase pathways in response to PM2.5 treatment (Cao et al., 2015). In addition, human lung epithelial cells exposed to PM2.5 in culture showed a marked increase in different apoptosis pathways, including increased caspase-3 cleavage, a shift in the ratio of bax to bcl-2 proteins in L132 cells (Dagher et al., 2006), and autophagy in A549 cells (Deng et al., 2013). Given the adverse consequences of PM2.5 on lung cells, it is important to identify treatment paradigms that can help prevent damage after fine particle exposure. In our study, we focused on the expression of caspase-3, bcl-2, bax and CHOP as a reflection of apoptosis. Bcl-2 and bax family members regulate mitochondrial outer membrane permeabilization (MOMP) to regulate apoptosis (reviewed in Kalkavan and Green, 2017). The expression levels of bcl-2 and bax in our model system suggested that the A549 cells responded to PM2.5 with increased apoptosis that might have been mediated, in part, by the mitochondrial apoptosis pathway. Moreover, levels of CHOP protein have been shown to play an important role in the response of the endoplasmic reticulum to stress (Gorman et al., 2012; Li et al., 2014; Liu et al., 2016). The elevation of CHOP linked to PM2.5 exposure was also alleviated by tea polyphenol treatment, indicating that stress effects mediated via the endoplasmic reticulum response pathway may be another target for reducing cell death.</p><p>Taken together, these results demonstrated that tea polyphenols at low concentrations could significantly inhibit oxidative stress and apoptosis induced by PM2.5. The green tea polyphenols contain catechin derivatives such epigallocatechin-3-gallate (EGCG; Renno et al., 2017); theaflavins (Leung et al., 2001) and thearubigins (Menet et al., 2004). Tea polyphenols, especially the derivative epigallocatechin gallate, are currently being explored in a variety of clinical and pre-clinical studies targeting neuropathic pain and cancer (reviewed in Bimonte et al., 2017; Fujiki et al., 2017; Sur et al., 2017; Lernoux et al., 2017). Previous studies have demonstrated that tea polyphenols are able to protect fibroblasts from peroxide-induced damage (Jie et al., 2006) and they can regulate oxidative stress by scavenging free radicals, chelating transition metal ions, and decreasing the accumulation of intracellular ROS (reviewed in Frei and Higdon, 2003; Raza et al., 2008). Moreover, the green tea catechin, EGCG, reduced the apoptosis of a human lung cancer line (PC-9) in combination with a synthetic retinoid, although oxidative stress was not assessed in that particular study (Oya et al., 2017). However, an investigation directed at determining tea polyphenols' effect on mitigating particulate matter toxicity, such as with PM2.5-induced toxicity, has not been reported in lung cells and our research sought to fill this gap in knowledge. Our data support an ameliorative effect of tea polyphenols on regulation of oxidative stress markers (ROS level, SOD activity and MDA level) and apoptosis proteins (caspase-3, bcl-2, bax and CHOP). In conclusion, we present here a novel application for tea polyphenols in reducing the cytotoxicity induced by PM2.5 in lung cells. Tea polyphenols can be extracted from either green tea or black tea (Lee et al., 2017; Seo et al., 2016) and its constituents have been carefully defined (Sang et al., 2011). We suggest that tea polyphenols should be further explored as a cost-effective and accessible method to help alleviate long-term damage to lung cells associated with exposure to PM2.5.</p>

PubMed Author Manuscript

A stepwise dechlorination/cross-coupling strategy to diversify the vancomycin \xe2\x80\x9cin-chloride\xe2\x80\x9d

In an effort to rapidly access vancomycin analogues bearing diverse functionality at the 6c-Cl (the \xe2\x80\x9cin-chloride\xe2\x80\x9d) position, a two-step dechlorination/cross-coupling protocol was developed. Conditions for efficient cross-coupling of the relatively unreactive 6c-Cl group were found that ensure high conversion with minimal product decomposition. A set of 2c-dechloro-6c-functionalized vancomycin derivatives was prepared, and antibiotic activities of the compounds were evaluated against a panel of vancomycin-resistant and vancomycin-susceptible strains. Results from biological testing further underscore the steric sensitivity of vancomycin\xe2\x80\x99s binding pocket.

a_stepwise_dechlorination/cross-coupling_strategy_to_diversify_the_vancomycin_\xe2\x80\x9cin-chlorid

<p>Glycopeptide natural product antibiotics, vancomycin (1) in particular, have seen widespread use and extensive study over the past half century.1,2 The glycopeptide antibiotics inhibit cell wall biosynthesis by coordinating the d-Ala-d-Ala peptide termini of peptidoglycan precursors, disrupting peptidoglycan cross-linking.3,4 Strong binding to d-Ala-d-Ala results from a network of five hydrogen bonds along with hydrophobic interactions within the glycopeptide binding pocket (Figure 1).5–10 For a period after its discovery, vancomycin was considered the "antibiotic of last resort;" however, clinical resistance was first observed in the 1980s and has become increasingly prevalent since then.11–14 In an effort to combat resistant infections and better understand resistance mechanisms, synthetic chemists have generated a diverse range of vancomycin analogues through both semisynthesis15 and total synthesis.16,17</p><p>The 6c-Cl group (the "in-chloride"), located in vancomycin's binding pocket, has inspired revealing studies over the past three decades. Harris and coworkers reported a method to prepare dechlorinated vancomycin derivatives via palladium-catalyzed hydrogenation and found 2c-Cl, oriented toward the convex face of the molecule, to be significantly more reactive than the in-chloride group.15a The authors prepared 2c-dechlorovancomycin (2) with 2c,6c-didechlorovancomycin (3) observed as the product of overreaction. More recently, Arimoto and coworkers utilized Suzuki-Miyaura (SM) coupling to access a library of 2c-functionalized and 2c,6c-difunctionalized vancomycin analogues, again observing substantially higher reactivity at 2c-Cl relative to 6c-Cl.15k Boger also reported an elegant 2c-borylation protocol that allowed unique access to organometallic substation reactions at the 2c-position.15l Moreover, although a synthesis of 6c-dechlorovancomycin has not yet been reported, Boger and coworkers recently prepared the aglycone of 6c-dechlorovancomycin, enabling binding studies and antibiotic measurements.15m</p><p>In recent years, our group has become interested in discovering approaches for the site-selective modification of vancomycin, and a single-step and site-selective synthesis of 6c-functionalized vancomycin has emerged as a goal.18 Toward this goal, we sought to better understand the reactivity of vancomycin's 6c-Cl position absent the competing, more favorable, reaction at 2c-Cl under SM coupling conditions. We identified 2 as a suitable substrate for such studies, and a modified method based on that of Harris et al. was found to furnish 2 with minimal overreaction to 3 (Scheme 1).19 Furthermore, we anticipated that products resulting from such reactivity studies could possess increased biological activity and inspire future research efforts toward 6c-Cl modification. Herein, we report a stepwise dechlorination/cross-coupling strategy to access 2c-dechloro-6c-functionalized vancomycin derivatives and antibiotic evaluation of novel reaction products featuring diverse functionality at the in-chloride position.</p><p>Due to the substrate's unique combination of reactivity, stability, and solubility properties, unusual challenges in the SM cross-coupling of 2 were encountered during reaction optimization. We found that aqueous conditions were required for substrate solubility, but that rapid decomposition of boronic acid coupling partners in water limited conversion.20–23 Additionally, product decomposition was unavoidable as a function of long reaction times; excess base (relative to boronic acid) was also found to accelerate this decomposition under the reaction conditions.24 The byproduct resulting from product decomposition was not identified; however, LCMS analysis revealed an identical isotope pattern to that of the product (see Supporting Information for more detail). Minimizing both types of decomposition (decomposition of boronic acid and decomposition of product) was found to be critical for efficient SM coupling of 2.</p><p>We began reaction optimization by investigating conditions utilizing high catalyst loading along with dropwise addition of a boronic acid/K2CO3 solution over a short reaction time (Table 1, Entry 1). Longer reaction times led to increased byproduct formation and lower product yield (Entry 2); yet, fewer equivalents of boronic acid and base led to decreased byproduct formation and higher product yield (Entry 3). We expected that dropwise addition of a boronic acid/K2CO3 solution would help avoid boronic acid decomposition and enable higher conversion; however, we observed nearly identical conversion upon switching to a "dump-and-stir" protocol (Entry 4; longer dropwise additions were not examined due to significant byproduct formation over longer reaction times). Next, we investigated conditions employing an excess of boronic acid relative to K2CO3 to further suppress base-promoted product decomposition, and a slight increase in product yield was, indeed, observed (Entry 5). Lower catalyst loading was also examined but afforded decreased yield due to low conversion, presumably because the slower coupling was unable to outcompete rapid boronic acid decomposition (Entries 6 and 7). Ultimately, the conditions in Entry 5 were best able to minimize decomposition of both boronic acid and product, leading to the highest product yield.</p><p>Once optimized SM reaction conditions were identified, a small library of 2c-dechloro-6c-functionalized vancomycin derivatives was prepared (Table 2). The antibiotic activities of vancomycin (1) and vancomycin derivatives 2–10 were then measured against vancomycin-sensitive S. aureus and E. faecalis strains and vancomycin-resistant strains of E. faecalis (VanA and VanB) using a standard microtiter plate-based antimicrobial assay (Table 3).25 In every case, 2 exhibited an approximately four-fold reduced activity compared to 1 (Entries 1 and 2), and the activity of 3 was reduced approximately eight-fold relative to 1 (Entries 1 and 3). These findings are consistent with those of Boger and coworkers, who measured similar reductions in activity against a vancomycin-sensitive strain of S. aureus for dechlorinated vancomycin aglycone derivatives relative to vancomycin aglycone.15l,15m In these prior studies, comparative binding of model cell wall ligands to 1–315a as well as dechlorovancomycin aglycone derivatives15l,15m suggested that the reduced antibiotic activity results from reduced binding efficiency between dechlorovancomycin analogues and cell wall precursors. The studies also indicate that decreases in binding affinity arising from dechlorination at 2c-Cl and 6c-Cl are additive in the case of 2c,6c-didechlorovancomycin analogues. Accordingly, the antibiotic activity of 2 is expected to be the most reliable baseline for comparison when considering the activities of novel 2c-dechloro-6c-functionalized vancomycin analogues 4–10.</p><p>We observed that 6c-trans-1-octenyl (4) and 6c-(4-biphenyl) substitution (5) reduced activity beyond the threshold of the assay (>64 µg/mL) for all strains tested, including vancomycin-sensitive strains (Table 2, Entries 4–5). These observations are consistent with MIC measurements of 2c,6c-difunctionalized vancomycin derivatives against vancomycin-sensitive and vancomycin-resistant strains of S. aureus, E. faecium, and E. faecalis reported by Arimoto and coworkers.15k The authors found that larger hydrocarbon-based substituents (trans-1-octenyl and trans-(5-phenyl)-1-pentenyl) reduced activity beyond the detection threshold of 64 µg/mL while the relatively smaller substituent trans-1-propenyl displayed an activity similar to that of vancomycin. As observed in the present study, no compounds exhibited measurably increased activity against vancomycin-resistant strains. Taken with MIC data for dechlorovancomycin derivatives, our data re-assert that vancomycin's antibiotic activity is highly sensitive to the binding pocket's steric environment.</p><p>Nevertheless, we hypothesized that hydrogen-bonding, nucleophilic, or charged functionality at the 6c-position might increase biological activity through conformational changes or through noncovalent or covalent interactions with cell wall precursors. Compounds 6–10 were prepared and tested for their potential effects. While 6c-(3-furanyl)-substituted analogue 6 inhibits vancomycin-sensitive S. aureus strains at high concentration (64 µg/mL), inhibition was not detected for any E. faecalis strains examined (>64 µg/mL; Table 2, Entry 6). Interestingly, compounds bearing primary amino (7), indolyl (8), amido (9), and fluorophenyl (10) functionality all failed to inhibit any strains examined in this study (>64 µg/mL; Table 2, Entries 7–10). As observed with dechloro and hydrocarbon functionality at the 6c-position, steric constraints play the predominant role in determining the antibiotic activity of compounds 6–10 despite their diverse functionality. Indeed, the only example of these 2c-dechloro-6c-functionalized vancomycin analogues that possesses any measurable activity is furanyl derivative 6, which bears the smallest substituent at the 6c-position.</p><p>In summary, we have developed a generalizable, stepwise approach to access 2c-dechloro-6c-functionalized vancomycin derivatives, and MIC measurements of novel compounds have further advanced understanding of the binding pocket's steric sensitivity. This stepwise approach will enable the preparation and evaluation of vancomycin derivatives beyond the scope of the present study. Selective chemical modification of the binding pocket of vancomycin continues to provide opportunities for creative analogue design.</p><p>This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.</p><p> Supplementary Material </p><p>Supplementary data associated with this article can be found, in the online version, at ________</p>

PubMed Author Manuscript

Delivering precision antimicrobial therapy through closed-loop control systems

AbstractSub-optimal exposure to antimicrobial therapy is associated with poor patient outcomes and the development of antimicrobial resistance. Mechanisms for optimizing the concentration of a drug within the individual patient are under development. However, several barriers remain in realizing true individualization of therapy. These include problems with plasma drug sampling, availability of appropriate assays, and current mechanisms for dose adjustment. Biosensor technology offers a means of providing real-time monitoring of antimicrobials in a minimally invasive fashion. We report the potential for using microneedle biosensor technology as part of closed-loop control systems for the optimization of antimicrobial therapy in individual patients.

delivering_precision_antimicrobial_therapy_through_closed-loop_control_systems

Introduction<!>Concept of closed-loop control for individualized antimicrobial therapy<!><!>Concept of closed-loop control for individualized antimicrobial therapy<!>Microneedles for continuous sensing of agents in the dermal interstitial fluid<!><!>Antimicrobial electrochemical sensing<!>Closed-loop control for drug delivery<!>PID control<!>ILC in closed-loop control<!>Additional PK/PD indices for individualizing therapy<!>Drug delivery<!>Conclusions

<p>Antimicrobial resistance (AMR) threatens to be a leading cause of death by 20501 making it a global patient safety issue. A major driver of AMR is the inappropriate use of antimicrobials in humans and animals.2 To date, research in this field has focused on optimizing the selection of antimicrobial agents. However, these strategies often fail to also consider optimization of the dose of the antimicrobial agent, which should aim to be sufficient to maximize bacterial killing whilst negating the harmful consequences of therapy, such as development of AMR and toxicity to the host.</p><p>Data are emerging within certain patient populations, such as critically ill patients, describing wide variations in how individuals handle antimicrobials (pharmacokinetics; PK).3–7 These wide variations in individual PK appear to be associated with increased variation in the effects of therapy, including outcomes of treatment and the development of AMR (their pharmacodynamics; PD).3–7 In response to the observed variations in individual PK, there has been a shift in the focus of therapeutic drug monitoring (TDM) away from primarily being used to prevent toxicity caused by antimicrobials with narrow therapeutic windows, towards enhancing the efficacy of less toxic agents such as the β-lactams, in order to optimize the outcomes of treatment.4,8–13 However, to achieve true individualization of therapy, we require a focus on not just the PK of antimicrobial agents. We must also understand the individual patient's physiology as well as the characteristics of the organism that we are treating. One method that has been explored widely is the use of Bayesian dose optimization platforms.3 Whilst TDM linked with Bayesian forecasting provides a powerful opportunity for delivering individualized care for patients,3,14 several gaps in current strategies for dose optimization of antimicrobials have hindered clinical implementation. Most notably, methods for more-continuous monitoring to allow real-time adaptive dosing of agents are still not available. Other challenges include difficulties in access to appropriate antimicrobial assays,12,15–20 poor integration of dosing software with electronic health records and decision support systems,3,21 challenges with collecting and handling PK samples,22,23 and failures of compliance with PK sampling protocols currently being used by healthcare professionals.24</p><p>Validation of novel methods for the monitoring and dose optimization of antimicrobial agents is required. Whilst several studies have explored the role of microfluidics,22,23,25 these are still hindered by many of the problems associated with routine antimicrobial TDM strategies, such as the need for laboratory analysis and transport of blood products. One potential method for avoiding these problems is the development of closed-loop systems based on minimally invasive, microneedle electrochemical sensor technology.26 This technology has been demonstrated to be applicable to the management of other conditions, such as diabetes control through individualized insulin delivery27–31 and anaesthesia control intra-operatively.32,33 This approach offers a potential avenue for enhancing the precision of antimicrobial therapy across a number of settings where invasive monitoring techniques may not be appropriate, including the community and non-critical care hospital settings. We report the current state of the art within the field of infection that offers a novel approach for the development of closed-loop systems for precision antimicrobial dosing.</p><!><p>There are several key concepts outlined in Figure 1 that must be considered for the development of closed-loop controllers for antimicrobial therapy. Ideally, monitoring of antimicrobials should be continuous and in a minimally invasive format that does not rely on blood sampling. The development of micro-needle array biosensor technology has provided an opportunity to achieve this, allowing for detection of antimicrobial concentrations in the dermal interstitial fluid (ISF).34,35 This technology has already been validated in the field of diabetes, demonstrating safety and tolerability in human clinical trials and accuracy in diabetic individuals who tend to have poor tissue perfusion due to underlying diabetic vasculopathy.26,34,35 Given that the free antimicrobial concentration in the ISF is generally in equilibrium with the plasma concentration this provides an opportunity for using this technology to monitor ISF concentrations as well as estimate plasma antimicrobial concentration in near real-time without requiring plasma sampling.36–38 This may be challenging in certain situations, such as during periods of tissue hypoperfusion in critically ill patients in the intensive care unit (ICU).39 However, it may also offer a novel option for supporting the optimization of antimicrobial dosing in these populations. This is because the majority of infections occur in tissue ISF.39,40 Therefore, this technology may provide a mechanism for monitoring antimicrobial concentrations in a compartment that is closer to the site where the infection is being treated when compared with plasma.39,40</p><!><p>Schematic for closed-loop control of antimicrobial delivery.</p><!><p>Data generated by this sensor can then be linked with machine-driven, closed-loop control algorithms such as Proportional-Integral-Derivative (PID)41 and Iterative Learning Controllers (ILC).42 These systems will allow for the optimization of both continuous and bolus (or oral) therapy to drive individualized target attainment of pre-defined PK/PD indices associated with maximal bacterial killing and/or suppression of the emergence of AMR.43,44 These may be current gold standard PK/PD targets45,46 or novel indices, such as AUC:EC50 ratio.47,48</p><p>Each of these concepts will individually be explored and critiqued within this manuscript.</p><!><p>Microneedle technology was first demonstrated as a suitable mechanism for drug monitoring and delivery over 20 years ago.49 Since then, microneedle technology has progressed rapidly with data supporting the use of microneedles to monitor glucose and lactate concentrations in humans34,35,50,51 as well as acting as delivery systems for drugs and vaccines.30,52 Microneedles work by penetrating the stratum corneum layer of the skin allowing access to the ISF, whilst avoiding the nerve fibres and blood vessels that are found within the dermis. Therefore, this offers a minimally invasive method for drug or metabolite monitoring.34,35,50,51 Side effects such as pain, bleeding, skin reactions, and infection risk have all previously been explored and shown to be minimal following application of such devices to the skin.34</p><p>One example of such technology was recently reported by Sharma and colleagues,28 who demonstrated high reproducibility when using microneedle technology to monitor glucose levels in healthy volunteers compared with capillary blood glucose measurements. The authors were able to demonstrate robustness of the device to sterilization using gamma-irradiation thus allowing the device to be sterilized and stored over time for use in monitoring human glucose concentrations.28 Furthermore, this technology can be reproduced reliably and at low cost through the development of scalable microneedle fabrication batch processing, producing up to 300 microneedles every hour.50</p><p>However, there are also challenges that remain in the development of microneedles within this field. Whilst microneedle-based methods of microdialysis have also been reported for the monitoring of vancomycin,53 this technique requires transfer of small volumes of ISF, which not only presents technical challenges in maintaining accuracy of the sensor but also leads to delays that mitigate against their application in real-time control.53 Moreover, in clinical trials for monitoring glucose using glucose oxidase-coated microneedles, the sensors appear to occasionally generate artefact during movements that cause them to be partially removed from the intradermal space.28 Whilst the artefact present in previous human studies had a shorter duration than changes in glucose concentration, this still requires consideration. Another challenge encountered with current microneedle sensors in humans has been accuracy of these devices at extreme ranges of glucose, especially hypoglycaemic ranges.28 It is likely therefore that sensor deployment for antimicrobial monitoring will encounter similar barriers for consideration.</p><p>In addition to microneedle-based sensing, other methods to facilitate continuous monitoring are also under consideration. Probably the most developed are attempts to perform real-time monitoring of drug concentrations in ambulatory animals using invasive vascular catheter insertion.54 These would only be acceptable in very specific situations in clinical practice, such as critical care or at the time of surgery, where tissue hypoperfusion may influence the ability of microneedle devices to accurately predict free drug concentrations in blood. However, invasive devices pose their own risks to the patient, including thrombosis.54 This type of invasive device would not be acceptable in the vast majority of individuals who receive antimicrobial therapy outside of critical care in hospital or in the community settings. A second consideration is the use of non-invasive, sweat-based monitoring systems as have been developed for glucose monitoring. However, to date very few data exist on whether this would be a viable option for monitoring antimicrobial concentrations.55</p><!><p>Current antimicrobial sensor classes reported in the literature</p><p>Spiked human urine</p><p>Water samples</p><p>Optimal analytical conditions</p><p>In spiked human urine:</p><p>0–2 μM (azithromycin)</p><p>Spiked human plasma</p><p>Spiked human urine</p><p>Milk</p><p>Optimal analytical conditions</p><p>In spiked human plasma:</p><p>0.05–100 μM (CIP)</p><p>0.1–100 μM (OFX)</p><p>0.1–40 μM (NOR)</p><p>0.06–100 μM (GAT)</p><p>Milk</p><p>Spiked human urine</p><p>Food samples</p><p>Optimal analytical conditions</p><p>In food samples:</p><p>0.08–1392 μM</p><p>LLD 0.015 μM</p><p>Spiked human urine</p><p>Optimal analytical conditions</p><p>Calibration in lab:</p><p>Linear range 0.8 pM–720 nM</p><p>In spiked urine samples reported recovery at concentrations 87, 96, 110, and 123 μM</p><p>Meat/feedstuff samples</p><p>Spiked honey</p><p>Optimal analytical conditions</p><p>In feedstuff</p><p>Linear range 0.3–52.0 μM (tetra) LLD 0.10 μM (tetra)</p><p>Optimal analytical conditions</p><p>Linear detection ranges:</p><p>0.006–10.0 mmol/L with an</p><p>LLD of 4.16 nmol/L</p><p>and 0.04–10 mmol/L with an</p><p>LLD of 2.34 nmol/L</p><p>Optimal analytical conditions</p><p>Food/milk samples</p><p>In spiked milk samples:</p><p>linear range 3–283 μM and</p><p>LLD 0.3 μM (Pen-G)</p><p>Recovery from spiked samples was 102±6%</p><p>In optimal conditions:</p><p>Km value 67±13 μM reported</p><p>using Michaelis Menten</p><p>kinetics equation (Pen-G)</p><p>Optimal analytical conditions</p><p>Ambulatory animals bloodstream</p><p>Spiked human serum</p><p>In spiked human serum:</p><p>Accurate within therapeutic range of 2–6 μM</p><p>Optimal analytical conditions</p><p>Foodstuff</p><p>Spiked human urine</p><p>In optimal conditions:</p><p>Linear detection range up to</p><p>1 mM and LLD of 0.08 μM</p><p>In spiked human urine:</p><p>Recovery in samples was 96.44% to 103.26%</p><p>Optimal analytical conditions</p><p>Milk</p><p>Spiked human urine</p><p>In optimal conditions:</p><p>Range of 0.1–10.0 mmol/L</p><p>with LLD of 60 nmol/L (TMP)</p><p>AND 1.0–10.0 mmol/L with</p><p>LLD of 38 nmol/L (SMX)</p><p>In spiked urine:</p><p>Recovery 91.3%-101%</p><p>CIP, ciprofloxacin; OFX, ofloxacin; GAT, gatifloxacin; NOR, norfloxacin; TMP, trimethoprim; SMX, sulfamethoxazole; Pen-G, penicillin G; LLD, lower limit of detection.</p><!><p>Enzymatic penicillin G sensors are some of the oldest antimicrobial sensors reported in the literature.57 These reactions can be detected through electrical, optical, or calorimetric methods.58 The majority of these techniques detect the hydrolysis of penicillin to penicillinoic acid and a hydrogen ion. One recent example of this technology is reported by Ro-Lee and colleagues utilizing field effect devices.59 The authors describe the high sensitivity of the enzyme-based device, its stability during storage, and re-usability over a 30 day period.59</p><p>These mechanisms for antimicrobial sensing have so far been demonstrated on microchips, disc electrodes, and nanotubes. This makes the devices small and highly transportable. This technology must now be transferred and tested on microneedle array devices to explore the sensitivity of such systems for real-time antimicrobial monitoring. However, based on current evidence provided by microdialysis of critically ill patients' tissue ISF concentrations, this approach is a potential avenue for estimation of antimicrobial concentrations and real-time monitoring.36–38 Preliminary in vitro work exploring the monitoring of β-lactam antibiotics (penicillin G, amoxicillin, and ceftriaxone) in artificial ISF using microneedles has demonstrated such devices provide plausible results.26 However, the major gap in the literature supporting translation currently is a paucity of human, in vivo studies with such biosensors to demonstrate their resistance to biofouling from proteins such as albumin and immunoglobulins.60,61 Furthermore, there remains limited data on the expected free antibiotic concentrations within the ISF for many antibiotics to predict the characteristics of tissue PK and allow accurate estimates of the linear range of response that such sensors will be required to work in before translation into human studies.</p><!><p>Closed-loop controllers have a broad application in the field of diabetes, being the cornerstone of novel developments, such as the artificial pancreas system.31,62 Furthermore, closed-loop control has been demonstrated as effective in controlling delivery of both intravenous and inhaled anaesthetic agents during surgery.32,63 This technology has been demonstrated in pre-clinical and in silico studies to be transferable to optimization of antimicrobial dosing.54,63 Two of the most widely used controllers for continuous and intermittent bolus infusions are the PID and ILC controllers, respectively.43,44 These controllers are algorithms that optimize the delivery of an agent against a pre-determined set point.</p><!><p>PID controllers depend on constant monitoring (e.g. every 5 minutes) and can be used to control continuous infusions maintaining drug concentrations at a set target (e.g. either target concentration or PK/PD index). As their name suggests, following data input the PID has three coefficients; the proportional, integral, and derivative. It alters these three coefficients to optimize the response against its target for therapy. The simplicity and robustness of PID algorithms make them extremely suitable for the range of operating conditions found in healthcare. This may be especially useful in critical care, where there is currently a drive towards continuous infusions of β-lactam antimicrobials and nephrotoxic agents, such as vancomycin, to optimize the PK exposure and PD properties.38,64–70 However, where current protocols require sporadic plasma TDM sampling this mechanism offers an opportunity for real-time response to changes in individual patient PK. For example, this would account for variations in PK caused by changes in the patient's inflammatory response, fluid shifts, augmented renal clearance, and in changing drain outputs in surgical patients that may currently be missed with sporadic TDM sampling.71–74</p><!><p>ILC provides the option for optimization of bolus or oral therapy, with data from continuous monitoring being used to optimize the amount, timing, and rate at which a bolus (or oral dose) is delivered. Like PID, ILC algorithms have wide applications but work on the assumption that during repetitive tasks (such as antimicrobial bolus dosing at regular intervals) there will be some level of error in target attainment (e.g. overshoot or undershoot). Therefore, the ILC aims to adjust the input, in this case the bolus dose, to reduce the transient error encountered during routine drug delivery to optimize the accuracy of such systems. This may be more applicable to non-critical care or the community setting (such as outpatient parenteral therapy or oral dosing) and in specialist populations, such as paediatrics and pregnancy, where rich data collection will allow for tailored therapy to be determined and adjusted for, based on real-time data and potentially previous experience housed within machine learning algorithms, as has been demonstrated by the use of Case-Based Reasoning in diabetes management.75</p><p>These systems can automatically control the delivery of an agent to optimize drug delivery to achieve defined PK/PD targets. If linked with Bayesian dose optimization software or Case-Based Reasoning platforms, which can provide individualized initial dose selection, and novel in vivo mechanisms of predicting antimicrobial PD, these could offer a powerful mechanism for reducing the errors that are commonly observed in the practice of current dose optimization strategies.</p><p>In terms of translating these into microneedle sensor-driven closed-loop control systems, the biggest challenge remaining is accurately describing the relationship for individual antimicrobials between tissue and plasma PK, especially during the initial phase of dosing, when the drug is not at steady state. This will be required to accurately describe the relationship between free concentrations of drug in both compartments and will likely require rich plasma and microdialysis PK sampling to enable development of accurate algorithms to support such controllers.</p><!><p>Currently, individualized PK/PD indices rely on factors such as the MIC to form part of time- and concentration-dependent measures for exposure response (such as AUC:MIC, Time>MIC, or Peak:MIC). MIC as a PD target requires isolation of the causative pathogen and determination of the individual organism's susceptibility. This causes a practical problem in cases where the invading pathogen is not identified, as is observed during the empirical phase of antimicrobial therapy, and in a significant proportion of cases of sepsis that remain culture-negative throughout the treatment period.76,77 Therefore, in the absence of microbiology results, population-level assumptions are made about the most likely organism causing the infection and the average MIC of this population. Thus this does not provide a truly individualized index on which to optimize antimicrobial therapy.</p><p>Furthermore, in place of an easily available individualized PK/PD index to guide the assessment of response to therapy, clinicians rely on clinical judgement, physiological parameters, and biochemical markers such as C-reactive protein (CRP) and procalcitonin (PCT) to assess individual patient response.78,79 In particular, CRP, an acute phase protein that is a non-specific marker of inflammation, is one of the most commonly used biomarkers during infection management in clinical practice.80–82 Despite its wide use in infection management, very little attempt has been made to link it directly to exposure–response using PK/PD modelling.</p><p>To address this, recent studies have reported the use of the ratio of the AUC to the EC50 in paediatric populations.47,48 The EC50 is the concentration of a drug (mg/L) that is estimated to induce a half-maximal antibacterial effect (such as reduction in serum CRP or galactomannan, a specific plasma marker in Aspergillus infection) for an individual patient. The AUC:EC50 ratio can provide an in vivo estimate of drug response by linking drug exposure with PD.47,48 Acting as an in vivo measure of potency, AUC:EC50 enables an estimate of the host immune response to the invading organism. This has the potential to circumvent the problems associated with in vitro MIC estimation and may provide data that can drive the development of real-time algorithms for the delivery and control of individualized antimicrobial therapy. With the clinical validation of tools such as the AUC:EC50 for predicting antimicrobial PD in individuals using markers such as CRP, future work must now explore the role of using newer infection-related biomarkers, such as procalcitonin and CD64 for improving the accuracy of these tools. Furthermore, exploration of similar methods for predicting toxicity (e.g. renal toxicity) may further enhance the individualization of therapy by including host, antimicrobial agent, and pathogen factors in estimations of the outcome of therapy.</p><!><p>Whilst intravenous and oral delivery of agents, via infusion pump and personalized dosing alerts respectively, may be the initial routes for antimicrobial delivery using such control systems there is also the potential for delivery via microneedle systems in the future. Such microneedles are now under investigation for drug and vaccine delivery that provide dual functions of sensing and also drug delivery.52 However, in the field of infection, the rate of drug delivery that can be achieved may be hindered by certain drug characteristics (such as hydrophilic versus hydrophobic agents) and the volume of agent required to be delivered. However, this technology may pose an interesting avenue for certain challenging cohorts, such as paediatric patients, as well as for local antimicrobial therapy delivery, such as skin and soft tissue infections or penetration of collections.</p><!><p>Novel systems are urgently required to individualize delivery of antimicrobial therapy, to address the wide variations in PK currently observed across a range of patient populations, and minimize the impact of sub-optimal dosing on clinical outcomes and AMR. Closed-loop control utilizing dermal antimicrobial sensing techniques offers a potential new avenue of applied research that addresses many of the current barriers associated with drug monitoring and dose optimization tools. Furthermore, the nature of minimally invasive sensor technology provides a platform that can be used across a range of settings from the community to those in intensive care. To achieve this there must be cross-disciplinary collaboration to explore the utility of such technologies to optimize the precision of antimicrobial therapy by addressing a number of the hurdles that remain to implementing this type of technology.</p>

PubMed Open Access

Antibacterial activity of 3-methylbenzo[d]thiazol-methylquinolinium derivatives and study of their action mechanism

AbstractThe increasing incidence of multidrug resistant bacterial infection renders an urgent need for the development of new antibiotics. To develop small molecules disturbing FtsZ activity has been recognized as promising approach to search for antibacterial of high potency systematically. Herein, a series of novel quinolinium derivatives were synthesized and their antibacterial activities were investigated. The compounds show strong antibacterial activities against different bacteria strains including MRSA, VRE and NDM-1 Escherichia coli. Among these derivatives, a compound bearing a 4-fluorophenyl group (A2) exhibited a superior antibacterial activity and its MICs to the drug-resistant strains are found lower than those of methicillin and vancomycin. The biological results suggest that these quinolinium derivatives can disrupt the GTPase activity and dynamic assembly of FtsZ, and thus inhibit bacterial cell division and then cause bacterial cell death. These compounds deserve further evaluation for the development of new antibacterial agents targeting FtsZ.

antibacterial_activity_of_3-methylbenzo[d]thiazol-methylquinolinium_derivatives_and_study_of_their_a

Introduction<!><!>General experimental<!>Synthesis of 4-chloro-1,2-dimethylquinolin-1-ium iodide (I1)<!>Synthesis of 1, 2-dimethyl–benzo[d]thiazol-1-ium iodide (I2)<!>Synthesis of (Z)-1,2-dimethyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl) quinolin-1-ium iodide (I3)<!>General procedure for the synthesis of 3-methylbenzo[d]thiazol-methylquinolinium derivatives (A1-A16)<!>Antimicrobial susceptibility assays<!>Time-killing curve determination<!>GTPase activity test<!>Light scattering assay<!>Transmission electron microscopy (TEM)<!>Bacterial morphology study<!>Molecular modeling<!>Cytotoxicity test of quinolinium derivatives<!>Synthesis of target compounds<!><!>In vitro antibacterial activity<!><!>In vitro antibacterial activity<!><!>Time-killing curve determinations<!><!>Effects of quinolinium derivatives on the GTPase activity of FtsZ<!><!>Effects of quinolinium derivatives on the FtsZ polymerization<!><!>Effects of quinolinium derivatives on the morphology of B. subtilis<!><!>Predicted binding mode of quinolinium derivative (A2) in FtsZ<!><!>Cytotoxicity of quinolinium derivatives<!><!>Conclusion<!>Acknowledgements<!>Disclosure statement

<p>Antimicrobial resistance is one of the major actual health plague. Due to the overuse and abuse of antibacterial drugs, bacteria develop resistance to conventional antibiotics at an alarming speed, and the treatment of antibiotic-resistant bacterial infections becomes more and more difficult1. Methicillin-resistant Staphylococcus auerus (MRSA) is a typical example of Gram-positive bacteria which have already shown resistance to the wildly prescribed antibiotics including methicillin as well as other β-lactam antibiotics, such as oxacillin and nafcillin. MRSA is responsible for many illnesses, ranging from skin infections to pneumonia. In 2011, the CDC estimated there were about 11,285 MRSA related deaths in United Stated2. This situation is also critical in Gram-negative bacteria infections. The WHO has released a list of the drug-resistant bacteria which new antibiotics are desperately needed. In this list, carbapenem resistant Gram-negative organisms are in the critical priority3. For example, the recently emerging New Delhi metallo-β-lactamase 1 (NDM-1) superbugs has made almost of the first-line clinical antibiotics ineffective4. Infections by antibiotic-resistant bacteria lead to high morbidity and mortality rates, however, there are limited treatment options for these infections to-date. There is an urgent need for the development of new antibacterial agents with innovative mechanisms of action to against the multidrug-resistant bacteria5.</p><p>Bacterial cell division is an essential process that has not yet been targeted by clinically approved antibiotics and thus it is a very important research area for antibacterial discovery. Bacterial cell division is believed to be critical in new antibiotic development because it is an essential process for bacterial survival and the bacterial divisome possesses a complex set of biochemical machinery that contains many proteins. The most important division proteins are widely conserved among bacterial pathogens and they are almost absent in eukaryotic cells6. Among these proteins, filamentous temperature sensitive protein Z (FtsZ) plays a critical role in cell division process. To initiate cell division, FtsZ assembles into protofilaments in a GTP dependent manner and forms a ring-like structure (Z-ring) at the division site7,8. Z-ring functions as a scaffold for the assembly of other cell division proteins to form bacterial divisome. Although the composition and the interdependency of divisome members may vary among different species, most bacteria depend on FtsZ as the central pacemaker protein9. Therefore, FtsZ is an attractive target for the development of novel antimicrobials.</p><p>Over the past decade, only few inhibitors of FtsZ have been reported showing the potency of disrupting FtsZ function and causing filamentation in bacteria10–12. However, these examples reveal that FtsZ targeting compounds inhibit bacterial growth through disrupting the dynamic polymerization and/or GTP hydrolysis of FtsZ. Among the FtsZ inhibitors, zantrin Z3 (Figure 1(A)) and its analogs which contain a benzo[g]quinazoline core can effectively inhibit the GTPase activity of FtsZ and display a broad-spectrum and modest antibacterial activity against a panel of bacteria13,14. Further SAR study revealed that replacing benzo[g]quinazoline by a smaller quinazoline, these molecules retain inhibitory activity on the FtsZ protein14. A quinoline derivative (Figure 1(B)) were reported to inhibited the growth of Mycobacterium tuberculosis through disrupting the polymerization of MtbFtsZ15. In addition, recent studies revealed that quinolinium derivatives (Figure 1(C)) have a potent antibacterial activity against drug resistant strains, including MRSA and Vancomycin-Resistant E. faecalis16,17. Based on our previous findings and experience on searching for novel potent anti-FtsZ agents through molecular design and synthesis18–24, we herein reported a new series of compounds (A1–A16) with a general molecular scaffold of 3-methylbenzo[d]thiazol-methylquinolinium (Figure 1(D)), in which we systemically varying the substituent groups linked at the ortho-position of 1-methylquinolinium core and investigated their antimicrobial activity with respect to different styryl substituents and mode of action targeting FtsZ.</p><!><p>Structures of zantrin Z3, quinoline derivative, quinolinium derivative, and 3-methylbenzo[d]thiazol-methylquinolinium derivatives.</p><!><p>Melting points (m.p.) were determined using a SRS Opti Mel automated melting point instrument without correction. 1H and 13C NMR spectra were recorded using TMS as the internal standard in DMSO-d6 with a Bruker BioSpin GmbH spectrometer at 400 and 100 MHz, respectively. Mass spectra (MS) were recorded on Bruker amaZon SL mass spectrometer with an ESI or ACPI mass selective detector. Reactions progress and compounds were checked by TLC with Merck silica gel 60F-254 glass plates. All chemicals were purchased from commercial sources unless otherwise specified, and all the solvents were analytical grade. The purities of synthesized compounds were confirmed by HPLC with a dual pump Shimadzu LC-20A system equipped with a photo-diode array detector and a C18 column (250 × 4.6 mm, 5 μM YMC) and eluted with acetonitrile/water (47:53) containing 0.5% acetic acid at flow rate of 1.0 ml/min.</p><!><p>Iodomethane (0.42 ml, 6.74 mmol) was added into the solution of 4-Chloro-2-methylquinoline (0.2 g, 1.12 mmol) in sulfolane (10 ml) was added. The reaction mixture was stirred at 50 °C for 20 h, cooled and anhydrous ether is added after the shock, suction filtration, the solid was washed with anhydrous diethyl ether, dried in vacuum to give of I1 (0.343 g, 95%): mp: 245–247 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.90 (s, 1H), 8.67 (d, J = 9.0 Hz, 1H), 8.26 (ddd, J = 8.7, 7.1, 1.4 Hz, 1H), 8.12 (t, J = 7.6 Hz, 1H), 8.05 (t, J = 7.5 Hz, 1H), 4.43 (s, 3H), 2.99 (d, J = 6.2 Hz, 3H). ESI-MS: m/z 192.1 [M − I]+.</p><!><p>A mixture of 2-methylbenzo[d]thiazole (0.25 g, 1.68 mmol), iodomethane (0.63 ml, 10.08 mmol) and anhydrous ethanol (10 ml) was stirred at reflux temperature for 15 h. After cooling, the mixture was dried over anhydrous ethanol and chloroform oscillating suction filtered. The precipitate was washed with chloroform and a small amount of ethanol, then vacuum dried to give I2 (0.447 g, 91.7%): mp: 232–235 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.44 (d, J = 8.1 Hz, 1H), 8.30 (d, J = 8.4 Hz, 1H), 7.90 (t, J = 7.8 Hz, 1H), 7.81 (t, J = 7.7 Hz, 1H), 4.20 (s, 3H), 3.17 (s, 3H). ESI-MS: m/z 164.4 [M − I]+.</p><!><p>I1 (0.5 g, 1.60 mmol), I2 (0.5 g, 1.75 mmol) and aqueous sodium bicarbonate solution (0.5 mol/l, 2 ml) were mixed with 10 ml methanol, and stirred at room temperature. After 1 h, 4 ml saturated KI solution was added to the reaction solution. After stirred another 15 min, I3 was obtained by washing with water and acetone, and dried in vacuum (0.475 g, 92%): mp: 268–271 °C. 1H NMR (400 MHz, DMSO-d6): δ 8.77 (d, J = 8.3 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.02–7.96 (m, 2H), 7.74 (d, J = 8.2 Hz, 2H), 7.59 (t, J = 7.7 Hz, 1H), 7.39 (t, J = 7.5 Hz, 1H), 7.33 (s, 1H), 6.84 (s, 1H), 4.06 (s, 3H), 3.98 (s, 3H), 2.87 (s, 3H). ESI-MS: m/z 319.0 [M − I]+.</p><!><p>A mixture of I3 (0.072 g, 0.16 mmol), 4-methylpiperidine (0.5 ml), n-butanol (10 ml) and selected aldehyde (0.32 mmol) were mixed and stirred at reflux temperature for 3 h. After the mixture was cooled down and filtered by suction, the solid was washed with n-butanol and purified by using column chromatography to obtain the pure target compounds A1–A16.</p><p>2-((E)-4-chlorostyryl)-1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-ylidene) methyl)quinolin-1-ium iodide (A1). Purple solid, yield 85%; mp 297–301 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.72 (d, J = 8.4 Hz, 1H), 8.24 (d, J = 8.68 Hz, 1H), 8.08 (d, 1H), 8.02 (d, J = 7.4 Hz,3H), 7.84 (d, J = 4.5 Hz, 1H), 7.75 (m, J = 9.5 Hz, 3H), 7.65 (s, 1H), 7.56 (s, 3H), 7.45 (t, J = 8.3 Hz, 1H), 6.95 (s, 1H), 4.27 (s, 3H), 4.02 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 159.31, 154.79, 148.40, 140.95, 139.64, 139.49, 133.58, 130.62, 129.43, 128.50, 126.83, 125.82, 124.67, 124.14, 123.91, 123.48, 123.17, 118.73, 113.10, 111.03, 87.33, 37.56, 34.09. ESI-MS: [M − I]+ (C27H22ClN2S+): m/z 441.0; HPLC retention time was 1.94 min.</p><p>2-((E)-4-fluorostyryl)-1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl) quinolin-1-ium iodide (A2). Rufous solid, yield 85%; mp 293–296 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.69 (d, J = 8.4 Hz, 1H), 8.01 (dd, J = 29.3, 10.1 Hz, 4H), 7.94–7.89 (m, 1H), 7.70 (d, J = 7.6 Hz, 1H), 7.63 (dd, J = 19.6, 11.2 Hz, 3H), 7.54 (d, J = 14.5 Hz, 1H), 7.44 (s, 1H), 7.34 (dd, J = 19.2, 8.3 Hz, 3H), 6.79 (s, 1H), 4.07 (s, 3H), 3.92 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 159.83, 152.07, 148.00, 140.82, 139.79, 139.32, 133.66, 132.22, 131.24, 131.16, 128.49, 126.90, 125.61, 124.66, 124.27, 123.85, 123.42, 121.96, 118.89, 116.47, 116.25, 113.09, 108.23, 88.29, 38.53, 34.13. ESI-MS: [M − I]+ (C27H22FN2S+): m/z 425.0; HPLC retention time was 3.63 min.</p><p>2-((E)-4-bromostyryl)-1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-ylidene) methyl)quinolin-1-ium iodide (A3). Rufous solid, yield 87%; Mp 307–309 °C; 1HNMR (400 MHz, DMSO-d6): δ 8.76 (d, J = 8.2 Hz, 1H), 8.18 (d, J = 8.2 Hz, 1H), 8.05 (d, J = 7.6 Hz, 1H), 8.00 (d, J = 7.3 Hz, 1H), 7.90 (d, J = 7.8 Hz, 2H), 7.86 – 7.70 (m, 5H), 7.63 (d, J = 19.0 Hz, 3H), 7.44–7.37 (m, 1H), 6.92 (s, 1H), 4.12 (d, J = 30.4 Hz, 3H), 4.01 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 159.57, 154.63, 148.40, 140.95, 139.64, 139.49, 133.58, 130.62, 129.43, 128.50, 126.83, 125.82, 124.67, 124.14, 123.91, 123.48, 123.17, 118.73, 113.10, 111.03, 87.33, 37.56, 34.09. ESI-MS: [M − I]+ (C27H22BrN2S+): m/z 486.9; HPLC retention time was 3.52 min.</p><p>2-((E)-2,4-dichlorostyryl)-1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (A4). Purple solid, yield 87%; Mp 271–275 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.74 (d, J = 7.8 Hz, 1H), 8.18 (d, J = 8.7 Hz, 1H), 8.10 (d, J = 8.5 Hz, 1H), 7.96 (d, J = 7.0 Hz, 2H), 7.85 (d, J = 15.8 Hz, 1H), 7.73 (t, J = 17.6 Hz, 4H), 7.59 (t, J = 10.7 Hz, 2H), 7.52 (s, 1H), 7.39 (d, J = 7.2 Hz, 1H), 6.91 (s, 1H), 4.11 (s, 3H), 3.98 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 159.90, 151.68, 148.25, 140.85, 139.37, 135.67, 134.83, 134.75, 133.87, 132.23, 130.11, 129.89, 128.67, 128.48, 127.11, 125.98, 125.67, 124.94, 123.97, 123.19, 118.98, 113.33, 108.68, 88.78, 38.68, 34.31. ESI-MS: [M − I]+ (C27H21Cl2N2S+): m/z 475.0; HPLC retention time was 4.29 min.</p><p>1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-2-((E)-4-methyl styryl)quinolin-1-ium iodide (A5). Red solid, yield 85%; mp 275–278 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.77 (d, J = 8.3 Hz, 1H), 8.19 (d, J= 8.8 Hz, 1H), 8.07 (d, J= 7.8 Hz, 1H), 8.00 (t, J= 7.9 Hz, 1H), 7.85 (d, J = 7.9 Hz, 2H), 7.75 (dd, J = 9.7, 5.6 Hz, 3H), 7.63 (dt, J = 15.6, 9.9 Hz, 3H), 7.43–7.34 (m, 3H), 6.92 (s, 1H), 4.17 (s, 3H), 4.00 (d, J = 8.1 Hz, 3H), 2.41 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 159.92, 152.60, 148.26, 141.32, 141.05, 140.79, 139.52, 133.74, 132.99, 130.03, 128.95, 128.55, 126.97, 125.69, 124.69, 124.27, 124.01, 123.48, 121.10, 119.03, 113.14, 108.44, 88.27, 38.53, 34.12, 21.57. ESI-MS: [M − I]+ (C28H25N2S+): m/z 421.2; HPLC retention time was 3.38 min.</p><p>2-((E)-4-(dimethylamino)styryl)-1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-y lidene)methyl)quinolin-1-ium iodide (A6). Purple solid, yield 89%; mp 301–304 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.70 (d, J = 8.3 Hz, 1H), 8.13 (d, J = 8.8 Hz, 1H), 8.03 (d, J = 7.7 Hz, 1H), 7.97–7.92 (m, 1H), 7.79 (d, J = 8.9 Hz, 2H), 7.71 (d, J = 7.8 Hz, 1H), 7.69–7.64 (m, 2H), 7.63 (s, 1H), 7.56 (t, J = 7.8 Hz, 1H), 7.43 (d, J = 15.7 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 6.79 (d, J = 9.1 Hz, 3H), 4.13 (s, 3H), 3.94 (s, 3H), 3.05 (s, 6H). 13C NMR (100 MHz, DMSO-d6): δ 158.77, 153.24, 152.30, 147.53, 142.83, 141.10, 139.57, 133.43, 130.97, 128.40, 126.63, 125.57, 124.30, 124.05, 123.92, 123.38, 123.11, 118.93, 115.10, 112.74, 112.16, 108.03, 87.58, 38.28, 33.91. ESI-MS: [M − I]+ (C29H28N3S+): m/z 450.1; HPLC retention time was 5.44 min.</p><p>1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-2-((E)-4-(methyl thio)styryl)quinolin-1-ium iodide (A7). Rufous solid, yield 90%; mp 293–295 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.73 (d, J = 7.8 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 8.06–8.02 (m, 1H), 8.00–7.95 (m, 1H), 7.87 (d, J = 8.5 Hz, 2H), 7.76–7.68 (m, 3H), 7.64 (s, 1H), 7.61–7.55 (m, 2H), 7.42–7.35 (m, 3H), 6.87(s, 1H), 4.13(s, 3H), 3.97 (d, J = 3.7 Hz, 3H), 2.56 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 159.81, 152.50, 148.16, 142.08, 141.04, 140.86, 139.51, 133.71, 132.08, 129.41, 128.54, 126.94, 126.07, 125.67, 124.68, 124.26, 123.98, 123.44, 120.95, 119.01, 113.11, 108.42, 38.53, 34.11, 14.77. ESI-MS: [M − I]+ (C28H25N2S2+): m/z 453.0; HPLC retention time was 3.45 min.</p><p>2-((E)-4-methoxystyryl)-1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-ylidene) methyl)quinolin-1-ium iodide (A8). Brown solid, yield 86%; mp 263–267 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.73 (d, J = 8.4 Hz, 1H), 8.08 (dd, J = 14.8, 8.3 Hz, 2H), 7.99–7.89 (m, 3H), 7.73 (t, J = 7.6 Hz, 1H), 7.66 (d, J = 8.3 Hz, 1H), 7.63–7.56 (m, 3H), 7.51 (s, 1H), 7.40 (t, J = 7.5 Hz, 1H), 7.09 (d, J = 8.5 Hz, 2H), 6.82 (s, 1H), 4.11 (s, 3H), 3.96 (s, 3H), 3.91 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 160.41, 158.29, 151.39, 146.68, 140.01, 139.75, 138.21, 132.41, 129.64, 127.29, 127.12, 125.65, 124.43, 123.36, 123.04, 122.70, 122.25, 118.06, 117.75, 113.67, 111.81, 106.95, 86.87, 54.80, 37.30, 32.91. ESI-MS: [M − I]+ (C28H25N2OS+): m/z 437.0; HPLC retention time was 2.95 min.</p><p>2-((E)-3-methoxystyryl)-1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-ylidene) methyl)quinolin-1-ium iodide (A9). Rufous solid, yield 87%; mp 252–256 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.50 (d, J = 8.5 Hz, 1H), 7.83–7.73 (m, 3H), 7.70 (d, J = 7.7 Hz, 1H), 7.56 (dd, J = 15.5, 11.8 Hz, 2H), 7.39 (dd, J = 16.2, 9.3 Hz, 3H), 7.31 (d, J = 8.2 Hz, 1H), 7.24 (t, J = 7.5 Hz, 1H), 7.09 (s, 1H), 6.99 (t, J = 7.4 Hz, 1H), 6.92 (d, J = 8.3 Hz, 1H), 6.52 (s, 1H), 3.81 (d, J = 12.7 Hz, 3H), 3.74 (s, 3H), 3.65 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 158.57, 157.74, 152.11, 147.18, 140.32, 138.85, 135.87, 133.43, 132.33, 128.64, 128.40, 126.69, 125.29, 124.49, 123.62, 123.54, 123.47, 122.65, 121.28, 121.09, 118.61, 112.79, 111.99, 107.86, 87.84, 56.28, 38.22, 34.02. ESI-MS: [M − I]+ (C28H25N2OS+): m/z 437.0; HPLC retention time was 3.14 min.</p><p>2-((E)-3,4-dimethoxystyryl)-1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (A10). Brown solid, yield 89%; mp 266–270 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.73 (d, J = 8.2 Hz, 1H), 8.14 (s, 1H), 8.05 (d, J = 7.8 Hz, 1H), 7.99–7.95 (m, 1H), 7.91 (d, J = 7.3 Hz, 1H), 7.69 (s, 1H), 7.62 (d, J = 6.7 Hz, 2H), 7.58 (s, 3H), 7.47 (d, J = 8.1 Hz, 1H), 7.39 (d, J = 7.6 Hz, 2H), 7.07 (d, J = 8.2 Hz, 1H), 6.85 (s, 1H), 6.65 (s, 1H), 4.16 (s, 3H), 3.97 (s, 3H), 3.91 (s, 3H), 3.85 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 158.57, 157.74, 152.11, 147.13, 140.32, 138.85, 135.87, 133.43, 132.33, 128.64, 128.40, 126.69, 125.29, 124.49, 123.62, 123.54, 123.47, 122.65, 122.41, 87.70, 56.28, 37.96, 33.94. ESI-MS: [M − I]+ (C29H27N2O2S+): m/z 467.0; HPLC retention time was 5.72 min.</p><p>1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-2-((E)-2-(pyridine-3-yl)vinyl)quinolin-1-ium iodide (A11). Rufous solid, yield 88%; mp 288–291 °C; 1H NMR (400 MHz, DMSO-d6): δ 9.10 (s, 1H), 8.77 (s, 1H), 8.66 (d, J = 3.7 Hz, 1H), 8.39 (d, J = 7.3 Hz, 1H), 8.17 (s, 1H), 8.06 (d, J = 7.5 Hz, 1H), 7.99 (s, 1H), 7.91 (d, J = 15.9 Hz, 1H), 7.74 (s, 2H), 7.67 (d, J = 18.3 Hz, 1H), 7.65–7.52 (m, 3H), 7.41 (s, 1H), 6.91 (s, 1H), 4.16 (s, 3H), 4.00 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 160.23, 151.88, 151.12, 150.40, 148.29, 140.98, 139.45, 137.63, 135.15, 133.81, 131.47, 128.59, 127.05, 125.70, 124.83, 124.41, 124.37, 124.29, 123.98, 123.45, 119.01, 113.27, 108.47, 88.58, 38.62, 34.22. ESI-MS: [M − I]+ (C26H22N3S+): m/z 408.0; HPLC retention time was 1.97 min.</p><p>2-((E)-2–(1H-indol-3-yl)vinyl)-1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (A12). Rufous solid, yield 85%; mp 298–303 °C; 1H NMR (400 MHz, DMSO-d6): δ 11.97 (s, 1H), 8.62 (d, J = 8.4 Hz, 1H), 8.20 (s, 1H), 8.09 (d, J = 7.3 Hz, 1H), 8.03 (d, J = 8.8 Hz, 1H), 7.98 (d, J = 7.8 Hz, 1H), 7.88 (dd, J = 15.5, 5.5 Hz, 2H), 7.63 (t, J = 7.6 Hz, 1H), 7.51 (q, J = 8.4 Hz, 4H), 7.33–7.23 (m, 4H), 6.70 (d, J = 8.5 Hz, 1H), 4.07 (s, 3H), 3.83 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 158.34, 153.65, 147.23, 140.96, 139.40, 136.84, 133.30, 128.29, 126.47, 125.49, 125.13, 124.14, 123.92, 123.80, 123.38, 123.26, 121.71, 120.70, 118.78, 114.21, 113.98, 113.05, 112.54, 107.63, 87.39, 38.20, 33.84. ESI-MS: [M − I]+ (C29H24N3S+): m/z 446.1; HPLC retention time was 2.81 min.</p><p>1-methyl-4-((E)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-2-((E)-2-(naphth alen-2-yl)vinyl)quinolin-1-ium iodide (A13). Black solid, yield 85%; mp 299–304 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.78 (d, J = 8.3 Hz, 1H), 8.41 (s, 1H), 8.18 (t, J = 8.1 Hz, 2H), 8.11–8.04 (m, 2H), 8.01 (d, J = 4.9 Hz, 3H), 7.91 (s, 1H), 7.85 (s, 1H), 7.75 (t, J = 8.6 Hz, 2H), 7.67 (s, 1H), 7.65–7.56 (m, 3H), 7.40 (t, J = 7.4 Hz, 1H), 6.94 (d, J = 9.8 Hz, 1H), 4.20 (s, 3H), 3.99 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 159.99, 152.42, 148.28, 141.24, 141.06, 139.55, 134.15, 133.77, 133.39, 133.34, 130.41, 129.05, 128.89, 128.56, 128.30, 127.90, 127.44, 126.99, 125.73, 124.72, 124.58, 124.32, 124.05, 123.52, 122.55, 119.06, 113.19, 108.54, 88.44, 38.63, 34.18. ESI-MS: [M − I]+ (C31H25N2S+): m/z 457.0; HPLC retention time was 6.90 min.</p><p>1-methyl-4-((Z)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)-2-((1E,3E)-4-phenylbuta-1,3-dien-1-yl)quinolin-1-ium iodide (A14). Brown solid, yield 80%; mp 254–257 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.73 (t, J = 10.7 Hz, 1H), 8.10 (dd, J = 18.2, 9.6 Hz, 2H), 7.97–7.92 (m, 1H), 7.74–7.68 (m, 2H), 7.67–7.61 (m, 2H), 7.60–7.51 (m, 3H), 7.45 (dd, J = 13.6, 5.9 Hz, 2H), 7.42–7.35 (m, 3H), 7.31 (d, J = 15.2 Hz, 2H), 6.83 (d, J = 9.2 Hz, 1H), 4.06 (d, J = 14.2 Hz, 3H), 3.97 (d, J = 7.1 Hz, 3H). 13C NMR (100 MHz, DMSO-d6): δ 159.64, 151.84, 147.88, 141.95, 141.06, 140.24, 139.50, 136.52, 133.65, 129.66, 129.53, 128.60, 128.54, 127.64, 126.90, 125.64, 125.09, 124.64, 124.24, 123.96, 123.48, 118.98, 113.11, 108.00, 88.23, 38.24, 34.12. ESI-MS: [M − I]+ (C29H25N2S+): m/z 433.2; HPLC retention time was 1.70 min.</p><p>2-((1E,3E)-4–(4-fluorophenyl)buta-1,3-dien-1-yl)-1-methyl-4-((Z)-(3-methylbenzo [d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (A15). Trovirens solid, yield 87%; mp 281–284 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.72 (d, J = 8.3 Hz, 1H), 8.09 (t, J = 9.5 Hz, 2H), 7.98–7.93 (m, 1H), 7.70 (dd, J = 15.8, 7.2 Hz, 4H), 7.61–7.56 (m, 1H), 7.55–7.49 (m, 2H), 7.42–7.37 (m, 1H), 7.31 (t, J = 8.8 Hz, 4H), 7.27 (s, 1H), 6.85 (s, 1H), 4.08 (s, 3H), 3.97 (s, 3H). 13C NMR (100 MHz, DMSO-d6): δ 164.19, 161.73, 159.63, 151.82, 147.89, 141.86, 141.05, 139.49, 138.96, 133.64, 133.20, 133.17, 129.74, 129.66, 128.53, 128.44, 126.89, 125.63, 124.99, 124.62, 124.23, 123.96, 123.47, 118.93, 116.60, 116.38, 113.09, 108.01, 88.24, 38.23, 34.14. ESI-MS: [M − I]+ (C29H24FN2S+): m/z 451.0; HPLC retention time was 1.71 min.</p><p>2-((1E,3E)-4–(4-(dimethylamino)phenyl)buta-1,3-dien-1-yl)-1-methyl-4-((Z)-(3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl)quinolin-1-ium iodide (A16). Atrovirens solid, yield 85%; mp: 289–294 °C; 1H NMR (400 MHz, DMSO-d6): δ 8.70 (d, J = 8.5 Hz, 1H), 8.12 (d, J = 8.9 Hz, 1H), 8.07 (d, J = 7.9 Hz, 1H), 7.95 (t, J = 7.8 Hz, 1H), 7.73–7.68 (m, 2H), 7.59 (dd, J = 13.5, 7.0 Hz, 3H), 7.49 (d, J = 8.7 Hz, 2H), 7.39 (t, J = 7.6 Hz, 1H), 7.17 (dd, J = 24.7, 13.8 Hz, 3H), 6.83–6.76 (m, 3H), 4.09 (d, J = 8.5 Hz, 3H), 3.96 (s, 3H), 3.00 (d, J = 11.7 Hz, 6H). 13C NMR (100 MHz, DMSO-d6): δ 158.75, 152.13, 151.49, 147.13, 143.68, 142.22, 140.98, 139.42, 133.35, 129.38, 128.39, 126.59, 125.49, 124.30, 124.08, 124.00, 123.81, 123.62, 123.41, 120.93, 118.77, 112.78, 112.53, 107.66, 87.68, 37.99, 33.94. ESI-MS: [M − I]+ (C31H30N2S+): m/z 476.1. HPLC retention time was 4.89 min.</p><!><p>Antimicrobial susceptibility tests were conducted in 96-well microplates using the broth microdilution procedure in accordance to the Clinical and Laboratory Standards Institute (CLSI) guidelines25. Cation-adjusted Mueller Hinton broth for all the S. aureus strains, including MRSA, or brain heart infusion broth for antibiotic-susceptible E. faecium ATCC 49624 and E. faecalis ATCC 29212, vancomycin-resistant E. faecium ATCC 700221 and E. faecalis ATCC 51575, or Mueller Hinton broth for the other strains were used in the assays. After incubation for 18 h at 37 °C, the absorbance at 600 nm (A600) was recorded using a microplate reader (Bio-Rad laboratory Ltd., UK) and the percentage of bacterial cell inhibition with respect to vehicles (1% DMSO) was calculated. The MIC was defined as the lowest compound concentration at which the growth of bacteria was inhibited by ≥90%. Three independent assays were performed for each test.</p><!><p>A growing culture of S. aureus ATCC 29213 or E. coli ATCC25922 were diluted to approximately 105 CFU mL−1 in volumes of Cation-adjusted Mueller Hinton broth or Mueller Hinton broth, respectively, containing various concentrations of tested compound. Cultures were incubated with shaking at 37 °C. At the appropriate time intervals, 100 μL samples were removed for serial dilution in 900 μL volumes of corresponding medium, and 100 μL volumes from three dilutions were spread on to MH agar. Cell counts (CFU ml−1) were enumerated after incubating the plates at 37 °C for 24 h.</p><!><p>FtsZ protein in the biological tests was prepared as our previous report21. The GTPase activity of SaFtsZ was measured in 96-well microplates using a phosphate assay Kit according to previous description20. FtsZ (3.5 μM) was preincubated with different concentrations of tested compounds in 20 mM Tris buffer (pH 7.4, 0.01%Triton X-100 to avoid compound aggregation) at room temperature. 5 mM of MgCl2 and 200 mM of KCl were added after 10 min incubation. Reactions were started with the addition of 500 mM GTP and incubated in a water bath at 37 °C. After 30 min, the reactions were quenched by adding 100 μL of Cytophos reagent for 10 min. Inorganic phosphate was quantified by measuring the absorbance at 650 nm with a microplate reader.</p><!><p>The FtsZ polymerization was measured using 90° light scattering in a fluorescence spectrometer at 37 °C. Excitation and emission wavelengths were set at 600 nm with a slit width of 2.5 nm. FtsZ (5 μM) in 20 mM of Tris buffer (pH 7.4, containing 0.01%Triton X-100 to avoid compound aggregation) was placed in a fluorometer cuvette, and the polymerization reaction was started by consecutive additions of 20 mM KCl, 5 mM MgCl2, 1 mM GTP, and different concentrations of tested compound.</p><!><p>FtsZ (13.5 μM) was incubated with different concentrations of tested compounds in 20 mM Tris buffer (pH 7.4) at room temperature. After 10 min incubation, 5 mM MgCl2, 50 mM KCl, and 1 mM GTP were added to the reaction mixtures and incubated at 37 °C for another 10 min. Then, 10 μL of the sample mixtures were dropped on a glow-discharged Formvar carbon-coated copper grid. The grids were subsequently subjected to negative staining using 10 μL of 0.5% phosphotungstic acid for 1 min, air-dried and digital images of the specimen were obtained from a transmission electron microscope (JEOL model JEM 2010).</p><!><p>The B. subtilis 168 was grown in LB medium. The cultures at an A600 of 0.01 from an overnight culture were inoculated in the same medium containing different concentrations of the tested compound and grown at 37 °C for 4 h. The cells for morphology studies were harvested and resuspended in 100 μL of PBS buffer containing 0.25% agarose. 10 μL of the suspension mixture were then placed on a microscopic slide and the morphology of the bacterial cells was observed under a light phase-contrast microscope.</p><!><p>The molecular modeling were performed using Discovery Studio 2016. The X-ray crystal structure of FtsZ was downloaded from the PDB database (PDB entry: 4DXD; resolution: 2.0 Å)26. Co-crystal ligands and water molecules were removed from the structure and the protein was prepared for docking using automated procedure of Discovery Studio. The structures of A2 was sketched and minimized using the Discovery Studio molecule preparation tools. The automated docking study was carried out using DS-CDocker protocol in the Discovery Studio. The highest scoring poses were visually inspected.</p><!><p>Selected quinolinium derivatives (A2 and A5) were tested with renal epithelial cells (HK-2) and Mouse fibroblasts cells (L929) to determine the cytotoxicity of eukaryotic mammalian cells. Cells were re-suspended in complete cell culture medium and the concentration was adjusted at approximately 1 × 105 cells mL−1. Cells seeded in the 96 wells microtitre plates for 24 h were used for the evaluation of the tested compounds. For the MTS assay, cells (5,000 per well) were seeded into 96-well plates. After treatment with compounds of different concentrations for 24h, cells were added with MTS at a final concentration of 0.3 mg/mL, followed by incubation for another 2h. The optical density (OD) of each well was determined at 490 nm (background subtraction at 690 nm) by a SpectraMax 340 microplate reader. The growth inhibitory ratio was calculated as follows: Growth inhibitory ratio = (Acontrol − Asample)/Acontrol (where A is the OD value per well).</p><!><p>In the present study, compounds A1–A16 listed in Table 1 were synthesized from 4-chloro-2-methylquinoline as outlined in Scheme 1. Intermediate I1 (4-chloro-1,2-dimethylquinolin-1-ium iodide) and Intermediate I2 (2,3-dimethylbenzo[d]thiazol-3-ium iodide) was obtained by the reaction of 4-chloro-2-methylquinoline and 2-methylbenzo[d]thiazole with iodomethane. Then the key intermediate (Z)-1,2-dimethyl-4-((3-methylbenzo[d]thiazol-2(3H)-ylidene)methyl) quinolin-1-ium iodide (I3) was prepared by the reaction of I1 and I2 by following the reported procedures27. The target compounds with different styryl substituents introduced at ortho-position of I3 were then achieved by the reaction of I3 with a variety of aromatic aldehydes. The target compounds were obtained with high isolated yields (80–90%) and were characterized by MS and NMR. All the spectral data were in agreement with the proposed structures. The purities of these compounds were confirmed to be above 95% using HPLC analysis.</p><!><p>Synthesis route of 3-methylbenzo[d]thiazol-methylquinolinium derivatives. Reagents and conditions: (a) iodomethane, tetramethylene sulfone, 50 °C, reflux; (b) iodomethane, absolute ethanol, 80 °C, reflux; (c) NaHCO3, methanol, room temperature; (d) aromatic aldehydes, 4-methylpiperidine, n-butanol, 135 °C, reflux.</p><p>List of 3-methylbenzo[d]thiazol-methylquinolinium derivatives (A1–A16).</p><!><p>The antibacterial activities of the compounds were first evaluated by using a standard two-fold micro-dilution assay in Mueller–Hinton broth against a panel of drug-sensitive bacterial strains, including B. subtilis 168, S. aureus (ATCC 29213), E. coli (ATCC 25922), E. faecium (ATCC 49624), E. faecalis (ATCC 29212) and S. epidermidis (ATCC 12228). Methicillin, vancomycin, and berberine were tested under the same assay conditions as reference compounds. The minimum inhibition concentration (MIC) results were summarized in Table 2. By comparing the results with berberine, all compounds tested exhibit a much superior antibacterial activity against the six selected drug-sensitive strains. It is noteworthy that compounds A2, A3, and A5 have the MIC values lower than 6 μg/mL, showing their efficacy is comparable to clinical antibiotics such as methicillin and vancomycin. From Table 2, the structural effects arising from different styryl substituents show strong influence on the antibacterial activity as indicated by their MIC values. It seems that the antibacterial activity of these new compounds is correlated with the size and the styryl substituents (R group). Compound A2 (para-fluorostyryl) and A5 (para-methylstyryl) were showing very similar MIC value in the experiment, while other substituents with increase molecular size, particularly A13–A16, the antibacterial activity were decreased. This is most probably due to the steric influence of the substituent in the binding domain of FtsZ protein.</p><!><p>Minimum inhibitory concentrations of compounds A1–A16 against a panel of drug-sensitive bacterial strains (μg/mL).</p><!><p>Based on the antibacterial results of the drug-sensitive strains, six compounds with the best MIC values (A1–A3 and A5–A7) were selected for further evaluation against an extended panel of clinically relevant drug-resistant bacterial strains including MRSA (ATCC BAA41, ATCC 43300), Vancomycin-Resistant E. faecalis (ATCC 51575) and E. faecium (ATCC 700221), NDM-1 E. coli (ATCC BAA2469), P. aeruginosa (ATCC BAA2108). As shown in Table 3, the selected compounds exhibited potent activity against three MRSA strains with MIC values of 1.5–4 μg/mL. The MIC values are comparable to that of vancomycin. Among these compounds, A2 was the most effective with a MIC value of 1.5 μg/mL against MRSA, and showed more than 100-fold better antibacterial activity than that of berberine and methicillin. The growth of vancomycin-resistant E. Faecalis and E. faecium (VREs) were inhibited with MIC values of 2–8 μg/mL. The antibacterial effects of these quinolinium derivatives on the VREs were much better that of vancomycin and berberine (MICs >64 μg/mL) and were comparable to that of methicillin. When tested on the Gram-negative strains NDM-1 E. coli and P. aeruginosa, methicillin shows little effect on the gowth of thesesuperbugs even at a high concentration (192 μg/mL). It is noteworthy that A2 and A5 can effectively inhibit the growth of NDM-1 E. coli with a much lower MIC value of 3 μg/mL. Moreover, P. aeruginosa which is resistant to most of the clinical antibiotics can be effectively inhibited by our quinolinium derivatives A2 and A5 with the MIC values of 6–8 μg/mL.</p><!><p>The antibacterial activity of selected compounds against drug-resistant bacterial strains (μg/mL).</p><p>Methicillin-resistant S. aureus.</p><p>Vancomycin-resistant strains.</p><p>E. coli expressing NDM-1 beta-lactamase.</p><p>A multidrug-resistant strain.</p><!><p>To further investigate whether the antibacterial activities of 3-methylbenzo[d]thiazol-methylquinolinium derivatives are bactericidal or not, the viable counts for the determination of killing curves were performed as previous report25. Time killing curves resulting from A2 against S. aureus ATCC 29213 and E. coli ATCC 25922 are presented in Figure 2. A significant reduction about 103 CFU mL−1 (99.9% of bacterial growth inhibited) was observed in 2 h at 2 × and 4 × MIC against S. aureus. These phenomena indicate that A2 can inhibit the growth of S. aureus quickly. Figure 2(A) showed that A2 at 1 × MIC concentration caused a reduction of 1 × 102 CFU mL−1 for S. aureus in 4 h and is below the lowest detectable limit (103 CFU mL−1) in 24 h. In the E. coli bacterial survival assay, 4 × MIC of A2 can rapidly reduce the viable counts below the lowest detectable limit after 2 h incubation, and the counts at MIC concentration were maintained under the lowest detectable limit for over 24 h (Figure 2(B)). These results indicate that quinolinium derivatives can inhibit the bacteria growth quickly through the bactericidal mode.</p><!><p>Time-killing curve of A2. (A) At time zero, samples of a growing culture of S. aureus ATCC 29213 were incubated with concentrations of A2 equivalent to 1 × (red), 2 × (green), or 4 × (blue) the MIC. (B) Samples of a growing culture of E. coli ATCC 25922 were incubated with concentrations of A2 equivalent to 1 × (red), 2 × (green), or 4 × (blue) the MIC. Vehicle (1% DMSO; black) was included. Samples were removed at the time intervals indicated for the determination of viable cell counts.</p><!><p>After examining the antibacterial activity of quinolinium derivatives, we further investigated their mode of action. Recent study reported that the antibacterial activity of zantrin Z3 and quinoline derivative (Figure 1) may due to their interferential effect on the GTPase activity of FtsZ13–15. To confirm whether the antibacterial activity of our quinolinium derivatives also follows this mechanism, we studied the effects with three selected compounds (A2, A5, and A15) which possess different antibacterial activities, on the GTPase activity of purified S. aureus FtsZ (SaFtsZ). The results shown that the quinolinium derivatives inhibit GTP hydrolysis of FtsZ in a concentration-dependent manner. For example, A2 at the concentration of 4 μg/mL showed an inhibition of 45% and achieved to 70% inhibitory effect when using 16 μg/mL (Figure 3). Athough A5 and A15 being less effective on the inhibition experiments, the results demonstrated that the compounds are able to inhibit the proliferation of bacteria via influencing the GTPase activity of FtsZ.</p><!><p>Inhibition of GTPase activity of SaFtsZ by quinolinium derivatives A2, A5 and A15.</p><!><p>Recent studies revealed that the dynamic polymerization of FtsZ was determined by its guanosine triphosphatase activity7,8. In order to understand the mechanism of antibacterial activities of these quinolinium derivatives, we used a light scattering assay to assess the impact of selected compounds on the polymerization dynamic of FtsZ. It was found that the selected compounds (A2, A5, and A15) at 4 μg/mL exhibited an obvious enhancement effect on FtsZ polymerization. Vancomycin (10 μg/mL) is also tested as a non-FtsZ-targeting control antibiotic. As expected, it dose not disrupt the FtsZ polymerization. As shown in Figure 4(A), A2 possesses a stronger impact than that of A5 and A15 on the FtsZ polymerization and this result is correlated with their effects on GTPase activity of FtsZ and the antibacterial activity. Figure 4(B) shows the time-dependent polymerization profiles of SaFtsZ in the absence and presence of A2 at a concentration range from 2 to 8 μg/mL. The results confirmed that A2 stimulates FtsZ polymerization in a concentration-dependent manner. The effect of A2 on FtsZ polymerization was also observed via transmission electron microscopy (TEM). It was found that the size of FtsZ polymers were sharply increased after the treatment with A2 at 4 μg/mL (Figure 5). These phenomena suggested that the inhibition of GTPase activity of FtsZ is attributed to the disruption of FtsZ polymerization dynamic.</p><!><p>Effect of quinolinium derivatives on the polymerization of FtsZ. (A) Effect on the polymerization of FtsZ in the absence or in the presence of 4 μg/mL of compound A2, A5, and A15, or 10 μg/mL of vancomycin. (B) The polymerizations of FtsZ in the presence of compound A2 at the concentrations of 2, 4, and 8 μg/mL.</p><p>Electron micrographs of FtsZ polymers in the absence (A) and in the presence (B) of 4 μg/mL of A2.</p><!><p>To further investigate the mechanism of the antibacterial activity of the quinolinium derivatives, we observed the bacterial morphology of B. subtilis incubated with/without A2 through an optical microscopy. In this assay, cetyltrimethylammonium bromide (CTAB, MIC is 1 μg/mL against B. subtilis), a membrane-targeting antiseptic agent and methicillin, a cell wall synthesis inhibitor were used as negative controls. Figure 6(A) shows that untreated B. subtilis cells have typical short rod morphology with cell lengths from 2 to 10 μm. After treatment with 1.5 μg/mL of A2, the cell morphology of B. subtilis was found to become long filament. Most of the cell lengths are longer than 20 μm (Figure 6(B)). On the other hand, cells treated with CTAB or methicillin at MIC concentration did not show any filamentation (Figures 6(C,D)), suggesting the antibacterial mechanism of these quinoliniium derivatives is different to that of CTAB and methicillin. It is noteworthy that similar cell elongation phenomena can also be found in other FtsZ inhibitors such as berberine and benzamide derivatives19,28–30. These results strongly indicated that our quinolinium derivatives inhibit cell division through disrupting GTPase activity and dynamic polymerization of FtsZ, then leading to cell death.</p><!><p>Inhibition of cell division by A2. Cells of B. subtilis 168 were grown in the absence (A), and presence of A2 (B), CTAB (C) and methicillin (D) at the MIC concentration. Scale Bar =10 μm.</p><!><p>To study the binding mode of our quinolinium derivatives in the FtsZ protein, molecular modeling was used to identify a potential binding site for these small molecules in the FtsZ. A 2.01 Å crystal structure of S. aureus FtsZ apo-form26 and A2 were used as models for this purpose. The highest docking score suggested that the ligand bind near the T7-loop and H7-helix of FtsZ (Figure 7(A)). Since the binding site is a relatively narrow cleft composed by T7-loop, H7-helix and a four-stranded β-sheet, the substrate requires some degree of planarity in its structure to fit in. The docking results suggested that a large number of favorable hydrophobic interactions occur between the molecule and the side chains of Asp199, Leu200, Met226, Ile228, Val297, Thr309 and Ile311. Moreover, the cationic pyridinium of A2 is predicted to participate in the charge interaction with the negative charged side chain of Asp199 (Figure 7(B)).</p><!><p>(A) Quinolinium derivative (A2) was predited to bind into the C-terminal interdomain cleft of FtsZ; (B) predicted interactions between A2 and the amino acids of FtsZ.</p><!><p>To probe for any potential mammalian cytotoxicity, the MTS tetrazolium assay was used to assess the cytotoxicity of selected quinolinium derivatives, which possess better antibacterial activity than other derivatives (A2 and A5), against two mammalian cell lines (HK-2, L929). Both tested compounds were found to be minimally toxic to these cell types, with 50% inhibitory concentrations (IC50s) higher than 40 μg/mL (Table 4), which are much higher than their MIC values (1.5 to 3.0 μg/mL) against MRSA and VRE bacterial strains (Table 3), indicating no significant toxicity towards normal mammalian cells.</p><!><p>Cytotoxicity of A2 and A5 on mammalian cells.</p><!><p>In conclusion, a series of new quinolinum derivatives were synthesized by systematically varying a styryl substituent at the ortho-position of 1-methylquinolinium core and their in vitro antibacterial activities were investigated comprehensively. The results indicate that these compounds possess significant antibacterial activity against the tested pathogens including the drug-resistant strains of MRSA, VRE, and NDM-1 E. coli. It is noteworthy that the MIC values of several compounds (A1, A2, A3, A5, A6, and A7) against MRSA strains are range from 1.5 to 4 μg/mL. The MIC values were found comparable to vancomycin and much lower than that of methicillin. In addition, these quinolinum derivatives exhibited potent antibacterial activity against VREs. In particular, A2 has the MIC values against vancomycin-resistant E. faecalis and E. faecium of 2 μg/mL, which are significantly lower than the MIC values of vancomycin (MICs >64 μg/mL). Moreover, A2 and A5 can effectively inhibit the growth of multidrug-resistant Gram-negative strains with the MIC values lower than 8 μg/mL, which is much lower than that of methicillin (MICs >192 μg/mL). In addition, the cytotoxicty values of A2 and A5 are much higher than their MIC values, suggesting these compounds possess little or low toxicity on the mammalian cells. The investigation on the mode of action revealed that the selected compounds can effectively disrupt the GTPase activity and polymerization of FtsZ in a dose-dependent manner. These results suggest that the interaction between quinolinum derivatives and the C-terminal interdomain cleft interferes with the GTPase activity of FtsZ, which in turn disrupts the polymerization of FtsZ, leading to the abnormal bacterial cell division and inhibition of cell proliferation. Therefore, the quinolinum derivatives could be useful in the development of antibacterial agents against drug resistant pathogens.</p><!><p>The authors are also grateful to the support from Nanshan Scholar Program of Guangzhou Medical University and Innovation and Technology Commission of Hong Kong, The Hong Kong Polytechnic University.</p><!><p>No potential conflict of interest was reported by the authors.</p>

PubMed Open Access

Asymmetric Synthesis of Inhibitors of Glycinamide Ribonucleotide Transformylase

Glycinamide ribonucleotide transformylase (GAR Tfase) catalyzes the first of two formyl transfer steps in the de novo purine biosynthetic pathway that require folate cofactors and has emerged as a productive target for antineoplastic therapeutic intervention. The asymmetric synthesis and evaluation of the two diastereomers of 10-methylthio-DDACTHF (10R-3 and 10S-3) and related analogues as potential inhibitors of GAR Tfase are reported. This work, which defines the importance of the C10 stereochemistry for this class of inhibitors of GAR Tfase, revealed that both diastereomers are potent inhibitors of rhGAR Tfase (10R-3 Ki = 210 nM, 10S-3 Ki = 180 nM) that exhibit effective cell growth inhibition (CCRF-CEM IC50 = 80 and 50 nM, respectively) which is dependent on intracellular polyglutamation by folylpolyglutamate synthetase (FPGS), but not intracellular transport by the reduced folate carrier.

asymmetric_synthesis_of_inhibitors_of_glycinamide_ribonucleotide_transformylase

Introduction<!>Inhibitor Synthesis<!>Results and Discussion<!>Conclusions<!>Experimental Section<!>Methyl 4-(1,3-Dithian-2-yl)benzoate (11)<!>Methyl 4-(2-(3-Chloropropyl)-1,3-dithian-2-yl)benzoate (12)<!>Methyl 4-(4-Chlorobutanoyl)benzoate (13)<!>Methyl (S)-4-(4-Chloro-1-hydroxybutyl)benzoate (S-14)<!>Methyl 4-((1S)-5-Cyano-6-ethoxy-1-hydroxy-6-oxohexyl)benzoate (S-15)<!>Methyl 4-((1R)-1-(Acetylthio)-5-cyano-6-ethoxy-6-oxohexyl)benzoate (R-16)<!>Methyl 4-((1R)-5-Cyano-6-ethoxy-1-mercapto-6-oxohexyl)benzoate (R-17)<!>Methyl 4-((1R)-5-cyano-6-ethoxy-1-(methylthio)-6-oxohexyl)benzoate (R-18)<!>Methyl (R)-4-(4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-(methylthio)butyl)benzoate (R-19)<!>(R)-4-(4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-(methylthio)butyl) benzoic Acid (R-20)<!>Di-tert-butyl (S)-2-(4-((R)-4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-(methylthio)butyl)benzamido)pentanedioate (10R-21)<!>(S)-2-(4-((R)-4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-(methylthio)butyl)benzamido)pentanedioic Acid (10R-3)<!>Methyl (S)-4-(4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-hydroxybutyl)benzoate (S-22)<!>(S)-4-(4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-hydroxybutyl)benzoic Acid (S-23)<!>Dimethyl (S)-2-(4-((S)-4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-hydroxybutyl)benzamido)pentanedioate (10S-24)<!>(S)-2-(4-((S)-4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-hydroxybutyl)benzamido)pentanedioic Acid (10S-7)<!>Methyl 4-((1S)-5-Cyano-6-ethoxy-1-methoxy-6-oxohexyl)benzoate (S-25)<!>Methyl (S)-4-(4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-methoxybutyl)benzoate (S-26)<!>(S)-4-(4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-methoxybutyl)benzoic Acid (S-27)<!>Di-tert-butyl (S)-2-(4-((S)-4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-methoxybutyl)benzamido)pentanedioate (10S-28)<!>(S)-2-(4-((S)-4-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)-1-methoxybutyl)benzamido)pentanedioic Acid (10S-8)<!>Methyl 4-(2-(4-Cyano-5-ethoxy-5-oxopentyl)-1,3-dithian-2-yl)benzoate (29)<!>Methyl 4-(2-(3-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)propyl)-1,3-dithian-2-yl)benzoate (30)<!>4-(2-(3-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)propyl)-1,3-dithian-2-yl)benzoic Acid (31)<!>Di-tert-butyl (S)-2-(4-(2-(3-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)propyl)-1,3-dithian-2-yl)benzamido)pentanedioate (32)<!>(S)-2-(4-(2-(3-(2,4-Diamino-6-oxo-1,6-dihydropyrimidin-5-yl)propyl)-1,3-dithian-2-yl)benzamido)pentanedioic Acid (9)<!>GAR and AICAR Tfase Assay<!>Cytotoxic Activity

<p>Glycinamide ribonucleotide transformylase (GAR Tfase) is a folate-dependent enzyme central to the de novo purine biosynthetic pathway.1,2 GAR Tfase utilizes the cofactor (6R)-N10-formyltetrahydrofolic acid (10-formyl-THF) to transfer a formyl group to the primary amine of its substrate, β-glycinamide ribonucleotide (β-GAR) (Figure 1). This one carbon transfer provides the C-8 carbon of the purines and is the first of two formyl transfer reactions enlisted in the biosynthesis of purines. Inhibitors of folate dependent enzymes including GAR Tfase have provided important compounds for cancer chemotherapy as a result of their inhibition of the biosynthesis of nucleic acid precursors.3,4 Validation of GAR Tfase as a useful anticancer target emerged with the discovery of the first potent and selective inhibitor, 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF).5 Its selective toxicity has been attributed in part to the reliance of sensitive tumor cells on de novo purine synthesis, whereas the salvage pathway is an available and primary source of purines in normal untransformed cells.6,7</p><p>In previous studies, we reported the synthesis and biological evaluation of two unique folate-based inhibitors of GAR Tfase. 10-Formyl-DDACTHF8 (1) and 10-CF3CO-DDACTHF9 (2), bearing a nontransferable formyl or trifluoroacetyl group, proved to be potent inhibitors of GAR Tfase (1, Ki = 0.014 μM against rhGAR Tfase; 2, Ki = 0.015 μM against rhGAR Tfase). Both inhibitors have been shown through X-ray and NMR studies to bind GAR Tfase as the gem-diols.9,10 The formation of the gem-diol mimics the formyl transfer reaction tetrahedral intermediate and provides strong stabilizing interactions between the inhibitor and the active site catalytic residues of the protein. In both instances, the inhibitors C10 diastereomers rapidly interconvert preventing independent assessment of the individual diastereomers.</p><p>In a more recent study designed to explore the ability of 10-methanesulfonyl-DDACTHF to also mimic such active site binding interactions, we reported the synthesis and biological activity of its precursor 3, 10-methylthio-DDACTHF, which proved to be a surprisingly potent inhibitor of GAR Tfase (IC50 = 100 nM, CCRF-CEM; Ki = 250 nM, rhGAR Tfase).11 Although the activity of 3 was unexpected since it does not contain a C10 tetrahedral intermediate mimic, the thiomethyl moiety does incorporate a potential hydrogen bond acceptor and presents a soft hydrophobic substituent potentially stabilizing active site binding. The introduction of sulfur within inhibitors of GAR Tfase is well established. The known inhibitors LY309887 (4), AG2034 (5), and AG2037 (6) all incorporate sulfur into their structures although none do so in a manner analogous to 3 (Figure 2),12-14 and it has been reported that analysis of the GAR Tfase active site using the program GRID suggested that sulfur atoms should have particular affinity for two regions of the folate cofactor binding site.13</p><p>The original synthesis of 3 was racemic and the derivative was evaluated as an equi-mixture of C10-diastereomers. However, and unlike 1 and 2, the individual C10-diastereomers of 3 would not be expected to easily interconvert and could be anticipated to be individually evaluated revealing the importance of the C10 stereochemistry to the properties of the candidate inhibitors. Herein, we report an asymmetric synthesis of 10-methylthio-DDACTHF (3) permitting this investigation into the importance of the C10 stereochemistry and report the biological properties of each C10-diastereomer of 10-thiomethyl-DDACTHF (3) along with that of a series of key analogues that further define the importance of the C10 substituent itself: 10-hydroxy-DDACTHF (10R-7 and 10S-7) and 10-methoxy-DDACTHF (10R-8 and 10S-8) and C10 substituted dithiane 9.</p><!><p>Starting from methyl 4-formylbenzoate, the aldehyde was converted to dithiane 11 upon reaction with 1,3-propanedithiol (Scheme 1). The resulting dithiane was alkylated with 1-chloro-3-iodopropane to give 12, and subsequent removal of the dithiane using bis(trifluoro)acetoxyiodobenzene (PIFA) provided 13. Ketone 13 was reduced using the R-Me-CBS15 catalyst in high yield (97%) and high enantiomeric excess (99% ee) to yield alcohol S-14. The absolute stereochemistry of S-14 and its subsequent derivatives were assigned based on the well-established stereochemical preferences of the reagent,16 and confirmed in X-ray structures of both 10R-3 and 10S-3 bound to recombinant human GAR Tfase. The details of these latter studies will be disclosed elsewhere. Similarly, the S-Me-CBS catalyst was utilized to provide the enantiomer of 14 (not shown) and characterization data for this enantiomeric series is provided in the Supporting Information.</p><p>The preformed sodium salt of ethyl cyanoacetate was alkylated with S-14 to give the common intermediate S-15 for all three optically active inhibitors. Treatment of S-15 with thiolacetic acid under modified Mitsunobu conditions provided R-16 with inversion of C10 stereochemistry (Scheme 2). Compound R-16 was subsequently deprotected and the free thiol was methylated to yield R-18. Intermediate R-18 was treated with the free base of guanidine under basic conditions to form the pyrimidine R-19 and this intermediate was subsequently saponified to give R-20. Compound R-20 was coupled with di-tert-butyl l-glutamate and subsequently deprotected using trifluoroacetic acid to provide final inhibitor 10R-3. The C10 diastereomer 10S-3 was prepared using an analogous route employing the enantiomeric R-Me-CBS catalyst to provide R-14 (See Supporting Information).</p><p>The inhibitor 10S-7 bearing a C10-hydroxyl substituent, was prepared from the common intermediate S-15 (Scheme 3). Compound S-15 was treated with guanidine hydrochloride to give the corresponding pyrimidone S-22 which subsequently was saponified using lithium hydroxide to afford S-23. Intermediate S-23 was then coupled with dimethyl l-glutamate and deprotected under basic conditions to provide the final inhibitor 10S-7.</p><p>A key compound for comparison is the C10-methoxy derivative 8 where an oxygen replaces the sulfur of 3 as a probe for its unique importance. The synthesis of inhibitor 10S-8, bearing the C10-methoxy substituent, was accomplished by methylation of the common intermediate S-15 using TMSCHN2 under acidic conditions17 to give S-25 (Scheme 4). Intermediate S-25 was carried forward employing an analogous route, starting with formation of pyrimidone S-26 and saponification to give S-27. Compound S-27 was coupled with di-tert-butyl l-glutamate hydrochloride and deprotected using trifluoroacetic acid to provide 10S-8.</p><p>Finally, 9, which incorporates two C10 sulfur atoms and, in a sense, simultaneously incorporates both C10-diastereomers of 3, was judged as an attractive additional derivative to examine that avoids the presence of two C10-diastereomers. Synthesis of inhibitor 9 started with alkylation of 12 using ethyl cyanoacetate to give 29 (Scheme 5). Intermediate 29 was converted to 9 using the same route detailed for the previous inhibitors, starting with formation of pyrimidine 30 and saponification to give 31. Compound 31 was coupled with di-tert-butyl l-glutamate and deprotected using trifluoroacetic acid to provide 9.</p><!><p>Each diastereomer of 3, 7, 8, and 9 was examined for inhibition of rhGAR Tfase and aminoimidazole carboxamide ribonucleotide transformylase (rhAICAR Tfase), and the results are summarized in Table 1. Remarkably, both diastereomers of the thiomethyl derivative 3 exhibited potent activity against rhGAR Tfase with 10S-3 being slightly more potent than 10R-3 (Ki = 180 nM vs Ki = 210 nM, respectively). This nearly indistinguishable activity of the two C10-diastereomers of 3 is only consistent with both possessing near equivalent capabilities for binding at the GAR Tfase active site and suggests that the flexible linker region joining the pyrimidine and phenyl ring conformationally adjusts to allow a comparable positioning of the thiomethyl substituent in the formyl transfer pocket of the GAR Tfase active site. Inhibitors 10R-7 and 10S-7 displayed very modest activity (Ki = 1060 nM and 1210 nM, respectively) against rhGAR Tfase. This represents a 6-fold reduction in activity relative to 3 and again both diastereomers proved active, although it was the C10-R diastereomer that was slightly more potent than the corresponding C10-S diastereomer. Even more interestingly, methoxy derivatives 10R-8 and 10S-8 were found to inhibit rhGAR Tfase with potencies nearly equivalent to the corresponding alcohols (Ki = 850 nM and 1240 nM, respectively). Again, while both diastereomers exhibited near equivalent potencies, the C10-R diastereomer was slightly more effective, and the 4-6 fold distinction relative to 3 highlights the special nature of the thiomethyl versus methoxy interaction at the enzyme active site. Inhibitor 9 also exhibited a reduced binding to rhGAR Tfase (Ki = 1060 nM), illustrating that optimally one, but not two, thioalkyl substituents may enhance binding at the enzyme active site. These results define a clear role and remarkable stereochemical accommodation of the C10-substituents on DDACTHF with a unique stabilization attributable to the C10-thiomethyl group.</p><p>All inhibitors were also examined for activity against rhAICAR Tfase. The only compound in the series to show any activity against the enzyme was dithiane 9 (Ki = 20 μM). Each diastereomer of the thiomethyl, hydroxyl, and methoxy inhibitors was inactive against rhAICAR Tfase (Ki >100 μM) illustrating that they disrupt de novo purine biosynthesis by selectively targeting GAR Tfase.</p><p>The compounds were also examined for cytotoxic activity (growth inhibition) both in the presence (+) and absence (-) of added hypoxanthine (purine) or thymidine (pyrimidine) against the CCRF-CEM cell line (Table 2). All the inhibitors exhibited activity that paralleled their potency against rhGAR Tfase. Of all the inhibitors, only 10R-3 and 10S-3 exhibited potent activity in this cell-based assay (IC50 = 80 and 50 nM, respectively), being only 3-5 times less potent than 2, the most potent inhibitor of GAR Tfase disclosed to date. While both C10-diastereomers displayed this potent cytotoxic activity, the 10S-diastereomer proved to be slightly more active than the 10R-diastereomer, and both display a potency that slightly exceeds that observed on the isolated enzyme. Compounds 10R-7, 10S-7, 10R-8, and 10S-8 were all approximately ten times less potent than the thiomethyl inhibitors. All still showed modest activity and there was little distinction between the individual C10 diastereomers. Inhibitor 9, on the other hand, showed no activity in this cell-based assay. An especially interesting set of comparisons are the activities of the two diastereomers of 3 and the related inhibitors with DDACTHF lacking a C10 substituent. These comparisons indicate that each of the substituents actually enhance, or at least maintain, the GAR Tfase inhibition of DDACTHF and enhance and maintain its corresponding cell growth inhibitory activity. Thus, the differences observed among one another and relative to DDACTHF may be attributed to relative binding enhancements provided by the key C10 substituents and not to destabilizing interactions they introduce at the enzyme active site. Finally, in the presence of thymidine, a pyrimidine, all inhibitors retained their activity, whereas the inhibitors were inactive in the presence of added hypoxanthine. This indicates that these compounds, like 2, inhibit cell growth by selectively inhibiting an enzyme within the purine biosynthetic (not pyrimidine) pathway consistent with their potent inhibition of GAR Tfase.</p><p>All seven inhibitors were subsequently examined in mutant CCRF-CEM cell lines that lack folypolyglutamate synthetase (CCRF-CEM/FPGS-) or the reduced folate carrier (CCRF-CEM/MTX) (Table 3). Like the past inhibitors of GAR Tfase that we have examined, all inhibitors including 3 lost activity against the cell line that lacks FPGS (ca. 100-fold for 3), indicating that they benefit from intracellular polyglutamation. This observation suggests that the potent activity of both diastereomers of 3 in the cell growth assays, which exceed their inhibition of GAR Tfase roughly 3-fold, may be attributed to their FPGS polyglutamation and the resulting intracellular accumulation or enhanced target affinity. In contrast, none of the inhibitors disclosed herein exhibited altered activity against the cell line lacking the reduce folate carrier indicating its transport of the inhibitors is not essential to their activity. Thus, cell lines and tumors that derive their resistance to antifolates through down regulation of the reduced folate carrier would be expected to remain sensitive to the two diastereomers of 3.</p><!><p>Examination of the importance of the C10 stereochemistry of DDACTHF-based inhibitors was addressed with the asymmetric synthesis of the two diastereomers of 10-methylthio-DDACTHF (3) and a series of key analogues incapable of in situ racemization of the C10 center. Remarkably, the activities of the C10-substituted DDACTHF derivatives proved to be essentially independent of the C10 stereochemistry and those bearing a C10-thiomethyl substituent were surprisingly potent and efficacious. Presumably this reflects the conformational flexibility of the linking region between the pyrimidine and phenyl group which contains the C10-substituent that allows a comparable positioning of the thiomethyl group in the formyl transfer pocket of the GAR Tfase active site. In addition to being substantially more potent than the corresponding C10-methoxy derivatives highlighting the unique properties embodied in 3, the two diastereomers of 3 proved especially efficacious in inhibiting cell growth (CCRF-CEM IC50 = 80 and 50 nM for 10R-3 and 10S-3, respectively) that could be rescued by added purine (but not pyrimidine) and that relies on intracellular FPGS polyglutamation, but not intracellular transport by the reduced folate carrier. Similarly, both diastereomers of 3 were found to be potent and essentially indistinguishable inhibitors of rhGAR Tfase (Ki = 210 and 180 nM for 10R-3 and 10S-3, respectively), albeit with potencies that are slightly lower than their actual cell growth inhibitory activity. This latter observation suggests that the required intracellular polyglutamation observed with 3 functionally enhances its potency in the cell-based assays.</p><!><p>Characterization of the compounds with the alternative C10-stereochemistry is provided in the Supporting Information. Our use of "concentrated" in the following experimental refers to evaporation to dryness on a rotary evaporator.</p><!><p>A solution of methyl 4-formylbenzoate (14.10 g, 85.9 mmol) in anhydrous CH2Cl2 (282 mL) was treated with 1,3-propanedithiol (9.48 mL, 94.5 mmol, 1.1 equiv) and the mixture was stirred for 1 h at room temperature. The solution was cooled to 0 °C and boron trifluoride diethyl etherate was added (11.9 mL, 94.5 mmol, 1.1 equiv). The reaction mixture was stirred and allowed to warm to room temperature overnight. The mixture was diluted with CH2Cl2 and quenched with the addition of saturated aqueous NaHCO3. The organic layer was washed with H2O and saturated aqueous NaCl, dried over Na2SO4, and concentrated (evaporated to dryness on a rotary evaporator). Column chromatography (SiO2, 25-50% EtOAc/hexanes gradient) yielded 11 (21.0 g, 96%) as a white solid: mp 132-134°C; 1H NMR (500 MHz, CDCl3) δ 8.02 (d, 2H, J = 8.3 Hz), 7.55 (d, 2H, J = 8.3 Hz), 5.22 (s, 1H), 3.92 (s, 3H), 3.10-3.04 (m, 2H), 2.96-2.92 (m, 2H), 2.22-2.19 (m, 1H), 1.99-1.95 (m, 1H); 13C NMR (125 MHz, CDCl3) δ 166.8, 144.2, 130.3 (2C), 130.2, 128.1 (2C), 52.4, 51.3, 32.1 (2C), 25.2; IR (film) νmax 2903, 1707, 1272, 1108 cm-1 ; ESI-TOF m/z 255.0507 (M + H+ , C12H14O2S2 requires 255.0508).</p><!><p>A solution of 11 (20.96 g, 82.4 mmol) in THF (629 mL) cooled to -78 °C was treated with NaHMDS (2 M in THF, 51.5 mL, 103 mmol, 1.25 equiv) dropwise. The solution was stirred for 30 min followed by the addition of 1-chloro-3-iodopropane (44.3 mL, 412 mmol, 5.0 equiv). The reaction mixture was allowed to warm to room temperature overnight and was quenched with the addition of saturated aqueous NH4Cl. The mixture was diluted with EtOAc and subsequently washed with saturated aqueous NaCl, dried over Na2SO4, and concentrated. Column chromatography (SiO2, 0-10% EtOAc/hexanes gradient) yielded 12 (21.73 g, 80%) as a white solid: mp 75-77 °C; 1H NMR (500 MHz, CDCl3) δ 8.05 (d, 2H, J = 8.7 Hz), 8.00 (d, 2H, J = 8.7 Hz), 3.92 (s, 3H), 3.40 (t, 2H, J = 6.4 Hz), 2.75-2.60 (m, 4H), 2.17-2.14 (m, 2H), 1.96-1.93 (m, 2H), 1.75-1.70 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 166.8, 147.2, 130.0 (2C), 129.2, 129.0 (2C), 58.1, 52.3, 44.7, 42.3, 27.8 (2C), 27.3, 25.0; IR (film) νmax 2944, 1723, 1277 cm-1 ; ESI-TOF m/z 331.0587 (M + H+ , C15H19ClO2S2 requires 331.0588).</p><!><p>A solution of 12 (10.79 g, 32.6 mmol) in 9:1 MeCN/H2O (163 mL) was treated with bis(trifluoro)acetoxyiodobenzene (21.0 g, 48.9 mmol, 1.5 equiv) and allowed to stir for 30 min. The reaction mixture was quenched with the addition of saturated aqueous NaHCO3 and diluted with EtOAc. The mixture was extracted with H2O (2×) and saturated aqueous NaCl, dried over Na2SO4, and concentrated. Column chromatography (SiO2, 20% EtOAc/hexanes) provided 13 (6.37 g, 81%) as a white solid: mp 45-46 °C; 1H NMR (500 MHz, CDCl3) δ 8.15 (d, 2H, J = 8.3Hz), 8.04 (d, 2H, J = 8.2 Hz), 3.96 (s, 3H), 3.70 (t, 2 H, J = 6.3 Hz), 3.22 (t, 2H, J = 6.9 Hz), 2.28-2.22 (m, 2H); 13C NMR (125 MHz, CDCl3) δ 198.6, 166.3, 140.0, 134.1, 130.0 (2C), 128.0 (2C), 52.6, 44.6, 35.8, 26.7; IR (film) νmax 2944, 1723, 1682, 1272, 1108 cm-1 ; ESI-TOF m/z 241.0624 (M + H+ , C12H13ClO3 requires 241.0626).</p><!><p>A solution of R-Me-CBS (1 M in toluene, 1.09 mL, 1.09 mmol, 0.1 equiv) in anhydrous THF (10.1 mL) cooled to 0 °C was treated with borane dimethysulfide complex (2 M in THF, 6.80 mL, 13.6 mmol, 1.25 equiv) and stirred for 15 min at 0 °C. A solution of 13 (2.62 g, 10.9 mmol) in THF (54.4 mL) was added dropwise to the reaction mixture and stirred for 1 h, allowing the reaction mixture to warm to room temperature. The reaction was quenched with the addition of MeOH and allowed to stir for 30 min at room temperature. The mixture was diluted with EtOAc and washed with 1 N HCl (3×), H2O (2×), dried over Na2SO4, and concentrated. Column chromatography (SiO2, 40% EtOAc/hexanes) afforded S-14 (2.55 g, 97%) as a clear oil: 1H NMR (500 MHz, CDCl3) δ 8.04 (d, 2H, J = 8.2 Hz), 7.44 (d, 2H, J = 8.4 Hz), 4.81 (t, 1H, J = 5.9 Hz), 3.92 (s, 3H), 3.60-3.53 (m, 2H), 1.97-1.82 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 167.0, 149.6, 130.0 (2C), 129.5, 125.8 (2C), 73.4, 52.2, 45.0, 36.3, 28.8; IR (film) νmax 2923, 1748, 1277 cm-1 ; ESI-TOF m/z 243.0781 (M + H+ , C12H15ClO3 requires 243.0782); [α]D -30 (c 1.0, CHCl3). Enantiomeric excess was determined (99% ee) using a Chiralcel AD-H analytical column (250 mm × 4.6 mm, 3% i-PrOH/hexane, 1 mL/min, α = 1.07).</p><!><p>A suspension of NaH (60% dispersion, 1.11 g, 27.7 mmol, 3.0 equiv) in anhydrous DMF (27.7 mL) was treated with ethyl cyanoacetate (2.96 mL, 27.7 mmol, 3.0 equiv) at 0 °C. The reaction mixture was stirred for 30 min while allowing the mixture to warm to room temperature, forming the sodium salt as a clear solution. A solution of S-14 (2.24 g, 9.24 mmol) in DMF (18.5 mL) was added at room temperature and the reaction mixture was stirred at 60 °C for 12 h. The reaction mixture was cooled to room temperature and quenched with the addition of saturated aqueous NH4Cl. The mixture was concentrated and then diluted with EtOAc. The organic layer was washed with H2O and saturated aqueous NaCl, dried over Na2SO4, and concentrated. Column chromatography (SiO2, 50% EtOAc/hexanes) provided S-15 (1.76 g, 60%) as a yellow oil: 1H NMR (500 MHz, CDCl3) δ 8.04 (d, 2H, J = 8.2 Hz), 7.43 (d, 2H, J = 8.2 Hz), 4.78 (t, 1H, J = 6.0 Hz), 4.26/4.25 (two q, 2H, J = 7.1 Hz), 3.92 (s, 3H), 3.49/3.48 (two t, 1H, J = 6.9 Hz), 2.00-1.97 (m, 2H), 1.85-1.65 (m, 4H), 1.31/1.30 (two t, 3H, J = 7.1 Hz); 13C NMR (125 MHz, CDCl3) δ 167.0, 166.1, 149.6, 130.0 (2C), 129.6, 125.8 (2C), 116.5, 73.6, 63.0, 52.2, 38.1, 37.6, 29.8, 23.2, 14.1; IR (film) νmax 3467, 2923, 1744, 1713, 1282, 1195, 1113 cm-1; ESI-TOF m/z 342.1309 (M + Na+, C17H21NO5 requires 342.1312).</p><!><p>A cooled (0 °C) solution of triphenylphosphine (358 mg, 1.36 mmol, 2.0 equiv) in THF (3.4 mL) was treated with DIAD (270 μL, 1.36 mmol, 2.0 equiv). The reaction mixture was stirred at 0 °C for 30 min before a solution of S-15 (218 mg, 0.68 mmol) and thiolacetic acid (121 uL, 1.36 mmol, 2.0 equiv) in THF (1.7 mL) was added dropwise. The mixture was stirred for 1 h, while allowing it to warm to room temperature. The reaction mixture was diluted with EtOAc, washed with saturated aqueous NaHCO3 (3×), dried over Na2SO4, and concentrated. Column chromatography (SiO2, 30% EtOAc/hexanes) yielded R-16 (246 mg, 95%) as a yellow oil: 1H NMR (500 MHz, CDCl3) δ 8.00 (d, 2H, J = 8.3 Hz), 7.36 (d, 2H, J = 8.2 Hz), 4.60 (t, 1H, J = 7.7 Hz), 4.25/4.24 (two q, 2H, J = 7.1 Hz), 3.91 (s, 3H), 3.47-3.43 (m, 1H), 2.31 (s, 3H), 2.02-1.94 (m, 4H), 1.54-1.43 (m, 2H), 1.29/1.28 (two t, 3H, J = 7.0 Hz); 13C NMR (125 MHz, CDCl3) δ 194.4, 166.8, 165.9, 146.6, 130.2 (2C), 129.5, 127.7 (2C), 116.4, 63.0, 52.3, 47.2, 37.4, 35.1, 30.6, 29.4, 24.7, 14.1; IR (film) νmax 3733, 2974, 1733, 1641, 1256, 1108 cm-1 ; ESI-TOF m/z 400.1191 (M + Na+ , C19H23NO5S requires 400.1189).</p><!><p>A solution of R-16 (2.01 g, 5.3 mmol) in EtOH (26.6 mL) was treated with K2CO3 (3.68 g, 26.6 mmol, 5.0 equiv) and stirred for 12 h. The reaction mixture was diluted with EtOAc and washed with H2O and saturated aqueous NaCl, dried over Na2SO4, and concentrated. Column chromatography (SiO2, 30% EtOAc/hexanes) yielded the unstable free thiol R-17 (291 mg, 16%) as a colorless oil: 1H NMR (600 MHz, CDCl3) δ 8.00 (d, 2H, J = 7.8 Hz), 7.38 (d, 2H, J = 8.2 Hz), 4.26-4.21 (m, 2H), 4.01-3.98 (m, 1H), 3.91 (s, 3H), 3.47-3.43 (m, 1H), 1.99-1.92 (m, 4H), 1.96 (d, 1H, J = 5.6 Hz), 1.66-1.58 (m, 1H), 1.49-1.40 (m, 1H), 1.30-1.26 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 166.7, 165.9, 149.4, 130.3 (2C), 129.4, 126.9 (2C), 116.4, 63.0, 52.2, 43.4, 38.6, 37.4, 29.4, 25.2, 14.1; IR (film) νmax 2933, 1718, 1436, 1271, 1184, 1102 cm-1 ; ESI-TOF m/z 336.1267 (M + H+ , C17H21NO4S requires 336.1264).</p><!><p>Free thiol R-17 (291 mg, 0.87 mmol) dissolved in MeOH (8.68 mL) was treated with MeI (542 μL, 8.68 mmol, 10 equiv) and NaHCO3 (80 mg, 0.97 mmol, 1.1 equiv) and the mixture was stirred for 3 h at room temperature. The reaction mixture was diluted with EtOAc and washed with H2O and saturated aqueous NaCl, dried over Na2SO4, and concentrated. Column chromatography (SiO2, 30% EtOAc/hexanes) provided R-18 (238 mg, 79%) as a colorless oil: 1H NMR (600 MHz, CDCl3) δ 8.01 (d, 2H, J = 7.3 Hz), 7.37 (d, 2H, J = 7.8 Hz), 4.26-4.20 (m, 2H), 3.91 (s, 3H), 3.69 (t, 1H, J = 7.4 Hz), 3.46-3.41 (m, 1H), 1.99-1.87 (m, 3H), 1.85 (s, 3H), 1.64-1.56 (m, 1H), 1.50-1.45 (m, 1H), 1.31-1.26 (m, 3H); 13C NMR (150 MHz, CDCl3) δ 166.9, 166.0, 147.4, 130.1 (2C), 129.3, 127.9 (2C), 116.4, 63.0, 52.2, 50.9, 37.5, 35.1, 29.6, 25.1, 14.4, 14.1; IR (film) νmax 3358, 2923, 1718, 1431, 1267, 1097, 1010 cm-1 ; ESI-TOF m/z 372.1252 (M + Na+ , C18H23NO4S requires 372.1240).</p><!><p>A solution of NaOMe (60 mg, 1.12 mmol, 3.0 equiv) in anhydrous MeOH (3 mL) was treated with guanidine hydrochloride (107 mg, 1.12 mmol, 3.0 equiv) at room temperature. After the reaction mixture was stirred for 30 min, a solution of R-18 (130 mg, 0.372 mmol) in MeOH (2.10 mL) was added. The reaction mixture was warmed at reflux for 16 h before being quenched with acetic acid after cooling to room temperature. After concentration, column chromatography (SiO2, 10% MeOH/CH2Cl2) gave R-19 (39 mg, 29%) as a white solid: mp 180-185 °C; 1H NMR (500 MHz, CD3OD) δ 7.96 (d, 2H, J = 8.4 Hz), 7.43 (d, 2H, J = 8.4 Hz), 3.89 (s, 3H), 3.81 (t, 1H, J = 6.7 Hz), 2.29 (t, 2H, J = 7.2 Hz), 1.97-1.86 (m, 2H), 1.84 (s, 3H), 1.53-1.47 (m, 1H), 1.42-1.34 (m, 1H); 13C NMR (125 MHz, CD3OD) δ 168.4, 165.2, 164.1, 154.8, 150.2, 130.6 (2C), 129.8, 129.2 (2C), 89.8, 52.6, 52.2, 36.3, 27.2, 23.1, 14.2; IR (film) νmax 3353, 2933, 1713, 1610, 1431, 1282, 1108 cm-1 ; ESI-TOF m/z 363.1489 (M + H+ , C17H22N4O3S requires 363.1485); [α]D +70 (c 0.1, MeOH).</p><!><p>A solution of R-19 (39 mg, 0.11 mmol) in MeOH (1.5 mL) was treated with LiOH monohydrate (23 mg, 0.54 mmol, 5 equiv) in water (840 μL), and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was diluted with H2O, washed with EtOAc, acidified to pH 4 by the addition of aqueous 1 N HCl, and concentrated. The resulting white solid, R-20 (38 mg, 100%), was used directly in the next step: mp 210 °C (dec); 1H NMR (500 MHz, CD3OD) δ 7.97 (d, 2H, J = 8.3 Hz), 7.43 (d, 2H, J = 8.4 Hz), 3.81 (t, 1H, J = 6.8 Hz), 2.35-2.31 (m, 2H), 1.97-1.87 (m, 2H), 1.85 (s, 3H), 1.55-1.48 (m, 1H), 1.43-1.36 (m, 1H); 13C NMR (125 MHz, CD3OD) δ 169.6, 164.3, 153.4, 152.6, 149.8, 130.8 (2C), 130.5, 129.1 (2C), 89.6, 52.0, 36.2, 26.9, 22.5, 14.3; IR (film) νmax3356, 1569, 1405, 1241 cm-1; ESI-TOF m/z 349.1336 (M + H+ , C16H20N4O3S requires 349.1329); [α]D +30 (c 0.1, MeOH).</p><!><p>A solution of R-20 (38 mg, 0.11 mmol), di-tert-butyl l-glutamate hydrochloride (48 mg, 0.16 mmol, 1.5 equiv) and NaHCO3 (27 mg, 0.32 mmol, 3.0 equiv) in DMF (1.08 mL) was treated with EDCI (62 mg, 0.32 mmol, 3.0 equiv). The reaction mixture was stirred for 24 h at room temperature before the addition of CHCl3. The resulting solution was washed with saturated aqueous NaHCO3 (2×), dried over Na2SO4, and concentrated. Column chromatography (SiO2, 10% MeOH/CH2Cl2) provided the protected glutamate intermediate 10R-21 (27 mg, 43%) as a white solid: mp 108-112 °C; 1H NMR (500 MHz, CD3OD) δ 7.80 (d, 2H, J = 8.3 Hz), 7.43 (d, 2H, J = 8.3 Hz), 4.52-4.49 (m, 1H), 3.81 (t, 1H, J = 6.8 Hz), 2.40 (t, 2H, J = 7.4 Hz), 2.30 (t, 2H, J = 7.6 Hz), 2.24-2.17 (m, 1H), 2.06-1.99 (m, 1H), 1.97-1.86 (m, 2H), 1.84 (s, 3H), 1.53-1.50 (m, 1H), 1.49 (s, 9H), 1.44 (s, 9H), 1.41-1.36 (m, 1H); 13C NMR (125 MHz, CD3OD) δ 173.9, 172.7, 170.2, 165.1, 164.2, 154.8, 148.6, 133.7, 129.2 (2C), 128.6 (2C), 89.8, 83.0, 81.9, 54.5, 52.1, 36.4, 32.9, 28.3 (6C), 27.6, 27.3, 23.1, 14.2; IR (film) νmax 2913, 1733, 1631, 1149 cm-1 ; ESI-TOF m/z 590.2999 (M + H+ , C29H43N5O6S requires 590.3007); [α]D +36 (c 0.1, MeOH).</p><!><p>A solution of 10R-21 (21 mg, 0.035 mmol) in CHCl3 (1.26 mL) was treated with trifluoroacetic acid (1 mL) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred overnight. The solution was concentrated and triturated with Et2O to give 10R-3 (20 mg, 100%) as a white solid: mp 190 °C (dec); 1H NMR (500 MHz, CD3OD) δ 7.82 (d, 2H, J = 8.4 Hz), 7.42 (d, 2H, J = 8.3 Hz), 4.66-4.63 (m, 1H), 3.81 (t, 1H, J = 6.6 Hz), 2.49 (t, 2H, J = 7.5 Hz), 2.35-2.28 (m, 3H), 2.15-2.07 (m, 1H), 1.98-1.91 (m, 1H), 1.88-1.82 (m, 1H), 1.84 (s, 3H), 1.54-1.47 (m, 1H), 1.42-1.33 (m, 1H); 13C NMR (125 MHz, CD3OD) δ 176.6, 175.1, 170.2, 164.6, 154.8, 153.0, 148.4, 133.8, 129.2, 128.6, 89.6, 53.8, 52.0, 36.3, 31.5, 27.6, 26.9, 22.6, 14.3; IR (film) νmax 2964, 1653, 1544, 1210 cm-1 ; ESI-TOF m/z 478.1752 (M + H+ , C21H27N5O6S requires 478.1755); [α]D +32 (c 0.1, MeOH). Agilent 1100 LC/MS: reverse phase (2-40% acetonitrile/water/0.1% formic acid, flow rate 0.75 mL/min); tR, 12.0 min; purity, 99.2%.</p><!><p>A solution of NaOMe (270 mg, 5.01 mmol, 3.0 equiv) in anhydrous MeOH (13.8 mL) was treated with guanidine hydrochloride (478 mg, 5.01 mmol, 3.0 equiv) at room temperature. After the reaction mixture was stirred for 30 min, a solution of S-15 (533 mg, 1.67 mmol) in MeOH (9.4 mL) was added. The reaction mixture was warmed at reflux for 16 h and quenched with acetic acid after cooling to room temperature. After concentration, column chromatography (SiO2, 10% MeOH/CH2Cl2) gave S-22 (336 mg, 61%) as a white solid: mp 120 °C (dec); 1H NMR (500 MHz, CD3OD) δ 7.97 (d, 2H, J = 8.4 Hz), 7.46 (d, 2H, J = 8.2 Hz), 4.73 (t, 1H, J = 7.4 Hz), 3.89 (s, 3H), 2.35-2.32 (m, 2H), 1.77-1.74 (m, 2H), 1.61-1.52 (m, 1H), 1.50-1.42 (m, 1H); 13C NMR (125 MHz, CD3OD) δ 168.0, 164.7, 163.7, 152.1, 152.0, 130.0 (2C), 129.9, 126.6 (2C), 89.5, 74.1, 52.0, 39.1, 24.9, 22.7; IR (film) νmax 3333, 2912, 1718, 1615, 1436, 1287, 1102 cm-1 ; ESI-TOF m/z 333.1562 (M + H+ , C16H20N4O4 requires 333.1557); [α]D -8 (c 0.1, MeOH).</p><!><p>A solution of S-22 (36 mg, 0.11 mmol) in MeOH (1.5 mL) was treated with LiOH monohydrate (23 mg, 0.54 mmol, 5 equiv) in water (840 μL), and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was diluted with H2O, washed with EtOAc, acidified to pH 4 by the addition of aqueous 1 N HCl, and concentrated. The resulting white solid, S-23 (35 mg, 100%), was used directly in the next step: mp 220 °C (dec); 1H NMR (500 MHz, CD3OD) δ 7.98 (d, 2H, J = 8.3 Hz), 7.45 (d, 2H, J = 8.4 Hz), 4.73 (t, 1H, J = 7.3 Hz), 2.36 (t, 2H, J = 7.4 Hz), 1.78-1.73 (m, 2H), 1.61-1.55 (m, 1H), 1.50-1.44 (m, 1H); 13C NMR (125 MHz, CD3OD) δ 169.8, 165.2, 163.5, 158.4, 152.2, 130.8 (2C), 130.5, 127.0 (2C), 89.9, 74.6, 39.4, 25.2, 22.8; IR (film) νmax 3374, 2923, 1636, 1390 cm-1; ESI-TOF m/z 319.1405 (M + H+ , C15H18N4O4 requires 319.1401); [α]D -12 (c 0.1, MeOH).</p><!><p>A solution of S-23 (29 mg, 0.090 mmol), dimethyl l-glutamate hydrochloride (29 mg, 0.14 mmol, 1.5 equiv) and NaHCO3 (23 mg, 0.27 mmol, 3.0 equiv) in DMF (0.90 mL) was treated with EDCI (52 mg, 0.27 mmol, 3.0 equiv). The reaction mixture was stirred for 24 h at room temperature before being concentrated. Column chromatography (SiO2, 20% MeOH/CH2Cl2) provided 10S-24 (31 mg, 72%) as a white solid: mp 105-110 °C; 1H NMR (600 MHz, CD3OD) δ 7.82 (d, 2H, J = 8.4 Hz), 7.46 (d, 2H, J = 7.8 Hz), 4.73 (t, 1H, J = 6.0 Hz), 4.65-4.63 (m, 1H), 3.75 (s, 3H), 3.65 (s, 3H), 2.50 (t, 2H, J = 7.2 Hz), 2.35-2.27 (m, 3H), 2.14-2.09 (m, 1H), 1.79-1.74 (m, 2H), 1.57-1.54 (m, 1H), 1.46-1.43 (m, 1H); 13C NMR (150 MHz, CD3OD) δ 174.9, 173.7, 170.3, 165.2, 164.3, 154.8, 151.2, 133.5, 128.5 (2C), 127.1 (2C), 90.0, 74.7, 60.4, 53.7, 52.8, 52.2, 39.7, 31.3, 27.4, 25.5, 23.3; IR (film) νmax 3364, 1723, 1631, 1441 cm-1 ; ESI-TOF m/z 476.2142 (M + H+ , C22H29N5O7 requires 476.2140); [α]D -18 (c 0.1, MeOH).</p><!><p>A solution of 10S-24 (26 mg, 0.055 mmol) in MeOH (0.55 mL) was treated with aqueous 1 N NaOH (0.22 mL, 4 equiv). The reaction mixture was stirred for 18 h and then acidified with the addition of Dowex 50X8-200. The solution was filtered and concentrated to give 10S-7 (21 mg, 87%) as a white solid: mp 200 °C (dec); 1H NMR (500 MHz, CD3OD) δ 7.82 (d, 2H, J = 8.3 Hz), 7.45 (d, 2H, J = 8.3 Hz), 4.72 (t, 1H, J = 6.1 Hz), 4.64-4.61 (m, 1H), 2.40-2.24 (m, 5H), 2.17-2.08 (m, 1H), 1.79-1.74 (m, 2H), 1.59-1.54 (m, 1H), 1.49-1.42 (m, 1H); 13C NMR (150 MHz, CD3OD) δ 176.7, 175.1, 174.3, 170.3, 163.7, 163.6, 151.0, 133.8, 128.5 (2C), 127.1 (2C), 89.9, 74.6, 53.8, 39.4, 35.4, 31.0, 25.2, 23.0; IR (film) νmax 3333, 1600, 1451, 1405, 1318, 1108 cm-1 ; ESI-TOF m/z 448.1836 (M + H+ , C20H25N5O7 requires 448.1827); [α]D +4 (c 0.1, MeOH). Agilent 1100 LC/MS: reverse phase (2-40% acetonitrile/water/0.1% formic acid, flow rate 0.75 mL/min); tR, 9.9 min; purity, 99.5%.</p><!><p>A solution of S-15 (246 mg, 0.77 mmol), 49% aqueous fluoroboric acid (97 μL, 1.54 mmol, 2 equiv) in CH2Cl2 (3.08 mL) was cooled to 0 °C. TMSCHN2 (2 M in hexanes, 0.385 mL, 0.77 mmol, 1 equiv) was added to the mixture and stirring was continued at 0 °C. Three further portions of TMSCHN2 (0.193 mL (0.5 equiv), 0.097 mL (0.25 equiv), and 0.097 mL (0.25 equiv)) were added dropwise at intervals of 20 min. The reaction mixture was stirred a further 30 min, poured into H2O and extracted with EtOAc. The organic layer was washed with H2O, dried over Na2SO4, filtered, and concentrated. Column chromatography (SiO2, 30% EtOAc/hexanes) gave S-25 (110 mg, 43%) as a colorless oil: 1H NMR (400 MHz, CDCl3) δ 8.04 (d, 2H, J = 8.2 Hz), 7.36 (d, 2H, J = 8.1 Hz), 4.28-4.22 (m, 2H), 4.18-4.15 (m, 1H), 3.92 (s, 3H), 3.49-3.45 (m, 1H), 3.22 (s, 1.5H), 3.21 (s, 1.5H), 1.98-1.93 (m, 2H), 1.82-1.78 (m, 1H), 1.71-1.63 (m, 2H), 1.54-1.48 (m, 1H), 1.32-1.28 (m, 3H); 13C NMR (125 MHz, CDCl3) δ 166.9, 166.0, 147.3, 129.9 (2C), 129.7, 126.5 (2C), 116.5, 83.1, 62.8, 57.0, 52.1, 37.6, 37.2, 29.7, 23.2, 14.0; IR (film) νmax 3405, 1708, 1272, 1103 cm-1 ; ESI-TOF m/z 356.1465 (M + Na+ , C18H23NO5 requires 356.1468).</p><!><p>A solution of NaOMe (44 mg, 0.81 mmol, 3.0 equiv) in anhydrous MeOH (2.2 mL) was treated with guanidine hydrochloride (77 mg, 0.81 mmol, 3.0 equiv) at room temperature. After the reaction mixture was stirred for 30 min, a solution of S-25 (90 mg, 0.27 mmol) in MeOH (1.53 mL) was added. The reaction mixture was warmed at reflux for 16 h and quenched with acetic acid after cooling to room temperature. After concentration, column chromatography (SiO2, 10% MeOH/CH2Cl2) gave S-26 (27 mg, 29%) as a white solid: mp 160-164 °C ; 1H NMR (600 MHz, CD3OD) δ 8.00 (d, 2H, J = 8.4 Hz), 7.41 (d, 2H, J = 7.8 Hz), 4.28-4.25 (m, 1H), 3.90 (s, 3H), 3.20 (s, 3H), 2.30 (t, 2H, J = 7.5 Hz), 1.84-1.78 (m, 1H), 1.68-1.62 (m, 1H), 1.56-1.52 (m, 1H), 1.43-1.36 (m, 1H); 13C NMR (150 MHz, CD3OD) δ 168.4, 165.1, 164.0, 154.8, 149.3, 130.7 (2C), 130.4, 127.9 (2C), 89.8, 84.4, 57.1, 52.6, 38.5, 25.4, 23.2; IR (film) νmax 3354, 2933, 1610, 1431, 1282 cm-1 ; ESI-TOF m/z 347.1729 (M + H+ , C17H22N4O4 requires 347.1714); [α]D -30 (c 0.1, MeOH).</p><!><p>The intermediate pyrimidine S-26 (30 mg, 0.086 mmol) in MeOH (1.2 mL) was treated with LiOH monohydrate (18 mg, 0.43 mmol, 5 equiv) in water (675 μL), and the reaction mixture was stirred at room temperature for 24 h. The reaction mixture was diluted with H2O, washed with EtOAc, acidified to pH 4 by the addition of 1 N aqueous HCl, and concentrated. The resulting white solid, S-27 (29 mg, 100%), was used directly in the next step: mp 175 °C (dec); 1H NMR (600 MHz, CD3OD) δ 7.95 (d, 2H, J = 8.2 Hz), 7.32 (d, 2H, J = 8.2 Hz), 4.24 (t, 1H, J = 5.8 Hz) 3.19 (s, 3H), 2.30 (t, 2H, J = 7.5 Hz), 1.84-1.78 (m, 1H), 1.70-1.63 (m, 1H), 1.56-1.52 (m, 1H), 1.42-1.36 (m, 1H); 13C NMR (150 MHz, CD3OD) δ 173.2, 165.1, 164.0, 154.9, 147.3, 135.4, 130.6 (2C), 127.4 (2C), 90.0, 85.2, 57.0, 38.5, 25.5, 23.3; IR (film) νmax 3385, 2923, 1626, 1395, 1246, 1087 cm-1 ; ESI-TOF m/z 333.1563 (M + H+ , C16H20N4O4 requires 333.1557); [α]D -20 (c 0.1, MeOH).</p><!><p>A solution of S-27 (29 mg, 0.086 mmol), di-tert-butyl l-glutamate hydrochloride (38 mg, 0.13 mmol, 1.5 equiv) and NaHCO3 (22 mg, 0.26 mmol, 3.0 equiv) in DMF (0.86 mL) was treated with EDCI (50 mg, 0.26 mmol, 3.0 equiv). The reaction mixture was stirred for 24 h at room temperature before the addition of CHCl3. The resulting solution was washed with saturated aqueous NaHCO3 (2×), dried over Na2SO4, and concentrated. Column chromatography (SiO2, 10% MeOH/CH2Cl2) provided 10S-28 (14 mg, 28%) as a white solid: mp 120-125 °C; 1H NMR (600 MHz, CD3OD) δ 7.83 (d, 2H, J = 7.8 Hz), 7.40 (d, 2H, J = 7.8 Hz), 4.51-4.49 (m, 1H), 4.27 (t, 1H, J = 7.4 Hz), 3.21 (s, 3H), 2.40 (t, 2H, J = 7.2 Hz), 2.30 (t, 2H, J = 7.2 Hz), 2.24-2.18 (m, 1H), 2.05-1.99 (m, 1H), 1.85-1.79 (m, 1H), 1.70-1.64 (m, 1H), 1.56-1.52 (m, 1H), 1.49 (s, 9H), 1.44 (s, 9H), 1.41-1.37 (m, 1H); 13C NMR (150 MHz, CD3OD) δ 173.9, 172.6, 170.3, 165.2, 164.3, 154.8, 148.1, 134.4, 128.6 (2C), 127.9 (2C), 90.0, 85.0, 83.0, 81.9, 57.0, 54.5, 38.5, 32.9, 28.3 (6C), 27.5, 25.4, 23.3; IR (film) νmax 3344, 2923, 1723, 1626, 1446, 1369, 1149 cm-1 ; ESI-TOF m/z 574.3234 (M + H+ , C29H43N5O7 requires 574.3235); [α]D -48 (c 0.1, MeOH).</p><!><p>The protected glutamate 10S-28 (8 mg, 0.015 mmol) in CHCl3 (0.52 mL) was treated with trifluoroacetic acid (0.5 mL) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred overnight. The solution was concentrated and triturated with Et2O to give 10S-8 (6 mg, 91%) as a white solid: mp 130 °C (dec); 1H NMR (600 MHz, CD3OD) δ 7.84 (d, 2H, J = 8.4 Hz), 7.40 (d, 2H, J = 8.4 Hz), 4.65-4.63 (m, 1H), 4.26 (t, 1H, J = 7.2 Hz), 3.20 (s, 3H), 2.50-2.47 (m, 2H), 2.33-2.30 (m, 3H), 2.14-2.08 (m, 1H), 1.85-1.79 (m, 1H), 1.70-1.64 (m, 1H), 1.58-1.51 (m, 1H), 1.44-1.37 (m, 1H); 13C NMR (150 MHz, CD3OD) δ 176.7, 175.1, 170.2, 165.0, 159.3, 154.0, 147.9, 134.4, 128.6 (2C), 127.9 (2C), 90.0, 85.0, 57.0, 53.8, 38.3, 31.6, 27.6, 25.1, 23.0; IR (film) νmax 3354, 2933, 1636, 1426, 1205 cm-1 ; ESI-TOF m/z 462.1997 (M + H+ , C21H27N5O7 requires 462.1983); [α]D -22 (c 0.1, MeOH). Agilent 1100 LC/MS: reverse phase (2-40% acetonitrile/water/0.1% formic acid, flow rate 0.75 mL/min); tR, 10.9 min; purity, 97.1%.</p><!><p>A suspension of NaH (60% dispersion, 9.19 g, 230 mmol, 3.5 equiv) in anhydrous DMF (202 mL) was treated with ethyl cyanoacetate (24.5 mL, 230 mmol, 3.5 equiv) at 0 °C. The reaction mixture was stirred for 30 min while allowing the mixture to warm to room temperature, forming the sodium salt as a clear solution. A solution of 12 (21.7 g, 65.7 mmol) in DMF (122 mL) was added at room temperature and the reaction mixture was stirred at 60 °C for 12 h. The reaction mixture was cooled to room temperature and quenched with the addition of saturated aqueous NH4Cl. The mixture was concentrated and then diluted with EtOAc. The organic layer was washed with H2O and saturated aqueous NaCl, dried over Na2SO4, and concentrated. Column chromatography (SiO2, 15-50% EtOAc/hexanes gradient) followed by vacuum distillation provided 29 (14.06 g, 53%) as a white solid: mp 54-56 °C; 1H NMR (500 MHz, CDCl3) δ 8.06 (d, 2H, J = 8.6 Hz), 8.00 (d, 2H, J = 8.6 Hz), 4.21 (q, 2H, J = 7.1 Hz), 3.93 (s, 3H), 3.37 (t, 1H, J = 7.6 Hz), 2.70-2.63 (m, 4H), 2.03 (t, 2H, J = 8.3 Hz), 1.97-1.93 (m, 2H), 1.84-1.81 (m, 2H), 1.57-1.42 (m, 2H), 1.26 (t, 3H, J = 7.2 Hz); 13C NMR (125 MHz, CDCl3) δ 166.8, 165.9, 130.1 (2C), 129.2, 129.0 (2C), 116.3, 62.9, 58.4, 52.3, 44.2, 37.4, 29.8, 27.8 (2C), 25.0, 21.6, 14.1; IR (film) 2924, 2360, 1719, 1276, 1105, 1017 cm-1 ; ESI-FTMS m/z 408.1292 (M + H+ , C20H25NO4S2 requires 408.1298).</p><!><p>A solution of NaOMe (3.26 g, 60.4 mmol, 3.5 equiv) in anhydrous MeOH (166 mL) was treated with guanidine hydrochloride (5.77 g, 60.4 mmol, 3.5 equiv) at room temperature. After the reaction mixture was stirred for 30 min, a solution of 29 (7.03 g, 17.25 mmol) in MeOH (97.5 mL) was added. The reaction mixture was warmed at reflux for 16 h and quenched with acetic acid after cooling to room temperature. After concentration, column chromatography (SiO2, 10% MeOH/CH2Cl2) gave 30 (6.04 g, 83%) as a white solid: mp dec 150 °C; 1H NMR (500 MHz, CD3OD) δ 8.00 (s, 4H), 3.91 (s, 3H), 2.74-2.59 (m, 4H), 2.19 (t, 2H, J = 7.2 Hz), 2.10-2.05 (m, 2H), 1.94-1.88 (m, 2H), 1.42-1.36 (m, 2H); 13C NMR (125 MHz, DMSO) δ 165.9, 162.3, 161.4, 152.9, 147.7, 129.3 (2C), 128.7 (2C), 128.1, 86.8, 58.1, 52.1, 43.2, 27.0 (2C), 24.6, 22.9, 21.9; IR (film) νmax 3361, 1711, 1646, 1435, 1282, 1111 cm-1; ESI-FTMS m/z 421.1364 (M + H+ , C19H24N4O3S2 requires 421.1363).</p><!><p>A solution of 30 (2.01 g, 4.78 mmol) in MeOH (36 mL) was treated with LiOH monohydrate (1.00 g, 23.9 mmol, 5 equiv) in water (12 mL), and the reaction was stirred at room temperature for 24 h. The reaction mixture was diluted with H2O, washed with Et2O, acidified to pH 4 by the addition of aqueous 1 N HCl, and concentrated. The resulting white solid, 31 (1.48 g, 76%), was used directly in the next step: 1H NMR (500 MHz, CD3OD) δ 8.04 (d, 4H, J = 16.6 Hz), 2.72-2.62 (m, 4H), 2.22 (t, 2H, J = 7.3 Hz), 2.07-2.04 (m, 2H), 1.95-1.89 (m, 2H), 1.41-1.38 (m, 2H); 13C NMR (125 MHz, DMSO) δ 167.8, 167.1, 151.0, 151.4, 147.2, 129.7 (2C), 129.5, 128.6 (2C), 87.2, 58.0, 42.9, 27.2 (2C), 24.7, 22.6, 21.3.</p><!><p>Compound 31 (1.48 g, 3.65 mmol), di-tert-butyl l-glutamate hydrochloride (1.62 g, 5.48 mmol, 1.5 equiv) and NaHCO3 (675 mg, 8.03 mmol, 2.2 equiv) in DMF (32 mL) were treated with EDCI (1.40 g, 7.30 mmol, 2.0 equiv). The reaction mixture was stirred for 24 h at room temperature before the addition of CHCl3. The resulting solution was washed with saturated aqueous NaHCO3 (2×), dried over Na2SO4, and concentrated. Column chromatography (SiO2, 10% MeOH/CH2Cl2) provided 32 (1.76 g, 75%) as a white solid: mp 120-124 °C; 1H NMR (500 MHz, CD3OD) δ 8.00 (d, 2H, J = 8.5 Hz), 7.84 (d, 2H, J = 8.5 Hz), 4.53-4.50 (m, 1H), 2.72-2.62 (m, 4H), 2.41 (t, 2H, J = 6.7 Hz), 2.37-2.30 (m, 2H), 2.19 (t, 2H, J = 7.5 Hz), 2.10-2.06 (m, 2H), 1.97-1.91 (m, 2H), 1.49 (s, 9H), 1.45 (s, 9H), 1.42-1.36 (m, 2H); 13C NMR (125 MHz, DMSO) δ 171.5, 171.0, 166.4, 161.4, 157.1, 153.0, 145.5, 132.5, 128.2 (2C), 127.6 (2C), 86.8, 80.6, 79.7, 58.2, 52.5, 43.3, 31.4, 30.9, 27.7 (3C), 27.6 (3C), 27.0, 25.9, 23.0, 22.0; IR (film) νmax 3349, 2961, 1728, 1617, 1440, 1364, 1183 cm-1 ; ESI-FTMS m/z 648.2890 (M + H+ , C31H45N5O6S2 requires 648.2884).</p><!><p>A solution of 32 (54 mg, 0.083 mmol) in CHCl3 (833 μL) was treated with trifluoroacetic acid (1 mL) at 0 °C. The reaction mixture was allowed to warm to room temperature and stirred overnight. The solution was concentrated and triturated with Et2O to give 9 (41 mg, 91%) as a white solid: mp dec 125-130 °C; 1H NMR (500 MHz, CD3OD) δ 7.98 (d, 2H, J = 8.6 Hz), 7.84 (d, 2H, J = 8.5 Hz), 4.68-4.65 (m, 1H), 2.72-2.62 (m, 4H), 2.51-2.48 (m, 2H), 2.43-2.39 (m, 2H), 2.19 (t, 2H, J = 7.1 Hz), 2.15-2.11 (m, 2H), 1.94-1.98 (m, 2H), 1.40-1.37 (m, 2H); 13C NMR (125 MHz, CD3OD) δ 176.9, 175.8, 170.0, 164.9, 154.0 (2C), 147.7, 133.7, 130.2 (2C), 128.5 (2C), 87.9, 59.5, 53.9, 44.8, 31.1, 28.6 (2C), 27.6, 26.9, 23.2, 22.7; IR (film) νmax 3380, 2961, 1718, 1634 cm-1 ; ESI-FTMS m/z 536.1625 (M + H+ , requires C23H29N5O6S2 536.1632). Agilent 1100 LC/MS: reverse phase (2-40% acetonitrile/water/0.1% formic acid, flow rate 0.75 mL/min); tR, 13.7 min; purity, 99.5%.</p><!><p>Enzyme assays for rhGAR and rhAICAR were performed as described previously.8,9 Kinetics of the enzyme reactions were monitored for 2 min after initiation of the reaction. Ki's of the inhibitors were calculated using Dixon plots.</p><!><p>The cytotoxic activity of the compounds was measured using the CCRF-CEM human leukemia cell lines as described previously.8,9,19</p>

PubMed Author Manuscript

Improved calibration of electrochemical aptamer-based sensors

Electrochemical aptamer-based (EAB) sensors support the real-time, high frequency measurement of pharmaceuticals and metabolites in-situ in the living body, rendering them a potentially powerful technology for both research and clinical applications. Here we explore quantification using EAB sensors, examining the impact of media selection and temperature on measurement performance. Using freshly-collected, undiluted whole blood at body temperature as both our calibration and measurement conditions, we demonstrate accuracy of better than ± 10% for the measurement of our test bed drug, vancomycin. Comparing titrations collected at room and body temperature, we find that matching the temperature of calibration curve collection to the temperature used during measurements improves quantification by reducing differences in sensor gain and binding curve midpoint. We likewise find that, because blood age impacts the sensor response, calibrating in freshly collected blood can improve quantification. Finally, we demonstrate the use of non-blood proxy media to achieve calibration without the need to collect fresh whole blood.

improved_calibration_of_electrochemical_aptamer-based_sensors

<!>Results and discussion<!>Conclusions<!>Materials and methods<!>Electrode preparation.<!>Measurements.

<p>Electrochemical aptamer-based (EAB) sensors are a specific class of aptasensors that, uniquely, support highfrequency 1,2 real-time molecular measurements 3,4 directly in complex biological media, including unprocessed, undiluted bodily fluids 5,6 . In this sensor architecture, a target-recognizing aptamer is modified with a redox reporter and covalently attached to a gold electrode using a self-assembled monolayer (Fig. 1A) 5,9 . Upon target binding, the aptamer undergoes a conformational change, producing an easily-measurable shift in electrochemical signal 1 . As these sensors include both a recognition element (the aptamer) and a signaling element (the redox reporter), they do not require washing steps or reagent addition. And because they maintain their signaling properties in even complex sample matrices, such as undiluted whole blood, they perform well even when placed in situ in the living body [2][3][4][5][6][7] . Indeed, their ability to perform real-time molecular measurements in vivo even supports closed-loop feedback-controlled drug delivery 8,9 . Their utility for in-vivo measurements makes this class of sensors particularly promising for clinical health monitoring and wearable devices.</p><p>While a number of electrochemical techniques 5,[10][11][12] have been used to interrogate EAB sensors, converting the resulting signals into estimated target concentrations always relies on the use of a calibration curve. When applying square wave voltammetry, the most commonly employed interrogation technique, a calibration curve is produced by collecting voltammogram peak currents over a range of target concentrations (Fig. 1C). Of note, because EAB sensor signaling varies with applied square wave frequency, square wave voltammetry can be tuned to yield an increase ("signal-on") or a decrease ("signal-off ") in peak current upon target addition (Fig. 1B) 1,[13][14][15] . To correct for drift and enhance gain during in vivo measurements, voltammograms are collected at two such frequencies and converted into "Kinetic Differential Measurement" (KDM) values. These KDM values are derived by subtracting the normalized peak currents seen at signal-on and signal-off frequencies, then dividing by their average (Fig. 1C) 16 . To generate a calibration curve, the averaged KDM values collected in vitro over a range of target concentrations are fitted to a Hill-Langmuir isotherm [Eq. 1, Fig. 1D] 17 :</p><p>where n H is the Hill coefficient (a measure of binding cooperativity), K 1/2 is the midpoint of the binding curve, KDM is the KDM value observed at the applied target concentration, KDM min is the KDM value seen in the absence of target, and KDM max is the KDM value expected at saturating target. These parameters extracted from a calibration curve, then, allow translation of EAB sensor output into estimates of target concentration [Eq. 2, Fig. 1E]: EAB calibration curves thus depend on both the affinity of the aptamer, K 1/2 (which, in the case of a noncooperative aptamer, simplifies to K D , the dissociation constant), the cooperativity (or anti-cooperativity) of binding, n H , and the sensor's signal gain, KDM max . Given that both of these parameters are influenced by environmental factors, such as temperature 18,19 , pH 20 , and ionic strength 20,21 , careful selection of calibration conditions is required in order to accurate and precise measurements. Here, we examine the calibration of EAB sensors in detail, with the aim of improving the accuracy of in vivo measurements performed using them. Specifically, we investigate the impact of media temperature, age, and composition on the observed calibration curves obtained using square wave voltammetry, and assess the accuracy of in vitro measurements performed in freshly-collected, body temperature blood. As our test bed, we examine the response for a sensor detecting vancomycin, an antibiotic target 22 of strong interest for therapeutic monitoring [23][24][25] .</p><!><p>EAB sensors calibrated in fresh whole rat blood under the same conditions employed during the measurement achieve clinically useful performance. To demonstrate this, we applied parameters obtained from a calibration curve collected in fresh, body temperature (37 °C) rat blood (Fig. 2A) to measurements performed in fresh, body temperature rat blood dosed with known concentrations of vancomycin (Fig. 2B). Doing so, we achieve mean accuracy of 1.2% or better over the drug's 6 to 42 µM clinically relevant range 26,27 and 10.4% or better at all concentrations (Fig. 2C). Here, we define accuracy as the mean of the relative difference between the estimated and applied concentrations (100*(expected -observed)/observed). Likewise, we achieve precision of 14% or better in the clinical range, which we define as the coefficient of variation (100*population standard deviation/ population mean). This level of performance is more than adequate for many clinical applications. For example, Figure 2. Vancomycin-detecting EAB sensors calibrated using a calibration curve collected in matched media and temperature easily achieve clinically useful measurement accuracy. (A) Here we created a calibration curve by fitting the average response of four sensors to a Hill-Langmuir isotherm using data collected at 37 °C in freshly-collected rat blood. We derive KDM values by subtracting the normalized peak heights collected at 300 Hz from those collected at 25 Hz, and dividing by their average 16 . We indicate the clinical range of vancomycin in grey. Specifically, the clinical window of vancomycin ranges from 6 to 42 µM, which reflects the minimum target concentrations to achieve clinical effect, to the mean maximum peak concentrations concentrations 26,27 . The error bars shown here and in the following figures reflect the standard deviation of replicate measurements performed using independent sensors (K 1/2 = 73 ± 4 µM, n = 4). The error reported for these and all other K 1/2 values reflect 95% confidence intervals. (B) We then apply this calibration curve to quantify measurements performed using the same four sensors in 37 °C fresh rat blood to which 10, 20, 50, 100, or 300 µM vancomycin has been added (dotted lines, red lines). We indicate the clinical range of vancomycin in grey. (C) From these measurements, we calculate mean estimated concentration, standard deviation, mean accuracy (defined as 100*(expected -observed)/observed), and coefficient of variation (100*population standard deviation/population mean). We observe better than 10% accuracy for all challenges.</p><p>Vol:.( 1234567890) www.nature.com/scientificreports/ accuracy of ± 20% over the therapeutic range is sufficient to achieve clinically relevant therapeutic monitoring of vancomycin 24,28 . This suggests that for measurements in the living body, applying a calibration curve captured in freshly collected, body temperature blood will yield clinically accurate and precise measurements of this drug.</p><p>In the studies described above, we calibrated each individual sensor using a single, common calibration curve obtained by averaging the sensor's calibration curve with those of other vancomycin-detecting sensors. That is, each sensor was calibrated using a calibration curve to which it itself had also contributed. If, however, we instead use an "out-of-set" calibration curve (a calibration curve to which the sensor under investigation did not contribute, Fig. 3A), we see no significant change in accuracy and only a slight reduction in precision over the clinically relevant range of vancomycin concentrations (Table S1). Likewise, we do not see significant improvement in accuracy or precision over the clinical range if we calibrate each sensor using its own, individual calibration curve (Fig. 3B, Table S2). These observations suggest that sensor-to-sensor variation is not a major contributor to the level of accuracy and precision we achieve with this sensor at clinical target concentrations.</p><p>Collecting calibration curves at the appropriate temperature is key to sensor calibration. Specifically, between room and body temperature, calibration curves differ significantly (Fig. 4A). Depending on interrogation frequency employed, this can lead to considerable under-or over-estimation of target concentrations. Interrogating at 25 and 300 Hz, for example, we observe up to a 10% higher KDM signal at room temperature than at body temperature over vancomycin's clinical concentration range. Consequently, applying a calibration curve collected at room temperature to those collected at body temperature causes substantial concentration underestimates (Figure S2). Interrogating using other square wave frequency pairs, likewise, can yield even more significant differences in KDM signal in the clinical range (Figure S3). These differences in sensor response occur because S3). (B) This occurs at least in part because the electron transfer rate from the redox reporter changes with temperature. Specifically, the peak charge transfer rate shifts toward higher frequencies at higher temperatures. To illustrate this, we plot here charge transfer versus frequency for a representative sensor in absence of target. We do so by determining square wave voltammogram peak current, and dividing it by its given interrogation frequency (additional sensor curves shown in Figure S1) 29 www.nature.com/scientificreports/ temperature changes can shift system properties, such as binding equilibrium coefficients and the electron transfer rate itself. For example, the electron transfer rate (indicated by the location of peak charge transfer, when plotting interrogation frequency versus charge transfer) increases with temperature for the vancomycin aptamer (Fig. 4B), as well as for EAB sensors against other targets (Figures S4-S6). This shift is great enough that it can affect the selection of signal-on and signal-off frequencies. For example, from room temperature to body temperature, 25 Hz changes from a weak signal-on frequency to a clear signal-off frequency (Figure S7). These results illustrate the importance of media temperature for both the accurate calibration of in vivo data, as well as the selection of the signal-on and signal-off frequencies used to perform KDM.</p><p>Obtaining bovine blood from commercial vendors is often more convenient than collecting fresh blood samples each time one needs to generate a calibration curve. Unfortunately, however, calibration curves obtained in the commercially sourced and freshly-collected blood samples differ (Fig. 5A). Specifically, vancomycin sensors challenged in commercially sourced bovine blood yielded lower signal gain, which would lead to overestimated vancomycin concentrations (Fig. 5A). This difference in gain could arise from differences in the species source of the blood (bovine versus rat), how the blood was processed, or its age. Due to shipping, for example, commercially sourced blood is at least a day old by the time we use it. To determine whether blood age (time after collection) is an important calibration parameter, we titrated sensors in commercial bovine blood one day after collection and again using the blood from the same blood draw 13 days later. While the two samples yielded similar signals over the drug's clinical range, the older sample produced lower signal at concentrations above this range (Fig. 5B). To assess the source of these differences, we examined the normalized square-wave signals at 25 and 300 Hz (Figs. 5C-D). Doing so, we find that while we observe a marked signal decrease at target concentrations below 1 µM (Fig. 5C-D), KDM effectively corrects for this. That there is still a difference in the 1 and 14 day old blood calibration curves suggests that blood age itself can impact the EAB sensor response. Given this, we believe measurements performed in the living body are best calibrated using curves obtained in the freshest possible blood.</p><p>Although it is the most accurate calibration matrix for in-vivo measurements, the collection of fresh blood for sensor calibration can be inconvenient, leading us to ask if there are other, more easily obtained media that Figure 5. (A) Titrations performed in fresh rat blood and commercially sourced, one-day-old bovine blood produce distinct calibration curves. (B) To determine whether this is driven by species-specific differences in blood, or the greater age of the commercially sourced bovine blood, we compared calibration curves obtained in a bovine blood sample 1 day (red, K 1/2 = 116 ± 15 µM) and 14 days (black, K 1/2 = 112 ± 9 µM) after it was collected. Doing so, we find that the KDM response does not change significantly in the clinical range. (C, D) Examining the signal at the two square-wave frequencies used to perform KDM (here, 25 Hz and 300 Hz) we see that, for the 14-day-old blood, there is a notable signal decrease at vancomycin concentrations far below the aptamer's dissociation constant. Given that no target-induced response should occur at these low concentrations, we believe this is an artifact due to time-dependent sensor degradation in the older blood.</p><p>Vol:.( 1234567890 ), the titration yields greater signal gain than that observed in rat blood (Fig. 6A).</p><p>Applying the resulting calibration curve, then, would lead to underestimation of vancomycin in rat blood. The PBS calibration curve, in contrast, exhibits higher response in the clinical range and a lower response above the clinical range. Thus, depending on the concentrations applied, this curve would lead to under-or overestimates of vancomycin in rat blood (Fig. 6B). A calibration curve collected in PBS with bovine serum albumin, however, reproduces the calibration curve obtained in fresh rat blood to a fair degree of accuracy (Fig. 6C). Enough so that, when we quantify the successive vancomycin additions in body temperature rat blood with this calibration curve, we obtain accuracies of better than 9% in the clinical range and better than 35% across all concentrations (Figure S8, Table S4). While poorer than the accuracy we achieve via calibration in rat blood, this calibration may still be acceptable in some applications. More broadly, these results suggest that buffered proxy systems can help guide development and application of sensors in vivo, and calibrate sensors when reduced accuracy is an acceptable tradeoff for improved convenience.</p><!><p>With the appropriate selection of media temperature, composition, and age, a vancomycin-detecting EAB sensor easily achieves clinically relevant accuracy. Specifically, when calibrated at the appropriate temperature in freshly collected blood, sensors achieve accuracy of better than 10% when challenged under those same conditions. This result holds for both sensors calibrated against themselves and for sensors calibrated against other sensors. In contrast, calibration curves collected at inappropriate temperatures or in aged blood samples deviated more significantly from those seen in fresh, body temperature blood. Thus, these conditions may produce substantial over-or under-estimates of target concentration. Finally, in some cases, it is possible to use simpler media, such as phosphate buffered saline with bovine serum albumin, as a convenient and proxy for freshly collected blood for applications in which reduced accuracy is an acceptable trade-off relative to improved ease of calibration. Here, we investigate whether simpler media can reproduce the response seen in freshly-collected rat blood. To do so, we compare titrations (KDM values derived from 25 and 300 Hz) collected in 37 °C freshly collected rat blood to (A) 37 °C Ringer's buffer with 35 mg/mL bovine serum albumin (BSA), (B) 37 °C phosphate buffered saline (PBS) with 2 mM MgCl 2 , and (C) 37 °C PBS with 2 mM MgCl 2 and 35 mg/mL bovine serum albumin. From these data we observe that calibration in 37˚C PBS with added BSA is a reasonable proxy for fresh rat blood. Of note, the close correspondence between the sensor's response in simple buffers and in whole blood indicate that the sensor does not respond significantly to the components naturally present in blood.</p><p>Vol</p><!><p>Electrode fabrication. We fabricated sensors by soldering a 5 cm long, 75 µm diameter, PFA-coated gold wire (A-M Systems, Sequim, WA) to a gold-plated pin connector (CH Instruments, Inc., Austin, TX) with 60/40 lead-selenium solder (Digikey, Thief River Falls, MN). Before soldering, we used a digital caliper and razor blade to isolate 3 mm of gold on both ends of the gold wire. One end forms the working electrode, and the other end must be exposed for successful soldering. To prevent breakage of the delicate wires, we coated the wire-solder interface with a coat of urethane conformal coating (MG Chemicals, Surrey CA).</p><p>Aptamer preparation. We first thawed a 2 μL, aliquot of 100 μM aptamer (sequence below) modified with a 6-carbon thiol linker on the 5′ end and methylene blue on the 3' end (Biosearch Technologies, Novato, CA, dual HPLC purification, stored at − 20 °C).</p><p>Because the manufacturer provides the DNA constructs in oxidized form, which does not effectively immobilize onto the gold surface, we must reduce the disulfide bond before deposition. To do so, we combined 2 μL of 10 mM Tris (2-carboxyethyl) phosphine (TCEP, Sigma-Aldrich, St. Louis, MO) with the aptamer sample for a 1 h thiol reduction in the dark at room temperature. We then diluted the sample with 96 μL 1X phosphate buffered saline (pH = 7.4). Here and elsewhere in our protocol, the 1X PBS is prepared by diluting 20X phosphate buffered saline (ChemCruz 20X phosphate buffered saline, Santa Cruz Biotechnology, Dallas, TX) to 1X using deionized water. , We quantified the concentration of the aptamer using the molar absorption coefficient at 260 nm provided by the supplier with UV-VIS spectroscopy (DU800, Beckman Coulter, Brea, CA). Using PBS, we then diluted the aptamer to 500 nM.</p><!><p>For electrode cleaning and in-vitro characterization, our electrochemical cell contained a PFA-wrapped gold sensor working electrode, a platinum counter electrode (CH Instruments, Inc, Austin, TX), and an Ag|AgCl reference electrode (CH Instruments, Inc., Austin TX). We secured our working, counter, and reference electrodes in a cell vessel "shot glass" with a custom-fabricated Teflon lid fixture (See Fig. 1C). First, we electrochemically cleaned the electrodes in 0.5 M NaOH (Sigma-Aldrich, St. Louis, MO) by performing repeated cyclic voltammetry scans between − 1 and − 1.6 V (all potentials versus Ag|AgCl) at 1 V s −1 scan rate for 300 cycles (Table 1) using a CH Multipotentiostat (CHI1040C, CH Instruments, Inc.) 31 . We rinsed the electrodes in deionized water, then increased the surface area of the gold wire electrode by electrochemically roughening in 0.5 M H 2 SO 4 (Sigma-Aldrich, St. Louis, MO) using a previously-described procedure that involves repeatedly stepping the potential between 0 and 2.2 V using chronoamperometry (Table 2) 31 . This electrochemical procedure, repeated 50 times using the CH instrument software, yields a 2 to fivefold increase in microscopic surface area 31 .</p><p>Sensor fabrication. Immediately after roughening, we rinse the electrodes thoroughly in deionized water and place them in the prepared aptamer solution for 1 h at room temperature in dark conditions 8 . We then rinsed the electrodes with deionized water and immersed them for 12 to 18 h at room temperature in a 10 mM 6-mercapto-1-hexanol (Sigma-Aldrich, St. Louis, MO) in 1X PBS to passivate the surface. Following a final rinse with deionized water, the sensor was ready for use. Blood collection. 12 mL of blood was collected from a single ~ 400 g adult male Sprague-Dawley Rat (Charles River Laboratory, Santa Cruz, CA). The rat was allowed to acclimatize to the colony for at least one week. The animal was placed under anesthesia using isoflurane gas and, following abolishment of toe pinch www.nature.com/scientificreports/ response, a horizontal incision was made below the rib cage. The incision was extended on each side to under the front legs, at which point vertical incisions were made through the ribcage. The diaphragm was then cut to reveal the heart and the septum pinned back using hemostats. A 20 mL syringe was prepared with 300 units of heparin in order to achieve a final concentration range of 20 units/mL (Sagent Pharmaceuticals, Schaumburg, IL). An 18G needle was attached to the top of the syringe and inserted into the left ventricle for blood collection (BD, Franklin Lakes, NJ). Very light pressure was placed upon the syringe in order to facilitate blood removal. The blood draw (15 mL total) was stopped when the flow of blood into the syringe finished after which point the animal was euthanized via isoflurane overdose. The Institutional Animal Care and Use Committee (IACUC) of the University of California at Santa Barbara approved our experimental protocol, which adhered to the guidelines provided by the American Veterinary Medical Association. Blood was transported immediately to a temperature-controlled bath at 37 °C, then used for experiments (Lauda Ecoline RE 106, Lauda-Brinkmann, Delran, NJ). This study adhered to ARRIVE guidelines, where applicable. Specifically, randomization, blinding, and exclusion criteria (ARRIVE guidelines 3, 4, and 5) are not applicable because we drew blood from a single animal.</p><!><p>We filled the electrochemical cell with PBS, rinsed the electrodes in deionized water, and secured them in the electrochemical cell's Teflon cap. Before each experiment, we collected three baseline cyclic voltammograms in PBS (− 0.1 to − 0.5 V versus Ag|AgCl, 0.1 V/s scan rate) to confirm successful aptamer deposition.</p><p>To perform a titration, we moved the electrode cap to a vessel of the selected media held in a temperature bath at the selected temperature (Lauda Ecoline RE 106, Lauda-Brinkmann, Delran, NJ). For bovine blood experiments, we commercially sourced blood from Hemostat Laboratories (Dixon, CA). For Ringer's buffer experiments, we prepared Ringer's buffer at pH 7.4 using 154 mM NaCl, 5.64 mM KCl, 2.16 mM CaCl2, 11.10 dextrose, 2.38 NaHCO3, 2 mM Trizma®, and 35 mg/mL bovine serum albumin (heat shock fraction, protease free, Sigma-Aldrich, St. Louis, MO). In the selected media, we collected square wave voltammetry scans (approximately − 0.2 to − 0.4 V versus Ag|AgCl, 25 mV amplitude) at 10, 25, and 300 Hz first in absence of target. We selected these frequencies because they were the originally reported signal-off, non-responsive, and signal-on frequencies for this particular aptamer. We observed, however, that at body temperature, 25 Hz yields a reliable signal-off response, and thus use this as the signal-off frequency for this work. Upon addition of incremental target concentrations, we back pipetted the electrochemical cell 15 times, allowed the solution to rest for two minutes, then proceeded with the measurement. To perform a spiking experiment, we used a CH software macro to repeatedly collect and store square wave voltammograms. We collected a 10 min baseline with no target, then sequentially added 10, 20, 50, 100, 300 µM vancomycin. For each addition, we pipetted rapidly, while taking care not to create bubbles in the blood, then measured the resulting signal for 5 to 10 min.</p><p>It is common to observe a downward drift in square wave voltammogram peak current, especially in complex media, such as whole blood. To correct for any peak signal loss, we applied a previously-described drift correction technique termed "Kinetic Differential Measurements" (KDM) 16 . Here, KDM denotes when normalized signal off-peak currents are subtracted from the normalized signal-on peak currents, then divided by the average of normalized signal-on and signal-off currents.</p>

Scientific Reports - Nature

New Thermal and Microbial Resistant Metal-Containing Epoxy Polymers

A series of metal-containing epoxy polymers have been synthesized by the condensation of epichlorohydrin (1-chloro-2,3-epoxy propane) with Schiff base metal complexes in alkaline medium. Schiff base was initially prepared by the reaction of 2,6 dihydroxy 1-napthaldehyde and o-phenylenediamine in 1 : 2 molar ratio and then with metal acetate. All the synthesized compounds were characterized by elemental, spectral, and thermal analysis. The physicochemical properties, viz., epoxy value, hydroxyl content, and chlorine content [mol/100 g] were measured by standard procedures. The antimicrobial activities of these metal-containing epoxy polymers were carried out by using minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods against S. aureus, B. subtilis (Gram-positive bacteria), and E. coli, P. aeruginosa (Gram-negative bacteria). It was found that the ECu(II) showed higher antibacterial activity than other metal-chelated epoxy resin while EMn(II) exhibited reduced antibacterial activity against all bacteria.

new_thermal_and_microbial_resistant_metal-containing_epoxy_polymers

1. Introduction<!>2.1. Materials and Reagents<!>Characterization for UV, FTIR, and NMR<!>2.3. Synthesis of Schiff Base Ligand (H2L)<!>2.4. Synthesis of Schiff Base Metal Complexes<!>2.5. Synthesis of Metal-Containing Epoxy Polymers<!>2.6. Antibacterial Assessments<!>3.1. Synthesis of Metal Complexes<!>3.2. Synthesis of Metal-Containing Epoxy Polymers<!>3.3. Antibacterial Activity<!>4. Conclusion

<p>Since the last two decades, several thermal and microbial resistant polymers have been synthesized by the immobilization of metal complexes into the polymers [1] and used as a thermal resistant, microbial resistant, scratch resistant, and flame retardant-coating materials. Some metal complexes commonly used in the synthesis of metal containing polymers are Schiff base, ferrocene, Imidazole, secondary and tresiory amine metal complexes, and so forth, [2, 3]. Among these metal complexes Schiff base metal complexes have been widely used due to their corrosive resistant, microbial as well as thermal resistant properties [4]. Epoxy polymers are one of the most important higher-performance polymer systems in use today, ranging from simple two-part adhesives and sports equipment to high-tech applications such as formula one racing cars and the aerospace industry. Epoxy polymers are capable of undergoing homopolymerisation, although this process generally yields products with inadequate properties for high-tech applications. Consequently, in many cases catalysts, additives, and cocuring-agents are formulated with the epoxy resin to significantly increase the storage stability, decrease the cure time, and improve the final properties [5, 6]. The use of metals to formulate resin systems with excellent storage stability is discussed, along with the use of coordination compounds to improve cured resin properties such as fracture toughness, thermal stability, and water absorption [7, 8]. Two approaches are generally used for the attachment of metal complexes with polymers. The first approach involves the introduction of the bifunctional metal complexes as a monomer, followed by their polymerization [9]. The second approach involves the linking of metal complexes directly onto preformed functional polymers [10]. The first approach has the advantage that the monomer can be polymerized with several other comonomers, and the composition can be varied easily. These facts propagated our interest at this time to synthesize new materials with antimicrobial and thermal resistance properties. In the present study, Schiff base metal complexes were reacted with epichlorohydrin in 1.25  :  1 molar ratios to produce a series of E-M(II) metal containing epoxy polymers. The characterization of the new epoxy polymers was done with the purpose of proposing their structures and determining their specific applications as thermally resistant and/or microbial resistant materials.</p><!><p>o-phenylenediamine, epichloro-hydrin, manganese(II) acetate tetrahydrate [Mn(CH3COO)2-4H2O], copper(II) acetate monohydrate [Cu(CH3COO)2-H2O], nickel(II) acetate tetrahydrate [Ni(CH3COO)2-4H2O], cobalt(II) acetate tetrahydrate [Co(CH3COO)2-4H2O], and zinc(II) acetate dihydrate [Zn(CH3COO)2-2H2O] (Sigma Aldrich) were used without furtherpurification. The solvents, such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO), ethyl alcohol, methan-ol, and acetone, were distilled before use. 2,6-hydroxy naphthaldehyde was prepared according to the literature [11].</p><!><p>The epoxy value (mol/100 g) of resin was determined by analytical method [12]. This method was based on the back titration. 0.2 g of resin was added to 30 cm3 of 0.1 M-HCl and mixed for 2 h. Then unreacted HCl was retitrated with phenolphthalein by standard alkali solution using the following formula: (1)w(EP)=(V2−V1)·c(NaOH)  ×  0.043mass  of  sample  , where V 1 is the volume of NaOH solution used for blank and V 2 is the volume of NaOH solution used for sample.</p><p>Hydroxyl content was determined by acetylation with acetyl chloride in pyridine. The excess of acetyl chloride was decomposed with water and the resulting acetic acid, formed both in hydrolysis and in the acetylation process, was titrated with standard alkali using the following formula: (2)w(OH)=mass  of  sample  (V1−V2)·  c(KOH)×170  , where V 1 is the volume of KOH solution used for blank and V 2 is the volume of KOH solution used for sample. The chlorine content was determined by treating the resin solution with alcoholic KOH and titrating it against standard HCl [13] (3)(Cl)=c(KOH)·V×0.0355mass  of  sample  .</p><!><p>Schiff base ligand was prepared by (1.88 g, 0.01 mol) of monoaldehyde and was dissolved in 8 mL of THF, and the solution was added to a THF/ MeOH (1;1 10 mL) solution of o-phenylenediamine (0.9 g, 0.005 mol) and refluxed for 6 hours. With continuous stirring the color of the solution has been changed reddish. The progress of reaction was monitored by thin layer chromatography (TLC). The reaction mixture was cooled and precipitated into 20 mL MeOH. The reddish purple colour precipitate was filtered, then washed with methanol, and dried in vacuum, Yield 45% (2.01 g).</p><!><p>A solution of 2.13 g (0.1 mol) of H2L and 0.05 mol of metal acetate hydrates in 25 cm3 of ethanol were stirred for 2 h at 70°C. Then, the mixture was cooled, filtered, and washed with methanol to give the colored metal complexes, and the pure product was obtained after recrystallization from methanol. All the metal complexes used in this study have been characterised using similar methods. The colour, yield, and spectral and elemental data of the complexes are given next.</p><p>MnL. Reddish purple, 3.64 g, 72% yield. FTIR [KBr pellets, υ(max), cm−1]: 3360, 3050,1640, 1530, 748, 620, 550, MALDI-TOF MS (m/z): 502.61 [M + H+], Anal. Calcd for C28H18O4N2 -Mn(II): C, 67.07%; H, 3.62%; N, 5.59%; Mn, 10.96%. Found: C, 67.08%; H, 3.63%; N, 5.57%; Mn, 10.94%.</p><p>CoL. Dark brown, 3.54 g, 70% yield. FTIR [KBr pellets, υ(max), cm−1]: 3360, 3050,1640, 1538, 748, 620, 540, MALDI-TOF MS (m/z): 506.03, Anal. Calcd for C28H18O4N2 -Mn(II): C, 66.54%; H, 3.59; N, 5.54%; Co, 11.66%. Found: C, 66.54%; H, 3.60%; N, 5.55%; Co, 11.68%.</p><p>NiL. Purple, 3.69 g, 73% yield. FTIR [KBr pellets, υ(max), cm−1]: 3360, 3055,1642, 1540, 745, 625, 540, MALDI-TOF MS (m/z): 505.12, Anal. Calcd for C28H18O4N2-Ni(II): C, 66.57%; H, 3.59; N, 5.55%; Co, 11.62%. Found: C, 66.58%; H, 3.61%; N, 5.56%; Ni, 11.63.</p><p>CuL. Dark purple, 3.52 g, 69% yield. FTIR [KBr pellets, υ(max), cm−1]: 3360, 3050,1645, 1535, 748, 620, 545, MALDI-TOF MS (m/z): 510.6, Anal. Calcd for C28H18O4N2-Cu(II): C, 65.94%; H, 3.56; N, 5.49%; Cu, 12.62%. Found: C, 65.95%; H, 3.57%; N, 5.51%; Co, 12.63%.</p><p>ZnL. Reddish purple, 3.62 g, 71% yield. 1H-NMR (300 MHz, DMSO, d): 9.56 (2H, OH), 7.52–6.45 (12 hours, Ar-H), 9.16 (2H, CH=N), FTIR [KBr pellets, υ(max), cm−1]: 3360, 3055,1645, 1540, 750, 620, 540, MALDI-TOF MS (m/z): 511.42, Anal. Calcd for C28H18O4N2 -Zn(II): C, 66.54%; H, 3.59; N, 5.54%; Co, 11.66%. Found: C, 66.54%; H, 3.60%; N, 5.55%; Co, 11.68%.</p><!><p>The epoxidation of metal complexes was carried out by the reaction of Schiff base metal complexes (ML) with epichlorohydrin [14]. A mixture of (0.01 mol) ML dissolved in 20 mL DMF and 10 mL of epichlorohydrin was refluxedin a three-round-bottom flaskin the presence of sodium hydroxide(10 mL of 2N) was added gradually for 4 h. The progress of the reaction was monitored by TLC technique, and epoxide value as the heat evolution was slowed; the solution was poured in to ice cooled ether. The resulting colour precipitate of metal-containing epoxy polymers was filtered, washed with water and methanol, respectively, and dried in vacuum oven at 100°C for 2 h.</p><!><p>The antibacterial activities of the chelated epoxy polymers were performed according to the National Committee for Clinical Laboratory Standards (NCCLS) to determine minimum inhibitory concentration (MIC) values [15].The microorganisms used in this study were S. aureus,B. subtilis (Gram-positive bacteria) and E. coli,P. aeruginosa (Gram-negative bacteria). The strains were all cultured on Tryptic Soy Agar (TSA) (Difco, USA) and Mueller-Hinton Broth (MHB) (Difco, USA), incubated aerobically at 35.5°C overnight. For the growth culture, one colony from culture on the TSA was inoculated into the MHB and incubated aerobically at 35.5°C for 24 hours. Then bacterial concentrations were determined by measuring optical density (OD) at λ = 600 nm at 0.2 (OD of 0.2 corresponded to a concentration of 108 CFU/mL) with a spectrophotometer.</p><p>The MIC90% of the-metal-containing epoxy polymers was determined by modification of the broth dilution method in 96-well microtiter plate. The growth of bacteria was determined at the difference in absorbance after 24 hours incubation at 35.5°C. The absorbance at 600 nm was then determined by using microplate reader. All experiments were performed in triplicates against each tested microorganisms. The lowest concentration which inhibited microbial growth was reported as MIC90% whereas minimal bactericidal concentration (MBC) was defined as the lowest concentration of the compound to kill the microorganisms [16].</p><!><p>It has been known that Schiff base ligand was synthesized by the reaction of carbonyl compound and primary amine. In this study, we have synthesized H2L from 2,4 dihydroxy 1-napthaldehyde and o-phenylenediamine. The metal complexes were prepared by adding the methanolic solution of metal acetate to the THF solution of H2L in 1  :  1 molar ratio as given in Scheme 1. The synthesized metal complexes were soluble in DMSO, THF, and DMF but insoluble in methanol, ethanol, acetone, and water. The formation of H2L and its metal complexes was supported by elemental analysis, FTIR and 1HNMR spectroscopy. The FTIR spectrum of H2L showed a strong peak at 1641 cm−1, which was assigned to the C=N stretching in the case of metal complexes this peak was shifted to lower frequency at 1605–1610 cm−1, due to metal ions coordination through imines nitrogen. Two addition peaks at 620–662 cm−1 and 540–550 cm−1 were found in the spectra of metal complexes corresponding to M-O and M-N bond, respectively, [17].</p><p>The 1HNMR spectrum of H2L showed a resonance signal at 9.84 ppm for HC=N group, which had actually shifted downfield from its position in the spectrum of metal complexes and showed resonance at 9.16 ppm. The overall profiles of metal complexes are similar and supported by elemental analysis.</p><!><p>Epoxy resins are prepared by the step-polymerization of a bisphenol, and epichlorohydrin [18]. Herein, E-M(II) was prepared by the reaction of Schiff base metal complexes (LM) with epichlorohydrin in the presence of a sodium hydroxide according to Scheme 2. The reaction mechanism is similar to that we describe in our previous work [14].All of the synthesized metal-containing epoxy polymers were colored solids insoluble in water, ethanol, and methanol but soluble in DMF and DMSO. E-M(II) was prepared in a molar ratio of 2  :  1 epichlorohydrin to Schiff base metal complexes, which was supported by the physicochemical properties (Epoxy value, hydroxyl value, and Chlorine value) and elemental analysis, as listed in Table 1. The epoxy value of all the polymers was found in the range 0.18–0.22 mol/100 g. Medium molecular weights in the range 2225–2300 were found by the reduction of the amount of excess epichlorohydrin. The chlorine content of all the epoxy polymers was found to be 0.01–0.012 mol/100 g due to many side reactions such as dehydrohalogenation [19]. The secondary hydroxyl group content (hydroxyl value) was found in the range of 0.24–0.28 mol/100 g, which was formed along the chain molecule after the epoxy group was reduced.</p><p>The spectra of all the chelated epoxy resin showed a broad band in the range 3345–3410 cm−1, assigned to υ(OH), which suggested the presence of hydroxyl groups. The presence of methylene groups in all the polymers was confirmed by the appearance of two strong bands at 2940 and 2860 cm−1 due to υC-H symmetric and asymmetric stretching and a band at 1415 cm−1 due to the δCH2 bending mode. All of the synthesized compounds showed additional absorption bands around 1260, 1165, and 890 cm−1 associated with epoxy groups although a band at 1260 cm−1 was identified with some reasonable certainty as being due to epoxy groups and a second band at 1155–1070 cm−1 was probably due to CH2-O vibrations when comparing their parental Schiff base ligand [20].</p><p>The 1H-NMR and 13C-NMR spectra of the diamagnetic metal-chelated epoxy resin were determined in DMSO-d6 and are given in Figures 1 and 2. The 1H-NMR spectra of these resins showed strong singlet signals at 9.20 ppm, which suggested azomethine protons (CH=N). The alcoholic protons (OH) showed a single resonance signal at 4.50 ppm in the case of E-Zn(II); this resonance signal was not found for ML. The chelated resin showed some other signals, assigned labels in Figure 1, at 2.20–3.02 ppm due to methylene protons in different environments. The number of protons calculated from the integration curves and those obtained from the values of the expected CHN analyses were in agreement. In the 13C-NMR spectra, Zn-chelated epoxy resin displayed signals assigned to CH=N carbons at 155 ppm. This signal appeared downfield in comparison with their original position (168 ppm), which indicated coordination with the central metal atom. A sharp peak at 62.2 ppm, assigned to the CH-OH function, was generated due to the reduction of oxirane groups with reactive hydrogen. Other resonance lines of these spectra fell into two main regions at 66.5–68.6 ppm for aliphatic carbons and 125–150.08 ppm for aromatic carbons [21].</p><p>A comparative study of the thermal behaviours of all the epoxy polymers was carried out in a nitrogen atmosphere with the purpose of examining the structure-property relationships at various temperatures, and results are given in Table 2. All of the polymers decomposed in two steps; the first step was faster than the second step as given in Figure 3. This may have been due to the fact that the non-coordinated part of the polymers decomposed first, and the actually coordinated part of the polymers decomposed later. The TGA trace of E-Cu(II) showed the initial decomposition at 450°C, about 10% weight loss was observed, which corresponded to an aliphatic portion/noncoordinated part such as CH2-CH-CH2 and epoxy groups per units of epoxy resin. Then, continued mass loss was observed up to 575°C, which indicated the decomposition and volatilization of the aromatic part into low-molecular-weight fractions, such as CH4, N2 and H2O. The thermogravimetric analysis (TG) of the chelated epoxy polymers revealed a mass loss in the temperature range 550–580°C, which corresponded to the formation of metal diisocyanate [M(OCN)2]. The next decomposition step occurred in the temperature range 610–800°C and corresponded to the thermal decomposition of M(OCN)2 to metal isocyanate [M(OCN)] and corresponded to the formation of MO [22]. The reduced masses of 34.20%, 32.05%, 28.50%, 25.21%, and 22.50% were found at 800°C, corresponded to E-Cu(II), E-Zn(II), E-Ni(II), E-Co(II) and E-Mn(II), respectively, and matched with Irvin-Williams order of stability of complexes of divalent metal ions. The observed reduced masses of all of the epoxy resin were greater than the calculated values; this was due to the formation of other compounds during the thermal reaction. Differential scanning calorimetry results of these epoxy resins revealed that the heat flow rate of the samples underwent a change during transition. The Tg values of all of the synthesized epoxy resins were computed from the results by the extrapolation of the pretransition and post transition line and by the calculation of the temperature when the heat flow rate was exactly in the middle of the pretransition and post transition rates. The Tg values of all of the synthesized polymers were in the range 180–220°C and are given in Table 2. All of the polymers showed a single Tg value due to the absence of any homopolymers, block polymers, and heterogeneous impurities [23].</p><!><p>The in vitro antibacterial activity of all the synthesized polymers was evaluated by using a minimum inhibitory concentration (MIC) and a minimum bactericidal concentration (MBC) procedure against S. aureus,B. subtilis (Gram-positive bacteria), and E. coli, P. aeruginosa (Gram-negative bacteria) bacteria accordance to methods of the National Committee for Clinical Laboratory Standards (NCCLSs) [15, 16]. The metal-chelated epoxy polymers inhibited growth of all the bacteria with MIC values ranging between 720 and 800 μg/mL whereas MBC value was 850 μg/mL displayed in Table 3. It was revealed that E. coli and P. aeruginosa were more sensitive to the chelated epoxy polymer than that of the other two bacteria due to the different component of bacterial cell wall. It was also observed that the E-Cu(II) showed higher MIC/MBC value 730/800, 740/810, 720/800 and 720/810 against S. aureus, B. subtilis,E. coli, andP. aeruginosa bacteria. On the other hand, the E-Mn(II) displayed lower antibacterial activity than that of other chelated epoxy polymers. In our previous study, we reported that chelation or coordination reduces the polarity of the metal ion by partial sharing of its positive charge with the donor groups and possibly p-electron delocalization within the whole chelate ring. This process thus increases the lipophilic nature of the compound, which, in turn, favors penetration through the bacterial wall of the microorganism. The Cu(II)-chelated resin showed the widest effective antibacterial due to a higher stability constant [24]. On the other hand, the difference in the magnitude of antimicrobial activity came from other factors, such as solubility, charge, and chirality of the polymers. Thus, it was apparent that the antibacterial activity of the chelated polymers of Schiff base was not only dependent on the charge but also on the chemical structure and nature of metal ions.</p><!><p>Metal-chelated epoxy polymers were successfully synthesized by the polycondensations of Schiff base metal complexes with epichlorohydrin in the basic medium and characterized by elemental, spectral, and thermal analysis. All the epoxy polymers in this study were colored solid, insoluble in common organic solvent but soluble in DMSO and DMF. The signal Tg value represents absence of any homopolymers, block polymers, and heterogeneous impurities. E-Cu(II) shows good thermal resistant as well as microbial resistant behaviour due to higher stability constant. Due to the promising thermal and microbial resistant behaviours, these polymers could be used as heat resistant-coating materials for aero space vehicles and antimicrobial coating materials in public palace and hospitals.</p>

PubMed Open Access

Enantioselective assembly and recognition of heterochiral porous organic cages deduced from binary chiral components

Chiral recognition and discrimination is not only of significance in biological processes but also a powerful method to fabricate functional supramolecular materials. Herein, a pair of heterochiral porous organic cages (HPOC-1), out of four possible enantiomeric products, with mirror stereoisomeric crystal structures were cleanly prepared by condensation occurring in the exclusive combination of cyclohexanediamine and binaphthol-based tetraaldehyde enantiomers. Nuclear magnetic resonance and luminescence spectroscopy have been employed to monitor the assembly process of HPOC-1, revealing the clean formation of heterochiral organic cages due to the enantioselective recognition of (S,S)binaphthol towards (R,R)-cyclohexanediamine derivatives and vice versa. Interestingly, HPOC-1 exhibits circularly polarized luminescence and enantioselective recognition of chiral substrates according to the circular dichroism spectral change. Theoretical simulations have been carried out, rationalizing both the enantioselective assembly and recognition of HPOC-1.

enantioselective_assembly_and_recognition_of_heterochiral_porous_organic_cages_deduced_from_binary_c

Introduction<!>Results and discussion<!>Conclusions<!>Conflicts of interest

<p>Porous organic cages (POCs) are newly emerging attractive crystalline molecular materials with great application potential in the elds of storage and separation, 1 sensing, 2 and catalysis. 3 Their advantages originate from the intrinsic and extrinsic voids together with the functional groups attached on POCs. Thus far, molecular POCs are mainly formed from the selfassembly of discrete reactive building blocks driven by dynamic covalent chemistry (DCC) including the reactions of boronic acid condensation, 4 imine condensation, 5 and alkyne/ alkene metathesis. 6 In particular, various amine-and aldehyde-decorated building blocks are available to assemble POC molecules with different cavities, dimensions, and topologies. 7 These discrete cage-like molecules obtained are engineered crystallographically to form porous assemblies (called POCs) through efficient supramolecular interactions, exhibiting huge adsorption capacity. In addition, the new application of POCs as a kind of unique synthon has been initiated by accommodating ne nanoparticles 8 and being fabricated into reticular frameworks 9 with the help of metal-coordination, covalent and hydrogen-bonding interactions.</p><p>Chirality is vital to biological processes and widely exists in various biological structures at the molecular level, including polysaccharides, proteins and DNA. 10 Incorporation of chirality into articial functional materials provides new objectives towards chiral separation, 11 stereospecic catalysis, 12 chiral recognition, 13 and unique chiroptical properties. 14 Selfassembly depending on chiral recognition and discrimination has been used to prepare chiral functional supramolecular materials through noncovalent interactions such as electrostatic interactions, 15 p-p interactions, 16 hydrogen bonding, 17 and metal-coordination bonds. 18 In quite recent years, DCCbased self-sorting of POCs has been achieved using the racemic mixtures of enantiomers as building blocks, generating a few homochiral and heterochiral cages. 19 It is worth noting that all the thus far obtained heterochiral POCs are made up of enantiomers from only one component. More complicated heterochiral systems derived from the enantiomers of two or more components still remain unreported, to the best of our knowledge. As a consequence, investigation of complicated heterochiral cages and the corresponding applications is surely of signicance for developing POCs and chirality chemistry.</p><p>Herein, we present the clean synthesis of heterochiral porous organic cages (HPOC-1) by enantioselective assembly of the enantiomer of a binaphthol-based tetraaldehyde with specic enantiopure cyclohexanediamine (CA). The corresponding self-assembly reaction kinetics has been tracked using luminescence and nuclear magnetic resonance (NMR) spectroscopy, revealing the clean formation of heterochiral porous organic cages due to the enantioselective recognition of the CA enantiomer towards the enantiomeric binaphtholderived building block 5,5 0 -(6,6 0 -dichloro-2,2 0 -diethoxy-[1,1 0binaphthalene]-4,4 0 -diyl)diisophthalaldehyde (DBD). The crystal structures of the pair of heterochiral HPOC-1 enantiomers show precise sorting of two kinds of chiral building blocks. Interestingly, HPOC-1 displays circularly polarized luminescence (CPL). Furthermore, heterochiral HPOC-1 is able to enantioselectively recognize chiral substrates through circular dichroism (CD) spectral change due to the host-guest supramolecular interactions.</p><!><p>The synthetic route to POCs from tetraaldehydes and CA created by Cooper's group has provided diverse [3 + 6] tubular organic cages. 20 Towards synthesizing more complex heterochiral POCs with two different kinds of enantiomers from more than one component, pure chiral binaphthol-bearing building blocks (DBD) shown in Scheme 1 were prepared to react with cyclohexanediamine (CA). Four sets of combination for these enantiomeric building blocks of two compounds, namely [(S,S)-DBD + (S,S)-CA], [(S,S)-DBD + (R,R)-CA], [(R,R)-DBD + (R,R)-CA] and [(R,R)-DBD + (S,S)-CA], are expected to generate four heterochiral organic cages. Unfortunately, only the reaction between (S,S)-DBD and (R,R)-CA or (R,R)-DBD and (S,S)-CA was able to deliver clean (S,R)-and (R,S)-HPOC-1, respectively, on the basis of MS and NMR spectroscopic data, Table S1 and Fig. S1-S11. †</p><p>In order to understand the assembly process, timedependent NMR spectra were collected for the system of (S,S)-DBD in 2.0 mL CDCl 3 containing 1.0 mL TFA with the addition of 2.0 equiv. of (R,R)-CA and (S,S)-CA, respectively, Fig. S12. † (S,S)-DBD showed one typical singlet peak at d 10.26 ppm due to the aldehyde protons. In addition, methyl and methylene protons displayed peaks at d 1.16 and 4.15 ppm, respectively. Following the addition of (R,R)-CA for 1.0 min, the proton signal of the aldehyde quickly disappeared. Instead, a doublet peak started to appear at d 8.38 ppm due to the formation of imine bonds. Aer 20.0 min, the methyl proton signals moved towards high eld of d 0.27 ppm and methylene proton signals migrated to d 3.00 and 3.28 ppm. The larger high-eld movement indicates an increase in the density of the electron cloud around the methyl group, which further illustrates the location of ethoxy groups inside the cage molecule rather than outside the cage. Aer the proceeding of the reaction for 24.0 h, the 1 H NMR spectrum of the reaction mixture is overall consistent with that of the as-synthesized (S,R)-HPOC-1, implying the clean generation of the heterochiral POC. This is veried by MALDI-TOF MS data with the observation of only one cage molecular ion peak at m/z ¼ 2495.6, Fig. 19a-d,f In addition, uorescence spectroscopy was used to trace the reaction dynamics of (S,S)-DBD with (R,R)-CA and (S,S)-CA, respectively, with a molar ratio of 1 : 2 in CH 2 Cl 2 , Fig. 1a-c. There is a slight emission band of DBD at ca. 502 nm. Aer the introduction of (R,R)-CA for 5.0 min, an obvious emission band was observed at ca. 418 nm. As time went on, the maximum position of the emission band with gradually increased intensity slightly moved to 406 nm. Aer 60.0 min, the intensity remained constant. Such a phenomenon is also observed in the reaction system of (S,S)-DBD and (S,S)-CA, Fig. 1d-f. Differently, a shorter time of 30.0 min enabled a constant emission intensity at 406 nm maximum for the latter system. It is worth noting that the emission intensity at maximum for the former system was almost twice as big as that of the latter one. An enantiomeric uorescence ratio (ef) value of 1.62 was therefore determined based on the division of maximum intensity of the former system by that of the latter one, conrming the higher enantioselectivity of (S,S)-DBD towards (R,R)-CA, rather than (S,S)-CA, to form pure [3 + 6] POC. When (R,R)-DBD was used, a similar phenomenon was found upon the addition of (S,S)-CA and (R,R)-CA, respectively, and the ef is calculated as 1.82, Fig. S16. † This value is similar to the ef of (S,S)-DBD. These results demonstrate the moderate enantioselectivity between these two chiral building blocks during the reaction. 13c,h Density functional theory (DFT) calculations were performed on (S,R)-HPOC-1 and (S,S)-HPOC-1 (assembled from (S,S)-DBD + (S,S)-CA) towards understanding the clean generation of the former heterochiral POC associated with enantioselective recognition between two kinds of enantiomeric building blocks. The simulated formation energy for (S,R)-HPOC-1 is À132.4 kcal mol À1 , much smaller than that for the latter species (À71.5 kcal mol À1 ), hinting at the more favorable formation of the former cage from a thermodynamics perspective. In the present case, the optimized structure of (S,R)-HPOC-1 exhibits less variation in bond length for the cyclohexanediimine moiety than that of (S,S)-HPOC-1 (Fig. S17 and Table S2 †), indicating the more stable structure of the former POC. To further conrm this point, theoretical simulation was also carried out on another heterochiral POC, HPOC-2 derived from the reaction between DBD and 1,2-diphenylethylamine (DPA) enantiomers. The formation energy of (S,R)-HPOC-2 (À137.3 kcal mol À1 ), derived from (S,S)-DBD and (R,R)-DPA, was revealed to be smaller than that of (S,S)-HPOC-2 (À131.7 kcal mol À1 ), assembled from (S,S)-DBD and (S,S)-DPA, further supporting the present clean generation of heterochiral POCs due to the enantioselective assembly mechanism. These results agree well with the experimental ndings that [3 + 6] topological (S,R)-HPOC-2 has been cleanly generated according to NMR and MS data, Fig. S18 and S19. † However, the small enantiomeric uorescence ratio of 1.17 for (S,S)-DBD toward (R,R)-DPA and (S,S)-DPA, Fig. 1f, indicates the weak selectivity of the enantioselective recognition process occurring in the generation of the latter heterochiral POCs in comparison with HPOC-1.</p><p>To detect the structural information of the heterochiral cages, colourless single crystal enantiomers of HPOC-1 were surveyed using a single crystal X-ray diffraction instrument, Fig. 2a and Table S3. † Both enantiomers crystallize in the monoclinic system with a chiral P2 1 space group, and each unit cell is made up of two cage molecules. The detailed structures of these two compounds were described with (S,R)-HPOC-1 assembled from (S,S)-DBD and (R,R)-CA as a typical representative. As expected, it inherits the [3 + 6] cage structural characteristics, possessing three binaphthol segments and six chiral diamines via imine connection. The length of this cage is about 24.4 Å, Fig. 2a. Although the cage cavity is crowded due to the ethoxy side chains, the solvent-accessible surface is computed to be 32.4% using Platon soware. 21 The Flack parameters for the resolved single crystal structures of (S,R)-HPOC-1 and (R,S)- HPOC-1 are 0.049(13) and 0.035(7), respectively, illustrating the enantiopurity of these two newly obtained POCs. As a result, the CD spectra were comparatively studied with reference to those of DBD, Fig. 2b and S20. † (S,S)-DBD shows a positive CD band from 240 to 260 nm and a negative band from 260 to 360 nm. The enantiomer exhibits mirror CD bands due to the completely inverse Cotton effect. Aer the formation of (S,R)-HPOC-1, three CD bands, namely a positive band in the range of 240-260 nm and two negative bands at 260-280 nm and 280-360 nm, respectively, were observed. The newly observed negative band at 260-280 nm might be the consequence of imine band formation from (R,R)-CA reacting with the (S,S)-DBD moiety. This is consistent with similar [3 + 6] POC analogues. 3b,5a,20 HPOC-1 in tetrahydrofuran (THF) displayed absorption bands at 295 and 357 nm, similar to those of DBD, Fig. S21. † The molar absorption coefficient of 3.9 Â 10 4 L mol À1 cm À1 at 357 nm for the cage is bigger than that of DBD (2.0 Â 10 4 L mol À1 cm À1 ). Upon excitation at 357 nm for DBD, a broad emission band with the maximum at 408 and 430 nm appears, Fig. S22, † corresponding to a low uorescence quantum yield of 1.6% (calculated with quinine sulfate as the standard). In contrast, a strong emission band at 403 nm was observed for HPOC-1 under the same excitation conditions with the uorescence quantum yield as high as 63.4% due to the formation of an imine bond, 13c,k exceeding that of most organic cage compounds. 3a,14b Additionally, transient uorescence spectra showed that HPOC-1 has a higher uorescence lifetime (1.67 ns) than DBD (1.09 ns), Fig. S23. † Since few POCs were revealed to show circularly polarized luminescence (CPL), a typical characteristic of the excited states of chiral systems with application potential in 3D display technology, optoelectronic devices and chiral sensing, 14d,f the present uorescence heterochiral HPOC-1 enantiomers composed of the binaphthol moiety therefore inspire us to investigate their CPL properties. As shown in Fig. 2c, (S,R)-HPOC-1 in THF exhibited a broad positive emission in the range of 380-500 nm with the maximum at 425 nm. However, the CPL prole of (R,S)-HPOC-1 shows a mirror-image with that of (S,R)-HPOC-1. The CPL dissymmetry factor (g lum ) was calculated to be AE3.3 Â 10 À4 . This value is similar to that of analogous POCs such as 6M-2 and T-FRP 1. 14a,b Incorporation of two kinds of chiral building blocks into HPOC-1 provides a new heterochiral host for enantioselective recognition of chiral molecules in solution. Herein, CD spectroscopy was employed to monitor the potential interaction between heterochiral HPOC-1 and chiral small molecules since this technique is able to directly discriminate molecules with different absolute congurations and ee quantitation according to visual signals, superior to uorescence and NMR techniques. 13a,b,13j In the present case, a series of chiral small molecules, including carvone, 1-phenylethanol, limonene, tartaric acid and pinene, were screened to probe the chiral recognition potential of HPOC-1. Gradual introduction of D-carvone resulted in the continuous decrease of the CD signal of (S,R)-HPOC-1 at 250 nm, Fig. 3a. In good contrast, a tiny CD change was found upon the introduction of L-carvone, Fig. 3b and c. These results imply the existence of specic interaction between (S,R)-HPOC-1 and D-carvone rather than L-carvone. In addition, upon increasing the D-carvone concentration from 0 to 240.0 mM, the CD signal at 250 nm for (S,R)-HPOC-1 solution decreased in a much faster manner than that of the control without POC molecules, supporting the presence of enantioselective recognition of this cage toward D-carvone, Fig. S24. † Such a phenomenon was also observed in the chiral recognition test using (R,S)-HPOC-1 towards L-carvone, Fig. 3d-3f. For the other four chiral substrates including 1-phenylethanol, tartaric acid, limonene and pinene, Fig. S25 and Table S4, † almost no obvious CD change was observed for (S,R)-HPOC-1, indicating the specicity of the chiral recognition of this POC towards Dcarvone. These results imply the existence of specic interaction between (S,R)-HPOC-1 and D-carvone rather than L-carvone. In addition, a titration experiment of carvone with (S,S)-DBD at three-times the concentration of (S,R)-HPOC-1 was carried out. As shown in Fig. S26, † the CD signal changes for the solution of monomer (S,S)-DBD upon adding D-and L-carvone are similar to those of the cage, indicating that this monomer can also enantioselectively recognize D-carvone rather than L-carvone. Similar enantioselective recognition phenomena of the cage and monomer towards carvone are due possibly to the presence of 1,1 0 -binaphthalene moieties. However, at the same concentration of 1,1 0 -binaphthalene moieties for the solution of (S,R)-HPOC-1 and (S,S)-DBD, the slope absolute value of the tting line between the CD signal intensity and the concentration of chiral D-carvone for the cage (À0.124) is much bigger than that of the monomer (À0.075), indicating the better enantioselectivity of the former species. This most probably originates from the superiority of the cage-like molecular structure which provides stronger interaction with the chiral substrate. Similar to (S,R)-HPOC-1, no obvious CD change is observed for (S,S)-DBD upon adding 1-phenylethanol enantiomers (as a representative of the used chiral substrates except carvone), Fig. S27. † These results indicate that cage (S,R)-HPOC-1 has better chiral recognition properties than monomer (S,S)-DBD.</p><p>To elucidate the monitored enantioselective recognition mechanism, molecular simulations were conducted on the (S,R)-HPOC-1 host and two enantiomeric analytes using DFT calculations. The optimized molecular structure of (S,R)-HPOC-1 bound with D-carvone shows that protons on the D-carvone point toward the face of the binaphthol moiety with atom-toplane distances of 2.620 and 3.046 Å (Fig. S28 †), respectively, indicating the presence of CH/p interactions between the cage and guest. This point was further supported by the observation of a proton signal shi in the 1 H NMR spectrum of a mixture of (S,R)-HPOC-1 and D-carvone in comparison with that of the control, Fig. S29. † The CH-p interaction may affect the p / p* transitions of the binaphthol moieties, leading to the change of the intensity of the CD peak at 250 nm. Furthermore, the binding energy of (S,R)-HPOC-1 and the D-carvone molecule is À102.5 kcal mol À1 , indicating the presence of binding interaction. Instead, a unstable state between (S,R)-HPOC-1 and the Lcarvone molecule is suggested by the high value of binding energy (807.3 kcal mol À1 ). In contrast to the obvious CD response of (S,R)-HPOC-1 towards D-carvone, we hypothesize that the non-enantioselective recognition effect of POC for Lcarvone might be attributed to the much weaker interaction strength between the host and chiral guest, as indicated by the absence of an optimized structure for (S,R)-HPOC-1 in the presence of L-carvone in a 1 : 1 ratio. However, the exact reason is still not clear at this stage.</p><p>At the end of this section, it is worth noting that a series of CD spectra of (S,R)-HPOC-1 with the addition of a mixture of rac-carvone with different ee values were collected, Fig. S30a. † A good linear relationship between CD signal intensity at 250 nm and ee value was observed, Fig. S30b. † According to this standard working curve, it is easy to determine the enantiomeric excess of carvone. This point is further supported by the fact that the experimental results for three rac-carvone samples are consistent with the theoretical values, Table S5. †</p><!><p>In summary, enantioselective assembly of new heterochiral functional materials from two kinds of enantiomeric building blocks has been clearly established. The enantioselective recognition mechanism involved in the self-assembly of building blocks has guaranteed the clean formation of a pair of heterochiral porous organic cage enantiomers with circularly polarized luminescence property. This new POC possesses enantioselective recognition capability towards chiral substrates according to the circular dichroism data. This work no doubt provides a new perspective towards constructing heterochiral POCs and therefore should benet the chemistry of chiral self-assemblies. performed theoretical calculations; J. Jiang, H. Wang, C. Liu and H. Ren linked experiments and analysis; and all the authors discussed and wrote the manuscript.</p><!><p>There are no conicts to declare.</p>

Royal Society of Chemistry (RSC)

Star PolyMOCs with Diverse Structures, Dynamics, and Functions by Three-Component Assembly

We report star polymer metal-organic cage (polyMOC) materials whose structures, mechanical properties, functionalities, and dynamics can all be precisely tailored through a simple three-component assembly strategy. The star polyMOC network is composed of tetra-arm star polymers functionalized with ligands on the chain ends, small molecule ligands, and palladium ions; polyMOCs are formed via metal-ligand coordination and thermal annealing. The ratio of small molecule ligands to polymer-bound ligands determines the connectivity of the MOC junctions and the network structure. The use of large M12L24 MOCs enables great flexibility in tuning this ratio, which provides access to a rich spectrum of material properties including tunable moduli and relaxation dynamics.

star_polymocs_with_diverse_structures,_dynamics,_and_functions_by_three-component_assembly

<p>Polymer networks are versatile materials with a wide range of structures and properties suitable for industrial and academic applications.[1–3] In a typical network, macromolecules of choice are connected to branched junctions of a particular type; the nature of these components determines the material's properties such as stiffness, toughness, responsiveness, etc.[2] Several strategies have been developed to tune polymer network structure in order to realize desirable properties. For example, interpenetrating networks,[4–5] nanocomposites,[6] and reversible and/or dynamic covalent bonds[7] are employed to yield materials with self-healing, stimuli-responsive, and other valuable behaviors.[7–8] In all of these cases, control over network structure and dynamics is critical. In this communication, we describe a versatile and simple strategy for controlling structure, function, and dynamics in a relatively new class of polymer networks that is based on the use of large metal-organic cages/polyhedra (MOCs).</p><p>MOCs are discrete 3D structures assembled from x metal ions and y organic ligands via coordination bonds.[9–22] By rational design of the ligands and proper choice of the metal ions, MOCs of different MxLy stoichiometries, sizes, and geometries can be synthesized.[23–29] Inspired by this structural versatility and potential applications such as mechanical enhancement, catalysis, encapsulation, sensing, etc., much effort has been recently devoted to installing MOCs into polymer networks to provide 'polyMOC' hybrid materials with tunable viscoelasticity and functionality.[30–37]</p><p>One scheme for polyMOC synthesis utilizes linear polymers linked by MOCs as crosslink junctions. For example, by using Pd-pyridine-based MOCs with distinct sizes, M12L24 and M2L4, we demonstrated that the mechanical properties of a poly(ethylene glycol) (PEG)-based polyMOC gel could be readily controlled, thereby offering a new strategy for regulating mechanics in constitutionally isomeric materials.[31] Nitschke and co-workers reported hydrogel polyMOCs crosslinked by tetrahedral MOCs that could selectively encapsulate and release small molecules.[32] Several other recent examples of MOC-containing polymeric materials highlight the potential of these systems[30, 38–42]; however, there is still a great need to understand how polyMOC microstructure translates to bulk material properties such as modulus and relaxation dynamics. To accomplish this goal, robust, modular polyMOC synthesis strategies that enable access to a wide range of structures and properties are needed.</p><p>Herein, we describe a three-component assembly approach for the modular synthesis of polyMOCs from ligand functionalized tetra-arm star polymers, small molecule ligands (SML), and Pd2+ ions (Figure 1). When these components are mixed in a desired ratio and annealed, a network of star polymers connected to MOCs is formed wherein each MOC possesses a mixture of polymer-bound ligands (PLs) and SMLs. By combining star polymers, which act as covalent network junctions, and large Pd12L24 MOCs, which serve as dynamic network junctions as well as reservoirs for SMLs, a wide spectrum of polyMOC structures, properties, and dynamics is accessible. This approach greatly expands the versatility of polyMOC chemistry, and provides a synthetic strategy that should be amenable to new classes of polyMOCs in the future.</p><p>The star polymer used in this study (Figure 1, Mn = 18k, Đ = 1.09) was prepared by atom transfer radical polymerization (ATRP) from pentaerythritol tetrakis(2-bromoisobutyrate) initiator followed by end-group substitution with a para-bispyridyl phenol (see Supporting Information, SI, for details). 1H NMR spectroscopy suggested greater than 95% chain end functionalization (Figure S1). The SML was an analogous para-bispyridyl methyl ether derivative (Figure 1, R = Me).</p><p>We synthesized a series of polyMOC gels where the concentration of polymer was held constant while the amounts of SML and Pd2+ were varied (the ratio of Pd2+ to pyridine groups was always 1:4). We name these polyMOC gels as Geln, where n is the molar ratio of SMLs to PLs (PL = equivalents of star polymer × 4) used in the synthesis. If we assume that all of the ligands are incorporated into M12L24 MOCs, then the average number of SMLs in each MOC is a = 24n/(n+1); the number of PLs per MOC is f = 24 - a. For example, Gel5 is a polyMOC made by mixing 1 equivalent of the star polymer (i.e., 4 PLs) and 20 equivalents of SML (20 / 4 = 5) with a stoichiometric amount of Pd2+. Thus, each MOC in this network has an average of 20 SMLs (a = 20) and 4 PLs (f = 4). PolyMOC gels with n values ranging from 0 (i. e. no SML) to 13 were prepared.</p><p>Figure 2 shows the aromatic region of the magic angle spinning (MAS) 1H NMR spectra for selected polyMOCs alongside the solution 1H NMR spectra for the SML and the M12L24 MOC formed from this SML. MAS NMR spectra for other polyMOCs are provided in Figure S2. From these data, it is clear that as n increases the MOCs become less restricted; the aromatic resonances in the MAS NMR spectra become more similar to those of the free MOC in solution.</p><p>To confirm that the SMLs were integrated within the polyMOC network, we thoroughly extracted the gels with fresh DMSO-d6 and analyzed the soluble fraction by 1H NMR. As shown in Table S1, as the amount of SML decreased, the percentage of soluble MOCs (MOCs not connected to the network) also decreased: ~15% for Gel11, 13% for Gel9, 10% for Gel5, and 4% for Gel3. When n ≤ 2, free MOCs were not detectable. Thus, the majority of the SMLs are incorporated into these polyMOCs. When n = 13, the average MOC contains only f = ~1.7 PLs. Thus, though gels were obtained, more than 25% percent of SML as well as some PL were extracted.</p><p>To investigate how the average number of PLs connected to each MOC (f) affects the MOC mobility, we measured the transverse relaxation time, T2, of a characteristic MOC peak (9.6 ppm) by MAS 1H NMR. We assume that MOCs with more SMLs (i.e., higher n and smaller f) will relax more rapidly (larger T2). The T2 values for the as made versus extracted Gel9 (f = 2.4) were 9.7 ± 0.6 ms and 8.5 ± 1.0 ms, respectively (Figure S3). The slight decrease in the T2 after extraction is likely due to the removal of soluble MOCs, which should have a higher T2 than the network-bound MOCs. For comparison, the T2 value for as-made Gel3 (f = 6) was 4.7 ± 0.6 ms, which confirms that the MOCs in this material are more restricted compared to Gel9. In a control experiment, free MOCs were added to Gel3; as expected, the measured T2 value increased (to 6.3 ± 0.6 ms).</p><p>The structure of these polyMOCs was also characterized by small-angle X-ray scattering (SAXS)(Figure 3). For Gel0, a broad and weak peak was observed at q = 0.065 Å−1. As n increases, three peaks emerge and sharpen. The q value for the first peak ranges from 0.79 for Gel3 to 0.90 Å−1 for Gel11, while the second and third peaks remain constant at q = 0.29 and 0.58 Å−1. The latter two peaks agree well with the form factors of a 3.5 nm nanoparticle; they are assigned to the MOCs embedded within the network.[37] The data suggests that as n increases, the fidelity of MOC formation also increases. The low q SAXS peak for each polyMOC is assigned as the average distance between adjacent MOCs linked by polymer chains (Figure 3, inset). This distance decreases as n, which is proportional to the MOC concentration, increases.</p><p>Next, we used shear oscillatory rheometry to investigate how n impacts the storage (G′) and loss (G″) moduli of these materials. First, we note that for all samples from n = 0 to 13 the G′ values were larger than the G″ values at all tested frequencies; these materials are elastic solids (Figure S4). A plot of G′ (at ω = 1 rad/s) versus n reveals a maximum at n = 3 (Figure 4a). Initially, G′ increases rapidly: from 400 ± 100 Pa for Gel0 to 3800 ± 200 Pa for Gel3; i.e., Gel3 was nearly 10 times stiffer than Gel0, which is remarkable given that the ~18 SMLs in each MOC in Gel3 are not elastically effective. As n increases beyond 3, the polyMOCs become softer: G′ dropped to 500 ± 50 Pa for Gel13. In these examples, a stoichiometric amount of Pd2+ was used to fully coordinate all PLs and SMLs. As expected, off-stoichiometry studies between Pd2+ and pyridine ligands led to decreases in G′, since the crosslinking density decreases when Pd2+ is in deficiency and f decreases when Pd2+ is in excess (See SI, Table S2).</p><p>The observed relationship between G′ and n can be rationalized by considering how n differentially affects the network crosslink density and f. As n increases, the MOC density, and therefore the crosslink density, increases (as confirmed by SAXS). At the same time, f decreases since each MOC must contain fewer polymer-bound ligands. Since n and f are inversely related, a plot of n × (f − 2) (assuming that linear junctions, f = 2, do not contribute to elasticity) closely resembles Figure 4a (Figure S5). Notably, the swelling ratios of these materials increased with n, suggesting that the network mesh size increases despite the fact that the MOC concentration increases (Figure S6).</p><p>Based on the data above, we can divide the structure of these polyMOCs into four characteristic classes defined by n (Figure 4). In class I, n < 1 (f > 12): the MOC concentration is low and the MOCs likely have a large fraction of topological defects[43–44] in order to pack >12 polymer chains around a single MOC (as indicated by broad peaks in NMR and SAXS). Thus, the materials are very soft. In class II, 1 ≤ n < 6, (3.4 < f ≤ 12): SMLs facilitate the formation of well-defined MOC junctions, the crosslink density increases and f is quite large, which results in large G′ values. In class III, 6 ≤ n < 11 (2 < f ≤ 3.4): the MOC concentration is high, but f is low, and the materials are soft. Finally, in class IV, n ≥ 11: the MOC stoichiometry is such that on average two or fewer polymer chains are attached to each MOC. Thus, some MOCs no longer serve as crosslink junctions, but instead as linear linkers between polymer chains or dangling ends. Cage-saturated star polymers may also be present.</p><p>Next, we sought to evaluate the stress relaxation dynamics of these polyMOC gels. Stress relaxation is a critical parameter in polymer network design that is exploited in synthetic and biological polymer networks to achieve unique time-dependent behaviors. To our knowledge, there are no reports on stress relaxation in polyMOC materials. However, Fujita and coworkers have shown that ligand exchange in analogous M12L24 MOCs in solution is very slow due to cooperativity effects.[45–46] At ambient temperature, the metal-coordination bonds in these MOCs are considered to be as static as covalent bonds; they become dynamic again only when heated to >70 °C. Since the dynamic nature of supramolecular networks is related to the rates of ligand exchange and polymer chain diffusion,[42, 47–48] we reasoned that stress relaxation in our polyMOCs could be dependent on n, and thus a diverse range of temperature-dependent mechanical timescales could be accessed.</p><p>Stress-relaxation studies were conducted to measure G′ under constant strain as a function of time at various temperatures for polyMOCs of varied n (Figure 5). As shown for Gel9 in Figure 5a, at 25 °C and 40 °C the gels show little relaxation; at 55 °C and 70 °C they relaxed progressively more rapidly. Following from these observations, the gels undergo self-healing when heated above 70 °C (Figure S7). Figure 5b–d and Figure S8 compare the relaxation behavior of polyMOCs with different n values at various temperatures. Gel1, Gel5, and Gel9 are chosen as they fall into the different aforementioned network classes (I, II, and III, respectively). At 40 °C, all the gels show similarly slow relaxation (Figure S8). At 55 °C, both Gel1 and Gel9 relaxed more rapidly than Gel5 (Figure 5b). Fitting using the Kohlrausch model[49] (solid lines in Figure 5b–c, see SI for details), provided the characteristic relaxation times τ: 2693 s, 11352 s, and 5804 s for Gel1, Gel5, and Gel9, respectively. A similar trend was observed at 70 °C, as shown in Figure 5c–d.</p><p>Interestingly, the trend for τ versus n follows the network classifications depicted in Figure 4 and mirrors the G' data in Figure S5. For Gel1 (class I), topological defects and ill-formed MOCs facilitate fast relaxation.[45–46] For Gel5 (class II), the MOCs are well-formed and thus the metal-ligand bonds are static. Furthermore, the crosslink density is high and polymer diffusion may be slow, leading to slowed relaxation even in the presence of SMLs. For Gel9 (class III), f is low and the high concentration of MOCs enables fast ligand exchange and enhanced relaxation (SMLs should diffuse faster than PLs). Taken together, these results reveal that polyMOCs have dynamic covalent nature, and that their dynamics can be easily tuned through addition of free SMLs.</p><p>Finally, having shown that SMLs can readily be incorporated into MOCs to provide a range of novel network structures and dynamics, we sought to use an alternative SML to selectively install functionality into the polyMOC junctions, thus demonstrating control over structure, dynamics, and function in this system. We prepared a series of Gel7 derivatives where the total amount of ligand was held constant, but 5%, 20%, or 60% of the methyl-ether SML was replaced with a pyrene-modified SML (Figure 6a). After swelling and extraction, the three polyMOCs had the same volume, which suggests that their network structures are similar as would be expected if the pyrene-SMLs were incorporated into the MOCs without otherwise changing the network connectivity. Fluorescence spectra of the gels confirmed that the increase in fluorescence intensity is qualitatively proportional to the amount of pyrene-SML incorporated (quantum yields were 0.08 for 5% and 60% gel, and 0.07 for 20% gel; see SI). The fluorescence can also be observed under a bench top UV lamp (Figure 6b, inset). These observations confirm that varied amounts of functional SMLs can be introduced to broaden the functional diversity of polyMOCs.</p><p>In conclusion, we have described a simple yet versatile approach –based on three-component assembly– that enables the synthesis of star polyMOC gels with diverse network structures, mechanical properties, dynamics, and functionality. Our strategy should translate to other potential polyMOC materials, e.g., ones based on the M30L60 cage recently reported by Fujita et al.[50] Importantly, our results show that MOCs can be used to precisely tune the structure and dynamics of polymer networks.</p>

PubMed Author Manuscript

A boron-transfer mechanism mediating the thermally induced revival of frustrated carbene–borane pairs from their shelf-stable adducts

Chemists have designed strategies that trigger the conformational isomerization of molecules in response to external stimuli, which can be further applied to regulate the complexation between Lewis acids and bases. We have recently developed a system in which frustrated carbene-borane pairs are revived from shelf-stable but external-stimuli-responsive carbene-borane adducts comprised of N-phosphine-oxide-substituted imidazolylidenes (PoxIms) and triarylboranes. Herein, we report the detailed mechanism on this revival process. A thermally induced borane-transfer process from the carbene carbon atom to the Nphosphinoyl oxygen atom initiates the transformation of the carbene-borane adduct. Subsequent conformational isomerization via the rotation of the N-phosphinoyl group in PoxIm moieties eventually leads to the revival of frustrated carbene-borane pairs that can cleave H 2 . We believe that this work illustrates an essential role of dynamic conformational isomerization in the regulation of the reactivity of external-stimuli-responsive Lewis acid-base adducts that contain multifunctional substituents.

a_boron-transfer_mechanism_mediating_the_thermally_induced_revival_of_frustrated_carbene–borane_pair

<!>Results and discussion<!>Conclusion.<!>Methods

<p>T here have been many recent developments in the chemistry of frustrated Lewis pairs (FLPs) that have been of note, for example, the activation of H 2 mediated by main-group elements [1][2][3][4][5][6][7][8][9] . In general, FLPs are transient and not shelf-stable species making their isolation challenging. Meanwhile, chemists have developed strategies that trigger the conformational isomerization of molecules in response to external-stimuli [10][11][12][13] . These strategies can also be used to generate transient FLP species from classical Lewis adducts (CLAs) that act like their shelf-stable precursors [14][15][16][17][18][19][20][21][22][23][24][25][26] . In 2015, we demonstrated a strategy to generate FLPs from shelf-stable CLAs (PoxIm•B 1 in Fig. 1) that are comprised of N-phosphine-oxide-substituted imidazolylidenes (PoxIms; 1) and B(C 6 F 5 ) 3 (B 1 ). Here, the revival of the FLP from the CLA is closely controlled by a thermally induced conformational isomerization of the N-phosphinoyl moiety 17,[27][28][29][30] . In 2018, Stephan et al. reported a system to control the generation of FLPs from CLAs via a light-induced E/Z isomerization of (C 6 F 5 ) 2 B((p-Tol)S)C = CCH( t Bu) 31 . Nevertheless, such FLP revival systems, including external-stimuli-responsive conformational isomerizations, are still underdeveloped. Thus, clarifying the relationship between external-stimuli-responsive conformational isomerizations and the interconversion that occurs between frustrated and quenched Lewis pairs is of great importance. This would allow a significant expansion of different strategies to design and apply FLP species 19 .</p><p>In our system that uses PoxIms, the revival mechanism has not been fully explained. A tentative mechanism in which a B(C 6 F 5 ) 3 moiety is repelled by the N-phosphinoyl group via a thermally induced isomerization from the syn to anti conformation had been proposed. In this case, the syn/anti conformation refers to the relative orientation of the carbene carbon atom and the N-phosphinoyl oxygen atom with respect to the N-P bond (Fig. 1a) 17 . Herein, we report the results of a combined experimental and theoretical mechanistic study that demonstrates the key role of a transfer step where the triarylborane (BAr 3 ) unit on the carbene carbon atom moves to the N-phosphinoyl oxygen atom (Fig. 1b). In this study, PoxIms with 2,6-i Pr 2 -C 6 H 3 , 2,4,6-Me 3 -C 6 H 2 , and 3,5-t Bu 2 -C 6 H 3 groups were studied and are herein referred to as 1a, 1b, and 1c, respectively.</p><!><p>Effects of Lewis acidity. To explore the impact of the Lewis acidity of BAr 3 on the formation and reactivity of the carbene-borane adducts, the reaction between 1a and B(p-HC 6 F 4 ) 3 (B 2 ) was undertaken (Fig. 2a). Full consumption of 1a was confirmed after 20 min, resulting in the formation of two CLAs, i.e., 2aB 2 , which contains a N-phosphinoyl oxygen-boron bond, and 3aB 2 , which contains a carbene-boron bond, in 61% and 29% yield, respectively. Previously, we have reported that, even at -30 °C, 2aB 1 could be converted to 3aB 1 and that full identification of 2aB 1 could therefore be achieved using NMR analysis conducted at -90 °C17 . In the present case, 2aB 2 exhibited a longer life-time at room temperature than 2aB 1 , which enabled us to prepare single crystals of 2aB 2 by recrystallization from the reaction mixture at -30 °C. The molecular structure of 2aB 2 was unambiguously confirmed using single-crystal X-ray diffraction (SC-XRD) analysis. A set of (R a ) and (S a ) atropisomers of 2aB 2 was identified in the asymmetric unit of the single crystal. The molecular structure of (R a )-2aB 2 is shown in Fig. 2b and demonstrates a rare example of complexation-induced N-P axial chirality 29 . As the reaction progressed, 2aB 2 was converted to 3aB 2 and 4a; 2aB 2 was fully consumed within 6 h to afford these compounds in 75% and 25% yield, respectively. It should be noted that 4a is likely furnished via the migration of the Nphosphinoyl group from the nitrogen atom to the carbene carbon atom. However, in the absence of B 2 , this migration only proceeded to 9% at 100 °C, even after 25 h 30 . The formation of 4a was therefore promoted by the enhancement of the electrophilicity of the P center via the coordination of the N-phosphinoyl moiety to B 2 . Regeneration of B 2 was observed along with the production of 4a. The molecular structure of 3aB 2 was also confirmed by SC-XRD analysis (Fig. 2c). Comparison of the structural parameters between the solid-state structures of 3aB 2 and 3aB 1 shows their similarity. For example, the C1-B distances in 3aB 2 and in 3aB 1 are 1.710(3) Å and 1.696(3) Å, respectively. The interatomic distance of 3.257(3) Å between the O and B atoms in 3aB 2 suggests the absence of a specific interaction between these atoms, similar to that in 3aB 1 (3.234(3) Å).</p><p>Thermolysis of 3aB 2 at 60 °C for 3 h resulted in the generation of 4a and B 2 in 77% and 73% yield, respectively, with concomitant formation of [1a-H][HO(B 2 ) 2 ] in 4% yield (conversion of 3aB 2 = 81%; Fig. 3a). Although 2aB 2 was not observed via NMR analysis of this reaction at 60 °C, the formation of 4a and B 2 indicates the in situ regeneration of 2aB 2 (vide supra). The formation of [1a-H][HO(B 2 ) 2 ] can be rationalized in terms of a reaction between contaminated H 2 O and the FLP species regenerated from 3aB 2 via 2aB 2 . The regeneration of the FLP species from 3aB 2 was then clearly confirmed by treating 3aB 2 with H 2 (5 atm) at 22 °C, resulting in the formation of</p><p>) 2 ] (8%) and 1a (6%) (Fig. 3b). Under identical conditions, no reaction occurred when 3aB 1 was used 17 . At 60 °C, 5aB 2 was generated in 90% yield after 3 h, which is almost comparable with the production of 5aB 1 (89%) from 3aB 1 . Thus, the lower Lewis acidity of B 2 relative to B 1 allowed a more facile revival of the FLP species from 3aB 2 than from 3aB 1 . However, the lower Lewis acidity did not affect the progress of the heterolytic cleavage of H 2 by FLPs at 60 °C. Kinetic studies. To gain further insight into the reaction mechanism, the initial rate constants for the generation of 5aB 1 , k int [10 -5 s -1 ], from the reaction between 3aB 1 and H 2 in 1,2dichloroethane-d 4 (DCE-d 4 ) at 60 °C were estimated by varying the H 2 pressure from 0.5 to 5.0 atm (Fig. 4a). It should be noted here that when H 2 was pressurized at 5.0 atm, an excess of H 2 (ca. 0.3 mmol) with respect to 3aB 1 (0.010 mmol) was added to the pressure-tight NMR tube. The concentration of H 2 clearly influenced the progress of the reaction, suggesting that the heterolytic cleavage of H 2 by the FLP species is involved in the ratedetermining events. Next, the reaction between 3aB 1 and H 2 at 5.0 atm of pressure was monitored in DCE-d 4 whilst the temperature was varied from 50 to 80 °C (Supplementary Figure 27). Pseudo-first order rate constants, k obs [10 -5 s -1 ], of 2.95(2), 11.2(8), 46.4(4) and 183(2) were estimated for the reactions at 50, 60, 70, and 80 °C, respectively. Thus, the activation energy and pre-exponential factor obtained from the plot based on the Arrhenius equation, lnk obs = -(E a /R)(1/T) + lnA, are E a = 31.2 [kcal mol -1 ] and A = 3.3(36) × 10 16 [s -1 ] (Fig. 4b). Given the close relation between E a and ΔH ‡ , the values obtained for E a suggest that the formation of 5aB 1 via the reaction between 3aB 1 and H 2 only occurs at temperatures higher than 25 °C32 .</p><p>Based on the results presented here and those previously reported 17 , the reaction between the carbene-borane adducts and H 2 to give [PoxIm-H][H-BAr 3 ] likely proceeds via the heterolytic cleavage of H 2 by the FLP species that are formed following the regeneration of the N-phosphinoyl oxygen-borane adducts. These steps are expected to be the rate-determining events because the concentration of H 2 (Fig. 4a), the steric bulk of the N-aryl group 17 and the Lewis acidity of the BAr 3 moiety (Fig. 3b) influence the reaction rates and/or the temperature required to initiate the reaction between the carbene-borane adducts and H 2 .</p><p>Theoretical studies. Density-functional theory (DFT) calculations were carried out at the ωB97X-D/6-311G(d,p), PCM (DCE)// ωB97X-D/6-31G(d,p) for H 2 and 6-31G(d) for all other atoms level of theory (Fig. 5a). The relative Gibbs free energies with respect to [1a + B 1 ] (0.0 kcal•mol -1 ) are shown. During the transformation of 3aB 1 (-17.2 kcal•mol -1 ) to 2aB 1 (-9.8 kcal•mol -1 ), both of which were experimentally confirmed, the formation of an intermediate 2a′ B 1 (-7.7 kcal•mol -1 ) was predicted via a C-to-O transfer of B 1 in 3aB 1 . This distinctive boron-transfer process takes place via saddle 2 ] and 1a were also observed in 8% and 6% yield, respectively. b [1a-H][HO(B 2 ) 2 ] was also observed in 7% yield. c Results obtained using 3aB 1 are reproduced from ref. 17 37) 33,34 . This AIM analysis demonstrates that several noncovalent interactions, including π-π and H•••F interactions, exist between the 1a and B 1 moieties to stabilize TS1a.</p><p>Two plausible mechanisms were evaluated for the FLPmediated cleavage of H 2 on the basis that the Lewis-basic center reacts with H 2 via cooperation with B 1 (Fig. 5b). One possibility is that the carbene carbon atom works as a Lewis base (path I; the right path in Fig. 5b) [1][2][3][4][5][6][7][8][9]35,36 , while the other is that the Nphosphinoyl oxygen functions as a Lewis base (path II; the left path in Fig. 5b) 37 . In path I, the heterolytic cleavage of H 2 takes places via TS4a (+11.4 kcal•mol -1 ), which arises from the insertion of H 2 into the reaction field around the carbene carbon and boron atoms in FLP-1aB 1 , affording 5aB 1 (-34.8 kcal•mol -1 ), a species more thermodynamically stable than 3aB 1 . In the optimized structure of TS4a (Fig. 5d), the dissociation of the H1-H2 bond (H1•••H2 = 0.84 Å) occurs with the partial formation of the H2-C1/H1-B bonds (H2•••C1 = 1.83 Å/H1•••B = 1.49 Å). Based on these results, the overall path from 3aB 1 to 5aB 1 via FLP-1aB 1 is substantially exothermic (ΔG°=-17.6 kcal•mol -1 ) and includes an overall activation energy barrier of +28.6 kcal•mol -1 required to overcome TS4a. In path II, which takes place via TS5a (a transition state for the insertion of H 2 into the O-P bond) and TS6a (a transition state for the cleavage of H 2 between the O and P atoms), a higher activation energy barrier of +32.7 kcal•mol -1 is predicted to yield intermediate 8aB 1 , which contains a P=O-H + and B-H − species. It should be noted that the potential energy of the optimized TS6a (-3633.288355 hartree) is almost identical to that of the optimized 7aB 1 (-3633.288363 hartree), which causes the reversed Gibbs energy levels as shown in Fig. 5b after the Gibbs energy correction and implementation of solvent effect. Therefore, the discussion on the activation energy barrier to overcome TS6a from 7aB 1 should be not essential. The subsequent transfer of H + from the Nphosphinoyl oxygen atom to the carbene carbon atom furnishes 5aB 1 , although the details of this process remain unclear at this point. The molecular structure of TS6a shows that the cleavage of the H1-H2 bond (H1•••H2 = 0.85 Å) by the N-phosphinoyl oxygen and boron atoms occurs in a cooperative fashion (Fig. 5d). Given the experimental and theoretical results reported here, we conclude that path I is the more likely one.</p><p>The impact of the N-aryl substituents on the activation energy barriers for the regeneration of [1 + B 1 ] was evaluated using calculations on 3bB 1 , which contains an N-2,4,6-Me 3 -C 6 H 2 group, as well as 3cB 1 , which contains an N-3,5-t Bu 2 -C 6 H 3 group. This afforded ΔG ‡ values of +28.3 and +32.8 kcal•mol -1 for 3bB 1 and 3cB 1 , respectively (Fig. 5a). These results are consistent with the experimental observations, i.e., that 3aB 1 −3cB 1 did not react in the presence or absence of H 2 under ambient conditions under the applied conditions. Furthermore, these results might rationalize the fact that temperature to induce the reaction between these CLAs and H 2 increases in the order 3aB 1 (60 °C) < 3bB 1 (80 °C) < 3cB 1 (120 °C) 17 .</p><!><p>In summary, the reaction mechanism for the revival of frustrated carbene-borane pairs from external-stimuliresponsive classical Lewis adducts (CLAs), comprised of N-phosphine-oxide-substituted imidazolylidene (PoxIm) and triarylboranes (BAr 3 ), is reported based on a combination of experimental and theoretical studies. Remarkably, a transfer of the borane moiety from the carbene carbon atom to the Nphosphinoyl oxygen atom was identified as a key step in the heterolytic cleavage of H 2 by the regenerated FLP species. The optimized transition-state structure for this borane-transfer process was confirmed to include no bonding interactions between the carbene carbon/phosphinoyl oxygen and boron atoms, albeit that it is stabilized by intermolecular non-covalent interactions between the PoxIm and BAr 3 moieties. The heterolytic cleavage of H 2 takes place via the cooperation of the carbene carbon and the boron atoms, and exhibits a lower overall activation energy barrier than that of the path in which a combination of the Nphosphinoyl oxygen and boron atom mediates the H 2 cleavage. These results demonstrate the essential role of dynamic conformational isomerization in the regulation of the reactivity of shelf-stable but external-stimuli-responsive Lewis acid-base adducts by multifunctional Lewis bases.</p><!><p>Synthesis of 3aB 2 . PoxIm 1a (154.8 mg, 0.40 mmol) and B(p-HC 6 F 4 ) 3 (B 2 ) (183.4 mg, 0.40 mmol) were mixed in toluene (10 mL) at room temperature to furnish the yellow solution. Stirring this mixture for 4 h resulted into the precipitation of a white solid that was collected via removal of the supernatant solution. The obtained solid was washed with hexane (5 mL) and dried in vacuo to afford 3aB 2 as a white solid (230.2 mg, 0.27 mmol, 68%). A single crystal suitable for X-ray diffraction analysis was prepared by recrystallization from CH 2 Cl 2 /hexane at -30 °C.</p><p>Synthesis of 5aB 2 . A solution of 3aB 2 (51.6 mg, 0.06 mmol) in CH 2 Cl 2 (3 mL) was transferred into an autoclave reactor, which was then pressurized with H 2 (5 atm). Subsequently, the reaction mixture was stirred at 60 °C for 4 h, before the solvent was removed in vacuo to give 5aB 2 as a white solid (51.8 mg, 0.06 mmol, >99%). A single crystal suitable for X-ray diffraction analysis was prepared by recrystallization from THF/hexane at -30 °C.</p><p>Reaction between 1a and B 2 giving 2aB 2 . A solution of 1a (7.4 mg, 0.02 mmol) and B 2 (9.3 mg, 0.02 mmol) in CD 2 Cl 2 (0.5 mL) was prepared at -30 °C and then transferred into a J. Young NMR tube. The quantitative formation of 2aB 2 was confirmed at -90 °C by 1 H, 13 C, 19 F, and 31 P NMR analysis (Supplementary Figs. 5-8). A single crystal suitable for X-ray diffraction analysis was prepared by recrystallization from toluene/hexane at -30 °C.</p>

Nature Communications Chemistry

STriatal-Enriched protein tyrosine Phosphatase (STEP) Regulates the PTP\xce\xb1/Fyn Signaling Pathway

The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr420) or inhibit (Tyr531) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTP\xce\xb1 dephosphorylates Tyr531 and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr420 and inactivates Fyn. Thus, PTP\xce\xb1 and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTP\xce\xb1 by directly dephosphorylating PTP\xce\xb1 at its regulatory Tyr789. Dephosphorylation of Tyr789 prevents the translocation of PTP\xce\xb1 to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction of STEP61 activity increased the phosphorylation of PTP\xce\xb1 at Tyr789, as well as increased translocation of PTP\xce\xb1 to synaptic membranes. Activation of PTP\xce\xb1 and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by ethanol administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by ethanol leads to the activation of PTP\xce\xb1, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTP\xce\xb1 and suggest that STEP and PTP\xce\xb1 coordinate the regulation of Fyn.

striatal-enriched_protein_tyrosine_phosphatase_(step)_regulates_the_ptp\xce\xb1/fyn_signaling_pathwa

Introduction<!>Materials and reagents<!>Animals<!>Tissue processing<!>Immunoblotting<!>GST fusion proteins and pull-down assays<!>Immunoprecipitation<!>Dephosphorylation of PTP\xce\xb1 by STEP in vitro<!>Primary neuronal cultures and treatments<!>Viral infection<!>Ethanol administration<!>Lipid rafts isolation<!>Statistical analysis<!>Increased phosphorylation of PTP\xce\xb1 at Tyr789 in STEP KO mouse brains<!>STEP61 binds to and dephosphorylates PTP\xce\xb1 at Tyr789<!>Regulation of PTP\xce\xb1 by STEP in primary cell cultures<!>Ethanol administration leads to phosphorylation and inactivation of STEP61, and subsequent translocation of PTP\xce\xb1 to lipid rafts fraction<!>Discussion

<p>PTPα is a member of the receptor-type protein tyrosine phosphatases (PTPs) family and is characterized by a transmembrane domain and two intracellular catalytic domains (Sap et al. 1990; Wang and Pallen 1991; Paul and Lombroso 2003; Tonks 2006). It is expressed in many tissues, including the brain (Sap et al. 1990; Sahin et al. 1995). Several reports implicate PTPα in the regulation of integrin signaling, neurite outgrowth, oligodendrocyte differentiation, and myelination through activation of its substrates Fyn and Src and modulation of signaling by NCAM (neural cell adhesion molecule) and CHL1 (close homolog of L1) (Bodrikov et al. 2005; Ye et al. 2008; Chen et al. 2006; Wang et al. 2009; Zeng et al. 2003).</p><p>PTPα activates Fyn by dephosphorylating its inhibitory Tyr residue (Y531), allowing full activation of Fyn by auto-phosphorylation at a second regulatory Tyr (Y420) (Engen et al. 2008, Ingley 2008). PTPα knockout (KO) mice have increased phosphorylation of Fyn at its inhibitory site (Y531) and decreased Fyn activity (Ponniah et al. 1999; Su et al. 1999). PTPα KO mice show deficits in LTP as well as in learning and memory, consistent with a role of Fyn in regulating NMDA receptor trafficking to synaptic membranes (Skelton et al. 2003; Petrone et al. 2003).</p><p>STriatal-Enriched protein tyrosine Phosphatase (STEP) is widely expressed in multiple brain regions including the striatum, where two major isoforms, STEP61 and STEP46, are expressed (Lombroso et al. 1991; Boulanger et al. 1995). STEP61 is enriched in membrane fractions while STEP46 is enriched in cytosol fractions (Lombroso et al. 1993; Bult et al. 1996). STEP normally opposes the development of synaptic strengthening through dephosphorylation and inactivation of several kinases, including Fyn, as well as endocytosis of both NMDARs and AMPARs (Snyder et al. 2005; Zhang et al. 2008; Zhang et al. 2010; Zhang et al. 2011). Both high and low activities of STEP disrupt synaptic plasticity, and dysregulation of STEP activity is implicated in several neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease (Zhang et al. 2010), schizophrenia (Carty et al. 2012), Parkinson's disease (Kurup et al. 2015), Huntington's disease (Saavedra et al. 2011; Gladding et al. 2012), post-traumatic stress disorder (Yang et al. 2012), and stress-induced anxiety disorders (Dabrowska et al. 2013).</p><p>Recent studies have indicated that PTPα is a critical determinant of ethanol consumption in rodent models (Gibb et al. 2011; Ben Hamida et al. 2013). PTPα, Fyn and STEP are all expressed in the striatum, and ethanol administration or binge drinking results in a PTPα-mediated activation of Fyn in the dorsomedial striatum (DMS) but not in the nearby dorsolateral striatum (DLS) or the nucleus accumbens (NAc). Moreover, viral-based knockdown of PTPα or Fyn in the DMS reduces ethanol intake in rats (Wang et al. 2007; Wang et al. 2010; Ben Hamida et al. 2013).</p><p>In contrast, recent findings have shown that STEP is phosphorylated and inactivated specifically in the DMS during ethanol administration, and that STEP KO mice or shRNA knockdown of STEP in the DMS increases ethanol consumption (Darcq et al. 2014; Legastelois et al. in press). STEP and PTPα act on one, and not the other, of the two regulatory tyrosines in Fyn (Bhandari et al. 1998; Nguyen et al. 2002). This led to our hypothesis that PTPα may be a novel substrate for STEP to coordinate the bidirectional regulation of Fyn by STEP and PTPα, which we test here using genetic, pharmacological, and molecular techniques. The results suggest that inactivation of STEP is required for activation of PTPα and Fyn both in rat primary corticostriatal cultures and in vivo after ethanol administration to mice.</p><!><p>All antibodies used in this study are listed in Table S1. Two dopamine D1 receptor agonists SFK-82958 and SKF-38393, the PKA inhibitor H-89, the PKA activator forskolin, and ethanol (190 proof) were purchased from Sigma-Aldrich (St. Louis, MO), while the selective phosphodiesterase 4 inhibitor rolipram was obtained from Tocris Biosciences (Ellisville, MO). Recombinant glutathione S-transferase (GST)-tagged PTPα protein was purchased from Sino Biological (Beijing, China), while active Fyn kinase was obtained from Millipore (Bedford, MA).</p><!><p>The wild type (WT) and STEP knockout (KO) male mice used in these experiments were 3–4 months of age, maintained on a C57BL/6 background, and generated at Yale University from heterozygous crosses as described previously (Venkitaramani et al. 2009). Experimental mice were group-housed with a maximum of 5 mice per cage in a climate-controlled facility with 12h light-dark cycle with access to food and water ad libitum. All experiments were carried out during the light phase of the cycle. All procedures were performed according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee at Yale University.</p><!><p>Total homogenates and crude synaptic membrane fractions (P2) were obtained from WT and STEP KO mouse striatum following previous protocols (Xu et al. 2012). Briefly, tissues were homogenized in ice-cold TEVP buffer (in mM): 10 Tris, pH 7.4, 5 NaF, 1 Na3VO4, 1 EDTA, 1 EGTA and 320 sucrose with protease inhibitor cocktail (Roche, Indianapolis, IN). Aliquots of samples were saved as total homogenates. The remaining homogenates were centrifuged at 1000 g for 10 min and the supernatants were further spun at 12,000 g for 20 min to isolate crude synaptic membrane fractions (P2). The pellets were resuspended and briefly sonicated in RIPA buffer (Pierce Biotechnology, Rockford, IL) with protease and phosphatase inhibitors (Roche). Total protein concentrations were determined using a BCA protein assay kit (Pierce Biotechnology).</p><!><p>All procedures were previously described (Xu et al. 2012). Briefly, 30 μg of samples were resolved on 8% SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Richmond, CA). Blots were blocked, incubated with primary and secondary antibodies (Table S1) and developed using a Chemiluminescent Substrate kit (Pierce Biotechnology). Densitometry was performed and analyzed using Genetools program (Syngene, Cambridge, UK). All phospho-protein levels were normalized to total protein levels and then to the loading control β-actin.</p><!><p>PCR-amplified open reading frames of WT STEP61, the substrate trapping STEP61 C472S, or the substrate trapping STEP46 C300S were inserted into pGEX4T1 vectors (GE Lifesciences, Piscataway, NJ). The substrate-trapping STEP protein has a point mutation at its critical cysteine within the phosphatase domain, rendering STEP inactive. This variant of STEP still binds to its substrates, but does not release them, as dephosphorylation is required for release; these constructs have been used to identify STEP substrates in the past (Nguyen et al. 2002; Paul et al. 2003; Xu et al. 2012). Glutathione S-transferase (GST) fusion proteins were expressed in E. coli BL21 (DE3) and purified on glutathione sepharose (GE Lifesciences) as described (Xu et al. 2012). For pull-down assays, GST fusion proteins were conjugated to glutathione sepharose beads and incubated with mouse striatal lysates overnight at 4 °C. STEP interacting proteins were probed with specific antibodies.</p><!><p>WT and STEP KO mouse striatum or rat primary corticostriatal neurons were lysed in immunoprecipitation (IP) buffer (in mM): 10 Tris-HCl pH 7.4, 150 NaCl, 1% Triton X-100, 1 EDTA and 1 EGTA with protease and phosphatase inhibitor cocktail (Roche). Lysates were incubated with anti-STEP or anti-PTPα antibodies overnight at 4 °C. On the second day, Protein A/G plus agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) were added for 4 h. Beads were washed 3 times and resuspended in 2×Laemmli buffer (Bio-Rad Laboratories) and subjected to SDS-PAGE and western blotting.</p><!><p>Recombinant GST-PTPα was phosphorylated at pY789 by Fyn kinase in vitro in kinase assay buffer (in mM): 50 Tris-HCl, pH 7.5, 0.1 EGTA, 10 MgCl2, 500 μM ATP for 30 min at 30 °C. Total reaction volume of kinase assay was 30 μl. Adding EDTA/EGTA mix to a final concentration of 5 mM stopped the phosphorylation reaction. GST-pY789 PTPα was then incubated with active WT GST-STEP61 or inactive GST-STEP61 C472S (0–200 nM) in phosphatase assay buffer (in mM): 25 HEPES pH 7.3, 5 EDTA, 10 DTT) for 30 min at 30 °C. The amount of pY789 PTPα remaining was visualized using the phospho-specific antibody to this site.</p><!><p>Primary corticostriatal cultures were derived from rat Sprague-Dawley E18 embryos (Jackson Laboratory, Bar Harbor, Maine) or from mouse STEP KO E18 embryos as described (Xu et al. 2012). Both male and female embryos were used in this study. Cultures were treated with SFK-82958 (10 μM, 30 min), SFK-38393 (10 μM, 30 min), forskolin (100 μM, 10 min) or rolipram (1 μM, 30 min). In some cases, cultures were pre-treated with the PKA inhibitor H-89 (10 μM, 30 min) followed by SKF-82958 or SKF-38393 stimulations. After treatments, neurons were lysed in RIPA buffer (Pierce Biotechnology) with protease and phosphatase inhibitors (Roche). Lysates were spun at 1000 g for 10 min and supernatants were saved for further analyses.</p><!><p>A recombinant adeno-associated virus of mixed serotype 1/2 (AAV1/2) was custom made (GeneDetect LTD, Auckland, New Zealand). Viruses contained either HA-tagged STEP61 or a HA-tagged empty vector, both with a hybrid chicken β-actin/CMV enhancer (CAG) promoter, rAAV2 inverted terminal repeat, a cis-acting woodchuck post-transcriptional regulatory element, and a bovine growth hormone polyadenylation signal sequence. The titers of the viral preparations were >1×1012 genomic particles/ml. At DIV (days in vitro) 5, STEP KO mouse cultures were infected with AAV1/2-STEP61 or AAV1/2-control vector for 10 days.</p><p>Lentivirus-based STEP shRNA (LV-STEP; a gift from Thomas Lanz, Pfizer Research & Development, Cambridge, MA) was made as described (Reinhart et al. 2014). The target sequence for STEP shRNA was: 5′-GCATGACTCTTTGGCAACATG-3′ using a loop sequence of 5′-TTCAAGAGA-3′. Control shRNA (5′-AATTCAGCGGGAGCCACCTGA-3′) was designed to target firefly luciferase, which is not homologous to any endogenous rat transcripts and therefore should work as a scrambled sequence. Validation of STEP shRNA and control shRNA was described (Reinhart et al. 2014; Lanz et al. 2013). Rat corticostriatal cultures were infected with LV-STEP or a luciferase control at DIV 7 for 7 days.</p><!><p>Male C57BL/6 mice (2–3 months, Jackson Laboratory) were injected intraperitoneally with saline or 20% ethanol (190 proof diluted in saline, 2 g/kg) and sacrificed 15 min post injection. For repeated treatment, mice were administrated with saline or 20% ethanol (2 g/kg, i.p.) once daily for 7 days and sacrificed 16 h after the last injection. In some experiments, STEP KO mice (C57BL/6 background) were administrated with saline or 20% ethanol (190 proof diluted in saline, 2 g/kg, i.p.) for 15 min. The DMS, DLS and NAc were microdissected and kept at −80 °C until use.</p><!><p>A detergent-free protocol was used to isolate lipid rafts from mouse brain as described (Persaud-Sawin et al. 2009). Briefly, WT or STEP KO mouse DMS were collected after ethanol administration. Tissues were homogenized in lysis buffer, as described above. Homogenates were centrifuged at 1000 g for 10 min and supernatants were saved. The pellets were resuspended in lysis buffer and passed through a 23-gauge needle, followed by 1000 g spin for 10 min. The second supernatant was pooled with the first one and 250 μl of pooled supernatant was mixed gently with equal amount of 80% sucrose in lysis buffer. Five hundred μl of 30% sucrose was layered on top, followed by a third layer of 5% sucrose. Gradients were centrifuged at 200,000 g in a Beckman Coulter TLA120.1 rotor for 18 h at 4 °C. Twelve sequential fractions (120 μl) were collected and assayed using immunoblotting.</p><!><p>All experiments were repeated at least three times. Data were expressed as means ± SEM. Statistical significance was determined by Student's t-test or one-way ANOVA with post hoc Tukey's test. For co-immunoprecipitation and rafts isolation experiments in WT and STEP KO mice, a two-way ANOVA with genotype and treatment as main factors followed by post hoc Tukey's test was used to determine statistical significance, with p values < 0.05 considered significant.</p><!><p>Previous studies have established that loss of STEP leads to elevated basal tyrosine phosphorylation of all STEP substrates identified to date (Venkitaramani et al. 2009; Nguyen et al. 2002; Xu et al. 2012). We reasoned that if PTPα is a novel substrate for STEP, there would be an increase in the tyrosine phosphorylation of PTPα at Y789 in STEP KO mouse brains. There was a significant increase in the phosphorylation of this site (1.47 ± 0.12 of WT, p < 0.05), with no change in total PTPα level (p > 0.05, Fig. 1a). We then examined the phosphorylation of the two regulatory tyrosine sites in Fyn. STEP KO brains showed elevated tyrosine phosphorylation of Fyn at pY420 (1.55 ± 0.13 of WT, p < 0.01), consistent with previous findings that this site is directly regulated by STEP (Nguyen et al. 2002). In contrast, there was a significant decrease in Fyn phosphorylation at the PTPα site (pY531: 0.60 ± 0.10 of WT, p < 0.05), consistent with an increase in PTPα activity (Ponniah et al. 1999; Su et al. 1999).</p><p>These results indicate that PTPα Y789 phosphorylation is increased in STEP KO samples. We next determined whether re-expressing STEP61 into STEP KO cultures was sufficient to reverse the increase in PTPα phosphorylation. Pilot studies using adeno-associated virus1/2 (AAV1/2)-STEP61 confirmed a robust STEP61 expression when corticostriatal cultures were infected with AAV1/2-STEP61 but not with vector alone (Fig. S1). The doublet present is due to differential phosphorylation of STEP61 (Paul et al. 2000). Restoration of STEP61 into STEP KO cultures led to significant decreases in the Tyr phosphorylation of PTPα and a concomitant increase in the phosphorylation of the PTPα site on Fyn (PTPα pY789: 0.71 ± 0.08 of control; Fyn pY531: 1.33 ± 0.10 of control, p values < 0.05, Fig. 1b). In addition, re-expression of STEP61 into these cultures resulted in a decrease in the Tyr phosphorylation of Fyn at the STEP61 site (Fyn pY420: 0.72 ± 0.06 of control, p < 0.05).</p><p>Phospho-Fyn antibodies also recognize other Src family kinases, including Src at equivalent phosphorylation sites. To determine whether the changes in Fyn phosphorylation were specific to Fyn, we immunoprecipitated Fyn and Src from STEP KO lysates with specific antibodies, followed by probing with phospho-antibodies. We found alterations in Fyn phosphorylation but not Src (Fig. S2). These results are in agreement with previous findings showing Src is not a direct target of STEP (Nguyen et al. 2002).</p><p>PTPα translocation to synaptic membrane fractions is required for full activation of Fyn (Gibb et al. 2011). Thus we next investigated the phosphorylation levels of PTPα and Fyn in synaptic fractions of striatum from WT and STEP KO mice. There was a significantly higher basal level of phospho-PTPα in the striatum of STEP KO mice (1.42 ± 0.15 of WT, p < 0.05), as well as an increase in total PTPα (Fig. 1c). The higher levels of pPTPα in synaptic membranes correlated with a decrease in the Tyr phosphorylation of the PTPα site in Fyn, while the Tyr phosphorylation of the STEP site was increased (PTPα site on Fyn (Y531): 0.75 ± 0.03 of WT; STEP site on Fyn (Y420): 1.44 ± 0.15 of WT, p values < 0.05), with no changes in total Fyn levels in P2 fractions. In addition, there were no changes in the Tyr phosphorylation of PTPα and Fyn in homogenates or P2 fractions from cerebellum (Fig. S3), a brain region in which STEP is not expressed.</p><!><p>We next examined whether there is physical association between STEP61 and PTPα. We took advantage of a substrate-trapping form of STEP61 that contains a mutation of the catalytic site cysteine to a serine residue that inactivates STEP61. This mutated isoform of STEP binds to its substrates but does not release them, as release requires dephosphorylation of substrates (Xu et al. 2012; Nguyen et al. 2002). The N-terminal unique sequence of STEP61 contributes to the binding of some substrates (Fyn and Pyk2) (Nguyen et al. 2002; Xu et al. 2012), while STEP46 preferentially binds to ERK2 and p38. Here we detected binding of endogenous PTPα to recombinant STEP61 C/S but not to STEP46 C/S. Positive controls included Fyn and ERK2 (Fig. 2a). We confirmed that there was no binding of either STEP isoform to Src (Nguyen et al. 2002). These results indicate that STEP61, and not STEP46, interacts with PTPα in vitro.</p><p>To investigate possible associations in vivo, we preformed reciprocal immunoprecipitation (IP) of STEP or PTPα from WT and STEP KO mouse striatal lysates. STEP IP co-precipitated PTPα and Fyn from WT lysates but not from STEP KO lysates (Fig. 2b, replicates shown in Fig. S4), while IgG alone and an antibody to Src were used as negative controls. A two-way ANOVA revealed that there was a main effect of treatment (IgG vs anti-STEP: F(1,8) = 44.22, p <0.001) and genotype (WT vs KO: F(1,8) = 40.46, p <0.001) with an interaction between these factors (F(1,8) = 38.77, p <0.001, Fig. S4a). Association was confirmed with the reciprocal IP; STEP61 co-precipitated with PTPα from WT lysates but not from STEP KO lysates (treatment: F(1,8) = 12.00, p <0.01); genotype: F(1,8) = 14.19, p <0.01) and interaction: (F(1,8) = 13.68, p <0.01, Fig. S4b). The absence of STEP did not affect the known interaction between PTPα and Fyn (Bhandari et al. 1998) (Fig. 2c, Fig. S4b). Reciprocal IP was also performed with rat primary corticostriatal cultures to confirm the interaction between STEP61 and PTPα (Fig. S5).</p><p>We next examined whether STEP61 could dephosphorylate PTPα. We phosphorylated recombinant PTPα using Fyn and confirmed the phosphorylation using a phospho-specific antibody to Y789 (Fig. 2d). We also assayed PTPα phosphatase activity using para-nitrophenyl phosphate (pNPP) and determined that phosphorylation of PTPα by Fyn did not alter PTPα activity (data not shown). We then incubated phosphorylated PTPα with active WT GST-STEP61 or inactive GST-STEP61 C/S proteins and saw a dose-dependent dephosphorylation of PTPα by active but not inactive STEP61 (Fig. 2e). Together, these results indicate that PTPα is a direct substrate of STEP61.</p><!><p>If PTPα is a substrate for STEP, we reasoned that the tyrosine phosphorylation level of PTPα would change as we modulated STEP61 activity. It is known that D1 dopamine receptor (D1R) stimulation results in a PKA-mediated phosphorylation of STEP at serine221 (Ser221) within the substrate-binding domain (kinase interacting motif, KIM) (Paul et al. 2000). Phosphorylation at this regulatory serine prevents STEP from interacting with its substrates. We treated corticostriatal cultures with two D1R agonists, SKF-82958 and SKF-38393, and showed the expected increase in STEP61 phosphorylation (SKF-82958: 1.46 ± 0.14 of control; SKF-38393: 1.43 ± 0.15 of control, p values < 0.05) (Fig. 3a). We also found an increase in pY789 PTPα levels when STEP61 was phosphorylated at Ser221 (inactive STEP61) (SKF-82958: 1.53 ± 0.19 of control; SKF-38393: 1.43 ± 0.11 of control, p values < 0.05). Phosphorylation levels of the STEP site in Fyn (Y420) was also increased (SKF-82958: 1.55 ± 0.17 of control; SKF-38393: 1.50 ± 0.06 of control, p values < 0.05). Moreover, there was a decrease in the phosphorylation of the PTPα site on Fyn (pY531) (SKF-82958: 0.72 ± 0.07 of control; SKF-38393: 0.64 ± 0.11 of control, p values < 0.05), presumably due to enhanced dephosphorylation by PTPα. Total protein levels did not alter during drug treatments. As expected, the PKA inhibitor H-89 blocked these effects (Fig. 3a).</p><p>To further confirm these results, we activated PKA using forskolin and rolipram in corticostriatal cultures. Both forskolin and rolipram induced robust increases in phosphorylation of STEP61 at Ser221 (Fsk: 2.13 ± 0.18 of control, p < 0.01; Rol: 1.45 ± 0.08 of control, p < 0.05) and subsequent increases in the Tyr phosphorylation of PTPα (Fsk: 1.46 ± 0.18 of control; Rol: 1.50 ± 0.18 of control, p values < 0.05) and Fyn at the STEP site (Y420) (Fsk: 1.76 ± 0.24 of control; Rol: 1.71 ± 0.21 of control, p values < 0.05) (Fig. 3b). In addition, forskolin and rolipram treatments led to a significant decrease in the phosphorylation of the PTPα site on Fyn (Y531), consistent with increased activation of PTPα (Fsk: 0.62 ± 0.09 of control; Rol: 0.59 ± 0.12 of control, p values < 0.05).</p><p>Next we used gene-specific knockdown to confirm the regulation of PTPα by STEP61. Lentiviral-STEP shRNA was added to cultures (DIV 5 days) for 7 days as described (Reinhart et al. 2014) and resulted in a significant decrease in STEP61 expression compared to control (0.24 ± 0.03 of control, p < 0.01, Fig. 4). We observed an increase in PTPα phosphorylation and a decrease in the Tyr phosphorylation of the PTPα site on Fyn (PTPα (pY789): 1.37 ± 0.08 of control; Fyn (pY531): 0.77 ± 0.08 of control, p values < 0.05). Moreover, the knockdown of STEP61 expression correlated with an increase in the Tyr phosphorylation of the STEP61 site on Fyn (pY420) (1.36 ± 0.09 of control, p < 0.05). Taken together, these knockdown experiments confirmed the earlier results of acute pharmacological inactivation of STEP.</p><!><p>We next examined the functional significance of the regulation of PTPα by STEP61. PTPα and Fyn both play a critical role in modulating ethanol (EtOH) intake. EtOH administration in rodents leads to activation of Fyn and an increase in the localization of PTPα within synaptic membranes specifically in the dorsomedial striatum (DMS) (Gibb et al. 2011; Ben Hamida et al. 2013). EtOH treatment also results in the phosphorylation and inactivation of STEP61, as well as activation of Fyn and the phosphorylation of the Fyn target GluN2B, again specifically in the DMS, but not in the adjacent dorsolateral striatum (DLS) or nucleus accumbens (NAc) (Darcq et al. 2014). Given these findings, we asked if STEP61 modulated PTPα phosphorylation and translocation during ethanol administration.</p><p>We employed two paradigms for these experiments: acute ethanol injection and repeated ethanol injections in mice followed by a withdrawn period (Gibb et al. 2011; Ben Hamida et al. 2013). C57BL/6 mice were acutely injected with ethanol (2 g/kg, i.p.) and sacrificed 15 min later. Synaptic membrane fractions from DMS, DLS or NAc were processed for levels of STEP61 and its substrates. Consistent with previous findings (Gibb et al. 2011), we observed an increased localization of PTPα in the synaptic membrane fractions in DMS (1.40 ± 0.18 of saline, p < 0.05) but not in DLS or NAc (Fig. 5a). We also confirmed an increase in STEP61 phosphorylation following ethanol injection in DMS (1.58 ± 0.27 of saline, p < 0.05) (Darcq et al. 2014). Both total and phospho-PTPα at Y789 increased in synaptic membrane fractions (Fig. 5a), supporting the observation that phosphorylation at this site is required for PTPα trafficking to membrane fractions (Maksumova et al. 2005; Gibb et al. 2011). Consistent with the enhanced trafficking of PTPα to synaptic fractions, we found a decrease in the phosphorylation of the PTPα site in Fyn (Y531: 0.66 ± 0.10 of saline, p < 0.05), and an increase in the phosphorylation of the STEP61 site in Fyn (Y420: 1.46 ± 0.18 of saline, p < 0.05), possibly due to inactivation of STEP61. We observed none of these changes in the DLS or NAc (Fig. 5b and c). These results suggest a model of synergistic regulation of Fyn by STEP and PTPα in DMS upon acute EtOH administration.</p><p>Previous studies showed that repeated ethanol exposure increased the phosphorylation of Fyn at Y420, but decreased the phosphorylation of Fyn at Y531 (Wang et al. 2010). We followed a similar protocol with repeated ethanol injection (2 g/kg, i.p. daily for 7 days followed by 16 h withdrawn) and examined the effects of ethanol treatment on STEP61, PTPα and Fyn in the DMS, DLS and NAc (Fig. S6). We confirmed the changes in phosphorylation of Fyn, finding increased pY420 Fyn and decreased pY531 Fyn only in the DMS (pY420: 1.46 ± 0.15 of saline; pY531: 0.66 ± 0.08 of saline, p values < 0.05). Moreover, we found increased phosphorylation (inactivation) of STEP61 and a concomitant increased phosphorylation of PTPα at Y789 and translocation of PTPα to synaptic fractions in DMS (pSTEP: 1.57 ± 0.21 of saline; PTPα: 1.39 ± 0.17 of saline; pY789 PTPα: 1.47 ± 0.19 of saline, p values < 0.05). These results suggest that inactivation of STEP61 may be a required for PTPα phosphorylation and translocation to synaptic membrane compartments, resulting in maximal activation of Fyn.</p><p>To follow translocation of PTPα into lipid rafts fraction upon EtOH administration, we used a detergent-free protocol (Persaud-Sawin et al. 2009). We first validated our preparation by two criteria commonly used in the field: (1) presence or absence of marker proteins and (2) high cholesterol and low protein content of the rafts fraction (Persaud-Sawin et al. 2009). Flotillin-1, a marker present in lipid rafts, was enriched and correlated with high cholesterol and low protein content in fractions 3 and 4 (Fig. S7). In contrast, the transferrin receptor (TfR), which is excluded from lipid rafts, was recovered mainly in fractions 10–12, and correlated with high protein and low cholesterol content (Fig. S7). Consistent with previous findings (Gibb et al. 2011), we confirmed the expression of Fyn in both lipid rafts and non-rafts fractions (Fig. 6a). In addition, we found that PTPα and STEP61 were primarily in non-rafts fractions at baseline in mouse DMS. We acutely administrated mice with EtOH (2 g/kg, i.p.) or vehicle for 15 min. EtOH administration led to PTPα re-distribution into rafts fractions, without altering the localization of STEP61 or Fyn (Fig. 6a).</p><p>If EtOH-induced phosphorylation and inactivation of STEP61 promoted PTPα translocation to the rafts fractions in WT mice, we reasoned that PTPα might be constitutively localized in the rafts fractions derived from STEP KO mice. We found that PTPα was present in both rafts and non-rafts fractions under baseline conditions (Fig. 6b). A two-way ANOVA analysis of PTPα expression in the rafts showed a main effect of treatment (Sal vs EtOH: F(1,16) = 17.46, p < 0.001) and genotype (WT vs KO: F(1,16) = 12.48, p < 0.01) with an interaction between these factors (F(1,16) = 17.46, p < 0.001, Fig. 6c). However, no changes were observed in PTPα levels in the non-rafts fractions (treatment: F(1,16) = 0.76, p = 0.39; genotype: F(1,16) = 0.74, p = 0.40; interaction: F(1,16) = 0.06, p = 0.80, Fig. 6c). Together, these results suggest STEP61 is involved in PTPα translocation to lipid rafts upon EtOH administration.</p><!><p>Here we establish that PTPα is a novel substrate for STEP61 and that STEP61 binds to and dephosphorylates PTPα at Y789. We used STEP KO mice, knockdown of STEP61, and pharmacological interventions to lower STEP activity, and find increased phosphorylation of at Y789 on PTPα, while overexpression of STEP61 results in a decreased phosphorylation of PTPα. The data indicate that inactivation of STEP61 contributes to the increased tyrosine phosphorylation of PTPα and subsequent translocation into lipid raft fractions, leading to the activation of Fyn. We also demonstrated the functional significance of this pathway by showing that it is activated specifically in the DMS during ethanol administration.</p><p>PTPα is a receptor-type protein tyrosine phosphatase that is widely expressed in many tissues. Within the CNS, PTPα is implicated in the development of synaptic plasticity and long-term potentiation (LTP) through its ability to activate Fyn and potentiate NMDA receptor signaling. In support of this, PTPα KO mice have decreased Fyn activity and deficits in memory consolidation and LTP (Skelton et al. 2003; Petrone et al. 2003). PTPα is regulated by several mechanisms, including oxidation-induced dimerization and inactivation (Jiang et al. 1999; Yang et al. 2007), phosphorylation (den Hertog et al. 1994; Zheng et al. 2000; Zheng et al. 2002), and translocation between cytoplasm and lipid rafts (Maksumova et al. 2005; Gibb et al. 2011). We find an elevation of PTPα pY789 in dorsal striatum but not in cerebellum of STEP KO mice, consistent with the absence of STEP protein in the cerebellum. It would be interesting to determine whether PTP-SL (Pulido et al. 1998), a closely related PTP to STEP that is present in cerebellum, similarly regulates PTPα in that brain region.</p><p>Early reports suggested that pY789 provides a binding site for the SH2 domain of Src/Fyn, thus facilitating the activation of Src/Fyn by removing its intramolecular inhibition (Zheng et al. 2000; Bhandari et al. 1998); however, there are conflicting results, as site-directed mutation did not affect downstream Src signaling, nor was the Y789 site involved in mediating the interaction of PTPα with Src/Fyn (Chen et al. 2006; Lammers et al. 2000; Vacaru and den Hertog 2010). Other studies suggest that the phosphorylation at Tyr789 is needed for Grb2 binding and subsequent activation of MAPK/ERK signaling (den Hertog et al. 1994; den Hertog and Hunter 1996; Su et al. 1996). The direct modulation of intrinsic phosphatase activity by phosphorylation at Y789 is also under debate. Some report that dephosphorylation of this site affects PTPα activity (Maksumova et al. 2007), while others suggest that it does not (den Hertog et al. 1994; Su et al. 1996; Zheng et al. 2000). Our in vitro assay indicate that phosphorylation of PTPα at Y789 does not alter its phosphatase activity using pNPP as substrate.</p><p>The possible regulation of PTPα translocation by pY789 is relevant to this study. Previous findings suggested that upon integrin activation, PTPα translocates to focal adhesions in an Y789 dependent-manner (Lammers et al. 2000; Sun et al. 2012), and translocation is critical for full activation of Fyn (Maksumova et al. 2005; Vacaresse et al. 2008). Ethanol administration was shown to induce translocation of PTPα to synaptic membrane fractions (Gibb et al. 2011). Consistent with these reports, we find elevated PTPα levels in synaptic membranes of STEP KO striatum, with a proportional increase in pY789. Acute or repeated ethanol administration also increased the phosphorylation and inactivation of STEP61, increased the phosphorylation of the STEP target PTPα, and increased trafficking of PTPα to synaptic membranes. Moreover, using a lipid rafts extraction protocol, we show that acute EtOH resulted in the re-distribution of PTPα to rafts fractions, where Fyn is enriched, in WT mice. In support of our hypothesis, we find increased localization of PTPα in rafts fractions under baseline conditions in STEP KO mice and EtOH administration did not induce a further translocation of PTPα in STEP KO mice.</p><p>These data suggest that decreased STEP61 activity leads to increased phosphorylation of PTPα at Y789 and subsequent translocation of PTPα to lipid rafts, facilitating its interaction with Fyn and NMDA receptors, both critical players in regulating EtOH-related behaviors (Wang et al. 2007; Ben Hamida et al. 2013). Interestingly, a recent report shows that STEP KO mice have more EtOH consumption (Legastelois et al. in press), possibly due to enhanced PTPα, Fyn and NMDAR signaling in rafts fractions in STEP KO mice. The molecular mechanisms that underlie PTPα translocation are not well understood. Several SH2-domain containing adaptor proteins are present at focal adhesions (Boivin et al. 2013; Shen and Guan 2004) or associated with raft targeting proteins (Liu et al. 2002; Kimura et al. 2001; Limpert et al. 2007), thus it is possible that phosphorylated PTPα is recruited by these adaptor proteins to neuronal lipid rafts via pY789-SH2 domain interaction during ethanol administration.</p><p>One unanswered question is what determines the brain region specificity of ethanol-induced phosphorylation and inactivation of STEP61 in the DMS, but not in DLS or NAc. STEP61 is phosphorylated by PKA at a regulatory serine residue within its KIM domain upon dopamine D1 receptor (D1R) activation (Paul et al. 2000), while PP2B/PP1 dephosphorylates and activates STEP61 (Snyder et al. 2005; Valjent et al. 2005). Activation of D1R and PKA by ethanol is well documented; however, activation of PKA by ethanol is not restricted to the DMS (Di Chiara and Imperato 1988; Asyyed et al. 2006; Ron and Messing 2013). Studies are needed to clarify the mechanism for the differential regulation of STEP in distinct brain regions, such as DMS, after EtOH administration. Although recent studies have suggested that subregions of the striatum are involved in the regulation of distinct aspects of alcohol abuse (Chen et al. 2011), the mechanisms by which this occurs remain unclear. The differential regulation of STEP within specific brain regions has been previously reported. For example, STEP levels are elevated in striatum in human sporadic Parkinson's disease and in MPTP-treated mouse models, but not in cortex (Kurup et al. 2015), while STEP levels are elevated in cortex of patients with Alzheimer's disease and schizophrenia (Zhang et al. 2010; Carty et al. 2012).</p><p>A-kinase anchoring proteins (AKAPs) represent a family of adapter proteins that bind to the regulatory subunits of PKA and provide temporal-spatial control by localizing PKA in proximity to substrates and optimal pools of cAMP (Wong and Scott 2004). AKAP proteins show distinct tissue and subcellular distribution, such as the enrichment of AKAP79/150 in neuronal postsynaptic densities. In addition to binding to PKA, it also binds to PP2B and PKC (Dell'Acqua et al. 2002; Klauck et al. 1996), both of which regulate STEP (Snyder et al., 2005 and unpublished data). Meanwhile, distinct combination of regulatory and catalytic subunits of PKA may also contribute to the region-specific regulation of STEP proteins (Gamm et al. 1996). It has been shown that different regulatory subunits of PKA display different sensitivity to cAMP levels. Moreover, RIIβ KO but not RIβ KO mice show increased EtOH consumption (Thiele et al. 2000). Thus it would be important to determine whether AKAP79/150 provides a platform to facilitate the convergent regulation of STEP by these signaling pathways and distinct combination of PKA holoenzyme in a region or compartmental-specific manner.</p><p>The synergistic regulation of Fyn by STEP and PTPα is of interest. STEP dephosphorylates and inactivates Fyn directly (Nguyen et al. 2002) and we demonstrate a parallel pathway by which STEP dephosphorylates and suppresses PTPα function also leading to Fyn inactivation (Engen et al. 2008; Ingley 2008). On the other hand, Fyn may provide a positive feedback by phosphorylating PTPα at Y789 and enhancing its signaling, which is inhibited by STEP61 at baseline and disinhibited upon ethanol exposure. Ethanol administration leads to activation of PKA (Ron and Messing 2013; Ortiz et al. 1995), and PKA phosphorylation of STEP isoforms results in the inability of STEP to interact with its substrates (Paul et al. 2000; Paul et al. 2003). At the same time, PKA phosphorylation of DARPP-32 results in inhibition of PP1, which is the phosphatase that dephosphorylates the PKA site in STEP isoforms (Paul et al. 2000). The inactivation of STEP61 results in an increase in the Tyr phosphorylation of PTPα (Y789) and a decrease in the Tyr phosphorylation of Fyn at its inhibitory site (Y531) by PTPα. In this example, the initial activation of PKA results in inactivation of STEP61 and a subsequent translocation of PTPα to synaptic membranes and the full activation of Fyn.</p><p>In addition to ethanol-related disorders, the cross-talk between STEP and PTPα has been implicated in several neuropsychiatric disorders including schizophrenia (SZ). Previous findings indicate an elevation of STEP61 level and activity in SZ postmortem brains and animal models of SZ (Carty et al. 2012). Interestingly, hypofunction of Fyn and PTPα are associated with neurobehavioral endophenotypes of SZ (Bjarnadottir et al. 2007; Takahashi et al. 2011), and the present results suggest a possible mechanism for these findings.</p><p>In summary, we show that PTPα is a novel substrate for STEP. The phosphorylation and inactivation of STEP61 upon ethanol administration facilitates the translocation of PTPα to lipid rafts and subsequent activation of Fyn-NMDA receptor signaling (Fig. 7). Further studies are needed to investigate possible signaling pathways that underlie the specific phosphorylation and inactivation of STEP61 in the DMS.</p>

PubMed Author Manuscript

Self-assembled Aptamer-based Drug Carriers for Bi-specific Cytotoxicity to Cancer Cells

Monovalent aptamers can deliver drugs to target cells by specific recognition. However, different cancer subtypes are distinguished by heterogeneous biomarkers, and one single aptamer was unable to recognize all clinical samples from different patients with even the same type of cancers. To address heterogeneity among cancer subtypes for targeted drug delivery, as a model, we developed a drug carrier with broader recognition range of cancer subtypes. This carrier (SD) was self-assembled from two modified monovalent aptamers. It showed bi-specific recognition abilities to target cells in cell mixtures, thus broadening the recognition capabilities of its parent aptamers. The self-assembly of SD simultaneously formed multiple drug loading sites for anticancer drug Doxorubicin (Dox). The Dox-loaded SD (SD-Dox) also showed bi-specific abilities of target cell binding and drug delivery. Most importantly, SD-Dox induced bi-specific cytotoxicity in target cells in cell mixtures. Therefore, by broadening the otherwise limited recognition capabilities of monovalent aptamers, bi-specific aptamer-based drug carriers would facilitate aptamer applications for clinically heterogeneous cancer subtypes which respond to the same cancer therapy.

self-assembled_aptamer-based_drug_carriers_for_bi-specific_cytotoxicity_to_cancer_cells

Introduction<!>Results and Discussion<!>Conclusion<!>General<!>Preparation of DNA<!>Cell culture<!>Aptamer binding assay<!>Detection of target cancer cells in cell mixtures using SD<!>Self-assembly and characterization of SD-Dox<!>Dox release study<!>Internalization of aptamers and uptake of Dox<!>Cytotoxicity assay

<p>Drug delivery systems that specifically recognize cancer cells and induce targeted cytotoxicity will reduce side effects caused by nonspecific drug toxicity. Specific recognition can be realized by using antibodies or aptamers[1].</p><p>Aptamers, which are selected through Systematic Evolution of Ligands by EXponential enrichment (SELEX), are single-stranded DNA or RNA molecules that can specifically and selectively bind to targets[1b, 1c]. The targets of aptamers range from metal ions, small molecules, to proteins, and even mammalian cells.[1b, 2] Recently, our group developed cell-SELEX to select aptamers against whole cells, using target cells for positive selection and non-target cells for negative selection[1c, 3]. With this technology, aptamers have been selected against cell lines such as CCRF-CEM (human T-cell acute lymphoblastic leukemia (ALL)) and Ramos (human B-cell Burkitt's lymphoma)[3a]. Compared with antibodies, nucleic acid aptamers have many distinct advantages, such as easy synthesis and modification, reproducible batch-to-batch fabrication, and low cytotoxicity and immunogenicity[1b, 1c, 4]. As such, aptamers are promising for future biomedical application such as targeted anticancer drug delivery.</p><p>However, recent aptamer binding tests with patient samples indicated that a single type of aptamer did not bind all samples from different patients with the same type of cancer[5], presumably resulting from the heterogeneity of cell surface biomarkers among different patient samples. This suggests that monovalent aptamers selected against cultured cancer cells may not be able to overcome the problem of heterogeneity among different patient samples. Yet, cancer heterogeneity has been widely reported [6], and more recently, it was further demonstrated by direct single-cell analysis such as genomic sequencing[7] and dissection of tumor cell transcription[8]. Therefore, improvement of aptamers for broader range of recognition capabilities would be highly significant for future clinical applications in targeted cancer therapy.</p><p>In this context, we propose developing multi-specific, aptamer-based drug carriers that are capable of recognizing and inducing targeted cytotoxicity in different subtypes of cancers. These carriers were designed to be self-assembled from modified monovalent aptamers. The assembly would simultaneously form drug loading sites in the double-stranded linker region. As a model, a bi-specific drug carrier, sgc8c-sgd5a (SD), was developed from monovalent aptamers sgc8c and sgd5a, and evaluated in this study. An anticancer drug Doxorubicin, which is used in chemotherapy of a wide range of cancers, including acute lymphoblastic and myeloblastic leukemias, malignant lymphomas, as well as breast cancer[9], was chosen in this study. Dox binds preferentially to dsDNA between adjacent GC or CG base pairs through intercalation, and the association of Dox with DNA is reversible.[10] Dox was loaded into the multiple intercalation sites designed in the dsDNA linker region of SD to study the bi-specific ability of SD for Dox delivery and target cell cytotoxicity. While the recognition abilities of monovalent aptamers are necessarily limited, the broader recognition capability of the bi-specific aptamer-based drug carrier, SD, allowed the cytotoxic effects of Dox to be bi-specifically directed to more types of target cells. Under these conditions, bi-specific aptamer-based drug carriers can sidestep the problem of cancer heterogeneity and, as a consequence, facilitate clinical aptamer applications in targeted therapy of many types and subtypes of cancers that respond to the same therapeutic methods.</p><!><p>In order to develop bi- or tri-specific aptamer-based drug carriers, we first constructed bi- and tri-specific aptamers (multi-aptamers) and studied their recognition capabilities. Engineering multi-aptamers is similar to multivalency engineering, which has been previously reported for antibodies[11] and aptamers[12] using chemical linkages or nanomaterials for binding affinity improvement, targeted therapy, cell-cell interaction, etc. In this study, chemical linkages were used. As an example shown in Figure. 1, two monovalent aptamers that recognize different cancer subtypes form a bi-specific aptamer via dsDNA linkage. Monovalent aptamers able to recognize different cultured cell lines were selected as "model building blocks": sgc8c (S) against CEM cells[3a, 13], TDO5 (T) against Ramos cells[14], and sgd5a (D) against Toledo cells[5a] (sequences in Table S1). To study the generality of linkers, different linkers including a polyT linker, a PEG linker and a dsDNA linker were used to construct S-T20-T, S-PEG-T, S-hyb-T and S-hyb-D (SD), respectively. Bi-specific aptamers with either polyT or PEG linkers were designed to be synthesized on a automated DNA synthesizer, and those with dsDNA linkers were self-assembled by hybridization of two complementary sequences designed to extend from the 3′ ends of monovalent aptamers. To further enhance the recognition range of drug carriers, a tri-specific aptamer, sgc8c-sgd5a-TDO5 (SDT), was developed using a Y-shaped dsDNA linker. The formation and purity of multi-aptamers linked by dsDNA were confirmed by agarose gel electrophoresis (Figure. S1).</p><p>For drug carriers to realize bi- or tri-specificity, each monovalent aptamer domain must retain its specific binding ability. To test this, an aptamer binding assay was performed with target cells of the parent aptamers. Either FITC or Cy5.5 was used to monitor aptamer binding. Using flow cytometry, specific binding abilities were confirmed at 4°C for bi-specific aptamers S-T20-T (Figure. S2A), S-hyb-T (Figure. S2B), S-PEG-T (Figure. S2C), S-hyb-D (SD) (Figure. 2A), and tri-specific aptamer S-D-T (SDT) (Figure. S3). Furthermore, the dissociation constants (Kd) to target cells were determined for S-T20-T, S-hyb-T and SD. As shown in Table S2, the binding affinities of these bi-specific aptamers to their target cells were comparable to those of the corresponding monovalent aptamers. Therefore, the binding specificity and affinity to both/all target cells were maintained in these multi-aptamers, indicating bi- or tri-specificity.</p><p>To develop drug carriers with multi-specific cytotoxicity using multi-aptamers, bi-specific aptamer SD was chosen as a model. It is well known that temperature change can result in aptamer conformation change and, hence, binding ability. Thus, the binding ability of SD was characterized and verified at physiological temperature (37°C) (Figure. S2D).</p><p>For future application of SD as a drug carrier in complicated clinical samples, the ability of SD to selectively detect both types of target cells was evaluated in cell mixtures containing non-target cells. To do so, SD binding assays were performed with cell mixtures containing a series of target CEM/Toledo cells of different concentrations into non-target NB4 cells of a fixed concentration. Because the morphology of NB4 cells is distinct from those of either CEM or Toledo cells, the populations of target and non-target cells in flow cytometric results can be easily distinguished and gated for respective fluorescence analysis (Figure. S4A). With as low as 5% target cells in a total mixture of 15,000 cells, both types of target cells were still easily detected, indicating bi-specificity of SD for sensitive cell detection in cell mixtures (Figure. S4).</p><p>Next, a quantitative analysis was performed to compare the recognition capabilities of monovalent aptamers sgc8c and sgd5a with that of SD in cell mixtures. Again, 50,000 CEM and/or 50,000 Toledo cells were spiked into 200,000 NB4 cells. Using a binding assay, these cell mixtures were tested with sgc8c, sgd5a and SD, respectively. Similarly as described above, flow cytometric data were analyzed to determine the percentage of cells with fluorescence signal enhancement resulting from aptamer binding. As shown in Figure. 2B, 15.35% and 14.4% cells tested with sgc8c and sgd5a, respectively, showed signal intensities located in M1 (marker for enhanced fluorescence signal range), while the percentage for SD was 26.89%, approximately twice that of either sgc8c or sgd5a alone. Overall, the results showed that SD had broader recognition capability to target cells than either sgc8c or sgd5a parent aptamers alone in the same cell mixtures. The broader recognition capability of SD is, in turn, expected to enhance the recognition range of SD-based drug carrier in targeted drug delivery, thus overcoming the problem of cancer heterogeneity discussed above.</p><p>Another key concern integral to intracellular drug delivery is the ability of drug carriers to be internalized. Regarding aptamer-based drug carriers, some aptamers[15] have already been reported to be specifically internalized into target cells. In our case, it is critical for SD to be internalized into both target cells for successful drug delivery. Therefore, the internalization capability of TAMRA-labeled SD was evaluated through confocal microscopy, and, as shown by the images in Figure. S5, SD was successfully internalized into both CEM and Toledo cells.</p><p>To develop a self-assembled drug carrier using SD for bi-specific drug delivery and target cell cytotoxicity, Doxorubicin (Dox) was used in this study. Dox is one of the most utilized chemotherapeutic drugs for a wide spectrum of cancers [16]. However, lack of specificity leads to many side effects, such as myelosuppression and mucositis[17]. Interestingly, many anthracycline drugs, including Dox and daunorubicin, can preferentially intercalate into tandem GC or CG sites in dsDNA, resulting in the quenching of Dox fluorescence[10, 18]. Accordingly, SD was designed with a dsDNA linker having 10 Dox intercalation sites (Table S1). As such, a drug carrier based on SD could be self-assembled through hybridization of the two complementary sequences modified on each aptamer, and the Dox intercalation sites would be simultaneously formed in the dsDNA linker, resulting in further self-assembly of SD-Dox.</p><p>The loading of Dox into drug carrier SD was studied using fluorescence spectrometry. As shown in Figure. 3A, Dox fluorescence signal was gradually quenched with increasing fractions of SD. At an SD/Dox molar ratio of 0.1, Dox fluorescence was significantly quenched. To assure the least amount of free Dox in solution, an SD/Dox molar ratio of 0.12 was used in cytotoxicity studies. By fluorescence titration, the overall dissociation constant (Kd) was determined to be 41±5.5 nM (Figure. 3A). By studying Dox intercalation with different SD components (sgc8, sgd5a and dsDNA linker only), we confirmed that the Dox intercalation sites were mostly localized in the linker (Figure. S6B). The loading of Dox into carrier was further confirmed by the enhancement of Dox fluorescence anisotropy with increasing SD fraction (Figure. 3B).</p><p>As a consequence of the rapid kinetics of DNA hybridization and Dox intercalation, the self-assembly of SD-Dox complexes is also fairly rapid, and intercalation equilibrium is achieved in less than 10 seconds (Figure. S6A). Moreover, the slow Dox release from SD-Dox in buffer indicated good stability for at least 5 days under our experiment condition(Figure. 3C).</p><p>Next, the bi-specificity of the complex SD-Dox was confirmed using flow cytometry, as indicated by the selective binding abilities to target CEM and Toledo cells, but not to NB4 cells (Figure. 4A). Furthermore, the ability of SD-Dox to selectively deliver Dox to target cells was studied by confocal microscopy. In this experiment, cells were incubated with 0.5 μM free Dox or SD-Dox with the equivalent Dox concentration. After 2-h incubation, cells were washed and observed for Dox fluorescence intensity by confocal microscopy. The uptake of Dox by cells treated with SD-Dox is presumably through two pathways: 1), binding of SD-Dox on target cell surfaces and then internalization of the entire SD-Dox before gradual Dox release inside cells; or 2), uptake of free Dox that diffuses from the highly concentrated SD-Dox on target cell surfaces. As shown in Figure. 4B, the Dox signal intensities in CEM and Toledo cells treated with SD-Dox were more comparable to those of the corresponding cells treated with free Dox, than those of non-target NB4 cells. The slightly lower Dox fluorescence intensities in target cells treated with SD-Dox than those treated with free Dox could be explained by 1), The efficiency of Dox transportation via a macromolecular drug carrier (SD) is lower than uptake of free Dox into cells; 2), The Dox that were not released from SD-Dox yet were still quenched, resulting in lower fluorescence. Overall, these results indicated that this aptamer-based drug carrier SD bi-specifically delivered Dox into target cells.</p><p>Finally, MTS assays were performed to study the specific cytotoxicity of Dox delivery by SD. First, the cytotoxicity of free Dox was investigated for CEM, Toledo and NB4 cells. CEM was reported to show dose-dependent response to free Dox[19], as did Toledo and NB4 (Figure. S7). Then, the cytotoxicity of SD-Dox to these cells was studied. Because the IC50s of Dox to these three cell types were not exactly the same, they were treated with Dox or SD-Dox at the respective Dox concentrations which could cause about 30% cell viability (CEM: 0.5 μM, Toledo: 0.7 μM, NB4: 0.35 μM). The resultant cell viabilities of each type of cells were then normalized, with 30% cell viability of the corresponding cells treated with free Dox. While free Dox induced 30% normalized cell viabilities, SD-Dox with the equivalent Dox concentrations induced approximately 50%, 50% and 75% normalized cell viability in CEM, Toledo, and NB4 cells, respectively (Figure. 5A). This indicated that Dox delivered by SD induced higher cytotoxicity in both target CEM and Toledo cells than in non-target NB4 cells. However, neither SD alone nor dsDNA linker-Dox showed significant cytotoxicity. We reason that the greater cell viabilities of CEM and Toledo cells treated with SD-Dox than those treated with free Dox were caused by less efficient Dox uptake via SD-Dox in the limited treatment time (2h), which is consistant with the Dox delivery efficiency discussed above.</p><p>Furthermore, to mimic clinical situation where target cells were in cell mixtures containing miscelleneous non-target cells, the cytotoxicity specificity of Dox delivered by SD was further examined in cell mixtures containing 50,000 CEM, 50,000 Toledo and 100,000 NB4 cells. Cell mixtures were treated with S-Dox, D-Dox and SD-Dox, respectively, with 0.5 μM Dox. The treated cell mixtures were stained with Propidium iodide (PI), which accumulated inside dead cells and could be analyzed by flow cytometry. As discussed before, the cell morphologies of CEM/Toledo cells can be easily distinguished from that of NB4 cells by flow cytometry. Again, target cell populations were gated for PI fluorescence intensity analysis. The results indicated that SD-Dox induced approximately the sum of target cell deaths induced by S-Dox and D-Dox, respectively (Figure. 5B).</p><p>Overall, these results clearly demonstrated the bi-specific cytotoxicity of Dox delivered by SD, thus broadening the range of targeted cytotoxicity in aptamer-based drug delivery. Previous studies showed that monovalent aptamer-directed drugs induced cytotoxicities only in the corresponding target cells[19–20]. However, because of the broadened recognition capability of SD, Dox delivered by SD could induce bi-specific cytotoxicities in the cells targeted by both apamers sgc8c and sgd5a.</p><!><p>In conclusion, we report an facilely self-assembled aptamer-based drug carrier for bi-specificity cytotoxicity. The bi-specificity resulted from the ability of the drug carrier SD to bi-specifically recognize target cells. In particular, SD was able to specifically bind and detect both target cells (CEM and Toledo), but not non-target cells (NB4), in cell mixtures. SD was also shown quantitatively to possess broader recognition capability to target cancer cells than its parent monovalent aptamers. This bivalent solution to the problem of heterogeneity in cancer subtypes is expected to overcome many diagnostic and therapeutic complications. As such, SD was then utilized to deliver Dox, an anticancer drug widely used for cancer chemotherapy, in order for bi-specific cytotoxicity. Through aptamer engineering, this carrier, SD, was designed for easy self-assembly and simultaneously forming multiple Dox intercalation sites on the dsDNA linker. The further drug loading and self-assembly of SD-Dox is rapid, easy to characterize via Dox fluorescence change, and the SD-Dox complex showed good stability for at least 5 days. The multiple Dox intercalation sites on each SD enabled high drug loading capacity. Furthermore, the Dox-loaded SD, SD-Dox, maintained bi-specific abilities in target cell binding and targeted Dox delivery. Most importantly, Dox delivered by SD induced bi-specific cytotoxicity in both seperate and mixed cancer cell solutions, indicating a broad recognition range of targeted therapy. While the recognition abilities of monovalent aptamers are necessarily limited, the broader recognition capability of bi-specific aptamer-based drug carriers allowed drug cytotoxicity to be specifically directed to more subtypes of cancer cells. Under these conditions, bi-specific aptamer-based drug carriers can sidestep the problem of cancer heterogeneity altogether and, as a consequence, facilitate clinical aptamer applications in targeted therapy of many subtypes of cancers that respond to the same therapeutic methods.</p><!><p>Washing buffer contained 4.5 g/L glucose and 5 mM MgCl2 in Dulbecco's PBS (Sigma Aldrich). Binding buffer was prepared by adding yeast tRNA (0.1 mg/mL) (Sigma Aldrich) and BSA (1 mg/mL) (Fisher Scientific) into the washing buffer to reduce background binding. Doxorubicin hydrochloride (Dox) was purchased from Fisher Scientific (Houston, TX).</p><!><p>All DNA synthesis reagents were purchased from Glen Research, and all DNA probes (Table S1; italicized sequences represent complementary sequence for duplex formation) were synthesized on an ABI3400 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA, USA). Coupled on the 5′ end of these DNA probes was Fluorescein (FITC), Biotin, or Cy5, unless otherwise noted. The completed sequences were then deprotected in AMA (ammonium hydroxide/40% aqueous methylamine 1:1) at 65°C for 30 min and further purified by reversed-phase HPLC (ProStar, Varian, Walnut Creek, CA, USA) on a C-18 column using 0.1 M trithylamine acetate (TEAA Glen Research Corp.) and acetonitrile (Sigma Aldrich, St. Louis, MO) as the eluent. The collected DNA products were dried, and detritylation was performed by dissolving and incubating DNA products in 200 μL 80% acetic acid for 20 minutes. The detritylated DNA product was precipitated with NaCl (3 M, 25 μL) and ethanol (600 μL). UV-Vis measurements were performed with a Cary Bio-100 UV/Vis spectrometer (Varian) for probe quantification.</p><!><p>Cell lines CCRF-CEM (Human T-cell ALL), Ramos (human B-cell Burkitt's lymphoma) and Toledo (CRL-2631, B lymphocyte, human diffuse large cell lymphoma) were obtained from the American Type Culture Collection (Manassas, VA). NB-4 (acute promyelocytic leukemia) was obtained from the School of Medicine, Department of Pathology, at the University of Florida. The cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) (heat-inactivated, GIBCO) and 100 IU/mL penicillin-streptomycin (Cellgro) at 37 °C in a humid atmosphere with 5% CO2. The cell density was determined prior to each experiment using a hemocytometer.</p><!><p>The binding abilities of aptamers were determined by incubating dye-labeled aptamers (400 nM, unless otherwise noted) with cells (2×105) on ice for 30 min, followed by washing twice with washing buffer (1 mL) and suspending in binding buffer (200 μL), before flow cytometric analysis. Random sequences (lib) were used as a negative control. The fluorescence intensities of cells were determined with a FACScan cytometer (BD Immunocytometry Systems). Data were analyzed with WinMDI software.</p><p>Binding affinities of aptamers were determined using a series of aptamer concentrations. As negative controls, similar assays were performed using random sequences at the same corresponding concentrations. The increased mean fluorescence intensities of cells bound by dye-labeled aptamers compared with those of random sequences were used to calculate the equilibrium dissociation constant (Kd) by fitting the dependence of fluorescence intensity (F) on aptamer concentration (L) to the equation F= Bmax[L]/(Kd+[L])[19], where Bmax represents binding capacity and reflects the density of binding sites. Bmax/2 was then used as the binding constant. The binding assay was repeated at least three times.</p><!><p>A series of different concentrations of CEM or Toledo cells was spiked into a fixed concentration of NB4 cells in binding buffer. Cell mixtures were then used for binding assay (as described above). The populations of CEM/Toledo cells were gated based on their distinct sizes and applied to fluorescence intensity analysis.</p><p>A similar assay was used to quantitatively compare the recognition capabilities of monovalent aptamers and SD. Specifically, 50,000 CEM/Toledo cells were spiked into 200,000 NB4 cells in binding buffer. Aptamers sgc8c, sgd5a, SD and random sequences were used for the binding assay to determine the percentage of cells that showed fluorescence signal enhancement.</p><!><p>Aptamer-based carrier-drug complex, SD-Dox, was formed by mixing Dox (Fisher Scientific, Houston, TX) and SD. The formation of SD-Dox was monitored (Ex: 480 nm, Em: 590 nm) on a Fluorolog-Tau-3 spectrofluorometer (Jobin Yvon, Edison, NJ). The titration experiment by fluorescence spectrometry was performed by sequential addition of increasing fractions of SD to Dox (1 μM) in PBS (Sigma). The fluorescence of the resultant solution was recorded to monitor Dox intercalation efficiency. The dissociation constant (Kd) was determined by fitting the recorded fluorescence intensities to F= Bmax[L]/(Kd+[L])[19], as explained above. Similarly, fluorescence anisotropy was studied with Dox (10 μM) and increasing SD fraction (Ex: 480 nm, Em: 590 nm).</p><!><p>Free Dox (20 μM, 200 μL) and SD-Dox complexes (20 μM Dox, [SD]: [Dox]=1.2:10, 200 μL) were prepared and transferred into MINI Dialysis Units (3.5 MWKO, Thermo Scientific, MA). The unit bottoms were immersed in 3 mL PBS buffer in an individual well of a 6-well plate, with a magnetic rod in each well. The plate was placed on a magnetic stirrer (150 rpm). At the indicated time points, a 120 μL aliquot from each well was collected for Dox fluorescence measurement and then returned to the corresponding well.</p><!><p>All cellular fluorescent images were collected on the FV500-IX81 confocal microscope (Olympus America Inc., Melville, NY) with a 60× oil immersion objective (NA=1.40, Olympus, Melville, NY). Excitation wavelength and emission filters: TAMRA, 543 nm laser line excitation, BP 580±20 nm filter; Dox: 488 nm laser line excitation, emission BP 580±20 nm filter. Cells (2×105 in 200 μL) were incubated at 37 °C with aptamers, Dox, or SD-Dox assembly for 2 h, followed by washing with washing buffer (1 mL) twice at 4 °C and suspending in binding buffer (200 μL) before imaging. Each experiment was repeated three times and analyzed with Fluoview software.</p><!><p>The cytotoxicity for each individual type of cells was determined using CellTiter 96 cell proliferation assay (Promega, Madison, WI, USA). Cells (5×104 cells/well) were treated with SD, Dox, linker-Dox or SD-Dox in medium without FBS (37°C, 5% CO2). After incubation for 2 h, cells were precipitated by centrifugation, 80% supernatant medium was removed, and fresh medium (10% FBS, 200 μL) were added for further cell growth (48 h) before removing cell medium. CellTiter reagent (20 μL) diluted in fresh medium (10% FBS, 100 μL) was added to each well and incubated for 1–2 h. The absorbance (490 nm) was recorded using a plate reader (Tecan Safire microplate reader, AG, Switzerland). Cell viability was determined as described by the manufacturer.</p><p>The cytotoxicity of aptamer-drug complexes to mixed cells was determined using PI (Invitrogen, Carlsbad, CA). Cell mixtures (50,000 CEM or Toledo cells, 100,000 NB4 cells, in 500 μL FBS-free medium) were treated with aptamer-drug complexes for 1.5 h, before replacing with fresh medium (10% FBS, 500 μL) for further cell growth (24 h). Cells were stained with 1 μg/mL PI at room temperature for 20 min to test target cell death by flow cytometry. Target cell populations were gated in flow cytometric results to analyze PI fluorescence intensity. Cell death percentage was defined as the ratio of dead cell amount (in the range of M1 in Fig. 5B) to total cell amount.</p>

PubMed Author Manuscript

Increased Mortality from Influenza Infection in Long-chain Acyl-CoA Dehydrogenase Knockout Mice

We previously showed that the mitochondrial fatty acid oxidation enzyme long-chain acyl-CoA dehydrogenase (LCAD) is expressed in alveolar type II pneumocytes and that LCAD-/- mice have altered breathing mechanics and surfactant defects. Here, we hypothesized that LCAD-/-mice would be susceptible to influenza infection. Indeed, LCAD-/- mice demonstrated increased mortality following infection with 2009 pandemic influenza (A/CA/07/09). However, the mortality was not due to increased lung injury, as inflammatory cell counts, viral titers, and histology scores all showed non-significant trends toward milder injury in LCAD-/- mice. To confirm this, LCAD-/- were infected with a second, mouse-adapted H1N1 virus (A/PR/8/34), to which they responded with significantly less lung injury. While both strains become increasingly hypoglycemic over the first week post-infection, LCAD-/- mice lose body weight more rapidly than wild-type mice. Surprisingly, while acutely fasted LCAD-/- mice develop hepatic steatosis, influenza-infected LCAD-/- mice do not. They do, however, become more hypothermic than wild-type mice and demonstrate increased blood lactate values. We conclude that LCAD-/- mice succumb to influenza from bioenergetic starvation, likely due to increased reliance upon glucose for energy.

increased_mortality_from_influenza_infection_in_long-chain_acyl-coa_dehydrogenase_knockout_mice

1. Introduction<!>2.1. Animals and influenza treatments<!>2.2. Bronchoalveolar lavage fluid (BALF) collection and analysis<!>2.3. Lung histology<!>2.4. Liver triglycerides<!>2.5. Western blotting<!>3.1 Increased mortality in LCAD-/- mice<!>3.2 Increased mortality in LCAD-/- mice is not due to lung injury<!>3.3 Metabolic effects of influenza infection

<p>Respiratory viral infections such as influenza have long been known to serve as triggers for metabolic decompensation and mortality in patients with genetic fatty acid oxidation (FAO) disorders [1]. The mechanisms behind the metabolic decompensation are not well understood. We previously showed that the alveolar type II pneumocyte (ATII), a key mitochondria-rich cell type in the lung, catalyzes FAO at high rates [2]. Both mouse and human ATII cells abundantly express the FAO enzyme long-chain acyl-CoA dehydrogenase (LCAD). LCAD-/- mice have increased lung epithelial permeability, altered breathing mechanics, and dysfunctional pulmonary surfactant [2]. Based on this, we hypothesized that LCAD-/- mice would show enhanced sensitivity to lung injury during a respiratory infection. To test this, LCAD-/- mice were infected with two different strains of influenza and evaluated for lung pathology as well as indicators of energy metabolism.</p><!><p>All protocols were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. LCAD+/- mice (B6.129S6-Acadltm1Uab) were purchased from the Mutant Mouse Regional Resource Center (University of Missouri, Columbia, MO) on a C57Bl/6 strain background. Due to infertility on the C57Bl/6 background, the LCAD-/- mice used here were maintained on a mixed C57Bl/6 and 129S6 background. Age and gender-matched wild-type B6/129S6 mice served as controls for all experiments. For influenza infection, the mouse-adapted A/PR/8/34 H1N1 virus (PR8) was propagated in chicken eggs as described [3]. The 2009 pandemic virus A/CA07/09 H1N1 (CA07) was propagated in MDCK cells [4]. Female mice age 6-8 weeks were given 80 plaque forming units (pfu) in 50 μl of sterile PBS by oropharyngeal aspiration. Body weights were tracked daily. Blood glucose and lactate were measured in unrestrained animals by nicking the tip of the tail and collecting droplets of blood into assay strips for handheld analyzers. A digital rodent rectal temperature probe (Physitemps Instruments, Clifton, NJ) was used to monitor core body temperature.</p><!><p>Mice were anesthetized, tracheotomized, and bronchoalveolar lavage performed using 1-ml of 0.9% NaCl. The amount of saline recovered was measured and recorded. The fluid was centrifuged at 300 × g for 10 min to pellet the cells, which were immediately resuspended in PBS, centrifuged onto glass slides, and stained for differential counting. The cell-free BALF supernatants were snap frozen in liquid nitrogen and stored at -80°C for later viral titer assays performed by standard MDCK plaque assay [4].</p><!><p>The lungs were inflated with 10% neutral-buffered formalin at a pressure of 25 cm of H2O for 10 min, then placed in fresh 10% neutral-buffered formalin for 24 h before processing for H&E staining. A pathologist blind to the genotypes scored lung injury on a severity scale of 1 (mild) to 5 (severe).</p><!><p>Snap-frozen liver tissue was assayed for triglyceride content exactly as described [5].</p><!><p>Western blotting was carried out as described. Primary anti-surfactant protein-A (SP-A) antibody (Proteintech, Inc) was used at a dilution of 1:300.</p><!><p>Based on our previous work demonstrating altered breathing mechanics, surfactant defects, and increased epithelial permeability [2], we hypothesized that LCAD-/- mice would be more sensitive to influenza infection. Indeed, 8-week old LCAD-/- female mice infected with the CA07 virus displayed significantly reduced survival compared to wild-type control mice (Fig 1A). LCAD-/- mice began to succumb by Day 5 post-infection compared to Day 8 for the wild-type controls. Weight loss is considered a good marker of disease severity in influenza-infected mice [6]; here, we observed significantly exacerbated weight loss in the LCAD-/- mice by Day 4 post-infection (Fig 1B).</p><!><p>Given the baseline lung defects previously established in LCAD-/- mice we hypothesized that the shortened survival time following infection was caused by enhanced lung injury. To test this, we repeated the CA07 infection but with tissue harvest on Day 5, just prior to the onset of mortality. The mice were subjected to bronchoalveolar lavage and lung tissue was processed for histopathology. Contrary to our hypothesis, the LCAD-/- mice did not show signs of enhanced lung injury following CA07 infection. There was a nonsignificant trend toward reduced inflammatory cells in bronchoalveolar lavage fluid (BALF) from LCAD-/- mice (Figs 2A), and the distribution of these cell counts across neutrophils, monocytes, and lymphocytes were not different between genotypes (Fig 2B). Equal titers of influenza virus in BALF indicated that viral replication and infectivity were not altered in LCAD-/- lungs (Fig 2C). In keeping with the lack of difference in these parameters, histological examination of lung tissue revealed no significant difference in severity of injury as scored by a pathologist blind to the genotypes (Fig 2D).</p><p>Seasonal H1N1 strains such as CA07 have been shown to cause less severe lung injury in mice than the more commonly used mouse-adapted PR8 strain [6,7]. We therefore infected a cohort of LCAD-/- and wild-type control mice with PR8. Compared to wild-type, LCAD-/- mice displayed significantly less lung tissue injury (Figs 3A-C). The protection against injury in LCAD-/- lungs was limited to the parenchyma, as histology scores were not significantly different between genotypes in the perivascular or peribronchial regions of the lung.</p><p>The protection of LCAD-/- lung parenchyma from PR8-induced injury was an unexpected finding. Our previous work implicated surfactant defects as the cause of the altered breathing mechanics in LCAD-/- mice [2]. LCAD-/- had significantly less total surfactant as well as relative changes in composition in terms of acyl chain lengths and degree of unsaturation [2]. Surfactant is known to be the first line of defense against pathogens, but this defensive property is mostly ascribed to the surfactant proteins A (SP-A) and D (SP-D), which are innate immune system collectins that opsonize pathogens and mark them for phagocytosis by alveolar macrophages [8]. The lipid component of pulmonary surfactant has actually been shown to promote viral infection of the alveolar epithelium, particularly dipalmitoylphosphatidylcholine (DPPC), a highly abundant surfactant phospholipid with two saturated acyl chains [8]. DPPC undergoes a constant cycle of cellular uptake and recycling. Viral particles can bind to DPPC and thereby obtain entry into epithelial cells [9]. In contrast to DPPC, phosphatidylcholine species with unsaturated acyl chains retard entry of viral particles into cells [9]. Our previous characterization of the surfactant profile in LCAD-/- lung indicated a paucity of DPPC with increased abundance of unsaturated phosphatidylcholine species [2]. This may be the mechanism that allows either normal (CA07) or suppressed (PR8) lung injury in the face of a viral infection. Here, we additionally show that the key surfactant collectin SP-A is increased in BALF from LCAD-/- mice under basal conditions (Fig 3D). Based on our previous and current observations we postulate that a combination of low DPPC and high SP-A helps protect LCAD-/- lungs against infection, particularly from the mouse-adapted PR8 virus.</p><!><p>The data discussed above suggested that extra-pulmonary factors must contribute to the increased mortality in LCAD-/- mice. LCAD-/- mice are known to be sensitive to fasting and develop fasting-induced hepatic steatosis, hypoglycemia, and hypothermia [10,11]. Given that influenza has been reported to severely reduce food intake in mice [12], we predicted that CA07 would drive severe hypoglycemia, fatty liver, and hypothermia in LCAD-/- mice. Surprisingly, while CA07 did indeed induce hypoglycemia, there was no difference in blood glucose values between genotypes (Fig 4A). Likewise, while LCAD-/- livers from uninfected mice had significantly more triglycerides at baseline and especially after an overnight fast (Fig 4B), the levels of triglycerides on Day 5 post-infection with CA07 were the same as wild-type. Core body temperatures were significantly lower in LCAD-/- mice after an acute 16-hr fast as well as on Day 5 post-infection with CA07 (Fig 4C).</p><p>Our metabolic data indicate that the effects of influenza are different than those of an acute, overnight fast. The course of influenza is that of a slow starvation rather than the rapid and massive mobilization of adipose-derived lipids seen in mice subjected to overnight fasting. The latter overwhelms the liver of humans and animals with FAO deficiencies resulting in hepatic steatosis and hypoketotic hypoglycemia [1,13]. LCAD-/- mice retain a partial ∼40% capacity for hepatic FAO due to very long-chain acyl-CoA dehydrogenase (VLCAD) which has overlapping substrate specificity [14]. We speculate that during influenza the presence of VLCAD in LCAD-/- mice is sufficient to maintain liver function, thereby explaining the lack of lipid deposition and the similar blood glucose values between LCAD-/- and wild-type mice during the course of infection. The cause of mortality appears to be wasting, as the enhanced weight loss was the most prominent difference we observed between genotypes of mice (Fig 1B). LCAD-/- mice have an increased reliance upon glucose for energy in peripheral tissues and thus a higher respiratory exchange ratio (RER)[15,16]. In the current study this is supported by the observation of higher blood lactate values in LCAD-/- mice after infection with CA07 (Fig 4D). We propose that the decreased metabolic efficiency in LCAD-/- mice leads to faster depletion of energy stores and muscle wasting in order to fuel gluconeogenesis, ultimately leading to more severe morbidity and death. Future studies are needed to determine the inter-relationships between inborn errors of metabolism, weight loss, and mortality from influenza.</p>

PubMed Author Manuscript

Palladium-catalyzed asymmetric allylic alkylation (AAA) with alkyl sulfones as nucleophiles

An efficient palladium-catalyzed AAA reaction with a simple a-sulfonyl carbon anion as nucleophiles is presented for the first time. Allyl fluorides are used as superior precursors for the generation of p-allyl complexes that upon ionization liberate fluoride anions for activation of silylated nucleophiles. With the unique bidentate diamidophosphite ligand ligated palladium as catalyst, the in situ generated a-sulfonyl carbon anion was quickly captured by the allylic intermediates, affording a series of chiral homo-allylic sulfones with high efficiency and selectivity. This work provides a mild in situ desilylation strategy to reveal nucleophilic carbon centers that could be used to overcome the pK a limitation of "hard" nucleophiles in enantioselective transformations.

palladium-catalyzed_asymmetric_allylic_alkylation_(aaa)_with_alkyl_sulfones_as_nucleophiles

Introduction<!>Optimization of conditions<!>Results and discussion<!>Conclusions

<p>Transition-metal-catalyzed asymmetric allylic alkylation (AAA) is a powerful tool for the enantioselective construction of stereogenic centers, enabling the elaboration of complex organic molecules and synthesis of pharmaceutical intermediates and bioactive natural products. 1 A variety of "so" carbon nucleophiles (Nu-H with pK a <25) and heteroatoms have been used in AAA reactions, generating the corresponding stereogenic centers with good to excellent selectivity. 2 However, transition-metal-catalyzed allylic substitution reactions with "hard" nucleophiles (Nu-H with pK a > 25) is mainly limited to non-enantioselective transformations. 3 During the past decades, our group and many other research groups have made tremendous efforts to engage "unstable" nucleophiles, such as ketones, acyclic amides and nitrogen-contained heteroarenes, etc. in the palladium-catalyzed AAA reactions. 4,5 Despite these achievements, a great number of "hard" carbon nucleophiles are still not compatible in palladium-catalyzed AAA reactions. One important example of such unexplored carbon based nucleophiles for AAA reactions is a-sulfonyl carbon anion (pK a ¼ 29 for (methylsulfonyl)benzene, Fig. 1a). 6 Sulfone represents an important moiety that is widely spread in many biologically active compounds and pharmaceutical intermediates. 7 Moreover; sulfones can be converted into a wide range of other groups at the a position via traceless transformations. 8 Efficient utilization of a-sulfonyl carbon anion in the palladium-catalyzed AAA reaction would lead to chiral homo-allylic sulfones, which would provide promising opportunities for the exploration of chiral sulfone containing compounds (Fig. 1c). Previous exploration to build chiral homoallylic sulfones via AAA reactions were limited to special sulfone reagents, an additional electron-withdrawing group was usually needed to enhance the acidity of a proton (Fig. 1a). 9 For example, the use of the ester group allowed removal aer the reaction of Krapcho demethoxycarbonylation; 10 however, Fig. 1 Representative methods for homo-allylic sulfones.</p><p>besides the moderate yield (60-80%), the harsh reaction conditions for demethoxycarbonylation would rule out many useful functional groups. Therefore, exploration of efficient method to realize the direct asymmetric allylic reaction of simple alkyl sulfones under mild reaction conditions is highly in needed.</p><p>To date, two elegant non-enantioselective reports on direct allylic alkylation of sulfones employed decarboxylation as the driving force. A thermodynamic decarboxylative Claisenrearrangement reaction reported by Craig et al. 11 under harsh reaction conditions (150 C) would restrict the diversity of functional groups (Fig. 1b). Another example is a palladiumcatalyzed intramolecular decarboxylative allylation of sulfonyl acetic esters with rac-BINAP as the ligated ligand (Fig. 1b). 12 Notably, the conditions developed by Tunge et al. still needed high reaction temperature or microwave conditions to get acceptable results. In most cases, the key nucleophiles were stabilized by both sulfonyl and phenyl or heteroatoms (pK a ¼ 23.4 for (benzylsulfonyl)benzene). 6 Herein, we report our endeavor and initial results on the palladium-catalyzed AAA reaction with simple a-sulfonyl carbon anion as the nucleophile (pK a > 25, Fig. 1c).</p><!><p>Recently, our and other groups have found that phosphoramidite and diamidophosphite ligands could facilitate transition-metal catalyzed transformations via in situ deprotonation of pro-nucleophiles. 4l,13 Notably, the Sawamura group found that the chiral phosphoramidite ligated palladium catalyst can facilitate the asymmetric allylic alkylation at the "hard"a position of 2-alkyl pyridines without additives. 4l The above achievements inspired us to utilize these unique ligands and exploit a-sulfonyl nucleophiles in AAA reactions. We started our research with commercial compound 1a-1 as a donor, and tert-butyl cyclohex-2-en-1-yl carbonate 2a-1 as the model counterpart (Fig. 2a). Ligand L1 (Fig. 2c) which has proven to be a suitable ligand for palladium-catalyzed transformations involving a deprotonation mechanism was selected as the ligated ligand to test different conditions. 14, 15 We soon realized that in the absence of additional base, the reaction gave no desired results. The tert-butoxide presumably generated in situ from Pd-mediated ionization of 2a-1 is incompetent to efficiently deprotonate the sulfone 1a-1 or perhaps the carboxylate leaving group never lost CO 2 to form tert-butoxide. We surmised that addition of an external base could lead to deprotonation. Addition of LiHMDS, KHMDS and NaO-tert-Bu to facilitate the desired deprotonation failed to give acceptable results. Perhaps, these strong bases interfered with the ionization event of 2a-1 and the moisture sensitive nature of these strong bases makes the reaction hard to handle. We turned our attention to nd mild conditions to generate the corresponding a-sulfonyl carbanion in a catalytic manner without additional stoichiometric base.</p><p>Recently, our group and the Hartwig group found that allylic uoride can be used as an excellent electrophilic precursor in transition metal-catalyzed asymmetric allylic alkylation, generating the nucleophile anion by in situ uoride induced desilylation, respectively. 16 The strategy invoked a synergistic interplay of the uoride leaving group to facilitate the generation of the electrophilic metal-allyl complex and delivery of a catalytically activated nucleophilic anion by desilylation. We sought to utilize this approach to engage asulfonyl 1a in palladium-catalyzed asymmetric allylic alkylation with allyl uoride 2a (Fig. 2b). Encouragingly, using L1 gave the desired product 3a with good yield (83%) and excellent enantioselectivity (91% ee). When t-BuOMe was used as the solvent, the product 3a was obtained in lower 74% yield but better 94% ee. When L2 was used as the supporting ligand, the product 3a was obtained in 51% yield and 83% ee. Some other phosphoramidite ligands were also tested. L3 afforded the product in a moderate 72% ee with a poor 35% yield. On the other hand, L4 17 and L5, which were successful ligands in our previous palladium-catalyzed transformations, did not afford the desired results. Nevertheless, Sphos L6 afforded 3a in moderate 61% yield, thus this ligand was selected as the supporting ligand for the non-enantioselective transformations. The solvent effect was very important for this transformation and only ethereal solvents gave the desired sulfone 3a with acceptable results. Other solvents such as toluene and DCE gave trace amounts of the desired product. t-BuOMe was proved to be the optimal solvent for the enantioselective transformations. Active CpPd(h 3 -C 3 H 5 ) was another key factor for this reaction; other palladium sources such as commonly used Pd(dba) 2 gave poor results.</p><!><p>To generate more elaborate chiral homo-allylic sulfones, we rst tested the scope of different sulfone donors with allyl uoride 2a as the reaction counterpart (Fig. 3). Aryl sulfones bearing an electrondonating (3b) or an electron-withdrawing (3c) group were suitable in our system, giving good to excellent results. The substrates bearing halogen atoms, such as uoro and chloro afforded corresponding products with good results (3e and 3f). Sterically hindered 2-naphthyl sulfone (3f) and bioactive 7-coumarinyl sulfone (3g) also gave rise to the desired products in good to excellent enantioselectivity. Additionally, different heteroaryl sulfones, 18 such as 2-pyridyl sulfone (3h), 4-pyridyl sulfone (3i), 1,3pyrimidinyl sulfone (3j) and benzothiazolyl sulfone (3k) all gave the desired products with excellent results (>94% ee). Besides the above aryl sulfones, simple alkyl sulfones were also tested with the optimized conditions, and the corresponding chiral products could be obtained with slightly diminished enantioselectivity compared to aryl sulfones (3l, 3m). Notably, sulfonamide which is a privileged functionality in modern drug discovery also could produce the corresponding homoallylic chiral sulfonamides with good results (3n, 3o). 19 An interesting anion shi was observed for the reaction with benzyl sulfone, which gave the expected product 3p in a good 63% yield and excellent 92% ee with a unseparated minor regioisomer 3p 0 . A similar shi is not exhibited in Tunge's decarboxylative allylations of benzyl alkyl sulfone. 12 Regrettably, vinyl and alkynyl sulfones did not give the desired products. The absolute conguration of 3h was determined by X-ray crystallography; the stereochemical outcome for all other homo-allylic sulfones was assigned by analogy.</p><p>Next we turned our attention to the substrate scope of allylic uorides. To make the detection and separation of product easier, 2-pyridyl sulfone 1h was selected as the standard nucleophile for most substrates (Fig. 4). Allylic uorides bearing 2-naphthyl (4a), more sterically hindered 1-naphthyl (4b), electron-rich aryl (4c) and electron-decient aryl (4d, 4e) all gave a range of chiral homo allylic sulfones with good yields and good to excellent ee values. Substrate bearing a chlorine atom, which is a good handle for further functionalization, was compatible with the reaction conditions (4f). Heteroarenes are also good choices for the reaction. Electron-rich indolyl (4g), thiophenyl (4h), and electron-decient quinolinyl (4i) were all successfully employed to give the desired products with excellent results (>89% ee). Besides the different aryls, alkenyl and alkyl substituted allylic uorides were also subjected to the optimized conditions. Alkenyl substituted acceptors gave the desired homo-allylic sulfones in good yield with slightly lower ee values compared to aryl substituted acceptors (4j, 4k). Simple benzyl substituted acceptor gave 57% yield but a poor 47% ee (4l). Added exibility of the benzyl substituent could account for the compromised selectivity. A nitrogen-containing heterocyclic acceptor was also tested and delivered the desired product 4m in 66% yield and an excellent 91% ee. Besides the six membered all carbon cyclic acceptors, medium-sized rings such as sevenmembered cyclic acceptors also proved to be good substrates for this reaction, giving the desired products with excellent ee values (4n, 4o). Unfortunately, ve-membered acceptors are not suitable for our current conditions, as the active allylic uoride preferentially eliminated HF thereby forming cyclopentadiene spontaneously. 20 To demonstrate the synthetic application of this transformation, the scale of the reaction was increased to 1.0 mmol (Fig. 5a). The palladium loading could be reduced to 2 mol% with 3 mol% of ligand L1 whereby the product 3a was obtained in 68% yield with 92% ee. Furthermore, the sulfone group in the products provides an enabling handle for further transformations (Fig. 5b). For example the sulfone group in 3a could be reductively cleaved with Na(Hg) to access chiral allylic methyl compound 5a in 70% yield. Notably, access to compound 5a is difficult by other methods. Here the sulfone donor acts as a formal methylation reagent. 21 Additionally, the sulfone group could be replaced by an ester group via a two-steps synthetic sequence in 82% yield (5b). Furthermore, the heteroaryl sulfonyl group in 3k could be used as a precursor for Julia-Kocienski olenation whereby upon reaction with benzaldehyde the skipped diene 5c forms in excellent yield and geometric selectivity (5c) for the E isomer. 22 The 1,3-diene unit in 4j is a good counterpart for an intermolecular Diels-Alder reaction, which afforded fused cyclic compound 5d in 63% yield (dr ¼ 3.5 : 1) upon reaction with N-methyl maleimide. 23</p><!><p>In conclusion, we realized the rst palladium-catalyzed AAA reaction with "hard" a-sulfonyl carbanions as the nucleophiles.</p><p>This transformation provides a rapid entry to chiral homo-allylic sulfones that otherwise are challenging to obtain. The presence of a sulfone motif provides a powerful handle for subsequent structural elaborations. The "outer sphere" reaction pathway is presumed to be involved in this transformation, 24 however, another possible reaction pathway involved anionic Si-F species couldn't be ruled out. 25 Detailed mechanistic studies and application of this novel AAA reaction in the synthesis of biologically active compounds and analogy of natural products are ongoing.</p>

Royal Society of Chemistry (RSC)

Improved syntheses of halogenated benzene-1,2,3,4-tetracarboxylic diimides

The preparation of halogenated benzene-1,2,3,4-tetracarboxylic diimide derivatives is challenging because of the possibility of competitive incorrect cyclizations and SNAr reactivity. Here, we demonstrate that the direct reaction of benzene-1,2,3,4tetracarboxylic acids with primary amines in acetic acid solvent successfully provides a range of desirable ortho-diimide products in good yields. Furthermore, we demonstrate that sterically challenging N-derivatizations can be readily achieved under microwave reactor conditions, and that SNAr reactivity is only observed when excess amine is used. The halogenated diimides described here are attractive building blocks for organic materials chemistry.

improved_syntheses_of_halogenated_benzene-1,2,3,4-tetracarboxylic_diimides

<p>Aromatic diimides, also known as bis(dicarboximide)s, are the linchpin of a diversity of organic materials encompassing vivid pigments and dyes, [1][2][3] thermally robust polymers, 4 and ntype electronic materials. 5,6 The electron-withdrawing nature of the cyclic imides lends itself to the creation of n-type semiconducting materials. 3 The annulation of aromatic rings with cyclic imides tends to lead to more significant LUMO-lowering effects than HOMO-lowering effects, thus resulting in red-shifted bandgaps and absorption profiles. Aromatic diimides derived from benzene (PMDI), naphthalene (NDI), and pyrene (PDI) (Figure 1a) have received significant research attention because of their facile syntheses and amenability to derivatization at both the core and imide N positions. [7][8][9][10] While changes in Nfunctionalization are often exploited for tuning solid-state packing behavior and solubility profiles, [11][12][13] core modification through substitution and metal-mediated cross-coupling reactions of halogenated aromatic diimides results in fine control over energy levels and electronic structure. 7,10 In recent years, researchers have developed new strategies for incorporating cyclic imides onto a growing number of aromatic scaffolds, resulting in interesting redox activity 14 and near-IR absorptions. 15 We recently began exploring the ortho-diimide structural isomer of the well-known pyromellitic diimide, which is known as mellophanic diimide (MDI), [16][17][18][19] and have discovered that it has significant potential as a building block in the construction of compounds with properties of interest to organic materials chemists (Figure 1b,c). Core dichlorinated N,N'-dihexyl MDI (Cl2-MDI-Hex), for example, readily undergoes both SNAr and Pd-catalyzed substitutions with aromatic ortho dinucleophiles to lead to a range of highly chromophoric and electron-accepting hetero-and azaacene structures. 20,21 The development of MDI as a building block is further attractive because it is derived from 1,2,3,4-tetramethylbenzene, which is a byproduct of durene synthesis and a constituent of petroleum extract that has no significant industrial use. 22 With these observations in mind, we set out to establish generalizable synthetic methods for obtaining differently halogenated MDI derivatives. Results and Discussion. Conventionally, aromatic diimides are synthesized by condensation between the relevant aromatic cyclic dianhydride and an amine, a process which proceeds through an amide-carboxylic (amic) acid intermediate. Extending this approach to the synthesis of ortho aromatic diimides such as mellophanic diimide, however, is complicated by the possibility of incorrect cyclizations to yield 3,6-dicarboxyphthalimide byproducts. Two strategies were identified by Fang et. al for overcoming this challenge: 1) room-temperature amic acid formation followed by acetic anhydride-mediated dehydration and 2) high-temperature equilibration of the reaction mixture to reach the MDI thermodynamic product (Fig 2a). 18 While both of these methods were successful for synthesizing N,N'-diaryl MDI compounds, we found that they could not be reliably extended toward either N,N'-dialkyl-or corechlorinated MDIs because of competing nucleophilic aromatic substitution reactions and significant 3,6-dicarboxyphthalimide formation. Inspired by the mellitic triimide synthesis developed by Rose et. al, 23 we were previously able to obtain N,N'-dihexyl-4,5-dichloro-MDI (Cl2-MDI-Hex) after the 3-day solidstate dehydration of an ammonium carboxylate salt (Fig. 2b). 20 In further explorations, however, we found that this solid-state method could not be consistently extrapolated to differently halogenated benzene tetracarboxylic acids, and furthermore was not successful with more sterically demanding amines. As a result of further synthetic exploration, here we report our findings that the direct solution-phase reaction between benzene-1,2,3,4tetracarboxylic acids and primary amines is a widely generalizable method for synthesizing MDI derivatives of a variety of N substitutions and core halogenations (Fig. 3c). Synthesis. The commercially available 1,2,3,4-tetramethylbenzene was chlorinated, 20 brominated, 24 or iodinated 25 following literature procedures to yield intermediates 1-X (X = Cl, Br, or I) which were then subjected to exhaustive oxidation by 10 eq. of KMnO4 in tBuOH/H2O (1/1:v/v) to provide the halogenated benzene-1,2,3,4-tetracarboxylic acids. Although many KMnO4 methylarene oxidation procedures use pyridine as a co-solvent to improve reactant solubility, we have found that the use of tBuOH co-solvent reduces the equivalents of KMnO4 required to achieve full oxidation and furthermore is less prone to exotherm during the addition of KMnO4. Complete removal of reaction solvent prior to acidification of the carboxylate intermediate is important for avoiding the formation of t-butyl ester impurities. Our largest scale oxidation (20.0 g, 98.5 mmol of 1-Cl) proceeded smoothly to provide the tetraacid 2-Cl in 85% isolated yield.</p><p>Although we initially followed our previously developed solid-state dehydration protocol for synthesizing 3-X-R, we were motivated by inconsistent yields and long reaction times to investigate a solution-phase method. Unexpectedly, simply heating the tetraacids 2-X with primary amines in acetic acid solvent followed, if necessary, by the precipitation of products with the addition of water or MeOH to the reaction mixture, provided MDIs 3-X-R in up to 93% isolated yield. In some cases, a small amount of additional product can be recovered by extraction of the aqueous filtrate with CH2Cl2. Although the reaction byproducts are often polar and soluble enough in AcOH to be removed during the filtration process, additional purification can be easily achieved by passing the crude product mixture through a SiO2 column. This method is successful with a variety of amines and there is no discernible trend between the identity of the halogen and the reaction yield. Hexylamine, aniline, and benzylamine react smoothly with 2-X (X = Cl, Br, and I) in good yields to produce a suite of 3-X-R compounds. Single crystal X-ray data for 3-Br-Ph confirms the MDI constitution of the products (Figure 3b). It is notable, as will be discussed below, that yields tend to be higher for the halogenated 3-X-R compounds than the nonhalogenated ones. Amino acid methyl esters can also be readily converted into MDIs, which suggests they may be interesting building blocks for self-assembling small molecules. 26,27 Attempts to perform imidization with 6-amino-1-hexanol, however, resulted in complex mixtures as a consequence of acetate ester formation with the free alcohol. With these results in hand, we attempted to install more sterically demanding Ngroups such as branched alkyl chains and 2,6-dialkylaryl groups since these are commonly used by organic materials chemists to manipulate crystal packing and solubility. Although 2ethylhexylamine reacted readily with 2-H to yield 3-H-EtHex, imidization of 2-H with the more sterically hindered nucleophiles such as (S)-1-phenylethan-1-amine and 2,6-diisopropylaniline proved to be more recalcitrant. We were unable to isolate any products after applying our standard reaction method, and heating (S)-1-phenylethan-1-amine with 2-H for 3 days at 110 °C resulted in only 11% formation of 3-H-1PhEt. These low yields for sterically hindered amines were also found when attempting these same reactions using our older solid-state approach (Figure 4).</p><p>To overcome this slow reaction rate, we turned to microwave reaction conditions. Gratifyingly, reacting 2-Cl and (S)-1-phenylethan-1-amine at 200 °C for 24 hours led to 58% isolated yield for 3-H-1PhEt. Under these forcing conditions, an important consideration is whether nucleophilic aromatic substitution (SNAr) reactions at the aryl halides start to take place when X = halogen. In our trials, we did not observe significant levels of SNAr reactivity when 2.1 equiv of amine was reacted with tetraacid 2-Cl. However, when 12 equivalents of (S)-1-phenylethan-1-amine were reacted with 2-Cl, we did observe core-diaminosubstituted derivatives in the resulting product mixture. Mechanistic Insights. Our first attempted synthesis of 3-H-1PhEt under microwave conditions was performed at 200 °C for 2 hr of reaction time and resulted in 11% isolated yield. Both running the reactions for longer (200 °C for 24 hours) or with more equivalents of nucleophile (12 equiv. amine, 200 °C for 2 or 24 hours) improved the isolated yield, to 58% and 46%, respectively. It is worth noting that the excess amine approach is not feasible under microwave conditions when X = halogen because of the competitive SNAr reactions. From a mechanistic perspective, these observations suggest that in AcOH solvent, the reaction is likely taking place under overall equilibrating conditions. To gain more insight into the reaction process, we performed a 1 H NMR time-course study of the reaction between 2-H and (S)-1-phenylethan-1-amine in d4-acetic acid at 110 °C. Upon dissolving only 2-H, the 1 H NMR spectrum reflects the presence of a complex mixture of anhydrides corresponding to intermediates with or without symmetry around the central benzene ring. Although we cannot rule out cyclic anhydride formation, we believe that mixed acetic anhydride formation is more likely based on the solubility of the reaction intermediates. Upon addition of the amine and heating for 5 minutes, a number of different intermediates are detected by 1 H NMR spectroscopy and formation of the desired MDI product is first observable after 20 minutes of reaction time. After 19 hours, the mixture resolves itself into being primarily four species, three with symmetry around the benzene core and one without. After 11 days of reaction time, the desired product constitutes only 18% of the product mixture, which highlights the value of microwave reaction conditions for achieving higher yields on a reasonable time scale. The NMR spectrum of the reaction after a total of 31.5 days at 110 °C shows that the product distribution reaches a roughly 1:1:1 ratio of 3-H-1PhEt, 4-H-1PhEt, and 5-H-1PhEt (Figure 5c).</p><p>We were able to tentatively assign the structure of the reaction intermediates by performing column chromatography on incomplete reaction mixtures and analyzing the filtrates collected during reaction workups. Of these, the most notable intermediates are dicarboxyphthalimides 4-X-R and 5-X-R, which correspond to the asymmetric and symmetric possibilities, respectively, for monophthalimide formation between the tetraacids 2-X and an amine. It is again worth noting that during the reaction, however, it is difficult to rule out whether mixed acetic acid anhydride derivatives are the dominant species. Liquid chromatography mass spectrometry analysis of crude reaction mixtures corroborates the 1 H NMR and preparatory observations of 4-X-R and 5-X-R as the primary reaction byproducts, but also reveal one other identifiable product corresponding to a diamidophthalimide species 6-X-R. We believe this isomer is more likely because the analogous diamido derivative of 4-X-R should be more likely to proceed to cyclize into 3-X-R. From a purely statistical perspective, intermediates 4-X-R and 5-X-R should be formed in a 3 to 1 ratio because both the 1-and 2-carboxamide derivatives of 2-X can dehydrate into intermediate 4-X-R, while only the 2-carboxamide can cyclize into intermediate 5-X-R. To add context to our understanding, we performed density functional theory calculations (M062X/6-31G(d)) with implicit acetic acid solvent to evaluate the energy landscape of the reaction for the reaction between methylamine and either 2-H or 2-Cl. In both cases, the formation of the less symmetric 4-X-Me is thermodynamically favorable compared to the formation of 5-X-Me. Interestingly, the transformation of 4-H-Me into 3-H-Me is found to be an uphill process by 3.5 kcal/mol, while the analogous conversion of 4-Cl-Me into 3-Cl-Me costs only 0.3 kcal/mol. Although these calculations do not account for the added complexities of mixed anhydride formation or R group identity, they do correlate with our experimental findings that 3-X-R formation is higher yielding when X = halogen. Taken together, it is reasonable to posit that the statistical advantage of monophthalimide formation balances against substrate-specific energetic differences to influence overall conversion into MDI products.</p><p>In conclusion, we have developed methods for the preparation of a wide range of 5,6-dihalobenzene-1,2,3,4-bis(dicarboximides) from the direct solution-phase condensation of benzene-1,2,3,4-tetracarboxylic acids and primary amines in acetic acid solvent. The preparation of compounds with sterically demanding groups at the N atoms of the imides can be achieved readily under microwave conditions. Experimental and computational studies suggest that the reaction can take place under equilibrium conditions that are favored both statistically and thermodynamically to yield the desired ortho-diimide compounds instead of the symmetric dicarboxyphthalimide. Importantly, it is possible to avoid perturbing the aryl halide positions under these reaction conditions, which sets the stage for exploring a broad range of chemistries available for producing imide-decorated aromatic compounds with tailored form and function.</p>

ChemRxiv

Isolation and partial characterization of Asian sea bass (Lates calcarifer) Vitellogenin

A study was conducted to isolate, partial characterize Asian sea bass (Lates calcarifer) vitellogenin (vtg). Two-year-old juvenile L. calcarifer (n = 10) were given three intraperitoneal injections of 17-β estradiol (E2) at a dose of 2 mg/kg body weight to induce vitellogenesis. Blood was collected 3 days after the last injection, and plasma was purified through gel filtration chromatography. A broad single symmetrical peak consisting of vtg molecule was produced. Protein concentration was 0.059 mg/ml as determined by Bradfrod assay using bovine serum albumin as a standard. The protein appeared as one circulating form in Native PAGE considering the dimeric form of putative vtg with molecular weight of 545 kDa. In SDS-PAGE under reducing conditions, two major bands appeared at 232.86 and 118.80 kDa and minor bands at 100.60, 85.80 and 39.92 kDa, respectively. The purified vtg was used to generate a polyclonal antibody, and the specificity of antibody was assessed by Western blot analysis. Two major bands were immunoreacted, but no cross-reactivity was observed with plasma from non-induced males. The protein was characterized as phosphoglycolipoprotein as it positively stained for the presence of lipid, phosphorus and carbohydrate using Sudan Black B, methyl green and periodic acid/Schiff reagent solution, respectively. The amino acid composition was analyzed by high sensitivity amino acid analysis that showed high percentage of non-polar amino acids (~48 %). The results suggest the potential utilization of vtg as a basis tool to further study about reproductive physiology of this important economical species.

isolation_and_partial_characterization_of_asian_sea_bass_(lates_calcarifer)_vitellogenin

Introduction<!>Experimental animals<!>Induction of vtg<!>Plasma preparation<!>Purification of vtg<!>Native PAGE<!>Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)<!>Production of polyclonal antibodies<!>Immunoblotting (Western blot)<!>Amino acid analysis<!><!>Discussion<!>Conclusion

<p>Lates calcarifer, known as barramundi, Asian sea bass or locally called siakap, is native to coastal Australia, Southeast and Eastern Asia, and India (Luna 2008). This species are farmed in cages, as well as in fresh water and salt water ponds (Webster and Lim 2002). In recent years, sea bass has gained growing importance in aquaculture both as recreational and commercial fish, with a high and fairly stable price (Luna 2008). Sea bass production and consumption in Malaysia has increased dramatically over the years from 49, 586.19 (DOF 2005) to 194, 623.11 tonnes (DOF 2009). In spite of its economic importance, knowledge about its reproductive physiology is limited. In recent years, the reproductive biology of many fish species has been studied by analyzing the vitellogenin (vtg) levels in the blood plasma (Susca et al. 2001).</p><p>Vitellogenin is an egg yolk precursor protein that is synthesized in the liver under estrogen control and secreted into the bloodstream (Celius and Walther 1998; Prakash et al. 2007). This large protein has high molecular weight ranging from 250 to 600 kDa depending on fish species (Utarabhand and Bunlipatanon 1996). In teleost oviparous fish, vtg is served as a nutritional source for growing oocyte and developing embryo in matured female through a process called vitellogenesis (Romano et al. 2004). Following oocyte growth, vtg will enzymatically cleave into smaller yolk proteins namely lapidated lipovitellin, phosphorylated phosvitin and β-components (Zhang et al. 2011). Mananos et al. (1994) noted that the main components that contribute to the circulation of vtg molecule are lipids, carbohydrates, phosphorus and various ions such as calcium and iron. The vtg was present in adult vitellogenic females but absent in male as well as in immature females (Nath 1999; Fenske et al. 2001). In vertebrate, vtg gene is expressed in male and immatured female, but insufficient circulating estrogen is incapable to enhance the production of this protein (Palumbo et al. 2007). However, these organisms will synthesize the vtg if they are administered with synthetic estrogens, mainly 17-β estradiol (Leonardi et al. 2010; Boucard et al. 2008). Estradiol was reported to successfully induce the synthesis of vtg in many fish species (Utarabhand and Bunlipatanon 1996; Mendoza et al. 2011). The levels of vtg in fish indicate the maturing stage in female individual under natural condition (Matsubara et al. 1994). Knowledge of reproductive physiology including vitellogenesis is very important in managing fish broodstock for reproduction in most farmed animals including fish.</p><p>Previous studies reported that vtg has been isolated and purified in many fish species including Senegalese sole (Solea senegalensis) (Guzman et al. 2008), grouper (Ephinephelus malabaricus) (Utarabhand and Bunlipatanon 1996), California halibut (Paralichthys californicus) (Palumbo et al. 2007), Chilean flounder (Paralichthys adpersus) (Leonardi et al. 2010) and sea bass (Dicentrarchus labrax L.) (Mananos et al. 1994) by using double chromatography, ion exchange followed by gel filtration chromatography. However, Watts et al. (2003) and Mendoza et al. (2011) have proven that gel filtration alone using Sepachryl 16/60 HR-300 column was able to completely isolate vtg in three teleost species.</p><p>Vitellogenin has never been purified and characterized in L. calcarifer, and knowledge about its reproduction and vitellogenesis is needed. A better understanding of vitellogenesis is very important for farm management as well as to determine the maturity status of this economically important species (Utarabhand and Bunlipatanon 1996). Thus, the aim of this study was to induce, purify and characterize the vtg from E2-treated juvenile L. calcarifer.</p><!><p>Sampling was done at Center of Marine Science, Universiti Putra Malaysia, Port Dickson, Malaysia. Two-year-old juvenile Asian sea bass (L. calcarifer) (n = 10), ranging in weight from 1.5 ± 0.5 kg, were obtained from commercial supplier, while matured males and vitellogenic females, weighing around 4.0–6.3 kg, were obtained from Fisheries Research Institute, Tanjung Demong, Terengganu, Malaysia. They were maintained for 2 weeks in 10-tonne tank with filtered, flow-through seawater at salinity of 29 ± 1 ppm, a temperature of 21 ± 2 °C, pH of 6.0–6.5 and supplied with dissolved oxygen (5.4 mg/ml). Fishes were tagged using microchips for further identification and fed daily at satiation with chopped fresh fish.</p><!><p>Each juvenile fish was injected with 17-β estradiol, E2 (Nacalai Tesque, Japan) to induce the vitellogenesis. They received a total of three intraperitoneal injections (i.p.) (dose of 2 mg E2/kg fish body weight), given every 2 days. Estradiol was dissolved in ethanol and 0.9 % NaCl solution (1:9, v/v) as a carrier. Control fishes (n = 6) were injected with saline only, and no foods were administered during the experiment.</p><!><p>Three days following the last injection, four millilitres of blood was collected from the caudal vein of each fish using heparinized syringe containing phenylmethylsolfonyl fluoride, PMSF (Roche, Germany) (100 μl, 1 mM). The blood was maintained on ice, allowed to clot at 4º C for 1 h and centrifuged at 8,000 rpm for 10 min at 5 °C (Braathen et al. 2009). The supernatant (plasma) was withdrawn and immediately stored in the presence of PMSF to avoid proteolysis of vtg at a ratio of 2: 1 v/v (plasma:PMSF) (Watts et al. 2003). It was stored at −80 °C prior to vitellogenic purification (Parks et al. 1999). At the same time, blood was collected from control group (vitellogenic females and non-induced males).</p><!><p>Plasma vtg was purified using gel filtration chromatography (Akta Prime) performed at room temperature according to Watts et al. (2003) with modifications. One millilitre of plasma from estradiol treated juvenile L. calcarifer was loaded into prepacked Sepachryl HR-300 column (HiPrep 16/60) (GE Healthcare Bio-Science, Uppsala, Sweden). The samples were eluted with 0.05 M Tris–HCl pH 8.0 (Nacalai tesque, Japan) at a flow rate of 0.4 ml/min. The elution profile was monitored at 280 nm, and the fractions containing vtg peak were collected at a final volume of 5 ml. The purified vtg consisting peak was pooled and directly concentrated using Vivaspin centrifuge tube (30 kDa Molecular weight cut-off, GE Healthcare Bio-Science, Uppsala, Sweden) at 4 °C, 10,000 rpm for 10 min. It was stored at −20 °C in aliquots before subjected to Native PAGE and SDS-PAGE analysis. The purified vtg was used as antigen for antibody production against vtg in rabbits. The protein concentration was determined by Bradford assay using bovine serum albumin (BSA) (Sigma Diagnostics, USA) as standard.</p><!><p>To determine the purity and molecular weight of circulating form of L. calcarifer vtg, purified plasma was subjected to Native gradient PAGE (4–8 % separating gel solution) (Sun and Zhang 2001) with constant current of 100 mA for 4 h on ice in an electrophoretic buffer (0.025 M Tris and 0.192 M glycine. The molecular weight was determined by comparing with Native markers (Serva, Heidelberg, Germany). Separated protein was identified as phosphoglycolipoprotein (vtg) after staining with Sudan Black B, periodic acid/Schiff reagent solution (PAS) and methyl blue (Nacalai tesque, Japan) to determine the presence of lipid, carbohydrate and phosphorus, respectively.</p><!><p>To determine the molecular weight of vtg subunits, the purified plasma vtg was electrophoresed (SDS-PAGE) under denaturing conditions. It was performed on 7.5 % separating gel and 4 % stacking gel. Prior to application on the gel, the purified vtg were diluted in SDS sample buffer (125 mM Tris–HCl, 10 % SDS, 20 % v/v glycerol, 5 % v/v β-mercaptoethanol, 0.02 % bromophenol blue) (15 μg/ml) and boiled for 5 min. Electrophoresis was run on ice in a buffer (50 mM Tris, 192 mM glycine and 0.1 % SDS) at a constant current of 100 mA, 50 V for 5 h and immediately subjected to Western blot analysis. The results obtained were viewed using gel imager (Alpha Innotech, Cell Bioscience, California). The molecular weights of purified vtg were estimated by comparing with molecular weight protein markers (Fermentas, USA).</p><!><p>Specific antiserum against L. calcarifer vtg was raised in seven-month-old (~3 kg) female New Zealand white rabbits (n = 4). Rabbits were initially injected with freshly purified vtg (0.059 mg/ml protein) emulsified in Freund's Complete Adjuvant (CFA, Calbiochem) (1:1 v/v, 1 ml) and then boosted up by four additional injections of 0.02 mg/ml protein emulsified in Freund's Incomplete Adjuvant (IFA, Calbiochem) (1:1 v/v, 1 ml) at multiple subcutaneous and intradermal sites. A total of five injections were given at weeks 1, 3, 5, 7 and 9. Blood (~2.0 ml) was collected at weeks 4, 6 and 10, and the serum was assayed for reactivity toward vtg by screening ELISA. When antibody titer was sufficient, blood (~10 ml) was withdrawn from the ear artery and allowed to clot overnight at 4 °C (Smith and Benfey 2002). Serum was separated by centrifuging the blood at 12,000×g at 4 °C for 10 min and then stored at −80 °C in small aliquots. For negative antibody control, the rabbits were bled before immunization (pre-immune serum).</p><!><p>The purity of antigen and specificity of antibodies were tested by using Western blot analysis. The proteins separated by Native PAGE and SDS-PAGE were electro-blotted onto polyvinylidene fluoride, PVDF membrane (Immobilon-P, Millipore). The unstained gels were soaked in transfer buffer (48 mM Tris, 39 mM glycine, 20 % methanol, pH 9.2) for 15 min, and the blot was run at constant current of 300 mA, 50 V for 2 h on ice cold using Bio-Rad Trans Blot Cell. Following the transfer, the membranes were stained with Coomassie brilliant blue R-250 (0.025 % Coomassie Blue R-250, 40 % methanol, 7 % acetic acid) (Hercules, Canada). PVDF membranes were blocked by incubating in TBS (50 mM Tris, 150 mM NaCl, pH 7.5) containing 3 % bovine serum albumin (BSA) for 2 h to prevent non-specific binding sites. For immunochemical detection, the membranes were then incubated with primary antibody (anti-Vtg in rabbits) at a dilution of 1:1,500 in blocking buffer. Bound antibodies were detected by incubating with secondary antibody, HRP (Horse-Reddish Peroxidase, conjugated goat anti-rabbit IgG) (Nacalai tesque, Japan) at a dilution of 1:2,000 in blocking buffer for 2 h at room temperature. For visualization, membrane was incubated with substrate solution, Opti-4CN™ Substrate Kit (Bio-Rad, Hercules, CA) for 30 min to reveal the location of vtg. The membranes were washed with TBST [50 mM Tris, 150 mM NaCl, pH 7.5 containing 0.05 % Tween-20 (v/v)] three times for 15 min after each incubation step. The developed membranes were photographed using gel imager.</p><!><p>For analysis of amino acid composition, the purified vtg (200 μl) was analyzed by high sensitivity amino acid analysis (AAA). The sample was lyophilized and resuspended in a solution of 20 % acetonitrile (ACN) and 0.1 % trifluoroacetic acid (TFA). Sample underwent 24-h gas-phase hydrolysis with 6 N HCl at 110 °C. After hydrolysis, all amino acids were analyzed using high performance liquid chromatography (HPLC) system (Waters AccQTag Ultra) chemistry. The analyses were carried out in duplicate, and results were expressed as means. Amino Acid Standard H (Thermo Scientific Pierce) was used as calibration standard for HPLC analysis.</p><!><p>Gel filtration chromatography from plasma of juvenile L. calcarifer before (a) and after treated with 17-β estradiol (b) using Sepachryl (16/60) HR 300 column (GE healthcare Bioscience, Uppsala, Sweden)</p><p>a, b Native PAGE electrophoretic pattern (a) and subsequent western blot analysis (b Lanes 1 and 4 purified plasma from 17-β estradiol treated fish, lanes 2 and 5 plasma from natural vitellogenic females, lanes 3 and 6 plasma from non-induced male. The proteins were stained with Coomassie Brilliant Blue R-250 (Bio-Rad, Hercules, Canada)</p><p>a, b SDS-PAGE (0.1 % SDS) (A) and corresponding Western blot analysis (B), stained with Coomassie Brilliant Blue. Lane A molecular weight markers (Fermentas, USA); lane B and E purified vtg; lanes C and F vitellogenic females; lanes D and G male plasma</p><p>a–c Native PAGE for determination of lipid phosphorus and carbohydrate components in vtg of L. calcarifer. Lane 1 male plasma, lane 2 E2-treated plasma. Gels were stained with Sudan black B (a), methyl green (b) and Periodic acid/Schiff reagent solution (PAS) (c), respectively</p><p>Total amino acid composition of vtg in Asian sea bass in comparison with other fish species by percentage of moles</p><p>ND Not determine</p><!><p>The induction of 17-β estradiol in juvenile L. calcarifer resulted in the production of high-molecular-weight protein (232.18 and 118.80 kDa). The same amount of high molecular weight protein appeared in vitellogenic female that is identified as vitellogenin (vtg). It is well established that vtg was present abundantly in blood plasma of natural vitellogenic females. However, it can be induced in males and juvenile fish by treating with estrogen hormone (Smith and Benfey 2002; Rankouhi et al. 2001).</p><p>There are a few steps involved in the isolation and separation of vtg molecules. In the present study, the vtg was isolated and purified by using gel filtration chromatography of Sepachryl HR-300 column. Chromatography was the best method to isolate vtg from blood plasma of induced fish as successfully proven by several studies (Watts et al. 2003; Roy et al. 2004; Palumbo et al. 2007; Braathen et al. 2009; Maltais and Roy 2009; Leonardi et al. 2010). A broad, single symmetrical peak (Fig. 1b) from purification of E2-treated juvenile L. calcarifer obtained in this study indicated that the peak was E2-inducible, and there was no sign of degradation occurred during the isolation process (Guzman et al. 2008). Norberg (1995) also obtained a single molecule after vtg purification in Atlantic halibut (Hippoglossus hippoglossus). Roy et al. (2004) suggested that the best condition to isolate and purify vtg was at lower temperature (4º C) so as to preserve the stability of the protein. However, in this present study, purification process was carried out at room temperature. Hence, to avoid degradations, it was run in the presence of protease inhibitor (PMSF), which contributes to the isolation of single vtg molecule (Mosconi et al. 1998). Similar result was also observed for vtg in carp, Cyprinus carpio and perch, Perca fluviatilis (Hennies et al. 2003). However, purification analysis alone cannot determine that the isolated protein was vtg molecule. It needs to be confirmed by cross-reactivity of anti-vtg raised in New Zealand white rabbits in immunoblotting analysis (Norberg 1995).</p><p>There are a few evidences proven that the purified protein was phosphoglycolipoprotein (vtg). Firstly, the plasma protein levels in induced L. calcarifer were increased compared to non-induced (0.008–0.059 mg/ml, data not shown). This is in agreement with the finding by Mananos et al. (1994). Secondly, the specificity of antiserum confirmed by Western blot analysis showed that polyclonal antibody have strong reactivity in estrogen exposed juvenile L. calcarifer as well as vitellogenic females but failed to show any reaction with plasma from non-induced males. This indicates that the polyclonal antibody was specific, which failed to react with any non-vitellogenic proteins. Similar result was also obtained by Tao et al. (1993) in striped bass (Morone saxatilis) and Zhang et al. (2011) in Amur sturgeon (Acipenser schrenckii). Thirdly, in native gradient PAGE (samples were run without SDS and β-mercaptoethanol), the protein was positively stained for the presence of phosphorus, carbohydrate and lipid using methyl blue, periodic acid/Schiff's reagent solution (PAS) and Sudan black B as previously reported (Egito et al. 2001; Sun and Zhang 2001; Roy et al. 2004). Fish vtg was generally known to have high phosphorus and lipid contents that normally present as phospholipid and triglycerides (Tao et al. 1993). Fourthly, the amino acid composition of L. calcarifer vtg was very similar to vtg as previously noted from other fish species (Utarabhand and Bunlipatanon 1996; Tao et al. 1993; Parks et al. 1999; Tyler and Sumpter 1990). In the present study, high percentage of non-polar amino acids (glycine, alanine, proline, valine, isoleucine, leucine, ~48 %) was found which contributed to its lipoprotein function for transportation of endogenous lipid (Parks et al. 1999; Tao et al. 1993; Kera et al. 2000).</p><p>The analysis of vtg on native gradient PAGE resulted only one band (Fig. 2) confirmed that the isolated protein was free from contaminants. Hence, it is proved that gel filtration alone was able to completely isolated vtg in L. calcarifer. Palumbo et al. (2007) showed that the analysis of vtg by Native PAGE also resulted one circulating band interpreted that the band was dimeric form of putative vtg, which was similar to the present study. Sun and Zhang (2001) reported that the vtg from fish, amphibians and birds, nematodes and arthropods also circulate as dimeric form. Native PAGE analysis resulted an apparent molecular weight 545 kDa of L. calcarifer vtg, which falls within the range of vtg dimeric form as shown in other fish species: 525 and 260 kDa in grouper (Utarabhand and Bunlipatanon 1996), 490 kDa in carp (Fukada et al. 2003), 454 kDa in sea bass (Mananos et al. 1994), 330 kDa in female Bluefin tuna (Susca et al. 2001) and 390 kDa in teleost sp. (Larsson et al. 1994).</p><p>The molecular weight of vtg (232.86 and 118.80 kDa) in Asian sea bass (L. calcarifer) was greater than other fish species: that is 205 kDa for Amur Sturgeon Acipenser schrenckii (Zhang et al. 2011), 183 kDa for Gag Mycteroperca microlepis (Heppel and Sullivan 1999), 220 kDa for Medaka Oryzias latipes (Shimizu et al. 2002), 150 kDa for carp Cyprinus carpio (Fukada et al. 2003), 172 kDa for S. senegalensis (Guzman et al. 2008) and 180 kDa for sea bass D. labrax (Mananos et al. 1994). The presence of minor bands represents the monomer form of vtg molecule probably due to the use of sodium dodecyl sulfate (SDS) and β-mercaptoethanol, which contributes to the degradation fragments of putative vtg and similar finding was noted by Bon et al. (1997). The difference in molecular weight of vtg in each species indicated the different structure of vtg molecule.</p><p>Previous study reported that vtg was unstable and easily degraded due to the storage and handling of plasma sample (Norberg 1995; Roy et al. 2004). Hence, in this study, plasma was stored in aliquots and purified in the presence of phenylmethylsulfonyl fluoride (PMSF) in a ratio of 2:1 v/v to prevent proteolysis of this labile protein (Watts et al. 2003).</p><!><p>In conclusion, the present study was successfully isolated and partial characterized vitellogenin in induced juvenile L. calcarifer by using gel electrophoresis, Western blotting and amino acid analysis. Further analyses of vtg levels using Abvtg proposed as indicator of maturing female stage for managing fish broodstock in captivity.</p>

PubMed Open Access

Selective inhibition of Rhizopus eumelanin biosynthesis by novel natural product scaffold-based designs caused significant inhibition of fungal pathogenesis

Melanin is a dark color pigment biosynthesized naturally in most living organisms. Fungal melanin is a major putative virulence factor of Mucorales fungi that allows intracellular persistence by inducing phagosome maturation arrest. Recently, it has been shown that the black pigments of Rhizopus delemar is of eumelanin type, that requires the involvement of tyrosinase (a copper-dependent enzyme) in its biosynthesis. Herein, we have developed a series of compounds (UOSC-1\xe2\x80\x9314) to selectively target Rhizopus melanin and explored this mechanism therapeutically. The compounds were designed based on the scaffold of the natural product, cuminaldehyde, identified from plant sources and has been shown to develop non-selective inhibition of melanin production. While all synthesized compounds showed significant inhibition of Rhizopus melanin production and limited toxicity to mammalian cells, only four compounds (UOSC-1, 2, 13, and 14) were selected as promising candidates based on their selective inhibition to fungal melanin. The activity of compound UOSC-2 was comparable to the positive control kojic acid. The selected candidates showed significant inhibition of Rhizopus melanin but not human melanin by targeting the fungal tyrosinase, and with an IC50 that are 9 times lower than the reference standard, kojic acid. Furthermore, the produced white spores were phagocytized easily and cleared faster from the lungs of infected immunocompetent mice and from the human macrophages when compared with wild-type spores. Collectively, the results suggested that the newly designed derivatives, particularly UOSC-2 can serve as promising candidate to overcome persistence mechanisms of fungal melanin production and hence make them accessible to host defenses.

selective_inhibition_of_rhizopus_eumelanin_biosynthesis_by_novel_natural_product_scaffold-based_desi

Introduction<!>General chemistry procedures<!>Organism and growth conditions<!>Inhibition of tyrosinase activity and calculation of IC50<!>Kinetic studies<!>Melanin staining of melanoma cell line<!>MTT cell viability assay<!>In vivo virulence studies of white versus wild (black) types Rhizopus spores<!>De-melanization of Rhizopus spores enhanced the phagocytosis and clearance activities of macrophages<!>Procedures of molecular modeling<!>Preparation of the target enzymes.<!>Preparation of the ligands.<!>Molecular docking.<!>Calculation of binding energy.<!>Molecular dynamics simulation.<!>Study approval<!>Statistical methods<!>Rational design of UOSC compounds<!>The newly designed compounds caused effective inhibition of R. delemar melanin production<!>UOSC-1, 2, 13, and 14 are potent candidates to inhibit R. delemar melanin production<!>UOSC-1, 2, 13, and 14 showed high tyrosinase-inhibitory activities with limited toxicity<!>Computational modeling predicted that UOSC-1, 2, 13, and 14 are selective inhibitors of fungal tyrosinase<!>UOSC-2 showed competitive reversible inhibition activity against fungal tyrosinase<!>De-melanized R. delemar (white) spores were phagocytized and cleared faster than the wild-type (black) spores<!>Discussion

<p>Melanin is predominantly dark-colored polymer widely distributed in bacteria, fungi, plants, and animals [1]. Several biological functions of melanin have been reported including its role as free radical scavenger, as cation-binding material, and as protection from UV radiation [1]. In fungi, melanin is more likely correlated with spore formation, virulence of pathogenic fungi, and evasion from host defense mechanisms or stressful environmental conditions [2,3]. In mammals, melanin is produced in specialized pigment-producing cells known as melanocytes [4]. In mammals, melanin pigments play several diverse and important roles, including thermoregulation, camouflage, and sexual attraction [5].</p><p>It has been reported that melanin can provide the pathogenic fungi including Rhizopus delemar with a protective shield from host defensive mechanisms and hence allow their persistence in the human body through antigen mimicry mechanism [6]. The presence of pathogenic fungi in the human body in a dormant state facilitates their pathogenesis once the conditions are appropriate.</p><p>Melanin can be classified into two major classes, eumelanins, and pheomelanins. R. delemar melanin has been identified as of eumelanin type [6]. Eumelanin is a black/brown colored cross-linked polymer of the monomer 5,6-dihydroxyindole (DHI) and 5, 6-dihydroxyindole-2-carboxylic acid (DHICA). The rate-limiting step in eumelanin biosynthesis is the enzymatic oxidation of tyrosine or l-3,4-dihydroxyphenylalanine (l-DOPA) to its corresponding O-dopaquinone [7]. It is the only step in eumelanin biosynthesis that is controlled by an enzyme, named tyrosinase [8,9]. Although the crystal structures of most tyrosinases are similar, there are key differences among tyrosinases from different sources. For instance, both fungal and human tyrosinases showed key differences [10–12]. Specifically, the mushroom tyrosinase is a cytosolic enzyme while the human tyrosinase is a membrane bound [13]. Furthermore, mushroom tyrosinase is a tetramer contains binuclear copper-binding site which is located in the bottom of the active pocket. Each copper ion co-ordinated by three histidine residues. The ligands of the first Cu-A are His 61, His85, and His94, while the second Cu-B surrounded with His259, His263, and His296 [14]. In contrast, the human tyrosinase is a monomer that is highly glycosylated during its complex maturation process. Human tyrosinase contains binuclear zinc site instead of the copper ions in the case of mushroom tyrosinase. Zn-A is co-ordinated with His192, His215, and His224, while Zn-B is co-ordinated with his377, His404, and His381 [15].</p><p>Numerous compounds were identified as tyrosinase inhibitor from natural and synthetic sources, such as kojic acid, hydroquinone, arbutin, 4-methoxycinnamic acid, and rhododendrol (Figure 1). However, they show instability and undesirable side effects including cytotoxicity, dermatitis, skin cancer, and neurodegenerative disorders because of interaction with human cells [16–18]. Furthermore, most identified inhibitors lack clinical efficacy since they were evaluated using mushroom tyrosinase as a target. On the other hand, despite the potent fungal tyrosinase inhibition activities of the naturally isolated aldehyde, cuminaldehyde (Figure 1), it shows significant toxic activities [19,20].</p><p>The aim of this research study is to design and synthesis safe and selective compounds with promising inhibitory activity on fungal melanin biosynthesis in particular R. delemar which is the main cause of the lethal infection, mucormycosis [6]. To achieve this goal, cuminaldehyde was employed as a lead structure to develop optimized inhibitors for fungal melanin biosynthesis with high activity and enhanced physicochemical properties and limited toxicity.</p><!><p>Most chemicals and solvents were of analytical grade and, when necessary, were purified and dried by standard methods. Reactions were monitored by thin-layer chromatography (TLC) using pre-coated silica gel plates (kiesel gel 60 F254, BDH), and spots were visualized under UV light (254 nm). Melting points were determined using a Gallenkamp melting point apparatus and are uncorrected. Column chromatography was performed with Merck silica gel 60 (40–60 μM). 1H-NMR and 13C-NMR spectra were recorded on a Bruker spectrometer at 500 MHz. Chemical shifts were expressed in parts per million (ppm) and coupling constant J values were represented in Hz. Mass spectroscopic data were obtained through electrospray ionization (ESI) mass spectrum. Detailed synthesis and spectroscopic data of UOSC-1–14 are described in Supplementary Information.</p><p>Cuminic acid was prepared from oxidation of cuminaldehyde following a reported procedure [21]. For the synthesis of ethyl 4-substituted benzoate (3), a mixture of appropriate 4-substituted benzoic acid 2 (10 mmol), absolute ethanol (20 ml), and concentrated sulfuric acid (2 ml) was refluxed for 3 h. Excess ethanol was distilled under reduced pressure and the resulting oil was rendered alkaline with aqueous sodium bicarbonate and extracted with methylene chloride (2 × 50 ml). The combined organic extract was dried over anhydrous sodium sulfate and distilled under reduced pressure to give the corresponding ester in a yield of 68%. For the synthesis of 4-substituted benzoic acid hydrazide (4), to a solution of ethyl 4-substituted benzoate (3) (10 mmol) in ethanol (10 ml), excess hydrazine monohydrate (5 ml) was added. The reaction mixture was refluxed for 24 h, and left to cool to room temperature. The formed precipitate was collected by filtration, washed with water followed by cold ethanol to remove excess hydrazine and left to dry to give the corresponding acid hydrazide. For the synthesis of 3-substituted-4-(4 substituted-benzyloxy) benzaldehyde (7), to a mixture of substituted benzyl chloride (5) (10 mmol), K2CO3 (12 mmol) and KI (trace amount) in 20 ml acetonitrile, 3-substituted-4-hydroxybenzaldehde (6) was added dropwise under inert nitrogen and stirred overnight, and then evaporate under reduced pressure. The crude mixture was quenched with water and the resulting un-dissolved solid was collected by filtration, washed with water, dried, and re-crystallized from aqueous ethanol to give the titled compound. For the synthesis of 4-substituted benzoic acid [3-substituted-4-(4-substituted-benzyloxy)-benzylidene]-hydrazide UOSC-1–14, a mixture of the acid hydrazide (4) (10 mmol) and appropriate aromatic aldehydes (10 mmol) (7) in glacial acetic acid (6 ml) was heated at reflux for 3–4 h until the reaction was completed. The reaction mixture was concentrated under reduced pressure, cooled and the solid obtained was filtered and crystallized.</p><!><p>The spore-forming fungus, Rhizopus delemar-9980, was maintained on potato dextrose agar (PDA) plates [33]. To test the effect of UOSC compounds on fungal melanin production, a fungal inoculum of 2 × 105 colony-forming unit (CFU)/ml was suspended in PD broth and streaked evenly on a PDA plates. The agar plates were cut into 1 cm discs using cork borer. The discs were suspended separately into a 12-well plate containing 1 ml PD broth mixed with different concentrations of UOSC-1–14 compounds. The plates were incubated for 3 days at 37°C in the dark. Kojic acid and cuminaldehyde were employed as positive controls, while DMSO (the solvent used to dissolve UOSC compounds) was used as negative control. Kojic acid is the common reference used to inhibit melanin production. Cuminaldehyde is the lead compound used as scaffold to synthesis UOSC compounds [20]. The fungal growth was monitored visually until the negative control wells showed the production of fully grown black spores. For melanin quantifications, clear pictures were taken for all wells at the same magnification power. The black color of Rhizopus spores were assessed by setting a 'threshold' of the black color using the tool followed by measuring the intensity of the black color using ImageJ (1.52n for Windows) [22]. In parallel, the fungal hyphae were collected, dried by cheese cloth followed by taking the weight.</p><p>To identify the melanin inhibitory concentration of the newly synthesized compounds, a pilot experiment was conducted. R. delemar spores were incubated in liquid media containing different concentrations (6.3, 12.5, 25, 50, and 100 μg/ml) of UOSC-1–14 compounds and kojic acid. The experiment was repeated at 5, 10, and 15 μg/ml for UOSC or 12.5, 25, and 50 μg/ml for kojic acid. The lowest concentration (10 μg/ml) of UOSC compounds that showed significant inhibition in melanin production and limited toxicity on normal fibroblasts was selected for subsequent experiments. However, 50 μg/ml kojic acid was selected since it is the only concentration caused persistent melanin inhibition activity.</p><!><p>The inhibitory activity of UOSC-1–14 compounds on the diphenolase activity of mushroom tyrosinase was investigated using l-DOPA as a substrate. Mushroom tyrosinase is commercially available from Agaricus bisporus (Cat. # T3824–25KU, Sigma–Aldrich). The spectrophotometric activity assay for tyrosinase was performed according to the previously reported method [23] with modifications. Briefly, 30 units (0.01 mg) of tyrosinase enzyme were pre-incubated with the compounds (dissolved in DMSO) in phosphate buffer (pH 6.8) at concentrations 1, 5, 10, 25, 50, 75, and 100 μg/ml for 20 min at 25°C. l-DOPA solution (0.5 mM) was added to the mixture and the reaction was monitored with the change in absorbance at 470 nm due to DOPA chrome formation. All measurements were in triplicate for each concentration. Kojic acid and DMSO were employed as positive and negative controls, respectively. The inhibition percentage was calculated from the below equation:</p><p>Inhibition ratio (%) = [(B − S)/B] × 100, where B and S are the absorbance for the blank and samples. The IC50 was calculated according to Ismaya et al. [23]. The IC50 values were calculated using GraphPad Prism 5.0 software.</p><p>To evaluate the activity of the developed compounds on R. delemar melanin production, fungal spores were treated with UOSC-1, UOSC-2, UOSC-13, UOSC-14, kojic acid (as positive control), cuminaldehyde (the lead compound), and DMSO as negative control for 3 days at 10 μg/ml. The same weight of the grown hyphae (0.9 g) was grinded using the mortar and pestle method. The total protein of fungal hyphae was extracted using 300 μl triton lysis buffer containing 30 μl protease inhibitor (50 mM Tris, 150 mM NaCl, 5 mM EGTA, 1% Triton-X100) followed by centrifugation at 14 000 rpm at 4°C for 10 min. The supernatant was separated and the extracted protein was quantified using Pierce BCA protein assay kit (Cat# 23227, Thermo Scientific). A pilot experiment was performed using protein extracted from untreated-fungal hyphae in order to determine the optimal concentration of protein and l-DOPA and incubation time. The same protein weight (50 μg) of treated-, control-treated, and non-treated fungi was employed in tyrosinase activity assay as mentioned before. Briefly, fungal protein extracts were mixed with 10 mM l-DOPA in 0.5 M sodium phosphate buffer (pH 6.8) and incubated in dark at 37°C for 3 h. The developed dark color due to melanin production was measured using microplate reader at 475 nm. Data obtained were expressed as mean ± standard error of the mean of three independent experiments.</p><!><p>A series of experiments were performed to determine the inhibition kinetics of compound UOSC-2. The inhibitor concentrations were 1, 2.5, and 5 μM, while the substrate (l-DOPA) concentrations were set at 0.625, 1.25, 2.5, and 5 mM in all kinetic studies. Pre-incubation and measurement time was as the same as mushroom tyrosinase inhibition assay. The tyrosinase inhibition rate was then calculated using Lineweaver-Burk plot using GraphPad prism. The Michaelis–Menten constant (Km) and the maximum reaction velocity (Vmax) were calculated at different concentrations of l-DOPA for 10 min [24].</p><!><p>Melanoma cells line (B16 cells) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 IU/ml penicillin, and 100 mg/ml streptomycin. Cells were passaged routinely and maintained at 37°C and 5% CO2. For each experiment, 5000 cells were seeded into each well of a clear flat-bottom 96-well plate and allowed to adhere for 24 h. The cells were then incubated with the tested compounds at a concentration of 10 μg/ml in triplicate. Kojic acid and DMSO were employed as positive and negative controls, respectively. The plates were then incubated for 24 h, followed by treatment with staining solution (1% aqueous ferric chloride (30 ml) and 1% aqueous potassium ferricyanide (10 ml), combine and mix well) by adding 0.1 ml to each well and stand for 10 min [25]. The excess solution was removed and washed with distilled water twice. An equal volume of phosphate buffer was added and photos using inverted microscope were taken prior to analysis.</p><!><p>The reduction of yellow tetrazolium salt 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was used to measure cellular metabolic activity as a proxy for cell viability [26]. To measure the toxicity of UOSC compounds, fibroblasts (HFF-1) cell lines were cultured until reached 80% confluency. A 96-well plate was seeded with 4000 cells per 100 μl media and incubated at 37°C for 24 h. All compounds at 100 μg/ml were added onto the cells and incubated for further 24 h. Freshly prepared MTT solution (5 mg/ml) was added to each well (20 μl per well) and followed by incubation for 2 h at 37°C. The supernatants were then removed and 100 μl DMSO was added and incubated until formazan violet crystals were developed and the OD540 were measured.</p><!><p>Immunocompetent mice were infected intratracheal with 106 fungal spores according to Andrianaki et al. [6]. The mice were anesthetized by intraperitoneal injection of 0.2 ml mixture of ketamine (82.5 mg/kg, Phoenix, St. Joseph, MO) and xylazine (6 mg/kg, Lloyd Laboratories, Shenandoah, IA). The intratracheal injection of the fungal spores was performed according to Luo et al. [27]. The mice were then euthanized by cervical dislocation at different time points including 4, 24, and 72 h, the lungs were homogenized, and fungal CFU counts were assessed. All animal studies have been taken place at the Lundquist Institute for Biomedical Innovations, Torrance, CA, U.S.A. Animal studies were approved by the IACUC of the Lundquist Institute for Biomedical Innovations at Harbor-UCLA Medical Center and according to the NIH guidelines for animal housing and care.</p><!><p>Peripheral blood was obtained in EDTA vacutainer collection tubes from healthy male and female individuals after signing an informed consent form approved by the Institutional Review Board (IRB) of the Lundquist Institute for Biomedical Innovations at Harbor-UCLA Medical Center. The blood was pooled in a 50 ml falcon tube. Peripheral blood mononuclear cells (PBMCs) were isolated from the whole blood by density gradient centrifugation through over layering 12.5 ml blood over 10 ml Histopaque-1077 (Sigma) followed by centrifugation at RCF 400 for 25 min at room temperature without brakes. Interfaces containing PBMCs were collected, washed in RPMI-1640 (Sigma), counted and seeded into 96-well tissue culture plates at a density of 10 000 cells per 200 μl of RPMI-1640 media supplemented with 10% fetal bovine serum (Sigma) and 1% penicillin/streptomycin (Sigma). At least six wells were employed for each treatment per each experiment. Plates were incubated for 24 h at 37°C and 5% CO2. Supernatants including floating non-monocytic cells were removed by gentle wash with pre-warmed PBS. Spores were added at a final ratio of 2 : 1 phagocyte. Co-cultures were incubated for 2, 5, and 16 h. At the end of each time point, non-phagocytosed spores were gently rinsed away with PBS and the remaining macrophages were incubated with cold water for 30 min to allow the lysis of phagocytes. The obtained lysates from each well were diluted and cultured on PDA (HiMedia) for 24 h at 37°C.</p><!><p>Molecular modeling for the binding of UOSC compounds with either the mushroom or the human tyrosinases was performed according to Hamdy et al. [28] and Debnath et al. [29].</p><!><p>The crystal structures of both mushroom (2Y9X) and human (5M8M) tyrosinases were downloaded from the Protein Data Bank at http://www.rcsb.org. The energy of the system was minimized using MOE 2008.10 (Molecular Operating Environment) available at http://www.chemcomp.com.</p><!><p>The compounds were prepared with MOE 2008.10. The structures of the compounds and the energy minimization were carried out using MOE 2008.10. The placement criteria were adjusted to be MMFF94× force field until RMSD gradient of 0.05 kcal mol−1 Å was reached.</p><!><p>Compounds UOSC-1, 2, 13, and 14 were docked within the binding site of mushroom and human tyrosinases. The placement criteria were adjusted to be Triangle Matcher. Rescoring 1 was selected to be London Gand Retain 10 poses. In our study, we prefer to make refinement with Force field and rescoring 2 was chosen to be London G.</p><!><p>The free binding energies of the ligands with the active sites of tyrosinase enzymes were calculated using PlayMolecule software available at https://www.playmolecule.org [30].</p><!><p>MD was carried out using GROMACS 5.1.2 software package [31]. The topology files of mushroom tyrosinase protein (PDB 2Y9X) were directly created by GROMACS, whereas Acpype software [32] was used to generate topology files of the compounds. Compounds UOSC-1, 2, 13, and 14 complexes and the enzyme alone were immersed in the center of the cubic box with margin of 1 nm, and then the boxes were filled with TIP3P water model. Sodium and chloride ions were added to neutralize the systems, then the complexes were relaxed via energy minimization using the steepest descent minimization algorithm until the maximum force is under1000 kcal/mol/nm. The complexes were equilibrated through two steps, first one was NVT ensemble (constant number of particles, volume, and temperature) for 100 picoseconds ( ps) to stabilize the system at 300 K, and the second step was NPT (constant number of particles, pressure, and temperature) running for 100 ps. Finally, after the equilibration of each system, 10 ns MD simulation was performed.</p><!><p>All procedures involving the use of mice and human were performed in accordance with the relevant guidelines and regulations. The use of mice was approved by the IACUC of The Lundquist Institute for Biomedical Innovations at Harbor-UCLA Medical Center, according to the NIH guidelines for animal housing and care. Human peripheral blood collection was approved by the IRB of The Lundquist Institute for Biomedical Innovations at Harbor-UCLA Medical Center under protocol #11671.</p><!><p>The data were graphed using Graph Pad 5.0 for Windows (GraphPad Software, La Jolla, CA, U.S.A.). The statistical significance was analyzed using one-way analysis of variance (ANOVA) using either Bonferroni's multiple comparisons test or Dunnett's multiple comparison test. P-value < 0.05 was considered significant.</p><!><p>Cuminaldehyde, a natural volatile oil extracted from a plant source, causes reduction of melanin production from R. delemar [20]. Cuminaldehyde has been identified as a non-specific tyrosinase inhibitor [16,19,33]. We have used cuminaldehyde as a scaffold to synthesize a library of compounds (UOSC-1–14) with specific potent and safe fungal tyrosinase inhibition activities, particularly against R. delemar, without affecting the human tyrosinase.</p><p>The newly developed compounds were designed based on the structural comparison of cuminaldehyde to ligands (Figure 1) known to inhibit fungal tyrosinase enzyme. The presence of 3D crystal structure of fungal tyrosinase enzyme with various inhibitory ligands and the help of modeling techniques provided an excellent opportunity for structure-based design of potential candidates to rationalize the structural elements responsible for the selective inhibitory activity of the enzyme. The toxic aldehyde group of cuminaldehyde was replaced with polar hydrophilic acid hydrazide moiety. The lone pair of electrons on N and carbonyl oxygen provided better hydrogen bond formation with the binuclear copper active site of the fungal tyrosinase enzyme. Furthermore, the introduction of the proper lipophilic group with different substitutions influenced the orientation and interaction of the compounds with the hydrophobic residues at the active site of fungal tyrosinase enzyme (Figure 2). The aforementioned designs resulted in the synthesis of novel UOSC-1–14 compounds (Figure 3) with enhanced physiochemical properties and selectively to inhibit R. delemar fungal tyrosinase.</p><!><p>To test for the melanin inhibition activity of the newly synthesized compounds, R. delemar spores were incubated for 3 days in liquid media containing 10 μg/ml of UOSC-1–14 compounds in comparison with 50 μg/ml kojic acid, a general de-pigmentation compound [34]. Out of the 14 compounds, UOSC-1, 2, 3, 4, 10, 13, and 14 showed significant inhibition (One-way ANOVA, P < 0.0001) of R. delemar melanin production (Figure 4A,B). While UOSC-1, 2, 3, 4, 10, and 14 did not reduce the growth of fungal hyphae (weight range = ~33.1 ± 0.6 − 33.5 ± 0.9 mg), UOSC-13 caused significant reduction in the growth of fungal hyphae (weight = 20.5 ± 2.5 mg) similar to kojic acid (Figure 4A). Thus, UOSC-1, 2, 3, 4, 10, and 14 were selected as potent anti-melanin candidates for the R. delemar fungus.</p><!><p>The inhibitory activities of all tested compounds on fungal tyrosinase enzyme were tested in comparison with kojic acid using a mushroom tyrosinase purified from A. bisporus and a fresh total cell protein extract of R. delemar. Although all compounds showed significant inhibition of mushroom tyrosinase activity when compared with kojic acid (One-way ANOVA, P < 0.0001), UOSC-1, 2, 8, 10, 13, and 14 showed the highest inhibition activity (Figure 5A). On the other hand, the inhibitory effect of the developed derivatives on the melanin biosynthesis of R. delemar was the highest with UOSC-1, 2, 13, and 14, kojic acid and cuminaldehyde (One-way ANOVA, P < 0.0001) (Figure 5B,C). Compound UOSC-2 showed the highest inhibition activity (Figure 5C).</p><p>Mushroom tyrosinase shows limited homology to mammalian ones (~30% similarity based on Smith–Waterman sequence alignment, Data not shown) and this renders it as a well-suited model for studies on melanogenesis [23,35]. Thus, any variation in the activities of the tested compounds between both mushroom and human tyrosinases will support the potent inhibition selectivity of UOSC compounds against fungal melanin biosynthesis but not the human, and hence offers minimal toxicity. Consequently, the inhibitory activities of UOSC compounds were tested on human tyrosinase (Cat. # T8455–1MG, Fragment 369–3771 Sigma–Aldrich) and melanoma cells (employed as melanin-producing human cells). Compared with kojic acid, UOSC-6, 7, 8, 12, and 14 showed significant inhibition of human tyrosinase (P < 0.0001) (Figure 6A). However, UOSC-7, 8, 10, 11, 12, and 14 caused significant inhibition of melanin production from melanoma cells (Figure 6B,C). Collectively, UOSC-1, 2, 13, and 14 can be considered as excellent candidates against R. delemar melanin production. Because UOSC-8 showed similar inhibitory activities on both fungal and human tyrosinase, the compound cannot be selected as candidate (Figures 5A and 6A). Although UOSC-10 showed significant inhibition to fungal tyrosinase but not human tyrosinase, the compound caused significant inhibition (P < 0.005) of melanin production from melanoma cells; thus the compound also excluded from the selection. On the other hand, UOSC-14 showed anti-melanin activities against both fungus and human (Figures 5C and 6C).</p><!><p>The toxicity of all developed compounds was tested on mammalian cells using the normal fibroblasts HFF-1 cell line. All tested compounds showed limited toxicity when compared with kojic acid toxicity (Figure 7, P < 0.005). In contrast, the inhibitory activities of all tested compounds to mushroom tyrosinase were tested at seven different concentrations (1, 5, 10, 25, 50, 75, and 100 μg/ml). Although all tested compounds showed variable inhibitory activities to mushroom tyrosinase, UOSC-1, 2, 13, and 14 showed the lowest IC50 of 0.01, 0.0074, 0.0086, and 0.009 μM, respectively (Table 1).</p><!><p>To confirm the selectivity of UOSC-1, 2, 13, and 14 to the fungal (represented by mushroom), but not the human tyrosinase, in-depth docking studies for the interaction of UOSC-1, 2, 13, and 14 within the hydrophobic binding pocket of mushroom tyrosinase (PDB:2Y9X) was performed in comparison with their interactions within human tyrosinase (PDB:5M8M) (Figure 8, Supplementary Figures S1–S3 and Supplementary Information) [37]. These binding interactions were further compared with the interactions of the enzyme with its native ligands, tropolone, and kojic acid. Furthermore, the binding mode and binding energies were investigated using Molecular Operating Environment (MOE) [38] and PlayMolecule [30], respectively.</p><p>Compared with the mushroom tyrosinase enzyme's binding modes with tropolone and kojic acid (Figure 8A), the enzyme's binding mode with UOSC-1, 2, 13, and 14 are buried beside the copper ions active site, while UOSC-13 and 14 are being displaced away by a small distance (Supplementary Figure S1 and Supplementary Information). The four compounds showed π-p stacking with copper ions ligands (His61, His 85, His259, and His263) which may lead to a change in the geometry of copper ions center and a disturbance in the redox reactions of tyrosinase. The four compounds illustrated strong interactions with the key and reserved residues of the hydrophobic pocket site including Hiss244, Val248, and Phe264. Moreover, the binding mode of UOSC-2 (Figure 8B) showed that it can occupy the active center similar to the native ligand. The compound was very close to the copper ions, while the remaining of the compound's structure was extended in the binding pocket, making UOSC-2 as the most promising mushroom tyrosinase inhibitors (Figure 8C) and may explain its superiority as anti-melanin when compared with kojic acid.</p><p>Co-crystallization of kojic acid with human tyrosinase shows a close contact to Zn atom active site in a distance of 3.3–3.6 Å, and a H-bond formed between O-atom of C=O group and the OH of the key residue Ser394 at distance 2.9 Å (Figure 8D). Compared with kojic acid, UOSC compounds including in particular UOSC-2 moved far away from the Zn binuclear active site of the human tyrosinase (Figure 8E,F, Supplementary Figure S2 and Supplementary Information).</p><p>To further predict the possible binding mode for each compound within the pocket site of mushroom tyrosinase, the binding free energy (ΔGBind) and the binding affinity pKd [39,40] were calculated (Table 2). It was found that the four compounds showed binding energies less than the reference compound kojic acid = −5.78 and higher binding affinity than kojic acid (pKd = 4.28). UOSC-2, which showed the best IC50 = 0.0074 μM, illustrated the lowest free binding energy (−9.13 Kcal/mol) and the best binding affinity (pKd = 6.76) (Table 2). This result is in agreement with the concept that a compound with the best IC50 should exhibit the lowest binding energy [40]. The results obtained confirmed that the newly designed derivatives exhibited good binding mode which conformed to their lowest binding energies and high binding affinity than kojic acid. Furthermore, the orientation of the four compounds within human tyrosinase was far away from the active site and this is conformed to the higher binding energies; explaining the selectively of the newly designed UOSC-1, 2, 13, and 14 compounds to the mushroom tyrosinase.</p><p>Molecular dynamics (MD) simulations was carried out to further confirm the accuracy of docking results and to obtain a more accurate ligand–protein binding model in a case close to the natural conditions [41]. Docking of UOSC compounds with the enzyme was tested using MD simulation for 10 ns. The MD simulations of the produced complexes with the mushroom tyrosinase were checked for root mean square deviation (RMSD) to confirm the stability of the protein–inhibitors in the solvent system. The produced complexes showed low RMSD values (0.15 nm) (Figure 8, Supplementary Figure S3 and Supplementary Information). Furthermore, the complex produced from UOSC-2 showed smooth RMSD curves (Figure 8G). The compound reached the equilibrium at 1 ns with ~0.14 nm RMSD which is less than the enzyme alone (Figure 8G). These results indicated that the most stable form of mushroom tyrosinase occurred in the presence of the aforementioned inhibitors in particular compound UOSC-2. Furthermore, the root mean square fluctuation (RMSF) of UOSC compounds–tyrosinase complexes were high (~0.4 nm) with the first 100 residues, indicating the high flexibility of this part. The remaining residues showed low RMSF value (~0.2 nm), particularly the binding pocket residues 200–300 (Figure 8, Supplementary Figure S3 and Supporting Information). The low flexibility of the binding pocket residues indicated the stability of the enzyme due to the presence of inhibitors [29]. Furthermore, the RMSF of UOSC-2 with residues between 240 and 250 in the binding pocket was lower than the protein alone (Figure 8H), which may be attributed to the H-bond between UOSC-2 and His244 and Val248.</p><p>The compactness of inhibitor–enzyme complexes was measured by the Radius of gyration (Rg). The Rg of the four inhibitor–protein complexes was ~2.055 nm and remained steady over the dynamics measurements (Figure 8, Supplementary Figure S3 and Supplementary Information). Studying the H-bonds over 10 ns indicated that UOSC-2, 13, and 14 showed stable and strong H-bonds with the tyrosinase enzyme much better than kojic acid (Figure 8, Supplementary Figure S3 and Supplementary Information). Compared with kojic acid (Figure 8I), UOSC-2 illustrated the most stable and maintained H-bonds (Figure 8J), supporting its superiority against fungal melanin.</p><!><p>The inhibition mechanism of UOSC-2 on mushroom tyrosinase enzyme was determined using l-DOPA as substrate. The relationship between enzyme activity and its concentration in the presence of different concentrations of UOSC-2 showed straight lines pass through the origin. The enzyme inhibition activity due to UOSC-2 is dose-dependent. As the concentration of UOSC-2 increased, the enzyme activity is reduced, while the amount of enzyme is not affected (Figure 9A). Moreover, Lineweaver–Burk plots were generated in order to determine the inhibition type. The plots of the enzyme activity (1/V) versus the concentration of substrate (1/[S]) at different inhibitor concentrations gave straight lines, which all passed through one point. The results indicated that compound UOSC-2 inhibited the diphenolase activity of tyrosinase in a dose-dependent manner [24]. With increasing the concentrations of the compound, Km value increased and Vmax value remained the same, while the enzyme activity was inhibited; confirming that the compound is a competitive reversible inhibitor (Figure 9B).</p><!><p>To determine the potential incorporation of anti-melanin activity of the inhibitors in fungal pathogenesis, we treated R. delemar spores with UOSC-2 at 10 μg/ml for 3 days. The generated white spores were compared for their infectivity with wild-type spores using the intratracheally infected immunocompetent mouse model as previously described [6]. Infection of mice with de-melanized R. delemar spores (Figure 10A) resulted in rapid fungal clearance from the lungs, when compared with infection with wild-type melanized fungal spores (Figure 10B). To discern if the white spores become prone to phagocytosis, peripheral human blood-enriched macrophages were co-cultured with white and wild-type spores (Figure 10A) and evaluated at 2, 5, and 16 h time points. The efficiency of fungal clearance by macrophages was also compared by culturing the macrophages lysates on fungal growth media (PDA) at the end of each time point. In contrast with wild-type black spores which persisted in the macrophages without being killed, white spores were phagocytized more efficiently and cleared faster within ~5 h. No spores clumping observed in association with macrophages co-cultured with white spores (Figure 10C). The growth of intracellular white (albino) conidia following macrophage lysis was significantly inhibited as compared with wild-type conidia (Figure 10D).</p><p>Collectively, the newly designed compounds, particularly UOSC-2, are promising candidates to overcome the persistence mechanism of the pathogenic R. delemar fungus due to melanin.</p><!><p>Fungal melanin exhibits efficient resistance mechanisms against most stressful conditions including human host defensive mechanisms [1]; thus allowing the persistence of the pathogenic fungi and development of pathogenesis mechanisms. Similarly, R. delemar which is the most common cause of mucormycosis (a life-threatening infection), demonstrates unique persistence mechanisms in animal models causing phagosome maturation arrest [6]. Furthermore, it has been reported that fungal melanin can bind to antifungal compounds including amphotericin B and caspofungin, thereby reducing their fungicidal activities [42].</p><p>Cuminaldehyde showed significant inhibition of fungal melanin biosynthesis including R. delemar melanin [20], and has been reported as potent tyrosinase inhibitor [33], but as any aldehyde, it has cytotoxic activities on mammalian cells and cannot be used for clinical applications [43]. Thus, the compound was employed in this study as scaffold to build potent and selective inhibitors against R. delemar melanin biosynthesis. We have successfully generated a series of compounds that showed potent inhibition of fungal melanin biosynthesis and significant inhibition of the rate-limiting melanin biosynthesis enzyme, tyrosinase, and limited toxicity on both melanoma and fibroblasts cell lines. Only compounds UOSC-1, 2, 13, and 14 were selected as promising candidates inhibitors for fungal melanin biosynthesis, since they showed limited effects on human melanin. In contrast with naturally identified anti-melanin compounds including cuminaldehyde and synthetic anti-melanin compounds, the newly designed compounds showed significant selectivity against fungal melanin, particularly R. delemar melanin. This activity may be attributed to the perfect binding of the aforementioned compounds with the fungal tyrosinase active site but not the human one.</p><p>UOSC-1, 2, 13, and 14 exhibited excellent binding with mushroom tyrosinase, as a representative to fungal tyrosinase particularly R. delemar, due to (i) the good binding mode of the compounds within the mushroom tyrosinase active site, similar to the enzyme's native ligand, (ii) the vicinity of the compounds to the catalytic copper ions of the active site and (iii) the interactions of the compounds with the key residues of the binding pocket as the four compounds illustrated strong H-bonds profile and hydrophobic interactions with the hydrophobic residues (Hiss244, Val248, and Phe264). These interactions stabilized the compounds within the active site of fungal tyrosinase. Additionally, the hydrophobic nature of the four compounds facilitates their hydrophobic interactions with the hydrophobic residues in the binding site of mushroom tyrosinase, whereas the active site in human tyrosinase were lining mostly with the hydrophilic residues of the compounds. Therefore, they are displaced away from the binuclear Zn binding site in the case of human tyrosinase.</p><p>The newly designed compounds differ in structures from other reported synthetic and natural anti-melanin compounds including cinnamic acid, thiosemicarbazone, umbeliferone, and resveratrol derivatives (Supplementary Figure S4). The free binding energy (ΔG) of UOSC compounds particularly compound UOSC-1, 2, 13, and 14 with mushroom tyrosinase was lower when compared with the aforementioned compounds except chalcone (Table 1). The results indicated that UOSC compounds and chalcone can strongly and efficiently bind with the pocket site of fungal tyrosinase. In contrast and compared with UOSC compounds, chalcone derivatives showed lower ΔG with human tyrosinase (Table 1), indicating in-selectivity of chalcone derivatives. Therefore, we can conclude that UOSC compounds showed efficient and selective binding capacities to fungal tyrosinase.</p><p>In conclusion, UOSC-1, 2, 13, and 14 showed significant inhibition activities against fungal melanin biosynthesis particularly against R. delemar melanin. The aforementioned compounds showed excellent binding capabilities within the fungal tyrosinase binuclear copper ions active site but not with the human tyrosinase binuclear zinc-binding site. This binding capability was attributed to the hydrophobic feature of the new designs, the substituent that facilitate the orientation of the compounds within the fungal tyrosinase active site, and the formation of hydrogen bond between the compounds and the fungal tyrosinase active site. Our new designs are considered as the first report for compounds that can distinguish between fungal (R. delemar) and human melanin biosynthesis. We propose that the newly designed compounds can be used effectively either alone or in combination therapy to ameliorate fungal infections in particular against R. delemar. Moreover, the newly designed compounds in particular compounds UOSC-1, 2, 13, and 14 can serve as promising lead drugs for not only Rhizopus infection but also other melanin-dependent serious fungal infections such as Cryptococcus.</p><p>Other examined inhibitors such as UOSC-6, 7, 8, and 14 significantly affecting human melanin production, thereby can be developed against diseases with hyper-pigmentation disorders such as actinic and senile lentigines, melasma, and post-inflammatory hyperpigmentation, that are considered as major cosmetic problems.</p>

PubMed Author Manuscript

Conformational Changes Involving Ammonia Tunnel Formation and Allosteric Control in GMP Synthetase

GMP synthetase is the glutamine amidotransferase that catalyzes the final step in the guanylate branch of de novo purine biosynthesis. Conformational changes are required to efficiently couple distal active sites in the protein; however, the nature of these changes has remained elusive. Structural information derived from both limited proteolysis and sedimentation velocity experiments support the hypothesis of nucleotide-induced loop- and domain-closure in the protein. These results were combined with information from sequence conservation and precedents from other glutamine amidotransferases to develop the first structural model of GMPS in a closed, active state. In analyzing this Catalytic model, an interdomain salt bridge was identified residing in the same location as seen in other triad glutamine amidotransferases. Using mutagenesis and kinetic analysis, the salt bridge between H186 and E383 was shown to function as a connection between the two active sites. Mutations at these residues uncoupled the two half-reactions of the enzyme. The chemical events of nucleotide binding initiate a series of conformational changes that culminate in the establishment of a tunnel for ammonia as well as an activated glutaminase catalytic site. The results of this study provide a clearer understanding of the allostery of GMPS, where, for the first time, key substrate binding and interdomain contacts are modeled and analyzed.

conformational_changes_involving_ammonia_tunnel_formation_and_allosteric_control_in_gmp_synthetase

Introduction<!>Experimental<!>Mutagenesis and expression vector preparation<!>Proteolysis and mass spectral analysis<!>Analytical Ultracentrifugation<!>Computational Models<!>Sequence Alignments<!>Kinetic and Stoichiometric Assays<!>Limited Proteolysis<!>Sedimentation Velocity<!>Molecular Modeling of a Closed Structure<!>A Second Closed Model<!>Computational Analyses of the Models<!>Probing the Role of the Interdomain Salt Bridge in the Allosteric Control of the Glutaminase Active Site<!>E383: The Synthetase Component of the Interdomain Salt Bridge<!>H186: The Interacting Residue Adjacent to the Glutaminase Catalytic Triad<!>H186A/E383A: Removing the Interdomain Salt Bridge<!>H186E/E383H: Inverting the Interdomain Salt Bridge<!>Discussion<!>A pathway for ammonia<!>XMP binding site<!>The Role of the Interdomain Salt Bridge in Glutaminase Activation<!>Mechanism of Glutaminase Signaling in GMPS<!>Allosteric Control in Triad Glutamine Amidotransferases<!>

<p>Guanosine monophosphate synthetase (GMPS) catalyzes the final step in the guanylate branch of purine biosynthesis. In this reaction, the glutamine amide is hydrolyzed and the resulting ammonia is incorporated into xanthosine monophosphate (XMP) to form GMP [1]. Glutamine hydrolysis is achieved by the glutaminase domain, a common protein fold that defines the class I glutamine amidotransferases (GATs) [1]. The glutaminase active site contains a catalytic cysteine (at position 86 in E. coli GMPS) and interacts with a histidine and glutamic acid 95 residues downstream [2]. Formation of GMP is achieved in a separate protein domain. A key feature of the class I GATs is tight regulation of glutamine hydrolysis [3]. The glutaminase site is inactive until an acceptor molecule binds and conveys an intramolecular activation signal that stimulates glutamine hydrolysis activity [3–5]. Another distinctive functional component of the amidotransferases is the ability to efficiently achieve interdomain transfer of the nascent ammonia via an intramolecular channel [3, 6]. Reviewing the seven class I GAT structures solved to date, [2, 7–12] a common face of the glutaminase domain docks to the acceptor domains. In many of these proteins, the docking forms a pathway for ammonia that allows sequestration of this reactive molecule from bulk solvent [6].</p><p>As can be deduced by the activity of GMPS, this protein is a metabolically critical enzyme. An adequate supply of cellular nucleotides is critical for nucleic acid production as well as other essential processes of growth and regulation. Cells of the immune system are especially stringent in their requirement of adequate nucleotide pools [13], and targeting the reduction of nucleotide levels in these cells with drugs has been a strategy for immunosuppressive therapies [14–16]. Recent studies of resistance in antimetabolite therapy have revealed a central role for GMPS in the activation of the prodrug thiopurines through similar amination events [17]. Individual resistance to thiopurines has been attributed to genetic polymorphisms in GMPS [18]. GMPS has also been shown to have an allosteric role in the enhanced activation of ubiquitin-specific protease 7, a key enzyme in the regulation of p53 and MDM2 levels [19].</p><p>The crystal structure of E. coli GMPS [2] provided one of the first views of the two-domain catalytic architecture of a glutamine amidotransferase. The enzyme is a homodimer, with two modular active sites and a third, noncatalytic domain involved in homodimerization. Unfortunately, the six structures of GMPS solved to date have only been captured as catalytically incompetent, with a long, solvent-exposed path between the active sites [2, 20–24]. Although ultracentrifugation analysis shows E. coli GMPS to exist predominantly as a homodimer in solution [25], all crystallization conditions have yielded an inactive dimer of dimers, with the active sites widely separated and important regions disordered [26], leaving questions of interdomain communication and ammonia transfer unanswered. The problems elicited by the high protein concentrations occurring under crystallization suggest that alternative, solution-based methods may be required to supplement the findings of x-ray crystallography. To this end, NMR studies have recently been performed using the heterodimer of GMPS from the archea bacterium Methanocaldococcus janaschii. Isotopic labeling of the glutaminase domain identified interface residues that interact with the synthetase domain [27].</p><p>Substrate-induced conformational changes are an important aspect of the activity of many enzyme families, including amidotransferases [28–30]. For example, E. coli PRPP amidotransferase contains a large nucleotide-binding loop that becomes ordered upon binding of substrate and forms part of the ammonia channel [31, 32]. Molecular dynamics studies showed IGP synthase undergoes a hinging motion upon binding of the nucleotide substrate. This domain motion in turn activated glutamine hydrolysis by reorienting the catalytic triad [33–35]. Further analysis using solution NMR techniques revealed the widespread induction of residue motion in IGP synthase focusing in the hinge region of the protein.[36] However, crystallography studies suggested there may be no subdomain motion in IGP synthase but that nucleotide binding may cause a reorientation of side chains at the subdomain interface 15 Å away that allows glutaminase activation [37].</p><p>The solvent-exposed domain organization observed in the six crystal structures of GMPS isoforms suggests that conformational transitions must occur to achieve efficient ammonia transfer. Biochemical evidence suggests that GMPS undergoes structural changes upon substrate binding [38]. Chemical analysis demonstrates that the enzyme adenylates its nucleotide substrate, XMP, after binding [39], and kinetic evidence suggests that ammonia is transferred to the adenylated XMP via an intramolecular path, sequestered from solvent [40, 41]. A conserved loop region nucleotide binding site undergoes a disordered-to-ordered transition [2, 24]. This 24 amino acid loop had been originally shown to be differentially susceptible to trypsinolysis when exposed to nucleotides [42]. More recently, kinetic and spectroscopic evidence supports a model that implicates coupling of the adenylation reaction and activation of glutaminase through a significant conformational change. [38]. The evidence from both biochemical and crystallographic data support the hypothesis that GMPS must undergo a large, substrate-induced conformational change to establish an intramolecular tunnel for ammonia to traverse the distance between active sites [2].</p><p>In the present study, we describe evidence from limited proteolysis, analytical ultracentrifugation, functional analysis of sequence variants, and computational modeling that support a model for a closed catalytically active structure of GMPS. In this closed structure, A conserved loop that was previously disordered in E. coli GMPS was modeled using the ordered density from human GMPS with bound substrate. Consistent with a hypothesis for a general allosteric role of interdomain contacts in controlling triad glutamine amidotransferases [34], an interdomain salt bridge between glutaminase residue H186 and synthetase residue E383 was verified in the closed model. The proximity of these two positions at the domain interface and the active sites implicates a functional role that links the XMP active site to the glutaminase active site. For the first time, a model reveals a plausible path taken by ammonia from the glutaminase to the adenylation site in the catalytically competent form of GMPS and establishes a structural basis for the coordinate binding and allosteric effects on the glutaminase active site.</p><!><p>Chemicals used in these experiments were of research grade or higher, and water was obtained by purification with a laboratory-grade filtration system. Trypsin (product code TRTPCK) and staphylococcal (V8, product code STAP) protease were obtained from Worthington Biochemicals.</p><p>The E. coli GMPS used in the analytical ultracentrifugation experiments was obtained by overexpression in E. coli according to an existing procedure [43]. For the proteolysis, mass spectrometry and mutagenesis experiments, a simplified method was developed by cloning the full-length GMPS gene into pET28 (Novagen), which imparts an N-terminal polyhistidine tag to the protein. The protein is highly purified by single-step purification through nickel sepharose (GE Healthcare Life Sciences), and the histidine tag appears to have no detrimental effect on the specific activity or oligomeric state of the protein (data not shown). This tag was not removed before use.</p><!><p>All site-directed mutagenesis was carried out using Pfu Turbo DNA polymerase with direct mutation in pguaA-tac[43]. Sequence confirmation was performed by the Purdue Genomics Core Facility. The mutated gene was then inserted into pET28 using ligation independent cloning. The LIC compatible expression vector pET-L8 was constructed by introduction of ligation-independent cloning sites and addition of TEV protease recognition site after N-term His tag into vector pET30a (Novagen).</p><p>The pET-L8 vector was linearized by digestion with Ssp1 restriction enzyme (New England BioLabs), followed by gel purification and then treated with T4 DNA Polymerase (Novagen) in the presence of dGTP (New England Biolab). The reaction was incubated at 22°C for 30 min, followed by heat inactivation at 75°C for 20 min..</p><p>Forward (5' TACTTCCAATCCAATGCCATGACGGAAAACATTCATAAGCATCG) and reverse (5' TTATCCACTTCCAATGCTATCATTCCCACTCAATGGTAGCTG) primers for guaA inserts were designed and analyzed for compatibility with each other using the program Clone Manager Professional Suite (Scientific and Educational Software, Cary, NC). Inserts for each of the five unique constructs were amplified by PCR from corresponding template plasmids (pguaA Wild Type (WT), pguaA A-A (H 186A/E383A), pguaA E-H (H186E/E383H, pguaA H186A, pguaA E383A) using the high-fidelity polymerase Platinum® Pfx DNA Polymerase (Invitrogen). The PCR products were treated with T4 DNA Polymerase in the presence of the dCTP thus generating 5' overhangs that are complementary to the 3' overhangs in the linearized and treated vector pET-L8.</p><p>To anneal the treated inserts to treated vectors, 0.02 pmol of each insert reaction mix was incubated with 0.01 pmol of vector in 3 μl reaction mix at 22°C for 10 min. The non-covalent interaction between DNA backbone and insert was stabilized by addition of 1 μL of 25 mM EDTA and incubation at 22°C for an additional 5 min.</p><p>Annealing reactions were directly transformed into X10Gold competent cells (Stratagene) and plated on LB-agar containing Kanamycin (50 μg/ml). Colony PCR analysis was performed to screen for constructs with correct size insert. Plasmids were isolated from selected colonies and verified by sequencing.</p><!><p>For limited proteolysis of GMPS, protease stocks were made at 1 mg/mL in 1% acetic acid (trypsin) or water (V8). The reactions without nucleotides consisted of 25 mg/mL GMPS in 20 mM buffer (for trypsin, EPPS pH8.5; for V8, Tris-PO4, pH 7.8) and 4 mM MgCl2. The nucleotide-containing reactions consisted of the same components, with the addition of 0.25 mM XMP and 1 mM ATP. The reactions were initiated by the addition of protease (4 μg) in a total volume of 100 μL and incubated at 37°C. Samples were removed at specific time intervals and quenched with the addition of AEBSF to 1 mM and acidification of formic acid to 4%. Standard discontinuous SDS-PAGE gels [44] were run on the samples after neutralization with NaOH and denaturation with loading buffer. Molecular weight estimations and band quantification from gels were performed using ImageJ software [45].</p><p>LC/MS experiments were performed with an HPLC (Agilent, model 1100) coupled to an API ion trap mass spectrometer (Bruker Esquire). Separations were performed with 2.1×40 mm C8 columns (Higgins Analytical) using gradient elution. Solvent A consisted of 5% acetonitrile and 0.01% TFA in water; solvent B of 0.01% TFA in pure acetonitrile. Elution was achieved by increasing the percentage of solvent B from 0% to 66% over 45 min. at 0.2 mL/min. The column was directly attached to the API ionization source of the mass spectrometer, and MS spectra were collected over the entire run. Deconvolution of the spectra were performed either manually with the instrument software or with the use of the program MoWeD [46].</p><!><p>Sedimentation velocity experiments were performed with a Beckman Optima XL-I analytical ultracentrifuge using the Rayleigh interference optics. Samples with or without nucleotide contained 25 mM EPPS pH 8.5, 125 mM NaCl, 0.5 mM DTT, and 1 mg/mL GMPS. Samples with nucleotides also contained 1 mM ATP, 0.5 mM XMP, and 4 mM MgCl2, mixed with the protein just prior to the run. Reference solutions were identical to the sample solution except for the presence of protein. Runs were performed at 20° C and 50,000 RPM after an initial 90-minute temperature equilibration period. Interference scans were obtained every 30 seconds for 5 h. The runs were repeated the following day, reversing the sample cells to minimize errors introduced by any rotor asymmetry. SEDFIT was used to generate the necessary files for the program SEPHAT, and each data set was fitted independently using the hybrid fitting algorithm [47].</p><p>The sedimentation properties of model GMPS structures were also calculated with a hydrodynamic bead modeling program HYDROPRO, version 7c [48]. The homodimeric form of GMPS was used in these calculations, as this is the predominant quaternary form in solution [25]. Automatic handling of bead radius was used, as was a value of 1.0 cp for solvent viscosity. Solution density was measured with an Anton Paar density meter.</p><!><p>Modeling of a closed form of GMPS was accomplished with a combination of software for predicting, manipulating, and minimizing the conformation of protein structures. The closed model was based on the existing crystal structure of the protein, with the addition of the 24-amino acid residue loop that was found to be disordered [2]. This loop, hereafter termed the LID loop, was placed in a random, highly solvent-exposed conformation, with the rest of the protein left unchanged. The secondary structure of the LID loop was predicted using the Robetta server [49] using as a query sequence the residues of the loop and the adjacent 25 residues on either side. Only one result both correctly modeled the structure of the adjacent sequence (as provided by the crystal structure of the protein) and secondary structure for the loop that was sterically compatible with the remainder of the protein. This result was incorporated into the model using the portions adjacent to the LID loop as anchors, leading to an E. coli GMPS structure with a complete nucleotide-binding domain, hereafter termed the Catalytic model. Second, the adenylated XMP intermediate was modeled into the nucleotide-binding site of the E. coli GMPS based on aspects of the E. coli 1GPM crystal structure [2]—including the location of AMP product as well as tetrahedral density that was modeled as belonging to the 5′-phosphate of XMP. Third, since the large-scale motions have been predicted to be involved in the conformational changes of GMPS, manipulation of the domain orientation was also necessary. These changes were accomplished primarily with the use of the program ProteinShop [50], which allowed manual manipulation of sections of the protein.</p><p>An assumption guiding this last manipulation was that the primary conformational changes in the protein upon nucleotide binding are experienced by the glutaminase domain. This domain was hinged around the helix marking the end of the domain (residues 210–225) to move it closer to the nucleotide-binding domain. Finally, the structure was energy minimized using Sybyl 7.0 (Tripos, Inc.). For the Catalytic model, cavity analysis and visualization were performed with the programs Voidoo/Flood [51] and CASTp [52]. The probe radius used in the calculations was 1.5 to 2.0 Å.</p><!><p>GMPS sequences were aligned using the sequence alignment program ClustalW (version 1.83) [53] as implemented in the program Bioedit (version 7.0.5.3) [54]. Analysis of conservation of the GMPS sequence was performed with the ConSurf server [55], using the default options for version 3.0 of the software, except for the value of the maximum number of homologues to be considered in the analysis, which was increased from 50 to the entire set of sequences with PSI-BLAST [56] E-values below a cutoff of 0.001. Since amidotransferases consist of modular domains [3], each domain was considered separately in the conservation analysis.</p><!><p>Glutamine-dependent synthetase assays were performed as previously described [57]. Steady-state kinetic assays of GMPS in the presence of ammonium were performed using the conditions described in the glutamine-dependent assays with the substitution of 200 mM ammonium chloride for glutamine. Steady-state kinetics studies of the glutaminase half-reaction or stimulated glutaminase and basal glutaminase activity were performed using a 96-well plate format. The initial reactions were built with a final volume of 100 µL in 96-well PCR plates with each well containing 0.1M EPPS, pH 8.5, 20 mM MgCl2, 2.5 mM EDTA, 0.1 mM DTT, 2 mM ATP, 200 µM XMP and varying concentrations of glutamine (eight replicates each). GMPS amounts used varied depending on the activity of the mutant. The remaining wells were used to establish a glutamic acid standard curve. The plate was incubated at 37°C with 200 RPM shaking for 15 min. and the reaction was quenched by placing the plate into a 110°C sand bath for 1 min. Analysis of formed glutamic acid proceeded as previously described [58]. Basal glutaminase assays differed from the above as follows: water replaced XMP, and the wells containing varying concentrations of glutamine were split such that four did not contain GMPS, while the other four were incubated with the enzyme. This allowed subtraction of contaminating glutamic acid in the glutamine stock solution from the results of each glutamine concentration. Analyses of the reaction stoichiometry were performed as end point assays. To assess GMP formation, we followed the protocol established by Sakamoto [59], a reaction containing 100 mM EPPS, pH 8.5, 1 mM EDTA, 100 µM DTT, 20 mM MgCl2, 2 mM ATP, 200 µM XMP and 20 mM glutamine were incubated for five min.. The reaction was quenched by the addition of 70% perchloric acid to a final concentration of 3.15%. The absorbance of the quenched reaction was measured at 290 nm using an extinction coefficient of 6 × 103 M−1 cm−1 for GMP. Background subtraction was achieved with a duplicate reaction excluding XMP. Glutamic acid formation was measured using the glutamate dehydrogenase coupled reaction analysis, where reaction mixtures, as described above, were incubated and quenched by boiling in a 110°C sand bath for three min., with analysis proceeding as previously described [58].</p><!><p>Limited proteolysis methods have been used to probe for regions of disorder in proteins, including loops [60, 61]. As an approach to probe the structural regions involved in the conformational changes in GMPS upon binding of substrates, limited digestion of the enzyme with trypsin or staphylococcal (V8) protease was performed in the presence or absence of substrate nucleotides, XMP and ATP, plus magnesium. Using the crystal structure data, the substrate specificity of trypsin (arginine and lysine) and V8 protease (aspartate and glutamate) and higher resolution mapping of the cleavage sites under limited conditions provided a structural basis for analysis of the conformational states. The qualitative protection of GMPS from trypsinolysis by substrates has been observed previously. [42], while the findings with V8 protease are reported here. Reverse-phase LC/MS analysis of the limited proteolysis reactions was used to reveal the exact positions of the differential cleavage. Although a number of bands observed in SDS-PAGE were not detected in the LC/MS experiments, key fragments were identified, and was consistent with the conformational model proposed.</p><p>A difference in proteolytic susceptibility between the nucleotide-bound and free states was evident from SDS-PAGE analysis of the time dependent proteolytic reaction samples (Figure 1). Quantification by densitometry of the band for the intact GMPS protein in the trypsin reaction gel also showed an effective stabilization by the addition of nucleotide substrates (Figure S1). A linear least-squares regression curve fit through the log of these data points indicates that the rate of disappearance of the nucleotide-bound form of the enzyme was nearly 10 times more stable against proteolysis than the unliganded form of the protein.</p><p>The clearest indication of the conformational differences induced in GMPS by nucleotides arose from the trypsinolysis reactions. The major protein fragments of GMPS from these experiments separated and analyzed by LC/MS had molecular weights of 19805, 19256, and 18229 Da. The corresponding cleavage sites occur in the C-terminal region of GMPS after lysine residues 351, 356, and 366 in the LID loop (Figure S2). Deconvolution of the LC/MS profiles for these three fragments allowed for their quantification in the reactions as a function of time, and the results are shown in (Figure S3). Importantly, the fragments arising from cleavage at lysine residues 351 and 356 appeared to increase in abundance at the early time interval under conditions where no nucleotides were present (Figure S3a–b). The presence of nucleotide led to the reduction in cleavage at these sites over the same time period. In contrast, there was little difference in the apparent rate of cleavage at K366 as a function of the substrate nucleotide (Figure S3c). The abundance of the resulting 18229 Da fragment was less at each time point when nucleotides were present, consistent with a reduction in the rate of cleavage at K366 when nucleotide was present.</p><p>A comparative analysis was pursued using a protease of different substrate specificity. Figure S3d shows the results of the time course of GMPS cleavage by V8 protease. Distinct differences in the banding patterns were observed depending upon the presence or absence of nucleotide substrates. A band in the V8 gel appeared to accumulate in the reactions with nucleotide only present at relatively low and unchanging levels in the absence of substrates (Figure 1, arrow). Secondary cleavage products are observed at lower molecular weights. Image analysis [45] of the gel band at 36 kDa was consistent with the LC/MS analysis of these digests, which revealed a fragment of mass 35709 Da (data not shown). This fragment was attributed to a portion of the protein (termed the "core fragment") between the V8 cleavage sites at glutamate residues 66 and 390 (Figure S3). While trypsinolysis demonstrated the effect of loop ordering by nucleotide binding, V8 proteolysis demonstrated the possibility for whole-domain motions leading to a compact, protease-resistant GMPS structure. This result was consistent with the resistance to nonspecific proteases or heat inactivation reported earlier [42]. In the presence of nucleotides, the core fragment appeared to remain intact and accumulate even after liberation by V8 cleavage at glutamic acids 66 and 390 (Figure S3d). However, in the absence of nucleotides, the interdomain association appeared diminished, as demonstrated by the lack of accumulation of the core fragment and the presence of lower molecular weight bands in the lanes without nucleotide (Figure 1B).</p><!><p>Sedimentation velocity experiments were performed in duplicate on GMPS in the presence or absence of nucleotide substrates. Apart from nucleotides, the solvent conditions were carefully matched between the two experiments to allow an assessment of sedimentation differences between the two states at an accuracy of +/− 0.1% for such side by side comparisons in the same run [62, 63]. Substrate nucleotides appeared to induce a reproducible 0.3 S difference in the sedimentation of GMPS (Table 1). Since only an approximately 0.07 S increase in sedimentation was predicted from the increase in mass imparted by binding of ATP and XMP substrates alone, without any conformational change (Table 1), the measured increase in S-value implied that the structure becomes more compact upon binding, giving rise to increased sedimentation rate. Moreover, the two major predicted aspects of the GMPS conformational change, loop ordering and domain motion, both appeared to be necessary to achieve the increase in sedimentation rate observed after mixing protein with nucleotide substrates (Table 1).</p><!><p>The crystal structure of E. coli GMPS pdb: 1gpm (Figure 2a) is incomplete for a region of high sequence conservation (Figure S4) at residues 344 to 368 (called the LID loop). The proximity of this loop region to the ATP binding site [2] implicates a functional role in substrate binding. Before any modeling of the missing density could be undertaken, certain protein-substrate interactions needed to be modeled. The crystal structure contained density for AMP in the nucleotide binding domain and this density served as the starting point to create an adenylated XMP intermediate bound in the active site of the synthetase domain. In addition, the protein crystalized in a largely solvent exposed conformation, with the subdomain interfaces several angstroms from each other. Large scale domain motions have been predicted for GMPS The glutaminase domain was therefore manually reoriented such that the glutaminase active-site docked against the nucleotide binding domain, creating an interdomain chamber similar to what is seen in other amidotransferase structures.</p><p>An important constraint for modeling the missing loop density is the resistance of the LID loop to trypsinolysis upon nucleotide binding. Since the loop contains several trypsin cleavage sites that would be highly accessible in the open form, a disorder-to-order transition of this loop is very likely to occur upon nucleotide binding, as seen in the human GMPS structure. Secondary structure for the LID loop was predicted using the Robetta server. Twenty-five crystalized residues on either side of the missing density were included to help verify the model. The model was chosen that showed those residues correctly modeled to reflect their crystalized secondary structure along with sterically compatible density for the LID loop.</p><p>The Robetta model generated for the LID loop is supported by the trypsinolysis data reported above. The two trypsin sites that are observed to be protected by nucleotide from cleavage, K351 and K356, are potentially involved in interactions with the substrate, while the third, unprotected site, K366, is not (Figure S5). The model was then subjected to a molecular dynamics simulations resulting in an energy minimized closed GMPS model (Figure 2b).</p><!><p>After the Robetta structure prediction model was created, a crystal structure of the human isoform of GMPS with density corresponding to the LID loop (E. coli numbering) was released (2VXO). Although the model was consistent with the trypsinolysis results, the critical role for the loop structure warranted further consideration. A model of the closed structure using this human isoform LID loop density was created (see supplemental data for method). In minimizing the model, however, a different force field algorithm was used (limited-memory Broyden-Fletcher-Goldfarb-Shanno (L-BFGS) algorithm in Desmond) (Figure 2c).</p><p>Comparing this second model with the Catalytic model just described, there are significant differences in LID loop interactions with the homodimer-binding domain (Figure 3). As a result, the homodimer-binding domain of GMPS is seen to be drawn away from the XMP binding site exposing the active site and interface domain to bulk solvent as if in product release. Henceforth, this model will be termed the Release model (Figure 3).</p><!><p>Intramolecular ammonia channeling is a recurring theme in amidotransfereases, and these channels are consistently lined with residues that are conserved and largely hydrophobic [9, 64–67]. The Catalytic model of GMPS satisfied these requirements. The domain closure brought the glutaminase active-site cysteine nearer to the nucleotide binding domain by approximately 13Å while also establishing an ammonia path. Analysis with Voidoo [51] and CASTp [52] revealed a large, essentially solvent-excluded cavity (termed the ammonia tunnel) between the adenylated XMP intermediate and the cysteine active site residue of the glutaminase in the Catalytic model structure (Figure 4a). When a 2 Å probe radius was used, no exits from this cavity to the surrounding solvent were found; however when the probe was reduced to 1.5 Å, a small number of exits were found, but they were distal to the path between the glutaminase active site and the nucleotide intermediate (data not shown).</p><p>With the Release model, Voidoo and CASTp analyses showed that the ammonia tunnel, including the interior of the nucleotide binding site was now solvent exposed (Figure 4b), with the nucleotide exiting the active site. These results support the model as showing product release. Consurf [55] conservation scores of the ammonia tunnel-lining residues were high (Figure 4a); the residues facing the tunnel had a propensity toward a hydrophobic or non-polar nature, with the exception of the highly conserved residues that may be involved in nucleotide binding.</p><p>The V8 protease digest data supported the closed nature of the Catalytic model (Figure 2b) When nucleotides were present in the digests, the model positions the glutaminase domain closer to the nucleotide-binding domain, while in the open, unbound form, the glutaminase is more mobile and distant from the synthetase domain. On average, an open form would display more proteolysis sites. The lower molecular weight of the proteolysis product bands in the V8 gel (Figure 1) is consistent with greater susceptibility to cleavage. In contrast, although V8 appeared to rapidly cut at two distal loops in the closed, nucleotide-bound enzyme, the remaining fragment remained resistant to further cleavage, likely due to the stabilization of a closed, compact conformation.</p><p>A final constraint on the Catalytic model came from the data provided by the differential sedimentation velocity experiments. The difference in sedimentation values seen in these experiments provided a basis for evaluation of the overall structure of the Catalytic model. Bead modeling software (HYDROPRO) allowed the calculation of hydrodynamic properties based on model structures to refine and corroborate the structural predictions for GMPS. The enzyme was observed to be a homodimer in solution [25], and the S-values of the closed homodimer model structures (Catalytic model) as given by bead modeling corresponded closely with the values seen in the experiment (Table 1).</p><!><p>The glutaminase active site resides at the interface of the two subdomains in GMPS. In the closed model, a salt bridge was formed between H186 in the glutaminase domain and E383 in the synthetase domain (Figure 5). Histidine 186 is two residues from the glutamic acid (E183) of the catalytic triad in the glutaminase active site. The glutaminase domain is highly conserved in all triad glutamine amidotransferases. Histidine 186 is in the same location as K196 identified in IGP synthase, a residue which has been shown to contribute a key interdomain contact conferring the acceptor substrate binding signal to the glutaminase active site through ionic interaction with a charged residue from the acceptor domain [34]. Similar contacts were identified in the crystal structures of CTP synthetase, FGAR-amidotransferase and anthranilate synthase [34].</p><!><p>Mutation of E383 to alanine disrupted the capacity of XMP to signal glutaminase activity by eightfold (Table 2), with a three-fold reduction in XMP turnover. Stoichiometric analysis of this mutant indicated a moderate uncoupling (2:1) of the two reactions (Table 2). The alanine mutation still allowed basal glutaminase activity as seen by the altered stoichiometry of two glutamine hydrolytic events to one XMP turnover. This result suggests that competency of the glutamine active site in the absence of XMP is enhanced, without disrupting the ammonia transfer.</p><!><p>The H186 residue proximal to the glutaminase active site in the closed model bridges to the acceptor domain through interactions with E383. Mutation to alanine disrupted the Km for glutamine by 50-fold and 34-fold. Any impact upon XMP turnover kinetics was not observed. As with wild type, there was no detectable glutamine turnover in the absence of XMP. The differences in the catalytic efficiency for the glutaminase half-reaction and XMP turnover are consistent with an uncoupling of the two functions (Table 2) as is seen with the 8:1 stoichiometry glutamine hydrolytic events to XMP turnover (Table 2).</p><!><p>To probe the effect of the interdomain salt bridge on the coupling of the two reactions, both residues were mutated to alanine to remove any possibility of non-specific interaction between the two subdomains by these residues. Of all the mutants, H186A/E383A exhibited poor solubility (data not shown). As anticipated, the glutaminase catalytic efficiency was significantly disrupted by 163-fold and 239-fold (Table 2). Basal glutaminase activity was detectable with the H186A/E383A double mutant; however, the degree of disruption of the Km for glutamine (130 mM, Table 2) prevented assays under condition of full saturation, due to limits of the solubility of the glutamine substrate. XMP turnover in this mutant was only slightly disrupted, with a three-fold reduction in the catalytic efficiency. Stoichiometric analysis revealed a significant uncoupling of the two reactions with 15:1 glutamine/XMP turnover (Table 2).</p><!><p>Finally, a "switch" mutant was created, where the amino acids of the interdomain salt bridge were interchanged. If these residues were interacting with only each other, then it was hypothesized this "switch" mutation would have similar kinetics to wild type. Significant disruption in the glutaminase activities for this mutant were observed by 114-fold and 111-fold changes in catalytic efficiency, while the XMP turnover remained relatively unchanged (Table 2). There was detectable basal glutaminase activity; however, the increased glutamine Km again did not allow for full saturation of the enzyme. The stoichiometry of the reactions for this mutant was 26:1, indicating significant uncoupling of the two half-reactions (Table 2). The fact that the results for this mutant were not similar to wild type indicates that these "switched" residues may have additional interactions within the protein structure that are greatly disrupted by changing the electrostatic nature of the amino acid. Taken together, the lines of evidence presented above all support the Catalytic model proposed here showing the active, closed form of GMPS.</p><!><p>The x-ray crystal structure of GMPS provided the first detailed view for both the triad-type glutaminase and N-type pyrophosphatase enzyme classes [2], and this structure was one of the earliest in the broader class of glutamine amidotransferases. However, the data represented an inactive, product-bound form. Key details in the function the enzyme were not directly addressed, including the path of ammonia during catalysis and the binding site of XMP substrate. Six additional structures of GMPS have been solved, and all show the protein in a catalytically unfavorable, solvent exposed conformation [2, 20–24]. The results presented here provide the basis for a closed, active enzyme form that addresses some of these remaining questions.</p><!><p>An important precedent for the modeling of a closed GMPS was the crystal structure of a closed form of E. coli GPATase bound to a nonhydrolyzable substrate analog [31, 64]. In this amidotransferase, a large loop orders upon binding its nucleotide substrate and thereby generates a transient, solvent-excluded tunnel for ammonia. The path taken by ammonia in GPATase is lined with conserved, hydrophobic or nonpolar residues, and ionic or polar interactions are involved in the binding of a nucleotide substrate at one end of the tunnel. In the closed model (Catalytic model) of GMPS, a very similar picture of enzyme function is seen, with a transient, solvent-excluded path being generated by the ordering of a substrate-binding loop and by hinging motions of the glutaminase domain toward the nucleotide-binding domain. Also similar to GPATase, the conservation of the proposed tunnel-lining residues is also high in the GMPS model, with a predominance of weakly interacting residues that would not impede the transmission of ammonia between active sites via hydrogen bonding. Water is thereby excluded from the path in both enzymes by the hydrophobic character of the tunnels which would favor the transfer of ammonia and not ammonium ion. Several GAT structures indicate flexible loops, or subdomain motions that help to shield the glutaminase active site upon ligand binding and create an ammonia tunnel within the protein [68–70]. For example, in 2-amino-2-desoxyisochorismate synthase, the binding of the substrate chorismic acid causes subdomain motions that form a 25Å ammonia tunnel between the glutaminase and acceptor active sites [71].</p><p>An additional section of the proposed ammonia tunnel is defined by the LID loop of the GMPS synthetase domain. While this loop was disordered in E. coli GMPS (1GPM), it was ordered in the human isoform crystal structure. The Catalytic model predicted secondary structure elements for this loop and modeled the loop into the closed version of the enzyme creating key protein contacts between the loop and the homodimer binding region, where two molecules of GMPS interact (Figure 3A). With the Release model, the random loop was defined using density from the human isoform of GMPS, which crystallized in an open form, and since no intramolecular contacts were defined for the loop, the homodimer binding region appears slightly open and more solvent exposed than the Catalytic model.</p><p>The Catalytic model for the closed form of GMPS reinforces the idea proposed earlier [64] regarding the apparent dichotomy in amidotransferase ammonia tunneling. The enzymes that generate a channel during each cycle with a conformational change require a lining of hydrophobic and nonpolar residues to minimize the presence of water in the ammonia tunnel, which could lead to inefficient hydrolysis of substrates. In contrast, proteins with pre-formed ammonia tunnels, such as carbamoyl phosphate synthetase [66], tend to have a reduced requirement for such hydrophobicity and may have other mechanisms in place to minimize the interference of water. Evidence of loop closure is seen in the trypsinolysis data. Domain motion and an increase in compactness are suggested by the V8 protease data, and generated computational models reflect the sedimentation velocity data and provide constraints for all proposed conformational changes in the models.</p><!><p>In the Catalytic model for the closed structure of GMPS, highly conserved residues within the LID loop (Figure S4) are in juxtaposition to the adenylated XMP (Figure 2b). Proteolysis experiments with this protein indicates that the LID loop is protected from proteolytic cleavage when nucleotide is bound, suggesting the LID loop participates in substrate binding. Given the fact that this region is highly conserved in GMP synthetase, it is proposed that the LID loop may participate in substrate recognition. With the Catalytic Model, this study suggests that the loop may participate in the communication between the active sites and facilitate ammonia transfer as the LID loop creates a wall of the putative ammonia tunnel in the interdomain region.</p><!><p>In the closed model, a salt bridge between H186 in the glutaminase domain and E383 on the acceptor domain makes up the outer edge of the ammonia cavity and mutations at these residues may allow bulk solvent access to the chamber. Both members of this salt bridge are found in highly conserved regions of the protein that include residues in each active site. H186 resides in the same location on the glutaminase domain as charged amino acids interacting in interdomain salt bridges identified in other triad glutamine amidotransferases (Table 3). Analysis of the roles of these charged residues in the coupling of the two half-reactions was pursued in IGP synthase, where K196 was mutated and analyzed in turnover kinetics [34] suggesting that K196 forms a key interaction between the subdomains and confers a signal upon acceptor substrate binding to the catalytic triad. In addition, K196 was seen to play a role in the competency of the glutaminase active site to adequately bind glutamine.</p><p>In a recent NMR study of the GMPS glutaminase domain, H186 (H168 in M. jannaschii) was identified as directly interacting with the ATPase domain [27]. Mutation of H186 and E383 in E, coli GMPS resulted in uncoupling of the two half-reactions of the enzyme; however, the degree of disruption (Table 2) indicated that the resulting ammonia may not be completely lost from the interdomain chamber as seen in similar studies with IGP synthase [34]. Mutations at H186 significantly altered the kinetic constants for glutamine in both synthetase and glutaminase half-reactions. Histidine 186 appears to act as a stabilizer of the glutaminase active site, and helps to orient the histidine (H181) and glutamic acid (E183) of the catalytic triad into a catalytically optimal conformation. Conversely, mutations at E383 resulted in moderate disruptions of the two half-reactions. As confirmed with the double mutant, as long as H186 remained intact, the binding signal could still be conferred to the glutaminase active site. Mutations at E383 allowed detectable basal glutaminase activity and indicate a role for this residue in the regulation of glutamine hydrolysis. In this study, only mutants with changes at this position allowed basal glutaminase activity, though E383A only moderately disrupted the glutaminase half-reaction. Adjacent to E383 is the highly conserved D382, which could interact with H186 in the E383A mutation. The side chain of D382 is within 7 Å of H186 in the model structure, and a slight shift of the random loop containing H186 may allow a compensating interaction with D382. Both E383 and D382 reside in a highly conserved region of the protein that begins with K381, which interacts with the pyrophosphate fragment from ATP in the crystal structure of E. coli GMPS (1GPM). A direct linkage to the nucleotide binding site with E383 is observed through this backbone connection (Figure 5).</p><!><p>Loops in the vicinity of the active site cysteine of the triad glutaminases may play a general role in modulating the glutamine hydrolysis activity [68].. The loop in GMPS that gives rise to the oxyanion hole appears to be in a favorable conformation, with the backbone nitrogen of G59 and Y87 positioned to interact with the negative oxyanion of the transition state [2]. These observations led to the hypothesis that the glutamine hydrolytic machinery is poised to perform catalysis, but full glutaminase activity is only achieved by formation of a complete binding site for glutamine [2].</p><p>Inspection of the glutamine binding pocket in the crystal structure of GMPS shows three loops that may be involved in modulation of glutaminase activity, one that provides part of the oxyanion hole (loop 1, residues 59–65), a second that lies on the opposite side of the glutamine binding cleft (loop 2, residues 142–147), and a third that lies more external to the other loops (loop 3, residues 100–108) (Figure 6). Comparison of the GMPS structure with that of the CPS glutamyl thioester adduct [7] reveals possibilities for loop motions that may be required to form a complete glutamine binding site and stimulate glutaminase activity. One possible motion is the orientation of loop 2, which would not provide optimum hydrogen bonding interactions with the alpha-carboxyl portion of glutamine. Another feature could involve an interaction between loops 1 and 3. In the E. coli GMPS crystal structure, an arginine in loop 3 (R106) is involved in hydrogen bonds with the backbone oxygens of P60 and E61 of loop 1, which may "cover" the glutamine binding site and preclude substrate from binding.</p><p>Conformational changes originating in the acceptor domain may also cause shifts of these loops, allowing activation of glutaminase binding site. In the Catalytic model for GMPS, the acceptor domain is within hydrogen-bonding distance of R106, which would allow residues in loop 1 to bind the glutamine substrate. In the Release model, R106 is hydrogen bonding with the phosphate group of GMP (Figure 7). It is plausible that R106 is functioning to help draw the product out of the nucleotide active site. Motion of loop 3 is also supported by the relatively high temperature factors reported in the crystal structure of the enzyme for this loop [2], which suggests high mobility for this segment [72]. Since they are connected via a hydrogen-bonding network, loop 3 may also induce a rearrangement in loop 2, bringing the latter into correct position for hydrogen bonding with glutamine and completing the binding site for the substrate. In this way, the conformational changes initiated by nucleotide substrate binding observed through tryptophan fluorescence analyses [38] may lead to the activation of the distal glutaminase active site by unblocking of the glutamine binding site.</p><!><p>The kinetic analyses described support the unified theory of triad glutamine amidotransferase allosteric control as being conferred in part through a conserved interdomain salt bridge. Direct contact between the two active sites in this family of proteins is achieved at the subdomain interface through ionic interactions between two highly conserved residues (Table 3). Disruption of these interactions results in diminished allostery. The results described here explain many of the features of GMPS that were unobtainable with crystallographic techniques. Biochemical data support a model of large-scale, substrate-induced conformational change that culminates in the fully active enzyme, capable of glutamine hydrolysis and intramolecular ammonia transfer.</p><!><p>This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.</p><p>Notes: The authors declare no competing financial interests</p>

PubMed Author Manuscript

The structure and glycolipid-binding properties of the nematicidal protein Cry5B

Crystal (Cry) proteins are globally used in agriculture as proteinaceous insecticides. They have also been recently recognized to have great potential as anthelmintic agents in targeting parasitic roundworms (e.g. hookworms). The most extensively characterized of the anthelmintic Cry proteins is Cry5B. We report here the 2.3 \xc3\x85 resolution structure of the proteolytically activated form of Cry5B. This structure, which is the first for a nematicidal Cry protein, shows the familiar three-domain arrangement seen in insecticidal Cry proteins. However, domain II is unusual in that it more closely resembles a banana lectin than it does other Cry proteins. This result is consistent with the fact that the receptor for Cry5B consists of a set of invertebrate-specific glycans (attached to lipids), and also suggests that domain II is important for receptor binding. We found that not only is galactose an efficient competitor for binding between Cry5B and glycolipids, but so too is N-acetylgalactosamine (GalNAc). GalNAc is one of the core arthroseries tetrasaccharides of the Cry5B receptor, and galactose an antennary sugar that emanates from this core. These and prior data suggest that the minimal binding determinant for Cry5B consists of a core GalNAc and two antennary galactoses. Lastly, the protoxin form of Cry5B was found to bind nematode glycolipids with equal specificity as activated Cry5B, but with lower affinity. This suggests that the initial binding of Cry5B protoxin to glycolipids can be stabilized at the nematode cell surface by proteolysis. These results lay the groundwork for the design of effective Cry5B-based anthelmintics.

the_structure_and_glycolipid-binding_properties_of_the_nematicidal_protein_cry5b

Introduction<!>Expression, purification and crystallization of Cry5B protein<!>Crystallization, data collection, structure determination, and refinement<!>Purification of upper phase glycolipids from nematodes<!>Glycolipid overlay assay<!>Overall structure of Cry5B<!>Domain I<!>Domain II<!>Domain III<!>Interactions of Cry5B with glycolipids<!>Discussion

<p>Crystal (Cry) proteins are widely used on a global scale as proteinaceous insecticides. These proteins target caterpillars, beetles, mosquitoes, and black flies, but have no effects on higher animals; they also lack the harmful side effects that small molecule pesticides often have on higher animals (1–5). Cry proteins are produced by the Gram-positive soil bacterium Bacillus thuringiensis (Bt), and over 200 varieties of such proteins are known (with sequence identities spanning <20% to >90%) (2). These proteins are produced as crystalline inclusions in sporulating Bt, and following ingestion by invertebrates, are solubilized in the invertebrate gut as monomeric protoxins. Monomeric protoxins are then activated by host proteases, which remove N-terminal and, in almost all cases, C-terminal regions of the protoxins to yield ~60–70 kDa activated Cry proteins. Activated Cry proteins bind specific receptors (2) on midgut epithelial cells and insert into the plasma membrane of these cells to form cytotoxic pores. In some cases, more than one receptor has been identified, and receptor binding has been noted to occur sequentially (6). Receptor binding has also been noted to promote oligomerization of activated Cry proteins, which appears to enhance subsequent pore formation (7).</p><p>The structures of seven insecticidal Cry proteins (Cry1Aa, Cry2Aa, Cry3Aa, Cry3Bb, Cry4Aa, Cry4Ba, and Cry8Ea1) have been determined and are available in the Protein Data Bank (8–14). These proteins have a characteristic compact three-domain architecture. Domain I is an all α-helical bundle responsible for pore formation (5). Evidence suggests that the pore is initiated by the insertion of an α-helical hairpin into the membrane, which is then followed by the insertion of the other helices (15). Domains II and III are rich in β-sheets and resemble lectins of the β-prism fold and jellyroll topology families, respectively (16). Domains II and III have been implicated in receptor binding (6, 17–22), and domain III also modulates pore activity (23).</p><p>Cry proteins are active not only against insects but also nematodes, including those that infest crops or are parasites of animals (24–29). The most extensively characterized nematicidal Cry protein is Cry5B, which has been shown to be a potent in vivo anthelmintic, providing therapeutic effects against the hookworm parasite Ancylostoma ceylanicum in hamsters and the intestinal roundworm parasite Heligmosomoides bakeri in mice (24, 25). The potential use of Cry5B as an anthelmintic has immense implications as intestinal roundworms infect ~2.3 billion people worldwide, and there is an urgent need for new and better anthelmintics (30–32). The receptor for Cry5B consists of a set of invertebrate-specific glycans that are attached to lipids on the surface of intestinal epithelial cells (33, 34). These glycans are composed of an arthroseries tetrasaccharide core (GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glc) decorated with antennary glycans (Gal, Glc, Fuc, and 2-O-Me-Fuc), or single sugars (Fig. S1) (33, 35).</p><p>Due to the importance of Cry5B as an anthelmintic and as a roundworm-active Cry protein (as opposed to an insect-active Cry protein), we undertook to characterize the structure of Cry5B and characterize its glycan-binding properties in further detail. We found that Cry5B is the most structurally divergent Cry protein described to date, with domain II being especially divergent. Domain II most closely resembles a banana lectin, suggesting that this domain is important for Cry5B glycolipid receptor binding. We found that GalNAc acts as an effective competitor for binding between Cry5B and nematode glycolipids, which in combination with prior results, suggests that the minimal binding determinant for Cry5B consists of a core GalNAc and two antennary galactoses. Lastly, we found that the protoxin form of Cry5B binds nematode glycolipids with the same specificity as activated Cry5B, but with weaker affinity. This suggests that the binding of Cry5B protoxin to the nematode cell surface may be strengthened by subsequent proteolysis. Our results provide a foundation for the design of effective anthelmintics based on Cry5B.</p><!><p>Cry5B protoxin was purified from the crystal protein-deficient B. thuringiensis (Bt) strain HD1 that had been transformed with a plasmid encoding cry5B (36). Cry5B protoxin was purified as previously described from spore crystal lysates (24) and stored as a precipitate in water at −80 °C. For experimental manipulations, aliquots of Cry5B protoxin were solubilized in 20 mM HEPES, pH 8.0 at a final concentration of 5 mg/mL. The protein concentration was determined using a calculated ε280 of 162,510 M−1cm−1.</p><p>For expression in E. coli, residues 1–772 of cry5B were cloned with an N-terminal His- tag into vector pQE9, and expressed in E. coli M15. Bacteria were grown at 37 °C to mid-log phase (OD600 0.6–0.8), and expression of Cry5B(1-772) was induced with 0.15 mM isopropyl β-D-1-thiogalactopyranoside. After induction, bacteria were grown at 25 °C for 10 hours, and then harvested by centrifugation (6328 × g, 20 min, RT). Bacteria were lysed by sonication in phosphate buffered saline (PBS), and the lysate was centrifuged (17,418 × g, 20 min, 4 °C). The supernatant, which contained Cry5B(1-772), was applied to a Ni2+-nitrilotriacetic acid (NTA) agarose column, the column was washed with 3 column volumes of NiC buffer (0.5 M NaCl, 20 mM HEPES, pH 8.0), and Cry5B(1-772) was eluted from the column with NiC buffer supplemented with 0.5 M imidazole. Cry5B(1-772) was dialyzed in 50 mM NaCl, 20 mM HEPES, pH 8.0 and concentrated by ultrafiltration to 5 mg/mL. The concentration of Cry5B(1-772) was determined using a calculated ε280 of 108,525 M−1 cm−1. For phase determination, selenomethionine (SeMet) was biosynthetically incorporated into Cry5B(1-772) as previously described (37), and SeMet-labeled Cry5B(1-772) was expressed and purified as above.</p><p>Cry5B protoxin produced in B. thuringiensis and Cry5B(1-772) produced in E. coli were activated by cleavage with elastase (overnight, RT, 200:1 Cry5B:elastase mass ratio) in 20 mM HEPES, pH 8.0. Activated Cry5B was purified by gel filtration chromatography (Superdex 200 26/60) in 200 mM NaCl, 20 mM HEPES, pH 8.0. Activated Cry5B was then dialyzed in 50 mM NaCl, 20 mM HEPES, pH 8.0 and concentrated to 5 mg/mL. The protein concentration of activated Cry5B was determined using a calculated ε280 of 72,825 M−1 cm−1. The site of elastase cleavage was determined by N-terminal sequencing. For this, activated Cry5B was run on SDS-PAGE and blotted onto a polyvinylidene fluoride membrane. The molecular mass of elastase cleaved Cry5B was determined by MALDI-TOF mass spectrometry to be 66,145 Da (calculated 65,815 Da for residues 112–170 and 173–698, the nicked protein that constitutes elastase-cleaved Cry5B).</p><!><p>Crystals of elastase-activated Cry5B produced in B. thuringiensis were grown at RT by the vapor diffusion, hanging drop method. One μL of 5 mg/mL activated Cry5B was mixed with 1 μL of 20% PEG 3350, 0.2 M NaCH3COO as the precipitant. Crystals of SeMet-labeled, elastase-activated Cry5B, which had been prepared from Cry5B(1-722) produced in E. coli, were obtained under the same condition. Crystals of activated Cry5B were soaked in the precipitant solution supplemented with 15% glycerol as a cryoprotectant, before being mounted and cooled to 100 K in a N2 stream.</p><p>Diffraction intensities from native and SeMet-labeled Cry5B crystals were collected at the Advanced Light Source (ALS, Berkeley, CA) beamline 4.2.2 (Table S1). Integration, scaling, and merging of intensities were carried out using Mosflm and Scala (38). Phases were determined by the single anomalous dispersion (SAD) method from crystals of SeMet-labeled Cry5B using Phenix (39), and were also refined using Phenix. Three selenomethionine positions were identified in the asymmetric unit, which contained a single molecule of elastase-activated Cry5B. The initial electron density map clearly showed an α-helical bundle corresponding to domain I and β-sheets corresponding to domains II and III. Residues 187–326 were built automatically with Phenix, and residues 112–161 (corresponding to the α4 helix in domain I) were manually built using Coot (40), as were domains II and III. The partial model encompassing ~450 residues, which was missing the loops connecting the β strands in domains II and III, was then refined against the higher resolution native data set. The electron density map generated from the native data enabled tracing of the rest of the chain, except for residues 171 and 172, which were removed by elastase and are missing in the model. Simulated annealing with torsion angle dynamics was carried out from 3000 K using the slow-cool protocol of CNS (41). Ten cycles of maximum likelihood restrained refinement was subsequently carried out using REFMAC (38), each cycle being followed by manual rebuilding into σA-weighted 2mFo-DFc and mFo-DFc maps using Coot. Waters were added in the later stages of the refinement using Phenix with default parameters (3σ peak height in mFo-DFc maps), followed by inspection of maps.</p><p>Structure validation was performed using Procheck (42) and Molprobity (43). In the final model, 93.1% and 98.8% of residues were in allowed and generously allowed Ramachandran regions, respectively. The final map had correlation coefficients of 0.997 and 0.995 for the main chain and side chains, respectively, as calculated with OVERLAPMAP (38). The Molprobity clash score was 19.4 (61st percentile) and the overall score was 2.57 (55th percentile). The atomic coordinates and structure factors have been deposited with the Protein Data Bank (accession code 4D8M).</p><p>Structure figures were produced with the program PyMOL (44). Structure-based sequence alignments were generated using Expresso (45) and displayed using ESPript (46). Calculations of structural superposition and sequence identity were carried out with Coot.</p><!><p>Caenorhabditis elegans N2 and Pristionchus pacificus PS312 were grown on high growth medium plates seeded with E. coli OP50. Once near starvation, nematodes were harvested from the plates, and pellets of mixed-life stage worms amounting to 0.5 mL were washed three times with water. The pellets were resuspended in three pellet volumes of water and sonicated five times for 2 min each at 11–14 watts, with chilling on ice between sonication steps. Upper phase glycolipids were purified based on the Svennerholm partitioning method (47–49).</p><!><p>The thin-layer chromatography (TLC) assay was carried out as previously described (49). For biotinylation, elastase-activated Cry5B (6 μL of a 2.5 mg/mL solution in 200 mM NaCl, 20 mM HEPES, pH 8.0) was treated with a 3.16-fold molar excess of N-Hydroxysuccinimido biotin in 10 μL total volume of 20 mM HEPES, pH 8.0. After 2 h at RT on a rocker, 90 μL of 20 mM HEPES, pH 8.0 was added. Cry5B protoxin was biotinylated similarly, except that a 22-fold molar excess of N-Hydroxysuccinimido biotin was used.</p><p>Developed HPTLC plates were fixed for 60 s in 40 mL hexanes (mixture of isomers, 99% pure, Acros Organics) and 40 mL hexanes containing 0.02 % polyisobutylmethacrylate. After drying for at least 5 min at 45 °C, the plates were blocked with 9.9 mL of blocking buffer (PBS containing 0.5 % bovine serum albumin and 0.02 % Tween). When monosaccharides were added to compete for binding, the HPTLC plates were blocked in 8.9 mL blocking buffer and 1 mL of 1 M monosaccharide. After 30 min on a rocker, 100 μL of labeled Cry5B was added. After a further 2 h on the rocker, the plates were washed with 10 mL blocking buffer for 1 min and then for a second time for 5 min. To visualize Cry5B, the plates were incubated for 1 h with 40 μL avidin DH and biotinylated alkaline phosphatase H (Reagent A and B, Vectastain ABC-AP kit) dissolved in 6 mL blocking buffer. The plates were washed with 10 mL blocking buffer three times for 1 min each, and then incubated in the alkaline phosphatase substrate NBT/BCIP (30 μL of each NBT and BCIP per 5 mL solution, Vector Labs) for 2–4 h. There were slight variations in the sharpness of bands from experiment to experiment, but the result that Gal and GalNAc blocked binding by Cry5B to glycolipids was consistent throughout.</p><!><p>The structure of elastase-activated Cry5B (residues 112–698) was determined by single anomalous diffraction (SAD) and refined to 2.3 Å resolution limit (Table S1). The electron density map was unambiguous and the entirety of the protein chain was clearly defined, except for residues 171 and 172, which are missing in the final model. Elastase-activated Cry5B is nicked at these residues, and as such is composed of two polypeptide chains (residues 112–170 and 173–698) connected by disulfide bonds. This conclusion was verified by N-terminal sequencing and MALDI-TOF mass spectrometry (data not shown). Although the nematicidal Cry5B protein shares only ~16–19% sequence identity with insecticidal crystal proteins, Cry5B has a three-domain structure that resembles that of insecticidal crystal proteins (Fig. 1). The insecticidal crystal proteins of known structure (i.e., Cry1Aa, Cry2Aa, Cry3Aa, Cry3Bb1, Cry4Aa, Cry4Ba, Cry8Ea1) are highly similar to one another. Their Cα positions vary by 1.0–2.1 Å root-mean-square deviation (rmsd) in pairwise comparisons, except for the most dissimilar member of this group, which is Cry2Aa (pairwise rmsd of 2.5–2.8 Å) (10). By comparison, Cry5B has an average rmsd of ~3 Å in pairwise comparisons with the insecticidal crystal proteins. This indicates that Cry5B is the most structurally divergent crystal protein described to date, in accordance with its differing host tropism.</p><!><p>Domain I is implicated in pore-formation (8), and in accord with its central mechanistic role, domain I is the most structurally conserved of the three domains of Cry5B. Cry5B domain I (residues 112–328), which is composed of a five α-helix bundle, is superimposable on domain I of the insecticidal Cry proteins with an average rmsd of 1.97 Å (153 Cα positions on average) (Fig. 2a), despite this domain sharing only 22% average sequence identity (Fig. 3). The five helices of activated Cry5B match the five helices of activated Cry4B, in agreement with the fact that these two proteins are cleaved at equivalent locations. In comparison, the protoxin Cry2Aa is uncleaved and therefore extends much farther in the N-terminal direction than does Cry5B; thus the five helices of Cry5B match the last five helices (α4–α8) of Cry2Aa. Similarly, some of the activated Cry proteins (i.e., Cry1Aa, Cry3Aa, Cry3Bb and Cry4Aa) are cleaved at positions N-terminal to activated Cry5B, and here again the five Cry5B helices match the last five helices (α3–α7) of these activated proteins.</p><p>The α6 helix of Cry5B is the central helix in the five-helix bundle and is surrounded by the four other helices. This central α-helix is the most conserved of the α-helices in domain I, containing three residues (Leu233, Ala240, and Leu244) that are absolutely conserved among the structurally characterized Cry proteins (Fig. 3). The central helix along with the preceding helix has been suggested to act as a 'helical hairpin' that inserts into the plasma membrane of host midgut epithelial cells and initiates the formation of a cytotoxic pore (15, 50). Mutagenesis studies have demonstrated the crucial role of this central α-helix role in toxicity (2, 51–53).</p><p>There are some small differences between Cry5B and the other Cry proteins in domain I. First, the α4 helix of Cry5B at ~45 residues is unusually long for Cry proteins. Second, Cry5B has two disulfide bridges (Cys163-Cys180, Cys177-Cys186) connecting the long loop between α4 and α5, whereas other crystal proteins lack these disulfides, or as in the case of Cry4Aa, have only one disulfide at this location. The long α4–α5 loop contains the nick generated by elastase, which results in the loss of residues 171 and 172. Residues 170 and 173 apparently shift position after the nicking, as deduced from the fact that these residues are too far apart for the missing residues to span the intervening space.</p><!><p>Domain II (residues 341–541) consists of a β-prism, as found in other Cry proteins, as well as in the jacalin-related superfamily of lectins (54) (Fig. 2b). β-prism domains generally consist of three four-stranded β-sheets, each with a Greek key topology. These sheets are all parallel to an approximate three-fold axis of symmetry and form the sides of a prism. In Cry5B, one of the β-sheets has four long strands (β5β4β3β6), whereas the other two have a mixture of two long and two short strands. These latter two are composed of β1β12β11β2, with β1 and β2 being short, and β9β8β7β10, with β9 and β10 being short; this latter sheet has an a-helix intervening between β9 and β10.</p><p>Domain II is the most structurally divergent of the three domains of Cry5B, with an average rmsd of 3.1 Å (122 average Cα, 8% average sequence identity) as compared to domain II of the insecticidal Cry proteins. A superposition of these domains makes this point clearly (Fig. 2b). In fact, a structural homology search revealed that Cry5B domain II is most similar not to a Cry protein domain II but to a banana lectin (2.7 Å rmsd, 114 Cα, Z-score 9.6, 14% sequence identity) (55) (Fig. 2c). Notably, the structure of the banana lectin (BanLec) has been determined with two different glycans bound, laminaribiose (Glcβ1-3Glc) and Xyl-β1,3-Man-α-O-Methyl (55). Two separate glycan binding sites were observed, both formed by loops at the tips of the β-sheets. The equivalent of the first site in Cry5B consists of the β1β2 and β11β12 loops (emanating from the β1β12β11β2 sheet), and the β3β4 and β5β6 loops (emanating from the β5β4β3β6 sheet) for the second site (Fig. 3). The closer relationship of Cry5B domain II with BanLec as compared to a crystal protein suggests that this domain has a primary role in glycan binding (17).</p><!><p>Domain III (residues 542–698) is a β-sandwich with a jellyroll topology, composed of two four-stranded antiparallel β-sheets (Fig. 2d). The β10β6β12β2 sheet packs against domain II, whereas the β5β11β7β8 sheet faces solvent. The β1 and β9 strands are short and part of the β5β11β7β8 and β10β6β12β2 sheets, respectively. Two additional β-strands (β3 and β4) and two short 310-helices lie rougly perpendicular to the β-sandwich and connect β2 to β5. In contrast to domain II, domain III is structurally most similar to domain III of other Cry proteins (2.2 Å average rmsd, 127 average Cα, 20% average sequence identity). As with other Cry proteins, domain III also has structural similarity to carbohydrate binding modules, such as the one in β-agarase (2.4 Å rmsd, 121 Cα, 15% sequence identity, Z-score 13.0). The structure of β-agarase bound to a hexasaccharide is known (56), and the glycan-binding site in β-agarase is equivalent in Cry5B to the solvent-exposed face of the β5β11β7β8 sheet (Fig. 2d).</p><!><p>Cry5B has been shown to bind several C. elegans glycolipid species (35), all containing a core arthroseries tetrasaccharide (GalNAcβ1-4GlcNAcβ1-3Manβ1-4Glc) and antennary glycans (Gal, Glc, Fuc, and 2-O-Me-Fuc) (Fig. S1). Significantly, interaction of Cry5B with these glycolipids is effectively blocked by one of the antennary glycans, galactose (35), suggesting that galactose serves at least in part as a Cry5B-binding determinant. To further characterize the interaction of Cry5B with glycolipids, we purified polar glycolipids from P. pacificus, a model nematode that is sensitive to Cry5B (29, 57), along with those from C. elegans. The polar glycolipids from these nematodes were separated by thin-layer chromatography (TLC) and visualized by orcinol staining (Figs. 4a and 4g). This staining showed that P. pacificus has a different set of glycolipids than does C. elegans. Next, Cry5B protoxin and elastase-activated Cry5B, which had both been biotinylated, were overlaid on the TLC plates and visualized. As shown previously, activated Cry5B bound to a number of the C. elegans glycolipids, including the B, C, E, and F species (Figs. 4b and S1), and these interactions were inhibited specifically by galactose (Fig. 4c) (35). For P. pacificus, a double band near the bottom of the TLC plate bound activated Cry5B, and this binding was also inhibited by galactose, demonstrating its specificity (Figs. 4b and 4c). The same pattern of interaction was seen with Cry5B protoxin (Figs. 4h and 4i), but these interactions were much weaker than for activated Cry5B. This was deduced from the fact that galactose completely eliminated glycolipid interactions in the case of Cry5B protoxin but only diminished them in the case of activated Cry5B (c.f. Figs. 4c and 4i). These results indicate that proteolytic activation is not required for the interaction of Cry5B with its glycolipid receptors, and does not change the specificity of such interactions. Activation does, however, increase the affinity of these interactions.</p><p>We next asked which other monosaccharides competed with C. elegans and P. pacificus glycolipid binding by Cry5B to get further insight into the binding of Cry5B with its glycolipid receptors. We found that neither glucose nor N-acetylglucosamine (GlcNAc) affected interaction to the same extent as galactose, although there was perhaps a slight diminution with glucose (Figs. 4d, 4e, 4j, 4k). Glucose is at the base of the arthroseries tetrasaccharide core (i.e., attached to ceramide) and is also one of the antennary sugars, and GlcNAc is part of the arthroseries tetrasaccharide core (Fig. S1). Notably, we found that the addition of N-acetylgalactosamine (GalNAc) substantially diminished binding of both Cry5B protoxin and activated Cry5B to the glycolipids (Figs. 4f and 4l). GalNAc is part of the arthroseries tetrasaccharide core and the sugar from which the antennary glycans emanate. These results provide evidence that Cry5B not only interacts with galactose but GalNAc as well.</p><!><p>We report here the first structure of a nematicidal Cry protein, and find it to be the most structurally divergent of the Cry proteins characterized to date. The previously characterized Cry proteins, which are all insecticidal, are highly similar in structure to one another (8, 9, 11–14). Of this insecticidal group, Cry2Aa is the most dissimilar (10), which may be related t the fact that it is toxic against both Lepidoptera and Diptera, while the other insecticidal Cry proteins act against only a single order, either Lepidoptera (Cry1Aa) (9), Diptera (Cry4Aa, Cry4Ba) (12, 13), or Coleoptera (Cry3Aa, Cry3Bb, Cry8Ea1) (8, 11, 14). Cry5B is structurally even more divergent than Cry2Aa, with the largest divergence between Cry5B and the insecticidal Cry proteins occurring in domain II. This domain has been implicated in receptor specificity in other Cry proteins (17, 19–22), and in Cry5B this activity would fit its divergent organismal specificity. In contrast, domain I is highly conserved between Cry5B and the insecticidal Cry proteins, which likely reflects its conserved role in pore formation (8). Domain III of Cry5B also bears a close relationship to insecticidal Cry proteins, although not as great as domain I but greater than domain II. This may reflect a role of domain III in modulating both receptor specificity and pore activity, as has been seen in other Cry proteins (6, 18, 21, 23).</p><p>The structure of Cry5B was modeled several years ago based on the structure of Cry1Aa (58). The modeled structure, however, is dissimilar in detail from the experimental one reported here, with an overall rmsd of 2.8 Å (401 Cα) between the two; the greatest differences occur in domain II (rmsd 3.4 Å, 131 Cα). This is not surprising given our finding that Cry5B is the structurally most divergent Cry protein characterized to date, and that domain II is the most dissimilar of all the domains.</p><p>We found that Cry5B domain II is most similar in structure not to another Cry protein, but instead to the lectin BanLec (55). BanLec is a member of the mannose-specific jacalin-related superfamily of lectins, and binds both glucose and mannose. The structure of BanLec with two different bound glycans is known, with each of these glycans being observed to bind at two separate sites (55). Site 1 is conserved in all lectins, and in BanLec is formed by the β1β2 loop, which contains a GG sequence, and the β11β12 loop, which contains a GXXXD sequence (Fig. 5). Site 2 is specific to BanLec and some related lectins, and is formed by the β3β4 loop, which contains a GXXXD sequence, and the β5β6 loop, with contains a GG sequence. In BanLec, most of the hydrogen bonds to the glycans are through main chain amides, while the Asp of the GXXXD sequence makes the only side chain hydrogen bonds. The equivalent of site 1 potentially exists in Cry5B. The β11β12 loop in Cry5B contains a GG sequence, and the β1β2 loop has a GXXXE sequence, closely approximating the GXXXD sequence (Figs. 3 and 5). These loops are considerably longer than in BanLec and may accommodate longer saccharide chain lengths. The equivalent of site 2 does not appear to exist in Cry5B, as the β3β4 and β5β6 loops lack GG and GXXXD sequences. The conservation of the BanLec glycan-binding motifs in Cry5B domain II provides further evidence that domain II is likely to be responsible for recognizing nematode glycolipids.</p><p>In contrast, domain III of Cry1Ac has been implicated in binding GalNAc moieties that decorate the surface of its putative receptor aminopeptidase N (59, 60). The GalNAc-binding site in Cry1Ac was suggested by mutational evidence to reside on the solvent-exposed face of the β-sandwich fold in domain III (equivalent to the solvent-exposed surface of the β5β11β7β8 sheet in Cry5B). This supposition was based on modeling, as no structure of Cry1Ac is available, despite the fact that Cry1Ac was reported to be crystallized a number of years ago (61). The putative GalNAc-binding site of Cry1Ac may be unique, in that the Cry1Ac residues identified to be involved in binding GalNAc are dissimilar from other Cry toxins, including the highly related Cry1Aa and the more distant Cry5B (59). Additionally, the functional significance of the interaction between Cry1Ac and GalNAc is questionable, as disruption of this interaction fails to reduce toxicity (59).</p><p>It is worth noting that other Cry proteins besides Cry5B are able to bind glycolipids. This includes some insecticidal Cry proteins. Members of the Lepidoptera-specific Cry1A family bind the glycolipids of Manduca sexta and Plutella xylostella (34, 62). This raises the question of why the BanLec-like domain II of Cry5B does not occur in Cry1Aa as well. One possibility is that the interaction of Cry proteins with roundworm and insect glycolipids are quantitatively different, e.g., that Cry5B has developed a more dominant reliance on glycans as receptors. This possibility is in accord with the observation that C. elegans lacking glycolipid receptors is resistant to as much as 1 mg/mL Cry5B (63). Alternatively, there may be a different, as yet unknown, carbohydrate receptor for Cry5B that is present in worms but not insects.</p><p>We also found that GalNAc, a part of the arthroseries tetrasaccharide core, is a competitor for interaction between Cry5B and its glycolipid receptors. The competition experiment required high monosaccharide concentration (i.e., 100 mM), indicative of the much higher affinity of Cry5B for its intact glycolipid receptor as compared to simple sugars. This is also consistent with the observation that Cry5B is effective in vivo as an anthelmintic (24, 25), demonstrating that dietary sugars and glycans do not inhibit it from interacting with its receptor. Along with our identification of GalNAc as a binding determinant, prior results provide further definition. These results have shown that Cry5B binds C. elegans glycolipid band E but not band D (Fig. S1) (34). Significantly, band E has two antennary galactoses attached to the tetrasaccharide core, while band D has only one antennary galactose attached to the tetrasaccharide core. Thus, this prior observation combined with our current results suggest that the minimal binding determinant for Cry5B consists of GalNAc from the arthroseries core decorated with at least two antennary galactoses.</p><p>Lastly, we found that the protoxin and activated forms of Cry5B bind nematode glycolipids with similar specificities but markedly differing affinities. The protoxin form has weaker receptor affinity, suggesting that the receptor-binding site is partially occluded by the N- or C-terminal regions, or both, that are present in the protoxin but absent in the activated protein. Furthermore, these results suggest that an initially weak binding event between the protoxin and glycolipid receptors could be stabilized by subsequent proteolysis of the protoxin on the nematode cell surface.</p><p>In summary, this first structure of a nematicidal Cry protein coupled with biochemical characterization of its interactions with nematode glycolipids have laid the groundwork for detailed dissection of Cry5B function, with the ultimate aim of devising an effective anthelmintic to combat parasitic worms.</p>

PubMed Author Manuscript

Controlled Detachment of chemically glued cells

We demonstrate a chemically-detachable cell-glue system based on linkers containing disulfide bonds as well as functional groups for metabolic glycoengineering and bioorthogonal click chemistry. The artificial cell-cell adhesion can be broken by the administration of glutathione (5 mM), which triggers the degradation of disulfide bonds. Both the gluing and detachment processes are rapid (< 10 min) and minimally cytotoxic.

controlled_detachment_of_chemically_glued_cells

<p>Adhesion between cells is a fundamental process enabling direct cell-cell interaction and tissue formation.1, 2 Cell-cell adhesion in vivo involves complex cell signaling and specific adhesion molecules.3 Recently, there have been interests in developing artificial methods to form cell-to-cell adhesion for biomedical applications. These methods are based on various artificial binding molecules on cell surface, such as avidin-biotin pairs,4 nucleotide with complementary sequences,5 aptamers,6 and antibody dimers.7 Recently, we have developed a robust cell-gluing system based on click chemistry and metabolic glycoengineering.8 The bioorthogonality, specificity, and fast reaction rate of click chemistry based on tetrazine (Tz) and trans-cyclooctene (TCO) allowed the gluing process to be completed in vitro in 10 min and remain stable in vivo in mice. Here, we describe an extension of this method to incorporate a mechanism to break the bonds between glued cells by simple administration of a chemical agent.</p><p>Two different approaches for detachable cell glue systems have been previously reported. Yousaf group used oxime-hydroquinone chemistry that can reversibly degrade via electrochemical stimuli.9 Although this technique is useful for two-dimensional cell patterning, electronic signal requires special plates, such as gold surface, and cannot be easily applied in vivo. Wagner group developed a genetic engineering-based method that forms cell-cell assembly by recombinant fusion protein of dihydrofolate reductase inhibitor methotrexate and disintegrates by treatment of trimethoprim, a bacterial dihydrofolate reductase inhibitor.10 By comparison, our chemical-based technique is simpler and applicable to general situations including in vivo environments.</p><p>As before,8 we used metabolic glycoengineering to introduce chemical functional groups to the cell surface with minimum perturbation on cellular viability and functions.11 After treatment of tetraacetylated N-azidoacetyl-d-mannosamine (Ac4ManNAz) to A549 cells, azide groups are generated on the cell surface (Fig. S3 in ESI).12 In this work, we used novel rationally-designed crosslinkers, dibenzocyclootyne disulfide tetrazine (DBCO-SS-Tz) and dibenzocyclootyne disulfide trans-cyclooctene (DBCO-SS-TCO), which have two functional groups for click chemistry—Tz and TCO—and degradable disulfide (SS) bonds in their backbone (Fig. S1 and S2 in ESI). The azide-modified cells are treated with these crosslinkers, so that the crosslinkers are conjugated to the azide groups by azide-DBCO click chemistry, and Tz- or TCO-modified cells are prepared;13 When the Tz-modified and TCO-modified cells are mixed together, cell gluing between these two cell groups is established by Tz-TCO click chemistry (Scheme 1). The disulfide bonds in the backbone are cleaved by glutathione (GSH).14 Therefore, the detachment of the glued cells can be achieved simply by administration of GSH (Scheme 1).</p><p>To determine the optimum concentration of GSH, we first evaluated the intrinsic toxicity of GSH on cells. A549 cells were treated with various concentrations of GSH and a cell viability assay (Prestoblue™) was performed. We found GSH at concentrations of 10 and 20 mM decreased cell viability significantly, but concentrations below 5 mM had negligible effects on cell viability (Fig 1a). Based on this result, we determine 5 mM to be the maximum concentration for non-toxic detachment.</p><p>To evaluate the efficiency for cleaving linkers, we added Cy3-TCO conjugates to the culture media of Tz-modified cells. The fluorescent probes are bound to the Tz groups on the cell surface, and the fluorescence intensity from Cy3 bound on the cell surface indicates the amount of Tz groups. We treated these cells with GSH at three different concentrations, 0.1, 1, and 5 mM. After washing the cells, the Cy3 fluorescence was measured. As expected, the fluorescence intensity decreased with the concentration of GSH (Fig. 1b). This supports the mechanism that the administered GSH breaks disulfide bonds and thus reduce the total amount of linkers on the cell surface. At a concentration of 5 mM, the Cy3 fluorescence intensity decreased to about 16.4 % compared to the control group without GSH treatment (0 mM). This indicates that at this condition the glue strength between cells would be degraded by a factor of 6. We obtained similar results with TCO-modified cells by using DBCO-SS-TCO crosslinkers and Cy3-Tz as fluorescent probes (Fig. S4 in ESI).</p><p>To analyse cell gluing strength, we cultured GFP-expressing A549 cells in a monolayer in a microfluidic chamber, and treated them to Tz-modified cells with the protocol described above. To produce glued cells, we added RFP-expressing TCO-modified Jurkat T cells in suspension into the chamber and incubated for 10 min. We injected PBS into the fluidic channels at different flow speeds and measured the number of TCO-modified Jurkat T cells that remained bound to the Tz-modified A549 cells adherent on the chamber under fluorescent microscopy. After applying flow at a speed of 60 ml/min for 10 min, we found about equal number of T cells to be attached on the A549 cells (Fig 2a), whereas almost all non-modified Jurkat T cells in a control group were washed away by the flow (Fig 2e).</p><p>We performed this flow assay on glued cells after GSH treatment at a concentration of 5 mM and found that the ratio of remaining Jurkat T cells to adherent A569 cells decreased significantly to about 10%; that is, about 90% of initially glued T cells were detached and washed away by the flow at a speed of 60 ml/min (Fig. 2b). At a reduced flow speed of 1 ml/min, 70% of T cells were washed away (Fig. 2c). These data show the effect of GSH on the degradation of the bonding strength of the glued cells.</p><p>To confirm that the mechanism of detachment is due to the GSH-induced breakage of the disulfide (S-S) bonds in the linkers, we prepared TCO-modified Jurkat T cells with DBCO-TCO (no disulfide bond) and glue the cells with A549 cells modified with DBCO-SS-Tz (Fig. S5 and S6 in ESI). In this case, there is only one S-S cleavage site in each cell-cell linker, as opposed to the double S-S bonds in the previous linkers. After GSH treatment and flow at 60 ml/min, we measured a remaining cell ratio of 45%, indicating only ~55% of the glued Jurkat T cells were washed away (Fig. 2d). It shows that two S-S bonds are advantageous for cleavage of the linkage and detachment of cells compared to one S-S bond. These results also evidence that the detachment involves the degradation of S-S bonds by GSH.</p><p>To evaluate our system for suspension cells, we prepared two groups of Jurkat T cells and modified them with DBCO-SS-Tz and DBCO-SS-TCO, respectively. After 10 min of incubation, fluorescence microscopy was used to confirm selective attachment of Tz- and TCO-modified Jurkat T cells (Fig. 3a). In flow cytometry, the ratio of double-positive counts was measured to be 57% (Fig. 3b). After incubating with GSH (5 mM), the ratio of glued cells decreased to 6.4% (Fig. 3b), indicating that nearly 90% (6.4/58) of the glued cells were dissociated by the GSH treatment.</p><p>After incubating with a cell viability probe, calcein AM, a cytometry analysis showed that 91% cells were alive after gluing, and that 87% cells remained vital after the gluing and de-gluing processes (Fig. 3c). This data confirms the low cytotoxicity of the detachable cell glue system.</p><p>In summary, we have demonstrated a rapid, efficient, non-toxic, artificial gluing and controlled detachment of cells by click chemistry and chemically-degradable bonds. In this study, we used disulfide bonds as the cleavable site in the cell-cell linkers and GSH as the chemical triggering agent. There are many other chemical bonds developed for degradation15 by various stimuli, such as chemicals,16 light,17 pH,18 or enzymes.19 Our scheme may be extended to different embodiments optimized for specific applications.</p>

PubMed Author Manuscript

The periodic transient kinetics method for investigation of kinetic process dynamics under realistic conditions: Methanation as an example

Due to rising interest for the integration of chemical energy storage into the electrical power grid, the unsteady-state operation of chemical reactors is gaining more and more attention with emphasis on heterogeneously catalyzed reactions. The transient response of those reactions is influenced by effects on different length scales, ranging from the active surface via the individual porous catalyst particle up to the full-scale reactor. The challenge, however, is to characterize unsteady-state effects under realistic operation conditions and to assign them to distinct transport processes. Therefore, the periodic transient kinetics (PTK) method is introduced, which allows for the separation of kinetic process dynamics at different length scales experimentally under realistic operation conditions. The methodology also provides the capability for statistical analysis of the experimental results and therefore improved reliability of the derived conclusions. Therefore, the PTK method provides the experimental basis for modelbased derivation of reaction kinetics valid under dynamic conditions. The applicability of the methodology is demonstrated for the methanation reaction chosen as an example process for heterogeneously catalyzed reactions relevant for chemical energy storage purposes.

the_periodic_transient_kinetics_method_for_investigation_of_kinetic_process_dynamics_under_realistic

Introduction<!>Terminology<!>Aperiodic step<!>Challenges for unsteady-state experiments<!>Experimental Setup<!>Implications of an internal and external standard<!>𝑛̇𝑖 ,out (𝑡) 𝑛̇I S,out (𝑡)<!>State space plot<!>SS II SS I<!>Derivation of the average limit cycle from experimental data<!>Calculation of transient molar flow rates<!>Experimental procedure for demonstrating examples<!>Examples for method application 4.1 Example I: CO2 Adsorption<!>Example II: CO methanation<!>Conclusion<!>Symbol Description Unit<!>𝐹

<p>The transition from a fossil-based energy sector to an energy supply based on renewables leads indispensably to fluctuations in the energy production [1][2][3]. In order to dampen these fluctuations, buffer systems need to be integrated into the energy supply chain, which are based on storing electrical, chemical or physical energy. One attractive option for chemical energy storage is the Power-to-Gas processes (PtG), which allows to convert electrical into chemical energy by electrolysis yielding hydrogen. Subsequently hydrogen can be further converted with carbon oxides into methane, which can directly be stored and distributed in the existing natural gas grid [3]. Due to cost and transport reasons efficient local PtG units are beneficial, allowing an implementation in proximity of the energy and carbon source [3][4][5]. In the same vein cost-efficient local PtG units are equipped with small buffer capacities to dampen feed fluctuations, which limits the possibility to ensure a steady-state operation of the chemical reactor. Hence, fluctuations of the power supply propagate into the chemical reactor and an unsteady-state operation of the methanation reactor is necessary [1][2][3].</p><p>Therefore, an efficient and safe operation of the methanation reactor is required under unsteady-state conditions, which needs a deep understanding of the underlying kinetic process dynamics at different length scales [1]. The challenge becomes evident considering the fact that the underlying balance equations for describing the reactor behavior lead to a system of non-linearly coupled ordinary differential equations, even for the most simplifying assumptions. More realistic assumptions even lead to a system of partial differential equations, e.g. for distributed systems. On top of this the reactor has to be distinguished into three different scales, whose intrinsic dynamic behavior is rather independent of each other [6,7]. For illustration the reactor dynamics at the reactor or macro scale is determined by the fluid dynamics and usually expressed by the residence time distribution (RTD). At the particle or meso scale diffusion processes between the bulk flow and the external catalyst surface, as well as within the porous system affect the dynamics. At the catalyst surface or micro scale, the sorption and surface reaction processes determine the dynamics. Therefore, the characteristic steps in heterogeneously catalyzed reactions, i.e. film and pore diffusion, ad-and desorption of reactants as well as surface reaction, can directly be linked to the length scales and dynamic behavior. Under realistic operating conditions the dynamic reactor response is an interplay of all those kinetic processes. This leads to a severe challenge for determination of the intrinsic dynamics at each individual length scale from the cumulative reactor response. Therefore, the scales are usually studied separately from each other, which requires sophistically designed experiments, though, in order to avoid mutual interaction.</p><p>For the investigation of the processes at the catalyst surface under fluctuating feed gas mixtures several experimental methods already exist. Happel et al., Bennet and Biloen et al. developed the steady-state isotopic transient kinetic analysis (SSITKA) method [8][9][10], which is capable to determine the exchange rate and surface coverage of individual species [11]. Therefore, the isotopic composition of the reactants is changed, while the catalyst surface is assumed to remain in a quasi-steady-state [12]. Im-portantly, this assumption means that the reactions rates are unaffected by the transient change in isotopic composition and thus remain in steady-state. Additionally, an inert tracer is usually used to correct the residence time distribution of the reactants within the reactor [11]. The capabilities and limitations of SSITKA as well as modeling approaches were summarized by Ledesman et al. comprehensively [13]. Temporal analysis of products (TAP) measurements introduced by Gleaves et al. are capable to extract kinetic information by avoiding changes of the catalyst surface during the experiment [14,15]. Therefore, small amounts of reactants compared to the number of active sites are pulsed into the system. The catalytic system experiences only small perturbations and the catalyst surface remains rather unchanged during the experiment [14][15][16]. As these measurements are conducted with small catalyst particles under vacuum atmosphere only Knudsen diffusion determines the RTD. Thus, intrinsic kinetic data of the reaction network can be received, with the limitation that the system is far from realistic conditions. Another dynamic method introduced by Mori et al. [17] exploits the benefits of single pulses applied to a reaction system. A defined number of molecules is pulsed into a carrier gas stream and the response signal is either analyzed with respect to the propagation in the falling edge [17] or with respect to an internal standard. The latter was used by Friedland et al. to investigate the storage capacity of a catalyst under dynamic conditions [18,19]. These methods, however, need welldefined conditions for meaningful results and are based on a limited degree of deflection of the catalyst from its original state for a small period of time.</p><p>The chemical transient kinetics (CTK) method is based on a step-shaped change in the feed gas mixture and "track of the construction (build-up) or scavenging (backtransient) of the catalytically active phase as a function of time" [12]. Therefore, the gas phase composition upon step-change is measured as function of time and linked to ad-and desorption processes between the catalyst surface and the gas phase. Since an internal standard is used as well, the RTD of the reaction system can be measured in-situ, which allows to separate the RTD and the dynamic processes at the catalyst surface in the measured signal [20][21][22]. Furthermore, an external standard allows for derivation of quantitative information, as well [12]. Finally, CTK provides the possibility to derive the coverage degree as function of time using the "surface atom counting technique" [12], which basically corresponds to the elemental balance of the gas phase. Interestingly, CTK has been applied successfully to CO hydrogenation reactions, such as Fischer-Tropsch (FT) synthesis [23], exhibiting the capability of deriving mechanistic insights and even information on the chain growth probability [24,25]. The method, however, is demonstrated for ambient pressure only [12], which is identified as an important limitation by Athariboroujeny et al. [25], in particular for deducing the effect of operating pressure on the mechanistic pathways in FT reaction. Recently, Chen et al. applied a combination of CTK and SSITKA to deduce the mechanism of CO methanation and Fischer-Tropsch reaction for cobalt catalysts [26,27]. Consequently, all methods described above are used to analyze the dynamic processes at the catalyst surface (micro scale) by eliminating the transport processes at macro and meso scale, but all are limited to model conditions (i.e. ambient operating pressure or below). An experimental methodology applicable to realistic conditions and capable to link the transient response of real reactors with the dynamic processes at all length scales, is thus still lacking.</p><p>In the present contribution we therefore introduce the method of periodic transient kinetics (PTK), which allows the investigation of kinetic process dynamics at different length scales separately of each other. It is based on the CTK method recently emphasized by Raub et al. [12], but provides some important improvements. First of all, periodic step-changes are performed in order to enhance the reliability by statistical analysis of the limit cycle. Secondly, the PTK method is demonstrated at higher pressures, which allows to close the gap between model and realistic conditions in future. We focus on a detailed introduction of the experimental implementation and the quantitative evaluation approach of the molar flow rates derived from the measured gas phase composition as function of time. The applicability of the PTK method will be demonstrated for the methanation reaction as an example for heterogeneously catalyzed gas phase reactions but can also be transferred to more complex multiphase reactions, such as FT synthesis. Furthermore, the methodology is flexible to be applied to various reactor types.</p><p>Since the present contribution focusses on the introduction of the methodology, it is thus based on comprehensive explanations of the relevant terms, as well as procedures for experimental conduction and data evaluation. We therefore structured the paper as follows: Because the terms used for unsteady-state reactor operation are often used inconsistently, we define and classify dynamic processes and the related terminology first. In section 3 we discuss the challenges related to the performance of dynamic experiments under realistic conditions and describe the developed methodology in detail, which comprises the experimental setup and procedure, as well as the respective model-based data evaluation. Finally, the methodology is demonstrated for two examples in the context of the methanation process in section 4, namely the CO2 adsorption on Al2O3 and the unsteady-state CO methanation reaction using a Ni/Al2O3 catalyst. The results of the latter example are briefly discussed in comparison to literature findings for illustration of the potential of the PTK methodology.</p><!><p>In the field of dynamically operated chemical reactors and unsteady-state processes a variety of different terms are required, in order to precisely correlate the experimental observations with the underlying mechanisms. For sake of clarity those terms relevant to the PTK methodology introduced in the present study are briefly defined below. The PTK method can be classified as a forced unsteady-state process according to Matros et al. [28], since the unsteady-state behavior is induced by an external fluctuating input signal. In particular, a step-shaped change in the feed molar flow rate is used as input signal in the present study. According to the scope of the study, the system is investigated for examples in the context of the methanation reaction either by modeling and simulation or by experiments. Therefore, self-oscillating systems and other types and shapes of forced input signals are not in the focus here, but comprehensively summarized by different authors [28][29][30][31][32].</p><p>A dynamic process is characterized by time dependent state variables, such as temperature or concentration, of the inlet and outlet streams, as well as of the reactor volume. Note that for forced unsteady-state reactor operation the inlet stream is usually used as the forced input, while the outlet stream is referred to as the reactor response. Each state variable can either be in steady-state or in unsteady-state, which is distinguished by the time derivative being zero or non-zero, respectively. Upon disturbance at time zero a state variable deflects from the initial state followed by a transient phase [31], which depends on the type of the disturbance and the dynamic behavior of the process. Therefore, the term 'unsteady-state' is defined strictly mathematical, while 'transient behavior' defines the trajectory of the variable in time.</p><p>The type respectively periodicity of a temporal change is usually classified as follows (for illustration of different system behaviors see Table 1): An aperiodic signal is characterized by a transient change of a variable from an initial to a final steady-state. A periodic signal, instead, changes from the initial state to a second state and finally reaches the initial state again, which can be repeated multiple times. Note, that the response induced by a consecutive positive and negative step change, such as applied in the CTK method [21,22], is designated with the terms build-up and back-transient phase, respectively.</p><p>The periodic operation allows the system to reach a limit cycle (LC), which is characterized by an invariant behavior for each period [33] and which is particularly advantageous for the evaluation of the dynamic response [18]. It has to be mentioned that the term LC refers to the response of a system only and characterizes its periodicity. The characteristics of a LC depends on the shape and cycle period 𝜏 (inverse frequency) of the input signal, as well as on the characteristic relaxation time 𝜏 res of the system, which can be determined from the system response [34,35]. According to literature [34][35][36] three types of the response being in a LC can be distinguished with respect to the 𝜏 /𝜏 res ratio. For large 𝜏 /𝜏 res ratios the response is in quasi-steady-state (qss), which means that the system response is fast compared to changes in the input signal. The system, thus, is nearly in a steady-state depending on the current input signal at each point in time. In the opposite case of small 𝜏 /𝜏 res ratios the relaxed steady-state (rss) is reached, which is characterized by fast changes of the forced input and a slow reaction by the inertial system. This leads to diminishing amplitudes of the response with increasing frequencies of the forced input. For high frequencies the system response even appears to be in steady-state, though it is still changing with time, since the response amplitude is smaller than the measurable threshold. Note that the apparent steady-state may differ from that under true steady-state conditions [34]. The fulltransient state (fts) is reached for 𝜏 𝜏 res ⁄ ≈ 1 between both boundary cases, where the response depends strongly on the applied frequency. Interestingly, the information content is high in this region [34], which makes it attractive for fundamental research. The main challenges arise from the strong non-linear relation between input and response, which results in a high complexity of the experimental procedure and data evaluation. Furthermore, the applied frequencies to reach the fts region can be quite high, which requires a high temporal resolution of the analytical system and demanding switching for periodic input signals.</p><!><p>Response Literature [37][38][39][40][41][42][43][44] [22, [45][46][47] Periodic step Response Literature quasi steady-state (𝜏 ≫ 𝜏 res ) [48][49][50][51] full transient-state (𝜏 ≅ 𝜏 res ) [47,[51][52][53][54][55][56][57] relaxed steady-state (𝜏 ≪ 𝜏 res ) [51,55] Table 1 illustrates the classification of forced input signals according to the periodicity and indicates references to the application in the context of the unsteady-state CO and CO2 methanation. The aperiodic step changes are mainly used to investigate the transient behavior of the reactants and to gain further insights into the underlying surface processes and reaction mechanisms [22,37,43,53,58]. The forced periodic operation, though, has been investigated for various reaction systems in order to achieve higher conversion [28,29,32,[59][60][61][62] or selectivity [29,63], while an increase in catalyst stability [32] has been reported for different reaction systems, as well. With emphasis on methanation experimental studies on forced periodic operation are performed aiming at kinetic investigations [47,53], as well as process intensification for CO [47,49,57] and CO2 [54,64] methanation. In addition to that theoretical approaches are introduced for the evaluation of the higher frequency response [56,61,65]. All these studies concentrate on the qss or fts region as the information content is high and experimental realization is feasible with reasonable effort.</p><!><p>With emphasis on periodic step-shaped experiments for the methanation reaction following real effects contribute to the observed signal and have to be considered in design of experimental procedures and data evaluation. At the macro scale an ideal step change in the composition is hardly possible, due to the duration of the switching event between two compositions. Furthermore, the RTD in the piping between the switching valve and the catalyst bed, as well as between the catalyst bed and the detector of the analytics may deviate from ideal plug flow behavior and thus affects the intrinsic response of the catalyst bed. In particular, laminar flow profiles can be expected given by the small diameters of the piping in lab-scale equipment and thus small Reynolds numbers. Finally, complex real effects on the RTD within the catalyst bed render the separation of the dynamics at each characteristic length scale more difficult. Specifically, axial and radial gradients in composition, flow rate and pressure induce dispersion effects and thus deviation from ideal plug flow behavior. The pressure drop over the fixed-bed differs for different feed gas compositions, as well. For methanation, in particular, the chemical reaction also causes volume contraction and thus changes in the volumetric flow rate can occur. At the meso scale heat and mass transfer limitations between the fluid bulk and the external catalyst surface, as well as within the porous pellet, lead to the formation of pronounced temperature and concentration profiles, which superimpose the dynamic behavior of the processes at the micro scale. At the micro scale the main challenges arise from slow dynamic processes, such as catalyst deactivation, which have to be separated from the very fast processes involved in the chemical surface reaction. In addition to that chromatographic effects may occur at all scales upon step-changes in the inlet composition [22,66,67].</p><!><p>The experimental setup is adapted from the CTK method [20][21][22]45,46]. A flowsheet of the experimental setup is shown in Figure 1 and the corresponding 3D model in Figure 2. Note, that the colors given in brackets behind the described parts of the experimental setup below correspond to the colors in Figure 1 and Figure 2. The setup is equipped with two independent gas lines (red and blue). By means of a pressure actuated high speed valve (Fitok, BOSS-4C) (orange) one gas line is fed into the reactor (green), while the other is directly injected into the bypass line (grey). With the high speed valve the switching process takes less than 0.2 s, which is below the temporal resolution of the MS analytics (0.5 s, see below). This provides a nearly ideal step change (see Appendix, Figure S9). The gases H2 (5.0 purity, MTI), CO/Ar (90 vol.-% CO of 3.8 purity in Ar of 5.0 purity, Air Liquide), CO2 (4.8 purity, MTI), Ar (5.0 purity, MTI) and He (5.0 purity, MTI) are supplied via mass flow controllers (MFC) (EL-FLOW Prestige, Bronkhorst), without further purification. Note, that Ar is used as an internal standard (IS) for all experiments.</p><p>Figure 1: Flowsheet of the experimental setup to investigate heterogeneously catalyzed gas-phase reactions under unsteady-state conditions (color code is used for explanation in the text and corresponds to Figure 2).</p><p>All lines consist of 1/8" tubing (inner diameter 2 mm) in order to minimize the volume of the reactor periphery. The section between the switching valve and reactor exhibits a volume of 3 mL, only and is equipped with an additional analytic port directly at the reactor inlet (light green). This allows to measure the deviation of the switching event from an ideal step and thus the apparent RTD of the switching event, which is considered for the data evaluation (see Appendix, Figure S9).</p><p>The experiments are conducted in a stainless-steel fixed-bed reactor (green) with an inner diameter of 4.5 mm and a total length of 30 cm, which results in a total volume of 7 mL. The reactor is equipped with a concentric 1/16-inch capillary containing a movable thermocouple (type K, TMH). The heating is accomplished by 4 heating cartridges (200 W Firerod, Telemeter Electronic) placed equally spaced inside an aluminum jacket, which provides an axial isothermal zone of around 10 cm. The catalyst particles are fractionated in a size range of 150 to 200 µm and are diluted with twice the amount of inert particles (200 µm, Al2O3, Sasol Puralox) of the same size. Therefore, internal heat and mass transfer limitations can be neglected, which was verified in our previous paper under identical conditions by applying the Mears, Anderson, and Weisz-Prater criteria [68]. The catalyst bed with a total length of 2 cm is placed in the center of the isothermal section, which ensures isothermal conditions for temperatures up to 600 K with deviations of < ± 1 K. The packing is fixed inside the reactor by solid, non-porous glass particles (silica beads, häberle) of the same particle size and glass wool (silica wool, VWR). The total length of the packing sums up to around 10 cm. Non-porous glass is used, since it provides a small surface area for potential interaction with the gas species. Furthermore, solid glass particles reduce the gas holdup in the reactor, as well. RTD measurements are performed, which confirm minor deviation from ideal plug flow behavior within the packed fixed-bed reactor (see Appendix, Figure S9). The operating pressure is controlled by two back pressure regulators (violet, LF1, Equilibar). The working principle is based on an equilibrium of forces between a flowing gas and a pilot gas at a membrane, which ensures negligible pressure fluctuations even at low flow rates. One regulator is placed at the reactor outlet and the second in the bypass line. In order to reduce pressure spikes during the switching between both gas lines to about 50 mbar, the reactor inlet pressure is used as pilot pressure for the bypass-line. At the reactor outlet a mixture of H2 (5.0 purity, MTI) and Ne (5.0 purity, MTI) (yellow line) is dosed via MFCs (EL-FLOW Prestige, Bronkhorst). The external standard (ES) mixture containing Ne as ES and additional H2 is fed directly at the reactor outlet in order to reduce the residence time between outlet and analytics below the temporal resolution of the analytics. The tubing downstream the reactor is heated to 70 °C to prevent condensation of H2O. Note that the maximum H2O partial pressure is significantly below the vapor pressure, due to the low conversion and dilution with inert gas.</p><p>The analytics consist of a process mass spectrometer (MS) (Cirrus 3-XD, MKS), which allows to analyze the mass-to-charge ratios 2 (H2), 4 (He), 15 (CH4), 18 (H2O), 20 (Ne), 28 (CO and CO2), 40 (Ar) and 44 (CO2) quantitatively with a temporal resolution of 0.5 s after calibration. The mass-to-charge ratios 26 (C2H6) and 43 (C3H8) are monitored qualitatively, as well. An auxiliary gas chromatograph (GC-2010, Shimadzu with RT-Q-Bond pre-column and molecular sieve 5A column), equipped with a thermal conductivity and flame ionization detector, is used to calibrate the MS results for CO, CO2, Ar, and hydrocarbons up to C3 under steady-state conditions. The analytics allow to minimize the error in the carbon mass balance based on MS measurements to < 5 % under steady-state and to < 10 % under unsteady-state conditions. Note, that higher hydrocarbons are negligible and not detected by means of MS, as indicated by steadystate experiments using the same catalyst and similar conditions previously reported [68].</p><!><p>The RTD of a reactor equipped with porous catalyst pellets is affected by transport processes at both the macro and the meso scale, since dispersion in the convective flow and diffusion in the porous structure contribute to the overall response. Since the RTD of the internal standard (IS) measured in-situ is assumed to be representative [12,19,22], it allows to provide each experimental data set together with the individual residence time behavior. If those transport phenomena are not selective towards certain components, the RTD is identical for all components 𝑖 and is just scaled with a proportional factor 𝐶 according to eq. ( 1), where 𝑛i refers to the measured molar flow rate of component 𝑖 as function of time. Hence, the obtained molar flow rate at the reactor outlet 𝑛̇𝑖 ,out of any compound 𝑖 can be plotted against 𝑛̇I S,out providing a linear dependency, if the above-mentioned assumptions are fulfilled. In other words, any deviation in experimental data from eq. ( 1) indicates compound selective effects in the system. For the example of heterogeneously catalyzed reactions, being in the focus of the present contribution, those effects are mainly caused by the interaction of compound 𝑖 with the solid surface. In particular, dissipation of species 𝑖 from the gas phase (𝑛̇𝑖 ,out /𝑛̇I S,out < 𝐶) indicates adsorption at the solid surface, while appearance (𝑛̇𝑖 ,out /𝑛İ S,out > 𝐶) may be caused by desorption.</p><!><p>= 𝐶 = const.</p><p>(</p><p>The ES is required to obtain the molar flow rates from MS data, in order to derive the material balance for all compounds 𝑖 quantitatively [12]. The ES also allows to identify unwanted fluctuations in the volumetric flow rate, in particular during the switching event between two different feed gas compositions, which might cause pressure fluctuations at the reactor inlet. Therefore, the ES is dosed into the reactor outlet stream with a constant molar flow rate 𝑛Ė S and the molar fraction 𝑥 ES is measured over time by the MS, in order to derive the outlet molar flow rate 𝑛ȯ ut according to eq. ( 2).</p><p>𝑛̇𝑖 ,out (𝑡) = 𝛼(𝑡) 𝑥 𝑖,out 𝑝 STP 𝑉 ̇in,STP 𝑅 𝑇 STP (4)</p><!><p>The graphical representation of eq. ( 1) is provided by the state-space plot, which displays the response of component 𝑖 as a function of that for the internal standard Ar in the present contribution. For heterogeneously catalyzed systems the state-space plot can unravel interactions between a gaseous component and the solid surface by sorption processes, for instance. Note that the state-space plot is not limited to specific types of transient signals and rather complements the typical plot of transient signals as function of time.</p><!><p>does not change with time during. Therefore, the transient processes during the periodic experiments become more prominent in the state-space plot and are thus easier accessible.</p><!><p>The PTK methodology provides periodic data of the system response, since the input signal is periodic. Considering that the transient outlet molar flow 𝑛̇𝑖 ,out of component 𝑖 is equivalent to the system response, it provides the experimental data basis for further analysis. The key property of the LC is that each point within a period 0 ≤ 𝑡 ≤ τ of the duration 𝜏 is reached in each period 𝑁 𝑚 and that the corresponding response 𝑛i ,𝑚,out is identical. We also assume that each measured data is affected by noise. Therefore, the results obtained within the LC are averaged over a certain number of periods 𝑁 Θ according to eq. ( 5), in order to improve the signal-to-noise ratio by statistical analysis. The obtained average molar flow rate at the reactor outlet 𝑛̅ 𝑖,out thus corresponds to the average LC. In addition, the variance Δ𝑛̅ 𝑖,out 2 of the average LC can be calculated by eq. ( 6), which can further be used to determine confidence intervals of the LC.</p><p>Δ𝑛̅ 𝑖,out</p><p>Figure 4a shows the periodic response of the system in form of the CH4 outlet molar flow rate as an example. In total the response for 50 periods is shown of which the last 25 periods (in red) except the very last are used for averaging, since the activity loss over those periods is less than 3 %. Figure 4b depicts the corresponding average LC together with the standard deviation (red shaded area).</p><!><p>The deviation of the system response for component 𝑖 from the RTD can be evaluated quantitatively by calculating the transient molar flow rates, which are identical to the net surface flows defined in the CTK method [12]. Therefore, the observed response in form of the measured outlet molar flow rate 𝑛i ,out of component 𝑖 during a transient process is assumed to be a superposition of two contributions according to eq. ( 7). The first contribution originates from the overall RTD, which results in the corresponding molar flow rate 𝑛̇𝑖 ,RTD . The second contribution quantifies the transient molar flow rate 𝑛i ,trans and thereby the deviation from the RTD during the transient process. Figure 5 visualizes this equation for CH4 as an example. It has to be mentioned that this approach requires the LC as necessary condition, since thereby 𝑛i ,trans corresponds to the deviation within one period only. If the LC is not achieved also previous deviations may accumulate in 𝑛̇𝑖 ,trans , which falsifies the analysis. Furthermore, it has to be mentioned that the transient molar flow rate is generally considered as a temporal source or sink term for each component, irrespective of the specific reason. Even though the main reason is probably the interaction of component 𝑖 with the catalyst surface by adand desorption, chemical reaction or irreversible processes may also contribute to 𝑛i ,trans . 𝑛i ,out (𝑡) = 𝑛i ,RTD (𝑡) + 𝑛i ,trans (𝑡) The contribution of the RTD for each component 𝑛i ,RTD is calculated by a function characterizing the RTD of the internal standard 𝐹 Ar and the outlet molar flow rates for each component at the steady-states for both feed gas mixtures 𝑛i ,out,ss,1 and 𝑛i ,out,ss,2 , according to eq. ( 8). Therefore, the function 𝐹 Ar is derived from the outlet molar flow rates of the internal standard Ar 𝑛̇A r,out normalized with the flow rates at both steady-states (see eq. ( 9)).</p><p>𝑛̇𝑖 ,RTD (𝑡) = (𝑛̇𝑖 ,out,ss,2 − 𝑛i ,out,ss,1 ) 𝐹 Ar (𝑡) + 𝑛i ,out,ss,1 (8)</p><p>The steady-state is assumed to be achieved, if no temporal gradients in the signal occur, which corresponds to the mathematical definition of time derivatives being zero. Under real conditions, however, long term processes monotonical affecting the response, such as catalyst deactivation, have to be considered. Since the investigation of those processes do not require periodic experiments, due to their long term and irreversible nature, a certain but very small temporal gradient in the signal is acceptable, depending on the individual case. In our case steady-state conditions are reached for the last 5 s in each half period. Hence, the respective outlet molar flow rates are used for the calculation of 𝑛̇𝑖 ,out,ss,1 and 𝑛i ,out,ss,2 by averaging.</p><p>The transient molar flow rates for each component can be applied in order to derive transient elemental balances, as shown for the carbonaceous species in eq. ( 10). For negligible formation of higher hydrocarbons (C2+), eq. ( 10) allows to calculate the amount of carbon stored at or released from the surface during the build-up and backtransient phase after integration over both half periods. Note, that the modification of carbon cannot be distinguished based on this equation without further surface specific analysis. In particular, various adsorbed carbon containing species may be present together with elemental carbon modifications and further carbon compounds [58].</p><p>It is noteworthy, that the proper choice of the experimental conditions is of eminent importance for calculation and interpretation of the transient molar flow rates. Care must especially be taken regarding the signal-to-noise ratio expressed by the relative transient molar flow rate 𝑛̇𝑖 ,trans 𝑛̇𝑖 ,out ⁄ . In particular, the transient molar flow rate needs to be distinguishable from the total molar flow rate of the respective component despite of the errors in the measured data and the mass balance.</p><p>The determination of the transient molar flow rate by the PTK method allows to link the analysis of the gas phase composition in continuous systems with the molar amount of adsorbed species at the surface measured with respective experimental operando methods (e.g. diffuse reflectance infrared fourier transform spectroscopy, DRIFTS) quantitatively using material balances as follows. The transient molar flow rate 𝑛̇ℎ ,trans of an element ℎ can be applied for calculation of the molar amount of substance of that element stored at the catalyst surface 𝑛 ℎ,surf , according to the elemental balance shown in eq. (11). The respective component balance (eq. ( 12)) requires to consider the amount of substance converted in a surface reaction 𝑅 𝑗,surf in addition to the transient molar flow rate 𝑛̇ℎ ,trans of component 𝑖, in order to calculate the amount of this component stored at the surface 𝑛 𝑖,surf . The stoichiometric coefficient 𝜈 𝑖,𝑗 describes the stoichiometry of the adsorbed species only and equals zero for non-adsorbed components. Eq. ( 12) therefore allows to deduce reaction kinetics under dynamic conditions, since it links model-based evaluation of experimental data with the transient surface reaction rates. The challenge is that the involved surface species and surface reactions depend on the specific reaction mechanism, which is not known a priori in most cases. Therefore, an iterative approach is meaningful by building up the complexity of the mechanism until the prediction of the model agree with the transient measurement results of gaseous and surface species. Eq. ( 13) connects the elemental balances with those of the components, using the number of atoms of element ℎ in component 𝑖 expressed by 𝛽 ℎ,𝑖 . Note that surface coverages can be obtained from the molar amount of substance stored at the catalyst surface, if the number of sorption sites is known.</p><p>𝑑𝑛 𝑖,surf 𝑑𝑡 = −𝑛i ,trans (𝑡) + ∑ 𝜈 𝑖,𝑗 𝑅 𝑗,surf 𝑗 (𝑡)</p><p>𝑛 ℎ = ∑ 𝛽 ℎ,𝑖 𝑛 𝑖 𝑖 and 𝑛̇ℎ = ∑ 𝛽 ℎ,𝑖 𝑛̇𝑖 𝑖 (13)</p><!><p>For the examples presented in this study the feed gas composition is switched 50 times either between H2/He (50/50 by volume) and CO2/Ar/H2/He (10/1/40/49 by volume) with a cycle period of 90 s for example I or between H2/He (40/60 by volume) and CO/Ar/H2/He (10/1/40/49 by volume) with a cycle period of 240 s for example II. Ar is chosen as the internal standard for the COx species and H2O, since it provides a similar diffusion coefficient and viscosity and is thus assumed to exhibit a representative RTD. The strong dilution with He is justified by the similarity to the physical properties of H2, the main component in the gas phase due to stoichiometric reasons. Therefore, the physical properties of all gas mixtures can be assumed to be rather similar, which provides the basis for a similarity in fluid dynamics in the system and thus in RTD. The total volumetric feed flow rate of 250 mLSTP min -1 was further diluted with the external standard mixture after the reactor outlet consisting of H2/Ne (245 mLSTP min -1 / 5 mLSTP min -1 ), which reduces the residence time between the reactor outlet and the analytics section to below the temporal resolution of the MS.</p><p>The CO2 adsorption experiment of example I is conducted with 100 mg Al2O3 particles (Puralox, Sasol) with a particle size of 150-200 µm. The methanation experiments from example II are conducted with 50 mg of a 5 wt-% Ni/Al2O3 catalyst of the same size diluted with 100 mg Al2O3 particles (Puralox, Sasol) at a temperature of 556 K and a pressure of 2 bar, which provides almost full conversion of the carbon oxides in equilibrium [69]. Furthermore, the reactor is operated under differential conditions in order to neglect the impact of concentration gradients by keeping the carbon oxide conversion below 20 % in all experiments [70]. Catalyst aging, e.g. due to deactivation, is monitored based on the steady-states and can be ruled out in the reported experiments [71,72].</p><!><p>For the example of CO2 adsorption at Al2O3 surface Figure 6a displays the observed system response in form of the measured outlet molar flow rate of CO2 𝑛̇C O 2 ,out . In addition, the theoretical response 𝑛̇C O 2 ,RTD is shown as well, which is expected from pure RTD according to eq. ( 8). Figure 6b depicts the transient molar flow rate 𝑛̇C O 2 ,trans , while Figure 6c shows the respective state-space plot for CO2. Due to the absence of catalytical active sites, the deviation of the CO2 response from the RTD can be attributed to interaction of CO2 with the solid surface by ad-and desorption processes. In Figure 6a a deviation in the build-up phase between the measured and theoretical CO2 response is observed, which causes a negative transient molar flow rate with a maximum of 2 µmol s -1 . In the state space plot the negative transient molar flow rate during the build-up phase causes a hysteresis below the linear relationship given by the dashed line. Note that a linear relationship would mean that the measured and the theoretical CO2 response is identical, indicating the CO2 signal being affected by RTD only. The negative transient molar flow rate is caused by a sink of CO2, which is thus removed from the gas phase due to adsorption at the surface. Furthermore, the maximum in transient molar flow rate in the early stage of the build-up phase indicates fast adsorption of CO2 at the alumina surface, which is still barely covered by adsorbent. With time the surface coverage increases resulting in the transient molar flow rate approaching zero towards the end of the build-up phase.</p><p>During the back-transient phase, in contrast, the transient molar flow rate is positive also exhibiting a maximum in the early stage. Hence, the hysteresis in the state-space plot exhibits a higher value than expected from RTD. This observation indicates that CO2 desorbs from the solid surface into the gas phase, which is more pronounced for high surface coverages early in the back-transient phase.</p><p>Further evaluation of the data can be performed by the integration of the transient molar flow rate during the back-transient phase, which yields the molar amount of CO2 desorbed. Therefore, the average back-transient phase and the last cycle is distinguished from each other and compared the sorption capacity measured volumetrically at an equal CO2 partial pressure and temperature (Triflex, micromeritics). After the last switch to the H2/He feed gas mixture the transient molar flow of CO2 is integrated over 180 s and sums up to 17 µmol, which is in reasonable agreement with the adsorption capacity of 24 µmol measured volumetrically. The deviation between the adsorbed amount determined by volumetric and flow methods, however, is in accordance with literature, where a lower adsorption capacity of CO2 during pulse experiments compared to volumetric measurements is reported [18,73]. The reason is that equilibrium between the gas phase and the adsorbed species is not reached during flow experiments, while volumetric measurements are performed in order to ensure equilibrium conditions to be fulfilled.</p><p>However, the amount of CO2 desorbing during the average back-transient phase amounts to 9 µmol and is thus significantly below the theoretical value and that of the last cycle. Hence, this difference between the average and the last cycle indicates incomplete desorption of CO2 during the half cycle period of 45 s. Note that CO2 is only weakly adsorbed at the surface, as found by temperature programmed desorption (TPD) experiments up to 400 °C (see Appendix, Figure S10), confirming the sorption process being completely reversible. Interestingly, the relative transient molar flow rate and thus the signal-to-noise ratio changes within each half-period and also differs for the build-up and back-transient phase. This becomes evident, if one considers that 𝑛̇𝑖 ,out follows the RTD and thus with time on the one hand. On the other hand, 𝑛i ,trans also changes with time depending on the underlying interaction mechanism with the catalyst. If ad-or desorption at the solid surface occurs, for instance, the surface coverage changes with time and therefore the sorption rate, as well. For illustration, the relative transient molar flow rate for one period is shown in Figure 7. Note that the time delay of the system amounts to 4 s, for which no meaningful data is available. It can be observed that the relative transient molar flow rate rises rapidly in the build-up phase and reaches a maximum within 10 s. During that phase the transient molar flow rate of CO2 is high, since CO2 In addition to that 𝑛Ċ O 2 ,trans is high in the early stage of the back-transient phase, due to high desorption rates given by the initially high surface coverages and decreases with time, as well. This gives raise to resolve slow processes in the interaction between gaseous species and the solid surface, which occur close to the steady-state. It is recommended to analyze and compare both half-periods in order to minimize the error in determination of the transient molar flow rates.</p><!><p>For the example of CO methanation the response in form of the measured outlet molar flow rate 𝑛̇𝑖 ,out for CO and CH4 are shown in comparison to the theoretical values 𝑛̇𝑖 ,RTD in Figure 8a, while the corresponding transient molar flow rates 𝑛̇𝑖 ,trans are displayed in Figure 8b. Note, that the CO signal is scaled in order to be displayed together with CH4. The comparison of the results shown in Figure 8 (a vs. b) exhibits that CO follows the RTD, though a significant transient molar flow rate is calculated. This discrepancy can be explained by the fact that the relative transient molar flow rate 𝑛̇C O,trans 𝑛̇C O,out ⁄ is small. We therefore focus on Figure 8b for further discussion. During the build-up phase (switch from H2/He to CO/Ar/H2/He) a significant negative transient molar flow rate for CO is observed shortly after the switching event, which indicates strong CO adsorption. At the same time a positive transient molar flow rate for CH4 indicates product formation and desorption into the gas phase. The interplay between both species can be evaluated by the transient molar flow rate of carbon species at the surface 𝑛̇C ,trans according to eq. ( 10). The negative value during the buildup phase indicates that the CO adsorbed at the surface is not only converted into CH4 but is also stored at the surface, which indicates that CO adsorption is a rather fast process. This is in accordance to Bundhoo et al., who reported a temporal delay of the formation of CH4 after the adsorption of CO on a Ni catalyst [22].</p><p>During the back-transient phase the transient molar flow rate of CH4 exhibits positive values, while that for CO and overall carbon is positive at first and turns negative before approaching zero. The positive transient molar flow rate of CH4 is attributed to the hydrogenation of a highly reactive intermediates adsorbed at the surface and subsequent desorption of the CH4 formed, according to literature [43,58,72,74]. Those intermediate are assigned as CHx species, which are converted into CH4 in the following two-step mechanism proposed by Bundhoo et al. [22]: First, metallic sites at the catalyst surface are released by desorption or reaction of adsorbed species, which enhances the atomic hydrogen supply. In the following step the adsorbed reactive intermediate reacts with the available hydrogen to gaseous CH4.</p><p>The positive transient molar flow rate of CO in the early stage of the back-transient phase indicates CO desorption from the surface, due to decreasing CO partial pressure in gas phase during the back-transient phase. Due to ongoing surface reaction further sorption sites become available with time, which leads to a (re-)adsorption of CO still available in the gas phase and therefore negative transient molar flow rates of CO. Obviously, this effect requires the surface reaction to be sufficiently fast, in order to release sorption sites, while CO is still present in the gas phase. The latter is governed by the residence time distribution of the reactor. The CO adsorbed in that later phase will also be converted into CH4 as long as sufficient H2 is available and thereby contributes to the positive transient molar flow rate of CH4 in that phase, as well. The transient molar flow rate of carbon corresponds to the carbon balance given in eq. ( 10), which is positive during the back-transient phase. This indicates that deposits of carbonaceous species formed during build-up phase are hydrogenated to CH4 due to the excess in hydrogen during the back-transient phase.</p><!><p>The PTK methodology is introduced in order to investigate the interplay of transient kinetic processes in heterogeneously catalyzed reactions under realistic conditions (i.e. elevated pressures). It allows to derive the molar flow rates between the gas phase and the catalyst surface quantitatively based on the transient reactor response with respect to gas phase composition and thus complements techniques focusing on analyzing the dynamics at the catalyst surface itself. In the contribution, the experimental procedure and equipment is described together with model-based evaluation of the experimental data. The strength of the PTK method relies on periodic step-shaped changes in feed gas composition, which induce a periodic reactor response, as well. Therefore, a limit cycle is reached for reversible interactions between the gas phase and the catalyst surface, which is evaluated statistically in order to provide reliable data for subsequent interpretation. In particular, the dynamic response with respect to the outlet molar flow rates of the reactive species present in the gas phase is evaluated in the limit cycle providing narrow confidence intervals. In order to distinguish the residence time behavior from transient processes related to interaction with the surface an internal standard is monitored and considered in result analysis. This approach provides the basis for evaluation of transient processes based on the material balances, exhibiting minor deviations only, and thus to resolve even small dynamic effects. Therefore, the presented PTK method is capable to link the transient behavior of single components to the dynamic response of the reactor. The evaluation procedure based on material balances of the catalyst surface and the overall reactor further provides the capability to deduce reaction kinetics valid under dynamic operation conditions from respective experimental results.</p><p>The methodology is demonstrated for two examples in the context of the methanation reaction. The adsorption of CO2 at Al2O3 represents a non-reactive reversible process, while for hydrogenation of CO into CH4 sinks and sources exist for reactants and products, respectively. The results reveal that the dynamics of the ad-and desorption processes can be resolved and quantitatively evaluated in terms of molar flow rates. Such information allows to deduce the kinetics of those processes under reaction conditions, which is very important for modeling and simulation of chemical reactors operated under dynamic conditions. The second example shows that experimental data can be obtained, which provide the basis to even derive the kinetics of sink and source terms under dynamic conditions. Since the PTK method is not limited to both examples reported, it allows to derive kinetic information for various heterogenously catalyzed gasphase reactions operated dynamically under realistic reaction conditions.</p><!><p>Latin letters 𝐶 Constant 1</p><!><p>Step response 1</p><p>𝑁 Θ Evaluated number of periods in limit cycle 1</p>

ChemRxiv

Synthesis and Electrocatalytic HER Studies of Carbene-Ligated Cu3-xP Nanocrystals

N-heterocyclic carbenes (NHCs) are an important class of ligands capable of making strong carbon-metal bonds. Recently, there has been a growing interest in the study of carbene-ligated nanocrystals, primarily coinage metal nanocrystals, which have found applications as catalysts for numerous reactions. The general ability of NHC ligands to positively affect the catalytic properties of other types of nanocrystal catalysts remains unknown. Herein, we present the first carbenestabilized Cu3-xP nanocrystals. Inquiries into the mechanism of formation of NHC-ligated Cu3-xP nanocrystals suggest that crystalline Cu3-xP forms directly as a result of a high-temperature metathesis reaction between a tris(trimethylsilyl)phosphine precursor and an NHC-CuBr precursor, the latter of which behaves as a source of both carbene ligand and Cu + . To study the effect of the NHC surface ligands on catalytic performance, we tested the electrocatalytic hydrogen evolving ability of the NHC-ligated Cu3-xP nanocrystals and found they possess superior activity to analogous oleylamine-ligated Cu3-xP nanocrystals. Density functional theory calculations suggest that the NHC ligands minimize unfavorable electrostatic interactions between the copper phosphide surface and H + during the first step of the hydrogen evolution reaction, which likely contributes to the superior performance of NHC-ligated Cu3-xP catalysts as compared to oleylamine-ligated Cu3-xP catalysts.Cu3-xP nanocrystal synthesis, characterization, and electrocatalytic HER studies will be discussed in detail, and density functional theory (DFT) calculations were carried out to elucidate the electronic effects of NHC ligands on catalysis. EXPERIMENTAL SECTIONMaterials and General Procedures. Reagents and solvents were purchased from commercial sources and used as received, unless otherwise noted. Benzimidazole (C7H6N2, 99%), 1-bromotetradecane (C14H29Br), copper(I) oxide (99.9%), potassium carbonate (K2CO3, anhydrous, 99%), and 1,4-dioxane were purchased from Alfa Aesar. Copper(I) bromide (98%) was purchased from Strem Chemicals. 1-octadecene (ODE, technical grade, 90%), oleylamine (technical grade, 70%), tris(trimethylsilyl)phosphine ((TMS)3P, 95%) and a phosphorus ICP standard (1000 ppm in 2% aqueous nitric acid) were purchased from Sigma-Aldrich. The copper ICP standard (1000 ppm in 2% aqueous nitric acid) was purchased from Perkin Elmer. Reactions involving air-or moisture-sensitive compounds were conducted under a nitrogen atmosphere by using standard Schlenk techniques. 1-Octadecene and oleylamine were degassed for 4 h at 105 °C and then overnight at room temperature prior to use. performance of the NHC-ligated Cu3-xP catalyst. Future studies should focus on leveraging the high synthetic tunability of the NHC surface ligands to confer better stability or improved activity. Specifically, the design of NHC ligands with variable electronic properties could be used to modulate their effect on electrocatalysis. ASSOCIATED CONTENT Supporting InformationThe Supporting Information is available free of charge on the ACS Publications website.Experimental details; UV-vis-NIR spectra; high-resolution TEM images; N 1s XPS spectrum; additional powder XRD patterns; TGA traces; EIS figures; double-layer capacitance figures; Tafel plots; controlled potential electrolysis figures; energy decomposition analysis from DFT; ligand density calculations (PDF)

synthesis_and_electrocatalytic_her_studies_of_carbene-ligated_cu3-xp_nanocrystals

INTRODUCTION<!>Synthesis of NHC-Stabilized Cu3-xP Nanocrystals. In a typical<!>RESULTS AND DISCUSSION<!>CONCLUSIONS

<p>N-Heterocyclic carbene (NHC) ligands have significant value as a result of their structural diversity, chemical stability, and strong electron donation that leads to a high coordinating ability with metals. 1 While NHCs are now commonly employed as neutral L-type ligands for molecular complexes, they are also gaining increasing utility as ligands to support the steric stabilization of nanocrystals. [2][3][4][5][6][7] And just as NHC ligands are widely utilized in molecular complexes for homogeneous catalysis as a result of their steric and electronic properties, 8 supporting NHC ligands are becoming of interest for heterogeneous nanocrystal catalysts. It has been demonstrated that NHC ligands have the ability to enhance nanocrystal catalyst stability and increase nanocrystal catalyst activity and/or selectivity. [9][10][11][12] NHC-stabilized nanocrystals have been shown to display catalytic activity toward, for example, hydrogenations and semihydrogenations, 9,12-14 lactonization, 10 asymmetric arylations, 15 Buchwald-Hartwig aminations, 16 and cross-coupling reactions. 15 NHC-stabilized nanocrystals have also been shown to be competent electrocatalysts for CO2 reduction. 11,17 For example, Cao et al. investigated the use of NHC-stabilized Au nanocrystals as electrocatalysts for the reduction of CO2 to CO. Interestingly, the Au nanocrystal catalyst with surface-bound carbene ligands exhibited a higher Faradaic efficiency for CO2 reduction to CO than did bare (or ligand-less) Au nanoparticles; it was further shown that the NHC affected the mechanistic pathway of catalysis. 17 Despite the proven benefits of applying NHC ligands to nanocrystal catalysts, they have thus far only been supported on metal nanocrystal catalysts (e.g., Ru, Au, Pt, Pd, Cu). [9][10][11][12][13][14][15][16][17][18] Therefore, the potential utility of NHC ligands as supporting ligands for other types of nanocrystal catalysts is currently unknown. Certain metal phosphide nanocrystals have emerged as an important class of catalysts for reactions such as hydrodesulfurization, [19][20][21] hydrodeoxygenation, 22 and the hydrogen evolution reaction (HER). 23 Along these lines, self-doped copper phosphide (Cu3-xP) nanocrystals have been shown to be efficient Janus catalysts for overall electrochemical water splitting. 24,25 Synthetic methods for the preparation of colloidal Cu3-xP nanocrystals remain underdeveloped, however. Early reports utilized trioctylphosphine (TOP) to phosphidize zero-valent Cu nanocrystals under relatively high-temperature conditions (320 °C). 26 More recently, reactive phosphines, such as tris(trimethylsilyl)phosphine ((TMS)3P), have been introduced as the phosphide source, which helped to lower reaction temperatures to 120 °C. 27,28 Herein, we present a direct synthetic route to NHC-stabilized Cu3-xP nanocrystals to explore the effect of the carbene ligand when using the resulting nanocrystals as HER electrocatalysts. The direct synthesis of NHC-stabilized Cu3-xP nanocrystals circumvents the need for a post-synthetic ligand exchange for the installation of carbenes on the nanocrystal surface. To the best of our knowledge, this is the first example of a metal phosphide nanocrystal supported by a carbene ligand. The NHC-stabilized Preparation of (TMS)3P Stock Solution. Caution! (TMS)3P is a pyrophoric material that must be handled with care. In a N2 glove box, an ampule containing 1 g of liquid (TMS)3P was opened and the contents were added to a Schlenk flask containing 40 mL ODE (dried as mentioned above) to make a 0.1 M stock solution that was used for all subsequent reactions.</p><!><p>Cu3-xP nanocrystal synthesis, a solution of NHC-CuBr (195 mg, 0.300 mmol) in 6 mL ODE was prepared. The mixture was subjected to vacuum and held for 1 h at 115 ˚C to remove any adventitious H2O. NHC-CuBr was completely dissolved in ODE after 1 h of stirring at 115 ˚C. Subsequently, the temperature was raised to 250 ˚C and 0.5 mL of the 0.1 M (TMS)3P stock solution was rapidly added into the NHC-CuBr solution. The solution immediately became black and turbid upon phosphine addition. After 2 min, the reaction was quenched by removing the heat and placing the flask in a room temperature water bath. To remove 1-octadecene and any unreacted precursors and by-products, the Cu3-xP nanocrystals were washed by splitting the crude reaction mixture into two 40-mL centrifuge tubes that were then filled to volume with acetone. The centrifuge tubes were sonicated for 10 min and centrifuged for 5 min at 6000 rpm. For long-term colloidal stability, particles were washed only once with acetone and redispersed in toluene or tetrachloroethylene (TCE). For electrochemical studies, an additional partial wash was performed in which the product of one centrifuge tube was redispersed in 5 mL of hexanes, washed with an additional 10 mL of acetone in the same manner as described above, and finally redispersed in 5 mL of hexanes. This hexanes suspension could then be dropcast directly onto a glassy carbon electrode for catalysis studies.</p><p>Characterization. UV-vis-NIR spectroscopy was carried out on a Perkin-Elmer Lamba 950 spectrophotometer equipped with a 150mm integrating sphere, using a quartz cuvette for liquid samples. Spectra were taken in tetrachloroethylene to reduce solvent contributions to the spectrum in the NIR region. Powder X-ray diffraction (XRD) data was collected using a Rigaku Ultima IV diffractometer in parallel beam geometry (2-mm beam width) using Cu Ka radiation (l = 1.54 Å). Samples were prepared by drop casting the nanocrystals onto zero-diffraction, single crystal Si substrates. X-ray photoelectron spectra (XPS) were obtained using a Kratos Axis Ultra X-ray photoelectron spectrometer with an analyzer lens in hybrid mode. High-resolution scans were performed using a monochromatic aluminum anode with an operating current of 6 mA and voltage of 10 kV using a step size of 0.1 eV, a pass energy of 40 eV, and a pressure range between 1-3 ´ 10 -8 Torr. The binding energies for all spectra were referenced to the C 1s core level at 284.8 eV. Transmission electron microscopy (TEM) analysis was performed on a JEOL JEM-2100 microscope at an operating voltage of 200 kV equipped with a Gatan Orius CCD camera. Samples for TEM analysis were prepared from dilute purified nanocrystal suspensions drop cast from hexanes dispersions onto 400 mesh carbon-coated copper grids (Ted Pella, Inc.). Thermogravimetric analysis (TGA) was performed on a TGA Q50 instrument with a heating rate of 10 °C•min −1 . 8-10 mg samples were used for all TGA studies, and samples were measured by TGA after one full wash and one partial wash, identical to the workup used for catalytic studies. More details on the ligand density calculations determined by TGA are provided in the Supporting Information. Fourier transform infrared spectroscopy (FT-IR) was performed on a Bruker Vertex 80 FT-IR spectrometer. Samples were prepared as a powder within a matrix of KBr. Inductively-coupled plasma optical emission spectroscopy (ICP-OES) was performed on an iCap 7400 ICP. All samples were digested with 2 mL of concentrated nitric acid and subsequently diluted to 25 mL with Millipore water in a volumetric flask. Phosphorus and copper ICP standards were prepared at different concentrations (0.1 ppm, 0.8 ppm, 2 ppm, 4 ppm, 10 ppm) to construct a five-point calibration curve from which the sample concentrations of Cu and P could be determined. NMR spectra were taken on a Varian VNMRS-600 in CDCl3.</p><p>Electrochemical Methods. Electrochemistry experiments were carried out using a VersaSTAT 3 potentiostat in a three-electrode configuration electrochemical cell under an inert N2 atmosphere. A rotating disk electrode (RDE, glassy carbon insert, 0.196 cm 2 surface area) was used as the working electrode. The glassy carbon surface was polished with 0.05 µm Al2O3 polish powder and sonicated in Millipore water prior to use. A graphite rod, purchased from Graphite Machining, Inc. (Grade NAC-500 Purified, < 10 ppm ash level), was used as the counter electrode. The reference electrode, placed in a separate compartment and connected by a porous Teflon tip, was based on an aqueous Ag/AgCl/1.0 M KCl electrode (purchased from CH Instrument, Inc.). All potentials reported in this paper were converted to the reversible hydrogen electrode (RHE) by adding a value of (0.235 + 0.059 × pH) V. 0.5 M H2SO4 aqueous solution was used as the electrolyte and was purged with nitrogen thoroughly prior to electrochemical testing. Cyclic voltammograms for double layer capacitance (Cdl) measurements were taken over a 100 mV potential window centered around the open circuit potential (OCP) at scan rates of 10, 20, 30, 40, 50, and 60 mV/s. The capacitance current obtained from the current difference (Δi = iaic) at OCP was plotted against the scan rate. The slope is twice the value of the double layer capacitance. Controlled potential electrolysis (CPE) measurements to determine long-term stability and Faradaic efficiency were conducted in a sealed twochambered H cell where the first chamber held the working and reference electrodes in 40 mL of 0.5 M H2SO4 aqueous solution and the second chamber held the counter electrode in 25 mL of 0.5 M H2SO4 aqueous solution. The two chambers were both under N2 and separated by a fine porosity glass frit. CPE experiments were performed with a glassy carbon plate electrode (6 cm × 1 cm × 0.3 cm; Tokai Carbon USA) as the working electrode and a graphite rod as the counter electrode. The reference electrode was a Ag/AgCl/1.0 M KCl (aq.) electrode separated from the solution by a porous Teflon tip. Using a gas-tight syringe, 2 mL of gas was withdrawn from the headspace of the H cell and injected into a gas chromatography instrument (Shimadzu GC-2010-Plus) equipped with a BID detector and a Restek ShinCarbon ST Micropacked column. To determine the Faradaic efficiency, the theoretical H2 amount based on total charge flowed was compared with the GCdetected H2 produced from CPE. Electrochemical impedance spectroscopy (EIS) measurements were carried out at different overpotentials in the frequency range of 100 kHz -1 Hz with 10 mV sinusoidal perturbations. Experimental EIS data were analyzed and fitted with the ZSimpWin software using a two-time constant parallel model. All of the polarization curves presented herein were corrected for iR loss according to the following equation:</p><p>where Ecorr is the iR-corrected potential, Emea is the experimentally measured potential, and Rs is the solution resistance extracted from the fitted EIS data.</p><p>Density Functional Theory. Density functional theory (DFT) calculations at the B97/def2-SVP level of theory 29,30 were performed using the ab initio quantum chemistry software Q-Chem 31 to probe the electronic effects of L-type NHC and alkylamine ligands on proton binding to a Cu3P catalyst.</p><!><p>Synthesis and Characterization. We previously reported that a metathesis reaction between bromo[1,3-(ditetradecyl)benzimidazol-2-ylidene]metal(I) (NHC-MBr, M = Ag, Cu) complexes and bis(trialkylsilyl) chalcogenides ((R3Si)2E, where E = S, Se) under ambient conditions yields monodisperse M2E nanocrystals, where the NHC ligands bearing long-chain alkyl substituents remain coordinated to the nanocrystal surface and provide excellent colloidal stability. 7 Here, we extended the use of the same NHC-CuBr precursor to the preparation of Cu3-xP nanocrystals by reaction with (TMS)3P. Initial attempts to synthesize Cu3-xP nanocrystals at room temperature resulted in an amorphous product, as revealed by powder X-ray diffraction (XRD). Therefore, the reaction temperature was raised to 250 °C and (TMS)3P was rapidly injected into a solution of NHC-CuBr in the high-boiling, non-coordinating solvent 1-octadecene. This resulted in the direct formation of Cu3-xP nanocrystals.</p><p>The as-synthesized Cu3-xP nanocrystals (1) were confirmed by powder XRD to be phase-pure hexagonal Cu3-xP, with lattice parameters determined by a Le Bail fit of a = 6.9376( 14 00-002-1263, Figure 1a). The UV-vis-NIR absorption spectrum displays a characteristic LSPR band at ~1360 nm for the resulting nanocrystals (Figure S1), as expected due to copper vacancies in the structure. 27,28 Inductively coupled plasma optical emission spectroscopy (ICP-OES) analysis of the as-synthesized product confirms a substoichiometric average composition of Cu2.30P, consistent with the copper-poor composition of Cu3-xP, which is known to allow a wide range of compositions from 0.1 < x < 0.7. 32 Figure 1c illustrates a representative transmission electron microscopy (TEM) image of the quasi-spherical Cu3-xP nanocrystals. The nanocrystals have a mean diameter of 6.7 ± 1.1 nm (N = 300 counts), and high-resolution TEM (HR-TEM) reveals their apparent single-crystalline nature with a lattice spacing of d = 0.20 nm corresponding to the (300) planes of the expected hexagonal phase (Figure S2).</p><p>Suspensions of 1 are colloidally stable under inert atmosphere, which can be ascribed to steric stabilization from legacy surface-bound NHC ligands, as evidenced by the presence of a N 1s peak from the NHC in the X-ray photoelectron spectrum (XPS) of the isolated nanocrystals (Figure S3), with further support from solution 1 H NMR spectroscopy (vide infra).The high-resolution XPS spectrum of the Cu 2p region confirms the presence of Cu + in the Cu3-xP nanocrystals with peaks centered at the expected binding energies of Cu 2p1/2 at 952.4 eV and 2p3/2 at 932.6 eV (Figure 2a). 25 Additionally, the high-resolution XPS spectrum of the P 2p region shows characteristic binding energies for P 3-(2p1/2 at 129.2 eV, 2p3/2 at 128.4 eV), with a small amount of oxidized P species (i.e., POx) observed at a binding energy of 132.5 eV (Figure 2b). 33 The oxidative stability of the Cu3-xP nanocrystals is in agreement with previously observed results. To further investigate the synthetic role of the NHC ligand, a control reaction was carried out in which the NHC-CuBr complex was replaced by CuBr in the presence of stoichiometric oleylamine, keeping the Cu:ligand molar ratio at 1:1. The reaction was otherwise performed identically. Interestingly, this control experiment yielded nearly amorphous copper phosphide nanoparticles (2) (Figure 1b, d), which also display a broad plasmon resonance feature in the NIR (Figure S1). Elemental analysis by ICP-OES of 2 reveals an average composition of Cu1.96P, deviating from the range of reported compositions for crystalline Cu3-xP. Transmission electron micrographs of the oleylamine-stabilized particles give a similar mean diameter of 5.5 ± 1.0 nm (N = 300 counts).</p><p>This control experiment provides two insights. First, it demonstrates that copper phosphide nanoparticles (albeit quasiamorphous) can be synthesized using oleylamine as a ligand as long as it is not used in excess (i.e., Liu et al. observe the reduction of Cu + to Cu nanocrystals without phosphidation by (TMS)3P when oleylamine is used as a solvent in the absence of TOP 28 ). Second, it indicates that the NHC ligand plays a significant role in directing the crystallinity of the resulting product. Carbene-influenced nanocrystal growth is not wholly unprecedented; there exists evidence that nanoparticles synthesized in imidazolium-based ionic liquids nucleate and grow in the presence of carbene ligands formed in-situ by deprotonation of the imidazolium cation. [34][35][36] In that vein, we previously observed that when imidazolium-based ionic liquids are used to synthesize nickel phosphide nanocrystals, phase-pure Ni2P forms, as compared to a mixture of phases that results from a different reaction pathway when using octadecene instead of the ionic liquid under otherwise identical conditions. 37 To explain the disparate degrees of crystallinity observed for 1 and 2, we noted that these two reactions visibly differ in the process of nucleation --while heating the oleylamine-CuBr mixture to the 250 ˚C reaction temperature, the solution transitions from a light-yellow color of dissolved oleylamine-CuBr at low temperatures to a dark blue-black color at temperatures greater than ~215 ˚C. Upon injection of the (TMS)3P precursor at 250 ˚C, the reaction mixture immediately turns black and becomes turbid, indicating the formation of Cu3-xP. We hypothesized that the color changes that precede the injection of (TMS)3P could be indicative of Cu nanoparticle formation induced by the reduction of Cu + by oleylamine. To test this hypothesis, we performed a control experiment in the absence of any phosphorus source, in which we heated the oleylamine-CuBr mixture to 250 ˚C and then quickly cooled the reaction mixture to room temperature. The electronic absorption spectrum of the product reveals a single, broad peak in the visible region centered at 700 nm (Figure S4), attributable to LSPR absorption of Cu nanoparticles. [38][39][40] Powder XRD of the product confirms it is amorphous in nature (Figure S4). This result sheds light on the mechanism of formation of 2; injection of the very reactive (TMS)3P precursor phosphidizes amorphous Cu nanoparticles and, despite the high temperature injection, leads to a persistent quasi-amorphous phase of Cu3-xP. It is known that amorphous materials are often stabilized when containing glass-forming elements including B, C, Si or P, which may explain why structural reorganization to crystalline copper phosphide is not observed. 41 In contrast, no such cascade of color changes occurs prior to injection of (TMS)3P in the presence of the NHC ligand, indicating that copper nanoparticles do not form as intermediates. This difference is likely due to the robust Cu-C bond within the NHC-CuBr precursor (i.e., Boehme and Frenking calculated via DFT that the bond dissociation energy of the Cu-C bond of a CuCl complex of imidazole-2-ylidene falls between 67-73 kcal/mol), 42 and the absence of the primary amine reducing agent. Therefore, rather than proceeding through phosphidation of an amorphous Cu nanoparticle intermediate, the carbene precursor produces crystalline Cu3-xP directly through a metathesis reaction between NHC-CuBr and (TMS)3P to produce crystalline Cu3-xP and TMS-Br. Thus, the NHC ligand serves dual purposes, namely, it affords colloidal stability to the resulting nanocrystals and it circumvents the formation of zero-valent Cu intermediates, allowing crystalline Cu3-xP to be accessed directly. Thus, in general, using metal-carbene precursors to make metal pnictide or metal chalcogenide nanocrystals may prove to be beneficial for chemistries that are susceptible to proceeding through undesirable zero-valent intermediates.</p><p>The carbene ligand used herein binds tightly to the nanocrystal surface but is more bulky than oleylamine. 3 To determine the density of ligands on the surface of 1 and 2, thermogravimetric analysis (TGA) was performed on isolated powders of both samples (Figure S5). The ligand density was determined to be ~1.0 ligands/nm 2 for 1 and ~2.5 ligands/nm 2 for 2 (see Supporting Information for calculation details). This finding demonstrates that, despite being a stronger binding L-type ligand, the steric bulk of the carbene prevents 1 from having a higher ligand density than 2.</p><p>Due to the utility of Cu3-xP electrocatalysts for water splitting, 24,25,43 we aimed to test these carbene-stabilized Cu3-xP nanocrystals for HER to evaluate both their potential as electrocatalysts and the effect the NHC ligand has on catalysis. To evaluate the effect of the carbene on catalysis, we sought to compare the HER activity of carbene-stabilized Cu3-xP nanocrystals to oleylamine-stabilized Cu3-xP nanocrystals. However, since 2 differs both in the identity of the supporting ligand and the degree of crystallinity of the nanocrystals, a straightforward comparison of the HER activity of 1 and 2 cannot be cleanly made. Therefore, we synthesized crystalline, carbene-stabilized Cu3-xP nanocrystals and then performed a post-synthetic ligand exchange under forcing conditions by dispersing 1 in an excess of oleylamine at 85 °C for 2 h.</p><p>The 1 H NMR spectrum of 1 prior to the ligand exchange clearly indicates the binding of the carbene to the nanocrystal surface, as the signature resonances of the NHC ligand are significantly broadened compared to the NHC-CuBr precursor (Figure 3a). The 1 H NMR spectrum of the ligand-exchanged Cu3-xP nanocrystals (3) indicates that ligand exchange is quantitative under these conditions, as no sign of the signature benzimidazole resonances remains in the aromatic region. Furthermore, the two peaks in the range of 5.26-5.46 ppm are diagnostic of a dynamic equilibrium of bound/free oleylamine on the surface of the nanocrystal (Figure 3b). 44,45 Interestingly, while oleylamine binding is a dynamic process on the NMR timescale, no resonances associated with a free carbene are observed in the 1 H NMR spectrum of 1, reiterating that the carbene binds more tightly to the nanocrystal surface than oleylamine.</p><p>TGA analysis of 3 returns a higher ligand density than that of 1 or 2, at 3.3 ligands/nm 2 , which is not surprising considering the ligand exchange step was performed with a large excess of oleylamine, whereas the preparations of 1 and 2 maintained Cu:ligand ratios of 1:1. Importantly, the nanocrystals maintain their crystallinity after ligand exchange and show no statistically significant change in nanocrystal size, as verified by TEM analysis (Figure S6). Electrocatalytic HER Studies. To investigate the electrocatalytic HER activity of 1 and 3, detailed electrochemical measurements were performed in a three-electrode setup with N2saturated 0.5 M H2SO4 aqueous electrolyte. 1 and 3 were deposited onto glassy carbon electrodes by directly drop-casting the respective hexane suspensions without further modifications. In general, 1 exhibits better overall HER activity than 3 (Figure 4 and Table 1); that is, the overpotential to reach 10 mA/cm 2 current density is 0.45 V for 1 and 0.86 V for 3, and 1 shows a much lower Tafel slope of 85 mV/dec as compared to the 228 mV/dec for 3. The superior activity of 1 is correlated with a lower charge transport resistance (Rct), as revealed by the electrochemical impedance spectroscopy (EIS). Figures S8 and S9 show the EIS responses of 1 and 3, which are described by a two-time constant parallel model (Figure S7). In both cases, the Rct value decreases at larger overpotentials (Table S1), as expected for an enhanced HER rate with increased catalytic driving force, and 1 exhibits lower Rct values in comparison to 3.</p><p>For instance, at an overpotential of 0.44 V, the Rct of 1 is only 19.7 W, whereas the Rct of 3 is 951 W at an overpotential of 0.55 V. The lower Rct and Tafel slope of 1 suggest that the HER kinetics are much more favored on the surface of 1 than 3.</p><p>Double layer capacitance (Cdl) measurements were performed to obtain the electrochemical active surface area (ECSA) of the catalyst-modified electrodes. As shown in Figure S10, 1 displays a much higher Cdl (125.6 µF) than 3 (25.0 µF), indicating a larger ECSA. This is consistent with the fact that the surface ligand density of 3 is higher than 1, as mentioned previously. To achieve a more equitable comparison between the two electrocatalysts, we normalized the catalytic current by the ECSA, instead of the geometric surface area of the electrode (0.196 cm 2 ). As shown in Figure 4b, upon normalization by ECSA, the superior activity of 1 is maintained -there is still a 0.33 V overpotential difference at 3 mA/cm 2 ECSA that is not accounted for by the ECSA alone. Therefore, 1 displays higher HER activity than 3 not only because of its larger number of active sites, but also due to the higher intrinsic activity of individual sites.</p><p>The superior HER activity of 1 suggests that the carbene ligands afford several benefits over oleylamine ligands for catalysis. First, the lower surface ligand coverage of 1 not only helps to expose more catalytically active sites, but also make the interfacial electron transfer between the electrode and the electrolyte more efficient. This is evidenced by a lower solution resistance (Rs) of 1 as compared to 3 (Tables S1, S2), which is affected by the electrical resistance of the catalyst layer. Second, it is also possible that the NHC ligands have a beneficial electronic effect at the nanocrystal 81.3 ± 0.5 72.1 ± 0.1 a ECSA = Cdl/Cs, Cs = 0.035 mF/cm 2 . 46,47 surface that helps to enhance the catalytic activity. Cao and coworkers previously reported on the enhanced electrocatalytic CO2 reduction activity of a NHC-functionalized Au nanoparticle catalyst. 17 Based on the different Tafel slopes of the carbenefunctionalized Au nanoparticles (72 mV/dec) and the parent Au nanoparticles (138 mV/dec), they concluded that the strong σdonation electronic effect from the carbenes alters the electron density at the gold surface, which induces a shift in the ratedetermining step for CO2 reduction. We observe a similar phenomenon here, where 1 exhibits a lower Tafel slope in comparison to 3 (85 mV/dec vs. 228 mV/dec, Figure S11). From this, we can deduce that for 1, the rate-determining step is likely the Heyrovsky or Tafel step, whereas for 3, the rate-determining step is likely the Volmer step. 48 However, it is noteworthy that the as-measured Tafel slopes might contain contributions from other sources that are not related to HER kinetics, such as the uncompensated resistance originating from the charge transfer between the electrode substrate and the catalyst. 49 Even so, such uncompensated contributions likely would not account for the large difference in the measured Tafel slopes of 1 and 3, maintaining that the rate-determining step of HER is different for these two catalysts.</p><p>Controlled potential electrolysis (CPE) experiments were performed to investigate the long-term stabilities of the materials as well as their Faradaic efficiency (FE) for hydrogen production. For 1, the current response remains relatively steady over the course of 1 h under an applied potential of -0.59 V vs. RHE (Figures S12a), with slight fluctuations caused by the evolution of bubbles on the electrode surface. However, when comparing the Cdl and EIS responses before and after CPE, it is found that the ECSA of the catalyst was reduced from 46.4 cm 2 to 6.8 cm 2 which was accompanied by a decrease in Rct from 14.3 W to 7.5 W (Figure S12, Table S3). The FE for hydrogen production is 81.3 ± 0.5% for 1, calculated by comparing the total charge passed and the amount of H2 produced over the period of 1 h. The change in ECSA and Rct during electrolysis and the less-than-unity FE is potentially a consequence of partial surface ligand desorption, a phenomenon that has been observed for other electrocatalytic nanomaterials. 50,51 Indeed, the TEM image of the post-electrolysis 1 reveals sintering of the nanoparticles (Figure S14a). On the other hand, the FE of 3 is only 72.1 ± 0.1%. The Rct value is massively reduced after electrolysis (from 241 W to 11.3 W), along with a minimal decrease in the ECSA (from 1.9 cm 2 to 1.6 cm 2 ). Similarly, these behaviors are attributed to the loss of surface ligands, but to a larger extent in comparison to 1. Since the oleylamine ligands have a higher surface ligand density and do not bind as tightly as the carbene ligands, a greater loss of the oleylamine ligands is expected, which leads to a lower FE. TEM images of the ligandexchanged nanocrystals (3) following CPE also reveals sintering (Figure S14b). While recent studies of metal phosphide nanocrystal HER catalysts lack reports of Faradaic efficiencies, 24,52 we believe another potential source of Faradaic losses for these catalysts may originate from the reduction of surface oxidized species that form upon exposure to air. Indeed, XPS of 1 indicates the presence of POx species on the nanocrystal surface prior to catalysis (Figure 2b). Density Functional Theory Calculations. Density functional theory (DFT) calculations were performed to probe the electronic effects of NHC and alkylamine ligands on proton binding to a Cu3P catalyst. A model Cu9P3 cluster with one surface ligand (either 1,3-(dimethyl)benzimidazol-2-ylidene or methylamine, see Figure S15) bound to a copper atom was used to represent catalysts 1 and 3, respectively. The energies of proton binding to an adjacent surface site on these clusters were then calculated and the physical origins of catalyst-proton interactions were examined using the second generation absolutely localized molecular orbital energy decomposition analysis (ALMO-EDA, referred to as EDA in this text). [53][54][55] Binding energies reveal that the proton prefers to bind at Cu-Cu bridge sites rather than P centers. When comparing the proton binding energies for NHC-and amine-ligated clusters, it was found that proton binding to the NHC-ligated cluster was favored by 29.7 kJ/mol (7.1 kcal/mol). EDA showed that cluster-proton interactions are dominated by charge transfer and polarization terms, with smaller, repulsive contributions from electrostatics (see Table S4). While charge transfer and polarization contributions are largely similar, electrostatic contributions are smaller for the NHC-ligated cluster than the amineligated cluster. This phenomenon likely arises as a consequence of stronger σ-donation from the NHC ligand to the Cu + center, which results in diminished electrostatic repulsion between Cu + and the proton.</p><p>The calculations suggest that the weaker electrostatic repulsions lead to more favorable proton binding for NHC-bound clusters. During catalysis, more favorable proton binding should speed up the rate of the Volmer step for NHC-ligated nanocrystals. This result aligns well with our experimental Tafel analysis wherein 1 exhibits a low Tafel slope, indicating that the Volmer step is fast and is not likely the rate-determining step. In contrast, 3 displays a higher Tafel slope characteristic of a slow, rate-determining Volmer step. Therefore, the nature of the L-type ligand on the surface of our Cu3-xP nanocrystal catalysts could indeed be contributing an electronic influence on catalytic activity and the rate-determining step.</p><!><p>In conclusion, we report a new synthesis that directly produces crystalline, NHC-stabilized Cu3-xP nanocrystals. This represents the first example of a carbene-stabilized metal phosphide nanocrystal. The NHC-stabilized Cu3-xP nanocrystals exhibit better HER activity compared to their oleylamine-capped counterparts with respect to the onset of catalysis, Tafel slope, and overpotential to reach 10 mA/cm 2 current density. DFT calculations suggest that the NHC ligands change the electronics of the copper phosphide catalyst surface by reducing unfavorable electrostatics, which may contribute to the superior catalytic for 10 min and centrifuged for 5 min at 6000 rpm. An additional partial wash as described above was performed before dispersing the particles in TCE for UV-vis-NIR or in hexanes to be dropcast for powder XRD.</p><p>Oleylamine-stabilized Cu3-xP nanocrystals via ligand exchange. NHC-capped Cu3-xP nanocrystals were synthesized as described in the main text and were then washed twice in 40 mL centrifuge tubes with acetone by sonication and centrifugation. Then, the product was redispersed in 5 mL of dry toluene. This suspension was added to 20 mL of oleylamine in a 100 mL three neck round bottom flask. The solution was then heated to 85 °C and was stirred for 2 h under a N2 atmosphere. The product was then washed in the same manner described in the main text prior to electrochemical testing and characterization.</p>

ChemRxiv

Minor Structural Variations of Small Molecules Tune Regulatory Activities toward Pathological Factors in Alzheimer\xe2\x80\x99s disease

Chemical tools have been valuable for establishing a better understanding of the relationships between metal ion dyshomeostasis, the abnormal aggregation and accumulation of amyloid-\xce\xb2 (A\xce\xb2), and oxidative stress in Alzheimer\xe2\x80\x99s disease (AD). Still, very little information is available to correlate the structures of chemical tools with specific reactivities used to uncover such relationships. Recently, slight structural variations to the framework of a chemical tool were found to drastically determine the tool\xe2\x80\x99s reactivities toward multiple pathological facets to various extents. Herein, we report our rational design and characterization of a structural series to illustrate the extent to which the reactivities of small molecules vary toward different targets as a result of minor structural modifications. These compounds were rationally and systematically modified based on consideration of properties, including ionization potentials and metal binding, to afford their desired reactivities with metal-free or metal-bound A\xce\xb2, reactive oxygen species (ROS), and free organic radicals. Our results show that although small molecules are structurally similar, they can interact with multiple factors associated with AD pathogenesis and alleviate their reactivities to different degrees. Together, our studies demonstrate the rational structure-directed design that can be used to develop chemical tools capable of regulating individual or interrelated pathological features in AD.

minor_structural_variations_of_small_molecules_tune_regulatory_activities_toward_pathological_factor

Introduction<!>Rational design consideration and characterization of chemical tools for regulating AD-related pathological factors<!>Modulation of metal-free A\xce\xb2 and metal-A\xce\xb2 aggregation<!>Metal-free and CuII-treated A\xce\xb2 monomers<!>Monomeric ZnII-A\xce\xb2<!>Regulation of oxidative stress<!>Abatement of toxicity induced by metal-free and metal-treated A\xce\xb2<!>Conclusions<!>Experimental Section

<p>Effective diagnostic tools and treatments for neurodegenerative diseases have been unavailable to date due to multiple aspects. One reason is the lack of our understanding of the underlying pathogenesis required to develop such medical interventions.[1] This is highlighted in Alzheimer's disease (AD) where, despite being one of the better-studied neurodegenerative diseases, the etiology is still unclear.[1] The current understanding of AD implicates multiple factors that could be interrelated.[1a–c,e,g,2] Recently, an area of particular high interest is the interconnection between metal ion dyshomeostasis, the abnormal aggregation and accumulation of an intrinsically disordered protein (IDP) [i.e., amyloid-β (Aβ)], and increased oxidative stress in the brain. Metal ions have been suggested to be central to this interrelationship as several biologically relevant metal ions [e.g., FeII/III, CuI/II, ZnII] have been shown in vitro to bind to Aβ and influence its aggregation and conformation.[1a–c,e,2a–e] Additionally, the coordination of Aβ to redox-active metal ions, FeII/III and CuI/II, has been shown to facilitate the production of reactive oxygen species (ROS) through Fenton-like reactions.[1a–c,e,2a–c,e,3]</p><p>To elucidate this interrelationship in depth, chemical tools have been developed to target individual or interrelated factors and modulate their reactivities.[1a,b,4] These tools and potential therapeutics include approaches that use organic, inorganic, peptide, and antibody frameworks.[1a–c,e–h,4] We recently reported the development of four small molecules (1–4; Table 1) which have different modes of action, despite being structurally similar, for targeting and controlling the aggregation of metal-free Aβ or metal-Aβ as well as the formation of ROS overall diminishing toxicity.[5] Herein, we report the overall investigations of the small molecules (1–9; Table 1) rationally designed to tune the reactivities with differing targets (i.e., metal ions, metal-free Aβ, metal-Aβ, ROS, free organic radicals) by performing slight structural modifications to a common structural framework. Through chemical, biochemical, biophysical, and computational studies, we demonstrate that 1–9 have structure-dependent capabilities to modulate the reactivities with their targets. The target specificity of these compounds (i.e., reactivity directed toward metal-free Aβ and/or metal-Aβ) have been indicated to be associated with their redox properties:[5] 1) The compounds that undergo oxidation relatively easily suppress the reactivity of metal-Aβ over metal-free Aβ (1, 3, 5, 6, 8, and 9) or both metal-free and metal-bound Aβ (4); 2) the compounds (2 and 7) that are more difficult to oxidize indicate limited abilities to control the reactivity of Aβ in the absence and presence of metal ions. In addition, the activity of our small molecules toward metal [CuII or ZnII]-Aβ was indicated to be correlated to the formation of compound-metal-Aβ ternary complexes. In particular, with respect to interactions with ZnII-Aβ, the metal binding affinity of the compounds for ZnII was found to be critical for promoting such reactivity. Moreover, the activity of 4 with both metal-free Aβ and metal-Aβ could be linked to its degradation to a known Aβ modulator, N,N-dimethyl-p-phenylenediamine (DMPD).[6] Taken together, our studies presented herein demonstrate the feasibility of developing chemical tools toward individual or interrelated pathological factors in AD, through considerations of the electrochemical, metal binding, and biological properties of a series of structurally similar molecules to generate a rational structure-directed design approach. The insight gained from these studies, particularly those relating the redox properties of our small molecules to their anti-amyloidogenic activity will open new, innovative approaches to devise new chemical reagents able to target and mitigate specific or intercommunicated pathogenic features shown in neurodegenerative diseases.</p><!><p>To develop small molecules able to interact with distinct and interrelated targets (i.e., metals, metal-free Aβ, metal-Aβ, ROS, free organic radicals) and modulate their reactivities (i.e., peptide aggregation, generation of toxic peptide conformations, oxidative stress), a library of small molecules was rationally designed based on the previously reported metal-Aβ-interacting framework (L2-b,[7] Table 1). In our initial studies of 1–4 (Table 1), the electron-donating properties of the aniline (in 1) and dimethylamine (in 3) groups were suggested to be important in the formation of their radical forms required for activity toward their targets.[5] In our complete chemical series, new compounds (5–9; Table 1) were included to contain electron-donating or electron-withdrawing groups to tune ionization potentials (IPs) of the basic framework affording distinct activities with targets: 1) a diethylamino (for 5) or 4-morpholino (for 6) group was installed in 5 and 6, respectively, compared with the dimethylamino component of 8, 9, and L2-b; 2) compound 7, composed of an electron-withdrawing nitro moiety, was constructed to decrease the compound's ability to oxidize similar to 2,[5] which contains a 3,5-dimethoxybenzene moiety and is relatively stable even in the presence of CuII. These compounds were relatively easily obtained and purified (see Supporting Information).</p><p>To confirm our rational design principle, computational calculations were first performed to predict the first and second electron ionization potentials (IP1 and IP2, respectively) of the two-electron oxidation of our compounds (1–9) as a method to compare their relative ability to undergo oxidation. As summarized in Table 1, the compounds (5, 6, 8, and 9) designed to undergo oxidation have similar or lower computed IPs to the previously determined values for 1,[5] 3,[5] 4,[5] and L2-b[8] suggesting that 1, 3–6, 8, and 9 can produce radicals to be necessary for activity. Furthermore, as expected, the computational studies indicate that 7, along with 2, are more difficult to be oxidized than the other compounds (Table 1). To experimentally support these computational findings, the electrochemical behavior of 1–9 was probed by cyclic voltammetry in dimethyl sulfoxide (DMSO). All compounds with the exception of 1 and 7 showed single irreversible oxidation waves (Figure 1). Compound 1 exhibited two irreversible oxidation waves with peak anodic potentials of 0.262 and 0.521 V (at a scan rate of 250 mVs−1), respectively (Figure 1 and Figure S1 in the Supporting Information). Due to the irreversible nature of the electrochemical waves in DMSO, E1/2 values could not be obtained; however, the peak anodic potentials of compounds 1, 3–6, 8, and 9 occurred at much lower potentials (i.e., 0.262, 0.284, 0.240, 0.228, 0.388, 0.286, and 0.238 V, respectively, at a scan rate of 250 mVs−1) than that of 2 (i.e., 0.923 V at a scan rate of 250 mVs−1) (Figure 1 and Table S1 in the Supporting Information). Compound 7 did not exhibit any oxidation waves over a potential window of −0.20 to 1.4 V. The compounds (i.e., 1, 3–6, 8, and 9) that are more easily oxidized have peak anodic potentials compared to a water-soluble analogue of vitamin E, Trolox (~0.3 V),[9a] as well as a structurally similar compound, p-phenylenediamine (~0.4 V).[9b,c] Likewise, the compounds, i.e., 2 and 7, that are more difficult to oxidize have higher peak anodic potentials more similar to acetaminophen (~0.6 V).[9d] As a way to compare 1–9 to other compounds, we also employed the commonly used the Trolox Equivalence Antioxidant Capacity (TEAC) assay (see below), directly correlated to the activity of Trolox.[5] Overall, our electrochemical studies are consistent with our calculation predictions that 2 and 7 are relatively more difficult to oxidize than 1, 3–6, 8, and 9; thus, they are less likely to generate the radical form required for reactivity with metal-free Aβ and metal-bound Aβ species.</p><p>In addition, because the oxidation of our compounds is associated with their binding to CuII, as indicated from our previous report,[5] 8 and 9 were constructed to modulate metal binding affinities to allow for more control of their activity. The steric hindrance from the methyl group on the bridging carbon of 8 can cause the pyridyl nitrogen and the secondary amine to align to provide a metal binding site, affording enhanced metal binding. Compound 3 with a quinoline group can have a slightly lower metal binding affinity than the compounds containing a pyridine moiety, while 9 was designed with a methylimidazole moiety to have a stronger metal binding affinity, on the basis of their relative Lewis basicities as reflected in their pKa values.[10]</p><p>Furthermore, the ability for small molecules to passively diffuse across the blood–brain barrier (BBB) was also considered in our tool design. The BBB permeability of 1–9 was predicted by adhering to Lipinski's rules, along with calculated logBB values, as well as using the in vitro parallel artificial membrane permeability assay adapted for assessing BBB permeability (PAMPA-BBB). All values for 1–9 were then compared with that of L2-b (Table S2 in the Supporting Information).[5,7b] The overall data indicate that our compounds can passively diffuse across the BBB, suggesting their potential application in the brain as chemical tools.</p><!><p>The ability of 1–9 to prevent Aβ aggregation (inhibition experiment; Figure 2 and Figure S2 in the Supporting Information) and disassemble preformed Aβ aggregates (disaggregation experiment; Figures S3 and S4 in the Supporting Information) in the absence and presence of metal ions [i.e., CuII and ZnII] were evaluated at both short (4 h) and longer (24 h) incubation time points. Note that 1–4 were previously studied only after the 24 h incubation time[5] and the new results of 1–4 at the 4 h incubation time interval were included in this present work for the purpose of a comprehensive comparison with those of the other compounds. Two isoforms of Aβ (Aβ40 and Aβ42) were used for our studies because their aggregation pathways have been suggested to occur through different mechanisms.[11] Size distributions and morphologies of the resulting Aβ species were analyzed by gel electrophoresis followed by western blot analysis (gel/western blot) and transmission electron microscopy (TEM), respectively.</p><p>Compound 4 was found to be the only molecule capable of interacting with both metal-free and metal-associated Aβ with more noticeable changes effected at the shorter time point (4 h incubation) in metal-free Aβ42 inhibition experiments and Aβ40 disaggregation experiments (Figure 2; Figures S2-S4 in the Supporting Information). For 3, 5, and 9, the aggregation of CuII-Aβ and ZnII-Aβ was redirected selectively over metal-free Aβ aggregation. Additionally, these compounds had a stronger effect at later time points with their activity toward ZnII-Aβ being negligible at the early incubation time point. This is similar to the previously reported activity of L2-b.[7] Compounds 1, 2, and 6 could only modulate CuII-Aβ aggregation to different extents. Compound 1 has a more noticeable effect on CuII-Aβ aggregation at the longer time point (24 h incubation) while 6 influences CuII-Aβ aggregation more noticeably at 4 h in both inhibition and disaggregation experiments (Figure 2; Figures S2–S4 in the Supporting Information). Compound 8 is shown to have the unique activity of having a more prominent action at the earlier incubation time point toward CuII-Aβ aggregation in the inhibition experiments but the opposite occurred in the disaggregation experiments where its ability to break up preformed CuII-Aβ aggregates was more detectable after 24 h. In addition, 8 is indicated to have influence on preformed ZnII-Aβ aggregates upon 24 h incubation (Figure S4 in the Supporting Information). In both the inhibition and disaggregation experiments, 2 does not have a prominent effect on CuII-Aβ aggregation with a stoichiometric ratio of CuII and Aβ; however, previous studies[5] indicate that 2 could control CuII-Aβ aggregation at higher CuII concentrations. Compound 7 also does not have significant effects on metal-free and metal-Aβ aggregation (Figure 2; Figures S2–S4 in the Supporting Information). Unlike 2, however, even at higher CuII and ZnII concentrations, 7 does not have an apparent influence on both metal-free and metal-induced Aβ aggregation (Figure S5 in the Supporting Information).</p><p>TEM studies were carried out to visualize the morphology of the resulting Aβ aggregates after 24 h incubation with 5, 6, and 8. These compounds were chosen for the further TEM analysis based on their differing activity toward metal-free and metal-Aβ in the gel/western blot experiments. Compounds 5 and 8 are shown to react with metal-Aβ while 6 is specific for CuII-Aβ. In agreement with the gel/western blot experiments, 5 does not have an impact on the Aβ forms in the absence of metal ions. In the presence of CuII and ZnII, however, 5 produces less structured, more amorphous Aβ aggregates (Figure 2; Figures S2–S4 in the Supporting Information). Similar results were obtained in the metal-free and CuII samples containing 6; however, there was not a significant effect on the Aβ species present in the ZnII-containing samples. In the case of 8, TEM was only conducted on the 8-treated CuII-Aβ samples to help understand the difference in the varied reactivity in both inhibition and disaggregation experiments (see above). Despite showing limited activity in the gel/western blot studies from the 24 h inhibition samples, long and thick fibrils, indicated in the control samples, are not present after treatment with 8 (Figure S2 in the Supporting Information). This suggests that 8 could redirect peptides into larger species that cannot penetrate into the gels. While in disaggregation samples, where gel/western blots showed activity, 8 could mainly produce shorter fibrils (Figure S4 in the Supporting Information). As a whole, our peptide aggregation experiments demonstrate that our small molecules have varying abilities to control metal-free and metal-induced Aβ aggregation to different extents.</p><!><p>15N-Labeled Aβ40 monomer was titrated with 1–9 (0 to 10 equiv) and monitored by two dimensional (2D) 1H-15N band-selective optimized flip-angle short-transient (SOFAST)-heteronuclear multiple quantum coherence (HMQC) nuclear magnetic resonance (NMR) spectroscopy to observe the amino acid residues of Aβ that are targeted by 1–9 (Figure 3 and Figure S6 in the Supporting Information). From our 2D SOFAST-HMQC NMR results, 1–9 are shown to trigger relatively low chemical shift perturbations (CSPs), similar to the previous studies with L2-b and L2-NO,[12] suggesting weak, nonspecific interactions with Aβ. This is also indicated for 4, which does show the reactivity toward metal-free Aβ, suggesting that its hydrolysis to produce DMPD[6] is required before interaction and subsequent aggregation modulation.[5]</p><p>To explore the interactions between Aβ40 and 5–9, nanoelectrospray ionization-mass spectrometry (nESI-MS) combined with ion mobility-mass spectrometry (IM-MS), optimized for the detection of non-covalent protein complexes, was used.[13] Data obtained from the CuII–treated peptide samples incubated with 5–9 for 30 min (37°C), presented in Figure 4, indicated that 5, 6 and 9 exhibited a metal-dependent interaction with Aβ40. All three of these compounds were capable of producing Cu-ligand-dependent signals corresponding to a mass 89 Da lighter than the apo Aβ40, with clear differences in product abundance, and are absent under the ligand-free conditions. These data are consistent with some small molecules reported previously.[5,7a] In the absence of CuII, no evidence of the compounds' interactions was observed, which is in agreement with the findings obtained from our aggregation experiments (Figure S7 in the Supporting Information). As expected from our gel/western blot studies (Figure 2; Figures S2–S5 in the Supporting Information), 7 is indicated to have no interactions with both metal-free Aβ and metal-Aβ, which could be related to its absence of noticeable anti-amyloidogenic activity in vitro. Along with other data presented here, these observations support that 8 likely targets higher order, more transient oligomeric species that cannot be resolved by IM-MS. Consistent with these observations, whilst attempts to observe CuII-small molecule-bound Aβ40 dimers were carried out, the data proved to be inconclusive due to poor signal-to-noise levels associated with increased metal adduct formation within the Aβ aggregate states.</p><!><p>Because 1[5] (only for CuII-Aβ) and L2-b[7] (for both CuII- and ZnII-Aβ) are demonstrated to bind ZnII but have differing activities toward modulation of ZnII-Aβ aggregation, their binding affinities (Kd) were compared. UV/Vis variable-pH spectrophotometric titrations were carried out with a mixture of ZnII and 1 (Figure S8 in the Supporting Information). This titration experiment indicates the presence of a 1:1 complex under the experimental conditions having a stability constant (logb) of 5.5(3). Based on this value and the previously determined pKa values of 1, the pZn (pZn=−log [Znunchelated]) was calculated to be 5.5, which is an approximate disassociation constant (Kd, 10−5.5m ≈ [Znunchelated]) (Figure S8 in the Supporting Information). Compared to 1, L2-b is shown to have an apparent Kd of 10−6·1m for ZnII indicating the generation of 1:1 and 1:2 ZnII-to-ligand complexes.[7b] The high micromolar binding affinity of 1 for ZnII is not enough to control ZnII-Aβ aggregation, since Aβ itself is indicated to have the micromolar to nanomolar Kd for ZnII.[1a–c] As previously reported, the minimum binding affinity of small molecules for ZnII is in the micromolar or lower range in order to control ZnII-Aβ aggregation.[1a–c] This is further supported by our previous studies with 2 which also does not modulate ZnII-Aβ aggregation where the Kd for CuII is reported to be micro-molar.[5] It is expected that 2 would have an even lower affinity for ZnII due to the trends observed in the Irving–Williams series.[14] Thus, 2, along with 1, has a binding affinity for ZnII that is too low to interact with ZnII-Aβ.</p><p>2D SOFAST-HMQC NMR studies were conducted to further understand the interaction of 1, 4, and 5 with ZnII-15N-labeled Aβ40 monomer (Figure 5 and Figure S9 in the Supporting Information). These compounds were chosen due to their differing abilities to interact with ZnII-Aβ. Compound 1 was indicated to have limited reactivity toward ZnII-Aβ, while 4 and 5 were shown to be able to redirect the aggregation of Aβ in the presence of ZnII (see below). As expected, 1 does not demonstrate an ability to interact with ZnII-Aβ as evidenced by limited changes in intensity upon treatment with the compound (Figure 5 and Figure S9 in the Supporting Information). Compound 4 also does not cause significant changes in the spectra, which could be due to this molecule having only a limited initial interaction with ZnII-Aβ or its degradation. Interestingly, unlike the other compounds which are able to reverse the decrease in signal intensity caused by ZnII-induced aggregation of Aβ, 5 had the opposite effect as addition to ZnII-Aβ further decreased the intensity of all residues. This could suggest that 5 may mediate the formation of NMR-invisible oligomers. This is in contrast to L2-b which demonstrates larger changes in the NMR spectra of ZnII-Aβ[7b,8] denoting that L2-b can directly interact with ZnII-Aβ possibly forming a ternary complex and subsequently control the ZnII-Aβ aggregation pathways.</p><p>Overall, the studies of our small molecules with ZnII or ZnII-Aβ suggest that our compounds which do not modulate ZnII-Aβ aggregation (1, 2, 6, and 7) do not have significant interactions with ZnII-Aβ most likely due to low binding affinities for ZnII. In contrast, the compounds which have an effect on ZnII-Aβ aggregation (3, 5, 8, and 9) could have the affinities for ZnII, which directs the formation of ternary complexes with ZnII-Aβ. In the case of 4, this compound can undergo hydrolysis in the presence of ZnII to produce DMPD,[5,6] a known ZnII-Aβ-interacting compound.</p><!><p>The activity of 5–9 to mediate oxidative stress was investigated (Figure 6a,b). First, the ability to scavenge free organic radicals was examined using the TEAC assay[5,7a,15] (Figure 6a). Compounds 5, 6, 8, and 9 display a greater antioxidant capacity than Trolox [5 and 9, slightly higher (~1.5 and 1.7, respectively); 6 and 8, much higher (~3.2 and 2.0, respectively)]. This is similar to the previously reported antioxidant capacities of 1–4[5] and L2-b.[7a,12] Conversely, 7 is not found to have an effect toward free organic radicals (Figure 6a) which can be attributed to its absence of observable anodic waves over a large potential window and its high calculated IPs as shown in Figure 1 and Table 1, respectively.</p><p>The ability of 5–9 to decrease the production of ROS by Fenton-like reactions promoted by CuI/II was examined using the 2-deoxyribose assay (Figure 6b).[16] Compounds 8 and 9 are found to better control the generation of hydroxyl radicals (·OH) than the previously studied 1–3[5] (~50% production inhibited) with 9 being the best compound studied (~75% production inhibited; Figure 6b). Compounds 5 and 7 have comparably lower abilities to prevent the formation of ROS being able to reduce the presence of ·OH by only ~30 and 20%, respectively. Therefore, our small molecules are able to quench free organic radicals as well as control ROS generation.</p><!><p>Based on the different activities toward redirection of metal-free Aβ and metal-Aβ aggregation and ability to mediate oxidative stress, 5–9 were chosen for further analysis of their toxicity in human neuroblastoma SK-N-BE(2)-M17 (M17) cells. Compounds 5 and 7 (20 μm) are found to decrease cell viability by ~30% in the absence or presence of metal ions [CuII (20 μm) or ZnII (20 mm)] (Figure 6c). Under the same conditions, 6, 8, and 9 are shown to be relatively nontoxic with 9 slightly decreasing cell viability to ~85% in the presence of CuII. Furthermore, 6, 8, and 9 are able to mitigate the toxicity of Aβ40 (Figure 6d) and Aβ42 (Figure 6e) in the absence (left) and presence of externally introduced CuII (middle) and ZnII (right). Thus, 6, 8, and 9 are observed to reduce the cytotoxicity of metal-Aβ.</p><!><p>Inspired by initial studies regarding molecular modes of action of structurally similar compounds toward multiple factors (i.e., metals, metal-free Aβ, metal-Aβ, ROS, free organic radicals) involved in AD pathogenesis, a chemical library of small molecules was constructed to elucidate how structural variations could direct their regulatory activities for pathological targets. Our studies indicate the specificity for tempering the reactivity of CuII-Aβ over ZnII-Aβ and metal-free Aβ for 1, 2, and 6; metal-Aβ versus metal-free Aβ for 3, 5, 8, 9, and L2-b; no specificity for 4 (modulating with Aβ regardless if metal ions are or are not present); having no detectable effects of 7 for both metal-free and metal-bound Aβ. We show that these reactivity behaviors can be imparted with consideration of the electrochemical characteristics and metal binding affinities of such small molecules. Furthermore, we present that these compounds have varying capabilities to diminish oxidative stress with 6 being the best antioxidant and 9 having the greatest control of ROS production from Fenton-like reactions, in contrast to 7 which has little antioxidant capacity and regulation of ROS formation. Additionally, the toxicity of metal-free Aβ and metal-Aβ was found to be diminished by 6, 8, and 9 in living cells.</p><p>Taken together, we demonstrate the feasibility of inventing chemical tools directed at investigating the involvement of specific facets of AD through tuning their oxidation and metal binding properties. The knowledge gained from our complete studies using a series of small molecules (1–9) will aid in the development of novel chemical tools with optimal properties. These optimal characteristics, in addition to good pharmacological features, include metal binding affinities that are strong enough to interact with metal-Aβ species but do not disrupt the activities of essential metalloproteins, the specificity for the particular metal ions of interest, as well as the ability to interact with metal-free and metal-bound Aβ species. By developing methods to rationally generate chemical tools that have different capabilities and combinations of capabilities, a more complete chemical tool set can be developed for identifying the pathogenesis of neurodegenerative diseases at the molecular level.</p><!><p>All reagents were purchased from commercial suppliers and used as received unless otherwise noted. Aβ40 and Aβ42 (the sequence of Aβ42: DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA) were purchased from Anaspec Inc. (Fremont, CA, USA). Trace metals were removed from buffers and solutions used in Aβ experiments by treating with Chelex overnight (Sigma–Aldrich, St. Louis, MO, USA). Optical spectra were recorded on an Agilent 8453 UV-visible (UV/Vis) spectrophotometer. Absorbance values for biological assays, including cell viability and antioxidant assays, were measured on a Molecular Devices SpectraMax 190 microplate reader (Sunnyvale, CA, USA). 1H and 13C 1D spectra were recorded using a 400 MHz Agilent NMR spectrometer. 2D NMR spectra were acquired on a Bruker Advance 600 MHz spectrometer equipped with a cryoprobe. More detailed experiments, including the preparation and characterization of the compounds, are described in the Supporting Information.</p>

PubMed Author Manuscript

In-Depth Lipidome Annotation Through an Operatively Simple Method Combining Cross-Metathesis Reaction and Tandem Mass Spectrometry

The in-depth knowledge of lipid biological functions calls for a comprehensive lipid structure annotation that implies implementing a method to locate fatty acids unsaturations, which remains a thorny problem. To address this challenge we have associated Grubbs' cross-metathesis reaction and liquid chromatography hyphenated to tandem mass spectrometry. Thus the pretreatment of lipids-containing samples by Grubbs' catalyst and an appropriate alkene generates substituted lipids through cross-metathesis reaction under mild, chemoselective, and highly reproducible conditions. A systematic LC-MS/MS analysis of the reaction mixture allows locating unambiguously the double bonds in fatty acid side chains. This method has been successfully applied at a nanomole scale to commercial standard mixtures consisting of 10 lipid subclasses as well as in lipid extracts from an in vitro model of corneal toxicity.

in-depth_lipidome_annotation_through_an_operatively_simple_method_combining_cross-metathesis_reactio

<p>Lipids are an essential class of metabolites playing a pleiotropic role in cells depending on their structures, the latter being actually very variable. [1,2] According to the international LipidMaps consortium, molecules arising from isoprene subunits condensation define the sterols or prenol lipid class, while lipids resulting from ketoacyl subunits condensation belong to the classes of fatty acids (FA), glycerolipids (GL), glycerophospholipids (PL) and sphingolipids (SL). [3,4] Within these latter classes, the structural diversity mainly originates from the nature of the fatty acids i.e. the chain length and the number and location of unsaturations. The location of double bonds in the FA chain is of prime relevance as in most cases it determines the biological activity of a given lipid. [5,6] For example, -3 polyunsaturated FAs (PUFA) display antiinflammatory properties, while in contrast, -6 ones mainly act as pro-inflammatory metabolites. In ocular surface diseases, -3 PUFA has been shown to increase corneal wound healing and nerve regeneration through docosanoids biosynthesis. [7,8] Indeed, the use of -3-based therapy has been purposed specifically in dry eye disease, both topically and per os. Regarding mono-unsaturated fatty acid (MUFA) species, a shift from Δ9 to Δ11 C=C isomers in phosphatidylcholines (PC) and phosphatidylethanolamines (PE) containing oleic acid (18:1), has been reported in the plasma of type 2 diabetic patients. [9] Consequently, to clarify the role of lipids in physiological processes, it is indispensable to determine unambiguously the location of the double bond in FA chains of lipids. For two decades, mass spectrometry (MS) has been increasingly used to study lipids. [10] Lipidomic analysis mainly uses liquid chromatography hyphenated to electrospray high resolution tandem mass spectrometry (LC-MS/MS). [11] Indeed, LC-MS/MS analysis is highly suitable to identify polar head groups, and therefore lipid subclasses but also FA chains. [11,12] However, LC-MS/MS alone can't determine position isomers when unsaturated FAs are involved, [13] unless using sophisticated analytical approaches implementing ion activation technics, such as ultraviolet-photodissociation (UVPD) or ozone-induced dissociation (OzID). [14,15] An online derivatization based on Paterno-Buchï (PB) photochemical reaction prior to MS analysis has also been successfully applied. [9,16] Nevertheless, these online sample preparation methods need technical adaptations of the mass spectrometer to be routinely used: UVPD, an hybrid MS 3 CID/UV mass spectrometer, OzID, the introduction of ozone within the collision cell and PB, the use of an UV lamp inside the ion source. Regarding offline sample preparation, a derivatization step based on an epoxidation reaction has been proposed to identify double bond lipid isomers. [17] Albeit very suitable for mono-unsaturated fatty acid (MUFA) species, this approach did not allow identifying double bond isomers in PUFA species readily. Furthermore, the above-mentioned methods implement harsh conditions that impede their chemoselectivity. In contrary, cross-metathesis (CM) is a highly chemoselective, functional group tolerant reaction using mild conditions, so it appeared to us particularly suitable for lipidomic analysis. Indeed, biologically occurring MUFA and PUFA species contain one or several but-2-en-1,4-ylidene moieties, these alkenes therefore belong with category II in the Grubbs' CM classification. [18] They are thus expected to react readily with Ru carbene complexes such as the commercially available secondgeneration Grubbs' catalyst giving CM product easily. It can be anticipated that from the MS analysis of CM products the location of lipid double bonds would be easily and unequivocally determine (Scheme 1). In a previous study, double bond location was determined in FA species using CM and mass spectrometry. [19] However, the method described in this study was designed for a use on pure FA samples at the mg scale, so it is sparsely adaptable to biological samples. The aim of our study was thus to explore the associating of CM and MS in order to devise a reproducible and routinely usable protocol allowing a universal identification of lipid isomers in biological samples, taking here, as example, a toxicological model of corneal damage. Scheme 1. Cross-metathesis reaction applied to PC (18:0/18:1 9).</p><p>We have first hypothesized that lipids could react with the Grubbs catalyst of second-generation under very mild conditions using (Z)-but-2-ene-1,4-diyl diacetate as CM partner. Indeed, this symmetrical alkene is highly reactive and allows preventing formation of ethylene and ethylene CM adducts. A first assay was performed at the mg scale with a commercial phospholipid. Briefly, 6.3 µmol (5.0 mg) of PC (18:0/18:1 9) diluted in CH2Cl2 (0.5 ml) were treated at 40 °C for 2 hours with 5 equivalents of (Z)-but-2-ene-1,4-diyl diacetate in the presence of 5 mol% of Grubbs' catalyst. The reaction mixture was subsequently diluted (1/10,000) with MeOH and analyzed through direct infusion on a Synapt G2 (Q-ToF) high resolution mass spectrometer. The resulting mass spectrum displayed an intense peak at m/z 770.4953 corresponding to the [M+Na] + adduct PC (18:0/AcO-11:1 9), the distal chain transformed into undec-2-en-1-yl acetate giving no signal (SI Figure S1). Therefore, the C10-C18 moiety of the unsaturated FA chain has been replaced through CM by a 2-acetoxyethyl-1-ylidene moiety. Given that in a typical biological sample the overall content in phospholipids is of about tens of nmol for 10 6 cells, a scale down of our method was needed. However, at such a scale the concentration usually used in a CM reaction would require an amount of CH2Cl2 so small that it would anyway evaporate immediately at 40 °C. This led us implementing neat reaction conditions using circa one equivalent of the catalyst compared to the lipids and 50 equivalents of the CM partner. Using 2 nmol of the previous PC (18:0/18:1 9), CM was performed as described here after. Briefly, Grubbs' catalyst (0.1 M) was pretreated with 50 equivalents of (Z)-but-2-ene-1,4-diyl diacetate in CH2Cl2 at 40 °C for 2 hours to afford a Ru carbene bearing the CM partner. This solution was diluted, then an appropriate volume was added to 2 nmol of PC (18:0/18:1 9) and the solvent was subsequently removed under a flux of argon at 40 °C (SI Figure S2). The reaction mixture was then heated in a sealed tube at 40 °C for 2 hours, diluted in 100 µl of a CHCl3/MeCN/H2O/i-PrOH (20/30/10/30, v/v) mixture and 5 µL were injected in the LC-MS/MS system, the analysis being performed using previously described conditions. [20][21][22] In positive ion mode, the MS extracted ion chromatograms (EIC) at m/z 748.5123 and m/z 788.618 showed a first peak at tR = 4.1 min and a second one at tR = 7.1 min corresponding to the [M+H] + of PC (18:0/OAc-11:1 9) and native PC (18:0/18:1 9), respectively (Figure 1A). The MS/MS spectra displayed an intense peak at m/z 184.07 characteristic of the PC polar head group (Figure 1B and 1C). In negative ion mode, the EIC at m/z 732.479 showed an intense peak assigned to the [M-CH3] ˗ PC (18:0/OAc-11:1 9) confirming information obtained under positive ion conditions. In addition, negative ion mode is required to identify the FA side chains. Indeed, the MS/MS spectrum displayed at m/z 283.26 a peak diagnostic of stearate in sn1 position and at m/z 241.1453, a second peak corresponding to 11-acetoxyundec-9-enoate (FA (OAc-11:1 9)) in sn2 position (Figure 1E). These product ions are indicative of an oleate in sn2 position and therefore allowed to unambiguously locate the double bond. On one hand, it has been long established that the Grubb's catalysts are poisoned by basic amines. [23] On the other hand, among the phospholipid subclasses commonly encountered in biological samples, phosphatidylethanolamine (PE) and phosphatidylserine (PS), display basic primary amines functions. Accordingly, when a mixture of 20 commercially available standard phospholipids (SI Table S1) including PE and PS subclasses is submitted to our CM conditions, it gave a very poor yield of the expected CM products. Indeed, CM afford PC (18:0/OAc-11:1 9) with approximatively 20% yield when it is performed on a pure PC (18:0/18:1 9) solution and dramatically decreases to 5% when this PL is included in the standard lipid mixture (Figure 2B deleterious effect of basic amines, the lipid mixture was pretreated with 1M HCl solution to give innocuous ammoniums. [23] CM performed under these conditions allowed to recover yields closed to those obtained with pure PC (18:0/18:1 9) whatever the PL considered (Figure 2C). Thus, HCl pretreatment proves essential for CM reactions performed on lipid of biological lipid mixtures. Our mixture of commercial lipid standards contains mono-and poly-unsaturated FA side chains representative of FAs commonly encountered in biological samples. While phospholipids such as PC (18:0/18:1 9) lead to a single CM product, all C=C bonds present in phospholipids containing PUFA chain are expected to react leading to several CM products. Indeed, when CM was performed on PC (18:0/20:4 5, 8, 11, 14) under the previous conditions, it led to 4 CM products: PC (18:0/OAc-16:4 5, 8, 11, 14) (C4), PC (18:0/OAc-13:3 5, 8, 11) (C3), PC (18:0/OAc-11:2 5, 8) (C2) and PC (18:0/OAc-9:1 5) (C1) (Figure 3A & 3B -SI Figure S3). Detecting simultaneously all possible CM products in the final reaction mixture is suitable as it allows locating all the double bonds and hence identifying phospholipids containing PUFA unambiguously. To ensure that the final reaction mixture contains all the CM products at concentrations letting optimal detection by mass spectrometry, a kinetic study was carried out by varying the temperatures with PC (18:0/20:4) and PC (16:0/20:4) (Figure 3D -3G). Whatever the reaction time, chromatographic peaks corresponding to C1 and C4 products exhibited the highest and the lowest AUC compared to C2 and C3 (Figure 3D & 3E). As expected, in the course of the reaction, C2, C3 and C4 decreased in favor of C1. Moreover, at 4 hours of reaction at 40°C C2, C3 and C4 displayed suitable AUC and thus represents the best compromise to identify all the C=C bonds position (Figure 3D and 3E). In contrast, CM carried out at 60°C mainly led to C1 products with a 4 times increased yield compared to 40°C, whatever the PC species (Figure 3F and 3G) while C2, C3 and C4 were sparsely detected in that case. min by calculating the ratio between peaks corresponding to the two fatty acid chains (SI Figure S4). On MS/MS spectra, an intense peak at m/z 184.07 corresponds to the PC polar head group (SI Figure S5). In negative ion mode, MS/MS spectrum exhibited peaks at m/z 337.31 and at m/z 297.21 due to respectively docos-13-enoate and 15-acetoxypentadec-13enoate. While m/z 337.31 is due to native PC (22:1/22:1 13/13), and m/z 297.21 associated to CM modified PC which allows pinpointing the C=C bond (Figure 4E -4G). Ring closure metathesis was also possible in that case, and interestingly PC Within PC subclass, CM has thus been successfully applied to identify isomers of lipid species commonly encountered in biological samples i.e., PL containing SFA/MUFA, SFA/PUFA and MUFA/MUFA side chains. The kinetic profiles we previously described for PC at 40 and 60°C were reproducibly observed for all lipid species whatever the phospholipid subclasses considered, i.e. PE, PS, PG and PI as well as with several sphingolipids and glycerolipids including Cer, SM, and DG, TG, respectively (Table S1 Figure S6-S25). Regarding the sensitivity of the method, CM was successfully applied on 10 pmol individual amount for species of the lipid standard mixture. We previously reported that benzalkonium chloride (BAK), a quaternary ammonium, have a major impact on the lipidome of human corneal epithelial (HCE) cell line, however we could not determine double bound position of lipids at that time. [24,25] So this complex example appeared particularly relevant to assess the efficiency of our method. BAK is the more often encountered preservative in multidose eye drops which have long term indication for glaucoma and dry eye, two diseases deeply impacting quality of life. [26] HCE cells exposed to BAK is also a well-established toxicological model of corneal damage, [26][27][28] for which we previously demonstrated a striking decrease in PC species whatever the FA side chainsconsidered. [24,25] So we were delighted to see that our method succeeded at fully determining the structure of the 5 following PC species revealing a homeostasis change following BAK exposure. Indeed, in PC (18:1/18:1), FA side chains were identified as an oleyl (18:1 9) and an octadec-11-enoyl (18:1 11) (SI Figure S26). In PC (16:1/18:1) FA chains were identified as palmitoleyl (16:1 9) and oleyl (18:1 9) in sn1 and sn2, respectively. PC (16:0/18:1) and PC (16:0/16:1) contain oleyl (18:1 9) and palmitoleyl (16:1 9) in sn2 position respectively. Finally, PC (18:0/20:3) contained an eicosatrienoyl (20:3 5 8 11) in sn2 position. Therefore, regarding PC subclass, our method has revealed BAK-induced changes mainly on lipid species containing -9 and -7 FA (Figure 5). It may thus be suggested that BAK induces ocular injury by targeting -9 and -7 FA. Indeed, stearoyl CoA desaturase (SCD), which is involved in the -9 FA metabolism, has been previously described as impacted by BAK exposure. [29] To confirm these results, the investigation of enzymes involved in FA elongation and desaturation metabolism remains to be carried out. In summary, we have demonstrated that associating Grubbs catalyzed CM reaction and LC-MS/MS analysis allows an accurate identification of a broad scope of lipid isomers through an operatively simple and reproducible procedure. We have optimized conditions to obtain all the possible CM products enabling a full identification of MUFA and PUFA with a level of sensitivity allowing the analysis of biological samples. We are currently pursuing this study testing CM partners that could transfer an ionizable functional group allowing the identification of the distal moiety of FA chains. Furthermore, any sophisticated adaptation of the LC-MS system previously developed is needed to analyze complex biological lipid samples with our method. Regarding MS acquisition, both low-and high-resolution mass spectrometry are suitable and CM products exhibit same fragmentation patterns in MS/MS as native lipids facilitating identification. A remaining key challenge is the postacquisition of the big amount of data in the case of biological sample. The data processing strategy is currently in progress and will be the subject of a dedicated study.</p>

ChemRxiv

The anti-inflammatory drug BAY 11-7082 suppresses the MyD88-dependent signalling network by targeting the ubiquitin system

The compound BAY 11-7082 inhibits IκBα [inhibitor of NF-κB (nuclear factor κB)α] phosphorylation in cells and has been used to implicate the canonical IKKs (IκB kinases) and NF-κB in >350 publications. In the present study we report that BAY 11-7082 does not inhibit the IKKs, but suppresses their activation in LPS (lipopolysaccharide)-stimulated RAW macrophages and IL (interleukin)-1-stimulated IL-1R (IL-1 receptor) HEK (human embryonic kidney)-293 cells. BAY 11-7082 exerts these effects by inactivating the E2-conjugating enzymes Ubc (ubiquitin conjugating) 13 and UbcH7 and the E3 ligase LUBAC (linear ubiquitin assembly complex), thereby preventing the formation of Lys63-linked and linear polyubiquitin chains. BAY 11-7082 prevents ubiquitin conjugation to Ubc13 and UbcH7 by forming a covalent adduct with their reactive cysteine residues via Michael addition at the C3 atom of BAY 11-7082, followed by the release of 4-methylbenzene-sulfinic acid. BAY 11-7082 stimulated Lys48-linked polyubiquitin chain formation in cells and protected HIF1α (hypoxia-inducible factor 1α) from proteasomal degradation, suggesting that it inhibits the proteasome. The results of the present study indicate that the anti-inflammatory effects of BAY 11-7082, its ability to induce B-cell lymphoma and leukaemic T-cell death and to prevent the recruitment of proteins to sites of DNA damage are exerted via inhibition of components of the ubiquitin system and not by inhibiting NF-κB.

the_anti-inflammatory_drug_bay_11-7082_suppresses_the_myd88-dependent_signalling_network_by_targetin

INTRODUCTION<!>Materials<!>Proteins and antibodies<!>Cell culture<!>Cell stimulation and cell lysis<!>Immunoblotting<!>Cell proliferation assays<!>Preparation of Halo-NEMO and NEMO ‘pull-down’ assays<!>E1 and E2 ubiquitin-loading assays<!>Assay of the endogenous LUBAC E3 ligase<!>Measurement of the molecular mass of Ubc13 by MALDI–TOF (matrix-assisted laser-desorption ionization–time-of-flight)-MS<!>Edman sequencing<!>Mass spectrometric analysis of tryptic peptides<!>Reaction of N-acetyl cysteine with BAY 11-7082 and analysis of the products of the reaction by NMR spectroscopy and MS<!>Immunofluorescence and induction of DNA damage<!>BAY 11-7082 and BAY 11-7085, but not other inhibitors of IKK activity, induce the death of HBL-1 lymphoma cells carrying the MyD88[L265P] mutation<!>BAY 11-7082 and BAY 11-7085, but not other inhibitors of the activity and activation of the canonical IKK complex, induce HBL-1 cell death<!>BAY 11-7082 does not inhibit the canonical IKK complex or the IKK-related kinases in vitro, but inhibits their activation by LPS and IL-1 in RAW and IL-1R cells respectively<!>BAY 11-7082 suppresses the activation of IKK family members and JNK<!>BAY 11-7082 does not affect the LPS-stimulated autophosphorylation of IRAK4<!>BAY 11-7082 does not inhibit the canonical IKK complex or the IKK-related kinases in vitro, but inhibits their activation by LPS and IL-1 in RAW and IL-1R cells respectively<!>BAY 11-7082 inhibits the formation of K63-pUb chains<!>BAY 11-7082 suppresses the LPS- or IL-1-stimulated formation of K63-pUb chains and the DNA damage response<!>BAY 11-7082 inhibits the recruitment of proteins to sites of DNA damage<!>BAY 11-7082 inhibits the loading of ubiquitin on to E2 conjugating enzymes<!>BAY 11-7082 prevents ubiquitin conjugation to Ubc13 and UbcH7, but not ubiquitin loading on to the E1 activating enzyme UBE1<!>The mechanism by which BAY 11-7082 inhibits Ubc13 and UbcH7<!>BAY 11-7082 forms a covalent adduct with Ubc13<!>The mechanism by which BAY 11-7082 inhibits Ubc13 and UbcH7<!>BAY 11-7082 inhibits LUBAC and the IL-1-stimulated formation of linear-pUb chains<!>BAY 11-7082 inactivates LUBAC and suppresses the IL-1-stimulated formation of linear-pUb chains<!>BAY 11-7082 inhibits LUBAC and the IL-1-stimulated formation of linear-pUb chains<!>BAY 11-7082 enhances K48-pUb chain formation in cells by inhibiting the proteasome<!>BAY 11-7082 enhances the formation of K48-pUb chains and prevents the degradation of HIF1α by the proteasome<!>BAY 11-7082 enhances K48-pUb chain formation in cells by inhibiting the proteasome<!>NSC697923 inactivates E2 conjugating enzymes and LUBAC similarly to BAY 11-7082<!>DISCUSSION<!>AUTHOR CONTRIBUTION<!>ACKNOWLEDGEMENTS<!>FUNDING

<p>MyD88 (myeloid differentiation factor 88) is an adaptor protein that plays an essential role in the signalling networks that are activated by PAMPs (pathogen-associated molecular patterns), as well as by IL (interleukin)-1, IL-18 and IL-33 [1]. The interaction of these agonists with their receptors induces the recruitment of MyD88 and members of the IRAK (IL-receptor-associated kinase) family of protein kinases to form a structure known as the Myddosome [2,3]. The activation of the IRAKs is followed by the activation of TRAF (tumour-necrosis-factor-receptor-associated factor) 6, an E3 ligase that facilitates the formation of K63-pUb [Lys63-linked pUb (polyubiquitin)] chains in the presence of the E2 conjugating complex Ubc (ubiquitin conjugating) 13-Uev1a [4]. Linear-pUb chains formed by the action of the E3 ligase LUBAC (linear ubiquitin assembly complex) also appear to play a key role in this pathway [5,6]. The K63-pUb chains have been proposed to be essential for the activation of the protein kinase TAK1 (transforming growth factor β-activated kinase 1) [7,8] and linear pUb chains for the activation of the canonical IKK {IκB [inhibitor of NF-κB (nuclear factor κB)] kinase} complex [9]. The interaction of K63-pUb chains with the TAB (TAK1-binding protein) 2 and TAB3 components of the TAK1 complex [10,11] is thought to induce a conformational change that permits the auto-activation of the TAK1 catalytic subunit [7,8]. Similarly, the binding of K63-pUb [12,13] and/or linear-pUb chains [14,15] to the NEMO (NF-κB essential modifier) component of the canonical IKK complex is believed to induce conformational changes that facilitate the activation of the IKKα and IKKβ components of this complex by TAK1. TAK1 also activates MAPK (mitogen-activated protein kinase) kinases that switch on p38 MAPKs and JNKs (c-Jun N-terminal kinases) [16,17], whereas the canonical IKK complex has multiple downstream targets, including not only the inhibitory IκBα component of the transcription factor NF-κB, but also the inhibitory p105/NF-κB1 component of the Tpl2 (tumour progression locus 2) kinase. The IKK-catalysed phosphorylation of these proteins leads to the activation of NF-κB and the MAPKs ERK (extracellular-signal-regulated kinase) 1 and ERK2 respectively [18,19]. Together the signalling networks initiated by PAMPs ultimately induce the production of many inflammatory mediators that are deployed to fight infection by invading microbes.</p><p>Recently, mutations in MyD88 that cause the constitutive activation of the MyD88 signalling pathway have been identified as a major cause of the activated B-cell subtype of DLBCL (diffuse large B-cell lymphoma), one of the least curable forms of this blood cancer. One MyD88 mutation in particular, in which Leu265 is changed to a proline residue, accounts for approximately a third of all cases of DLBCL [20]. These findings raised the question of whether inhibitors of protein kinases that are activated downstream of MyD88, when used alone or in combination, might prevent the proliferation of these lymphoma cells or even induce their destruction. We therefore tested a number of compounds reported to inhibit the protein kinases in this pathway that are known to suppress inflammatory mediator production. However, only BAY 11-7082 and the closely related BAY 11-7085 induced the death of a B-cell lymphoma line carrying the MyD88[L265P] mutation. BAY 11-7082 has been reported to inhibit the phosphorylation of IκBα in cells and for this reason has been used in over 350 published research papers to implicate the canonical IKK complex and NF-κB in many cellular events. However, we found that other inhibitors of the canonical IKK complex or its activator TAK1 did not induce the apoptosis of the B-cell lymphoma carrying the L265P mutation. This led us to discover that BAY 11-7082 is not a direct inhibitor of the canonical IKK complex, but prevents its activation by targeting components of the ubiquitin system. These include Ubc13, the E2 conjugating enzyme that directs the formation of K63-pUb chains, as well as UbcH7 and LUBAC, which generate linear pUb chains. BAY 11-7082 also stimulates the production of K48-pUb (Lys48-linked polyubiquitin) chains in cells, probably by inhibiting the proteasome.</p><!><p>NG25 [21], BI 605906 [22], MLN4924 [23] and NSC697923 [24] were synthesized as described previously. BAY 11-7082 and BAY 11-7085 were purchased from Merck-Millipore, 5Z-7-oxozeaenol [25] was from BioAustralis, LPS (lipopolysaccharide; Escherichia coli 055:B5) was from Alexis Biochemicals (catalogue number ALX-581-001), Resazurin and MG132 were from Sigma and N-acetyl cysteine was from Tokyo Chemical Industry.</p><!><p>Human IL-1β was expressed as a glutathione transferase fusion protein in E. coli and cleaved with PreScission proteinase to release IL-1β[117–268], which was purified by gel filtration on Superdex 200. The human UBE1 (ubiquitin-activating enzyme), the E2 ubiquitin-conjugating enzyme Ubc13 (also called UBE2N) and UbcH7 (also called UBE2L3) were expressed as His6-tagged fusion proteins followed by a PreScission proteinase cleavage tag. Each protein therefore started with the sequence MGSSHHHHHHSSGLEVLFQGPGS, followed by the amino acid residue after the initiating methionine residue of each protein. The E2scan™Kit was purchased from Ubiquigent Ltd. Immunoprecipitating antibodies against bacterially expressed human HOIP (haem-oxidized IRP2 ligase-1-interacting protein) (S174D, 3rd bleed) and human IRAK4 (S522C, 3rd bleed) were raised in sheep at Diagnostics Scotland and the antisera were affinity purified on antigen–agarose columns by the Antibody Production Team (Division of Signal Transduction Therapy, Medical Research Council Protein Phosphorylation Unit, University of Dundee, Dundee, U.K.). Antibodies that recognize ubiquitin were purchased from Dako (catalogue number Z0458) and Enzo Life Sciences (catalogue number BML-PW8810-0500). Antibodies that recognize GFP (green fluorescent protein) (Abcam), K63-pUb chains (eBioscience), K48-pUb chains, IRAK4 and histone γH2AX (Merck-Millipore) were purchased from the sources indicated. Antibodies that recognize IKKβ phosphorylated at Ser177 and Ser181, p105 phosphorylated at Ser933, TBK1 (TRAF-associated NF-κB activator-binding kinase 1) phosphorylated at Ser172, IRAK4 phosphorylated at Thr345 and Ser346, p38α MAPK phosphorylated at its Thr-Gly-Tyr motif, and all forms of IκBα and GAPDH (glyceraldehyde-3-phosphate dehydrogenase) were purchased from Cell Signaling Technology. The antibody recognizing HIF1α (hypoxia-inducible factor 1α) was from R&D Systems, whereas the antibodies recognizing Cullin 2 and JNK phosphorylated at its Thr-Pro-Tyr motif were from Invitrogen. Secondary antibodies with fluorophores 488 and 594 for the detection of GFP and γH2AX respectively, were obtained from Alexa Fluor.</p><!><p>HBL-1 cells (provided by Louis Staudt, National Cancer Institute, Bethesda, MD, U.S.A.) were cultured in RPMI medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). HEK (human embryonic kidney)-293 cells stably expressing IL-1R (IL-1 receptor) (hereafter called IL-1R cells) (provided by Xiaoxia Li and George Stark, Case Western Reserve University, Cleveland, OH, U.S.A.) and the RAW 264.7 macrophage cell line (hereafter called RAW cells) were maintained in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% fetal bovine serum, 2 mM L-glutamine and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin). U2OS cells were cultured in McCoy's 5A growth medium supplemented as described for DMEM. U2OS cells were transfected using Lipofectamine™ (Invitrogen) according to the manufacturer's instructions. All cells were cultured at 37°C in a 10% CO2 humidified atmosphere.</p><!><p>All cells were incubated for 1 h with or without inhibitors prior to stimulation with agonists. IL-1R cells were stimulated with 0.5 ng/ml IL-1β and RAW cells with 100 ng/ml LPS. Cells were rinsed in ice-cold PBS and extracted in lysis buffer [50 mM Tris/HCl, pH 7.5, 1 mM EGTA, 1 mM EDTA, 1% (v/v) Triton X-100, 1 mM sodium orthovanadate, 50 mM NaF, 5 mM sodium pyrophosphate, 0.27 M sucrose, 10 mM sodium 2-glycerophosphate, 0.2 mM PMSF and 1 mM benzamidine]. For the experiments in which pUb chains were captured on Halo-NEMO, or in which the expression of HIF1α was studied, the lysis buffer included 100 mM iodoacetamide to inactivate deubiquitylases. Cell lysates were clarified by centrifugation (14000 g; 30 min; 4°C) the supernatants (cell extracts) were removed and protein concentrations were determined by the Bradford procedure.</p><!><p>Protein samples were denatured in LDS (lithium dodecyl sulfate) or SDS and subjected to SDS/PAGE on 4–12% gradient polyacrylamide gels (NuPAGE; Invitrogen). After transfer to PVDF membranes and blocking with 5% (w/v) non-fat dried skimmed milk powder in 50 mM Tris/HCl, pH 7.5, 0.15 M NaCl and 0.1% Tween 20, proteins were visualized by immunoblotting using the ECL (enhanced chemiluminescence) system (GE Healthcare).</p><!><p>HBL-1 cells were seeded into a black 96-well plate at 25000 cells per well in a total volume of 0.1 ml of RPMI medium. The pharmacological inhibitors (at 10 mM in DMSO) were diluted appropriately in RPMI medium and 50 μl was added to each well. All assays were performed in triplicate. To assess cell viability and proliferation, 15 μl of a 0.11 mg/ml solution of resazurin in water was then added to each well. The solution was incubated for 3 h at 37°C before reading the emitted fluorescence at 590 nm after excitation at 540 nm on a SpectraMax M2 Fluorescence Plate Reader. A blank reaction in which 0.15 ml of RPMI medium was incubated with 15 μl of 0.11 mg/ml rezaurin and used as the control.</p><!><p>The pUb-binding protein NEMO was expressed in E. coli as a Halo-tagged protein. The cells were lysed in 50 mM Tris/HCl, pH 7.5, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 0.1% 2-mercaptoethanol, 1 mM benzamidine and 0.2 mM PMSF, sonicated and the lysate was centrifuged to remove cell debris. The supernatant was coupled to the HaloLink resin (Promega) by incubation for 5 h at 4°C as described by the manufacturer. The HaloLink resin (1.0 ml) was added to 10 ml of cleared lysate. The resin was washed with 50 mM Tris/HCl, pH 7.5, 0.5 M NaCl, 0.1 mM EDTA, 270 mM sucrose, 0.03% Brij 35, 0.1% 2-mercaptoethanol, 0.2 mM PMSF and 1 mM benzamidine and stored at 4°C. To capture K63-pUb and linear-pUb chains from cell extracts, 3–6 mg of cell extract protein was incubated for 16 h at 4°C with Halo-NEMO beads (30 μl packed volume). The beads were washed three times with 1 ml of lysis buffer containing 500 mM NaCl and once with 1 ml of 10 mM Tris/HCl, pH 8.0. K63-pUb chains captured by the Halo-NEMO were released by denaturation in SDS and identified by immunoblotting.</p><!><p>UBE1 (0.17 μM) in 22.5 μl of 20 mM Hepes, pH 7.5, containing 10 μM ubiquitin was incubated for 45 min at 21°C with 1 μl of DMSO or 1 μl of BAY 11-7082 in DMSO. A 2.5 μl solution of 10 mM magnesium acetate and 0.2 mM ATP was added, incubated for 10 min at 30°C, and the reactions were terminated by the addition of 2.5 μl of 10% (w/v) SDS and heating for 6 min at 75°C. The samples were subjected to SDS/PAGE in the absence of any thiol. The gels were stained for 1 h with Coomassie Instant Blue and destained by washing with water. The loading of ubiquitin to E2 conjugating enzymes was carried out in an identical manner, except that UBE1 (0.17 μM) was mixed with Ubc13 (2.4 μM) or UbcH7 (2.9 μM) prior to incubation with BAY 11-7082.</p><!><p>Anti-HOIP antibody (1 μg) was incubated for 2 h at 4°C with Protein G–Sepharose (10 μl packed beads) in 0.5 ml of 50 mM Tris/HCl, pH 7.5, and 0.2% Triton X-100. The beads were washed three times with cell lysis buffer and incubated for 16 h at 4°C with 1 mg of cell extract protein. The beads were collected by brief centrifugation, washed three times with 0.5 ml of 50 mM Tris/HCl, pH 7.5, 1% (v/v) Triton X-100, 0.05% 2-mercaptoethanol and 0.2 M NaCl and once with 50 mM Tris/HCl, pH 7.5, and 5 mM MgCl2. The immunoprecipitated LUBAC complex was resuspended in 20 μl of 20 mM Tris/HCl, pH 7.5, 2 mM DTT, 0.1 μM UBE1, 0.4 μM UbcH7, 10 μM ubiquitin, 5 mM MgCl2 and 2 mM ATP. After incubation for 1 h at 30°C, reactions were stopped by denaturation in SDS. The formation of linear-pUb chains was analysed by immunoblotting with an anti-ubiquitin antibody.</p><!><p>Ubc13 (2.6 μM) was incubated for 30 min at 21°C without or with 10 μM BAY 11-7082 in 25 mM Hepes, pH 7.5, and then exchanged into 5 mM Tris/HCl, pH 7.5. An aliquot of the reaction (2 μl) was added to 2 μl of the matrix (2,5-dihydroxyacetophenone) and 2 μl of 2% (v/v) trifluoroacetic acid was added before spotting 1 μl of the sample on to a steel target. The analysis was performed manually in reflector positive mode using an UltrafleXtreme (Bruker Daltonics) MALDI–TOF mass spectrometer. For external calibration, two mono-isotopic masses were used: cytochrome c [M+H]+ (12361 Da) and myoglobin [M+H]+ (16952 Da).</p><!><p>This was performed on an Applied Biosystems ProCis e494c sequencer according to the manufacturer's instructions.</p><!><p>Tryptic peptide analysis using LC (liquid crystallography)-MS/MS (tandem MS) was performed on an Easy nLC HPLC coupled to an LTQ-Orbitrap Classic (Thermo) and data was analysed using the Mascot search program (http://www.matrixscience.com). Peptides were analysed for modification by BAY 11-7082 by the addition of a cysteine-specific modification of +C(3) H(1) N(1) (+51.011 Da) to Mascot. Gluconylation, +C(6) H(10) O(6) (+178.048 Da), was a standard Mascot modification.</p><!><p>A solution of BAY 11-7082 (0.0037 g, 0.018 mmol) in DMSO (0.2 ml) was added at 20°C to a solution of N-acetyl cysteine (0.003 g, 0.018 mmol) and Tris (2-carboxyethyl) phosphine hydrochloride (0.011 g, 0.038 mmol) in 1 M phosphate buffer, pH 8.5 (1 ml). The reaction was vortex-mixed for 2 min, allowed to stand for 15 min, diluted with 1 ml of 5:95 acetonitrile/water (0.1% formic acid) and applied to a Waters Xbridge 19 μm×100 μm diameter, 5 μm particle size C18 column. The column was developed with a gradient from 5% to 95% acetonitrile in water (0.1% formic acid) at a flow rate of 25 ml/min. The appropriate fractions were pooled and concentrated, and the structures of the products (R,E)-2-acetamido-3-[(2-cyanovinyl)thio] propanoic acid (Compound 1) (2.0 mg, 0.009 mmol, 50%) and 4-methylbenzene-sulfinic acid (Compound 2) (0.002 g, 0.0128 mmol, 71%) deduced from NMR spectra were recorded on a Bruker AVANCE II 500 spectrometer. HRMS (high-resolution mass spectra) were obtained on a microTOF Bruker Daltonics instrument.</p><!><p>Cells plated on sterile coverslips were fixed for 10 min at 21°C using 4% paraformaldehyde. Soluble proteins were removed before fixation by extraction for 5 min in ice-cold 10 mM Pipes, pH 7.0, 300 mM sucrose, 100 mM NaCl, 3 mM MgCl2, 1 mM EGTA and 0.5% Triton X-100. Cells were permeabilized for 20 min at 21°C with PBS containing 0.2% Triton X-100, washed several times in PBS and then incubated in blocking solution (PBS containing 5% donkey serum, 0.1% fish skin gelatin and 0.05% Tween 20). Cover slips were incubated for 1 h at 21°C with primary antibodies in blocking solution, then washed in PBS and incubated for 1.5 h with the fluorophore-conjugated secondary antibodies. The nuclei were stained for 30 min with DAPI (4′,6-diamidino-2-phenylindole) before coverslips were mounted on to glass slides. Slides were viewed using a Nikon eclipse Ti inverted microscope. Exposure of the cells to ionizing radiation was carried out at 2 Grays with a 137Cs radiation source.</p><!><p>We examined whether compounds reported to inhibit the activity or activation of the canonical IKK complex also inhibited the proliferation of HBL-1 lymphoma cells expressing the MyD88[L265P] mutation. BI605906, a potent and selective inhibitor of IKKβ [22] and two structurally unrelated inhibitors of TAK1, 5Z-7-oxozeaenol [25] and NG25 [21], only slowed cell growth modestly (Figure 1). We were therefore surprised to find that BAY 11-7082, another compound reported to inhibit the phosphorylation of IκBα and activation of NF-κB, induced the death of HBL-1 cells (Figure 1). BAY 11-7085, a closely related molecule, had a similar effect (Figure 1). Since neither the IKKβ inhibitor BI605906 nor the TAK1 inhibitors that prevent the MyD88-dependent activation of the IKK complex in fibroblasts or macrophages had this effect [22,26], it seemed that BAY 11-7082 must be inducing HBL-1 cell death by another mechanism and prompted us to explore which proteins BAY 11-7082 and BAY 11-7085 might be targeting.</p><!><p>HBL-1 cells were incubated without any inhibitor (■) or in the presence of the TAK1 inhibitors 5Z-7-oxozeaenol (1 μM, ●) and NG25 (1 μM, ○), the IKKβ inhibitor BI605906 (10 μM, ∆), BAY 11-7082 (3 μM, ▲) or BAY 11–7085 (3 μM, □). Values are means±S.D. for three experiments each performed in triplicate.</p><!><p>We found that BAY 11-7082 did not inhibit IKKα, IKKβ and the IKK-related kinases TBK1 and IKK∊ when assayed at 10 μM in vitro (Supplementary Table S1 at http://www.biochemj.org/bj/451/bj4510427add.htm). Nevertheless, BAY 11-7082 completely suppressed the LPS-stimulated (Figure 2A) and IL-1-stimulated (Figure 2B) phosphorylation of the activation loop of IKKβ. As a consequence, the phosphorylation of its substrate p105/NF-κB1 and the degradation of IκBα (which is triggered by the IKK-catalysed phosphorylation of IκBα) were also prevented. The protein kinase TAK1 was partially inhibited by BAY 11-7082 in vitro (Supplementary Table S1), but BAY 11-7082 also suppressed the IL-1-stimulated activation of TBK1 in IL-1R cells (Figure 2C), which is dependent upon the expression of TRAF6, but independent of the expression or catalytic activity of TAK1 [22]. BAY 11-7082 additionally prevented the LPS- or IL-1-stimulated activation of JNK in RAW or IL-1R cells. BAY 11-7082 did not inhibit IRAK4 or IRAK1 in vitro (Supplementary Table S1), which are the most 'upstream' protein kinases in the MyD88 signalling network, and nor did it prevent the autophosphorylation of IRAK4 induced by LPS in RAW macrophages (Figure 3A) or IL-1 in IL-1R cells (Figure 3B).</p><!><p>(A) Murine RAW 264.7 macrophages were incubated for 1 h with the indicated concentrations of BAY 11-7082 and then stimulated for 15 min with 100 ng/ml LPS. The cells were lysed and aliquots of the cell extract (20 μg of protein) were denatured in SDS, subjected to SDS/PAGE, and immunoblotted with antibodies that recognize the active phosphorylated form of IKKβ, the IKKβ substrate p105 phosphorylated at Ser933 and all forms of IκBα and GAPDH. (B) Same as (A), except that human IL-1R cells were used and the cells stimulated for 15 min with 0.5 ng/ml IL-1β. (C) Same as (B), except that IL-1R cells were immunoblotted with antibodies that recognize TBK1 phosphorylated at Ser172 and an antibody that recognizes all forms of TBK1. (D) As in (A), except that the RAW macrophages were incubated for 1 h with 15 μM BAY 11-7082, then stimulated with LPS and the gels were immunoblotted for JNK phosphorylated at Thr183 and Tyr185, p38 MAPK phosphorylated at Thr180 and Tyr182 and GAPDH. (E) As in (D), except that IL-1R cells were stimulated with 0.5 ng/ml IL-1. p-, phospho-.</p><!><p>(A) Murine RAW 264.7 macrophages were incubated for 1 h without (−) or with (+) 15 μM BAY 11-7082 and then stimulated for the times indicated with 100 ng/ml LPS. The cells were lysed and aliquots of the cell extract (20 μg of protein) were subjected to SDS/PAGE and immunoblotting (bottom three panels) with the antibodies used in Figure 2. IRAK4 was immunoprecipitated (IP) from 2 mg of cell extract protein using 2 μg of anti-IRAK4 antibody using the procedure used to immunoprecipitate HOIP (see the Experimental section). The immunoprecipitates were denatured in SDS, subjected to SDS/PAGE and immunoblotted with an antibody that recognizes IRAK4 phosphorylated at Thr346 and Ser346 (p-IRAK4) and an antibody that recognizes all forms of IRAK4. (B) Same as (A), except that IL-R cells were used and stimulated with 0.5 ng/ml IL-1. p-, phospho-.</p><!><p>BAY 11-7082 induced near-maximal activation of p38α MAPK in unstimulated cells that could not be enhanced further by stimulation with LPS or IL-1 (Figures 2D and 2E), suggesting that this compound induces the activation of one or more stress-response pathways that are known to activate p38α MAPK.</p><!><p>The experiments described above indicated that BAY 11-7082 exerted its effects on the MyD88-dependent signalling network 'downstream' of the IRAK family of protein kinases, but 'upstream' of IKKα, IKKβ and TBK1, which suggested that it might be affecting the ability of TRAF6 and/or other E3 ubiquitin ligases to generate K63-pUb chains. To investigate this possibility, we used NEMO immobilized on Halo beads to capture the K63-pUb chains formed upon stimulation with LPS or IL-1 [27]. These experiments showed that BAY 11-7082 prevented the LPS- or IL-1-stimulated formation of K63-pUb chains at concentrations similar to those that suppressed the activation of IKKβ (Figures 4A and 4B).</p><!><p>(A and B) The experiment was carried out as in Figure 2, except that the K63-pUb chains formed in response to LPS (A) or IL-1β (B) were captured on Halo-NEMO from 6 mg (RAW cells) or 3 mg (IL-1R cells) of cell extract protein as described in the Experimental section. The K63-pUb chains were identified by immunoblotting with a specific antibody. Further aliquots of the cell extract were immunoblotted for IKKβ phosphorylation, p105 phosphorylation and GAPDH as in Figure 2. (C) Indirect immunofluorescence images of U2OS cells transiently expressing GFP–RAP80[1–200]. Cells were incubated for 1 h with or without BAY 11-7082 (15 μM) and either exposed to ionizing radiation (IR) or not exposed. GFP–RAP80 or γH2AX were visualized using anti-GFP and anti-γH2AX antibodies respectively, and nuclei were stained with DAPI.</p><!><p>K63-pUb chains are not just involved in IL-1 and TLR (toll-like receptor)-stimulated signalling networks, but also in other cellular events, including the recruitment of components of the DNA-repair machinery to sites of DNA damage [28], including the RAP80 component of the BRCA1 (breast cancer early-onset 1) complex [29]. The recruitment of RAP80 to DNA damage-induced sub-nuclear 'foci' has been shown to require Ubc13 [29]. If BAY 11-7082 inactivates Ubc13, we reasoned that this compound should also suppress the recruitment of RAP80 to DNA damage foci. When U2OS cells were exposed to ionizing radiation, GFP–RAP80 became localized to sites of DNA damage (Figure 4Ci), which was prevented by BAY 11-7082 (Figure 4Cii). In contrast, in cells that were not exposed to ionizing radiation (Figure 4Ciii), RAP80 did not form foci in the absence (Figure 4Ciii) or presence (Figure 4Civ) of BAY 11-7082. As a control experiment, the formation of the phosphorylated form of histone H2AX (γH2AX) at sites of DNA damage (Figure 4Cv), which is not dependent on the formation of K63-pUb chains [28], was studied. As expected, γH2AX foci were little affected by BAY 11-7082 (Figure 4Cvi). In contrast, cells that had not been exposed to ionizing radiation did not form foci and so no γH2AX foci were observed (Figure 4Cvii). However, some γH2AX foci were observed in cells that had not been exposed to ionizing radiation, but had been incubated with BAY 11-7082 (Figure 4Cviii), probably because the inhibition of K63-pUb chain formation by BAY 11-7082 prevents the repair of DNA damage that occurs spontaneously in cells at a low rate. Figures 4(Cix)–4(Cxii) are control experiments showing DAPI staining and merged images of Figures 4(Ci)–4(Civ) with 4(Cv)–4C(viii).</p><!><p>The generation of pUb chains is initiated by the MgATP-dependent transfer of ubiquitin to a cysteine residue on the ubiquitin-activating enzyme UBE1, which is followed by transfer of the ubiquitin to a cysteine residue on E2 conjugating enzymes. It was therefore possible that BAY 11-7082 might be suppressing K63-pUb chain formation by preventing the conjugation of ubiquitin to UBE1 or the transfer of ubiquitin from UBE1 to the active-site cysteine residue on an E2. To examine these possibilities we initially used Ubc13, which directs the formation of K63-pUb chains by TRAF6 [4] and other E3 ubiquitin ligases [30] when it is complexed to the inactive 'pseudo-E2' Uev1a. These experiments demonstrated that BAY 11-7082 did not affect the loading of ubiquitin to UBE1 under the conditions tested (Figure 5A), but completely blocked the transfer of ubiquitin from UBE1 to Ubc13 (Figure 5B). BAY 11-7082 also prevented the transfer of ubiquitin from UBE1 to UbcH7 (Figure 5C), which is the E2 thought to act with RBR (RING-between-RING) E3 ligases [31] such as LUBAC (see below). BAY 11-7085 similarly prevented the conjugation of ubiquitin to Ubc13 and UbcH7, although it was slightly less potent than BAY 11-7082 (results not shown). These findings led us to test whether BAY 11-7082 affected the loading of other E2 conjugating enzymes that accept ubiquitin from UBE1. We found that 24 E2 conjugating enzymes were inactivated by incubation with 10 μM BAY 11-7082 in vitro, but the conjugation of ubiquitin to UBE2G1 and UBE2H was only partially reduced by this concentration of BAY 11-7082 (Supplementary Figure S1A at http://www.biochemj.org/bj/451/bj4510427add.htm).</p><!><p>UBE1 and ubiquitin (A), UBE1, Ubc13 and ubiquitin (B) or UBE1, UbcH7 and ubiquitin (C) were incubated for 60 min with the indicated concentrations of BAY 11-7082 and the ubiquitin-loading reactions were then started by the addition of MgATP. Reactions were terminated by denaturation in SDS, proteins were subjected to SDS/PAGE and the gels were stained with Coomassie Instant Blue, followed by destaining in water. Ub, ubiquitin.</p><!><p>The Ubc13 preparation used in these experiments (see the Experimental section) was expressed in E. coli as a His6-tagged protein. When this preparation was subjected to MALDI–TOF-MS, two components were observed with molecular masses of 19.32 kDa (major component) and 19.50 Da (minor component) (Figure 6A). The mass of the major component was approximately 130 Da less than that predicted from the molecular mass of the expressed protein. N-terminal sequencing of the preparation revealed that it lacked the initiator methionine residue, the sequence starting with the next amino acid, glycine. The absence of the N-terminal methionine residue accounted for the difference between the determined and the predicted molecular mass of the protein. The minor component in the preparation, accounting for approximately 25% of the total material, was shown by mass spectrometric analysis of tryptic peptides to be Ubc13 in which the α-amino group of the N-terminal glycine residue was gluconoylated, explaining why its molecular mass was 178 Da greater than that of the major component. N-gluconoylation is a frequently encountered modification when N-terminally His6-tagged proteins lose their N-terminal methionine residue to start with the N-terminal sequence GSSHHHHHH [32].</p><!><p>(A) Ubc13 was incubated without or with BAY 11-7082 and subjected to MALDI–TOF-MS as described in the Experimental section. Incubation with BAY 11-7082 increased the molecular mass of Ubc13 by 51 Da. (B) Proposed mechanism for how BAY 11-7082 covalently modified Ubc13 and UbcH7 by reacting with the cysteine residue that accepts ubiquitin from UBE1. (C) N-acetyl cysteine was incubated with BAY 11-7082 and the two products of the reaction, (R,E)-2-acetamido-3-[(2-cyanovinyl)thio]propanoic acid (Compound 1) and 4-methylbenzene-sulfinic acid (Compound 2) were identified by NMR and MS as described in the Experimental section. Splitting patterns of spectral multiplets are indicated as: s, singlet; d, doublet; dd, doublet of doublets; m, multiplet. (R,E)-2-acetamido-3-[(2-cyanovinyl)thio]propanoic acid (1): 1H-NMR (500 MHz, [2H4]methanol) δ 7.63 (d, J=15.8 Hz, 1 H), 5.57 (d, J=15.8 Hz, 1 H), 4.70 (dd, J=7.7, 5.0 Hz, 1 H), 3.43 (dd, J=14.1, 5.0 Hz, 1H), 3.24 (dd, J=14.0, 7.8 Hz, 1H), 2.02 (s, 3H). 13C-NMR (126 MHz, CD3OD) δ 173.4, 172.6, 153.9, 118.5, 93.0, 53.1, 34.7, 22.4. HRMS-TOF (+): m/z=215.0499, expected for C8H11N2O3S 215.0490 [M+H]+. 4-Methylbenzene-sulfinic acid (2): 1H-NMR (500 MHz, [2H4]methanol) δ 7.61 (m, 2 H), 7.40 (m, 2 H), 2.44 (s, 3 H).13C-NMR (126 MHz, [2H4]methanol) δ 146.5, 143.7, 130.7, 125.7, 21.4. HRMS-TOF (+): m/z=157.0327, expected for C7H9O2S 157.0323 [M+H]+.</p><!><p>When Ubc13 was mixed with BAY 11-7082, the molecular masses of the major and minor components of Ubc13 both increased by 51 Da, as judged by MALDI–TOF-MS (Figure 6A). This suggested that the thiol group of the only cysteine residue in Ubc13 had reacted with BAY 11-7082, forming a covalent bond by Michael addition at the C3 carbon atom of BAY 11-7082, with the elimination of 4-methylbenzene-sulfinic acid by the mechanism depicted in Figure 6(B).</p><p>To establish whether the putative covalent adduct in Figure 6(B) had really been formed, Ubc13 and UbcH7, that had been inactivated by incubation with BAY 11-7082, were digested with trypsin and the digest was analysed using an Orbitrap Classic mass spectrometer (Thermo Scientific). The tryptic digest of Ubc13 that had been reacted with BAY 11-7082 generated peptides with molecular masses of 867.5, 1110.6 and 2062.1 Da, corresponding to the tryptic peptides ICLDILK, ICLDILKDK and ICLDILKDKWSPALQIR plus 51 Da respectively. MS/MS analysis of the peptides confirmed that the site of this 51 Da modification was the single cysteine residue in each peptide. These tryptic peptides contain the only cysteine residue present in Ubc13 (Cys87), the two longer peptides arising from partial tryptic cleavage of the lysine-aspartate and lysine-tryptophan bonds between amino acid residues 92/93 and 94/95 of Ubc13. The unmodified forms of these peptides with molecular masses of 816.5, 1059.6 and 2011.1 could not be detected in this experiment, but were identified when Ubc13 that had not been incubated with BAY 11-7082 was digested with trypsin. Similarly, tryptic digestion of UbcH7 generated peptides with molecular masses of 1593.8, 1991.0 and 3100.6 Da, corresponding to the molecular masses of the peptides GQVCLPVISAENWK, GQVCLPVISAENWKPATK and IYHPNIDEKGQVCLPVISAENWKPATK plus 51 Da respectively. These tryptic peptides contain the cysteine residue in UbcH7 (Cys86) that accepts ubiquitin from ubiquitin-loaded UBE1. The two longer peptides arise from partial tryptic cleavage of the lysine-proline and lysine-glycine peptide bonds between residues 96/97 and 82/83 of UbcH7. The unmodified forms of these peptides with molecular masses of 1542.8, 1940.0 and 3049.6 could not be detected after reaction with BAY 11-7082, but were identified when UbcH7 that had not been exposed to BAY 11-7082 was digested with trypsin. Taken together, these experiments establish that BAY 11-7082 inactivates E2-conjugating enzymes in the way depicted in Figure 6(B).</p><p>To further establish the mechanism by which cysteine residues react with BAY 11-7082, we incubated N-acetyl cysteine with BAY 11-7082, purified the reaction products as described in the Experimental section and solved their structures by NMR (Figure 6C). These experiments established that the reaction mechanism postulated in Figure 6(B) was correct and also confirmed that 4-methylbenzene-sulfinic acid had not been generated in the ion source of the mass spectrometers.</p><!><p>LUBAC is required for the activation of the canonical IKK complex by the MyD88 signalling network [5,6], raising the question of whether BAY 11-7082 also suppressed the formation of linear-pUb chains by HOIP, the catalytic component of LUBAC. HOIP is an RBR E3 ligase in which ubiquitin is first transferred from the E2 conjugating enzyme UbcH7 to a cysteine residue on the E3 ligase before transfer to the substrate [31]. This suggested that BAY 11-7082 might not only have suppressed the formation of linear-pUb chains by preventing the conjugation of ubiquitin to UbcH7, but also the transfer of ubiquitin from UbcH7 to the active-site cysteine residue on HOIP [9,33]. We therefore assayed the endogenous LUBAC activity in RAW 264.7 macrophages after incubating the cells with and without BAY 11-7082. These experiments showed that BAY 11-7082 irreversibly inactivated LUBAC in either RAW 264.7 macrophages (Figure 7A) or IL-1R cells (Figure 7B) at concentrations similar to those that suppress the activation of IKKβ and TBK1.</p><!><p>RAW 264.7 macrophages (A) or IL-1R cells (B) were incubated for 1 h with the indicated concentrations of BAY 11-7082. The cells were then lysed and LUBAC was immunoprecipitated (IP) from 1.0 mg of cell extract protein using anti-HOIP as described in the Experimental section. The immunoprecipitates were washed and LUBAC-catalysed linear-pUb chain formation was initiated by the addition of UBE1, UbcH7, ubiquitin and MgATP. After incubation for 60 min at 30°C, the reactions were terminated by denaturation in SDS. Following SDS/PAGE, pUb chain formation was detected by immunoblotting (IB) with an anti-ubiquitin antibody (Dako). (C) In lanes 1–6, IL-1R cells were incubated for 1 h with (+) or without (−) 15 μM BAY 11-7082, then stimulated for 10 min with (+) or without (−) 0.5 ng/ml IL-1β. Following cell lysis, linear-pUb chains and K63-pUb chains were captured from 6 mg of cell extract protein using Halo-NEMO (see the Experimental section). After denaturation in SDS, the pUb chains were separated by SDS/PAGE and transferred on to PVDF membranes. The membranes were cut into two pieces and immunoblotted for 10 s (upper half of gel) or 120 s (lower half of gel) with an anti-ubiquitin antibody (Enzo Life Sciences). Authentic linear ubiquitin oligomers (lane 7) and Lys63-linked ubiquitin oligomers (lane 8) were used as markers to demonstrate that the small ubiquitin oligomers formed in response to IL-1β and captured by Halo-NEMO were linear-pUb chains and not K63-pUb chains. Ub, ubiquitin.</p><!><p>LUBAC is thought to be the E3 ligase that generates linear-pUb chains in cells. Therefore the finding that BAY 11-7082 inactivates LUBAC implied that it should also have prevented the IL-1-stimulated formation of linear-pUb chains in IL-1R cells. To investigate whether this was so, we captured the linear-pUb chains on Halo-NEMO (see the Experimental section) and identified them by their characteristic electrophoretic mobility on SDS/PAGE, which differs from K63-pUb oligomers of equivalent length. These experiments established that BAY 11-7082 completely suppressed the IL-1-stimulated formation of linear-pUb oligomers comprising two to seven ubiquitin molecules (Figure 7C). These linear pUb oligomers could be detected most clearly with the anti-ubiquitin antibody from Enzo Life Sciences, which we found to be more sensitive than the anti-ubiquitin antibody from Dako that was used to assay LUBAC in vitro (Figure 7A). The detection of these small linear-pUb oligomers additionally required exposure of the immunoblots for 2 min, compared with the 5–15 s needed to detect the much longer pUb chains formed in response to IL-1 (compare also Figures 4 and 7C). Small K63-pUb oligomers were not detected in these experiments, either because they are not formed or are not captured by NEMO as efficiently as the small linear-pUb oligomers, as reported by others [34].</p><!><p>The finding that BAY 11-7082 not only prevented the loading of ubiquitin on to Ubc13 and UbcH7, but also the conjugation of ubiquitin to many other E2 conjugating enzymes (Supplementary Figure S1) raised the possibility that it might inhibit every cellular ubiquitylation event. We therefore investigated its effect on the formation of K48-pUb chains in cells. Interestingly, BAY 11-7082 did not suppress, but increased the formation of K48-pUb chains considerably in RAW cells (Figure 8A) and IL-1R cells (Figure 8B). It further increased the formation of K48-pUb chains in RAW macrophages that had been elevated by incubation with the proteasome inhibitor MG132 (Figure 8A), but had little effect on MG132-stimulated K48-pUb chain formation in IL-1R cells (Figure 8B). These observations imply that E2 conjugating enzymes that direct the formation of K48-pUb chains are still active at the concentrations of BAY 11-7082 used in these experiments. For example, UBE2G1 and UBE2H, which are reported to direct the formation of K48-pUb chains [35], were only inhibited partially by BAY 11-7082 under conditions where the conjugation of ubiquitin to other E2 ligases was abolished.</p><!><p>RAW 264.7 macrophages (A) or IL-1R cells (B) were incubated for 1 h without (−) or with (+) 15 μM BAY 11-7082, and then for a further 1 h without (−) or with (+) the proteasome inhibitor MG 132. The cells were lysed and aliquots of the cell extract (20 μg of protein) were denatured in SDS, subjected to SDS/PAGE and immunoblotted with antibodies that recognize K48-pUb chains specifically. (C and D) RAW macrophages (C) or IL-1R cells (D) were incubated for 2 h without or with 15 μM BAY 11-7082, 25 μM MG132 or 1 μM MLN4924. The cells were then lysed in the presence of 50 mM iodoacetamide to inhibit de-ubiquitylases and de-NEDDylases and immunoblotted with antibodies that recognize HIF1α and Cullin 2, and GAPDH as a loading control. IB, immunoblot.</p><!><p>The observation that BAY 11-7082 enhanced the formation of K48-pUb chains in cells suggested that this compound might also inhibit the proteasome. We therefore incubated IL-1R cells (Figure 8C) and RAW 264.7 macrophages (Figure 8D) in the absence or presence of BAY 11-7082 or MG132 followed by immunoblotting of the cell extracts with antibodies that recognize HIF1α. In normoxic cells, HIF1α is barely detectable because it undergoes Cullin-2-mediated Lys48-linked polyubiquitylation followed by proteasomal degradation [36]. We found that BAY 11-7082 induced the appearance of HIF1α similarly to MG132 and polyubiquitylated species of HIF1α could be detected, as well as unmodified HIF1α (Figures 8C and 8D). These results are consistent with BAY 11-7082 inhibiting the proteasome. However, it was possible that BAY 11-7082 had inactivated UbcH12, the E2 conjugating enzyme for NEDDylation, thereby preventing the NEDDylation and activation of Cullin 2 [36]. This possibility was excluded by showing that the proportion of the slower migrating NEDDylated form and the faster migrating de-NEDDylated form of Cullin 2 was not altered by treatment with BAY 11-7082 or MG132. In contrast, the formation of the NEDDylated species was blocked by MLN4924 (Figures 8C and 8D), a specific inhibitor of NAE1 [NEDD8 (neural-precursor-cell-expressed developmentally down-regulated 8)-activating enzyme E1 subunit 1], the E1 activating enzyme for NEDDylation [23]. These results show that BAY 11-7082 does not inhibit the E1 activating enzyme for NEDD8 or the E2 conjugating enzyme UbcH12 under the conditions tested and indicate that BAY 11-7082 is likely to inhibit the proteasome. However, the inhibition of deubiquitylases by BAY 11-7082 may also contribute to the enhanced formation of K48-pUb chains. Most deubiquitylases are cysteine proteinases and we have observed that several of these enzymes are partially inhibited by BAY 11-7082 in vitro if thiols are excluded from the assays (A. Knebel, unpublished work).</p><!><p>Recently, the compound NSC697923 was reported to prevent the survival of DLBCL cell lines, including HBL-1 cells, and to inhibit Ubc13/UBE2N [37]. We noticed that its structure had marked similarity to BAY 11-7082. We therefore incubated Ubc13 with NSC697923 and found that a covalent adduct was formed with a molecular mass 95 Da greater than that of Ubc13/UBE2N (Supplementary Figure S2A at http://www.biochemj.org/bj/451/bj4510427add.htm). This indicated that the cysteine residue in Ubc13/UBE2N had reacted with NSC697923 to form the derivative shown in Supplementary Figure S2(B) and that, as occurred with BAY 11-7082, 4-methylbenzene-sulfinic acid had been eliminated. It had been reported that NSC697923 did not inhibit the E2 conjugating enzyme UbcH5c/UBE2D3 [37], but we found that this E2 was inhibited partially under the conditions we used (Supplementary Figure S1B). NSC697923 also prevented the transfer of ubiquitin to a number of other E2 conjugating enzymes, including UbcH7/UBE2L3, UBE2D2, UBE2G1, UBE2G2, UBE2L6, UBE2R2, UBE2S and UBE2T (Supplementary Figure S1B). However, NSC697923 appears to be more selective than BAY 11-7082 as a number of other E2 conjugating enzymes were unaffected by this compound (Supplementary Figure S1B). Similar to BAY 11-7082, NSC697923 irreversibly inhibited LUBAC, increased the formation of Lys48-linked pUb chains and blocked the IL-1-stimulated formation of K63-pUb chains in IL-1R cells (Supplementary Figure S3 at http://www.biochemj.org/bj/451/bj4510427add.htm). Similar results were obtained using RAW 264.7 macrophages and LPS as the stimulus (results not shown).</p><!><p>BAY 11-7082 has been reported to induce the necroptotic death of precursor-B acute lymphoblastic leukaemic blasts (pre-B ALL) [38] and the death of natural killer/T-cell lymphomas [39]. It was found to destroy HTLV-1 (human T-cell lymphotropic virus 1) T-cell lines, but not HTLV-1-negative T-cells, and was shown to induce the apoptosis of primary adult leukaemic cells more readily than normal peripheral blood mononuclear cells [40]. BAY 11-7082 and the closely related BAY 11-7085 also induced the apoptosis of colon cancer cells and inhibited tumour implantation in the liver after the intra-peritoneal delivery of HT-29 colon cancer cells [41]. These effects have all been attributed to the inhibition of NF-κB. In the present study we found that BAY 11-7082 and BAY 11-7085 also induced the death of HBL-1 lymphoma cells expressing the MyD88[L265P] mutation, but other inhibitors of the canonical IKK complex and its activator TAK1 did not (Figure 1), which suggested that BAY 11-7082 and BAY 11-7085 were exerting their effects on HBL-1 cells by alternative/additional mechanisms and led us to investigate what the mechanism might be.</p><p>BAY 11-7085 was originally described as a potent anti-inflammatory drug, which reduced oedema formation in the rat carrageenan paw oedema assay and reduced paw swelling in a rat adjuvant arthritis model [42]. It was also shown to suppress irreversibly the TNFα (tumour necrosis factor α)-stimulated phosphorylation of IκBα, and hence the activation of the transcription factor NF-κB [42]. For these reasons it was assumed to exert its anti-inflammatory effects by suppressing the activation of NF-κB. It has been used in more than 350 papers to implicate the canonical IKK complex and NF-κB in many cellular events. However, we found that BAY 11-7082 did not inhibit IKKα, IKKβ or the IKK-related kinases in vitro and nor did it inhibit the activity or activation of IRAK1 and IRAK4 (Supplementary Table S1 and Figure 3). These observations led us to discover that BAY 11-7082 prevented the IL-1-stimulated and LPS-stimulated formation of K63-pUb and linear-pUb chains by irreversibly inhibiting E2 conjugating enzymes required for the formation of these pUb chains (Ubc13, UbcH7) and the E3 ubiquitin ligase HOIP, the catalytic component of LUBAC that directs the formation of linear-pUb chains. The suppression of K63-pUb chains and/or linear-pUb chains presumably explains how BAY 11-7082 prevents the activation of the IKK subfamily of protein kinases by LPS and IL-1.</p><p>Although the inhibition of NF-κB may contribute to the BAY 11-7082/5-induced death of leukaemic and lymphoma cells, the present study has suggested several other ways in which these molecules may induce cell death. For example, we found that BAY 11-7082 is likely to inhibit the proteasome (Figure 8) and the proteasome inhibitor bortezomib/velcade also induced the death of HBL-1 cells (Supplementary Figure S4 at http://www.biochemj.org/bj/451/bj4510427add.htm). Moreover, BAY 11-7082 prevented the response to DNA damage by blocking the formation of K63-pUb chains leading to the gradual accumulation of DNA lesions (Figure 4Cviii) that may culminate in apoptosis. Finally, BAY 11-7082 inactivated many E2 conjugating enzymes in vitro (Supplementary Figure S1), suggesting that the inhibition of multiple ubiquitylation events may contribute to HBL-1 cell death. The present study has also established that the recently described compound NSC697923 exerts its effects on DLBCL cell lines, including HBL-1 cells, by the same mechanism as BAY 11-7082 (Supplementary Figures S1B, S2 and S3).</p><p>Consistent with the findings reported in the present study, BAY 11-7082 and BAY 11-7085 have been reported to inhibit the NALP3 inflammasome in macrophages by an NF-κB-independent mechanism [43]. The NALP3 inflammasome processes pro-IL-1β and pro-IL-18 into the active pro-inflammatory cytokines IL-1β and IL-18 respectively. The anti-inflammatory effects of these compounds could therefore result from the combined inhibition of the NALP3 inflammasome, the inhibition of NF-κB and JNK and other branches of the MyD88 signalling network. Although BAY 11-7082 was reported to inhibit NALP3 ATPase activity in vitro [43], the way in which this compound suppresses the processing of pro-IL-1β and pro-IL-18 by the inflammasome is unclear. An intriguing possibility is that BAY 11-7082 also blocks activation of the inflammasome by targeting components of the ubiquitin system that affect the NALP3 ATPase.</p><p>BAY 11-7085 was found to be as effective as dexamethasone in reducing paw swelling in a rat model of adjuvant-induced arthritis when it was injected intraperitoneally once a day at 20 mg/kg [42]. It is remarkable that a compound like BAY 11-7085, which has such a profound effect on the ubiquitin system, could be used daily for 2 weeks to reduce inflammation in an animal model of arthritis, without significant side effects being noted. It will clearly be of great interest in the future to see whether more specific inhibitors of LUBAC and particular E2 conjugating enzymes can be developed, and whether they also show efficacy in the treatment of inflammatory diseases, as well as lymphomas and other cancers of immune cells.</p><!><p>Sam Strickson performed the experiments presented in Figures 1–4, 5(A), 5(B), 7 and 8 and Supplementary Figures S1, S3 and S4. Maria Stella Ritorto performed the experiments in Figure 6(A) and Supplementary Figure S2(A). Axel Knebel performed the experiments in Figure 5(C) and Natalia Shpiro performed the experiments in Figure 6(C). Axel Knebel also provided purified UBE1, Ubc13 and UbcH7, and Natalia Shpiro synthesized NSC697923. Lorner Plater performed the experiments shown in Supplementary Table S1, and David Campbell performed the MS analysis that identified the cysteine residues in Ubc13 and UbcH7 that were covalently modified by BAY 11-7082. Christoph Emmerich developed the Halo-NEMO method for capturing and identifying K63-pUb and linear-pUb chains, the assay of the endogenous LUBAC and contributed ideas. Sam Strickson and Philip Cohen designed the experiments and wrote the paper.</p><!><p>We are grateful to our colleagues Thimo Kurz, John Rouse, Mathew Stanley and Satpal Virdee for valuable suggestions, and Christophe Lachaud for help with the DNA damage experiments. We thank Louis Staudt (National Cancer Institute, Bethesda, MD, U.S.A.) for HBL-1 cells, Xiaoxia Li (Cleveland Research Clinic, Cleveland, OH, U.S.A.) for IL-1R cells, and Stephanie Panier and Daniel Durocher (Samuel Lunenfeld Institute, Toronto, Canada) for the RAP80 construct. The MRC Protein Phosphorylation Unit's (PPU) DNA Sequencing Service (co-ordinated by Nicholas Helps), DNA cloning team (co-ordinated by Mark Peggie and Rachel Toth), tissue culture team (co-ordinated by Kirsten Airey), Protein Production Team (co-ordinated by Hilary McLauchlan and James Hastie) and International Centre for Kinase Profiling (kinase-screen@dundee.ac.uk) provided outstanding technical support.</p><!><p>This work was supported by the UK Medical Research Council, and by the pharmaceutical companies that support the Division of Signal Transduction Therapy at Dundee (AstraZeneca, Boehringer-Ingelheim, GlaxoSmithKline, Janssen Pharmaceutica, Merck KGaA and Pfizer).</p>

PubMed Open Access

Glutamine-chitosan modified calcium phosphate nanoparticles for efficient siRNA delivery and osteogenic differentiation

RNA interference (RNAi)-based therapy using small interfering RNA (siRNA) exhibits great potential to treat diseases. Although calcium phosphate (CaP)-based systems are attractive options to deliver nucleic acids due to their good biocompatibility and high affinity with nucleic acids, they are limited by uncontrollable particle formation and inconsistent transfection efficiencies. In this study, we developed a stable CaP nanocarrier system with enhanced intracellular uptake by adding highly cationic, glutamine-conjugated oligochitosan (Gln-OChi). CaP nanoparticles coated with Gln-OChi (CaP/Gln-OChi) significantly enhanced gene transfection and knockdown efficiency in both immortalized cell line (HeLa) and primary mesenchymal stem cells (MSCs) with minimal cytotoxicity. The osteogenic bioactivity of siRNA-loaded CaP/Gln-OChi particles was further confirmed in three-dimensional environments by using photocrosslinkable chitosan hydrogels encapsulating MSCs and particles loaded with siRNA targeting noggin, a bone morphogenetic protein antagonist. These findings suggest that our CaP/Gln-OChi nanocarrier provides an efficient and safe gene delivery system for therapeutic applications.

glutamine-chitosan_modified_calcium_phosphate_nanoparticles_for_efficient_sirna_delivery_and_osteoge

Introduction<!>Synthesis of Gln-OChi<!>Preparation of Gln-OChi coated CaP nanoparticles incorporating siRNA<!>Characterization of nanoparticles<!>Cell culture<!>Cytotoxicity assay<!>Cellular uptake and GFP silencing studies in 2D monolayer cultures<!>Cellular uptake in 3D hydrogels<!>Noggin suppression in 3D hydrogels<!>Osteogenic activity in 3D hydrogels<!>Statistical analysis<!>Nanoparticle preparation and characterization<!>Cellular uptake and gene knockdown<!>HeLa cell line<!>Primary ADSCs<!>Bioactivity of nanoparticles in 3D hydrogels<!>Conclusions

<p>Gene therapy has been extensively investigated to treat diseases by delivering nucleic acids into cells to induce or silence specific gene expression.1–3 In particular, RNA interference (RNAi)-based therapy, mediated by small interfering ribonucleic acids (siRNA), exhibits great potential for cancer treatment4 or tissue engineering applications by suppressing expression of a gene of interest and directing cell behaviors.5 Given that naked siRNA scarcely has cellular penetration due to its large size and anionic nature, various carriers have been developed for efficient delivery of siRNA.6–10 Although viral vectors provide highly efficient gene transfer in genetic engineering of cells,11 the risk of viral insertional mutagenesis and immunogenicity limits their clinical potential.12–14 Therefore, non-viral delivery systems have been investigated to transfer siRNA in a safe and efficient manner.5, 8, 13</p><p>Calcium phosphate (CaP) precipitates have been utilized as DNA transfer vehicles to mammalian cells for a long time due to their good biocompatibility, high affinity with DNA, ease of use and cost-efficiency.15–21 In addition, CaP occurs as natural bone mineral and its pH-dependent solubility enables release of encapsulated nucleic acids into the cytoplasm by endosomal acidification.21, 22 These characteristics make CaP an attractive option to deliver nucleic acids.21–24 However, a major drawback of current CaP-based carriers is their inconsistent transfection efficiencies. This is mainly due to their uncontrollable particle size and formation of large aggregates during rapid CaP precipitation.25</p><p>Cell-based therapy using stem or progenitor cells with their genetic modification is an attractive option to regenerate damaged tissues and treat various diseases. In particular, mesenchymal stem cells (MSCs) are promising candidates for clinical applications due to their ability to be expanded to large cell numbers and differentiated into various cell lineages.26–28 Non-viral gene delivery was efficient at transferring genes into cancer cells or other fast dividing cell lines, but less effective at transferring genes to MSCs or primary cells.29–31 Thus, investigations have been exploited to stabilize CaP colloidal systems to increase its transfection efficiency.22, 25</p><p>In this study, we developed a strategy to stabilize the formation of CaP particles and enhance their cellular uptake for efficient delivery of siRNA molecules using glutamine (Gln)-conjugated oligochitosan (OChi). Chitosan is a naturally occurring polysaccharide and is widely used in biomedical applications due to its biocompatibility, biodegradability, and low immunogenicity.32–34 Moreover, abundant primary amine groups in chitosan facilitate the formation of electrostatic complexes with negatively charged siRNA. Chitosan derivatives such as OChi exhibit lower viscosity and higher solubility at physiological pH than chitosan, making them more attractive to be applied in biomedical formulations. It has been demonstrated that positive surface charges induced a great binding with genes and enhanced cellular uptake of the nanoparticles.35–38 We further modified OChi using highly cationic Gln (pKa = 9.13) to create a particle with high net positive charge under normal physiological conditions (pH = 7.4). We evaluated the transfection and gene silencing efficiency of the CaP particles modified with Gln-conjugated OChi (CaP/Gln-OChi) in both HeLa cells and adipose-derived MSCs (ADSCs) expressing green fluorescent proteins (GFP). We further investigated the feasibility of the CaP/Gln-OChi particles to induce osteogenic differentiation of MSCs by loading Noggin targeting siRNA (siNOG) into the particles and subsequently embedding them into three-dimensional (3D) hydrogels with ADSCs.</p><!><p>Gln-OChi was synthesized using EDC chemistry.39 Briefly, Gln (Invitrogen, Carlsbad, CA) was added at a concentration of 5% (w/v) to OChi (Mw 5 kDa, 86% deacetylated, Sigma-Aldrich, St. Louis, MO) aqueous solution (1% (w/v)) under stirring. To this solution, EDC (Sigma-Aldrich) was added drop wise at a final concentration of 0.1 M. The reaction was continued for 15 h and the resultant solution was dialyzed against distilled water with four changes, subsequently lyophilized and stored at 4 °C. The substitution degree of Gln (DSGln) in Gln-OChi was assessed using 1H NMR (D2O for Gln and OChi, D2O/acetic acid for Gln-OChi).</p><!><p>siRNA-loaded CaP particles were prepared as described previously40 with some modifications. Briefly, a solution of 2.5 M CaCl2 was diluted in 10 mM Tris/HCl buffer (pH 7.5) (2 : 23 μL) (solution A). Another solution (10 μL) of 50 mM Hepes buffer containing 1.5 mM Na3PO4 and 140 mM NaCl (pH 7.5) was mixed with siRNA (10 μM, 10 μL) (solution B). Solution A was mixed with solution B by short vortex. Chitosan (OChi or Gln-OChi) solution (0.02% w/v, pH 6, 35 μL) was subsequently added during vortex mixing (1,500 rpm) at a weight ratio of 5 to siRNA. The pH of the final reaction solution was approximately 7. Commercially available Lipofectamine® 2000 (Invitrogen, Carlsbad, CA) was used as a positive control. Lipoplexes were prepared as described in the manufacturer's protocol at a Lipofectamine® 2000/siRNA weight ratio of 7.5. Each sample solution was used immediately after preparation. Chitosan NPs incorporating siRNA without CaP (OChi/siRNA or Gln-OChi/siRNA) were prepared at a weight ratio of 50 as previously described.41, 42 The incorporation efficiency (%) of siRNA in the particles was determined using fluorescently labeled siRNA (Cy3-siRNA, Ambion Inc., Austin, TX). After Cy3-siRNA was loaded with the particles as stated above, particles were centrifuged at 13,000 rpm for 40 min, and then the amount of residual Cy3-siRNA in the supernatant was measured using fluorescent microplate reader (n = 3). The incorporation efficiency (%) was calculated using the following Equation: (1)Incorporation efficiency (%)=T−ST×100%, where T is the total Cy3-siRNA and S is the amount of free Cy3-siRNA in the supernatant.</p><!><p>To observe morphology of particles, a drop of each particle-dispersed solutions as stated in section 4.2 was placed on silicon wafer and air-dried for 24 h, and then scanning electron microscopy (SEM; Nova NanoSEM230, FEI, Hillsboro, OR) was performed in low vacuum mode. The particle size was measured immediately after preparation and after incubation for 0.5, 1, 2, 3, and 20 h, respectively. The size and zetapotential of particles were determined using Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) (n = 3). Measurements were performed in water at an angle of 173° and 25 °C.</p><!><p>HeLa cell line and eGFP expressing HeLa cell line (HeLa-GFP) were kind gifts from Dr. Kamei, University of California, Los Angeles. HeLa and HeLa-GFP cell lines were grown in the basal culture medium of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin/streptomycin. ADSCs and eGFP expressing ADSCs (ADSC-GFP) were isolated from inguinal fat pads in C57BL/6 mice at age of 4 – 8 weeks according to methods previously described.43, 44 Briefly, adipose tissues collected from mice were washed in sterilized phosphate-buffered saline (PBS); cut into small pieces and digested with collagenase type I (0.1% w/v in PBS) for 2 h. The digested solution was centrifuged at 1,200 rpm for 5 min to collect cells, and then cell-pellet was resuspended in the basal culture medium. The resuspended cells were seeded onto tissue culture flasks.</p><!><p>Cytotoxicity of nanoparticles was determined by alamarBlue assay (Invitrogen, Carlsbad, CA). Briefly, HeLa cell line or ADSCs were seeded in 96-well plates at a density of 3 × 104 cells/well or 1 × 104 cells/well, respectively. After 24 h incubation, medium was replaced by 100 μL transfection medium (DMEM medium containing 10% FBS) with various concentrations of nanoparticles and incubated for another 5 h for HeLa cell line and 15 h for ADSCs. Then, the medium was replaced with 100 μL of 10% (v/v) alamarBlue solution in basal medium. After 3 h incubation, the fluorescence intensity (FI) of alamarBlue was assayed at 600 nm (emission) with an excitation wavelength of 535 nm. Cells without nanoparticles (blank group) were taken as positive controls with 100% viability. As an untreated group, the 10% (v/v) alamarBlue solution was added in an empty well without cells and incubated together. The relative cell viability (%) was calculated using the following Equation: (2)Relative cell viability (%)=FIs−FIuntreatedFIblank–FIuntreated×100%, where FIs, FIuntreated, and FIblank are fluorescence intensity of sample, untreated group, and blank group, respectively.</p><!><p>HeLa cell line or ADSCs were seeded onto 8-well chambered glass for 2D cellular uptake study. For GFP silencing study, HeLa-GFP or ADSC-GFP was used. When cells were grown to 70% confluence, the basal growth medium was changed with the transfection medium. The cells were then incubated with the transfection media containing siRNA incorporated NPs for 6 h for HeLa cells or 15 h for ADSCs in a 37 °C humidified atmosphere with 5% CO2. Fluorescently labeled siRNA (Cy3-siRNA) was used for cellular uptake study and anti-GFP siRNA (siGFP) (Life Technologies, Grand Island, NY) was used for GFP silencing study. Following this incubation, the media was aspirated and the cells were washed with PBS to remove any free-floating NPs. Then, cells were fixed with 10% neutral buffered formalin (NBF) solution and fluorescence images were observed using an Olympus IX71 microscope (Olympus, Tokyo, Japan). Transfection efficiency (%) was analyzed by counting transfected cells in relation to the total number of cells (n = 5).45 Relative GFP intensity (%) was quantified by using NIH-ImageJ software (http:/rsb.info.nih.gov/ij/) and normalized by total cell number as determined by the DAPI staining (n = 5).</p><!><p>Cy3-siRNA was incorporated into CaP/Gln-OChi to visualize cellular uptake of NPs inside hydrogel. Photocrosslinkable methacrylated glycol chitosan (MeGC) was prepared as previously described.46 Mix ADSCs at a density of 2 × 106 cells/mL and CaP/Cy3-siRNA/Gln-OChi (final concentration of 625 nM for siRNA) in MeGC solution (final concentration of 2% w/v). The hydrogel was formed by exposing 40 μL of the solution to visible blue-light (400 – 500 nm, 500 – 600 mW/cm2, Bisco Inc., Schaumburg, IL) in the presence of riboflavin (final concentration 6 μM), as a photoinitiator. Prepared hydrogels were incubated in 1 mL of transfection media for each time point. Following this incubation, the hydrogel was fixed with 10% NBF solution and images were taken using an Olympus IX71 microscope. Transfection efficiency (%) was quantified by counting transfected cells in relation to the total number of cells (n = 5).45</p><!><p>siNOG incorporated CaP/Gln-OChi nanoparticles (CaP/siNOG/Gln-OChi) were prepared and encapsulated with ADSCs in MeGC hydrogels as described above. After 24 h of transfection, total RNA was extracted from the sample following the protocol developed by Flynn with a few modifications using TRIZOL (Invitrogen, Carlsbad, CA) and RNeasy Mini kit (Qiagen, Valencia, CA).47 Total RNA was reverse transcribed to cDNA using cDNA transcription kit (Invitrogen, Carlsbad, CA). NOG expression was evaluated by quantitative real-time PCR using LightCycler 480 PCR system (Roche, Indianapolis, IN) with SYBR Green (n = 3). The housekeeping gene (GAPDH) expression was used to normalize NOG gene expression levels. The following primers were used. GAPDH: TGTGTCCGTCGTGGATCTGA (forward), CCTGCTTCACCACCTTCTTGA (reverse); NOG: GCCAGCACTATCTACACATCC (forward), GCGTCTCGTTCAGATCCTTCTC (reverse).</p><!><p>CaP/siNOG/Gln-OChi was encapsulated with ADSCs in MeGC hydrogels. After 24 h of transfection, transfection medium was replaced by osteogenic medium (basal medium supplemented with 50 μg/mL L-ascorbic acid, 10 mM glycerophosphate, 100 nM dexamethasone, and 100 ng/mL BMP2) and cultured for 3 days. Osteogenic differentiation was confirmed using alkaline phosphatase (ALP) staining and ALP activity assay. ALP staining was performed as previously described.48 Briefly, cells were fixed in 10% NBF for 20 min, washed with PBS, and incubated in a solution consisting of Nitro Blue Tetrazolium (Sigma, St. Louis, MO) and 5-Brom-4-chlor-3-indoxylphosphat (Sigma, St. Louis, MO) stock solutions in AP buffer (100 mM Tris, 50 mM MgCl2, 100 mM NaCl, pH 8.5) for 2 h. Macroscopic images were taken using Olympus SZX16 Stereomicroscope and magnified images were observed using an Olympus IX71 microscope. ALP expression appeared in blue. For ALP activity assay, samples were washed with PBS, incubated in a lysis buffer consisting of 0.01% Tween-20 diluted in PBS at 4 °C for 5 min. ALP activity was determined colorimetrically using p-nitrophenol phosphate (Sigma, St. Louis, MO) as a substrate and measured at 405 nm. Measurements were performed in triplicates and normalized to total protein contents determined by the MicroBCA protein assay kit (Thermo Scientific, Rockford, IL).</p><!><p>Statistical analysis was performed using the analysis of variances (ANOVA) followed by Tukey's post hoc test. A value of p < 0.05 was considered as significant.</p><!><p>Gln was covalently conjugated to OChi by EDC mediated chemical conjugation to increase its amino group content (Figure 1a). 1H NMR spectra clearly showed the presence of Gln peaks at around 2.5 ppm and 2.2 ppm in the Gln-OChi, which were not detected in OChi. The DSGln was assessed by integration of the peaks at δ2.0 – 2.5 (-NH(CO)CH3 of acetyl in OChi and -CH2CH2(CO)NH2 of Gln) and δ2.9 – 3.2 (protons at the C2 position in OChi) (Figure 1b). The DSGln and the molecular weight of Gln-OChi determined by 1H NMR were 9% and 5.4 kDa, respectively.</p><p>siRNA-loaded CaP nanoparticles (NPs) with (CaP/siRNA/OChi, CaP/siRNA/Gln-OChi) or without (CaP/siRNA) chitosan coating were prepared to examine the effects of Gln-OChi on NP formation and characteristics as illustrated in Figure 2a. The hydrodynamic size and zeta potential of particles were analyzed using Zetasizer Nano ZS. The initial mean diameter of CaP/siRNA NPs was 443 nm with a broad size distribution (PDI = 0.863). The addition of OChi or Gln-OChi significantly reduced the particle size to 89 nm or 119 nm, with a reduced PDI of 0.153 or 0.216, respectively. It is known that particles in size ranging from tens to one hundred nanometers are optimal for efficient endocytic uptake.49 siNRA-loaded CaP precipitates rapidly formed large aggregates (~1.5 μm in diameter) in 1 h incubation at room temperature, while chitosan-modified CaP NPs were stable in dispersion for 24 h with a diameter of 110 nm and 172 nm for CaP/siRNA/OChi and CaP/siRNA/Gln-OChi, respectively (Figure 2b). The zeta potential of CaP/siRNA NPs was +13.0 ± 1.3 mV, while the modification of the particles with OChi or Gln-OChi greatly increased the zeta potentials to +32.3 ± 0.9 mV or +41.9 ± 1.2 mV, respectively. These results indicate that chitosan coating enhanced the surface charge and prevented particle aggregation because of electrostatic repulsion. In general, particles are physically stable when their zeta potential values are higher than 30 mV, while lower zeta potential values can lead to unstable particle dispersion.50 The SEM observations revealed that chitosan-coated NPs are relatively spherical, while unmodified CaP/siRNA particles without chitosan coating formed large agglomerates during preparation (Figure 2c).</p><p>Although positive surface charge can improve cellular uptake, meanwhile it may present greater cytotoxicity.41, 42, 51 The cytotoxicity of siRNA-loaded NPs was evaluated in HeLa cell line and primarily harvested ADSCs using an alamarBlue assay (Figure 3). CaP-based NPs with or without chitosan modification (CaP/siRNA/OChi, CaP/siRNA/Gln-Ochi, and CaP/siRNA) were found to be minimally cytotoxic and no significant decrease on cell viability was observed for both HeLa cells and ADSCs. Chitosan-based NPs (OChi/siRNA and Gln-OChi/siRNA) without CaP also displayed minimal cytotoxicity (Figure S1).</p><!><p>The siRNA incorporation efficiency was found to be 80 ~ 83% for CaP related NPs used in this study, and 68% for Lipofectamine® 2000.</p><!><p>To test the potential of the NPs for intracellular delivery, HeLa cells were incubated with NPs containing fluorescently labeled model siRNA (Cy3-siRNA) for 6 h. Fluorescent microscopic observation showed that fluorescence was detected in the cells treated with NPs but not in the cells treated with free siRNA or blank NPs (Figure 4a). The transfection efficiency of CaP NPs was greatly increased from 65 ± 4% to 80 ± 2% after OChi modification (Figure 4b). Modification of CaP NPs with Gln-OChi further increased the transfection efficiency to 91 ± 6%, which was comparable to the Lipofectamine® 2000-mediated transfection (98 ± 3%). These data indicate that particle surface charges played a dominant role in membrane penetration and cellular internalization. The observed higher transfection efficiency may be attributed to the increased positive charge of the particles by chitosan or Gln moiety. It is well known that positively charged particles can facilitate the nonspecific attachment to the negatively charged cellular membranes and promote subsequent uptake by cells.52 It has been demonstrated that the particles with higher positive charges possessed stronger affinity with negatively charged proteoglycans of cell membrane, which led to higher cellular internalization.35–38 It was also reported that the covalent conjugation of Gln to chitosan increased the mucoadhesivity and permeation capacity across intestinal tissue.39, 53</p><p>To determine the gene knockdown efficiency, NPs were loaded with GFP targeting siRNA (siGFP) and incubated with HeLa cells expressing GFP (Figure 4c and d). CaP-based NPs greatly suppressed the expression of GFP with the highest GFP suppression (83% ± 5%) from CaP/siGFP/Gln-OChi NPs, which was comparable to GFP suppression mediated by Lipofectamine® (89% ± 4%). No significant GFP suppression was observed in cells treated with free siGFP or blank NPs. These results indicate that increased surface charges by the chitosan modification induced a stable suspension of the NPs via electrostatic stability and improved cellular transfection efficiency, thus enhancing target gene silencing in HeLa cells. Since the chitosan NPs without CaP displayed strong positive charge of +27 – +29 mV (Table S1), their transfection efficiency were evaluated as well. The transfection efficiency of chitosan based NPs was 64 ± 2% and 84 ± 7% for OChi/Cy3-siRNA and Gln-OChi/Cy3-siRNA NPs, respectively, which was similar to those observed with CaP/Cy3-siRNA/OChi and CaP/Cy3-siRNA/Gln-OChi (Figure S2). However, the gene knockdown efficiency was significantly lower with the chitosan-based NPs compared with CaP-based NPs (Figure S3). Although positively charged surface of NPs facilitated cellular uptake in a charge dependent manner, these results indicate that the increased transfection efficiency does not necessarily lead to higher knockdown efficiency. The possible reasons could be that rapid dissociation of the ployplex before lysosome escape for low deacetylate chitosan and highly stable and inefficient polyplex formed by high deacetylate chitosan which did not dissociate after 24 h.54, 55 The relative low gene knockdown efficiency of our chitosan-based NPs probably resulted from the high deacetylate chitosan used in the present study.</p><!><p>Non-viral mediated gene delivery methods are often less efficient in primary cells. However, primary cells will be more clinically relevant in tissue engineering than immortalized cells. We verified the transfection efficiency and gene knockdown efficiency of the NPs using primary ADSCs. The transfection efficiency of Cy3-siRNA-loaded CaP was 28 ± 8% in ADSCs (Figure 5a and b), which was significantly lower (p < 0.01) than that in HeLa cell line (65 ± 4%). In contrast, CaP NPs modified with chitosan showed efficient cellular uptake in ADSCs, which was comparable to that observed in HeLa cell line. Specifically, the transfection efficiency was 73 ± 6% or 87 ± 5% with CaP/Cy3-siRNA/OChi or CaP/Cy3-siRNA/Gln-OChi NPs, respectively.</p><p>We also examined the GFP knockdown efficiency of the NPs in ADSC expressing GFP (Figure 5c and d). As expected, CaP/siGFP showed lower GFP silencing in ADSCs compared to HeLa cells. However, chitosan-modified CaP NPs (CaP/Cy3-siRNA/OChi or CaP/Cy3-siRNA/Gln-OChi) revealed similar GFP-silencing efficiency in primary ADSCs to that in HeLa cell line. This result is consistent with previous studies demonstrating that the transfection through nonviral vectors was less efficient in primary cells.29–31 These results also indicate that sophisticated controls in sizes and surface charge density of NPs are required for effective gene transfection in primary cells.</p><!><p>To investigate the feasibility of CaP/Gln-OChi NPs as an efficient gene carrier for bone tissue engineering, we evaluated the NPs in in vitro 3D setting by loading siRNA into the NPs and subsequently embedded them into photocrosslinkable chitosan hydrogels with ADSCs. We have previously developed the injectable hydrogel system using visible blue-light inducible chitosan (MeGC) and riboflavin initiator that supported proliferation and osteo- or chondrogenic differentiation of encapsulated MSCs.46, 56, 57 Localization of siRNA-loaded NPs into the cells encapsulated in the hydrogel was visualized by incorporating fluorescent model siRNA (Cy3-siRNA) into the NPs (Figure 6). Fluorescent microscopy showed that the Cy3-siRNA loaded NPs were rapidly localized to the cells over time and cellular uptake was reached up to 85% after 24 h of incubation in the hydrogels (Figure 6b).</p><p>We further evaluated bioactivity of the NPs in the 3D hydrogel system by loading siNOG into CaP/Gln-OChi NPs. NOG is a specific antagonist of bone morphogenetic proteins (BMPs) and prevents BMPs from binding to their cell surface.58–60 Our previous studies demonstrated that knockdown of NOG expression enhanced osteogenesis in vitro48 and bone formation in vivo.44 The expression of NOG mRNA was examined by qRT-PCR analysis (Figure 7a). Lipofectamine® 2000 was used as a positive control. After 3 days in culture, mRNA level of NOG was significantly decreased to 49% and 45% in the hydrogels containing CaP/Gln-OChi and Lipofectamine® 2000, respectively, whereas no significant change was observed in the hydrogels containing naked siNOG, blank NPs without siRNA, or NPs with control-siRNA. The osteogenic effect of NOG suppression was evaluated by ALP expression in ADSCs (Figure 7b). Our results showed that ADSCs incubated with siNOG-loaded CaP/Gln-OChi NP significantly increased expression of ALP to the similar extent to Lipofectamine®-mediated ALP expression. ALP expression was further confirmed by ALP staining as shown in Figure 7c. Live/Dead staining showed a high level of viability of encapsulated cells (>90%) during transfection in the hydrogel (Figure 7d). The collective results indicate that CaP/Gln-OChi NPs not only successfully delivered siNOG into primary ADSCs but also suppressed the expression of target gene (NOG), which induced osteogenic differentiation of encapsulated cells in 3D hydrogel environment.</p><!><p>The formation of CaP NPs was stabilized via Gln-conjugated chitosan (Gln-OChi) coating which introduced electrostatically stable (> +30 mV) particles without aggregation. Gln-OChi coating significantly enhanced the transfection efficiency of the NPs in both immortalized cell line and primary cells with no significant cytotoxicity. Moreover, the Gln-OChi coated CaP NPs loaded with siRNA targeting BMP antagonist NOG greatly suppressed the expression of NOG in MSCs and subsequently enhanced their osteogenic differentiation in in vitro 3D hydrogel microenvironments. These results indicate that our nanocarrier system prepared with synthetic bone mineral and natural polysaccharide may be an effective and safe vehicle for therapeutic drug/gene delivery.</p>

PubMed Author Manuscript

Dual Wavelength RP-HPLC Method for Simultaneous Determination of Two Antispasmodic Drugs: An Application in Pharmaceutical and Human Serum

A reverse phase stability indicating HPLC method for simultaneous determination of two antispasmodic drugs in pharmaceutical parenteral dosage forms (injectable) and in serum has been developed and validated. Mobile phase ingredients consist of Acetonitrile : buffer : sulfuric acid 0.1 M (50 : 50 : 0.3 v/v/v), at flow rate 1.0 mL/min using a Hibar μBondapak ODS C18 column monitored at dual wavelength of 266 nm and 205 nm for phloroglucinol and trimethylphloroglucinol, respectively. The drugs were subjected to stress conditions of hydrolysis (oxidation, base, acid, and thermal degradation). Oxidation degraded the molecule drastically while there was not so much significant effect of other stress conditions. The calibration curve was linear with a correlation coefficient of 0.9999 and 0.9992 for PG and TMP, respectively. The drug recoveries fall in the range of 98.56% and 101.24% with 10 pg/mL and 33 pg/mL limit of detection and limit of quantification for both phloroglucinol and trimethylphloroglucinol. The method was validated in accordance with ICH guidelines and was applied successfully to quantify the amount of trimethylphloroglucinol and phloroglucinol in bulk, injectable form and physiological fluid. Forced degradation studies proved the stability indicating abilities of the method.

dual_wavelength_rp-hplc_method_for_simultaneous_determination_of_two_antispasmodic_drugs:_an_applica

1. Introduction<!>2. Experimental<!>2.1. Apparatus and Materials<!>2.2. Chromatographic Conditions<!>3.1. Standard Preparation<!>3.2. Sample Preparation of Injectable Solutions<!>3.3. Sample Preparation of Serum<!>3.4. Sample Preparation for Degradation Studies<!>3.5. Stability Studies<!>3.6. Method Validation<!>4. Results and Discussion<!>4.1. Method Development and Optimization<!>4.2. Validation Studies<!>4.3. Degradation Studies<!>4.4. Stability Studies<!>5. Conclusions

<p>Chemically, phloroglucinol (1,3,5-trihydroxybenzene, PG) and its methylated derivative tri-O-methylphloroglucinol (TMP), Figure 1, are established pharmaceutical agents inhibit the action of catechol-O-methyl transferase, inducing relaxation of smooth muscles, and decreasing glycerol-induced abdominal pain and are also characterized by a swift and strong spasmolytic activity, hence relieving pain. Therefore, PG is often used in combination with trimethylphloroglucinol as an antispasmodic drug and is regarded to be effective in decreasing smooth muscle spasm. PG/TMP combination is recommended against biliary calculi, severe pain of urinary or gastrointestinal tract, pain of abdominal region, spastic conditions of the female genital system, and pain in gynecology [1–7].</p><p>Literature survey reveals that some of the analytical methods for phloroglucinol are available including extraction and high-performance liquid chromatography (HPLC) [8–10], HPLC-mass spectrometry [11, 12], gas chromatography-mass spectrometry [13], and spectrophotometry [14]. Other reported methods include titrimetry, spectrophotometry, paper chromatography, and flow injection analysis [15–20].</p><p>However, there is no simple and sensitive method to be followed on industrial basis especially in general quality control laboratories. Previously mentioned methods involve complicated instrumentation and serialization. Therefore, they cannot be followed in the laboratories particularly those of third world countries. Hence our group already developed a cost effective method for its fixed dose composition in tablet form [21], but still there was a need for an analytical method which would help to determine the active pharmaceutical ingredients (APIs) in parenteral products and physiological fluid. Accordingly, the purpose of this write-up is to suggest a systematic approach for the development of a validated simple, sensitive, and stability indicating RP-HPLC method that should meet the current ICH and regulatory requirements [22].</p><!><p>The present method was designed to be easy to use, sensitive, and rapid. Separation and quantification of PG and TMP in pharmaceutical drug formulations and blood were achieved with an isocratic elution and with dual wavelength technique.</p><!><p>SIL 10A autoinjector HPLC system comprising of SCL 10A system, controller, SPD 20A prominence UV/VIS detector, and Shimadzu LC 20 AT pump with LC Solutions software, was used. Separation was performed on a Hibar μBondapak ODS C18 HPLC column (4.6 × 250 mm; 10 μm bead size) and maintained at 25°C. A UV-visible Shimadzu 1650 PC spectrophotometer with UV Probe software, ultrasonic cleaner (Elmasoni E 60 H), Jenway 3240 pH meter, and Sartorius TE2145 analytical balance were used in the research work. Throughout the work only amber glass flasks were used to avoid light effect on the solution of PG and TMP standards and samples.</p><p>Trimethylphloroglucinol and phloroglucinol were kind gifts from a National Pharmaceutical company, and sulphuric acid (Merck, Germany) and acetonitrile (HPLC grade) were purchased from Fisher Scientific. The injectable containing PG and TMP were obtained from commercial source (SPASFON Injection, SPADIX Injection, FUROSINOL Injection, and ANAFORTAN PLUS Injection) labeled to contain 10 mg/mL and 0.01 mg/mL of PG and TMP, respectively. Distilled water was procured through RO plant (Waterman, Pakistan).</p><!><p>The HPLC analysis was carried out at ambient temperature. The compound was chromatographed isocratically with a mobile phase consisting of acetonitrile (HPLC grade): sodium n-heptane sulphonate (0.005 M)  : sulfuric acid 0.1 M (50 : 50 : 0.3,  v/v/v) with the pH adjusted if required to 3.0 ± 0.1 using 0.1 M sulfuric acid or 0.1 M NaOH. While for sample and standard preparation diluent was prepared from acetonitrile (HPLC grade): distilled water (50 : 50, v/v%). The mobile phase was filtered by passing through a 0.45 µm membrane filter (Millipore, Bedford, MA, USA) and degassed via sonication. The flow rate was 1.0 mL/min, and the injected volume was 20 µL. The effluent was monitored at dual wavelength of 266 and 205 nm.</p><!><p>In a 100 mL volumetric flask, about 10 mg of TMP reference standard was weighed accurately and dissolved in diluent to have a stock solution of 100 µg/mL. Similarly in another 100 mL volumetric flask, accurately 500 mg of PG reference standard was weighed and dissolved in diluent, sonicated for 2 minutes, and let to cool to room temperature. Then, 5 mL of TMP stock solution was added, and volume was made up to the mark with the same diluent. Finally 2 mL of this solution was diluted in 100 mL of diluent to get a 100 µg/mL of PG and 0.1 µg/mL of TMP working standard solution. The standard was then filtered through 0.45 µm filter paper and injected into the HPLC system.</p><!><p>For making a sample of 100 µg/mL of PG and 0.1 µg/mL of TMP, 10 ampoules were broken, and content was mixed to get an evenly homogenized stock sample. The sample volume was taken accurately equivalent to 10 mg of PG & 0.01 mg of TMP in 100 mL volumetric flask, and 50 mL of diluent was added. The sample was sonicated for 1 minute and then diluent was added up to the mark and placed on stirrer for 5 minutes. The sample was then filtered through 0.45 µm filter paper and injected into the HPLC system.</p><!><p>Blood samples were collected from healthy volunteers in evacuated glass tube through an indwelling cannula placed on forearm vein by a trained clinical laboratory technician. The volunteers were not involved in any medication, smoking, and strenuous activity. The blood was shaken and centrifuged at 6,000 rpm for 30 min to separate out plasma. 9 mL acetonitrile was added to 1 mL plasma and centrifuged at 6,000 rpm for 30 min to deproteinate it. The supernatant serum thus obtained was stored at 20°C and filtered for subsequent analysis. For making a working sample solution of 12.5 mL, stock sample was taken in 50 mL flask followed by 30 mL of serum addition. The sample thus obtained was stirred for 10 minutes and then diluent was added up to the mark. Further 2 mL of this solution was diluted in 50 mL of flask with the aid of diluent. All samples prepared were filtered through 0.45 µm membrane filter and injected in triplicate into the HPLC system.</p><!><p>For this purpose 5 mL of the stock sample was diluted in 100 mL diluent to make a stock sample solution. For working purpose, 10 mL of stock sample solution was diluted in four individual 50 mL volumetric flasks, and 15 mL of degrading agent were added to each flask, with the exception of one to which only diluent was added; these included 0.1 N HCl, 0.1 N NaOH, and 30% H2O2, and then to each flask diluent was added up to the mark. All the four samples were placed in water bath at 60°C for one hour. The samples were then filtered through 0.45 µm membrane filter and injected into the HPLC system. Intentional degradation was attempted with stress conditions exposing the drugs to acid (0.1 N HCl), alkali (0.1 N NaOH), hydrogen peroxide (30%), and heat (60°C) to evaluate the ability of the proposed method to separate drugs from its degradation products. For all conditions the temperature was kept constant at 60°C for a period of one hour.</p><!><p>For stability studies the commercially available injection (parenteral) samples in ampoules were placed at accelerated conditions of temperature that is at 40°C with 75% relative humidity and at ambient conditions of 30°C temperature with 65% relative humidity in environmental chamber for six months. The stability protocol in Table 6 was followed for six months.</p><!><p>ICH guidelines [22] were used to perform method validation studies. Various procedures including specificity, linearity, range, accuracy, and intraday and interday precision were evaluated.</p><p>To study linearity, twenty dilutions of standard were made to prepare standard solution in range from 10% to 200%, that is, from 10 µg/mL to 200 µg/mL of PG and from 0.01 µg/mL to 0.2 µg/mL of TMP, respectively, drugs content. The standard calibration curve was generated using regression analysis with online help (http://wessa.net/). For specificity commonly used excipients in injection preparation were spiked in a preweighed quantity of drugs to determine the effect and interference of excipients in quantification of the drugs.</p><p>In order to find out the repeatability and reproducibility of the method, precision was studied to find out intra- and interday variations in the test method of PG and TMP in the concentration range of 80–120 μg/mL for PG and 0.08–0.12 μg/mL for TMP, respectively. Precision was determined by analyzing corresponding bulk sample daily for a period of three days and three times a day with an interval of 8 hours against a freshly prepared standard. For determining accuracy the PG and TMP reference standard were accurately weighed and spiked to the injection sample at three different concentration levels to gain 110%, 120%, and 130% of both APIs. At each level, samples were prepared in triplicate, and the recovery percentage was determined.</p><p>Limit of detection and quantification (LOD and LOQ) for the method were established by sequentially diluting the standard solutions at decreasing concentrations, in the range of 100–1 pg/mL for PG and 10–1 pg/mL for TMP. The limit of detection was defined as the concentration for which a signal-to-noise ratio of 3 was obtained, and for quantification limit, a signal-to-noise ratio of 10 was considered. The standards were injected in LC system, and measured signals from the diluted standards were compared with those of blank samples.</p><p>To study robustness, samples of injections were assayed with deliberate variation in the method parameters, such as in the chromatographic conditions, like mobile phase, flow rate, and temperature so forth. Justification of system suitability was established by calculating % relative standard deviation of replicate injections and analyzing the symmetry, resolution of the standard peaks, and theoretical plates of the column.</p><!><p>The HPLC method development and its validation are the prioritized requirements for any drug available in the market to ensure the quality of the products. A few methods are available for determination of the APIs as described earlier, but many of them are used only for certain definite objectives and lack generalization for simultaneous analytical applications in form of pharmaceutical products and serum. Similarly none of them are as much sensitive as ours in terms of their precision, accuracy, limit of detection (LOD), and limit of quantification (LOQ) especially as compared to [7, 9, 12, 14] whose LOD or LOQ was in microgram range only, while ours method is sensitive enough to be used for pharmacokinetic studies. Here, the LOD and LOQ for both APIs are 10 pg/mL and 33 pg/mL, respectively; however considering their ratio in sample formulation, the LOD and LOQ for PG are taken as 10 ng/mL and 33 ng/mL, respectively, and hence LOQ and LOD for TMP can also be considered as the levels of sensitivity of the method.</p><!><p>For developing an efficient method for the simultaneous analysis of PG and TMP, parameters such as detection wavelength, mobile phase composition, optimum pH, and concentration of the standard solutions were comprehensively studied. Both PG and TMP were diluted in dilution solvent and then run through UV spectrophotometer in UV range of 190 nm–400 nm to get maximal wavelengths, Figure 2, where maximum absorbance was gained, that is, 266 nm for both APIs.</p><p>However considering the difference of concentration in both APIs that is 10 to 0.01 mg/mL for PG and TMP, respectively, therefore 205 nm was used for TMP and 266 nm for PG.</p><p>Mobile phase was selected in terms of its components and their proportions. The chromatographic parameters were evaluated using a Hibar μBondapak ODS C18 column the mobile phase composed of acetonitrile : buffer of given proportion promoted a short run time (10 min) without any interference, so this condition was adopted in subsequent analysis.</p><p>The literature survey also revealed that almost all the methods developed so far have utilized acetonitrile as a major component in mobile phases. Acetonitrile is always preferred due to the supreme solubility properties and UV absorbance characteristics, and there is no counterpart substitute for acetonitrile in the reverse phase HPLC and UV application. Therefore, keeping in view the chromatography type and the detection wavelengths in use, acetonitrile was chosen for analysis, though in our previous work [21] we used methanol for its cost effectiveness.</p><!><p>The linearity ranges were found to be 10–200 µg/mL for PG and 0.01–0.2 µg/mL for TMP. The assay was judged to be linear, as the correlation coefficient was 0.9999 and 0.9992 for PG and TMP, respectively, as calculated by the least-square method. A linear correlation was found between the peak areas and the concentrations of APIs, in the assayed range. The regression analysis data are presented in Table 1.</p><p>Chromatogram shown in Figure 3 proves specificity or selectivity of the assayed method, as the chromatograms in samples were found identical with standard chromatogram and no interference peak was observed in sample chromatogram. Peak purities higher than 98.0% were obtained in the chromatograms of sample solutions, demonstrating that other compounds did not coelute with the major peaks. The chromatogram obtained with the mixture of the injection excipients proves that there is no interference from excipients and peak of interest that fulfills all the requirements of symmetrical peak, and hence, the specificity is confirmed.</p><p>The precision of an analytical method is the degree of agreement among individual test results when the method is applied repeatedly to multiple sampling of homogeneous bulk. Intraday precision of the method was evaluated at three different independent concentrations that are, 80%, 100%, and 120% for both drugs (n = 3) by determining their assays. Interday precision of the method was tested for 3 days at the same concentration levels. Solutions for calibration curves were prepared every day on fresh basis. Since the interday and intraday precision obtained %RSD were less than 2% it is assured that the proposed method is quite precise and reproducible, as shown in Table 2.</p><p>The accuracy was investigated by spiking reference standards to a mixture of the injection excipients at three different concentration levels, that is, multiple level recovery studies, and subjected to the proposed HPLC method. The obtained recovery (n = 9) was 98.86–100.96% (RSD% = 0.488) for PG and 98.56%–101.24% (RSD% = 0.139) for TMP, demonstrating the accuracy of the method. Percentage recoveries for marketed products were found to be within the limits, Table 3.</p><p>The statistical analysis showed no significant difference between results obtained by employing the analytical conditions established for the method and those obtained in the experiments in which variations of some parameters were introduced. The parameters used in system suitability test were symmetry of peaks, tailing factor, resolution, and RSD% of peak area for replicate injections. Thus, the method showed to be robust for changes in mobile phase acetonitrile proportion, mobile phase pH, flow rate, and column temperature, Table 4.</p><!><p>During the degradation study, it was observed that upon treatment of PG and TMP with base (0.1 M NaOH), acid (0.1 M HCl), hydrogen peroxide (30%), and heat, maximum degradation was observed in acid and H2O2. Table 5 shows the extent of degradation of both drugs under various stress conditions.</p><p>It is a fact that phenolic compounds, such as phloroglucinol, are known to undergo ready oxidation in basic solutions (due to dissolved oxygen) and by agents such as hydrogen peroxide which increases the absorbance capacity of the molecules, causing increased peak area and hence % calculated amount. And the same is revealed by the data given that PG is more vulnerable to stress conditions especially alkaline and oxidation treatments as compared to TMP. Further, Figures 4(a)–4(d) show the chromatograms of forced degraded samples. Degraded peaks are observed in case of acid hydrolysis, and peak had been broadened due to extra absorbance caused by oxidation of the PG, whereas no appreciable degradation was observed in heat treated sample; hence the drug is stable under heat stress conditions.</p><!><p>Stability testing is an important part of the process of drug product development. The purpose of stability testing is to provide evidence of how the quality of a drug substance or drug product varies with time under a variety of environmental conditions, like temperature, humidity, and light and enable recommendation of storage conditions, retest periods, and shelf life to be established. The two main aspects of drug product that play an important role in shelf-life determination are assay of the active drug and the degradation products generated during stability studies.</p><p>The proposed assay method was applied to the stability study of commercially available injections, for which the samples were placed at 30°C with relative humidity of 65% and at 40°C with relative humidity of 75%. Stability study was performed according to stability protocol as described in the previous section. Samples were analyzed and percentage of contents was measured, Table 6. According to the results obtained both APIs were found to be stable at applied conditions of temperature and relative humidity and were accurately analyzed with the proposed method.</p><!><p>The proposed new HPLC method described in this paper provides a simple, universal, convenient, and reproducible approach for the simultaneous identification and quantification of phloroglucinol and trimethylphloroglucinol in human serum and pharmaceutical formulations (injectable) with good separation and resolution. In addition, this method has the potential application to clinical research of drug combination. Analytical results are accurate and precise with good recovery and lowest detection limit values. In short, the developed method is simple, sensitive, easy, and efficient having short chromatographic time and can be used for routine analysis in QC laboratory and therapeutic monitoring.</p>

PubMed Open Access

Folded Small Molecule Manipulation of Islet Amyloid Polypeptide

SUMMARY Islet amyloid polypeptide (IAPP) is a hormone co-secreted with insulin by pancreatic \xce\xb2-cells. Upon contact with lipid bilayers, it is stabilized into a heterogeneous ensemble of structural states. These processes are associated with gains-of-function including catalysis of \xce\xb2-sheet rich amyloid formation, cell membrane penetration, loss of membrane integrity and cytotoxicity. These contribute to the dysfunction of \xce\xb2-cells, a central component in the pathology and treatment of diabetes. To gain mechanistic insight into these phenomena, a related series of substituted oligoquinolines were designed. These inhibitors are unique in that they have the capacity to affect both solution and phospholipid bilayer catalyzed IAPP self-assembly. Importantly, we show this activity is associated with the oligoquinoline\xe2\x80\x99s capacity to irreversibly adopt a non-covalently fold. This suggests that compact foldamer scaffolds, such as oligoquinoline, are an important paradigm for conformational manipulation of disordered protein states.

folded_small_molecule_manipulation_of_islet_amyloid_polypeptide

INTRODUCTION<!>RESULTS<!>DISCUSSION<!>SIGNIFICANCE<!>Chemical Compounds<!>Materials<!>Synthesis of Oligoquinolines<!>Preparation of IAPP<!>Preparation of Unilameller Vesicles<!>Kinetic assay<!>Circular Dichroism spectroscopy (CD)<!>Electron Microscopy (EM)<!>Fluorescence correlation spectroscopy (FCS)<!>NMR Titration

<p>The aggregation of proteins is implicated in the pathology of numerous diseases(Buxbaum, and Linke, 2012). For example, amyloid-β, α-synuclein, and IAPP underpin pathology in Alzheimers's disease (AD), Parkinson's disease, and type 2 diabetes respectively(Hebda, and Miranker, 2009). Proteins specific to these disorders undergo a conformational change from disordered to a cross-β sheet rich state. Transient intermediates of this process are associated with the toxic gains of function that define disease pathology.</p><p>Membrane-bound oligomeric intermediates of the amylodogenic protein IAPP are hypothesized to contribute to β-cell pathology in diabetes(Haataja, Gurlo, et al, 2008) as well as disease progression in AD(Walsh, Klyubin, et al, 2002) and Parkinson's(Winner, Jappelli, et al, 2011). Such oligomers additionally display cell-penetration and mitochondrial dysfunction gains-of-function(Magzoub, and Miranker, 2012), which may account for data suggesting an intracellular location for toxic potential(Gurlo, Ryazantsev, et al, 2010). Small molecule approaches have been employed to probe the pathways of IAPP self-assembly. One approach is protein mimetics which can serve as template to match IAPP:IAPP, helix:helix interactions and thereby obstruct protein:protein interactions(Cummings, and Hamilton, 2010). Indeed, several compounds have been identified that bind to a membrane stabilized, α-helical sub-domain of IAPP(Hebda, Saraogi, et al, 2009). The scaffold, based on oligopyridine (OP) (Fig. 1A), was designed to project chemical moieties in a linear fashion with spacing that corresponds to the rise per turn of an α-helix. Here, we have taken an alternative approach in which a scaffold based on oligoquinoline (OQ) is used instead with the intended goal of it binding and acting as a perturbant of the target protein structure (Kumar, and Miranker, 2013). In essence, we are comparing the capacity of a foldamer versus a mimetic to affect the activity of an intrinsically disordered system.</p><p>The goal in foldamer design is to recapitulate properties evident in proteins(Gellman, 1998). Namely, the small molecule should cooperatively fold, have a defined and hierarchical structure, and be formed from a discrete length polymer capable of variation without affecting the first two properties. OQs have these properties and present alternative functional groups with a density that does not mimic an α-helix. We hypothesize that display of comparable moieties on OQ versus OP would create a state requiring a bound IAPP to change conformation. The size of the change in IAPP is unimportant except in the requirement that it be sufficient to affect its capacity to self-assemble. The role of structure formation by OQ and the molecular mechanism of perturbation of IAPPs gains of function are directly evaluated in this work.</p><!><p>A series of oligoquinolines (Fig. 1), were synthesized and used to make direct comparisons to oligopyridine scaffolds previously reported by us for the inhibition of membrane-catalyzed IAPP self-assembly(Hebda, Saraogi, et al, 2009). The pentameric oligoquinoline, OQ5, inhibits large unilamellar vesicle (LUV) catalyzed conversion of IAPP to a β-sheet rich state. Upon exposure to 630 μM LUVs formed from a 1:1 mixture of anionic [dioleoylphosphatidylglycerol (DOPG)] and zwitterionic [dioleoylphosphatidylcholine (DOPC)] lipids, 30 μM IAPP undergoes a transition from a predominantly random coil conformation to one that includes strong spectroscopic contributions from α-helical structures (Fig. 2A). After ~1 hour, the protein converts to β-sheet rich species evident by a single Cotton effect minimum near ~218 nm. The presence of equimolar OQ5 prohibits this conversion with α-helical states still dominant after 2 hr (Fig. 2A, S6). Imaging studies further show filamentous aggregate end-products, but only for reactions conducted in the absence of OQ5 (Fig. 2B, C). The presence of OQ5 plainly results in the delayed conversion of IAPP to a filamentous β-sheet rich state without diminishing the sampling of α-helical conformations.</p><p>Lipid catalyzed IAPP fibrillation is more inhibited by OQ5 than by OP5. Reactions were initiated by dilution of a 1 mM stock solution of IAPP into a standard buffer (see Materials and Methods). Changes in fluorescence intensity of an exogenously added fluorophore, thioflavin T (ThT) allowed for real-time monitoring of the fibrillation(Wolfe, Calabrese, et al, 2010). Under these conditions, conversion of 10 μM IAPP to an aggregated state occurs with a reaction time midpoint (t50) of 22±2 hr. In contrast, fibrillation is strongly accelerated by the presence LUVs (Fig. 2D). The t50 for 10 μM IAPP catalyzed by LUVs is 1.0±0.1 hr, consistent with our previously published reports(Hebda, Saraogi, et al, 2009). Addition of 100 μM OP5 to an otherwise matched reaction results in inhibition of aggregation t50 by a factor of ~3 (Fig. 2D). Addition instead of 100 μM OQ5 resulted in no detection of fibers. Using light scatter instead of ThT gave comparable results (Supporting Information Fig. S1 and Fig. S3). OQ5 is plainly a more potent inhibitor of lipid catalyzed fiber formation than OP5.</p><p>Reaction inhibition is sensitive to the repeat number of the mimetic subunit. For both the OP and OQ compound series, inhibition increases with increasing length (n=1–5) (Fig. 2E). The effect of OP compounds saturate at n=4–5 and display inhibition of 3–4 fold at an OP:IAPP ratio of 10:1. In contrast, OQ compounds do not behave methodically for n<5. At n=5, inhibition is abruptly complete (i.e. >20 hr). Lowering the small molecule:IAPP ratio to 1:1 exaggerates this observation with only OQ5 showing any apparent inhibition (Fig. 2F).</p><p>Oligoquinolines affect IAPP via a mechanism that is distinct from oligopyridyls. IAPP self-assembly was observed under buffer only conditions (no LUVs). At 40 μM IAPP, the t50 for IAPP fibrillation is 1.9±0.1 hr (Fig. 3A). The presence of equimolar OP5 instead results in a t50 of 1.1±0.1 hr, i.e. OP5 is a mild agonist under bilayer-free conditions. In contrast, OQ5 is a strong inhibitor. At a ratio of only 10:1, (IAPP:OQ5) 3–4 fold inhibition is apparent (Fig. 3A). At 1:1, no conversion is apparent, even after 22 hr. Observations using light scatter, CD and TEM imaging give comparable results (Supporting Information Fig. S1). In part, these observations can be a consequence of OQ5 effects on fiber elongation. In the presence of LUVs, a 10 μM reaction of IAPP is accelerated 5–6 fold by the addition of 0.1 μM IAPP as preformed fibers. One equivalent of OQ5 is sufficient to negate this acceleration while 10 equivalents eliminates fiber formation (Supporting Information Fig. S4A). Similarly, in the absence of a bilayer catalyst, 0.1 equivalent of OQ5 is sufficient to negate seeded acceleration (Supporting Information Fig. S4B). This stands in marked contrast to OP5 in which 1 equivalent of small molecule is shown to have little effect on seeded reactions(Hebda, Saraogi, et al, 2009). Thus, under matched conditions, OP5 is either neutral or acts as an agonist while OQ5 acts as a potent antagonist.</p><p>Numerous small molecules have previously been reported as inhibitors of IAPP fiber formation. Here, we assess epigallocatechin-3-gallate (EGCG) and acid fuchsin. EGCG inhibits amyloid formation of many proteins including α-synuclein and Aβ-peptide and is currently under phase 2 clinical trials for Alzheimer's disease(Bieschke, Russ, et al, 2010). These molecules show significant (3–4 fold) inhibitory capacity in the absence of bilayer at equimolar protein:compound ratio (Fig. 3B). For both compounds however, no inhibition of IAPP assembly is apparent for lipid catalyzed fibrillation. To our knowledge OQ5 is the first reported inhibitor of IAPP that functions effectively under both solution phase and lipid bilayer catalyzed conditions.</p><p>OQ5 binds to the α-helical sub-domain of IAPP. OQ5 was titrated into a solution of 15N rIAPP and assessed for structural changes by NMR (Fig. 4A). Because of hIAPP's amyloidogenic nature under the NMR conditions, a sequence variant of IAPP from rodents (rIAPP) was used. We have previously shown that disruption of β-sheet formation strongly diminishes cytotoxic potential using rIAPP and its helical subdomain variant (H18R). This result has been recently confirmed(Cao, Abedini, et al, 2013). Importantly, elevation of peptide concentration to 200 and 50 μM respectively not only results in the restoration of colorimetrically assessed toxicity, but also a concentration dependent mitochondrial localization phenotype(Magzoub, and Miranker, 2012). By NMR, the residues affected by OQ5, 3–20 (Fig. 4B), have been previously identified by us and others to be part of the region of IAPP that adopts a helical structure upon interaction with bilayer surfaces (Apostolidou, Jayasinghe, and Langen, 2008; Williamson, Loria, and Miranker, 2009). Thus, OQ5 likely interacts with IAPP in a manner that stabilizes and/or perturbs one or more forms of its α-helical sub-domains. The NMR study supports our hypothesis that OQ5 alters IAPP structure. Titration was limited to a stoichiometric ratio of 4:1 (OQ5:rIAPP) as a result of limited solubility of oligoquinoline (OQ5) at the higher IAPP concentrations required for NMR. The absence of titration sensitivity across central regions of the rIAPP sequence is not the result of mutation. A key subpeptide outside the helical subdomain of IAPP, hIAPP20-29, was independently examined. This sub-region is the hypothesized site of amyloid nucleation, likely catalyzed by high local concentrations created by helical assembly of IAPP's N-terminal domain(Ruschak, and Miranker, 2009). The kinetic profile of 200 μM hIAPP20-29 shows nucleation dependent kinetics with a t50 of 6.2±0.1 hr (Fig. 4C) consistent with previous reports assessing the independent behavior of this peptide. Inclusion of stoichiometric OQ5 in an otherwise matched reaction results in no change in the fiber formation profile. OQ5 inhibition of full length hIAPP at 1:1 stoichiometry (Fig. 3A) is therefore unlikely to be the result of interactions with the 20–29 sub-domain.</p><!><p>The development of synthetic foldamers is inspired by the capacity of proteins to fold to a conformationally restricted state, and from that state, achieve strongly specific molecular interactions (Guichard, and Huc, 2011). Oligoquinoline, a recent addition to the foldamerome (Gellman, 1998), has recently been identified by us to have the capacity to affect IAPP amyloid formation. The mechanism and effects of this interaction remained elusive. Here, we have made a series of foldamers and made comparisons both to a series of comparably derivitized protein mimetics as well as literature compounds identified through screening. Our findings show that i) OQs specifically and uniquely inhibit lipid catalyzed as well as solution phase amyloid nucleation and elongation processes, ii) perform this inhibition by interacting with a specific structural subunit of IAPP and iii), acquire these capacities in a manner that is strongly nonlinear in terms of its dependence on oligoquinoline subunit length. Our observations suggest that folded and not unfolded oligoquinolines represents the paradigm for targeting weakly ordered states IAPP.</p><p>A plethora of small molecules have previously been identified which inhibit IAPP fibrillation, e.g. polyphenols(Meng, Abedini, et al, 2010b), resveratrol(Mishra, Sellin, et al, 2009), phenolsulfonphthalein(Levy, Porat, et al, 2008), EGCG derivatives(Meng, Abedini, et al, 2010b),acid fuchsin (Meng, Abedini, et al, 2010a), and IAPP derivatives(Yan, Tatarek-Nossol, et al, 2006). Typically, such molecules are identified using assays that measure only solution phase fibrillation. The collective result appears to be a set of molecules dominated by planar aromatic scaffolds. In the case of N-methyl derivatives of IAPP(Yan, Tatarek-Nossol, et al, 2006), the likely mechanism is not intercalation, but rather a crystal poisoning-like interaction that halts efficient elongation. In both sets of mechanisms, β-sheet structure formation represents the macromolecular target.</p><p>The capacity of OQ5 to inhibit lipid-catalyzed assembly reflects the contribution of α-helical intermediate states to IAPP gains-of-function. Far UV-CD and NMR suggests that OQ5 is interacting directly with the membrane bound helical sub-states of IAPP (Fig. 4) (Williamson, Loria, and Miranker, 2009). In addition, we performed assays looking for inhibition of IAPP20-29 fibrillar assembly. This subunit assembles by direct nucleation from a random coil to a β-sheet state (Ruschak, and Miranker, 2007). No inhibition by OQ5 was observed (Fig. 4C). Additionally, FCS data suggests that IAPP interactions with membranes are weakened by interactions with OQ5 (Supporting Information Fig. S5). In solution, it is plausible that the inhibitory mechanism involves an alternate structure such as β-sheet. Random coil still dominates for IAPP:OQ5 in free solution potentially masking a low population of structured states (Supporting Information Fig. S6D). However, we have shown that a subset of IAPP residues weakly samples α-helical states even in the absence of membrane. Addition of a membrane model simply stabilizes this sub- population (Williamson, Loria, and Miranker, 2009). The fact that OQ5 operates substoichiometrically for IAPP in free solution might reasonably be attributed to interactions with this small population. In total, these observations strongly support our assertion that OQ5 interacts and affects amyloid assembly through interactions with the α-helical state of IAPP.</p><p>The adoption of a folded compact state underpins OQ capacity to affect IAPP. OQs undergo a dramatic change in their efficacy upon reaching a subunit length of five (Fig. 2E, F). This change is not accounted for by charge pairing as human IAPP contains only 2–3 positive charges. Five units is the minimum required for OQ to complete one unit of its non-covalent fold (Fig. 1). In addition, previous studies have shown that pentameric and not shorter polymers of OQ collapse into left and right handed states that do not interconvert in aqueous solution (Qi, Maurizot, et al, 2012). We therefore suggest that only at five units is OQ able to adopt the folded structure that is essential for its function.</p><p>In conclusion, a new scaffold has been identified which affects amyloid self-assembly by IAPP via a mechanism that differs from earlier reported compounds. To our knowledge, carboxylate substituted OQ5 is the most effective IAPP self-assembly inhibitor when considering both lipid free and lipid catalyzed solution conditions. This represents a useful and new paradigm in which a folded small molecule serves to conformationally capture an off-pathway state of an intrinsically disordered peptide toxin. Indeed, this goal is central to many other systems including Aβ, α-synuclein, and others that sample a diverse set of conformational states. Oligoquinolines are a vital new class of compound to add to these important efforts.</p><!><p>Heterogeneous disorder-to-order transitions are central to the development of cytotoxicity in many diseases including Alzheimer's, Parkinson's, and type 2 diabetes. The intrinsically disordered protein precursors in these systems lack stable secondary structure and exist as an ensemble of rapid fluctuating intermediates states. Nevertheless, they have the capacity to make structure specific interactions. Islet Amyloid Polypeptide (IAPP), a 37 residue hormonal peptide cosecreted by pancreatic β-cells samples a series of intermediate states contributing to gain of toxic function in type 2 diabetes. There are few tools, however, that can illuminate the subset of structures that give rise to any particular protein activity. A foldamer based scaffold, here based on oligoquinoline, offers an opportunity which in effect inverts the problem of molecular recognition. A designed, non-covalent interaction based on a folded and compact small molecule becomes the object about which a flexible and dynamic protein can bind. This pairing of synthetic chemistry with molecular biophysics offers a new paradigm for addressing dynamic protein targets.</p><!><p>ThT was purchased from Acros Organics (Fair Lawn, New Jersey). Lipids [dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylcholine (DOPC)] were purchased from Avanti Polar Lipids, Inc. (Alabaster, Al). The 96-well plates (coated, non-binding surface, black, flat bottom) were bought from Greiner Bio-One (Monroe, NC). All of the chemicals were purchased from commercial suppliers and used without further purification. Silica plates (w/UV254, aluminum backed, 200 micron) and silica gel (standard grade, particle size = 40–63 micron, 230 × 400 mesh) for flash column chromatography were purchased from Sorbent Technologies (Atlanta, GA). Dry solvents were purchased from Sigma Aldrich (St. Louis, MI) or VWR (Bridgeport, NJ). Triethylamine (dry), 2-chloro-1-methylpyridinium iodide, tert-Butyl bromoacetate, 2-nitroaniline, dimethylacetylene dicarboxylate, and polyphosphoric acid were purchased from Sigma Aldrich (St. Louis, MI).</p><!><p>15N Wild-type rIAPP was prepared by recombinant E. coli purification as described previously (Williamson, and Miranker, 2007). Human islet amyloid polypeptide (IAPP) was synthesized by t-Boc methods and purified by the W. M. Keck facility (New Haven, CT). Fluorescently labeled protein was prepared using amine coupling, by reacting rIAPP with TAMRA-SE (5-carboxytetramethyl rhodamine succinimidyl ester; Life Technologies, Carlsbad, CA) for 2 h at room temperature in 10 mM potassium phosphate pH 7.2. Labeled rIAPP was separated from free dye using a HiTrap Sephadex G-25 column (GE Healthcare, Piscataway, NJ).</p><!><p>The synthesis of oligoquinolines was carried out using linear solution phase iterative amide coupling with a slight modification in the previously reported procedure (Jiang, Lèger, and Huc, 2003)(Jiang, Lèger, and Huc, 2003). The starting material, methyl 8-nitro-(1H)-4-quinolinone-2-carboxylate, was synthesized according to the known procedure which is then functionalized by introducing O-tert butyl ester as side chain. Chain elongation was achieved using successive amide coupling (in the presence of 2-chloro-1-methylpyridinium iodide) and reduction of nitro group. The acid labile tert-butyl ester derivatized oligoquinolines were then treated with TFA cocktail [TFA/TES/DCM (dichloromethane) (95:2.5:2.5, v/v)] to afford the carboxylate substituted oligoquinolines in modest yield. The oligquinolines were purified using HPLC and characterized for identity using NMR, MS ESI, and elemental analysis (see synthesis and characterization of oligoquinolines for details).</p><!><p>IAPP (~2 mg) was solubilized in 7 M guanidinium chloride solution. The solution was filtered (0.2 micron) and transferred to C-18 spin column, washed twice with water (400 μL each) and 10% acetonitrile in water, 0.1% formic acid (v/v) and then eluted with 200 μL of 50% acetonitrile in water, 0.1% formic acid (v/v). The concentration of IAPP (oxidized form) was calculated using absorbance measurements at 280 nm (ε = 1400 M−1cm−1). The IAPP solution was divided into several aliquots (50–100 μL, 1–2 mM), lypholized, and stored at −80°C. Fresh stock solutions of IAPP were prepared in water for each experiment.</p><!><p>LUVs were composed from a mixture of DOPG/DOPC in a 1:1 molar ratio. The solution of DOPG and DOPC in chloroform (25 mg/ml each) was first mixed and then dried with argon gas for 1 hr followed by drying on lyophilizer for 2 hr. The mixture was hydrated in 100 mM KCl, 50 mM sodium phosphate, and pH 7.4 for 20 minutes. A 20 mg/mL solution of lipid in buffer was passed 21 times through an extrusion film (size = 100 nm). The phospholipid content of the final material was also confirmed by measurement of total phosphorous (Chen, Toribara, and Warner, 1956).</p><!><p>Unless otherwise stated, kinetic reactions were conducted in buffer contains liposome (500 μg/mL) and 20 μM ThT in a Microfluor 1 black 96-well plate (Thermo Electron Corp). This was followed by addition of the small molecule dissolved in DMSO (final DMSO conc.: 0.1%, v/v). Fiber formation was initiated by addition of IAPP stock solution (final conc.:10 μM). Final volume in each well was 200 μL. The kinetics of IAPP fibrillation was monitored by ThT fluorescence (Ex 450 nm and Em 485 nm) using a FluoDia T70 fluorescence plate reader (Photon Technology International). The data were blank subtracted and renormalized to the maximum intensity of reactions containing only IAPP. All the ThT assays were performed in triplicate.</p><p>Reaction profiles were fit using the built in sigmoidal fit in Origin 5.0 separately for each run. This was used to extract separate t50 (time required to reach 50% fluorescence) from which average and standard deviations were calculated. All presented fits were normalized for clarity. Kinetic assays under lipid free condition were repeated in the same way as mentioned above except there was no lipid present.</p><!><p>Circular dichroism measurements were made at room temperature on an Aviv 215 spectrometer using 1 mm path length cuvettes. The stock solution of IAPP (1–2 mM) was diluted to 30 μM IAPP in 100 mM KCl, 50 mM sodium phosphate, pH 7.4. Spectra were collected at 0.5 or 1 nm intervals from 195 to 260 nm with 10 s averaging time. The lipid catalyzed CD spectra were recorded using similar method as described above except in presence of liposome (500 mg/mL, DOPG:DOPC, 1:1, 100 nm)</p><!><p>IAPP (40 μM) was incubated in buffer (100 mM KCl, 50 mM sodium phosphate, pH 7.4) both in the absence and presence of OQ5. Aliquots of these samples were then applied on glow-discharged (25 mA and 30 sec.) carbon-coated 300-mesh copper grids for 1 min. and then dried. Grids were then negatively stained for 1 min. with uranyl acetate (2%, w/v) and dried. Micrographs of grids were examined on a Phillips Tecnai 12 transmission electron microscope at 120 kV accelerating voltages. All conclusions drawn from images in this work include at least one repeat in which the sample identity was withheld from the investigator preparing and analyzing images.</p><!><p>FCS measurements were collected on a home-built instrument as previously described (Nath, Trexler, et al, 2010). Autocorrelation traces were analyzed using custom Matlab scripts.</p><p>FCS samples contained 10 nM TAMRA-labeled rIAPP in 20 mM Tris pH 7.4, 100 mM NaCl, and varying concentrations of compounds of interest added from aqueous (OQ5) or DMSO (all other compounds, final DMSO conc.:0.5%, v/v) stocks. For IAPP-lipid binding experiments (Supporting Information Fig. S5), 5 μM of liposome (DOPG:DOPC, 1:1, 100 nm) was added to samples. Between ten and thirty 10-second measurements were averaged for each FCS observation.</p><p>All data were fit to a single-component, 3D diffusion model to extract an apparent diffusion time parameter τD that quantified changes in the hydrodynamic properties of IAPP: (1)G(τ)=1N(1+ττD)-1(1+τs2τD)-½</p><p>Here, G(τ) represents the normalized autocorrelation at lag time τ, N represents the mean number of molecules in the observation volume, s is a structure factor that describes the prolate shape of the observation volume, and τD is the diffusion time.</p><!><p>All data were collected using a Varian 600 MHz wide bore spectrometer with a triple resonance HCN probe. All experiments were performed under similar solution conditions (10% D2O in 150 mM KCl, 50 mM sodium phosphate, pH 6.5). Titration of OQ5 was performed into a 120 μM 15N-rIAPP sample. The dilution of rIAPP by the titration of OQ5 was less than 6%. Reported Δ ppm values were obtained by scaling the contributions for the proton and nitrogen dimensions (Grzesiek, Stahl, et al, 1996).</p>

PubMed Author Manuscript

(S)-N-(2,5-Dimethylphenyl)-1-(quinoline-8-ylsulfonyl)pyrrolidine-2-carboxamide as a Small Molecule Inhibitor Probe for the Study of Respiratory Syncytial Virus Infection

A high-throughput, cell-based screen was used to identify chemotypes as inhibitors for human respiratory syncytial virus (hRSV). Optimization of a sulfonylpyrrolidine scaffold resulted in compound 5o that inhibited a virus-induced cytopathic effect in the entry stage of infection (EC50 = 2.3 \xc2\xb1 0.8 \xc2\xb5M) with marginal cytotoxicity (CC50 = 30.9 \xc2\xb1 1.1 \xc2\xb5M) and reduced viral titer by 100-fold. Compared to ribavirin, sulfonylpyrrolidine 5o demonstrated an improved in vitro potency and selectivity index.

(s)-n-(2,5-dimethylphenyl)-1-(quinoline-8-ylsulfonyl)pyrrolidine-2-carboxamide_as_a_small_molecule_i

INTRODUCTION<!>CHEMISTRY<!>RESULTS AND DISCUSSION<!>CONCLUSION<!>Chemistry<!>Synthesis of (S)-N-(2,5-Dimethylphenyl)-1-(quinolin-8-ylsulfonyl)pyrrolidine-2-carboxamide (5o)<!>Time of Addition Assay<!>

<p>Human respiratory syncytial virus (hRSV) is the most common cause of bronchiolitis and pneumonia among infants and children under one year of age.1 In the United States there are approximately 125,000 yearly hRSV-related hospitalizations, and of those, 500 young children could die due to the infection or its complications each year.2 The virus is highly contagious and affects those with compromised cardiac, pulmonary (COPD), and immune systems.3 As such, the elderly are also a highly susceptible population to hRSV. Treatment options are limited. Due to infant mortality associated with attempted vaccination, vaccine development is proceeding cautiously.4,5 Synagis® (Palivizumab), a humanized monoclonal antibody, is a prophylactic, injectable therapeutic used only with high risk pediatric patients.6 Ribavirin, a nucleoside antimetabolite, is approved for acute infection7,8 and infected, immunocompromised patients,9 but has a long half-life and accumulates in erythrocytes, thus requiring regeneration of the affected red blood cells to eliminate the drug. These issues, coupled with embryocidal and teratogenic effects constitute severe toxicological liabilities that limit the use of ribavirin, especially in infants,7 around administering pregnant medical personnel, and in treated partners of pregnant women (Figure 1).</p><p>To address the absence of clinically relevant and safe hRSV therapies, many investigators have pursued a target-based drug design approach. Ribavirin 5′-monophosphate resembles GMP and can decrease cellular GTP pools due to the inhibition of the enzyme inosine monophosphate dehydrogenase (IMPDH).10,11 Nevertheless, this decrease does not completely account for the observed antiviral activity as inhibitory effects have been noted on RNA capping12 and direct inhibition of viral polymerase activity for influenza viruses.13 Literature disclosed IMPDH inhibitors have reported in vitro therapeutic indices that are not competitive with ribavirin.14 Several other inhibitors that target the fusion protein,15–20 ribonucleoprotein (RNP) complex,21 guanylylation events,22 and N-protein23 have been discovered. Of these, some demonstrate potency and limited toxicity in animal models.24–26 However, due to formulation for oral bioavailability,19 strategic reasons,27 and observed loss of activity in vivo,28 many have not progressed to the clinic to combat hRSV disease.</p><p>In an effort to identify lead compounds acting through new mechanisms, we developed, optimized and validated a high-throughput cell-based screen that measures the respiratory syncytial virus-induced cytopathic effect (CPE) in HEp-2 cells (unpublished results). CPE was measured using the Cell Titer-Glo™ viability assay in which the luminescent signal generated is directly proportional to the amount of cellular ATP present which is also proportional to the number of metabolically active cells. A total of 313,816 compounds from the Molecular Libraries Small Molecule Repository (MLSMR) were screened in single dose against hRSV (strain Long) at a concentration of 10 µM. Hits (2,465 compounds) were evaluated for their antiviral activity and cell toxicity in dose response experiments, and 409 compounds produced a protective effect of at least 50% CPE inhibition. Based on potency, selectivity and chemical tractability, 51 hits were selected for verification in an in vitro titer reduction assay to assess their effect on the production of infectious progeny virus. Many chemotypes of interest emerged, including several compounds containing a sulfonylpyrrolidine moiety. Hit compound 2 displayed a CPE EC50 of 5.0 µM, a CC50 (HEp-2 cellular cytotoxicity) of 31.5 µM, and a selectivity index (CC50/EC50) of 6.3 (Figure 2). Profile improvements were explored by modulating various structural elements (shaded regions, Figure 2).</p><!><p>Analogs were prepared by treating substituted sulfonyl chlorides 3 with proline or another suitable amino acid to generate sulfonamide carboxylates 4. Coupling of the acids to various amines via traditional methods afforded the desired products 5 (Scheme 1).</p><!><p>Attention was first placed on the proline unit between the aryl sulfone and aniline groups. The hit compound 2 was tested as a racemic mixture; therefore, each enantiomer was individually prepared from L- or D-proline to determine the more pharmacologically active constituent. The S-enantiomer, derived from L-proline, was found to be the more active component of the racemic mixture, delivering a selectivity index of 11.8 when cytotoxicity was accounted for (entry 4, Table 1). This compound showed a 1 log reduction in virus titer in a plaque reduction assay. Acyclic variants of the linker region, including those that probed the methylation of the amide nitrogen, were found to have EC50 values > 50.0 µM.</p><p>Several variants of the quinoline moiety were pursued but offered no benefit in potency. Replacing the nitrogen atom with –CH- afforded an inactive analog (EC50 > 50.0 µM), as did migrating the nitrogen to alternate positions of the quinoline substructure. The replacement of the quinoline with a 4-linked-benzooxadiazole, a phenyl ring or a simple methyl group was also not advantageous. Substitution of the sulfonyl group (SO2) for a carbonyl functionality or its replacement with –CH2- resulted in complete loss of potency (EC50 > 50.0 µM, see supporting information, Tables S1 and S2, respectively). Focus was then shifted to the modification of the 2,4-dimethylanilide. Following the results noted above, all analogs in this study were prepared as the S-enantiomer (Table 2). Simplifying the 2,4-dimethylphenyl substitution pattern down to monomethyl substituted phenyl derivatives revealed that the observed potency was not due to the presence of one group alone (entries 3–5). Increased steric bulk at C2 marginally improved potency vs. monomethyl substitution at the same position (cf. entries 8 to 7). Mimicking the 2,4-substitution pattern with chlorine atoms in place of methyl groups resulted in complete loss of potency (5n, entry 11). As the 2-alkyl substituent appeared to be necessary in combination with other substituents to preserve potency, this dynamic was explored further to reveal that the 2,5-dimethylphenyl moiety of analog 5o was slightly more beneficial in terms of potency and maintained the cytotoxicity threshold (entry 12, EC50 = 2.3 µM, CC50 = 30.9 µM). For select compounds assessed for aqueous solubility, no effect was observed on CPE potency. Solubility and stability were determined for 5o, revealing an acceptable solubility measurement of 92.7 µg/mL in PBS buffer and stability of 95.4% (unchanged parent remaining) after 48 h in 50% PBS/50% acetonitrile.29</p><p>To probe the mechanism of action of the sulfonylpyrrolidines, the window of inhibitory activity in the cell-based assay was refined. Potency of compounds over time following infection was examined to ascertain early (entry) or late (replication) antiviral activity in the virus life cycle.30</p><p>In the time of addition study, HEp-2 cells were infected with hRSV strain Long at an MOI of 3.0 at time point 0 and incubated for 6 days and test compounds, 5b, 5t, 5o, or ribavirin were added to plates at −1, 0, 1, 2, 3, 5, 7, 21 and 24 h post infection (p.i.). CPE was assessed using Cell-Titer Glo as an endpoint reagent. Controls without test compound included HEp-2 cells with no hRSV exposure (cell control) and hRSV-infected cells (virus control). To evaluate cellular toxicity attributed to test compound alone, uninfected HEp-2 cells were treated with 5b, 5t, 5o, or ribavirin at 25 µM concentration at time point 0, and cell viability was assessed after 144 hours.</p><p>There was less than 1% cell viability for the hRSV-infected cells without addition of any test compound or ribavirin. Uninfected cells treated with ribavirin, 5t or 5o displayed 90%, 88% and 95% cell viability, respectively, indicating low cellular toxicity inherent to these compounds. However, uninfected cells treated with 5b exhibited only 46% cellular viability, suggesting moderate toxicity due to the test compound alone.31 Ribavirin treatment protected cells from hRSV induced CPE for up to 7 h p.i., indicating that it targets the period of infection during which viral replication is in progress. Compound 5o protected cells from hRSV induced CPE (> 50%) from 1 – 3 h p.i., and at 24 h p.i. cell viability was only 26%. (Figure 3).</p><p>Two analogs of similar profile to 5o, 5b and 5t, demonstrated a decrease in efficacy when added at each time point from 0–5 hours p.i. This profile could be due to the inhibition of one or more early virus life cycle steps (entry, post-entry, or early-stage infection processes), a hypothesis that is supported by an inability of these compounds to affect processes later in the viral replication cycle. This data lead us to conclude that 5o was inhibiting early infection events, characterized by viral attachment, uptake, fusion or initial transcription. The sulfonylpyrrolidine scaffold analogues were evaluated for their ability to reduce the amount of infectious virus produced in cell culture. These measurements of compound-mediated viral titer reduction were used to complement the cytoprotection assay results. A standard plaque-reduction assay was used as a secondary assay to determine the ability of this class of compounds to reduce the amount of infectious virus produced in HEp-2 cells.32 Cells were infected with hRSV in the presence of 25 µM test compound (5b, 5o, or 5t). Compounds 5b and 5t each showed 1 log reduction in virus titer, or 10-fold, as compared to ribavirin which reduced viral titer by 2.5 log units, or ~300-fold. Analog 5o showed a 100-fold, or 2 log, reduction. Improvements in cell protection against hRSV did not translate to significant improvement in the plaque assay as was seen with ribavirin. Consequently, the titer reduction assay was not used to drive SAR efforts.</p><!><p>In summary, the HTS and chemistry optimization efforts produced a series of enantiomerically pure, sulfonylpyrrolidine-based compounds that are effective in vitro inhibitors of hRSV in the low micromolar range. Many of these compounds were shown to reduce the in vitro viral titer by 100-fold. The therapeutic index for the series was maximized at 13.4-fold and is an issue for further refinement preceding in vivo assessment.</p><!><p>All final compounds were confirmed to be of >95% purity based on HPLC analysis. 1H and 13C NMR spectra were recorded on a Bruker AM 400 spectrometer (operating at 400 and 101 MHz respectively) or a Bruker AVIII spectrometer (operating at 500 and 126 MHz respectively) in CDCl3 with 0.03% TMS as an internal standard or DMSO-d6. The chemical shifts (δ) reported are given in parts per million (ppm) and the coupling constants (J) are in Hertz (Hz). The spin multiplicities are reported as s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublet and m = multiplet. The LCMS analysis was performed on an Agilent 1200 RRL chromatograph with photodiode array UV detection and an Agilent 6224 TOF mass spectrometer. The chromatographic method utilized the following parameters: a Waters Acquity BEH C-18 2.1 × 50mm, 1.7 µm column; UV detection wavelength = 214 nm; flow rate = 0.4ml/min; gradient = 5 – 100% acetonitrile over 3 minutes with a hold of 0.8 minutes at 100% acetonitrile; the aqueous mobile phase contained 0.15% ammonium hydroxide (v/v). The mass spectrometer utilized the following parameters: an Agilent multimode source which simultaneously acquires ESI+/APCI+; a reference mass solution consisting of purine and hexakis(1H, 1H, 3H-tetrafluoropropoxy) phosphazine; and a make-up solvent of 90:10:0.1 MeOH:Water:Formic Acid which was introduced to the LC flow prior to the source to assist ionization. Melting points were determined on a Stanford Research Systems OptiMelt apparatus.</p><!><p>To a mixture of L-proline (0.50 g, 4.34 mmol) in 10% K2CO3 (10 mL) and THF (10 mL) was added 8-quinolinesulfonyl chloride (1.98 g, 8.68 mmol), and the resulting mixture was stirred at 50 °C for 5 h. After cooling to room temperature, the reaction mixture was acidified with 3 N aqueous HCl to pH 2 and then extracted with EtOAc (3 × 30 mL). Separation and drying of the combined organic extracts (MgSO4), followed by removal of solvent under reduced pressure afforded (S)-1-(quinolin-8-ylsulfonyl)pyrrolidine-2-carboxylic acid as a white solid (0.80 g, 60% yield) that did not require further purification and was used in the next step. To a solution of (S)-1-(quinolin-8-ylsulfonyl)pyrrolidine-2-carboxylic acid (0.060 g, 0.20 mmol) in DMF (0.75 mL) was added 2,5- dimethylaniline (0.024 mL, 0.20 mmol), HATU (0.082 g, 0.22 mmol), and DIPEA (0.097 mL, 0.59 mmol). The reaction mixture was stirred for 2 h at room temperature, then diluted with CH2Cl2 (5 mL) and washed sequentially with aqueous 10% HCl (2 × 5 mL), saturated aqueous NaHCO3 (2 × 5 mL), and water (2 × 5 mL). The separated organic extracts were dried (MgSO4), and evaporated to give the crude product which was purified by silica gel flash column chromatography (2% MeOH in CH2Cl2) to afford (S)-N-(2,5-dimethylphenyl)-1-(quinolin-8-ylsulfonyl)pyrrolidine-2-carboxamide as a colorless oil (0.050 g, 62% yield). 1H NMR (500 MHz; CDCl3): δ (ppm) 9.54 (s, 1H), 8.88 (dd, J = 4.3 and 1.8 Hz, 1H), 8.62 (dd, J = 7.4 and 1.4 Hz, 1H), 8.28 (dd, J = 8.4 and 1.7 Hz, 1H), 8.11 (dd, J = 8.2 and 1.3 Hz, 1H), 7.69 (t, J = 7.8 Hz, 1H), 7.63 (s, 1H), 7.53 (dd, J = 8.3 and 4.3 Hz, 1H), 7.11 (d, J = 7.7 Hz, 1H), 6.93 (d, J = 7.6 Hz, 1H), 5.41 (dd, J = 7.9 and 2.0 Hz, 1H), 3.44-3.32 (m, 2H), 2.52-2.40 (m, 1H), 2.33 (s, 6H), 1.96-1.82 (m, 2H), 1.82-1.72 (m, 1H). 13C NMR (126 MHz; CDCl3): δ (ppm) 170.66, 151.58, 143.91, 137.12, 136.37, 135.88, 135.29, 134.92, 134.62, 130.45, 129.29, 127.65, 126.34, 125.84, 124.23, 122.51, 63.20, 49.29, 30.19, 24.98, 21.24, 17.65. LCMS purity (214 nm) =100%. HRMS: m/z calcd for C22H23N3O3S (M + H+) 410.1533, found 410.1532. Enantiomeric excess was determined by HPLC analysis: [α]D25 −31.5 (c 0.0039 CHCl3), > 99% ee.</p><!><p>HEp-2 cells were plated in 96 well black tissue culture plates at 10,000 cells per well in 100 µL and incubated 24 h at 37 °C, 5% CO2. Test compounds were diluted in media to give a final concentration of 25 µM and added to plates in triplicate at −1, 0, 1, 2, 3, 5, 7, 21 and 24 h post-infection. Cells were infected with hRSV strain Long at an MOI of 3.0 at time point 0 and incubated for 6 days at 37 °C, 5% CO2. Following a six day incubation period, the assay plates were equilibrated to room temperature for 30 min. An equal volume (100 µL) of Cell Titer-Glo reagent (Promega Inc.) was added to each well using a WellMate (Matrix, Hudson, NH) and the plates were incubated for an additional 10 min at room temperature. At the end of the incubation, luminescence was measured using a multi-label reader (PerkinElmer, Wellesley, MA) with an integration time of 0.1 s. Ribavirin was used as a control compound.</p><!><p>Supporting Information. Assay details and experimental characterization for select compounds. This material is available free of charge via the internet at http://pubs.acs.org.</p>

PubMed Author Manuscript

Light-Activated Reversible Imine Isomerization: Towards a Photochromic Protein Switch

Mutants of cellular retinoic acid-binding protein II (CRABPII), engineered to bind all-trans-retinal as an iminium species, demonstrate photochromism upon irradiation with light at different wavelengths. UV light irradiation populates the cis-imine geometry, which has a high pKa, leading to protonation of the imine and subsequent \xe2\x80\x9cturn-on\xe2\x80\x9d of color. Yellow light irradiation yields the trans-imine isomer, which has a depressed pKa, leading to loss of color because the imine is not protonated. The protein-bound retinylidene chromophore undergoes photoinduced reversible interconversion between the colored and uncolored species, with excellent fatigue resistance.

light-activated_reversible_imine_isomerization:_towards_a_photochromic_protein_switch

Introduction<!>Identification of the isomeric states of retinal<!>Thermal isomerization of the retinylidene chromophore<!>Elucidation of the thermal isomerization mechanism<!><!>Elucidation of the thermal isomerization mechanism<!>Photochemical isomerization of the retinylidene chromophore<!>Application to another iLBP\xe2\x80\x94hCRBPII<!>Conclusions<!>General<!>HPLC analysis of extracted R111K:R132l:Y134F:T54V:R59W\xc2\xb7retinal complexes<!>UV/Vis measurements<!>Time-dependent changes in pKa<!>Time-dependent changes in pKa in D2O versus H2O<!>Time-dependent changes in pKa at various pH values<!>Iterative UV and visible light irradiations

<p>Photochromic proteins, although rare in the totality of the proteome, play key roles in essential biological processes, such as photosynthesis, phototaxis, energy production, and visual perception.[1] Amongst these proteins, the rhodopsins share a unique characteristic: the photoisomerization of the retinylidene chromophore (or one of its closely related analogues).[2] Principally, these carbon–carbon double-bond isomerization events are coupled to the ultimate function of the protein. Interestingly, the iminium species (otherwise known as the protonated Schiff base, PSB) formed between the retinal aldehyde functionality and an active site Lys residue undergoes a drastic pKa change as the consequence of the isomerization in most systems. A well-studied example is bacteriorhodopsin: a 26 kDa photosynthetic protein that functions as a light-driven proton pump in Halobacterium halobium.[3] The all-trans-retinal chromophore, responsible for the absorption of light, is bound through a PSB to Lys216.[4] Illumination with visible light initiates an ultrafast isomerization of the bound all-trans-retinal to the 13-cis isomer (Scheme 1A).[2f,5] The change in the environment of the iminium species, as a result of the isomerization, leads to a dramatic change in its pKa (13.3 for all-trans isomer, reduced by 4–5 pKa units upon isomerization[6]), with the eventual translocation of a proton across the membrane.[7]</p><p>Rhodopsin, the light-activated G protein-coupled receptor, the engine of vertebrate vision, possesses a similar mechanism. The difference, however, is that rhodopsin activation relies on the isomerization of the PSB of 11-cis-retinal, bound to Lys296,[8] which yields a strained all-trans-retinal configuration in the sub-nanosecond time regime (Scheme 1B).[5f,9] Subsequent thermal relaxations eventually lead to the metarhodopsin II state (which initiates the visual transduction pathway), in which the initial PSB, with a high estimated pKa>16, is deprotonated.[2e,9,10] The significant change in the pKa of the imine functionality is again the consequence of the isomerization that changes the environment of the imine nitrogen atom. As described below, our recent efforts in generating rhodopsin protein mimics[11] have also provided the opportunity to produce engineered proteins that recapitulate the change in the pKa of the iminium functionality as a function of isomerization.[12] Here we disclose a protein system that is capable of light-activated isomerization, with concomitant changes to the pKa of the PSB. Specifically, we report for the first time a rhodopsin mimic that can be reversibly photocycled between a protonated cis-iminium species and its deprotonated trans-imine with >4 pKa unit change (Scheme 1C).</p><p>Our efforts began with an observation reported previously during our optimization studies for the design of a colorimetric proteinaceous pH sensor.[12] Cellular retinoic acid-binding protein II (CRABPII) was reengineered to bind all-trans-retinal as a PSB. We were not only able to show wavelength regulation in absorption, as a function of perturbing electrostatic potential across the length of the chromophore, but were also successful in modulating the pKa of the iminium species from 2.4 to 8.1. Unexpectedly, a series of mutants exhibited apparent time-dependent conversion of the PSB (λmax>450 nm) to the Schiff base (SB, λmax=360 nm); this was ascribed to an event that must have changed the pKa of the iminium species. In most cases, the observed deprotonation occurred quickly. Fortuitously, the retinal-bound R111K:R132L:Y134F:T54V:R59W-CRABPII penta-mutant exhibited a slow conversion (complete within 2 h), thus enabling pKa measurement of each state to be made independently (Figure 1A). The pKa of the initial species formed with the addition of retinal was 6.6. Gradual loss of the red-shifted PSB peak, concurrent with the rise of the SB absorption, led to a new species, the pKa of which was 2.5.</p><p>The previously obtained crystal structure of the matured protein complex (pKa=2.5),[12] shows a trans imine geometry (Figure 1B). The imine nitrogen atom is surrounded with aliphatic amino acid residues, presumably leading to the low observed pKa (2.5) to avoid buildup of charge in a hydrophobic environment. The latter observations led us to hypothesize that the initial complex exhibiting high pKa must be an isomeric variant (kinetic product), which over time yields the crystallographically observed thermodynamic product. This presumes that the initial isomeric geometry places the imine nitrogen in an environment that can support a positive charge.</p><!><p>We hypothesized that the conversion between the two forms stems from the cis–trans isomerization of the imine, as shown in Scheme 2. This was further supported by HPLC analysis of the chromophore. Similar retention times were obtained when all-trans-retinal was incubated in 0.2m citric acid buffer and when incubated with R111K:R132L:Y134F:T54V:R59W-CRABPII, thus indicating that the isolate was all-trans-retinal in both cases (see below). This further supports the hypothesis of varying isomeric states at the imine, rather than a C=C bond isomerization, which would have led to isomeric retinal species with dissimilar retention times in HPLC. Figure 2 details the HPLC traces of all-trans-retinal incubated in the absence or presence of R111K:R132L:Y134F:T54V:R59W-CRABPII.</p><!><p>The observed loss of the PSB peak for the R111K:R132L:Y134F:T54V:R59W-CRABPII·retinal complex occurs in the absence of light. Therefore, the presumed imine isomerization, concurrent with the loss of a proton, is thermally activated. In fact, as depicted in Figure 3, the rate of isomerization, in the dark, is temperature-dependent, with longer t1/2 at 6.9°C and shorter t1/2 at 30.5°C (Figure S2 in the Supporting Information).</p><!><p>Further examination of the initially observed thermal isomerization led to a potential mechanism for isomerization from the presumed cis-iminium species 1a-H to the trans-imine 1b, depicted in Scheme 3. We propose the reversible addition of water to 1a-H, leading to the hemiaminal 2, which, upon single bond rotation to 3 and loss of water, yields the trans-iminium species 1b-H. This is promptly deprotonated as a result of the highly nonpolar nature of the new imine environment. The following lines of evidence support the mechanism described above.</p><!><p>As illustrated in Figure 3, rates of thermal isomerization are temperature-dependent. The Arrhenius plot for the temperature dependence of k (pseudo-first-order) yields the following thermodynamic parameters (see Figures S3 and S4 for graphs): Ea=15.4 kcalmol−1, A=9.5×109 and ΔH*=+14.8 kcalmol−1, ΔS*=+14.8 calmol−1K−1, and ΔG*=+10.4 kcalmol−1 (at pH 5.2 and 25°C). These values are in good agreement with previously reported thermal isomerization parameters of various other synthetic imines in aqueous solvent,[13] thus suggesting that enzyme-like catalysis is most probably not at play.</p><p>Figure 4A depicts a pH/rate profile for the thermal decay of the PSB to the SB, measured in solutions of constant ionic strength (0.2M citric acid; note that these are specific acid catalysis conditions; see Figure S6 for kinetic data at different pH values) and temperature (25°C). The increase in rate, apparent below pH 5, suggests that the change in the protonation state of a nearby acidic residue (pKa≈4) could be responsible for accelerating the isomerization. Note that the imine is protonated at pH 5 and below (pKa=6.6).[12] The only acidic residue with its side chain pointing into the interior of the binding cavity is Glu73. One can surmise that the protonation of Glu73 could reduce the barrier to the hydration of the iminium species. As depicted in Figure 4B, this could be the result of anionic stabilization originating from the glutamate–iminium interaction, which is reduced upon protonation of Glu73. Interestingly, all E73A mutants investigated exhibit low iminium pKa values.[12] Putatively, the absence of Glu73 reduces the barrier for isomerization such that the trans-imine is the only observable species.</p><p>On the basis of the suggested mechanism in Scheme 3, one would not expect a large deuterium isotope effect because deprotonation is not presumed to be rate-determining. In fact, rates of thermal isomerization measured in H2O- and D2O-based buffers showed no significant differences (Figure S5).[14]</p><!><p>Taken together, these observations suggest that addition of water to the imine to generate the tetrahedral intermediate is rate-determining. Nonetheless, the photoinduced isomerization of either the SB or PSB (see below) certainly is not bound by the mechanistic discussions above, and is currently the focus of further studies.</p><!><p>We were delighted to discover that the thermal isomerization, requiring 2 h at 25°C for completion, could be greatly accelerated with visible light irradiation. As depicted in Figure 5A, 15 min irradiation with yellow light fully eradicated the PSB peak at 556 nm, concomitantly with maximum growth of the SB peak at 362 nm. This observation illustrates a light-induced isomerization pathway that is specific and unique to the imine functionality.</p><p>Gratifyingly, UV light irradiation (<360 nm) of the thermally equilibrated protein complex (low pKa, trans-imine) led to a fast restoration of the kinetically observed high-pKa species (complete within 1 min), evident from the change in the absorption spectrum (loss of the SB peak and gain of the red-shifted PSB peak, Figure 5A). The opposite behavior was observed when the SB-maximized complex, obtained through UV light irradiation as described above, was photoisomerized with yellow light (>500 nm). These observations led us to propose the cycle depicted in Scheme 2. The cis isomer 1a-H, exhibiting a high pKa, and thus protonated with an absorption above 500 nm, is thermally unstable and converts into the trans-iminium species 1b-H. The trans-iminium species is not able to support the protonated state at pH 7.2 (low-pKa isomer) and therefore reverts to the SB form 1b that absorbs at <380 nm. The latter thermal isomerization is photoinducible, with yellow light irradiation (>500 nm). The cycle is completed with UV light irradiation that restores the original cis-imine 1a, which now protonates to form 1a-H as a result of the change in the pKa of the system.</p><p>We next investigated the possibility of repeated cycling of the complex to operate as a photoinducible switch. Fatigue resistance is crucial in assessing the potential usefulness of photochromic material.[15] It is desirable to see a reproducible return to the original state of a system upon repeated cycling, without significant loss in signal, or otherwise maintaining a large difference in signal between the two interconverting states. Rapid and complete cycling with UV and yellow light was reproduced several times (as measured by the intensity of absorption, Figure 5B), demonstrating that little light-induced degradation occurs in each cycle. The initial cycle leads to a higher percentage loss as measured by the absorption of the PSB peak (9.2%); however, the average loss is 3.2% per cycle thereafter. More importantly, as can be seen in Figure 5B, the difference in absorption between each state remains large, and thus could be differentiated with ease. We attribute the larger initial loss of intensity to singlet-oxygen-mediated bleaching due to the presence of dissolved molecular oxygen. The protein solutions utilized for fatigue resistance studies had been crudely degassed. In the absence of degassing, the initial drop in intensity was 18.9%, with subsequent average losses of 3.8% per cycle.</p><p>Subsequent to the multiple rounds of photoisomerization, the chromophore was extracted at both the SB and PSB stages and analyzed by HPLC (Figure 2, blue and purple traces). The results show only the presence of all-trans-retinal, thus further verifying the lack of C=C bond isomerization and the structural resilience of the chromophore during the process.</p><!><p>Having shown that the photoisomerization of the retinylidene SB can be achieved photochemically with UV and visible light in CRABPII mutants, we sought to determine whether the same process could be observed in a related protein, human cellular retinol binding protein II (hCRBPII). Both CRABPII and hCRBPII belong to the superfamily of intracellular lipid-binding proteins (iLBPs). These two proteins possess similar tertiary structures, each consisting of a ten-stranded antiparallel β-barrel enclosing an internal ligand-binding pocket, with two α-helices capping the pocket. As such, we hypothesized that with variants engineered to bind the same chromophore retinal, the light-induced isomerization of the imine could occur in hCRBPII, as similarly observed in CRABPII.</p><p>Q108K:K40L:T51V:R58F hCRBPII tetra-mutant, optimized to form a PSB with all-trans-retinal,[11k] showed remarkably similar behavior. Green light irradiation of the PSB populated the blue-shifted SB state, whereas UV irradiation of the SB maximized the levels of PSB. Nonetheless, in contrast to the behavior discussed above for CRABPII, retinal-bound hCRBPII complex possessed a stable PSB that did not convert over time into the SB. Each state is thermally stable (at room temperature); the trigger for imine isomerization requires light. Cycling of the retinal-bound hCRBPII tetra-mutant showed good fatigue resistance for cycling between the trans-imine and the cis-iminium species (Figure S7).</p><p>Although there could be multiple reasons for the difference in behavior, we are tempted to attribute the thermally stable nature of hCRBPII mutants to the greater distance between E72 (E73 in the CRABPII series) and the iminium nitrogen atom. The distance between E72 and the iminium species is 10.9 Å (in comparison with 9.3 Å in CRABPII), thus attenuating its electrostatic influence (see Figure S8 for an overlay of the two proteins).</p><!><p>The results disclosed here demonstrate temperature- and light-dependent isomerization of an iminium bond formed between all-trans-retinal and an active-site Lys residue in an engineered rhodopsin protein mimic. The isomerization can be cycled between each state repetitively. Evidence gathered thus far has not indicated any C=C bond isomerization, thus suggesting selective isomerization of the imine bond. This system provides a unique platform to study the isomerization of an imine bond with complete control. This SB/PSB interconversion might also show potential as a photoswitchable protein quencher or in artificial proton pump designs.</p><!><p>UV/Vis spectra were recorded with a Cary 100 Bio WinUV, Varian spectrophotometer. All-trans-retinal was purchased from Sigma–Aldrich and was used as received. pET17b plasmid containing the previously designed R111K:R132L:Y134F:T54V:R59W-CRABPII penta-mutant was expressed in BL21(DE3)pLysS competent cells, and the protein was purified as described before.[12] The absorption extinction coefficient (ε) for R111K:R132L:Y134F:T54V:R59W-CRABPII was reported previously (25038M−1cm−1).[12]</p><p>The pET-17b plasmid including the Q108K:K40L:T51V:R58F-hCRBPII tetra-mutant was obtained by site-directed mutagenesis of the pET-17b plasmid containing Q108K:K40L:T51V-hCRBPII with use of the following primers: forward primer 5′-CTAGC ACATT CTTCA ACTAT GATGT G-3′ and reverse primer 5′-CACAT CATAG TTGAA GAATG TGCTA G-3′, under PCR conditions previously reported.[11k] The tetra-mutant was expressed in BL21(DE3)pLysS competent cells, and the protein was purified as described before.[11k] The e value for Q108K:K40L:T51V:R58F-hCRBPII was found to be 25038M−1cm−1, measured by a method previously reported by Gill and von Hippel.[16]</p><p>For light irradiation of samples, an Oriel Illuminator (Model 66142, Oriel Instruments) connected to a power supply [Model 668820, Oriel Instruments, 500 W Mercury (Xenon) lamp] was used. For all light irradiations, a combination of two filters was used. One of these was always a glass filter (6 mm thickness, Figure S1C) to filter UV light below ≈320 nm, and the second was either of the following: for UV irradiations, a U-360 (UV) 2″ square band-pass filter [center wavelength (CWL)=360 nm, full width at half-maximum height (FWHM)=45 nm, purchased from Edmund Optics, Figure S1A] was used, whereas for visible light yellow irradiations a Y-50 2″ square long-pass filter (cut-off position=500±6 nm, purchased from Edmund Optics, Figure S1B) was employed. The transmittance of the filters was verified before use.</p><!><p>HPLC analysis was used to verify the identity of all-trans-retinal after the following treatments: 1) incubation of all-trans-retinal (10 μm) in citric acid buffer (pH 5.2, 0.2m) for 2 min, 2) incubation of all-trans-retinal (10 μM) in citric acid buffer (pH 5.2, 0.2M) for 2 h, 3) incubation of all-trans-retinal (10 μM) with R111K:R132L:Y134F:T54V:R59W-CRABPII (20 μM) in citric acid buffer (pH 5.2, 0.2M) for 2 h, 4) incubation of all-trans-retinal (10 μM) with R111K:R132L:Y134F:T54V:R59W-CRABPII (20 μM) in citric acid buffer (pH 5.2, 0.2M) for 2 h, followed by white light irradiation with the UV band-pass filter for 1 min, and 5) incubation of all-trans-retinal (10 μM) with R111K:R132L:Y134F:T54V:R59W-CRABPII (20 μM) in citric acid buffer (pH 5.2, 0.2M) for 2 h, followed by white light irradiation with the LP 500 nm yellow filter for 15 min.</p><p>Similar retention times in all instances indicate that all-trans-retinal was not modified during incubation in citric acid buffer (0.2M), either in the presence or in the absence of R111K:R132L:Y134F: T54V:R59W-CRABPII or after white light irradiation with either the UV band-pass filter or the LP 500 nm yellow filter. This further supports the hypothesis of imine isomerization, rather than a C=C isomerization, which would have led to isomeric retinal species with dissimilar retention times in HPLC.</p><p>To prepare samples for HPLC, the solution was extracted with ethyl acetate, and the organic layer was transferred to an Eppendorf tube (1.5 mL), dried with sodium sulfate, and then concentrated to dryness under a nitrogen stream. The sample was then dissolved in hexane/ethyl acetate (90:10, 100 μL). The resulting solution was analyzed by normal-phase HPLC (silica column, Zorbax Rx-SIL, 9.4 mm×25 cm) after manual injection. The sample was eluted with hexane/ethyl acetate (90:10) at 3 mLmin−1. The products were detected at 325 nm.</p><!><p>The CRABPII-R111K:R132L:Y134F:T54V:R59W-CRABPII·retinal PSB formation (556 nm) in citric acid buffer (pH 5.2, 0.2M) was followed by UV/Vis. The pH was verified every time before the spectrum was recorded. The experiment was performed with a final protein concentration of 20 μm (from a stock of 1.6 mM in Tris buffer), and retinal (0.5 equiv.) was added (from a stock solution of 0.6 mM in ethanol). Peaks with λmax=556 nm are considered PSB peaks, whereas deprotonated imine peaks (SB) appear at ≈360 nm. Free retinal absorbs at ≈380 nm.</p><!><p>Time-dependent PSB deprotonation of the CRABPII-R111K:R132L:Y134F:T54V:R59W penta-mutant was monitored over time by UV/Vis at varying temperatures. To ensure that only the PSB deprotonation to the SB event was being kinetically analyzed, the CRABPII·retinal complex was allowed to convert into the SB thermally over time, and then the PSB was restored upon irradiation with a UV band-pass filter, at which point the sample was continuously scanned over time until the sample had again reverted to the SB.</p><p>For each experiment, a solution of the protein (20 μM) and retinal (0.5 equiv, 10 μM) was incubated at room temperature for three hours. The sample was then scanned by UV/Vis to ensure complete conversion into the SB. The solution was then irradiated with white light and with use of a UV band-pass filter for 1 min (cuvette cooled with a circulating solution of saturated NaCl in water for the sample cooled to 6.9°C and ice-water for the samples cooled to 16.4 and 24.9°C), followed by UV/Vis monitoring in two-minute intervals at the desired temperature. The UV/Vis chamber was purged with N2 during the course of measurements to avoid condensation buildup on the sides of the cuvette. Note that the temperature of the protein solution was in each case verified by use of a calibrated thermo probe immersed in the cuvette (6.9, 16.4, 24.9, and 30.5°C). The actual temperature settings of the Cary temperature controller were set to 4, 15, 25, and 32°C, respectively.</p><p>The absorption of the PSB at 556 nm was plotted as a function of time, and fit to an exponential decay (pseudo first order) in KaleidaGraph to the following Equation (1). (1)A=A0×(e−kt)+cwhere A is the absorbance at each recorded time point, A0 is the final absorbance value after complex formation, k is the rate constant, t is the time after addition, and c is a free constant. The equation was rewritten in KaleidaGraph as Equation (2). (2)y=m1×(e−m2×m0)+m3where m2 is the rate constant. For kinetic plots, see Figure S2.</p><!><p>Citric acid (0.84 g) was dissolved in D2O (20 mL) to give a 0.2M solution. The pH was then adjusted to 5.2 by addition of the required amount of a NaOH (10M) in D2O solution. Note that the measured pH of a D2O solution with a pH probe requires the following adjustment, as previously reported.[14b] (3)pH=0.929pH∗+0.42where pH* is the pH of the solution measured in D2O with a pH probe calibrated in H2O. pH is the calculated pH value in H2O after application of Equation (3).</p><p>A solution of the protein (20 μM) and retinal (10 μM, 0.5 equiv) was incubated at room temperature for three hours. The sample was then scanned by UV/Vis to ensure complete conversion into the SB. The solution was irradiated with white light and with use of a UV band-pass filter for 1 min, followed by UV/Vis monitoring at two-minute intervals at 25°C. The absorption of the PSB at 556 nm was plotted as a function of time and fit to an exponential decay (pseudo first order) in KaleidaGraph according to Equation (2). For kinetic plots, see Figure S5.</p><!><p>Samples of the protein (20 μM) and retinal (10 μM, 0.5 equiv) were incubated at room temperature in citric acid buffer (pH 5.2, 0.2M) for three hours. The sample was then scanned by UV/Vis to ensure complete conversion into the SB. The solution was then adjusted to the desired pH by addition of HCl or NaOH. The solution was then irradiated with white light and with use of a UV band-pass filter for 1 min, followed by UV/Vis monitoring at two-minute intervals at 25°C. The absorption of the PSB at 556 nm was plotted as a function of time and fit to an exponential decay (pseudo first order) in KaleidaGraph according to Equation (2). For kinetic plots, see Figure S6.</p><!><p>For the R111K:R132L: Y134F:T54V:R59W-CRABPII·retinal complex, samples were incubated in citric acid buffer (pH 5.2, 0.2M) until full SB formation was observed by UV/Vis spectroscopy. The sample was then irradiated with white light with use of a UV band-pass filter for 1 min, followed by UV/Vis scanning of the sample. The sample was then irradiated with white light through a LP 500 nm yellow filter (while the cuvette was circulated with water) for 15 min. Cycling of UV and yellow irradiations was repeated 20 times. For degassed samples, argon was bubbled through the citric acid buffer for 1 h before incubation of the CRABPII mutant and retinal.</p><p>For the Q108K:K40L:T51V:R58F-hCRBPII·retinal complex, a sample of Q108K:K40L:T51V:R58F-hCRBPII (20 μM) and retinal (0.5 equiv) in PBS buffer (pH 7.3) was scanned by UV/Vis spectroscopy until complete formation of the PSB was observed. The sample was then irradiated with white light and with use of a VG-9 (VIS) 2″ square colored glass band-pass filter (CWL=526 nm, FWHM=53 nm, purchased from Edmund Optics) for 12 min. Upon conversion into the SB, the sample was irradiated with white light and with use of a UV band-pass filter for 1 min. Successive cycling was repeated 15 times.</p>

PubMed Author Manuscript

Visualizing Cholesterol in the Brain by On-Tissue Derivatization and Quantitative Mass Spectrometry Imaging

Despite being a critical molecule in the brain, mass spectrometry imaging (MSI) of cholesterol has been under-reported compared to other lipids due to the difficulty in ionizing the sterol molecule. In the present work, we have employed an on-tissue enzyme-assisted derivatization strategy to improve detection of cholesterol in brain tissue sections. We report distribution and levels of cholesterol across specific structures of the mouse brain, in a model of Niemann-Pick type C1 disease, and during brain development. MSI revealed that in the adult mouse, cholesterol is the highest in the pons and medulla and how its distribution changes during development. Cholesterol was significantly reduced in the corpus callosum and other brain regions in the Npc1 null mouse, confirming hypomyelination at the molecular level. Our study demonstrates the potential of MSI to the study of sterols in neuroscience.

visualizing_cholesterol_in_the_brain_by_on-tissue_derivatization_and_quantitative_mass_spectrometry_

<!>Materials and Methods<!>Chemicals and Reagents<!>Experimental Models<!>Tissue Sectioning<!>Histology<!>Region of Interest (ROI) Analysis and Quantitative Morphometry<!>Stereology<!>Deposition of the Standard and On-Tissue EADSA<!>Mass Spectrometry Imaging<!>Vacuum MALDI-TOF-MSI<!>AP-MALDI-MSI<!>Vacuum-MALDI-Q-IM-TOF MSI<!>DESI-Q-IM-TOF MSI<!>Quantification<!>Statistics<!>Results and Discussion<!>Quantitative MSI of Sterols in WT Mouse Brain<!><!>Quantitative MSI of Sterols in WT Mouse Brain<!><!>Quantitative MSI of Sterols in WT Mouse Brain<!><!>Quantitative MSI of Sterols in WT Mouse Brain<!>MSI of Cholesterol in the Developing Mouse<!><!>MSI of Cholesterol in the Developing Mouse<!>MSI of Cholesterol in the Niemann-Pick Disease Type C1 Shows a Lack of Cholesterol in Hypomyelinated Fibers Tracts<!><!>MSI of Cholesterol in the Niemann-Pick Disease Type C1 Shows a Lack of Cholesterol in Hypomyelinated Fibers Tracts<!>EADSA-MSI with Multiple Ionization Modes and Analyzers<!>Conclusions and Perspective<!><!>Author Contributions<!>

<p>Cholesterol is the most abundant individual molecular species in plasma membranes of animals, accounting for approximately 20–25% of the lipid molecules in the plasma membrane of most cells,1 with only a small proportion of cellular cholesterol embedded in organelles. Within membranes, cholesterol influences bilayer fluidity and permeability and lipid and protein sorting in membrane trafficking.2 In the brain, cholesterol makes up about 15% of the dry weight of white matter (WM) and is a major component of myelin sheaths.3 However, little is known about how sterol concentrations vary in different anatomical locations or at sites of focal pathology.4 Cholesterol is metabolized to oxysterols, steroid hormones, and bile acids. These metabolic pathways are at least partially operative in the brain, and their metabolic products and intermediates serve as biologically active signaling molecules.5 In light of this, it is not surprising that impairment in sterol homeostasis and signaling is implicated in a number of human disorders including neurodegenerative and neurodevelopmental conditions.6,7 Dysregulation of cholesterol homeostasis has been implicated in Alzheimer's disease8 and multiple sclerosis,9 while inborn errors of cholesterol biosynthesis, metabolism, and transport can result in neurological disorders,10 such as Smith–Lemli–Opitz syndrome (SLOS, 7-dehydrocholesterol reductase deficiency) and Niemann-Pick disease types C1 and C2 (NPC1 and NPC2, respectively).</p><p>Traditionally, cholesterol analysis in tissue begins with homogenization followed by lipid extraction, leading to loss of spatial information. To better understand sterol biochemical and physiological roles, there is a need to match molecular abundance with the exact location. To this end, brain dissection can be coupled to gas chromatography (GC)—mass spectrometry (MS) or to liquid chromatography (LC)—MS.8,11,12 An alternative method to map sterol concentrations in the brain is by exploiting mass spectrometry imaging (MSI), for example, time-of-flight (ToF) secondary-ion MS (SIMS)—MSI, where cholesterol has been detected with high intensities, even at subcellular resolutions. However, a drawback with this approach is that ToF-SIMS is a surface-sensitive technique, and cholesterol has been shown to migrate to and crystallize on the surface, covering up all colocalizing species in the tissue. Matrix-assisted laser desorption/ionization (MALDI)-MSI has been employed to detect and identify multiple molecular species and simultaneously map their distribution in tissue sections.13,14 It can generate pixelated MS data at near-cellular resolution, providing spatial mapping of protein, peptide, and lipid molecules according to X–Y position on a tissue section.15,16 MALDI-MSI has been used to image lipids in the brain;17 however, cholesterol and other sterols tend to be poorly ionized by conventional MALDI and are discriminated against when compared to lipid classes that are easily ionized. Cholesterol has been detected in MALDI-MSI studies,18 but to enhance ionization, other desorption methods have been employed, including nanostructure-initiator MS,19 sputtered silver MALDI,20 and silver nanoparticle MALDI.21 Silver ions coordinate with carbon–carbon double bonds, providing cationic adducts of sterols in the MALDI matrix. Recently, "MALDI-2"-MSI has been developed, where a postdesorption second-tuneable laser enhances the ionization of neutral lipid species including cholesterol, allowing improved visualization in tissue sections.22 Alternatively, derivatization strategies can be utilized to enhance sterol ionization. For in-solution studies, we and others have exploited enzyme-assisted derivatization for sterol analysis (EADSA)23,24 where the sterol molecule is reacted first with the cholesterol oxidase enzyme to oxidize the 3β-hydroxy group to 3-oxo and then with Girard-P (GP) hydrazine to give a charge-tagged sterol hydrazone (Figure 1). This strategy enhances the MS signal and provides unique structural information upon multistage fragmentation (MSn) which, together with the retention time and accurate mass measurements, can provide unambiguous identification, even of isomeric species. Of note, others have similarly exploited a Girard-T hydrazine to derivatize and visualize by MSI steroid molecules, already possessing an oxo function.25,26</p><p>MSI of cholesterol in WT mouse brain exploiting on-tissue EADSA. (A) EADSA process occurs in two steps where the 3β-hydroxy-5-ene group is first converted to a 3-oxo-4-ene by the enzyme cholesterol oxidase and then charge-tagged with the GP hydrazine. (B) Typical mass spectrum generated in an EADSA-MALDI-MSI experiment for a single pixel. The spectrum, in the m/z range 500–550, is dominated by the signals of derivatized endogenous cholesterol at m/z 518.4 and sprayed-on standard [2H7]cholesterol at m/z 525.4. In each pixel, the peak at 518.4 is normalized to the peak at 525.4, and an MS image of the distribution of cholesterol across the mouse brain tissue section is created as shown in (C). MSI data were acquired on a vacuum-MALDI-TOF MS at a pixel size of 50 μm and visualized with an isolation window width of 0.5 m/z. See Supporting Information, Table S1 for instruments used in each figure.</p><p>We have adapted EADSA to MSI in order to image cholesterol in the developing and adult mouse brain and in a mouse model of Niemann-Pick C1 disease (Npc1 null) at 30–50 μm pixel size. We demonstrate the use of isotope-labeled standards to determine the absolute quantity of cholesterol in different anatomical regions of the mouse brain. A quantitative MSI of the adult wild-type (WT) mouse in sagittal sections was determined by identifying the pons and medulla of the brain stem as the regions with the highest cholesterol level. The WT mouse was compared to the Npc1 null mouse showing a significant reduction of cholesterol in the corpus callosum. In the WT mouse brain at birth, cholesterol is the highest in the pontine hind brain that will develop into the cholesterol-rich pons region in the adult mouse. The derivatization-based method has potential to be expanded to low abundance sterols, while simultaneously detecting other nonderivatized lipid classes.</p><!><p>The aim of the study was to develop an MSI method suitable to map the distribution and to determine the concentration of cholesterol in different anatomical regions of mouse brain.</p><!><p>HPLC-grade methanol, propan-2-ol, acetonitrile, ethanol, xylene, and industrial methylated spirit were from Fisher Scientific (Loughborough, UK). Glacial acetic acid was from VWR (Lutterworth, UK). [25,26,26,26,27,27,27-2H7]Cholesterol was from Avanti Polar Lipids (Alabaster, AL). Cholesterol oxidase from Streptomyces sp., and potassium dihydrogen phosphate, Luxol Fast Blue (LFB), Cresyl Violet (CV), DPX mountant, paraformaldehyde (PFA), lithium carbonate, and α-cyano-4-hydroxycinnamic acid (CHCA) were from Merck (Dorset, UK). GP-hydrazine was from TCI (Zwijndrecht, Belgium).</p><!><p>In the present study, adult WT and Npc1–/– mice and the phenotypically normal newborn Dhcr7T93M/+ mouse were employed. Details can be found in Supporting Information Methods.</p><!><p>Brain tissue, mounted on and only partially embedded in the optimal cutting temperature (OCT) compound, was cryosectioned using a Leica Cryostat CM1900 (Leica Microsystems, Milton Keynes, UK) at a chamber temperature of −18 °C into 10 μm-thick sections which were thaw-mounted onto optical microscope slides for histology or onto indium tin oxide (ITO)-coated glass slides for MSI and stored at −80 °C until use. ITO-coated glass slides (8–12 Ohm/Sq) were from Diamond Coatings (Halesowen, UK). Three sections were mounted on each glass slide, and each section was separated by 100 μm from the adjacent section, that is, the nine sections in between were placed on other consecutive slides.</p><!><p>Tissue sections adjacent to sections analyzed by MSI were thawed, fixed in PFA to preserve anatomy, and subjected to LFB histology with cresyl violet as the counterstain27 (Supporting Information Methods). Histological data were analyzed by QuPath28 and ImageJ (NIH) following whole-section digitization at 400× magnification using a Zeiss AxioScanner.</p><!><p>Quantitative analysis of histological data was carried out as follows. To assess fiber myelination, the caudate-putamen ROI was outlined on the digitized images with QuPath. Images of defined ROI were cropped and converted into an 8-bit format with ImageJ to mark and measure specific areas. The threshold was adjusted to exclude cell nuclei and automatically outline WM areas exclusively, and the total WM area was measured per section. These data were used to calculate the percentage of myelinated fibers in the selected ROI. Cerebellar area and length of the Purkinje cell layer were also defined with QuPath, and Purkinje cells were manually counted to determine cell linear density.</p><!><p>Stereological methods were employed to identify ROI within mouse brain sagittal tissue sections. The defined ROI was employed to analyze both the MSI and the histology data. For determination of ROI, we referred to the Allen Mouse Brain Atlas (AMBA, Allen Institute for Brain Science) (sagittal sections, P56, https://atlas.brain-map.org/atlas?atlas=2).29 Details can be found in Supporting Information Methods.</p><!><p>This was performed as described in ref (30) with minor modifications. Mouse brain sections mounted on an ITO-coated glass slide were transferred from a −80 °C freezer to a vacuum desiccator. After 15 min dessication, [2H7]cholesterol (200 ng/μL in ethanol) was sprayed from a SunCollect sprayer (SunChrom, Friedrichsdorf, Germany, supplied by KR Analytical Ltd, Cheshire, UK) at a flow rate of 20 μL/min at a linear velocity of 900 mm/min with a 2 mm line distance and height of 30 mm from the section in a series of 18 layers. The resulting density of the deuterated standard was 40 ng/mm2 (see below). The sprayer was thoroughly flushed with about 2 mL of methanol after which cholesterol oxidase (0.264 units/mL in 100 μM KH2PO4 pH 7) was sprayed for 18 layers. The first layer was applied at 10 μL/min, the second was applied at 15 μL/min, and then all the subsequent layers were applied at 20 μL/min to give an enzyme density of 0.05 munits/mm2. Thereafter, the enzyme-coated slide was placed on a PTFE bed in a glass jar (11 cm × 11 cm × 7.5 cm) above 30 mL of warm water (37 °C) and then incubated at 37 °C for 1 h. The slide was removed, and the tissue was dried in a vacuum desiccator for 15 min. GP (5 mg/mL in 70% methanol, 5% acetic acid) was sprayed on the dried slide with the same spray parameters as used for spraying of cholesterol oxidase. The resulting GP density was 1.00 μg/mm2. The slide was then placed in the custom-made humidity chamber as mentioned above containing 10 mL of prewarmed (37 °C) 50% methanol and 5% acetic acid and incubated at 37 °C for 1 h. The slide was removed and dried in a vacuum desiccator and then stored in a cold room (4 °C) until MSI analyses. Desorption electrospray ionization (DESI)-MSI experiments were performed without any further pretreatment. For MALDI-MSI, on the next day, the desiccator was allowed to reach room temperature, and then, the slide was removed and sprayed with the CHCA matrix. CHCA was sprayed from an HTX TM-sprayer (HTX Technologies, NC, USA) at 5 mg/mL in water/propan-2-ol/acetonitrile (3:4:3, v/v/v) at a flow rate of 80 μL/min and a linear velocity of 1200 mm/min, with 2 mm line distance and a criss-cross deposition method which alternates vertical and horizontal passes, for a total of 8, with an offset of 1 mm, resulting in a matrix density of 1.33 μg/mm2. The sprayer nozzle was heated at 70 °C to enhance the solvent evaporation rate.</p><!><p>Following EADSA treatment, tissues sections were analyzed using different mass spectrometers. Optimized instrumental parameters are described below.</p><!><p>Experiments were carried out on an ultrafleXtreme MALDI TOF/TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) equipped with a Smartbeam Nd:YAG laser emitting at 355 nm (2 kHz) and operated in the reflectron mode and positive polarity. Each mass spectrum was automatically acquired using the autoexecute method in FlexControl (Bruker) software in the m/z range of 400–1000. The pixel size was set at 50 μm using flexImaging 4.1 software (Bruker), setting laser focus to "small". The laser spot size was about 50 μm, according to factory specifications and as verified by visual inspection with the instrument camera. Each raster was sampled with 200 shots in five steps for a total of 1000 shots per raster. The total acquisition time was typically about 11.5 h for a total of ∼27,000 positions. The MALDI instrument was calibrated using a mixture of phosphatidylcholine and lysophosphatidylcholine (Avanti Polar Lipids). After measurement, imaging spectra were recalibrated using the batch process in flexAnalysis. On-tissue, mass accuracy was typically within ∼100 ppm of the theoretical mass. Data were analyzed and visualized using flexImaging 3.0 (Bruker) and SCiLS Lab 2014b (SCiLS, Bremen, Germany) without any processing step. Data were visualized using normalization to [2H7]cholesterol at m/z 525.5. Mass selection windows for ions of interests were chosen with a width of ±0.25 Da in flexImaging 3.0 and of ±0.125% in SCiLS Lab 2014b. A mass resolution of 20,000 (fwhm) was typically achieved in a single pixel. An optical image of each tissue section was acquired prior to the MS acquisition by means of a flatbed scanner.</p><!><p>MSI was carried out in the positive-ion mode with an Orbitrap Elite hybrid linear ion trap (LIT)-Orbitrap mass spectrometer (ThermoFisher Scientific) coupled with an AP-MALDI UHR source (MassTech, Maryland USA, supplied by KR Analytical Ltd) equipped with a Nd:YAG laser emitting at 355 nm. Full scan (MS1) imaging analysis was performed with m/z measurement in the Orbitrap over the m/z range of 400–1200 at 60,000 resolution (fwhm at m/z 400), MALDI laser energy was set at 45% of the maximum, and frequency was 1.5 kHz. Data were acquired in the constant speed raster (CSR) mode at a scan speed of 2.8 mm/min and a pixel size of 30 μm. A lock mass for [2H7]cholesterol at m/z 525.4544 was employed. The acquisition of one mouse brain tissue section was achieved in about 15 h. In MS3 experiments, the MALDI laser energy was set at 14% and frequency was 1.5 kHz. Data were acquired in the CSR mode at a scan speed of 3 mm/min and a pixel size of 40 μm. In the LIT, precursor ions were isolated and fragmented with an isolation width of 2 and an arbitrary collision-induced dissociation (CID) energy of 35%. The most intense fragment ion produced in MS2 was selected with an isolation width of 2 and fragmented with a CID energy of 40% to produce an MS3 spectrum. MS3 spectra of cholesterol and [2H7]cholesterol were acquired in each pixel. The MS3 transitions for cholesterol and [2H7]cholesterol were 518.4 → 439.4→ and 525.4 → 446.4 →, respectively.</p><p>Data were analyzed and visualized using ImageQuest (ThermoFisher Scientific). Alternatively, after exporting the file into an imzml format, data were analyzed by SCiLS Lab MVS 2014c (SCiLS, Bremen, Germany) without any processing step. MS1 data were normalized to [2H7]cholesterol at m/z 525.454. Ions of interests were extracted with a width of 7 mDa and 0.3 Da for MS1 and MS3 scans, respectively.</p><!><p>Experiments were carried out on two Synapt G2-Si instruments (Waters, Wilmslow, UK) exploiting Waters HDI 1.4 software for acquisition and image visualization. Images were generated from spectra acquired in the positive-ion mode in the m/z range 400–1000. The laser frequency was 1 kHz, and power was kept at 100 arbitrary units. The scan time was 0.5 s, and the pixel size was 50 μm. IMS cell wave velocity was from 1000 to 300 m/s, and transfer wave velocity was 281 m/s. In all experiments, the cholesterol signal was measured to better than 5 ppm mass accuracy.</p><!><p>Experiments were carried out on a Synapt G2-Si. Spectra were acquired in the positive ion mode in the m/z range 100–1200, with a needle voltage of 4.5 kV. The DESI solvent flow rate was 1.25 μL/min. The scan time was 0.25 s, and the pixel size was 25 μm. IMS cell wave velocity was from 1000 to 300 m/s, and transfer wave velocity was 281 m/s. In all experiments, the cholesterol signal was measured with a mass accuracy better than 8 ppm.</p><!><p>Known amounts of [2H7]cholesterol were sprayed on tissue prior to the EADSA process. This procedure corrects for variation in the matrix effect and MS response. The linearity of the on-tissue response of sprayed-on [2H7]cholesterol verses endogenous cholesterol was determined by spraying eight consecutive tissue sections with [2H7]cholesterol at varying densities (endogenous cholesterol areal density is assumed to be constant for a given ROI across the consecutive slices). Quantification was made from [M]+ ion signal intensities, averaged in each brain region. Brain regions of interest were defined according to AMBA. The areal density of cholesterol in defined regions of interest was calculated by correlating signal intensity to that of known density of [2H7]cholesterol sprayed on tissue. All quantitative measurements were analyzed employing SCiLS Lab MVS 2019c (SCilS, Bremen, Germany).</p><!><p>To determine statistical difference in cholesterol areal density between defined regions of interest in five adult WT mice, two-way ANOVA was performed with cholesterol areal density as the dependent variable and the mouse and brain region as factors. The interaction between the mouse and brain region was used as error variance. The residuals representing the interaction deviations were approximately normally distributed. Tukey's multiple comparisons test was used to identify significant differences between brain regions. Statistical analysis was applied for the assessment of myelinated fiber density and specific cell counts, in defined brain regions of WT and Npc1–/– mouse brain. Five WT and three Npc1–/– brains were employed, analyzing three or more sections for each mouse.</p><p>To determine statistical differences in cholesterol areal density in defined regions of interest between WT and Npc1–/– mouse brain, a Shapiro–Wilk test for normality was performed, followed by an unpaired t-test for significance. The analyses were performed using GraphPad Prism 8.2.1 software (GraphPad Software, Inc, CA, USA). A P-value of less than 0.05 was considered statistically significant. P < 0.05, *; P < 0.01, **; and P < 0.001, ***. All whiskers on bar graphs represent 1 standard deviation. Note that one of the five control mice was not considered in the calculation of the average cholesterol areal density for the caudate-putamen as it was not sectioned on an equivalent anatomical plane.</p><!><p>In MALDI-MS and electrospray ionization (ESI)-MS, cholesterol is poorly ionized and is often detected as the ammonium adduct [M + NH4]+ at m/z 404.39 or as the dehydrated protonated molecule [M + H–H2O]+ at m/z 369.35.31 In this study, to enhance ionization of sterols, we exploit the EADSA method, previously used for in-solution analysis of sterols. Once the sterol analyte is specifically and effectively charge-tagged by EADSA (Figure 1A), it is readily analyzed by MSI, thereby allowing its detection and identification (e.g., by MS3) and the mapping of its distribution. The advantage of this methodology is fourfold in that it (i) greatly increases sensitivity, (ii) allows for absolute quantification, (iii) enhances structural information, and, equally importantly, (iv) increases analytical specificity. Here, we report how EADSA has been adapted to work on brain tissue sections for MSI studies.</p><!><p>Initial studies were performed using sagittal mouse brain sections with a MALDI-TOF instrument. GP-tagged cholesterol gives an intense [M]+ signal, as does sprayed-on [2H7]cholesterol, and dominates the resulting mass spectrum (Figure 1B). An MS image of cholesterol distribution, normalized in each pixel to [2H7]cholesterol sprayed-on standard, is shown in Figure 1C.</p><p>To confirm the identity of the signals assigned to cholesterol, we separately carried out MS3 ([M]+ → [M – Py]+→, where Py corresponds to the pyridine component of the GP-tag, see Supporting Information, Figure S1) analysis of the peaks at m/z 518.41 (cholesterol) and m/z 525.45 ([2H7]cholesterol) using AP-MALDI on an Orbitrap Elite, see Figure 2. In Supporting Information, Figure S1, structures of the major fragment ions observed in Figure 2 are described. The fragment ion at m/z 163 (*b3-28) is formed by cleavage of the A/B ring and is devoid of the CD rings and the side chain. It is present in MS3 spectra of both cholesterol (Figure 2C) and [2H7]cholesterol (Figure 2D) authentic standards and can thus be exploited in a multiple reaction monitoring (MRM)-like experiment to confirm the location of tissue-endogenous cholesterol and sprayed-on [2H7]cholesterol in each pixel (Figure 2A,B, respectively). Figure 2E shows that the MRM transition 518.4 → 439.4 → 163 is essentially absent off tissue, while most notably enriched in the midbrain, pons, medulla, and WM tracts of the cerebellum. Conversely, Figure 2F shows that the MRM transition 525.5 → 446.4 → 163 is saturated off tissue, while being quite evenly distributed on tissue. This transition does show some variation on tissue due to matrix effects. For MS3 data, the current software does not allow automated normalization of cholesterol signals to the internal standard.</p><!><p>MS3 fragmentation patterns of (A) tissue-endogenous cholesterol and (B) sprayed-on [2H7]cholesterol, in a single pixel obtained in the LIT of an AP-MALDI-Orbitrap Elite after on-tissue EADSA of a mouse brain tissue section and of (C) cholesterol and (D) [2H7]cholesterol reference standards obtained in the LIT of an ESI-Orbitrap Elite following in-solution EADSA. The MS3 spectra were obtained for the transitions [M]+ → [M – Py]+→. (E,F) MSI of the distribution of the MS3 fragment ion at m/z 163 from (E) cholesterol and (F) [2H7]cholesterol in a sagittal brain section of WT adult mouse.</p><!><p>Using MS1, areal densities were determined against a known density of the sprayed-on internal standard in WT mouse brain sections, for selected brain structures (Figure 3A). The linearity of the on-tissue response of endogenous cholesterol versus the sprayed-on deuterated standard was determined by spraying eight consecutive tissue sections with [2H7]cholesterol at varying known densities (Supporting Information, Figure S2A). Examples of calibration curves obtained on whole-brain sections and considering the cerebellum as an ROI are shown in Supporting Information, Figure S2B,C, respectively. R2 for the whole brain was determined to be 0.94, and for the cerebellum, it was determined to be 0.97. Our quantitative data reported in Figure 3B and in Supporting Information, Table S2 (ng/mm2, mean of five biological replicates ± standard deviation) indicate that cholesterol abundance is the highest in the pons (681.6 ± 123.9 ng/mm2) and cerebellar white matter (652.0 ± 119.8 ng/mm2) and the lowest in the olfactory traits, cortex, and hippocampus (348.5 ± 52.4, 327.8 ± 32.5, and 326.3 ± 31.6 ng/mm2, respectively). Note that cholesterol was quantified via MSI in the whole cerebellum and separately in its WM tracts, whereas cholesterol content in cerebellar grey matter (GM) is estimated from the difference of the total and WM. In previous reports,11 cholesterol synthesis and concentration were found to be higher in regions of the central nervous system (CNS) containing heavily myelinated fiber tracts such as the brain stem (medulla and pons) and the midbrain.</p><!><p>Quantitation of cholesterol in WT adult mouse brain via MSI. (A) LFB/CV staining for myelin of a sagittal mouse brain section adjacent to a section undergoing MSI. Major anatomical structures were identified by comparison with the AMBA29 and are outlined with dashed lines: olfactory traits, OLF; cortex, CTX; corpus callosum, CC; caudate-putamen, CP; thalamus, TH; hypothalamus, HY; hippocampus, HP; midbrain, MB; pons, P; medulla, MY; cerebellum, CBX; and cerebellar white matter, CBX WM. (B) Areal density (ng/mm2) of cholesterol in brain regions from five WT mice, averaged over different slices (see the Statistics section). Values for individual mice are given by separate histogram bars. The three dots within each bar correspond to region averages for each brain slice employed. The height of each bar represents the mean of the region average for each mouse across the three slices. The error bars indicate the SD of all the sections per mouse. Data were acquired on a vacuum-MALDI-TOF MS instrument.</p><!><p>In an early report,8 it was shown that the concentration of cholesterol in the pons is ∼2.5 times more than in the cortex, which is also in agreement with our data showing a ratio of 2.1. In earlier MSI studies, cholesterol was visualized in mouse brain in coronal or horizontal sections.18,20,32 Sagittal MS images have the advantage that the brain stem can be easily differentiated into the midbrain, pons, and medulla regions (Figures 2E and 3A). These brain stem structures show high cholesterol content (Figure 3 and Supporting Information, Table S2), in agreement with previous GC–MS and LC–MS studies.8,11</p><p>Interestingly, the distribution of gene transcripts of late-stage cholesterol biosynthetic enzymes matches regions of high cholesterol abundance, that is, midbrain, medulla, and pons regions. Please see mRNA expression data of Dhcr24, entrez ID (EID) 74754; Dhcr7, EID 13360; and Sc5d, EID 235293, provided by the AMBA.29 Of note, the abundance of cholesterol in the corpus callosum and in the fiber tracts of the caudate-putamen mirrors the distribution of transcripts unique to myelinating oligodendrocytes. See Mbp, EID 17196; Plp1, EID 18823; and Cnp, EID 12799 provided by the AMBA.29</p><p>When MS1 data obtained by AP-MALDI are visualized in a peripheral sagittal section taken at a plane about 3 mm from the midline, the distribution of GP-derivatized cholesterol at m/z 518.4103 is clearly enhanced in specific regions of the brain (Figure 4A). These are either WM tracts such as the corpus callosum and cerebellum or brain regions (deep GM structures) containing myelinated fibers, such as the pons, medulla of the brain stem, and caudate-putamen of the diencephalon. In Figure 4, the selected sagittal plane does not include the midbrain but shows hippocampal features.</p><!><p>AP-MALDI-MSI of cholesterol in sagittal sections of WT adult mouse brain after on-tissue EADSA. The data were obtained on an Orbitrap instrument. (A) MSI of cholesterol. (B) MSI of PC 32:0. (C) Typical AP-MALDI-Orbitrap MS spectrum averaged over the entire MSI data set showing signals of sterol and other brain lipids that can be detected simultaneously. (D) Anatomical layering of the cerebellum (CBX, yellow). Image from AMBA: Adult Mouse, P56, Sagittal, Image 7 of 21 id = 100883846.29 In (A, B), the isolation window width was 7 mmu and the pixel size was 30 μm. Images normalized against sprayed-on [2H7]cholesterol.</p><!><p>Notably, using our method for on-tissue cholesterol derivatization and in contrast to ToF-SIMS, other lipids can be mapped simultaneously, particularly when experiments are carried out with API (i.e., AP-MALDI—Orbitrap and DESI—Q-TOF). To show the potential of our approach, we report the MS image of PC 32:0 at m/z 734.5702 (about 1 ppm deviation from the theoretical m/z), normalized to [2H7]cholesterol sprayed-on standard (Figure 4B), but many other peaks could be similarly imaged. Note that the peak at m/z 734.5702 could also be assigned to PE 35:0. However, phospholipids containing fatty acids with an odd number of carbon atoms are minor species in animals.</p><p>Interestingly, in Figure 4A, a continuous gradient of cholesterol concentration is observed going out from the corpus callosum, where cholesterol is at an areal density of about 520 ng/mm2, decreasing on moving through the overlying layers of the neocortex. The continuous cholesterol gradient is mirrored by the distribution of PC 32:0 in the same brain regions (Figure 4B). Within the cerebellum, a decreasing concentration of cholesterol is observed going from the WM of the cerebellum (CBX WM, 652.0 ± 119.8 ng/mm2) to the granule cell layer and molecular layer of the GM (Figure 4D shows reference anatomy). Note that cholesterol density in cerebellar GM can be estimated (CBX—CBX WM = CBX GM) to be about 260 ng/mm2. Here, the cholesterol smooth gradient contrasts with the step gradient shown by PC 32:0 (Figure 4B) which is deficient in the granule cell layer of the cerebellum but more evident in the molecular layer. Further description of MSI of cholesterol in cortical layers and hippocampus can be found in Supporting Information, Figure S3.</p><p>The EADSA-MALDI-MSI quantitative assessment of cholesterol areal density in defined brain regions of the WT mouse can be compared with measurements previously obtained with a similar but different experimental approach exploiting low-spatial-resolution (400 μm pixel size) liquid extraction for surface analysis (LESA), that is, EADSA-LESA-LC-MS.30Supporting Information, Table S2 reports the values obtained and their standard deviations, in each defined brain region of WT mouse, for both the present and the EADSA-LESA-LC-MS study. The agreement was >90% for very homogenous regions such as the cortex and thalamus, it was about 80% for caudate-putamen, hippocampus, pons, and cerebellar white matter, and it was >67% for heterogeneous midbrain and medulla.</p><!><p>To illustrate how our EADSA-MSI method can be used to monitor brain cholesterol distribution during development, we compared tissues from mice at 1 day and 10 weeks. At birth, myelination is in its very early stage, while at 10 weeks, it is nearly completed.33 During development, the cholesterol content in the whole brain goes from about 4 mg/g at birth up to about 15 mg/g in the adult at 26 weeks34 and comes from local synthesis only.1 During the first 3 weeks of life, when myelin sheaths are being generated, the rates of cholesterol synthesis and accumulation in brain are high at about 250 μg/day11 and drop rapidly beyond 3 weeks of age.34 Most strains reach sexual maturity between 6 and 8 weeks, so postnatal week 10 can be defined as a young adult mouse.35</p><p>EADSA-MSI was employed to visualize cholesterol distribution in the mouse brain at 1 day and at 10 weeks (Figure 5). We compared MSI of cholesterol with LFB chemical stain and CV as a counterstaining, histological stain for myelin,27 as shown in Figure 5C (newborn) and Figure 5D (10 weeks). Figure 5E shows the MSI of cholesterol at 1 day around the time when oligodendrocytes start to contribute to cholesterol synthesis,36 and Figure 5F shows the distribution at 10 weeks.</p><!><p>Quantitative MSI of cholesterol in mouse brain at birth and at 10 weeks. In (A,C,E), 1 day-old newborn, and in (B,D,F), 10 week-old adult mouse. (A,B) AMBA images depicting mouse brain sagittal sections with annotations of anatomical structures. The 1 day-old newborn is matched with the E18.5-day embryo atlas image (A). Pallium, Pal; telencephalic vesicle, Tel; diencephalon, D; peduncular hypothalamus, PHY; midbrain, M; pontine hindbrain, PH; pontomedullary and medullary hindbrain, PMH + MH; prepontine hindbrain, PPH. (B) Adult mouse, abbreviations as in Figure 3. (C,D) LFB/CV staining of sagittal mouse brain sections adjacent to sections undergoing MSI. (E,F) MSI of cholesterol after on-tissue EADSA by vacuum-MALDI-TOF MS, normalized against sprayed-on [2H7]cholesterol, at a pixel size of 50 μm. (G) Areal density (ng/mm2) of cholesterol in brain regions from two newborn WT mice, each averaged over four slices. The mean of the region average for individual mice is given by separate histogram bars, and region averages in each slice are represented by dots. The error bars indicate SD. Image from AMBA: Developing Mouse, E18.5, Image 16 of 19 id = 100740373 and Adult Mouse, P56, Sagittal, Image 15 of 21 id = 100883867.29</p><!><p>As measured by quantitative EADSA-MSI, the newborn (Figure 5G and Supporting Information, Table S3) shows the highest cholesterol level in the pontine hindbrain (193.4 ± 28.4 ng/mm2) and in the medullary and pontomedullary hindbrain (180.3 ± 25.6 ng/mm2) that will develop into the cholesterol-rich pons and medulla of the adult mouse (Figure 3B).33 The lowest levels of cholesterol are detected in the telencephalic vesicle (111.7 ± 13.9 ng/mm2) and in the pallium (125.1 ± 15.3 ng/mm2) which will develop into the cortex, olfactory tracts, hippocampus, and caudate-putamen.33 Similar to the newborn, these are regions with low cholesterol in the adult (Figure 3 and Supporting Information, Table S2) except for the caudate-putamen which contains some fiber tracts in the adult that are not yet formed in the newborn.33 The pro-hypothalamic region (peduncular hypothalamus), which begets the adult hypothalamus and associated fiber tracts, shows a diffused enrichment in cholesterol in the newborn (Figure 5A,E), while the hypothalamus in the adult accumulates cholesterol only in surrounding fibers (Figure 5B,F). A striking difference between 1 day and 10 week animals is the lack of a visible corpus callosum (CC) in the newborn. In the mouse, myelination of the CC is reported to begin at 11 days after birth37 and CC is detected by histological methods at around 16–17 days of age.37 In contrast to the newborn, in the 10 week adult, the CC is fully formed.35 In particular, in the adult, the thicker regions of the CC show enrichment in cholesterol, namely, the rostrum-genu (frontal, 504.3 ± 57.7 ng/mm2, see Supporting Information, Figure S4), the body (central, 546.2 ± 66.3 ng/mm2), and the splenium (back, 500.4 ± 35.3 ng/mm2), while the thinnest part of the CC which is the isthmus connecting the body and the splenium is the CC structure with the lowest cholesterol abundance (457.8 ± 51.2 ng/mm2).</p><p>Worthy of note, the nonspecific LFB myelin stain of the newborn provides little distributional information when compared to the MSI heat map for cholesterol (Figure 5C cf 5E). Importantly, MSI, as applied here, is specific for cholesterol, while the exact molecular species bound by LFB remain uncertain.38 Notably, the cholesterol distribution in the newborn mouse imaged by vacuum MALDI-TOF (Figure 5E) is consistent with the image of an adjacent brain section produced by DESI-Q-TOF (Supporting Information, Figure S5E), proving the robustness of the EADSA-MSI approach.</p><p>Finally, as measured by EADSA-MSI, the whole-brain areal density of cholesterol in the newborn is about 160 ng/mm2, while it is about 480 ng/mm2 in the adult at 10 weeks, showing a threefold increase. Our data are in good agreement with previous reports11 where the cholesterol content in the newborn was determined to be about 4 mg/g at birth and to increase to about 10 mg/g at 10 weeks, showing a 2.5-fold increase. In summary, the present data demonstrate that EADSA-MSI can be used effectively to monitor cholesterol abundance in brain structures during development.</p><!><p>Niemann-Pick disease, type C is a neurodegenerative, lysosomal storage disorder, characterized by accumulation of unesterified cholesterol and sphingolipids in the endo-lysosomal system.39 The disease is caused by mutations in the encoding region of genes either for the lysosomal transmembrane protein, NPC1 (95% of cases), or the cholesterol-binding soluble glycoprotein, NPC2 (∼4% of cases).39 These two proteins work together to transport cholesterol through the late endosomal–lysosomal membrane into the metabolically active cholesterol pool. Patients with NPC disease show extensive hypomyelination that manifest in cerebral and cerebellar atrophy and WM hypoplasia.40</p><p>In the present study, we analyzed the cholesterol content and distribution in the brain of the Npc1–/– mouse.41 In the brain of this mouse, at the 7 week time point, cellular dysfunction translates into loss of many large neurons. Purkinje cells of the cerebellum are particularly sensitive to NPC pathology and are largely lost in patients42 and in the mouse model.43 Moreover, the brain of the Npc1–/– mouse generally shows severe dysmyelination of fiber tracts with impairment of oligodendrocyte maturation.36 As in patients,40 oligodendrocyte loss and dysmyelination may result in hypoplasia of the corpus callosum in this mouse model.44 When NPC1/2 proteins are lacking, cholesterol and other lipids remain in the late endosomes/lysosomes and are not transported into the endoplasmic reticulum (ER) and, therefore, sterol homeostasis is undermined by the lack of feedback regulatory mechanisms, that is, free cholesterol accumulates in the late endosome/lysosome compartment, while the rest of the cell perceives a shortage of sterol. Recently, one MSI study has assessed lipid changes in this Npc1–/– mouse but was limited to the cerebellum.18</p><p>In the present study, we have exploited MSI to study the whole brain. The chosen time point was of 10 weeks when the phenotype is severe but not yet lethal: Npc1–/– mice die around 12 weeks of age.45Figure 6A,B shows the MSI heat maps of cholesterol distribution in WT and Npc1–/– mouse brain, respectively. These heat maps can be compared with Figure 6C,D, showing LFB/CV staining for myelin of the corresponding adjacent brain tissue sections. For further comparison of MSI with histology, the density of myelinated fibers in the caudate-putamen (Figure 6E and Supporting Information S6A) and the number of Purkinje cells in the cerebellum (Figure 6F and Supporting Information S6B) of WT and Npc1–/– mouse were determined from the histological data obtained via LFB/CV staining. Figure 6G shows cholesterol levels in selected brain ROI, as quantified by MSI for the Npc1–/– and WT mice. The quantitative data reported in Figure 6G and in Supporting Information, Table S2 indicate that in Npc1–/– brain, cholesterol is most abundant in the cerebellar white matter, medulla, and pons (462.1 ± 58.7, 461.6 ± 111.3, and 454.2 ± 49.5 ng/mm2, respectively) and least abundant in the cortex and hippocampus (307.4 ± 36.9 and 288.9 ± 36.5 ng/mm2, respectively).</p><!><p>Histology-matched quantitative MSI displaying cholesterol distribution and quantification in brain tissue sagittal sections of WT and Npc1–/– mice. (A,B) MSI of cholesterol in WT (A) and in Npc1–/– (B) mouse brain tissue. The cholesterol signal was normalized to the signal of sprayed-on [2H7]cholesterol. Data acquired using vacuum-MALDI-TOF MS at 50 μm pixel size. (C,D) LFB/CV staining of sections adjacent to MSI. Major anatomical structures were identified by comparison with the AMBA31 and are outlined with dashed lines. Abbreviations are the same as in Figure 3. (E) Myelinated fiber density in the CP and (F) Purkinje cell counts in the CBX of WT and of Npc1–/– mice, as assessed by quantitative morphometry of stained sections. (G) Areal density (ng/mm2) of cholesterol in brain regions from five WT and three Npc1–/– mice. Group averages are given by separate histogram bars. Dots within each bar correspond to the individual mouse average. (■CTRL or •KO). The error bars indicate the SD. Data were acquired on a vacuum-MALDI-TOF MS instrument.</p><!><p>Supporting Information, Table S2 also reports the % difference in cholesterol areal density between the Npc1–/– and WT animals. The regions showing the highest reduction of cholesterol in the Npc1–/– mouse compared to the WT are the corpus callosum (34.4%), pons (33.4%), cerebellar WM (29.1%), and midbrain (27.8%) (p-values are indicated in Figure 6G).</p><p>A comparison of histological and MSI data in WT and Npc1–/– mouse brain reveals structural differences that can be correlated with compositional changes in cholesterol distribution and abundance. The most striking difference is in the CC.</p><p>Figure 6C shows that the CC in the WT mouse is heavily myelinated and highlighted by the LFB dye. In contrast, Figure 6D shows that the CC is apparently nonmyelinated in the Npc1–/– brain with the LFB stain showing this structure as mostly white. This correlates well with our MSI data where cholesterol areal density is significantly higher in the WT as compared to the Npc1–/– CC (Figure 6G, ***p-value < 0.001).</p><p>Other than in the CC, the significantly higher cholesterol areal density as determined by MSI in the caudate-putamen region and in the cerebellar WM of the WT mouse as compared to the Npc1–/– mouse (*p-values < 0.05, Figure 6G) also relates to known histological markers.44 This prompted us to further analyze histological data by assessing the percentage of myelinated fibers in the caudate-putamen (Figure 6E and Supporting Information S6A) and the number of Purkinje cells in the cerebellar GM (Figure 6F and Supporting Information S6B) of these mice. In the caudate-putamen, myelinated fiber density assessed in LFB/CV-stained sections was found to be significantly reduced in the Npc1–/– mouse as compared to WT (Figure 6E and Supporting Information S6A, **p-value < 0.01), agreeing with MSI measurement of cholesterol areal density in the same brain region (Figure 6G, *p-value < 0.05).</p><p>Focusing on the cerebellum, the MSI measurements show a significant difference when cerebellar WM is considered (Figure 6G, *p-value < 0.05). This is in agreement with a previous MSI study,18 similarly showing a reduced cholesterol signal intensity, as normalized by the total ion current (TIC), in the cerebellum of the same Npc1–/– mouse compared to WT. Interestingly, there is significant reduction of the number of Purkinje cells in the GM of the Npc1–/– cerebellum (Figure 6F and Supporting Information S6B, ***p-value < 0.001). Loss of Purkinje cells is a known phenotypic marker of Niemann-Pick type C patients42 and animal models.43 These neuronal cells have their cell bodies residing in the cerebellar GM, but their myelinated axons establish postsynaptic connections with cerebellar deep nuclei in the WM.46 Therefore, a reduction in the cholesterol content of the cerebellar WM of the Npc1–/– mouse could be explained in part by the loss of Purkinje cell efferent and afferent connections.</p><p>A significant reduction in the cholesterol areal density of the hypothalamus, midbrain, and pons in the Npc1–/– mouse compared to WT was revealed by MSI (Figure 6G, *p-value < 0.05 and ** p-value < 0.01). Histological staining shown in Figure 6C,D illustrates reduced LFB stain density in these same areas of the Npc1–/– mouse, correlating with MSI heat maps of cholesterol in Figure 6A,B, respectively.</p><!><p>We assessed the robustness of our EADSA-MSI method on different mass spectrometers having different sources (vacuum-MALDI, AP-MALDI, and DESI) and analyzers (Q-TOF with and without ion mobility, linear TOF, and Orbitrap). Our data are consistent as shown for the cholesterol distribution in adult mouse in Figures 1C, 5F, 6A, and Supporting Information S2A all generated by vacuum-MALDI-TOF, in Supporting Information Figure S5A generated by MALDI-Q-IM-TOF, in Supporting Information Figure S5C generated by DESI-Q-TOF, and in Figure 4A generated by AP-MALDI-Orbitrap. Altogether, these data illustrate the reproducibility and robustness of our EADSA-MSI methodology.</p><!><p>The EADSA-MSI method presented provides a tool for the quantitative imaging of cholesterol in mouse brain tissue sections. On-tissue EADSA was successfully employed to improve the analytical power of MSI toward sterols, allowing quantitative mapping of cholesterol at pixel sizes down to 30 μm. Different MS platforms were utilized including vacuum-MALDI-TOF, vacuum-MALDI-Q-IM-TOF, AP-MALDI-Orbitrap, and DESI-Q-TOF, demonstrating the robustness of the method toward different ionization sources, analyzers, and detectors. With atmospheric pressure ionization-MSI (AP-MALDI-Orbitrap and DESI-Q-TOF), the method allowed detection of other lipid classes (phospholipids) in parallel to derivatized sterols, thereby extending the reach of the methodology to the characterization of diverse lipid markers simultaneously. Experiments are underway to extend the reach of the methodology to less abundant and isomeric sterols, including oxysterols, by integrating MS3 analyses in the imaging workflow. MSI is a rapidly advancing technology that can reach cellular resolution,47 thereby providing information on changes happening both at the structural and cellular level. Bridging MS-based lipidomics with histopathology will allow the correlation of quantitative molecular information with the anatomical location, opening a further window for the entry of MSI into clinical chemistry. The EADSA-MSI method described here for imaging of cholesterol directly on tissue can be easily applied to a number of scientific fields including neuroscience, pharmacology, biochemistry, and pathology. Particularly, its application to the study of diseases such as Alzheimer's and multiple sclerosis has the potential to unveil the role of cholesterol in these important neuropathologies.</p><!><p>Experimental details on animal models; histology; stereology; calculation of areal densities; MSI file size and computational resources used; MS3 fragmentation patterns; calibration curves; high-resolution capability as a measure of homogeneity of the sprayed-on isotope-labeled standard; robustness; quantitative morphometry; comparison of normalization strategies; tables relating MSI instruments to figures; comparison of MALDI and LESA data; and cholesterol areal densities in developing mouse brain (PDF)</p><p>ac0c05399_si_001.pdf</p><!><p>The manuscript was written through contributions of all authors. All authors have approved the final version of the manuscript.</p><!><p>The authors declare the following competing financial interest(s): WJG and YW are listed as inventors on the patent Kit and method for quantitative detection of steroids US9851368B2. WJG, EY and YW are shareholders in CholesteniX Ltd. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.</p>

PubMed Open Access

Shedding new light on an old molecule: quinophthalone displays uncommon N-to-O excited state intramolecular proton transfer (ESIPT) between photobases

Excited state dynamics of common yellow dye quinophthalone (QPH) was probed by femtosecond transient absorption spectroscopy. Multi-exponential decay of the excited state and significant change of rate constants upon deuterium substitution indicate that uncommon nitrogen-to-oxygen excited state intramolecular proton transfer (ESIPT) occurs. By performing density functional theory (DFT) and time-dependent density functional theory (TDDFT) calculations, we found that adiabatic surface crossing between the S 1 and S 2 states takes place in the photoreaction. Unlike most cases of ESIPT, QPH does not exhibit tautomer emission, possibly due to internal conversion or back-proton transfer. The ESIPT of QPH presents a highly interesting case also because the moieties participating in ESIPT, quinoline and aromatic carbonyl, are both traditionally considered as photobases.ESIPT, where migration of a proton takes place within a molecule after photoexcitation, is a fundamental photophysical process that garnered decades of interest in varying fields of chemistry. Widespread attention to the phenomenon stems from the fact that molecules that undergo ESIPT may exhibit emission with a large Stokes' shift, which minimizes self-absorption and opens up possibilities of diverse applications 1 . Some examples utilizing this property include molecular sensors 2 , optical memory 3 , and white light sources 4 .Most research on ESIPT utilizes tautomer fluorescence for probing dynamics [5][6][7] and therefore lack of such emission, as is the case with our subject molecule QPH, poses challenge to investigation of ESIPT. Another problem is that it is difficult to recognize ESIPT at first sight if the tautomer is non-emissive. Possibility of fully non-radiative ESIPT has only recently been recognized by Yin et al. 8 and we suspect that this is the reason why ESIPT dynamics of QPH went unnoticed until now.QPH, or more commonly known as quinoline yellow, is a commercial dye with distinctive greenish yellow color. It has three possible tautomers (Fig. 1), of which the enaminone (E) form is confirmed to be the most stable one by NMR 9, 10 and calculation 11 . Unlike the ketoenol (K) and zwitterion (Z) forms, the bond connecting phthalone and quinoline rings is a double bond, making the molecule nearly coplanar 12 . An intramolecular hydrogen bond (IMHB) exists between hydrogen attached to the nitrogen of quinoline and the carbonyl group of the phthalone moiety. Previous researchers acknowledged possible contribution of the Z form in the ground state, whereas the K form does not appear in NMR and is believed to be too energetically unstable (~30 kJ/mol) to exist in the ground state.QPH has a long history of usage since it was discovered in 1882 13 . Because of its many desirable traits such as high solubility, significant resistance against photodegradation, and easy synthesis, QPH and its derivatives are used as coloring agents for various materials including polyester fibers, wools, paper, wax, paraffin, paints, and

shedding_new_light_on_an_old_molecule:_quinophthalone_displays_uncommon_n-to-o_excited_state_intramo

<!>Results<!>Transient absorption spectroscopy.<!>Deuterium isotope effect.<!>P E-S2<!>Discussions<!>Methods

<p>silk [13][14][15][16] . Certification of QPH and its sulfonated derivative by the European Union and FDA allowed more diverse applications.</p><p>Until now the majority of research on QPH has been limited to identifying potential cellular toxicity and developing assays for analysis. Especially the matter of its biocompatibility has long been a subject of debate [17][18][19] . However, to the best of our knowledge, there exists only one photodynamics study of the molecule in pH 12 water conducted in the nanosecond scale 15 . Results in organic solvent and neutral pH water were not reported because the authors found no interesting signal. This most likely indicates that de-excitation dynamics of QPH is completed within sub-nanosecond scale.</p><p>We hypothesized that QPH's notable stability against light may originate from its ultrafast relaxation to the ground state after photoexcitation and attempted to carry out femtosecond pump-probe spectroscopy in order to unravel the long-neglected picture of QPH photochemistry. In doing so we have unexpectedly discovered signs of intramolecular proton transfer occurring in the excited state.</p><p>In the following sections, we report evidence of QPH displaying an unprecedented case of ESIPT between two groups known to be photobases (quinoline and aromatic carbonyl) and provide theoretical basis for its occurrence in QPH.</p><!><p>Absorption and emission spectra. QPH is known for negative solvatochromism, i.e., absorption peaks shifting to shorter wavelengths in polar solvents 14,20 . It is a common behavior for many polar organic molecules that have environment-sensitive energy levels. Strong absorption below 460 nm gives QPH its characteristic yellow hue. Although emission in polar solvent is virtually nonexistent, we could observe weak photoluminescence in highly nonpolar solvents such as cyclohexane (Fig. 2). Even then, the emission signal is very low, reaffirming that significant non-radiative process is involved in relaxation dynamics of the molecule. A small Stokes' shift and near-mirror image symmetry of the absorption vs. emission peaks indicate that the emission is most likely fluorescence from the lowest singlet excited state 21 .</p><!><p>To probe the ultrafast relaxation process of QPH, we conducted femtosecond transient absorption measurement. Figure 3 shows transient absorption spectrum of QPH in cyclohexane excited with 400 nm pump and probed with supercontinuum light in the range of 480 to 660 nm. It is apparent from the picture that the decay kinetics cannot be represented by a single exponential curve, indicating the possibility of relaxation involving more than one excited state. We also take note of the fact that the spectra in the shorter timescales (Fig. 3c) show a "dynamic isosbestic point" near 590 nm, where absorbance remains the same regardless of the temporal evolution. It is generally regarded as a sign that a transient species is evolving into another over the course of time, rather than there being two unrelated excited species 22 . After rapid initial decay, the time profile shows a complex trace for tens of ps, after which the decay becomes nearly single exponential (Fig. S1). Almost no signal is found after 300 ps, indicating that most species has returned to the ground state. Lifetimes that best explain temporal behavior at four different wavelengths of 500, 530, 600, and 620 nm are acquired by global nonlinear fit (Fig. S1) and presented in Table 1. As the current decay profiles are not compatible with simple exponential fit, we truncated their middle part and divided them into a single exponential head (<2 ps) and a biexponential tail (>30 ps) for separate analysis. The initially populated state shows a strong negative signal below 580 nm, which may be interpreted as either disappearance of the ground state species or increased photon output due to stimulated emission 23 . Because the absorption spectrum of ground state QPH ends around 450 nm and the emission spectrum has peaks at similar wavelengths, it is most likely to be stimulated emission signal. Therefore the initially decaying species is presumably the lowest singlet excited state, which is an observable emissive state. From the rapid decay, we know that the emissive state lasts very short, which may explain the low quantum yield of the molecule. From the aforementioned isosbestic point, we assume that it evolves into a different excited state. The excited state absorption signal peaked at 610 nm lasts longer (until 300 ps), suggesting that at least two or more states are involved in the process.</p><!><p>To identify the evolutionary course of the excited state, we carried out comparative experiments using deuterium substituted QPH and unsubstituted QPH. As the hydrogen participating in IMHB (H 1 ) is known to be acidic 14 , we substituted it with deuterium (d-QPH) by following the protocols in the Methods section and acquired time profile at several wavelengths. The same procedure was carried out using water (h-QPH) and compared against QPH solution in cyclohexane to ensure that it was not the effect of hydrate formation. Overall spectral features of d-QPH remained similar to those of h-QPH, including stimulated emission and the isosbestic point, with their only difference being the decay rate. Comparison of the time profiles between d-QPH and h-QPH readily shows significantly slower kinetics of the former (Fig. 4). Lifetimes in Table 1 indicate that a primary kinetic isotope effect may be present in both τ 1 and τ 3 . Table 1. Lifetimes (in ps) of h-QPH and d-QPH and their ratios. τ 1 was determined from single exponential fitting of the sub-2 ps profile, while τ 2 and τ 3 were determined from biexponential fitting of over-30 ps profile.</p><p>(*Standard error in parenthesis).</p><p>Since isotopic mass change does not affect the force field of the molecule, we expect that only the nuclear motions involving the substituted atom will change 23 . In fact, deuterium substitution has been extensively used to verify ESIPT 6 . As the mass of H 1 hydrogen comprises a very small fraction of the mass of QPH, it is unlikely that single deuterium substitution can significantly affect any motion other than the N-H vibration. Since the vibrational frequency of an oscillator is inversely proportional to the square root of the reduced mass of the oscillator if the force constant remains unchanged, we expect the ratio τ D /τ H ( = k H /k D ) of Table 1 should be nearly equal to the square root of the ratio of the reduced masses between N-D and N-H, or {[(14 × 2)/(14 + 2)]/[(14 × 1)/ (14 + 1)]} 1/2 = (15/8) 1/2 = 1.37, which is actually in very good accord with our measured values for τ 1 and τ 3 , indicating that it is indeed the motion of hydrogen such as occurring in ESIPT that governs the relaxation of the excited state.</p><p>Quantum calculation using DFT/TDDFT. Since ESIPT in systems like QPH is unexpected and rare as previously mentioned, we investigated the dynamics of ESIPT by DFT/TDDFT calculations. In order to enlist different QPH species involved in the reaction, we first define the optimized geometry of the singlet ground state (S 0 ) of the E tautomer as P E (Fig. 5(a)). This is the geometry where the HOMO, LUMO and LUMO+1 shown in the leftmost side of Fig. 6 constitutes a major MO for the S 0 , S 1 and S 2 states, respectively. Optimizing the first excited state at the Franck-Condon point P E yields P E-S1 (Figs 5(b) and 6), the state property of which differs markedly from that of the experimentally expected K tautomer, P K (Fig. 5(d)). This led us to suspect a missing link and search for other, more relevant local minima on the S 1 potential energy surface along the reaction coordinate. We eventually found another local minimum located between P E-S1 and P K , which is denoted P E-S2 (Fig. 5(c)) as it is associated with the S 2 state (see below).</p><p>Each geometry possesses distinctive structural and electronic properties as described below.</p><p>• The LUMO at P E-S1 is very similar to that at P E , indicating that the former is a relaxed conformation of S 1 after vertical excitation, hence the designation P E-S1 . The bond lengths of N 1 −H 1 and O • P K is the proton-transferred geometry (K form). Striking similarities between its LUMO and the LUMO+1 at P E and the LUMO at P E-S2 are apparent in Fig. 6.</p><!><p>is the key intermediate geometry between P E and P K , as its tautomeric structure is similar to the geometry at P E but its LUMO is similar to that at P K . Also, the LUMO at P E-S2 lies lower in energy than LUMO at P E-S1 but higher than that at P K , making it a more likely candidate for a geometry immediately preceding proton transfer.</p><p>Assigning the S 1 state at P E-S2 as the reaction intermediate leads us to two assumptions about the potential energy surface. First of all, because the electron density of the LUMO shifts from the quinoline ring at P E and P E-S1 to the phthalone ring at P E-S2 and P K , we may suspect the presence of an adiabatic surface crossing between the S 1 and S 2 excited states, which is also consistent with the similarity between the LUMO+1 at P E and the LUMO at P E-S2 (see also Figure S2 to observe apparent 'flip' in molecular orbitals). From the state property of each species, we deduce that the lowest singlet excited states of P E and P E-S1 are on a same adiabatic surface while those of P E-S2 and P K are on another adiabatic surface. In order to see the possibility of the nonadiabatic transition, we searched for a conical intersection of the S 1 and S 2 surfaces and found one at P E-S1/S2(CI) (Fig. 5(e)). Although the calculated value did not take the solvent effect into account, the transition is still shown to be energetically feasible when considering the excess excitation energy. Also the geometry at P E-S1/S2(CI) is quite similar to those at P E-S1 and P E-S2 (Fig. 5), which implies no additional barriers to the conical intersection.</p><p>Another assumption is that the transition state (TS) from E to K is expected via the P E-S2 geometry. By placing H 1 midway between N 1 and O 15 , we found what appears to be a transition state (P E/K(TS) in Fig. 5(e)) that has a low energy barrier of ~0.13 eV (Fig. 6). Intrinsic reaction coordinate (IRC) path has been determined starting from P E/K(TS) by the steepest descent method, which led to minima that correspond to P E-S2 and P K (Fig. S3). Therefore P E-S2 is deemed the doorway geometry for the proton transfer.</p><p>From the results above we construct a rudimentary potential energy surface diagram shown in Fig. 6. QPH photoexcited at the Franck-Condon point (P E ) will vibrate about the local minimum P E-S1 on the S 1 adiabatic surface, during which a nonadiabatic transition to the S 2 state occurs at P E-S1/S2(CI) , which leads to the ESIPT process toward the point P K via P E-S2 and P E/K(TS). We also note that we could not find a conical intersection between S 1 and S 0 of the K tautomer.</p><p>Natural bond orbital (NBO) charge analysis reveals that N 1 becomes electrostatically less negative (−0.520 to −0.488) and O 15 more negative (−0.617 to −0.656) in the S 1 state at P E-S2 relative to those of the S 1 state at P E (Fig. S4). Such a change in electrostatic charge of atoms involved in IMHB should be a preceding sign of ESIPT.</p><!><p>Molecules known for ESIPT typically display either a large Stokes'-shifted emission or dual emission 7 . Despite the fact that both characteristics are absent from the spectrum in Fig. 1, our experiment on deuterium substitution of H 1 proton verified the occurrence of ESIPT in QPH. Therefore we may conclude that after ESIPT, the K form of QPH undergoes a significant nonradiative decay. There are some notable examples for ESIPT that do not display tautomer emission. One is hypericin, for which it was speculated that either energy levels remain unchanged upon tautomerization or that tautomers are mixed in the ground state 24 . Another is 1-hydroxypyrene-2-carbaldehyde, for which intersystem crossing to a triplet state happens 8 . However, from aforementioned calculations and NMR results, we may rule out the possibility of the ground state tautomerization. For the latter possibility, we found the time profile of QPH insensitive to nitrogen purging (Fig. S5) and thus intersystem crossing to a triplet state seems unlikely.</p><p>From our experimental results for deuterium substitution and the lack of conical intersection between S 1 and S 0 of the K tautomer, we suspect that the vibrational mode related to IMHB is involved when the K tautomer returns to the ground state. It has been discussed in depth that the presence of hydrogen bond is associated with enhanced internal conversion back to the ground state and that the isotope effect may appear if the associated hydrogen is deuterated [25][26][27][28] . For ESIPT, some cases are known where enhanced internal conversion lowers photoluminescence quantum yield after tautomerization 6,29 . Another possibility is that ultrafast relaxation occurs via back proton transfer process 5 . Currently we cannot differentiate between the two possibilities and will leave it for future studies.</p><p>It is also interesting to note that the biexponential global fit after 30 ps brings about a τ 2 component of ~15 ps that is seemingly unaffected by deuteration. This observation, along with the complex nature of kinetic traces before 30 ps, suggests that there might be another relaxation pathway other than ESIPT.</p><p>Although IMHB is an important factor in facilitating excited state intramolecular hydrogen transfer, not every molecule with IMHB undergoes ESIPT. It has been well-established that ESIPT occurs readily in conjugated ketoenol system (O•••HO) or, similarly, N•••HN system and frequently reported, but ESIPT between heteroatoms are less known 30 . Especially, ESIPT from nitrogen to oxygen is a rare case, but not completely unheard of. Some molecules known for displaying N-to-O ESIPT include o-acetylaminoacetophenone 25 and 1-(acylamino)anthraquinone with appropriate functional groups 31 . More cases are known if dimerized molecules are considered 32 . However, what makes our case unique is that the proton donor of QPH is quinoline, which is better known for its photobasicity, meaning that it is more likely to accept a proton in the excited state 33 .</p><p>The driving force for ESIPT has been traditionally explained by the change in electron density at atoms involved in ESIPT. Excited state tautomerization is usually accompanied by electron redistribution after vertical excitation 34 , and the resulting change in the relative acidity and basicity is explained by the change in electron density at 'heavy atoms' (nitrogen or oxygen) 35 . Calculations of electrostatic charge at atoms related to photoacidity have been presented as supporting evidence in several studies of established systems 33,36 . Results of our NBO calculation may be interpreted in similar way. In the transient P E-S2 state, the increased electron density renders carbonyl oxygen O 15 photobasic, and the decreased electron density makes N 1 photoacidic. Because acidity and basicity are only relative terms, we suggest that if the relative strengths differ, ESIPT between photobases may occur.</p><p>Usually the magnitude of deuterium isotope effect is assessed by parameter k H /k D , whose value may lie anywhere between 1 and 50, depending on the shape of potential energy surface and the size of energy barrier 34,37 . Furthermore, the kinetic isotope effect may not appear at all when the system reaches the adiabatic limit. Generally, such lack of isotope effect is regarded as a sign of unsubstantial potential energy barrier, and happens in cases when the distance between heavy atoms involved in ESIPT is sufficiently short 5,29 . Since our system displays a clear isotope effect, the possibility of a barrierless potential energy surface may be excluded. Also the k H / k D value of 1.39 and 1.35 in our system is nearly identical to the square root of the reduced mass ratio between hydrogen and deuterium, suggesting the crucial role of H 1 vibration on the overall kinetics.</p><p>To summarize, we conducted ultrafast transient absorption spectroscopy of QPH and observed signs of ESIPT. This study is the first report to observe the ESIPT of QPH that involves a very rare case of proton transfer from nitrogen to oxygen. Also, this study reports the first case of ESIPT between two traditionally known photobasic moieties, quinoline and aromatic carbonyl 35 . Calculated results show that an S 1 -S 2 adiabatic surface crossing occurs before undergoing ESIPT.</p><!><p>Quinophthalone (Quinoline Yellow 2SF, ≥97%, #01354) was purchased from Sigma-Aldrich and used without further purification. All solutions were prepared using ACS grade cyclohexane (Sigma-Aldrich, #179191) with 100 μM concentration.</p><p>The d-QPH sample was prepared by sonicating the 1:2 volume mixture of QPH solution and D 2 O (Sigma-Aldrich, #151882) in the same vial. Then we waited for separation and took the upper layer of cyclohexane. We tested several experimental conditions and found that 15 minutes of sonication and 90 minutes of waiting for separation were enough to reproduce the results. The same procedure was adopted with HPLC grade water (JT Baker, #4218-03) to ensure that the change originates from the isotope effect. 1 H NMR spectrum of the solutions (Fig. S6) show that the majority of H 1 proton is deuterated by the protocol.</p><p>Steady-state UV/Vis absorption spectra and photoluminescence spectra were recorded using Lambda 25 (Perkin-Elmer) and QM-3/2004SE (PTI), respectively.</p><p>Femtosecond transient absorption spectroscopy was conducted using conventional pump-probe setup. The optical source was a regeneratively-amplified Ti:Sapphire laser (Spectra-Physics) with an average power of 0.65 mJ/pulse, pulse width of 130 fs FWHM, and repetition rate of 1 kHz at 800 nm output wavelength. Pump pulses were generated using a second harmonic generator (TP1A, Spectra-Physics), filtered down by neutral density filter and used at 1.5 mW. A continuum light, which was used as the probe pulse, was generated by focusing the 800 nm light using a UV-fused silica plano convex lens (CVI Melles Griot, f = 100 mm) onto a sapphire plate (WG31050, Thorlabs). Diameters of the pump and probe beams at the sample position were 390 μm and 295 μm, respectively. The IRF (~250 fs FWHM) was estimated by the cross-correlation between the pump with the probe. Solvent background subtraction and GVD-correction were done according to the known methods 38 . The time delay between the pump and the probe pulses was scanned using Daedal 404300XRMP delay stage (Parker). The pump pulses were modulated using an optical chopper (MC1000, Thorlabs) synchronized to the laser. The probe pulses of the signal were detected by photodiodes (2031, New Focus) after wavelength selection using a monochromator (250 IS/SM, Chromex, 600 grooves/mm grating blazed at 750 nm). Transient absorption signals were obtained using a lock-in amplifier (SR830, Stanford Research Systems). Temperature was controlled at 23 °C.</p><p>Calculations were executed using the DFT and TDDFT methods at B3LYP/6-31 G(d) level of theory. Structure optimization was performed using quadratic approximation with gradient convergence tolerance of 0.0001 Hartree/Bohr (default value) using General Atomic and Molecular Electronic Structure System (GAMESS) 39,40 and Gaussian 09 41 . Transition state and conical intersection were acquired using the default method in GAMESS. It must be noted that the branching plane update method for CI search requires TAMMD approximation and therefore was applied only on this value. It is known that it gives a better intermediate geometry during photophysical processes 42 . NBO charge analysis was performed with the NBO version 3.1 included in Gaussian 09 43 . Molecular structures and orbitals were visualized with GaussView 5.0.8 44 and wxMacMolPlt 45 .</p>

Scientific Reports - Nature