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Figure 1Open in figure viewerPowerPointMolecular structure (displacement ellipsoids set at the 50 % probability level) and 2D fingerprint plot (showing all contributions of intermolecular contacts) of 2,6-dibromophenol (1) in the crystal. Selected bond lengths [Å]: C1−O1 1.361(8), Br1−C2 1.883(7), Br2−C6 1.893(7). Intramolecular hydrogen bond: O1−H1 0.773 Å, H1⋅⋅⋅Br1 2.518 Å, C1−O1−H1 103.15°, O1−H1⋅⋅⋅Br1 132.44°.

2,6-Dibromophenol (1) crystallized from hexane at −30 °C in the orthorhombic crystal system, space group P212121 (Figure #slct202101723-fig-0001#1 and Table 1), which is the same as for all other known crystal structures of 2,6-dihalophenols. The Br−C bond lengths with 1.883(7) Å and 1.893(7) Å differ only slightly. The molecule is absolutely flat, with the O−H bond pointing to the Br1 atom (H1⋅⋅⋅Br1 2.518 Å), which might be due to the large polarizable bromine atom in the ortho position. The C1−O1−H1 angle is noticeably reduced to 103.15°. Figure #slct202101723-fig-0001#1, right, shows the 2D fingerprint diagram of 2,6-dibromophenol (1). Three main structural pattern can be identified, which are characteristic for the description of the intermolecular interactions. There are 1) the distinct spikes, i. e. the point on the Hirshfeld surface where di≈1.3 Å and de≈1.0 Å, 2) the blue-green area around di=de≈1.8 Å, which is traversed by 3) a dark red stripe. What is most striking about compound 1 is the dark red stripe in the fingerprint plot with the closest contacts at di=de≈1.8 Å, which roughly corresponds to the van der Waals radius of bromine and can be assigned to Br⋅⋅⋅Br interactions (18.2 %) (Figure #slct202101723-fig-0001#1). The closest distances that can be found are 3.568 Å (Br1⋅⋅⋅Br1) and 3.647 Å (Br1⋅⋅⋅Br2) (Figure #slct202101723-fig-0002#2). Halogen bonds have both a directional electrostatic and a dispersion component. A more detailed analysis of the halogen⋅⋅⋅halogen interaction pattern in compound 1 shows that they can be classified as electrostatically-driven highly directional type-II C−Br⋅⋅⋅Br−C interactions. θ1 and θ2, i. e. the two C−Hal⋅⋅⋅Hal angles, are diagnostic for the nature of the halogen bond and ideally have values of θ1≈180° and θ2≈90° in σ-hole type-II C−Hal⋅⋅⋅Hal−C interactions. The σ-hole is the result of an anisotropic charge density distribution around the C−Hal bond leading to a positive electrostatic potential at the outermost region along the extension of the C−Hal bond (charge depletion) and concomitantly to an equatorial belt of negative electrostatic potential (charge concentration) around the halogen atom. The θ1 (170.97° and 163.56°) and θ2 (116.13° and 104.08°) angles in our simplyfied halogenated model system 1 nicely reflect this kind of highly directional and attractive interaction mode (Figure #slct202101723-fig-0002#2). The comparison with the lighter homologues 2 and 3 clearly shows how this type of interaction is becoming less directional and less important with incrasing electronegativity of the halogen substituents (clearly recognizable by the lack of strong Hal⋅⋅⋅Hal interactions in the fingerprint diagrams, Figure #slct202101723-fig-0003#3), while the O−H⋅⋅⋅O hydrogen bond becomes all the more important in the same direction (visible at the sharp spikes, Figure #slct202101723-fig-0003#3). The geometrical parameters (θ1 and θ2) of the two shortest F⋅⋅⋅F contacts in 2,6-difluorophenol (2) are θ1=76.65° and θ2=77.93° (F⋅⋅⋅F 3.217 Å) and θ1=105.77° and θ2=110.88° (F⋅⋅⋅F 4.246 Å). This indicates that the F⋅⋅⋅F contacts in 2 (2.8 %) are more of a dispersion-like nature, since the θ angles meet the conditions for type-I C−Hal⋅⋅⋅Hal−C interactions (θ1≈θ2) quite well. In 2-bromo-6-chlorophenol (3), the dihalogen contacts are exclusively unsymmetrical type-I Br⋅⋅⋅Cl interactions (3.2 %), although a σ-hole-type C−Br⋅⋅⋅π contact (Br⋅⋅⋅C 3.362 Å, C−Br⋅⋅⋅C 177.06°) can also be found. The molecular electrostatic potentials (ESPs) of compounds 1–3 within the crystal packings give a nice impression of the anisotropic charge distribution around the C−Br bonds and of the isotropic negative electrostatic potential around the C−F bonds (see the Supporting Information). A characteristic packing feature of all three halogenated phenols is the offset parallel π-stacking arrangement, which is least pronounced in the 2,6-difluoro compound. The blue-green area in the 2D fingerprint plot of 2,6-dibromophenol (1) centered around di=de≈1.8 Å (Figure #slct202101723-fig-0001#1) is close to the van der Waals radius of carbon and represents the π⋅⋅⋅π interactions in the stacking arrangement. Our investigation of interplaying intermolecular interactions in halogenated phenols may contribute to a better understanding of the tunability of noncovalent interactions.

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Figure 2Open in figure viewerPowerPointHirshfeld surface of 2,6-dibromophenol (1) highlighting the O−H⋅⋅⋅O hydrogen bonds, the Br⋅⋅⋅Br interactions, and the offset parallel π-stacking in the crystal packing. Selected distances [Å] and angles [°]: H1⋅⋅⋅O1 2.314, O1⋅⋅⋅O1 2.813, O1−H1⋅⋅⋅O1 123.27, C2−Br1⋅⋅⋅Br1−C2 3.568 (θ1=170.97°, θ2=116.13°), C2−Br1⋅⋅⋅Br2−C6 3.647 (θ1=163.56°, θ2=104.08°). Symmetry transformations used to generate equivalent atoms: (i) −0.5 + x, 0.5 − y, 1 − z; (ii) −0.5 + x, 1.5 − y, 1 − z; (iii) −1 + x, 1 + y, z; (iv) −1 + x, y, z.

The spikes are typical for hydrogen bonds and belong to intermolecular O−H⋅⋅⋅O contacts between the hydroxyl groups (actually found in the crystal structure: H1⋅⋅⋅O1 2.314 Å, O1⋅⋅⋅O1 2.813 Å) (Figure #slct202101723-fig-0002#2). Although the distances are below the sum of the van der Waals radii, this hydrogen bond is more likely located on the weaker end of the strength scale according to Jeffrey's classification, with a more electrostatic rather than covalent contribution. The reasons may be attributed to the competition between intra- and intermolecular hydrogen bonding, which is also reflected by the small O−H⋅⋅⋅O angle of 123.27° for the intermolecular hydrogen bond. The influence of the fluorine substitution on the O−H⋅⋅⋅O hydrogen bond is considerable and causes sharp spikes in the 2D fingerprint plot, which are more pronounced here than in compound 1 (Figure #slct202101723-fig-0003#3, left). In the crystal structure of 2,6-difluorophenol (2), the O−H bond is slightly turned out of the ring plane by approximately 25°. The bond parameters of the O−H⋅⋅⋅O hydrogen bond in compound 2 (O−H 0.814 Å, H⋅⋅⋅O 2.002 Å, O⋅⋅⋅O 2.805 Å, O−H⋅⋅⋅O 168.77°) show a significant shortening of the H⋅⋅⋅O contact together with a strong expansion of the O−H⋅⋅⋅O angle in the direction of linearity, and are thus in a range that is also found in O−H⋅⋅⋅O hydrogen bonds of halogenated aliphatic alcohols. One explanation for the stronger O−H⋅⋅⋅O hydrogen bonding in 2 may be the increased acidity of the hydroxyl group due to fluorine substitution. This effect is supported by our DFT calculations on the M062X/6-311+G(d,p) level of theory, which gave insight into the mobility of the hydroxyl hydrogen atom in 2,6-dihalogenated phenols (for details, see the Supporting Information). The activation barrier for the exchange between the two intramolecular O−H⋅⋅⋅Hal hydrogen bonds was found to be 4.3 kcal mol−1 (1-TS≠) for the dibromo compound 1 and 3.3 kcal mol−1 (2-TS≠) for the difluoro derivative 2. This can be regarded as an indirect measure of the ability of the hydroxyl group to participate in hydrogen bonding and proves a higher flexibility of the O−H bond in compound 2. What is most striking about compound 1 is the dark red stripe in the fingerprint plot with the closest contacts at di=de≈1.8 Å, which roughly corresponds to the van der Waals radius of bromine and can be assigned to Br⋅⋅⋅Br interactions (18.2 %) (Figure #slct202101723-fig-0001#1). The closest distances that can be found are 3.568 Å (Br1⋅⋅⋅Br1) and 3.647 Å (Br1⋅⋅⋅Br2) (Figure #slct202101723-fig-0002#2). Halogen bonds have both a directional electrostatic and a dispersion component. A more detailed analysis of the halogen⋅⋅⋅halogen interaction pattern in compound 1 shows that they can be classified as electrostatically-driven highly directional type-II C−Br⋅⋅⋅Br−C interactions. θ1 and θ2, i. e. the two C−Hal⋅⋅⋅Hal angles, are diagnostic for the nature of the halogen bond and ideally have values of θ1≈180° and θ2≈90° in σ-hole type-II C−Hal⋅⋅⋅Hal−C interactions. The σ-hole is the result of an anisotropic charge density distribution around the C−Hal bond leading to a positive electrostatic potential at the outermost region along the extension of the C−Hal bond (charge depletion) and concomitantly to an equatorial belt of negative electrostatic potential (charge concentration) around the halogen atom. The θ1 (170.97° and 163.56°) and θ2 (116.13° and 104.08°) angles in our simplyfied halogenated model system 1 nicely reflect this kind of highly directional and attractive interaction mode (Figure #slct202101723-fig-0002#2). The comparison with the lighter homologues 2 and 3 clearly shows how this type of interaction is becoming less directional and less important with incrasing electronegativity of the halogen substituents (clearly recognizable by the lack of strong Hal⋅⋅⋅Hal interactions in the fingerprint diagrams, Figure #slct202101723-fig-0003#3), while the O−H⋅⋅⋅O hydrogen bond becomes all the more important in the same direction (visible at the sharp spikes, Figure #slct202101723-fig-0003#3). The geometrical parameters (θ1 and θ2) of the two shortest F⋅⋅⋅F contacts in 2,6-difluorophenol (2) are θ1=76.65° and θ2=77.93° (F⋅⋅⋅F 3.217 Å) and θ1=105.77° and θ2=110.88° (F⋅⋅⋅F 4.246 Å). This indicates that the F⋅⋅⋅F contacts in 2 (2.8 %) are more of a dispersion-like nature, since the θ angles meet the conditions for type-I C−Hal⋅⋅⋅Hal−C interactions (θ1≈θ2) quite well. In 2-bromo-6-chlorophenol (3), the dihalogen contacts are exclusively unsymmetrical type-I Br⋅⋅⋅Cl interactions (3.2 %), although a σ-hole-type C−Br⋅⋅⋅π contact (Br⋅⋅⋅C 3.362 Å, C−Br⋅⋅⋅C 177.06°) can also be found. The molecular electrostatic potentials (ESPs) of compounds 1–3 within the crystal packings give a nice impression of the anisotropic charge distribution around the C−Br bonds and of the isotropic negative electrostatic potential around the C−F bonds (see the Supporting Information).

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Figure 3Open in figure viewerPowerPoint2D fingerprint plots (showing all contributions of intermolecular contacts) of 2,6-difluorophenol (2)54 and 2-bromo-6-chlorophenol (3).56

The spikes are typical for hydrogen bonds and belong to intermolecular O−H⋅⋅⋅O contacts between the hydroxyl groups (actually found in the crystal structure: H1⋅⋅⋅O1 2.314 Å, O1⋅⋅⋅O1 2.813 Å) (Figure #slct202101723-fig-0002#2). Although the distances are below the sum of the van der Waals radii, this hydrogen bond is more likely located on the weaker end of the strength scale according to Jeffrey's classification, with a more electrostatic rather than covalent contribution. The reasons may be attributed to the competition between intra- and intermolecular hydrogen bonding, which is also reflected by the small O−H⋅⋅⋅O angle of 123.27° for the intermolecular hydrogen bond. The influence of the fluorine substitution on the O−H⋅⋅⋅O hydrogen bond is considerable and causes sharp spikes in the 2D fingerprint plot, which are more pronounced here than in compound 1 (Figure #slct202101723-fig-0003#3, left). In the crystal structure of 2,6-difluorophenol (2), the O−H bond is slightly turned out of the ring plane by approximately 25°. The bond parameters of the O−H⋅⋅⋅O hydrogen bond in compound 2 (O−H 0.814 Å, H⋅⋅⋅O 2.002 Å, O⋅⋅⋅O 2.805 Å, O−H⋅⋅⋅O 168.77°) show a significant shortening of the H⋅⋅⋅O contact together with a strong expansion of the O−H⋅⋅⋅O angle in the direction of linearity, and are thus in a range that is also found in O−H⋅⋅⋅O hydrogen bonds of halogenated aliphatic alcohols. One explanation for the stronger O−H⋅⋅⋅O hydrogen bonding in 2 may be the increased acidity of the hydroxyl group due to fluorine substitution. This effect is supported by our DFT calculations on the M062X/6-311+G(d,p) level of theory, which gave insight into the mobility of the hydroxyl hydrogen atom in 2,6-dihalogenated phenols (for details, see the Supporting Information). The activation barrier for the exchange between the two intramolecular O−H⋅⋅⋅Hal hydrogen bonds was found to be 4.3 kcal mol−1 (1-TS≠) for the dibromo compound 1 and 3.3 kcal mol−1 (2-TS≠) for the difluoro derivative 2. This can be regarded as an indirect measure of the ability of the hydroxyl group to participate in hydrogen bonding and proves a higher flexibility of the O−H bond in compound 2. What is most striking about compound 1 is the dark red stripe in the fingerprint plot with the closest contacts at di=de≈1.8 Å, which roughly corresponds to the van der Waals radius of bromine and can be assigned to Br⋅⋅⋅Br interactions (18.2 %) (Figure #slct202101723-fig-0001#1). The closest distances that can be found are 3.568 Å (Br1⋅⋅⋅Br1) and 3.647 Å (Br1⋅⋅⋅Br2) (Figure #slct202101723-fig-0002#2). Halogen bonds have both a directional electrostatic and a dispersion component. A more detailed analysis of the halogen⋅⋅⋅halogen interaction pattern in compound 1 shows that they can be classified as electrostatically-driven highly directional type-II C−Br⋅⋅⋅Br−C interactions. θ1 and θ2, i. e. the two C−Hal⋅⋅⋅Hal angles, are diagnostic for the nature of the halogen bond and ideally have values of θ1≈180° and θ2≈90° in σ-hole type-II C−Hal⋅⋅⋅Hal−C interactions. The σ-hole is the result of an anisotropic charge density distribution around the C−Hal bond leading to a positive electrostatic potential at the outermost region along the extension of the C−Hal bond (charge depletion) and concomitantly to an equatorial belt of negative electrostatic potential (charge concentration) around the halogen atom. The θ1 (170.97° and 163.56°) and θ2 (116.13° and 104.08°) angles in our simplyfied halogenated model system 1 nicely reflect this kind of highly directional and attractive interaction mode (Figure #slct202101723-fig-0002#2). The comparison with the lighter homologues 2 and 3 clearly shows how this type of interaction is becoming less directional and less important with incrasing electronegativity of the halogen substituents (clearly recognizable by the lack of strong Hal⋅⋅⋅Hal interactions in the fingerprint diagrams, Figure #slct202101723-fig-0003#3), while the O−H⋅⋅⋅O hydrogen bond becomes all the more important in the same direction (visible at the sharp spikes, Figure #slct202101723-fig-0003#3). The geometrical parameters (θ1 and θ2) of the two shortest F⋅⋅⋅F contacts in 2,6-difluorophenol (2) are θ1=76.65° and θ2=77.93° (F⋅⋅⋅F 3.217 Å) and θ1=105.77° and θ2=110.88° (F⋅⋅⋅F 4.246 Å). This indicates that the F⋅⋅⋅F contacts in 2 (2.8 %) are more of a dispersion-like nature, since the θ angles meet the conditions for type-I C−Hal⋅⋅⋅Hal−C interactions (θ1≈θ2) quite well. In 2-bromo-6-chlorophenol (3), the dihalogen contacts are exclusively unsymmetrical type-I Br⋅⋅⋅Cl interactions (3.2 %), although a σ-hole-type C−Br⋅⋅⋅π contact (Br⋅⋅⋅C 3.362 Å, C−Br⋅⋅⋅C 177.06°) can also be found. The molecular electrostatic potentials (ESPs) of compounds 1–3 within the crystal packings give a nice impression of the anisotropic charge distribution around the C−Br bonds and of the isotropic negative electrostatic potential around the C−F bonds (see the Supporting Information).

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Figure 1Open in figure viewerPowerPointXRD patterns of (A) wet-kneaded (WK), co-precipitated (CP), and natural talc (NT) samples and (B) N2 adsorption isotherms of the samples. Adsorption and desorption branches are indicated by full and open symbols, respectively.

Compositional and textural characteristics of the three MgO−SiO2 materials, used in the present study, are given in Table 1. The Si to Mg weight ratio of the catalysts is close to the 1.54 Si-to-Mg weight ratio in the chemical formula of natural talc (Mg3Si4O10(OH)2). Comparison of the bulk and surface compositions indicate a slight surface enrichment of Mg for the natural talc and for the co-precipitated material. This is not surprising as the surface of most magnesium silicates tends to contain more Mg than the bulk. The exception is the wet-kneaded sample, which is somewhat Mg-deficient both at the surface and in the bulk with respect to the natural talc. The SSA of the NT sample is significantly lower than that of the synthesized samples. According to the XRD and nitrogen adsorption measurements summarized in Figures #cctc202001007-fig-0001#1A and B, respectively, this material is non-porous and is well crystallized. Figure #cctc202001007-fig-0001#1B shows that the CP and WK samples have micro and mesoporosity.

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Figure 2Open in figure viewerPowerPointSolid state NMR spectra natural talc (NT), wet-kneaded (WK) and co-precipitated (CP) samples. (A) 1H single pulse MAS NMR spectra, (B) 29Si{1H} cross-polarization MAS NMR spectra, and (C) 1H-29Si FSLG HETCOR representation of the results.

Due to the low level of crystallization XRD could not provide adequate information about the bulk structure of the samples. Therefore, 1H MAS NMR, 29Si CP MAS NMR, and 1H-29Si FSLG HETCOR NMR spectra were recorded to evaluate the environment of the Si atoms and the possible formation of magnesium silicate phases. Figure #cctc202001007-fig-0002#2A shows that all the hydroxyl groups have identical environment in the crystal structure of the NT sample resulting in a single 1H MAS NMR signal at 0.3 ppm. The relatively low chemical shift suggests that the hydroxyl groups are isolated from each other. The CP sample contains less hydrogen atoms than the NT sample. Two different signals could be distinguished. The signal at 0.5 ppm was assigned to “talc-like” and terminal hydroxyl groups. The broader signal at 4.1 ppm belongs most probably to H-bonded hydroxyl groups and strongly absorbed/structural water molecules. The WK sample contains even less hydrogen atoms. The signals are similar to those obtained for the CP sample, but their intensity is much lower and the broad signal has a chemical shift value of about 3.3 ppm indicating less pronounced H-bonding. The 29Si CP MAS NMR chemical shift depends on the environment of the silicon atoms. The NT sample shows a single well defined signal at −98 ppm (Figure #cctc202001007-fig-0002#2B). Pure SiO2 has a chemical shift around −110 ppm which decreases by replacing the neighboring atoms of the SiO4 tetrahedra by H or Mg. In order to get spectra, which are easier to interpret direct polarization 29Si spectra were recorded with relatively short relaxation delay (5 sec) to filter out the signals of pure crystalline or semi-crystalline SiO2 phases, having long T1 relaxation time. Nevertheless, the spectrum of the WK sample remained dominated by Q3 and Q4 signals of the SiO2 phase. Signal belonging to Si(OSi)3OH (Q3) phase was clearly shown by the 1H-29Si FSLG HETCOR spectrum (Figure #cctc202001007-fig-0002#2C). No Q4 phase is detectable in the spectrum of CP sample (Figure #cctc202001007-fig-0002#2B). According to the correlation spectra (Figure #cctc202001007-fig-0002#2C), signals between −70 and −99 ppm originate from different magnesium hydroxy silicate species.

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Figure 3Open in figure viewerPowerPointFTIR spectra of adsorbed (A) CDCl3 and (B) pyridine. The pellets were pre-treated in vacuum at 450 °C for 1 h. The spectra were recorded at room temperature (A) in the presence of CDCl3 at about 933 Pa pressure, and (B) after adsorption of pyridine at 666 Pa pressure at 200 °C and evacuation at the same temperature for 30 min.

The order of basicity of the neat MgO−SiO2 samples is also supported by the CDCl3 adsorption measurements shown in Figure #cctc202001007-fig-0003#3A. The presented spectra do not show bands at 2139 and 2086 cm−1, which Angelici et al. assigned to CDCl3, bound to strong basic sites. The spectra in Figure #cctc202001007-fig-0003#3A are more similar to those reported by Paukshtis et al. in their pioneering work. The νCD frequency of free CDCl3 molecules is at 2260 cm−1. Interaction of CDCl3 molecules and basic surface sites shifts the νCD stretching band to lower wavenumbers. The band displacement reflects the strength of interaction and, thereby, the base strength of the adsorbing surface sites. The integrated band area gives an approximate measure of the amount of adsorption sites. Figure #cctc202001007-fig-0003#3A shows that the WK sample contains the highest number of basic sites, whereas the NT sample of low surface area has only small amount of similar sites. This is in accordance with the results of the CO2-TPD study. Unlike the spectra of the NT and CP samples the FTIR spectrum of the WK sample exhibits two νCD component peaks, suggesting the presence of two kinds of adsorption sites having different base strengths (Figure #cctc202001007-fig-0003#3A). The broad band at 2234 cm−1 indicates the presence of sites having stronger basicity than the sorption sites of the other samples. The adsorption of CO2 on these sites can be responsible for the pronounced tailing of the CO2-TPD peak at its high-temperature side (Figure S4C). Figure #cctc202001007-fig-0003#3A shows that addition of metal oxide significantly decreased the relative and absolute amounts of strong base adsorption sites. While the total peak area increased only slightly, the area ratio of the peaks at about 2230 and 2260 cm−1 changed significantly. The ratio that was 4.94 for the WK sample dropped to 0.42, 0.30, and 0.21 for the In/WK, Zn/WK and Ga/WK samples, respectively. It is clear that the additive decreased the basicity of the WK catalysts. Vibrations of the aromatic ring of adsorbed Py molecules can be used to distinguish Py, coordinatively bonded to Lewis acid sites or to Brønsted acid sites in protonated form, (PyH+) (Figure #cctc202001007-fig-0003#3B). The band, diagnostic for PyH+ should appear at about 1545 cm−1. The MgO−SiO2 catalysts did not have Brønsted acid sites, strong enough to protonate Py. The infrared adsorption band of pyridine bound to Lewis acid sites is around 1445 cm−1. Comparing the spectra of Py bound to neat MgO−SiO2 samples after identical sample treatments, it can be concluded that the NT sample does not bind any Py under the experimental conditions used, whereas the WK sample can bind and retain slightly more Py than the CP sample (Figure S4A). Figure #cctc202001007-fig-0003#3B shows that addition of only 1 wt.% dopant significantly increases the Py uptake of the WK sample and also increases its Py bonding strength. Based on the thermal stability of the Py species giving the band at 1445 cm−1, the following order of acidity can be established: In/WK >Zn/WK >Ga/WK (Figure #cctc202001007-fig-0003#3B). The spectra of the entire series of experiments and the integrated absorbance values of selected peaks are summarized in Figure S6 and Table S2. The Nicolet Impact Type 400 FT-IR spectrometer was used for the determination of the adsorbed Py and CDCl3 molecules. All of the spectra were recorded at room temperature either with or without adsorptive in the cell. The difference spectra were obtained from the difference between the spectrum of a wafer containing CDCl3 or Py and the spectrum of a wafer without adsorptive. Spectra were recorded as an average of 64 scans using a resolution of 2 cm−1. (For experimental details confer the legends of Figure #cctc202001007-fig-0003#3and Figure S6.)

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Figure 4Open in figure viewerPowerPointFormation of crotyl alcohol in the ETB process.

The initiation step of the ETB process is the dehydrogenation of ethanol to acetaldehyde. It is generally believed that the hydrogen formed during the dehydrogenation of ethanol hydrogenates the crotonaldehyde, formed from the acetaldehyde by aldol coupling, to get the crotyl alcohol intermediate of BD formation. Our results (vide infra) supports the idea that the formation of the unsaturated alcohol takes place predominantly by hydrogen transfer between ethanol and crotonaldehyde according to the Meerwein-Ponndorf-Verley (MPV) mechanism to give acetaldehyde side product. Regarding the ETB reaction the MPV process is favourable compared to the direct hydrogenation by H2 because it is highly selective in the reduction of the carbonyl group and lives the double bound untouched. Moreover, the process generates acetaldehyde from the reducing reactant ethanol, which aldehyde can then participate then in the aldol coupling reaction step of the ETB process. The heterogeneous MPV process can take place either on Lewis acidic or basic sites. Niiyama et al. found that over MgO−SiO2 catalysts the basic sites are responsible for the MPV activity of the ethanol. Ordomsky et al. compared the activity of ZnO2/SiO2 and MgO/SiO2 catalysts in acetaldehyde condensation and found that over ZnO2 containing catalyst the initial acetaldehyde conversion is somewhat higher. Over MgO−SiO2 catalysts the aldol condensation step is considered to take place on an acid-base pair sites by concerted mechanism. Nevertheless, the catalyst of the one-step Lebedev-process must have also hydrogenation-dehydrogenation activity (Figure #cctc202001007-fig-0004#4).

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Figure 5Open in figure viewerPowerPointConversion of ethanol over (A) natural talc, (B) co-precipitated, and (C) wet-kneaded MgO−SiO2 catalysts as function of reaction temperature. The WHSV was 0.5 gethanol gcat−1 h−1 at a total gas flow rate of 30 cm3 min−1. The partial pressure of ethanol was about 15 kPa in He. The conversion curve is labelled by letter C. Selectivity curves are given for butadiene (BD), acetaldehyde (AA), ethylene (EE), diethyl ether (DEE), butanol (BOL) and butanes (BUE).

Figure #cctc202001007-fig-0005#5shows the catalytic performance of neat MgO−SiO2 preparations in the ETB transformation. As function of temperature the conversion over the CP and WK catalysts was quite similar. The NT catalyst, having much lower surface than latter catalysts, was much less active. Over each of the catalysts the main reaction products were diethyl ether at lower temperatures, and ethylene at higher temperatures. This temperature dependence is understandable regarding that the bimolecular dehydration of ethanol is exothermic, whereas the monomolecular reaction is endothermic. The best performing ETB catalyst was the WK sample having BD selectivity around 30 % in almost the whole examined temperature region. The data in Tables 3, S1, and S2 show that this catalyst has the strongest basic properties and that the number and strength of Lewis acid sites on it are also higher than those of the other two samples. The favourable catalytic activity of the WK sample is most probably due to cooperation of relatively strong basic and acidic sites on its surface. Figures #cctc202001007-fig-0005#5A and B show that especially at low temperatures the acetaldehyde was the main reaction product over the NT and CP catalysts, poorly performing in the ETB reaction. The high acetaldehyde selectivity is associated with a low yield of C4 products. This suggests that over these two catalysts the aldehyde coupling catalytic function is weak. Besides generating BD, the WK catalyst also initiates formation of butanol and butenes in significant amounts, confirming that this catalyst is highly active in aldol coupling and dehydration reactions. This activity can be attributed to the strong basicity of this catalyst (Table 3).

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Figure 6Open in figure viewerPowerPointEffect of 1 wt.% (A) Ga2O3, (B) In2O3, and (C) ZnO promoter on the conversion of ethanol to butadiene. The WHSV was 0.5 gethanol gcat−1 h−1 at a total gas flow rate of 30 cm3 min−1. The partial pressure of ethanol was about 15 kPa in He. See the Figure 5. for the legends.

Interestingly, butanol was a side product that appeared in clearly detectable amount (Figures #cctc202001007-fig-0006#5–#cctc202001007-fig-0007#7) according to Scalbert et al. the crotyl alcohol can be hydrogenated to butanol or dehydrated to BD depending on the reaction conditions and the properties of the used catalyst. Figure #cctc202001007-fig-0006#6shows that the conversion of ethanol and the selectivity of BD formation were significantly increased by all three applied metal oxide additives. Because the WK catalyst showed the best activity in the ETB reaction the promoting effect of Ga-, In-, and Zn-oxide was investigated using this material as support. In general, the In and Zn promoters suppressed dehydration reactions and increased both ethanol conversion and BD selectivity (Figure 6). The Ga changed only the conversion, but the product selectivities were very similar to those obtained using the non-promoted WK sample. The positive effect of In dopant is significant at temperatures 250–350 °C. The Zn/WK performs about 50 % BD selectivity in the 250–375 °C temperature range. At higher temperatures the full analysis was difficult because a number of different C5−C8 dienes, trienes, and aromatics were formed.

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Figure 7Open in figure viewerPowerPointEffect of 1 wt.% ZnO additive on the activity and selectivity of (A) NT and (B) CP samples in the ETB reaction. The WHSV was 0.5 gethanol⋅gcat−1 h−1 at a total gas flow rate of 30 cm3 min−1, The partial pressure of ethanol was about 15 kPa in He. Section C shows the effect of WHSV on the product yield and conversion over 1 wt.% ZnO/WK sample at 300 °C, and ethanol partial pressure of about 15 kPa. The total flow rate was varied in the 5–120 cm3 min−1 range. See the Figure 5. for the legends.

Interestingly, butanol was a side product that appeared in clearly detectable amount (Figures #cctc202001007-fig-0006#5–#cctc202001007-fig-0007#7) according to Scalbert et al. the crotyl alcohol can be hydrogenated to butanol or dehydrated to BD depending on the reaction conditions and the properties of the used catalyst. The increasing aldehyde concentration as function of space time (Figure #cctc202001007-fig-0007#7C) must be paralleled by an increasing surface concentration of the aldehyde activated for aldol coupling, most probably in the form of surface enolate. The rate of aldol coupling increases with increasing space time but it remains the rate-controlling process step (Figure #cctc202001007-fig-0007#7C). The steady-state product composition varies as the concentration of the surface species changes with the conversion. Figure #cctc202001007-fig-0010#10shows the product distribution over CP supported metal oxide catalysts at conversion levels of 10 to 50 %. The results show that higher C4 and BD selectivities were achieved in all cases at higher conversions, i. e., at longer space is times.

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Figure 8Open in figure viewerPowerPointCatalytic conversion of crotyl alcohol over (A) NT, (B) CP, and (C) WK catalysts. The WHSV was 0.125 galcohol⋅gcat−1 h−1 at a total gas flow rate of 30 cm3 min−1. The partial pressure of crotyl alcohol was about 4 kPa in He. See the Fig.5. for the legends. CA=crotonaldehyde and BAL=butanal.

In order to better understand the formation and transformation processes of C4 intermediates, we studied the reaction of crotyl alcohol over unpromoted MgO−SiO2 catalysts (Figure #cctc202001007-fig-0008#8). Full conversion was reached at around 325 °C. Over each catalyst the main reaction products were BD, formed by crotyl alcohol dehydration, and butanol. Interestingly, crotonaldehyde was also obtained. The highest amount of crotonaldehyde was obtained over the NT catalyst probably because this catalyst was hardly active in its further conversion. It cannot be excluded that butanol formation could happen by hydrogenation of crotyl alcohol consuming the hydrogen evolved in aromatization side process. GC-MS measurements showed traces of C8 aromatics, such as, xylenes and styrene, were formed in low-temperature reactions. The results, obtained studying the crotonaldehyde conversion, did not support this notion. In the reaction over neat MgO−SiO2 preparations formation of polyolefins and aromatics poisoned the catalysts very quickly. Addition of hydrogen to the reactant did not stop rapid poisoning. It was observed that butanol and crotonaldehyde were obtained together with similar selectivites. Results seem to support that disproportionation of crotyl alcohol (Figure #cctc202001007-fig-0009#9) could have been responsible for the butanol formation during the ETB process and not a direct crotonaldehyde hydrogenation.

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Figure 9Open in figure viewerPowerPointDisproportionation reaction of crotyl alcohol.

In order to better understand the formation and transformation processes of C4 intermediates, we studied the reaction of crotyl alcohol over unpromoted MgO−SiO2 catalysts (Figure #cctc202001007-fig-0008#8). Full conversion was reached at around 325 °C. Over each catalyst the main reaction products were BD, formed by crotyl alcohol dehydration, and butanol. Interestingly, crotonaldehyde was also obtained. The highest amount of crotonaldehyde was obtained over the NT catalyst probably because this catalyst was hardly active in its further conversion. It cannot be excluded that butanol formation could happen by hydrogenation of crotyl alcohol consuming the hydrogen evolved in aromatization side process. GC-MS measurements showed traces of C8 aromatics, such as, xylenes and styrene, were formed in low-temperature reactions. The results, obtained studying the crotonaldehyde conversion, did not support this notion. In the reaction over neat MgO−SiO2 preparations formation of polyolefins and aromatics poisoned the catalysts very quickly. Addition of hydrogen to the reactant did not stop rapid poisoning. It was observed that butanol and crotonaldehyde were obtained together with similar selectivites. Results seem to support that disproportionation of crotyl alcohol (Figure #cctc202001007-fig-0009#9) could have been responsible for the butanol formation during the ETB process and not a direct crotonaldehyde hydrogenation.

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Figure 10Open in figure viewerPowerPointEffect of the ethanol conversion on the distribution of the reaction products over doped CP samples, doped by 1 wt.% Zn, In, or Ga-oxide doped. The reaction temperature was 350 °C, the WHSV was varied in the 0.2–1.0 galcohol gcat−1 h−1 range. The partial pressure of ethanol in He was about 8 kPa. Over ZnO/CP the low conversion levels were achieved by diluting of 0.50 g catalyst with 0.50 g inert SiC.

The rate of aldol coupling increases with increasing space time but it remains the rate-controlling process step (Figure #cctc202001007-fig-0007#7C). The steady-state product composition varies as the concentration of the surface species changes with the conversion. Figure #cctc202001007-fig-0010#10shows the product distribution over CP supported metal oxide catalysts at conversion levels of 10 to 50 %. The results show that higher C4 and BD selectivities were achieved in all cases at higher conversions, i. e., at longer space is times. In general the applied metal additives were found to increase the activity and modify selectivity (Figure #cctc202001007-fig-0010#10). The dopant increased not only the number active sites but also the rate constants of the different reactions. As a consequence the concentration of surface intermediates can increase and the relative amounts of the surface intermediates of different products can change. The composition of the product mixture changes accordingly. We needed each rate and adsorption equilibrium constant (k and K) and the temperature dependence of these constants to give full description of the complex network and give the activation energy (Ea) of each reaction. Wang et al. made an attempt to determine the activation energy of the ethanol conversion and the formation of the main products over MgO−SiO2 and Zn/MgO−SiO2 catalysts. The reported Arrhenius plots suggest that all the reactions were handled as processes of zero order kinetics. In this case only the apparent rate constants (kapp) are equal with the initial rate of the reaction that can be experimentally determined. Anyhow, the obtained kapp includes the intrinsic k values of all the processes that contribute to forming and consuming the surface intermediate of a transformation and determines its concentration. Obviously, no strong mechanistic conclusions can be established on the values of the apparent activation energies (Eapp), derived from such Arrheniius plots. Getting deeper knowledge of complex reaction networks like the ETB reaction requires theoretical and microkinetic analysis.

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Scheme 1Open in figure viewerPowerPointSynthetic strategy for the preparation of a library of substituted 3-hydroxyisoquinolines and structure and photophysical properties of compound ISO-1.

Control larvae as well as those developed in Artificial Sea Water-HEPES (ASWH) plus 0.02 % DMSO displayed the typical larval morphology. They were characterized by an elongated trunk with two pigmented sensory organs, clearly visible in the sensory vesicle, and a straight long tail (Figure #cbic202100058-fig-0001#1A). ISO-1 concentration significantly affected Ciona intestinalis development in terms of number of normal developed individuals (ANOVA: F=351.72; P=<0.001), number of mildly affected individuals (ANOVA: F=57.64; P=<0.001), and number of severely damaged individuals (ANOVA: F=641.56; P=<0.001). Larvae exposed to the lowest tested concentration of ISO-1 (1 μM; Figure #cbic202100058-fig-0001#1B) were similar to the control ones and the incidence of adverse effects (either mildly or severely affected larvae) was not significantly different from controls (Tukey's post hoc test: P>0.05). Exposure to 5 μM ISO-1 caused a statistically significant increase of mild malformations (Tukey's post hoc test: P<0.001; Figure #cbic202100058-fig-0002#2): almost 50 % of larvae showed short and bent tail, round trunk with a small sensory vesicle with close pigmented cells (Figure #cbic202100058-fig-0001#1C). Exposure to higher concentrations caused a significant increase of severe malformations (Tukey's post hoc test: P<0.001) characterized by a curled tail, round trunk, small sensory vesicle with no pigmented organs (Figure #cbic202100058-fig-0001#1D).

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Scheme 2Open in figure viewerPowerPointGram-scale synthesis of ISO-1.

Control larvae as well as those developed in Artificial Sea Water-HEPES (ASWH) plus 0.02 % DMSO displayed the typical larval morphology. They were characterized by an elongated trunk with two pigmented sensory organs, clearly visible in the sensory vesicle, and a straight long tail (Figure #cbic202100058-fig-0001#1A). ISO-1 concentration significantly affected Ciona intestinalis development in terms of number of normal developed individuals (ANOVA: F=351.72; P=<0.001), number of mildly affected individuals (ANOVA: F=57.64; P=<0.001), and number of severely damaged individuals (ANOVA: F=641.56; P=<0.001). Larvae exposed to the lowest tested concentration of ISO-1 (1 μM; Figure #cbic202100058-fig-0001#1B) were similar to the control ones and the incidence of adverse effects (either mildly or severely affected larvae) was not significantly different from controls (Tukey's post hoc test: P>0.05). Exposure to 5 μM ISO-1 caused a statistically significant increase of mild malformations (Tukey's post hoc test: P<0.001; Figure #cbic202100058-fig-0002#2): almost 50 % of larvae showed short and bent tail, round trunk with a small sensory vesicle with close pigmented cells (Figure #cbic202100058-fig-0001#1C). These kinds of malformations have been reported after exposure to different chemicals and are considered to be due to toxic effects caused by a pleiotropic action of the molecule. Twenty per cent of larvae treated with 10 μM ISO-1 showed severe malformations. This percentage gradually increased with ISO-1 doses reaching 100 % in larvae exposed to 25 μM ISO-1 (Figure #cbic202100058-fig-0002#2). Embryos exposed to 50 μM ISO-1 died at early developmental stages (data not showed).

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Figure 1Open in figure viewerPowerPointPhenotypes of larvae developed from embryos exposed to different concentrations of ISO-1. A) Control larvae exposed to DMSO. B) Larvae exposed to 1 μM ISO-1 showing a normal phenotype. C) Larvae exposed to 5 μM ISO-1. D) Larvae exposed to 10 μM ISO-1. E) Larvae exposed to 20 μM ISO-1. F) Larvae exposed to 25 μM ISO-1.

Performing Probit analysis, we determined ISO-1 toxicological features during Ciona intestinalis embryogenesis (Figure #cbic202100058-fig-0003#3). The concentration of ISO-1 that caused adverse effects in 50 % of exposed sample (median effective concentration, EC50) was 5.69 μM. The median lethal concentration (LC50) predicted by the analysis was 30.19 μM.

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Figure 2Open in figure viewerPowerPointGraph showing the incidence of different phenotypes (normal, mildly, severely affected) in larvae developed from embryos treated with different concentrations of ISO-1. Mean values of three replicates and standard errors are indicated. Legend of symbols: *= differences from control, °=different from 1 μM; +=different from 5 μM; #=different from 10 μM; §=different from 20 μM. The repetition of each symbol indicates the level of significance according to ANOVA significance codes: p<0.001 ***; p<0.01 **; p<0.05 *.

Hatched larvae developed in ASWH were exposed to different concentrations of ISO-1 for 1 hour. After this time, a fluorescence signal could be observed using a microscope equipped with a UV lamp and a DAPI filter (band pass 352 to 477 nm). The intensity of the signal appeared proportional to the tested concentrations and was detectable only in larvae exposed to concentrations higher than 1 μM. At high magnification, fluorescent signal was clearly localized in the nuclei of the cells (Figure #cbic202100058-fig-0004#4).

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Figure 3Open in figure viewerPowerPointEC50 and LC50 predicted values calculated with Probit analysis.

Exposure to ISO-1 interfered with larval ability to swim. After 1 hour, 90 % of larvae exposed to 1 μM ISO-1 could swim, a percentage comparable to that of larvae exposed to DMSO alone. On the contrary, only 40 % of larvae exposed to 5 μM ISO-1 showed a normal swimming behaviour (Figure #cbic202100058-fig-0005#5). Concentrations of ISO-1 higher than 5 μM blocked tail movements resulting in an increasing number of larvae that appeared resting on the bottom of Petri dishes (Figure #cbic202100058-fig-0005#5).

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Figure 4Open in figure viewerPowerPointA living larva exposed to 1 μM ISO for 15 minutes. A) Larva observed at the light microscope. B) The same larva of A observed with a UV light and a DAPI filter. The fluorescence signal is concentrated in the trunk. C) Higher magnification of the tail of B shows the signal in the nuclei.

These effects were partially reversible. In fact, after two short rinses in ASWH, all larvae exposed to 5 μM ISO-1 were able to swim again (Table 2) even if they maintained a faint fluorescence visible mainly in the trunk, where the cells are smaller and densely packed (Figure #cbic202100058-fig-0006#6).

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Figure 5Open in figure viewerPowerPointGraph showing the percentage of swimming and resting larvae after an exposure of 15 minutes to different concentrations of ISO-1.

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Figure 6Open in figure viewerPowerPointLarvae rinsed in ASWH after exposure to ISO-1 observed at light (A, C, E) and fluorescent microscope (B, D, F). A, B) Larvae exposed to 1 μM ISO-1, the fluorescent signal disappeared completely. C, D) A faint signal was still present in larvae exposed to 5 μM ISO-1. E, F) The fluorescent signal persisted in larvae exposed to 10 μM ISO-1.

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Scheme 1Open in figure viewerPowerPointPreparation of cobalt oxide electrocatalysts by pulsed laser fragmentation in water.

To confirm the oxidation process and obtain more accurate information on the crystal structure, high-resolution XRD patterns were measured by using synchrotron radiation. As starting models for structure refinements, the crystal structures given in a previous study were used. The results from the Rietveld analysis are listed in Table S1. The as-prepared CW-Co3O4 sample was phase pure with the standard spinel structure, whereas a mixture of Co3O4 (5 wt %) and CoO (95 wt %) phases was observed in the ethanol-reduced sample, confirming the reduction effect from the ethanol treatment (Figure #cssc201903186-fig-0001#1 a,b). It should be noted that laboratory XRD using CuKα1/2 radiation is not able to detect such a low concentration of Co3O4 (5 wt % or 1.61 mol %). Cobalt-containing samples are excited by the Cu radiation, which causes fluorescence radiation. This also increases the noise of the measured data and is one reason that cobalt phases of low content cannot be seen clearly. After the PLFL process, similar XRD patterns were observed on Co3O4-L compared with pristine Co3O4, apart from a small amount of CoO generated owing to laser radiation (Figure #cssc201903186-fig-0001#1 c). The formation of CoO was also observed when treating commercial CoFe2O4 with the same PLFL process, with thermal decomposition proposed as the reason. For CoO-L, substantial oxidation had occurred in the sample, with only 5 wt % of the CoO phase remaining (Figure #cssc201903186-fig-0001#1 d). More importantly, the oxidized phase was determined by structure refinement to be Co2+0.9Co3+2O2−4−x, which possesses 10 % fewer Co2+ cations in the tetrahedral sites of the perfect spinel structure, suggesting the formation of Co2+ defects as well as oxygen vacancies. It has been reported that instantaneous temperature elevation upon picosecond laser irradiation can initiate the formation of vacancy defects, including metal and oxygen vacancies, which become trapped in the crystal with subsequent quenching in water.

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Figure 1Open in figure viewerPowerPointRietveld refinement analysis of synchrotron diffraction patterns of CW-templated Co3O4 (a), Co3O4-L (b), CoO (c), and CoO-L (d). The reflections marked by the circles and triangles are indexed to spinel Co3O4 and cubic CoO, respectively.

The particle size and morphology were then examined by transmission and scanning electron microscopy (TEM, SEM). Consistent with the XRD result, a similar morphology was exhibited for Co3O4-L, where nanoparticles of approximately 8 nm were interconnected to form a mesoporous structure (Figure S3). Owing to the mild reduction conditions (270 °C for 2 h), particles were not grown or aggregated on CoO, as shown in Figure #cssc201903186-fig-0002#2 a. A closer examination at higher magnification confirms the high crystallinity with clear lattice fringes (Figure #cssc201903186-fig-0002#2 b). The space between lattice fringes was measured to be 0.213 nm and 0.246 nm, corresponding to the (2 0 0) and (1 1 1) planes of cubic CoO, respectively. After the PLFL process, much smaller particles were formed on CoO-L, as shown in Figure #cssc201903186-fig-0002#2 c, compared with the TEM image of the initial CoO. In Figure #cssc201903186-fig-0002#2 d, the observed lattice fringes in the CoO-L crystallites correspond to planes in spinel Co3O4, supporting the XRD result on the laser-induced oxidation effect. Meanwhile, vacancy sites (marked with yellow circles in Figure S4) were observed in the crystal structure, suggesting the laser-induced formation of defects on CoO-L. This is in line with the Rietveld analysis of XRD patterns obtained by synchrotron measurements. To check the fragmentation effect of PLFL, the average particle size was then calculated based on 120 particle counts (Figure S5). As seen in Figure #cssc201903186-fig-0002#2 g, the average particle size of 8 nm was dramatically decreased to 4.2 nm for CoO after laser irradiation. Furthermore, SEM images provide a visualized downsizing effect comparing the particles of CoO (Figure #cssc201903186-fig-0002#2 e) and CoO-L (Figure #cssc201903186-fig-0002#2 f), which were interconnected to form a mesoporous structure. Normally, particle size reduction contributes to a higher specific surface area, which can be examined by nitrogen physisorption measurements. The mesoporous structure of these oxides was confirmed from the typical type IV isotherms (Figure #cssc201903186-fig-0002#2 h and Figure S6). The same Brunauer–Emmett–Teller (BET) surface area of 46 m2 g−1 was measured for Co3O4 and Co3O4-L, slightly lower than that of CoO (49 m2 g−1). CoO-L showed a very high BET surface area of 136 m2 g−1, agreeing well with the XRD and TEM results. For OER catalysts, such a high surface area is highly desirable as more catalytic sites on the surface could be exposed to the electrolyte.

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Figure 2Open in figure viewerPowerPointTEM (a, c), high-resolution TEM (b, d), and SEM (e, f) of CoO and CoO-L, respectively. (g) Particle size distribution histograms. (h) Nitrogen sorption isotherms. Insets in (b) and (e) are the corresponding close-up of the marked rectangles (white color) showing the lattice fringes.

To investigate the surface chemical state, X-ray photoelectron spectroscopy (XPS) was carried out to study the oxidation state of atoms in the top few layers. The binding energies of fitted peaks in the Co 2p and O 1s regions are summarized in Tables S2 and S3, respectively. In the high-resolution Co 2p XPS spectra (Figure #cssc201903186-fig-0003#3), characteristic peaks of Co2+ and Co3+ were fitted fo the Co 2p3/2 and Co 2p1/2 peaks of Co3O4, Co3O4-L, and CoO-L, in good agreement with the XRD result that these oxides mainly show the spinel structure of Co3O4. The peaks at approximately 779.7 and 794.9 eV are ascribed to Co3+, with the peaks located at about 781.4 and 796.6 eV corresponding to Co2+. For the reduced oxide CoO, the main peaks at 780.1 and 795.8 eV were assigned to only Co2+, accompanied by satellite peaks at 786.1 and 802.5 eV.

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Figure 3Open in figure viewerPowerPointCo 2p XPS spectra of Co3O4, Co3O4-L, CoO, and CoO-L.

The OER catalytic performance of the oxides was measured in 1 m KOH by following the protocol proposed by Jaramillo and co-workers, with the electrochemical results listed in Table S4. As a reference catalyst, ordered mesoporous Co3O4 was prepared through nanocasting (labeled as OM-Co3O4), and its structural characterization is presented in Figure S17. As shown in the linear sweep voltammetry (LSV) curves (Figure #cssc201903186-fig-0004#4 a), OM-Co3O4 exhibited higher OER activity over CW-templated Co3O4, on account of its larger surface area (113 m2 g−1 vs. 46 m2 g−1). As predicted, the highest OER activity was observed for CoO-L, which even outperformed the model catalyst (OM-Co3O4). For a straightforward comparison, Figure #cssc201903186-fig-0004#4 b summarizes the overpotential and the current density of these cobalt oxides electrocatalysts. To reach a current density of 10 mA cm−2, an overpotential of 369 mV is required for CoO-L, which is considerably smaller than those of Co3O4 (402 mV), Co3O4-L (400 mV), CoO (403 mV), and OM-Co3O4 (392 mV). Additionally, significantly higher current density of CoO-L was achieved at an applied voltage of 1.7 V (vs. reversible hydrogen electrode, RHE), implying that oxygen was generated much faster compared with that from other cobalt oxide electrocatalysts under the same potential. The catalytic kinetics of the cobalt oxides were evaluated by Tafel plots, which were derived directly from the LSV curves, as shown in Figure #cssc201903186-fig-0004#4 c. The calculated Tafel slope of CoO-L is 46 mV dec−1, which is lower than those of other cobalt oxides (ca. 52 mV dec−1). Despite the same active species (cobalt hydroxide/oxyhydroxide) formed on all these cobalt oxides, the high porosity of CoO-L allows for superior mass transport, which contributes to its lower Tafel slope (Table S4). In addition, the formation of structural defects should also be taken into consideration, as defect-induced electronic delocalization could optimize the adsorption energy for the OER intermediates. A lower Tafel slope suggests more favorable OER kinetics, which is especially desirable for practical applications as the current increases dramatically with applying a higher voltage to the electrode. Based on the values of the two most important parameters of OER activity, that is, overpotential at 10 mA cm−2 and the Tafel slope, we find that CoO-L displays a competitive catalytic performance compared with the benchmark cobalt electrocatalysts (Table S5). Next, we employed different electrochemical approaches to illustrate the outstanding OER activity of CoO-L. The catalytic performance is determined by the active site density of a catalyst. The double-layer capacitance (Cdl) was measured by using cyclic voltammetry (CV) to estimate the electrochemical surface area (ECSA) of the cobalt oxide catalysts. The CV curves were collected in a non-Faradic region with different scan rates (Figure S18). As shown in Figure #cssc201903186-fig-0004#4 d, the capacitance current differences were plotted against the scan rate, where the slope of the plot is proportional to the value of Cdl. The largest Cdl was obtained on CoO-L (0.26 mF), which was significantly higher than those for Co3O4 (0.13 mF), Co3O4-L (0.14 mF), and CoO (0.09 mF). By dividing the specific capacitance of the metal oxide (0.04 mF cm−2), the corresponding ECSA were obtained (Table S4). The largest ECSA on CoO-L resulted from the highly porous structure and provided more active sites exposed to the electrolyte for catalyzing electrochemical OER. Next, the LSV curves were normalized based on ECSA to compare the intrinsic activity of cobalt oxides. The largest normalized current density was obtained with CoO-L (Figure S19), suggesting the amount of exposed active sites is not the sole promotor for OER activity. As another key parameter to evaluate an electrocatalyst, the charge transfer rate of the cobalt oxides was measured by performing electrochemical impedance spectroscopy (EIS) at an overpotential of 350 mV (Figure #cssc201903186-fig-0004#4 e). The corresponding Nyquist plots can be fitted into a simplified Randles circuit (Figure S20). A similar resistance (ca. 6 Ω) under high frequency was observed for all the cobalt oxides, which is assigned to the electrolyte resistance (1 m KOH solution). The diameter of the semi-circle is related to the charge transfer resistance (Rct), with the lower values indicative of faster charge transport kinetics. In comparison with the initial oxides, the samples after laser irradiation display lower Rct, which may be attributed to the formation of structural defects. Specifically, the Rct of Co3O4 (48.8 Ω) was slightly decreased to 38.3 Ω, whereas a dramatic drop was exhibited in the value of Rct from CoO (25.8 Ω) to CoO-L (12 Ω). The different changes in Rct can be explained by the contribution of the much smaller particles to a higher conductivity in CoO-L. The reduction treatment led to a lower resistance on Co3O4 as well, in line with the other studies that reported that cobalt monoxide has a faster charge transfer rate than that of cobalt spinel oxide. Among these cobalt oxides, the lowest Rct of CoO-L enables much faster transfer of electrons from catalytic sites to the glassy carbon electrode, improving the reaction rate of OER. Beside an efficient catalytic performance, the operating stability of an OER catalyst is essential to its application, especially in terms of practical water electrolysis. A stability test was carried out by using chronopotentiometry on CoO-L loaded onto conducting Ni foam (1 mg cm−2, Figure #cssc201903186-fig-0004#4 f). During delivery of a static current density of 10 mA cm−2 for 13 h, a slight increase in potential was observed, which is due to partial blocking of the catalyst surface by evolved oxygen. After removing the oxygen bubble, the potential dropped immediately and stayed constant for the following electrolysis. In addition, post-testing characterization was done to check the structural stability of catalyst. As shown in the TEM image of the catalyst after electrolysis (inset of Figure #cssc201903186-fig-0004#4 f), ultra-small particles were maintained in the CoO-L catalyst without severe particle growth or aggregation. These results suggest the robust durability of CoO-L as an OER catalyst.

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Figure 4Open in figure viewerPowerPoint(a) The LSV curves of various cobalt oxides. The current density was determined by the geometry surface area of the glassy carbon electrode (0.196 cm2). (b) Comparison of the overpotential required to reach 10 mA cm−2 (left axis) and the current density at 1.7 V vs. RHE (right axis). (c) Tafel plots of cobalt oxides derived from their LSV curves correspondingly. (d) Capacitive current differences (Δj=janode−jcathode) at 1.05 vs. RHE against different scan rates. (e) The Nyquist plots measured at 1.6 V vs. RHE. (f) Chronopotentiometric curve of CoO-L at a current density of 10 mA cm−2 and the inset shows a TEM image of CoO-L scratched from the electrode after long-term electrolysis.

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Scheme 1Open in figure viewerPowerPointPreparation of chiral β-aminoalkylzinc halides of type 4 from optically pure amino-alcohols of type 1. [a] Zinc insertion conditions: LiCl (1.0 equiv), Zn dust (1.5 equiv, activated with DBE/TMSCl) in THF at 25 °C. [b] Complexed with LiCl. [c] Determined by titration against iodine.7

Additionally, the crystal structures of (S,R)-15 e and (R,S)-15 g were obtained by X-ray diffraction of single-crystals. The structure of (S,R)-15 e (Figure #chem202000870-fig-0001#1) confirmed the trans-configuration of the formamide group to the aryl moiety which was determined for pyrrole derivative (S,R)-12 c via NMR-spectroscopy.

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Scheme 2Open in figure viewerPowerPointPalladium-catalyzed Negishi cross-coupling and acylation reactions of 4 a with various aryl halides and acid chlorides leading to functionalized pyrrole derivatives 5 a–l. [a] Yield of analytically pure isolated products.

Furthermore, the crystal structure of (R,S)-15 g (Figure #chem202000870-fig-0002#2) showed an anti-configuration. This was in accordance with the results of the Mosher's ester analysis using NMR-spectroscopy described above.

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Scheme 3Open in figure viewerPowerPointNegishi cross-coupling reactions of chiral β-N-pyrrolyl alkylzinc reagents 4 b–e with various functionalized aryl halides. [a] Yield of analytically pure isolated products.

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Scheme 4Open in figure viewerPowerPointPalladium- and copper-catalyzed acylation reactions using chiral organozinc reagents 4 b–e with various acyl chlorides. [a] Palladium-catalyzed acylation. [b] Copper-catalyzed acylation. [c] Yield of analytically pure isolated products.

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Scheme 5Open in figure viewerPowerPointPreparation of a chiral pyrrole-protected alkylzinc reagent ((R)-4 f) derived from tryptophanol (R)-1 f bearing an unprotected indolyl-moiety and its use in Negishi cross-coupling reactions.

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Scheme 6Open in figure viewerPowerPointPreparation of a chiral N-pyrrolyl-alkylzinc iodide ((S)-4 g) derived from l-tyrosine and its use in Negishi cross-coupling and acylation reactions.

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Scheme 7Open in figure viewerPowerPointPreparation of a chiral N-pyrrolyl-alkylzinc reagent starting from the 1,2-cis substituted iodocyclohexyl derivative (S,R)-3 h providing exclusively trans-configured products using palladium-catalysis.

18495_chem202000870-fig-5008.jpg

Scheme 8Open in figure viewerPowerPointPreparation of the 2-N-pyrrolyl-cyclopentlyzinc reagent (R)-4 i. Exclusively trans-configured products of type 13 were obtained after palladium-catalyzed Negishi cross-coupling.

18495_chem202000870-fig-5009.jpg

Scheme 9Open in figure viewerPowerPointEnantioselective CBS-reduction of the ketones (R)-8 f and (S)-8 f provides a pathway towards the 1,3-substituted N-pyrrolyl-alcohols 14 a–d.

18495_chem202000870-fig-0001.jpg

Figure 1Open in figure viewerPowerPointMolecular structure of the deprotected formamide (S,R)-15 e. Thermal ellipsoids are drawn at 50 % probability level. The fluorine atoms of both CF3 groups are disordered.

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Figure 2Open in figure viewerPowerPointMolecular structure of the deprotected formamide (R,S)-15 g. Thermal ellipsoids are drawn at 50 % probability level.

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Scheme 1Open in figure viewerPowerPointEthylene copolymerization with 1-decene (DC), 1-dodecene (DD), and with 2-methyl-1-pentene (2M1P) using Cp*TiCl2(O-2,6-iPr2-4-R-C6H2)−MAO catalyst systems [R=H (1), tBu (2), Ph (3), CHPh2 (4), CPh3 (5), SiMe3 (6), SiEt3 (7), 4-tBuC6H4 (8), 3,5-Me2C6H3 (9)].

Ethylene (E) copolymerizations with 1-decene (DC), 1-dodecene (DD) (called long-chain α-olefins) using complexes 1–9 were conducted in toluene in the presence of AlMe3-free MAO white solid (d-MAO). Table 1 summarizes results of the E/DC copolymerization by 1–9 – MAO catalyst systems. It turned out that the catalytic activities (on the basis of polymer yields) at 25 °C were affected by the para-substituent in the phenoxide ligand, and the activity increased in the order (also shown in Figure #open202100047-fig-0001#1): R=Ph (3)<H (1)<tBu (2)<Ph3C (5)<4-tBuC6H4 (8)<3,5-Me2C6H3 (9)<SiEt3 (7)<Ph2CH (4)<SiMe3 (6). It also turned out that the activities by 2–9 at 50 °C are higher than those conducted at 25 °C, whereas a slight decrease in the activity was observed by 1 (3.46-3.50×105→2.42×105 kg-polymer/mol-Ti⋅h). In particular, the SiMe3 analogue (6) showed a notable increase in the activity at 50 °C (8.04×105→1.44×106 kg-polymer/mol-Ti⋅h). As reported previously, the notable activities were observed by the SiMe3 (6) and SiEt3 (7) analogues at 50 °C even under the low catalyst concentration conditions (runs 17 vs 18, 21 vs 22). The resultant polymers were poly(E-co-DC)s that possess relatively high molecular weights with unimodal molecular weight distributions (Mn=1.38-1.97×105; Mw/Mn=1.48-1.85) as well as with high DC contents (20.1–21.4 mol %). Importantly, as shown in Figure #open202100047-fig-0001#1, the activities by 2–9 increased at 50 °C in all cases, whereas slight decrease in the activity was observed by 1 at 50 °C (runs 28, 29). As described above, both the SiMe3 (6) and the SiEt3 (7) analogues showed the highest activities, and the 3,5-Me2C6H3 analogue (9) also showed a notable increase in the activity at 50 °C (run 27, activity 1.10×106 kg-polymer/mol-Ti⋅h; run 49, activity 1.04×106 kg-polymer/mol-Ti⋅h). The resultant poly(E-co-DD)s possessed rather high molecular weights with unimodal molecular weight distributions (Mn=1.41–1.89×105; Mw/Mn=1.52–1.85) as well as with high DD contents (15.3–18.3 mol %). Significant differences in the DD incorporation (DD contents in the copolymers) were not observed in the resultant poly(E-co-DC)s and poly(E-co-DD)s prepared by 1–9-MAO catalysts systems. Moreover, no significant differences in the DC/DD contents in the copolymers prepared between 25 °C and 50 °C, although 1-hexene content in poly(ethylene-co-1-hexene)s prepared by 1-MAO catalyst system slightly increased at 50 °C probably due to decrease in solubility of ethylene in toluene.

20851_open202100047-fig-5002.jpg

Scheme 2Open in figure viewerPowerPointSynthesis of Cp*TiCl2(O-2,6-iPr2-4-R-C6H2) [R=4-tBuC6H4 (8), 3,5-Me2C6H3 (9)].

Figure #open202100047-fig-0002#2 shows selected 13C NMR spectra (in 1,1,2,2-tetrachloroethane-d2 at 110 °C) of poly(ethylene-co-DC)s prepared by 1 (at 25 °C), 9 (at 50 °C), and Figure #open202100047-fig-0003#3 shows the spectra of poly(ethylene-co-DD)s by 1, 2 – MAO catalyst systems (at 25 °C). Additional 13C NMR spectra of poly(ethylene-co-DC)s and poly(ethylene-co-DD)s by 4–7, 9 at 25 and 50 °C are also shown in Figures S2-1-S2-11 in the Supporting Information. All resonances could be assigned according to the previous reports, and the resultant copolymers [poly(ethylene-co-DC)s, poly(ethylene-co-DD)s] possessed resonances ascribed to the isolated DC or DD insertion in addition to resonances ascribed to the alternating sequence [assigned as Cββ, Cαγ and TECE]. Moreover, the resonance ascribed to repeated comonomer insertion were also observed (TECC+CCE, Cαα). The resultant polymers thus possessed random α-olefin (DC, DD) incorporation as also described below on the basis of analysis of monomer sequence distributions (Table 3).

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Figure 1Open in figure viewerPowerPointEffect of para-substituent in ethylene copolymerization with 1-decene (DC), 1-dodecene (DD) using Cp*TiCl2(O-2,6-iPr2-4-R-C6H2) [R=H (1), tBu (2), Ph (3), CHPh2 (4), CPh3 (5), SiMe3 (6), SiEt3 (7), 4-tBuC6H4 (8), 3,5-Me2C6H3 (9)]−MAO catalyst systems (ethylene 6 atm, DC 0.88 M or DD 0.75 M in toluene at 25 or 50 °C).

Figure #open202100047-fig-0002#2 shows selected 13C NMR spectra (in 1,1,2,2-tetrachloroethane-d2 at 110 °C) of poly(ethylene-co-DC)s prepared by 1 (at 25 °C), 9 (at 50 °C), and Figure #open202100047-fig-0003#3 shows the spectra of poly(ethylene-co-DD)s by 1, 2 – MAO catalyst systems (at 25 °C). Additional 13C NMR spectra of poly(ethylene-co-DC)s and poly(ethylene-co-DD)s by 4–7, 9 at 25 and 50 °C are also shown in Figures S2-1-S2-11 in the Supporting Information. All resonances could be assigned according to the previous reports, and the resultant copolymers [poly(ethylene-co-DC)s, poly(ethylene-co-DD)s] possessed resonances ascribed to the isolated DC or DD insertion in addition to resonances ascribed to the alternating sequence [assigned as Cββ, Cαγ and TECE]. Moreover, the resonance ascribed to repeated comonomer insertion were also observed (TECC+CCE, Cαα). The resultant polymers thus possessed random α-olefin (DC, DD) incorporation as also described below on the basis of analysis of monomer sequence distributions (Table 3).

20851_open202100047-fig-0002.jpg

Figure 2Open in figure viewerPowerPoint13C NMR spectra (in 1,1,2,2-tetrachloroethane-d2 at 110 °C) for poly(ethylene-co-DC)s prepared by a) Cp*TiCl2(O-2,6-iPr2C6H3) (1, run 1, DC 21.4 mol %) and b) Cp*TiCl2(O-2,6-iPr2-4-(3,5-Me2C6H3)-C6H2) (9, run 27, DC 21.1 mol %).

In contrast, a trend in the activities at 80 °C were affected by the para-substituents employed; the activity by the SiEt3 analogue (7) further increased at 80 °C (run 85, 53800 kg-polymer/mol-Ti h), whereas decreases in the activities by 1, 2, 6 were observed. Moreover, effect of 2M1P concentration toward the activities also seemed to be affected by the para substituents. The activities by 2 and 6 increased upon the increasing 2M1P concentration charged (runs 57 vs 58, runs 73 vs 75), whereas the opposite trend was observed by 7 (runs 85 vs 87). It was revealed that the activities by 6, 7 on the basis of polymer yields were not dependent on the polymerization time between 5–15 minutes (runs 72–74, 80–83), suggesting no significant catalyst deactivations were occurred during the copolymerization. The activity was affected by MAO charged; the activities by 2, 4, 6 and 7 in the presence of 5.0 mmol of MAO were higher than those in the presence of 1.0 or 3.0 mmol of MAO (runs S5, 64, 71, 79 vs 56, 63, 69–70, 77–78, respectively). These complexes afforded high molecular weight poly(ethylene-co-2M1P)s with unimodal molecular weight distributions (Mn=2.02–13.3×104; Mw/Mn=1.36-2.05) and their compositions are uniform confirmed by DSC thermograms as observed sole Tm (Figure #open202100047-fig-0004#4) with efficient 2M1P incorporations (2M1P 1.7–3.9 mol %). Moreover, no apparent differences in the Tm, Mn, Mw/Mn values and the 2M1P contents were observed in the resultant copolymers prepared by 1–9 under the same conditions, except that the Tm value in the copolymer by 5 (CPh3) at 25 and 50 °C was rather high (run 66, 67, Tm=113, 120 °C, less 2M1P content) compared to those by the others.(Figures #open202100047-fig-0004#4, S3-1 in the Supporting Information). One probable reason we may take into consideration that these results would be due to an electronic effect of the CPh3 substituent. Importantly, the Tm value in the copolymer by the Ph analogue (3) and 3,5-Me2C6H3 analogue (9) prepared at 25 °C, 50 °C and 80 °C (Figure S3-2 in the Supporting Information) seems rather low (run 59–61, 91–93), and the results also suggest a possibility that an electronic factor play a role toward the 2M1P incorporation. The Mn values were slightly decreased upon increasing the reaction temperature (25–80 °C) with decrease in the 2M1P contents, which are corresponded to the increases in the Tm values in the copolymers at 50 and 80 °C consistent with possessing their uniform compositions confirmed by their DSC thermograms (Figure #open202100047-fig-0005#5).

20851_open202100047-fig-0003.jpg

Figure 3Open in figure viewerPowerPoint13C NMR spectra (in 1,1,2,2-tetrachloroethane-d2 at 110 °C) for poly(ethylene-co-DD)s prepared by a) Cp*TiCl2(O-2,6-iPr2C6H3) (1, run 28, DD 17.6 mol %) and b) Cp*TiCl2(O-2,6-iPr2-4-tBu-C6H2) (2, run 30, DD 18.3 mol %).

Moreover, no apparent differences in the Tm, Mn, Mw/Mn values and the 2M1P contents were observed in the resultant copolymers prepared by 1–9 under the same conditions, except that the Tm value in the copolymer by 5 (CPh3) at 25 and 50 °C was rather high (run 66, 67, Tm=113, 120 °C, less 2M1P content) compared to those by the others.(Figures #open202100047-fig-0004#4, S3-1 in the Supporting Information). One probable reason we may take into consideration that these results would be due to an electronic effect of the CPh3 substituent. Importantly, the Tm value in the copolymer by the Ph analogue (3) and 3,5-Me2C6H3 analogue (9) prepared at 25 °C, 50 °C and 80 °C (Figure S3-2 in the Supporting Information) seems rather low (run 59–61, 91–93), and the results also suggest a possibility that an electronic factor play a role toward the 2M1P incorporation. The Mn values were slightly decreased upon increasing the reaction temperature (25–80 °C) with decrease in the 2M1P contents, which are corresponded to the increases in the Tm values in the copolymers at 50 and 80 °C consistent with possessing their uniform compositions confirmed by their DSC thermograms (Figure #open202100047-fig-0005#5). Figure #open202100047-fig-0005#5 shows the selected DSC thermograms of poly(ethylene-co-2M1P)s prepared by Cp*TiCl2(O-2,6-iPr2-4-tBu-C6H2) (2)-MAO catalyst system (at 25, 50 and 80 °C). As described above, the Tm values in the resultant poly(E-co-2M1P)s increased at higher temperature (50 and 80 °C) along with decrease in the 2M1P contents [Tm 104→111→120 °C, content 3.4→2.4→2.3 mol % (run 55–57)] as well as consisting with uniform compositions. The similar trends in the DSC thermograms (and 2M1P contents) in the copolymers prepared were observed by the other complexes (1 and 3–4, 6–9, Figures S3-3–S3-5, S3-7–S3-10, in the Supporting Information), whereas the slight decrease of Tm value in the copolymer by complex 5 at 80 °C was observed (Tm 120→117 °C; see Figure S3-6, in the Supporting Information). The Tm values were not affected by the amount of MAO charged and the polymerization time (see Figures S3-11–S3-17 in the Supporting Information). The Tm values decreased upon increasing 2M1P concentration charged along with increase the 2M1P contents in the copolymers (see Figures S3-18–S3-21 in the Supporting Information). The observed temperature dependence is unique contrast to those observed in the ethylene copolymerization with 1-hexene, 1-decene and with 1-dodecene.

20851_open202100047-fig-0004.jpg

Figure 4Open in figure viewerPowerPointDSC thermograms of poly(ethylene-co-2M1P)s prepared by Cp*TiCl2(O-2,6-iPr2-4-R-C6H2) [R=H (1), tBu (2), Ph (3), CHPh2 (4), CPh3 (5), SiMe3 (6), SiEt3 (7), 4-tBuC6H4 (8), 3,5-Me2C6H3 (9)]–MAO catalysts systems at 50 °C. Detailed results are shown in Table 4 (runs 51, 56, 60, 63, 67, 73, 81, 89, 92).

Figure #open202100047-fig-0006#6 shows typical 13C NMR spectrum in poly(ethylene-co-2M1P) prepared by 2–MAO catalyst system (run 55 1,1,2,2-tetrachloroethane-d2 solution at 110 °C). Selected 13C NMR spectra in poly(ethylene-co-2M1P)s by 2, 6, 7 – MAO catalyst systems are also shown in Figures S2-12–S2-17 in the Supporting Information. All resonances could be assigned according to the previous report, and the resultant copolymer possessed resonances ascribed to the isolated 2M1P inserted unit in addition to resonances due to alternating 2M1P incorporations (assigned as Cββ and Cαγ). No resonances ascribed to the repeated 2M1P insertion were observed, and the fact could explain that negligible or no catalytic activity was observed in an attempted 2M1P homopolymerization by the 2-MAO catalyst system. The results also explain that 2M1P incorporation is less efficient compared to DC and DD incorporations in this catalysis, as observed in the ordinary metallocenes (in the ethylene/isobutene copolymerization) and the linked half-titanocenes like [Me2Si(C5Me4)(NtBu)]TiCl2.

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Figure 5Open in figure viewerPowerPointDSC thermograms of poly(ethylene-co-2M1P)s prepared by Cp*TiCl2(O-2,6-iPr2-4-tBu-C6H2) (2)–MAO catalyst system at 25, 50 and 80 °C. Detailed results are shown in Table 4 (runs 55–57).

20851_open202100047-fig-0006.jpg

Figure 6Open in figure viewerPowerPoint13C NMR spectrum (in 1,1,2,2-tetrachloroethane-d2 at 110 °C) for poly(ethylene-co-2M1P) prepared by Cp*TiCl2(O-2,6-iPr2-4-tBu-C6H2) (2)-MAO catalyst system (run 55, 2M1P 3.4 mol %).

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Figure 1Open in figure viewerPowerPointStructures of the flavanones eriodictyol and naringenin, the flavone luteolin, the flavonol quercetin and the isoflavone genistein. Ring assignment and backbone atoms numbering are shown in the structure of eriodictyol.

Flavonoids are a large group of natural polyphenols and secondary metabolites from plants. They attract a lot of attention due to their nutritional, health-beneficial and pharmacological properties including free radical-scavenging antioxidative activities, anti-inflammatory, antimicrobial and anticancer activities. Flavonoids are classified into flavonoids (2-phenylbenzopyrans), isoflavonoids (3-phenylbenzopyrans) and neoflavonoids (4-phenylbenzopyrans) based on the position of the phenyl ring. Flavonoids are further divided into flavanes, flavanones, flavonols, flavones and anthocyanins (Figure #cctc202000471-fig-0001#1). Various modification reactions such as oxidation, hydroxylation, glycosylation and methylation lead to a huge variety of flavonoids. In particular, O-methylation of free hydroxyl groups on dietary flavonoids greatly increases their absorption and oral bioavailability through the improvement of their metabolic stability and better membrane transportation. Methylation of flavonoids can also bring new biological activities. For example, chrysoeriol (4′,5,7-trihydroxy-3′-methoxyflavone, 3′-methylluteolin) and isohamnetin (3,4′,5,7-tetrahydroxy-3′-methoxyflavone, 3′-methylquercetin) show strong and selective inhibition on the formation of a carcinogenic estrogen metabolite related to breast cancer. Besides, eriodictyol (3′,4′,5,7-tetrahydroxyflavanone) and homoeriodictyol (4′,5,7-trihydroxy-3′-methoxyflavanone, 3′-methyleriodictyol) are known by their remarkable bitter masking effect. Methylation reactions mostly take place at the 7-, 3′- and 4′-hydroxyl groups of flavonoids, the 7- and 4′-hydroxyl groups of isoflavonoids and the 3- and 4-hydroxyl groups of phenylpropanoids. In order to discover the factors determining the substrate discrimination of plant OMTs, we chose 21 plant OMTs from different plant species with different substrate preferences and regioselectivities for comparison. Sequence alignment shows that the sequences are extremely diverse between different plant OMTs, with only 4.6 % identity (Figure #cctc202000471-fig-0002#2, Figure S1). Since several IOMT crystal structures have been resolved but no FOMT structure is available, we chose MeSa-7/4′-IOMT which has been obtained in a catalytic conformation (PDB code: 1FP2) and investigated based on it the substrate-enzyme interactions. The ligand isoformononetin, the 7-methylated product of the isoflavonone daidzein, is situated in the enzyme active site and well-stabilized by multiple interactions. Residues critical for substrate binding are highlighted in Figure #cctc202000471-fig-0001#1. Met183 and Met323 constrain the aromatic A-ring and help positioning the 7-hydroxyl group to the catalytic residue His272 and SAM. These two residues are conserved throughout the plant OMT superfamily. Zubieta et al. suggested that the interaction of the ketone group in the C-ring is stabilized by the amide side chain of Asn322, a residue that is only conserved among the 7-IOMTs. Other OMTs rather have middle size hydrophobic residues, namely Ile, Val or Met, at this position. Leu326 also interacts with the C-ring of isoformononetin, but it locates closer to the ether oxygen. Residues at this position are quite different between the selective OMTs. They are either the hydrophobic Leu, Val, Ile or Met, or the basic residues Arg or His. In the absence of Asn322, the basic residues might play an important role in stabilizing the C-ring ketone group. The accommodation of the isoflavone B-ring is achieved by Cys133 and Val134. Although these two residues in other plant OMTs are quite diverse, they mostly have Gly/Leu/Met and Ala/Val/Asn at these two positions, respectively. These substrate binding residues bring proper binding patterns to their preferred substrates and thus determine substrate specificity and regioselectivity of different plant OMTs. In order to investigate the influence of these residues, we constructed mutants L322H/N/M and Y326H/R/L using the IeOMT-T133M variant as template. IeOMT is a phenylpropanoid 4-OMT isolated from Clarkia breweri and the variant T133M has been proved to expand the substrate scope and to enhance the regioselectivity. Flavonoids are classified into flavane, flavanone, flavone and flavonol, while isoflavonoids are divided into isoflavone and isoflavanone, depending on their structures. We have chosen several commonly known compounds eriodictyol and naringenin (flavanones), luteolin (flavone), quercetin (flavonol) and genistein (isoflavone) as substrates (Figure #cctc202000471-fig-0001#1). Since optically pure flavanones will racemize in aqueous solution, we used racemic eriodictyol and naringenin as substrates and obtained racemic products. Activities of the wild type IeOMT and designed mutants were tested against these substrates. In each reaction, a molar excess of SAM and 25 % (v/v) Escherichia coli (E. coli) cell lysate, which contains S-adenosyl-l-homocysteine (SAH) nucleosidase, were provided in order to increase the yield. Products were confirmed by either comparing their retention times on HPLC to commercial standards or structurally characterized via NMR and MS. Catalytic performance of the mutants are presented in area percentages calculated by peak area of both substrates and products measured by HPLC (Figure #cctc202000471-fig-0003#3, Figure S2). The accession numbers of plant OMTs chosen for sequence alignment in Figure #cctc202000471-fig-0001#1 are as follows: MePi_7-FOMT1 from Mentha piperita (AAR09598.1), MeTr_7-FOMT7 from Medicago truncatula (ABD83946.1), OrSa_7-FOMT from Oryza sativa (BAM13734.1), ArTh_3′-FOMT from Arabidopsis thaliana (AAB96879.1), ChAm_3′-FOMT from Chrysosplenium americanum (AAA80579.1), MePi_3′-FOMT3 from Mentha piperita (AAR09601.1), OrSa_3′-FOMT from Oryza sativa (XP_015650053.1), CaRo_4′-FOMT from Catharanthus roseus (AAR02420.1), GlMa_4′-FOMT/IOMT from Glycine max (C6TAY1.1), MePi_4′-FOMT4 from Mentha piperita (AAR09602.1), CiAr_7-IOMT from Cicer arietinum (XP_004489528.1), MeTr_7-IOMT1 from Medicago truncatula (AAY18582.1), MeSa-7/4′-IOMT from Medicago sativa (AAC49928.1), GlEc_4′-IOMT from Glycyrrhiza echinate (BAC58011.1), LoJa_4′-IOMT from Lotus japonicus (BAC58013.1), MeTr_4′-IOMT5 from Medicago truncatula (AAY18581.1), CaRo_3-POMT from Catharanthus roseus (AAK20170.1), ClBr_3-POMT from Clarkia breweri (O23760.1), LoPe_3-POMT from Lolium perenne (AAD10253.1), MeSa_3-POMT from Medicago sativa (AAB46623.1), ClBr_4-POMT from Clarkia breweri (O04385.2).

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Figure 2Open in figure viewerPowerPointPart of the sequence alignment of plant flavonoid OMTs (FOMTs), isoflavonoid OMTs (IOMTs) and phenylpropanoid OMTs (POMTs) performed with Geneious 10.0.2 (the whole sequence alignment is shown in Figure S1). Residues involved in substrate binding are highlighted in blue. Numbering of residues are based on the sequence of the Clarkia breweri isoeugenol OMT (IeOMT). OMTs are named by their original organisms and regioselectivity. MePi, Mentha piperita; MeTr, Medicago truncatula; OrSa, Oryza sativa; ArTh, Arabidopsis thaliana; ChAm, Chrysosplenium americanum; CaRo, Catharanthus roseus; GlMa, Glycine max; CiAr, Cicer arietinum; MeSa, Medicago sativa; GlEc, Glycyrrhiza echinate; LoJa, Lotus japonicus; ClBr, Clarkia breweri; LoPe, Lolium perenne. MeTr_7-FOMT7 is a putative IOMT but it has higher preference against naringenin (flavanone) than isoflavonoids.

Methylation reactions mostly take place at the 7-, 3′- and 4′-hydroxyl groups of flavonoids, the 7- and 4′-hydroxyl groups of isoflavonoids and the 3- and 4-hydroxyl groups of phenylpropanoids. In order to discover the factors determining the substrate discrimination of plant OMTs, we chose 21 plant OMTs from different plant species with different substrate preferences and regioselectivities for comparison. Sequence alignment shows that the sequences are extremely diverse between different plant OMTs, with only 4.6 % identity (Figure #cctc202000471-fig-0002#2, Figure S1). Since several IOMT crystal structures have been resolved but no FOMT structure is available, we chose MeSa-7/4′-IOMT which has been obtained in a catalytic conformation (PDB code: 1FP2) and investigated based on it the substrate-enzyme interactions. The ligand isoformononetin, the 7-methylated product of the isoflavonone daidzein, is situated in the enzyme active site and well-stabilized by multiple interactions. Residues critical for substrate binding are highlighted in Figure #cctc202000471-fig-0001#1. Met183 and Met323 constrain the aromatic A-ring and help positioning the 7-hydroxyl group to the catalytic residue His272 and SAM. These two residues are conserved throughout the plant OMT superfamily. Zubieta et al. suggested that the interaction of the ketone group in the C-ring is stabilized by the amide side chain of Asn322, a residue that is only conserved among the 7-IOMTs. Other OMTs rather have middle size hydrophobic residues, namely Ile, Val or Met, at this position. Leu326 also interacts with the C-ring of isoformononetin, but it locates closer to the ether oxygen. Residues at this position are quite different between the selective OMTs. They are either the hydrophobic Leu, Val, Ile or Met, or the basic residues Arg or His. In the absence of Asn322, the basic residues might play an important role in stabilizing the C-ring ketone group. The accommodation of the isoflavone B-ring is achieved by Cys133 and Val134. Although these two residues in other plant OMTs are quite diverse, they mostly have Gly/Leu/Met and Ala/Val/Asn at these two positions, respectively. These substrate binding residues bring proper binding patterns to their preferred substrates and thus determine substrate specificity and regioselectivity of different plant OMTs. In order to investigate the influence of these residues, we constructed mutants L322H/N/M and Y326H/R/L using the IeOMT-T133M variant as template. IeOMT is a phenylpropanoid 4-OMT isolated from Clarkia breweri and the variant T133M has been proved to expand the substrate scope and to enhance the regioselectivity.

3365_cctc202000471-fig-0003.jpg

Figure 3Open in figure viewerPowerPointProduct composition of each reaction catalyzed by the wild-type IeOMT and its mutants. Area percentages were calculated by the peak areas of both substrates and products as determined by HPLC (Figure S2). 3′-Me-E, 3′-methyleriodictyol; 3′,4′-DiMe-E, 3′,4′-dimethyleriodictyol; 3′-Me-L, 3′-methylluteolin; 3′,4′-DiMe-L, 3′,4′-dimethylluteolin; 3′-Me-Q, 3′-methylquercetin; 3′,4′-DiMe-Q, 3′,4′-dimethylquercetin. Structures of these products are confirmed by NMR and LC-MS (see supporting information). 7-Me-N, 7-methylnaringenin; 7-Me-G, 7-methylgenistein; 4′-Me-G, 4′-methylgenistein. These products were confirmed by comparing their retention times on HPLC to commercial standards as well as LC-MS. Racemic eriodictyol and naringenin were used as substrates and racemic products were obtained. All experiments were performed in triplicates and standard deviations were provided.

Flavonoids are classified into flavane, flavanone, flavone and flavonol, while isoflavonoids are divided into isoflavone and isoflavanone, depending on their structures. We have chosen several commonly known compounds eriodictyol and naringenin (flavanones), luteolin (flavone), quercetin (flavonol) and genistein (isoflavone) as substrates (Figure #cctc202000471-fig-0001#1). Since optically pure flavanones will racemize in aqueous solution, we used racemic eriodictyol and naringenin as substrates and obtained racemic products. Activities of the wild type IeOMT and designed mutants were tested against these substrates. In each reaction, a molar excess of SAM and 25 % (v/v) Escherichia coli (E. coli) cell lysate, which contains S-adenosyl-l-homocysteine (SAH) nucleosidase, were provided in order to increase the yield. Products were confirmed by either comparing their retention times on HPLC to commercial standards or structurally characterized via NMR and MS. Catalytic performance of the mutants are presented in area percentages calculated by peak area of both substrates and products measured by HPLC (Figure #cctc202000471-fig-0003#3, Figure S2).

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Figure 4Open in figure viewerPowerPointModels of Clarkia breweri IeOMT variants with SAM (grey sticks with elemental coloring at the left side) and substrates in the binding pocket. The three targeted residues are given as lines. For clarity, non-polar hydrogens are removed. A) (S)-eriodictyol (green), luteolin (magenta) and quercetin (blue) docked in the binding pocket of the wild-type IeOMT, exposing the 3′-hydroxyl group for methylation. B) the same substrates in the binding pocket of IeOMT_T133M/Y326L, exposing the 4′-hydroxyl group for methylation. C) Genistein binding on IeOMT_T133M/Y326H provides the 7-position for methylation. D) (S)-Naringenin binding pattern in IeOMT_T133M/L322M also exposes the 7-position, however, with a totally different binding pattern.

To gain an insight into the structural differences that lead to the different regioselectivity of the variants, we performed in silico analysis. As seen in Figure #cctc202000471-fig-0004#4A, the wild-type IeOMT can accommodate eriodictyol, luteolin and quercetin in an orientation that the methylation of the 3′-position is favored. In all three cases the distance of the oxygen of 3′-hydroxyl group to the methyl group to be transferred is between 3.2 and 3.5 Å. It is interesting to note that eriodictyol and luteolin bind in a similar orientation, however, quercetin seems to be a little tilted in comparison to SAM, which brings also the 4′-position in closer proximity to the transfer group (3.3 Å to the 3′-OH and 3.4 Å to the 4′-OH group) and thus the wild-type IeOMT can also produce some dimethylated quercetin. In the case of the double mutant T133M/Y326L, the double methylation is increased for all three substrates. As seen in Figure #cctc202000471-fig-0004#4B, the double mutation enabled the inverse binding of these three flavonoids in the binding pocket, bringing the 4′-hydroxyl group in the proximity of the methyl group of the SAM and thus the double methylation is favored. The reason for this inversed binding seems to be the more hydrophobic character of the introduced Y326L, which cannot accommodate the carbonyl of 5-position. In the case of genistein, the T133M mutation increased the activity to a detectable level, but the mutations at positions 322 and 326 do not further increase the activity of the enzyme. However, the double mutant T133M/Y326H has a shift of its regioselectivity to position 7. As seen in Figure #cctc202000471-fig-0004#4C, the substrate is bound with ring A facing SAM, and the position 7 is closer to the methyl group for the transfer (3.7 Å). It seems that the histidine at position 326 can interact with the hydroxyl group at position 5 of genistein (3.0 Å distance) and thus stabilizes the substrate in this orientation to complete the catalysis. Naringenin differs from eriodictyol only by the lack of the 3′-hydroxyl group. Thus, although it can bind the same way in the active site of the wild type, the B-ring cannot be methylated to a 3′-methoxy derivative and thus the wild type is almost inactive. However, the mutation T133M (and the T133M/L322M mutations) enabled a different binding pattern, where the position 7 of the ring A is accessible to the SAM and the 7-hydroxyl group at catalytic distance (3.4 Å). The binding pattern differs for genistein, for which the enzyme exhibited the same regioselectivity. In the case of variant T133M/L322M (Figure #cctc202000471-fig-0004#4D), the reason for this can be the lack of the histidine at position 326 that could interact with the 5-OH, in combination with the methionine in position 322 that pushes away the substrate. As naringenin is not planar, the ring B may cause steric clashes with 322 and thus the substrate binds in a different orientation.

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FIGURE 1Open in figure viewerPowerPoint(a) Absorbance spectra of various binary doped PANI samples. (b) Tauc plot showing band gap energies of binary doped PANI samples and (c) FTIR transmittance spectra of (a) P-1.9, (b) P-2.1, (c) P-2.3, and (d) P-2.5

Dried PANI (0.5 g) was dispersed in 1 ml of NMP and was stirred for 2 h to attain a uniform dispersion. The slurry was coated onto the surface of cleaned FTO glass substrates by the Doctor Blade technique.[] The PANI-coated FTO substrate was dried at 50°C for 10 min. The thickness of the film was kept at 8 μm (Figure #elsa202100155-fig-0001#1g,h). A film of TiO2 paste (20 nm opaque) was deposited on an FTO glass substrate by screen printing and was heated at 80°C for 10 min. The same screen printing and heating process were repeated eight times to get 12μm thick TiO2 film as shown in Figure #elsa202100155-fig-0001#1(f). After this, TiO2 coated FTO was subjected to rapid annealing at 150, 300, 350, 400, 450, and 500 °C for 10, 15, 10, 15, 10, and 15 min, respectively. TiO2 film was obtained into a circle with an effective area of 0.28 cm2. Thereafter, TiO2 coated substrates were immersed in dye solution (0.5 mM N719 dye in ethanol) for 24 h. The dye adsorbed TiO2 films were then rinsed with ethanol followed by cool air drying. The absorption curves of all the samples (Figure #elsa202100155-fig-0001#1a) display a similar shape with three absorption bands at about 338 nm, 418 nm and 790 nm. The band ranging from 338–346 nm can be attributed to the electronic π to π* transitions due to the excitation of nitrogen in the benzenoid rings. A shoulder at 418–428 nm is ascribed to the polaron to π* transitions indicating incorporation of dopants in polymer backbone.[] The band at infrared region ranging from 790 to 822 nm originates from the charged cationic species, known as π-localized polaron transition. This broadband may be caused by an interband charge transfer from benzenoid to quinoid rings of conjugated PANI. Stronger absorption of this band represents the protonation of the synthesized material.[] The band positions, intensity, and intensity ratios (A2/A1) of the second band (A2) to the first band (A1) of the synthesized PANI samples are listed in Table 2. The values of A2/A1 for P-2.3 are larger compared to others indicating the high doping level of this sample. It is illustrious that lambda 2 exhibits a significant redshift with an increase in dopant ration from P-1.9 to P-2.3. This might be assigned to the selective site for the interactions between the ALS dopant and the quinoid ring of emeraldine salt (ES), facilitating the charge transfer between the quinoid unit of ES and the dopant via highly reactive imine groups. Possibly some template effects are produced by dopant ions which improve the ordering of PANI chains. This confirms effective incorporation of counter ions in a polymer structure.[] Figure #elsa202100155-fig-0001#1c depicts the FTIR transmittance spectra of synthesized PANI samples. Vibrations for the building units of PANI i-e benzene and quinone rings can be observed in the vicinity of 1545–1563 cm−1 and 1468–1470 cm−1 []. Their intensity ratio could be used as an indicator of the oxidation state of PANI i-e demonstrating the comparative content of quinoid diamine and benzene ring structure. The (IQ/IB) ratio calculated for all spectra is listed in Table 2. The intensity ratio of PANI is in order of P-1.9 < P-2.1 < P-2.3 > P-2.5. The higher intensity ratio reveals the greater conductivity and reflects the emeraldine salt state of PANI.[] A band at 3219–3226 cm−1 represents the stretching vibrations of NH part present in the polymer. Band at 2906–2914 cm−1 is related to NH2+ part in C6H4NH2+C6H4– groups.[] The presence of the band at 2836–2850 cm−1 is related to symmetrical and asymmetrical stretching of alkyl substituent of ALS, which is used in the polymerization reaction.[] The C-N bending and stretching modes of various benzenoid, quinonoid, and polaronic forms are observed at 1206–1220 cm−1 and 1283–1290 cm−1, respectively. These polaronic forms also indicate the conducting protonating process of PANI. The bands located in the range of 1109–1114 cm−1 are attributed to the vibration mode of the –NH+ = structure and are associated with the vibrations of the charged polymer quinonoid structure. This indicates the existence of positive charges on the polymer chain. The bands observed in the range of 751–785 cm−1 are assigned to 1,4-substituted aromatic rings of polymer.[] The bands at 1631 and 1348 in all the samples, reveal effective polaron formation (C-N+).[] Slight shifting in the band position is observable.[] Literature reveals that a higher Ired value manifests better electrocatalytic ability and conductivity for the catalytic material and the more striking observation was the separation of the anodic and the cathodic peak potentials (Epp) that is negatively correlated with the electrochemical rate constant of a redox reaction. The Epp values can be used to estimate their redox reaction resistances.[] The peak current density of reduction (Jred) and oxidation (Jox), and peak separation (Epp) of the five electrodes are listed in Table 1. It is observed that the variation of ALS content caused variance in electrocatalytic activity (ECA) of the PANI CEs. It can be seen from Figure #elsa202100155-fig-0007#7a, the reduction process of P-1.9 is not obvious as it displays smaller current density as well as high Epp compared to others. This clearly depicts relatively weak electrocatalytic ability and lower conductivity of P-1.9 as seen in conductivity analysis.[] With the increasing content of ALS, the current densities of the electrodes (P-2.1 and P-2.3) increase gradually, indicating that more active sites have been created, which might be due to the role of the ALS that induces different morphologies in binary doped PANI samples with different concentrations as exemplified by Figure #elsa202100155-fig-0001#1. According to experimental data, P-2.3 represents the highest value of Jred and Jox, and the smaller Epp value but larger than Pt, reflecting its largest surface area for efficient catalytic reduction of I3- and fastest charge transfer at electrolyte/PANI nanofibers interfaces, which contributes to its high conductivity and electrocatalytic behavior to the I−/I3−redox couple[] as represented in Table 2.

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SCHEME 1Open in figure viewerPowerPointFabrication of DSSC based on PANI CE

The XRD curves of PANI samples are shown in Figure #elsa202100155-fig-0002#2. It can be noted that the P-1.9 shows three main peaks at 2θ = 15°, 20°, and 25°, representing that the sample is partly crystalline and these peaks are characteristics of the emeraldine state of binary doped PANI.[]

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FIGURE 2Open in figure viewerPowerPointXRD patterns of (a) P-1.9, (b) P-2.1, (c) P-2.3, (d) P-2.5

Figures #elsa202100155-fig-0003#3 and #elsa202100155-fig-0004#4 display the SEM- EDX spectra along with the elemental composition of PANI salts (inset table of respective spectra) and surface mapping of elements of P-1.9, P-2.1, P-2.3, P-2.5. From the SEM-EDX spectra, the elements like C, N, O, and S were examined in a selected area of SEM shown as an inset in Figures #elsa202100155-fig-0003#3 and #elsa202100155-fig-0004#4. The signals from C, N, O, and S show good agreement with each other and reveal the successful incorporation of binary dopants into the polymer backbone. The experimental results showed that compared to other samples, P-2.3 has a high content of carbon which is the main element present in the polymer backbone. Moreover, an increase in S content with dopant amount is observed. A high percentage of the sulfur (S) in P-2.3 is assumed to be responsible for high dopant amounts.[] It can be noticed that all the spectra exhibited three peaks of S at 2.3 KeV, 2.47 KeV, and 0.014 KeV with different intensities. These peaks can be associated with Kα1, Kedge, and L1edge, respectively.[] For a specific absorber, a sharp increase in intensity was observed when the energy of the X-rays coincides with the energy of an electron shell (K, L, or M) in the absorber. The absorbed energy (also called “critical excitation” or “edge”) creates a vacancy in the specific shell. This vacancy is then occupied by an electron transferred from another shell. As a result, specific X-Ray lines are generated from K, L, M, N, or O shells, corresponding to the excitation of the K, L, M, N, and O levels. The mapped images also confirmed the distribution of S contents along with other elements.

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FIGURE 3Open in figure viewerPowerPoint(a and b) SEM-EDX spectrum and Mapping of P-1.9 and P-2.1

Figures #elsa202100155-fig-0003#3 and #elsa202100155-fig-0004#4 display the SEM- EDX spectra along with the elemental composition of PANI salts (inset table of respective spectra) and surface mapping of elements of P-1.9, P-2.1, P-2.3, P-2.5. From the SEM-EDX spectra, the elements like C, N, O, and S were examined in a selected area of SEM shown as an inset in Figures #elsa202100155-fig-0003#3 and #elsa202100155-fig-0004#4. The signals from C, N, O, and S show good agreement with each other and reveal the successful incorporation of binary dopants into the polymer backbone. The experimental results showed that compared to other samples, P-2.3 has a high content of carbon which is the main element present in the polymer backbone. Moreover, an increase in S content with dopant amount is observed. A high percentage of the sulfur (S) in P-2.3 is assumed to be responsible for high dopant amounts.[] It can be noticed that all the spectra exhibited three peaks of S at 2.3 KeV, 2.47 KeV, and 0.014 KeV with different intensities. These peaks can be associated with Kα1, Kedge, and L1edge, respectively.[] For a specific absorber, a sharp increase in intensity was observed when the energy of the X-rays coincides with the energy of an electron shell (K, L, or M) in the absorber. The absorbed energy (also called “critical excitation” or “edge”) creates a vacancy in the specific shell. This vacancy is then occupied by an electron transferred from another shell. As a result, specific X-Ray lines are generated from K, L, M, N, or O shells, corresponding to the excitation of the K, L, M, N, and O levels. The mapped images also confirmed the distribution of S contents along with other elements.

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FIGURE 4Open in figure viewerPowerPoint(c and d) SEM- EDX spectrum and Mapping of P-2.3 and P-2.5

Figure #elsa202100155-fig-0005#5a-d shows SEM images of P-1.9, P-2.1, P-2.3, and P-2.5 in powder form. An irregular morphology can be seen in P-1.9 (Figure #elsa202100155-fig-0005#5a), since there is no sign of elongation, we can conclude that fibers are not formed. SEM micrographs reveal that there are agglomerations of particles with the formation of a cauliflower-like structure. It is expected that the counterions from the dopant interact with the cation-radical of aniline might act as a driving force for the self-assembling cauliflower type shape and be responsible for the highly random aggregation of particles in the cauliflower-like structure. This type of aggregation results in low electron delocalization and therefore increases the penetration and charge transfer resistance for exchange of I−/I3−redox couples.[] With an increase in ALS amount, an evolution from dense irregular morphology observed in P-1.9 to microsized (1 μm) short needle-like morphology with large flakes is observed in P-2.1 (Figure #elsa202100155-fig-0005#5b). Here, selective types of aggregation process result in the formation of short needle formation. In case of P-2.3 (Figure #elsa202100155-fig-0005#5c) further increase in the amount of ALS induced long nanosized (diameter range 43-50 nm) needle-like morphology with some porosity. Similar morphology of PANI is also reported by Katarzyna Krukiewicz et al.[] They synthesized PANI in m-cresol using camphor sulphonic acid (CSA) as a surfactant with improved optical and morphological properties. Such a structure is known to be of great value to photovoltaic applications such as in DSSCs. The needle-like and some porous structure of the material is beneficial for the enhancement of electrocatalytic activity for I3−/I− redox reaction and is expected to facilitate the migration of redox couples within the CE by adsorption of the liquid electrolyte by trapping the liquid in the nanoporomerics.[] However, with further increase in ALS amount, the needle-like morphology of P-2.3 is getting closure with the formation of large flakes that reduce the porosity in P-2.5 as shown in (Figure #elsa202100155-fig-0005#5d). The reason for observing different morphology in binary doped PANI system is related to the micelle formation which is directly related to the concentration of counterions. We can say that an appropriate amount of counterions is necessary to assist the cation-radical of aniline to remarkably alter to the other shape.[] In order to evaluate the performance of PANI CEs in solar cell, the J-V curves for the DSSC were measured under standard light irradiation and are shown in Figure #elsa202100155-fig-0008#8a. The device parameters are summarized in Table 3. DSSCs-based on P-1.9 CE exhibited poor conversion efficiency (2%) with low values of Jsc, which manifests low conductivity of the polymer. The low value of FF and Voc depicts the low catalytic activity of P-1.9 CE as these two parameters are largely affected by the electrocatalytic activity of the CE.[] It may be assumed from the fact that this CE has insufficient pores (as explained in SEM) for the diffusion of redox species resulting in the increased availability of recombination between I3− ions and the photoinjected electrons at the CE.[] When using P-2.1 and P-2.3, all photovoltaic parameters increase with a conversion efficiency of 2.7% and 4.54 %, which may arise due to increase in ALS content that gives significant morphology to the material. While P-2.5 (3%) depicts lower efficiency as further increase in ALS content results in the formation of flakes by decreasing porosity in P-2.5 as represented in Figure #elsa202100155-fig-0005#5d. Comparing P-2.3 CE-based DSSC with Pt electrode (4.02%), all the parameters of the DSSC with P-2.3 CE are enhanced. The better photoelectric performances of DSSC with P-2.3 CE can be envisaged to come from three aspects. First, the increased content of ALS induces needle-like and porous morphology in P-2.3 (Figure #elsa202100155-fig-0005#5c) engenders a large surface area on the electrode. This is beneficial for the efficient reduction reaction in the I−/I3−system.[] Second, the small resistance of P-2.3 CE is responsible for ease in electron transfer and enhancement in Jsc. Thirdly, the higher conductivity and electrocatalytic activity of P-2.3 results in an increase in FF and hence the efficiency of the cell.[]

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FIGURE 5Open in figure viewerPowerPoint(a-d) SEM images of P-1.9, P-2.1, P-2.3 and P-2.5, respectively

Figure #elsa202100155-fig-0006#6a,b* displays top-view SEM images of P-2.3 and TiO2 films. Here, it is observed that needle-like morphology with small pores of P-2.3 and rough surface of TiO2 are beneficial for better electrocatalytic activity as a porous structure of films can easily adsorbed liquid electrolyte by trapping it in porous sides.[] Figure #elsa202100155-fig-0006#6c,d FIB cross-sectional image of P-2.3 and TiO2 films shows the thickness of 8 and 12 μm, respectively.

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FIGURE 6Open in figure viewerPowerPointTop view SEM image of P-2.3 CE (a and a*) at 8000 and 50,000 magnification, Top view SEM image of TiO2 CE (b and b*) 8,000 magnification and 50,000 magnification, (c and d) FIB-SEM cross section of P-2.3 and TiO2 films, respectively

Literature reveals that a higher Ired value manifests better electrocatalytic ability and conductivity for the catalytic material and the more striking observation was the separation of the anodic and the cathodic peak potentials (Epp) that is negatively correlated with the electrochemical rate constant of a redox reaction. The Epp values can be used to estimate their redox reaction resistances.[] The peak current density of reduction (Jred) and oxidation (Jox), and peak separation (Epp) of the five electrodes are listed in Table 1. It is observed that the variation of ALS content caused variance in electrocatalytic activity (ECA) of the PANI CEs. It can be seen from Figure #elsa202100155-fig-0007#7a, the reduction process of P-1.9 is not obvious as it displays smaller current density as well as high Epp compared to others. This clearly depicts relatively weak electrocatalytic ability and lower conductivity of P-1.9 as seen in conductivity analysis.[] With the increasing content of ALS, the current densities of the electrodes (P-2.1 and P-2.3) increase gradually, indicating that more active sites have been created, which might be due to the role of the ALS that induces different morphologies in binary doped PANI samples with different concentrations as exemplified by Figure #elsa202100155-fig-0001#1. According to experimental data, P-2.3 represents the highest value of Jred and Jox, and the smaller Epp value but larger than Pt, reflecting its largest surface area for efficient catalytic reduction of I3- and fastest charge transfer at electrolyte/PANI nanofibers interfaces, which contributes to its high conductivity and electrocatalytic behavior to the I−/I3−redox couple[] as represented in Table 2. Figure #elsa202100155-fig-0007#7b shows the effect of the scan rates on the CV of the P-2.3 CE in I−/I3−electrolyte. With the increase of scan rate, the cathodic peak current densities gradually shift negatively, and the corresponding anodic peak current densities shift positively with increasing scan rates. A good linear relationship between cathodic and anodic peak current densities and the square root of the scan rates for the P-2.3 CE is illustrated in Figure #elsa202100155-fig-0007#7c. The result indicates that the adsorption of iodide species has little influence on the P-2.3 CEs' surface, thus demonstrating no species interaction between the I−/I3−redox couple and the P-2.3 CE as well as the Pt CE.[]

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FIGURE 7Open in figure viewerPowerPoint(a) Cyclic voltammograms of iodide species on PANI films and Pt at scan rate of 50 mV/s, (b) CV curves of P-2.3 at scan rates of 20, 50, 100 and 125 mV/s and (c) relation of cathodic and anodic peaks of P-2.3 vs (scan rate)1/2

In order to evaluate the performance of PANI CEs in solar cell, the J-V curves for the DSSC were measured under standard light irradiation and are shown in Figure #elsa202100155-fig-0008#8a. The device parameters are summarized in Table 3. DSSCs-based on P-1.9 CE exhibited poor conversion efficiency (2%) with low values of Jsc, which manifests low conductivity of the polymer. The low value of FF and Voc depicts the low catalytic activity of P-1.9 CE as these two parameters are largely affected by the electrocatalytic activity of the CE.[] It may be assumed from the fact that this CE has insufficient pores (as explained in SEM) for the diffusion of redox species resulting in the increased availability of recombination between I3− ions and the photoinjected electrons at the CE.[] When using P-2.1 and P-2.3, all photovoltaic parameters increase with a conversion efficiency of 2.7% and 4.54 %, which may arise due to increase in ALS content that gives significant morphology to the material. While P-2.5 (3%) depicts lower efficiency as further increase in ALS content results in the formation of flakes by decreasing porosity in P-2.5 as represented in Figure #elsa202100155-fig-0005#5d. Comparing P-2.3 CE-based DSSC with Pt electrode (4.02%), all the parameters of the DSSC with P-2.3 CE are enhanced. To investigate the durability of DSSC/PANI, the start-up behavior and multiple start/stop (ON/OFF) capability are important parameters. Solar cell performance was monitored for 600 s with the start/stop switching of the DSSCs using P-1.9, P-2.1, P-2.3, and P-2.5 by alternating between turning ON and OFF the illumination, the performance of DSSC/Pt is also examined for comparison (Figure #elsa202100155-fig-0008#8b). On turning ON the illumination, the current density of cell based on P-2.3 CE increases sharply as compared to others and there is no time delay in starting the cell in all CEs. This confirms a rapid light response and hence high electrocatalytic activity of P-2.3 CE toward redox electrolyte. About 96% of the initial photocurrent density was measured after eleven cycles, implying a superior capability of the P-2.3 CE to start multiple times.[]

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FIGURE 8Open in figure viewerPowerPoint(a) Photocurrent density–photovoltage (J–V) characteristics of DSSCs fabricated using different CEs and (b) start-stop switches of DSSC assembled with P-1.9, P-2.1, P-2.3, P-2.5 and Pt CE

EIS has been carried out to elucidate further the electrochemical catalysis of different CEs on the reduction of I3−, by using symmetrical cells fabricated with the fabricated PANI CEs. For comparison purpose, measurements with Pt as a CE were also performed. The Nyquist impedance of DSSCs based on various CEs are shown in Figure #elsa202100155-fig-0009#9. There is a well-defined semicircle for the plots of binary-doped PANI and Pt-based cells. The semicircles in the high-frequency region are assigned to impedance (Rct) related with charge transfer processes occurring at the CE/electrolyte interface. The high-frequency intercept on the real axis represents the series resistance (Rs) of the electrode which describes mainly the resistance of the two identical electrodes and the electrolytic resistance.[]

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FIGURE 9Open in figure viewerPowerPointNyquist plots for the electrochemical cells assembled using various electrode materials and the equivalent circuit

As prepared photoanodes were assembled with Pt and binary doped CEs into sandwich-type cells using hot-melt Surlyn (25μm) (Scheme #elsa202100155-fig-0010#1). A drop of the redox electrolyte (0.04 M I2, 0.4 M LiI, and 0.4 M tetrabutylammonium iodide (TBAI) in 3 methoxypropionitrile (MPN) and acetonitrile (ACN) mixture (1:1) were injected into the cells.

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Figure 1Open in figure viewerPowerPoint(a) Cartoon showing the mechanism of the different behavior of the wild type (1) and the variants (2) of [NiFeSe] hydrogenase when exposed to O2. In the wild-type [NiFeSe] hydrogenase, oxygen reaches the active center of the enzyme (1), whereas in the variants G491A and G491S, the pathway to the active center is partially blocked by altered amino acid residues, which hamper the access of O2 (2). For a detailed structural and mechanistic description, see Ref. 27. (b) Schematic of the high-current-density carbon cloth gas-diffusion H2-oxidation bioanode equipped with a polymer/hydrogenase layer. For the immobilization of the active P(N3MA-BA-GMA)-vio/hydrogenase layer, a carbon cloth-based gas-diffusion layer was first modified with an adhesion layer, that is, the redox polymer P(GMA-BA-PEGMA)-vio, which shows a higher hydrophobic monomer content (for a detailed description of the electrode architecture and electrochemical gas diffusion cell, see Ref. 12). By combining the low-potential redox polymer with a more O2-tolerant hydrogenase variant, a stable high-current-density bioanode is obtained, which can be operated in a membrane-free H2/O2 biofuel cell. (a, b) The structure of the wt-[NiFeSe] from D. vulgaris Hildenborough (5JSH)28 was used as a representative enzyme model; not drawn to scale.

Recently, it was shown that variants (G491A and G491S) of the [NiFeSe] hydrogenase from D. vulgaris Hildenborough with modification in a specific amino acid close to the active site led to enhanced stability of the biocatalyst in the presence of O2 while retaining a high activity for H2-oxidation [G491A: up to (4080±80) s−1; G491S: up to (2810±150) s−1, wild type: ≈(4850±260) s−1] and a redox potential that is still close to the H2/2 H+ couple [approximately −450 mV vs. standard hydrogen electrode (SHE) at pH 7]. The enhanced stability of the altered proteins was attributed to a physical blocking effect of the O2 molecule in a hydrophilic channel that connects the active site of the protein with the enzyme surface, thus preventing oxidation of a specific active-site cysteine ligand (Figure #cssc202000999-fig-0001#1 a; for a more comprehensive description of the structural changes inside the protein shell of the enzyme that lead to the desired O2-blocking effect, see Ref. ). This effect was evidenced by protein film electrochemistry conducted in a direct electron transfer (DET) regime and in the presence and absence of O2. However, the DET mode does not provide any protection against high-potential inactivation or against high O2 concentrations and is hence impractical for potential applications. Nevertheless, it shows that variants of this type of hydrogenase can be prepared with enhanced O2 stability. Figure #cssc202000999-fig-0003#3 shows chronoamperometric experiments at an applied potential (Eappl) of +160 mV (vs. SHE) under alternating gas-mixture atmospheres. The O2 content in the gas feed was stepwise increased after each H2 cycle. To ensure that all H2 had been removed before the O2 was added to the gas feed, the cell was purged with argon. After the background current was reached, the film was exposed to an O2/Ar mixture with varying O2 content (5, 10, and 15 %, gray shaded areas in Figure #cssc202000999-fig-0003#3). The wild type shows a steady decrease of the H2-oxidation activity over all O2/Ar cycles. After exposure to 15 % O2, the electrode remains inactive when switching the gas feed back to H2. This is consistent with a fast in-diffusion of O2 to the active center of the enzyme (Figure #cssc202000999-fig-0001#1 a). In contrast, both variants show a rather constant current output after the 5 and 10 % O2 cycle. Moreover, even after exposure to 15 % O2, both electrodes still show a remarkable activity towards H2-oxidation (G491A: ≈20 % of the initial H2-oxidation current; G491S: ≈35 %). The results demonstrate that the variants indeed exhibit an increased O2 tolerance compared with the wild-type enzyme owing to a partial blocking of molecular oxygen (hampered access) based on the altered amino acid residues in the variants (Figure #cssc202000999-fig-0001#1 a) and, by this, that the variants provide an additional protection for the proposed H2-oxidation bioanodes. The electrochemical results obtained with the polymer/enzyme films are in line with the results reported for operating the same hydrogenases in the DET regime. However, strong variations in the residual currents were observed after exposure to O2, which is attributed to variations in film thickness and inhomogeneities of the catalytic layers, leading to different diffusion profiles of O2. However, in all experiments the variants showed a higher stability towards O2. Recently, we showed that the use of gas-diffusion layers modified with polymer/wt-[NiFeSe] and polymer/[NiFe] films displayed enhanced power output owing to an enhanced mass transport of the gaseous substrate H2 towards the bioanode. Current densities for the bioanode of close to 8 mA cm−2 and power densities of 3.8 mW cm−2 for biofuel cells with a bilirubin oxidase-modified gas-diffusion biocathode were observed. To demonstrate the relevance of the O2-tolerant [NiFeSe] variants, carbon cloth-based gas-diffusion layers were first modified with P(GMA-BA-PEGMA)-vio [poly(glycidyl methacrylate-co-butyl acrylate-co-poly(ethylene glycol)methacrylate)-vio; for the structure and synthesis of this polymer, see Figure S1 in the Supporting Information and Ref. , respectively] films followed by the immobilization of an active P(N3MA-BA-GMA)-vio/G491S layer [Figure #cssc202000999-fig-0001#1 b; for a detailed description of the preparation process see the Experimental Section; electrodes are denoted as P(GMA-BA-PEGMA)-vio//P(N3MA-BA-GMA)-vio/G491S; owing to the limited amount of enzyme, only the variant G491S was used for the preparation of a H2-oxidation gas-diffusion layer]. Cyclic voltammograms (Figure S8 in the Supporting Information) measured before and after biofuel cell operation showed similar values for the bioanode, with the slightly higher currents after the biofuel cell test most likely as a result of changed diffusion properties inside the polymer/enzyme layer, for example, owing to swelling and/or slightly changed local pH values, which will affect the overall activity of the enzyme. In contrast, the current of the biocathode was slightly decreased after the biofuel evaluation (Figure S7 in the Supporting Information). This again highlights the high stability of the bioanode in a membrane-free biofuel cell under anode-limiting conditions. The operational stability of the biofuel cell was tested at a constant load of 0.7 V (Figure #cssc202000999-fig-0005#5 c). After 10 h of continuous operation, 75 % of the initial current density remained. Cyclic voltammograms measured after the long-term experiment showed significantly lower currents for the bioanode (Figure S8 in the Supporting Information) and the biocathode (Figure S7 in the Supporting Information). We want to emphasize that the amplitudes of the polymer signals (Figure S7 in the Supporting Information, dashed black curve) were also decreased compared with the voltammograms measured with the freshly prepared electrode. Thus, not only does deactivation/decomposition of the enzyme contribute to the decreased activity after long-term operation, but the loss of immobilization matrix may also have an effect. Nevertheless, the bioanode shows an outstanding performance and demonstrates the potential applicability of G941S (enhanced O2 tolerance) as a highly active and stable catalyst in a membrane-free biofuel cell device. Moreover, the proposed H2-oxidation bioanodes combine the advantages of the protection matrix (O2 quenching; no high-potential deactivation) and the enhanced enzyme stability of the hydrogenase variants (blocking of O2 access) in accordance with the mechanism depicted in Figure #cssc202000999-fig-0001#1 b and thus demonstrate a triple-protection system for the high-current-density H2-oxidation bioanodes.

21510_cssc202000999-fig-0002.jpg

Figure 2Open in figure viewerPowerPointCyclic voltammograms recorded at a scan rate of 10 mV s−1 of (a) P(N3MA-BA-GMA)-vio/G491A and (b) P(N3MA-BA-GMA)-vio/G491S films immobilized on glassy carbon disk electrodes (3 mm Ø) in the absence (black lines, 100 % Ar, purged through solution) and presence of H2 (red lines, 100 % H2, purged through solution). Working electrolyte: phosphate buffer, 0.1 m, pH 7.3, room temperature.

Indeed, cyclic voltammograms of drop-cast P(N3MA-BA-GMA)-vio/G419A and P(N3MA-BA-GMA)-vio/G419S films measured under turnover conditions, that is, under H2 atmosphere (Figure #cssc202000999-fig-0002#2 a, b, red curves), showed pronounced catalytic H2-oxidation waves with half-wave potentials (a and b: approximately −0.32 V vs. SHE), which matches closely the mid-point potential of the polymer-bound viologen units (≈0.34 V vs. SHE, black curves). The behavior is in line with the results measured for the wild type (Figure S2 in the Supporting Information). We hence conclude that the [NiFeSe] variants can also be productively wired through the redox polymer P(N3MA-BA-GMA)-vio in a mediated electron-transfer regime. Moreover, long-term chronoamperometric measurements over 7 h under continuous turnover conditions showed similar operational stability for the wild type and the two variants (Figure S3 in the Supporting Information). The steady-state current observed at high potentials (>−0.2 V vs. SHE, Figure #cssc202000999-fig-0002#2, red lines) under turnover conditions indicates that the variants can also be effectively protected against high-potential inactivation in contrast to the operation under DET conditions for this type of hydrogenases. Oxygen-tolerant hydrogenases typical display higher redox potentials, which will decrease the maximum OCV of a corresponding biofuel cell compared with their O2-sensitive analogues. However, because the O2-tolerant variants G491A and G941S show similar H2-oxidation potentials as the wild type, the electrical wiring is possible with the same polymer (Figure #cssc202000999-fig-0002#2). Hence, we expect similar OCV values for related biofuel cells as for those based on the wild-type hydrogenase. To evaluate the performance of the bioanodes in a biofuel cell, polymer/hydrogenase-modified glassy carbon electrodes were combined with a gas-diffusion O2-reducing bilirubin oxidase-based biocathode. The use of a gas-diffusion system at the cathode side, in which mass transport is not limiting, ensures bioanode-limiting conditions. The biocathode was prepared with bilirubin oxidase from Bacillus pumilus (Bp-BOD), a stable multi-copper oxidase used previously in biofuel cells, by drop-casting a Bp-BOD stock solution (borate buffer, 50 mm, pH, pH 9, 54.75 mg mL−1) onto a carbon cloth-based gas-diffusion layer equipped with a conducting microporous Nafion/Teflon/carbon layer with enhanced surface area (for a detailed description of the immobilization process, see the Experimental Section). In cyclic voltammograms, maximum absolute currents for O2 reduction of approximately 180 μA were observed when the gas-diffusion electrode was exposed to air (Figure S5 in the Supporting Information). These values are significantly higher than those obtained for the polymer/hydrogenase-modified glassy carbon electrodes exhibiting maximum absolute currents <80 μA for all hydrogenases. Under gas-diffusion conditions, the bioanode showed absolute H2-oxidation currents of approximately 0.8 mA (Figure #cssc202000999-fig-0005#5 a). The modified surface area of the carbon cloth-based bioanode has a diameter of approximately 4 mm, which results in a surface area of the active layer of approximately 0.126 cm−2, and thus maximum current densities of 6.3 mA cm−2 were achieved. The values are similar to previously reported polymer-based gas-diffusion systems equipped with wt-[NiFeSe] and [NiFe] hydrogenases (Table S1 in the Supporting Information). However, care must be taken when comparing current densities measured with porous electrodes. Because of the 3D structure of the electrodes, the real surface is often unknown. Hence, the catalyst loading is a better value for comparison. For the G491S-based electrodes, the catalyst loading is 8.4 nmol cm−2/1.06 nmol electrode−1. Interestingly, for the wt-[NiFeSe] hydrogenase, current densities of only 5.3 mA cm−2 were observed with a substantially higher catalyst loading of 27.0 nmol cm−2/3.4 nmol electrode−1 as reported in our previous work (see Ref.  and Table S1 in the Supporting Information), which largely exceeds the values of the G491S variant at almost identical overall polymer loading [wt-[NiFeSe]: 230 μg electrode−1 (previous work, Ref. ); G491S: 260 μg electrode−1]. At a lower catalyst loading of 12.1 nmol cm−2/1.53 nmol electrode−1 (polymer loading 230 μg electrode−1), which is only slightly higher than the loading of the G491S enzyme, the wt-[NiFeSe] shows a Jmax value of only 3.6 mA cm−2 (see Ref. ). This effect might be related to an improved incorporation of the G941S variant in the polymer film when immobilized on the rather hydrophobic carbon cloth-based electrodes. A stronger interaction prevents leaching of the enzyme and thus ensures a higher local concentration of the biocatalyst during the experiment. In addition, a loss of activity in the immobilized state for the wild type may also contribute to a reduced electrode activity. An effect of different polymer-to-enzyme ratios can be ruled out because almost identical polymer loadings were used for all experiments. However, the effect seems to be specific for the porous, hydrophobic carbon cloth electrodes because the wild type shows a higher activity on flat glassy carbon electrodes (see Figure #cssc202000999-fig-0002#2 and Figure S2 in the Supporting Information).

21510_cssc202000999-fig-0003.jpg

Figure 3Open in figure viewerPowerPointRepresentative chronoamperometric experiments with (a) P(N3MA-BA-GMA)-vio/wt-[NiFeSe], (b) P(N3MA-BA-GMA)-vio/G491A, and (c) P(N3MA-BA-GMA)-vio/G491S films immobilized on glassy carbon disk electrodes (3 mm Ø) under alternating gas feeds (90 % H2/10 % Ar, 100 % Ar as well as 5 % O2/95 %Ar, 10 % O2/90 % Ar, and 15 % O2/85 % Ar). Working conditions: phosphate buffer, 0.1 m, pH 7.3, room temperature; Eappl=+160 mV vs. SHE.

Figure #cssc202000999-fig-0003#3 shows chronoamperometric experiments at an applied potential (Eappl) of +160 mV (vs. SHE) under alternating gas-mixture atmospheres. The O2 content in the gas feed was stepwise increased after each H2 cycle. To ensure that all H2 had been removed before the O2 was added to the gas feed, the cell was purged with argon. After the background current was reached, the film was exposed to an O2/Ar mixture with varying O2 content (5, 10, and 15 %, gray shaded areas in Figure #cssc202000999-fig-0003#3). The wild type shows a steady decrease of the H2-oxidation activity over all O2/Ar cycles. After exposure to 15 % O2, the electrode remains inactive when switching the gas feed back to H2. This is consistent with a fast in-diffusion of O2 to the active center of the enzyme (Figure #cssc202000999-fig-0001#1 a). In contrast, both variants show a rather constant current output after the 5 and 10 % O2 cycle. Moreover, even after exposure to 15 % O2, both electrodes still show a remarkable activity towards H2-oxidation (G491A: ≈20 % of the initial H2-oxidation current; G491S: ≈35 %). The results demonstrate that the variants indeed exhibit an increased O2 tolerance compared with the wild-type enzyme owing to a partial blocking of molecular oxygen (hampered access) based on the altered amino acid residues in the variants (Figure #cssc202000999-fig-0001#1 a) and, by this, that the variants provide an additional protection for the proposed H2-oxidation bioanodes. The electrochemical results obtained with the polymer/enzyme films are in line with the results reported for operating the same hydrogenases in the DET regime. However, strong variations in the residual currents were observed after exposure to O2, which is attributed to variations in film thickness and inhomogeneities of the catalytic layers, leading to different diffusion profiles of O2. However, in all experiments the variants showed a higher stability towards O2.

21510_cssc202000999-fig-0004.jpg

Figure 4Open in figure viewerPowerPointChronoamperometric experiments with aerobically deactivated (a) P(N3MA-BA-GMA)-vio/G491A and (b) P(N3MA-BA-GMA)-vio/G491S films immobilized on glassy carbon disk electrodes (3 mm Ø). First, a potential of −440 mV vs. SHE was applied for 500 s to fully reduce the viologen-modified polymer (the enzyme is reactivated during reduction via the polymer). After switching the potential to +160 mV vs. SHE (t>500 s), H2-oxidation currents indicate successful reactivation. Working conditions: phosphate buffer, 0.1 m, pH 7.3, room temperature, electrodes were deactivated by extensive exposure to O2 until any H2-oxidation current was absent.

To evaluate a possible reactivation behavior of the two [NiFeSe] hydrogenase variants, glassy carbon electrodes modified with P(N3MA-BA-GMA)-vio/G491A and P(N3MA-BA-GMA)-vio/G491S films were exposed to O2 until complete inactivation occurred, as evidenced by the current dropping back to background values. Application of a negative potential of −440 mV (vs. SHE; polymer is fully reduced, inactive mediator form) for 500 s leads to reactivation of the enzyme (Figure #cssc202000999-fig-0004#4) as indicated by the oxidative currents, which were observed again when the potential was stepped back to +160 mV (vs. SHE; t>500 s, mediator is oxidized, active form). Both potentials were applied under a 90 % H2/10 % Ar gas feed. The wild type shows the same behavior (see Figure S4 in the Supporting Information and Ref. ).

21510_cssc202000999-fig-0005.jpg

Figure 5Open in figure viewerPowerPointCharacterization of (a) the P(GMA-BA-PEGMA)-vio//P(N3MA-BA-GMA)-vio/G491S-based gas-diffusion bioanode and (b, c) biofuel cells equipped with a Mv-BOD-based gas-diffusion O2-reducing biocathode (operated in 100 % O2) in 0.1 m phosphate buffer, pH 7.4. (a) Cyclic voltammograms of the gas-diffusion P(N3MA-BA-GMA)-vio/G491S bioanode in the absence (black curve) and presence of H2 (red curve); scan rate=5 mV s−1. (b) Current density (red squares, right ordinate) and power density (black squares, left ordinate, with respect to the geometric surface area of the modified part of the bioanode, ≈0.126 cm−2). (c) Operational stability of the biofuel cell over 10 h at 0.7 V. Nominal biocatalyst loading: 8.4 nmol cm−2/1.06 nmol electrode−1.

Under gas-diffusion conditions, the bioanode showed absolute H2-oxidation currents of approximately 0.8 mA (Figure #cssc202000999-fig-0005#5 a). The modified surface area of the carbon cloth-based bioanode has a diameter of approximately 4 mm, which results in a surface area of the active layer of approximately 0.126 cm−2, and thus maximum current densities of 6.3 mA cm−2 were achieved. The values are similar to previously reported polymer-based gas-diffusion systems equipped with wt-[NiFeSe] and [NiFe] hydrogenases (Table S1 in the Supporting Information). However, care must be taken when comparing current densities measured with porous electrodes. Because of the 3D structure of the electrodes, the real surface is often unknown. Hence, the catalyst loading is a better value for comparison. For the G491S-based electrodes, the catalyst loading is 8.4 nmol cm−2/1.06 nmol electrode−1. Interestingly, for the wt-[NiFeSe] hydrogenase, current densities of only 5.3 mA cm−2 were observed with a substantially higher catalyst loading of 27.0 nmol cm−2/3.4 nmol electrode−1 as reported in our previous work (see Ref.  and Table S1 in the Supporting Information), which largely exceeds the values of the G491S variant at almost identical overall polymer loading [wt-[NiFeSe]: 230 μg electrode−1 (previous work, Ref. ); G491S: 260 μg electrode−1]. At a lower catalyst loading of 12.1 nmol cm−2/1.53 nmol electrode−1 (polymer loading 230 μg electrode−1), which is only slightly higher than the loading of the G491S enzyme, the wt-[NiFeSe] shows a Jmax value of only 3.6 mA cm−2 (see Ref. ). This effect might be related to an improved incorporation of the G941S variant in the polymer film when immobilized on the rather hydrophobic carbon cloth-based electrodes. A stronger interaction prevents leaching of the enzyme and thus ensures a higher local concentration of the biocatalyst during the experiment. In addition, a loss of activity in the immobilized state for the wild type may also contribute to a reduced electrode activity. An effect of different polymer-to-enzyme ratios can be ruled out because almost identical polymer loadings were used for all experiments. However, the effect seems to be specific for the porous, hydrophobic carbon cloth electrodes because the wild type shows a higher activity on flat glassy carbon electrodes (see Figure #cssc202000999-fig-0002#2 and Figure S2 in the Supporting Information). To evaluate the performance of the gas-diffusion P(GMA-BA-PEGMA)-vio//P(N3MA-BA-GMA)-vio/G491S electrode in an all-gas-diffusion membrane-free H2/O2 biofuel cell, the bioanode was combined with an O2-reducing biocathode modified with bilirubin oxidase from Myrothecium verrucaria (Mv-BOD, for comparison purposes because it was used in our previously reported experiments). For the immobilization of Mv-BOD, the carbon cloth was first modified with 2-ABA (2-amino benzoic acid) to ensure a proper orientation of the enzyme on the electrode surface. The modifier was anchored in an electrochemical grafting process by applying an oxidative potential pulse. The Mv-BOD was then immobilized by means of a conventional drop-casting process and was operated in the DET regime. A high catalyst loading was used to ensure anode-limiting conditions (nominal enzyme loading: 1.2 mg electrode−1). Absolute currents under gas-diffusion conditions (100 % O2) reached approximately 2 mA (Figure S7 in the Supporting Information), which largely outperforms the bioanode (≈0.8 mA, Figure #cssc202000999-fig-0005#5 a). The fully assembled H2/O2 biofuel cell (Figure #cssc202000999-fig-0005#5 b) showed an OCV of 1.14 V, which is slightly higher than the values obtained on glassy carbon electrodes (1.05–1.06 V); this might be attributed to the slightly lower overpotential for O2 reduction of Mv-BOD compared with Bp-BOD. The maximum power density was reached at 0.7 V and was estimated to be 4.4 mW cm−2. This value even outperforms our previously reported value for the [NiFe]-based biofuel cell (3.6 mW cm−2) and—to the best of our knowledge—sets a new benchmark for a biofuel cell using redox-polymer-based bioanodes (Table S1 in the Supporting Information). Moreover, the catalyst loading is significantly lower than the [NiFe] system (31.8 nmol cm−2/4 nmol electrode−1) reported previously (Table S1 in the Supporting Information). Cyclic voltammograms (Figure S8 in the Supporting Information) measured before and after biofuel cell operation showed similar values for the bioanode, with the slightly higher currents after the biofuel cell test most likely as a result of changed diffusion properties inside the polymer/enzyme layer, for example, owing to swelling and/or slightly changed local pH values, which will affect the overall activity of the enzyme. In contrast, the current of the biocathode was slightly decreased after the biofuel evaluation (Figure S7 in the Supporting Information). This again highlights the high stability of the bioanode in a membrane-free biofuel cell under anode-limiting conditions. The operational stability of the biofuel cell was tested at a constant load of 0.7 V (Figure #cssc202000999-fig-0005#5 c). After 10 h of continuous operation, 75 % of the initial current density remained. Cyclic voltammograms measured after the long-term experiment showed significantly lower currents for the bioanode (Figure S8 in the Supporting Information) and the biocathode (Figure S7 in the Supporting Information). We want to emphasize that the amplitudes of the polymer signals (Figure S7 in the Supporting Information, dashed black curve) were also decreased compared with the voltammograms measured with the freshly prepared electrode. Thus, not only does deactivation/decomposition of the enzyme contribute to the decreased activity after long-term operation, but the loss of immobilization matrix may also have an effect. Nevertheless, the bioanode shows an outstanding performance and demonstrates the potential applicability of G941S (enhanced O2 tolerance) as a highly active and stable catalyst in a membrane-free biofuel cell device. Moreover, the proposed H2-oxidation bioanodes combine the advantages of the protection matrix (O2 quenching; no high-potential deactivation) and the enhanced enzyme stability of the hydrogenase variants (blocking of O2 access) in accordance with the mechanism depicted in Figure #cssc202000999-fig-0001#1 b and thus demonstrate a triple-protection system for the high-current-density H2-oxidation bioanodes.

12500_cmtd202100058-fig-0001.jpg

Figure 1Open in figure viewerA) Schematics of the combined XPS-Raman setup based on long-range Raman measurements comprised of: I – Hardware control, II – XPS chamber, III – Raman probe head with laser-focusing camera objective, IV – optical excitation/signal collection fibers, V – optics laser entrance, VI – laser focusing lens, VII – solid-state laser, VIII – Peltier chiller, IX – Raman spectrometer. B) The combined XPS-Raman setup in use. C) Finite difference time domain simulations of the laser spot on the sample surface for 532 nm excitation, a distance of 285 mm, and a 33 mm lens (left), as well as for a casual 0.5x magnification, 532 nm excitation, and 285 mm distance (right). The bimodal laser spot is a combination of the double-objective lens and the incident angle, which results in two focal points f2d and f1a. Images of the experimental laser spots resemble the simulated bimodal distribution (see Figure 2).

The longest Raman working distance from the objective to the sample reported to date in specialized systems of mirrors and UHV is 120 mm. Here, we present an inexpensive and straightforward approach for combined XPS and Raman spectroscopic analysis without the need for constructional changes, where the XPS and Raman foci can be adjusted separately. Raman spectra are recorded over a distance of 285 mm from the sample from outside the XPS analysis chamber, as illustrated in Figure #cmtd202100058-fig-0001#1A/B. The pathway of the laser beam and its divergence were simulated by the finite difference time domain (FDTD) method and were found to be in good agreement with the experimental results (see Figure #cmtd202100058-fig-0001#1C). With the novel setup, powder materials with Raman cross-sections of at least 10−30 cm2 sr−1 are accessible, enabling the analysis of a large variety of battery, catalytic, and sensing materials. Here, the applicability of our approach is illustrated in the context of Li-ion batteries by detailed analysis of the cathode material γ-LixV2O5 prior to and after electrochemistry. We also present an optimization procedure of the Raman-XPS signal adjustment based on the use of a (400) single crystal (SC) diamond, performed outside the analysis chamber. Media of various refractive indices influence the divergence of the laser light along its path to the sample and back. In the present case shown in Figure #cmtd202100058-fig-0001#1, the laser beam emitted by the probe-head (III) is focused by the camera objective into the XPS-chamber under UHV (10−8 mbar). Thereby, the laser beam travels from the source (VII) to the Raman beam-splitting system (III) and then changes the medium from air to the XPS-flange borosilicate glass and finally to vacuum when entering the analysis chamber. Figure #cmtd202100058-fig-0001#1C shows results of FDTD simulations of the Maxwell equations describing the 532 nm laser beam in a distance of 285 mm from the lens (at the end of III) to the sample at an angle of incidence of 35°. The simulations considered two refractive indices, that is, that of the air outside the chamber, and that of the vacuum within the chamber. The presence of a borosilicate window in the XPS-flange causes additional deflection of the beam, as illustrated in Figure #cmtd202100058-fig-0001#1C, and adds to signal loss. The second simulation, based on a casual long-range objective (0.5x, NA=0.02) shows the expected result, that is, the failure of focusing the laser beam in one spot due to the angle of incidence. In addition, the intensity of the scattered laser light is significantly lower as compared to the focus conditions present in our setup. One of the characteristics of our measurement system is the difference in environments. The laser passes through media of various refractive indices. The scattered light comes into contact with the camera objective by passing through the glass-protected flange, allowing a transmission of 80 % and up to 550 nm as shown in Figures #cmtd202100058-fig-0001#1B and C. At this incident point, which is most probably responsible for the majority of intensity loss in the Raman spectrum, we were able to obtain a spectrum matching that of silicon dioxide. This might be the source of the observed fluorescence background, which was subtracted from Figure #cmtd202100058-fig-0002#2 for clarity. The raw spectrum is included in the Supporting Information (see Figure S2). where θ stands for the divergence. Df and Di are the beam diameters outside the focal points and l is the distance between those points. Additionally, it is possible to narrow the focal laser spot to smaller values and therefore to reach higher Raman intensities by changing the distance between the flange and lens (with simultaneous variation of the focal points f1 and f2), as shown in Figure #cmtd202100058-fig-0001#1, according to the principle of the lens equation. Once the distance from the focal point was increased, the intensity of the D band dropped monotonically. Please note that the intensity of the D band was normalized by the intensity at 220 mm distance from the sample where the intensity was the highest. We observed a dependence of the incidence angle on Raman intensity, where an angle of 15° turned out to yield a higher intensity than 10° at the same distance. We relate these observations to the presence of the bimodal laser spot distribution shown in Figure #cmtd202100058-fig-0001#1, which possibly generates two Raman hot spots that contribute synergistically to the gathered signal. We have also performed the measurements of the full width at half maximum (FWHM), which was found to increase with the distance from the source. This behavior may be explained by spectral diffusion, a well-known phenomenon in long-distance Raman measurements. It stands to reason that the target geometry is associated with the highest intensity and lowest FWHM, which according to Figure #cmtd202100058-fig-0003#3, at a distance of 285 mm, is obtained for incidence angles of 30° and 45°. As demonstrated above, the combination of Raman and XP spectroscopy provides a unique possibility for both surface (photoelectrons) and bulk (phonon) analysis of functional materials. We employed green laser excitation, which penetrates the XPS-machine shielding glass (Figure #cmtd202100058-fig-0001#1A), yielding, as a compromise, a signal with some fluorescence background. By the combined XP-Raman spectroscopy analysis, the electrode-electrolyte interaction has been elucidated in detail. Besides the potential-dependent polymorphization, as evidenced by the Raman spectra in Figure #cmtd202100058-fig-0004#4D, the decomposition of electrolyte has been accessible by XPS surface analysis as shown in Figure #cmtd202100058-fig-0004#4C and Tables 1/2. Owing to its potential for the characterization of energy storage materials, further optimization towards in situ/operando XP-Raman spectroscopy with electrochemistry would be desired in the future in order to monitor the potential-dependent changes directly. For combined XP-Raman spectroscopy analysis the material was pressed on an Al2O3 mesh and inserted into the XPS analysis chamber with a base pressure of 2 ⋅ 10−8 mbar and equipped with standard vacuum viewport windows (see Figure #cmtd202100058-fig-0001#1).

12500_cmtd202100058-fig-0002.jpg

Figure 2Open in figure viewerA) Raman spectrum of of γ-LixV2O5 recorded over a 285 mm distance and at an angle of 35°. The inset shows a picture of the laser spot inside the analysis chamber. B) Profile of the long-distance laser spot. C) Profile of a laser spot in conventional Raman microscopy. For details, see main text.

As an example, Figure #cmtd202100058-fig-0002#2A shows the experimental result of the laser beam focused at an angle of 35°, exhibiting a similar bimodal distribution as predicted by the FDTD simulations. In addition, the order of magnitude of the sizes of laser spots in the simulations showed similar trends to those measured in experiment. The spot size of roughly 500 μm necessitates using higher laser powers of up to 35 mW, as the intensity for such a large spot will be about 100 times than that of common Raman microscopes using typical spot sizes of a few μm. One of the characteristics of our measurement system is the difference in environments. The laser passes through media of various refractive indices. The scattered light comes into contact with the camera objective by passing through the glass-protected flange, allowing a transmission of 80 % and up to 550 nm as shown in Figures #cmtd202100058-fig-0001#1B and C. At this incident point, which is most probably responsible for the majority of intensity loss in the Raman spectrum, we were able to obtain a spectrum matching that of silicon dioxide. This might be the source of the observed fluorescence background, which was subtracted from Figure #cmtd202100058-fig-0002#2 for clarity. The raw spectrum is included in the Supporting Information (see Figure S2). The size of the material under investigation was in the range of several hundreds of micrometers, matching the size of the spot achieved by the 35°-tilted laser beam depicted in Figure #cmtd202100058-fig-0002#2. If the size of the material is considerably smaller, for example, several micrometers, the recording of long-distance Raman spectra becomes a challenge due to the decreased intensity of the incident radiation. This limitation may be overcome by the use of a laser source with higher power. In our experiments, we set the laser power to 35 mW, which may be detrimental at short distance, but did not lead to any laser-induced effects of the sample due to the large reduction in laser intensity (see also above). An example of such a Raman spectrum of γ-LixV2O5 is shown in Figure #cmtd202100058-fig-0002#2A. The corresponding XPS spectra from the combined experiment are presented in Figure #cmtd202100058-fig-0004#4 and will be discussed below. The Raman-active modes of γ-LixV2O5 were elucidated from a standard symmetry adapted linear combination (SALC) approach, in good agreement with the literature. To further specify the aspects relevant to the recording of long-distance Raman spectra, we have performed a series of measurements as a function of the geometry of incidence (see also below). Regarding the spot size, we found a dependence of the incidence angle on the shape and on the spot diameter. For example, at 35°, we found the spot size to be 552.3±15.2 μm, approximately matching the size of the particles in the cathode material. Based on our results, we postulate that the applicability of long-distance Raman spectroscopy is facilitated by matching the measurement spot size with a homogenously composed piece of material. Established strategies to transform the size of a material and improve the homogenization are ball-milling, ultrasonication, and focused ion beam (FIB)-induced transformation. Please note that all these techniques require pure conditions and bear a jeopardy of cross contamination (FIB: foreign ions, for example, Cr; ball milling: Fe compounds; ultrasonication: incorporation of Ti). An influence of the spot size on sample properties was not observed in conventional short-range Raman measurements, which were performed as a control (see Figure #cmtd202100058-fig-0002#2). The S/N ratio of the spectrometer readout of the long-distance measurements (285 mm, diameter: 552.3±15.2 μm) compared to standard Raman measurements (spot diameter: 4.1±0.5 μm) decreased by a factor of 10, which was confirmed by utilizing the same laser power and acquisition time for the material in focus, that is, γ-LixV2O5 and (400)-CVD grown SC diamond. The S/N ratio was established by consecutive acquisition of three spectra and dividing the band intensity by the mean of the band intensity over three measurements. The lower S/N ratio in the long-distance measurements is caused by the relatively large laser spot as verified by experiment (see Figure #cmtd202100058-fig-0002#2). The laser fluence on a spot of the same size (552.3 μm) was estimated outside the XPS chamber under ambient conditions and was found to be 1461 W cm−2.

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Figure 3Open in figure viewerComparison of the geometries of laser incidence on the sample based on measurements of the Raman D-band of (400)-CVD grown SC diamond outside the ultrahigh vacuum chamber with fitted 95 % confidence intervals. The intensity I0 refers to the D-band intensity at 1332 cm−1 at 90° incidence. We note the fact that the root-mean square roughness (rms) of the sample will have an influence on the incidence and reflection angle of the laser/Raman signal. In case of the diamond sample, the rms was estimated to be less than 1 nm. Therefore this statement corresponds to materials where the grain size reaches the same order as the Abbé limit (meaning roughly 200 nm).33

The results of angle-dependent measurements are shown in Figure #cmtd202100058-fig-0003#3. For flexibility, the measurements were performed outside the XPS analysis chamber, allowing to use a wide range of angles in several geometries. In the upright geometry, the beam was directly reflected back to the spectrometer, corresponding to an angle of incidence of 90°. Once the distance from the focal point was increased, the intensity of the D band dropped monotonically. Please note that the intensity of the D band was normalized by the intensity at 220 mm distance from the sample where the intensity was the highest. We observed a dependence of the incidence angle on Raman intensity, where an angle of 15° turned out to yield a higher intensity than 10° at the same distance. We relate these observations to the presence of the bimodal laser spot distribution shown in Figure #cmtd202100058-fig-0001#1, which possibly generates two Raman hot spots that contribute synergistically to the gathered signal. We have also performed the measurements of the full width at half maximum (FWHM), which was found to increase with the distance from the source. This behavior may be explained by spectral diffusion, a well-known phenomenon in long-distance Raman measurements. It stands to reason that the target geometry is associated with the highest intensity and lowest FWHM, which according to Figure #cmtd202100058-fig-0003#3, at a distance of 285 mm, is obtained for incidence angles of 30° and 45°.

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Figure 4Open in figure viewerElectrochemical and spectroscopic data of LixV2O5 recorded by the combined XP-Raman spectroscopy setup. A) Chronopotentiometric curves of 2 μA charging over time. B) Differential pulse voltammograms showing the redox activity of the sample. C) XPS O 1s and V 2p photoemissions together with the results of a fit analysis. D) Long-distance Raman bands showing the symmetric stretching mode of vanadium oxide (normalized to the V=O intensity in V2O5). For details, see the main text.

The size of the material under investigation was in the range of several hundreds of micrometers, matching the size of the spot achieved by the 35°-tilted laser beam depicted in Figure #cmtd202100058-fig-0002#2. If the size of the material is considerably smaller, for example, several micrometers, the recording of long-distance Raman spectra becomes a challenge due to the decreased intensity of the incident radiation. This limitation may be overcome by the use of a laser source with higher power. In our experiments, we set the laser power to 35 mW, which may be detrimental at short distance, but did not lead to any laser-induced effects of the sample due to the large reduction in laser intensity (see also above). An example of such a Raman spectrum of γ-LixV2O5 is shown in Figure #cmtd202100058-fig-0002#2A. The corresponding XPS spectra from the combined experiment are presented in Figure #cmtd202100058-fig-0004#4 and will be discussed below. The Raman-active modes of γ-LixV2O5 were elucidated from a standard symmetry adapted linear combination (SALC) approach, in good agreement with the literature. Prior to applications, we validated the use of the novel XPS-Raman setup by experiments on commercial V2O5, the separate XP and Raman spectra of which are well documented in the literature. While V2O5, in the current context, is mainly employed as a methodical/spectroscopic standard, it has been widely applied, for example in electrochemistry, heterogeneous catalysis, and gas sensing. In order to investigate the potential of the coupling between XP and long-distance Raman spectroscopy for applications in the context of Li-ion batteries, we performed a series of measurements on γ-LixV2O5-based cathode materials, comparing their state prior to charging (reference) to that after charging. To this end, γ-LixV2O5 was charged up to 4.87 V versus Li/Li+ in a two-electrode combination (see Figure #cmtd202100058-fig-0004#4A), while the reference state of γ-LixV2O5 was left uncharged and without electrolyte contact. The results from the combined XPS-Raman measurements are summarized in Tables 1 and 2 and Figure #cmtd202100058-fig-0004#4 and will be discussed in the following. Additional data is provided in the Supporting Information. The electrochemical measurements on the γ-LixV2O5 show two distinct peaks responsible for the vanadium activity, as proven by differential pulse voltammetry (DPV, see Figure #cmtd202100058-fig-0004#4B). The lack of binder did not affect the electrochemical measurements, which yielded results expected from the literature. The anodic peak at 3.15 V versus Li/Li+ corresponds to the γ-ϵ phase transformation upon lithium exertion from LixV2O5 and the anodic peak at 3.4 V versus Li/Li+ to the V4+ to V5+ oxidation due to electron stripping. In the course of the potentiostatic measurement shown in Figure #cmtd202100058-fig-0004#4A, the Li+ was exerted from the γ-LixV2O5 bi-layered structure and the Raman spectrum was slowly moving towards a V2O5 state. This was observed as a blue-shift of the vanadyl stretching mode (see Figure #cmtd202100058-fig-0004#4D). However, upon Li+ de-intercalation, the sample did not adopt the pure V2O5 state but rather underwent a polymorphic transformation from γ-LixV2O5 to ϵ-LixV2O5, that is, the Li-impoverished polytype of LixV2O5. As demonstrated above, the combination of Raman and XP spectroscopy provides a unique possibility for both surface (photoelectrons) and bulk (phonon) analysis of functional materials. We employed green laser excitation, which penetrates the XPS-machine shielding glass (Figure #cmtd202100058-fig-0001#1A), yielding, as a compromise, a signal with some fluorescence background. By the combined XP-Raman spectroscopy analysis, the electrode-electrolyte interaction has been elucidated in detail. Besides the potential-dependent polymorphization, as evidenced by the Raman spectra in Figure #cmtd202100058-fig-0004#4D, the decomposition of electrolyte has been accessible by XPS surface analysis as shown in Figure #cmtd202100058-fig-0004#4C and Tables 1/2. Owing to its potential for the characterization of energy storage materials, further optimization towards in situ/operando XP-Raman spectroscopy with electrochemistry would be desired in the future in order to monitor the potential-dependent changes directly.

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Scheme 1Open in figure viewerPowerPointThree synthetic routes to substituted pyrido[3,2-d]pyrimidines 1 and structures of bis-pyrido[3,2-d]pyrimidines 8 and polyazapentacene 9.

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Scheme 2Open in figure viewerPowerPointSynthesis of aminopyrimidine carbaldehydes 14 and 15.

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Scheme 3Open in figure viewerPowerPointHWE-reaction of aldehyde 14 resulted in Z-olefin 17-(Z) which underwent isomerization and condensation to pyridopyrimidine 18 a.

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Scheme 4Open in figure viewerPowerPointScope of the HWE-reaction/photoisomerization/cyclization sequence to produce various pyrido[3,2-d]pyrimidines 18. All phosphonates except for carbamate-functionalized phosphonate 16 b (conditions b)), were treated with conditions a).

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Scheme 5Open in figure viewerPowerPointFunctionalization in C6-position of pyridopyrimidones 18 via deoxyhalogenation, sulfonation, or PyBrop mediated SNAr.

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Scheme 6Open in figure viewerPowerPointHartwig–Buchwald coupling enabled amination in 7-position.

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Scheme 7Open in figure viewerPowerPointMiscellaneous reactions to valuable building blocks 24–26.

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Scheme 8Open in figure viewerPowerPointAccess to bis-pyridopyrimidines was enabled by Hartwig-Buchwald coupling of amines 27 with the respective aryl bromide.

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Figure 1Open in figure viewerPowerPointStructure of cationic trithiolato diruthenium(II)⋅arene complexes presenting high cytotoxicity against cancer cells and efficacy against various parasites.

The use of ruthenium complexes as potential chemotherapeutics has been an active area of research for almost two decades. Developed initially as a potential alternative to platinum-based anticancer drugs, ruthenium(II)⋅arene complexes were also considered for other pharmacological properties, particularly as antiparasitic and antibacterial compounds. A special class of ruthenium(II)⋅arene complexes is constituted by symmetric and “mixed” cationic trithiolato-bridged dinuclear ruthenium(II)⋅arene complexes (general formulae [(η6-arene)2Ru2(μ2-SR)3]+ and [(η6-arene)2Ru2(μ2-SR1)2(μ2-SR2)]+, respectively). The high cytotoxicity against human cancer cells shown by this type of compounds and, more interestingly, their apparent ability to circumvent platinum-drug resistance, encouraged the development of several libraries of complexes containing this scaffold as potential biological active compounds. For example, IC50 values as low as 30 nM against A2780 (human ovarian cancer) cells and also against their cisplatin-resistant variant A2780cisR were measured for compounds B and C (Figure #cbic202000174-fig-0001#1), whereas the less lipophilic complex A exhibited lower cytotoxicity (IC50 of 130 and 80 nM on A2780 and A2780cisR, respectively).

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Figure 2Open in figure viewerPowerPointStructures of various organometallic moiety⋅coumarin conjugates.

In the particular case of traceable organometallic drugs, two strategies can be considered: 1) direct coordination of the (coumarin) fluorophore to the metal center or 2) anchoring of the dye to more elaborated ligands.[32,43, 44] The nature of the metal center as well as the type of the ligand can strongly affect the photophysical properties of the hybrid molecule. Some data on the development of trackable anticancer agents based on metal complexes were recently reviewed. Conjugates combining metalorganic units with covalently linked coumarins have been shown to be versatile tools for imaging in the case of various fluorophore-tagged platinum,[43,44, 46] ruthenium, gold[28,47, 46] or iridium complexes. Some examples of coumarin modified organometallic compounds are presented in Figure #cbic202000174-fig-0002#2.

10633_cbic202000174-fig-5001.jpg

Scheme 1Open in figure viewerPowerPointSynthesis of the dinuclear dithiolato 1 a/b and OH-, NH2-, CH2CO2H-functionalized trithiolato ruthenium(II)⋅arene intermediates 2 a/b, 3 a/b and 4 a/b.

The solid-state structure of symmetric intermediate 21 containing three free hydroxy groups was established by single crystal X-ray diffraction analysis (Figure #cbic202000174-fig-0003#3), confirming the expected molecular structure. The crystal structure of the symmetric complex 21 was established in the solid state by single-crystal X-ray diffraction (ORTEP representations shown in Figure #cbic202000174-fig-0003#3), confirming the expected structure. Selected bond lengths and angles are presented in Table S2. Data collection and refinement parameters are given in Table S1.

10633_cbic202000174-fig-5002.jpg

Scheme 2Open in figure viewerPowerPointSynthesis of the coumarin precursors containing an amino spacer 8, 9 and 10.

The photophysical properties of coumarin containing compounds investigated in this study, namely the starting Dye1-CO2H and Dye2-CO2H, coumarin-based amino intermediates 5–7, and ester and amide conjugates with the trithiolato ruthenium(II)-p-cymene scaffold 11–17 a/b, 20 and 22, were studied in CHCl3 and EtOH at room temperature and are summarized in Tables 1 and S4. No solvatochromism was observed. The absorption and emission spectra of representative compounds for 10 μM solutions in CHCl3 and/or EtOH are comparatively presented in Figures #cbic202000174-fig-0004#4, #cbic202000174-fig-0005#5 and S5–S7. The absorption spectra of all diruthenium unit/coumarin conjugates 11–17 a/b, 20 and 22 present a similar profile (Figures #cbic202000174-fig-0004#4, #cbic202000174-fig-0005#5 and S5–S7). In both solvents, strong peaks corresponding to the coumarin fragment are observed in the 410–460 nm region. The signals in the 200–300 nm range, associated with the trithiolato dinuclear ruthenium(II)⋅arene moiety, are better resolved in the spectra measured in CHCl3 compared to those in EtOH. For 11 and 12 a/b, the direct attachment of the trithiolato diruthenium moiety to coumarins by ester bonds induced no shifts of the absorption peaks, whereas in case of amides 13 and 14 a/b a slight bathochromic shift (Δλ ≈10 nm) was observed. In contrast, the presence of the di-amino linker in conjugates 15–17 a/b led to a slender hypsochromic shift (Δλ ≈12–16 nm). When excited at 405 nm, all coumarin-containing compounds 5–17 a/b, 20 and 22 emit in the blue range (450–490 nm). The emission spectra of conjugates 11–17 a/b, presenting a 1 : 1 ratio organometallic unit: coumarin, show similar profiles (Figures #cbic202000174-fig-0004#4, #cbic202000174-fig-0005#5 and S5–S7) with almost complete fluorescence quenching, only slightly less pronounced for solutions in CHCl3 compared to those in EtOH. This loss of fluorescence efficacy was independent of the nature of coumarin (Dye1-CO2H or Dye2-CO2H), the type of bond (ester or amide) or presence of a di-amino linker between the two moieties. From this compound library, the highest calculated fluorescence quantum yields remain very modest (ΦF ≈3 %). Of note, similar but less pronounced quenching effects were observed in other organometallic coumarin-based conjugates.[26,32, 43]

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Scheme 3Open in figure viewerPowerPointSynthesis of the coumarin-based ester 11, 12 a/b (top) and amide 13, 14 a/b (bottom) conjugates.

The photophysical properties of coumarin containing compounds investigated in this study, namely the starting Dye1-CO2H and Dye2-CO2H, coumarin-based amino intermediates 5–7, and ester and amide conjugates with the trithiolato ruthenium(II)-p-cymene scaffold 11–17 a/b, 20 and 22, were studied in CHCl3 and EtOH at room temperature and are summarized in Tables 1 and S4. No solvatochromism was observed. The absorption and emission spectra of representative compounds for 10 μM solutions in CHCl3 and/or EtOH are comparatively presented in Figures #cbic202000174-fig-0004#4, #cbic202000174-fig-0005#5 and S5–S7. The absorption spectra of all diruthenium unit/coumarin conjugates 11–17 a/b, 20 and 22 present a similar profile (Figures #cbic202000174-fig-0004#4, #cbic202000174-fig-0005#5 and S5–S7). In both solvents, strong peaks corresponding to the coumarin fragment are observed in the 410–460 nm region. The signals in the 200–300 nm range, associated with the trithiolato dinuclear ruthenium(II)⋅arene moiety, are better resolved in the spectra measured in CHCl3 compared to those in EtOH. For 11 and 12 a/b, the direct attachment of the trithiolato diruthenium moiety to coumarins by ester bonds induced no shifts of the absorption peaks, whereas in case of amides 13 and 14 a/b a slight bathochromic shift (Δλ ≈10 nm) was observed. In contrast, the presence of the di-amino linker in conjugates 15–17 a/b led to a slender hypsochromic shift (Δλ ≈12–16 nm). When excited at 405 nm, all coumarin-containing compounds 5–17 a/b, 20 and 22 emit in the blue range (450–490 nm). The emission spectra of conjugates 11–17 a/b, presenting a 1 : 1 ratio organometallic unit: coumarin, show similar profiles (Figures #cbic202000174-fig-0004#4, #cbic202000174-fig-0005#5 and S5–S7) with almost complete fluorescence quenching, only slightly less pronounced for solutions in CHCl3 compared to those in EtOH. This loss of fluorescence efficacy was independent of the nature of coumarin (Dye1-CO2H or Dye2-CO2H), the type of bond (ester or amide) or presence of a di-amino linker between the two moieties. From this compound library, the highest calculated fluorescence quantum yields remain very modest (ΦF ≈3 %). Of note, similar but less pronounced quenching effects were observed in other organometallic coumarin-based conjugates.[26,32, 43]

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Scheme 4Open in figure viewerPowerPointSynthesis of the coumarin conjugates 15, 16 a/b and 17 a/b containing an amino spacer.

In both solvents, CHCl3 and EtOH, a linear dependence of the absorbance and emission intensity with concentration was determined (data not shown). This is observed even in the case of the conjugate 22 bearing three Dye1-CO2H units (spectra of 22 at various concentrations in CHCl3 are summarized in Figure #cbic202000174-fig-0006#6 and Figure S7).

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Scheme 5Open in figure viewerPowerPointSynthesis of conjugates 20 and 22 bearing two and three fluorophore units, respectively.

In the emission spectra, notable changes were observed only for ester conjugates 12 b, 20, and 22 (data not shown). A similar effect was noticed for the amide conjugates 14 b and 17 b (Figure #cbic202000174-fig-0007#7), but only after one week of light exposure, while no spectral changes were observed for amide 16 b. This increase of the emission signal can be attributed to a partial solvolysis of the ester or amide bonds present on the conjugates with the release of the respective coumarin dyes. Nevertheless, considering the time scale, and seen the very high fluorescence efficacy of Dye1-CO2H, Dye2-CO2H and corresponding coumarin intermediates 5–10 compared to the emission of the conjugates (fluorescence almost entirely quenched in the hybrid molecule), we can conclude that the coumarin-organometallic conjugates present high stability in the conditions used for this experiment.

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Figure 3Open in figure viewerPowerPointORTEP representation of complex 21 (thermal ellipsoids are 50 % equiprobability envelopes, and H atoms are spheres of arbitrary diameter; the asymmetric unit contains also one CH2Cl2 molecule).

The compounds presented in this study were screened for biological activity in vitro against T. gondii β-gal, a transgenic strain that constitutively expresses β-galactosidase, which is grown in HFF monolayers. In addition, the effects on uninfected HFF host cells were assessed. For the primary screening, cell cultures were exposed during 3 days to 1 and 0.1 μM of each compound (including unmodified thiolato-bridged dinuclear ruthenium(II)⋅arene complexes 2 a/b, 3 a/b, 4 a/b, coumarin-labeled conjugates 11–17 a/b, 20 and 22, free dyes Dye1-CO2H and Dye2-CO2H, and corresponding coumarin-based intermediates). The viability of HFF cultures following drug treatments was measured by alamarBlue assay, and the proliferation of T. gondii was quantified by measuring β-galactosidase activity. The results of this primary screening are presented as percentage in relation to untreated control cultures in Table S5. The results obtained at concentration of 0.1 and 1 μM of tested compound for T. gondii and HFF are presented in Figure #cbic202000174-fig-0008#8, in relation to controls (CTR), namely HFF treated with 0.1 % DMSO exhibiting 100 % viability, and T. gondii β-gal tachyzoites treated with 0.1 % DMSO showing 100 % proliferation. Dye1-CO2H -functionalized compounds 13, 15, and 16 a presented a similar cytotoxicity profile for HFF at 0.1 and 1 μM. The introduction of the linker augmented the measured activity against the parasite. For compounds bearing Dye2-CO2H moieties, the introduction of a linker between the diruthenium scaffold and the coumarin led to a substantially increased antiparasitic activity in the case of tBu analogue 17 a compared to 14 a; however, a similar effect was not observed for CF3 analogues 17 b and 14 b. Compounds 13, 14 a and 22 are only poorly active against T. gondii β-gal at 1 μM, and the last two compounds did not notably affect HFF cell viability. In the series of Dye1-CO2H functionalized ester compounds 11, 20 and 22 no noteworthy correlation between the number of coumarin units and the measured biological activity was observed. At 1 μM, 11 displayed substantial HFF toxicity, while 20 and 22 did not. As shown in Figure #cbic202000174-fig-0008#8, Dye1-CO2H and Dye2-CO2H did not affect viability of HFF when applied at 0.1 or 1 μM. However, proliferation of T. gondii relative to the rate of β-galactosidase activity was decreased following treatment with 0.1 μM of Dye1-CO2H (53 %) and Dye2-CO2H (69 %; Table S5). These results might suggest that the in vitro anti-toxoplasma activity of these coumarin dyes may be lost due to solubility issues.

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Figure 4Open in figure viewerPowerPointUV/Vis absorption (left) and emission (right) spectra of rhodamine 6G, Dye2-CO2H, intermediate 7, and the corresponding ester 12 a and amide 14 a, 17 a conjugates at 10 μM in CHCl3.

Three selected compounds (2 a, 3 a and 12 a), applied at their respective IC50 against T. gondii, were further assessed with respect to their potential to interfere in splenocyte proliferation in vitro. Isolated murine splenocytes from healthy mice were stimulated with concanavalin A (ConA) to induce T-cell proliferation or with bacterial lipopolysaccharide (LPS) to induce B-cell proliferation, either in the presence or absence of tested compounds 2 a, 3 a and 12 a. Measurements of proliferation were done using an assay that quantifies the incorporation of 5-bromo-2’-deoxy-uridine (BrdU) into the DNA of replicating cells. As seen in Figure #cbic202000174-fig-0009#9, 3 a and 12 a significantly interfered with T-cell proliferative responses, which resulted in a reduction of BrdU incorporation by 25 and 31 %, respectively, whereas 2 a did not affect T-cell proliferation. Compounds 2 a and 3 a significantly impacted the proliferation of B cell (only 48 and 68 % BrdU incorporation, respectively, compared to the control), whereas 12 a did not exhibit significant proliferation inhibition of antibody producing cells. Thus, 3 a affected both B- and T-cell proliferation, whereas 2 a and 12 a impaired the proliferative capacity of only one type of immune cells; B cells for 2 a (humoral immunity) and T cells for 12 a (cellular immunity).

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Figure 5Open in figure viewerPowerPointUV/Vis absorption (left) and emission (right) spectra of rhodamine 6G, Dye2-CO2H, intermediate 7 and the corresponding ester 12 b and amide 14 b, 17 b conjugates, at 10 μM in CHCl3.

A reduction in the capacity of T and B cells to respond to external signals by proliferative responses could also indicate a potential risk of impaired immunity. However, a reduction in cellular proliferation does not automatically imply a reduced metabolic activity or reduction in cellular viability. Thus, in addition to proliferative responses of B and T cells upon LPS and ConA stimulation, we also assessed the effects of 2 a, 3 a and 12 a on the viability of splenocytes employing the alamarBlue assay, which allows, similar to what we have tested in HFF, to quantify metabolic activity (Figure #cbic202000174-fig-0010#10). Measurements taken each hour during the first 5 h after adding the substrate resazurin showed that none of the compounds impaired the metabolic activity of ConA-stimulated T cells, but compounds 2 a and 3 a significantly impaired the viability of B cells upon stimulation with LPS. 12 a did not show any interference in the metabolic activity of either B or T cells. The lack of viability impairment of splenocytes due to exposure with 12 a observed here indicates that this compound is a promising drug candidate for future in vivo studies in the mouse model, to combat T. gondii infection without impairing the cellular and humoral immune response.

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Figure 6Open in figure viewerPowerPointUV/Vis absorption (left) and emission (right) spectra of tricoumarin ester conjugate 22 at various concentrations in CHCl3.

The ultrastructural changes induced by compounds 12 a (IC50 of 0.105 μM against T. gondii, HFF viability of 58 % at 2.5 μM) and 17 a (IC50 of 0.243 μM against T. gondii, HFF viability of 24 % at 2.5 μM) were further studied by TEM. HFF monolayers were infected with T. gondii β-gal tachyzoites and after 24 h of drug treatment (500 nM of each compound, a concentration that did not notably affect the host cell) were initiated. Samples were fixed and processed after 6, 24, and 48 h. Untreated control cultures are shown in Figure #cbic202000174-fig-0011#11. Part A shows a sample fixed 6 h post-invasion, B and C were fixed 36 and 60 h post-invasion, respectively, and the increase in number of parasites illustrates the proliferation that takes place within the host cell. Once invaded, T. gondii tachyzoites are localized within a parasitophorous vacuole (PV), surrounded by a parasitophorous vacuole membrane (PVM), which is essentially derived from host cell-surface membrane modified by the parasite following invasion. The mitochondrion exhibits a characteristic electron-dense matrix containing numerous cristae. Tachyzoites treated with 500 nM 12 a are shown in Figure #cbic202000174-fig-0012#12. At 6 h after initiation of treatment, parasites do not exhibit massive alterations, however, PVs usually contained only 1–3 tachyzoites. The PVM was still clearly discernible, and the secretory organelles such as rhoptries, micronemes, and dense granules, as well as the mitochondria remained largely unaltered (Figure #cbic202000174-fig-0012#12 A, B). At 24 h, first ultrastructural changes were noted within the mitochondrial matrix, which started to lose its characteristic electron-dense matrix. These mitochondrial alterations became progressively more pronounced at 24 (Figure #cbic202000174-fig-0012#12 D and E) and 48 h (Figure #cbic202000174-fig-0012#12 F, G), resulting in tachyzoites that were completely devoid of a mitochondrion, but exhibited large, seemingly empty, vacuoles instead. Although it is not clear whether these effects were reversible, it is conceivable that this extensive vacuolization would eventually lead to parasite death. Similar results were seen for 17 a, (Figure S10), although the effects after 24 and 48 h were slightly less pronounced. Overall, these findings mirror previously reported structural alterations induced by ruthenium complexes reported in T. gondii, N. caninum and in T. brucei, and in the latter it was recently shown that active complexes strongly impaired the mitochondrial membrane potential; this indicates that ruthenium complexes interfere in the energy metabolism of these parasites. However, although oxidative phosphorylation resulting in the generation of ATP is the major function of mitochondria, these organelles are also involved in other crucial processes, including cell-cycle regulation, tRNA and protein import, mitochondrial protein translation, alternative oxidase, acetate production for cytosolic and mitochondrial fatty acid biosynthesis, amino acid metabolism, and calcium homeostasis, they are also involved in the steps leading to programmed cell death. How, and to what extent, these compounds actually target mitochondrial functions or whether other targets are also involved remain to be elucidated

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Figure 7Open in figure viewerPowerPointUV/Vis absorption (left) and emission (right) spectra of the trithiolato ruthenium(II)⋅p-cymene complex 14 b after 0 h, 24 h, 48 h, and one week (168 h) of exposure to indoor light at 10 μM in EtOH.

The ultrastructural changes induced by compounds 12 a (IC50 of 0.105 μM against T. gondii, HFF viability of 58 % at 2.5 μM) and 17 a (IC50 of 0.243 μM against T. gondii, HFF viability of 24 % at 2.5 μM) were further studied by TEM. HFF monolayers were infected with T. gondii β-gal tachyzoites and after 24 h of drug treatment (500 nM of each compound, a concentration that did not notably affect the host cell) were initiated. Samples were fixed and processed after 6, 24, and 48 h. Untreated control cultures are shown in Figure #cbic202000174-fig-0011#11. Part A shows a sample fixed 6 h post-invasion, B and C were fixed 36 and 60 h post-invasion, respectively, and the increase in number of parasites illustrates the proliferation that takes place within the host cell. Once invaded, T. gondii tachyzoites are localized within a parasitophorous vacuole (PV), surrounded by a parasitophorous vacuole membrane (PVM), which is essentially derived from host cell-surface membrane modified by the parasite following invasion. The mitochondrion exhibits a characteristic electron-dense matrix containing numerous cristae. Tachyzoites treated with 500 nM 12 a are shown in Figure #cbic202000174-fig-0012#12. At 6 h after initiation of treatment, parasites do not exhibit massive alterations, however, PVs usually contained only 1–3 tachyzoites. The PVM was still clearly discernible, and the secretory organelles such as rhoptries, micronemes, and dense granules, as well as the mitochondria remained largely unaltered (Figure #cbic202000174-fig-0012#12 A, B). At 24 h, first ultrastructural changes were noted within the mitochondrial matrix, which started to lose its characteristic electron-dense matrix. These mitochondrial alterations became progressively more pronounced at 24 (Figure #cbic202000174-fig-0012#12 D and E) and 48 h (Figure #cbic202000174-fig-0012#12 F, G), resulting in tachyzoites that were completely devoid of a mitochondrion, but exhibited large, seemingly empty, vacuoles instead. Although it is not clear whether these effects were reversible, it is conceivable that this extensive vacuolization would eventually lead to parasite death. Similar results were seen for 17 a, (Figure S10), although the effects after 24 and 48 h were slightly less pronounced. Overall, these findings mirror previously reported structural alterations induced by ruthenium complexes reported in T. gondii, N. caninum and in T. brucei, and in the latter it was recently shown that active complexes strongly impaired the mitochondrial membrane potential; this indicates that ruthenium complexes interfere in the energy metabolism of these parasites. However, although oxidative phosphorylation resulting in the generation of ATP is the major function of mitochondria, these organelles are also involved in other crucial processes, including cell-cycle regulation, tRNA and protein import, mitochondrial protein translation, alternative oxidase, acetate production for cytosolic and mitochondrial fatty acid biosynthesis, amino acid metabolism, and calcium homeostasis, they are also involved in the steps leading to programmed cell death. How, and to what extent, these compounds actually target mitochondrial functions or whether other targets are also involved remain to be elucidated

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Figure 8Open in figure viewerPowerPointIn vitro activities of all compounds at 0.1 and 1 μM on A) HFF viability and B) T. gondii β-gal tachyzoites proliferation, in relation to treatments with 0.1 % DMSO. For each assay, standard deviations were calculated from triplicate experiments.

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Figure 9Open in figure viewerPowerPointInhibitory effect of selected compounds 2 a, 3 a and 12 a on A) ConA- and B) LPS-induced proliferative activity of mouse splenocytes. Bars represent standard deviation from the mean of four replicates. 100 % proliferation is attributed to the control (ConA or LPS); values are percentage proliferation compared to control, * p <0.01 in relation to controls.

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Figure 10Open in figure viewerPowerPointDetermination of cytotoxic effects of 2 a, 3 a and 12 a in A) ConA- and B) LPS-induced splenocytes by alamarBlue assay. Bars represent the mean emission of four replicates ± standard deviation. In both experiments and for each time point, 100 % metabolic activity was attributed to the control sample (splenocytes induced with ConA or LPS), then the percentage of metabolic activity in relation to the controls was calculated, and is indicated in the graph for each sample. Significance with p <0.001 was observed between LPS and LPS+2 a, and LPS+3 a for all five time points.

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Figure 11Open in figure viewerPowerPointTEM of untreated T. gondii-β-gal tachyzoites fixed at A) 6, B) 24, and C) 48 h post infection. (A) shows a single tachyzoite, located within a PV that is delineated by a PVM (arrows). (B) Proliferation of tachyzoites takes place within the vacuole, which occupies a substantial part of the host-cell cytoplasm. (C) shows two neighboring PVs, delineated by arrows, located within a HFF host cell, both containing numerous newly formed tachyzoites. Note the mitochondrion (mito) with an electron dense matrix in (A); nuc=tachyzoite nucleus, dg=dense granule, hcc=host cell cytoplasm; hcn=host cell nucleus.

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Figure 12Open in figure viewerPowerPointTEM of T. gondii-β-gal tachyzoites treated with 500 nM of 12 a for A), B) 6, C)–E) 24, and F)–H) 48 h. No alterations (A) or only very slight changes in the electron dense mitochondrial matrix (mito in (B)) are detected after 6 h. (C)–(E) show that alterations were much more pronounced after 24 h of treatment; (C) is a low-magnification overview, (D) and (E) represent distinct parts of (C) shown at higher magnification. (F) is a low-magnification view of a PV of tachyzoites treated for 48 h, (G) and (H) are high magnifications. Note the increased vacuolization (marked with *) and the absence of any mitochondrial matrix after 24–48 h of treatment. con=conoid; rop=rhoptries; mic=microneme; arrows point towards the PVM.

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Figure 1Open in figure viewerIllustration of SARS-CoV-2 structural proteins. Created with BioRender.com.

Four main structural proteins define SARS-CoV-2 as a whole (Figure #anse202200012-fig-0001#1): envelope (E), membrane (M), nucleocapsid (N), and spike (S), each of which could potentially serve as a diagnostic and therapeutic target.

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Figure 2Open in figure viewerSchematic outline of SELEX procedure, with the SARS-CoV-2 S protein as the target for identifying DNA aptamers.

The primary and most popular method by which aptamers are created is through an in vitro selection technique known as SELEX (Systematic Evolution of Ligands through EXponential enrichment), which was first reported in 1990. Conventional SELEX for isolating DNA aptamers, for example, begins with a library of 1014-1015 random single-stranded DNA sequences, as illustrated in Figure #anse202200012-fig-0002#2 with a general scheme for selecting DNA aptamers that bind the S protein of SARS-CoV-2. Potential aptamers from a library are progressively enriched through many repetitive rounds of binding-mediated partitioning and amplification. One round may include a negative selection step to eliminate sequences binding to non-target molecules and a positive selection step to retain high-affinity sequences for the intended target. Selected sequences at the end of each cycle are amplified to create an enriched DNA pool. Iterative cycles of this procedure are performed to let the molecules compete for survival based on their affinity and/or specificity. Once the evolving pool exhibits satisfactory binding properties, sequencing is used to reveal the identities of the remaining sequences in the pool.

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Figure 3Open in figure viewerSummary of in vitro selection conducted by the Yang Lab to obtain high-affinity SARS-CoV-2 receptor-binding domain (RBD) aptamers.42 Reprinted with permission from ref [42]. Copyright (2020) American Chemical Society.

Implementation of additional strategies has also been reported to achieve different goals. For example, Schmitz et al. used a robotic-assisted selection procedure that was capable of performing 12 consecutive, automated selection cycles, without manual interference. Song et al. included ACE2 competition within their selection process (Figure #anse202200012-fig-0003#3) where ACE2 was incubated with bead-bound aptamers in order to select aptamers that compete with ACE2 for S protein binding so that the aptamers could be further developed as potential therapeutic agents to block viral binding to ACE2. Our McMaster team combined two complex separation strategies in tandem to achieve aptamer selection: bead-based selection was used for the first 3 rounds and gel-based selection for the next 10 rounds. Using multiple partitioning methods for the isolation of functional nucleic acids, which had been implemented in several previous reports, can help eliminate selfish DNA sequences that can survive the use of a single physical separation method. Finally, Peinetti et al. used “active” pseudotyped viruses as the positive selection target and “deactivated” pseudotyped viruses as the counter selection target with the goal of isolating aptamers that bind only to actively transmitting viruses.

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Figure 4Open in figure viewer(A) The pre-engineered secondary structure of the DNA library used for the SELEX experiment by Li et al. (B) The hairpin structures of the minimized mutants of two top ranked DNA aptamers MSA1 and MSA5. Adapted with permission from ref [43]. Copyright (2021) Oxford University Press.

Most of the SELEX studies used completely random libraries. However, our group took a different approach: the two flanking constant regions were designed with specific sequences for the creation of a pairing element that places the random-sequence domain into a hairpin structure (Figure #anse202200012-fig-0004#4A), which is often found in many published aptamers. In this way, we significantly increased the potential of finding more aptamers from the structured library. Indeed, after 13 rounds of selection, we obtained several aptamers that exhibited excellent affinity. Another advantage of such a library design is rapid identification of minimized sequences and secondary structures of these aptamers. For example, we were able to quickly identify the shortest sequences as well as the secondary structures of two top ranking aptamers from our SELEX experiment (Figure #anse202200012-fig-0004#4B).

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Figure 5Open in figure viewer(A) Schematic of engineering dimeric aptamers by ligation of two monomeric aptamers with a polythymidine linker. (B) Determination of binding affinity of heterodimeric aptamer DSA1N5 binding for the spike proteins and pseudotyped viruses of the original SARS-CoV-2 and alpha variant. WHPV and UKPV: lentiviruses pseudotyped with the spike protein of the wildtype SARS-CoV-2 and the alpha variant. WHTS and UKTS: trimeric spike protein of the wildtype SARS-CoV-2 and the alpha variant. CV: control lentivirus. DMC: inactive mutant dimeric aptamer control. Adapted with permission from ref [78]. Copyright (2021) Wiley-VCH.

We recently constructed a series of dimeric aptamers by linking two monomeric aptamers with a polythymidine linker. These include two homodimers DSA1N1 and DSA5N5 as well as a heterodimer DSA1N5 (Figure #anse202200012-fig-0005#5A). We found that the heterodimeric aptamer DSA1N5 had the highest affinity among reported dimeric or trimeric aptamers for recognizing pseudo viruses of both the original SARS-CoV-2 and the Alpha variant with Kd values of 2.1 pM and 2.3 pM, respectively (Table 2 and Figure #anse202200012-fig-0005#5B). The affinities for viral recognition were more than 57 and 126-fold better than for their spike proteins (Kd of 120 pM and 290 pM, respectively; Figure #anse202200012-fig-0005#5B), highlighting the advantage of engineering bivalent aptamers for viral particle recognition.

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Figure 6Open in figure viewerA universal aptamer for the S protein. (A) Pictorial representation of the reselection process for the discovery of universal aptamers. (B) Secondary structure MSA52. (C) Binding between MSA52 and pseudotyped lentiviruses expressing the spike of the wildtype (WH) and 7 variants of SARS-CoV-2. CV: control lentivirus; MC: inactive mutant of MSA52 as the control sequence. Adapted with permission from ref [87]. Copyright (2022) Wiley-VCH.

To address this issue, we recently made an effort to select for universal DNA aptamers that recognize several VoCs. Our previous DNA aptamers, mainly MSA1 and MSA5, were targeted specifically to the S1 subunit of the wildtype spike protein. However, it was realized that the epitopes recognized by these two aptamers were sensitive to the changes caused by the mutations to the S protein. To reselect aptamers that can function as universal affinity agents, we conducted five parallel, one-round SELEX experiments with our previously established Round 13 aptamer pool and five VoC spike proteins (Figure #anse202200012-fig-0006#6A). One particular aptamer, named MSA52 (Figure #anse202200012-fig-0006#6B), displayed a significant increase in the pools selected using the variant spike proteins. Upon binding analysis, we found that this aptamer indeed displayed high affinity for pseudotyped lentiviruses expressing each variant protein (Figure #anse202200012-fig-0006#6C). Furthermore, to test its universality, MSA52 was also assessed with the Kappa, Delta and Omicron variants, which were not yet available at the time of the original reselection experiment. The binding assays demonstrated that MSA52 could also recognize these variants, thus confirming MSA52 as a “universal” aptamer for SARS-CoV-2 S protein. Perhaps what is most impressive is that the entire discovery process took place in less than a week, which is a testament to the rapidity of the SELEX process.

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Figure 7Open in figure viewerElectrochemical aptamer-based (EAB) sensors exploit the binding-induced conformational change of a covalently attached, redox reporter-modified aptamer to generate an easily measurable electrochemical signal. Reprinted with permission from ref [106]. Copyright (2021) American Chemical Society.

There are several recent examples of aptamer-based electrochemical sensors for SARS-CoV-2. For example, Idili et al. developed an electrochemical sensor by simply immobilizing an S protein-specific DNA aptamer on electrodes for quantitative detection of SARS-CoV-2 (Figure #anse202200012-fig-0007#7). The platform utilized conformational changes of the immobilized aptamers upon binding with the S protein to bring a redox probe into close proximity to the electrode surface, producing an increase in current. This sensor was easy to manufacture, rapid (within minutes), and could conduct direct testing in clinical samples (serum and saliva). However, this direct immobilization strategy generated a high background signal in the absence of targets, leading to low sensitivity and a relatively high false-positive rate.