bluuebunny/crossref_metadata_2025 huggingface dataset:
Cysteic‐ and homocysteic acid‐S‐(aminoiminomethyl) amides: a new class of modified arginines useful in peptide synthesis
Authors: Buchinska Todorka V.
Published: May 2003

A novel, practical synthesis of the title compounds and their derivatives are described. The protecting groups for amino, guanidino and carboxylic functions of substituted amides of cysteic and homocysteic acid were selected with the aim of making the amino acid derivatives synthons for peptide synthesis both in solution and by the solid phase method. Studying the structure–activity relationship some new kyotorphin, [Leu1] kyotorphin and MIF‐1 analogues, containing the unusual amino acid cysteic acid‐S‐(aminoiminomethyl) amide (sArg) in position two, have been prepared. It is a very promising compound, a structural analogue of arginine and an efficient antagonist in its metabolism.
Structure–activity studies at the rat tachykinin NK2 receptor: effect of substitution at position 5 of neurokinin A
Authors: Comis A. , Burcher E.
Published: May 2003

A series of analogues of neurokinin A(4–10) was synthesized using solid phase techniques with Chiron pins, and purified by HPLC. The potencies of 10 peptides with substitution at Ser5 were assessed at rat fundus NK2 receptors. In membrane binding studies with [125I]‐[Lys5,Tyr(I2)7,MeLeu9,Nle10]‐NKA(4–10), all compounds except [Asp5]NKA(4–10) showed reasonable affinity, and analogues with Lys and Arg substitutions were five‐fold more potent than NKA(4–10). In functional studies, all peptides were able to contract the rat isolated fundus strips. Analogues with Phe, His and Asn substitutions were substantially weaker in functional than in binding studies, whereas there was an excellent correlation (r = 0.95) between binding and functional potency for the remaining seven peptides. [Phe5]NKA(4–10) is in fact neurokinin B(4–10) and this residue may be critical in determining selectivity between NK2 and NK3 receptors. Analogues with a basic residue (Lys, Arg) at position 5 showed both increased affinity and functional potency, whereas the neutral [Asn5]NKA(4–10) was equally as weak in contractile studies as the acidic [Asp5]NKA(4–10). However, [Glu5]NKA(4–10) and [Gln5]NKA(4–10) were no different from NKA(4–10). Our results could indicate the presence of a negative charge on the NK2 receptor, close to position 5 of NKA. This would facilitate interaction with positively charged side chains and impede interaction with negatively charged side chains, particularly the inflexible side chain of aspartic acid. Thus, not only the charge, but also the length of the side chain of the residue at position 5, seems to be important for interaction with the rat NK2 receptor.
Purification and characterization of huwentoxin‐II, a neurotoxic peptide from the venom of the Chinese bird spider Selenocosmia huwena
Authors: Shu Q. , Liang S.
Published: May 2003

Abstract: A neurotoxic peptide, huwentoxin‐II (HWTX‐II), was purified from the venom of the Chinese bird spider Selenocosmia huwena by ion exchange chromatography and reversed phase HPLC. The toxin can reversibly paralyse cockroaches for several hours, with an ED50 of 127 ± 54 µg/g. HWTX‐II blocks neuromuscular transmission in an isolated mouse phrenic nerve diaphragm preparation and acts cooperatively to potentiate the activity of huwentoxin‐I. The complete amino sequence of HWTX‐II was determined and found to consist of 37 amino acid residues, including six Cys residues. There is microheterogeneity (Ile/GIn) in position 10, and mass spectrometry indicated that the two isoproteins have a tendency to dimerize. It was determined by mass spectrometry that the six Cys residues are involved in three disulphide bonds. The sequence of HWTX‐II is highly homologous with ESTX, a toxin from the tarantula Eurypefina californicum.
Refined solution structure of ω‐conotoxin GVIA: implications for calcium channel binding
Authors: Pallaghy P.K. , Norton R.S.
Published: May 2003

The polypeptide ω‐conotoxin GVIA (GVIA) is an N‐type calcium channel blocker from the venom of Conus geographus, a fish‐hunting cone shell. Here we describe a high‐resolution solution structure of this member of the ‘inhibitor cystine knot’ protein family. The structure, based on NMR data acquired at 600 MHz, has mean pairwise RMS differences of 0.25 ± 0.06 and 1.07 ± 0.14 Å over the backbone heavy atoms and all heavy atoms, respectively. The solvent‐accessible side chains are better defined than in previously published structures and provide an improved basis for docking GVIA with models of the calcium channel. Moreover, some side chain interactions important in GVIA folding in vitro and in stabilizing the native structure are defined clearly in the refined structure. Two qualitatively different backbone conformations in the segment from Thr11 to Asn14 persisted in the restrained simulated annealing calculations until a small number of lower bound constraints was included to prevent close contacts from occurring that did not correspond with peaks in the NOESY spectrum. It is possible that GVIA is genuinely flexible at this segment, spending a finite time in the alternative conformation, and this may influence its interaction with the calcium channel.
Conformational changes of urea‐denatured colicin E1 induced by phospholipid membranes
Authors: Wu Y. , Sui Sen‐Fang , Wu Y.
Published: May 2003

Abstract: The membrane insertion of urea‐denatured colicin E1 was studied by using fluorescence spectroscopy, circular dichroism and monolayer techniques. The results showed that the denatured colicin E1 taking mainly the ‘random coil’ conformation may recover its orderliness to a certain extent under the induction of the phospholipid membrane and insert spontaneously into phospholipid membrane, indicating that unfolding of colicin E1 does not inhibit its membrane insertion. Among the four tryptophan residues of the membrane‐bound colicin E1 molecules, at least two were accessible by the quenchers, i.e. not inserted into the membranes. Although urea‐denatured colicin E1 interacted preferentially with negatively charged phospohlipids, it seems less dependent on the negatively charged lipid than colicin A. The addition of urea increased the speed of the adsorption of colicin E1 to the membrane, but did not affect obviously its membrane insertion ability.
Binding of cations to individual gamma‐carboxyglutamate residues of conantokin‐G and conantokin‐T
Authors: Blandl T. , Warder S.E. , Prorok M. , Castellino F.J.
Published: May 2003

Abstract: Conantokin‐G (con‐G) and conantokin‐T (con‐T) are naturally occurring γ‐carboxyglutamate (Gla)‐containing peptides that interact with multivalent cations in functionally relevant manners. Selective 13C‐enrichment of Cγ and Cδ in each of the Gla residues has allowed metal binding affinities to be measured at individual side chains. Con‐T possesses two metal binding sites, one with high affinity at Gla10/Gla14 and another with weak binding at Gla3/Gla4. Con‐G contains two sites of comparable low affinity for Ca2+. Analysis of the 13C line‐widths of con‐G in the presence of Mg2+ allowed the order of metal binding to be determined, with Gla10/Gla14 loading before the Gla3/Gla4/Gla7 cluster. While the variant peptide, apo‐con‐T[Lys7Gla], was shown to have a very low α‐helical content, this peptide binds a second metal with much greater affinity than wild‐type con‐T. This provides additional evidence that Gla7 in con‐G is primarily responsible for destabilizing the apo‐form, but is an important ligand for metal chelation. The residue‐specific α‐helical stabilities of con‐G and con‐T in their metal‐free and metal‐loaded states were estimated by determining rates of proton exchange from backbone peptide bond amides with deuterium atoms from 2H2O‐containing solvents. For both peptides, the lifetimes of protons on several peptide bond amides increased as metals of higher affinity were bound to the peptides, with the longest half‐lives found in the region of the α‐helical turn stabilized by the Gla10/Gla14 metal coordination site. We propose that Gla10 and Gla14 constitute the primary tight metal ion binding site in both peptides. This detailed analysis with physiologically relevant metal cations is crucial for deciphering the roles of critical amino acids in the bioactivity of the conantokin peptides.
Purification and characterization of an acid proteinase from mesophilic Mucor sp. solid‐state cultures
Authors: Fernandez‐Lahore H.M. , Auday R.M. , Fraile E.R. , Cascone O. , Biscoglio de Jimenez Bonino M. , Pirpignani L. , Machalinski C.
Published: May 2003

Abstract: The fourth‐day extract of a solid‐state culture of the mesophilic Mucor sp. (M‐105) strain showed a high milk‐clotting activity and a clotting/proteolytic activity ratio similar to that of commercial preparations from microbial origin used in cheese manufacture. After ultrafiltration of the crude extract, the milk‐clotting proteinase was purified in two steps: ion‐exchange followed by size‐exclusion chromatography. Enzyme homogeneity was assessed by HPLC, SDS‐PAGE and N‐terminal residue determination. A pI value of 4.21 was obtained and a molecular weight of 33 kDa was calculated from size‐exclusion chromatography and SDS‐PAGE data. The optimum pH for proteolytic activity towards dimethylcasein was in the 3.0–3.5 range. The proteinase retained 26 and 13% of its proteolytic activity after a 30‐min incubation period, at pH 5.0 and 50 and 60°C, respectively. This evidenced a lower heat stability than that of the thermophilic enzymes currently used in the cheese industry and also than that of bovine chymosin. The enzyme was fully inhibited by pepstatin A and no effect was observed with PMSF, p‐CMPS or EDTA. The N‐terminal amino acid sequence: GTGTVPVTDDGNLNEYYXTVTVGXP was compared with those from other fungal enzymes.
Control of ditryptophan cross‐linking: dihydrotryptophan as a tryptophan precursor in peptide synthesis
Authors: Dinh T.D. , Van Vranken D.L.
Published: May 2003

Abstract: In neat trifluoroacetic acid, tryptophan side chains cross‐link to form a diastereomeric mixture of tryptophan dimers. Convergent oxidation with 2,3‐dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ) converts tryptophan dimers to ditryptophan. Since cross‐link formation is under thermodynamic control, there has been no simple way of controlling the regiochemistry of the cross‐linking process when more than one tryptophan side chain is present. Here, we show that dihydrotryptophan (Dht) can be incorporated into peptides as a tryptophan precursor, which reforms tryptophan upon treatment with DDQ. Dihydrotryptophan was prepared as a mixture of γS and γR diastereomers and the indoline nitrogen was protected with a Cbz group. The resulting amino acid, Nα‐BOC‐Dht(Cbz)‐OH, was then incorporated into peptides as a mixture of diastereomers. Dht was resistant to tryptophan cross‐linking in neat trifluoroacetic acid and was converted back to tryptophan during convergent oxidation of tryptophan dimers. While Dht is useful for control of ditryptophan regiochemistry and as a potential tryptophan analog, it is not a general strategy for Trp protection since DDQ is unlikely to be compatible with easily oxidized amino acids such as cysteine.
Solid‐phase total synthesis of polymyxin B1
Authors: Sharma S.K. , Wu A.D. , Chandramouli N. , Fotsch C. , Kardash G. , Bair K.W.
Published: May 2003

Abstract: The total solid‐phase synthesis of polymyxin B1 (PMB1) has been achieved in 20% yield using the orthogonal protecting group N‐1‐(4,4‐dimethyl‐2,6‐dioxocyclohexylidene)ethyl‐(Dde). This report demonstrates that a complex peptide macrocycle can be synthesized in high yields using solid‐phase synthesis. According to MS and HPLC, the synthetic peptide was identical to the naturally occurring antibiotic.
Unique molecular conformation of aureobasidin A, a highly amide N‐methylated cyclic depsipeptide with potent antifungal activity: X‐ray crystal structure and molecular modeling studies
Authors: In Y. , Ishida T. , Takesako K.
Published: May 2003

Abstract: A structural feature of aureobasidins, cyclic depsipeptide antibiotics produced by Aureobasidium pullulans R106, is the N‐methylation of four out of seven amide bonds. In order to investigate possible relationship between the molecular conformation and the amide N‐methylation, aureobasidin A (AbA), which exhibits the potent antifungal activity, was subjected to X‐ray crystal analysis. The crystal, recrystallized from ether (orthorhombic, space group P212121, a = 21.643 (3) Å, b = 49.865(10) Å, c = 12.427 (1) Å, Z = 8), contained two independent conformers per asymmetric unit and they took on a similar arrowhead‐like conformation. The conformation consisted of three secondary structures of antiparallel β‐sheet, and β‐ and γ‐turns, and was stabilized by three intramolecular and transannular N‐H…O=C hydrogen bonds. The β‐hydroxy‐N‐methyl‐l‐valine residue, which is indispensable for its bioactivity, was located at the tip of the corner. Since a nearly identical conformation has been observed for aureobasidin E, a related cyclic depsipeptide, this arrowhead‐like conformation may be energetically stable and important for biological activity. The contribution of the amide N‐methylation to the conformation was investigated by model building and energy calculations. The energy‐minimizations of AbA analogs, in which some (one to four) of four N‐methylated amide bonds were replaced with usual amide bond, led to some conformers which are fairly different from the arrowhead form of AbA, although they are stabilized by three intramolecular N‐H…O=C hydrogen bonds. This result explains the reason why four out of the seven amide bonds have to be methylated to manifest biological activity, i.e. the high N‐methylation of aureobasidin is necessary to form only one well‐defined conformation.
Combinatorial peptide on‐resin analysis: optimization of static nanoelectrospray ionization technique for sequence determination
Authors: Biederman K.J. , Lee H. , Haney C.A. , Kaczmarek M. , Buettner J.A.
Published: May 2003

The optimizations of static nanoelectrospray parameters to determine peptide or mimetic sequences released from resin were explored. Several different manufacturers of probe tips were utilized and a method was developed for the direct analysis of bead‐bound peptides by nanoelectrospray. The method involved minimum sample handling to assure maximum recovery from individual beads. Parameters that were explored included an inside and outside wash of the probe tip, the distance from the probe housing to the probe tip, source temperature, drying gas flow, individual tips and presence of beads. The same soluble synthetic peptide was used in all comparisons, which had a molecular weight of 717 amu. The discovery of the sequence of a bead‐bound peptide was achieved. The parameters that were found to effect signal were outside wash, presence of bead and distance. There was the need for pneumatic assist to initiate electrospray on some occasions, although this generally resulted in unsatisfactory performance.
A 30‐residue fragment of the carp granulin‐1 protein folds into a stack of two β‐hairpins similar to that found in the native protein
Authors: Vranken W.F. , Chen Z.G. , Xu P. , James S. , Bennett H.P.J. , Ni F.
Published: May 2003

Abstract: Upon air oxidation, a peptide corresponding to the 30‐residue N‐terminal subdomain of carp granulin‐1 spontaneously formed the disulfide pairing observed in the native protein. Structural characterization using NMR showed the presence of a defined secondary structure within this peptide. The chemical shifts for most of the αCH protons of the peptide and the protein are very similar, and the observed NOE contacts of the peptide strongly resemble those in the protein. A structure calculation of the peptide using NOE distance constraints indicates that the peptide fragment adopts the same conformation as formed within the native protein. The 30‐residue N‐terminal peptide of carp granulin‐1 is the first example of an independently folded stack of two β‐hairpins reinforced by two interhairpin disulfide bonds. Two key areas of the structure show a clustering of hydrophobic residues that may account for its exceptional conformational stability.
In situ generation of Fmoc‐amino acid chlorides using bis‐(trichloromethyl)carbonate and its utilization for difficult couplings in solid‐phase peptide synthesis
Authors: Falb E. , Yechezkel T. , Salitra Y. , Gi1on C.
Published: May 2003

Abstract: This paper reports procedures for the straightforward in situ generation of Fmoc‐amino acid chlorides using bis‐(trichloromethyl)carbonate (BTC) and their utilization for difficult couplings during solid‐phase peptide synthesis. The BTC‐mediated coupling of all Fmoc‐protected proteinogenic amino acids to a large variety of N‐alkylated amino acid‐peptidyl‐resin was studied. The majority of the couplings proceeded with quantitative conversion and without racemization. The utilization of BTC‐mediated coupling for facile solid‐phase synthesis of backbone cyclic peptides is presented.
Pepsin as a catalyst for peptide synthesis: formation of peptide bonds not typical for pepsin substrate specificity
Authors: Malak Camelia Adly Abdel
Published: May 2003

Abstract: Porcine pepsin in water solutions containing 15–28% of dimethylformamide at pH 5 and 20–37°C catalysed the formation of peptide bonds between Z‐Ala‐Ala‐Phe‐OH and various amino acid or peptide derivatives. Substrate binding subsite S′1 of pepsin demonstrated broad specificity in these reactions but revealed a certain preference for hydrophobic amino acid residues, including non‐proteinous homophenylalanine, p‐nitrophenylalanine, S‐methylcysteine, as well as for those that contained, in addition to the hydrophobic elements, a group capable of donating a hydrogen bond, e.g. o‐nitrotyrosine. This observation increases the range of peptides that might be prepared by pepsin‐catalysed synthesis.
Not only the nature of peptide but also the characteristics of cell membrane determine the antimicrobial mechanism of a peptide
Authors: Liang Jun Feng , Sun  , Kim Chang
Published: May 2003

Abstract: The mechanisms of antimicrobial actions of magainin 2, buforin II and poly l‐lysine against various Escherichia coli strains were studied. Poly l‐lysine inhibited BL21, ad 434 and GroE+/DnaK+ growth without lysing the cell. Magainin 2 had a pore‐forming activity on BL 21 and ad 434 membrane but could not inhibit the GroE+/DnaK+ growth in a nutrient‐rich medium. Buforin II, which killed BL21 and ad 434 without cell membrane damage, lysed GroE+/DnaK+ to death. Once they were introduced into the cell by electroporation, all three peptides were able to inhibit cell growth at concentrations of 10 times lower than their MICs. These results indicate that the nature of the peptide and also the characteristics of the cell membrane determine the antimicrobial actions of a peptide.
Solution stability of linear vs. cyclic RGD peptides
Authors: Bogdanowich‐Knipp Susan J. , Chakrabarti Soma , Siahaan Teruna J. , Williams Todd D. , Dillman Robert K.
Published: May 2003

Abstract: Arg‐Gly‐Asp (RGD) peptides contain an aspartic acid residue that is highly susceptible to chemical degradation and leads to the loss of biological activity. Our hypothesis is that cyclization of RGD peptides via disulphide bond linkage can induce structural rigidity, thereby preventing degradation mediated by the aspartic acid residue. In this paper, we compared the solution stability of a linear peptide (Arg‐Gly‐Asp‐Phe‐OH; 1) and a cyclic peptide (cyclo‐(1, 6)‐Ac‐Cys‐Arg‐Gly‐Asp‐Phe‐Pen‐NH2;2) as a function of pH and buffer concentration. The decomposition of both peptides was studied in buffers ranging from pH 2–12 at 50°C. Reversed‐phase HPLC was used as the main tool in determining the degradation rates and pathways of both peptides. Fast atom bombardment mass spectrometry (FAB‐MS), electrospray ionization mass spectrometry (ESI‐MS), matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) mass spectrometry, liquid chromatography‐mass spectrometry (LC‐MS), and one‐ and two‐dimensional nuclear magnetic resonance spectroscopy (NMR) were used to characterize peptides 1 and 2 and their degradation products. In addition, co‐elution with authentic samples was used to identify degradation products. Both peptides displayed pseudo‐first‐order kinetics at all pH values studied. The cyclic peptide 2 appeared to be 30‐fold more stable than the linear peptide 1 at pH 7. The degradation mechanisms of linear (1) and cyclic (2) peptides primarily involved the aspartic acid residue. However, above pH 8 the stability of the cyclic peptide decreased dramatically due to disulphide bond degradation. Both peptides also exhibited a change in degradation mechanism upon an increase in pH. The increase in stability of cyclic peptide 2 compared to linear peptide 1, especially at neutral pH, may be due to decreased structural flexibility imposed by the ring. This rigidity would prevent the Asp side chain carboxylic acid from orientating itself in the appropriate position for attack on the peptide backbone.
Synthesis and biological evaluation of a pyoverdin‐β‐lactam conjugate: a new type of arginine‐specific cross‐linking in aqueous solution
Authors: Kinzel O. , Budzikiewicz H.
Published: May 2003

Abstract: Arginine specific reagents such as phenylglyoxal and other α‐dioxo compounds react with arginine side chains by forming adducts with a stoichiometry of 2 : 1 or a mixture of 2 : 1 and 1 : 1. These adducts are labile in neutral and slightly alkaline aqueous solution. We developed a new type of cross‐linking reaction with aliphatic β‐dioxo compounds. They can be used for the well‐defined, irreversible covalent attachment of molecules carrying a primary amino group to arginyl residues of water soluble peptides. The reaction proceeds under mild conditions in aqueous solution, essentially without the formation of side products. A pyoverdin‐cephalexin conjugate was synthesized in order to promote its cellular uptake by Pseudomonas aeruginosa. Preliminary biological investigations of the conjugate indicated that it enters the bacterial cell via the pyoverdin‐mediated iron uptake pathway.
Preparation and properties of novel gramicidin S analogs possessing a tri‐, tetra‐ or pentamethylene bridge between ornithine side chains
Authors: Yamada K. , Ando K. , Takahashi Y.‐I. , Oda Y. , Yamamura H. , Araki S. , Kobayashi K. , Katakai R. , Kato F. , Kawai M.
Published: May 2003

Abstract: Gramicidin S (GS) analogs in which the Nδ atoms of the two Orn side chains are linked by an oligomethylene bridge [‐(CH2)n‐; n=3–5] were prepared via the bis(p‐nitrobenzenesulfonyl) derivative [Orn(NBS)2,2′]GS. For comparison the nonbridged secondary amino group‐containing analog [Orn(Me)2,2′]GS was also prepared. 1H NMR and CD spectral analysis indicated that these analogs adopt the same β‐sheet conformation as GS. The antimicrobial activities of these analogs were very similar, but were slightly dependent on the bridge chain length, the trimethylene‐bridged analog being the most potent.
The effect of conformation on the solution stability of linear vs. cyclic RGD peptides
Authors: Bogdanowich‐Knipp S.J. , Jois D.S.S. , Siahaan T.J.
Published: May 2003

Abstract: The objective of this study was to evaluate the relationship between conformational flexibility and solution stability of a linear RGD peptide (Arg‐Gly‐Asp‐Phe‐OH; 1) and a cyclic RGD peptide (cyclo‐(1, 6)‐Ac‐Cys‐Arg‐Gly‐Asp‐Phe‐Pen‐NH2; 2); as a function of pH. Previously, it was found that cyclic peptide 2 was 30‐fold more stable than linear peptide 1. Therefore, this study was performed to explain the increase in chemical stability based on the preferred conformation of the peptides. Molecular dynamics simulations and energy minimizations were conducted to evaluate the backbone flexibility of both peptides under simulated pH conditions of 3, 7 and 10 in the presence of water. The reactive sites for degradation for both molecules were also followed during the simulations. The backbone of linear peptide 1 exhibited more flexibility than that of cyclic peptide 2, which was reflected in the rotation about the phi and psi dihedral angles. This was further supported by the low r.m.s. deviations of the backbone atoms for peptide 2 compared with those of peptide 1 that were observed among structures sampled during the molecular dynamics simulations. The presence of a salt bridge between the side chain groups of the Arg and Asp residues was also indicated for the cyclic peptide under simulated conditions of neutral pH. The increase in stability of the cyclic peptide 2 compared with the linear peptide 1, especially at neutral pH, is due to decreased structural flexibility imposed by the ring, as well as salt bridge formation between the side chains of the Arg and Asp residues in cyclic peptide 2. This rigidity would prevent the Asp side chain carboxylic acid from orienting itself in the appropriate position for attack on the peptide backbone.
Enzymatic synthesis of vasoactive intestinal peptide analogs by transglutaminase
Authors: Esposito C. , Cozzolino A. , Mariniello L. , Stiuso P. , De Maria S. , Metafora S. , Ferranti P. , Cartenì‐Farina M.
Published: May 2003

Abstract: Vasoactive intestinal peptide is an amino acceptor and donor substrate for tissue transglutaminase (TGase) in vitro. This peptide contains a single glutamine residue, Gln16, which was identified as the amino acceptor substrate. Different γ(glutamyl16)amine derivatives of vasoactive intestinal peptide were synthesized enzymatically in vitro. The modification is very fast when compared with that of many native substrates of TGase. The analogs 1,3‐diaminopropane, putrescine, cadaverine, spermidine, spermine, glycine ethyl ester and mono‐dansyl‐cadaverine of the peptide were purified by high‐performance liquid chromatography on a reverse‐phase column and were analyzed by electrospray mass spectrometry. When amines were absent in the assay mixture as an external amino donor, lysine residue occurring in the peptide was an effective amino donor site for TGase. Only one of the three lysine residues of vasoactive intestinal peptide, namely Lys21, was demonstrated to be involved in both inter‐ and intramolecular cross‐link formation.
Fibril formation by amyloid‐β proteins may involve β‐helical protofibrils
Authors: Lazo N.D. , Downing D.T.
Published: May 2003

Abstract: We have proposed that amyloid fibrils contain subunits (protofibrils) that are formed from β‐strands wound into continuous 2–3 nm‐diameter β‐helices. Subsequent lateral aggregation of the β‐helices to form the widely observed 5–12 nm‐diameter fibrils could be promoted by hydrophobic residues on the exterior of the postulated β‐helix. A number of short peptide fragments of the amyloid‐β (Aβ) proteins, such as Aβ34–42 [LMVGGVVIA], the nine‐residue, carboxyl‐terminal portion of Aβ1–42, can also form amyloid fibrils. In the present study, it was found that a β‐helix formed from Aβ34–42 accounts for features suggested by published rotational resonance solid‐state NMR data, including an anomalous conformation about the Gly‐37–Gly‐38 region and exaggerated pleating. An analogue of Aβ34–42 was synthesized in which the hydrophobic groups on the exterior of the postulated β‐helix were replaced with glutamates, giving LEVGGVEIE. The analogue was completely soluble at pH 7, but at pH 2.5 it produced 2–2.5 nm‐diameter fibrils which did not associate into larger‐diameter bundles. The results of this study support the proposal that amyloid fibrils are formed from β‐helical subunits.
Serotonin uptake stimulating peptide found in plasma of normal individuals and in some autistic urines
Authors: Pedersen O.S. , Liu Y. , Reichelt K.L.
Published: May 2003

Abstract: We have isolated a tripeptide from normal plasma and autistic urines which stimulates the uptake of serotonin (5‐HT) into platelets. This peptide was purified by high‐performance liquid chromatography (HPLC) and characterized by sequenation and mass‐spectrometry. Synthetic peptide showed co‐chromatography with the biological sample in the HPLC systems used. Close to 60% of the autistic children diagnosed using the Diagnostic Statistical Manual III‐R had an increased HPLC peak eluting like this peptide in their urines compared with controls.
A cleavage cocktail for methionine‐containing peptides
Authors: Huang H. , Rabenstein D.L.
Published: May 2003

Abstract: A new cocktail has been developed for cleavage and deprotection of methionine‐containing peptides synthesized by 9‐fluorenylmethoxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis methodology. The cocktail (trifluoroacetic acid 81%, phenol 5%, thioanisole 5%, 1,2‐ethanedithiol 2.5%, water 3%, dimethylsulphide 2%, ammonium iodide 1.5% w/w) was designed to minimize methionine side‐chain oxidation. Application of the new cocktail (Reagent H) is demonstrated with the synthesis of a model pentadecapeptide from the active site of DsbC, a periplasmic protein involved in protein disulphide bond formation. The model peptide, which contains one methionine and two cysteine residues, was cleaved with several cleavage cocktails, including Reagent H. The crude peptides obtained with the widely used cocktails K, R and B were found to be 15% to 55% in the methionine sulphoxide form, whereas no methionine sulphoxide was detected in the crude peptide obtained by cleavage and deprotection with Reagent H. Also, no methionine sulphoxide was detected when 1.5% w/w NH4I was added to cocktails K, R and B; however, the yield of the desired peptide was less than with Reagent H. A second 28 amino acid model peptide of the active site of DsbC was also cleaved and deprotected with Reagent H. The reduced dithiol form of the peptide was found to be the major component (51% yield) of the crude peptide obtained by cleavage for 3 h. When the cleavage time was extended to 10 h, the peptide was converted to the intramolecular disulphide form (35% yield). A proposed mechanism for the in situ oxidation of cysteine with Reagent H is presented.
Solid‐phase synthesis of ovine Leydig cell insulin‐like peptide – a putative ovine relaxin?
Authors: Dawson N.F. , Macris M. , Tregear G.W. , Wade J.D. , Tan Y.Y. , Summers R.J. , Otvos L.
Published: May 2003

Abstract: The primary structure of ovine Leydig cell insulin‐like peptide (Ley I‐L) was recently deduced from the corresponding cDNA sequence. It consists of two peptide chains and three disulphide bonds in an arrangement similar to both relaxin and insulin. As in relaxin B‐chain, an Arg‐X‐X‐X‐Arg sequence exists within the Ley I‐L B‐chain although it is located four residues towards the C‐terminus from the corresponding position within relaxin. This sequence of amino acids is known to be essential for relaxin biological activity and its presence in Ley I‐L suggested that the peptide might possess a relaxin‐like function. Ovine Ley I‐L was assembled by Fmoc‐solid‐phase synthesis of the separate chains followed by their combination in solution at high pH. The purity and identity of the chain‐combined peptide was confirmed by chemical characterization including mass spectrometry. At physiological concentrations, the peptide was shown not to possess relaxin‐like activity in the rat isolated atrial chronotropic and inotropic assay. This strongly suggests that Ley I‐L is not a relaxin in the sheep. In order to explore further a possible structural relationship between Ley I‐L and relaxin, we prepared a synthetic analogue of ovine Ley I‐L containing a single replacement of B‐chain residue 12, His, with Arg. This was found to possess significant relaxin‐like chronotropic and inotropic activity demonstrating that the tertiary structure of Ley I‐L is similar to that of relaxin and highlighting the key requirement for the five‐residue sequence, Arg‐X‐X‐X‐Arg, to be present in position B12–16 for characteristic relaxin activity.
PATIC: a conformationally constrained photoisomerizable amino acid
Authors: Zhang J. , James D.A. , Woolley G.A.
Published: May 2003

Abstract:The synthesis of a conformationally constrained photoisomerizable amino acid, phenylazo‐1,2,3,4‐tetrahydro‐3‐isoquinolinecarboxylic acid (PATIC), is described. This amino acid can be incorporated into peptides using standard Fmoc procedures and can be accommodated within α‐helical structures albeit with some loss of stability of the structure. PATIC can serve as a useful building block for the synthesis of photoregulated peptides and proteins.
V3 loop‐derived peptide SPC3 inhibits infection of CD4− and galactosylceramide− cells by LAV‐2/B
Authors: Moulard M. , Mabrouk K. , Van Rietschoten J. , Rochat H. , Sabatier J.‐M. , Martin I.
Published: May 2003

Abstract: SPC3, a synthetic multibranched peptide including the GPGRAF consensus motif of the human immunodeficiency virus type 1 (HIV‐1) gp120 V3‐loop is a potent inhibitor of HIV infection of human CD4+ lymphocytes, macrophages and CD4‐/galactosylceramide+ human colon epithelial cells and is currently tested in phase II clinical trials (FDA protocol 257 A). The antiviral property of SPC3 was further investigated for its ability to inhibit LAV‐2/B, an HIV‐2 clone with a CD4‐independent tropism. SPC3 inhibited the LAV‐2/B‐mediated infection of B‐cell line which does not express the CD4 and the galactosylceramide molecules on their cell surface, suggesting an SPC3‐sensitive CD4/galactosylceramide‐independent pathway of viral infection in HIV susceptible cells. The molecular mechanism of the peptide inhibition was also investigated. The data suggested that the SPC3‐mediated inhibition does not result from a direct compe0tition between SPC3 and gp120 binding to the cell surface of the target cell.
N‐myristoylation of a peptide substrate for Src converts it into an apparent slow‐binding bisubstrate‐type inhibitor
Authors: Ramdas L. , Obeyesekere N.U. , Sun G. , McMurray J.S. , Budde R.J.A.
Published: May 2003

Abstract: The conversion of a peptide substrate to a potent inhibitor by chemical modification is a promising approach in the development of inhibitors for protein tyrosine kinases. N‐acylation of the synthetic peptide substrate NH2‐Glu‐Phe‐Leu‐Tyr‐Gly‐Val‐Phe‐Asp‐CONH2 (EFLYGVFD) resulted in synergistic inhibition of Src protein kinase activity that was greater than the inhibition by either free peptide and/or free acyl group. Synergistic inhibition was dependent upon the peptide sequence and the length of the acyl chain. The minimum length of the fatty acyl chain to synergistically inhibit Src was a lauryl (C11H23CO) group. N‐myristoylated EFLYGVFD (myr‐EFLYGVFD) inhibited the phosphorylation of poly E4Y by Src with an apparent Ki of 3 µm, whereas EFLYGVFD and myristic acid inhibited with Ki values of 260 and 35 µm, respectively. The nonacylated EFLYGVFD was a substrate for Src with Km and Vmax values of 100 µm and 400 nmol/min/mg protein, respectively. However, upon myristoylation, the peptide was no longer a substrate for Src. Both the acylated and non‐acylated peptides were competitive inhibitors against the substrate poly E4Y. The non‐acylated free peptide showed mixed inhibition against ATP while the myristoylated peptide was competitive against ATP. Myristic acid was uncompetitive against poly E4Y and competitive against ATP. Further analysis indicated that the myristoylated peptide acted as a reversible slow‐binding inhibitor with two binding sites on Src. The myristoylated 8‐mer peptide was reduced in size to a myristoylated 3‐mer without losing the affinity or charactersitics of a bisubstrate‐type inhibitor. The conversion of a classical reversible inhibitor to a reversible slow‐binding multisubstrate analogue has improved the potency of inhibition by the peptide.
Analogs of arginine vasopressin modified in position 3 with (R)‐α‐ hydroxymethylphenylalanine
Authors: Łempicka E. , Lammek B. , Slaninova J. , Olma A.
Published: May 2003

Abstract: This study describes the synthesis and some pharmacological properties of three new analogs of arginine vasopressin (AVP) substituted in position 3 with (R)‐α‐hydroxymethylphenylalanine ([R]‐HmPhe). All new peptides were tested for vasopressor and antidiuretic as well as uterotonic activity. None of the 3 analogs showed any pressor activity and their uterotonic activity was negligible. Only analog [Mpa1,(R)‐HmPhe3]AVP exhibited significant antidiuretic activity.
Synthesis and secondary structural studies of penta(acetyl‐Hmb)Aβ(1–40)
Authors: Clippingdale A.B. , Macris M. , Wade J.D. , Barrow C.J.
Published: May 2003

Abstract: The Fmoc solid phase synthesis of Aβ(1–40), a strongly aggregating peptide found in Alzheimer’s disease brain, was performed using 2‐hydroxy‐4‐methoxybenzyl (Hmb) backbone amide protection. Hmb‐Gly residues were incorporated using Nα‐Fmoc‐Hmb‐Gly‐OH rather than N,O‐bisFmoc‐Hmb‐Gly‐OPfp. Amino acid acylation of the sterically hindered Hmb‐amino acids was monitored using ‘semi‐on‐line’ MALDI‐TOF‐MS in a novel application of this technique which significantly simplified the successful incorporation of these residues. Standard coupling conditions in N,N‐dimethylformamide (DMF) were used throughout the synthesis. Comparative structural studies of acetyl‐Hmb‐protected and native Aβ(1–40) were performed to investigate the structural basis of Hmb‐mediated disaggregation. The incorporation of backbone amide protection was observed by circular dichroism spectroscopy and gel electrophoresis to strongly affect the solution structure of Aβ(1–40). Despite the reported structure‐breaking activity of Hmb groups, penta(acetyl‐Hmb)Aβ(1–40) was found to adopt both α‐helix and intermolecular β‐sheet conformations. In 100% TFE a mixed α‐helix/random coil structure was formed by the protected peptide indicating reduced α‐helical propensity relative to Aβ(1–40). The protected peptide formed β‐sheet structures in aqueous buffer. Gel electrophoresis indicated that, unlike native Aβ(1–40), penta(acetyl‐Hmb)Aβ(1–40) did not form large aggregate species.
Hydrophilic interaction/cation‐exchange chromatography for the purification of synthetic peptides from closely related impurities: serine side‐chain acetylated peptides
Authors: Litowski J.R. , Semchuk P.D. , Mant C.T. , Hodges R.S.
Published: May 2003

Abstract: Mixed‐mode hydrophilic interaction/cation‐exchange chromatography (HILIC/CEC) is a novel HPLC technique which has excellent potential for peptide separations. Separations by HILIC/CEC are carried out by subjecting peptides to linear increasing salt gradients in the presence of high levels of acetonitrile, which promotes hydrophilic interactions overlayed on ionic interactions with the cation‐exchange matrix. Complex peptide mixtures produced by solid‐phase synthesis are a frequently encountered and challenging purification problem. In the present study a two‐step protocol, consisting of HILIC/CEC followed by RPC, was required for the successful purification of a 21‐residue synthetic amphipathic α‐helical peptide from serine side‐chain acetylated impurities, with HILIC/CEC proving to be highly sensitive to subtle differences in hydrophilicities between the acetylated peptides and the desired product. Investigation of the three potential sites of serine acetylation through solid‐phase synthesis of acetylated analogues of the desired peptide (peptides of the same sequence and secondary structure, but acetylated at different positions on the hydrophilic face of the α‐helix) demonstrated that acetylation was occurring at different sites on the peptide. HILIC/CEC was able to take advantage of very subtle changes in environment around the acetylation sites and thus effect a separation of these analogues not achievable by RPC or CEC alone.
NMR structural characterization of cecropin A(1–8) – magainin 2(1–12) and cecropin A(1–8) – melittin(1–12) hybrid peptides
Authors: Oh D. , Kim Y. , Shin S.Y. , Kang J.H. , Hahm K. ‐S. , Kim K.L.
Published: May 2003

Abstract: In order to elucidate the structure–antibiotic activity relationships of the peptides, the three‐dimensional structures of two hybrid peptides, CA(1–8) – MA(1–12) and CA(1–8) – ME(1–12) in trifluoroethanol‐containing aqueous solution were investigated by NMR spectroscopy. Both CA(1–8) – MA(1–12) and CA(1–8) – ME(1–12) have strong antibacterial activity but only CA(1–8) – ME(1–12) has hemolytic activity against human erythrocytes. CA(1–8) – MA(1–12) has a hydrophobic 310‐helix of only two turns combined with one short helix in the N‐terminus with a flexible hinge section in between. CA(1–8) – MA(1–12) has a severely bent structure in the middle of the peptide. These structural features as well as the low hydrophobicity of CA(1–8) – MA(1–12) seem to be crucial for the selective lysis against the membrane of prokaryotic cells. CA(1–8) – ME(1–12) has an α‐helical structure of about three turns in the melittin domain and a flexible structure with one turn in the cecropin domain connected with a flexible hinge section in between, and these might be the structural features required for membrane distruption against prokaryotic and eukaryotic cells. The central hinge region (Gly9‐Ile10‐Gly11) in an amphipathic antibacterial peptide is considered to play an important role in providing the conformational flexibility required for ion channel formation of the C‐terminal hydrophobic α‐helix on cell membrane.
An insulin analogue with γ‐amino butyric acid substitution for A13Leu‐A14Tyr
Authors: Huang Y.‐D. , Feng Y.‐M. , Yang S.‐Z. , Niu C.‐I.
Published: May 2003

Abstract: An insulin A chain analogue, [A13–14 GABA, A21 Ala]A chain, for which the dipeptide Leu‐Try at A13‐A14 was substituted by a non‐coded amino acid, γ‐amino butyric acid (GABA) and A21 Asn by Ala, was prepared by stepwise Fmoc solid‐phase manual synthesis and then combined with the natural B chain of porcine insulin to yield an insulin analogue, [A13–14 GABA, A21Ala] porcine insulin (GABA substituted insulin). This insulin analogue still retains 50%in vivo biological activity and 59% in receptor binding capacity. It can also be crystallized. These results indicate that its overall conformation is similar to the native form and that the side chains of A13Leu and A14Tyr are not essential for insulin activity. In addition, the replacement of a normal C–N peptide bond by an unnatural C–C bond may have general meaning in structure and function studies of other proteins.
Characterization and comparison of leuprolide degradation profiles in water and dimethyl sulfoxide
Authors: Hall S.C. , Tan M.M. , Leonard J.J. , Stevenson C.L.
Published: May 2003

Abstract: The effect of solvent on the rate of leuprolide degradation and on the structure of the degradation products was explored. Leuprolide solutions (370 mg/mL) were prepared in water and dimethyl sulfoxide (DMSO) for delivery in DUROSTM osmotic implants. Both solvent systems demonstrated better than 90% stability after 1 year at 37°C, where the DMSO formulation afforded better stability than the aqueous formulation and was used in subsequent clinical trials. The rate of leuprolide degradation in DMSO was also observed to accelerate with increasing moisture content, indicating that the aprotic solvent minimized chemical degradation. Interestingly, leuprolide degradation products varied with formulation vehicle. The proportions of leuprolide degradation products observed to form in water and DMSO at 37°C were hydrolysis > aggregation > isomerization > oxidation and aggregation > oxidation > hydrolysis > isomerization, respectively. Specifically, more N‐terminal hydrolysis and acetylation were observed under aqueous conditions, and increased Trp oxidation and Ser β‐elimination were seen under non‐aqueous conditions. Furthermore, the major chemical degradation pathway changed with temperature in the DMSO formulation (decreasing oxidation with increasing temperature), but not in the aqueous formulation.
Synthesis and evaluation of the physicochemical properties of esterase‐sensitive cyclic prodrugs of opioid peptides using an (acyloxy)alkoxy linker
Authors: Bak A. , Gudmundsson O.S. , Gangwar S. , Friis G.J. , Siahaan T.J. , Borchardt R.T.
Published: May 2003

Abstract: The objective of this work was to synthesize the cyclic prodrugs 1 and 2 of [Leu5]‐enkephalin (Tyr‐Gly‐Gly‐Phe‐Leu‐OH) and DADLE (Tyr‐D‐Ala‐Gly‐Phe‐D‐Leu‐OH), respectively, using an (acyloxy)alkoxy linker. The cyclic prodrugs 1 and 2 were synthesized via a convergent method using the (acyloxy)alkoxy promoiety that connected the C‐ and N‐terminus of the peptides. The key intermediates were compounds 6a and 9a for cyclic prodrug 1 and compounds 6b and 9b for cyclic prodrug 2. The key intermediates 6a and 9a (or 6b and 9b) were coupled to give compound 10a (or 10b). The N‐ and C‐terminus protecting groups were removed from 10a and 10b to give compounds 11a and 11b, respectively, which were then treated with HBTU to give 1 and 2 in 40% and 53% yields, respectively. The cyclic prodrugs 1 and 2 exhibited Stokes–Einstein molecular radii similar to those of [Leu5]‐enkephalin and DADLE; however, the cyclic prodrugs were shown to be significantly more lipophilic than the corresponding opioid peptides, as determined by partitioning experiments using immobilized artificial membrane (IAM) column chromatography. In addition, the cyclic prodrugs exhibit stable solution conformations, which reduce their hydrogen bonding potentials. Based on these physicochemical characteristics, the cyclic prodrugs 1 and 2 should have exhibited better transcellular flux across the Caco‐2 cell monolayer than [Leu5]‐enkephalin and DADLE, respectively. However, the cyclic prodrugs 1 and 2 were shown in separate studies to be substrates for P‐glycoprotein, which significantly reduced their ability to permeate across Caco‐2 cell monolayers. When P‐glycoprotein was inhibited, the permeability characteristics of prodrugs 1 and 2 were consistent with their physicochemical properties.
Synthesis and evaluation of the physicochemical properties of esterase‐sensitive cyclic prodrugs of opioid peptides using coumarinic acid and phenylpropionic acid linkers
Authors: Wang B. , Wang W. , Zhang H. , Shan D. , Nimkar K. , Gudmundsson O. , Gangwar S. , Siahaan T. , Borchardt R.T.
Published: May 2003

Abstract: In an attempt to improve the membrane permeabilities of opioid peptides, we have synthesized cyclic prodrugs of [Leu5]‐enkephalin and DADLE using a coumarinic acid or a phenylpropionic acid linker. The synthesis of the coumarinic acid‐ and phenylpropionic acid‐based cyclic prodrugs followed similar strategies. Key intermediates were the compounds with the C‐terminal amino acids of opioid peptides (L‐Leu, [Leu5]‐enkephalin; D‐Leu, DADLE) attached to the phenol hydroxyl group and the remaining amino acids of the peptide linked via the N‐terminal amino acid (L‐Tyr) attached to the carboxylic acid groups of the prodrug moieties (coumarinic acid or propionic acid). Cyclization of these linear precursors gave the cyclic prodrugs in 30–50% yields. These cyclic prodrugs exhibited excellent transcellular permeation characteristics across Caco‐2 cell monolayers, an in vitro model of the intestinal mucosa. To correlate the cellular permeabilities of these cyclic prodrugs with their physicochemical properties, we calculated their Stokes–Einstein molecular radii from their diffusion coefficients which were determined by NMR and we determined their membrane interaction potentials using immobilized artificial membrane (IAM) column chromatography. The cyclic prodrugs exhibited molecular radii similar to those of the parent compounds, [Leu5]‐enkephalin and DADLE. However, these cyclic prodrugs were shown to have much higher membrane interaction potentials than their corresponding opioid peptides. Therefore, the enhanced cellular permeation of the cyclic prodrugs is apparently due to the alteration of their lipophilicity and hydrogen bonding potential, but not their molecular sizes.