NMR and CD conformational studies of the C‐terminal 16‐peptides of Pseudomonas aeruginosa c551 and Hydrogenobacter thermophilus c552 cytochromes

Abstract: The 16‐amino acid sequences of the C‐terminal helices of the homologous bacterial cytochromes c₅₅₁ from Pseudomonas aeruginosa and c₅₅₂ from Hydrogenobacter thermophilus were synthesized and their solution structure studied. Circular dichroism and NMR experiments in aqueous solution have shown the presence of α‐helices and 3₁₀‐helices. The populations of helical structures in phosphate buffer, pH 3.5, 293 K, were 21% for c₅₅₁ and 20% for c₅₅₂, but increased to 56.7 and 48%, respectively, in 50% aqueous 2,2,2‐trifluoroethanol. An isodichroic point was observed at 203 nm in CD spectra for the helix/coil transition in mixtures of water/2,2,2‐trifluoroethanol. NMR spectra in phosphate buffer show the presence of both α‐ and 3₁₀‐helical structures. In water/2,2,2‐trifluoroethanol (50 : 50) α‐helices are predominant. CD temperature‐dependency studies indicate that both peptides exhibit the same cooperativity for the transition in water/2,2,2‐trifluoroethanol (50 : 50). The experimental data show that the amino acid substitutions do not favor heat resistance of the secondary structure of the c₅₅₂ C‐terminal helix at the local level. Instead, they optimize nonlocal contacts of the polypeptide chain, which stabilize the tertiary structure in the native protein.     

Divergent solid‐phase synthesis and candidacidal activity of MUC7 D1, a 51‐residue histidine‐rich N‐terminal domain of human salivary mucin MUC7

Abstract: Domain 1 of the low‐molecular‐weight human salivary mucin, designated MUC7 D1, spans the 51 N‐terminal amino acid residues. This domain contains a 15‐residue basic histidine‐rich subdomain (R3‐Q17) which has 53% sequence similarity to histatin 5 (Hsn‐5), a salivary molecule known to exert potent in vitro cidal activity against Candida albicans and many other medically important fungi. The MUC7 D1‐15mer and its derivatives have previously been synthesized in our laboratory and their candidacidal activities have been found to be inferior to that of Hsn‐5. We were therefore intrigued to explore the candidacidal potency of the full‐length MUC7 D1 (51‐mer). Linear solid‐phase synthesis of this domain has been accomplished following standard Fmoc chemistry. The problems of partial coupling, owing to the peptide chain length, at several stages of the solid‐phase step‐by‐step synthesis were circumvented either by double‐coupling techniques or efficient coupling procedures. The MUC7 D1 peptide was purified to homogeneity by conventional reverse‐phase HPLC using two columns connected in series. Secondary structure of the purified peptide was assessed by circular dichroism (CD) spectroscopy in phosphate buffer and trifluoroethanol and compared to that of MUC7 D1‐15mer and Hsn‐5. The MUC7 D1 candidacidal activity was assessed against azole‐sensitive and azole‐resistant C. albicans strains and was found, unlike that of the MUC7 D1‐15mer, to be comparable with that of Hsn‐5, indicating that in addition to Hsn‐5, MUC7 D1 could provide an attractive alternative to the classical antifungal agents. The candidacidal potency of MUC7 D1, like that of MUC7 D1‐15mer, and of Hsn‐5, appears to be largely dependent on peptide charge, irrespective of α‐helical structure.     

Design and function of a conformationally restricted analog of the influenza virus fusion peptide

Abstract: A conformationally restricted analog of the N‐terminal 12‐residue peptide segment of the HA2 subunit of the PPV/34, PR/8/34, and Jap/57 strains of influenza virus hemagglutinin was synthesized containing three residues of C^α‐methyl‐valine. This peptide has a higher content of helical structure in the presence of 50% trifluoroethanol or when interacting with liposomes of egg phosphatidylcholine compared with the conformationally more flexible control peptide with the native sequence. The control and analog peptides had opposite effects on membrane curvature as measured by shifts in the bilayer‐to‐hexagonal phase transition temperature of a synthetic phosphatidylethanolamine derivative. The control peptide promoted more negative curvature, particularly at acidic pH and was also more potent than the analog in promoting lipid mixing. The results indicate that the ability of the peptide to sample a broader range of conformations is required for the influenza fusion peptide to destabilize membranes and that a rigid helical structure is less fusogenic. The difference between the two peptides and between pH 7.4 and pH 5.0 show a correlation between the ability to promote negative curvature and to accelerate lipid mixing.     

Structural study of the sodium channel inactivation gate peptide including an isoleucine–phenylalanine–methionine motif and its analogous peptide (phenylalanine/glutamine) in trifluoroethanol solutions and SDS micelles

In order to gain insight into the gating mechanisms of Na⁺ channels, in particular their inactivation mechanisms, we studied the structures of the Na⁺ channel inactivation gate related peptide which includes the IFM (Ile‐Phe‐Met) motif (Ac‐KKKFGGQDIFMTEEQKK‐NH₂; K1480–K1496 in rat brain type‐IIA Na⁺ channels, MP‐3A) and its F/Q(Gln) substituted one (MP‐4A) in trifluoroethanol (TFE) solutions and sodium dodecyl sulfate (SDS) micelles using circular dichroism (CD) and ¹H‐NMR spectroscopies. Based on observed nuclear Overhauser effect constraints, three‐dimensional structures of MP‐3A and MP‐4A were determined using simulated annealing molecular dynamics/energy minimization calculations. In TFE solutions, no appreciable differences in the structure were observed using either CD or NMR spectra. In SDS micelles, however, the two peptides exhibited definitely different structures from each other. It was found that in MP‐3A, residues I1488 and T1491 were spatially proximate with each other owing to hydrogen bonding between the amide proton of I1488 and the hydroxyl oxygen atom of T1491, whereas in MP‐4A, F/Q substitution separated them owing to conformational changes. The solvent‐accessible surfaces calculated for the structures of MP‐3A and MP‐4A showed that the former has a smoother interaction surface to the hydrophobic docking site than the latter. In conclusion, the conformational changes, as well as decreased hydrophobicity around the IFM motif owing to the F/Q mutation, may be one reason why F1489Q mutated channels cannot inactivate almost completely.     

Selective and facile cyclization of N‐chloroacetylated peptides from the C4 domain of HIV Gp120 in LiCl/DMF solvent systems

Lithium salts have been reported to mediate the solubilization of peptides in organic solvents in 1989 (Seebach, D., Thaler, A. & Beck, A. K. Helv. Chim. Acta 1989; 72 , 857–867). The use of Li salts in an organic solvent to influence cyclization of a reactive peptide that only polymerizes in an aqueous solvent, has not been reported. Here, the selective and facile cyclization of N‐chloroacetylated, C‐cysteine amide peptides from the C4 domain of HIV‐1 gp120 in LiCl/DMF solvent systems is demonstrated. The addition of stoichiometric amounts of Tris base to 1 mg/mL peptide in LiCl/DMF solutions was sufficient to drive the cyclization to completion within 3 h at ambient temperatures. Cyclic peptides were the only detectable reaction products and these were confirmed using reversed‐phase HPLC and mass spectrometric analyses of the final products. In aqueous solutions at pH 7.4, only polymers were obtained as judged by HPLC and SDS–PAGE. The method of using Li salts in an organic solvent to enhance the cyclization of unprotected amphipathic peptides may be useful in many situations beyond those described here.     

Baeyer‐Villiger oxidation tuned to chemoselective conversion of non‐activated [18F]fluorobenzaldehydes to [18F]fluorophenols

A reaction pathway via oxidation of [¹⁸F]fluorobenzaldehydes offers a very useful tool for the no‐carrier‐added radiosynthesis of [¹⁸F]fluorophenols, a structural motive of several potential radiopharmaceuticals. A considerably improved chemoselectivity of the Baeyer‐Villiger oxidation (BVO) towards phenols was achieved, employing 2,2,2‐trifluoroethanol as reaction solvent in combination with Oxone or m‐CPBA as oxidation agent. The studies showed the necessity of H₂SO₄ addition, which appears to have a dual effect, acting as catalyst and desiccant. For example, 2‐[¹⁸F]fluorophenol was obtained with a RCY of 97% under optimised conditions of 80°C and 30‐minute reaction time. The changed performance of the BVO, which is in agreement with known reaction mechanisms via Criegee intermediates, provided the best results with regard to radiochemical yield (RCY) and chemoselectivity, i.e. formation of [¹⁸F]fluorophenols rather than [¹⁸F]fluorobenzoic acids. Thus, after a long history of the BVO, the new modification now allows an almost specific formation of phenols, even from electron‐deficient benzaldehydes. Further, the applicability of the tuned, chemoselective BVO to the n.c.a. level and to more complex compounds was demonstrated for the products n.c.a. 4‐[¹⁸F]fluorophenol (RCY 95%; relating to 4‐[¹⁸F]fluorobenzaldehyde) and 4‐[¹⁸F]fluoro‐m‐tyramine (RCY 32%; relating to [¹⁸F]fluoride), respectively.     

A new B‐chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B‐chain structure

Abstract: The solution structure of a new B‐chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D‐NMR and molecular modeling. This structure is compared with that of the oxidized B‐chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res. 46 , 424–433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B‐chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B‐chain. 2D‐NMR experiments confirmed, among multiple conformations, that the B‐chain mutant presents defined secondary structures such as a α‐helix between residues B9 and B19, and a β‐turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B‐chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B‐chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B‐chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B‐chains.     

Sensory irritation mechanisms investigated from model compounds: trifluoroethanol,hexafluoroisopropanol and methyl hexafluoroisopropyl ether

Quantitative structure-activity relationships (QSAR) have suggested the importance of hydrogen bonding in relation to activation of the sensory irritant receptor by nonreactive volatile organic chemicals. To investigate this possibility further, three model compounds with different hydrogen bond acidity, trifluoroethanol, hexafluoroisopropanol and methyl hexafluoroisopropyl ether, were selected for study. The potency of each chemical is obtained from the concentration necessary to reduce respiratory rate in mice by 50% (RD50). The RD50 values obtained were: methyl hexafluoroisopropyl ether (?160?000?ppm), trifluoroethanol (11?400–23?300?ppm), and hexafluoroisopropanol (165?ppm). QSAR showed that trifluoroethanol and methyl hexafluoroisopropyl ether behaved as predicted as nonreactive sensory irritants, whereas hexafluoroisopropanol was much more potent than predicted. The higher than predicted potency of hexafluoroisopropanol could be due to a coupled reaction, involving both strong hydrogen bonding and weak Brönsted acidity. A concerted reaction could thus be more efficient in activation of the receptor. Hydrogen bonding properties and concerted reactions may be important in the activation of the sensory irritant receptor by nonreactive volatile organic chemicals.     

Design and characterization of a membrane permeable N‐methyl amino acid‐containing peptide that inhibits Aβ1–40 fibrillogenesis

Abstract: Alzheimer's disease, Huntington's disease and prion diseases are part of a growing list of diseases associated with formation of β‐sheet containing fibrils. In a previous publication, we demonstrated that the self‐association of the Alzheimer's β‐amyloid (Aβ) peptide is inhibited by peptides homologous to the central core domain of Aβ, but containing N‐methyl amino acids at alternate positions. When these inhibitor peptides are arrayed in an extended, β‐strand conformation, the alternating position of N‐methyl amino acids gives the peptide two distinct faces, one exhibiting a normal pattern of peptide backbone hydrogen bonds, but the other face having limited hydrogen‐bonding capabilities due to the replacement of the amide protons by N‐methyl groups. Here, we demonstrate, through two‐dimensional NMR and circular dichroic spectroscopy, that a pentapeptide with two N‐methyl amino acids, Aβ16–20m or Ac‐K(Me)LV(Me)FF‐NH₂, does indeed have the intended structure of an extended β‐strand. This structure is remarkably stable to changes in solvent conditions and resists denaturation by heating, changes in pH (from 2.5 to 10.5), and addition of denaturants such as urea and guanindine‐HCl. We also show that this peptide, despite its hydrophobic composition, is highly water soluble, to concentrations > 30 mm , in contrast to the nonmethylated congener, Aβ16–20 (Ac‐KLVFF‐NH₂). The striking water solubility, in combination with the hydrophobic composition of the peptide, suggested that the peptide might be able to pass spontaneously through cell membranes and model phospholipid bilayers such as unilamellar vesicles. Thus, we also demonstrate that this peptide is indeed able to pass spontaneously through both synthetic phospholipid bilayer vesicles and cell membranes. Characterization of the biophysical properties of the Aβ16–20m peptide may facilitate the application of this strategy to other systems as diverse as the HIV protease and chemokines, in which there is dimerization through β‐strand domains.     

Molecular dynamics simulation on the inhibition mechanism of peptide‐based inhibitor of islet amyloid polypeptide (IAPP) to islet amyloid polypeptide (IAPP22–28) oligomers

Aggregation of islet amyloid polypeptide (IAPP) is implicated in the development of type 2 diabetes. The modified NFGAIL with double N‐methylated at Gly24 and Ile26 has the property of soluble, non‐amyloidogenic, non‐cytotoxic, and the ability of inhibiting amyloid formation and cytotoxicity of IAPP. To discover the inhibition mechanism of this peptide inhibitor and provide useful information to design more potential peptide inhibitors, molecular dynamics simulations in explicit solvent were performed. The simulation results reveal that Gly24 and Ile26 are of importance in IAPP aggregation, and N‐methylation at these two key residues will disrupt the stability of formed oligomer and prevent the conformation transition of free monomer near the oligomer template. The origin of the N‐methylated peptide inhibitor inhibiting IAPP aggregation is that it can keep good binding with IAPP template by stable hydrogen bonding interaction. Furthermore, it cannot induce the conformational transition of free monomer by preventing the hydrogen bond interaction between free monomer and boundary peptide. The structural environment can largely affect the stacking of free monomers to the template. Our study sheds light on the inhibition mechanism of peptide inhibitor at molecular level and may provide guidance for the future design and discovery of new peptide inhibitors.     

An asymmetric conformation of 1,3,5‐benzene tricarbonyl [Aib4OMe]3*

Abstract: Molecules of 1,3,5‐benzene tricarbonyl [Aib₄OMe]₃ do not possess any internal symmetry, neither exact nor approximate, in the crystalline state. The Aib₄OMe moieties each form a 3₁₀‐helix with an appropriate pair of hydrogen bonds but the sense of rotation is right‐handed for two of the helices and left‐handed for the third one. The helices are not evenly positioned around the benzene ring, and their helix axes are inclined toward one side of the plane of the benzene ring, giving the molecule the shape of a shallow bowl with an irregular periphery. The molecules are largely surrounded by water and dimethyl sulfoxide (DMSO) solvent molecules that form hydrogen bonds with the CO and NH moieties that protrude from the surfaces of the peptide molecule. The space group is Cc with a = 23.618(4) Å, b = 19.708(6) Å, c = 17.939(7) Å and β = 100.09(3)°.     

Effect of viscosity on the deamidation rate of a model Asn‐hexapeptide

Abstract: The effect of viscosity on the deamidation rate of a model Asn‐containing hexapeptide (l ‐Val‐l ‐Tyr‐Pro‐l ‐Asn‐Gly‐l ‐Ala) was assessed in aqueous solution and in solids containing varying amounts of poly(vinyl pyrrolidone) (PVP) and water. Stability studies were conducted at 0.1 mg/mL peptide and 0–50% PVP (w/w) in aqueous solution, and at 5% (w/w) peptide and different relative humidities (31.6, 53.1, 74.4 and 96%) in the solid state. The parent peptide and its deamidation products were analysed by reverse‐phase high‐performance liquid chromatography. Deamidation rates decreased with increasing solvent viscosity in a manner described by a semi‐empirical mathematical model developed to describe this relationship. The results suggest that the motion of the Asn side‐chain along the reaction coordinate is a function of the macroscopic solvent viscosity. However, the apparent energy barrier for the diffusive movement of the side‐chain appears to be less than the energy barrier for that associated with macroscopic viscosity. The dependence of the deamidation rate on viscosity in both viscous solution and hydrated solids further demonstrates the importance of mobility in peptide deamidation.     

Mixtures of trifluoroethanol or hexafluoroisopropanol and dimethylformamide are not of general applicability for peptide condensations catalyzed by trypsin

Mixtures of a good hydrogen bond donor, 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3-hexafluroisopropanol, and an acceptor, dimethylformamide (DMF) (1:1,v/v), containing 4% buffer have been described as adequate solvent systems for trypsin-catalyzed peptide fragment condensations [Mihara et al. (1993) Int. J. Pept. Protein Res. 41 , 405]. Thus, we decided to study the behaviour of trypsin in such solvent systems. We investigated whether this protease would efficiently catalyze condensations between fragments derived from an analogue of the gp-41 capsid protein of HIV virus or from cholecystokinin-22. None of the reactions carried out yielded the desired condensation products. However, when Fmoc-NLQNLDPSHR-OH and cholecystokinin-12 (H-ISDRDYMGWMDF-NH₂) were used as substrates, the last had its R-D peptide bond hydrolyzed producing cholecystokinin-8. The proteolytic activity of this enzyme measured against a fluorogenic peptide derivative was 50 times lower in DMF/TFE containing 5% of aqueous phase than in buffer. Steady-state fluorescence studies in DMF/TFE buffer were performed to examine the structure of this protease in these media. Steady-state spectra obtained with increasing proportions of these two organic solvents in buffer showed that the emission intensities built up. Quenching studies with iodide revealed that the I_o/I ratio (where I_o and I are the fluorescence emission intensities in the absence and presence of quencher, respectively) changed from 1.2 in aqueous media to 2.2 in DMF/TFE (1:1, v/v) containing 11% 0.2 m Tris-HCI buffer, pH 8.0, for 0.5 m iodide. The complete data indicated a higher exposure of tryptophan residues to the quencher in organic media, probably because of the partial unfolding of the enzyme.     

O‐Linked glycopeptides retain helicity in water

Abstract: A 17‐residue O‐linked glycopeptide model incorporating a central α‐mannosyl serine residue, and its unglycosylated analog both demonstrate substantial helicity in water. The peptide sequence was derived from previous studies in which differences in overall helicity as a function of single amino acid substitutions were measured by circular dichroism (CD). The helical content was predicted by molecular modeling, and confirmed by CD and NMR. Moreover, the glycopeptide retained its helicity in the presence of SDS micelles, whereas the native peptide lost secondary structure in the presence of micelles. The inference is that the peptide sequence is a more important helix determinant than glycosylation per se.     

Solvent effects on coupling yields during rapid solid‐phase synthesis of CGRP(8‐37) employing in situ neutralization*

Abstract: The success of solid‐phase peptide synthesis is often dependent upon solvation of the resin and the growing resin‐bound peptide chain. We investigated the relationship between solvent properties and solvation of the resin and peptide‐resin in order to obtain satisfactory coupling yields for the rapid solid‐phase peptide synthesis, using butyloxycarbonyl‐(Boc)‐amino acid derivatives, of human‐α‐calcitonin gene‐related peptide(8‐37) (CGRP(8‐37)). Solvation of (p‐methylbenzhydrylamine)copoly(styrene–1% divinylbenzene (DVB) (resin) and resin covalently bound to the fully protected amino acid sequence of CGRP(8‐37) (peptide–resin) was correlated to solvent Hildebrand solubility (δ) and hydrogen‐bonding (δ_h) parameters. Contour solvation plots of δ_h vs. δ revealed maximum solvation regions of resin and peptide–resin. Maximum resin solvation occurred with N‐methylpyrrolidinone (NMP), NMP : dimethylsulfoxide (DMSO) (8 : 2) and DMSO. Inefficient solvation of the peptide–resin occurred with these solvents and resulted in poor syntheses with average coupling yields of 78.1, 88.9 and 91.8%, respectively. Superior peptide–resin solvation was obtained using dimethylacetamide (DMA) and dimethylformamide (DMF), resulting in significantly higher average coupling yields of 98.0 and 99.5%, respectively. Thus, the region of maximum peptide–resin solvation shifts to solvents with higher δ_h values. DMF provided the most effective peptide–resin solvation and was the only solvent from which CGRP(8‐37) was obtained as a single major product in the crude cleaved material.     

Relationship between the tertiary structures of mastoparan B and its analogs and their lytic activities studied by NMR spectroscopy

Abstract: Mastoparan B (MP‐B), an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis, is an amphiphilic α‐helical peptide. MP‐B possesses a variety of biological activities, such as mast cells degradation histamine release, erythrocyte lysis and inhibition of the growth of Gram‐positive and Gram‐negative bacteria. In order to study the relationship between the structure and the biological activity of MP‐B, we used four analogs by replacing amino acids with alanine. Tertiary structures of MP‐B and its analogs in 2,2,2‐trifluoroethanol (TFE)‐containing aqueous solution have been determined by NMR spectroscopy and molecular modeling. The results indicate that [Ala⁴]MP‐B and [Ala¹²]MP‐B with higher hydrophobicity adopt a higher content of amphiphilic helical structures, and have better antimicrobial and hemolytic activities than MP‐B. However, [Ala³]MP‐B and [Ala⁹]MP‐B with lower hydrophobicity have disordered structures. [Ala³]MP‐B and [Ala⁹]MP‐B have low antimicrobial activity and much less hemolytic activity relative to MP‐B. It is likely that tryptophan residue in MP‐B and appropriate hydrophobicity of MP‐B to induce α‐helical structure is essential for the antibacterial and hemolytic activity of MP‐B. This study can aid understanding of the structure–activity relationship of MP‐B and to design peptides to possess lytic activity.     

气相色谱法测定硫酸壮观霉素有机溶剂残留量

目的建立硫酸壮观霉素中有机溶剂残留量的测定方法。方法采用顶空进样毛细管气相色谱法,色谱柱为Elite-5毛细管柱,载气为氮气,FID检测器,正丙醇为内标物质,纯净水为溶解介质,检测了硫酸壮观霉素中丙酮的残留量。结果丙酮和正丙醇完全分离,在所考察的浓度范围内具有良好的线性,r=0.9998,丙酮的定量限为:6.88×106g·mL-1。精密度RSD分别为1.5%、0.8%、0.5%均小于5%,平均回收率为97.15%～100.63%,均介于97%～103%。结论本试验建立的方法简便灵敏,结果准确可靠,适用于硫酸壮观霉素中有机溶剂残留量的检测。     

Studies on analogs of a peptide derived from alpha‐fetoprotein having antigrowth properties

Abstract: A 34‐amino acid portion of the third domain of alpha‐fetoprotein possesses antigrowth and anticancer activities. Three analogs of this sequence were chemically synthesized, in which the two cysteines of the original sequence were replaced by alanines, glycines or serines. The original cysteine and alanine peptides formed trimers at 0.20 g/L in pH 7.4 phosphate buffer, and the glycine and serine peptides formed dimers. Trimer preparations were more potent in inhibiting estrogen‐induced growth in the mouse uterine assays than the two dimeric oligomers. Of salient importance is that the alanine peptide retained its trimeric form in solution much longer than the cysteine peptide. Antigrowth assays were performed starting with stock solutions at a peptide concentration of 0.20 g/L, because at very high peptide concentration (8.0 g/L) the peptides aggregated extensively. All the peptides, although differing in biological activity, had almost identical secondary structures. Unlike alpha‐fetoprotein, the three peptides have low amounts of α‐helix. Trifluoroethanol has the ability to convert peptides into a helical conformation when they have a propensity for that structure. At trifluoroethanol concentrations of 20% and higher, the alanine and glycine peptides were changed into highly helical structures.     

Studies on the conformational properties of CP‐1042−55, the hinge region of CP‐10, using circular dichroism and RP‐HPLC

Abstract: The conformational properties of CP‐10^42?55, a peptide corresponding to the hinge region of CP‐10, were investigated using circular dichroism spectroscopy and reverse‐phase high‐performance liquid chromatography (RP‐HPLC). The circular dichroism studies indicated that CP‐10^42?55 formed considerable secondary structure in the presence of hydrophobic solution environments including 50% acetonitrile, 50% trifluoroethanol and 200 mm sodium dodecyl sulfate, which comprised a mixture of α‐helix and β‐sheet. The effect of temperature on the conformation of CP‐10^42?55 was investigated between 5 and 40°C, with very small changes in the spectra being observed.RP‐HPLC was then used to investigate the effect of temperature on the conformation of CP‐10^42?55 in the presence of a hydrophobic surface. Using a C₁₈‐adsorbent, CP‐10^42?55 exhibited a conformational transition at 25°C, which was associated with an increase in the chromatographic contact area and the binding affinity of the peptide for the stationary phase. In addition, near‐planar bandbroadening behaviour indicated that conformational species interconverted with rapid rate constants compared with the chromatographic time scale. These results indicated that the conformational change at 25°C in theRP‐HPLC system most likely corresponds to the unfolding of an α‐helical and/or β‐sheet structure to an extended coil structure. Therefore, the strong chemotactic properties of this peptide may be attributed to its ability to form considerable secondary structure in the presence of a hydrophobic environment.     

Deamidation of a model hexapeptide in poly(vinyl alcohol) hydrogels and xerogels

Abstract: Polymeric controlled release systems have been proposed to prolong the half‐lives of protein and peptide drugs in vivo and to deliver active drug at a controlled rate. These systems are ineffective, however, if the drug is not stable during storage and release. This study addresses the effect of poly(vinyl alcohol) on the stability and release of an incorporated hexapeptide, VYPNGA, which undergoes deamidation. Two types of peptide‐loaded poly(vinyl alcohol) matrices were formed, a semisolid hydrogel and a lower water content ‘xerogel’, and stored at 50°C for up to 122 days. The hexapeptide was less stable in both poly(vinyl alcohol) matrices than in aqueous buffer or lyophilized polymer‐free powders. The type of poly(vinyl alcohol) matrix appeared to influence the degradation mechanism, since the product distributions differ in the hydrogel and the xerogel. The results suggest that, rather than stabilizing this peptide, incorporation in poly(vinyl alcohol) matrices reduces stability relative to solution and lyophilized controls.