Asparagine coupling in Fmoc solid phase peptide synthesis

To investigate side reactions during the activation of side chain unprotected asparagine in Fmoc-solid phase peptide synthesis the peptide Met-Lys-Asn-Val-Pro-Glu-Pro-Ser was synthesized using different coupling conditions for introduction of the asparagine residue. Asparagine was activated by DCC/HOBt, BOP (Castro's reagent) or introduced as the pentafluorophenyl ester. The resulting peptide products were analyzed by HPLC, mass spectrometry and Edman degradation. In the crude products varying amounts of beta-cyano alanine were found, which had been formed by dehydration of the side chain amide during carboxyl activation of Fmoc-asparagine. A homogeneous peptide was obtained by using either side chain protected asparagine derivatives with BOP mediated activation or by coupling of Fmoc-Asn-OPfp. Fmoc-Asn(Mbh)-OH and Fmoc-Asn(Tmob)-OH were coupled rapidly and without side reactions with BOP. For the side chain protected derivatives the coupling was as fast as that of other Fmoc-amino acid derivatives, whereas couplings of Fmoc-Asn-OH proceed more slowly. However, during acidolytic cleavage both protection groups, Mbh and Tmob, generate carbonium ions which readily alkylate tryptophan residues in a peptide. Tryptophan modification was examined using the model peptide Asn-Trp-Asn-Val-Pro-Glu-Pro-Ser. Alkylation could be reduced by addition of scavengers to the TFA during cleavage and side chain deprotection. A homogeneous peptide containing both, asparagine and tryptophan, was obtained only by coupling of Fmoc-Asn-OPfp.     

Fmoc-Asn(Trt)-OH与Wang树脂的合成研究

研究了Fmoc-Asn(Trt)-OH与Wang树脂的酯化反应工艺。探讨了反应策略、溶剂体系、投料比、反应时间以及反应温度等条件对手工合成Fmoc-Asn(Trt)-Wang树脂反应的影响。并用微波辅助方法与常规方法进行了对比。实验结果表明了采用HBTU/HOBt/DIEA方法的连接效率最高。对反应的优化结果为:NMP/DCM体积比为1:7,体系、摩尔比为3:1,每次反应时间4 h,温度40℃。微波对本酯化反应影响不大。     

Synthesis of phosphopeptides by the Multipinast method: Evaluation of coupling methods for the incorporation of Fmoc- Tyr(PO3Bzl,H)-OH,Fmoc- Ser(PO3Bzl,H)-OH and Fmoc- Thr(PO3Bzl,H)-OH

Summary The efficiency of various coupling methods for the incorporation of the three monobenzyl phosphorodiesterprotected derivatives, Fmoc-Tyr(PO₃Bzl,H)-OH, Fmoc-Ser(PO₃Bzl,H)-OH and Fmoc-Thr(PO₃Bzl,H)-OH, was examined through the test synthesis of Ala-Ser-Gln-Gly-Xxx(PO₃H₂)-Leu-Glu-Asp-Pro-Ala-NH₂ (Xxx=Tyr, Ser, Thr) using the Multipin method of multiple peptide synthesis. The coupling methods examined were (1) PyBrop/DIEA; (2) BOP/HOBt/NMM; (3) BOP/HOBt/DIEA; (4) HBTU/HOBt/DIEA; (5) HATU/HOAt/DIEA; (6) HATU/DIEA; (7) DIC/HOBt; (8) DIC/HOBt/DIEA; (9)DIC/HOAt; (10) DIC/HOAt/DIEA. While all four DIC-based coupling procedures resulted in incomplete incorporation, both the HBTU/HOBt/DIEA and HATU/HOAt/DIEA coupling procedures provided most efficient incorporation of the three Fmoc-Xxx (PO₃Bzl,H)-OH derivatives. In the subsequent synthesis of the α-helical Tyr(P)-peptide, Glu-Thr-Gly-The-Lys-Ala-Glu-Leu-Leu-Ala-Lys-Tyr(PO₃H₂)-Glu-Ala-Thr-His-Lys-NH₂, analysis of the crude peptide by electrospray MS confirmed that several residue deletions had occurred but that complete incorporation of the Tyr(P)-residue had been accomplished using HBTU/HOBt/DIEA coupling of Fmoc-Tyr(PO₃Bzl,H)-OH. Multipin is a trademark of Chiron Technologies Pty. Ltd., Clayton, Victoria, Australia.     

Solid-phase synthesis of reactive peptide crosslinker by selective deprotection

An effective and simple strategy for preparing peptide crosslinkers is described. An MMP-13 degradable peptide QPQGLAK-NH(2) was prepared on the solid-phase using Fmoc chemistry. The peptide crosslinker was synthesized on-bead by the coupling reaction between acrylic acid and the amine groups of glutamine and lysine residues. The synthetic procedure employed the acid-labile Fmoc-Lys (Mtt)-OH and base-labile Fmoc-AA-OH derivatives. Selective deprotection, of -Mtt and -Fmoc groups on-bead, freed the amine end-groups on glutamine and lysine residues for coupling reaction with acrylic acid while maintaining the peptide attached to the resin. Subsequent cleavage from the resin yielded a peptide crosslinker with two unsaturated acrylate end-groups with high yield and purity. This method can be generally employed for the synthesis of a wide range of peptides with one or more reactive groups for grafting in the fabrication of biomimetic scaffolds in tissue engineering applications.     

The deprotection of Lys(Mtt) revisited.

The selective deprotection of Lys(Mtt)-containing peptidyl resins was successfully monitored by RP-HPLC using very short linear gradients. RP-HPLC analyses of the acidic filtrates also revealed the partial cleavage of the Trt groups and of the peptide-resin bond. The absorbance of the Mtt carbocation at 470 nm is only twice that of the Trt cation. Thus, the UV monitoring at 470 nm seems to be inappropriate, especially at the end of the deprotection, when the Mtt and the Trt levels are comparable.     

Synthesis of phosphopeptides by the Multipinast method: Evaluation of coupling methods for the incorporation of Fmoc- Tyr(PO3Bzl,H)-OH, Fmoc- Ser(PO3Bzl,H)-OH and Fmoc- Thr(PO3Bzl,H)-OH

astMultipin is a trademark of Chiron Technologies Pty. Ltd., Clayton, Victoria, Australia.The efficiency of various coupling methods for the incorporation of the three monobenzyl phosphorodiester-protected derivatives, Fmoc- Tyr(PO3Bzl,H)-OH, Fmoc-Ser(PO3Bzl,H)-OH and Fmoc-Thr(PO3Bzl,H)-OH, was examined through the test synthesis of Ala-Ser-Gln-Gly-Xxx(PO3H2)-Leu- Glu-Asp-Pro-Ala-NH2 (Xxx = Tyr, Ser, Thr) using the Multipin method of multiple peptide synthesis. The coupling methods examined were (1) PyBrop/DIEA; (2) BOP/HOBt/NMM; (3) BOP/HOBt/DIEA; (4) HBTU/HOBt/DIEA; (5) HATU/HOAt/DIEA; (6) HATU/DIEA; (7) DIC/HOBt; (8) DIC/HOBt/DIEA; (9) DIC/HOAt; (10) DIC/HOAt/DIEA. While all four DIC-based coupling procedures resulted in incomplete incorporation, both the HBTU/HOBt/DIEA and HATU/HOAt/DIEA coupling procedures provided most efficient incorporation of the three Fmoc- Xxx(PO3Bzl,H)-OH derivatives. In the subsequent synthesis of the -helical Tyr(P)-peptide, Glu-Thr-Gly-The-Lys- Ala-Glu-Leu-Leu-Ala-Lys-Tyr(PO3H2)-Glu-Ala-Thr- His-Lys-NH2, analysis of the crude peptide by electrospray MS confirmed that several residue deletions had occurred but that complete incorporation of the Tyr(P)-residue had been accomplished using HBTU/HOBt/DIEA coupling of Fmoc- Tyr(PO3Bzl,H)-OH.     

Biosensor analysis of anti-citrullinated protein/peptide antibody affinity

Anti-citrullinated protein/peptide antibodies (ACPAs) are detected in rheumatoid arthritis (RA) sera and because of their strict association with the disease are considered marker antibodies, probably endowed with pathogenic potential. Antibody affinity is one of the parameters affecting pathogenicity. Three diagnostic citrullinated peptides—viral citrullinated peptide 1 (VCP1) and VCP2 derived from Epstein–Barr virus (EBV)-encoded proteins and histone citrullinated peptide 1 (HCP1) derived from histone H4—were synthesized as tetrameric multiple antigen peptides and immobilized on sensor chips CM5 type in a Biacore T100 instrument. Specific binding of purified antibodies from RA patients to the three peptides was analyzed by surface plasmon resonance using two arginine-containing sequences as controls. Employing a 1:1 binding model for affinity constant calculation, ACPAs interacted with VCP1 and VCP2 with lower apparent affinity (10⁻⁶ M > K_D > 10⁻⁷ M) and interacted with HCP1 with higher apparent affinity (K_D = 10⁻⁸ M). The results indicate that the binding to citrullinated peptides is characterized by wide differences in affinity, with slower association and faster dissociation rates in the case of antibodies to viral citrullinated peptides as compared with antibodies specific for the histone peptide. This biosensor analysis shows the high cross-reactivity of purified ACPAs that bind other citrullinated peptides besides the one used for purification.     

The kinetics of the removal of the N-methyltrityl (Mtt) group during the synthesis of branched peptides.

The purpose of this short communication is to describe the reaction rate for the removal of the N-methyltrityl (Mtt) protecting group that is used in solid-phase peptide synthesis for the production of branched and cyclic peptides. The reaction rate was observed to follow zero-order kinetics, and we suggest the optimal conditions for the removal of the Mtt group in batchwise synthesis.     

Fmoc‐Sec(Xan)‐OH: synthesis and utility of Fmoc selenocysteine SPPS derivatives with acid‐labile sidechain protection

We report here the synthesis of the first selenocysteine SPPS derivatives which bear TFA‐labile sidechain protecting groups. New compounds Fmoc‐Sec(Xan)‐OH and Fmoc‐Sec(Trt)‐OH are presented as useful and practical alternatives to the traditional Fmoc‐Sec‐OH derivatives currently available to the peptide chemist. From a bis Fmoc‐protected selenocystine precursor, multiple avenues of diselenide reduction were attempted to determine the most effective method for subsequent attachment of the protecting group electrophiles. Our previously reported one‐pot reduction methodology was ultimately chosen as the optimal approach toward the synthesis of these novel building blocks, and both were easily obtained in high yield and purity. Fmoc‐Sec(Xan)‐OH was discovered to be bench‐stable for extended timeframes while the corresponding Fmoc‐Sec(Trt)‐OH derivative appeared to detritylate slowly when not stored at ?20 °C. Both Sec derivatives were incorporated into single‐ and multiple‐Sec‐containing test peptides in order to ascertain the peptides' deprotection behavior and final form upon TFA cleavage. Single‐Sec‐containing test peptides were always isolated as their corresponding diselenide dimers, while dual‐Sec‐containing peptide sequences were afforded exclusively as their intramolecular diselenides. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Preparation of O-phosphotyrosine-containing peptides by Fmoc solid-phase synthesis: Evaluation of several Fmoc-Tyr(PO3R2)-OH derivatives

Summary The synthesis of two model Tyr(P)-containing peptides using Fmoc-Tyr(PO₃ tBu₂)-OH, Fmoc-Tyr(PO₃Bzl₂)-OH and Fmoc-Tyr(PO₃H₂)-OH established that the t-butylphosphate-protected derivative was the preferred derivative for use in Fmoc solid-phase peptide synthesis, since it afforded phosphopeptides in high purity and with the lowest amount of Tyr-peptide contamination. In addition, this study confirmed that commercially available Fmoc-Tyr(PO₃H₂)-OH is also suitable for use in Fmoc solid-phase synthesis but gives less pure phosphopeptides, along with the generation of 1–4% of the tyrosine-containing peptide for the model sequences studied. In view of the good performance of Fmoc-Tyr(PO₃ tBu₂)-OH, a large-scale three-step synthetic procedure was developed which involved phenacyl protection of the carboxyl group, phosphite-triester phosphorylation of the tyrosyl hydroxyl using di-t-butyl N,N-diethylphosphoramidite, and final removal of the phenacyl group by zinc reduction in acetic acid.Abbreviations BOP benzotriazol-1-yl-oxy-tris(dimethylamino)phosphonium hexafluorophosphate - tBu t-butyl - Bzl benzyl - DBU 1,8-diazabicyclo[5,4,0]undec-7-ene - DMF N,N-dimethylformamide - EDT ethanedithiol - Fmoc 9-fluorenylmethoxycarbonyl - HOBt N-hydroxybenzotriazole - HPLC high performance liquid chromatography - NMM N-methylmorpholine - Pac phenacyl - TFA trifluoroacetic acid - THF tetrahydrofuran - Tyr(P) O-phosphotyrosine     

Preparation of protected peptidyl thioester intermediates for native chemical ligation by Nalpha-9-fluorenylmethoxycarbonyl (Fmoc) chemistry: considerations of side-chain and backbone anchoring strategies, and compatible protection for N-terminal cysteine.

Native chemical ligation has proven to be a powerful method for the synthesis of small proteins and the semisynthesis of larger ones. The essential synthetic intermediates, which are C-terminal peptide thioesters, cannot survive the repetitive piperidine deprotection steps of N(alpha)-9-fluorenylmethoxycarbonyl (Fmoc) chemistry. Therefore, peptide scientists who prefer to not use N(alpha)-t-butyloxycarbonyl (Boc) chemistry need to adopt more esoteric strategies and tactics in order to integrate ligation approaches with Fmoc chemistry. In the present work, side-chain and backbone anchoring strategies have been used to prepare the required suitably (partially) protected and/or activated peptide intermediates spanning the length of bovine pancreatic trypsin inhibitor (BPTI). Three separate strategies for managing the critical N-terminal cysteine residue have been developed: (i) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-(N-methyl-N-phenylcarbamoyl)sulfenylcysteine [Fmoc-Cys(Snm)-OH], allowing creation of an otherwise fully protected resin-bound intermediate with N-terminal free Cys; (ii) incorporation of N(alpha)-9-fluorenylmethoxycarbonyl-S-triphenylmethylcysteine [Fmoc-Cys(Trt)-OH], generating a stable Fmoc-Cys(H)-peptide upon acidolytic cleavage; and (iii) incorporation of N(alpha)-t-butyloxycarbonyl-S-fluorenylmethylcysteine [Boc-Cys(Fm)-OH], generating a stable H-Cys(Fm)-peptide upon cleavage. In separate stages of these strategies, thioesters are established at the C-termini by selective deprotection and coupling steps carried out while peptides remain bound to the supports. Pilot native chemical ligations were pursued directly on-resin, as well as in solution after cleavage/purification.     

Basic peptides in bee venom, II. Synthesis of two pentapeptides from the sequence of the mast-cell-degrading peptide (author's transl)]

The synthesis of two protected pentapeptides is described. The peptides are fragments of the sequence of a mast-cell degranulating peptide from bee venom. The fragments Boc-Lys(Z)-Ile-Cys(SiPr)-Gly-Lys(Z)(I) and Boc-Pro-His(Trt)-Ile-Cys(Trt)-Arg(Tos) (II) were synthesized conventionally. The deprotection of the alpha-amino group by HCl/acetic acid of Boc-Ile-Cys(SiPr)-Gly-Lys(Z) was accompanied by a disulfide exchange at the cysteine residue. After the hydrolysis of fragment II with 6N HCl, allo-isoleucine could be detected by gas chromatography and amino-acid analysis.     

Efficient solution-phase synthesis of multiple O-phosphoseryl-containing peptides related to casein and statherin.

The multiple Ser(P)-containing peptides, H-Ser(P)-Ser(P)-Ser(P)-Glu-Glu-NHMe.TFA, H-Asp-Ser(P)-Ser(P)-Glu-Glu-NHMe.TFA and H-Glu-Ser(P)-Ser(P)-Glu-Glu-NHMe.TFA were prepared by the use of Boc-Ser(PO3Ph2)-OH in the Boc mode of solution phase peptide synthesis followed by platinum-mediated hydrogenolytic de-protection of the Ser(PO3Ph2)-containing peptides. The protected peptides were assembled using the mixed anhydride coupling methods with 40% TFA/CH2Cl2 used for removal of the Boc group from intermediate Boc-protected peptides.     

Evaluation of acid‐labile S‐protecting groups to prevent Cys racemization in Fmoc solid‐phase peptide synthesis

Phosphonium and uronium salt‐based reagents enable efficient and effective coupling reactions and are indispensable in peptide chemistry, especially in machine‐assisted SPPS. However, after the activating and coupling steps with these reagents in the presence of tertiary amines, Fmoc derivatives of Cys are known to be considerably racemized during their incorporation. To avoid this side reaction, a coupling method mediated by phosphonium/uronium reagents with a weaker base, such as 2,4,6‐trimethylpyridine, than the ordinarily used DIEA or that by carbodiimide has been recommended. However, these methods are appreciably inferior to the standard protocol applied for SPPS, that is, a 1 min preactivation procedure of coupling with phosphonium or uronium reagents/DIEA in DMF, in terms of coupling efficiency, and also the former method cannot reduce racemization of Cys(Trt) to an acceptable level (<1.0%) even when the preactivation procedure is omitted. Here, the 4,4′‐dimethoxydiphenylmethyl and 4‐methoxybenzyloxymethyl groups were demonstrated to be acid‐labile S‐protecting groups that can suppress racemization of Cys to an acceptable level (<1.0%) when the respective Fmoc derivatives are incorporated via the standard SPPS protocol of phosphonium or uronium reagents with the aid of DIEA in DMF. Furthermore, these protecting groups significantly reduced the rate of racemization compared to the Trt group even in the case of microwave‐assisted SPPS performed at a high temperature. © 2013 The Authors. European Peptide Society published by John Wiley & Sons, Ltd.     

Membrane association and activity of 15/16-membered peptide antibiotics: Zervamicin IIB, ampullosporin A and antiamoebin I

Permeabilization of the phospholipid membrane, induced by the antibiotic peptides zervamicin IIB (ZER), ampullosporin A (AMP) and antiamoebin I (ANT) was investigated in a vesicular model system. Membrane-perturbing properties of these 15/16 residue peptides were examined by measuring the K⁺ transport across phosphatidyl choline (PC) membrane and by dissipation of the transmembrane potential. The membrane activities are found to decrease in the order ZER > AMP >> ANT, which correlates with the sequence of their binding affinities. To follow the insertion of the N-terminal Trp residue of ZER and AMP, the environmental sensitivity of its fluorescence was explored as well as the fluorescence quenching by water-soluble (iodide) and membrane-bound (5- and 16-doxyl stearic acids) quenchers. In contrast to AMP, the binding affinity of ZER as well as the depth of its Trp penetration is strongly influenced by the thickness of the membrane (diC_16:1PC, diC_18:1PC, C_16:0/C_18:1PC, diC_20:1PC). In thin membranes, ZER shows a higher tendency to transmembrane alignment. In thick membranes, the in-plane surface association of these peptaibols results in a deeper insertion of the Trp residue of AMP which is in agreement with model calculations on the localization of both peptide molecules at the hydrophilic-hydrophobic interface. The observed differences between the membrane affinities/activities of the studied peptaibols are discussed in relation to their hydrophobic and amphipathic properties.     

Effects of sphingomyelin on melittin pore formation

The effect of sphingomyelin (SM), one of the main lipids in the external monolayer of erythrocyte plasma membrane, on the ability of the hemolytic peptide melittin to permeabilize liposomes was investigated. The peptide induced contents efflux in large unilamellar vesicles (LUV) composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC)/SM (1:1 mole ratio), at lower (>1:10,000) peptide-to-lipid mole ratios than in pure POPC (>1:1000) or POPC/1-palmitoyl-2-oleoylphosphatidylglycerol (POPG) (1:1 mole ratio) (>1:300) vesicles. Analysis of the leakage data according to a kinetic model of pore formation showed a good fit for hexameric-octameric pores in SM-containing vesicles, whereas mediocre fits and lower surface aggregation constants were obtained in POPC and POPC/POPG vesicles. Disturbance of lateral separation into solid (s_o) and liquid-disordered (l_d) phases in POPC/SM mixtures increased the peptide-dose requirements for leakage. Inclusion of cholesterol (Chol) in POPC/SM mixtures under conditions inducing lateral separation of lipids into liquid-ordered (l_o) and l_d phases did not alter the number of melittin peptides required to permeabilize a single vesicle, but increased surface aggregation reversibility. Partitioning into liposomes or insertion into lipid monolayers was not affected by the presence of SM, suggesting that: (i) melittin accumulated at comparable doses in membranes with different SM content, and (ii) differences in leakage were due to promotion of melittin transmembrane pores under coexistence of s_o-l_d and l_o-l_d phases. Our results support the notion that SM may regulate the stability of size-defined melittin pores in natural membranes.     

Chilled storage of Asian seabass,Lates calcarifer Bloch semen: Effects of ions,extenders and storage periods on sperm quality and fertilization ability

The development of a chilled storage protocol of fish sperm requires an understanding of sperm biology and function as the activation/inhibition of fish sperm is greatly affected by several environmental factors. This study aimed to determine the effects of ionic and non-ionic solutions, extender types (Ringer's solution, Ca-F HBSS solution, HBSS solution, He and Wood solution, Saline solution, and Modified Cortland solution), and chilled-storage period on sperm quality and fertilization ability of Asian seabass, Lates calcarifer semen. Regulation of Asian seabass sperm motility was dependent on the osmolality of both ionic and non-ionic activation media. The threshold levels on the initiation of sperm motility were detected in KCl (>100 mM), NaCl (>50 mM), CaCl₂ (>50 mM), glucose (>300 mM), and mannitol (>100 mM) solutions. Relatively high percentages of sperm motility (>80%) were observed when activated with KCl, NaCl, CaCl₂, glucose, and mannitol solutions at above 700, 600, 350, 1,000, and 1,000 mM, respectively. Ringer's solution was the most optimal extender for chilled storage of Asian seabass semen at 2–4°C supported by the retention of sperm motility and viability for 6 days. Semen diluted in Ringer's solution and chilled-stored for 2 days exhibited acceptable fertilization (66.1% ± 6.2%) and hatching (56.4% ± 2.9%) rates. This report, for the first time, describes the ionicity and non-ionicity effects on the motility of Asian seabass sperm.     

Comparison of the stepwise and convergent approaches in the solid-phase synthesis of rat Tyr0-atriopeptin II

Summary Tyr^o-Atriopeptin II was synthesized on a 2-chlorotrityl resin by both, the stepwise and the convergent approach. For both methods an Fmoc/tBu(Trt)-based protection scheme was used. The convergent methodology utilizes the sequential condensation of four protected peptide fragments. These were chosen so that after every condensation reaction, the amino-terminal region of the newly formed resin-bound peptide did not contain a β-turn. This ‘designed’ convergent synthesis gave the target peptide in much higher yield and purity than the conventional stepby-step synthesis. HOAc, acetic acid; Boc,tert-butyloxycarbonyl; DCC, dicyclohexylcarbodiimide: DCM, dichloromethane; DIC, diisopropylcarbodiimide; DIEA,N,N-diisopropylethylamine; DMFN,N-dimethylformamide; DMSO, dimethylsulfoxide; EDT. ethanedithiol; FAB-MS, fast atom bombardment mass spectrometry; Fmoc, 9-luorenylmethoxycarbonyl; HOBt, 1-hydroxybenzotriazole; HPLC, high-performance liquid chromatography; i-PrOH, isopropanol; Mmt, 4-methoxytrityl; PEG-PS, polvethyleneglycol grafted polystyrene; Pme, 2,2,5,7,8-pentamethylchroman-6-sulfonyl; RP, reversed phase; rt, room temperature; SPPS, solid phase peptide synthesis;tBu,tert-butyl; TFA, trifluoroacetic acid; TFE, trifluoroethanol; TLC, thin layer chromatography; Trt, triphenylmethyl, trityl. Abbreviations used for amino acids follow the rules of the IUPAC-IUB Commission of Biochemical Nomenclature [J. Biol. Chem. 247 (1972), 977]. All amino acids are of the L-configuration.     

Secondary structure-improved bioaffinity correlation in elongated and modified synthetic epitope peptides from p24 HIV-1 core protein

Summary In order to study the correlation between secondary structure and bioaffinity of long and modified sequences of p24 protein from HIV-1, three peptides containing the minimal size epitope from region 208–217 (EAAEWDRVHP) were prepared. It was found that peptide p24-1n, an elongated native sequence, has the lowest K_D and a predominant α-helix structure in the presence of trifluoroethanol. Three peptides containing another epitope from region 293–302 (FRDYVDRFK) were also synthesized. We have observed that modifications on the native sequence p24-2n (region 287–308) increased the α-helix content and this was correlated with an improvement of the recognition by antibodies in ELSA. Abbreviations: CD, circular dichroism; DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; ELSA, enzyme linked immunosorbent assay; EDT, 1,2-ethanedithiol; Fmoc, fluorenylmethoxycarbonyl; FAB-MS, fast atom bombardment mass spectrometry; HPLC, high pressure liquid chromatography; MeCN, acetonitrile; NMM, N-methylmorpholine; PyBOP, benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate; SELCOM, self consistent method; TMB, 3,3′,5,5′-tetramethylbenzidine; TFA, trifluoroacetic acid; TFE, trifluoroethanol; Aminoacid symbols denote the L-configurations. Abbreviations for amino acids and nomenclature of peptides follow the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature (Eur. J. Biochem., 138 (1984) 9).     

Coupling of peptides to protein carriers by mixed anhydride procedure

Carboxyl groups of succinylated bovine serum albumin were activated by isobutylchloroformate in dimethylformamide solution. Subsequent reaction of the mixed anhydride with amino groups of the added peptide provided rapid and efficient coupling of peptide to protein. For different peptides the yield of coupling was equal to 40-100%. These values corresponded to 20-50 mol peptide bound/mol protein. Immunization of rabbits with these conjugates produced antisera to peptides with titers of 1:1000-1:3000 (estimated by enzyme-linked immunosorbent assay).