Configuration and racemization determination of cysteine residues in peptides by chiral derivatization and HPLC: application to oxytocin peptides.

An improved RP-HPLC method was developed for the determination of the configuration and stereochemical purity of cysteine residues in peptides. The method consists of oxidation of cysteine and cystine residues to cysteic acid, followed by hydrolysis and pre-column chiral derivatization with Val-Marfey's reagent.

Determination of the absolute configuration of synthetic pterocarpans by chiral HPLC using on-line CD detection.

Resolution of synthetic racemic pterocarpans was achieved by CD monitored HPLC using a chiral stationary phase. No sign of exciton coupling was seen in the CD spectra of the pterocarpan enantiomers. The predominance of the chromane chromophore was indicated by the high intensity of the (1) L(b) band. The chirality of both the chromane and dihydrobenzo[b]furane chromophores was found to be governed by the second chiral sphere. An oxygen atom in pseudoaxial position at the benzylic atom of the chroman ring system gave rise to an increased third-sphere contribution and the positive sign of the (1) L(b) and negative sign of the (1) L(a) band. This resulted in a general positive-negative-negative sign pattern of the (1) L(b), (1) L(a) and (1) B(b) bands of the second-eluted enantiomer of P helicity suggesting homochirality and the same absolute configuration of the chiral centers.

Coloured peptides: synthesis, properties and use in preparation of peptide sub-library kits.

Several methods were developed for the solid-phase synthesis (SPPS) of coloured peptides and peptide libraries. At first a bifunctional red compound, 4-(4-(N-ethyl-N-(3-(tert-butyloxycarbonyl)aminopropyl)amino)phenylazo)be nzoic acid (Boc-EPAB), was coupled with chloromethyl resin to obtain a new solid support suitable for SPPS using Boc chemistry. Peptides synthesized on this coloured resin had the chromophore at their C-termini. N-terminally coloured peptides were synthesized on a traditional solid support, coupled with chromophoric carboxylic acid before cleavage. A model pentapeptide, Phe-Ala-Val-Leu-Gly, and its ten derivatives were synthesized and their properties studied. It was found that the presence of chromophores decreases the water solubility of peptides. However, insertion of solubilizing tags (penta-lysine sequences or polyoxyethyl chains) into the molecule of any coloured derivative resulted in enhancement of the solubility. The RP-HPLC hydrophobicity indexes (phi0) of the coloured peptides were also determined because phi0 values are closely related to their water solubility. A coloured pentapeptide library was synthesized using the portioning-mixing method. Each component of this library contained the red azo dye (EPAB) and the penta-lysine tag. Before the last coupling step the samples were not mixed. All of the 19 sub-libraries obtained after cleavage were readily soluble in water, giving intense red solutions. The effect of chromophore (EPAB) and/or penta-lysine solubilizing tag on the biological activity was also studied. Potencies of the bovine neurotensin 8-13 fragment and its different coloured and penta-lysine derivatives were compared in isolated longitudinal muscle strips of guinea pig ileum. It was shown that the hexapeptide with penta-lysine tag had almost the same activity as the 8-13 fragment itself. The activity of the EPAB-derivative was found to be rather low. However, the presence of the solubilizing tag in the coloured hexapeptide compensated the negative effect of the chromophore.

Cryochemistry: freezing effect on peptide coupling in different organic solutions.

The freezing effect on peptide coupling in organic solutions of different polarity has been investigated and compared with the results obtained in liquid phase. The model reaction of DCC-activated coupling of Boc-Ala-Phe-OH with H-Ala-OBu(t) has been carried out in dioxane, dimethylsulfoxide and formamide, as well as in mixtures (90%/10%, v/v) of dioxane with acetonitrile, dimethylformamide, dimethylsulfoxide and formamide. The reactions have been traced and evaluated by RP-HPLC analysis. Freezing the reaction mixture resulted in all cases in a significant suppression of the N-dipeptidylurea side-product formation together with a slight decrease of tripeptide epimerization. The coupling yields and the side effects depended on the solvent, with the dioxane and dioxane/acetonitrile mixture produced the best results. The role of freezing and solvent in the improved results is discussed.

Structure determination and synthesis of lysine isopeptides influencing on cell proliferation.

The clavicepamines are lysine-rich basic proteins isolated from saprophytic culture of ergot (Claviceps purpurea), having human pharmacological importance. Based on structure determinations, it was demonstrated that the epsilon-lysine (poly)peptides are the fundamental structural units of clavicepamines. To study the relationship between chemical structure and biological effect, solution and solid-phase synthesis of lysine isopeptides were performed. Poly-epsilon-lysines were synthesized with polycondensation via application of p-nitrophenylester temporarily protecting groups together with simultaneous activation. The biological investigations of poly-epsilon-lysines showed a cell-proliferation retarding effect, so they inhibit growth of some animal tumors, practically without toxic side effects.

Side chain distribution and enantiomer composition of biodegradable branched polypeptides with polylysine backbone.

A detailed investigation is reported about the primary structure of biodegradable branched polypeptides suitable for carrier function. The side chain distribution of poly[Lys(DL-Alam)], m approximately 3.5, the common inside area of a branched polypeptide model system, was determined by automated Edman degradation. These data indicate that 65% of the branches consist of 2-4 Ala residues and 25% of the side chains are longer than 4 amino-acid residues. The enantiomer composition of poly[Lys(Xi-DL-Alam)], (X = Glu, D-Glu, Leu, D-Leu) and of poly[Lys(DL-Ala2.9-Leu0.79)] polypeptides was investigated by HPLC analysis of their hydrolysates following derivatization with N-(5-fluoro-2,4-dinitrophenyl)-L-alanine amide (Marfey's reagent). The results proved that the synthesis methods applied for the preparation of branched polypeptides produce compounds whose enantiomer compositions are in good agreement with calculated expectations. These findings could be useful for the reliable interpretation of various biological phenomena observed in connection with the presence of L- or D-amino-acid residues in the side chains.

The influence of the side-chain sequence on the structure-activity correlations of immunomodulatory branched polypeptides. Synthesis and conformational analysis of new model polypeptides.

New branched polypeptides were synthesized for a detailed study of the influence of the side-chain structure on the conformation and biological properties. The first subset of polypeptides were prepared by coupling of tetrapeptides to poly[L-Lys]. These polymers contain either DL-Ala3-X [poly[Lys-(X-DL-Ala3)n]] or X-DL-Ala3 [poly[Lys-(DL-Ala3-X)n] (n less than or equal to 1)] tetrapeptide side chains. Another group of branched polymers comprise a mixture of DL-Alam and of DL-Alam-X oligomeric branches in a random distribution [poly[Lys-(DL-Alam-Xi)] (i less than 1, m approximately 3)]. In each subset the X = Leu or Phe derivatives were prepared. The N-protected tetrapeptides were synthesized by conventional liquid phase methods and were coupled as active esters. The degree of racemization was found relatively high both for active esters and coupled derivatives, when optically active amino acids were in the C-terminal position of the tetrapeptides. In the case of the poly[Lys-(Leu-DL-Ala3)n] derivative, comparative experiments were carried out using various methodical alterations. The highest stereochemical homogeniety could be achieved when the tetrapeptide active ester was synthesized by the "backing off" method. CD spectra of poly[Lys-(Xi-DL-Alam)] (i less than 1, m approximately 3) and of poly[Lys-(X-DL-Ala3)n] were analyzed and compared to those of poly[Lys-(DL-Alam-Xi)] and of poly[Lys-(DL-Ala3-X)n]. All measurements were performed in water solutions of varying pH values and ionic strengths. The data obtained suggest that branched polypeptides containing a mixture of two different types of oligomeric side chains (DL-Alam and DL-Alam-Xi or Xi-DL-Alam) distributed randomly adopt an almost identical conformation to those that comprise only the respective tetrapeptide (DL-Ala3-X or X-DL-Ala3) branches. The results also indicate that the tendency to form an ordered structure is determined by the identity and the position of the chiral amino acid X (Phe or Leu) in the side chain.

Application of Marfey's reagent in racemization studies of amino acids and peptides.

Reversed-phase high-performance liquid chromatography and pre-column derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent) were used for monitoring racemization in peptides, amino acids and their derivatives by separation of optical isomers of amino acids. The technique was applied to the analysis of biologically active peptides, amino acids, their N- and C-protected derivatives, branched polypeptides based on polylysine, and endothiopeptides, and to the detection of stereochemical consequences of side reactions and hydrolysis. The chromatographic samples were mixtures of L- and D-amino acids obtained by hydrolysis of different peptides and derivatives. Baseline separations could be achieved on an ODS-Hypersil column with methanol-acetonitrile-acetate buffer (pH 4) mixtures as the eluents. The rates of racemization were calculated.

Monoclonal antibodies directed to two groups of viral proteins neutralize human cytomegalovirus in vitro.

Monoclonal antibodies (MAbs) that neutralized human cytomegalovirus (HCMV) were produced by ten hybrid cells lines, generated from BALB/c mice immunized with HCMV-infected human fibroblasts. By immunoblot technique six antibodies detected a set of HCMV glycosylated polypeptides which, when separated under reducing conditions, migrated with apparent molecular weights (m.wt.) of 47.5K, 51K, 54K, 58K, and 60-69K. One other antibody reacted only with the 47.5 and 51K polypeptides and the 60-69K broad band. Under nonreducing conditions, these antibodies showed no reactivity with any polypeptide. The three remaining MAbs reacted with two high-m.wt. polypeptides of approximately 200K and greater than 200K when separated under nonreducing conditions. One of these antibodies showed no clear reactivity with the polypeptides, one detected a 58K and 92-94K species and one detected a 58K and 130K species, when separated under reducing conditions.

A synthetic peptide induces long-term protection from lethal infection with herpes simplex virus 2.

Immunization against viral pathogens is generally directed toward the induction of virus neutralizing antibody (VNA) and the maintenance of the potential for a second-set (IgG) response. Indeed, an elevated level of specific antibody is considered a reliable clinical indicator that a state of immunity exists in the host. However, in the case of herpes simplex virus (HSV), the presence of circulating VNA does not necessarily correlate with protection. Thus, it has been found that secondary infections occur in individuals even with high neutralizing titers to HSV, suggesting that antibody to the virus may be useless or even deleterious. In consideration of these facts, we were interested in inducing a T cell response to HSV. We had already shown that synthetic peptides corresponding to the NH3-terminal region of the glycoprotein D (gD) molecule of HSV could induce a strong T cell response when injected into mice, but did not, by themselves, confer protection. In this report, we examined the ability of peptides, covalently coupled to palmitic acid and incorporated into liposomes, to induce virus-specific T cell responses that confer protection against a lethal challenge of HSV-2. We have demonstrated that long-term protective immunity is achieved with a single immunization in the absence of neutralizing antibody when antigen is presented in this form. Furthermore, T cells but not serum from such immune mice can adoptively transfer this protection.

High-performance liquid chromatographic detection of side reactions in peptide synthesis.

High-performance liquid chromatographic systems with chemically bonded packing materials were elaborated for monitoring synthetic stages during peptide coupling, purity control of peptides produced by solid-phase methods and detection of impurities formed as a consequence of side reactions such as oxidation, desulphation, racemization, transpeptidation, alkylation and decomposition. The technique was applied to the analysis of peptide hormones, neuropeptides, cyclopeptides, isopeptides and branched polypeptides based on poly-lysine. The mobile phases were methanol-acetonitrile-water mixtures containing phosphate, acetate, carbonate buffers or trifluoroacetic acid (pH 2-9). After optimization, baseline separations could be achieved for some critical peptide separations.

High-performance liquid chromatography of isopeptides.

High-performance liquid chromatographic (HPLC) methods are described for the structural analysis of clavicepamines, their analogues and branched-chain polypeptides, the analysis in synthetic stages of isopeptides (analysis of half-protected derivatives and purity control of active esters) and the differentiation between alpha- and iso-peptides (such as alpha- and gamma-glutamyl peptides). Pre-column derivatization was used to label the free amino groups for the structural investigation. Dansylated and hydrolysed isopeptides were analysed by HPLC methods based on isocratic separation of alpha-, epsilon- and bis-Dns-lysines using Hypersil, ODS-Hypersil and Partisil PAC columns. For the analysis of peptide active esters, an RP-HPLC method was developed, with methanol-acetonitrile-water mobile phases containing an acidic buffer. Peptides containing alpha-glutamyl residues were analysed for gamma-isomer content in the same system.

Synthesis of gamma-L-glutamyl-taurine.

Glu(Tau), a bioactive substance previously isolated from the protein free aqueous extract of bovine parathyroid powder, has been synthesized. The intermediate derivative Z-Glu(Tau)-OBzl was prepared in three different ways from Z-Glu-OBzl and (1) cystamine by using the mixed anhydride method followed by oxidation, (2) Tau by the active ester procedure via Z-Glu-(ONp)-OBzl, (3) Tau applying mixed anhydride coupling. The protecting groups were removed by hydrogenolysis.