Biomedical subjects
Publications and source records attributed to G Szókán.
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.
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.
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.
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.
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.
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.
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.
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 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.
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.
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