A. M. Nikitina

Synthesis of stereoisomer analogs of the amino end sequence of human growth hormone releasing factor

A new method has been developed for the synthesis of previously unreported stereoisomer analogs of the amino end sequence of the human growth hormone releasing factor.

Synthesis of structural analogs of the 13 – 19 sequence of human growth hormone releasing factor

Development of the principles of synthesis of bioregulators responsible for the secretion of natural hormones in the organism is among currently important tasks. One particular basic problem consists in finding previously unknown peptide agents stimulating the secretion of insulin, a hormone playing a key role in the regulation of metabolic processes in higher mammals and humans. One possible approach to solving this problem is via the synthesis of peptide structures capable of participating in hormone – receptor interactions and enhancing insulin secretion by cells in Largenhans’ islets in the pancreas. A real pathway to such peptide stimulators of insulin secretion can be based on the synthesis of previously unknown structural analogs of the amino end region of somatoliberin, the hypothalamic releasing factor of human growth hormone [1, 2]. Our previous investigations showed that this R-factor is capable of indirectly influencing insulin biosynthesis and secretion by pancreatic islet cells. However, from the standpoint of pharmacology, a significant disadvantage of using natural somatoliberin as a bioregulator of insulin secretion is its relatively low activity and insufficient selectivity. In addition to weakly stimulating the secretion of insulin, natural somatoliberin enhances (to a much greater extent) production of growth hormone and activates the secretion of glucagon. For this reason, much better prospects in solving the problem under consideration can be related to the amino end region of somatoliberin. This conclusion agrees with the results of computer simulation of hormone – receptor interactions involved in the biosynthesis and secretion of insulin. In this context, we have undertaken a special investigation aimed at the development of a method for the synthesis of previously unknown structural analogs of the amino end region of somatoliberin, which can be of interest as potential stimulators of insulin secretion. After a series of preliminary experiments and a comparative analysis of the results, it was concluded that the most promising approach is that based on the use of activated esters of N-protected amino acids. This approach leads to a high yield of the target compounds, excludes undesired side reactions, allows effective control over the racemization processes, and is less tedious than the other possible schemes. As a result, we have developed an effective method for the synthesis of previously unreported structural analogs of the 13 – 19 somatoliberin peptide sequence, which is a part of the amino end of somatoliberin. Distinguishing features of the proposed method are as follows: (i) use of benzyl esters of oligopeptides as amino components; (ii) use of p-nitrophenyl or pentafluorophenyl esters of N-protected amino acids as carboxy components; (iii) use of an acid-labile tert-butyloxycarbonyl protection for masking -amino groups in the carboxy components; (iv) stepwise peptide synthesis in the C N direction. An important advantage of the proposed method is the possibility of using protected derivatives of trifunctional amino acids for the synthesis of hydrophobic analogs of somatoliberin. It was also established that the isolation of target compounds from the reaction mixtures is considerably simplified by conducting the condensation stage in DMF. In this case, the target peptides can be isolated in a crystalline state and purified by recrystallization. The structure of peptides obtained using the proposed scheme is uniquely determined by the scheme of synthesis. The synthetic analogs of the terminal region of somatoliberin can be obtained in an analytically pure form.

Synthesis of Structural Analogs of Thyroliberin with Single-Point Replacements of Amino Acid Residues

In the course of investigation of the quantitative relationship between chemical structure and biological activity of the peptide bioregulators of prolactin secretion, we have synthesized a series of previously unreported structural analogs of thyroliberin [1 – 3]. The new compounds (I and II) differ from the natural hormone by single-point replacements of L-histidine by L-alanine and L-phenylalanine, respectively:

Synthesis of Thyroliberin-Analogous Tripeptidamide

An analysis and experimental evaluation of various pathways for the synthesis of tripeptidamide I showed that a promising method is offered by a solution synthesis using the 2 + 1 scheme. Based on this approach, we have developed an effective procedure for the complete chemical synthesis of L-pyroglutamyl-L-seryl-D-leucine isopropylamide (I). A distinguishing feature of the proposed method is the possibility of introducing a D-leucine derivative with modified carboxamide fragment in the final stage of the peptide synthesis. It was established that the peptide bonds can be successfully formed using a combination of the activated ether method and the azide method. The isolation of both intermediate compounds and the target peptide from the reaction mixture is facilitated if the condensation stages are conducted in DMF. The synthesized peptides can be isolated in the crystalline state and purified by recrystallization. The structures of all new peptides obtained using the proposed method is uniquely determined by the scheme of synthesis; the final tripeptidamide I can be obtained in an analytically pure form. The initial compounds in our synthesis were L-pyroglutamic acid pentachlorophenyl ester of (II), L-serine methyl ester hydrochloride (III), and N-tert-butyloxycarbonyl-D-leucine (IV). Intermediate products included L-pyroglutamyl-L-serine methylate (V), L-pyroglutamyl-L-serine hydrazide (VI), N-tert-butyloxycarbonyl-D-leucine isopropylamide (VII), and D-leucine isopropylamide (VIII). Particular experimental procedures are described in the experimental part below. EXPERIMENTAL PART

Preparation and properties of phenylalanine-B30-(human insulin)

In a directed experimental evaluation of the influence of the replacement of an aliphatic amino acid residue in the C-terminal part of the B-chain of insulin by an aromatic amino acid residue, we have prepared the previously unknown phenylalanine-BS°-(human insulin) and have investigated the biological activity of this analog, which differs from the natural hormone by the replacement of a L-threonine residue by a L-phenylalanine residue.

Preparation and properties of a cysteine analog of human insulin

characteristic absorption spectrum in the visible region with a maximum at 470 nm also permitted it to be identified as lactoferrin. Since transferrin, which is present in milk in definite amounts, possesses similar properties we made a qualitative analysis of the N-terminal amino acid. In the protein analyzed this was glycine, which is characteristic for lactoferrin, and not valine, which is characteristic for transferrin.

Preparation and properties of the methyl ester of\(Willardine^{B_{3 0} }\)-(human insulin)

To study the molecular mechanisms of the interaction of human insulin with the receptors, it is necessary to have available structural analogs of this hormone that retain a high biological activity and are readily detectable in complex mixtures by methods of nondestructive analysis. One such method is spectrometry in the near ultraviolet region. However, human insulin and related animal insulins possess extremely limited UV absorption because of the presence in the A and B chains of insulin of residues of the aromatic protein amino acids tyrosine and phenylalanine, which are relatively weak chromophores.

Preparation and properties of a new structural analog of human insulin — AsparagineB30-insulin

The trypsin-catalyzed transamination [i] of porcine insulin (II) takes place on the reaction of the latter with the tert-butyl ester of L-asparagine (III) in an aqueous organic med• . • o r lum (water--dlmethylformamlde) at 24 C and pH 6.3. Unde these conditions, the tryptic transamination reaction proceeded only at the Lys D29 residue, and the undesirable side reaction of the Arg B22 residue did not occur.

Preparation and properties of leucine-B30-insulin

The hydrolysate was separated on a column (i.i × 70 cm) of Sephadex G-50 fine equilibrated with 30% CH3COOH. As in the case of cyanogen bromide cleavage a peptide of low molecular mass was obtained for which the N-terminal amino acid was found to be Val and the amino acid composition: Vall, Asps, Serl, Glu3, Glyl, Alsl, Leul, Phel, Argl. The yield of peptide was 15%. The compositions of the peptides were determined after hydrolysis in 5.7 N HCI at II0°C for 24 h on a LKB 4101 analyser.