Solid‐phase synthesis of somatostatin and glucagon‐selective analogs in gram quantities

Abstract

SynopsisSomatostatin (SS) and two glucagon selective analogs -sS and [D-Trp8,D-Cys14] -ss have been synthesized in gram quantities by the solid-phase procedure. A general modification of Monahan and Gilon's procedure for esterification of the first protected amino acid onto the chloromethylated resin as well as a general protocol for solid-phase peptide synthesis on Beckman 990 automatic synthesizer are described. A new general procedure for disulfide formation, which involves the adaptation of the "high-dilution" principle to the ferricyanide oxidation and the optimization of the sequence of purification steps as applied to somatostatin and its analogs, yields highly purified peptides (199% pure) as checked by reverse-phase high-pressure liquid chromatography-which is shown to be a highly sensitive, resolutive, and quantitative analytical tool for evaluation of the homogeneity of peptides.

Interaction of tachykinins with their receptors studied with cyclic analogues of substance P and neurokinin B.

“…SP, NKB, NKA, and the cyclic analogues of SP were synthesized by solid-phase methodology and purified by gel filtration and by ion-exchange chromatography (24). The disulfide bridge cyclizations were done either by classical high-dilution ferricyanide oxidation (25) or by a new procedure involving a polymeric support (26). The physical parameters of the cyclic compounds, whose syntheses have not been previously described, are listed in Table 2.…”

“…After removal of the last N-a-Boc protecting group, one half of the'peptidyl resin was treated with 1.5 ml of anisole, 0.25 ml of dimethyl sulfide, and 10 ml of HF per g of resin for 30 min at -20°C and 30 min at 0°C. The peptide solution was diluted to 400 ml with degassed water and added dropwise to a ferricyanide solution to form the disulfide bridge as described by Rivier et al (25). After oxidation, the solution was chromatographed on an anion-exchange resin, AG 3-X4 (acetate form), to remove the ferri-and ferrocyanide anions and was lyophilized to yield 1.26 g of crude material, which was purified by batch.…”

Peptídeos de conformação restrita relacionados ao sítio 2 do fator de crescimento de fibroblastos ácido humano (hFGF-1): estudo sobre estrutura e atividade

“…A existência destes compostos como móleculas íntegras ativas e não apenas como produtos de degradação de proteínas, foi evidenciada após vários terem sido isolados de fontes naturais diversas. Dentre os peptídeos biologicamente ativos pode-se citar as encefalinas (Lord et al, 1977), a angiotensina (Cushman & Cheung, 1971), a bradicinina (Rocha e Silva, 1963), a ocitocina (Nestor et al, 1975), a vasopressina (Dyckes et al, 1974, a somatostatina (Verber et al, 1981), o glucagon (Rivier et al, 1978), as colecistocininas (Liddle et al, 1986), α-melanotropina (Sawyer et al, 1982), as conotoxinas de veneno de Conus geografus (Rivier et al, 1987, Oliveira et al, 1990, as fitoquelatinas (Rauser, 1995) e as equinocandinas (Kurokawa & Ohfune, 1993). Com a descoberta de que estes compostos poderiam ser sintetizados, tornou-se possível a obtenção dos mesmos em quantidades suficientes para o estudo de suas propriedades biológicas, dos mecanismos de ação (Wrigthon et al, 1996, Leconte et al, 1998 e para o desenvolvimento de diversos agentes terapêuticos.…”

“…A ciclização foi feita imediatamente após a desproteção total e clivagem da peptidilresina com base em Rivier et al (1978).…”

“…Obviamente que a sua escolha determina a estratégia a ser empregada para a construção de cadeia peptídica a ser ciclizada, Optamos inicialmente, pela ciclização por oxidação de grupos sulfidrilas de cisteínas pelo oxigênio do ar (Rivier et al, 1978) ou por K 3 Fe(CN) 6 (Misicka & Hruby, 1994). A ligação dissulfeto é um elemento estrutural natural presente em um grande número de proteínas e peptídeos nativos.…”