Assuntos
Antineoplásicos/química , Hidrocarbonetos Aromáticos com Pontes/química , Paclitaxel/análogos & derivados , Paclitaxel/química , Taxoides , Alcaloides/síntese química , Alcaloides/química , Alcaloides/toxicidade , Animais , Antineoplásicos/síntese química , Antineoplásicos/toxicidade , Hidrocarbonetos Aromáticos com Pontes/síntese química , Cristalografia por Raios X , Feminino , Humanos , Melanoma Experimental/tratamento farmacológico , Camundongos , Camundongos Endogâmicos , Conformação Molecular , Estrutura Molecular , Neoplasias Ovarianas , Paclitaxel/síntese química , Paclitaxel/toxicidade , Células Tumorais CultivadasRESUMO
The solid-phase synthesis of the N alpha-Fmoc analog of protein kinase C substrate (PKCS, Lys-Arg-Ala-Lys-Ala-Lys-Thr-Thr-Lys-Lys-Arg) was characterized by low recovery from the resin and the concomitant appearance of four impurities. FAB-MS revealed molecular weights for two of these impurities that corresponded to the desired peptide plus Tos or Bzl. The other two were justified by invoking a CO2 elimination of the Clz protecting group to yield: 1) peptide plus 2-chlorobenzyl (ClBzl) and 2) peptide plus ClBzl and Tos. A CF-FAB analysis of carboxypeptidase digestions allowed observation of peptide cleavage down to an ion corresponding to lysine, Fmoc, and the corresponding protecting group(s). These data revealed that the impurities were not the result of incomplete deprotection but the result of migration of the protecting groups to the N-terminal end of the peptide. NMR experiments were subsequently performed and revealed the exact site of substitution: the meta positions of the N-terminal Fmoc. These impurities are presumed to arise by electrophilic aromatic substitution of the fluorene group during HF treatment. The desired Fmoc analog served as a convenient, albeit low-yielding, intermediate for purification of the highly charged PKCS by preparative self-displacement HPLC.
Assuntos
Aminoácidos/química , Carboxipeptidases/química , Fluorenos/química , Ácido Fluorídrico/química , Peptídeos/síntese química , Sequência de Aminoácidos , Radioisótopos de Carbono , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/isolamento & purificação , Peptídeos/isolamento & purificação , Espectrometria de Massas de Bombardeamento Rápido de ÁtomosRESUMO
The use of N alpha-tert.-butyloxycarbonyl-N pi-benzyloxymethylhistidine in peptide synthesis resulted in significant levels of several different side products attributable to the generation of formaldehyde during the hydrogen fluoride cleavage reaction. Methylated impurities in a decapeptide were isolated and identified. These methylated impurities were attributed to the use of the benzyloxymethyl protecting group for the histidines, since the impurities did not form when the dinitrophenyl protecting group was used. Also, peptides containing benzyloxymethyl-protected histidines in addition to N-terminal cysteines quantitatively yielded their respective N-terminal thiazolidine derivatives upon isolation from standard hydrogen fluoride cleavage mixtures. Thiazolidine ring formation was circumvented by including in the cleavage reaction a formaldehyde scavenger such as cysteine hydrochloride or resorcinol.
