Use of the same polymer for synthesis and purification of peptides

Abstract

Este trabalho revela uma estratégia biotecnológica incomum mas valiosa e relacionada com o uso de um mesmo polímero (benzidrilamino-resina, BHAR) para fins de síntese e purificação por troca aniônica de peptídeos. Inicialmente, o octapeptídeo DRVYIHPF-NH 2 foi sintetizado em BHAR com 1% e 3% de intercruzamento e contendo 2,5 mmol g -1 de grupos amônio. Devido certamente a uma menor rigidez da sua estrutura polimérica, um maior rendimento na síntese (cerca de 80%) foi obtido com a primeira resina. A seguir, os peptídeos DEVYEHPF-NH 2 e DEVYEDPF-NH 2 carregados negativamente (-1 e -3 em pH neutro, respectivamente), ambos sintetizados em 1% BHAR, foram submetidos a um teste de separação cromatográfica no mesmo tipo de resina (1% e 3%). Concordante com resultados da síntese de peptídeos e de valores de inchamento dos grãos das resinas, obtidos por microscopia, uma melhor separação entre ambos os peptídeos ocorreu com o lote de 1% BHAR. Estes achados demonstraram que a BHAR, aplicada até o momento somente para a síntese peptídica, se contiver elevado teor de grupos amônio positivos, pode ser utilizada alternativamente como suporte sólido para purificação cromatográfica deste tipo de molécula biológica.This work reveals an uncommon but valuable biotechnological approach regarding the use of a same polymer (benzhydrylamine-resin, BHAR) for synthesis and anion exchange purification of peptides. Initially, the octapeptide DRVYIHPF-NH 2 was synthesized in 1% and 3% cross-linked BHAR, attaching 2.5 mmol g -1 ammonium groups. Due certainly to its less rigid polymeric backbone, higher synthesis yield (about 80%) was achieved with the former resin. Next, the negatively charged peptides DEVYEHPF-NH 2 and DEVYEDPF-NH 2 (-1 and -3 in neutral pH, respectively), both synthesized in 1% BHAR were submitted to chromatographic separation test in this same type of resin (1% and 3%). Following comparative results of peptide synthesis and swelling data of resin beads obtained by microscopy, an improved separation of both peptides occurred with 1% BHAR batch. These findings demonstrated that BHAR applied so far for peptide synthesis, when containing high amount of positively charged ammonium groups, can be also used alternatively as a solid support for chromatographic purification of this type of biological molecule.Keywords: peptide synthesis, resin, anion exchange chromatography, polymer, solvent polarity IntroductionThe inception of solid phase peptide synthesis (SPPS) 1 almost four decades ago 2 represented a landmark in the use of cross-linked beaded polymers, which were, until that time, restricted to stationary phases in column chromatography. Since then, various studies have been conducted in order to develop more varied applications for such polymeric materials. In addition to the peptide synthesis method itself, the concept of performing chemical reactions on an insoluble polymeric structure has, for instance, been successfully extended to oligonucleotides 3 and polysaccharides. 4 In recent years, a process denoted solid phase o...

Preparation of a Library of EDTA Amide x-Aminenaphthalene-y-sulfonic Acid Derivatives on Solid Phase and Their Fluorescence Behavior toward Transition Metals

“…Different fluorescence responses of the materials arw and drw , may be attributed to the cross-linking degree. In resin a the cross-linking degree is higher than in d , which gives a greater rigidity of the structure matrix and prevents expansion of the polymer backbone, discouraging response from the fluorescent material supported on resin a toward metals …”

“…In resin a the cross-linking degree is higher than in d, which gives a greater rigidity of the structure matrix and prevents expansion of the polymer backbone, discouraging response from the fluorescent material supported on resin a toward metals. 31 The best properties as sensor were found in the materials supported on resin a, since the changes in the fluorescence response with the metals studied were greater. In the case of rz supported on d, the inability to discriminate between the two metals (Ni 2+ and Cu 2+ ) suggests that the properties are not the most appropriate.…”