Síntese de peptídeos em fase sólida: reavaliação do uso do ácido trifluorometanosulfônico na etapa de clivagem peptídica da resina

• Autor(a) principal: Pinto, Marcelo Rodrigo Silva [UNIFESP]
• Data de Publicação: 2017
• Tipo de documento: Dissertação
• Idioma: por
• Título da fonte: Repositório Institucional da UNIFESP
• Texto Completo: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=5010275; http://repositorio.unifesp.br/handle/11600/50534
• topic: Peptide; Method of peptide synthesis; Cleavage with trifluoromethanesulfonic acid; Solid phase; Peptídeo; Método da síntese peptídica; Clivagem com ácido trifluorometanosulfônico; Fase sólida

The main objective of the present work was to reavaluate the strategy of the use of trifluoromethanesulfonic acid (TFMSA) in the step of final cleavage of peptides from the support resin. The main reason for investing in this topic is due to the great difficulty of operationalization that occurs with the more conventional method used in Boc (tert-butyloxycarbonyl) chemistry, which is cleavage in anhydrous HF. Because it is toxic, extremely corrosive, it requires imported equipment made with special Teflon, difficult to handle and that currently in Brazil, is unviable to be used due to the inexistence of a chemical company that produces this acid. And the option for importation (in special stainless steel cylinders) has so far not been feasible. In this context, the study of this alternative method of cleavage with the strong acid TFMSA had already been preliminarily initiated in our laboratory some years ago and we had observed that, in addition to evaluating the efficiency of the cleavage method in terms of the purity of the removed material, it was fundamental to quantitatively determine this step, ideally with the integral removal of chains from the resin matrix and also the protecting groups of the reactive amino acid side chains. In this way, we decided to investigate deeply the TFMSA´s cleavage method. Briefly, we have observed that a solution composed of TFMSA: TFA: DMS: o-cresol, in the ratio 20: 70: 5: 5 (v / v), appeared to be suitable for the integral removal of peptide chains, even those containing hydrophobic amino acids linked to the resin and which we have shown to be difficult to cleave due to the high stability of the covalent bond between them and the resin backbone in an acid medium. The aforementioned protocol with 20% TFMSA in solution was more efficient when compared to the literature solutions that employ this acid in amounts always around 10%. On the other hand, kinetic studies of cleavage with TFMSA Summary 18 also showed that at 8 ° C (in cold room), the time required for integral peptide cleavage was 6 h. Preliminary studies of the synthesis efficiency between the traditional Boc (tert-butyloxycarbonyl) chemistry and the Fmoc (9-fluorenylmethyloxycarbonyl) alternative indicated the influence of the peptide sequence in this comparison between these two synthetic techniques. The use of this mixture containing 20% (v / v) TFMSA was also fortunately viable (as is in the anhydrous HF) for use in cleavage the procedure of peptide sequences bearing amino acid – type spin probes such as the known Toac (acid 2, 2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid). No irreversible degradation occurred with this type of nitroxide-group paramagnetic compounds during this strongly acid treatment. In addition, important investigation of the lability degree of some protecting groups of amino acid side chains of peptide sequences has been developed and indicated for example that the Nα-Fmoc group is surprisingly removed in this acidic cleavage medium with TFMSA. The Arg´s p-toluenesulfonyl (Tos) protecting was also shown to be susceptible to total removal in this acid medium, unlike the formyl group (For) of Trp, which remained stable against the deprotection reaction. In this case, we successfully tested cleavage of this indolic ring protecting group by pretreatment of the peptidyl resin in 4-methylpiperidine followed by the cleavage in the standard condition of 20% TFMSA. The last and most problematic item herein examined involved a more careful study of the reported cyclization reaction occurring in the side chain of Asp residue, resulting in a a succinimidic-type cyclic side-product formation (elimination of a water molecule) with – 18 Da lower molecular weight than the expected for the peptide of interest. Initial studies of this item, performed with the hypertensive peptide AII and its Gly8-AII derivative indicated that in the standard cleavage mixture used (20% TFMSA for 6 h at 8 °C); the percentage of Summary 19 the analogue containing the Asp-succinimide residue was about 14%, in comparison with respective native peptides. Thus, among the several items that will require continuity, this is one of the most extensive, with the possibility of alterations of several parameters during the acid cleavage stage, including not only the modification in the composition of the acid mixture but also the temperature and time of this hydrolytic reaction. Nevertheless, the present study based on experiments with several types of peptide sequences, indicated the feasibility of using the TFMSA: TFA: DMS: o-cresol solution in the ratio of 20: 70: 5: 5 (v / v) to 8 ° C in 6 h as a protocol to be used in Boc-SPFS chemistry, replacing the traditional cleavage process in anhydrous HF. The importance of this fact should be emphasized, since the TFMSA cleavage method is much simpler, practical and with more potential for application of greater scale of synthesis due to the possibility of handling bigger glass flasks and bottles in comparison with small Teflon reaction flasks used in the anhydrous HF peptide cleavage procedure.