Liquid-Phase Peptide Synthesis Optimisation: Selection of Quenching Reagents and Peptide Solubility Estimation

• Autor(a) principal: Rosa, Marta Filipa Calvinho
• Data de Publicação: 2021
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10362/112039
• topic: Peptide Synthesis; Process Optimisation; Quenching Reaction; Solubility; Síntese de Peptídeos; Otimização do Processo; Inativação Aminoáci-dos; Solubilidade; Domínio/Área Científica::Engenharia e Tecnologia

With the increasing demand on peptides market, the peptide synthesis process must be kept upgraded. Therefore, the focus of this project is the optimisation of liquid-phase peptide synthesis. During peptide synthesis, the by-products and the amino acid excess must be kept controlled to not interfere with the main reactions and, consequentially, with the final product yield and purity. In order to achieve this, a quenching reagent is added to inactivate the amino acid in excess. An optimisation study was performed for the quenching reaction, where it was found that 2 or 3 amino acids equivalents reacting with 2.1 quenching reagent equivalents allowed the best conditions for a controlled reaction. From there, with the conditions established, three reagents – piperidine, aniline, thiomalic acid – were used in the quenching reaction, where the quenching efficiency was evaluated by the quenching rate constant (kq). The kq values for piperidine, thi-omalic acid and aniline we deduced to 2.962, 1.849, and 0.020 min-1 respectively. Piperidine was then determined as the best quenching reagent, followed by thiomalic acid and aniline, respec-tively. The second problem of peptide synthesis that this project approached, was the determina-tion of the peptide solubility. Currently, the solubility of peptides is measured experimentally, which is a time-consuming and wasteful process once it needs to be measured in different sol-vents, thus increasing the process cost. The second part of the project investigated two theoretical approaches – Solubility Param-eters and COSMO-RS – to predict the best and worst solvents for free peptides and peptides attached to a hub (wang and rink amide nanostar). DMSO was found the best solvent for most of the peptides, followed by NFM, which also presented itself as the best green solvent. The valida-tion of the COSMO-RS method was tested and exhibited a RMSE of between 1.10 – 0.74 loga-rithmic units. Therefore, this model is presented as a tool to be implemented in determining the solubility of the amino acids.