Desenvolupament de processos per a la síntesi de pèptids d’interès terapèutic a escala industrial

Abstract

[cat] Investigacions recents en el camp de la biomedicina han demostrat que les interaccions entre pèptids i macromolècules com ara les proteïnes, modulen i regulen processos biològics importants com la memòria, la resposta a l’estrès, el dolor, la resposta immunitària, entre d’altres. El desenvolupament de pèptids com a possibles candidats farmacològics que tenen com a diana els receptors que regulen aquestes funcions biològiques, és un dels principals objectius en els camps de la biologia i la medicina. El Fexapotide (FT) és un pèptid terapèutic amb propietats apoptòtiques. Actualment, es troba en fase III d’assajos clínics per al tractament de la hiperplàsia benigna de la pròstata (BPH). En aquest context, el treball de tesi va consistir en un estudi preliminar vers el desenvolupament d’una ruta sintètica per a la síntesi de FT i la posterior producció a escala pilot en el marc d’una col·laboració amb una empresa farmacèutica. Els pèptids es poden sintetitzar en solució o en fase sòlida. Tots dos enfocaments sintètics han demostrat ser eficaços, però cada un té les seves limitacions. D'una banda, la síntesi en solució requereix un procés d’optimització molt llarg. D'altra banda, l’escalat de la síntesi en fase sòlida és generalment difícil. La possibilitat d’utilitzar resines altament sensibles a àcids com la 2-CTC, és possible sintetitzar pèptids i fragments protegits que es poden alliberar de la resina sense la necessitat d’eliminar cap grup protector de les cadenes laterals. Això ha permès plantejar un enfocament híbrid, també conegut com a síntesi convergent. Aquesta estratègia permet la síntesi de seqüències complexes de pèptids mitjançant la síntesi de fragments peptídics protegits curts del pèptid objectiu mitjançant la síntesi en fase sòlida i, a continuació, la unió d'aquests fragments en solució. En aquest treball, es van proposar tres rutes sintètiques diferents per a la síntesi del Fexapotide. La primera proposta es va basar en la síntesi lineal mitjançant la metodologia de fase sòlida. No obstant això, aquesta estratègia va presentar inconvenients com la baixa puresa del producte sintetitzat. Per solucionar aquest problema es van plantejar dues aproximacions convergents, a partir de tres i quatre fragments protegits. La síntesi en fase sòlida dels fragments de pèptids protegits es va estudiar àmpliament realitzant diverses síntesis de cada fragment. Les condicions d’eliminació del grup Fmoc i les reaccions d’acoblament en solució també es van estudiar exhaustivament, així com també la purificació dels pèptids protegits resultants.

[eng] Recent investigations in the biomedical field have shown that peptide-macromolecular interactions modulate and control the main biological processes such as memory, response to stress, pain, immune response, kidney and cardiovascular function and many others. Development of peptide drug candidates that can target the receptors that regulate these biological functions is one of the main concerns in biology and medicine. In the past, peptides were considered to be poor drug candidates because of their low oral bioavailability and their susceptibility of being rapidly metabolised by proteolytic enzymes of the digestive system and blood plasma. Nonetheless, peptides also present many advantages over small organic molecules that led to a revival of interest of these molecules as potential active pharmaceutical ingredients (APIs) such as greater efficiency, selectivity and specificity, and less systemic toxicity. Moreover, few peptide drugs accumulate in tissues because of their short half-life, thus decreasing the risk of complications. Fexapotide triflutate (FT) is a new therapeutic peptide with selective pro-apoptotic properties. Currently, it is in phase III of clinical trials for the treatment of benign prostatic hyperplasia (BPH). In this context, the thesis has been focused on the development of a synthetic route for the synthesis of FT compatible with pilot plant scale-up. The work has been carried out within the framework of a collaboration between the Inorganic and Organic Department of the University of Barcelona and a pharmaceutical company. Peptides can either be synthesised in solution or in solid-phase. Both synthetic approaches have proven to be effective but, each approach has its limitations. On one hand, solution-phase synthesis requires long time development of the procedure. On the other hand, scale-up of a solid-phase synthesis protocol is generally difficult. Nonetheless, with the introduction of new acid sensitive resins such as 2-CTC, it is possible to synthesise protected peptides that can be released from the resin without the necessity of removing any side chain protecting groups. This has introduced the possibility of an hybrid approach, also known as convergent synthesis that allows the production of complex peptide sequences by the synthesis of short protected peptide fragments of the target peptide using the solid-phase approach and then, the assembly of these fragments either by solution or solid-phase methods. This strategy circumvents difficulties encountered during the solid-phase synthesis of long peptides, becoming a promising approach for the commercial-scale production of large peptides. Three different synthetic routes were proposed for the synthesis of Fexapotide. The first approach is based on the use of the traditional solid-phase methodology following a linear approach. However, this strategy presented some disadvantages, and two different convergent approaches were designed and studied as alternatives, differing on the size of the fragments. The solid-phase synthesis of the protected peptide fragments was extensively studied by performing several syntheses of each fragment under different conditions. Moreover, the solution-phase coupling conditions were deeply examined as well as the purification of different impurities that were generated during the synthetic process to find suitable conditions to avoid them. Special attention was given to the removal of the Fmoc group with the aim of searching protocols suitable for peptide purification. With this goal, the isolation and characterization of the by-products generated was carried out.