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Title: Synthesis of biaryl bicyclic peptides for recognition of protein surfaces
Author: Garcia Pindado, Júlia
Director/Tutor: Giralt Lledó, Ernest
Keywords: Pèptids
Síntesi en fase sólida
Solid-phase synthesis
Issue Date: 12-May-2017
Publisher: Universitat de Barcelona
Abstract: [eng] The present thesis is based on the development and optimization of a solid-phase strategy to prepare biaryl bicyclic pentapeptides. Suzuki-Miyaura cross-coupling reactions were used to obtain the biaryl bridge. The metholodogy was compatible with the introduction of amino acids with functional groups in their side-chains (lysine, arginine and serine). The bicyclic pentapeptides were considered privileged structures regarding the evaluated properties. The introduction of the biaryl stapled enhanced passive diffusion permeability across the blood-brain barrier (BBB). Furthermore, protease resistance in human serum was also improved due to the presence of the biaryl. Moreover, no cytotoxicity was observed in HeLa cells at 500µM concentration. The developed methodology was expanded to a new kind of compounds. While the compounds synthesized in chapter 1 displayed a phe-phe stapled, the peptides prepared in chapter 3, presented the stapling between two tryptophans (trp-trp). Permeability studies demonstrated that the nature of the stapled was critical to improve the passive transport across the BBB. Cyclic peptides with bromine at different positions (5, 6 or 7) of the indole group of tryptophan were also synthesized. These compounds showed high passive permeability across the BBB. High cell survival rates in HeLa cells were observed for these peptides, except for the analog with bromine at position 5 of the indole. We aimed to evaluate the recovery of the self-assembly of a mutated p53 protein with some of the previously described peptides. Protein p53 is known as the “genome guardian”. The alteration of the balance of p53 pathway is mainly related with cancer. We were interested in the region that controls the proper folding of the protein, named the tetramerization domain. This domain is a dimer of dimer that contains 37 residues. The most relevant point mutation in this region is R337H. This mutant is mainly associated with the atypically frequent cases of pediatric adrenocortical carcinoma (ACC) in southern Brazil. Moreover, it is also associated with high prevalence of breast cancer in women from this region. The native p53TD protein, as well, as the R337H mutant were synthesized and characterized by circular dichroism (CD). The tetramer formed by R337H mutant was unstable, especially at higher pH. In order to stabilize the folding of this mutant, we used two different peptides prepared during the synthesis as ligands for this protein. Previous studies in our group demonstrated that calixarenes displaying guanidinium groups were able to recover the self-assembly of R337H mutant. Therefore, the selected peptides were from the arginine family. The bicyclic peptide, cyclo(Arg- (4&)Phe-Arg-(4&)Phe-D-Pro), was used as ligand of the protein R337H mutant. Unfolding experiments by CD did not show a relevant stabilization of the tetramer formed by the mutant in the presence of this ligand. Nevertheless, a more promising result was obtained for the linear stapled peptide, H-Arg-(4&)Phe-Arg-(4&)Phe-D-Pro- OH. The thermal stability of the protein R337H mutant in the presence of the linear stapled ligand was increased, transition temperature was 7ºC higher. Therefore, we could find an interesting application of one of these peptides synthesized by our previously optimized methodology.
[cat] La present tesis es centra en el desenvolupament i optimització d’una metodologia per a la obtenció de pèptids biaril bicíclic mitjançant una estratègia de fase sòlida. Per tal d’obtenir l’anell biaril es van emprar les reaccions de Suzuki-Miyaura. La correcta selecció del grup protectors va permetre expandir la metodologia incorporant aminoàcids tals com lisina, arginina i serina. Es va decidir avaluar algunes propietats farmacològiques dels compostos per tal de conèixer l’efecte d’introduir l’anell biaril. Pel que fa a la permeabilitat per difusió passiva a través de la barrera hematoencefàlica (BHE), va resultar beneficiós incorporar la grapa biaril a l’estructura peptídica. Atenent a la resistència del pèptids a les proteases presents en el sèrum humà, els pèptids que incorporen l’anell biaril van mostrar major resistència. En els assajos de permeabilitat cel·lular duts a terme amb cèl·lules HeLa no es va observar una mortalitat rellevant a una concentració de 500µM. Emprant triptòfans halogenats a diferents posicions del grup indole, es va dur a terme la síntesis de pèptids bicíclic amb una unió carboni-carboni entre les diferents posicions dels triptòfans, trp-trp. Aquests compostos van mostrar valors rellevants de permeabilitat per difusió passiva a través de la BHE. Tanmateix, els valor obtinguts pels pèptids units entre fenilalanines van ser majors, denotant la importància del aminoàcids involucrats en l’enllaç carboni-carboni d’aquests pèptids. Estudis previs del nostre grup van demostrar que l’ús de calixarens amb guanidinis permetia recuperar l’autoensamblatge d’un mutant de la proteïna p53. La proteïna p53 està altament relacionada amb el càncer. El mutant R337H és el més freqüent en el domini de tetramerització i tot i ser capaç de formar un tetràmer, aquest no és estable. Es van seleccionar dos pèptids de la família d’arginines, d’entre ells, el pèptid grapa H-Arg-(4&)Phe-Arg-(4&)Phe-D-Pro, va donar lloc a un resultat interessant. Per dicroisme circular, es va observar una major estabilització del tetràmer format pel mutant en presència del pèptid grapa. De manera que es va obtenir un resultat prometedor amb un compost sintetitzat amb la metodologia prèviament descrita.
Appears in Collections:Tesis Doctorals - Departament - Química Orgànica

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