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Doctoral Thesis
DOI
https://doi.org/10.11606/T.46.2007.tde-13112007-093304
Document
Author
Full name
Patrícia Barrientos Proti
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2007
Supervisor
Committee
Miranda, Maria Teresa Machini de (President)
Andrade, Leandro Helgueira de
Cilli, Eduardo Maffud
Ferreira, Ana Maria da Costa
Nascimento, Maria da Graça
Title in Portuguese
Preparação de ésteres e tioésteres de peptídeos protegidos através de solvólise da ligação peptidil-resina mediada por íons metálicos
Keywords in Portuguese
Biocatálise
Ca+2
Lipase
Metanólise
Peptídio
Resina de Kaiser
Tiólise
Tripsina
Abstract in Portuguese
Os objetivos do presente trabalho foram: i) aprimorar o procedimento alternativo de mediação por íons metálicos da alcoólise da ligação peptidil-resina com vistas à obtenção de ésteres metílicos de peptídeos protegidos (Nα-acil-peptídeo protegido-OMe) em condição reacional branda e com alta eficiência; ii) investigar a aplicabilidade do procedimento para a preparação de Nα-acil-peptídeo protegido-SR e de Nα-acil-aminoácido-OR; iii) verificar se os Nα-acil-peptídeo-OMe obtidos atuariam como doadores de acila em reações de formação de ligação peptídica catalisadas por lipases. Para tanto, na busca da melhor condição de metanólise e comparação com os procedimentos usuais de alcoólise de ligação peptidil-resina, foram usados: o fragmento 22-24 da colecistocinina-33 humana (tripeptídeo modelo), Ca2+, Zn+2, Co+2 e Cu+2 (mediadores), as resinas oxima de Kaiser (KOR), p-hidroximetilfenil acetamidometil, ácido p-hidroximetilbenzóico e álcool p-benziloxibenzil (suportes poliméricos), misturas de MeOH com DCM, DMSO, NMP, THF ou DMF (solventes) e 25, 37, 50 ou 60°C. A condição ótima encontrada [KOR, Ca+2 (1 eq./eq. de peptídeo), 50% MeOH/DMF, 50°C] foi empregada com sucesso na preparação do Nα--acil-heptapeptídeo protegido-OMe, fragmento do peptídeo quimiotático M de Vespa mandarinia. Variações dessa condição foram usadas com sucesso nas preparações dos Nα-acil-tripeptídeo protegido-S(CH2)2COOEt e Nα-acil-Ala-OR (R: Me; Bzl), pois eles foram gerados com boas qualidades e rendimentos similares ou superiores aos obtidos via procedimentos usuais. Após desproteção de cadeias laterais, os Nα-acil-tripeptídeo-OMe e Nα-acil-heptapeptídeo-OMe foram usados em reações de acoplamento com Gly-NH2 em presença de preparações lipásicas comerciais. Estes ensaios inéditos também foram bem sucedidos, pois após adequação das condições reacionais, os Nα-acil-tetrapeptídeo-NH2 e Nα-acil-octapeptídeo-NH2 foram obtidos com boas qualidades e rendimentos de 65% (1 h) e 55% (24 h), respectivamente.
Title in English
Preparation of protected peptide esters and thioesters through peptide-resin linkage solvolysis mediated by metal ions
Keywords in English
Biocatalysis
Ca+2
Kaiser oxime resin
Lipase
Methanolysis
Thiolysis
Trypsin
Abstract in English
The present work aimed to: i) improve the alternative procedure based on mediation by metal ions of peptide-resin linkage alcoholysis to obtain fully protected peptide methyl esters (Nα-acyl-protected peptide-OMe) under mild reaction condition and with high efficiency; ii) investigate the usefulness of the alternative procedure for preparing Nα-acyl-protected peptide-SR and Nα-acyl-amino acid-OR; iii) verify whether the resulting Nα-acyl-peptide-OMe would act as acyl donors in peptide bond formation catalyzed by lipases. Thus, in the search for the best methanolysis condition and comparison with the usual procedures for that, we used: fragment 22-24 of human cholecystokinin-33 (model tripeptide), Ca+2, Zn+2, Co+2 and Cu+2 (mediators), Kaiser oxime resin (KOR), p-hydroxymethylphenylacetamido methyl resin, p-hydroxymethylbenzoic acid resin and p-benzyloxy benzyl alcohol resin (polymeric supports), mixtures of MeOH and DCM, DMSO, NMP, THF or DMF (solvents) and 25, 37, 50 or 60°C. The optimal condition found [KOR, Ca+2 (1 eq./eq. of peptide), 50% MeOH/DMF, 50°C] was used successfully for preparing Nα-acyl-protected heptapeptide-OMe, fragment 1-7 of the chemotactic peptide M produced by Vespa mandarinia. Variations of this condition were employed successfully for preparing Nα-acyl-protected tripeptide-SR and Nα-acyl-Ala-OR (R: Me, Bzl): indeed, these compounds were obtained in good quality and with similar or superior yields than those provided by usual procedures. After side chain deprotections, the Nα-acyl-tripeptide and Nα-acyl-heptapeptide methyl esters obtained were used in coupling reactions with Gly-NH2 in the presence of commercial lipase preparations. Those pioneer reactions were also successful, since after optimizing the conditions, Nalfa-acyl-tetrapeptide-NH2 and Nα-acyl-octapeptide-NH2 were obtained in good qualities with yields of 65% (1 h) and 55% (24 h), respectively.
 
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Publishing Date
2007-12-05
 
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