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Title: Novel Disulfide Formation Strategies in Peptide Synthesis
Author: Postma, Tobias Maria
Director: Albericio Palomera, Fernando
Keywords: Pèptids
Enllaços químics
Química combinatòria
Chemical bonds
Combinatorial chemistry
Issue Date: 21-Mar-2014
Publisher: Universitat de Barcelona
Abstract: [spa] La cisteína (Cys) es un aminoácido único debido a su capacidad para formar enlaces disulfuro que son reversibles. El capítulo 1 describe la gran variedad de métodos que existen para preparar este tipp de péptidos. La mayoría de éstos comportan protocolos tediosos y/o requieren reactivos agresivos. En esta misma parte, describimos los objetivos de nuestra investigación que ha estado enfocada a aumentar y a enriquecer el arsenal de herramientas químicas para preparar péptidos ricos en puentes disulfuro. . En el capítulo, el 2 trimetoxifeniltio (S-Tmp) se describe como un nuevo protector de la Cys compatible con la estrategia de síntesis de péptidos en fase sólida basada en el grupo Fmoc. El S-Tmp ha estado diseñado para sustituir el grupo terc-butiltio, que es difícil de eliminar. El grupo S-Tmp se puede eliminar en 5 min con agentes reductores suaves. Los crudos de los péptidos que se obtienen con el grupo S-Tmp son de gran pureza. En el capítulo 3, se describe la N-clorosuccinimida como un reactivo eficaz para la formación de enlaces disulfuro sobre resina. La formación de enlaces disulfuro tiene lugar en 15 minutos, utilizando DMF como disolvente. La validez de esta estratregia se ha demostrado mediante la sínntesis de varios péptidos. Este método es compatible con la presencia de metionina y de triptófano que son propensos a la oxidación. En el capítulo 4 se describe la utilización de la N-clorosuccinimida para la formación eficiente de enlaces disulfuro en condiciones acuosas. El proceso tiene lugar en 15 minutos . En el capítulo 5, se decribe una resina que lleva inmovilizada N-clorosuccinimida, que ha resultado ser muy conveniente para la formación de enlaces disulfuro en la preparación de bibliotecas combinatorias de péptidos. La formación del puente disulfuro es rápida y limpia. Este reactivo inmovilizado debe encontrar una gran utilización en química combinatoria . En el capítulo 6, se describe la utilización de pseudoprolinas derivadas de Cys en la síntesis de péptidos que contengan Cys. La formación de la Cys a partir de la pseudoprolina de Cys tiene lugar con tratamientos cortos de TFA. En este capítulo se ha descrito que las pseudoprolinas de Cys pueden favorecer las reacciones de macrociclación. En resumen, creemos que los métodos que presentamos son una aportación importante a los ya existentes para la preparación péptidos con puentes disulfuro.
[eng] Cysteine (Cys) is a unique amino acid due to its ability to form reversible covalent disulfide bonds. These bonds cause conformational constraints which make peptides or proteins more rigid and stable. In addition, the conformational constraints induced by disulfide bonds can result in highly potent molecules as illustrated by the vast number of disulfide-rich plant and animal peptide toxins. Furthermore, disulfide-rich peptides are gaining importance as pharmaceuticals and therefore powerful new synthetic strategies are needed. Disulfide-rich peptides can be prepared synthetically by several methods of which the state-of-the-art is described in chapter 1. However, most methods are time consuming or use harsh reagents. Herein, we describe the results of our research with the main goal of introducing and enriching novel molecular tools to aid in the preparation of disulfide-rich peptides. In chapter 2 trimethoxyphenylthio (S-Tmp) is described as a novel Cys protecting group in Fmoc solid phase peptide synthesis replacing, the difficult to remove, tert-butylthio. S-Tmp and dimethoxyphenylthio (S-Dmp) were successfully used for Cys protection in a variety of peptides. Moreover, both groups can be removed in 5 min with mild reducing agents. S-Tmp is recommended for Cys protection as it yields crude peptides of high purity. In chapter 3 N-chlorosuccinimide is described as a widely applicable on-resin disulfide forming reagent. Disulfide bond formation was completed within 15 minutes in DMF. This strategy was successfully used in the synthesis of oxytocin, and a regioselective synthesis of an α–conotoxin. Moreover, disulfide formation with N-chlorosuccinimide was found to be compatible with oxidation prone methionine and tryptophan. In chapter 4 a novel method has been developed for the efficient formation of peptide disulfide bonds under aqueous conditions using N-chlorosuccinimide. Complete disulfide bond formation is achieved in 15 minutes with solvent mixtures containing water and acetonitrile. This method has applicability when on-resin disulfide formation gives poor results. In chapter 5 a novel immobilized N-chlorosuccinimide resin was developed for peptide disulfide bond formation in combinatorial libraries. The resin is prepared in a simple two-step process from commercial starting materials. Disulfide formation is initiated by adding a peptide solution to the resin, and excess reagent is removed by a convenient filtration upon completion of disulfide formation. Completion of disulfide formation is rapid and clean, as demonstrated by the oxidation of a small nonapeptide library. This immobilized reagent allows a wider scope for the use of N-chlorosuccinimide based disulfide formation in combinatorial chemistry. In chapter 5 we describe Cys pseudoproline-containing peptides with short deprotection times in TFA, contrary to other studies. The deprotection times fell in the same range as other protecting groups commonly used in SPPS. Moreover, a novel application for Cys pseudoprolines is disclosed in which this protecting group acts as a peptide macrocyclization-enhancing moiety that considerably reduces reaction time. In summary, a novel Cys protecting group was introduced; N-chlorosuccinimide was found to be an efficient disulfide-forming reagent on-resin as in solution; an Immobilized derivative of N-chlorosuccinimide was described for combinatorial libraries; and Cys pseudoprolines with short deprotection times were observed which enhanced macrocyclization. These methods are a useful addition to the molecular tools available to prepare complex disulfide-rich peptides.
Appears in Collections:Tesis Doctorals - Facultat - Química

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