CYSTEINE-FREE NCL · Development of a new methodology for the synthesis of mercapto amino acids for protein synthesis by cysteine-free native chemical ligation
FP7 — People (Marie Curie Actions)
- Duration
- 2014-04-14 → 2016-04-13
- EU contribution
- €173,371
- Participants
- 1
- Scheme
- MC-IIF
Lines connect the coordinator with its partners.
Results in brief
Development of a new methodology for the synthesis of mercapto amino acids for protein synthesis by cysteine-free native chemical ligation.
To improve the understanding of disease states, scientists need to investigate the cellular pathways involved in their development. Stapled peptides not only represent a valuable tool to study protein-protein interactions and to identify new drug targets, they also potentially make powerful therapeutics. Despite the importance of stapled peptides in nowadays drug discovery programs, very few methods currently feature in the chemists's toolbox to prepare these precious entities. Simultaneously C-H activation has emerged as a practical method to readily functionalised natural products and pharmaceuticals. This project aimed at the development of a new stapling technology using state-of-the-art C-H activation chemistry. During the course of this project an efficient protocol for the stapling of peptides was developed in-solution. The scope of the reaction was evaluated. A library of 25 stapled peptides was prepared using the optimized reaction conditions identified, thus demonstrating the compatibility of the reaction with a broad range of substrates. Parameters such as the size and the length of the staple were also evaluated. Next, a more practical on-resin protocol was established which should allow chemists to readily access these novel entities. Finally, the stability of these novel cross-linked peptides to protease degradation was evaluated as well as their propensity to generate specific secondary structures. These new stapled peptides were found to dramatically increase the half-life of the molecules, thus addressing one of the major drawbacks of standard peptides. In conclusion, the new technology developed is expected have a tremendous impact on drug discovery, by providing an efficient method to readily access novel stapled peptide entities, useful for the understanding of the biological processes involved in the development of diseases and for the design of novel powerful drugs.
Data: CORDIS, © European Union
Project objective
Proteins are in the heart of almost every single biological function and therefore are essential tools for drug discovery. Although the use of recombinant DNA techniques has been successfully applied to the synthesis of many proteins, the access to proteins bearing non natural amino acids, naturally obtained through post-translational modifications, remains limited. However, with the invention of Solid Phase Peptide Synthesis (SPPS) by Merrifield, the total chemical synthesis of peptide containing non-proteinogenic amino acids can be easily achieved. Nonetheless, the SPPS technique is only suitable for peptide chains of a length below 50 amino acids. The problem related to the synthesis of large peptide or even proteins was overcome by Kent et al. who were able to link peptide fragments together by Native Chemical Ligation (NCL). NCL involves the reaction between the N terminus cysteine of a first fragment and the thioester C-terminus of a second fragment. After the transthioesterification, a S -> N acyl transfer yields the desired native amide bond. Despite this ground-breaking discovery, NCL remains limited to cysteine-containing proteins. In 2001, Dawson et al. introduced the ligation-desulfurisation technique. This latest advance paved the way to cysteine free NCL and prompted many research groups to focus their efforts on the preparation of beta- and gamma-mercapto amino acids for use in ligation-desulfurisation strategy. Unfortunately, the 7 to 16 steps required for their synthesis considerably restrain their uses by peptide chemists. The proposed project is directed towards the elaboration of a straightforward synthetic route to access beta-thiol amino acids from a single precursor using the latest breakthroughs in C-H activation technique. In order to demonstrate the usefulness of these novel building blocks for the synthesis of proteins, these cysteine surrogates will be employed in the synthesis of the cysteine-free anti-HIV protein, Griffithsin.
Original text from CORDIS.
Participants
- FUNDACIO INSTITUT DE RECERCA BIOMEDICA (IRB BARCELONA) · BarcelonaCoordinatorSpain
Links
Data: CORDIS, © European Union