Solid phase total synthesis of cyclic proteins – CyProt

The first part of this project is mainly synthetic organic chemistry in solution and on the solid phase which aims at producing novel dichalcogenoazepane structures and characterizing novel reactivities in aqueous solution.
In the meantime, we have further optimized different phenotypic assays relying on HGF/MET signalling to be ready to test the new proteins synthesized in this project.

We have prepared successfully four different derivatives derived from the dichalcogenoazepane structure. These compounds have an additional functionality, i.e. a carboxylic acid group, for their attachment to the water compatible solid support. They constitute the starting point for the solid phase work and allow also at the end of the process the intramolecular ligation process leading to the release in solution of the target cyclic protein. Before starting the solid phase work we wanted to validate their reactivity and potential in solution using short model peptides. We found that the dichalcogenoazepane homologous to the SEA group and featuring a disulfide bond displayed the appropriate reactivity to be further used on the solid phase. The dichalcogenoazepane initially described in the projet and having a diselenide bond in its structure was found to partially decompose during the ligation. However and interestingly, the mixed Se,S dichalcogenoazepane is highly promising and should constitute a potential alternative to the diselenide derivative, given the similar redox potential between diselenides and mixes selenosulfides.

Although we encoutered some difficulties with the diselenide dichalcogenoazepane structure initially targetered in the proposal, we found two potential alternatives that allow us to continue the project in the best conditions.

One manuscript has been submitted (in revision)

One of the great challenges in the development of small protein drugs is the introduction of modifications which allow an increased the stability of the protein, its half-life in biological fluids, the yield of production and folding. The cyclization of small peptides is well developed, whereas the cyclization of large polypeptides or proteins is still in its infancy, primarily due to the difficulty in producing cyclic polypeptides or proteins using living systems. Given the fast growing importance of long polypeptides or proteins in the drug market, the development of processes enabling the efficient total synthesis of cyclic analogue libraries is of utmost importance. The aim of this project is to develop an innovative self-purifying process enabling the synthesis of large cyclic polypeptides or proteins by performing several sequential ligations of purified and unprotected peptide segments on a water compatible solid support. The method will be automated by adapting a commercially available multiple peptide synthesizer. The technology will be used for optimizing a lead cyclic protein (< 100 amino acids) currently developed in Lille for its capacity to act as a potent mimic of hepatocyte growth factor (HGF), which is the ligand of the MET receptor tyrosine kinase. Such potent MET agonists have a great potential in regenerative medicine. In particular, potent MET agonist can prevent the fulminant hepatic failure, an application which is mainly targeted in this project.