Source: http://cbm.cnrs-orleans.fr/en/research/research-teams/molecular-structural-and-chemical-biology/synthetic-protein-and-bioorthogonal-chemistry/
Timestamp: 2019-04-24 04:50:11+00:00

Document:
The production of proteins by total synthesis is a growing field of research. This approach is very complementary to recombinant techniques for applications to the deciphering of biological mechanisms at the molecular level (chemical biology) or the development of new drugs. This alternative route is particularly useful for accessing modified proteins (post-translational modifications, selective labeling, introduction of probes, non-natural amino acids, modification of the peptide backbone, etc.) or difficult to produce (cytotoxic proteins, disulfide-rich miniproteins etc.).
Current technologies focus on the modular assembly of unprotected peptide fragments, through very selective reactions called "chemical ligation". This approach revolutionized the field some thirty years ago and is gradually being democratized for the synthesis of small proteins (50-100 amino acids). However, access to more ambitious targets in terms of size or molecular complexity remains a real challenge still reserved for rare specialists, often at the cost of considerable effort and investment. Three major technological bottlenecks remain to be overcome: (1) the assembly of numerous fragments by successive ligations, that requires repeated purifications steps, ultimately leading to very low overall yields, (2) the handling of poorly soluble or aggregation-prone fragments, a very frequently encountered problem, and (3) the development of more efficient ligation methods, from both the points of view of the nature of the chemical reactions used and of the synthesis of appropriately functionalized peptide fragments.
One of the main focuses of our group relies on the conception and development of original solutions to each of the three above-mentioned bottlenecks. We have thus gradually established a coherent molecular toolkit, aiming to be robust and versatile.
Particular emphasis has been placed on the development of techniques allowing a solid support assembly in order to avoid intermediate purifications. To this end, we have introduced a range of new linkers allowing the specific immobilization of a first pure and unprotected peptide fragment on a suitable solid support. Similar linkers have also been applied to the temporary modification of hydrophobic or aggregation-prone fragments, in order to render them soluble by the addition of charged residues (e.g., hexalysine label). The main challenge is to control the conditions necessary for the cleavage of these arms under very mild conditions, in order to release the protein assembled by successive ligations from the solid support or to eliminate the solubilizing groups.
The most commonly used reaction for protein synthesis was discovered some 20 years ago and is called native chemical ligation (NCL). It consists in the reaction of peptide fragments equipped respectively with a thioester function and an N-terminal cysteine. If the synthesis of the cysteinyl moiety can be carried out routinely, that of the thioester moiety is still very problematic. Indeed, the reaction conditions most commonly used in solid phase peptide synthesis (Fmoc / tBu strategy) are intrinsically incompatible with the thioester function.
Inspired by the spontaneous formation of a thioester during protein splicing, a non-enzymatic maturation process mediated by protein domains called inteins, we designed a molecular device programmed to in situ form a thioester under NCL conditions: so-called peptide crypto-thioesters. This bio-inspired process has the advantage of not requiring an additional chemical step after solid phase peptide synthesis. It can be totally automated at low cost, which makes its implementation by non-specialists very affordable. The peptide crypto-thioesters thus obtained are stable and can be handled, analyzed and purified by conventional techniques, and can be directly used in an NCL reaction, with very fast kinetics of transformation into a reactive thioester.
While seeking alternative reactions to NCL, we pioneered the use of the copper(I)-catalyzed alkyne/azide cycloaddition (CuAAC) in the context of chemical protein synthesis. This highly chemoselective reaction leads to the formation of a 1,4-disubstituted 1,2,3-triazole which is recognized as an excellent mimic of a trans amide bond: CuAAC can thus be considered as a peptidomimetic ligation technique.
Additionally, triazoles cannot be hydrolyzed by proteases, and this feature has been exploited for the development of peptidomimetics whith enhanced in vivo stability for drug discovery purposes.
Disulfide rich peptides (DRP) are ubiquitous natural products composed of 10 to 90 amino acids, characterized by evolutionary-conserved cysteines reticulated by a dense disulfide bond network. These remarkably compact and stable compounds are active in adverse environments, exhibiting incredibly diverse host-defense or predation-related functions. DRPs are potent and selective binders of various targets, making them attractive compounds as pharmacological tools or new drug leads.
Our group has a long-lasting expertise in the synthesis of these compounds, including their oxidative folding to achieve the natural disulfide connectivity. The ligation-based synthetic methodologies we developed now allow us to synthesize very long DRPs, particularly difficult to access through standard synthetic means.
◊ New strategies for cancer immunotherapy.
Glycan antigens carried by mucins present on cancer cells are tumor markers which are in part responsible for the immune system anergy regarding tumors. Structure and density of mucin glycans have an important impact on immune cells inaction. The mucins most often used in order to study those mechanisms are a mixture of heterogeneous glycoproteins with multiple glycan structures. But, for a better understanding of the relationship between glycan structure and immune response, it is mandatory to have access to mucins of variable size with well-defined glycan structures. Therefore, in order to produce those synthetic glycoproteins, we developed solid support synthesis methodologies combining chemical and enzymatic glycosylation steps. This allowed the preparation of homogenous and structurally well-defined mucins (up to 160 aminoacids and 24 short O-glycans) useful to study with precision the role of glycans carried by those glycoproteins in endocytosis and antigen presentation by dendritic cells and macrophages.
Many tumor antigens could in theory be used as targets for cancer immunotherapy. However, they usually don’t provoke a strong response because they have already been present on normal cells during embryonic development. In order to circumvent that problem, we use monosaccharide analogs of N-acetylgalactosamine or N-acetylmannosamine which can be readily incorporated into tumor cell glycans, due to the abnormal and very active carbohydrate metabolism of cancer cells, and exposed on cell surface. This approach targets specifically tumor cells and the reactive groups on the glycans can be used as handles to covalently link through bioorthogonal ligation reactions, strong immunostimulants specifically to the tumor cell surface where they will activate the anergic immune cells inside the tumor.
Jacobsen M. T., Petersen M. E., Ye X., Galibert M., Lorimer G. H., Aucagne V. and Kay M. S.
Terrier V. P., Adihou H., Arnould M., Delmas A. F. and Aucagne V.
Galibert M., Piller V., Piller F., Aucagne V. and Delmas A. F.
Chuh K. N., Zaro B. W., Piller F., Piller, V. and Pratt M. R.
Aucagne V., Valverde I. E., Marceau P., Galibert M., Dendane N. and Delmas A. F.
Valverde I., Lecaille F., Lalmanach G., Aucagne V. and Delmas A. F.

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