Solid-supported synthesis of peptidoglycan fragments and their biological evaluation

In this work, a new methodology for the synthesis of peptidoglycan fragments containing (2S,6R)-A2pm is proposed. Thanks to a solid phase platform, cross metathesis was achieved between two easily manufactured chiral synthons, allylglycine anchored to a resin and vinylglycine derivatives. Due to the need of fragments for the study of the peptidoglycan recycling, this modular efficient platform could be useful to synthesize diverse derivatives of other peptides and muropeptides containing (2S,6R)-A2pm. In the framework of this project, (S)-Ala-γ-(R)-Glu-(2S,6R)-A2pm-(R)-Ala and γ-(R)-Glu-(2S,6R)-A2pm were synthesized. Their interactions with the R39 peptidase were studied by X-ray diffraction. According to the crystallographic results, (S)-Ala-γ-(R)-Glu-(2S,6R)-A2pm-(R)-Ala 1 is a reversible covalent binding inhibitor of R39. These data are supported by an enzymatic study using the thioesterase activity of the R39 peptidase on (R)-S2d. Because of the specific affinity for the R enantiomer of thioesters, the enantioselective synthesis of (R)-S2d is described that makes use of the coupling agent T3P. A chromatographic method is proposed to evaluate the degree of thioester racemization. The kinetics of the interaction between the thioester and different PBPs were studied and a protocol for microtiter plate-based assays is developed allowing the screening of compound libraries for inhibitors of PBPs. In the future, the synthesized peptidoglycan fragments will be used as reference compounds in the framework of the NetRBI project.