Structural Characterization of the Dimers and Selective Synthesis of the Cyclic Analogues of the Antimicrobial Peptide Cm-p5.

Abstract

Background/Objectives: Cm-p5 and its cyclic monomeric and dimeric analogues are known for their antifungal, antibacterial, antiviral, and antibiofilm activities. Previously, our cyclization method produced a mixture of peptides that were difficult to separate, which was then improved by a selective synthesis of the parallel dimer and its differentiation from the antiparallel by comparison of the retention times in RP-HPLC. Methods: Here, we developed a more reliable identification method for the Cm-p5 dimer identification, which included chymotrypsin proteolytic digestion and sequencing of the different fragments by ESI-MSMS. We also improved our cyclization methods to specifically produce higher amounts of the desired cyclic variant, either cyclic monomer or dimer. Results: We show that liquid phase oxidation with 20% DMSO or iodine oxidation yields only the cyclic analogue. However, the on-resin oxidation with iodine showed greater efficacy and efficiency. Additionally, liquid phase cyclization yields the antiparallel dimer in high EtOH or peptide concentration, indicating a kinetic control. On the other hand, the parallel dimer was preferentially produced in 5% of TFE and low peptide concentration without the formation of the cyclic analogue indicating a thermodynamic control. Conclusions: In conclusion, we report that chymotryptic digestion combined with ESI-MS and MS/MS allows an unambiguous differentiation of Cm-p5 dimers. Here, we develop more selective and efficient methods for the synthesis of cyclic and dimeric analogues of Cm-p5.