Unusual thionation of a cyclic hexapeptide

One carbonyl oxygen of the cyclic hexapeptide cycle(-Gly¹-Pro²-Phe³-Val⁴-Phe⁵-Phe⁶-) (A) can be selectively exchanged with sulphur using Yokoyama's reagent. Surprisingly it was not the C=O of Gly¹ but that of Phe⁵ which was substituted and cyclo(-Gly¹-Pro62-Phe³-Va1⁴-Phe⁵ψCS-NH]Phe⁶-) (B)was obtained. Thionation results in a conformational change of the peptide backbone although the C=O of Phe⁵ and the corresponding C=S are not involved in internal hydrogen bonds. Two isomers in slow exchange, containing a CIS Gly¹-Pro² bond in a βVIa-turn (minor) and a trans Gly-Pro bond in a βII′-turn (major), were analyzed by restrained molecular dynamics in vacuo and in DMSO as well as using time dependent distance constraints. It is impossible to fit all experimental data to a static structure of each isomer. Interpreting the conflicting NOES, local segment flexibility is found. MD simulations lead to a dynamic model for each structure with evidence of an equilibrium between a βI- and βII-turn about the Val⁴-Phe⁵ amide bond in both the cis and trans isomers. Additionally proton relaxation rates in the rotating frame (R_1p) were measured to verify the assumption of this fast βI/βII equilibrium within each isomer. Significant contributions to R_1p-rates from intramolecular motions were found for both isomers. Therefore it is possible to distinguish between at least four conformers interconverting on different time scales based on NMR data and MD refinement. This work shows that thionation is a useful modification of peptides for conformation-activity investigations.