20. Mechanism for the enhanced reactivity of 4-mercaptoprolyl thioesters in native chemical ligation

Ali Shah, M. I.; Xu, Z.-Y.; Liu, L.; Jiang, Y.-Y.;* Shi, J.*  RSC Adv. 2016, 6, 68312–68321. Download Link

Abstract: The introduction of a 4-mercaptan substituent was recently reported to be effective to improve the reactivity of C-terminal prolyl thioesters in native chemical ligation (NCL). To elucidate the origin of the high reactivity of 4-mercaptoprolyl thioesters, a theoretical study was performed on the transthioesterification of NCL herein with the aid of density functional theory (DFT) methods. The calculation results support a transthioesterification mechanism involving two stages. The first stage is the intramolecular transthioesterification of 4-mercaptoprolyl thioesters to form a bicyclic thiolactone intermediate and the second stage is the intermolecular transthioesterification of the thiolactone intermediate with N-terminal cysteine. The two stages proceed both via nucleophilic attack of the thiolate and proton-donor-assisted thiolate release. The thiolate release in the first stage is the ratedetermining step of the whole transthioesterification of 4-mercaptoprolyl thioesters. Based on the mechanistic details, further analysis was conducted to gain a deeper insight with natural bond orbital (NBO) analysis, non-covalent interaction plots (NCIplots), distortion analysis, etc. The n / p* interaction between the N-carbonyl oxygen and thioester carbonyl was found to be detrimental to the first stage while a previously unnoticed n / p* interaction between the amide nitrogen and the thiolactone carbonyl slightly benefits the second stage. The entropy effect was the main driving force for the first stage because this stage is an intramolecular one-to-two process overall. On the other hand, the release of the ring stain precluded in the bicyclic thiolactone intermediate is mainly responsible for the high reactivity of the thiolactone in the second stage compared with other prolyl thioesters. Meanwhile, the intramolecular hydrogen bond between the thiolactone carbonyl oxygen and the protonated amine of N-terminal cysteine also contributes to the transthioesterification.