Role of Solvent on Nonenzymatic Peptide Bond Formation Mechanisms and Kinetic Isotope Effects Katarzyna S ́ widerek, †,‡ Iñ aki Tuñ ó n,* ,‡ Sergio Martí, § Vicent Moliner,* ,§ and Juan Bertra ́ n ∥ † Institute of Applied Radiation Chemistry, Faculty of Chemistry, Lodz University of Technology, Zeromskiego 116, 90-924 Lodz, Poland ‡ Departament de Química Física, Universitat de Vale ̀ ncia, 46100 Burjassot, Spain § Departament de Química Física i Analítica, Universitat Jaume I, 12071 Castelló , Spain ∥ Departament de Química, Universitat Autò noma de Barcelona, 08193 Bellaterra, Spain * S Supporting Information ABSTRACT: Based on the hypothesis that similar mecha- nisms are involved in the peptide bond formation in aqueous solution and in the ribosome, the aminolysis of esters in aqueous solution has been the subject of numerous studies as the reference reaction for the catalyzed process. The mechanisms proposed in the literature have been explored in the present paper by hybrid QM/MM molecular dynamics simulations. The free energy profiles have been computed with the QM region of the system described at semiempirical AM1 level and by DFT within the M06-2X functional. According to the results, the formation of adduct zwitterion species is a preliminary step required for all possible mechanisms. Then, from different conformers of this species, four different paths were found: three of them taking place through concerted mechanisms of four-, six- and eight-membered ring transition states, and a stepwise mechanism through a neutral intermediate. Comparison of the free energy profiles indicates that the concerted mechanisms would be kinetically favored, with free energy barriers in very good agreement with experimental data. Calculations of kinetic isotope effects, when including the solute interactions with the first solvation shell, show that the 8-membered ring TS renders values in better agreement with available experimental data. Quantitative discrepancies can be attributed to different employed models in experiments and calculations. ■ INTRODUCTION Ribosomes are exquisitely complex molecular machines used by living organism to flow the genetic information encoded within genes into proteins. The ribosome catalyzes the peptide bond formation by the nuclephilic attack of an aminoacyl-tRNA in the A-site on peptidyl-tRNA in the P-site. Despite great progress in the study of ribosome function in the elongation step, 1−3 the mechanism of this process and the origin of the catalytic power of this ancient enzyme are still an unsolved puzzle. In this regard, many experimental and theoretical studies have focused on the aminolysis of esters in aqueous solution based on the hypothesis that similar mechanisms are involved in both media. 4−6 Experimental studies based on liner free-energy relation- ships 7−12 and isotope substitution effects 13−18 in solution apparently support a stepwise mechanism with participation of different kind of intermediates, ranging from anionic, cationic, neutral, and zwitterionic species. Reactions of esters with aliphatic amines generally show breaks in their pH-rate profiles at pH 6.3−8.7, indicating a change in rate-limiting step and therefore the presence of intermediates. 19 Computational studies have suggested the existence of basically three different mechanisms, as depicted in Scheme 1: (1) a concerted mechanism in which the nucleophilic attack on the amino group, the C−O cleavage bond, and the proton transfer take place simultaneously; (2) a stepwise mechanism through a neutral intermediate; and (3) a stepwise mechanism through a zwitterionic intermediate. 20−22 Mechanisms through positive or negative intermediates will not be explored, since our simulations are carried out at neutral pH, in the absence of additional bases or acids species. The mechanism through the zwitterionic intermediate has been the subject of controversy since separated charges species cannot be located in gas phase or in solution by traditional continuum models. 20−24 The addition of a reduced number of explicit water molecules in the model has shown to be crucial to stabilize this intermediate, 24−26 as well as the use of the more recently developed solute electron density model (SMD) 27 that has shown to be capable of stabilizing zwitterionic species. 28 In this sense, Warshel and co-workers located a zwitterionic intermediate in the catalyzed methanolysis of formamide (formally the reverse reaction of the one studied in the present paper) by combined ab initio/Langevin dipoles calculations. 29 Thus, most of the theoretical studies have been focused on the first two mechanisms, the concerted and the stepwise Received: March 26, 2013 Article pubs.acs.org/JACS © XXXX American Chemical Society A dx.doi.org/10.1021/ja403038t | J. Am. Chem. Soc. XXXX, XXX, XXX−XXX