Authors
Nathalie OllivierEliott RoyRémi DesmetVangelis AgouridasVincent DiemerOleg Melnyk
Topics
Chemical Synthesis and AnalysisChemical Synthesis and ReactionsSynthetic Organic Chemistry Methodsa Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 9017 - CIIL - Center for Infection and Immunity of Lille, F-59000 Lille, France. b Centrale Lille ; F-59000 Lille, France.ABSTRACT: 4-mercaptophenylacetic acid (MPAA) is a popular catalyst of the native chemical ligation (NCL) but has to be used in large excess for achieving practically useful rates (up to 50-100 equivalents). We report here that the catalytic potency of MPAA can be boosted by introducing a stretch of arginines in the departing thiol from the thioester. By doing so, the electrostatically-assisted NCL reaction proceeded rapidly by using sub-stoichiometric concentrations of MPAA, an advantage that enabled useful synthetic applications.The native chemical ligation (NCL) reaction is recognized as a leading tool for the semi or total chemical synthesis of proteins. 1 NCL involves the coupling of a peptide thioester with a cysteinyl peptide to produce a peptide bond to cysteine (Fig. 1). Peptide alkylthioesters are reagents appreciated for their ease of synthesis and resistance to hydrolysis and epimerization during their preparation and storage. However, their modest acyl donor properties requires using them in combination with nucleophilic catalysts, 2 among which thiols are the most popular. 3, 4 In particular, the water soluble arylthiol MPAA (4mercaptophenylacetic acid) is frequently used as an NCL catalyst. 4 It is highly water-soluble at neutral pH owing to the presence of a carboxylate group in its structure, and acts by converting in situ the starting peptide alkylthioester into a peptide arylthioester (I IV in Fig. 1), which is a more powerful acylating species. Under classical experimental conditions using mM peptide concentrations, the MPAA-thioester exchange process leading to the formation of the arylthioester IV is rate limiting and requires large excesses of the catalyst to achieve practical ligation rates. We report here that the rate of peptide arylthioester IV formation and hence the rate of NCL using MPAA as the catalyst is dramatically enhanced when the alkyl thioester is derived from a Cys peptide featuring several arginines in its sequence (Fig. 1). The thiol thioester exchange is accelerated to such an extent that MPAA could be used at submillimolar concentrations. This property enabled useful synthetic applications such as performing kinetically controlled ligations (KCL 5) or NCL/desulfurization processes in one-pot. 6Figure 1. Principle of the NCL reaction and its catalysis by MPAA.The enhanced reactivity of peptide thioester 1 derived from Ac-Cys-Arg6-L-NH2 thiol toward MPAA is revealed by a series of thiol-thioester exchange reactions shown in Fig. 2. The reaction mixtures were analyzed by HPLC using UV detection at 215 nm and the peak areas of peptide thioester corrected for the absorbance of the mercaptoaryl moiety to determine the fraction of aryl thioester formed over time (see Supplementary Information). The formation of peptide thioester 4 from the reaction of peptide thioester 1 with MPAA proceeded with a second order rate constant of 4.24 0.31 M -1 s -1. This rate constant is ~ 1000 times that reported for the exchange between MPAA and a model peptide alkylthioester derived from 3mercaptopropionic acid (MPA) (4.3 10 -3 M -1 s -1), 7 a type of acyl donor that is frequently used in the field. Under the dilute conditions used for the exchange experiment, the formation of peptide thioester 6 from MPA peptide thioester 3 was found to be indeed insignificant.Figure 2. Electrostatic assistance of MPAA-alkylthioester exchange reactions. a) Model reactions studied. b) Time course of peptide arylthioester formation (HPLC, 215 nm). Reaction conditions: [peptide thioester] = 50 µM, [arylthiol] = 250 µM, 10 mM sodium phosphate buffer, pH 7, 19 °C. The dots correspond to the experimental data, the dashed lines to the fitting curves. The kinetic data presented in Fig. 2b also show that substituting the arginines in the departing thiol from the thioester by uncharged amino acid residues (thioester 2 + MPAA) or masking the negatively charged carboxylate from MPAA (thioester 1 + arylthiol 7) resulted in a significant reduction of the exchange rate compared to the reaction involving the thioester 1 and MPAA. A severe reduction of the exchange rate was also observed when peptide thioester 1 and MPAA were reacted in 6 M guanidine hydrochloride (Gn∙HCl) or in the presence of 250 mM NaCl. Gn∙HCl competes likely with the guanidinium groups displayed by arginine residues, while adding NaCl is expected to alter electrostatic interactions. Taken together, the data presented in Fig. 2. show the critical role played by the electrostatic interactions between the arginine guanidinium groups and MPAA carboxylate in the assistance of the thiol-thioester exchange reaction (Fig. 3). The pKa of MPAA thiol is 6.6, 2 meaning that MPAA is mostly present as a dianion at the working pH of the exchange process. Thus, the thiophenolate might potentially have a dual role here by being the nucleophilic species in the thiol-thioester exchange reaction but also by contributing to the negative charge on MPAA catalyst sensed by the oppositely charged thioester 1, thereby favoring encounter complex VII formation (Fig. 3). Note that the tetrahedral intermediate VIII might be stabilized by electrostatic interactions as well. Whether electrostatic assistance proceeds by one of these mechanisms or both remains to be established. In any case, all reactions were performed in 10 mM phosphate buffer, showing that phosphate monoanion H2PO4 - and dianion HPO4 2-, i.e. the phosphate anion species dominating at pH 7, apparently do not interfere significantly with the electrostatic assistance even when they are present in large excess compared to MPAA.Figure 3. The thiol-thioester exchange between peptide thioester 1 and MPAA is assisted by electrostatic interactions. We next examined if the above reported accelerations for the thiol-thioester exchange reaction would translate into a rate increase of an NCL reaction. In the series of experiments described in Fig. 4, we compared the reactivity of peptide thioester 1 and MPA peptide thioester 3 at different MPAA catalyst concentrations. The ligation experiments performed in the absence of MPAA show that thioester 1 is intrinsically more reactive than MPA thioester
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PublishedMar 10, 2023
TypePreprint
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