Theoretical study to gain fundamental insight into reaction mechanism of N-S acyl transfer of N-sulfanylethylanilide-based protein labeling reagent on protein surface.

Elucidation of protein function is one of the central issues in the field of life sciences. To study the function of proteins not in isolation, but in a cell or its lysate, thus, it is necessary to selectively label the target protein in a mixture. Affinity labeling is one of several widely used methods for selective labeling; however, this method has the disadvantage that the labeling reagent is always activated, albeit weakly. Therefore, fine-tuning of the reactivity and/or reaction conditions is generally required for successful target-selective labeling. We previously developed a new affinity labeling reagent with N-sulfanylethylanilide (SEAlide) as a key reactive unit. It was designed based on the following hypotheses. SEAlide is less reactive and does not label in the absence of a target protein. Upon target binding, amino acid side-chain functional groups on the target surface convert SEAlide into a thioester form via N-S acyl transfer, allowing the target to be labeled. However, no evidence has been obtained so far to directly prove the hypothesis. In this study, we examine whether amino acid side-chain functional groups can activate SEAlide from the viewpoint of theoretical chemistry. The theoretical studies show that the activation free energy and enthalpy of the acyl transfer of SEAlide are reduced in the presence of methylammonium, which is a model for the protonated side chain of Lys, and acetate, which is a model for the deprotonated side chain of Asp/Glu. It suggests that Lys and Asp/Glu side chains could potentially stabilize the activation transition states to accelerate the thioester formation. Furthermore, the significant decrease in the activation enthalpy indicates that the contribution of entropy to the transition state is large. This result supports the original hypothesis that the SEAlide-based labeling reagent is efficiently activated by binding to the target protein.