Multicomponent Oxidative Nitrile Thiazolidination Reaction for Selective Modification of N-terminal Dimethylation Posttranslational Modification

[Image: see text] Protein α-N-terminal dimethylation (Nme(2)) is an underexplored posttranslational modification (PTM) despite the increasing implications of α-N-terminal dimethylation in vital physiological and pathological processes across diverse species; thus, it is imperative to identify the sites of α-N-terminal dimethylation in the proteome. So far, only ∼300 α-N-terminal methylation sites have been discovered including mono-, di-, and tri-methylation, due to the lack of a pan-selective method for detecting α-N-terminal dimethylation. Herein, we introduce the three-component coupling reaction, oxidative nitrile thiazolidination (OxNiTha) for chemoselective modification of α-Nme(2) to thiazolidine ring in the presence of selectfluor, sodium cyanide, and 1,2 aminothiols. One of the major challenges in developing a pan-specific method for the selective modification of α-Nme(2) PTM is the competing reaction with dimethyl lysine (Kme(2)) PTM of a similar structure. We tackle this challenge by trapping nitrile-modified Nme(2) with aminothiols, leading to the conversion of Nme(2) to a five-membered thiazolidine ring. Surprisingly, the 1,2 aminothiol reaction with nitrile-modified Kme(2) led to de-nitrilation along with the de-methylation to generate monomethyl lysine (Kme(1)). We demonstrated the application of OxNiTha reaction in pan-selective and robust modification of α-Nme(2) in peptides and proteins to thiazolidine functionalized with varying fluorescent and affinity tags under physiological conditions. Further study with cell lysate enabled the enrichment of Nme(2) PTM containing proteins.