Identification of alternative products and optimization of 2-nitro-5-thiocyanatobenzoic acid cyanylation and cleavage at cysteine residues

The reagent 2-nitro-5-thiocyanatobenzoic acid (NTCB) is commonly used to cyanylate and cleave proteins at cysteine residues, but this two-step reaction requires lengthy incubations and produces highly incomplete cleavages. In previous reports, incomplete cleavage was attributed to a competing beta-elimination reaction that converts cyanylated cysteine to dehydroalanine. In this study, previously unidentified side reactions of the NTCB cleavage were discovered and beta-elimination was not the major reaction competing with peptide bond cleavage. A major side reaction was identified as carbamylation of lysine residues. Carbamylation could be minimized by desalting the cyanylation reaction before cleavage or by reducing the reactant concentrations, but both methods suffered from further reductions in cleavage efficiency. Based on model peptide studies, poor cleavage was primarily caused by a mass neutral rearrangement of the cyanylated cysteine which produced a cleavage-resistant, nonreducible product. The formation of this product could be minimized by using stronger nucleophiles for the cleavage reaction. We discovered that base-catalyzed nucleophilic cleavage could be achieved with many amino-containing compounds. Most notably, glycine is capable of promoting efficient cleavage. In addition, efficient NTCB cleavage can be performed in a simple one-step method without a prior cyanylation step, rather than the previously described two-step reaction.