Selectivity of labeled bromoethylamine for protein alkylation

Alkylation of cysteine residues has been used extensively for characterization of proteins and their mode of action in biological systems, research endeavors that are at the core of proteomics. Treatment with a simple alkylating agent such as [2-(13)C] bromoethylamine would result in labeled thialysine at the ε-position. This chemical modification of proteins would allow investigations via both (13)C NMR spectroscopy and mass spectrometry. However [2-(13)C] labeled bromoethylamine is not available commercially. We investigated its synthesis at acid pH with the goal of obtaining singly labeled bromoethylamine and understanding the mechanistic details of the reaction. Based on our experimental and theoretical results, bromination of [2-(13)C] labeled ethanolamine in acidic conditions takes place via exclusive attack of the nucleophile (HBr) at the hydroxyl bearing C. Moreover, hydrogen bonding guides the nucleophilic attack, resulting in no label scrambling of the bromoethylamine product. Protein alkylation at cysteine residue with the synthesized Br(13)CH(2)CH(2)NH(2)-HBr is successful. Ab initio calculations in which CH(3)SH serves as a model for the cysteine residue suggest that in gas phase intermolecular attack by the sulfur bearing nucleophile is favored over the intramolecular substitution by the amino group by 15.4 kJ mol(-1). Solution modeling shows that the trend is preserved at basic pH, which is the experimental one, but is reversed at neutral pH.