3-Bromo-1,1,1-trifluoroacetone (BTFA) is a widely used probe for labeling cysteine (Cys) residues to study protein structure by 19F NMR. We evaluated the mechanism by which BTFA labels transthyretin (TTR). In aqueous solution, BTFA rapidly undergoes hydration to its corresponding geminal diol (gem-diol), thereby establishing an equilibrium that disfavors the ketone, which is the reactive form of BTFA. The -84 ppm 19F signal commonly observed in BTFA-substructure-labeled biomolecules is attributable to the gem-diol, not the ketone as too often claimed. We demonstrate that the BTFA-substructure-Cys conjugate can undergo further reaction with a nearby nucleophile to produce cyclized products, particularly when the targeted Cys is the most N-terminal residue. If the initially labeled Cys is internal to the protein primary structure, 19F NMR analysis reveals that subsequent intramolecular cyclization reactions with nearby residues are possible (exacerbated by lyophilization or reduced pressure associated with mass spectrometric analysis), which can perturb the native structure, complicating 19F NMR structural interpretations. We sought an alternative probe whose conjugates do not undergo hydration and are stable enough not to undergo further reactions after Cys alkylation. We introduce 2-iodo-N-(2,2,2-trifluoroethyl)acetamide (ITFEA) for this purpose, which displays similar labeling efficiency as BTFA without the side product risk.
Norman et al. (Wed,) studied this question.