Diazoalkane decay kinetics from UVA-active protein labelling molecules: trifluoromethyl phenyl diazirines

Abstract Trifluoromethyl phenyl diazirine (TPD) molecules are relatively stable carbene precursors, that readily form carbon covalent bonds with proteins. The stability of the diazoalkane intermediates are unknown, as are the factors which control carbene/diazoalkane ratios. This leads to incomplete carbene insertion onto desired compounds. Herein, stability and decay kinetics of diazoalkanes are evaluated from TPDs with various electron drawing groups, including 3-Phenyl-3-(trifluoromethyl)-3H-diazirine (TPD-H), p-benzyl alcohol (TPD- CH2OH), p-4-benzoic acid (TPD-COOH), and p-benzyl bromide (TPD-CH2Br). The spectroscopic analysis before and after UVA activation is performed both in dilute chloroform and neat by 19F NMR and ATR-FTIR, respectively. The increase of diazoalkane concentration after UVA exposure was in the order of: TPD-H > TPD- CH2Br > TPD- CH2OH > TPD-COOH. Indirect carbene/diazoalkane ratios ranged from 6: 1 to 3:1. Diazoalkane was relatively stable over the evaluation period of 30 min (post-UVA activation) in all compounds except TPD- CH2Br, which exhibits an 11 min half-life.