SYNTHESIS OF C-13, N-15-ENRICHED ALPHA-DICARBONYL MODEL ADDUCTS TO DETERMINE THE UTILITY OF C-13 AND N-15 NMR FOR STUDYING MECHANISM-BASED INACTIVATION OF CYTOCHROMES-P-450 BY SUBSTITUTED DICHLOROACETAMIDES

To determine the feasibility of using NMR to study the inactivation of cytochromes P450 by dichloroacetamide-containing mechanism-based inac tivators, C-13, N-15-enriched compounds were synthesized, modeling add ucts between the nucleophilic side-chains of cysteine, lysine, serine (threonine) and tyrosine and the alpha-aminooxoacyl chloride that pres umably results from enzymatic conversion of a dichloroacetamide-contai ning inhibitor. C-13 chemical shifts of the carbonyls attached to the nucleophiles easily distinguish the model cysteine adduct (delta 192.3 ) from the other adducts (delta 159.6-161.8). Although the other adduc ts cannot be distinguished at this carbon, the carbonyl attached to th e N-15 two bonds away can distinguish the lysine mimic (delta 161.2 or 161.3) from the serine (threonine) and tyrosine mimics (delta 156.1-1 58.1). These assignments were made by comparing N-15/C-13 J values and were confirmed by solid-state rotational ecbo double resonance (REDOR ) studies. Finally, aminooxo-N-15 chemical shifts three bonds removed from the nucleophile can distinguish adducts formed to lysine (delta 1 08.3), cysteine (delta 104.8 and oxygen-containing side-chains (delta 112.8-113.9).