3,4-Dihydro-3-methyl-6-nitro-2H-1,3-benzoxazin-2-one, a Reagent for Labeling p-Nitrophenyl Esterases with a Chromophoric Reporter Group - Synthesis and Reaction with Chymotrypsin

We report the synthesis of 3,4-dihydro-3-methyl-6-nitro-2H-l,3-benzoxazin-2-one (DMNB), a close structural analogue of p-nitrophenyl dimethylcarbamate. DMNB is unstable in aqueous solution when exposed to light, but is stable in the dark. The compound reacts slowly with chymotrypsin as an analogue of the substrate, p-nitrophenyl acetate, and this results in the incorporation of a p-nitrophenol-containing "reporter group" within the enzyme′s active site. The pKa of this group is two pH units higher than that of free p-nitrophenol, a result consistent with a hydrophobic environment around the enzymic binding site. Denaturation of the enzyme returns the pKa to its unperturbed value. DMNB has the promise of being a useful active site-directed reagent for other enzymes with p-nitrophenyl esterase activity.