Different Mechanisms of Alkaline and Enzymatic Hydrolysis of the Insensitive Munition Component 2,4-Dinitroanisole Lead to Identical Products

The emerging use of 2,4-dinitroanisole (DNAN) in insensitive munitions formulations has caused concern about future contamination of subsurface environments, generating significant interest in understanding and identifying its transformation processes. Here we characterized the C and N isotope fractionation associated with abiotic and biological DNAN hydrolysis through alkaline hydrolysis at high pH as well as enzymatic hydrolysis by Nocardioides sp. JS1661 and partially purified DNAN O-demethylase. Whereas both reactions generated 2,4-dinitrophenol (DNP), compound-specific isotope analysis (CSIA) of DNAN and DNP revealed that these reactions occur by different mechanisms. Alkaline hydrolysis was associated with apparent C-13 and N-15 kinetic isotope effects (C-13-AKIE and N-15-AKIE) of 1.044 +/- 0.003 and 1.0027 +/- 0.0004, respectively, reflecting the previously postulated nucleophilic aromatic substitution mechanism. Conversely, enzyme-catalyzed DNAN hydrolysis exhibited a C-13-AKIE of 1.027 +/- 0.005 and a N-15-AKIE of 1.0032 +/- 0.0003. On the basis of these AKIE values and the C and N isotope fractionation of DNP, our results imply that enzymatic O-demethylation of DNAN occurs through a nucleophilic substitution reaction at the aliphatic C of the methoxy group. This work provides a basis for the assessment of DNAN transformation by CSIA, as the C and N isotope fractionation patterns observed in this work are distinct from other hypothesized degradation pathways.