Bamberger rearrangement during TNT metabolism by Clostridium acetobutylicum

Studies were conducted to isolate and identify polar and oxygen-sensitive intermediates of 2,4,6-trinitrotoluene (TNT) transformation by Clostridium acetobutylicum. Studies conducted in anaerobic cell extracts demonstrated that a polar product formed from the transformation of 2,4-dihydroxylamino-6-nitrotoluene by a mechanism known as the Bamberger rearrangement. The product was stabilized by derivatization with acetic anhydride, and the structure was confirmed by mass spectroscopy, (1)H NMR, and IR spectroscopy techniques. The reaction occurred in the presence of cell extract and H(2) but did not occur in cell extract-free controls. From spectroscopic data, the product of 2,4-dihydroxylamino-6-nitrotoluene rearrangement was identified as either 2-amino-4-hydroxylamino-5-hydroxyl-6-nitrotoluene (4-amino-6-hydroxylamino-3-methyl-2-nitrophenol) or 2-hydroxylamino-4-amino-5-hydroxyl-6-nitrotoluene (6-amino-4-hydroxylamino-3-methyl-2-nitrophenol). Acid-catalyzed rearrangement of 2,4-dihydroxylamino-6-nitrotoluene resulted in a single product, which after derivatization, was identical to a derivatized product from cell extracts. Acid-catalyzed Bamberger rearrangement occurs with the hydroxyl addition para to the participating hydroxylamine, indicating that the 2-amino-4-hydroxylamino-5-hydroxyl-6-nitrotoluene (4-amino-6-hydroxylamino-3-methyl-2-nitrophenol) was the product isolated form cell extracts. This product was also confirmed in whole cell systems that had been fed TNT. Following derivatization of the culture broth, a product was isolated that was identical to hose isolated from crude cell extracts and acid catalysis experiments.