2-amino-4-formamido-6-nitrotoluene

The experiment was then repeated with chemically synthesized 4-formamido-2,6-dinitrotoluene instead of 4-amino-2,6-dinitrotoluene. The reduction product isolated was identified as 2-amino-4-formamido-6-nitrotoluene. Using this compound as substrate, very slow formation of 2,4-diamino-6-nitrotoluene (detectable only after 96 h of incubation) could be observed. The sequence of reactions that follows 4-amino-2,6-dinitrotoluene is depicted in Figure 5. We could not obtain any evidence for a direct reduction of 4-amino-2,6-dinitrotoluene to 2,4-diamino-6-nitrotoluene. 

Our next goal was to find out which compound serves as the substrate for the ligninolytic system. It is apparent from Figure 6B that 2-amino-4-formamido-6-nitrotoluene disappeared rapidly in ligninolytic cultures, but not in non-ligninolytic cultures (Fig. 6A). 

Figure 5. Reduction of 4-amino-2,6-dinitrotoluene (4Am-DNT) to 2,4-diamino-6-nitrotoluene (2,4DAm-NT) by non-ligninolytic cultures of R chrysosporium. The formylation of the amino group of 4Am-DNT led to 4-formamido-2,6-dinitrotoluene (4FAm-DNT). The latter is reduced to 2-amino-4-formamido-6-nitrotoluene (2Am4FAm-NT) which is subsequently deformylated.

Therefore, we assume that Stahl and Aust (45) detected the diamino-derivative only in non-ligninolytic cultures because its precursor 2-amino-4-formamido-6-nitrotoluene is preferentially oxidized by the ligninolytic enzymes. The purified lignin peroxidase isozyme H8 was used to confirm that 2-amino-4-formamido-6-nitrotoluene serves as a substrate for this enzyme. In contrast to the oxidation of hydroxylaminodinitrotoluene by the lignin peroxidase (6, 32) veratryl alcohol is not involved in this reaction. 

The identification of 2-amino-4-formamido-6-nitrotoluene as a substrate of the lignin peroxidase raises questions about the role of 2,4-diamino-6-nitrotoluene. Its formation from 2-amino-4-formamido-6-nitrotoluene under non-ligninolytic conditions can be expected, since acylation reactions (formylation and acetylation) of aromatic amino goups are clearly reversible (29). 

Research is now being conducted to identify the oxidation products of 2-amino-4-formamido-6-nitrotoluene. Also the metabolic fate of 2,4-diamino-6-nitrotoluene under non-ligninolytic conditions is of interest, since some non-ligninolytic mineralization of TNT by R chrysosporium was demonstrated (32, 44). 

Acetylation of 2-nitro-4-aminotoluene by Mucrosporium sp. was observed in the course of 2,4-dinitrotoluene degradation (31). The isomer 2-acetamido-4-nitrotoluene could not be detected as a transformation product. This indicates that acetylation of an aromatic amino group is affected by its position on the ring. In this case the methyl residue ortho to the amino group could hinder the acetylation of 2-amino-4-nitrotoluene. Since our attempts to synthesize an authentic standard of 2-formamido-4,6-dinitrotoluene have failed so far, we could not investigate a formylation reaction of 2-amino-4,6-nitro-toluene by the fungus. 

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