4- formamido-2,6-dinitrotoluene

Since ligninolytic activity was required for extensive mineralization of TNT, the accumulation of substrates of the ligninolytic enzymes originating from aminodinitrotoluenes was expected under non-ligninolytic conditions. Nitrogen-sufficient (12 mM ammonium tartrate) agitated cultures were incubated with 4-amino-2,6-dini-trotoluene for 48 h, and a transformation product was extracted, purified by preparative thin layer chromatography and analyzed by mass spectroscopy. By comparison with chemically synthesized reference compounds it was identified as 4-formamido-2,6-dinitrotoluene. 

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. 

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.

Figure 6. Further degradation of the formylated intermediates in TNT breakdown by non-ligninolytic (A) or ligninolytic cultures (B) of P. chrysosporium. Shallow stationary cultures (7 ml) were incubated for 6 days at 39°C in 100 ml serum bottles. The intermediates were added to separate cultures to a final concentration of 20 pg/ml. 4-Amino-2,6-dinitrotoluene, 4-formamido-2,6-dinitrotoluene, A 2-amino-4-formamido-6-nitro-toluene, .

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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See also in sourсe #XX -- [ , , ]

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