Microbiotic synthesis of 14C-ringlabelled aminodinitrotoluenes (ADNT) and diaminonitrotoluenes (DANT)

Article abstract of DOI:10.1002/jlcr.556

The four ¹⁴C-ringlabelled TNT-metabolites 2-aminodinitrotoluene (2-ADNT), 4-aminodinitrotoluene (4-ADNT), 2,4-diaminonitrotoluene (2,4-DANT) and 2,6-diaminonitrotoluene (2,6-DANT) were synthesized in one step from TNT by reduction with baker's yeast (Saccharomyces cervisiae). Copyright

Full text of DOI:10.1002/jlcr.556

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2002; 45: 249–255.
DOI: 10.1002/jlcr.556
Research Article
14
Microbiotic synthesis of C-ringlabelled
aminodinitrotoluenes (ADNT) and
diaminonitrotoluenes (DANT)
.
Mario Kroger and Gregor Fels*
.
Universitat Paderborn, FB 13-Organische Chemie, Warburger Str.100,
D-33098 Paderborn, Germany
Summary
The four 14C-ringlabelled TNT-metabolites 2-aminodinitrotoluene (2-ADNT),
4-aminodinitrotoluene (4-ADNT), 2,4-diaminonitrotoluene (2,4-DANT) and
2,6-diaminonitrotoluene (2,6-DANT) were synthesized in one step from TNT
by reduction with baker’s yeast (Saccharomyces cervisiae). Copyright # 2002
John Wiley & Sons, Ltd.
Key Words: baker’s yeast; TNT; microbiotic 14C-synthesis; aminodinitroto-
luenes; diaminonitrotoluenes
Introduction
Extensive production of the explosive 2,4,6-trinitrotoluene (TNT) over
the last century, particularly during World War II, has caused severe
contamination of former TNT-manufacturing and –handling sites.1 The
toxic, cancerogenic and mutagenic properties of TNT as well as the
chemical persistency have turned it into a major environmental hazard
and the effective degradation of TNT is still a challenge for
environmental research.2
.
*Correspondence to: G. Fels, Universitat Paderborn, FB-13 Organische Chemie, Warburger Str.
100, D-33098 Paderborn, Germany. E-mail: gf@chemie.uni-paderborn.de
Copyright # 2002 John Wiley & Sons, Ltd.
Received 10 September 2001
Revised 20 November 2001
Accepted 21 November 2001

250
M. KROGER AND G. FELS
We are investigating the degradation of TNT in aqueous environ-
ments by chemical and biological processes.3,4 In order to determine the
mineralization of TNT and its metabolites we were in need of the
reduced, 14C-ring-labelled TNT-transformation products 2-aminodini-
trotoluene (2-ADNT), 4-aminodinitrotoluene (4-ADNT), 2,4-diamino-
nitrotoluene (2,4-DANT) and 2,6-diaminonitrotoluene (2,6-DANT).
These compounds occur as natural TNT metabolites and their
reactivity results in a higher toxicity as compared to TNT itself.2 On the
other hand, the compounds are more susceptible to degradation reactions,
and there is a growing interest in their role as possible intermediates in the
conversion of TNT into commercially interesting derivatives.5,6
So far, 4-ADNT and 2,4-DANT were prepared by way of reduction
by hydrogen sulfide in the presence of catalytic amounts of ammonia as
reducing agent. Using dioxane as solvent this has led to a high
regioselectivity for para-reduction (99:1).7 Until recently, 2,6-DANT
could only be obtained via a six-step synthesis starting from 2,6-
dichlorotoluene.8 A new procedure now allows us to selectively reduce
TNT at the ortho-nitro group by iron powder in acetic acid. Thus
2-ADNT and 2,6-DANT are now available by direct reduction of TNT.6
Considering our demand for all four ADNT- and DANT-isomers, a
separate synthesis for each compound would have been inefficient.
Hence we looked for an alternative method to obtain all four
compounds in one step.
Many microorganisms are known to reduce TNT, which has already
been used to prepare 14C-labelled aminodinitrotoluenes on a microscale
level.9 In addition baker’s yeast was successfully employed to convert
dinitronbenzenes into aminonitro-benzenes on a preparative scale.10
Baker’s yeast is advantageous for such reactions as it is cheap, readily
available, easy to handle and quite versatile. Furthermore it is not toxic
and as a whole cell system no cofactors have to be added.11
All these facts encouraged us to investigate the reaction of TNT with
baker’s yeast, which eventually yielded a procedure for the preparation
of all four desired ring-labelled aminoaryl compounds in a single step
staring from 14C-TNT and yeast.
Results and discussion
The reduction of nitroaromatic to arylamine compounds is a six-
electron reduction sequence which includes two-electron steps via a
Copyright # 2002 John Wiley & Sons, Ltd.
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MICROBIOTIC SYNTHESIS OF 14C-RINGLABELLED
251
nitroso and a hydroxylamino intermediate. HPLC analysis of the
reaction products showed that baker’s yeast can convert TNT into the
diaminonitrotoluenes within 3–4 days. Since the nitroso intermediates
are unstable, the first observable intermediates are hydroxyl-aminoto-
luenes (OH-ADNTs) which are easily reduced and under oxidative
conditions are transferred into azoxy-dimers.
OH-ADNTs can be observed after a few hours, but these
intermediates are completely reduced to ADNTs after 3 days. Finally
the diaminonitrotoluenes build up without any formation of detectable
intermediates (see Schemes 1 and 2).
Scheme 1. Reduction pathways from TNT to DANTs
Scheme 2. Progress of TNT reduction over a period of 3 days
Copyright # 2002 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2002; 45: 249–255

252
M. KROGER AND G. FELS
In order to arrive at the ADNTs and DANTs we monitored the
reaction to when all four compounds are present in approximately equal
amounts, while almost no TNT and OH-ADNT are remaining. If a
single batch of yeast did not produce enough DANT, the reaction was
extended by simply adding more yeast.
Separation of all four products was achieved by preparative TLC,
using a toluene: methanol mixture to separate amino from diamino
compounds followed by a second chromatography step with ethylace-
tate:hexane to isolate each of the regioisomers. Detection of the
products was facilitated by their colour. With a growing level of
reduction, the intensity of the colour increases: hydroxylaminodinitro
compounds are pale yellow, aminodinitrotoluenes are yellow and
diaminotoluenes are orange. All analytical data are listed in Table 3
in the experimental section. In the ‘hot’ run of the reaction the
separation was also documented by autoradiography.
The most crucial point regarding control and reproducibility of the
reaction turned out to be the regioselectivity of the reduction. Use of
different batches of baker’s yeast yielded different ratios of ortho and
para products (see Table 1).
Though in a few experiments (R21, R20) 2-ADNT exceeded 4-
ADNT, usually para-reduction was favoured over ortho. Similar results
with an ortho:para ratio of 1:3 were found for an electrochemical
reduction of TNT by a Russian group.12 The excess of 2,4-DANT over
2,6-DANT is expected, as further reduction of 4-ADNT exclusively
yields 2,4-DANT, while reduction of 2-ADNT leads to both regio-
isomers (Scheme 1). Based on the data in Table 1, batch 37 was most
appropriate for our needs and chosen for the radioactive synthesis.
Employing recrystallized 14C-TNT synthesized earlier in our labora-
tory13 we obtained the desired radioactively labelled four amino-
derivatives with yields as given in Table 2.
Table 1. Yields (%) from selected batches
Batch no.
37
32
30
22
21
20
16
13
4-ADNT
2-ADNT
2,4-DANT
2,6-DANT
17.3
10.2
28.7
4.8
37.6
1.5
28.7
9.6
26.4
8.1
21.6
13.2
42.8
2.9
6.2
1.5
16.1
20.3
10.8
15.6
13.2
23.4
15.6
9.6
20.3
9.2
30.0
11.0
16.3
10.2
19.2
12.0
Total
61
77
69
53
63
62
70
57
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MICROBIOTIC SYNTHESIS OF 14C-RINGLABELLED
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Table 2. Results of the radiolabelled synthesis
Compound
Yield (%)
Radioactivity (MBq)
Radioactivity (mCi)
4-ADNT
2-ADNT
2,4-DANT
2,6-DANT
25
6
19
2
148
40
114
11
4.0
1.1
3.1
0.2
Total
52
512
8.4
Chemical and radiochemical purities of the separated compounds
were >95% as determined by HPLC.
Experimental
HPLC analysis was performed with a water–methanol gradient from
55:45 to 100% methanol on a Nucleosil RP18 column and Merck-
Hitachi equipment (655A pump, L-5000 controller, L-3000 diode array
detector). Before injection each sample was neutralized and extracted
with CH2Cl2.
NMR measurements were performed in CDCl3 on a Bruker 200 MHz
spectrometer.
Synthesis of 14C-2-ADNT, 14C-4-ADNT, 14C-2-4-DANT and 14C-2,
6-DANT
Commercially available baker’s yeast (6 g, RuF Lebensmittelwerk,
batch JAN 019303) was suspended in tap water (40 ml) and stirred
carefully at 328C. To start the reaction [ring-14C]-TNT (0.5 mmol,
114 mg)13 in hot ethanol (1.6 ml) was quickly added. The course of the
reaction was controlled by HPLC. To obtain an equal ratio of ADNT
to DANT another 1.5 g of yeast was added on day four. The reaction
was stopped after 5 days. Extraction with dichloromethane (250 ml)
yielded 119 mg of raw material. The four products were applied to two
preparative TLC plates (Macherey-Nagel, SIL G-200) and separated by
consecutive use of a toluene:methanol 9:1 and a hexane:ethylacetate 1:1
solvent mixture, respectively (for Rf -values see Table 3). Products were
detected via their color and by autoradiography. For the latter a
photographicfilm was applied to the TLC plates for 1 min and
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J Label Compd Radiopharm 2002; 45: 249–255

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M. KROGER AND G. FELS
Table 3. Analytical data
Compound
Colour
Rt (HPLC)
Rf
NMR: chemical shifts
(min)
(TLC) Ar-H
Ar-H
8.85
CH3
2.69
NH2
TNT
}(UV)
23.3
21.1
21.1
26.3
25.5
5.7
0.90
0.54
0.54
0.60
0.66
0.32
0.39
8.85
2-OH-ADNT Pale yellow
4-OH-ADNT Pale yellow
2-ADNT
4-ADNT
2,4-DANT
2,6-DANT
Yellow
Yellow
Orange
Orange
8.03
7.28
6.56
6.96
7.72
7.28
6.23
6.96
2.36
2.44
2.16
2.00
4.21
4.22
3.79
5.17
5.1
developed as usual. The products were eluted from silica with
ethylacetate and the purity of each product was determined by HPLC.
Conclusion
The reduction of TNT with baker’s yeast is a new and effective synthetic
approach towards the aminodinitro- and diaminonitrotoluenes. All four
compounds can be obtained in satisfactory yields. Together with 14C-
TNT13 the 14C-labelled TNT metabolites will now be employed in
combined biological and photochemical degradation experiments, a
procedure that has been shown to represent a very effective degradation
procedure for nitroaromatic compounds.14,15 The aim of this investiga-
tion is to study the synergistic effects of such a combined technique and
to quantify the transformation products with particular emphasis on the
mineralization of starting material.
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