Preparation of deuterium-labeled biotransformation products of 2,4,6-trinitrotoluene

Article abstract of DOI:10.1002/jlcr.3040

Methods for the preparation of deuterium-labeled analogs to six prominent biotransformation products of the explosive 2,4,6-trinitrotoluene were developed. These are useful as reference standards for stable isotope dilution techniques and for solid state

Full text of DOI:10.1002/jlcr.3040

Research Article
Received 14 December 2012,
Accepted 07 February 2013
Published online 12 April 2013 in Wiley Online Library
(wileyonlinelibrary.com) DOI: 10.1002/jlcr.3040
Preparation of deuterium-labeled
biotransformation products of
2,4,6-trinitrotoluene
a
b
*
Thomas Junk and Jason A. Carr
Methods for the preparation of deuterium-labeled analogs to six prominent biotransformation products of the explosive
2,4,6-trinitrotoluene were developed. These are useful as reference standards for stable isotope dilution techniques and for solid
state 2H NMR spectroscopic studies. Although syntheses for most of the target compounds in protiated form had been reported
in the past, most of those were found to be poorly suited for the preparation of the deuterated materials. Selective reduction of
[2H5]trinitrotoluene furnished [2H5]-4,6-dinitro-2-hydroxylaminotoluene, [2H5]-2,6-dinitro-4-hydroxylaminotoluene, [2H5]-
2-amino-4,6-dinitrotoluene, and [2H5]-4-amino-2,6-dinitrotoluene. The syntheses of [2H10]-2,20-azo-4,40,6,60-tetranitrotoluene
and [2H10]-4,40-azo-2,20,6,60-tetranitrotoluene were accomplished by selective oxidation of [2H5]-2-amino-4,6-dinitrotoluene
and [2H5]-4-amino-2,6-dinitrotoluene, respectively.
Keywords: trinitrotoluene; explosives; biotransformation; degradation
contrast, no methods for the preparation of 4 or its nonlabeled
analog could be found in the literature.
Introduction
Contamination of soils with 2,4,6-trinitrotoluene (TNT) results in the
formation of transformation products including hydroxylamino-,
amino-, azo-, and azoxydinitrotoluenes, which constitute a
Experimental
significant environmental hazard and are confirmed or suspected
General
TNT degradation products.1–3 Samples of the deuterated analogs
All [2H5]-TNT required for the synthesis of transformation products were
shown in Figure 1 were required as reference materials for isotope
prepared from commercial (Sigma-Aldrich) [2H8]-toluene, IP >99%, by
dilution mass spectrometry (MS), as well as for the elucidation of
2
soil binding characteristics by solid state H NMR spectroscopy.4
stepwise nitration following a published procedure for the preparation
of nonlabeled TNT.11 MS analyses were performed on an Agilent
The preparation of the targeted products by reduction with baker’s
Technologies ESI TOF 6210 mass spectrometer using a capillary voltage
yeast in analogy to a procedure reported for 14C-labeled TNT
of 4200 V and nitrogen drying gas at 325ꢀ at 8 L/min. The fragmentor
metabolites5 was considered but found to be less suitable for the
preparation of deuterated products than targeted synthesis due
to its tendency to form complex mixtures.
voltage was set to 150 V. The sample was delivered by the flow through
method by an Agilent 1200 HPLC using a mobile phase of acetonitrile
and water (90:10) with 0.1% formic acid at a flow rate of 0.4 mL/min.
Nuclear magnetic resonance spectra were recorded on a Jeol Eclipse
300 MHz spectrometer. Sigma-Aldrich brand basic and acidic alumina,
Brockmann, 150 mesh were used for column chromatography. All other
chemicals employed in this study were purchased from Sigma-Aldrich
and used as received. Melting points were taken on a Mel-Temp II.
Protiated analogs of 3, 5, 6, and 7 used as reference materials were
prepared according to published procedures.12 Products were char-
acterized by 1H NMR spectroscopy (to verify deuteration at >95%), 13C NMR
spectroscopy, MS, and comparison (thin layer chromatography, mp) to
authentic protiated analogs.
Methods for the preparation of 4-hydroxylamino-2,6-
dinitrotoluene 5 and 4-amino-2,6-dinitrotoluene 6 described
in the literature6–8 could be adapted for the synthesis of the
deuterated analogs after some modification. In contrast, an
adaptation of the synthesis of 2-amino-4,6-dinitrotoluene 3 from
2-methyl-3,5-dinitrobenzoic acid by Hoffmann degradation2 was
considered impractical, because of the extensive synthetic effort
that would be required to obtain the precursor acid in labeled
form. The selective ortho reduction of TNT has been reported,7,9
but the published procedures were not found to be satisfactory
for the small-scale preparation of high purity reference material.
Likewise, the only published preparation of 2-hydroxylamino-4,
6-dinitrotoluene 210 was regarded as unsuitable for the deuterated
analog, because of the complexity of the reported procedure
aDepartment of Chemistry, University of Louisiana at Lafayette, Lafayette
LA 70504, USA
and its low yield. Consequently, novel methods for the synthesis bDepartment of Chemistry, Nazarbayev University, School of Science and
of 2, 3, 5, and 6 by selective ortho reduction of [2H5]-TNT 1 were
Technology, Astana 010000, Kazakhstan
developed. The synthesis of the nonlabeled analog of 7 in low
*Correspondences to: Thomas Junk, Department of Chemistry, University of
Louisiana at Lafayette, Lafayette, LA 70504, USA.
yield by partial electrolytic reduction of 1 has been reported8
and a laborious procedure for its purification described.2 In E-mail: txj9137@louisiana.edu
J. Label Compd. Radiopharm 2013, 56 344–346
Copyright © 2013 John Wiley & Sons, Ltd.

T. Junk and J. A. Carr
[2H5]-2,6-Dinitro-4-hydroxylaminotoluene 5 and [2H5]-4-amino-2,6-
dinitrotoluene 6
A total of 10 g (44 mmol) of 1, dissolved in 20 mL dioxane, was amended
with 0.2 mL concentrated aqueous ammonia and the mixture saturated
with gaseous hydrogen sulfide. After a 1- to 5-min initiation period, the
mixture warmed spontaneously, and sulfur began to precipitate. No
attempts were made to moderate the reaction, which was considered
complete when hydrogen sulfide was no longer absorbed (10–15 min).
Sulfur was removed by filtration and dioxane by rotary evaporation.
The remaining material was slurred with 10 mL dichloromethane, and
solids were collected by filtration (total yield, 10.5 g). A total of 1 g
batches of this mixture was chromatographed on basic alumina.
Elution of 6 with dichloromethane–acetonitrile 10:1 v/v was followed
by elution of 5 with acetonitrile–ethanol (1:1). The combined fractions
of 5 were taken to dryness and recrystallized from approximately
50 mL hot toluene. Orange crystals, 2.6 g (30%), mp 171–172ꢀ;
literature, protiated analog: 172ꢀ.13 (EMI) m/z: 219.0769 [M + H].
Compound 6 was purified by crystallization from ethanol–water 1:1 v/v,
followed by crystallization from toluene. Yellow needles, 2.7 g (28%);
mp 145–146ꢀ; literature, protiated analog: 135–136ꢀ, 141ꢀ, 144–146ꢀ.2,6,14
MS (EMI) m/z: 203.0826 [M+ H].
Figure 1. Deuterated 2,4,6-trinitrotoluene biotransformation products referenced
in this study.
[2H5]-4,6-Dinitro-2-hydroxylaminotoluene 2
A total of 10 g (43 mmol) of 1 was dissolved in 15 mL dioxane. This
solution was added to 11.0 g (72 mmol) of stannous chloride dihydrate
suspended in 200 mL absolute ethanol. The mixture warmed
spontaneously and was set aside for 5 h. All ethanol was then removed
by rotary evaporation. The mixture was added to 200 mL water and the
product extracted with 2 ꢁ 100 mL dichloromethane. The organic phase
was dried over sodium sulfate, and solvents were removed by rotary
evaporation. Crude 2 was taken up in 80 mL absolute ethanol, leaving
an oily residue behind. The solvent was removed, and the remaining,
partially solidified material was treated with 50 mL dichloromethane–
hexane 1:1 v/v. The resulting solid was collected on a Buchner funnel,
washed with approximately 5 mL dichloromethane, and recrystallized
from toluene and then from acetic acid–water 1:10 v/v. Pale yellow
needles, yield: 1.9 g (20%); mp 126–128ꢀ. MS (EMI) m/z: 219.0773 [M + H].
[2H10]-2,20-Azo-4,40,6,60-tetranitrotoluene 4 and isomer 7
The respective amines 3 or 6 (1.01 g, 5 mmol) were dissolved in 50 mL
dichloromethane, stirred, and amended with an equimolar amount of
iodobenzene diacetate. The reaction mixtures gradually turned orange
and were set aside for 6 h, and volatiles were then allowed to evaporate
under a hood in an open beaker. The remaining, partially crystallized
products were purified by flash chromatography (acidic alumina,
dichloromethane) and subsequently recrystallized from approximately
5 mL hot toluene. Compound 4, orange crystals, 0.24 g (24%); mp
244–246ꢀ. (EMI) m/z: 401.1256 [M + H]. Compound 7, orange crystals,
0.81 g (81%); mp 267–268ꢀ; literature: 248–250ꢀ, 266–268ꢀ.2,8 MS (EMI) m/z:
401.1260 [M + H].
[2H5]-2-Amino-4,6-dinitrotoluene 3
A total of 10 g (44 mmol) of 1 was dissolved in 150 mL dioxane. The
mixture was placed in an ice bath and saturated with hydrogen chloride
gas. To the continuously stirred and chilled mixture, 30.0 g stannous
chloride dihydrate (196 mmol) was added in portions of approximately
3 g. The reaction mixture was stirred at ambient temperature for exactly
30 min and then added to 500 g crushed ice and 500 mL water. The
hydrochloride of 3 hydrolyzes under these conditions, and a mixture of
3 and unreacted 1 was collected by filtration. For further purification,
crude 3 was dissolved in 250 mL dichloromethane, precipitated as the
hydrochloride salt by saturation with hydrogen chloride gas, and
collected by filtration. Unreacted 1 remained in solution and was
collected by rotary evaporation for further use (approximately 1.5 g).
The collected hydrochloride was suspended in 50 mL water, and sodium
bicarbonate was added until pH 7 was reached. For further purification, 3
was recrystallized from benzene. Yellow needles, yield: 1.8g (24% based on
consumed 1); mp 173–174ꢀ; literature, protiated analog: 155ꢀ, 173–174ꢀ.13,2
MS (EMI) m/z: 203.0826 [M + H].
Results and discussion
It was found that the preparation of [2H5]-TNT 1 from commer-
cial [2H8]-toluene by stepwise nitration following a published
procedure11 could be carried out in protic media. By comparison
with a single batch of [2H5]-TNT prepared in perdeuterated
media, it was found that no loss of isotopic purity occurred,
greatly reducing the cost of the nitration procedure. Compounds
2 and 3 are accessible by selective ortho reduction of 1 with
stannous chloride in empirically optimized molar ratios. Highly
acidic reaction conditions were chosen for this step to assure
the immediate formation of the hydrochloride salts of the
Table 1. 13C NMR chemical shifts of deuterated 2,4,6-trinitrotoluene transformation products in [2H6]-DMSO
Compound
13C chemical shifts, ppm
[2H5]-4,4,6-Trinitrotoluene 1
15.0a, 122.2b, 134.2, 145.7, 151.6
[2H5]-2-Hydroxylamino-4,6-dinitrotoluene 2
[2H5]-2-Amino-4,6-dinitrotoluene 3
12.4a, 107.9b, 122.6, 146.6, 150.5, 151.5, 152.8
12.9a, 105.1b, 110.1b, 121.0, 146.2, 150.3, 151.4
13.5a, 114.8b, 121.7, 139.2, 146.8, 151.6, 151.9
13.4a, 111.0b, 114.7, 151.5, 151.8
[2H10]-2,20-Azo-4,40,6,60-tetranitrotoluene 4c
[2H5]-4-Hydroxylamino-2,6-dinitrotoluene 5
[2H5]-4-Amino-2,6-dinitrotoluene 6
13.6a, 110.7, 111.5b, 148.6, 152.1
[2H10]-4,40-Azo-2,20,6,60-tetranitrotoluene 7c
14.5a, 121.7b, 130.1, 150.0, 152.3
aPeak appears as septet.
bPeak appears as triplet.
cSample heated to 100ꢀ during acquisition to increase solubility.
J. Label Compd. Radiopharm 2013, 56 344–346
Copyright © 2013 John Wiley & Sons, Ltd.
www.jlcr.org

T. Junk and J. A. Carr
mono-reduction products, which resisted further reduction. It is
noteworthy that numerous attempts to modify the procedure
used for the preparation of 3 to improve its yield resulted in
Acknowledgements
We gratefully acknowledge the late Dr. W. James Catallo for his
drastically reduced, rather than increased, yields. Oxidation of support and advice during the early stages of this project.
Furthermore, we thank Ms. L. Dana Civils and Dr. F. R. Fronczek
for their assistance during the synthesis and characterization of
TNT transformation products.
3 to 4 and 6 to 7 represented a synthetic challenge, as 3 and
6 are readily oxidized to the corresponding azoxy compounds
if conditions are not chosen carefully. Trials employing lead
tetraacetate, m-chloroperbenzoic acid, and peroxide-based
oxidizers under varying conditions did not produce the desired
results. In contrast, treatment of 3 and 6 with hypervalent
iodobenzene diacetate resulted in the relatively clean forma-
tion of 4 and 7. Conflicting information exists about the
Conflict of Interest
The authors did not report any conflict of interest.
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J. Label Compd. Radiopharm 2013, 56 344–346