Conversion of 2-(4-carboxyphenyl)-6-nitrobenzothiazole to 4-(6-amino-5-hydroxybenzothiazol-2-yl)benzoic acid by a recombinant E. coli strain

Article abstract of DOI:10.1039/b516032d

E. coli C43(DE3)pNbzAHabA, expressing nitroreductase and mutase enzymes, converts 2-(4-carboxyphenyl)-6-nitrobenzothiazole to 4-(6-amino-5- hydroxybenzothiazol-2-yl)benzoic acid. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b516032d

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Conversion of 2-(4-carboxyphenyl)-6-nitrobenzothiazole to 4-(6-amino-
5-hydroxybenzothiazol-2-yl)benzoic acid by a recombinant E. coli
strain{
Lloyd J. Nadeau,a Jim C. Spain,*b Ramamurthi Kannanc and Loon-Seng Tanc
Received (in Cambridge, MA, USA) 10th November 2005, Accepted 28th November 2005
First published as an Advance Article on the web 21st December 2005
DOI: 10.1039/b516032d
necessitated additional esterification/de-esterification steps in order
to rigorously establish the identity and purity of the target
nitrobenzothiazolecarboxylic acid. The detailed preparative proce-
dures for 7a–8b are provided as supplementary information.
Previously, we reported that nitroarenes are converted to
o-aminophenols by nitroreductase and mutase enzymes.5 The
nitroreductase reduces nitroaromatic compounds to hydroxyl-
aminoarenes6 and hydroxylaminobenzene mutase catalyzes a
regio-specific reaction converting hydroxylaminoarenes to the
corresponding o-aminophenols by an intramolecular transfer of
the hydroxyl group.7 Furthermore, an E. coli containing
nitroreductase and mutase converts simple nitroarenes regio-
specifically to o-aminophenols at high yields.8
E. coli C43(DE3)pNbzAHabA, expressing nitroreductase and
mutase enzymes, converts 2-(4-carboxyphenyl)-6-nitroben-
zothiazole to 4-(6-amino-5-hydroxybenzothiazol-2-yl)benzoic
acid.
4,6-Diaminoresorcinol (1) and 2,5-diamino-1,4-benzenedithiol (2)
(usually stored as dihydrochloride salts) are key co-monomers for
the synthesis of strong, thermally resistant rigid-rod poly(p-phe-
nylenebenzobisoxazole) (PBO) and poly(p-phenylenebenzobisthia-
zole) (PBT) polymers (Scheme 1) for lightweight structural,
nonlinear optical and electronic applications.1,2 More recently,
PBO has been considered for use in the proton-exchange
membranes of fuel cells.3 While an AB-monomer 4-[5-amino-6-
hydroxybenzoxazol-2-yl]benzoic acid (3) has been prepared and
utilized in the synthesis of the corresponding PBO polymer,4 the
analogous AB-monomer for PBT (4) has not been synthesized. In
addition to having an intrinsically perfect stoichiometry that helps
to promote high molecular weight polymers in polycondensation
processes, AB-monomers are also useful starting materials for the
synthesis of AB diblock copolymers, ABA triblock copolymers
and star polymers. They can also be grafted onto appropriately
functionalized surfaces. The availability of the above aminophenol
deriviative is limited because the synthesis is complex and yields are
low. Recent advances in biologically converting aromatic nitro
compounds to the corresponding o-aminophenols suggest the
possibility of synthesizing novel AB-monomers such as 4-(6-
amino-5-hydroxybenzothiazol-2-yl)benzoic acid (5) and in turn,
the corresponding rigid-rod polymer that is a hybrid of both PBO
and PBT with respect to the chemical structure.
In preliminary experiments, cells of E. coli C43(DE3)-
pNbzAHabA (i.e. strain JS995) were grown and induced as
described previously.8 2-(4-Carboxyphenyl)-6-nitrobenzothiazole
7a (49 mM) was incubated with the cells and HPLC analysis of
the reaction mixture revealed a transformation rate for the parent
compound of 0.9 nmoles min21 mg21 protein and the accumula-
tion of one product (Fig. 1). LC/MS (+APCI) analysis of the
product revealed a 287 m/z as expected of an aminophenol. The
conversion efficiency was 100%. The transformation was scaled up
Although the requisite starting nitro compounds (either as
carboxylic acid, 7a or ethyl ester 7b) are relatively simple
molecules, they have not been reported to our knowledge. In
principle, 5 could be synthesized in two steps: (i) a condensation
reaction between 2-aminothiophenol and 4-carboxybenzaldehyde,
followed by (ii) nitration of the resulting 2-(4-carboxyphenyl)ben-
zothiazole (7a). In practice, the poor solubility of 7a and 8a has
aAFRL/MLQL, Tyndall AFB, 139 Barnes Dr., Building 1117, FL 32403,
USA. E-mail: lloyd.nadeau@tyndall.af.mil; Fax: 850-283-6090;
Tel: 850-283-6018
bGeorgia Institute of Technology, 311 Ferst Dr., Atlanta,
GA 30332-0512, USA. E-mail: jspain@ce.gatech.edu;
Fax: 404-894-8266; Tel: 404-894-0628
cAFRL/MLBP, Wright-Patterson AFB, 2941 Hobson Way, B654 R309,
OH 45433-7750, USA. E-mail: loon-seng.tan@wpafb.af.mil;
Fax: 937-255-9157; Tel: 937-255-9141
{ Electronic supplementary information (ESI) available: experimental
section. See DOI: 10.1039/b516032d
Scheme 1 Chemical structures of pertinent monomers, polymers, sub-
strate and intermediates.
564 | Chem. Commun., 2006, 564–565
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Fig. 1 Transformation of 2-(4-carboxyphenyl)-6-nitrobenzothiazole
to 4-(6-amino-5-hydroxybenzothiazol-2-yl)benzoic acid by E. coli
C43(DE3)pNbzAHabA.
Fig. 2 Proton NMR spectrum of aminophenol product in DMSO-d6.
For clarity, only the aromatic proton region is shown.
to a 2 L bioreactor containing M9 medium9 (30 uC) supplemented
with sorbitol (20 g/L), ampicillin (100 mg/L) and isopropylthio-b-D-
galactoside (1 mM). Induced cells were added to the bioreactor
(A600 5 8.0) and 7a (5 mM) dissolved in NH4OH (2 N) at 65 uC
was delivered repeatedly to the reactor. The disappearance of
reactant and the accumulation of product were monitored by
HPLC. Over a 2 hour period, 200 mg of 7a were converted. The
cells were removed by centrifugation and the product was
precipitated by adjusting the pH of the supernatant to 2.7 with
HCl. Green crystals were recovered by centrifugation. The crystals
were dissolved in NH4OH, filtered and recrystallized by lowering
the pH to 2.7. The pelleted crystals were dried overnight under
vacuum, washed with water, and then with acetone. The melting
point was 326–328 uC.
The proton NMR spectrum (270 MHz) of the purified
compound in DMSO-d6 showed that in the aromatic proton
region, there were three singlet peaks at d (ppm)7.117, 7.309, and
8.012 at relative intensities of 1 : 1 : 4. The fact that only two
distinct singlets were observed from the two protons on the phenyl
ring with tetrasubstitution ruled out the isomeric structure 6 or
mixture of 5 and 6 (Fig. 2). The FT-IR (KBr) spectrum (ESI) is
consistent with the NMR data, indicating the presence of n(CLO)
of carboxylic acid at 1692 cm21 and a strong, broad band centered
at y3431 cm21 that is attributable to the hydroxyl-group
vibrations of the carboxylic acid and the phenol moieties. The
symmetrical and asymmetrical NH2 stretches, typically detected as
a doublet at y3400 and y3500 cm21 respectively, are most likely
hidden beneath the broad n(OH) band. Electron-impact mass
spectroscopy gave a molecular ion with m/z 5 285.96 (100%
relative abundance). Thus, all the available spectroscopic data
confirm the structure of the product as 4-(6-amino-5-hydroxyben-
zothiazol-2-yl)benzoic acid, 5.
conversions using traditional organic chemistry would be prohibi-
tively complex and expensive. We are currently exploring the
ability of the biocatalyst to transform a variety of other complex
molecules.
We thank Michael Matuszewski and Ann Dombroskie for their
assistance in the preparation of the nitrobenzothiazole-based
substrates, Glenn Johnson and Ion Ghiriviga for helpful discus-
sions, and the Air Force Office of Scientific Research for funding.
Notes and references
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The biocatalyst, E. coli C43(DE3)pNbzAHabA converts 2-(4-
carboxyphenyl)-6-nitrobenzothiazole to a potentially useful o-
aminophenolic synthon for the synthesis of novel polymers. Our
previous work indicated that the combination of the reductase and
mutase enzymes could catalyze the transformation of very simple
nitroaromatic compounds to the corresponding ortho-aminophe-
nols. The results presented here indicate that the biocatalyst can
transform more complex and potentially useful nitroaromatic
compounds stoichiometrically to the ortho-aminophenols. Such
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