Continuous synthesis of aminophenols from nitroaromatic compounds by combination of metal and biocatalyst

Article abstract of DOI:10.1039/b413519a

The combined action of immobilized hydroxylaminobenzene mutase and zinc in a flow-through system catalyzes the conversion of nitroaromatic compounds to the corresponding ortho-aminophenols, including a novel analog of chloramphenicol.

Full text of DOI:10.1039/b413519a

View Article Online / Journal Homepage / Table of Contents for this issue
COMMUNICATION
www.rsc.org/chemcomm | ChemComm
Continuous synthesis of aminophenols from nitroaromatic compounds
by combination of metal and biocatalyst{
Heather R. Luckarift, Lloyd J. Nadeau and Jim C. Spain*
Received (in Cambridge, MA, USA) 2nd September 2004, Accepted 4th October 2004
First published as an Advance Article on the web 29th November 2004
DOI: 10.1039/b413519a
nitrobenzene (1 mM) was pumped through the two columns at a
flow rate of 0.25 ml min21, ortho-aminophenol (0.89 ¡ 0.095 mM)
was produced continuously for over 5 h (conversion efficiency of
89 ¡ 1.45%). Small quantities of HAB (66 ¡ 22 mM) and aniline
(97 ¡ 13 mM) (byproduct of the initial zinc reaction) were detected
throughout (Fig. 1). When the flow rate and substrate concentra-
tion were increased to 0.5 ml min21 and 5 mM, 3.56 mM (¡0.24)
ortho-aminophenol was produced for over 8 h with no loss in
activity indicating a conversion efficiency of 71.2% (¡5.83). HAB
(0.89 ¡ 0.31 mM), aniline (0.37 ¡ 0.07 mM) and nitrosobenzene
(0.25 ¡ 0.024 mM) were detected in the effluent, which suggested
that the capacity of the mutase column was exceeded at the higher
flow rate.
The combined action of immobilized hydroxylaminobenzene
mutase and zinc in a flow-through system catalyzes the
conversion of nitroaromatic compounds to the correspond-
ing ortho-aminophenols, including
a
novel analog of
chloramphenicol.
Aminophenols are important precursors for the synthesis of high
performance polymers and biologically active compounds but
applications are limited by the complexity and low yields of
synthetic routes.1 With nitrobenzene as a model substrate we
describe a continuous reaction system that consists of an initial
reduction of nitrobenzene to hydroxylaminobenzene using zinc,
followed by enzymatic conversion to ortho-aminophenol.
In Pseudomonas pseudoalcaligenes JS45, nitrobenzene is reduced
to hydroxylaminobenzene (HAB) by a nitrobenzene reductase
enzyme. A second enzyme, HAB mutase (EC 5.4.4.1), then
catalyzes the rearrangement of HAB to ortho-aminophenol which
is biodegraded to support growth of the bacteria.2 Pseudomonas
pseudoalcaligenes JS45 encodes two distinct enzymes, HAB mutase
A and HAB mutase B.2b The activity of nitrobenzene reductase
and HAB mutase in concert can be exploited to catalyze the
conversion of a range of nitroaromatic compounds to yield novel
ortho-aminophenols,1b,c but the use of whole cell systems is limited
by product toxicity, poor substrate uptake rates and the substrate
specificity of the enzymes. The use of nitrobenzene nitroreductase
is limited by the requirement for the cofactor, NADPH. In
addition, it is difficult to recover enzymes following catalysis.
Nitroaromatic compounds can alternatively be reduced to the
corresponding hydroxylamines by zinc.3 The hydroxylamine can
then be enzymatically rearranged to ortho-aminophenol by the
HAB mutase.2b The activities of enzymes and metal catalysts are
often optimal under dissimilar conditions, which limits their use in
a single reaction system. The use of combined metal catalysts and
biocatalysts however, has recently started to receive attention and
a few reports demonstrate the applicability of the approach.4
We recently developed a method of enzyme immobilization in
biomimetically-derived silica (biosilica) that increases the mechan-
ical stability of the immobilized enzyme and facilitates their
application in flow-through reactor systems.5 Here we immobilized
HAB mutase B in biosilica by the same method.5 A column
containing immobilized mutase enzyme was connected in series to
a column containing zinc (Fig. 1). When an aqueous solution of
{ Electronic Supplementary Information (ESI) available: S1: Purification
stategy for mutase enzymes HabA and HabB; S2: 1H and 13C NMR
structural data for aminophenol analog of chloramphenicol. See http://
www.rsc.org/suppdata/cc/b4/b413519a/
Fig. 1 Transformation of nitrobenzene (1 mM) to ortho-aminophenol by
a sequential zinc-reduction and mutase-catalyzed reaction system.
*jim.spain@tyndall.af.mil
This journal is ß The Royal Society of Chemistry 2005
Chem. Commun., 2005, 383–384 | 383

View Article Online
When we investigated the efficiency of the mutase column alone
with a feed of 1 mM HAB, the substrate was unstable due to auto-
oxidation, which made quantification of the conversion efficiency
difficult. One of the advantages to a continuous reaction system
is the rapid conversion of the unstable HAB intermediate into
ortho-aminophenol.
(1.04 ¡ 0.029 mM) was obtained continuously for 24 h,
demonstrating 100% conversion efficiency and a product forma-
tion rate of 0.24 mg h21 mg21 total protein. The zinc becomes
oxidized over time. When the column was repacked with new zinc,
the system could be continued for a further 24 h.
The flow-through system described can be applied to the
transformation of a wide variety of nitroarene substrates into the
corresponding aminophenolic products while bypassing many of
the current limitations of whole cell biocatalysis. The transforma-
tion of antibiotics with nitro functional groups to the correspond-
ing aminophenols may provide a simple method for synthesising
novel antibiotic analogs. The high efficiency and regiospecificity of
the reported system provides an attractive alternative to conven-
tional chemical synthesis. In addition, the immobilized enzyme can
be recovered and subsequently reused.{
The biosynthesis of antibiotics using intact bacterial cells is
inherently limited due to the biocidal properties of the product. An
immobilized enzyme reaction system therefore provides an
attractive alternative. The use of the zinc/mutase cascade was
therefore investigated with the antibiotic chloramphenicol (N-[2-
hydroxy-1-(hydroxymethyl)-2-(4-nitrophenyl) ethyl]-2,2-dichloro-
acetamide) as a model system (Fig. 2). Analogs of chloramphenicol
that lack the nitro substituent have been investigated,6 but the
formation of an aminophenol analog has not been reported.
Preliminary investigation demonstrated that the nitro group of
chloramphenicol was reduced to a hydroxylamino derivative by
reaction with zinc and the identity of the product was confirmed
by LC–MS analysis (data not shown). When the hydroxylamino
derivative of chloramphenicol was incubated with partially purified
HAB mutase B, the product was not converted to the expected
aminophenol. HAB mutase A, however, converted the hydro-
xylamino derivative to the corresponding aminophenol (N-[2-(4-
amino-3-hydroxy-phenyl)-2-hydroxy-1-hydroxy methyl-ethyl]-2,
2-dichloro-acetamide). This was the first observation of a
difference in substrate specificity between the two enzymes. The
aminophenol was purified from this reaction by HPLC and
characterised by LC–MS (molecular ion, m/z 308) and NMR.{
The success of the batch reaction with chloramphenicol led us to
investigate the synthesis of the product using the continuous zinc/
mutase column system as described above for the nitrobenzene
model system. When an aqueous solution of chloramphenicol
(1 mM) was pumped through the two columns at a flow rate
of 0.25 ml min21, the corresponding aminophenol analog
This work was funded by the US Air Force Office of Scientific
Research. HRL was supported by a postdoctoral fellowship from
the Oak Ridge Institute for Science and Education (US
Department of Energy).
Heather R. Luckarift, Lloyd J. Nadeau and Jim C. Spain*
Air Force Research Laboratory, 139 Barnes Drive, Suite #2, Tyndall
AFB, FL, 32403-5323, USA. E-mail: jim.spain@tyndall.af.mil;
Fax: 850 283 6090; Tel: 850 2836058
Notes and references
{ Biosilica immobilization of partially purified mutase enzyme ({) was
performed as described previously.5 Mutase enzyme activity was deter-
mined as described previously.2b For continuous flow experiments, columns
(XK-16/20, Pharmacia Biotech) were packed with (a) zinc (5 g, 40 mesh)
(total 1 ml volume), (b) immobilized mutase from a 5 ml reaction mixture
(containing approx. 10 mg protein) and 5 g of glass beads (60/80 mesh,
Alltech) (total 10 ml volume). Substrate was pumped through the system at
a fixed flow rate and the eluate collected for analysis. The entire apparatus
was maintained at 30 uC. Substrates were dissolved in water containing
NH4Cl (40 mM) and sparged with argon to ensure an anaerobic
environment. The pH of the reaction remained at approximately pH 7.4
throughout. Reactants and products of nitrobenzene conversion were
monitored by reverse-phase HPLC on a Supelco ABZ column with an
acetonitrile/water (+0.1% trifluoroacetic acid) gradient. Reactants and
products of chloramphenicol transformation were resolved by ion pair
chromatography on a Luna C18 column (150 6 2 mm, Phenomenex) with
an acetonitrile/triethylamine (10 mM, adjusted to pH 5 with acetic acid)
gradient.
1 (a) M. Keitmann, R. Sezi and A. Weber, US Patent 6 320 081; (b)
L. J. Nadeau, Z. He and J. C. Spain, J. Ind. Micro. Biotech., 2000, 24,
301; (c) V. Kadiyala, L. J. Nadeau and J. C. Spain, Appl. Environ.
Micro., 2003, 69, 11, 6520.
2 (a) S. F. Nishino and J. C. Spain, Appl. Environ. Micro., 1993, 59, 8,
2520; (b) J. K. Davis, G. C. Paoli, Z. He, L. J. Nadeau, C. C. Somerville
and J. C. Spain, Appl. Environ. Micro., 2000, 66, 7, 2965.
3 B. S. Furniss, A. J. Hannaford, P. W. G. Smith and A. R. Tatchell,
Vogel’s Textbook of Practical Organic Chemistry, 1989, Longman
Scientific and Technical and John Wiley & Sons, New York.
4 F. Gelman, J. Blum and D. Avnir, J. Am. Chem. Soc., 2002, 124, 48,
14460; O. Pamies and J. E. Backvall, Curr. Opin. Biotech., 2003, 14, 407.
5 H. R. Luckarift, J. C. Spain, R. R. Naik and M. O. Stone, Nat. Biotech.,
2004, 22, 2, 211; R. R. Naik, M. M. Tomczak, H. R. Luckarift,
J. C. Spain and M. O. Stone, Chem. Commun., 2004, 1684.
6 M. D. Corbett and B. R. Chipko, Antimicrob. Agents Chemother., 1978,
13, 2, 193; T. Izard, Protein Sci., 2001, 10, 1508.
Fig. 2 Structure of chloramphenicol (A) and an aminophenol analog (B).
384 | Chem. Commun., 2005, 383–384
This journal is ß The Royal Society of Chemistry 2005