Enzymatic conversion of 6-nitroquinoline to the fluorophore 6-aminoquinoline selectively under hypoxic conditions

Article abstract of DOI:10.1021/tx300483z

There is substantial interest in small molecules that can be used to detect or kill the hypoxic (low oxygen) cells found in solid tumors. Nitroaryl moieties are useful components in the design of hypoxia-selective imaging agents and prodrugs because one-electron reductases can convert the nitroaryl group to nitroso, hydroxylamino, and amino metabolites selectively under low oxygen conditions. Here, we describe the in vitro, cell free metabolism of a pro-fluorescent substrate, 6-nitroquinoline (1) under both aerobic and hypoxic conditions. Both LC-MS and fluorescence spectroscopic analyses provided evidence that the one-electron reducing enzyme system, xanthine/xanthine oxidase, converted the nonfluorescent parent compound 1 to the known fluorophore 6-aminoquinoline (2) selectively under hypoxic conditions. The presumed intermediate in this reduction process, 6-hydroxylaminoquinoline (6), is fluorescent and can be efficiently converted by xanthine/xanthine oxidase to 2 only under hypoxic conditions. This finding provides evidence for multiple oxygen-sensitive steps in the enzymatic conversion of nitroaryl compounds to the corresponding amino derivatives. In a side reaction that is separate from the bioreductive metabolism of 1, xanthine oxidase converted 1 to 6-nitroquinolin-2(1H)-one (5). These studies may enable the use of 1 as a fluorescent substrate for the detection and profiling of one-electron reductases in cell culture or biopsy samples. In addition, the compound may find use as a fluorogenic probe for the detection of hypoxia in tumor models. The occurrence of side products such as 5 in the enzymatic bioreduction of 1 underscores the importance of metabolite identification in the characterization of hypoxia-selective probes and drugs that employ nitroaryl units as oxygen sensors.

Full text of DOI:10.1021/tx300483z

Article
pubs.acs.org/crt
Enzymatic Conversion of 6‑Nitroquinoline to the Fluorophore
6‑Aminoquinoline Selectively under Hypoxic Conditions
Anuruddha Rajapakse,† Collette Linder,∥ Ryan D. Morrison,§ Ujjal Sarkar,† Nathan D. Leigh,†
Charles L. Barnes,† J. Scott Daniels,§,∥ and Kent S. Gates*,†,‡
†Department of Chemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, United States
‡Department of Biochemistry, University of Missouri, 125 Chemistry Building, Columbia, Missouri 65211, United States
§Department of Pharmacology, Vanderbilt Center for Neuroscience Drug Discovery, Vanderbilt University Medical Center, Nashville,
Tennessee 37232, United States
∥Pfizer, Inc., 700 Chesterfield Parkway, Chesterfield, Missouri 63017, United States
ABSTRACT: There is substantial interest in small molecules that can be used to
detect or kill the hypoxic (low oxygen) cells found in solid tumors. Nitroaryl
moieties are useful components in the design of hypoxia-selective imaging agents
and prodrugs because one-electron reductases can convert the nitroaryl group to
nitroso, hydroxylamino, and amino metabolites selectively under low oxygen
conditions. Here, we describe the in vitro, cell free metabolism of a pro-fluorescent
substrate, 6-nitroquinoline (1) under both aerobic and hypoxic conditions. Both LC-MS and fluorescence spectroscopic analyses
provided evidence that the one-electron reducing enzyme system, xanthine/xanthine oxidase, converted the nonfluorescent
parent compound 1 to the known fluorophore 6-aminoquinoline (2) selectively under hypoxic conditions. The presumed
intermediate in this reduction process, 6-hydroxylaminoquinoline (6), is fluorescent and can be efficiently converted by
xanthine/xanthine oxidase to 2 only under hypoxic conditions. This finding provides evidence for multiple oxygen-sensitive steps
in the enzymatic conversion of nitroaryl compounds to the corresponding amino derivatives. In a side reaction that is separate
from the bioreductive metabolism of 1, xanthine oxidase converted 1 to 6-nitroquinolin-2(1H)-one (5). These studies may
enable the use of 1 as a fluorescent substrate for the detection and profiling of one-electron reductases in cell culture or biopsy
samples. In addition, the compound may find use as a fluorogenic probe for the detection of hypoxia in tumor models. The
occurrence of side products such as 5 in the enzymatic bioreduction of 1 underscores the importance of metabolite identification
in the characterization of hypoxia-selective probes and drugs that employ nitroaryl units as oxygen sensors.
Scheme 1. Enzymatic Reduction of Nitroaryl Compounds
INTRODUCTION
■
More than 70 years ago, it was suggested that poor
vascularization and high cell densities combine to create a
hypoxic (low oxygen) environment in tumors.1,2 It has now
been well established that many tumors contain substantial
regions of hypoxia and that these hypoxic cells play a significant
role in cancer biology.3−7 For example, the hypoxic environ-
ment may select for cells that are incapable of undergoing
apoptosis.6,8 In addition, there is evidence that the cancer stem
cells thought to be responsible for metastases are harbored in
the hypoxic niche of tumors.9−14 Therefore, it is not surprising
that tumor hypoxia correlates with poor patient prognosis.3,4,15
Accordingly, there is substantial interest in agents that detect or
kill the hypoxic cells found in tumor tissue.
Nitroaryl moieties are useful components in the design of
hypoxia-selective imaging agents and prodrugs because one-
electron reductases can convert nitroaryl compounds to nitroso,
hydroxylamino, and amino metabolites selectively under low
oxygen conditions (Scheme 1).16−25 In normal tissue,
molecular oxygen inhibits the enzymatic generation of reduced
metabolites via back-oxidation of radical anion intermediates
(reverse reactions, Scheme 1).21 It is generally assumed that the
initially generated nitro radical anion is the key oxygen-sensitive
intermediate that confers hypoxia selectivity to these metabolic
processes.19−21 Importantly, hypoxia-selective conversion of the
electron-withdrawing nitro group to the electron-donating
hydroxylamino or amino substituents constitutes an “electronic
switch” that has been exploited for the development of
anticancer drug candidates,26,27 radiochemical imaging agents,28
and immunohistochemical stains of hypoxic tissue.29
Fluorescent probes for the direct detection of cellular
hypoxia would provide a useful complement to existing
radiochemical and immunohistochemical imaging methods.
One strategy for the development of fluorogenic probes of
cellular hypoxia involves identification of nonfluorescent
Received: December 7, 2012
Published: March 14, 2013
© 2013 American Chemical Society
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Scheme 2
Scheme 3
solution diluted with water (2 mL) and extracted with ethyl acetate (2
× 10 mL). The combined organic extracts were washed with brine,
dried over sodium sulfate, and concentrated by rotary evaporation.
Column chromatography on silica gel eluted with ethyl acetate
followed by MeOH/CH2Cl2 gave 6 as a yellow solid (100 mg, 25%
yield, Rf = 0.1 in MeOH/CH2Cl2 4:96). This compound is unstable
nitroaromatic compounds that can be metabolized to a
fluorescent amine derivative.30−34 Molecules of this type
could also serve as fluorescent substrates for the detection
and profiling of the one-electron reductases involved in
bioactivation of hypoxia-selective drugs. Indeed, recent work
suggests that bioreductively activated, hypoxia-selective drugs
will benefit from the development of matching probe molecules
capable of offering surety that the necessary reductase enzymes
are expressed in the target tumor tissue.35,36 Therefore, we set
out to characterize the hypoxic metabolism of a pro-fluorescent
nitroaryl substrate, 6-nitroquinoline (1). Enzymatic metabolism
of this compound has the potential to generate the known
fluorophore, 6-aminoquinoline (2, Scheme 2). Compound 2
displays fluorescence emission at 530 nm and a remarkable 205
nm Stokes shift37 and has found use as a fluorescent reporter in
a number of applications.38−42 We examined the hypoxic, cell-
free, in vitro metabolism of 1 by the xanthine/xanthine oxidase
enzyme system. Xanthine oxidase is important in human drug
metabolism43,44 and has been employed for the in vitro one-
electron reduction of various hypoxia-selective com-
pounds.45−53 Here, we describe the hypoxia-selective con-
version of 1 to the fluorescent product 2 by the xanthine/
xanthine oxidase enzyme system. These results stand in
contrast to the previous finding that the hypoxic metabolism
of 1 by NADPH/cytochrome P450 reductase produced the
unexpected helicene compound, pyrido[3,2-f ]quinolino[6,5-
c]cinnoline 3-oxide 4 (Scheme 3).54
1
upon standing in organic solvents. H NMR (CD3OD, 300 MHz) δ
8.53 (d, J = 5.0 Hz, 1H), 8.07 (d, J = 8.0 Hz, 1H), 7.82 (m, 1H), 7.33
(m, 3H). 13C NMR (CD3OD, 75.5 MHz) δ 151.4, 147.6, 144.8, 136.7,
131.1, 129.2, 122.5, 121.0, 107.7; HRMS (ESI, M + H+) m/z calcd for
C9H9N2O 160.0715; found, 160.0707. The structure of this compound
was confirmed by single crystal X-ray crystallographic analysis
(Rajapakse, A., Barnes, C. L., and Gates, K. S., manuscript in
preparation).
Enzymatic Synthesis of 6-Nitroquinolin-2(1H)-one (5). The
compound 6-nitroquinoline (1, 250 mg, 1.32 mmol) was dissolved in
warm DMF (1 mL) and sprayed into warm water (300 mL at 70 °C)
with vigorous stirring. To this mixture, warm sodium phosphate (100
mL, pH 7.4, 500 mM, 70 °C) was added with stirring. After cooling to
∼40 °C, xanthine oxidase (100 μL, 0.005 U/mL) was added every 12
h over the course of 3 days. The mixture was extracted with ethyl
acetate (3 × 30 mL) and the combined organic layers extracted with
brine (3 × 30 mL), dried over sodium sulfate, and concentrated by
rotary evaporation. Column chromatography on silica gel eluted with
ethyl acetate gave 5 as a yellow solid (4 mg, 2% yield, Rf = 0.55, 1%
methanol/ethyl acetate). X-ray quality crystals were obtained by
dissolving the pure compound in a minimum amount of warm ethyl
1
acetate followed by slow evaporation over the course of 3 days. H
NMR (CD3OD, 500 MHz) δ 8.64 (d, J = 2.5 Hz, 1H), 8.36 (dd, J =
9.0 Hz, J = 2.5 Hz, 1H), 8.09 (d, J = 9.0 Hz, 1H), 7.46 (d, J = 9.0 Hz,
1H), 6.73 (d, J = 9.0 Hz, 1H); 13C NMR (CD3OD, 125.77 MHz) δ
165.0, 144.1, 144.1, 142.3, 126.3, 125.4, 124.3, 120.6, 117.4; HRMS
(ESI, M + H+) m/z calcd for C9H7N2O3 191.0457; found, 191.0456.
Our crystal structure of this compound was deposited at the
Cambridge Crystallographic Data Centre (CCDC 913304).
General Procedure for in Vitro Hypoxic Metabolism. For
anaerobic reactions, all reagents except xanthine oxidase were degassed
by three freeze−pump−thaw cycles in Pyrex tubes. The glass tubes
were torch sealed and then opened inside an argon-purged glovebag
and bubbled with argon for 5 min. In a typical enzymatic reaction, 1 (4
μL of a 50 mM solution in DMF; final concentration, 0.8 mM) or the
nonfluorescent electron acceptor, 1,2,4-benzotriazine 1,4-di-N-oxide,
(24 μL from 50 mM in 15% DMF/water; final concentration, 6.4
mM), was mixed with xanthine (20−160 μL of 10 mM; final
concentrations, 0.8−6.4 mM), xanthine oxidase (20 μL of a 20 U/mL
stock solution; final concentration, 2.4 U/mL), sodium phosphate
buffer (6 μL from a 50 mM stock solution; final concentration, 12
mM, pH 7.4), and HPLC grade water to obtain the final solution (0.25
mL final volume, with all reactions containing less than 2% DMF by
volume). After mixing, the containers were covered with aluminum foil
to prevent exposure to light, and incubations were carried out at room
temperature (24 °C) inside the inert atmosphere glovebag. Following
EXPERIMENTAL PROCEDURES
■
Materials and Methods. Materials were from the following
sources: sodium phosphate, DMF, Raney nickel, silica gel plates for
thin layer chromatography, and silica gel (0.04−0.063 mm pore size)
for column chromatography, xanthine oxidase from bovine milk (CAS:
9002-17-9), Sigma X4500-25UN, 24.9 mg protein/mL, 1.3 units/mg
protein, and 18O-water (H218O) (CAS: 14314-42-2), Sigma 329878,
97 atom % 18O were obtained from Sigma-Aldrich (St. Louis, MO), 6-
aminoquinoline (2), 6-nitroquinoline (1), and hydrazine hydrate from
Alfa-Aesar (Ward Hill, MA), deuterated NMR solvents from
Cambridge Isotope Laboratories (Andover, MA), and ethyl acetate,
dichloromethane, methanol, hexane, ethanol, HPLC grade water, and
acetonitrile from Fischer. The nonfluorescent electron acceptor, 1,2,4-
benzotriazine-1,4-di-N-oxide, was prepared using literature methods.55
Azoxy compound 3 was prepared as described previously.54
Synthesis of 6-Hydroxylaminoquinoline (6). To a stirred
solution of 6-nitroquinoline (1, 0.5 g, 2.87 mmol) in EtOH/CH2Cl2
(1:1, 20 mL) at 0 °C was added a slurry of Raney nickel (0.5 mL). To
this mixture under an atmosphere of nitrogen gas, hydrazine hydrate
(10 equiv based on 1) was added dropwise with stirring over the
course of 1 h. The solid was removed by filtration and the resulting
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incubation, anaerobic reactions were opened to air and after
approximately 1 h diluted to 1 mL with aerobic sodium phosphate
buffer (50 mM, pH 7.4), and the fluorescence measurements were
then carried out in a cuvette open to air.
Incorporation of 18O-Labeled Water in to 6-Nitroquinolin-
2(1H)-one (5) during Xanthine Oxidase-Mediated Oxidation of
6-Nitroquinoline (1). Xanthine oxidase from bovine milk was diluted
4:1 with 18O-labeled water and was incubated at 37 °C in borosilicate
glass test tubes under ambient oxygenation for 1 h. Protein was
precipitated by the addition of two volumes of MeCN with subsequent
centrifugation (3000 rcf, 10 min). The supernatant was dried under a
stream of nitrogen and reconstituted in 85:15 (v/v) ammonium
formate (10 mM, pH 4.1)/MeCN in preparation for LC-MS analysis.
LC-MS Analysis. In vitro enzymatic metabolism of 1 and 6 was
carried out as described above. The reaction mixtures were extracted
with ethyl acetate, the organic layer extracted with brine, and the
solvent removed by rotary evaporation. The resulting solid was
redissolved in methanol and analyzed by LC-MS in the positive ion
mode. LC-MS analyses were carried out using two different methods.
In the analysis of the in vitro metabolism of 6 shown in Figure 5, the
separation of metabolites was carried using a C18 reverse phase
Phenomenex Luna column (5 μm particle size, 100 Å pore size, 150
mm length, 2.00 mm i.d.) and a ThermoSeparations liquid
chromatograph (TSP4000), and the metabolites were detected by
their UV-absorbance at 254 nm. The elution started with a gradient of
A, 99% HPLC water (0.1% acetic acid), and B, acetonitrile (0.1%
formic acid), followed by a linear increase to 90% B over the course of
30 min. The elution was continued at 90% B for 3 min and decreased
to 1% over the next 8 min. A flow rate of 0.25 mL/min was used.
Products were monitored at both 214 and 240 nm. The LC-ESI-MS
analyses were carried out in the positive ion mode on a Finnigan TSQ
7000 triple quadrupole instrument using a 4.5 kV needle voltage and at
a capillary temperature of 250 °C. The analyses shown in Figures 2−4
were carried out using an Agilent 1100 HPLC system was coupled to a
Supelco Discovery C18 column (5 μm, 2.1 × 150 mm; Sigma-Aldrich
Chemical Co., St. Louis, MO). Solvent A was 10 mM (pH 4.1)
ammonium formate, and solvent B was MeCN. The initial mobile
phase was 85:15 A/B (v/v) and by linear gradient transitioned to
20:80 A/B over 20 min. The flow rate was 0.400 mL/min. The HPLC
eluent was first introduced into an Agilent 1100 DAD (single
wavelength selected, 254 nm) followed by electrospray ionization-
assisted introduction into a Finnigan LCQ Deca XPPLUS ion trap
mass spectrometer (Thermo Scientific Corp., San Jose, CA) operated
in negative ionization mode. Ionization was assisted with sheath and
auxiliary gases (ultra pure nitrogen) set at 60 and 40 psi, respectively.
The electrospray voltage was set at 5 kV with the heated ion transfer
capillary set at 300 °C and 30 V. Relative collision energies of 25−35%
were used when the ion trap mass spectrometer was operated in the
MS/MS or MSn mode.
Figure 1. Enzymatic conversion of 1 into a fluorescent product
selectively under hypoxic conditions. (A) Fluorescence emission at
530 nm (λex 340 nm). Each set of assays depicted in the bar graph
consists of (from left to right) a control sample of compound 1 alone
(0.8 mM), navy blue square; a control reaction composed of xanthine
oxidase (2.4 U/mL) and xanthine (2.4 mM, 3.2 mM, 4.0 mM, and 6.4
mM) under aerobic conditions, orange square; a control reaction
composed of xanthine oxidase (2.4 U/mL) and xanthine (2.4 mM, 3.2
mM, 4.0 mM, and 6.4 mM) under anaerobic conditions, light blue
square; a control reaction composed of xanthine oxidase (2.4 U/mL),
xanthine (2.4 mM, 3.2 mM, 4.0 mM, and 6.4 mM), and the
nonfluorescent electron acceptor, 1,2,4-benzotriazine 1,4-dioxide52
(6.4 mM), under aerobic conditions, maroon square; a control
reaction composed of xanthine oxidase (2.4 U/mL), xanthine (2.4
mM, 3.2 mM, 4.0 mM, and 6.4 mM), and the nonfluorescent electron
acceptor, 1,2,4-benzotriazine 1,4-dioxide (6.4 mM), under anaerobic
conditions, green square; a reaction composed of xanthine oxidase (2.4
U/mL), xanthine (2.4 mM, 3.2 mM, 4.0 mM, and 6.4 mM), and 1
under aerobic conditions, bright blue square; and a reaction composed
of xanthine oxidase (2.4 U/mL), xanthine (2.4 mM, 3.2 mM, 4.0 mM,
and 6.4 mM), and 1 under anaerobic conditions, purple square.
Reactions were incubated for 18 h in sodium phosphate buffer at (12
mM, pH 7.4) at 24 °C, diluted with aerobic sodium phosphate buffer
(12 mM, pH 7.4), and the fluorescence measured (λex, 340 nm; λem,
530 nm). (B) Fluorescence spectra of reaction mixtures generated in
the aerobic and anaerobic metabolism of 1 by xanthine oxidase (2.4
U/mL) and xanthine (6.4 mM) carried out as described in the
Experimental Procedures section and fluorescence spectrum of 6-
aminoquinoline (2, 0.1 mM, λex 340 nm, in sodium phosphate buffer,
10 mM, at pH 7.4).
RESULTS AND DISCUSSION
■
Conversion of 1 into Fluorescent Metabolites by the
Xanthine/Xanthine Oxidase Enzyme System under
Hypoxic Conditions. In these studies, we used the
xanthine/xanthine oxidase enzyme system to carry out the
reductive metabolism of 1.43−53 For reactions carried out under
anaerobic conditions, molecular oxygen was removed from the
solutions by three cycles of freeze−pump−thaw degassing and
assay mixtures assembled and incubated in an inert atmosphere
glovebag.
distinct from that of the fluorescent helicene, pyrido[3,2-
f ]quinolino[6,5-c]cinnoline 3-oxide 4 (Scheme 3) observed
previously in the hypoxic metabolism of 1 by NADPH/
cytochrome P450 reductase.54 Specifically, compound 4
displayed emission maxima at 440 and 460 nm,54 while the
hypoxic metabolism of 1 by xanthine/xanthine oxidase
described here generated fluorescence with a broad emission
maximum at 530 nm and a shoulder at 445 nm. The emission
spectrum resembled that of an authentic standard of 6-
aminoquinoline (2) synthesized by catalytic hydrogenation of 1
or obtained from commercial sources (Figure 1B). Importantly,
The compound 6-nitroquinoline (1) is not fluorescent in
aqueous solution.54 However, incubation of 1 with xanthine
(6.4 mM)/xanthine oxidase under hypoxic conditions led to a
12-fold increase in fluorescence emission at 530 nm (versus
anaerobic xanthine (6.4 mM)/xanthine oxidase control, Figure
1A). The fluorescence yield increased with increasing
concentration of the enzyme substrate xanthine, and the
fluorescence spectrum of the reaction mixture was clearly
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relatively little fluorescence increase at 530 nm was observed
when 1 was incubated with xanthine/xanthine oxidase under
aerobic conditions (Figure 1B). Control assays containing only
xanthine/xanthine oxidase under aerobic or anaerobic con-
ditions, or xanthine/xanthine oxidase with a nonfluorescent
electron acceptor in place of 1, similarly displayed little
fluorescence at 530 nm (Figure 1A). The modest increase in
background fluorescence centered near 450 nm in the control
assays containing only xanthine and xanthine oxidase may be
due to a previously reported byproduct resulting from
decomposition of the enzyme’s pterin cofactor.56 Overall, the
results suggest that 1 is converted selectively under hypoxic
conditions to a product(s) with a fluorescence spectrum
matching that of authentic 2.
LC-MS Analysis of the Products Generated by
Hypoxic Metabolism of 1 by Xanthine/Xanthine
Oxidase. To better understand the molecular origin of the
fluorescence generated in the hypoxic metabolism of 1 by the
xanthine/xanthine oxidase enzyme system, we employed LC-
MS to examine the reaction mixtures. We first characterized the
mixture generated by the incubation of 1 under hypoxic
conditions in the presence of five equivalents of xanthine
(Figure 2A). A major product eluting at 2.5 min in the HPLC
3 prepared by reduction of 1 with hydrazine hydrate in the
presence of Raney nickel54,59,60 matched the 13 min metabolite
generated in the hypoxic metabolism of 1.59,60 Compound 3 is
not fluorescent in aqueous buffered solution54 indicating that
this product did not contribute to the fluorescence observed in
Figure 1. We note that the azoxy compound 3 was observed
using thin-layer chromatography prior to concentration of the
reaction mixtures. Thus, the generation of this dimerization
product is not dependent upon evaporation of the samples
prior to LC-MS (though we cannot rule out the possibility that
the concentration of the samples alters the yield of 3).
We observed an additional product eluting at about 11.5 min,
slightly ahead of the parent probe 1. This compound produced
an [M + H]+ at m/z 191, an increase of 16 Da relative to 1.
Xanthine oxidase is able to oxidize a number of nitrogen
heterocycles, usually at a position adjacent to an endocyclic
nitrogen atom,44,50,53,61−63 leading us to suspect that the 11.5
min product might be 6-nitroquinolin-2(1H)-one 5 (Scheme
4). In order to generate quantities of the putative product 5
Scheme 4
sufficient for NMR analysis, we incubated 1 with xanthine
oxidase (no xanthine substrate) under aerobic conditions
(Scheme 4). Spectroscopic analyses of the resulting product
were consistent with the 6-nitroquinolin-2(1H)-one structure
proposed for the metabolite 5. Specifically, the 1H NMR
spectrum of this product was lacking a resonance for the proton
adjacent to the endocyclic nitrogen in the parent quinoline
heterocycle, and the 13C-spectrum showed a new resonance at
165 ppm diagnostic for the carbonyl carbon of the quinolin-
2(1H)-one system. Ultimately, X-ray crystallographic analysis
confirmed the structure of 5 (Scheme 4).64 With an authentic
standard of 5 in hand, we were able to confirm that this product
was indeed generated in the metabolism of 1 by xanthine/
xanthine oxidase. We further used LC-MS analysis to show that
incubation of 1 with xanthine oxidase in H218O led to the
formation of an isotopomer of 5 increased by 2 Da (Figure 3),
consistent with the incorporation of an oxygen atom from
water as expected for the oxidation of nitrogen heterocycles by
xanthine oxidase.43,50,53,61−63
LC-MS analysis revealed that, at lower ratios of xanthine/1
under anaerobic conditions, the same products were generated
but in different relative amounts. For example, when only 1
equiv of xanthine was present, the yield of the amino derivative
2 was low, while larger relative amounts of the starting probe 1,
the azoxy compound 3, and the 6-nitroquinolin-2(1H)-one 5
were observed (Figure 2B).
Under aerobic conditions at either 1 or 5 equiv of xanthine,
the major products were the starting probe 1 and the 6-
nitroquinolin-2(1H)-one 5, alongside small amounts of the
azoxy derivative 3 (Figure 4). Our LC-MS analyses did not
detect 2 in the aerobic incubations of 1 with xanthine/xanthine
oxidase, in line with our finding that the incubation of 1 with
the enzyme system did not produce fluorescent products under
aerobic conditions (Figure 1).
Figure 2. LC-MS analysis of the reaction mixture generated by
anaerobic metabolism of 1 (0.8 mM) by xanthine oxidase (2.4 U/mL)
and xanthine. The reaction shown in panel A contained 4 mM
xanthine and that shown in panel B contained 0.8 mM xanthine.
Compound 1, retention time (RT) 12.3 min, [M + H]+ 175, fragments
145, 129, 117; 2, RT 2.2 min, [M + H]+ 145, fragments 128, 103; 3,
13.2 min, [M + H]+ 301, fragments 283, 273, 146, 128, 117; 5, RT
11.3 min, [M + H]+ 191, fragments 174, 161, 145, 133.
chromatogram displayed the m/z of 145 expected for the [M +
H+] ion of 2. As described above, compound 2 adequately
accounts for the fluorescence emission at 530 nm generated by
the hypoxic metabolism of 1 by xanthine/xanthine oxidase. A
second, relatively minor product eluting around 13 min
produced an [M + H]+ at m/z 301 consistent with the
compound 6,6′-azoxyquinoline (3, Scheme 3). This compound
can be envisioned to arise from the condensation of 6-nitroso
and 6-hydroxylamino intermediates generated in the reductive
metabolism of 1.57,58 Indeed, LC-MS of an authentic sample of
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Figure 3. Incubation of 1 with xanthine oxidase in H218O yields 18O-5. LC-ESI(−)-MS of 5 generated by incubation of 1 with xanthine oxidase in
H2O. The inset shows LC-ESI(−)-MS of 5 generated by incubation of 1 with xanthine oxidase in a buffered solution containing 80% H218O (97%
isotopic purity) and 20% H2O.
hydroxylaminoquinoline (6) by xanthine/xanthine oxidase
under both aerobic and hypoxic conditions.
Compound 6 was prepared by the reduction of 6-
nitroquinoline using hydrazine hydrate in the presence of
Raney nickel.66 LC-MS analysis revealed that incubation of 6
with xanthine/xanthine oxidase under anaerobic conditions
generated a mixture of 2 and 3 (Figure 5). Amino compound 2
likely arises from the expected enzymatic reduction of 6. Azoxy
compound 3 was unexpected and may result from spontaneous
oxidation of the arylhydroxylamine 6 to the corresponding
nitroso compound during aerobic reaction workup and
analysis,67 followed by condensation of the nitroso with
remaining 6.57,58 Compound 6 was fluorescent, displaying
emission maxima at 442 and 470 nm similar to the helicene 4,54
but clearly distinct from the emission spectrum of 6-
aminoquinoline 2 (Figure 6B). In line with the LC-MS
analysis, we found that anaerobic metabolism of 6 by the
Figure 4. LC-MS analysis of the reaction mixture generated by aerobic
xanthine/xanthine oxidase enzyme system generates a strong
fluorescence emission peak centered at 530 nm, consistent with
generation of 2 (Figure 6). In contrast, incubation of 6 with
xanthine/xanthine oxidase under aerobic conditions afforded
almost no increase in emission at 530 nm. Rather, a modest
increase in fluorescence at 450 nm was observed. The emission
at 450 nm may represent a combination of fluorescence
emission signals from xanthine oxidase degradation products56
and unreacted 6. Overall, these results provide evidence that
the conversion of hydroxylamino compound 6 to amino
derivative 2 mediated by xanthine/xanthine oxidase occurs
selectively under hypoxic conditions.
metabolism of 1 (0.8 mM) by xanthine oxidase (2.4 U/mL) and
xanthine. The reaction shown in panel A contained 4 mM xanthine,
and that in panel B contained 0.8 mM xanthine.
Hypoxia-Selective Conversion of 6-Hydroxylamino-
quinoline (6) to 6-Aminoquinoline (2). The generation of
arylamines in the hypoxia-selective metabolism of nitroaryl
compounds proceeds via nitroso and hydroxylamino inter-
mediates (Scheme 1).16−18,20,22,23 Indeed, azoxy compound 3
observed in our reactions likely arises via the condensation of
the nitroso and hydroxylamino intermediates.57,58 Under
physiological conditions, nitroso intermediates can undergo
rapid thiol-mediated reduction to the corresponding hydrox-
ylamino compound,65 meaning that the enzymatic reduction of
nitroso compounds to hydroxylamino derivatives may not be
important in vivo. In contrast, the enzymatic transformation of
arylhydroxylamines to arylamines is a key step in the hypoxic
conversion of nitroaryl compounds to arylamines, but this
process has not been well studied. To better understand the
overall bioreduction of 1 leading to fluorescent compound 2,
we set out to examine the biotransformation of 6-
CONCLUSIONS
■
We found that the xanthine/xanthine oxidase enzyme system
converted 6-nitroquinoline (1) to 6-aminoquinoline (2)
selectively under hypoxic conditions. The metabolism of
nitroaryl compounds by the xanthine/xanthine oxidase enzyme
system is expected to proceed via a series of one-electron
reduction steps that generate nitroso and hydroxylamino
intermediates, as shown in Scheme 1.16−24 Indeed, the azoxy
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Figure 5. LC-MS analysis of the reaction mixture generated by
anaerobic metabolism of 6 (0.8 mM) by xanthine oxidase (2.4 U/mL)
and xanthine (6.4 mM). The enzymatic reduction of 1 was carried out
as described in the Experimental Procedures section. Panel A: HPLC
trace of the reaction mixture monitoring absorbance at 254 nm. Panel
B: LC-MS spectrum of the product eluting at 4.70 min. Panel C: LC-
MS spectrum of the product eluting at 19.20 min.
Figure 6. Enzymatic conversion of 6 into a fluorescent product
selectively under hypoxic conditions. (A) Fluorescence emission at
530 nm (λex 340 nm). Each set of assays depicted in the bar graph
consists of (from left to right) a control sample of compound 6 alone
(0.8 mM), navy blue square; a control reaction composed of xanthine
oxidase (2.4 U/mL) and xanthine (2.4 mM, 3.2 mM, 4.0 mM, and 6.4
mM) under aerobic conditions, orange square; a control reaction
composed of xanthine oxidase (2.4 U/mL) and xanthine (2.4 mM, 3.2
mM, 4.0 mM, and 6.4 mM) under anaerobic conditions, light blue
square; a control reaction composed of xanthine oxidase (2.4 U/mL),
xanthine (2.4 mM, 3.2 mM, 4.0 mM, and 6.4 mM) and the
nonfluorescent electron acceptor, 1,2,4-benzotriazine 1,4-dioxide (6.4
mM), under aerobic conditions, maroon square; a control reaction
composed of xanthine oxidase (2.4 U/mL), xanthine (2.4 mM, 3.2
mM, 4.0 mM, and 6.4 mM) and the nonfluorescent electron acceptor,
1,2,4-benzotriazine 1,4-dioxide (6.4 mM), under anaerobic conditions,
green square; a reaction composed of xanthine oxidase (2.4 U/mL),
xanthine (2.4 mM, 3.2 mM, 4.0 mM, and 6.4 mM) and 6 under
aerobic conditions, purple square; and a reaction composed of
xanthine oxidase (2.4 U/mL), xanthine (2.4 mM, 3.2 mM, 4.0 mM,
and 6.4 mM) and 6 under anaerobic conditions, teal square. Reactions
were incubated for 18 h in sodium phosphate buffer at (12 mM, pH
7.4) at 24 °C, diluted with aerobic sodium phosphate buffer (12 mM,
pH 7.4), and the fluorescence measured (λex, 340 nm; λem, 530 nm).
(B) Fluorescence spectra of aerobic and anaerobic reaction mixtures
containing 6 (0.8 mM), xanthine oxidase (2.4 U/mL), and xanthine
(3.2 mM) and the fluorescence spectrum of 6 alone (0.05 mM, λex 340
nm, in sodium phosphate buffer, 10 mM, at pH 7.4).
product 3 observed in these experiments likely arises via
condensation of 6-nitroso and 6-hydroxylaminoquinoline
intermediates during the enzymatic reaction or subsequent
analysis. Synthesis of the 6-hydroxylaminoquinoline intermedi-
ate 6 allowed us to provide evidence that xanthine/xanthine
oxidase converted this aryl hydroxylamine to fluorescent
product 2 selectively under hypoxic conditions. This result is
striking because the initial one-electron reduction step in the
nitro-to-nitroso conversion typically is thought to be the key
oxygen-sensitive step that confers hypoxia selectivity to
nitroreduction processes.19−21 Our work with 6 provides
evidence for an additional oxygen-sensitive step, beyond the
initial one-electron reduction of the nitroaryl group, in the
hypoxia-selective enzymatic conversion of some nitroaryl
compounds to arylamines.
The results described here combined with our previous
work54 reveal striking differences in the products generated in
the hypoxic metabolism of 1 by the xanthine/xanthine oxidase
and NADPH/cytochrome P450 reductase enzyme systems.54
Here, we demonstrated that xanthine/xanthine oxidase
converted 6-nitroquinoline (1) to 6-aminoquinoline (2)
selectively under hypoxic conditions, while our earlier work
showed that NADPH/cytochrome P450 reductase generates
the helicene product 4 (Scheme 2).54 The mechanism by which
the helicene 4 forms remains uncertain, but it seems that
NADPH/cytochrome P450 reductase lacks the ability to
rapidly reduce the 6-nitroso and/or 6-hydroxylamino inter-
mediates derived from 1 (Scheme 1), thus leaving these species
to combine in a manner that generates 4.
that xanthine oxidase converts 1 to the 6-nitroquinolin-2(1H)-
one derivative 5 under both aerobic and anaerobic conditions
(Scheme 5). This reaction is not altogether unexpected given
that xanthine oxidase can oxidize a wide range of nitrogen
heterocycles.43,44,50,53,61−63 In principle, reductive metabolism
of 5 could generate 6-aminoquinolin-2(1H)-one (7), which is a
much brighter fluorophore than 2, with an emission maximum
of 485 nm (data not shown); however, we observed no
evidence for the formation of 7 in either our LC-MS or
fluorescence experiments. Nonetheless, 5 clearly has the
Another difference between xanthine oxidase and NADPH/
cytochrome P450 reductase is highlighted by our observation
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Chemical Research in Toxicology
Article
Scheme 5. Overview of the Fluorescent Products That Can Be Generated in the Hypoxic Metabolism of 1
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potential to serve as a bioreductively activated, hypoxia-selective
fluorescent probe. The relative yields of 5 were lower when
greater amounts of xanthine were present in the reaction
mixture to drive the reduction of 1 by xanthine oxidase
suggesting that, under our anaerobic reaction conditions, the
reduction of 1 by the xanthine/xanthine oxidase system was
faster than the oxidation of 1 to 5 by xanthine oxidase.
Our work may enable the use of 6-nitroquinoline (1) as a
fluorescent substrate for the detection and profiling of one-
electron reductases in cell culture or biopsy samples. The
compound also could find use as a fluorogenic probe for the
visualization of hypoxia in tumor models, though information
regarding the potential toxicity of 1 will be required for this
application. The occurrence of side products such as 3, 4, and 5
in enzymatic bioreduction of 1 underscores the importance of
metabolite identification in the characterization of hypoxia-
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oxygen-sensing units.
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A., Meletis, K., Wolter, M., Sommerlad, D., Henze, A.-T., Nister, M.,
́
Reifenberger, G., Lundeberg, J., Frisen, J., and Acker, T. (2010) A
AUTHOR INFORMATION
■
hypoxic niche regulates glioblastoma stem cells through hypoxia
inducible factor 2 alpha. Brain 133, 983−995.
Corresponding Author
*Phone: (573) 882-6763. Fax: (573) 882-2754. E-mail:
gatesk@missouri.edu.
(14) Charafe-Jauffret, E., Ginestier, C., Iovino, F., Wicinski, J.,
Cervera, N., Finetti, P., Hur, M.-H., Diebel, M. E., Monville, F.,
Dutcher, J., et al. (2009) Breast cancer cell lines contain functional
cancer stem cells with metastatic capacity and a distinct molecular
signature. Cancer Res. 69, 1302−1313.
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R., Caron, H. N., Molenaar, J. J., Ora, I., Eva, A., Puppo, M., and
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gene signature as a predictor of the outcome of neuroblastoma
patients. Mol. Cancer 9, 185.
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Camalier, R., and Lewis, A. D. (1996) Reductase enzyme expression
across the National Cancer Institute tumor cell line panel: correlation
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Funding
We thank the National Institutes of Health for partial support
of this work (CA 100757).
Notes
The authors declare no competing financial interest.
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