Design of a bioreductively-activated fluorescent pH probe for tumor hypoxia imaging

Article abstract of DOI:10.1016/j.bmc.2009.08.037

We have designed and evaluated UTX-12 as a novel fluorescent pH probe for tumor hypoxia imaging. UTX-12 consists of a p-nitro benzyl moiety, which is a latent hypoxia-selective leaving group activated by nitro reduction, directly linked to SNARF. Although

Full text of DOI:10.1016/j.bmc.2009.08.037

Bioorganic & Medicinal Chemistry 17 (2009) 6952–6958
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design of a bioreductively-activated fluorescent pH probe for tumor
hypoxia imaging
a
a
a
a
a
Eiji Nakata a, , Yoshihiro Yukimachi , Hirokazu Kariyazono , Seongwang Im , Chiaki Abe , Yoshihiro Uto ,
*
Hiroshi Maezawa b, Toshihiro Hashimoto c, Yasuko Okamoto c, Hitoshi Hori a,
*
a Department of Life System, Institute of Technology and Science, Graduate School, The University of Tokushima, Minamijosanjimacho 2, Tokushima 770-8506, Japan
b Faculty of Medicine, The University of Tokushima, Tokushima 770-8509, Japan
c Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro-cho, Tokushima 770-8514, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
We have designed and evaluated UTX-12 as a novel fluorescent pH probe for tumor hypoxia imaging.
UTX-12 consists of a p-nitro benzyl moiety, which is a latent hypoxia-selective leaving group activated
by nitro reduction, directly linked to SNARF. Although UTX-12 itself is colorless and non-fluorescent in
aqueous solution, nitro reduction triggers the release of SNARF which has well-characterized long wave-
length absorption and fluorescence that is sensitive to pH. The resultant SNARF, released intracellularly
by enzymatic reduction of UTX-12, allows measurement of pH by pH-dependent dual emission shifts.
UTX-12 showed clear differences in fluorescence behavior between hypoxic and aerobic conditions in
liver microsomes and inside V79 cells. These data are confirmation that UTX-12 is biologically reduced
inside tumor cells and the released SNARF should monitor intracellular pH of tumor cells selectively with
reduced background signal.
Received 16 June 2009
Revised 6 August 2009
Accepted 7 August 2009
Available online 21 August 2009
Keywords:
Fluorescent pH probe
Tumor hypoxia
Fluorescent imaging
Bioreductive activation
Nitro reduction
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
including high resolution, high sensitivity and noninvasiveness.7,10
Included in this approach are a few examples of a strategy to iden-
Robust tumor growth requires the presence of a local vascular
network that supplies both oxygen and nutrients to tumor cells.
However, a highly proliferative mass of tumor cells develops faster
than the vasculature, and new tumor cells are formed with an
avascular environment deficient in oxygen, a condition known as
tumor hypoxia. Tumor hypoxia is characterized by low oxygen ten-
sion, low nutrient level, low pH and an over-expression of angio-
genic factors. This is a fundamentally important character of the
tumor environment because it has been associated closely with
the malignant phenotype of cancer disease, resistance to cancer
therapies, and the high mortality rate of cancer patients.1,2 For
these reasons the specific characteristics of tumor hypoxia are
attractive targets in the development of anticancer drugs.3–6 In
addition, there has been an increasing clinical interest in hypox-
ia-specific molecular probes which can characterize the hypoxic
cells fraction before, during and after therapy.7
tify hypoxic cells by using fluorescent probes which become more
fluorescent after biological reduction resulting from tumor hypox-
ia.11–16 Although these are promising candidates as tumor hypox-
ia-selective fluorescent probes, there are two drawbacks. The first
is that the maximal absorption wavelength of most of the fluores-
cent probes used in this approach is less than 500 nm, a complica-
tion for bio-imaging because there are many biomolecules that
absorb at such a short wavelength. In general, fluorophores with
long absorption and fluorescence wavelengths are more suitable
as fluorescent probes for in vivo usage. The second drawback is
that all of these fluorescent probes were activated irreversibly
under hypoxic conditions, a factor that could possibly complicate
the monitoring of any time-dependent change during therapy.
The fluorescent probe which has a reversible functionality would
be more useful because it would have the potential to monitor
the therapeutic effect in real time.17 These considerations
prompted us to investigate the design of a fluorescent pH probe
which would be selectively and irreversibly activated under tumor
hypoxia and subsequently able to monitor the pH in its local
environment reversibly, an important determination since pH
measurements in tumors have significant diagnostic value.18,19
Thus, tumor cells have a very high capacity to produce lactic acid,
which is generated through glucose metabolism and inefficient
vascular clearing, resulting in an acidic microenvironment in solid
To measure tumor oxygenation in experimental or clinical tu-
mors, different analytical probes have been used in various meth-
ods.8,9 Among these, fluorescent detection has several advantages
* Corresponding authors. Tel./fax: +81 88 656 7517 (E.N.); tel.: +81 88 656 7514;
fax: +81 88 656 9164 (H.H.).
E-mail addresses: nakata@bio.tokushima-u.ac.jp (E. Nakata), hori@bio.tokushima-u.
ac.jp (H. Hori).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.037

E. Nakata et al. / Bioorg. Med. Chem. 17 (2009) 6952–6958
6953
tumors.1,2 Therefore, the monitoring of tumor pH plays a signifi-
corresponding values of kmax, extinction coefficient at kmax (e),
cant role in cancer treatment.20–25 In this article, we describe the
design and synthesis of new fluorescent pH probes to detect tumor
hypoxia selectively and to monitor the pH of the tumor in a revers-
ible manner.
kem, and quantum yield (/) at pH 5.0 are listed in Table 1. The rel-
ative intensities of these compounds were determined by calculat-
ing the product of extinction coefficient and quantum yield and
then normalizing these values to those of SNARF. These values also
are given in Table 1.26 As compared to SNARF, UTX-38 had only
1.6% and UTX-12 had less than 0.5% of the intensity of SNARF. It
is well known that SNARF exists as an equilibrium mixture of the
quinoid form that absorbs visible light and is fluoresce and the lac-
tone form that is colorless and non-fluorescent.26,27 The absorption
and fluorescent spectral data indicate that UTX-12 and UTX-38 ex-
ist mainly as the lactone form in aqueous solution and that the
fluorescence of UTX-12 is also quenched by the nitro group, a well
known quencher of fluorophores.11–14
2. Results and discussion
2.1. Molecular design and synthesis
UTX-12 was designed as the hypoxia-selective fluorescent pH
probe. This molecule is comprised of a p-nitrobenzyl moiety di-
rectly linked to SNARF (seminaphthorhodafluors) through an ether
linkage (Fig. 1). SNARF belongs to the group of asymmetric xan-
thene derivatives which are well known as long-wavelength fluo-
rophores. It is, in particular, an example of a pH indicator that
shows a dual-emission change (typically 583 nm and 627 nm) in
the neutral pH region (pKa = 7.5). It is therefore useful for measur-
ing pH changes around the physiological pH range (pH 6.5–8.5).26
In our strategy, the phenolic substituent of SNARF, which is the
critical substituent responsible for the unique fluorescent proper-
ties, was masked by the nitro benzyl moiety. This moiety is often
used as the leaving group activated by biological reduction in hy-
poxia-selective prodrug strategies.5,6 As depicted in Figure 2, the
nitro benzyl moiety is reduced via a series of one-electron reduc-
tion processes to form the hydroxylamino (4eꢀ) and amino (6eꢀ)
intermediates that lead to the release of SNARF. However the rad-
ical anion product of the first electron reduction step is re-oxidized
efficiently to UTX-12 in the presence of oxygen, making further
reduction dependent on hypoxic conditions. This selective reduc-
tion of the nitro benzyl moiety under hypoxia is the key to giving
UTX-12 the potential of being a hypoxia-selective fluorescent pH
probe (Fig. 2). UTX-38 was designed as a control compound that
is lacking the nitro substituent on the benzyl moiety (Fig. 1). The
synthetic routes to UTX-12 and UTX-38 are shown in Scheme 1.
As shown, treatment of SNARF with excess K2CO3 and p-nitroben-
zyl bromide or benzyl bromide in dry DMF gave UTX-12 or UTX-38,
respectively.
2.3. Nitro reduction of UTX-12 catalyzed by nitroreductase
To test the selective deprotection of UTX-12 via nitro reduction,
enzymatic reduction of the p-nitrobenzyl moiety was carried out
using nitroreductase from Escherichia coli (NTR), an example of
an oxygen-insensitive nitroreductase.28 When the absorption spec-
trum and fluorescent spectrum of UTX-12 were measured in the
NTR, a time dependent absorption and fluorescence increase orig-
inating from SNARF was observed (Fig. 4a and b). The conversion
yield (95%) from UTX-12 to SNARF and the maximal fluorescence
change (40 times) after 6 h treatment were determined by the
absorption spectrum and fluorescence spectrum, respectively. In
contrast, no SNARF fluorescent increase was observed in the ab-
sence of NTR (Fig. 4c). In addition, no increase was observed using
UTX-38 instead of UTX-12 (Fig. 4c). The decrease of UTX-12 and
production of SNARF were confirmed by HPLC analysis (Fig. S-1).
All of these results indicate that the reduction of the nitro substitu-
ent of the p-nitro benzyl moiety triggered the release SNARF from
UTX-12. To demonstrate the property of pH response of UTX-12
after biological reduction of the nitro substituent, the UTX-12 solu-
tion prepared with or without NTR in different pH conditions (pH
5.0, pH 7.0, pH 9.0) were observed by using a transilluminator and
the fluorescent spectrometer. The dependence of fluorescent
enhancement by nitro reduction and fluorescent color change on
the pH was confirmed (Fig. 4d). Based on these data, UTX-12 was
demonstrated to function as a fluorescent pH probe mediated by
nitro reduction.
2.2. Spectral analysis
We first compared the absorption and the fluorescence spec-
trum of UTX-12 and UTX-38 with that of the reference compound
SNARF (Fig. 3). SNARF produced maximal absorption and strong
fluorescence at 544 nm and 583 nm (kex = 534 nm), respectively.
In contrast, UTX-12 and UTX-38 displayed no significant absorp-
tion above 400 nm and no fluorescence excitation at 534 nm. The
2.4. Biological reduction of UTX-12 under different oxygen
concentrations
To further evaluate the potential of the UTX-12 as a hypoxia-
selective pH probe, we used fluorescence to monitor its biological
reduction in different oxygen concentrations. Preparations of liver
microsomes of chick embryo were used as a metabolic enzyme
cocktail. This includes cytochrome P450 reductase, an electron-
donating protein that catalyzes the one-electron reduction of nitro
derivatives to nitro anion radicals.29–31 UTX-12 was incubated with
the microsomes in different oxygen concentrations, including hyp-
oxic (without oxygen), aerobic (20% v/v) and oxic conditions (95%
v/v), respectively. The time course of fluorescent spectral change
under hypoxic conditions is shown in Figure 5a. Intense fluores-
cence emission originating from SNARF and its time dependent in-
crease (sixfold after 20 h incubation) were observed (inset of
Fig. 5a). In contrast, under both aerobic and oxic conditions after
20 h incubation, the extent of enhanced fluorescence intensity
diminished significantly as a result of the competitive scavenging
by molecular oxygen (Fig. 5b and inset). The hypoxic-aerobic or
oxic fluorescence differential became almost five to sixfold after
20 h incubation. As measured by HPLC (Fig. S-2), the differences
in the fluorescence intensities of these samples correlate well with
Figure 1. The structure of UTX-12, UTX-38 and SNARF.

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E. Nakata et al. / Bioorg. Med. Chem. 17 (2009) 6952–6958
Figure 2. Presumed reaction schemes of UTX-12 as the fluorescent pH probe activated under hypoxia.
Figure 3. Absorption (black) and emission (red) spectra of SNARF derivatives at
pH7.0 used in this study. (solid line: SNARF, dashed line: UTX-12, dotted line: UTX-
38).
Scheme 1. Synthesis of SNARF, UTX-12 and UTX-38. Reagents and conditions: (i)
(1) toluene, reflux; (2)1,6-dihydroxynaphthalene, ZnCl2, 160 °C; (ii) p-nitrobenzyl-
bromide, K2CO3, dry DMF, rt; (iii) benzylbromide, K2CO3, dry DMF, rt.
medium, confocal laser scan microscopy (CLSM) was performed.
As shown in Figure 6a, strong fluorescence in the cytosol of V79
cells which were incubated under hypoxic conditions was ob-
served. In contrast, negligible fluorescence was observed in the
cells treated under aerobic conditions (Fig. 6b). Figure 6c shows
the time-course of fluorescence intensity of UTX-12 incubated un-
der different conditions. The hypoxic-aerobic fluorescent differen-
tial increased fourfold after incubation for 6 h.32 These results
indicate that UTX-12 responds to the oxygen concentration and
that UTX-12 has a potential to use as a fluorescent pH probe in cell.
the hypoxia-selective release of SNARF from UTX-12 upon treat-
ment with the liver microsomes of chick embryo. This indicates
that UTX-12 is stable to other metabolizing enzyme in liver micro-
somes which function independent of oxygen concentration. This
is an important requirement for hypoxia-activated fluorescent
probes in order to reduce background signal. All of these results
demonstrated that UTX-12 was reduced and SNARF was produced
under hypoxic conditions, selectively.
2.5. Fluorescent imaging of hypoxic cell by using UTX-12
3. Conclusion
To further assess the potential applications of UTX-12, fluores-
cent imaging inside the hypoxic cell using UTX-12 was examined.
V79 cells (Chinese hamster lung) were incubated with UTX-12 un-
der hypoxic or aerobic conditions, respectively. After exchanging
In this study, we have demonstrated the design and evaluation
of a novel fluorescent pH probe for tumor hypoxia. SNARF, which
has long wavelength fluorescence emission and has the potential
to monitor pH in neutral range, was released from UTX-12 after ni-

E. Nakata et al. / Bioorg. Med. Chem. 17 (2009) 6952–6958
6955
Table 1
ible factor (HIF) mediated up-regulation and activation of a num-
ber of membrane located transporters, exchanges, pumps and
ecto-enzymes.18 Inhibiting such selectively up-regulated functions
in tumor cells, for example, carbonic anhydrase IX which is a high
activity tumor-associated membrane enzyme predominantly
found in hypoxic tumor tissues and is absent from most normal tis-
sues, may suffice to allow acidic metabolites to build up intracellu-
larly within a tumor and inhibit proliferation. Thus, acidic pHi has
been shown to affect cell function, growth and division, and can be
clastogenic and even induce apoptosis.19,22 For the purpose of anti-
cancer drug development based on this strategy, selective pHi
measurement in tumor cells would be very useful for high
throughput screening and direct monitoring of the effect of an
anticancer drug.
Spectroscopic properties of SNARF, UTX-12 and UTX-38
Abs
a
Em
Compounds
k
(e
ꢁ 10ꢀ3
)
k
(nm)b
U
(ꢁ10ꢀ2
)
e ꢁ / (rel)
max
max
(nm) (Mꢀ1 cmꢀ1
)
SNARFc
UTX-12
UTX-38
515 (17.7)
544 (21.6)
520 (2.07)
545 (1.82)
520 (2.16)
545 (1.88)
583
3.0
100%
<0.5%
1.6%
d
d
—
—
579
0.5
a
b
c
Measured in pH 5.0 10 mM acetate buffer.
Excited at 534 nm.
Ref. 26.
d
Precise values cannot be determined because of low fluorescence.
In summary, we have demonstrated that UTX-12 can be selec-
tively activated by reductive enzymes under the conditions of tu-
mor hypoxia. This activation in turn triggers to release SNARF
inside tumor cells where it should monitor the pHi selectively with
reduced background signal. Although SNARF derivatives have pre-
viously been used to monitor intra- or extracellular compartments
for different purposes,22,23 we note that UTX-12 is the first example
of selective translocation of SNARF as a fluorescent pH probe into
tumor cells. On these points, we believe that UTX-12 should be a
promising candidate for further evaluation as a fluorescent pH
probe for tumor hypoxia in vivo. In addition to monitoring the
oxygen environment, potential applications include following the
effects of drug treatment through pHi measurement.
tro reduction. The selective biological reduction under tumor hyp-
oxic conditions and release of SNARF thus enable UTX-12 to be
used to measure the proximal pH of the tumor. Furthermore,
UTX-12 produced a clear fluorescence differential between hypoxic
and aerobic conditions in the liver microsomes of chick embryo
and inside V79 cells. Based on these data, we conclude that UTX-
12 can produce fluorescence under tumor hypoxia selectively
and should monitor the pH of tumor hypoxia reversibly.
To describe the tumor pH environment in detail, the extracellu-
lar fluid of tumor cells is acidified (pH 6.2–6.8; pHe) in order to
maintain the intracellular pH (pHi) at a relatively normal pH (pH
7.0–7.4). This condition is reported to result from hypoxia induc-
Figure 4. (a and b) Real time spectral change of NTR-catalyzed nitro reduction. (a) Absorption spectral change. (b) Fluorescence spectral change. (c) Time profile of the
relative emission intensity (black circle: UTX-12 with NTR, white circle: UTX-38 with NTR, asterisk: UTX-12 without NTR). (d) The fluorescence spectra of UTX-12 in the
presence (black) or absence (red) of NTR in pH 5.0 (dot line), pH 7.0 (solid line) and pH 9.0 (dashed line), respectively. Inset: Photograph of the ratiometric fluorescent
response in different pH conditions with or without NTR.

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E. Nakata et al. / Bioorg. Med. Chem. 17 (2009) 6952–6958
Figure 5. (a) The spectral change of the UTX-12 incubated with liver microsomes of
chick embryo under hypoxic condition at different incubation times (0, 2.5, 5, 10
and 20 h). Inset: time profile of the relative emission intensity (kem = 627 nm)
caused by incubation under hypoxic conditions. (b) Fluorescence spectra of UTX-12
resulting from incubation under different oxygen concentration. (solid line: UTX-12
was incubated with liver microsomes for 20 h under hypoxic (red), aerobic (green)
and oxic (blue) conditions, dashed line (black): UTX-12 was incubated with liver
microsomes for 0 h, dotted line (black):UTX-12 in 0.1 M phosphate buffer (pH 7.4)
without liver microsomes. Inset: The relative emission intensity of UTX-12
(kem = 627 nm) incubated in the different oxygen concentration.
Figure 6. (a–d) Fluorescence microphotograph of V79 cells incubated with UTX-12
for 6 h under (a and b) hypoxic conditions (incubated in 95% N2 and 5% CO2) or (c
and d) aerobic conditions (incubated under atmosphere gas). (a and c) Transmission
channel. (b and d) fluorescence channel. The scale bar (50 lM) are shown in the
photograph. (e) The time courses of accumulation of fluorescent intensity in V79
cells incubated with UTX-12 under hypoxic (d) or aerobic (s) conditions.
4.2. Synthesis
4. Experimental
10-(Dimethylamino)-3-hydroxy-Spiro[7H-benzo[c]xanthene-
7,10(30H)-isobenzofuran]-30-one (seminaphthorhodafluor, SNARF)
was synthesized according to the literature procedure.33,34
4.1. General procedures
1H NMR spectra were recorded on JEOL JNM-EX400 spectrome-
ter (400 MHz) with tetramethylsilane as the internal standard.
Chemical shifts are reported in ppm. Coupling constants are re-
ported in Hz. IR spectra were measured in KBr pellets with a Per-
kin–Elmer 1600 spectrometer. HRMS were measured on a JOEL
JMS-700 mass spectrometer using the FAB method. Elemental
analyses were performed with a Yanako CHN recorder MT-5. Reac-
tions were monitored by analytical TLC using Merck Silica Gel 60
F254 aluminium plates. Column chromatography was performed
on Kanto Chemical Silica Gel 60 N (230–400 mesh). Absorption
spectra were recorded on a Hitachi U3300 spectrometer. Fluores-
cent spectra were recorded on a Hitachi F-4500 spectrometer.
4.2.1. 10-(Dimethylamino)-3-[(4-nitrophenyl)methoxy]-spiro-
[7H-benzo[c]xanthene-7,10(30H)-isobenzofuran]-30-one (UTX-
12)
A mixture of SNARF (58.2 mg, 142
l
mol), K2CO3 (68.5 mg,
mol) in dry
496 mol), 4-nitrobenzyl bromide (50.3 mg, 233
l
l
DMF (2.5 ml) was stirred for 6.5 h at room temperature. The reac-
tion mixture was diluted with CH2Cl2 and washed with satd NaH-
CO3 aq followed by drying over anhydrous Na2SO4. The solvent was
removed in vacuo, and the residue was purified by column chro-
matography on silica gel (CH2Cl2/AcOEt: a linear gradient from
1:0 to 1:1(v/v)) to give UTX-12 as a pale pink powder in 40% yield
(30.6 mg):1H NMR (CDCl3, 400 MHz): dH 8.54 (d (9.3 Hz),1H, Ar–H),
8.27 (d (8.8 Hz), 2H, Ar–H) 8.05 (dd (6.2, 1.6 Hz), 1H, Ar–H), 7.59–
7.68 (m, 4H, Ar–H), 7.36 (dd (9.1, 2.3 Hz), 1H, Ar–H), 7.30 (d
(8.8 Hz), 1H, Ar–H), 7.14–7.15 (m, 2H, Ar–H), 6.74 (d (8.8 Hz), 1H,
Ar–H), 6.67–6.70 (m, 2H, Ar–H), 6.48 (dd (8.9, 2.6 Hz), 1H, Ar–H),
5.30 (s, 2H, Ar–CH2-Ar), 3.03 (6H, s, N–CH3) FT-IR(KBr) 3478,
HPLC was performed on
a
lRPC C2/C18 SC column
(2.1 ꢁ 100 mm, Pharmacia) with a Pharmacia Biotech SMART sys-
tem. All chemicals were purchased from Wako Pure Chemical
Industries, Ltd (Osaka, Japan), Kanto Chemical Co., Inc. (Tokyo, Ja-
pan), Tokyo Chemical Industry Co., Ltd (Tokyo, Japan), or Sigma–Al-
drich Japan (Tokyo, Japan).

E. Nakata et al. / Bioorg. Med. Chem. 17 (2009) 6952–6958
6957
3079, 2911, 1749, 1627, 1521, 1419, 1361, 1344, 1250, 1110,
water containing 0.1% trifluoroacetic acid (TFA) (solvent A) and
water containing 0.1% TFA (solvent B). HPLC conditions were as fol-
lows: solvent A: solvent B = 0:100 (0 min)–0:100 (5 min)–100:0
852 cmꢀ1; FAB-HRMS (m-NBA): calcd for C33H25N2O2 ([M+H]+)
þ
545.1713, found 545.1729. Anal. Calcd for C33H24N2O6: C, 72.78;
H, 4.44; N, 5.14. Found: C, 72.55; H, 4.61; N, 5.12.
(55 min), flow rate 200
charts were shown in Supplementary data).
The reaction solution of UTX-12 (50 M) and NADPH (2 mM),
ll/min, detection by UV (280 nm) (HPLC
4.2.2. 10-(Dimethylamino)-3-phenylmethoxy-Spiro[7H-
benzo[c]xanthene-7,10(30H)-isobenzofuran]-30-one (UTX-38)
l
which was incubated with or without NTR (2.0 U/ml) for 22 h un-
der darkness, was adjusted pH 5.0, pH 7.0 and pH 9.0 was observed
by using UV transilluminator (Vilber Lourmat TFX-20.M,
kex = 312 nm) and fluorescence spectrometer.
A
mixture of SNARF (50.9 mg, 124 mol) K2CO3 (60 mg,
l
434
l
mol), benzyl bromide (29.5 l, 248 lmol) in dry DMF (2 ml)
l
was stirred for 19 h at room temperature. The reaction mixture
was diluted with CH2Cl2 and washed with satd NaHCO3 aq fol-
lowed by drying over anhydrous Na2SO4. The solvent was removed
in vacuo, and the residue was purified by column chromatography
on silica gel (CH2Cl2 only and then CH2Cl2/MeOH = 50:1 (v/v)) to
give UTX-38 as a pale pink powder in 76% yield (46.8 mg): 1H
NMR (CDCl3, 400 MHz) dH 8.52 (d (9.0 Hz), 1H, Ar–H), 8.05 (dd
(6.3, 1.7 Hz), 1H, Ar–H), 7.58ꢀ7.66 (m, 2H, Ar–H), 7.49 (d
(6.8 Hz), 2H, Ar–H), 7.41 (t (6.6 Hz), 2H, Ar–H), 7.36 (dd (9.3,
6.8 Hz), 2H, Ar–H), 7.31 (d (8.5 Hz), 1H, Ar–H), 7.19 (d (2.4 Hz),
1H, Ar–H), 7.15 (dd (6.5, 1.3 Hz), 1H, Ar–H), 6.72 (d (8.8 Hz), 1H,
Ar–H), 6.67–6.70 (m, 2H, Ar–H), 6.47 (dd (8.8, 2.4 Hz), 1H, Ar–H),
5.20 (s, 2H, Ar–CH2–Ar), 3.03 (s, 6H, N–CH3); FT-IR(KBr) 3430,
2926, 2855, 2365, 1752, 1626, 1422, 1252, 1115 cmꢀ1; FAB-HRMS
4.4.2. Bioreductive activation of UTX-12 by microsomes of
chick embryo under different oxygen concentration
The liver microsomes were prepared according to the method
reported previously with a slight modification.35 Chick embryo li-
ver microsomes were prepared from a pooled set of fourteen liver
samples (4.899 g). After homogenization of the liver samples in
three volume of 50 mM phosphate 0.25 M sucrose pH 7.4, micro-
somes (0.777 g wet weight) were isolated by centrifugation at
10,000g and 105,000g, and resuspended in 6.0 ml of 0.1 M phos-
phate buffer pH 7.4. The samples were prepared with microsomes
(64.8 mg/ml), UTX-12 (50 lM), NADPH (2 mM) and MgCl2 (3 mM)
in 0.1 M phosphate buffer pH 7.4. To establish hypoxic conditions,
the suspension was degassed with three freeze-pump-thaw cycles
under nitrogen gas (without oxygen). To generate oxic conditions,
the suspension was vigorously bubbled for 20 s with a gas mixture
containing 95% oxygen and 5% carbon dioxide. For control, similar
compositions as in the foregoing samples were prepared without
UTX-12 or microsomes, respectively. All these samples were incu-
bated at 37 °C. After the appropriate incubation time (hypoxic con-
ditions: 0, 2.5, 5, 10, 20 h, the others: 20 h), these samples were
centrifuged at 105,000g and the fluorescent spectra of the superna-
tants were measured.
+
(m-NBA): calcd for C33H26NO4 ([M+H]+) 500.1862, found
500.1884. Anal. Calcd for C33H25NO4: C, 79.34; H, 5.04; N, 2.80.
Found: C, 79.08; H, 5.29; N, 2.87.
4.3. Spectroscopic assessment
4.3.1. Measurement of extinction coefficients
The extinction coefficients (e) of UTX-12 and UTX-38 were cal-
culated according to the Lambert–Beer law. Absorption spectra of
the samples were measured in pH 5.0 10 mM acetate, HEPES and
Tris buffer.
The supernatants were diluted with equal amounts of DMSO
and were filtered with Millipore Millex-GV filters (pore size
4.3.2. Fluorescence spectrophotometry
0.22 lM). The filtrates were analyzed by HPLC under the same con-
Fluorescence spectra of UTX-12, UTX-38 and SNARF were mea-
sured with excitation at 534 nm in pH 5.0 or pH 7.0 10 mM acetate,
HEPES and Tris buffer at 20 1 °C. The slit widths of the excitation
and emission were set to 10 nm and 10 nm, respectively.
ditions as described above (detection by UV at 534 nm, HPLC charts
are shown in Supplementary data).
4.4.3. Bioimaging of V79 cells incubated under different oxygen
conditions by using UTX-12
4.3.3. Measurement of fluorescence quantum yield
V79 cells were cultured in Eagle minimum essential medium
without phenol red containing 12.5% (v/v) fetal bovine serum
and kanamycin (60 mg/l) at 37 °C in 5% CO2. Cells (5 ꢁ 104 cells)
in late log phase were seeded in a 35-mm glass base dish. After
growth in a CO2 incubator for 12 h, the culture medium was ex-
The fluorescence quantum yield (U) was determined by inte-
gration of corrected emission spectra, compared to a SNARF stan-
dard with a known value of 0.03 of the same optical density at
534 nm.26 Under these conditions, quantum yields were calculated
using Eq. 1.
changed to drug containing medium (UTX-12 50 lM). The cells
ꢀ
Z
ꢁ
ꢂ
Z
were then placed in a hypoxic chamber flushed with 5% CO2 and
95% N2 at 25 °C for 2, 4, 6 h at flow rates of 1.5–2.0 NL/min for hyp-
oxic conditions. For the aerobic conditions they were placed at
25 °C under atmosphere gas. After treatment, the culture medium
was exchanged to fresh medium and observed by confocal laser
scanning microscopy (CLSM, Nikon C1si-Ready). Fluorescence at
the emission wavelength of 605 nm was measured at room tem-
perature by exciting SNARF at 544 nm. The fluorescence intensities
of the CLSM images were obtained by the averaged value of at least
ten independent points of the photos. The scanning speed and the
laser intensity were adjusted to avoid photobleaching.
Usample
¼
Ustandard
Fem;
Fem;
ð1Þ
sample
standard
4.4. Biological analysis
4.4.1. Bioreductive activation of nitroaromatic residue by
nitroreductase
Nitroreductase from E. coli (NTR) was purchased from SIGMA
co. ltd. The preincubated solution of UTX-12 (5.0 M) and NADPH
(500 M) in the assay buffer (50 mM Tris–HCl buffer (pH 7.0)) at
l
l
20 1 °C was mixed with NTR (final concentration: 0 or 2.0 U/
ml). The fluorescence spectrum (excitation wavelength:
kex = 534 nm) and UV–vis spectrum then was monitored from 0
Acknowledgements
to 300 min. As the control experiment, UTX-38 (5.0
of UTX-12 was used. The conversion yield from UTX-12 to SNARF
was calculated by UV–vis spectrum (
534 nm = 25 750 Mꢀ1 cmꢀ1).
The reaction solutions were analyzed by HPLC after centrifuga-
tion. The HPLC solvents employed were 95% acetonitrile and 5%
lM) instead
This work was supported in part by Grant-in-Aid for Young Sci-
entist (B) (No. 21710232) from the Ministry of Education, Culture,
Sports, Science and Technology, Japan. The authors thank Professor
Itaru Hamachi (Kyoto University) for usage of the fluorescent spec-
tra measurement, and Ms. Maki Nakamura, Ms. Yuka Sasaki, Ms.
e

6958
E. Nakata et al. / Bioorg. Med. Chem. 17 (2009) 6952–6958
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Emiko Okayama and the staff of our Faculty for measurement of
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Supplementary data
Supplementary data associated with this article can be found, in
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