A highly sensitive long-wavelength fluorescence probe for nitroreductase and hypoxia: Selective detection and quantification

Article abstract of DOI:10.1039/c3cc45367g

A novel long-wavelength fluorescence probe NBP has been developed for the detection of nitroreductase (NTR) and hypoxia. NBP could be activated by NTR at 0.1 μM to release the fluorophore NBF and significant changes in fluorescence emission at 658 nm were

Full text of DOI:10.1039/c3cc45367g

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A highly sensitive long-wavelength fluorescence probe
for nitroreductase and hypoxia: selective detection and
quantification†
Cite this: Chem. Commun., 2013,
49, 10820
Received 16th July 2013,
Accepted 24th September 2013
Ting Guo, Lei Cui, Jiaoning Shen, Weiping Zhu, Yufang Xu* and Xuhong Qian*
DOI: 10.1039/c3cc45367g
www.rsc.org/chemcomm
A novel long-wavelength fluorescence probe NBP has been developed various advantages, including deeper penetration and minimized
for the detection of nitroreductase (NTR) and hypoxia. NBP could autofluorescence.13 Nile Blue is widely used in biological imaging
be activated by NTR at 0.1 lM to release the fluorophore NBF and because of its excellent photochemical stability, long wavelength
significant changes in fluorescence emission at 658 nm were excitation/emission maxima and modest Stokes shifts.14 Herein,
observed. This feature makes it advantageous for imaging hypoxic we describe an NIR fluorescence probe (NBP) based on Nile Blue
cells with minimal endogenous interference.
for nitroreductase and tumor hypoxia with minimized inter-
ference from absorption and autofluorescence of bio-species.
Our strategy for the construction of this NIR probe NBP relies
Hypoxia, regions of low oxygen tension, is characteristic of solid
tumors.1 Hypoxia renders tumor cells higher resistance towards on tethering the p-nitrobenzyl moiety to a Nile Blue fluorophore
cancer therapies and is associated with metastasis.2 Compared via a self-collapsible carbamate linkage (Scheme 1).15 The
to normal cells, hypoxic cells are found to display an elevated p-nitrobenzyl moiety as a nitroreductase-sensitive moiety has
level of reductive enzymes, such as nitroreductase, DT-diaphor- been frequently used in prodrugs and detection of tumor
ase and azoreductase, which result in reductive stress.3,4 Thus, hypoxia.4,16 To improve the cell permeability of the probe, we
hypoxia and these enzymes represent a viable target for tumor introduced two methyl esters into the Nile Blue scaffold. Under
cell detection and treatment. Nitroreductases (NTRs), a family hypoxic conditions, the p-nitrobenzyl moiety of the probe NBP
of flavin-containing enzymes, reduce nitroaromatics to corre- was reduced to form the p-aminobenzyl derivative, which is
sponding nitroso, hydroxylamine or amino derivatives, which unstable and spontaneously collapses to release the Nile Blue
are often mutagenic or carcinogenic.5,6 Recently, NTRs were fluorophore NBF, and the fluorescence was restored.
employed in development of gene- or virus-directed enzyme
prodrug cancer therapies and drug screening.7,8 Therefore, the
detection of NTRs activity and hypoxia is significant for drug
development and tumor diagnosis.
Hypoxia or NTRs have been detected by many approaches.9
Because of their high sensitivity and spatiotemporal resolution,
fluorescence probes are favorable tools for noninvasive detection
of enzymes and disease diagnosis.10 Recently, several fluores-
cence probes against hypoxia have been developed based on
various reductases such as nitroreductase, azoreductase and
quinone reductase.11 A few classic examples include a p-nitro
benzyl moiety functionalized SNARF dye reported by Nakata and
co-workers, the azo bond tethered BHQ–Cy5 pair reported by
Nagano et al. and an indolequinone unit employed as the hypoxia-
sensitive moiety by Komatsu et al.12 Near-infrared probes offer
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of
Chemical Biology, School of Pharmacy, East China University of Science and
Technology, 130 Meilong Road, Shanghai, 200237, China.
E-mail: xhqian@ecsut.edu.cn, yfxu@ecust.edu.cn; Fax: +86 21-64252603
Scheme 1 Synthetic scheme of the fluorescence probe NBP and the deduced
mechanism of NBP activated by the enzyme NTR.
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c3cc45367g
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The probe NBP was readily synthesized using p-aminophenol
NB-2 as the starting material (Scheme 1) and its structure was
confirmed by 1H- and 13C-NMR and HRMS. The reactive azo
compound NB-3 was synthesized by the reaction of substituted
p-aminophenol NB-2 with 1 equivalent of an aryl diazonium salt.
The condensation reaction of NB-3 with 1-aminonaphthalene
was accomplished by stirring in perchloric acid at 160 1C to
afford the Nile Blue fluorophore (NBF). NBF was stirred with
4-nitrophenyl chloroformate in anhydrous THF–DCM for 2 hours
to yield the probe NBP.17
Fig. 2 Fluorescent spectra of NBP with respect to concentrations of nitroreductase.
(a) 1 mM NBP was incubated with different concentrations of nitroreductase and
50 eq. NADH. (b) A linear correlation between fluorescence response and concen-
trations of nitroreductase. The excitation wavelength was 580 nm.
The fluorescence properties of fluorophore NBF with respect
to pH were investigated (Fig. S1, ESI†). NBF exhibits a pKa of
9.6, which is two units lower than the physiological pH. There-
fore, NBF can be used in biological systems. Spectroscopic
evaluation of NBP and NBF was carried out in PBS buffer
(pH 7.4, 0.01 M) with 1% DMSO as cosolvent. The probe NBP
has a strong absorption peak at 525 nm and is essentially non-
fluorescent. The fluorophore NBF shows maximal absorption
and a strong fluorescence peak at around 613 nm and 658 nm,
respectively (Fig. S2, ESI†). When NBP was incubated with the
enzyme NTR, a drastic enhancement of fluorescence intensity
at 658 nm was observed.
Next, we studied the reactivity of the probe NBF toward NTR
reduction.18 The fluorescence spectra were collected in the
presence of NADH (50 mM, as a cofactor of NTR) in PBS buffer
(pH 7.4, 0.01 M, 1% DMSO) at 37 1C. Under these conditions,
the free probe NBP (1 mM) is nearly non-fluorescent when
excited at around 580 nm. Upon addition of 12.5 mg mLÀ1 of
oxygen-sensitive NTR, a time dependent fluorescence enhance-
ment was observed and the maximal fluorescence intensity was
enhanced at around 658 nm in 1 h (Fig. 1a). This demonstrated
that the p-nitrobenzyl moiety of the probe NBP was reduced and
the fluorophore NBF was released as expected. The formation
of NBF was also confirmed by HPLC analysis (Fig. S3, ESI†). We
also found that the fluorescence enhancement is related to
the initial concentrations of NBP (0–1 mM) (Fig. 1b). As shown
in Fig. 2a, the fluorescence enhancement of NBP was dose-
dependent with respect to NTR. A linear response was found at
2.5–12.5 mg mLÀ1 and the detection limit was deduced to be
180 ng mLÀ1 (Fig. 2b). Therefore, NBP is a sensitive fluores-
cence probe for detection of nitroreductase not only because of
the detection limit but also because of the low concentration
of the probe. This implies its potential for application in bio-
logical systems.
To further study the versatility of NBP, we investigated the
selectivity of NBP toward other biological reducing agents such as
thiols, which have been reported to reductively activate various
antitumor prodrugs.19 The probe NBP (1 mM) was treated with the
various biological reductants in PBS buffer (pH 7.4, 0.01 M, 1%
DMSO) at 37 1C, such as dithiothreitol (DTT), glutathione (GSH),
cysteine (Cys) and homocysteine (Hcy). As shown in Fig. 3,
biological thiols (2 mM) induced nearly no fluorescence
responses within 70 min at 37 1C. And the impact of NADH
on the probes NBF and NBP was also studied. Upon addition of
50 equiv. of NADH (50 mM), the fluorescence intensity of NBF
decreased slightly (Fig. S4, ESI†) and the fluorescence intensity
of NBP remained negligible, even after 70 min at 37 1C (Fig. S5,
ESI†).20 In contrast, incubation of the probe NBP (1 mM) with
NTR (12.5 mg mLÀ1) and NADH (50 mM) together induced
distinguished fluorescence enhancement at around 658 nm.
These results suggested that the fluorescence changes of NBP
were only induced by nitroreductase and NADH, which can be
used to selectively monitor NTR without interferences from
other biological reductants.
The MTT assay showed that NBP did not exhibit cytotoxicity
at 1 mM and at 37 1C for 48 h (Fig. S6, ESI†). The application
of the probe NBP for intracellular hypoxia detection was also
investigated. A549 cells were incubated under normoxic (20% pO2)
and hypoxic (5% and 1% pO2) conditions for 8 h at 37 1C. Then the
Fig. 1 Fluorescent spectra of NBP to NTR. (a) NBP (1 mM) was cultured with
12.5 mg mLÀ1 NTR and NADH (50 mM). (b) Fluorescence response (I658) with varied
concentrations of NBP: 0 (control), 0.1, 0.3, 0.5 and 1 mM. The measurements were
performed in 0.01 M PBS buffer (pH 7.4, 1% DMSO) with 12.5 mg mLÀ1
nitroreductase and 50 equivalents of NADH at 37 1C over an incubation time
of 70 min. The fluorescence intensity data were collected after a certain time at
around 658 nm. The excitation wavelength was 580 nm.
Fig. 3 Fluorescence response of NBP to the biological reductants. Control: the free
probe NBP. Fluorescence response of NBP (1 mM) treated with various biological
thiol reductants (2 mM) and NADH (50 eq.) in PBS buffer (0.01 M, pH 7.4 with 1%
DMSO) at 37 1C. The fluorescent intensity data at around 658 nm were collected
after reaction for 70 min. Fluorescent excitation was provided at 580 nm.
c
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The authors are grateful for the financial support from
the State Key Program of National Natural Science of China
(21236002), the National Basic Research Program of China
(2010CB126100), the National High Technology Research
and Development Program of China (2011AA10A207), and the
Fundamental Research Funds for the Central Universities.
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