7-((5-Nitrothiophen-2-yl)methoxy)-3H-phenoxazin-3-one as a spectroscopic off-on probe for highly sensitive and selective detection of nitroreductase

Article abstract of DOI:10.1039/c3cc42610f

A new spectroscopic off-on probe, 7-((5-nitrothiophen-2-yl)methoxy)-3H- phenoxazin-3-one, is developed and applied to real-time detection of nitroreductase produced by Escherichia coli.

Full text of DOI:10.1039/c3cc42610f

ChemComm
View Article Online
View Journal | View Issue
COMMUNICATION
7-((5-Nitrothiophen-2-yl)methoxy)-3H-phenoxazin-
3-one as a spectroscopic off–on probe for highly
sensitive and selective detection of nitroreductase†
Cite this: Chem. Commun., 2013,
49, 5859
Received 10th April 2013,
Accepted 13th May 2013
Zhao Li, Xinghui Gao, Wen Shi, Xiaohua Li and Huimin Ma*
DOI: 10.1039/c3cc42610f
www.rsc.org/chemcomm
A new spectroscopic off–on probe, 7-((5-nitrothiophen-2-yl)methoxy)-
3H-phenoxazin-3-one, is developed and applied to real-time detection
of nitroreductase produced by Escherichia coli.
Nitroreductase plays an important role in chemotherapy and
biodegradation. In the presence of reduced nicotinamide
adenine dinucleotide (NADH) as an electron donor, nitroreductase
can catalyze the reduction of a broad range of nitroaromatic
compounds, and this behavior has been used not only to
activate nitrofuran antibiotics but also to eliminate pervasive
nitroaromatic pollutants.1 Moreover, decrease of oxygen levels
Scheme 1 Synthesis of probe 1 and its reaction with nitroreductase.
in tumor cells is often accompanied by the apparent increase of was designed by connecting 5-nitrothiophene as a masking
endogenous nitroreductase activity.2 Thus, detection of nitro- and recognizing moiety to resorufin through an ether bond.
reductase is of great significance for biological and environ- Resorufin is chosen as a signaling unit owing to its long
mental studies.
analytical wavelength (l570/585nm), good water solubility, and
For the nitroreductase assay,2,3 fluorescence spectroscopy efficient fluorescence quenching via alkylation of the 7-hydroxy
has attracted much attention because of its great temporal group. On the other hand, the usage of thiophene instead of
and spatial sampling capability as well as high sensitivity. furan2a is anticipated to produce more effective fluorescence
Several fluorescent nitroreductase probes have been developed quenching because the electron-donating ability of the S atom
by employing the enzymatic reduction of nitroaromatic is stronger than that of the O atom. By such design, a latent
compounds.2,3 For instance, Huang et al. synthesized a fluores- fluorescent probe3a,4 has been prepared, which is desired for
cence off–on probe by attaching 4-nitrobenzene to coumarin achieving a low background signal. Reaction of 1 with nitro-
with a diamine linker, and Qian et al. prepared a novel fluorescent reductase in the presence of NADH would result in the
probe by linking a p-nitrobenzyl moiety to naphthalimide via a reduction of the 5-nitrothiophene moiety, followed by the
carbamate group for nitroreductase detection.2b,3a In spite of 1,6-rearrangement-elimination reaction and thus the release
these advances, fluorescent probes with superior properties of resorufin (Scheme 1). As a result, both color and fluorescence
such as high sensitivity and selectivity are still rare for nitro- of resorufin are restored, which leads to the establishment of a
reductase analysis.
highly sensitive and selective method for real-time monitoring
We have recently presented a resorufin-based fluorescent probe of nitroreductase produced by Escherichia coli.
for imaging the hypoxic status of tumor cells via the detection of
Probe 1 can be readily synthesized by treating resorufin
endogenous nitroreductase.2a Here we report ((5-nitrothiophen- sodium salt with 2-(bromomethyl)-5-nitrothiophene (2); the
2-yl)methoxy)-3H-phenoxazin-3-one (1; Scheme 1) as an excellent raw material 2 was prepared starting from 5-nitrothiophene-2-
spectroscopic off–on probe for the nitroreductase assay. The probe carbaldehyde, as depicted in Scheme 1. Detailed synthetic steps
and characterization of 1 were described in the ESI.†
Absorption and fluorescence spectra of 1 before and after
reaction with nitroreductase are shown in Fig. 1. As can be seen
from Fig. 1A, probe 1 exhibits a weak absorption in the long-
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Analytical
Chemistry for Living Biosystems, Institute of Chemistry, Chinese Academy of
Sciences, Beijing 100190, China. E-mail: mahm@iccas.ac.cn
wavelength region, but its reaction solution with nitroreductase
shows a strong absorption at about 570 nm, accompanied by a
† Electronic supplementary information (ESI) available: Apparatus and reagents,
and other supporting data. See DOI: 10.1039/c3cc42610f
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 5859--5861 5859

View Article Online
Communication
ChemComm
Next, the kinetic parameters for the enzymatic cleavage
reaction of 1 were determined. Fig. S6 (ESI†) shows the
Lineweaver–Burk plot of 1/V (here V is the initial reaction rate)
versus the reciprocal of the probe concentration. By fitting the
data with the Michaelis–Menten equation, the corresponding
Michaelis constant (Km) and maximum of initial reaction rate
(Vmax) for the enzyme-activated reaction were found to be
34.7 mM and 0.03 mM sÀ1, respectively. Furthermore, a docking
study was performed to evaluate the binding ability of nitro-
reductase with 1. The docking score (Àlog Kd) is found to be
7.07, meaning that the binding action between 1 and nitro-
reductase is strong.7 This is also supported by the result of the
ribbon model created by Pymol. As is seen from Fig. S7 (ESI†),
the potential hydrogen bonds between 1 and the four amino
Fig. 1 (A) Absorption spectra of 1 (5 mM) before (a) and after (b) reaction with
nitroreductase (0.20 mg mLÀ1) in the presence of 50 mM NADH at 37 1C for 20 min.
The color changes of 1 before and after reaction are shown in the inset of Fig. 1A.
(B) Fluorescence spectra of 1 (5 mM) reacting with nitroreductase at varied
concentrations (0, 0.0025, 0.005, 0.025, 0.1, 0.15, 0.2, 0.3, 0.4, 0.6 and 1 mg mLÀ1
in the presence of 50 mM NADH at 37 1C for 20 min. lex = 550 nm.
)
distinct color change from nearly colorless to pink (see the inset acids residues (Ser12, Arg14, Thr74 and Ala79) in the resulting
of Fig. 1A). Furthermore, probe 1 itself has almost no emission complex may contribute to the strong binding affinity as well.
at 585 nm (Fig. 1B), with a quantum yield of F E 0.01,5 which is
To explore the selectivity of the reaction, various potential
four times lower than that of its analogue bearing the furan interfering species, such as inorganic salts (KCl, CaCl2, MgCl2),
moiety.2a This lower background signal is attributed to the glucose, vitamin C, vitamin B6, human serum albumin (HSA),
stronger fluorescence quenching of the thiophene moiety, and reactive oxygen species (H2O2, ꢀOH), glutamic acid, arginine,
is conducive to affording sensitive detection.
serine, and biothiols (glutathione, cysteine, homocysteine and
Effects of pH and temperature on the fluorescence of 1 dithiothreitol) were examined in parallel under the same con-
were studied (Fig. S3, ESI†), which reveals that the probe ditions. As shown in Fig. S8 (ESI†), the probe exhibits high
functions well under physiological conditions (about pH 7 selectivity for nitroreductase over the other species tested, even
and 37 1C). Besides, the influence of NADH on the present including reductive biothiols at a high concentration, which
reaction system was also examined, and the result showed may be attributed to the specific reduction of the substrate
that 50 mM NADH was suitable for nitroreductase of no more (5-nitrothiophene) by the enzyme.
than 1 mg mLÀ1
.
The effect of a common inhibitor of nitroreductase,
Fluorescence kinetic curves of 1 reacting with nitroreductase dicoumarin,8 on the activity of the enzyme was also studied.
at varied concentrations are given in Fig. S4 (ESI†), which As shown in Fig. S9 (ESI†), the fluorescence intensity in the
indicates that higher concentrations of nitroreductase result presence of 0.1 mM dicoumarin is much less than that in
in faster cleavage reaction and stronger fluorescence intensity. the absence of the inhibitor. This indicates that the enzyme
For nitroreductase of r1 mg mLÀ1, this fluorescence increase activity can be effectively inhibited by dicoumarin, and thus the
could reach a plateau in about 20 min. In contrast, the fluorescence off–on response of 1 to nitroreductase indeed
fluorescence of 1 in the absence of nitroreductase (control) arises from the enzyme-catalyzed cleavage reaction.
scarcely changes during the same period of time, also implying
that the probe is stable in the detection system.
It is known that Escherichia coli can generate nitroreductase,
and this bacterial enzyme has been considered to be useful
Under the optimized conditions (reaction at 37 1C for 20 min for the removal of nitroaromatic pollutants.1,9 However, the
in 10 mM PBS of pH 7.4 in the presence of 50 mM NADH), the specific level of nitroreductase produced by Escherichia coli
fluorescence response of 1 to nitroreductase at varied concen- remains unclear, and solving this problem would thus be
trations is shown in Fig. 1B. As can be seen, the fluorescence helpful for better understanding the biodegradation efficiency
intensity is increased with increasing nitroreductase concen- of nitroaromatic compounds as well as the activation of nitro-
tration, and a good linearity is obtained in the concentration furan prodrugs. In view of high sensitivity and selectivity of
range of 0.005–0.3 mg mLÀ1 nitroreductase, with a linear probe 1, here, using it we made such an attempt. In this
equation of DF = 4.2 Â 103 C (mg mLÀ1) À 11.8 (R = 0.997), experiment, Escherichia coli (DH5a) was grown at 37 1C for 12 h
where DF is the difference of fluorescence intensity of 1 in the in Luria–Bertani (LB) culture media (pH 7.4). Then, the bacterial
presence and absence of nitroreductase. The detection limit colonies were harvested using a sterile swab and inoculated into
(3S/m, in which S is the standard deviation of blank measure- 50 mL of fresh LB culture media with an OD600 of 0.2.10 An
ments, n = 11, and m is the slope of the linear equation) is appropriate volume (typically 3 mL) of the 50 mL culture media
determined to be 0.1 ng mLÀ1 nitroreductase, which is the was taken and then pre-incubated at 37 1C in a rotary shaker for
lowest one to our knowledge.2,3a It is noted that both the different periods of time (0–4 h). After reaction with probe 1
absorption and fluorescence spectra from the reaction system (5 mM) for 20 min, both fluorescence and absorbance of the
resemble those2a,6 of resorufin, suggesting that the enzyme- reaction solutions were measured (see ESI† for details). As
triggered cleavage reaction causes the release of free resorufin shown in Fig. 2A, the fluorescence of the reaction solutions is
(Scheme 1). The generation of resorufin was further proved by increased with increasing pre-incubation time of Escherichia coli,
electrospray ionization mass spectral analysis (m/z = 211.9 [M À H]À; which may be because the generation of more nitroreductase
see Fig. S5, ESI†).
leads to the release of more resorufin; the formation of
c
5860 Chem. Commun., 2013, 49, 5859--5861
This journal is The Royal Society of Chemistry 2013

View Article Online
ChemComm
Communication
In summary, we have developed a highly sensitive and
selective spectroscopic off–on probe for nitroreductase detec-
tion. The detection limit of the probe for nitroreductase is
down to 0.1 ng mLÀ1. Moreover, the probe can be applied
to real-time monitoring of nitroreductase produced by
Escherichia coli.
We are grateful for the financial support from the NSF of
China (No. 21275146, 21275147, 20935005 and 21105104), the
Ministry of Science and Technology (2011CB935800), and the
Chinese Academy of Sciences (KJCX2-EW-N06-01).
Fig. 2 (A) Fluorescence change of probe 1 (5 mM) reacting with Escherichia coli
that was pre-incubated in the LB culture media (pH 7.4) for different periods of
time (0, 1, 2, 3 and 4 h); lex/em = 550/585 nm. (B) Absorbance change of the LB
culture media (pH 7.4) containing only Escherichia coli with incubation time. In
these experiments, Escherichia coli has an initial OD600 of 0.2, and the data are
the mean Æ standard deviation of three separate measurements.
Notes and references
1 (a) J. C. Spain, Annu. Rev. Microbiol., 1995, 49, 523–555;
(b) P. R. Race, A. L. Lovering, R. M. Green, A. Ossor, S. A. White,
P. F. Searle, C. J. Wrighton and E. I. Hyde, J. Biol. Chem., 2005, 280,
13256–13264; (c) J. Whiteway, P. Koziarz, J. Veall, N. Sandhu,
P. Kumar, B. Hoecher and I. B. Lambert, J. Bacteriol., 1998, 180,
5529–5539; (d) A. Caballero, J. J. Lazaro, J. L. Ramos and A. Esteve-
Nunez, Environ. Microbiol., 2005, 7, 1211–1219; (e) A. Celik and
G. Yetis, Bioorg. Med. Chem., 2012, 20, 3540–3550.
2 (a) Z. Li, X. H. Li, X. H. Gao, Y. Y. Zhang, W. Shi and H. M. Ma, Anal.
Chem., 2013, 85, 3926–3932; (b) L. Cui, Y. Zhong, W. Zhu,
Y. Xu, Q. Du, X. Wang, X. Qian and Y. Xiao, Org. Lett., 2011, 13,
928–931.
3 (a) H. C. Huang, K. L. Wang, S. T. Huang, H. Y. Lin and C. M. Lin,
Biosens. Bioelectron., 2011, 26, 3511–3516; (b) E. Johansson,
G. N. Parkinson, W. A. Denny and S. Neidle, J. Med. Chem., 2003,
46, 4009–4020; (c) A. L. James, J. D. Perry, C. Jay, D. Monget,
J. W. Rasburn and F. K. Gould, Lett. Appl. Microbiol., 2001, 33,
403–408; (d) M. Cellier, O. J. Fabrega, E. Fazackerley, A. L. James,
S. Orenga, J. D. Perry, V. L. Salwatura and S. P. Stanforth, Bioorg.
Med. Chem., 2011, 19, 2903–2910.
4 (a) X. Q. Chen, M. Sun and H. M. Ma, Curr. Org. Chem., 2006, 10,
477–489; (b) W. Shi and H. M. Ma, Chem. Commun., 2012, 48,
8732–8744; (c) Y. Meyer, J.-A. Richard, B. Delest, P. Noack, P.-Y.
Renard and A. Romieu, Org. Biomol. Chem., 2010, 8, 1777–1780;
(d) C. D. Sun, W. Shi, Y. C. Song, W. Chen and H. M. Ma, Chem.
Commun., 2011, 47, 8638–8640; (e) D. Feng, Y. Y. Zhang, T. T. Feng,
W. Shi, X. H. Li and H. M. Ma, Chem. Commun., 2011, 47,
10680–10682; ( f ) L. A. Montoya and M. D. Pluth, Chem. Commun.,
2012, 48, 4767–4769.
5 C. Bueno, M. L. Villegas, S. G. Bertolotti, C. M. Previtali,
M. G. Neumann and M. V. Encinas, Photochem. Photobiol., 2002,
76, 385–390.
6 (a) W. Chen, Z. Li, W. Shi and H. M. Ma, Chem. Commun., 2012, 48,
2809–2811; (b) Y. Y. Zhang, W. Chen, D. Feng, W. Shi, X. H. Li and
H. M. Ma, Analyst, 2012, 137, 716–721.
7 (a) A. N. Jain, J. Med. Chem., 2003, 46, 499–511; (b) W. B. Hu, Q. Luo,
K. Wu, X. C. Li, F. Y. Wang, Y. Chen, X. Y. Ma, J. P. Wang,
J. A. Liu, S. X. Xiong and P. J. Sadler, Chem. Commun., 2011, 47,
6006–6008.
8 R. L. Koder and A. F. Miller, Biochim. Biophys. Acta, 1998, 1387,
395–405.
Table 1 Real-time detection of nitroreductase produced by Escherichia coli
(DH5a) with an initial OD600 of 0.2
Growth time (h)
0
1
2
3
4
Concentration of 8.4 Æ 0.1 9.7 Æ 0.3 11.3 Æ 0.4 17.1 Æ 0.4 36.6 Æ 0.8
nitroreductase in
the culture mediaa
(ng mLÀ1
)
a
Mean of three determinations Æ standard deviation.
resorufin in the LB culture media was also verified by electro-
spray ionization mass spectral analysis (m/z = 212 [M À H]À;
Fig. S10, ESI†). On the other hand, addition of dicoumarin
largely inhibits the activity of the Escherichia coli nitroreductase
and thus decreases the fluorescence of the reaction solutions
(Fig. S11, ESI†), which also indicates that the fluorescence
generation of the reaction solutions containing Escherichia coli
and probe 1 may be due to the action of nitroreductase.
Furthermore, a corresponding calibration curve of 1 reacting
with nitroreductase in the cell-free LB culture media containing
50 mM NADH was constructed to eliminate any possible inter-
ference, and with this calibration curve the concentration of
nitroreductase produced by Escherichia coli has been quantita-
tively determined in real time. As shown in Table 1, the concen-
tration of nitroreductase in the culture media is increased with
increasing growth time. Interestingly, it is noted that this increase
is nonlinear. For example, the concentration (36.6 ng mLÀ1) of the
Escherichia coli nitroreductase under the 4 h growth condition is
not two times higher than that (11.3 ng mLÀ1) under the 2 h
growth condition. The above results provide the first information
about the levels of nitroreductase produced by Escherichia coli,
and show that probe 1 is suited for real-time and quantitative
determination of nitroreductase in biosystems.
9 (a) R. E. Asnis, Arch. Biochem. Biophys., 1957, 66, 208–216; (b) W. D.
Won, R. J. Heckly, D. J. Glover and J. C. Hoffsommer, Appl. Microbiol.,
1974, 27, 513–516.
10 (a) G. Sezonov, D. Joseleau-Petit and R. D’Ari, J. Bacteriol., 2007, 189,
8746–8749; (b) A. M. Sanchez, G. N. Bennett and K.-Y. San,
J. Biotechnol., 2005, 117, 395–405; (c) S. J. Berrios-Rivera,
G. N. Bennett and K.-Y. San, Metab. Eng., 2002, 4, 217–229;
(d) M. D. Roldan, E. Perez-Reinado, F. Castillo and C. Moreno-
Vivian, FEMS Microbiol. Rev., 2008, 32, 474–500; (e) H. E. Kubitschek,
Biophys. J., 1974, 14, 119–123.
Fig. 2B shows the growth of Escherichia coli with time. As can
be seen, the growth curve of Escherichia coli is consistent with
the fluorescence change curve of Fig. 2A, suggesting that probe 1
may also be used as a microbial growth indicator.
c
This journal is The Royal Society of Chemistry 2013
Chem. Commun., 2013, 49, 5859--5861 5861