A new prodrug-derived ratiometric fluorescent probe for hypoxia: High selectivity of nitroreductase and imaging in tumor cell

Article abstract of DOI:10.1021/ol102975t

Based on the hypoxia prodrug moiety of p-nitrobenzyl, a selective ratiometric fluorescent sensor (RHP) for the detection of microenvironment hypoxia was designed and synthesized. RHP can be selectively activated by bioreductive enzymes (NTR) and results i

Full text of DOI:10.1021/ol102975t

ORGANIC
LETTERS
2011
Vol. 13, No. 5
928–931
A New Prodrug-Derived Ratiometric
Fluorescent Probe for Hypoxia:
High Selectivity of Nitroreductase
and Imaging in Tumor Cell
Lei Cui,† Ye Zhong,† Weiping Zhu,† Yufang Xu,*,† Qingshan Du,†
Xin Wang,† Xuhong Qian,*,† and Yi Xiao‡
State Key Laboratory of Bioreactor Engineering, Shanghai Key Laboratory of Chemical
Biology, School of Pharmacy, East China University of Science and Technology
(ECUST), Shanghai 200237, China, and Dalian University of Technology (DLUT),
Zhongshan Road 158, Dalian, China
yfxu@ecust.edu.cn; xhqian@ecust.edu.cn
Received December 8, 2010
ABSTRACT
Based on the hypoxia prodrug moiety of p-nitrobenzyl, a selective ratiometric fluorescent sensor (RHP) for the detection of microenvironment
hypoxia was designed and synthesized. RHP can be selectively activated by bioreductive enzymes (NTR) and results in an evident blue to green
fluorescent emission wavelength change in both solution phases and in cell lines, which might be the first fluorescent ratiometric probe for
hypoxia in solid tumors.
Hypoxia is usually observed in solid tumors and affects
the therapy of tumor cells by preventing the proper
metabolism of various anticancer drugs.1 Clinical research
found that a hypoxic environment in tumor cells has a
positive correlation with their resistance to treatment and
metastasis.2 In cervix cancer and head and neck squamous
cancer, hypoxia has been consideredasa poor progression-
free survival signal for detection.3 Therefore, it is of con-
siderable clinical significance to measure the hypoxic frac-
tion in tumors.4
(3) (a) Fyles, A.; Milosevic, M.; Pintilie, M.; Syed, A.; Levin, W.; Manchul,
L.; Hill, R. P. Radiother. Oncol. 2006, 80, 132–137. (b) Kaanders, J. H.;
Wijffels, K. I.; Marres, H. A.; Ljungkvist, A. S.; Pop, L. A.; van den Hoogen,
F. J.; de Wilde, P. C.; Bussink, J.; Raleigh, J. A.; van der Kogel, A. J. Cancer
Res. 2002, 62, 7066–7074.
(4) (a) He, F.; Deng, X.; Wen, B.; Liu, Y.; Sun, X.; Xing, L.; Minami,
A.; Huang, Y.; Chen, Q.; Zanzonico, P. B.; Ling, C. C.; Li, G. C. Cancer
Res. 2008, 68, 8597–8606. (b) Iglesias, P.; Costoya, J. A. Biosens.
Bioelectron. 2009, 24, 3126–3130. (c) Lehmann, S.; Stiehl, D. P.; Honer,
M.; Dominietto, M.; Keist, R.; Kotevic, I.; Wollenick, K.; Ametamey,
S.; Wenger, R. H.; Rudin, M. Proc. Natl. Acad. Sci. U.S.A. 2009, 106,
14004–14009.
† ECUST.
‡ DLUT.
(1) (a) Williams, K. J.; Albertella, M. R.; Fitzpatrick, B.; Loadman,
P. M.; Shnyder, S. D.; Chinje, E. C.; Telfer, B. A.; Dunk, C. R.; Harris, P. A.;
Stratford, I. J.. Mol. Cancer Ther. 2009, 8, 3266–3275. (b) Shinohara, E. T.;
Maity, A. Curr. Mol. Med. 2009, 9, 1034–45.
(2) (a) Hockel, M.; Schlenger, K.; Aral, B.; Mitze, M.; Schaffer, U.;
Vaupel, P. Cancer Res. 1996, 56, 4509–4515. (b) Rofstad, E. K.; Rasmussen,
H.; Galappathi, K.; Mathiesen, B.; Nilsen, K.; Graff, B. A. Cancer Res. 2002,
62, 1847–1853.
r
10.1021/ol102975t
Published on Web 01/26/2011
2011 American Chemical Society

Scheme 1. Synthesis of the Probe RHF and RHP
aqueous solution based on an intramolecular charge trans-
fer (ICT) mechanism.10
The p-nitrobenzyl moiety is used frequently in the devel-
opment of prodrugs for hypoxia.11 Therefore, we designed
our hypoxia probe RHP by connecting a p-nitrobenzyl
moietyand a signaling moietyRHF via a carbamate group.
The electron-withdrawing carbamate group weakens the
ICT effect and results in a fluorescent emission wavelength
blue shift. Upon reduction, the amino group of RHF will
be released and the fluorescence emission will be restored12
(Figure 1). Both probe RHP and bioreduction product
RHF were efficiently synthesized (Scheme 1) and well
characterized.
Next, we studied their spectral characteristics in chemi-
cal, enzymatic, and cell media. Spectroscopic evaluation of
RHP and RHF was carried out under physiological con-
ditions at 37 °C in PBS buffer (pH=7.0, 0.01 M) with 1%
DMSO (Figure S1). The absorption spectra of RHP and
RHF have an intersection at a wavelength of 410 nm
implying that they could be equally excited at this wave-
length. Thus, wechose 410 nm asthe excitationwavelength
for ratiometric studies. The maximum emission wave-
length of the RHP locates at 475 nm. And that of RHF is
longer by ca.75 nm and falls at 550 nm (Figures S2, S3).
Thus, the probe exhibited a favorable ratiometric signal
change13 (Table S1).
Figure 1. Proposed detection mechanism of RHP.
Various methods for hypoxia detection are available.5
However, their practical applications are restricted due to
the invasiveness. Fluorescence based techniques havesome
obvious advantages since they are noninvasive, display
high sensitivity, and provide high spatiotemporal resolu-
tion. They have also been viewed as a powerful and
versatile toolbox in the field of life sciences, environmental
monitoring, and disease diagnosis.6 Based on nitro groups,
which can be reduced to amino groups under hypoxic
conditions, several fluorescent probes for hypoxia were
reported.7 Fraser et al. reported an elegant sensing plat-
form that emits both fluorescence and phosphorescence
for hypoxia imaging.8 Probes with dual emission are
particularly viable because they enable a built-in calibra-
tion for environmental effects.9 Herein, we describe a novel
prodrug-derived ratiometric fluorescent probe RHP in
(5) (a) Dasu, A.; Denekamp, J. Int. J. Radiat. Oncol. 1999, 43, 1083–
1094. (b) Stern, S.; Guichard, M. Radiother. Oncol. 1996, 41, 143–149.
(6) (a) Elfeky, S. A.; Flower, S. E.; Masumoto, N.; D’Hooge, F.;
Labarthe, L.; Chen, W.; Len, C.; James, T. D.; Fossey, J. S. Chem.
;
Asian J. 2010, 5, 581–588. (b) Jiang, W.; Cao, Y.; Liu, Y.; Wang, W.
Chem. Commun. 2010, 46, 1944–1946. (c) Zhang, X.; Chi, L.; Ji, S.; Wu,
Y.; Song, P.; Han, K.; Guo, H.; James, T. D.; Zhao, J. J. Am. Chem. Soc.
2009, 131, 17452–17463. (d) Lee, Y. H.; Liu, H.; Lee, J. Y.; Kim, S. H.;
We then tested the uncaging sensitivity of the p-nitrobenzyl
moiety under enzymatic reduction. The nitro group of RHP
was reduced by an oxygen-sensitive nitroreductase (NTR)
expressed in Escherichia coli. The fluorescence spectra of
RHP were collected in the presence of NTR (3.2 unit of
NTR), and a time dependent fluorescence change originated
from RHP was observed (Figure 2). Within 10 min, the
maximal fluorescence intensity at a wavelength of 550 nm
Kim, S. K.; Sessler, J. L.; Kim, Y.; Kim, J. S. Chem. Eur. J. 2010, 16,
;
5895–5901.
(7) (a) Zhu, W.; Dai, M.; Xu, Y.; Qian, X. Bioorg. Med. Chem. 2008,
16, 3255–3260. (b) Dai, M.; Zhu, W. P.; Xu, Y. F.; Qian, X. H.; Liu, Y.;
Xiao, Y.; You, Y. J. Fluoresc. 2008, 18, 591–597. (c) Tanabe, K.; Hirata,
N.; Harada, H.; Hiraoka, M.; Nishimoto, S. ChemBioChem 2008, 9,
426–432. (d) Hodgkiss, R. J.; Parrick, J.; Porssa, M.; Stratford, M. R.
J. Med. Chem. 1994, 37, 4352–4356. (e) Kiyose, K.; Hanaoka, K.;
Oushiki, D.; Nakamura, T.; Kajimura, M.; Suematsu, M.; Nishimatsu,
H.; Yamane, T.; Terai, T.; Hirata, Y.; Nagano, T. J. Am. Chem. Soc.
2010, 132, 15846–15848.
was enhanced by more than 4-fold and the ratio (F550/F475)
of RHF to RHP emission upon excitation at 410 nm exhibited
(8) Zhang, G.; Palmer, G. M.; Dewhirst, M. W.; Fraser, C. L. Nat.
Mater. 2009, 8, 747–751.
(11) (a) Brown, J. M.; Wilson, W. R. Nat. Rev. Cancer 2004, 4, 437–
447. (b) Xu, G.; McLeod, H. L. Clin. Cancer Res. 2001, 7, 3314–3324.
(c) Denny, W. A. Lancet Oncol. 2000, 1, 25–29. (d) Denny, W. A. Eur. J.
Med. Chem. 2001, 36, 577–595.
(12) Jaffar, M.; Williams, K. J.; Stratford, I. J. Adv. Drug Delivery
Rev. 2001, 53, 217–228.
(9) Tsien, R. Y.; Poenie, M. Trends Biochem. Sci. 1986, 11, 450–455.
(10) (a) Srikun, D.; Miller, E. W.; Domaille, D. W.; Chang, C. J.
J. Am. Chem. Soc. 2008, 130, 4596–4597. (b) Cui, L.; Zhong, Y.; Zhu, W.;
Xu, Y.; Qian, X. Chem. Commun. 2010, 46, 7121–7123. (c) Bozdemir, O. A.;
Sozmen, F.; Buyukcakir, O.; Guliyev, R.; Cakmak, Y.; Akkaya, E. U. Org.
Lett. 2010, 12, 1400–1403. (d) Ojida, A.; Miyahara, Y.; Wongkongkatep, J.;
(13) The quantum yield (ΦF) is determined by using Fluorescein with
0.1 M NaOH solution (ΦF =0.95) as a standard.
Tamaru, S.; Sada, K.; Hamachi, I. Chem. Asian. J. 2006, 1, 555–563.
;
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Figure 3. Ratio changes (F550/F475) with the λex = 410 nm at
37 °C over an incubation time of 10 min at varied concentra-
tions of RHP: 0 (control), 5, 10, 20, 50, and 100 μM. The
measurements were performed in PBS buffer with 10 μg/mL
nitroreductase and 500 μM of NADH.
Figure 2. Fluorescent spectra of RHP (10 μM) in PBS buffer
with 1% DMSO treated with nitroreductase (10 μg/mL) and
NADH (500 μM) with excitation at 410 nm. After addition of
RHP to the nitroreductase buffer, the fluorescent intensity data
were collected after certain time intervals as indicated in the
figure. Inset: the emission ratio (F550/F475) verse time.
a notable dynamic range from 0.3 to 1.2 (inset of Figure 2).
It was calculated, using their photophysical parameters,
that around 80% of RHP was converted to RHF. After the
bioreaction for 10 min, the fluorescence intensity of RHP
wasdecreasedbutnotfullyquenched, asshowninFigure2;
this could be attributed to the uncompleted biological
reaction and relatively high fluorescence quantum yield
(Table S1) of RHP. Fluorescence and naked eye images of
RHP to NTR also showed a huge color difference before
and after reaction (Figure S4). We also certified that the
nitroreductase metabolite is RHF using HPLC analysis
(FigureS5).TheaboveresultsprovedthatRHP was a success-
ful ratiometric fluorescent probe mediated by nitroreduction.
The kinetics of RHF release from RHP triggered by
nitroreductase inthe presence of NADHwere measuredby
adding a known concentration of RHP (5-100 μM) to the
reaction mixture. The fluorescence change was recorded at
a wavelength of 475 and 550 nm, respectively. The rate was
calculated using the fluorescence emission intensity ratio
at a wavelength of 475 and 550 nm. All assays were per-
formed in triplicate, and the results reported are the
average of three experiments (Figure 3).
Fluorescence response assays by the biological reduc-
tants were also carried out because biological thiols have
been suggested to be the electron providers for reductive
activation of various hypoxia sensitive antitumor prodrugs.14
Therefore, the fluorescence response of RHP to other bio-
relevant thiols, such as dithiothreitol (DTT), glutathione
(GSH), cysteine (Cys), homocysteine (Hcy), and β-Nicotin-
amide adenine dinucleotide (NADH), was investigated.
Under normal physiological conditions, the concentration
of reduced biological reductants is far lower than 1 mM.
As shown in Figure 4, incubation of the probe RHP with
up to 100 equiv of biological reductants did not induce
any noticeable signal modulation. The selectivity was
Figure 4. Fluorescence response assays by the biological reduc-
tants. Control: no addition compound. Control-1: all of the
biological reductants and NTR. Fluorescence responses of RHP
(10 μM) coincubated with various biological thiol reductants in
phosphate buffered saline at pH 7.0. Bars represent the final
fluorescence intensity at the wavelength 550 nm after the reaction
for 30 min (I550) over final fluorescence intensity at the wavelength
475 nm after reaction for 30 min (I475) at λex = 410 nm.
also tested in the mixture of NTR with 100 equiv of other
biological reductants, and the changes in fluorescence inten-
sity were also negligible. The competition experiments
showed that in the mixture of NTR and NADH with 100
equiv of other biological reductants, no obvious influence
was found in comparison to the experiments containing
only NTR and NADH (Figure 4). The above results demon-
strated that the thiols employed in these experiments
exhibited no interferences and our probe RHP showed a
very high selectivity for NTR. Additionally, this observa-
tion could potentially benefit the development of hypoxia
targeted prodrugs based on nitroreduction.
To further demonstrate the potential of the probe for
practical application, we also applied RHP in living cells
for ratiometric fluorescenceimaging of hypoxia. A549cells
were incubated with RHP under hypoxic and normal
physiological conditions, respectively, at 37 °C for 7 h
and subsequently washed 3 times with PBS buffer to
remove the extracellular probe (containing 0.5% DMSO,
(14) (a) Paz, M. M. Chem. Res. Toxicol. 2009, 22, 1663–1668. (b) Paz,
M. M.; Tomasz, M. Org. Lett. 2001, 3, 2789–2792. (c) Wolkenberg, S. E.;
Boger, D. L. Chem. Rev. 2002, 102, 2477–2495.
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collection windows using ImageJ software gave an average
emission value of the fluorescent photos.
From the images above, we could see that there was a
notable increase between 540 to 580 nm (green) (Figure 5a
and 5d) of fluorescence under aerobic and hypoxic condi-
tions. The data in Figure 5c and 5f show clear increases in
green-to-blue emission ratios under normal conditions
(<45) compared to the cells under hypoxic conditions
(>45). Finally, we evaluated the cytotoxicity of RHP and
RHF, and the results showed that when the concentrations
of RHP and RHF were less than 1 mM, the death rate of
RHP and RHF were as low as 5% when both of the
compounds RHP and RHF were at the concentration
10 μM (Figure S7). These data have clearly demonstrated
that RHP is capable of in vitro imaging of hypoxia in solid
tumors. Moreover, the ratiometric readout provided by
this probe allows for detection of a highly imperceptible
change of oxygen tension in a solid tumor.
In summary, we have developed a selective ratiometric
fluorescent probe (RHP) for microenvironment hypoxia
detection. RHP can be selectively activated by reductive
enzymes (NTR) and results in an evident blue to green
fluorescent emission wavelength change in both solution
phases. It was also successfully applied in in vitro imaging
of hypoxia in an A549 cell line, displayed no cytotoxicity,
and is highly promising for real world applications such as
in tumor diagnosis via hypoxia imaging. In addition to
monitoring the oxygen environment, other potential ap-
plications including monitoring the effect of drug treat-
ment are being investigated.
Figure 5. Fluorescence microphotographs of A549 cells incu-
bated with RHP (1) at 37 °C in F-12 Nutrient Mixture (Ham).
The top row was at aerobic condition (a, b, and c). The bottom
row was under hypoxic conditions (d, e, and f). All cells
were incubated with 10 μM RHP (1) for 7 h. (a) and (d) images
were taken in optical windows between 540 and 580 nm
(green). (b) and (e) phase contrasts, (c) and (f) displayed
in pseudocolor represent the ratio of emission intensities
collected in optical windows between 540 and 580 (green)
and 430-495 nm (blue), respectively. (g) showed the mean
fluorescent photons and relative ratio. ImageJ software
gave an average emission value of fluorescent photos. Bl:
mean fluorescent intensity (MFI) of single cell between
430 and 495 nm. Gr: MFI of single cell between 540 and
580 nm. GrHy/GrAr means the green ratio of hypoxic to
aerobic; BlHy/BlAr means the blue ratio of hypoxic to aerobic.
Acknowledgment. This work was financially supported
by the Key New Drug Creation and Manufacturing
Program (2009ZX09103-102), the National High Technol-
ogy Research and Development Program of China (863
Program 2011AA10A207), the National Basic Research
Program of China (973 Program, 2010CB126100), the
China 111 Project (Grant B07023), and the Shanghai
Leading Academic Discipline Project (B507). We appreciate
Professor Eric V. Anslyn (Department of Chemistry &
Biochemistry, The University of Texas at Austin) for the
directions for the proposed detection mechanism. We also
appreciate Professor Youjun Yang (School of Pharmacy,
East China University of Science and Technology) for the
improvements in this paper.
Ra (black): the ratio of GrHy/GrAr to BlHy/BlAr
.
pH=7.0).15 The changes in the fluorescence intensity were
measured using an inverted fluorescence microscope
(Figures 5 and S6). The ratio of fluorescence intensity
was collected with a 4 s exposure time from 540 to 580 nm
(green) and with a 800 ms exposure time from 430 to
495 nm (blue). The ratio image constructed from 540 to
580 nm (green) and from 430 to 495 nm (blue) fluorescence
Supporting Information Available. Synthesis, experi-
mental details, and additional spectroscopic data. This
material is available free of charge via the Internet at http://
pubs.acs.org.
(15) Grigoryan, R.; Keshelava, N.; Anderson, C.; Reynolds, C. P.
Chemosensitivity 2005, 110, 87–100.
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