Rational design of fluorescent probes for targeted: In vivo nitroreductase visualization

Article abstract of DOI:10.1039/d0ob00082e

Nitroreductase (NTR) has been recognized as a biomarker for identifying the hypoxic status of cancers. Therefore, it is of high scientific interest to design effective fluorescent probes for tracking NTR activity. However, studies on elucidation of the structure-performance relationship of fluorescent probes and those providing valuable insight into optimized probe design have rarely been reported. Three BODIPY based fluorescent probes were made by conjugation of para-, ortho-, and meta-nitrobenzene to the BODIPY core via a thiolether bond, respectively. Our study revealed that the linkage and nitro substituent position significantly influence the capability of nitroreductase detection.

Full text of DOI:10.1039/d0ob00082e

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DOI: 10.1039/D0OB00082E
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Rational Design of Fluorescent Probes for Targeted in vivo
Nitroreductase Visualization
Jie Gaoa$, Xiaofan Yin a$, Mimi Lia, Ji-An Chena, Jiahui Tana, Zhen Zhao a*, Xianfeng Gua*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Nitroreductase (NTR) has been recognized as a biomarker for NTR-catalysed reduction of the nitro group in substrate into an
identifying the hypoxic status of cancers. Therefore, it is of high amino group. Particularly, fluorescence imaging is attractive on
scientific interest to design effective fluorescent probes for tracing the changes and activities of biological targets due to
tracking NTR activity. However, studies are rarely reported to their exceptional sensitivity. Therefore, several fluorescent
disentangle the structure−performance relationship of fluorescent probes for detection of NTR activities in vitro and in vivo have
probes and provide valuable insight into optimized probe design. been reported by employing a para-nitroaromatic group as
Three BODIPY based fluorescent probes were made by reaction and recognition group.4b, 7b, 8 For example, Li’s group
conjugation of para-, ortho-, and meta-nitrobenzene to BODIPY reported a series of NTR probes with a nitro group at para-,
core via thiolether bond, respectively. Our study revealed that the ortho-, and meta-phenyl position, respectively. It was found
linkage and nitro substituent position significantly influence the that only the probe with a para-nitrophenyl group via an ester
capability of Nitroreductase detection.
linkage to fluorescent dye displayed significant fluorescent
response to NTR.7b Despite these advancements, few studies
have focused on the structure−performance relationship of
fluorescent probes wherein the linkage between fluorophores
and aromatic nitro group as well as nitro substituent position
can dramatically induce the change of fluorescence, thus
sensitively influencing the NTR activity assay.
Tumor hypoxic microenvironments are caused by abnormal
angiogenesis and rapacious demands of oxygen by fast dividing
cancer cells. Hypoxia microenvironments are usually observed
in most of solid tumors where the median oxygen
concentration has been reported to be around 4-0 % locally,
adapted and exploited by cancer cells for their survival and
invasion.1 The worst thing about tumor hypoxic
microenvironments is that it has been regarded as one of the
major causes of the resistance to various therapies including
immunotherapy.2 Therefore, estimating the tumor issue and
cells hypoxia degree is of great importance in predicting
anticancer efficacies.
In tumor hypoxic microenvironments, tumor cells display
enhanced endogenous nitroreductase (NTR) activities,
enabling NTR an attractive target for selectively and efficiently
measuring hypoxia in tumor cells or tumor tissue.3 NTR is
flavoenzymes that catalyze the reduction of a variety of
nitroaromatic compounds using NAD(P)H as source of reducing
equivalents.4 To date, several imaging techniques have been
To deeply disentangle such issues, we herein designed and
synthesized three BODIPY based fluorescent probes (BpS, BoS
and BmS) by conjugation of para-, ortho-, and meta-
nitrobenzene to BODIPY core via thiolether bond, respectively.
We expected that the nitro groups in the probes could be
reduced into amino groups under the catalyzation of NTR, and
the amino group resulting from BoS rather than from BpS or
BmS would trigger an intramolecular nucleophilic substitution
through five-membered cyclic transition state, finally yielding
an amino substituted BODIPY. As sulfenyl-substituted BODIPYs
showed markedly different optical properties from amino-
substituted BODIPYs, thus detection of NTR activities could be
achieved by monitoring fluorescence changes of BoS. As
expectedly, these three probes showed different fluorescent
responsiveness to NTR: BpS displayed a fluorescence turn off
response to NTR; a weak turn on fluorescent signal was
observed for BmS; and BoS exhibited a significant turn on
fluorescent response to NTR. Due to the unique fluorescence
light up, BoS was successfully used for the detection of NTR
activities in living HeLa and HepG-2 cells under different
hypoxic conditions. Importantly, BoS realized the selective
identification of tumor hypoxia by real time monitoring of NTR
activity in vivo.
developed, such as NMR,5 PET6 and fluorescence3e,
monitoring NTR activities in biological system based on the
changes of physical and chemical properties caused by the
to
4b,
7
a. School of Pharmacy & Minhang Hospital, Fudan University, Shanghai 201301,
China. E-mail :. xfgu@fudan.edu.cn；zhaozhen72@126.com.
Electronic Supplementary Information (ESI) available: Procedures for synthesis,
characterization data, and supplementary figures. See DOI: 10.1039/x0xx00000x
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BpS, BoS and BmS were prepared via the aromatic substitution
between the key intermediate 2 and para-, ortho-, and meta-
nitrobenzene thiol (Scheme 1). Compound 2 was obtained via
the condensation of compound 5 with compound 8, followed
by boron insertion with BF3·Et2O (Scheme 1 and S1).9 para-,
ortho-, and meta-nitrobenzene thiol 1 were synthesized from
the reduction of the corresponding nitrophenyl disulfane
To evaluate the photophysical properties of BpS, BmS and BoS
in the presence/absence of NTR and NADH, the absorption and
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DOI: 10.1039/D0OB00082E
fluorescence emission spectra of these three probes in Tris-HCl
buffer (pH 7.4; 25 % MeCN) were investigated. Impressively,
the three probes displayed different fluorescent responses to
NTR. The addition of NTR to the solution of BoS resulted in an
obvious fluorescence turn-on response with maximum
emission at 540 nm when the excitation wavelength was 450
nm (Figure 1C), producing 13-fold increase. And the “turn on”
effect by BOS can be supressed by the inhibitor of NTR which
indicate the essential of NTR for activation the probe (Figure
S3). However, NTR only triggered a weak fluorescent increase
around 565 nm in the solution of BmS (Figure 1A). In contrast,
upon addition of NTR in the solution of BpS, a significant
fluorescence turn-off response around 580 nm was observed
(Figure 1B). In view of the excellent fluorescence
responsiveness of BoS to NTR, its linear fluorescent response
NTR was further investigated. The kinetic profiles of BoS
treated with the various concentrations of NTR were shown in
Figure 1D. Notably, the fluorescence intensity increased
linearly with concentrations of NTR increased from 0 to 11
μg/mL, contributing to a detection limit of 0.022 μg/mL (Figure
1D). These results indicate that BoS is a promising probe for
detecting NTR under physiological conditions.
To understand the probes interactive behavior with
nitroreductase, molecular docking simulations were
performed to dock probes BoS, BpS, BmS into NTR (PDB code
4DN2) using the Glide module encoded in Schrodinger 3.5
software package, with Extra Precision (GlideXP) algorithm.
Probe BoS tends to approach the hydrophobic interspace of
NTR by the aromatic rings π-π interactions. Hydrogen bonds
are also constructed between the O atom of BoS and the NTR
amino acid residues. The probe forms five hydrogen bonds
with NTR. Three is for the Arg 10 amino acid residues of NTR
with the O atom of the nitro group of BoS, and the other two
hydrogen bonds are for the Arg 172 residues with the O atom
of the nitro group, which indicated the strong binding energy
between BoS and NTR (Figure 2A). In contrast, although
probes BmS and BpS can approach the hydrophobic interspace
of NTR by the aromatic rings π-π interactions, only two
hydrogen bonds were formed in BmS, BpS counterparts
(Figure 2B and 2C).
1
(scheme 1). All the structures were fully characterized by H
NMR, 13C NMR and HRMS (Figure S6-S11).
NO2
i
S
N
N
O2N
S
B
O2N
SH
S
F
F
1
BoS
NO2
O
+
H
N
Cl
5
ii,iii
iv
N
N
O2N
B
+
N
N
S
F
F
B
H
Cl
N
F
F
BmS
2
8
N
N
B
S
F
F
O2N
BpS
Scheme 1. The synthetic procedures of BoS, BmS, BpS. (i)
NaBH4, THF; (ii) POCl3, CH2Cl2; (iii) Et3N, BF3·Et2O (iv)) DMAP,
MeCN.
Figure 1. Time-dependent spectra changes of BmS, BpS, BoS
(10 μM) in the presence of NTR (10 μg/mL) and NADH (0.5
mM) in buffer (MeCN/Tris-HCl, v/v, 1:3, pH 7.4) at 37 °C. (A)
fluorescence spectral changes of BmS (λex = 510 nm, λem = 565
nm), (B) fluorescence spectral changes of BpS (λex = 510 nm,
λem = 580 nm), (C) fluorescence spectral changes of BoS (λex
=
450 nm, λem = 540 nm). (D) The linear relationship between the
fluorescence intensity of BoS (10 μM) at 540 nm and the
concentrations of NTR (0-11 μg/mL) in the presence of NADH
(0.5 mM, λex = 450 nm). (D) The linear relationship between
the fluorescence intensity of BoS (10 μM) at 540 nm and the
concentrations of NTR (0-11 μg/mL) in the presence of NADH
(0.5 mM, λex = 450 nm) for 180 min.
Figure 2. Calculated binding model of BoS (A), BmS (B), BpS (C)
with NTR. The C, N and O atoms of probes structures are
shown in blue, cyan and red, respectively. Hydrogen bonds are
2 | J. Name., 2012, 00, 1-3
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indicated with yellow dotted lines, aromatic rings π-π intensity (Figure 4). In contrast, addition of dicoumarol, an NTR
interactions are indicated with red dotted lines.
inhibitor, led to weaker emission in greDeOnI:c1h0a.1n0n3V9ei/elDsw.0AOArtBilcl0let0Oh0ne8lis2neEe
results indicated that the BoS can discriminate hypoxic tumor
cells from normal cells based on the NTR expression levels.
Costaining experiments were employed to identify the location
of BoS utilizing commercially available Mito-Tracker Deep Red
FM and Lyso-Tracker Deep Red (Figure S7). The overlapped
fluorescence images of red fluorescence from Mito-Tracker
Deep Red FM/ Lyso-Tracker Deep Red and NTR induced green
fluorescence from BoS indicated that probe BoS possessed
nonspecific intracellular localization.
The deduced detection mechanism of BoS activated by NTR
was shown in Figure 3. The NTR catalysed reduction of nitro
group into amino group initiated an intramolecular
nucleophilic aromatic substitution (SNAr) via a five-membered
cyclic transition state, which finally yield an amino substituted
BODIPY with a free thiol group. As the sulfur-substituted and
amino-substituted BODIPYs have the same molecular weight, it
is difficult to identify the deduced detection mechanism by
HRMS spectrometric analysis. Considering that there is a free
thiol group in the amino substituted derivative, the thiol
scavenger (N-methylmaleimide) was added in the reaction
solution. The further HRMS spectrometric analysis indicated
occurrence of the reaction between thiol and thiol scavenger,
which confirmed our deduced mechanism (Figure 3).
Figure 4. Confocal fluorescence images of living Hela and
HepG-2 cells. (A-D) The Hela and HepG-2 cells were grown
under normoxic condition (20 % O2) for 24 h, and then
incubated with 10 μM probe BoS for 30 min. (E-H) Hela and
HepG-2 cells were grown under hypoxic condition (1 % O2) for
12 h, and then incubated with 10 μM probe BoS for 30 min. (I-
L) Hela and HepG-2 cells were grown under hypoxic condition
(1 % O2) for 24 h, and then incubated with 10 μM probe BoS
for 30 min. (M-P) Hela and HepG-2 cells were grown under
hypoxic condition (1 % O2) for 24 h, and then incubated with 1
mM dicoumarol for 1 h, followed with probe BoS (10 μM) for
30 min. Green channel at 500-580 nm with λex = 488 nm. The
scale bar is 10 μm.
Figure 3. The deduced detection mechanism of BoS to NTR
confirmed by HRMS. (A) Mass spectrum of reduced products
(BoS-A or BoS-S), HRMS (ESI, m/z): calculated for C25H24BF2N3S
[M+Na]+: 470.1650, found: 470.1644. (B) The deduced
detection mechanism of BoS to Nitroreductase. (C) Mass
spectrum of BoS-SN, HRMS (ESI, m/z): calculated for
C30H29BF2N4O2S [M-H]-:557.1994, found: 557.2003.
Before using BoS for its potential application in living cell
imaging, the specificity and cytotoxicity of BoS have been
explored. BoS exhibited high specificity towards NTR even in
presence of bio thiols (Figure S2). And the cytotoxicity towards
the HeLa and HepG-2 cells was determined by a CCK-8 assay
(Figure S6). In the presence of BoS at 0−50 μM, the cellular
viabilities were estimated to be >70 % after incubation for 24
hours. The results indicated that BoS has very low toxicity.
The capability of BoS for the intracellular NTR imaging was
evaluated on hypoxia HeLa and HepG-2cells by confocal
fluorescence microscopy. The HeLa and HepG-2 cells were
cultured under normoxic conditions (20 % O2) and hypoxic
condition (1 % O2) for 12 h and 24 h, and then treated with 10
μM BoS for 30 min. As shown in Figure 4, negligible
fluorescence increases under the normoxic conditions were
observed both in HeLa and HepG-2 cells. When the cells were
cultured under the hypoxic conditions for 24 h, both HeLa and
HepG-2 cells showed the great increase in the fluorescence
Encouraged by the results of living cell imaging, the capability
of BoS as a fluorescent probe for animal bioimaging in vivo was
further investigated. After direct injection of BoS (100 nmol) in
Tris-HCl buffer into a HepG-2-tumor-bearing nude mice,
images were recorded at various times by using IVIS spectrum
imaging system. The tumor site displayed a significant
enhancement fluorescence intensity within minutes. The signal
eventually reached a plateau at 60 min post-injection. In
contrast, there is no obvious fluorescence increasing in normal
site after injection of BoS. The results demonstrated that the
probe BoS is suitable to bioimaging hypoxic tumor in vivo
(Figure 5).
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Figure 5. Time dependent in vivo fluorescence imaging of
HepG-2 tumor-bearing mice model. (A) The time dependent
fluorescence signals in tumor and normal sites of the tumor-
bearing mice with injection of probe BoS (100 nmol) in Tris-HCl
via intratumoral injection. (B) The fluorescence intensity
changing of tumor (red) and normal injection sites (green). The
fluorescence signals were collected between 560 nm-580 nm
using λex = 460 nm.
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Conclusions
In summary, we have designed and synthesized a series of
probes with para-nitrophenyl sulfide, meta-nitrophenyl sulfide
and ortho-nitrophenyl sulfide respectively as recognition
group for NTR detection. BoS, the probe with ortho-
nitrophenyl sulfide displayed high sensitivity and excellent
selectivity, with a distinct fluorescence off-on response to NTR.
HRMS validated the occurrence of the sulfur-nitrogen
translocation reaction. The efficient NTR detection ability of
BoS was further validated by fluorescence imaging of hypoxic
cells (Hela and HepG-2 cells) with overexpressed NTR.
Importantly, BoS realized the selective identification of tumor
hypoxia by real time monitoring of NTR activity in vivo. All the
results demonstrate that ortho-nitrophenyl sulfide also can be
the recognition group of the probe for detection of NTR.
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Conflicts of interest
There are no conflicts to declare.
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4 | J. Name., 2012, 00, 1-3
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