A Cofactor-Substrate-Based Supramolecular Fluorescent Probe for the Ultrafast Detection of Nitroreductase under Hypoxic Conditions

Article abstract of DOI:10.1002/anie.201915040

Identifying the location and expression levels of enzymes under hypoxic conditions in cancer cells is vital in early-stage cancer diagnosis and monitoring. By encapsulating a fluorescent substrate, L-NO₂, within the NADH mimic-containing metal–organic capsule Zn-MPB, we developed a cofactor-substrate-based supramolecular luminescent probe for ultrafast detection of hypoxia-related enzymes in solution in vitro and in vivo. The host–guest structure fuses the coenzyme and substrate into one supramolecular probe to avoid control by NADH, switching the catalytic process of nitroreductase from a double-substrate mechanism to a single-substrate one. This probe promotes enzyme efficiency by altering the substrate catalytic process and enhances the electron transfer efficiency through an intra-molecular pathway with increased activity. The enzyme content and fluorescence intensity showed a linear relationship and equilibrium was obtained in seconds, showing potential for early tumor diagnosis, biomimetic catalysis, and prodrug activation.

Full text of DOI:10.1002/anie.201915040

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Accepted Article
Title: Cofactor-substrate-based Reporter for Enhancing Signalling
Communications towards Hypoxia Enzyme Expression
Authors: Yang Jiao, Lei Zhang, Xu Gao, Wen Si, and Chunying Duan
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201915040
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
Cofactor-substrate-based Reporter for Enhancing Signalling
Communications towards Hypoxia Enzyme Expression
Yang Jiao, Lei Zhang, Xu Gao, Wen Si and Chunying Duan*
[9]
Abstract: Identifying the location and expression levels of hypoxic
probes) , and the cofactor levels have high effect on enzyme
kinetics and the detection precision of hypoxia enzyme[
content. Only if the concentration and diffusion processes of the
cofactors were maintained at sufficient level, the enzyme content
and the luminescent responses would potentially exhibit a linear
relationship. A large excess of cofactor related to the substrate-
based probe was always added in the solution to enable the
precise and quick detection of enzyme content[11]. However, in
different living cells and even in the same cells with different
hypoxia conditions, the expression levels of hypoxia enzymes
and corresponding cofactors are diverse and indefinite[12], such
that the detection of enzyme content in vivo was highly
dependent on the content of cofactors[13], that is also affected by
the hypoxic condition. The combination of substrate-based
probe and cofactor into one supramolecular system with clarified
stoichiometric ratio between them would render the probe
reacting rate almost independent from the cofactor supply, which
is beneficial for rapid quantitative detection.
10]
enzymes in cancer cells is vital in early-stage cancer diagnosis and
2
efficacy monitoring. By encapsulating a fluorescent substrate, L-NO ,
within the NADH mimic-containing metal-organic capsule Zn-MPB,
we developed a new reporter platform named as the cofactor-
substrate-based supramolecular luminescent probe for ultrafast
detection of hypoxic enzymes in solution in vitro and in vivo. The
host-guest structure directly fuses the coenzyme and substrate into
one supramolecular probe to avoid control by NADH, switching the
catalytic process of nitroreductase from a complex double-substrate
mechanism to a single-substrate one. This probe promotes natural
enzyme efficiency by altering the substrate catalytic process and
enhances the electron transfer efficiency via a promising intra-
molecular pathway with increased activity. The enzyme content and
fluorescence intensity showed a linear relationship and ultrafast
equilibrium was obtained in seconds, showing potential for early
tumor diagnosis, biomimetic catalysis, and prodrug activation.
To echo the remarkable properties of enzymes for achieving
efficient chemical conversions, chemists have used metal-
Introduction
organic cages with defined hydrophobic cavities as unique hosts
to catalyze unique chemical transformations[
14,15]
. The direct
Hypoxia, defined as a reduction in the average tissue
modulation of mimics of cofactors into the backbone of the
capsules is postulated to be a promising approach to enforce
active sites into close proximity with substrates[16] and promote
the electron transfer process between reactants efficiently. The
oxygen tension, is an important indicator of some cancers that
disturb the microcirculation[1] and is closely related to various
physiological activities of tumors[2,3]. Identifying the location and
expression levels of hypoxic enzymes in live cancer cells using
molecularly targeted hypoxia-activated methods is essential in
encapsulation of
a substrate-based luminescent probe in
[17]
[
4]
stoichiometric cofactor-backbone ‘molecular containers’ would
be an important tool for distinguishing and quantitatively
detecting different degrees of the enzymatic activity between
healthy and diseased states. The new host-guest cofactor-
substrate-based supramolecular probe approach enabled the
redox reaction of luminescent substrate, cofactor and enzyme to
occur efficiently with excellent compatibility between kinetics and
early-stage cancer diagnosis and efficacy monitoring . Recent
advances in these fields have demonstrated several outstanding
substrate-based enzymatic fluorescent probes[5] for detecting
and imaging abnormal levels of hypoxic enzymes in solution and
live cancer cells through their high sensitivity, non-destructive
[
6]
analysis, and real-time detection abilities . Notably, in early-
stage diagnoses of cancers, the concentration and expression
levels of the hypoxic enzymes often varied widely in different
reaction intermediates[18]
.
[
7]
cells and tumors . Quantitatively detecting and accurately
distinguishing the changes in enzyme activity over a wide range
between healthy and diseased states remains challenging.
Because the different and unknown levels of cofactors and
enzymes cause considerable variations in probe emission over
time, the fluorescence response rarely shows a distinct and
stable linear relationship with the enzyme content.
These cofactor-dependent hypoxia enzymes universally
need small-molecule cofactors, redox carriers of biosynthetic
[8]
reaction , to accomplish enzymatic reactions with substrates (or
[*]
Dr. Jiao, L. Zhang, X. Gao, W. Si, Prof. C. Duan
State Key Laboratory of Fine Chemicals
Dalian University of Technology,
Dalian City, 116024, China
E-mail: cyduan@dlut.edu.cn
Scheme 1. Schematic of the cofactor-substrate-based supramolecular probe
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201915040
for NTR imaging process.
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RESEARCH ARTICLE
By incorporating the NADH mimics, the most common
cofactor[19] in the hypoxic enzymes, into the backbone of a
metal-organic capsule Zn-MPB, herein we present a cofactor-
Tris-HCl solution (Figure S25E), and the calculation result of K
a
value was 1.70x106 M , which matched with the
−1
microcalorimetry titration result and indicated Zn-MPB⊃L-NO
2
substrate-based supramolecular probe (Zn-MPB⊃L-NO
2
) for
could exist stably in aqueous solution.
biochemical imaging of hypoxia enzyme nitroreductase (NTR) in
aqueous solution and living systems.[20,21] We envisioned that
the stoichiometric supply of the cofactor would modify the
original multi-substrate enzymatic process of a substrate-based
probe into a simpler pseudo-single-substrate kinetic, following
the Michaelis–Menten equation. The linear relationship between
fluorescence intensity and enzyme content was expected to
reach an ultrafast equilibrium in hypoxia enzyme detection with
removal of the cofactor content effect (Scheme 1)[13]. In the
meantime, the inclusion of the substrate within the NADH–
containing host would reduce the transport time of the external
NADH and increase the proximity between substrate and the
active sites of the cofactor[22], thus enabling the rapidly signalling
communication towards hypoxia enzyme expression.
Results and Discussion
2
The guest molecular probe, L-NO , comprised a nitrobenzene
fragment and phenanthro[9,10-d]imidazole group[23], transducing
the reduction reaction to fluorescence signals (Scheme S1,
3 3
Figure 1B). Zn-MPB, an M L capsule, was synthesized by a
combination of Zn2+ with the two-arm tridentate ligands which
contained niacinamide skeletons for efficiently promoting
electron transfer (Scheme S2 and Figure 1A). It comprised three
alternating connected ligands and three Zn ions that each
coordinated to two tridentate chelators from two different ligands.
The amide moieties of the ligands provide hydrogen bonding
Figure 1. A) Self-assembly of ligands with Zn2+ to form Zn-MPB. B) Strategy
for detection of NTR with cofactor-substrate-based supramolecular
luminescent probe. C) ESI mass spectrum of Zn-MPB⊃L-NO . D) Binding
model of Zn-MPB⊃L-NO with NTR. E) H-H interaction between H-5 of L-NO
2
+
a
2
2
2
and other H of Zn-MPB in deuterated DMSO-d6. F) The MALDI-TOF MS of
sites for stable binding of L-NO
2
inside Zn-MPB and a
+
[
24]
Zn-MPB-NTR complex showed [M+H ] = 25159.
hydrophilic environment in cell-targeted delivery . The three
NADH mimics in the ligand backbones of Zn-MPB acted as
electron carriers with bioaffinity, providing adequate electrons
Docking calculations[20b] were carried out to obtain additional
information regarding the intermolecular interaction between the
host-guest platform and NTR. The substrate-based luminescent
and protons to reduce the nitro group[25] of L-NO
2
, since
reduction of one nitro group requires six-electrons from three
NADH cofactors.
The electrospray ionization-mass spectrometry results for
Zn-MPB in acetonitrile showed an intense peak at m/z = 942.54
probe L-NO
and other intermolecular interactions. Hydrogen bonds formed
between the O and N atoms of L-NO and amino acid residues
generating intermediate state that exhibited free energy of
binding about -8.78 kcal/mol between NTR and L-NO
Comparatively, Zn-MPB⊃L-NO was attached to the surface of
2
was bound in the pocket of NTR via hydrophobic
2
2
+
(
Figure 1A) assigned to [H
2
Zn
3
(MPB)
3
]
species, suggesting the
high stability of capsule [ . After adding an equimolar amount of
26]
2
.
)]2+ species appeared at m/z =
2
L-NO
166.17, revealing the formation of a 1:1 stoichiometric host-
guest species, Zn-MPB⊃L-NO (Figure 1C). Moreover, the
formation of Zn-MPB⊃L-NO species was demonstrated by the
significant H-H interactions between the H atom (N-H) of L-NO
2
, an H Zn (MPB) (L-NO
2 3 3 2
NTR binding pocket, giving a free energy of binding of -11.03
kcal/mol (Figure 1D), which showed the formation of Zn-
1
2
MPB⊃L-NO
2
-NTR and
a
strong electron-donating system.
2
+
Moreover, the MALDI-TOF MS of NTR showed a [M+H ] peak at
2
2
4715 (Figure S27). After binding with Zn-MPB, the MS dates
showed a new peak 25159 (Figure 1F) that was attributed to the
NTR species with the losing of an FMN (C17 NaO P, M =
78.3), an NADH (C21 Na , M = 709.4) and 14 H O,
indicating the stable binding behaviour of Zn-MPB with NTR.
and other H atoms from Zn-MPB (Figure 1E, S24). Next, we
conducted the microcalorimetry titration of Zn-MPB (0.1 mM)
with L-NO (1 mM) in DMSO Tris-HCl solution (v/v, DMSO 90%)
2
H
20
N
4
9
4
H
27
N
7
O
2 14
P
2
2
(
Figure S26A). The titration results showed formation of the 1:1
[27], for which the free energy
inclusion complex Zn-MPB⊃L-NO
2
Besides, the UV-vis absorption titration (Figure S25A-S25C)
and the microcalorimetry titration (Figure S26B) tests of NTR
with Zn-MPB were conducted to verify the sable binding of NTR
and Zn-MPB, of which the binding constants were calculated as
2
of binding between Zn-MPB and L-NO was calculated as -9.43
) was calculated as 8.33×106
kcal/mol. The binding constant (K
a
−
1
M , which indicated the preferred formation and stable
[
28]
existence of host-guest systems . UV-vis titration experiment
of Zn-MPB and L-NO was also conducted in the same DMSO
6
−1
6
−1
1
.30x10 M or 1.92×10 M , respectively. The result confirmed
2
the stability of the Zn-MPB contained NTR, demonstrating the
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RESEARCH ARTICLE
possible
application
of
the
cofactor-substrate-based
significant changes at equilibrium emission intensity, which
means NTR detection is severely dependent on NADH supply.
supramolecular luminescent probe in vitro and in vivo.
2
When excited at 468 nm in Tris-HCl buffer (10.0 mM, pH 7.4, While, the reaction between Zn-MPB⊃L-NO and NTR hardly
2
5℃), fluorescence spectra of Zn-MPB⊃L-NO
no obvious emission from 490 to 670 nm. After adding NTR to
Zn-MPB⊃L-NO an emission peak rapidly appeared and
increased at 530 nm with the increasing addition of NTR (Figure
B). The biomimetic catalytic reaction quickly reached
2
or NTR exhibited
changed with/without addition of NADH before or after the
reaction (Figure S29, S30B). Such an NADH independent
2
,
2
behavior of Zn-MPB⊃L-NO system was postulated to be
beneficial the quickly detecting and accuracy imaging NTR in
vivo.
2
equilibrium within 5 s (Figure 2D, S29, S30B, S31A). The
corresponding emission band was assigned to L’-OH according
It is well-known that the reduction of nitro group by NADH
and NTR is a typically double-substrate enzymatic reaction[13]
.
to the reported the reduction conversion of L-NO
2
into L’-OH
2
While, the new reporter, Zn-MPB⊃L-NO , responded to the NTR
(
Figure S33).[20a,29] Zn-MPB⊃L-NO
2
showed good linear
concentration rarely affected by the NADH supply, and signal
communication occurred in a pseudo-intramolecular manner,
relationship with an approximately equal slope between enzyme
content and fluorescence intensity independent of time changes
[
30]
simplifying the double-substrate mechanism
into a single-
substrate[ free collision mechanism. In the presence of various
31]
(
Figure 2F), ensuring quantitative analysis of NTR.
concentrations of L-NO
2
2
or Zn-MPB⊃L-NO with a specific
content of NTR (Figure S31, S32), the kinetic parameters
[
32]
Zn-
calculated by the Michaelis–Menten equation exhibited Vmax
MPB⊃L-NO2/
V
max
L-NO2
and kcat
Zn-MPB⊃L-NO2
/kcat
L-NO2
values of about 100.
Our new single-substrate free collision mechanism showed that
the maximum reaction rate and TON of our Zn-MPB⊃L-NO
were enhanced by ca. 100-fold. Compared to a natural catalytic
system, Zn-MPB⊃L-NO improved catalytic efficiency by
2
2
changing the catalytic kinetics.
Considering the complexity of the intracellular environment,
L-NO
2
2
and Zn-MPB⊃L-NO were treated with various potential
interfering analytes in Tris-HCl buffer, such as glucose (50 mM),
dithiothreitol (DTT 10 mM), amino acids (1 mM D-Glu, L-Tyr, L-
Pro, L-Arg, L-Asp, and H-Cys-OH·HCl respectively) and bovine
serum albumin (BSA 1 μg/mL) (Figure 3D). These interfering
species induced no obvious fluorescence changes, showing a
high selectivity and specificity of L-NO
2
2
and Zn-MPB⊃L-NO for
NTR over other intracellular species. Noted that the addition of
NTR to the solution containing interfering analyte and Zn-
MPB⊃L-NO
response to that of Zn-MPB⊃L-NO
detection for NTR under complex conditions. Moreover, Zn-MPB
and Zn-MPB⊃L-NO were tested in cell media and plasma, the
2
immediately caused the similar fluorescent
2
solution, indicating the good
2
dates showed that the fluorescence intensity (λex = 375 nm) of
Zn-MPB changed little over times (Figure S28). These results
demonstrated that the cofactor-substrate-based supramolecular
probe might be a promising tool to be applied for ultrafast
detection of NTR in living organisms.
Figure 2. A, B) Fluorescence spectra showing reaction of NTR with L-NO
μM) and NADH (15 μM) or Zn-MPB⊃L-NO
intensity variation of L-NO (5 μM) with NADH (15 μM) or Zn-MPB⊃L-NO
μM) in NTR. E, F) Intensity vs. NTR levels in 5 μM L-NO
Zn-MPB⊃L-NO (5 μM) at different times.
2
(5
(5
2
(5 μM). C, D) Time-dependent
2
2
Further CCK-8 (Cell Counting Kit-8) assays[33] were carried
out to evaluate biocompatibility. The cell viability of MCF-7,
MDA-MB-231, and A549 cells was over 80% after pre-incubation
2
2
and 15 μM NADH or
2
In contrast, control experiments (Figure 2A, 2C, 2E) based
on traditional probe L-NO , by adding different amounts of NTR
to L-NO and NADH mixed solution, displayed similar emission
peaks as the solution above. Nevertheless, L-NO reached
equilibrium over 10 min (Figure S30A, S31B), which spent over
00-fold reaction time longer than that of Zn-MPB⊃L-NO . The
slower reaction of L-NO with NTR made the plots and slopes of
with various concentrations of the substrate L-NO and Zn-MPB
2
(1–20 μM) for 24 h, revealing the low cytotoxicity of L-NO and
2
2
Zn-MPB in organisms (Figure S34). These results indicated the
capabilities of L-NO and Zn-MPB⊃L-NO for NTR imaging and
2
2
2
kinetic monitoring in vivo. To study intracellular fluorescence
1
2
imaging of NTR, cells were incubated at 37℃ under normoxic
(20% O ) or hypoxic conditions (8% or 0.1%) [
34,35]
and imaged at
2
2
the intensity vs. NTR content keep altering over time (Figure 2E), various reaction times. As shown in Figure 3A, MCF-7 cells were
which made it hard to work out the enzyme concentration. Zn-
MPB⊃L-NO improved NTR detection with significantly shorter
reaction time and higher sensitivity. Further tests found increase
of NADH had significant influences on L-NO reduction by NTR
Figure S30A), expectedly showing increasing in initial rates and
incubated in different oxygen levels treatment for 6 h before the
tests. Because DMPDD(dimethyl 1-(2-morpholinoethyl)-4-phenyl
-1,4-dihydro-pyridine-3,5-dicarboxylate, named compound 4 in
Scheme S2) has the similar NADH mimics structure and didn’t
contain any coordinating groups, it could not formed capsule
2
2
(
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RESEARCH ARTICLE
structure and guaranteed as the reference compound in the bio-
imaging experiments. After MCF-7 cells were incubated with Zn-
with Co2+ treatment[36] which showed that the addition of Co2+
caused moderate hypoxia (approximately similar effects with
that of 8% O
2
groups) as a result of the binding of Co2+ with
MPB⊃L-NO
2
or L-NO
2
or L-NO
2
-DMPDD (Figure 3) for 1 min,
cells were subjected to confocal microscopy. The fluorescence
intensity of cells increased with the degree of hypoxia.
hypoxia-inducible factor (HIF). These phenomena were also
consistently observed in A549 and MDA-MB-231 cells (Figure
S35, S36), showing the better intracellular NTR imaging
Additionally, intensity of cells incubated with Zn-MPB⊃L-NO
was much stronger than that incubated with L-NO or L-NO
2
2
2
-
2
capability of Zn-MPB⊃L-NO compared to the traditional probe
DMPDD at the same oxygen content, which could be identified
platform L-NO
2
.
by direct observation. We also did the cell control experiments
Figure 3. A) Imaging of MCF-7 cells treated with 1 μM L-NO
different hypoxia or CoCl conditions. B) Imaging of MCF-7 cells in 0.1% O
MPB⊃L-NO for different times (0, 1, 3, 5, 10 min). C) Cellular fluorescence intensity changes after treatment with 1 μM L-NO
μM DMPDD (green columns) or 1 μM Zn-MPB⊃L-NO (blue columns) for different times. D) Fluorescence intensity changes for 5 μM L-NO
purple columns) or Zn-MPB⊃L-NO (5 μM, blue columns) in Tris-HCl solution among the interfering analytes (date were recorded for 10 min, until L-NO
2
, 1 μM L-NO
2
with 3 μM DMPDD or 1 μM Zn-MPB⊃L-NO
groups incubated with 1 μM L-NO , 1 μM L-NO
(wine columns), 1 μM L-NO
(with 15 μM NADH,
groups
2
(5 μM) in present of various interfering analyte mixture solutions, the fluorescence
2
for 1 min after the 6 h incubation in
with 3 μM DMPDD or 1 μM Zn-
with 3
2
2
2
2
2
2
2
2
2
2
2
reached equilibrium); And after adding NTR (5 μg/ml) to the Zn-MPB⊃L-NO
intensity reached equilibrium in seconds (wine columns).
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2 2 2
Figure 4. A, C, E) Bio-imaging of MCF-7 tumor-bearing mice at various times after injection with L-NO , L-NO -DMPDD or Zn-MPB⊃L-NO . B, D, F) Bio-imaging
of MDA-MB-231 tumor-bearing mice at various times after injection with L-NO , L-NO -DMPDD or Zn-MPB⊃L-NO .
2 2 2
Further studies on intracellular catalytic kinetics[37,38] of NTR
in MCF-7 cells with Zn-MPB⊃L-NO or L-NO or L-NO -DMPDD
were conducted under hypoxic conditions (0.1% with
Additionally, mice were used to monitor and trace the NTR
2
2
2
catalytic kinetics with Zn-MPB⊃L-NO
2
.
Mice fluorescent
39,40]
O
2
)
imaging[
tests were excited at 470 nm and collected at 530
different incubation times (Figure 3B, 3C). Fluorescence images
of cells were obtained in the red channel at 508–608 nm and λex
nm by Bruker In Vivo F Pro system. MCF-7 and MDA-MB-231
tumor-bearing mice were used to conduct the small animal
=
488 nm. Surprisingly, the fluorescence in Zn-MPB⊃L-NO
2
fluorescence imaging experiments of Zn-MPB⊃L-NO
and explore the fluorescence signal changes over time (Figure
4A-4F). While, L-NO or L-NO -DMPDD were used as control
groups for the investigation of imaging capability of our cofactor-
substrate-based supramolecular probe. Rapid fluorescence
2
in vivo
experimental groups rapidly reached equilibrium even with 1 min
incubation time, and the fluorescence images of cells exhibited
no apparent changes during longer incubation time (1–10 min)
2
2
(
Figure 3B and 3C). In contrast, the fluorescence of L-NO
2
or L-
NO -DMPDD control groups was only slightly enhanced with
2
2
enhancement of Zn-MPB⊃L-NO groups was shown in the
increasing cell incubation time, and even after 10 min of
incubation, the fluorescence intensity of cells remained
tumor region of tumor-bearing mice, which reached equilibrium
within 10 min, and exhibited considerable increase in intensity
unequilibrium and was weaker than that of Zn-MPB⊃L-NO
2
. A
compared with L-NO
2
2
groups or L-NO -DMPDD groups.
similar tendency was also exhibited by MDA-MB-231 and A549
cells (Figure S35, S36). These results demonstrated that Zn-
MPB⊃L-NO was a rapid and efficient method for intracellular
2
Alternatively, the latter two control groups showed much weaker
fluorescence imaging in the tumor region and the intensity
reached equilibrium for over 10 min, showing a longer time than
NTR imaging and verified that the cofactor-substrate-based
supramolecular could help improve the double-substrate
mechanism to a single-substrate one.
that of Zn-MPB⊃L-NO
MPB⊃L-NO exhibited excellent performance for rapid NTR
imaging and enzymatic kinetics monitoring in vivo.
2
. These results demonstrated that Zn-
2
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RESEARCH ARTICLE
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MPB⊃L-NO forced the NADH mimic capsule into proximity with
L-NO , facilitated a unique NADH-independent NTR's detection,
2
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Angewandte Chemie International Edition
10.1002/anie.201915040
RESEARCH ARTICLE
Entry for the Table of Contents (Please choose one layout)
RESEARCH ARTICLE
We propose a cofactor-substrate-
based supramolecular probe strategy
for ultrafast (within seconds) and
quantitative hypoxia detection of NTR
Yang Jiao, Lei Zhang, Xu Gao, Wen Si
and Chunying Duan*
Page No. – Page No.
Cofactor-substrate-based Reporter
for Enhancing Signalling
Communications towards Hypoxia
Enzyme Expression
This article is protected by copyright. All rights reserved.