A fluorescent ESIPT-based benzimidazole platform for the ratiometric two-photon imaging of ONOO-: In vitro and ex vivo

Article abstract of DOI:10.1039/d0sc02347g

In this work, we have developed an ESIPT-based benzimidazole platform (MO-E1 and MO-E2) for the two-photon cell imaging of ONOO- and a potential ONOO-activated theranostic scaffold (MO-E3). Each benzimidazole platform, MO-E1-3, were shown to rapidly detect ONOO- at micromolar concentrations (LoD = 0.28 μM, 6.53 μM and 0.81 μM respectively). The potential theranostic MO-E3 was shown to release the parent fluorophore and drug indomethacin in the presence of ONOO- but unfortunately did not perform well in vitro due to low solubility. Despite this, the parent scaffold MO-E2 demonstrated its effectiveness as a two-photon imaging tool for the ratiometric detection of endogenous ONOO- in RAW264.7 macrophages and rat hippocampus tissue. These results demonstrate the utility of this ESIPT benzimidazole-based platform for theranostic development and bioimaging applications. This journal is

Full text of DOI:10.1039/d0sc02347g

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DOI: 10.1039/D0SC02347G
ARTICLE
A Fluorescent ESIPT Benzimidazole Platform for the Ratiometric
Two-Photon Imaging of ONOO- In Vitro and Ex Vivo
Maria. L. Odyniec,a Sang-Jun Park,b Jordan E. Gardiner,a Emily C Webb,a Adam C. Sedgwick,c Juyoung
Received 00th January 20xx,
Accepted 00th January 20xx
Yoon,d Steven D. Bull,*a Hwan Myung Kim,*b Tony. D. James.*a
DOI: 10.1039/x0xx00000x
In this work, we have developed an ESIPT benzimidazole-based platform (MO-E1 and MO-E2) for the two-photon cell
imaging of ONOO- and a potential ONOO- -activated theranostic scaffold (MO-E3). Each benzimidazole platform, MO-E1-3,
were shown to rapidly detect ONOO- at micromolar concentrations (LoD = 0.28 µM, 6.53 µM and 0.81 µM respectively).
Theranostic MO-E3 was shown to release the parent fluorophore and drug indomethacin in the presence of ONOO- but did
not perform well in vitro, due to low solubility. Despite this, MO-E2 demonstrated its effectiveness as a two-photon imaging
tool for the ratiometric detection of endogeneous ONOO- in RAW264.7 macrophages and rat hippocampus tissue. These
results demonstrate the utility of this ESIPT benzimidazole-based platform for theranostic development and bioimaging
applications.
Excited state intramolecular proton transfer (ESIPT) probes are
attractive systems for developing reaction-based fluorescence
systems since they have excellent photophysical properties,
Introduction
Fluorescent probes using bespoke chemical architectures are
rationally designed to elicit a fluorescent response after reacting with
a target molecule. Peroxynitrite (ONOO-) is a reactive nitrogen
species (RNS) of interest. Low levels of ONOO- are constituently
produced under basal conditions, which are tolerated through thiol-
dependent antioxidant detoxification pathways.1 However, high
levels of ONOO- are implicated in the pathological and physiological
processes of multiple oxidative stress-related diseases, including;
neurodegenerative and inflammatory diseases.2 ONOO- is a strong
oxidant with a short half-life which can react with various
biomolecules such as DNA and proteins; causing major oxidative
injury.3 Due to this high reactivity and short lifetime accurate
detection of ONOO- remains a challenging task.
including; good photostability and large stokes shifts.10 In addition
ratiometric responses can also be observed. A two colour
ratiometric response is desirable as it can provide direct information
about concentration of a target analyte using internal calibration,
which is not possible with a single colour system.10 ESIPT probes
often incorporate; 2-(2′-hydroxyphenyl)benzimidazole (HBI), 2-(2′-
hydroxyphenyl)benzoxazole
(HBO)
or
2-(2′-
hydroxyphenyl)benzothiazole (HBT) in the framework.
Four ESIPT fluorescent probes (1-4) for ONOO- detection are
illustrated in Fig 1. Each of these probes use an HBT core. The first
reported example, probe 1 designed by Kim et al., used a
benzothiazolyl iminocoumarin scaffold and
a boronate ester
activating group to enable endogenous imaging of ONOO- in J774A.1
macrophages.11 Probes 2 and 3 designed by the James group
incorporated the boronate ester onto a more simple benzothiazole
scaffold for detection of ONOO- in vitro. Moreover, probe 3 was
targeted to the endoplasmic reticulum (ER).5,12 Following a different
mechanism of activation, probe 4 designed by Li et al. was used for
visualisation of ONOO- in neurovascular ischemia progression in the
brain of a live mouse.13 As such, benzothiazoles have been shown to
be good fluorescent probes for ONOO- detection, with high
selectivity, good cell permeability and potential to transport across
the blood brain barrier.13
One successfully implemented approach for the detection of ONOO-
uses boronate-based fluorescent probes.4-5 Previously; such probes
have been used to detect hydrogen peroxide (H2O2). However; given
the reaction rates of boronate esters with ONOO- are 106 times faster
than H2O2, probes for the former have recently been developed.6 A
variety of probes to detect ONOO- via different mechanisms are
reported and are discussed elegantly in reviews by Shen et al. and
Chan et al.7, 8 Prehaps one of the most useful non-boronate probes
for monitoring ONOO- is the reversible Near-IR cyanine dye
modulated by a PET processes through selenium oxidation; designed
by the Han Group. A reversible signal is particularly useful for
continuous monitoring real time changes in the levels of a biological
analyte.9
a.Department of Chemistry, University of Bath, BA2 7AY, UK.
Email: T.D.James@bath.ac.uk, S.D.Bull@bath.ac.uk
b.Department of Chemistry, Ajou University, 16499, Suwon, Korea.
Email: kimhm@ajou.ac.kr
c. Department of Chemistry, University of Texas at Austin, 105 E, 24th Street, A5300,
Austin, USA.
d.Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-
750, Korea.
Electronic Supplementary Information (ESI) available: See DOI: 10.1039/x0xx00000x
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chosen drug indomethacin using EDCI, in DMF to give MO-E3 in 40 %
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DOI: 10.1039/D0SC02347G
yield (Scheme S1, ESI)
Figure 3- Structures of MO-E1-3
Figure 1 – (a) Structures of previously reported ONOO- activatable probes.
Due to the favourable properties of ESIPT based probes, we were
interested in developing
a potential ESIPT-based theranostic.
Theranostics are the portmanteau of diagnostics and therapy.
Typically; a theranostic will include; an activating group, therapeutic
group and a targeting group connected via a single linker.12, 14
A common feature of theranostic linker building blocks are multiple
primary alcohol functional groups, which need to be derivatised
independently. This can lead to long tedious synthetic routes with
multiple protection and deprotection steps. However, with our
design strategy, we use the fluorophore as the ‘linker’ (Fig. 2).15,16 In
this work, we have used HBI as the fluorophore ‘core’ and as the
theranostic linker. Indomethacin was chosen as the therapeutic
agent. Prominent HBI-based examples in the literature are probes
which detect changes in pH. The Kim group developed a two-photon
benzimidazole system to estimate acidic pH values in lysosomal
compartments in vitro and in vivo.17 Liang and colleagues developed
3-benzimidazole-7-hydroxycoumarin which was able to detect two
distinct pH ranges and monitor pH changes in the mitochondria of
HeLa cells.18 From this work, we anticipated that our proposed
benzimidazole system would provide a suitable platform for two-
photon imaging of peroxynitrite and provide the potential for
theranostic applications (Fig. 2).
Figure 2 – Design strategy towards ESIPT theranostics.
Results and discussion
In brief, for the synthesis of MO-E1-3 , commercially available HBI
was subjected to duff reaction conditions (hexamethylenetetramine
(HTMA) and trifluoroacetic acid (TFA)) to form intermediate 4 in
reasonable yield (35 %); Next, a mixture of 4, 4-(4,4,5,5-tetramethyl-
1,3,2-dioxaborolan-2-yl)benzyl bromide, and potassium carbonate in
dry DMF afforded MO-E1 in 35 % yield. The aldehyde functionality
of MO-E1 was reduced using NaBH4 to afford the corresponding
alcohol MO-E2 in 62 % yield. Finally, MO-E2 was conjugated to our
2 | J. Name., 2012, 00, 1-3
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Figure 4 – (a-c) Fluorescence spectra of MO-E1-3 (5 µM) in the presence of ONOO- (a) MO-E3: 0-30 µM, (b) MO-E2: 1-7 µM and (c) MO-E1: 1-5 µM. The dashed line represents sensor
only and measurements were taken instantly after ONOO- addition. (d-e) Fluorescence selectivity of MO-E1-3 (5 µM) in the presence of ONOO- (10 µM), ·OH (100 µM), O2·- (100 µM),
1O2 (100 µM), ClO- (100 µM), ROO· (100 µM) and H2O2 (100 µM). The fluorescence intensity was taken at λmax= 450 nm. Fluorescence measurements were recorded after 30 mins
incubation with all ROS/RNS. All fluorescence measurements were measured in PBS buffer pH = 8.2 (52 % w/w H2O: MeOH) at ambient temperature where λex= 325 (bandwidth: 16)
nm on a BMG Labtech CLARIOstar® plate reader. (e) ESIPT excited state diagram of HBI.
Fluorescence Analysis.
The synthetic route to MO-E3 provided a novel theranostic
compound and two new fluorescent probes; MO-E1 and MO-E2.
With these molecules in hand, spectroscopic analysis was
performed. Data was collected in PBS buffer pH = 8.2 (52 % w/w H2O:
MeOH) at ambient temperature. Measurements were taken
immediately after ONOO- addition. The UV-VIS spectra of MO-E1-3
(5 µM) were shown to have an absorption peak at 325 nm. The
addition of ONOO- led to an increase in absorption at 325 nm (Fig S2-
4 – ESI).
1.4
MO-E3
MO-E3+ONOO_
1.2
MO-E2
MO-E2+ONOO_
1.0
0.8
MO-E1
MO-E1+ONOO_
0.6


0.4
0.2
Figure 6 - TPM ratiometric images of RAW264.7 macrophages labelled with MO-E2 (5 μM)
for 30 min. (a) Control image. (b‒f) Cells were pre-treated with (b) exogenous ONOO- (50
0.0
μM, 30 min), (c) SIN-1 (50 μM, 30 min), (d) SIN-1 and ebselen (50 μM, 30 min), (e) LPS (1
700
750
800
850
900
μg mL-1, 4h) and IFN-γ (50 ng mL-1, 1h), (e) LPS, IFN-γ, and ebselen (f) Average ratio values
in the corresponding TPM ratiometric images. Excitation wavelength was 740 nm and
emission windows of each regions were 380–430 nm (Fblue) and 470–520 nm (Fgreen). Scale
bars = 20 μm. Asterisks stand for the statistical significance (p < 0.001) and n is number of
counted cells.
TP excitation (nm)
Figure 5 - Two-photon action spectra of MO-E probes (5 μM) without and with ONOO-
(50 μM) in PBS buffer pH = 8.2 (52 % w/w H2O: MeOH) at 25 °C.
This journal is © The Royal Society of Chemistry 20xx
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The mechanism of activation requires ONOO- induced oxidation of an the benzyl boronate ester. As the concentration of ONOO- increased,
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DOI: 10.1039/D0SC02347G
aryl boronate and subsequent elimination of the therapeutic. The (0-7 µM), a decrease in fluorescence emission at 355 nm with a
boron atom of the boronate ester is sp2 hybridised and Lewis acidic. concomitant increase at 450 nm was observed; characteristic of the
ONOO- attacks the boron atom resulting in the formation of a ESIPT-based emission of HBI (Fig 3e). The limit of detection for MO-
peroxyborate intermediate. Subsequently aryl migration to oxygen E1-2 were calculated to be 6.53 µM and 0.81 µM respectively (Fig
gives the aryl boronate, which in the presence of water, undergoes S15-18). To date, the estimated steady state concentration of ONOO-
quantitative hydrolysis to the corresponding phenol. Finally; a is in the nanomolar range.20-22 There are limited fluorescence
spontaneous 1,4-elimination results in release of the therapeutic methods for direct quantitation of ONOO-. Recently Wang and
leaving a reactive quinone methide, which on reaction with water colleagues used a ratiometric BODIPY probe which measured
forms the desired fluorescent adduct. The full mechanism can be average basal ONOO- levels as 550-700 nM.23 Hence, MO-E3 and
found in the supplementary information (Fig S5 – ESI) Mass MO-E2 are well placed to monitor endogenous basal changes in
spectrometric analysis confirmed the formation of the expected ONOO- as well as oxidative stress events where the concentration of
fluorescent molecule (Fig S6-S9 – ESI).
ONOO- is likely to increase significantly.
Following confirmation of the expected products; fluorescence To test the probes ability to be activated by ONOO- over other
studies were performed. Each probe (5 µM) was incubated with reactive oxygen species (ROS), selectivity studies were performed
varying concentrations of ONOO- and the fluorescence response was (Fig. 3d-e). MO-E1-3 were tested against; ROS (100 µM), ·OH, O2 ,
·-
monitored (Fig.3a-c). Initially, MO-E3 was shown to be non- 1O2, ClO-, ROO· and H2O2. As expected, the most significant
fluorescent. As the concentration of ONOO- increased, the fluorescence response was shown towards ONOO-. A 98- fold- 20-
fold and 10-fold fluorescent enhancement was observed for MO-E3,
5.0
(a)
(d)
(b)
(e)
(c)
100
80
60
40
20
0
3.75
2.5
1.25
0.0
***
(n = 192)
6
5
4
3
2
1
0
***
Control 5 M
10 M 25 M 50 M
(n = 198)
Figure 7 - Cytotoxicity assays of MO-E2 labelled RAW264.7 macrophages using CCK-8.
Cells were incubated with 0‒50 μM of MO-E2 for 2 h.
(n = 168)
(n = 166)
fluorescence response increased proportionally up to a plateau of 20
µM. The maximum fluorescence response was observed at λmax = 450
nm (Fig.3a). The limit of detection for ONOO- is 0.28 µM (Fig S13-14.-
ESI). To ensure indomethacin was released from the system, LC-MS
Control
SIN-1
PMA
PMA
+Ebselen
Figure 9 - TPM ratiometric images of rat hippocampal slices stained with MO-E2 (50
μM) for 1.5 h. (a) Control image. (b‒d) Tissues were pre-treated with (b) SIN-1 (200 μM,
studies were performed using H2O2 as the reactive species. The data 30 min), (c) PMA (20 μM, 30 min) (d) PMA and ebselen (200 μM, 30 min), (e) Average
ratio values in the corresponding TPM ratiometric images. Excitation wavelength was
740 nm and emission windows of each regions were 380–430 nm (Fblue) and 470–520
nm (Fgreen). Scale bars = 35 μm. Asterisks stand for the statistical significance (p <
0.001) and n is number of ROI from three samples for each imaging condition.
revealed the release of indomethacin, thus confirming MO-E3 as a
potential theranostic platform (Fig. S10- S12 - ESI). The cleavage of
the ester was as expected from previous research in the James
group.19
MO-E2 and MO-E1 respectively at 450 nm (Fig. S19-21).
Unlike MO-E3, MO-E1-2 elicited ratiometric responses in the
Unfortunately, MO-E1-3 were unsuitable for traditional cell imaging
presence of ONOO- (Fig. 3b-c). Initially, both probes displayed a
and confocal imaging experiments due to low excitation wavelengths
fluorescence emission intensity at 355 nm. This emission can be
(< 360 nm). Therefore, to overcome this limitation each probe was
attributed to an ESIPT ‘enol’ form as the ESIPT process is blocked by
evaluated towards two-photon microscopy (TPM). Unlike traditional
fluorescence microscopy; in which the excitation wavelength is
shorter than the emission wavelength, in TPM the two photons
excite at a longer wavelength than the resulting emitted light. This
TPM strategy reduces short-wavelength induced phototoxicity and
increases light penetration through living tissues with minimal light
scattering.24 Before conducting TPM cell imaging experiments, two-
photon action cross sections (TPA) of each ESIPT probe was
measured (Fig. 5). TPA spectra for each probe before and after the
addition of ONOO- (50 μM) in PBS buffer (pH = 8.2, 52 % w/w H2O:
Figure 8 - (a) TPM fluoresce images of RAW264.7 macrophages labelled with MO-E2.
MeOH) were measured over a 700 nm–880 nm wavelength range.
MO-E3 and MO-E2 reacted with ONOO- and an increase in TPA was
observed. All probes exhibited the highest TPA at 740 nm. However,
(b) The fluorescence intensities of areas A–C as a function of time from Figure (a). The
fluorescence intensity was obtained for 60 min at 2 sec intervals. Excitation wavelength
and detection windows were 740 nm and 380–600 nm, respectively.
2 | J. Name., 2012, 00, 1-3
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excellent photophysical properties of TPM and comVpieawtibAritliictlye Ownliitnhe
DOI: 10.1039/D0SC02347G
MO-E2, we turned our attention towards the ex vivo imaging of
MO-E1 was unsuccessful for TPM imaging as only a minimal change
was observed before and after the addition of ONOO-. The TPA
values of each probe are shown in table S1 - ESI.
ONOO- using rat hippocampal tissue slices (Fig. 9). The average ratio
values of tissues that had been stained with MO-E2 (50 μM) for 1.5
h was 2.0, which was comparable to the macrophage control (Fig 9a).
When the hippocampal slice was pre-treated with SIN-1 (200 uM) for
30 min, the average ratio value was dramatically increased to 4.8
(Fig. 9b). When endogenous ROS was generated by treatment with
PMA (20 μM, 30 min), the average ratio value was significantly
increased to 3.6. While the addition ebselen suppressed the value
similar-to that observed for the control (Figure 9c-d). These results
demonstrate the potential of MO-E2 to be used as a two-photon
ratiometric tool for the imaging of endogenous ONOO- in living
systems.
TPM imaging of MO-E3 was performed using RAW264.7
macrophages. RAW264.7 macrophages were incubated with MO-E3
(5 μM) for 30 min and with a 740 nm excitation wavelength, MO-E3
was successfully imaged (Fig S22). However, intense spots on the
outside of the cells were observed suggesting poor cell
permeability/solubility for MO-E3. In addition, fluorescence
intensities of intracellular MO-E3 were almost unchanged, despite
pre-treatment with 3-morpholinosydnonimine (SIN-1, 50 μM, 30
min), a ONOO- donor, and exogenous ONOO- (50 μM, 30 min). SIN-1
is the most widely used ONOO- generator under physiological
conditions. The sydnonimine ring of SIN-1 hydrolyses under aerobic
aqueous conditions and releases nitric oxide and superoxide radicals,
these two molecules the generate the ONOO-.25 Overall, we believe
the limited solubility of MO-E3 has resulted in its unsuccessful in vitro
ONOO- imaging. Nevertheless, MO-E3 represents a proof of principle
system which can allow for development and exploration into novel
ratiometric ESIPT-based theranostics.
Conclusions
With this work; we demonstrate the utility of benzimidazoles for
two-photon fluorescence imaging and as a reactive linker scaffold for
the design of theranostic systems. Our approach used a simple
synthetic route to prepare two-photon activatable probes, MO-E1-3
The probes were evaluated using two-photon microscopy. MO-E1
did not produce large fluorescence changes on the addition of
ONOO- and as such was not evaluated further. Unfortunately,
despite MO-E3 illustrating the successful release of the fluorophore
Meanwhile, blue (380−430 nm, Fblue) and green (470−520 nm, Fgreen
)
regions were chosen as the detection windows for the ratiometric
images (Fgreen/Fblue) of MO-E2. Unlike MO-E3, MO-E2 (5 μM, 30 min)
reacted with ONOO- and displayed a large fluorescence change (Fig. and therapeutic agent (mass spectrometry and LC-MS analysis), the
6). When pre-treated with exogenous ONOO- (50 μM, 30 min) and
SIN-1 (50 μM, 30 min), the average ratio values were significantly
enhanced from 2.0 to 5.0 and 3.5, respectively (Fig. 6a–c).
Additionally, cells were pre-treated with lipopolysaccharide (LPS, 1
μg mL-1, 4 h) and interferon gamma (INF-γ, 50 ng mL-1, 1 h), which are
known to produce nitric oxide and superoxide to generate ONOO-
through induced iNOS expression and activated NADPH oxidase,26
and the average ratio value increased to 2.6 (Fig. 6e). Ebselen, an
organoselenium compound, is a known ONOO- scavenger that
rapidly catalyses the reduction of ONOO-.27 When the stimulated
macrophages were treated with ebselen (50 μM, 30 min), the
average ratio values were unchanged and were at a similar level to
those of the control (Fig. 6d-f). These results successfully
demonstrate that MO-E2 can be used to visualise ONOO- in living
cells. Furthermore, a CCK-8 assay demonstrated that MO-E2
exhibited almost no cytotoxicity. When RAW264.7 cells were treated
with MO-E2 (50 μM) 80 % of the cells survived (Fig. 7). The IC50 of
MO-E2 in RAW 264.7 cells for 24, 48, and 72 h were 164, 125 and 75
μM (Fig. S23) respectively, confirming that MO-E2 has negligible
cytotoxicity under the imaging conditions. In addition, MO-E2 has
high photostability (Fig. 8). This was shown by incubating RAW264.7
cells with MO-E2 followed by irradiation. The fluorescence intensities
were obtained from 1800 signals with 2 sec intervals for 1 h.
Remarkably, the fluorescence emission intensity remained constant
at 740 nm excitation wavelength over 1 h.
system did not perform well in vitro. We believe this was due to the
highly hydrophobic nature of indomethacin facilitating aggregation
in the cell medium before the probe could enter cells. However, MO-
E2 excelled in visualising ONOO- endogenously in RAW 264.7
macrophages and rat hippocampus tissue. The success of MO-E2 in
imaging ONOO- using two-photon excitation; indicates that a
benzimidazole ‘linker’ is suitable for the construction of novel
theranostic molecules incorporating more suitable drug candidates.
Towards that end, we are currently exploring the development of
such improved theranostic systems.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
MLO, JEG, SDB and TDJ would like to thank the University of
Bath for support. MLO and JEG thank the EPSRC for
studentships. TDJ wishes to thank the Royal Society for a
Wolfson Research Merit Award. H.M.K. acknowledges a grant
from the National Leading Research Lab Program of the
National Research Foundation of Korea (NRF), funded by the
Korean
government
(2019R1A2B5B03100278).
NMR
Characterisation facilities were provided through the Chemical
Characterisation and Analysis Facility (CCAF) at the University of
Bath (www.bath.ac.uk/ccaf). All data supporting this study are
provided as supplementary information accompanying this
paper.
For the detection of ONOO- in tissue, TPM is a powerful tool because
of its lower excitation energy (> 700 nm), deep tissue penetration
depth, low photo damage and longer observation times. Due to the
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