Coumarin-based fluorescent probe for the detection of glutathione and nitroreductase

Article abstract of DOI:10.1016/j.tet.2020.131890

With this research we set out to develop a coumarin-based novel fluorescent probe NTR-AHC for the detection of biological thiols and nitroreductase (NTR). Probe NTR-AHC was constructed by attaching the NTR trigger moiety (p-nitrobenzyl) and biothiol accep

Full text of DOI:10.1016/j.tet.2020.131890

Tetrahedron 82 (2021) 131890
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Coumarin-based fluorescent probe for the detection of glutathione
and nitroreductase
,
,
, c, **
Xue Tian a 1, Lokesh K. Kumawat b 1, Steven D. Bull a, Robert B.P. Elmes b
,
Luling Wu a ***, Tony D. James a
a Department of Chemistry, University of Bath, Bath, BA2 7AY, UK
,
, d, *
b Department of Chemistry and the Kathleen Lonsdale Institute for Human Health Research, Maynooth University, The National University of Ireland,
Maynooth, Co., Kildare, Ireland
c Synthesis and Solid-State Pharmaceutical Centre, Maynooth University, Ireland
d School of Chemistry and Chemical Engineering, Henan Normal University, Xinxiang, 453007, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
With this research we set out to develop a coumarin-based novel fluorescent probe NTR-AHC for the
detection of biological thiols and nitroreductase (NTR). Probe NTR-AHC was constructed by attaching the
NTR trigger moiety (p-nitrobenzyl) and biothiol acceptor (maleic acid monoamide), to the core fluo-
rophore AHC. In the presence of both glutathione (GSH) and NTR, probe NTR-AHC produced a >8-fold
increase in fluorescence intensity at 463 nm.
Received 27 October 2020
Received in revised form
14 December 2020
Accepted 16 December 2020
Available online 28 December 2020
© 2020 Published by Elsevier Ltd.
Keywords:
Fluorescent probe
Nitroreductase
Glutathione
1. Introduction
fluorescent probes for the detection of GSH [13e15]. While, many of
these fluorescent probes have focused on the detection of a single
Biothiols play many crucial roles in intracellular antioxidant
defense systems [1]. Glutathione (GSH), for example, the most
prevalent intracellular non-protein thiol ranges in concentration
from 1 to 15 mM depending on the cell type [2e5]. It is a natural
biomarker (GSH), many processes associated with GSH often
involve more than one biochemical species. For example, while
hypoxia is reported to decrease cellular GSH stores [16], maternal
hypoxia has been reported to facilitate fetal liver GSH production
[21]. Hypoxia is caused by an insufficient supply of oxygen [9] and
can result in increased reductive stress; a consequence of the
upregulation of various reductive enzymes such as nitroreductase
(NTR) [17]. Indeed, upregulation of NTR has been associated with
various health problems, including inflammatory disease, solid
tumors, and stroke [18e20]. Hence, the development of methods
that help explore the relationships between GSH and NTR are of
great importance in terms of pathological analysis and early
diagnostics.
tripeptide (g-L-glutamyl-cysteinyl-glycine) that exists in the thiol
reduced form (GSH) and disulphide-oxidised (GSSG) form [6]. GSH
serves many cellular functions, including the protection of cellular
components against oxidative stress, redox homeostasis, and
intracellular signal transduction [1,7e9]. However, an imbalance of
GSH is observed for a wide range of pathologies, including cancer,
Parkinson's disease, HIV and aging [10e12]. Therefore, significant
effort has been devoted towards the development of efficient
As a useful analytical tool, fluorescence spectroscopy is of broad
interest in bioanalysis [22]. Fluorescence-based small-molecule
probes are able to selectively sense and image analytes through the
utilization various chemical reactions, furthermore, fluorescent
probes are often less invasive and more convenient than many
other methods for the detection of biologically-relevant cellular
targets [23,24]. Recently, a number of fluorescence-based probes
for dual or multi-analyte detection have been developed [25e27].
* Corresponding author. Department of Chemistry, University of Bath, Bath, BA2
7AY, UK.
** Corresponding author. Department of Chemistry and the Kathleen Lonsdale
Institute for Human Health Research, Maynooth University, The National University
of Ireland, Maynooth, Co., Kildare, Ireland.
*** Corresponding author.
E-mail addresses: Robert.Elmes@mu.ie (R.B.P. Elmes), lw960@bath.ac.uk (L. Wu),
t.d.james@bath.ac.uk (T.D. James).
1
Equal contribution.
https://doi.org/10.1016/j.tet.2020.131890
0040-4020/© 2020 Published by Elsevier Ltd.

X. Tian, L.K. Kumawat, S.D. Bull et al.
Tetrahedron 82 (2021) 131890
Scheme 1. Synthesis of target probe NTR-AHC.
These types of fluorescent probes have been used for the con-
struction of molecular logic gates and/or for medical diagnostics
[28]. AND logic-based fluorescent probes require both analytes to
be simultaneously present or work in tandem in order to elicit a
fluorescence response, and provide different information to that
available from serial measurements of two analytes within the
same biological system.
In our group, we have recently focused on developing dual an-
alyte activated probes [29e33]. With this design principle in mind,
we identified a suitable fluorophore based on coumarin __3-amino-
7-hydroxy-2H-chromen-2-one (AHC). We anticipated that its free
phenol and amine functionalities could provide an excellent op-
portunity for differential derivatization (Scheme 1). Furthermore,
coumarins are an important structural element of various com-
pounds with biological activities including compounds with anti-
cancer [34], antiproliferative [35], antioxidant [36], and antifungal
activities [37]. Previous literature reports indicate that the protec-
tion of AHC with a maleic acid monoamide group results in fluo-
rescence quenching of the coumarin's fluorescence due to a
photoinduced electron transfer (PeT) process, the fluorescence is
rapidly restored in the presence of biological thiols due to their fast
addition to this functional group [38]. Therefore, we envisioned
that functionalization of the free phenol of this probe using a p-
nitrobenzyl should further quench the fluorescence, whilst serving
as a selectivity reporter unit toward NTR over other various ana-
lytes [39,40]. Consequently, with this research we anticipated that
the attachment of a NTR and GSH differentiating motif to AHC could
produce a selective fluorescent probe NTR-AHC for the simulta-
neous detection of GSH and NTR (Scheme 2).
2. Results and discussion
We evaluated the ability of NTR-AHC to detect GSH ‘AND’ NTR in
PBS buffer solution (10 mM, pH 7.4, containing 1% DMSO). The
maximum absorption of NTR-AHC at 344 nm shifted to 340 nm
upon addition of GSH with a significant increase at 340 nm
following the addition of NTR (Fig. S1). As shown in Fig. 1, probe
NTR-AHC was initially non-fluorescent, and exhibited a small
Scheme 2. Reaction mechanism of NTR-AHC with GSH and nitroreducase (NTR) and NADPH.
2

X. Tian, L.K. Kumawat, S.D. Bull et al.
Tetrahedron 82 (2021) 131890
NTR-AHC for the detection of biological thiols and NTR, respec-
tively, however, an optimum response was only obtained if GSH
was added first followed by NADPH and NTR, while, the reverse
sequence does not show significant fluorescence changes, which
we attribute to a large initial increase to the background fluores-
cence by NADPH and potential oxidation of the GSH by NADPþ
[42,43] generated during the NTR/NADH catalyzed reduction of
NTR-AHC [44]. In summary, AHC is a useful core unit for the
development of coumarin-based dual-analyte ‘AND’ logic fluores-
cent probes.
4. Experimental section
4.1. Chemicals and reagents
All starting materials and reagents were purchased from Sigma
Aldrich, Alfa Aesar, Fluorochem, or Acros Organics, and used as
received without any further purification. Unless otherwise stated,
all solvents used were reagent grade and were used without
distillation. All water was deionized which is generated by a reverse
osmosis (Ultra-Purified Type I, 18.2 Megohm water). Thin-layer
chromatography was performed using commercially available
Fluorochem aluminum-backed plates coated with a layer of silica
gel (60 Å) with fluorescent indicator UV254. These plates were
visualized using ultraviolet light with a wavelength of either 254 or
365 nm. Silica gel column chromatography was carried out using
Sigma Aldrich 60 Å silica gel (200e400 mesh). All NMR spectra
were obtained using an Agilent ProPulse 500 and analyzed using
MestreNova. LC-MS analyses were performed using an Agilent
QTOF 6545 mass spectrometer, IR spectra were obtained using a
Thermo Fisher Nicolet iS5 FTIR spectrometer and analyzed using
OMNIC (version 9.7.7).
Fig. 1. Fluorescence spectra of NTR-AHC (10.0
incubated for 20 min, followed by the addition of NADPH (400
m
M) with initial addition of GSH (5
m
M)
mM) and NTR
(10
m
g mLꢀ1) monitored for a further 90 min in PBS buffer solution (pH ¼ 7.4, 10 mM,
containing 1% DMSO). lex ¼ 365 nm. Ex slit: 5 nm and em slit: 5 nm. Dashed line
represents NTR-AHC and GSH addition only. Blue line represents highest intensity after
addition of NTR.
4.2. Synthesis of NTR-AHC
Compounds 1e4 were synthesized using adapted literature
procedures (Scheme S1) [29,38,45]. Compound
4 (300 mg,
1.04 mmol) was dissolved in dry DMF (5 mL) and K2CO3 (215 mg,
3.15 mmol) was added with stirring. 4-nitrobenzyl bromide
(249 mg, 1.157 mmol) was then added and the reaction was stirred
for 4 h at room temperature under a N2 atmosphere. The reaction
mixture was diluted with EtOAc (90 mL) and washed with brine
(90 mL ꢁ 3), dried over anhydrous Na2SO4 and concentrated in
vacuo. The crude product obtained was purified by flash chroma-
tography (SiO2, 30% EtOAc in petroleum ether) to afford the desired
yellow solid NTR-AHC (198 mg, 48%) (Scheme S1). M.p.127e129 ꢂC;
1H NMR (500 MHz, DMSO‑d6) dH 10.12 (s, 1H), 8.63 (s, 1H), 8.27 (d,
J ¼ 8.5 Hz, 2H), 7.74 (d, J ¼ 8.4 Hz, 2H), 7.71 (d, J ¼ 8.6 Hz, 1H), 7.11 (s,
1H), 7.07 (d, J ¼ 8.6 Hz, 1H), 6.73 (d, J ¼ 11.8 Hz, 1H), 6.51 (d,
J ¼ 11.7 Hz, 1H), 5.39 (s, 2H), 3.69 (s, 3H). 13C NMR (126 MHz,
DMSO‑d6) dC 167.1, 163.3, 159.5, 157.5, 151.4, 147.1, 144.2, 131.1, 129.4,
129.2, 128.4, 125.7, 123.6, 121.7, 113.4, 113.1, 101.6, 68.6, 51.6. FTIR
(ATR, cmꢀ1): 3309 (NeH), 2955 (CeH, alkyl), 1721 (C]O), 1708 (C]
O), 1605 (C]C), 1247 (CeN). HRMS (ESIþ): calculated [MþNa]þ
Fig. 2. Fluorescence intensity changes at 463 nm of NTR-AHC (10.0 mM) with initial
addition of GSH (5.0
mM) incubated for 20 min, followed by the addition of NADPH
(400 M) and NTR (2.0e15.0
m
m
g mLꢀ1) in PBS buffer (pH ¼ 7.4, 10 mM, containing 1%
DMSO). lex ¼ 365 nm. Ex slit: 5 nm and em slit: 5 nm.
fluorescence increase upon addition of GSH (5
m
M) (Fig. S2), how-
ever, the successive addition of NTR (10
(400 M) led to a significant and time dependent increase in fluo-
m
g mLꢀ1) and NADPH
m
rescence intensity (>8-fold). These results illustrated the require-
ment for both GSH ‘AND’ NTR to obtain a significant turn ‘on’
fluorescence response. We then evaluated the kinetic behavior of
NTR-AHC with both GSH and NTR (Figs. 2 and 3) and determined
that NTR-AHC exhibits a dose dependent fluorescence increase in
response of both GSH and NTR.
To ensure both analytes were required, the same fluorescence
experiments were then carried out in reverse order, NTR and
447.0799 m/z, found 447.0798 m/z,
(chemical formula of NTR-AHC).
M represents C21H16N2O8
NADPH was kept constant (10 m mM, respectively)
g mLꢀ1 and 400
resulting in a large fluorescence increase (Fig. 4), attributed to the
background fluorescence of NADPH [41]. Subsequent addition of
4.3. Equipment and spectral measurements
GSH (200
m
M) led to a 1.2-fold increase in fluorescence intensity.
All Fluorescence emission spectra were recorded on a Jasco FP-
6300 spectrofluorometer. Excitation and emission slit widths were
both set at 5.0 nm. The UVevisible absorption spectra were
measured at room temperature on a Jasco V-630-Bio Spectrometer.
Nuclear Magnetic Resonance (NMR) spectroscopy experiments
were performed in deuterated solvent at 298 K on an Agilent
3. Conclusions
We have developed a novel coumarin-based fluorescent probe
3

X. Tian, L.K. Kumawat, S.D. Bull et al.
Tetrahedron 82 (2021) 131890
Fig. 3. Fluorescence spectra of NTR-AHC (10.0 mM) with initial addition of GSH (5.0 mM) incubated for 20 min (dashed line), followed by the addition of NADPH (400 mM) and NTR
((a) 2
slit: 5 nm.
m
g mLꢀ1, (b) 7
m
g mLꢀ1, (c) 10
m
g mLꢀ1, (d) 15
m
g mLꢀ1) monitored for a further 90 min in PBS buffer (pH ¼ 7.4, 10 mM, containing 1% DMSO). lex ¼ 365 nm. Ex slit: 5 nm and em
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgments
XT wishes to thank the University of Bath for supporting her
PhD in the UK. LKK wishes to acknowledge the Irish Research
Council for
a Government of Ireland Postdoctoral Research
Fellowship (GOIPD/2017/1091) and Science Foundation Ireland are
acknowledged for the funding of Advion Mass Spec facilities
through the Opportunistic Infrastructure Fund (16/RI/3399). RBPE
would like to thank Science Foundation Ireland (SFI) and the Eu-
ropean Regional Development Fund (12/RC/2275_P2). LW wishes to
thank China Scholarship Council and the University of Bath for
supporting his PhD in the UK. We would like to thank the EPSRC
and the University of Bath for funding. TDJ wishes to thank the
Royal Society for a Wolfson Research Merit Award and the Open
Research Fund of the School of Chemistry and Chemical Engineer-
ing, Henan Normal University for support (2020ZD01). NMR char-
acterisation facilities were provided by the Material and Chemical
Characterisation Facility (MC2) at the University of Bath (http://go.
bath.ac.uk/mc2). The EPSRC UK National Mass Spectrometry Facil-
ity at Swansea University is thanked for analyses. All data sup-
porting this study are provided as ESIy accompanying this paper.
Fig. 4. Fluorescence spectra of NTR-AHC (10.0
m
M) with initial addition of NADPH
(400
(200
m
M) and NTR (10 m
g mLꢀ1) incubated for 90 min, followed by the addition of GSH
mM) monitored for a further 40 min in PBS buffer (pH ¼ 7.4, 10 mM, containing 1%
DMSO). lex ¼ 365 nm. Ex slit: 5 nm and em slit: 5 nm. Dashed line represents NTR-AHC
and NTR addition. Blue line represents highest intensity after addition of GSH.
ProPulse 500 MHz spectrometer.
4.4. Preparation of samples for analysis
NTR-AHC was dissolved in DMSO and a stock solution (0.1 mM)
prepared in PBS buffer (pH ¼ 7.4). NTR stock solutions were pre-
pared by dissolving 1 mg of enzyme in 1 mL of PBS buffer solution
and individual ampules were stored in the freezer before being
thawed and used in each experiment. Stock solutions of NADPH
(2 mM) and GSH (0.1 mM) were prepared in PBS buffer (pH ¼ 7.4).
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2020.131890.
4

X. Tian, L.K. Kumawat, S.D. Bull et al.
Tetrahedron 82 (2021) 131890
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