An isophorone-fused near-infrared fluorescent probe with a large Stokes shift for imaging endogenous nitroxyl in living cells and zebrafish

Article abstract of DOI:10.1016/j.saa.2019.117765

Nitroxyl (HNO) plays an important role in multiple physiological and pathological processes, but the detailed generation mechanism of the endogenous HNO still remained to explore and perfect further. There is an urgent need to develop an excellent fluorescent probe for selective recognition and sensitive detection of HNO in biological systems. Near-infrared (NIR) fluorescent probes with a large Stokes shift are an ideal tool for bioimaging applications. Here, we have developed a NIR fluorescent probe with a large Stokes shift, namely, NIR-HNO, to monitor HNO in cells and zebrafish. NIR-HNO consists of an isophorone-fused NIR fluorescence reporter and a diphenylphosphinobenzoyl HNO-responsive unit. Based on an aza-ylide intramolecular ester aminolysis reaction, NIR-HNO showed a rapid selective NIR fluorescent turn-on response for HNO, high sensitivity (detection limit was 39.6 nM), and large Stokes shift (265 nm). The biological imaging results indicate that NIR-HNO is a good candidate for imaging of endogenous HNO in living systems.

Full text of DOI:10.1016/j.saa.2019.117765

SAA-117765; No of Pages 6
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Short Communication
An isophorone-fused near-infrared fluorescent probe with a large Stokes
shift for imaging endogenous nitroxyl in living cells and zebrafish
Chao Wei a,b, , Xiaofen Wang a,b, Xueyan Li a,b, Xu Jia a,b, Xinya Hao b, Jiangyan Zhang b,
⁎
Pingzhu Zhang a,b, Xiaoliu Li a,b,
⁎
a
Key Laboratory of Medicinal Chemistry and Molecular Diagnosis of Ministry of Education, Key Laboratory of Chemical Biology of Hebei Province, College of Chemistry and Environmental Science,
Hebei University, Baoding 071002, China
b
College of Chemistry and Environmental Science, Hebei University, Baoding 071002, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 11 July 2019
Received in revised form 4 November 2019
Accepted 4 November 2019
Available online xxxx
Nitroxyl (HNO) plays an important role in multiple physiological and pathological processes, but the detailed
generation mechanism of the endogenous HNO still remained to explore and perfect further. There is an urgent
need to develop an excellent fluorescent probe for selective recognition and sensitive detection of HNO in biolog-
ical systems. Near-infrared (NIR) fluorescent probes with a large Stokes shift are an ideal tool for bioimaging ap-
plications. Here, we have developed a NIR fluorescent probe with a large Stokes shift, namely, NIR-HNO, to
monitor HNO in cells and zebrafish. NIR-HNO consists of an isophorone-fused NIR fluorescence reporter and a
diphenylphosphinobenzoyl HNO-responsive unit. Based on an aza-ylide intramolecular ester aminolysis reac-
tion, NIR-HNO showed a rapid selective NIR fluorescent turn-on response for HNO, high sensitivity (detection
limit was 39.6 nM), and large Stokes shift (265 nm). The biological imaging results indicate that NIR-HNO is a
good candidate for imaging of endogenous HNO in living systems.
Keywords:
Nitroxyl
Isophoron
Near-infrared fluorescence
Large Stokes shift
Fluorescent imaging
© 2019 Elsevier B.V. All rights reserved.
1. Introduction
[8–10]. Although HNO has the above versatile roles, its specific mecha-
nism is still unclear. As a result, it is very necessary to develop a reliable
Reactive nitrogen species (RNS) is one class of highly chemical active
species in living systems. Nitroxyl (HNO) is a well-known member of
RNS, which is the one-electron reduced and further protonated form
of nitric oxide (NO). HNO plays important roles during a series of phys-
iological and pathological processes [1]. Endogenous HNO can come
from the reaction of S-nitrosoglutathione with glutathione [2], the re-
duction of L-arginine by nitric oxide synthase [3], and oxidation of hy-
droxylamine by peroxidases. In addition [4], it is also reported that
HNO has unique biological activity and medicinal potential. For exam-
ple, HNO was used in research to treat heart failure because it could en-
hance the myocardial contractile force and promote the contraction of
heart cells [5,6]. HNO could also inhibit the activity of acetaldehyde de-
hydrogenase to be used, as a potential drug for treating alcoholism [7].
Recently, HNO has been reported to possess anti-inflammatory effects
method for selective recognition of HNO in biological systems.
At present, fluorescence probe assay is a fast-growing technique for
the recognition and detection of different analysts because of its unique
characteristics, such as high selectivity, good sensitivity, non-invasion,
and spatiotemporal resolution, and has been basically mature and
used in the fields such as life sciences, biotechnology, drugs analysis,
etc. [11–18]. To date, based on the redox reaction and Staudinger reac-
tion, multiple HNO fluorescent probes have been reported [19–21].
Among them, the Staudinger reaction has been widely applied for de-
veloping HNO fluorescent probes because of its better biocompatibility
[22–40]. However, many reported fluorescent probes with short excita-
tion and emission wavelengths, suffer from the potential photo-
bleaching, background fluorescence interference, and photo-damage
to cells and tissues, which could be used for detecting HNO in living
cells, but not suitable for in vivo applications [22,32–37]. To make up
for these deficiencies, many near-infrared (NIR, N650 nm) fluorescent
probes have been developed for imaging HNO in biological specimens
[23–28,30,31]. As is well-known, fluorescent probes with large Stokes
shift could be free from interference from excited light scattering,
which leads to more suitable for practical application in vivo. Thus,
⁎
Corresponding authors at: Key Laboratory of Medicinal Chemistry and Molecular
Diagnosis of Ministry of Education, Key Laboratory of Chemical Biology of Hebei
Province, College of Chemistry and Environmental Science, Hebei University, Baoding
071002, China.
E-mail addresses: weichao@hbu.edu.cn (C. Wei), lixl@hbu.cn (X. Li).
https://doi.org/10.1016/j.saa.2019.117765
1386-1425/© 2019 Elsevier B.V. All rights reserved.
Please cite this article as: C. Wei, X. Wang, X. Li, et al., An isophorone-fused near-infrared fluorescent probe with a large Stokes shift for imaging
endogenous..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117765

2
C. Wei et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
Scheme 1. (A) The synthetic scheme of NIR-HNO; (B) the possible mechanism of sensing HNO by NIR-HNO.
new NIR fluorescent HNO probes with large Stokes shifts are urgently
needed.
(DMAP) (367 mg, 3.00 mmol) was dissolved in dry N,N-
dimethylformamide (DMF) (10 mL). After stirring for 30 min, com-
pound 1 [41] (145 mg, 0.50 mmol) was added into the above solution
and further stirred overnight at room temperature. After the reaction
completely, the solvent was poured into ice water (50 mL), the forming
precipitation was filtered, and the crude product was purified by silica
gel column chromatography to give NIR-HNO as a light yellow solid
(217 mg, 75%). m.p.: 186–188 °C; 1H NMR (600 MHz, CDCl3): δ =
8.27–8.24 (m, 1H), 7.51–7.45 (m, 4H), 7.38–7.31 (m, 11H), 7.04–6.90
(m, 5H), 6.84 (s, 1H), 2.60 (s, 2H), 2.46 (s, 2H),1.08 (s, 6H). 13C NMR
(151 MHz, CDCl3): 169.16, 164.91, 153.60, 151.54, 141.42, 141.15,
137.40, 1.37.30, 135.93, 134.49, 134.13, 133.92, 133.28, 133.10, 132.67,
131.32, 129.17, 128.83, 128.71, 128.60, 128.53, 128.45, 128.38, 123.63,
122.29, 113.43, 112.64, 78.82, 42.97, 39.19, 31.99, 27.99. HRMS calcu-
lated for C38H32N2O2P [M + H]+ 579.2201, found: 579.2198.
Herein, we report a NIR fluorescent probe with a large Stokes shift,
namely, NIR-HNO, for detection HNO in living systems (Scheme 1A).
NIR-HNO consists of an isophorone-fused dye 1 as a NIR fluorophore
and a diphenylphosphinobenzoyl group as the HNO responsive group.
The fluorescence of NIR-HNO was inhibited by disrupting the intramo-
lecular charge transfer (ICT) effect. Upon reaction with HNO, aza-ylide
and further intramolecular aminolysis reaction occur, and the resulting
fluorophore 1 emits NIR fluorescence (Scheme 1B). NIR-HNO could se-
lectively and sensitively detect HNO with a rapid fluorescent response, a
low detection limit (39.6 nM), and a remarkable large Stokes shift
(265 nm). The results for the detection of the endogenous generation
of HNO demonstrate the suitability of the probe in practical applications
in living systems.
2. Materials and methods
2.2. Procedure of fluorescence measurement
Unless especially noted, all chemical agents were purchased from
J&K Scientific. NMR spectra were determined using Advance 600 MHz
(Bruker, Germany). High-Resolution Mass Spectrum (HRMS) were col-
lected using Varian 7.0 T FTICR-MS. UV–Vis absorption spectra and fluo-
rescence spectra were recorded on Cary 5000 Bio (Agilent, USA) and F-
7000 (Hitachi, Japan) spectrophotometer, respectively. Bioimaging of
cells and zebrafish were acquired on a confocal microscope (Olympus
Fluoview FV1000 and Zeiss LSM 880).
The stock solution of NIR-HNO (2 mM) was prepared using DMSO and
was used with a concentration of 10 μM. Different analytes with a concen-
tration of 1 M were prepared in PBS solution. These analytes include NaF,
NaCl, NaBr, NaI, KCl, MgCl2, CuCl2, ZnCl2, FeCl3, Na2SO4, Na2SO3, NaNO3,
NaNO2, NaClO, H2O2, KO2, Sodium nitroprusside (source of NO), ONOO−,
CySNO, GSNO, Na2S (source of H2S), cysteine (Cys), homocysteine (Hcy),
glutathione (GSH) and Angeli's salt (source of HNO, AS). All spectroscopic
measurement was performed in PBS (10 mM, pH 7.4, 50% DMF, 25 °C)
with excitation at 556 nm and slit width of 5.0 nm.
2.1. Synthesis
2.3. Cell culture and fluorescence microscopy imaging
A mixture of 2-(diphenylphosphanyl)benzoic acid (230 mg,
0.75 mmol), N-(3-dimethylaminopropyl)-N′-ethyl carbodiimide hydro-
chloride (EDCI) (289 mg, 1.50 mmol) and 4-dimethylamino pyridine
HeLa cells were cultured in DMEM supplemented with 10% fetal
calf serum at 37 °C in 5% CO2. The cytotoxicity was measured
Fig. 1. Time-dependent optical changes in absorption (a) and emission (b) of NIR-HNO (10 μM) in the presence of AS (75 μM) in PBS solution (10 mM, pH = 7.4, 50% DMF) at 25 °C.
(c) Time dependence of emission intensity at 688 nm for NIR-HNO (10 μM) in the absence and presence of AS (75 μM).
Please cite this article as: C. Wei, X. Wang, X. Li, et al., An isophorone-fused near-infrared fluorescent probe with a large Stokes shift for imaging
endogenous..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117765

C. Wei et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
3
Fig. 2. (a) Emission spectra of NIR-HNO (10 μM) upon addition of AS (0–80 μM). (b) The linearity of fluorescent response at 688 nm between 0 and 80 μM AS concentration.
through MTT assays [36]. Cells were incubated with different con-
centrations of NIR-HNO for 12 h in a 96-well plate than remove
the medium, MTT (0.5 mg/mL) media was added to cells. After in-
cubation for 4 h, the produced formazan was dissolved in DMSO
(150 μL) and read at OD 490 nm with a Spectramax microwell
plate reader.
For imaging exogenous HNO, cells were firstly incubated with NIR-
HNO (5 μM) for 30 min, washed with PBS, and then treated with HNO
(20 μM and 50 μM) for another 30 min. For imaging of NaASc and
SNP-induced endogenous HNO, cells were first co-incubated with
NaASc (2 mM) and SNP (2 mM) for 2 h, washed with PBS, and then
treated with NIR-HNO (5 μM) for another 1 h. The above-treated cells
were imaged on a confocal microscope (Zeiss LSM 880). Emission was
collected at the red channel (662–737 nm) with 561 nm excitation
and a 20× objective lens.
3. Results and discussion
3.1. Design and synthesis
The fluorophore is a crucial component of a fluorescent probe. Re-
cently, isophorone-fused fluorophore 1 has received extensive attention
due to its excellent NIR emission fluorescence and large Stokes shift, and
the protection and deprotection of hydroxyl group of 1 can regulate the
switch of fluorescence [41,42]. Thus, it was expected that probe NIR-
HNO could be a NIR HNO fluorescent probe with large Stokes shift by
the protection of the hydroxyl group with triarylphosphine. NIR-HNO
was prepared by only one step with high yield, and its structure was
characterized by NMR spectroscopy and HRMS in Supporting informa-
tion (Figs. S1–3).
3.2. Spectral response
2.4. Fluorescence imaging of zebrafish
The absorption spectra of NIR-HNO and its reaction with HNO was
firstly measured in PBS buffer solution (10 mM, pH 7.4, 50% DMF) at 25
°C. Angeli's salt (AS), a water-soluble HNO releasing molecule, was used
as the source of HNO. The probe displayed a maximal absorption band at
406 nm. After incubating with AS, two absorption peaks appeared at
423 nm and 556 nm, which is because of the phenolic hydroxyl group of
compound 1 could partly form a deprotonated phenate anion in DMF/
PBS (1:1) solution (Fig. 1a) [43]. The fluorescence spectra of NIR-HNO
were then tested. The probe itself exhibited weak fluorescence (Φ b
0.0006). After reaction with AS, an obvious NIR fluorescence with the
Zebrafish (3 days postfertilization) were purchased from Eze-Rinka
Company (Nanjing, China). The zebrafish were treated with NIR-HNO
(10 μM) for 4 h. After removed the embryo medium and washed with
PBS (10 mM, pH 7.4) three times, the zebrafish were further incubated
with HNO (50 μM) for 1 h at 28 °C. The imaging was collected on a con-
focal microscope (Olympus Fluoview FV1000). Emission was collected
at the red channel (570–670 nm) with 559 nm excitation and a 10× ob-
jective lens.
Fig. 3. Fluorescence responses of NIR-HNO (10 μM) in the presence of HNO (75 μM) and various relevant analytes (200 μM) in PBS solution (10 mM, pH = 7.4, 50% DMF) at 25 °C for
15 min, λex = 556 nm. (1–18: NIR-HNO, NaF, NaCl, NaBr, NaI, KCl, MgCl2, CuCl2, ZnCl2, FeCl3, Na2SO4, Na2SO3, NaNO3, NaNO2, NaClO, H2O2, KO2, NO, 200 μM; 19: ONOO−, 100 μM;
20–21: CySNO, GSNO, 50 μM; 22: H2S, 200 μM; 23–25: GSH, Cys, Hcy, 1 mM; 26: HNO, 75 μM).
Please cite this article as: C. Wei, X. Wang, X. Li, et al., An isophorone-fused near-infrared fluorescent probe with a large Stokes shift for imaging
endogenous..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117765

4
C. Wei et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
Fig. 4. Fluorescent imaging of exogenous HNO in HeLa cells. Cells were incubated with NIR-HNO (5 μM) for 30 min (a), and then treated with 20 μM (b) and 50 μM (c) AS for another
30 min. (Up) Red fluorescence channels. (Down) Merged fields. Scale bars = 20 μm. (d) Average fluorescence intensity of a–c. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
maximal value at 688 nm appeared with the excitation at 556 nm. To our
knowledge, the remarkable Stokes shift for NIR-HNO is up to 265 nm,
which is the largest among the NIR fluorescent probes reported so far for
HNO (Table S1). The fluorescence quantum yield of compound 1 in the
PBS and in PBS/DMF (1:1) solution is approximately 0.0095 and 0.0231, re-
spectively, which indicate the potential applicability of the probe. HRMS of
the reaction system was used to analyze the recognition mechanism. The
peak at m/z 313.1318 [M + Na]+ and 344.0811 [M + Na]+ corresponding
to compound 1 and amide derivative, respectively, was observed (Fig. S4).
Therefore, NIR-HNO most likely undergoes the mechanism as the reported
literature [32] and shown in Scheme 1B.
The time-dependent fluorescent response of the probe solution to-
ward AS was performed. As shown in Fig. 1c, 75 μM AS could cause
the reaction to be completed within 10 min. The pseudo-first-order
rate constant (kobs) was calculated as 0.29 min−1 and the reaction rate
(k2) was determined to be 64.78 M−1·s−1, which is comparable with
other phosphine-based HNO probes [37,39,44]. The rapid fluorescence
response indicated that NIR-HNO was suitable for the detection of
HNO. A time of 15 min after the addition of AS was selected in the sub-
sequent experiments.
3.3. Sensitivity and detection limit
To evaluate the sensitivity of NIR-HNO to HNO, the fluorescence ti-
tration experiments were further carried out. As shown in Figs. 2 and
S5, with the increase of AS concentration, the fluorescence signal grad-
ually increased. The fluorescence signal at 688 nm was linearly related
to the concentration of AS from 0 to 80 μM (R = 0.9990), indicating a
large dynamic range for the detection of HNO. The detection limit was
calculated to be 39.6 nM based on the 3σ/k method, where k is the
slope between fluorescence intensity versus HNO concentration and σ
is the relative standard deviation of the blank measurements. These re-
sults demonstrated that NIR-HNO is highly sensitive toward HNO in
buffer solution.
Fig. 5. Fluorescent imaging of NaASc and SNP-induced endogenous HNO in HeLa cells. Cells were incubated with NIR-HNO (5 μM) for 30 min (a), pre-treated with NaASc (2 mM) (b) or
SNP (2 mM) (c) for 2 h, pre-treated with NaASc (2 mM) and SNP (2 mM) (d) for 2 h, and then treated with NIR-HNO (5 μM) for another 1 h. (Up) Red fluorescence channels. (Down)
Merged fields. Scale bars = 50 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Please cite this article as: C. Wei, X. Wang, X. Li, et al., An isophorone-fused near-infrared fluorescent probe with a large Stokes shift for imaging
endogenous..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117765

C. Wei et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
5
indicated the possible capability of this probe for long time monitoring
intracellular HNO changes in living HeLa cells.
3.6. Bioimaging of HNO in zebrafish
To further investigate the application of NIR-HNO in the living sys-
tem, a zebrafish imaging experiment was also carried out. As shown in
Fig. 6, zebrafish were first treated with NIR-HNO (10 μM) for 4 h, and al-
most no fluorescence could be observed. After further treated with AS
(50 μM) for another 1 h, the red fluorescence increased dramatically.
These results demonstrated that NIR-HNO can track HNO in a living
system.
4. Conclusions
In summary, a new isophoron-fused NIR fluorescent probe was de-
veloped for the detection of HNO. This probe shows a fast response,
good selectivity and high sensitivity. The remarkable large Stokes shift
makes the probe more suitable for fluorescence detection of HNO in bi-
ological samples.
Fig. 6. Fluorescent imaging of exogenous HNO in zebrafish. Zebrafish were incubated with
NIR-HNO (10 μM) for 4 h (a), and then treatment with AS (50 μM) for another 1 h (b).
(Up) Red fluorescence channels. (Down) Merged fields. Scale bars = 100 μm. (For
interpretation of the references to color in this figure legend, the reader is referred to
the web version of this article.)
Declaration of competing interest
3.4. Selectivity
We declare that we do not have any commercial or associative inter-
est that represents a conflict of interest in connection with the work
submitted.
Because of the complexity of biological systems, the selectivity is ex-
tremely important for the fluorescent probe. To test the selectivity of
NIR-HNO, the reactivity of NIR-HNO toward different biological
analytes was also carried. These biologically species include anions
Acknowledgments
(F−, Cl−, Br−, I−, SO42−, SO32−, NO−3 , NO−2 ), cations (K+, Mg2+, Cu2+
,
This work was supported by the National Natural Science Founda-
tion of China (21605034, 21778013), the Natural Science Foundation
of Hebei Province (B2017201184, B2018201234), the Foundation of He-
bei Education Department (QN2017015).
Zn2+, Fe3+), reactive oxygen species (ClO−, H2O2, KO2), reactive sul-
phur species (Cys, Hcy, GSH, H2S), and reactive nitrogen species (NO,
ONOO−, CySNO, GSNO). Various biological species were respectively
added into NIR-HNO solutions to test their fluorescence response. As
shown in Fig. 3, of all the tested species, GSNO only triggered small fluo-
rescence enhancement, HNO caused an obvious increase of fluores-
cence, while other analysts did not trigger dramatic fluorescence
enhancement. The excellent selectivity of NIR-HNO to HNO suggested
it can be a powerful tool for specifically detecting HNO in biological en-
vironments. In addition, NIR-HNO could function over a wide range of
pH from 6.5 to 10.0 (Fig. S7).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.saa.2019.117765.
References
[1] V. Shafirovich, S.V. Lymar, Proc. Natl. Acad. USA 99 (2002) 7340–7345.
[2] P.S.Y. Wong, J. Hyun, J.M. Fukuto, F.N. Shirota, E.G. DeMaster, D.W. Shoeman, H.T.
Nagasawa, Biochemistry 37 (1998) 5362–5371.
[3] S. Adak, Q. Wang, D.J. Stuehr, J. Biol. Chem. 275 (2000) 33554–33561.
[4] S.I. Liochev, I. Fridovich, Free Radicals Biol. Med. 34 (2003) 1399–1404.
[5] G. Tocchetti Carlo, W. Wang, P. Froehlich Jeffrey, S. Huke, A. Aon Miguel, M. Wilson
Gerald, G. Di Benedetto, B. O’Rourke, D. Gao Wei, A. Wink David, P. Toscano John, M.
Zaccolo, M. Bers Donald, H. Valdivia Hector, H. Cheng, A. Kass David, N. Paolocci, Circ.
Res. 100 (2007) 96–104.
[6] M. Eberhardt, M. Dux, B. Namer, J. Miljkovic, N. Cordasic, C. Will, T.I. Kichko, J. de la
Roche, M. Fischer, S.A. Suárez, D. Bikiel, K. Dorsch, A. Leffler, A. Babes, A. Lampert, J.K.
Lennerz, J. Jacobi, M.A. Martí, F. Doctorovich, E.D. Högestätt, et al., Nat. Commun. 5
(2014) 4381.
3.5. Bioimaging application in living cells
Before bioimaging, HeLa cells were chosen to evaluate the cytotoxic-
ity of NIR-HNO. MTT assay results show NIR-HNO has only minimal cy-
totoxicity at 20 μM concentration, implying that the probe is suitable for
imaging HNO in living cells (Fig. S7). HeLa cells were firstly incubated
with NIR-HNO (5 μM) for 30 min, washed with PBS to remove the ex-
cess probe, and then treated with AS (20 and 50 μM) for another
30 min. As shown in Fig. 4, cells only treated with NIR-HNO displays
very weak red fluorescence (Fig. 4a). For the cells treated with AS, a re-
markable red fluorescence increase can be observed (Fig. 4b–d).
NIR-HNO was next used to imaging endogenous HNO. It is reported
that intracellular reactions of L-ascorbate and NO could produce endog-
enous HNO [45]. HeLa cells were treated only with NIR-HNO, negligible
fluorescence can be observed (Fig. 5a). By contrast, for HeLa cells were
pretreated with L-ascorbate (NaASc, 2.0 mM) and sodium nitroprusside
(SNP, NO donor, 2.0 mM) for 2 h, then incubated with NIR-HNO (5 μM)
for another 1 h, the bright red fluorescence intensity was gathered
(Fig. 5d). However, cells were pretreated with NaASc (2.0 mM) or SNP
(2.0 mM) for 2 h and then incubated with NIR-HNO (5 μM) for 1 h,
slightly brighter fluorescence imaging appeared (Fig. 5b and c).
Whereafter, the photostability of compound 1 was also studied through
fluorescence confocal measurements in living HeLa cells (Fig. S9), which
[7] M.J.C. Lee, H.T. Nagasawa, J.A. Elberling, E.G. Demaster, J. Med. Chem. 35 (1992)
3648–3652.
[8] A.C. Zarpelon, G.R. Souza, T.M. Cunha, I.R.S. Schivo, M. Marchesi, R. Casagrande, P.
Pinge, F.Q. Cunha, S.H. Ferreira, K.M. Miranda, W.A. Verri, Neuropharmacology 71
(2013) 1–9.
[9] Y.B. Zhou, Z.Y. Wu, X. Cao, L. Ding, Z.S. Wen, J.S. Bian, Pharmacol. Res. 111 (2016)
885–895.
[10] L. Staurengo-Ferrari, A.C. Zarpelon, D.T. Longhi-Balbinot, M. Marchesi, T.M. Cunha,
J.C. Alves-Filho, F.Q. Cunha, S.H. Ferreira, R. Casagrande, K.M. Miranda, W.A. Verri,
Pharmacol. Rep. 66 (2014) 691–698.
[11] L. Yuan, W. Lin, K. Zheng, L. He, W. Huang, Chem. Soc. Rev. 42 (2013) 622–661.
[12] X. Chen, F. Wang, J.Y. Hyun, T. Wei, J. Qiang, X. Ren, I. Shin, J. Yoon, Chem. Soc. Rev.
45 (2016) 2976–3016.
[13] D. Wu, A.C. Sedgwick, T. Gunnlaugsson, E.U. Akkaya, J. Yoon, T.D. James, Chem. Soc.
Rev. 46 (2017) 7105–7123.
[14] W. Chen, C. Liu, B. Peng, Y. Zhao, A. Pacheco, M. Xian, Chem. Sci. 4 (2013)
2892–2896.
[15] W. Chen, E.W. Rosser, D. Zhang, W. Shi, Y. Li, W.-J. Dong, H. Ma, D. Hu, M. Xian, Org.
Lett. 17 (2015) 2776–2779.
Please cite this article as: C. Wei, X. Wang, X. Li, et al., An isophorone-fused near-infrared fluorescent probe with a large Stokes shift for imaging
endogenous..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117765

6
C. Wei et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy xxx (xxxx) xxx
[16] W. Chen, E.W. Rosser, T. Matsunaga, A. Pacheco, T. Akaike, M. Xian, Angew. Chem.
Int. Ed. 54 (2015) 13961–13965.
[17] W. Chen, A. Pacheco, Y. Takano, J.J. Day, K. Hanaoka, M. Xian, Angew. Chem. Int. Ed.
[33] C. Liu, H. Wu, Z. Wang, C. Shao, B. Zhu, X. Zhang, Chem. Commun. 50 (2014)
6013–6016.
[34] X. Zhu, M. Xiong, H.-w. Liu, G.-j. Mao, L. Zhou, J. Zhang, X. Hu, X.-B. Zhang, W. Tan,
55 (2016) 9993–9996.
[18] W. Chen, S. Xu, J.J. Day, D. Wang, M. Xian, Angew. Chem. Int. Ed. 56 (2017)
16611–16615.
Chem. Commun. 52 (2016) 733–736.
[35] F. Ali, S. Sreedharan, A.H. Ashoka, H.K. Saeed, C.G.W. Smythe, J.A. Thomas, A. Das,
Anal. Chem. 89 (2017) 12087–12093.
[19] X. Jing, F. Yu, L. Chen, Prog. Chem. 26 (2014) 866–878.
[20] P. Rivera-Fuentes, S.J. Lippard, Acc. Chem. Res. 48 (2015) 2927–2934.
[21] B. Dong, X. Kong, W. Lin, ACS Chem. Biol. 13 (2018) 1714–1720.
[22] K. Zheng, H. Chen, S. Fang, Y. Wang, Sensor. Actuat. B-Chem. 233 (2016) 193–198.
[23] X. Jing, F. Yu, L. Chen, Chem. Commun. 50 (2014) 14253–14256.
[24] P. Liu, X. Jing, F. Yu, C. Lv, L. Chen, Analyst 140 (2015) 4576–4583.
[25] Y. Tan, R. Liu, H. Zhang, R. Peltier, Y.-W. Lam, Q. Zhu, Y. Hu, H. Sun, Sci. Rep. 5 (2015)
16979.
[26] B. Dong, K. Zheng, Y. Tang, W. Lin, J. Mater. Chem. B 4 (2016) 1263–1269.
[27] X. Gong, X.-F. Yang, Y. Zhong, Y. Chen, Z. Li, Dyes Pigments 131 (2016) 24–32.
[28] B. Dong, X. Song, X. Kong, C. Wang, N. Zhang, W. Lin, J. Mater. Chem. B 5 (2017)
5218–5224.
[36] Y. Zhou, X. Zhang, S. Yang, Y. Li, Z. Qing, J. Zheng, J. Li, R. Yang, Anal. Chem. 89 (2017)
4587–4594.
[37] H. Li, Q. Yao, F. Xu, N. Xu, X. Ma, J. Fan, S. Long, J. Du, J. Wang, X. Peng, Anal. Chem. 90
(2018) 4641–4648.
[38] W. Xiaofen, W. Chao, L. Xueyan, Z. Xueyang, G. Xiaowei, Z. Pingzhu, L. Xiaoliu, Chin. J.
Org. Chem. 39 (2018) 469–474.
[39] P. Zhang, P. Lian, X. Wang, X. Li, C. Wei, X. Li, Anal. Methods 11 (2019) 1299–1303.
[40] D. Zhang, W. Chen, Z. Miao, Y. Ye, Y. Zhao, S.B. King, M. Xian, Chem. Commun. 50
(2014) 4806–4809.
[41] S. Gong, J. Hong, E. Zhou, G. Feng, Talanta 201 (2019) 40–45.
[42] J.X. Hong, E.B. Zhou, S.Y. Gong, G.Q. Feng, Dyes Pigments 160 (2019) 787–793.
[43] K. Wang, C.X. Zhao, T.H. Leng, C.Y. Wang, Y.X. Lu, Y.J. Shen, W.H. Zhu, Dyes Pigments
151 (2018) 194–201.
[29] S. Yuan, F. Wang, G. Yang, C. Lu, J. Nie, Z. Chen, J. Ren, Y. Qiu, Q. Sun, C. Zhao, W.-H.
Zhu, Anal. Chem. 90 (2018) 3914–3919.
[44] K. Sunwoo, K.N. Bobba, J.-Y. Lim, T. Park, A. Podder, J.S. Heo, S.G. Lee, S.L. Bhuniya, J.S.
[30] Y. Huang, X. Zhang, N. He, Y. Wang, Q. Kang, D. Shen, F. Yu, L. Chen, J. Mater. Chem. B
7 (2019) 305–313.
[31] T. Wang, Y. Chai, S. Chen, G. Yang, C. Lu, J. Nie, C. Ma, Z. Chen, Q. Sun, Y. Zhang, J. Ren,
F. Wang, W.-H. Zhu, Dyes Pigments 166 (2019) 260–265.
[32] K. Kawai, N. Ieda, K. Aizawa, T. Suzuki, N. Miyata, H. Nakagawa, J. Am. Chem. Soc. 135
(2013) 12690–12696.
Kim, Chem. Commun. 53 (2017) 1723–1726.
[45] S.A. Suarez, N.I. Neuman, M. Muñoz, L. Álvarez, D.E. Bikiel, C.D. Brondino, I. Ivanović-
Burmazović, J.L. Miljkovic, M.R. Filipovic, M.A. Martí, F. Doctorovich, J. Am. Chem.
Soc. 137 (2015) 4720–4727.
Please cite this article as: C. Wei, X. Wang, X. Li, et al., An isophorone-fused near-infrared fluorescent probe with a large Stokes shift for imaging
endogenous..., Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy, https://doi.org/10.1016/j.saa.2019.117765