Washington Red (WR) dyes and their imaging applications

Article abstract of DOI:10.1016/bs.mie.2020.04.027

Near-infrared (NIR) fluorescent dyes with optimum photophysical properties are highly useful for bioimaging in living systems, but such dyes are still rare. Recently, our laboratory developed a unique class of NIR fluorescent dyes with high quantum yields and large Stokes shifts. These dyes, named as Washington Red (WR), were found to be useful scaffolds to develop NIR fluorescent imaging probes upon easy modifications on their phenolic hydroxyl group. In this chapter, we provide a comprehensive summary of this work, including the design and synthesis of WR dyes, their photophysical properties studies, and applications in developing NIR probes to image H₂S in cells. The detailed experimental protocols are described.

Full text of DOI:10.1016/bs.mie.2020.04.027

ARTICLE IN PRESS
Washington Red (WR) dyes
and their imaging applications
Wei Chen, Ming Xian∗
Department of Chemistry, Washington State University, Pullman, WA, United States
∗Corresponding author: e-mail address: mxian@wsu.edu
Contents
1. Introduction
1
3
4
5
5
2. Rational design and chemical structures of WR dyes
3. Photophysical properties of WR dyes
3.1 Materials and experimental methods
3.2 Safety precautions
4. Applications of WR dyes in developing NIR probes to image H2S in cells
4.1 Rational design and synthesis of NIR H2S probe based on WR dyes
4.2 Chemistry and properties of NIR H2S probes
4.3 Applications of SeSP-NIR in H2S imaging in cell-based experiments
5. Conclusions
6
6
8
11
13
14
14
Acknowledgments
References
Abstract
Near-infrared (NIR) fluorescent dyes with optimum photophysical properties are highly
useful for bioimaging in living systems, but such dyes are still rare. Recently, our labo-
ratory developed a unique class of NIR fluorescent dyes with high quantum yields and
large Stokes shifts. These dyes, named as Washington Red (WR), were found to be useful
scaffolds to develop NIR fluorescent imaging probes upon easy modifications on their
phenolic hydroxyl group. In this chapter, we provide a comprehensive summary of this
work, including the design and synthesis of WR dyes, their photophysical properties
studies, and applications in developing NIR probes to image H2S in cells. The detailed
experimental protocols are described.
1. Introduction
Fluorescent probes with optimum photophysical properties are essential
research tools for bioimaging due to their high sensitivity and fast response.
So far, a great variety of fluorescent dyes have been used to construct various
#
Methods in Enzymology
ISSN 0076-6879
https://doi.org/10.1016/bs.mie.2020.04.027
2020 Elsevier Inc.
All rights reserved.
1

ARTICLE IN PRESS
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Wei Chen and Ming Xian
types of fluorescent probes (Chan, Dodani, & Chang, 2012; Li, Gao,
Shi, & Ma, 2014). In particular, xanthene-based dyes such as fluorescein,
rhodamine, and rhodol are very popular fluorophore scaffolds because of
their excellent photophysical properties (high quantum yields and extinction
coefficients, excellent water solubility and photostability) (Beija, Afonso, &
Martinho, 2009; Chen, Pradhan, Wang, Kim, & Yoon, 2012). While many
xanthene-based fluorescent probes have been developed in the past several
years, several challenging issues still need to be addressed: (1) Most xanthene
dyes emit green to red fluorescence (500–600nm). By contrast, NIR emis-
sion (650–900nm) has distinct advantages including low photodamage
to biological samples, deep tissue penetration, and minimum interference
from background autofluorescence by biomolecules in living systems
(Escobedo, Rusin, Lim, & Strongin, 2010; Hilderbrand & Weissleder,
2010). Therefore, more xanthene dyes with NIR emission need to be devel-
oped. (2) Considerable efforts have been devoted in recent years to modify
xanthene core by the extension of π-conjugation or the replacement of the
endocyclic oxygen atom with other elements (such as C and Si). These have
yielded some novel red-emitting and NIR xanthene dyes (Butkevich et al.,
2016; Grimm et al., 2016; Grimm et al., 2017; Sibrian-Vazquez, Escobedo,
Lowry, Fronczek, & Strongin, 2012; Uno et al., 2014; Yuan, Lin, Yang, &
Chen, 2012). However, like traditional xanthene dyes, they still suffer from
the limitations of small Stokes shifts (typically less than 30nm), which can
result in serious self-quenching of fluorescence and measurement error
from the excitation backscattering (Peng et al., 2005). (3) It is well-known
that ideal NIR dyes suited for constructing sensitive imaging probes should
not only have high fluorescence quantum yields and large Stokes shifts in
aqueous solution, but also bear optically tunable groups which can be readily
modified to obtain all sorts of reaction-based fluorescent probes for target
analytes (Chan et al., 2012; Li et al., 2014). Unfortunately, current xanthene
dyes that meet the aforementioned criteria are still quite limited. To address
this problem, our laboratory recently developed a unique class of NIR
xanthene-based dyes named Washington Red (WR). These dyes were
found to be useful scaffolds to develop NIR fluorescent imaging probes
through easy modifications on their phenolic hydroxyl groups (Chen,
Xu, Day, Wang, & Xian, 2017). In this chapter, we provide a comprehen-
sive summary of this work, including the design and synthesis of WR
dyes, their photophysical properties studies, and applications in developing
NIR probes to image H2S in cells. The detailed experimental protocols
are described.

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Washington Red (WR) dyes and their imaging applications
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2. Rational design and chemical structures of WR dyes
In the design of WR dyes, we were considering two main factors: (1) it
is known that the free –OH or –NH2 groups in xanthene core are potential
optically tunable groups, and their substitution is liable to cause the fluores-
cence changes (Beija et al., 2009; Chan et al., 2012; Chen et al., 2012; Li
et al., 2014). Therefore, a free –OH group was retained in xanthenes core
so that it could function as a key fluorescence switch. (2) It is well-established
that introducing rigid rings or increasing the electron-donating capability of
the substituents on the nitrogen atom generally induced bathochromic shifts
in rhodamine and rhodol dyes (Peng & Yang, 2010; Vogel, Rettig, Sens, &
Drexhage, 1988). Herein we selected rhodols as our framework because they
contain a free –OH group and a substitutable –NH2 group. We expected the
incorporation of heteroatom containing fused rings would be favorable for
improving the spectroscopic properties. As the first proof-of-concept, an
electron-rich and rigid ring, tetrahydroquinoxaline, was used to study the role
of substituent group on the changes of properties. Based on this idea, a series
of WR dyes containing tetrahydroquinoxaline unit were synthesized
(Scheme 1). Their structures were confirmed by NMR and HRMS.
Scheme 1 The structures of rhodols and WR dyes.

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Wei Chen and Ming Xian
3. Photophysical properties of WR dyes
The photophysical properties of WR1–WR10 were tested in 50mM
PBS buffer solution (pH 7.4, containing 15% ethanol). The results are sum-
marized in Table 1. As expected, compared with common rhodols, the
absorption and emission maxima of WR dyes have distinct bathochromic
shifts. Their aqueous solutions displayed pink color and NIR fluorescence
emission with large Stokes shifts (>110nm). Interestingly, when the meta-
position of endocyclic oxygen atom has a pyrrolidine ring, it is helpful for
enhancing fluorescence quantum yields (for WR3–6). The different alkyl-
ations on the para-nitrogen atom only had minor effects on their fluorescence
quantum yields. However, when the pyrrolidine unit was at the para-position
of the endocyclic oxygen atom (WR7 and WR8), or was replaced by piper-
idine ring (WR9), the quantum yields of resultant dyes were not improved
but their emission wavelength showed obvious bathochromic shifts.
Table 1 Photophysical data of WR1–WR10 in 50mM PBS buffer solution (pH 7.4
containing 15% ethanol).
Dye
b
λabs/nma
εmax
λem/nmc
668
Stokes shiftd
129
Φfe
WR1
WR2
WR3
WR4
WR5
WR6
WR7
WR8
WR9
WR10
539
31,000
30,000
35,500
44,500
32,500
42,000
29,500
30,000
39,000
30,500
0.06
0.07
0.20
0.21
0.19
0.20
0.06
0.05
0.04
0.06
545
668
123
540
656
116
543
656
113
545
662
117
551
662
111
539
676
137
545
676
131
557
706
149
542
666
124
aThe maximal aꢀb1sorption of the dye.
bUnit: Mꢀ1 cm
cThe maximal emission of the dye.
dUnit: nm.
eΦf is the relative fluorescence quantum yield estimated by using seminaphthofluorescein-10 (Φf ¼ 0.35
in pH 11 Glycine-NaOH buffer) as a reference standard (Azuma et al., 2012).
Reprinted with permission from Chen, W., Xu, S., Day, J. J., Wang, D., & Xian, M. (2017). A general
strategy for development of near-infrared fluorescent probes for bioimaging. Angewandte Chemie I-
nternational Edition, 56, 16611–16615. Copyright 2017 John Wiley & Sons, Inc.

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Washington Red (WR) dyes and their imaging applications
5
Adding another condensed ring (WR10) or introducing a chloride to the
xanthene core (WR6, WR9) also did not enhance fluorescence quantum
yields. Below, we describe the detailed experimental protocols that were used
to evaluate these WR dyes.
3.1 Materials and experimental methods
(1) All WR dyes are stable in solid form and can be stored for at least 1 year
at 4°C. They can dissolve easily in dimethyl sulfoxide (DMSO), meth-
anol, and ethanol. The dyes may be sensitive to light. So both solid and
stock solutions should be kept away from light by storing in dark. The
stock solutions of WR1–WR10 (1mM) were prepared in ethanol,
respectively. The stock solutions need to be freshly prepared before use.
(2) PBS buffer (50mM, pH 7.4) was prepared by dissolving NaCl (20g),
KCl (0.5g), Na2HPO4 (3.6g), and KH2PO4 (0.6g) in 500mL double
deionized water. Then pH was adjusted to 7.4 by 1M HCl or 1M
NaOH. PBS buffers with different pH can also be obtained by adding
1M HCl or 1M NaOH. All aqueous solutions were prepared using
double deionized water from a Nano pure ultrapure water system.
(3) The photophysical properties of WR1–WR10 were tested in 50mM
PBS buffer solution (pH 7.4 containing 15% ethanol). All the measure-
ments were carried out at room temperature according to the following
procedure. In a test tube, 3.4mL of PBS buffer and 400μL of ethanol
were mixed, followed by the addition of 8μL of testing WR dyes stock
solution. The final volume of the mixture solution was adjusted to 4mL
with ethanol. After mixing and then standing for 1min at room tem-
perature, a 3.5-mL portion of the mixture solution was transferred to
measure absorbance and fluorescence intensity. The testing solutions
should be optically clear and without particulates.
(4) Absorption spectra were recorded on a Thermo 300 UV/VIS spectro-
photometer. Fluorescence excitation and emission spectra were mea-
sured on Cary Eclipse fluorescence spectrophotometer.
3.2 Safety precautions
Because ethanol is always used in the photophysical properties studies of
WR1–WR10. Experiments should be kept away from any ignition sources,
and be performed with sufficient ventilation. The preparation of all stock
solutions and testing solutions should be performed in a fume hood
whenever possible.

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4. Applications of WR dyes in developing NIR probes
to image H2S in cells
As a proof-of-concept, we verified the usefulness of WR dyes as effec-
tive fluorophore templates by developing new “turn-on” NIR fluorescent
probes. H2S is a gasotransmitter that modulates diverse cellular function. In
the past several years the development of fluorescence probes for H2S has
attracted considerable attention. Although many H2S probes have been
reported for bioimaging, the maximum emission of most probes are below
600nm with small Stokes shifts. Only a few can be applied to real biological
detection (Hartle & Pluth, 2016; Lin, Chen, Xian, & Chang, 2015; Xuan,
Sheng, Cao, He, & Wang, 2012; Zhou, Lee, Xu, & Yoon, 2015). These
problems could be addressed by new NIR fluorescent probes based on
WR dyes.
4.1 Rational design and synthesis of NIR H2S probe based
on WR dyes
In 2011 our laboratory developed the first nucleophilic reaction-based H2S
probes (WSP, Scheme 2). 2-(Pyridin-2-yldisulfaneyl)benzoate was used as
the recognition unit for H2S (Liu et al., 2011; Peng et al., 2014). In this
design, pyridyl disulfide reacts with H2S to form a persulfide intermediate,
which then undergoes an intramolecular cyclization to release a fluorophore
containing free –OH group. However, these probes can be consumed by
thiols (RSH), which could lead to decreased sensitivity for H2S in the pres-
ence of high concentrations of RSH. To address this problem we then
developed diselenide-based SeP probes in 2015 (Peng et al., 2015). The fast
reaction between SeP and H2S (Scheme 2) made the presence of biothiol not
a problem. However, two identical fluorophores are needed in SeP. That
does not allow much flexibility to tune their reactivity toward H2S, and also
results in the poor water solubility of probes. To further optimize the SeP
template, and inspired by the intermediate of SeP reacting with RSH, we
reasoned that the selenenyl sulfide (SedS) linkage should have a similar
reactivity to that of SeP toward H2S. It should react with H2S rapidly to pro-
duce ꢀSedSH. The following cyclization should release the fluorophore
and turn-on fluorescence. While the SedS bond could also react with
RSH, the product should still have the -SedS- linkage and therefore be eas-
ily turned on by H2S. This new SeSP probe should have high selectivity to

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Washington Red (WR) dyes and their imaging applications
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Scheme 2 Design strategies of WSP/SeP/SeSP probes.
Scheme 3 Synthesis and structures of NIR H2S probes.
H2S, and not be consumed by thiols. This new strategy allowed the probes
more structurally tunable and synthetically feasible.
Based on our design, WR6 was used as the fluorophore to construct
two NIR H2S probes (WSP-NIR; SeSP-NIR) for comparison. WR6 was
separately conjugated with compound 1 and 2 to provide WSP-NIR and
SeSP-NIR under mild conditions (Scheme 3).

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4.2 Chemistry and properties of NIR H2S probes
4.2.1 Time-course assays
With WSP-NIR and SeSP-NIR in hand, we first tested their fluores-
cence responses to H2S. As expected, WSP-NIR and SeSP-NIR showed
non-fluorescent (quantum yields <0.01) themselves while gave obvious
fluorescence enhancements in the presence of H2S. The maximum emission
intensities of WSP-NIR and SeSP-NIR were reached within 2 and 8min
respectively, indicating their fast and sensitive response to H2S. Here, we
describe the detailed experimental protocols:
(1) WSP-NIR and SeSP-NIR are stable in solid form and can be stored for
at least 1 year at 4°C. They can dissolve easily in DMSO. The probes
may be sensitive to light, so both solid and stock solutions should be
kept away from light. The stock solutions (0.4mM) of WSP-NIR
and SeSP-NIR were prepared in DMSO. The stock solutions need
to be freshly prepared before use.
(2) Na2S stock solution was prepared freshly by dissolving 9.61mg sodium
sulfide nonahydrate (ACROS ORGANICS, 98+%, ACS reagent) in
4mL PBS buffer (50mM, pH 7.4), concentration: 10mM. This oper-
ation should be performed in a fume hood whenever possible, due to
potential toxic issue of H2S.
(3) CTAB (hexadecyltrimethylammonium bromide/cetyltrimethyl-
ammonium bromide, ACROS, 99+%) stock solution was prepared
freshly by dissolving 7.29mg CTAB in 4mL ethanol (200 proof ),
concentration: 5mM.
(4) The fluorescence responses of the probes to H2S were tested in 50mM
PBS buffer solution (pH 7.4) containing 100μM CTAB. All the
measurements were carried out at room temperature according to
the following procedure: in a test tube, 3mL of 50mM PBS buffer
(pH 7.4) and 80μL of the stock solution of CTAB were mixed, followed
by the addition of a requisite volume of Na2S stock solution. The final
volume of the reaction solution was adjusted to 3.98mL with 50mM
PBS buffer. Then 20μL of the stock solution of WSP-NIR or SeSP-
NIR was added into this mixture. After mixing at room temperature,
a 3.5-mL portion of the mixture solution was transferred to measure
fluorescence intensity changes at different time (λex/em ¼571/656nm).
The testing solutions should be optically clear and without particulates.
Results are shown in Fig. 1.

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Washington Red (WR) dyes and their imaging applications
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100
75
50
25
0
(1)
(2)
0
5
10
15
20
Time (min)
Fig. 1 Time-dependent fluorescence intensity changes of the probes in the presence of
Na2S: (1) 2μM SeSP-NIR+20μM Na2S; (2) 2μM WSP-NIR+5μM Na2S.
4.2.2 Selectivity experiments
A series of reactive sulfur species including glutathione (GSH), cysteine (Cys),
homocysteine (Hcy), glutathione disulfide (GSSG), Na2S, Na2SO3, and
Na2S2O3 were used to investigate the selectivity of WSP-NIR and SeSP-
NIR for H2S. As shown in Fig. 2, no significant fluorescence enhancement
was observed except for Na2S. Other common amino acids also showed no
response to the probes. We also tested the responses of the probes to H2S in
the presence of high concentrations of RSH. The results showed that SeSP-
NIR still produced strong fluorescence, but WSP-NIR displayed dramatically
decreased fluorescence. These results clearly demonstrated the advantage of
the SeSP template in the development of H2S probes. Experimental proce-
dure is as following:
(1) The stock solutions (100mM) of GSH, Cys and Hcy were prepared in
PBS buffer. The stock solutions (10mM) of GSSG, Na2SO3, Na2S2O3,
alanine, arginine, glutamine, glycine, proline, serine were prepared in
PBS buffer. All the stock solutions need to be freshly prepared before use.
(2) 18 empty vials (4mL) were numbered #1 to #18. To each vial was
added 3mL PBS buffer.
(3) 80μL CTAB stock solution was added into each vial (#1 to #18).
Final concentration of CTAB was 100μM.
(4) 900μL PBS buffer was added to vial #1. This was the control (blank
sample).

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Wei Chen and Ming Xian
90
60
30
0
WSP-NIR
SeSP-NIR
1
2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Fig. 2 Fluorescence response of WSP-NIR (2μM) or SeSP-NIR (2μM) in the presence of
various reactive sulfur species and amino acids: (1) probe alone; (2) 100μM GSSG; (3)
100μM Na2S2O3; (4) 100μM Na2SO3; (5) 100μM alanine; (6) 100μM arginine; (7) 100μM
glutamine; (8) 100μM glycine; (9) 100μM proline; (10) 100μM serine; (11) 2mM GSH;
(12) 10mM GSH; (13) 1mM Cys; (14) 1mM Hcy; (15) 2mM GSH+50μM Na2S; (16)
1mM Cys+50μM Na2S; (17) 1mM Hcy+50μM Na2S; (18) 50μM Na2S. Reprinted with
permission from Chen, W., Xu, S., Day, J. J., Wang, D., & Xian, M. (2017). A general strategy
for development of near-infrared fluorescent probes for bioimaging. Angewandte Chemie
International Edition, 56, 16611–16615. Copyright 2017 John Wiley & Sons, Inc.
(5) 40μL stock solution of GSSG, Na2S2O3, Na2SO3, alanine, arginine,
glutamine, glycine, proline, serine was added to vials #2 to #10,
respectively. Final concentration of them was 100μM.
(6) 80μL and 400μL stock solution of GSH was added to vial #11 and
#12, respectively. The corresponding final concentration of GSH
was 2mM and 10mM.
(7) 40μL stock solutions of Cys and Hcy were added to vial #13 and #14,
respectively. Final concentration was 1mM.
(8) 80μL stock solution of GSH and 20μL stock solution of Na2S were
added to vial #15. The final concentrations of GSH and Na2S were
2mM and 50μM, respectively.
(9) 40μL stock solution of Cys and 20μL stock solution of Na2S were
added to vial #16. The final concentrations of Cys and Na2S were
1mM and 50μM, respectively.
(10) 40μL stock solution of Hcy and 20μL stock solution of Na2S were
added to vial #17. The final concentrations of Hcy and Na2S were
1mM and 50μM, respectively.

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Washington Red (WR) dyes and their imaging applications
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(11) 20μL stock solution of Na2S was added to vial #18. The final concen-
tration of Na2S was 50μM.
(12) The final volume of the reaction solution was adjusted to 3.98mL with
50mM PBS buffer (pH 7.4) in each vial. Then 20μL stock solution of
WSP-NIR or SeSP-NIR was added. The final concentration of the
probe was 2μM. After mixing and standing for 10min at room temper-
ature, a 3.5-mL portion of the reaction solution was transferred into a
1-cm quartz cuvette to measure fluorescence with λex/em ¼571/656nm.
Results are shown in Fig. 2.
4.2.3 The model reaction of SeSP with H2S
To validate the fluorescence turn-on mechanism of SeSP-NIR by H2S,
a model reaction using phenyl 2-((pyridin-2-ylthio)selenyl)benzoate was
carried out in a mixed solution of THF/PBS (Scheme 4). As expected,
the products 2,1-benzothiaselenol-3-one and phenol were obtained with
high yields, which supported the reaction mechanism we proposed.
The following are the detailed experimental procedures:
(1) Phenyl 2-((pyridin-2-ylthio)selenyl)benzoate (38mg) was dissolved in
2mL THF. Na2S (120mg) was dissolved in 2mL PBS buffer.
(2) These two solutions were mixed and stirred at room temperature
for 3h.
(3) The reaction mixture was extracted by CH2Cl2 (10mLꢁ3).
(4) The combined organic layers were dried by MgSO4, filtered, and con-
centrated. The crude products were purified by flash chromatography
(20% ethyl acetate in hexane) to afford the products shown in Scheme 4.
4.3 Applications of SeSP-NIR in H2S imaging in cell-based
experiments
4.3.1 Materials
The stock solutions of SeSP-NIR and CTAB were prepared using the same
protocols described in Section 4.2.1.
Scheme 4 The reaction between Phenyl 2-((pyridin-2-ylthio)selenyl)benzoate and H2S.

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Wei Chen and Ming Xian
(1) Growth media: Dulbecco’s modified Eagle’s Medium (DMEM,
Cellgro) supplemented with 10% fetal bovine serum, 4mM glutamine,
100U/mL penicillin, and 100μg/mL streptomycin.
(2) Washing media: Dulbecco’s modified Eagle’s Medium (DMEM,
Cellgro) supplemented without glutamine and phenol red, Dulbecco’s
phosphate-buffered saline (DPBS, Hyclone).
(3) HeLa cells were grown on glass-bottom culture dishes (Corning Inc.) in
DMEM supplemented with 10% (v/v) FBS, penicillin (100U/mL) and
streptomycin (100μg/mL) at 37°C under a humidified atmosphere
containing 5% CO2. Before use, the adherent cells were washed one
time with FBS-free DMEM.
(4) H2S donor N-(benzoylthio)benzamide (Zhao, Wang, & Xian, 2011)
stock solution was freshly prepared by dissolving 2.57mg the donor in
1mL DMSO, concentration: 10mM.
(5) H2S biosynthesis inhibitor DL-propargylglycine (PAG) stock solution
was freshly prepared by dissolving 1.13mg inhibitor in 1mL DMEM,
concentration: 10mM.
(6) A solution of 4μM SeSP-NIR was prepared by adding 10μL probe
stock solution to 990μL DMEM and mixing thoroughly; a solution
of 60μM PAG was prepared by adding 6μL PAG stock solution to
994μL DMEM and mixing thoroughly; a solution of 100μM H2S
donor N-(benzoylthio)benzamide was prepared by adding 10μLN-
(benzoylthio)benzamide stock solution to 990μL DMEM and mixing
thoroughly.
4.3.2 Fluorescence imaging of H2S in HeLa cells
To investigate the capability of SeSP-NIR in imaging H2S in cells, the
following three different group experiments were carried out:
(1) Group a: All cell media was removed and exchanged with 500μL 4μM
SeSP-NIR solution. Then cells were incubated for 20min at 37°C.
After removal of excess probe and washed with PBS buffer (pH 7.4),
cells were incubated with PBS buffer (pH 7.4, containing 100μM
CTAB) at room temperature for 15min. Cell imaging was carried
out after removal of the culture solution.
(2) Group b: All cell media was removed and exchanged with 500μL
60μM PAG solution. Then cells were incubated for 40min at 37°C.
All cell media was next removed and exchanged with 500μL 4μM
SeSP-NIR solution, then cells were incubated for 20min at 37°C.

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Washington Red (WR) dyes and their imaging applications
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Fig. 3 Fluorescence imaging of H2S in HeLa cells. Cells were imaged after incubation
with 4μM SeSP-NIR for 20min after pretreatment with (A) no pretreatment, (B) 60μM
PAG for 40min, or (C) 100μM H2S donor for 60min. Reprinted with permission from
Chen, W., Xu, S., Day, J. J., Wang, D., & Xian, M. (2017). A general strategy for development
of near-infrared fluorescent probes for bioimaging. Angewandte Chemie International
Edition, 56, 16611–16615. Copyright 2017 John Wiley & Sons, Inc.
After removal of excess probe and washed with PBS buffer (pH 7.4),
cells were incubated with PBS buffer (pH 7.4, containing 100μM
CTAB) at room temperature for 15min. Cell imaging was carried
out after removal of the culture solution.
(3) Group c: All cell media was removed and exchanged with 500μL
100μM H2S donor N-(benzoylthio)benzamide solution. Then cells
were incubated for 60min at 37°C. All cell media was next removed
and exchanged with 500μL 4μM SeSP-NIR solution, then cells were
incubated for 20min at 37°C. After removal of excess probe and
washed with PBS buffer (pH 7.4), cells were incubated with PBS buffer
(pH 7.4, containing 100μM CTAB) at room temperature for 15min.
Cell imaging was carried out after removal of the culture solution. As
can be seen in Fig. 3, compared to group a (control cells), a significant
reduced fluorescence was observed in cells pretreated with PAG.
However, cells pretreated with a H2S donor showed obvious fluores-
cence enhancement. These results showed SeSP-NIR had good cell
permeability and was sensitive to image H2S in cells.
5. Conclusions
In this chapter we described the design and evaluation of a unique class
of NIR dyes (named as Washington Red-WR) with large Stokes shifts
and high quantum yields. Among them, WR3–WR6 displayed excellent
photophysical properties in aqueous solutions. Moreover, their fluorescence
properties were found to be tunable by easy modifications on the

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Wei Chen and Ming Xian
phenolic –OH group. Based on WR6, two probes (WSP-NIR and SeSP-
NIR) were developed for the detection of H2S. SeSP-NIR overcame a
major limitation of our previously reported WSP and SeP probes, and
showed much improved sensitivity to H2S. SeSP-NIR was also used for
imaging intracellular H2S.
Acknowledgments
This work is supported by NIH (R21DA046386) and NSF (CHE1738305).
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