An ESIPT-based highly selective and sensitive probe for the detection of hydrogen sulfide

Article abstract of DOI:10.1016/j.tetlet.2015.04.113

Abstract A new excited state intramolecular proton transfer dye with red keto emission (620 nm) and remarkably large Stokes shift (240 nm) was discovered. Based on its unique red emission and large Stokes shift, it was developed to a novel fluorescent probe which can highly selectively and sensitively detect hydrogen sulfide.

Full text of DOI:10.1016/j.tetlet.2015.04.113

Tetrahedron Letters xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
An ESIPT-based highly selective and sensitive probe for the detection
of hydrogen sulfide
⇑
Pengfei Xu, Meihui Liu, Tang Gao, Hailiang Zhang, Ziwei Li, Xinyi Huang, Wenbin Zeng
School of Pharmaceutical Sciences, Central South University, Changsha 410078, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A new excited state intramolecular proton transfer dye with red keto emission (620 nm) and remarkably
large Stokes shift (240 nm) was discovered. Based on its unique red emission and large Stokes shift, it was
developed to a novel fluorescent probe which can highly selectively and sensitively detect hydrogen
sulfide.
Received 13 March 2015
Revised 24 April 2015
Accepted 28 April 2015
Available online xxxx
Ó 2015 Elsevier Ltd. All rights reserved.
Keywords:
Fluorescent probes
Keto emission
Thiolysis
Gasotransmitter
Red emission
Hydrogen sulfide (H2S), well-known for its unpleasant rotten
egg smell and traditionally considered as a toxic gas,1 can be
endogenously produced by enzymes such as cystathionine b-syn-
considerable attention because of their separated dual fluorescence
in a four-level cycle.11 In the ground state, HBT adopts the enol (E)
form stabilized by intramolecular hydrogen bonding Upon pho-
toexcitation at 320 nm, the excited enol (E*) is quickly converted
into the excited keto (K⁄) tautomer by ESIPT on a subpicosecond
time scale, which gives rise to an emission band (ꢀ500 nm) with
a large Stokes shift (ꢀ180 nm). Nevertheless, the emission color
of HBT/HBO is limited in the blue and green region, which may
restrain the potential of their biological applications due to poor
penetration and high photodamage.12 Therefore, it is of great value
to modify the structure of HBT/HBO and make the emission wave-
length into the red region. On the other hand, many reported
HBT/HBO derivatives still suffer from various shortcomings involv-
ing short emission wavelength, obvious hypochromatic shift, or the
blockage of ESIPT process resulting in small Stokes’ shift in strong
polar solvents such as acetonitrile, methanol, and water.13 Thus, it
is challenging to tune the emission of HBT/HBO derivatives to
longer wavelength, and still retain the ESIPT process for large
Stokes’ shift. To the best of our knowledge, among all the probes
for H2S detection based on ESIPT mechanism, rare of them have
been found to have an emission exceed 600 nm, and most of them
are at below 500 nm which may restrict their potential applica-
tions.14 In this work, we present the development of HBTPP-S as
a novel red fluorescent H2S sensor based on ‘turn-on’ ESIPT mech-
anism (Scheme 1).
thase, cystathionine c-lyase, and 3-mercaptopyruvate sulfurtrans-
ferase in mammalian systems.1,2 Nevertheless, recent studies have
demonstrated that H2S can work as gaseous signaling compound
(gasotransmitter) with importance on a par with that of the other
two known endogenous gasotransmitters, nitricoxide (NO) and
carbon monoxide (CO).3 Moreover, H2S has been recognized for
mediating a wide range of physiological effects, such as vasodila-
tion,4 anti-oxidation,5 anti-apoptosis,6 and antiinflammation.7
Any imbalance in H2S has also been recognized to connect to var-
ious diseases such as Alzheimer’s and Down’s syndrome.8
Therefore, accurate and reliable measurement of H2S concentra-
tions is needed and can provide useful information to study the
function of H2S in depth.
A number of techniques including polarography, gas chro-
matography, and colorimetry are available for the detection of
H2S.9 However, these techniques require complicated procedures
which do not allow for temporal monitoring and are destructive
in nature. In recent years, fluorescent sensing has received great
attention due to its simple operation and high sensitivity.10 Thus,
a fluorophore with a high quantum yield and large Stokes shift
draws our interests. Recently, 2-(2-hydroxyphenyl) benzothiazole
(HBT) and 2-(20-hydroxyphenyl) benzoxazole (HBO) have received
In a typical ESIPT process, upon photoexcitation, HBT can
undergo fast proton transfer reaction in a sub picosecond time
scale and imino nitrogen is converted into an amine group which
⇑
Corresponding author.
E-mail address: wbzeng@hotmail.com (W. Zeng).
http://dx.doi.org/10.1016/j.tetlet.2015.04.113
0040-4039/Ó 2015 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Xu, P.; et al. Tetrahedron Lett. (2015), http://dx.doi.org/10.1016/j.tetlet.2015.04.113

2
P. Xu et al. / Tetrahedron Letters xxx (2015) xxx–xxx
+
H
-
N
O
N
OH
NH
O
EWG
EWG
EWG
ICT
S
S
S
ESIPT
Scheme 1. Our design strategy.
SH
OH
N
OH
N
OH
H2N
1 N NaOH
86%
F3C
HMT, CF3COOH
53%
CHO
+
S
S
NO2
O
HBT
DIEA
HBTQ
I
N
NO2
N
S
OH
N
N
piperidine
78%
S
I
N
I
Cl
NO2
HBTPP
83%
HBTPP-S
O2N
Scheme 2. Synthetic scheme of HBTPP-S.
Figure 1. (a) Absorption and fluorescence spectra of HBTPP. (b) Fluorescence spectra of HBTPP and HBTPP-S. All absorption and fluorescence were recorded in a mixed
solution of CH3CN/PBS (50:50, v/v, pH = 7.4, 10 mM).
can serve as an electron donor group.15 As we all know, when a dye
possesses an electron rich group conjugated to an electron defi-
cient group as a push–pull structure, intramolecular charge trans-
fer (ICT) from the donor to the acceptor will proceed upon
excitation.16 Thus, if we appended an electron acceptor group in
the ortho-position of phenol through double bond, it is logical to
expect that the amine group formed by ESIPT process is capable
of effective charge transfer into the electron acceptor unit and trig-
gers the strong push–pull ICT interaction. This ESIPT coupled ICT
process may lead to dramatic changes in fluorescent properties
involving red shift keto emission and large Stokes shift.
Furthermore, the ESIPT process can be hampered by a specific pro-
tecting group. Such a protected phenol can be used as a ‘turn-on’
molecular probe for detection or imaging of an analyte that can
react with the probe to remove the protecting group (Scheme 2).
To test the above-mentioned hypothesis, we judiciously con-
structed HBTPP by the condensation reaction between HBTQ and
1,2-dimethylpyridin-1-ium iodide. HBTQ was prepared by the
modified Duff reaction starting from HBT, which was synthesized
by the established literature procedure.17 With HBTPP in hand,
we developed a fluorescent probe by connecting with 4-chloro-
1,2-dinitrobenzene known as the protecting group for tyrosine in
peptide synthesis.18 These compounds synthesized were fully
Figure 2. Emission spectra of probe HBTPP-S (5.0
lM) in a mixed solution of
CH3CN/PBS (50:50, v/v, pH = 7.4, 10 mM) upon addition of NaHS with kex = 380 nm.
characterized by the standard NMR and mass spectrometry.
Please cite this article in press as: Xu, P.; et al. Tetrahedron Lett. (2015), http://dx.doi.org/10.1016/j.tetlet.2015.04.113

P. Xu et al. / Tetrahedron Letters xxx (2015) xxx–xxx
3
Figure 4. Fluorescence intensities of probe HBTPP-S upon addition of various
biologically relevant species in a mixed solution of CH3CN/PBS (50:50, v/v, pH = 7.4,
10 mM). Red bars representative anions, metal ions, reactive oxygen species,
reactive nitrogen species, reducing agents, small-molecule thiols, and NaHS. Fꢁ, Brꢁ,
Figure 3. Fluorescence intensity changes at 620 nm of the probe HBTPP-S (5.0 lM)
with the amount of NaHS in a mixed solution of CH3CN/PBS (50:50, v/v, pH = 7.4,
10 mM).
Iꢁ, NO3ꢁ, AcOꢁ, CO23ꢁ, and H2PO4ꢁ, and CNꢁ (0.25 mM for each); K+, Ca2+, Mg2+, and
Zn2+ (1 mM for each); H2O2, ClOꢁ, OH, 1O2, and NO (50
l
M for each); SO23ꢁ, S2O32ꢁ
Å
(100
lM for each); 1 mM for ascorbic acid, cysteine, N-acetyl-cysteine(NAC),
C6H5NH2, C6H5CH2NH2, H2NCH2CH2NH2, HOCH2CH2NH2; 10 mM for GSH, and
The UV–vis spectrum of HBTPP exhibits major absorption peaks
with maximum at 380 nm (Fig. 1). As expected, the fluorescence
spectrum indeed shows emission peak in the red region at a wave-
length of 620 nm, which is preferable for in vivo bioimaging due to
the deep penetration ability and low background autofluores-
cence.19 The obtained large Stokes shift between the excitation
and the emission wavelengths of about 240 nm is a desirable fea-
ture for fluorescence probes, which assists in increasing the sig-
nal-to-noise ratio. Next, we measured the fluorescence spectrum
of HBTPP-S in comparison with that of HBTPP. Since the phenolic
hydroxyl group was masked by dinitrophenyl group resulting in
the lost of ESIPT, as predicted, non-fluorescence was observed for
HBTPP-S.
50 l
M for NaHS. 1, probe HBTPP-S alone; 2, Fꢁ; 3, Brꢁ; 4, Iꢁ; 5, NO3ꢁ; 6, ACOꢁ; 7,
CO23ꢁ; 8, CNꢁ; 9, H2PO4ꢁ; 10, K+; 11, Ca2+; 12, Mg2+; 13, Zn2+; 14, H2O2; 15, ClOꢁ; 16,
ÅOH; 17, 1O2; 18, NO; 19, ascorbic acid; 20, SO32ꢁ; 21, S2O23ꢁ; 22, cysteine; 23, GSH;
24, N-acetyl-cysteine(NAC); 25, C6H5NH2; 26, C6H5CH2NH2; 27, H2NCH2CH2NH2;
28, HOCH2CH2NH2; 29, NaSH.
In conclusion, we have designed and synthesized HBTPP-S as a
new ESIPT fluorescent probe for detection of H2S based on thiolysis
of dinitrophenyl ether. Due to the rapid conversion to the fluores-
cent compound HBTPP by H2S, the solution of HBTPP-S shows
remarkably red keto emission (620 nm) and large Stokes shift
(240 nm), which is preferable for in vivo bioimaging. It is expected
that HBTPP will be useful as a new platform for the development
of various fluorescent probes and provided insight into the
development of novel fluorescent chromophores based on ESIPT
process.
Subsequently, we performed the fluorescence titration studies
of HBTPP-S toward NaSH (commercially available H2S donor) in a
mixed solution of CH3CN/PBS (50:50, v/v, pH = 7.4, 10 mM). The
absorption and fluorescence spectra of the solution of HBTPP-S
treated with a series of NaHS (0–100 lM) were recorded. As shown
in Figure 2, upon addition of NaHS, the initial absorption peak cen-
tered at 320 nm decreased gradually, along with a simultaneous
emergence of the red-shifted new absorption peak at 380 nm.
Moreover, the isosbestic point at 348 nm was observed. The free
HBTPP-S displayed quite weak fluorescence. Importantly, with
the addition of NaHS, as shown in Figure 3, the fluorescence inten-
sity of HBTPP-S increased significantly at 620 nm due to the thiol-
ysis of the dinitrophenyl ether by H2S. In addition, the increase in
fluorescence intensity is in a concentration dependent manner,
Acknowledgments
This research was supported in part by the National Natural
Science Foundation of China (30900377, 81271634), the
Fundamental Research Funds for the Central Universities, and
Hunan Provincial Natural Science Foundation of China (12JJ1012).
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