Water-soluble BODIPY derivative as a highly selective "turn-on" fluorescent probe for hydrogen sulfide in living cells

Article abstract of DOI:10.1246/cl.150631

A novel BODIPY derivative incorporating quaternized 4-pyridinium group as a self-immolative linker was synthesized and studied for the selective detection of hydrogen sulfide. Such a probe exhibits excellent selectivity to allow differentiation of H₂S from biologically essential thiol-containing amino acids and other common analytes. It can be used to visualize NaSH in living cells.

Full text of DOI:10.1246/cl.150631

CL-150631
Received: July 3, 2015 | Accepted: August 19, 2015 | Web Released: August 27, 2015
Water-soluble BODIPY Derivative as a Highly Selective “Turn-on” Fluorescent Probe
for Hydrogen Sulfide in Living Cells
Jian Zhang,1 Fangfang Peng,2 Xiaochun Dong,*1 and Weili Zhao*1,2
1School of Pharmacy, Fudan University, Shanghai 201203, P. R. China
2Key Laboratory for Special Functional Materials of the Ministry of Education, Henan University, Kaifeng 475004, P. R. China
(E-mail: xcdong@fudan.edu.cn, zhaoweili@fudan.edu.cn)
A novel BODIPY derivative incorporating quaternized 4-
pyridinium group as a self-immolative linker was synthesized
and studied for the selective detection of hydrogen sulfide. Such
a probe exhibits excellent selectivity to allow differentiation of
H2S from biologically essential thiol-containing amino acids and
other common analytes. It can be used to visualize NaSH in
living cells.
moiety was intrinsically fluorescent,34,35 fluorescence of BODIPY
dye fragments functionalized with the meso-pyridinium-4-yl
group was extensively quenched through the quaternization
process and the pyridinium unit resulted in improved water
solubility.37,38 Once the N-substituent of pyridinium was re-
moved, regeneration of fluorescence was realized (Φfl = 0.78).37
It was believed that such “Turn-On” of fluorescence was the
result of the interruption of photoinduced electron-transfer (PET)
process, which was supported by the density functional theory
(DFT) calculation as reported.37 In the current communication,
the self-immolative strategy was used, wherein the reduction of
azide by H2S4,39 led to the production of the p-aminobenzyl
moiety, which was able to self-immolate through an intra-
molecular 1,4-elimination and release pyridine derivative.38
BODIPY-based probe 1 was prepared conveniently as shown
in Scheme S1 (Supporting Information (SI)). Probe 1 exhibits
87.4 ¯g mL¹1 solubility in water and possesses very low fluores-
cence quantum yield (Φfl = 0.003) due to the photoinduced
electron-transfer (PET) process. Upon addition of H2S to probe 1,
a significant fluorescence enhancement was noticed. The primary
response of probe 1 to H2S prompted us to investigate the
behavior of probe 1 in detail.
Probe 1 displays absorption maxima at 508 nm in PBS
(10 mM, pH 7.4), and a very weak emission centered at 517 nm
(Figure S1, SI). Upon addition of NaSH (500 ¯M) to the buffered
solution of probe 1 (10 ¯M), the absorption maxima at 508 nm
remained almost unchanged, however with the intensity decreas-
ing gradually over time (Figure S2, SI). The time-dependent
spectra changes of probe 1 to NaSH are shown in Figure 1. For
the fluorescence response, the addition of NaSH caused a steady
increase in the intensity of fluorescence up to 27-fold at 517 nm.
We subsequently evaluated the responses of probe 1 toward
various concentrations of NaSH. Figure 2 showed that with the
increase in the NaSH concentration, the fluorescence intensity at
517 nm enhanced accordingly. The fluorescence intensity was
Hydrogen sulfide (H2S) gas is a member of reactive sulfur,
which is regarded as the third gasotransmitter in the body after
nitric oxide (NO) and carbon monoxide (CO).1-3 Endogenous
H2S is produced inside the cells from the cysteine (Cys) substrate
or its derivatives by at least four separate enzymes: cystathionine
β-synthase (CBS), cystathionine γ-lyase (CSE), 3-mercapto-
pyruvate sulfurtransferase (3-MST), and cysteine aminotransfer-
ase (CAT). The physiologically relevant H2S concentration is
estimated to be nano- to millimolar levels.4,5 At physiological
levels, H2S is involved in a diverse array of biological functions,
including neurotransmission,6 vasodilation,7 apoptosis,8 inflam-
mation,9 ischemia/reperfusion-induced injury,10 and insulin
secretion.11 Abnormal H2S production was shown to correlate
with various human diseases such as Alzheimer’s disease (AD),12
Down’s syndrome,13 hypertension,14 and liver cirrhosis.15 To
obtain valuable information on the functions of H2S in biology,
new techniques for accurate detection of H2S levels are in high
demand.
Conventional approaches to H2S detection, such as elec-
trochemical assays, gas chromatography, colorimetric, and
sulfide precipitation, often require complicated sample process-
ing and destruction of tissues or cells.16-20 A fluorescence
imaging probe is more desirable for the noninvasive and real-
time detection of H2S.21-26
4,4-Difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) dyes
are known to be highly fluorescent, very stable, and exceptionally
insensitive to the polarity of solvents as well as to pH. BODIPYs
are widely applied as fluorophore in probes and sensors.27-29
Since 2011, several BODIPY-based fluorescent probes for H2S
have been developed on the basis of nucleophilic addition
of H2S,30 reduction of azides to amines mediated by H2S,31
reduction of selenoxide,32 as well as thiolysis of dinitrophenyl
ether by H2S.33
Recently, several BODIPY derivatives possessing a pyridi-
nium moiety in the meso-position have been reported for various
potential applications.34-37 In this paper, we present a novel
water-soluble BODIPY derivative 1 containing pyridinium-4-yl
moiety quaternized with a para-azido benzyl moiety as the H2S-
recognizing group for the selective detection of H2S.
Figure 1. Time-dependent fluorescence spectra of probe 1 (10 ¯M,
The design of probe 1 was inspired by the recent discoveries
that although BODIPY dye carrying a meso-pyridinium-2-yl
-
ex = 450 nm) and the corresponding responses (inset, -em = 517 nm)
in the present of 50 equiv of NaSH in PBS (10 mM, pH 7.4, 25 °C).
1524 | Chem. Lett. 2015, 44, 1524–1526 | doi:10.1246/cl.150631
© 2015 The Chemical Society of Japan

Figure 2. Fluorescence intensity changes (-em = 517 nm) of probe 1
(10 ¯M, -ex = 450 nm) in the presence of increasing concentration of
NaSH (final concentration: 0, 1, 5, 10, 15, 20, 30, 40, 50, 100, 200,
300, 400, and 500 ¯M) after incubation at 25 °C in PBS (10 mM,
Figure 3. The fluorescence intensities (-em = 517 nm) of probe 1
(10 ¯M, -ex = 450 nm) in PBS (10 mM, pH 7.4, 25 °C) in the presence
of 200 equiv of various analytes (black bars) and after addition of
50 equiv of NaSH to the solution (red bars): (1) free; (2) GSH; (3) Cys;
¹
2¹
2¹
2¹
2¹
pH 7.4, 25 °C). Inset:
A linear calibration curve between the
(4) Hcy; (5) HSO3 ; (6) SO3 ; (7) S2O3 ; (8) S2O4 ; (9) S2O5
;
¹
¹
¹
¹
¹
fluorescent intensity changes (-em = 517 nm) of probe 1 and the
concentration of NaSH in the range of 0 to 30 ¯M. Each spectrum was
recorded 150 min after addition of NaSH.
(10) Cl ; (11) Br ; (12) I ; (13) NO2 ; (14) N3 ; (15) H2O2; (16)
2¹
¹
CO3 ; (17) CH3COO ; (18) Mn2+; (19) Fe3+; (20) Mg2+; (21) Cu2+
;
2¹
(22) Zn2+; (23) SO4 . Data were taken 150 min after addition of
analyte(s).
linearly proportional to the amount of NaSH ranging from 1 to
30 ¯M with a detection limit of 0.8 ¯M (3·/k, wherein · is the
standard deviation of blank measurement, and k is the slope
between the fluorescence intensity versus NaSH concentration).
To evaluate the selectivity of probe 1 for NaSH (500 ¯M)
¹
2¹
2¹
over various analytes, the responses of probe 1 to HSO3 , SO3
,
,
2¹
2¹
2¹
¹
¹
¹
¹
¹
S2O3 , S2O4 , S2O5 , Cl , Br , I , NO2 , N3 , H2O2, CO3
¹
2¹
CH3COO , Mn2+, Fe3+, Mg2+, Cu2+, Zn2+, and SO4 , as
well as biothiols, e.g., cysteine (Cys), homocysteine (Hcy), and
glutathione (GSH) were investigated (Figure S4, SI). As shown
in Figure S4, only addition of NaSH to the solution of probe 1
resulted in a significant enhancement of the fluorescence
intensity. None of the interferences, including biothiols, metal
cation, common anion, nucleophile, or oxidant, caused any
noticeable fluorescence enhancement.
Scheme 1. The proposed fluorescence sensing mechanism of probe
1 towards NaSH.
To further demonstrate the excellent selectivity of probe 1
to NaSH, the competition experiments were carried out by the
addition of 50 equiv of NaSH to the solution of probe 1 in the
presence of 200 equiv of the interference (GSH, Cys, Hcy,
¹
2¹
2¹
2¹
2¹
¹
¹
¹
¹
HSO3 , SO3 , S2O3 , S2O4 , S2O5 , Cl , Br , I , NO2
,
,
N3 , H2O2, CO3 , CH3COO , Mn2+, Fe3+, Mg2+, Cu2+, Zn2+
¹
2¹
¹
2¹
and SO4 ), and the results are shown in Figure 3. As shown
in Figure 3, the coexistence of 4-fold of other analytes did not
have any obvious impact on the detection of H2S. These results
suggested that probe 1 displayed excellent selectivity toward
H2S.
Figure 4. 1H NMR analysis of the reaction between probe 1 and
NaSH in a mixture of CD3CN/D2O (1/1, v/v): (a) probe 1 only; (b)
150 min after addition of 50 equiv of NaSH; (c) 1H NMR of 2 in a
mixture of CD3CN/D2O (1/1, v/v).
The effects of pH on the fluorescence response of probe 1 to
H2S were then investigated. As showed in Figure S5, probe 1
responded nicely to NaSH over the pH range varying from 6.0
to 7.5, which fit well the physiological pH requirements for
biological applications.
for 150 min, as displayed in Figure 4b, a series of characteristic
proton signals of H4¤¤ (¤ = 9.25, d), H5¤¤ (¤ = 8.00, d), and H6¤¤
(¤ = 6.79, d) appeared identical with those of compound 2
shown in Figure 4c. However, the characteristic proton signals of
compound 4 were not identified presumably due to its instability
for self-immolative reactions. Aza-quinone-methide 5 was not
clearly identified in the NMR spectra. It is known that reactant 5
is prone to attacks by water and various nucleophiles.38 The
NMR studies suggested an initial reduction reaction of azide
by NaSH, which produced intermediate 4. Subsequently, 1,4-
elimination of 4 took place, leading to aza-quinone-methide 5
and compound 2.38 The formation of compounds 2 and 5 was
further confirmed from ESI-MS measurements (Figures S6 and
S7, SI).
The fluorescence sensing mechanism for probe 1 toward
NaSH was believed to follow a sequential reduction-elimination
process, as shown in Scheme 1. The proposed mechanism was
1
supported by H NMR studies and ESI-MS measurements.
NMR studies of the sensing mechanism were performed in
CD3CN/D2O (1/1, v/v), and the resulted are shown in Figure 4.
In Figure 4a, the characteristic proton signals of probe 1
designated as shown in Scheme 1 were H4 (¤ = 9.56, d), H5
(¤ = 8.69, d), H1 (¤ = 8.02, d), H2 (¤ = 7.70, d), H6 (¤ = 6.72,
s), and H3 (¤ = 6.36, s). After the addition of 50 equiv of NaSH
Chem. Lett. 2015, 44, 1524–1526 | doi:10.1246/cl.150631
© 2015 The Chemical Society of Japan | 1525

10 J. W. Elrod, J. W. Calvert, J. Morrison, J. E. Doeller, D. W. Kraus, L.
Tao, X. Jiao, R. Scalia, L. Kiss, C. Szabo, H. Kimura, C.-W. Chow,
D. J. Lefer, Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 15560.
11 Y. Kaneko, Y. Kimura, H. Kimura, I. Niki, Diabetes 2006, 55, 1391.
12 C. Yu, X. Li, F. Zeng, F. Zheng, S. Wu, Chem. Commun. 2013, 49,
403.
13 P. Kamoun, M.-C. Belardinelli, A. Chabli, K. Lallouchi, B.
Chadefaux-Vekemans, Am. J. Med. Genet., Part A 2003, 116A, 310.
14 G. Yang, L. Wu, B. Jiang, W. Yang, J. Qi, K. Cao, Q. Meng, A. K.
Mustafa, W. Mu, S. Zhang, S. H. Snyder, R. Wang, Science 2008, 322,
587.
15 S. Fiorucci, E. Antonelli, A. Mencarelli, S. Orlandi, B. Renga, G.
Rizzo, E. Distrutti, V. Shah, A. Morelli, Hepatology 2005, 42, 539.
16 M. W. Warenycia, L. R. Goodwin, C. G. Benishin, R. J. Reiffenstein,
D. M. Francom, J. D. Taylor, F. P. Dieken, Biochem. Pharmacol. 1989,
38, 973.
17 A. Tangerman, J. Chromatogr. B 2009, 877, 3366.
18 T. Ubuka, J. Chromatogr. B 2002, 781, 227.
19 J. E. Doeller, T. S. Isbell, G. Benavides, J. Koenitzer, H. Patel, R. P.
Patel, J. R. Lancaster, Jr., V. M. Darley-Usmar, D. W. Kraus, Anal.
Biochem. 2005, 341, 40.
Figure 5. Fluorescence imaging of NaSH in Human Hepatoma
SMMC-7721 incubated at 37 °C with 10 ¯M of probe 1. (a) and (b)
show that cells were incubated with probe 1 only for 60 min. (c) and
(d) show that cells were pre-incubated with probe 1 for 30 min, and
then incubated with 500 ¯M of NaSH for 60 min. (a) and (c) are bright
field images, (b) and (d) are fluorescence images of (a) and (c),
respectively.
20 T. Nagata, S. Kage, K. Kimura, K. Kudo, M. Noda, J. Forensic Sci.
1990, 35, 706.
21 F. Yu, X. Han, L. Chen, Chem. Commun. 2014, 50, 12234.
22 V. S. Lin, W. Chen, M. Xian, C. J. Chang, Chem. Soc. Rev. 2015, 44,
4596.
To further investigate the biological application of probe 1,
the fluorescence imaging experiment was carried out. Probe 1
exhibited very low cytotoxicity toward human hepatoma SMMC-
7721, which was evaluated by classic MTT assays, after the cells
were incubated for 24 h in the presence of 80 ¯M probe 1
(Figure S8, SI). When human hepatoma SMMC-7721 cells were
incubated with 10 ¯M of probe 1 only in a culture medium
at 37 °C for 1 h, no fluorescence was observed (Figure 5). In
contrast, addition of NaSH to the cells pre-incubated with probe 1
produced green fluorescence.
In conclusion, we report a water-soluble BODIPY-based
fluorescent probe for the detection of H2S. The detection limit
of probe 1 for H2S was 0.8 ¯M. Probe 1 exhibits excellent
selectivity to allow the differentiation of H2S from biologically
essential thiol-containing amino acids and other common
analytes. The probe had low toxicity and was successfully used
to detect H2S in living cells.
23 J. Li, C. Yin, F. Huo, RSC Adv. 2015, 5, 2191.
24 L. Wei, Z. Zhu, Y. Li, L. Yi, Z. Xi, Chem. Commun. 2015, 51, 10463.
25 D.-T. Shi, D. Zhou, Y. Zang, J. Li, G.-R. Chen, T. D. James, X.-P. He,
H. Tian, Chem. Commun. 2015, 51, 3653.
26 J. Cao, R. Lopez, J. M. Thacker, J. Y. Moon, C. Jiang, S. N. S. Morris,
J. H. Bauer, P. Tao, R. P. Mason, A. R. Lippert, Chem. Sci. 2015, 6,
1979.
27 A. Loudet, K. Burgess, Chem. Rev. 2007, 107, 4891.
28 G. Ulrich, R. Ziessel, A. Harriman, Angew. Chem., Int. Ed. 2008, 47,
1184.
29 J. Karolin, L. B.-A. Johansson, L. Strandberg, T. Ny, J. Am. Chem.
Soc. 1994, 116, 7801.
30 a) R. Hong, G. Han, J. M. Fernández, B. Kim, N. S. Forbes, V. M.
Rotello, J. Am. Chem. Soc. 2006, 128, 1078. b) T. Cheng, Y. Xu, S.
Zhang, W. Zhu, X. Qian, L. Duan, J. Am. Chem. Soc. 2008, 130,
16160. c) Y. Qian, J. Karpus, O. Kabil, S.-Y. Zhang, H.-L. Zhu, R.
Banerjee, J. Zhao, C. He, Nat. Commun. 2011, 2, 495. d) Y. Qian, L.
Zhang, S. Ding, X. Deng, C. He, X. E. Zheng, H.-L. Zhu, J. Zhao,
Chem. Sci. 2012, 3, 2920. e) X. Li, S. Zhang, J. Cao, N. Xie, T. Liu, B.
Yang, Q. He, Y. Hu, Chem. Commun. 2013, 49, 8656. f) Y. Qian, B.
Yang, Y. Shen, Q. Du, L. Lin, J. Lin, H. Zhu, Sens. Actuators, B 2013,
182, 498.
This research was supported by NSFC (No. 21372063) and
Program for Changjiang Scholars and Innovative Research Team
in University, No. PCS IRT1126.
31 T. Saha, D. Kand, P. Talukdar, Org. Biomol. Chem. 2013, 11, 8166.
32 a) B. Wang, P. Li, F. Yu, P. Song, X. Sun, S. Yang, Z. Lou, K. Han,
Chem. Commun. 2013, 49, 1014. b) B. Wang, P. Li, F. Yu, J. Chen, Z.
Qu, K. Han, Chem. Commun. 2013, 49, 5790.
Supporting Information is available electronically on J-STAGE.
References
1
Y. Han, J. Qin, X. Chang, Z. Yang, J. Du, Cell. Mol. Neurobiol. 2006,
26, 101.
C. Szabó, Nat. Rev. Drug Discovery 2007, 6, 917.
O. Kabil, R. Banerjee, J. Biol. Chem. 2010, 285, 21903.
A. R. Lippert, E. J. New, C. J. Chang, J. Am. Chem. Soc. 2011, 133,
10078.
J. Furne, A. Saeed, M. D. Levitt, Am. J. Physiol. Regul. Integr. Comp.
Physiol. 2008, 295, R1479.
L.-F. Hu, M. Lu, P. T. Hon Wong, J.-S. Bian, Antioxid. Redox
Signaling 2011, 15, 405.
33 X. Cao, W. Lin, K. Zheng, L. He, Chem. Commun. 2012, 48, 10529.
34 N. Jiang, J. Fan, T. Liu, J. Cao, B. Qiao, J. Wang, P. Gao, X. Peng,
Chem. Commun. 2013, 49, 10620.
35 S. Zhang, T. Wu, J. Fan, Z. Li, N. Jiang, J. Wang, B. Dou, S. Sun, F.
Song, X. Peng, Org. Biomol. Chem. 2013, 11, 555.
36 J. Xu, Q. Li, Y. Yue, Y. Guo, S. Shao, Biosens. Bioelectron. 2014, 56,
58.
37 J. Xu, S. Sun, Q. Li, Y. Yue, Y. Li, S. Shao, Anal. Chim. Acta 2014,
849, 36.
38 S. Gnaim, D. Shabat, Acc. Chem. Res. 2014, 47, 2970.
39 a) H. Peng, Y. Cheng, C. Dai, A. L. King, B. L. Predmore, D. J. Lefer,
B. Wang, Angew. Chem., Int. Ed. 2011, 50, 9672. b) F. Yu, P. Li, P.
Song, B. Wang, J. Zhao, K. Han, Chem. Commun. 2012, 48, 2852.
c) L. A. Montoya, M. D. Pluth, Chem. Commun. 2012, 48, 4767.
2
3
4
5
6
7
8
9
D. J. Lefer, Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 17907.
G. Yang, L. Wu, R. Wang, FASEB J. 2006, 20, 553.
R. C. O. Zanardo, V. Brancaleone, E. Distrutti, S. Fiorucci, G. Cirino,
J. L. Wallace, FASEB J. 2006, 20, 2118.
1526 | Chem. Lett. 2015, 44, 1524–1526 | doi:10.1246/cl.150631
© 2015 The Chemical Society of Japan