Fluorogenic detection of hydrogen sulfide via reductive unmasking of o-azidomethylbenzoyl-coumarin conjugate

Article abstract of DOI:10.1039/c2cc34682f

Selective detection of hydrogen sulfide was achieved with 7-o-2′-(azidomethyl)benzoyl-4-methylcoumarin via analyte mediated reductive removal of the 2′-(azidomethyl)benzoyl moiety and concurrent generation of fluorescent 7-hydroxy-4-methylcoumarin. This journal is The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc34682f

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COMMUNICATION
Fluorogenic detection of hydrogen sulfide via reductive unmasking
of o-azidomethylbenzoyl-coumarin conjugatew
Zhisheng Wu,a Zhu Li,a Liu Yang,a Jiahuai Hanb and Shoufa Han*a
Received 30th June 2012, Accepted 29th July 2012
DOI: 10.1039/c2cc34682f
Selective detection of hydrogen sulfide was achieved with 7-o-20-
(azidomethyl)benzoyl-4-methylcoumarin via analyte mediated
reductive removal of the 20-(azidomethyl)benzoyl moiety and con-
current generation of fluorescent 7-hydroxy-4-methylcoumarin.
chloride which was obtained from treatment of o-(azidomethyl)-
benzoic acid with thionyl chloride under reflux.
To access the applicability and mechanism of hydrosulfide
assay with AzMB-coumarin, AzMB-coumarin was incubated
with sodium hydrosulfide in acetonitrile. The reaction solution
was analyzed by high resolution mass spectrometry (HRMS)
and fluorometry. A major peak located at 156.0422 corres-
ponding to compound 1 (C8H7NO, [M+Na+]: 156.0420) was
observed (Fig. 1). Formation of fluorescent 7-hydroxy-4-
methylcoumarin in the assay solution was confirmed by
fluorescence emission spectrum analysis of the assay solution,
which was identical to the authentic sample, and mass spectro-
metry analysis where a major peak at 175.0408 was identified,
which was consistent with the theoretical molecular weight of
7-hydroxy-4-methylcoumarin (C10H8O3, [MꢀH]ꢀ: 175.0401)
(Fig. S2 and S3, ESIw). In contrast, the proposed product due
to direct cleavage of the ester by hydrosulfide was absent in the
assay solution (Scheme S1 and Fig. S3, ESIw). Collectively,
these results provide strong support for the hydrosufide
mediated reductive cleavage of the o-(azidomethyl)benzoyl
group of AzMB-coumarin as outlined in Scheme 2.
Hydrogen sulfide (H2S) is a toxic gas that is often present in
environments and industrial processing. Exposure to gaseous
H2S can often cause detrimental effects on human health.1 On
the other hand, endogenous H2S in living systems, recently
recognized as a gaseous transmitter, exhibits beneficiary effects
in a number of pathophysiological conditions, e.g. vasodilation,
antioxidant and anti-inflammation.2 The roles of H2S in the
aforementioned physiological events have recently spurred
significant efforts in the development of chemosensors for
detection of hydrosufide in biological settings.3
Thiols, e.g. cysteine and glutathione, are ubiquitous and
abundant in biological systems. To discriminate H2S or
hydrosulfide (HSꢀ) from the interfering biological thiols, the
double nucleophilicity or the reductive nature of hydrosulfide
are often employed in the design of chemosensors.3 One
elegant strategy, based on the reduction of the azido moiety
of the corresponding amine by hydrosufide, has been success-
fully developed for fluorogenic sensing of hydrosulfide
(Scheme 1A and B). Aryl azides are subjected to photolysis4
while sulfonyl azides are potentially reactive in selected condi-
tions.5 To complement this strategy further, we present the
fluorogenic detection of H2S with 7-o-20-(azidomethyl)-
benzoxy-4-methylcoumarin (AzMB-coumarin) featuring an
aliphatic azide group of superior stability (Scheme 2).
To validate the dependence of the o-(azidomethyl)benzoyl
moiety of AzMB-coumarin in sensing hydrosulfide, 7-acetoxy-
4-methylcoumarin was synthesized and tested in parallel for its
efficacy to detect hydrosulfide. Analysis showed that no fluor-
escent species were produced in the assay solution of 7-acetoxy-
4-methylcoumarin incubated with sodium hydrosulfide (Fig. 2).
The differential responses of 7-acetoxy-4-methylcoumarin and
AzMB-coumarin to hydrosulfide highlighted the essential role
of the o-(azidomethyl)benzoyl moiety in AzMB-coumarin
based detection of hydrosulfide.
Inspired by the utilization of o-(azidomethyl)benzoyl as the
hydroxyl protecting group in synthetic organic chemistry,6 we
designed AzMB-coumarin as the chemodosimeter of hydro-
sulfide. AzMB-coumarin was readily synthesized by esterification
of 7-hydroxy-4-methylcoumarin with o-(azidomethyl)benzoyl
a Department of Chemical Biology, College of Chemistry and
Chemical Engineering, and the Key Laboratory for Chemical Biology
of Fujian Province, Xiamen, Fujian 361005, China
b State Key Laboratory of Cellular Stress Biology and School of Life
Sciences, Xiamen University, Xiamen, Fujian 361005, China.
E-mail: shoufa@xmu.edu.cn; Tel: +86-0592-2181728
w Electronic supplementary information (ESI) available: Synthesis
and characterization of AzMB-coumarin and 7-acetoxy-4-methyl-
coumarin, characterization of the assay mechanism and detailed assay
procedures. See DOI: 10.1039/c2cc34682f
Scheme 1 Reported fluorogenic chemosensors for H2S based on
analyte specific reduction of the azido moiety.3c,g
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Fig. 3 Kinetic profiles of the reaction between AzMB-coumarin and
NaHS. Formation of 7-hydroxy-4-methylcoumarin was monitored by
fluorescence emission intensity at 450 nm (lex@365 nm) in acetonitrile
containing AzMB-coumarin (20 mM) and NaHS (0 mM, in dark;
100 mM, in red, A) or in sodium phosphate buffer (pH 7.4, 100 mM)
containing acetonitrile (20%, v/v) spiked with AzMB-coumarin
(100 mM) and NaHS (0 mM, in dark; 1 mM, in red, B).
Fig. 1 HRMS confirmed formation of compound 1 in the reaction of
AzMB-coumarin with hydrosulfide.
The solutions were incubated at room temperature for 1 h and
then analyzed by fluorometry. Fig. 4 revealed that fluorescence
emission, centered at 450 nm, intensified as a function of
hydrosulfide concentrations in aqueous acetonitrile with a
detection limit of 100 mM hydrosulfide. In contrast, as low
as 10 mM hydrosulfide can be detected in acetonitrile (Fig. S5,
ESIw), suggesting that the AzMB-coumarin based assay was of
a similar sensitivity as a previously reported chemodosimeter
of hydrogen sulfide.3e
Scheme 2 Detection of hydrosulfide with AzMB-coumarin via ana-
lyte triggered reduction of the azido group into an amine which leads
to the release of 7-hydroxy-4-methyl-coumarin by intramolecular
substitution.
To image hydrosulfide in biological settings, it is critical that
AzMB-coumarin can effectively recognize hydrosuflide in the
presence of numerous biologically relevent analytes, e.g. thiol
containing glutathione. Hence, the reactivity of AzMB-
coumarin towards representative biological thiols, reactive
oxygen species, reactive nitrogen species, and a variety of
cations were investigated under identical assay conditions. It
was shown that AzMB-coumarin efficiently detected hydro-
sulfide, whereas no formation of 7-hydroxy-4-methylcoumarin
was observed in the presence of glutathione, cysteine, homo-
cysteine, or b-mercaptoethanol in aqueous acetonitrile (Fig. 5)
or acetonitrile (Fig. S6, ESIw), suggesting the stringent
selectivity of AzMB-coumarin for hydrosulfide over mono-
substituted biological thiols. AzMB-coumarin sharply
responded to hydrosulfide while remaining inert to all the
cations, representative reactive oxygen species and reactive
nitrogen species examined, which could be generated in cells
under certain circumstances (Fig. S7, ESIw). Collectively, these
finding demonstrated the high selectivity of AzMB-coumarin
towards hydrosufide.
Fig. 2 Differential fluorescence responses of AzMB-coumarin to
hydrosulfide as compared to 7-acetoxy-4-methylcoumarin. The
fluorescence emission at 450 nm was recorded for 7-acetoxy-4-methyl-
coumarin (20 mM, in blue) or AzMB-coumarin (20 mM, in dark) in
acetonitrile in the absence (ꢀ) or presence (+) of sodium hydrosulfide
(100 mM) (lex@365 nm). The insert shows the chemical structure of
7-acetoxy-4-methylcoumarin.
To probe the optimal assay conditions, the rates of 7-hydroxy-
4-methylcoumarin formation under a variety of solvents were
screened by fluorometry. It was shown that the assay was
efficient in acetonitrile relative to that in ethanol, dimethyl-
formamide, or aqueous acetonitrile (Fig. S4, ESIw). Time
course studies revealed that the reaction between AzMB-
coumarin and hydrosulfide was completed at about 20–40 minutes
in acetonitrile or sodium phosphate (pH 7.4, 100 mM)
buffered acetonitrile (20%, v/v) (Fig. 3). H2S, with a pKa of
6.9, reversibly dissociates into hydrosulfide (HSꢀ) at physio-
logical pH (7.4). pH titration showed that the fluorogenic
assay in aqueous acetonitrile was much more efficient at pH
7.4 as compared to that at pH 6.4–5.4 (Fig. S5, ESIw),
indicating that the hydrosilfide ion was more effective than
hydrogen sulfide in reductive cleavage of the o-azidomethyl-
benzoyl group of AzMb-coumarin at physiological pH.
To assess the sensitivity of the AzMB-coumarin based
assay, various amounts of NaHS and AzMB-coumarin were
spiked into acetonitrile or sodium phosphate buffered acetonitrile.
Fig. 4 Fluorescence emission spectra of AzMB-coumarin (100 mM)
in sodium phosphate buffer (pH 7.4, 100 mM) containing acetonitrile
(20%, v/v) in the presence of NaHS (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7,
0.8, 0.9, 1.0 mM, from bottom to top) (A). The titration curve was
plotted by fluorescence emission intensity at 450 nm vs. analyte
concentration (B). (lex@365 nm).
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and capability of AzMB-coumarin in sensing of hydrosulfide
in living cells.
In summary, detection of hydrosulfide was achieved with an
o-(azidomethyl)benzoyl-coumarin conjugate where o-(azido-
methyl)benzoyl can be efficiently deblocked via hydrosulfide
triggered tandem reduction of the azido moiety, intra-
molecular amidation of the in situ generated amine group,
and simultaneous release of highly fluorescent 7-hydroxy-4-
methylcoumarin. AzMB-coumarin, featuring a stable aliphatic
azide as the analyte responsive element, exhibited stringent
selectivity for hydrosulfide over a broad variety of chemical
and biological species, e.g. cysteine and glutathione, suggest-
ing the utility of the 2-azidomethylbenzoyl group in the design
of sensors for studies of hydrosulfide mediated biomedical
events.
Fig. 5 Selectivity of AzMB-coumarin for hydrosulfide over endogen-
ous biological thiols. Fluorescence responses of AzMB-coumarin
(100 mM) in sodium phosphate buffer (100 mM, pH 7.4) containing
acetonitrile (20%, v/v) with no addition (1), or with addition of
glutathione (1 mM) (2), cysteine (1 mM) (3), homocysteine (1 mM)
(4), b-mercaptoethanol (1 mM) (5), or sodium hydrosulfide (1 mM) (6).
Dr S. Han was supported by grants from NSF China
(21072162), Natural Science Foundation of Fujian Province
of China (2011J06004), and the Fundamental Research Funds
for the Central Universities (2012121018); Dr J. Han was
supported by grants from NSF China (30830092, 30921005,
91029304, 81061160512) and 973 program (2009CB522200).
To access the feasibility of hydrosulfide detection in living
cells, HeLa cells preloaded with AzMB-coumarin were
incubated in phosphate buffered saline (PBS) supplemented
with or without sodium hydrosulfide for 60 minutes. The
cells were washed to remove extracellular reagents and then
visualized by fluorescence microscopy. As shown in Fig. 6A,
control cells treated with AzMB-coumarin remained non-
fluorescent in the absence of sodium hydrosulfide, suggesting
that AzMB-coumarin is immune to metabolism of various
intracellular species under the assay conditions, e.g. hydrolysis
by cytosolic esterases. In contrast, the fluorescence of
7-hydroxy-4-methylcoumarin was clearly observed in cells
treated with hydrosulfide (Fig. 6B), suggesting the selectivity
Notes and references
1 P. A. Patnaik, Comprehensive Guide to the Hazardous Properties of
Chemical Substances, Wiley, New York, 3rd edn, 2007.
2 (a) M. Lavu, S. Bhushan and D. J. Lefer, Clin. Sci., 2011, 120, 219;
(b) L. Li, P. Rose and P. K. Moore, Annu. Rev. Pharmacol. Toxicol.,
2011, 51, 169; (c) C. Szabo, Nat. Rev. Drug Discovery, 2007, 6,
917–935; (d) M. Whiteman and P. K. Moore, J. Cell. Mol. Med.,
2009, 13, 488.
3 (a) F. Hou, J. Cheng, P. Xi, F. Chen, L. Huang, G. Xie, Y. Shi,
H. Liu, D. Bai and Z. Zeng, Dalton Trans., 2012, 41, 5799;
(b) F. Hou, L. Huang, P. Xi, J. Cheng, X. Zhao, G. Xie, Y. Shi,
F. Cheng, X. Yao, D. Bai and Z. Zeng, Inorg. Chem., 2012, 51, 2454;
(c) A. R. Lippert, E. J. New and C. J. Chang, J. Am. Chem. Soc.,
2011, 133, 10078; (d) C. Liu, J. Pan, S. Li, Y. Zhao, L. Y. Wu,
C. E. Berkman, A. R. Whorton and M. Xian, Angew. Chem., Int.
Ed., 2011, 50, 10327; (e) C. Liu, B. Peng, S. Li, C. M. Park,
A. R. Whorton and M. Xian, Org. Lett., 2012, 14, 2184;
(f) L. A. Montoya and M. D. Pluth, Chem. Commun., 2012,
48, 4767; (g) H. Peng, Y. Cheng, C. Dai, A. L. King,
B. L. Predmore, D. J. Lefer and B. Wang, Angew. Chem., Int.
Ed., 2011, 50, 9672; (h) Y. Qian, J. Karpus, O. Kabil, S. Y. Zhang,
H. L. Zhu, R. Banerjee, J. Zhao and C. He, Nat. Commun., 2011,
2, 495; (i) K. Sasakura, K. Hanaoka, N. Shibuya, Y. Mikami,
Y. Kimura, T. Komatsu, T. Ueno, T. Terai, H. Kimura and
T. Nagano, J. Am. Chem. Soc., 2011, 133, 18003.
Fig. 6 Imaging of hydrosulfide in HeLa cells with AzMB-coumarin.
HeLa cells were seeded on glass coverslips and then incubated with
AzMB-coumarin (1 mM) for 10 min. The cells were washed and then
treated with (B) or without (A) NaHS (1 mM) in PBS buffer for
60 min. The cells were rinsed and then analyzed by confocal fluores-
cence microscopy. The fluorescence emission at 420–500 nm was
recorded using an excitation wavelength of 405 nm.
4 M. Das and C. F. Fox, Annu. Rev. Biophys. Bioeng., 1979, 8, 165.
5 J. Kubicki, H. L. Luk, Y. Zhang, S. Vyas, H. L. Peng, C. M. Hadad
and M. S. Platz, J. Am. Chem. Soc., 2012, 134, 7036.
6 T. Wada, A. Ohkubo, A. Mochizuki and S. Sekine, Tetrahedron
Lett., 2001, 42, 1069.
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