Letter pubs.acs.org/ac
Bioluminescence Probe for Detecting Hydrogen Sulfide in Vivo Bowen Ke,*,†,§ Wenxiao Wu,‡,§ Wei Liu,† Hong Liang,† Deying Gong,† Xiaotong Hu,† and Minyong Li*,‡ †
Laboratory of Anaesthesiology & Critical Care Medicine, Translational Neuroscience Center, West China Hospital, Sichuan University, Chengdu, Sichuan 610041, China ‡ Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (MOE), School of Pharmacy, Shandong University, Jinan, Shandong 250012, China S Supporting Information *
ABSTRACT: Considering that hydrogen sulfide (H2S) is an endogenous signaling molecule involved in numerous biological processes, a method for monitoring H2S as a powerful tool for investigating its complicated functions and mechanisms is urgently demanded. Herein, a bioluminescent turn-on probe was reported based on caged strategy for the detection of H2S in vitro and in vivo. This probe will help us understand the intricate contribution of H2S to a variety of physiological and pathological processes.
t first sight, hydrogen sulfide (H2S) is best characterized by the well-known malodorous smell and high toxicity that makes it seem incompatible with any physiological functions and therapeutic roles. However, since H2S was first observed to present as a neuromodulator in the brain in 1996,1 an emerging body of research has changed this traditional view and indicated that this gaseous small molecule is correlated with various biological processes.2−4 It has been demonstrated that H2S is generated endogenously from cysteine or cysteine derivatives in reactions catalyzed by three enzymes, including cystathionine β-synthase (CBS), cystathionine-lyase (CES), and 3-mercaptopyruvate sulfurtransferase (MPST).5 As the third member of the gasotransmitter family along with its congeners nitric oxide (NO) and carbon monoxide (CO), H2S is widespread in a diversity of tissue types and proposed as an indispensable component of signaling pathways which are involved in several physiological functions, such as vasorelaxation regulation6 and neuronal transmission modulation.7 On the other hand, dysregulated generation and level aberrance of H2S have been recognized to characterize the occurrence and development of various diseases, as discovered in models of diabetes8 and Down’s syndrome.9 In addition, exogenous H2S can exert potential therapeutic effects, such as anti-inflammation10 and protection against oxidative stress,11 for the treatment of wound healing,12 cancer,13 inflammation,14 and neurodegenerative disease.15 The realization of H2S as a vital signaling molecule sparked interest in its molecular mechanism underlying physiology and pathology. To better understand how this gasotransmitter contributes to convoluted biological processes, accurate and reliable determination of biologically relevant concentrations of H2S in live cells, tissues, and whole animals is directly
A
© 2015 American Chemical Society
requested. However, real-time detection of H2S in vivo still remains a critical challenge, mainly due to fast anabolism and catabolism of this transient chemical species. It is well established that concentration of endogenous H2S may rapidly fluctuate in the wide range of nanomolar to submillimolar level in signaling processes. In this context, traditional analytical methods such as gas chromatography16 and electrochemical assay17 may yield inconsistent results so that they hardly provide a sensitive detection for tracking endogenous H2S in living biological systems. Compared with traditional detection systems, optical imaging techniques are highly sensitive, convenient, and easily handled, which makes it suitable for nondestructive, real-time spatial detection of this volatile, gaseous molecule with readily available instruments. Among them, fluorescent and chemiluminescent probes have been widely developed for H2S detection in various biological samples, ranging from cell culture specimens to blood serum.18−26 However, because of autofluorescence and poor tissue penetration, these detection techniques seem to be unsuitable for reporting in deep tissues and whole animals, which leaves a big gap in the ability to visualize location and dynamic change of H2S across large space or time scales. Bioluminescent imaging (BLI) is a well-established optical technique that has become indispensable for noninvasive imaging of biological phenomena in vivo in the past decade. As a significant alternative for fluorescence imaging, BLI provides valuable insights into biological findings and processes Received: September 24, 2015 Accepted: December 4, 2015 Published: December 4, 2015 592
DOI: 10.1021/acs.analchem.5b03636 Anal. Chem. 2016, 88, 592−595
Letter
Analytical Chemistry in culture, such as protein−protein interaction and gene expression.27 Moreover, it helps in facilitating drug identification and evaluating subsequent treatment efficacy in preclinical studies.28−30 BLI simply relies on the detection of photons produced from the catalytic firefly luciferin−luciferase reaction without the requirement of excitation light so as to avoid the intrinsic disturbances from fluorescence, such as autofluorescence and photobleaching, thus resulting in worthy signal-to-noise ratio in the long-time study. In addition, favorable properties of firefly luciferin, including biological inertness, cell permeability, and nontoxicity, also render BLI a desirable biocompatibility. However, to the best of our knowledge, BLI-based H2S probe still remains unavailable. To address this issue, herein we undertake the efforts in developing a bioluminescent turn-on probe for H2S detection as well as studying its initial applications in living cells and animal models. Caging the luciferase substrates is a versatile and wellestablished strategy for selective detection of analyte and has been successfully employed for designing turn-on bioluminescent probes for detecting numerous analytes.31−36 As a highly specific substrate for firefly luciferase, firefly luciferin or aminoluciferin could be appropriately caged by chemical modification at the 4- or 6′-position of the scaffold that results in prohibiting the response to luciferase and suppressing the light production.37 With a cleavage process triggered by the target analyte, the probe could release the free luciferin, which subsequently activates the catalytic luciferin−luciferase reaction to generate a light emission. In the current case, unique chemical characteristics of H2S, such as reducing potency and nucleophilicity, could provide an excellent opportunity to offer probes valuable selectivity toward this reactive gaseous molecule. Using chemoselective reduction of azide to amine, reaction-based probes can selectively and sensitively detect H2S in vitro and in vivo (Scheme 1). In addition, extensive
Figure 1. Chemical structure of hydrogen sulfide bioluminescent probes.
10 mM MgCl2 was chosen to be an optimum solution condition for determining enzyme activity. This buffer system was employed in all the experiments described in this report. Initially, we evaluated the turn-on responsiveness of probes to H2S and measured luminescence produced in a concentrationdependent manner. As expected, only weak luminescence was recorded by a plate reader in the absence of H2S. However, the reaction of probes upon H2S can reduce the N3 group to the NH2 group and subsequently release free luciferin or aminoluciferin, thus triggering a significant luminescence increase within 60 min. Incubated with a series of concentrations of NaHS, three probes, respectively, exhibited different extent of luminescence enhancements (Figure S1). These promising results indicated that all probes possess the considerable ability for detecting H2S at diverse concentrations in the aqueous environment. Subsequently, we moved forward to examine the selectivity of probes toward H2S over other relevant species. Addition of a range of the representative reducing anions and compounds including HS−, NO3−, NO2−, H2PO4−, HSO3−, SO42−, NAC, and D-cysteine at 10 mM to probes 1−3 (20 μM), respectively, did not elicit any significant bioluminescence increase in the presence of ATP and luciferase at 37 °C for 60 min. Moreover, these probes were also proved to have nonreactivity toward small-molecule thiols such as NAC and D-cysteine, even when their concentrations were high up to 10 mM. In comparison, the marked bioluminescence enhancements were induced immediately and varying degrees of signal increases were easily measured (8-, 4-, and 2-fold increasing for probes 1−3 within 60 min, respectively) after the addition of 10 mM NaSH (Figure 2). On the basis of the above bioluminescence characterizations of all probes, compound 1 was determined as the best potent probe for further investigation toward in cellular and in vivo imaging owing to its fastest response and highest sensitivity toward H2S compared with the other two probes. We continued to examine whether the performance of probe 1 in aqueous buffer translated to cell culture that could represent a promising ability for visualizing fluctuations in H2S level in living cells. Standard MTT assays of probe 1 were first performed for 48 h to investigate its biocompatibility. It was shown that probe 1 was low cytotoxic (IC50 = 623 μM) to the cultured cells under the experimental conditions. Therefore, we went forward to test the permeability of probe 1 by means of penetrating the lipophilic cell membrane and the responsiveness to alterations in H2S level in living cells. First, living ES-2Fluc cells loaded with 10 μM of probe 1 for 1 h at 37 °C showed only weak bioluminescence signal. In contrast, incubating the cells with a series of concentrations of NaHS ranging from 0 to 1000 μM for 30 min, followed by removing
Scheme 1. Schematic of Bioluminescent Probes for Selective Detection of H2S
application of azido group as a biorthogonal functional group in chemical biology also proves this functional group compatible with living systems. On the basis of these collective reasons, three H2S bioluminescent probes 1−3 were well designed and synthesized via introducing a self-immolative linker into luciferin scaffold at 6′-position or via replacing the 6′-NH2 group of aminoluciferin with an azido group (Figure 1). The detailed synthesis protocols and structural characterizations are provided in the Supporting Information. With these compounds in hand, we first examined if H2S probes can be carried out in aqueous systems. As a H2S donor, NaHS was used in this study, because its solution is more accurate and reproducible to define H2S concentrations than the solution made by bubbling H2S gas. To be biologically compatible, the Tris-HCl buffer (50 mM, pH 7.4) containing 593
DOI: 10.1021/acs.analchem.5b03636 Anal. Chem. 2016, 88, 592−595
Letter
Analytical Chemistry
After establishing that probe 1 acquires the ability to monitor H2S fluctuations in living cells, we next sought to apply it for visualizing H2S flux in the whole animal. As a control group, transgenic FVB-luc+ mice with ubiquitous firefly luciferase expression were pretreated with saline followed by i.p. injection of probe 1 to determine bioluminescence signal generated from the animal model in the absence of exogenous H2S. As a result, a weak but measurable bioluminescence background could be detected throughout a sensitive CCD camera, which indicated probe 1 may be sensitive enough to detect basal levels of endogenous H2S in living animals. Next, mice were pretreated with injection of NaSH at a dose of 20 μmol via the tail vein 5 min prior to subsequent injection of 20 μmol of probe 1 into the i.p. cavity of mice. The bioluminescence signal produced from living animals was measurably recorded in real-time every 5 min for 40 min after injection of probe 1. The integrated total photon flux for whole animals (except tails) revealed timedependent differences in bioluminescence signal. When the bioluminescence intensity reached a maximum value within 25 min, a ∼50% increase in bioluminescence relative to control animals was observed (Figure 4). Notably, the bioluminescence
Figure 2. (a) The relative bioluminescence intensity of probes with various reducing ions and compounds (HS−, NO3−, NO2−, H2PO4−, HSO3−, SO42−, NAC, and D-cysteine) after adding luciferase and ATP; (b) The relative bioluminescence intensity of probes with various concentrations of NaHS solution after adding luciferase and ATP. All assays were performed in triplicate and represented as the mean ± SEM.
the medium prior to 10 μM probe 1 treatment 60 min later results in remarkable luminescence increases in a concentration-dependent manner (Figure 3). We also demomstrated
Figure 3. Total flux of bioluminescence imaging of probe 1 with NaHS solution in cell. All assays were performed in triplicate and represented as the mean ± SEM.
Figure 4. Bioluminescence imaging of probe 1 in FVB-luc+ mice. (a) Representative bioluminescence imaging of probe 1 in the mice for (b) quantification of the total flux (photon/s) from the whole body area except tail, in the presence or absence of NaHS, (c) integrated bioluminescence emission for mice injected with probe 1, in the presence or absence of NaHS. All assays were performed in triplicate and represented as the mean ± SEM ** P < 0.05.
the H2S production and detemined the H2S concentrations in cells by using a colormetric method (Figure S2). Taken together, these results not only indicated the presence of living cells has no effect on the bioluminescence signal produced from probe 1 in this biological environment but also established that probe 1 possesses a significant dose-dependent responsiveness in living cells.
increase could be monitored within the first few minutes after probe injection, providing a valuable approach for rapid detection of H 2 S levels in vivo. Compared to other fluorescence-based or chemluminescence-based in vivo imaging, bioluminescent probe 1 offers a highly sensitive method for whole-animal H2S detection in a long-term study. 594
DOI: 10.1021/acs.analchem.5b03636 Anal. Chem. 2016, 88, 592−595
Letter
Analytical Chemistry
(11) Szabó, G.; Veres, G.; Radovits, T.; Gerő , D.; Módis, K.; MieselGröschel, C.; Horkay, F.; Karck, M.; Szabó, C. Nitric Oxide 2011, 25, 201−210. (12) Liu, F.; Chen, D.-D.; Sun, X.; Xie, H.-H.; Yuan, H.; Jia, W.; Chen, A. F. Diabetes 2014, 63, 1763−1778. (13) Lee, Z. W.; Teo, X. Y.; Tay, E. W.; Tan, C. H.; Hagen, T.; Moore, P.; Deng, L. W. Br. J. Pharmacol. 2014, 171, 4322−4336. (14) Tamizhselvi, R.; Koh, Y.-H.; Sun, J.; Zhang, H.; Bhatia, M. Exp. Cell Res. 2010, 316, 1625−1636. (15) Hu, L. F.; Lu, M.; Tiong, C. X.; Dawe, G. S.; Hu, G.; Bian, J. S. Aging Cell 2010, 9, 135−146. (16) Zhang, M.; Deng, M.; Ma, J.; Wang, X. Chem. Pharm. Bull. 2014, 62, 505−507. (17) Doeller, J. E.; Isbell, T. S.; Benavides, G.; Koenitzer, J.; Patel, H.; Patel, R. P.; Lancaster, J. R.; Darley-Usmar, V. M.; Kraus, D. W. Anal. Biochem. 2005, 341, 40−51. (18) Bailey, T. S.; Pluth, M. D. J. Am. Chem. Soc. 2013, 135, 16697− 16704. (19) Cao, J.; Lopez, R.; Thacker, J. M.; Moon, J. Y.; Jiang, C.; Morris, S. N.; Bauer, J. H.; Tao, P.; Mason, R. P.; Lippert, A. R. Chem. Sci. 2015, 6, 1979−1985. (20) Lippert, A. R.; New, E. J.; Chang, C. J. J. Am. Chem. Soc. 2011, 133, 10078−10080. (21) Liu, C.; Pan, J.; Li, S.; Zhao, Y.; Wu, L. Y.; Berkman, C. E.; Whorton, A. R.; Xian, M. Angew. Chem., Int. Ed. 2011, 50, 10327− 10329. (22) Peng, H.; Cheng, Y.; Dai, C.; King, A. L.; Predmore, B. L.; Lefer, D. J.; Wang, B. Angew. Chem., Int. Ed. 2011, 50, 9672−9675. (23) Qian, Y.; Karpus, J.; Kabil, O.; Zhang, S. Y.; Zhu, H. L.; Banerjee, R.; Zhao, J.; He, C. Nat. Commun. 2011, 2, 495. (24) Sasakura, K.; Hanaoka, K.; Shibuya, N.; Mikami, Y.; Kimura, Y.; Komatsu, T.; Ueno, T.; Terai, T.; Kimura, H.; Nagano, T. J. Am. Chem. Soc. 2011, 133, 18003−18005. (25) Li, W.; Sun, W.; Yu, X.; Du, L.; Li, M. J. Fluoresc. 2013, 23, 181− 186. (26) Yu, F.; Han, X.; Chen, L. Chem. Commun. 2014, 50, 12234− 12249. (27) Badr, C. E.; Tannous, B. A. Trends Biotechnol. 2011, 29, 624− 633. (28) McMillin, D. W.; Delmore, J.; Weisberg, E.; Negri, J. M.; Geer, D. C.; Klippel, S.; Mitsiades, N.; Schlossman, R. L.; Munshi, N. C.; Kung, A. L.; Griffin, J. D.; Richardson, P. G.; Anderson, K. C.; Mitsiades, C. S. Nat. Med. 2010, 16, 483−489. (29) Badr, C. E.; Niers, J. M.; Morse, D.; Koelen, J. A.; Vandertop, P.; Noske, D.; Wurdinger, T.; Zalloua, P. A.; Tannous, B. A. Gene Ther. 2011, 18, 445−451. (30) Carlon, M.; Toelen, J.; Van der Perren, A.; Vandenberghe, L. H.; Reumers, V.; Sbragia, L.; Gijsbers, R.; Baekelandt, V.; Himmelreich, U.; Wilson, J. M.; Deprest, J.; Debyser, Z. Mol. Ther. 2010, 18, 2130− 2138. (31) Wu, W.; Li, J.; Chen, L.; Ma, Z.; Zhang, W.; Liu, Z.; Cheng, Y.; Du, L.; Li, M. Anal. Chem. 2014, 86, 9800−9806. (32) Li, J.; Chen, L.; Wu, W.; Zhang, W.; Ma, Z.; Cheng, Y.; Du, L.; Li, M. Anal. Chem. 2014, 86, 2747−2751. (33) Van de Bittner, G. C.; Dubikovskaya, E. a.; Bertozzi, C. R.; Chang, C. J. Proc. Natl. Acad. Sci. U. S. A. 2010, 107, 21316−21321. (34) Mofford, D. M.; Adams, S. T., Jr.; Reddy, G. S.; Reddy, G. R.; Miller, S. C. J. Am. Chem. Soc. 2015, 137, 8684−8687. (35) Porterfield, W. B.; Jones, K. A.; McCutcheon, D. C.; Prescher, J. A. J. Am. Chem. Soc. 2015, 137, 8656−8659. (36) Ke, B.; Wu, W.; Wei, L.; Wu, F.; Chen, G.; He, G.; Li, M. Anal. Chem. 2015, 87, 9110−9113. (37) Li, J.; Chen, L.; Du, L.; Li, M. Chem. Soc. Rev. 2013, 42, 662− 676.
In conclusion, we well described the design, synthesis, optical properties, and preliminary biological application of H2S bioluminescent probes. On the basis of the interaction of azido group with H2S, probe 1 exhibited a robust “turn-on” response to the analyte with high sensitivity and selectivity. Furthermore, H2S-dependent responsiveness of bioluminescent probe 1 was successfully applied for cell- and animal-based imaging, thus promising a marked improvement over other fluorescence imaging as well as providing an opportunity to understand the complicated contributions of H2S to a variety of physiological and pathological processes.
■
ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.analchem.5b03636. Full experimental procedure and NMR, MS, fluorescence, and cell imaging data (PDF)
■
AUTHOR INFORMATION
Corresponding Authors
*Phone/fax: +86-28-8518-8632. E-mail: bowenke80@hotmail. com. *Phone/fax: +86-531-8838-2076. E-mail:
[email protected]. Author Contributions §
B.K. and W.W. contributed equally. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We are grateful for financial support from the National Natural Science Foundation of China (Grant 21402130), the National Program on Key Basic Research Project (Grant 2013CB734000), the Program of New Century Excellent Talents in University (Grant NCET-11-0306), the Shandong Natural Science Foundation (Grant JQ201019), and the Fundamental Research Funds of Shandong University (Grant 2014JC008).
■
REFERENCES
(1) Abe, K.; Kimura, H. J. Neurosci. 1996, 16, 1066−1071. (2) Fiorucci, S.; Distrutti, E.; Cirino, G.; Wallace, J. L. Gastroenterology 2006, 131, 259−271. (3) Li, L.; Moore, P. K. Trends Pharmacol. Sci. 2008, 29, 84−90. (4) Szabo, C. Nat. Rev. Drug Discovery 2007, 6, 917−935. (5) Kimura, H. Amino Acids 2011, 41, 113−121. (6) Ariyaratnam, P.; Loubani, M.; Morice, A. H. Microvasc. Res. 2013, 90, 135−137. (7) Zhang, L.-J.; Tao, B.-B.; Wang, M.-J.; Jin, H.-M.; Zhu, Y.-C.; Yang, C. PLoS One 2012, 7, e44590. (8) Yang, W.; Yang, G.; Jia, X.; Wu, L.; Wang, R. J. Physiol. 2005, 569, 519−531. (9) Kamoun, P.; Belardinelli, M. C.; Chabli, A.; Lallouchi, K.; Chadefaux-Vekemans, B. Am. J. Med. Genet. 2003, 116A, 310−311. (10) Li, L.; Bhatia, M.; Zhu, Y. Z.; Zhu, Y. C.; Ramnath, R. D.; Wang, Z. J.; Anuar, F. B. M.; Whiteman, M.; Salto-Tellez, M.; Moore, P. K. FASEB J. 2005, 19, 1196−1198. 595
DOI: 10.1021/acs.analchem.5b03636 Anal. Chem. 2016, 88, 592−595
