Rhodamine–propargylic esters for detection of mitochondrial hydrogen sulfide in living cells

Bioorganic & Medicinal Chemistry Letters 23 (2013) 5295–5299

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Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl

Rhodamine–propargylic esters for detection of mitochondrial hydrogen sulfide in living cells Xi Chen a, , Shuqi Wu a, , Jiahuai Han b, Shoufa Han a,⇑ a Department of Chemical Biology, College of Chemistry and Chemical Engineering and the Key Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen 361005, China b State Key Laboratory of Cellular Stress Biology and School of Life Sciences, Xiamen University, Xiamen 361005, China

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Article history: Received 30 May 2013 Revised 29 July 2013 Accepted 31 July 2013 Available online 9 August 2013

a b s t r a c t Flow cytometric detection of mitochondrial H2S was achieved with propargylic esters of rhodamine B which selectively react with H2S via cationic rhodamine-moiety directed thiolysis of the propargylic esters to give nonfluorescent rhodamine thio-spirolactone. Ó 2013 Elsevier Ltd. All rights reserved.

Keywords: Rhodamine Propargylic ester Hydrogen sulfide Mitochondria Bioimaging Flow cytometry

Hydrogen sulfide (H2S) is an endogenous gaseous messenger in biological systems and regulates a broad variety of biological events, for example vasodilation and inflammation.1 Generally considered to be synthesized in cytosol, H2S was recently shown to be produced in mitochondria in vascular smooth-muscle cells due to the translocation of cystathionine-b-synthase, a H2S-producing enzyme, from the cytosol into mitochondria under hypoxic conditions.2 Despite the cytotoxicity of high levels of H2S,3 physiological levels of H2S exhibited distinct effects on mitochondria ranging from sustaining energy production, depolarization, to inhibition of cellular respiration.3,4 As such, agents that could selectively report the levels of intra-mitochondrial H2S is highly desired to probe the aforementioned roles of H2S. Current reaction based probes rely on the double nucleophilicity or the reducing potential of H2S to distinguish the analyte from interfering biological thiols such as cysteine (Cys) and glutathione (GSH).5 Despite the advances in fluorescent imaging of H2S, probes suitable for mitochondrial H2S have been largely unexplored. Recently, a coumarin–cyanine diad was reported for imaging of H2S in mitochondria for the first time.6 In the investigation of Au(III) catalyzed rearrangement of propargylic esters,7 we serendipitously found that the propargylic esters of rhodamine B (RB-PEs) quickly react with H2S to give a colorless product (Scheme 1). The ⇑ Corresponding author. Tel.: +86 592 2181728.  

E-mail address: [email protected] (S. Han). These authors contribute equally to this work.

0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bmcl.2013.07.072

documented accumulation of cationic rhodamine derivatives in mitochondria8 prompted us to explore the feasibility to detect mitochondrial H2S with RB-PEs by dose-dependent ‘turn-off’ fluorescence. The red color of RB-PEs quickly disappeared upon treatment with sodium sulfide (Na2S), which is used as the donor of H2S, in dimethylformamide (DMF). Analysis of the reaction media by TLC revealed the formation of a colorless product. Mass spectrometry analysis revealed a major peak located at 459.2 (Fig. S1, Supplementary data), which is consistent with the theoretical molecular weight of the proposed product (C28H30N2O2S, MH+: 459.2) (Scheme 1). The product was further isolated by silica gel column chromatography and characterized by 1H NMR and 13C NMR (Supplementary data). The analytical data were found to be identical to that of reported rhodamine thiospirolactone,9 confirming the molecular identity of the product as described in Scheme 1. To probe the influences of propargylic moieties, three rhodamine B derivatives containing different propargylic moieties were prepared (Scheme 1) and assayed for their relative reaction rates with Na2S by monitoring the fluorescence emission as a function of time (Fig. 1). It was shown that the fluorescence of RB-PE-1, RB-PE-2 and RB-PE-3 quickly faded in DMF upon addition of Na2S whereas the methyl ester of rhodamine B (RB-ME) was largely unaffected (Fig. 1), suggesting the critical role of the propargylic moieties in sensing of H2S. In a separate experiment, the propargylic ester of benzoic acid was found to inactive to Na2S in DMF under the assay conditions, revealing the requirement of

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Scheme 1. Reaction of H2S with RB-PEs.

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reaction time (min) Figure 1. Kinetic profiles of the reactions between RB-PE-1, RB-PE-2, RB-PE-3 or RB-ME (5 lM) and Na2S (20 lM) in DMF. The reaction rates were monitored by recording the fluorescence emission@590 nm as a function of time (kex@560 nm).

the rhodamine B moiety in detection of H2S. Taken together, these results revealed the synergistic effects of the rhodamine moiety and the propargylic group in effective sensing of the analyte. Albeit the exact reaction mechanism is not clear, it is likely that the cationic rhodamine moiety could direct the anionic suifide to nucelophilic attack the carbonyl moiety of the propargylic ester to give the colorless product (Scheme 2).

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With a few exceptions,5f,6,10 tens of minutes or hours are required for the assays of many reported probes. As H2S is volatile and poised to air oxidation, the quick response of RB-PE-3 to Na2S suggests its utility for real-time studies of H2S-generating biological processes (Fig. 1). To access the detection range, various amounts of Na2S was spiked into DMF containing RB-PE-1, RB-PE-2 or RB-PE-3. The solutions were mixed and then analyzed by fluorometry. Figure 3A showed that the fluorescence emission@590 nm decreased as a function of Na2S concentrations. As expected, RB-ME failed to react with Na2S under identical conditions (Fig. S4, Supplementary data). In mitochondria, cytochrome C is half maximally inhibited by 20 lM H2S.11 The titration revealed that 0–20 lM H2S can be effectively detected by RB-PEs where subtle alterations of H2S concentrations can be discerned (Fig. 2A), suggesting that the applicability of these probes for detection of H2S in mitochondria. Biological thiols are ubiquitous within mammalian cells. For instance, cytosol contains high levels of glutathione (GSH) while mitochondria contain abundant cysteine (Cys).12 It is essential that RB-PEs are immune to these thiols. Hence, the reactivity of these probes was investigated towards representative biological thiols. Figure 2B showed that all the RB-PEs could efficiently detected Na2S whereas no obvious changes on fluorescence emission were observed for RB-PEs cultured with GSH, Cys, or homocysteine (HCY), suggesting their stringent selectivity for H2S over these biological thiols.

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Scheme 2. Possible sensing mechanism of RB-PEs for H2S.

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X. Chen et al. / Bioorg. Med. Chem. Lett. 23 (2013) 5295–5299

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Wavelength(nm) Figure 2. Sensitivity and selectivity of RB-PEs for H2S. (A) Fluorescence emission spectra of RB-PEs (5 lM) in DMF containing Na2S (0, 2, 4, 6, 8, 10, 20 lM, from top to bottom); the inserts showed the titration curves plotted by fluorescence emission intensity@590 nm versus analyte concentrations (kex@560 nm); (B) selectivity of RB-PEs for Na2S over selected endogenous biological thiols. The fluorescence emission@590 nm of the DMF solutions of RB-PEs (5 lM) containing GSH (100 lM), Cys (100 lM), HCY (100 lM), Na2S (10 lM), or no addition was recorded using kex@560 nm and normalized.

Cationic dyes, for example rhodamine 123, effectively accumulate in mitochondria owing to the negative transmembrane potential of mitochondria.8 To probe the capability of RB-PEs to target mitochondria, HeLa cells were respectively incubated with RB-PE-1, RB-PE-2, RB-PE-3 and RB-ME in DMEM spiked with rhodamine-123 which is a mitochondria specific dye. Confocal fluorescence images revealed colocalization of rhodamine-123 fluorescence and that of RB-PEs (Fig. 3A), demonstrating the

regio-selective accumulation of RB-PEs in mitochondria in living cells. We then evaluated the efficacy of RB-PEs to sense mitochondrial H2S. Cells pre-stained with RB-PEs were separately cultured in DMEM supplemented with 0–1 mM Na2S for 30 min. The cells were rinsed and then visualized by confocal fluorescence microscopy. As shown in Figure 3B, cells treated with Na2S exhibited dramatic decreases in fluorescence emission intensity. In contrast, no fluores-

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Figure 3. Detection of mitochondrial H2S with RB-PEs in living cells. (A) Images of HeLa cells co-stained with rhodamine 123 (1 lg ml 1) and RB-PEs/RB-AE (1 lg ml 1); (B) images of HeLa cells that were stained with RB-PEs/RB-ME and then treated with or without Na2S (1 mM); cells were visualized by fluorescence confocal microscopy. The fluorescence of rhodamine 123 was shown in green and that of rhodamine derivatives was shown in red. Merged images revealed colocalization of rhodamine 123 with RBPEs/RB-AE where yellow color was observed. (C) Quantitation of mitochondrial H2S by flow cytometry. HeLa cells stained with RB-PEs or RB-AE (1 lM) were incubated in PBS with Na2S (0, 0.5, 1 mM) and then analyzed flow cytometry.

cence change was observed on RB-ME loaded cells upon treatment with Na2S, further confirming the dependence of the propargylic moieties in the reaction based detection of mitochondrial H2S. The overall intra-mitochondrial fluorescence intensity of HeLa cells treated with or without Na2S were quantitated by flow cytometry. Figure 3C showed that the fluorescence emission of cells pre-stained with RB-PEs decreased markedly as a function of H2S concentrations whereas that of cells satined with RB-ME remained constant. Collectively, these data established the efficacy of RB-PEs for fluorescence detection of mitochondrial H2S in living cells. In summary, a novel reaction based fluorescent detection of mitochondrial H2S was achieved with propargylic esters of rhodamine B (RB-PEs) which selectively react with the analyte to form nonfluorescent rhodamien B thio-spirolactone. The dose-dependent ‘turn-off’ fluorescence of RB-PEs enabled facile quantitaion of H2S levels in mitochondria by flow cytometry. As rhodamines are routinely utilized for bioimaging of mitochondria in cell biology,8 RBPEs are compatible with the existing instruments and protocols and would be of utility in the studies of H2S metabolism, for example biogenesis of H2S in mitochondria under hypoxia conditions. Acknowledgments Dr. S.H. was supported by Grants from NSF China 21272196, 21072162, 973 program 2013CB933901, the Fundamental

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