Development of a highly selective H₂S fluorescent probe and its application to evaluate CSE inhibitors

Abstract

In this paper, we developed a novel fluorescent probe C359 for highly selective detection of H₂S over other relevant biothiols. C359 is designed to contain a thiol-specific cleavable disulfide bond. H₂S-mediated the disulfide cleavage and subsequent intramolecular cyclization released the masked 7-hydroxyl coumarin, displaying a remarkable fluorescence enhancement. With the promising features in hand, C359 has been applied to detect the activity of CSE (one of H₂S-producing enzyme) and build up an assay for screening CSE inhibitors. We anticipated that the enzyme assay using C359 could provide a powerful methodology for screening more potent and selective enzyme inhibitors.

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Introduction

Hydrogen sulfide (H₂S) has been recognized as the third gaseous transmitter of signaling molecules in biological systems, succeeding nitric oxide (NO) and carbon monoxide (CO). Therefore, H₂S is a topic of great interest in chemistry and biology. In 1996, the role of H₂S in human neuromodulation was first reported by Abe and Kimura, which had become a research prelude for the biological signaling function of H₂S.¹ Since then, all aspects of the research on the biological functions of H₂S has been unfolded, such as cardioprotective,^2–5 neuroprotective^6–8 and gastroprotective effects,⁹ the regulation of insulin release¹⁰ and anti-inflammatory effects,¹¹ which make it known that H₂S also plays an important, probably even pivotal role in human and other biological systems.

In mammalian systems, the generation of H₂S derives from several enzymatic pathways. For example, both cystathionine γ-lyase (CSE, EC 4.4.1.1) and cystathionine β-synthase (CBS, EC4.2.1.22), two pyridoxal 5′-phosphate (PLP) dependent enzymes, are able to convert cysteine into H₂S within different organs and tissues.^12–17 Recently, it was reported that 3-mercaptopyruvate sulphurtransferase (3-MST, EC2.8.1.2) and cysteine aminotransferase (CAT cysteine lyase (CL, EC4.4.1.10)) can also catalyzed the production of H₂S.¹⁸ To understanding the biological roles of H₂S, the inhibitors of these enzymes would be valuable tools. Up to now, some inhibitors of these enzymes have been reported, such as PAG, BCA and AOAA, which are the inhibitors of CSE and CBS. Methylene blue assay is a colorimetric method for H₂S detection,¹⁹ which has been used as a common enzyme activity assay to evaluate inhibitors of H₂S-producing enzymes. However, by using this method, complicated sample processing is often required, and variable results are often yielded. To obtain more potent and selective inhibitors, new and efficient inhibition assays are needed. In recent years, fluorescence-based assays are fast emerging in the field of biological molecule detection because of their high sensitivity and convenience.²⁰ As the studies on H₂S biological functions are proceeding, more and more fluorescent probes for H₂S detection have been reported.²¹ However, most of these probes are only applied to cell imaging, and rare of them used to the research on enzyme activities, which is of great value in understanding the role of H₂S. Herein, a highly selective H₂S fluorescence probe over other thiols was designed and synthesized. Furthermore, an inhibition assay of CSE by using this fluorescent probe was developed.

Results and discussion

Design and synthesis of probe C359

CSE, a PLP-dependent enzyme, is able to catalyze the production of H₂S using cysteine as a substrate. Therefore, interference from cysteine could occur in the assay of CSE activity when using a H₂S fluorescent probe as an assay tool, due to the similar reactivity of thiol-containing compounds.²² Thus, it is highly desirable to construct a selective fluorescent probe for detecting H₂S over thiols. Recently, Xian's group reported a series of selective probes for H₂S with 2-(2-pyridinyldithio)-benzoic group.^21g These probes can be triggered by H₂S to undergo a tandem nucleophilic substitution–cyclization reaction, and then to release the fluorophores and render the fluorescence turn-on. Although these probes exhibit highly selectivity between thiols and H₂S, poor water solubility and slow reaction rate limit their biological application. In order to improve water solubility and fluorescence turn-on rate of these probes, they added surfactant CTAB. However, the need for a surfactant may also limit their application in living systems. With this consideration in mind, a selective and fast reactive disulfide containing probe (C359) was designed and synthesized. C359 was readily synthesized in two steps using the procedure shown in Scheme 1.

Scheme 1 The synthesis of C359.

7-Hydroxyl coumarin (C169) is a commercial available fluorescent dye with a broad absorption band and an emission band centered around 350 nm (ε = 1.7 × 10⁷ M⁻¹ cm⁻¹, in EtOH) and 450 nm (Φ_f = 0.09), respectively. By introducing a 3-(pyridin-2-yldisulfanyl)-propionyl group into C169, we tend to build up a highly selective fluorescent probe for H₂S over other related thiols. Notably, the introduction of this cage unit further reduced the fluorescent quantum yield to be 0.005, making C359 a promising probe with low background interference. Moreover, C359 exhibits fast reactivity with H₂S due to its good solubility in buffer system under experimental conditions and the less steric effect of 3-(pyridin-2-yldisulfanyl)-propionyl group. As shown in Scheme 2, the probe C359 could react with H₂S to afford an intermediate with a nucleophile SSH, then a spontaneous intramolecular nucleophilic reaction would be triggered as the electrophile carbonyl is present in a suitable site, to release a five membered cyclic lactone ring and the fluorescent product C169. In sharp contrast, the reaction between thiols and C359 yielded a compound with disulfide bond, and this compound could not undergo an intramolecular nucleophilic attack to release C169, which leads to a high discriminative detection of H₂S over thiols.

Scheme 2 Proposed mechanism of the reaction of C359 with H₂S and thiols.

To validate the proposed mechanism of the reaction between C359 and H₂S, ¹H NMR and HPLC analysis were further performed to confirm the production of C169 in the reaction. The partial ¹H NMR spectra of C359 in the absence and presence of H₂S, and that of C169 are shown in Fig. 1. Upon addition of H₂S to the solution of C359, new peaks at 7.9, 7.5, 6.8, 6.7 and 6.2 ppm assigned to C169 were observed, suggesting the formation of C169. In the HPLC spectra, the retention time of standard C359 and C169 are around 5.8 min and 2.3 min respectively. Upon addition of H₂S to the solution of C359, the peak around 5.8 min decreased, while a new peak with the retention time around 2.3 min appeared simultaneously (Fig. S1†). These observations confirm that treatment of C359 with H₂S led to formation of C169.

Fig. 1 Partial ¹H NMR spectra of C359, C359 + H₂S and C169.

The spectroscopic response of C359 to H₂S

Initially, we evaluated the optical properties of C359 toward H₂S in 200 mM Tris HCl buffer (pH = 7.4) by monitoring the changes in the absorption and fluorescence spectra. In the absence of H₂S, C359 displayed a strong absorption at 280 nm with a shoulder band around 310 nm. Introduction of H₂S led to buildup of a new band at 350 nm. Two well-defined isosbestic points at 305 and 275 nm were noted. The absorption band at 350 nm is the characteristic feature of C169, indicating the formation of C169 (Fig. 2a). In the fluorescence spectra, free C359 exhibited a weak emission feature centered on 450 nm. Upon gradual addition of H₂S to the solution of C359 in 200 mM Tris HCl buffer (pH = 7.4), a remarkable enhancement of fluorescence intensity at 450 nm was observed (Fig. 2b). Notably, the fluorescence intensity increased linearly with concentrations of H₂S increased from 0 to 35 μM, indicating that C359 is a promising probe for detecting H₂S at micromolar concentration level under physiological conditions. Based on the titration experiments, the detection limits were evaluated to be 5.0 × 10⁻⁸ M (Fig. S3†), which are comparable to previously reported probes.

Fig. 2 (a) Absorption spectra of C359 (5 × 10⁻⁶ M) in the presence of different concentrations of NaHS (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 equiv.) in Tris HCl buffer (200 mM, pH = 7.4). (b) Fluorescence spectra of C359 (λ_ex = 305 nm) in the presence of different concentrations of NaHS (0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 equiv.).

Interference from other related analytes was then investigated under the same condition. As shown in Fig. 3, no obvious fluorescence change was observed upon addition of 50 equiv. of F⁻, Cl⁻, Br⁻, I⁻, SO₄²⁻, SO₄²⁻, NO₃⁻, CO₃²⁻, SCN⁻, S₂O₅²⁻, N₃⁻, and NO₂⁻, as well as relevant thiols and cofactor PLP. More interestingly, only addition of H₂S introduced remarkable fluorescent enhancement. These results indicate the excellent capacity of C359 for selective detection of H₂S over the other competitive anions, thiols and enzyme cofactor.

Fig. 3 Fluorescence response of C359 (450 nm) in the absence and presence of 50 equiv. of various anions, thiols and PLP in Tris HCl buffer (200 mM, pH = 7.4). Each measuring was performed after 2 min of mixing. I₀ represents the fluorescence intensity of C359 only, and I_a represents the intensity in the presence of various anions, PLP and thiols.

The application of C359 to evaluation of CSE inhibitors

As shown in Fig. 3, no fluorescence response was observed upon the incubation of C359 with CSE/CBS substrate (Cys) or cofactor (PLP), enabling C359 a promising probe for testing activity of H₂S-producing enzymes and screening their inhibitors. To test our proposal, CSE was chosen in our enzyme inhibition assay due to the commercial availability of recombinant CSE (GST-CSE). To assess the ability of C359 for monitoring activity of CSE, treatment of CSE with C359 was performed firstly. As shown in Fig. 4, significant fluorescent signal over the background was recorded when Cys was used as a substrate. In the absence of Cys, significantly reduced fluorescence intensity was noted. The weak fluorescence response of C359 toward Cys in the absence of CSE further confirmed the role of CSE in production H₂S. These observations indicated that C359 can be employed to monitor CSE catalyzing Cys to produce H₂S. With this result in hand, we then assessed the capability of C359 for the evaluation of CSE inhibitors. Two known inhibitors, BCA and PAG, were used to test the validity of the inhibition assay. As shown in Fig. 5, two inhibition curves of BCA and PAG against CSE were obtained using C359 as a reporter. Interestingly, IC₅₀ of BCA and PAG was determined to be 16.75 μM and 40.81 μM respectively, which are comparable to the literature data by using methylene blue as a reporter (Table 1). These results indicated that C359 is an ideal fluorescent probe for monitoring enzyme activity, which further allows the buildup of a useful methodology to screen more potent and selective inhibitors of H₂S-producing enzyme.

Fig. 4 Utility of C359 as a H₂S probe and assay for CSE activity. I₀ represents the fluorescence intensity of C359 only, and I_a represents the intensity in the presence of Cys, PLP and enzyme.

Fig. 5 Inhibition curves for BCA and PAG against CSE. Data are presented as mean ± SEM; n = 3; *P < 0.05 versus control.

Table 1 IC₅₀ of CSE inhibitors

Inhibitors	IC50 (μM)	IC50^a (μM)
a Literature data (ref. 19).
BCA	16.75	14.0
PAG	40.81	40.0

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Conclusions

In conclusion, a fluorescent probe C359 was designed and synthesized, which show high selectivity for H₂S. The reaction between C359 and H₂S triggered the disulfide bond cleavage and subsequent intramolecular cyclization, releasing 7-hydroxyl coumarin and resulting in a remarkable fluorescence enhancement. Other relevant thiols introduce no observable fluorescent response. More importantly, C359 is capable of monitoring CSE activity, which further allows the buildup of an inhibition assay of CSE by using this fluorescent probe.

Experimental section

General method

All chemical reagents and solvents for synthesis were purchased from commercial suppliers and were used without further purification. Sodium hydrogen sulfide (NaSH) was purchased from Sigma. cystathionine γ-lyase (CSE), BCA and PAG were purchased from Cayman Chemical. ¹H NMR and ¹³C NMR spectra were recorded on a Bruker AV-400 spectrometer with chemical shifts reported in ppm (in DMSO-d₆) at room temperature. The analytical HPLC was performed on Waters 600E HPLC system. Mass spectra were measured on a HP 1100 LC-MS spectrometer. UV-vis absorption spectra were recorded on a Varian Cary 100 spectrophotometer. Fluorescence spectra were measured with a Varian CARY Eclipse Fluorescence spectrophotometer. Spectral-grade solvents were used for measurements of UV-vis absorption and fluorescence.

Synthesis of C359

3,3′-Dithiodipropionic acid (0.500 g, 2.38 mmol) and triethylamine (431 μl, 3.09 mmol) were dissolved in dichloromethane (40 mL). After the solution was cooled to 0 °C, oxalyl chloride (264 μl, 3.09 mmol) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour. After removed solvent under vacuum, the resulting mixture added in the stirring solution of 7-hydroxyl coumarin (771 mg, 4.76 mmol) and triethylamine (1 mL) in dichloromethane (40 mL), the reaction mixture was stirred at room temperature overnight. The solvent was removed under vacuum, and the residual solid was purified by flash chromatography (silica gel) to afford C498 2.13 g (90%). ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 8.05 (d, 2H), 7.75 (d, 2H), 7.23 (s, 1H), 7.14 (m, 2H), 6.46 (d, 2H), 5.74 (s, 1H), 3.07 (m, 8H). ¹³C NMR (100 MHz, DMSO-d₆, δ ppm): 170.5, 160.3, 154.7, 153.3, 144.5, 130.1, 119.2, 117.4, 116.3, 110.6, 34.3, 32.9. C498 (666 mg, 1.34 mmol) and 2-thiol pyridine (180 mg, 1.62 mmol) were dissolved in ethyl acetate (20 mL). After the addition of 3 drops of BF₃ ether solution, the reaction mixture was stirred at room temperature for 3 days. White solid was appear, and after filtering, filter cake was washed by cold ethyl acetate to afford 390 mg of C359 (81%). ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 8.47 (d, 1H), 8.06 (d, 2H), 7.84–7.75 (m, 3H), 7.26 (m, 2H), 7.14 (d, 1H), 6.47 (d, 1H), 3.17 (m, 2H), 3.05 (m, 2H). ¹³C NMR (100 MHz, DMSO-d₆, δ ppm): 169.9, 159.6, 158.6, 153.9, 152.5, 149.5, 143.7, 137.9, 129.3, 121.4, 119.5, 118.5, 116.7, 115.6, 109.9, 33.4, 32.6. HR-MS (ESI-TOF) (m/z): C₁₇H₁₄N₁O₄S₂ calcd, 360.0359; found, 360.0356 [M + 1]⁺.

UV and FL spectroscopic measurements

Stock solutions of probe C359 (5 × 10⁻³ M) and NaHS (1.5 × 10⁻² M) were prepared in deionized H₂O. 3 mL Tris HCl buffer (200 mM, pH = 7.4) was firstly added to a 5 mL cuvette, and then 3 μL of probe stock solutions and 0–16 μL of NaHS stock solutions were added. The resulting solution was thoroughly shaken before recording the spectra.

CSE enzyme inhibition assay using C359

The standard reaction was performed in the presence of 2 μg of CSE, 6 μM PLP, 0.5 mM of L-cysteine as substrate, and 10 μM of C359 as the probe in 100 μL Tris HCl buffer (200 mM, pH = 7.4). The concentration of inhibitors were varied from 0–5 mM, and the assays were incubated for 75 minutes before fluorescence was measured. Data are presented as mean ± SEM; n = 3; *P < 0.05 versus control.

Acknowledgements

We gratefully acknowledge the financial support by the National Science Foundation of China (no. 21001013).

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Footnote

† Electronic supplementary information (ESI) available: NMR spectra, HPLC spectra. See DOI: 10.1039/c4ra09066g

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