Chinese Chemical Letters  2017, Vol. 28 Issue (2): 218-221   PDF    
Citation
Li-Li Zhang, Hui-Kun Zhu, Chun-Chang Zhao, Xian-Feng Gu. A near-infrared fluorescent probe for monitoring fluvastatin-stimulated endogenous H2S production[J]. Chin. Chem. Lett., 2017,28(2): 218-221  
A near-infrared fluorescent probe for monitoring fluvastatin-stimulated endogenous H2S production
Li-Li Zhanga, Hui-Kun Zhub, Chun-Chang Zhaoa, Xian-Feng Gub     
a Key Laboratory for Advanced Materials and Institute of Fine Chemicals, East China University of Science and Technology, Shanghai 200237, China;
b Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai 201203, China
Received 03 June 2016, Received in revised form 17 June 2016, Accepted Jun 29, 2016, Available online 16 July 2016
* Corresponding author. E-mail addresses: zhaocchang@ecust.edu.cn (C.-C. Zhao). E-mail addresses: xfgu@fudan.edu.cn (X.-F. Gu);
Abstract: Most reported fluorescent probes have limitations in practical applications in living systems due to the strong autofluorescence background, construction of probes with near-infrared (NIR) fluorescence emission is an accessible approach for addressing this challenge. We here designed a NIR fluorescent probe for monitoring the endogenous production of H2S in living cells. The designed probe showed significant NIR fluorescence turn-on response to H2S with high selectivity, enabling the sensitive detection H2S. Importantly, the probe could be applied in monitoring the endogenous production of H2S in raw264.7 macrophages. This study showed that fluvastatin can promote the activity of cystathionine γ-lyase (CSE) for generation H2S.
Key words: Fluorescent probe     Near-infrared fluorescence emission     Endogenous H2S detection     Fluvastatin    
1. Introduction

Endogenous hydrogen sulfide, generated enzymatically by cystathionine γ-lyase (CSE), cystathionine β-synthase (CBS), and 3-mercaptopyruvate sulfurtransferase, is an important gasotransmitter which is closely involved in a number of physiological functions and pathophysiological processes [1]. Therefore, development of valuable methods to interrogate the physiological and pathological functions of H2S is in high demand [2]. In this context, fluorescent detection with H2S-responsive fluorescent probe has been established as a promising tool due to its simplicity and high spatio-temporal resolution [3-5]. However, most of reported probes suffer from limitations imposed by their short emission wavelengths and thus retard their practical applications in living system due to the strong autofluorescence background from living tissues. To address this challenge, construction of probes with near-infrared (NIR) fluorescence emission is an accessible approach due to some advantages with NIR imaging, such as decreased autofluorescence, low light scattering, and high penetration depth [6].

Here, we developed a novel NIR probe (NIRDCM-H2S) for H2S, composed of dicyanomethylene-4H-pyran (DCM) chromophore as a NIR fluorescence reporter and a pyridine-disulfide-propionate group as the responsive site toward H2S. The H2S active trigger was specifically incorporated into DCM through esterification of a phenol conjugated into the dicyanopyran moiety through a vinyl bond. We reasoned that the cleavage of the disulfide (S-S) bond in NIRDCM-H2S by H2S could selectively liberate the phenol-based DCM (Scheme 1) . Since ester is an electron withdrawing group while phenol is a strong electron donating unit, the transformation of ester to phenol would induce large spectral shifts into NIR region via modulation of the electron-donor ability of DCM derivatives. Furthermore, NIRDCM-H2S was expected to be a promising probe in trapping endogenous H2S produced by upregulation of CSE activity upon stimulation with fluvastatin.

2. Experimental 2.1. Synthesis of NIRDCM-H2S

Synthesis of DCM-OH and PSSAcid were prepared according to our reported procedures [7, 9].

To a solution of DCM-OH (34 mg, 0.11 mmol) and PSSAcid (25 mg, 0.11 mmol) in dry CH2Cl2 were added DPTS (218 mg, 0.70 mmol) and DIPC (88 mg, 0.70 mmol). The resulting mixture was stirred for 10 h at room temperature, then CH2Cl2 was removed under vacuum, and the product was purified by silica chromatograph to afford 50 mg NIRDCM-H2S (90%). 1 H NMR (400 MHz, CDCl3): δ 8.91 (d, 1H, J = 8.0 Hz), 8.50 (d, 1H, J = 4.0 Hz), 7.78-7.54 (m, 7H), 7.46 (t, 1H, J = 8.0 Hz), 7.19 (d, 2H, J = 8.0 Hz), 7.15-7.10 (m, 1H), 6.87 (s, 1H), 6.78 (d, 1H, J = 16.0 Hz), 3.17 (t, 2H, J = 8.0 Hz), 3.07 (t, 2H, J = 8.0 Hz). 13C NMR (100 MHz, CDCl3) : δ 169.88, 159.48, 157.15, 152.77, 152.30, 152.07, 149.84, 137.64, 137.15, 134.75, 132.47, 129.07, 126.05, 125.83, 122.35, 121.01, 119.99, 118.99, 118.64, 117.80, 116.68, 115.61, 107.05, 63.13, 34.01, 33.11, 29.70, 23.47; HRMS (ESI+ ) calcd. for C28H19N3O3S2 [M + H]+: 510.0946; Found: 510.0940.

2.2. Confocal image of cells

Raw264.7 macrophage cells were cultured within a humidified atmosphere of 5/95 CO2/air incubator in Roswell Park Memorial Institute 1640 medium (RPMI-1640) supplemented with 10% fetal bovine serum (FBS) at 37 ℃. Then the cells were seeded in a glass bottom dish for 24 h, followed by loading with NIRDCM-H2S (5 μmol/L) in culture medium for 30 min at 37 ℃, washed with DHanks. For imaging exogenous H2S, Cells incubated with NIRDCMH2S (5μmol/L) for 30 min at 37 ℃, followed by the addition of 1 μmol/L NaHS for another 30 min. For imaging endogenous H2S, Cells pretreated with 2 μmol/L fluvastatin for 48 h were loaded with 5 μmol/L NIRDCM-H2S for 30 min at 37 ℃. For evaluation the inhibitory effect, Cells were firstly incubated with 2 μmol/L fluvastatin and 1 μmol/L PAG for 48 h, then treated with 5 μmol/L NIRDCM-H2S for 30 min at 37 ℃. The excitation wavelength was 488 nm, images were collected at 620-750 nm.

3. Results and discussion

NIRDCM-H2S was readily prepared in two steps commencing fromDCMby knownchemistry including Knoevenagel condensation and esterification in the presence of p-(dimethylamino)pyridinium p-toluenesulfonate (DPTS) and N, N’-diisopropylcarbodiimide (DIPC) [7]. The yield is 90% and NIRDCM-H2Swas fully identified by 1HNMR and 13C NMR and high-resolution mass spectrometry (HRMS).

The reaction of NIRDCM-H2S between H2Swas firstly monitored with fluorescence spectra. As shown in Fig. 1, when 10 μmol/L NIRDCM-H2S in CH3CN-PBS buffer (1:1, v/v, pH 7.4) was treated with NaHS as a H2S donor, the dramatic enhancement of an NIR emission falling into the range of 620-750 nm was observed. Obviously, such time-dependent NIR fluorescence increase resulted from H2S-triggered modulation of the electronic characteristics of the substituents, which is well known in controlling the optical properties of DCMderivatives [8]. According to our previous results and NMR as well as HRMS characterization [9], it can be deduced that the S-S bond in NIRDCM-H2S could be selectively cleaved by H2S, followed by an intramolecular nucleophilic attack on the carbonyl function by the strong nucleophilic SH (Scheme 1, Fig. S1 and S2 in Supporting information), inducing the liberating DCM-OH through a five-membered cyclic transition state.

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Scheme1. The synthesis of NIRDCM-H2S and the proposed reaction mechanism with H2S.

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Figure 1. Time-dependent fluorescence profiles of NIRDCM-H2S (10 μmol/L) in the presence of 100 μmol/L NaHS in CH3CN-PBS buffer (1:1, v/v, pH 7.4) . Spectra were acquired at 0, 2, 4, 6, 8, 10, 15, 20, 25, 30, 40, 45 min after addition of NaHS.λex = 450 nm.

NIRDCM-H2S was found to afford high selectivity toward H2S over other biologically relevant species (Fig. S3 in Supporting information). Only H2S led to a significant NIR emission increase, while minimal NIR fluorescence enhancement was observed upon incubation of NIRDCM-H2S with various analytes, including glutathione, cysteine, glycine, Cl- , Br- , H2O2, O2.-, ClO4-, ClO-, NO2-, NO3- , N3-, SO42-, S2O32-, S2O32-, HCO3-, HPO42-. These results indicated that the good selectivity of NIRDCM-H2S toward H2S, which facilitates the accurate detection under complex biosystem.

It was also noticed that the NIR emission response to H2S is dose dependent (Fig. 2) . There was a good linear relationship between the fluorescence intensity and H2S concentration in the 5-60 μmol/L range, resulting in a detection limit to be 2.5 μmol/L. These results inferred that NIRDCM-H2S is sufficiently sensitive for detection endogenous H2S in living systems.

Next, we explored the NIR emission characteristic of NIRDCMH2S for detection of cellular H2S (Fig. 3) . It is known that enzyme CSE is responsible to generate H2S in macrophages [10], we here applied the probe to monitor the endogenous production of H2S in raw264.7 macrophages under fluvastatin stimulation. When cells incubated with NIRDCM-H2S (5 μmol/L) at 37 ℃ for 30 min, faint cell image was observed. In contrast, bright red image was noticed when the cells loaded with the probe were further treated with NaHS for 30 min. These results indicated that NIRDCM-H2S is suitable for monitoring H2S within living cells. Then NIRDCM-H2S was utilized to underline the activation of CSE for generation ofH2S upon stimulation by fluvastatin. Macrophages cells pretreated with fluvastatin (2.0 μmol/L) for 48 h followed by incubation with NIRDCM-H2S for 30 min gave strong red fluorescence signals. However, such red cellular fluorescence in stimulated cells was greatly attenuated with DL-propargylglycine (PAG, 1 μmol/L), a commercial CSE irreversible inhibitor for inhibiting the activity of CSE. Collectively, NIRDCM-H2S has the capability for evaluation of endogenous H2S generation under drug stimulation.

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Figure 2. (a) Fluorescence spectra of NIRDCM-H2S (10 μmol/L) upon addition of various concentrations NaHS (0, 5, 10, 15, 20, 30, 40, 50, 60, 80, 100 μmol/L). (b) Plots of fluorescence intensity changes as a function of NaHS concentrations. Rt represents the fluorescence intensity of NIRDCM-H2S at 650 nmin the presence of NaHS, R0 represents the fluorescence intensity of NIRDCM-H2S at 650 nm in the absence of NaHS.

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Figure 3. Confocal microscopy images of H2S in live raw264.7 macrophages cells: (A) Cells were incubated with NIRDCM-H2S (5 μmol/L) for 30 min at 37 ℃. (B) Cells incubated with NIRDCM-H2S (5 μmol/L) for 30 min at 37 ℃, followed by the addition of 1 μmol/L NaHS for another 30 min.(C) Cells pretreated with 2 μmol/L fluvastatin for 48 h were loaded with 5 μmol/L NIRDCM-H2S for 30 min at 37 ℃. (D) Cells were firstly incubated with 2 μmol/L fluvastatin and 1 mmol/L PAG for 48 h, then treated with 5 μmol/L NIRDCM-H2S for 30 min at 37 ℃. (E) Average fluorescence intensities of images. The excitation wavelength was 488 nm, images were collected at 620-750 nm. The scale bar is 20 μm.

4. Conclusion

In summary, we developed a NIR probe for monitoring the endogenous production of H2S. This probe was composed of a NIR DCM chromophore as a reporter and pyridine-disulfidepropionate group as the responsive site. NIRDCM-H2S showed specific response to H2S accompanied by the transforming ester function to phenol unit, thereby inducing large spectral shifts into NIR region. Moreover, NIRDCM-H2S has also been successfully applied in monitoring of endogenous H2S generation in raw264.7 macrophages cells under fluvastatin stimulation.

Acknowledgment

We gratefully acknowledge the financial support by the National Natural Science Foundation of China (Nos. 21190033, 21372083, 21572039) and National 973 Program (No.2013CB733700) .

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