Construction of a turn-on probe for fast detection of H₂S in living cells based on a novel H₂S trap group with an electron rich dye

Abstract

A turn-on probe (ANR) for fast detection of H₂S is constructed based on a 2-(azidomethyl)-4-nitrobenzoate moiety as a trap group. This group is very effective for the design of H₂S probes especially with electron rich dyes. The potential biological applications of ANR were proved by employing it for fluorescence imaging of H₂S in living cells.

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Hydrogen sulfide (H₂S), known as a toxic pollutant, has been recently recognized as the third gaseous transmitter after nitric oxide and carbon monoxide.¹ Several endogenous enzymes in mammalian systems, including cystathionine β-synthase (CBS), cystathionine λ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (MPST), make a contribution to the production of H₂S.² These enzymes convert cysteine or its derivatives into H₂S in different organs and tissues, which play important roles in several pathophysiological processes, such as vasodilation, angiogenesis, regulation of cell growth, mediation of neurotransmission, inhibition of insulin signalling and regulation of inflammation.³ Recent studies have shown that the deregulation of H₂S has been correlated with the symptoms of Alzherimer's disease, Down's syndrome, diabetes, and liver cirrhosis.⁴ Obviously, accurate and real-time detection of H₂S concentrations in biological samples is highly required and would provide important information to understand the functions of H₂S.

Currently, several methods for H₂S detection have been established including colorimetric and electrochemical assays, gas chromatography, sulfide precipitation⁵ and fluorescence-based assays.⁶ Among these methods, fluorescence-based assays were useful because of their high sensitivities, non-destructive detection, and high spatiotemporal resolutions. A few fluorescent probes designed for H₂S detection in living systems have been reported since 2011.⁷ Several significant characteristic properties of H₂S, such as its dual nucleophilicity,⁸ excellent reducing property,⁹ high binding affinity towards copper ions,¹⁰ efficient thiolysis of dinitrophenyl ethers¹¹ as well as specific addition reactions toward unsaturated double bonds,¹² have been exploited for the design of fluorescent probes.

The fluorescent probes designed based on the strategy of the dual nucleophilicity of H₂S are especially attractive, which contain a potential fluorescent reporter and a H₂S trap group with two electrophilic reaction sites.⁸ Another strategy which draws our attention is by using the reducing property of H₂S and the nucleophilicity of the produced amine. A designed trap group would be triggered by the reduction of an azido moiety via H₂S, and the resulting amine would attack the adjacent electrophilic reaction site through an intramolecular nucleophilic substitution (SN_i).¹³ Han used o-(azidomethyl)benzoate¹⁴ as the probe trigger for their H₂S probe which can easily discriminate H₂S from the interfering biological thiols such as cysteine and glutathione (Scheme 1).^13a The azido moiety in the probe 7-o-2′-(azidomethyl)benzoyl-4-methylcoumarin was reduced to the amino group which then attacked the benzoate, releasing the fluorescent 7-hydroxy-4-methylcoumarin.

Scheme 1 The design of H₂S fluorescent probe based on a novel H₂S trap group with an electron rich dye.

Inspired by Han's design, we developed a fluorescent probe for discriminating detection of H₂S over thiols containing 2-(azidomethyl)-4-nitrobenzoate as the trigger (Scheme 1). This probe was synthesized from N,N-diethylrhodol, a new platform for the construction of fluorescent probes.

Firstly, a molecule named AR (Fig. 1) was synthesized following Han's design. However, when treated with 80 eq. of Na₂S in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5/95), no obvious fluorescence enhancement was observed. Some other interfering species containing sulfur were also examined. Unfortunately, the results showed that AR was not a proper probe for any of them (Fig. 1). A more extensive screening made to test AR was not promising as well (Fig. S1 and S2, ESI†).

Fig. 1 (A) Fluorescence response of AR (5 μM) upon addition of various species (80 eq.) in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5/95). (B) Bar graph and the structure of AR. (1) Blank; (2) Na₂S; (3) Cys; (4) HCys; (5) GSH; (6) NaHSO₃. λ_ex = 519 nm, λ_em = 550 nm. Slits: 5/5 nm.

Perhaps the azido moiety was reduced to an amino group by H₂S, while the resulting amine was not able to undergo the subsequent substitution reaction (Scheme 2). We assumed that compound 1 was formed during this process. An HRMS-ESI test was taken to confirm our speculation. However compound 1 was not found when AR (5 μM in CH₃OH/PBS buffer, 10 mM, pH = 7.4, 5 : 95) was treated with 80 eq. of Na₂S. Although the test failed to confirm our speculation, it proved that there neither 3 nor 4 were generated except a little AR ([M + H]⁺ calcd for C₃₂H₂₇N₄O₅⁺, 547.1976, found: 547.1901) was left in the solution (Fig. S3, ESI†).

Scheme 2 Speculation for the result of AR and ANR treated with H₂S and conformation for the result of ANR.

With this result in hand, we began to explore why molecule AR cannot act as a H₂S probe in contrast to AzMB-coumarin.^13a The reason is perhaps that Han used an electron-withdrawing coumarin group as dye, which reduces the electron density of the ester carbonyl, facilitating the SN_i reaction. However, the rhodol moiety is an electron rich ring, which we postulate will decrease the kinetics of the SN_i reaction. We believe that introducing an electron-withdrawing group in the other aryl ring would solve this problem (Scheme 3). 2-(Azidomethyl)-4-nitrobenzoyl chloride 5 was synthesized according to a modified method.¹⁴ Using this method, the probe ANR was obtained in 70% yield.

Scheme 3 Design and synthesis of probe ANR and possible explanation for the different results of probe AzMB-coumarin and AR.

When ANR (5 μM) was treated with 80 eq. of Na₂S in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5 : 95), the probe showed excellent response to H₂S. What's more, it could easily detect H₂S over biothiols and other nucleophiles (Fig. 2). As shown, the free probe ANR exhibited almost no fluorescence (fluorescence quantum yield: Φ = 0.0270, in CH₃OH/PBS buffer, 10 mM, pH = 7.4, 5/95, ESI†). When treated with Na₂S it elicited obvious fluorescence turn-on at 550 nm (fluorescence quantum yield: Φ = 0.3520, in CH₃OH/PBS buffer, 10 mM, pH = 7.4, 5/95, ESI†). Then we evaluated the effect of pH on the fluorescence of ANR which showed the probe was very stable from pH 6 to 8, even in the presence of Na₂S (Fig. S4, ESI†).

Fig. 2 (A) Fluorescence spectra of ANR (5 μM) upon addition of various species (80 eq.) in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5/95). (B) Bar graph. (1) Blank; (2) Na₂S; (3) GSH; (4) Cys; (5) HCys; (6) NaHSO₃; (7) H₂O₂; (8) Na₂S₂O₃; (9) NH₄Cl; (10) Na₂CO₃; (11) NaHCO₃; (12) KI; (13) CaCl₂; (14) NaBr; (15) CH₃COONa; (16) KF; (17) NaClO; (18) Na₂SO₄. λ_ex = 519 nm, λ_em = 550 nm. Slits: 5/5 nm.

The HRMS-ESI test proved that compound 3 ([M + H]⁺ calcd for C₂₄H₂₂NO₄⁺, 388.1543, found: 388.1547, Fig. S5, ESI†) was generated in the solution, which contributed to the fluorescence enhancement (Scheme 2).

The above results proved that ANR was a candidate for H₂S probe. The ester bond in ANR was stable enough with most of the nucleophiles. Only when the azido was reduced to an amine by H₂S, an SN_i reaction would happen and break the ester bond which released the fluorophore (Scheme 2). While the ester bond in AR was too stable to be broken even coexistent with H₂S. Considering the rhodol moiety is an electron rich ring, it is obvious that 2-(azidomethyl)-4-nitrobenzoate is a good trap for the design of H₂S probe with electron rich dyes.

Since the catabolism of H₂S is extremely fast in vivo, time-based experiments were performed to study the kinetics of ANR reacting with H₂S. As was expected, the reaction time was as short as 4 minutes to reach a satisfied fluorescent intensity (Fig. 3). When extended to 9 minutes, the fluorescent intensity only increased a minimal amount, thus we chose 4 minutes as test time.

Fig. 3 (A) Time-dependent fluorescence spectral changes of ANR (5 μM) with H₂S (80 eq. Na₂S, 400 μM) in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5/95). Time points represent 2, 4, 6, 8, 9 and 10 min. (B) Line chart. λ_ex = 519 nm, λ_em = 550 nm. Slits: 5/5 nm.

Subsequently, we examined the reactivity of ANR (5 μM) towards different concentrations of Na₂S in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5/95) at 25 °C. It turned out that the increasing of the probe's fluorescence intensity in PBS solution was linear to the concentration of Na₂S up to 600 μM, which indicated that ANR could monitor H₂S quantitatively in a wide concentration range (Fig. 4). Specifically, the detection limit of ANR was determined to be 0.4327 μM based on the 3σ/slope method (ESI†).

Fig. 4 (A) Fluorescence spectra of ANR (5 μM) upon addition of Na₂S (0–120 eq., 0–600 μM) in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5/95). Spectra were recorded after incubation with different concentrations of Na₂S for 4 min. (B) Linear fitting chart. λ_ex = 519 nm, λ_em = 550 nm. Slits: 5/5 nm.

Next, we performed competition experiments in the presence of biothiols and other interfering species (Fig. 6). ANR was still able to respond to H₂S with strong fluorescence enhancements in the coexistence of other biothiols or other interfering species. What is worthy mentioning is that 4.0 mM of Cys or GSH had little interference to ANR (Fig. 5, the blue colour bars). The above results demonstrated the high selectivity of ANR towards H₂S and its feasibility to detect H₂S in the presence of other biologically relevant biothiols and other interfering species.

Fig. 5 Fluorescence responses of ANR (5 μM, 4 min after incubation) to Na₂S and other interfering species (80 eq. for most of them except 2 samples with 800 eq.) in CH₃OH/PBS buffer (10 mM, pH = 7.4, 5/95). (1) Blank; (2) Na₂S; (3) GSH; (4) 800 eq. of GSH; (5) Cys; (6) 800 eq. of Cys; (7) HCys; (8) NaHSO₃; (9) H₂O₂; (10) Na₂S₂O₃; (11) NH₄Cl; (12) Na₂CO₃; (13) NaHCO₃; (14) KI; (15) CaCl₂; (16) NaBr; (17) CH₃COONa; (18) KF. λ_ex = 519 nm, λ_em = 550 nm. Slits: 5/5 nm.

Encouraged by the above results, we subsequently explored the potential applications of ANR in biological systems. Firstly, the cytotoxicity of ANR was evaluated using MCF-7 cells and 3T3 cells by MTT assay (Fig. S6, ESI†). Probe ANR showed almost no cytotoxicity in the 0.1–30 μM range for MCF-7 cells (cancer cells, a human breast adenocarcinoma cell line, IC₅₀, 69.6 μM) and 3T3 cells (healthy cells, a standard fibroblast cell line, IC₅₀, 91.5 μM), implying that the probe is probably suitable for bioimaging of H₂S in living cells. Considering the regulation of H₂S on cancer cells, MCF-7 cells were chosen for the biology tests. MCF-7 cells incubated with ANR (5 μM) in culture medium for 30 min at 37 °C, showed almost no fluorescence (Fig. 6C). However, if the MCF-7 cells were pretreated with ANR (5 μM) for 30 min and then incubated with Na₂S (400 μM) for 30 min, strong fluorescence was observed (Fig. 6D). This result indicated that probe ANR has the potential to visualize H₂S levels in living cells.

Fig. 6 Bright-field (A) and fluorescence image (C) of MCF-7 cells incubated with ANR (5 μM) for 30 min. Bright-field (B) and fluorescence image (D) of MCF-7 cells incubated with ANR (5 μM) for 30 min and washed with PBS three times. After replacement of the medium, cells were incubated with Na₂S (400 μM) for another 30 min.

Conclusions

In summary, a novel reaction-type fluorescent probe ANR for fast detection of H₂S in aqueous solution was developed based on a novel H₂S trap group 2-(azidomethyl)-4-nitrobenzoate and an SN_i reaction mechanism. The novel H₂S trap group is very effective for the design of H₂S fluorescent probes especially with electron rich dyes. While the other trap group 2-azidomethylbenzoate was failed to be introduced to H₂S fluorescent probes with electron rich dyes. This probe shows high selectivity and sensitivity for H₂S even in the presence of micromole amounts. Probe ANR shows a linear fluorescence intensity enhancement with a wide range of concentrations of Na₂S. Preliminary fluorescence imaging experiments in cells indicate its potential to probe H₂S in biological systems.

Acknowledgements

This work was supported by NSFC (21402064).

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra21041k

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