DOI:
10.1039/C5RA21041K
(Communication)
RSC Adv., 2015,
5, 106156-106160
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†
Received
11th October 2015
, Accepted 8th December 2015
First published on 10th December 2015
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.
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 (SNi).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. λex = 519 nm, λem = 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 SNi reaction. However, the rhodol moiety is an electron rich ring, which we postulate will decrease the kinetics of the SNi 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. λex = 519 nm, λem = 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 SNi 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. λex = 519 nm, λem = 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. λex = 519 nm, λem = 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. λex = 519 nm, λem = 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 SNi 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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