A fluorescent probe for rapid detection of hydrogen sulfide in blood plasma and brain tissues in mice

Abstract

We describe the design, synthesis and application of a sensitive, selective, and fast fluorescent probe for H₂S detection. A linear relationship between emission intensity and sulfide concentration was observed in biological bovine plasma systems. With this probe, we were able to estimate the sulfide concentration in mouse blood plasma and brain tissues.

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Introduction

Hydrogen sulfide (H₂S) is a toxic gas best known for its rotten egg smell. Paradoxically, our body produces it in small amounts and H₂S may play key roles in the health of the heart and other organs. H₂S was recently suggested as the third signaling gasotransmitter, along with nitric oxide (NO) and carbon monoxide (CO).¹ Altered levels of H₂S has been linked to many diseases, such as Down syndrome² and Alzheimer's diseases. Previous studies have identified a few enzymes, including cystathionine β-synthase (CBS),³ cystathionine γ-lyase (CSE),⁴ and 3-mercaptopyruvate sulfurtransferase (3MST),⁵ which produce endogenous H₂S in mammals. It was reported that mitochondrial sulfide quinone oxidoreductase (SQR) and persulfide dioxygenase (ETHE1)⁶ are involved in the consumption of H₂S. These findings highlight importance of H₂S homeostasis.

However, the levels of H₂S in biological settings has been reported to span over a broad range of concentrations in the literature.^1b,7 To monitor the fluctuation of H₂S in the body and its cellular site of action, probes with high sensitivity, selectivity, and real-time capability to measure intracellular H₂S are desirable.

Specifically, several challenges are present: (1) the chemistry needs to be bioorthogonal to native cellular processes; (2) due to the transient nature of H₂S and low concentration in most tissues,⁸ fast-responding probes are in demand to track this small molecule; (3) one major challenge of developing H₂S-selective probes is thiols (millimolar) in cells, which often interfere with the reaction of H₂S (micromolar or lower). To overcome these obstacles, several groups including us adopted different molecular frameworks to design selective fluorescence probes for H₂S.⁹ While the Chang and Wang groups took advantage of the unique reduction reaction between an azide group and H₂S to accomplish selectivity,^9a,9c,9f,9g our group and the Xian group utilized a bis-electrophilic strategy that took advantage of the double nucleophilic character of H₂S.^9b,9e Most recently, Nagano et al. reported a copper-based coordination complex in which the Cu²⁺ center can be released by binding H₂S to induce fluorescence.^9d,9h

Our recent strategy to develop H₂S probe^9e featured: (a) 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene (BODIPY) as the reporter template for its high brightness and photostability;¹⁰ (b) a benzene ring substituted by an acrylate methyl ester moiety and an aldehyde (–CHO) functional group at the ortho position as the H₂S targeting site for double nucleophilic reactions.

Though the probe is highly selective, one drawback of this probe is that the reaction time tends to be a little long. Faster response with high sensitivity is highly desirable and practical. Herein, we report a fast response fluorescent probe SFP-3 for the detection of H₂S in blood plasma, which maintains good selectivity and sensitivity. Noting the fact that the nucleophilic SH⁻ group adds faster to an electron-poor C=C double bond,¹¹ we hypothesized that, by tuning the electronics, the reaction rate could accelerate while retaining the selectivity. Therefore, we set out to prepare the BODIPY-based probe second-generation reagent SFP-3, replacing the acrylate ester with an α,β-unsaturated phenyl ketone. In this work, we report the synthesis and application of SFP-3 for the measurement of H₂S concentration in mice blood plasma and brain tissues.

Results and discussion

We synthesized the BODIPY-based probe SFP-3 (8) as shown in Fig. 1. The synthesis is quite straightforward. The final probe, SFP-3, was characterized by NMR spectroscopy and mass spectrometry (see the ESI†). We then tested the SFP-3 probe (10 μM) with Na₂S (100 μM) as an aqueous sulfide source at 25 °C in 20 mM PBS buffer (pH 7.4) (Fig. 2a and ESI, Fig. S2†). Gratifyingly, SFP-3 showed a fast, robust increase of the fluorescence intensity (>70-fold) in the emission maximum at 515 nm when excited at 500 nm (ε = 1.12 × 10⁵ M⁻¹ cm⁻¹, Φ = 0.10), which was completed within 20 min. We also isolated the H₂S addition product 8a and confirmed its molecular formula by high resolution mass spectrometry (ESI, Fig. S1†).

Fig. 1 Probe SFP-3 synthesis: (a) TBSCl (1.2 equiv.), imidazole (2 equiv.), DMF, 25 °C, 6 h, 82%; (b) n-BuLi (2 equiv.), THF, −78 °C, 1 h; then DMF (4 equiv.), −78 °C–25 °C , 2 h, 67%; (c) acetophenone (1 equiv.), MeOH, NaOH, 25 °C, 2 h, 74%; (d) TBAF (1 equiv.), THF, 25 °C, 30 min, 82%; (e) PCC (1.5 equiv.), celite, CH₂Cl₂, 25 °C, 1 h, 93%; (f) (1) 2,4-dimethylpyrrole (2 equiv.), TFA (one drop), CH₂Cl₂, 25 °C, 12 h. (2) DDQ (1.1 equiv.), CH₂Cl₂, 25 °C, 1.5 h. (3) Et₃N (5 equiv.), BF₃·Et₂O (5 equiv.), CH₂Cl₂, 25 °C, 2 h, 22%; (g) HCl, acetone, 25 °C, 2 h, 81%. TBSCl, tert-butyl(chloro)dimethylsilane; DMF, N,N-dimethylformamide; n-BuLi, n-butyllithium; TBAF, tetrabutylammonium fluoride trihydrate; PCC, pyridinium chlorochromate; Et₃N, triethylamine; TFA, trifluoroacetic acid; DDQ, 2,3-dichloro-5,6-dicyano-p-benzoquinone; THF, tetrahydrofuran.

Fig. 2 Fluorescence spectra of the SFP-3 probe (10 μM) in PBS buffer (20 mM, pH 7.0, 40% CH₃CN) at 25 °C. Excitation: 500 nm, emission: 505–600 nm. The data represents the average of three independent experiments. (a) Incubated with 100 μM Na₂S after 5, 10, 15, 20, 25, 30 min. (b) Incubated with different concentrations of Na₂S (10, 20, 30, 40, 50, 60, 80 and 100 μM) for 20 min. (c) Incubated with Na₂S and various thiols or amino acids for 20 min: 1) Na₂S (0 μM ); 2) Na₂S (50 μM ); 3) GSH (100 μM ); 4) GSH (100 μM) + Na₂S (50 μM ); 5) Cystine (100 μM ); 6) Lys (100 μM ); 7) Ala (100 μM ); 8) Cys (100 μM ). (d) Incubated with 1 μl H₂S buffer (bubbling H₂S 10 min–saturated solution) at 25 °C from 5 s–300 s.

Next, we examined the sensitivity of SFP-3 for sulfide via varying concentrations of Na₂S (10–100 μM). The fluorescent intensity increased about 20–73 fold with the addition of Na₂S (Fig. 2b and Fig. S3†). Moreover, the turn-on fluorescence response was also found to be selective for sulfide over other various biologically relevant thiols and amino acids in the PBS buffer (Fig. 2c and Fig. S4†). SFP-3 probe displayed ∼35-fold greater response toward Na₂S than cysteine, and ∼8-fold more selective for Na₂S than glutathione. Cystine, lysine, and alanine did not lead to a fluorescent turn-on response. Additionally, exposing SFP-3 to a mixture of GSH and Na₂S still yielded a significant fluorescence signal increase (Fig. 2c). SFP-3 showed good selectivity towards Na₂S in the presence of physiological concentrations (5 mM and 10 mM) of GSH (Fig. 2c insert). The direct response of SFP-3 toward H₂S was also tested. After the addition of 1 μL H₂S buffered solution (10 min H₂S bubbling), a significant fluorescence increase was observed between 5 s to 300 s after mixing, and the reaction was completed in 120 s at 25 °C (Fig. 2d). A smaller amount of H₂S can still induce significant responses, further confirming that SFP-3 is a sensitive probe for H₂S detection (Fig. S5†) under physiological conditions.

To further establish the utility of SFP-3 for the determination of sulfide in a biological sample, we evaluated SFP-3 (100 μM) in commercially available bovine plasma with Na₂S (100 μM) after 30–300 s (Fig. 3a and Fig. S6†). Importantly, the SFP-3 probe was able to retain its significant fluorescence response to sulfide in plasma, and the reaction was completed in 120 s at room temperature. Then, we treated SFP-3 with different Na₂S concentrations (20–200 μM) to obtain a linear relationship of emission intensity versus sulfide concentration (Fig. 3b and Fig. S7†). An excellent linear correlation between the added Na₂S concentrations and the fluorescence emission responses was observed in the bovine plasma system. The fluorescence intensity response in plasma was lower than the signal observed in pure PBS buffer system. Interestingly, a small emission wavelength shift was observed, which may due to the fast metabolism of sulfide in plasma. Nevertheless, the fast responses and excellent linear relationship provided a real-time quantitative detection method for sulfide in biological samples.

Fig. 3 (a) Fluorescence spectra of the SFP-3 probe (100 μM) incubated with 100 μM Na₂S after 30 s, 60 s, 90 s, 120 s, 180 s, 240 s, 300 s in bovine serum (40% CH₃CN) at 25 °C. (b) SFP-3 probe (100 μM) incubated with 0, 20, 40, 60, 80, 100, 150, 200 μM Na₂S after 3 min in bovine serum (40% CH₃CN) at 25 °C. Excitation: 500 nm, emission: 505–600 nm. The data represents the average of four independent experiments.

Finally, we applied SFP-3 to the measurement of sulfide concentration in mouse blood. Spiked Na₂S was used as internal standard in the blood and we discovered that a significant portion of sulfide was rapidly removed, leading to inconsistent data. These failed attempts confirmed the previous findings, in which sulfide rapidly disappeared in blood by binding to hemoglobin or metabolising.⁸

We next used blood plasma to determine the sulfide concentration. The mouse blood was first centrifuged, Na₂S was then spiked into the blood plasma as an internal standard. The spiked plasma samples were subsequently precipitated by acetonitrile to remove proteins.¹² To the supernatant of the spiked plasma was added SFP-3 (20 μM). The mixture was incubated in PBS buffer at 37°C for 30 min and then monitored. We found that the average sulfide concentration in mice blood plasma is ∼36.3 μM (Fig. S8†).

Inspired by the report from the group of Wang,^9c we conducted our measurements in bovine plasma and were delighted to find that the reaction was significantly faster. The reaction typically completed in 3 min. Considering the short half-life nature of sulfide in plasma,⁸ we believe that this fast-response probe could be highly useful in obtaining the accurate value of sulfide concentration in blood. To this end, we tried to directly measure the sulfide concentration in fresh plasma. Fresh mice blood plasma was added to bovine plasma and incubated directly with SFP-3 using Na₂S as the internal standard (see the ESI for details†) at 37 °C. A fluorescence signal increase was observed after 3 min. The average sulfide concentration in four mice blood plasma was 56.0 ± 2.5 μM (Table 1, 2 and Fig. S9†), very close to other reports on sulfide concentrations in blood plasma.¹³ Overall, these findings demonstrated that SFP-3 is suitable to detect sulfide in real biological samples in a rapid manner.

Table 1 Determination of H₂S concentration in different mice blood samples with different preparation

Mouse	Spiked blood^a	Spiked plasma^b	Plasma^c
a Spiked blood: plasma from whole mouse blood spiked with Na2S before centrifugation. b Spiked plasma: plasma spiked with Na2S after centrifugation and then precipitated protein to detect sulfide in PBS buffer. c Plasma: direct measurement of sulfide in fresh plasma with bovine plasma using Na2S as internal standard.
C57BL6/J	Inconsistent	36.3 μM	56.0 ± 2.5 μM

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Table 2 Measurement of H₂S concentration in mouse blood plasma and brain tissue

Mouse	Blood Plasma (μM)	Brain Tissue (μmol g^−1 protein)
1	55.4	6.7
2	57.9	8.5
3	52.6	7.7
4	58.0	5.3
Average	56.0 ± 2.5	7.1 ± 1.4

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Importantly, hydrogen sulfide is recognized as a neuromodulator as well as neuroprotectant in the brain.¹⁴ Several methods have been employed for H₂S measurement in the brain, such as methylene blue colorimetric assay, polarographic H₂S sensor,¹⁵ and gas/ion chromatography.¹⁶ H₂S concentration in the brain were reported as a wide range, from undetectable to more than 100 μM, indicating that methods to obtain accurate measurements are in high demand. We applied SFP-3 in determining the sulfide level in C57BL6/J mice brain tissues. Using a similar method to measuring blood H₂S concentration, the average sulfide concentration in four mice brains was estimated to be 7.1 ± 1.4 μmol g⁻¹ protein (Table 1 and Fig. S10†). This is the first probe that could allow parallel measurements of H₂S concentrations in both blood and brain tissues.

In blood, the levels of thiols can be low. While in brain tissues, thiol levels can be relatively high. We repeated the measurement with probe SFP-2^9e which is more selective than SFP-3 but with a slower response rate. The sulfide concentration was determined to be 5.0 ± 2.0 μmol g⁻¹ protein, confirming the results from the SFP-3 probe.

Conclusions

To conclude, we have developed a new sensitive and selective probe that shows a significant emission increase in the fast response to sulfide over the biologically relevant pH range. The excellent linear relationship between emission intensity and sulfide concentration was obtained in a bovine plasma system. This probe was successfully applied for the parallel measurement of sulfide concentration in mice blood plasma and brain tissues. This fast, sensitive and selective probe has the potential to be a useful tool for the fast and real-time detection of sulfide in more types of biological samples. Current efforts are directed at developing more specific fluorescent probes for optical imaging and mechanistic studies of H₂S metabolism in biological systems.

Acknowledgements

This work is financially supported by grants of the National Basic Research Program of China (2010CB923303 to J. Z.) and the Program for New Century Excellent Talents in University. J. Z. thanks the National Natural Science Foundation of China (20902045 and 91013009) and the Natural Science Foundation of Jiangsu Province (BK2011547) for support.

Notes and references

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Footnotes

† Electronic supplementary information (ESI) available: experimental details and supplemental data. See DOI: 10.1039/c2sc20537h

‡ These authors contributed equally to this work.

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