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. Author manuscript; available in PMC: 2013 May 16.
Published in final edited form as: Chem Commun (Camb). 2012 Apr 4;48(39):4767–4769. doi: 10.1039/c2cc30730h

Selective turn-on fluorescent probes for imaging hydrogen sulfide in living cells

Leticia A Montoya 1, Michael D Pluth 1,*
PMCID: PMC3340910  NIHMSID: NIHMS369603  PMID: 22473176

Abstract

Hydrogen sulfide (H2S) is an important biological messenger but few biologically-compatible methods are available for its detection. Here we report two bright fluorescent probes that are selective for H2S over cysteine, glutathione and other reactive sulfur, nitrogen, and oxygen species. Both probes are demonstrated to detect H2S in live cells.


Hydrogen sulfide (H2S)1 has emerged as the most recent biosynthetic gasotransmitter along with nitric oxide (NO) and carbon monoxide (CO).27 Although historically known for its toxicity and characteristic rotten egg smell, H2S has gained substantial interest as an established signaling molecule. Despite this interest, few methods for its detection offer the spatial or temporal resolution required for typical cell-based or live tissue experiments. Only recently have fluorescence-based methods emerged for the study of H2S.817 Based on our interest in developing chemical tools to study the biological roles of H2S, we have developed two new turn-on fluorescent probes for H2S that can be used to image H2Sin live cells.

Hydrogen sulfide is biosynthesized endogenously by three enzymes: cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfur transferase (3MST).1821 Much like how NO production in different tissues results from different isoforms of nitric oxide synthase (NOS),2224 H2S genesis in different tissues is thought to differ depending on which enzymes are present. Unlike NO and CO, H2Sishighly water soluble, with an aqueous solubility of approximately 90 mM at 37 °C.25 Despite this heightened solubility, biological concentrations of H2S vary by approximately five orders of magnitude although exact H2S concentrations remain controversial.6

Hydrogen sulfide has potent cardioprotective and neuroprotective effects and is an active neuromodulator.18,2629 High endogenous levels of neuronal H2S suggest that H2S has an important physiological function in the central nervous system (CNS). Moreover, H2S is thought to be involved in long-term potentiation (LTP), calcium homeostasis, suppression of oxidative stress, and modulation of neurotransmission.27 In humans, H2S is involved in blood pressure potentiation and abnormal concentrations have been correlated with CNS diseases such as Down syndrome, Alzheimer’s disease, and other diseases of mental deficiency.21,3034 Notably, much higher concentrations of exogenous H2S are typically required to elicit the same physiological response as endogenous H2S, suggesting that local concentrations of H2S may be higher than those measured in mean bulk tissue experiment,35 thus underscoring the need for selective probes for intracellular H2S detection.

When coupled with fluorescence microscopy techniques, small-molecule fluorescent probes offer high spatiotemporal resolution and are important tools used to study biological signaling pathways.3638 In our design of fluorescent probes able to detect H2S, the amino-naphthalimide fluorophore platform was particularly attractive due to its ease of synthetic modification at both the amide and 4-position of the dye, ease of fluorescence modulation by functionalization of the amine moiety, and large Stokes shift. Furthermore, naphthalimide dyes, along with other members of the Lucifer Yellow family, have been used for a variety of fluorescence and colorimetric sensing applications.39,40 Based on previous reports of using H2StoreduceeitherRNO2 or RN3 to the parent amine moieties,4144 we sought to exploit this chemistry for H2S sensing. By masking a fluorogenic amine as a nitro or azido group, mild reduction with H2S regenerates the parent amine and results in fluorescence turn-on.

Both developed probes were prepared as outlined in Scheme 1. Treatment of naphthalic anhydride 1 with 2-methoxyethylamine in ethanol afforded Hydrosulfide Naphthalimide-1 (HSN1) in good yield (Scheme 1). Prior to reaction with H2S, HSN1 is weakly fluorescent (λmax = 356 nm, ε = 13100 ± 300 M−1 cm−1, Φ = 0.0020 ± 0.0005). Reaction of naphthalic anhydride 2 with 2-methoxyethylamine in ethanol afforded substituted naphthalimide bromide 3. Subsequent azide incorporation using NaN3 in NMP at elevated temperature resulted in formation of Hydrosulfide Naphthalimide-2 (HSN2) (Scheme 1). HSN2 is also weakly fluorescent in its unreacted state (λmax = 376 nm, ε = 13 500 ± 200 M−1 cm−1, Φ = 0.0020 ± 0.0005). Upon treatment with H2S, both probes are converted efficiently to fluorescent amine 4max = 432 nm, ε =11700 ± 300 M−1 cm−1, λem =542nm, Φ =0.096 0.001). Product 4 was also prepared independently from ±4-amino naphthalic anhydride and does not show any photobleaching during the typical timecourse of an experiment (see SI).

Scheme 1.

Scheme 1

Synthesis of HSN1 and HSN2. Both probes are converted to fluorescent amine 4 upon treatment with H2S.

Treatment of a 5 μM solution of HSN1 or HSN2 in aqueous buffer (50 mM PIPES, 100 mM KCl, pH 7.4) with100 equiv. of H2S resulted in a marked increase in fluorescence. For HSN1, a 15-fold turn-on was observed after 90 min,45 and for HSN2 a 60-fold turn-on was observed after 45 min (Fig. 1).46 The detection limits of HSN1 and HSN2 for H2S after 60 min incubation are 5–10 μM and 1–5 μM, respectively. Although not instantaneous, the rate of fluorescence turn-on is comparable to that of commonly-used nitric oxide fluorescent probes based on o-diamine scaffolds, which have been used for many biological studies of NO.47 The large change in fluorescence of HSN2 upon exposure to H2S should allow for fluorescence detection of H2S in biological samples even if reaction with the probe has not proceeded to completion.

Fig. 1.

Fig. 1

Fluorescence turn-on of (a) HSN1 and (b) HSN2 (5 μMprobe, 50 mM PIPES, 100 mM KCl, pH 7.4, 37 °C, λex = 435 nm.) after treatment with 100 equiv. of H2S. The insets show the time dependence of the fluorescence enhancement.

Based on the large fluorescence turn-on observed with H2S, the selectivity of the developed probes for H2S over other reactive sulfur, oxygen, and nitrogen species (RSONs) including cysteine, glutathione, α-lipoic acid (ALA), nitric oxide (NO), hydrogen peroxide (H2O2), sulfite (SO32−), and thiosulfate (S2O32−) was examined. In these experiments, 100 equiv. of each RSON was added to the probe and the integrated fluorescence response was monitored over time (Fig. 2). In addition, because cysteine and glutathione (GSH) are often found in millimolar concentrations in biological milieu, these analytes were also tested at 10 mM (2 000 equiv.). HSN1 shows good selectivity for H2S over other RSONs, but addition of 2 000 equiv. of cysteine or GSH eroded much of the selectivity. By contrast, HSN2 showed both greater fluorescence turn-on and increased selectivity for H2S over other RSONs by comparison to HSN1. Most importantly, the selectivity for H2S over cysteine and GSH was maintained even when 2 000 equiv. were added. Furthermore, we demonstrated that the fluorescence response of HSN2 for H2S is maintained in the presence of cysteine or GSH (see SI).

Fig. 2.

Fig. 2

Selectivity of (a) HSN1 and (b) HSN2 with other reactive oxygen, nitrogen, and sulfur species. (5 μM probe, 100 equiv. of RSONS, 50 mM PIPES, 100 mM KCl, pH 7.4, 37 °C, λex = 435 nm, λscan = 450–750 nm). Data were acquired before analyte addition and 1, 5, 10, 15, 30, 45, 60, 75, and 90 min after analyte addition for HSN1. Time points at 75 and 90 min are omitted for HSN2 due to the faster reactivity. Comparisons of 2,000 equiv. (10 mM) of cysteine and GSH are shown at right. The data shown are the average of at least three independent runs.

Having demonstrated the selectivity of HSN1 and HSN2 for H2S, we next tested the ability of the developed probes to be used to visualize H2S in live cells. HeLa cells were incubated with 5 μM HSN1 or HSN2 for 30 min and then treated with either buffer or buffer containing 250 μMH2S; a concentration of H2S comparable with physiological H2S levels. After further incubation for 30 min, the cells were imaged. As shown in Fig. 3, addition of H2S to the cells resulted in distinct change in the observed fluorescence, thus demonstrating the ability of the developed probes for use as tools to visualize H2S in live cells. Although both probes are able to effectively image H2S in live cells, HSN2 provides a more robust intensity change consistent with the selectivity and turn-on studies.

Fig. 3.

Fig. 3

Fluorescence imaging of H2S in HeLa cells incubated with 5 μM HSN1 or HSN2. Cells were incubated with HSN1 or HSN2 for 30 min, after which either buffer or 250 μMH2S was added. After further incubation for 30 min the cells were imaged. (a) HSN1; (b) HSN1 with H2S; (c) HSN2; (d) HSN2 with H2S. Top: DIC image with nuclear stain overlay. Bottom: fluorescence image. Scale bars = 25 μm.

In summary, we have reported two fluorescent probes for H2S detection that exhibit a robust response both in vitro and in live cells. The high selectivity of HSN1 and HSN2 for H2S over other reactive sulfur, oxygen, and nitrogen species highlight the utility of these probes. We are currently pursuing these and other strategies for developing selective fluorescent probes for H2S and using such probes to study the physiological roles of endogenous H2S.

Supplementary Material

Supporting Information

Acknowledgments

This work was supported by the National Institute of General Medical Sciences (R00 GM092970) and funding from the University of Oregon (UO). The NMR facilities at the UO are supported by CHE-0923589. We thank Christopher Wreden for assistance with tissue culture and microscopy techniques.

Footnotes

Electronic supplementary information (ESI) available: Experimental details, full selectivity data, NMR spectra. See DOI: 10.1039/c2cc30730h

Notes and references

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