Fluorescent probes for sensing and imaging biological hydrogen sulfide

Abstract

Hydrogen sulfide (H₂S) has long been recognized as a toxic molecule in biological systems. However, emerging studies now link controlled fluxes of this reactive sulfur species to cellular regulation and signaling events akin to other small molecule messengers, such as nitric oxide, hydrogen peroxide, and carbon monoxide. Progress in the development of fluorescent small-molecule indicators with high selectivity for hydrogen sulfide offers a promising approach for studying its production, trafficking, and downstream physiological and/or pathological effects.

Introduction

For centuries, hydrogen sulfide (H₂S) has been viewed primarily as a noxious chemical species that is naturally produced by geological and microbial activities [1]. Exposure to this colorless, flammable gas, which gives rotten eggs their distinctive odor, can trigger eye and respiratory tract irritation [2]. Inhalation of excess H₂S can result in loss of consciousness, respiratory failure, cardiac arrest, and, in extreme cases, death [3]. On the other hand, more recent studies have challenged this traditional view of H₂S as a toxin and have shown that mammals can also produce H₂S in a controlled fashion [4], suggesting that this reactive sulfur species is important in maintaining normal physiology [5]. H₂S may arise from non-enzymatic processes, including release from sulfur stores and metabolism of polysulfides [6,7]. In mammalian systems, H₂S may also be produced by two pyridoxal-5′-phosphate (PLP)-dependent enzymes, cystathionine gamma lyase (CSE) and cystathionine beta synthase (CBS), as well as cysteine aminotransferase and mercaptopyruvate sulfurtransferase (CAT/MST). These enzymes catalyze an assortment of reactions that produce H₂S from sulfur-containing biomolecules such as cysteine and homocysteine (Fig. 1a) [8,9]. The presence of these enzymes in human tissues ranging from the heart and vasculature [10,11], brain [12,13,14], kidney [15], liver [16], lungs [17,18], and pancreas [19] presages widespread physiological roles for H₂S in the body (Fig. 1b). Moreover, a variety of disease phenotypes have been linked to inadequate levels of H₂S, including Alzheimer’s disease [20], impaired cognitive ability in CBS-deficient patients [21], and hypertension in CSE knockout mice [22]; excessive H₂S production in vital organs may be responsible for the pathogenesis of other diseases such as diabetes [23,24,25]. These seminal studies have patently established H₂S as an essential physiological mediator and cellular signaling species [26,27], but our understanding of H₂S chemistry and its far-ranging contributions to physiology and pathology is still in its infancy.

Figure 1.

Biology of H₂S in the human body. (a) Selected major biochemical pathways for H₂S production. Two PLP-dependent enzymes, cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), are found in the cytoplasm and synthesize H₂S. Biochemical studies show that CBS primarily catalyzes the formation of cystathionine from homocysteine and cysteine, whereas CSE facilitates the α,β-elimination of cysteine by water, producing serine and H₂S. Two additional enzymes located in the cytoplasm and mitochondria, cysteine aminotransferase (CAT) and mercaptopyruvate sulfur transferase (MST), have also been identified as sources of H₂S. CAT acts upon keto acids and cysteine to yield mercaptopyruvate, from which H₂S is released by MST. (b) Selected physiological effects and biological roles of H₂S in the human body and enzymes responsible for H₂S production in various tissue types.

The complex biological roles of H₂S and potential therapeutic implications provide compelling motivation for devising new ways to monitor its production, trafficking, and consumption in living cells, tissues, and whole organisms. Traditional methods for H₂S detection [28], including colorimetric assays [29,30], polarographic sensors [31], and gas chromatography [32,33], typically result in sample destruction and/or are limited to extracellular detection. As such, fluorescent molecular probes offer an appealing approach for the detection of H₂S and other reactive small molecules because of their cell permeability and high sensitivity. A key challenge for selective detection of H₂S within the cellular milieu is the comparatively high concentrations of biological sulfur species such as glutathione as well as cysteine residues. In this review we provide a brief survey of recent chemical strategies for fluorescence detection and imaging of H₂S in biological systems (Table 1) [34].

Table 1.

Summary of fluorescent H₂S probes.

Azide-based	λex (nm)	λem (nm)	Detection limit	Response in vitro
SF1	490	525	5–10 μM	7-fold turn on after 60 min at 25 °Ca (10 μM probe, 100 μM NaHS, 20 mM HEPES, pH 7.4)
SF2	492	525	5–10 μM	9-fold turn-on after 60 min at 25 °Ca (10 μM probe, 100 μM NaHS, 20 mM HEPES, pH 7.4)
DNS-Az	340	535	1 μM	40-fold turn-on after 3 min (30 μM probe, 30 μM NaHS, 20 mM PBS, 0.5% Tween-20, pH 7.5)
Cy-N3	625	710 (–N3) 750 (–NH2)	0.08 μM	F750nm/F710nm = 0.6 before reaction to 2.0 after complete conversion to amine after 20 min at 37 °C (10 μM probe, 100 μM NaHS, 40 mM HEPES, pH 7.4)
HSN2	432	542	1–5 μM	60-fold turn-on after 45 min at 37 °C (5 μM probe, 500 μM NaHS, 50 mM PIPES, 100 mM KCl, pH 7.4)
cpGFP- Tyr66pAzF	480	510	10 μM	1.7-fold turn-on after 15 min at RT (5 μM probe with 50 μM NaHS, PBS, pH = 7.4)
FS1	363	548	5–10 μM	21-fold TPEF enhancement after 120 min at 37 °C (5 μM probe, 100 μM Na2S, 20 mM HEPES, 100 mM KCl, pH 7.2)

H2S trapping	λex (nm)	λem (nm)	Detection limit	Response in vitro
SFP1	300	388	--	>10-fold turn-on after 60 min at 37 °C (10 μM probe, 50 μM Na2S, 9:1 10 mM PBS:CH3CN, pH 7.4)
SFP2	465	510	5 μM	>13-fold turn-on after 60 min at 37 °Ca (5 μM probe, 50 μM Na2S, 20 mM PBS, pH 7.0)
Xian et al. Probe 1	465	515	1–10 μM	55–70-fold turn-on after 60 min at 25 °C (100 μM probe, 50 μM NaHS, 9:1 PBS:CH3CN, pH 7.4)
Xian et al. Probe 5	476	513	1 μM	11-fold turn-on after 30 min at 25 °C (5 μM probe, 100 μM NaHS, 10 mM PBS, pH 7.4)
Xian et al. Probe 6	476	513	1 μM	160-fold turn-on after 30 min at 25 °C (5 μM probe, 100 μM NaHS, 10 mM PBS, pH 7.4)

CuS precipitation	λex (nm)	λem (nm)	Detection limit	Response in vitro
HSip-1	491	516	10 μM	50-fold turn on immediately at 37 °C (1 μM probe, 10 μM NaHS, 30 mM HEPES, pH 7.4)
L1Cu	495	534	~5 μM	>4-fold turn-on immediately (20 μM probe, 40 μM S2−, 1:1 PBS:CH3CN v/v, pH 7.2)
L1Cu′	494	523	1.7 μM	25–30-fold turn-on after 1 min at RT (10 μM probe, 20 μM H2S, 6:4 HEPES:CH3CN v/v, pH 7.0)

^aReaction not complete at indicated time.

H₂S-mediated reduction of azides to amines

To expand our laboratory’s program in the area of redox biology, particularly on reaction-based detection methods for studying the chemistry and biology of the oxidative signaling molecule H₂O₂ via chemospecific boronate [35–45] and ketoacid [46] oxidations, we noted that the selective reduction of azides by H₂S could provide amines under mild conditions [47,48] and sought to utilize this reaction-based switch as a new approach to molecular H₂S probes. Specifically, we found that by masking a rhodamine with an azide functional group we could generate Sulfidefluor-1 (SF1) and Sulfidefluor-2 (SF2) probes that give fluorescent turn-on responses upon H₂S-mediated reduction of the aryl azide to the corresponding aniline (Fig. 2a). These probes show high selectivity for H₂S over a range of reactive sulfur species, including abundant cellular thiols such as glutathione and cysteine, as well as a host of reactive oxygen and nitrogen species. Moreover, these first-generation probes are capable of monitoring changes in H₂S levels in living cells. Exogenous addition of micromolar NaHS produces a robust fluorescent response in HEK293T cells and related mammalian cell lines (Fig. 3) [49]. We have now developed a series of next generation of H₂S probes that are capable of monitoring endogenous H₂S fluxes and are actively pursuing biological studies in a variety of models. Concomitant to our work, Wang showed that nonfluorescent DNS-Az, a dansyl fluorophore modified with a sulfonyl azide, reacts rapidly with H₂S concentrations as low as 1 μM in phosphate buffer, releasing strongly-fluorescent dansyl amide [50]. Upon ultraviolet excitation, DNS-Az can detect H₂S concentrations from 5 to 100 μM in bovine serum, presaging that this promising platform and related motifs can be applied to bioanalytical assays.

Figure 2.

Selected molecular probes for the detection of hydrogen sulfide. (a) The H₂S-mediated reduction of azides to amines has been applied to rhodamine (SF1 and SF2), dansyl (DNS-Az), cyanine (Cy-N₃), 8-naphthalimide (HSN2), protein (cpGFP-Tyr66pAzF), and fluorene (FS1) scaffolds which display a range of fluorescent responses and sensitivities. (b) Attack of H₂S at the aldehyde group and subsequent intramolecular Michael addition converts SFP-1 and SFP-2 to fluorescent dyes. Nucleophilic addition of H₂S to Xian et al. probe 1 results in displacement of 2-thiopyridine and persulfide formation; intramolecular cyclization due to nucleophilic attack of the thiol on the ester group generates benzodithiolone and releases 3-O-methoxyfluorescein. Two probes containing benzylidienemalonate and cyanoacrylate as Michael acceptors (Xian et al. probe 5 and probe 6) react to trap H₂S in a similar manner. (c) Reaction of H₂S with Cu²⁺ precipitates copper sulfide from the weakly fluorescent copper complex HSip-1, releasing Cyclen-AF which exhibits enhanced fluorescent properties. L1Cu and L1Cu′ also produce a fluorescent turn-on response following CuS precipitation.

Figure 3.

Confocal microscopy images demonstrating detection of changes in H₂S levels in living cells by SF1. (a) SF1 alone incubated in HEK293T cells. (b) Cells loaded with SF1 followed by incubation with NaHS. (c) Brightfield images of the field of cells in (b) overlaid with images of Hoescht stain at 37 °C. Scale bar represents 50 μm. Adapted with permission from the American Chemical Society. [49]

Shortly after our initial publication appeared, we were pleased to see that the concept of H₂S-mediated reduction of azides to amines was extended to other fluorophore scaffolds by several laboratories (Fig. 2a), establishing the generality of this reaction-based switch for selective H₂S detection. Han reported Cy-N₃, an azido-heptamethine cyanine dye that functions as a unique ratiometric internal charge transfer (ICT) probe for H₂S [51]. Cy-N₃ displays a 40 nm red-shift in fluorescence emission wavelength upon azide reduction. With a detection limit of 80 nM H₂S, Cy-N₃ can respond to changes in H₂S levels in RAW264.7 macrophages. To the best of our knowledge, this probe is the first ratiometric fluorescent indicator for H₂S. Pluth reported a 4-azido-1,8-naphthalimide probe, Hydrosulfide Naphthalimide-2 (HSN2), along with a nitro analog, which can react selectively with H₂S to give a turn-on response and can operate in living cells with good signal-to-noise ratios [52]. Recently, Ai and coworkers developed the first genetically-encoded H₂S probe by substituting p-azidophenylalanine (pAzF) for Tyr66 in circularly-permuted green fluorescent protein (cpGFP) [53]. Conversion of the azide to the amine in cpGFP-Tyr66pAzF facilitates formation of the mature chromophore and elicits a fluorescent turn-on response. Finally, Cho and colleagues prepared the first two-photon H₂S probe, featuring an azide-modified benzothiazole-fluorene that was used to image H₂S in rat hippocampal slices at depths of 90–190 μm [54]. Taken together, this selection of probes illustrates the versatility of the azide functionality as an H₂S trigger, and further elaborations of this strategy will allow modulation of emission colors, targetability, imaging modalities, and probe reactivity.

Trapping of H₂S via nucleophilic addition

In parallel to the work on azide reduction strategies, several other reaction-based H₂S probes detect the analyte by acting as electrophiles. Initial nucleophilic addition of H₂S to the probe results in a thiol which performs a subsequent nucleophilic addition to a second electrophilic site and leads to cyclization to generate a fluorescent molecule (Fig. 2b). Using this general strategy, He, Jiao, and co-workers have designed elegant probes which position an aldehyde group ortho to an αβ, -unsaturated acrylate methyl ester on an aryl ring [55]. Following nucleophilic attack of H₂S on the aldehyde functionality, the trapped thiol undergoes an intramolecular Michael addition to the unsaturated ester to produce a dihydrobenzothiophene derivative. While other thiols can reversibly add to the aldehyde, the resulting thioacetal product cannot perform a second nucleophilic addition, imparting the probe with selectivity for H₂S. Two reagents—the blue-emitting SFP-1, based on a 1,3,5-triaryl-2-pyrazoline fluorophore, and SFP-2, based on a BODIPY dye—utilize this Michael addition trigger and displays selective reaction with H₂S. SFP-2 exhibits a 13-fold increase in fluorescence intensity upon reaction with 50 μM Na₂S and demonstrates fluorescent properties suitable for imaging in living cells, with a H₂S detection range in cells from 0–200 μM.

Another clever probe by Xian’s laboratory employs a related strategy, using a disulfide and an ester group as the two electrophilic sites for reaction with H₂S [56]. H₂S displaces 2-thiopyridine, resulting in a persulfide where the terminal sulfur attacks the ester, cyclizing to form a benzodithiolone and simultaneously releasing methoxyfluorescein. The probe responds to 50–500 μM H₂S in bovine plasma and 250 μM H₂S in cells. This reagent demonstrates selectivity for H₂S over other biological thiols in vitro, because displacement of the thiopyridine by a substituted thiol results in a mixed disulfide which does not undergo cyclization. Xian and coworkers reported two additional probes that feature benzylidienemalonate or cyanoacrylate moieties as sites for Michael addition; these probes also release methoxyfluorescein upon reaction with H₂S [57].

Copper sulfide precipitation

The classic gravimetric precipitation of CuS from Cu²⁺ complexes has been successfully employed as a strategy to detect H₂S by a fluorescence response (Fig. 2c). Nagano reported H₂S imaging probe 1 (HSip-1), which consists of a cyclen macrocycle attached to fluorescein [58]. Binding of Cu²⁺ to the cyclen receptor quenches fluorescence. Upon reaction with a sulfide donor such as Na₂S or NaHS, CuS precipitates and releases unbound cyclen-AF, which displays enhanced fluorescence. HSip-1 rapidly detects sulfide concentrations as low as 10 μM in vitro, and the membrane-permeable diacetate derivative can be loaded into cells to detect 100 μM changes in Na₂S. Zeng and Bai have utilized a similar approach in the design of their L1 sulfide probe, which combines a fluorescein with a pendant 8-hydroxyquinoline ligand for copper binding [59]. Coordination of copper to L1 quenches fluorescence and produces a spectral shift when the probe is converted to the L1Cu metal complex. Addition of sulfide anion triggers precipitation of CuS and regeneration of L1, resulting in a fluorescent turn-on as well as a colorimetric change from pink to yellow in the presence of sulfide at 10–100 μM concentrations. Utilizing this strategy, Zeng and Bai prepared another copper-containing probe with a lower detection limit of 1.7 μM [60]. This L1Cu′ probe displays 25–30-fold fluorescence enhancement upon reaction with H₂S. In these reports the authors have demonstrated that HSip-1 and L1Cu undergo CuS precipitation preferentially in the presence of sulfide (S²⁻) over other sulfur-containing compounds such as sulfates, biologically-relevant thiols, and selected reactive oxygen and reactive nitrogen species. However, as alternative Cu²⁺ complexes may react with other sulfur species such as reduced glutathione [58], appropriate chelating groups must be screened and selectivity studies performed to confirm specific H₂S detection.

Conclusions and outlook

The rapid development of several first-generation molecular H₂S probes by a wide range of chemical approaches illustrates the promise for studying the roles of H₂S in intact biological systems with unprecedented spatial and temporal resolution. As the field continues to advance and a wider series of H₂S indicators become available, we can envision probes tailored for specific models of interest including cells, tissues, and whole organisms, allowing the elucidation of intricate and dynamic inter- and intracellular events involving H₂S production or depletion. Further exploration of H₂S biology in a variety of systems will undoubtedly reveal how this reactive sulfur species modulates cellular and physiological conditions, offering deeper insight into redox biology and the dynamic interplay that occurs between reductive and oxidative species in complex cellular and in vivo environments.

Highlights.

• Hydrogen sulfide (H₂S) can mediate physiology and disease pathology in mammalian systems.

• The development of molecular probes for H₂S detection allows H₂S to be studied in intact cells, tissues, or whole organisms.

• Current probes employ a variety of reaction-based strategies to achieve selectivity for H₂S over other biological thiols.