H2S donors with optical responses

Abstract

Reactive sulfur species, including hydrogen sulfide (H₂S), are important biological mediators and play key roles in different pathophysiological conditions. Small molecules that release H₂S on demand, often referred to as "H₂S donors," constitute a key investigative tool for H₂S-related research. A significant challenge, however, is correlating the rate of H₂S release from such donors in complex systems with biological outcomes, because release rates are commonly perturbed by different biological environments. In this chapter, we outline an approach to use H₂S donors that provide a fluorescent response upon H₂S release to address this problem. These compounds leverage the intermediate release of carbonyl sulfide (COS), which is quickly converted to H₂S by the endogenous enzyme carbonic anhydrase (CA), to provide activatable donors with an optical response. The described donors are activated by biological thiols and provide a fluorescence response that correlates directly with H₂S delivery, which allows for delivered H₂S levels to be measured in real time by fluorescence techniques.

Keywords: Donors; Fluorescence; H(2)S; Hydrogen sulfide; Reactive sulfur species.

Scheme 1.

Activation and response mechanism for the sulfenyl thiocarbonate donors. Reaction with cellular thiols results in disulfide reduction and COS release, which is quickly converted to H₂S by carbonic anhydrase (CA).

Scheme 2.

Synthesis of FLD-1 and FLD-2.

Figure 1.

(a) Fluorescence response of FLD-1 (10 μM) in PBS (pH 7.4, 10 mM) containing Cys (100 μM) and CA (25 μg/mL). (b) Cys-dependent (0 – 200 μM) fluorescence turn on of FLD-1 (10 μM) in PBS. General fluorescence acquisition parameters: λ_ex = 490 nm, λ_em = 500 – 650 nm. The data shown is the average of three replicates and errors are shown as mean ± SD (n = 3).

Figure 2.

Fluorescence turn on of FLD-1 (grey) and FLD-3 (blue). General conditions: 10 μM FLD in PBS (pH 7.4, 10 mM) with Cys (100 μM). λ_ex = 490 nm for FLD-1, λ_ex = 454 nm for FLD-3, λ_em = 500 – 650 nm. The data shown is the average of three replicates and errors are shown as mean ± SD (n = 3).

Figure 3.

Fluorescence response (red) and H₂S release (blue) after treatment of FLD-3 (10 μM) in PBS (pH 7.4, 10 mM) with Cys (100 μM) and CA (25 μg/mL). No H₂S was detected in the absence of CA (black). λ_ex = 454 nm, λ_em = 500 – 650 nm. (b) Correlation between quantified H₂S released and fluorescence response. The data shown is the average of three replicates and errors are shown as mean ± SD (n = 3).

Figure 4.

H₂S delivery from FLD-1 in HeLa cells. HeLa cells were treated with the H₂S-responsive probe C7-Az (50 μM) in DMEM only (top row) or DMEM containing FLD-1 (50 μM) (bottom row) for 30 min. Cells were then washed with PBS and cell images were taken in PBS using a fluorescent microscope. Bar scale: 50 μm

Figure 5.

Effects of FLD-1 on LPS-induced NO₂^– accumulation. RAW 264.7 cells were pretreated with FLD-1 (0 – 25 μM) for 2 h, followed by a 24-h treatment of LPS (0.5 μg/mL). Results are expressed as mean ± SD (n = 4). *** p < 0.001 vs the control group; ## p < 0.01 vs vehicle-treated group; ### p < 0.001 vs vehicle-treated group.