P55 Selective turn-on fluorescent probes for detection of hydrogen sulfide in living cells

Abstract

Background Hydrogen sulfide (H2S) is an important gasotransmitter that mediates a wide range of physiological effects. Although H2S is known for its toxicity and rotten egg smell, it has recently been established to play a role in neurological degradation and is correlated with central nervous system diseases, including Alzheimer’s disease, Down syndrome, and multiple sclerosis. H2S is produced endogenously by three enzymes: cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfur transferase (3MST). Although its biological genesis is well understood, the lack of techniques and methods for selectively monitoring H2S has led to a poor understanding of its multifaceted roles in biology. The development and use of selective fluorescent probes offers a technique to monitor endogenous small molecules. Our interest in developing chemical tools to study the biological roles of H2S has led us to synthesize two new compounds for H2S detection based on the 4-amino-1,8-naphthalimide fluorophore. Methods Hydrogen sulfide can be used to reduce nitro (RNO2) and azido (RN3) functional groups to the parent amine. Based on these chemical properties, we sought to design H2S-selective fluorescent probes in which H2S selectively reduces a nitro (HSN1) or azide (HSN2) analog of the parent naphthalimide probe. In addition to testing the response to H2S, the developed probes were tested under anaerobic conditions with various reactive species including cysteine, glutathione, α-lipoic acid, nitric oxide, hydrogen peroxide, sulfite, and thiosulfate. Results Treatment of a 5 μM solution of HSN1 or HSN2 in aqueous buffer (50 mM PIPES, 100 mM KCl, pH 7.4) with 100 equivalents of H2S resulted in a 15 and 60-fold fluorescence turn-on, respectively. Reduction of a nitro or azido analog of the parent naphthalimide probe generates the parent fluorescent amine, resulting in a large fluorescence turn-on. The developed fluorescent probes are able to selectively detect H2S in the presence of reactive sulfur, oxygen, nitrogen, species (RSONs) including cysteine, glutathione, α-lipoic acid, nitric oxide, hydrogen peroxide, sulfite, and thiosulfate. Furthermore, HSN2 remains highly selective for H2S even in the presence of 2000 equivalents (10 mM) of cysteine or glutathione. HSN1 and HSN2 were also used to detect exogenous H2S in live HeLa cells. After incubating HeLa cells with 5 μM HSN1 or HSN2, addition of 250 μM H2S resulted in a significant fluorescence enhancement by comparison to the control cells. Summary HSN1 and HSN2 are highly-selective fluorescent probes for H2S. Reactions of these probes with H2S resulted in a 15–60-fold fluorescence turn-on and were demonstrated to be viable H2S-detection methods in live HeLa cells.