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 is an important biological signalling molecule and an important environmental target for detection. A major challenge in developing H2S detection methods is separating the often similar reactivity of thiols and other nucleophiles from H2S. To address this need, the nucleophilic aromatic substitution (SNAr) reaction of H2S with electron-poor aromatic electrophiles was developed as a strategy to separate H2S and thiol reactivity. Treatment of aqueous solutions of nitrobenzofurazan (7-nitro-1,2,3-benzoxadiazole, NBD) thioethers with H2S resulted in thiol extrusion and formation of nitrobenzofurazan thiol (λmax = 534 nm). This reactivity allows for unwanted thioether products to be converted to the desired nitrobenzofurazan thiol upon reaction with H2S. The scope of the reaction was investigated using a Hammett linear free energy relationship study, and the determined ρ = +0.34 is consistent with the proposed SN2Ar reaction mechanism. The efficacy of the developed probes was demonstrated in buffer and in serum with associated sub-micromolar detection limits as low as 190 nM (buffer) and 380 nM (serum). Furthermore, the sigmoidal response of nitrobenzofurazan electrophiles with H2S can be fit to accurately quantify H2S. The developed detection strategy offers a manifold for H2S detection that we foresee being applied in various future applications.
Hydrogen
sulfide (H2S) is a critical gaseous signaling
molecule emerging at the center of a rich field of chemical and biological
research. As our understanding of the complexity of physiological
H2S in signaling pathways evolves, advanced chemical and
technological investigative tools are required to make sense of this
interconnectivity. Toward this goal, we have developed an azide-functionalized O-methylrhodol fluorophore, MeRho-Az, which
exhibits a rapid >1000-fold fluorescence response when treated
with
H2S, is selective for H2S over other biological
analytes, and has a detection limit of 86 nM. Additionally, the MeRho-Az scaffold is less susceptible to photoactivation than
other commonly used azide-based systems, increasing its potential
application in imaging experiments. To demonstrate the efficacy of
this probe for H2S detection, we demonstrate the ability
of MeRho-Az to detect differences in H2S levels
in C6 cells and those treated with AOAA, a common inhibitor of enzymatic
H2S synthesis. Expanding the use of MeRho-Az to complex and heterogeneous biological settings, we used MeRho-Az in combination with light sheet fluorescence microscopy
(LSFM) to visualize H2S in the intestinal tract of live
zebrafish. This application provides the first demonstration of analyte-responsive
3D imaging with LSFM, highlighting the utility of combining new probes
and live imaging methods for investigating chemical signaling in complex
multicellular systems.
Hydrogen sulfide (H2S) is an essential biological signaling molecule in diverse biological regulatory pathways. To provide new chemical tools for H2S imaging, we report here a fluorescent H2S detection platform (HSN2-BG) that is compatible with subcellular localization SNAP-tag fusion protein methodologies and use appropriate fusion protein constructs to demonstrate mitochondrial and lysosomal localization. We also demonstrate the eӽcacy of this detection platform to image endogenous H2S in Chinese hamster ovary (CHO) cells and use the developed constructs to report on the subcellular H2S distributions provided by common H2S donor molecules AP39, ADT–OH, GYY4137, and diallyltrisulfide (DATS). The developed constructs provide a platform poised to provide new insights into the subcellular distribution of common H2S donors and a useful tool for investigating H2S biochemistry.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.