2015 Beckman Symposium   

Loveprit Singh

Presentation Date:
August-8-2014

Presenter:
Loveprit Singh

Title:
23 - The Development of Novel Fluorescent Hydrogen Sulfide Detectors and Donors

Institution:
University of Oregon

Department:
Chemistry

Description:
In recent years, our view on hydrogen sulfide (H2S) has evolved from that of a toxic, odiferous gas to an important biomolecule that participates in a variety of functions within the human body. Biocompatible tools have been developed with the goal of further illuminating the physiological roles of H2S. My research focuses on contributing to this array of biological tools with the development of novel fluorescent H2S detectors and donors. Although the general biological roles of H2S are known, the concentration-dependent effects of this gaseous molecule on cellular mechanisms remain challenging to determine. This difficulty is due, in part, to the lack of accurate quantification techniques that are selective for H2S over cellular thiols and are compatible with living systems. One solution to this problem is by the use of ratiometric fluorescent probes to report on relative concentrations of H2S and biological thiols, such as cysteine, within a living system. We proposed that the probe NBD-Naph, the coupled product of NBD-Cl to a naphthalimide fluorophore, would be able to differentiate H2S and cysteine and give their relative concentrations. Upon reaction with H2S, NBD-Naph produces only one fluorescent product, Naph-OH. Conversely, reaction of NBD-Naph with cysteine produces two fluorescent products, NBD-Cys and Naph-OH. Using the distinct emission intensities of the products allows for ratiometric quantification of H2S and cysteine within a given system. Unfortunately, the reaction products Naph-OH and NBD-Cys have similar spectral characteristics and are not easily distinguishable via absorbance or fluorescence spectroscopy, thus rendering NBD-Naph unsuitable as a ratiometric fluorescent probe. Ongoing investigations with other dual-fluorophore combinations, however, have shown that NBD-Coumarin is a viable ratiometric probe and produces easily identifiable fluorescent products upon reaction with H2S and cysteine. Researchers studying the physiological effects of H2S typically use the sodium salt NaSH as their H2S source for experiments. However, NaSH delivers SH-, the biologically dominant form of H2S, extremely rapidly, contrasting the slow physiological production of the gas. Highlighting this problem, biological studies using NaSH often produce contradictory results from otherwise equivalent studies using slower H2S-releasing donors, which suggests that NaSH is not a reliable method for H2S delivery in a biological context. A fluorescent slow-releasing H2S donor would allow for a traceable H2S source which could illuminate the biological effects of H2S over a concentration range. A common moiety used for the slow release of H2S is 5-(4-hydroxyphenyl)-3H-1,2-dithiole-3-thione (ADT-OH), which models the biological release of H2S more accurately than NaSH. We have coupled ADT-OH to NBD-Cl to produce the fluorescent H2S donor NBD-ADT, which demonstrated a 6.7 fold fluorescence turn-on in 10% FBS/PIPES buffer over the course of an hour. However, we determined that NBD-ADT fragments upon reaction with thiols, and this result has led us to investigate other fluorophores that could be appended to ADT-OH to form a donor that is more chemically resistant to biological nucleophiles. Currently we are screening possible fluorophores and conducting experiments with model compounds.


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