An Esterase Activated Persulfide Donor with a Fluorescence Reporter

Abstract

Persulfides are found physiologically in prokaryotes, eukaryotes and mammalian tissues, which are abundant in quantities. The persulfides are formed by a process called persulfidation or S-sulfhydration, which is now recognized as the primary pathway for signal transduction. Along with the H2O2 scavenging ability, it is also known to activate or inhibit protein activity by regulating the structures and thereby the functions. Recent signalling studies the potential importance of persulfides (RSSH) was revealed in redox biology. This prompted further investigation of their chemical properties for providing a basic understanding of their probable functioning. This led us to the development of a triggerable persulfide donor. In this project, we aim to design a persulfide (RSSH) donor, which releases persulfide (RSSH) upon activation along with the release of a fluorophore, and the fluorescent signal was used for the direct quantification of the RSSH. Here, we attempted to study the release as well as the quantification of RSSH with esterase as an enzyme trigger and 3-(allyloxy)-6H-benzo[c]chromen-6-one as a fluorescence reporter. Due to synthetic challenges, we modified the ester trigger as well as the persulfide and was characterized by NMR, Mass spectrometry, and HPLC. Upon activation with esterase, the compound undergoes lactonization to form a lactone (fluorophore), and the fluorescence was exploited for the quantification of the persulfide. The fluorescence signal that is produced should correlate with the persulfide released or is an estimate of the persulfide released. Encouragingly, HPLC analysis showed that there is a 1:1 correlation between the fluorophore and the persulfide released. Although excellent correlation between the signal and yield of persulfide was seen, the yield observed was 50%. The rationale for diminished yield is not clear, but could be due to poor solubility in buffer. The fluorescence studies also showed that the fluorophore yield was less than 50%, which was not quantitative. We hypothesize that this could be due to the solubility issues of both the persulfide donor as well as the fluorophore. The outlook of the project is to increase the solubility of the persulfide donor as well the fluorophore by changing the functional groups to achieve a quantitative yield of both.