Self-reporting polymeric hydrogen sulfide donors and their bioactivity

2015 Fiscal Year Research-status Report

• Project/Area Number: 15K01286
• Research Institution: Osaka University
• Principal Investigator: ファンデルフリース アンドレ 大阪大学, 工学(系)研究科(研究院), 研究員 (80648593)
• Project Period (FY): 2015-04-01 – 2017-03-31
• Keywords: Hydrogen sulfide (H2S) / H2S donor / self-reporting / fluorescence

Outline of Annual Research Achievements

After several experiments the intended compound was isolated during the last year. The compound consists of a phenyl ring with two CH(SCOCH3)2 groups in ortho position relative to each other and its structure was confirmed by NMR and HRMS measurements. The compound was able to release H2S in the presence of glutathione as detected in the gas phase by lead sulfide. More important it could be shown that in presence of glutathione, the major thiol present inside the cell a time-dependent increase in fluorescence was measured showing that the proposed concept is working. To show that this fluorescence is due to the proposed adduct formed by reaction of glutathione with the formed dialdehyde after H2S release the fluorescence emission spectrum was measured and found to be the same as that measured for the product obtained after reacting glutathione with the dialdehyde directly. Furthermore the isomer having the CH(SCOCH3)2 groups in para position did release H2S but no increase in fluorescence was detected after reaction with glutathione. This results support the proposed reaction mechanism. Cell experiment in mouse macrophages showed both compounds not to be toxic up to 100 M. Unfortunately confocal laser scanning electron microcopy was not able to detect fluorescence because of auto-fluorescence of the cells.

Current Status of Research Progress

3: Progress in research has been slightly delayed.

Reason

The fluorescence characteristics of the synthesized self-reporting donor were not compatible with the confocal settings used to detect fluorescence in live cells. Furthermore in model experiments the observed fluorescence intensity was much less than that can be expected from the model reaction between the dialdehyde and glutathione.

Strategy for Future Research Activity

The next effort is to introduce functional groups onto the ring and to shift the fluorescence emission spectra to longer wavelengths to remove the interference of the auto-fluorescence of the cells. To understand what is the reason for the lower fluorescence intensity model reactions that will be analyzed by HPLC.At this point it may be necessary to redesign the self-reporting H2S donor.

Causes of Carryover

At the beginning it was anticipated that more money would be spent on chemicals for synthesis and fluorescence detection dyes.

Expenditure Plan for Carryover Budget

To further confirm H2S release from the donors in cells fluorescent detection dyes for H2S will be purchased. Since the final goal is to prepare polymeric self-re[porting H2S donors polymers will be purchased along with the reagents to modify them.