Synthesis of Sulfur-35-Labeled Trisulfides and GYY-4137 as Donors of Radioactive Hydrogen Sulfide

Hydrogen sulfide has emerged as a key gasotransmitter in humans and in plants, and the addition of exogenous hydrogen sulfide has many beneficial effects in vivo and in vitro. A challenge in investigating the effect of exogenous hydrogen sulfide is tracking the location of exogenous hydrogen sulfide on an organism and cellular level. In this article, we report the synthesis of three key chemicals (cysteine trisulfide, glutathione trisulfide, and GYY-4137) that release radiolabeled 35S as hydrogen sulfide. The synthesis started with the reduction of Na235SO4 mixed with Na2SO4 to generate hydrogen sulfide gas that was trapped with aq NaOH to yield radiolabeled Na2S. The Na2S was converted in one step to GYY-4137 at 65% yield. It was also converted to bis(tributyltin) sulfide that readily reacted with N-bromophthalimide to yield a monosulfur transfer reagent. Trisulfides were synthesized by reaction with the monosulfur transfer reagent and the corresponding thiols. The levels of radioactivity of the final products could be varied on a per gram basis to alter the radioactivity for applications that require different loadings of hydrogen sulfide donors.

To the trapped sulfide solution, 1.35 g of Na2S • 9H2O was added. In a separate flask, 6.00 g of tributyltin chloride was dissolved in 60 mL of THF and added to the flask containing sulfide. The tributyltin chloride flask was washed with 10 mL of THF and 22 mL of water and added to the reaction flask. The solution was heated to 65 °C and stirred for 16 h. THF was removed under reduced pressure and the aqueous solution was washed 3 times with 20 mL of Et2O. The organic layers were combined, washed with 15 mL of brine and dried over MgSO4. The solvent was removed under reduced pressure to give bis(tributyltin) sulfide as a colorless oil in a 67% yield. Bis(tributyltin) sulfide was used in the next step without further purification.
To the bis(tributyltin) sulfide, 2.80 g of phthalimide dissolved in 25 mL of DMF was added in one portion. Immediately following, 4.25 g of N-bromophthalimide in 20 mL of DMF was added dropwise over 5 min. The solution was stirred for 48 h. The reaction was filtered and washed with 50 mL of toluene yielding MSTR as a white solid in a 46% yield.
The synthesized MSTR (0.427 g) was suspended in 4.7 mL of isopropanol. In a separate flask, 0.412 g of L-cysteine was dissolved in 3.5 mL of H2O. Using a syringe pump, the cysteine solution was added to MSTR with a flow rate of 15 mL/h. Upon full addition of L-cysteine, the solution was stirred for an additional 14 h. The solution was filtered and the solid was washed with 30 mL of acetone followed by 30 mL of DCM, affording Cys-TriS as a white solid (625 mg) in a 73% yield.
The remaining MSTR (0.426 g) was suspended in 9.5 mL of isopropanol. In a separate flask, 0.807 g of glutathione was dissolved in 9.5 mL of H2O. Using a syringe pump, the glutathione solution was added to MSTR with a flow rate of 15 mL/h. Upon full addition of glutathione, the solution was stirred for an additional 25 min. The solution was filtered and the solid was washed with 30 mL of acetone followed by 30 mL of DCM, affording Glu-TriS as a white solid (333 mg) in a 75% yield.
Synthesis of radioactive GYY-4137. Radioactive GYY-4137 was synthesized using the optimized reactions. A 0.5 mCi solution of Na2 35 SO4 in 0.5 mL of water was added to 12 mL of degassed HI/NaH2PO2 reducing solution. An additional 0.305 g of anhydrous Na2SO4 was added. The gas was lead into two washing traps with 15 mL of MilliQ optima water, which lead to the trapping solution of 40 mL of 0.1 M NaOH in MilliQ optima water. Thereafter, it lead to 15 mL of 2 M NaOH and then 15 mL of bleach in order to trap any remaining sulfide. The solution was degassed for 10 min, and heated to 130 °C and stirred for 3 h. UV-Vis spectroscopy showed a sulfide concentration of 52.1 mM, giving a yield of 95%.
The trapped sulfide solution was added to 1.57 g of GYY-Cl in 30 mL of ethyl acetate. 0.770 g of Na2S • 9H2O and 3.15 g of tetrabutylammonium bromide were added, and the solution was stirred at 40 °C and monitored by 31 P NMR spectroscopy. The reaction was 70% complete after 6 h with the other byproduct being unreacted GYY-Cl. Additional 0.500 g of Na2S • 9H2O was added and the reaction was stirred at room temperature for an additional 16 h. The ethyl acetate layer was extracted and the aqueous layer was washed 3x with 20 mL of ethyl acetate. The organic layers were combined and dried over Na2SO4 and concentrated under reduced pressure to give the tetrabutylammonium salt of GYY-4137 as an off white solid (1.80 g) in a 65% yield. Figure S1. A schematic of the reduction of Na2SO4 to H2S is shown. Teflon tubing (g, 1/16" ID) was pierced through a rubber stopper to bubble the N2 carrier gas (3 psi) into a 25 mL 2-neck reaction flask (a) that was heated to 130 °C and contained 15 mL 57% HI, 2.0 g NaH2PO2, and 450 mg Na2SO4. One end of the reaction flask was connected to the Teflon tubing (h, 1/4" ID) and also to two 40 mL Schlenk bubblers (b and c) in series that each contained 15 mL MilliQ optima grade water. The second bubbler was connected to a 50 mL round bottom trapping flask (d) equipped with a gas bubbler adapter and that contained 40 mL of 0.1 M NaOH. Finally, the round bottom flask was connected to two 40 mL Schlenk bubblers containing 15 mL 2 M NaOH (e) and 15 mL bleach (f) using tygon tubing (h).   Figure S3. 1 H and 13 C NMR spectra of bis(tributyltin) sulfide in CDCl3, and HRMS of bis(tributyltin) sulfide. Figure S4. 1 H and 13 C NMR spectra of 2,2'-thiobis(isoindoline-1,3-dione) (MSTR) in CDCl3, and HRMS of 2,2'-thiobis(isoindoline-1,3-dione) (MSTR). S9 S10 Figure S5. 1 H and 13 C NMR spectra of cysteine trisulfide in D2O, and HRMS of cysteine trisulfide. S11 Figure S6. 1 H NMR spectrum of glutathione trisulfide in D2O, and HRMS of glutathione trisulfide. S12 S13 Figure S7. 1 H, 13 C, and 31 P NMR spectra of GYY-Cl in CDCl3, and HRMS of GYY-Cl. S14 Figure S8. 1 H, 13 C, and 31 P NMR spectra of the tetrabutylammonium salt of GYY-4137 in CDCl3, and HRMS of the tetrabutylammonium salt of GYY-4137.