Synthesis and Hydrogen Sulfide Releasing Properties of Diaminodisulfides and Dialkoxydisulfides

Heterosubstituted disulfides are an understudied class of molecules that have been used in biological studies, but they have not been investigated for their ability to release hydrogen sulfide (H2S). The synthesis of two sets of chemicals with the diaminodisulfide (NSSN) and dialkoxydisulfide (OSSO) functional groups was reported. These chemicals were synthesized from commercially available sulfur monochloride or a simple disulfur transfer reagent. Both the diaminodisulfide and dialkoxydisulfide functional groups were found to have rapid rates of H2S release in the presence of excess thiol. The release of H2S was complete with 10 min, and the only byproducts were conversion of the thiols into disulfides and the amines or alcohols originally used in the synthesis of the diaminodisulfide or dialkoxydisulfide functional groups. These results will allow the design of H2S releasing chemicals that also release natural, biocompatible alcohols or amines. Chemicals with the diaminodisulfide and dialkoxydisulfide functional groups may find applications in medicine where a controlled, burst release of H2S is needed.


cis-1,2,3,4,5,6-Hexahydrophthalimide:
We followed a procedure previously described in the literature. 1 Pd/C (10% w/w) (0.51 g, cat.) was added to a solution of cis-1,2,3,6-Tetrahydrophthalimide (25.03 g, 165 mmol) in CH 2 Cl 2 (200 mL) in a Parr reactor equipped with a stir bar. The Parr reactor was assembled and charged with hydrogen gas (1000 psi) and allowed to stir at room temperature for 6 days. The reaction was filtered and the filtrate evaporated to yield a white crystalline solid (23.65 g, 94%). 1  Diethanolamine disulfide (0.27 g, 1 mmol) was dissolved in MeOH (20 mL). 2-Mercaptoethanol (1.17 g, 15 mmol) was added in one portion and allowed to stir for 24 h. Three products were observed by 1 H NMR spectroscopy, and they were isolated by silica gel column chromatography using 5% MeOH in DCM. Diethanolamine and excess 2-mercapto ethanol were identified by known 1 H NMR and 13 C NMR chemical shift values. The third isolated product bis(2-hydroxyethyl)disulfide was additionally confirmed by HRMS. Individual data for each isolated product given below.

Degradation studies of diethoxy disulfide (10) and 2-mercapto ethanol:
Diethoxy disulfide (0.15 g, 1 mmol) was dissolved in MeOH (30 mL). 2-Mercapto ethanol (1.17 g, 15 mmol) was added in one portion with MeOH (20 mL). The reaction was stirred at room temperature. Aliquots were extracted at various time points and solvent removed under reduced pressure. 1 H NMR spectra were taken at these time points (24 h, 48 h, 72 h). The NMR spectra was compared to the data from the previously isolated 2-mercaptoethoxy disulfide to confirm products. These products were not isolated due to ethanol's volatility. Individual data for each observed product is given below. Stability studies of bis(triethylene glycol monomethyl ether) disulfide (12) and diethanolamine disulfide (16) in phosphate buffered solutions (PBS) (pH = 5.5, 6.7, 7.4) Compounds 10 or 12 (0.05 mmol) were added to a vial and dissolved in 20 mL of phosphate buffer solution (0.1 M, pH = 5.5, 6.7, or 7.4). At 24 hours an aliquot (0.50 mL) was extracted and water was removed under a stream of N 2 . 1,3-Dinitrobenzene (0.0167 g, 0.05 mmol) was added to the NMR tube as an internal standard for 1 H NMR spectroscopy analysis. The percent degradation was determined by tracking the α-hydrogens to the disulfide with the internal standard. A stock solution of diethanolamine disulfide (27.2 mg, 0.1 mmol) in water (10 mL) was prepared. A portion of the diethanolamine disulfide stock solution (0.30 mL) was diluted in 0.1 M phosphate buffer (75 mL, 40 µM, pH= 6.7) in a 100 mL jar equipped with a stir bar. The concentration of total sulfide (H 2 S + HS -) was recorded for 1h while stirring, and then a stock solution of L-cysteine (0.56 mL of an 80 mM stock solution) was added to yield a final concentration of L-cysteine of 0.60 mM (15 eq. to diethanolamine disulfide). The concentration of total sulfide (H 2 S + HS -) was recorded for an additional 17 h ( Figure S1). Figure S1. The total sulfide release of 16 and 12 (40 μM) in phosphate buffer (0.1 M, pH= 6.7) is shown. Cysteine (6 mM, 15 equiv.) was added after 1 h as indicated by the red dot.

Stability studies of diethoxy disulfide (10) and bis(triethylene glycol monomethyl ether) disulfide (12) (Table S2):
Compounds 10 or 12 (0.1 mmol) were added to a vial and dissolved in 0.5 mL CDCl 3 . 1,3-Dinitrobenzene (0.0167 g, 0.1 mmol) was added to the NMR tube as an internal standard for 1 H NMR analysis. If an additive was used, 1 equivalent (0.1 mmol) was added to the experiment immediately before the initial NMR. 1 H NMR spectra were taken at 2 h and 24 h to track degradation by comparison of the α-hydrogens to the disulfide with the internal standard.

Stability studies of diethanolamine disulfide (16) (Table S3):
Diethanolamine disulfide (0.0272 g, 0.1 mmol) was added to a vial and dissolved in 0.5 mL d 6 -DMSO. 1,3-Dinitrobenzene (0.0167 g, 0.1 mmol) was added to the NMR tube as an internal standard for NMR analysis. If an additive was used, 1 equivalent (0.1 mmol) was added to the experiment S-5 immediately before the initial NMR spectrum. 1 H NMR spectra were taken at 2 h and 24 h to track degradation by comparison of the α-hydrogens to the disulfide with the internal standard.   Figure S2. The proposed degradation mechanisms in the presence of a thiol (2-mercaptoethanol) of 16 is shown.
S-8 Figure S4. General reaction scheme for HRMS studies.  Table S4. HRMS calculated and found values for starting materials used in all reactions. Values reported for observed cation pairings (Na + , K + , DEA-H). Table S5. HRMS calculated and found values for observed intermediates for experiment with 2 equiv. NAC. Values reported for observed cation pairings (Na + , K + , DEA-H). Full HRMS data below (Figure S6-S10) Table S6. HRMS calculated and found values for observed intermediates for experiment with 5 equiv. NAC. Values reported for observed cation pairings (Na + , K + , DEA-H). Full HRMS data below (Figure S11-S15)   Figure S21. Synthetic scheme, 1 H NMR spectrum and 13 C NMR spectrum of 12.