Enol-mediated delivery of H2Se from γ-keto selenides: mechanistic insight and evaluation

Like hydrogen sulfide (H2S), its chalcogen congener, hydrogen selenide (H2Se), is an emerging molecule of interest given its endogenous expression and purported biological activity. However, unlike H2S, detailed investigations into the chemical biology of H2Se are limited and little is known about its innate physiological functions, cellular targets, and therapeutic potential. The obscurity surrounding these fundamental questions is largely due to a lack of small molecule donors that can effectively increase the bioavailability of H2Se through their continuous liberation of the transient biomolecule under physiologically relevant conditions. Driven by this unmet demand for H2Se-releasing moieties, we report that γ-keto selenides provide a useful platform for H2Se donation via an α-deprotonation/β-elimination pathway that is highly dependent on both pH and alpha proton acidity. These attributes afforded a small library of donors with highly variable rates of release (higher alpha proton acidity = faster selenide liberation), which is accelerated under neutral to slightly basic conditions—a feature that is unique and complimentary to previously reported H2Se donors. We also demonstrate the impressive anticancer activity of γ-keto selenides in both HeLa and HCT116 cells in culture, which is likely to stimulate additional interest and research into the biological activity and anticancer effects of H2Se. Collectively, these results indicate that γ-keto selenides provide a highly versatile and effective framework for H2Se donation.


Contents
Page Number I. General S2 II. Safety and Handling S2 III. Synthesis S2 IV. Kinetic Studies S8 V. Trapping Experiments with Benzyl Bromide S10 VI. Confirmation of Se 0 Formation with Triphenylphosphine S13 VII. Formation of Selenodiglutathione VIII: Gas Trapping Experiments S14 S14 IX. Cell Culturing and Cytotoxicity Studies S15 X. References S16 XI. NMR Spectra S17 Electronic Supplementary Material (ESI) for Chemical Science. This journal is © The Royal Society of Chemistry 2022 S2

I. General
Commercial reagents were used without further purification unless stated otherwise. All glassware was oven or flame-dried and reactions were performed under an N 2 (g) atmosphere. Dichloromethane (DCM) and tetrahydrofuran (THF) were dried over a column of alumina. Flash chromatography was performed with columns of 40-63 Å silica from Silicycle (Québec City, Canada). Thin-layer chromatography (TLC) was performed on plates of EMD 250-m silica 60-F254. Preparative thin-layer chromatography (PTLC) was performed on Whatman silica gel (500-m) F254 glass plates. The term "concentrated under reduced pressure" refers to removal of solvents and other volatile materials using a rotary evaporator while maintaining the water-bath temperature below 40 °C. Residual solvent was removed from samples at high vacuum (<0.1 torr) using an Edwards RV5 pump. All NMR spectra were acquired at ambient temperature with a Bruker Ascend 400 MHz spectrometer and referenced to TMS or residual protic solvent. High resolution mass spectra were acquired using a Thermo Orbitrap LTQ XL (ESI). Adjustments in pH/pD for buffered solutions were accomplished with a Fisher Scientific Accumet AE150 pH meter. Cell viability was assessed spectrophotometrically using a BioTek SYNERGY-HTX multi-mode 96-well plate reader.

II. Safety and Handling
Hydrogen selenide is highly toxic and proper safety precautions must be in place when handling selenide salts and synthetic donors. All reactions and assays were performed in a well-ventilated fume hood and solutions containing donor we immediately quenched with silver nitrate prior to their disposal.

III. Synthesis
To a solution of sodium borohydride (40 mg, 1.1 mmol) in a 1:1 solution of degassed ethanol and DI water (20 mL) was added selenium powder (39 mg, 0.5 mmol). The mixture was then heated to 50 °C under an atmosphere of nitrogen for 30 minutes, at which point the solution turned colorless and the selenium had completely dissolved. Next, 3-chloropropiophenone (168 mg, 1.0 mmol) was added and the reaction continued to stir at 50 °C until the starting material was observed to be consumed by TLC. The reaction was then cooled to room temperature and extracted with dichloromethane (50 mL). The organic layer was isolated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The desired product was isolated by silica gel chromatography (gradient of 100% hexanes to 10% ethyl acetate in hexanes) to yield 1 as a white solid (158 mg, 91% yield). Phosphorous tribromide (475 μL, 5.0 mmol) was added dropwise to a cooled solution (using an ice bath) of 4-hydroxybutan-2-one (861 μL, 10.0 mmol) in dry dichloromethane (10 mL). After two hours, consumption of the starting material was inferred by TLC, and the mixture was directly concentrated under reduced pressure via rotary evaporation. The resultant oil was loaded onto a silica gel column and eluted (using a gradient of 100% hexanes to 25% ethyl acetate in hexanes) to afford 4-bromobutan-2-one as an oil (1.40 g, 93% yield).

S4
To a round bottom flask containing sodium borohydride (79 mg, 2.1 mmol) in a 1:1 solution of degassed ethanol and DI water (20 mL) was added selenium powder (78 mg, 1.0 mmol). The reaction mixture was heated to 50 °C under a nitrogen atmosphere for 30 minutes, at which point the solution turned colorless and the selenium had dissolved. Next, 4-bromobutan-2-one (300 mg, 2.0 mmol) was added, and the reaction was allowed to stir at 50 °C until the starting material was deemed to be consumed by TLC. The reaction was cooled to room temperature, diluted with DI water (20 mL) and extracted with dichloromethane (20 mL). The organic layer was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the resultant residue was purified by silica gel chromatography (gradient of 100% hexanes to 25% ethyl acetate in hexanes) to yield 4 as an oil (123 mg, 56% yield). To a solution of 4,4,4-trifluorobutane-1,3-diol 2 (641 mg, 4.4 mmol) in dry dichloromethane (20 mL) was added p-toluenesulfonic anhydride-freshly recrystallized from hot hexanes-(1.83 g, 5.6 mmol), 3 and pyridine (1.8 mL, 22.3 mmol), and the resultant mixture was allowed to stir at room temperature overnight. Upon confirming the consumption of starting material by TLC, the crude mixture was concentrated under reduced pressure and purified by silica gel chromatography (slow gradient of 100% hexanes to 25% ethyl acetate in hexanes) to afford 4,4,4-trifluoro-3-hydroxybutyl 4methylbenzenesulfonate as a yellow oil (1.03 g, 79%).

S5
To a solution of 4,4,4-trifluoro-3-hydroxybutyl 4-methylbenzenesulfonate (260 mg, 0.9 mmol) in dry dichloromethane (10 mL  To a round bottom flask containing sodium borohydride (11 mg, 0.3 mmol) in a 1:1 solution of degassed ethanol and DI water (10 mL) was added selenium powder (9 mg, 0.1 mmol). Once the vigorous bubbling had subsided, the mixture was heated to 50 °C under nitrogen until the reaction turned colorless and the selenium had completely dissolved. At this point, the reaction was cooled to room temperature using a water bath and acetic acid (200 L) and 4,4,4-trifluoro-3-oxobutyl 4-methylbenzenesulfonate (78 mg, 0.26 mmol in degassed ethanol (3 mL) were both quickly added. After stirring for an additional hour, the mixture was extracted with dichloromethane (20 mL) and the organic layer was dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was quickly purified via a silica gel plug (50% ethyl acetate in hexanes) and, after further evaporation under high vacuum, afforded 5 (15 mg, 45% yield). Analytically pure samples of 5 for kinetics and cell testing were obtained by preparative thin layer chromatography (1:3 hexanes:ethyl acetate) immediately prior to use. To a solution of sodium borohydride (76 mg, 2.0 mmol) in degassed DI water (5 mL) and ethanol (5 mL) was added selenium powder (80 mg, 1.0 mmol). The mixture was then heated to 50 °C under nitrogen for 30 minutes, at which point the solution turned colorless and the selenium had completely dissolved. Next, neat benzyl bromide (283 μL, 2.4 mmol) was introduced and the reaction continued to stir at 50 °C until the starting material was observed to be consumed by TLC. The reaction was then cooled to room temperature and extracted with dichloromethane (30 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The product was isolated by silica gel chromatography (gradient of 100% hexanes to 10% ethyl acetate in hexanes) to yield 6 as a yellow oil (247 mg, 95% yield). To a solution of sodium borohydride (40 mg, 1.1 mmol) in a 1:1 solution of degassed ethanol and DI water (20 mL) was added selenium powder (40 mg, 0.5 mmol). The mixture was then heated to 50 °C under nitrogen for 30 minutes, at which point the solution turned colorless and the selenium had completely dissolved. A second portion of selenium powder (40 mg, 0.5 mmol) was then delivered, and the mixture was allowed to react at 50 C for an additional 30 minutes. At this point, the reaction was cooled to room temperature using a water bath and acetic acid (60 L) and 3-chloropropiophenone (168 mg, 1.0 mmol) in degassed THF (5 mL) were both quickly added. The reaction was then left to stir at room temperature until the starting material was deemed to have been consumed by TLC. The reaction was then diluted with DI water (20 mL) and extracted with ethyl acetate (20 mL). The organic layer was isolated, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The resultant crude product was S7 purified by silica gel chromatography (gradient of 100% hexanes to 10% ethyl acetate in hexanes) to yield 9 as a yellow solid (67 mg, 32% yield). To a solution of sodium borohydride (79 mg, 2.1 mmol) in degassed DI water (5 mL) was added a portion selenium powder (83 mg, 1.05 mmol). The mixture was then heated to 50 °C under nitrogen for 30 minutes, at which point the solution turned colorless and the selenium had completely dissolved. A second portion of selenium powder (83 mg, 1.05 mmol) was then delivered, and the mixture was allowed to react at 50 C for an additional 30 minutes. Benzyl bromide (238 μL, 2.0 mmol) in THF (10 mL) was then added and the reaction was allowed to stir overnight at 50 °C. Upon cooling to room temperature, the reaction mixture was diluted with dichloromethane (20 mL) and washed with DI water (20 mL). The organic layer was isolated, dried over anhydrous sodium sulfate, and concentrated under the reduced pressure. The resultant oil was purified by silica gel chromatography (100% hexanes) to afford 10 as a yellow oil (201 mg, 59% yield).

H NMR (400 MHz
4-hydroxybutan-2-one (861 μL, 10.0 mmol) was added neat to a round bottom flask with p-toluenesulfonic acid monohydrate (48 mg, 0.25 mmol) and heated to 90 °C under argon overnight. The mixture was cooled to room temperature, diluted with diethyl ether (40 mL) and washed with deionized water (40 mL). The organic layer was isolated, dried over anhydrous sodium sulfate, and concentrated under the reduced pressure. The resultant oil was purified by silica gel chromatography (gradient of 100% hexanes to 50% diethyl ether in hexanes) to yield 4EC as an oil (169 mg, 21% yield).

IV. Kinetics Studies
Prior to the start of the experiment, deuterated buffer was prepared from a 50 mM solution of Na 2 HPO 4 in D 2 O and the respective pD values (8.5, 7.4, 6) were achieved with the addition of deuterium chloride (DCl). A standard solution of 1,4-dioxane was prepared at 31.25 mM in D 2 O. Donor stock solution (1 or 9) was prepared at 75 mM in deuterated acetonitrile (acetonitrile-d3). For each experiment, acetonitrile-d3 (350 μL) was added to a conical tube followed by the dioxane standard solution (15 μL) and donor stock solution (25 μL). Deuterated phosphate buffer (50 mM, 360 μL) was then added to bring the final concentration of 1 (or 9) to 2.5 mM and the dioxane standard to 0.625 mM. At this point, the solution was immediately transferred to an NMR tube and the cap was fastened with a small strip of parafilm to prevent evaporation. Experiments were run at ambient temperature and for 64 scans at each respective timepoint. To monitor reaction progress, the integration value for 1,4-dioxane (3.75 ppm) was normalized to 1 proton in each spectrum. Figure S1. Time-course for the conversion of ketone 1 (2.5 mM) into enone 2 at ambient temperature and in a 1:1 mixture of CD 3 CN and deuterated phosphate buffer (50 mM, pD 7.4). Reaction progress was monitored by 1 H NMR using 1,4-dioxane as an internal standard.

V. Trapping Experiments with Benzyl Bromide
Acetonitrile-d3 (321 μL) was added to a conical tube followed by a The solution was immediately transferred to an NMR tube and the cap was fastened with a small strip of parafilm to prevent evaporation. Experiments were run at ambient temperature and for 64 scans at each respective timepoint. To monitor reaction progress, the integration value for 1,4-dioxane (3.75 ppm) was normalized to 1 proton in each spectrum.

VI. Confirmation of Se 0 Formation with Triphenylphosphine
When kinetic experiments were deemed complete by 1 H NMR, solvent was decanted from the elemental selenium (Se 0 ), and the NMR tube was placed in an oven at 115 °C for approximately 30 minutes. After cooling to room temperature, a solution of triphenylphosphine in deuterated chloroform (700 L, 5 mM) was then added to the NMR tube and allowed to react for 12 hours prior to analysis by 31 P NMR (Figure 3c).
To confirm the above results, 10 mg of elemental selenium (Alfa Aesar, 325 mesh, 99.5%) was added to an NMR tube and 700 μL of triphenylphosphine solution (5 mM) in deuterated chloroform was added. The reaction was allowed to progress for 6 hours prior to analysis by 31 P NMR ( Figure S4).

VII. Formation of Selenodiglutathione
To a 0.1 M solution of ammonium bicarbonate buffer (pH 7.4, 1455 μL) was added donor 1 (15 μL of a 10 mM solution in ethanol), glutathione (27 μL of a 10 mM solution in DI water) and glutathione disulfide (3 μL of a 10 mM solution in DI water) to give a final concentration of 100 μM 1, 180 μM glutathione, and 20 μM glutathione disulfide. The reaction was warmed to 37 °C and, after reacting for 30 min, a 250 μL aliquot was removed and diluted with 750 μL of methanol with 0.1% formic acid. The resultant solution was analyzed via direct infusion mass spectrometry to observe the selenodiglutathione sodium adduct (Figure 6).

VIII: H 2 Se Gas Trapping Experiments
Adopted procedure from Newton et al. 4 Donor 1 (1.0 g) was placed in a 15 mL Falcon® tube and dissolved in a mixture of THF (1 mL), deionized water (1 mL), and 4M HCl in 1,4-dioxane (10 mL). In a second tube, iodoacetamide (186 mg, 1 mmol) was dissolved in 10 mL of ammonium bicarbonate buffer (1.0 M, pH 8.5). Both tubes were then capped with septa and wrapped with parafilm. A vent needle was inserted into the septa S15 containing the trapping solution and two 4" cannula needles were used to connect the two tubes, ensuring that the tips of both needles remained in the headspace at all times. The first tube containing donor 1 was then pressurized with argon using a disposable needle, and the reaction was heated to 37 C for three days. Aliquots of the trapping solution were monitored by mass spectrometry to confirm formation of trapped hydrogen selenide with iodoacetamide. Figure S5. High-resolution mass spectrum of volatilized hydrogen selenide gas trapped with iodoacetamide.