Synthesis of thiol precursors

G3MH was prepared in two step synthesis³³. A mixture of acetonitrile (5.5 mL) and water (2.5 mL) was purged with argon, and L-glutathione (750 mg, 2.44 mmol), trans-2-hexenal (300 μL, 2.56 mmol) and pyridine (393 μL, 4.88 mmol) were then added. After stirring in an inert atmosphere at room temperature for 45 h, the mixture was concentrated under reduced pressure, water (10 mL) was added to the residue and aqueous solution was washed with dichloromethane (3 × 10 mL). The organic layers were discarded. Sodium borohydride (190 mg, 7.32 mmol) in water (7 mL) was added dropwise to the aqueous layer at 0 °C. After stirring at room temperature for 3 h, the mixture was acidified with 1 M HCl to pH 2–3, concentrated under reduced pressure and dried with a vacuum pump at room temperature. The solid residue was dissolved in water (3.5 mL) and purified on a C18 semi-prep column with mobile phases of (A) water and (B) acetonitrile; the gradient was: 1–5% B from 0–20 min, 5–15% B from 20–40 min and 15–40% B from 40–60 min. Fractions from 26–35 min were evaporated to provide pure G3MH as a white solid (55 mg, 6%).

¹H NMR (500 MHz, D₂O): δ = 0.86 (m, 3 H), 1.38 (m, 2 H), 1.54 (m, 2 H), 1.72 (m, 1 H), 1.84 (m, 1 H), 2.14 (m, 2 H), 2.45–2.58 (m, 2 H), 2.78–2.88 (m, 2 H), 3.05 (dd, J = 13.5, 5 Hz, 1 H), 3.66–3.75 (m, 2 H), 3.78 (t, J = 6.5 Hz, 1 H), 3.93 (s, 2 H), 4.54 (m, 1 H) ppm (Supplementary Material: Fig. ^S4).

High resolution mass spectra (HRMS) (UHPLC/TOF-MS), [M + H]⁺ calculated for C₁₆H₂₉N₃O₇S 408.1799 Da, detected 408.1800 Da (Fig. ^S5).

Cys3MH was synthesized in a three-step reaction starting from trans-2-hexenal and Boc-protected L-cysteine^21,34 with slightly modified protocols to reduce the formation of undesirable by-products and the presence of impurities in the final product³⁵. trans-2-Hexenal was added in three portions (470 μL (4.05 mmol) at t = 0 h, 340 μL (2.93 mmol) at t = 12 h and 180 μL (1.55 mmol) at t = 24 h) to a solution of N-(tert-butoxycarbonyl)-L-cysteine (430 mg, 1.94 mmol) and triethylamine (620 μL, 4.45 mmol) in 1,4-dioxane (9 mL). After stirring in an inert atmosphere at room temperature for additional 24 h, the mixture was evaporated under reduced pressure and water (10 mL) was added to the residue. The aqueous solution was acidified with 5% HCl to approximately pH 3 and washed with dichloromethane (2 × 10 mL). The combined organic phases were washed with water (10 mL) and evaporated under reduced pressure. The oily residue was rinsed with pentane (10 × 5 mL), evaporated under reduced pressure and dissolved in phosphate buffer (Na₂HPO₄/NaH₂PO₄, 10 mL, pH 8, 1 M), to which sodium borohydride (250 mg, 6.61 mmol) in water (4 mL) was added dropwise. After stirring at room temperature for 3 h, the mixture was diluted with water (3 mL) and washed with dichloromethane (3 × 10 mL). The organic layers were discarded and the aqueous layer was acidified with 25% acetic acid to approximately pH 6 and extracted with ethyl acetate (3 × 20 mL). The combined organic phases were dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The residue was dissolved in dichloromethane (6 mL), and triethylsilane (135 μL, 0.845 mmol) and trifluoroacetic acid (856 μL, 11.19 mmol) were added successively at 0 °C. After stirring in an inert atmosphere at 0 °C for 20 min, the volatiles were evaporated under reduced pressure. The residue was dissolved in water (5 mL) and washed with ethyl acetate (3 × 20 mL). The organic layers were discarded and the aqueous layer was evaporated under reduced pressure. The oily residue was treated with diethyl ether (5 mL), evaporated again under reduced pressure and dried overnight with a vacuum pump at room temperature to give white product Cys3MH as trifluoroacetate salt (150 mg, 23%).

¹H NMR (500 MHz, D₂O): δ = 0.88 (t, J = 7.5 Hz, 3 H), 1.40 (m, 2 H), 1.57 (m, 2 H), 1.71 (m, 1 H), 1.86 (m, 1 H), 2.88 (m, 1 H), 3.00–3.18 (m, 2 H), 3.71 (m, 2 H), 4.06 (m, 1 H) ppm (Fig. ^S6).

¹⁹F NMR (470 MHz, D₂O): δ = –75.6 ppm (Fig. ^S7).

HRMS (UHPLC/TOF-MS), [M + H]⁺ calculated for C₉H₁₉NO₃S 222.1158 Da, detected 222.1149 Da (Fig. ^S8).

G4MMP was prepared in a base-catalysed reaction between glutathione and mesityl oxide as described⁶ with some modifications. Water (4 mL) was purged with argon, and L-glutathione (150 mg, 0.488 mmol), pyridine (267 μL, 3.308 mmol) and mesityl oxide (84 μL, 0.734 mmol) were then added. After stirring in an inert atmosphere at room temperature for 48 h, the mixture was diluted with water (10 mL) and washed with dichloromethane (5 × 10 mL). The organic layers were discarded and the aqueous layer was evaporated under reduced pressure. The solid residue was suspended in a mixture of water/ethanol (1 mL/13 mL). After cooling for 24 h the precipitate was filtered off to give pure G4MMP as a white solid (55 mg, 28%).

¹H NMR (300 MHz, D₂O): δ = 1.46 (s, 6 H), 2.22 (m, 2 H), 2.30 (s, 3 H), 2.59 (m, 2 H), 2.90 (s, 2 H), 3.00 (dd, J = 13.0, 8.4 Hz, 1 H), 3.15 (dd, J = 13.0, 5.4 Hz, 1 H), 3.88 (t, J = 6.3 Hz, 1 H), 4.03 (s, 2 H), 4.64 (dd, J = 8.4, 5.4 Hz, 1 H) ppm (Fig. ^S9).

HRMS (UHPLC/TOF-MS), [M + H]⁺ calculated for C₁₆H₂₇N₃O₇S 406.1642 Da, detected 406.1637 Da (Fig. S10).

For the preparation of deuterium labelled G4MMP-d ₁₀ mesityl oxide-d ₁₀ was used instead of mesityl oxide¹³. Water was purged with argon, then L-glutathione (150 mg, 0.488 mmol) and pyridine (267 μL, 3.308 mmol) were added. Mesityl oxide-d ₁₀ was added to the mixture in three portions: 50 μL (0.437 mmol) at t = 0 h, 20 μL (0.175 mmol) at t = 8 h and 15 μL (0.131 mmol) at t = 20 h. After stirring in an inert atmosphere at room temperature for an additional 24 h, the mixture was diluted with water (10 mL) and washed with dichloromethane (4 × 10 mL). The organic layers were discarded and the aqueous layer was evaporated under reduced pressure. The solid residue was suspended in a mixture of H₂O/EtOH (1 mL/13 mL). After cooling for 24 h the precipitate was filtered off to give pure G4MMP-d ₁₀ as a white solid (52 mg, 26%).

¹H NMR (500 MHz, D₂O): δ = 2.14 (m, 2 H), 2.45–2.56 (m, 2 H), 2.81 (m, 1 H), 2.93 (dd, J = 13.0, 8.5 Hz, 1 H), 3.08 (dd, J = 13.0, 5.0 Hz, 1 H), 3.79 (t, J = 6.3 Hz, 1 H), 3.93 (s, 2 H), 4.56 (dd, J = 8.5, 5.0 Hz, 1 H) ppm (Fig. ^S11).

HRMS (UHPLC/TOF-MS), [M + H]⁺ calculated for C₁₆H₁₇N₃O₇SD₁₀ 416.227 Da, detected 416,2289 Da (Fig. ^S12).

Cys4MMP was prepared in two-step synthesis according to the literature³⁴ with some modifications. Boc-Cys-OH (200 mg, 0.904 mmol) and mesityl oxide (385 μL, 3.366 mmol) were added to a solution of Na₂CO₃ (101 mg, 0.953 mmol) in water (6 mL). After stirring in an inert atmosphere at room temperature for 48 h, the mixture was washed with diethyl ether (4 × 10 mL). The organic layers were discarded and the aqueous layer was acidified with 25% acetic acid to approximately pH 5 and extracted with ethyl acetate (2 × 10 mL). The combined organic phases were dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The residue was dissolved in dichloromethane (2 mL), and triethylsilane (40 μL, 0.250 mmol) and trifluoroacetic acid (360 μL, 4.704 mmol) were added successively at 0 °C. After stirring in an inert atmosphere at 0 °C for 20 min, the volatiles were evaporated under reduced pressure. The residue was dissolved in water (5 mL) and washed with ethyl acetate (3 × 10 mL). The organic layers were discarded and the aqueous layer was evaporated under reduced pressure. The oily residue was treated with diethyl ether (10 mL), evaporated again under reduced pressure and dried overnight with a vacuum pump at room temperature to give white product Cys4MMP as trifluoroacetate salt (60 mg, 20%).

¹H NMR (500 MHz, D₂O): δ = 1.41 (s, 6 H), 2.24 (s, 3 H), 2.86 (m, 2 H), 3.09 (dd, J = 7.5, 14.0 Hz, 1 H), 3.21 (dd, J = 14.0, 4.2 Hz, 1 H), 4.11 (m, 1 H) ppm (Fig. ^S13).

¹⁹F NMR (470 MHz, D₂O): δ = –75.6 ppm (Fig. ^S14).

HRMS (UHPLC/TOF-MS), [M + H]⁺ calculated for C₉H₁₇NO₃S 220.1002 Da, detected 220.1015 Da (Fig. ^S15).

The procedure was the same as described for the synthesis of Cys4MMP except that mesityl oxide-d ₁₀ was used instead of mesityl oxide³⁴. Boc-Cys-OH (200 mg, 0.904 mmol) and mesityl oxide-d ₁₀ (385 μL, 3.366 mmol) were added to a solution of Na₂CO₃ (101 mg, 0.953 mmol) in water (6 mL). After stirring in an inert atmosphere at room temperature for 48 h, the mixture was washed with diethyl ether (4 × 10 mL). The organic layers were discarded and the aqueous layer was acidified with 25% acetic acid to approximately pH 5 and extracted with ethyl acetate (2 × 10 mL). The combined organic phases were dried over anhydrous Na₂SO₄ and evaporated under reduced pressure. The residue was dissolved in dichloromethane (2 mL), and triethylsilane (40 μL, 0.250 mmol) and trifluoroacetic acid (360 μL, 4.704 mmol) were added successively at 0 °C. After stirring in an inert atmosphere at 0 °C for 20 min, the volatiles were evaporated under reduced pressure. The residue was dissolved in water (5 mL) and washed with ethyl acetate (3 × 10 mL). The organic layers were discarded and the aqueous layer was evaporated under reduced pressure. The oily residue was treated with diethyl ether (10 mL), evaporated again under reduced pressure and dried overnight with a vacuum pump at room temperature to give white product Cys4MMP as trifluoroacetate salt (97 mg, 31%).

¹H NMR (500 MHz, D₂O): δ = 2.22 (s, 3 H), 2.84 (m, 2 H), 3.05 (dd, J = 13.5, 7.5 Hz, 1 H), 3.18 (dd, J = 13.5, 4 Hz, 1 H), 3.96 (m, 1 H) ppm (Fig. ^S16).

¹⁹F NMR (470 MHz, D₂O): δ = –75.7 ppm (Fig. ^S17).

HRMS (UHPLC/TOF-MS), [M + H]⁺ calculated for C₉H₁₁NO₃SD₆ 226.1379 Da, detected 226.1402 Da (Fig. ^S18).