Morin and Kobert 1010.1073/pnas.0710366105 |
Fig. 5. TCEP does not cleave CTX-Mal. KCNQ1-KCNE1T14C channels are irreversibly inhibited by 10 nM CTX-Mal treatment, and they remain inhibited upon treatment with 5 mM TCEP. Current was monitored at +20 mV every 15 s.
Fig. 6. CTX-Clv electrospray mass spectrum (positive mode). Expected molecular weight for CTX-Clv: 4,933.0 (methionine oxidized); +3, 1,646; +4, 1,234; +5, 988. CTX-Clv: 5157 (methionine oxidized, 2 TFA); +3, 1,718; +4, 1,289.
SI Materials and Methods
3-Maleimido-N-[2-(phenylamino)ethyl]propanamide (2).
This compound was synthesized by dissolving 1.33 g (5 mmol) of 3-maleimidopropionic acid N-hydroxysuccinimide ester (Aldrich) in 100 ml of methylene chloride. N-phenylethyldiamine (0.72 ml, 5.5 mmol) and triethylamine (0.78 ml, 5.5 mmol) were added and the solution turned yellow and a white precipitate formed. After 4 h at room temperature, the precipitate was filtered and the solvents were removed in vacuo to afford a yellowish-orange oil. The crude material was purified by silica gel chromatography, eluting with a gradient of 20:1 to 10:1 methylene chloride/ethyl acetate to yield 0.83 g (58%). 1H NMR (400 MHz, CDCl3) d 2.53 (app t, 2H, J = 7.0), 3.24-3.27 (m, 2H), 3.47-3.51 (m, 2H), 3.84 (app t, 2H, J = 7.0), 6.61 (d, 2H, J = 8.5), 6.62 (s, 2H), 6.71 (t, 1H, J = 7.3), 7.18 (t, 2H, J = 8.5); 13C NMR (100 MHz, CDCl3) d 34.26, 34.80, 39.00, 43.64, 112.7, 117.5, 129.3, 134.1, 147.9, 170.4, 170.6; HRMS (ESI) calculated for C15H17O3N3 (M+H)+ 288.1348, found 288.1344.
Bis(chlorocarbonyl)disulfane.
This compound was synthesized by literature precedent (1, 2) with several modifications to simplify the procedure for small-scale preparations. Ethoxythiocarbonyl chloride was synthesized as described in ref. 2 and used without distillation. An equimolar amount of chlorocarbonylsulfenyl chloride (Alfa Aesar) was added to ethoxythiocarbonyl chloride and the reaction mixture was stirred overnight at room temperature. The mixture was chilled to 0°C with an ice bath, a catalytic amount of FeCl3 was added, and the mixture was allowed to warm slowly to room temperature overnight. The low-boiling contaminants were removed by rotary evaporation at aspirator pressure and then the product was purified by distillation as described in ref. 1.
Cleavable Bismaleimide (1b).
A solution of 0.83 g (2.9 mmol) of compound 2 in dry chloroform was chilled to -20°C, and a solution of 175 ml (1.5 mmol) of bis(chlorocarbonyl)disulfane in 5 ml of chloroform was slowly added to this solution. The cooling bath was removed and the reaction mixture was stirred at room temperature for 1 h. The mixture was diluted with chloroform and washed twice with 0.1 M HCl, and the organic layer was dried over Na2SO4. The solvents were removed in vacuo and the crude product was purified by silica gel chromatography, eluting with a shallow gradient from CHCl3 to 33:1 CHCl3/methanol to afford 226 mg (23%). 1H NMR (400 MHz, CDCl3) d 2.56 (app t, 4H, J = 7.0), 3.44-3.48 (m, 4H), 3.84-3.88 (m, 8H), 6.62 (s, 4H), 7.37-7.46 (m, 10H); 13C NMR (100 MHz, methanol-d4 with 10% CDCl3) d 33.26, 33.61, 36.56, 50.06, 128.9 (br s), 129.1, 133.5, 138.6, 164.8, 170.0, 170.8; HRMS (ESI) calculated for C32H32O8N6S2 (M+H)+ 693.1801, found 693.1814.
1. Barany G, Merrifield RB (1977) A new amino protecting group removable by reduction. Chemistry of the dithiasuccinoyl (Dts) function. J Am Chem Soc 99:7363-7365.
2. Barany G, Schroll AL, Mott AW, Halsrud DA (1983) A general strategy for elaboration of the bithiocarbonyl functionality, -(C=O)SS-: Application to the synthesis of bis(chlorocarbonyl)disulfane and related derivatives of thiocarbonic acids. J Org Chem 48:4750-4761.