Sodium Hydroxide → HCID 10178874, compound

Participants

Reaction Steps

6.4 g (15.658 mmol) of N-tert-butoxycarbonyl-3-(4-alloxycarbonyl-2-aminophenylthio)-L-alanine methyl ester (99%) was dissolved in 480 ml TBME and emulsified in 1.5 L buffer solution (0.1M sodium chloride; 160 mM sodium phosphate pH 7.5) under vigorous stirring. 12.0 ml Alcalase 2.4 L [a subtilisin Carlsberg from Novo Nordisk] was added and the pH maintained at 7.5 under vigorous stirring by the controlled addition (pH-static) of 1.0N sodium hydroxide solution. After 48.5 h, 16.6 ml of 1.0N sodium hydroxide solution was consumed and the conversion degree was >97% (HPLC analysis). After phase separation, the aqueous phase was extracted once with 1 L TBME. The combined TBME phases were extracted with 2×0.4 L 0.1 M potassium phosphate buffer pH 7.6. The combined aqueous phases were acidified to pH 2 with 32% hydrochloric acid and extracted with 3×0.5 L ethyl acetate. In case a stabile emulsion was formed, the phase separation was achieved by filtration on Dicalite. The combined ethyl acetate phases were dried on anhydrous sodium sulfate and evaporated. The residue was dissolved in dichloromethane, evaporated and dried at HV to give 5.85 g N-tert-butoxycarbonyl-3-(4-alloxycarbonyl-2-aminophenylthio)-L-alanine as a yellow highly viscous oil (yield: 94.6%). Analytical data: 1H-NMR (DMSO, 400 MHz) 1.338 (s, 9H, OC(CH3)); 3.077 (dd, 1H, SCH2, J=12.8 Hz, J=6.8 Hz); 3.238 (dd, 1H, SCH2, J=13.8 Hz, J=4.6 Hz); 3.867 (m, 1H, COCHNH); 4.753 (d, 2H, COOCH2, J=5.2 Hz); 5.526 (d, 1H, CH═CH2, J=10 Hz); 5.538 (d, 1H, CH=CH2, J=17.6 Hz); 5.595 (m, 2H, NH2); 6.018(ddtr, 1H, CH2CH═CH2, J=16.6 Hz, J=10.6 Hz, J=5.6 Hz); 6.519 (m, 1H, CONHCH); 6.876 (d, 1H, arom. CSCH, J=8.4 Hz); 7.330 (d, 1H, arom. CSCHCH, J=8.4 Hz); 7.338 (s, 1H, arom. CHCNH2). IR (ATR-IR) 3420 and 3355 cm−1 (—NH, —NH2); 2980 and 2935 cm−1 broad (—COOH); 1705 cm−1 (COOH —C═O, carbamate —C═O); 1510 cm−1 (amide —CO—NH); 1486 cm−1 aromate; 1229 and 1160 cm−1 (COOH); 982 and 932 cm−1 (vinyl, —C═CH2). MS (ESI-positive ionisation) m/z=397.1 [M+H+]; m/z=419.4 [M+Na+]. OR [α]D=+16.16° (CHCl3; c=1.0). HPLC analysis: column: ABZ+plus; mobile phase: A: 0.1% TFA in H20; B: MeCN; gradient B: 30–80% 0–15 min, 80–30% 15–16 min, 30% 16–19.5 min; flow; 1 ml/min; pressure:50–80 bar; detection: UV, 300 nm; retention times: 11.2 min (product-acid); 12.7 min (substrate-ester).

Provenance & Evidence