Sodium Hydroxide → HCID 10222027, compound

Participants

Reaction Steps

19.9 g (48.02 mmol) of N-tert-butoxycarbonyl-3-(2-amino-4-bromophenylthio)-L-alanine methyl ester (97.8%) was dissolved in 750 ml TBME and emulsified in 3 L buffer solution (0.1M sodium chloride, 20 mM sodium phosphate pH 7.5) under vigorous stirring. 12.0 ml Alcalase 2.4 L and 30 mg Substilisin A [both enzyme preparations are subtilisin Carlsberg from Novo Nordisk] were added and the pH was maintained at 7.5 under vigorous stirring by the controlled addition (pH-static) of 1.0N sodium hydroxide solution. After 7d 44.75 ml of 1.0N sodium hydroxide solution was consumed and the conversion degree was >99% (HPLC analysis). The biphasic reaction mixture was separated. The aqueous phase was washed briefly with 1 L TBME for the separation of small amounts of lipophilic impurities and traces of the remaining substrate. The combined organic phases were extracted with 1×250 ml 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 1.5 L ethyl acetate. The resulting emulsion was separated by the addition of 10% Dicalite under stirring and a subsequent filtration. The aqueous phase was extracted with 2×1 L ethyl acetate. 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 18.79 g N-tert-butoxycarbonyl-3-(2-amino-4-bromophenylthio)-L-alanine as a pale yellow solid (yield: 81.8%). Analytical data: 1H-NMR (CDCl3, 400 MHz) 1.399 (s, 9H, OC(CH3)); 3.184 (dd, 1H, SCH2, J=14.2 Hz, J=6.6 Hz); 3.235 (dd, 1H, SCH2, J=14.2 Hz, J=6.6 Hz); 4.515 (m, 1H, COCHNH); 5.517 (d, 1H, CONHCH, J=6.8 Hz); 6.796 (dd, 1H, arom., J=2 Hz, J=8.4 Hz); 6.876 (d, 1H, arom., J=2 Hz); 7.250 (d, 1H, arom., J=8.4 Hz). IR (ATR-IR) 3445 and 3355 cm−1 (—NH, —NH2); 2978 and 2929 cm−1 broad (—COOH); 1693 cm−1 (COOH —C═O, carbamate —C═O); 1508 cm−1 (amide —CO—NH); 1247 and 1157 cm−1 (COOH). MS (ESI-positive ionization) m/z=391.1 [M+H+] with 79Br; m/z=393.1 [M+H+] with 81Br; m/z=413.2 [M+Na+] with 79Br; m/z=415.2 [M+Na+] with 81Br. OR [α]D=+53.3° (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: 12.1 min (product-acid) 13.5 min (substrate-ester).

Provenance & Evidence