2.3 Diethyl 2-thienylmalonate was used as the starting material, and the saponification reaction was first carried out with 2.3 equivalents of aqueous sodium hydroxide. Subsequently, L-acylase, 9-fluorenylmethyl-N-succinimidyl carbonate (Fmoc-OSu) and acetone were added, and the pH of the reaction system was adjusted to 8-9 with aqueous sodium carbonate.After completion of the reaction, Fmoc-Thi-OH was isolated (stored separately), and the mother liquor was frozen and stored.2.4 About 2 L of the above mother liquor was transferred to a 5 L three-necked flask, and 1 L of a mixture of hydrochloric acid and methanol mixture. The reaction mixture was heated to reflux for 60 hours. The reaction was monitored by nuclear magnetic resonance (NMR) and thin layer chromatography (TLC). When the reaction was essentially complete, the Fmoc-OSu solid was removed by filtration and the reaction process continued to be followed by TLC. After the reaction was complete, the reaction system was washed four times with 500 mL of a solvent mixture of ethyl acetate and petroleum ether. Subsequently, the reaction system was acidified with hydrochloric acid to a pH of about 1. Cooling and stirring resulted in 48 g of brown crystalline product.
