Enzymatic Resolution Of Racemic Butylphthalide In Organic Medium

Butylphthalide is a national significant pharmaceutical, which can be used to cure cerebrovascular disease. Moreover, (S)-enantiomer of racemic butylphthalide plays a main role. In this study, highly efficient resolution of racemic butylphthalide was obtained by choice of available scheme and construction of correspondingly optimal reaction system.Racemic butylphthalide with above 98% purity was identified by GC—MS and NMR. Then standards of (S)-butylphthalide and (R)-butylphthalide were prepared by chiral HPLC. Afterward the method to determine of butylphthalide was constructed by HPLC using a Chiral OD-H column. Enantiomers were eluted with hexane and 2-propanol (98:2) at a flow rate of 0.5 mL/min. The effluent was monitored at 256 nm. Theoretically 15 schemes to resolution of racemic butylphthalide were designed.However, only three schemes of them were available. The three schems were: enantioselctive acidolysis of racemic butylphthalide, enantioselctive acylation of racemic butylphthalide with enol ester and enantioselctive acylation of racemic butylphthalide with anhydride catalyzed by Novozyme 435 in organic solvent.Effects of characteristic parameters of organic solvent, water activity, butylphthalide concentration, molar ratio of acetic acid to butylphthalide, enzyme dosage, agitation velocity and reaction time on the enantioselctive acidolysis of racemic butylphthalide were investigated using Novozyme 435 in organic solvent.The optimum conditions were: solvent hexane, water activity 0.54, butylphthalide concentration 0.5% (v/v), acetic acid to butylphthalide molar ratio 10:1, enzyme dosage 8 mg/mL, agitation velocity 150 r/min and reaction time 64 h as for the reaction. Under the conditions, 14.7% conversion of butylphthalide was achieved.Effects of single solvent, mixture of two solvents, water content, butylphthalide concentration, molar ratio of acyl donor to butylphthalide, enzyme dosage, agitation velocity and reaction time on the enantioselctive transesterification and acylation were investigated using Novozyme 435. Then optimal reaction media were constructed: binary miture of solvents comprising 65% (mol/mol) tetrahydrofuran (THF) and 35% (mol/mol) hexane. Other optimum reaction conditions: water content 0.4% (v/v), butylphthalide concentration 25.0 mM, acyl donor (vinyl acetate or acetic anhydride) concentration 0.1 M, enzyme dosage 8 mg/mL, agitation velocity 150 r/min and reaction time 40 h (48 h for the acylation). Under the conditions, 49.2% conversion of butylphthalide with 96.8% enatiomeric excess was achieved for the enantioselective tansesterification of butylphthalide; 49.0% conversion of butylphthalide with 95.7% enatiomeric excess was achieved for the enantioselective acylation of butylphthalide.Water played a crucial role in the resolution of butylphthalide. In this section, different methods to control water content in reaction system were used to study enantioselective acidolysis of butylphthalide, enantioselective transesterification of butylphthalide and enantioselective acylation of butylphthalide. The results showed that the method to control water content in reaction system with preequation using salthydrate (or saturated salt solution) was available for the acidolysis. The methods to control water content in reaction system with direct addition of water to reaction system and to control water content in reaction system with preequation using salt hydrate before direct addition of salthydrate to reaction system were all available for both of the enantioselective transesterification of butylphthalide and the enantioselective acylation of butylphthalide. The method to control water content with preequation using salt hydrate before direct addition of salt hydrate to reaction system was available for the enantioselective transesterification of butylphthalide and the enantioselective acylation of butylphthalide, because salt hydrate could continually free water to meet the continuous consumption of water in the reaction.Among the three routes above, the optimal route was Novozyme 435-catalyzed enantioselective transesterification reaction of butylphthalide. In the reaction, it could be obtained that the conversion of butylphthalide was above 49% and the enantiomeric excess of butylphthalide was above 96%.