The Separation Of Chiral Compound By HPLC And The Application To Resolution By Lipase

The current advances in chiral pharmaceuticals research, the methods for resolution of chiral pharmaceuticals and separation and measurement by high performance liquid chromatography(HPLC) were reviewed. The research and application of the biocatalysis in organic media and the enzyme-catalyzed transesterification were outlined. Chrysanthemic acid and a-Cyano-3-phenoxybenzyl alcohol (CPBA) are important intermediates for synthesis of pyrethriod insecticides, which have high level pesticide activity. In this article, research of separation and measurement of chiral Chrysanthemic acid and CPBA content by HPLC chiral stationary phases method and the separation mechanism were conducted. After the reliable and quick measurement method for CPBA was set up, this method was applied to monitor CPBA in the course of lipase catalyzed asymmetric transesterification of a-Cyano-3- phenoxybenzyl acetate(CPBAc) to S-CPBA.First, chiral intermediates of Chrysanthemic acid and CPBA were separated by HPLC. Different chiral columns were selected for different samples, Detection wave length ,mobile phase composition, sample flow rate and column temperature were also chosen to optimized. The results showed that low temperature facilated separation effect, according to thermodynamic formula the AH6 for S-CPBA.was -3.406kJ.mol-1, and for R-CPBA was -3.719 kJ mol-1. AH6 for (+)-trans, (-)-trans, (+)-cis, (-)-cis -Chrysanthemic acid were- 4.179 kJ-mol"1, -4.297 kJ-mol"1, -4.906 kJ-mol"1, -4.937 kJ. mol-1 respectively. Finally the optimized separation conditions were followed: for CPBA, chiral column: Sumichiral OA-4400(25cmx4.6mm, 5um), mobile phase: n-hexane-l,2-Dichloroethane-ethanol (87:10:3, volume ratio), flow rate: 1.0 ml.min-1, UV detection wavelength 254nm, column temperature: 15C, sample injection volume: 5ul; for Chrysanthemic acid, chiral column: two Sumichiral OA-2200 (25cmx4.6mm, 5um) were used in series, mobile phase: n-hexane-acetic acid (99.9:0.1, volume ratio), flow rate: 0.8 ml min-1, UV detection wavelength 254nm, column temperature: 10C, sample injection volume: 5ul. Under these conditions, experimental results show that linear relationship and repeatablity are good, then their separation mechanism was analysed briefly.The method for separation and measurement of CPBA enantiomer was applied to lipase catalyzed asymmetric transesterification of CPBAc to S-CPBA. The suitable enzyme catalyzed reaction system was chosen. 36 Upases from different sources were screened, 10 of them showed higher catalytic activity and were screened again, Upases of 7^ 10#^ 13# showed higher enantioselectivity. 15 different organic solvents include mixed solvents and 14 different alcohols were compared, THF and dichloromethanewere the best organic media, and methanol was the best acyl accepter. Thus the preferable system for the asymmetric reaction were: 7#, THF, methanol and 7#> dichloromethane, methanol. When the reaction carried out in the optimum systems above, several factors that affecting the reaction were investigated in detail. The results indicated that trace water(0-1.0%) is desired in the reaction; >1:0.5 is the ideal molar ratio of CPBAc: alcohol for the reaction; 35-45C is a nice choice for the reaction ; Shorten the reaction time (<4days) would decreased the decomposition of the product; when the rate of the shaker >200rpm, the external diffusion limitation of the reaction would be omitted; 10mg-ml-1 lipase is enough when 337.08mmol.l-1 CPBAc is used ; The inhibitions of substrate and product were observed; >90% of the catalytic activity of the lipase was loss after 4 and 24 runs of use in dichloromethane and THF respectively. When the reaction carried out in THF for preparation a 43.85% yield of S-CPBA with >98%e.e. was obtained.