Study On Lipase-catalyzed Resolution Of Chiral Drug Ibuprofen

Ibuprofen is an important kind of drugs from 2-aryl propionic acid derivative family with good anti- inflammatory, analgesic, antipyretic effects. It is the only children antipyretic common recommended by World Health Organization and FDA. However, there is a chiral ?-carbon in the molecule, so it has two optical enantiomers:(S)-enantiomer and(R)-enantiomer. The two optical enantiomers have a great difference. The bioactivity of ibuprofen is mainly due to the(S)- ibuprofen, which has been reported to have 160- fold activity more than that of its antipode in the synthesis of prostaglandin ‘in vitro'. Whearas,(R)-ibuprofen causes the main side-effects of racemic ibuprofen. Therefore, production of highly pure(S)- isomer is urgent for reducing dosage and the possibility of adverse effects.So far, there are many resolution ways for chiral drugs, such as crystallization method, chemical resolution method, membrane method, chromatography method, and enzymatic resolution method. Compared with other resolution methods, lipase-catalyzed resolution is the most advantageous because of its mild reaction conditions, high enantioselectivity and environment- friendly processes. Therefore, in this study, Candida rugosa lipase(CRL) was chosen as a catalyst to catalyze the resolution of racemic ibuprofen. Solvent engineering, reverse micelles, enzyme immobilization and other new technologies were employed to improve the activity and enantioselectivity of C. rugosa lipase. The main work and results of this paper were summarized as follows.1. The process of resolution ibuprofen has been investigated in non-aqueous medium. The effects of organic solvents with different log P values on the catalytic efficiency and ees were examined. Under the optimal reaction conditions of using 1- isooctanol as the acyl acceptor, substrate molar ratio 6:1, water content less than 2%, reaction temperature 50 °C, reaction time 12 h, the conversion and ees respectively attained 47.2 % and 84.8%. In 13 different organic solvents and three kinds of ionic liquids, isooctane is the best reaction medium, and ionic liquid [Emim] PF6 ranked the second best. Secondary structure analysis by FT-IR showed that the variation in secondary structure element content s was probably responsible for the enchancement of enzyme activity and ees. In addition, the change in fluorescence intensity indicated that, to some extent, there occurred the location alteration of fluorescence aminos, wich could explains the correlation between variation of tertiary structure and enzyme activity.2. The reaction parameters were optimized. Through the optimization of single factorial test and response surface methodology(RSM), the optimal key reaction parameters(temperature, water content, and Triton X-100 content) have been obtained. They were temperature 46.77°C, water content 2.17, and Triton X-100 content 14.26%. Under the reaction conditions, the conversion rate and ees attained 38.83% and 65.45%, respectively. While the conversion rate was only 13.93% and ees was 20.31% in isooctane system under the same conditions.3. Three kinds of immobilized enzyme—PCMCs, CL-PCMCs and CLEAs were prepared. For PCMCs, using acetone as the precipitation reagent, K2SO4: enzyme solution =5:1, pH =9.0, water content=20%, it exhibited the highest enzyme activity and ees(49.18% and 97.74%, respectively). Based on PCMCs, CL-PCMCs was prepared. The best conditions were 25% glutaraldehyde 150?L, temperature 4 °C and reactive time 60 min. Under the reaction conditions, the conversion and ee s attained 46.78% and 88.98%. While in term of CLEAs, under the conditions of water content of 6%, 80 mg of glucose as a stabilizing protein additive, glutaraldehyde 200?L, it had the ideal result, the conversion rate and ees reached 40.41% and 62.34%, respectively.4. A comparison was conducted between the three kinds of immobilized CRL on enzymatic characteristics and resulotion of ibuprofen. Scanning electron microscopy(SEM) analysis showed significant morphological distinctions existed among free CRL, K2SO4 crystals, CRL-PCMCs, CRL-CL-PCMCs CRL-CLEAs and CRL- glucose-CLEAs, which revealed the mechanism of immobilized enzyme formation. The optimal reactio n temperature of the free enzyme and immobilized enzyme were both 50 °C, and the catalytic ability of resolution ibuprofen was PCMCs > CL-PCMCs > CLEAs > free lipase. However, compared with the free lipase, the temperature stability of the immobilized forms had been greatly improved. Being incubated at 70 oC for 3 h, only 25.68% conversion was left for the free lipase, while the retained conversion of CRL-PCMCs, CRL-CL-PCMCs CRL-CLEAs were 75.98% ? 72.79% and 57.71%, respectively. Additionally, immobilized forms demonstrated much higher activity than free CRL after incubation in different organic solvents for 3 h. Moreover, the operational stability of the three immobilized forms was also investigated. They still remained 83.04%, 89.56%, 70.38% of relative conversion respectively for CRL-PCMCs, CRL-CL-PCMCs CRL-CLEAs after running 15 batches, indicating good operational stability.