Optical resolution of (R, S)-ibuprofen in organic solvent by porcine pancreatic lipase catalyzed enantioselective esterification

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1 Indian Journal of Biotechnology Vol 7, January 2008, pp Optical resolution of (R, S)-ibuprofen in organic solvent by porcine pancreatic lipase catalyzed enantioselective esterification Ganesh Ramachandran and Laxmi Ananthanarayan* Food Engineering and Technology Department, Institute of Chemical Technology (Formerly UDCT) University of Mumbai, Matunga, Mumbai , India. Received 19 April 2006; revised 4 April 2007; accepted 10 May 2007 Porcine pancreatic lipase (PPL), a comparatively inexpensive enzyme, was used in the optical resolution of (R, S)- ibuprofen in methanol through enantioselective esterification. The different reaction parameters during the esterification of (R, S)-ibuprofen using PPL was studied with respect to temperature, time, enzyme concentration, substrate concentration and water content. The percentage conversion initially was 13%, which increased to 33% after optimization of the different reaction conditions. Surfactant coating of PPL resulted in a 2.5-fold increase in the hydrolytic activity of PPL. Surfactant coated PPL, when used in the esterification reaction slightly increased the percentage conversion from 21 to 22.5%. In all the reactions, PPL showed preference for catalyzing the esterification of S-(+)-ibuprofen. Keywords: Enantioselectivity, esterification, ibuprofen, NSAIDS, optical resolution, porcine pancreatic lipase, racemates Introduction Since the mid 1980 s, there has been an increasing trend to produce and market chiral drugs composed of single active enantiomers rather than as racemates, which contain both active and inactive isomers. Due to this revolution, there is now an absolute need to produce new drugs with a high optical purity or old drugs are enantio-enriched so that they show a high optical purity 1. Ibuprofen is an important member of the nonsteroidal, anti-inflammatory drugs (NSAIDS) consisting of the 2-arylpropionic acids (profens). The racemic mixture of its two enantiomers S-(+) and R-(-) is being used, but its biological activity is mainly from the S-enantiomer 1. It is widely believed that it will soon be marketed as a single isomer drug. Studies project that over the next few years, (S)-ibuprofen has the potential of becoming a one billion dollar per year over the counter medication. 1 In recent years, lipases have been used for chiral resolution of ibuprofen through catalyzing enantioselective hydrolysis of its chemically synthesized racemic ester or direct enantioselective esterification in organic media 2. In organic solvents, lipases are stable, can be more enatioselective and the solubility of hydrophobic substrate can be enhanced. *Author for correspondence: Tel & Fax: laxmi@udct.org Esterification in such solvents thus seems more promising. Optical resolution of ibuprofen has been carried out by using lipases from Candida antartica, C. rugosa and Rhizomucor miehei. The most widely used lipases are from C. rugosa and C. antarfica, where the former preferentially catalyzes the esterification of S- ibuprofen and the latter the esterification of R- enantiomer. Ideally, the lipase selectively catalyzing the transformation of R-ibuprofen should be so used, that the required S-ibuprofen left unreacted. However, the enantioselectivity of C. antartica lipase is very low 3. C. rugosa lipase has a high enantioselectivity for the esterification of the S-ibuprofen. Also a very high enantiomeric excess of the produced S-ester can be obtained at a conversion close to 50% 2. For such resolutions to be carried out on a large-scale, the lipases have to be inexpensive so that the process becomes cost-effective. Almost all the commercially available lipases are made available from genetically engineered organisms, which in turn make the enzyme expensive. Thus use of cheaper and effective lipases in enantioselective separations, may be from plant or animal sources, is the need of the hour. Porcine pancreatic lipase (PPL) is one of the widely available mammalian lipases, which is cheaper as compared to the other commercial fungal/bacterial lipases. To our knowledge, the reaction of ibuprofen and methanol being catalyzed by PPL has not been reported so far; hence, the different reaction

2 RAMACHANDRAN & ANANTHANARAYAN: OPTICAL RESOLUTION OF (R, S) IBUPROFEN BY ENANTIOSELECTIVE ESTERIFICATION 95 parameters were optimized using this enzyme. Surfactant coating is known to increase the activity of lipases, mainly by the opening of the lid, which covers the active site. Apart from increase in the inherent activity of the enzyme, surfactant coating will also aid in enhancing the percentage conversion of the reaction. Hence, in the present study PPL was surfactant coated using SPAN 80 (sorbitan monooleate) and the surfactant coated enzyme was used in the esterification reaction between (R, S)-ibuprofen and methanol. Results of this study will be useful in the development of a process for preparing S- ibuprofen from its racemate by using an inexpensive and easily available lipase, viz. PPL. Materials and Methods Materials PPL was obtained from Advanced Biochemicals Ltd., Mumbai, India. (R, S)-Ibuprofen was a free gift sample from Shasun Chemicals and Drugs Ltd., Mumbai, India. Tributyrin and gum acacia were purchased from Hi Media Ltd. Mumbai, India. High Performance Thin Layer Chromatography (HPTLC) plates, precoated with Silica gel F254 and having dimensions of 20 cm 20 cm were obtained from Merck. All other reagents, viz. n-heptane, n-hexane, methanol, ethanol, benzene, chloroform, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium bicarbonate, sodium sulphate and SPAN 80 were of the purest grade available and were procured from Merck. Assay of Lipase Activity Lipase activity was estimated by its hydrolytic action on tributyrin. The lipase activity was determined as follows: 1 ml of tributyrin was mixed with 2 ml of 7.5% gum acacia solution on a cyclomixer for 10 min till an emulsion was formed. To this emulsion was added 10 ml of sodium phosphate buffer (50 mm, ph 7.0) and 0.5 g of the lipase powder. The mixture was kept in an incubator shaker at 40 C. 0.5 ml aliquots were removed at regular intervals, i.e. at 0, 10, 20 and 30 min, and the liberated fatty acids was titrated against 0.01 M NaOH solution, using phenolphthalein as an indicator, after the reaction was quenched using 1 ml methanol. The total reaction volume was maintained at 13 ml. One unit of lipase activity is the amount of enzyme required to liberate one micromole of butyric acid (by the hydrolysis of tributyrin) per minute. Chemical Synthesis of Racemic Methyl Ester of Ibuprofen To 20 ml of methanol in a 100 ml round bottom flask, equipped with a condenser (with a calcium chloride guard tube), was added 2 g of racemic iibuprofen and 0.1 ml of concentrated sulphuric acid. The reaction mixture was refluxed for 10 h. Samples were withdrawn periodically and the reaction was monitored by thin-layer chromatography on precoated Silica gel F254 plates using benzene:chloroform (9:1) as a mobile phase. After 10 h of refluxing, the reaction mixture was poured into 250 ml of distilled water and extracted with n-hexane (50 ml 2). Combined organic layers were washed with saturated sodium bicarbonate solution (50 ml 3) to remove the acid. Organic layers were dried over anhydrous sodium sulphate and solvent evaporated to afford a colourless liquid ester with a characteristic odour. This chemically synthesized ester, viz. methyl ester of (R, S)-ibuprofen was used as a standard in the further work. HPTLC Analysis Methyl ester of ibuprofen formed during the lipase catalyzed reaction was determined by HPTLC. Samples of the reaction mixtures were applied onto the precoated Silica gel F254 plates using the CAMAG Linomat IV applicator. The mobile phase used for developing the plates was composed of benzene:chloroform (9:1). After the plates were run, they were dried and then quantified using the densitometer (CAMAG TLC Scanner II) at wavelength 254 nm. The plates appeared fluorescent green and the spots were dark pink in colour due to quenching of fluorescence. The acid spot had an R f value of 0.246, while the ester had an R f value of Initially, a calibration curve was obtained by applying known concentrations of the racemic ester, developing the plate and determining the area under the curve obtained on scanning in the densitometer. PPL Catalyzed Esterification: Effect of Time To study the optimum time for the reaction, stoppered flasks containing 1 mm (R, S)-ibuprofen, 3.8 mm of methanol and 42 units of the enzyme in 10 ml of n-heptane were placed in an incubator shaker at 40 C for a period of 72 h. Samples were withdrawn at 10, 24, 34, 48 and 72 h and analyzed by HPTLC. Effect of Temperature The reactants, in concentrations mentioned above, were incubated for 48 h at different temperatures, viz.

3 96 INDIAN J BIOTECHNOL, JANUARY , 40, 50 and 60±3 C. The enzyme was filtered off and the reaction mixture was analyzed for per cent conversion of ibuprofen. Effect of PPL Concentration 18, 36, 45, 54, 73 and 91 U of PPL were added to the flasks containing the reactants in n-heptane. The flasks were incubated at 40 C for 48 h. At the end of the incubation period, the enzyme was filtered off and the samples were analyzed by HPTLC. Effect of (R, S)-Ibuprofen Concentration The concentration of racemic ibuprofen added was varied from 0.48 to 2.4 mm, keeping the amount of other reactants as mentioned earlier. The reaction was halted after 48 h and the samples were subjected to HPTLC analysis. Effect of Water Content To study the effect of water content, different volumes of phosphate buffer (ph 7.0, 50 mm), viz. 1, 2.5, 5 and 10% (v/v) was added to each flask. Other parameters were kept constant. At the end of 48 h, the reaction mixture was passed through sodium sulphate bed to remove traces of water and then analyzed by HPTLC. Surfactant Coating of PPL For surfactant coating of PPL, 1 g of the lipase preparation was dispersed in 250 ml of sodium phosphate buffer (100 mm, ph 6.9). 0.5 g of SPAN 80 (sorbitan mono-oleate) dissolved in 10 ml of ethanol was added drop-wise to the stirred enzyme solution and sonicated in an ultrasonic bath for 30 min at 8-10 C. After stirring the mixture on a magnetic stirrer for 3 h at 8-10 C, the solution was centrifuged at 10,000 rpm for 30 min. The pellet obtained was dried under vacuum and used in the esterification reaction. The hydrolytic activity of surfactant-coated lipase was determined as mentioned earlier. The esterification reaction mixture consisted of 1 mm (R, S)-ibuprofen, 3.8 mm of methanol and 42 units of the surfactant-coated enzyme in 10 ml of n- heptane, which was placed in an incubator shaker at 40 C for a period of 48 h. Separation of Acid (Ibuprofen) and Ester after Esterification The separation of the acid (ibuprofen) and the ester was done by column chromatography 5 using silica gel ( mesh) as the adsorbent. 20 g of silica gel was suspended in 75 ml of benzene. This was packed in a glass column (1.5 cm diam 20 cm height) and the column was kept in benzene. The reaction mixture was concentrated to 2 ml and it was loaded onto the column. 50 ml of benzene was used to elute the ester and later 75 ml of methanol was used to elute the acid. The methanol fractions containing the acid were pooled and the optical rotation of the separated acid was determined using a Jasco DIP 370 Digital polarimeter. Results and Discussion In case of PPL catalyzed esterification reaction of (R, S)-ibuprofen and methanol, a maximum conversion of 28% of ibuprofen was achieved in 48 h (Table 1). After 48 h, the reaction stopped proceeding further. However, around 40% conversion in 6 h has been reported in the reaction between 66 mm of ibuprofen and 66 mm of propanol using 100 mg of CAL-B as a catalyst 3. In the enantioselective resolution of racemic ibuprofen catalyzed by commercial R. miehei lipase (Lipozyme ) in isooctane, using butanol as esterificating agent, an enantiomeric excess of 93.8% and total conversion of 49.9% has been reported after 112 h of reaction 6. On using PPL, a maximum ibuprofen conversion of 27.9% was obtained when the temperature of esterification reaction was kept at 40 C (Table 2). However, using immobilized lipase from C. antartica, the S-(+) form of aryl propionic acids has been prepared by enantioselective esterification of the racemate at temperatures below 24 C and at conversions greater than 50% 3. In case of PPL catalyzed trans-esterifications of 2, 2, 2-trifloroethyl butyrate with racemic 2-octanol and 1-phenylethanol, around 46% conversion with ee of 100% was Table 1 Effect of time on esterification reaction of Time (h) Table 2 Effect of temperature on esterification reaction of Temperature ( C)

4 RAMACHANDRAN & ANANTHANARAYAN: OPTICAL RESOLUTION OF (R, S) IBUPROFEN BY ENANTIOSELECTIVE ESTERIFICATION 97 obtained for (R)-2-octylbutyrate at 25 C 7. PPL being a mammalian lipase might be performing better at 40 C as this temperature is closer to the physiological temperature of mammals. In an esterification reaction of (R, S)-ibuprofen and methanol at 18 U/10 ml concentration of PPL, an ibuprofen conversion of about 15.2% was achieved (Table 3). Further increase in the concentration of the enzyme showed gradual increase in the per cent ibuprofen conversion and the highest conversion of 29.4% was achieved when the enzyme concentration was 54 U/10 ml. However, further increase in the concentration of PPL did not increase the ibuprofen conversion. Instead, the apparent enzyme activity decreases at high lipase concentrations. This phenomenon could be due to diffusional limitations because at higher mass of enzyme preparation, the thickness of the solid phase would be higher in which the reactants diffuse. In case of increasing ibuprofen concentrations (0.48 to 2.4 mm) in PPL catalyzed reactions, the overall ester synthesized increased but the per cent conversion reduced (32.2 to 16.8%) as evident from Table 4. For esterification reactions carried out in organic media, two new parameters not present in aqueous solutions may be of paramount importance, viz. the nature of solvent and its water content 1. It has been recognized that the amount of water present is very important for both thermal stability and the activity of enzymes in non-aqueous media. Changes in stereoselectivity and even stereo-selectivity reversal caused by change in solvent or water content have been reported 8. The lack of influence of water activity has also been reported. In the present study, when the amount of water added to the esterification reaction increased from 0 to 2.5% (v/v), the per cent ibuprofen conversion increased from 29.5 to 33 % (Table 5). However, further increase in the volume of water from 5 to 10% drastically decreased the per cent conversion. This may be because of the problems in mass transfer. Also, the reaction doesn t proceed in the forward direction due to the water formed during the course of reaction. It has been shown that a certain amount of water is necessary to maintain the activity of enzymes in organic solvents. In case of the commercial enzyme Lipozyme, an immobilized enzyme from R. miehei, about 10% moisture content is optimal and the product is delivered with this amount of water 9. There are reports of C. cylindracea lipase (CCL) and PPL being used in nearly anhydrous Table 3 Effect of PPL concentration on esterification reaction of (R, S)-ibuprofen and methanol Enzyme (U/10 ml) Table 4 Effect of Ibuprofen concentration on esterification reaction of Ibuprofen (mm) Table 5 Effect of water content on esterification reaction of Water content (%, v/v) Table 6 Effect of surfactant coating on hydrolytic and esterification activity of PPL Free PPL Surfactant-coated PPL Hydrolytic activity (U/g) Esterification activity (%) organic solvents for the production of numerous optically active alcohols, carboxylic acids and their esters in a stereo-selective manner 10. Surfactant coating of lipases is known to increase the lipase activity by a mechanism which opens-up the lid covering the active site of the enzyme. In the present study, it was observed that the activity of lipase on surfactant coating increased 2.5-folds with respect to its hydrolytic action on tributyrin (Table 6). There are reports on the reaction rate of surfactantcoated lipase being more than 100 times to that of the powder lipase 11 ; similar rates with surfactant-coated lipase AY was also obtained 12. To the best of our knowledge, there have been no reports on the surfactant coating of PPL and its use in the optical resolution of racemic ibuprofen. Further, the per cent conversion of ibuprofen improved from 21 to 22.5% by the surfactant-coated enzyme in comparison to free enzyme (Table 6).

5 98 INDIAN J BIOTECHNOL, JANUARY 2008 Thus, under optimized conditions, if surfactant coated lipase is used, the percentage conversion could be achieved greater than 33%, which was the highest conversion obtained in this study. The optical rotation of Ibuprofen (acid) after esterification reaction and subsequent separation on silica gel was found to be 39.6, which indicated that the R-enantiomer remained unreacted. Thus, in all these experiments, PPL showed a preference for catalyzing the esterification of S-(+)-ibuprofen. PPL is a mammalian enzyme which is cheaper than most of the commercially available enzymes. Therefore, either the free enzyme or the surfactantcoated enzyme can be used for the optical resolution of (R, S)-ibuprofen at a large scale. Acknowledgement The present work was supported by grants from the Department of Biotechnology, Government of India, New Delhi. References 1 Trani M, Ducret A & Lortie R, Lipase catalyzed enantioselective esterification of Ibuprofen in organic solvents under controlled water activity, Enzyme Microb Technol, 22 (1998) Xie Y C, Liu H Z & Chen J Y, Candida rugosa lipase catalyzed esterification of racemic ibuprofen and chemical hydrolysis of S-ester formed, Biotechnol Lett, 20 (1998) Arroyo M & Sinisterra J V, High enantioselective esterification of 2-arylpropionic acids catalyzed by immobilized lipase from Candida antartica: A mechanistic approach, J Org Chem, 59 (1994) Prabhu A V, Tambe P S, Gandhi N N, Savant S B & Joshi J B, Rice bran lipase: Extraction, activity and stability, Biotechnol Prog, 15 (1999) Nashine V, Studies in microbial lipases. M Sc (Tech) Thesis, University of Mumbai, Sanchez A, Valero F, Lafuente J & Sole C, Highly enantioselective esterification of racemic ibuprofen in a packed bed reactor using immobilized Rhizomucor miehei lipase, Enzyme Microb Technol, 27 (2000) Kanerva L T, Viharto J, Halme M H, Loponan J M & Euranto E K, Solvent effects in lipase catalyzed transesterification reactions, Acta Chem Scand, 44 (1990) Fitzpatrick P A & Klibanov A M, How can solvent affect enzyme enantioselectivity?, J Am Chem Soc, 113 (1991) Neidleman, Enzyme reactions under stress conditions, Crit Rev Biotechnol, 9 (1990) Kirchner G, Scollar M P & Klibanov A M, Resolution of racemic mixtures via lipase catalysis in organic solvents, J Am Chem Soc, 107 (1985) Kamiya N, Goto M & Nakashio F, Surfactant-coated lipase suitable for the enzymatic resolution of menthol as a biocatalyst in organic media, Biotechnol Prog, 11 (1995) Babali B, Ayse A, Tuter M & Ustun G, Enzymatic esterification of (-) menthol with lauric acid in isooctane by sorbitan monostearate coated lipase from Candida rugosa, J Am Oil Chem Soc, 78 (2001)

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