Current Organic Chemistry, 2012, 16, 000-000 1 1385-2728/12 $58.00+.00 © 2012 Bentham Science Publishers Application of Lipases from Candida rugosa in the Enantioselective Esterification of (R,S)-Ibuprofen Tomasz Siódmiak 1 , Jan K. Rumiski 2 and Micha P. Marsza 1* 1 Department of Medicinal Chemistry, Collegium Medicum in Bydgoszcz, Jurasza 2, 85-094 Bydgoszcz, Poland 2 Department of Organic Chemistry, Nicolaus Copernicus University, Gagarina 7, 87-100 Toru, Poland Abstract: Three commercially available lipases from Candida rugosa (OF and MY from Meito Sangyo Co., and CRL from Sigma- Aldrich Co.) were used for the enantioselective esterification reaction of (R,S)-ibuprofen with 1-propanol and 2-propanol in saturated cy- clohexane as reaction medium. All tested lipases preferentially catalysed the esterification of the S-enantiomer of ibuprofen. However, each one of the analysed lipases demonstrated differences in the catalytic activity. Lipase OF showed the highest conversion degree, and the best enantioselectivity was observed for MY and CRL lipases. The influence of temperature, reaction time and addition of N,N’- dicyclohexylcarbodiimide (DCC) on the enantioselectivity and on the conversion degree in the enzymatic esterification was studied and the optimal condition for enantioselective esterification was evaluated. Moreover, the application of new commercial cellulose-based tris(3,5-dimethylphenylcarbamate) HPLC chiral column was demonstrated for effective separation, qualification and quantification of both substrates and products within one chromatographic analysis. Keywords: Esterification, biocatalytic reaction, resolution, (R,S)-ibuprofen, Candida rugosa lipase. INTRODUCTION Lipases are very suitable enzymes for organic synthesis thanks to their capacity of catalysing different reactions such as asymmet- ric esterification, asymmetric hydrolysis and asymmetric trans- esterifiaction [1,2]. These enzymes have been applied in the resolu- tion of racemic mixture for the preparation of optically pure com- pounds. The active centre of these enzymes builds a specific envi- ronment, that is able to distinguish between the enantiomers [3]. It is demonstrated, that lipases can exist in two different forms, closed and open. The first one is inactive form, where by a polypeptide chain called ‘lid’ the active centre of the lipase is secluded from the reaction medium. The second one is considered to be active, be- cause the ‘lid’ is displaced and the active centre exposed to the reaction medium [4]. Over the application of these enzymes, it has been proved that lipases act at the interface between hydrophobic and hydrophilic regions and water content is one of the most impor- tant factors affecting the enantioselectivity of lipases. The small amount of water is needed to retain their active three-dimentional conformation state, stability and active site polarity [5]. Further- more, it should be considered, that lipases tolerate a great number of non-natural substrates, are stable and active in organic solvents and necessitate no cofactors. Based on the previous reports, it seems to be obvious, that use of these enzymes as biocatalysts in the industry will be continuously increasing due to their high activ- ity and low price [6-9]. 2-Arylopropionic acids (profens) are known as major nonster- oidal anti-inflammatory drugs (NSAID) used in the treatment of headache, rheumatoid arthritis, cephalgia, muscular strain [10-12]. All those profen drugs have the chiral carbon atom within the propionic acid moiety. One of the most frequently used drugs within this therapeutic group is ibuprofen [13]. The pharmacologi- cal activity of ibuprofen results mainly from the (S)-enantiomer, *Address correspondence to this author at the Collegium Medicum in Bydgoszcz, M. Skodowskiej-Curie 9, 85-094 Bydgoszcz, Poland; Tel: +48 525853540; Fax: +48 52 585 3804; E-mail: mmars@cm.umk.pl which is 160 times more active than its antipode in the in vitro in- hibiting prostaglandin synthesis [14,15]. What is more, from the pharmacological studies results, that the accumulation of R- ibuprofen will cause serious side effects on the human organism such as gastrointestinal pain [16,17], as well as production of ‘hy- brid’ triglycerides between (R)- ibuprofen and Coenzyme A, which disturb normal membrane function and lipid metabolism. Thus, an important effort has been done to synthesise optically pure (S)- enantiomer [18]. Facing these facts, the two enantiomers of ibupro- fen can be regarded as two different drugs thanks to the difference in their pharmacological properties [19]. Moreover, the application of the pure (S)-enantiomer instead of racemic ibuprofen allows to reduce the amount of total drug prescribed to achieve the expected therapeutic effect [20]. Nowadays, immobilization methods of lipases on different sup- ports are developed, due to the possibility to reuse them since they can be easily recovered from the reaction medium. Additionally, immobilization of lipases improves their stability and facilitates to control water activity, which is very important factor in activity of these enzymes. From economical point of view the use of immobi- lized lipases as biocatalyst is favorable, because it allows to reduce cost of the enzymatic reactions [21-23]. However, the application of crude lipases is better in the preliminary research of the enzymatic activity in various reaction medium, because it gives a possibility to select enzymes with high catalytic activity, which can be immobilized on different supports. Therefore, this step is very important in the development new procedures with the use of immobilized lipases as biocatalysts. In this work, we compared catalytic activity of 3 Candida rugosa lipases, used to obtain the S-enantiomer of ibuprofen. We reported on the enantioselective HPLC sampling of lipase-catalysed enantioselective access to enantiomerically pure ibuprofen. We showed the ability of these lipases to resolve (R,S)-ibuprofen enan- tiomers by esterification reaction with primary and secondary alco- hols. Additionally, we presented the influence of temperature, reac-