Biotechnology Letters, Vol 20, No 12, December 1998, pp. 1145–1148
Enantioselective esterification of 2-arylpropionic acids and trans2-phenyl-1-cyclohexanol: Comparison between immobilised lipases from Candida rugosa and Rhizomucor miehei Antoni Sánchez, Francisco Valero*, Javier Lafuente and Carles Solà Departament d’Enginyeria Química. ETSE. Universitat Autònoma de Barcelona. 08193, Bellaterra (Barcelona), Spain E-mail:
[email protected], Fax number: 34–93–5812013 2-Phenyl propionic acid, ibuprofen and trans-2-phenyl-1-cyclohexanol were resolved using commercial Rhizomucor miehei lipase (Lipozyme IM20) and Candida rugosa lipase produced in our laboratory immobilised on EP100 polypropylene powder. Important differences were found on the enantioselectivity of both lipases in esterification reactions. Candida rugosa lipase was more enantioselective in the resolution of the tested substrates, especially with trans-2-phenyl-1-cyclohexanol, whereas the lipase from Rhizomucor miehei did not show catalytic activity with this substrate. Keywords: Candida rugosa and Rhizomucor miehei lipases, 2-phenyl propionic acid, ibuprofen, trans-2-phenyl1-cyclohexanol, esterification, enantioselectivity.
Introduction Lipases have been widely used for the production of enantiomerically pure compounds, resolving racemic alcohols and organic acids via hydrolysis, esterification and transesterification reactions (Schudok and Kretzschmar, 1997). Among them, 2-aryl propionic acids are used as intermediates for the synthesis of non-steroidal antiinflammatory drugs (Shen, 1972; Stratman et al., 1997) whereas trans-2-phenyl-1-cyclohexanol is considered an excellent chiral auxiliary in a variety of asymmetric reactions (Basavaiah and Rao, 1994). Two of the most common lipases used in the resolution of 2-aryl propionic acids are from Candida rugosa (Mustranta, 1992; Kim and Lee, 1996; Tsai et al., 1997) and Rhizomucor miehei (López-Belmonte et al., 1997) with excellent enantioselectivity results in the case of ibuprofen although worse results were obtained with 2-phenyl propionic acid (García et al., 1993). For our knowledge, the resolution of trans-2-phenyl1-cyclohexanol has only been accomplished with Lipase PS30 on Celite (Carpenter et al., 1996) or with crude chicken liver esterase (Basavaiah and Rao, 1994). © 1998 Chapman & Hall
In this paper, we have studied and compared in terms of enantioselectivity the synthesis of 2-phenyl propionic acid and ibuprofen with 1-butanol and trans-2-phenyl-1cyclohexanol with propionic acid in isooctane using a commercial lipase from R. miehei (Lipozyme IM20) and lipase from C. rugosa immobilised on EP100 produced in our laboratory. Materials and methods Lipases C. rugosa lipase was obtained by fermentation fed-batch operation using oleic acid as a carbon source (Gordillo et al., 1998). The culture broth was first centrifuged at 3000g and the supernatant microfiltered through a 0.45 mm to eliminate the biomass. The filtrate was then concentrated by ultrafiltration (10000 D cut-off) with a Minitan Millipore system equipped with 4 plaques PMNL and afterwards dialysed with tris-HCl buffer (20 mM, pH57.4) at the same cut-off. This concentrated liquid lipase was frozen using liquid nitrogen. Afterwards, the frozen product was introduced in a lyophilizer (Vintris Sentry 5L) for 24 hours. C. rugosa lipase was immobilised on EP100 microporous polypropylene powder from Akzo Nobel according to Gitlesen et al. (1997). Biotechnology Letters ⋅ Vol 20 ⋅ No 12 ⋅ 1998
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A. S´anchez et al. In the case of R. miehei lipase, the immobilised preparation Lipozyme IM20 from Novo-Nordisk was used directly. Esterification reactions Esterification of 2-phenyl propionic acid and ibuprofen were carried out using 1-butanol, whereas in the case of trans-2-phenyl-1-cyclohexanol, propionic acid was used. Concentrations of all the reagents were initially 25 mM. In all the reactions isooctane was used as solvent. Reaction volume was 10 ml, temperature 40°C and orbital stirring of 250 rpm. 100 mg of each enzyme preparation were used. The monitoring of the reaction and the determination of enantiomeric excess of the product were conducted by HPLC analysis using Chiralcel OD column (Daicel Chemical Co.) with the mobile phase, hexane/2-propanol (98/2 v/v). Results and discussion The three esterification reactions were tested using isooctane as solvent. Both enzymes had the same enantiopreference for the S(1) enantiomer of 2-phenyl propionic acid and ibuprofen and for the 1R,2S-(2)-trans-2-phenyl1-cyclohexanol. The evolution of the enantiomeric excess of the remaining substrate and the total conversion for the three reactions for the two enzyme preparations are shown in Fig. 1, 2 and 3, respectively. A summary of the final results (96 h) of total conversion (X), enantiomeric excess (EE) and enantiomeric ratio (E) calculated according to Chen et al. (1982) is shown in Table 1.
In the case of 2-phenyl propionic acid and ibuprofen, the enantiomeric ratio using Lipozyme was 2 and 8 respectively, similar to that obtained by López-Belmonte et al. (1997) under different conditions. C. rugosa lipase showed a better behaviour in terms of enantioselectivity reaching enantiomeric ratios of 25 for 2-phenyl propionic acid and 39 for ibuprofen. These differences were mainly due to the fact that in the esterification reaction of ibuprofen using lipase from C. rugosa, the relation between rate of S(1) and R(2) enantiomers is higher than the observed with R. miehei lipase and this explains the higher enantiomeric ratio (E) observed. Under our conditions, the S(1) enantiomer of ibuprofen did not disappear entirely from the reaction mixture before the esterification of the other enantiomer started as reported by Mustranta (1992). In the case of 2-phenyl propionic acid, the value of enantiomeric ratio achieved is 25, which is considerably higher than previously obtained with commercial C. rugosa lipase. From the point of view of its industrial application in large volume scales, it is also significant to have achieve rate synthesis that are similar to commercial Lipozyme R. miehei lipase, using a C. rugosa lipase entirely produced, isolated and immobilised in our laboratory. These results were also confirmed in synthesis reactions where non-chiral substrates were used and the possibilities of reutilization of the enzyme seemed at least as good as in the case of commercial R. miehei lipase (data not showed).
(b)
(a)
Figure 1 Stereoselective esterification of racemic 2-phenyl propionic acid with 1-butanol in isooctane. a) C. rugosa lipase, b) R. miehei lipase.
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Enantioselective esterification using lipases
(a)
(b)
Figure 2 Stereoselective esterification of racemic ibuprofen with 1-butanol in isooctane. a) C. rugosa lipase, b) R. miehei lipase.
this case, C. rugosa lipase seemed to be an excellent option to carry out this resolution, in spite of the fact that conversion only reached a 40 % value. This problem might be due to the formation of water, which is a common problem in esterification (Wehtje et al., 1997). This problem may be corrected by introducing some system of water removal in the medium. In conclusion, C. rugosa lipase immobilised on EP100 has shown to be more enantioselective in front of the ester synthesis of 2-aryl-propionic acid compared with Lipozyme and specially in the resolution of trans-2-phenyl-1-cyclohexanol, where practically a perfect enantiomeric separation was achieved.
Figure 3 Stereoselective esterification of racemic trans2-phenyl-1-cyclohexanol with propionic acid in isooctane using C. rugosa lipase.
In the case of trans-2-phenyl-1-cyclohexanol, only C. rugosa lipase appeared to be a suitable enzyme to obtain a good resolution. Practically a complete enantiomeric resolution with an enantiomeric ratio over 230 was reached. However, lipase from R. miehei showed no appreciable catalytic activity for this substrate, with conversions under 5 %. In
Acknowledgements This work has been supported by the European Program on Biotechnology (project BIO-4–96–0005) and the Spanish Program on Chemical Processes Technologies (CICYTQUI97–0506-C03). A. Sánchez is recipient of a predoctoral fellowship from the CIRIT (Generalitat de Catalunya). The Department of Chemical Engineering (UAB) is member as unit of Biochemical Engineering of the Centre de Referència en Biotecnologia de Catalunya (Generalitat de Catalunya). Biotechnology Letters ⋅ Vol 20 ⋅ No 12 ⋅ 1998
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A. S´anchez et al. Table 1 Summary of the obtained results at 96 h in the three esterification processes using both lipases. X (%) Esterification 2-phenyl propionic acid Ibuprofen trans-2-phenyl-1-cyclohex.
EE (%)
E
C. rugosa R. miehei C. rugosa R. miehei C. rugosa R. miehei 53 54 40
73 66 4
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Received: 18 September Revisions requested: 22 October Revisions received: 10 November Accepted: 11 November
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