Financial support: Research grant from the National Institutes of Health (GM1073-01A1).

Keywords: enantioselectivity, esterification, flurbiprofen, lipase, solvent dependence, water activity.

Many drugs exhibit stereoisomerism, i.e., the molecules may be oriented in space in two possible ways around a chiral center, such that these two different configurations rotate the plane of plane-polarized light in opposite directions. If there is only one chiral center on the molecule, the two stereoisomers are known as enantiomers (the R-enantiomer rotates plane-polarized light to the right, the S-enantiomer to the left). Enantiomers of a drug have identical chemical properties except when they interact with other molecules which also exhibit stereoisomerism. The activity and toxicity of drugs are associated with their interaction with proteins, genetic material, and other biological molecules, all of which exhibit stereoisomerism. As a consequence, the enantiomers of drugs often have different therapeutic and toxicological profiles. Resolution of the enantiomers refers to separating the enantiomers from each other.

Non-steroidal, anti-inflammatory drugs (NSAIDs) often have one chiral center and are administered as a racemic mixture, i.e. a mixture containing equal proportions of the two enantiomers. Flurbiprofen (2-fluoro-a-methyl-[1,1'-biphenyl]-4-acetic acid) is a potent NSAID with a structure similar to that of ibuprofen. There is evidence that the R-enantiomer of flurbiprofen is less potent than the S-enantiomer, but it causes little toxicity in comparison to the other enantiomer (Lotsch et al. 1995). An earlier study, found that the R-enantiomer enhances the gastrointestinal toxicity caused by the S-enantiomer (Wechter et al. 1993). There is clearly an advantage to be gained by administering the R-enantiomer and excluding the S-enantiomer.

When drugs are synthesized in the normal course, without regard to their stereospecificity, the end product is invariably a racemic mixture. Synthesis of a specific enantiomer may be achieved by using chiral reagents, but such techniques are difficult in practice. It may be more efficient to synthesize the racemic mixture of a drug, and then separate the two enantiomers. A technique which is gaining increasing popularity for this purpose uses the natural enantioselectivity of enzymes. Enzymes discriminate between the enantiomers of a compound, and the rate of the enzyme-catalyzed reaction of one enantiomer with a reagent may differ vastly from that of its antipode. As a consequence, the reaction mixture becomes rich in one enantiomer, while the other is converted to a new compound. This reaction is often conducted in organic media because enzymes are more stable in organic solvents, and not all potential substrates are soluble in water.

One approach that may be used to obtain a pure enantiomer of flurbiprofen from its racemic mixture is to use lipase in an organic medium to catalyze the esterification of the flurbiprofen with an alcohol, such as n-butanol. In principle, lipase would preferentially catalyze the reaction of one enantiomer of flurbiprofen, resulting in the formation of its butyl ester and water. The butyl ester could be separated from the unreacted enantiomer by partitioning that acidic flurbiprofen into a dilute alkaline aqueous solution. The butyl ester, without the free acidic group on it, would remain in the organic solvent. Thus, the two enantiomers will be resolved.

There are several factors that influence the effectiveness of lipases as biocatalysts in organic media. These include the water content (or water activity) of the medium, the source of the lipase, and the nature of the solvent. Lipases are active in organic solvents as long as there is enough water present for them to remain in their native folded state. Water activity can be controlled by adding hydrated pairs of an inorganic salt to the reaction medium. As long as there is enough of each hydrate, the water activity in a closed system will be maintained at a value characteristic for the pair. Lipases appear to be more stable and more active in solvents with a high log P (octanol/water partition coefficient), apparently because these solvents are less likely to pull water away from the enzyme. Lipases occur in organisms from bacteria to human beings, and while they all serve similar functions they are often very different in their primary structure, shape, and size. Consequently, they differ in their capacity to act as biocatalysts, their stability in various media, and their purity.

The purpose of our study was to investigate some factors that contribute to the improvement of a lipase-catalyzed reaction for the resolution of flurbiprofen enantiomers. The results of this study may aid in optimizing such a reaction for preparing pure R-flurbiprofen. The following parameters were varied.

1. Solvent: Toluene, n-heptane, isooctane, and n-nonane. These solvents were chosen to determine a limiting log P value beyond which the rate of the reaction would not improve.

2. Source of lipase: Commercially available lipases from Candida rugosa (Crl), Mucor javanicus (Mjl), and porcine pancreas (Ppl) were used in this study.

3. Water activity: The water activity of the reaction medium was controlled at 0.18, 0.37, 0.65, and 0.85 using various salt hydrate pairs.

The reaction between 0.6 mM flurbiprofen and 12 mM n-butanol was catalyzed by 300 mg of lipase in the desired organic solvent, and the reaction mixture was agitated in a constant temperature shaker batch maintained at 30°C. Samples were taken at periodic intervals and analyzed by an enantioselective HPLC assay for flurbiprofen using an a₁-acid glycoprotein column.

The solvent effect was not similar for lipases from Candida rugosa, Mucor javanicus, and porcine pancreas. The lipase-catalyzed reaction rates in different solvents across this range of water activities revealed that the Ppl-catalyzed reaction exhibited no enantioselectivity and no substantial water activity or solvent dependence. The Mjl-catalyzed reaction proceeded faster, preferring the R-enantiomer reaction over that of the S-enantiomer, but had little solvent or water activity dependence. The Crl-catalyzed reactions in n-heptane and n-nonane had similar water activity dependence and demonstrated a preference for the S-enantiomer. The reaction was considerably faster and more enantioselective (still preferring the S-enantiomer reaction) in isooctane, a solvent whose hydrophobicity is intermediate between that of the other two alkanes. Substrate enantiomeric excess, for the Crl-catalyzed reaction at 96 hours and at a water activity of 0.65, in n-heptane, isooctane, and n-nonane was 40.9, 93.0, and 50.0%, respectively. Since the three solvents possess similar physical properties, the explanation for this anomalous behavior might be the effect of the solvent structural characteristics on Crl, since isooctane is the only branched alkane.

The results of this study suggest that the enantioselective esterification of flurbiprofen catalyzed by lipases is significantly influenced by the nature of the solvent, the source of the lipase, and the activity of water in the reaction medium. Esterification reactions with C. rugosa lipase and M. javanicus lipase exhibited preferences for opposite enantiomers, a phenomenon which may be exploited to prepare the enantiomer of choice. In order to optimize the reaction, careful consideration should be given to the reaction conditions considered in this study.

Wechter, W.J., Bigornia, A.E., Murray, E.D., Levine, B.H. and Young, J.W. (1993). Rac-flurbiprofen is more ulcerogenic than its (S)-enantiomer. Chirality 5:492-494.