Syntheses of Enantiopure Aliphatic Secondary Alcohols and Acetates by Bioresolution with Lipase B from Candida antarctica

The lipase B from Candida antarctica (Novozym 435®, CALB) efficiently catalyzed the kinetic resolution of some aliphatic secondary alcohols: (±)-4-methylpentan-2-ol (1), (±)-5-methylhexan-2-ol (3), (±)-octan-2-ol (4), (±)-heptan-3-ol (5) and (±)-oct-1-en-3-ol (6). The lipase showed excellent enantioselectivities in the transesterifications of racemic aliphatic secondary alcohols producing the enantiopure alcohols (>99% ee) and acetates (>99% ee) with good yields. Kinetic resolution of rac-alcohols was successfully achieved with CALB lipase using simple conditions, vinyl acetate as acylating agent, and hexane as non-polar solvent.


Introduction
The use of enzymes for organic synthesis has become an interesting area for organic and bio-organic chemists. Since many enzymes have been demonstrated to possess activity against non-natural substrates in organic media they have become widely use to carry out synthetic transformations. Hydrolases are the most frequently used enzymes due to their broad substrate spectrum and considerable stability. Additionally, many of them are commercially available and they work under OPEN ACCESS mild reaction conditions and without the necessity for cofactors [1]. Enantiomerically pure alcohols and esters are key intermediates for asymmetric synthesis of pharmaceutical and agrochemical compounds [2,3]. Among the numerous synthetic methods for asymmetric synthesis, enzyme-catalysed kinetic resolution of racemic alcohols through transesterification is an attractive route. For example, the enantiomeric resolution of atenolol by lipase B from Candida antarctica [4]. Transesterification reactions catalysed by hydrolytic enzymes have been extensively studied in conventional nonaqueous solvents, producing excellent results [5][6][7][8][9]. In addition, the dynamic kinetic resolution (DKR) is a useful methodology for the conversion of racemic substrates to single enantiomer [10,11].
The resolution of a racemic substrate can be achieved with a range of hydrolases including lipases and esterases. Kinetic resolution (KR) of racemic secondary alcohols with lipases has been shown the dependence between the structure of the substrate and the enantioselectivity of enzymatic transesterification in according to the empirical Kazlauskas' rule [16].
Recently we reported the use of enzyme-catalysed transesterifications of secondary alcohols by kinetic resolution using different compounds to produce enantiomerically pure or enriched alcohols and acetates [17][18][19][20]. In this work, we report an efficient protocol for the KR of aliphatic secondary alcohols using mild conditions to produce enantiopure compounds by lipase CALB.

Results and Discussion
Rocha et al. reported the kinetic resolution of secondary iodophenylethanols using lipase B from Candida antarctica (CALB) to produce alcohols and acetates with high enantiomeric excesses [17]. Efficient KR of rac-alcohols is successfully achieved by lipase under mild friendly conditions, vinyl acetate as acylating agent, and hexane as non-polar solvent. To extend this biocatalytic methodology, a series of aliphatic secondary alcohols 1-6 were used in order to obtain enantiopure products. Initially, the racemic alcohols 1-6 and acetates 7-12 were obtained with good yields by conventional methods (Scheme 1).
In general, the KR of aromatic secondary alcohols produced enantiopure compounds with CALB. However, the enzymatic kinetic resolution of aliphatic secondary alcohols did not show an effective enantioresolution, and low enantiomeric excesses are frequently obtained. In this study, our focus on the KR enabled a series of aliphatic secondary alcohols containing different groups attached to the stereogenic center. The results are summarized in Table 1. The kind of group attached to the stereogenic center is crucial for the useful kinetic resolution of secondary alcohols by CALB. The racemic substrates 1, 3-4 were efficiently resolved by CALB producing the enantiopure (S)-alcohols (1, 3-4) and (R)-acetates 7, 9-10 with high enantiomeric excesses (>99% ee, Table 1). The studies, of the rac-alcohols 1, 3-4 contain the methyl group attached to the stereogenic center, which were responsible for the high selectivities obtained by CALB.   Table 1. Enzymatic transesterification of (±)-secondary alcohols 1-6 by lipase B from Candida antarctica a .
In addition, we investigated the selectivity of lipase by increasing the steric bulk attached to the stereogenic center in other racemic alcohols. For the aliphatic alcohol 2, with n-propyl and isopropyl groups linked to the stereogenic center, the enzymatic KR for the production of acetate 8 not occurred. These results confirm that the n-propyl and isopropyl are large groups for the KR of secondary alcohols by lipase B from C. antarctica.
However, the KR for alcohols 5-6 were highly efficient, producing enantiopure products (>99% ee, Table 1). In these cases, the substituent groups attached to the stereogenic center were n-butyl and n-ethyl for 5 and n-pentyl and vinyl for 6. Moreover, for the aliphatic alcohol 5 the reaction occurred slowly and the time necessary to reach 50% conversion was 7 hours (Table 1). In this investigation, the alkyl and vinyl groups were accepted by CALB, giving alcohols and acetates with excellent optical purities. In general, high selectivity for these reactions was obtained (E > 200). Recently, the chemoenzymatic resolution of racemic allylic alcohol 6 was described using CALB [21,22]. Lipase from Pseudomonas cepacia catalysed esterification of allylic alcohols with different selectivities, but in several cases not exceeding 98% ee to the compounds [23].
According to the empirical Kazlauskas rule, lipase stereoselectivity is mainly set by steric interactions between enzyme and substrate. The small difference in the steric bulk of ethyl and vinyl groups on aliphatic alcohols 5-6 compared with the methyl group on alcohols 1, 3-4, showed the occurrence of non-steric interactions, and these rac-alcohols reacted by lipase CALB.
The assignment of the absolute configuration for (S)-alcohols 1, 4-5, (R)-alcohol 6, (R)-acetate 7 and (S)-acetate 12 were done on the basis of their specific rotation values and compared with literature values. In addition, these data were confirmed with the empirical Kazlauskas rule. This rule shows the enantiopreference of the esterification of secondary alcohols by lipase and suggests the attribution of the absolute configuration of products. The absolute configurations of (R)-acetates 10-11 were suggested by the empirical Kazlauskas rule [5,24].
The enantiomeric excesses of alcohol 1 and acetates (7,(9)(10)(11) were determined by chiral column chromatography. The rac-alcohols 3-6 did not show enantioseparation on the chiral column chromatography used. In these cases, these alcohols were derivatized with pyridine/anhydride acetic to produce the corresponding acetates 9-12. The enantiomeric excesses of its acetate derivatives were determined by gas chromatography with an FID detector using chiral column (Table 1).

Biocatalyzed Enzymatic Reactions
Racemic alcohols 1-6 (0.5 mmol) were added to a 50 mL Erlenmeyer flask containing 10 mL of hexane (HPLC grade), 0.5 mL of vinyl acetate and 80 mg of immobilized lipase B from C. antarctica. The reaction mixture was stirred in an orbital shaker (32 C, 150 rpm) until the consumption of the reagents ( Table 1). The mixture was filtered and the solvent evaporated. The residue was purified by silica gel column chromatography using hexane and AcOEt producing in good yields the enantiopure alcohols 1, 3-6 and acetates 7, 9-12 ( Table 1). The progress of the reactions was monitored by collecting samples (0.1 mL) and these were analyzed by GC-FID (1.0 µL) in a chiral capillary column. The products were compared with the previously analyzed racemic mixtures. A similar procedure was repeated to obtain the isolated yield (Table 1). In this case, a 50 mL Erlenmeyer flask was used containing 10 mL of hexane, 1.0 mL of vinyl acetate, 100 mg of lipase CALB and 2.0 mmol of racemic alcohols 1, 3-6.

GC-FID Analyses
The reaction products were analyzed using a Hewlett Packard (Palo Alto, CA, USA) model HP-5890 gas chromatograph and using a Shimadzu GC 241 gas chromatograph equipped with an AOC 20i auto injector equipped with a Varian CP-Chiralsil-DEX -Cyclodextrin column (25 m × 0.25 mm i.d.; 0.39 µm). The programs used for the GC-FID analyses of rac-alcohols 1-6 and rac-acetates 7-12 are described in Table 2. The injector and detector were maintained at 200 C, the split ratio of the injector was 1:20, and the carrier gas was N 2 at 60 kPa. The ee values of alcohols and acetates were determined by GC-FID analyses (Figures 1-3 and Table 2).

Assignment of the Absolute Configuration
The optical rotation of the products from the biocatalytic reaction was measured in a Perkin-Elmer (Waltham, MA, USA) model 241 polarimeter using a 1 dm cuvette and referenced to the Na-D line. The absolute configurations of compounds (1, 4-6, 12) were determined comparing the specific rotation signs measured for the products with that reported in the literature (Table 3) [14,[25][26][27][28][29]. The compounds 3, 7, 9-11 were suggested by empirical rule of Kazlauskas [5,24].    Table 2. Programs used for identification of the alcohols 1-6 and acetates 7-12 by GC-FID analyses a .

Enantiomeric Ratio (E)
The enantiomeric ratio (E) for the kinetic resolutions of racemic alcohols was calculated for catalysed reactions by equations formulated by Sih and coworkers [31]. The calculation of the selectivity of the kinetic resolution expressed as enantiomeric ratio (E) for irreversible reactions was obtained by a computer program described by Faber [32].