Preparation of (S)-1-Halo-2-octanols Using Ionic Liquids and Biocatalysts †

Preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol was carried out by the enzymatic hydrolysis of halohydrin palmitates using biocatalysts. Halohydrin palmitates were prepared by various methods from palmitic acid and 1,2-octanediol. A tandem hydrolysis was carried out using lipases from Candida antarctica (Novozym® 435), Rhizomucor miehei (Lipozyme IM), and “resting cells” from a Rhizopus oryzae strain that was not mycotoxigenic. The influence of the enzyme and the reaction medium on the selective hydrolysis of isomeric mixtures of halohydrin esters is described. Novozym® 435 allowed preparation of (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol after 1–3 h of reaction at 40 °C in [BMIM][PF6].


Introduction
Racemic halohydrins and haloesters can be obtained using diverse approaches [1]. Among them, our research group has developed a direct methodology for halohydrin ester formation using ionic liquids (ILs) and potassium halides [2]. These compounds can be stereoselectively hydrolyzed using an enzymatic approach [3][4]. Enantiomerically pure halohydrins are useful intermediates in the preparation of agrochemicals, pharmaceuticals, and liquid crystals [5][6].
Enzymes can catalyze a variety of reactions with high specificity and under mild conditions such as low temperature and atmospheric pressure; biotransformations are looked on as a way of resolving environmental problems involved in chemical procedures [7][8][9][10]. Furthermore, ILs have a very low vapor pressure and are considered a greener alternative to volatile organic solvents. Their solvent properties may be easily controlled by altering the nature of their cations and anions [11]. Although the application of ILs as a solvent in biocatalysis is very recent and only three articles had been published up to 2002 [12][13][14], many enzymatic reactions and their applications have been described since then [15][16][17]. Combining both technologies allows the design of an environmentally friendly process.
The aim of this work was to evaluate the suitability of commercial lipases and resting cells prepared from a nonmycotoxigenic strain of Rhizopus oryzae in the regio-and stereoselective hydrolysis of halohydrin esters for preparing (S)-1-bromo-2-octanol and (S)-1-chloro-2-octanol. These compounds have been used for the synthesis of epoxides [18], tetrathiafulvalene derivatives [19] and highermolecular-weight aliphatic sulfonic acids [20]. The compound ONO-2506, an agent that suppresses astrocyte activation [6], and some adenosine deaminase inhibitors [21] have also been synthesized from these halohydrins.

Results and Discussion
In a previous paper, we described a chemoenzymatic method for preparing (R)-4-chloro-2-butanol from 1,3-butanediol [3]. First, the synthesis of isomeric mixtures of chlorohydrin esters was carried out using this diol and several alkyl carboxylic acids. The isomeric mixtures were then hydrolyzed selectively using both fungal resting cells and commercial lipases in a tandem enzymatic system. The regioselectivity and enantioselectivity of these hydrolyses depended on the solvent, the temperature, the acyl alkyl chain, and the biocatalyst used. The best results were obtained using t-butanol as solvent and C 16 and C 18 carboxylic acids, and by carrying out the hydrolytic reactions at 40 °C. Considering these previous results and our interest in preparing enantiomers of 1-halo-2-octanols, a set of experiments was conducted to study the extension of the described method to 1-halo-2-octyl palmitates prepared according to the methods already described. Using these methods, isomeric mixtures of 1chloro-2-octyl (1a) and 2-chloro-1-octyl (2a) palmitates (regioisomeric ratio 90:10) and 1-bromo-2octyl (1b) and 2-bromo-1-octyl (2b) palmitates (regioisomeric ratio 75:25) can be obtained. The tandem enzymatic reaction was then studied comparing t-butanol, the best organic solvent found in our previous study, with some ILs. Table 1 shows the influence of the solvents on the regioselectivity of various biocatalysts in the presence of an isomeric mixture of chlorooctyl (1a, 2a) and bromooctyl (1b, 2b) palmitates. The hydrolyses were performed at 40 °C for 24 h unless otherwise indicated in the  4 ] than in t-butanol. These results differ from those obtained with chlorobutyl esters, where resting cells of R. oryzae and t-butanol had been shown to be the best combination for the regioisomeric hydrolysis of the corresponding esters [3]. Moreover, t-butanol is also a nonideal solvent for regioisomeric hydrolysis of bromooctyl palmitates with any of the biocatalysts studied, showing that the introduction of a more bulky halogen group than chloro dramatically diminishes the enzyme regioselectivity in this solvent. Although [BMIM][PF 6 ] has only been tested using Novozym, a considerable increase in the hydrolytic activity for the four isomers was observed. These results prompted us not to test this IL with the other biocatalysts. Table 2 shows the influence of the solvents on the enantioselectivity of various biocatalysts in the presence of an enantiomeric mixture of 1-chloro-2-octyl (1a) and 1-bromo-2-octyl (2a) palmitates. The hydrolysis was performed at 40 °C for 24 h unless another time is indicated in the Table. The biocatalysts and solvents showing hydrolysis above 30% of 1-halo-2-octyl regioisomers (1a and 2a) in the previous experiments were the only ones studied in this case. In all cases, the (S)-isomers were hydrolyzed more quickly than the corresponding (R)-isomers. These results were the opposite of those observed previously in the hydrolysis of chlorobutyl esters [3], but in accordance with the results described by Rotticci et al. [4] for similar esters of the same haloalcohols. The best results were obtained for Novozym ® 435 and [BMIM][PF 6 ] in terms of ee and E values. Both values are better than the values described previously for similar approaches. In contrast, reduction of α−haloketones with recombinant whole cells bearing an alcohol dehydrogenase and a glucose dehydrogenase [22], and dehalogenation of haloalcohols using dehalogenases gave the corresponding haloalcohols with similar ee [23]. Thus, kinetic resolution of 1-chloro-2-octanol and 1-bromo-2-octanol with Candida antarctica lipase B and hexane gave the two (S)-alcohols with an ee/E of 41%/14 and 56%/7.6 respectively [4]. Moreover, kinetic resolution of 1-chloro-2-octanol using PS-C "Amano" II and toluene produced the (R)-isomer with an ee/E of only 28%/3 [23].  6 ]. If the reaction time was increased, the percentage of hydrolysis increased but the percentage ee s of the reaction product decreased (data not shown). Several publications have already shown that using this IL and C. antarctica lipases for the resolution of enantiomeric alcohols by esterifications [24][25], transesterifications [26][27], and alcoholysis reactions [13] gave better results than using conventional organic solvents.

General Procedures
Reactions were performed in 3 ml Eppendorf vials by shaking the mixture at 1,400 rpm and 40 °C. 1 H-and 13 C-NMR spectra were recorded on a Varian Unity 400 spectrometer (at 400 MHz for 1 H-NMR and at 100 MHz for 13 C-NMR) using CDCl 3 as solvent, as indicated. Chemical shifts are reported in ppm (δ) relative to the TMS signal. Chiral gas chromatography analyses were carried out with a capillary column CP Chirasil-DEX CB (Varian) (25 m × 0.25 mm diameter, 0.25 μm film thickness). Hydrogen (2.8 mL/min) was used as the carrier gas. T injector = 230 °C, T detector = 250 °C. The GC temperature was programmed at 60 °C and held for 30 min, ramped at 10 °C/min to 180 °C and held for 2 min. Flash column chromatography (FCC) and analytical thin-layer chromatography (TLC) were performed using silica gel 230-400 mesh and precoated silica gel 60 F254 Merck plates, respectively. 1,2-Octanediol was obtained from Acros Organics. (R)-(+)-1,2-octanediol and immobilized lipase from Mucor miehei (Lipozyme ® ) were purchased from Sigma-Aldrich.
[BMIM][PF 6 ] was obtained from Merck. Palmitic acid was obtained from Riedel-de Haën. Immobilized lipase from Candida antarctica (Novozym ® 435) was a gift from Novo Nordisk. Resting cells of Rhizopus oryzae were obtained by the method described in Méndez et al [3].
[OMIM] [PF 4 ] was synthesized at Imperial College, London, U.K. All chemicals were used without further purification.

Procedure for the Syntheses of Halohydrin Standards
(R)-(+)-1,2-octanediol was used to obtain the corresponding haloesters as described above. The synthesis of the corresponding halohydrin mixtures was carried out by reduction of the parent esters as described in Wilen et al. [30]. The absolute configuration was assigned assuming an S N 2 process for the synthesis of the corresponding halohydrin esters [15]. These haloalcohols were used as external standards in the chiral chromatographic analyses.

General Procedure for Optimizing the Enzymatic Hydrolysis of Halohydrin Esters
Hydrolysis in t-butanol. Thirty milligrams of either lyophilized mycelium or commercial enzyme, water (5 μL) and a 0.17 M solution (0.5 mL) of each halohydrin palmitate in t-butanol were added to each vial. The reaction mixtures were incubated as described above for 24 h. Final mixtures were centrifuged at 3,000 rpm for 2 min and the clear supernatant was recovered by decantation. The organic solvent was evaporated with a nitrogen stream and halohydrin esters and haloalcohols were quantified by 1 H-NMR using 1,4-dichlorobenzene as the internal standard. Chiral GC-FID was used to determine the corresponding enantioselectivity of the process. All reactions were carried out in duplicate.
Hydrolysis in ionic liquids. One-half milliliter of a 0.17 M solution of each halohydrin palmitate in dichloromethane was added to each vial. The solvent was evaporated with a nitrogen stream, and 30 mg of either lyophilized mycelium or commercial enzyme, water (5 μL) and a solution of each IL (0.4 g) were added to each vial. The reaction mixtures were incubated as described above for 24 h or 3 h, depending on the ionic liquid and biocatalyst (see Tables 1 and 2). Final mixtures were extracted with hexane (4 × 0.4 mL). The hexane extracts were joined together and evaporated with a nitrogen stream. Halohydrin esters and haloalcohols were quantified by 1 H-NMR using 1,4-dichlorobenzene as the internal standard. Chiral GC-FID was used to determine the corresponding enantioselectivity of the process. All reactions were carried out in duplicate.

Conclusions
An enzymatic tandem method was developed to prepare (S)-1-halo-2-octanols with >98% ee from racemic 1,2-octanediol. First, the synthesis of regioisomeric mixtures of halohydrin esters was carried out using described methods. The regioisomeric mixtures were then hydrolyzed selectively using both resting cells of R. oryzae and commercial lipases. The regioselectivity of these hydrolyses depended on the solvent and the biocatalyst used. Finally, Novozym ® 435 was used to carry out a kinetic resolution of the almost pure racemic mixture obtained previously. Using palmitic acid and the corresponding enzymes and ionic liquids, (S)-1-chloro-2-octanol and (S)-1-bromo-2-octanol were obtained with 26% and 15% overall yield, respectively, from 1,2-octanediol.