Enzymatic process for the preparation of optically active alcohols from ketones using tuberous root Daucus carota

This invention relates to an enzymatic process for the preparation of optically active chiral alcohols using tuberous root Dallcus carota . More particularly, the present invention relates to an enzymatic process for the preparation of optically active alcohols by enantioselective reduction of corresponding ketones using tuberous root Daucus carota.

In recent years, great attention has been paid to asymmetric synthesis of chiral synthons that are used in developing modern drugs and agrochemicals. Chiral alcohols are one of the well-known synthons that can be obtained from corresponding prochiral ketones by asymmetric reduction.

However, numerous reduction reactions were carried out using different chemical and biocatalyst reductions (Corey. E. J., Helal, G. J. Angewantha Chern Int. Eng. (1998) 91 (1986) and Csuk, R. et al. Chern. Rev. (1991) 91 46; these reactions have some difficulties in attaining high chemical yield and optical purity under ecofriendly conditions. Asymmetric reduction by means of chemical methods involves use of expensive chiral reagents and environmental hazardous heavy metals. On the contrary use of biocatalyst microorganisms or plant cells for reduction of prochiral ketones yielding the corresponding optically active alcohols with excellent enantioselectivity are of present day interest.

The applicants now for the first time describe the novel methodology in oxido reduction of some of the pro-chiral functional keto functionalities e.g., acetophenones, cyclic ketones, β-keto esters, azidoketones and aliphatic ketones etc. along with their corresponding substituents using crude reductive enzymes isolated from tuberous roots carrot (D.corta) with yields ranging from 70–98%, with Ees' 92–98%, most preferably showing ‘S’ configurations. The final reduction products in all the cases leading to valuable chiral intermediates, which can be further used for total synthesis of various drugs and agrochemicals.

The main object of the present invention is to provide a process for the preparation of optically active chiral alcohol from corresponding ketones using a crude extract of Daucus carota or an enzyme reductase isolated from Daucus carota.

Another object of the invention is to provide a process of reducing optically active chiral ketones using a crude extract of Daucus carota or an enzyme reductase isolated from Daucus carota to produce corresponding optically active chiralalcohols.

Accordingly, the present invention provides an enzymatic process for the preparation of optically active chiral alcohols using tuberous root Daucus carota . More particularly, the present invention relates to an enzymatic process for the preparation of optically active alcohols by enantioselective reduction of corresponding ketones using tuberous root Daucus carota.

This invention involves the enantioselective reduction of ketones using a reducing enzyme (reductase) isolated from tuberous roots for example carrot and beet root. Stereochemistry is one of the most important parameters governing the biological activity of organic compounds. Therefore, chirality is emerging as a key for both academic and industrial laboratories in synthesis of organic chemicals in the area of pharmaceuticals and agrochemicals. Benefits from the use of single enantiomers include avoidance of gratuitous environmental contamination, separation of interfering activity or toxicity and less material to be processed with reduced cost and effluent treatment.

Accordingly, the present invention provides a process for the preparation of optically active chiral alcohols from ketones, said process comprising reducing the ketones to corresponding optically active chiral alcohols using a crude extract of Daucus carota or an enzyme reductase isolated from Daucus carota

One embodiment of the invention provides a process comprising adding a ketone to a crude extract of Daucus carota in about 0.1 M to 2.0 M buffer of pH ranging between 6.0 to 8.0, incubating the reaction mixture at a temperature of 25 to 40° C. for a period ranging from 10 to 110 hours, and isolating the product followed by purification to obtain the desired product.

In another embodiment of the invention, the incubation is preferably carried out at a temperature in the range of 35 to 40° C.

In another embodiment of the invention, the ketone used is selected from a group consisting of unsubstituted/substituted ketones, from alkyl aryl ketones, cyclic ketones, β-ketoesters, azido ketones and aliphatic ketones.

In still another embodiment, the alkylaryl ketone is selected from the group consisting of p-chloro acetophenone, p-bromo acetophenone, p-fluoro acetophenone, p-nitro acetophenone, p-methyl acetophenone, p-methoxy acetophenone, p-hydroxy acetophenone, 1-(2-naphthyl)-1-ethanone, 1-(6-methyl-2-naphthyl)-1-ethanone and 1-(2-furyl)-1-ethanone.

In still another embodiment, the cyclic ketone is selected from a group consisting of -tetralone, 2-tetralone, 6-methoxy-1-tetralone and 1-indalone.

In yet another embodiment, the p-ketoester is selected from a group consisting of ethylacetoacetate, ethyl-4-chloro-3-oxobutanoate, ethyl-4-bromo-3-oxobutanoate, ethyl-4-azido-3-oxobutanoate, ethyl-3-oxo-3-phenylpropanoate, ethyl-4,4,4 trichloro-3-oxobutanoate, 4,4,4 trifluoro-3-oxobutanoate, ethyl-3-oxo-4-phenylsulfonylbutanoate, ethyl-2-oxo-1-cyclopentanecarboxylate and ethyl-2-oxo-1-cyclohexanecarboxylate.

In yet another embodiment, the substituted azidoketone is selected from a group consisting 2-azido-1-phenyl-1-ethanone, 2-azido-1-(4-chlorophenyl)-1-ethanone, 2-Azido-1-(4-methylphenyl)-1-ethanone, 2-azido-1-(4-methoxlphenyl)-1-ethanone, 2-azido-1-(4-fluorophenyl)-1-ethanone, 2-azido-1-(4-fluorophenyl)-1-ethanone, 2-azido-(4-tertbutyldimethyl-silyloxyphenyl)-1-ethanone, 2-azido-1-(2-furyl)-1-ethanone, 2-azido-1-(2-thienyl)-1-ethanone and 2-azido-1-(2-napthyl)-1-ethanone.

In still another embodiment, the substituted open chain ketone is selected from a group consisting 2-butanone, 2-pentanone, 2-hexanone, 4-methyl-2-pentanone, 3,3-dimethyl-2-butanone and 2-heptanone.

In yet another embodiment, the substituted ketone is selected from a group consisting acetophenone, tetralone, ethylacetoacetate, 2-azido-1-phenyl-1-ethanone and 2-hexanone.

Another embodiment of the present invention provides a process, wherein the buffer used is selected from sodium phosphate, sodium acetate, potassium phosphate, potassium acetate, and tris HCl.

In another embodiment, the incubation period used is preferably in the range of 30 to 100 hours.

In another embodiment, the reductase enzyme after completion of the reduction is filtered, washed with a buffer solution and again used for carrying out a reduction reaction.

In another embodiment, the percentage yield of optically active chiral alcohol consisting of aliphatic and aromatic alcohol obtained from the corresponding ketone is in the range of 30–95 wt percent;

Yet another embodiment, the optical purity (ee) of chiral alcohols obtained is in the range of 90 to 100%.

FIG. 1 : Flowsheet of a novel enzymatic process for enantioselective reductions of ketones using tuberous root Daucus carota.

FIGS. 2 & 2 a represent conversion charts for some of the ketones obtained using Daucus carota root.

Reduction of ketones with Daucus carota root: Ketones (50–200 mg) were added to a stirred suspension of freshly cut/homogenized tuberous root Daucus carota (5–25 gm, protein content 9–15 gm/ml Biuret method) in 50–100 ml of 0.1 to 2 M sodium phosphate buffer pH 6 to 8 and the reaction mixtures containing substrate (100–500 mg) were stirred at temperatures 25 to 35° C. for the incubation time of 10 to 20 hrs to obtain the appropriate conversion. Filtrates containing products were extracted in polar organic solvents. The organic phase was dried in anhydrous Na 2 SO 4 and then concentrated in vacuum. The final products were purified by flash chromatography and the compound structure was confirmed by spectral data.

For the mini preparative scale reactions, the substrate ketones (5–10 gm) were taken in a conical 2 L flask and water (500–800 ml) was added to it followed by addition of freshly cut/isolated reductase from carrot (100–500 g). The reaction mixture was stirred in an incubator shaker for the required time (10–90 hrs), later the product alcohol was extracted and the structures were confirmed by known spectral data.

Preparative scale production: Several ketones that afforded high enantioselectivity for the reduction on a small scale were taken up for a large batch (30–50 gm) synthesis of chiral alcohols. The isolated yields and Ees of the reduction were summarized in Table 6.

The applicants have also performed the reduction of some ketones with Daucus carota root in an aqueous-organic biphasic reaction system. Common organic solvents, which were immiscible in water, have been used e.g., ethylacetate, hexane, cyclohexane, etc. However, the rate of reaction decreased significantly, due to enzyme unstability in organic solvents.

Recycling of the Daucus Carota root: After reaching the completion of the reduction reaction the mixture was filtered and the crude enzyme of D. carota washed successively with buffer pH 7.4. The crude enzyme was again reused to carry out a reduction reaction. It was observed that the activity of the enzymes was decreased significantly; only 20–30% conversion was achieved for acetophenone, after four repeated experiments.

The following examples are given by the way of illustration and should not be construed to limit the scope of the invention.

Advantages of a novel enzymatic process using tuberous root Daucus carota in catalyzing the enantioselective reduction of ketones.

1. The reaction conditions employed to develop chiral alcohols from corresponding ketones are simple, economical and eco-friendly conditions with high enantioselectivity and yields. 2. The advantages to this reduction over the traditional yeast mediated reduction are:

a) Easy availability b) Low cost of the enzyme source c) Easy isolation/separation of the product from the reaction medium, useful in scale-up process