WO2008074506A1

WO2008074506A1 - Optical resolution of a mixture of enantiomers of butynol or butenol

Info

Publication number: WO2008074506A1
Application number: PCT/EP2007/011267
Authority: WO
Inventors: Stefaan Marie André WILDEMAN DE; Wolfgang Skranc; Henricus Martinus Maria Gerardus Straatman
Original assignee: Dsm Ip Assets B.V.
Priority date: 2006-12-21
Filing date: 2007-12-20
Publication date: 2008-06-26

Abstract

In the present invention a desired enantiomer of a compound selected from the group consisting of 3-butyn-2-ol and 3-buten-2-ol can be prepared from a racemic mixture containing the desired enantiomer thereof by selective oxidation of the non-desired enantiomer and isolation of the desired enantiomer. Such selective oxidation preferably can be carried out using a suitable enzyme system. Such an enzyme system may contain a suitable alcohol dehydrogenose and a cofactor and a cofactor regeneration enzyme.

Description

OPTICAL RESOLUTION OF A MIXTURE OF ENANTIOMERS OF BUTYNOL

OR BUTENOL

The invention relates to a process for the preparation of a desired enantiomer of a compound selected from the group of 3-butyn-2-ol and 3-buten-2-ol, from a (racemic) mixture of enantiomers of the compound by selective oxidation of the non-desired enantiomer and isolation of the desired enantiomer.

The optical resolution of said compounds from a (racemic) mixture poses serious problems to the man skilled in the art. Due to minimal sterical difference between both methyl and ethynyl ethenyl respectively groups within said compounds neither the use of an enzyme such as lipase nor the use of classical chemical methods for optical resolution of these alkynol compounds do provide preferred processes in terms of selectivity for and yield of the desired enantiomer. Derivatization at the β-position of the triple or double bonds of the C₄-compounds forming derivatives such as trimethylsilyl-alkynols, partially can solve this problem, but this adds chemical steps to the synthesis route.

The present invention provides a solution to these problems avoiding derivatization of the compound or other additional steps while maintaining a high stereoselectivity due to the recognition of only one of both enantiomers by certain dehydrogenase enzymes. Although the resolution of 3-butyn-2-ol has never been shown to proceed well in literature, the present invention surprisingly provides a solution for this problem.

According to the present invention the desired enantiomerically enriched compound selected from the group of 3-butyn-2-ol and 3-buten-2-ol can be prepared from a (racemic) aqueous mixture of the enantiomers by selectively oxidating the non-desired enantiomer of the compound in the presence of an alcohol dehydrogenase to 3-butyn-2-one or 3-buten-2one, respectively, and in the presence of a cofactor and cofactor regeneration enzyme, and isolating the desired enantiomerically enriched compound. In a preferred embodiment, the present invention relates to optical resolution of a racemic mixture containing an alkynol compound of the general formula [1]

According to a preferred embodiment the invention results in the separation of the desired enantiomeric form of 3-butyn-2-ol, or 3-buten-2-ol, respectively, from 3-butyn-2-one or 3-buten-2-one, respectively, and the target enantiomer of 3-butyn-2-ol or 3-buten-2-ol may subsequently be isolated and purified to the desired purity.

A cofactor suitable for use in the process of the present invention includes any oxidized cofactor, for instance an oxidized nicotinamide cofactor, preferably nicotinamide adenine dinucleotide (NAD⁺) or nicotinamide adenine dinucleotide phosphate (NADP⁺). During the process of the invention, these cofactors will be reduced, for example in the case of NAD⁺ to NADH and for example in the case of NADP⁺ to NADPH.

In principle the concentration of cofactor used in the process of the present invention is not critical. Preferably, cofactor concentrations of between

0.01 mol/l and 10 mmol/l are used, more preferably between 0.1 mmol/l and 1 mmol/l, in particular between 0.2 mmol/l and 0.5 mmol/l.

In the framework of the present invention, alcohol dehydrogenase is defined as an enzyme capable of catalyzing the oxidation of an alcohol to the corresponding ketone or corresponding aldehyde, preferably, also capable of catalyzing the reduction of a ketone or an aldehyde to the corresponding alcohol.

Alcohol dehydrogenases suitable for the invention include: alcohol dehydrogenase from EC class: 1.1 , preferably from EC class 1.1.1. Alcohol dehydrogenases are abundant and may for instance be isolated from living organisms, preferably microorganisms, such as yeasts, bacteria and fungi. Examples of alcohol dehydrogenases include lactate dehydrogenases.

An alcohol dehydrogenase may for example be selected for the process of the invention by screening several enzymes or host cells expressing genes encoding alcohol dehydrogenases. Suitable ADH enzymes can for example be selected from the group of ADH enzymes represented by the sequences SEQ ID NO. 2 and SEQ ID NO. 4. During the enzymatic process of selective oxidation of the compound, the reduced form of the cofactor will be formed. In the process according to the invention the reduced cofactor is being oxidized to the oxidized form of the cofactor by a cofactor regeneration enzyme during the enzymatic resolution reaction. More preferably this oxidation of reduced cofactor, for example

NAD(P)H, into oxidized co-factor, for example NAD(P)+, is effected by a cofactor regeneration enzyme, for example a NAD(P)H oxidase. Suitable NAD(P)H oxidases are for example derived from Bacillus sp., Lactobacillus sp.and Streptococcus sp. In another preferred embodiment of the invention this oxidation of reduced cofactor is catalyzed by a cofactor regeneration enzyme in the presence of a ketone forming a second phase with water.

Enzymes may be used as cell free extracts or as part of a whole cell catalytic system co-expressing both required enzymes. In a preferred embodiment, a whole cell catalytic system co-expressing both required enzymes is used. In principle the concentration of the compound used in the process of the present invention is not critical. Preferably, the compound is used in a concentration of at least 1 mmol/l, more preferably at least 10mmol/l, in particular at least 0.1 mol/l, more in particular at least 1 mol/l.

Preferably the compound is used in a concentration of not more than 5 mol/l, more preferably not more than 4 mol/l.

The amount of enzyme (alcohol dehydrogenase and/or NAD(P)H oxidase) used is in principle not critical. For purpose of the invention with Unit (U) is meant 1 micromole substrate converted per minute at 37°C at pH 7.0 and at 1 bar pressure. Preferably, between 100 and 100,000 U per liter reaction mixture is used, more preferably at least 200 U per liter reaction mixture and most preferably at least 1000 U per liter reaction mixture is used.

The alcohol dehydrogenase and the cofactor regeneration enzyme may be used in any form. For example, the alcohol dehydrogenase and the cofactor regeneration enzyme may be used - for example in the form of a dispersion, emulsion, a solution or in immobilized form - as crude enzyme, as a commercially available enzyme, as an enzyme further purified from a commercially available preparation, as an enzyme obtained from its source by a combination of known purification methods, in whole (optionally permeabilized and/or immobilized) cells that naturally or through genetic modification possess alcohol dehydrogenase and/or cofactor regeneration enzyme activity, or in a lysate of cells with such activity. In a preferred embodiment of the invention, both alcohol dehydrogenase and cofactor regeneration enzyme are coexpressed in one host allowing whole cell application shuttling the cofactor within the cell and avoiding difficult work-up procedures.

The process of the present invention may be performed in a batch process. Alternatively, the process of the present invention is performed in a

(semi)continuous process, in effect a process wherein the compound and/or at least one of the enzymes is (semi) continuously fed to the reactor and wherein the formed enantiomerically enriched compound and/or the 3-butyn-2-one or 3-buten-2-one, hereinafter referred to as "the oxidized compound" is (semi)continuously removed from the reactor.

In a preferred embodiment of the invention, the enantiomerically enriched compound is removed from the reaction mixture, for instance by distillation. It is known to the person skilled in the art which conditions are suited for distilling the enantiomerically enriched compound. In principle, in the present invention, solvents may be chosen from a wide range of solvents . For example, a ketone different from 3-butyn-2-one may function both as a solvent and as a cofactor oxidizing agent, for example: formaldehyde, acetaldehyde, acetone, 2-butanone, 4-methyl-2-pentanone, 2- pentanone, 3-pentanone, 2-hexanone, 3-hexanone, cyclo-hexanone, methyl iso-butyl ketone, 2-heptanone, 2-octanone. Water may also be chosen as the sole solvent, which is advantageous from a practical and environmental point of view. Of course it is also possible to use combinations of solvents, for example combinations of solvents with water and a solvent as mentioned above. The use of alcohols is not recommended because they can compete with the compound as a substrate for the alcohol dehydrogenase present in the process. In a preferred embodiment of the process according to the invention, the reaction is carried out in a two phase system. More preferably, the process is carried out in a system comprising water and a ketone having a boiling point exceeding 135⁰C at atmospheric pressure, for example 2-octanone.

The water concentration is preferably at least 1 vol%, more preferably at least 5 vol%, even more preferably at least 10 vol%, in particular at least 20 vol%, more in particular at least 30 vol%. Using a two-phase system has as an advantage that it allows higher compound concentrations to be used, exceeding 1wt% of compound, mor prefereably exeding 2wt% of compound, relative to the total weight of the reaction mixture, while still achieving conversions exeeding 50% and enantiomeric excess exceeding 60%, preferably 70%, more preferably 90%. Most preferred, the ee of the process according to the invention exceeds 95%.

The choice of the reaction conditions of the process of the invention depends on the choice of the enzyme system used for the optical resolution. Usually, the temperature of the process is chosen between 0 and 90⁰C, in particular between 10 and 70⁰C, more in particular between 20 and 50⁰C; usually the pH of the process is chosen between 5 and 12, more preferably between 6 and 11. For the ADH according to SEQID NO2, the pH is preferally chosen between 8-10, more preferably between 8.50-9.50.

In case an NAD(P)H oxidase is used as cofactor regeneration enzyme, preferably the used oxygen concentration is as high as possible in view of solubility and safety. Depending on the pH, the temperature and the chosen solvent, more or less oxygen will dissolve in the reaction mixture. Oxygen transfer to the reaction mixture can be enhanced using methods known to the person skilled in the art, for example by oxygen transfer systems used in large-scale fermentors, for example by the use of nozzles.

Isolation of the enantiomerically enriched compound may be performed by methods known to the person skilled in the art. For example, it may be isolated by evaporation of the organic phases, such as the organic solvents that may be present and the reduced form of the ketone. Such conditions are known to the person skilled in the art. In a preferred embodiment, the process according to the invention is a process wherein 3-butyn-2-one or 3-buten-2-one, respectively, is wholly or partially removed from the reaction mixture, or wholly or partially neutralized.

Neutralization of 3-butyn-2-one and 3-buten-2-one may for example be carried out by adding NaHSO₃. Enantiomerically enriched compound according to the present invention may be used as a building block in e.g. pharmaceutical and agro products.

Example 1

Preparation of enantiomerically enriched (R)(+)-3-butyn-2-ol by resolution ofracemic 3-butyn-2-ol with (ADH) enzyme co-expressed with NADH oxidase (NOX)

Materials and methods

Synthetic E. coli codon-optimized gene constructs of (S)-selective alcohol dehydrogenase (ADH) of Pseudomonas aeruginosa (SEQ ID No. 1) and NADH oxidase (NOX) of Streptococcus mutans (SEQ ID No. 5) were prepared for co- expression in a single E. coli host cell. Cloning was done with Gateway technology (Invitrogen) towards pBAD-DEST expression vectors. The E. coli host cells were TOP10 cells; competent cells were used in transformation experiments (purchased at Invitrogen). The expression clones were transformed via the standard heat-shock transformation protocol of Invitrogen. Medium used in fermentation experiments was Luria Bertani broth, applying 100 mg/l carbenicillin as antibiotic and 0.02 wt%

L-arabinose as inducer. Induction of cells occurred at OD₆₂o 0.6 under fermentation conditions. Cell densities of co-expressing E. coli cells reached 30 gcww/l- Cells were harvested after centrifugation.

Racemic 3-butyn-2-ol and NAD⁺ were purchased at Sigma-Aldrich. LB broth was purchased at Difco (BD).

Biooxidation reaction

Ingredients needed: 1 liter of water containing racemic 3-butyn-2-ol (50 g/l), 500 mM NaHSO₃ (diluted from 2 M NaHSO₃ stock solution set at pH = 8), NAD⁺ (700 mg), 75 gram of cell wet weight of cells co-expressing ADH (SEQ 1) + NOX (SEQ 5) (75 gcww/l). Second phase is 1 liter of 2-octanone.

Process conditions: pH was set at 9.0 (pH stat dosing 1 M NaHSO₃), oxidation of (S)-enantiomer was followed in time, till high enantiomeric excess of (fi)-enantiomer was obtained. Open Erlenmeyer flasks (5 liter) contained 2 liter reaction mixture well stirred at 500 rpm with a magnetic stirrer to oxygenate sufficiently. After reaching high enantiomeric excess (about 24 hours), both phases are separated after which the aqueous phase is extracted twice with 100 mL 2-octanone and (R)(+)-3-butyn-2-ol is harvested from the resulting 700 mL 2-octanone phase by distillation.

Comparative example 1

Preparation of weakly enantiomerically enriched (R)(+)-3-butyn-2-ol by resolution of racemic 3-butyn-2-ol with (ADH) enzyme

Materials and methods Synthetic E. coli codon-optimized gene constructs of (S)-selective alcohol dehydrogenase (ADH) of Pseudomonas aeruginosa (SEQ ID No. 1 ) were prepared for co-expression in a single E. coli host cell. Cloning was done with Gateway technology (Invitrogen) towards pBAD-DEST expression vectors. The E. coli host cells were TOP10 cells; competent cells were used in transformation experiments (purchased at Invitrogen). The expression clones were transformed via the standard heat-shock transformation protocol of Invitrogen. Medium used in fermentation experiments was Luria Bertani broth, applying 100 mg/l carbenicillin as antibiotic and 0.02 wt% L-arabinose as inducer. Induction of cells occurred at OD₆₂₀ 0.6 under fermentation conditions. Cell densities of expressing E. coli cells reached 40 gcww/l- Cells were harvested after centrifugation.

Biooxidation reaction Ingredients needed: 1 liter of water containing racemic 3-butyn-2-ol

(10 g/l), 500 mM NaHSO₃ (diluted from 2 M NaHSO₃ stock solution set at pH = 8), NAD⁺ (700 mg), 150 gram of cell wet weight of cells expressing ADH (SEQ 1 ) (150 gcww/l), 50 ml. of acetone.

Process conditions: pH was set at 9.0 (pH stat was not needed because of pH neutral reaction), oxidation of (S)-enantiomer was followed in time. No high enantiomeric excess of (R)-enantiomer was obtained (5% e.e.). Open Erlenmeyer flasks (5 liter) contained 1 liter reaction mixture well stirred at 500 rpm with a magnetic stirrer to oxygenate sufficiently. After reaching enantiomeric excess of 5% (about 96 hours), the experiment was stopped and retested with 200 mL acetone in stead of 50 mL acetone. No increase in e.e. could be obtained, although acetone is an excellent oxidative substrate for ADH (SEQ 1 ).

The same results were obtained by substituting acetone by 2- octanone.

Claims

1. Process for the preparation of a desired enantiomerically enriched compound selected from the group of 3-butyn-2-ol and 3-buten-2-ol, from an aqueous mixture of enantiomers of the compound by selectively oxidating the non- desired enantiomer of the compound in the presence of an alcohol dehydrogenase to 3-butyn-2-one or 3-buten-2-one, respectively, and in the presence of a cofactor and cofactor regeneration enzyme, and isolating the desired enantiomerically enriched compound.

2. Process according to claim 1 , wherein the cofactor is a nicotinamide and wherein the cofactor regeneration enzyme is a NAD(P)H oxidase.

3. Process according to claim 1 or 2, wherein 3-butyn-2-one or 3-buten-2-one, respectively, is removed from the reaction mixture or neutralized.

4. Process according to any one of claims 1-3, whereby the process is carried out within a two phase system.

5. Process according to any one of claims 1-4, wherein the compound and/or enzymes are fed to the process over time.

6. Process according to any one of claims 1-5, wherein the alcohol dehydrogenase and the cofactor regeneration enzyme are co-expressed.

7. Process according to any one of claims 1 - 6, wherein the alcohol dehydrogenase is selected from the group of alcohol dehydrogenases represented by the sequences according to SEQ ID NO. 2 and SEQ ID NO. 4.

8. Process according to any one of claims 2 - 6, wherein the NAD(P)H oxidase is represented by the sequence according to SEQ ID NO. 6.