WO2009153325A1

WO2009153325A1 - Optical resolution of a mixture of enantiomers of butynol or butenol

Info

Publication number: WO2009153325A1
Application number: PCT/EP2009/057633
Authority: WO
Inventors: Stefaan Marie André WILDEMAN DE
Original assignee: Dsm Ip Assets B.V.
Priority date: 2008-06-19
Filing date: 2009-06-18
Publication date: 2009-12-23

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, which is the same or a different alcohol dehydrogenase.

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 respectively ethynyl or ethenyl 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 (ADH) to 3-butyn-2-one or 3-buten-2one, respectively, and in the presence of a cofactor being regenerated by this or another alcohol dehydrogenase, and isolating the desired enantiomerically enriched compound. The present invention relates to a 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 a first alcohol dehydrogenase to 3-butyn-2-one or 3-buten-2-one, respectively, and in the presence of a cofactor and cofactor regeneration enzyme, which cofactor regenerating enzyme is an alcohol dehydrogenase which is the same as the first alcoholdehydrogenase or which is a second dehydrogenase, and isolating the desired enantiomerically enriched compound. In an embodiment, the process according to the invention is a 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 a first alcohol dehydrogenase to 3-butyn-2-one or 3-buten-2-one, respectively, and in the presence of a cofactor and cofactor regeneration enzyme, which cofactor regenerating enzyme is an alcohol dehydrogenase which is the same as the first alcoholdehydrogenase or which is a second dehydrogenase, and optionally isolating the desired enantiomerically enriched compound, with the proviso that the process is not a process wherein 10 g/l of racemic 3-butyn-2-ol, 500 mM NaHSO₃, 700 mg NAD⁺, 150 g of cell wet weight of cells expressing (S)-selective alcohol dehydrogenase of Pseudomonas aeruginosa (SEQ ID No. 1 ) and 50 ml. or 200 ml. of acetone or 2-octanone is used.

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 (ADH's) 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, SEQ ID NO. 4, SEQ ID NO. 6, SEQ ID NO. 8 and SEQ ID NO. 10. Preferably, proteins having a homology of at least 40%, more preferably at least 70%, even more preferably at least 90% with the sequence represented by SEQ ID NO:2 or SEQ ID NO: 4 are applied. To calculate the homology percentages of proteins, the following parameters Matrix: BLOSUM62, Open gap: 5, extension gap: 2, Penalties gap x_dropoff: 11 , Expected: 10, word size: 1 1 are used, e.g at http://www.ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi. The method of calculating homology percentages are described in detail by Tatiana A. Tatusova and Thomas L. Madden (1999), "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences", FEMS Microbiol Lett. 174:247-250). Most preferably ADH enzymes of the sequence represented by SEQ ID NO: 2 or SEQ ID NO: 4 are selected for the process of the invention.

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 which is the same or a different alcohol dehydrogenase as used for the oxidation of the non-desired enantiomer during the enzymatic resolution reaction. More preferably this oxidation of reduced cofactor, for example - A -

NAD(P)H, into oxidized co-factor, for example NAD(P)+, is effected by the same ADH that catalyses the oxidation of the undesired substrate enantiomer.

The process according to the invention is preferably carried out using only one or more ADH enzymes, more preferably, using only one type of ADH enzyme. Thus, the process according to the invention is preferably carried out without any other type of cofactor regeneration enzyme present. Thus, the cofactor regeneration enzyme consists of one or more ADH's only.

In another preferred embodiment of the invention this oxidation of reduced cofactor is catalyzed by an alcohol dehydrogenase in the presence of a ketone forming a second phase with water. More preferably, the reaction medium is chosen so that an emulsion is formed. Typically, to get an emulsion formed, the conditions for the reaction are chosen at a volumetric ratio of water over organic solvent, which organic solvent forms a two-phase system with water, exceeding the number of 1. More preferably, the volumetric ratio of water over the organic solvent is equal to or higher than 1.5.

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 10 mmol/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) 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. Most preferably, only one alcohol dehydrogenase is expressed in a host, which alcohol dehydrogenase is capable of oxidizing the non-desired enantiomer and of regenerating the co-factor. 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 water unsoluble 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. Prefered solvents are 4-methyl-2-pentanone, 2-pentanone, 3-pentanone, 2-hexanone, 3-hexanone, cyclo-hexanone, methyl iso-butyl ketone, 2-heptanone, 2-octanone. 2-Octanone is most preferred. 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.

When a two-phase system is formed with water and a co-solvent, the water concentration is preferably at least 40 vol%, more preferably at least 50 vol%, even more preferably at least 60% and preferably at most 80%, more preferably at most 70%, even more preferably at most 65% relative to the total volume of water and the co-solvent at the start of the process. An optimal range for the water concentration in the two-phase system is between 50 and 70% at the start of the process. When the water concentration during the process rises over 80% relative to the total volume of water and co-solvent, for example the butynol is taken up in the co-solvent at too low amounts, therewith increasing the toxicity to the alcohol dehydrogenase. Using a two- phase system has as an advantage that it allows higher compound concentrations to be used, exceeding 1wt% of compound, more 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%. When using racemic butynol or butenol as a starting material, the concentration preferably does not exceed 7.5 wt%, since higher concentrations would result in substrate-inhibition of the enzyme or enzymes used to an undesirable extent. 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 1 1. For the ADH according to SEQ ID NO 4, the pH is preferably chosen between 7-9, more preferably between 7.50-8.50.

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. The amount of NaHSO₃ or other neutralizing compounds may vary between wide ranges. The optimum amount can easily be determined by a person skilled in the art.

In a preferred embodiment, the process according to the invention is a process for the preparation of a desired enantiomerically enriched compound selected from the group of 3-buty-2-ol and 3-buten-2-ol, from an aqeous mixture of enantiomers of the compound by selectively oxidating the non-desired enantiomer of the compound in the presence of a first alcohol dehydrogenase to 3-butyn-2-one or 3-buten-2-one, respectively, and in the presence of a cofactor and cofactor regeneration enzyme, which cofactor regeneration enzyme is the same alcohol dehydrogenase or a different alcohol dehydrogenase than the first alcohol dehydrogenase, and wherein the reaction is carried out in a mixure of water and a ketone that forms a two-phase system with water, and which mixture comprises more than 50 vol% water relative to the total volume of water and ketone, and in the presence of NaHSO₃. Enantiomerically enriched compounds obtained with the process according to the present invention may be used as a building block in e.g. pharmaceutical and agro products.

The invention also relates to all possible combinations of the embodiments and/or preferred features as described above.

Example 1

Confirming appropriate enzyme candidates for enzymatic resolution of racemic

3-butyn-2-ol

Materials and methods Synthetic E. coli codon-optimized gene constructs of alcohol dehydrogenases (ADH's) (SEQ ID No. 1 , 3, 5, 7, 9) were prepared for expression in E. coli host cells. 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 OD_62O 0.6 under growth conditions. Cell densities of E. coli cells reached 30 gcww/l- Cells were harvested after centrifugation and frozen for 18 hours. Lysates of these cells were prepared after thawing by mixing 5 mg cell wet weight with 500 μl_ of lysate buffer containing 50 mM MOPS buffer (pH 7.5), 1.5 mg/mL dithiothreitol, 2 mg/mL lysozyme, 0.1 mg/mL DNAse and 1.2 mg/mL MgSO₄.

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

Ingredients needed and mixed: 150 μl_ of buffer (potassium phosphate buffer 50 mM at pH 7.5) containing 1 mM of each NAD⁺ and NADP⁺, 40 μl_ of the lysates prepared from each enzyme tested, and 20 μl_ of a 100 mM stock solution of racemic butynol. Cofactor consumption was followed in time, and samples were analyzed for conversion and enantiomeric excess. Enriched (R)-butynol was found for enzymes with SEQ ID NO 2, 4 and 6, while enriched (S)-butynol was found for enzymes with SEQ ID NO 8 and 10.

Example 2 Preparation of enantiomericallv enriched (R)(+)-3-butyn-2-ol by resolution of racemic 3-butyn-2-ol with (ADH) enzyme Materials and methods

A synthetic E. coli codon-optimized gene construct of (S)-selective alcohol dehydrogenase (ADH) of Pseudomonas aeruginosa (SEQ ID No. 3) was prepared for expression in E. coli host cells. 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 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 and mixed into a 1 liter flask: 275 mL of water, 30 g of Na₂SO₃, 3 g of Na₂S₂O₅, 30 mL of aqueous stock solution of NAD⁺ (15 g/L), 184.5 g 2-octanone, 54.3 g racemic butynol (97%). The pH was corrected from 7.8 to 8.0 with 1 M NaOH (6 mL). Cell suspension was added (108 mL suspension containing 54 gram demi water and 54 gram E. coli cell wet weight). Extra 30 ml. of water were used to rinse the cell suspension vessel, and this was added to the 1 liter flask. pH dropped to 7.94, and was corrected again with 1 ml. of NaOH. Start concentration of racemic butynol was 7.52 wt%, and the total weight of the 1 liter flask content reached 722 gram.

The reaction mixture was stirred at 500 rpm, and titration was performed with 4 M NaHSO₃ to keep the pH at 8.0. After 24 hours of reaction time and adding 198 ml. of 4 M NaHSO₃ solution, the enantiomeric excess of (R)-butynol reached 99%. The reactor was rinsed with water, and 990 gram final reaction mixture was collected. The enriched (R)-butynol was harvested after atmospheric distillation of this reaction mixture. Isolated yield of (R)-butynol was 24.7 g.

Example 3

Preparation of enantiomericallv 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. 3) were prepared for expression in E. coli host cells. 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 expressing E. coli cells reached 30 gcww/l- Cells were harvested after centrifugation.

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 3) (150 gcww/l), 50 ml. of acetone. Process conditions: pH was set at 9.0 and 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. After reaching enantiomeric excess of 5% (about 96 hours), the experiment was stopped. The experiment was then repeated 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 3).

Reason for the lower yield in the desired enantiomer is believed to be the toxicity of acetone and butynol towards the ADH. In contrast to example 1 , the bio-oxidation reaction described in this example does not provide efficient protection of the ADH against butynol since no sufficient amount of second phase is present lowering the aqueous concentration of butynol.

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 a first alcohol dehydrogenase to 3-butyn-2-one or 3-buten-2-one, respectively, and in the presence of a cofactor and cofactor regeneration enzyme, which cofactor regenerating enzyme is an alcohol dehydrogenase which is the same as the first alcoholdehydrogenase or which is a second dehydrogenase, and optionally isolating the desired enantiomerically enriched compound.

2. Process according to claim 1 , wherein the cofactor is a nicotinamide .

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 expressed in the same host.

7. Process according to any one of claims 1 - 6, wherein the alcohol dehydrogenase and the co-factor regeneration enzyme are each independently 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 any one of claims 1-7, wherein the cofactor regeneration enzyme consists of one or more alcohol dehydrogenases.