IE903437A1

IE903437A1 - Enantioselective enzymatic synthesis of s(-)- and¹r(+)-esters of 4-hydroxy-2-cyclopenten-1-one and its ketal¹formed with 2,2-dimethyl- propane-1,3-diol

Info

Publication number: IE903437A1
Application number: IE343790A
Authority: IE
Original assignee: Europ Economic Community
Priority date: 1989-09-25
Filing date: 1990-09-24
Publication date: 1991-04-10
Also published as: JPH06500458A; CA2066689A1; DK37392D0; PT95404A; EP0419988A1; LU87593A1; DK37392A; WO1991004337A1

Abstract

The S(-) and R(+) esters of 4-hydroxy-cyclopentene-1-one and its 2',2'-dimethylpropane 1',3'-diol-ketal are produced by direct, enantioselective, enzymatic synthesis by reacting the corresponding 4-hydroxy compounds with an ester as acyl donor in the presence of an enzyme. The esters obtained are valuable intermediate products for stereospecific synthesis, in particular for the stereoselective synthesis of chemical compounds, in particular stable, storable, chiral prostaglandin derivatives, especially storable chiral prostaglandinsynthone.

Description

Enantioselective enzymatic synthesis of S(—)— and R( + )esters of 4-hydroxy-2-cyclopenten-l-one and its ketal formed with 2,2-dimethylpropane-l,3-diol The invention relates to the enantioselective enzymatic synthesis of S(—)— and R(+)-esters of 4hydroxy-2-cyclopenten-l-one and its ketal formed with 2,2-dimethylpropane-l,3-diol, and it particularly relates to a process for the enantioselective preparation of the said S(—)— and (R(+)-esters of the general formula (la) and (lb), respectively, indicated below (la) (lb) which are valuable intermediates for the targeted, that is to say stereo- and enantioselective, synthesis of chemical compounds, especially chiral prostaglandin derivatives which are stable on storage. - 2 German Offenlegungsschrift 37 24 721 has disclosed the preparation from racemic ketal acetate by hydrolytic ester cleavage in the presence of an enzyme, that is to say by enzymatic hydrolysis, of the corres5 ponding S(-)-alcohol, with the R(+)-ester being left behind. However, the S(-)-alcohol contained in the reaction mixture (pH 7) is unstable in the aqueous reaction system and the further processing thereof to the S(-)-ester by chemical esterification is extremely technically complicated, and the desired final product is obtained in only relatively low yields.

Hence the object of the invention was to find a way for selectively preparing the S(-)- and R(+)-esters of 4-hydroxy-2-cyclopenten-l-one and its ketal formed with 2,2-dimethylpropane-l,3-diol, which is industrially straightforward and provides the desired final product in high purity and yield.

It has now been found that this object can be achieved according to the invention by preparing in a reversal of the already disclosed enantioselective enzymatic cleavage of the racemic ketal acetate in an aqueous medium buffered to pH 7 the S(-)- and R(+)-esters of 4-hydroxy-2-cyclopenten-l-one and its ketal formed with 2,2-dimethylpropane-l,3-diol by targeted, that is to say stereo- and enantioselective, enzymatic synthesis from the corresponding racemic hydroxyl compound and from an acid ester as acyl donor in the presence of an enzyme in an organic solvent.

The invention relates to a process for the - 3 enantioselective preparation of S(—)— and R(+)-esters of 4-hydroxy-2-cyclopenten-l-one and its ketal formed with 2,2-dimethylpropane-l,3-diol, of the general formula (la) and (lb) respectively (la) (lb) which is characterized in that a) a racemic mixture of 4-hydroxy-2-cyclopenten-l-one or its ketal formed with 2,2-dimethylpropane-l,3diol, of the formula (II), is reacted in the presence of an enzyme with an ester of the general formula (III) as acyl donor in organic phase, preferably in anhydrous organic phase, at room temperature or elevated temperature, with the formation of the S(-)-ester (la) and with the R( + )alcohol of the formula (lib) being left behind, in accordance with the reaction equation: (II) (la) (lib) + R - OH (IV) in which acyl denotes any desired acid residue, preferably the residue of an organic Ci-Caacarboxylic acid, and R denotes an optionally substituted, unbranched or branched alkyl, alkenyl or alkynyl group with 1 to 22, preferably 1 to 12, in particular 1 to 6, carbon atoms, an optionally substituted aryl group with 6 to 12, preferably 6 to 10, carbon atoms, or an optionally substituted heterocyclic group with 5 to 10, preferably 5 to 7, ring atoms, which contains at least one nitrogen, oxygen and/or sulphur atom as hetero atom(s), b) the product mixture obtained in stage (a) is fractionated by chromatography or extraction into the S(-)-ester of the formula (Ia), which is obtained as first final product, and into the R(+)-alcohol of the formula (lib), which - 5 c) is converted by chemical or enzymatic esterification with the ester of the formula (III) into the R(+)ester of the formula (lb) which in turn is obtained by chromatography or extraction in pure form as second final product, in accordance with the reaction equation: + Acyl-OR ~> (III) 0. 0 OAo/l (lb) + R - OH (IV) It is possible by the process according to the invention to prepare in an industrially straightforward and economic manner, by direct enantioselective enzymatic 10 synthesis, avoiding the unstable S(-)-hydroxy compound, the S(-)- and R(+)-esters of 4-hydroxy-2-cyclopenten-lone and its ketal formed with 2,2-dimethylpropane-1,3diol, which are valuable intermediates for the synthesis of a chiral prostaglandin synthon which is stable on 15 storage. In particular, 4-acetoxycyclopentenone and its ketal are key substances in the synthesis of cyclopentanoid natural substances (compare German - 6 Of fenlegungsschrift 37 24 721, R. Noyori et al, Angewandte Chemie, 96 . 854 (1984), and E. Winterfeldt et al, Angewandte Chemie, 94., 496 (1982)).

When carrying out the process according to the 5 invention, the desired final products are obtained in stable form and extremely high purity and yield. Furthermore, the process according to the invention has the following industrial advantages over the process disclosed in German Offenlegungsschrift 37 24 721: besides the smaller number of synthetic steps, there is a distinct reduction in the solvent requirement; the reaction takes place in very small volumes; it is unnecessary to use toxic or carcinogenic extracting solvents because direct working up is possible, which is also associated with an energy saving; the enzyme can easily be removed (for example by filtration) and no immobilization is necessary; continuous use with an enzyme cartridge (fixed bed) is directly possible; and no use of solubilizers is necessary.

When carrying out the process according to the invention, the acyl donor which is preferably used is an ester of the general formula R1 - C - 0R2 V in which Rx and R2, which can be identical or different, each have the meanings indicated above for R.

It is very particularly preferred to use the ethyl ester of acetic acid (ethyl acetate) and a glycerol - Ί triester of an organic acid with 1 to 22 carbon atoms, especially triacetin, tributyrin and the like, as acyl donor, the latter being distinguished by their nontoxicity and their high reaction rate.

When carrying out the process according to the invention, conventional flash column chromatography is preferable to extractive working up (compare Still et al, J. Org. Chem., 43., 2 923 (1978)), as has also been used in the examples hereinafter. When carrying out stage (c) of the process according to the invention, the enzymatic esterification is preferable to standard chemical esterification (with an acid chloride/acid anhydride and pyridine and an alcohol). The procedure for this process corresponds to the procedure for the enantioselective esterification. Direct enantioselective enzymatic esterification according to the present invention is particularly advantageous for the ketalized compound, it being possible to achieve complete separation of the enantiomers with the ketal formed with 2,2-dimethylpropane-l,320 diol (= ketal alcohol), in which case it is easy to obtain, by subsequent deketalization, the enantiomerically pure ester of non-ketalized 4-hydroxycyclopentenone (= keto alcohol) (for example with addition of catalytic acetic acid or formic acid at room temperature by shaking for 1 hour or leaving to stand for 48 hours on Merck silica gel No. 9385).

Whereas in the case of the keto substrate of the formula (II) in stage (a) of the process according to the invention the enzyme which is preferably used is pig - 8 liver esterase, in the case of the ketal substrate of the formula (II) the enzyme which is preferably used is lipase, in particular a lipase which has been obtained from Pseudomonas fluorescens (Amano lipase P, batch no.

LPL 05518).

A 60 % enantioselective enzymatic esterification of the keto substrate is achieved according to the invention with Pseudomonas fluorescens lipase (for example Amano lipase P, batch no. LPL 05518 or Rohm, EL 220-88). In the case of the ketalized compound, complete separation of enantiomers can be achieved with the following lipases; lipases from Pseudomonas fluorescens (for example Amano lipase P, batch no. LPL 05518 or Rdhm, EL 220-88), Candida cylindracea (manufacturer, inter alia, Amano lipase AY, batch no. LAY MO 3517 or Sigma, cat. no. L-1754, batch no. 34F-0621), porcine pancreas (manufacturer, inter alia, Rohm, batch no. 7023 C, Sigma, cat. no. L-3126, batch no. 74F-0470) and Mucor miehei (Gist-Brocades, batch no. 0282).

The enzymes are preferably employed in excess.

Enzyme/alcohol ratios of from 0.5:1 to 10:1 by mass have proved advantageous, depending on the activity (U) of the enzyme (reference reaction: hydrolysis of a triglyceride or solvent ester, U = μ mol of fatty acid equivalent per minute at constant pH in aqueous emulsion, also in accordance with the statements of the enzyme manufacturers). The immobilized Pseudomonas lipase from Rohm has, for example, a 6-times lower specific activity, and thus a correspondingly higher dose is necessary. - 9 When carrying out the process according to the invention, the organic solvent preferably used in stage (a) is a hydrocarbon such as n-heptane, i-octane etc., or an ester, especially cyclohexyl acetate, specifically the ester employed as acyl donor, especially tributyrin.

The transesterification carried out in stage (a) of the process according to the invention can be carried out at room temperature or elevated temperature, preferably at a temperature in the range from 40 to 75°C, in particular 58 to 62°C, specifically at 60°C.

The organic solvent and the alcohol of the formula (II) are preferably employed in stage (a) of the process according to the invention in a ratio of from 4:1 to 100:1, in particular from 5:1 to 10:1, by mass, with the ratio of solvent to alcohol also being crucially determined by the solubility of the alcohol used.

The enzyme and the alcohol of the formula (II) are preferably employed in stage (a) of the process according to the invention in a ratio of from 0.5:1 to :1, specifically 0.5:1 to 1.5:1, in particular 0.7:1 to 0.9:1, by mass .

According to a particularly preferred embodiment of the process according to the invention it is possible, in place of the esterification of the R(+)-alcohol of the formula (lib) in stage (c), to carry out a racemization of the alcohol and recycling thereof to stage (a), which makes it possible to achieve complete enantioselective esterification of the racemic hydroxy compound of the formula (II) to the S(-)-ester of the formula (la). - 10 The course of the reaction, and the enantiomer ratio can be determined by use of high-pressure liquid chromatography (HPLC with the chiral Daicel OA HPLC column, eluent n-hexane/isopropanol 10:1) or capillary gas chromatography (Lipodex* A column from Macherey & Nagel). The details of these processes correspond to the general laboratory standard (taking account of the manufacturers' manuals). Finally, the enantiomer ratio and the absolute configuration can be confirmed in a standard way by H-NMR of the Mosher esters (compare J.A. Dale et al, JACS, 95, 512-519 (1973), and German Offenlegungsschrift 37 24 721).

The enzyme used according to the invention can be employed in free form, that is to say dissolved or suspended in the organic reaction medium, or in immobilized form, for example in a form entrapped in a polymer substrate.

The formulae Ia, lb, II and lib indicated above represent combined formulae in which the structural formulae of 4-hydroxy-2-cyclopenten-l-one and its ester derivatives on the one hand, and its ketal formed with 2,2-dimethylpropane-l,3-diol and its ester derivatives on the other hand, are combined in order to simplify the reaction equations. Thus, for example, the formula (II) represents a combination of formulae (II') and (II): (II) (II') (II) Taking into account the above definition, the course of the process according to the invention when ethyl acetate is used as acyl donor for the two starting compounds 4-hydroxy-2-cyclopenten-l-one (A) and its ketal formed with 2,2-dimethylpropane-l,3-diol (B) can be represented diagrammatically as follows (Ac = acetyl): - 12 0 A) OH Esterase +Ac-GR 70% conversion after OAc 20 h or alkaline/acid reracemization OH + Ac-OR B) 0. 0 Lipase OAc 0. 0 0. 0 +AC-OR 70% conversion after OH 20 h OAC I or alkaline/acid reracemization OH +AC-OR 0. 0 OAc - 13 The reaction parameters suitable for the acetylation by the process according to the invention depend on the varying marginal conditions, for example on the ratios by mass or the particular activity of the enzyme used in each case, on the choice of the desired acid residue (for example acetic acid, butyric acid or oleic acid residue) and the like. The optimal parameters for the acetylation by the process according to the invention are the following, in particular: Enzyme: Solvent (S): Dried ester (S)/ketal alcohol ratio by mass: Enzyme/ketal alcohol ratio by mass: Reaction temperature: Water content of the enzyme: Pig liver esterase or lipase from Pseudomonas fluorescens (Amano lipase P, batch no. LPL 05518) Triacetin (glycerol triacetate, completely environmentally neutral because a natural substance) 6:1 1:1 40°C + 1°C 3.17 % (lyophilized enzyme, employed for the reaction directly from the turer) manufac- The invention is explained in more detail by the examples which follow, without being limited to them. - 14 Example 1 521.3 mg (= 2.83 mmol) of ketal alcohol, 511.3 mg of Amano lipase P (batch no. LPL 05518) in 3 ml (= 3.1 g = 9 mmol) of tributyrin were reacted in a 10 ml round-bottomed flask in an oil bath at 40°C on a magnetic stirrer.

After 45 h, the reaction was stopped, the enzyme was filtered off and washed with acetone, and the entire filtrate was fractionated on a 50 g silica column (Merck flash silica gel 60, No. 9385) with 2:1 petroleum ether/ ether as mobile phase. 240.1 mg (= 0.98 mmol) of S(-)-ketal butyrate (enantiomeric purity > 95 %ee, according to HPLC) and 281.2 mg (= 1.53 mmol) of R(+)-ketal alcohol (> 95 %ee according to HPLC) were obtained.

Example 2 ml of dried cyclohexyl acetate (13.6 mmol), 250 mg of Amano lipase P (as in Example 1) and 308.0 mg (= 1.68 mmol) of ketal alcohol were reacted in a 10 ml round-bottomed flask in an oil bath at 60 °C on a magnetic stirrer.

After 20 h, the reaction was stopped, the enzyme was filtered off and washed with acetone, and the filtrate was fractionated on a 50 g silica column (Merck, No. 9385), with a petroleum ether/ether (1:1) mixture.

Yield: 140.2 mg (0.76 mmol) of R(+)-ketal alcohol (> 95 %ee according to HPLC and H-NMR of the Mosher ester) and 203.4 mg (= 0.83 mmol) of S(-)-ketal butyrate (> 95 %ee according to HPLC). - 15 Enantioselective enzymatic synthesis of S(—)— and R( + )esters of 4-hydroxy-2-cyclopenten-l-one and its ketal formed with 2,2-dimethylpropane-l,3-diol

Claims

1. Patent Claims

1. Process for the enantioselective preparation of S(-)- and R(+)-esters of 4-hydroxy-2-cyclopenten-l-one and its ketal formed with 2,2-dimethylpropane-l,3-diol of the general formula (la) and (lb) respectively (lb) (la) characterized in that a) a racemic mixture of 4-hydroxy-2-cyclopenten-l-one or its ketal formed with 2,2-dimethylpropane-l,3diol, of the formula (II) below, is reacted in the presence of an enzyme with an ester of the general formula (III) below as acyl donor in organic phase, with the formation of the corresponding S(-)-ester of the general formula (la) and with the R( + )alcohol of the formula (lib) being left behind, in accordance with the reaction equations (II) (la) (lib) + R - OH (IV) in which acyl denotes any desired acid residue, preferably the residue of an organic C 1 -C 22 carboxylic acid, and R denotes an optionally substituted, unbranched or branched alkyl, alkenyl or alkynyl group with 1 to 22, preferably 1 to 12, in particular 1 to 6, carbon atoms, an optionally substituted aryl group with 6 to 12, preferably 6 to 10, carbon atoms, or an optionally substituted heterocyclic group with 5 to 10, preferably 5 to 7, ring atoms, which contains at least one nitrogen, oxygen and/or sulphur atom as hetero atom(s), b) the product mixture obtained in stage (a) is fractionated by chromatography or extraction into the S(-)-ester of the formula (la), which is obtained as first final product, and into the R(+)-alcohol of the formula (lib), which c) is converted by chemical or enzymatic esterification - 17 with the ester of the formula (III) into the R( + )ester of the formula (lb) which in turn is obtained by chromatography or extraction in pure form as second final product, in accordance with the reaction equation: A OH (lib) (III) + R - OH (IV)

2. Process according to Claim 1, characterized in that the acyl donor used is an ester of the general formula: in which R x and R 2 , which can be identical or different, each have the meanings indicated for R in Claim 1.

3. Process according to Claim 1 or 2, characterized - 18 in that the ethyl ester of acetic acid (ethyl acetate) or a glycerol triester of an organic acid with up to 22 carbon atoms, especially triacetin or tributyrin, is used as acyl donor.

4. Process according to one of Claims 1 to 3, characterized in that pig liver esterase is used as enzyme in the case of the keto substrate of the formula (II).

5. Process according to one of Claims 1 to 3, characterized in that lipase from Pseudomonas fluorescens is used as enzyme in the case of the ketal substrate of the formula (II).

6. Process according to one of Claims 1 to 5, characterized in that a hydrocarbon and/or an ester, preferably the ester employed as acyl donor, is used as organic solvent in stage (a).

7. Process according to one of Claims 1 to 6, characterized in that the transesterification in stage (a) is carried out at room temperature or elevated temperature, preferably at a temperature in the range from 40 to 75°C, in particular 58 to 62°C, specifically at 60°C.

8. Process according to one of Claims 1 to 7, characterized in that the organic solvent and the alcohol of the formula (III) are employed in stage (a) in a ratio of from 4:1 to 100:1, preferably 5:1 to 10:1, by mass.

9. Process according to one of Claims 1 to 8, characterized in that the enzyme and the alcohol of the formula (III) are employed in stage (a) in a ratio of from 0.5:1 to 10:1, specifically 0.5:1 to 1.5:1, in particular 0.7:1 to 0.9:1, by mass.

10. Process according to one of Claims 1 to 9 characterized in that in place of the esterification of the R( + )-alcohol of the formula (lib) in stage (c), a racemization of the R(+)-alcohol and a recycling thereof to stage (a) are carried out for complete enantioselective conversion of the racemic compound of the formula (II) into the S(-)-ester of the formula (la).