DE3724721A1 - Method for the enzymatic racemate resolution of 1-acyloxy-2-cyclopenten-4-one 2,2-dimethylpropanediol ketal - Google Patents

Abstract

Racemic 1-acyloxy-2-cyclopenten-4-one 2,2-dimethylpropanediol ketal can be fractionated in high yield and enantiomeric purity with the aid of lipases from Pseudomonas fluorescens and/or from Mucor miehei or Rhizomucor miehei.

Description

Optically active racemate cleavage products of 1-acyloxy-2-cyclopenten-4-one provide a key substance for manifold chemical syntheses. In particular, the Acetoxy compound as a chiral synthon Prostaglandin synthesis can be used. The stake microbiological conversions to produce optically active Structures in this area have been varied Starting products or intermediate stages examined [J. Jiu "Microbial Reactions in Prostaglandin Chemistry" Adv. Biochem. Engineer., Ed. A. Fiechter, Springer Verlag Berlin, Vol. 17, pp. 37-62 (1980)].

In 1976, Takano first described the manufacture of a Lactons (3) as a prostaglandin intermediate on the way a microbiological hydrolysis. Cis- (1,4) -diacetoxy-2-cyclopentene (1) was caused by Bacillus subtilis var. Niger an optically active monoacetate (2a) in 56.1% yield hydrolyzed, which in two reaction stages in the optical active lactone (3) can be converted [p. Takano, K. Tanigawa, K. Ogasawara, J. Chem. Soc. Chem. Comm. P. 189 (1976)].

However, the optical purity of the lactone (3) is only 35%.  

At the same time, Miura et al. [P. Miura, S. Kurozomi, T. Toru, T. Tanaka, M. Kobayashi, S. Matsubara, S. Ishimoto, Tetrahedron 32, p. 1893 (1976)] the hydrolysis a mixture of the cis and trans isomers (3 R), 5 R), (3 S, 5 S) and (3 R, 5 S) of the diacetate (1) with baker's yeast. Man however, only receives a separable mixture of trans- (3 R) -acetoxy- (5 R) -hydroxy-cyclopentene with 95% more optical Purity, optically active (3 R, 5 R) -di-acetoxy-cyclopentene and the diol as a 1: 1 mixture of (3 R, 5 R) - and (3 R, 5 S) -dihydroxy-cyclopentene. The additionally tested enzymes Wheat germ lipase, citrus acetylesterase and lipase Aspergillus niger showed an even less favorable one Stereoselectivity. The enantiomerically pure trans (1 S, 4 S) diacetate showed a faster reaction with baker's yeast than the R-acetate portion of the cis-trans mixture.

The Sih [Y. F. Wang, CH. S. Chen, G. Girdaukas, C.J. Sih "Bifunctional Chiral Synthons via Biochemical Methods. 3. Optical Purity Enhancement in Enzymatic Asymmetric Catalysis "J. Am. Chem. Soc. 106, Pp. 3695-3696 (1984)] used for the hydrolysis of enantiomerically pure cis-1,4-diacetate (1) for the first time Pig liver esterase and received the (1 S) -acetoxy- (4 R) -hydroxy-2-cyclopentene in an optical yield of 80.3% or after recrystallization from 96%. The analytical yield was 83%.

With the same enzyme and comparatively with α- chymotrypsin, acetylesterase and baker's yeast, Läum and Schneider also received optically active synthons with a cyclopentene structure [K. Läum and M. Schneider "Enzymatic Hydrolysis of Prochiral cis-1,4-diacyl-2-cyclopentenediols: Separation of (1 S, 4 R) - and (1 R, 4 S) -4-hydroxy-2-cyclopentenyl derivatives, Versatile Building Blocks for Cyclopentanoid Natural Products "Tetrahedron Letters 25, 5875-5878 (1984)]. The best values are obtained with the pig liver esterase with an optical yield of 86% and a chemical yield of likewise 86% after recrystallization from 59%. The dipropionate and dibutyrate instead of the diacetate showed significantly less favorable optical and chemical yields.

Lipases from Candida cylindracea and from Rhizopus sp. in contrast to the esterases, this preferentially hydrolyzes S-acetate with the formation of (1 R, 4 S) -4-hydroxy-2-cyclopentenyl acetate (50% ee; 82% chemical yield).

Pork liver esterase was later used on ®Eupergit (Phenol / formaldehyde resin) immobilized for the hydrolysis of the cis-diacetate (1) used, which in 68% yield 1 S-monoacetate (2a) with an optical purity of 98% ee was won [K. Läum, E. H. Reimerdes, M. Schneider "Immobilized Porcine Liver Esterase: A Convenient Reagent for the Preparation of Chiral Building Blocks "tetrahedron Letters 26, 407-410 (1985)].

After testing numerous enzymes, Deardorff et al. [D. R. Deardorff, A. J. Matthews, D. S. McKeekin and C. L. Craney "A Highly Enantioselective Hydrolysis of cis-3,5-Diacetoxycyclopent-1-ene. An Enzymatic Preparation of 3  (R) -acetoxy-5 (S) -hydroxycyclopent-1-ene "tetrahedron Letters 27, 1255-1256 (1986)] that acetylcholinesterase again in the same way as lipases, (1 R, 4 S) -4-hydroxy-2-cyclopentenyl acetate forms, the product in high optical purity (<99% ee) in chemical Yields of 94% arise.

The optically active 4-hydroxy-2-cyclopentenylacetates that can be prepared in this way require a subsequent chemical oxidation to the chiral synthon 1-acetoxy-2-cyclopenten-4-one. However, this oxidation becomes milder Conditions accompanied by undesirable side reactions, what with a certain instability of 1-acetoxy-2-cyclopenten-4-one, only at low temperatures can be stored and transported.

In contrast, the connection with a protection of Keto group through ketal formation and associated Blocking any isomerization is a major advantage So represents an acyl-2-cyclopenten-4-one [2,2-dimethyl-propanediol] ketal a stable connection. Even the corresponding alcohol is much more stable than that relatively unstable 1-hydroxy-2-cyclopenten-4-one. A such racemic acetate is by simple chemical Synthesis easily accessible. For example, you get that Cyclopentenediol or the corresponding diketone first Precursors according to Organic Synthesis Vol. V, p. 324 or p. 414. Then the diketone can with 2,2,5,5-tetramethyl-1,3-dioxane under BF₃ etherate catalysis be converted to the monoketal. Then the remaining keto group reduced with NaBH₄ according to Luche (JACS 100, 2226) and acetylation with acetic anhydride / pyridine be performed. The manufacturing offers of the ketal-protected ketoacetate despite the higher number of stages due to the better stability and the higher yield Advantages over the synthesis of a cis-diacetate.  

For the production of an optically active component of the ketal as a chiral, stable synthon is also one Racemate separation required.

Among the numerous literature examples [J. B. Jones "Enzymes in Organic Synthesis" Tetrahedron 42, 3351-3403 (1986)] was previously the enzymatic separation of racemates 1-acyloxy-2-cyclopenten-4-one [2,2-dimethylpropanediol] ketal or a structurally analogous substrate described.

Only acyl hydrolysis of racemic 2-substituted 3-acyloxy-5-oxo-1-cyclopenten-1-heptanoic acid derivatives (4) with Saccharomyces cerevisiae is known (FB Colton, WJ Marsheeck, M. Miano "Process for the preparation of 2-substituted- 3- α- hydroxy-5-oxo-1-cyclopentene-1-heptanoic acid derivatives (5) by stereospecific microbiological hydrolysis "DE 2 31 859).

R = alkyl C₁-C₇
R ′ = styryl or the like
R ′ ′ = acyl C₁-C₇

It has now been found that racemic 1-acyloxy-2-cyclopenten-4-one [2,2-dimethylpropanediol] ketal with help of lipases from Pseudomonas fluorescens and / or from Mucor miehei or Rhizomucor miehei in high yield and Enantiomeric purity can be separated. This was particularly surprising given the steric conditions  such a changed substrate structure is not known and due to a lack of knowledge about the "active site "even simple hydrolases cannot be predicted may whether stereoselective hydrolysis is actually possible is.

The invention thus relates to a method for Racemate resolution of 1-acyloxy-2-cyclopenten-4-one [2,2-dimethylpropanediol] ketal of the general formula I,

in the R an acyl group with a chain length of C₁ to C₁₆, which is characterized in that the said Substrate with a lipase available from Pseudomonas fluorescens and / or from Mucor miehei or Rhizomucor miehei is incubated.

The invention is described in detail below. especially in its preferred embodiment. Further the invention is defined in the claims.

Suitable substrate for the lipases is 1-acyloxy-2-cyclopenten-4-one [2,2-dimethylpropanediol] ketal, being the Acyl group has a chain length of C₁ to C₁₆, preferably C₁ to C₈. Racemic is particularly preferred 1-acetoxy-2-cyclopenten-4-one [2,2-dimethylpropanediol] ketal used as a substrate. The lipases can be the substrate can be added both individually and in a mixture. It will expediently worked in an aqueous medium, a organic solvent, for example ethanol or DMSO, can be used as a solubilizer. The substrate may be the reaction in portions or the total be fed once.  

The reaction runs at 10 to 50 ° C, preferably 20 to 35 ° C from. At higher temperatures, the enzymes increase deactivated. It runs at lower temperatures Response too slow. The pH is between 6 and 8, preferably between 6.5 and 7.5. The enzyme concentration can fluctuate in wide ranges. Appropriately choose one at a substrate concentration of 1 to 100 mmol / l, preferably 5 to 50 mmol / l an enzyme concentration of 1000 to 100,000 U / mmol substrate, preferably 10,000 to 50,000 U / mmol substrate. One unit (U) of enzyme activity is called the amount per minute at pH 7.0 and a temperature of 37 ° C, from a triglyceride 1.0 microequivalents Split off the fatty acid hydrolytically.

The enzymes can be in free form or immobilized be used. The immobilization happens after conventional methods, such as, for example, in Laum et al. Tetrahedron Letters 26, 407 (1985). Because of is sufficient lifespan of the immobilized system use in continuous processes is also possible. It is also possible to remove whole cells of the microorganisms from which the enzymes can be obtained, in particular Pseudomonas fluorescens and Mucor or Rhizomucor miehei.

The invention will be further detailed in the examples explained. The percentages refer to the Weight unless otherwise stated.

example 1 General reaction formula:

In two 2 l Erlenmeyer flasks 500 ml of 0.2 molar potassium phosphate buffer (pH 7.0), 0.5 g racemic 1-acetoxy-2-cyclopenten-4-one [2,2-dimethylpropanediol] ketal (6) and 2.5 g lipase (1 g 32 700 U) from Pseudomonas fluorescens Type P (Amano, Nagoya, Japan, batch number LPJ 12517) given.

The flasks are shaken at 120 rpm for 16 h at 27 ° C.

Then the contents of both pistons are combined and extracted three times with 500 ml dichloromethane. The organic Phase is separated, dried over Na₂SO₄ and on Volume of about 3 ml concentrated. The extract is on a Si-60 column (type "Lobar B"; from Merck, Darmstadt) separated (eluent n-hexane / ethyl acetate gradient from 0-20% ethyl acetate) and for quantitative determination balanced. The enantiomeric purity (ee) becomes Production of the ester derivatives with mosheric acid chloride by NMR determined.

Exploit:
a) Acetate (8): 433.4 mg ee _R : 96%
b) Alcohol (7): 170 mg ee _S : 95%

Example 2

500 ml of 0.2 in each case are placed in a 2 l Erlenmeyer flask molar potassium phosphate buffer (pH 7.0), 600 mg racemic 1-acetoxy-2-cyclopenten-4-one [2,2-dimethylpropanediol] ketal and 2.5 g Amano lipase type P (batch no.LPJ 12517) given.

The flask is shaken at 27 ° C at 120 rpm for 5 h.

The flask contents are extracted three times with 500 ml dichloromethane. The organic phase is separated, dried over Na₂SO₄ and concentrated to a volume of about 1.5 ml. The alcohol is obtained by separating the extract with a Si-60 column (type "Lobar B", Merck) (eluent n-hexane / ethyl acetate gradient from 0-20% ethyl acetate).
Yield: 124 mg S-alcohol (7) ee _S : 97%

Example 3

In a 1 liter Erlenmeyer flask, 200 ml becomes 0.2 molar Potassium phosphate buffer (pH 7.0), 200 mg racemic 1-acetoxy-2-cyclopenten-4-one [2,2-dimethylpropanediol] ketal and 1 g of lipase / esterase from Mucor miehei (Gist-Brocades, Delft, Netherlands, batch no. 055-6-LRE).

The flask is shaken at 27 ° C at 120 rpm for 22 h.

The flask contents are extracted three times with 100 ml dichloromethane. The organic phases are separated, combined and dried over sodium sulfate. After concentration, the extract is separated using a Si-60 column (type "Lobar A", Merck) (eluent n-hexane / ethyl acetate gradient of 0-10% ethyl acetate).
Yield: 31.4 mg of S-alcohol ee _(S) : 95%

Claims (5)

1. Process for the resolution of 1-acyloxy-2-cyclopenten-4-one [2,2-dimethylpropanediol] ketal of the general formula I, in which R is an acyl group with a chain length of C₁ to C₁₆, characterized in that said substrate is incubated with a lipase obtainable from Pseudomonas fluorescens and / or Mucor miehei or Rhizomucor miehei. 2. The method according to claim 1, characterized in that is incubated at 10 to 50 ° C. 3. The method according to claim 2, characterized in that is incubated at 20 to 35 ° C. 4. The method according to one or more of claims 1 to 3, characterized in that at a pH of 6 until 8 is incubated. 5. The method according to claim 4, characterized in that is incubated at a pH of 6.5 to 7.5.