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  • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
  • C12P41/003—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions
  • C12P41/005—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by ester formation, lactone formation or the inverse reactions by esterification of carboxylic acid groups in the enantiomers or the inverse reaction
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  • C07C327/00—Thiocarboxylic acids
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  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P11/00—Preparation of sulfur-containing organic compounds
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins

Definitions

• the present invention relates to a novel process for producing chiral 3-acylthio-2-methylpropionic acid derivatives. Specifically, it relates to a process for the enzymatic enantiospecific hydrolysis of racemic 3-acylthio-2-methylpropionic acid esters to give chiral 3-acylthio-2-methylpropionic acids.
• Capoten (captopril, see Physicians Desk Reference, 1985, page 1986) is designated chemically as 1-[(2S) )-3-mercapto-2-methylpropionyl]-L-proline (1).
• captopril (1) as an inhibitor of ACE depends critically on the configuration of the mercaptoalkanoyl moiety and the compound with the S-configuration is about 100 times more active than its corresponding R-enantiomer [M. A. Ondetti et al., Biochemistry, 16, 5484 (1977)].
• Captopril (1) has been prepared by direct coupling of racemic 3-acetylthio- or 3-benzoylthio-2-methyl propionyl chloride to L-proline to give two diastereomers having the formulae 2 and 3 [Japanese Patent KoKai (Laid-Open) No. 52-116457; see also J. Fisher et al., U.S. P. 4,332,725 (1982)].
• captopril 1, was prepared by coupling 3-acetylthio- or 3-benzoylthio-2S-methylpropionic acid or the acyl chloride of either compound with L-proline followed by deacylation of the product [N. Ohashi et al., U.S. P. 4,297,282 (1981); U.S. P. 4,325,886 (1982)].
• these chemical resolution procedures are very expensive, cumbersome to carry out and the yield of the product is relatively low.
• an asymmetric synthesis of captopril was developed [M. Shimazaki et al., Chem. Pharm. bull.. 30, 3139 (1982)].
• the requisite 3-mercapto-2S ⁇ methylpropionic acid moiety was prepared from the microbiologically-derived chiral 3-hydroxy-2R-methylpropionic acid, which is obtained via an expensive bacterial hydroxylation of isobutyric acid [C. T. Goodhue and J. R. Schaeffer, Biotechnol Bioeng. , 13, 203 (1971); J. Hasegawa et al . , J. Ferment. Technol., 59, 203 (1981)].
• this invention comprises the use of extracellular lipases of microbial origin (EC 3.1.1.3) to catalyze the enantiospecific hydrolysis of 3-acylthio-2-methylpropionic acid esters as hereinbelow defined.
• R 1 is a radical in straight chain, branched chain, or cyclic configuration selected from the class consisting of alkane radicals having from 1 to about 12 carbon atoms with or without electronegative substituents at C-2 ' ; cycloalkane radicals having from about 5 to 7 carbon atoms; phenyl and benzyl radicals having from 6 to 8 carbon atoms; (examples of electronegative substituents of the alkane radicals referred to above are radicals such as halogens, nitro groups, nitriles and carboxylates);
• R 2 is an acyl radical in straight chain, branched chain or cyclic configuration having 1 to about 12 carbon atoms, cycloalkane radicals having 5 to 7 carbon atoms, benzoyl, naphthoyl, biphenoyl and carbobenzoxy radicals containing substituents such as nitro, halogen, methyl or alkoxy groups on the aromatic ring. It is an object of this invention to produce opticallyactive 3-acylthio-2-methylpropionic acids in excellent yields using an enzymatic resolution process.
• Another object of the present invention is to provide an improved catalytic process for preparing the chiral mercaptoalkanoyl moiety of 1 using extracellular inexpensive microbial lipases.
• the process of the invention comprises subjecting said 3-acylthio-2-methylpropionic ester to the hydrolytic action of a microbial lipase (EC 3.1.1.3) and recovering the desired optically-active 3-acylthio-2-methylpropionic acid and derivatives.
• the desired optically active 3-acylthio-2-methylpropionic acid can be readily coupled with L-proline followed by removal of the R 2 group to obtain the desired captopril (Capoten).
• extracellular microbial lipases are capable of functioning to catalyze the desired enantiospecific hydrolysis.
• Particularly suitable are those extracellular lipases derived from the microorganisms of the genera Candida, Rhizopus. Mucor, Aspergillus, Penicillium, Geotrlchlum, Humicola, Pseudomonas and Chromobacterium.
• Extracellular microbial lipases are well known and many of these are available commercially (see M. Iwai and Y. Tsujsaka, page 443, and M. Sugiura, page 505, in Lipases, edited by B. Borgstrom and H. L. Brockman, Elsevier, N.Y., 1984). For example, they are used industrially for the transesterification of natural oils, removal of oil stains/spots and lipids, meat and fish processing, etc. (see M. Iwai and Y. Tsujsaka, page 466).
• M. Iwai and Y. Tsujsaka, page 466 One outstanding feature of these microbial lipases that distinguishes them from intact microorganisms is that they can tolerate high substrate and product concentrations.
• the 3-acylthio-2-methyl propionic ester substrate may be added in solid or liquid form at concentrations of 0.1-5 M to a suitable buffer solution containing the lipase to effect the enantiospecific hydrolysis.
• the substrate may be dissolved in a suitable organic solvent such as carbon tetrachloride, cyclohexane, carbon disulfide, or hexane, so long as the solvent does not denature the enzyme.
• the substrate may be emulsified by the use of polyvinyl alcohol or propylene glycol.
• the temperature and pressure conditions under which the contact of the ester substrate with the lipase are interdependent as will be apparent to those skilled in the art. Generally, at atmospheric pressure, the temperature can range from about 10°C to about 40oC and the pH of the medium can range from 3-8.5.
• acylthio-2-methylpropionic acids are prepared by reaction of their corresponding thiol acid with methacrylic acid [see J. T. Suh et al., J . Med . Chem . , 28, 57 (1985); and U.S. P. 4,256,761 (1981)].
• the acylthio-2-methylpropionic esters are prepared by esterification via conventional methods (see I. T. Harrison and S. Harrison in Compendium of Organic Synthetic Methods, Chapter 8, John Wiley & Sons, N.Y., 1971, page 271).
• Acylthio-2-methylpropionic esters are also prepared by reaction of their corresponding acyl chloride with 3-aercapto-2-methylpropionic ester [M. Shimazaki et al., Chem. Phara. Bull,, 30, 3139 (1982)].
• the process may be made continuous wherein the enzyme is immobilized and recycled several times to reduce cost; the (+)-ester or (+)- acid can be recovered, racemized [N. Ohashi et al., U.S. P. 4,411,836 (1983)] and reused.
• it may be possible to dissolve the ( ⁇ )-substrate and a racemization agent in a suitable solvent so only the ester will be continuously raceraized in situ without cleaving the ester grouping. In this case, this process is tantamount to an asymmetric synthesis.
• activators and stabilizers of the lipase [N.
• Mucor meihei lipase Mano, 10,000 ILu/gm, MAP
• Chromobacterium viscosum lipase U.S. Biochemicals cat. 18492, 3,400 u/mg
• Rhizopus arrhizus lipase Boehringer-Mannheim
• Example 10 The procedure of Example 1 was repeated except that 50 mg of Candida cylindracea lipase (Sigma L1754 Type VII, 500 units per mg solid) was used as the enzyme and (-)-S-3-benzoylthio-2- methylpropionic acid and (+)-R-3-benzoylthio-2-methylpropionic methyl ester were recovered.
• Candida cylindracea lipase Sigma L1754 Type VII, 500 units per mg solid
• EXAMPLE 12 The procedure of Example 4 was repeated except that 176 mg of ( ⁇ )-3-acetylthio-2-methylpropionic methyl ester was used as the substrate and optically-active 3-mercapto-2-methylpropionic aethyl ester and (-)-S-3-acetylthio-2-methylpropionic acid were recovered.
• EXAMPLE 13 The procedure of Example 9 was repeated except that 176 mg of ( ⁇ )-3-acetylthio-2-methylpropionic methyl ester was used as the substrate and optically-active 3-mercapto-2-raethylpropionic aethyl ester and (-)-S-3-acetylthio-2-methylpropionic acid were recovered.
• EXAMPLE 14 The procedure of Example 7 was repeated except that 176 mg of ( ⁇ )-3-acetylthio-2-methylpropionic methyl ester was used as the substrate and optically-active 3-mercapto-2-raethylpropionic aethyl ester, 3-acetylthio-2-methylpropionic methyl ester, 3-acetylthio-2-methylpropionic acid and 3-mercapto-2-methylpropionic acid were recovered.
• EXAMPLE 15 The procedure of Example 3 was repeated except that 200 mg of ( ⁇ )-3-octylthio-2-methylpropionic methyl ester was used as the substrate and optically-active 3-mercapto-2-methylpropionic aethyl ester, 3-octylthio-2-methylpropionic methyl ester, 3- octylthio-2-methylpropionic acid and 3-mercapto-2-methylpropionic acid were recovered.
• Rhizopus sp. lipase (30 mg ⁇ (Serva, 35 u/mg) in 0.87 ml of 0.2 M phosphate buffer, pH 7.0, was added 250 mg of (+)-3-1'-naphthoylthio-2-methylpropionic methyl ester.
• the reaction mixture was stirred with a magnetic stirrer for 24 hours at 22oC.
• the contents were then acidified to pH 3.0 with IN HCl and exhaustively extracted with ethyl acetate three times.
• the combined organic extract was dried over sodium sulfate and evaporated to dryness to give an oily residue.
• the residue was chromatographed over a silica gel (MN Kieselgel 60) column (1 x 25 cm).
• Mucor meihei lipase Mano, 10,000 ILu/gm, MAP
• Rhizopus oryzae lipase (30 mg) (Amano 750,000 Lu/gm, FAP) in 1 ml of 0.2 M phosphate buffer, pH 7.0, was added 250 mg of ( ⁇ )-3-2',6'-dimeth ⁇ xybenzoylthio-2-methylpropionic methyl ester.
• the reaction mixture was stirred with a magnetic stirrer for 24 hours at 22°C.
• the contents were then acidified to pH 3.0 with IN HCl and exhaustively extracted with ethyl acetate three times.
• the combined organic extract was dried over sodium sulfate and was then evaporated to dryness to yield an oily residue.
• Mucor meihei lipase Amano, 10,000 ILu/gm MAP
• Candida cylindracea lipase Sigma Type VII L-1754
• EXAMPLE 51 The procedure of Example 1 was repeated using 100 mg of crude lipase of Penicillium cvclopium ATCC 34613 [M. Iwai et al., Agr. Biol. Chem. , 39, 1063 (1975)] as the enzyme to obtain optically active 3-benzoylthio-2-methylpropionic acid.
• Example 52 The procedure of Example 1 was repeated using 50 mg of crude Geotrichum candidum (ATCC 34614) lipase [Y. Tsujisaka et al., Agr. Biol. Chem., 37, 1457 (1973)] as the enzyme to obtain optically active 3-benzoylthio-2-methylpropionic acid.
• ATCC 34614 crude Geotrichum candidum
• lipase Y. Tsujisaka et al., Agr. Biol. Chem., 37, 1457 (1973)
• EXAMPLE 54 The procedure of Example 41 was repeated using 250 mg of ( ⁇ )-3-pivaloylthio-2-methylpropionic cyanomethyl ester as the substrate. (-)-S-3-pivaloylthio-2-methylpropionic acid was recovered.
• EXAMPLE 55 The procedure of Example 41 was repeated using 250 mg of ( ⁇ )-3-pivaloylthio-2-methylpropionic-2'-nitropropyl ester as the substrate. (-)-S-3-pivaloylthio-2-methylpropionic acid was recovered.
• Example 1 The procedure of Example 1 was repeated using 280 mg of ( ⁇ )-3-benzoylthio-2-methylpropionic trichloroethyl ester as the substrate. (-)-S-3-benzoylthio-2-methylpropionic methyl ester was recovered.
• Example 41 The procedure of Example 41 was repeated using 250 mg of ( ⁇ )-3-pivaloylthio-2-methylpropionic carboethoxy methyl ester as the substrate. (-)-S-3-pivaloylthio-2-methylpropionic acid was recovered.
• Example 41 The procedure of Example 41 was repeated using 250 mg of ( ⁇ )-3-pivaloylthio-2-methylpropionic methoxy methyl ester as the substrate. (-)-S-3-pivaloylthio-2-methylpropionic acid was recovered.
• Rhizopus oryzae lipase (30 mg) (Amano, 750,000 Lu/gm FAP) in 1 ml of 0,2 M phosphate buffer, pH 7.0, was added 232 mg of ( ⁇ )-3-tert-butylacetylthio-2-methylpropionic methyl ester.
• the reaction mixture was stirred with a magnetic stirrer for 24 hours at 22°C.
• the contents were then acidified to pH 3.0 with IN HCl and exhaustively extracted with ethyl acetate three times.
• the combined organic extract was dried over sodium sulfate and was then evaporated to dryness to yield an oily residue.
• Example 61 The procedure of Example 61 was repeated using 40 mg of

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