PROCESS FOR PREPARING OPTICALLY-ACTIVE 3-ACYLTHIO-2-METHYLPROPIONIC ACID DERIVATIVES

Abstract: A process for resolving racemic 3-acylthio-2-methylpropionic acid esters by enantiospecific hydrolysis utilizing extracellular lipases of microbial origin (EC 3.1.1.3). The desired chiral products can be readily converted to captopril.

Description: Description Process for Preparing Optically-active 3-Acylthio-2-methylpropionic Acid Derivatives Technical Field The present invention relates to a novel process for pro¬ ducing chiral 3-acylthio-2-methylpropionic acid derivatives. Specifically, it relates to a process for the enzymatic enantio- specific hydrolysis of racemic 3-acylthio-2-mβthylpropionic acid esters to give chiral 3-acylthio-2-methylpropionic acids. Background Capoten (captopril, see Physicians Desk Reference, 1985, page 1986) is designated chemically as 1-[(2SJ )-3-mercapto-2- aethylpropionyl]-L-proline (1J. Although its mechanism of action has not yet been fully defined, it appears to act as an antihypertensive and as an adjunct in the therapy of heart failure primarily through sup¬ pression of the renin-angiotensin-aldosterone system. Captopril prevents the conversion of angiotensin I to angiotensin II by inhibition of ACE (angiotensin converting enzyme), a peptidyl dipeptide carboxy hydrolase [M. A. Ondetti et al., Science, 196, 441 (1977)]. The potency of captopril (3J as an inhibitor of ACE depends critically on the configuration of the mercapto- alkanoyl 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 race¬ mic 3-acetylthio- or 3-benzoylthio-2-methyl propionyl chloride to L-proline to give two diastereomers having the formulae 2_aπd 3 [Japanese Patent KoKai (Laid-Open) No. 52-116457; see also J. Fisher et al., U.S.P. 4,332,725 (1982)]. After isolating the SS-diastereomer, 2_, the acyl group repre¬ sented by R-C0- is removed to obtain captopril, 1_. The above method has a serious drawback because an equal amount of the undesired RS-diastereomer 3_was formed. The expensiveness of the L-proline and the tedious separation procedure led to the development of improved procedures. To circumvent these problems, 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)]. However, these chemical resolution procedures are very expensive, cumbersome to carry out and the yield of the product is relatively low. In an attempt to avoid this tedious chemical resolution, an asymmetric synthesis of captopril was developed [M. Shimazaki et al., Chem. Pharm. bull.. 30. 3139 (1982)]. The requisite 3-mercapto-2S- aethylpropionic acid moiety was prepared from the microbiologi- cally-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)]. Disclosure of the Invention Broadly, this invention comprises the use of extracellular lipases of microbial origin (EC 3.1.1.3) to catalyze the enan- tiospecific hydrolysis of 3-acylthio-2-methylpropionic acid esters as hereinbelow defined. Wherein: Rj 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' ; cyclo- alkane 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) ; R2 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 optically- active 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 mercapto- alkanoyl moiety of 1_ using extracellular inexpensive microbial lipases. These and other objects and advantages of the invention will become more apparent from the following detailed description. The process of the invention comprises subjecting said 3- acylthio-2-methylpropionic ester to the hydrolytic action of a ■icrobial lipase (EC 3.1.1.3) and recovering the desired opti¬ cally-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 R2 group to obtain the desired captopril (Capoten). It has been found that extracellular microbial lipases are capable of functioning to catalyze the desired enantiospeci ic 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 exam¬ ple, 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). One outstanding feature of these microbial lipases that distin¬ guishes them from intact microorganisms is that they can toler¬ ate high substrate and product concentrations. For example, no Barked substrate and product inhibitions were noted. Hence, these enzymatic hydrolytic reactions can be carried out in high concentrations (0.1-5 M) with an unusually high degree of enan- tioselectivity. Moreover, they are remarkably stable under the described reaction conditions, so that they may be recycled. 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. Alternatively, the substrate may be dissolved in a suitable organic solvent such as carbon tetra- chloride, cyclohexane, carbon disulfide, or hexane, so long as the solvent does not denature the enzyme. In addition, the substrate may be emulsified by the use of polyvinyl alcohol or propylene glycol. Of course, the temperature and pressure con¬ ditions 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 40 β C and the pH of the medium can range from 3-8.5. Detailed Description of the Invention The following examples are presented to illustrate this invention and are not to be considered as limiting the scope of the appended Claims. The 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-methyl- propionic 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)]. It is obvious to those skilled in the art that further improvement of the process can be made. For example, the pro¬ cess 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. Moreover, it may be possible to dissolve the (+)-substrate and a racemization agent in a suit¬ able 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. Also, activators and stabilizers of the lipase [N. Tomizuka et al., Agr. Biol. Chem.. 30, 576 (1966)] may be introduced into the incubation mixture or substrates possessing many different types of activated esters (Bodansky et al., Peptide Synthesis, Seconα Ed., Wiley, 1976, pp. 99-108) may be used to enhance the rate of conversion. In addition, active site directed mutagenesis or chemical modification of the enzyme may be used to prepare enzymes with improved V maχ /κ m and/or stability. EXAMPLE 1 To a suspension of Aspergillus niger lipase (50 mg) (A ano K-10 10,000 ILu/gra) in 1 ml of 0.2 M phosphate buffer, pH 7.0 was added 238 mg of (+j3-benzoylthio-2-methylpropionic methyl ester. The reaction mixture was stirred with a magnetic stirrer for 44 hours at 22 β C. The contents were then acidified to pH 3.0 with IN HC1 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. The residue was chroraatographed over a silica gel (MN Kieselgel 60) column (1 x 25 cm), Elution of the column with dichloromethane.hexane (25:1) gave (-)-S_-3-benzoylthio-2-methyl- ropionic methyl ester, [α] ,2 25 p 'D' = -27.2" (c, 6.00, CHC1 3 ) and (+)- _R-3-benzoylthio-2-methylpropionic acid, [α] 2 p 5 = +60.5° (c, 4.31, CHCI3). EXAMPLE 2 The procedure of Example 1 was repeated except that 20 mg of Pseudomonas lipase (Amano Lipo-protein Lipase 80, 800 u/mg) was used as the enzyme and (-)-S-3-benzoylthio-2-methylpropionic aethyl ester, [«] Q 5 - -30.97" (c. 6.51, CHC1 3 ) and (+)-R-3- 25 benzoylthio-2-methylpropionic acid, [α]j ~ +50.42" (c, 6.51, CHCI3) were recovered. EXAMPLE 3 The procedure of Example 1 was repeated except that 50 mg of Rhizopus niveus lipase (Amano, 45,000 ILu/gm, N) was used as the enzyme and (+)-R-3-benzoylthio-2-methylpropionic methyl OR ester, [α]* = +3.52" (c, 4.15, CHC1 3 ) and (-)-S-3-benzoylthio- 2-aethylpropionic acid, [~ ]^° = -20.28" (c, 1.78, CHC1 3 ) were recovered. EXAMPLE 4 The procedure of Example 1 was repeated except that 40 mg of Mucor meihei lipase (Amano, 10,000 ILu/gm, MAP) was used as the enzyme and (+)-R.-3-benzoylthio-2-methylpropionic methyl 25 ester, [«]J = +15.5" (c, 5.8, CHCI3) and (-)-S-3-benzoylthio-2- 25 methylpropionic acid, [cc] j = -26.6" (c, 4.59, CHCI3) were recovered. EXAMPLE 5 The procedure of Example 1 was repeated except that 1 mg of Chromobacteriu viscosum lipase (U.S. Biochemicals cat. 18492, 3,400 u/mg) was used as the enzyme and (-)-S-3-benzoylthio-2- 25 πethylpropionic methyl ester, [o jj ~ -31.25° (c, 5.77, CHCI3) and (+)-R-3-benzoylthio-2-methylpropionic acid, [α]? 5 ~ +46.49* (c, 5.35, CHCI3) were recovered. EXAMPLE 6 The procedure of Example 1 was repeated except that 1 mg of Rhizopus arrhizus lipase (Boehringer-Mannheim) was used as the enzyme and (+)-R-3-benzoylthio-2-methylpropionic methyl ester, 25 [α] Q = +6.75" (c, 5.51, CHCI3) and (-)-S-3-benzoylthio-2-meth- 25 ylpropionic acid, [α]^ = -15.13° (c, 4.89, CHC1 3 ) were recovered . EXAMPLE 7 The procedure of Example 1 was repeated except that 50 mg of Aspergillus niger lipase (Amano AP 120,000 Lu/gm) was used as the enzyme and (+)-R-3-benzoylthio-2-raethylpropionic methyl 25 —— ester, [α]' = +0.174" (c, 6.89, CHC1 3 ) and (-)-S-3-benzoylthio- 25 2-raethylpropionic acid, [«]« - -6.0" (c, 0.6, CHCI3) were recovered. EXAMPLE 8 The procedure of Example 1 was repeated except that 50 mg of Humicola lanuginosa lipase (Amano) was used as the enzyme and 25 (+)-R-3-benzoylthio-2-methylpropionic methyl ester, [α] n = +0.934" (c, 5.36, CHCI3) and (-)-S-3-benzoylthio-2-methylpro- pionic acid, [α]^° = -8.59° (c, 1.42, CHC1 3 ) were recovered. EXAMPLE 9 The procedure of Example 1 was repeated except that 40 mg of Rhizopus oryzae lipase (Amano, 750,000 Lu/gm FAP) was used as the enzyme and (+)-R-3-benzoylthio-2-methylpropionic methyl 25 ester, [α] = +13.56" (c, 3.09, CHCI3) and (-)-S-3-benzoylthio- 25 2-methylpropionic acid, [α]' = -13.8° (c. 1.6, CHCI3) were recovered. 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. EXAMPLE 11 The procedure of Example 1 was repeated except that 25 mg of Rhizopus sp. lipase (Serva, 35 u/mg) was used as the enzyme and (+)-R-3-benzoylthio-2-methylpropionic methyl ester , [<x ] D 25 τ9.66" (c. 5.67, CHC1 3 ) and (-)-S-3-benzoylthio-2-methylpro- 25 pionic acid, [α]' = -19.81° (c. 7.40, CHCI3) were recovered. 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 (+J-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-methylpro- pionic acid were recovered. EXAMPLE 15 The procedure of Example 3 was repeated except that 200 mg of (+J-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-methylpro- pionic acid were recovered. EXAMPLE 16 To a suspension of 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-l'-naphthoylthio-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 HC1 and exhaustively extracted with ethyl acetate three times. The combined organic extract was dried over sodium sul- fate and evaporated to dryness to give an oily residue. The residue was chroaatographed over a silica gel (MN Kieselgel 60) column (1 x 25 cm). Elution of the column with a solvent systjm consisting of ethyl acetate-hexane (1:3.5) gave (+)-R-3-l '-naph- thoylthio-2-methylpropionic methyl ester, [«]« = + 12- 8δ ° (c, 4.93, CHC1 3 ) and (-)-S-3-l'-naphthoylthio-2-methylpropionic acid, [ ]^ 5 = -41.68" (c, 2.98, CHCI3). EXAMPLE 17 The procedure of Example 16 was repeated except that 30 mg of Mucor meihei lipase (Amano, 10,000 ILu/gm, MAP) was used as the enzyme and (+)-R-3-l'-naphthoylthio-2-methylpropionic methyl 25 ester, [α]^ = +12.4" (c, 4.75, CHC1 3 ) and (-)-S-3-l '-naphthoyl- thio-2-methylpropionic acid, [α]jj 5 = -46.13" (c, 2.38, CHC1 3 ) were recovered. EXAMPLE 18 The procedure of Example 16 was repeated except that 40 mg of Aspergillus niger lipase (Amano K-10, 10,000 ILu/gm) was used as the enzyme and (-)-S-3-l'-naphthoylthio-2-methylpropionic methyl ester, [αlH 5 * -8.36" (c, 5.18, CHCI3) and (+)-R-3-l'- 25 naphthoylthio-2-methylpropionic acid, [θ_] = +51-43° (c, 1.4, CHCI3) were recovered. EXAMPLE 19 The procedure of Example 16 was repeated using 50 mg of Rhizopus niveus lipase (Amano, 45,000 ILu/gm) as the enzyme and the incubation period was extended to 48 hours. (+)-R-3-l'- 'aphthoylthio-2-methylpropionic methyl ester, [θi ] - +7.89" (c, 5.27, CHC1 3 ) and (-)-S-3-l*-naphthoylthio-2-methylpropionic 25 acid, [ ]^ = -42.44° (c, 1.6, CHCI3) were recovered. EXAMPLE 20 The procedure of Example 16 was repeated using 25 mg of Rhizopus oryzae lipase (Amano, 750,000 Lu/gm, FAP) as the enzyme and (+)-R-3-l'-naphthoylthio-2-methylpropionic aethyl ester, [α] 5 = +17.29" (c. 6.17, CHCI3) and (-)-S-3-l'-naphthoylthio-2- aethylpropionic acid, [α]jj 5 ~ - -4 .66" (c, 3.28, CHC1 3 ) were recovered. EXAMPLE 21 To a suspension of Mucor meihei lipase (40 mg) (Amano, 10,000 ILu/gm, MAP) in 1 ml of 0.2 M phosphate buffer, pH 7.0, was added 272 mg of (+ -3-2 1 -chlorobenzoylthio-2-methylpropionic aethyl 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 HC1 and exhaustively extracted with ethyl acetate three times. The combined organic extract was dried over sodium sulfate and was then evaporated to drynβss to yield an oily residue. The residue was chroraatographed over a silica gel (MN Kieselgel 60) column (1 x 25 cm). Elution of the column with a solvent system consisting of ethyl acetate-hexane (1:3.5) gave (+)-R-3-2'-chlorobenzoylthio-2-methylpropionic methyl 25 ester. [α]J" = +6.78" (c, 5.16, CHCI3) and (-)-S-3-2'-chloro- 25 benzoylthio-2-methylpropionic acid, [<*]« = -13.83° (c, 3.68, CHCI3). EXAMPLE 22 The procedure of Example 21 was repeated using 50 mg of Rhizopus niveus lipase (Amano) and (+)-R-3-2'-chlorobenzoylthio- 25 2-aethylpropionic methyl ester, [α]„ = +10.64" (c, 4.87, CHCI3) 25 and (-)-S-3-2'-chlorobenzoylthio-2-methylpropionic acid, [OS] Q = -21.45" (c, 4.82, CHClg) were recovered. EXAMPLE 23 The procedure of Example 21 was repeated using 30 mg of Rhizopus sp. lipase (Serva) and (+)-R-3-2'-chlorobenzoylthio-2- aethylpropionic methyl ester, [α]j: 5 = +15.73° (c, 4.36, CHCI3) 25 and (-)-S-3-2'-chlorobenzoylthio-2-methylpropionic acid, [α] D -17.35" (c, 5.44, CHCI3) were recovered. EXAMPLE 24 The procedure of Example 21 was repeated using 30 mg of Rhizopus oryzae lipase (Amano) as the enzyme and (+)-J?-3-2'- chlorobenzoylthio-2-methylpropionic methyl ester, [a]" = +11.68" (c, 4.82, CHCI3) and (-)-S-3-2'-chlorobenzoylthio-2- πethylpropionic acid, [α] \g 5 = -22.52° (c, 1.59, CHCI3) were 'D ~l recovered. EXAMPLE 25 The procedure of Example 21 was repeated using 40 mg of Aspergillus niger lipase (Amano K-10) as the enzyme and (-)-S-3- 25 2'-chlorobenzoylthio-2-methylpropionic methyl ester, [<*]« * -7.53" (c, 6.39, CHCI3), and (+)-R-3-2'-chlorobenzoylthio-2 25 mmeetthhyyllpprroopionic acid, {<x ] - +38.45" (c, 1.29, CHCI3), were recovered EXAMPLE 26 To a suspension of 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 (+J-3-2' ,6"-dimethαxybenzoylthio-2-methyl¬ propionic 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. The residue was chromatographed over a 13 silica gel (MN Kieselgel 60) column (1 x 25 cm). Elution of the column with a solvent system consisting of ethyl acetate-hexane (1:2) gave (+)-R-3-2' ,6'-dimethoxy-benzoylthio-2-methylpropionic methyl ester, [α]^ 5 = +1.39" (c, 8.26, CHC1 3 ) and (-)-S-3-2* ,6'- dimethoxy-benzoylthio-2-raethylpropionic acid, [<*] « a -9.10° (c, 1.72, CHC1 3 ). EXAMPLE 27 The procedure of Example 26 was repeated using 30 mg of Mucor meihei lipase (Amano, 10,000 ILu/gm MAP) as the enzyme and (+)-R-3-2* ,6'-dimethoxy-benzoylthio-2-methylpropionic methyl ester, [α]g 5 , +1 Λ2 - (C( 6-96> C HC1 3 ) and (-)-S-3-2' ,6'-dimeth- 25 oxy-benzoylthio-2-methylpropionic acid, [«] p = -8.21 (c, 1.4, CHCI3) were recovered. EXAMPLE 28 To a suspension of Mucor meihei lipase (40 mg) (Amano, 10.000 ILu/gm, MAP) in 1 ml of 0.2 M phosphate buffer, pH 7.0, was added 250 mg of (+ -3-2'-benzoylbenzoylthio-2-methylpro- pionic 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. The residue was chromatographed over a silica gel (MN Kieselgel 60) column (1 x 25 cm). Elution of the column with a solvent system consisting of ethyl acetate-hexane (1:2) gave (+)-R-3-2'-benzoylbenzoylthio-2-methylpropionic methyl ester, [α]p 5 = +3.08" (c, 5.85, CHC1 3 ), and (-)-S-3-2'- benzoylbenzoylthio-2-methylpropionic acid, [<x]|? 5 = -18.8° (c, 3.09, CHC1 ). EXAMPLE 29 The procedure of Example 28 was repeated using 40 rag of Rhizopus sp. lipase (Serva) and (+)-R-3-2'-benzoylbenzoylthio-2- aethylpropionic methyl ester, [α]^ 5 = +9.62° (c, 7.05, CHCI3), and (-)-S-3-2'-benzoylbenzoylthio-2-methylpropionic acid, [α]jj 5 = -26.94" (c, 2.16, CHC1 3 ) , were recovered. EXAMPLE 30 The procedure of Example 28 was repeated except 40 mg of Rhizopus oryzae lipase (Amano) and (+)-R-3-2'-benzoylbenzoyl- thio-2-aethylpropionic methyl ester, [α]j *5 - +1.70° (c, 9.36, CHCI3), and (-)-S-3-2'-benzoylbenzoylthio-2-methylpropionic acid. [α-]j 5 =_ -16.9° (c. 1.16, CHC1 3 ), were recovered. EXAMPLE 31 The procedure of Example 28 was repeated except 250 mg of (+)-3-4'-chloro-2 , -benzoylbenzoylthio-2-methylpropionic methyl ester was used as the substrate and (+)-R-3-4'-chloro-2'-ben- zoylbenzoylthio-2-methylpropionic methyl ester, [α]^ 5 = +1.56° (c, 8.42, CHCI3), and (-)-S-3-4'-chloro-2'-benzoylbenzoylthio- 2-aethylpropionic acid, Cα]^ 5 = -10.48" (c, 2.31, CHC1 3 ) , were recovered. EXAMPLE 32 To a suspension of Mucor meihei lipase (40 mg) (Amano) in 1 al of 0.2 M phosphate buffer, pH 7.0, was added 230 mg of (+J- 3-furoylthio-2-methylpropionic methyl ester. The reaction mix¬ ture 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. The resi¬ due was chromatographed over a silica gel (MN Kieselgel 60) column (1 x 25 cm). Elution of the column with a solvent system consisting of ethyl acetate-hexane (1:2.5) gave (+)-R-3-furoyl- thio-2-methylpropionic methyl ester, [α]^ 5 = +15.68° (c, 4.63, CHCI3), and (-)-S-3-furoylthio-2-methylpropionic acid, [α]^ 5 - -20.65° (c, 2.32, CHCI3). EXAMPLE 33 The procedure of Example 32 was repeated using 40 mg of Rhizopus oryzae lipase (Amano, MAP) as the enzyme and (+)-R.-3- furoylthio-2-methylpropionic methyl ester, [α ^ ~ +7.95° (c, 8.21, CHC1 3 ), and (-)-S-3-furoylthio-2-methylpropionic acid, 25 [α]p = -5.52° (c, 5.10, CHCI3), were recovered. EXAMPLE 34 The procedure of Example 32 was repeated using 40 mg of Rhizopus sp. lipase (Serva) as the enzyme and (+)-R-3-furoyl- 25 thio-2-aethylpropionic methyl ester, [«]' =• +8.66* (c, 6.72, 25 CHCI3). and (-)-S-3-furoylthio-2-methylpropionic acid, [α]p ' -8.66" (c, 5.75, CHCI3), were recovered. EXAMPLE 35 The procedure of Example 28 was repeated except 240 mg of (+ -3-2' ,4'-dichlorobenzoylthio-2-methylpropionic methyl ester was used as the substrate and (+)-R-3-2' ,4'-dichlorobenzoylthio- 25 2-methylpropionic methyl ester, [α]« = +5.79" (c, 4.85, CHC1 3 ), and (-)-S-3-2' ,4'-dichlorobenzoylthio-2-methylpropionic acid, 25 [α]* - -8.58" (c, 3.72, CHCI3), were recovered. EXAMPLE 36 The procedure of Example 35 was repeated using 50 mg of Aspergillus niger (Amano K-10) as the enzyme and (-)-S-3-2' ,4' - 25 dichlorobenzoylthio-2-methylpropionic methyl ester, [α] n = -5.8° (c, 7.14, CHCI3), and (+)-R-3-2' ,4*-dichlorobenzoylthio- 25 2-methylpropionic acid , [α] * = +31.34° (c , 1.2, CHC1 3 ) , were recovered. EXAMPLE 37 The procedure of Example 36 was repeated except 244 mg of ( χ_)-3-cyclohexanoylthio-2-methylpropionic methyl ester was used as the substrate and (-)-S-3-cyclohexanoylthio-2-methylpropionic methyl ester, [ ]^ 5 = -20.27" (c , 7.2 , CHCI3) , and (+)-R-3- 25 cyclohexanoylthio-2-methylpropionic acid, [α] = +45.1° (c, 1.9, CHCI3), were recovered. EXAMPLE 38 The procedure of Example 16 was repeated using 50 mg of Candida cylindracea lipase (Sigma Type VII L-1754) as the enzyme and (+ R-3-l I -naphthoylthio-2-methylpropionic methyl ester, [α]j? 5 = +9.04° (c, 6.26, CHC1 3 ), and (-)-S-3-l'-naphthoylthio- 25 2-methylpropionic acid, [ot]jj = -34.0° (c, 2.82, CHC1 3 ), were recovered. EXAMPLE 39 To a suspension of Mucor meihei lipase (40 mg) (Amano, 10,000 ILu/gm, MAP) in 1 ml of 0.2 M phosphate buffer, pH 7.0, was added 220 mg of (+J-3-3 1 ,4' ,5'-trimethoxybenzoylthio-2-meth- ylpropionic methyl ester. The reaction mixture was stirred with a magnetic stirrer for 48 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. The residue was chromatographed over a silica gel (MN Kieselgel 60) column (1 x 25 cm). Elution of the column with a solvent system consisting of ethyl acetate-hexane (1:2) gave (+)-R-3-3* ,4' ,5'-trimethoxybenzoylthio-2-methylpro- pionic methyl ester, [oclH 5 = +9.38° (c, 7.25, CHC1 3 ), and (-)-S- 3-3' ,4' ,5'-trimethoxybenzoylthio-2-methylpropionic acid, [(X] Q 5 = -37.0° (c. 2.61, CHCI3). EXAMPLE 40 The procedure of Example 39 was repeated using 20 mg of Candida cylindracea lipase (Meito Sangyo 0F-360) as the enzyme and (+)-R-3-3' ,4' ,5'-trimethoxybenzoylthio-2-methylpropionic methyl ester, [ ~ = +5.97" (c, 4.37, CHCl 3 ). and (-)-S-3- 3 1 ,4' ,5'-trimethoxybenzoylthio-2-methylpropionic acid, [α]" = -20.1" (c, 1.31, CHCI3) were recovered. EXAMPLE 41 To a suspension of Rhizopus niveus lipase (50 mg) (Amano, 45,000 ILu/gm, N) in 1 ml of 0.2 M phosphate buffer, pH 7.0, was added 200 mg of (+J-3-pivaloylthio-2-methylpropionic methyl ester. The reaction mixture was stirred with a magnetic stirrer for 48 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. The residue was chromatographed over a silica gel (MN Kieselgel 60) column (1 x 25 cm). Elution of the column with a solvent system consisting of ethyl acetate-hexane (1:5) gave 25 (+)-R-3-pivaloylthio-2-methylpropionic methyl ester, [α] n ■ +13.64° (c, 5.72, CHC1 3 ), and (-)-S-3-pivaloylthio-2-methylpro- pionic acid, [α]^ 5 = -45.1" (c, 1.04, CHCI3). EXAMPLE 42 The procedure of Example 41 was repeated using 50 mg of .Mucor meihei lipase (Amano) as the enzyme and (+)-R-3-pivaloyl- 25 thio-2-methylpropionic methyl ester, [α] = +13.13° (c, 6.51, 25 CHCI3), and (-)-S-3-pivaloylthio-2-methylpropionic acid, [α] D -32.27° (c, 1.41, CHCI3) were recovered. EXAMPLE 43 The procedure of Example 41 was repeated using 50 mg of Rhizopus oryzae lipase (Amano, FAP) as the enzyme and (+)-R-3- 25 pivaloylthio-2-raethylpropionic methyl ester, [tt]fT = +26.5° (c, 3.73, CHCI3), and (-)-S-3-pivaloylthio-2-methylpropionic acid, 25 [<X ] Q = -36.4° (c , 1 .47 , CHCI3) were recovered . EXAMPLE 44 The procedure of Example 28 was repeated except 220 mg of (+_)-3-3'-methylbenzoylthio-2-methylpropionic methyl ester was used as the substrate and (+)-R-3-3'-methylbenzoylthio-2-methyl- 25 propionic methyl ester, [OC] Q = +8.95° (c, 4.09, CHC1 3 ), and 25 (-)-S-3-3'-methylbenzoylthio-2-methylpropionic acid, C«] D = -28.71° (c, 2.56, CHC1 3 ) were recovered. EXAMPLE 45 The procedure of Example 26 was repeated except 200 mg of (+_)-3-2'-chloro-2* ,2'-diphenacetylthio-2-methylpropionic methyl ester was used as the substrate and (+)-R-3-2'-chloro-2 ! ,2'- 25 diphenylthio-2-raethylpropionic methyl ester, 1<~ ]Z - +6.63° (c, 6.44, CHCI3), and (-)-S-2'-chloro-2',2 » -diphenylthio-2-methyl- 25 propionic acid, [«] Q = -36.79" (c, 0.53, CHCI3) were recovered. EXAMPLE 46 The procedure of Example 26 was repeated except 200 mg of (+)-3-3' ,5'-dinitrobenzoylthio-2-methylpropionic methyl ester was used as the substrate and (+)-R-3-3* ,5'-dinitrobenzoylthio- 25 2-aethylpropionic methyl ester, [ t] . = +5.92° (c, 5.2, CHC1 3 ) , and (-)-S_-3-3' ,5'-dinitrobenzoylthio-2-methylpropionic acid, [α]p 5 = -13.54° (c, 3.14, CHCI3) were recovered. EXAMPLE 47 The procedure of Example 39 was repeated except 250 mg of (+J-3-3' ,4' ,5'-triethoxybenzoylthio-2-methylpropionic methyl ester was used as the substrate and (+J-R-3-3 1 ,4' ,5'-triethoxy- 25 benzoylthio-2-methylpropionic methyl ester, [«]-, = +4.9° (c, 5.14, CHCI3), and (-)-S-3-3' ,4* ,5'-triethoxybenzoylthio-2- 25 methylpropionic acid, [α] I'D n 2 = -22.0° (c, 2.88, CHC1 3 ) were recovered. EXAMPLE 48 The procedure of Example 41 was repeated except 250 mg of (+J-3-3* ,4' ,5'-triethoxybenzoylthio-2-methylpropionic methyl ester was used as the substrate and (+)-R-3-3' , ' ,5*-triethoxy- 25 benzoylthio-2-methylpropionic methyl ester, [cc ^ ■ +2.69° (c, 6.39, CHCI3), and (-)-S-3-3' ,4' ,5'-triethoxybenzoylthio-2- 25 methylpropionic acid, [α]" = -15.41° (c, 1.46, CHC1 3 ) were recovered. EXAMPLE 49 The procedure of Example 41 was repeated except 204 mg of (+ -3-isobutylthio-2-methylpropionic methyl ester was used as the substrate and (+)-R-3-isobutylthio-2-methylpropionic methyl ester, [ ]^ 5 = +9.69° (c, 6.10, CHC1 3 ), and (-)-S-3-isobutyl- thio-2-methylpropionic acid, [α]^ 5 - -26.82° (c, 2.42, CHC1 3 ) were recovered. EXAMPLE 50 The procedure of Example 1 was repeated except 300 mg of (+_)-3-benzoylthio-2-methylpropionic chloroethyl ester was used as the substrate and (-)-S-3-benzoylthio-2-methylpropionic chloroethyl ester, [ ] ,2 25 'D -29.14° (c, 3.84, CHCI3), and (+)-R- 3-benzoylthio-2-methylpropionic acid, [α]^ 5 = +41.29° (c, 4.09, CHCI3) were recovered. 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. EXAMPLE 53 The procedure of Example 41 was repeated except 250 mg of (+J-3-pivaloylthio-2-methylpropionic chloroethyl ester was used as the substrate and (+)-R-3-pivaloylthio-2-methylpropionic chloroethyl ester, [a. ] 5 = +10.73° (c, 2.88, CHC1 3 ), and (-)-S- 25 3-pivaloylthio-2-methylpropionic acid, [«]„ = -27.16° (c, 1.94, CHCI3), were recovered. EXAMPLE 54 The procedure of Example 41 was repeated using 250 mg of (+J-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 56 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 57 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 58 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. EXAMPLE 59 The procedure of Example 41 was repeated using 2,000 units of Rhizopus delemar lipase (Chemical Dynamics Corp. 55-7220-00, 5000 units/mg) as the enzyme and (+)-R-3-pivaloylthio-2-mβthyl- propionic methyl ester, [α]^ 5 = +15.1° (c, 3.98, CHC1 3 ), and (-)-S-3-pivaloylthio-2-methylpropionic acid, [α] 25 = -45.84° (c, 2.81, CHCI3)- were recovered. EXAMPLE 60 The procedure of Example 16 was repeated using 2,000 units of Rhizopus delemar lipase (Chemical Dynamics Corp., 5000 units/ mg) as the enzyme and (+)-R-3-l'-naphthoylthio-2-methylpropionic methyl ester, [α]^ 5 - +11.34" (c, 6.05. CHC1 3 ), and (-)-S-3-l'- ~"~ naphthoylthio-2-methylpropionic acid, [~. )Z ~ -48.99° (c, 2.77, CHCI3), were recovered. EXAMPLE 61 TO a suspension of 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. The residue was chromatographed over a silica gel (MN Kieselgel 60) column (1 x 25 cm). Elution of the column with a solvent system consisting of ethyl acetate-hexane (1:3.5) gave (+)-R-3-tert-butylacetylthio-2-methylpropionic methyl ester, [α][J 5 = +6.91° (c, 6.58, CHC1 3 ), and (-)-S-3-tert-butyl- acetylthio-2-methylpropionic acid, [ot]j: 5 = -41.66° (c, 2.11, CHClg). EXAMPLE 62 The procedure of Example 61 was repeated using 40 mg of Rhizopus niveus lipase (Amano, N) as the enzyme and (+)-R-3- 25 tert-butylacetylthio-2-methvlpropionic methyl ester, [α] j = -7.91° (c, 4.83, CHC1 3 ), and (-)-S-3-tert-butylacetylthio-2- 25 nethylpropionic acid, [<*■ ] = -36.47° (c, 1.84, CHC1 3 ), were recovered. EXAMPLE 63 The procedure of Example 61 was repeated using 40 mg of Mucor meihei (Amano, MAP) as the enzyme and (+)-R-3-tert-butyl- 25 acetylthio-2-methylpropionic methyl ester, [α] j = +7.21° (c, 6.57, CHCI3), and (-)-S-3-tert-butylacetyithio-2-methylpropionic 25 acid, [α] j j = -27.83" (c, 1.29, CHCI3), were recovered. EXAMPLE 64 The procedure of Example 61 was repeated using 40 mg of Candida cylindracea (Meito Sangyo, My) as the enzyme and (+)-£_- 25 3-tert-butylacetyIthio-2-methylpropionic methyl ester, [α] D = +12.53° (c, 3.52, CHClg), and (-)-S-3-tert-butylacetylthio-2- 25 methylpropionic acid, [α] * = -15.31° (c, 5.80, CHCI3), were recovered. EXAMPLE 65 The procedure of Example 61 was repeated using 2,000 units of Rhizopus delemar lipase (Chemical Dynamics Corp., 5000 units/ ag) as the enzyme and (+)-R-3-tert-butylacetylthio-2-methylpro- 25 pioπic methyl ester, [<x] = +15.40* (c, 5.07 , CHC1 3 ) , and (-)- 25 S-3-tert-butylacetylthio-2-methylpropionic acid, [α]^ = -36.95° (c, 3.11, CHCI3), were recovered. EXAMPLE 66 The procedure of Example 62 was repeated except 250 mg of ( +)-3-3' -chloro-2 l , 2' -dimethylpropionylthio-2-methylpropionic aethyl ester was used as the substrate and (+)-R-3-3 ' -chloro- 2 ' , 2 ' -diraethylpropionylthio-2-methylpropionic methyl ester, [α ]^ 5 = +13.22° (c , 6.95 , CHCI3 ) , and (-)-S-3-3 ' -chloro-2 ' , 2 * - 25 dimethylpropionylthio-2-methylpropionic acid, [α]^ = -29.96° (c, 2.60, CHCI3), were recovered. EXAMPLE 67 The procedure of Example 63 was repeated except 250 mg of (+J-3-3 ' -chloro-2 ' , 2 ' -dimethylpropionylthio-2-methylpropionic methyl ester was used as the substrate and (+)-R-3-3'-chloro- 2' ,2'-dimethylpropionylthio-2-methylpropionic methyl ester, [ex] 25 = +12.52" (c, 5.40, CHC1 3 ), and (-)-S-3-3'-chloro-2 ' ,2'- diraethylpropionylthio-2-methylpropionic acid, [α]^ 5 ~~ -18.94° (c, 5.09, CHC13), were recovered.

Claims: Claims 1. A process for preparing optically-active 3-acylthio-2-meth- ylpropionic acid, which comprises subjecting esters of racemic 3-acylthio-2-methylpropionic acid to the hydrolytic enzymatic action of a microbial lipase (EC 3.1.1.3) and recovering the desired optically-active compounds. 2. The process of Claim 1 wherein the racemic esters are rep¬ resented by compounds having the formula Where R^ is a radical in straight chain, branched chain, or cyclic configuration selected from the class consisting of: alkane radicals having 1 to about 12 carbon atoms with or without electronegative substituents at C-2'; cycloalkane radicals having from 5 to about 7 carbon atoms; and, phenyl and benzyl radicals having from 6 to 8 carbons. Where R2 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 carbobenzoxyl radicals containing substituents such as nitro, halogens, methyl, and alkoxyl groups on the aromatic ring(s). 3. The process of Claim 1 wherein the racemic ester substrate ( l) is an activated ester. 4. The process of Claim 1 wherein an activator for the lipase is present. 5. The process of Claim 1 wherein the enzyme is immobilized. 6. The process of Claim 1 wherein the lipase is an extracel¬ lular lipase derived from microorganisms selected from the genera consisting of Candida. Rhizopus. Mucor, Aspergillus, Pseudomonas, Chromobacterium, Geotrichiuπt. Humicola and Penicillium. 7. The process of Claim 6 wherein the lipase (EC 3.1.1.3) is that derived from a microorganism of the genus Mucor. 8. The process of Claim 7 wherein the lipase is derived from Mucor meihei. 9. The process of Claim 6 wherein the lipase (EC 3.1.1.3) is that derived from a microorganism of the genus Rhizopus. 10. The process of Claim 9 wherein the lipase is derived from Rhizopus niveus. 11. The process of Claim 9 wherein the lipase is derived from Rhizopus arrhizus. 12. The process of Claim 6 wherein the lipase (EC 3.1.1.3) is that derived from a microorganism of the genus Aspergillus. 13. The process of Claim 12 wherein the lipase is derived from Aspergillus niger. 14. The process of Claim 6 wherein the lipase (EC 3.1.1.3) is that derived from a microorganism of the genus Candida. 15. The process of Claim 14 wherein the lipase is derived from Candida cyllndracea. 8 26 16. The process of Claim 2 wherein the substrate ester is (+}- 3-pivaloylthio-2-methylpropionic methyl ester and chloro¬ ethyl ester. 17. The process of Claim 2 wherein the substrate ester is (+)- 3-pivaloylthio-2-methylpropionic chloroethyl ester. 18. The process of Claim 16 wherein the lipase is derived from Rhizopus niveus. 19. The process of Claim 17 wherein the lipase is derived from Rhizopus niveus. 20. The process of Claim 17 wherein the lipase is derived from Rhizopus delemar. 21. The process of Claim 2 wherein the substrate ester is (+_)- 3-3' ,4' ,5'-trimethoxybenzoylthio-2-methylpropionic methyl ester. 22. The process of Claim 21 wherein the lipase is derived from Mucor meihei. 23. The process of Claim 2 wherein the substrate ester is (+J- 3-l'-naphthoylthio-2-methylpropionic methyl ester. 24. The process of Claim 23 wherein the lipase is derived from Mucor meihei. 25. The process of Claim 23 wherein the lipase is derived from Rhizopus delemar. 26. The process of Claim 2 wherein the substrate ester is (+J- 3-benzoylthio-2-methylpropionic methyl ester. 27. The process of Claim 26 wherein the lipase is derived from Aspergillus niger. 28. (-)-S-3-pivaloylthio-2-methylpropionic acid. 29. (-)-S-3-3' ,4' ,5'-trimethoxybenzoylthio-2-methylpropionic acid. 30. (-)-S-3-l '-naphthoylthio-2-methylpropionic acid. 31. (-)-S-3-tert-butylacetylthio-2-methylpropionlc acid.

Inventors: SIH CHARLES J, SIH, CHARLES, J.
Applicants: WISCONSIN ALUMNI RES FOUND, WISCONSIN ALUMNI RESEARCH FOUNDATION
Application year: 1987
Main classification: C12P11
IPC codes: C12P7, C12P11, C12P19, C12P41