Abstract:
A process for the microbiological or enzymatic hydrolytic resolution of racemic trans-2-(alkoxycarbonylethyl) lactams of the formula I: ##STR1## wherein R is C 1  -C 7  alkyl, 2,2,2-trifluoroethyl or methoxyethoxyethyl and R 1  is hydrogen or a protecting group is disclosed, whereby an optically enriched compound of the formula Ib or IIa: ##STR2## is obtained.

Description:
BACKGROUND OF THE INVENTION 
     Trans-2-(alkoxycarbonylethyl)lactams and trans-2-(carboxyethyl)lactams are intermediates in the synthesis of 1-(4-fluorophenyl)-3(R)- 3(S)-hydroxy-3-(4-fluorophenyl)-propyl)!-4(S)-(4-hydroxyphenyl)-2-azetidinone, a cholesterol lowering agent disclosed in U.S. Pat. No. 5,767,115. 
     U.S. Pat. No. 5,618,707 discloses stereoselective microbial reduction of a keto intermediate (4-(4-fluoro-benzoyl)butyric acid or a phenyloxazolidinone conjugate thereof) to the corresponding hydroxy intermediate used in the preparation of the azetidinone. Preferred microorganisms used in the process are Zygosaccharomyces bailii or Schizosaccharomyces octosporus. 
     SUMMARY OF THE INVENTION 
     The process of the present invention relates to microbiological or enzymatic hydrolytic resolution of a racemic trans-2-(alkoxycarbonylethyl)lactam of the formula I: ##STR3## wherein R is C 1  -C 7  alkyl, 2,2,2-trifluoroethyl or methoxyethoxyethyl and R 1  is hydrogen or a protecting group selected from the group consisting of benzyl, trimethylsilyl, t-butyldimethylsilyl (TBDMS) and acetyl, to obtain an optically enriched compound of the formula Ib or IIa: ##STR4## 
     When a carboxylic acid ester of formula Ib is obtained, the process further comprises hydrolysis of the resulting compound of formula Ib to obtain an acid of formula IIa. The resulting 3R,4S lactam acid is useful as an intermediate in the preparation of 1-(4-fluorophenyl)-3(R)- 3(S)-hydroxy-3-(4-fluorophenyl)propyl)!-4(S)-(4-hydroxyphenyl)-2-azetidinone. 
     The resolution comprises the use of microorganisms (obtained from environmental sources and culture collections, e.g., the American Type Culture Collection (ATCC)) in medium, medium and buffer, medium and solvent, or medium and a mixture of buffer and solvent, or the use of enzymes in buffer, solvent or a mixture thereof, to which a racemic trans-2-(alkoxycarbonylethyl)lactam is added so that a compound having an ester or acid group of desired stereochemistry can be formed, accumulated and isolated. The resolution is either direct or subtractive, depending on the microorganism or enzyme used. 
     Microorganisms selected from the group consisting of the following genera have been found to be useful in the direct resolution: Aspergillus, Bacillus, Candida, Cunninghamella, Debaryomyces, Mycobacterium, Paecilomyces, Penicillium, Rhodobacter, Streptomyces and Trichothecium. The following species of the above genera are preferred: Aspergillus alliaceus, niger, niveus and terreus; Bacillus sphaericus; Candida parapsilosis and rugosa; Cunninghamella homothallica; Debaryomyces hansenii; Mycobacterium fortuitum; Paecilomyces marquandii; Penicillium implicatum; Rhodobacter sphaeroides; Streptomyces spectabilis; and Trichothecium roseum. 
     Microorganisms selected from the group consisting of the following genera have been found to be useful in the subtractive resolution: Comamonas, Curvularia, Mucor, Nocardia and Rhodococcus. The following species of the above genera are preferred: Comamonas testosteroni; Curvularia brachyspora and geniculata; Mucor circinelloides and racemosus; Nocardia corallina; and Rhodococcus erythropolis, rhodochrous and species. 
     Commercially available enzymes suitable for use in the resolution of this invention include Amano Lipase D (Rhizopus delemar); Amano Lipase FAP-15 (Rhizopus javanicus); Amano Lipase MAP-10 (Mucor javanicus); Amano Lipase N (Rhizopus niveus); Interspex Bacterial Esterase/Lipase BE1-Supported (Pseudomonas mandocino); Nagase Lipase A-10 (Rhizopus japonicus); Novo SP 525 (Candida antarctica, type B); Toyobo Lipoprotein lipase LPL-701 and LPL-311, type A (Pseudomonas sp.); Seikagaki Lipase (Rhizopus delemar); Kinzi &amp; Payne Lipase WT (Rhizopus sp.); Svedas Lipase (Rhizopus oryzae); Sawa Lipase A-10 (Rhizopus japonicus); Sawa LPL-701 and LIP 301 (Pseudomonas sp.); Boehringer-Mannheim Chirazyme™ L2 (Candida antarctica lipase, type B); Boehringer-Mannheim Chirazyme™ L4 and L6 (Pseudomonas sp.); Interspex Lipase/Esterase ICS-16-FL1 Fungal (Rhizopus oryzae); Fluka Lipase (Aspergilius niger); Toyobo LIP-300/301 and LIP-321 (Pseudomonas sp.); Toyobo Lipoprotein lipase LPL 311 Type A (Pseudomonas sp.); Novo Lipozyme IM-60 (Mucor miehei); and Sigma Lipase Type XI (Rhizopus arrhizus). 
     Preferred enzymes are hydrolases of Pseudomonas sp. (Toyobo LPL 311 Type A, Toyobo LIP-301/LIP 300, Toyobo LPL 701, Boehringer-Mannheim Chirazyme™ L6). 
     In particular, the present invention relates to direct resolution of trans-1-(4-fluorophenyl)-3-(alkoxycarbonylethyl)!-4(S)-(4-hydroxyphenyl)-2-azetidinone comprising adding said compound to a microorganism in medium, medium and buffer, medium and solvent, or medium and a mixture of buffer and solvent, especially wherein the microorganism is Aspergillus terreus or alliaceus, or Candida parapsilosis, incubating the resulting mixture, and isolating a compound of the formula IIa ##STR5## wherein R 1  is as defined above. 
     In particular, the present invention also relates to subtractive resolution of trans-1-(4-fluorophenyl)-3-(alkoxycarbonylethyl)!-4(S)-(4-hydroxy-phenyl)-2-azetidinone comprising adding said compound to a microorganism in medium, medium and buffer, medium and solvent, or medium and a mixture of buffer and solvent, especially wherein the microorganism is Rhodococcus rhodochrous or Rhodococcus species, or to an enzyme in a solvent, buffer or a mixture thereof, especially wherein the enzyme is a hydrolase from Pseudomonas sp., incubating the resulting mixture, and isolating a compound of the formula Ib: ##STR6## wherein R and R 1  are as defined above. The compound of formula Ib is then hydrolysed to remove the carboxylic acid ester group, R, to obtain a compound of formula IIa. 
     DETAILED DESCRIPTION 
     The hydrolytic resolution of the present invention is summarized in the following reaction scheme: ##STR7## 
     This scheme shows a method for performing a direct hydrolysis using a microorganism or enzyme, where racemic lactam ester I is hydrolyzed to generate enantiomerically enriched acid (3R,4S)-IIa which is easily separated from unreacted carboxylic acid ester (3S,4R)-Ia. Alternatively, a subtractive resolution of racemic lactam ester I yields acid IIb and enantiomerically enriched carboxylic acid ester (3R,4S)-Ib which is subsequently hydrolyzed to generate (3R,4S)-IIa. The enantiomerically enriched (3R,4S)-IIa is subsequently used to synthesize 1-(4-fluorophenyl)3(R)- 3(S)-hydroxy-3-(4-fluorophenyl)propyl)!-4(S)-(4-hydroxy-phenyl)-2-azetidinone using procedures known in the art, for example by converting the acid of formula IIa to the corresponding acid chloride, reacting the acid chloride with a 4-fluorophenyl derivative, and reducing the ketone to the alcohol as described in Method H of U.S. Pat. No. 5,767,115. 
     The hydrolytic resolution is carried out by adding a racemic trans-2-(alkoxycarbonylethyl) lactam I to medium, medium and buffer, medium and solvent, or medium and a mixture of buffer and solvent containing microorganisms, or to solvent, buffer, or a mixture thereof, containing enzymes. The bioconversion may be conducted at temperatures in the range from between about 20° C. to about 40° C.; the microbial reaction is preferably conducted at ambient temperature to 30° C. and the enzymatic reaction is preferably conducted at ambient temperature to 37° C. The initial pH value of the reaction is adjusted to be in the range from between about pH 5.0 to about 9.0, preferably pH 7.0. 
     The initial concentration of racemic trans lactam ester I in the microbial reaction may vary from between about 0.5 g/l to about 5 g/l, and is preferably 0.5 g/l. The duration of the microbial hydrolysis may vary from about 18 to about 96 hours, and is preferably about 48 hours. 
     The initial concentration of trans lactam ester I in the enzyme mediated reaction may vary from between about 5 mg/ml to about 200 mg/ml, and is preferably 25 mg/ml. The duration of the enzymatic hydrolysis may vary from about 24 to about 192 hours. 
     Suitable fermentation media, buffers and solvents are known to those skilled in the art. Fermentation media typically contain a carbon and nitrogen source or mixtures thereof, using such ingredients as yeast extract, nutrient broth, dextrose (cerelose), white potato dextrin, soy flour, peptone and other components known in the art. Typical buffers are phosphate buffer (e.g., 0.1 M at pH 7), MES (2- N-morpholino!ethanesulfonic acid), Bis-Tris (bis 2-hydroxyethyl!iminotris hydroxymethyl!methane), PIPES (1,4-piperazine-diethanesulfonic acid), HEPES (N- 2-hydroxyethyl!piperazine-N&#39;- 2-ethanesulfonic acid!), TRIS (tris(hydroxymethyl)aminomethane) and MOPS (3- N-morpholino!propanesulfonic acid) buffer (e.g., 0.1 M at pH 7). Typical solvents are acetonitrile, acetone, ethyl ether, isopropanol, t-butanol, isoamyl alcohol, p-dioxane, isopropyl ether, dimethyl sulfoxide, t-butyl methyl ether (TBME), toluene, tetrahydrofuran and CH 2  Cl 2 . Preferably, the microbial resolutions are carried out in fermentation media, and the enzymatic resolutions preferably are carried out in a buffer with a co-solvent; a preferred co-solvent for enzymatic resolutions is TBME. 
     At the end of the hydrolysis, optically enriched acids or esters may be extracted using organic solvents such as ethyl acetate (EtOAc), TBME, CH 2  Cl 2  and the like. Adsorption to resins, chromatography, and other physical methods known in the art may also be used for the isolation of optically enriched acids or esters. 
     The carboxylic acid ester of formula Ib can be hydrolysed to the corresponding acid of formula IIa using methods well known in the art, for example by treatment with a suitable base, e.g., LiOH, as described in U.S. Pat. No. 5,767,115. 
     The examples below demonstrate the evaluation of microorganisms and enzymes in the hydrolysis of this invention and the preparation of milligram quantities of compounds of formulas IIa and Ib. 
    
    
     EXAMPLE 1 
     The general method for identifying the microbial hydrolysis of racemic trans lactam methyl ester I for use in generating acid IIa is described below. 
     Seed cultures of yeast, filamentous fungi, and bacteria were grown in 125 ml or 300 ml flasks containing 25 ml or 50 ml of YPD (1% yeast extract, 2% peptone, 2% dextrose; pH 5.5), SIM6 (3.5% soy flour, 5% white potato dextrin, 0.5% cerelose, 2 mg/l cobalt chloride, 0.5% calcium carbonate; pH 6.0) and NYC (0.8% nutrient broth, 2% yeast extract, 2% cerelose; pH 7.0) media respectively, for 72 hours at 30° C. with agitation (175-250 rpm) prior to inoculation (4% v/v) into flask fermentations (25 ml YPD/125 ml flask for yeast and filamentous fungi or 25 ml NYC/125 ml flask for bacteria) which were incubated at 30° C. with agitation (250 rpm). In all fermentations, medium pH was adjusted prior to inoculation but was not controlled during culture propagation and substrate hydrolysis. Microbial resolution was initiated by adding 0.5 g/l of racemic trans lactam methyl ester I dissolved in ethanol (25 mg/ml), directly to cultures following 24 hours of growth. Samples of fermentation broth were extracted with TBME following 24-72 hours incubation with substrate and were analyzed by reverse-phase HPLC. Preferred cultures demonstrating selective hydrolysis generating acid IIa are summarized in Table 1. 
     
                       TABLE 1______________________________________Direct resolution of racemic trans lactammethyl esters using microorganisms   Strain #                         %Culture (ATCC)  Substrate Product   % ee Yield______________________________________A. terreus   10020   benzyl    benzyl protected                               100  9   20542   protected IIa: (3R, 4S) acid                               91   23   24839   racemate I          91   35Penicillium   SPR     benzyl    benzyl protected                               100  6implicatum   938*    protected IIa: (3R, 4S) acid           racemate IAspergillus   9029    unprotected                     IIa: (3R, 4S) acid                               100  14niger           racemate IAspergillus   1024    unprotected                     IIa: (3R, 4S) acid                               100  29alliaceus       racemate ICandida 7330    unprotected                     IIa: (3R, 4S) acid                               100  29parapsilosis   16632   racemate I          100  26   22019                       100  28   34078                       100  20Candida 14830   unprotected                     IIa: (3R, 4S) acid                               100  20rugosa          racemate I______________________________________ *Schering-Plough Research: Biotransformations Culture Collection 
    
     EXAMPLE 2 
     Milligram quantities of acid IIa derived from the microbial hydrolysis of benzyl protected racemic trans lactam methyl ester I was prepared as described below. 
     Microbial resolution of methyl ester I (0.5 g/l) to generate acid IIa was conducted as described in Example 1 using multiple flask fermentations employing Aspergillus terreus strain ATCC #24839. Following 48 hours of incubation, fermentation broths of each of the cultures were pooled prior to centrifugation to separate the cells from the fermentation broth. Cell pellets were pulverized in liquid nitrogen using a mortar and pestle prior to three sequential extractions with TBME (1-2 volumes/wet weight). Fermentation broth was extracted separately with TBME. The TBME extracts contained both the (3R,4S)-acid and the (3S,4R)-ester, each in &gt;99% enantiomeric excess. Anhydrous MgSO 4  was added to the TBME extracts to remove residual water, the extracts were filtered and the filtrate concentrated by evaporation. Extract concentrate was subjected to purification by preparative thin layer chromatography employing multiple 10-20 GF silica plates (20 cm×20 cm×1000 micron) and developed with a solution of EtOAc:hexane (50:50). Material comigrating with the desired product was scraped from each of the silica plates, pooled and eluted from the silica with TBME. The eluate was evaporated to yield the (3R,4S)-acid IIa: 170 mg, 17% yield; 86% enantiomeric excess;  α! D   25  =-13.0° (c=0.123, ethanol). 
     EXAMPLE 3 
     The general method for identifying the microbial resolution of benzyl protected racemic trans lactam methyl ester I for use in generating ester Ib is described below. 
     Seed cultures of yeast, filamentous fungi, and bacteria were grown in 125 ml or 300 ml flasks containing 25 ml or 50 ml of YPD (1% yeast extract, 2% peptone, 2% dextrin; pH 5.5), SIM6 (3.5% soy flour, 5% white potato dextrose, 0.5% cerelose, 2 mg/l cobalt chloride, 0.5% calcium carbonate; pH 6.0) and NYC (0.8% nutrient broth, 2% yeast extract, 2% cerelose; pH 7.0) media respectively, for 72 hours at 30° C. with agitation (175-250 rpm) prior to inoculation (4% v/v) into flask fermentations (25 ml YPD/125 ml flask for yeast and filamentous fungi or 25 ml NYC/125 ml flask for bacteria) which were incubated at 30° C. with agitation (250 rpm). In all fermentations, medium pH was adjusted prior to inoculation but was not controlled during culture propagation and substrate hydrolysis. Microbial resolution was initiated by adding 0.5 g/l of racemic trans lactam methyl ester I dissolved in ethanol (25 mg/ml), directly to cultures following 24 hours of growth. Samples of fermentation broth extracted with TBME following 24-72 hours incubation with substrate were analyzed by reverse-phase HPLC. Cultures yielding optically enriched ester Ib are summarized in Table 2. 
     
                       TABLE 2______________________________________Subtractive resolution of racemic trans lactam methyl estersusing microorganisms    Strain #          Product        %Culture  (ATCC)  Substrate (methylester)                                % ee Yield______________________________________R. erythropolis     4277   benzyl    benzyl    100  17    11048   protected protected 100  17    19369   racemate I                      Ib: (3R, 4S)                                100  11R. rhodochrous    29670   benzyl    benzyl    100  30    19150   protected protected 100  22    29675   racemate I                      Ib: (3R, 4S)                                100  24R. species    19148   benzyl    benzyl    100  31    19071   protected protected 100  31            racemate I                      Ib: (3R, 4S)C. testosteroni    33083   benzyl    benzyl    100  12            protected protected            racemate I                      Ib: (3R, 4S)N. corallina    31338   benzyl    benzyl    100  11            protected protected            racemate I                      Ib: (3R, 4S)______________________________________ 
    
     EXAMPLE 4 
     Milligram quantities of methyl ester Ib derived from the hydrolysis of benzyl protected racemic trans lactam methyl ester I (0.5 g/l) was prepared as described in Example 3 using multiple flask fermentations employing Rhodococcus species ATCC #19071. Following 48 hours of incubation, fermentation broths of each of the flasks were pooled prior to centrifugation to separate the cells from the fermentation broth. Cell pellets were disrupted by sonication prior to three sequential extractions with TBME (1-2 volumes/wet weight). Fermentation broth was extracted separately with TBME. Anhydrous MgSO 4  was added to the TBME extracts to remove residual water, the extracts were filtered and the filtrate concentrated by evaporation. Extract concentrate was subjected to purification by preparative thin layer chromatography employing multiple 10-20 GF silica plates (20 cm×20 cm×1000 micron) and developed with a solution of EtOAc:hexane (50:50). Material comigrating with the desired product was scraped from each of the silica plates, pooled and eluted from the silica with TBME. The eluate was evaporated to yield the (3R,4S)-ester; 360 mg, 36% yield; &gt;99% enantiomeric excess;  α! D   25  =-7.5° (c=0.133, ethanol). 
     EXAMPLE 5 
     The general method for identifying the enzymatic resolution of benzyl protected racemic trans lactam methyl or trifluoroethyl esters I for use in generating optically enriched acid and ester is described below. 
     Enzyme screening reactions were conducted using a two-phase system of 0.6 ml TBME with 1.0 ml of 0.1 M phosphate buffer (pH 7.0). Enzyme, typically 50-200 mg or 100-200 μL, was added to the suspension followed by 14.4 mg of methyl ester. The mixture was agitated (350 rpm) at room temperature. Some deviations from these reaction conditions were evaluated as indicated in Table 3. Material was recovered by separating the phases by centrifugation and product and unreacted starting material were analyzed by chiral HPLC. Enzymes demonstrating selective hydrolysis of racemic trans lactam methyl ester I yielding acid IIb and ester Ib are summarized in Table 3. 
     
                       TABLE 3______________________________________Enzymatic resolution of benzyl protected racemic trans lactammethyl ester I yielding optically enriched ester Ib and acid IIb.               (3S, 4R) (3R, 4S)       Time    IIb      Ib     Conver-Enzyme      (hour)  ee.sub.p ee.sub.s                               sion  E______________________________________Amano Lipase D       64.5    0.71     0.67   0.485 11Rhizopus delemarAmano Lipase FAP-15       44.25   0.76     0.49   0.392 12Rhizopus javanicusAmano Lipase       64.5    0.68     0.39   0.364  8MAP-10Mucor javanicusAmano Lipase N       64.5    0.75     0.34   0.312 10Rhizopus niveusInterspex Bacterial       44.25   0.11     &gt;0.95  0.897 n/dEsterase/LipaseBE1 -SupportedP. mandocinoNagase Lipase A-10       44.25   0.72     0.73   0.504 13R. japonicusNovo SP 525 Lipase,       122     0.77     0.18   0.191  9type BC. antarcticaToyobo Lipoprotein       46.5    0.95     0.62   0.395 68lipase (LPL-701)       90*     0.957    0.486  0.337 74Pseudomonas sp.Seikagaki Lipase       122     0.69     0.63   0.478 10Rhizopus delemarToyobo Lipoprotein       46.5    0.97     &gt;0.97  0.507 n/dLipase      90*     0.975    0.709  0.421 165(LPL-311) Type APseudomonas sp.Kinzie &amp; Payne       138.25  0.72     0.41   0.359  9Lipase WTRhizopus sp.Svedas Lipase       119     0.70     0.73   0.511 12Rhizopus oryzaeSawa Lipase A-10       47      0.68     0.82   0.547 13Rhizopus japonicusSawa LPL-701       90*     0.967    0.467  0.326 93Pseudomonas sp.Boehringer-Mannheim       47      0.82     0.05   0.060 11Chirazyme ™ L2,lipase BCandida antarcticaBoehringer-Mannheim       119     0.94     0.24   0.206 38Chirazyme ™ L4Pseudomonas sp.Boehringer-Mannheim       47      0.95     0.77   0.450 86Chirazyme ™ L6       90*     0.97     0.46   0.321 103Pseudomonas sp.Interspex   119     0.73     0.64   0.465 12Lipase/EsteraseICS-16-FL1FungalRhizopus oryzaeFluka Lipase       71      0.44     0.43   0.495  4Aspergillus nigerNovo Lipozyme IM-60       141.5   0.49     0.15   0.235  3Mucor miecheiSigma Lipase Type XI       136.5   0.75     0.35   0.316 10Rhizopus arrhizus______________________________________ *Conditions: Ester (50 mg), Enzyme (50 mg), TBME/Phosphate Buffer (pH 7) (1 mL:1 mL), 300 rpm, RT. #Conditions: Ester (19 mg), Enzyme (1 mg), Tetrahydrofuran/0.5M MOPS buffer pH 7.0 (0.2/1.0 mL) 
    
     A similar procedure was conducted using benzyl protected racemic trans lactam trifluoroethyl ester I. Enzyme reactions were conducted using a two-phase system of 1.0 ml TBME with 1.0 ml of 0.1 M phosphate buffer (pH 7.0). Approximately 50 mg of enzyme and 50 mg of ester were added to the suspension and mixed with agitation (300 rpm) at room temperature for up to 186 hours. Material was recovered by separating the phases by centrifugation and product and unreacted starting material were analyzed by chiral HPLC. Enzymes demonstrating selective hydrolysis of racemic trans lactam trifluoroethyl ester I yielding acid IIb and ester Ib are summarized in Table 
     
                       TABLE 4______________________________________Enzymatic resolution of benzyl protected racemic trans lactamtrifluoroethyl ester I yielding optically enriched ester Ib and acidIIb.               (3R, 4S) (3S, 4R)       Time    Ib       IIb    Conver-Enzyme      hour    ee.sub.s ee.sub.p                               sion  E______________________________________Toyobo LIP-301       186     0.755    0.987  0.433 360Pseudomonas sp.Toyobo LPL-701       48      0.713    0.774  0.480 16Pseudomonas sp.Toyobo LPL 311       48      0.994    0.628  0.613 23(Type A)    24      0.991    0.788  0.557 44Pseudomonas sp.Boehringer-Mannheim       48      0.680    0.796  0.461 18Chirazyme ™ L6Pseudomonas sp______________________________________ 
    
     EXAMPLE 6 
     Milligram quantities of ester Ib derived from the enzymatic resolution of benzyl protected racemic trans lactam methyl ester were prepared as described below. ##STR8## 
     Toyobo LPL-311 (Type A) (Pseudomonas sp.) (202 mg) was dissolved in 0.1M phosphate buffer (pH 7) (8 mL) at room temperature. A solution of racemic methyl ester (199.5 mg, 0.46 mmol) in TBME (8 mL) was added. The two-phase mixture was shaken at 37° C. at 250 rpm for 187 h. The reaction mixture was acidified with 0.5 M H 2  SO 4  (1 mL), diluted with water (15 mL) and placed in two centrifuge tubes. EtOAc (20 mL) was added to each tube and the tubes shaken, then centrifuged at 3000 rpm for 0.5 h. The organic layer was removed and the extraction/centrifugation repeated twice. The combined organic extracts were evaporated and the crude product was placed on a silica gel column (Selecto 32-63 mesh; 20 g) and eluted with 30% (300 mL) and 50% (400 mL) EtOAc/heptane, collecting fractions of ˜20 mL. Fractions 4-6 were combined and evaporated to yield the (3R,4S)-methyl ester: 89 mg, 44.6%; 95.1% enantiomeric excess;  α! D   25  =-14.15° (c=0.89, ethanol). Fractions 11-19 provided the (3S,4R)-acid: 39 mg, 20.2%; 84.5% enantiomeric excess;  α! D   25  =+14.87° (c=0.39, ethanol). 
     EXAMPLE 7 
     Milligram quantities of acid IIa derived from the enzymatic resolution of benzyl protected racemic trans lactam trifluoromethyl ester, followed by hydrolysis of the trifluoromethyl ester, were prepared as described below. 
     Step 1 ##STR9## 
     Toyobo LPL-311 (Type A) (Pseudomonas sp.) (365 mg) was dissolved in 0.1M phosphate buffer (pH 7) (16 mL) at room temperature. A solution of racemic trifluorethyl ester (428 mg, 0.85 mmol) in TBME (16 mL) was added. The two-phase mixture was shaken at 37° C. at 250 rpm for 7.75 h, then stored in a refrigerator overnight. The reaction mixture was acidified with 0.5 M H 2  SO 4  (1 mL), diluted with water (50 mL) and placed in four centrifuge tubes. EtOAc (15 mL) was added to each tube and the tubes shaken, then centrifuged at 3000 rpm for 0.5 h. The organic layer was removed and the extraction/centrifugation repeated twice. The combined organic extracts were evaporated and the crude product was placed on a silica gel column (Selecto 32-63 mesh; 35 g) and eluted with 30% (450 mL) and 50% (600 mL) EtOAc/heptane, collecting fractions of ˜20 mL. Fractions 5-7 were combined and evaporated to yield the (3R,4S)-trifluoroethyl ester: 191 mg, 44.6%; 99.0% enantiomeric excess;  α! D   25  =-9.31° (c=1.88, ethanol). Fractions 18-36 provided the (3S,4R)-acid: 100 mg, 27.9%; 88.3% enantiomeric excess;  α! D   25  =+15.96° (c=0.99, ethanol). 
     Step 2 ##STR10## 
     (3R,4S)-Trifluoroethyl ester (181 mg, 0.36 mmol) (99.0% ee) was dissolved in THF (4 mL) and cooled to 0° C. in an ice bath. A solution of LiOH (52.5 mg, 1.25 mmol) was added and the mixture stirred at 0° C. for 3.25 h, by which time HPLC indicated complete hydrolysis. The reaction mixture was acidified with 0.5 M H 2  SO 4  (12 mL) and extracted with EtOAc (2×15 mL). The combined organic extracts were washed with sat&#39;d. NaCl solution (10 mL), dried (Na 2  SO 4 ), filtered and evaporated: 146 mg, 96.4%; 98.2% enantiomeric excess. 
     A sample of the crude product was purified by preparative TLC (Analtech Uniplate Silica Gel GF; 20×20 cm; 1000 μm) eluting with 50% EtOAc/heptane:  α! D   25  =-16.52° (c=0.66, ethanol).