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Practical Enzymatic Resolution of Racemic Alcohols and Amines

in Organic Solvents" ARIE L. GUTMAN, ELEONORA SHKOLNIK,

ELAZAR MEYER, FELIX POLYAK, DOV BRENNER, AND AVIV BOLTANSKI

Department of Chemishy Israel Institute of Technology

Technion City Haifa 32,000, Israel

INTRODUCTION

One of the most important properties of enzymes is their ability to catalyze reactions in a stereoselective manner. This has been used for many years by organic chemists, who have exploited enzymes as catalysts in asymmetric synthesis and resolution for the preparation of optically pure compounds.' It is now well estab- lished that hydrolytic enzymes such as lipases, esterases, and proteases are highly stable in organic solvents and can be used for certain types of transformations that are difficult or impossible to carry out in water.2 The most common reactions are lipase-catalyzed stereoselective esterifications and transesterifications, which have been extensively used for preparative kinetic resolutions of chiral acids and alco- hols: as well as for the synthesis of chiral lactones4 and polyester^.^ It has also been reported that some lipases and proteases are effective in catalyzing the reaction between carboxylic esters and amines in anhydrous organic solvents? and these reactions have been recently used in intermolecular or intramolecular fashion for kinetic resolution of racemic amines' and for the synthesis of lactams.* In the present paper, we wish to report on novel applications of enzymes in organic solvents for the synthesis of chiral alcohols and amines with an emphasis on the development of large-scale processes useful for industrial production.

ENZYMATIC SYNTHESIS OF CHIRAL ALCOHOLS VIA ACYLATION IN ORGANIC SOLVENTS

Chiral secondary alcohols are common as intermediates and as valuable chiral auxiliaries in organic synthesis and are of great synthetic utility for the pharmaceuti- cal and the fine-chemical industries. Unlike the chiral acids or amines, which can be resolved via diastereomeric crystallization of appropriate salts, alcohols do not form salts. Hence, chiral alcohols cannot be obtained easily by this direct method. It is

aThis work was supported by a fund for the promotion of research at the Technion and by a Minerva Foundation grant from the Otto Meyerhof Biotechnological Laboratories.

620

GUTMAN et al.: ENZYMATIC RESOLUTION 62 1

therefore not surprising that much effort has been devoted to the kinetic resolution of raccmic secondary alcohols via enzymatic acylation in organic solvents. The acylating agents included carboxylic acids, various activated and enol esters, or acyclic aliphatic acid anhydrides, and the enzymatic reactions amounted to conver- sion of one enantiomer of the alcohol (1) into an ester (2) with the other one remaining as the unreactive alcohol (3) (SCHEME 1). Separation of the unreacted alcohol ( 3 ) from the ester (2) (followed by hydrolysis of the latter) enabled the resolution of racemic mixtures into their enantiomers.

A major problem with this approach is the difficulty of separating the ester from the unreacted alcohol. In many cases, both have similar boiling points, making it impossible to separate them by distillation, and tedious chromatographic separation becomes necessary. This makes the procedure impractical for large-scale prepara- tion.

We developed a principal solution to this problem by using succinic anhydride as an acylating agent.y The reaction depicted in SCHEME 2 was shown to be highly stereospecific with a whole range of secondary alcohols (4a-4e) using several commercially available lipase preparations. Particularly productive from the point of

0

H 4' Enzyme

+ R-C, - X R' R"

1 2 3

Scheme 1

view of enzyme efficiency and stereospecificity were the reactions with succinic anhydride catalyzed by the lipases, Amano P, Amano PS, and Amano Sam 2, in tert-butyl methyl ether. This enabled us to convert the (R)-enantiomer of the alcohol into the half-acid, which could be separated from the unreacted (S)-alcohol by simple extraction with aqueous base. The unreacted (S)-alcohol was recovered by evaporating the organic solution, whereas the (R)-enantiomer was obtained in quantitative yield by straightforward hydrolysis of the half-acid. This method was shown to be effective for the prcparation of several pharmaceutically important alcohol intermediates with higher than 98% optical purity on a multikilo scale.

Reactions catalyzed by lipase Amano Sam 2 required only small quantities of enzyme and, in preparative experiments, we worked with a substrate:enzyme ratio of 100:3. Because the price of this enzyme is less than $l/gram, $30 worth of enzyme enabled us to carry out a reaction on 1 kg of sec-phenethyl alcohol (4e) to obtain 280 grams and 420 grams of the optically pure (R)- and (S)-enantiomers, respectively (see EXPERIMENTAL WORK below). This high substrate-to-enzyme ratio coupled with the extreme ease of separation between the reacted and unreacted enantiomers

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4a 4b 4c 4d 4e

Scheme 2

make the economics of the process plausible for industrial preparation of various optically pure phenyl-alkyl and naphthyl-alkyl secondary alcohols.

Although this approach was useful for the preparation of several chiral secondary alcohols, it cannot apply to highly sterically hindered alcohols, such as 2,2,2-trifluoro- 1-(9-anthryl)ethanol (7), which remained unreactive under all enzymatic acylation conditions tried. The inefficiency of enzymatic acylation of this alcohol can be explained by steric and electronic considerations affecting the mechanism of the enzymatic process. In the first step, an acyl-enzyme intermediate is formed between the enzyme and the acylating agent (step I, SCHEME 3). In the second (critical) step, the carbonyl of this intermediate has to undergo a nucleophilic attack by the alcohol oxygen of (7), which is accompanied by the breakup of the acyl-enzyme link, resulting in the release of the free enzyme and the formation of the desired ester. It is at this second stage, which is critical from the point of view of enantiodiscrimination, that the steric hindrance around the hydroxyl group plays a crucial role by blocking the approach of the nucleophile to the carbonyl that is positioned deep inside the enzyme pocket (SCHEME 3). The electronic effect of the CF3 group, which strongly

GUTMAN el 01.: ENZYMATIC RESOLUTION 623

reduces the nucleophilicity of the alcohol oxygen, is also responsible for the ineffi- ciency of step 11.

To resolve this problem. we developed an alcoholysis approach, according to which the easily prepared racemic butyrate ester (8) is enzymatically reacted with butanol. In this case, enantiodiscrimination is exercised during the first enzymatic step and the reacting ester (and hence the released alcohol) is one enantiomer, whereas the other one is left behind as the unreactive ester. The second step along this route simply involves the breakup of the acyl-enzyme intermediate by an achiral nuclcophile (butanol) and is of no stereochemical interest. According to this approach, the electron-withdrawing effect of the CF3 group should help as it creates a better leaving group (SCHEME 4).

We exploited this approach for preparative purposes. Using crude porcine pancrcatic lipase (PPL) immobilized on aluminum oxide in n-butanol, it was possible to conveniently carry out this reaction on a large scale and to obtain in one laboratory batch hundreds of grams of the (R)-alcohol and of the unreacted (S)-butyrate ester (SCHEME 5) . After a facile separation procedure between the (R)-alcohol and the unreacted (S)-ester, the latter was hydrolyzed and both enantiomers of the alcohol were obtained.

The very high cost of the enantiomerically pure isomers of (7) ($33 for 100 mg from Aldrich Chemical) and its important analytical applications as chiral shift reagents and as key ingredients of stationary phases for chiral chromatography columns emphasize the practical usefulness of this enzymatic process. Furthermore, it was shown that the stability of PPL and the ratc of the enzymatic reaction could be

I

a b c

a -formation of acyl-enzyme intermediate via nucleophilic attack of serine oxygen on the acylating agent: b - acyl-enzyme intermediate: c - sterically hindered interaction between acyl-enzyme and racemic alcohol.

Scheme 3

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. 0 @-OH /p FIR + H G - E T E , A R L O - - C , + R a

(RS) - 7 H

(R)-7 a -free enzyme; b - acyl-enzyme intermediate.

E-OH

Scheme 4

enhanced considerably by simple dispersion of the enzyme on neutral aluminum oxide. The combination of an inexpensive and stable enzyme and of a relatively efficient reaction makes the economics of the process feasible for the practical preparation of this and other sterically hindered alcohols. Work in our laboratory is

\

Scheme 5

GUTMAN er al.: ENZYMATIC RESOLUTION 625

currently under way to determine the generality of this approach to other sterically hindered alcohols.i0

RESOLUTION OF RACEMIC AMINES VIA ENZYMATIC AMINOLYSIS IN ORGANIC SOLVENTS

Practical methods for the synthesis of chiral amincs usually involve multistep transformations starting from chiral natural amino acids, or resolution of racemic mixtures via reaction with a chiral acid to form a diastereomeric mixture of salts, followcd by consecutive fractional crystallizations and recovery of the amine. As an efficient alternative to these classical methods, we developed a facile large-scale continuous process for enzyme-catalyzed aminolysis in organic solvents that enabled us to prepare a whole range of optically active amines. We also addressed several technological aspects pertaining to enzyme immobilization and to development of a continuous enzymatic reactor.

FIGURE 1. Structures (R-9 ) and (R-10).

(R)-10

Our target chiral amines included the pharmaceutically important intermediate, (R)-I-aminoindan (R-9) (see FIGURE 1). The latter is an immediate synthetic precursor of the heretofore unknown (R)-N-propargyl- I-aminoindan (PAI) (R-10) (see FIGURE I ) , which was shown to be a potent irreversible inhibitor of the B form of monoaminc oxidasc and could therefore he used in the treatment of Parkinson's disease, memory disorders, dementia of the Alzheimer type, depression, and the hyperactive syndrome." Recently, very considerable differences in MAO-B- inhibiting activity have been reported between the two cnantiomers of PAI: it was shown that the (R) - ( +) enantiomer is nearly four orders of magnitude more active than the ( S ) - ( - ) enantiomer.l2

We found that certain enzymes catalyzed the stcrcoselective aminolysis reaction of the raccmic I-aminoindan (9) with esters, in particular, with the active ester 2,2,2-1rilluorocthyI butyratc, in various organic solvents. The (S)-enantiomcr of

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0 Immobilized subtilisin

(S)-11

Scheme 6

I-aminoindan reacted in preference to give the corresponding amide (12), whereas the (R)-enantiomer remained as a free amine (SCHEME 6).

Unfortunately, the desired stereospecific enzymatic reaction was accompanied by a significant nonenzymatic reaction between the active ester and amine. Because the latter reaction undermined the stereospecificity of the enzymatic reaction, it was important to slow it down. This could be done by an appropriate choice of solvent and by shortening the time of contact between the amine and active ester. When tert-amyl alcohol or 3-methyl-3-pentanol was used as solvent, the nonenzymatie reaction is slowest. Furthermore, we succeeded in reducing the time of contact between the reaction components by performing the reaction in a continuous flow reactor with noncovalently immobilized enzyme (FIGURE 2).13

A slurry of the immobilized enzyme in 3-methyl-3-pentanol was packed onto a gravitational column equipped with a water jacket for temperature control. Solutions of 1 -aminoindan and of 2,2,2-trifluoroethyl butyrate in 3-methyl-3-pentanol were delivered t o the mouth of thc column in parallel, through separate channels of a dual-piston pump, and the mixing took place just before loading onto the column. The enzymatic aminolysis reaction took place on the column and the eluant was allowed to drip directly into a solution of aqueous hydrochloric acid; this immedi- ately converted the unreacted (R)-1 -aminoindan to its hydrochloride and thus prevented any possibility of further nonenzymatic reaction between the chiral (9) and the remaining active cstcr.

The extent of the enzymatic reaction and of the optical purity of the desired (R)-aminoindan is a function of several parameters: (a) the flow rate through the column, which determines the time of contact between the reactants and thc enzyme; (b) concentrations of the rcactants, I-aminoindan and 2.2,2-trifuoroetliyI butyratc; (c) temperature; and (d) the content of water in the solvcnt. All thcsc parameters ;ire easily controlled and they wcrc modified while periodically monitoring thc optical purity of the eluting amine by HPLC on a chiral column. At steady-state operating conditions, a column constructed with 3.7 g of subtilisin on 370 g of glass bcads

GUTMAN el al.: ENZYMATIC RESOLUTION 627

operated continuously for 95 hours at 36 “C to elute 330 g of racemic (9) and to give, in 40% yield, (R)-1-aminoindan of optical purity higher than 98%. In this continuous flow procedure, the enzyme-to-substrate ratio was 1:90. Furthermore, it is important to note that at the end of the process the immobilized enzyme was “clean” of any contaminants and its activity was only very slightly lower than at the beginning. Clearly, it could be used to resolve further quantities of 1-aminoindan. To check the lifetime of the immobilized subtilisin in a packed column, a small column was constructed and used approximately once a month over a period of 10 months to resolve a small quantity of the reactants. After each experiment, the column was washed with 3-methyl-3-pentanol and left “wet” with solvent a t room temperature until the next run. The performance of this column was assessed on the basis of optical purity of the eluting (R)-1-aminoindan, and it was revealed that no detectable loss of enzymatic activity occurred on the column after 10 months of such intermit- tent operation and storage.

The enzymatic process described in this work may be used as a general method for the resolution of chiral primary amines. It can be readily scaled up for industrial application because it is highly efficient, yields optically pure (R)-amines, and withstands long-term operation. The high substrate-to-enzyme ratios and the possi- bility for efficient recycling of the solvent and active ester make the economics of the process plausible. The method, therefore, represents a valid alternative to the classical chemical resolutions of amines and it should be particularly useful in cases where resolution via diastereomeric crystallizations is not readily accomplished.

\f TD \n v

FIGURE 2. Continuous enzymatic reactor: ( 1 ) solution of active ester; (2) solution of amine; (3) dual-piston pump; (4) column-packed suhtilisin immobilized on glass beads; (5) stirred solution o f aqueous hydrochloric acid.

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EXPERIMENTAL WORK

General Methods

‘H-NMR spectra were recorded on a Bruker AM 200-MHz spectrometer in CdC13. All chemical shifts were reported in ppm with tetramethylsilane as the internal standard. Enantiomeric excess (ee) for the chiral alcohols was determined by Merck Hitachi HPLC on a chiral column (Chiralcel OJ, Daicel) with a mixture of hexane and 2-propanol(95:5) as the mobile phase at a flow rate of 0.9 mL/min, using detection at 258 nm. Enantiomeric excess for the chiral amines was determined on a chiral column [Crownpak CR( +), 4 x 150 mm (Daicel)] eluting with a mixture of 15% methanol and 85% perchloric acid (pH 2) at a flow rate of 1.3 mL/min, with detection at 222 nm. Optical rotations were determined on a JASCO digital polarimeter, DIP-370. Column chromatography and “filtration through silica” were performed on silica gel kieselgel40,0.063-0.200 mm (E. Merck). Solvent extracts of aqueous solutions were dried over anhydrous Na2S04. Solutions were concentrated under reduced pressure on a rotary evaporator. Chemical purity of substrates and products was determined either by HPLC as described above or by HP 5890 Series I1 GC on a DB-210 column. Freeze-drying was performed on a 1-L LYPH-LOCK Labconco lyophilizer. Determinations of water in organic solvents and in immobi- lized enzyme preparations were performed using the Karl-Fischer method on a Metrohm titration instrument consisting of dosimat Model 665 and processor Model 678 EP/KF.

The lipases, Amano PS and Amano Sam 2, were purchased from Amano Pharmaceutical. Porcine pancreatic lipase (PPL) was purchased from Sigma Chemi- cal. Subtilisin A (batch A-4008-75) was kindly provided by Novo Nordisk, A/S Denmark. Aluminum oxide (A1203) used for enzyme dispersion was Aluminum oxide 90 active neutral (E. Merck). Glass beads were acid-washed ( < 106 Fm) and purchased from Sigma Chemical ((3-4649). Unless otherwise stated, all solvents and other chemicals were obtained from commercial suppliers and were used without further purification.

Preparative Resolution of sec-Phenethyl Alcohol (4e)

One liter (1.01 kg) of sec-phenethyl alcohol (4e) in 5 liters of t-butyl methyl ether (TBME) was placed in a polyethylene wide-mouth bottle equipped with a powerful magnetic stirrer. To the solution were added 0.994 kg of succinic anhydride and 30 g of lipase SAM 2. A suspension was formed because succinic anhydride is poorly soluble in TBME and the enzyme is insoluble in this or other organic solvents. The suspension was stirred at room temperature and progress of the reaction was followed by NMR: (4e) has a quartet at 4.77 ppm corresponding to the benzylic hydrogen, whereas the quartet of this hydrogen in the product (6) appears at 5.90 ppm. Accurate integration of the appropriate signals enabled us to determine the ratio between starting material and product and thus determine the degree of conversion. After 38 hours, the enzymatic reaction reached 30.4% conversion and was stopped by filtering off the solids, which were washed with TBME, dried at room temperature in the air, and kept aside (to reuse the enzyme). The filtrates were

GUTMAN el al.: ENZYMATIC RESOLUTION 629

extracted with a 1 M solution of sodium carbonate (3 x 1 L) and with 1 L of water. Thc aqueous phase containing (6) was washed with TBME (2 x 600 mL) and then hydrolyzed by adding 300 g of sodium hydroxide and stirring for 6 hours at room temperature. Then, the basic aqueous solution was extracted with 3 x 500 mL dichloromethane; the organic phase was washed with water (500 mL), dried over Na2S04, and evaporated; and the residue was distilled (98 T / 2 0 mmHg) to give 280 g of R-( +)-sec-phenethyl alcohol, [a]: = +40.8" (ncat). The ee of this alcohol was determined to be 98% by HPLC on a chiral column. The combined organic phases (which contained the unreacted S-enriched alcohol) were poured into the 10-L polyethylene wide-mouth bottle; the enzyme and the succinic anhydride recovered from the previous experiment, together with 15 g of fresh enzyme and 50 g of succinic anhydride, were added; and the suspension was stirred at room temperature. Periodically, aliquots were withdrawn and the increase in optical purity of the unreactcd (S)-enantiomer was monitored by HPLC on the chiral column. The reaction was continued for 3 days until the complete disappearance of (R)-alcohol. The insoluble enzyme and succinic anhydride were then filtered off, the organic solution was washed with 1 M sodium carbonate (3 x 1 L), and the organic solvent was removed under reduced pressure. The residue was distilled (98 "C/20 mmHg) to give 420 g of (S)-(-)-sec-phenethyl alcohol, [a]:' = -42.7" (neat). The ee of this alcohol was determined to be 99.6%.

Preparation of Dispersed Porcine Pancreatic Lipase (PPL)

PPL (370 g) was added to neutral aluminum oxide (370 g) and thoroughly mixed in a stoppered Erlenmeyer flask by gentle inversions of the flask for about 15 rninutcs. The resultant homogeneous white powder was stored in the cold and used in the preparative enzymatic resolution experiments.

Preparative Resolution of (R,S)-2,2,2-Trifluoromethyl-l-(Panthryl)ethanol(7) by Enqmatic Alcoholysis

(R,S)-2,2,2-Trifluoromethyl-l-(9-anthryl)ethyl butyrate (8) (370 g, 1.03 mol) was dissolved in 2.5 L of boiled n-butanol in a 5-L flat-bottom flask equipped with a powerful magnetic stirrer. The solution was cooled to 40 "C and 400 g of the dispersed PPL preparation (containing 200 g PPL) was added. The resultant suspension was vigorously stirred at 40 "C and the reaction progress was followed by periodically examining aliquots of the suspension by HPLC on the chiral column. Integration of the appropriate HPLC signals enabled us to determine the ratio between starting material and product, as well as their optical purity. Thus, both the reaction rate and the ee values of the remaining ester and formed alcohol were followed simultaneously during the course of the reaction. On the fourth day of the reaction, an additional 340 g of the dispersed enzyme and 500 mL of butanol were added and the reaction was Continued for another 6 days (altogether 10 days). After 9 days, it was observed that the reaction came to a virtual standstill at 47% conversion, at which point the remaining (S)-ester had an enantiomeric excess value of ee = 98.3% and the resultant alcohol had ee = 96.5%. The butanol solution was

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filtered off, the solid dispersed PPL was washed with 5 L of hot acetone, and the combined filtrates were evaporated at reduced pressure to remove most of the acetone. To the remaining butanol solution, hexane (350 mL) was added and evaporated to remove azeotropically part of the butanol. This operation was re- peated four times and most of the butanol was removed in this way. An additional 500 mL of hexane was then added to the remaining butanol solution, and the new solution was left overnight at -15 "C. Crystallization occurred and the precipitate was filtered off and washed with hexane to give the crystalline alcohol (R-7) (55 g, 39%), with ee = 100%. The mother liquor was evaporated to dryness to give 281 g of light-brown oily residue containing the ester (S-8) and the alcohol (R-7) at a ratio of approximately 251 . This was mixed with 250 g of silica gel in 400 mL of hexane and partially evaporated until a thick brown slurry was obtained. The slurry was placed onto a 300-g silica gel column and eluted with hexane. TLC and HPLC examination indicated that the eluant (7 L) contained only the ester (S-8). After all the ester was eluted, the column was washed with diethyl ether (1.5 L) to remove the alcohol (R-7). Evaporation of the hexane fractions resulted in the crude ester (S-8), which was crystallized from ethanol to give (S)-2,2,2-trifluoromethyl-l-(9-anthryl)ethyl buty- rate (S-8) (171 g, 92%), as white crystalline powder. The ee of the ester as determined by HPLC was 99.2%. Evaporation of the ether fractions resulted in the crude alcohol (R-7), which was washed with hexane to give (R)-2,2,2-trifluoromethyl- 1-(9-anthryl)ethanol (R-7) (62 g, 43%). Together with the first crop, 117 g of alcohol (R-7) was obtained (yield of 82%) with an optical purity of ee = 99.4% by HPLC, [a];" = -32" (C = 6, CHC13).

Hydrolysis of (S-8)

To a solution of the ester (S-8) (171 g, 0.475 mol) in 600 mL of methanol was added KOH (27 g, 0.49 mol) in 60 mL of water and the mixture was stirred at room temperature for 1 h. Water (400 mL) was then added, forming a white suspension that was stirred for another 1 h and filtered to afford white precipitate, which was dried and washed with hexane to give a white crystalline powder of (S-7) (130 g, 99%). Optical purity of ee = 99% was obtained by HPLC, [a];" = +32.5" (c = 6, CHC13).

Preparation of Immobilized Subtilisin and Packing of the Column

First, 3.7 g of subtilisin A was dissolved in 130 mL of 0.1 M phosphate buffer (pH 7.8) and the solution was mixed with 370 g of glass beads (Sigma, G-4649, acid- washed, < lo6 microns). The slurry was then gently stirred and freeze-dried to constant weight during 24 h. The resultant solid material was crushed to a thin powder, which was suspended in 3-methyl-3-pcntanol to a thin slurry, and that slurry was loaded onto a gravitational column (3.8 cm x 56 cm) equipped with a jacket for maintenance of the desired temperature. When the slurry settled down, the packed support had a density of 1.25 g/mL and its dimensions were 26 x 3.8 cm (occupying volume of approximately 300 mL). In another experiment, 5.7 g of subtilisin immobilized on 570 g of glass beads was packed on the same reactor. In that case, thc

GUTMAN el nl.: ENZYMATIC RESOLUTION 631

volume of the support material was 450 mL and the dimensions of the packed reactor wcrc 40 x 3.8 cm. The immobilized enzyme could be kept on the column with 3-methyl-3-pentanol for at least 10 months without any detectable deterioration.

Enzymatic Aminolysis of I-Aminoindan (9)

The solution of I-aminoindan (9) was prepared by mixing 318 mL (330 g, 2.5 mol) of the amine (R,S)-9 with 3174 mL of 3-methyl-3-pentanol (solution A). The solution of 2,2,2-trifluoroethyl butyrate was prepared by mixing 1330 mL (1443 g, 8.5 mol) of the active ester with 2160 mL of 3-methyl-3-pentanol (solution B). Solutions A and B were placed in glass bottles and each was connected with the top of the column (26 x 3.8 cm) (containing 3.7 g of subtilisin on 370 g of glass beads) by Teflon tubing through separate channels of a dual-piston pump, Gearmotor type NYC-1303, purchascd from Bodine Electric (Chicago). The column was fitted with a water jacket for temperature control at 36 “C. The two solutions were fed into the column in parallel, each at a rate of 35 mL/h. The rate of outflow from the column was controlled by a valve at the bottom adjusted to maintain the outflow at about 70 mL/h, thus keeping the layer of the solution above the packed bed at levels not higher than 2-5 cm. rhis was intended to minimize the time of contact between the reactants before elution through the column. After 95 hours of continuous opera- tion, the column was washed for a period of 10 hours with 3-methyl-3-pentanol at a flow rate of 70 mL/h. All the eluant from the column (approximately 7.5 L) was dripping into a reservoir containing a total of 2.5 L of 1 M hydrochloric acid, which was vigorously stirred to immediately convert the unreacted free (R)-amine (R-9) into the hydrochloride salt and to prevent any nonenzymatic reaction with remaining active ester after passage through the column. The acidic aqueous layer contained (R)-1-aminoindan hydrochloride. The layers were separated and the organic layer was distilled to leave a “cakelike” residue containing the amide (1 1) and some of the (R)-amine hydrochloride, which was moderately soluble in 3-methyl-3-pentanol. This “cake” was dissolved in a mixture of dichloromcthane ( 1 L) and water (1 L) and the layers wcrc separated. The dichloromethanc layer was evaporated to give (S)-( -)-N-butyrylindanamide (11) (247 g, 49%). The aqueous layer containing the amine hydrochloride was combined with the original acidic aqueous layer and its HPLC on a chiral column showed an cc > 98%. To recover the free (R-9), water was evaporated, the hydrochloride salt was suspended in dichloromethane, and the suspension was treated with a solution of ammonia in dichloromethane. Ammonium chloride was filtcrcd off and washed with dichloromcthane, the solvent was evapo- rated, and the residue was distilled at 80 “C and 3 mmHg to give (R-9) (132 g, 40% ).

CONCLUSIONS

This paper exemplified some applications ol‘ enzymatic catalysis to the prcpara- tivc synthesis o f chiral alcohols and amincs required and used as pharmaceutical intermediates and chiral auxiliaries. I t is clcar that thc methods can be scaled up and applied to commercial production of a wide variety o f these and other chiral compounds.

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(a) GUTMAN, A. L., K. Zuoei & A. BOLTANSKI. 1987. Enzymatic lactonization of y-hydroxyesters in organic solvents: synthesis of optically pure y-methylbutyrolactone and y-phenylbutyrolactone. Tetrahedron Lett. 2 8 3861-3864; (b) MAKITA, A., T. NIHIRA & Y. YAMADA. 1987. Lipase-catalyzed synthesis of macrocyclic lactones in organic solvents. Tetrahedron Lett. 2 8 805-808; (c) Guo, Z-W. & C. J. SIH. 1988. Enzymatic synthesis of macrocyclic lactones. J. Am. Chem. SOC. 1 1 0 1999-2001; (d) Guo, Z-W., T. K. NGOOI, A. SCILIMATI, G. FULLING & C. J. SIH. 1988. Macrocyclic lactones via biocatalysis in non-aqueous media. Tetrahedron Lett. 2 9 5583-5586; (e) GUTMAN, A. L. & T. BRAVDO. 1989. Enzyme-catalyzed enantioconvergent lactonization of y-hydroxy diesters in organic solvents. J. Org. Chem. 54 42634265; (f) GUTMAN, A. L., K. ZUOBI & T. BRAVDO. 1990. Lipase-catalyzed preparation of optically active y-butyrolactones in organic solvents. J. Org. Chem. 55: 3546-3552.

(a) GUTMAN, A. L., D. OREN, A. BOLTANSKI & T. BRAVDO. 1987. Enzymatic oligomeriza- tion versus lactonization of w-hydroxyesters. Tetrahedron Lett. 28: 5367-5368; (b) MARGOLIN, A. L., J-Y. CRENNE & A. M. KLIBANOV. 1987. Stereoselective oligomeriza- tions catalyzed by lipases in organic solvents. Tetrahedron Lett. 2 8 1607-1610; (c) WALLACE, J. S. & C. J. MORROW. 1989. Biocatalytic synthesis of polymers: synthesis of an optically active, epoxy-substituted polyester by lipase-catalyzed polymerization. J. Polym. Sci. Part A 27: 2553-2567; (d) GUTMAN, A. L. & T. BRAVDO. 1989. Enzyme- catalyzed enantioconvergent polymerization of p-hydroxyglutarate in organic solvents. J. Org. Chem. 545645-5646; (e) KNANI, D., A. L. GUTMAN & D. KOHN. 1993. Enzymatic polyesterification in organic media: enzyme-catalyzed synthesis of linear polyesters. J. Polym. Sci. Part A 31: 1221-1232.

ZAKS, A. & A. M. KLIBANOV. 1985. Enzyme-catalyzed processes in organic solvents. Proc. Natl. Acad. Sci. U.S.A. 82: 3192-3196.

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