Application of lipases in kinetic resolution of racemates

Application of Lipases in Kinetic Resolutionof Racemates

ASHRAF GHANEM1,2* and HASSAN Y. ABOUL-ENEIN2

1Department of Organic Chemistry, University of Geneva, Geneva, Switzerland2Pharmaceutical Analysis Laboratory, Biological and Medical Research Department (MBC-03-65),

King Faisal Specialist Hospital and Research Centre, Riyadh, Saudi Arabia

Dedicated to Prof. Volker Schurig on the occasion of his receiving the chirality medal 2004.

ABSTRACT Lipases have been well established as valuable catalysts in organicsynthesis. This review article focuses on some of the recent developments in the rapidlygrowing field of lipase-catalyzed kinetic resolution of racemates as a versatile method forthe separation of enantiomers. The literature search dates back to the last five years andcovers some comprehensive examples. The main emphasis is on the use of lipases inorganic solvents. Chirality 17:1–15, 2005. A 2004 Wiley-Liss, Inc.

KEY WORDS: biocatalysis; chiral separation; enantioselectivity; kinetic resolution; lipase

ENZYMES IN ORGANIC SOLVENTS:GENERAL ASPECTS

Enzymatic catalysis in nonaqueous media significantlyextents conventional aqueous-based biocatalysis.1 – 18

Water is a poor solvent for nearly all applications in in-dustrial chemistry, since most organic compounds ofcommercial interest are very sparingly soluble and aresometimes unstable in aqueous solutions. Furthermore,the removal of water is tedious and expensive due to itshigh boiling point and high heat of vaporization. In con-trast, biocatalysis in organic solvents offers several advan-tages. Among these advantages are: the use of low boilingpoint organic solvents facilitates the recovery of the prod-uct with better overall yield, nonpolar substrates areconverted at a faster rate due to their increased solubilityin the organic solvent,15 deactivation and/or substrate orproduct inhibition is minimized, side reactions are largelysuppressed, immobilization of enzymes is not required,denaturation of enzymes (loss of the native structure andthus catalytic activity) is minimized, and shifting thermo-dynamic equilibria favors synthesis over hydrolysis.

CATALYSIS IN ORGANIC SYNTHESIS BY LIPASES

Lipases (triacylglycerol acyl hydrolases, EC 3.1.1.3)have been well established as valuable catalysts in organicsynthesis.12 They are usually distinguished from carboxylesterases (EC 3.1.1.1) by their substrate spectra, i.e., ester-ases prefer water-soluble substrates and lipases show sig-nificantly higher activity towards their natural substrates,triglycerides. Since the hydrolytic reaction is reversible innonaqueous systems, these biocatalysts can also catalyzethe formation of esters from acyl donors and alcohols.

Lipases do not require cofactors. A range of enzymesare commercially available in free and immobilized form.Most lipases accept a broad range of nonnatural sub-strates and are thus very versatile for applications inorganic synthesis. In many cases they exhibit good toexcellent stereoselectivity.

Lipases have been widely used in three main types ofreactions yielding optically pure compounds. These arekinetic resolution of racemic carboxylic acids or alcohols,enantioselective group differentiations of meso dicarboxylicacids or meso diols, and enantiotopic group differentiationof prochiral dicarboxylic acid and diol derivatives.3

In kinetic resolutions of racemic alcohols via trans-esterification, the acyl donors of choice are enol esterssuch as vinyl acetate or isopropenyl acetate. The vinylalcohol formed as a byproduct when using vinyl acetateundergoes keto-enol tautomerization yielding the corre-sponding carbonyl compound (acetaldehyde), while theisopropenyl alcohol released when using isopropenylacetate tautomerizes to acetone, making the reactionpractically irreversible in both cases. Thus, these trans-esterifications are much faster compared to reactions usingfree carboxylic acids or simple esters such as ethyl acetate.

In contrast to asymmetric synthesis, a kinetic resolutionyields at maximum 50% of the desired enantiomer.19 Toachieve higher yields, the unwanted enantiomer can beseparated and re-racemized in a second step. Alternatively,this can be achieved by a dynamic kinetic resolution(DKR).20 Several methods have been described,21 and arereviewed below.

METHODS USED TO ACCESS OPTICALLYPURE COMPOUNDS

The methods used to access enantiomeric compoundscan be divided into three categories depending on the type

Contract grant sponsors: Fonds der Chemischen Industrie and the Grad-uate College Analytical Chemistry, University of Tubingen, Swiss Chem-ical Society, Analytical Division.*Correspondence to: Dr. Ashraf Ghanem, Pharmaceutical Analysis Lab-oratory, Biological and Medical Research Dept. (MBC 03-95), King FaisalSpecialist Hospital and Research Center, P.O. Box 3354, Riyadh 11211,Saudi Arabia. E-mail: [email protected] or [email protected] for publication 29 June 2004; Accepted 6 August 2004

Review Article

A 2004 Wiley-Liss, Inc.

CHIRALITY 17:1–15 (2005)

DOI: 10.1002/chir.20089Published online in Wiley InterScience (www.interscience.wiley.com).

of starting material used22 (Fig. 1). Two methods are usedfor the preparation of enantiomers using enzymes23,24: 1)stereoselective synthesis, and 2) resolution of the racemate.

Stereoselective Synthesis

Stereoselective synthesis versus resolution of racematesis shown in Figure 2.

Resolution of Racemates

Despite the impressive new progress in asymmetricsynthesis, the dominant production method to obtain asingle enantiomer in industrial synthesis consists of theresolution of racemates.25 – 29 The resolution of enan-tiomers can be divided into four categories: direct pref-erential crystallization, crystallization of diastereomericsalts, chromatography, and kinetic resolution.

Preferential crystallization. Also referred to as reso-lution by entrainment, preferential crystallization is widelyused on the industrial scale, e.g., in the manufacture ofchloramphenicol30 and a-methyl-L-dopa.31

Haarmann & Reimer, the market leader in synthetic(–)-menthol, utilizes the preferential crystallization ofmenthyl benzoate enantiomers. This can be induced byseeding the bulk with one of the pure enantiomers and isused in the production of (–)-menthol.32 This process istechnically feasible only with racemates that form con-glomerates (ones that consist of mechanical mixtures ofcrystals of the two enantiomers in equal amounts). Un-fortunately, less than 20% of all racemates are conglom-erates, the rest comprising true racemic compounds thatcannot be separated by preferential crystallization. Thesuccess of preferential crystallization depends on the factthat for a conglomerate the racemic mixture is moresoluble than either of the enantiomers.22

Diastereomer crystallization. When the racemate is atrue racemic mixture, it cannot be separated by prefer-

ential crystallization, but can be resolved using diaster-eomer crystallization, developed by Pasteur in 1848. Asolution of the racemic mixture in water or methanol isallowed to react with a pure enantiomer (resolving agent),thereby forming a mixture of diastereomers that can beseparated by crystallization.

Kinetic resolution. The third method used in theresolution of racemates is kinetic resolution. The successof this method depends on the fact that the two enan-tiomers react at different rates with a chiral entity. Thechiral entity should be present in catalytic amounts; itmay be a biocatalyst (enzyme or a microorganism) or achemocatalyst (chiral acid or base or even a chiral metalcomplex). Kinetic resolution of racemic compounds is byfar the most common transformation catalyzed by lipases,in which the enzyme discriminates between the two enan-tiomers of racemic mixture, so that one enantiomer isreadily transferred to the product faster than the other.1 – 18

(Fig. 3). Kinetic resolution occurs when kR p kS and thereaction is stopped somewhere between 0 and 100% con-version. Ideally, one enantiomer reacts much faster thanthe other; for example, if the reactant (R) is the onlyreacting enantiomer (kS = 0). In this case, 50% conversionof the initial 50/50 mixture leads to a final mixture of 50%reactant (S) and 50% product (P). This route has the ad-vantage of easy separation of both enantiomers by using asingle enzyme.

Dynamic kinetic resolution. The conventional kineticresolution reported above often provides an effective routeto access the enantiomerically pure/enriched compounds.However, a limitation of such a process is that the reso-lution of two enantiomers will provide a maximum 50%yield of the enantiomerically pure materials. Such a limita-tion can be overcome in several ways. Among these waysare: the use of meso compounds or prochiral substrates,33

inversion of the stereochemistry (stereoinversion) of theunwanted enantiomer (the remaining unreacted sub-strate),34 racemization and recycling of the unwanted en-antiomer, and dynamic kinetic resolution (DKR).21 In bothconventional (Fig. 4a) and dynamic (Fig. 4b) kinetic res-

Fig. 1. Methods to obtain enantiomerically pure compounds.

Fig. 2. Stereoselective synthesis (a) versus resolution of a racemate (b).

Fig. 3. Catalytic kinetic resolution.

Fig. 4. (a) Conventional kinetic resolution (max. 50% conv.). (b)Dynamic kinetic resolution with theoretical 100% yield.

2 GHANEM AND ABOUL-ENEIN

olution, the enantiomer (R)-substrate is transformed to (R)-product faster than the enantiomer(S)-substrate (kR > kS).The only difference is that in conventional kinetic resolu-tion the enantiomer (S)-substrate is left behind as un-reacted starting material, while in DKR the substrate iscontinuously isomerized during the resolution process;thus, (R)- and (S)-substrates are in equilibrium, whichallows for the possibility of converting all starting materialsof (R)-substrate into (R)-product. Several conditions shouldbe applied and are reviewed in literature.21 For instance,Backvall and co-workers20 used a combination of enzymeand transition metal complex (Ru-catalyst) to perform theDKR of a set of secondary alcohols (Fig. 5). Depending onthe substrate, the chemical yield ranged from 60–88% withmore than 99% ee.20,35

ENANTIOSELECTIVITY OF LIPASES INORGANIC SOLVENTS

Two important concepts should be understood in theenzyme-catalyzed reactions; the enantiomeric excess (ee)and the enantiomeric ratio E.

The enantiomeric purity of any compound is expressedin terms of its ee value, defined as:

% eeR ¼ R � S

R þ S� 100 For R > S

Where R is the concentration of the (R)-enantiomer and Sis the concentration of (S)-enantiomer. Thus, for a racemiccompound the ee value is zero, whereas for an enantio-merically pure compound the ee value is 1 (or 100% ee).

Since lipases are chiral, they possess the ability todistinguish between the two enantiomers of a racemicmixture. The parameter of choice to describe the stereo-selectivity or the enantioselectivity of lipase-catalyzedreaction is the enantioselectivity, which is also called theenantiomeric ratio, E. The E-value is defined as the ratio ofspecificity constant for the two enantiomers:

ERS ¼ ðkcat=kMÞR

ðkcat=kMÞS

where kcat is the rate constant or the turnover numberand kM is the Michaelis-Menten constant. Sih andcolleagues36,37 developed this equation in terms of theee of the product (eep), the unreacted substrate (ees), and

the conversion (c). Thus, for a reversible enzymatic reac-tion, the E value is expressed by:

E ¼ ln½1 � ð1 þ KÞcð1 þ eepÞ�ln½1 � ð1 þ KÞcð1 � eepÞ�

¼ ln½1 � ð1 þ KÞðcþ eesf1 � cgÞ�ln½1 � ð1 þ KÞðc� eesf1 � cgÞ�

where K is the equilibrium constant. When the reaction isirreversible or the reverse reaction is negligible (K = 0),this equation is reduced to:

E ¼ ln½1 � cð1 þ eepÞ�ln½1 � cð1 � eepÞ�

¼ ln½ð1 � cÞð1 � eesÞ�ln½ð1 � cÞð1 þ eesÞ�

Where (c) is expressed by:

c ¼ ees

ees þ eep

E can also be expressed in terms of ees and eep only by:12

E ¼

Thus, to calculate the E value one can measure twoof the three variables: ees, eep, and the extent of con-version (c). A nonselective reaction has an E-value of 1,while an E-value above 20 is the minimum for an accept-able resolution.12

Chiral Recognition by Lipases

An enzyme model always describes the mechanism ofthe enantioselectivity in an enzymatic reaction. Thesimplest models, more accurately referred to as rules, donot attempt to predict the degree of enantioselectivity, butonly predict which enantiomer reacts faster. The earliestexample of such a model is Prelog’s rule,38 which predictsthe enantioselectivity of the reduction of ketones by yeastalcohol dehydrogenases based on the size of the twosubstituents on the carbonyl group. Other models arebased on pockets, which give an indication of the size andshape of the molecules tolerated in the active site.Examples of such models are the model of Jones andcolleagues39 for pig liver esterase (PLE), subtilisin,40 andseveral lipases.41 – 43 One example is the empirical rule ofKazlauskas et al.44 for chiral recognition by lipases. Thisrule predicts, as exemplified for lipase from Pseudomonascepacia, enantiopreference towards a certain substrate, butcannot predict the degree of enantioselectivity. It istranslated into an active site model for lipases consistingof two pockets of different sizes, a large one and a smallone (Fig. 6).

The stereoselectivity for substrates bearing a small anda large substituent (e.g., a secondary alcohol, as shownin Fig. 6) is explained by assuming that when the sec-ondary alcohol is subjected to resolution by a lipase,Fig. 5. Dynamic kinetic resolution of secondary alcohols.

ln1 � ees

1 þ ðees=eepÞ

� �

ln1 þ ees

1 þ ðees=eepÞ

� �

3LIPASES IN KINETIC RESOLUTION OF RACEMATES

the fast-reacting enantiomer binds to the active side inthe manner shown in Figure 6a; however, when the otherenantiomer reacts with the lipase, it is forced to accom-modate its large substituent into the smallest pocket(Fig. 6b). This rule works well for secondary alcohols.However, for primary alcohols the rule is only applicableif an oxygen atom is attached to the stereocenter. Asimilar rule was also proposed for the resolution ofcarboxylic acids.

In addition, a range of lipase structures have beensolved by X-ray crystallography or are available by homol-ogy modeling. This information together with sequencedata in public databases (e.g., www.led.uni-stuttgart.de)allows further insight into the structure–function relation-ships of lipases. In addition, rational protein designallowed alteration of the enantioselectivity of lipases. Thus,the directed evolution method combining random muta-genesis and high-throughput screening has been used as aversatile tool for tuning or engineering of the enantiose-lectivity of lipases. At this particular point of research,Reetz et al.45,46 reported that the combination of error-prone PCR and DNA shuffling gave a lipase variant ofPseudomonas aeruginosa having completely invertedenantioselectivity. Recently, Koga et al.47 reported inver-sion of the enantioselectivity of another thermostablelipase from Burkhorderia cepacia KWI-56 using a novelin vitro technique for construction and screening of aprotein library by single-molecule DNA amplification byPCR followed by an in vitro coupled transcription/trans-lation system termed single-molecule-PCR-linked in vitroexpression (SIMPLEX).

ANALYTICAL METHODS: DETERMINATIONOF ENANTIOMERIC EXCESS (ee)

The development of accurate methods for the deter-mination of enantiomeric purity, which began in the late1960s, has been critical for the assessment of enantiose-lective synthesis. Thus, a prerequisite in the enzyme-catalyzed kinetic resolution of racemates is a precise andreliable assessment of the degree of enantioselectivity(E), enantiomeric excess (ee), and conversion (c).Among these methods are: 1) polarimetric methods, 2)gas chromatographic methods, 3) liquid chromato-graphic methods, and 4) NMR spectroscopy. The mostconvenient and sensitive methods used are chiral GCand HPLC.

Gas Chromatographic Methods

An attractive method for the determination of the ee ofsubstrates and products resulting from the enzyme-catalyzed kinetic resolution of secondary alcohols is chiralgas chromatography (GC).48,49 This sensitive method isquick, simple to carry out, and unaffected by the presenceof impurities in the analyzed sample; therefore, isolationand purification of the analyzed sample is not required.Very small sample size is required for the analysis; hence,reactions can be done on a small scale.

This method is based on the fact that molecularassociation may lead to an efficient chiral recognition,leading to enantiomeric separation when a chiral sta-tionary phase (e.g., cyclodextrins) is used in GC. The gas(mobile phase, e.g., hydrogen, helium, nitrogen) iscarrying the chiral analyte through the stationary phase.The enantiomers to be analyzed undergo rapid andreversible diastereomeric interactions with the chiralstationary phase, and hence may be eluted at differenttimes. One of the limitations associated with this method isthat the sample should be sufficiently volatile, thermallystable, and resolvable on the chiral stationary phase used.The measurement of ee using GC is linked with a highdegree of precision (F0.05%), so that reliable data may beobtained.50 It should be noted that high ee (up to 99%) maybe detected.51 – 58

HPLC Methods

HPLC methods follow the same principles and advan-tages as GC analysis. The major difference is that morepolar and also nonvolatile compounds can be analyzed.

PRACTICAL APPLICATIONS OF LIPASES IN THERESOLUTION OF RACEMATES

Resolution of racemates via lipase-catalyzed kineticresolution is one of the most attractive methods used toaccess to enantiomerically pure compounds. Of allmethods used in kinetic resolution, transesterification inorganic solvents catalyzed by lipase is the most dominantone. Thus, in the presence of a suitable acyl donor, anenzyme as well as the appropriate solvent, and at theoptimum temperature, one enantiomer of the racemicmixture is selectively transferred to the correspondingester, leaving the second unreacted enantiomer inenantiomerically pure form.51 – 58 If a good leaving groupis present on the acyl donor, as in the case of trichlo-roethyl or trifluoroethyl esters (Fig. 7a), the reaction of thehalogenated alcohol with the formed ester (the backwardreaction) is minimized, allowing the shift of equilibrium toproduct formation. Oxime esters have been proposed asacyl transfer agents (Fig. 7b) for irreversible process;however, this approach is limited due to some disadvan-tages related to cosubstrate inhibition and reversibility ofthe reaction. The best method for an irreversible trans-esterification procedure is achieved when using enolesters (Fig. 7c) where the back reaction is suppresseddue to the tautomerization of the resulting enol alcohol (toacetaldehyde or acetone, depending on whether a vinyl

Fig. 6. The fast reacting enantiomer (a) and the slow reacting one (b)in the active side model for lipases derived from Kazlauskas’ rule.


or an isopropenyl ester serve as acyl donors), therebyshifting the equilibrium to the required product. However,acetaldehyde may have some detrimental effects on someenzymes.8 Isopropenyl acetate was proposed as aninnocuous and more suitable acyl donor in lipase-catalyzedirreversible transesterification in organic solvents (Fig. 7d).The use of different reagents in irreversible acylationcatalyzed by lipase has been recently reviewed.59

Kinetic Resolution of Primary Alcohols

Homochiral primary alcohols are useful buildingsblocks for the synthesis of a wide range of biologicallyactive compounds. While lipase-catalyzed enantioselectiveaccess to enantiomerically pure secondary alcohols arevery efficient tools in organic synthesis, the kineticresolution of racemates of primary alcohols by the samemethod is more difficult to achieve. This is due to lowerenantioselectivity of lipases toward chiral primary alco-hols. Lipase from Pseudomonas cepacia (PSL) is the most

efficient lipase, which shows high enantioselectivitytowards a broad range of primary alcohols.60 Nordinet al.61 studied the enantioselectivities of lipases from

Fig. 10. Selected examples showing the kinetic resolution of primaryalcohols.

Fig. 9. Lipase-catalyzed kinetic resolution of 3-substituted 2-methyl-propan-1-ols by transesterification with vinyl acetate.61

Fig. 8. Lipase-catalyzed kinetic resolution of 2-substituted 2-methyl-ethanols by transesterification with vinyl acetate.61

Fig. 7. Lipase-catalyzed irreversible transesterification.


Pseudomonas cepacia toward a series of primary 2-methyl-substituted alcohols using vinyl acetate as acyl donor intransesterifications in organic solvents (Figs. 8, 9).

In terms of enantioselectivity, the best results werefound for 3-aryl-2-methylpropan-1-ols with an enantiomericratio (E) over 100 in most cases, whereas other 3-substi-tuted primary 2-methylpropan-1-ols generally displayedlower enantioselectivities: 3-cycloalkyl-2-methylpropan-1-ols (E f20) and 2-methylalkan-1-ols (E f10). Movingthe aryl group closer or further away from the chiralcenter resulted in low enantioselectivities: 2-arylpropan-1-ols (E < 10), 2-methyl-4-(2-thienyl)butan-1-ol (E = 12),2-methyl-5-(2-thienyl)pentan-1-ol (E = 3.2), and 2-methyl-6-(2-thienyl)hexan-1-ol (E = 3.8). Other primary alcoholshave been also successfully used as substrates for otherlipases (Fig. 10).

Kinetic Resolution of Secondary Alcohols

Secondary alcohols are by far the most frequently usedtargets in lipase-catalyzed resolutions. This is due to theirimportance in organic synthesis, but also since lipasesusually show much higher enantioselectivity in resolutionscompared to primary and tertiary alcohols. Numerousexamples can be found in the literature and only a fewselected examples are included in this survey. Schurig andcolleagues51 – 57 published a series of reports about theutility of isopropenyl acetate as an innocuous acyl donor inthe lipase-catalyzed transesterification of secondary alco-hols (Fig. 11). The nonreacting alcohol enantiomers wereobtained in >99% ee (Figs. 12, 13).

In the transesterification of (R,S)-secondary alcohols, the(R)-alcohol was the faster-reacting enantiomer, yieldingthe (R)-acetate in high ee and leaving the (S)-alcohol asan enantiomerically pure unreacted enantiomer. Trans-4-phenyl-3-buten-2-ol 50 (Fig. 14), another substrate pos-sessing an allylic strain, has been successfully resolved onthe gram scale via lipase-catalyzed enantioselective acyla-

tion of the alcohol (Fig. 15) and hydrolysis of its acetate(Fig. 16).57

The lipase-catalyzed asymmetric transesterification wasperformed using isopropenyl acetate in organic mediaaffording the (S)-alcohol in high ee (>99%). The reversereaction consisted of lipase-catalyzed hydrolysis of theracemic acetate, affording the (R)-alcohol in high ee (>99%).

In order to reduce the time needed to perform acomplete kinetic resolution, Lindner et al.53 reported theuse of the allylic alcohol 50 in enantiomerically enrichedform rather than a racemic mixture in kinetic resolution.Thus, the kinetic resolution of 50 was performed starting

Fig. 14. Lipase-catalyzed kinetic resolution of racemic 50 usingisopropenylacetate as acyl donor in toluene as organic solvent.57

Fig. 13. Gas chromatographic chiral separation of: (left) racemic (dl)-menthol 48 and its corresponding acetate 48a (reference) and (right)lipase-catalyzed transesterification of (dl)-menthol (15 hrs) using isopro-penyl acetate as acyl donor in toluene as organic solvent: ees = 85.2%, eep =88%, conv. = 49%, E = 42.

Fig. 12. Gas chromatographic chiral separation of: (left) racemic1-(4-methoxyphenyl)ethanol 42 and its corresponding acetate 42a (ref-erence) and (right) lipase-catalyzed transesterification of 1-(4-methoxy-phenyl)ethanol 42 (4 hrs) using isopropenyl acetate as acyl donor intoluene as organic solvent: ees = 99.9%, eep = 87%, conv. = 53.4%, E = 141.

Fig. 11. Lipase-catalyzed transesterification of secondary alcoholsusing isopropenyl acetate as acyl donor in toluene.51 – 57


from the enantiomerically enriched alcohol (R) or (S)-50(45%) ee obtained by the ruthenium-catalyzed asymmetricreduction of 52 with the aim of reaching f100% ee ina consecutive approach (Fig. 17). Several lipases werescreened in resolving the enantiomerically enriched 50either in the enantioselective transesterification of (S)-50(45% ee) using isopropenyl acetate as an acyl donor intoluene in nonaqueous medium, or in the enantioselectivehydrolysis of the corresponding acetate (R)-51 (45% ee),using a phosphate buffer (pH = 6) in aqueous medium.An E value of 300 was observed and the reaction wasterminated after 3 h, yielding (S)-50 >99% ee and the ester(R)-51 was recovered with 86% ee determined by capillaryGC after 50% conversion.

Instead of two steps reaction, Kamal et al.72 reported theone-pot lipase-catalyzed synthesis of enantiopure second-ary alcohols starting from a carbonyl compounds. Thus,

the reduction of acetophenones with sodium borohydridein the presence of neutral alumina in hexane followedby enantioselective acylation catalyzed by lipases wasperformed in one pot (Fig. 18). Other secondary alcoholscontaining benzofuran (60), azide (61), alkylthio (62), car-boxylic acid ethyl ester (63), and a-methylene-b-hydroxyesters(64)moietyhavebeensuccessfullyresolved(Fig.19).

The rapid screening of different hydrolases for theenantioselective hydrolysis of esters of the difficult toresolve substrates such as pentalactone 70, 1-methoxy-2-propanol 71, 3-butyn-2-ol 72, and 3-hydroxy-tetrahydro-fuarn 73 was studied by Baumann et al.81 The screeningwas performed in a pH-indicator-based format in microtiterplates (Fig. 19).

Fig. 19. Selected examples of kinetic resolution of secondary alcoholsand difficult to resolve substrates (70–73).

Fig. 18. Selected examples of the one-pot lipase-catalyzed synthesis ofenantiopure secondary alcohols.72

Fig. 17. Ruthenium/lipase-catalyzed separation of enriched (R,S)-50.53

Fig. 16. Lipase-catalyzed enantioselective hydrolysis of racemic (51)using phosphate buffer (pH = 6) and toluene as organic solvent.57

Fig. 15. Gas-chromatographic separation of the enantiomer of bothsubstrate (50) (as carbamate) and product (51) on heptakis-(2,3-di-O-methyl-6-O-tert-butyldimethylsilyl)-b-cyclodextrin of the Pseudomonas fluo-rescens lipase (PFL) catalyzed transesterification of (50) in toluene at t =9 h, ees = 99.9%, eep = 92.2%, conv. = 52%, E = 284.


Kinetic Resolution of Tertiary Alcohols

Krishna et al.82 reported the enantioselective trans-esterification of a tertiary alcohol 74 using lipase A fromCandida antarctica (CAL-A) and vinyl acetate as acyldonor in organic solvent. Attempts to resolve other tertiaryalcohols (75,83 76,84 7785) are documented in theliterature (Fig. 20).

Kinetic Resolution of Chiral Carboxylic Acids

2-Arylpropionic acids are important class of nonsteroidalantiinflammatory drugs (NSAID). Their pharmacologicalactivity is mainly in one of both enantiomers. Thus, effortshad been made to access to the enantiomerically puresubstance. The kinetic resolution of racemic 2-(2-fluoro-4-biphenyl)propanoic acid (Flurbiprofen) 78 and 2(4-iso-butylphenyl)propanoic acid 79 (Ibuprofen) was performedvia enzymatic esterification and transesterification usingan alcohol and vinyl acetate, respectively, in a membranereactor. The unreacted acid is obtained in highly enan-tiomerically enriched form. A consecutive approach con-sisting of the enzymatic hydrolysis of the resulted estersis needed to achieve the alcohol in optically pure form(Fig. 21).86

Henke et al.87 reported the resolution of 2(4-isobutyl-phenyl)propanoic acid 79 (ibuprofen) by transesterifica-tion of its corresponding vinylester using lipase fromCandida antarctica. Depending on the nucleophile, thevinylester was recovered with 8–99% ee, while the alkylester or the free acid 79 is recovered with 16–75% ee.

Kinetic Resolution of Diols

Desymmetrization of prochiral substrates. The de-symmetrization of prochiral substrates including meso and

P-stereogenic substrates has become a powerful method inasymmetric synthesis.26 The advantage of desymmetriza-tion over conventional kinetic resolution is the potentialability to achieve high ee even at high conversion, with atheoretical yield of 100%.22 Among the widely usedsubstrates in this approach, prochiral ketones and alcoholsreceived much attention. For ketones, the known BaeyerVilliger oxidation or deprotonation using chiral lithiumamide bases serves to differentiate the two-prochiralgroups attached to the carbonyl of the ketone. Apart fromthe chiral amide approach, Baeyer Villiger monooxygen-ase enzymes were used successfully in the desymmetri-zation of prochiral and meso cyclohexanones.27 In acomplementary method, lipases have been used in thedesymmetrization of enol esters derived from two syntheti-cally important classes of cyclic and bicyclic prochiralketones26 and in the desymmetrization of prochiralalcohols or acetate.28

Desymmetrization of racemic diols. Diols such as theoptically active 1,1V-binaphthyl-2-2V-diol (BINOL) havebeen used as versatile templates and chiral auxiliaries incatalysts employed successfully in asymmetric synthesis.The application of enzymes in the enantioselective accessto axially dissymmetric compounds was first reported byFujimoto et al.92 In aqueous media, the asymmetrichydrolysis of the racemic binaphthyl dibutyrate (the ester)using whole cells from bacteria species afforded the (R)-diol with 96% ee and the unreacted substrate (S)-ester with94% ee at 50% conversion. Recently, in nonaqueous medialipases from Pseudomonas cepacia and Ps. fluorescens havebeen employed in the enantioselective resolution anddesymmetrization of racemic 6,6V-disubstituted BINOLderivatives using vinyl acetate.93 The monoacetate (R)-85 (product) was obtained in 32–44% chemical yields and78–96% ee, depending on the derivatives used. Theunreacted BINOL (S)-84 was obtained in 30 – 52%chemical yield and 55–80% ee (Fig. 22).

Biphenyls are recognized as stable analogs of BINOL.They are found in numerous natural products. Sanfilippo

Fig. 23. Lipase-catalyzed kinetic resolution of 2,2V-dihydroxy-6,6V-dime-thoxy-1,1V-biphenyl 86.94

Fig. 22. Lipase-catalyzed stereoselective resolution and desymmetri-zation of binaphthols 84.93

Fig. 21. Selected examples of lipase-catalyzed kinetic resolution ofchiral carboxylic acids.

Fig. 20. Lipase-catalyzed enantioselective transesterification of 2-phe-nylbut-3-yn-2-ol 74.82


et al.94 reported the Pseudomonas cepacia lipase-catalyzedkinetic resolution of 2,2V-dihydroxy-6,6V-dimethoxy-1,1V-biphenyl 86 using vinyl acetate as acyl donor in tert-butylmethyl ether as organic solvent. (R)-87 is obtained with anee up to 98%, while (S)-86 is recovered with an ee up to96% (Fig. 23).

Diols of different structures such as the meso-diol 88(Fig. 24), the C2-symmetric diol rac-91 (Fig. 25), the diolrac-94 in which the primary hydroxy group is protected(Fig. 26), and the unprotected diol rac-96 with a primaryand secondary hydroxy group (Fig. 27) were used assubstrates in the lipase-catalyzed transesterification usingvinyl acetate as acyl donor in organic solvents with theaim of preparing chiral buildings blocks of high enantio-meric purity.95

Apart from vinyl acetate, vinyl benzoate was used asacylating agent in the Mucor miehei lipase (MML) andCandida antarctica lipase (CAL)-catalyzed benzoylation of1,2-diols 99 in organic solvents.96 The reaction proceededwith high regioselectivity and moderate enantioselectivity(Fig. 28). An efficient synthesis of (R)- and (S)-1-amino-2,2-difluorocyclopropanecarboxylic acid (DFACC) 103 vialipase-catalyzed desymmetrization of prochiral diols 101and prochiral diacetates 104 was recently reported(Fig. 29).28 Thus, the lipase-catalyzed transesterificationof 101 using vinyl acetate as acyl donor in benzene:di-i-propyl ether (20:1) as organic solvent afforded (R)-102with 91.3% ee and 96.5% chemical yield. The reverse enan-tioselective hydrolysis of 104 in a mixed solvent of ace-tone and phosphate buffer afforded (S)-102 with 91.7% eeand 86.2% chemical yield.

The first enzymatic desymmetrizations of prochiralphosphine oxides was recently reported by Kielbasinskiet al.97 Thus, the prochiral bis(methoxycarbonylmethyl)phenylphosphine oxide 105 was subjected to the PLE-mediated hydrolysis in buffer affording the chiral mono-acetate (R)-106 in 72% ee and 92% chemical yield. In turn,the prochiral bis(hydroxymethyl)phenylphosphine oxide107 was desymmetrized using either lipase-catalyzed acet-ylation of 107 with vinyl acetate as acyl donor in organicsolvent or hydrolysis of 109 in phosphate buffer and sol-vent affording the chiral monoacetate 108 with up to 79%ee and 76% chemical yield (Fig. 30).

Neri and Williams98 reported the desymmetrization ofN-Boc-serinol 110 by the selective mono-acetylation using

Fig. 29. Lipase-catalyzed desymmetrization of prochiral diols 101 anddiacetates 104.28

Fig. 28. Lipase-catalyzed benzoylation of propane-1,2-diol 99.96

Fig. 27. Lipase-catalyzed kinetic resolution of (R,S)-3-(4-methoxyphe-noxy)propane-1,2-diol (rac-96).95

Fig. 26. Lipase-catalyzed kinetic resolution of trans-2-(tert-butyldimeth-ylsiloxymethyl) cyclopentanol (rac-94).95

Fig. 25. Lipase-catalyzed kinetic resolution of endo-endo-cis-bicy-lo[3.3.0]octane-2,6-diol (rac-91).95

Fig. 24. Lipase-catalyzed desymmetrization of cis-2-cyclopentene-1,4-diol 88.95


PPL (porcine pancreas lipase) and vinyl acetate as theacylating agent in organic solvent. The mono-acetylatedproduct (R)-111 was obtained after 2 h with 99% ee andisolated in 69% chemical yield (Fig. 31). Traces of the di-acetylated product 112 were observed. The cyclization of(R)-111 in basic medium afforded the racemic oxazoli-dinone 113. The latter was subjected to enzymatic hydrol-ysis in phosphate buffer, affording (R)-114 in up to 93% eeand isolated in chemical yield up to 42%. To avoid basicconditions, (S)-113 was also obtained in one step by cycli-zation of (R)-111 with thionyl chloride. The reactionproceeded with >98% ee and 72% yield. The enzymatichydrolysis of (S)-113 afforded (R)-114 in >98% ee and77% yield (Fig. 32).

The kinetic resolutions of a series of racemic trans-cycloalkane-1,2-diol monoacetates rac-115a–d were re-ported using the enantioselective transesterificationmode with vinyl acetate as acyl donor and commercialas well as self-prepared fungal lipases affording the di-acetates (R,R)-116a–d and monoacetates (S,S)-115a–din high enantiomeric purity (up to 99% ee) (Fig. 33). Themonoacetates (R,R)-115a–d were also prepared startingfrom the racemic diacetates rac-116a-d by lipase-cata-lyzed hydrolysis.99

Miscellaneous Cases

Allenes, another class of compounds having interestingproperties, have also been resolved by lipases. The kinetic

resolution of a variety of racemic 1-ethenyl and ethynyl-substituted 2,3-allenols was reported using a lipase fromCandida antarctica type B (CAL-B) with vinyl acetate asacyl donors in organic solvent. The biocatalytic resolutionafforded (S)-2,3-allenols (S)-117 and (R)-2,3-allenyl ace-tates (R)-118 in chemical yields up to 55% and an ee up to99% for both enantiomers, depending on the substituents(Fig. 34).100 Faure et al.101 reported the first enzymaticresolution of phosphane-borane complex. Thus, theborane adduct of (2-hydroxypropyl)diphenylphosphane119 was resolved using the lipase CAL-B and vinylacetate as acyl donor in organic solvent. The remainingunreacted substrate (S)-119 was recovered with 91% ee(Fig. 35). The use of Candida antarctica lipase B in thekinetic resolution of a series of bicyclic 1-heteroarylprimary amines 121 using ethyl acetate as acyl donor inisopropyl ether as organic solvent was studied bySkupinska et al.102 High yields and ee of either enantiomercould be obtained. The undesired enantiomer could be insome cases recycled by thermal racemization (Fig. 36).

Irurre et al.103 reported the enzymatic resolution oftrans-10-azido-9-acetoxy-9,10-dihydrophenanthrene 123 ingram-scale using Candida cyclindracea lipase-catalyzed en-antioselective hydrolysis in phosphate buffer. The sub-strate 123 (the ester) was obtained in 89% yield and83% ee, while the product 124 (the alcohol) was ob-tained in 90% yield and 98% ee (Fig. 37). A practical meth-od for the synthesis of chiral pyridazinone bearing apyrazolopyridine ring via lipase-catalyzed resolution of2-(acyloxymethyl)-4,5-dihydro-5-methylpyridazin-3(2H)-

Fig. 33. Enzymatic acylation of trans-2-acetoxycycloalkan-1-ols 115.99

Fig. 34. Lipase-catalyzed kinetic resolution of a variety of racemic 1-ethenyl and ethynyl-substituted 2,3-allenols 117.100

Fig. 35. Lipase-catalyzed kinetic resolution of a phosphane-boranecomplex 119.101

Fig. 32. Synthesis of (S)-4-acetoxymethyl-2-oxazolidinone 113 and itsenzymatic hydrolysis.98

Fig. 31. Desymmetrization of N-Boc-serinol 110 by PPL.98

Fig. 30. Lipase-catalyzed desymmetrizations of prochiral phosphineoxides.97


one derivatives 125 was reported by Yoshida et al.(Fig. 38).104

Forro et al.105 reported a very simple method for thesynthesis of enantiopure b-amino acids 126a–129a (e.g.,cispentacin) and b-lactams 126b–129b via lipase-cata-lyzed enantioselective ring opening of unactivated alicyclicb-lactams 126–129 in organic media. High enantiose-lectivity (E >200) was observed when using CAL-Bcatalyzed reaction with H2O (1 equiv) in diisopropyl etherat 60jC. The products (126a–129a) and the substrates126b–129b were obtained in up to 99% ee with chemicalyields ranging from 36–47% (Fig. 39). Other approachesfor the resolution of compounds containing larger alicyclicrings were recently reported.106

APPLICATION OF LIPASES IN INDUSTRY

As the use of lipases for industrial chemical synthesisbecomes easier, several chemical companies have begunto significantly increase their biocatalytic process used insynthetic application. Among these companies is BASF, inwhich enantiomerically pure alcohols and amines areproduced on an industrial scale (Fig. 40).107 The enantio-selective hydrolysis of the racemic acetamide (R,S)-133was developed at Bayer in the middle 1990s. The reactionwas performed using Candida antarctica lipase B (CAL-B)to afford the free amine (R)-134 in high ee (>99.5%)(Fig. 41).108 However, the requirement of a high concen-tration of the catalyst limits the exploitation of the processon an industrial scale.109

Apart from amines and secondary alcohols, Ladner andWhitesides110 developed a procedure to resolve racemicglycidylbutyrate (135) with porcine pancreatic lipase(PPL) to afford (R)-135 in 89% of the theoretical yield

and 92% ee. This process was further developed and usedby Andeno-DSM to produce the epoxy alcohol (R)-glycidol[(R)-136] and (R)-135 on a multitone scale (Fig. 42).22 Inthe pharmaceutical industry, salt-activated biocatalystshave been used to synthesize a library of paclitaxel (taxol)derivatives. CAL was used in the hydrolysis of the terminalvinyl ester in taxol 2V-vinyladipate 137. The resulting taxol2V-adipic acid derivative 138 was nearly 1,700 times moresoluble in water than the native taxol, a result in the designof taxol prodrugs with increased bioavailability (Fig. 43).107

A broader overview of the industrial methods used forthe production of optically active intermediates was recent-ly reviewed by Breuer et al.109

CONCLUSIONS AND PERSPECTIVES

The lipase-catalyzed access to enantiomerically purecompounds remains a versatile method for the separationof enantiomers. The selected examples shown in this

Fig. 37. Kinetic resolution of azido acetate 123.103

Fig. 38. Lipase-catalyzed resolution of 2-(acyloxymethyl)-4,5-dihydro-5-methylpyridazin-3(2H)-one derivatives 125.104

Fig. 39. Lipase-catalyzed enantioselective ring opening of unactivatedalicyclic b-lactams.105

Fig. 40. Some of the biocatalytic steps using lipase developed at BASF:Lipase-catalyzed kinetic resolution of (a) phenyl ethanol 18 using succinicanhydride, (b) secondary amine 131 using ethyl methoxyacetate as acyldonor.107

Fig. 36. Lipase-catalyzed enantioselective acetylation of bicyclic 1-heteroaryl primary amines (rac-121).102


survey demonstrate the broad applicability of lipases interms of substrate structures and enantioselectivity. Morerecently, modern molecular biology methods such asrational protein design and especially directed evolution111

will further boost the development of tailor-made lipasesfor future applications in the synthesis of optically purecompounds. It has been shown that a virtually non-enantioselective lipase (E = 1.1 in the resolution of 2-methyldecanoate) could be evolved to become an effectivebiocatalyst (E >50). Furthermore, variants were identifiedwhich showed the opposite enantiopreference.

Abbreviations

Ac acetylANL Aspergillus niger LipaseBINOL 1,1V-binaphthyl-2-2V-diolBoc tert-butyloxycarbonylCAL-A Candida antarctica lipase ACAL-B Candida antarctica lipase B (Novozyme 435)Cbz benzyloxycarbonylCCL Candida cylindracea lipaseCD cyclodextrinConv. conversionCRL Candida rugosa lipaseDFACC 1-amino-2,2-difluorocycloropanecarboxylic acidDKR dynamic kinetic resolutionDMF dimethylformamideDMSO dimethylsulfoxide

E enantiomeric ratioee enantiomeric excesseep enantiomeric excess of productees enantiomeric excess of substrateEq. equationES enzyme-substrate complexEtOH ethanolGC gas chromatographyGPCL PCL from Genzyme diagnosticsHIV humane immune deficiency virusHPLC high-performance liquid chromatographyi-Pr2O diisopropyl etherkcat rate constantKM Michaelis-Menten constantMML Mucor miehei lipaseMTMS methyltrimethoxysilanen.d. not determinedNMR nuclear magnetic resonanceNovozyme 525 L liquid version of novozyme 435Novozyme IM immobilized CAL-BNSAID nonsteroidal antiinflammatory drugsP productPFL Pseudomonas fluorescens lipasePLE pig liver esterasePPL Porcine pancreas lipasePCL=PSL Pseudomonas cepacia lipasePCL-C=PS-C Pseudomonas cepacia immobilized on ceramicsPCL-D Pseudomonas cepacia immobilized on

diatomaceous earthrac. racemicrec. recombinantS substratet timeTBDMS tert-butyl dimethyl silylTBME tert-butyl methyl etherTHF tetrahydrofuranTMOS tetramethoxysilane

ACKNOWLEDGMENTS

A.G. thanks Prof. Volker Schurig (Institute of OrganicChemistry, University of Tubingen) for help and sup-port and Prof. Uwe Bornscheuer (Department of Techni-cal Chemistry & Biotechnology, University of Greifswald)for advice and for revising as well as writing parts ofthe manuscript.

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Application of lipases in kinetic resolution of racemates

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