Enzyme Technologies (Metagenomics, Evolution, Biocatalysis, and Biosynthesis) || Enzyme Catalysis in the Synthesis of Active Pharmaceutical Ingredients

PART B

BIOCATALYTIC APPLICATIONS

125

5ENZYME CATALYSIS IN THESYNTHESIS OF ACTIVEPHARMACEUTICAL INGREDIENTS

Animesh GoswamiProcess Research and Development, Bristol-Myers Squibb, New Brunswick, New Jersey

I. INTRODUCTION

The active pharmaceutical ingredient (API) is the component responsible for thepharmaceutical activity of a drug. An API can be a small molecule (molecularweight <2000 Da) or a large molecule. Small molecules have been used to treatvarious human diseases for centuries. The modern drug industry was establishedaround the late nineteenth century based on small molecules as active pharma-ceutical ingredients [1]. Although a few large API molecules (e.g., insulin) havebeen known for quite some time, the large-molecule API industry started to growin the 1980s and has seen exponential growth over the last three decades. Theuse of enzymes as catalysts in the synthesis of small-molecule APIs is the subjectof this chapter.

The synthesis of APIs typically involves a multistep synthesis consisting ofmany different types of reactions, starting from a commercially available startingmaterial. Enzyme catalysis has been used to perform one or more of these steps.The fact that the API synthesis is a multistep process and enzyme catalysis ofteninvolves only one of these steps is an important factor to remember during thedesign and execution of the process. Process steps that utilize enzyme catalysis

Enzyme Technologies: Metagenomics, Evolution, Biocatalysis, and Biosynthesis,Edited by Wu-Kuang Yeh, Hsiu-Chiung Yang, and James R. McCarthyCopyright © 2010 John Wiley & Sons, Inc.

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128 ENZYME CATALYSIS IN THE SYNTHESIS OF API

often require somewhat different strategic and tactical considerations for scale-upversus traditional organic chemistry synthetic transformations. There are manyexcellent books and reviews on the application of enzymes in organic synthesis[2,3]. The purpose of this chapter is not to provide a thorough chronologicalreview of the applications of enzymes for the synthesis of API, but to provideexamples of applications, issues, solutions, and future possibilities. The synthesisof all pharmaceutical intermediates in the synthetic sequence as well as the laststep leading to the API is covered in this chapter. Although many commerciallyavailable raw materials (e.g., amino acids) have been synthesized using enzymecatalysis, they are not discussed in this chapter. We focus on the applicationof enzymes for the synthesis of APIs and pharmaceutical intermediates startingfrom commercially available raw materials.

Enzymes are catalysts for biological process. All living cells perform reactionscatalyzed by enzymes. Hence, the terms biocatalyst and biocatalysis are oftenused for enzyme and enzyme-catalyzed processes, respectively. The source of anenzyme could be any living cell (e.g., microorganism, plant, or animal). Microor-ganisms are relatively easy to cultivate and serve as the predominant source ofenzymes for organic synthesis applications. Some plant-derived enzymes (e.g.,papain from papaya) are available and are used for organic synthesis. Animal-derived enzymes (e.g., porcine liver esterase) are also available and are used fororganic synthesis. Enzymes of animal origin can cause regulatory issues for phar-maceutical applications and are discussed later. The enzyme can be isolated ornonisolated. Some isolated enzymes are available from commercial sources. Themost notable isolated enzymes are the various hydrolytic enzymes (e.g., lipase,esterase, and protease). Some enzymes are either not available in isolated formor cannot be isolated in stable form to carry out organic reactions. The reactionswith these nonisolated enzymes are done either with intact cells or with cell-freeextract containing the enzyme.

II. REQUIREMENTS IN DIFFERENT PHASES OF PHARMACEUTICALDEVELOPMENT

The development of a drug candidate from discovery to final approval and com-mercialization spans several years and involves different phases of development.The first step is the discovery of an API with a desired in vitro activity. Usually, afew milligrams of an API are required for this initial identification process. Oncea drug candidate has been identified, the initial evaluation with multiple grams ofAPI is performed in animals to establish the safety and to evaluate pharmacoki-netic properties. The first studies in humans, the phase I clinical trial, providesan initial evaluation of the safety of the drug candidate in humans. After phaseI, studies to evaluate the efficacy of the drug (proof of concept, phase IIa/IIb) inhumans are performed. These early phases of drug development typically requirevery rapid development of a process to supply up to tens of kilograms of an API.During the drug discovery phase and this initial development phase, speed isextremely important. Usually, parallel approaches of several synthetic schemes,

REQUIREMENTS IN DIFFERENT PHASES OF PHARMACEUTICAL DEVELOPMENT 129

each involving multistep processes, are pursued. The synthetic sequence withthe potential of safely providing the earliest delivery of an adequate quantity ofhigh-quality API is typically selected as the process to make the API to supportphase I and II studies.

In these early phases of development, enzyme-catalyzed transformations arefrequently considered in the analysis of the most efficient means to synthesize thetarget API. The availability of enzymes as a platform technology for immediatescreening is essential to their successful application in these early API synthesiscampaigns. In considering any step of the multistep synthesis of an API it isextremely important to consider the impact of each step on the quality of theAPI. The quality of API is extremely crucial, as based on ICH guidance [4],any new impurity usually above 0.15 area percent by high-performance liquidchromatography (HPLC) must be qualified by toxicity assessment. For somehighly toxic impurities or mutagenic (or potentially mutagenic) impurities, themaximum allowable limit can be significantly lower (ppm levels). Each step inthe synthesis scheme can make a contribution to the impurity profile of the finalAPI, often in an interdependent fashion. During the selection of an API syntheticsequence for scale-up, each step is carried out in the laboratory and the productobtained in one step is subjected to the next step, and so on, ultimately generatingthe API. The multistep synthesis carried out in the lab explores the generationof low levels of impurities in different steps, the conversion of one impurity toanother in the next step, and the purging ability of successive synthetic steps.Once the synthetic route is set, changing one step may result in the presence ofhigher than the allowable limit of an impurity in the API. Although the earlydevelopment phases are carried out fairly rapidly, the strategic decision taken onselecting the synthetic route at this stage may be profound and may make laterchange challenging. Thus, for enzyme catalysis to play a competitive role in thesynthesis of an API it is important to move rapidly. Once the synthetic route isselected, it is important that a large quantity of the enzyme of quality similar tothat used for screening should be available immediately to make several kilogramsof products. In practical terms, the enzyme used for screening should be availablein kilogram quantities within several weeks of route selection for scale-up.

After successful achievement of proof of concept, a drug candidate is evalu-ated in large-scale clinical trials, phase III, for detailed evaluation of safety andefficacy. During these studies it is typically necessary to produce hundreds of kilo-grams to tons of an API for clinical trials and supporting studies. The commercialprocess for manufacture of the API is established in this phase of development.All synthetic steps, including enzyme-catalyzed steps, are finalized at this stage.The critical and key process parameters should be established and their effects onthe yield and quality should be clearly demonstrated. It is extremely important toestablish that the enzyme catalysis step can use the starting material obtained ona large scale with a preestablished quality and can provide the product of enzymereaction with a preestablished quality. The quality and certainty of supply of theenzyme from commercial sources should be established. These criteria are thesame for other steps, chemical reagents, or catalysts. It is important to remember


that for API synthesis applications, enzymes are treated essentially the same asare other chemical catalysts; they just happen to have larger molecular massesand are derived from biological sources.

One factor of potential concern for the use of enzymes in the synthesis of APIsis the issues associated with animal origin. For materials originating from animalsources, there is the risk of prion contamination, resulting in transmissible spongi-form encephalopathy (TSE) and bovine spongiform encephalopathy (BSE). Forpharmaceutical applications, enzyme manufacturers are required to provide certi-fication as to whether any material of animal origin is used for making the enzymeand provide a TSE/BSE-free certification. As part of a TSE/BSE evaluation, itis necessary to consider both direct and indirect animal sources. A direct sourceis involved when an enzyme has been obtained from an animal directly (e.g.,porcine liver esterase, porcine pancreatic lipase). An animal source can also beindirect, for example, an enzyme is from a microbial source but an animal product(e.g., beef extract) was used as a component of the fermentation medium. Inter-estingly, sometimes there are remote connections to animal sources. For example,isopropyl thiogalactoside (IPTG) is often used to induce synthesis of enzymes inrecombinant organisms. IPTG is made chemically from galactose, which in turnis obtained from the hydrolysis of lactose. Lactose is generally obtained frommilk, an animal source. Although this connection of an animal source to IPTG isremote, enzyme suppliers should consider switching to IPTG made from galac-tose originating from plant-derived lactose. Applications of novel technology canprovide unique solutions for the animal source and TSE/BSE issue. For example,porcine liver esterase (PLE) is an extremely efficient enzyme for the hydrolysis ofsterically hindered esters to acids and alcohols. The enzyme, as the name implies,is from pig liver and has been known for more than a century. Although theenzyme is very efficient, its application in API production is often limited, due toits animal origin. The enzyme is a mixture of several isozymes that have recentlybeen cloned and expressed in microorganisms [5]. The cloned enzyme is madeby microbial fermentation, hence completely eliminating the risk associated withthe animal origin and TSE/BSE issues of the original pig liver–derived enzyme.

III. SELECTIVITY AND TYPES OF ENZYME-CATALYZEDREACTIONS

The selectivity of enzymatic transformations is their most attractive feature forapplication to organic synthesis. Selectivity can be group specific, regiospecific,or stereospecific. Group specificity is reaction on a specific group without affect-ing a chemically similar group (e.g., hydrolysis of an ethyl ester without affectinga butyl ester). Regiospecificity is reaction at a specific position among multiplepossible sites. Stereospecificity is production of only one stereoisomer amongvarious possible stereoisomers. API molecules typically are a single enantiomer,as in most cases only a single enantiomer possesses the desired pharmacologi-cal activity. The specificity of enzymatic transformations is the most importantattribute for the application of enzymes to the synthesis of APIs.

SELECTIVITY AND TYPES OF ENZYME-CATALYZED REACTIONS 131

Enzymes are known to catalyze a wide array of reactions in biological systems.For application to the synthesis of APIs, the following types have been the mostutilized to date [6]: hydrolysis, esterification, transesterification, and amidationof ester, amide, alcohol, acid, and amine-resolution of enantiomer; reduction ofketone to alcohol; keto acid to amino acid conversion; hydroxylation reaction;and C—C bond-forming reaction (aldolase, pyruvate decarboxylase). In addition,there are a variety of other enzyme-catalyzed reactions that have been recentlyutilized in API synthesis, including epoxide hydrolysis, nitrile hydrolysis, andcyanohydrin formation.

A. Hydrolysis–Esterification–Transesterification–Amidation: Resolution

Alcohols, acids, esters, or amides can all be utilized as substrates for enzyme-catalyzed transformations. For example, esters can be hydrolyzed, transesterifiedby reaction with a different alcohol, or converted to an amide by reaction with anamine. The enzymatic reaction of a racemic substrate often proceeds faster withone enantiomer, leading to a resolution process known as kinetic resolution . Theconversion and enantiomeric excess in such a process depends on the enantiomerratio [7], representing how much one enantiomer is preferred over the other.Enzyme-catalyzed hydrolysis, esterification, acylation, and amidation reactionsare often used for kinetic resolution. Since there is only 50% of each enantiomerin a racemic mixture, the maximum theoretical yield of the product in the kineticresolution process is 50%, and at the end of the process as much as 50% of theundesired enantiomer of the remaining substrate is also present in the reactionmixture. In a dynamic resolution process, the undesired enantiomer of the startingmaterial is simultaneously racemized during the enzymatic transformation andcan provide the desired product enantiomer in potentially 100% yield.

R1 OHEsterification

R1 O

O

R2Hydrolysis

R1 NHAcylation

R1 N

O

R2

HydrolysisX X

O

R2HO

O

R2HO

+

+

Amidation

R1 NH

X

R3 O

O

R2R1 OH +

Trans-Esterification

1 2 3 1 4

5

65 2

SCHEME 1

A similar enzymatic hydrolysis–esterification–amidation of a meso startingmaterial leads to the formation of one product enantiomer in 100% maximumtheoretical yield by desymmetrization.


R1

R1

O

O

O

O

R2

R2

Meso

2

R1

R1

OH

O

O

R

One Enantiomer

R1

R1

OH

OH

Meso

EsterificationHydrolysis

7 8 9

SCHEME 2

Hydrolytic enzymes are widely distributed in various microorganisms, ani-mals, and plants. Many isolated hydrolytic enzymes are available from variouscommercial sources. The common hydrolytic enzymes are lipases, esterases, andproteases. Lipases operate at the interface for the hydrolysis of water-insolubleesters [e.g., fat (glycerides of fatty acids)]. Esterases hydrolyze water-solubleesters. Proteases hydrolyze proteins. Lipases, esterases, and proteases are usedextensively in the food and detergent industries.

B. Reduction of Ketone to Alcohol

The enzyme-catalyzed reduction of ketones typically provides selectively eitherthe R- or the S-alcohol. The enzymes, commonly referred to as alcohol dehydro-genase (ADH) or ketoreductase (KRED), are widely distributed in yeast, bacteria,and fungi.

R1 R2

O

R1 R2

OHAlcohol Dehydrogenase

Ketoreductase

NAD(P)H NAD(P)

N

X

H HCONH2

N+

X

CONH2

H10 11

12 13

SCHEME 3

The reduction requires the participation of the cofactor nicotinaide adeninedinucleotide (NAD) or its phosphate (NADP). The reduced form of the cofactor


NAD(P)H delivers the necessary hydrogen to the ketone, converting it to analcohol. The cofactor, in turn, transforms to the oxidized form NAD(P), whichneeds to be regenerated to the reduced form for the reaction cycle to continue.Cofactors are expensive and regeneration of cofactor is necessary to developa cost-efficient process. The cofactors are regenerated within a growing cellby various cofactor-regenerating enzymes in the cell. With isolated ADH orKRED, the cofactors are regenerated by either a substrate- or an enzyme-coupledsystem.

C. Conversion of Keto Acid to Amino acid

Many applications of enzymes in organic synthesis involve the conversion of aketo acid to an amino acid. The reaction is carried out by two distinct classes ofenzymes: amino acid dehydrogenase and transaminase or aminotransferase.

R2

O

OH

OR3

NH2

OH

O

+

Aminotransferase/Transaminase

R2

NH2

OH

O

R3

O

OH

O

+

R1

O

OH

O

Amino acid dehydrogenase

R1

NH2

OH

O14 15

16 17 18 19

SCHEME 4

An amino acid dehydrogenase carries out the oxidation of amino acids to ketoacids or the synthetically more useful reverse reaction, converting the keto acidsto amino acids. The substrate keto acid must be an α-keto acid except for theenzyme lysine ε-dehydrogenase. An aminotransferase/transaminase carries outthe transfer of an amino group between two keto acids and thereby converts amixture of an amino acid and a keto acid to a different amino acid and ketoacid. Substrates for aminotransferases are typically α-amino acids, except forω-aminotransferase, which accepts other keto acids as substrates. The amino-transferase reaction is reversible and requires a large amount of one substrate orremoval of one product to drive the reaction to completion.

An amino acid oxidase can also oxidize an amino acid to a keto acid. Kineticresolution can be carried out with a stereospecific amino acid oxidase. Indeed, itis possible to perform dynamic resolution of a racemic amino acid by combiningamino acid oxidase with a chemical reduction or with an amino acid dehydroge-nase or a transaminase. Similarly, an amine oxidase could be used for the kineticor dynamic resolution of a racemic amine.


R1 R2

NH2

20, R1 = Alkyl, Aryl, R2 = COOH, Amino acid 21, R1 = Alkyl, Aryl, R2 = Alkyl, Aryl, Amine

Unreacted enantiomer

R1 R2

NH2

Amino acid oxidase/amine oxidase

R1 R2

X

X = NH, AmineX = O, Amino acid

Chemical reduction ofenzyme-bound imine

Amino acid dehydrogenaseor transaminase

R1 R2

NH2

Enantiomersubstrate ofthe enzyme

22

23 24

SCHEME 5

D. Hydroxylation Reaction

One of the early successful applications of enzymes for synthesis in the phar-maceutical industry is the microbial hydroxylation to make corticosteroids [8].Hydroxylations of both aliphatic and aromatic substrates are known.

R1 R3

H

R2R1 R3

OH

R2

25 26

R1 = R2 = R3 = Alkyl, Aryl, H

Ar-H Ar-OH

27 28

Ar = Aryl

SCHEME 6


The cytochrome P450 enzyme systems are mostly responsible for this reaction.Metabolic transformation of most drugs in humans involves similar hydroxylationby human cytochrome P450 systems, and these transformations are important toan understanding of drug metabolism. The cytochrome P450 systems are oftenmulticomponent and bound to the cell membranes. These enzymes are difficultto isolate and unstable in the pure isolated form. Thus, enzymatic hydroxyla-tion reactions are generally carried out with whole cell systems. Recently, somerecombinant P450 enzymes became commercially available. However, they areavailable only in small quantities and are quite expensive; their use is mostlyrestricted to studies on understanding drug metabolism.

E. C—C Bond-Forming Reaction

The most important enzymatic C—C bond-forming reactions are those involvingaldolases and pyruvate decarboxylases. The aldolase enzyme catalyzes an aldolcondensation reaction.

H

O

R2+R1 H

O

R1 H

OH

R2

O

29 30 31

Aldolase

SCHEME 7

The pyruvate decarboxylase enzyme catalyzes an aldol condensation betweenan aldehyde and the product of decarboxylation of pyruvic acid.

H

O

R2

R1 H

O

R1 H

OH

R2

O

R1

O

OH

O

32 29 31

30−CO2

SCHEME 8

C—C bond-forming reactions using pyruvate decarboxylase, benzoyl formatedecarboxylase, and phenylpyruvate decarboxylase have been reported in the lit-erature.

The hydroxynitrile lyase enzymes catalyze the C—C bond-forming reactionbetween an aldehyde and HCN to provide a chiral hydroxynitrile which can beconverted to a chiral hydroxyacid.


R H

O HCN

R CN

OH

H

33 34

R COOH

OH

H

35HydroxynitrileLyase

SCHEME 9

Both R- and S-selective enzymes are reported in the literature. Hydroxynitrilelyase enzyme from bitter almond has been known for a long time. The safetyissues associated with handling HCN are a significant obstacle for large-scaleapplication of the hydroxynitrile lyase enzyme.

F. Other Reactions

Enzymes catalyzing other reactions have been reported in the literature with lessfrequent application to the synthesis of APIs. Epoxide hydrolase enzymes can beused for the resolution of racemic epoxide.

O

R

O

R R

OH

OH+

36 37 38

EpoxideHydrolase

SCHEME 10

Hydrolysis of nitriles to amides and acids is catalyzed by nitrile hydratasesand nitrilases, respectively. Amidases hydrolyze amides to acids.

R

R

N

OH

O

R NH2

O

R OH

O

Nitrilase

NitrileHydratase

Amidase

39

40

41 40

SCHEME 11

ISSUES WITH ENZYME-CATALYZED REACTIONS 137

Halohydrin dehalogenase enzymes catalyze interconversion of halohydrins andepoxides.

ClR2

OH

R1

O

R1

R2

42 43

nHalohydriDehalogenase

SCHEME 12

IV. ISSUES WITH ENZYME-CATALYZED REACTIONS

Besides selectivity, environmental friendliness is the most important beneficialattribute of the application of enzymes in organic synthesis. Enzyme-catalyzedreactions are typically carried out in water under ambient conditions. Traditionalorganic reactions often require the use of organic solvents possessing more envi-ronmental liability than that of water. Organic reactions often require higher orlower temperatures and therefore utilize more energy. Thus, enzyme-catalyzedreactions are often classified as greener or more environmentally friendly thantraditional organic reactions. Although this advantage could be true in manycases, it is important to analyze the situation on a case-by-case basis. It is nec-essary to account for the entire process instead of the reaction part alone. Formany processes, the actual reaction portion may be relatively small comparedto the entire process: comprising the reaction, workup, isolation, and purifica-tion. Although enzymes are prepared and often function best in aqueous systems,some of the major applications of enzymes in organic synthesis require non-aqueous systems. Enzyme-catalyzed esterification is one of the most importantsynthetic applications of enzymes for organic synthesis, which requires the useof organic solvents. For the vast majority of enzymatic reactions, water is the pri-mary solvent. There is, however, a dilemma. Although enzymes are soluble andhave the highest activity in water, most organic compounds, especially those com-monly encountered for the synthesis of APIs, are not soluble in water. To deliverthe organic starting material to the reactor, and to have a reasonable amount ofstarting material in bulk liquid medium, the starting material is usually dissolvedin an organic solvent. The choice and amount of organic solvents depends onthe combination of solubility of the organic compound in the particular solvent,the stability of the enzyme, and the activity of the enzyme. Usually, the reactionis carried out with the highest effective ratio of solvent in water to optimize thesolubility, stability, and activity. Many reactions are carried out with a slurry ofsolid (reactant, product, enzyme) in an organic–aqueous liquid medium, and notnecessarily in water alone as the liquid medium. After the desired reaction, theproduct typically has to be extracted into an organic solvent from water. Thus,


many enzyme-catalyzed organic synthesis processes use significant quantities oforganic solvents. For isolation of the product, the solvent typically is removedby distillation requiring energy. It is necessary to consider the entire process indefining the environmental impact of the enzyme-catalyzed step.

Enzyme-catalyzed reactions often require high dilution conditions. Such reac-tions have very low volumetric productivity and require a large volume and hencelarge reactors to make a relatively small quantity of product. In addition, a highvolume of aqueous reaction mixture may require a high volume of organic sol-vents for extraction, which diminishes the process greenness. High volumetricproductivity is very important, and in many cases is essential for maintaining theappropriate throughput and process economics.

The availability of suitable biocatalysts is an important issue. A large num-ber of hydrolytic enzymes (e.g., lipases, esterases, proteases) were originallydeveloped and commercialized for the food and detergent industries and havebeen available from commercial sources for quite some time. Only a very smallfraction of the total volumes of these enzymes are used for applications in thesynthesis of APIs. As a consequence, very few hydrolytic enzymes have beendeveloped solely for application in the pharmaceutical industry by large enzymeproducers. On the contrary, as the need in the food and detergent industrieschanges, some existing enzymes may be replaced by new enzymes and/or newerversions of the enzyme, which can have important implications for applicationsin synthesis by the pharmaceutical industry. For example, recently, a commercialenzyme producer changed the process for production of one of their well-knownlipase enzymes for the food industry. Although the change in the process pro-vided an enzyme with the same activity as that defined by the hydrolysis ofolive oil, it was found to have much less activity in a hydrolysis process underdevelopment for a pharmaceutical intermediate [9]. A stable and steady sup-ply of a predefined quality of any reagent and catalyst, including enzymes, isan essential requirement for the pharmaceutical industry. Several other classesof enzymes have been commercialized in recent years: ketoreductases, aminoacid dehydrogenases, aminotransferases, amino acid oxidases, nitrile hydratases,nitrilases, and others. It must be noted that unlike the hydrolytic enzymes, theoff-the-shelf commercial supplies of these new enzymes are still relatively smallscale (<1 kg). During the early stages of process research, laboratory scientistsare scouting various approaches and order commercially available enzymes forscreening activities. Therefore, unavailability of the enzyme for screening oftenhas the ultimate result of not using a specific enzyme, or in the worst case, avoid-ing the enzyme catalysis path as a whole. For the broad application of enzymesin API synthesis, greater availability of larger numbers of various categories ofenzymes is one of the opportunities for future growth. In addition to the lim-ited number of enzymes available on a large scale, the scope of the availableenzymes is also limited. Most enzymes are useful for only a limited numberof substrates. Indeed, there is a dichotomy of our need for having enzymeswith broad specificity to cover a large variety of substrates while delivering ahigh specificity for the desired reaction. There are very few, if any, enzymes

SYNTHESIS OF ACTIVE PHARMACEUTICAL INGREDIENTS 139

with broad substrate specificity, yet high selectivity. Some naturally occurringenzymes have been optimized for high specificity for a specific substrate. Unfortu-nately, the optimized enzyme often lacks generality even with a slightly differentsubstrate.

V. TECHNOLOGICAL IMPROVEMENTS IN RECENT YEARS

Traditionally, the search for new biocatalytic systems involved searching forthe appropriate enzymes in nature. Typically, microorganisms were collectedfrom various sources, grown in the laboratory, and screened for their ability tocatalyze the desired reaction. Although a vast number of microorganisms havebeen cataloged, it is estimated that only a minute fraction of Earth’s microor-ganisms have been evaluated to date. Of these known microorganisms, a smallfraction can be grown in the laboratory, and only a small portion of those cul-turable microorganisms have been screened for their biocatalytic activity. Thecollection of microorganisms from various sources, growing them in the lab-oratory, and screening them for activity is a laborious, time consuming, andresource-intensive activity. Recent developments in molecular biology, analyti-cal methodology, automation, and information technology have made the taskof screening and optimization significantly more efficient. Advances in biochem-istry have enabled the rapid identification of an enzyme responsible for catalysisof the desired reaction. Molecular biologists can quickly clone the gene, expressit in a suitable host, and provide the enzyme within a short period of time.Many different variants of enzymes can be prepared very quickly by applicationof modern molecular biology techniques: for example, site-directed mutagene-sis, saturation mutagenesis, and DNA shuffling. Powerful analytical methodologyand automation technology can be used to rapidly analyze very large numbersof variants. Advances in information technology have enabled the rapid trans-formation of the enormous data set into knowledge of what is the best enzymefor a desired reaction. In essence, what nature could have done in a long time(e.g., millions of years) to evolve an enzyme can be done in the laboratory by“directed evolution” in a short period of time [10–12]. Although the technolo-gies are powerful, so far only limited applications of these recent advances havebeen realized commercially. Broad applications of these technologies have thepotential of significantly increasing the impact of biocatalysis for the synthesisof APIs.

VI. APPLICATIONS OF ENZYME CATALYSIS TO THE SYNTHESISOF ACTIVE PHARMACEUTICAL INGREDIENTS

In this section we provide examples of the application of enzymes to catalyzevarious reactions in the synthesis of APIs.


A. Hydrolysis–Esterification–Transesterification–Amidation: Resolution

Hydrolytic enzymes have been utilized most frequently for the synthesis of APIsin both degradative (hydrolysis) and synthetic (esterification–transesterification–amidation) reactions. The greatest frequency of utilization is for the resolution ofstereoisomers via selective reaction of a single enantiomer. Hydrolytic enzymeshave also been utilized for desymmetrization, selective reaction of only one of theseveral identical groups (e.g., diester to monoester), and selective reaction of onegroup in a multifunctional compound (e.g., selective hydrolysis of an O-acetylwithout hydrolysis of an N -acetyl).

Alcohols Enzyme-catalyzed hydrolysis of racemic secondary esters has fre-quently been utilized to resolve chiral secondary alcohols. Enzymatic hydrol-ysis was employed to prepare 3R-hydroxy-4S-phenyl-2-azetidinone 44, a keychiral intermediate for the anticancer drug Taxol [13,14]. The racemic cis-3-acetoxy-4-phenyl-2-azetidinone 45 was hydrolyzed by lipase PS-30 enzyme fromPseudomonas cepacia to provide the 3S-alcohol 46 and remaining unreacted3R-acetate 47 in high yields and enantiomeric excess (e.e.). The alcohol 46 andacetate 47 were separated by extraction. Hydrolysis of the acetate 47 under basicconditions gave the desired chiral alcohol 44 in high yield (45%, maximum theo-retical yield 50%) and e.e. (99%). The process was demonstrated successfully ona large scale. Such enzymes can also catalyze the reverse reaction of esterificationof an alcohol with an acyl donor. Vinyl or isopropenyl acetate is generally usedfor enzymatic acetylation of alcohol. The reaction by-product, vinyl alcohol, orisopropenyl alcohol isomerizes to acetaldehyde or acetone, respectively, drivingthe reaction toward completion.

NH

O

O

O

Lipase

NH

O

O

ONH

HO

O

+

45 46 47

Solvent extractionto separate the alcoholand acetate

NH

O

O

O

47

NaHCO3

NH

HO

O

44

Racemic cis-acetate

SCHEME 13


The kinetic resolution of an alcohol or an ester always provides a mixture ofan alcohol and an ester. In the best case, the reaction mixture at the end of kineticresolution is a 1 : 1 mixture of the product and unreacted starting material, whichmust be separated. In the previous case, the product alcohol 46 and the remainingstarting acetate 47 were easily separated by solvent extraction. This ease of sep-aration is very important for industrial application. Often, the starting ester andproduct alcohol are very similar in polarity and solubility, which precludes theirseparation by simple methods (e.g., solvent extraction or crystallization). In sucha case, a more tedious separation method (e.g., chromatography) is necessary.Lipase AK20 from Pseudomonas fluorescens catalyzed acetylation of racemic(3–R,S) alcohol 48 + 49 by vinyl acetate in methyl isobutyl ketone provided areaction mixture containing the (3R,5)-hydroxy-49, and (3S,5)-acetoxy-50 com-pounds [15]. The alcohol 49 and acetate 50 were separated by chromatography.The alcohol 49 is an intermediate for the synthesis of an androgen receptorantagonist. When chromatography is required to separate the products, the incre-mental benefit of the biocatalytic process must be considered carefully, as inmany cases it is possible to resolve the enantiomers directly by chromatogra-phy on a chiral column. For the resolution of an alcohol, a clever process wasdeveloped which involves acylation with a cyclic anhydride instead of an acylgroup [16,17]. Acylation with an anhydride affords a half ester acid which can beseparated from the desired alcohol by extraction with base. For example, enzyme-catalyzed acylation of (3-R,S) alcohol 48 + 49 by succinic anhydride in methylt-butyl ether provided a reaction mixture containing the 3R,5-hydroxy-49, and3S,5-hemisuccinate-51 compounds. The alcohol 49 and hemisuccinate 51 wereeasily separated by extraction of the hemisuccinate 51 in dilute NaHCO3. Thehemisuccinate 51 was hydrolyzed to the corresponding 3S-alcohol 48 by use ofa strong base (e.g., NaOH) (15).

NO

HO

H

O

O

CN

CF3

NO

HO

H

O

O

CN

CF3

NO

O

O

CN

CF3

HO

H

NO

O

O

CN

CF3

O

H

Lipase AK

++

48

49

R

O

OAc

O

O

O

or

50, R = CH3

51, R = CH2-CH2-COOH

49

SCHEME 14

Enzyme-catalyzed acylation with succinic anhydride was also usedfor the resolution of a key chiral alcohol S-N -(tert-butoxycarbonyl)-3-hydroxymethylpiperidine 52 [18]. Alcohol 52 is a key intermediate for the


asymmetric synthesis of a tryptase inhibitor candidate. Although the reactiveprimary alcohol 52 is one carbon away from the chiral center, a reasonablyeffective biocatalyst was identified for its resolution. Lipase from Pseudomonascepacia was found to be the best catalyst for the resolution of the racemicalcohol 53 to the S-hemisuccinate 54 and remaining R-alcohol 55. TheS-hemisuccinate 54 was easily separated by extraction with a weak base 5%NaHCO3. Hydrolysis of S-hemisuccinate 54 with a stronger base, 5 N NaOH,gave the S-alcohol 52. The selectivity determined by the enantiomer ratio wasnot high [7], with S-alcohol 52 being produced in 23% yield and about 95%e.e. The yield and e.e. were improved by resubjecting the product alcohol tothe process to afford the desired S-alcohol 52 in 32% overall yield (maximumtheoretical yield 50%) with 98.9% e.e.

N

Boc

OH

53

O

O

O

Lipase

N

Boc

O

54

COOH

O

N

Boc

OH

55

+

Extraction5% NaHCO3

N

Boc

O

54

COOH

O

5N NaOH

N

Boc

OH

52

SCHEME 15

Enzymatic hydrolysis of esters of tertiary alcohols is often difficult. A largenumber of commercial hydrolytic enzymes were found to be ineffective inhydrolyzing the 4-acetoxy group of 10-deacetylbaccatin 56. A microorganismable to hydrolyze 56 to the 4-tertiary alcohol 57 was isolated from soil byenrichment culture. The microorganism was identified as Stenotrophomonasmaltophilia , and the hydrolytic enzyme was isolated [19]. Although not availablecommercially, microorganisms from soil and other natural sources should bescreened to identify enzymes to catalyze the hydrolysis of tertiary alcoholesters.


HO

O

O

HO

H

OHO

O

OO

OH

10

4

H

HO

O

O

HO

H

OHHO OO

OH

10

4

H

56 57

SCHEME 16

Recently, enzymes from hyperthermophiles [20] and Bacillus subtilis and Can-dida antarctica [21,22] were reported to be effective catalysts for the hydrolysisof tertiary alcohol esters. It was reported that the presence of the amino acidsequence glycine–glycine–glycine (alanine)–Xaa in these enzymes is necessaryfor activity toward esters of tertiary alcohols. A recent application from our labswas to utilize enzymatic hydrolysis of tertiary butyl ester 58 to afford a late-stageintermediate 59 for the synthesis of a 3-hydroxy-3-methylglutaryl–coenzyme A(HMG-CoA) reductase inhibitor candidate [23].

N N

N

F

O

HO

O

O

N

N N

CH3

CH3

CH3

CH3

N N

N

F

O

HO

O

OH

N

N N

CH3

CH3

CH3

CH3

Enzyme

58 59

SCHEME 17

Protecting groups are often used in the synthesis of APIs. The enzymes fromB. subtilis and C. antarctica have been reported to catalyze selective hydrolysisof a tertiary butyl ester without removal of a t-butoxycarbonyl (Boc) protectinggroup [24].

Hydrolytic removal of a CBz protecting group by an enzyme from Sph-ingomonas paucimobilis has been reported [25,26]. The initial study was onthe removal of the CBz-group from CBz protected L-amino acid derivatives 63(R = PhCH2, R1 = alkyl, aryl, R2 = COOH, X = O).


OHN

O

O

O

OHN

OH

O

O

HO+

60 61 62

SCHEME 18

RXN

H

O

R1

R2

NH2R1

R2

63 64

SCHEME 19

A CBz-deprotecting enzyme with selectivity for CBz-protected D-amino acidderivative was later identified. The structure–activity relationships of bothenzymes for the hydrolysis of carbamates and amides 63 with varying R(PhCH2, H), R1 (alkyl, aryl), R2 (COOH, H), and X (O, NH, CH2) groups havebeen reported [27].

The kinetic resolution of alcohols affords a maximum of 50% yield of thedesired product enantiomer. Enzyme-catalyzed dynamic resolution of an alcoholwherein the undesired enantiomer is racemized could theoretically provide 100%yield. Enzyme-catalyzed esterification coupled with a transition metal–catalyzedracemization has been employed for the dynamic resolution of alcohols [28–30].

R1 R2

OH

R1 R2

OH

Enzyme

R1 R2

O

R1 R2

OH

R

O

Racemization

+

65

66

65

67

SCHEME 20

Transition metal–catalyzed racemization involves an oxidation–reduction(alcohol–ketone–alcohol) sequence. The substrate for the enzymatic dynamicresolution is practically limited, only secondary allyl or benzyl alcohols haveso far been known to be useful. A handful of enzymes are stable enough tobe practically useful under the high temperature condition required for the


racemization reaction, and thus the dynamic resolution of alcohols has beenused only in a limited number of cases to date. New catalysts were developedrecently to carry out the racemization at ambient temperature [31,32]. A fewexamples of enzyme-catalyzed dynamic resolution of alcohols where both theoxidation and reduction are catalyzed by enzymes have been reported. In thisdynamic resolution, often referred to as deracemization by stereoinversion , oneenantiomer of the alcohol is enzymatically oxidized to the ketone while theother enantiomer of the alcohol remains unchanged. The ketone is reducedto the opposite enantiomer of the alcohol by another enzyme. The net resultis conversion of the racemic alcohol to a single enantiomer of the alcohol inpotentially 100% yield. Deracemization of the racemic diol 68 by stereoinversionwas carried out by Candida and Pichia species providing 60 to 70% yield and90 to 100% e.e. of the chiral diol 69, a key pharmaceutical intermediate [33].

O

OH

OH

O

OH

OH

O

OH

OH

O

OH

O

UnreactedEnantiomer

68

Enantiomer-Substrate ofan oxidaseenzyme

69

70

EnzymaticOxidation

71

EnzymaticReduction

SCHEME 21

Carboxylic Acids The application of enzymes to the resolution of 2-arylpropionic acids has been studied extensively. A number of 2-arylpropionicacids were introduced as nonsteroidal anti-inflammatory drugs (NSAIDs)starting in the late 1960s (e.g., ibuprofen, naproxen, ketoprofen). Although the2-arylpropionic acids all possess a chiral center, only naproxen was originallyapproved and marketed as the single S-enantiomer. The other 2-arylpropionicacids were initially approved and sold as racemic mixtures. The anti-inflammatory activity is due to cyclooxygenase inhibition and it was shownthat only the S-enantiomers of 2-arylpropionic acids are responsible forcyclooxygenase inhibition [34]. There was a flurry of activities in the late1970s and 1980s to develop processes for making pure chiral enantiomers


of 2-arylpropionic acids, which included numerous efforts on the applicationof enzymes for the resolution of enantiomers of 2-arylpropionic acids. Manyefficient applications of enzymatic hydrolysis for the resolution of esters of2-arylpropionic acids were reported [35–37].

ArO

R

O

ArOH

O

72 73

SCHEME 22

Several commercial enzymes marketed for the food and detergent industries(e.g., lipases from Candida rugosa) were found to be very effective catalysts,and processes for many 2-arylpropionic acids using commercial enzymes werereported. The commercial enzymes often are mixtures of various isozymes. Onecommercial lipase from C. rugosa (Lipase MY from Meito-Sangyo) was reportedto contain two lipase isozymes, and one of them was shown to be excellent forthe enzymatic hydrolysis of esters of ketoprofen [38].

Enzyme-catalyzed resolutions of other carboxylic acids have found utility inthe synthesis of several other APIs. A stereospecific Pseudomonas lipase cat-alyzed esterification with methanol in organic solvent was developed for the res-olution of racemic 3-benzoylthio-2-methylpropionic acid 74 to S-3-benzoylthio-2-methylpropionic acid 75, a key intermediate for the antihypertensive drugCaptopril 76 [39]. The lipase from Pseudomonas species was immobilized onpolypropylene and the immobilized enzyme was reused for 23 cycles withoutany loss of activity. Toluene and methanol were found to be the optimal solventand alcohol, respectively, for this esterification at a very high substrate concen-tration (1 M). The desired S-acid 75 was obtained in 40% yield (50% theoreticalmaximum) with >94% e.e.

Ph S OH

O O

PseudomonasLipase

CH3OH

Ph S O

O O

Ph S OH

O O

HS N

O

76

HOOC

74

75

77

SCHEME 23


Another enzyme-catalyzed synthesis of 75 has been reported based on theresolution of the racemic 3-halo-2-methylpropionic acid ester 78 [40]. Enzymatichydrolysis of the racemic ester 78 provided the S-ester 79 and R-acid 80. Furtherchemical hydrolysis of S-ester 79 provided the S-acid 81, which was convertedto the key Captopril intermediate 75.

X O

O

X OH

O

Ph S OH

O O

75

80

X O

O79

REnzyme

R

78

X = Cl, Br

X OH

O

81

SCHEME 24

The isomeric 2-chloropropionic acid is a key intermediate for the synthesis ofmany bioactive compounds. In 1980s and early 1990s, there were several reportson the utilization of biocatalysis for the synthesis of chiral 2-halopropionic acid[41–43].

X

OH

O

X

OH

O

82

83

X

O

O

R

Lipase

Unreacted Enantiomer

84

X = Cl, Br

ROHHexane

SCHEME 25

An interesting combination of enzyme catalysis, novel reactor technology,and product derivatization to prevent enzyme inhibition was developed for theenzymatic resolution of the racemic epoxy ester 85 to the desired (2R,3S)-epoxy ester 86, an important intermediate for the synthesis of the potent calciumchannel blocker Diltiazem 87. Many lipases catalyzed the hydrolysis of only


one enantiomer of the racemic epoxy ester 85 with the (2R,3S)-epoxy ester86 remaining unreacted. The (2S,3R)-epoxy acid 88 formed is unstable anddecarboxylates to afford p-methoxybenzaldehyde 89 which inhibits the activ-ity of the enzyme. In order to minimize the inhibitory activity, the aldehyde89 was removed from the reaction mixture by forming the sodium bisulfiteadduct 90. A multiphase/extractive membrane bioreactor system was used [44].The high-molecular-weight enzyme stayed inside the membrane while the low-molecular-weight reactants and products passed through, thus enabling a con-tinuous enzymatic resolution process [45]. The hydrolysis of the racemic epoxyester 85 by a lipase from Serratia marcescens using a hollow fiber reactor wascommercialized for the production of the 2R,3S-epoxy ester 86 [46,47].

H3CO

H3CO

H3CO H

3CO

H3CO

O COOCH3

COOCH

COOH

3O

O

86

85

Enzyme

88

H

O

H

OH

SO3NaNaHSO3

89 90

N

S

ON

O

OCH3

O

87

−CO2

SCHEME 26

As mentioned earlier, kinetic resolution provides a 50% theoretical maximumyield of one chiral isomer of the product acid. The remaining ester (50%) ofopposite chirality can be recovered, racemized, and recycled in the enzymatichydrolysis step to increase the overall yield. However, such processes wouldbe multistep. A dynamic kinetic resolution where the remaining ester can beracemized concurrently with the enzymatic step is of great value. The desiredS-isomer 91 of the anti-inflammatory drug ketorolac was prepared via dynamicresolution of the conjugated racemic ester 92 by a Streptomyces griseus protease-catalyzed hydrolysis under slightly alkaline conditions [48].

N

O

CO2Et

91

N

O

CO2H

92

Protease

SCHEME 27

Most other nonconjugated esters can only be racemized under strongly basicconditions, where enzymes do not work efficiently. Thioesters can be racemized


under mild conditions, and enzyme-catalyzed dynamic resolution of thioestersunder ambient conditions has been reported [49].

SPh

O

S

Lipase

SPh

O

OH

SPh

O−

S

SPh

O

S

93

95

96

94

SCHEME 28

An ingenious combination of enzymatic hydrolysis of thioester coupled with aretro-Michael/Michael equilibrium with a mild base (trimethylamine) for racem-ization was employed to effect dynamic resolution for the synthesis of a key chiralpharmaceutical intermediate for the synthesis of a platelet glycoprotein receptorantagonist [50]. Hydrolysis of the racemic thioester 97 catalyzed by the lipasefrom Pseudomonas cepacia led to the R-acid 98. A mild base (trimethylamine)was used to racemize the unreacted S-ester 99 via a retro-Michael/Michael reac-tion sequence during the enzymatic hydrolysis. The net result was a dynamicresolution providing the desired R-acid 98 in high yield. The reaction has beenscaled up and 28 kg of the R-acid 98 was obtained in one batch in 80% yieldand 99.9% e.e.

NCN O

S

O

Lipase fromPseudomonas cepacia

NCN O

OH

O

NCN O

S

O

Me3N

NCN O

S

ONC

N O

S

ONCN O

S

O

97

98

99

100101102

SCHEME 29


Enzymatic hydrolysis of meso-diesters usually stops at the monoester–monoacid stage and is an efficient way to prepare chiral monoester–monoacidin high yield. An efficient process was developed for making the monoester(1S,2R)-2-(methoxycarbonyl)cyclohex-4-ene-1-carboxylic acid 103 by C.antarctica lipase (Novozym 435)-catalyzed desymmetrization of the correspond-ing diester, dimethyl-cyclohex-4-ene-cis-1,2-dicarboxylate 104. The monoester103 is a key chiral intermediate for the synthesis of a potential drug candidatefor the modulation of chemokine receptor activity [51]. Interestingly, theopposite enantiomer of the monoester, (1R,2S)-2-(methoxycarbonyl)cyclohex-4-ene-1-carboxylic acid 105, was prepared by porcine liver esterase-catalyzedhydrolytic desymmetrization of the dimethyl ester 104 [52,53].

H

H

CO2Me

CO2Me

CO2H

H

H

CO2H

CO2Me

CO2Me

H

H

105

103

Lipase from Candida antarctica

Porcine Liver Esterase

104

SCHEME 30

An efficient enzyme-catalyzed hydrolysis of a diester has recently beendeveloped for a second-generation manufacturing process for S-3-(aminomethyl)-5-methylhexanoic acid 106, Pregabalin, the active ingredient of Lyrica [54]. Inthe first-generation synthesis of pregabalin 106, the racemic acid was resolvedby crystallization of diastereomeric salt at the end of the API synthesis step,providing the desired S-isomer 106 in 25% yield with an ee of 99.5%. Theundesired enantiomer could not be recycled [55]. For economic reasons, it ispreferable to carry out resolution at the earliest possible step. In a recentlyreported enzymatic process, racemic 2-carboxyethyl-3-cyano-5-methylhexanoicacid ethyl ester 107, a regulatory starting material for the first-generationsynthesis, was resolved by Thermomyces lanuginosus lipase (Lipolase fromNovozymes)-catalyzed hydrolysis to 2-carboxyethyl-3S-cyano-5-methylhexanoicacid 108. The acid 108 was decarboxylated thermally, hydrolyzed, and reduced


to Pregabalin 106. The unreacted 2-carboxyethyl-3R-cyano-5-methylhexanoicacid ethyl ester 109 from the enzymatic step was racemized and recycled in theprocess. This new route improved the process efficiency and provided 40 to45% higher yields of Pregabalin 106 than did the first-generation manufacturingprocess. In addition, the second-generation process reduced the process wastesignificantly.

CO2Et

CO2Et

CO2Et

CO2Et

CO2Et

CO2

NH2

HCO2H

CN

Lipolase

CN

CN

106

107

108

109

SCHEME 31

Amines Chiral amines constitute an important class of pharmaceutical interme-diates. Vinyl and isopropenyl acetate, generally used for enzymatic acetylationof alcohols, react rapidly with most amines in the absence of enzyme. Alkylesters react slowly with amines and are generally used for enzymatic acylationof the amines. There are many reports in the literature on the resolution of aminesby enzymes. In most cases the substrate is a primary amine adjacent to a sec-ondary carbon chiral center (∗CH—NH2). The lipase from C. antarctica hasbeen used extensively for the resolution of primary amines [56]. The enzymewas shown to be especially good for the resolution of arylalkylamines (e.g., α-phenylethylamine 110). The chiral α-phenylethylamine is not only an importantpharmaceutical intermediate by itself, but is also used frequently for the asym-metric synthesis of APIs (e.g., resolution of chiral acids via diastereomeric saltsand as a chiral auxiliary for the synthesis of other chiral amines).

NH2

NH

NH2

NH3 OOC

LipaseC. antarctica

HOOC R2

R1

R3

O

NH R2

R1H2/Catalyst

H2N R2

R1

R1

R2R3110

111

112

113

114 115

SCHEME 32


For the resolution of alkylamines, especially short-chain alkylamines, theenantiospecificity of the lipase from C. antarctica is low. C. antarctica lipase-catalyzed acylation gave only 70% e.e. for the resolution of sec-butylamine 116[57], which is too low for synthetic purposes. The e.e. was increased by usingethyl esters of long-chain fatty acids. S-sec-Butylamine 117 of high e.e. (>99%)was obtained by C. antarctica lipase-catalyzed acylation with ethyl decanoate118 in methyl t-butyl ether [58].

NH2

+

116

C9H19 O

O

118

Candida antarctica Lipase

Unreacted EnantiomerNH2

117

NH

119

C9H19

OMTBE

SCHEME 33

BASF reported a novel acylating agent, alkyl 2-alkoxyacetate 120, for theenzymatic resolution of amines. The unreacted chiral amine enantiomer 111and the product chiral amide 121 are separated by distillation or extraction.The chiral amide 121 is hydrolyzed to amine 122 without racemization,thus generating both chiral isomers of the amine. BASF commercial-ized the process in 2002 and reported the production of several metrictons per year of chiral α-phenylethylamine and 1-methoxy-2-propylamine[59,60].

NH2

NH

NH2

Lipase

O

OR2O

O

R1+

OR1

NH2

110 120

111

121 122

SCHEME 34

There are limited examples of enzymatic resolution of secondary amines.One of the few examples of enzymatic resolution of an atropisomeric sec-ondary amine was reported in the synthesis of a key piperidine intermediate123 for a farnesyl protein transferase inhibitor SCH6636 124. A lipase from


Pseudomonas aeruginosa (Tyobo LIP-300) and trifluoroethyl isobutyrate 126were found to be the best enzyme and acylating agent, respectively, for res-olution of the racemic amine 125. The unreacted amine 128 was recovered,thermally racemized, and recycled in the resolution process. The enzyme wasrecovered and reused [61]. The N -acetyl derivative 127 of the desired chiralatropisomer was hydrolyzed to the desired chiral amine 123 and reduced in thesynthetic scheme to the final product 124. The enzymatic resolution also workson the corresponding reduced compound 129, but the undesired enantiomer ofthe reduced compound could not be racemized, and hence that approach wasabandoned.

N

N

N

N

NH

NH

NH

Cl

Br O

O

CF3+LIP-300

N

N N

Cl

Br

O

N

H

Cl

Br

Hydrolysis

Cl

Br

Racemization by Heating

N

Cl

Br

O

N NH2

O

Cl

Br

125126

129

128

123

124

127

SCHEME 35

B. Reduction of Ketones to Alcohols

Enzymatic reduction of ketones is one of the best methods of preparingchiral alcohols. A key chiral pharmaceutical intermediate, S-1-(2-bromo-4-fluorophenyl)ethanol 130 for a gamma-secretase inhibitor 131, a potentialAlzheimer’s drug candidate was prepared by the enzymatic reduction ofthe corresponding ketone, 2-bromo-4-fluoroacetophenone 132. A completereduction of the ketone 132 with very high e.e. was seen with many yeasts,bacteria, and fungi. Reduction by baker’s yeast was the simplest to performand most cost-efficient. The reaction was optimized and carried out on akilogram scale to provide the S-alcohol 130 in 65% isolated yield and >99%e.e. [62].


BrF

O

BrF

OH

132

Baker's Yeast

F

N

O

OH

S

F

F

O

O

Cl

130131

SCHEME 36

To simplify the overall synthesis of the API, it was necessary to change thesynthetic route and carry out the reduction of the keto ester 133 to the hydroxyester 134.

F

O

CO2R

CO2H CO2H

CO2RF

OH

F

O

F

OH

133

a, R = Meb, R = Etc, R = t-Bu

135 136

134

SCHEME 37

Only a limited number of microorganisms showed reasonable yield in the reduc-tion of the keto ester 133 (a or b) to the hydroxy ester 134 (a or b). Threespecies of Pichia methanolica were the best, providing 33 to 41% conversionto the hydroxy ester in >95% e.e. Hydrolysis of the keto ester 133 to the ketoacid 135 by the hydrolytic enzymes present in the whole cell was a major sidereaction. Unfortunately, keto acid 135 is not a substrate of the ketoreductasespresent in these microorganisms. An attempt to prevent the hydrolysis by usingthe t-butyl ester 133c, although successful in stopping the hydrolysis to the ketoacid 135, failed to provide the hydroxy t-butyl ester 134c, suggesting that thet-butyl ester 133c is not a good substrate for the ketoreductase enzymes. Theketoreductase enzyme from the best P. methanolica culture was purified, cloned,and expressed in E. coli . It was possible to prevent the hydrolysis and carry out


reduction of the keto ester 133b with the cloned enzyme to the hydroxy ester134b in 86% yield and >99.9% e.e.

Whole-cell systems were used in the past when few ketoreductase enzymeswere commercially available. The whole-cell system did not require theaddition of an expensive cofactor for reduction. As shown in the previousexample, reduction with a whole-cell system can suffer from side reactionsby other enzyme systems present in the cells. In many cases, the wholecells contain many ketoreductases with different specificities and show lowerproduct selectivity (low e.e. of product alcohol). The reaction with growingwhole cells is often limited to low substrate input, resulting in low volumetricproductivity. There could also be other complicating issues, such as difficultyin product isolation and purification due to interference with other cellularcomponents. Fortunately, many ketoreductase enzymes have recently becomeavailable commercially, and a large number of enzymatic reductions of ketoneshave been reported with isolated ketoreductase enzymes [63]. Both NAD andNADP cofactor-dependent ketoreductase enzymes are available. Processeshave been developed for the efficient regeneration of cofactors, enabling thecost-effective use of commercial ketoreductases for reduction of ketone toalcohol [64]. There are two different methods for cofactor regeneration: substratecoupled or enzyme coupled. In a substrate-coupled system, the NAD(P)H isregenerated by oxidation of another alcohol to a ketone, typically isopropanolto acetone.

NAD(P)H NAD(P)

R1 R2

O

R1 R2

OH

Ketoreductase

O OH

AlcoholDehydrogenase

10 11

12 13

137138

SCHEME 38

In an enzyme-coupled system, another enzyme, formate dehydrogenase (FDH)or glucose dehydrogenase (GDH), is used for regeneration. GDH regenerates bothNAD and NADP cofactors. FDH regenerates only NAD, although regenerationof NADP by a modified FDH has been reported [65].


NAD(P)H NAD(P)

R1 R2

O

R1 R2

OHKetoreductase

GlucoseGluconolactone

or

HCOOH

or

CO2

GDH

or

FDH

10 11

12 13

C6H12O6C6H10O6

140 139

142

Gluconic acid

C6H12O7

H2O

141

SCHEME 39

The reduction of ketones to alcohols can be carried out in the presence ofother functional groups in the molecule. Reduction of α-chloroketone 143 bydifferent microorganisms gave either enantiomer of α-chlorohydrin, 144 or 145,which was easily converted to the chiral epoxide pharmaceutical intermediate146 or 147 [66].

Cl

OCl

OH

Cl

OH O

O O O O O

O

143 144145 146147

SCHEME 40

Microbial reduction of the ketosulfone 148 is used commercially to make thecorresponding S-alcohol 149, a key intermediate for synthesis of the carbonicanhydrase inhibitor Trusopt 150, marketed for the treatment of glaucoma. Thefungus Neurospora crassa showed complete reduction of 148 to the desiredalcohol 149 in >85% yield with about 100% e.e. [67].

S

HN

150

S SSO2NH2

SO2NH2

SO2NH2

O O

SO O

148

O

SO O

S

149

OH

Neurospora crassa

.HCl

SCHEME 41


Enzymatic reduction of many α- or β-keto esters and some γ-, δ-, or ω-ketoesters to their corresponding hydroxy esters have been reported for the synthesisof APIs. Several strains of P. methanolica were found to be best for the reductionof the δ-keto ester ethyl 5-oxo-hexanoate 151 to ethyl 5S-hydroxyhexanoate 152,a pharmaceutical intermediate [68].

OEt

O O

OEt

OH O

151 152

SCHEME 42

The β-ketoester 153 was reduced completely by a number of microorgan-isms and gave the (3R,4R)-hydroxy ester 154 in >99% enantiomeric and >97%diastereomeric excess [69]. The high yield and high diastereomeric excess suggestthat only the 4R-carboxylate 155 is undergoing the reduction, and the unreacted4S-carboxylate 156 is racemized via the enol form 157 under the reaction con-dition.

N

Ph

CO2Et

O

N

Ph

CO2Et

OH

N

Ph

CO2Et

O

N

Ph

CO2Et

O

N

Ph

CO2Et

OH

Reduction

153

155

154

157 156

SCHEME 43

Reduction of alkyl esters of 4-halo-3-oxo-butanoic acid 158 by cellsuspensions of Geotrichum candidum gave the corresponding S-4-halo-3-hydroxybutanoic acid 159, which was converted to the corresponding S-epoxide


160, a key intermediate for a HMG CoA reductase inhibitor [70]. The eein the reduction of methyl 4-chloro-3-oxobutanoate to the corresponding3S-hydroxy compound was increased from 92% to 98% by heat treatment ofthe cell suspension prior to reduction. The NADP-dependent ketoreductaseenzyme responsible for the stereospecific reduction was isolated, purified andcharacterized.

O

OO

RXO

OOH

RX

X = Cl, BrR = Me, Et, i-Pr, t-Bu

Geotrichum candidum

158 159

O

O

R

160

O

SCHEME 44

Microbial reduction of the 3,5-diketoester 161 by an Acinetobacterculture gave the (3R,5S)-dihydroxy ester 162, which is a precursor of akey starting material 163 for many statins, HMG CoA reductase inhibitors[71]. Three different ketoreductase enzymes were isolated from the cul-ture. Reductase I showed preferential selectivity for the 5-keto group andprovided 3-keto-5-hydroxy 164 as the major and 5-keto-3-hydroxy 165 asthe minor products of reduction. Reductase II reduced the monohydroxyesters 164 and 165 to the dihydroxy ester 162. Reductase III reduced thediketo ester 161 directly to the dihydroxy compound 162 and is the onenecessary to make the 3R,5S-compound 162 in high enantiomeric excess[72]. The desired reductase III has been cloned and expressed in Escherichiacoli [73].

OPh

O OOPh

OH OH

OPh

O OHOPh CO2Et

CO2EtCO2Et

CO2Et

CO2Et

OH O

+

Reductase III

Reductase IIReductase I

HO

O O

161 162163

164 165

SCHEME 45

An interesting example of chemoselectivity was seen in the reduction of m-and p-trifluoroacetyl acetophenone 166 [74]. Reduction of either 167 or 168or both (169) keto groups with high enantiomeric excess was accomplished byreadily available commercial ketoreductase enzymes.


O

166

F3C

O

OH

F3C

O

O

F3C

HO

OH

F3C

HO

167

168

169

SCHEME 46

Reduction of α,β-unsaturated ketone 170 by Candida chilensis provided theR-allylic alcohol 171 which is a key chiral intermediate for a drug candidatefor the treatment of osteoporosis. Developments of fermentation for the growthof microorganism and biocatalytic reduction conditions led to an efficient large-scale process providing the product R-allylic alcohol 171 in >80% yield with>95% e.e. with a negligible amounts (<5%) of saturated ketone 172 and saturatedalcohol 173 side products [75]. The side products were produced by enoatereductase enzyme-catalyzed reduction of a double bond and were substantiallydiminished by fermentation development.

HN

HN NN

NN

O

N N

HN N

N N

OH O

HN N

N N

OH

170 172 173171

SCHEME 47

Enoate reductase enzymes for the reduction of the carbon–carbon doublebond of α,β-unsaturated ketones occur in many microorganisms. The whole cellscontain not only the enoate reductase but also many ketoreductase enzymes,resulting in the formation of saturated ketone and saturated alcohol side


products. It was possible to control the reaction to minimize the side productsin the baker’s yeast reduction of a cyclic α,β-unsaturated diketone 174 andobtain >80% yield of R-levodione 175, a key intermediate in the synthesis ofcarotenoids [76].

O

O

O

O

175174

SCHEME 48

The enoate reductase enzymes, often referred to as “old yellow enzyme” fam-ily, add two hydrogens in trans-fashion on the double bond and have long beenknown [77]. Enantioselective reduction of the α,β-unsaturated compound 176 bythe yeast Rhodotorula rubra to the R-isomer 177 was reported for the synthesisof candidates for the treatment of non-insulin-dependent diabetes [78]. The reduc-tion of compound 176 to 177 is not easily achievable by conventional chemicalreduction methods.

N NO

S

NHO

ON N

O

S

NHO

O

176 177

SCHEME 49

The enoate reductase reaction was carried out in the past by whole-cellsystems. Recently, the genes for the enzymes have been cloned, overexpressed[77], and some isolated enoate reductase enzymes are now availablecommercially.

C. Keto Acids to Amino Acids

Chiral α-amino acids are key intermediates for many active pharmaceuticalingredients. Many nonnatural chiral amino acids have been synthesized byemploying enzyme catalysis by a variety of methods. Many of them requirethe preparation or in situ production of the corresponding α-keto acid asexemplified in the case of the antihypertensive drug candidate Omapatrilat178 [79].


N

SH

NH O O

OH

O

HS

178

N

SH

NH O O

OHR

NorleucineHomocysteine

179

SCHEME 50

The key chiral intermediate 179 of Omapatrilat contains a norleucine fragmentand a homocysteine fragment. The norleucine fragment, S-6-hydroxynorleucine180 was prepared by reductive amination of the corresponding α-keto acid, 2-keto-6-hydroxyhexanoic acid 181, by glutamate dehydrogenase enzyme from beefliver. The cofactor (NADH) was regenerated by glucose dehydrogenase enzyme.

HOOH

O

O Glutamate Dehydrogenase+ NH3

HOOH

O

NH2

NADH NAD

GlucoseGluconic acid

Glucose Dehydrogenase

180181

SCHEME 51

The synthesis of the starting material 2-keto-6-hydroxyhexanoic acid 181was quite lengthy and an alternative process was sought. The synthesis ofracemic 6-hydroxynorleucine 182 was relatively facile and was utilized asthe starting material in a second-generation process. D-Amino oxidase fromTrigonopsis variabilis converts only the R-6-hydroxynorleucine 183 of theracemic mixture to 2-keto-6-hydroxyhexanoic acid 185. The hydrogen peroxideproduced in the reaction was decomposed to water by a catalase enzyme. Asthe reaction proceeds, the e.e. of S-6-hydroxynorleucine 184 increases. Whenall R-6-hydroxynorleucine 183 was converted to 2-keto-6-hydroxyhexanoicacid 185, the e.e. of S-6-hydroxynorleucine 184 reached a value of >99%.At this point, the reaction mixture contained 50% S-6-hydroxynorleucine184 and 50% 2-keto-6-hydroxyhexanoic acid 185. The reductive aminationof 2-keto-6-hydroxyhexanoic acid 185 was then initiated by the addition ofglutamate dehydrogenase enzyme. The net result of the process is the completeconversion of racemic 6-hydroxynorleucine 182 to S-6-hydroxynorleucine 184in 98% yield with >99% e.e.


O Glutamate Dehydrogenase+ NH3

NADH NAD

GlucoseGluconic acidGlucose Dehydrogenase

HO HOOH

O

OH

O

NH2

H2O2

NH2 NH2

182

S-EnantiomerNo Reaction withD-Amino acid oxidase

HOOH

O

OH

O

NH2

HO

184

+

D-Amino acid Oxidase

H2O + O2

Catalase

HOOH

O

183

185184

SCHEME 52

In the downstream synthesis to combine the norleucine and homocysteinefragment to make the chiral intermediate 179, it was necessary to oxidizethe 6-hydroxy group of S-6-hydroxynorleucine 184, which required aprotection–deprotection sequence. A third-generation enzymatic process startingfrom 186 with the appropriate oxidation level at the 6-position of norleucinewas developed to simplify the synthesis of the key chiral intermediate 179.Reductive amination of 186 with phenylalanine dehydrogenase (PDH) fromThermoactinomyces intermedius provided the corresponding amino acid 187.The phenylalanine dehydrogenase enzyme was cloned and overexpressedin E. coli and Pichia pastoris . The cofactor was regenerated by formatedehydrogenase enzyme (FDH) from Candida boidinii or P. pastoris . Using thePDH and FDH enzyme systems, multikilogram quantities of 187 were preparedin >94% yield and >98% e.e.

OH

O

O Phenylalanine dehydrogenase+ NH3 OH

O

NH2

NADH NAD

Formate Dehydrogenase

O

O

O

O

186

HCOO−CO2

187

SCHEME 53

The norleucine derivative 187 was combined with the homocysteine 188 frag-ment for an efficient synthesis of the chiral intermediate 179.


187

SS

H2N

H2N

RHN

H3COH3CO OCH3

NH2

OCH3

COOCH3

COOCH3

OH

OH

O

O

SS

RHN

O

O

188

OH

O

O

O

NH

HN

OCH3

OCH3

COOCH3O

HN

SH

RHN

N

SH

NH O O

OHR

179

NH2OH

O

O

O

190 191

189

SCHEME 54

An improved synthesis of the intermediate 179 from a readily available start-ing material was developed using another enzyme-catalyzed process. This pro-cess utilized readily available and inexpensive L-lysine 192 for synthesizing thenorleucine portion of 179. The dipeptide derivative 193 was prepared from homo-cystine 194 and L-lysine 192. Dipeptide monomer 195 was easily obtained fromthe dimer 193. Oxidation of the ε-amino group of the dipeptide monomer 195by L-lysine-ε-aminotransferase (LAT) enzyme provided the aldehyde 198, whichwas converted readily to the key chiral intermediate 179 for Omapatrilat. TheL-lysine-ε-aminotransferase (LAT) originally from Sphinghomonas paucimobiliswas cloned and overexpressed in E. coli . In the conversion of 195 to 198 by theL-lysine-ε-aminotransferase (LAT) enzyme, the amino group was transferred to α-ketoglutarate 196, which was converted to glutamate 197. The α-ketoglutarate 196was regenerated by glutamate oxidase enzyme (GOX) from Streptomyces noursei .

SS

OH

OH

O

O

O194

NH

COOH

COOH

L-Lysine Aminotransferase

HOOC

O

COOH

HOOC COOH

Glutamate Oxidase

198

196

H2N

H2N

H2N NH2

NH2

SS

NH

O

OH2N

H2N

COOH

NH2SH

NH

COOH

NH2

OCbz–HN

COOH

SH

NH

OCbz–HN

Cbz–HN

N

SH

NH O O

OHR

N

NH O O

OHR

HS HO

193

195

197

199 179

192

SCHEME 55


The synthesis of Omapatrilat exemplifies the application of multiple types ofenzymatic transformations to develop the most convenient, inexpensive, and bestchemoenzymatic synthesis for the molecule, which requires judicious applica-tions of synthetic organic chemistry, biocatalysis, biochemistry, microbiology,genetic engineering, fermentation, and chemical and biochemical engineeringtechnologies and a joint team working in various disciplines for a common goal.

The amino acid and keto acid starting materials for Omapatrilat synthesis,although nonnatural, are structurally closely related to natural amino acids. Aminoacid oxidase, aminotransferase, and amino acid dehydrogenase enzymes are alsofrequently utilized to prepare complex nonnatural amino acids. The S-aminoacid 200 is a key intermediate for a glucagon-like-peptide-1 (GLP-1) receptormodulator for the treatment of diabetes. The S-amino acid 200 was prepared fromthe corresponding racemic amino acid 201 by an enzymatic process. Only the R-isomer 202 of the racemic amino acid 201 was oxidized by a D-amino acid oxidaseto the keto acid 204 (via the imine intermediate 203), resulting in a mixture ofS-amino acid 200, and the keto acid 204. The keto acid 204 was converted to theS-amino acid 200 by an aminotransferase enzyme. L-Aspartate 205 was the donorof the amine group for the reaction, which in turn was converted to oxaloacetate206 in the reaction. L-Aspartate 205 was not regenerated during the reaction.The aminotransferase reaction is an equilibrium reaction, and a large excess ofL-aspartic acid 205 was used to drive the reaction to the direction of S-aminoacid 200 in 78% yield and 100% e.e. The product 200 is insoluble in water andwas easily separated from water-soluble aspartate 205 and oxaloacetate 206 [80].

N

H N

O OH

200

N

H N

O OH

N

H N

O OH

202

201

N

O

O OH

204

D-Amino acidOxidase

No Reaction with D-Amino acid Oxidase

N

HN

O OH

Aminotransferase

HO CCO H

NH

HO CCO H

O

203

205

206

SCHEME 56

An alternative process, a chemoenzymatic dynamic resolution, was also devel-oped. Oxidation by D-amino acid oxidase produced an imine intermediate 203bound to the enzyme. The imine intermediate 203 was intercepted directly and


reduced by a chemical reducing agent, borane ammonia, to the racemic aminoacid 201. The dynamic resolution process gave the S-amino acid 200 in 80%yield and 100% e.e.

N

H2N

O OH200

N

H2N

O OH

N

H2N

O OH

202

201

D-Amino acidOxidase

No Reaction withD-Amino acid Oxidase

N

HN

O OH

BH3-NH 3

203

SCHEME 57

D. Enzymatic Hydroxylation

Peterson and Murray from the Upjohn Company first reported the hydroxylationof progesterone 207 to 11α-hydroxyprogesterone 208 by Rhizopus species [8].

O

O

207

O

O

208

HO

SCHEME 58

The 11-hydroxy derivatives corticosteroids are an important class of hormones.There was no convenient chemical synthetic method for preparing the corticos-teroids. Peterson and Murray’s discovery ultimately led to the development ofa commercial microbiological hydroxylation process and commercialization ofcorticosteroid hormones. This is one of the earliest examples of the commercialapplication of biocatalysis in the synthesis of active pharmaceutical ingredients.


The microbiological hydroxylation process is used commercially today withmany subsequent modifications and optimizations for the preparation of varioussteroids.

Pravastatin (Pravachol) 209 is a potent HMG CoA reductase inhibitor for thetreatment of hyperlipidemia. A related compound, Mevastatin 210, was obtainedfrom Penicillium citrinum . Chemical conversion of Mevastatin 210 to Pravastatin209 was attempted, but an economical commercial chemical synthesis processwas not feasible. Instead, an enzymatic hydroxylation of Mevastatin 210 toPravastatin 209 by Streptomyces carbophilus was discovered, developed, com-mercialized, and is still being used [81].

O

O

O

H

O OH

H

210

O

O

O

H

O OH

H

209

HO

SCHEME 59

The two classic examples above demonstrate the power of enzymatic hydrox-ylation. Enzymatic hydroxylation can be carried out with remarkable selectivityon an unreactive site far away from any other functional group. Often, thereis no alternative chemical path, at least no simple chemical path, to carry outsuch a transformation. These attributes make the enzymatic hydroxylation animportant method for introducing hydroxyl group with high regio- and stere-oselectivity. There are, however, some potential issues with the hydroxylationprocess. Unlike the reduction of ketone or hydrolysis of ester, where the enzymecan act only on a functional group of the molecule at a specific site, it is oftendifficult to predict a specific hydroxylation site except for some specific cases(e.g., benzylic hydroxylation). Another problem with the enzymatic hydroxyla-tion is the nature of the enzymes involved in the hydroxylation reaction. Thehydroxylation is carried out primarily by the cytochrome P450 enzyme systems.These enzymes are often multicomponent systems, membrane bound, difficult toisolate, and unstable as free enzymes. Generally, the biocatalytic hydroxylationreaction has to be carried out by a whole-cell system and often with growingcells. The substrate input is usually low, resulting in low volumetric productivity.The process-related cost can be high for a low volumetric productivity process.For high-value, low-volume product, even a low volumetric productivity processcan be useful if there is no alternative economical process for achieving thedesired hydroxylation. Enzymatic hydroxylation of epothilone B 211 exemplifiesa case in point. Epothilones are secondary metabolites of microorganisms and


several members are under consideration as potential anticancer drugs. Microbialhydroxylation of epothilone B 211 resulted in the hydroxylation at the benzylic(thiazole ring) site to provide epothilone F 212, which is an intermediate forthe synthesis of a potential anticancer drug candidate, 21-aminoepothilone 213.Hydroxylation of epothilone B 211 by growing cells of Actinomyces sp. gave a30% yield of epothilone F 212 [82].

N

S

O

OH O

O

OH

Me

O

211

N

S

O

OH O

O

OH

Me

O

212

OH

N

S

O

OH O

O

OH

Me

O

213

NH2

SCHEME 60

The cytochrome P450 enzyme responsible for hydroxylation by the wild-typeculture was cloned in Streptomyces species. The yield was still low (40%), evenwith the cloned organism, due to other degradative reactions carried out by thewild-type hydroxylase enzyme. Mutagenesis guided by molecular modeling ledto the development of a mutated enzyme, providing a significantly improvedyield (85%) of epothilone F 212 [83–86].

Dihydroxylation of double bonds by microbial dioxygenase and monooxyge-nase enzymes results in the formation of cis- and trans-diols, respectively [87].Enantiomerically pure cis-1S-amino-2R-indanol 214 is a key chiral intermediatefor indinavir sulfate 215, the active ingredient of Crixivan, a protease inhibitorfor the treatment of AIDS. The intermediate 214 was synthesized from either cis-(1S,2R)-indandiol 216 or trans-(1R,2R)-indandiol 217. Microbial oxidation ofindene 218 by Pseudomonas putida and Rhodococcus sp. provided cis-(1S,2R)-indandiol 216 and trans-(1R,2R)-indandiol 217 in high enantiomeric excess withother by-products [88].


OH

OH

218

216 NH2

OH

214

OH

OH

217

N

N

NHN

ONH

OHOH

O

+ H2SO4

215

SCHEME 61

To reduce the undesirable by-products, the toluene dioxygenase enzyme fromP. putida was expressed in E. coli and further evolved by directed evolution. Amutant with threefold reduction in 1-indenol by-product formation and providing40% increase in yield of the desired product 216 has been identified [89]. Oxida-tion of indene 218 by the monooxygenase system of Rhodococcus sp. resulted information of trans-(1R,2R)-indandiol 217, probably via an epoxide. Crystalliza-tion provided trans-(1R,2R)-indandiol 217 with improved stereoisomeric purity,which was then converted to cis-1S-amino-2R-indanol 214 [90].

As mentioned above, hydroxylation of double bonds often proceeds via epoxi-dation. Epoxides are important synthetic intermediates for further transformation.A large number of microorganisms are known to add oxygen to the double bondsto form epoxides [91]. However, the synthetic utility of the microbial epoxida-tion is limited for several reasons. The epoxides are toxic to microorganisms andare often converted to other products during the biotransformation. An exampleof enzymatic epoxidation is the conversion of methyl 4-allyloxyphenylacetate219 by Pseudomonas oleovorans to the S-epoxide 220, an intermediate of theβ-blocker Atenolol 221 [92,93].

O

H3COOC

219

O

H3COOC

220

OPseudomonas oleovorans

O

H2NOC

OH HN

221

SCHEME 62


E. C—C Bond-Forming Reactions

The acyloin condensation between benzaldehyde 222 and pyruvate 223 infermenting yeast to form R-phenacetylcarbinol 224 was discovered in 1921.R-Phenacetylcarbinol 224 was used for the synthesis of L-ephedrine 225.L-Ephedrine 225, originally isolated from plants (several Ephedra species), iswidely used as a bronchodialating agent and decongestant. This enzyme-catalyzedacyloin condensation process for making L-ephedrine was commercialized andis one of the earliest examples of a commercial chemoenzymatic industrialprocess [94]. It has since been recognized that a pyruvate decarboxylaseenzyme is involved in the acyloin condensation, and the acetaldehyde 226produced in the decarboxylation of pyruvate 223 undergoes condensation withbenzaldehyde 222 to provide the product R-phenacetylcarbinol 224. The processhas been modified, and many new microbial strains with improved activityand stability have been developed [95–98]. Currently, most of the world’ssupply of synthetic ephedrine derivatives is made by this chemoenzymaticmethod [60].

H

O

O

OH

OH

O

OH

O222

223

224

OH

HN

225

226

CH3NH2

H2/Catalyst

SCHEME 63

Similar condensations catalyzed by benzoylformate decarboxylase [99] andphenylpyruvate decarboxylase [100] have been reported and have potential foruse in the synthesis of pharmaceutical intermediates (Scheme 64).

The aldol condensation is a well-known and efficient synthetic methodfor forming carbon–carbon bonds. Many aldolase enzymes catalyzingstereospecific formation of C—C bonds are known [101]. The sequentialaldol condensation between 1 equivalent of 2-chloroacetaldehyde 231 and2 equivalents of acetaldehyde 232 catalyzed by a deoxyribose-5-phosphatealdolase (DERA) enzyme afforded the lactol 233 in a one-pot process. Thelactol 233 can easily be oxidized to lactone 234, which is a key intermediatefor many statins, HMG-CoA reductase inhibitors for the treatment of highcholesterol.


R2 H

O

R1

O

OH

OR1 H

O

R2

OH

R1

O227

228

230

229

R1 = Ph, Benzoylformate decarboxylase

R1 = PhCH2, Phenylpyruvate decarboxylase

SCHEME 64

HCl

H

O O

+ 2

OCl

OH

OHO

Cl

OH

O

DERA

231 232

233 234

SCHEME 65

The initial DERA enzyme from E. coli had several issues limiting thepracticality of the process. The activity was low, requiring a high loading (20wt%) of the enzyme, the reaction time was long (on the order of several days),and the maximum allowable 2-chloroacetaldehyde 231 concentration was low(100 mM), which meant that the volumetric productivity was low (2 g/L perday). Screening of genomic libraries from environmental DNA samples collectedaround the world resulted in the discovery of an improved version of DERAenzyme. A fed-batch process was developed to overcome the inhibition at higherconcentrations of 2-chloroacetaldehyde 231. The catalyst load was decreased10-fold (2 wt%), and volumetric productivity was improved by almost 400-fold,to 30.6 g/L per hour. The downstream chemistry for oxidation of lactol 233 tolactone 234 was also developed, with an overall yield of 45% for the two steps.The e.e. and diastereomeric excess (d.e.) of crystallized product lactone 233was >99.9% and 99.8%, respectively. The net result was the development ofa commercially attractive process for lactone 234 [102]. A different approach,directed evolution, was also used to overcome the issues of the initial DERAenzyme. Several improved mutants were identified by directed evolution of theDERA enzyme from E. coli , and combination of the most beneficial mutations


led to an improved mutant with a tenfold increase in activity over the wild typefor the synthesis of the lactol 233 [103].

Hydroxynitrile lyase enzyme-catalyzed addition of hydrogen cyanide tocarbonyl compounds is another method of forming a new stereospecificcarbon–carbon bond. The hydroxynitrile lyase enzyme from bitter almonds haslong been known [104,105]. A large number of aldehydes and some ketoneswere converted to chiral cyanohydrins with R-, and S-specific hydroxynitrilelyase enzymes. The addition of hydrogen cyanide to 2-chlorobenzaldehyde 235catalyzed by the immobilized hydroxynitrile lyase from bitter almonds (Prunusamygdalus) in an organic solvent–water mixture gave the R-cyanohydrin236. Hydrolysis of the R-cyanohydrin 236 with acid in an aqueous–organicsolvent mixture proceeded without racemization to provide R-hydroxy acid 237[106–108]. The R-hydroxy acid 237 is a key intermediate for the potent oralantiplatelet agent Clopidogrel 238, the active ingredient of Plavix [109].

H

O

Cl

Hydrxynitrile lyase

HCN

CN

OH

Cl

COOH

OH

Cl

N

SCl

O OH

238235 236 237

SCHEME 66

F. Other Reactions

Epoxide Hydrolase Resolution of racemic epoxides by enzymatic hydrolysisis an important way to make chiral epoxides and chiral diols. Stereoselectivehydrolysis of the racemic monosubstituted epoxide derivative 239 gave a highyield with excellent e.e. of the chiral epoxide 240, a pharmaceutical intermediate.The e.e. of enzymatic hydrolysis by Rhodotorula glutinis was improved by theaddition of methyl t-butyl ether, and the S-epoxide 240 was obtained in 48%yield (theoretical yield 50%) and >99% e.e. [110].

O

O

O

O

O

OH

OH

+

239 240 70

SCHEME 67

For the nonterminal epoxide racemic indan 1,2-oxide (241 + 242), the1R,2S-enantiomer 242 was hydrolyzed by fungal cells of Diplodia gossipina tothe diol 243, and the remaining 1S,2R-indanoxide 241 was obtained in highe.e. [111]. The enantiomerically pure 1S,2R-indanoxide 241 was converted to


cis-1S-amino−2R-indanol 214, a key chiral intermediate for indinavir sulfate215 (Crixivan), a protease inhibitor for the treatment of AIDS.

242

NH2

OH

214241N

N

NHN

ONH

OH OH

O

+ H2SO4

215O

O

Diplodia gossipina

243

241

O

OH

OH

+

SCHEME 68

The enzymatic hydrolysis of epoxides is a kinetic resolution process, andthe maximum theoretical yield is 50%. The enzymatic hydrolysis of epoxidesby Rhodotorula glutinis and Aspergillus niger proceeds with retention of con-figuration. Beauveria bassiana catalyzed hydrolysis of epoxides proceeds withinversion of configuration. By judicious combination of two enzyme systems orcombining enzymatic and chemical hydrolysis, it is possible to effectively der-acemize some epoxides and generate significantly higher (50 to 100%) yield ofchiral epoxide or chiral diol in high e.e. [112].

Nitrilase, Nitrile Hydratase, and Amidase As mentioned before, classicaldiastereomeric salt resolution of a racemic acid was used for the first-generationsynthesis of S-3-(aminomethyl)-5-methylhexanoic acid 106, Pregabalin,the active ingredient of Lyrica. Another novel enzyme-catalyzed approachfor its synthesis has been reported using nitrilase. Hydrolysis of racemic2-isobutylsuccinonitrile 244 by nitrilase from Arabidopsis thaliana resulted inthe hydrolysis of only one enantiomer to S-3-cyano-5-methylhexanoic acid245 in 43% yield and ≥99% e.e. The unreacted R-2-isobutylsuccinonitrile246 can be racemized under basic conditions in 84% yield. The S-3-cyano-5-methylhexanoic acid 245 was converted to S-3-(aminomethyl)-5-methylhexanoicacid 106, Pregabalin [113]. The nitrilase enzyme from A. thaliana was clonedand improved by mutagenesis. The best mutant showed a threefold improvementin activity for the hydrolysis desired [114].

CNNitrilase

CO2H

NH2

106

244

CN

CN

246

CN

CN

245

CO2H

SCHEME 69


An efficient and scalable chemoenzymatic process using a key nitrilase-catalyzed step was developed for the conversion of epichlorohydrin 247 to ethylR-4-cyano-3-hydroxybutyrate 248 [115]. Ethyl R-4-cyano-3-hydroxybutyrate248 is a key intermediate for many statins used for the treatment of highcholesterol and atherosclerosis. Indeed, 248 is a regulatory starting materialfor atorvastatin (Lipitor), the world’s largest-selling drug. Reaction of cyanidewith inexpensive epichlorohydrin 247 afforded 3-hydroxyglutaronitrile 249.The key step is a nitrilase enzyme-catalyzed desymmetrization of 249 toR-4-cyano-3-hydroxybutyric acid 250. The nitrilase enzyme was identified byscreening genomic libraries of DNA collected from environmental sourcesaround the world [116]. The best nitrilase gave complete conversion of 100mM input of 249 to 250 in 24 h with an e.e. of 95%. At higher substrate input,the e.e. decreased significantly with the nitrilase enzyme identified initially, forexample, at 300 mM input of 249 the e.e. of 250 was 87.6%. The nitrilaseenzyme identified initially was improved by the gene site saturation mutagenesis(GSSM) directed-evolution technique. The most active GSSM mutant gavecomplete conversion of 2.25 M input of 249 to 250 in 15 h with an e.e. of 98%[117]. The biocatalyst production was optimized by expression in Pseudomonasfluorescens (Pfenex expression technology). The optimized nitrilase hydrolysisreaction was carried out at 3 M (330 g/L) input of 249 and 6 wt% enzymeloading, giving complete conversion to 250 in 16 h with an e.e. of 99%. Theacid 250 was esterified to ethyl R-4-cyano-3-hydroxybutyrate 248 in 98.8% e.e.and 97% purity with an overall yield of 23% from epichlorohydrin 247.

OClCNNC

OH

247 249

Nitrilase CO2HNCOH

250

CO2EtNCOH

248

EtOH/H+

1. HCN, Base

2. NaCN

SCHEME 70

The nitrilase enzyme hydrolyzes nitriles directly to acids. On the other hand,hydrolysis of nitriles by the nitrile hydratase enzyme stops at the amide stage,which can, in turn, be hydrolyzed to an acid by an amidase enzyme. Nitrilehydratase-catalyzed conversion of acrylonitrile to acrylamide is one of the largest-scale industrial applications of a biocatalytic process and is used for the pro-duction of about 40,000 tons per year of the commodity chemical acrylamide[118,119]. Lonza has developed a nitrile hydratase-catalyzed conversion of 3-cyanopyridine 251 to nicotinamide 252 and commercialized the process in 1998,producing about 3400 tons per year [120].

N

CN

N

CONH2Nitrile Hydratase

251 252

SCHEME 71


A stereospecific amidase-catalyzed hydrolysis was developed for the resolutionof racemic 2-piperazinecarboxamide 253 to S-piperazine-2-carboxylic acid 254,a key chiral intermediate for indinavir sulfate 215 (Crixivan), a protease inhibitorfor the treatment of AIDS [121].

N

N

NHN

ONH

OHOH

O

+ H2SO4

215

HN

NH

CONH2

HN

NH

CO2H

Amidase

253 254

SCHEME 72

Halohydrin Dehalogenase The interconversion of halohydrins and epoxides iscatalyzed by halohydrin dehalogenase (HHDH) enzymes [122]. The enzymesaccept other nucleophiles and generate β-hydroxynitrile in the presence of cyanide[123]. The HHDH enzyme from Agrobacterium radiobacter converts ethyl S-4-chloro-3-hydroxybutyrate 255 to ethyl R-4-cyano-3-hydroxybutyrate 248. Theenzyme was improved by the protein sequence activity relationship (ProSAR)approach. The HHDH enzyme evolved resulted in a 4000-fold improvementof volumetric productivity and development of a practical process for ethyl R-4-cyano-3-hydroxybutyrate 248, a regulatory starting material for atorvastatin,Lipitor [124].

ClOH

OEt

O

OEt

OOHHDH HHDH

HCNNC

OH

OEt

O

255 248256

SCHEME 73

VII. CONCLUDING REMARKS

The application of enzymes in organic synthesis is no longer considered to bean area of merely academic interest, but instead, is frequently being utilized forthe synthesis of active pharmaceutical ingredients [60,125–128]. Biocatalysis isbecoming a viable alternative for many key selective steps in the synthesis ofcomplex molecules, particularly the synthesis of single-enantiomer APIs.

It is important to recognize that compared to the large numbers of traditionalorganic synthetic chemical reactions, only a limited number of biocatalytic trans-formations have thus far been reported for synthetic applications. Nature carriesout a large number of transformations catalyzed by a wide variety of enzymes,

CONCLUDING REMARKS 175

and only a few of those natural biocatalytic reactions have been applied in thelaboratory for the synthesis of organic compounds. For example, enzymes catalyz-ing fluorination reactions in the biosynthesis of naturally occurring fluorinatedcompounds are known [129]. Many active pharmaceutical ingredients containfluorine, and the development and application of enzymes to carry out selec-tive fluorination in API synthesis would be extremely valuable. There have beenreports of other important reactions catalyzed by enzymes (e.g., the Diels–Alderreaction) [130]. The discovery of enzymes for new reactions and their develop-ments for novel synthetic applications would be of immense value in the synthesisof API.

The enzymes identified initially often are not optimal for large-scale produc-tion. In addition to process optimization, it is often necessary to improve thecatalyst for turnover number, volumetric productivity, selectivity, solvent toler-ance, temperature, and other factors. Recently, there have been many reports onimproving the activity of enzymes by application of molecular biology techniques(e.g., directed evolution, rational design) [10,11,131–133]. Both directed evolu-tion and rational design strategies have been applied successfully for developingenzymes, and different strategies may be important in different cases [134]. Withthe advances in molecular biology, analytical methodology, and information tech-nology, a large number of mutants can be generated, analyzed for the desiredactivity(ies), and the data can be processed efficiently to select the best mutant.The time required for such an effort has been reduced significantly. However, theapproach is still based primarily on trial and error and requires screening a largenumber of mutants. The structure–activity relationship knowledge being devel-oped with various mutants can be utilized for further reductions of developmenttime in the future. One wonders if it will be feasible in future to look at a specificsynthetic step and design the best enzyme by theoretical prediction of the aminoacid sequence and synthesis of the DNA de novo, requiring no screening andonly the process development work for scale-up and manufacturing!

Enzyme catalysis is one of the many synthetic methodologies available forthe synthesis of active pharmaceutical ingredients. A molecule can often beprepared by multiple techniques (e.g., chemical synthesis, chromatography, crys-tallization). These techniques are both competitive and complementary. Eachspecific technique has both pros and cons. These complementary technologiesare important and in many cases necessary to explore to develop a manufactur-ing process. In the end, the entire process has to be critically assessed based onsome fundamental issues (e.g., safety, environmental, legal, economics, control,throughput) [135,136]. Ultimately, the best process will be selected for manu-facturing and commercialization. There is, will be, and should be competitionamong possible synthetic pathways and techniques. Neither biocatalysis nor anyother technology will be the best answer in all cases. In this chapter we havedocumented the important recent contributions of biocatalysis to the synthesisof APIs. With the advancements in identifying new enzymes and techniques forimproving enzymes, it is anticipated that the further evolution of biocatalysis willlead to novel synthetic approaches to active pharmaceutical ingredients.


Acknowledgments

The author is indebted to Ronald L. Hanson and Robert E. Waltermire for review-ing the manuscript and providing valuable suggestions.

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Enzyme Technologies (Metagenomics, Evolution, Biocatalysis, and Biosynthesis) || Enzyme Catalysis in the Synthesis of Active Pharmaceutical Ingredients