Enzyme Technologies (Metagenomics, Evolution, Biocatalysis, and Biosynthesis) || Enzymatic Processes for the Production of Pharmaceutical Intermediates

6ENZYMATIC PROCESSESFOR THE PRODUCTIONOF PHARMACEUTICALINTERMEDIATES

David RozzellSustainable Chemistry Solutions, Burbank, California

Jim LalondeCodexis, Inc., Redwood City, California

I. INTRODUCTION: EXPANDING THE APPLICATIONSOF BIOCATALYSIS

Research activity in biocatalysis is at an all-time high in terms of the increasednumbers of peer-reviewed articles being published. There has also been a steadyincrease in biocatalysis tracks at major scientific conferences. Reports throughoutthe fine chemicals industry indicate that an increasing number of enzyme-basedprocesses are under active development [1]. Furthermore, biocatalytic processeshave the advantage of being “greener.” Enzyme-catalyzed reactions are typicallymore atom efficient, achieve higher stereoselectivity, operate at or near ambientconditions, and generate fewer by-products and less waste: advantages that leadto processes that are both environmentally friendly (“green”) and lower in cost.As a result, interest in biocatalysis is now surging.

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

185

186 PRODUCTION OF PHARMACEUTICAL INTERMEDIATES

Codexis has been an active participant in the development of improvedenzymes for chemical processing and in the use of improved enzymes for theproduction of key chemical products. Enzymes catalyzing a wide range ofchemical reactions are now readily available from commercial sources [2] orusing established cloning and expression methods. Chemists can now combinethe advantages of the remarkable selectivity of enzymes with an unprecedentedbreadth of enzymatic reaction scope, allowing the development of a wider rangeof biocatalytic reactions than in the past [3]. Through its shuffling technologyplatform, Codexis has achieved rapid, large improvements in the activity,stability, and selectivity of enzymes, enabling biocatalysis to meet the rigorousdemands of efficient chemical processes [4].

Codexis has developed newer, more powerful methodologies for the improve-ment and optimization of enzymes that go beyond classical shuffling methods.One of the most important developments is a proprietary bioinformatics pro-gram called ProSAR [5] (protein-sequence activity relationships). As a highlyrefined statistical model, ProSAR enables predictions to be made about whichresidues in a protein are most important in causing favorable changes in func-tion, reducing library size, and shortening the time required for optimization of anenzyme. Typical optimization programs focus on improving rate, stereoselectiv-ity, thermal stability, and robustness in the presence of organic solvents. Codexisscientists are able to go quickly beyond natural evolutionary boundaries and cre-ate enzymes that are tailormade for specific process applications. The implicationsof achieving rapid, large improvements in enzyme function and stability are sig-nificant. Rather than being constrained to the optimization of a process aroundan available enzyme, Codexis is able to take the opposite approach. First designthe ideal process, and make it “green” by design; then create the enzyme thatenables this process . A major focus of Codexis’s efforts has been in the produc-tion of key pharmaceutical intermediates and active pharmaceutical ingredients.We describe three such processes that have recently been commercialized byCodexis.

II. PRODUCTION OF TBIN: THE KEY ADVANCED INTERMEDIATEFOR ATORVASTATIN

Atorvastatin (trade name: Lipitor), a cholesterol-lowering agent, is the largest-selling drug in the world, with sales near $14 billion annually. Codexis hasdeveloped enzymatic steps to replace two traditional chemical steps in the overallsynthetic process, leading to a route for the manufacture of the key advancedintermediate in the synthesis of atorvastatin, a compound known as TBIN. Thesynthetic route for atorvastatin is shown in Scheme 1.

PRODUCTION OF TBIN 187

NC CO2Et

OH

Hydroxynitrile (HN)Produced by Codexis in multi-tonamounts by a biocatalytic process

OLi

O-tBuNC

OH O

CO2tBu

NaBH4CH3OBOEt2

Cryogenic (−70°C)NC

OH

CO2tBu

OH

"C7Diol"

NC

O

CO2tBu

O

TBINFirst Crystalline Intermediate

O

CO2tBu

O

H2N

NH

O

N

F

CO2Ca0.5

OH

OH

Atorvastatin

SCHEME 1 Synthetic route for atorvastatin.

Given the large manufacturing scale for atorvastatin, reducing the cost andenvironmental footprint of its manufacture have significant implications. Theimpact of the biocatalytic step to produce TBIN, the key advanced intermediatein the manufacture of atorvastatin, is described here. TBIN, the first crystallineintermediate, represents an important point of control in the synthesis, as it is akey point in the synthesis where product is purified and characterized. TBIN alsocontains both of the chiral centers that are present in the final drug substance, andtherefore the stereochemical purity of atorvastatin depends on the stereochemicalpurity of the TBIN intermediate.

The chemical process for the production of TBIN goes through a diol inter-mediate and uses a nonrenewable boronate reagent under cryogenic conditions(−70◦C) to introduce the chiral center via borohydride reduction (Scheme 2). Theboronate reagent is used in stoichiometric amounts, resulting in a large volume ofwaste in the form of boronate salts and solvents [6]. The cryogenic conditions areboth energy and capital intensive but are required to improve the stereoselectivityof the asymmetric reduction. Even at −70◦C, the stereochemical purity of theproduct generated by this chemical step is below the specification needed in thefinal drug, requiring further recrystallization to upgrade the purity, and leadingto yield losses and additional costs in energy and solvent.


OOHN

OtBu

O OHOHN

OtBu

OBEt3NaBH4

hydroxyketone ("HK") "diol"

−75°C

SCHEME 2 Chemical route to the key diol precursor of TBIN.

In contrast, the enzymatic reduction is catalyzed by an enzyme created specif-ically for this process using Codexis’s gene shuffling and directed evolutiontechnologies and adapted to the conditions of the process desired, which is oper-ated at ambient temperature and pressure (Scheme 3). The cost in capital andenergy of cryogenic chemistry is completely eliminated. Sodium borohydride isreplaced by glucose as a source of reducing equivalents. Glucose is a nontoxicand completely biodegradable substance. The pyrophoric and toxic triethyl boranereagent, used in stoichiometric quantities, is replaced by a biocatalyst used at 1%by weight. The biocatalyst system is comprised of a ketoreductase (KRED) forthe stereoselective reduction of the ketone and glucose dehydrogenase (GDH) forregeneration of the reduced nicotinamide cofactor. As the biocatalyst is composedof entirely biodegradable proteins, the resulting waste stream is nonhazardous andreduced dramatically in volume. In addition, the product is a single enantiomer,which requires no further recrystallization to upgrade the stereochemical purity ofthe diol, further saving on energy and solvent use, and the overall yield is higher.Codexis currently produces TBIN using this process at the multiton scale.

OOHN

OtBu

O OHOHN

OtBu

O

diolHK

glucoseNa+-gluconate−

NaOH

OON

OtBu

O

"ATS-8"NADPH NADP

KRED

GDH

SCHEME 3 Biocatalytic route to the key diol precursor of TBIN.

Table 1 shows comparative data from both the chemical and biocatalyticprocesses. The enzymatic process operates at higher concentration—300 g/L com-pared to approximately 100 g/L for the boronate process—with approximatelya 1% biocatalyst loading compared to a stoichiometric amount of the boronatereagent. The expense and energy of cryogenic conditions are eliminated. Anotherimportant advantage of the biocatalytic process is the large reduction in solvent:The biocatalytic process uses almost 90% less solvent than the chemical pro-cess, resulting in far lower waste generation. The process is not only greener

SYNTHESIS OF MONTELUKAST 189

TABLE 1 Comparison of the Biocatalytic and Chemical Processes for Productionof the TBIN Precursor

Parameter Enzymatic Process Chemical Processa

Crude substrate load 300 g/L 100 g/LSubstrate/catalyst (w/w) Approximately 100 : 1 1 : 1Conversion 99.3% Not providedStereochemical purity 99.99% ∼94%Reaction conditions required Ambient temperature and pressure Cryogenic: −70◦CSolvent use 3.2 L/kg 27.5 L/kg

aU.S. Patent 5,155,251 to Warner-Lambert.

xx xx xx xx xx xx xx xx xx xxxx

FIGURE 1 Comparison of the stereochemical purity of TBIN produced via biocatalyticreduction compared to TBIN produced by asymmetric borohydride reduction. The blackchromatogram tracing shows the purity of enzymatically produced TBIN sampled fromthe crude reaction mixture without any purification; the green tracing shows TBIN pro-duced by borohydride reduction after purification by recrystallization. (See insert for colorrepresentation of the figure.)

and lower in cost but also produces a purer product. Conversion is near 100% oftheoretical yield, with essentially a single enantiomer. Figure 1 shows a compar-ison of chromatograms of enzymatically produced, crude TBIN sampled directlyfrom the reaction mixture compared to chemically produced TBIN sampled afterrecrystallization. The chemically produced material shows the presence of theundesired diastereomer even after recrystallization; the enzymatically producedproduct contains no detectable amount of the undesired diastereomer.

III. PRODUCTION OF THE KEY CHIRAL ALCOHOL INTERMEDIATEFOR THE SYNTHESIS OF MONTELUKAST

By eliminating the use of hazardous reagents and producing purer products,biocatalysis is bringing multiple advantages into chemical manufacturing. Thesebenefits are well illustrated in a new biocatalytic process for the production ofthe key chiral alcohol intermediate in the synthesis of montelukast, the active


ingredient in Merck’s blockbuster drug Singulair. Montelukast is an orally activeselective leukotriene receptor antagonist used for the treatment of asthma. Withan annual volume of approximately 20 metric tons per year and a syntheticroute that requires a large amount of solvents and a toxic, difficult-to-handlechiral reducing agent, montelukast represents an attractive target for biocatalysisas a way to implement a greener, more efficient process. The synthetic routefor the production of montelukast is shown in Scheme 4. The key step is thestereoselective reduction of ketone IV to the S-configured alcohol [7]. The alcoholsubsequently undergoes an SN2 displacement with a thiol to give the R-configuredfinal product.

NCl

HX

O

NCl H

O

(D)-DIP-Cl

xxxxxx

xxxxxx

N

OHX

O OMe

NCl

OOMeCl

NCl

OHOMeCl

NCl

OH

NCl

OMe

MLK-II MLK-III

OH OH

HO2C

N

MeO2C

OHSH

Cl

S

SCHEME 4 Chemical route for the synthesis of montelukast.

The key reduction step highlighted in the synthetic scheme, the reduction ofthe ketone MLK-II to produce the chiral alcohol MLK-III, requires stoichiometricamounts of the chiral reducing agent (−)-β-chlorodiisopinocampheylborane [(−)-DIP-chloride]. While (−)-DIP-chloride is a selective reducing agent that avoidsthe side reactions and overreduction products generated by most metal hydridereductions, this reagent causes a number of significant problems. In addition tobeing expensive, (−)-DIP-chloride is hazardous, causing burns if allowed to con-tact the skin. The reagent is also both corrosive and moisture-sensitive, makingit difficult to ship, store, and handle. The reaction also requires cryogenic con-ditions; reduction with (−)-DIP-chloride must be carried out at −20 to −25◦Cto achieve the best stereoselectivity. Furthermore, the exothermicity of the reac-tion requires the use of energy-consuming chilling equipment. The quench andextractive workup generate large volumes of waste solvent, due to the low solu-bility of the product. Finally, according to published data, to obtain a complete


reaction, at least 1.8 equivalents of (−)-DIP-chloride is required, increasing thecost and creating large volumes of borate salt waste that must be removed andtreated. Even under the best conditions, the (−)-DIP-chloride reaction producesan alcohol product with only 97% enantiomeric excess (e.e.), below the requiredspecification for the product. Further crystallization of the product from aqueousmethanol is necessary to raise the stereochemical purity of the alcohol from 97%to 99.5% (S), with an isolated yield of 87%.

In conceptualizing the ideal process, it was envisioned that an enzyme-catalyzed reduction of the ketone would lead to a greener, more economicalprocess, potentially eliminating many if not all of the problems associated withthe chemical process [8]. The biocatalytic route desired is shown in Scheme 5.The stereoselective reduction of the ketone precursor MLK-II is catalyzed bya ketoreductase enzyme. In this case, recycling of the reduced nicotinamidecofactor is accomplished by reducing equivalents transferred from isopropanol,catalyzed by the same ketoreductase that catalyzes the ketone reduction. In thisway only a single enzyme is needed. Another advantage of using isopropanol asthe source of reducing equivalents in this case is the need for partial dissolutionof the ketone starting material. MLK-II is extremely insoluble, and even thoughit was envisaged that the reaction would be carried out in a slurry mode, areaction mixture containing approximately 50% isopropanol would both help toshift the equilibrium in favor of the alcohol product desired and incrementallysolubilize the highly insoluble MLK-II.

NCl

OMeOOH

NCl

OMeOKRED

O

O OH

NAD(P)H NAD(P)+MLK-II MLK-III

SCHEME 5 Enzyme-catalyzed reduction of MLK-II to produce the desired chiral alco-hol MLK-III.

Biocatalysis offers numerous benefits over the current chemical process. Aketoreductase enzyme is a nontoxic catalyst that is easily shipped, stored, andhandled. In contrast to the chemical reagent (−)-DIP-chloride, the enzymaticreaction would not require cryogenic conditions; ambient or near-ambient condi-tions could be used. Waste would also be reduced dramatically. The generationof borate salt waste would be eliminated entirely. Enzymatic reduction furtheroffers the potential for higher stereochemical purity and yield.

Codexis’s directed evolution technology enabled the rapid development of aketoreductase variant that was improved 2000-fold over the starting enzyme using


FIGURE 2 Three-dimensional model of the final ketoreductase developed for produc-tion of the key alcohol intermediate in the synthesis of montelukast. The ketone substrateis docked into the putative active site, and key amino acid residues are identified. (Seeinsert for color representation of the figure.)

an iterative gene shuffling procedure. In addition to large improvements in activ-ity, the enzyme was improved in terms of thermal robustness and stability in thepresence of >50% isopropanol and smaller amounts of tetrahydrofuran and/ortoluene. Stereoselectivity was also evolved to produce pure (S)-alcohol. Reac-tivity at low concentrations of dissolved ketone, and therefore an enzyme with alow Km value, was also an important property achieved in the final enzyme. Thefinal enzyme included a total of 19 mutations that were accumulated through mul-tiple evolution steps, including three enabling mutations predicted by ProSAR.Equally important, one-third of the amino acids within 7.5 AA of the dockingsite of the substrate were mutated (Fig. 2).

Process chemists at Codexis focused on a slurry-to slurry reaction as the ide-alized process mode. Critical to the process design was the low solubility of thecrystalline monohydrate of the product (the anhydrous product is viscous oil) inthe reaction medium. By designing a process where the product precipitated fromthe reaction mixture, it was not necessary to distill off the acetone by-productto drive the reaction to completion; the equilibrium was driven by precipitation


FIGURE 3 Slurry-to-slurry reaction for the biocatalytic reduction of MLK-II to thechiral alcohol MLK-III. (See insert for color representation of the figure.)

of the highly insoluble product. Furthermore, because of the extremely low sol-ubility of the substrate in 1 : 1 IPA/water (� 0.2 g/L), a cosolvent was deemedto be necessary to achieve a “respectable” substrate concentration (ca. 1 g/L).Tetrahydrofuran and toluene were identified as suitable cosolvents, and optimiza-tion experiments were carried out to determine the best solvent mixture for thereaction.

The final process was carried out as a slurry-to-slurry reaction, with thesparingly soluble ketone being converted to an almost equally insoluble alco-hol product at a concentration of 100 g/L in aqueous isopropanol and toluene(Fig. 3). This type of reaction format greatly simplified the process; no specialequipment was needed, and the product was isolated by direct filtration of thereaction mixture. No organic extraction was required, eliminating a large volumeof solvent from the overall process. A further advantage of the biocatalytic routeover the chemical route was an elimination of the need for a quench of the reac-tion, which was required for the (−)-DIP-chloride-mediated reduction but notnecessary for the enzyme-catalyzed reduction. The conversion of MLK-III fromMLK-II catalyzed by the ketoreductase was close to 100%, and the stereochemi-cal purity was essentially perfect. The undesired enantiomer of MLK-III was notdetectable in the crude product stream. The main waste stream from this processstep consists of water, isopropyl alcohol, acetone, and toluene.

Comparative data for the (−)-DIP-chloride and biocatalytic processes to pro-duce MLK-III are shown in Table 2. Codexis’s biocatalytic reduction processtechnology provides several economic advantages over the existing process,


TABLE 2 Comparison of Biocatalytic and (–)-DIP-Cl Process Metricsfor MLK-II to MLK-III

Parameter Biocatalytic Process (–)-DIP-Cl Process

Ketone concentration 100 g/L 100 g/LChiral agent/ketone Catalytic 1.25 (DIP-Cl, 1.8 Eq)Temperature 45◦C –25◦CConversion 99.3% Not providedProduct isolation Direct filtration Extraction with high dilutionEnantiomeric excess >99.9% (enantiomer not

detected)99.2% (after recryst.)

Solvent/MLK-III (l/kg) 6 30–50Solvents used Isopropanol, water,

tolueneDichloromethane, THF

Other waste generation Biodegradable enzyme,cofactor

Nonbiodegradable borate salts,other inorganics, 3.6 Eq ofα-pinene

resulting from its simplicity and environmental friendliness. The biocatalyticprocess avoids the hazardous boron reagents and the use and control of low-temperature reaction conditions. The (−)-DIP-chloride process generates about164 g of sodium borate (anhydrous basis) per kilogram of MLK-III, which isdiscarded with the wastewater. In contrast, the biocatalytic process generatesno inorganic by-products. Solvent consumption is markedly lower for the bio-catalytic process, further reducing waste generation and cost. The biocatalyticprocess has sixfold increased volumetric productivity over the (−)-DIP-chlorideprocess. Furthermore, it is not necessary to clean the vessel between runs. Afterdraining the reactor of its contents, the next reaction can be initiated immediately(demonstrated for four cycles). In addition to being lower in cost and generatingfar less waste, the enzyme-catalyzed step produces the desired alcohol in greateryield and higher stereochemical purity. The process has been scaled to batchesof 100+ kg at Arch Pharmalabs (Mumbai, India) in preparation for commercialmanufacture to be initiated in 2009.

IV. BIOCATALYTIC PROCESSES FOR THE PRODUCTION OF KEYCYCLIC AMINE INTERMEDIATES FOR BOCEPREVIRAND TELAPREVIR

Boceprevir is an NS3 serine protease inhibitor under active development bySchering Plough for the treatment of hepatitis C, a disease affecting 170 millionpeople worldwide with no known cure. Promising results were reported in April2008 from phase II clinical trials in the United States. The structure of bocepreviris shown in Figure 4. A key intermediate for boceprevir is a bicyclic prolineanalog containing a fused dimethylcyclopropyl ring. The compound has three

BOCEPREVIR AND TELAPREVIR 195

O

HN

(S) O

N

(S)

(S) (R)

O

HN

O

NH2

HN N

H

(S)

(S) (R)

CO2Me

Key Chiral Intermediate

O

Boceprevir

FIGURE 4 Structure of boceprevir, a potential first-in-class hepatitis C proteaseinhibitor.

contiguous chiral centers. The best apparent nonenzymatic route to this compoundproceeds via a chemical hypochlorite oxidation followed by cyanide additionand hydrolysis to bring in the additional carbon atom. Cyanide adds from thetrans face of the bicyclic ring system. Resolution of the racemic compound isaccomplished by diastereomeric crystallization using a tartaric acid derivative.The complete synthetic route is shown in Scheme 6.

NH

1. bleach

2. hydroxideMeOH/HCl

NH

N NH

1. NaHSO3

2. NaCN/HCl CN CO2Me

racemic

NH

D-Ditolulyltartarate

(D-DTTA)

(R)(S)

NH·HCl

(S)HCl

·D-DTTA1:1

CO2Me CO2Me

40-45% yield from racemate95-98% e.e.

SCHEME 6 Chemical route for the synthesis of the key intermediate for boceprevir.

Codexis devised an alternative biocatalytic route using a stereoselectiveoxidation catalyzed by a monoamine oxidase (MAO). MAO enzymes areflavin-dependent oxidases that catalyze the oxidative deamination of primaryamines to produce aldehydes after spontaneous hydrolysis of the intermediateimine. The reaction with secondary amines has not been well studied. Theproposed amine-oxidase route to the boceprevir precursor was based on theassumption that an enzyme could be found that would catalyze the parallel


reaction on the bicyclic amine shown as the starting material in Scheme 6, anddo so in a stereoselective fashion.

Based on published sequences, several microbial amine oxidase genes weresynthesized and cloned, and the resulting enzyme was screened for the desiredreaction. Two MAOs were identified with activity on the desired substrate,although the activity was low. In addition, the reaction was found to be stereose-lective. Subsequent evolution through the application of shuffling methods guidedby ProSAR led to a biocatalyst that was robust, highly active, and completelystereoselective in the oxidation of the amine to the imine.

Interestingly, this reaction sequence generates three chiral centers from anachiral starting material. The first step, stereoselective oxidation of the cyclicamine, produces an intermediate imine with two chiral centers. The oxidation ofthe meso bicyclic amine produces a diastereomerically pure imine intermediate.The imine is trapped as a bisulfite adduct. The subsequent addition of cyanide,which adds selectively to the trans face of the ring, creates the third chiral cen-ter. The biocatalytic route for synthesis of the stereochemically pure boceprevirprecursor is shown in Scheme 7.

NH

CNNH

CO2MeNH

1. Amine oxidase, air /NaHSO3

2. NaCN

MeOH

acid

Symmetrical

Stereoselective Oxidation

> 99% d.e.

SCHEME 7 Biocatalytic route to a stereochemically pure boceprevir precursor: creatingthree chiral centers.

As with boceprevir, telaprevir (Figure 5) is currently in phase III clinicaltrials. Vertex Pharmaceuticals is developing this hepatitis C protease inhibitor,

N

N

HN

NH

O O

N

O NH

H

H

NH

O

O

MeO

Telaprevir

P

NO

OHH H

Key Chiral Intermediate

FIGURE 5 Structure of telaprevir, a potential first-in-class hepatitis C protease inhibitor.

CONCLUSIONS 197

which also contains a bicyclic proline analog that is problematic to synthesizeby traditional means.

Taking advantage of the diverse library of amine oxidase enzymes createdin other programs, we were able to find a suitable starting point for evolution;a mutated amine oxidase that had trace activity on the telaprevir precursor andthe desired stereoselectivity. Using an exhaustive evolution program consistingof structure-guided evolution and ProSAR-guided combinatorial recombinationof diversity, we were able to create a biocatalyst with the targeted activity. Theresulting process (Scheme 8) employs less than 5% biocatalyst and providescomplete conversion of the amine in less than 24 h, giving the resulting aminoacid with >99% diastereomeric excess (d.e.). The efficiency of the biocatalyticroutes for both telaprevir and boceprevir is such that they provide a 2.5× higheryield from symmetrical amine over the late-stage resolution routes.

NH

NH

CO2HNH

CN

not isolated

HCl/H2O

·HCl

1. tBuOAc/MsOH

2. oxalic acid

NH

·HO2CCO2H1:1

CO2tBu

1. air/MAO/catalase2. aq HCl

3. NaCN4. passage through organic phase5. aq HCl ·HCl

symmetrical

SCHEME 8 Biocatalytic route to a stereochemically pure telaprevir precursor.

V. CONCLUSIONS

Opportunities for the use of biocatalysis are expanding, in large part due to recenttechnological advances enabling dramatic modifications in enzyme characteristicsto meet the demands of efficient chemical processes. As the montelukast casestudy shows, investment in the development of optimized enzymes for chiralsynthesis can return a number of advantages, including improved yield, higherstereochemical purity, and lower cost. Processes can also be made “green-by-design,” reducing the waste burden and simplifying the overall manufacturingoperation.

The biocatalytic stereoselective reduction of ketones has become well estab-lished, with a number of current examples and many more under development.Codexis is broadening the scope of enzyme-catalyzed reactions to include a rangeof new chemistries: C C reduction, reductive amination, transamination, nitrilehydrolysis, nucleophilic epoxide opening, chiral sulfoxidation, Baeyer–Villigeroxidation, and many others [2]. As we continue to expand the range of enzymaticreactions that can be used at the commercial scale, biocatalysis will become amainstream approach for chemical synthesis.

Acknowledgments

The authors would like to acknowledge the hard work and creativity of the manyscientists involved in this research. They include Lori Giver, Gjalt Huisman,


Chris Davis, Anke Krebber, Jack Liang, Steve Ma, John Gruber, Hyo Lee,Sheela Muley, Xiyun Zhang, Mike Clay, Tara Gurtler, Amritha Appaswami,John Munger, Jun Zhu, Richard Fox, Emily Mundorff, Birthe Borup, SarenaTam, Behnaz Behrouzian, Stephan Jenne, Ben Mijts, Lisa Newman, VesnaMitchell, Matt Tobin, Les Partridge, Kyle Leopold, Justin Kittell, Na Trinh,Jon Postlethwaite, Roger Sheldon, and John Grate. They would also like toacknowledge Roger Sheldon (TU Delft), Alex Zaks, Tao Li, and George Wong,for their helpful discussions, and the Schering-Plough Research Institute, for itsgenerous support of a portion of this work.

REFERENCES

1. Chiral catalysis. C&E News 83:40, Sept 5, 2005.

2. Codexis 2008 Enzyme Catalog. Available at http://www.codexis.com.

3. S Kambourakis, D Rozzell. Broadening the scope of biocatalysis to new reactions. sp2Mag, Dec, 2005.

4. G Huisman. Biocatalysis: giving nature a helping hand. sp2 Mag, Aug, 2007.

5. RJ Fox, GW Huisman. Enzyme optimization: moving from blind evolution to statisticalexploration of sequence function space. Tibtech 26:132–138, 2008.

6. AO King, RD Larsen, TR Venhoeven, M Zhao. Process for the preparation ofdiisopinocamphenylchloroborane. US Patent 5,693,816, 1996.

7. I Shinkai, AO King, RD Larsen. A convenient and economical method for the prepara-tion of DIP-chloride and its application in the asymmetric reduction of aralkyl ketones.Tetrahedron Lett 38:2641–2644, 1998.

8. A Shafiee, H Motamedi, A King. Purification, characterization, and immobilizationof an NADPH-dependent enzyme involved in the chiral specific reduction of theketo ester M, an intermediate in the synthesis of an anti-asthma drug montelukast,from Microbacterium campoquemadoensis (MB5614). Appl Microbiol Biotechnol49:709–717, 1998.

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Enzyme Technologies (Metagenomics, Evolution, Biocatalysis, and Biosynthesis) || Enzymatic Processes for the Production of Pharmaceutical Intermediates