COVER STORY
OPTIMIZED Degussa has invested in developing enzymatic reactions as a technology platform and has now made its Service Center Biocatalysis part of its Exclusive Synthesis & Catalysts business unit.
ENZYMES AT WORK Rapid screening and optimization of enzymatic activity, along with available, easy-to-use enzymes, are making biocatalysis a handy tool for chiral synthesis ANN M. THAYER, C&EN HOUSTON
C
HEMISTS HAVE BEEN PRACTICING ORGANIC CHEMISTRY
for hundreds of years; microbes have been at it even longer. Microbial and other enzymes are superbly enantio-, chemo-, and regioselective across a diverse range of reactions under mild conditions of pH, temperature, and pressure. Why, then, has it taken chemists so long to put aside a dislike of "bugs" and use their enzyme catalysts? The question is especially pertinent when it comes to making pharmaceuticals. When scientists at GlaxoSmithKline, AstraZeneca, and Pfizer examined 128 syntheses from their own companies,
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theyfoundthat as many as half of the drug compounds made by their process R&D groups are not only chiral but also contain an average of two chiral centers each (Org. BiomoL Chem. 2006,4,2337). And to meet regulatory requirements, enantiomeric purities of 99.5% were found to be necessary. "When it comes to wanting selectivities of 98% or higher, you are probably bound to a bioprocess, because getting beyond 95% otherwise isreallytough," says Kurt Faber, professor of chemistry at the University of Graz and a member of the Research Center for Applied Biocatalysis (RCAB) in Austria. Biocatalysis can also open up new or even greener reaction routes (J. MoL Catal. A
2006,25166). The pharmaceutical researchers also C & E N / AUGUST 14. 2006
15
COVER STORY pointed but that more than half the time process chemists purchased chiral starting materials rather than generating the chiral centers themselves. Putting two and two together, fine chemicals producers are taking advantage of biocatalysis to produce the needed high-purity chiral intermediates. They do so on both a custom and catalog basis; BASF, for example, has its ChiPros chiral building blocks, and DSM offers intermediates under the Chiralitree label. BASF and DSM, Degussa, Lonza, NPIL Pharma (through its acquisition of Avecia's business), and manyJapanese companies actually have long histories of using largescalefermentationsand biotransformations. Enzymes are end products and processing aids in marry industries and are used to make bulk food ingredients and specialty chemicals (C&EN, April 3, pagp 69). Other fine chemicals companies, such as Dowpharma, Cambrex, and Archimica (theformerQariant pharmaceutical fine chemicals unit), have added biocatalysis capabilities largely through acquisitions. Within the past 10 or so years, fine chemicals firms have begun integrating biocatalysis into their custom synthesis of-
ferings. "We see it as one part of the toolbox to address our customers' problems as broadly as possible," says Wolfgang Wienand, head of Degussa's Service Center Biocatalysis. SCB was created in 2004 after the company invested for three years in one of its Project House technology development initiatives. In February, SCB became part of the company's Exclusive Synthesis & Catalysts business unit. Most have built technology platforms around several enzyme types to offer different chemistries. For example, resolution using hydrolase enzymes, such as lipases, is a well-developed approach to separating racemic mixtures, although its inherent 50%
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page 27). In other work on epoxides, BASF has collaborated with Dick B. Janssen's group at the University of Groningen, in the Netherlands, whose work also supported Enzis, on halohydrin dehalogenases. These enzymes typically catalyze the reversibleringclosure ofvicinal haloalcohols to yield epoxides and halide ions. But the enzyme can accept other small negatively charged nucleophiles, such as azide, cyanide, and nitrite ions, to yield p-substituted alcohols and nitriles (Adv. Synth. Catal. 2006,348,579).
CATALYTIC Enzymes fall into different classes depending on the reactions they catalyze CLASS
EXAMPLES
REACTION TYPES
Hydrolases
Hydrolytic cleavage of C-0P C-N, C-C, and other bonds Oxidoreductases Oxidation and reduction reactions Transferases Lyases
Ligases
,acamas •Laboratories iL,
metal-cataryzedJacobsen hydrorytic kinetic resolution in resolving epoxides with high enantioselectivity (C&EN, Oct. 24,2005,
Isomerases
Transfer of group from donor molecule to an acceptor molecule Cleavage by elimination (leaving double bonds) or addition to double bonds Molecular coupling with simultaneous splittinq of an energy-rich bond Isomerization and racemization
Lipases, acylases, nitrilases, amidases, proteases Dehydrogenases, oxidases, ketoreductases Amino acid transaminases Aldolases, oxynitrilases, hydroxynitrile lyases Synthetases Racemases
NOTE: Classes listed in general order of availability and prevalence.
maximum yield can be a drawback (see page 29). Enzymatic reactions that generate chiral centers are valuable, because they convert prochiral substrates into single-enantiomer products and offer 100% theoretical yield. For more than a decade, BASF has used lipases to resolve racemic alcohols and amines by selective enzymatic acylation of one enantiomer to yield easily separable products. More recently, the company has been using new dehydrogenase enzymes to make optically active styrene oxides and aliphatic alcohols. And it is expanding its access to other new enzymes through a relationship with Diversa. BASF, Enzis (recently acquired by Codexis), the start-up company Oxyrane, and others have also been exploring epoxide hydrolasesformaking chiral intermediates. These enzymes have generated interest because they can compete, for example, with
C-C bond-forming reactions are fundamental synthetic tools, and DSM has a strong position using lyases and aldolases, says Marcel Wubbolts, DSM's program director for white (industrial) biotechnology. For example, DSM researchers have modified a deoxyribose aldolase enzyme to increase its activity and substrate tolerance (PioteehnolJ. 2006,1,537) and thereby make it more synthetically useful for producing an intermediateforthe blockbuster drug Lipitor (see page 26). DSM also makes enantiopure cyanohydrins on a large scale from hydrogen cyanide and aldehydes or ketones using optimized (R)- and CS)-hydroxynitrile lyases (HNLs). These cyanohydrins then go into intermediates such as CR)-2-chloromandelic acid for a cardiovascular drug and (R)-2-hydroxy-4phenylbutyric acidforangiotensin-converting enzyme inhibitors, Wubbolts explains.
Some of the drug developers will look at biocatalysis sooner, but by and large it still is a niche technology/' WWW.CEN-0NLINE.ORG
Other desirable intermediates include DSM and RCAB researchers also have relative position of functional groups (JEur. amino acids, especially nonnatural variapplied HNLs to the kilogram-scale pro- J.Org.Chem.2006,1904). duction of 1,2-amino alcohol intermediates. Earlier this year, DSM started collaborat- ants that can't be made by fermentation. They have developed a chemoenzymatic ing with enzyme developer IEP, which has Many fine chemicals companies produce process for making (#)-2-amino-l-(2- commercialized more than 10 bioreduction these on several-hundred-kilogram or ton furyOethanol to demonstrate a convenient processes. Whereas cxidoreductases are now scales via hydrolytic resolution. Other engeneral route {Org. Process Res. Dev. 2006,appearing in industrial processes to more zymatic approaches are reductive anima10,618). An HNL enzyme stereoselectively efficiently make chiral alcohols, Wubbolts tion or amino-group transfer. Excelsyn's generates the chiral center in one step from a believes that "there is still a lot ofwork to do biocatalytic capabilities, which came with cheap starting material and avoids the need on very selective hydroxylations or oxidations its acquisition of the former Great Lakes for protection/deprotection steps. within complex molecules and on addition fine chemicals business, include making nonnatural amino acids using transamiand elimination reactions, as with lyases.,, ON THE OTHER HAND, to produce (i?)- or (5)-alcohols on an industrial scale, Degussa has engineered a two-enzyme, whole-cell system containing either an (J?)- or (S)-alcohol dehydrogenase and either formate This unique catalog with specifications and dehydrogenase (FDH) or glucose dehyprices for over 3,000 chemicals for drogenase (GDH), Wienand says. The company collaborated with BRAIN AG Scale-Up and Production - Now Available. (Biotechnology Research & Information Network) and German universities and government organizations to develop the technology under a government grant for sustainable bioproduction methods. Ketoreductases or alcohol dehydrogenases are attractive means to synthesize a chiral center directly, but they require nicotinamide adenine dinucleotide (NADH) or 2007/081 f i n e c h e m i c a , s a n d intermediates ^ f l NAD phosphate (NADPH) as a hydrogen ' I for scale-up & production ^^H source. Two-enzyme systems, either in cells or using isolated enzymes, have become the prevailing solution to recycle catalytic amounts of the expensive cofactors. As the second enzyme, FDH oxidizes formate to C0 2 or GDH converts glucose to ghiconolactone to regenerate NADH or NADPH, respectively. Daicel Chemical Industries has created a recombinant whole-cell system containing GDH and an optimized tropinone reductase, originally isolatedfroma plant rather thanfrommore typical microbial sources, explainsJohn R. Peterson, president ofThesis Chemistry, DaicePs U.S. representative. Daicel uses it to reduce 3-quinuclidone to (#)-3-quinuclidinol, an intermediate for urinary incontinence drugs. Improving on the yield and selectivity of competing chemocatarytic and enzymatic approaches, the process is now industrially usefulformaking Over this and other intermediates, he adds. 800 NEW Although converting a ketone to a single Chemicals chiral hydroxy! group is very useful, a greater challenge is achieving regioselective reactions in multifunctional molecules while avoiding the need for protecting groups. Faber's group and collaboratorsfromCiba Specialty Chemicals have investigated the reduction of diketones and oxidation of diols using an alcohol dehydrogenase they or visit www.spectrufrt * * >i
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like Codexis for more sophisticated optimization and process development. "Our target market, for better or worse, has been relatively slow to adopt biotechnological approaches to traditional chemical process R&D," he continues. "It's probably not surprising since there's been a lot ofvery good chemistry done over the past 30-40 years within pharmaceutical companies, and layered on top of that is the fact that, historically, biocatalysis hasn't delivered the promise." He anticipates there will be less dissatisfaction and more results now that the tools exist to make enzymes work with most substrates and in most reactions. "People have to have faith that this is actually going to be fruitful," Gianakakos explains. The company's technology platform includes millions of mutated enzymes and a dozen or more different reaction platforms. "We can screen compounds against actual enzymes and our databases," he says. Bioinformatics capabilities allow Codexis scientists to accelerate its directed evolution process by predicting the amount of gene shuffling and specific mutations needed, along with the likelihood and length of time for reaching a target. "Awareness is increasing as chemists take the first step by bringing in off-the-shelf enzymes to use," he says. "It just takes time for disruptive technologies to get adopted." To shorten the adoption curve, Codexis tries to make its biocatalytic processes user-friendly, familiar, and efficient. "We make our processes look just like chemical processes: The substrate concentrations are extremely high, the enzyme catalyst loading is very low, and separations are very fast," Gianakakos says. "There's a confluence now where some of the drug developers will look at biocatalysis sooner, but by and large it still is a niche technology," he continues. Success in the marketplace will be another measure to demonstrate value: After about five years of work, Codexis' technology is now used in seven commercial products. These include two Pfizer products, the animal health product doramectin and the side-chain intermediate for Lipitor, and with DSM the 7-amino-3-deacetoxycephalosporanicacid intermediate for (5-lactam antibiotics. Contributing to the growth of biocatalysis are cost pressures, lower R&D productivity, the need to address selectivity and complex chemistry for high-purity chiral compounds, and a desire to access greener technologies. Codexis believes biocatalysis can often help shorten syntheses from as many as 12 steps to just three or four, cut manufacturing costs 40-60%, and reduce capital expenditures by more than 25%,
while significantly reducing the environmental footprint. There is also the potential to create new intellectual property by tailor-making enzymes and routesforgiven substrates. And new enzymes catalyzing more and different reactions are expected to emerge.
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