COVER STORY the N-acyl-L-amino acid untouched; after separation, the latter can be racemized and returned to thereaction.Using a D-acylase forms the alternate product likewise, DSM and others have an amidase process that works on the same principle: D^-Amino acid amides are selectrvery hydroryzed, and the remaining l>amino acid amide can be either racemized or chemically hydroryzed. Dowpharma is experienced with hydrolases and prepares enantiomerically ANN M. THAYER, C&EN HOUSTON pure a- and p-amino acids from racemic HARMACEUTICAL PROCESS CHEM"Nowadays, the competition is so tough mixtures of the related esters via selective hydrolysis with acylases and lipases, as well ists use resolutions, or the that you try to save every single cent in the as by asymmetric chemoenzymatic routes. separation of enantiomers from production." The company makes several pharmaceutiracemic mixtures, more than A number ofmukiton industrial processes cal intermediates this way at large scale, 60% ofthe time to generate chiral still use enzymatic resolution, often with licompounds. Among resolution methods, pases that tolerate different substrates. BASF, says Karen Holt-Tiffin, Dowpharma's head stereoselective salt formation followed by for example, makes a range of chiral amines of biocatalysis. For example, using a lipase crystallization accounts for two-thirds of by acylaring racemic amines with proprietary to hydrofyze the corresponding ethyl ester, Dowpharma prepares 99% enantiopure pthe cases, chemists from three leading drug esters. Only one enantiomer is acylated to phenylalanines from racemic mixtures on a companies found in analyzing dozens of an amide, which can be readily separated scale of hundreds of kilograms. syntheses (Org. Bwmol. Chem. 2006,4,2337). from the unreacted amine. Although not Other common approaches involve chromaa DKR process, the unreacted amine can The company also has a technology plattography ami dynaWc kinetic be racemized off-line and fed back into the form of (5)- or (R)-nitrilases to make chiral resolutions. Although resolutions are not process to increase the final yield. carboxylic acids via either conventional or new, recent advances in tailoring enzymes dynamicresolutions.A dynamic resolution Many fine chemicals producers also emfor high activity and coupling them with is possible if one starts with cyanohydrins ploy acylases and amidases to resolve chiral chemocatarytic reactions are expanding the amino acids on a large scale. L-Acylases, for because these racemize rapidly enough in role ofbiocatalysis in producing enantiopure situ. Enantioselective hydrolysis by the niexample, can resolve acyl D,L-amino acids compounds from racemic mixtures. by producing the L-amino acids and leaving trilase produces one a-hydroxycarboxylic acid enantiomer, the unreacted cyaEnzymes can be extremely enantiose< nohydrin racemizes, and the desired lective, achieving greater than 99% enacid accumulates in high yield and high antiomeric excess (ee) in reactions. They enantiomeric excess. BASF uses this to can catalyze the formation of chiral cenmake (R)-mandelic acid and derivatives ters from prochiral substrates (see page on a multiton scale (Angew. Chem. Int. 15) or selectively discriminate between Ed. 2004,43,788). enantiomers in a racemic mixture. This latter property makes enzymes effective Dowpharma has developed lacresolving agents. A drawback is that the tamases as wellforbioresohitions. Using maximum theoretical yield of the dean optimized ^-lactamase, the company sired enantiomer inresolutionsis 50%, produces (-)-2-azabicyclo{2.2.1}heptand half may be waste material. 5-en-3-one, an intermediate for carbocyclic nucleosides, on the multiton A solution offering a potential 100% scale. Companyresearchersalso have yield is dynamic kinetic resolution developed an immobilized microbial (DKR). It combines enantioselective esterase, replacing one of animal oriresolution and in situ racemization to gin, to prepare enantiopure (-)-2',3'recycle the unwanted enantiomer. A dideoxy-S-fluoro-S'-thiacytidine, the similar alternative is a deracemization active ingredient in the anti-HIV drug process that, instead of separating enEmtriva, by selective hydrolysis of the antiomers, turns one into the other. Albutyrate ester (Org. Process Res. Dev. though biocatarysts needn't be involved, 2006,10,670). their enantioselectivity and proclivity for mild operating conditions may be Other established processes, such as advantageous over chemical resolutions the hydantoinase technology practiced or synthesis. by Kaneka, DSM, and Degussa, take "Resolutions were state-of-the-art 20 advantage of spontaneous racemizayears ago because at that time nobody tion. Starting withreadilyavailable racared if you threw away 50% of the cemic hydantoins, D- or L-hydantoinase material," says Kurt Faber, professor enzymes yield only the D- or the L-hyof chemistry at the University of Graz RESOLVED Using biocatalysts, BASF can dantoin byformingthe corresponding and member of the Research Center make more than 1,000 metric tons per year of N-carbarrKjylamino acid; the remaining for Applied Biocatalysis, in Austria. chiral amines at its plant in Ludwigshafen. hydantoin racemizes at dilute alkaline
BIOCATALYSIS HELPS REACH A RESOLUTION
Enzymatic reactions combined with racemization can generate enantiopure materials in high yields
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COVER STORY pH. The free amino acid is generated by treatment with a D- or L-carbamoylase. Kaneka uses immobilized recombinant hydantoinase and carbamoylase enzymes to make several thousand metric tons per year of D-amino acids.
has applied directed-evolution methods to develop amine oxidases with R enantioselectivity, thus enabling the production of enantiopure (5)-amines. "We have adapted enzymes toward a number of targets of industrial interest and are currently implementing our process at INSTEAD OF using base to racemize the multiton scale for the production of one remaining hydantoin, a hydantoin racesuch unnatural amino acid," he says. Using mase will work. Degussa has succeeded this basic technology platform and statistiin combining this racemase and either the cal experimental design methods, Ingenza L- or i>hydantoinase/carbamoylase pairing evolves and optimizes the enzyme and proin a whole-cell system to make nonnatural cess conditionsforparticular customer subD- or L-oc-amino acids at industrial scale. strates. CWcago-basedRidimond Chemical "We could do it as well with three isolated is the exclusive commercialization partner enzymes, but it would be much more labor for Ingenza's proprietary technology. and cost intensive to produce and Although the technology has poisolate all of them separately," says tential applicability to other classes Resolution Dynamic kinetic Enantioconvergence Wolfgang Wienand, head of Deresolution or deracemization of compounds, Fotheringham beA ^P A ^P A ^P gussa's Service Center Biocatalysis. lieves amines and amino acids offer "Having them engineered into one large market opportunities due in cell, we only have to ferment once Racemization . part to the technical challenges in and end up with an economically current chemical syntheses. "Chiral attractive biocatalyst that accomamines and unnatural amino acids B--X-—Q B Q •Q B plishes all three transformations in are two of the largest and most sigA, B = substrate enantiomers _ _ . . _ . A T C I one working step." nificant classes ofpharmaceutical inP, Q = product enantiomers T R A N 5 L A T E In a plane of symmetry resolution, enantiomers termediates, so there are more than Meanwhile, Kaneka has develenough targets to address," he says. oped a one-pot deracemization (Ap B) in a racemic mixture react differently and "There are several proven biocataprocess for producing nonnatural selectively to form separable products (P, Q), lytic and chemical routes to prepare amino acids in greater than 90% each in 50% yield. Dynamic kinetic resolution unnatural amino acids with high opyield and 99% ee. The approach uses enantioselective resolution coupled with tical purity, but no single method has integrates four enzymes. To make in situ racemization so that one enantiomer ever dominated commercially; each the L-amino acid, a D-amino acid accumulates in 100% theoretical yield. Through has only really been applied to just oxidase converts the D-isomer to a enantioconvergence or deracemization, the two one or two compounds." ketoacid and leaves the L-enantioenantiomers are turned into the same product. mer untouched. Then an L-amino Chiral alcohols are another imacid dehydrogenase (AADH) converts the compared with chiral carboxylic acids or al- portant class of intermediates, and although ketoacid to the L-amino acid as well. Two cohols, so we focused there.'' Turner and co- it's popular to use ketoreductases to reduce enzymes, a highly durable formate dehyworkers haverecentlyadapted the deracemi- carbonyls directly to make them, they can drogenase and a catalase, are present for be easily prepared by lipase-catalyzed acylazation process to work with tertiary amines recycling cofactors. Because the AADH is tion. A practical DKR method combining (J. Am. Cham. Soc. 2006,128,2224). substrate specific, the company has develThis deracemization approach, which can an immobilized lipase and in situ ruthenium oped a library of enzymes to handle differcatalyst to racemize enantiomers was develbe applied to more than 100 different natuent aromatic or aliphatic amino acids. ral and nonnatural amino acids and amines, oped byJan-Eriing Backvall and coworkers, then at Sweden's Uppsala University, in 1997. Similarly, Nicholas J. Turner, chemistry is one technology being implemented by Ingenza, an Edinburgh, Scotland-based bioDSM has since scaled up the process, exprofessor at the University of Manchester, process company. Ingenza has developed the plains Marcel Wubbolts, DSM's program in England, and research program director technology from an academic bench-scale director for white (industrial) biotechnolat the new Centre of Excellence for Biocareaction to a more robust, economical, and ogy, and by "tweaking" it has lowered the talysis, Biotransformations & Biocatalytic broadly applicable process, currently unenzyme and catalyst loadings to produce Manufacturing there, has developed a dergoing scale-up, Ingenza President Ian chiral alcohols on the ton scale in 77% yield practical deracemization to separate chiral Fotheringham says. and99%ee. amino acids or amines. Backvall's group and others—including Although lipase-catalyzed resolution IN CONTRAST TO inefficient reductants Mahn-Joo Kim,Jarwook Park, and coworkworks well for primary amines, it can be such as sodium borohydride, employed in ers at South Korea's Pohang University of applied to only some secondary amines and 1992 by Kenji Soda at Kyoto University, in Science & Technology—have applied the doesn't work for tertiary ones. And DKR DKR method to a variety of functionalized methods often fail since conditions needed Japan, in initial work deracemizing proline and later modified by Turner and collaboalcohols. Changes in ruthenium catalyst to racemize amines are inhospitable to enligands and reaction conditions have dezymes. For these reasons, Turner's two-step, rators, Ingenza's current process uses even more cost-effective supported-metal catacreased DKR times by orders of magnitude one-pot process uses an enantioselective lysts, Fotheringham explains. "It's one of to just a few hours. Also key to the progress amine oxidase and a nonselective chemical the key changes that has permitted our apis a catalyst that racemizes alcohols in less reducing agent, such as ammonia-borane. proach to be scaled up." The company also than 10 minutes, discovered by Backvall The enzyme oxidizes only the S enantiomer to the corresponding imine, which is then reduced back to the racemic amine. Repeated cycles lead to the R enantiomer accumulating in high yield and enantiomeric excess. Turner's collaborators have included GlaxoSmithKline for modifying enzymes for broader substrate specificity and enantioselectivity and Great Lakes Fine Chemicals, now part of Excelsyn, for finding practical reducing agents. "We talked to a number ofpharmaceutical companies and found that they probably had more problems making chiral amines than pretty much any other type of chiral intermediate,"Turner says. "There were for fewer technologies available for chiral amines
J
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mers into the same steteoisomeric product via independent routes. For this to work, the stereochemistry ctfc^ie enantk»ner isretainedwhile the configuratkxi ofthe other undergoes inversion. Just within the past few years, they have unearthed sulfatases that catalyze the hydrolysis of sulfate esters; whether one of these
enzymes inverts or retains the configuration depends on the microbial source. Such enzymes maybe even rarer than the racemases, Faber suggests. Along with alkyl sulfatases, epoxide hydrolases and haloalkane dehalogenases can affect stereochemistry in similar controlled ways (JBiochem. Soc. Trans. 2006,34,296). "Hopefully we will see more industrial applications within the nextfiveyears," he remarks, calling DKR, deracemization, and enantioconvergence methods the front line of biocatalysis. •
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