R e p r i n t e d f r o m t h e J o u r n a l o f t h e A m e r i c a n C h e m i c a l S o c i e t y , 1 9 E 5 ,I 0 2 , 6 9 9 9 . Copyright O 1985by the American Chemical Society and reprinted by permission of the copyright owner.

Enzyme-Catalyzed OrganicSynthesis:A Comparison of for in Situ Regeneration Strategies of NAD from NADH Linda G. L€e and George M. Whitesides* Contributionfrom the Departmentof Chemistry,Haru-ardUniuersity, Cambridge,Massachusetts02138,and Massachusetts Institute of Technology, Cambridge,Massachusetts 02139. Receiued February25, 1985

Abstnct: This papercomparesthreedifferenttypesof enzvmaticsvstemsfor in situ regeneration of NAD from NADH for usein practical-scale enzymc-catalyzed organicsynthesis.The first. and the mostgenerallyuseful.usesan organicoxidant quantities(2-oxoglutarate, in stoichiometric with catalysisby glutamatedehydrogenase); the secondinvolvesdioxygenas the terminaloxidant.with an intermediateelectroncarrierdye (methylcneblue,with a diaphorase as catalyst):the third is based on a stoichiometric inorganicoxidant(fenicyanide,with diaphorase as catalyst).The relativementsof theseand other NAD regeneration systemsare discussed with particularreferenceto intrinsickinetic and thermodynamiclimitationsto practical application. The pap€r includes representativeexamplesof oxidationsusing each regenerationsystem. For 2-oxoglutantef glutamatedehydrogenase and methyleneblue/diaporase/O2,the conversionof cis-cyclohexanedimethanol to (+)-(lR,6.5)'cis-8-oxabicyclo[4.3.0]nonan-7-one catalyzedby horseliveralcoholdehydrogenase wascarriedout on 70- and 30-mmolscales.respcctively.The lessusefulferricyanide/diaphorase systemwas testedon a S-mmolscalein the oxidation of glucse to gluconatecatalyzedby glucoscdehydrogenasc.For many dehydrogenas€-catalyzed oxidations,the most important limitationsto syntheticapplicationseemto lie not in NAD regeneration but in the unrelatedproblemof noncompetitive inhibition by product. The paperdescribesempincal relationship betweenthe equilibrium constantsfor the oxidationor reductionreactions beingconsidered and the valuesof Michaelisand productinhibitionconstants.Theserelationships are usefulin identifying reactionswhich are plausiblecandidatesfor practical-scale enzymaticcatalysis.

Introduction The NAD(PXH)-requiring oxidoreductasesare porentially useful catalystsin chiral synthesis.r.2Practical use of this class of enzymeshas been inhibited by severalfactors: the cost of the enzymes, the requirement for efficient cofactor regeneration proccdures,and thc frequent requirement for opcration using dilute solutionsof reactantsor products. [n addition, oxidoreductasecatalyzed reactionshave often proved lesseffrcient on a preparative scale than might have bcen expccted from analytical-scalereactions. The reasonsfor this inefficiency have not been clearly defined, and one function of this paper is to suggcstthe importance of product inhibition in determining efficiency. The cost of an enzyme used in synthesisis a function both of its initial c6t (that is, the c6t to purchaseor prepare the enzyme) and the turnover number (TN = mol of product/mol of cnzyme) achicvedin reaction. For large-scalepreparations,reactor size and productivity also become important. Improved methods of enzyme stablization, especiallyimmobilization in suitablc polymer matricesl or on solid supports,ahave dramatically increasedthe lifetimes (i.e., turnover numbers) obtainable for the oxidoreductasesunder the conditions used for organic synthesis. Regeneration of NAD(P)H from NAD(P) is now relatively straightforward.5'6 Thc rcverseregeneration-that of NAD(P) from NAD(P)H-remains a more difficult problem for three reasons: first, most enzymatic oxidations are thermodynamically unfavorable; se@nd, oxidations are often strongly inhibited by products; third, many of the organic oxidants of potential use in enzyme-qrtalyzd oxidations are unstable at the higher pH values required for maximal activity of the enzFnatic catalysts (pH =9). t Harvard Univcnity. ( I ) For rwiew articlcs, scc: Findcis, M. A.; Whitcsidcs,G. M. Aruru. Rep. L{ed. Chcm. l9S, 19,263-272. Whitcsidcs,G. M.: Wong, C.-H. Aldrichimica Acta 19t3, 16, 27-34. Jones,J. B. In 'Asymmctric Synthesis"; Morrison. J. D., Ed.: Acadcmic Press: Ncw York, 19t5. (2) Hirschbcin,B. L.: Whitcsidcs,G. M. "/. Am. Chem. Soc.19t2. 104. 445t-.1450. Wandrcy, C.: Buckmann,A. F.; Kula, M. R. Biotech. Bioeng. t9tl. 2J. 2789-2802. (3) Pollak,A.: Blumcnfeld,H.; Wax, M.; Baughn,R. L.; Whitcsidcs,G. M. J. Am. Chem. Soc. 19t0. 102. 63244336. ( 4 ) K f i b a n o v ,A . M . S c i e n c e1 9 t 3 , 2 1 9 , ' 1 2 2 - 7 2 7 . C h i b a t a . I . t n 'lmmobilized Enzymcs-Rcscarchand Dcvclopmcnt":HalstcdPress:Ncw York. 1978. (5) Shakcd, Z.; Whitcsidcs.G. M. J. Am. Chem. Soc. l9t{t. /02. 7 1 0 4 - 7 1 0 5 . L c v y , H . R . ; L o c w u s ,F . A . ; V e n n c s l a n dB, . I b i d . 1 9 5 7 , 7 9 . 2949-2953. ( 6 ) W o n g ,C . - H . ;W h i t e s i d c sG, . M . J . A m . C h e m .S o c .1 0 3 , 4 8 9 f 4 8 9 9 .

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SchemeI. Methodsfor rn Situ Regeneration of NAD from NADH R^CHOH R^CO z \ l1 t /luuyno a NAD NADH l./

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