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Convenient Syntheses of Cytidine 5'-Triphosphate, Guanosine

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Convenient Syntheses of Cytidine 5'-Triphosphate, Guanosine Reprinted from The Journal of Organic Chemistry, 1990,Vol. 55. Copyright O 1990by the American Chemical Society and reprinted by permission of the copyright owner. Convenient Synthesesof Cytidine 5'-Triphosphate,Guanosine 5'-Triphosphate,and Uridine S'-Triphosphateand Their Use in the Preparation of UDP-glucose,UDP-glucuronic Acid, and GDP-mannose Ethan S. Simon,l Sven Grabowski,2 and George M. Whitesides* Department of Chemistry,Haruard Uniuersity,Cambridge, Massachusetts 02138 ReceiuedSeptember 20, 1989 This paper comparesenzymatic and chemical methods for the synthesisof cy'tidine 5'-triphosphate, guanosine 5'-triphosphate,and uridine 5'-triphosphate from the correspondingnucleoside monophosphates on scalesof - 10 g. Thesenucleoside triphosphates are important as intermediatesin l,eloir pathwavbiosl'ntheses of complex carboh'n'drates;the nucleosidemonophosphates are readilvavailairle <'onrmerciallr'. The bestroute to CTP is basedon phosphi-rn'lationof C\{P usingadenvlate kinase (FlC:.;.-1.:l): the ror.tteto (}TP involvesphosphorylation of GI\{Pusing guanl'late kinase (EC 2.;..1.81:chemicaldeaniination ol ('T}) tpreparedenzvmaticallv from CMP) is the bestsynthesis of UTP. For the 10-200-mmol-scalereactions described in this paper.it is moreconvenient to preparephosphoenolpyruvate (PEP), usedin the enz-vmaticpreparations. from o-(-)-3-phosphoglycericacid (3-PGA) in the reaction mixture rather than to synthesizePEP in a separatechemical step. The in situ conversion of 3-PGA to PEP requiresthe coupledaction of phosphoglyceratemutase (EC 2.7.5.3)and enolase(EC 4.2.L.L1). The enzyme-catalyzedsyntheses of uridine 5'-diphosphoglucose(UDP-Glc), uridine 5'-diphosphoglucuronicacid (UDP-GIcUA), and guanosine5'-diphosphomannose (GDP-Man) illustrate the use of the nucleosidetriphosphates. Introduction that generatethe NTPs and nucleosidephosphate sugars As part of a broad program3to developsynthetic tech- as stoichiometricreagents, rather than relying on their niquesbased on glycosyltransferases for the preparation generationin situ, for five reasons. First, this type of of glycoproteins,glycolipids, and proteoglycans.4we wished approachis the most practical. Developingcomplex sys- to developconvenient routes to cytidine 5'-triphosphate tems of coupledenzymes is difficult. If the synthesesof (CTP),guanosine 5'-triphosphate (GTP), and uridine5'- the NTPs and nucleosidephosphate sugars can be de- triphosphate (UTP). Enzyme-calafyzedreactions of these veloped and optimized separately,the final systemsare three compoundswith monosaccharidesare central reac- simpler. Second,this approachhas greatergenerality. If tions in the biosynthesisof the nucleosidephosphate sugars convenient routes to all of the NTPs and nucleoside required by glycosyl transferasesin mammalian biochem- phosphatesugars can be developed,these compounds are istry (CMP-NeuAc, GDP-Fuc, GDP-Man, UDP-Gal, then available for the full range of oligo- and poly- UDP-GalNAc, UDP-GIc, UDP-GIcNAc, UDP-GIcUA, and saccharidesyntheses. Third, this approach is the most UDP-Xyl).3 flexible. By conducting synthesesof these compounds An important issue in planning synthetic tactics con- separately,it is possibleto use whatever synthetic method cernsthe method of synthesizingthe NTPs and nucleoside works best for each,without concern for the compatibility phosphate sugarsfor use in enzyme-catalyzedreactions: of these methods. Fourth, separating synthesesof the should they be synthesized independently and used as nucleosidephosphate sugarsfrom the steps involving use stoichiometric reagents(in which casechemical, enzymatic of these compoundsin forming glycosidicbonds permits or fermentation syntheseswould all, in principle, be ac- the latter reactions to be conducted in a way that optimizes ceptable)or should they be generatedand usedin situ (in the use of the glycosyl transferases(normally the most which caseonly enzymaticsyntheses would be acceptable)? difficult enzymesto obtain and use).3 Finally, this ap- We have decided initially to develop synthetic methods proach is more likely to be successfulfor the synthesisof unnatural compounds, where analoguesof the natural reactants may have to be synthesizedchemically. (1) DuPont Fellow 1986-87. (2) NATO Postdoctoral Fellow 1988-89 (administered by the CTP, GTP, and UTP are all available from commercial DeutscherAkademischer Austauschdienst). sources,but their cost precludestheir use in multigram- (3) Toone, E. J.; Simon, E. S.; Bednarski, M. D.; Whitesides,G. M. scalereactions. We do not discussin detail the synthesis Tetrahedron 19E9,45, 5365. (4) Sharon, N. Complex Carbohydrates;Addison-Wesley: Reading, of adenosine 5'-triphosphate (ATP) here becauseit is MA. 19?5. relatively inexpensivecompared with other NTPs,5 and 0022-3263lg0 11955-1834$02.50/0O 1990American Chemical Societv ConvenientSyntheses of Nucleoside5'-Triphosphates 1990 1835 Table I. Scale and Yields for Enzymatic Synthesis of Nucleoside Triphosphates NTP enzymeo phosphoryldonorb ,,1 reaction time (days) 1lll,r.lr,t )I'Pjlfj!.? CTP AdK 3-PGA I li tltjt 3 GTP GK 3-PGA ll t.jt 3 GK PEP ll r,rrir 3 AdK (Mn2+) PEP 3, no reactn AdK (Mn2+lMg'*) PEP ll. no reactn UTP AdK 3-PGA 12rf)l I 5 NMPK 3-PGA I()(;8r 1L):in6srnplsls NMPK PEP 6 (92) I AdK (Mnz+) PEP 0.92' :l AdK PEI) 1.16' :l AdK (Mn2+lMe'*) PEP 0.92, ,) ATP AdK 3-PGA e (91) I 'Magnesium(ll) was present in all teactions unlessnoted otherwise. AdK = adenylatekinase (EC 2.?.4.3);GK = guanr.larekinase (EC 2.7.4.8);NMPK = nucl€osidemonophosphatekinase (EC 2.7.4.4). 63-PGA = D-(-)-3-phosphoglycericacid; PEP = phosphoenolpl-ruvate. 'Product was not aBsayed. becauseit has already been synthesizedenzymatically on Scheme I.o Enzymatic Synthesis of Nucleoside a 50-mmoi scale.6 Triphosphates Objective. Our objectivein this work was to develop convenientsyntheses on - 10-gscale of CTP. CiT'I',and UTP of suf'ficientpurity for use in enzyme-catall'zedre- actions. Four strategiescan be usedto produceN'fPs: (l ) enzynatic svnthesis(using cell-free enzt'rnes). (2) chemical s5trthesis,(ll) fermentation,and (4) isolationfrom natural d O'l H OP sources"We consideredthe latter two methodsto be too rpo / 'coo- - 2HO ,1. unfamiliar to be useful in classicalsynthetic organic coo- chemistry laboratoriesand did not investigatetheir merits. , i"oo- We provide conclude that enzymatic methods the most "(i) Phosphoglyceratemutase (EC, 2.7.5.3);(ii) enolase(ECl convenientroutes to CTP and GTP. Chemicaldeamina- ,t.2.L.11);(iii) pyruvatekinase (EC 2.7.I.40);(iv) adenylatekinase tion of CTP (producedenzymatically) is the best route to {8C2.1.4.3,X = A, C, U), guanylatekinase (EC 2.?.4.8,X = G) or UTP. nucleosidemonophosphate kinase (EC 2.7.4.4,X = U). P - phos- Methods of Enzymatic Synthesis. Enzymatic con- phate. Table I listsscales and yields. versionof a NMP to a NTP requirestwo kinases: one for kina-ses.cvtidvl kinase (EC 2.7.4.14)and uridyl kinase7(EC NMP and one fbr NDP (eq 1). The synthesisof N'I'Ps :.;. 1.-iil)ai'e nr)1 t.'()ntmercial prodtrcts. Nucleosidemono- KiNASC1. 1r[1i;.1vi1;1tr.kina.e rr:e:A'l'l) to phosphorvlateAMP, CMP, NMP NDP \TP {1i ( ;\11'..rritl [ \ll'. frcimNDPs is straightforw'ard.Three kinasesare available A:eriou> clriirrbackto the useot N\IPK is its instabiiity that convertall tour of the NDPs (ADP. CDP. GDP. and and cost. F urtlierrnore. preparations of NMPK are not UDP) to the correspondingNTPs: pyruvatekinase; (PK. homogeneous, and a mixture of kinases may actually be EC 2.7.1.40)uses phosphoenolpyruvate (PEP) as a phos- present. Because adenylate kinase is the least expensive phoryl donor, acetate kinase?(EC 2.7.2.1)uses acetyl and most stable of these three kinases, we tried to use it phosphate,ffi d nucleosidediphosphatekinase? (EC 2.7.4.6) to phosphorylate UMP and GMP. We were able to con- usesATP. We chosepyruvate kinaseas kinase2 because vert UMP to UDP using adenylate kinase, but not GMP PEP is more stable than acetyl phosphate and pyruvate to GDP. kinaseis lessexpensive than nucleosidediphosphate ki- Many kinasesuse A'IP as a phosphorylatingagent. nase.5 ATP usuallyis recvcledfrom ADP by using p,vruvateki- The preparation of NDPs from NMPs is more difficult naseand PEPl2'r3or acetatekinase and acetvlphosphate.la than the preparation of NTPs from NDPs. No one, stable, A recentrevie$ sunrrnarizesthe relativemerits of these inexpensive enzyme is known that converts all of the two methodstr) regenerateAl'P in organicsynthesis.5 NMPs to NDPs. We examined three commerciallv PEP is more stablein soluttr)nthan is acetylphosphate availablekinases: adenylatekinase8 (AdK, EC 2.7.4.3), and is thernrodvnanrirallva strongerphosphoryl donor.s guanylatekinase? (GK, EC 2.7.4.8),and nucleosidemono- Commercial['Fl[' i.. hos'ever.too expensive($a800/mol) phosphatekinase? (NMPK, EC 2.7.4.q. In vivo, adenyiate t() usein re'actions,rltii preparative scale (>50 mmol of kinasephosphorylates AMP and guanylatekinase7 phos- PEP is reqtrireclfirr the largerreactiorls described in this phorylates GMP. Adenylate kinase also phosphorylates paper),so il must be svnthesizedin a separatestep. For CMP at synthetically useful rates.e,loTwo other specific this work, we developeda convenientmethod (the PGA method, SchemeI t lirr the enzymaticsynthesis of PEP from the relativeiv inexpensiveo-(-)-3-phosphoglyceric (5) Chenault,H. K.; Simon, E. S.;Whitesides, G. M.In Biatechnologl, acid (il-PGA,$2501mol).15 & Genetic Engineering Reuiews;Russell, G. E., Ed.; Intercept: Wim- borne,Dorset, 1988; Vol.6, Chapter6. (6) Kim, M.-J.; Whitesides,G. M. Biotechnol,Bioeng.
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    1184 BIOCHEMISTRY: HOAGLAND ET AL. PROC. N. A. S. and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 549. 3 Speyer, J., P. Lengyel, C. Basilio, A. J. Wahba, R. S. Gardner, and S. Ochoa, in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 559. 4 Doctor, B. P., J. Apgar, and R. W. Holley, J. Biol. Chem., 236, 1117 (1961). 6 Weisblum, B., S. Benzer, and R. W. Holley, these PROCEEDINGS, 48, 1449 (1962). 6 von Ehrenstein, G., and D. Dais, these PROCEEDINGS, 50, 81 (1963). 7 Sueoka, N., and T. Yamane, these PROCEEDINGS, 48, 1454 (1962). 8 Yamane, T., T. Y. Cheng, and N. Sueoka, in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 569. 9 Benzer, S., personal communication. 10 Bennett, T. P., in Synthesis and Structure of Macromolecules, Cold Spring Harbor Symposia on Quantitative Biology, vol. 28 (1963), p. 577. 11 Yamane, T., and N. Sueoka, these PROCEEDINGS, 50, 1093 (1963). 12 Berg, P., F. H. Bergmann, E. J. Ofengand, and M. Dieckmann, J. Biol. Chem., 236, 1726 (1961). 13Bennett, T. P., J. Goldstein, and F. Lipmann, these PROCEEDINGS, 49, 850 (1963). 14 Keller, E. B., and R. S. Anthony, Federation Proc., 22, 231 (1963). ASPECTS OF CONTROL OF PROTEIN SYNTHESIS IN NORMAL AND REGENERATING RA T LIVER, I. A MICROSOMAL INHIBITOR OF AMINO ACID INCORPORATION WHOSE ACTION IS ANTAGONIZED BY GUANOSINE TRIPHOSPHATE* BY MAHLON B. HOAGLAND, OSCAR A. SCORNIK, AND LORRAINE C. PFEFFERKORN DEPARTMENT OF BACTERIOLOGY AND IMMUNOLOGY, HARVARD MEDICAL SCHOOL, BOSTON Communicated by John T.
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