Reprinted from the Journal of the American Chemical Society, 1985, 107-,7008. Copyright O l98B by the American Chemical Society and reprinted by permission of the copyright owner.

Glycerol Kinase: SubstrateSpecificityl

Debbie C. Crans and George M. Whitesides*

Contribution from the Department of Chemisty, Haruard Uniuersity, Cambridge, Massachusetts 02138. ReceiuedMarch I l, 1985

Abstract: Glycerolkinase (8.C. 2.7.1.30,ATP: glycerol-3-phosphotransferase)catalyzes the phosphorylationof 28 nitrogen, sulfur,and alkyl-substitutedanalogues of glycerol.A surveyof 66 analogueshas defined qualitatively the structuralcharacteristics nffissary for acceptabilityas a substrateby this enzyme. This surveywas conducted using an assaybased on 3lP NMR spectroscopy whichdetected products produced even by slowreactions. These studies indicate that glycerolkinase accepts a rangeof substituents in placeof oneterminal hydroxyl group (that whichis not phosphorylated),and that the hydrogenatcm atC-2 can be replaced by a methylgroup. Replacementof the secondterminal hydroxyl group (that whichis normallyphosphorylated) by other nucleophiliccenters usually results in lossof activity. Glycerolkinase is a usefulcatalyst for the synthesisof chiralorganic substances,especially starting materials for the preparationof phospholipidsand analogues.Comparisons of kineticconstants for enzymesfrom four microorganisms(Candida mycoderma,Saccharomyces cereuisiae, Escherichia coli, and Bacillus stearothermophilus)indicate little variationamong them. All phosphorylatedproducts have stereochemistry analogous to that of sn-glycerol-3-phosphate.

Introduction alcoholdehydrogenasel0) has demonstratedthat certain enzymes We and others are developing practical synthetic methods based can accepta broad range of substratestructures and still retain on enzymaticcatalysis for the selectivefunctionalization of organic catalytic ratesand enantioselectivitiesuseful in organicsynthesis. molecules.2-5 Much of the work concernedwith enzymes as These enzymes are, however, believed to function in vivo as catalysts,both in mechanisticenzymology and in catalytic organic broad-spectrumcatalysts, and it is perhapsneither unexpected synthesis,has centeredon reactionsinvolving naturally occurring nor representativethat they accept a range of substrates. The substances.As a result, enzymeshave developedthe reputation work reported in this and the following paperrr was intended to 'highly of being specific",that is, by implication, of limited utility provide complementary information concerning the substrate *unnatural' for reactionsrequiring transformationsof substrates specificity of a representativeenzyme (glycerol kinase) chosen (substratesnot encounteredin vivo or not consideredas primary from the group of enzymesbelieved, on the basisof assignedin substratesfor the enzymesof interest). Recent work on enan- vivo function (reactionsin major metabolic pathways), to have tioselectivetransformations involving esterases (especially lipase and hog liver acylaseH) and oxoreductases(especially horse liver (6) Ladner, W. E.; Whitesides,G. M. J. Am. Chem. Soc. 1984, 106, 7250-1. (l) Supportedby the National Institutesof Health, Grant GM 30367. (7) Cambou,B.; Klibanov,A. M. J. Am. Chem.Soc. 1984,106,2687-92. (2) Whitesides,G. M.; Wong,C.-H. AldrichimicaActa 1983,16,27-34. (8) Wang,Y.-F.;Chen, C.-S.;Girdaukas, G.;Sih, C. J. J. Am. Chem.Soc. Whitesides,G. M.; Wong, C.-H. Angew.Chem., Int. Ed. Engl. 1985,24, 1984.t06.3695-6. 617-638. (9) Arita, M.; Adachi,D.; Ito, Y.iSawai,H.;Ohno, M. J. Am. Chem.Soc. (3) Findeis,M.; Whitesides,G. M. Annu. Rep. Med. Chem. 1984, 18, 1983.105.4049-55. 263-72. (10) Takemura,T.; Jones,J. B. J. Org. Chem. 1983,48, 791-6, and (4) Jones,J. B. In'Asymmetric Synthesis";Morrison, J. O.; Ed.;Academic referencestherein. Press: New York, 1983. (l l) Crans,D. C.; Whitesides,G. M., J. Am. Chem.Soc., following paper (5) Battersby,A. R. Chem.Brit. 1984,20,611-6. in this issue.

0002-7863l8s lrs07-7008$01.50/0 @1985 American Chemical Society Glycerol Kinase: Substrate Specificity r985 7009 naruowspecificity.r2 The objectivesof this work were to establish the range of unnatural substratesaccepted by this enzyme,and to determine if synthetically useful differencesin substratese- lectivity characterizedenzymes derived from different sources. We selectedglycerol kinase (E.C. 2.7.1.30,ATP: glycerol-3- phosphotransferase)for four reasons. l. Glycerol kinases derived from four microbial sources PEP (Candida mycoderma, Saccharomycescereuisiae, Escherichia coli, and,Bacillus stearothermophilus) are commercially available. This enzymeis inexpensiveand stablewhen immobilized. It has high specificactivity. 2. The assignedfunction of glycerol kinaser3-enantiospecific phosphorylationof glycerol and generationof sn-glycerol-3- phosphate'a-providesa route to an intermediateimportant in the synthesisof phospholipids.The ability to synthesizechiral analoguesof sn-glycerol-3-phosphatewould be useful in the preparationof new phospholipids.rs 3. Organic phosphatesare relativelyinconvenient to synthesize usingclassical chemical methods, especially if enantiomerically enrichedmaterials are required. Synthetic methodsbased on glycerolkinase do not duplicateexisting synthetic methods. 4. The supporting technology required to use glycerol kinase-especiallythat for in situ regenerationof ATP and for immobilizationof the enzyme-is already fully developed.2.3The successfuluse of glycerolkinase in kilogram-scalesynthesis of ASSAY sn-glycerol-3-phosphate and dihydroxyacetonephosphate has i.5 h previouslyestablished the practicality of syntheticprocedures based on this enzyme.l6-18 ASSAY Glycerol kinasesfrom C. mycoderma,re-24E. coli,t2'2s'26and l=?4h B. stearothermophilus2Thave been reported to accept a total of l_ eight compoundsas substrates.Gancedo et aI.20and Eisenthal -to -20 et aI.23have proposedstereochemical models for the active site(s) PPM of theseenzymes. We have confirmed the results from theseprior Figurel. Proton-decoupledrrPNMR spectraof phosphorus-containing studiesand examineda number of additionalpotential substrates. species.,All spectra were recorded in deuteriumoxide at pFI7.5. cxcept Basedon theseexaminations. we were able to definethe limits thatfor thcphosphoramidatc which was recorded at pH 9.5.The spectra of syntheticutility of glycerolkinase and proposea simplemodel labeled"assa\ " \\'ereobtained using ot.-3-chloropropane- 1,2-diol as which summarizesthe structuralfeatures required for activitl as substratefor glrccrol krnasc uith PEPas the phosphoryl donor for the .{TP regencrall()n \\ stenr. f hcas5a\ \\'as carried out using triethanol- a substrate. Comparisonof substrateselectivity for the four - enzymesderived from different sourcesindicates that only minor amineas the but't'cr .rt pl l 5 variationin substrateselectivity is observedand that. with certain exceptions,kinetic characteristics of theseenzr mes arc similar. We concludefrom thesestudies that glycerolkinase should be (12) Thorner,J. W.; Paulus,H. In "The Enzymes";Boyer, P. D., Ed.; useful in the preparation of a number of analoguesof sn- AcademicPress: New York, 1973;Vol. 8, pp 487-508. glycerol-3-phosphate,and that the most usefulenzyme for syn- (13) In higherorganisms the primaryrole of glycerolkinase is to salvage thetic applicationsis presentlythat from S. cereuisiae. the glycerolreleased upon lipolysis. In microorganismsthe primary function Glycerolkinase consists of four similaror identicalsubunits. of the enzymeis to utilize glycerolas a carbonsource: Lin, E. C. C. Annu. Rec.Biochem. 1977, 46,765-95. The enzymefrom E. coli hasa molecularweight of 217000,26 (14) The IUPAC chemicalname for the compoundis n-propane-1,2,3- and that from C. nt)'codermahas a molecularweight of 251000.1e triol-l-phosphate.It is knownas sn-glycerol-3-phosphate(or l-glycerol-3- Glycerolkinase has been obtained in crystallineform from both phosphate)in the biochemicalliterature. The analoguesof sn-glycerol-3- E. coli)6and ('. nr)'t'odernta,rebut to our knowledgeno crystal phosphatewill be referredto by their systematicchemical names in this work. We useoL nomenclaturerather than RS nomenclatureto indicateabsolute structureis available.Gly'cerol kinase contains sulfhydryl groups configuration. The namesof the following substratesfor glycerol kinase and is stableonly' in oxi/gen-freesolution.l2 Glycerol is essential illustrateour choiceof nomenclaturesystem: D-propane-1,2-diol((R)- for the stabilityof glycerolkinase in solution.r2Immobilization propane-I o-3-fluoropropane-I ((S)-3-fluoropropane-I,2-diol), ,2-diol), ,2-diol of glycerolkinase in PAN gel28markedly increasesits stability; o-3-aminopropane-I,2-diol((R)-3-aminopropane-I,2-diol), and o-3- mercaptopropane-1,2-diol ((S)-3-mercaptopropane-1,2-diol). immobilizedenzyme (from either E. coli or S. cererisiae)lost no (15) Radhakrishnan,R.; Robson,R. J.; Takagaki,Y.; Khorana,H. G. activity on storagefor 6 months at 4 oC, and lost only 30Voof MethodsEnzymol. 1982,72,408-33. its activity on use for I month at 25 oC. ( V. Whitesides, l6) Rios-Mercadillo, M.; G. M. "/.Am. Chem.Soc. 1979. High specificactivity of enzymesused as catalystsin practi- t01.5828-9. (17) Crans,D. C.; Kazlauskas,R. J.; Hirschbein,B. F.; Wong, C.-H.; cal-scaleorganic synthesis permits the constructionof compact Abril, O.; Whitesides,G. M. MethodsEnzymol., submitted for publication. reactors;low cost for the enzymeis critical when poor substrates ( I 8) Wong.C.-H.; Whitesides,G. M. "/. Org. Chem.1983, 48, 3199-205. are beingtransformed and whenlarge quantities of enzymemust ( l9) Bergmeyer,H. Holtz,G.; Kauder,E. M.; Mollering,H.; Wieland, U.; be employed. Glycerol kinasesfrom the four commercialsources O. Biochem.Z.1961. JJ-i.471-80. (20) Gancedo,C.; Gancedo,J. M.; Sols,A. Eur. J. Biochem.1968,5, examinedhave specific activities of 50-100 u/mg (measuredwith t65-12. glycerolas substrate)and cost$50-125 per 1000U (l U = I prmol (21)Janson, C. A.; Cleland,W. W. J. Biol. Chem.1974,249,2562-6. of product formed per min; 700 U produces- I mol of product (22) Grunnet,N.; Lundquist,R. Eur. J. Biochem.1967,3,78-84. per day). (23) Eisenthal,R.; Harrison,R.; Lloyd,W. J.;Taylor,N. F. Biochem.J. 1972.1 30, 199-205. Results (24) Eisenthal,R.; Harrison,R.; Lloyd,Yl. J. Biochem.J.1974, 141, 305-7. QualitativeStudies of SubstrateReactivity. Previousstudies (25) Thorner,J. W. Ph.D.Dissertation, Harvard University, Cambridge, of the substratespecificity of glycerolkinase have relied on en- Mass.l972. (26) Hayashi.S.-1.; Lin, E. C. C. -/. Biol. Chem.1967.242, 1030-,5. (27) Comer,M. J.; Bruton,C. J.; Atkinson,T. J. Appl. Biochem.1979, (28) Pollak.A.; Blumenfeld,H.; Wax,M.; Baughn.R. L.:Whitesides. G. t.259-10. M. J. Am. Chem.Soc. 1980. 102.6324-36. 7010 J. Am. Chem. Soc., Vol. 107, No. 24, 1985 (-rans and Whitesides

zymatic assays.le-27Enzymatic assaysare unreliablefor poor or otherwiseidentified. All of the compoundslisted in Table I, substratesbecause these assays may not functionreliably in the exceptD-propane- I ,2-diol,23 o-3-fluoropropane- 1,2-diol,2r and concentratedsolutions of reactantsrequired to achievemeasurable 2-fluropropane-1,2-diol,23were examined in this work; certainof 27 rates,and becausethey do not distinguisha slow reactiondue to them havealso been examined previously by others.le The a poor substratefrom a slow reactiondue to low concentrations resultsof our studiesand theseprevious reports are in good of reactiveimpurities in a nonsubstrate.In this work we have qualitativeagreement. relied on a semiquantitativeprocedure based on rrP NMR The most importantconclusion from Table I is that a number spectroscopyto follow the rates of phosphorylationof substrates (28) of compoundsother than glyceroland dihydroxyacetoneare and to identify the structuresof the phosphorylatedproducts. substratesfor glycerolkinases. Because the enzymeis inexpensive. PhosphorusNMR spectroscopyis particularly well suited to follow evenrelatively unreactive substrates (r = ++) can be considered enzyme-catalyzedphosphorylation reactions for severalreasons. for practical-scale(>10 g) preparationsusing this enzyme. A First, enzymaticactivity in phosphorylationof substrateis clearly secondobservation is that the phosphorylationof substrates differentiated from ATPase activity. Second,the severaltypes containingnucleophilic substituents (nitrogen and sulfur) is not of phosphoruscompounds of interestin thesestudies (nucleoside dictatedsolely by the nucleophilicityof the substituent.Dt.-3- phosphates,inorganic phosphate, phosphoenolpyruvate (PEP), Aminopropane-1,2-diolis phosphorylatedon nitrogeneven at pH organicphosphates, amidates, and thiophosphates)have distinct 7.5, at which pH 99Voof the amino groupsare protonated.3l and characteristicchemical shifts.2e Third, 3lP NMR assaysare ot--3-Mercaptopropane-1,2-diol is phosphorylatedon the hydroxyl applicableto studiesin the concentratedsolutions of interestin group evenat pH 10.5,at which pH 9O%of the thiol groupsare organic synthesisbecause small concentrationsof contaminating deprotonated.3lA third observationconcerns the greaterflexibility reactivesubstrates are easilyidentified. TheseNMR assayscan in substratestructures that are acceptedby the glycerolkinase also be carried out at high or low valuesof pH. Fourth, other from S. cereri.riaerelative to that from E. coli. Both enzymes typesof spectroscopicinformation (phosphorus-proton coupling show high substrateactivity with glycerol, ot--3-mercapto- constants,changes in chemicalshift with changesin pH) can be propane-I ,2-diol. and ol-3-methoxypropane-1,2-diol. The inac- helpfulin assigningstructure. The centraladvantage of the riP tivity (r = 0) of glycerol kinasefrom E. coli with compounds NMR assayis, however.that it unambiguousllfollous thc for- having R groupslarger than CH2OCH, contrastswith the greater mationof the productand is not misledb1 competingside re- toleranceof the glycerolkinase from S. ceret,isiaefor substituents actions. in this position. The S. cereuisiaeenzyme acceptsDL-3-thio- Figure I summarizesllP NMR spectraof a number of com- methylpropane-1,2-diol (r = +++), ol-3-ethoxypropane-1,2-diol poundsimportant in theseassays, together with spectraobserved (r = **), or--3-thioethylpropane-1,2-diol(r = **). ol-butane- during one representativeassay procedure. We emphasizethat 1,2-diol(r = **), and ol-1V-acetyl-3-aminopropane-1,2-diol(r the resultsobtained using this assayprocedure are qualitative; = +). The glycerolkinase from E. coli does,however, selectively it is possibleto differentiatebetween substrates that react rapidly, phosphorylatepr--butane- 1,2,3-triol at C- I , while the enzymefrom thosethat react slowly, and thosethat do not react under the S. cereuisiaeappears to be lessselective. A fourth observation conditionsemployed. Quantitative comparisonof rates depends concernsthe greater flexibility in substratestructures at C-2 upon classicalkinetic techniquesreported in a subsequentsection. acceptedby the glycerol kinase from C. mycodermc relative to Table I summarizesthe resultsof thesesurvey experiments carried that of E. coli and S. cereuisiae. Substrate activity of nl-2- out by I'P NMR spectroscopy.Triethanolamine was used as methylpropane-L,2,3-triolwith glycerolkinase from C. mycoderma buffer at pH 7-8;glycine was usedat pH 9-10.30 All potential has previouslybeen reported2a (and confirmed by our 3rPNMR substratescontaining nitrogen were examinedboth at pH 7.5 and assay). We observedno substrateactivity with glycerolkinase pH 9.5-10 in order to test for reactivityof protonated(RNH3+) from E. coli and S. cereuisiae. and unprotonated(RNH2) materials. Certain of the substrates Certain of the observationsin this table require brief expla- listedin this tablewhich showlittle or no activitywere relativelv nation. First.a racemicmixture of propane-l,2-diolis inactive impure(impurity >90%,). We believethat the low activitiesare even though the pure D enantiomeris phosphorylated.23The real,but we noteexplicitly' that they might, in principle,reflect inactivity of the racemic material reflectsthe weak substrate inhibition of the enzymeby impurities. activityof the o enantiomerand the stronginhibitory capability The entriesin Table I are classifiedrelative to the structure of the L enantiomer.23The substrateactivity (r = **) of or-- and reactivityof glycerol(eq I and 2). The structuralfeatures butane-I ,2-diolcontrasts with the inactivityof ot--propane-1,2-diol with glycerolkinase from S. cereuisiae,and probably reflectsthe YZ YZ fact that L-butane-1,2-diol does not bind as stronglyto the enzyme \ / ArP \"' (l) (^ ----+ as doesL-propane-1,2-diol. Activities reportedfor dihydroxy- ,/" \ GK ,zc\ I acetoneand derivativesrequire caution in interpretation: di- R CH XH R 2 cHzxP(o-L hydroxyacetoneexists in aqueoussolution primarily as its hy- drate;33a-hydroxy ketonesare also hydrated. The actual substrate for the enzymatic reaction is probably the hydrated form of these HOH HOH compounds,20although this mechanistichypothesis has not been ArP \'c / '-'------\.t e) proven experimentally. GK A number of other substancesnot fitting convenientlyinto the ,/"\ ,/"\ I organization of Table I were also not R CH2OH R c H2OP(O-)z established to act as sub- stratesfor glycerol kinase: cis- and trans-cyclohexane-1,2-diol, - listed in the table are those that differ from thoseof glycerol. An cis- and trans-cyclohexane-1,3-diol, 2,2-bis(hydroxymethyl)- l entry r = *** indicatesthat the reactivity of the indicated substanceis > 10% that of glycerol;r = ** indicatesthat the (31) The pK" valuesfor protonatedol-3-aminopropane-1,2-diol and for ) ) = reactivity is llVo r 0.17othat of glycerol; r * indicatesthat ot--3-mercaptopropane-1,2-diolwere determined from their titration curves the reactivity is <0.lVo that of glycerolbut still detectable. A to be respectively9.4 and 9.6. reactivity of 0 indicatesthat no detectablereaction occurred over (32) Eisenthalet a1.23determined the K. for l-propane-1,2-diol,r--3- 72h. The productsincluded under the group r = * were char- fluoropropane-1,2-diol,and 2-fluoropropane-1,3-diolto be respectively45, 165, 3rP and 100 mM. They also determinedK, for o-propane-1,2-dioland o-3- acterizedonly by NMR spectroscopyand were not isolated fluoropropane-1,2-diolusing glycerol as substrateto be respectively4.6 and 8.6 mM (usingdihydroxyacetone as substrate,the K, valuesare 1.2and 3.6 mM). Their work wascarried out usingglycerol kinase from C. mycoderma. (29) Mark, V.; Dungan,C. H.;Crutchfield,M. M.;Van Wazer,J. R. In We confirmedwith the 3'P NMR assaythat glycerolkinase *Topics lrom S. cereuisiae in PhosphorusChemistry"; Grayson, M., Griffith, E. J., Eds.; In- and f. coli catalyzed the phosphorylationof glycerol in the presenceof tersciencePublishers: New York, 1967;Vol. 5, pp 227-457. or-propane-1,2-diol ( l0 mM). (30) Ktihne,H.;Lehman, H.-A.;Topelmann, W. Z. Chem.1916,16,23-4. (33) Befl, R. P. Adu. Phys.Org. Chem. 1966,4, l-29. Glycerol Kinase: Substrate Specificity J Am Chem. Soc.' Vol. 107' No.24, 1985 7011

TableI. QualitativeSubstrate Activities of CompoundsHaving the StructurexCHICYZR (Eq l) for Glycerol Kinasefrom s. celerlrlae

activity notcs ref OH OH cH2oH +++ t7-24 H ++ A-fPasc b CH, + (D onlv)c 2I CH,F + {D onlv)c 2I cH,cl +-++ 19,33

cH, Br T'- CH]SH +r i 24 cH2NH2 Tta cHzocHs T TT cH,ococH. +++(0)e cHrocH2cH3 ++ +(0)e CH,SCH3 +++(0)e cH2scH2cH3 + + (0)€ CH,NHCHs gd cHrN(CH3)2 0d cH,NHCOCH3 +(0)€,/ CH=O -rr (D ATPase)c iB,l9 cH(ocH3)' gh b 1^ COOII r ATPase L1 b cFI,CI{3 -r + (0)e ATPase cHrcl{roH (.H(CH3)OH --(--)€ cHOHCFITOII {l CH,CN cHrcH2cH3 0 (:O) + t-r i 8. 19.21 (:O) H 0 (:O) CH, - ,\ I'Purc f (=o) cH2cH2oH 0 (ocH,) H + ATPasc / H H 0 H CH., 0 H TT I 8,19, 21 H cHrcl 0 H CII.Br (l H (-HrNH2 0d H CH,CX-H.CII. H CII.(-I I. {l H cr{.cH, o}{ il H ct{(cHr)oH (l D.L rll tt H CH,C}I,CHJ 0 CH. 6i )2 CH,CH3 0t cH2 oH cHroH 0 cHroH CH,CH3 0 j I] +-1 21 SH H 0 SH CH,SH 0 NHt pd NHt FI gd NHt CH, 6d D.L all 0 NH, .' td NH, It t)d NHt CH, od D,Dr, all 0 NH, cH2NH2 11d NH, cH,cl gd NH, cH,ocH. gd r\TPase f H cH(cH.)oH 0 a Thc structuralfeatures listed are thoscthat drtar from glycerol. Unlessindicated otherwise, thc astivitieswere indistinloishable using th€ gl\ecrolkinases from,S. cercfisiae and, I:..o/i. Anentryr=+++indicrtcsthatthereactivityofthcsubstanceisr>l0Tthatofglycerol; | = ++ indicatcsthnt thereactivity is l0% > / > 0.1%that ofglycerol;/= + indicatcsthrl the rcactivityisr < 0.t% that ofdycerol or thrt the rc clivityis accompanicdby significantside reactions. Seefootnotes.,/, o.te\tfor det.riledinfornrution on spccificsubstrates; r = 0 indi_ cltcsthat no dctectirblercaction occurred ovcr 72 h. Unlcssindioated othcrwise, thc reactionswcrc cardcd out underthese conditions; tri 'C), cthanolaminebuffer (0.J M, pH 7.5.30 mirgnesirmchloride (12.5 mM), PEP (65 mM). ATP (6.5mM). and substrate (250 mM). The o solutioncontained DzO as intefn,rl NMR lock. Thcactivity in subslrdteFhosphorylation $as accompaniedby ATPaseactivity (<407 of lotalnctivity). c Thcunrcnctive enantionrer (L) is sucha potentinhibitor that only the pureD enrntiomcris phosphorylated.Racemic rrri\trfrcsdo not showsubstrrte ilctivity. Thescstudies were carried out usingglyccrol kinasc from C n4.coderna.1tThe in ctivityof r!ce- nic proprnc-1,2-diolwrs confirmcd for glyccrol kinasctrom both ,s.reretisia? ,tnd l.:...r/i. d The relcdons werecarried out at pH 7.5 (de- seribcdin footnote./).rndnH 9.5 in orderto e\arninethe substratcclivity ofboth the prolonatedand nonprolonated forms. Al pH 9.5 thc fcrctkrnsolution con![ncd thc following:glycinc (0.3 M), malnesiumchloride (12.5 nrM). PI]P (65 nrM).ATP (6.5mM), and substrare (250 e nrM). Thc .rctivit! ()f thc indiciltcd substanccwas lbund to bc diffcrcnt with dyccrol kin selrom ^!'.@ret'isiae a')d [:. coll. The rcactivily irdiculedin plrcnthcscsis thatwith kinaseliom Z. col1. / tlvdrolysisof ATP wrs a rLrbstantialside reilction (>407 of totnlirctivity). !lyccrol g Ihc ATPrscactivity nlay be induccdby rhc substanceor n1ir!,rcflcct r.rpid hydrolysis of rcictionproducts, Only ATPIscitclivity \,_us ob_ \ervcd. n Theirclivitv wits dctermined in theprcscncc ol hydf.lzine(0.4 M) in ordcr1o removc residu l D-glyccraldehyde.I 2-Methylpropanc- bul nol \r'ith!:l)cerol kin sesfroD I;. kinlscitonr(.'It1!0|l(|1)]d'Th!'ilctivi!yol.1hisfon]p{}tInd$lsn.)te\:ln]incdwith!ltccrolkinnseI()In,/: 7012 J. Am. Chem. Soc.. Vol. 107, No. 24, 1985 Crans and Whitesides

I tl

ll 75 70

I . \,J.. - _ rlrrrrlrrrrl I 75 70

ll d ,*,* **-{|Jl,**l lt,-,* .-,*,., *-

'r I l, J-_^-_..-=_J* trlrrtltr 7570--LrJ-- c

'1ty1,1-.,^a-f-sf .tr^-t'-r-zlff+----r.*ag

loo o PPM Figure4. c:pu.ison of oro,on-o.loupled3rp NMR .J;1"- or the Figure 2. Proton-decoupledr3C NMR spectra of o-3-chloropropane- phosphoramidateand the phosphate ester of or--3-aminopropane-1,2-diol: 1,2-diol-I -phosphateand related species: (a) nr--3-chloropropane-1,2- (a) n-3-aminopropane-1.2-diol-3-phosphate, (b) o-3-aminopropane- 1,2- diol, (b) n-3-chloropropane-1,2-diol- I -phosphateprepared by phospho- diol-3-phosphateand o-3-aminopropane- 1,2-diol- I -phosphate, (c) o-3- rylation catalyzed by glycerol kinase, (c) authentic or--3-chloro- aminopropane-1.2-diol- I -phosphate. propane-1,2-diol-l-phosphate, (d) off-resonancedecoupled spectrum of (b), (e) authenticsn-glycerol-3-phosphate, (f) authenticor-gly'cidol-l - Identification of the Structures of Phosphorylatedhoducts. For phosphate. many of the substrateslisted in Table [, it was possibleto establish the structuresof the phosphorylatedproducts directly by exam- ination of lrP NMR spectra. For others,more detailedexami- nation was required. In many cases,even though the structure of a product could be deduceddirectly from knowledgeof the structure of the substrate and from the 3lP spectra,the phos- h phorylatedproduct was isolatedand examinedusing other tech- //, 4'*ll niques. Carbon-13 NMR spectroscopyproved particularly useful rlrrll in this context. Specific characterizationsfollow. 76.8 76.5 o*'_o'.:l-l*l _ The isolationand identificationof the phosphorylatedproduct from ol-3-chloropropane-1,2-diolare of particular interest,since ii this substanceis currently in use as an antifertility agent for ll ll rats.l4'3s The activity of this compound as a substratefor glycerol '"'-l l*" kinase has previously been ascribed to glycerol impurities.36 Glycerol is presentas an impurity in or--3-chloropropane-1,2-diol,36 77 76 and it is also formed in aqueoussolutions under basicconditions,36 presumablyvia glycidol as an intermediate. It is thereforedifficult ll to distinguishwhether activity detectedby enzymaticassay is due ll o to ot--3-chloropropane-1,2-diolor to the product of hydrolysis. *"**-**JUL*.J{******* Figure 2 summarizesrelevant '3C NMR spectra. The assignment of the structure 3-chloropropane-1,2-diol-l-phosphateto the phosphorylatedproduct restson two piecesof information. First, the r3CNMR spectrumis indistinguishablefrom that of authentic -f-*,-r* ot--3-chloropropane-1,2-diol-l-phosphate.37Second, both r3Cand 3rP spectra of this material are clearly distinct from those of authentic sn-glycerol-3-phosphate(or or,-glycerol-3-phosphate) and ol-glycidol-l -phosphate. 1oo o The compound isolated following phosphorylationof or--3- PPM aminopropane-1,2-diolat pH 10.0(eq 3) wasassigned the unusual Figure 3. Proton-decoupledr3C NMR spectra of n-3-aminopropane- 1,2-diol-3-phosphateand relatedspecies: (a) nl-3-aminopropane-1,2- HOH HOH O diol, (b) o-3-aminopropane-1,2-diol-3-phosphate prepared by phospho- \/ ATBGK rylation catalyzedby glycerol kinase, (c) ur--3-aminopropane-1,2-diol- Ho.=X=_ruH, Ho_,LNHP(o-L (3) l-phosphate prepared chemically (eq 4), (d) off-resonance #;- decoupled 75 "/" spectrumof (b), (e) l-3-aminopropane-1,2-diol-l-phosphateprepared chemically(eq 5) structure of the correspondingphosphoramidate based on five piecesof evidence.First, the 3lPandl3C spectra(Figures 3 and butanol (2-ethyl-2-hydroxymethylpropane- 1,3-diol), D-ribose, 4) of this material were indistinguishablefrom thoseof authentic o-arabinose, DL-3-mercaptopropane-1,2-diol disulfide, and or--S- acetyl-3-mercaptopropane-1,2-diol. or--S-Acetyl-3-mercapto- (34) propane-1,2-diol was not recovered after the reaction; its inactivity Lobl, T. J. Clin. Androl.1980, J, 109-22. (35) Jones,A. R. Aust.J. Biol. Sci. 1983,-t6, 333-50. may reflect acetyl migration from sulfur to oxygen under the (36) Brooks,D. E. ,/. Reprod.Fertil. 1979,56,593-9. reaction conditions. (31) Zetzsche,F.; Aeschlimann,F. Helu. Chim. Acta 1926.9.708-14. Glt'cerol Kinase: Substate Specificity 107, No. 24, 1985 7013 racemicmaterial (eq 4).18 Second,the observationof a 2,/pNc

l)Pocl3' a Ho H PY Ho H o \/ cHacN,o"c \/ ,l t. I HO_V_ NHz HO_v_ NHP(O-)2@l ;ffi do* b = 1.6Hz (+0.3) for the carbonattached to the nitrogenin the rrC NMR spectrum is compatible with N-phosphorylation.3e Third. this substanceis stablein basicsolution and decomposes rapidly in acidic solution.ao No decompositionwas observedfor chemicallyprepared ot--3-aminopropane- 1,2-diol- I -phosphateafter ,t r l, 2 weeksat ambient temperatureat pH ( I . Phosphoramidates 73 70 are basestable and acid labile.ar Fourth,the 3rPand r3CNMR spectra of o-3-aminopropane-1,2-diol- I -phosphate prepared chemically(eq 5) are distinct from thoseof the product from the ,*...,,J*^-*,,ir** HOH o NH3,Hzo Ho H o -fr? (5) 1oo o c,---h-,oilro-r, NHz--\.-oi,to-r, ppM Figure5. Proton-decoupledrrC NMR spectraof o-butane-1,2.4-triol- phosphorylationcatalyzedby glycerol kinase (Figures 3 and 4). | -phosphateand relatedspecies: (a) ol-butane-1.2,4-triol. (b) off-reso- Fifth, the o-3-aminopropane-1,2-diol-1-phosphateprepared nancespectrum of (a), (c) o-butane-I,2,4-triol- I -phosphateprepared by chemicallydoes not transfera phosphorylgroup to nitrogento phosphorvlationcatalyzed by glycerolkinase, (d) off-resonancedecoupled form o-3-aminopropane-1,2-diol-3-phosphate in aqueoussolutions spectrumof-(c). (pH l-14, ambienttemperature). The phosphoramidateisolated from the enzymaticreaction is thus not formed by rearrangement TableII. Lstinratcsrri [--nantionrericand ChemicalPurities of the ( TOTPXCHTCYZR. of an initially formed phosphateester. .\nalogucsol' rrr-(ilrr,crul-.1-phtrsphirtL cq I) Preparedby Cirecrr)l Klna\c ( atalrzcd Phosphorr.lation as Phosphorylationof ot--3-mercaptopropane-1,2-diol was con- Determined*rth (jirccrtrl--i-phosphatcDchrdrogcnasc (GDH. eq 6)d firmed to be on the C-l hydroxyl group by ''C NMR spectroscopy: 'rP and Quantitativc \ \1R Spectrorcopr^ in particular,the two-bondP-C couplingconstant was that ex- assa y' pected('"/poc = 3.J H.).0' In addition, this substanceoxidized rrP to the correspondingdisulfide on standing overnight in solution GDH(%,)" NMR(%)b at pH l3 in contactwith air. Addition of sodiumborohydride OH OH cH20H >97 >95 to a solutionof the disulfide regeneratedthe thiol quantitatively cH2cl >97 >95 (as observedby both r3C and 3rPNMR spectroscopy). CH2Br 95 >95 The siteof phosphorylationof or--butane-1,2.4-triol was assigned cH:ocH, 94' >95. as C-l on the basisof its l3C spectra(Figure 5). The most c'HrsH 9-5d' >95" >91 >95 importantobservation is that of a three-bondP{ couplingto the \H. ('lJ:('H:()ll >95 carbon atom bearing the secondaryhydroxyl group expectedfor I phosphorylationat C-1 ('/pocc = 9.2 Hz). "Thc enriln;;t; n..,t^ rl ,I*' by enzynraticassa\ usrng sn-glr cerol--1-phosphatc dchrdrogenase. The The site of phosphorylationof ot -butane-I,2,3-triol was assigned ^ assayconditions are describedin thc L.rperimentalSection. The to the C- I hydroxylon the basisof 3rPspectroscopy; a triplet is 3rP l3C chemicalpurity of the organic phosphateswas determined b1'quanti- observedin the proton-coupled NMR spectrum. The tative rrP NMR. Sodium phosphatewas usedas the internalstandard. NMR spectraconfirmed that the phosphorylationtook placeat The recordingparameters are describedin the E,xperimentalSection. C- l; in particular, the two-bond P-C coupling constantwas that 'lnorganic phosphate(4%) was observedby quantitativelrP NMR. expected(t/ooc = 3.7 Hz). dDithiothreitol(DTT, I mM) was added to the assay. "lnorganic The assignmentof structuresto other compoundslisted in Table phosphate(2%\ wasobserved by quantitative3rP NMR. /o-Butane- I is basedon similar, althoughless complex, 3rP and l3C NMR 1.2,4-triol-I -phosphate was not a substratefor .rn-glycerol-3-phosphate data. Thesedata are summarizedin the ExperimentalSection dehydrogenase (TableIV). having thc opposite cont'rgurationat C-2 is well documented.a3'45 Assignmentof AbsoluteConfiguration. Assignmentof absolute The assar is carricd out in the prcsenceof hydrazine in order to configurationto the productsobtained from phosphorylations drive the cquilibrium 1eq6 ).r" catalyzedby glycerolkinase was basedon activity of thesesub- stancesas substratesfor glycerol-3-phosphatedehydrogenase (8.C. H, ,N 1.1.1.8.sn-glycerol-3-phosphate: NAD 2-oxidoreductase).This H0 .H O GDH,NAD N O assavdepends upon the stereospecificoxidation of sn-glycerol- t.' ; .-r-- tt i HO OP(O-L HO u Op(O-) (6) 3-phosphateand structuralanalogues (o-3-fluoropropane-1,2- _* _., _, * _ _ 22 diol-I -phosphate)to dihydroxyacetonephosphate (and ana- logues).al'44The lack of substrateactivity of the enantiomer Our results are summarized in Table IL The close agreement between estimatesof enantiomeric and chemical purities of the samples indicates high enantioselectivityfor the reactions. For (38) Horner,L.; Gehring,R. PhosphorusSulfur 1981,1 l. 151-76. example, for 3-chloropropane-1,2-diol- I -phosphate, )97% of the (39) Under theseconditions no couplingwas observedfor the carbon was by NAD/GDH, and is, as a result, assigned attachedto the primaryhydroxyl group. sample oxidized (40) The half-livesfor nl- 3-aminopropane-1,2-diol- 3-phosphate at various the structure D-3-chloropropane-I ,2-diol- I -phosphate1')957o of pH areas follows (pH, l'72.6): 7.0,5; 8.0, 18;9.0, 5l; 10.0,336;11.0,960; the sample contained,by ''P NMR, 3-chloropropane-1,2-diol-l- 12.0.>5000. (41) Fluck,E.; Haubold,W. In "OrganicPhosphorus Compounds"; Ko- solapoff,G. M., Maier,L. Eds.;Wiley-lnterscience: New York, 1973;Vol. (43) Michal,G.; l-ang,G. In "Methodsof EnzymaticAnalysis".2nd ed.: 6. Chapter16, pp 580-831. Bergmeyer,H. Li.,Ed.; Verlag Chemie: Weinheim, 1974; Vol. 3, pp l4l5-8. (42) McFarlane,W.; Proc.R. Soc.London, Ser. A 1968,306,185-99. (44) Ghangas.G. C.; Fondy,T. P. Biochemistry'1971, 10,3204-10. Curzon.E. H.; Hawkes,G. E.;Randall. E. W.; Britton,H. G.; Fazakerley, (45) Fondy,T. P.: Pero,R. W.; Karker,K. L.; Ghangas,G. S.; Batzold, G. V. J. Chem.Soc., Perkin Trans. 2 1981.494-9. For reference,the coupling F. H. J. Med. Chem.1974. 17.69'7-702. constantsfor.rn-glycerol-3-phosphate are respectively(Hz): :../6ep= 5.-5, (46) Fondy,T. P.; Ghangas,G. S.; Reza,M. J. Biochemisty1970,9, t"/..op= 7.3(pH -l):2Jcop = 3'5,r"/('('op = 6.2(pH -12)' 3272-80. 7014 J. Am. Chem.Soc.. Vol. 107. Nct.24. 1985 Crans and Whitesides

Trble IIl, ApparentValues of K. and RelativeValues of Z** for Phosphorylationof CompoundsHaving the StructureXCHTCYHR Catalyzed by GlycerolKinaseo

C. myco S. cere E. coli B. stearo.

K. (mM) v^ *b (Vo) K. (mM) V^^^'(vn) K. (mM) V^^*D(Vo) K. (mM) V^u^b(7o) OH OH cH2oH 0.044 100 0.041 100 0.042 100 0.054 r00 0.049 t0'7, 0.047 I 07' cH2cl 8.0 84 t2.9 r03 5.7 12 1.2 87 't4 CH2Br 5.5 66 t2.1 87 8.7 74 3.4 cH2NH3* d 5.0 30 2.2 43 2.9 65 1.4 38 cH2NH2" t.l 30 CH25H t.2 97 1.4 il0 z. -) l0l 2.1 95 cH2ocH3 1.7 104 4.0 95 2.9 104 1.5 92 a 86 0.5 51 I 21 0.5 80 J cH2cH20H 6.4 2 1.6 0.6 H l8 0.04 'Reaclions oC. w€re carried out under theseconditions unless indicated otherwise: pH 7.6, 25 [ATP] = 5 mM, [Mg'?+]= 15.6mM, [tri- ethanolaminel= 50 mM, INADH] = 0.16mM, [K+] = 1.03mM, pyruvatckinasc = 6 U/mL, andlactate dehydrogenase = 12 U/mL. Boththe (m and ,/sii valuesare in mo6tcases reproducible within | 0%. In the caseof DL-l-ch loropropane- I ,2-diol,DL- 3-bromopropane- | ,2-diol and the weak substrates,the (m and ,/i,r valuesare reproduciblewithin 20?. 6valuesof I/.!, are relativeto ,/**{ry--r = 100as determined by.eduction of NAD ar pH 9.8 and 37 "C catalyzedby rn-Slycerol-3-phosphatedchldrogenase- Absolurc values of l/.,*,sly--l obtained(as describedin footnoted) were: 0.50OforC.mycodermo,0.203lotS.cereLisia?.0.823forE.coli.andO.49lforB.stearothermophilus.'IATP]=lmM.dThepredominantform of the aminegroup at pH 7.6 is indicated(p,(" = 9.4). "pH 9.5tThe triethanolaminebuffer (50 mM) wassubstituted with glycinebuffer (50 mM). Assayconditions are otherwiseas describedin footnotea.

phosphate(configuration undetermined). Thesetwo numbers agree within experimentalerror and suggestthat the sample containsonly one (*-ZVo) enantiomerof the organicphosphate. (D HOH\,/ HSXOH Most of the organic phosphatesexamined were substratesfor o \,/ v glycerol-3-phosphatedehydrogenase and were, as a result,assumed n Q ns, to havethe o configuration. In two cases(3-chloropropane-L,2- '= 8o o diol-I -phosphateand 3-aminopropane-1,2-diol-3-phosphate), we ao n o ao o c) a o establishedexplicitly that the opposite(presumably r-) enantiomer a 6O - was not acceptedby glycerol-3-phosphatedehydrogenase by' aa o m t a demonstratingthat a racemicmixture of l and o enantiomerswas o.o I oxidizedonly to the extentof 50% by the enzyme. In one case roo 200 300 (o-butane-I ,2,4-triol-I -phosphate)the organicphosphate was not v/[s] a substratefor glycerol-3-phosphatedehydrogenase and enan- tiomeric pure triols were preparedfrom >' and r--malicacid. Only t.o o-butane-l,z,4-triol derived from o-malic acid was phosphorylated c) in the reaction catalyzeAby glycerol kinase. We have not explicitly + o tr examinedthe absoluteconfiguration of phosphorylatedproducts *r.o{o, listed in Table I but not listed in Table IL We assume.however, G) tr that all fall in the samestereochemical series.aT UN o o."o Kinetics. The mechanismof phosphorylationof glycerolcat- oo (D ol alyzedby glycerolkinase from rat liver is believedto follow an o a oo ordered bi-bi mechanismin which glycerol binds first to the a enzyme,followed by ATP.Mg; sn-glycerol-3-phosphateleaves oo last.r2The patternof productinhibition observed in glycerolkinase o roo 200 300 from f. coli suggestsa similar orderedmechanism.r2 Although straightforward in many details,the phosphorylationof glycerol v/[s] hasone peculiarity: previousexaminations have generated biphasic Figure6. Eadie-Hofsteeplots for phosphorylationof oL-3-mercapto- doublereciprocal plots, with the implicationof different values propane-1,2-diol(upper) and ol-J-methoxypropane-1,2-diol(lower) of K* for ATP at low and high concentrationsof 41p.t2'2s A catalyzedby glycerolkinase. The sources of theglycerol kinase used in (O), (a), relatedeffect has recentlybeen observed with glycerolitself with theseexperiments were from C. ntye'oderma S. c'ererisiae B. stearothermophilus(O), andE. coli (tr). glycerolkinase from adiposetissues.a8 The origin of thesekinetic peculiaritieshas not beenestablished in detail, but they havebeen ferencesin kinetic behavioramong theseenzymes which could suggestedto be due to two typesof sitesthat differ in their affinity be usedto advantagein organicsynthetic procedures. In fact, for ATP.r2 We havechosen conditions such that the ATP con- the valuesobserved for the different enzymeswere very similar centrationused is higher than the highestreported value of K..4e (Table III). We thereforeconclude that the availabilityof Michaelis-Menten parameterswhich are not limited by cofactor enzymesfrom differentmicrobial sources provides no advantage concentrationsare more relevantfor use in enzyme-catalyzed in this particularsystem, and that the mostattractive enzyme for organicsynthesis than thoseobtained using dilute reagents.Under most syntheticapplications is simply the leastexpensive, thesecircumstances, we observedgood linear behaviorin Ea- Severalfeatures of thesekinetic data dcservebrief comment. die-Hofsteeplots (Figure 6). We havecompared the kinetics First, Clelandand co-workershave observed for glycerolkinase observedfor phosphorylationof glyceroland severalof its ana- from C. mycoderntathat K* for MgATP changedfrom 0.009 to loguesusing glycerol kinase from four different microbialsources. 0.02mM whenglycerol was replaced by dihydroxyacetone.2r's0 We hopedin making this comparisonthat we might detectdif- We determinedK. tor ATP usingglycerol kinase from E. coli and pl-3-chloropropane-1,2-diolassubstrate (Kn, = 0.5mM). The (47) Prelog,U. PureAppl. Chem.1964,9, 119-30. similaritybetween this valueand that observedfor glycerol(K. (48) Barrera,L. A.; Ho. R.-J.Biochem. Biophys. Res. Commun.1979,86, t45-52. (49) The highestK,lor ATP is reportedfor glycerolkinase from E. c'oli. (50) The K, for ATP for the followingsubstrates are: l-glyceraldehyde. Thevalues are in theranse 0.1-4 mM. Seeref 12 and25. 0.04 mM; o-glyceraldehyde.0.007 mM; and o-propane-1,2-diol,0.1 mM. Glvcerol Kinase: Substrate Specificity J. Am. Chem.Soc., Vol. 107,.\o. :1, 198-\ 7015

Y= OH I Z= H tionssufficiently efficient to generate100-g quantitics of product; FI thoseclassified as r -- ** can be phosphorylatedon l0-g scale HD _\3H. without major difficulty, and thoseclassified as reactivit\ r = * _/ YZ\ are not practicalfor syntheticuses. Practicalillustrations giving \.s experimentaldetails for procedureson the largerscales are sum- CO marizedin the accompanyingpaper.r' The examplesin this paper '/ \ are restrictedto smallscales (<5-g), but the ratesgiven in Table crr, oto;l I correlatewith practicalsynthetic experience. The group classified 3=,$moll, , X=O r = * comprisessubstrates from three groups. In the first group preferoblypolor group NH (e.g.,ol-propane- 1,2-diol and ol-3-fluoropropane-1,2-diol), no (CHzOH,CH2Cl, etc.) substrateactivity is observedfor a oL mixture becauseweak Figure7. Structuralcharacteristics of substrates for glycerolkinase. activity of the oL enantiomerwas suppressedby inhibition due to the L enantiomer.In the secondgroup (e.g.,acetol and ol- = 0.3 mM) suggeststhat such effectsare not likely to be large glycericacid), the substrateinduces significant ATPase activity enoughto influenceour data. (>40% of total activity) in glycerolkinase concurrently with The substratesincluded in Table III cover the range from the substrate phosphorylation. In the third group (e.9., DL-l/- most activeobserved in Table I to thosewith relatively low activity acetyl-3-aminopropane-1,2-diol and glycidol),the substrateand (ol-butane-1,2,4-trioland ethyleneglycol). In going from highly its hydrolysisproduct generate a mixture in which phosphorylation reactivesubstrates to inactive substrates,the valuesof K- increase catalyzed by glycerol kinases produces nonspecific mixtures. from 0.04to 100mM, while thevaluesof V^u*decreaseby ap- Practicalexamples of synthesesare summarizedin an accom- = proximatelya factor of 1000. The increasein K,n is not a matter panyingpaper.rr The group classifiedr 0 includesthe crude of great syntheticconcern; the enzymeis still active in highly preparationsof compoundsdescribed in the Experimental Section concentratedsolutions of organic substrates,and the rates of which contain impurities. It is, of course,possible that small conversionare only greatly reducedat concentrationsconsiderably quantitiesof adventitiousimpurities in the crude preparations lower than K.. The increase in K. can, however, become a testedfor substrateactivity act as inhibitors. ln the caseof rH problem for the few racemicsubstrates (ot--propane- l .2-diol and ot--,N-methylpropane-1.2-diol.bascd on the NMR spectrum, ot--3-fluoropropane-1,2-diol),where the enantiomerthat is not we estimatethc anrountol'inrpuritl to be lessthan l0'7: fbr other a substrateinhibits the enzyme. The decreasein 2.,. is of more compoundslo cl: of inrpuritics(unlc:s othcrrr isc indicatcd ) were importancefor syntheticuses; this decreasecorresponds to a renl loue r. decreasein the fastestpossible rate of reactionand necessitates .'\nintcrestrng rc:ull that cnrcrgc:I-rorn thr: *ork is the ob- largerquantities of enzymeto maintainuseful reaction rates. It servationthat hrghlr:clcctrr c phtr:phtrrrlation on nitrogencan beachiered in ot --l-enrinopropanc-l.l-drol. Thi. :ubstrateactivity is, thus, ultimately the valuesof V^ * which limit the utility of this enzymeas a catalyst for practical-scalesynthesis with weak was not duplicatedb1 otherstructuralll analogous amines al- -methoxl substrates. thoughot--3-amino- I propanc-l-olinduced ..\TPase ac- tivity.s2 The interestin this observationis lessin thc utility of Conclusions the reactionitself than in the implicationthat an enz\me may The data summarized in Table I permit the hypothesisof a haveuseful activity under nonphysiologicalconditions *'hich is usefully detailed model summarizing the structural requirements masked under physiologicalconditions by protonationof nu- for acceptabilityas a substratewith glycerol kinase (Figure 7). cleophiliccenters. The position to which phosphatetransfer occurscan be either a ln summary,this study bearson two concerns. First, it es- CH2OH group or a CH2NHT group, since it appearsthat the tablishesthat the range of structureswhich are in substratesfor enzyme is reluctant to accept groups other than hydrogen on the glycerol kinaseis wider than has been suggestedin previouswork. carbon attached to the phosphorylatednucleophile. No secondary In particular,the substrateactivity of or--3-chloropropane-1,2-diol hydroxyl group was phosphorylated.Compounds with multiple is relevantto the antifertility activity of this substance.34'35This sites-if substrates-orient themselvessuch that the primary work alsosuggests that glycerolkinase should be a usefulcatalyst hydroxyl group is phosphorylated. The group Y should clearly in syntheticorganic chemistry. It is possiblein one stepto prepare be a hydroxyl group for highest activity. Substitution of hydroxyl a number of phosphorylated,chiral analoguesof glycerolfrom by fluorine or hydrogenleads to poor substrates;substitution by the racemicmixtures. Thesematerials should find usein synthesis NH2 or NHI* eliminatessub'strate activity. The rangeof variation of phosphorylatedorganic substances(especially analogues of accepted in the group Z is dependenton the enzyme source. phospholipidsrs). Hydrogen is obviously acceptable;the observationthat di- Experimental Section hydroxyacetonephosphate reacts (probably in the hydrated form) Materials.Glycerol kinase from E. coli wasobtained as lyophilized suggeststhat OH is also acceptablefor enzyme from all four powderfrom Sigma. Glycerol kinase from C. mycodermawas obtained sources.The activity of ol-2-methylpropane-1,2,3-trioland 2- asa crystallinesuspension in ammonium sulfate from Sigma and from (hydroxymethyl)butane-1,2-diol (2-ethylpropane- 1,2, 3-triol) with BoehringerMannheim. No differenceswere observed in the kinetic glycerol kinase from C. mycoderma suggeststhat small alkyl parametersfor the C. mycodermaenzyme from thesetwo sources. groupsare acceptablefor this enzyme,whereas even small alkyl Glycerolkinase from B. stearothermophiluswas obtained as a solution groups are too large for glycerol kinase from both E. coli and S. in Tris buffer from BoehringerMannheim. Glycerolkinase from S. cereuisiae. Other groups have not been systematicallyexamined. cereuisiaewas obtained from Genzyme.Other enzymes and biochemicals The enzymewill accepta significant amount of variation in the wereobtained from Sigma. All enzymeswere used as received; absolute puritieswere werereagent grade group R, although small, preferablypolar groupslead to highest notestablished. Chemicals andused withoutfurther purification unless otherwise indicated. ol-3-Chloro- rates. Variations are observedin substrateactivities for different propane-1,2-diol and ol-3-bromopropane- 1.2-diol were distilled before enzyme sour@s;the enzymesfrom S. cereuisiaeand C. mycoderma usefor kineticmeasurements (distillation was not necessary for thellP acceptlarger substituentsthan the enzymesfrom E. coli and B. NMR assays);aqueous solutions of or-3-chloropropane-1,2-dioland st e arot he rmophi I us.sl The rangeof ratessummarized in Table I is approximatelyl0a (52) in phosphorylation from reactivity r = *** to reactivity r -- *. Compoundsshowing The activityof o-6-amino-6-deoxyglucose cata- hexokinasewas a modification = lyzedby examinedby of the assaydescribed reactivity r *** providethe basisfor enzymatictransforma- in the ExperimentalSection; glycerol kinase was substituted with hexokinase from yeast. After 24 h in a signalat 9 ppm wasobserved in the rrP NMR spectrum. The reactivitywas lessthan 20 times the ATPase activity of (5 I ) ot--3-Thiomethylpropane-1,2-diolshowed substrate activity with gly- hexokinasefrom yeast(as determinedby integrationof the 3rPNMR peak cerol kinasefrom C. mycoderma,whereas no activity was observedfor glycerol heightsof the phosphoramidateand the inorganicphosphate produced in the kinasefrom ^8.stearothermophilus. reaction).No isolationswere carried out. 7016 J. Am. Chem. Soc., Vol. 107, No. 24, 1985 Crans and Whitesides oL-3-bromopropane-1,2-diolwere freshly preparedbefore use. Water deuteriumoxide (pH 7-9) usinga pulseangle of 90o and a pulsedelay was distilledtwice. the secondtime from glass. of 2 min.5a The data were processedtwice and the averagewas reported. Methods. Spectrophotometricmeasurements were performed at 25 A representativesample solution was prepared as follows: n-3-thio- -phosphate "C usinga Perkin-ElmerModel 552 spectrophotometerequipped with methylpropane-I ,2-diol-I barium salt ( I 24 mg) was dissolved a constant-temperaturecell. PhosphorusNMR spectroscopywas rou- in water (3 mL) by additionof excesssodium sulfate (150 mg). The tinelydone on a 40.48-MHt (23-T) Varian instrumentin l2-mm NMR precipitated barium sulfate was removed by centrifugation, and the tubes. Samplescontained 20Va deuterium oxide as internalNMR lock. precipitatewas washedwith deuteriumoxide (0.8 mL). After the pre- Chemical shifts for phosphorus-containingcompounds were reported as cipitate was removedfrom the deuterium oxide, the supernatantand the 3rP positiveupfield relativeto external85% phosphoricacid; the temperature deuteriumoxide wash were placedin a l2-mm NMR tube and a was 30 oC unlessnoted otherwise. Accumulation parametersused for NMR spectrumwas recorded. The data were processedtwice (without assayswere: pulseangle. 2lo; pulsedelay, 1.5 s; accumulationtime, 1.02 sensitivity enhancement)and the average was reported. An internal rlP (in s. Quantitative NMR measurementswere carriedout using a 90o standard,inorganic phosphate,was added the caseof ol-3-thio- pulseangle and a 2-min pulsedelay, and an internalstandard of known ethylpropane-1,2-diol-l-phosphate,0.5mL of 0.4 M monosodiumphos- rrP concentration(sodium phosphate). Carbon-13 NMR spectroscopywas phate solution) and another NMR spectrumwas recorded. The 3rP routinelydone on a 67.8-MHz (63 T) Jeol spectrometerin a 5-mm NMR intensitiesin the NMR spectrumwere measuredand the purity of tube. Samples( 100-200mg/mL) were dissolvedin deuteriumoxide and the organicphosphate was calculated based on the inorganicphosphate rrC chemical shifts were reported relative to external sodium 2,z-di- used as internal standard. The inorganicphosphate used as internal methyl-2-silapentane-5-sulfonate(DSS). The spectrawere completely standard had previouslybeen calibrated against or--glycerol-3-phos- proton decoupledand the accumulationparameters used for the spectra phate.55 The sensitivity'of the method was examinedby adding in- were: pulseangle,90o; pulse delay, 1.45 s; accumulationtime,0.55 s. creasingamounts of inorganicphosphate to a samplesolution containing Mass spectra were recorded on an AEI MS9 mass spectrometer at knoun amountsof ot--glycerol-3-phosphatedisodium salt. This assaywas 50-100'c. able to detect inorganic phosphateat a concentrationequal to 2 mol Vo 'rP I'P NMR Assayfor SubstrateActivity with Glycerol Kinase. The of the major phosphatecomponent (ot--glycerol-3-phosphate). Impurities NMR assaycarried out in a l2-mm NMR tube couldconrcnientlr *ere thcreforedetected at the levelof2 mol % but the overallaccuracy observeI to l0 i.tnrolof phosphorus-containingcompound. Thc bufier ol the quantitativedeterminations was not higher than 5Vo. lor the riP \\{R assal at pl{ l.-i uas prcparedas folloirs. Tri- Kinetic f)eterminations. Initial rates of enzymatic reactionswere all ethanolaminehydrochloride 1-s.57 g. -j0mmol). magnesium chloride t0 l5 deternrrnedbv UV spectroscopyat 25 "C. The phosphorylgroup transfer g, 1.2-smmol), PEP trisodiumsalt (1.3ag.6.5 mmol).and.'\TP tri- from ATP to substratewas coupledto the oxidationof NADH using sodiumsalt (0.39g,0.65 mmol) *ere dissolvedin uater (100 mL) and phosphoenolplruvate,pyruvate kinase (8.C. 2.1.1.40),and t--lacticde- the pH was adjustedto 7.5 with 2 M NaOH. The rrP NMR assal hy'drogenase(E.C. 1.1.1.27).The formationof NAD (or the disap- carriedout at high pH (when substratescontain amine groups)used a pearanceof NADH) was monitoredat 340 nm. Solutionscontained modified buffer in which the triethanolamine hydrochloride was replaced triethanolaminehydrochloride (50 mM, pH 7.6), ATP disodiumsalt (5.0 by glycine (2.25 g,30 mmol) and the pH was adjustedto 9.5. mM), NADH (0.16mM), PEP trisodiumsalt (3.04mM), magnesium A representativeassay for oL-3-mercaptopropane-1,2-diol followed this sulfate(15.6 mM), potassiumchloride (1.04 mM), pyruvatekinase (20 course. Triethanolaminebuffer (0.3 M, 3.2 mL) containingmagnesium U), t--lacticdehydrogenase (40 U), and 0.01-l U of glycerolkinase'2i chloride(12.5 mM), PEP (65 mM), and ATP (6.5 mM) wasadded to The total assayvolume was 2.5 mL. The measurementsof rates of deuteriumoxide (0.7 mL). or--3-Mercaptopropane-1,2-diol(0.I mL, 0.8 reaction for glycerol analogueswere done at the following substrate mmol) was added to the buffer solution and generateda substratecon- concentrations:0.5, 1,2,3, 5, 10, 15,and 20 mM. In two cases(pro- centrationof 0.25 M. The assaywas started by addition of pyruvate pane-1,3-dioland glycerol).the K. valueswere significantlydifferent kinase(l U) and glycerolkinase (5 U), and was left for 24 h at room from thoseof the other glycerolanalogues. The substrateconcentrations temperature.The 3rPNMR chemicalshifts of the assaysolution without were varied from l0 to 500 mM for propane-1,3-dioland from 0.01 to -10 -19 substrateare: ATP t-5 (d), (d), (t)1,PEP [-1 (s)]. After 24 0.2 mM for glvcerol. ATP and the magnesiumion were presentin excess trP h the followingchemical shifts were observed in the NMR spcctrum at all times. Reactionswere startedby additionof substrate.Control -10 of the ol-3-mercaptopropane-1.2-diolassar solution: ADP [-5 (d). crperirncntsuerc carriedout to ensurethat the rate-limitingstep was (d)], the organic phosphateproduced b1 phosphorllationoi or-3- that catallzedbr glrcerolkinase. The glycerolanalogues had no effect ''l-.*, mercaptopropane-l.l-diol[-] 5 (t. = 6.-1Hz)1. For reference.thc on thc ratesot'reaction of pyruvatekinase and t.-lacticdehydrogenase; 3rPchemical shift oi inorganicphosphatc uas l.-i ppnr. rrtcs (rf rcactiondid not changeby the additionof glycerolanalogues in Purity. The enantiomericpuritr oi the isolatedniatcrials iras deter- .r\\.r\s in uhich glyceroland glycerolkinase had beensubstituted with minedby quantitativeenzymatic analysis. The chemicalpuritl of the glucoseand hexokinase.A standardassay for glycerolkinase activity organicphosphates *cre determinedby quantitativerrP \ \{R. The using 0..1m\{ gl1'cerolas substratewas performedbefore and after each characterizationsuere carried out as follows. scriesof crperiments. One unit of glycerolkinase activity is definedas EnzymaticAnalysis. The procedurefor quantitativedeterminations the anrountof enzy'me that is requiredto catalyzethe transformationof of .sn-gly'cerol-3-phosphatewas basedon the glycerol-3-phosphatede- I pmol ol glvcerolper min to sn-glycerol-3-phosphateat pH 9.8 and 37 oC. hydrogenasccatalyzed reduction of NAD. Since most of the sn- The commerciallyavailable glycerol kinase from E. coli wasassayed glycerol-3-phosphateanalogues are substratesfor glycerol-3-phosphate accordingto the aboveprocedure.56 Assay of glycerolkinase from other dehydrogenase,the procedurefor assayof sn-glycerol-3-phosphatewas sourceslollowed this standard. The assayconcentrations were as follows: expandedto include theseanalogues. The assaywas carried out as hydrazine(0.88 M), glycine(0.18 M), magnesiumchloride (0.25 mM), describedpreviously' with minor modifications.43'45A representative assay ATP (1.4mM), NAD (0.25mM), glycerol(1.5 mM), and glycerol-3- for o-3-aminopropane-1,2-diol-3-phosphate follows. A buffer containing phosphatedehydrogenase (10 U). hydrazineand glycine(2.00 mL, 0.04 M hydrazine,0.5 M glycine,pH Glycerol Kinase Catalyzed Phosphorylationof Glycerol Analogues: 9.8) and NAD (0.1 mL,3l mM) was placedin a 3-mL cuvette.The General. The synthesesof phosphorylatedglycerol analogueswere car- cuvettewas equilibratedfor 2-4 min at 25 oC, glycerol-3-phosphate ried out on scalesfrom 0.2 to I mmol using in situ ATP regencrationand dehydrogenase(20 pL,20 U) was added,and the absorbanceat 340 nm solubleenzymes. PEP was usedas the ultimate phosphorylgroup donor. was recorded(exnoH = 6220M-lcm l). When a steadyabsorbance level The glycerolkinase used for theseenzyme-catalyzed syntheses was from (baseline)had been reached,a small volume (0.1 mL) of the sample S. cerecisiaeor from E. coli. The syntheseswere performed under similar solutionwas addedto the assaysolution and the increasein absorbance conditionsfor all the sn-glycerol-3-phosphateanalogues. Threc repre- was recorded. Aiter 5 to l0 min no further increasein the absorbance sentative syntheses(n-3-chloropropane- I ,2-diol- I -phosphate,o-3- was observed. The samplesolution contained o-3-aminopropane-1,2- aminopropane-1,2-diol- 1-phosphate, and ethyleneglycol phosphate)il- diol-3-phosphatebarium salt (32 mg) dissolvedin water (25 mL). The lustratethe variationsin the reactions.Table IV summarizesthe data assa)sl\ere run in duplicates(or triplicatesif more than 3% variation for other sn-glycerol-3-phosphateanalogues. was observedin the first two determinations).The absorbanceat 340 o-3-Chloropropane-1,2-diol-l-phosphate.nl-3-Chloropropane- I .2-diol nnr was mcasuredagainst a blank,although this controlappeared not to (reagentgrade from Aldrich,0.l0 mL, 1.2mmol), PEP monopotassium be critical for the determinations.In the blank, water was substituted salt (83 mg, 0.40mmol), and ATP disodiumsalt (25 mg, 0.040mmol) for the samplesolution. weredissolved in a 0.1 M solutionof triethanolamine(3 mL) containing rrP Quantitatire NMR. The phosphorus-containingcompounds were magnesiumchloride (10 mM) and deuteriumoxidc (0.7 mL). The pH assayedquantitatively' by integrationof their lrP NMR signalsand comparisonof thc observedintensities with that of an internalstandard (54) Glonek.T.; Van Wazer,J. R. Phys.Chem. 1976. 80, 639-43. of knownconcentration.5l The 3lPNMR sDectrawere recorded in 20% "/. (55)Glonek.T. J. Am. Chem.Soc. 1976, 98,1090-2. (56)Glycerol kinase, Sigma, Product No. G-4509,Lot No. l0lF-6805. (53) Barany.M.; Glonek,T. MethodsEnzymol. 1982, 8J, 624-16. Assayand productare describedon a productchart. GlycerolKinase: SubstruteSpecificity J. Am. Chem.Soc., Vol. 107,No. 24, 1985 7Ol1

Trbl€IV. Summaryof IsolatedYields and Experimental Conditions for PhosphorylationCatalr'zed b\ (;lycerolKinase' product rrC NMR datad scale RTD GK'/PK speciaI (mmol) (days) (U/U) conditions '/r*. '/r*.. yield' (%) -2OoP OH cH20H 0.22 I 20110 7.3 95 H 0.47 6 1s0l2o Pi, 7.3 60 cH2cl 0.42 2 20lt0 1.3 96 CH2Br 0.53 2 201t0 6.1 95 11 ch2sH 0.61 2 I 00/20 Pi' t.-) 12 cH2ocH3 0.25 2 25120 Pi' t.J IJ A cH2scH3 0.35 t l 00i20 92 cH2ocH2cH3 0.23 A 251t0 88 ? cH2cH3 0.22 150120 P.', .S. r't're t.) 94 cH2cH2oH 0.48 200/r 00 9.2 96 CH2CN 0.10 100120 P,' 15 cH(cHr)oH 0.20 l soi r00 [,. t'rtlis 37 -5.5 87 ro.PN 0.29 100/30 pt{ 10.0/' 1.6 7.8 81 'The reactionwas carried out asdescribed for DL-3-chloropropane- I ,2-diol unless otherwise indicared lspecial conditions). 'The indicatedreaction times(RT) are upperlimits fo. completionof the reactions."The reactionswere carried out wilh glycerolkinase from S. rer€r,rideunless othe.wise indicaled. Similar resultsare obtainedwith glycerolkinase from E. ro1i. r'The productstructure indicared was verified by observingthe two- and three-bondcouplingconstanlsofth€cartrcnsaandptophosphorusintherrcsp€ctrum.'Theisolatedlieldsarecalculatedbasedonthetotal phosphorylgroup donorsadded in the reaction- /During the reactioninorganic phosphate formed. The inorganicphosphat€ was .emovedby precipitationas barium phosphatebefore the organicphosphate was isolated(as describedfor ethyle e glycol phosphate).ronly the indicated ghcerol kinasecatalyzed the formationof product. tThe reactionwas carried out at pH 10.0.

was adjustedto 7.5 and the reaction was started by addition of glycerol cipitatewas allowedto settlefor 30 min at 0 oC and centrifuged.The kinase(20 U) and pyruvatekinase (10 U). After 3 daysthe reactionwas supernatantwas decantedand the solid was washedonce with acetone complete;PEP and ATP were no longer observedin the 3rP NMR (20 mL). The resultingbarium ethyleneglycol phosphate (160 mg) was spectrum. The solutionwas passedthrough charcoal(0 I g) to remove driedover CaSO. at I torr overnight.As determinedby quantitativerrP ADP and enzymes.Barium chloride(0.2a g, 1.0mmol) and ethanol12-5 \MR. the puritr uas 8l'7 andcorresponded to a.,'ieldt-tf 60(7, based on mL, 95Vo)were added to the solution to precipitaterhe o--l-chloro- conrbincdphosphtrrr i uroup dontlrs Thc ntrrlorimpurit;. as dctermined propane-1,2-diol-l-phosphatebarium salt. After the soliduas removed b) t'P \\lR. \\ir\in,)rsilni. ph(r\phittc (li'l ): :tl tf.{R (D.O.pH 7.5) -a -i by centrifugationand decantingthe supernatant.the solid uas *a:hed o tl)SS)-1 I \) rni.I tli. I6: I rnr.I Fl):r'C \MR (D20,pH -.5); - oncewith ethanol(25 mL, 95Vo)and dried over CaSOoar I rorr o\cr- (DSS)6-Q r:./r,,,. =.r Hz).6J-(t/Po,,, =1.3H2):3lPNMR - 'J './1,,,, night. The white solid was977o pure as determinedby enzrmaric assa\ rD-O.pll it I rt ri = i r Ilzt and contained more than 95Vaorganic phosphate as determined b1 or-3-('hloropropane-1.2-diol-l-phosphate\\'rls prepared from epi- rrP quantitative NMR (no other phosphate-containingcompound u'as chlorohrdrinand pho'phorie.rcrd accordinp to the procedureof Zetzsche r'C observed). The yield basedon combined phosphorylgroup donors was andAeschlimann:'- \\1R (D:O.pti - tr; tDSS)74.2 (d,3J = tH 'iP 96Vo: NMR (DzO,pH -l) 6 (DSS) 3.95-4.15(m, I H),3.60-3.8,s 7.,1Hz).70.6 (d.:J = 3.1Hz)..19 9 (.): \MR (D:O.pH9.8) 6 4.4 (m,4 H); r3CNMR (D2O,pH -l) 6 (DSS)75.3 (d,3J = 7.3H2),i0.4 (t. r.r = 6.8 Hz). I'pNMR (d,2"r= 3.7Hz),49.9 (s); (DzO,pH g.2) 64.2(t,3J = 6.5 or--3-Aminopropane-1,2-diol-3-phosphate. Phosphorus oxychloride ( I .9 Hz). mL, 20 mmol) and ol-3-aminopropane-1,2-diol(1.9 g, 20 mmol) were o-3-Aminopropane-1,2-diol-3-phosphate.ol-3-Aminopropane- 1,2-diol dissolvedin acetonitrile (25 mL) and cooled in a salt water-ice bath. (0.10mL, 1.3mmol), PEP monopotassiumsalt (80 mg,0.39 mmol), and Triethylamine (2.0 g, 20 mmol) was addeddropwise to the solutionover ATP disodiumsalt (25 mg, 0.040 mmol) were dissolvedin 0.1 M glycine a period of 30 min. After being stirred for t h at 0 oC the solutionwas (4 mL) containingmagnesium chloride (10 mM) and deuteriumoxide slowly addedto an aqueoussolution of NaOH (25 mL, 2 M) at 0 'C. (0.7 mL). The pH was adjustedto 10.3and the reactionwas started by The reactionmixture was stirredfor 30 min at 0 oC and for an additional additionof glycerolkinase (100 U) and pyruvatekinase (30 U). The 2 h at ambient temperature. Barium chloride (2.4 g, l0 mmol) was reaction was complete after 2 days; the lrP NMR spectrum showedno addedto precipitatethe inorganicphosphate. The precipitatewas re- remaining PEP or ATP. The solutionwas passedthrough charcoal (0.1 movedby filtration. Additionalbarium chloride (2.4 e,l0 mmol) and g) to removeADP and enzymes.Barium chloride(0.12 g, 0.50 mmol) ethanol(75 ml-. 957.) wereadded to the filtrate to precipitatethe barium and ethanol(25 mL,957o) wereadded to precipitatethe organicphos- nl-3-aminopropane-I .2-diol-3-phosphate. The suspensionwas allowed oC phate. The suspensionwas stirred at 0 "C for 30 min and the solid was to settlefor 3 h at 0 beforethe solidwas separated by filtration and separatedby centrifugation. The supernatantwas discardedand the solid dried over KOH at I torr overnight.The isolatedsolid (3.2 g) was8,5% was washedonce with ice-cold ethanol (50 mL, 95Vo). The suspended pure barium ot.--l-anrinopropane-1,2-diol-3-phosphate (8.8 mmol,44% precipitatewas isolatedby centrifugationand the supernatantdiscarded. reactionyield) as determined by' quantitative 3rP NMR, and contained The solid was dried over CaSOaat I torr overnight. The resulting barium 45%,o-3-aminopropane- I .2-diol-3-phosphate as determinedby enzymatic o-3-aminopropane-1,2-diol-l-phosphate(20 mg, 0.38 mmol) was 98Vo assay:rF{ NMR (D,o. pH -12) 6 (DSS)3.7-3.8 (m, I H),3.50-3.65 pure as determined by enzymatic assayand contained more than 95% (m, 2 H). 2.7_s-:.9-s(m. I H): r'C NMR (D,O,pH -12) 6(DSS) 76.6 organic phosphateas determined by quantitative 3rP NMR (no other (d, r"r= 7.8Hz), 68.1(s). 19 I (d. 2J = 1.9Hz);3'P NMR (D2O,pH phosphate-containingcompound was observed). The yield based on -12) a 9.1(r, ]J = 8.1Hz). tH combinedphosphoryl group donorswas 87Vo: NMR (DzO, pH I l) o-3-Aminopropane-1.2-diol- 1-phosphate. o-3-Chloropropane- I ,2- 6 (DSS) 3.70-3.80(m, I H),3.50-3.65(m,2 H),2.75-2.90(m,2 H); diol-l-phosphatebarium salt (600 mg, 1.8mmol) wasadded to a water rrC NMR (DrO,pH ll) 6 (DSS)76.8 (d,3J = i.3 Hz),68.1(s), a9.0 solution(5 mL). and cooledin an ice bath. Dowex 50W-X8 wasadded IP @,2J = 1.6Hz); NMR (DzO,pH l1) 6 9.4 (t,3J = 8.9 Hz). until the organic phosphatedissolved (pH <2). The Dowex 50W-X8 EthyleneGlycol Phosphate. Ethylene glycol (0.25 mL, 0.45 mmol), beadsand the faint yellow color of the solutionwere removedby passing PEP monopotassiumsalt (150 mg,0.70 mmol), and ATP (60 mg,0.090 the solutionthrough charcoal. The Dowex 50W-X8 resinwas washed mmol) were dissolvedin 0.1 M triethanolamine (6 mL) containing by passingan additional 20 mL of water through the charcoal. The magnesiumchloride (10 mM) and deuteriumoxide (l mL). The pH was o-3-chloropropane-1.2-diol-l -phosphate solution was added dropwise to adjustedto 7.8 and solubleglycerol kinase(150 U) and pyruvate kinase a stirredsolution of ammonium hydroxide(25 mL) placedin an ice bath. (20 U) were added to the solution. After 2 days excessethylene glycol After the end of this addition the solutionwas stirred for 30 min at 0 oC, (0.25 mL, 0.45 mmol) was added to the reaction mixture, and after 4 the ice bath was removed,and the reactionsolution was stirredfor I h more days the reaction was complete; 3lP NMR spectrum showed no at ambient temperature. The excessammonia and water were removed remaining ATP or PEP. The solutionwas passedthrough charcoal (0.1 by evaporationat 40-50 oC and the resultingoil wasdissolved in 2 mL g) to removeADP and enzymes.Barium chloride(24 mg,0.l0 mmol) of water. Bariumchloride Q. a g, I .8 mmol) and ethanol(20 mL.95%) was added to the reaction mixture and most of the inorganic phosphate were added to precipitatethe barium o-3-chloropropane-1.2-diol-l- precipitated as barium phosphate. The precipitate was removed by phosphate.The suspensionwas stirred for 15 min at 0 oC, and the solid centrifugation, and then barium chloride (240 mg, 1.0 mmol) and 5 was separatedby centrifugation.The supernatantwas discarded.The volumes of ice-cold acetonewere added to the supernatant. The pre- solid was washedwith ethanol( l0 mL. 95%,\.and after centrifusation 7018 J. Am. Chem. Soc., Vol. 107, No. 24, 1985 Crans and Whitesides

the solid was dried over CaSOaat I torr overnight. The resultingbarium mixture was placedin an ice bath. Acroleindimethyl acetal(l 1.8mL, o-3-aminopropane-1.2-diol- 1-phosphate (0.50 g, 94Vo)was more than 100 mmol) was added to the solution,and the mixture was slowly 957o pure as determinedby quantitative 3rP NMR (no other phos- equilibratedto ambienttemperature (4-6 h). The solutionwas stirred phate-containingcompound was observed): r3C NMR (DuO,pH 13) 6 overnight. Sodium hydrosulfite(0.5 g) and magnesiumsilicate (Woelm, (DSS)76.8 (d. r/ = 5.5Hz),70.5 (d,2J = 3.7Hz),47.6 (s);3rP NMR Eschwege,6 g) were addedto the black solution,and the suspenslonwas (D:O. pH l0) 6 -1.5(1. U = 6.3 Hz). stirred for 20 min beforethe solidswere removedby filtration. This step A similar preparationwas carried out for ol-3-chloropropane-1,2- was repeateduntil all osmiumesters were removed and the filtrate was diol-l-phosphateon a l-mmol scale. The isolatedol-3-aminopropane- red brown. The solventand N-methylmorpholinewere removed by 1,2-diol-l-phosphatebarium salt (0.28g) was more than 95% pure (no evaporation.The residualvolatiles were removedovernight over CaSOo other phosphate-containingcompound was observed)as determinedby at I torr; a colorlessliquid (9.6 g) resulted. The liquid containedN- quantitative3rP NMR (88% reactionyield). methylmorpholine(-25Vo), but was usedwithout further purification to ot--S-Acetyl-3-mercaptopropane-1,2-diol. ol-3-Mercaptopropane- test substrateactivity with glycerolkinase: rH NMR (DzO) d (DSS) 4.3 = '3C 1,2-diol(l.l g, 10 mmol) wasdissolved in water(25 mL) and cooledin (d,"l 6.0Hz),3.65-3.70 (m,3 H), 3.61(s,3 H),3.62(s, 3 H); an ice bath. The solutionwas stirred for 30 min at 0 oC and acetic NMR (DzO) d (DSS) 109.3(s), 75.8 (s), 66.4 (s), 60.2 (s), 59.7 (s) anhydride(2.8 mL, 30 mmol) was slowly added to the solution. The (1V-methylmorpholine;rrC NMR (D:O) 6 (DSS) 69.1(s), 57.9 (s),48.2 mixture was stirred lor 2 h at 0 oC beforethe water, aceticacid, and (s)); massspectrum, mlz 136 (M), 135 (M - 1), 104 (M - CH2OH- residualacetic anhydride were removedby evaporationat I torr. (The (ocHr)). t0s (M - I - cH2oH(ocH3)), 75 (M - CH2OHCHOH), 3l temperatureof the solutionmust not exceed20 oC during thesemanip- (CHTOH). The rH NMR spectrumis in accordwith a partialspectrum ulationssince acetvl migration occurs verl readih'.) The colorlessliquid publishedpreviously.6r (0.65g, 43% reactionyield) was usedwithout further purifications:IR or-3-Amino-l-chloropropan-2-olHydrochloride. nl-3-Amino-l- (neat) 1690 (S-C:O); rH NMR (acetone-du)D 3.6-3.7(m, I H), chloropropan-2-olwas preparedby acidichydrolysis of l-benzalimino- 3.4-3.5(m, 2 H), 2.85-3.1(m, 2 H), 2.3 (s, 3 H); r3CNMR (acetone-d6) 3-chloropropan-2-olaccording to the procedureof Paul et al.:62 mp 'H 196.1(s), 70.4 (s),64.3 (s), 31.5 (s),29.9 (s). The'H NMR and IR data l0l-102 "c ilit.62l0l-102 "cl; NMR (D2o,pH 7) 6 (DSS)3.9-4.2 correspondto thoseof o-S-acetyl-3-mercaptopropane-I,2-diol prepared (m, 1 H), 3.50-3.65(m, 2 H)8 3.0-3.2(m, 2 H): rrC NMR (D2O,pH from o- 1,2-isopropylidene-3-S-acetyl-3-mercaptopropane-1,2-diol.57 On 7) 6 (DSS) 71.7(s), sO-ss (s), 46.6 (s) oC storageat 4 the acetyl group migratesfrom sulfur to oxygenover the or-3.4-Dihydroxybutanonitrilewas prepared from ot--3-chloro- courseof 2 months. No thioesterbond was observedat the end of this propane-l .2-diol by nucleophilicsubstitution of the chloridewith sodium time. cyanide.6r ot -J-Chloropropane-1,2-diol (2 2 g,20 mmol) wasdissolved ol-f/-Methyl-3-aminopropane-1,2-diol Hydrobromide. The DL- in water(-r mt ) and cooledin an ice bath. Sodiumcyanide (1.0 g,2l methyl-3-aminopropane-1,2-diol hydrobromide was preparedby a mod- mmol) w'asadded to the solutionand stirred for 3 h at 0 oC. The ificationof the procedureof Knorr and Knorr.58 or.-3-Bromopropane- rcaction mlxture was passedthrough an ion-exchangecolumn (AG 1,2-diol(0.a5 g,3.2 mmol) wasdissolved in ethanol(5 mL.9-s?). The -i0l\\'-X8.60 g of'resin)and washedwith ice-coldmethanol (100 mL). ethanol mixture was slowly added to a vigorouslr stirred solutionof The cornbincdfractions were evaporatedto dryness. The resultingoil aqueousmethylamine (404..5.0g) u'hich had beenplaced in an icebath. (l -1g)uas uscdu'ithout further purification: IR (neat) 2260(C N); rH After l5 min of stirringat 0 oC. thesolution uas heatedior -i h at;10-50 \\{R (D.O) , (DSS)3.2-3.6 (m,3 H),2.5-2.7(m,2 H): IrC NMR oC. The solventand excessnrethrlamine \\erc removedb1 rotarr (DrO) (DSS) 123.9(s). 71.9 (s), 68.7 (s), 26.4 (s); mass spectrum, mf z evaporation.Residual volatiles \\'ere removed at I torr over KOH re- 102(M + 1).l0l (M), 84(M + r -H2O), 70 (72)(M + I -CH2OH), sultingin 0.35 g of colorlessoil. The or-,\-methyl-3-aminopropane- 6r (M - CH2CN),4l (M + r - CHOHCH2OH), 31 (CH2OH). The 1,2-diolhydrobromidc was usedwithout further purification: rH NMR spectraldata correspondto thosereported by Jung and Shaw.6a (DzO)6 (DSS) 3.9-4.2(m, I H), 3.6-3.7(m,2 H), 3.1-2.3(m.2 H), ot--3-Amino-l-methoxypropan-2-olHydrochloride. nl-3-Amino-l - 2.72(s, 3 H). methoxypropan-2-olwas prepared in two steps. First, acid-catalyzed DL-N,lg-Dimethyl-3-aminopropane-1,2-diolhydrobromide was pre- methanolysisof epichlorohydrinformed ot--3-chloro-I -methoxypropan- parcd by a modificationof the procedureof Knorr and Knorr.58 or--3- 2-ol.6s Second.nucleophilic displacement of the chlorideby ammonia Bromopropane-1,2-diol(0.59 g,3.8 mmol) wasdissolved in ethanol(5 producedot-- 3-amino- I -methoxypropan-2-ol.ot-- Epichlorohydrin (0.9 3 mL). The ethanolsolution was added to a stirredsolution ol aqueous g. l0 mmol) uas dissolvedin dry methanol(10 mL), and cation-exchange dimethllaminc(5.0 g). The mixtureuas heatedfor 6 h at:10 50 oC rcsin(Do*'cr 50W-X8. 100mg) wasadded to the mixture. The solution The solrentand excessdinrethrlamine ucre rentovedbr rotarr cvapo- urrs hcatedat -\0 oC for 1,5h. The solutionwas filtered to removethe ration.and residualrolatiles \\ere removed at I torr overKOH o\ernight. cati()n-c\changeresin, and the solventwas removed by evaporation.The The crude ot -.\. \ -dimethrl--l-aminopropane- I .l-diol hr drobrontide rcsultingoil wasdissolved in water(5 mL) and this mixturewas added (y'ellouoil.0.-1.1 g) uas used*ithout furtherpurification l'l \\1R dropuise to an ice-coldstirred aqueous solution of ammoniumhydroxide (D2O)6 (DSS)-1.7-31.9 (m. I H).3.3-3.,1(m.2 H).2.8-:.9(ni. I H). tl0 ml . l;1\{)which hadbeen placed in an icebath. The reactionwas 2.55 (s,6 H). The spectraldata are in accordwith thosepreriouslr stirrcdat 0 o(- l'orI h. After evaporationof the solventand washingof published.5e thc solidirith ether.0.6 g (as a colorlessoil) oi crudeor-3-amino-l- ol-N-Acetyl-3-aminopropane-1,2-diol.ot--3-Aminopropane- 1,2-diol nrethorrpropan-2-olhydrochloride resulted. The compoundwas used (9.1g, 100mmol) wasdissolved in aceticanhydride (25 mL) and stirred withoutfurthcr purification: rH NMR (D:O. pH 7) 6 (DSS)1.0-4.2 overnightat ambient temperature. The aceticanhydride and aceticacid (m, I H).3.5--1.6(m.2 H),3.4(s,3 H).3.1--r.3 (m,2 H): ''C NMR wereremoved by rotary evaporation.and the resultingoil was dissolved (D2O,pH 7) 6 (DSS) 78.3(s), 70.5 (s). 63.3 (s), 46.5 (s); mass spectrum, in methanol. Solid ootassiumcarbonate was added to the methanol mf z 106(M + l), 105(M),88 (M + I - HrO),87 (M - H:O). solutionand the suspensionwas stirred for 2 days. After the solventwas 2-Methylpropane-1,2,3-triolwas preparedaccording to the procedure removed by' rotarl evaporation,the liquid contained ol-A-acetyl-3- of Eisenthaler al.:24rH NMR (D:O) 6 (DSS) 3.3(s, 4 H). 0.7 (s.3 H). aminopropane-1,2-diol,residual sodium acetate, and methanol(total 9.5 2-(Hydroxymethyl)butane-1,2-diol (2-Ethylpropane-1,2,3-triol) was g). This mixture containingot--'V-acetyl-3-aminopropane- 1,2-diol was preparedaccording to the procedureof Eisenthalet al.:la ltl NMR usedwithout lurther purification: 'H NMR (DzO) D(DSS) 3.63-3.73 (DzO)6 (DSS) 3.4(s.4 H), 1.3(q,2 H). I I (t,3 H); massspectrum, (m, I H), 3.3s-3.5(m. 2 H), 3.05-3.25(m, 2 H), 1.9(s, 3 H); r3CNMR mlz tl7 (M - 3). 103(M - OH),9l (M - CH2CH3),8e(M - (DzO)6 (DSS) 178.6(s),74.6 (s),67.7 (s),46.2 (s),26.3 (s). cH2oH),43 (M - OH - CH2OH - CH2CHT),3l (CH2OI{). ol-GlyceraldehydeDimethyl Acetal was preparedby osmium tetroxide catalyzed oxidation of acroleindimethyl acetal.60 N-Methylmorpholine Supplementary Material Available: Tables of Vm ^ and K* for 1V-oxide(1a.8 g, 107 mmol) and osmium tetroxide(70 mg) were dis- a number of substrates for glycerol kinase for C. mycoderma and (40 (20 solvedin a mixtureof water mL) and acetone mL). The reaction B. stearothermophilus (2 pages). Ordering information is given on any current masthead page. (57) Gronowitz,S.; Herslof, B.; Michelsen, P.;Aakesson,B. Chem. Phys. Lipids 1978,2 2, 307-21. (58) Knorr,L.; Knorr,E. Chem.Ber.1899,32,750-7. (61)Capon, B.; Thacher, D. J. Chem.Soc.81967. 1322-6. *The (59) Pouchert,C. J. Aldrich Library of NMR Spectra",2nd ed.; (62) Paul,R.: Williams, R. P.;Cohen, E. J. Org.Chem.1975,40.1653-6. AldrichChemical Co.: Milwaukee,Wi., 1984. (63) Koelsch,C. F. J. Am. Chem.Soc. 1943,65,2460-5. (60) VanRheenen,V.: Cha,D. Y.; Hartley,W. M. In "OrganicSyntheses"; (64)Jung, M. E.:Shaw, T. J. J. Am. Chem.Soc. 1980, 102.6304-ll. Sheppard,W. A., Ed.;Wiley: New York. 1978;Vol.58, pp 43-52. (65) Fourneau.E,.; Ribas, L Bull. Soc.Chim. Fr. 1926.1584-9.