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Home , Aldol reaction, Sorbose

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Synthesis of by Aldolase-Catalyzed, Condensation Reactionsl

Chi-Huey Wong and George M. Whitesides*

Departmentsof Chemistry,Massachusetts Institute of Technologyand Haruard Uniuersity, Cambridge,Massachusetts 02138

ReceiuedMarch 15. 1983

Dihydroxyacetone phosphate was prepared in 2OGmmol scale from by two procedures: reaction with phosphorus oxytrichloride and glycerol kinase catalyzed phosphorylation using ATP with in situ regeneration of ATP by phosphoenolp5rruvateor acetyl phosphate. Dihydroxyacetone phosphate was converted to 6-phosphatein 80% yield by exposureto a mixture of co-immobilizedtriosephosphate isomerase and aidolase followed by acid hydrolysis of the condensation product fructose 1,6-bisphosphate. Fructose 6-phosphate was subsequentlyconverted by chemical and enzymatic schemesinto fructose, glucose6-phosphate, and . Practical procedures are described for the preparation of o- and l- 3-phosphate and foiseveral hexoseslabeled with 13Cin the C-2 and C-2,5 positions.

Introduction reactions as a route to isotopically labeled and uncommon The work reported in this manuscript is part of a pro- sugarson scalesfrom 0.01to 1 mol. The aldolaseused in gram to evaluate the potential of aldolase-catalyzedaldol this work (from rabbit muscle,E.C. 4.1.2.13)is commer- cially available. It is inexpensive,and it has high specific (1) Supported by the National Institutes of Health Grant GM-26b48. activity and good stability. It requiresdihydroxyacetone Inquiries should be addressedto G.M.W. at Harvard Universitv. phosphate(DHAP)2 as one substrate,but will accept a

0022-3263i 8:r i 1948-iti99$01 .5f) i 0 .c 1983American Chemical Societr \\'r 'ng \\'hitesides 3200 J. Org.Chem., Vol. 48, No. 19,1983 ancl

Table I. Kinetic Parameters of Aldolase from N{uscle antl Y eiLst " rabbit muscle Yeast

substrate K-, mM v K*, mNI Aldol Cleavage b 1" -fructose 1, 6-bisphosphate 0.006 1c 0.36 D 0.13 0.002 -sorbose 1,6-bisPhosPhate 0.o44 0.1 L 0.0002 1-phosphate 0.6 0.02 0.2 L-sorbose 1.0 0.000.1 D -fructose 1 -phosPhate 6.3 0.01 D -fructose 6-phosPhate 0.0001 1.0(0.4)" 2.4 dihydroxyacetone PhosPhate 2.0 3.0 * D-glYceraldehYde-3-P 4.0 2.8 t D .t, -giYceraldehYde 0.1 0.1f i- D,L-lactaldeirYde f I D.L -.r-hydroxybutyraldehycie 0.1 dihydrnxyacetone sulfate i3 ) r + D,L -glYceraldehYde-3-P < 0.0001 dihydroxyacetone .< -t D -glyceraldehYde-3-P 0.0001 phosphate (2) acetol 0.0006 + o -glyceraldehyde-3-P <0.0003 -phosphonate 4-l'rydroxy-3-oxo-1, i 1 ) o.4e '' + tr -glyceraldehyde-3-P 0.L l-9-A 3185-92) a numben cited are from: Richardsand Pottet (Richar&, o. C-; Rutter' w. J "I Broi gt:,l] The -glyceraldehyde3-phoaphate' ii6" unlessotherwise indicated. The f- OH,qi rn* aetlrmined in the presenceof D ' D "i i.-8. Biochem.Biophls ResCommun lil6' 69' 844-51 rrmolmin v - v^-.. Mariensen,';il;o;;i;i;-;T. Irl.; vra,isout-. r.(-42 in I'his ,"J??;'r;'. ;;'' oi 'stfiblins,D B.iichem't ts14, I4t' ?25-8 Determined ;; (DHAP or diirvdroxy- i"i""r-t"ir.' 'ift" ufa"ivO" *"."ptoi".,,v-. *as iO -l,i'.na tn" concentmtion of donor acetonesulfate) was ".n-"."it"iio"20 mM, PH "r ?.0

li i'ltr.'tiilai irn,t i',itz.5'nttrttt'I\t'parati'i.rtrs

? \ QPO1H riO^-=. ---R ! t{ fr PEP HO-!-_{'-CP \ pCC I -!2 l:( a, H r ,____=x I - z Ac \ I:^c^ \..'r\ v . ^- - ll ^ Av' H03PO--,'xt'!rh ' DHAP Acp R WidelY vorioble {R'l R 60% f rom POCI3(not !sololed ) R'= H,OH { a') d--l rom ATP (isoloted) + HOi 807" f -oH o- Norrow specificirY Po- previously in small-scale (-2 mmol) svntheses ut'1:r(l-la- { I PO-"{"' beled fructoseand glucose.s Its utilitv for larger scalework has not been explored. wide variety of as the second.3 The stereo- These methods should be useful in the svnthesisof chemistry of the aldol condensation catalyzed by this en- certain simple sugarsand in the preparation of isotopically zyme is indicated in Scheme I; the two new chiral centers labeledsugars for use in biochemistry,metabolism, and are formed enantiospecifically.4 Aldolase has been used physiology.

Results (2) Abbreviations: AcP, acetyl phosphate; DHA, dihydroxyacetone; -P, FDP, o-fructose 1,6-bisphosphate;F-O-p, o-fructose 6-phosphate;F- 1 The aldolase from rabbit muscle or yeast is o-fructose 1-phosphatb; G-6-P, n-glucose 6-phosphat".;-.Gq'glycer- - Table I summarizesdata on Gd-^3-P,'glyceraldehyde3-phosphate; Gr- 1-P, (B) - g-lycerol1 highly specific for DHAP. "ld"hydu; -P, 1-plo-sptrate; change in the f norptt"t"; PEP, p"hosphoenolpyruvate;-S- 1 L-sorbose su-bstratespecificity.3 In general, any bK, gly""rokinase; GPDH, glycerophosphatedehydrogenase; HK, hexo- of DHAP greatly reduces reactivity in the al- phosph-oglucoisomerase; structure tinaJe; GluDH, glutamic dehydrogenase;PGI, reaction. The phosphonate analogue Pase, potato acid"phosphatase;PK, pyruvate kinase; T?I, triosephosphate dolase-catalyzed isomerase. 4-hydroxy-3-oxol-phosphonate' 1, providesan exception: (3) In addition to the substrates described in Table I, the aldolase from this substanceis 10% as active as DHAP. Acetol phos- ,"u'bit muscle also accepts the following aldehydes using DHAP as as DHAP, while di- not available for the reactions: form- phate 2 is about 0.017oas active reactant, but kinetic data are the , ,t--glycidaldehyde (O'Connell, E' L'; Rose, I' A' irydroxyacetone monosulfate (3) is not a substrate for J. Bi;t. Ciem.1973, i't8,22t541'1, t--(Jones, J. K. N.; Matheson, muscle enzvme. N. K. Con. J. Chem.1959, 37, 1?54-6),n-, L-, D-, and n- (Jones,J. K. N.; Stephton, H. H. Ibtd. 1960,38, 753-60)' ih" from peas has been reported to accept: glycolic aldehyde Soc. 1958,80' 5835'4' (Hough,L.;Jones,"trry*. i. X. f. J. Chern.Soc. 1952,4A47-52), o,i--lactaldehyde (4) Rose,l. A. J. Am. Chem. A. S.; Cadman,E.; Pierce,J.; Haves,M L'; Barker, R' igo"gt, L.';.Iones, J. K. N. Ibid. 1952,4052-5),and o-(Hough, i5) Serianni, 1982,89, 83:92 and referencescited therein' L.: Jones.J. K. N. Ibid. 1952,342'-5) Methods Enzvmol. Aldolase-Catalyzed Condensations J. Org. Chem., Vol.48, ftlo.19, 1983 3201

III. Synthesesof Fructose 1'6'Bisphosphate, oa RR Scheme H O--,.$ HaP (OH )2 Fructose 6-Phosphate, Fructose, Glucose x o-,2\zoii t ox ), G-Phosphate,and Glucose DHAP I a rpr ^ It P x o-t=\-oe +-i--+ o\,.:\-.o e9 AR --- H-._,A..--.OP(0H)2 HO-_-x.-,.osoH DHAP 0 (. o'oo,or" : I ,;t":-t-t OP Dihydroxyacetone Phosphate. We have examined /oH two routes from dihydroxyacetone (DHA) to DHAP FDP "oas-t/xo{4 'oP (SchemeIi). Fcir most preparationscarried out on scales procedure on chemical I H+(oowexso) up tri several moles, the based I phosphorylationis the more convenienl (especiallyfor i 86% preparationof PEP or Acl) are not OH laboratoriesin which OP /oN routine). The deficienciesof the c:hemicaltrlroi:edttre are Pqse H ,'o'^.- -p -.--- r: a-- r that it generatesrelatively dilute sohitionso1'prodttct and r i.-r-. uo-\-i iCC"/" Lox f-hatimpurities remaining from the chemicalsteps n'ra1' -ctl comJlhcateworkup. F urther, becausethe product solutirltr ti r PGl,66% from this chemicairoute containsphosphate, isolation and I purificationof the DHAP from it are ir,convenient.It is Pose (o"-o '*'ititout ,o' -- bestused directly irr subsequenttransfortnatiorts G-6-P ,o--(-5,,_Ho\4_oH rdFv"'[Nft-o, isolation. The puritl' of the I)HAI' prepared bv this procedureseerns. however, to be perfectly'acceptable in the enzymaticreact.ions we have explored. SchemeIV. Synthesesof (2''3C,)Glucose and The biochemicalroute to DHAP is basedon the enzy- (2,5-1'C.)Glucose anti Intermediates(Heary Circles matic phosphorylationof DHA bv ATP with in situ co- lndicate Positionsof '3C Label) factor regeneration. This proceduregives a product that oH ,i *3* ,^, ^ r) xr can either be isoiated in relatively high purity (80-90% ) HCHO o:^\n \^--'H or used in situ. The enzymatic procedure using PEP as 2)H;ti' ffik," pH t.7 pH r.7 I the ultimate phosphorylating agent is somewhat more Prl,/Bo3O4 Pd,/Bq3O4 | ^ convenient than that based on AcP, becausePEP has eo% sz% | NoBHo | 82 greater stability in solution than AcP.6'7 The procedures "t" are otherwise comparable. ADP'PEP oH rF.r,€r-re r-,--\-./.\-,/v. I -1p p* , HVSJH D- and l-Glyceraldehyde and I)- and t.-Glycer- ----. Of Gt,, <--. - NAD 89% aldehyde 3-Phosphate. Thesethree-carbon substances '),,,oruou I GPDH condensewith DHAP in aldolase-catalvzedreactions and NH: ) \ruADn/Y yield hexosephosphates: the stereochemistrvat C-5 i-s crKG "' definedby the of the glyceraldehyde.Procedures for preparing each are summarized in Table II. D- Glyceraldehyde(D-Gd) can be prepared in 82% enan- tiomeric excess(ee) by selectivephosphorylation of r,-Gd in a mixture of D-Gd and l-Gd using glycerokinase(GK). It can also be prepared by treatment of o-glyceraldehyde 3-phosphate(D-Gd-3-P) with acid phosphatase(Pase, E.C. OP 3.1.3.1).L-Gd is best preparedin 100% ee by Pase-cata- /9L lyzed hydrolysis of l-Gd-3-P prepared enzymatically by 'ofr12 ,.ffi# FDP --- -oP using GK. D-Gd-3-P is easily obtained by isomerization oP I of DHAP with triosephosphate isomerase (TPI, E.C. + I H*(Do*"x50),86% 5.3.1.1)and used in situ, although the equilibrium mixture contains 96% DHAP.8 If a separate preparation is re- quired, it can be obtained by oxidative cleavageof fructose ] af-sn# \-sH JI &phosphate with lead tetraacetate. l-Gd-3-P is most easily +l ", *l PGt,66"a obtained by enantiospecific phosphorylation of the L-Gd Y + present in o,l-Gd using glycerokinase. fP 2P Fructose G-Phosphate and Derivatives. The con- *o*N4Fo, *gSo, version of DHAP to fructose 1,6-bisphosphate(FDP) and I fructose 6-phosphate (F-6-P) and from this latter inter- l | 'o" ' looo/o (G-6-P) mediate to glucose 6-phosphate and glucose is PH ,oH outlined in Scheme III. The initial step is the equilibrium *lffi$"* *3N4\-o, conversion of DHAP to o-glyceraldehyde 3-phosphate (o-Gd-3-P) bV TPI. The two are then condensedto FDP by aldolase. The FDP was converted to F-6-P by acid- catalyzedhydrolysis. The conversion of this substanceto fructose,to G-6-P, and (by way of G-6-P) to glucosefollows enzymatic and acid-catalyzedroutes outlined in the scheme. Theseconversion are all reiatively'straightforward.'fhe pnzvmesrelir ; i reii ar(, (:omtnerr:ia iiv avaiia ir'ie. inerpensive. 1983 Wong and Whitesides

Table iI. Freparation of

lrrc-ciitct. ^vield, 7o \ee, Vc) gll;ceralciehr ile glyceraldehvde 3-P reactant reagent, cataiysts Di Di, _ D, L -glyceraldehyde GK, ATP 45" i82) 4i, irbo)_ 'fPI dihydroxyacetone i:hosphate -! (100) D -fructose 6-phosphate Pb(OAc|.4erluiv 85 r100\ n-glyceraldehyde-3-P Pase _qirI i0t) 1 t,-giyceraldehyde-3-P Pase ,J,'rii-ti): D -fructose FbtOAc i . + €rquli :16 ( I 0(i tjr L -sorbose Pb(G;\cr . ,1orluiv liti r iii0 I' ( ' Based on o.t,-glyceralciehyde" Perlin. \ S -l/t'lirc.,,ls()uritr;tirrii' ('lrt'trr 1gfi2.'. tii-3

Scheme \'. P16'paration of i-i'Pructose 6-Plrosphal,e ancl t.'Sorbose

OH Ho.--..!..-,,oe oor\-oH fH f' Hod,-o\ no-Nz-or xo-\-i$r\-ox Ho+----+-\ \r.____-__/ Aldolose ./ ,'t'6t ---t/ | ( 9.F" OHa I xa-\t-Lz Ho-t\-i2 | tovroo Ho-\2f' I L \o)-.-op J

--,r'":x 5o)

l_ ''I H oH^ ,)i 1 - o t. Hc t-.' *?Na'' ,l i '--^) s'z rrom l, 'ro.-\/ '*69 I DHAP i- : \ frt -oH

r) HK,eEp,pK

{ S.ocrolton OP t oH '-q /oa \ Ho'\r-tz Ho'{-(, ,/ HG-\}f xo-\-,{_ | \--oH OH \oH

F-6-P , 42* L-9orborr , 394

the C-2 and C-2,5 positions. Figure 1 contains the riJC NlvlR spectraof glucoseprepared bv using theseproce- dures. We note that thesesequences of'reactions are also di- l,l'l,i rectlt' applicableto the synthesisof glucoseand fructose l?C tOO 80 60 4C (PPmi having dift'erent labeling patterns and (using L-glycer- 13C 13C aldehydeand t,-glyceraldehyde3-phosphate) to sorbose Figure l. NMR spectra of glucose with enrichment at tBC C-2 (A) and at C-2 and C-5 (B). derivatives. For example,introduction of the by the secondcyanohydrin reduction (to generate(1-13Cr)-n,L-Gd) and stable in the immobilized forms. No inhibition has would yield glucosecontaining label in the C-1,C-3, C-4. been observedin a reaction with reactants present at 0.1 and C-6 positions (or the C-l and C-3 positions,if D-Gd-3-P M concentrations. One technical problem in the prepa- is suppliedexogenously). D- or L-Gd labeledat C-1 (pre- ration of G-6-P is that the equilibrium mixture achieved pared from n,l,-Gd,enzymatically as describedabove), after in the PGl-catalyzed reaction contains 70% G-6-P and aldolase-catalyzedcondensation with DHAP, would yield 30% F-6-P;sthis mixture complicatesisolation of the prue glucoseor t,-sorbose,respectively, containing label in the sugars. To separatethese two species,barium acetateor L--4position. (1-13C1)-D,L-Gdwould react with DHAP and barium chloride was added to the solution. G-6-P pre- generatea mixture containingF-1-P and sorbose-1-PIa- cipitated as its barium sal| F-6-P remained in the solution. beled at the C-4 positions. After acid-catalyzedhydrolysis Preparation of Isotopically Labeled Fructose and to remove the phosphate moiety from both sugars,the Glucose Derivatives. In preparing isotopically labeled fructose in the mixture can be converted to F-6-P with sugars, it is useful to differentiate between the two C, hexokinase(HK, 8.C.2.i.1.1) and thus separatedfrom the pieceswhich are assembledto make the C6sugar. We take unreactedl-sorbose (Scheme V). advantageof the ability of aldolaseto accept a range of aldehydesas reactants and use DHAP as only one com- Conclusion ponent of the aldol reaction. SchemeIV outlines a syn- r3C Aldolase-catalyzedcondensation provides a practical thesis of glucose(and intermediates) labeled with at route to a wide variety of simple sugarsand deriv- atives,and especiallyto those (glucose,fructose, and de- (9) Wong, C.-H.; Whitesides,G. M. J. Am. Chem. Soc. 1981,103, rivatives) centrally important in intermediary metabolism. 4890-9. The procedurehas the advantagethat it is applicable to Aldoiase-CatalvzedL'r)nd en-sat ions unprotectedsugars in aqueoussolution at pH 7. Bpimeric nihydrox, ^"t ;ff t:: ;: t: *:r', :t::r:l: mixtures can, in favorable cases,be separatedwithout Phosphorylation. "r',,J"To a l-L solution containing dihydroxyacetone chromatographybv taking advantageof substrate-seiective t18g,0.2 mol), ATP (2 mmol),MgCl2 (5 mmol),and PEP (asits enzvmaticphosphorylation. The sugarphosphates pre- monopota^ssiumsalt, K+PEP-, 41.2g,0.2 mol);6pH ?.0,was addeci PAN-immobilized (150 gel) pK (1000 pared here are of 80-90% purity. The impurities are GK U, 8 mL of and U. 15 mL of gel). The mixture wasstirred at roorn lemperatureunder mainly inorganicphosphates, and are of no consequence argon ancithe pH wasautomatically controlled at 7.0 by adding in most further enzymatic transformations. Using im- aqueousKOH (1 N) using peristaltic pump. Enzymatic anaiysis mobilized enzy'rnesand in situ cofactor regeneration,these indicated that the reactionwas completein 12 h. After recovery methods should be applicableto preparationscarried out of the enzyrne-contarninggels, the solution was treated with active on mole quantities. Although the enantioselectivityof charcoal (10 g) and filtered. The colorlessfiltrate was treated aidolase-catalyzedreactions has been examinedexplicitly with Dowex 50 (H+ form) until the pH reached4.2 (-2 g of resin), with only a few unnatural substrates.it is probably high: then filtered, and concentrated to a volume of 200 mL. Ethanol we have, for example,prepared 6-deoxy-D-fruct,oseand (1 L) was added to precipitate DHAP as a monopotassiumsalt. 6-rieoxy-t.-sorbosel0with high enantioselectivityusing o- After being ciriedin vacuoover KOH, the solid (37 g) contained 90% rveightDHAP-K* (160 yield), and t,-iactaldehyde,respectively, as substrates. by mmol,80Vo as determined enzymaticaily by using GPDH and NADH. Turnover numbers A limitation of the enzymatic method is that di- (anrdrecovereci activities for the componentsof the system) were hydroxyacetonephosphate is required as one reactantin as {oliows.(;K,.1 x 10r i86%);PK,2 x 10?88To); ATP, 100 the aldol condensation,although the secondcomponent i-qb%i. can be varied over a range of structures. A seconddis- ^{ srrnilarrt,actior.l on the sarnescale was carried

; Verlag (14) DHAP is not stable at valuesof pH higher than 6. The half-life of DHAP (10 mM) in Lriethanolaminebuffer (0.1 M, pH 7.0) is 65 h. Whitesides 3204 J. Org. Chem., VoI. 48, No. 19, 1983 Wong and r.-(]C and treated with Dowex 50-X8 (H+) until the solution had pH argonfor 4 h until no further lncreirsein the concentrationoi -2. The resin was removed by filtration, and the filtrate was was observed.After separatirrn0f enzrrne-containinggel, the final (85% yieldt concentratedto an oily residue under reduced pressurearoung solution (24 mL) contained0.70 mnrol of'r,-Gd de- 40 oC; this procedure removed acetic acid. The residue,after termined enzymaticalll'. (1 L) dilution with water, was used directly in further reactions without o-Fructose 6-Phosphate. To a deorvgenatedsolution (ir IvIgCl2 additional purification: the ion-exchangechromatographlF was containingo-fructose (200 mmol. 0.2 \lr, ADI' mlM), (5 of gel eliminated. The progressof hydrogenationswas monitored by (10mM), and mercaptoethanoi mM)wa. arldeci4 nrl r.150 enzymatic determination of the produced by use of al- containingco-immobilized HK (600U) and Acl., lri solid (90% dehyde dehydrogenaseand NAf).13 diammonium acetyl phosphate purity. 210 mmolt wa. added Dihydroxyacetone monosulfate was prepared in a prcrcedure in ten equal portionsto the mixture with stirring over a I'rt'ri,'d pH lrr similar to that for glucose6-sulfate.l5 P1'ridine-sulfur trioxide of 20 h. The solution was automatically controlled at ? I (1.+g, 43 mmol) in DMF (7 mL) was added with stirring to a addition of ! N NaOH. Enzyrnaticanalysis indicated that rt solution of dihydroxyacetone(3'9 g, 43 mmol) dissolvedin DMF ctrntainedlgfi rnmoi (98%\ of P-6-P. After separationof tht g of ac' (10 mL) over 3 min at 25 "C. The mixture was stirred for another enzvme-c()ntarninggel. the solutionwas treated with 5 'C) hour and concentrated(0.1 mm Hg, 35 to removeDMP and iir,aterj1'11|;on anr-i :'iitereci, and the filtrate was concentraterj o(- -100 pyridine. The oilv residue was dissolvedin water, adjusted tfructose (retention 74% (22mmol) basedon ftrrmaldehydeas starling material. This time 8 min) and L-sorbose(retention time 7 min). compoundwas dissolvedin 100mL of water and addeddroprvisp periodof 30 min to a stirred.t'r'ld. aque()us soltrtion (10(i Hexokinase-Catalyzed Selective Phosphorylation of n- overa 'fhe Fructose in the Presence of t -Sorbose. To the solution pre- mL) containingNaI3Ho (2.3 g. 60 mm