Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Copyright Reprinted from the Journal of the American Chemical Society, 1989, 111,624. O 1989 by the American Chemical Society and reprinted by permission of the copyright owner. A Combined Chemical-EnzymatrcSynthesisof I 3-Deoxy-D-arabino-heptulosonic Acid 7-Phosphate NicholasJ. Turner2and GeorgeM. Whitesides* Contribution from the Department of Chemistry, Haruard Uniuersity, Cambridge, Massachusetts 02138. ReceiuedDecember8, 1987 Abstract: 3-Deoxy-o-arabino-heptulosonic acid 7-phosphate(DAHP) has been synthesizedfrom N-acetyl-n/l-aspartate p-semialdehyde and dihydroxyacetone phosphatein four stepswith an overallyield of 13%. The key stepgeneratesthe required threo stereochemistry by usingrabbit musclealdolase(E.C.4.1.2.13)as catalysl. 3-Deoxy-o-arabino-heptulosonic acid 7-phosphate(DAHP, l) is an important intermediate in the shikimic acid pathway, standing at the crossoverpoint from the sugar phosphatesto the cyclitols that eventually are transformed into the aromatic amino acids.3-5 This cascadeof transformationsis initiated by the conversionof DAHP (l) to dehydroquinate(DHQ, 2), catalyzedbydehydroquinate synthase(DHQ synthase)(eq I ) SchemeI. Synthesisof DA[{P, I Babbit Muscle Aldolase OH I - Y- i- ^ . Po//\n// co2fl HO 3 tr I ---\ AcOH CO2H PO I dehydroquinate synthase co.M" NHlc O M e r N ' B l - r{ C A c l r HO - ll o l o H malor rsomer (a:1) ( r) 6NHcr {HAc The inhibitionof this reaction,and the resultingdisruprionof aromatic amino acid biosynthesisin plants,providesthe basisfor the activity of certain herbicides.6The developmentof new plant growth regulatorsof this type would be facilitated by a convenient synthetic route to DAHP; such a route might also allow for the preparation of structural analoguesof DAHP. Most of the previous chemical synthesesof DAHPT-rl have employed 2-deoxy-o-glucose(a relatively costly carbohydrate at $17/g) as the starting material. The most efficient of these synthesesr0requires eight stepsand proceedsin 6Vooverall yield. Recently, Frost and co-workersl2have used an enzymatic system to produce DAHP from o-fructose. Despite the efficiency of this methodology(85Voconversionof o-fructose to DAHP) the cost and sensitivity of the enzymesinvolved are such that this method is not a practical one for the production of DAHP. This paper exploresa new approach to the synthesisof DAHP that usesrabbit musclealdolasel3-tt(8.C. 4.1.2.13)to catalyzeformationof the C4-C5 bond. This aldol condensationgeneratesthesetwo chiral (l ) Supportedby NIH Grant GM 30367. (2) N.J.T. was a Royal SocietyResearchFellow, 1985-1987. (3) Haslam, E. The ShikimatePathway. Wiley: New York, 1974. (4) Ganem,B. Tetahedron 1978,34,3353. (5) Robinson,J. A.; Gani, D. Nat. Prod. Rep. 1985,2,293. (6) Hardy, R. W. F.;Giaquinta, R, T. Bioessays19t4, l,l52. (7) Sprinson,D. B.; Rothschild,J.; Sprecher,M. J. Biol. Chem.1963, 238, 3170. ( 8 ) H e r m a n n ,K . M . ; P o l i n g ,M . D . J . B i o l . C h e m . 1 9 7 5 , 2 5 0 , 6 8 t l . . (9) Trigalo, F.; Level,M.; Szabo,L. J. Chem. Soc.,perkin Trans. I 1975, 600. Trigalo, F.; Szabo,L. Methods Enzymol.1975,41, pt. 8,9j. (10) Frost, J. W.; Knowles,J. R. Biochemistry1984,23,4465. (l l) Adlersberg,M.; Sprinson,D. B. Carbohydr. Res.1984,127, g. (12) Reimer,L. M.; Corley,D. L.; Pompliano,D. L.; Frost,J. W. J. Am. Chem.Sac. 1986./08. 8010. (13) Wong, C.-H.; Whitesides,G. M. J. Org. Chem. 1983, 48, 3199. (14) Durrwachter,J. R.; Drueckhammer,D. G.;Nozaki, K.; Sweers,H. M . ; W o n g , C . - H . , I . A m . C h e m .S o c . 1 9 8 6 , 1 0 8 , i 8 1 2 . (15) Bednarski,M. D.;Waldmann,H. J.: Whitesides. G.M. Tetrahedron Irtt. 19t6, 27. 5807. // '/, OH I t OH NH3'C|' malor lsomor (4:1) 5 At3' -oP I €> O H 2. lon-Exchange Chromaloqraphy DIJO (2) I - oo.\-\-.-.y'to'" \ l. Sodlum glyoxylate DAHP (1) 1 o oo c ,-"o,n corH O H Table I. Diastereoselectivin o 'f R e d u c t i o no f 4 reagent solvent temp (oC) time (h) anti:syno'' Na+BHa HzO 25 I 45:55 Na*BHa-/CeCl3 HzO 25 I 50:50 Na*BH(OAc)3AcOH 25 2 75 : 2 5 Me4N+BH(OAc)3- AcOH 25 2 80:20 MeaN*BH(OAc)r- AcOH" 0 24 78:22 oDiastereomericratios were determined by tH NMR spectroscopy (500 MHz). Integration of the N-acetyl signalsdue to the 2R isomer are reported although the 25 isomer gave essentiallythe same ratios. bThe assignmentof anti and syn was made as follows: the product from entry 4 was converted to the diastereomericmixture of amino acids 6 (seeScheme I). Treatment with sodium glyoxylate generateda mixture of DAHP (l) together with its C-6 epimer (ratio 80:20 respectively) thus confirming the predominantly anti reduction. "This experiment was carried out on the tetramethylammonium salt of 4. Acetonitrile was used as a cosolvent to prevent freezing of t..e solution. centers disasteriospecifically (eq 2). The successful execution of this approach is summarized in Scheme I. H I o4* rabbit muscle aldolase HO HO OP (2) OH il u HO _ _ -OP Results and Discussion Treatment of N-acetyl-o/r--allylglyciner6with diazomethane afforded the correspondingmethyl ester in quantitative yield. Ozonolysis,followed by reductive workup with dimethyl sulfide, gave the aldehyde 3 in 75Voyield after chromatography. In (16) Black,S.; Wright, N. G. ,r. Biol. Chem. t955, 213,39. 3- Deoxy-o- arabino- heptulosonicAcid 7- Phosphate J. Am. Chem. Soc., Vol. ll1, No. 2, 1989 625 practice,the crude aldehydewas usedin the enzymaticreactions sincethe major byproduct of the ozonolysis(dimethyl sulfoxide) did not influencethe activity of aldolaseat concentrationsbelow approximately 20Vo.t7 The reactionof aldehyde3r8with dihydroxyacetonephosphatere in the presenceof aldolase was monitored by rH NMR spectroscopy. Conversionreacheda maximum of 40Vo. Ion-exchange chromatographyof the product mixture gave4 in 37Voyield. In addition to the expectedcarbon-carbon bond formation, hydrolysis of the methyl ester also occurred.2o Both 31Pand tH NMR spectroscopyshowedthe product to be a mixture of diastereomers, e p i m e r i ca t C - 2 ( 2 R : 2 5 = L 5 : 1 . 0 ; . z t ' z z The sodium salt of 4 was reducedby using a variety of reagents and conditions(eq 3; Table I, ExperimentalSection). Since the 1.,".-lY, Syn Anti anti disastereomerwas required, we focusedour attention on the use of the triacetoxyborohydrides.23Although the diastereoselectivities were lower than had previously been observed23'2a (possiblydue to the influenceof the neighboringphosphategroup), both sodium and tetramethylammoniumtriacetoxyborohydride gave the desired anti configuration as the dominant isomer (anti:syn - 4:1).zs No attempt was made to separatethese (17) Bischofberger, N.; Whitesides,G. M. Unpublishedresults. ( l8) Although our initial plan had beento usethe aldehydeA (eq i) as the precursor,we observedno reactionof A with dihydroxyacetonephosphatein the presenceof aldolase.This unreactivityis most likely due to the high enol contentof A (a,6-Unsaturatedcarbonylcompoundsinactivatealdolase. Effenberger,F.; Straub, A. TetrahedronLett. 1987, 28, 164l). A (19) Crans,D. C.;Whitesides, G. M. J. Am. Chem.Soc. 1985,107,7019. (20) It is likely that hydrolysisof the methyl esteroccurredafter carboncarbonbond formation sincca solutionof the aldehyde3 in D2O showedonly slight hydrolysis(lessthan SVo)after 72 h at 25 oC. The relative lability of the methyl ester group is probably due to intramolecularnucleophilicassistancc from the C(4) hydroxyl (eq ii). Further support for this rationale comes from the easewith which thesecompoundswere found to undergo lactonization under acidic conditions. oH oH diastereomers. Hydrolysis of the crude reaction product containing 5 was carried out at 100 oC in 6 N HCI for 90 min. Under these conditions removal of the N-acetyl group occurred with only minimal cleavageof the phosphate(as evidencedby TLC). Purification on ion-exchangeresin gave a diastereomericmixture of amino acids 6. The final transformationof 6 to the correspondingketoacidwas troublesome. We had hoped to use the commercially available o- and L-transaminasesto accomplishthis transformation, but the mixture of amino acids 6 showed no reaction with either enzyme. We then turned to someconventionalchemical methods, namely trifluoroacetic anhydride26 and 3,S-di-tert-butyl-obenzoquinone,2T but again were only able to recovereither starting material or unidentified products from thesereactions. The final step was eventually accomplishedby a transamination reaction with sodium glyoxylate.28 Addition of only I equiv of sodium glyoxylate gave rise to an equilibrium mixture of starting material and two diastereomericproducts. Five equivalentswere ne@ssary to causecomplete conversionto products. Separation of the two diastereomersby ion-exchangechromatography yielded pure DAHP l. tH NMR indicatedthat the samplewas -907o pure. Repurificationof an aliquot (- l0 mg) of the syntheticDAHP by chromatographyon DE-52 increasedthe purity to -95Vo as judgedby 'H NMR. The syntheticDAHP (from DE-52 column) was a substrate for DHQ synthase.The valueof the initial rate obtained(0.123 AU/min) was, however.significantlybelow that recordedin a p a r a l l e le x p e r i m e n tw i t h a u t h e n t i cD A H P ( 0 . 3 3 4A U / m i n ) . 2 e This low valuewas established kineticallyto be due to the presence of a competitiveinhibitor in the syntheticDAHP. Although we did not determinethe structureof this compound,its influence on the rate of reactionof pure DAHP demonstratedthat it was a potent inhibitor. Determinationof its structure might suggest leadsto new inhibitorsof DHQ synthase. In summary this route to DAHP has four attractive features. First, the protection and deprotectionstepsare minimized, and all reactionsare carried out in aqueousmedia. Second,the introduction of the phosphategroup, often a step proceedingin low yield, is accomplished simultaneously with formation of the carbon--carbonbond. Third, the preparationof isotopicallylabeled DAHP I can be readily accomplishedby using this method with appropriatelylabeledaldehyde3 or dihydroxyacetonephosphate. Fourth, the high toleranceof rabbit musclealdolasefor the aldehydecomponentsuggeststhat this route offers a potentially useful method for synthesizinganaloguesof DAHP, with the possibilityof structural modificationat any centersother than C-4 and C-5 (the centers formed in the aldolase-catalyzedreaction). The synthesisof DAHP by the sequenceof reactionsin Scheme I demonstratesthe use of rabbit muscle aldolaseto synthesize amino sugars4, 5, and 6 from an aldehydederived from an amino acid and suggeststhe use of this enzyme for the preparation of other amino sugars. NHAc {ri) -- -oH r\ I : (21) The assignmentof the diastereomerswas made by repeating the aldolase reaction with the aldehyde derived from l-allylglycine, thereby generatingthe 25 isomer of 4. (22) We havepreviouslyobservedkinetic selectivityby aldolasein similar r e a c t i o n s :B e d n a r s k iM , . D . ; L e e s , W . ; K i m , M . J . ; W h i t e s i d e sG, , M . U n publishedresults. ( 2 3 ) E v a n s ,D . A . ; C h a p m a n ,K . T . T e t r a h e d r o nl * t t . 1 9 8 6 , 2 7 , 5 9 3 9 . (24) Saksena,A. K.; Mangiaracina,P.TetrahedronLett. 1983,24,273. (25) Examinationof the rH NMR spectrum(D2O,pH 1.5)of the crude product containing 5 indicated the presenceof a new set of resonancesin additionto thosesignalsarisingfrom 5 (iatio of unknown/S = l0:l). Adjustment of the pH to 7.5 causedthe disappearance of thesesignals,and the spectrumnow consistedentirely of 5. We tentatively assignthesesignalsto diastereomeric a mixture of lactones. Experimental Section GeneralMethods.TLC plateswerevisualizedbyimmersionin anisaldehydestain(by volume: 93Voethanol,3.SVo sulfuric acid,l%oglacial aceticacid,and2.5Vo anisaldehyde) followedby heating.AG l-X8 and AG 50W-X8werepurchased from Bio-Rad(100-200mesh).Aldolase (rabbit muscle,E.C. 4.1.2.13),wasobtainedin lyophilizedform from phosphate SigmaChemicalCo. Dihydroxyacetone was preparedaccordingto the methodof Wonget al.l3 Satisfactory analyses werenot obtainedfor compounds 4, 5, and 6 owingto the difficulty experienced in preparingstablederivatives.The purity of eachwasjudgedto be ( 2 6 ) W i l s o n ,M . L . ; C o s c i a C , . J. J. Org.Chem.1979,44,302. (27) Anderson,V. E.; Weiss,P. M.; Cleland,W. W. Biochemistry1984, 23,2',t79. (28) Metzler,D. E.; Olivard,J.; Snell,E. E. "/. Am. Chem.Soc. 1934,76, 644. (29) We thank our colleagueProfessorJ. R. Knowles,Harvard University, for the gift of an authenticsampleof DAHP. 626 J. Am. Chem. Soc., Vol. lll, No. 2, 1989 -95Vo by 'H NMR and r3CNMR. N- Acetyl- o / l-allylglycine Methyl Ester. N- A cetyl-o I t- allyl g lyci ne7 (8.0 g, 50 mmol) was dissolvedin ethyl acetate (100 mL) and cooled to 0 oC. Diazomethane (a solution in ether) was added dropwise to the stirred solution until the yellow color persisted for 5 min. The excess diazomethanewas destroyedwith glacial acetic acid, and the solvent was removedin vacuo to give the methyl ester (8.7 g,l00%o): IR (film) 3270 ( s ) , 3 0 7 0 ( m ) , 2 9 4 0 ( m ) , 1 7 4 0( s ) , 1 6 5 5( s ) , 1 5 3 0( s ) , 1 4 3 5( m ) , 1 3 6 5 (m), l2l0 (s), I145 (m), 990 (m), 915 (m) cm-r;rH NMR (250MHz, C D C I 3 ) 6 1 . 9 8( s , 3 H ) , 2 . 3 9 - 2 . 6 2 ( m , 2 H ) , 3 3 0 ( s , 3 H ) , 4 . 5 9 - 4 . 6 7 ( m , I H ) , 5 . 0 2 - 5 . 1 4( m , 2 H ) , 5 . 5 8 - 5 . 7 4( m , I H ) , 6 . 1 8 - 6 . 3 2( d , , I = 5 H z , I H); t3C NMR (125J MHz, CDCI3) 6 22.63,36.12, 51.52, 52.03, 1 1 8 . 6 9 ,1 3 2 . 1 2 , 1 6 9 . 8 9 ,1 7 2 . 1 3 ;M S ( C I , i s o b u t a n e ) ,1 7 2 ( M H + , 1 0 0 ) , 1 4 0 ( 7 ) , 1 3 0 ( 8 ) , l l 2 ( a ) : e x a c t m a s sc a l c d f o r C r H r l N O 3 + 1 i 2 . 0 9 i 3 6 , found l'12.09742. Methyl N-Acetyl-o/l-aspartate p-Semialdehyde (3). The protected amino acid (8.7 g, 50 mmol) was dissolvedin CH2CI2 (40 mL) and CH3OH (10 mL) and cooled to -78 oC. Ozone was bubbled through until the solution became pale blue. Nitrogen was passed through to remove the excessozone. Dimethyl sulfide (3 mL) was added, and the solution was stirred overnight. The solvent was removed in vacuo, and the crude product was chromatographed on silica (ethyl acetate) to give the aldehyde3 (400 mg75Vo): IR (film) 3280 (s), 2950 (m), 1740 (s), 1 6 5 0( s ) , 1 5 3 0( s ) , 1 4 3 5( m ) , 1 3 7 0( m ) , 1 2 2 0( s ) , I 1 4 0 ( m ) , 1 0 4 0( m ) , " 1 2 5( m ) c m - r ; r H N M R ( 5 0 0 M H z , C D C I 3 ) 6 2 . 0 2 ( s , 3 H ) , 3 . 0 6 - 3 . 1 9 ( m , 2 H ) , 3 . 7 6 ( s , 3 H ) , 4 . 8 3 - 4 . 8 7( m , 1 H ) , 6 . 4 4 - 6 . 5 2 ( d , J = 7 H z , I H ) , 9 . 7 2 ( s , I H ) ; r 3 C N M R ( t 2 5 . 7 M H z , C D C I 3 )6 2 2 . 5 0 , 4 5 . 1 7 , 4 6 . 9 8 ,5 2 . 4 8 , 1 7 0 . 1 81, 7 1 . 0 5 ,1 9 9 . 3 4 M ; S ( C I , i s o b u t a n e )1, 7 4 ( M H + , 1 0 0 ) , 1 3 2 ( 1 3 ) , l l 4 ( 5 ) : e x a c tm a s sc a l c d f o r C 7 H r 2 N O 4 +\ i 4 . 0 7 6 6 3 , found 174.07655. (2RS, 4R, 5^S)-2-Acetamido-6-oxo-4,5,7-trihydroxyheptanoic Acid, 7-(Dihydrogen phosphate) (4). A solution of dihydroxyacetone phosphater3(19.5 mmol) in distilled water (250 mL) was adjustedto pH 6.8 with 2 N NaOH. To this solution was added the crude product cont a i n i n g t h e a l d e h y d e3 ( 3 . 0 0 g , 7 5 V o p u r e , 1 3 . 0 m m o l ) , t h e p H w a s readjustedto 6.8, and the solution was then purged with N, for 30 min. Aldolase (45 mg, 500 U) was added,and the reaction mixture was shaken ^t 125 rpm and 27 oC. After 22 h the pH was readjustedto 6.8, and additional aldolase( l6 mg, 200 U) was added. The degreeof conversion wasestimated by using the following protocol: an aliquot (l mL) of the reaction was removed,lyophilized, and resuspendedin D2O (0.60 mL). A n a c c u r a t e l y m e a s u r e dq u a n t i t y ( 0 . 0 5 m L ) o f a s o l u t i o n o f 0 . i S V c sodium 3-(trimethylsilyl) propion ate-2,2,3,3,-d4(TSP-d1) was added, and the lH NMR spectrum was measuredat 500 MHz. Integration of the resonancesof the product against TSP-d4 gave the following values for t h e e x t e n t o f c o n v e r s i o n( e r r o r S V a ) : 4 . 5 h ( l l V o ) , 2 2 h ( 2 0 V o ) , 2 8 . 5h ( 3 2 V o ) , 4 7h ( 3 7 V o ) , 7 1 h ( 4 0 V o ) .T h e r e a c t i o nw a s t e r m i n a t e da f t e r 7 l h, diluted to 500 mL with distilled water, and applied to a column of AG l-X8 resin (HCO3- form, 75 mL), followed by elution with 250 mL each of the following concentrations of triethylammonium bicarbonate: 150, 200,300,350 mM. Fractions (volume = l8 mL, total no. of fractions = 70) were collected and examined by TLC. Fractions 23-50 contained the desired product and were pooled and lyophilized; excessbuffer was removed by the addition of water (15 mL) to the residueand reevaporation (3 times). The white crystalline solid thus obtained was redissolved in water (150 mL) and passeddown a column of AG 50W-X8 resin (Na+ form, 50 mL). A further 100 mL of water was added. and the combined eluant was concentrated in vacuo to give the trisodium salt of 4 ( L88 g, 3 7 V o ) : f a l s + 8 . 8 o ( c 1 . 0 0 ,H z O ) ; I R ( N u j o l ) 3 2 5 0 ( s ) , 1 7 3 0 ( m ) , l 6 l 0 ( s ) , 1 0 7 0( s ) , 9 7 0 ( m ) , 9 2 0 ( m ) c m - t ; ' H N M R ( 5 0 0 M H z , D 2 O ,p H 6 . 5 ) 6 l . ' 1 9 - 2 . 2 2( c o m p l e xm , 2 H ) , 2 . 0 5 ( s , 1 . 2H ) , 2 . 0 6 ( s , 1 . 8 H ) , 4 . t 4 - 4 . 1 7 ( m , 0 . 6 7 H ) , 4 . 1 9 - 4 . 2 4( m , 0 . 6 7 H ) , 4 . 3 1 ( d d , , f = 3 . 5 , l l H 2 , 0 . 6 7 H ) , 4 . 4 2 ( d , J = 2 H 2 , 0 . 6 7 H ) , 4 . 4 5 ( d , J = 2 H 2 , 0 . 3 3 H ) , 4 . 4 7 - 4 . 8 6( m , 2 H ) ; ' 3 C N M R ( 1 2 5 . 7M H z , D 2 O , p H 6 . 5 ) 6 2 2 . 7 0 , 3 5 . 6 43, 5 . 8 2 ,5 2 . 9 4 , 5 3 . 4 4 ,6 9 . 0 8 ,6 9 . 2 3 ( " / p o c= 7 H z ) , 7 0 . 0 3 ,7 7 . 6 2 , 7 8 . 73 , 1 74 . 15 , 1 74 . 6 3 , 1 7 9 . 1 8 ,1 7 9 . 8 13; r PN M R ( t 2 1 . 4 7 M H z , D 2 O ,p H 6 . 5 ) 6 0 . 7 9 , 0 . 8 3( r a t i o 1.0:1.5); mlz (positive argon, fast atom bombardment) 396 (MH*). General Procedure for the Reduction of 4. (A). In Water. To a solution of the trisodium salt of 4 (30 mg, 0.070 mmol), dissolvedin water (3 mL), was added the appropriate borohydride reagent. After stirring for I h, the reaction was quenched by lowering the pH to 5 with 0.1 M HCl. The pH was readjustedto 7 with 0.1 M NaOH and then applied to a column of AG l-X8 resin (HCO3- form, 5 mL). After washing the column with triethylammonium bicarbonate (100 mM, 100 mL), the product was eluted with triethylammonium bicarbonate (300 mM, 100 mL). Lyophilization of the 300 mM fraction gave the triethylammonium salt of 5. (B). In Acetic Acid. To a solution of the trisodium salt of 4 (30 mg, 0.070 mmol), dissolvedin anhydrous glacial acetic acid (3 mL), was added the triacetoxyborohydride (3 equiv), and the mixture was stirred for 2-24 h. The solvent was removed in vacuo (azeotropically by Turner and Whitesides the addition of heptane, 3 x 5 mL), the residue was redissolvedin water, and the pH was adjusted to 7 with 0.1 M NaOH. Purification on AG lX-8 resin was carried out as describedin A. Results of reductionsdesignedto test the stereoselectivityof several reduction proceduresare summarized in Table I. (2RS,4R,5S,6RS )-2-Acetamido-4,5,6,7-tetrahydroxyheptanoicAcid, 7-(Dihydrogen phosphate)(5). The trisodium salt of the aldol product 4 (800 mg,2.03 mmol) was dissolvedin anhydrous glacial acetic acid (10 mL). To this solution was added tetramethylammonium triacetoxyborohydride ( I .58 g, 6.0 mmol, 3 equiv) in anhydrous glacial acetic acid (8 mL), and the mixture was stirred at room temperature for 2 h. Excess acetic acid was removed in vacuo (by azeotropicdistillation with heptane, 3 X 5 mL), and the resulting solid was taken up in water (50 mL). Sufficient AG 50W-X8 resin (H+ form) was then added to adjust the pH to 1.5, and the resin was removed by filtration. To the filtrate was added methanol (50 mL). The solvent was removed in vacuo at a water bath temperature of 40 oC, and the resulting gummy solid redissolved in water (25 mL) and methanol (25 mL) followed by evaporation to dryness. Drying under high vacuum for 3 h yielded the hydrogen form of 5 as a yellow solid (660 mg,98Vo). For characterization,a sample ( 100 mg) was removedand dissolvedin water (5 mL), and the pH was readjusted to 7 with dilute NaOH and followed by dilution to 20 mL with water (50 mL). The solutionwas applied to a column of AG l-X8 resin (HCO3- form, l0 mL) and eluted with the following concentrations (volume) of triethylammonium bicarbonate: 100 nM (100 mL) and 300 mM (100 mL). Concentration of the latter fraction, evaporationwith 2-propanol(3 times. l0 mL). gave the triethylammonium salt of 5. This w h i t e s o l i d w a s r e d i s s o l v e di n w a t e r ( 2 0 m L ) a n d a p p l i e d t o a c o l u m n o f A G 5 0 W - X 8 r e s i n( N a + f o r m , l 0 m L ) , e l u t e dw i t h w a t e r ( 2 0 0 m L ) , and lyophilizedto give 5 as the trisodium salt (107 mg,90Vo): IR (Nujol) 3 2 5 0( s ) , 1 5 9 0( s ) , 1 3 0 0( w ) , 1 0 7 0( s ) ,9 6 5 ( m ) , 9 2 0 ( m ) c m - r ; r H N M R ( 5 0 0 M H z , D 2 O . p H 6 . 0 ) 6 I . 7 1 - 2 . 2 2 ( c o m p l e xm , 2 H ) , 2 . 0 3 6 ( s , 0 . 9 6 H ) , 2 . 0 3 9 ( s , 0 . 2 4H ) , 2 . 0 5 4 ( s , 0 . 3 6H ) , 2 . 0 5 5 ( s , 1 . 4 4H ) , 3 . 4 8 - 4 . 0 8 , 4 . 2 0 - 4 . 2 5 , 4 . 3 2 - 4 . 3 8( c o m p l e xm , 6 H ) ; ' 3 C N M R ( t 2 5 . 7 M H z , D 2 O , p H 6 . 0 ) 6 2 2 . 76 , 3 5 . 9 4 ,3 6 . 0 8 ,3 6 . 4 6 ,5 2 . 9 5 ,5 3 .I 9 , 5 3 . 74 , 5 3 . 8l , 6 6 , 5 0 , 66.75,66.93, 68.05, 69.09, 70.02, 71.02, 71.72, 72.06, 73.49, 74.20, 1 7 1 . 8 4 , 1 7 4 . 2 3 , l ' 1 4 . 6 4 , 1 7 9 . 6 9 , 1 7 9 . 7 9 ,1 8 0 . 2 2 . 1 8 0 . 3 0 :3 r P N M R ( 1 2 1 . 4 7M H z , D 2 O ,p H 6 . 5 ) 6 2 . 2 5 , 2 . 6 9 :m l z ( p o s i t i v ea r g o n ,f a s t a t o m b o m b a r d m e n t )1 9 8 ( M H * ) . ( 2RS,4R,5.S.6RS) - 2-Amino-4,5,6,7-tetrehydroxyheptanoic Acid, 7(Dihydrogen phosphate)(6). The crude product containing 5 (600 mg, 1 . 8 m m o l ) w a s d i s s o l v e di n 6 N H C I ( 3 0 m L ) a n d h e a t e da t 1 0 0 o C f o r 60 min (TLC indicated complete removal of starting material). Removal of water gave a dark yellow solid that was redissolvedin water (100 mL). The pH was adjusted to 7.0 with I M NaOH, and the solution was applied to a column of AG l-X8 resin (HCO3- form, 30 mL). The column was sequentiallyeluted with the following concentrations(volume) of triethylammonium bicarbonate: 100 mM (200 mL), 150 mM (200 mL), 200 mM (200 mL), and 250 mM (200 mL). The product was located with ninhydrin in both the 150- and 200-mM fractions. These were concentrated,redissolvedin water (50 mL), and passeddown a column of AG 50W X-8 resin (H+ form, 30 mL), followed by elution with water (50 mL). The combined fractions were lyophilized and redissolvedin water (50 mL), the pH was adjustedto 7.0 with I M NaOH, and the fractions were lyophilized to give the disodium salt of 6 (390 mg, 6 5 V o ) :l R ( N u j o l ) 3 2 0 0 ( s ) , 1 6 2 5( s ) , 1 5 3 0( w ) , 1 0 7 0( s ) , 9 6 5 ( m ) , 7 2 0 ( w ) c m - ' ; ' H N M R ( 5 0 0 M H z , D 2 O , p H 7 . 0 ) 6 1 . 9 6 - 2 . 3 3( c o m p l e xm , 2 H ) , 3 , 1 9 - 4 . 1 2 ( c o m p l e xm , 6 H ) ; ' 3 C N M R ( 1 2 5 j M H z , D 2 O , p H 7 .0) 6 34.21,34.37, 34.89,53.26,53.49,54.37,65.95, 67.92,69.99,69.30, 7 1 . 1 7 ,7 1 . 8 6 ,7 2 . 9 1 ,7 3 . 2 9 ,74 . 4 1 , L ' ,5l . 4 2 ; 3 r pN M R ( 1 2 1 . 4 7M H z , D 2 O , p H 7 . 0 ) 6 4 . 0 6 , 4 . 5 0 , 4 . 5 4 , 4 . 8 0 ; m l z ( p o s i t i v ea r g o n , f a s t a t o m b o m bardment)334 (MH+). 3-Deoxy-o-arahino-heptulosonicAcid 7-Phosphate(l). To a solution of the disodium salt of 6 (210 mg, 0.63 mmol), dissolvedin water (30 mL), were added sodium glyoxylate (350 mg, 3.15 mmol, 5 equiv) and potassiumaluminum sulfate (30 mg, 0.063 mmol, l0 mol Vo). The pH was adjusted to 5 with I M HCI solution, and the mixture was heated at 100 oC. After 2 h, TLC indicated the complete disappearanceof starting material. The crude solution containing DAHP was allowed to cool to room temperature, neutralized to pH 7 with I M NaOH, and applied to a column of AG l-X8 resin (HCO3- form, 20 mL). The column was eluted first with triethylammonium bicarbonate(100 mM, 200 mL) followed by a linear gradient of triethylammonium bicarbonate (300 mL of 150 mM solution to 300 mL of 250 mM solution). Fractions containing DAHP (located by TLC against an authentic sample) were pooled and lyophilized. Excess buffer was removed by the addition of water and reevaporation(10 mL, 3 times). The resulting white solid was redissolvedin water (20 mL) and passeddown a column of AG 50W-X8 resin (H* form, 20 mL). The eluant was brought to pH 5.0 by the careful addition of 0.I M lithium hydroxide solution and then lyophilized 627 to give the dilithium salt of I (104 mg,55Vo)(-90Vo pure by 'H NMR): tH NMR (500 MHz, [ a ] o + 1 9 . 8 o ( c 0 . 5 0 ,H 2 O ) , a u t h e n t i c 3*o1 8 . 6 o ; D 2 O ,p H 5 . 0 ) d 1 . 8 0( d d , J = 1 2 , 1 3 H z , I H ) , 2 . 1 9 ( d d , " / = 5 , 3 H z , I H ) , 3 . 5 3 ( o v e r l a p p i n gd d , " / = l 0 H z ) , 3 . 8 2 - 3 . 8 7( m , I H ) , 3 . 9 2 - 3 . 9 7 ( m , I H ) , 4 . 0 ' l - 4 . 1 7( m , I H ) ; t 3 C N M R ( 1 2 5 . 7M H z , D 2 O , p H 5 . 0 ) 6 3 9 . 8 7 ,6 4 . 9 1 , 6 9 , 3 8 7, 1 . 1 4 , 7 3 . 5 2( d , . I p o c = 7 H z ) , 9 7 . 1 2 , 1 7 7 . 1 7 ; 3 t P NMR (121.47MHz, D2O, pH 5.0) 6 1.09. A sample of I (5 mg) was applied to a column of SephadexDEAE A-25 ion-exchangeresin (HCO3- form, l0 mL) and eluted with a linear gradient to triethylammonium bicarbonate(150 mL of 100 mM to 150 mL of 350 mM). The DAHP containing fractions were pooled and lyophilized to give a white solid that was redissolvedin water (10 mL) and passeddown a column of AG 50W-X8 resin (H+ form, l0 mL). Adjustment of the eluant to pH 5.0 with 0.1 M lithium hydroxide, followed by lyophilization, gave the dilithium salt of I (3 mg) ('H NMR indicated -95Vo purity). This samplewas used for the assaywith dehydroquinate synthase. Assay of Synthetic DAHP (l) with Dehydroquinate Synthase. The assay procedure for DAHP used a coupled enzyme system of dehydroquinate synthase and dehydroquinasewith subsequentmonitoring of dehydroshikimateproduction. Assay solutions(1.00 mL) containing 50 m M M O P S b u f f e r , p H 7 . 5 0 ,c o b a l t s u l f a t e( 5 0 l t M ) , N A D + ( 1 5 s . M ) , DAHP (500 pM), and 2 units of dehydroquinasewere incubated at 20 oC in quartz cuvettes. The reaction was initiated by the addition of 800 milliunits of DHQ synthase,and the production of dehydroshikimatewas monitored al.234 nm. Initial rates obtained from the first -20 s after mixing were as follows: synthetic DAHP, 0.123 AU/min; authentic D A H P , 0 . 3 3 4A U / m i n . Acknowledgment. We thank our colleagues Ethan Simon and Mark Bednarski, both of whom provided assistance with the initial aldolase experiments, and Keith Chenault, who ran the mass spectra. Dr. Steven Bender and Professor Jeremy Knowles provided an authentic sample of DAHP and conducted the enzymatic assays.