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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.