Probing the stereochemistry of E. coli 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (phenylalanine-sensitive)-catalyzed synthesis of KDO 8-P analogues

Article abstract of DOI:10.1021/jo991529l

The five-carbon phosphorylated monosaccharide analogues, D-arabinose 5-phosphate, D-ribose 5-phosphate, and 2-deoxy-D-ribose 5-phosphate, were separately condensed with (Z)- and (E)-[3-²H]-phosphoenolpyruvate (PEP) in the presence of Escherichia coli 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH 7-P) synthase (phe) to give in the case of (Z)-[3-²H]-PEP (3S)-[3-²H]-3-deoxy-D-manno-octulosonate 8-phosphate, (3S)-[3-²H]-3-deoxy-D-altro-octulosonate 8-phosphate, and (3S)-[3-²H]-3,5-dideoxy-D-altro-octulosonate 8-phosphate, respectively, whereas incubation with (E)-[3-²H]-PEP gives the corresponding (3R)-monosaccharides. These results are in complete agreement with the observed facial selectivity of DAH 7-P synthase for its normal substrates D-erythrose 4-phosphate and PEP and provide direct evidence that DAH 7-P synthase (phe) catalyzes the si face addition of the C3 of PEP to the re face of C1 of the phosphorylated monosaccharides tested. Products formed by DAH 7-P synthase (phe)-catalyzed condensation of (Z)- and (E)-[3-F]-PEP with E 4-P were completely characterized by ¹H and ¹⁹F NMR analysis for the first time. Results of our studies suggest that disappearence of the double bond between C2 and C3 of PEP and formation of a bond between C3 of PEP and C1 of the phosphorylated monosaccharide tested occur in concert during the DAH 7-P synthase-catalyzed condensation reaction.

Full text of DOI:10.1021/jo991529l

J . Org. Chem. 2000, 65, 5891-5897
5891
P r obin g th e Ster eoch em istr y of E. coli
3-Deoxy-D-a r a bin o-h ep tu loson a te 7-P h osp h a te Syn th a se
(P h en yla la n in e-Sen sitive)-Ca ta lyzed Syn th esis of KDO 8-P
An a logu es
Appavu K. Sundaram and Ronald W. Woodard*
Interdepartmental Program in Medicinal Chemistry, College of Pharmacy, University of Michigan,
Ann Arbor, Michigan 48109-1065
rww@umich.edu
Received September 28, 1999
The five-carbon phosphorylated monosaccharide analogues, D-arabinose 5-phosphate, D-ribose
5-phosphate, and 2-deoxy-D-ribose 5-phosphate, were separately condensed with (Z)- and (E)-[3-
2H]-phosphoenolpyruvate (PEP) in the presence of Escherichia coli 3-deoxy-D-arabino-heptulosonate
7-phosphate (DAH 7-P) synthase (phe) to give in the case of (Z)-[3-2H]-PEP (3S)-[3-2H]-3-deoxy-D-
manno-octulosonate 8-phosphate, (3S)-[3-2H]-3-deoxy-D-altro-octulosonate 8-phosphate, and (3S)-
[3-2H]-3,5-dideoxy-D-altro-octulosonate 8-phosphate, respectively, whereas incubation with (E)-[3-
2H]-PEP gives the corresponding (3R)-monosaccharides. These results are in complete agreement
with the observed facial selectivity of DAH 7-P synthase for its normal substrates D-erythrose
4-phosphate and PEP and provide direct evidence that DAH 7-P synthase (phe) catalyzes the si
face addition of the C3 of PEP to the re face of C1 of the phosphorylated monosaccharides tested.
Products formed by DAH 7-P synthase (phe)-catalyzed condensation of (Z)- and (E)-[3-F]-PEP with
1
E 4-P were completely characterized by H and 19F NMR analysis for the first time. Results of our
studies suggest that disappearence of the double bond between C2 and C3 of PEP and formation
of a bond between C3 of PEP and C1 of the phosphorylated monosaccharide tested occur in concert
during the DAH 7-P synthase-catalyzed condensation reaction.
In tr od u ction
methyl]-PEP,23-39 and/or (Z)- and (E)-[3-2H or 3-3H]-
PEP40-50 have been used in mechanistic studies of
pyruvate kinase,1,23,24,28,36,43 PEP carboxylase,5,35,38,43 PEP
carboxy-kinase,4,6,8,10,31,32 5-enolpyruvylshikimate 3-phos-
phate (EPSP) synthase,12,18 UDP-N-acetylglucosamine
enolpyruvyl transferase (EPTase),14-21,30,39 and 3-de-
oxy-D-manno-octulosonate 8-phosphate (KDO 8-P) syn-
thase.13,50 In the case of EPTase and EPSP synthase,
stable, fluorine-containing tetrahedral intermediates of
Regio- and stereospecifically labeled substrate ana-
logues are commonly used to probe enzyme reaction
mechanisms. A number of regiospecifically labeled phos-
phoenolpyruvate (PEP) analogues have been prepared
and utilized to study both the stereoselectivity and the
stereospecificity of several PEP-utilizing enzymes. The
analogues (Z)- and (E)-[3-F]-PEP,1-22 (Z)- and (E)-[3-
* To whom correspondence should be addressed.
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10.1021/jo991529l CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/22/2000

5892 J . Org. Chem., Vol. 65, No. 19, 2000
Sundaram and Woodard
the enzyme-catalyzed reactions have been isolated and
characterized using [3-F]-PEP as a pseudo-substrate.12,14-21
The breakdown of the fluorinated tetrahedral intermedi-
ates into the final products is not observed due to the
presence of the fluorine atom.51 The PEP analogues (Z)-
and (E)-[3-F]-PEP as well as (Z)- and (E)-[3-2H]-PEP have
been incubated with KDO 8-P synthase in the presence
of arabinose 5-phosphate (A 5-P), and the labeled KDO
8-Ps obtained have been isolated and characterized by
NMR analysis.13,50 The two regioisomers of [3-F]-PEP
serve as true substrates for KDO 8-P synthase, albeit at
different rates.13
ment of the stereochemistry was based soley on the study
described above by Floss and not on data from their own
study.
Later, Duggan and co-workers22 utilized (Z)- and (E)-
[3-F]-PEP (88:12) and E 4-P as substrates for DAH 7-P
synthase in the first step of a one-pot multistep enzy-
matic synthesis of [6-F]-shikimic acid. In this study each
enzyme involved in the biosynthetic pathway was added
successively to the reaction mixture and the reaction
progress monitored by 19F NMR. Based on the stereo-
chemistry of the final product, [6-F]-shikimic acid, and
the coupling constant values obtained from the mixture
of 3S and 3R [3-F]-DAH 7-Ps, the previously reported
stereochemical course of the DAH 7-P synthase catalyzed
reaction appears to be correct, although the individual
[3-F]-DAH 7-Ps were neither isolated nor completely
characterized. Sheflyan et al. have recently shown that
the five-carbon monosaccharides, A 5-P, 2-deoxy-D-ribose
5-phosphate (dR 5-P), and D-ribose 5-phosphate (R 5-P)
are substrates for DAH 7-P synthase (phe).52 Since
binding of these alternate substrates (longer by one
carbon atom) could potentially perturb the architecture
of the enzyme active site and hence may alter the binding
of PEP and/or alter the relative orientations of the two
substrates in the active site, we were interested in
studying the stereochemical course of the reaction using
these alternate monosaccharide substrates as potential
mechanistic probes.
3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAH
7-P) synthase, a pivotal enzyme in the biosynthesis of
aromatic amino acids, catalyzes an aldol-type condensa-
tion reaction similar to that catalyzed by KDO 8-P
synthase except between D-erythrose 4-phosphate (E 4-P)
and PEP. Floss and co-workers41,42 have utilized (Z)- and
(E)-[3-3H]-PEP, derived via a series of enzymatic trans-
formations of [1-3H] glucose and [1-3H] mannose, respec-
tively, to study the stereochemical course of DAH 7-P
synthase. The enzymatically obtained [3-3H]-DAH 7-Ps
were converted to [3-3H]-malates via a series of enzymatic
and chemical reactions. The stereochemistry of the
labeled malates was determined utilizing fumarase [EC
4.2.1.2]. On the basis of the known stereochemistry of
the enzymatic and chemical reactions involved in both
the synthesis of the [3-3H]-PEPs, as well as the degrada-
tion of DAH 7-P, a si face attack of the C3 of PEP on the
C1 re face of E 4-P was postulated. Pilch and Somerville3
reported an apparent Km of (Z)-[3-F]-PEP for DAH 7-P
synthase less than twice that of PEP (65 µM versus 38
µM) and an apparent Vmax 1% that of PEP. Although (E)-
[3-F]-PEP was completely converted to [3-F]-DAH 7-P,
the kinetics of the reaction were nonlinear. The authors
report that they isolated and obtained the NMR spectra
for the condensation product (3S)-[3-F]-DAH 7-P; how-
ever, no data or spectra were presented. Their assign-
In an effort to directly compare as well as to confirm
the results from previous studies of DAH 7-P synthase
to those for KDO 8-P synthase with various PEP ana-
logues, we have chemically synthesized (Z)- and (E)-[3-
F]-PEP as well as (Z)- and (E)-[3-2H]-PEP and utilized
them to investigate the stereoselectivity of the reaction
catalyzed by DAH 7-P synthase with its natural substrate
E 4-P as well as with several five-carbon analogues. The
KDO8P synthase condensation reaction with A 5-P and
both [3-F]-PEPs have also been repeated. The studies
involved the isolation and NMR characterization of the
isotopically labeled analogues obtained from the DAH 7-P
synthase-catalyzed condensation reactions.
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Resu lts a n d Discu ssion
The initial stereochemical studies of DAH 7-P synthase
focused on the condensation of (Z)-[3-F]-PEP with E 4-P.
The purified reaction product obtained from incubation
of (Z)-[3-F]-PEP and E 4-P with DAH 7-P synthase was
1
characterized by both H and 19F NMR spectroscopy, to
completely characterize, for the first time, the product.
The 19F NMR spectrum (Figure 1) shows one major and
two minor signals (each one as a doublet of a doublet).
The two-dimensional NMR spectrum (1H-19F HETCOR)
recorded for the product shows a correlation between the
major fluorine signal at δ ) -130.2 and the proton signal
at δ ) 4.8 as well as a correlation between the same
fluorine signal and the proton signal at δ ) 3.9 (data not
1
shown). The H NMR spectrum of the product confirms
that the fluorine on C3 is coupled to the C3 proton at δ
) 4.8 (J ) 49.1 Hz) as well as to the C4 proton at δ ) 3.9
(J ) 30.2 Hz) (Table 1). Incubation of PEP and E 4-P, A
5-P, R 5-P, or dR 5-P with DAH 7-P synthase results in
a phosphorylated monosaccharide elongated by three
carbons in which the orientation of the C4 proton is axial,
indicating that the stereochemistry at C4 of all the
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1996, 3, 83-91.

E. coli DAH 7-P Synthase-Catalyzed Synthesis of KDO 8-P Analogues
J . Org. Chem., Vol. 65, No. 19, 2000 5893
F igu r e 1. The 19F NMR spectrum of (3S)-[3-F]-DAH 7-P obtained from incubation of (Z)-[3-F]-PEP with E 4-P in the presence
of DAH 7-P synthase.
Ta ble 1. 1H NMR Da ta for (3S)-[3-F ]-DAH 7-P ]
substrate as well as to further confirm the above stereo-
chemical results, it was necessary to prepare (E)-[3-F]-
PEP. A 60:40 mixture of (Z)-[3-F]-PEP and (E)-[3-F]-PEP
was prepared by irradiating a 0.4 M solution of (Z)-[3-
F]-PEP in a Rayonet photochemical reactor at 254 nm
for 24 h.3 The progress of the photoisomerization was
monitored by 19F NMR (Figure 2) over a period of 24 h.
At equilibrium, the solution contained a 60:40 mixture
of (Z:E)-[3-F]-PEP, as determined from the 19F NMR as
well as the 1H NMR spectra. This mixture of regioisomers
and A 5-P were incubated with E. coli KDO 8-P synthase,
and the progress of the reaction was monitored by 19F
NMR. The results shown in Figure 3 confirm the earlier
report by Kohen et al.13 that KDO 8-P synthase shows a
preference for the E-isomer over the Z-isomer. However,
when an identical mixture of (Z)-[3-F]-PEP and (E)-[3-
F]-PEP (60:40) was incubated with E 4-P in the presence
of DAH 7-P synthase, both the isomers were consumed
at equal rates as evidenced by 19F NMR (Figure 4). The
19F NMR spectrum of the DAH 7-P synthase reaction
mixture shows four sets of signals (two major and two
minor signals, each one as a doublet of a doublet) in the
product region. The major signal at δ ) -130.2 and the
minor signal at δ ) -140.4 are identical to those observed
for the product obtained from the condensation of (Z)-
[3-F]-PEP and E 4-P (shown in Figure 1) and are hence
assigned to the R- and â-pyranose forms of (3S)-[3-F]-
DAH 7-P, respectively. The other major signal at δ )
-123.4 is identical to that of the minor signal at δ )
-123.4 in Figure 1 and is assigned to the R-pyranose form
of (3R)-[3-F]-DAH 7-P obtained from the condensation
of (E)-[3-F]-PEP with E 4-P. The minor signal at δ )
-125.1 with a coupling constant values of J ) 47.8 Hz
between the fluorine on C3 and the C3 proton and J )
15 Hz between the C3 fluorine and the C4 proton
(indicating an equatorial orientation for the C3 fluorine)
is assigned to the â-pyranose form of the product (3R)-
[3-F]-DAH 7-P. Therefore, in the case of DAH 7-P
synthase-catalyzed condensation of [3-F]-PEP with E 4-P,
the Z-isomer forms (3S)-[3-F]-DAH 7-P while the E-
isomer forms (3R)-[3-F]-DAH 7-P. These results clearly
indicate that the si face of PEP attacks the re face of E
4-P during the DAH 7-P synthase-catalyzed condensation
reaction. Unlike KDO 8-P synthase, DAH 7-P synthase
does NOT show any prefernce for one regioisomer over
the other, at least in the case of [3-F]-PEP.
C
δ, ppm
no. (multiplicity)
J values, Hz
3
4
4.8 (dd)
3.9 (ddd)
2.5 (C3-Heq-C4-Hax), 49.1 (C3-Heq-C3-Fax)
2.5 (C4-Hax-C3-Heq), 9.8 (C4-Hax-C5-Hax),
30.2 (C4-Hax-C3-Fax
9.8 (C5-Hax-C4-Hax), 9.8 (C5-Hax-C6-Hax
)
5
6
7
3.7 (dd)
3.8 (ddd)
4.1 (mult)
)
9.8 (C6-Hax-C5-Hax)
nd
a
nd ) not determined.
products is R.52 The coupling constant values of J ) 2.5
Hz between the C3 and C4 protons and J ) 9.8 Hz
between C4 and C5 protons observed here is consistent
with an axial orientation of C4 proton and an equatorial
orientation of C3 proton in the product. The three-bond
coupling constant value of J ) 30.2 Hz observed between
C4 proton (axial) and the C3 fluorine indicates a trans-
diaxial orientation of both the atoms in the product.53,54
The major fluorine signal at δ ) -130.2 is thus assigned
to the R-pyranose form of (3S)-[3-F]-DAH 7-P. The minor
signal at δ ) -140.4 with similar coupling constant
values of J ) 51.3 Hz between the C3 fluorine and the
C3 proton and J ) 29 Hz between the C3 fluorine and
the C4 proton (axial) indicates a similar axial orientation
of the C3 fluorine and hence is assigned to the minor
â-pyranose form of (3S)-[3-F]-DAH 7-P. Therefore, incu-
bation of (Z)-[3-F]-PEP and E 4-P in the presence of DAH
7-P synthase forms (3S)-[3-F]-DAH 7-P. The other minor
signal at δ ) -123.4 shows a coupling constant value of
J ) 50 Hz between the C3 fluorine and the C3 proton
and J ) 12.9 Hz between the C3 fluorine and the C4
proton (axial). The smaller three-bond coupling constant
value of J ) 12.9 Hz between the axial proton on C4 and
the fluorine on C3 indicates a gauche arrangement of the
two atoms, and thus the C3 fluorine in this product is
equatorial. The minor signal at δ ) -123.4 is assigned
to the minor product (3R)-[3-F]-DAH 7-P obtained from
condensation of (E)-[3-F]-PEP (8% contaminant in (Z)-
[3-F]-PEP) and E 4-P.
To determine if DAH 7-P synthase demonstrates a
preference for one regioisomer over the other as a
(52) Sheflyan, G. Y.; Howe, D. L.; Wilson, T. L.; Woodard, R. W. J .
Am. Chem. Soc. 1998, 120, 11027-11032.
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1986, 154, 127-144.
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1989, 187, 43-66.

5894 J . Org. Chem., Vol. 65, No. 19, 2000
Sundaram and Woodard
F igu r e 2. The 19F NMR spectra of 0.4M (Z)-[3-F]-PEP solution at various time intervals during UV irradiation at 254 nm to
form a mixture of (Z)- and (E)-[3-F]-PEP.
F igu r e 3. The 19F NMR spectra, at various time intervals, of the reaction mixture containing A 5-P, a 60:40 mixture of (Z)-and
(E)-[3-F]-PEP (-65 ppm and -73 ppm, respectively) and KDO 8-P synthase in 0.2 M Tris-acetate buffer (pH 7.5) and 10% D2O
at 37 °C. Chemical shifts of (Z)- and (E)-[3-F]-PEP are different from those in Figure 2 due to the prsence of the buffers and the
difference in the pH.
F igu r e 4. The 19F NMR spectra of the reaction mixture containing E 4-P, a 60:40 mixture of (Z)- and (E)-[3-F]-PEP and
manganese(II) chloride in BTP buffer (pH 6.8) and 10% D2O at various time intervals after addition of DAH 7-P synthase at room
temperature. Chemical shifts of (Z)-and (E)-[3-F]-PEP are different from those in Figure 2 due to the prsence of the buffers and
the difference in the pH.
Since there is an electronic perturbation in [3-F]-PEP,
caused by the substitution of F for H, we wanted to utilize
a PEP analogue that is electronically more similar to
PEP. Therefore, to substantiate the results obtained from
the studies of [3-F]-PEP with DAH 7-P synthase as well
as to probe the stereochemistry of the enzyme-catalyzed
reaction when the five-carbon alternate substrates are
incubated with DAH 7-P synthase, reactions employing
(Z)- and (E)-[3-2H]-PEP as PEP analogues were per-
formed. Condensation products obtained from incubation
of either (Z)- or (E)-[3-2H]-PEP with E 4-P (Scheme 1), A
5-P, R 5-P, and dR 5-P in separate reactions were purified
and then characterized by H NMR spectroscopy. Incuba-
1
tion of (Z)-[3-2H]-PEP with E 4-P, A 5-P, and dR 5-P
forms (3S)-[3-2H]-DAH 7-P, (3S)-[3-2H]-KDO 8-P and
(3S)-[3-2H]-3,5-dideoxy-D-altro(manno)-octulosonate 8-phos-
phate, respectively, whereas incubation of (E)-[3-2H]-PEP
with E 4-P, A 5-P, and dR 5-P forms (3R)-[3-2H]-DAH
7-P, (3R)-[3-2H]-KDO 8-P and (3R)-[3-2H]-3,5-dideoxy-D-
altro(manno)-octulosonate 8-phosphate, respectively. The
NMR analysis of the products obtained from the incuba-
tion of (Z)- and (E)-[3-2H]-PEP with R 5-P in the presence

E. coli DAH 7-P Synthase-Catalyzed Synthesis of KDO 8-P Analogues
J . Org. Chem., Vol. 65, No. 19, 2000 5895
Sch em e 1
of J ) 11.5 Hz between the C3 proton and C4 axial proton
suggests a trans-diaxial relationship between the two
protons, consistent with incorporation of deuterium into
the C3 equatorial position, indicating an R configuration
1
at C3 for the product. The H NMR spectrum recorded
for the product obtained from incubation of (Z)-[3-2H]-
PEP with R 5-P (Figure 5c) shows the disappearance of
the signal for the C3 axial proton and the presence of a
simple doublet (with a coupling constant value of J ) 5
Hz between C3 and C4 protons) which would correspond
to the signal for C3 equatorial proton. A coupling constant
value of 5 Hz between the C4-axial proton and the proton
on C3, indicative of a gauche orientation between the two
protons, suggests that the C3 proton is in an equatorial
orientation, consistent with incorporation of deuterium
into the C3 axial position in the product, indicating an S
configuration at C3 of the product. Therefore, the con-
densation of (Z)-[3-2H]-PEP with R 5-P forms (3S)-[3-2H]-
D-altro-octulosonate 8-phosphate while the condensation
of (E)-[3-2H]-PEP with R 5-P forms (3R)-[3-2H]-D-altro-
octulosonate 8-phosphate.
F igu r e 5. The partial 1H NMR spectra (signals for the C3
protons) of the products obtained from DAH 7-P synthase-
catalyzed condensation reactions between R 5-P and (a) PEP,
(b) (E)-[3-2H]-PEP, and (c) (Z)-[3-2H]-PEP.
In this paper, we report the isolation, purification, and
characterization of the products formed from the DAH
7-P synthase (phe)-catalyzed condensation of various
stereospecifically labeled PEP analogues and four differ-
ent phosphorylated monosaccharides. Characterization
of these products confirms that the DAH 7-P synthase-
catalyzed condensation of PEP analogues with E 4-P
analogues proceeds via the same facial selectivity and
hence follows the same stereochemical course, irrespec-
tive of the substrate. These results further confirm that
the relative orientations of both the PEP and E 4-P
analogues in the enzyme active site and that the reaction
mechanism for alternate substrates is the same as for
the normal substrates, E 4-P and PEP. These results
provide direct evidence for the si face addition of PEP to
the re face of the aldehyde group of the respective
monosaccharide substrate during the DAH 7-P synthase-
catalyzed condensation reaction, which is the same facial
selectivity observed for KDO 8-P synthase. This adds to
the list of PEP-utilizing enzymes that have been shown
to be si-face specific, suggesting that there may be a
common PEP binding motif for all these enzymes. Our
observations indicate that both KDO 8-P synthase and
of DAH7P synthase is presented below as a typical
example of product characterization.
The regions of the 1H NMR spectra containing the
signals for C3 protons of the C3 deuterated monosaccha-
rides obtained from incubation of [3-2H]-PEPs with R 5-P
1
are shown in Figure 5. The partial H NMR spectrum of
the protio-compound (D-altro-octulosonate 8-phosphate)
is also shown in Figure 5. In the case of D-altro-
octulosonate 8-phosphate (Figure 5a), the C3 axial proton
signal appears as a triplet at δ ) 1.72, while the C3
equatorial proton signal appears as a doublet of a doublet
at δ ) 2.1.52 The 1H NMR spectra recorded for the product
obtained from incubation of (E)-[3-2H]-PEP with R 5-P
(Figure 5b) shows the disappearance of the signal for the
C3 equatorial proton and the appearance of a simple
doublet (with a coupling constant values of J ) 11.5 Hz
between C3 and C4 protons) that would correspond to
the signal for C3 axial proton. A coupling constant value

5896 J . Org. Chem., Vol. 65, No. 19, 2000
Sundaram and Woodard
UV Isom er iza tion of (Z)-[3-F ]-P EP a n d (E)-[3-F ]-P EP
CHA Sa lts. A 60:40 mixture of (Z)-[3-F]-PEP and (E)-[3-F]-
PEP was obtained by irradiating a 0.4 M solution of (Z)-[3-
F]-PEP in D2O (0.5 mL). The solution in a 5 mm quartz NMR
tube (WILMAD) was irradiated at 254 nm at room tempera-
ture, in a Rayonet photochemical reactor for 24 h.3 The
conversion of (Z)-[3-F]-PEP to (E)-[3-F]-PEP was monitored
by 19F NMR. At equilibrium, the solution contained a 60:40
mixture of (Z)-[3-F]-PEP and (E)-[3-F]-PEP, as evidenced by
19F NMR and 1H NMR.
DAH 7-P synthase may have similar binding sites for
both the substrates and some mechanistic commonalties.
The results suggest that if a “carbanion-like species” is
formed at C3 of PEP that attacks the electrophilic C1
aldehyde carbon of the phosphorylated monosaccharide,
as hypothesized earlier by Floss and co-workers for DAH
7-P synthase and by our group for KDO 8-P synthase,
that the lifetime of this anionic species must be shorter
than the time required for the rotation of a methyl group
(<10-10 s). Although it is possible that the free rotation
of the carbanion is significantly restricted in the enzyme
active site, it seems rather unlikely suggesting that
disappearance of the double bond between C2 and C3 of
PEP and formation of a bond between C3 of PEP and C1
of the phosphorylated monosaccharide occur in concert.
Finally, DAH 7-P synthase utilizes both the regioisomers
of [3-F]-PEP at equal rates, unlike KDO 8-P synthase
which exhibits a preference for (E)-[3-F]-PEP over (Z)-
[3-F]-PEP.
(Z)-[3-2H]-P EP a n d (E)-[3-2H]-P EP CHA sa lts. (Z)-[3-2H]-
PEP was prepared as previously reported.49
(E)-[3-2H]-PEP was synthesized by the method reported by
Dotson et al.50 Ethyl bromopyruvate was brominated using
N-bromosuccinimide to give ethyl 3,3-dibromopyruvate, which
upon treatment with trimethyl phosphite under standard
Perkow-type reaction conditions gave a 72:28 mixture of ethyl
(Z)-3-bromo-2-[(dimethoxyphosphinyl)oxy]propenoate and eth-
yl (E)-3-bromo-2-[(dimethoxy-phosphinyl)oxy]propenoate. This
mixture of Z- and E-isomers was treated with tetrakis-
(triphenylphosphine)palladium(0) (1 equiv with respect to the
E-isomer) to give an 80:20 mixture of (E)- and (Z)-[2-
((dimethoxyphosphinyl)oxy)-3-ethoxy-3-oxo-1-propenyl]bromo-
bis(triphenylphosphine)palladium. The E-vinyl palladium com-
plex was separated from the Z-vinyl palladium complex by
flash chromatography on silica gel 60 (230-400 mesh, E.
Merck) using a stepwise gradient of 0, 1, 2, and 3% methanol
in dichloromethane.50
Exp er im en ta l Section
Gen er a l. 1H NMR spectra were acquired on a Bruker
Avance DRX 500 (operating at 500.132 MHz for 1H) with a 5
mm multinuclear inverse gradient probe using the water
suppression program, WATERGATE Gradient Suppression.57
19F NMR spectra were recorded on a Bruker Avance 300
(operating at 282.36 MHz for 19F) and referenced externally
to trifluoroacetic acid (δ ) 0). E 4-P, A 5-P, R 5-P, dR 5-P,
manganese (II) chloride, and trifluoroacetic anhydride were
obtained from Sigma Chemical Co. Bromine and N-bromo-
succinimde were purchased from Fisher Scientific. Cyclohexyl-
amine, diethyl oxalate, ethyl bromopyruvate, ethyl fluoro-
acetate, trimethyl phosphite, and tetrakis(triphenylphos-
phine)palladium(0) were purchased from Aldrich Chemical Co.
Recombinant DAH 7-P synthase (phenylalanine-sensitive) and
KDO 8-P synthase were isolated and purified as previously
reported.52
The purified E-vinyl palladium complex was treated with a
mixture of trifluoroacetic acid-D (99.5 atom % D, Aldrich) and
trifluoroacetic anhydride, under anhydrous conditions to give
ethyl (E)-3-deuterio-2-[(dimethoxyphosphinyl)oxy]propenoate,
which was hydrolyzed using 1 N KOH and immediately loaded
onto a Biorex-70 (BioRad) cation exchange column (1 × 5 cm,
in the proton form). Cyclohexylamine (1 equiv with respect to
the E-vinyl palladium complex) was added to the eluent from
the column, and the mixture was lyophilized to give the CHA
salt of (E)-[3-2H]-PEP.
Gen er a l En zym a tic Syn th esis of [3-2H]-DAH 7-P s a n d
[3-2H]-KDO 8-P An a logu es. To a Chemtube (BioRad, 12 ×
75 mm), containing a solution of 1,3-bis[tris(hydroxymethyl)-
methylamino]propane (BTP) (42.35 mg, 0.15 mmol) and man-
ganese(II) chloride (0.3 mg, 1.5 µmol) in water, was added 13
mg of one of the following monosaccharides, E 4-P (0.044
mmol), A 5-P (0.056 mmol), R 5-P (0.056 mmol), or dR 5-P (0.06
mmol) and 12 mg of either (Z)- or (E)-[3-2H]-PEP CHA salt
(0.044 mmol), and the pH was adjusted to 6.8 using 1 N NaOH.
DAH 7-P synthase (3 mg, 79 nmol) was then added to initiate
the reaction, and the final volume of the reaction mixtures
were made up to 2 mL. Reaction mixtures were incubated at
37 °C for 2 h. The enzymatic reactions were quenched by
adding 0.5 mL of 10% trichloroacetic acid (TCA), vortexed for
30 s, and finally centrifuged for 30 min (1500g) to remove the
precipitated protein. The supernatants were loaded onto 5 mL
Econo-Pac HighQ (BioRad) anion-exchange columns (chloride
form), preequilibrated with water. The columns were washed
(Z)-[3-F ]-P EP Cycloh exyla m m on iu m Sa lt. The title
compound was prepared by the procedure originally reported
by Bergmann and Shahak58 and modified by Stubbe and
Kenyon.1 In brief, diethyl oxalate was treated with sodium
ethyl fluoroacetate to give the sodium enolate of diethyl
fluorooxaloacetate which was directly brominated to give
diethyl bromofluorooxaloacetate. After purification by distil-
lation, the diethyl bromofluorooxaloacetate (86 °C at 0.7
mmHg) was heated at 80 °C for 8 h in concentrated HCl.
Removal of the excess acid in vacuo followed by distillation
gave pure bromofluoropyruvic acid which solidified upon
cooling. The bromofluoropyruvic acid was converted into (Z)-
3-F-2-[(dimethoxyphosphinyl)oxy]propenoic acid under stand-
ard Perkow-type reaction conditions (reaction with trimethyl
phosphite at 0 °C followed by slow warming to room temper-
ature over a 5 h period). This dimethyl ester was hydrolyzed
by dissolving it in water and stirring at room temperature for
8 h. One equivalent of cyclohexylamine was then added, and
the solution was immediately lyophilized to give the mono-
cyclohexylammonium (CHA) salt of (Z)-[3-F]-PEP (92% Z-
with 30 mL of water at
a flow rate of 1 mL/min. The
phosphorylated monosaccharides were eluted from the column
using a linear gradient of 0 to 0.5 M LiCl solution over a period
of 1 h. Fractions containing 3-deuterated DAH 7-P and KDO
8-P analogues, as identified by the thiobarbituric acid as-
say,60,61 were pooled and lyophilized.
1
isomer and 8% E-isomer as determined by H and 19F NMR).
NOTE: the addition of cyclohexylamine to the aqueous solu-
tion of the diester followed by stirring at room temperature,
as in the standard procedure for the preparation of unlabeled
PEP,59 leads to total decomposition.
En zym a tic Syn th esis of (3S)-[3-F ]-DAH 7-P a n d (3R)-
[3-F ]-DAH 7-P . (Z)-[3-F]-PEP or a mixture of (Z)- and (E)-[3-
F]-PEP (60:40) (15 mg, 0.052 mmol) was dissolved in 100 µL
of 0.5 M BTP (pH ) 11.0) in a 1.5 mL microcentrifuge tube.
To these solutions was added 15 mg of E 4-P (0.056 mmol),
and the pH was adjusted to 6.8 using 0.5 M BTP solution (pH
) 11.0). After the addition of 30 µL of 50 mM manganese(II)
chloride (1.5 µmol) solution, 50 µL of D2O was added for
deuterium lock. The reaction mixtures were transferred to
(55) Hansen, P. E. In Annual Reports on NMR Spectroscopy; Webb,
G. A., Ed.; Academic Press: London, 1983; Vol. 15, pp 118-119.
(56) Hansen, P. E. In Progress in NMR Spectroscopy; Pergamon-
Elsevier Science Ltd.: Oxford, U.K., 1988; Vol. 20, pp 207-255.
(57) Piotto, M.; Saudek, V.; Sklenar, V. J . Biomol. NMR 1992, 2,
661-665.
(58) Bergmann, E. D.; Shahak, I. J . Chem. Soc. 1960, 462-463.
(59) Clark, V. M.; Kirby, A. J . Biochim. Biophys. Acta 1963, 78, 732.
(60) Aminoff, D. Biochem. J . 1961, 81, 384-392.
(61) Ray, P. H. J . Bacteriol. 1980, 141, 635-44.

E. coli DAH 7-P Synthase-Catalyzed Synthesis of KDO 8-P Analogues
J . Org. Chem., Vol. 65, No. 19, 2000 5897
NMR tubes. DAH 7-P synthase (3 mg, 79 nmol) was added to
the NMR tubes to initiate the reactions, and the final volumes
were adjusted to 500 µL. The reaction mixtures were incubated
at room temperature, and the reaction progress was followed
by 19F NMR. After 4 h, the reactions were quenched by the
addition of 500 µL of 10% TCA. [3-F]-DAH 7-P was purified
as described above for [3-2H]-DAH 7-P. Fractions containing
[3-F]-DAH 7-P, as identified by 19F NMR, were pooled and
lyophilized.
W. Taylor for their critical reading and discussion of the
manuscript. The contents of this paper are solely the
responsibility of the authors and do not necessarily
reflect the official views of NIGMS.
Su p p or tin g In for m a tion Ava ila ble: The region of 1H
NMR spectra containing the signals for C3 protons of (3R)-
[3-2H]-DAH 7-P, (3S)-[3-2H]- DAH 7-P, (3R)-[3-2H]-3,5-dideoxy-
D-altro(manno)-octulosonate 8-phosphate, and (3S)-[3-2H]-3,5-
dideoxy-D-altro(manno)-octulosonate 8-phosphate. This material
is available free of charge via the Internet at http://pubs.acs.org.
Ack n ow led gm en t . This investigation was sup-
ported by U. S. Public Health Service Grant GM 53069.
We thank M. Birck, D. Howe, and Drs. H. Duewel and
J O991529L