Synthesis of 2-Bromo- and 2-Fluoro-3-dehydroshikimic Acids and 2-Bromo- and 2-Fluoroshikimic Acids Using Synthetic and Enzymatic Approaches

Article abstract of DOI:10.1021/jo971858i

The syntheses of 2-bromo-3-dehydroshikimic acid¹ (5) and 2-bromoshikimic acid² (8), are described. Their formation from (2R)-2-bromo-3-dehydroquinic acid (2) using dehydroquinase and shikimate dehydrogenase is also reported. The corr

Full text of DOI:10.1021/jo971858i

J . Org. Chem. 1998, 63, 1591-1597
1591
Syn th esis of 2-Br om o- a n d 2-F lu or o-3-d eh yd r osh ik im ic Acid s a n d
2-Br om o- a n d 2-F lu or osh ik im ic Acid s Usin g Syn th etic a n d
En zym a tic Ap p r oa ch es
C. Gonza´lez-Bello,†,‡ M. K. Manthey,†,§ J . H. Harris,†,| A. R. Hawkins, J . R. Coggins,X and
C. Abell*,†
Cambridge Centre for Molecular Recognition, University Chemical Laboratory, Lensfield Road,
Cambridge CB2 1EW, UK, Department of Biochemistry and Genetics, University of Newcastle upon Tyne,
Newcastle upon Tyne NE2 4HH, UK, Department of Biochemistry, University of Glasgow,
Glasgow G12 8QQ, UK
Received October 8, 1997
The syntheses of 2-bromo-3-dehydroshikimic acid1 (5) and 2-bromoshikimic acid2 (8), are described.
Their formation from (2R)-2-bromo-3-dehydroquinic acid (2) using dehydroquinase and shikimate
dehydrogenase is also reported. The corresponding enzymatic formation of 2-fluoro-3-dehydro-
shikimic acid1 (6) and 2-fluoroshikimic acid2 (9) from (2R)-2-fluoro-3-dehydroquinic acid (3) are
reported.
Sch em e 1a
In tr od u ction
The shikimate pathway is the biosynthetic pathway
utilized by plants, fungi, and microorganisms3 for the
synthesis of the amino acids L-phenylalanine, L-tryp-
tophan, and L-tyrosine, as well as precursors to the folate
coenzymes, alkaloids, and vitamins. Analogues of shiki-
mate pathway intermediates are of interest as potential
enzyme inhibitors, and consequently a lot of synthetic
effort has been directed to this area.4 However, it is only
recently that much attention has been focused on ana-
logues substituted at the 2-position with the publication
of syntheses of 2-chloro-5 and 2-fluoroshikimic acids,6 and
(2R)-bromo- and (2R)-2-fluoro-3-dehydroquinic acids.7
Syntheses of (2R)- and (2S)-2-hydroxyquinic acids8 and
(2R)-2-bromoquinic acid had been published previously.9
In this paper we report the first syntheses of the 2-bromo-
3-dehydroshikimic acid (5), 2-fluoro-3-dehydroshikimic
acid (6), 2-bromoshikimic acid (8), and a novel synthesis
of 2-fluoroshikimic acid (9), using a combination of
synthetic and enzymatic approaches (Scheme 1).
a
Enzymes: (i) dehydroquinase; (ii) shikimate dehydrogenase.
Syn th esis of 2-Br om o-3-Deh yd r osh ik im ic Acid .
We have previously published a synthesis of (2R)-2-
bromo-3-dehydroquinic acid (2).7 The bromolactone 11
was a key intermediate in that synthesis, formed in six
steps and 29% from quinic acid (10) (Scheme 2). This
compound is the starting point for the synthesis of
2-bromo-3-dehydroshikimic acid (5).
Methyl dehydroquinate has been reported to undergo
quantitative conversion to methyl dehydroshikimate
upon treatment with triethylamine in dichloromethane.10
We therefore initially attempted to prepare 2-bromo-3-
dehydroshikimic acid in the same way, after first con-
verting the lactone 11 into the methyl ester 12 (Scheme
2). Opening the lactone 11 with a catalytic quantity of
sodium methoxide in methanol afforded the expected
methyl ester 12 in 68% yield, along with a small quantity
of the epoxide 13 (6%). This reaction could not easily be
optimized further as use of more sodium methoxide
increased the proportion of epoxide formed, and using
greater than a stoichiometric amount of sodium meth-
oxide or extended reaction times led to aromatization.
Treatment of 12 with triethylamine in dichloromethane
did not result in the expected formation of 16 (Scheme
3), but rather the rapid formation of 14 (84%). An
alternative two-step procedure was therefore devised,
involving initial elimination of water from 12 using
TFAA/pyridine. This proceeded well in 90% yield but
resulted in unwanted trifluoracetylation at C-5. The
* Corresponding author. tel 01223 336405. FAX 01223 336362.
e-mail ca26@cam.ac.uk.
† Cambridge Center for Molecular Recognition.
‡ Present address: Departamento de Quimica Orga´nica, Univer-
sidade de Santiago de Compostela, 15706 Santiago de Compostela,
Spain.
§ Present address: Department of Biochemistry, The University of
Sydney, Sydney NSW 2006, Australia.
| Present address: Department of Chemistry, University of Alberta,
Edmonton, Alberta, Canada T6G 2G2.
University of Newcastle upon Tyne.
X University of Glasgow.
(1) IUPAC names: (4S,5R)-2-bromo-4,5-dihydroxy-3-oxocyclohex-1-
enecarboxylic acid, (4S,5R)-2-fluoro-4,5-dihydroxy-3-oxocyclohex-1-en-
ecarboxylic acid, respectively.
(2) IUPAC names: (3R,4S,5R)-2-bromo-3,4,5-trihydroxycyclohex-1-
enecarboxylic acid, (3R,4S,5R)-2-fluoro-3,4,5-trihydroxycyclohex-1-en-
ecarboxylic acid, respectively.
(3) Bentley, R. CRC Crit. Rev. Biochem. 1990, 25, 307.
(4) Campbell, M. M.; Sainsbury, M.; Searle, P. A. Synthesis, 1993,
179.
(5) Rich, R. H.; Lawrence, B. M.; Barlett, P. A. J . Org. Chem., 1994,
59, 693.
(6) Rich, R. H.; Barlett, P. A. J . Org. Chem. 1996, 61, 3916.
(7) Manthey, M. K.; Gonza´lez-Bello, C.; Abell, C. J . Chem. Soc.,
Perkin Trans. 1, 1997, 625.
(8) Adlersberg, M.; Bondinell, W. E.; Sprinson, D. B. J . Am. Chem.
Soc., 1973, 95, 887.
(9) Grewe, R.; Lorenzen, W. Chem. Ber. 1953, 86, 928.
(10) Delfourne, E.; Despeyroux, P.; Gorrichon, L.; Veronique, J . J .
Chem. Res., Synop. 1991, 3, 56.
S0022-3263(97)01858-6 CCC: $15.00 © 1998 American Chemical Society
Published on Web 02/07/1998

1592 J . Org. Chem., Vol. 63, No. 5, 1998
Gonza´lez-Bello et al.
Sch em e 2a
Sch em e 4a
a
Reagents and conditions: (i) p-CH3OPhCH2OH, n-BuLi, THF,
room temp; (ii) TFAA, pyridine, room temp; (iii) K2CO3, H2O, THF,
room temp; (iv) AcOH, THF, H2O (4:1:1), 40 °C.
a
Reagents, conditions, and yields: (i) ref 7; (ii) CH3ONa,
H2SO4/H2O. In these reactions either no reaction was
observed or the starting material decomposed with none
of the required product formed.
methanol (74%).
Sch em e 3a
Because of the problems of removing the methyl ester,
the strategy was revised so that the final step involved
removal of the more labile, acid-sensitive p-methoxyben-
zyl ester. The ester 18 was formed easily in 80% yield
by opening the lactone 11 with the anion of p-methoxy-
benzyl alcohol (Scheme 4). The subsequent elimination
and trifluoracetylation proceeded in 72% yield to give 19,
from which the trifluoracetyl group was smoothly re-
moved to form p-methoxybenzyl 2-bromo-3-dehydroshiki-
mate (20) (78%). Final removal of both the TBDMS and
p-methoxybenzyl protecting groups from 20 was readily
achieved employing HOAc/H2O/THF at 40 °C to give the
required 2-bromo-3-dehydroshikimic acid 5 in 92% yield.
En zym a tic Con ver sion of (2R)-Br om o- a n d (2R)-
2-F lu or od eh yd r oqu in ic Acid s. An alternative ap-
proach to the synthesis of 2-bromo-3-dehydroshikimic
acid (5) is by enzyme-catalyzed elimination of water from
(2R)-2-bromo-3-dehydroquinic acid (2). This would be
analogous to the conversion of 3-dehydroquinic acid (1)
to 3-dehydroshikimic acid (4) catalyzed by the third
enzyme on the shikimate pathway, dehydroquinase (3-
dehydroquinate dehydratase).3
a
Reagents and conditions: (i) Et3N, dichloromethane, room
temp; (ii) TFAA, pyridine, room temp; (iii) K2CO3, H2O, THF, room
temp.; (iv) AcOH, THF, H2O, (4:1:1), 40 °C.
There is a subtlety in the enzymology of dehydro-
quinase that we needed to take into account before
proceding. The most studied form of dehydroquinase is
the type I enzyme from E. coli. This enzyme catalyses
the conversion of 1 to 4 (Scheme 1) by a mechanism
involving Schiff base formation between the substrate
and an active site lysine and involves loss of the equato-
rial pro-R hydrogen from C-2, corresponding to an overall
syn elimination.15 The structurally distinct type II dehy-
droquinases, which are involved in both the biosynthetic
shikimate pathway and the catabolic quinate pathway,16
catalyze the same conversion but with the opposite
trifluoracetyl group was removed from 15 by using
potasssium carbonate in water-THF, affording 16 in 81%
yield, which upon desilylation (THF/HOAc/H2O) gave a
92% yield of the methyl 2-bromo-3-dehydroshikimate
(17). Unfortunately conditions could not be found for the
removal of the methyl ester despite a number of attempts,
by using inter alia K2CO3/H2O/THF, BBr3,11 LiI/pyri-
dine,12 pig liver esterase,13 rabbit liver esterase14 and
(11) Williard, P. G.; Fryhle, C. B. Tetrahedron Lett., 1980, 21, 3731.
(12) Scheiner, P. Org. Synth., 1965, 45, 7.
(13) (a) Chen, C.-S.; Fujimoto, Y.; Girdankas, G.; Sih, C. J . J . Am.
Chem. Soc., 1982, 104, 7294. (b) Wang, Y.-F.; Chen, C.-S.; Girdankas,
G.; Sih, C. J . J . Am. Chem. Soc., 1984, 106, 3695. (c) Patel, D. V.;
VanMiddlesworth, F.; Donaubauer, J .; Gannett, P.; Sih, C. J . J . Am.
Chem. Soc., 1986, 108, 4603. (d) Bjo¨rkling, F.; Boutelje, F.; Gatenbeck,
S.; Norin, T.; Szmulik, P. Bioorg. Chem., 1986, 14, 176.
(14) Whitesides, G. M.; Wong, C.-H. Angew. Int., 1985, 24, 617.
(15) Chauduri, C.; Ducan, K.; Graham, L. D.; Coggins, J . R. Biochem.
J ., 1990, 275, 1.

Synthesis of 2-Haloshikimic Acids
J . Org. Chem., Vol. 63, No. 5, 1998 1593
Ta ble 1. Kin etic P a r a m eter s for th e Con ver sion of
Deh yd r oqu in ic Acid (1), (2R)-2-Br om o-3-d eh yd r oqu in ic
Acid (2) a n d (2R)-2-F lu or o-3-d eh yd r oqu in ic Acid (3) w ith
Typ e II Deh yd r oqu in a ses fr om M. tu ber cu losis a n d A.
n id u la n sa
Km
(µM)
kcat
kcat/Km
source
substrate
(s-1
)
(M-1 s-1) × 105
M. tuberculosis
1
2
3
1
2
3
13
15
8
122
26
86
3.3
5.5
0.7
290
70
80
2.5
3.67
0.86
23.9
26.9
9.3
A. nidulans
a
Enzyme assays were performed at
a
range of substrate
concentrations, typically 0.2-5 Km, measuring the initial rate of
the reaction at 234 nm for 1, 264 nm for 2, and 249 nm for 3.
Reactions were performed at 25 °C in Tris/HCl buffer (50 mM,
pH 7.0).
stereochemistry,17 by a mechanism which is thought to
involve initial proton abstraction followed by stabilization
of the carbanion intermediate as an enolate.18
As the conversion of (2R)-2-bromo-3-dehydroquinic acid
(2) into 2-bromo-3-dehydroshikimic acid (5) involves an
anti elimination of water, the type II dehydroquinase was
used as a catalyst. In fact the M. tuberculosis enzyme
from the biosynthetic shikimate pathway and the A.
nidulans enzyme from the catabolic quinic acid pathway
were both tested as they are known to have quite distinct
kinetic properties.18 Parallel studies were also carried
out using (2R)-2-fluoro-3-dehydroquinic acid 3 as a
substrate.7 The steady-state kinetic parameters for both
2 and 3 together with the normal substrate 3-dehydro-
quinic acid (1) for reaction with both type II dehydro-
quinases are shown in Table 1.
F igu r e 1. 1H NMR spectrum (D2O, 500 MHz) of (a) (2R)-2-
bromo-3-dehydroquinic acid (2) and (b) an equilibrium mixture
of (2R)-2-bromo-3-dehydroquinic acid (2) and 2-bromo-3-de-
hydroshikimic acid (5) formed in potassium phosphate buffer
(50 mM, pH 7.0, 26 °C), by incubation over 48 h with type II
dehydroquinase from M. tuberculosis (15 µL, 2.6 U) in a total
volume of 0.6 mL. Peaks labeled with ( ) belong to 2-bromo-
*
3-dehydroshikimic acid (5).
The conversion of 2 was followed by UV spectrometry,
monitoring the increase in absorbance at 264 nm (ꢀ/M-1
cm-1 6 300) due to formation of the extended chro-
mophore in 5. The transformation could also be followed
1
by H NMR spectroscopy (Figure 1) by monitoring the
decrease of the singlet at δ 5.55 due to the proton at C-2
of 2. Similarly, the conversion of (2R)-2-fluoro-3-dehy-
droquinic acid (3) was followed by UV spectrometry at
249 nm (ꢀ/M-1 cm-1 9 600), or by the decrease of the
doublet at δ 5.64 due to the C-2 proton of 3 in the 1H
NMR spectrum (Figure 2). The reaction could addition-
ally be followed by 19F NMR spectroscopy, monitoring the
appearance of the broad singlet at δ -133 due to the
vinylic fluorine at C-2 of 6 (Figure 3).
The equilibrium constant between (2R)-2-bromo-3-
dehydroquinic acid (2) and 2-bromo-3-dehydroshikimic
acid (5) was measured by 1H NMR spectroscopy, by
letting the enzymatic conversion run to equilibrium in
the NMR tube (Figure 1). A value of 7.3 in favor of 5
was determined. The corresponding equilibrium be-
tweeen (2R)-2-fluoro-3-dehydroquinic acid (3) and 2-fluoro-
3-dehydroshikimic acid (6) is 4.0 (Figure 2). These values
compare with an equilibrium constant between dehy-
droquinic acid (1) and 3-dehydroshikimic acid (4) of 15.19
The decrease in the equilibrium constant may reflect
increasing destabilization of the 3-dehydroshikimic acid
F igu r e 2. 1H NMR spectrum (D2O, 500 MHz) of (a) (2R)-2-
fluoro-3-dehydroquinic acid (3) and (b) an equilibrium mixture
of (2R)-2-fluoro-3-dehydroquinic acid (3) and 2-fluoro-3-dehy-
droshikimic acid (6) formed in potassium phosphate buffer (100
mM, pH 7.0, 26 °C), by incubation over 21 h with type II
dehydroquinase from M. tuberculosis (15 µL, 2.6 U) in a total
volume of 0.6 mL. Peaks labeled with ( ) belong to 2-fluoro-
*
3-dehydroshikimic acid 6.
form by the progressively increasing inductive effects in
moving from a hydrogen to bromine and then a fluorine
substitutent at C-2.
(16) Kleanthous, C.; Davis, K.; Kelly, S. M.; Cooper, A.; Harding, S.
E.; Price, N. C.; Hawkins, A. R.; Coggins, J . R. Biochem. J ., 1992, 282,
687.
(17) Harris, J .; Kleanthous, C.; Coggins, J . R.; Hawkins, A. R.; Abell,
C. J . Chem. Soc., Chem. Commun., 1993, 1080.
(18) Harris, J .; Gonza´lez-Bello, C.; Kleanthous, C.; Hawkins, A. R.;
Coggins, J . R.; Abell, C. Biochem. J ., 1996, 319, 333.
(19) Mitsuhashi, S.; Davis, B. D. Biochim. Biophys. Acta, 1954, 15,
54.

1594 J . Org. Chem., Vol. 63, No. 5, 1998
Gonza´lez-Bello et al.
F igu r e 3. 19F NMR spectra (D2O, 235 MHz) showing the conversion of (2R)-2-fluoro-3-dehydroquinic acid (3) into 2-fluoroshikimic
acid (9) in potassium phosphate buffer (100 mM, pH 7.0, 26 °C). Type II dehydroquinase from M. tuberculosis (15 µL, 2.6 U) in
a total volume of 0.6 mL was added inmediately after spectrum (a). NADPH and shikimate dehydrogenase (10 µL, 10 U) was
added inmediately after spectrum (b): (a) t ) 0, (b) t ) 25 h. (c) t ) 48 h.
Time-dependent irreversible inhibition of the type II
dehydroquinases from M. tuberculosis and A. nidulans
by (2R)-2-bromo-3-dehydroquinic acid (2) and (2R)-2-
fluoro-3-dehydroquinic acid (3) was also investigated. In
each case two parallel incubations were set up with type
II dehydroquinase in Tris/HCl buffer 50 mM at pH 7.0.
The first contained 2 (or 3), and the second was a control
that lacked potential inhibitor. Both enzyme mixtures
were identically incubated at 25 °C, and the enzyme
activity was periodically monitored by assaying an
aliquot with 3-dehydroquinic acid (1). There was no time
dependent inhibition of either type II dehydroquinase
with 2. Likewise, the M. tuberculosis enzyme was stable
in the presence of 3. It was therefore somewhat surpris-
ing that incubation with 3 resulted in slow irreversible
inhibition of the A. nidulans enzyme (50% over 4 h).
The above enzymatic studies were a prelude to using
the type II dehydroquinases to synthesize 2-halo-3-
dehydroshikimates. 2-Bromo-3-dehydroshikimic acid (5)
was synthesized enzymatically in 88% yield using either
the type II dehydroquinase from A. nidulans or M.
tuberculosis. Because of the problems of inhibition of the
A. nidulans enzyme by 3, the type II dehydroquinase
from M. tuberculosis was used for the conversion of (2R)-
2-fluoro-3-dehydroquinic acid into 2-fluoro-3-dehydro-
shikimic acid (6) in 88% yield. The incubations were
carried out in 100 mM ammonium bicarbonate buffer at
pH 7.8 and 25 °C. After the reactions had reached equi-
librium, the products were purifed by HPLC.
In conclusion, 2-bromo-3-dehydroshikimic acid (5) has
been synthesized in nine steps in 11% overall yield from
quinic acid. This improves to an overall yield of 25% if
(2R)-2-bromo-3-dehydroquinic acid (2) is first synthesized
and then converted enzymatically to (5). The corre-
sponding synthesis of 6 via 3 gives 2-fluoro-3-dehydro-
shikimic acid 6 in an overall yield of 23% from quinic
acid.
Syn th esis of 2-Br om osh ik im ic Acid 8. The avail-
ability of the 2-halo-3-dehydroshikimates 5 and 6 opened
up the possibility of a route to the 2-haloshikimic acids
by enzymatic reduction using shikimate dehydrogenase
(Scheme 1), albeit no C-2 substituted analogues of
dehydroshikimic 4 or shikimic acid 7 had previously been
reported as substrates for this enzyme. Shikimate de-
hydrogenase catalyzes the stereospecific reduction of
3-dehydroshikimic acid 4 to shikimic acid 7 and requires
NADPH as a cofactor (Scheme 1).20 The gene for the E.
coli enzyme has been cloned and overexpressed.21 Pre-
(20) Dansette, P.; Azerad, R. Biochimica, 1974, 56, 751.

Synthesis of 2-Haloshikimic Acids
J . Org. Chem., Vol. 63, No. 5, 1998 1595
sium phosphate buffer (50 mM, pH 7.0), DTT (1 mM) was
filter-sterilized through a 0.2 µm filter and stored at 4 °C under
which conditions it was stable for at least 9 months. When
required for assays, aliquots of the enzyme stocks were diluted
into water and stored on ice. Shikimate dehydrogenase was
purified as described previously23 and the concentrated solu-
tion (2.3 mg mL-1) stored in Tris/HCl buffer (50 mM, pH 7.5),
DTT (0.4 mM), KCl (50 mM) and 50% (v/v) glycerol at -20 °C.
When required for assays aliquots of the enzyme stocks were
diluted into water and stored on ice.
One unit (1 U) of enzyme is defined as the amount of enzyme
required to convert 1 µmol of substrate to product in 1 min.
Buffer reagents were purchased from Sigma Chemical Com-
pany, and pH values of prepared buffers were adjusted using
HOAc or HCl (c). All pH measurement were made at 25 °C.
Deuterated buffer was made up in 99.9% D2O and pD adjusted
with DCl or DOAc. pD values have been quoted where pH )
meter reading + 0.4.
liminary UV assays of shikimate dehydrogenase with
2-bromodehydroshikimic acid (5) and NADPH in potas-
sium phosphate buffer at pH 7.0 indicated that 5 was a
substrate. Detailed kinetic studies were performed to
determine a kcat of 2.7 s-1 and a Km of 4.0 mM. Although
the specificity constant (kcat/Km ) 6.8 × 102 M-1 s-1) is
103 lower than for 3-dehydroshikimic acid 4, 5 is still a
good enough substrate for shikimate dehydrogenase to
be used preparatively to make 2-bromoshikimic acid (8).
1
This transformation was followed by H NMR spectros-
copy, monitoring the upfield shift of the proton at C-5
from δ 4.06 in 5 to δ 3.94 in 8, and the appearance of a
double doublet at δ 2.26 due to the axial proton at C-6 in
8. The other peaks were obscured by the signals due to
NADPH and NADP+. After HPLC, an 83% yield of
2-bromoshikimic acid (8) was obtained.
Assa ys of Deh yd r oqu in a se. Dehydroquinase was as-
sayed in the forward or reverse direction by monitoring the
increase or decrease in absorbance at 234 nm in the UV
spectrum due to the absorbance of the enone-carboxylate
chromophore of 3-dehydroshikimic acid (4) (ꢀ/M-1 cm-1 12 000).
Standard assay conditions for type II dehydroquinase were pH
7.0 at 25 °C in Tris/HCl (50 mM) unless otherwise indicated.
A typical assay of type II dehydroquinase contained 50 mM
Tris/HCl at pH 7.0, 0.5 mM dehydroquinic acid (1) and 0.7 U
type II dehydroquinase. Each assay was initiated by addition
of the enzyme. Solutions of dehydroquinic acid (and ana-
logues) were calibrated by equilibration with type II dehyd-
roquinase and measurement of the change in the UV absor-
bance at 234 nm due to the formation of the enone-carboxylate
chromophore of dehydroshikimic acid.24
Assa y of Sh ik im a te Deh yd r ogen a se. Shikimate dehy-
drogenase was assayed in its forward or reverse direction by
monitoring the increase or decrease in absorbance at 340 nm
in the UV spectrum due to the absorbance of NADPH (ꢀ/M-1
cm-1 6 200). Standard assay conditions for shikimate dehy-
drogenase were in the forward direction pH 7.0 at 25 °C with
200 µM NADPH in potassium phosphate buffer (50 mM)
unless otherwise indicated.25 A typical assay of shikimate
dehydrogenase contained 50 mM potassium phosphate buffer
at pH 7.0, 50 µM dehydroshikimic acid and 1 U of shikimate
dehydrogenase. Each assay was initiated by addition of the
enzyme. Solutions of shikimic acid (and analogues) were
calibrated by equilibration with shikimate dehydrogenase and
measurement of the change in the UV absorbance at 340 nm
due to the disappearance of NADPH.
Meth yl (1S,2R,4S,5R)-2-Br om o-4-[(ter t-bu tyld im eth yl-
silyl)oxy]-1,5-d ih yd r oxy-3-oxocycloh exa n eca r b oxyla t e
(12). To a solution of 11 (100 mg, 0.27 mmol) in methanol (3
mL) was added sodium methoxide (0.03 mmol as a 10%
solution in methanol), and the resultant solution was stirred
for 15 min. The reaction mixture was diluted with diethyl
ether (20 mL) and then washed successively with 5% HCl (20
mL), water (20 mL), and brine (20 mL). The organic layer
was dried (Na2SO4), filtered, and concentrated under reduced
pressure to give an oil, which was purified by flash chroma-
tography eluting with ethyl acetate-hexane (1:2) to yield the
epoxide 13 (5 mg, 6%), followed by the bromide 12 (74 mg,
68%), both as amorphous solids.
An analogous study was carried out using 2-fluoro-3-
dehydroshikimic acid (6), which proved an even better
substrate for shikimate dehydrogenase than 5. Kinetic
studies in potassium phosphate buffer at pH 7.0 and 25
°C, were used to determine a Km of 235 µM, and a kcat of
103 s-1. This gives a specificity constant (kcat/Km ) 4.4
× 105 M-1 s-1) only five times lower than that for the
natural substrate.
A transformation from (2R)-2-fluoro-3-dehydroquinic
acid (3) to 2-fluoroshikimic acid (9) was carried out by
sequential use of type II dehydroquinase and shikimate
dehydrogenase. The transformation was monitored by
19F NMR spectroscopy (Figure 3). In the 19F NMR
spectrum the fluorine in (2R)-2-fluoro-3-dehydroquinic
acid (3) gives rise to a double doublet at δ -206. Addition
of type II dehydroquinase results in a reduction of this
signal and appearance of a broad singlet at δ -133 due
to 6 as the equilibrium mixture of 3 and 6 is formed. At
this point, NADPH and shikimate dehydrogenase were
added. After several hours the only signal in the 19F
NMR spectrum is a quartet at δ -112 due to 2-fluo-
roshikimic acid (9). Under the conditions of the incuba-
tion, the equilibrium is completely pulled over toward
product. 2-Fluoroshikimic acid (9) was isolated in 91%
yield from this transformation.
A synthesis of 2-fluoroshikimic acid (7) has recently
been published.6 The synthesis required 10 steps from
shikimic acid and proceeded in an overall 4% yield. Our
approach, using a combination of synthetic chemistry and
enzymatic transformations, proceeds in eight steps from
quinic acid in an overall 21% yield.
Exp er im en ta l Section
Gen er a l. Where quoted, carboxylic acids were analyzed or
purified by HPLC which was carried out on either a semi-
preparative (300 mm × 8 mm), or preparative (300 × 16 mm)
Bio-Rad Aminex Ion Exclusion HPX-87H Organic Acids col-
umn. The eluent used for these columns was 50 mM aqueous
formic acid, at a flow rate of 0.6 mL min-1 (semipreparative
column) or 1.2 mL min-1 (preparative column). FPLC was
carried out using a Mono Q HR 10/10 column and eluting with
a gradient of ammonium bicarbonate at 1.0 mL min-1 with
the UV detector set at 254 nm. [R]D values are given in 10-1
deg cm2 g-1. UV spectra and enzyme assays were performed
at 25 °C (using either a temperature control thermostat or a
thermostated water bath), using 1 cm path quartz cells.
Type II dehydroquinases from M. tuberculosis and A.
nidulans were purified as described previously:22 a concen-
trated solution (1.5 and 0.08 mg mL-1, respectively) in potas-
Data for 12: mp 111-112 °C (from diethyl ether-hexane);
1H NMR (250 MHz; CDCl3) δ 5.03 (1 H, d, J ) 1.1, 2-H), 4.23
(1 H, dd, J ) 1.1 and 9.0, 4-H), 4.08 (1 H, ddd, J ) 5.0, 9.0
(22) (a) Moore, J . D.; Lamb, H. K.; Garbe, T.; Servos, S.; Dougan,
G.; Charles, I. G.; Hawkins, A. R. Biochem. J ., 1992, 287, 173. (b)
Gourlay, D. G.; Coggins, J . R.; Isaacs, N. W.; Moore, J . D.; Charles, I.
G.; Hawkins, A. R. J . Mol. Biol., 1994, 241, 488. (c) Hawkins, A. R.;
Smith, M. Eur. J . Biochem., 1991, 196, 717. (d) Krell, T.; Pitt, A. R.;
Coggins, J . R. FEBS Lett., 1985, 50, 888.
(23) Chaudhuri, S.; Anton, I. A.; Coggins, J . R. Methods in Enzymol.,
1987, 142, 315.
(24) 264 nm for (2R)-2-bromo- (2) and 249 nm for (2R)-2-fluoro-3-
dehydroquinic acid (3).
(21) Anton, A. I.; Coggins, J . R. Biochem. J ., 1988, 249, 319.
(25) Chaudhuri, S.; Coggins, J . R. Biochem. J ., 1985, 226, 217.

1596 J . Org. Chem., Vol. 63, No. 5, 1998
Gonza´lez-Bello et al.
and 11.4, 5-H), 3.87 (3 H, s, OCH3), 3.79 (1 H, s, OH), 3.47 (1
-5.8 (each, SiCH3); υmax (CCl4)/cm-1 2900, 1765, 1698. Anal.
Calcd for C16H22BrF3SiO6: C, 40.42; H, 4.63. Found: C, 40.25;
H, 4.60.
H, s, OH), 2.52 (1 H, dd, J ) 5.0 and 13.7, 6eq-H), 2.34 (1 H,
t
dd, J ) 11.4 and 13.7, 6ax-H), 0.92 (9 H, s, Bu), 0.16 and 0.04
(each 3 H, s, SiCH3); 13C NMR (63 MHz; APT; CDCl3) δ 194.4
(C), 171.7 (C), 82.9 (CH), 77.1 (C), 71.5 (CH), 60.7 (CH), 54.0
(OCH3), 38.3 (CH2), 25.8 (C(CH3)3), 18.4 (C(CH3)3), -4.4 and
-5.4 (each, SiCH3); υmax (Nujol)/cm-1 3640 (OH), 3590 (OH),
1740 (CdO). Anal. Calcd for C14H25BrSiO6: C, 42.32; H, 6.30.
Found: C, 42.23; H, 6.40.
Met h yl (4S,5R)-2-Br om o-4-[(ter t-b u t yld im et h ylsilyl)-
oxy]-5-h yd r oxy-3-oxocycloh ex-1-en eca r boxyla te (16). To
a solution of the ester 15 (60 mg, 0.13 mmol) in tetrahydro-
furan (2 mL) was added 1 mL of an aqueous solution of
K2CO3 (17 mg, 0.13 mmol). The resultant mixture was stirred
for 2 h, diluted with diethyl ether (25 mL), and washed
successively with 1 M HCl (10 mL), water (10 mL), and brine
(10 mL). The organic phase was dried (Na2SO4), filtered, and
evaporated to afford the alcohol 16 (39 mg, 81%) as an
amorphous solid: mp 65-66 °C (from petroleum ether bp 40-
Data for methyl (1S,2R,4S,5R)-4-[(tert-butyldimethylsilyl)-
oxy]-1,2-epoxy-1,5-dihydroxy-3-oxocyclohexanecarboxylate
(13): mp 121-122 °C (from hexane); 1H NMR (250 MHz;
CDCl3) δ 3.87 (1 H, ddd, J ) 4.9, 9.6 and 10.2, 5-H), 3.79 (3 H,
s, OCH3), 3.74 (1 H, d, J ) 9.6, 4-H), 3.56 (1 H, d, J ) 0.8,
2-H), 2.89 (1 H, ddd, J ) 0.8, 4.9 and 15.5, 6eq-H), 2.46 (1 H,
dd, J ) 10.2 and 15.5, 6ax-H), 2.38 (1 H, br s, OH), 0.90 (9 H,
s, tBu), 0.17 and 0.07 (each 3 H, s, SiCH3); 13C NMR (63 MHz;
APT; CDCl3) δ 198.2 (C), 167.8 (C), 80.8 (CH), 66.3 (CH), 57.3
(C + CH), 53.2 (OCH3), 29.7 (CH2), 25.8 (C(CH3)3), 18.4
(C(CH3)3), -4.3 and -5.4 (each, SiCH3); υmax (CCl4)/cm-1 3680
(OH), 2920 and 1730 (CdO). Anal. Calcd for C14H25BrSiO6:
C, 53.16; H, 7.59. Found: C, 52.86; H, 7.55.
1
60 °C); H NMR (250 MHz; CDCl3) δ 4.15 (1 H, d, J ) 10.4,
4-H), 4.02 (1 H, ddd, J ) 5.1, 9.4 and 10.4, 5-H), 3.88 (3 H, s,
OCH3), 3.05 (1 H, dd, J ) 5.1 and 18.3, 6eq-H), 2.66 (1 H, dd,
J ) 9.4 and 18.3, 6ax-H), 2.52 (1 H, br s, OH), 0.91 (9 H, s,
tBu), 0.20 and 0.08 (each 3 H, s, SiCH3); 13C NMR (63 MHz;
APT; CDCl3) δ 190.4 (C), 166.3 (C), 146.0 (C), 121.6 (C), 80.2
(CH), 70.3 (CH), 53.0 (OCH3), 35.0 (CH2), 25.8 (C(CH3)3), 18.4
(C(CH3)3), -5.5 and -4.4 (each, SiCH3); υmax (Nujol)/cm-1 3550
(OH), 1710 (CdO). Anal. Calcd for C14H23BrSiO5: C, 44.33;
H, 6.07. Found: C, 44.30; H, 6.06.
Employing greater quantities of sodium methoxide (>0.2
equiv) and extended reaction times (>30 min) resulted in the
formation of a white precipitate (purified as described above)
of methyl 3-[(tert-butyldimethylsilyl)oxy]gallate (40-80%): mp
116-117 °C (from ethyl acetate-hexane); 1H NMR (250 MHz;
CDCl3) δ 7.30 (1 H, d, J ) 1.9, ArH), 7.14 (1 H, d, J ) 1.9,
ArH), 5.65 (1 H, s, OH), 5.52 (1 H, br.s., OH), 3.85 (3 H, s,
Meth yl (4S,5R)-2-Br om o-4,5-d ih yd r oxy-3-oxocycloh ex-
1-en eca r boxyla te (17). A solution of the silyl ether 16 (45
mg, 0.12 mmol) in tetrahydrofuran (0.3 mL), acetic acid (0.4
mL), and water (0.4 mL) was stirred at 35 °C for 72 h. It was
then lypholized to afford 17 (29 mg, 92%) as a fine yellow
powder. Recrystallization afforded pure diol 17 as clear light
yellow plates: mp 125-126 °C (from dichloromethane-hex-
ane); 1H NMR (250 MHz; d6-acetone) δ 5.0-4.6 (2 H, br s, 2 ×
OH), 4.26 (1 H, d, J ) 10.0, 4-H), 4.06 (1 H, ddd, J ) 5.1, 8.7
and 10.0, 5-H), 3.86 (3 H, s, OCH3), 3.01 (1 H, dd, J ) 5.1 and
18.1, 6eq-H), and 2.71 (1 H, dd, J ) 8.7 and 18.1, 6ax-H); 13C
NMR (63 MHz; APT; d6-acetone) δ 192.4 (C), 167.0 (C), 148.7
(C), 120.6 (C), 79.1 (CH), 70.6 (CH), 53.0 (OCH3), 36.4 (CH2);
υmax (Nujol)/cm-1 3500-3200 (OH), 1740, 1710 (each CdO).
Anal. Calcd for C8H9BrO5: C, 36.23; H, 3.40. Found: C, 36.33;
H, 3.42.
t
OCH3), 1.00 (9 H, s, Bu) and 0.28 (6 H, s, 2 × SiCH3); 13C
NMR (63 MHz; APT; CDCl3) δ 166.8 (C), 143.7 (C), 142.3 (C),
138.9 (C), 121.6 (C), 112.0 (CH), 111.0 (CH), 52.1 (OCH3), 25.7
(C(CH3)3), 18.2 (C(CH3)3) and -4.3 (2 × SiCH3). Anal. Calcd
for C14H22SiO4: C, 56.38; H, 7.38. Found: C, 56.16; H, 7.22.
Meth yl (1S,5R)-3-[(ter t-Bu tyld im eth ylsilyl)oxy]-1,5-d i-
h yd r oxy-4-oxocycloh ex-2-en eca r boxyla te (14). To a solu-
tion of the alcohol 12 (10 mg, 24.94 µmol) in dichloromethane
(2 mL) was added triethylamine (15 µL, 99.75 µmol). The
resultant mixture was stirred at room temperature for 8 h and
then was diluted with dichloromethane (10 mL) and washed
with 1 M HCl (2 × 5 mL). The organic layer was dried (Na2-
SO4), filtered, and concentrated under reduced pressure to give
an oil, which was purified by flash chromatography eluting
with ethyl acetate-hexane (1:2) to yield the enone 14 (7 mg,
84%), an amorphous white solid: mp 94-95 °C (from hexane);
1H NMR (250 MHz; CDCl3) δ 5.86 (1 H, d, J ) 1.8, 2-H), 4.71
(1 H, ddd, J ) 5.7, 2.1 and 12.1, 5-H), 3.85 (3 H, s, OCH3),
3.41 (1 H, s, OH), 3.36 (1 H, d, J ) 2.1, OH), 2.45 (1 H, ddd,
p -Met h oxyb en zyl (1S,2R,4S,5R)-2-Br om o-4-[(ter t-b u -
t yld im et h ylsilyl)oxy]-1,5-d ih yd r oxy-3-oxocycloh exa n e-
ca r boxyla te (18). To a stirred solution of the lactone 11 (760
mg, 2.08 mmol) in tetrahydrofuran (30 mL) was added p-
methoxybenzyl alcohol (218 mg, 1.58 mmol). To this was
added 10 mmol aliquots of lithium p-methoxybenzyloxide
(prepared by addition of n-BuLi to a solution of p-methoxy-
benzyl alcohol in tetrahydrofuran) in 15 min intervals until
reaction was complete. The reaction mixture was diluted with
ethyl acetate (150 mL) and washed with 1 M HCl (20 mL) and
water (20 mL). The organic phase was dried (MgSO4), filtered,
and concentrated under reduced pressure to give a residue
which was purified by flash chromatography eluting with ethyl
acetate-hexane (4:1) to yield the ester 18 as an oil (660 mg,
80%): 1H NMR (250 MHz; CDCl3) δ 7.31 (2 H, d, J ) 8.7, ArH),
6.90 (2 H, d, J ) 8.7, ArH), 5.23 and 5.17 (each 1 H, d, J )
11.7, CHHAr), 4.99 (1 H, d, J ) 1.1, 2-H), 4.20 (1 H, dd, J )
1.1 and 9.0, 4-H), 4.07 (1 H, ddd, J ) 5.0, 13.8 and 11.5, 5-H),
3.81 (3 H, s, OCH3), 3.63 (1 H, br s, OH), 2.58 (1 H, br s, OH),
2.47 (1 H, dd, J ) 5.0 and 13.8, 6eq-H), 2.31 (1 H, dd, J ) 11.5
J ) 1.8, 5.7 and 12.8, 6eq-H), 2.32 (1 H, dd, J ) 12.1 and 12.8,
t
6
ax-H), 0.93 (9 H, s, Bu), 0.18 and 0.15 (each 3 H, s, SiCH3);
13C NMR (63 MHz; APT; CDCl3) δ 196.6 (C), 174.7 (C), 147.8
(C), 123.3 (CH), 71.4 (C), 69.4 (CH), 53.8 (OCH3), 41.2 (CH2),
25.5 (C(CH3)3), 18.3 (C(CH3)3), -4.7 and -4.8 (each, SiCH3);
υ
max (CHCl3)/cm-1 3600-3500, 1740, 1700, 1630. Anal. Calcd
for C14H24SiO6: C, 53.16; H, 7.59. Found: C, 52.93; H, 7.70.
Met h yl (4S,5R)-2-Br om o-4-[(ter t-b u t yld im et h ylsilyl)-
oxy]-5-(t r iflu or oa ce t yl)-3-oxocycloh e x-1-e n e ca r b oxy-
la te (15). To a solution of the alcohol 12 (15 mg, 38 µmol) in
dichloromethane (2 mL) and pyridine (100 µL, 1.26 mmol) was
added trifluoroacetic anhydride (12 µL, 83.3 µmol). The
resultant mixture was stirred for 2 h after which time it was
diluted with dichloromethane (15 mL). The mixture was
washed with 1 M HCl (10 mL) and brine (10 mL). The organic
layer was dried (MgSO4), filtered, and evaporated. The residue
was purified by flash chromatography eluting with ethyl
acetate-hexane (1:9) to afford the trifluoroester 15 (16 mg,
90%) as an amorphous solid: mp 74-75 °C (from petroleum
t
and 13.8, 6ax-H), 0.91 (9 H, s, Bu), 0.14 and 0.02 (each 3 H, s,
SiCH3); 13C NMR (63 MHz; APT; CDCl3) δ 194.5 (C), 171.2
(C), 160.1 (C), 130.7 (CH), 126.3 (C), 114.1 (CH), 82.6 (CH),
77.0 (C), 71.7 (CH), 68.9 (CH2), 60.6 (CH), 55.3 (OCH3), 38.4
(CH2), 25.8 (C(CH3)3), 18.4 (C(CH3)3), -4.5 and -5.4 (each
SiCH3); υmax (CCl4)/cm-1 3600, 2800-3000, 1720, 1590.
p -Met h oxyb en zyl (4S,5R)-2-Br om o-4-[(ter t-b u t yld i-
m e t h ylsilyl)oxy]-5-(t r iflu or oa ce t yl)-3-oxocycloh e x-1-
en eca r boxyla te (19). To a solution of the alcohol 18 (500
mg, 1.26 mmol) in dichloromethane (40 mL) and pyridine (300
µL, 3.78 mmol) was added trifluoroacetic anhydride (375 µL,
2.65 mmol), and the resultant mixture was stirred at room
temperature for 4 h. The reaction mixture was washed
successively with 1 M HCl (2 × 40 mL), water (40 mL), and
then brine (40 mL). The organic phase was dried (MgSO4),
filtered, and evaporated to leave a light yellow oil. Purification
1
ether bp 30-40 °C); H NMR (250 MHz; CDCl3) δ 5.38 (1 H,
ddd, J ) 5.4, 9.1 and 10.3, 5-H), 4.42 (1 H, d, J ) 10.3, 4-H),
3.90 (3 H, s, OCH3), 3.24 (1 H, dd, J ) 5.4 and 18.0, 6eq-H),
t
2.84 (1 H, dd, J ) 9.1 and 18.0, 6ax-H), 0.88 (9 H, s, Bu), 0.16
and 0.09 (each 3 H, s, SiCH3); 13C NMR (63 MHz; APT; CDCl3)
δ 188.9 (C), 165.4 (C), 156.4 (COCF3, q, J ) 43), 144.0 (C),
122.5 (C), 114.2 (CF3, q, J ) 285), 75.9 (CH), 74.6 (CH), 53.1
(OCH3), 32.3 (CH2), 25.4 (C(CH3)3), 18.1 (C(CH3)3), -4.6 and

Synthesis of 2-Haloshikimic Acids
J . Org. Chem., Vol. 63, No. 5, 1998 1597
by flash chromatography (ethyl acetate-hexane, 1:4) afforded
the desired ester 19 (416 mg, 72%) as an oil: 1H NMR (250
MHz; CDCl3) δ 7.34 (2 H, d, J ) 8.7, ArH), 6.90 (2 H, d, J )
8.7, ArH), 5.37 (1 H, ddd, J ) 5.5, 9.4 and 10.5, 5-H), 5.24 (2
H, s, CH2Ar), 4.41 (1 H, d, J ) 10.5, 4-H), 3.81 (3 H, s, OCH3),
3.19 (1 H, dd, J ) 5.5 and 18.0, 6eq-H), 2.82 (1 H, dd, J ) 9.4
acids HPLC column (eluting with 50 mM formic acid at 1.2
mL min-1 detection at 277 nm). Fractions eluting with
retention time of 17 min were lyophilized to give the acid 6 as
an oil: 1H NMR (500 MHz; D2O) δ 4.20 (1 H, d, J ) 11.1, 4-H),
3.94 (1 H, m, 5-H), 2.84 (1 H, dt, J ) 5.0 and 17.4, 6eq-H), 2.64
(1 H, m, 6ax-H); 19F NMR (235 MHz; D2O) δ -133 (1 F, br s);
λmax (H2O)/nm 249 (ꢀ/M-1 cm-1 9 600); MS (FAB +ve) m/z (%)
191 (MH+); HRMS calcd for C7H8FO5: MH+, 191.0320.
Found: MH+, 191.0314.
(3S,4S,5R)-2-F lu or o-3,4,5-t r ih yd r oxycycloh ex-1-en e-
ca r boxylic Acid [2-flu or osh ik im ic a cid ] (9).6 To a solution
of (2R)-2-fluorodehydroquinic acid (3) (6 mg, 29 µmol) in
potassium phosphate buffer at pH 7.0, 26 °C (0.6 mL, includes
10% D2O, 100 mM) was added type II form M. tuberculosis
(15 µL, 2.6 U). The reaction was monitored by 19F NMR
spectroscopy. After 24 h, the reaction had reached equilibri-
um, and NADPH (29 mg, 35 µmol) and shikimate dehydroge-
nase (10 U, 10 µL) were added and after 24 h it was observed
that all (2R)-2-fluorodehydroquinic acid (3) had been trans-
formed into 2-fluoroshikimic acid (9). The crude mixture was
purified by FPLC using a Mono Q HR 10/10 column. Eluting
with a gradient of 0-0.3 M ammonium bicarbonate over 140
mL and then 0.3-1 M over 110 mL, at 1.0 mL min-1 at 254
nm. Fractions eluting at 220 mM ammonium bicarbonate
contained 2-fluoroshikimic acid (9) (5 mg, 91%), 1H NMR (500
MHz; D2O) δ 4.34 (1 H, dd, J ) 8.7 and 4.5, 3-H), 3.82 (1 H,
m, 5-H), 3.63 (1 H, dd, J ) 9.9 and 4.5, 4-H), 2.61 (1 H, dt, J
) 16.7 and 5.6, 6eq-H), 2.12 (1 H, m, 6ax-H); 19F NMR (235 MHz;
D2O) δ -112 (1 F, q, J ) 7).
t
and 18.0, 6ax-H), 0.87 (9 H, s, Bu), 0.17 and 0.05 (each 3 H, s,
SiCH3); 13C NMR (63 MHz; APT; CDCl3) δ 188.7 (C), 165.0
(C), 160.1 (C), 156.4 (COCF3, q, J ) 43), 144.7 (C), 130.7 (CH),
126.2 (C), 122.4 (C), 114.2 (CF3, q, J ) 285), 114.1 (C), 76.0
(CH), 74.6 (CH), 68.3 (CH2), 32.4 (CH2), 25.4 (C(CH3)3), 18.2
(C(CH3)3), -4.6 and -5.8 (each SiCH3); υmax (CCl4)/cm-1 2880,
1770 and 1698; MS (CI+) m/z (%) 485 and 483 ([M + NH4]+
-
COCF3); HRMS calcd for C21H28BrSiO6: MNH4+, 485.0818.
Found: MNH4+, 485.0813.
p -Met h oxyb en zyl (4S,5R)-2-Br om o-4-[(ter t-b u t yld i-
m eth ylsilyl)oxy]-5-h yd r oxy-3-oxocycloh ex-1-en eca r box-
yla te (20). To a stirred solution of the ester 19 (340 mg, 0.6
mmol) in tetrahydrofuran (15 mL) and water (15 mL) was
added 10 mL of an aqueous solution of K2CO3 (84 mg, 0.61
mmol) over a 30 min period. After stirring for a further 15
min, dichloromethane (50 mL) was added and washed succes-
sively with 1 M HCl (20 mL), water (20 mL), followed by brine
(20 mL). The organic phase was dried (MgSO4) and filtered.
The solvent was removed under reduced pressure to afford the
alcohol 20 as an oil, which was purified by flash chromatog-
raphy (ethyl acetate-hexane, 1:4) to yield 20 as an oil (220
mg, 78%): 1H NMR (250 MHz; CDCl3) δ 7.34 (2 H, d, J ) 8.6,
ArH), 6.89 (2 H, d, J ) 8.6, ArH), 5.24 (2 H, s, CH2Ar), 4.13 (1
H, d, J ) 10.5, 4-H), 4.00 (1 H, dddd, J ) 1.5, 5.2, 9.5 and
10.5, 5-H), 3.81 (3 H, s, OCH3), 3.04 (1 H, dd, J ) 5.2 and
18.3, 6eq-H), 2.65 (1 H, dd, J ) 9.5 and 18.3, 6ax-H), 2.54 (1 H,
(3S,4S,5R)-2-Br om o-3,4,5-t r ih yd r oxycycloh ex-1-en e-
ca r boxylic Acid [2-br om osh ik im ic a cid ] (8). To a solution
of 2-bromodehydroshikimic acid (5) (6 mg, 22 µmol) in deu-
terated potassium phosphate buffer at pD 7.0, 26 °C (0.5 mL,
100 mM) were added NADPH (30 mg, 36 µmol) and shikimate
dehydrogenase (10 U, 10 µL). The reaction was followed by
1H NMR spectroscopy. After 24 h, approximately 78% conver-
sion had occurred, and the reaction was assumed to have
reached equilibrium. NADPH/NADP+ were removed by anion
exchange chromatography using a SAX HPLC column (eluting
with 50 mM NH4OAc, pH 6.9 at 4.00 mL min-1, UV detection
at 260 nm). Fractions eluting between 4 and 6 min were
collected and further purified using a semipreparative organic
acids HPLC column (eluting with 50 mM formic acid at 1.2
mL min-1, UV detection at 240 nm). Fractions eluting with a
retention time between 16 and 18 min were combined and
lyophilized to give 2-bromoshikimic acid 8 (5 mg, 90%) as an
oil: 1H NMR (400 MHz; D2O) δ 4.39 (1 H, d, J ) 4.4, 3-H),
3.94 (1 H, ddd, J ) 10.2, 9.0 and 5.8, 5-H), 3.75 (1 H, dd, J )
10.2 and 4.4, 4-H), 2.76 (1 H, dd, J ) 17.2 and 5.8, 6eq-H), 2.27
(1 H, dd, J ) 17.2 and 9.0, 6ax-H); 13C NMR (100 MHz; APT;
D2O) δ 175.5 (C), 137.9 (C), 128.6 (C), 67.7 (3 × CH) and 37.5
(CH2); υmax (thin film)/cm-1 3368 (OH), 2921 (CH), 1725 (CdO)
t
br d, J ) 1.5, OH), 0.92 (9 H, s, Bu), 0.21 and 0.08 (each 3 H,
s, SiCH3); 13C NMR (63 MHz; APT; CDCl3) δ 190.5 (C), 165.8
(C), 160.1 (C), 146.2 (C), 130.6 (C), 126.5 (C), 121.5 (C), 114.1
(CH), 80.2 (CH), 70.3 (CH), 68.0 (CH2Ar), 55.3 (CH3), 35.1
(CH2), 25.9 (C(CH3)3), 18.4 (C(CH3)3), -4.3, and -5.5 (each
SiCH3); υmax (CHCl3)/cm-1 3560 and 1710.
(4S,5R)-2-Br om o-4,5-d ih yd r oxy-3-oxocycloh ex-1-en e-
ca r boxylic a cid [2-br om osh ik im ic a cid ] (5). A solution
of 20 (220 mg, 0.43 mmol) in tetrahydrofuran (0.5 mL), acetic
acid (2 mL), and water (0.5 mL) was stirred at 50 °C for 72 h.
The solvent was removed under reduced pressure, and the
residue was partitioned between water (30 mL) and diethyl
ether (30 mL). The aqueous fraction was washed further with
diethyl ether (30 mL) and lyophilized to afford 5 (100 mg, 92%).
Recrystallization gave pure acid 5 as white needles: mp 176-
178 °C (from ethyl acetate-hexane); 1H NMR (250 MHz; D2O)
δ 4.33 (1 H, d, J ) 11.2, 4-H), 4.06 (1 H, ddd, J ) 5.5, 9.9 and
11.2, 5-H), 3.03 (1 H, dd, J ) 5.5 and 18.0, 6eq-H) and 2.76 (1
H, dd, J ) 9.9 and 18.0, 6ax-H); 13C NMR (63 MHz; APT; D2O)
δ 196.8 (C), 174.2 (C), 156.1 (C), 116.4 (C), 80.6 (CH), 72.4 (CH),
38.3 (CH2); υmax (Nujol)/cm-1 3100-3650 (br), 1720, 1690; λmax
(H2O)/nm 264 (ꢀ/M-1 cm-1 6 300). Anal. Calcd for C7H7-
BrO5‚H2O: C, 31.23; H, 3.34. Found: C, 31.52; H, 3.26.
(4S,5R)-2-F lu or o-4,5-d ih yd r oxy-3-oxocycloh ex-1-en e-
ca r boxylic Acid (6). To a solution of the (2R)-2-fluoro-3-
dehydroquinic acid (3) (1 mg, 4.2 nmol) in 0.5 mL ammonium
bicarbonate (100 mM) at pH 7.8 and 25 °C was added type II
dehydroquinase from M. tuberculosis (10 µL, 1.7 U). After 24
h, approximately 88% conversion had occurred by reaching the
equilibrium. The enzyme was removed by centrifugation on
Amicon Centricon-10 microconcentrator,and lyophilized, and
the resultant mixture was purified using a preparative organic
and 1640 (CdC); λmax (H2O)/nm 196; MS (CI+) m/z (%) 272 and
270 ([M + NH4]+); HRMS calcd for C7H13BrNO5: MNH4
,
+
269.9977. Found: MNH4+, 269.9977.
Ack n ow led gm en t. We thank the BBSRC for pos-
doctoral funding (C.G.B.), the Royal Society for an
Endeavor Research Fellowship (M.K.M.), and Zeneca
Pharmaceuticals and the SERC for a CASE studentship
(J .M.H.). We thank C. Goddard for running the 19F
NMR spectra.
J O971858I