Chemoenzymatic Synthesis of Isotopically Labeled Chorismic Acids

Full text of DOI:10.1021/jo9823792

J . Org. Chem. 1999, 64, 4935-4938
4935
Sch em e 1
Ch em oen zym a tic Syn th esis of Isotop ica lly
La beled Ch or ism ic Acid s
Darin J . Gustin and Donald Hilvert*
Department of Chemistry, The Scripps Research Institute,
La J olla, California 92037 and Laboratorium fu¨r
Organische Chemie, Swiss Federal Institute of Technology
(ETH), Universita¨tstrasse 16, CH-8092 Zu¨rich, Switzerland
Sch em e 2
Received December 3, 1998
The rearrangement of chorismate to prephenate, cata-
lyzed by chorismate mutase (CM), is a key step in the
biosynthesis of aromatic amino acids in bacteria and
plants.1 Although CM has been much studied, details
concerning its mechanism remain elusive.2-5 Recently,
as part of an investigation6 to probe the structure of the
enzymatic transition state with heavy-atom isotope ef-
fects,7 we required a synthesis of the isotopically labeled
chorismic acids 1a -c.
Thus, starting with aldehyde 2,12 Knoevenagel con-
densation (TiCl4, NMM, THF, -10 to 0 °C)13 with the
commercially available 13C-labeled triethyl phosphono-
acetates 3b or 3c allowed introduction of 13C labels at
either or both the C(1) and C(7) positions (Scheme 2).
For the synthesis of [7-12C]shikimate (6a ), methyl [1-12C]-
diethylphosphonoacetate (3a ) was prepared in 38% yield
by carboxylation of diethyl methylphosphonate with
12CO2, followed by Fisher esterification (Scheme 1). The
moderate yield of the latter sequence is probably the
result of in situ anion exchange. Addition of a solution
of lithiated diethyl methylphosphonate to an excess of
12CO2 in THF did not significantly improve the efficiency
of the reaction, however.
Catalytic hydrogenation of vinyl phosphonate 4 with
concomitant removal of the benzyl ether, followed by
intramolecular Horner-Wadsworth-Emmons reaction,
provided 3,4-(isopropylidene)shikimate esters 5a -c in
25-49% from 2 without purification of the intermediates
(Scheme 2). Subsequent acid-catalyzed hydrolysis of the
isopropylidene acetals with Dowex 50W-X8 resin gave
labeled shikimate esters 6a -c in 68-82% yield. The 18O
label required for 1c was introduced by a two-step
procedure via 4,5-anhydroshikimate 714 (Scheme 3). Acid-
catalyzed epoxide opening with 18OH2 is â-selective14 and
provides the doubly labeled shikimate ester 6d in 74%
overall yield.
To date there have been two reported total syntheses
of chorismic acid.8,9 In principle, we could have used one
of these routes to prepare 1a -c, but incorporation of the
labels would have necessitated cumbersome modifica-
tions unique to each derivative. A more flexible strategy
for introducing the remote label7 at the C(10) carboxyl
group and the reporter groups at the site of C-C bond
formation (1b) or the site of C-O bond cleavage (1c) was
highly desirable. For this reason, we turned to a chemoen-
zymatic approach involving the synthesis of appropriately
labeled shikimic acids, followed by enzymatic conversion
to chorismic acid. Shikimic acid has been prepared in
several ways.10 Of these, we were particularly attracted
to the synthesis described by Mirza11 in which both C(1)
and C(7) (corresponding, respectively, to the C(1) and
C(10) positions in chorismate) are introduced by a Knoe-
venagel condensation on a relatively advanced interme-
diate, allowing preparation of each of the labeled choris-
mic acids from the same carbohydrate precursor.
(1) Haslam, E. Shikimic acid: metabolism and metabolites; J ohn
Wiley & Sons: New York, 1993.
(2) Lee, A. Y.; Stewart, J . D.; Clardy, J .; Ganem, B. Chem. Biol. 1995,
2, 195-203.
(3) Addadi, L.; J affe, E. K.; Knowles, J . R. Biochemistry 1983, 22,
4494-4501.
(4) Guilford, W. J .; Copley, S. D.; Knowles, J . R. J . Am. Chem. Soc.
1987, 109, 5013-5019.
With shikimates 6a -d in hand, we turned to the
enzymatic conversion of shikimate to chorismate. Al-
though this transformation has been described previously
using the potentially pathogenic chorismate mutase-
(5) Rajagopalan, J . S.; Taylor, K. M.; J affe, E. K. Biochemistry 1993,
32, 3965-3972.
(6) Gustin, D. J .; Mattei, P.; Kast, P.; Wiest, O.; Lee, L.; Cleland,
W. W.; Hilvert, D. J . Am. Chem. Soc. 1999, 121, 1756-1757.
(7) O’Leary, M. H. Methods Enzymol. 1980, 64, 83-104.
(8) Ganem, B.; Ikota, N.; Muralidharan, V. B.; Wade, W. W.; Young,
S. D.; Yukimoto, Y. J . Am. Chem. Soc. 1982, 104, 6787-6788.
(9) McGowan, D. A.; Berchtold, G. A. J . Am. Chem. Soc. 1982, 104,
7036-7041.
(12) Brimacombe, J . S.; Hunedy, F.; Tucker, L. C. N. J . Chem. Soc.
C 1968, 1381-1384.
(13) Reetz, P.; Peter, R.; von Itzstein, M. Chem. Ber. 1987, 120, 121-
(10) Campbell, M. M.; Sainsbury, M.; Searle, P. A. Synthesis 1993,
179-193.
(11) Mirza, S.; Harvey, J . Tetrahedron Lett. 1991, 32, 4111-4114.
122.
(14) McGowan, D. A.; Berchtold, G. A. J . Org. Chem. 1981, 46,
2381-2383.
10.1021/jo9823792 CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/06/1999

4936 J . Org. Chem., Vol. 64, No. 13, 1999
Notes
Sch em e 3
isotopic composition of the chorismate mixtures simply
by mixing appropriately labeled shikimates prior to the
enzymatic step. This is especially noteworthy given the
instability of chorismate and in light of the fact that the
remote label method for determination of heavy-atom
isotope effects requires only trace amounts of the doubly
labeled chorismate relative to “unlabeled” 1a in which
the indicator position is depleted in 13C. The use of the
engineered KA12/pKAD50 strain provides chorismate in
approximately 50% yield from 500 mg of shikimate and
represents a significant improvement over previously
reported procedures.3,15 Finally, this route can be easily
adapted to the preparation of other doubly labeled
chorismate derivatives for investigations of the enzymatic
and nonenzymatic reactions. Studies along these lines
will be reported in due course.
Sch em e 4a
a
Key: (a) NaOH, THF, H2O; (b) KA12/pKAD50 extract in 50
mM Tris-HCl (pH 8.1) containing PEP, ATP, FMN, KCl, MgSO4,
and Na2S2O4. Yield over two steps: 49-54%.
deficient Klebsiella pneumoniae strain 62-1 (American
Type Culture Collection no. 25306),3,15 yields are typically
low, and the experiments require a relatively large ratio
of cell extract to shikimate for optimal results. We
imagined that the yield of chorismate could be greatly
improved by overexpressing the genes for the three
enzymes on the biosynthetic pathway from shikimate to
chorismate in KA12, a previously described chorismate
mutase-deficient Escherichia coli strain.16,17 The desired
production strain was obtained by transforming KA12
with plasmid pKAD50,18 which carries the aroA, aroC
and aroL genes (encoding EPSP synthase, chorismate
synthase, and shikimate kinase, respectively) under the
control of the lac promoter. Dell and Frost showed
previously that introduction of pKAD50 into E. coli
increases the activities of EPSP synthase, chorismate
synthase, and shikimate kinase 4- to 40-fold.18
Because the experimental protocol for measuring heavy-
atom isotope effects via the remote label method involves
a competition between unlabeled and doubly labeled
substrates,7 with the isotopic composition of the remote
label [here the C(10) carboxylate of chorismate] set close
to natural abundance (13C ) 1.1%), chorismates were
prepared as isotopic mixtures. Thus, purified 6a was
combined with 6b or 6d at a ratio of ca. 99:1. These
mixtures were saponified individually, and the crude
sodium shikimates were converted to chorismate at 23
°C by incubation with phosphoenolpyruvate (PEP) and
cell extracts from KA12/pKAD50 supplemented with
ATP, flavin mononucleotide (FMN), magnesium sulfate,
and sodium dithionite (Scheme 4).18 Typical reaction
times were 2 h. Under these conditions, labeled chorismic
acid could be prepared on a several hundred milligram
scale in 49-53% overall yield.
Exp er im en ta l Section
All reactions were carried out in flame-dried glassware. THF
and Et2O were dried by distillation from sodium-benzophenone
ketyl. CH2Cl2 was dried by distillation from CaH2. All other
commercially available reagents and solvents were used as
received. High-resolution mass spectral data were recorded by
the Scripps Research Institute mass spectrometry laboratory.
Chromatographic purifications were performed with Kieselgel
60, 230-400 mesh silica gel. Eluent mixtures are reported as
v:v percentages of the minor constituent in the major constitu-
ent.
Meth yl [1-12C]Dieth ylp h osp h on oa ceta te (3a ). Methyl di-
ethylphosphonate (10.0 mL, 68.4 mmol) was added to a -78 °C
solution of n-BuLi (2.5 M in hexanes, 28 mL, 70 mmol) in THF
(300 mL). After the addition was complete, the cooling bath was
removed, and the solution was allowed to warm to -20 °C
(internal temperature) over 30 min and then recooled to -78
°C. In a separate vessel, THF (75 mL) was degassed under
vacuum and sparged with 12CO2 (99.99% atom, Aldrich) at -78
°C, until approximately 10 g of 12CO2 had been added (estimated
gravimetrically). The 12CO2 solution was added via cannula to
the -78 °C solution of lithiated diethyl methylphosphonate. The
resulting white slurry was allowed to warm to -10 °C over 2 h
and then concentrated to dryness. The residue was dissolved in
H2O (15 mL) acidified to pH 1 using concentrated HCl and
extracted with CH2Cl2 (5 × 40 mL). The combined extracts were
washed with brine (15 mL), dried over Na2SO4, and concen-
trated. The concentrate was dissolved in MeOH (400 mL) and
treated with AcCl (0.50 mL, 7.0 mmol). The mixture was stirred
for 17 h, treated with concentrated NH4OH (0.50 mL), and
concentrated to near dryness. The concentrate was partitioned
between CH2Cl2 (150 mL) and saturated aqueous NaHCO3 (20
mL). The CH2Cl2 layer was washed with brine (20 mL), dried
over Na2SO4, and concentrated. This gave a ca. 1:2.5 mixture of
the desired methyl diethylphosphonoacetate and unreacted
starting material. Purification of this mixture via careful distil-
lation (45 °C at 0.15 mm Hg and then 92-96 °C at 0.15 mm
Hg) gave 3a (5.47 g, 38%): 1H NMR (500 MHz, CDCl3) δ 4.16
(apparent quintet, J ) 7 Hz, 4 H), 3.73 (s, 3 H), 2.96 (d, J )
21.5 Hz, 2 H), 1.33 (t, J ) 7 Hz, 6 H); 13C NMR (125 MHz, CDCl3)
δ 62.7, 52.6, 34.1 (d, J ) 130 Hz), 16.3 (d, J ) 6.3 Hz).
In summary, we have prepared the (doubly) labeled
chorismic acids 1a -c via a flexible chemoenzymatic route
involving the chemical synthesis of shikimate esters 6a -
d , followed by enzymatic conversion to chorismic acid
using an engineered chorismate mutase-deficient E. coli
strain. This route has the advantage that each of the
shikimate precursors are prepared from the readily
available lyxose 2, which allows complete control of the
Meth yl 3,4-[1-12C](O-Isopr opyliden e)sh ikim ate (5a). TiCl4
(8.1 mL, 24.7 mmol) was added to THF (150 mL) at 0 °C. The
solution was stirred for 5 min before adding a solution of 3a
(8.55 g, 40.7 mmol) and 2 (10.35 g, 37.0 mmol) in THF (75 mL)
via cannula. The resulting mixture was stirred for 10 min. Then
a solution of N-methylmorpholine (NMM) (16.2 mL, 147 mmol)
in THF (30 mL) was added dropwise via cannula over a period
of 30 min. After the addition was complete, the dark red-brown
mixture was stirred for an additional 30 min before saturated
aqueous NaHCO3 (150 mL) was added. The resulting mixture
was extracted with Et2O (5 × 300 mL). The combined ethereal
extracts were washed with H2O (3 × 100 mL) and brine (100
mL), dried over Na2SO4, and concentrated. The crude product
was filtered through a pad of silica using 80% EtOAc/hexanes
as eluent, and the filtrate was concentrated. The material so
(15) Ife, R. J .; Ball, L. F.; Lowe, P.; Haslam, E. J . Chem. Soc., Perkin
Trans. 1 1976, 1776-1783.
(16) Kast, P.; Asif-Ullah, M.; Hilvert, D. Tetrahedron Lett. 1996, 37,
2691-2694.
(17) Grisostomi, C.; Kast, P.; Pulido, R.; Huynh, J .; Hilvert, D.
Bioorg. Chem. 1997, 25, 297-305.
(18) Dell, K. A.; Frost, J . W. J . Am. Chem. Soc. 1993, 115, 11581-
11589.

Notes
J . Org. Chem., Vol. 64, No. 13, 1999 4937
obtained was dissolved in 0.5% AcOH/MeOH (120 mL) sparged
with N2 and treated with 10% Pd/C (1.96 g, 1.8 mmol). The
mixture was shaken in a Parr apparatus under 50 atm of H2 at
23 °C. After 17 h, an additional portion of 10% Pd/C (1.96 g,
1.85 mmol) was added, and shaking was continued for an
additional 17 h. The reaction mixture was filtered through a pad
of silica gel over Celite. The filter pad was washed with several
portions of MeOH, and the filtrate was concentrated. The residue
was dissolved in MeOH (100 mL) and added dropwise over 40
min to a 0 °C solution of Na metal (3.07 g, 134 mmol) dissolved
in MeOH (200 mL). The mixture was stirred for an additional 2
h and then poured into saturated aqueous NaHCO3 (350 mL).
The aqueous mixture was extracted with Et2O (5 × 400 mL).
The combined extracts were divided into two portions, and each
was washed with H2O (3 × 100 mL) and brine (100 mL). The
combined extracts were dried over Na2SO4 and concentrated.
Purification of the crude product by silica gel chromatography
(35% EtOAc/hexanes) gave shikimate 5a (3.59 g, 43%): 1H NMR
(500 MHz, CDCl3) δ 6.93 (s with fine splitting, 1 H), 4.75 (dd, J
) 6.0, 5.0 Hz, 1 H), 4.08 (dd, J ) 7.5, 6.5 Hz, 1 H), 3.88 (dt, J )
4.5, 8.5 Hz, 1 H), 3.73 (s, 3 H), 2.81 (dd, J ) 17.5, 4.5 Hz, 1 H),
2.55 (s (br), 1 H), 2.23 (ddt, J ) 17.5, 8.5, 2 Hz, 1 H), 1.46 (s, 3
H), 1.40 (s, 3 H); 13C NMR (125 MHz, CDCl3) δ 133.9, 130.6,
109.7, 77.9, 72.2, 68.7, 52.1, 29.3, 27.9, 25.7.
mg, 82%): 1H NMR (500 MHz, CDCl3) δ 6.89 (s (br), 1 H), 4.47
(t (br), J ) 5 Hz, 1 H), 4.22 (qd, J ) 7.0, 3.0 Hz, 2 H), 3.98 (td,
J ) 9.0, 5.5 Hz, 1 H), 3.63 (dd, J ) 9.0, 4.5 Hz, 1 H), 2.95 (d (br),
J ) 17.5 Hz, 1 H), 2.72 (s (br), 1 H), 2.40 (s (br), 1 H), 2.27 (s,
(br), 1 H), 2.22 (dd, J ) 17.5, 8.5 Hz, 1 H), 1.30 (t, J ) 7.0 Hz,
3 H); 13C NMR (125 MHz, CDCl3) δ 166.5 (100% 13C), 136.0,
130.5 (d, J ) 75 Hz), 73.0, 66.6, 66.2 (d, J ) 6.5 Hz), 61.1, 32.2,
14.1; HRMS (FAB, NBA) calcd for C813CH14O5 (M+ + H)
204.0953, found 204.0948.
Eth yl 4,5-[7-13C]An h yd r osh ik im a te (7).14 DEAD (0.18 mL,
1.1 mmol) was added to a 0 °C solution of 6c (187 mg, 0.92 mmol)
and Ph3P (286 mg, 1.1 mmol) in THF (7.5 mL). The solution
was stirred for 2 h and concentrated, and the residue was heated
to 120 °C in a Kugelrohr oven at 0.150 mmHg for 15 min. The
volatile components were collected at -78 °C. The distillation
flask was cooled, combined with the contents of the receiver
flask, and concentrated. Purification of the residue by silica gel
chromatography (20% Et2O/hexanes) gave the epoxyalcohol 7
(132 mg, 78%): 1H NMR (500 MHz, CDCl3) δ 6.71 (d (br), J )
6.5 Hz, 1 H), 4.56 (s, (br), 1 H), 4.20 (qd, J ) 7.0, 3.0 Hz, 2 H),
3.57-3.54 (m, 2 H), 3.02 (d with fine splitting, J ) 19.5 Hz, 1
H), 2.48 (dd with fine splitting, J ) 19.5, 4.5 Hz, 1 H), 2.20 (s
(br), 1 H), 1.29 (t, J ) 7.0 Hz, 3 H); 13C NMR (125 MHz, CDCl3)
δ 166.1 (100% 13C), 135.7, 126.6 (d, J ) 75 Hz), 65.4 (d, J ) 6.3
Hz), 60.9, 54.6, 52.1, 24.2, 14.1; HRMS (FAB, NBA) calcd for
C813CH12O4Na (M+ + Na) 208.0667, found 208.0674.
Eth yl 3,4-[1,7-13C](O-Isopr opyliden e)sh ikim ate (5b). Com-
pounds 2 (1.35 g, 4.86 mmol) and 3b (1.21 g, 5.35 mmol) were
condensed in the presence of TiCl4 (1.20 mL, 10.9 mmol) and
NMM (2.4 mL, 21.8 mmol) as above. Hydrogenation of 4b over
10% Pd/C (562 mg) in 0.5% AcOH/MeOH (55 mL), cyclization
(508 mg Na0 in 75 mL EtOH), and chromatographic purification
gave 5b (278 mg, 25%): 1H NMR (500 MHz, CDCl3) δ 6.93 (s
(br), 1 H), 4.75 (d (br), J ) 4.5 Hz, 1 H), 4.22 (qd, J ) 7.0, 3.0
Hz, 2 H), 4.08 (dd, J ) 7.5, 6.5 Hz, 1 H), 3.88 (m, 1 H), 2.82 (dt
(br), J ) 17.0, 3.0 Hz, 1 H), 2.23 (m, 1 H), 2.00 (s (br), 1 H), 1.46
(s, 3 H), 1.41 (s, 3 H), 1.30 (t, J ) 7.0 Hz, 3 H); 13C NMR (75
MHz, CDCl3) δ 166.4 (d, J ) 73 Hz, 100% 13C), 133.9 (d, J ) 66
Hz), 131.3 (d, J ) 73 Hz, 100% 13C), 109.9, 78.2 (d, J ) 2.5 Hz),
72.4 (dd, J ) 6.1, 2.5 Hz), 69.0 (d, J ) 3.7 Hz), 61.1, 29.5 (dd, J
) 44, 2.4 Hz), 28.0, 25.8, 14.2; HRMS (EI) calcd for C1013C2H18O5
(M+) 244.1222, found 244.1223.
Eth yl [5-18O,7-13C]Sh ik im a te (6d ). Trifluoromethanesulfon-
ic acid (15 mL, 0.17 mmol) was added to a solution of 7 (52 mg,
0.28 mmol) in freshly distilled MeCN (0.80 mL) and 18O-labeled
H2O (99% atom, Cambridge Isotopes) (0.30 mL). After stirring
for 48 h, the resulting mixture was concentrated in vacuo.
Purification of the residue by silica gel chromatography gave
the doubly labeled ethyl shikimate 6d (55 mg, 95%): 1H NMR
(500 MHz, CDCl3) δ 6.89 (m, 1 H), 4.47 (t, J ) 4.5 Hz, 1 H), 4.22
(qd, J ) 7.0, 2.5 Hz, 2 H), 3.97 (dt, J ) 9.0, 5.5 Hz, 1 H), 3.62
(dd, J ) 9.0, 4.5 Hz, 1 H), 2.95 (dd with fine splitting, J ) 18.0,
3.0 Hz, 1 H), 2.81 (s (br), 1 H), 2.52 (s (br), 1 H), 2.39 (s (br), 1
H), 2.20 (dd with fine splitting, J ) 18.0, 8.5 Hz, 1 H), 1.30 (t,
J ) 7.0 Hz, 3 H); HRMS (FAB, NBA) calcd for C813CH14O418O
(M+ + H) 206.0996, found 206.0989.
Eth yl 3,4-[7-13C](O-Isop r op ylid en e)sh ik im a te (5c). Com-
pounds 2 (1.35 g, 4.85 mmol) and 3c (1.20 g, 5.33 mmol) were
condensed in the presence of TiCl4 (1.20 mL, 10.9 mmol) and
NMM (2.4 mL, 21.8 mmol) as above. Subsequent hydrogenation
of 4c (H2, 505 mg Pd/C, 45 mL 0.5% AcOH/MeOH) and
cyclization (508 mg Na0 in 75 mL EtOH) yielded 578 mg of 5c
after purification (49%): 1H NMR (500 MHz, CDCl3) δ 6.92 (s
with fine splitting, 1 H), 4.74 (dd, J ) 5.0, 4.5 Hz, 1 H), 4.21
(qd, J ) 7.5, 3.0 Hz, 2 H), 4.08 (dd, J ) 7.5, 6.5 Hz, 1 H), 3.88
(td, J ) 8.0, 4.5 Hz, 1 H), 2.80 (ddd, J ) 17.0, 4.0, 3.0 Hz, 1H),
2.41 (s (br), 1 H), 2.23 (ddq, J ) 17.0, 8.5, 1.5 Hz, 1 H), 1.45 (s,
3 H), 1.40 (s, 3 H), 1.29 (t, J ) 7.5 Hz, 3 H); 13C NMR (125 MHz,
CDCl3) δ 166.0 (100% 13C), 133.5, 130.9 (d, J ) 75 Hz), 109.6,
77.9, 72.2, (d, J ) 6.3 Hz), 68.8 (d, J ) 5 Hz), 61.0, 29.3, 27.9,
25.7, 14.1; HRMS (FAB, NBA) calcd for C1113CH19O5 (M+ + H)
244.1266, found 244.1273.
P r ep a r a tion of th e Cr u d e Cell Extr a ct fr om E. coli
Str a in KA12/p KAD50. Miller’s LB broth (100 mL) containing
tryptophan (4 mg) was inoculated with a single colony of E. coli
strain KA12 transformed with plasmid pKAD50.18 The culture
was shaken at 37 °C and 250 rpm for 24 h. Six 2-L Erlenmeyer
flasks were each charged with Miller’s LB broth (1 L) and Trp
(20 mg/L) and sterilized by autoclaving. After cooling, each flask
was provided with a solution of IPTG in EtOH (0.5 M, 0.4 mL)
and a solution of chloramphenicol in EtOH (30 mg/mL, 2.0 mL).
Finally, each flask was inoculated with 15 mL of the above
prepared KA12/pKAD50 culture. The broth was shaken at 37
°C and 250 rpm for 24 h. After cooling to 23 °C, the cells were
collected by centrifugation at 5000 rpm (4200 × g). The cell pellet
was resuspended in Tris-HCl (50 mM, pH 8.2) containing
dithiothreitol (DTT, 10 mM). The cells were again collected by
centrifugation at 5000 rpm (4200 × g) to give 30 g of cells. The
cell pellet so obtained was resuspended in Tris-HCl (100 mL,
50 mM, pH 8.2) containing DTT (10 mM), and the cells were
ruptured by passing them through a French Press (three times).
Cell debris was removed from the lysate by centrifugation at
14 500 rpm (30 000 × g). The supernatant was dialyzed against
Tris-HCl (2 L, 50 mM, pH 8.2) containing DTT (10 mM) and
phenylmethanesulfonyl fluoride (50 mg) for 8 h and then
dialyzed 3 × 17 h against Tris-HCl (2 L, 50 mM, pH 8.2)
containing DTT (10 mM). The crude cell extracts were stored at
4 °C prior to use. Note: best results in the following preparations
of chorismate were obtained with freshly prepared cell extract.
Mixtu r e of [10-12C]Ch or ism ic Acid (1a ) a n d [5-18O,10-13C]-
Ch or ism ic Acid (1c). NaOH (1 M, 2.8 mL, 2.8 mmol) was added
to a solution of 6a (6.2 mg, 0.030 mmol) and 6d (514 mg, 2.73
mmol) in a 1:1 mixture of THF and H2O (41 mL). The mixture
was stirred at 23 °C for 8 h. After the removal of THF by
distillation in vacuo, the aqueous mixture was lyophilized. ATP
(monosodium salt) (2.29 g, 4.14 mmol) was carefully dissolved
in Tris-HCl (80 mL, 50 mM) while the pH of the solution was
maintained between 6.5 and 9.0. To this solution was added
FMN (142 mg, 0.276 mmol), phosphoenolpyruvate (PEP, tripo-
Meth yl [7-12C]Sh ik im a te (6a ). Dowex 50W-X8 acidic ion-
exchange resin (4.5 g) was added to a solution of 5a (2.71 g, 12.0
mmol) in MeOH (300 mL) and H2O (15 mL). The mixture was
stirred at 23 °C for 36 h. The resin was then removed via
filtration, and the filtrate was concentrated. Purification of the
crude product by silica gel chromatography (10% MeOH/CH2-
Cl2) gave methyl shikimate 6a (1.85 g, 82%).
Eth yl [1,7-13C]Sh ik im a te (6b). Hydrolysis of 5b (198 mg,
1.66 mmol) with Dowex 50W-X8 (1.5 g) in 95% EtOH (50 mL)
gave 113 mg of ethyl shikimate 6b (68%) after chromatogra-
phy: 1H NMR (500 MHz, CDCl3) δ 6.88 (m, 1 H), 4.46 (s (br), 1
H), 4.21 (qd, J ) 7.0, 3.0, 2 H), 3.97 (td, J ) 9.0, 5.5 Hz, 1 H),
3.62 (dd, J ) 9.0, 4.0 Hz, 1 H), 3.30 (s (br), 1 H), 2.94 (ddd, J )
18.0, 5.5, 2.5 Hz, 1 H), 2.74 (s (br), 1 H), 2.64 (s (br), 1 H), 2.21
(dd with fine splitting, J ) 18.0, 8.5 Hz, 1 H), 1.30 (t, J ) 7.0
Hz, 3 H); 13C NMR (125 MHz, CD3OD) δ 168.2 (d, J ) 75 Hz,
100% 13C), 139.2 (d, J ) 75 Hz), 130.4 (d, J ) 75 Hz, 100% 13C),
72.6, 68.4, 67.2 (d, J ) 6.3 Hz), 61.8, 31.4 (d, J ) 43 Hz), 14.5;
HRMS (EI) calcd for C713C2H14O5 (M+) 204.0909, found 204.0919.
Eth yl [7-13C]Sh ik im a te (6c). Hydrolysis of 5c (403 mg, 1.66
mmol) with Dowex 50W-X8 (2.5 g) in 95% EtOH gave 6c (275

4938 J . Org. Chem., Vol. 64, No. 13, 1999
Notes
tassium salt) (1.95 g, 8.33 mmol), a solution of KCl (1.0 M
aqueous, 6.9 mL), a solution of MgSO4 (1.0 M aqueous, 0.69 mL),
and a solution of the sodium shikimates (obtained above) in Tris-
HCl (15 mL, 50 mM, pH 8.2). KA12/pKAD50 extract (35 mL,
corresponding to the lysate of ca. 10 g of cells, estimated total
protein concentration ca. 40 mg/mL) was added, and the pH of
the resulting mixture was adjusted to 8.1 using HCl. Finally,
solid sodium dithionite (272 mg, 1.56 mmol) was added. The
resulting solution was sealed and gently stirred at 23 °C for 2.5
h. The reaction mixture was then filtered through an Amicon
10K cutoff ultrafiltration membrane at 4 °C. When nearly dry,
the membrane was washed with Tris-HCl (100 mL, 50 mM, pH
8.2). The combined filtrate was acidified to pH 1.5 and extracted
with EtOAc (6 × 150 mL). The combined extracts were dried
over Na2SO4 and carefully concentrated in vacuo (while the bath
for the rotary evaporator was maintained at ca. 20 °C). The
residue was dissolved in a 1:1 mixture of H2O and MeOH and
filtered through a pad of charcoal. The filtrate was lyophilized
to provide the mixture of chorismic acids 1a and 1c (310 mg,
49%), containing only minor impurities as judged by HPLC and
1H and 13C NMR (see Supporting Information for representative
data). This material can be further purified by C18 reverse phase
silica gel flash chromatography17 as needed.
chorismic acid mixture 1a and 1c. A solution of ATP (2.76 g,
5.01 mmol), FMN (162 mg, 0.334 mmol), PEP (2.35 g, 10.0
mmol), KCl (1.0 M aqueous, 8.4 mL), and MgSO4 (1.0 M aqueous,
0.84 mL) was prepared in Tris-HCl (100 mL, 50 mM, pH 8.2) as
described above. The solution of sodium shikimates in Tris-HCl
(25 mL, 50 mM, pH 8.2) was then added. The crude cell extract
of KA12/pKAD50 (35 mL) was added, and the pH was adjusted
to 8.1. Finally, solid Na2S2O4 (321 mg, 1.84 mmol) was added,
and the reaction mixture was sealed and gently stirred at 23 °C
for 2.5 h before being processed as described above to give the
chorismic acid mixture 1a and 1b (403 mg, 53%).
Ack n ow led gm en t. This work was supported in part
by grants from the National Institutes of Health
(GM38723 to D.H. and a postdoctoral fellowship to
D.J .G.). We are grateful to J ohn D. Frost for the
generous gift of plasmid pKAD50, to Patrizio Mattei for
careful reading of the manuscript, and to Peter Kast
for discussion and for preparing the E. coli strain KA12/
pKAD50.
Su p p or t in g In for m a t ion Ava ila b le: 1H and 13C NMR
spectra for compounds 3a , 5c, 6c, 7, and a representative 1a /
1b mixture. This information is free of charge via the Internet
at http://pubs.acs.org.
Mixtu r e of [10-12C]Ch or ism ic Acid (1a ) a n d [1,10-13C]-
Ch or ism ic Acid (1b). A mixture of shikimate esters 6a (620
mg, 3.29 mmol) and 6b (10.8 mg, 0.052 mmol) were saponified
in 1:1 THF/H2O (41 mL) as described for the production of the
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