Synthesis of (E)-4-hydroxydimethylallyl diphosphate. An intermediate in the methyl erythritol phosphate branch of the isoprenoid pathway

Article abstract of DOI:10.1021/jo0258453

The syntheses of (E)-1-hydroxy-2-methyl-2-buten-4-yl diphosphate ((E)-4-hydroxydimethylallyl diphosphate, HDMAPP), an intermediate in the methyl erythritol phosphate pathway, and (E)-[4-²H]HDMAPP were accomplished in two steps from (E)-4-chloro-2-methyl-2-butenal. The synthetic route is easily adaptable for the facile incorporation of tritium at C-4 of the diphosphate.

Full text of DOI:10.1021/jo0258453

Syn th esis of (E)-4-Hyd r oxyd im eth yla llyl
Dip h osp h a te. An In ter m ed ia te in th e
Meth yl Er yth r itol P h osp h a te Br a n ch of th e
Isop r en oid P a th w a y
David T. Fox and C. Dale Poulter*
Department of Chemistry, University of Utah,
Salt Lake City, Utah 84112
poulter@chem.utah.edu
Received April 22, 2002
Abstr act: The syntheses of (E)-1-hydroxy-2-methyl-2-buten-
4-yl diphosphate ((E)-4-hydroxydimethylallyl diphosphate,
HDMAPP), an intermediate in the methyl erythritol phos-
phate pathway, and (E)-[4-2H]HDMAPP were accomplished
in two steps from (E)-4-chloro-2-methyl-2-butenal. The
synthetic route is easily adaptable for the facile incorporation
of tritium at C-4 of the diphosphate.
Isoprenoid compounds constitute one of the largest and
most diverse groups of natural products, with greater
than 35 000 identified members to date.1 These molecules
are derived from the simple five-carbon precursors iso-
pentenyl diphosphate (IPP) and dimethyallyl diphosphate
(DMAPP). Until recently, IPP and DMAPP were believed
to originate from acetate by the mevalonate pathway.2
However, pioneering studies by Rohmer3 and Arigoni4
revealed an alternative pathway that operates in plant
chloroplasts, algae, and bacteria.5 As outlined in Figure
1, pyuvate and glyceraldehyde 3-phosphate are con-
densed in a thiamine diphosphate-dependent reaction
catalyzed by 1-deoxy-D-xylulose 5-phosphate (DXP) syn-
thase to give DXP.6 The branched skeleton typical of
isoprenoid compounds is then constructed from the linear
deoxysugar by a reversible rearrangement-NADPH-
dependent reduction catalyzed by 2-C-methyl-D-erythritol
4-phosphate (MEP) synthase.7 MEP is converted to 2-C-
methyl-D-erythritol-2,4-cylcodiphosphate (cMEPP) by three
successive enzymes, 4-diphosphocytidyl-2-C-methyl-D-
erythritol (CDPME) synthase,8 4-diphosphocytidyl-2-C-
methyl-D-erythritol-2-phosphate (CDPME) kinase,9 and
cMEPP synthase.10 Recently, Wolff et al.11 reported that
the cyclic diphosphate is converted to (E)-1-hydroxy-2-
methyl-2-buten-4-yl diphosphate ((E)-4-hydroxydimeth-
F IGURE 1. Methyl erythritol phosphate (MEP) pathway for
isoprenoid biosynthesis.
ylallyl diphosphate, HDMAPP) by the protein encoded
by the Escherichia coli gcpE gene and established the
structure of HDMAPP by synthesis. We now report a
short synthesis of HDMAPP that accommodates the
introduction of an isotopic tag in the penultimate step.
The procedure of Miyakado et al.12 was used to convert
pyruvic aldehyde dimethyl acetal 1 and vinylmagnesium
bromide to (E)-4-chloro-2-methyl-2-butenal (3), as out-
1
lined in Scheme 1. Both H NMR and 13C NMR spectra
indicated that only one double bond isomer was present,
and the NMR data were in full agreement with those
(1) Buckingham, J . In Dictionary of Natural Products on CD-ROM
Version 6.1; Chapman and Hall: New York, 1998.
(2) Bochar, D. A.; Freisen, J . A.; Stauffacher, C. V.; Rodwell, V. W.
In Comprehensive Natural Products Chemistry; Elsevier: Oxford, 1999;
Vol. 2, pp 15-44.
(3) Flesch, G.; Rohmer, M. Eur. J . Biochem. 1988, 175, 405.
(4) Arigoni, D.; Sagner, S.; Latzel, C.; Eisenreich, W.; Bacher, A.;
Zenk, M. H. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 10600.
(5) Eisenreich, W.; Schwarz, M.; Cartayrade, A.; Arigoni, D.; Zenk,
M. H.; Bacher, A. Chem. Biol. 1998, 5, R221.
(6) Lois, L. M.; Campos, N.; Putra, S. R.; Danielson, K.; Rohmer,
M.; Boronat, A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2105.
(7) Koppisch, A. T.; Fox, D. T.; Blagg, S. J . B.; Poulter, C. D.
Biochemistry 2002, 41, 236.
(8) (a) Rohdich, F.; Wungsintaweekul, J .; Fellermeier, M.; Sagner,
S.; Herz, S.; Kis, K.; Eisenreich, W.; Bacher, A.; Zenk, M. H. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 11758. (b) Kuzuyama, T.; Takagi, M.;
Kaneda, K.; Dairi, T.; Seto, H. Tetrahedron Lett. 2000, 41, 703.
(9) (a) Lu¨ttgen, H.; Rohdich, F.; Herz, S.; Wungsintaweekul, J .;
Hecht, S.; Schuhr, C. A.; Fellermeier, M.; Sagner, S.; Zenk, M. H.;
Bacher, A.; Eisenreich, W. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1062.
(b) Kuzuyama, T.; Takagi, M.; Kaneda, K.; Watanabe, H.; Dairi, T.;
Seto, H. Tetrahedron Lett. 2000, 41, 2925.
(10) (a) Herz, S.; Wungsintaweekul, J .; Schuhr, C. A.; Hecht, S.;
Lu¨ttgen, H.; Sagner, S.; Fellermeier, M.; Eisenreich, W.; Zenk, M. H.;
Bacher, A.; Rohdich, F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2426.
(b) Takagi, M.; Kuzuyama, T.; Kaneda, K.; Watanabe, H.; Dairi, T.;
Seto, H. Tetrahedron Lett. 2000, 41, 3395.
(11) Wolff, M.; Seemann, M.; Grosdemange-Billiard, C.; Tritsch, D.;
Campos, N.; Rodriguez-Concepcion, M.; Boronat, A.; Rohmer, M.
Tetrahedron Lett. 2002, 43, 2555.
(12) Miyakado, M.; Ohno, N.; Yoshioka, H.; Mabry, T. J . Phytochem-
istry 1978, 17, 143.
10.1021/jo0258453 CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/31/2002
J . Org. Chem. 2002, 67, 5009-5010
5009

SCHEME 1a
dry acetonitrile (2 × 5 mL) at 40 °C. (E)-4-Chloro-2-methyl-2-
buten-1-ol (40 mg, 0.33 mmol) in 200 µL of dry acetonitrile was
added via syringe to a stirred solution of 634 mg (0.663 mmol)
tris(tetra-n-butylammonium) hydrogen pyrophosphate in 1 mL
dry acetonitrile at room temperature. After being stirred for 2
h, the suspension was concentrated by rotary evaporation. The
residue was dissolved in 1 mL of cation-exchange buffer (49:1
(v/v) 25 mM NH4HCO3 (pH 8.0)/2-propanol) and passed over 120
mequiv of Dowex 50WX8-200 cation-exchange resin (ammonium
form) preequilibrated with two column volumes of the same
buffer. The diphosphate was eluted with two column volumes
(120 mL) of the same buffer, flash frozen, and lyophilized.15 The
residue was resuspended in 1 mL of a 50 mM ammonium
bicarbonate solution to which 1 mL of 2-propanol and a few drops
of acetonitrile were added. After vortexing and centrifugation
at 2000 rpm for 2 min, the supernatant was decanted. This
procedure was repeated three times, and the supernatants were
combined, concentrated, frozen, and lyophilized. The residue was
resuspended in 1 mL of 53:47 (v/v) 2-propanol/50 mM NH4HCO3
(pH 8.0) and loaded onto a cellulose column (4 × 15 cm)
preequilibrated with the same buffer. Fractions containing
HDMAPP were pooled, frozen, and lyophilized to give 86 mg
(83%) of a white solid: Rf 0.47 (53:47 (v/v) 2-propanol/50 mM
a
Reagents and conditions: (a) 1.0 M vinylmagnesium bromide
in THF, THF, -20 °C; (b) cat. CuCl2, NaCl, concd HCl, benzene
0 f 50 °C, overnight, 25% (two steps); (c) NaBH(D)4, THF, -20
°C, 80%; (d) tris(tetra-n-butylammonium) hydrogen pyrophos-
phate, MeCN, Dowex (NH4+), 83%.
reported by Miyakado et al. The (E)-geometry of the
double bond was originally established by Miyakado et
al. by using 3 in a total synthesis of tricholin, a natural
product isolated from Trichocline incana that contains
the (E)-4-hydroxydimethylallyl13 moiety in the side chain
of the furocoumarin. Wolff et al. confirmed the (E)-
geometry of synthetic HDMAPP from nuclear Over-
1
NH4HCO3 (pH 8.0)); H NMR (300 MHz, D2O) δ 5.62 (dt, 1 H,
J ) 6.8, 0.98 Hz), 4.50 (t, 2 H, J ) 7.3 Hz), 3.99 (s, 2 H), 1.68 (s,
3 H); 13C NMR (75 MHz, D2O/CD3OD) δ 140.63, 121.76 (d, J )
7.5 Hz), 67.56, 63.38 (d, J ) 5.0 Hz), 14.08; 31P NMR (121 MHz,
D2O) δ -10.5 (d, J ) 20.8 Hz), -8.65 (d, J ) 21.4 Hz); HRMS
(FAB, M - H) calcd for C5H11P2O8 260.9929, found 260.9924.
(E)-4-Ch lor o-1-d eu ter io-2-m eth yl-2-bu ten -1-ol ([1-2H]-5).
A solution (100 mg, 0.84 mmol) of 3 in 0.25 mL of THF was added
via syringe to 35 mg (0.84 mmol) of NaB2H4 in 0.5 mL of THF
at -20 °C. The mixture was allowed to stir for 5 h at the same
temperature before ice-cold MeOH was added. The reaction
mixture was filtered through a plug of silica with diethyl ether,
and solvent was removed by rotary evaporation. The residue was
purified by flash silica chromatography (7:3 hexanes/ether) to
yield 82 mg (80%) of a pale yellow oil: Rf 0.17 (7:3 hexanes/
12
hauser effects. Reduction of aldehyde 3 with NaBH4
or NaB2H4 gave 4 and [1-2H]-5. (E)-4-Chloro-2-methyl-
2-buten-1-ol (4) and [1-2H]-5 were readily converted to
the corresponding diphosphates by displacement of chlo-
ride with tris(tetra-n-butylammonium) hydrogen pyro-
phosphate. The tetrabutylammonium counterion was
replaced by cation-exchange over Dowex (ammonium
form) and purification of the crude ammonium diphos-
phates over cellulose gave HDMAPP11 and [4-2H]-HD-
MAPP.
1
ether); H NMR (300 MHz, CDCl3) δ 5.74 (tt, 1 H, J ) 8.0, 1.5
Hz), 4.14 (d, 2 H, J ) 8.0 Hz), 4.06 (br s, 1 H), 1.76 (s, 3 H); 13
C
NMR (75 MHz, CDCl3) δ 141.41, 120.57, 67.35 (t, J ) 21.9 Hz),
The (E)-chloroaldehyde is a versatile intermediate. The
compound is sufficiently stable to be purified by chro-
matography, and reduction with commercially available
NaB2H4 or NaB3H4 allows one to easily attach an isotopic
label to C-1 in the penultimate step of the synthesis. The
overall yield for reduction of chloroaldehyde 3 to chloro
alcohol 4 and the subsequent phosphorylation to give
HDMAPP, with purification of the intermediate olefin,
were 66%. This procedure should be readily amenable
to the synthesis of tritium labeled HDMAPP by modifying
the procedure to omit purification of [1-3H]-4 by flash
chromatography in order to minimize handling volatile
radioactive materials before the phosphorylation.
40.38, 13.68.
(E)-1-Hyd r oxy-1-d eu ter io-2-m eth yl-2-bu ten -4-yl Dip h os-
p h a te ((E)-[4-2H]-4-Hyd r oxyd im eth yla llyl Dip h osp h a te,
[4-2H]-HDMAP P ). Following the procedure described for HD-
MAP P , 40 mg (0.33 mmol) of 5 was converted to 80 mg (83%)
of [4-2H]-HDMAP P : Rf 0.47 (53:47 (v/v) 2-propanol/50 mM NH4-
HCO3 (pH 8.0)); 1H NMR (300 MHz, D2O) δ 5.64 (t, 1 H, J ) 6.8
Hz), 4.51 (t, 2 H, J ) 7.1 Hz), 3.98 (br s, 1 H), 1.69 (s, 3H); 13C
NMR (75 MHz, D2O/CD3OD) 140.37, 122.11 (d, J ) 7.6 Hz),
67.25 (t, J ) 21.9 Hz), 63.15 (d, J ) 5.0 Hz), 14.10; 31P NMR
(121 MHz, D2O) δ -10.25 (d, 1 P, J ) 22.0 Hz), -6.53 (d, 1 P,
J
) 22.6 Hz); HRMS (FAB, M - H) calcd for C5DH10P2O8
261.9992, found 261.9982.
Ack n ow led gm en t. We thank Dr. A. T. Koppisch for
insightful discussions.
Exp er im en ta l Section
(E)-1-Hyd r oxy-2-m eth yl-2-bu ten -4-yl Dip h osp h a te ((E)-
4-Hyd r oxyd im eth yla llyl Dip h osp h a te, HDMAP P ). Tris-
(tetra-n-butylammonium) hydrogen pyrophosphate14 (2.0 g) was
dried by azeotropic removal of water by rotary evaporation of
Su p p or tin g In for m a tion Ava ila ble: 1H, 13C, and 31P
NMR spectra for HDMAP P and [4-2H]HDMAP P . This
material is available free of charge via the Internet at
http://pubs.acs.org.
J O0258453
(13) In IUPAC nomenclature, the correct numbering scheme of the
carbon framework has the methyl group at the C-2 position and the
hydroxyl at C-1. The common name given to the isoprenoid precursor,
HDMAPP, places the methyl group at the C-3 position and the hydroxyl
moiety at C-4.
(14) Davisson, V. J .; Woodside, A. B.; Neal, T. R.; Stremler, K. E.;
Muehlbacher, M.; Poulter C. D. Biochemistry 1986, 25, 4768.
(15) If more than 5% of the tetra-n-butylammonium salt remains,
a second pass through freshly prepared Dowex is necessary; otherwise,
the subsequent chromatography on cellulose is not efficient.
(16) Davisson, V. J .; Woodside, A. B.; Poulter, C. D. Methods
Enzymol. 1984, 110, 130-144.
5010 J . Org. Chem., Vol. 67, No. 14, 2002