Chemical synthesis of enantiopure 2-C-methyl-D-erythritol 4-phosphate, the key intermediate in the mevalonate-independent pathway for isoprenoid biosynthesis

Article abstract of DOI:10.1016/S0040-4020(00)00065-X

Optically pure 2-C-methyl-D-erythritol 4-phosphate, a key intermediate in the mevalonate-independent route for isoprenoid biosynthesis, is conveniently synthesized from 1,2-O-isopropylidene-α-D-xylofuranose, including the possibility of radiolabeling of t

Full text of DOI:10.1016/S0040-4020(00)00065-X

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 1485–1489
Chemical Synthesis of Enantiopure 2-C-Methyl-d-Erythritol
4-Phosphate, the Key Intermediate in the
Mevalonate-Independent Pathway for Isoprenoid Biosynthesis
*
Jean-Franc¸ois Hoeffler, C. Pale-Grosdemange and Michel Rohmer
´
Laboratoire de Chimie et Biochimie des Microorganismes, Universite Louis Pasteur, Institut Le Bel, 4 rue Blaise Pascal,
67043 Strasbourg Cedex, France
Received 31 August 1999; accepted 24 January 2000
Abstract—Optically pure 2-C-methyl-d-erythritol 4-phosphate, a key intermediate in the mevalonate-independent route for isoprenoid
biosynthesis, is conveniently synthesized from 1,2-O-isopropylidene-a-d-xylofuranose, including the possibility of radiolabeling of this
substrate. ᭧ 2000 Elsevier Science Ltd. All rights reserved.
Introduction
by an intramolecular rearrangement of DXP followed by the
reduction of the resulting aldehyde 4.4 The key role of 2-C-
methyl-d-erythritol derivatives was first evidenced by the
incorporation of the free tetrol into the prenyl chain of
ubiquinone and menaquinone from E. coli.5 The corre-
sponding monophosphate 5 (MEP) is however the precursor
of IPP, as it is directly formed from DXP by the DXP
isomero-reductase.4 Due to the probable absence of an effi-
cient kinase, free ME is only poorly incorporated into
isoprenoids by the wild-type E. coli and not incorporated
at all by all other tested organisms.5b An efficient synthesis
of MEP is therefore required in order to investigate its
conversion into IPP in cell-free systems.
Two biosynthetic pathways are leading to isopentenyl
diphosphate 6 (IPP), the universal precursor of isoprenoids:
the well known mevalonate (MVA) pathway and the long
overlooked 2-C-methyl-d-erythritol 4-phosphate 5 (MEP)
route starting from triose phosphate derivatives,1 recently
discovered in eubacteria and later found in the plastids of
phototrophic eukaryotes (Scheme 1).1,2 Only two C5 inter-
mediates are known: 1-deoxy-d-xylulose 5-phosphate 3
(DXP) is formed from the condensation of (hydroxy-
ethyl)thiamin diphosphate on glyceraldehyde 3-phosphate
2,3 and 2-C-methyl-d-erythritol 4-phosphate 5 is obtained
Scheme 1. 2-C-Methyl-d-erythritol 4-phosphate pathway for isoprenoid biosynthesis.
Keywords: biosynthesis; isoprenoids; 2-C-methyl-d-erythritol 4-phosphate.
* Corresponding author. Tel.: 33-(0)3-88-41-61-02; fax: 33-(0)3-88-41-61-01; e-mail: mirohmer@chimie.u-strasbg.fr
0040–4020/00/$ - see front matter ᭧ 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00065-X

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J.-F. Hoeffler et al. / Tetrahedron 56 (2000) 1485–1489
Results and Discussion
carried out by azeotropic distillation of the water with
toluene. The required configuration for the 2-C-methyl-d-
erythritol was expected to be obtained by introducing a
methyl group at the C-3 carbon atom of the 1,2-O-iso-
propylidene-a-d-xylofuranose 1. Consequently, the key
step of this approach was the stereoselective nucleophilic
addition of methylmagnesium chloride on the carbonyl
group of ketone 9. Treatment of ketone 9 with methyl-
magnesium chloride resulted in the addition of the methyl
group from the less hindered b-face and yielded the tertiary
alcohol 10 with the desired stereochemistry.10 The structure
assigned to product 10 was verified using 1H-1H COSY and
NOESY NMR experiments. The NOEs between H-1 and
H-2 confirmed the anomeric a configuration. The position
of the methyl group at C-3 on the b-face of the furanose ring
was supported on the one hand by strong NOEs between
H-2 and the protons from the C-3-methyl group, and on the
other hand by medium NOEs between the protons of the C-3
methyl protons and H-4 was very weak. Benzylation of the
tertiary alcohol 10 could only be performed using benzyl
2,2,2-trichloroacetimidate in acidic catalytic conditions.11
The acetonide protection in 11 was removed in the presence
The 3-oxo group of the ketone obtained from protected 1,2-
O-isopropylidene-a-d-xylofuranose exhibits an excellent
stereoselectivity in addition reactions to yield 3-C-alkyl-
ribofuranoses with the alkyl group on the b-face of the
carbohydrate ring.6,7 The configurations of these 3-alkyl
ribofuranosides at C-3 and C-4 are respectively identical
with those at C-3 and C-2 of 2-C-methyl-d-erythritol
4-phosphate. The synthesis of 2-C-methyl-d-erythritol
4-phosphate from commercially available 1,2-O-isopropyl-
idene-a-d-xylofuranose 7 was therefore attempted using a
methodology based on these literature data (Scheme 2).
The first step of the synthetic route is the selective pro-
tection of the primary alcohol 7 with dibenzylphosphoro-
chloridate8 in pyridine to give 5-dibenzylphosphate-1,2-O-
isopropylidene-a-d-xylofuranose 8 in 95% yield. Oxidation
of the free alcohol with pyridinium dichromate (PDC) in the
presence of acetic acid9 gave the corresponding ketone 9,
best isolated and stored as a hydrate. The gem-diol had to be
dehydrated back to the ketone 9: this could be conveniently
˚
Scheme 2. Chemical synthesis of 2-C-methyl-d-erythritol 4-phosphate. (i) (BnO)2P(O)Cl, pyridine (95%); (ii) PDC, AcOH, 3 A molecular sieves, CH2Cl2
(90%); (iii) MeMgCl, THF (84%); (iv) benzyl 2,2,2-trichloroacetimidate, CF3SO3H (cat), cyclohexane/CH2Cl2 2/1 (70%); (v) 90% aq. TFA, Ϫ10ЊC (80%);
(vi) NaIO4, MeOH–H2O (93%); (vii) NaBH4, MeOH (92%); (viii) Ac2O, TEA, DMAP, CH2Cl2 (95%); (ix) H2, 10% Pd/C, EtOH (95%).

J.-F. Hoeffler et al. / Tetrahedron 56 (2000) 1485–1489
1487
of 90% aqueous trifluoroacetic acid. The resulting mixture
of the two anomers of hemicetal 12 underwent a sodium
metaperiodate mediated glycol oxidative cleavage into the
tribenzyl derivative 13 of 2-C-methyl-d-erythrose 4-phos-
phate. Sodium borohydride reduction of aldehyde 13
followed by hydrogenolysis of the benzyl groups12 yielded
free 2-C-methyl-d-erythritol-4-phosphate 5, which was
characterized either directly4a or via its tribenzyl derivative
14 and the corresponding diacetate 15.
1H NMR (400 MHz, CDCl3): dˆ1.32 (3H, s, CH3); 1.50
(3H, s, CH3); 4.02 (1H, m); 4.24 (4H, m); 4.55 (1H, d,
Jˆ3.7 Hz, 3-H); 5.04 (m, 4H, 2×CH2Ph); 5.88 (d, 1H,
Jˆ3.3 Hz, 1-H); 7.33–7.48 (10H, m, 2×Ph). 13C NMR
(50 MHz, CDCl3): dˆ26.16 (CH3); 26.84 (CH3); 63.54
2
2
(CH2, d, JC,Pˆ4.9 Hz); 69.80 (CH2, d, JC,Pˆ4.9 Hz);
2
69.92 (CH2, d, JC,Pˆ6.6 Hz); 73.72 (CH); 78.78 (CH,
3
C-4, d, JC,Pˆ4.9 Hz); 85.03 (CH); 105.00 (CH, C-1);
111.78 (quaternary C); 127.94, 128.04, 128.63, 128.72,
135.18, 135.25 and 135.38 (aromatic C). 31P NMR
(162 MHz, CDCl3): dˆ2.09 (m). HRMS (FABϩ):
(MϩH)ϩ calculated for C22H28O8P 451.1530, found
451.1522.
This straightforward synthesis easily affords optically pure
2-C-methyl-d-erythritol 4-phosphate
5 in satisfactory
yields. It can be extended to the obtention of free methyl-
erythritol. Radiolabeling can be performed with 14C at the
level of the addition of the Grignard reagent using
1,2-O-isopropylidene-a-d-erythro-pentofuranose-3-
ulose 5-dibenzylphosphate (9). To a stirred solution of 5-
dibenzylphosphate-1,2-O-isopropylidene-a-d-xylofuranose
8 (3.1 g, 7 mmol, 1 equiv.) in dry CH2Cl2 (60 ml) were
3
[14C]methylmagnesium halide, and more easily with H
by reduction of the tribenzylated 2-C-methyl-d-erythrose
4-phosphate 13 using tritiated sodium borohydride.
˚
slowly added at 0ЊC activated molecular sieves (3 A, 3 g),
pyridinium dichromate (6.6 g, 17.5 mmol, 2.5 equiv.) and
acetic acid (0.72 ml, 12.6 mmol, 1.8 equiv.). The reaction
was stirred at room temperature for 3 h. Chromium salts
were eliminated by adding Celite (3 g), MgSO4 (3 g) and
diethyl ether (50 ml).14 The mixture was stirred for addi-
tional 30 min, filtered through a bed of silica gel (about
5 cm thickness) on a sintered-glass funnel. The solid cake
was washed with diethyl ether (3×30 ml), and the filtrate
was concentrated in vacuum to give 9 as oil. Remaining
chromium salts were removed by repeating the diethyl
ether treatment. The hydrated ketone was obtained after
evaporation of the solvent as colorless syrup (2.85 g,
90%). (Rfˆ0.42, ethyl acetate/hexane, 70:30). 1H NMR
(400 MHz, CDCl3): dˆ1.39 (3H, s, CH3); 1.45 (3H, s,
Experimental
General methods and materials
Most analytical techniques were as previously described.13
All non-aqueous reactions were run in dry solvents under an
argon atmosphere. ‘Dried and concentrated’ refers to
removal of residual amounts of water with anhydrous
Na2SO4 followed by evaporation of solvent on a rotary
evaporator. Flash chromatography was performed on
Merck silica gel (40–63 mm) with the indicated solvent
system.14 Thin-layer plates were developed with an ethanol
solution of p-anisaldehyde (2.5%), sulfuric acid (3.5%) and
acetic acid (1.6%) by heating up to 100ЊC. NMR spectra
were recorded on Bruker AC200, AV400 or ARX500
spectrometers at 200 and 400 MHz for 1H NMR,
50.32 MHz and 125.77 MHz for 13C NMR and 162 MHz
for 31P NMR. NMR experiments were carried out in
CDCl3 or D2O using as internal standard CHCl3 (dˆ
3
4
CH3); 4.18 (1H, dd, J1,2ˆ4.4 Hz, J2,4ˆ1.1 Hz, 2-H);
2
3
3
4.19 (1H, ddd, J5a,5bˆ11.2 Hz, J5a,Pˆ6.2 Hz, J4,5a
ˆ
2
3
2.6Hz, 5-Ha); 4.24 (1H, ddd, J5a,5bˆ11.2 Hz, J5b,P
ˆ
3
3
2.6 Hz, J4,5bˆ5.7 Hz, 5-Hb); 4.43 (1H, ddd, J4,5bˆ5.5 Hz,
3J4,5aˆ2.6 Hz, J2,4ˆ1.1 Hz, 4-H), 5.01 (1H, dd, 2Jˆ
4
2
11.7 Hz, JH,Pˆ8.4 Hz, CH2Ph), 5.02 (1H, dd, Jˆ11.7 Hz,
JH,Pˆ8.4 Hz, CH2Ph), 5.04 (1H, dd, 2Jˆ11.7 Hz, JH,P
ˆ
1
7.26 ppm) or DHO (dˆ4.65 ppm) for H NMR and CDCl3
2
(dˆ77.03 ppm) or t-butanol (dˆ31.60 ppm) for 13C NMR.
31P NMR spectra were calibrated against an external H3PO4
standard (dˆ0.00 ppm). Negative mode electrospray mass
spectrometry was performed on a Hewlett Packard 1100MS
spectrometer using acetonitrile/water (1:1) as solvent,
and high-resolution mass spectrometry on a ZAB-HF
spectrometer with an acceleration potential of 8 keV using
m-nitrobenzyl alcohol as matrix and xenon as ionization
gas. All intermediates as well as the final 2-C-methyl-d-
erythritol 4-phosphate (5) were found to be pure by the 1H-,
13C- and 31P NMR (in the latter case after H-decoupling) and
TLC criteria.
8.4 Hz, CH2Ph), 5.05 (1H, dd, Jˆ11.7 Hz, JH,Pˆ8.4 Hz,
3
CH2Ph); 5.96 (1H, d, J1,2ˆ4.4 Hz, 1-H); 7.38 (10H, m,
2×Ph). 13C NMR (50 MHz, CDCl3): dˆ26.97 (CH3);
2
27.40 (CH3); 66.15 (CH2, d, JC,Pˆ5.2 Hz); 69.54 (2×CH2,
2
3
d, JC,Pˆ5.0 Hz); 76.15 (CH, C-2); 78.00 (CH, d, JC,P
ˆ
8.2 Hz, C-4); 103.25 (CH, C-1); 114.24 (quaternary C);
127.94, 128.53, 128.63, 135.38 and 135.58 (aromatic C);
207.46 (C-3). 31P NMR (162 MHz, CDCl3): dˆϪ0.13
(m). HRMS (FABϩ): (MϩH)ϩ calculated for C22H26O8P
449.1365, found 449.1386. The gem-diol was dried back
to the ketone 9 by evaporation with dry toluene three times.
1,2-O-isopropylidene-3-C-methyl-a-d-ribofuranose 5-
dibenzylphosphate (10). The ketone 9 (850 mg, 1.9
mmol, 1 equiv.), dissolved in dry THF (30 ml), was treated
at Ϫ10ЊC with a 3 M solution of methylmagnesium chloride
(0.82 ml, 2.46 mmol, 1.3 equiv.) in THF. The reaction
mixture was stirred at room temperature for 4 h. A saturated
ammonium chloride solution (20 ml) was added to the
cooled (Ϫ10ЊC) solution. After addition of diethyl ether
(30 ml), the reaction mixture was extracted with ethyl ace-
tate (3×50 ml). The combined extracts were washed with
brine, dried, and filtered. After concentration of the filtrate,
1,2-O-isopropylidene-a-d-xylofuranose 5-dibenzylphosphate
(8). Dibenzylphosphorochloridate8 (5.56 g, 19 mmol,
2.1 equiv.) dissolved in anhydrous CCl4 (5 ml) was added
under stirring to a solution of 1,2-O-isopropylidene-a-d-
xylofuranose 7 (1.7 g, 8.9 mmol, 1 equiv.) in pyridine
(25 ml) at 0ЊC. The reaction was stirred at rt for 3 h,
quenched by addition of water (2 ml), and the solvents
were removed under vacuum. Flash chromatography
(ethyl acetate/hexane, 70:30) gave a colorless oil (3.84g,
95%) (Rfˆ0.52, ethyl acetate/hexane, 70:30).

1488
J.-F. Hoeffler et al. / Tetrahedron 56 (2000) 1485–1489
flash chromatography of the residue (ethyl acetate/hexane,
70:30) afforded product 10 (740 mg, 84%). (Rfˆ0.48, ethyl
acetate/hexane, 70:30). 1H NMR (400 MHz, CDCl3):
dˆ1.10 (3H, s, CH3); 1.28 (3H, s, CH3); 1.55 (3H, s,
consumed, and the reaction mixture was diluted with water
(5 ml), neutralized with calcium carbonate and extracted
with CHCl3 (3×20 ml). The combined extracts were dried,
filtered and concentrated to give in 80% yield a colorless oil
corresponding to the two anomers of 12 in a ca. 3:1 ratio
according to the integration of the quaternary methyl signals
(280 mg, Rfˆ0.66, ethyl acetate). 1H NMR (400 MHz,
CDCl3): dˆ1.38 (3/4 of 3H, s, CH3); 1.43 (1/4 of 3H, s,
CH3); 2.72 (3/4 of 1H, d, 3Jˆ8.6 Hz, OH); 2.79 (1/4 of 1H,
3
3
CH3); 3.95 (1H, dd, J4,5aˆ7.8 Hz, J4,5bˆ3.0 Hz, 4-H);
2
3
3
4.06 (1H, ddd, J5a,5bˆ11.1 Hz, J4,5aˆ7.8 Hz, JH5a,P
ˆ
3
7.8 Hz, 5-Ha); 4.09 (1H, d, J1,2ˆ3.8 Hz, 2-H); 4.19 (1H,
2
3
3
ddd, J5b,5aˆ11.1 Hz, J5b,Pˆ7.0 Hz, J4,5bˆ3.0 Hz, 5-Hb);
5.04 (1H, dd, 2Jˆ11.8 Hz, JH,Pˆ8.0 Hz, CH2Ph); 5.05
3
2
3
3
(1H, dd, Jˆ11.8 Hz, JH,Pˆ8.1 Hz, CH2Ph); 5.07 (1H, dd,
d, Jˆ8.4 Hz, OH); 3.72–5.20 (11H; m, 1-H, 2-H, 4-H,
2Jˆ11.8 Hz, JH,Pˆ8.1 Hz, CH2Ph); 5.08 (1H, dd, 2Jˆ
5-Ha, 5-Hb and 3 benzylic CH2); 7.28–7.36 (15H, m,
3
3
3
11.8 Hz, JH,Pˆ8.0 Hz, CH2Ph); 5.76 (1H, d, J1,2ˆ3.8 Hz,
1-H); 7.32–7.37 (10H, m, 2×Ph). 13C NMR (50 MHz,
CDCl3): dˆ18.12 (CH3); 26.52 (2×CH3); 65.93 (CH2, d,
3×Ph). 13C NMR (50 MHz, CDCl3) dˆ15.67 and 16.19
2
(CH3); 65.59, 66.08, 66.66 (d, Jˆ4.9 Hz), 69.49, 69.56,
69.62 and 69.92 (CH2 signals); 75.78, 79.73 (C-4, d,
3JC-4,Pˆ8.2 Hz), 81.42, 81.91, 82.27, 96.67 and 102.76
(CH signals); 127.41, 127.64, 127.97, 128.59, 135.48,
135.61 and 137.31 (aromatic C). 31P NMR (162 MHz,
CDCl3): dˆ0.18 (m); 0.46 (m). HRMS (FABϩ): (MϩH)ϩ:
calculated for C27H32O8P 515.1835, found 515.1842.
2JC,Pˆ4.9 Hz); 69.35 (CH2, d, JC,Pˆ3.7 Hz); 69.45 (CH2,
2
2
d, JC,Pˆ4.9 Hz); 76.74 (quaternary C, C-3); 80.21 (CH, d,
3JC,Pˆ6.6 Hz, C-4); 84.08 (CH, C-2); 103.48 (CH, C-1);
112.66 (quaternary C); 127.94, 128.00, 128.49, 135.64 and
135.77 (aromatic C). 31P NMR (162 MHz, CDCl3): dˆ0.31
(hept, JH,Pˆ7.9 Hz). HRMS (FABϩ): (MϩH)ϩ calculated
3
for C23H30O8P 465.1678, found 465.1687.
2-O-Benzyl-2-C-methyl-d-erythrose 4-dibenzylphosphate
(13). To a solution of 12 (280 mg, 0.55 mmol, 1 equiv.) in
methanol (3 ml) was added a solution of sodium meta-
periodate (150 mg, 0.7 mmol, 1.2 equiv.) in water (3 ml).
The resulting suspension was stirred for 30 min at room
temperature, neutralized with sodium hydrogen carbonate
and then extracted with CHCl3 (3×10 ml). The combined
extracts were dried, filtered and concentrated to gave a
colorless oil 13 (250 mg, 93%) which was not further
purified. The NMR spectra of the crude product, which
corresponded to a 1:1 mixture of the aldehyde and the corre-
sponding hydrate according to the integration of the H-1
signals, were satisfactory (Rfˆ0.90, ethyl acetate). 1H
NMR (400 MHz, CDCl3): dˆ1.35 (3H, s, CH3); 4.16 (1H,
3-O-Benzyl-1,2-O-isopropylidene-3-C-methyl-a-d-ribo-
furanose 5-dibenzylphosphate (11). To a stirred solution
of the tertiary alcohol 10 (450 mg, 0.97 mmol, 1 equiv.) and
benzyl 2,2,2-trichloroacetimidate (0.49 g, 1.94 mmol,
2 equiv.) in cyclohexane/CH2Cl2 (2:1, 3 ml) under an
argon atmosphere was added trifluoromethanesulfonic acid
(30 ml). The mixture was stirred at room temperature and
monitored by TLC (ethyl acetate/hexane, 70:30) until the
starting material had completely reacted. The crystalline
trichloroacetamide was removed by filtration, and the
filtrate was successively washed with a saturated solution
of sodium hydrogencarbonate (20 ml) and with water
(20 ml). The mixture was extracted with CH2Cl2
(3×20 ml). The combined extracts were dried, filtered and
concentrated. Finally, the residue was purified by flash
chromatography to afford a colorless oil (380 mg, 70%).
(Rfˆ0.40, ethyl acetate/hexane, 50:50). 1H NMR (400
MHz, CDCl3): dˆ1.18 (3H, s, CH3); 1.36 (3H, s, CH3);
2
3
3
ddd, J4a,4bˆ11.4 Hz, J4a,Pˆ7.4 Hz, J3,4aˆ7.3 Hz, 4-Ha);
4.26
(1H,
ddd,
2J4b,4aˆ11.4 Hz,
3J4b,Pˆ6.6 Hz,
3J3,4bˆ3.3 Hz, 4-Hb); 4.44 (1H, d, Jˆ11.4 Hz, CH2Ph);
2
4.55 (1H, d, 2Jˆ11.4 Hz, CH2Ph); 4.96–5.06 (4H, m,
3
3
2×CH2Ph); 5.50 (1H, dd, J3,4aˆ7.3 Hz, J3,4bˆ3.3 Hz,
3-H); 7.3–7.5 (15H, m, 3×Ph), 7.99 (0.5 H, s, 1-H of the
aldehyde hydrate); 9.55 (0.5 H, s, 1-H of the aldehyde). 13C
NMR (50 MHz, CDCl3): dˆ13.63 and 14.39 (2×CH3);
2
1.58 (3H, s, CH3); 4.11 (1H, ddd, J5a,5bˆ11.0 Hz,
3
3J4,5aˆ7.9 Hz, J5a,Pˆ6.6 Hz, 5-Ha); 4.21 (1H, ddd,
3
3
2
2J5a,5bˆ11.0 Hz, J5b,Pˆ6.6 Hz, J4,5bˆ2.8 Hz, 5-Hb); 4.30
64.95 (CH2, d, JC,Pˆ4.9 Hz); 66.34 and 66.57 (2×CH2);
3
3
2
3
(1H, dd, J4,5aˆ7.9 Hz, J4,5bˆ2.8 Hz, 4-H); 4.33 (1H, d,
3J1,2ˆ3.9 Hz, 2-H); 4.54 (1H, d, 2Jˆ11.0 Hz, CH2Ph);
4.61 (1H, d, 2Jˆ11.0 Hz, CH2Ph); 5.02 (2H, 2d,
69.59 (CH2, d, JC,Pˆ6.6 Hz); 72.38 (CH, d, JC,Pˆ6.6 Hz,
C-3); 81.95 (quaternary C, C-2 of the aldehyde hydrate);
98.86 (quaternary C, C-1 of the aldehyde hydrate);
127.48, 128.00, 128.53, 128.63, 135.61 and 137.25
(aromatic C); 159.60 (quaternary C, C-2 of the aldehyde);
200.84 (C-1 of the aldehyde). 31P NMR (162 MHz, CDCl3):
dˆ1.66 (m). HRMS (FABϩ): (MϩH)ϩ: calculated for
C26H30O7P 485.1729, found 485.1708.
2Jˆ11.4 Hz, CH2Ph); 5.04 (2H, 2d, Jˆ11.4 Hz, CH2Ph);
2
3
5.78 (1H, d, J1,2ˆ3.9 Hz, 1-H); 7.24–7.38 (15H, m,
3×Ph). 13C NMR (50 MHz, CDCl3): dˆ16.22 (CH3);
2
26.68 (CH3); 26.84 (CH3); 66.16 (CH2, d, JC,Pˆ4.9 Hz);
2
66.74 (CH2); 69.25 (CH2, d, JC,Pˆ4.9 Hz); 69.35 (CH2, d,
2JC,Pˆ4.9 Hz); 77.26 (quaternary C, C-3); 79.67 (CH, d,
3JC,Pˆ8.2 Hz, C-4); 82.77 (CH, C-2); 104.34 (CH, C-1);
113.02 (quaternary C); 127.58, 127.68, 127.90, 128.00,
128.23, 128.36, 128.46, 135.77, 135.94 and 138.36
(aromatic C). 31P NMR (162 MHz, CDCl3) dˆ0.21 (m).
HRMS (FABϩ): (MϩH)ϩ calculated for C30H36O8P
555.2148, found 555.2158.
2-O-benzyl-2-C-methyl-d-erythritol 4-dibenzylphosphate
(14). To an ice-cooled solution of the aldehyde 13
(250 mg, 0.52 mmol, 1 equiv.) in methanol (10 ml) was
added sodium borohydride (24 mg, 0.62 mmol, 1.2
equiv.). Stirring was continued at room temperature, and
after 2 h the reaction mixture was diluted with water
(5 ml), treated with 0.1N HCl and extracted with CHCl3
(3×10 ml). The combined extracts were dried, filtered and
concentrated, and the residue afforded after flash chroma-
tography 2-O-Benzyl-2-C-methyl-d-erythritol 4-dibenzyl-
phosphate as a colorless oil (230 mg, 92%). (Rfˆ0.60,
3-O-Benzyl-3-C-methyl-d-ribofuranose 5-dibenzylphos-
phate (12). To a solution of 11 (380 mg, 0.68 mmol) in
CCl4 (0.5 ml) was added at Ϫ10ЊC a 90% aqueous solution
of TFA (5 ml). After 30 min, all the starting material was

J.-F. Hoeffler et al. / Tetrahedron 56 (2000) 1485–1489
1489
1
ethyl acetate/hexane, 90:10). H NMR (400 MHz, CDCl3:
D2O containing traces of t-BuOH): dˆ21.48 (CH3); 67.67
3JC-3,Pˆ6.2 Hz, C-3); 77.15 (quaternary C, C-2). 31P NMR
(162 MHz, CDCl3): dˆ5.50 (s). Electrospray MS: m/zˆ215
(M-H, molecular ion of the MEP mono-anion).
dˆ1.16 (3H, 1s, CH3); 3.66 (1H, d, 2J1a,1bˆ12.0 Hz, 1-Ha);
(CH2, JC-4,Pˆ4.7 Hz, C-4); 72.33 (CH2, C-1); 76.88 (CH,
2
3.70 (1H, d, 2J1a,1bˆ12.0 Hz, 1-Hb); 3.99–4.11 (2H, m, 4-Ha
2
and 4-Hb); 4.33 (1H, m, 3-H); 4.47 (1H, d, Jˆ11.0 Hz,
CH2Ph); 4.52 (1H, d, 2Jˆ11.0 Hz, CH2Ph); 5.00–
5.11 (4H, m, 2×CH2Ph); 7.28–7.34 (15H, m, 3×Ph). 13C
NMR (50 MHz, CDCl3): dˆ15.40 (CH3); 64.28 (CH2);
2
64.80 (CH2); 69.64 (3×CH2, d, JC,Pˆ4.9 Hz); 72.98 (CH,
Acknowledgements
3
d, JC,Pˆ4.9 Hz, C-3); 78.30 (quaternary C, C-2); 127.38,
127.58, 128.04, 128.43, 128.63, 135.58, 135.67 and 138.59
(aromatic C). 31P NMR (162 MHz, CDCl3): dˆ0.11 (m).
HRMS (FAB)ϩ: (MϩH)ϩ calculated for C26H32O7P
487.1886, found 487.1899.
We thank Mr J.-D. Sauer for all NMR measurements and Mr
R. Huber for the FAB MS analyses. This investigation was
supported by a grant to M.R. from the ‘Institut Universitaire
de France’.
Diacetate of 2-O-benzyl-2-C-methyl-d-erythritol 4-
dibenzylphosphate (15). The diol 14 (30 mg,
6.22×10Ϫ2 mmol, 1 equiv.) dissolved in CH2Cl2 (2 ml)
was acetylated at room temperature for 30 min with acetic
anhydride (13 ml, 0.131 mmol, 2.1 equiv.) in the presence of
DMAP (0.05 equiv.) and triethylamine (19 ml, 0.131 mmol,
2.1 equiv.). The reaction mixture was quenched with an
aqueous solution of sodium hydrogencarbonate and
extracted with CH2Cl2 (3×5 ml). The combined extracts
were dried, filtered and concentrated. The residue was
finally purified by chromatography to afford compound 15
as a colorless oil (33 mg, 95%) (Rfˆ0.50, ethyl acetate/
hexane, 50:50). 1H NMR (400 MHz, CDCl3): dˆ1.25
(3H, s, CH3); 2.00 (3H, s, CH3COO–); 2.04 (3H, s,
References
1. (a) Rohmer, M.; Knani, M.; Simonin, P.; Sutter, B.; Sahm, H.
Biochem. J. 1993, 295, 517–524. (b) Rohmer, M.; Seemann, M.;
Horbach, S.; Bringer-Meyer, B.; Sahm, H. J. Am. Chem. Soc. 1996,
118, 2564–2566.
2. (a) Rohmer, M. In Comprehensive Natural Product Chemistry;
Cane, D. E., Ed.; Pergamon: Oxford, UK, 1999; Vol. 2, pp 45–47.
(b) Schwarz, M., Arigoni, D. In Comprehensive Natural Product
Chemistry; Cane, D. E., Ed.; Pergamon: Oxford, UK, 1999; Vol. 2,
pp 367–400. (c) Eisenreich, W.; Schwarz, M.; Cartayrade, A.;
Arigoni, D.; Zenk, M. H.; Bacher, A. Chem. Biol. 1998, 5,
R221–R233. (d) Lichtenthaler, H. K. Annu. Rev. Plant Physiol.
Plant Mol. Biol. 1999, 50, 47–65.
2
CH3COO–); 4.07 (1H, d, J1a,1bˆ12.6 Hz, 1-Ha); 4.14 (1H,
2
2
¨
d, J1a,1bˆ12.6 Hz, 1-Hb); 4.21 (1H, ddd, J4a,4bˆ11.2 Hz,
3. (a) Sprenger, G. A.; Schorken, U.; Wiegert, T.; Grolle, S.;
3J3,4aˆ7.9 Hz, J4a,Pˆ7.7 Hz, 4-Ha); 4.39 (1H, ddd,
Sahm, H. Proc. Natl. Acad. Sci. USA 1997, 94, 12857–12862.
(b) Lange, B.; Wildung, M. R.; McCaskill, D.; Croteau, R. Proc.
Natl. Acad. Sci. USA 1998, 95, 2100–2104. (c) Lois, L.-M.;
Campos, N.; Rosa Putra, S.; Danielsen, K.; Rohmer, M.; Boronat,
A. Proc. Natl. Acad. Sci. USA 1998, 95, 2105–2110.
4. (a) Kuzuyama, T.; Takahashi, S.; Watanabe, H.; Seto, H.
Tetrahedron Lett. 1998, 39, 4509–4512. (b) Takahashi, S.;
Kuzuyama, H.; Watanabe, H.; Seto, H. Proc. Natl. Acad. Sci.
USA 1998, 95, 9879–9884.
5. (a) Duvold, T.; Bravo, J.-M.; Pale-Grosdemange, C.; Rohmer,
M. Tetrahedron Lett. 1997, 38, 4769–4772. (b) Duvold, T.; Cali,
P.; Bravo, J.-M.; Rohmer, M. Tetrahedron Lett. 1997, 38, 6181–
6184.
6. Nakatani, K.; Arai, K.; Terashima, S. Tetrahedron 1993, 49,
1901–1912.
7. Nielsen, P.; Pfundheller, H. M.; Olsen, C. E.; Wengel, J.
J. Chem. Soc., Perkin Trans. 1 1997, 3423–3433.
8. Hall, R. H.; Khorana, H. G. J. Am. Chem. Soc. 1954, 76, 5056–
5060.
9. Czernecki, S.; Ezzitouni, A.; Krausz, P. Synthesis 1990, 651,
653.
10. Brimacombe, J. S.; Rollins, A. J.; Thompson, S. W.
Carbohydr. Res. 1973, 31, 108–113.
11. (a) Iversen, T.; Bundle, D. R. J. Chem. Soc. Chem. Commun.
1981, 1240–1241. (b) Widmer, U. Synthesis 1987, 568–570.
12. Sim, M. M.; Kondo, H.; Wong, C.-H. J. Am. Chem. Soc. 1993,
115, 2260–2267.
13. Duvold, T.; Rohmer, M. Tetrahedron 1999, 55, 9847–9858.
14. Still, W. C.; Khan, M.; Mitra, M. J. Org. Chem. 1978, 43,
2923–2925.
3
2J4a,4bˆ11.2 Hz, J4b,Pˆ6.6 Hz, J3,4bˆ2.6 Hz, 4-Hb); 4.49
3
3
2
3
(2H, s, CH2Ph); 4.97 (1H, dd, Jˆ11.7 Hz, JH,Pˆ8.1 Hz,
2
3
CH2Ph); 5.00 (1H, dd, Jˆ11.7 Hz, JH,Pˆ8.1 Hz, CH2Ph);
5.02 (1H, dd, 2Jˆ11.7 Hz, 3JH,Pˆ8.1 Hz, CH2Ph); 5.04 (1H,
dd, 2Jˆ11.7 Hz, JH,Pˆ8.1 Hz, CH2Ph); 5.43 (1H, dd,
3
3J3,4aˆ7.9 Hz, J3,4bˆ2.6 Hz, 3-H); 7.32–7.34 (15H, m,
3
3×Ph). 13C NMR (50 MHz, CDCl3): dˆ16.52 (CH3);
20.81 (CH3); 20.88 (CH3); 64.05 (CH2); 64.64 (CH2);
2
2
66.20 (CH2, d, JC,Pˆ4.9 Hz); 69.39 (2×CH2, d, JC,P
ˆ
3
6.6 Hz); 72.05 (CH, d, JC,Pˆ6.6 Hz, C-3); 76.74 (quater-
nary C, C-2); 127.28, 127.58, 127.94, 128.40, 128.56,
135.74, 135.90 and 138.23 (aromatic C); 169.83 (CO);
170.58 (CO). 31P NMR (162 MHz, CDCl3): dˆ0.35 (m).
HRMS (FABϩ): (MϩH)ϩ: calculated for C30H36O9P
571.2097, found 571.2104.
2-C-methyl-d-erythritol 4-phosphate (5). The diol 14
obtained after the reduction of aldehyde 13 was hydro-
genated (200 mg, 0.42 mmol) over 10% Pd/C (20 mg) in
EtOH (20 ml) for 2 h at room temperature and atmospheric
pressure. The mixture was filtered, and the filtrate concen-
trated. The residue was diluted in water (5 ml), treated with
a NaOH solution (1 M) until the pH 9 was reached and
neutralized with HCl (0.5 M) to pH 7.5. The mixture was
lyophilized to give the di-sodium salt of 5 (102 mg, 95%).
1H NMR (200 MHz, D2O): dˆ0.98 (3H, s, CH3); 3.32 (1H,
2
2
d, J1a,1bˆ11.8 Hz, 1-Ha); 3.43 (1H, d, J1a,1bˆ11.8 Hz,
1-Hb); 3.87 (2H, m, 4-Ha and 4-Hb); 4.07 (1H, ddd,
Jˆ2.4 Hz, 6.9 and 9.3 Hz, 3-H). 13C NMR (125 MHz,