Isoprenoid biosynthesis via the MEP pathway. Synthesis of (3,4)-3,4-dihydroxy-5-oxohexylphosphonic acid, an isosteric analogue of 1-deoxy-D-xylulose 5-phosphate, the substrate of the 1-deoxy-D-xylulose 5-phosphate reducto-isomerase.

Article abstract of DOI:10.1039/b312193c

(3,4)-3,4-Dihydroxy-5-oxohexylphosphonic acid, an isosteric analogue of 1-deoxy-D-xylulose 5-phosphate (DXP), was obtained in enantiomerically pure form from (+)-2,3--benzylidene--threitol by a seven-step sequence. This phosphonate did not affect the growth of. It did not inhibit the 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR), but was converted by this enzyme into (3,4)-3,4,5-trihydroxy-3-methylpentylphosphonic acid, an isosteric analogue of 2-C-methyl-D-erythritol 4-phosphate. The enzyme was, however, less efficient with the methylene phosphonate analogue than with the natural substrate.

Full text of DOI:10.1039/b312193c

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Isoprenoid biosynthesis via the MEP pathway. Synthesis of
(3R,4S )-3,4-dihydroxy-5-oxohexylphosphonic acid, an isosteric
analogue of 1-deoxy-D-xylulose 5-phosphate, the substrate
of the 1-deoxy-D-xylulose 5-phosphate reducto-isomerase
Odile Meyer, Catherine Grosdemange-Billiard, Denis Tritsch and Michel Rohmer*
Université Louis Pasteur, CNRS UMR 7123, Institut Le Bel, 4 rue Blaise Pascal,
67070 Strasbourg Cedex, France. E-mail: mirohmer@chimie.u-strasbg.fr;
Fax: ϩ33 3 90 24 13 45; Tel: ϩ33 3 90 24 13 46
Received 1st October 2003, Accepted 21st October 2003
First published as an Advance Article on the web 6th November 2003
(3R,4S)-3,4-Dihydroxy-5-oxohexylphosphonic acid, an isosteric analogue of 1-deoxy--xylulose 5-phosphate
(DXP), was obtained in enantiomerically pure form from (ϩ)-2,3-O-benzylidene--threitol by a seven-step sequence.
This phosphonate did not affect the growth of Escherichia coli. It did not inhibit the 1-deoxy--xylulose 5-phosphate
reductoisomerase (DXR), but was converted by this enzyme into (3R,4R)-3,4,5-trihydroxy-3-methylpentylphosphonic
acid, an isosteric analogue of 2-C-methyl--erythritol 4-phosphate. The enzyme was, however, less efficient with the
methylene phosphonate analogue than with the natural substrate.
(DXR), affords 2-C-methyl--erythritol 4-phosphate (MEP) 2
Introduction
(Scheme 1(a)).5 Further steps include the formation of methyl-
erythritol 4-diphosphocytidine, its 2-phosphate derivative,
methylerythritol 2,4-cyclodiphosphate,6 and 4-hydroxy-3-meth-
ylbut-2-enyl diphosphate,7 which is converted by a single
enzyme (LytB) either into IPP or into DMAPP.8
Isoprenoids are synthesized by all living organisms and include
essential compounds (e.g. sterols as membrane stabilizers or
steroid precursors, carotenoids of the plant chloroplasts, prenyl
chains of ubiquinones, plastoquinone or prenylated proteins)
as well as a wealth of secondary metabolites (e.g. mono- and
sesquiterpenes from essential oils). They are synthesized from
two precursors: isopentenyl diphosphate (IPP) and dimethyl-
allyl diphosphate (DMAPP). Two different metabolic routes
leading to IPP have been identified. In animals, fungi, plant
cytoplasm, archaebacteria and some eubacteria, IPP is syn-
thesized through the mevalonate pathway,1 while the recently
discovered methylerythritol phosphate (MEP) pathway is
present in most bacteria, in some unicellular eukaryotes,
including Plasmodium falciparum, the parasite responsible for
malaria, and in the chloroplasts of all phototrophic organisms.2
As the inhibition of the biosynthesis of essential isoprenoids is
lethal, each enzyme of the MEP pathway is a potential target
for antibacterial or antiparasitic drugs.3 The MEP pathway
is now nearly fully elucidated. The initial step of this pathway
is the formation of 1-deoxy--xylulose 5-phosphate (DXP) 1
(Scheme 1) by condensation of hydroxyethylthiamine diphos-
phate derived from pyruvate decarboxylation and -glycer-
aldehyde 3-phosphate mediated by the DXP synthase.4 An
intramolecular rearrangement of DXP followed by a NADPH
dependent reduction, catalysed by the DXP reductoisomerase
Due to the rather recent elucidation of the MEP pathway,
only few inhibitors of its enzymes were reported. Fosmido-
mycin (3-(N-formyl-N-hydroxyamino)propylphosphonate) and
the related FR900098 specifically inhibit the DXP reducto-
isomerase (DXR) from the bacteria E. coli9 and Zymomonas
mobilis10 and led to the inhibition of the MEP pathway in
higher plants11 and in Plasmodium falciparum.12 Its efficiency as
an antimalarial drug was verified.13 The recently determined
crystal structure of the E. coli DXR complexing manganese
and fosmidomycin may contribute to the development of
specific inhibitors of this enzyme.14 Two DXP analogues
respectively lacking the hydroxyl group at C(4) and C(3) were
found to be reversible mixed type inhibitor of the DXR.15 Con-
cerning other enzymes of the MEP pathway, fluoropyruvate16
and 5-ketoclomazone, a breakdown product of the herbicide
clomazone,17 were reported to inhibit DXP synthase.
In the search of novel drugs acting on the MEP pathway, we
synthesized (3R,4S)-3,4-dihydroxy-5-oxohexylphosphonic acid
(DXPN) 3 (Scheme 1(b)), an isosteric analogue of 1-deoxy-
-xylulose 5-phosphate and checked its action on the E. coli
DXR.
Scheme 1 DXP reducto-isomerase catalysed reactions: (a) in the MEP pathway, (b) for the conversions of the phosphonate isostere 3 of DXP 1.
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 3
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 3 6 7 – 4 3 7 2
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Scheme 2 Synthesis of (3R,4S)-3,4-dihydroxy-5-oxohexylphosphonic acid 3. (i) TBDMSCl, NaH, DME (96%); (ii) Tf2O, NEt3, CH2Cl2 (95%);
(iii) MePO(OBn)2, n-BuLi, HMPA, THF (70%); (iv) Bu4NF, THF, (98%); (v) (COCl)2, DMSO, NEt3, MeMgCl, THF, (86%); (vi) TPAP, NMO, 3 Å
molecular sieves, CH2Cl2 (80%), (vii) H2, 10% Pd/C, MeOH–H2O (9 : 1) (quantitative).
Table 1 Kinetic constants of DXR for DXP 1 and DXPN
3
Results and discussion
Substrate
Km/µM
kcat/minϪ1
(kcat/Km)/minϪ1 µMϪ1
Synthesis of DXPN
DXPN was synthesized following the reaction sequence
shown in Scheme 2. The commercially available -threitol
benzylidene derivative 5 (Scheme 2) was a starting material
of choice because it possesses the required configurations of
the carbon atoms bearing hydroxyl groups. The benzylidene
protection of the vicinal diol was preferred, despite the
resulting complication of the NMR spectra. Indeed, this
benzylidene group was simultaneously removed with the
phosphonate benzyl protecting group by one single catalytic
hydrogenation, requiring no further purification of the free
phosphonate 3.
The first step of the synthesis was the protection of one
of the two primary alcohol groups by an O-silyl group. This
was completed with tert-butyldimethylsilyl chloride in di-
methoxyethane, after formation of the alcoholate using sodium
hydride. The remaining hydroxyl group was then activated by
conversion into a triflate by action of triflic anhydride in
the presence of triethylamine. The triflate 7 was displaced by the
methylphosphonate anion, formed by action of n-butyl lithium
on freshly prepared methylphosphonate18 to give 8 in 95%
yield. After deprotection of the O-silyl protective group
with tetrabutylammonium fluoride, the resulting alcohol 9 was
oxidized using the Swern oxidation modified by Ireland and
Norbeck.19 In these conditions, the methyl group was directly
introduced by addition of methyl magnesium chloride with-
out isolation of the aldehyde intermediate. The new C6 carbon
framework 10 was obtained in 86% yield. The following
mild oxidation with TPAP/NMO gave rapidly after silica gel
chromatography the ketone 11 in 80% yield.20 Finally, the
DXP 1
30
120
740
74
24.7
0.6
DXPN
3
Kinetic studies
Incubation of DXPN and NADPH in the presence of DXR led
to the formation of NADPϩ, as evidenced by the decrease of
the NADPH absorbance at 340 nm. The enzyme seemed thus
to catalyse the conversion of DXPN 3 into MEPN 4, the
phosphonate isostere of MEP (Scheme 1(b)). To confirm this
hypothesis, the enzymatic reaction was followed by 31P NMR in
the presence of Na2HPO4 as an internal standard (Fig. 1). The
31P NMR spectrum of the DXPN 3 in the conditions of the
enzymatic reaction displays a signal at 24.7 ppm (Fig. 1(a),
t = 0). The enzyme was incubated for 24 h in the presence of
DXPN and NADPH. Bovine serum albumin was added to the
reaction medium to stabilize the enzyme21 as well as a NADPH
recycling system.22 The decrease in the intensity of the signal of
the DXPN 3 (δ 24.7 ppm) was accompanied by the concomitant
increase of a novel signal at 25.8 ppm corresponding to the
MEPN 4 (Fig. 1(a), t = 12 h). The structural assignment for
the novel reaction product was supported by the addition of
synthetic MEPN in the NMR tube, which lead to an increase
of the intensity of the signal at 25.8 ppm (Fig. 1(b)). DXR
catalysed not only the rearrangement of DXPN to the phos-
phonate analogue of methylerythrose phosphate, but also the
reduction of the latter aldehyde intermediate to MEPN (Scheme
1). The kinetic constants (Km and kcat) of DXR for DXP and
DXPN were compared (Table 1). The increase of the Km for
DXPN and the diminution of the rate of the enzymatic reaction
led to a considerable decrease of the catalytic efficiency
(40-fold) of DXR towards DXPN.
phosphonate
3 was quantitatively obtained by catalytic
hydrogenation with palladium over charcoal in a mixture
of methanol and water. This step required no purification.
The benzylidene protection of -threitol and the dibenzyl
protection of methylphosphonate were chosen for this
reason.
This synthesis afforded optically pure phosphonate 3 in
satisfactory yields. It can be extended to the synthesis of
α-halogenated phosphonate.
Some compounds with C–P bonds have attracted attention
owing to their antibacterial, antiviral and herbicidal activities.23
This is essentially valid in the case of enzymes catalysing the
hydrolytic cleavage of the phosphate group. The methylene
phosphonate analogues, isosteric to phosphate ester substrates,
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 3 6 7 – 4 3 7 2
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Fig. 1 31P NMR spectra of DXR enzyme assays: (a) enzymatic assays, (b) enzymatic assay ϩ synthetic MEPN. *Na2HPO4; 3: DXPN; 4: MEPN.
are often potent inhibitors, owing to the stability of the phos-
phonate group towards hydrolysis.24 When the phosphate group
is not directly implied in the enzymatic reaction, analogues
mimicking the natural substrates are often transformed by
the enzymes.25–27 As DXPN mimics DXP, it was not too sur-
prising that the analogue was recognized and transformed
by DXR. As reported in other studies, the efficiency of conver-
sion is weaker with the phosphonate analogues than with the
natural substrate. There are some examples where the analogue
was not or almost not transformed by the parent enzyme,
notably in the case of the isomerization of the phos-
phonomethyl analogues of glyceraldehyde 3-phosphate and of
dihydroxyacetone phosphate catalysed by the triose phosphate
isomerase.26
agar diffusion method. Depositing the compound on the paper
discs (6 mm diameter), even at the highest amount tested (100
µg/disc) did not prevent the bacterial growth. In contrast, in the
presence of fosmidomycin, a phosphonate, which is known to
efficiently inhibit the DXR and to prevent the growth of E. coli
in vitro,9,21 multiplication of the bacteria was strongly inhibited,
when only small amounts of fosmidomycin (10 µg) were applied
on the paper disc (30 mm diameter growth inhibition area).
Such results were not fully unexpected. Even if DXPN is taken
up by the cells, DXR has a much lower affinity for DXPN than
for DXP, and a high concentration of the phosphonate would
be required to inhibit the enzyme.
Experimental
Several factors could contribute to the differences of the
kinetic properties observed for the natural phosphate substrate
and the phosphonomethyl analogues. Among them is the
perturbation of the electronic properties upon the introduction
of an electron-donating methylene group instead of an electro-
negative oxygen atom. The electronegativity loss was assessed
from the pKa value of the second deprotonation of phos-
phonate groups, which is one pKa unit less acidic.24 The
non-recognition of the phosphonomethyl analogues of di-
hydroxyacetone phosphate by triose phosphate isomerase was
attributed to the fact that the enzyme binds the substrate in the
doubly ionised form and that the methylene phosphonate
analogue is rather in a mono ionised form owing to the increase
of the second pKa.25,28 To circumvent this problem, the replace-
ment of the methylene group by a monofluoro or a difluoro-
methylene group was proposed to render the analogues not only
isosteric but also isopolar. If ameliorations of the catalytic
efficiency were reported in some cases,26 it is not a common fact,
clearly showing that factors other than phosphonate ionisation
state may contribute to the differential binding of the analogues
to the enzymes. For instance the bridging oxygen itself of the
phosphate group may be implied in interactions with amino
acid(s) of the substrate binding site. Its replacement by a meth-
ylene group could be detrimental for an ideal binding of the
phosphonate analogue leading to a decrease of the rate.
General methods
All non-aqueous reactions were run in dry solvents under an
argon atmosphere. Thin-layer chromatography was performed
on analytical silica gel Si 60 F254 silica plates (Merck) and flash
chromatography on silica gel Si 60 230–400 mesh (Merck) with
the indicated solvent system.29 TLC plates were developed with
an ethanol solution of p-anisaldehyde (2.5%), sulfuric acid
(3.5%) and acetic acid (1.6%) or by an ethanol solution of
phosphomolybdic acid (20%) by heating up to 100 ЊC. NMR
spectra were recorded on a Bruker AC200, AC300 and AV300
spectrometers. NMR experiments were carried out in CDCl3
or D2O using as an internal standard CHCl3 (δ = 7.26 ppm) or
1
DHO (δ = 4.65 ppm) for H NMR and CDCl3 (δ = 77.0 ppm)
for 13C NMR. In the description of the NMR spectra corre-
sponding to diastereomer mixtures, signals for different
diastereomers are distinguished by a *, # or § sign added to the
assignments. If only one signal is described, it is common to
all diastereomers. Infrared spectra were recorded on a Nicolet
Avatar 320 FT-IR spectrometer using KBr discs or in CHCl3
solution in 0.2 mm pathlength NaCl cells. Optical activity was
measured with a Perkin Elmer 341 polarimeter. UV spectro-
scopy for enzyme tests was performed on an Uvikon 933
spectrophotometer (Kontron instruments). All compounds
were found to be pure by 1H and 13C NMR spectroscopy.
The effect of DXPN on the growth of E. coli was tested by the
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 3 6 7 – 4 3 7 2
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Protein concentrations were measured using the method of
Bradford with bovine serum albumin as the standard.
All reagents and solvents used were reagent grade. 1-Deoxy-
-xylulose 5-phosphate was prepared chemically. A sample of
(3R,4R)-3,4,5-trihydroxy-3-C-methylpentylphosphonate
(MEPN) 4 was synthesized in the laboratory by G. Hirsch
(unpublished results).
of water (30 mL) and diluted with diethyl ether (25 mL). The
aqueous layer was extracted with diethyl ether (4 × 30 mL). The
combined extracts were dried over Na2SO4, filtered and concen-
trated. The residue (9 g) was purified by flash chromatography
to afford a colourless oil (8.49 g, 98%). (Rf = 0.32, ethyl acetate–
hexane, 80 : 20). δH (200 MHz, CDCl3) 1.47 (3H, d, JH–P
=
17 Hz, CH3), 4.90–5.12 (4H, m, 2 × CH2Ph), 7.27–7.39 (10H,
m, 2 × Ph); δC (50 MHz, CDCl3) 11.7 (CH3, d, JC–P = 144 Hz),
67.1 (CH2); 67.2 (CH2), 127.9, 128.4, 128.6, 136.3 and 136.4
(aromatic C); δP (121.5 MHz, CDCl3) 29.0 (s).
(2R,
3S )-O-Benzylidene-4-O-tert-butyldimethylsilyl-D-
threitol (6). To a cold (0 ЊC) solution of (ϩ)-2,3-O-benzylidene-
-threitol 5 (3.0 g, 14.3 mmol, 1 eq.) in 1,2-dimethoxyethane
(35 mL) sodium hydride (0.4 g, 16.7 mmol, 1.1 eq.) was added
in portions. The mixture was stirred at 0 ЊC for 15 min before
addition of tert-butyldimethylsilyl chloride (2.2 g, 14.4 mmol,
1 eq.). After 3 h, the starting material was consumed, and the
reaction was quenched by addition of a saturated aqueous
ammonium chloride solution (40 mL). The aqueous layer was
extracted with diethyl ether (3 × 40 mL), and the combined
extracts were dried over anhydrous Na2SO4, filtered and con-
centrated to give an orange oil (5.9 g). The residue was purified
by flash-chromatography to afford 6 as a colourless oil (4.45 g,
96%) and a 1 : 1 mixture of two diastereomers (Rf = 0.20, ethyl
acetate–hexane, 20 : 80). δH (200 MHz, CDCl3) 0.08 (1/2 of 6H,
s, 2 × CH3), 0.10 (1/2 of 6H, s, 2 × CH3*); 0.90 (1/2 of 9H, s,
t-Bu), 0.92 (1/2 of 9H, s, t-Bu*), 2.25 (1/2 of 1H, dd, J1a–OH = 5.4
Hz, J1b–OH = 7.3 Hz, OH), 2.32 (1/2 of 1H, t, J1-OH = 6.1 Hz,
OH*), 3.69–4.18 (6H, m, 1-, 2-, 3- and 4-H), 5.96 (1/2 of 1H, s,
CHPh), 5.97 (1/2 of 1H, s, CHPh*), 7.34–7.51 (5H, m, Ph); δC
(50 MHz, CDCl3) Ϫ5.3 (2 × CH3), 18.4 (quaternary C, t-Bu),
26.0 (3 × CH3, t-Bu), 62.6 (CH2, C-1), 63.0 (CH2, C-1*), 63.6
(CH2, C-4), 63.7 (CH2, C-4*), 78.2 (CH, C-2), 79.3 (CH, C-2*),
80.4 (CH, C-3), 80.7 (CH, C-3*), 103.9 (CHPh), 104.3
(CHPh*), 126.1, 126.7, 128.5, 128.6, 129.1, 129.5, 137.6 and
137.8 (aromatic C). IR (CHCl3) νmax (cmϪ1): 3591, 3447, 1602,
1462, 1381, 1256, 1225, 1093, 839. MS (FABϩ) m/z: 325.3
(M ϩ H)ϩ. HRMS (FABϩ) m/z: calc. for C17H29O4Si 325.1835,
found 325.1822.
Dibenzyl
[(3R,4S )-O-benzylidene-5-O-tert-butyldimethyl-
silylpentyl]phosphonate (8). A solution of dry dibenzyl methyl-
phosphonate (2 g, 7.2 mmol, 2.6 eq.) and HMPA (1.25 mL, 7.2
mmol, 2.6 eq.) in THF (10 mL) was deoxygenated by argon
bubbling at Ϫ78 ЊC for 30 min. A solution of n-butyllithium
(4.5 mL, 1.6 M in hexane, 7.2 mmol, 2.6 eq.) was added, and the
solution turned to orange. After stirring for 30 min at Ϫ78 ЊC, a
previously deoxygenated solution of dry triflate 7 (1.29 g, 2.8
mmol, 1 eq.) in THF (6 mL), was added via a cannula at Ϫ78
ЊC. When the starting material disappeared, the reaction was
quenched with saturated aqueous ammonium chloride solution
(25 mL) and diluted with diethyl ether (10 mL). The aqueous
layer was further extracted with diethyl ether (4 × 25 mL) and
the combined organic extracts were dried over anhydrous
Na2SO4, filtered and evaporated. Flash chromatography
afforded 8 as a colourless oil (1.14 g, 70%) and a 1 : 1 mixture
of two diastereomers (Rf = 0.46, ethyl acetate–hexane, 50 : 50).
δH (300 MHz, CDCl3) 0.05 (1/2 of 6H, s, 2 × CH3), 0.08 (1/2 of
6H, s, 2 × CH3*), 0.88 (1/2 of 9H, s, t-Bu), 0.90 (1/2 of 9H, s,
t-Bu*), 1.77–2.19 (4H, m, 1- and 2-H), 3.61–3.85 (3H, m, 3- and
5-H), 3.96–4.15 (1H, m, 4-H), 4.88–5.17 (4H, s, 2 × CH2Ph),
5.83 (1/2 of 1H, s, CHPh), 5.86 (1/2 of 1H, s, CHPh*), 7.30–
7.44 (15H, m, Ph); δC (75 MHz, CDCl3) Ϫ5.3 (2 × CH3), 18.4
1
(quaternary C, t-Bu), 22.5 (CH2, d, JC–P = 143 Hz, C-1), 22.7
(CH2, d, 1JC–P = 143 Hz, C-1*), 25.9 (3 × CH3, t-Bu), 26.4 (CH2,
2
2
d, JC–P = 4 Hz, C-2), 26.6 (CH2, d, JC–P = 4 Hz, C-2*), 63.3
(CH2, C-5), 63.7 (CH2, C-5*), 67.2 (2 × CH2Ph), 67.4 (2 ×
CH2Ph*), 79.3 (CH, d, 3JC–P = 18 Hz, C-3), 79.4 (CH, d, 3JC–P
=
(2R,3S )-O-Benzylidene-4-O-tert-butyldimethylsilyl-1-O-tri-
fluoromethanesulfonyl-D-threitol (7). To a solution of 6 (0.4 g,
1.2 mmol, 1 eq.) and triethylamine (0.48 mL, 3.4 mmol, 2.9 eq.)
in CH2Cl2 (15 mL) at Ϫ40 ЊC was added dropwise triflic
anhydride (0.38 mL, 2.3 mmol, 2 eq.). The reaction mixture was
stirred for 1 h at Ϫ40 ЊC, allowed to warm up to 0 ЊC and
treated with a saturated aqueous solution of sodium bicarb-
onate solution (20 mL). The organic layer was washed with
water (20 mL) and with brine (20 mL). Each aqueous layer was
extracted with chloroform (3 × 20 mL). The combined extracts
were dried over anhydrous Na2SO4, filtered and concentrated.
The residue (0.9 g) was purified by flash chromatography to
afford 7 as a colourless oil (0.50 g, 95%) and a 1 : 1 mixture of
two diastereomers (Rf = 0.57, ethyl acetate–hexane, 20 : 80).
Due to the instability of compound 7, only NMR analysis were
performed. δH (200 MHz, CDCl3) 0.08 (1/2 of 6H, s, 2 × CH3),
0.11 (1/2 of 6H, s, 2 × CH3*), 0.90 (1/2 of 9H, s, t-Bu), 0.92
(1/2 of 9H, s, t-Bu*), 3.70–4.14 (3H, m, 2,4-H), 4.38–4.78 (3H,
m, 1- and 3-H), 5.97 (1/2 of 1H, s, CHPh), 6.02 (1/2 of 1H, s,
CHPh*), 7.33–7.51 (5H, m, Ph); δC (50 MHz, CDCl3) Ϫ5.4
(2 × CH3), 18.3 (quaternary C, t-Bu), 25.9 (3 × CH3, t-Bu), 63.3
(CH2, C-4), 63.4 (CH2, C-4*), 75.4 (CH2, C-1), 77.1 (CH, C-2),
77.3 (CH, C-2*), 77.8 (CH, C-3), 104.5 (CHPh), 105.1
(CHPh*), 126.7, 128.5, 129.8 and 136.7 (aromatic C).
18 Hz, C-3*), 80.8 (CH, C-4), 81.9 (CH, C-4*), 102.9 (CHPh),
103.8 (CHPh*), 126.3, 126.7, 127.1, 127.6, 128.0, 128.4, 128.5,
128.7, 129.1, 129.4, 136.4, 136.5, 137.6 and 137.7 (aromatic C);
δP (121.5 MHz, CDCl3) 33.9 (s). IR (CHCl3) νmax (cmϪ1): 1602,
1498, 1456, 1380, 1256, 1240, 1224, 1091, 1044, 998, 839. MS
(FABϩ) m/z: 583.4 (M ϩ H)ϩ. HRMS (FABϩ) m/z: calc. for
C32H44O6SiP 583.2645, found 583.2649.
Dibenzyl
[(3R,4S )-O-benzylidene-5-hydroxypentyl]phos-
phonate (9). To a solution of 8 (1.12 g, 1.9 mmol, 1 eq.) in THF
(25 mL) was added Bu4NF (0.90 g, 2.9 mmol, 1.5 eq.). After
stirring overnight, the solvent was removed under vacuum and
the residue was purified by flash chromatography to afford a
colourless oil (0.88 g, 98%) as a 1 : 1 mixture of two dia-
stereoisomers (Rf = 0.31, ethyl acetate). δH (300 MHz, CDCl3)
1.77–2.12 (4H, m, 1- and 2-H), 3.60–3.89 (3H, m, 3- and 5-H),
4.01–4.17 (1H, m, 4-H), 4.89–5.12 (4H, s, 2 × CH2Ph), 5.83 (1/2
of 1H, s, CHPh), 5.86 (1/2 of 1H, s, CHPh*), 7.33–7.43 (15H,
m, Ph); δC (75 MHz, CDCl3) 22.5 (CH2, d, 1JC–P = 150 Hz, C-1),
22.7 (CH2, d, 1JC–P = 150 Hz, C-1*), 25.9 (CH2, d, 2JC–P = 4 Hz,
2
C-2), 26.1 (CH2, d, JC–P = 4 Hz, C-2*), 62.3 (CH2, C-5), 62.6
(CH2, C-5*), 67.4 (2 × CH2Ph), 67.5 (2 × CH2Ph*), 77.9 (CH,
3
3
d, JC–P = 16 Hz, C-3), 78.5 (CH, d, JC–P = 16 Hz, C-3*), 81.2
(CH, C-4), 82.4 (CH, C-4*), 103.1 (CHPh), 103.7 (CHPh*),
126.4, 126.7, 127.7, 128.1, 128.6, 128.8, 129.1, 129.6, 136.3,
136.4 and 137.4 (aromatic C); δP (121.5 MHz, CDCl3) 33.9 (s).
IR (CHCl3) νmax (cmϪ1): 3595, 3405, 1602, 1498, 1456, 1379,
1237, 1221, 1095, 1047, 998. MS (FABϩ) m/z: 469.2 (M ϩ H)ϩ.
HRMS (FABϩ) m/z: calc. for C26H30O6P 469.1780, found
469.1777.
Dibenzyl methylphosphonate
To a suspension of sodium hydride (1 g, 41.7 mmol, 1.3 eq.) in
THF (35 mL) at 0 ЊC, was slowly added dibenzyl phosphite
(7 mL, 31.5 mmol, 1 eq.) and the mixture was stirred for 1 h. To
this was added iodomethane (2.7 mL, 43.4 mmol, 1.3 eq.). The
solution was stirred for an additional 2 h, quenched by addition
O r g . B i o m o l . C h e m . , 2 0 0 3 , 1, 4 3 6 7 – 4 3 7 2
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Dibenzyl
[(3R,4S )-O-benzylidene-5-hydroxyhexyl]phos-
(quaternary C, C-5), 208.5 (quaternary C, C-5*); δP (121.5
phonate (10). To a stirred solution of oxalyl chloride (300 µL,
3.5 mmol, 7 eq.) in THF (8 mL) at Ϫ78 ЊC was added dimethyl
sulfoxide (300 µL, 4.2 mmol, 9 eq.). The solution was allowed to
warm up to Ϫ35 ЊC for 5 min and was cooled again to Ϫ78 ЊC.
A solution of alcohol 9 (0.21 g, 0.5 mmol, 1 eq.) in THF (4 mL)
was then added to the reaction mixture via a cannula. The
resulting solution was allowed to warm up to Ϫ35 ЊC and after
15 min was treated with triethylamine (0.5 mL, 3.6 mmol,
8 eq.). The reaction mixture was allowed to warm up to room
temperature for 1 h and was then cooled to Ϫ78 ЊC. A 3 M
diethyl ether solution of methyl magnesium chloride (0.4 mL,
1.2 mmol, 2.7 eq.) was then added dropwise. The reaction was
followed by TLC (ethyl acetate) until the aldehyde had com-
pletely disappeared. The solution was diluted with ethanol (10
mL), saturated aqueous ammonium chloride solution (20 mL),
water (5 mL) and diethyl ether (10 mL). The aqueous phase was
extracted with diethyl ether (4 × 25 mL). The combined organic
phases were dried over anhydrous Na2SO4, filtered and evapor-
ated to dryness under reduced pressure. The yellow residue
(0.30 g) was purified by flash chromatography to afford 10 as a
colourless oil (186 mg, 86%) and a 14 : 14 : 4 : 1 mixture of four
diastereomers (Rf = 0.42, ethyl acetate). δH (300 MHz, CDCl3)
1.18 (1/33 of 3H, d, J5–6 = 6.4 Hz, 6-H), 1.19 (14/33 of 3H, d,
J5–6 = 6.4 Hz, 6-H*), 1.24 (14/33 of 3H, d, J5–6 = 6.4 Hz,
6-H§), 1.25 (4/33 of 3H, d, J5–6 = 6.4 Hz, 6-H#), 1.75–2.18 (4H,
m, 1- and 2-H), 3.54–3.64 (1H, m, 5-H), 3.88–3.98 (1H, m,
4-H), 4.16–4.25 (1H, m, 3-H), 4.91–5.11 (4H, s, 2 × CH2–Ph),
5.79 (14/33 of 1H, s, CHPh), 5.81 (4/33 of 1H, s, CHPh*), 5.83
(14/33 of 1H, s, CHPh§), 5.86 (1/33 of 1H, s, CHPh#), 7.30–7.46
(15H, m, Ph); δC NMR (75 MHz, CDCl3) 19.2 (CH3, C-6), 19.8
MHz, CDCl3) 33.2 (s) and 33.3 (s). IR (CHCl3) νmax (cmϪ1):
1718, 1657, 1598, 1498, 1456, 1407, 1378, 1359, 1244, 1225,
1094, 998. MS (FABϩ) m/z: 481.2 (M ϩ H)ϩ. HRMS (FABϩ)
m/z: calc. for C27H30O6P 481.1780, found 481.1780.
(3R,4S )-3,4-Dihydroxy-5-oxohexylphosphonic acid (3). The
protected DXPN 11 (90 mg, 0.19 mmol) was hydrogenated over
15% Pd/C (14 mg) in MeOH–H2O (95 : 5, 10 mL) for 1 h at
room temperature and atmospheric pressure. The mixture was
filtered through Celite and the filtrate was concentrated. The
residue (39 mg) was dissolved in water (1 mL) and the solution
was neutralized by addition of 1 M sodium hydroxide. The
solution of the monosodium salt of DXPN 3 was lyophilised to
give a colourless vitrous solid, which decomposed at 120 ЊC.
[α]2D0 = ϩ 37 (c 1.8, H2O). δH (300 MHz, D2O) 1.51–1.89 (4H, m,
1- and 2-H), 2.26 (3H, s, 6-H), 4.08–4.18 (1H, m, 4-H), 4.31–437
(1H, m, 3-H); δC (75 MHz, D2O) 24.8 (CH2, d, 1JC–P = 137 Hz,
C-1), 25.7 (CH3, C-6), 27.3 (CH2, s, C-2), 71.9 (CH, d, 3JC–P = 18
Hz, C-3), 78.9 (CH, s, C-4), 213.2 (quaternary C, C-5); δP (121.5
MHz, D2O) 25.9 (s). IR (KBr) νmax/cmϪ1 : 3409, 1714, 1651,
1417, 1354, 1146, 1118, 1064. MS (ESϪ) m/z: 211 (M Ϫ Hϩ).
Purification of 1-deoxy-D-xylulose 5-phosphate
reductoisomerase
E. coli cells transformed with a plasmid bearing the dxr gene15
were grown in a LB medium containing ampicillin (0.1 mg/mL).
DXS expression was induced by the addition of IPTG to a final
concentration of 0.4 mM at an OD600
of 0.6. Cells were
nm
grown for an additional 3 h at 37 ЊC and then harvested by
centrifugation (4.2 g, cell paste from a 1.5 L culture). Cells were
disrupted at 0 ЊC by sonication (40 W, 8 × 30 s, with 30 s rest
period) in a 50 mM Tris/HCl buffer (pH 7.5) containing 2 mM
DTT and 0.5 mM PMSF. After centrifugation (20000 rpm,
20 min), the supernatant was applied on a Sepharose Q
(Amersham Biosciences) column (2 × 10 cm) pre-equilibrated
with the lysis buffer. After washing with 50 mM Tris/HCl buffer
(pH 7.5) containing 50 mM NaCl, 2 mM DTT and 0.5 mM
PMSF until the OD280nm decreased to about 0.1, the enzyme
was eluted by applying a linear gradient of NaCl (50 to 500
mM) in 50 mM Tris/HCl buffer (pH 7.5) containing 2 mM DTT
and 0.5 mM PMSF (total volume 200 mL). Fractions contain-
ing DXR activity were pooled and dialysed against 2 × 1 L
25 mM imidazole, 2 mM DTT buffer pH 7.2. After centrifug-
ation to discard precipitated proteins, the enzymatic solution
was applied on a PBE94 column (2 × 11 cm) equilibrated with
the dialysis buffer. After washing with the same buffer (50 mL),
the pH gradient was started by applying a Polybuffer 74
solution (1/8 dilution, pH 4.0, volume 450 mL). Fractions
containing DXR activity (pH fractions 4.8–5.0) were >90%
pure as shown by PAGE/SDS and Coomassie Blue coloration.
These fractions were pooled and dialysed against 50 mM Tris/
HCl–2 mM DTT buffer (pH 8.0) by ultrafiltration on Centricon
30 units. The enzymatic solution was diluted 4-fold with buffer
before the centrifugation to ensure an efficient elimination of
Polybuffer. The enzymatic solution was then applied on a 2Ј5Ј
ADP Sepharose 4B column (0.8 × 2.5 cm) equilibrated with a
25 mM Tris/HCl–100 mM NaCl–DTT 2 mM buffer (pH 8.0).
After washing with the equilibration buffer (8 mL), the enzyme
was eluted by applying a gradient of NADPH (0 to 3 mM, total
volume 16 mL). Fractions containing DXR activity were
pooled, concentrated and dialyzed against 50 mM Tris/HCl–2
mM DTT buffer (pH 7.5) by ultrafiltration on Centricon 30
units. As shown by PAGE/SDS and Coomassie Blue coloration
the enzyme was pure at this stage.
1
(CH3, C-6*), 22.5 (CH2, d, JC–P = 142 Hz, C-1), 22.8 (CH2, d,
1JC–P = 142 Hz, C-1*), 27.0 (CH2, d, JC–P = 4 Hz, C-2), 27.1
2
2
(CH2, d, JC–P = 4 Hz, C-2*), 67.3 (2 × CH2Ph), 67.4 (2 ×
CH2Ph*), 67.5 (2 × CH2Ph§), 67.6 (CH, C-5), 68.1 (CH, C-5*),
3
3
77.7 (CH, d, JC–P = 20 Hz, C-3), 77.8 (CH, d, JC–P = 20 Hz,
C-3*), 79.0 (CH, d, 3JC–P = 20 Hz, C-3§), 79.1 (CH, d, 3JC–P = 20
Hz, C-3#), 84.3 (CH, C-4), 85.7 (CH, C-4*), 102.6 (CHPh),
103.7 (CHPh*), 126.7, 128.0, 128.1, 128.5, 128.6, 128.7, 129.6,
129.7, 136.3, 136.4, 137.2 and 137.7 (aromatic C); δP (121.5
MHz, CDCl3) 33.8 (s), 33.9 (s), 34.3 (s) and 34.4 (s). IR (CHCl3)
νmax (cmϪ1): 3598, 3408, 1602, 1498, 1456, 1380, 1236, 1225,
1090, 1047, 998. MS (FABϩ) m/z: 483.3 (M ϩ H)ϩ. HRMS
(FABϩ) m/z: calc. for C27H32O6P 483.1937, found 483.1941.
Dibenzyl [(3R, 4S )-O-benzylidene-5-oxohexyl]phosphonate
(11). To a solution of the diastereomeric alcohols 10 (75 mg,
0.16 mmol, 1 eq.) in dichloromethane (5 mL) were added
activated 4 Å molecular sieves (0.17 g), N-methylmorpholine
N-oxide (37 mg, 0.27 mmol, 1.7 eq.) and solid TPAP (4 mg,
0.01 mmol, 0.07 eq.). The mixture was stirred at room temper-
ature and monitored by TLC (ethyl acetate) until the starting
material completely disappeared. On completion, the reaction
mixture was filtered through a pad of silica on a sintered-glass
funnel. The solid cake was washed with ethyl acetate, and the
filtrate was evaporated. The residue (60 mg) was purified by
flash chromatography to afford 11 as a colourless oil (57 mg,
79%) and a 1 : 1 mixture of two diastereomers (Rf = 0.46, ethyl
acetate). δH (300 MHz, CDCl3) 1.18–2.15 (4H, m, 1- and 2-H),
2.21 (1/2 of 3H, s, 6-H), 2.29 (1/2 of 3H, s, 6-H*), 4.01 (1/2 of
1H, d, J3–4 = 7 Hz, 4-H), 4.04 (1/2 of 1H, d, J3–4 = 6 Hz, 4-H*),
4.05–4.17 (1H, m, 3-H), 4.92–5.11 (4H, s, 2 × CH2Ph), 5.86 (1/2
of 1H, s, CHPh), 5.89 (1/2 of 1H, s, CHPh*), 7.32–7.48 (15H,
m, Ph); δC (75 MHz, CDCl3) 22.3 (CH2, d, 1JC–P = 143 Hz, C-1),
22.6 (CH2, d, 1JC–P = 143 Hz, C-1*), 26.6 (CH3, C-6), 26.9 (CH3,
C-6*), 26.7 (CH2, d, 2JC–P = 4 Hz, C-2), 67.4 (2 × CH2Ph), 67.5
3
(2 × CH2Ph*), 78.9 (CH, d, JC–P = 22 Hz, C-3), 79.0 (CH, d,
Determination of the DXR activity
3JC–P = 22 Hz, C-3*), 84.7 (CH, C-4), 85.9 (CH, C-4*), 103.8
(CHPh), 104.8 (CHPh*), 126.7, 126.8, 128.1, 128.8, 129.8,
129.9, 136.2, 136.4, 136.5 and 136.6 (aromatic C), 207.4
Standard assays were routinely performed at 37 ЊC in 50 mM
Tris/HCl buffer (pH 7.5) containing 1 mM MgCl2 and 2 mM
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M. Rohmer and A. Boronat, Proc. Natl. Acad. Sci. USA, 1998, 95,
2105–2110.
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Acad. Sci. USA, 1998, 95, 9879–9884.
6 F. Rohdich, J. Wungsintaweekul, M. Fellermeier, S. Sagner, S. Hertz,
K. Kis, W. Eisenreich and A. Bacher, Proc. Natl. Acad. Sci. USA,
1999, 96, 11758–11763; T. Kuzuyama, M. Takagi, K. Kaneda,
T. W. Dairi and H. Seto, Tetrahedron Lett., 2000, 41, 703–706;
H. Lüttgen, F. Rohdich, S. Herz, J. Wungsintaweekul, S. Hecht,
C. A. Schuhr, M. Fellermeier, S. Sagner, M. H. Zenk, A. Bacher and
W. Eisenreich, Proc. Natl. Acad. Sci. USA, 2000, 97, 1062–1067;
S. Hertz, J. Wungsintaweekul, S. Hecht, H. Lüttgen, M. Fellermeier,
S. Sagner, M. H. Zenk, A. Bacher and W. Eisenreich, Proc. Natl.
Acad. Sci. USA, 2000, 97, 2486–2490.
DTT (total volume 1 mL) in the presence of 0.2 mM NADPH
and 3 mM DXP. The enzymatic activity was monitored by
following the decrease of the absorbance at 340 nm due to the
oxidation of the cofactor. The reaction was initiated by the
addition of enzyme.
To determine the kinetic constants (Km and Vmax) for DXP
and DXPN, assays were performed in duplicate at six different
concentrations of the variable substrate, DXP (40 to 200 µM)
and DXPN (100 to 500 µM). The reaction was initiated by the
addition of enzyme, 4 µg with DXP as the substrate or 6 µg with
DXPN. The graphical representation of Lineweaver–Burk was
used to determine the kinetic constants.
7 S. Hecht, W. Eisenreich, P. Adam, S. Amslinger, K. Kis, A. Bacher,
D. Arigoni and F. Rohdich, Proc. Natl. Acad. Sci. USA, 2001, 98,
14837–17842; M. Wolff, M. Seemann, C. Grosdemange-Billiard,
D. Tritsch, N. Campos, M. Rodriguez-Conception, A. Boronat and
M. Rohmer, Tetrahedron Lett., 2002, 43, 2555–2559; M. Hintz,
A. Reichenberg, B. Altincicek, U. Bahr, R. M. Gschwind,
A. K. Kollas, E. Beck, J. Wiesner, M. Eberl and H. Jomaa, FEBS
Lett., 2001, 509, 317–322; F. Rohdich, S. Hecht, K. Gärtner,
P. Adam, C. Krieger, S. Amslinger, D. Arigoni, A. Bacher and
W. Eisenreich, Proc. Natl. Acad. Sci. USA, 2002, 99, 1158–1163;
M. Seemann, B. Tse Sum Bui, M. Wolff, D. Tritsch, N. Campos,
A. Boronat, A. Marquet and M. Rohmer, Angew. Chem., Int. Ed.,
2002, 41, 4337–4339.
8 B. Altincicek, E. C. Duin, A. Reichenberg, R. Hedderich,
A. K. Kollas, M. Hintz, S. Wagner, J. Wiesner, E. Beck and
H. Jomaa, FEBS Lett., 2002, 532, 432–436; F. Rohdich, F. Zepeck,
P. Adam, S. Hecht, J. Kaiser, R. Laupitz, T. Gräwert, S. Amslinger,
W. Eisenreich, A. Bacher and D. Arigoni, Proc. Natl. Acad. Sci.
USA, 2003, 1007, 1586–1591; M. Wolff, M. Seemann, B. Tse Sum
Bui, Y. Frapart, D. Tritsch, A. Garcia Estrabot, M. Rodríguez-
Concepción, A. Boronat, A. Marquet and M. Rohmer, FEBS Lett.,
2003, 541, 115–120.
Identification of the reaction product of DXPN with DXR by 31
NMR
P
The reaction was performed in a 50 mM Tris/HCl buffer (pH
7.5) containing 1 mM MgCl2, 2 mM DTT in the presence of
3 mM DXPN and 0.4 mM NADPH. The reaction volume was
600 µL. The medium contained BSA (600 µg) to increase the
stability of DXR,21 3 mM Na2HPO4 as an internal standard,
and D2O (150 µL) to lock the spectrometer. The regeneration
of NADPH was realized by adding 3 mM -glutamate and
glutamate dehydrogenase.22 DXR (30 µg) was added to initiate
the enzymatic reaction. A 31P NMR spectrum was taken after
an incubation of 12 h at 37 ЊC. A sample of MEPN (3 mM final
concentration) was added to the NMR tube, and the spectrum
was taken again. Blank experiments were performed with
DXPN and MEPN in the same medium but in the absence of
enzymes.
Influence of DXPN on E. coli growth
9 T. Kuzuyama, T. Shimizu, S. Takahashi and H. Seto, Tetrahedron
Lett., 1998, 39, 7913–7916.
10 S. Grolle, S. Bringer-Meyer and H. Sahm, FEMS Microbiol. Lett.,
2000, 191, 131–137.
The susceptibility of E. coli cells to DXPN was tested by the
agar diffusion method. LB agar plates (5 cm diameter) were
inoculated with bacteria (100 µL, OD600 = 0.68, 5 × 107 cells).
Whatman No. 1 paper discs of 6 mm diameter were impreg-
nated with DXPN (50 and 100 µg). The dried discs were then
placed on the agar surface. The plates were incubated over-
night at 30 ЊC. Assays with fosmidomycin (10 to 50 µg) were
performed. Antibacterial activity was indicated by the presence
of clear growth inhibition zones around the discs.
11 J. Zeidler, J. Schwender, C. Müller, J. Wiesner, C. Weidemeyer,
E. Beck, H. Jomaa and H. K. Lichtenthaler, Z. Naturforsch., Teil C,
1998, 53, 980–986; M. Fellermeier, K. Kis, S. Sagner, U. Maier,
A. Bacher and M. Zenk, Tetrahedron Lett., 1999, 40, 2743–2746.
12 A. Reichenberg, J. Wiesner, C. Weidemeyer, E. Dreiseidler,
S. Sanderbrand, B. Altincicek, E. Beck, M. Schlitzer and H. Jomaa,
Bioorg. Med. Chem Lett., 2001, 11, 833–835.
13 M. A. Missinou, S. Borrmann, A. Schindler, S. Issifou,
A. A. Adegnika, P.-B. Matsiegui, R. Binder, B. Lell, J. Wiesner,
T. Baranek, H. Jomaa and P. G. Kremsner, Lancet, 2002, 360,
1941–1942.
Acknowledgements
14 S. Steinbacher, J. Kaiser, W. Eisenreich, R. Huber, A. Bacher and
F. Rohdich, J. Biol. Chem., 2003, 278, 18401–18407.
15 J. F. Hoeffler, D. Tritsch, C. Grosdemange-Billiard and M. Rohmer,
Eur. J. Biochem., 2002, 269, 4446–4457.
16 B. Altincicek, M. Hintz, S. Sanderbrand, J. Wiesner, E. Beck and
H. Jomaa, FEMS Microbiol. Lett., 2000, 190, 329–333.
17 C. Mueller, J. Schwender, J. Zeidler and H. K. Lichtenthaler,
Biochem. Soc. Trans., 2000, 28, 792–793.
We thank Mr J.-D. Sauer for all NMR measurements,
Mr R. Huber for the MS analysis and Dr Eilers (Saintt Louis,
Missouri) for the gift of fosmidomycin. This investigation was
supported by a grant to M. R. from the ‘Institut Universitaire
de France’. O. M. was a recipient of a grant from the ‘Ministère
de la Jeunesse, de l’Education Nationale et de la Recherche’.
18 D. B. Berkowitz, M. Bose, T. J. Pfannenstiel and T. Doukov, J. Org.
Chem., 2000, 65, 4498–4508.
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23 R. L. Hilderbrand and T. O. Henderson, in The Role of
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