Synthesis and evaluation of a mechanism-based inhibitor of KDO8P synthase

Article abstract of DOI:10.1016/j.carres.2003.10.007

The enzyme 3-deoxy-D-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the condensation reaction between phosphoenolpyruvate (PEP) and D-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate. In attempts to investigate the lack of

Full text of DOI:10.1016/j.carres.2003.10.007

Carbohydrate
RESEARCH
Carbohydrate Research 339 (2004) 385–392
Synthesis and evaluation of a mechanism-based inhibitor of
KDO8P synthase
Valery Belakhov,a Ekaterina Dovgolevsky,a Emilia Rabkin,a Smadar Shulami,a,b
Yuval Shohamb,c and Timor Baasova,c,
*
aDepartment of Chemistry, Technion––Israel Institute of Technology, Haifa 32000, Israel
bDepartment of Food Engineering and Biotechnology, Technion––Israel Institute of Technology, Haifa 32000, Israel
cInstitute of Catalysis Science and Technology, Technion––Israel Institute of Technology, Haifa 32000, Israel
Received 30 April 2003; revised 7 October 2003; accepted 8 October 2003
Abstract—The enzyme 3-deoxy-
tween phosphoenolpyruvate (PEP) and
D
-manno-2-octulosonate-8-phosphate (KDO8P) synthase catalyzes the condensation reaction be-
-arabinose 5-phosphate (A5P) to produce KDO8P and inorganic phosphate. In attempts to
D
investigate the lack of antibacterial activity of the most potent inhibitor of KDO8P synthase, the amino phosphonophosphate 3, we
have synthesized its hydrolytically stable isosteric phosphonate analogue 4 and tested it as an inhibitor of the enzyme. The synthesis of
4 was accomplished in a one step procedure by employing the direct reductive amination in aqueous media between unprotected
sugar phosphonate and glyphosate. The analogue 4 proved to be a competitive inhibitor of KDO8P synthase with respect to both
substrates A5P and PEP binding. In vitro antibacterial tests against a series of different Gram-negative organisms establish that both
inhibitors (3 and 4) lack antibacterial activity probably due to their reduced ability to penetrate the bacterial cell membrane.
ꢀ 2003 Elsevier Ltd. All rights reserved.
Keywords: KDO8P synthase; Lipopolysaccharide; Enzyme inhibitors; Isosteric phosphonate; Antibacterial drug
1. Introduction
prompted us to a further design of synthetic molecules
exhibiting selective activity against Gram-negative bac-
terial cells.
The rapid spread of antibiotic resistance in Gram-neg-
ative bacteria has prompted a continuing search for new
agents against this important class of bacterial patho-
gens.1 Because the biosynthesis of lipopolysaccharide is
unique to Gram-negative bacteria2 and required by
them for growth and virulence,3 the site-specific sugar
found almost exclusively in all bacterial lipopolysac-
For this purpose we have selected the 3-deoxy-D-
manno-2-octulosonate-8-phosphate (KDO8P) synthase,
which is a key enzyme that controls the carbon flow in
the biosynthetic formation of KDO.4 The enzyme cat-
alyzes the unusual aldol-type condensation of phos-
phoenolpyruvate (PEP) with D-arabinose 5-phosphate
(A5P) to produce KDO8P and inorganic phosphate (Pi,
Scheme 1).6
The mechanism of this reaction is still uncertain. Most
recent biochemical7 and structural8 studies suggest a
reaction pathway involving the formation of an acyclic
bisphosphate intermediate 2, however to date there is no
direct evidence for the existence of 2 as a true enzymatic
intermediate. Recently, the first indirect support to the
concept of this mechanism was provided. The stable
analogue of the putative oxocarbenium 1, the amino
charides, 3-deoxy-
D
-manno-2-octulosonic acid (KDO),
has been identified as a target and most efforts to design
synthetic inhibitors have been aimed towards inhibiting
this sugarꢀs biosynthesis and processing.4 Indeed, some
of the potent inhibitors of CMP-KDO synthetase have
shown in vivo antibacterial activity.5 These results have
* Corresponding author. Tel.: +972-4-829-2590; fax: +972-4-829-5703;
e-mail: chtimor@techunix.technion.ac.il
0008-6215/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2003.10.007

386
V. Belakhov et al. / Carbohydrate Research 339 (2004) 385–392
H
HO
O
OH
OH
OTBDMS
OTBDMS
OBn
2-
OPO3
OH
OH
O
a
b
HO
HO
2-
O
OPO3
OH
O
HO
HO
HO
HO
BnO
BnO
-
2
+
A5P
3
H
KDO8P
Synthase
OPO
2-
6
OMe
OMe
5
7
OMe
-
OPO3
2
O
HO
a
O 2
H
3
2
+
C
3
-
si
CO2
b
Et2O3P
H
PEP
re
OH
1
O
H
H
OBn
f
c
OBn
d,e
O
BnO
BnO
O
BnO
BnO
OMe
OMe
8
9
HO
HO
HO
HO
HO
HO
2-
2-
OPO3
OPO3
H
Pi
a
OH
PO3Et2
OH
PO3H2
OH
O
2-
-
OPO3
CO2
g
h
3
2
OH
O
O
3
4
HO
HO
H
OH
-
HO
HO
2
b
OH
CO2
KDO8P
2
OMe
10
11
Scheme 2. Reagents and conditions: (a) TBDMSCl, pyridine, DMAP,
rt; (b) BnBr, NaH, DMF, rt; (c) 1 M H2SO4 (cat.), MeOH, rt; (d) PCC,
CH2Cl2, rt; (e) CH2(PO3Et2)2, n-BuLi, THF, )78 ꢁC; (f) H2, Pd/C,
EtOH, rt; (g) i: (Me)3SiBr, CH2Cl2, rt, ii: Dowex Hþ, H2O, reflux;
(h) glyphosate, NaBH3CN, MeOH/H2O, pH 6.2, 80 ꢁC.
HO
HO
HO
HO
2-
2-
OPO3
CH PO3
2
HO
HO
OH
+
OH
2-
2-
+
PO3
PO3
NH
NH
-
-
3
4
CO2
CO2
Scheme 1. Proposed mechanism for KDO8P-synthase-catalyzed
reaction.
alogues 3 and 4 are similar and their structures can be
divided in two parts: sugar part and N-phosphono
methyl glycine (glyphosate) part. Therefore, it was as-
sumed that the direct reductive amination in aqueous
media between unprotected sugar part (with a free re-
ducing end) and the commercial glyphosate would
yield the desired structures. Indeed, using this strategy,
we previously reported on a very efficient, one-step
synthesis of 39b and its various 13C and 15N-labeled
analogues, which were successfully used for structure–
phosphonophosphate 3, was synthesized and tested as
inhibitor of the synthase.9a Compound 3 was found to
be a potent mechanism-based inhibitor of KDO8P
synthase,9b but failed to inhibit bacterial growth.10 This
lack of antibacterial activity was suspected to be the
result of either the inability of the inhibitor 3 to cross the
cytoplasmic membrane,5 or the hydrolysis of its crucial
C-6 phosphate monoester by various phosphatases.11
While both of these reasons could explain why 3 does
not have antibacterial activity, we decided first to focus
on the hydrolysis issue, which is indeed a major draw-
back with small molecule drugs containing phosphate
monoesters.12 For this purpose, we designed the amino
bisphosphonate 4, which is an isosteric phosphonate
analogue of 3 and should be stable towards the action of
bacterial phosphatases. In this paper we describe the
synthesis of the analogue 4 and its evaluation as inhib-
itor of KDO8P synthase. The examination of 4 against a
series of different Gram-negative bacteria is also re-
ported.
function investigation of the KDO8P synthase.8b;13
A
same approach led us to synthesize the target amino
bisphosphonate 4 in a one-step procedure by directly
coupling the isosteric phosphonate analogue of man-
nose-6-phosphate (compound 11) with glyphosate. The
overall synthetic pathway to 4, as illustrated in Scheme
2, starts from the commercial methyl a-D-mannopyr-
anoside 5 and includes eight chemical steps with an
overall yield of 16%.
Treatment of 5 with tert-butyldimethylsilyl chloride in
pyridine, in the presence of 4-DMAP as a catalyst,
selectively protected primary hydroxyl to afford the 6-
silyloxy mannoside 6 in 85% isolated yield. Benzylation
of 6 with benzyl bromide (BnBr) yielded fully protected
derivative 7 (76%), which was desilylated under mild
acidic condition (catalytic amount of 1 M H2SO4 in
MeOH) to give the primary alcohol 8 (97%).
The introduction of the phosphonomethylene moiety
at the side chain of the mannose derivative 8 was ac-
complished in three chemical steps. The primary alcohol
8 was oxidized with pyridinium chlorochromate (PCC)
to afford the corresponding aldehyde (65% isolated
2. Results and discussion
2.1. Preparation of 4
Our approach in this search for new inhibitors of
KDO8P synthase was to develop synthetic strategy that
avoids lengthy protection/deprotection steps. Both an-

V. Belakhov et al. / Carbohydrate Research 339 (2004) 385–392
387
yield), which was found to be stable and could be iso-
lated in pure form by column chromatography on silica
gel. Treatment of this aldehyde with tetraethyl meth-
ylenediphosphonate ylide by Wittig–Horner reaction
afforded the vinyl phosphonate 9 as an E isomer in 83%
yield.14 Hydrogenolysis (H2, Pd/C in MeOH) of 9 al-
lowed the reduction of the double bond and the simul-
taneous removal of the benzyl groups to afford the
phosphonate 10 in almost quantitative yield.
Treatment of 10 with trimethylsilyl bromide was fol-
lowed by treatment with Dowex (Hþ) cation exchange
resin to afford the completely deprotected phosphonate
11 as a mixture of anomers in 88% yield.15 The direct
reductive amination between 11 and glyphosate
[NaBH3CN in MeOH/H2O (1:1), pH 6.2, 80 ꢁC]9b was
monitored by 31P NMR. The product was purified on
AG 1x8 ion-exchange column (triethylammonium bi-
carbonate buffer, pH 7.5, 0–0.5 M linear gradient), fol-
lowed by treatment with Dowex (Kþ) cation exchange
column to afford the target amino bisphosphonate 4 as a
potassium salt in 53% yield. The spectral characteriza-
1
tion of purified 4 using 2D-COSY, H, 13C, 31P NMR,
and mass spectra were all consistent with this structure.
2.2. Inhibition of KDO8P synthase with 4
The analogue 4 was evaluated as an inhibitor of homo-
geneous KDO8P synthase by using a well established
procedure reported earlier.9 In this procedure, the inhi-
bition constant is measured from the initial velocity
studies, while the substrates (A5P and PEP) and the in-
hibitor 4 are preincubated at constant temperature and
the reaction is initiated by addition of the enzyme. The
kinetic measurements by this procedure assume that
substrates and inhibitor are in a rapid equilibrium with
the enzyme and that the steady-state conditions are es-
tablished instantaneously within the initiation of the re-
action. Using this experimental procedure, we found that
4 is a competitive inhibitor against PEP binding, and the
apparent inhibition constant was estimated to be
50 5 lM (Fig. 1a). Since the structure 4 combines the
key features of both substrates, A5P and PEP, into a
single molecule, it was expected that 4 should be com-
petitive inhibitor with respect to both PEP and A5P.
Examination of 4 as inhibitor against A5P binding (Fig.
1b) reveals that it serves as a competitive inhibitor with
apparent inhibition constant of 50 8 lM.
Comparison of the Ki value of 4 against PEP binding
(50 lM) with that of the amino phosphonophosphate 3
(3.3 lM) determined under the same experimental con-
ditions,9a reveals that 4 is more than 15-fold weaker in-
hibitor than 3. A similar preference in binding of
phosphate esters against their phosphonate mimics is
precedent.16 In general, enzymes bind isosteric phos-
phonate analogues several times less tightly than the
naturally occurring phosphates.17 This has been attrib-
Figure 1. Inhibition of KDO8P synthase by the analogue 4. Double-
reciprocal plots of initial velocities are given as a function of (a) PEP,
when the A5P concentration was 500 lM, and the inhibitor concen-
trations were none ð.Þ, 10 lM (h), 20 lM ðdÞ and 40 lM ð ; (b)
ꢀÞ
A5P, when the PEP concentration was 200 lM, and the inhibitor
concentrations were none ðdÞ, 20 lM ð , 40 lM ðjÞ, and 60 lM (h).
ꢀÞ
The assays were carried out at 37 ꢁC, pH 7.3 in a reaction buffer con-
sisting 0.1 M Tris/HCl, BSA (0.1 mg/mL), PEP, A5P, inhibitor 4 and
ꢁ30 mU of homogeneous KDO8P synthase in a final volume of 1 mL.
All samples were assayed in triplicate and analogous results were ob-
tained in two to four different experiments.
uted to several contributing factors: small differences in
bonding geometries; steric interactions with the methyl-
ene hydrogens; deletion of a hydrogen bond to the
phosphate oxygen; and incomplete ionization of phos-
phonate diacids. Each of these factors could plausibly
explain the observed reduced affinity of 4 to that of 3.
Nevertheless, both of these inhibitors were further sub-
jected to in vitro antibacterial tests against a series of
different Gram-negative bacteria.
2.3. Antibacterial activity test
In vitro antibacterial activities of 3 and 4 were evaluated
by using disc diffusion tests18 on wild-type Escherichia
coli strain K12, and on various E. coli strains mutated in

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V. Belakhov et al. / Carbohydrate Research 339 (2004) 385–392
the lpxA and lpxC genes including: SM101,19a R477-
10,19b and D22.19c These mutant strains are defective in
lipid A biosynthesis and therefore show increased sen-
sitivity to a range of antibiotics. Clinically important
antibiotics, ampicillin, and kanamycin, were used for the
comparison and for the control. Unfortunately, no de-
tectable inhibition was measured with both 3 and 4 even
at concentrations >1 mg per disc, while a considerable
large zone of inhibitions was observed with both ampi-
cillin and kanamycin even at concentrations <10 lg per
disc. Therefore, we conclude that the problem associated
with the lack of antibacterial activity of these inhibitors
(3 and 4) lies in their low permeability through the
bacterial cell membrane, and not due to the hydrolysis
of the crucial C-6 phosphate monoester of 3 as it would
be anticipated before this work. This observation indi-
cates that in order to redesign these inhibitors (as well as
other future analogues of the putative intermediate 2) as
potential antibacterials, their impermeability through
the bacterial cell membrane should be solved. Indeed, in
an attempt to circumvent this barrier, several transport
devices in which the inhibitor to be transported is at-
unless noted. THF was dried over sodium and freshly
distilled in the presence of benzophenone prior to use.22
Flash column chromatography23 was performed on
Silica Gel 60 (70–230 mesh). Reactions were monitored
by TLC on Silica Gel 60 F254 (0.25 mm, Merck) and
detected by charring with a yellow solution containing
Na2MoO4Æ4H2O (5 g) and cerric ammonium nitrate (5 g)
in 10% H2SO4 (300 mL). Soviet spray solution24 was
used for the detection of the compounds containing
phosphate monoesters.
3.2. Spectral methods
Spectrophotometric measurements were made on a
Hewlett-Packard 8452A diode array spectrophotometer
using 1 cm path-length cells with a thermostatic cell
holder, and a circulating water bath at the desired
temperature. 1H NMR spectra were recorded on a
Bruker AM-400 and Bruker Avance-500 spectrometers,
and chemical shifts reported (in ppm) are relative to
internal tetramethylsilane ðd ¼ 0:0Þ with CDCl3 as the
solvent and relative to HOD ðd ¼ 4:63Þ with D2O
as the solvent. 13C NMR spectra were recorded on a
Bruker AM-400 (100.6 MHz) and Bruker Avance-500
(125.8 MHz) spectrometers, and the chemical shifts re-
ported (in ppm) are relative to the external sodium 2,2-
dimethyl-2-silapentane sulfonate ðd ¼ 0:0Þ in D2O. 31P
NMR spectra were recorded on a Bruker AC-200
spectrometer at 81.0 MHz and the chemical shifts re-
ported (in ppm) are relative to external orthophosphoric
acid ðd ¼ 0:0Þ in D2O. All the coupling constants ðJÞ are
in Hz. Mass spectra were obtained on a TSQ-70B mass
spectrometer (Finnigan Mat) by negative chemical ion-
ization (NCI) and by fast-atom bombardment (FAB) in
glycerol matrices or on a Bruker Daltomics Apex-III
using the method of electrospray ionization (ESI).
tached to L
-amino acid,20a to a small peptide,5 or is not
peptide-linked,11;12;20b have been reported. At this stage,
each of these devices might serve as a possible candidate
to overcome the impermeability problem of KDO8P
synthase inhibitors. Solution of this problem should be
one of the most important issues for the design of new
antibacterial drugs that target the KDO biosynthesis.
In summary, we have synthesized an isosteric phos-
phonate analogue of the most potent inhibitor of
KDO8P synthase and tested it as an inhibitor of the
enzyme. The new analogue binds to the enzyme with an
affinity lower than the phosphate inhibitor, and in vitro
antibacterial tests establish that both the inhibitors lack
antibacterial activity probably due to their reduced
ability to penetrate the bacterial cell membrane. The
synthesis of isosteric phosphonate analogues conjugated
to various transport systems is now being considered.
3.3. Inhibition study
3.3.1. Enzyme assays. Unless otherwise stated, the en-
zyme activity was assayed in a 1.0 mL reaction buffer
consisting of 0.1 M Tris/HCl (pH 7.3), A5P (0.5 mM,
>50Km), PEP (0.5 mM, >50Km), and BSA (0.1 mg/mL).
All solutions except enzyme were filtered through Mil-
lipore type-HA filters (0.45 lm) before use. Following
equilibration at 37 ꢁC for 2 min, KDO8P synthase
(20 lL, at a final concentration of approximately 10 nM)
was added. The decrease in the absorbance difference
between 232 and 350 nm (as internal reference) was
monitored as a function of time (MS-DOS UV/VIS
software). This method25 is based on the absorbance
3. Experimental
3.1. General
The homogeneous KDO8P synthase (specific catalytic
activity of 16 U/mg) was isolated from the overproduc-
ing strain E. coli BL21 (DE3), following the procedure
of Ray6 with some modifications.16b A5P was prepared
enzymatically according to the procedure of Whitesides
and co-workers.21 All other chemicals were received
from Aldrich or from Sigma and used without further
purification, unless noted. In all the synthetic work, re-
actions were performed under argon atmosphere unless
otherwise noted. All solvents used in the synthetic work
were anhydrous and received from Aldrich or Sigma,
difference at 232 nm between PEP (e ¼ 2840 Mꢂ1Æcmꢂ1
)
)
and the other substrates and products (e < 60 Mꢂ1Æcmꢂ1
under the assay conditions. The concentrations of PEP
and A5P were determined precisely by quantitative as-
saying of the Pi released by alkaline phosphatase.26 In

V. Belakhov et al. / Carbohydrate Research 339 (2004) 385–392
389
each case, to ensure complete hydrolysis of the phos-
phate monoester, the aliquots of the incubation mixture
with alkaline phosphatase were tested by 31P NMR. One
unit of the enzyme activity is defined as the amount that
catalyzes the consumption of 1 lmol of PEP per minute
at 37 ꢁC. The protein concentration of the enzyme
fractions was determined using the Bio-Rad protein
assay with bovine serum albumin as a standard. During
the enzyme purification, the enzyme activity was assayed
by thiobarbituric acid assay method.6
agarose. The 6 mm discs (Schleicher & Schuell, Ger-
many) were saturated with 500 and 1000 lg of either
inhibitor 3 or 4 dissolved in water and after 10 min
drying at room temperature the discs were placed on the
freshly plated cells and incubated overnight at 37 ꢁC.
The control experiments with ampicillin and kanamycin
were prepared in a same manner, but only 10–30 lg of
each antibiotic were used. The diameter of growth in-
hibition was detected as a zone clearing around each
disc after overnight incubation.
3.3.2. Enzyme inhibition study. In order to determine the
Ki value of inhibitor 4 against PEP binding, the reaction
solutions were prepared in 1.0 mL of a reaction buffer
consisting of 0.1 M Tris/HCl (pH 7.3), BSA (0.1 mg/
mL), the constant saturating concentration of A5P
(0.5 mM >50Km) and variable concentrations of PEP (4–
50 lM). Four inhibitor concentrations were examined
(0, 10, 20, 40 lM) and for each inhibitor concentration
variable concentrations of PEP (4–50 lM) were used.
Following equilibration at 37 ꢁC for 2 min, KDO8P
synthase (20 lL) was added. The rate measurements
were made as described above, while a 5 s delay was
allowed following initiation of the reaction. The initial
rate was then determined by a least squares fitting of the
first 10% of the progress curve (between 20 and 200 s,
depending on the initial concentration of PEP) to a
straight line. All samples were assayed in triplicate and
analogous results were obtained in two different exper-
iments. The data were fitted to the competitive model
using the equation: Y ¼ V ½Sꢃ=½Kð1 þ ½Iꢃ=KiÞ þ ½Sꢃꢃ, em-
ploying the commercial software GraFit5 program. The
Ki value was calculated either from the above treatment,
or from the secondary replots of the slopes from initial
double-reciprocal plots (1=V vs 1=[S]) versus inhibitor
concentration27 and found to be 50 5 lM against PEP
(Fig. 1a).
3.4. Synthetic procedures
3.4.1. Methyl 6-O-tert-butyldimethylsilyl-a-D-mannopyr-
anoside (6). To a solution of compound 5 (10 g,
51.5 mmol) in 20 mL pyridine were added TBDMSCl
(8.53 g, 56.65 mmol), and catalytic amount of 4-di
methylaminopyridine at 0 ꢁC. The reaction mixture was
stirred at room temperature and the reaction progress
was monitored by TLC (MeOH–CHCl3, 9:1). After
completion, reaction mixture was diluted with EtOAc,
and then washed with 3% H2SO4, saturated NaHCO3,
water, and saturated NaCl. The organic layer was dried
(MgSO4) and concentrated under reduced pressure. The
crude material was purified by flash chromatography to
give 6 as a white solid (13.42 g, 85%). 1H NMR (CDCl3,
500 MHz): d 0.07 (s, 6H, OSi(CH3)2t-Bu), 0.87 (s, 9H,
OSiC(CH3)3Me2), 3.32 (s, 3H, OCH3), 3.52 (ddd, 1H,
0
J5;6 5.5 Hz, J5;6 4.5 Hz, J4;5 9.5 Hz, H-5), 3.66 (t, 1H,
J3;4 ¼ J4;5 ¼ 9:5 Hz, H-4), 3.75 (dd, 1H, J3;4 9.5 Hz, J2;3
0
3.5 Hz, H-3), 3.80 (dd, 1H, J6;6 11 Hz, J5;6 5.5 Hz, H-6),
3.87 (dd, 1H, J1;2 1.5 Hz, J2;3 3.5 Hz, H-2), 3.89 (d, 1H,
J1;2 1.5 Hz, H-1). 13C NMR (CDCl3, 125.8 MHz): d 4.7
(OSi(CH3)2t-Bu),
19.0
(OSiC(CH3)3Me2),
25.8
(OSiC(CH3)3Me2), 54.7 (OMe), 64.3 (C-6), 69.5 (C-4),
70.4 (C-2), 71.7 (C-5), 72.0 (C-3), 100.7 (C-1); CIMS m=z
309.1 (MHþ, C13H28O6Si requires 308.4).
The determination of Ki value of inhibitor 4 against
A5P binding was carried out in a similar manner, while
the constant saturating concentration of PEP (0.5 mM,
>50Km) and variable concentrations of A5P (5–100 lM)
were used. Four inhibitor 4 concentrations were exam-
ined (0, 20, 40, 60 lM) and for each inhibitor concen-
tration, variable concentrations of A5P (5–100 lM) were
used. The rate measurements were made as described
above. From received data, the Ki value was calculated
as described above and found to be 50 8 lM against
A5P (Fig. 1b).
3.4.2. Methyl 2,3,4-tri-O-benzyl-6-O-tert-butyldimethyl-
silyl-a-D-mannopyranoside (7). To a solution of com-
pound 6 (10.12 g, 32.8 mmol) in DMF (20 mL) was
added NaH (5.67 g, 49.2 mmol). The reaction mixture
was stirred at room temperature for 1 h. BnBr
(14.04 mL, 39.36 mmol) was added to this mixture at
0 ꢁC and the reaction progress was monitored by TLC
with two solvent systems: 100% EtOAc and EtOAc–
hexane (1:9). After completion, the reaction mixture was
diluted with EtOAc and then washed with water and
saturated NaCl solution. The organic layer was dried
(MgSO4) and concentrated under reduced pressure. The
crude material was purified by flash chromatography to
give 7 (14.37 g, 76%). 1H NMR (CDCl3, 500 MHz):
d 0.04, 0.05 (2s, 2 · 3H, OSi(CH3)2t-Bu), 0.86 (s, 9H,
OSiC(CH3)3Me2), 3.28 (s, 3H, OCH3), 3.55 (m, 1H,
H-5), 3.74 (t, 1H, J1;2 ¼ J2;3 ¼ 2 Hz, H-2), 3.79–3.84
(m, 2H, H-6, and H-3), 3.86–3.90 (m, 2H, H-60, and
3.3.3. In vitro antibacterial tests. The Gram-negative
bacterial strains used in this study were wild-type E. coli
K12 and its mutants: SM101,19a R477-10,19b and D22,19c
which are known as holding increased sensitivity to a
range of antibiotics. The test strains were grown to A600
0.2–0.4 at 37 ꢁC in LB broth. A volume of 100 lL culture
of each strain was plated on LB agar plate using top

390
V. Belakhov et al. / Carbohydrate Research 339 (2004) 385–392
H-4), 4.60 (d, 1H, J 11 Hz, C0HHPh), 4.61 (s, CH2Ph),
4.65 (d, 1H, J 12.5 Hz, C00HHPh), 4.70 (d, 1H, J1;2 2 Hz,
H-1), 4.72 (d, 1H, J 12.5 Hz, C00HHPh), 4.90 (d, 1H, J
11 Hz, C0HHPh), 7.24–7.34 (m, 15H, 3C6H5). 13C NMR
(CDCl3, 125.8 MHz): d )5.3, )5.2 (2 · s, OSi(CH3)2t-Bu),
25.9 (OSiC(CH3)3Me2), 54.4 (OMe), 62.8 (C-6), 72.1,
72.5, 75.0 (3 · s, 3 · CH2Ph), 73.1 (C-2), 74.9 (C-3),
77.2 (C-4), 80.2 (C-5), 98.6 (C-1), 127.4–128.3 (m,
15 · Carom:), 138.5, 138.7, 138.8 (3 · s, 3 · Carom:). CIMS
m=z 579.4 (MHþ, C34H46O6Si requires 578.8).
2.8 Hz, H-1), 7.20–7.41 (m, 15H, 3C6H5), 9.71 (d, 1H,
J5;CHO 0.47 Hz, CHO). 13C NMR (CDCl3, 100.6 MHz): d
55.5 (OMe), 72.4 (m, 2 · CH3CH2OP), 73.0 (s,
CH3CH2OP), 74.1 (C-2), 74.6 (C-4), 76.2 (C-5), 79.2 (C-
3), 99.6 (C-1), 127.6–128.5 (m, 15 · CHarom:), 137.0,
138.7, 139.0 (3 · s, 3 · Carom:), 197.8 (CHO). FABMS
m=z 463.1 (MHþ, C28H30O6 requires 462.5). To a solu-
tion of tetramethyl methylenediphosphonate (0.64 g,
2.21 mmol) in THF (5 mL) at )78 ꢁC was added n-bu-
tyllithium (0.28 mL of 1.6 M solution in hexane). To this
mixture was added a solution of the above aldehyde
(0.68 g, 1.47 mmol) in THF (5 mL) and stirring was
continued for about 30 min at )78 ꢁC. The mixture was
allowed to warm to 0 ꢁC and the reaction progress was
monitored by TLC EtOAc–CH2Cl2 (1:9). The reaction
was quenched by the addition of a few drops of AcOH
solution in THF. The reaction crude was diluted with
EtOAc, and then washed with saturated NaHCO3, wa-
ter, and saturated NaCl. The organic layer was dried
(MgSO4) and concentrated under reduced pressure. The
crude material was purified by flash chromatography to
give 9 (0.73 g, 83%). 1H NMR (CDCl3, 400 MHz): d 1.29
(t, 6H, J 7.2 Hz, 2 · CH3CH2OP), 3.27 (s, 3H, OCH3),
3.70 (t, 1H, J3;4 ¼ J4;5 ¼ 9:3 Hz, H-4), 3.77 (dd, 1 H, J1;2
1.7 Hz, J2;3 3.0 Hz, H-2), 3.88 (dd, 1H, J2;3 3.0 Hz, J3;4
9.3 Hz, H-3), 4.01–4.17 (m, 5H, 2ꢄCH3CH2OP, and H-
5), 4.56 (d, 1H, J 10.6 Hz, CHHPh), 4.59 (d, 1H,
J 10.6 Hz, C0HHPh), 4.63 (d, 1H, J 10.6 Hz, C0HHPh),
4.68 (d, 1H, J 12.4 Hz, C00HHPh), 4.71 (d, 1H, J1;2
1.7 Hz, H-1), 4.75 (d, 1H, J 12.4 Hz, C00HHPh), 4.87 (d,
1H, J 10.6 Hz, CHHPh), 6.11 (ddd, 1H, J5;7 1.8 Hz, J6;7
17.2 Hz, J7;P 21.3 Hz, H-7), 6.94 (ddd, 1H, J5;6 4.3 Hz, J6;7
17.2 Hz, J6;P 22.4 Hz, H-6), 7.23–7.35 (m, 15H, 3C6H5).
13C NMR (CDCl3, 100.6 MHz): d 16.3 (s, CH3CH2OP),
16.7 (s, C0H3CH2OP), 55.0 (s, OMe), 61.8 (m,
2ꢄCH3CH2OP), 71.2 (d, J5;P 21.4, C-5), 72.3 (s,
C0H2Ph), 72.9 (s, C00H2Ph), 74.8 (C-2), 75.3 (CH2Ph),
78.3 (C-4), 80.1 (C-3), 99.3 (C-1), 118.1 (d, J7;P 188.8 Hz,
C-7), 127.6–128.4 (m, 15 · CHarom:), 148.0, 148.4, 148.7
(3 · s, 3 · Carom:), 148.9 (d, J6;P 5.94, C-6). 31P NMR
(CDCl3, 81.0 MHz): d 16.4. CIMS m=z 597.3 (MHþ,
C33H41O8P requires 596.6).
3.4.3. Methyl 2,3,4-tri-O-benzyl-a-D-mannopyranoside
(8). To a stirred solution of compound 7 (5.51 g,
9.52 mmol) in 15 mL of MeOH was added catalytic
amount of 1 M solution of H2SO4 in MeOH at 0 ꢁC. The
reaction mixture was stirred at room temperature and
the reaction progress was monitored by TLC using two
solvent systems: EtOAc–hexane (1:9) and EtOAc–hex-
ane (1:1). After completion, the reaction mixture was
diluted with EtOAc, and then washed with saturated
NaHCO3, water, and saturated NaCl. The organic layer
was dried (MgSO4) and concentrated under reduced
pressure. The crude material was purified by flash
chromatography to give 8 (4.29 g, 97%). 1H NMR
(CDCl3, 500 MHz): d 3.31 (s, 3H, OCH3), 3.64 (ddd, 1H,
0
0
J5;6 4 Hz, J5;6 3 Hz, J4;5 9.5 Hz, H-5), 3.79 (dd, 1H, J6;6
12 Hz, J5;6 4 Hz, H-6), 3.82 (m, 1H, J2;3 3 Hz, H-2), 3.87
(dd, 1H, J6;6 12 Hz, J5;6 3 Hz, H-60), 3.93 (dd, 1H, J3;4
9.5 Hz, J2;3 3 Hz, H-3) 3.40 (t, 1H, J3;4 ¼ J4;5 ¼ 9:5 Hz,
H-4), 4.65 (s, CH2Ph), 4.68 (d, 1H, J 11 Hz, C0HHPh),
4.71 (d, 1H, J ¼ 12:5 Hz, C00HHPh), 4.74 (s, H-1), 4.79
(d, 1H, J 12.5 Hz, C00HHPh), 4.96 (d, 1H, J 11 Hz,
C0HHPh), 7.18–7.38 (m, 15H, 3C6H5). 13C NMR
(CDCl3, 125.8 MHz): d 54.8 (OMe), 62.3 (C-6), 69.5 (C-
5), 72.2 (CH2Ph), 73.0 (C00H2Ph), 74.2 (C-2), 74.9 (C-4),
75.6 (C0H2Ph), 80.2 (C-3), 99.3 (C-1), 127.6–128.4 (m,
15 · CHarom:), 138.5, 138.8, 138.9 (3 · s, 3 · Carom:). CIMS
m=z 465.0 (MHþ, C28H32O6 requires 464.5).
0
0
3.4.4. Methyl 2,3,4-tri-O-benzyl-6-deoxy-6-diethoxyphos-
phinylmethylene-a-D-mannopyranoside (9). Solution of
compound 8 (1.06 g, 2.28 mmol) in dichloromethane
(5 mL) was added to the mixture of PCC (2.0 g,
4.67 mmol), and activated molecular sieves (5 g) in di-
chloromethane (20 mL) at 0 ꢁC. The reaction mixture
was stirred at room temperature and the reaction pro-
gress was monitored by TLC EtOAc–CH2Cl2 (1:9).
After completion, the reaction crude was passed through
Celite, and the solvent was removed under reduced
pressure. The crude material was purified by flash chro-
matography to give the corresponding aldehyde (0.68 g,
65%). 1H NMR (CDCl3, 400 MHz): d 3.36 (s, 3H,
OCH3), 3.74 (t, 1H, J1;2 ¼ J2;3 ¼ 2:8 Hz, H-2), 3.92 (dd,
1H, J2;3 2.8 Hz, J3;4 7.7 Hz, H-3), 4.03 (dd, 1H, J4;5
8.8 Hz, J3;4 7.7 Hz, H-4), 4.07 (dd, 1H, J4;5 8.8 Hz, J5;CHO
0.47 Hz, H-5), 4.56–4.69 (m, 3CH2Ph), 4.83 (d, 1H, J1;2
3.4.5. Methyl 6-deoxy-6-diethoxyphosphinylmethyl-a-D-
mannopyranoside (10). A mixture of compound 9 (0.62 g,
1.05 mmol), and Pd/C (178 mg) in EtOH (10 mL) was
stirred under H2 (1 atm) and the reaction progress was
monitored by TLC using two solvent systems: EtOAc–
hexane (7:3) and MeOH–CHCl3 (1:4). After completion,
the catalyst was removed by filtration through Celite,
and filtrate was concentrated under reduced pressure.
The crude material was purified by flash chromatogra-
phy to give 10 in (0.34 g, 100%): 1H NMR (CDCl3,
400 MHz): d 1.30 (t, 6H, J 7.1 Hz, 2 · CH3CH2OP),
1.71–1.90 (m, 2H, H-6, and H-7), 2.0–2.2 (m, 2H, H-60,
and H-70), 2.25 (s, 1H, OH), 3.30 (s, 3H, OCH3), 3.47

V. Belakhov et al. / Carbohydrate Research 339 (2004) 385–392
391
0
(td, 1H, J5;6 ¼ J4;5 ¼ 9:4 Hz, J5;6 ¼ 2:4 Hz, H-5), 3.56
(t, 1H, J3;4 ¼ J4;5 ¼ 9:4 Hz, H-4), 3.71 (dd, 1H, J2;3
2.9 Hz, J3;4 9.4 Hz, H-3), 3.89 (dd, 1H, J2;3 2.9 Hz, J1;2
1.1 Hz, H-2), 4.01–4.11 (m, 4H, 2 · CH3CH2OP), 4.41
(s, 1H, OH), 4.55 (s, 1H, OH), 4.64 (d, 1H, J1;2 1.1 Hz,
H-1). 13C NMR (CDCl3, 100.6 MHz): d 16.1 (CH3-
CH2OP), 16.6 (C0H3CH2OP), 20.7 (d, J7;P 140.8 Hz,
C-7), 24.1 (C-6), 54.9 (OMe), 61.3 (d, JC;P 5.9 Hz, 2 ·
CH3CH2OP), 62.0 (d, JC;P 6.02 Hz, 2 · CH3C0H2OP),
70.2 (C-2), 70.7 (C-4), 71.2 (d, J5;P ¼ 15:05, C-5), 71.8
(C-3), 100.9 (C-1). 31P NMR (CDCl3, 81.0 MHz): d 31.8.
CIMS m=z 329.0 (MHþ, C12H25O8P requires 328.2).
hydride (103.5 mg, 1.6 mmol) and stirred at 80 ꢁC. The
pH of the mixture was kept constant (pH 6.2) by addi-
tion of 5% solution of acetic acid whenever the pH was
higher than that. The reaction progress was monitored
by 31P NMR and after the completion, the solvent was
removed under reduced pressure. The residue was di-
luted to 50 mL H2O, applied to AG 1 · 8 ion-exchange
column (16 · 2.8 cm), eluted with H2O to elute inorganic
salt and by-products and then with a linear gradient of
triethylammonium bicarbonate (1.2 L, 0–0.5 M, pH 7.5).
Fractions containing the product were combined and
then lyophilized. The residue was passed through a
Dowex (Kþ) column to give pure 4 as a potassium salt
1
(89.5 mg, 53%). H NMR (D2O, 500 MHz): d 1.45–1.53
3.4.6. 6-Deoxy-6-dihydroxyphosphinylmethyl-a-D-manno-
(m, 2H, H-60, and H-70), 1.65–1.74 (m, 1H, H-7), 1.89–
pyranoside potassium salt (11). To a solution of
compound 10 (0.32 g, 0.97 mmol) in 12 mL of dichlo-
romethane was added trimethylsilyl bromide (3.30 mL,
25.21 mmol). The reaction mixture was stirred at room
temperature and the reaction progress was monitored by
TLC MeOH–CHCl3 (1:4). After completion, the solvent
was then removed under reduced pressure. The residue
was diluted with 15 mL of H2O and stirred for 30 min.
Then Dowex Hþ was added and the solution was heated
at reflux for 12 h. The Dowex Hþ was removed by fil-
tration and the filtrate was concentrated under reduced
pressure. The residue was diluted with H2O, applied to
AG 1 · 8 ion-exchange column (16 · 2.8 cm) and eluted
with H2O to elute inorganic salt and then with a linear
gradient of triethylammonium bicarbonate (1.2 L,
0–0.5 M, pH 7.5). Fractions containing the product was
combined and lyophilized. The residue was passed
through a Dowex (Kþ) column to give anomeric mixture
1.97 (m, 1H, H-6), 3.09–3.19 (m, 2H, NCH2PO23ꢂ), 3.39
(dd, 1H, J1;1 13 Hz, J1 ;2 10.5 Hz, H-10), 3.52–3.58 (m,
0
0
0
2H, H-4, and H-5), 3.61 (dd, 1H, J1;1 13 Hz, J1;2 3 Hz, H-
0
1), 3.74 (d, 1H, J2;3 8 Hz, H-3), 3.81 (d, 1H, JH;H 16 Hz,
NCHH0COꢂ2 ), 4.03 (m, 2H, JH;H 16 Hz, NCHH0CO2ꢂ,
0
and H-2). 13C NMR (D2O, 125.8 MHz): d 23.9 (d, J7;P
134.6 Hz, C-7), 27.3 (d, J6;P 3.77 Hz, C-6), 52.7 (d, JC;P
125.7 Hz, NCH2PO23ꢂ), 58.4 (d, JC;P 4 Hz, NCH2COꢂ2 ),
59.2 (d, JC;P 5 Hz, C-1), 64.9 (C-2), 70.7 (d, JC;P 16.35 Hz,
C-5), 71.3 (C-3), 71.9 (C-4), 173.2 (COꢂ2 ). 31P NMR
(D2O, 81.0 MHz): d 7.7 (P from glyphosate moiety), 26.8
(P at side chain of molecule). ESIMS m=z 563.1 (MHþ,
C10H18O12P2K3 requires 562.6).
Acknowledgements
1
of 11 (0.85 g, 88%): H NMR (D2O, 400 MHz): d 1.21–
This work was supported by the United States––Israel
Binational Science Foundation (BSF), Jerusalem, Israel
(Grant no. 97-00356 to T.B.). V.B. acknowledges the
financial support by the Center of Absorption in Sci-
ence, the Ministry of Immigration Absorption, and the
Ministry of Science and Technology, Israel (Kamea
Program).
1.35, 1.45–1.65, 1.85–2.02 (m, 8H, H-6a, H-60a, H-6b,
H-60b, H-7a, H-70a, H-7b, H-70b), 3.19 (td, 1H,
0
J4b;5b ¼ J5b;6 b ¼ 9:6 Hz, J5b;6b 2.5 Hz, H-5b), 3.38 (t, 1H,
J4b;5b ¼ J3b;4b ¼ 9:6 Hz, H-4b), 3.46 (t, 1H, J4a;5a
¼
J3a;4a ¼ 9:7 Hz, H-4a), 3.52 (dd, 1H, J3b;4b 9.6 Hz, J2b;3b
0
3.3 Hz, H-3b), 3.64 (td, 1H, J5a;4a ¼ J5a;6 a ¼ 9:7 Hz, J5a;6a
2.6 Hz, H-5a), 3.71 (dd, 1H, J4a;3a 9.7 Hz, J2a;3a 3.4 Hz,
H-3a), 3.83 (dd, 1H, J2a;3a 3.4 Hz, J1a;2a 1.4 Hz, H-2a),
3.84 (d, 1H, J2b;3b 3.3 Hz, H-2b), 4.77 (s, 1H, H-1b), 5.04
(d, 1H, J1a;2a 1.4 Hz, H-1a). 13C NMR (D2O,
100.6 MHz): d 70.4 (C-4b), 71.0 (C-4a), 71.6 (C-3a), 72.4
(C-2a), 72.9 (C-2b), 73.6 (d, J5a;P 15.9 Hz, C-5a), 74.4
(C-3b), 77.5 (d, J5b;P 15.9 Hz, C-5b), 94.1 (C-1b), 94.9
(C-1a). 31P NMR (D2O, 81.0 MHz): d 22.8. ESIMS m=z
335.2 (MHþ, C7H13O8PK2 requires 334.4).
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