Synthesis and biological evaluation of novel lipid A antagonists

Article abstract of DOI:10.1016/j.bmc.2005.08.047

A mimetic of Lipid A with a β-N(OMe) glycosidic linkage, four linear C-14 hydrophobic chains and without phosphate groups has been prepared together with its β-O-linked analogue. Both these molecules were active in inhibiting the inflammatory action of Es

Full text of DOI:10.1016/j.bmc.2005.08.047

Bioorganic & Medicinal Chemistry 14 (2006) 190–199
Synthesis and biological evaluation of novel lipid A antagonists
Francesco Peri,a,* Chiara Marinzi,a Marek Barath,b Francesca Granucci,a
Matteo Urbanoa and Francesco Nicotraa
aDepartment of Biotechnology and Biosciences, University of Milano-Bicocca, P.zza della Scienza 2, 20126 Milan, Italy
bInstitute of Chemistry, Slovak Academy of Sciences, Dubravska cesta 9, 845 38 Bratislava, Slovakia
Received 5 April 2005; accepted 2 August 2005
Available online 3 October 2005
Abstract—A mimetic of Lipid A with a b-N(OMe) glycosidic linkage, four linear C-14 hydrophobic chains and without phosphate
groups has been prepared together with its b-O-linked analogue. Both these molecules were active in inhibiting the inflammatory
action of Escherichia coli lipid A on MT2 macrophages in a dose-dependent manner, while they were completely devoid of inflam-
matory activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
interactions and for elucidating the entire LPS signal
transduction system, synthetic inhibitors of LPS are
interesting lead compounds for the development of
anti-sepsis drugs.
Septic shock is a serious syndrome associated with
high mortality rates and is caused by a systemic
inflammatory and immune response due to the pres-
ence of lipopolysaccharides (LPS) in the blood of
affected patients.1–5 LPS, a component of Gram-nega-
tive bacteria cell wall, consist of a hydrophilic hetero-
polysaccharide and a covalently bound disaccharide
phospholipid component, termed Lipid A.6,7 Lipid A
(Fig. 1A), the biologically active part of the entire
LPS, initiates the production of multiple endogenous
mediators of the inflammatory response, including
cytokines (e.g., tumour necrosis factor TNFa), arachi-
donic acid metabolites and tissue factors. The molecu-
lar pathway associated to LPS action is initiated by
the extra-cellular association of LPS with membrane-
bound CD148 followed by formation of the LPS
receptor complex by recruitment of MD-29,10 and
TLR4 receptors, the latter being responsible for the
intracellular transmission of the signal leading to the
induction of cytokine gene expression.11,12 Lipid A
has received worldwide scientific attention being one
of the most potent pro-inflammatory substances
known and several synthetic lipid A mimetics have
been developed with both agonistic and antagonistic
properties. While synthetic agonists have been used
as important tools for characterizing LPS–receptor
Depending on the different bacterial origin, Lipid A ex-
ists in a variety of natural forms that differ by slight
chemical modifications mainly involving the lipophilic
chains. Some structural features have been recognized
to be essential for the endotoxic activity of different Lip-
id A species, including the presence of a b(1-6)-linked
glucosamine backbone, diphosphorylation at the ano-
meric C-1 and C-40 positions and a suitable number
and location of appropriately long 3-acyloxyacyl groups
per disaccharide. These general rules have allowed the
design of synthetic analogues that have shown potent
LPS agonist activities. Compound ER-112022
(Fig. 1B) is an interesting but unique exception, being
a phospholipid dimer connected via an acyclic spacer.
Although lacking the disaccharide scaffold, it presents
a potent pro-inflammatory activity.13 Recently, the bio-
activity of Lipid A has been related to its three-dimen-
sional conformation, that is, mainly influenced by the
length, number and symmetry of acyl chains, as well
as the number and distribution of negative charges.14
Lipid A species having an asymmetric (4+2) distribution
of lipophilic chains adopt a conical shape and are highly
active, whereas lipid A species with a symmetric (2+2)
distribution have a cylindrical conformation and have
antagonistic properties. Conical-shaped lipid A species
are believed to trigger the conformational change that
*
Corresponding author. Tel.: +39 02 6448 3453; fax: +39 02 6448
3565; e-mail: francesco.peri@unimib.it
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.08.047

F. Peri et al. / Bioorg. Med. Chem. 14 (2006) 190–199
191
NH
HN
OH
O
O
O
O
HO
P
O
HO P O
O
O
HO
O
O
O P OH
O
HO
NH
O
NH
O
O
H
N
O
O
P
O
O
NH
O
OH
O
O
HO
O
O
OH
O
O
OH
9
O
O
11
O
11
10
9
10
9
9
O
O
O
O
6
6
A
3
3
B
5
5
O
O
OH
O
HO
P
O
O
HO
HO
O
O
O
HO
O
O
HO
O
NH
9
O
O
O
NH
O
O
O
NH
O
NH
P
O
O
O
O
HO
12
OH
HO
HO
O
8
O
12
O
6
10
O
12
12
5
26
D
C
E
OH
OH
O
O
HO
O
N
HO
O
HO
O
O
HO
O
O
O
O
O
O
O
O
O
13
O
13
13
13
13
13
13
13
2
1
Figure 1. (A): Escherichia coli lipid A; (B): ER-112011 (synthetic agonist); (C): E5564 (synthetic antagonist); (D): synthetic antagonist from R. sin-1
lipid A; (E): molecules 1 and 2.
allows the intracellular signal transmission, and thus
interact with the TLR4 binding site in an optimal way.
Cylindrical Lipid As bind to the same receptor without
causing downstream target activation and cytokine pro-
duction. Finally, lipid As adopting an intermediate
slight conical shape, deriving from a (3+2) chain distri-
bution such as in Porphyromonas gingivalis, have weak
pro-inflammatory activity deriving from TLR2 activa-
tion, and in some cases, for example, in Rhodobacter
sphaeroides and Rhodobacter capsulatus, lipid As have
antagonistic properties. The two latter species of lipid
A, the so-called non-toxic lipid A, lack a pro-inflamma-
tory activity but are able to inhibit the action of Esche-
richia coli lipid A in vitro and in vivo.15,16 The chemical
structures of both these compounds inspired the design
of lipid A antagonists with a symmetrical (2+2) chain
arrangement, among which included compound E5564
(Fig. 1C) that showed a potent LPS antagonist activity
and is now under development as a therapeutic com-
pound.17 Boons and co-workers have recently shown
that a synthetic analogue of the lipid A from the nitro-
gen-fixing bacterium R. sin-1 (Fig. 1D) is able to antag-
onize the effect of E. coli lipid A in in vitro experiments
on human monocytic cell lines.18 Noteworthy, the disac-
charide has unique and interesting features, being
devoid of phosphate groups and containing a 2-aminog-
luconolactone monosaccharide unit.
The described data prompted towards the design and
synthesis of two new lipid A mimetics, a b(1-6) disaccha-
ride with a polar methoxyamino group between two glu-
cose units (1) and its O-linked analogue (2) with
potential LPS antagonistic properties (Fig. 1). In physi-
ological condition, at neutral pH values, compound 1
would be protonated to the intra-glycosidic nitrogen
thus presenting a group with a positive charge between
the sugar units.19 As addressed above, the presence of
negatively charged phosphate groups is a common fea-

192
F. Peri et al. / Bioorg. Med. Chem. 14 (2006) 190–199
ture in the majority of mimetics synthesized so far. On
the other hand, the influence of positively charged
groups in lipid A mimetics is to our knowledge a new
interesting feature still to be explored and investigated.
for the first time and probably takes place through the
intermediate formation of the positively charged oxi-
iminium ion.21 The b isomer being the thermodynamic
favoured product gets accumulated in time course. In
the case of the O-linked disaccharide 2, intermediate
13 was activated as anomeric trichloroacetimidate 16
and used for the glycosylation reaction with glycosyl
acceptor 5 in the presence of trimethylsilyltriflate as cat-
alyst. The reaction was found to be regioselective in fa-
vour of the thermodynamic more stable b isomer (ratio
a:b = 1:2). Final deprotection of the hydroxyl group on
C-4 and de-acetylation afforded the target disaccharide
in good overall yields.
2. Results and discussion
2.1. Synthesis
Disaccharide 1 possesses an oxymethyl interglycosidic
bridge that can be introduced in a chemoselective fash-
ion by reacting two unprotected monosaccharide units
bearing, respectively, an aldehyde and a hydroxylamine
group in the proper position. The methodology, recently
described as a very efficient and convergent route for the
synthesis of b(1–6) disaccharide analogues,20,21 leads to
the formation of N(OMe) disaccharides with good gly-
cosylation yields and total stereoselectivity in favour of
the b-anomeric configuration. In order to exploit the
straightforward synthetic approach for the preparation
of lipid A mimetics, monosaccharides 9 and 11 derived
from D-glucose were prepared, each bearing two hydro-
phobic chains on C-2 and C-3. Monosaccharide 11 has a
free anomeric carbon and 9 has a methoxyamino group
on C-6 (Scheme 1). Unfortunately, and in contrast with
previous observation on monosaccharides with free C-2
and C-3 hydroxyls, the condensation between the two
monosaccharides with lipidic chains did not take place
in a reasonable time (up to 24 h) in a series of experi-
mental conditions of solvents and temperature. Unlike
analogue compounds, saccharides 9 and 11 showed to
be mutually unreactive probably because of the presence
of the long lipophilic chains on C-2 and C-3.22 There-
fore, the desired N-linked disaccharide product 1, as
well as its O-linked analogue 2, was synthesized accord-
ing to more traditional condensation strategies. Both
synthetic pathways share the common key intermediate
4 that bears a p-methoxybenzylidene on C-4,6 and two
lipophilic chains on C-2 and C-3 (Scheme 2) that can
easily give access to the required monomers through a
series of functional group interconversions and selective
deprotection reactions. In the case of target molecule 1,
the key glycosylation step was successfully achieved with
a Koenigs–Knorr condensation of bromide 12 with the
hydroxylamino glycoside 8, with a 100% conversion
yield (Scheme 3).23 Noteworthy, the stereochemical out-
come of the reaction gave interesting mechanistic infor-
mation. The disaccharide was formed as a mixture of a
and b anomers, in a 1:2 ratio in favour of the b, as deter-
mined by NMR characterization of the two products,
isolated by chromatography. While the major (b) ano-
mer showed good stability, its a counterpart spontane-
ously interconverted into the b when dissolved in
solvents in neutral environment.à The interconversion
of a into b-N(OMe) disaccharide was observed here
2.2. Disaccharide antagonistic activities
To test the antagonistic activity of the disaccharides 1
and 2, we took advantage of the well-characterized mac-
rophage cell line MT2 from mice.24,25 MT2 cells express
TLR4 and, upon stimulation with LPS, produce TNFa,
one of the principal mediators of the septic shock, and
the anti-bacterial product nitrogen oxide (NO). We first
investigated whether the lipid A itself could be sufficient
to activate MT2 cells. As shown in Figure 2, lipid A used
at a concentration ranging from 2 to 6 ng/mL was able
to stimulate TNFa and NO production by MT2 cells,
while it was toxic at higher concentrations. Since the lip-
id A showed its maximal activity at the concentration of
2 nM, we used this concentration in the subsequent
experiments. Before testing if the two disaccharides
could interfere with the lipid A activity, we excluded
the fact that they could show any possible inflammatory
function. For this reason we directly incubated them
with MT2 cells and evaluated their capacity to elicit
TNFa and NO. As expected no activation of macro-
phages could be observed in these conditions (Fig. 3a).
We then analyzed the antagonistic function of the two
disaccharides. MT2 cells were, thus, stimulated with
the lipid A after a pre-exposure to the compounds 1
and 2, and the amounts of NO and TNFa produced
in these conditions were measured. As shown in Figure
3 both the disaccharides interfered with the lipid A func-
tion in a dose-dependent manner. The antagonistic
activity shown by the two disaccharides specifically in-
volved the TLR4 since no effect on TNFa and NO pro-
duction was observed when the MT2 cells were activated
in the presence of an oligo-DNA containing the unme-
thylated CpG motif of bacterial DNA that is recognized
by TLR926 and the synthetic double-stranded RNA poly
I:C that exerts its function through the TLR327(Fig. 4).
Both disaccharides 1 and 2 antagonized the inflammato-
ry effect of E. coli Lipid A on MT2 macrophages, while
they were devoid of pro-inflammatory effects on the
same cell lines In the same experiments, monosaccha-
rides 9 and 11 were tested on murine MT2 cells (data
not shown) and, as expected, were inactive both as ago-
nist and antagonist. Taken together, these results sug-
gest that the disaccharide core is essential to the
antagonistic activity. Anyway, the chemical structures
of disaccharides 1 and 2 are quite different from those
of natural Lipid As and other known synthetic antago-
nists, so the finding that both these compounds lacking
phosphate groups inhibited cytokine production initiat-
No degradation product was detected in a sample solution in organic
solvents after one week at room temperature and light exposure.
As a consequence, no 13C NMR of the pure a-N(OMe) disaccharide
à
could be recorded, the interconversion rate being higher than the time
required for spectra acquisition.

F. Peri et al. / Bioorg. Med. Chem. 14 (2006) 190–199
193
O
O
HO
HO
HO
RO
NH
O
O
O
HO
N
OH +
HO
RO
O
RO
HO
RO
R=H : 98%
R=C14H29 : n.r.
OR
OR
RO
O
RO
O
Scheme 1. Chemoselective aminooxy-aldehyde condensation for N(OMe) disaccharide preparation.
H
N
R'
HO
PMBO
O
O
O
PMBO
iv.
vii.
O
O
O
O
HO
O
O
iii.
O
O
13
13
13
O
13
O
OH
O
O
13
6 R' = CHO
13
5
O
i.
O
v.
HO
HO
RO
7 R' = CH=NOCH3
8 R' = CH2NHOCH3
9
vi.
RO
HO
OCH3
H3CO
OCH3
3 R = H
4 R = C14H29
ii.
viii.
x.
HO
HO
ix.
AcO
AcO
O
O
OH
O
O
O
13
O
13
OAc
13
AcO
AcO
O
13
O
11
10
xi.
O
Br
13
13
AcO
AcO
O
12
OH
O
O
13
13
13
Scheme 2. Reagents: (i) anisaldehyde dimethylacetal, CSA, DMF, 94%; (ii) C14H29Br, NaH, DMF, 74%; (iii) LiAlH4, AlCl3, CH2Cl2, Et2O, 86%; (iv)
Dess Martin periodinane, CH2Cl2; (v) H2NOCH3, pyridine, 65% (over two steps); (vi) NaBH3CN, AcOH, 92%; (vii) TFA, CH2Cl2, 90%; (viii) Ac2O,
TFA, 83%; (ix) NaOCH3, CH3OH, 87%; (x) HBr, AcOH, CH2Cl2, quant.; (xi) H2NNH3 AcOꢀ, DMF, quant.
+
H
N
R1O
R1O
O
O
AcO
AcO
O
R1O
R1O
O
O
O
i.
O
N
R2O
N
PMBO
O
O
O
+
O
O
O
O
O
R2O
Br
O
O
O
13
O
13
O
13
13
O
13
O
13
O
O
13
13
13
13
13
13
8
12
14: R1=Ac; R2=p-methoxybenzyl
15: R1=Ac; R2=H
1: R1=H; R2=H
ii.
iii.
AcO
AcO
O
OH
O
O
13
13
13
iv.
O
R1O
R1O
HO
PMBO
AcO
AcO
O
O
v.
O
+
O
CCl3
NH
O
O
O
O
α:β = 1:2
O
O
O
O
R2O
13
13
13
13
13
13
O
O
O
5
16
13
13
17: R1=Ac; R2=p-methoxybenzyl
18: R1=Ac; R2=H
2: R1=H; R2=H
vi.
vii.
Scheme 3. Reagents and conditions: (i) AgCO3, AgOTf, CH2Cl2 (quant.); (ii) DDQ, H2O, CH2Cl2 (90%); (iii) NaOCH3, MeOH (quant.);
(iv) CCl3CN, CsCO3, CH2Cl2, then (v) TMSOTf, CH2Cl2, ꢀ30 ꢁC (41%); (vi) DDQ, H2O, CH2Cl2 (83%); (vii) NaOCH3, MeOH (quant.).

194
F. Peri et al. / Bioorg. Med. Chem. 14 (2006) 190–199
a
b
TNFα
NO
2,5
2
6
4
1,5
1
0,5
0
2
0
NT LPS 2nM 4nM 6nM 8nM
LipA
NT LPS 2nM 4nM 6nM 8nM
LipA
Figure 2. Lipid A inflamꢀmatory activity. MT2 cells (2.5 · 105) were incubated with LPS (10 ng/mL) or in presence of the indicated concentration of
ꢀ
lipid A. After 24 h NO2 accumulation (a) or TNFa (b) production were tested in the supernatants. NO2 accumulation was measured as NO
production by Greiss assays. NT: untreated cells; Lip A: cells treated with lipid A; LPS: cells treated with LPS.
a
Disaccharide inflammatory activity
1,4
5
4
3
2
1
0
1
0,6
0,2
0
NT
NT
LipA
LipA
(10µM)
(20µM)
(10µM)
(20µM)
b
Disaccharide antagonistic activity
0,6
4,5
3,5
0,4
0,2
2,5
1,5
0,5
0
0
NT LipA
NT LipA
Lipid A +2
Lipid A + 2
4,5
3,5
0,6
0,4
0,2
2,5
1,5
0,5
0
0
NT LipA
NT LipA
Lipid A + 1
Lipid A + 1
Figure 3. Disaccharide antagonistic activities. (a) The inflammatory activity of the two disaccharides was tested by incubating MT2 cells (2.5 · 105)
in the presence of the indicated concentration of disaccharides 1 and 2 or lipid A as control. After 24 h, NO2ꢀ accumulation or TNFa production was
measured in the supernatants. (b) Inhibition of the lipid A function induced by the disacꢀcharides 1 and 2. MT2 cells were treated with lipid A alone or
lipid A in the presence of increasing concentrations of the two disaccharides. NO2 accumulation or TNFa production was measured in the
supernatants 24 h later. NT: untreated cells; Lipid A: cells treated with lipid A alone; Lipid A + 2: cells treated with lipid A together with increasing
concentrations of disaccharide 2; Lipid A + 1: cells treated with lipid A together with increasing concentrations of disaccharide 1.
ed by E. coli Lipid A is significant. The fact that N- and
O-linked disaccharides have very similar activities indi-
cates that the chemical nature of the interglycosidic
bridge does not influence in a relevant way their antag-
onistic activities. The N-linked disaccharide was much
more soluble in water and in aqueous buffers that its
O-linked counterpart, this property due to the presence
of a positive charge in the protonated methoxyamino

F. Peri et al. / Bioorg. Med. Chem. 14 (2006) 190–199
195
NO
0,2
0,3
0,25
0,2
0,15
0,15
0,1
0,1
0,05
0,05
0
0
-
-
NT
NT
Poly I:C
CpG
TNFα
0,025
0,02
0,015
0,01
0,005
0
0,02
0,015
0,01
0,005
0
NT
NT
-
-
Poly I:C
CpG
Figure 4. Specificity of disaccharide 1 and 2 antagonistic activities. MT2 cells weꢀre incubated for 24 h with CpG (1lM) or Poly I:C (20 ng/mL) in the
presence or absence of disaccharide 1 or 2 (20 nM each). The amounts of NO2 or TNFa were then measured in the supernatants. NT: untreated
cells; -: cells treated with CpG or poly I:C alone; 2: cells treated with CpG or poly I:C in the presence of the disaccharide 2; 1: cells treated with CpG
or poly I:C in the presence of the disaccharide 1.
group at neutral pH, facilitated solubilization of
compound 1 and its use in biological tests. We are cur-
rently investigating the activity of both disaccharides on
dendritic cells (DCs), a special type of leukocyte able to
alert the immune system for the presence of infections
and responsible for the activation of both innate and
adaptive immune responses. Given the central role of
DCs in priming immunity, blocking their activation
could result in a profound downregulation of initial
inflammatory responses.
ment, using gentisic acid (DHB) as matrix. Optical
rotations were measured at ambient temperature on
a Perkin-Elmer 241 polarimeter.
3.1.1. Methyl 4,6-O-(4-methoxybenzylidene)-a-D-gluco-
pyranoside (3).28,29 To a solution of methyl a-D-gluco-
pyranoside (10 g, 51.5 mmol) in DMF (50 mL),
camphorsulfonic acid (cat.) and anisaldehyde dimethy-
lacetal (10 mL, 51.5 mmol) were added and the resulting
mixture was stirred for 40 min, concentrated and
quenched by addition of NaHCO3 (satd aq solution,
100 mL) and stirring for an additional hour. The precip-
itate was filtered and washed with cold NaHCO3 (satd
aq, 100 mL). Trituration with hexane afforded 3 (15 g,
94%). Compound 3: Rf = 0.31 (EtOAc/MeOH/H2O
7:2:1).
3. Experimental
3.1. General
All solvents were dried over molecular sieves (Fluka),
for at least 24 h prior to use. When dry conditions
were required, the reactions were performed under
Ar atmosphere. Thin-layer chromatography (TLC)
was performed on Silica Gel 60 F254 plates (Merck)
with detection with UV light when possible, or char-
ring with a solution containing concd H2SO4/EtOH/
H2O in a ratio of 5/45/45 followed by heating at
180 ꢁC. Column flash chromatography was performed
on silica gel 230–400 mesh (Merck). The boiling range
of petroleum ether used as eluent in column chroma-
3.1.2. Methyl 4,6-O-(4-methoxybenzylidene)-2,3-di-O-
tetradecyl-a-D-glucopyranoside (4).30 To a solution of
3 (8 g, 25.6 mmol) in DMF (80 mL), NaH (60% suspen-
sion in mineral oil, 6.4 g, 160 mmol) was carefully added
in small portions. Tetradecylbromide (38 mL,
128 mmol) was added dropwise and the resulting mix-
ture was stirred at 60 ꢁC overnight. After cooling to
room temperature, methanol (20 mL) was carefully
added and the solution was stirred for 20 min to hydro-
lyze excess of sodium hydride. Solvents were then evap-
orated and the residue diluted with AcOEt (500 mL).
Citric acid (satd aq soln, 400 mL) was added, the layers
were separated, the organic layer was washed with water
(3 · 400 mL), dried (Na2SO4) and evaporated. Flash
column chromatography on silica gel of the residue
(petroleum ether/AcOEt 85:15) afforded 4 (13.3 g,
74%). Compound 4: Rf = 0.32 (petroleum ether/AcOEt
1
tography is 40–60 ꢁC. H and 13C NMR spectra were
recorded at 400 MHz on a Varian MERCURY instru-
ment at 300 K. Chemical shifts are reported in parts
per million downfield from TMS as an internal stan-
dard. In the proton assignment, H-a and H-b refer
to the positions of C-14 chains. Mass spectra were re-
corded on a MALDI2-TOF Kompakt Kratos instru-

196
F. Peri et al. / Bioorg. Med. Chem. 14 (2006) 190–199
1
9:1); [a]D = +23 (c 1 in CHCl3); H NMR (400 MHz,
CDCl3): d (ppm) = 7.40–6.85 (A2X2q, 4H, aromatics),
5.48 (s, 1H, OCH3), 4.77 (d, J = 3.7 Hz, H-1), 4.24
(dd, J = 9.7, 4.4 Hz, 1H, H-6a), 3.80 (s, 3H, OCH3),
3.77–3.60 (m, 8H), 3.47 (t, J = 9.3 Hz, 1H), 3.42 (s,
3H, OCH3), 3.34 (dd, J = 9.3, 3.7 Hz, 1H, H-2), 1.5–
1.6 (m, 4H, H-b), 1.22 (br s, 44H, CH2), 0.85 (t,
J = 5.8 Hz, 6H, CH3). 13C NMR (400 MHz, CDCl3): d
(ppm) = 161.2 (Car), 127.4 (Car), 128.4 (Car),
114.0(Car), 105.1, 98.4 (C-1), 83.4, 80.8, 74.5 (OCH2),
73.5 (OCH2), 71.6 (OCH2), 71.2, 70.2 (C-6), 69.6, 57.7
(OCH3), 55.6 (OCH3), 32.4, 30.8, 30.5, 30.1, 30.1, 30.0,
29.9, 29.8, 26.4, 23.1, 14.6. MS (MALDI-TOF): m/z:
705.6 [M+H]+, 727.5 [M+Na]+.
1.5 h, the solvent was evaporated. Flash column
chromatography of the residue (petroleum ether/AcOEt
9:1) afforded 7 (540 mg, 65%). Compound 7: Rf = 0.70
1
(petroleum ether/AcOEt 7.5:2.5); H NMR (400 MHz,
CDCl3): d (ppm) = 7.17 (d, 1H, J = 7.6 Hz, H-6), 7.13–
6.77 (A2X2q, 4H, J = 8.6 Hz, aromatic), 4.71 (d, 1H,
J = 3.4 Hz, H-1), 4.65, 4.44 (ABq, 2H, J = 10.6 Hz,
CH2–(4-OMePh)), 4.10 (dd, 1H, J = 9.9, 6.3 Hz, H-3),
3.82 (s, 3H, OCH3), 3.72 (s, 3H, OCH3), 3.33 (s, 3H,
OCH3), 3.45–3.78 (m, 5H), 3.20–3.28 (m, 2H), 1.50–
1.60 (m, 4H, H-b), 1.22 (br s, 44H, CH2), 0.85 (t, 6H,
J = 5.8 Hz, CH3). 13C NMR (400 MHz, CDCl3): d
(ppm) = 159.4 (Car), 147.3 (Car), 130.3 (Car), 129.9
(Car), 113.9 (C6), 98.4 (C1), 81.4, 80.4, 79.7, 76.0, 76.5,
72.1, 68.5, 62.1 (NOCH3), 55.9 (OCH3), 55.9 (OCH3),
32.3, 31.0, 30.4, 30.1, 30.1, 30.1, 30.0, 30.0, 29.9, 29.8,
26.7, 26.4, 23.1, 14.6 (O(CH2)13CH3). MALDI-TOF
(DHB): m/z: 757.1 [M+Na]+, 773.3 [M+K]+.
3.1.3. Methyl 4-O-(4-methoxybenzyl)-2,3-di-O-tetrade-
4
cyl-a-D-glucopyranoside (5). Compound
(1.0 g,
1.41 mmol) was dissolved in a mixture of diethylether/
CH2Cl2 2:1 (70 mL) under argon atmosphere. LiAlH4
(1 M in THF, 7.2 mL) and AlCl3 (1.16 g, 8.72 mmol) in
diethylether (25 mL) were added dropwise and the result-
ing mixture was refluxed for 4 h. After cooling to room
temperature, AcOEt (300 mL) and water (300 mL) were
added and the layers separated. The organic layer was
washed with brine (3 · 200 mL), dried over sodium sul-
fate and evaporated. Flash column chromatography of
the residue (petroleum ether/AcOEt 7:3) afforded 5
(860 mg, 86%). Compound 4: Rf = 0.25 (petroleum
ether/AcOEt 8.5:1.5). [a]D = +46 (c 1 in CHCl3); 1H
NMR (400 MHz, CDCl3): d (ppm) = 7.36 (m, 2H, aro-
matic), 6.92 (m, 2H, aromatic), 4.86–4.57 (ABq, 2H,
J = 10.6 Hz, CH2–(4-OMePh)), 4.75 (d,1H, J = 3.5 Hz,
H-1), 3.90–3.54 (m, 11H, H-3, H-5, H-6a, H-6b, H-a),
3.41 (dd, 1H, J = 9.8, 9.0 Hz, H-4), 3.37 (s, 3H, OCH3),
3.27 (dd, 1H, J = 3.6, 9.7 Hz, H-2), 1.5–1.6 (m, 4H, H-
b), 1.22 (br s, 44H, CH2), 0.85 (t, 6H, J = 5.8 Hz,
CH3). 13C NMR (400 MHz, CDCl3): d (ppm) = 159.2
(Car), 130.2 (Car), 129.47 (Car), 113.9(Car), 98.3 (C-1),
81.3, 80.7, 73.9 (OCH2), 73.5 (OCH2), 71.6 (OCH2),
71.1, 70.2 (C-6), 69.6, 55.6 (OCH3), 55.6 (OCH3), 32.4,
30.8, 30.5, 30.1, 30.1, 30.1, 30.0, 29.9, 29.8, 26.4, 23.2,
14.6 (CH2). MS (MALDI-TOF): m/z: 707.4 [M+H]+,
729.4 [M+Na]+, 745.3 [M+K]+.
3.1.6. Methyl 6-methoxyamino-4-O-(4-methoxybenzyl)-
2,3-di-O-tetradecyl-6-deoxy-a-D-glucopyranoside (8). To
a solution of O-methyloxime 7 (500 mg, 0.68 mmol) in
glacial acetic acid (35 mL), sodium cyanoborohydride
(214 mg, 3.4 mmol) was added and the resulting mixture
was stirred for 4 h. The solvent was evaporated and the
residue was purified by flash column chromatography
on silica gel (petroleum ether/AcOEt 8:2) to yield 8
(460 mg, 92%). Compound 8: Rf = 0.42 (petroleum
1
ether/AcOEt 7.5:2.5); H NMR (400 MHz, CDCl3): d
(ppm) = 7.25 (m, 2H, aromatic), 6.83 (m, 2H, aromatic),
5.60 (br s, 1H, NH), 4.82, 4.55 (ABq, 2H, J = 10.6 Hz,
CH2–(4-OMePh)), 4.72 (d, 1H, J = 3.5 Hz, H-1), 3.90–
3.55 (m, 5H, 4H-a, H-3), 3.80 (s, 3H, OCH3), 3.49 (s,
3H, OCH3), 3.38 (s, 3H, OCH3), 3.30–3.20 (m, 4H, H-
2, H-4, H-5, H-6a), 2.84 (dd, 1H, J = 13.2, 7.7 Hz, H-
6b). 13C NMR (400 MHz, CDCl3): d (ppm) = 159.4,
130.7, 129.8, 114.5, 98.0, 97.9, 82.0, 81.3, 79.9, 74.9,
74.1, 72.1, 67.1, 61.6, 61.6, 55.6, 55.6, 55.4, 55.4, 52.7,
32.3, 31.0, 30.5, 30.1, 30.1, 29.9, 29.7, 26.46, 23.15,
14.6. MALDI-TOF: m/z: 758.1[M+Na]+, 774.1 [M+K]+.
3.1.7. Methyl 6-methoxyamino-2,3-di-O-tetradecyl-6-
8
deoxy-a-D-glucopyranoside (9). Monosaccharide
(200 mg, 0.27 mmol) was dissolved in TFA and CH2Cl2
(1/1, 20 mL) at 0 ꢁC and the solution was stirred for 1 h
at this temperature. The solvent was evaporated, and
the residue was purified by flash column chromatography
on silica gel (35% ethyl acetate in petroleum ether)
(150 mg, 90%). Compound 9: Rf = 0.42 (petroleum
3.1.4. Methyl 4-O-(4-methoxybenzyl)-2,3-di-O-tetrade-
cyl-a-D-gluco-hexodialdo-1,5-pyranoside (6). To a solu-
tion of compound 5 (800 mg, 1.13 mmol) in dry
dichloromethane (35 mL), Dess Martin periodinane
(720 mg, 1.70 mmol) was added under argon atmo-
sphere. After 1 h, crude was diluted with dichlorome-
thane (200 mL) and saturated solutions of NaHCO3
and Na2S2O3 (1/1, 200 mL) were added. The layers were
separated, the organic layer was washed with water
(200 mL), dried on sodium sulfate and evaporated.
The residue was used without further purification for
the next reaction. Compound 6: Rf = 0.30 (petroleum
ether/AcOEt 7.5:2.5).
1
ether/AcOEt 7.5:2.5); H NMR (400 MHz, CDCl3): d
(ppm) = 4.76 (d, 1H, J = 3.5 Hz, H-1), 3.89 (m, 1H, H-
a), 3.82 (m, 1H, H-5), 3.63 (m, 1H, H-a), 3.60–3.50 (m,
2, H-3, H-4), 3.54 (s, 1H, OCH3), 3.32 (dd, 1H, J = 3.1,
13.5 Hz, H-6a), 3.27 (dd, 1H, J = 3.5, 9.5 Hz, H-2), 3.00
(dd, 1H, J = 7.2, 13.5 Hz, H-6b), 1.5–1.6 (m, 4H, H-b),
1.22 (br s, 44H, CH2), 0.85 (t, 6H, J = 5.8 Hz, CH3).
13C NMR (400 MHz, CDCl3): d (ppm) = 98.2 (C-1),
81.2, 80.9, 74.9, 73.4, 72.1, 67.3, 61.8, 55.5, 53.7, 32.3,
31.0, 30.5, 30.1, 30.1, 29.9, 29.7, 26.46, 23.15, 14.6. MAL-
DI-TOF (DHB): m/z: 639.4 [M+Na]+, 655.1 [M+K]+.
3.1.5. Methyl 4-O-(4-methoxybenzyl)-2,3-di-O-tetrade-
cyl-a-D-gluco-hexodialdo-1,5-pyranose-6-O-methyloxime
(7). Crude aldehyde 6 (800 mg, 1.13 mmol) was dis-
solved in pyridine (12 mL) and O-methyl hydroxylamine
hydrochloride (142 mg, 1.7 mmol) was added. After
3.1.8. 1,4,6-Tri-O-acetyl-2,3-di-O-tetradecyl glucopyr-
anoside (10). The 4-methoxybenzylidene derivative 4

F. Peri et al. / Bioorg. Med. Chem. 14 (2006) 190–199
197
(200 mg, 0.28 mmol) was dissolved in acetic anhydride
(12 mL) in the presence of TFA (3 mL). After 4 h, the
reaction was quenched by adding water and ice
(150 mL). Chloroform (200 mL) was added and the
layers were separated. The organic layer was washed
with saturated aqueous NaHCO3 (200 mL), water
(2 · 200 mL), then dried over sodium sulfate and evapo-
rated. Flash column chromatography of the residue on
silica gel (petroleum ether/AcOEt 8.5:1.5) afforded 10
(162 mg, 83%). Compound 10: Rf = 0.35 (petroleum
ether/AcOEt 9:1), mixture of a and b anomers (1:2 ratio
in favour of the a-anomer): [a]D = +32 (c 0.50 in
added. The layers were separated, the organic phase
was washed with 0.1 M hydrochloric acid (150 mL),
water (3 · 150 mL), dried over sodium sulfate and evap-
orated. Flash column chromatography on silica gel of
the residue (petroleum ether/AcOEt 7.5:2.5) afforded
13 (139 mg, quant. yield). Compound 13: Rf = 0.30
(petroleum ether/AcOEt 8:2). MALDI-TOF (DHB):
m/z: 657.8 [M+H]+, 680.2 [M+Na]+.
3.1.12. Methyl 6-methoxyamino-4-O-(4-methoxybenzyl)-
2,3-di-O-tetradecyl-6-deoxy-6-N-(4,6-di-O-acetyl-2,3-di-
O-tetradecyl-b-D-glucopyranosyl)-a-D-glucopyranoside
(14). Silver carbonate (55 mg, 0.2 mmol) and silver tri-
flate (8 mg, 0.03 mmol) were dissolved in dry CH2Cl2
1
CHCl3); H NMR (400 MHz, CDCl3): d (ppm) = 6.30
(d, 1H, J = 3.5 Hz, H-1a anomer), 5.51 (d, 1H,
J = 8.0 Hz, H-1b anomer), 5.00 (t, 1H, J = 9.5 Hz, H-
4), 4.23 (dd, 1H, J = 4.4, 12.4 Hz, H-6a), 4.00 (dd, 1H,
J = 2, 12.3 Hz, H-6b), 3.94 (m, 1H, H-5), 3.80–3.70
(m, 2H, H-2, H-3), 3.60–3.30 (m, 4H, H-a), 2.05 (3s,
9H, CH3CO), 1.5–1.6 (m, 4H, H-b), 1.22 (br s, 44H,
CH2), 0.85 (t, 6H, J = 5.8 Hz, CH3). 13C NMR
(400 MHz, MeOH-d): d (ppm) = 94.9 (C-1), 88.6, 87.3,
85.2, 84.8, 79.9, MALDI-TOF (DHB): m/z: 721.8
[M+Na]+, 737.6 [M+K]+.
˚
(5 mL) in the presence of 4 A molecular sieves under ar-
gon atmosphere and the resulting mixture was stirred
for 40 min in the absence of light. A solution of bromide
12 (90 mg, 0.14 mmol) in dry CH2Cl2 (5 mL) was added
dropwise, followed by a solution of the methoxyamino
monosaccharide 8 (55 mg, 0.075 mmol) in dichloro-
methane (5 mL) and the resulting mixture was stirred
overnight. CH2Cl2 (50 mL) was added and the organic
layer was washed with brine (2 · 100 mL), dried over so-
dium sulfate and evaporated. Flash column chromatog-
raphy of the residue (petroleum ether/AcOEt 9:1)
afforded 14 (66 mg of a-anomer, 36 mg of b-anomer,
quant.). The a-anomer was converted into b by dissolv-
ing it in CHCl3 and stirring the solution for 24 h. Evap-
oration of the solvent and filtration on silica gel afforded
14, Rf = 0.42 (petroleum ether/AcOEt 8:2). Compound
14: 1H NMR (400 MHz, CDCl3): d = 7.20,6.80
(A2X2q, 4H, Harom), 4.83 (t, 1H, J = 9.5 Hz, H-40),
4.78, 4.44 (ABq, 2H, CH2–PMB), 4.66 (d, 1H,
J = 3.4 Hz, H-1), 4.16 (dd, 1H, J = 12.0, 5.0 Hz, H-
60a), 4.08 (d, 1H, J = 8.2 Hz, H-10), 4.02 (dd, 1H,
J = 12.0, 2.5 Hz, H-60b), 3.80 (m, 1H, H-a), 3.71 (s,
1H, OCH3–PMB), 3.65 (m, 2H, H-5 and H-a), 3.60 (t,
1H, J = 9.4 Hz, H-3), 3.60–3.50 (m, 2H, H-6), 3.51 (s,
3H, NOCH3), 3.40 (m, 1H, H-50), 3.35 (m, 1H, H-30),
3.33 (s, 3H, OCH3), 3.28 (m, 1H, H-20), 3.20 (dd, 1H,
J = 9.7, 3.4 Hz, H-2), 3.09 (t, 1H, J = 8.9 Hz, H-4),
1.60 (m, 8H, H-b), 1.30 (m, 38H, CH2), 0.90 (t, 12H,
J = 8.4 Hz, CH3). 13C NMR (400 MHz, CDCl3): d
(ppm): 98.6 (C-1), 95.1 (C-10), 84.4, 82.4, 81.5, 80.5,
79.2, 75.6, 74.0, 74.1, 73.9, 73.6, 72.4, 71.7, 70.8, 63.5
(C-6), 61.0, 56.1, 56.0. MALDI-TOF (DHB): m/z:
1376.4 [M+H]+, 1398.5 [M+Na]+.
3.1.9. 2,3-Di-O-tetradecyl glucopyranoside (11). The tri-
acetyl derivative 10 (150 mg, 0.21 mmol) was dissolved
in methanol (15 mL). Sodium (catalytic) was added
and the mixture was stirred for 20 min. At the end of
the reaction, the pH was adjusted to 7 by addition of
IR 120 H+. After filtration, the solvent was evaporated
and the product was isolated by flash column chroma-
tography on silica gel (petroleum ether/AcOEt 1:1)
(104 mg, 87%). Compound 11: Rf = 0.20 (petroleum
ether/AcOEt 1:1). Compound 11 was almost exclusively
1
in the a-anomeric configuration. H NMR (400 MHz,
MeOH-d): d (ppm) = 4.80 (d, 1H, J = 3.5 Hz, H-1),
4.00–3.40 (m 9H), 3.52 (t, 1H, J = 9.3 Hz, H-3), 3.28
(dd, 1H, J = 3.5, 9.3 Hz, H-2), 1.5–1.6 (m, 4H, H-b),
1.22 (br s, 44H, CH2), 0.85 (t, 6H, J = 5.8 Hz, CH3).
MALDI-TOF (DHB): m/z: 573.5 [M+H]+, 596.3
[M+Na]+, 612.2 [M+K]+.
3.1.10. 4,6-Di-O-acetyl-2,3-di-O-tetradecyl glucopyrano-
syl bromide (12). The triacetyl derivative 10 (150 mg,
0.21 mmol) was dissolved in dichloromethane (5 mL).
At 0 ꢁC, hydrobromic acid (33% in acetic acid, 220 lL,
1.26 mmol) was added dropwise and the resulting mix-
ture was stirred for 1.5 h. Dichloromethane (100 mL)
was added and the reaction was quenched by careful
addition of ice and water (50 mL). The layers were sep-
arated, the organic layer was washed with NaHCO3
(satd aq soln, 100 mL), brine (100 mL), dried over so-
dium sulfate and evaporated. The crude product was
pure according to TLC analysis and was used without
further purification for the next reaction. Compound
12: Rf = 0.50 (petroleum ether/AcOEt 8:2).
3.1.13. Methyl 6-methoxyamino-2,3-di-O-tetradecyl-6-
deoxy-6-N-(4,6-di-O-acetyl-2,3-di-O-tetradecyl-b-D-
glucopyranosyl)-a-D-glucopyranoside (15). To a solution
of disaccharide 14 (pure b-anomer) (70 mg, 0.051 mmol)
in CH2Cl2 (10 mL), DDQ (10 mg, 0.044 mmol) and
water (0.2 mL) were sequentially added and the resulting
mixture was stirred overnight. Dichloromethane
(20 mL) and NaHCO3 (satd aq soln, 30 mL) were added
and the layers were separated. The organic layer was
washed with brine (2 · 50 mL), dried over sodium sul-
fate and evaporated. Flash column chromatography of
the residue on silica gel (petroleum ether/AcOEt 9:1)
afforded 15 (58 mg, 90%). Compound 15: Rf = 0.25
(petroleum ether/AcOEt 8:2). 1H NMR (400 MHz,
CDCl3): d = 4.84 (t, 1H, J = 9.5 Hz, H-40), 4.66 (d, 1H,
3.1.11. 4,6-Di-O-acetyl-2,3-di-O-tetradecyl glucopyra-
nose (13). To a solution of triacetyl derivative 10
(150 mg, 0.21 mmol) in DMF (8 mL), hydrazynium ace-
tate (40 mg, 0.42 mmol) was added and the resulting
mixture was stirred for 40 min. AcOEt (150 mL) and a
saturated aqueous solution of NaHCO3 (150 mL) were

198
F. Peri et al. / Bioorg. Med. Chem. 14 (2006) 190–199
J = 3.5 Hz, H-1), 4.16 (dd, 1H, J = 11.6, 4.6 Hz,
H-60a), 4.10 (d, 1H, J = 8.7 Hz, H-10), 3.99 (dd, 1H,
J = 11.6, 2.2 Hz, H-60b), 3.78 (m, 1H, H-a), 3.65 (m,
2H, H-6), 3.70 (m, 1H, H-5), 3.51 (s, 3H, NOCH3),
3.50 (m, 1H, H-50), 3.40 (m, 2H, H-30, H-20), 3.33 (s,
3H, OCH3), 3.24 (m, 1H, H-2), 3.20 (dd, 1H, J = 9.7,
3.5 Hz, H-2), 1.60 (m, 8H, H-b), 1.30 (m, 38H, CH2),
0.90 (t, 12H, J = 8.4 Hz, CH3). 13C NMR (400 MHz,
CDCl3): d (ppm): 105.4 (C-1), 100.4 (C-10), 91.2, 87.9,
87.4, 85.4, 81.5, 80.9, 80.8, 80.7, 80.2, 78.8, 77.2, 76.2,
70.9, 69.8, 67.6, 62.7. MALDI-TOF (DHB): m/z:
1256.2 [M+H]+, 1277.5 [M+Na]+.
3.80 (s, 3H, OCH3–PMB), 3.38 (s, 3H, OCH3), 1.60
(m, 8H, H-b), 1.30 (m, 38H, CH2), 0.90 (t, 12H,
J = 8.4 Hz, CH3). 13C NMR (only the signals of 17-b
carbons have been reported) (400 MHz, CDCl3): d
(ppm): 104.5 (C-10), 98.4 (C-1), 82.0 (C-20), 81.8, 81.7,
80.9, 78.2, 77.0, 68.2, 68.0, 66.4, 66.3, 65.8, 65.3, 65.2,
64.8, 62.2 (C-60), 52.8 (OCH3).
3.1.16. Methyl 2,3-di-O-tetradecyl-6-O-(2,3-di-O-tetrade-
cyl-4,6-di-O-acetyl-b-D-glucopyranosyl)-a-D-glucopyr-
anoside (18). To a solution of the disaccharide 17
(mixture of a and b isomer, 50 mg, 0.037 mmol) in
CH2Cl2 (4 mL), DDQ (20 mg, 0.088 mmol) and water
(60 lL) were sequentially added and the resulting mixture
was stirred for 3 h. Dichloromethane (40 mL) and NaH-
CO3 (satd aq soln, 50 mL) were added and the layers were
separated. The organic layer was washed with brine
(2 · 50 mL), then dried over sodium sulfate and evapo-
rated. Flash column chromatography of the residue
(petroleum ether/AcOEt 9:1) afforded 18a (14 mg) and
18-b (28 mg) (83%). MALDI-TOF (DHB): m/z: 1376.4
[M+H]+, 1398.5 [M+Na]+ 18-a: Rf = 0.30 (petroleum
ether/AcOEt 8:2). Compound 18-b: Rf = 0.25 (petroleum
ether/AcOEt 8:2). 1H NMR (400 MHz, CDCl3): d (ppm):
4.84 (t, 1H, J = 9.9 Hz, H-40), 4.71 (d, 1H, J = 3.5 Hz, H-
1), 4.28 (d, 1H, J = 7.9 Hz, H-10), 4.12 (dd, 1H, J = 11.6,
4.6 Hz, H-60a), 4.02 (dd, 1H, J = 11.6, 2.2 Hz, H-60b),
4.01 (m, 1H, H-6a), 3.80 (m, 2H, H-a), 3.70 (m, 2H, H-
6b, H-a), 3.60–3.30 (m, 9H), 3.33 (s, 3H, OCH3), 3.20
(dd, 1H, J = 9.7, 3.5 Hz, H-2), 3.15 (t, 1H, J = 7.9 Hz,
H-20), 1.60 (m, 8H, H-b), 1.30 (m, 38H, CH2), 0.90 (t,
12H, J = 8.4 Hz, CH3). 13C NMR (400 MHz, CDCl3): d
(ppm): 104.5 (C-10), 98.4 (C-1), 82.0 (C-20), 81.1, 80.5
(C-2), 70.0 (C-40), 68.9 (C-6), 62.2 (C-60), 55.4.
3.1.14. Methyl 2,3-di-O-tetradecyl-6-deoxy-6-methoxya-
mino-6-N-(2,3-di-O-tetradecyl-b-D-glucopyranosyl)-a-D-
glucopyranoside
(1).
Disaccharide
15
(20 mg,
0.016 mmol) was dissolved in methanol (3 mL). A freshly
prepared solution of sodium methanoate (1 M, 20 lL,
0.02 mmol) was added dropwise. After 20 min the reac-
tion was quenched by adding IRA 120 H+ to a pH value
of 7. The resin was filtered and the solvent was evapo-
rated. Flash column chromatography of the residue
(petroleum ether/AcOEt 6:4) afforded 1 (18 mg, quant.).
1: Rf = 0.45 (petroleum ether/AcOEt 1:1). [a]D = ꢀ58.26
(c 0.30 in CHCl3). 1H NMR (400 MHz, CDCl3): d = 4.64
(d, 1H, J = 2.6 Hz, H-1), 4.14 (d, 1H, J = 7.4 Hz, H-10),
4.0–3.3 (m, 17H), 3.44 (s, 3H, NOCH3), 3.36 (s, 3H,
OCH3), 3.20 (m, 1H, H-20), 3.10 (dd, 1H, J = 7.9,
2.7 Hz, H-2), 1.60 (m, 8H, H-b), 1.30 (m, 38H, CH2),
0.90 (t, 12H, J = 8.4 Hz, CH3). 13C NMR (400 MHz,
CDCl3): d (ppm): 98.0 (C-1), 93.8 (C-10), 86.0, 81.8,
80.9, 79.7, 76.8, 73.2, 70.1, 69.7, 62.7, 56.0 (OCH3).
MS(ESI): m/z: 1193.02 [M+Na]+.
3.1.15. Methyl 2,3-di-O-tetradecyl-4-O-p-methoxybenzyl-
6-O-(2,3-di-O-tetradecyl-4,6-di-O-acetyl-b-D-glucopyr-
anosyl)-a-D-glucopyranoside (17). To a solution of
monosaccharide 13 (100 mg, 0.15 mmol) in dry CH2Cl2
(6 mL), trichloroacetonitrile (60 lL, 0.6 mmol) and cae-
sium carbonate (25 mg, 0.075 mmol) were added and the
resulting mixture was stirred for 1.5 h at room tempera-
ture. AcOEt (100 mL) was added and the organic layer
was washed with NaHCO3 (satd aq soln, 100 mL), brine
(3 · 100 mL), dried over sodium sulfate and evaporated.
To a solution of the crude trichloroacetimidate 16
(116 mg, 0.14 mmol) and glycosyl donor 5 (98 mg,
0.14 mmol) in dry CH2Cl2 (6 mL), trimethylsilyl triflate
(10 lL) was added at ꢀ30 ꢁC and the resulting mixture
was stirred for 2 h. Ethyl acetate was added and the
organic layer was washed with NaHCO3 (satd aq
soln, 50 mL), brine (3 · 50 mL), then dried over sodium
sulfate and evaporated, to yield disaccharide 17 as a
mixture of a and b isomers. Flash column chromatogra-
phy on silica gel (petroleum ether/AcOEt 9.5:0.5) affor-
ded 17 as a mixture of a and b isomers (102 mg, 55%).
Rf = 0,45 (petroleum ether/AcOEt 9:1). MALDI-TOF
(DHB): m/z: 1346.8 [M+H]+, 1370.0 [M+Na]+,
3.1.17. Methyl 2,3-di-O-tetradecyl-6-O-(2,3-di-O-tetrade-
cyl-b-D-glucopyranosyl)-a-D-glucopyranoside (2). Disac-
charide 18 (pure b isomer, 20 mg, 0.014 mmol) was
dissolved in dry methanol (3 mL) under argon atmo-
sphere. A freshly prepared solution of sodium methano-
ate (1 M, 20 lL, 0.02 mmol) was added dropwise and
the resulting mixture was stirred for 20 min, after which
time IRA 120 H+ was added until pH = 7. The resin was
filtered and the solvent was evaporated. Flash column
chromatography (petroleum ether/AcOEt 7:3) afforded
1 (16 mg, quant.). Compound 2: Rf = 0.30 (petroleum
ether/AcOEt 8:2). 1H NMR (400 MHz, CDCl3): d
(ppm): 4.80 (d, 1H, J = 3.3 Hz, H-1), 4.40 (d, 1H,
J = 7.5 Hz, H-10), 4.10 (bd, 1H, J = 10.0 Hz, H-6a),
4.0–3.3 (m, 16H, H-30, H-40, H-50, 2H-60, H-4, H-5, H-
6b, 8H-a), 3.43 (s, 3H, OCH3), 3.29 (dd, 1H, J = 8.4,
3.0 Hz, H-2), 3.20 (t, 1H, J = 9.0 Hz, H-3), 3.15 (t, 1H,
J = 7.9 Hz, H-20), 1.60 (m, 8H, H-b), 1.30 (m, 38H,
CH2), 0.90 (t, 12H, J = 8.4 Hz, CH3). 13C NMR
(400 MHz, CDCl3): d (ppm): 104.5 (C-10), 97.8 (C-1),
85.0 (C-3), 83.2 (C-20), 82.0, 81.0 (C-2), 76.8, 75.2 (C-
a), 75.1 (C-a), 73.8 (C-a), 69.5 (C-6), 62.7 (C-60), 55.4
(OCH3). MS(ESI): m/z: 1163.99 [M+Na]+.
1
[a]D = +21 (c 0.5 in CHCl3); The H NMR spectrum is
composed by the signals of both a and b anomers in
1:3 ratio, we report here the diagnostic signals for each
isomer: 17-a: d = 5.10 (d, 0.25H, J = 3.0 Hz, H-1), 4.72
(d, 0.25H, J = 2.8 Hz, H-10); 17-b: d = 4.79 (d, 0.75H,
J = 3.5 Hz, H-1), 4.26 (d, 0.75H, J = 7.9 Hz, H-10),
3.1.18. Chemoselective coupling of 7 and 9. Equimolar
(50 lmol) amounts of the two monomers were dissolved
in THF (0.25 mL). Acetic acid (0.4 mL) was added and
the mixture was stirred for 6 days.

F. Peri et al. / Bioorg. Med. Chem. 14 (2006) 190–199
199
6. Westphal, O.; Luderitz, O. Angew. Chem. 1954, 66, 407–
417.
3.2. Biological assays
7. Rietschel, E. T.; Kirikae, T.; Schade, F. U.; Mamat, U.;
Schmidt, G.; Loppnow, H.; Ulmer, A. J.; Zahringer, U.;
Seydel, U.; Di Padova, F.; Schreier, M.; Brade, H. FASEB
J. 1994, 8, 217–225.
8. Hulevitch, R. J.; Tobias, P. S. Annu. Rev. Immunol. 1995,
13, 437–457.
9. Shimazu, R. J. Exp. Med. 1999, 187, 1777–1782.
10. Visintin, A.; Mazzoni, A.; Spitzer, J. A.; Segal, D. M.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 12156–12161.
11. Beutler, B. Curr. Opin. Immunol. 2000, 12, 20–26.
12. Poltorak, A.; Ricciardi-Castagnoli, P.; Citterio, S.; Beu-
tler, B. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2163–2167.
13. Lien, E.; Chow, J. C.; Hawkins, L. D.; McGuinness, P. D.;
Miyake, K.; Espevik, T.; Gusovsky, F.; Golenbock, D. T.
J. Biol. Chem. 2001, 19, 1873–1880.
14. Netea, M. G.; van Deuren, M.; Kullberg, B. J.; Cavaillon,
J.-M.; Vander Meer, J. W. M. Trends Immunol. 2002, 23,
135–139.
15. Seydel, U. Chem. Immunol. 2000, 74, 5–24.
16. Loppnow, H. Infect. Immun. 1990, 58, 3743–3750.
17. Ingalls, R. R.; Monks, B. G.; Savedra, R. J.; Christ, W. J.;
Delude, R. L.; Medvedev, A. E.; Espevik, T.; Golenbock,
D. T. J. Immunol. 1998, 161, 5413–5420.
3.2.1. Culture medium and reagents. MT2 cells were cul-
tured in IscoveÕs modified DulbeccoÕs medium (IMDM,
Sigma, St. Louis, MO) containing 10% heat-inactivated
fetal bovine serum (Gibco-BRL, Gaithersburg, MD),
100 IU of penicillin, 100 lg/mL of streptomycin, 2 mM
L-glutamine (all from Sigma Chem. St. Louis, MO)
and 50 lM b-mercaptoethanol.
LPS (E. coli 026:B6, used at 10 lg/mL) and lipid A
were obtained from Sigma. Oligo CpG (TCCATGA
CGTTCCTGATGCT) was purchased from Primm
(Milan, Italy) and used at a concentration of 1 lM.
Poly I:C (used at 20 lg/mL) was purchased from
Amersham.
3.2.2. Determination of nitrite accumulation. NO2ꢀ accu-
mulation was measured as NO production by Greiss
assays, with a sodium nitrite standard.
3.2.3. TNFa ELISA. TNFa ELISA was performed using
the DuoSet kit (R&D, Minneapolis, MN) and following
the manufacturer recommendations.
18. Demchenko, A. V.; Wolfert, M. A.; Santhanam, B.;
Moore, J. N.; Boons, G.-J. J. Am. Chem. Soc. 2003, 125,
6103–6112.
19. pKa values of about 13 units have been calculated for
similar N(OMe) disaccharides (published in Ref. 20) using
Advanced Chemistry Development (ACD/Labs) Software
Solaris V4.76 (1994-2005 ACD/Labs).
20. Peri, F.; Deutman, A.; La Ferla, B.; Nicotra, F. Chem.
Commun. 2002, 1504–1505.
Acknowledgments
ˇ
We thank Professor Ladislav Petrus for helpful discus-
sion. Financial support for this work was provided by
the European CommunityÕs Human Potential Program
under contract HPRN-CT-2002-00173 and in part by
the MURST (FIRB project, Design, preparation and
biological evaluation of new organic molecules as poten-
tial innovative drugs).
´
´
21. Peri, F.; Jimenez-Barbero, J.; Garcıa-Aparicio, V.; Tvar-
oska, I.; Nicotra, F. Chem. Eur. J. 2004, 10, 1433–1444.
22. As observed for the synthesis of N(OMe) oligosaccharide
mimetics as well as of N(OMe) glycopeptides, the chemo-
selective ligation methodology based on the condensation
between the methoxyamino and the anomeric carbon of a
reducing sugar is very sensitive to steric effects.
Supplementary data
23. Renaudet, O.; Dumy, P. Tetrahedron 2002, 58, 2127–2135.
24. Alberati-Giani, D.; Ricciardi-Castagnoli, P.; Kohler, C.;
Cesura, A. M. J. Neurochem. 1996, 66, 996–1004.
25. Altenschmidt, U.; Ricciardi-Castagnoli, P.; Modolell, M.;
Otto, H.; Wiesmuller, K. H.; Jung, G.; Simon, M. M.
Immunol. Lett. 1996, 50, 41–49.
Supplementary data associated with this article can be
found in the online version at doi:10.1016/
j.bmc.2005.08.047.
26. Hemmi, H.; Takeuchi, O.; Kawai, T.; Kaisho, T.; Sato, S.;
Sanjo, H.; Matsumoto, M.; Hoshino, K.; Wagner, H.;
Takeda, K.; Akira, S. Nature 2000, 408, 740–745.
27. Alexopoulou, L.; Holt, A. C.; Medzhitov, R.; Flavell, R.
A. Nature 2001, 413, 732–738.
References and notes
1. Roberts, F. J.; Geere, I. W.; Coldman, A. Rev. Infect Dis.
1991, 13, 34–46.
2. Pittet, D.; Tarara, D.; Wenzel, R. P. J. Am. Med. Assoc.
1994, 271, 1589–1601.
28. Oikawa, Y.; Nishi, T.; Yonemitsu, O. Tetrahedron Lett.
1983, 24, 4037–4040.
3. Morrison, D. C.; Ryan, J. L. Annu. Rev. Med. 1987, 38,
417–432.
29. Chen, J.; Dorman, G.; Prestwich, G.; Glenn, D. J. Org.
Chem. 1996, 61, 393–397.
4. Hurley, J. C. Clin. Microbiol. 1994, 32, 2120–2127.
5. Hurley, J. C. Clin. Microbiol. Rev. 1995, 8, 268–292.
30. Barath, M.; Petrusova, M.; Bystricky, S.; Krˇen, V.; Petrusˇ,
L. Chem. Pap. 2003, 57, 125–130.