Synthesis of a dimeric monosaccharide lipid A mimic and its synergistic effect on the immunostimulatory activity of lipopolysaccharide

Article abstract of DOI:10.1016/j.carres.2011.05.018

Bacterial endotoxin lipopolysaccharide (LPS) is a potent immune stimulant, with the recognition of LPS and its active principal lipid A mediated by the Toll-like receptor 4 (TLR4)/MD-2 receptor complex. Due to the broad downstream implications of TLR4-med

Full text of DOI:10.1016/j.carres.2011.05.018

Carbohydrate Research 346 (2011) 1705–1713
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of a dimeric monosaccharide lipid A mimic and its synergistic
effect on the immunostimulatory activity of lipopolysaccharide
Jordan D. Lewicky a, Marina Ulanova b, Zi-Hua Jiang a,
⇑
a Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, Ontario, Canada P7B 5E1
b Medical Sciences Division, Northern Ontario School of Medicine, 955 Oliver Road, Thunder Bay, Ontario, Canada P7B 5E1
a r t i c l e i n f o
a b s t r a c t
Article history:
Bacterial endotoxin lipopolysaccharide (LPS) is a potent immune stimulant, with the recognition of LPS
and its active principal lipid A mediated by the Toll-like receptor 4 (TLR4)/MD-2 receptor complex.
Due to the broad downstream implications of TLR4-mediated signalling, TLR4 ligands show great poten-
tial for immunotherapeutic manipulations. In this paper a dimeric monosaccharide lipid A mimic (3) has
been designed as a potential TLR4 ligand. The chemical synthesis and the preliminary biological studies
are described. Compound 3 shows a significant synergistic effect on LPS-induced ICAM-1 expression in
human monocytic THP-1 cells.
Received 14 March 2011
Received in revised form 29 April 2011
Accepted 15 May 2011
Available online 26 May 2011
Keywords:
Lipid A mimic
Toll-like receptor 4
Synthesis
Ó 2011 Elsevier Ltd. All rights reserved.
ICAM-1 expression
Synergistic effect
1. Introduction
chain is exposed to the surface of MD-2, forming a hydrophobic
interaction with the conserved phenylalanines of TLR4.
Lipopolysaccharide (LPS) is a component of the outer mem-
brane of Gram-negative bacteria, and is perhaps the most effective
trigger of the innate immune response.1 The active principal
responsible for most of the biological activities of LPS lies in the
terminal lipophilic moiety of the molecule, known as lipid A.2,3
The core structure of lipid A is conserved regardless of bacterial
Immediately distal to TLR4 activation are two intracellular signal-
ing cascades, each dependent on different adaptor proteins. A
MyD88-dependent pathway leads to the production of inflamma-
tory cytokines such as tumor necrosis factor-
a (TNF-a),
interleukin-1 (IL-1) and IL-6, whereas a TRIF-dependent pathway
results in interferon-b (IFN-b) and nitric oxide production.15,16
Given its broad downstream implications, the TLR4 signaling
pathway has attracted considerable attention for potential immu-
notherapeutic manipulation both in terms of vaccine adju-
vants,17,19 as well as anti-sepsis treatments.8,18 Due to the
potential toxicity of the TLR4 induced inflammatory response, a
great deal of work has focused on the structure–activity profile
of lipid A molecules in order to harness potential beneficial immu-
nostimulatory properties from those pro-inflammatory character-
istics.8,19 For example, the removal of the anomeric phosphoryl
group of lipid A could significantly reduce its toxicity while still
retaining its immunostimulatory activity,20 as exemplified by the
recently approved vaccine adjuvant MPLÒ which is a monophos-
phoryl lipid A product derived from bacterial LPS.21 On the other
hand, the removal of some fatty acyl chains can also dramatically
affect the activity profile of lipid A molecules. The lipid A derivative
OM-174 (Chart 1), isolated from partially degraded E. coli LPS, car-
ries three fatty acyl chains and is an effective immunoadjuvant
with potent antitumor activity and very low toxicity.22,23 Previ-
ously, we have reported the synthesis of several lipid A-based
molecules as inmunostimulatory vaccine adjuvants.24–26 Herein,
we report a dimeric monosaccharide framework42 (2 and 3, Chart
species, and consists of a b-(1?6) glycosidically linked di-D-gluco-
samine backbone bisphosphorylated at the 1-O- and 40-O-posi-
tion,4,5 for example, Escherichia coli lipid A (1, Chart 1). This
disaccharide core is acylated by up to seven lipid chains through
both ester and amide linkages, with differences in the number,
length, and composition of said chains accounting for the highly
variable in vitro and in vivo host responses observed among differ-
ent bacterial species.6–8
Toll-like receptor 4 (TLR4) has been identified as the receptor of
bacterial LPS and lipid A,9 with significant effort having gone into
the precise delineation of the molecular events occurring upon its
activation. TLR4 is expressed on the cell surface as a stable complex
with obligate accessory protein MD-2.10–12 Recently, the crystal
structures of TLR4/MD-2 complexes with bound agonist LPS and
antagonist lipid IVa, the biosynthetic precursor of E. coli lipid A with
four acyl chains, have been resolved by Park et al.13 and Ohto et
al.,14 respectively. Five of the six lipid chains of LPS are buried deep
inside the large hydrophobic pocket in MD-2 and the remaining
⇑
Corresponding author. Tel.: +1 807 766 7171; fax: +1 807 346 7775.
E-mail address: zjiang@lakeheadu.ca (Z.-H. Jiang).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.05.018

1706
J. D. Lewicky et al. / Carbohydrate Research 346 (2011) 1705–1713
OH
OH
O
O
P
O
P
OH
O
O
O
O
HO
RO
HO
O
OH
O
OR
HO
P
O
R1O
O
O
RO
OH
O
HO
HO
NH
NH
O
P
NH
O
NH
O
O
O
OH
O
O
O
OH
O
O
HO
O
O
1 R = (R)-3-hydroxytetradecanoyl
R1 = (R)-3-tetradecanoyloxytetradecanoyl
2 R = (R)-3-hydroxytetradecanoyl
3 R = H
OM-174 R = R1 = H
Chart 1. Structures of E. coli lipid A (1), OM-174 and the dimeric monosaccharide lipid A mimics 2 and 3.
1) as an alternative to the archetypical disaccharide framework of
lipid A molecules. Hexa-acylated analog (2) has been designed to
mimic E. coli lipid A (1),4,5 while the tetra-acylated analog (3)
resembles those lipid A structures with fewer acyl chains, such
as lipid IVa27 and OM-174.22 The chemical synthesis and prelimin-
ary biological investigation of the dimeric monosaccharide lipid A
mimic 3 is described in this paper.
a two step procedure in which isomerization of the allyl double
bond is first brought about using the iridium complex, [bis(meth-
yldiphenylphosphine)](1,5-cyclooctadiene) iridium(I) (hexafluoro-
phosphate),34 followed by the hydrolysis of the isomerized moiety
in the presence of N-bromosuccinimide (NBS). Finally, trichloro-
acetimidate glycosyl donor 9 was obtained as a single
a-isomer
(1H NMR, d 6.43, d, J 4.0 Hz, H-1) in 81% yield via the treatment
of 8 with trichloroacetonitrile and a catalytic amount of sodium
hydride in dichloromethane.
2. Results and discussion
The desired dimeric backbone in compound 11 was envisioned
to be formed through a one step, di-glycosylation reaction between
ethylene glycol and donor 9 (Scheme 2). The trimethylsilyl trifluo-
romethanesulfonate (TMSOTf) catalyzed glycosylation reaction of
9 with ethylene glycol in solely dichloromethane solvent produced
a complex profile of multiple reaction products. As such, both di-
meric 11 and the mono-glycosylated intermediate 10 were isolated
in poor yields, the cause of which was later attributed to poor gly-
col solubility in dichloromethane. This solubility issue was allevi-
ated via the inclusion of THF in the glycosylation reaction
solvent, yet conditions could not be optimized to ensure complete
transformation of 10 into 11, with the best yield obtained for 11
being 47%. The newly formed b-linkage and symmetry of the dimer
was confirmed by 1H NMR data (d 4.76, d, J = 8.0 Hz, H-1). How-
ever, significant chromatographic difficulties were encountered,
thus preventing the isolation of sufficient quantities of pure 10
or 11 to continue the synthesis.
2.1. Syntheses of lipid A mimic 3
Preparation of the dimeric monosaccharide backbone began
with the synthesis of glycosylation donor 9 (Scheme 1). Readily
available glucosamine derivative 428,29 was chosen as the synthetic
starting point, with the 2,2,2-trichloroethoxycarbonyl (Troc)
amine-protecting group being employed for its widely known
b-glycoside directing capability through neighboring group
participation.24,30,31 The protection of the 3-O-position as the 9-flu-
roenylmethoxycarbonyl (Fmoc) ester was utilized to allow for
selective deprotection and incorporation of acyl lipid functional-
ities at a later point in the synthesis. This transformation was
brought about through reaction of 4 with 9-fluorenylmethyl chlo-
roformate (Fmoc-Cl) in the presence of 4-N,N-dimethylaminopyri-
dine (DMAP) to furnish 5 in 89% yield. Regioselective ring opening
of the 4,6-di-O-benzylidene group in 5 via treatment with sodium
cyanoborohydride (NaBH3CN) and an HCl(g)-infused diethyl ether
solution at 0 °C32 gave free 4-OH derivative 6 in 85% yield. Reaction
of 6 with phosphoramidite and tetrazole, followed by an m-chloro-
peroxybenzoic acid (m-CPBA) oxidation33 produced phosphate 7.
Compound 7 was converted to reducing end derivative 8 through
Given the difficulties encountered in forming the dimeric
monosaccharide backbone via the one step approach, an alternate
synthetic route was employed. Monosaccharide 10 was first pre-
pared, from which a further glycosylation with acceptor 9 would
yield the desired 11 (Scheme 3). As such, the ethylene glycol was
OBn
O
OBn
Ph
O
O
O
O
O
(BnO)2PO
b
d
RO
FmocO
X
RO
FmocO
NH
NH
Troc
OAll
a
NH
Troc
Y
Troc
6 R = H
7 R = P(O)(OBn)2
OAll
4 R = H
8 X, Y = H, OH
e
c
5 R = Fmoc
9 X = H, Y = OC(NH)CCl3
Scheme 1. Reagents and conditions: (a) Fmoc-Cl, DMAP, pyridine 0 °C, 89%; (b) NaBH3CN, HCl(g)–Et2O, THF, 0 °C, 85%; (c) (i) (BnO)2PN(iPr)2, 5-Ph-tetrazole, CH2Cl2; (ii) m-
CPBA, CH2Cl2, ꢀ20 °C, 98%; (d) (i) [bis(methyldiphenylphosphine)](1,5-cyclooctadiene) iridium(I) hexafluorophosphate, THF, rt; (ii) NBS, THF–H2O, 79%; (e) Cl3CCN, NaH,
CH2Cl2, rt, 81%.

J. D. Lewicky et al. / Carbohydrate Research 346 (2011) 1705–1713
HOCH2CH2OH
a or b
1707
9
+
11
a
O
O
OBn
O
O
OBn
O
OBn
O
O
O
OP(OBn)2
OFmoc
(BnO)2PO
FmocO
(BnO)2PO
FmocO
O
O
10
OH
NH
NH
R
R
NH
+
Troc
14 R = H
n-C13H27
OBn
O
O
OBn
O
O
b
O
O
OP(OBn)2
OFmoc
(BnO)2PO
FmocO
CO
O
15 R =
n-C11H23
O
NH
NH
Troc
Troc
n-C13H27
O
O
11
c
n-C11H23
OH
Scheme 2. Reagents and conditions: (a) TMSOTf, CH2Cl2, rt, 20% for 10 and 10% for
11; (b) TMSOTf, CH2Cl2–THF, rt, 11% for 10 and 47% for 11.
3
16
Scheme 4. Reagents and conditions: (a) Zn dust, THF–HOAc, rt; (b) 16, DCC, CH2Cl2,
rt, 65%; (c) (i) H2, Pd/C, THF–HOAc, rt; (ii) piperidine, THF, rt, 65% for two steps.
selectively protected as mono-silyl ether 12 through reaction with
the tert-butyldimethylsilyl chloride (TBDMS-Cl) in the presence of
imidazole. The TMSOTf catalyzed glycosylation with imidate 9 fur-
nished protected monosaccharide 13 in 81% yield. Subsequent
deprotection in the presence of tetrabutylammonium fluoride
(TBAF) and acetic acid provided pure monosaccharide 10 in 73%
yield. Degradation of 13 was observed when the reaction was car-
ried out without the addition of acetic acid. Compound 10 was
then further glycosylated with imidate 9 to provide the desired
11 in 83% yield.
Removal of the N-Troc protecting group in 11 by treatment with
zinc dust in acetic acid–tetrahydrofuran afforded di-amine 14,
which was coupled with lipid acid 1634 (Scheme 4) in the presence
of N,N0-dicyclohexylcarbodiimide (DCC) to provide 15 in 65%. For
the preparation of lipid A mimic 3, compound 15 was subjected
to a Pd/C-catalyzed hydrogenation under atmosphere pressure in
THF–HOAc (4:1) (Scheme 4). Although Fmoc protection group
can in general by cleaved via catalytic hydrogenation,23 the cleav-
age of Fmoc in 15 was incomplete even after three days of hydro-
genation reaction. Therefore, the initial hydrogenolysis product of
15 was further subjected to a simple piperidine treatment to give
3, which was structurally confirmed by high resolution MALDI-
MS analysis. 1H NMR spectra of 3 were acquired in different sol-
vents such as DMSO-d6 and CD3OD–CDCl3 (1:1); however, due to
self-aggregation of the molecule, no visible signals were detected
in the carbohydrate region (see Supplementary data).
tial deprotection attempt with diazabicylo[5,4,0]undec-7-ene
(DBU) as a base in THF formed an inseparable mixture of three
products, presumably 17–19 (Scheme 5). The 1H NMR spectrum
of the mixture showed four sets of doublets of amide-NH protons
in an approximate ratio of 5:5:2:4 (Fig. 1). Compound 17 and 19
each gives one NH signal due to their symmetric nature, whereas
the asymmetric 18 gives two NH signals. The formation of 17–19
was rationalized through the migration of the phosphotriester
group at the 4-O- and/or 40-O-position to 3-O- and/or 30-O-position,
respectively, via an SN2(P) mechanism forming a cyclic, penta-va-
lent oxyphosphorane intermediate.36 Additional attempts at
Fmoc-deprotection were undertaken, the likes of which involved
both the inclusion of MeOH as a protic solvent and TBAF as a base,
however similar results were noted.
Given the apparent lability of the 4-O-phosphotriester group,
further attempts at Fmoc-deprotection and the preparation of
15
a, b, or c
OBn
O
O
OBn
O
O
OP(OBn)2
OH
(BnO)2PO
O
In order to introduce additional fatty acyl chains onto the 3- and
30-O-position for the preparation of structures such as 2, Fmoc-
deprotection in 15 was attempted. Fmoc cleavage is typically
achieved through a base-promoted b-elimination reaction. The ini-
O
HO
NH
NH
R
R
17
+
OBn
O
OBn
O
O
O
OH
(BnO)2PO
O
a
OP(OBn)2
O
HO
HOCH2CH2OH
12
HOCH2CH2OTBDMS
NH
NH
R
R
18
9
b
+
OBn
O
OBn
O
O
OBn
O
O
(BnO)2PO
FmocO
OH
HO
(BnO)2PO
O
O
OR
OP(OBn)2
O
NH
O
NH
NH
Troc
R
R
19
O
13 R = TBDMS
10 R = H
c
9
n-C13H27
O
11
CO
R =
n-C11H23
b
Scheme 3. Reagents and conditions: (a) TBDMS-Cl, DMF, imidazole, 0 °C, 70%; (b)
TMSOTf, CH2Cl2, 81% for 13 and 83% for 11; (c) TBAF, HOAc, THF, 0 °C, 73%.
Scheme 5. Reagents and conditions: (a) DBU, THF, rt; (b) DBU, THF–MeOH, rt; (c)
TBAF, THF–MeOH, 0 °C to rt.

1708
J. D. Lewicky et al. / Carbohydrate Research 346 (2011) 1705–1713
Figure 1. Part of 1H NMR spectrum for the Fmoc-deprotected product, the mixture of 17–19.
compound 2 were abandoned. In order to circumvent this issue, a
different synthetic strategy would need to be employed, one in
which the 3-O-acyl installation occurs prior to the phosphate
group installation or a different 3-O-protecting group is used.
structure of lipid A. Since compound 3 is antagonistically inactive
against LPS and there are other TLRs expressed by THP-1 cells,37
it is likely that 3 may not compete directly with LPS binding and
its synergistic effect may involve receptors other than TLR4. Earlier
studies have shown that TLR7, TLR8 and TLR9 cooperate with TLR4
to synergistically induce cytokine secretion in murine and human
dendritic cells.46,47 Further studies are needed to demonstrate
the full immunological activity of compound 3 and the mechanistic
details of its synergistic effect on LPS stimulation.
In conclusion, we have synthesized a dimeric monosaccharide
lipid A mimic 3 linked through an ethylene moiety as a potential
TLR4 ligand. Preliminary biological studies indicate that compound
3 is agonistically inactive in terms of stimulating ICAM-1 expres-
sion in human monocytic THP-1 cells. Compound 3 is also antago-
nistically inactive against LPS, yet exerts significant synergistic
effect on LPS-induced ICAM-1 expression by THP-1 cells. Studies
on the synthesis of 3,30-di-O-acylated analogs such as structure 2
and their immunological properties are in progress.
2.2. Biological evaluation
The activation of TLR4 by specific ligands leads to the release of
pro-inflammatory cytokines and cellular adhesion molecules.
Studies have shown that human monocytic THP-1 cells express
TLR4 and other TLRs.37 It is well established that LPS, a TLR4 ago-
nist, induces the expression of intercellular adhesion molecule-1
(ICAM-1) by THP-1 cells.38,39 Here we have evaluated the immuno-
stimulatory/modulatory activity of compound 3 in its capacity of
affecting the expression level of ICAM-1 by THP-1 cells. Basically,
compound 3 alone does not show any stimulatory effect on
ICAM-1 expression when THP-1 cells are treated with 3 at a con-
centration up to 32 lM. In an attempt to study its antagonistic
activity against LPS, we have found that 3 shows significant syner-
gistic effect on the LPS-induced expression of ICAM-1 by THP-1
cells (Fig. 2). Up to a 66% increase in ICAM-1 expression is observed
3. Experimental
when THP-1 cells are treated with compound 3 (at 16.0
prior to stimulation by LPS (at 0.1 g/mL). Further increase in the
concentration of 3 (at 32 M) resulted in a dramatic decrease in
lM) for 1 h
3.1. General methods
l
l
All air and moisture sensitive reactions were performed under
nitrogen atmosphere. All commercial reagents were used as sup-
plied. Anhydrous pyridine was distilled over potassium hydroxide
and dichloromethane over calcium hydride. Anhydrous N,N-
dimethylformamide (DMF) was purchased from Aldrich. ACS grade
solvents were purchased from Fisher Scientific and used for chro-
the expression level of ICAM-1. The decrease in ICAM-1 expression
at a higher concentration of 3 may be the result of the decreased
viability of THP-1 cells rather than the inhibitory effect of 3 on
LPS stimulation. At a 32 lM concentration of 3, an approximately
40% decrease in cell viability was observed as measured through
the exclusion of Trypan Blue, while no significant effect on cell via-
bility was observed for any of the lower concentrations of 3 tested
(data not shown).
matography without distillation. TLC plates (Silica Gel 60 F254
,
thickness 0.25 mm) and Silica Gel 60 (40–63 m) for flash column
l
chromatography were purchased from SILICYCLE Inc., Canada. 1H
and 13C NMR spectra were recorded on a Varian Unity Inova
500 MHz spectrometer. Tetramethylsilane (TMS, d 0.00 ppm) or
solvent peaks were used as internal standards for 1H and 13C
NMR spectra. The chemical shifts were given in ppm and coupling
constants in Hertz indicated to a resolution of 0.5 Hz. Multiplicity
of proton signals is indicated as follows: s (singlet), d (doublet), dd
(double doublet), t (triplet), q (quartet), m (multiplet), br (broad).
Structural assignments were made using standard gCOSY and
gHSQC. MALDI mass spectra were measured on an Applied Biosys-
tems 4700 TOF instrument at the University of Western Ontario,
Canada, whereas ESI mass spectra were measured on the Applied
Biosystems Mariner Biospectrometry Workstation at the Univer-
sity of Alberta, Canada. Optical rotations were measured with
Perkin–Elmer 343 Polarimeter at 22 °C. Elemental analyses were
carried out on a CEC (SCP) 240-XA Analyzer instrument by Lake-
head University Instrumentation Laboratory (LUIL).
ICAM-1 expression is NF-jB-dependent and induction of high
levels of ICAM-1 occurs in response to various inflammatory medi-
ators, including bacterial LPS, and pro-inflammatory cytokines,
such as tumor necrosis factor-
a (TNF-a), interleukin-1b, and c-
interferon (
c
-IFN).40,41 It will be interesting to measure a range
of cytokines induced by 3. LPS- or lipid A-induced cellular activa-
tion through TLR4/MD-2 is complex. In recent years, a number of
studies have addressed the differential induction of innate immune
responses by synthetic lipid A derivatives,19,43 most notably from
Boons’ group.35,44,45 Their studies have confirmed that structural
features such as the presence of 3-deoxy-D-manno-octulosomic
acid moiety, anomeric phosphate, lipid length, and acylation
pattern are important for pro-inflammatory activity. Boons and
co-workers45 have also reported that the dosage of lipid A and
the differentiation state of the immune cells (macrophages and
dendritic cells) are important determinants of the pattern of cyto-
kine secretion which, unexpectedly, is not modulated by the

J. D. Lewicky et al. / Carbohydrate Research 346 (2011) 1705–1713
1709
140.00
120.00
100.00
80.00
60.00
40.00
20.00
0.00
3
+ LPS
0.1
1
2
4
8
16
32
LPS
Cells Alone
Concentration (µM)
Figure 2. ICAM-1 expression in human monocytic THP-1 cells induced by 3 plus LPS. THP-1 cells were incubated for 1 h with increasing concentrations of 3, and then
stimulated with 0.1 g/mL LPS for a further 18 h. The resulting ICAM-1 expression was measured via immunostaining and flow cytometry analysis. The results are expressed
as the mean fluorescence intensity and shown as the average of three separate experiments.
l
3.2. Allyl 4,6-O-benzylidene-2-deoxy-3-O-(9-
fluorenylmethoxycarbonyl)-2-(2,2,2-
trichloroethoxycarbonylamino)-a-D-glucopyranoside (5)
extracted with EtOAc (3 ꢂ 200 mL), and the combined organic
phase was washed with a saturated sodium chloride solution
(150 mL), dried over Na2SO4, and concentrated. The resulting resi-
due was purified by flash chromatography (hexane/EtOAc,
3.5:1?2:1) to give 6 as a white solid (6.34 g, 85%). Rf 0.34 (hex-
To a cooled solution (ice water bath) of 4 (8.05 g, 16.68 mmol)
and DMAP (204 mg, 1.67 mmol) in dry pyridine (35 mL), 9-fluore-
nylmethoxychloroformate (6.50 g, 25.00 mmol) was added. After
stirring at room temperature for 4 h, the reaction was quenched
with MeOH. Pyridine was removed in vacuo via co-distillation with
toluene and the residue was purified by flash column chromatog-
raphy (hexane/EtOAc, 3:1) to give 5 (10.46 g, 89%) as a white solid.
ane/EtOAc, 2.5:1), ½a D22
ꢁ
+55.4 (c 1.0, CHCl3); 1H NMR (500 MHz,
CDCl3): d 2.76 (br s, 1H, OH), 3.33 (dd, 1H, J 10.0, 4.0 Hz, H-6b),
3.80–3.87 (m, 2H, H-5, H-6a), 3.92 (dd, 1H, J 10.0, 10.0 Hz, H-4),
4.01 (dd, 1H, J 12.8, 6.5 Hz, Fmoc-CHH), 4.09 (ddd, 1H, J 10.0,
10.0, 3.5 Hz, H-2), 4.18–4.26 (m, 2H, Fmoc-CH, Fmoc-CHH), 4.35–
4.42 (m, 2H, OCH2CH@CH2), 4.56–4.66 (m, 4H, Troc-Ha, Troc-Hb,
Ph-CH2), 4.93 (d, 1H, J 3.5 Hz, H-1), 5.02 (dd, 1H, J 10.0, 10.0 Hz,
H-3), 5.21–5.36 (m, 3H, NH, OCH2CH@CH2), 5.86–5.92 (m, 1H,
OCH2CH@CH2), 7.30–7.77 (m, 13H, Ar-H); 13C NMR (125 MHz,
CDCl3): d 46.47 (Fmoc-CH), 53.80 (C-2), 68.48 (Fmoc-CH2), 69.43
(C-6), 69.89 (C-5), 70.24 (C-4), 70.43 (OCH2CH@CH2), 73.64 (Ph-
CH2), 74.46 (Troc-CH2), 77.83 (C-3), 95.24 (Troc-CCl3), 96.34 (C-
1), 118.34 (OCH2CH@CH2), 120.01 (CH-Ar), 125.18 (CH-Ar),
127.19 (CH-Ar), 127.64 (CH-Ar), 127.84 (CH-Ar), 127.87 (CH-Ar),
128.44 (CH-Ar), 133.09 (OCH2CH@CH2), 137.58 (C-Ar), 141.19 (C-
Ar), 143.05 (C-Ar), 143.17 (C-Ar), 154.20 (C@O), 155.74 (C@O).
Anal. Calcd for C34H34Cl3NO9: C, 57.76; H, 4.85; N, 1.98. Found: C,
57.52; H, 4.73; N, 2.01.
Rf 0.31 (hexane/EtOAc, 3.5:1); ½a D22
ꢁ
+44.7 (c 1.0, CHCl3); 1H NMR
(500 MHz, CDCl3): d 3.79–3.85 (m, 2H, H-6a, H-6b), 3.95–4.04 (m,
2H, H-4, H-5), 4.17–4.25 (m, 3H, H-2, Fmoc-CH, OCHHCH@CH2),
4.30–4.35 (m, 3H, Fmoc-CH2, OCHHCH@CH2), 4.53 (d, 1H,
J
12.0 Hz, Troc-Ha), 4.64 (d, 1H, J 12.0 Hz, Troc-Hb), 4.96 (d, 1H, J
3.5 Hz, H-1), 5.21–5.26 (m, 2H, OCH2CH@CHH, H-3), 5.29–5.33
(m, 1H, OCH2CH@CHH), 5.43 (d, 1H, J 10.0 Hz, NH), 5.57 (s, 1H,
Ph-CH), 5.85–5.93 (m, 1H, OCH2CH@CH2), 7.18–7.75 (m, 13H, Ar-
H); 13C NMR (125 MHz, CDCl3): d 46.65 (Fmoc-CH), 54.90 (C-2),
63.24 (C-5), 68.96 (C-6), 69.02 (OCH2CH@CH2), 70.69 (Fmoc-CH2),
74.42 (C-3), 74.77 (Troc-CH2), 79.13 (C-4), 95.45 (Troc-CCl3),
97.27 (C-1), 101.86 (Ph-CH), 118.93 (OCH2CH@CH2), 120.25 (CH-
Ar), 125.37 (CH-Ar), 125.44 (CH-Ar), 126.41 (CH-Ar), 127.42 (CH-
Ar), 128.10 (CH-Ar), 128.45 (CH-Ar), 129.35 (CH-Ar), 133.16
(OCH2CH@CH2), 137.03 (C-Ar), 141.39 (C-Ar), 141.42 (C-Ar),
143.32 (C-Ar), 143.42 (C-Ar), 154.49 (C@O), 155.37 (C@O). Anal.
Calcd for C34H32Cl3NO9: C, 57.93; H, 4.58; N, 1.99. Found: C,
57.81; H, 4.52; N, 1.73.
3.4. Allyl 6-O-benzyl-2-deoxy-4-O-(di-O-benzylphosphono)-3-O
-(9-fluorenylmethoxycarbonyl)-2-(2,2,2-
trichloroethoxycarbonylamino)-a-D-glucopyranoside (7)
To a solution of 6 (6.34 g, 8.96 mmol) in dry CH2Cl2 (50 mL), 5-
phenyltetrazole (2.30 g, 15.69 mmol) and dibenzyl N,N-dii-
sopropylphosphoramidite (5.2 mL, 15.69 mmol) were added. The
mixture was stirred at room temperature for 1 h and then cooled
to ꢀ20 °C. m-Chloroperbenzoic acid (4.03 g, 77%, 17.92 mmol)
was added and the mixture was stirred at ꢀ20 °C for 1 h. The reac-
tion mixture was then poured into a saturated sodium bicarbonate
solution (100 mL), extracted with dichloromethane (3 ꢂ 100 mL)
and concentrated in vacuo. Flash column chromatography on the
residue (hexane/EtOAc, 2:1) afforded 7 (8.53 g, 98% over two steps)
3.3. Allyl 6-O-benzyl-2-deoxy-3-O-(9-
fluorenylmethoxycarbonyl)-2-(2,2,2-
trichloroethoxycarbonylamino)-a-D-glucopyranoside (6)
A solution of 5 (7.43 g, 10.54 mmol) in dry THF (50 mL) and
molecular sieves (4 Å, 5 g) was stirred at room temperature under
nitrogen for 20 min. Sodium cyanoborohydride (6.65 g,
105.40 mmol) was added and the mixture was cooled to 0 °C, fol-
lowed by the drop wise addition of dry ethereal-HCl(g) until no
gas was evolved. The mixture was poured into a saturated sodium
bicarbonate solution (100 mL) and the solids were filtered out be-
fore the removal of THF in vacuo. The resulting solution was
as a white solid. Rf 0.24 (hexane/EtOAc, 2.5:1); ½a D22
ꢁ
+ 59.4 (c 1.0,
CHCl3); 1H NMR (500 MHz, CDCl3): d 3.71–3.77 (m, 2H, H-6a, H-
6b), 3.94–3.97 (m, 2H, OCH2CH@CH2), 4.01–4.08 (m, 2H, H-5,
Fmoc-CHH), 4.14–4.23 (m, 2H, H-2, Fmoc-CH), 4.28 (dd, 1H, J 9.8,

1710
J. D. Lewicky et al. / Carbohydrate Research 346 (2011) 1705–1713
7.5 Hz, Fmoc-CHH), 4.44–4.60 (m, 4H, Troc-Ha, Troc-Hb, Ph-CH2),
4.71 (ddd, 1H, J 9.5, 9.5, 9.0 Hz, H-4), 4.88–4.98 (m, 5H, H-1,
(PhCH2O)2P), 5.22–5.43 (m, 4H, NH, H-3, OCH2CH@CH2), 5.85–
5.93 (m, 1H, OCH2CH@CH2), 7.19–7.77 (m, 23H, Ar-H); 13C NMR
(125 MHz, CDCl3): d 46.48 (Fmoc-CH), 54.24 (C-2), 68.17 (C-6),
68.88 (Fmoc-CH2), 69.70 (d, J 5.5 Hz, (PhCH2O)P), 69.78 (d, J
5.5 Hz, (PhCH2O)P), 70.10 (C-5), 70.66 (OCH2CH@CH2), 73.54 (d, J
ꢃ 7 Hz, C-4), 73.60 (PhCH2), 74.72 (Troc-CH2), 76.13 (C-3), 95.36
(Troc-CCl3), 96.28 (C-1), 118.78 (OCH2CH@CH2), 120.13 (CH-Ar),
125.35 (CH-Ar), 125.43 (CH-Ar), 127.22 (CH-Ar), 127.29 (CH-Ar),
127.67 (CH-Ar), 127.69 (CH-Ar), 127.93 (CH-Ar), 128.00 (CH-Ar),
128.39 (CH-Ar), 128.45 (CH-Ar), 128.54 (CH-Ar), 128.59 (CH-Ar),
133.04 (OCH2CH@CH2), 135.56 (d, J 7.25 Hz, C-Ar), 135.68 (d, J
7.25 Hz, C-Ar), 137.99 (C-Ar), 141.22 (C-Ar), 141.26 (C-Ar), 143.20
(C-Ar), 143.23 (C-Ar), 154.25 (C@O), 155.34 (C@O). Anal. Calcd
for C48H47Cl3NO12P: C, 59.61; H, 4.90; N, 1.45. Found: C, 59.86; H,
5.07; N, 1.56.
a white fluffy solid. Rf 0.29 (hexane/EtOAc, 3:1); ½a D22
ꢁ
+41.4 (c 1.0,
CHCl3); 1H NMR (500 MHz, CDCl3): d 3.71–3.77 (m, 2H, H-6a, H-
6b), 3.99–4.11 (m, 3H, Fmoc-CHH, Fmoc-CH, H-2), 4.31–4.37 (m,
2H, Fmoc-CHH, H-5), 4.46–4.66 (m, 5H, Troc-Ha, Troc-Hb, H-4, Ph-
CH2), 4.86–5.01 (m, 4H, (PhCH2O)2P), 5.29–5.33 (m, 2H, NH, H-3),
6.43 (d, 1H, J 4.0 Hz, H-1), 7.18–7.74 (m, 23H, Ar-H), 8.75 (s, 1H,
NH); 13C NMR (125 MHz, CDCl3); d 46.26 (Fmoc-CH), 54.23 (C-2),
67.60 (C-6), 69.80 (d, J 5.5 Hz, (PhCH2O)P), 69.99 (d, J 5.5 Hz,
(PhCH2O)P), 70.93 (Fmoc-CH2), 72.63 (C-5), 72.69 (d, J 6.7 Hz, C-4),
73.69 (Ph-CH2), 74.84 (Troc-CH2), 75.32 (C-3), 90.76 (CCl3 imidate),
94.82 (C-1), 95.3 (Troc-CCl3), 120.26 (CH-Ar), 125.40 (CH-Ar),
125.53 (CH-Ar), 127.44 (CH-Ar), 127.85 (CH-Ar), 127.91 (CH-Ar),
128.19 (CH-Ar), 128.56 (CH-Ar), 128.69 (CH-Ar), 128.77 (CH-Ar),
135.67 (d, J 7.25 Hz, C-Ar), 135.75 (d, J 7.25 Hz, C-Ar), 138.03 (C-
Ar), 141.44 (C-Ar), 143.20 (C-Ar), 143.22 (C-Ar), 154.25 (C@O),
155.67 (C@O), 160.61 (C@NH). Anal. Calcd for C47H43Cl6N2O12P: C,
52.68; H, 4.04; N, 2.61. Found: C, 52.79; H, 4.21; N, 2.46.
3.5. 6-O-Benzyl-2-deoxy-4-O-(di-O-benzylphosphono)-3-O-(9-
3.7. 1-[6-O-Benzyl-2-deoxy-4-(di-O-benzylphosphono)-3-O-(9-
fluorenylmethoxycarbonyl)-2-(2,2,2-
fluorenylmethoxycarbonyl)-2-(2,2,2-
trichloroethoxycarbonylamino)-
a
/b-D
-glucopyranose (8)
trichloroethoxycarbonylamino)-b-D-glucopyranosyloxy]-ethan-
2-ol (10)
Into a suspension of [bis(methyldiphenylphosphine)](1,5-cyclo-
octadiene) iridium(I) hexafluorophosphate (338 mg, 0.40 mmol) in
dry THF (10 mL), hydrogen gas was bubbled in for 5 min to give a
yellowish solution, which was added to a solution of 7 (8.25 g,
8.53 mmol) in dry THF (40 mL). The mixture was stirred at room
temperature for 2 h. Water (3 mL) and freshly recrystallized N-bro-
mosuccinimide (2.36 g, 12.80 mmol) was added and the mixture
was stirred for a further 1 h. The residue obtained after solvent re-
moval was dissolved in EtOAc (200 mL) and washed with a satu-
rated sodium bicarbonate solution (2 ꢂ 50 mL). The combined
organic phase was dried over Na2SO4, concentrated, and purified
by repeated flash column chromatography (hexane/EtOAc, 3:2) to
Method 1: See the preparation of 11 in Section 3.8.
Method 2: To a mixture of 13 (165 mg, 0.15 mmol) and glacial
acetic acid (0.86 mL, 15.20 mmol) in dry THF (3 mL) cooled to
0 °C (ice water bath), a tetrabutylammonium fluoride solution
(1.0 M in THF, 0.19 mL, 0.19 mmol) was added. The mixture was
stirred at 0 °C for 4 h, and at room temperature for a further 4 h,
upon which the solvent was removed in vacuo. The residue was
dissolved in dichloromethane (50 mL) and washed with a satu-
rated sodium bicarbonate solution (25 mL). The organic phase
was dried over Na2SO4, concentrated, and purified via flash column
chromatography (hexane/EtOAc, 1:1.25) to afford 10 (108 mg, 73%)
afford white solid 8 (6.45 g, 79%) as an
a
/b mixture in a ratio of
as a white solid. Rf 0.26 (hexane/EtOAc, 1:1.25); ½a D22
ꢁ
+15.2 (c 1.0,
ꢄ10:1. Rf 0.22 (hexane/EtOAc, 3:2); ½a D22
ꢁ
+36.3 (c 1.0, CHCl3); 1H
CHCl3); 1H NMR (500 MHz, CDCl3): d 3.11 (br s, 1H, OH), 3.65–
3.86 (m, 8H, H-6a, H-6b, H-2, H-5, OCH2CH2OH, OCH2CH2OH),
4.01 (dd, 1H, J 9.5, 7.5 Hz, Fmoc-CHH), 4.11 (dd, 1H, J 7.5, 7.5 Hz,
Fmoc-CH), 4.33 (dd, 1H, J 7.5, 7.5 Hz, Fmoc-CHH), 4.47–4.65 (m,
5H, Troc-Ha, Troc-Hb, H-4, Ph-CH2), 4.76 (d, 1H, J 8.0 Hz, H-1),
4.91–4.95 (m, 4H, (PhCH2O)2P), 5.29 (dd, 1H, J 10.0, 10.0 Hz, H-3),
5.61 (d, 1H, J 8.0 Hz, NH), 7.15–7.74 (m, 23H, Ar-H); 13C NMR
(125 MHz, CDCl3); d 46.58 (Fmoc-CH), 56.83 (C-2), 68.66 (2C, OCH2-
CH2OH, OCH2CH2OH), 69.81 (d, J 5.5 Hz, (PhCH2O)P), 69.92 (d, J
5.5 Hz, (PhCH2O)P), 70.75 (Fmoc-CH2), 73.58 (C-6), 73.75 (Troc-
CH2), 74.05 (C-5), 74.11 (d, J 6.6 Hz, C-4), 74.67 (Ph-CH2), 76.75
(C-3), 95.53 (Troc-CCl3), 101.46 (C-1), 120.24 (CH-Ar), 125.41
(CH-Ar), 125.53 (CH-Ar), 127.39 (CH-Ar), 127.45 (CH-Ar), 127.87
(CH-Ar), 128.00 (CH-Ar), 128.12 (CH-Ar), 128.20 (CH-Ar), 128.64
(CH-Ar), 128.74 (CH-Ar), 128.81 (CH-Ar), 128.85 (CH-Ar), 135.62
(d, J 7.25 Hz, C-Ar), 135.68 (d, J 7.25 Hz, C-Ar), 137.84 (C-Ar),
141.44 (C-Ar), 143.35 (C-Ar), 154.40 (C@O), 155.25 (C@O). Anal.
Calcd for C47H47Cl3NO13P: C, 58.12; H, 4.88; N, 1.44. Found: C,
58.27; H, 4.97; N, 1.52; HRESI-MS (m/z) calcd for C47H47Cl3NO13P-
Na [M+Na]+: 992.1751, found: 992.1743.
NMR (500 MHz, CDCl3): d 3.62–3.77 (m, 3H, H-6a, H-6b, OH),
3.95 (dd, 1H, J 10.5, 8.5 Hz, Fmoc-CHH), 4.04–4.12 (m, 2H, H-2,
Fmoc-CH), 4.19–4.23 (m, 1H, H-5), 4.28 (dd, 1H, J 10.5, 7.5 Hz,
Fmoc-CHH), 4.43–4.60 (m, 5H, Troc-Ha, Troc-Hb, H-4, Ph-CH2),
4.86–4.98 (m, 4H, (PhCH2O)2P), 5.23–5.28 (m, 2H, H-1, H-3), 5.42
(d, 1H,
J
9.5 Hz, NH), 7.18–7.77 (m, 23H, Ar-H); 13C NMR
(125 MHz, CDCl3); d 46.51 (Fmoc-CH), 54.54 (C-2), 68.74 (C-6),
69.50 (C-5), 69.62 (d, J 5.5 Hz, (PhCH2O)P), 69.85 (d, J 5.5 Hz,
(PhCH2O)P), 70.70 (Fmoc-CH2), 73.69 (Ph-CH2), 74.06 (d, J 6.6 Hz,
C-4), 74.76 (Troc-CH2), 75.89 (C-3), 91.66 (C-1), 95.39 (Troc-CCl3),
120.17 (CH-Ar), 125.50 (CH-Ar), 125.64 (CH-Ar), 127.37 (CH-Ar),
127.44 (CH-Ar), 127.85 (CH-Ar), 128.08 (CH-Ar), 128.12 (CH-Ar),
128.62 (CH-Ar), 128.70 (CH-Ar), 128.76 (CH-Ar), 135.46 (d, J
7.25 Hz, C-Ar), 135.56 (d, J 7.25 Hz, C-Ar), 137.61 (C-Ar), 141.37
(C-Ar), 141.40 (C-Ar), 143.32 (C-Ar), 143.38 (C-Ar), 154.33 (C@O),
155.33 (C@O). Anal. Calcd for C45H43Cl3NO12P: C, 58.29; H, 4.67;
N, 1.51. Found: C, 58.67; H, 4.92; N, 1.42.
3.6. 6-O-Benzyl-2-deoxy-4-O-(di-O-benzylphosphono)-3-O-(9-
fluorenlymethoxycarbonyl)-2-(2,2,2-
trichloroethoxycarbonylamino)-
trichloroacetimidate (9)
a
-D
-glucopyranosyl
3.8. 1,2-Di-[6-O-benzyl-2-deoxy-4-O-(di-O-benzylphosphono)-
3-O-(9-fluorenylmethoxycarbonyl)-2-(2,2,2-
trichloroethoxycarbonylamino)-b-D-glucopyranosyloxy]-
To a solution of 8 (5.95 g, 6.42 mmol) in dry CH2Cl2 (10 mL), tri-
chloroacetonitrile (3.22 mL, 32.1 mmol) and sodium hydride
(23 mg, 0.22 mmol) were added successively. The mixture was stir-
red at room temperature for 1 h upon which the solvent was re-
moved in vacuo, followed by co-distillation with EtOAc
(2 ꢂ 20 mL). The residue was purified by flash column chromatogra-
phy (hexane/EtOAc, 2.5:1 with 0.5% Et3N) to afford 9 (5.57 g, 81%) as
ethane (11)
Method 1: A solution of 9 (400 mg, 0.37 mmol), ethylene glycol
(11 mg, 0.18 mmol), and molecular sieves (4 Å, 2.0 g) in dry CH2Cl2
(1.5 mL) was stirred under nitrogen at room temperature for
20 min. A solution of TMSOTf (0.01 M in dry dichloromethane,
0.7 mL) was added drop wise, and the mixture stirred at room tem-

J. D. Lewicky et al. / Carbohydrate Research 346 (2011) 1705–1713
1711
perature for 20 min. The reaction was quenched by the addition of
a saturated sodium bicarbonate solution (10 mL), before being fil-
tered and extracted with CH2Cl2 (3 ꢂ 25 mL). The combined organ-
ic phase was dried over Na2SO4, concentrated, and purified via
flash column chromatography (hexane/EtOAc, 3:2) to yield 11
(68 mg, 10%) and 10 (70 mg, 20%) as fluffy white solids.
0.70 mmol) was performed in dry CH2Cl2 (4 mL), promoted by
the dropwise addition of a TMSOTf solution (0.01 M in dichloro-
methane, 0.56 mL). The remaining residue after aqueous workup
was purified via flash column chromatography (hexane/acetone,
3.75:1) to yield 13 (196 mg, 81%) as a colorless syrup. Rf 0.28 (hex-
ane/EtOAc, 2.5:1); ½a D22
ꢁ
+12.9 (c 1.0, CHCl3); 1H NMR (500 MHz,
Method 2: In a similar way as described above, the glycosylation
of 9 (400 mg, 0.37 mmol) and ethylene glycol (11 mg, 0.18 mmol
was performed in dry CH2Cl2 (1.5 mL) and dry THF (1.5 mL), pro-
moted by the dropwise addition of a TMSOTf solution (0.01 M in
dry dichloromethane, 0.7 mL). The remaining residue after workup
was purified via flash column chromatography (hexane/EtOAc, 3:2)
to yield 11 (163 mg, 47%) and 10 (39 mg, 11%) as fluffy white solids.
Method 3: In a similar way as described above, the glycosylation
of 10 (121 mg, 0.13 mmol) and 9 (180 mg, 0.17 mmol) was per-
formed in dry CH2Cl2 (3 mL), promoted by the dropwise addition
of a TMSOTf solution (0.01 M in dichloromethane, 0.25 mL) The
remaining residue after workup was purified via flash column
chromatography (hexane/EtOAc, 1.25:1) to yield 11 (195 mg,
CDCl3): d 0.06 (s, 6H, Si(CH3)2), 0.89 (s, 9H, SiC(CH3)3), 3.65–3.83
(m, 7H, H-6a, H-5, H-2, OCH2CH2OTBDMS, OCH2CH2OTBDMS),
3.89 (dd, 1H, J 10.0, 5.0 Hz, H-6b), 3.97 (dd, 1H, J 9.5, 7.5 Hz,
Fmoc-CHH), 4.07 (dd, 1H, J 7.5, 7.5 Hz, Fmoc-CH), 4.28 (dd, 1H, J
7.5, 7.5 Hz, Fmoc-CHH), 4.47–4.60 (m, 5H, Troc-Ha, Troc-Hb, H-4,
Ph-CH2), 4.76 (d, 1H, J 8.0 Hz, H-1), 4.89–4.95 (m, 4H, (PhCH2O)2P),
5.22 (dd, 1H, J 10.0, 10.0 Hz, H-3), 5.31 (d, 1H, J 7.0 Hz, NH), 7.16–
7.71 (m, 23H, Ar-H); 13C NMR (125 MHz, CDCl3); d ꢀ5.09 (Si(CH3)),
ꢀ5.03 (Si(CH3)), 18.55 (SiC(CH3)3), 26.16 (SiC(CH3)3), 46.63 (Fmoc-
CH), 56.80 (C-2), 62.89 (OCH2CH2OTBDMS), 68.74 (OCH2-
CH2OTBDMS), 69.78 (d, J 5.5 Hz, (PhCH2O)P), 69.85 (d, J 5.5 Hz,
(PhCH2O)P), 70.65 (Fmoc-CH2), 71.26 (C-6), 73.74 (Troc-CH2),
73.98 (C-5), 74.50 (d, J 6.7 Hz, C-4), 74.70 (Ph-CH2), 77.19 (C-3),
95.55 (Troc-CCl3), 100.92 (C-1), 120.20 (CH-Ar), 120.49 (CH-Ar),
125.58 (CH-Ar), 127.39 (CH-Ar), 127.47 (CH-Ar), 127.83 (CH-Ar),
127.87 (CH-Ar), 128.10 (CH-Ar), 128.14 (CH-Ar), 128.57 (CH-Ar),
83%) as a white fluffy solid. Rf 0.35 (hexane/EtOAc, 1:1), ½a D22
ꢁ
+5.5
(c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d 3.61–3.69 (m, 3H, H-
6a, H-6b, H-5), 3.78–3.83 (m, 2H, H-2, (OCHHCHHO)/2), 3.91–
3.94 (m, 2H, Fmoc-CHH, (OCHHCHHO)/2), 4.05 (dd, 1H, J 7.5,
7.5 Hz, Fmoc-CH), 4.26 (dd, 1H, J 7.5, 7.5 Hz, Fmoc-CHH), 4.46–
4.75 (m, 6H, Troc-Ha, Troc-Hb, Ph-CH2, H-4, H-1), 4.88–4.96 (m,
4H, (PhCH2O)2P), 5.13–5.16 (dd, 1H, J 10.0, 10.0 Hz, H-3), 5.67 (d,
1H, J 7.5 Hz, NH), 7.14–7.72 (m, 23H, Ar-H); 13C NMR (125 MHz,
CDCl3); d 46.53 (Fmoc-CH), 56.73 (C-2), 68.59 (C-6), 69.11 (OCH2-
CH2O)/2), 69.77 (d, J 5.5 Hz, (PhCH2O)P), 69.83 (d, J 5.5 Hz,
(PhCH2O)P), 70.70 (Fmoc-CH2), 73.63 (Troc-CH2), 73.93 (C-5),
74.14 (d, J 6.6 Hz, C-4), 74.63 (Ph-CH2), 77.33 (C-3), 95.74 (Troc-
CCl3), 100.97 (C-1), 120.17 (CH-Ar), 125.49 (CH-Ar), 125.61 (CH-
Ar), 127.38 (CH-Ar), 127.43 (CH-Ar), 127.82 (CH-Ar), 127.87 (CH-
Ar), 128.08 (CH-Ar), 128.14 (CH-Ar), 128.60 (CH-Ar), 128.70 (CH-
Ar), 128.75 (CH-Ar), 135.52 (d, J 7.25 Hz, C-Ar), 135.57 (d, J
7.25 Hz, C-Ar), 138.12 (C-Ar), 141.40 (C-Ar), 141.43 (C-Ar), 143.37
(C-Ar), 154.73 (C@O), 155.25 (C@O). Anal. Calcd for
128.70 (CH-Ar), 128.75 (CH-Ar), 135.76 (d,
J 7.25 Hz, C-Ar),
135.82 (d, J 7.25 Hz, C-Ar), 138.23 (C-Ar), 141.40 (C-Ar), 141.47
(C-Ar), 143.42 (C-Ar), 154.27 (C@O), 155.17 (C@O). Anal. Calcd
for C53H61Cl3NO13PSi: C, 58.64; H, 5.66; N, 1.29. Found: C, 58.27;
H, 5.36; N, 1.28.
3.11. 1,2-Di-[6-O-benzyl-2-deoxy-4-O-(di-O-benzylphosphono)-
3-O-(9-fluorenylmethoxycarbonyl)-2-((R)-3-
tetradecanoyloxytetradecanamido)-b-D-glucopyranosyloxy]-
ethane (15)
To a solution of 11 (1.20 g, 0.64 mmol) in dry THF (8 mL) and
acetic acid (2 mL), zinc powder (1.0 g) was added. The mixture
was stirred at room temperature for 30 min and then filtered.
The solid was washed with acetic acid (30 mL) and the filtrate con-
centrated in vacuo. The residue was dissolved in dichloromethane
(150 mL) and washed with a saturated sodium bicarbonate solu-
tion (75 mL). The combined organic phase was dried with Na2SO4
and concentrated to obtain free amine 14 (1.1 g) as a white fluffy
solid.
C92H88Cl6N2O24P2: C, 58.76; H, 4.72; N, 1.49. Found: C, 58.52; H,
4.65; N, 1.52; HRESI-MS (m/z) calcd for C92H88Cl6N2O24P2Na
[M+Na]+: 1899.3210, found: 1899.3232.
3.9. 1-tert-Butyldimethylsilyloxy-ethan-2-ol (12)
A mixture of amine 14 (1.1 g), fatty acid 16 (870 mg, 1.91 mmol)
and DCC (760 mg, 3.83 mmol) in dry CH2Cl2 (5 mL) was stirred at
room temperature for 20 h. Water (0.50 mL) was added and the
reaction mixture was stirred for a further 20 min. The solid was
then filtered through a sintered glass funnel with a bed of Na2SO4.
The filtrate was concentrated and the residue purified by repeated
flash column chromatography (hexane/acetone, 3:1) to afford 15
(997 mg, 65% over two steps) as a colorless syrup. Rf 0.26 (hex-
A mixture of ethylene glycol (3.82 mL, 68.4 mmol) and imidaz-
ole (1.45 g, 21.27 mmol) in dry N,N-dimethylformamide (8 mL)
was cooled to 0 °C (ice water bath). tert-Butyldimethylsilyl chloride
(2.57 g, 17.01 mmol) was added and the mixture was stirred at the
reduced temperature for 4 h before being quenched via the addition
of cold water (200 mL). The mixture was extracted with EtOAc
(3 ꢂ 200 mL), with the combined organic layers dried over Na2SO4
and concentrated. The residue was purified by flash column chro-
matography (hexane/EtOAc, 15:1) to give 12 (2.1 g, 70%) as a color-
less syrup. Rf 0.25 (hexane/EtOAc, 9:1); 1H NMR (500 MHz, CDCl3): d
0.21 (s, 6H, Si(CH3)2), 0.82 (s, 9H, SiC(CH3)3), 1.99 (t, 1H, J 6.0 Hz,
OH), 3.55–3.63 (m, 4H, 2 CH2); 13C NMR (125 MHz, CDCl3); d
ꢀ5.16 (Si(CH3)2), 18.52 (SiC(CH3)3), 26.08 (SiC(CH3)3), 63.85
(HOCH2CH2OTBDMS), 64.34 (HOCH2CH2OTBDMS). Anal. Calcd for
C8H20O2Si: C, 58.64; H, 11.43. Found: C, 58.62; H, 11.40.
ane/acetone, 3:1); ½a D22
ꢁ
+5.9 (c 1.0, CHCl3); 1H NMR (500 MHz,
CDCl3): d 0.86–0.89 (m, 6H, 2 ꢂ CH3, lipid), 1.08–1.36 (br m, 38H,
0
19 ꢂ CH2, lipid), 1.50–1.60 (m, 4H, H-4L, H-3L ), 2.21–2.25 (m, 2H,
0
H-2L ), 2.32 (dd, 1H, J 14.5, 6.5 Hz, H-2La), 2.44 (dd, 1H, J 14.5,
6.5 Hz, H-2Lb), 3.66–3.89 (m, 6H, H-6a, H-6b, H-2, H-5, (OCH2-
CH2O)/2), 3.95 (dd, 1H, J 10.0, 8.0 Hz, Fmoc-CHH), 4.10 (dd, 1H, J
8.0, 8.0 Hz, Fmoc-CH), 4.28 (dd, 1H, J 10.0, 8.0 Hz, Fmoc-CHH),
4.45–4.62 (m, 3H, Ph-CH2, H-4), 4.84–4.97 (m, 5H, H-1,
(PhCH2O)2P), 5.07 (m, 1H, H-3L), 5.32 (dd, 1H, J 9.5, 9.5 Hz, H-3),
6.60 (d, 1H, J 5.0 Hz, NH), 7.13–7.72 (m, 23H, Ar-H); 13C NMR
(125 MHz, CDCl3); d 14.34 (CH3, lipid), 22.89 (CH2, lipid), 25.16
(CH2, lipid), 25.23 (CH2, lipid), 25.46 (CH2, lipid), 25.81 (CH2, lipid),
29.44 (CH2, lipid), 29.57 (CH2, lipid), 29.66 (CH2, lipid), 29.77 (CH2,
lipid), 29.83 (CH2, lipid), 29.87 (CH2, lipid), 29.90 (CH2, lipid), 29.91
(CH2, lipid), 32.12 (CH2, lipid), 34.14 (CH2, lipid), 34.21 (CH2, lipid),
34.71 (CH2, lipid), 41.63 (C-2L), 46.60 (Fmoc-CH), 55.28 (C-2), 68.76
3.10. 1-[6-O-Benzyl-2-deoxy-4-O-(di-O-benzylphosphono)-3-O-
(9-fluorenylmethoxycarbonyl)-2-(2,2,2-
trichloroethoxycarbonylamino)-b-D-glucopyranosyloxy]-2-tert-
butldimethylsilyloxy-ethane (13)
In a similar manner as described for the preparation of 11, the
glycosylation of (240 mg, 0.22 mmol) and 12 (124 mg,
9

1712
J. D. Lewicky et al. / Carbohydrate Research 346 (2011) 1705–1713
(C-6), 69.00 ((OCH2CH2O)/2), 69.65 (d, J 5.5 Hz, (PhCH2O)P), 69.73
(d, J 5.5 Hz, (PhCH2O)P), 70.52 (Fmoc-CH2), 71.40 (C-3L), 73.55
(Ph-CH2), 74.08 (C-5), 74.15 (d, J 6.6 Hz, C-4), 77.12 (C-3), 100.52
(C-1), 120.04 (CH-Ar), 120.06 (CH-Ar), 125.51 (CH-Ar), 125.67
(CH-Ar), 127.38 (CH-Ar), 127.76 (CH-Ar), 127.80 (CH-Ar), 127.89
(CH-Ar), 127.97 (CH-Ar), 128.06 (CH-Ar), 128.11 (CH-Ar), 128.51
(CH-Ar), 128.52 (CH-Ar), 128.61 (CH-Ar), 128.65 (CH-Ar), 128.68
(CH-Ar), 135.58 (d, J 7.25 Hz, C-Ar), 135.64 (d, J 7.25 Hz, C-Ar),
138.19 (C-Ar), 141.35 (C-Ar), 141.37 (C-Ar), 143.45 (C-Ar), 143.52
(C-Ar), 155.05 (C@O), 170.47 (C@O), 174.00 (C@O); HRESI-MS (m/
incubated at 4 °C for 20 h in the dark. After incubation, cells were
washed twice with PBS, resuspended in 500 L PBS, and subjected
to flow cytometry analysis on FACSCalibur with CELLQUEST PRO soft-
ware (BD Biosciences), acquiring 15,000 events. The results were
presented as the mean fluorescence intensity (MFI) on the FL2
channel.
l
Acknowledgments
Authors are grateful for the kind gifts of compounds 4 and 16
from Biomira Inc. that were used as the starting materials for the
synthesis. This work was supported by Natural Sciences and Engi-
neering Research Council of Canada (NSERC, Grant 312630) and
Lakehead University.
z) calcd for
C
142H190N2O26P2Na [M+Na]+: 2424.2931, found:
2424.2980 and 1223.6429 [M+2Na]2+
.
3.12. 1,2-Di-[2-deoxy-4-O-phosphono-2-((R)-3-
tetradecanoyloxytetradecanamido)-b-D-glucopyranosyloxy]-
ethane (3)
Supplementary data
To a solution of 15 (60 mg, 0.03 mmol) in THF–HOAc (4:1,
40 mL) was added palladium on charcoal (5%, 20 mg). The mixture
was stirred at room temperature under hydrogen atmosphere for
24 h. The mixture was filtered and the filtrate concentrated in va-
cuo. The resulting residue was dissolved in THF (4 mL) and piper-
idine (1 mL) was added. The mixture was stirred at rt for 2 h
before being concentrated. The residue was dissolved in CHCl3/
MeOH (4:1, 3 mL), and the product was precipitated out via the
addition of EtOAc (20 mL). The precipitate was collected after cen-
trifugation and the crude product was further purified by repeated
precipitation for two more times as described. The resulting solid
was finally freeze-dried from a dioxane–CHCl3 mixture (95:5) to
afford 3 (27 mg, 65%) as white fluffy solid. Rf 0.47 (CHCl3/MeOH/
H2O/NH4OH, 50:15:4:2); HR MALDI-TOF-MS (m/z) calcd for
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2011.05.018.
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