Monophosphoryl lipid A analogues with varying 3-O-substitution: synthesis and potent adjuvant activity

Article abstract of DOI:10.1016/j.carres.2007.01.012

Structurally defined immunostimulatory adjuvants play important roles in the development of new generation vaccines. Here described are the syntheses of three monophosphoryl lipid A analogues (1-3) with different substitution at 3-O-position of the reduci

Full text of DOI:10.1016/j.carres.2007.01.012

Carbohydrate Research 342 (2007) 784–796
Monophosphoryl lipid A analogues with varying
3-O-substitution: synthesis and potent adjuvant activity
Zi-Hua Jiang,*,ꢀ Wladyslaw A. Budzynski, Dongxu Qiu,ꢁ
Damayanthi Yalamati and R. Rao Koganty*
Biomira Inc., 2011-94 Street, Edmonton, Alberta, Canada T6N 1H1
Received 4 November 2006; received in revised form 18 January 2007; accepted 19 January 2007
Available online 26 January 2007
Abstract—Structurally defined immunostimulatory adjuvants play important roles in the development of new generation vaccines.
Here described are the syntheses of three monophosphoryl lipid A analogues (1–3) with different substitution at 3-O-position of the
reducing sugar and their potent immunostimulatory adjuvant activity. The syntheses involve the preparation of glycosylation accep-
tors benzyl 3,4-di-O-benzyl-2-deoxy-2-[(R)-3-tetradecanoyloxytetradecanamido]-b-D-glucopyranoside (16) and benzyl 3-O-allyl-4-
O-benzyl-2-deoxy-2-[(R)-3-tetradecanoyloxytetradecanamido]-b-D-glucopyranoside (17). The glycosylation reactions between the
donor 4,6-di-O-benzylidene-2-deoxy-2-(20,20,20-trichloroethoxycarbonylamino)-a-D-glucopyranosyl trichloroacetimidate (21) and
acceptors 16 and 17 provide the desired b-(1!6)-linked disaccharides 22 and 23, respectively. Selective reductive ring opening of
the 4,6-di-O-benzylidene group, installation of a phosphate group to the 40-hydroxyl group, and the final global debenzylation pro-
duce the designed monophosphoryl lipid A analogues 1–3. All three synthetic analogues induce antigen specific T-cell proliferation
and interferon-gamma (IFN-c) production in ex vivo experiments with a totally synthetic liposomal vaccine system. The immuno-
stimulatory potency of compound 1–3 is in the same order of magnitude as that of the detoxified natural lipid A product isolated
from Salmonella minnesota R595 (R595 lipid A). The substituent at the 3-O-position of the reducing sugar does not have much effect
on the adjuvant activity of monophosphoryl lipid A analogues. The preliminary lethal toxicity study indicates that the 3-O-acylated
hepta-acyl monophosphoryl lipid A may not be more toxic than its 3-O-deacylated hexa-acyl analogue.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Vaccine adjuvant; Immunostimulant; Monophosphoryl lipid A; Glycolipid; Cytokine
1. Introduction
of vaccine adjuvants is that they serve as danger signals,
which are detected by a group of pattern recognition
receptors such as Toll-like receptors (TLRs) expressed
on macrophages and dendritic cells.1 The activation of
TLRs triggers the activation of antigen presentation
cells (APCs) and the secretion of inflammatory cyto-
kines and chemokines, leading to the subsequent devel-
opment of a strong and specific acquired immunity.
Interests in developing novel vaccine adjuvants continue
to grow in recent years,2–4 particularly in the context of
developing new type of vaccines capable of eliciting TH1
immune responses.5,6
Successful vaccination against infectious or neoplastic
diseases is to prime the host’s immune system to
generate an efficient defence and memory response.
Generation of strong immune responses to poorly
immunogenic antigens requires the help of an immuno-
stimulatory adjuvant. Current understanding of the role
*
Corresponding authors. Tel.: +1 807 766 7171; fax: +1 807 346
7775; e-mail: zjiang@lakeheadu.ca
ꢀ Present address: Department of Chemistry, Lakehead University, 955
Oliver Road, Thunder Bay, Canada ON P7B 5E1.
ꢁ Present address: Beijing Joinn Pharmaceutical Center, No. 5 Rong-
jingdong Street, Beijing, Economic-Technological Development
Area, PR China.
Lipid A is the active principle of lipopolysaccharide
(LPS), the outer membrane component of Gram-nega-
tive bacteria. Lipid A has strong immunostimulatory
activity, but its high toxicity prevents its use in clinical
0008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.01.012

Z.-H. Jiang et al. / Carbohydrate Research 342 (2007) 784–796
785
practice. However, the endotoxic effect of lipid A can be
largely reduced by selective hydrolysis of the anomeric
phosphate group while the immunostimulatory property
of the molecule remains unaffected.7 The promising
immunostimulatory adjuvant MPLꢁ, monophosphoryl
lipid A, is the natural lipid A product isolated from Sal-
monella minnesota R595 and detoxified by selective
hydrolysis of the anomeric phosphate group.8 Currently,
MPLꢁ is under extensive clinical evaluation for both
prophylactic and therapeutic human vaccine use.9
Lipid A preparations purified from bacterial cultures
suffer from lack of consistency both in composition
and performance. Its heterogeneity is a major cause of
large batch-to-batch variations both in composition
and activity. As a result, its use in vaccine formulations
adds to the compliance requirements. On the other
hand, synthetic lipid A analogues are pure material of
single molecule, which are advantageous in achieving
reproducibility and consistency with respect to product
manufacturing and performance. In order to develop
synthetic lipid A adjuvants with reduced toxicity and
enhanced beneficial immunostimulatory effect, various
lipid A analogues have been designed and synthesized
by us10,11 and many others.12–17 As part of our continu-
ous cancer vaccine program,4,18,19 we report here the
syntheses and immunostimulatory property of three
monophosphoryl lipid A analogues (1–3) (Chart 1) with
different 3-O-substitute groups.
ation donor.24 The preparation of glycosylation accep-
tors 16 and 17 is shown in Scheme 1. The readily avail-
able glucosamine derivative 625 is selectively protected
with the trityl group at the 6-O-position to give 8, which
is then treated with benzyl bromide and sodium hydride
to provide 10 in high yield. The removal of the 6-O-trityl
group (!12) and the phthalimide function affords the
free amine 14, which is then coupled with fatty acid
1826 in the presence of N,N0-dicyclohexylcarbodiimide
(DCC) to provide the glycosylation acceptor 16 in 82%
yield. In order to attach a lipid chain at the 3-O-position
for the synthesis of compound 3, glycosylation acceptor
17 with an allyl group at the 3-O-position has been pre-
pared. This allyl group can be converted to a propyl
group upon catalytic hydrogenation at the final debenz-
ylation step, allowing for the straightforward prepara-
tion of compound 2. Thus, glucosamine derivative 425 is
treated with allyl bromide and sodium hydride to give
5, which upon treatment with aqueous acid at 65 ꢂC pro-
vides the 3-O-allyl protected intermediate 7. Following
the same reaction sequence as described for the synthesis
of 16, compound 7 is converted to acceptor 17 in an
overall good yield through the following intermediates:
6-O-trityl-protected 9, 4-O-benzylated 11, 6-O-detrity-
lated 13 and the free amine 15.
The 2,2,2-trichloroethoxycarbonyl (Troc) group is an
efficient amine-protection group, which can be cleaved
by reductive b-elimination.27 The Troc group has been
widely employed in the syntheses of b-glycosides of glu-
cosamine derivatives because of its neighbouring group
participating capacity.11,28,29 Here we have prepared
the glycosyl donor 21 with N-Troc protection for the
stereoselective synthesis of b-(1!6)-linked diglucos-
amine unit (Scheme 2). The previously reported building
block 1911,22 is converted to the reducing end derivative
20 in 82% yield following the two-step procedure: first
the isomerization of the allyl double bond using the
iridium complex,26 [bis(methyldiphenylphosphine)](1,5-
cyclooctadiene) iridium(I) hexafluorophosphate, and
then hydrolysis of the isomerized aglycone in the pres-
ence of N-bromosuccinimide (NBS).
2. Results and discussion
2.1. Syntheses of monophosphoryl lipid A analogues 1–3
Synthetic strategies for lipid A molecules15,20–23 incorpo-
rate different protecting groups and glycosylation meth-
ods in constructing the b-(1!6)-linked diglucosamine
unit. Here we use the benzyl group as the global protect-
ing group and the trichloroacetimidate as the glycosyl-
O
P
OH
O
The conversion of compound 20 to trichloroacetimi-
date 2121 is effected by treating with trichloroacetonitrile
and diazabicyclo[5,4,0]undec-7-ene (DBU) in 81% as a
single a-isomer (1H NMR, d 6.42, d, J 4.0 Hz, H-1).
The glycosylation reaction of 21 with either 16 or 17
in the presence of BF3ÆOEt2 as the catalyst gives the de-
sired disaccharide 22 or 23 in good yield. The newly
formed b-linkage is confirmed by 1H NMR data in both
22 (d 4.52, d, J 8.0 Hz, 1H, H-10; d 4.89, d, J 8.0 Hz, 1H,
H-1) and 23 (d 4.51, d, J 8.0 Hz, 1H, H-10; d 4.88, d, J
8.0 Hz, 1H, H-1). The allyl group at the 3-O-position
in 23 is then removed to give 24, which is subsequently
coupled with (R)-3-benzyloxytetradecanoic acid 3530 in
the presence of DCC and 4-N,N0-dimethylaminopyri-
dine (DMAP) to afford 25 in 71%.
O
O
HO
O
1
2
3
R = H
HO
O
O
NH
HO
RO
OH
O
O
NH
R = CH3CH2CH2
O
O
O
O
O
OH
CO
R =
n-C11H23
O
Chart 1. Monophosphoryl lipid A analogues (1–3) with different
3-O-substitution.

786
Z.-H. Jiang et al. / Carbohydrate Research 342 (2007) 784–796
OTrt
O
OTrt
O
OH
Ph
O
O
RO
c
b
d
O
O
HO
RO
BnO
RO
HO
RO
OBn
OBn
OBn
OBn
NPhth
NPhth
NPhth
NPhth
R = H
6
7
10 R = Bn
11 R = All
4
5
R = H
R = All
8
9
R = H
R = All
a
R = All
OH
OH
OH
O
O
O
g
f
BnO
RO
e
BnO
RO
n-C13H27CO
n-C11H23
O
BnO
RO
OBn
OBn
OBn
COOH
NH
NH2
NPhth
O
O
18
16 R = Bn
17 R = All
12 R = Bn
13 R = All
14 R = Bn
15 R = All
O
n-C11H23
n-C13H27
Scheme 1. Reagents and conditions: (a) AllBr, NaH, DMF, 82%; (b) HOAc–H2O, 65 ꢂC, 95% for 7; (c) Trt–Cl, DMAP, pyridine, 40 ꢂC, 90% for 8
and 79% for 9; (d) BnBr, NaH, DMF, 90% for 10 and 57% for 11; (e) HOAc–H2O–AllOH, 110 ꢂC, 70% for 12 and 87% for 13; (f) H2NNH2ÆH2O,
EtOH, reflux, 97% for 14 and 77% for 15; (g) 18, DCC, CH2Cl2, 82% for 16 and 80% for 17.
Ph
O
O
O
Ph
Ph
O
O
O
e
O
O
16 or 17
O
O
O
O
O
O
X
c
O
O
O
O
O
BnO
RO
BnO
RO
NH
NH
NH
Y
OBn
OBn
Troc
n-C11H23
n-C13H27
Troc
O
O
O
O
O
O
O
NH
O
n-C11H23
NH
n-C11H23
O
O
O
O
n-C13H27
n-C13H27
O
n-C11H23
n-C13H27
O
O
n-C11H23
n-C11H23
n-C13H27
OBn
CO
19 X = H Y = All
20 X, Y = H, OH
21 X = H Y = OC(NH)CCl3
a
b
n-C13H27
22 R = Bn
23 R = All
24 R = H
26 R = Bn
27 R = All
OBn
a
d
CO
28 R = n-C11H23
25 R =
n-C11H23
f
1
OBn
O
OBn
O
O
O
(BnO)2P
g
HO
h
O
O
O
O
2
3
O
O
O
O
O
O
BnO
RO
BnO
RO
NH
NH
OBn
OBn
O
O
O
O
O
n-C11H23
O
NH
n-C11H23
NH
O
O
O
O
n-C13H27
n-C13H27
O
O
n-C11H23
n-C11H23
n-C13H27
O
n-C13H27
O
n-C11H23
n-C11H23
n-C13H27
OBn
n-C13H27
COOH
n-C11H23
29 R = Bn
30 R = All
32 R = Bn
33 R = All
34 R = n-C11H23
OBn
OBn
CO
35
CO
31 R =
n-C11H23
Scheme 2. Reagents and conditions: (a) (i) [bis(methyldiphenylphosphine)](1,5-cyclooctadiene) iridium(I) hexafluorophosphate, THF; (ii) NBS,
THF–H2O, 82% for 20 and 62% for 24; (b) Cl3CCN, DBU, CH2Cl2, 81%; (c) BF3ÆOEt2, CH2Cl2, 60% for 22 and 88% for 23; (d) 35, DCC, DMAP,
CH2Cl2, 71%; (e) (i) Zn dust, HOAc; (ii) 18, DCC, CH2Cl2, 60% for 26, 56% for 27 and 72% for 28; (f) NaBH3CN, HCl(g)–Et2O, THF, 0 ꢂC, 83% for
29, 67% for 30 and 71% for 31; (g) (i) (BnO)2PN(iPr)2, tetrazole, CH2Cl2; (ii) m-CPBA, CH2Cl2, 0 ꢂC, 85% for 32, 61% for 33, and 59% for 34; (h) H2,
Pd/C, THF–HOAc, 62% for 1, 95% for 2, and 75% for 3.
Removal of the N-Troc protecting group in 22, 23 and
25, followed by coupling with fatty acid 18 (Scheme 1) in
the presence of DCC, provides compounds 26, 27 and
28, respectively, in 56–72%. With all the acyl chains in-
stalled in proper positions of the disaccharide backbone,
the next task is to introduce the phosphate group at 40-
O-position. Regioselective reductive ring opening of the
4,6-di-O-benzylidene group in 26–28 by treating with
NaBH3CN and HCl(g)-saturated diethyl ether solu-
tion31 at 0 ꢂC results in the formation of 40-free hydroxyl
derivatives 29–31. Treatment of compounds 29–31 with
phosphoramidite and tetrazole, followed by oxidizing
with m-chloroperbenzoic acid (m-CPBA), furnishes
phosphates 32–34. The two-step phosphorylation proce-

Z.-H. Jiang et al. / Carbohydrate Research 342 (2007) 784–796
787
dure,32 through phosphite to phosphate, is highly effi-
cient for all three transformations, as indicated by
TLC profile of these reactions. The typical isolated yield
for this phosphorylation reaction is in the range of 59–
85%. The reduced yield in some cases is the result of
multiple chromatographic purification procedures,
which are often required in order to obtain highly pure
material. Finally, the removal of all benzyl groups in 32–
34 and the reduction of the allyl double bond in 33 are
facilitated by catalytic hydrogenation over Pd/C in
THF–HOAc to give target compounds 1–3, respectively,
in 62–95% yield. The synthesis and immunostimulant
activity of structure 1 as its triethylammonium salt was
reported earlier by Johnson et al.15
most active one in terms of inducing antigen specific
T-cell response while compound 2 with a propyl group
at the 3-O-position has lower activity. The differences
in both CPM and IFN-c levels induced by these com-
pounds indicate that the substituent at the 3-O-position
of monophosphoryl lipid A molecules affects the po-
tency of their immunostimulating activity. However,
the differences in both CPM and IFN-c levels are rela-
tively small; therefore, the effect exhibited by this 3-O-
substituent on the adjuvant activity of these molecules
is probably not significant.
The 3-O-acylated monophosphoryl lipid A analogue 3
shows very good adjuvant activity, and structurally it
has seven fatty acyl chains of uniform 14 carbon length.
Thus, compound 3 is of particular interest as a vaccine
adjuvant for further evaluation. A preliminary lethal
toxicity study was carried out for compound 3 in com-
parison with natural product R595 lipid A of which
the main component is the 3-O-deacylated hexa-acyl
monophosphoryl lipid A with its six fatty acyl chains
each having 12–16 carbon atoms.15 The actinomycin
D-sensitized33 C57 black mice are injected with different
doses of monophosphoryl lipid A analogue 3 or R595
lipid A. The mice injected with 50 lg of compound 3 have
all survived while the mice injected with the same dose of
R595 lipid A have all died. Two thirds of the mice have
also died when they were injected with a 10 lg dose of
R595 lipid A. This finding is a bit surprising since the re-
moval of the 3-O-acyl group of structurally diverse lipid
A molecules is believed to reduce lipid A toxicity.34,35
The manufacturing process of R595 lipid A includes a
basic hydrolysis step, which is supposed to have selec-
tively removed the (R)-3-hydroxytetradecanoyl group
at the 3-O-position of the main component.35 The re-
duced toxicity of R595 lipid A has been partially attrib-
uted to the removal of this 3-O-acyl group during the
manufacturing process. Our data suggest that the 3-O-
acylated hepta-acyl monophosphoryl lipid A may not
be more toxic than the 3-O-deacylated hexa-acyl ana-
logue. In order to unravel the effect of the 3-O-substitu-
ent on the toxicity profile of monophosphoryl lipid A
molecules, further toxicology investigation is needed.
In summary, we have described a straightforward syn-
thesis of three monophosphoryl lipid A analogues with
different substitution groups at the 3-O-position of the
reducing sugar. The strategy is geared towards the incor-
poration of different acyl groups at 2-N-, 3-O-, 20-N-
and 30-O-positions of the lipid A disaccharide backbone.
In a totally synthetic liposomal vaccine formulation, all
three monophosphoryl lipid A analogues (1–3) show
strong adjuvant activity in promoting T-cell prolifera-
tion and IFN-c production. Their immunostimulatory
potency is in the same level as that of the detoxified lipid
A product purified from S. minnesota R595. The preli-
minary lethal toxicity study indicates that 3-O-acylated
hepta-acyl monophosphoryl lipid A molecules may not
2.2. Biological evaluation
The immuno-adjuvant activity of monophosphoryl lipid
A analogues 1–3 has been evaluated in a totally syn-
thetic BLP25 liposomal vaccine system in comparison
with the natural lipid A product, R595 lipid A, which
is purified from the bacteria S. minnesota R595. The
BLP25 liposomal vaccine formulation contains
a
MUC1-derived 25 amino acid lipopeptide as the anti-
gen,18 with one of the synthetic compounds (1–3) as
adjuvant. Mice are immunized with a single dose of this
vaccine formulation containing 40 lg of the antigen and
20 lg of the adjuvant. Lymphocytes obtained from
draining lymph nodes of the sacrificed mice are re-acti-
vated by the same antigen and immune responses are
measured by T-cell proliferation (blastogenesis) and
the secretion of the cytokine interferon-gamma (IFN-
c) (Fig. 1). All three synthetic analogues 1–3 demon-
strate potent adjuvant activity in the same order of mag-
nitude as R595 lipid A in promoting antigen specific T-
cell response (CPM, counts per minute) and IFN-c pro-
duction (pg/mL). The same vaccine construct without a
monophosphoryl lipid A analogue as an adjuvant fails
to induce antigen specific T-cell proliferation and IFN-
c production. Compound 3 with an (R)-3-hydroxytetra-
decanoyl group at the 3-O-position appears to be the
T Cell Proliferation and IFN- Production
70000
CPM
60000
IFN- (pg/mL)
50000
40000
30000
20000
10000
0
1
2
3
R595
lipid
No lipid Saline
A
A
Adjuvant
Figure 1. T-Cell proliferation and IFN-c production.

788
Z.-H. Jiang et al. / Carbohydrate Research 342 (2007) 784–796
be more toxic than their 3-O-deacylated hexa-acyl
analogues.
C40H35NO7Æ1.3H2O (641.72): C, 72.23; H, 5.70; N,
2.10. Found: C, 72.24; H, 5.92; N, 1.83.
3.1.3. Benzyl 3-O-allyl-2-deoxy-6-O-triphenylmethyl-2-
phthalimido-b-D-glucopyranoside (9). The soln of 4
(2.02 g, 4.14 mmol) in dry DMF (15 mL) was added drop-
wise within 10 min to a mixture of sodium hydride
(230 mg, 9.58 mmol), allyl bromide (0.75 g, 0.50 mL,
6.21 mmol) and dry DMF (20 mL). The reaction mixture
was stirred at room temperature for 3 h and then MeOH
(1.0 mL) was added and stirred for 15 min. DMF was re-
moved under high vacuo, followed by aqueous work-up.
The residue was purified by flash chromatography (hex-
ane–EtOAc, 5:1) to give 5 as syrup (1.79 g, 82%). Com-
pound 5 (5.79 g, 11.0 mmol) was treated with acetic
acid–water (4:1, 130 mL) at 65 ꢂC for 6 h. The solvent
was removed and the residue was purified by flash chro-
matography (hexane–EtOAc, 1:2) to give 7 as syrup
(4.91 g, 95%). In a similar way as described for the prep-
aration of 8, compound 7 (4.79 g, 10.91 mmol) was con-
verted to 9 (5.87 g, 79%). Rf 0.66 (hexane–EtOAc, 1:2);
3. Experimental
3.1. Synthesis
3.1.1. General methods. All air and moisture sensitive
reactions have been performed under nitrogen atmo-
sphere. Anhydrous tetrahydrofuran (THF), N,N-
dimethylformamide (DMF), acetonitrile and CH2Cl2
are purchased from Aldrich, and other dry solvents
are prepared in accordance with standard procedures.
ACS grade solvents are purchased from Fisher and used
for chromatography without distillation. TLC plates
(Silica Gel 60 F254, thickness 0.25 mm, E. Merck) and
flash Silica Gel 60 (35–75 lm) for column chromatogra-
phy are purchased from Rose Scientific, Canada. 1H
NMR spectra are recorded on Brucker AM 300 MHz,
Varian Unity 500 MHz or Brucker DRX 600 MHz spec-
trometer with tetramethylsilane as internal standard.
Chemical shifts are reported in parts per million (d),
and signals are expressed as s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet) or br (broad). Pro-
tons of disaccharide backbone are indicated by regular
number (1–6) for the reducing end sugar, while for the
non-reducing end sugar they are indicated by prime
(10–60). Optical rotations are measured on a Perkin–
Elmer 241 Polarimeter at room temperature (20–22 ꢂC).
Elemental analysis data are obtained from the Micro-
analytical laboratory in the University of Alberta, Can-
ada. Electron-spray ionization mass spectrometric anal-
yses (ESIMS) are performed either on MS50B or MSD1
SPC mass spectrometer, and the data are reported in
m/z.
22
½aꢀD ꢁ37.2 (c 1.0, CHCl3); 1H NMR (300 MHz, CDCl3):
d 2.71 (d, J 2.8 Hz, 1H, OH), 3.46 (m, 2H, H-6a/b), 3.59
(m, 1H, H-5), 3.80 (m, 1H, H-4), 3.95 (m, 1H,
CHHCH@CH2), 4.15 (dd, J3,2 10.0, J3,4 8.5 Hz, 1H, H-
3), 4.16 (m, 1H, CHHCH@CH2), 4.25 (dd, J3,2 10.0, J2,1
8.0 Hz, 1H, H-2), 4.55 (d, J 12.0 Hz, 1H, CHHPh), 4.84
(d, J 12.0 Hz, 1H, CHHPh), 4.85 (m, 1H, CHH@CH),
5.02 (m, 1H, CHH@CH), 5.19 (d, J1,2 8.0 Hz, 1H, H-1),
5.59 (m, 1H, CH2@CH), 7.09–7.90 (m, 24H, Ar–H).
Anal. Calcd for C43H39NO7 (681.78): C, 75.75; H, 5.76;
N, 2.04. Found: C, 75.37; H, 5.67; N, 2.04.
3.1.4. Benzyl 2-deoxy-3,4-di-O-benzyl-2-phthalimido-6-
O-triphenylmethyl-b-D-glucopyranoside (10). The soln
of 8 (1.34 g, 2.09 mmol) in dry DMF (8 mL) was added
dropwise to the mixture of sodium hydride (120 mg,
5.02 mmol) and benzyl bromide (0.86 g, 0.60 mL,
5.02 mmol) in dry DMF (10 mL). The reaction mixture
was stirred at room temperature for 1 h and treated fur-
ther with an additional amount of benzyl bromide
(0.43 g, 0.30 mL, 2.51 mmol) and sodium hydride
(60 mg, 2.51 mmol). The reaction mixture was allowed
to stir for another 2 h. Methanol (2 mL) was then added
and the mixture was stirred for 10 more minutes. The
reaction was then poured into ice water (100 mL) and
extracted with diethyl ether (60 mL · 3). The combined
ether layer was washed with ice water (15 mL · 3), dried
with sodium sulfate and concentrated. The residue was
purified by flash chromatography (hexane–EtOAc, 5:1)
to give 10 (1.55 g, 90%). Rf 0.60 (hexane–EtOAc, 3:1);
3.1.2.
Benzyl
2-deoxy-2-phthalimido-6-O-triphenyl-
methyl-b-D-glucopyranoside (8). To a soln of 6 (1.0 g,
3.50 mmol) in dry pyridine (10 mL), triphenylmethyl
chloride (836 mg, 3.0 mmol) and DMAP (30.5 mg,
0.25 mmol) were added. The mixture was stirred at
room temperature for 20 h. Additional trityl chloride
(418 mg, 1.25 mmol) and DMAP (30.5 mg, 0.25 mmol)
were added and the mixture was stirred at 40 ꢂC for
4 h. The solvent was removed by co-distillation with tol-
uene and the residue was purified by flash chromatogra-
phy (hexane–EtOAc, 1:1) to give 8 (1.42 g, 88%). Rf 0.36
22
(hexane–EtOAc, 1:1); ½aꢀD ꢁ48.3 (c 0.6, CHCl3); 1H
NMR (300 MHz, CDCl3): d 2.70 (br s, 1H, OH), 3.00
(br s, 1H, OH), 3.44–3.53 (m, 2H, H-6a/b), 3.59 (m,
1H, H-5), 3.65 (dd, J4,5 9.5, J4,3 9.0 Hz, 1H, H-4), 4.21
(dd, J2,3 9.5, J2,1 8.0 Hz, 1H, H-2), 4.33 (dd, J2,3 9.5,
J4,3 9.0 Hz, 1H, H-3), 4.55 (d, J 12.0 Hz, 1H, CHHPh),
4.95 (d, J 12.0 Hz, 1H, CHHPh), 5.23 (d, J1,2 8.0 Hz,
1H, H-1), 7.10–7.80 (m, 24H, Ar–H). Anal. Calcd for
22
1
½aꢀD +5.5 (c 0.8, CHCl3); H NMR (300 MHz, CDCl3):
d 3.32 (dd, J 10.0, J6a,5 4.5 Hz, 1H, H-6a), 3.60 (m, 1H,
H-5), 3.68 (dd, J 10.0, J6b,5 1.8 Hz, 1H, H-6b), 4.03 (dd,
J2,3 9.5, J2,1 8.2 Hz, 1H, H-2), 4.33 (m, 2H, H-3, H-4),
4.43 (d, J 12.0 Hz, 1H, CHHPh), 4.47 (d, J 10.0 Hz,

Z.-H. Jiang et al. / Carbohydrate Research 342 (2007) 784–796
789
1H, CHHPh), 4.61 (d, J 12.0 Hz, 1H, CHHPh), 4.72 (d,
J 10.0 Hz, 1H, CHHPh), 4.80 (d, J 12.0 Hz, 1H,
CHHPh), 4.94 (d, J 12.0 Hz, 1H, CHHPh), 5.20 (d,
J1,2 Hz, 1H, H-1), 6.84–7.80 (m, 34H, Ar–H). Anal.
Calcd for C54H47NO7Æ1.3H2O (821.97): C, 76.72; H,
5.91; N, 1.66. Found: C, 76.56; H, 6.13; N, 1.52.
12.0 Hz, 1H, CHHPh), 4.68 (d, J 10.5 Hz, 1H, CHHPh),
4.79 (d, J 12.0 Hz, 1H, CHHPh), 4.80 (m, 1H,
CHH@CH), 4.84 (d, J 10.5 Hz, 1H, CHHPh), 5.00 (m,
1H, CHH@CH), 5.23 (d, J1,2 8.0 Hz, 1H, H-1), 5.55
(m, 1H, CH2@CH), 7.10–7.85 (m, 14H, Ar–H). Anal.
Calcd for C31H31NO7Æ0.7H2O (529.59): C, 68.67; H,
6.02; N, 2.58. Found: C, 68.46; H, 5.93; N, 2.53.
3.1.5. Benzyl 3-O-allyl-4-O-benzyl-2-deoxy-6-O-tri-
phenylmethyl-2-phthalimido-b-D-glucopyranoside (11). In
a similar way as described for the preparation of 10, com-
pound 9 (3.80 g, 5.57 mmol) was converted to 11 (2.45 g,
3.1.8. Benzyl 2-amino-2-deoxyl-3,4-di-O-benzyl-b-D-gluco-
pyranoside (14). To the soln of 12 (0.60 g, 1.04 mmol)
in 95% ethanol (40 mL) was added hydrazine mono-
hydrate (2.06 g, 2.0 mL, 41.2 mmol). The mixture was
refluxed for 2 h and then the solvent was removed under
diminished pressure. The residue was purified by flash
chromatography (1–2% MeOH in CH2Cl2) to give 14
22
57%). Rf 0.67 (hexane–EtOAc, 2:1); ½aꢀD ꢁ37.2 (c 1.0,
CHCl3); 1H NMR (300 MHz, CDCl3): d 3.32 (dd, J
10.0, J6a,5 3.5 Hz, 1H, H-6a), 3.62 (m, 1H, H-5), 3.69
(dd, J10.0, J6b,5 1.0 Hz, 1H, H-6b), 3.93 (m, 1H,
CHHCH@CH2), 3.96 (m, 1H, H-4), 4.25 (m, 1H,
CHHCH@CH2), 4.27 (dd, J2,3 10.5, J2,1 8.5 Hz, 1H, H-
2), 4.42 (dd, J2,3 10.5, J3,4 8.5 Hz, 1H, H-3), 4.44 (d,
J 10.0 Hz, 1H, CHHPh), 4.66 (d, J 12.0 Hz, 1H,
CHHPh), 4.70 (d, J 10.0 Hz, 1H, CHHPh), 4.83 (m,
1H, CHH@CH), 4.99 (d, J 12.0 Hz, 1H, CHHPh), 5.02
(m, 1H, CHH@CH), 5.27 (d, J1,2 8.5 Hz, 1H, H-1), 5.59
(m, 1H, CH2@CH), 6.92–7.90 (m, 29H, Ar–H). Anal.
Calcd for C50H45NO7Æ0.5H2O (771.91): C, 76.90; H,
5.94; N, 1.79. Found: C, 76.72; H, 6.11; N, 1.78.
22
(450 mg, 97%). Rf 0.20 (2% MeOH in CH2Cl2); ½aꢀD
1
ꢁ9.4 (c 0.35, CHCl3); H NMR (300 MHz, CDCl3): d
1.75 (br s, 3H, OH, NH2), 2.92 (dd, J2,3 9.0, J2,1
8.0 Hz, 1H, H-2), 3.43 (m, 1H, H-5), 3.49 (dd,
J4,3 = J4,5 9.5 Hz 1H, H-4), 3.66 (dd, J3,4 9.5, J3,2
9.0 Hz, 1H, H-3), 3.76 (dd, J 12.0, J6a,5 5.0 Hz, 1H, H-
6a), 3.91 (dd, J 12.0, J6b,5 2.5 Hz, 1H, H-6b), 4.39 (d,
J1,2 8.0 Hz, 1H, H-1), 4.63 (d, J 11.5 Hz, 1H, CHHPh),
4.70 (d, J 11.0 Hz, 1H, CHHPh), 4.74 (d, J 11.0 Hz,
1H, CHHPh), 4.86 (d, J 11.0 Hz, 1H, CHHPh), 4.88
(d, J 11.5 Hz, 1H, CHHPh), 4.99 (d, J 11.0 Hz, 1H,
CHHPh), 7.35 (m, 15H, Ar–H). Anal. Calcd for
C27H31NO5 (449.55): C, 72.14; H, 6.95; N, 3.15. Found:
C, 72.34; H, 7.15; N, 3.12.
3.1.6. Benzyl 2-deoxy-3,4-di-O-benzyl-2-phthalimido-b-D-
glucopyranoside (12). The soln of 10 (1.42 g,
1.73 mmol) in acetic acid–water (4:1, 60 mL) was stirred
at 110 ꢂC for 1 h. The solvent was removed by co-distil-
lation with toluene and the residue was purified by flash
chromatography (hexane–EtOAc, 2:1) to give 12
3.1.9. Benzyl 3-O-allyl-2-amino-4-O-benzyl-2-deoxy-b-D-
glucopyranoside (15). In a similar way as described for
the preparation of 14, compound 13 (0.90 g, 1.70 mmol)
22
(700 mg, 70%). Rf 0.31 (hexane–EtOAc, 2:1); ½aꢀD
1
+16.0 (c 0.25, CHCl3); H NMR (300 MHz, CDCl3): d
was converted to 15 (525 mg, 77%). Rf 0.28 (3% MeOH
22
1.90 (dd, J 6.5, J 6.5 Hz, 1H, OH), 3.54 (m, 1H, H-5),
3.73 (dd, J4,5 9.5, J4,3 9.0 Hz, 1H, H-4), 3.78 (m, 1H,
H-6a), 3.93 (m, 1H, H-6b), 4.19 (dd, J2,3 10.0, J2,1
8.5 Hz, 1H, H-2), 4.36 (dd, J3,2 10.0, J3,4 9.0 Hz, 1H,
H-3), 4.43 (d, J 12.0 Hz, 1H, CHHPh), 4.50 (d, J
12.0 Hz, 1H, CHHPh), 4.73 (d, J 11.0 Hz, 1H, CHHPh),
4.76 (d, J 12.0 Hz, 1H, CHHPh), 4.79 (d, J 12.0 Hz, 1H,
CHHPh), 4.90 (d, J 11.0 Hz, 1H, CHHPh), 5.20 (d, J1,2
8.5 Hz, 1H, H-1), 6.80–7.80 (m, 19H, Ar–H). Anal.
Calcd for C35H33NO7Æ0.8H2O (579.65): C, 70.76; H,
5.87; N, 2.35. Found: C, 70.74; H, 6.14; N, 2.20.
in CH2Cl2); ½aꢀ ꢁ17.0 (c 0.5, CHCl3); 1H NMR
D
(300 MHz, CDCl3): d 1.75 (s, 3H, NH2, OH), 2.87 (dd,
J 9.5, 8.0 Hz, 1H, H-2), 3.35 (dd, J 9.5, 9.5 Hz, 1H, H-
4), 3.36 (m, 1H, H-5), 3.57 (dd, J 9.5, 9.5 Hz, 1H, H-
3), 3.72 (dd, J 12.0, 4.0 Hz, 1H, H-6a), 3.88 (dd, J
12.0, 2.5 Hz, 1H, H-6b), 4.24 (m, 1H, CHHCH@CH2),
4.36 (d, J 8.0 Hz, 1H, H-1), 4.42 (m, 1H, CHHCH@
CH2), 4.62 (d, J 11.5 Hz, 1H, CHHPh), 4.64 (d, J
1.0 Hz, 1H, CHHPh), 4.82 (d, J 11.0 Hz, 1H, CHHPh),
4.88 (d, J 11.5 Hz, 1H, CHHPh), 5.18–5.33 (m, 2H, CH2@
CH), 5.97 (m, 1H, CH2@CH), 7.30 (m, 10H, Ar–H).
3.1.7. Benzyl 3-O-allyl-4-O-benzyl-2-deoxy-2-phthalim-
ido-b-D-glucopyranoside (13). In a similar way as de-
scribed for the preparation of 12, compound 11
(1.50 g, 1.94 mmol) was converted to 13 (0.90 g, 87%).
3.1.10. Benzyl 2-deoxy-3,4-di-O-benzyl-2-[(R)-3-tetra-
decanoyloxytetradecanamido]-b-D-glucopyranoside (16).
To the soln of compound 14 (410 mg, 0.913 mmol) in
dry CH2Cl2 (30 mL), compound 18 (623 mg, 1.37 mmol)
and DCC (564 mg, 2.74 mmol) were added. The mixture
was stirred at room temperature for 24 h. The solid was
filtered off and washed with CH2Cl2 (4 mL). The filtrate
was concentrated and the residue purified by flash chro-
matography (0.5–1% MeOH in CH2Cl2) to give 16
22
Rf 0.33 (hexane–EtOAc, 2:1); ½aꢀD ꢁ16.3 (c 1.0, CHCl3);
1H NMR (300 MHz, CDCl3): d 1.90 (dd, J 6.0, J 6.0 Hz,
1H, OH), 3.52 (m, 1H, H-6a), 3.65 (dd, J 9.5, J 8.5 Hz,
1H, H-4), 3.75 (m, 1H, H-5), 3.90 (m, 2H, H-6b,
CHHCH@CH2), 4.20 (m, 2H, H-3, CHHCH@CH2),
4.28 (dd, J2,3 10.0, J2,1 8.0 Hz, 1H, H-2), 4.52 (d, J
22
(664 mg, 82%). Rf 0.33 (2% MeOH in CH2Cl2); ½aꢀD

790
Z.-H. Jiang et al. / Carbohydrate Research 342 (2007) 784–796
1
ꢁ3.2 (c 0.6, CHCl3); H NMR (300 MHz, CDCl3): d
0.90 (t, J 7.0 Hz, 6H, 2CH3), 1.25 (m, 38H, 19CH2),
1.55 (m, 4H, 2CH2), 1.89 (dd, J 7.0, 6.0 Hz, 1H, OH),
2.15 (m, 2H, CH2), 2.27 (dd, J 15.0, 5.5 Hz, 1H,
CHH), 2.36 (dd, J 15.0, 6.0 Hz, 1H, CHH), 3.46 (m,
1H, H-5), 3.52 (m, 1H, H-4), 3.59 (dd, J 10.0, 9.0 Hz,
1H, H-3), 3.70 (m, 1H, H-6a), 3.86 (m, 1H, H-6b),
4.10 (dd, J 10.0, 8.0 Hz, 1H, H-2), 4.60 (d, J 12.0 Hz,
1H, CHHPh), 4.64 (d, J 11.5 Hz, 1H, CHHPh), 4.65
(d, J 11.5 Hz, 1H, CHHPh), 4.81 (d, J 11.5 Hz, 2H,
2CHHPh), 4.83 (d, J 12.0 Hz, 1H, CHHPh), 4.95 (d, J
8.0 Hz, 1H, H-1), 5.04 (m, 1H, lipid-3-H), 5.92 (d, J
8.0 Hz, 1H, NH), 7.30 (m, 15H, Ar–H). Anal. Calcd
for C55H83NO8 (886.26): C, 74.47; H, 9.44; N, 1.58.
Found: C, 74.25; H, 9.44; N, 1.64.
dried with sodium sulfate and concentrated. The residue
was purified by flash chromatography (hexane–EtOAc,
4:1 and 3:1) to give 20 (314 mg, 82%) as an anomeric
22
mixture (a/b, 4:1). Rf 0.36 (hexane–EtOAc, 3:1); ½aꢀD
1
ꢁ9.6 (c 1.0, CHCl3); H NMR (300 MHz, CDCl3) for
the a-isomer: d 0.88 (t, J 6.5 Hz, 6H, 2CH3), 1.24 (m,
38H, 19CH2), 1.50 (m, 4H, 2CH2), 2.16 (t, J 7.5 Hz,
2H, CH2), 2.49 (dd, J 15.0, 5.0 Hz, 1H, CHH), 2.60
(dd, J 15.0, 7.0 Hz, 1H, CHH), 3.65 (d, J 4.0 Hz, 1H,
OH), 3.70 (dd, J 9.5, 9.5 Hz, 1H, H-4), 3.77 (dd, J
10.0, 10.0 Hz, 1H, H-6a), 4.03 (m, 1H, H-2), 4.17 (m,
1H, H-5), 4.28 (dd, J = 10.0, 4.5 Hz, 1H, H-6b), 4.67,
4.75 (2d, J = 12.0 Hz, each 1H, Troc–CH2), 5.15 (m,
1H, lipid-3-H), 5.35 (dd, J 4.0, 4.0 Hz, 1H, H-1), 5.43
(dd, J 9.5, 9.5 Hz, 1H, H-3), 5.51 (s, 1H, CHPh), 5.81
(d, J 10.0 Hz, 1H, NH), 7.32–7.47 (m, 5H, Ar–H). Anal.
Calcd for C44H70Cl3NO10 (879.39): C, 60.10; H, 8.02; N,
1.59. Found: C, 60.11; H, 8.09; N, 1.61.
3.1.11. Benzyl 3-O-allyl-4-O-benzyl-2-deoxy-2-[(R)-3-tetra-
decanoyloxytetradecanamido]-b-D-glucopyranoside (17).
In a similar way as described for the preparation of 16,
compound 15 (510 mg, 1.28 mmol) was coupled with 18
(870 mg, 1.92 mmol) in the presence of DCC (659 mg,
3.20 mmol) to give 17 (853 mg, 80%) after flash chro-
matographic purification (2–5% acetone in CHCl3). Rf
3.1.13. 2-Deoxy-4,6-di-O-benzylidene-3-O-[(R)-3-tetra-
decanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycar-
bonylamino)-a-D-glucopyranosyl trichloroacetimidate (21).
To the soln of 20 (2.50 g, 2.88 mmol) in dry CH2Cl2
(30 mL), trichloroacetonitrile (8.64 g, 6.0 mL, 60.0
mmol) and DBU (10 drops) were added. The mix-
ture was stirred at room temperature for 2 h and con-
centrated under diminished pressure (not to dryness).
The residue was purified by flash chromatography (hex-
ane–EtOAc–Et3N, 6:1:1% and 5:1:1%) to give 21 (2.40 g,
22
0.38 (2% MeOH in CH2Cl2); ½aꢀD ꢁ6.0 (c 1.0, CHCl3);
1H NMR (300 MHz, CDCl3): d 0.89 (t, J 6.5 Hz, 6H,
2CH3), 1.25 (br s, 38H, 19CH2), 1.59 (m, 4H, 2CH2),
1.86 (t, J 7.0 Hz, 1H, OH), 2.23 (t, J 7.5 Hz, 2H,
CH2), 2.37 (dd, J 15.0, 5.5 Hz, 1H, CHH), 2.48 (dd, J
15.0, 5.5 Hz, 1H, CHH), 3.40 (m, 2H, H-2, H-5), 3.52
(dd, J 9.5, 8.5 Hz, 1H, H-4), 3.70 (m, 1H, H-6a), 3.85
(m, 1H, H-6b), 4.00 (dd, J 10.0, 8.5 Hz, 1H, H-3), 4.14
(m, 1H, CHHCH@CH2), 4.26 (m, 1H, CHHCH@CH2),
4.59 (d, J 11.5 Hz, 1H, CHHPh), 4.63 (d, J 11.0 Hz, 1H,
CHHPh), 4.82 (d, J 11.0 Hz, 1H, CHHPh), 4.83 (d, J
11.5 Hz, 1H, CHHPh), 4.96 (d, J = 8.0 Hz, 1H, H-1),
5.08 (m, 1H, lipid-3-H), 5.13 (m, 1H, CHH@CH), 5.23
(m, 1H, CHH@CH), 5.88 (m, 1H, CH@CH2), 6.00 (d,
J 8.0 Hz, 1H, NH), 7.35 (m, 10H, Ar–H). Anal. Calcd
for C51H81NO8Æ0.7H2O (836.20): C, 72.17; H, 9.78; N,
1.65. Found: C, 72.07; H, 9.81; N, 1.72.
22
81%). Rf 0.25 (hexane–EtOAc, 8:1); ½aꢀD +35.0 (c 1.0,
CHCl3); 1H NMR (300 MHz, CDCl3): d 0.90 (t, J
7.0 Hz, 6H, 2CH3), 1.25 (m, 38 Hz, 19CH2), 1.50 (m,
4H, 2CH2), 2.20 (t, J 7.5 Hz, 2H, CH2), 2.56 (dd, J
15.5, 5.5 Hz, 1H, CHH), 2.65 (dd, J 15.5, 7.0 Hz, 1H,
CHH), 3.81 (dd, J 10.0, 10.0 Hz, 1H, H-4), 3.83 (dd, J
10.0, 10.0 Hz, 1H, H-6a), 4.06 (m, 1H, H-5), 4.25
(ddd, J 10.0, 9.0, 4.0 Hz, 1H, H-2), 4.36 (dd, J 10.0,
5.0 Hz, 1H, H-6b), 4.63, 4.78 (2d, J 12.0 Hz, each 1H,
Troc–CH2), 5.18 (m, 1H, lipid-3-H), 5.45 (dd, J 10.0,
10.0 Hz, 1H, H-3), 5.56 (d, J 9.0 Hz, 1H, NH), 5.58 (s,
1H, CHPh), 6.42 (d, J 4.0 Hz, 1H, H-1), 7.30–7.45 (m,
5H, Ar–H), 8.73 (s, H, NH). Anal. Calcd for
C46H70Cl6N2O10 (1023.78): C, 53.97; H, 6.89; N, 2.74.
Found: C, 53. 80; H, 6.77; N, 2.80.
3.1.12. 2-Deoxy-4,6-di-O-benzylidene-3-O-[(R)-3-tetra-
decanoyloxytetradecanoyl]-2-(2,2,2-trichloroethoxycar-
bonylamino)-a/b-D-glucopyranose (20). [Bis(methyldi-
phenylphosphine)](1,5-cyclooctadiene) iridium(I) hexa-
fluorophosphate (37 mg, 0.044 mmol) was suspended
in dry THF (5 mL) and hydrogen gas was bubbled in
for 5 min to give a yellowish soln, which was added to
the soln of 19 (400 mg, 0.44 mmol) in dry THF
(5 mL). The mixture was stirred at room temperature
for 2 h. Water (0.5 mL) and N-bromosuccinimide
(NBS, 117 mg, 0.66 mmol) were then added and the
reaction was stirred for 1 hour longer. The remainder
obtained from solvent removal was dissolved in EtOAc
(200 mL) and washed with saturated sodium bicarbon-
ate soln (20 mL · 2). Combined organic layers were
3.1.14. Benzyl 2-deoxy-6-O-{2-deoxy-4,6-di-O-benzyl-
idene-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-(2,2,2-
trichloroethoxycarbonylamino)-b-D-glucopyranosyl}-3,4-
di-O-benzyl-2-[(R)-3-tetradecanoyloxytetradecanamido]-
b-D-glucopyranoside (22). To the soln of 16 (290 mg,
0.328 mmol) and 21 (503 mg, 0.492 mmol) in dry
˚
CH2Cl2 (6 mL) were added molecular sieves (4 A,
0.5 g). The mixture was stirred under nitrogen at room
temperature for 20 min. Trifluoroboron etherate soln
(0.1 M in CH2Cl2, 1.3 mL) was added dropwise within
20 min. The mixture was stirred for 1 h and then poured

Z.-H. Jiang et al. / Carbohydrate Research 342 (2007) 784–796
791
into saturated sodium bicarbonate soln (10 mL) and
extracted with CH2Cl2 (20 mL · 3). Combined organic
layers were dried with sodium sulfate and concentrated.
The residue was purified by silica gel chromatography
(0.5–1% MeOH in CH2Cl2) to give 22 (457 mg, 80%).
was converted to 24 (200 mg, 62%). Rf 0.30 (5% acetone
22
in CHCl3); ½aꢀ ꢁ25.7 (c 0.83, CHCl3); 1H NMR
D
(300 MHz, CDCl3): d 0.90 (t, J 6.5 Hz, 12H, 4CH3),
1.25 (br s, 76H, 38CH2), 1.55 (m, 8H, 4CH2), 1.70 (s,
1H, OH), 2.17 (t, J 7.0 Hz, 2H, CH2), 2.26 (t, J
7.0 Hz, 2H, CH2), 2.43 (m, 2H, CH2), 2.50 (dd, J 14.0,
5.5 Hz, 1H, CHH), 2.60 (dd, J 15.0, 7.5 Hz, 1H,
CHH), 3.40–3.90 (m, 9H, H-2, H-3, H-4, H-5, H-6a,
H-20, H-40, H-50, H-60a), 4.13 (d, J 10.0 Hz, 1H, H-
6b), 4.34 (dd, J 10.0, 5.0 Hz, 1H, H-60b), 4.51, 4.52
(2d, J 8.5 Hz, each 1H, H-1, H-10), 4.60 (d, J 12.5 Hz,
1H, CHHPh), 4.66 (m, 3H, CHHPh, Cl3CCH2O), 4.90
(d, J 12.5 Hz, 1H, CHHPh), 4.98 (d, J 11.5 Hz, 1H,
CHHPh), 5.04–5.25 (m, 3H, H-30, 2 lipid-3-H), 5.49 (s,
1H, CHPh), 6.02 (d, J 5.0 Hz, 1H, NH), 7.40 (m, 15H,
Ar–H). Anal. Calcd for C92H145Cl3N2O17Æ0.8H2O
(1657.52): C, 66.09; H, 8.84; N, 1.67. Found: C, 66.06;
H, 8.84; N, 1.64.
22
Rf 0.21 (3% acetone in CHCl3); ½aꢀD ꢁ17.8 (c 0.6,
CHCl3); 1H NMR (300 MHz, CDCl3): d 0.90 (t, J
7.0 Hz, 12H, 4CH3), 1.25 (m, 76H, 38CH2), 1.52 (m,
8H, 4CH2), 2.15 (m, 4H, 2CH2), 2.26, 2.35 (2dd, J
14.0, 6.0 Hz, each 1H, CH2), 2.48 (dd, J 15.0, 5.5 Hz,
1H, CHH), 2.58 (dd, J 15.0, 7.0 Hz, 1H, CHH), 3.34–
3.78 (m, 8H, H-2, H-3, H-4, H-5, H-6a, H-20, H-40, H-
60a), 4.02–4.13 (m, 2H, H-6b, H-50), 4.30 (dd, J 10.5,
5.0 Hz, 1H, H-60b), 4.52 (d, J 8.0 Hz, 1H, H-10), 4.57–
4.90 (m, 8H, 3CH2Ph, Troc–CH2), 4.89 (d, J 8.0 Hz,
1H, H-1) 5.02 (m, 1H, lipid-3-H), 5.15 (m, 3H, NH,
H-30, lipid-3-H), 5.55 (s, 1H, CHPh), 6.00 (d, J 8.0 Hz,
1H, NH), 7.25–7.45 (m, 20H, Ar–H). Anal. Calcd for
C99H151Cl3N2O17 (1747.64): C, 68.04; H, 8.71; N, 1.60.
Found: C, 67.92, H, 8.85, N, 1.64.
3.1.17. Benzyl 4-O-benzyl-3-O-[(R)-3-benzyloxytetra-
decanoyl]-2-deoxy-6-O-{2-deoxy-4,6-di-O-benzylidene-3-
O-[(R)-3-tetradecanoyloxytetradecanoyl]-2-(2,2,2-trichlo-
roethoxycarbonylamino)-b-D-glucopyranosyl}-2-[(R)-3-tet-
radecanoyloxytetradecanamido]-b-D-glucopyranoside (25).
To the mixture of compound 24 (670 mg, 0.405 mmol),
35 (270 mg, 0.81 mmol), DCC (208 mg, 1.01 mmol) and
DMAP (25 mg, 0.20 mmol) was added dry CH2Cl2
(10 mL). The reaction mixture was stirred at room tem-
perature for 72 h. The solid was filtered and washed with
CH2Cl2. The filtrate was concentrated under diminished
pressure and the residue was purified by flash chroma-
tography (CH2Cl2–hexane–acetone, 2:1:3%; and 1%
MeOH in CH2Cl2) to give 25 (570 mg, 71%). Rf 0.60
3.1.15. Benzyl 3-O-allyl-4-O-benzyl-2-deoxy-6-O-{2-
deoxy-4,6-di-O-benzylidene-3-O-[(R)-3-tetradecanoyloxy-
tetradecanoyl]-2-(2,2,2-trichloroethoxycarbonylamino)-b-
D-glucopyranosyl}-2-[(R)-3-tetradecanoyloxytetradecan-
amido]-b-D-glucopyranoside (23). In a similar method
as described for the preparation of 22, compound 23
was prepared by reacting imidate 21 (1.15 g, 1.12 mmol)
and the glycosylation acceptor 17 (652 mg, 0.75 mmol) in
the presence of catalyst BF3ÆOEt2 (0.15 M in CH2Cl2,
3.5 mL). Purification by flash chromatography (1–2%
acetone in CHCl3) yielded 23 (1.30 g, 83%). Rf 0.36
22
1
(6% acetone in CHCl3); ½aꢀD ꢁ18.6 (c 0.5, CHCl3); H
NMR (300 MHz, CDCl3): d 0.86 (t, J 6.5 Hz, 12H,
4CH3), 1.22 (br s, 76H, 38CH2), 1.53 (m, 8H, 4CH2),
2.15 (t, J 7.5 Hz, 2H, CH2), 2.20 (t, J 7.5H, 2H, CH2),
2.32 (dd, J 14.0, 5.5 Hz, 1H, CHH), 2.42 (dd, J 14.0,
6.0 Hz, 1H, CHH), 2.47 (dd, J 15.0, 5.0 Hz, 1H, CHH),
2.57 (dd, J 15.0, 7.0 Hz, 1H, CHH), 3.34–4.21 (m, 12H,
H-2, H-3, H-4, H-5, 2H-6, H-20, H-40, H-50, H-60a,
CH2CH@CH2), 4.30 (dd, J 10.0, 5.0 Hz, 1H, H-60b),
4.51 (d, J 8.5 Hz, 1H, H-10), 4.57 (m, 4H, 2CHHPh,
Cl3CCH2O), 4.78 (d, J 11.0 Hz, 1H, CHHPh), 4.85 (d,
J 11.5 Hz, 1H, CHHPh), 4.88 (d, J 8.0 Hz, 1H, H-1),
5.00–5.25 (m, 5H, H-30, 2 lipid-3-H, CH2@CH), 5.45
(s, 1H, CHPh), 5.85 (m, 1H, CH@CH2), 6.00 (d, J
8.0 Hz, 1H, NH), 7.30 (m, 15H, Ar–H). Anal. Calcd
for C95H149Cl3N2O17 (1697.58): C, 67.21; H, 8.85; N,
1.65. Found: C, 66.99; H, 8.96; N, 1.65.
22
(CH2Cl2–hexane–acetone, 10:5:1); ½aꢀD ꢁ20.0 (c 1.0,
CHCl3); 1H NMR (600 MHz, CDCl3): d 0.88 (t, J
7.0 Hz, 15H, 5CH3), 1.25 (m, 74H, 37CH2), 1.53 (m,
10H, 5CH2), 2.17 (t, J 7.5 Hz, 2H, CH2), 2.22 (dd, J
15.0, 6.0 Hz, 1H, CHH), 2.24 (t, J 7.5 Hz, 2H, CH2),
2.34 (dd, J 15.0, 6.5 Hz, 1H, CHH), 2.45 (dd, J 16.0,
5.0 Hz, 1H, CHH), 2.50 (dd, J 15.5, 5.5 Hz, 1H, CHH),
2.55 (dd, J 16.0, 7.5 Hz, 1H, CHH), 2.59 (dd, J 15.5,
7.5 Hz, 1H, CHH), 3.38 (ddd, J 10.0, 10.0, 5.0 Hz, 1H,
H-50), 3.56 (m, 2H, H-4, H-5), 3.62 (m, 1H, H-2), 3.64
(dd, J 10.0, 10.0 Hz, 1H, H-40), 3.69 (dd, J 11.0,
5.0 Hz, 1H, H-6a), 3.76 (dd, J 10.0, 10,0 Hz, 1H, H-
60a), 3.83 (m, 1H, lipid-3-H), 3.95 (m, 1H, H-20), 4.05
(br d, J 11.0 Hz, 1H, H-6b), 4.32 (dd, J 10.0, 5.0 Hz,
1H, H-60b), 4.45 (d, J 11.0 Hz, 1H, CHHPh), 4.48 (d, J
11.0 Hz, 2H, 2CHHPh), 4.51 (d, J 8.0 Hz, 1H, H-1),
4.59 (d, J 8.0 Hz, 1H, H-10), 4.60–4.67 (m, 4H, 2CHHPh,
Cl3CCH2O), 4.85 (d, J 12.0 Hz, 1H, CHHPh), 5.01 (m,
1H, lipid-3-H), 5.12 (d, J 9.0 Hz, 1H, NH), 5.19 (m,
4H, H-3, H-30, 2 lipid-3-H), 5.48 (s, 1H, CHPh), 5.71
(d, J 8.0 Hz, 1H, NH), 7.20–7.45 (m, 20H, Ar–H). Anal.
Calcd for C113H177Cl3N2O19 (1974.00): C, 68.76; H, 9.04;
N, 1.42. Found: C, 68.68; H, 9.10; N, 1.39.
3.1.16. Benzyl 4-O-benzyl-2-deoxy-6-O-{2-deoxy-4,6-di-
O-benzylidene-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-
2-(2,2,2-trichloroethoxycarbonylamino)-b-D-glucopyranos-
yl}-2-[(R)-3-tetradecanoyloxytetradecanamido]-b-D-gluco-
pyranoside (24). In a similar way as described for the
preparation of 20, compound 23 (350 mg, 0.195 mmol)

792
Z.-H. Jiang et al. / Carbohydrate Research 342 (2007) 784–796
3.1.18. Benzyl 2-deoxy-6-O-{2-deoxy-4,6-di-O-benzyl-
idene-2-[(R)-3-tetradecanoyloxytetradecanamido]-3-O-[(R)-
3-tetradecanoyloxytetradecanoyl]-b-D-glucopyranosyl}-
3,4-di-O-benzyl-2-[(R)-3-tetradecanoyloxytetradecanam-
ido]-b-D-glucopyranoside (26). Compound 22 (224 mg,
0.126 mmol) was treated with zinc dust (5.0 g) in acetic
acid–EtOAc (4:1, 300 mL) at room temperature for
24 h. The solid was filtered and washed with CH2Cl2,
and the filtrate concentrated under diminished pressure.
The residue was re-dissolved in CH2Cl2 (200 mL) and the
soln washed with saturated aq bicarbonate soln (20 mL).
The organic layer was dried with sodium sulfate and con-
centrated to give the free amine (192 mg, 95%).
H-1), 4.85 (d, J 11.0 Hz, 1H, CHHPh), 4.99–5.30 (m, 6H,
H-30, CH2@CH, 3 lipid-3-H), 5.48 (s, 1H, CHPh), 5.87
(m, 1H, CH2@CH), 5.91 (d, J 8.5 Hz, 1H, NH), 6.07 (d,
J 8.0 Hz, 1H, NH), 7.35 (m, 15H, Ar–H). Anal. Calcd
for C120H200N2O18 (1958.90): C, 73.58; H, 10.30; N,
1.43. Found: C, 73.40; H, 10.70; N, 1.39.
3.1.20. Benzyl 4-O-benzyl-3-O-[(R)-3-benzyloxytetra-
decanoyl]-2-deoxy-6-O-{2-deoxy-4,6-di-O-benzylidene-2-
[(R)-3-tetradecanoyloxytetradecanamido]-3-O-[(R)-3-tet-
radecanoyloxytetradecanoyl]-b-D-glucopyranosyl}-2-[(R)-
3-tetradecanoyloxytetradecanamido]-b-D-glucopyranoside
(28). In a similar way as described for the preparation
of 26, compound 25 (550 mg, 0.279 mmol) was con-
verted to free amine (500 mg, 100%). The free amine
(270 mg, 0.15 mmol) was coupled with 18 (205 mg,
0.45 mmol) in the presence of DCC (139 mg, 0.68 mmol)
to give 28 (240 mg, 72%). Rf 0.36 (5% acetone in
The free amine (175 mg, 0.11 mmol), 18 (101 mg,
0.22 mmol) and DCC (68 mg, 0.33 mmol) were dissolved
in dry CH2Cl2 (5 mL) and the mixture was stirred at
room temperature for 48 h. The solid was filtered and
washed with CH2Cl2. The filtrate was concentrated
under diminished pressure and the residue purified by
repeated flash chromatography (CH2Cl2–hexane–acetone,
2:1:3%, and CH2Cl2–MeOH, 100:1) to give 26 (150 mg,
22
CHCl3); ½aꢀD ꢁ19.6 (c 0.7, CHCl3); 1H NMR
(300 MHz, CDCl3): d 0.89 (t, J 6.5 Hz, 21H, 7CH3),
1.25 (br s, 132H, 66CH2), 1.50 (m, 14H, 7CH2), 2.14–
2.65 (m, 14H, 7CH2), 3.40–4.50 (m, 10H, H-2, H-4, H-
5, H-6a/b, H-20, H-40, H-50, H-60a, lipid-3-H), 4.31
22
67%). Rf 0.27 (2% MeOH in CH2Cl2); ½aꢀD ꢁ14.2 (c 0.5,
1
CHCl3); H NMR (300 MHz, CDCl3): d 0.90 (t, J 7.0
(dd, Jgem 10.5, J6 b,5 4.5 Hz, 1H, H-60b), 4.44 (d, Jgem
11.0 Hz, 1H, CHHPh), 4.50 (d, Jgem 11.0 Hz, 2H,
2CHHPh), 4.58 (d, J1,2 8.0 Hz, 1H, H-1), 4.59 (d, 1H,
CH2Ph), 4.63 (d, Jgem 12.0 Hz, 1H, CHHPh), 4.75 (d,
0
0
Hz, 18H, 6CH3), 1.25 (m, 114H, 57CH2), 1.53 (m, 12H,
6CH2), 2.15 (t, J 7.0 Hz, 4H, 2CH2), 2.23–2.39 (m, 6H,
3CH2), 2.56 (dd, J 15.5, 5.5 Hz, 1H, CHH), 2.60 (dd, J
15.5, 7.0 Hz, 1H, CHH), 3.37–4.00 (m, 9H, H-2, H-3,
H-4, H-5, H-6a, H-20, H-40, H-50, H-60a), 4.09 (dd, J
11.0, 2.0 Hz, 1H, H-6b), 4.27 (dd, J 11.0, 4.5 Hz, 1H,
H-60b), 4.58–4.88 (m, 7H, 3CH2Ph, H-10), 4.82 (d, J
7.5 Hz, 1H, H-1), 5.00–5.09 (m, 2H, 2 lipid-3-H), 5.16
(m, 1H, lipid-3-H), 5.26 (dd, J 10.0, 10.0 Hz, 1H, H-30),
5.47 (s, 1H, CHPh), 5.93 (d, J 8.5 Hz, 1H, NH), 6.06
(d, J 8.0 Hz, 1H, NH), 7.25–7.45 (m, 20H, Ar–H). Anal.
Calcd for C124H202N2O18Æ0.5H2O (2008.96): C, 73.80; H,
10.14; N, 1.39. Found: C, 73.64; H, 9.88; N, 1.41.
J1 ;2 8:5 Hz, 1H, H-10), 4.85 (d, 1H, CHHPh), 5.04 (m,
0
0
2H, 2 lipid-3-H), 5.15 (m, 2H, H-3, lipid-3-H), 5.27
(dd, J3 2 ¼ J3 ;4 10:0 Hz, 1H, H-30), 5.48 (s, 1H, CHPh),
5.80 (d, J 9.0 Hz, 1H, NH), 5.93 (d, J 8.5 Hz, 1H, NH),
7.20–7.45 (m, 20H, Ar–H). Anal. Calcd for
0
0
0
0
C
138H228N2O20 (2235.32): C, 74.15; H, 10.28; N, 1.25.
Found: C, 74.00; H, 10.63; N, 1.40.
3.1.21. Benzyl 2-deoxy-6-O-{6-O-benzyl-2-deoxy-2-[(R)-
3-tetradecanoyloxytetradecanamido]-3-O-[(R)-3-tetradeca-
noyloxytetradecanoyl]-b-D-glucopyranosyl}-3,4-di-O-benz-
yl-2-[(R)-3-tetradecanoyloxytetradecanamido]-b-D-gluco-
pyranoside (29). To the soln of 26 (135 mg, 0.067
mmol) in dry tetrahydrofuran (8.0 mL), molecular sieves
3.1.19. Benzyl 3-O-allyl-4-O-benzyl-2-deoxy-6-O-{2-
deoxy-4,6-di-O-benzylidene-2-[(R)-3-tetradecanoyloxytetra-
decanamido]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-
b-D-glucopyranosyl}-2-[(R)-3-tetradecanoyloxytetradeca-
namido]-b-D-glucopyranoside (27). In a similar way as
described for the preparation of 26, compound 23
(350 mg, 0.206 mmol) was converted to free amine
(314 mg, 100%), which was coupled with 18 (191 mg,
0.42 mmol) in the presence of DCC (130 mg, 0.62 mmol)
˚
(4 A, 0.5 g) were added and the mixture was stirred at
room temperature for 20 min. Sodium cyanoboro-
hydride (211 mg, 3.36 mmol) was added and the mixture
was cooled to 0 ꢂC and HCl(g)–Et2O soln was added
dropwise till no gas was evolved. Additional sodium
cyanoborohydride (211 mg, 3.36 mmol) was added, fol-
lowed by slow addition of HCl(g)–Et2O until no gas was
formed. The mixture was poured into satd aq NaHCO3
soln (50 mL) and extracted with EtOAc (100 mL · 3).
The organic layer was washed with satd sodium chloride
soln (50 mL), dried over sodium sulfate, and concen-
trated. The residue was purified by flash chromato-
graphy (2–5% acetone in CHCl3) to afford 29 (112 mg,
to give 27 (226 mg, 56%). Rf 0.25 (5% acetone in CHCl3);
22
½aꢀ ꢁ16.0 (c 0.5, CHCl3); 1H NMR (300 MHz, CDCl3): d
D
0.89 (t, J 6.5 Hz, 18H, 6CH3), 1.23 (br s, 114H, 57CH2),
1.55 (m, 12H, 6CH2), 2.13–2.48 (m, 10H, 5CH2), 2.50
(dd, J 15.0, 5.5 Hz, 1H, CHH), 2.59 (dd, J 15.0, 7.5 Hz,
1H, CHH), 3.38–4.24 (m, 12H, H-2, H-3, H-4, H-5, 2H-
6, H-20, H-40, H-50, H-60a, CH2CH@CH2), 4.30 (dd, J
10.5, 5.5 Hz, 1H, H-60b), 4.58 (d, J 11.0 Hz, 1H, CHHPh),
4.59 (d, J 12.0 Hz, 1H, CHHPh), 4.73 (d, J 8.5 Hz, 1H, H-
10), 4.77 (d, J 12.0 Hz, 1H, CHHPh), 4.83 (d, J 8.0 Hz, 1H,
22
83%). Rf 0.20 (2% MeOH in CH2Cl2); ½aꢀD ꢁ13.5 (c
1
0.6, CHCl3); H NMR (300 MHz, CDCl3): d 0.89 (t, J

Z.-H. Jiang et al. / Carbohydrate Research 342 (2007) 784–796
793
6.5 Hz, 18H, 6CH3), 1.25 (m, 114H, 57CH2), 1.50 (m,
12H, 6CH2), 2.14 (t, J 7.0 Hz, 2H, CH2), 2.23–2.60 (m,
10H, 5CH2), 3.33 (d, J 3.3 Hz, 1H, OH), 3.44–3.96 (m,
10H, H-2, H-3, H-4, H-5, H-6a, H-20, H-40, H-50, 2H-
60), 4.09 (dd, J 10.0, 2.0 Hz, 1H, H-6b), 4.49–4.86 (m,
9H, 4CH2Ph, H-10), 4.80 (d, J 7.5 Hz, 1H, H-1), 4.92–
5.18 (m, 4H, H-30, 3 lipid-3-H), 5.80 (d, J 9.0 Hz, 1H,
NH), 5.95 (d, J 8.5 Hz, 1H, NH), 7.30 (m, 20H, Ar–
H). Anal. Calcd for C124H204N2O18ÆH2O (2010.98): C,
73.40; H, 10.23; N, 1.38. Found: C, 73.40; H, 10.04;
N, 1.38.
4.47–4.61 (m, 8H, H-1, H-10, 6CHHPH), 4.83 (d, Jgem
12.0 Hz, 1H, CHHPh), 4.93–5.18 (m, 5H, H-3, H-30, 3
lipid-3-H), 5.72 (d, J 9.5 Hz, 1H, NH), 5.81 (d, J
9.0 Hz, 1H, NH), 7.30 (m, 20H, Ar–H). Anal. Calcd
for C138H230N2O20 (2237.34): C, 74.08; H, 10.37; N,
1.25. Found: C73.75; H, 10.41; N, 1.41.
3.1.24. Benzyl 2-deoxy-6-O-{6-O-benzyl-4-O-(di-O-benz-
yl-phosphono)-2-deoxy-2-[(R)-3-tetradecanoyloxytetra-
decanamido]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-
b-D-glucopyranosyl}-3,4-di-O-benzyl-2-[(R)-3-tetradeca-
noyloxytetradecanamido]-b-D-glucopyranoside (32). To
the soln of 29 (61 mg, 0.030 mmol) in dry CH2Cl2
(3 mL) were added 1H-tetrazole (12.6 mg, 0.18 mmol)
and dibenzyl diisopropylphosphoramidite (42 mg,
0.041 mL, 0.12 mmol). The mixture was stirred at room
temperature for 30 min and then cooled to 0 ꢂC. m-
Chloroperbenzoic acid (m-CPBA, 75 mg, 55%,
0.24 mmol) was added and the mixture was stirred for
30 min at 0 ꢂC. The mixture was then poured into 10%
aq NaHSO3 soln (10 mL) and extracted with CH2Cl2
(10 mL · 3). The combined organic layer was washed
with satd aq NaHCO3 soln (10 mL), dried with sodium
sulfate and concentrated. The residue was purified by re-
peated flash chromatography (1–5% acetone in CHCl3
and then toluene–acetone, from 18:1 to 12:1) to afford
3.1.22. Benzyl 3-O-allyl-4-O-benzyl-2-deoxy-6-O-{2-
deoxy-6-O-benzyl-2-[(R)-3-tetradecanoyloxytetradecan-
amido]-3-O-[(R)-3-tetradecanoyloxytetradecanoyl]-b-D-glu-
copyranosyl}-2-[(R)-3-tetradecanoyloxytetradecanamido]-
b-D-glucopyranoside (30). In a similar way as described
for the preparation of 29, compound 27 (194 mg,
0.10 mmol) was converted to 30 (130 mg, 67%). Rf
22
0.22 (8% acetone in CHCl3); ½aꢀD ꢁ9.6 (c 0.5, CHCl3);
1H NMR (600 MHz, CDCl3): d 0.89 (t, J 7.0 Hz, 18H,
6CH3), 1.25 (br s, 114H, 57CH2), 1.49–1.58 (m, 12H,
6CH2), 2.21 (t, J 8.0 Hz, 2H, CH2), 2.26 (m, 3H, CH2,
CHH), 2.27 (t, J 8.0 Hz, 2H, CH2), 2.33 (dd, J 14.0,
6.0 Hz, 1H, CHH), 2.36 (dd, J 15.0, 6.5 Hz, 1H,
CHH), 2.43 (dd, J 15.0, 6.5 Hz, 1H, CHH), 2.50 (dd, J
16.5, 6.50 Hz, 1H, CHH), 2.53 (dd, J 16.5, 8.0 Hz, 1H,
CHH), 3.28 (d, J 3.0 Hz, 1H, OH), 3.43 (m, 2H, H-4,
H-50), 3.54 (m, 2H, H-2, H-6a), 3.62 (ddd, J 10.0, 9.0,
3.0 Hz, 1H, H-40), 3.71 (m, 3H, H-5, 2H-60), 3.84 (m,
2H, H-3, H-20), 4.06 (dd, J 11.0, 2.5 Hz, 1H, H-6b),
4.10–4.20 (m, 2H, CH2CH@CH2), 4.52 (d, J 12.0 Hz,
1H, CHHPh), 4.58 (m, 4H, H-10, 3CHHPh), 4.74 (d, J
10.5 Hz, 1H, CHHPh), 4.80 (d, J 8.0 Hz, 1H, H-1),
4.83 (d, J 11.5 Hz, 1H, CHHPh), 4.95 (dd, J 10.0,
9.0 Hz, 1H, H-30), 5.02 (m, 1H, lipid-3-H), 5.10 (m,
3H, 2 lipid-3-H, CHH@CH), 5.22 (m, 1H, CHH@CH),
5.77 (d, J 9.0 Hz, 1H, NH), 5.85 (m, 1H, CH2@CH),
5.98 (d, J 8.0 Hz, 1H, NH), 7.30 (m, 15H, Ar–H). Anal.
Calcd for C120H202N2O18 (1960.92): C, 73.50; H, 10.38;
N, 1.43. Found: C, 73.25; H, 10.95; N, 1.60.
22
32 (58 mg, 85%). Rf 0.17 (1% acetone in CHCl3); ½aꢀD
1
ꢁ3.1 (c 0.35, CHCl3); H NMR (300 MHz, CDCl3): d
0.87 (t, J 6.5 Hz, 18H, 6CH3), 1.24 (m, 114H, 57CH2),
1.40–1.57 (m, 12H, 6CH2), 2.11–2.50 (m, 12H, 6CH2),
3.52–3.94 (m, 9H, H-2, H-3, H-4, H-5, H-6a, H-20, H-
50, 2H-60), 4.09 (dd, J 11.0, 2.0 Hz, 1H, H-6b), 4.44
(m, 3H, CH2Ph, H-40), 4.56–4.90 (m, 12H, 6CH2Ph),
4.78 (d, J 8.0 Hz, 1H, H-10), 4.98 (d, J 8.0 Hz, 1H, H-
1), 5.05 (m, 2H, 2 lipid-3-H), 5.16 (m, 1H, lipid-3-H),
5.39 (dd, J 10.0, 9.0 Hz, 1H, H-30), 5.88 (d, J 8.5 Hz,
1H, NH), 6.08 (d, J 8.0 Hz, 1H, NH), 7.25 (m, 30H,
Ar–H). Anal. Calcd for
C138H217N2O21PÆ0.5H2O
(2271.21): C, 72.69; H, 9.63; N, 1.22. Found: C, 72.45;
H, 9.32; N, 1.19.
3.1.25. Benzyl 3-O-allyl-4-O-benzyl-2-deoxy-6-O-{2-
deoxy-6-O-benzyl-4-O-(di-O-benzyl-phosphono)-2-[(R)-3-
tetradecanoyloxytetradecanamido]-3-O-[(R)-3-tetradeca-
noyloxytetradecanoyl]-b-D-glucopyranosyl}-2-[(R)-3-tetra-
decanoyloxytetradecanamido]-b-D-glucopyranoside (33).
In a similar way as described for the preparation of 32,
compound 30 (117 mg, 0.060 mmol) was converted to
33 (81 mg, 61%) and purified by repeated flash chroma-
tography (1–3% acetone in CHCl3; toluene–acetone,
from 15:1 and 12:1; hexane–acetone, 6:1 and 5:1). Rf
3.1.23. Benzyl 4-O-benzyl-3-O-[(R)-3-benzyloxytetra-
decanoyl]-6-O-{6-O-benzyl-2-deoxy-2-[(R)-3-tetradecano-
yloxytetradecanamido]-3-O-[(3R)-3-tetradecanoyloxytet-
radecanoyl]-b-D-glucopyranosyl}-2-deoxy-2-[(R)-3-tetra-
decanoyloxytetradecanamido]-b-D-glucopyranoside (31).
In a similar way as described for the preparation of 29,
compound 28 (233 mg, 0.104 mmol) was converted to 31
22
(166 mg, 71%). Rf 0.32 (8% acetone in CHCl3); ½aꢀD
1
ꢁ14.0 (c 0.5, CHCl3); H NMR (300 MHz, CDCl3): d
22
0.88 (t, J 6.5 Hz, 21H, 7CH3), 1.24 (br s, 132H,
0.46 (9% acetone in CHCl3); ½aꢀD ꢁ4.8 (c 0.33, CHCl3);
66CH2), 1.57 (m, 14H, 7CH2), 2.18–2.62 (m, 14H,
1H NMR (300 MHz, CDCl3): d 0.89 (t, J 6.5 Hz, 18H,
6CH3), 1.25 (br s, 114H, 57CH2), 1.45–1.55 (m, 12H,
6CH2), 2.19–2.51 (m, 12H, 6CH2), 3.45–4.23 (m, 12H,
H-2, H-3, H-4, H-5, 2H-6, H-20, H-50, 2H-60,
0
7CH2), 3.32 (d, J4 ;OH 3:0 Hz, 1H, OH), 3.44–4.05 (m,
11H, H-2, H-4, H-5, H-6a/b, H-20, H-40, H-50, H-60a/
b, lipid-3-H), 4.42 (d, Jgem 12.0 Hz, 1H, CHHPh),

794
Z.-H. Jiang et al. / Carbohydrate Research 342 (2007) 784–796
CH2CH@CH2), 4.50 (m, 3H, H-40, CH2Ph), 4.58 (d, J
12.5 Hz, 2H, 2CHHPh), 4.75 (d, J 11.0 Hz, 1H, CHHPh),
4.80 (d, J 8.0 Hz, 1H, H-1), 4.88 (m, 5H, 5CHHPh), 4.99
(d, J 8.0 Hz, 1H, H-10), 5.05–5.26 (m, 5H, 3 lipid-3-H,
CH2@CH), 5.41 (dd, J 10.0, 9.0 Hz, 1H, H-30), 5.86 (m,
1H, CH2@CH), 5.93 (d, J 8.0 Hz, 1H, NH), 6.09 (d, J
7.5 Hz, 1H, NH), 7.30 (m, 25H, Ar–H). Anal. Calcd for
verted to 2 (55 mg, 95%). Rf 0.35 (CHCl3–MeOH–water,
22
3:1:0.1); ½aꢀD +6.0 (c 0.1, CHCl3–MeOH, 4:1); ESIMS
Calcd for C99H187N2O21P: 1771.3. Found (negative
mode): 1770.3 [MꢁHꢁ], 1771.3 ([MꢁHꢁ], M+1 isotope
peak).
3.1.29. 2-Deoxy-6-O-{2-deoxy-4-O-phosphono-2-[(R)-3-
tetradecanoyloxytetradecanamido]-3-O-[(R)-3-tetradeca-
noyloxytetradecanoyl]-b-D-glucopyranosyl}-3-O-[(R)-3-
hydroxytetradecanoyl]-2-[(R)-3-tetradecanoyloxytetradeca-
namido]-a/b-D-glucopyranose (3). In a similar way as
described for the preparation of 1, compound 34 (85
mg, 0.034 mmol) was converted to 3 (50 mg, 75%). Rf
C
134H215N2O21P (2221.15): C, 72.46; H, 9.76; N, 1.26.
Found: C, 72.21; H, 9.92; N, 1.27.
3.1.26. Benzyl 4-O-benzyl-3-O-[(R)-3-benzyloxytetra-
decanoyl]-6-O-{6-O-benzyl-4-O-(di-O-benzyl-phosphono)-
2-deoxy-2-[(R)-3-tetradecanoyloxytetradecanamido]-3-O-
[(R)-3-tetradecanoyloxytetradecanoyl]-b-D-glucopyranos-
yl}-2-deoxy-2-[(R)-3-tetradecanoyloxytetradecanamido]-
b-D-glucopyranoside (34). In a similar way as described
for the preparation of 32, compound 31 (150 mg,
0.067 mmol) was converted to 34 (98 mg, 59%) after re-
peated purification by flash chromatography (CHCl3–
acetone, 100:3; toluene–acetone, 16:1 then 12:1; hex-
ane–acetone, 8:1 then 6:1). Rf 0.27 (5% acetone in
20
0.46 (CHCl3–MeOH–water, 3:1:1); ½aꢀD ꢁ6.6 (c 0.1,
CHCl3–MeOH, 4:1); ESIMS Calcd for C110H207
-
N2O23P: 1955.5. Found (negative mode): 1954.5
[MꢁHꢁ].
3.2. Biological evaluation
3.2.1. General method for preparation of liposomal
vaccine formulation. Typically, the liposomal formula-
tion is composed of MUC1 mucin-derived 25-mer lipo-
peptide BLP25,18 monophosphoryl lipid A analogue or
R595 lipid A, and lipids such as cholesterol, dimyristoyl
phosphatidylglycerol (DMPC), and dipalmitoyl phos-
phatidylcholine (DPPC) in saline (0.9% NaCl soln).
The liposomal construct is formulated by first dissolving
the phospholipids, cholesterol and lipid A analogue in
tert-butanol at 50–60 ꢂC. Lipopeptide and water (5%,
v/v) are then added to the tert-butanol soln. The result-
ing tert-butanol soln is injected into about 4 vol of rap-
idly stirred water at 50–55 ꢂC, using a glass syringe with
an 18-gauge needle. The small unilamellar vesicles
(SUV) formed in this process are cooled, sterilized by fil-
tration through a 0.22 lm membrane filter, filled into
vials and lyophilized. The dry powder is re-hydrated
with sterile saline before injection, resulting in the for-
mation of multi-lamellar large vesicles (MLV). The so
formed BLP25 liposomal vaccine formulation is used
to immunize mice (injection dose, 100 lL).
22
CHCl3); ½aꢀD ꢁ8.5 (c 0.33, CHCl3); 1H NMR
(300 MHz, CDCl3): d 0.88 (t, J 6.5 Hz, 21H, 7CH3),
1.24 (br s, 132H, 66CH2), 1.54 (m, 14H, 7CH2), 2.16–
2.55 (m, 14H, 7CH2), 3.52–4.06 (m, 10H, H-2, H-4, H-
5, H-6a/b, H-20, H-50, H-60a/b, lipid-3-H), 4.38–4.91
(m, 14H, H-1, H-40, 6CH2Ph), 5.00 (d, J1 ;2 8:0 Hz,
0
0
1H, H-10), 5.03–5.22 (m, 4H, H-3, 3 lipid-3-H), 5.38
(dd, J3 ;2 10:0; J3 ;4 9:0 Hz, 1H, H-30), 5.66 (d, J
9.0 Hz, 1H, NH), 6.07 (d, J 8.0 Hz, 1H, NH), 7.30 (m,
0
0
0
0
30H, Ar–H). Anal. Calcd for
C152H243N2O23P
(2497.57): C, 73.09; H, 9.81; N, 1.12. Found: C, 72.83;
H, 9.96; N, 1.13.
3.1.27. 2-Deoxy-6-O-{2-deoxy-4-O-phosphono-2-[(R)-3-
tetradecanoyloxytetradecanamido]-3-O-[(R)-3-tetradeca-
noyloxytetradecanoyl]-b-D-glucopyranosyl}-2-[(R)-3-tetra-
decanoyloxytetradecanamido]-a/b-D-glucopyranose (1).
To the soln of 32 (64 mg, 0.028 mmol) in THF–HOAc
(10:1, 60 mL) was added palladium on charcoal (5%,
60 mg). The mixture was stirred at room temperature
under hydrogen atmosphere for 24 h. The solid was fil-
tered off and the filtrate was concentrated under dimi-
nished pressure. The residue was purified by flash
chromatography (CHCl3–MeOH–water, 4:1:0 and then
3:1:0.1) to give compound 1 (30 mg, 62%). Rf 0.35
3.2.2. Immunization of mice with liposomal vac-
cines. Groups of C57Bl/6 mice are immunized intra-
dermally with BLP25 liposomal vaccine containing
40 lg of MUC1 mucin-based 25-mer lipopeptide as an
antigen and 20 lg of one monophosphoryl lipid A ana-
logue (1–3) or the reference R595 lipid A as an adju-
vant. Nine days after vaccine injection, mice are
sacrificed and lymphocytes are taken from the draining
lymph nodes to determine the immune responses by
measuring the antigen specific T-cell proliferation
in vitro.
22
(CHCl3–MeOH–water, 3:1:0.1); ½aꢀD ꢁ10.0 (c 0.1,
CHCl3–MeOH, 4:1); ESIMS Calcd for C96H181N2O21P:
1729.3. Found (negative mode): 1728.3 [MꢁHꢁ].
3.1.28.
2-Deoxy-6-O-{2-deoxy-2-[(R)-3-tetradecanoyl-
oxytetradecanamido]-4-O-phosphono-3-O-[(R)-3-tetradeca-
noyloxytetradecanoyl]-b-D-glucopyranosyl}-3-O-propyl-2-
[(R)-3-tetradecanoyloxytetradecanamido]-a/b-D-glucopyr-
anose (2). In a similar way as described for the prepa-
ration of 1, compound 33 (73 mg, 0.035 mmol) was con-
3.2.3. Measurement of T-cell proliferation. T-Cell pro-
3
liferation is evaluated using a standard H thymidine

Z.-H. Jiang et al. / Carbohydrate Research 342 (2007) 784–796
795
incorporation assay. Briefly, nylon wool passed ingui-
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