Synthesis of 8-azidooctyl glycoside derivatives of the O-chain repeating unit of Escherichia coli O9a lipopolysaccharide and a methylated analog

Article abstract of DOI:10.1016/j.carres.2008.02.025

Described is the synthesis of 8-azidooctyl glycoside derivatives of the Escherichia coli serotype O9a O-chain tetrasaccharide repeating unit and the terminal tetrasaccharide motif in this polysaccharide, which contains a methyl group on O-3 of the distal mannopyranose residue. The assembly of these compounds involved the sequential addition of monosaccharide residues from the reducing to the nonreducing end of the molecule using glycosyl trichloroacetimidate donors. Both compounds were initially prepared as p-methoxyphenyl glycosides, which were converted to the corresponding 8-azidooctyl derivatives at a late stage in the synthesis.

Full text of DOI:10.1016/j.carres.2008.02.025

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1778–1789
Synthesis of 8-azidooctyl glycoside derivatives of the O-chain
repeating unit of Escherichia coli O9a lipopolysaccharide
and a methylated analog
Dianjie Hou, Fredrik Skogman and Todd L. Lowary*
Department of Chemistry and Alberta Ingenuity Centre for Carbohydrate Science, Gunning-Lemieux Chemistry Centre,
University of Alberta, Edmonton, AB, Canada T6G 2G2
Received 29 January 2008; received in revised form 21 February 2008; accepted 26 February 2008
Available online 4 March 2008
Abstract—Described is the synthesis of 8-azidooctyl glycoside derivatives of the Escherichia coli serotype O9a O-chain tetrasaccha-
ride repeating unit and the terminal tetrasaccharide motif in this polysaccharide, which contains a methyl group on O-3 of the distal
mannopyranose residue. The assembly of these compounds involved the sequential addition of monosaccharide residues from the
reducing to the nonreducing end of the molecule using glycosyl trichloroacetimidate donors. Both compounds were initially
prepared as p-methoxyphenyl glycosides, which were converted to the corresponding 8-azidooctyl derivatives at a late stage in
the synthesis.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Lipopolysaccharide; Synthesis; Oligosaccharide; Escherichia coli; Neoglycoconjugate
1. Introduction
gram quantities of analogs of both 1 and 2, which could
be used as substrates and authentic standards in assays
of the mannosyltransferase and methyltransferase
enzymes. Although this tetrasaccharide repeating unit
had been previously synthesized,4 it was prepared as
its methyl glycoside, which does not easily allow its con-
version to neoglycoconjugates (e.g., fluorescently
labeled analogs) that would facilitate these investiga-
tions. To the best of our knowledge, the synthesis of
tetrasaccharide 2 has not been reported. We therefore
describe here the synthesis of tetrasaccharides 3 and 4
(Fig. 2), both functionalized at the reducing end with a
group (8-azidooctyl) that will allow for conjugation to
other species either through classical methods (e.g.,
reduction to the amine and subsequent amidation) or
through more recently developed approaches (e.g., an
azide–alkyne 2+3 cycloaddition5).
Gram-negative bacteria produce, as a component of their
outer membrane lipopolysaccharide (LPS), a glycoconju-
gate containing three structural domains: Lipid A, a core
oligosaccharide, and the O-chain.1 LPS has potent endo-
toxic properties arising from the Lipid A domain.1 The O-
chain, which contains multiple copies of a repeating unit
with typically 1–4 monosaccharide residues, is antigenic,
and a number of these glycans have been studied as
vaccine candidates.2 In Escherichia coli serotype O9a,
the O-chain repeating unit is a tetrasaccharide containing
only mannopyranoside residues (1, Fig. 1). This system
has been the focus of a number of biosynthetic investiga-
tions,1 and a recent report3 has established that the length
of the O-chain is mediated by a methylation event, lead-
ing to structure 2, which prevents further polymerization.
As part of a collaboration on the biosynthesis of the
LPS O-chain in E. coli serotype O9a, we required milli-
2. Results and discussion
In designing a route to 3 and 4, we envisioned that the
former could be obtained from monosaccharides 5–7,
*
Corresponding author. Tel.: +1 780 492 1861; fax: +1 780 492
7705; e-mail: tlowary@ualberta.ca
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.02.025

D. Hou et al. / Carbohydrate Research 343 (2008) 1778–1789
1779
OH
HO
HO
O
HO
HO
HO
O
O
CH3O
OH
HO
HO
HO
HO
HO
O
OH
HO
HO
HO
HO
HO
O
O
O
O
O
O
O
O
O
O
HO
O
HO
O
HO
HO
OH
OH
2
n
1
Figure 1. O-Chain repeating unit of LPS from E. coli serotype O9a (1) and the structure of the methylated repeating unit (2) that terminates the
O-chain.
OBn
O
OBn
O
OH
BnO
BnO
HO
BnO
BnO
AllO
HO
HO
RO
O
D
OH
OPMP
OPMP
HO
HO
HO
HO
HO
O
O
O
5 (Ring A)
6 (Ring B)
C
A
O
O(CH2)8N3
O
OBz
OBz
B
BnO
BnO
BnO
BzO
HO
O
O
O
BzO
CH3O
HO
OH
OPMP
7 (Ring C; Ring D in 3)
3, R = H
4, R = CH3
OPMP
8 (Ring D in 4)
Figure 2. Target tetrasaccharides (3 and 4) and the building blocks (5–8) required for their synthesis.
while the preparation of the latter would require these
compounds in addition to another building block, 8
(Fig. 2). The approach would involve the sequential
assembly of the targets from the reducing to the nonre-
OH
OH
BnO
HO
HO
RO
O
O
BnO
RO
OPMP
OPMP
ducing end via imidate donors derived by the p-
methoxyphenyl glycosides shown in Figure 2. In a final
sequence, the reducing-end p-methoxyphenyl aglycone
would be exchanged for an 8-azidoctyl moiety. Of the
building blocks chosen, 5 and 6 were prepared as
described earlier,6 and the preparation of 7 and 8 was
accessed straightforwardly as described below. In the
previous synthesis of the tetrasaccharide repeating unit
of the E. coli serotype O9a O-chain by Kong and co-
workers,4 the molecule was obtained via a 2+2 coupling
strategy in which the key glycosylation reaction pro-
ceeded in excellent yield. While this approach could
have also been used to access 3 and 4, as these two
molecules differed in the identity of the terminal sugar
residue, we viewed the step-wise approach as equally
efficient in that the same trisaccharide (18, see below)
could be used as the precursor to both molecules.
9, R = H
11, R = H
a, b
d, e
10, R = Bn
12, R = CH3
f
c
8
7
Scheme 1. Reagents and conditions: (a) n-Bu2SnO, toluene, 105 °C; (b)
BnBr, n-Bu4NI, toluene, 105 °C, 92% over two steps; (c) BzCl,
pyridine, DMAP, 0 °C?rt, 97%; (d) n-Bu2SnO, toluene, 105 °C; (e)
CH3I, n-Bu4NI, toluene, 105 °C, 83% over two steps; (f) BzCl,
pyridine, DMAP, 0 °C?rt, 96%.
steps. Heating 9 with di-n-butyltin oxide in toluene gave
the intermediate stannylidene acetal,7 which was subse-
quently reacted with benzyl bromide and tetra n-butyl-
ammonium iodide to give alcohol 10 in 92% yield.
The regioselectivity of the process was established
As illustrated in Scheme 1, the synthesis of 7 was
achieved from the previously reported diol 96 in three

1780
D. Hou et al. / Carbohydrate Research 343 (2008) 1778–1789
by 13C NMR spectroscopy; as expected, the chemical
shift of C-3 moved downfield upon alkylation (dC3
the p-methoxyphenyl glycoside 6 was cleaved upon reac-
tion with ceric ammonium nitrate (CAN) in wet CH3CN
to afford the corresponding reducing sugar, which was
then converted to glycosyl trichloroacetimidate (13), in
84% overall yield. Glycosylation of 5 with 13 under
the promotion of trimethylsilyl trifluoromethanesulfo-
nate (TMSOTf) provided a 74% yield of the expected
disaccharide 14, together with 15% of the corresponding
b-isomer, which could be separated by chromatography.
The allyl group in 14 was then removed upon reaction
with palladium chloride in methanol to give 15 in 86%
yield. We observed none of the Wacker oxidation pro-
ducts. This disaccharide alcohol was the acceptor for a
=
71.6 in 9 and dC3 = 80.0 in 10). Benzoylation of 10
under standard conditions afforded building block 7 in
97% yield. A similar series of reactions was used to
convert p-methoxyphenyl a-D-mannopyranoside (11)8
to building block 8 in 80% overall yield in a three-step
route that proceeded via intermediate 12. Again, the
regioselectivity of the methylation could be ascertained
by comparing the C-3 chemical shift in the 13C NMR
spectrum of 11 (dC3 = 72.5) and 12 (dC3 = 82.2).
With the monosaccharide precursors in hand, tetra-
saccharide 3 was assembled as shown in Scheme 2. First,
OBn
BnO
OBn
BnO
O
BnO
c
a, b
O
BnO
AllO
O
OPMP
O
6
O
NH
CCl3
AllO
BnO
BnO
13
OBn
14
d
OBn
O
OR
O
OBn
BnO
BnO
BnO
BnO
BnO
BnO
OBz
BnO
BnO
BnO
O
BnO
e
O
O
O
+
OPMP
O
OPMP
HO
BnO
BnO
O
NH
CCl3
BnO
O
O
16
BnO
OBn
OBn
15
17, R = Bz
f
18, R = H
g
OBz
O
OBz
O
BzO
BzO
BzO
RO
RO
RO
OBz
OR
BzO
BzO
BzO
BzO
RO
RO
RO
RO
RO
O
O
O
j
O
O
O
BzO
O
O
OR
OPMP
O
O
BzO
RO
O
O
BzO
RO
OBz
OR
22, R = H
19, R = Bn
20, R = H
21, R = Bz
k
h
23, R = C(=NH)CCl3
24, R = (CH2)8N3
l
i
m
3
Scheme 2. Reagents and conditions: (a) CAN, H2O, CH3CN, rt; (b) DBU, CCl3CN, CH2Cl2, rt, 84% over two steps from 6; (c) 5, TMSOTf, CH2Cl2,
0 °C, 74%; (d) PdCl2, CH3OH, rt, 86%; (e) TMSOTf, CH2Cl2, 0 °C, 87%; (f) NaOCH3, CH3OH, rt, 93%; (g) 16, TMSOTf, CH2Cl2, 0 °C, 92%; (h) H2,
Pd(OH)2–C, EtOAc/CH3OH, rt, 100%; (i) BzCl, pyridine, DMAP, 50 °C, 96%; (j) CAN, H2O, CH3CN, rt, 97%; (k) DBU, CCl3CN, CH2Cl2, rt,
100%; (l) HO(CH2)8N3, TMSOTf, CH2Cl2, rt, 79%; (m) NaOCH3, CH3OH, rt, 85%.

D. Hou et al. / Carbohydrate Research 343 (2008) 1778–1789
1781
second glycosylation reaction, this time with trichloro-
acetimidate donor 16, which was prepared as previously
described.6 Upon reaction of 15 and 16 in the presence
of TMSOTf, an 87% yield of trisaccharide 17 was ob-
tained. Debenzoylation of 17 with sodium methoxide
in methanol afforded trisaccharide alcohol 18 in 93%
yield. The addition of the final monosaccharide residue
was achieved by a TMSOTf-promoted glycosylation
reaction between 18 and 16, which gave a 92% yield of
tetrasaccharide 19.
Having assembled the tetrasaccharide, all that
remained was the introduction of the azidooctyl agly-
cone. The inclusion of an azido group into the target
necessitated a change in protecting groups, as the
removal of benzyl protecting groups while leaving the
azide group intact would be problematic, if not impossi-
ble. Therefore, the benzyl ethers in 19 were removed by
hydrogenolysis over palladium hydroxide on carbon,
and the resulting product, 20, was benzoylated. This
benzoylation proved sluggish, and the successful
completion required that the reaction mixture be heated
at 50 °C for 15 h. Nevertheless, desired product 21 was
obtained in 96% yield over the two steps. Next, the p-
methoxyphenyl glycoside in 21 was reacted with CAN
in wet CH3CN, which gave the reducing sugar 22 in
97% yield. Conversion of 22 into trichloroacetimidate
23 was carried out under standard conditions, and this
donor was then reacted with 8-azido-1-octanol9 and
TMSOTf to give a 79% yield of the fully protected
trisaccharide 24. Removal of the benzoyl groups with
sodium methoxide proceeded in excellent overall yield
giving target 3 in 85% yield. In 3, the stereochemistry
of all mannopyranoside residues was established by
1
the measurement of the JC1,H1 for each glycosidic
linkage. These values ranged from 168.0 to 172.2 Hz,
thus clearly confirming the a-stereochemistry.10
An analogous series of transformations was used in
the preparation of the methylated tetrasaccharide 4
(Scheme 3). First, p-methoxyphenyl glycoside 8 was con-
verted to the corresponding imidate 25 in two steps. This
donor was then used to glycosylate trisaccharide alcohol
19 in the presence of TMSOTf, which afforded tetrasac-
OBz
BzO
O
BzO
CH3O
OR
OBz
BzO
RO
RO
RO
RO
RO
O
O
O
c
a, b
O
BzO
CH3O
8
O
O
NH
CCl3
OPMP
O
RO
O
RO
25
OR
26, R = Bn
27, R = H
28, R = Bz
d
e
OBz
O
BzO
BzO
CH3O
f
OBz
O
BzO
BzO
BzO
BzO
O
O
BzO
O
OR
O
BzO
BzO
O
OBz
29, R = H
g
i
30, R = C(=NH)CCl3
31, R = (CH2)8N3
h
4
Scheme 3. Reagents and conditions: (a) CAN, H2O, CH3CN, rt; (b) DBU, CCl3CN, CH2Cl2, rt, 78% over two steps from 8; (c) 18, TMSOTf,
CH2Cl2, 0 °C, 79%; (d) H2, Pd(OH)2–C, EtOAc, CH3OH, rt, 100%; (e) BzCl, pyridine, DMAP, 50 °C, 93%; (f) CAN, H2O, CH3CN, rt, 91%; (g)
DBU, CCl3CN, CH2Cl2, rt, 91%; (h) HO(CH2)8N3, TMSOTf, CH2Cl2, rt, 72%; (i) NaOCH3, CH2Cl2/CH3OH, rt, 83%.

1782
D. Hou et al. / Carbohydrate Research 343 (2008) 1778–1789
charide 26 in 79% yield. Replacement of the benzyl
ethers in 26 with benzoate esters proceeded via 27 to give
the expected product 28 in 93% yield over the two steps.
CAN-mediated cleavage of the aglycone in 28 afforded a
91% yield of reducing sugar 29, which was then con-
verted to imidate 30, also in 91% yield.
Matrix-assisted laser-desportion ionization mass spectra
(MALDI MS) were obtained on samples suspended in a
DCTB (2-[(2E)-3-(4-tert-butylphenyl)-2-methylprop-2-
enylidene]malononitrile) matrix using the delayed-
extraction mode and positive-ion detection.
Glycosylation of 8-azido-1-octanol9 with 30 was
achieved in 72% yield upon treatment with TMSOTf.
Reaction of the product of this reaction, 31, with so-
dium methoxide provided, in 83% yield, target molecule
4. The 1JC1,H1 for each monosaccharide residue in 4 ran-
ged from 168.2 to 172.3 Hz, which would be expected
for a molecule containing four a-mannopyranoside
residues.10
In conclusion, we have described the synthesis of 8-azi-
dooctyl glycoside derivatives of the E. coli serotype O9a
O-chain repeating unit, and the terminal tetrasaccharide
motif in this polysaccharide, which contains a methyl
group on O-3 of the distal mannopyranose residue. Both
compounds were assembled by the addition of single
monosaccharide units via glycosyl trichloroacetimidate
donors, from the reducing to the nonreducing end of
the molecule. The 8-azidooctyl aglycone will facilitate
the preparation of neoglycoconjugates from these oligo-
saccharides, and their use in studying the biosynthesis of
the O-chain in E. coli serotype O9a is in progress.
3.2. 8-Azidooctyl a-D-mannopyranosyl-(1?2)-a-D-man-
nopyranosyl-(1?3)-a-D-mannopyranosyl-(1?3)-a-D-
mannopyranoside (3)
To a solution of compound 24 (49 mg, 0.02 mmol) in 1:1
CH2Cl2–CH3OH (10 mL) was added M NaOCH3 in
CH3OH (0.29 mL). After stirring for 16 h at rt, the reac-
tion mixture was neutralized with Amberlite IR-120
(H+) resin, filtered, and concentrated. The resulting oil
was purified by chromatography in 2:1 CH2Cl2–CH3OH
to afford compound 3 (16 mg, 85%) as a white foam. Rf
0.38 (3:1, CH2Cl2–CH3OH); [a]D +77.6 (c 0.2, CH3OH)
1H NMR (500 MHz, CD3OD, dH) 5.41 (d, 1H,
J = 1.5 Hz, H-1), 5.02 (d, 1H, J = 1.9 Hz, H-10), 4.98
(d, 1H, J = 1.6 Hz, H-100), 4.71 (d, 1H, J = 1.8 Hz, H-
1000), 4.20–4.17 (dd, 1H, J = 3.2, 1.9 Hz, H-20), 4.01
(dd, 1H, J = 3.4, 1.5 Hz, H-2), 3.99 (dd, 1H, J = 2.9,
1.8 Hz, H-2000), 3.98–3.50 (m, 22H, H-0200, H-3, H-4,
H-5, H-6a, H-6b, H-30, H-40, H-50, H-6a; H-60b, H-300,
H-400, H-500, H-6a00; H-6b00, H-3000, H-4000, H-5000,
H-6000; H-6000, octyl OCH2), 3.41 (ddd, 1H, J = 9.6, 6.4,
a
b
6.4 Hz, octyl OCH2), 3.27 (dd, 2H, J = 6.9, 6.9 Hz,
CH2N3), 1.63–1.54 (m, 4H, octyl CH2), 1.42–1.31 (m,
8H, octyl CH2); 13C NMR (125 MHz, CD3OD, dC)
3. Experimental
1
1
3.1. General methods
104.2 (C-100, JCH = 169.2), 103.9 (C-10, JCH = 169.8),
1
1
101.8 (C-1, JCH = 172.2), 101.6 (C-1000, JCH = 168.0),
80.8 (C-200), 80.5 (C-20), 80.2 (C-2), 75.2 (C-30), 75.1
(C-2000), 75.0 (C-4), 74.7 (C-3000), 72.5 (C-400), 72.1 (C-3),
71.9 (C-4000), 71.5 (C-40), 71.3 (C-300), 69.4 (C-5), 68.8
(C-50), 68.7 (octyl OCH2), 67.7 (C-5000), 67.6 (C-500),
63.3 (2C, C-60, C-600), 62.9 (C-6000), 62.8 (C-6), 52.5
(CH2N3), 30.6 (octyl CH2), 30.4 (octyl CH2), 30.2 (octyl
CH2), 29.9 (octyl CH2), 27.8 (octyl CH2), 27.3 (octyl
CH2). ESIMS: m/z calcd for [C32H57O21N3]Na+:
842.3377. Found: 842.3375.
Solvents used in the reactions were purified by successive
passage through columns of alumina and copper under
an argon atmosphere. All reagents used were purchased
from commercial sources and used without further puri-
fication unless noted otherwise. 1H NMR and 13C NMR
spectra were recorded at 500 MHz and 125 MHz,
1
respectively. H NMR chemical shifts are referenced to
(CH3)4Si (d 0.0, CDCl3) or CD3OD (d 3.30, CD3OD).
13C NMR chemical shifts were referenced to CDCl3 (d
77.23, CDCl3) or CD3OD (d 48.90, CD3OD). Optical
rotations were measured at 21 2 °C. Unless stated
otherwise, all reactions were carried out at room temper-
ature under a positive pressure of argon and monitored
by TLC on Silica Gel 60 F254 (0.25 mm, E. Merck).
Spots were detected under UV light and/or by charring
with 10% H2SO4 in EtOH, anisaldehyde, or CeSO4.
Organic solutions of crude products were dried over an-
hyd Na2SO4. Solvents were evaporated under reduced
pressure and below 40 °C. Column chromatography
was performed on Silica Gel 60 (40–60 lm); the ratio
between silica gel and crude product ranged from
100:1 to 20:1 (w/w). Electrospray-ionization mass spec-
tra (ESIMS) were recorded on samples suspended in
mixtures of THF with CH3OH and added NaCl.
3.3. 8-Azidooctyl 3-O-methyl-a-D-mannopyranosyl-
(1?2)-a-D-mannopyranosyl-(1?3)-a-D-mannopyranosyl-
(1?3)-a-D-mannopyranoside (4)
To a solution of 31 (43 mg, 0.02 mmol) in 1:1 CH2Cl2–
CH3OH (10 mL) was added 1M NaOCH3 in CH3OH
(0.25 mL). After stirring for 16 h at rt, the reaction mix-
ture was neutralized with Amberlite IR-120 H+ resin, fil-
tered, and concentrated. The resulting oil was purified
by chromatography in 2:1 CH2Cl2–CH3OH to afford 4
(14 mg, 83%) as a white foam. Rf 0.41 (3:1, CH2Cl2–
CH3OH); [a]D +73.9 (c 0.25, CH3OH); 1H NMR
(500 MHz, CD3OD, dH) 5.42 (d, 1H, J = 1.6 Hz, H-1),
5.03 (d, 1H, J = 1.8 Hz, H-10), 5.01 (d, 1H, J = 1.8 Hz,

D. Hou et al. / Carbohydrate Research 343 (2008) 1778–1789
1783
H-100), 4.71 (d, 1H, J = 1.8 Hz, H-1000), 4.21–4.26 (m, 2H,
H-20, H-200), 4.07 (dd, 1H, J = 3.4, 1.6 Hz, H-2), 3.99
(dd, 1H, J = 2.9, 1.8 Hz, H-2000), 3.95–3.51 (m, 21H,
H-3, H-4, H-5, 2 ꢀ H-6, H-30, H-40, H-50, 2 ꢀ H-60,
H-300, H-400, H-500, 2 ꢀ H-600, H-3000, H-4000, H-5000, 2 ꢀ
H-6000, octyl OCH2), 3.45 (s, 3H, OCH3), 3.41 (ddd,
1H, J = 9.7, 6.3, 6.3 Hz, octyl OCH2), 3.36 (dd, 1H,
J = 9.3, 3.1 Hz, H-300), 3.27 (t, 2H, J = 6.9 Hz,
CH2N3), 1.63–1.55 (m, 4H, octyl CH2), 1.43–1.31 (m,
8H, octyl CH2); 13C NMR (125 MHz, CD3OD, dC)
m/z calcd for [C41H40O8]Na+: 683.2615. Found:
683.2616.
3.5. p-Methoxyphenyl 2,4,6-tri-O-benzoyl-3-O-methyl-a-
D-mannopyranoside (8)
To a solution of pyridine (10 mL), DMAP (11 mg,
0.09 mmol), and 12 (260 mg, 0.87 mmol) was added
BzCl (0.36 mL, 3.13 mmol) dropwise. The reaction mix-
ture was stirred at rt for 4 h before being diluted with
CH2Cl2 (50 mL) and washed with N HCl (2 ꢀ 50 mL),
satd aq NaHCO3 (50 mL), and brine (50 mL). The
organic layer was dried and concentrated, and the crude
product was purified by chromatography (4:1 hexane–
EtOAc) to give 8 (490 mg, 96%) as a colorless oil. Rf
0.71 (2:1, hexane–EtOAc); [a]D +21.2 (c 0.4, CH2Cl2);
1H NMR (500 MHz, CDCl3, dH) 8.12–8.06 (m, 4H,
Ar), 8.02–7.98 (m, 2H, Ar), 7.59–7.54 (m, 3H, Ar),
7.47–7.36 (m, 6H, Ar), 7.12–7.07 (m, 2H, Ar), 6.80–
6.75 (m, 2H, Ar), 5.87–5.81 (m, 2H, H-2, H-4), 5.62
(d, 1H, J = 1.9 Hz, H-1), 4.64–4.59 (m, 1H, H-6a),
4.47–4.38 (m, 2H, H-5, H-6b), 4.19 (dd, 1H, J = 9.7,
3.3 Hz, H-3), 3.75 (s, 3H, ArOCH3), 3.46 (s, 3H,
OCH3); 13C NMR (125 MHz, CDCl3, dC) 166.1
(C@O), 165.7 (C@O), 165.5 (C@O), 155.4 (Ar), 149.8
(Ar ꢀ 2), 133.4 (Ar), 133.3 (Ar), 133.0 (Ar), 130.0
(Ar ꢀ 2), 129.9 (Ar ꢀ 2), 129.8 (Ar ꢀ 2), 129.5 (Ar),
129.4 (Ar), 128.50 (Ar ꢀ 2), 128.48 (Ar ꢀ 2), 128.3
(Ar ꢀ 2), 118.0 (Ar ꢀ 2), 114.7 (Ar ꢀ 2), 97.0 (C-1),
77.2 (C-3), 69.4 (C-5), 68.45 (C-4), 68.42 (C-2), 63.2
(C-6), 58.1 (OCH3), 55.6 (ArOCH3). ESIMS: m/z calcd
for [C35H32O10]Na+: 635.1888. Found: 635.1892.
1
1
104.2 (C-100, JCH = 171.5), 103.9 (C-10, JCH = 170.4),
1
1
101.8 (C-1, JCH = 172.3), 101.6 (C-1000, JCH = 168.2),
82.1 (C-300), 80.8 (C-2), 80.7 (C-200), 80.3 (C-2000), 75.2
(C-4), 75.0 (2C, C-400, C-20), 74.7 (C-30), 72.0 (C-3000),
71.5 (C-3), 71.3 (C-40), 69.4 (C-4000), 68.7 (octyl OCH2),
67.9 (C-500), 67.7 (2C, C-5, C-50), 67.5 (C-5000), 63.3 (2C,
C-60, C-600), 62.9 (C-6000), 62.8 (C-6), 57.4 (OCH3), 52.5
(octyl CH2N3), 30.6 (octyl CH2), 30.4 (octyl CH2),
30.2 (octyl CH2), 29.9 (octyl CH2), 27.8 (octyl CH2),
27.3 (octyl CH2). ESIMS: m/z calcd for [C33H59O21N3]-
Na+: 856.3533. Found: 856.3535.
3.4. p-Methoxyphenyl 2-O-benzoyl-3,4,6-tri-O-benzyl-a-
D-mannopyranoside (7)
Alcohol 10 (1.31 g, 2.35 mmol) was dissolved in CH2Cl2
(20 mL), pyridine (1.0 mL, 11.88 mmol), and benzoyl
chloride (0.33 mL, 2.82 mmol) was added at 0 °C. The
reaction mixture was stirred for 2 h at rt before being di-
luted with CH2Cl2 (50 mL) and sequentially washed
with 1N HCl (2 ꢀ 50 mL), satd aq NaHCO3 (50 mL),
and H2O (50 mL). The organic layer was dried and con-
centrated, and the crude product was purified by chro-
matography (6:1 hexane–EtOAc) to give 7 (1.51 g,
97%) as a colorless oil. Rf 0.54 (4:1 hexane–EtOAc);
3.6. p-Methoxyphenyl 3,4,6-tri-O-benzyl-a-D-manno-
pyranoside (10)
1
[a]D +37.0 (c 0.5, CHCl3); H NMR (500 MHz, CDCl3,
dH) 8.12–8.08 (m, 2H, Ar), 7.59–7.55 (m, 1H, Ar), 7.40–
7.21 (m, 17H, Ar), 7.04–7.02 (m, 2H, Ar), 6.83–6.80 (m,
2H, Ar), 5.80 (dd, 1H, J = 3.2, 1.9 Hz, H-2), 5.59 (d, 1H,
J = 1.9 Hz, H-1), 4.91 (d, 1H, J = 10.8 Hz, PhCH2),
4.86 (d, 1H, J = 11.4 Hz, PhCH2), 4.71 (d, 1H,
J = 11.8 Hz, PhCH2), 4.66 (d, 1H, J = 11.4 Hz, PhCH2),
4.58 (d, 1H, J = 10.8 Hz, PhCH2), 4.50 (d, 1H,
J = 11.8 Hz, PhCH2), 4.32 (dd, 1H, J = 3.2, 9.7 Hz,
H-3), 4.20 (dd, 1H, 9.7, 9.7 Hz, H-4), 4.03 (ddd, 1H,
J = 2.0 Hz, J = 9.7, 3.9, 1.0 Hz, H-5), 3.90 (dd, 1H,
J = 3.9, 10.9 Hz, H-6a), 3.77–3.75 (m, 4H, OCH3,
H-6b); 13C NMR (125 MHz, CDCl3, dC) 165.7 (C@O),
155.1 (Ar), 150.0 (Ar), 138.41 (Ar), 138.36 (Ar), 138.0
(Ar), 133.2 (Ar), 130.0 (2 ꢀ Ar), 129.8 (Ar), 128.4
(2 ꢀ Ar), 128.33 (2 ꢀ Ar), 128.32 (2 ꢀ Ar), 128.30
(2 ꢀ Ar), 128.04 (2 ꢀ Ar), 127.96 (2 ꢀ Ar), 127.66
(Ar), 127.65 (Ar), 127.51 (2 ꢀ Ar), 127.47 (Ar), 117.8
(2 ꢀ Ar), 114.6 (2 ꢀ Ar), 97.0 (C-1), 78.1 (C-3), 75.3
(PhCH2), 74.2 (C-4), 73.4 (PhCH2), 72.1 (C-5), 71.8
(PhCH2), 68.9 (C-6), 68.8 (C-2), 55.6 (OCH3). ESIMS:
Diol 96 (1.20 g, 2.57 mmol) was dissolved in toluene (50
mL), and n-Bu2SnO (0.92 g, 3.08 mmol) was added. The
reaction mixture was heated to 105 °C and stirred for
2 h, then cooled for 30 min before n-Bu4NI (1.14 g,
3.09 mmol) and benzyl bromide (3.06 mL, 25.7 mmol)
were added. The reaction mixture was then heated to
105 °C again and stirred for 16 h. The reaction mixture
was cooled and concentrated, and the crude product was
purified by chromatography (4:1 hexane–EtOAc) to give
10 (1.31 g, 92%) as an oil. Rf 0.33 (2:1 hexane–EtOAc);
1H NMR (500 MHz, CDCl3, dH) 7.41–7.24 (m, 13H,
Ar), 7.21–7.19 (m, 2H, Ar), 7.02–6.99 (m, 2H, Ar),
6.83–6.81 (m, 2H, Ar), 5.53 (d, 1H, J = 1.7 Hz, H-1),
4.88 (d, 1H, J = 10.8 Hz, PhCH2), 4.76–4.81 (m, 2H,
PhCH2), 4.62 (d, 1H, J = 11.9 Hz, PhCH2), 4.56 (d,
1H, J = 10.8 Hz, PhCH2), 4.48 (d, 1H, J = 11.9 Hz,
PhCH2), 4.22 (br s, 1H, H-2), 4.09 (dd, 1H, J = 3.3,
8.4 Hz, H-3), 3.99–3.93 (m, 2H, H-4, H-5), 3.78–3.75
(m, 4H, H-6a, OCH3), 3.67 (dd, 1H, J = 10.9, 1.0 Hz,
H-6b), 2.56 (d, 1H, J = 2.6 Hz, 2-OH); 13C NMR

1784
D. Hou et al. / Carbohydrate Research 343 (2008) 1778–1789
(125 MHz, CDCl3, dC) 155.0 (Ar), 150.1 (Ar), 138.3
(Ar), 138.2 (Ar), 137.9 (Ar), 128.6 (2 ꢀ Ar), 128.34
(2 ꢀ Ar), 128.26 (2 ꢀ Ar), 128.0 (Ar), 127.89 (2 ꢀ Ar),
127.86 (2 ꢀ Ar), 127.8 (2 ꢀ Ar), 127.7 (Ar), 127.5 (Ar),
117.8 (2 ꢀ Ar), 114.6 (2 ꢀ Ar), 98.2 (C-1), 80.0 (C-3),
75.2 (PhCH2), 74.2 (C-4), 73.4 (PhCH2), 72.2 (PhCH2),
71.6 (C-5), 68.8 (C-6), 68.4 (C-2), 55.6 (OCH3). ESIMS:
m/z calcd for [C34H36O7]Na+: 579.2353. Found:
579.2356.
3.9. p-Methoxyphenyl 3-O-allyl-2,4,6-tri-O-benzyl-a-D-
mannopyranosyl-(1?3)-2,4,6-tri-O-benzyl-a-D-manno-
pyranoside (14)
Alcohol 56 (380 mg, 0.68 mmol) and imidate 13 (621 mg,
0.89 mmol) were dissolved in dry CH2Cl2 (20 mL), and
crushed molecular sieves (1 g) were added. The mixture
was stirred at rt for 30 min before it was cooled to 0 °C.
A solution of 10% TMSOTf in dry CH2Cl2 (124 lL,
0.07 mmol) was added dropwise. The reaction mixture
was stirred for 1 h at 0 °C, it was then quenched by
the addition of Et3N. After filtration and concentration
of the reaction mixture, the crude product was purified
by chromatography (8:1 hexane–EtOAc) to give 14
(513 mg, 74%) as a colorless oil. Rf 0.32 (4:1 hexane–
EtOAc); [a]D +41.6 (c 0.5, CHCl3); 1H NMR
(500 MHz, CDCl3, dH) 7.37–7.16 (m, 30H, Ar), 6.97–
6.92 (m, 2H, Ar), 6.79–6.74 (m, 2H, Ar), 5.94–5.86 (m,
1H, CH2@CH), 5.42 (d, 1H, J = 1.8 Hz, H-1), 5.32 (br
s, 1H, H-10), 5.30–5.24 (m, 1H, CH2@CH), 5.15–5.10
(m, 1H, CH2@CH), 4.90 (d, 1H, J = 11.0 Hz, PhCH2),
4.75–4.48 (m, 10H, 10 ꢀ PhCH2), 4.44 (dd, 1H,
J = 11.9 Hz, PhCH2), 4.35 (dd, 1H, J = 9.5, 3.0 Hz,
H-3), 4.12–3.85 (m, 8H, H-2, H-4, 2 ꢀ CH2@CHCH2O,
H-5, H-40, H-50, H-6a), 3.82–3.62 (m, 8H, H-20, Ar-
OCH3, H-30, H-6b, H-6a0 ; H-6b0 ); 13C NMR (125 MHz,
CDCl3, dC) 154.9 (Ar), 150.4 (Ar), 138.9 (Ar), 138.44
(2 ꢀ Ar), 138.43 (Ar), 138.37 (Ar), 138.2 (Ar), 135.1
(CH@CH2), 128.40 (Ar), 128.38 (Ar), 128.31 (Ar),
128.24 (Ar), 128.22 (Ar), 128.19 (4 ꢀ Ar), 127.74
(2 ꢀ Ar), 127.69 (4 ꢀ Ar), 127.66 (4 ꢀ Ar), 127.56
(Ar), 127.5 (2 ꢀ Ar), 127.44 (2 ꢀ Ar), 127.42 (2 ꢀ Ar),
127.37 (2 ꢀ Ar), 127.0 (2 ꢀ Ar), 118.0 (2 ꢀ Ar), 116.6
(CH2@CH), 114.5 (2 ꢀ Ar), 100.3 (C-10), 96.6 (C-1),
79.7 (C-3), 77.6 (C-20), 75.6 (C-2), 75.1 (C-5), 75.0
(C-50), 74.8 (PhCH2), 74.6 (PhCH2), 73.5 (PhCH2),
73.3 (PhCH2), 72.7 (C-3’), 72.5 (C-40), 72.42 (PhCH2),
72.39 (C-4), 72.36 (PhCH2), 71.1 (CH2@CHCH2O),
69.8 (C-6), 69.1 (C-60), 55.7 (ArOCH3). ESIMS:
m/z calcd for [C64H68O12]Na+: 1051.4603. Found:
1051.4606.
3.7. p-Methoxyphenyl 3-O-methyl-a-D-mannopyranoside
(12)
Compound 118 (300 mg, 1.05 mmol) was dissolved in
toluene (30 mL), and n-Bu2SnO (380 mg, 1.27 mmol)
was added. The reaction mixture was heated to 105 °C
and stirred for 4 h, then cooled to room temperature
before n-Bu4NI (460 mg, 1.25 mmol) and iodomethane
(0.65 mL, 10.5 mmol) were added. The reaction mixture
was then heated to 105 °C again and stirred for 24 h.
The reaction mixture was cooled and concentrated,
and the crude product was purified by chromatography
(10:1, CH2Cl2–CH3OH) to give 12 (260 mg, 83%) as a
colorless oil. Rf 0.39 (10:1, CH2Cl2–CH3OH); [a]D
1
+105.3 (c 0.8, CH3OH); H NMR (500 MHz, CD3OD,
dH) 7.06–7.02 (m, 2H, Ar), 6.85–6.81 (m, 2H, Ar), 5.36
(d, 1H, J = 2.0 Hz, H-1), 4.20 (dd, 1H, J = 3.3, 2.0 Hz,
H-2), 3.82–3.63 (m, 7H, ArOCH3, H-4, H-5, H-6a,
H-6b), 3.54 (dd, 1H, J = 9.4, 3.3 Hz, H-3), 3.50 (s, 3H,
OCH3); 13C NMR (125 MHz, CD3OD, dC) 156.7 (Ar),
152.0 (Ar), 119.2 (2 ꢀ Ar), 115.7 (2 ꢀ Ar), 101.1 (C-1),
82.2 (C-3), 75.2 (C-5), 68.1 (C-2), 67.3 (C-4), 62.7
(C-6), 57.6 (OCH3), 56.1 (ArOCH3). ESIMS: m/z calcd
for [C14H20O7]Na+: 323.1101. Found: 323.1102.
3.8. 3-O-Allyl-2,4,6-tri-O-benzyl-D-mannopyranosyl
trichloroacetimidate (13)
A solution of compound 66 (560 mg, 0.94 mmol) and
ceric ammonium nitrate (2.58 g, 4.7 mmol) in 4:1
CH3CN–H2O (25 mL) was stirred at rt for 1 h, diluted
with EtOAc (40 mL), and washed with water (40 mL),
satd aq NaHCO3 (40 mL), and brine (40 mL). The
organic layer was dried and concentrated, and the
residue was purified by chromatography (3:1 hexane–
EtOAc) to afford the expected monosaccharide
(393 mg, 85%) as a colorless oil. The reducing monosac-
charide (393 mg, 0.71 mmol) was then dissolved in dry
CH2Cl2 (20 mL), and then CCl3CN (0.85 mL, 8.52
mmol) and DBU (31.7 lL, 0.21 mmol) were sequentially
added at 0 °C. The solution was stirred at rt for 2 h and
then concentrated. The resulting oil was purified by
chromatography (4:1 hexane–EtOAc and 1% Et3N) to
afford compound 13 (469 mg, 99%) as a colorless oil,
which, following drying under vacuum overnight, was
used immediately in the next step.
3.10. p-Methoxyphenyl 2,4,6-tri-O-benzyl-a-D-manno-
pyranosyl-(1?3)-2,4,6-tri-O-benzyl-a-D-mannopyr-
anoside (15)
Compound 14 (634 mg, 0.63 mmol) was dissolved in 9:1
CH3OH–CH2Cl2 (30 mL) and PdCl2 (28 mg, 0.16
mmol) was added. The reaction mixture was stirred at
rt for 2 h before it was filtered and concentrated. The
crude product was purified by chromatography (4:1 hex-
ane–EtOAc) to give 15 (524 mg, 86%) as a colorless oil.
Rf 0.24 (4:1 hexane–EtOAc); [a]D +50.4 (c 1.2, CHCl3);
1H NMR (500 MHz, CDCl3, dH) 7.38–7.15 (m, 30H,
Ar), 6.98–6.94 (m, 2H, Ar), 6.82–6.76 (m, 2H, Ar),
5.44 (d, 1H, J = 1.7 Hz, H-1), 5.37 (br s, 1H, H-10),

D. Hou et al. / Carbohydrate Research 343 (2008) 1778–1789
1785
4.87 (d, 1H, J = 11.3 Hz, PhCH2), 4.79–4.36 (m, 11H,
11 ꢀ PhCH2), 4.17–4.04 (m, 4H, H-2, H-4, H-6a,
H-30), 3.97–3.90 (m, 2H, H-3, H-5), 3.80–3.64 (m, 9H,
ArOCH3, H-6b, H-20, H-40, H-50, H-6a0 ; H-6b0 ), 2.31 (d,
1H, J = 9.5 Hz, 30-OH); 13C NMR (125 MHz, CDCl3,
dC) 154.9 (Ar), 150.4 (Ar), 138.7 (Ar), 138.5 (Ar),
138.3 (2 ꢀ Ar), 138.1 (Ar), 137.8 (Ar), 128.4 (4 ꢀ Ar),
128.3 (4 ꢀ Ar), 128.2 (3 ꢀ Ar), 127.75 (Ar), 127.70
(2 ꢀ Ar), 127.67 (4 ꢀ Ar), 127.63 (4 ꢀ Ar), 127.59
(2 ꢀ Ar), 127.53 (2 ꢀ Ar), 127.4 (2 ꢀ Ar), 126.9
(2 ꢀ Ar), 117.8 (2 ꢀ Ar), 114.6 (2 ꢀ Ar), 99.2 (C-1),
96.6 (C-10), 78.9 (C-20), 78.7 (C-30), 77.5 (C-2), 76.7 (C-
40), 75.1 (C-4), 74.5 (2 ꢀ PhCH2), 73.5 (PhCH2), 73.3
(PhCH2), 72.6 (C-3), 72.37 (PhCH2), 72.36 (PhCH2),
71.8 (C-50), 71.7 (C-5), 69.6 (C-6), 69.1 (C-60), 55.7
(ArOCH3). ESIMS: m/z calcd for [C61H64O12]Na+:
1011.4290. Found: 1011.4290.
0.31 (4:1 hexane–EtOAc); [a]D +21.7 (c 0.7, CHCl3);
1H NMR (500 MHz, CDCl3, dH) 8.06–8.03 (m, 2H,
Ar), 7.57–7.54 (m, 1H, Ar), 7.38–7.12 (m, 47H, Ar),
6.98–6.94 (m, 2H, Ar), 6.79–6.76 (m, 2H, Ar), 5.74 (br
s, 1H, H-200), 5.46 (s, 1H, H-1), 5.35 (br s, 2H, H-10,
H-100), 4.88 (d, 1H, J = 11.1 Hz, PhCH2), 4.81 (d, 1H,
J = 11.0 Hz, PhCH2), 4.77–4.33 (m, 17H, 16 ꢀ PhCH2,
H-3), 4.28 (dd, 1H, J = 8.7, 2.8 Hz, H-30), 4.15–3.86
(m, 9H, H-4, H-2, H-6a, H-6b, H-40, H-20, H-300, H-400,
H-5), 3.78–3.60 (m, 8H, ArOCH3, H-50, H-6a0 ; H-6b0 ;
H-60a0; H-6b00), 3.55–3.48 (m, 1H, H-500); 13C NMR
(125 MHz, CDCl3, dC) 165.4 (C@O), 154.9 (Ar), 150.4
(Ar), 138.7 (Ar), 138.4 (Ar), 138.35 (Ar), 138.33 (Ar),
138.28 (Ar), 138.1 (Ar), 138.05 (Ar), 138.0 (2 ꢀ Ar),
133.0 (Ar), 130.0 (Ar), 128.42 (2 ꢀ Ar), 128.4 (4 ꢀ Ar),
128.32 (4 ꢀ Ar), 128.29 (4 ꢀ Ar), 128.28 (4 ꢀ Ar),
128.26 (4 ꢀ Ar), 128.24 (2 ꢀ Ar), 128.17 (2 ꢀ Ar),
128.0 (2 ꢀ Ar), 127.9 (2 ꢀ Ar), 127.73 (2 ꢀ Ar), 127.72
(2 ꢀ Ar), 127.68 (2 ꢀ Ar), 127.67 (2 ꢀ Ar), 127.58
(2 ꢀ Ar), 127.56 (2 ꢀ Ar), 127.45 (2 ꢀ Ar), 127.42
(2 ꢀ Ar), 127.2 (2 ꢀ Ar), 127.1 (Ar), 117.9 (2 ꢀ Ar),
114.5 (2 ꢀ Ar), 99.6 (2C, C-10, C-100), 96.6 (C-1), 78.1
(2C, C-20, C-2), 78.0 (C-4), 77.6 (C-300), 75.4 (C-400),
75.0 (PhCH2), 74.6 (2 ꢀ PhCH2), 74.2 (2C, C-30, C-5),
73.5 (PhCH2), 73.4 (PhCH2), 73.3 (PhCH2), 72.5 (3C,
C-40, C-3, C-500), 72.4 (PhCH2), 72.0 (PhCH2), 71.6
(PhCH2), 69.5 (C-600), 69.12 (2C, C-200, C-50), 69.07
(C-60), 68.9 (C-6), 55.7 (ArOCH3). ESIMS: m/z calcd
for [C95H96O18]Na+: 1547.6489. Found: 1547.8496.
3.11. 2-O-benzoyl-3,4,6-tri-O-benzyl-D-mannopyranosyl
trichloroacetimidate (16)
A solution of compound 7 (200 mg, 0.3 mmol) and ceric
ammonium nitrate (830 mg, 1.5 mmol) in 4:1 CH3CN–
H2O (25 mL) was stirred at rt for 1 h, diluted with
EtOAc (40 mL), and washed with water (40 mL), satd
aq NaHCO3 (40 mL), and brine (40 mL). The organic
layer was dried and concentrated, and the residue was
purified by chromatography (3:1 hexane–EtOAc) to
afford reducing monosaccharide (145 mg, 86%) as a
colorless oil. A portion of this material (119 mg,
0.21 mmol) was then dissolved in dry CH2Cl2 (10 mL),
and then CCl3CN (0.27 mL, 2.57 mmoL) and DBU
(9.9 lL, 0.06 mmol) were sequentially added at 0 °C.
The solution was stirred at rt for 2 h and then concen-
trated. The resulting oil was purified by chromatogra-
phy (5:1 hexane–EtOAc and 1% Et3N) to afford 16
(132 mg, 88%) as a colorless oil, which, following drying
under vacuum overnight, was used immediately in the
next step.
3.13. p-Methoxyphenyl 3,4,6-tri-O-benzyl-a-D-manno-
pyranosyl-(1?3)-2,4,6-tri-O-benzyl-a-D-mannopyrano-
syl-(1?3)-2,4,6-tri-O-benzyl-a-D-mannopyranoside (18)
Compound 17 (714 mg, 0.47 mmol) was dissolved in 1:1
CH3OH–CH2Cl2 (50 mL), and 1M NaOCH3 (0.5 mL)
was added. The reaction mixture was stirred at rt for
16 h. Amberlite IR-120 (H+) resin was added, and the
solution was filtered and concentrated. The crude prod-
uct was purified by chromatography (3:1 hexane–
EtOAc) to give 18 (618 mg, 93%) as a colorless oil. Rf
0.55 (2:1, hexane–EtOAc); [a]D +50.3 (c 0.4, CHCl3);
1H NMR (500 MHz, CDCl3, dH) 7.42–7.13 (m, 45H,
Ar), 7.02–6.97 (m, 2H, Ar), 6.83–6.78 (m, 2H, Ar),
5.49 (br s, 1H, H-1), 5.36 (br s, 1H, H-10), 5.30 (br s,
1H, H-100), 4.85 (d, 1H, J = 10.0 Hz, PhCH2), 4.74–
4.58 (m, 8H, 8 ꢀ PhCH2), 4.56–4.35 (m, 10H,
9 ꢀ PhCH2, H-3), 4.26 (dd, 1H, J = 5.4, 2.3 Hz, H-30),
4.11–3.86 (m, 10H, H-2, H-4, H-5, H-6a, H-6b, H-20,
H-200, H-300, H-40, H-50), 3.78 (s, 3H, ArOCH3), 3.76–
3.48 (m, 6H, H-60a0, H-6b00, H-500, H-400, H-6a0 ; H-60b),
2.33 (br s, 1H, OH); 13C NMR (125 MHz, CDCl3, dC)
154.9 (Ar), 150.4 (Ar), 138.7 (Ar), 138.4 (Ar), 138.32
(Ar), 138.30 (Ar), 138.27 (Ar), 138.24 (Ar), 138.16
(Ar), 138.1 (Ar), 138.0 (Ar), 128.50 (2 ꢀ Ar), 128.47
(2 ꢀ Ar), 128.4 (2 ꢀ Ar), 128.38 (2 ꢀ Ar), 128.32
3.12. p-Methoxyphenyl 2-O-benzoyl-3,4,6-tri-O-benzyl-a-
D-mannopyranosyl-(1?3)-2,4,6-tri-O-benzyl-a-D-manno-
pyranosyl-(1?3)-2,4,6-tri-O-benzyl-a-D-mannopyrano-
side (17)
Disaccharide 15 (524 mg, 0.52 mmol) and imidate 16
(562 mg, 0.80 mmol) were dissolved in dry CH2Cl2
(20 mL), and crushed molecular sieves (1 g) were added.
The mixture was stirred at rt for 30 min and then it was
cooled to 0 °C. A solution of 10% TMSOTf (98 lL,
0.05 mmol) in dry CH2Cl2 was added dropwise. The
reaction mixture was stirred for 1 h at 0 °C, and then
it was quenched by the addition of Et3N. After filtration
and concentration of the reaction mixture, the crude
product was purified by chromatography (6:1 hexane–
EtOAc) to give 17 (714 mg, 87%) as a colorless oil. Rf

1786
D. Hou et al. / Carbohydrate Research 343 (2008) 1778–1789
(2 ꢀ Ar), 128.28 (2 ꢀ Ar), 128.25 (2 ꢀ Ar), 128.2
(4 ꢀ Ar), 127.9 (2 ꢀ Ar), 127.83 (2 ꢀ Ar), 127.79
(2 ꢀ Ar), 127.72 (2 ꢀ Ar), 127.70 (4 ꢀ Ar), 127.68
(4 ꢀ Ar), 127.50 (2 ꢀ Ar), 127.47 (Ar), 127.46 (Ar),
127.44 (Ar), 127.37 (Ar), 127.24 (2 ꢀ Ar), 127.20
(2 ꢀ Ar), 127.1 (Ar), 117.9 (2 ꢀ Ar), 114.6 (2 ꢀ Ar),
99.8 (C-10), 96.6 (2C, C-1, C-100), 80.0 (2C, C-300, C-30),
78.1 (C-3), 77.6 (C-5), 75.4 (2C, C-40, C-500), 75.0 (2C,
C-4, C-400), 74.8 (PhCH2), 74.6 (PhCH2), 74.2 (C-50),
73.5 (PhCH2), 73.4 (PhCH2), 73.3 (2 ꢀ PhCH2), 72.5
(C-20), 72.3 (PhCH2), 72.1 (PhCH2), 72.0 (PhCH2),
71.9 (C-2), 69.5 (C-6), 69.0 (C-60), 68.9 (C-600), 68.7
(C-200), 55.6 (ArOCH3). ESIMS: m/z calcd for
[C88H92O17]Na+: 1442.6232. Found: 1443.6234.
(4 ꢀ Ar), 127.1 (2 ꢀ Ar), 118.1 (2 ꢀ Ar), 114.7
(2 ꢀ Ar), 100.9 (C-10), 99.8 (C-100), 99.6 (C-1000), 97.1
(C-1), 79.6 (C-2), 78.9 (C-200), 78.6 (C-3000), 77.9 (C-20),
77.3 (C-40), 76.1 (C-4), 75.6 (C-300), 75.4 (C-30), 75.2
(PhCH2), 74.9 (C-3), 74.8 (PhCH2), 74.7 (C-400), 74.4
(C-4000), 73.6 (PhCH2), 73.5 (PhCH2), 73.4 (PhCH2),
73.3 (2 ꢀ PhCH2), 73.0 (PhCH2), 72.8 (C-500), 72.7
(C-50), 72.59 (C-5000), 72.55 (2 ꢀ PhCH2), 72.48 (C-5),
72.2 (PhCH2), 71.7 (PhCH2), 69.8 (C-6), 69.6 (C-6000),
69.43 (C-60), 69.36 (C-2000), 68.9 (C-600), 55.7 (ArOCH3).
MALDI MS: m/z calcd for [C122H124O23]Na+:
1979.8426. Found: 1979.8427.
3.15. p-Methoxyphenyl 2,3,4,6-tetra-O-benzoyl-a-D-
mannopyranosyl-(1?2)-3,4,6-tri-O-benzoyl-a-D-manno-
pyranosyl-(1?3)-2,4,6-tri-O-benzoyl-D-mannopyranosyl-
(1?3)-2,4,6-tri-O-benzoyl-a-D-mannopyranoside (21)
3.14. p-Methoxyphenyl 2-O-benzoyl-3,4,6-tri-O-benzyl-a-
D-mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-a-D-manno-
pyranosyl-(1?3)-2,4,6-tri-O-benzyl-a-D-mannopyrano-
syl-(1?3)-2,4,6-tri-O-benzyl-a-D-mannopyranoside (19)
To a solution of compound 19 (782 mg, 0.41 mmol) in
CH3OH–EtOAc 3:1 (24 mL) was added 20% Pd(OH)2-
on-carbon (100 mg), and the reaction mixture was stir-
red for 24 h under a hydrogen atmosphere. The reaction
mixture was filtered through Celite and then concen-
trated; an 1H NMR spectrum of the crude product
revealed that all of the benzyl groups had been removed.
The resulting oil (20) was dissolved in pyridine
(10 mL). To this solution were added DMAP (5 mg)
and benzoyl chloride (0.86 mL, 7.43 mmol) dropwise
at rt. The reaction mixture was stirred at 50 °C for
15 h, and it was then diluted with CH2Cl2 (50 mL).
The organic layer was washed by N HCl (2 ꢀ 50 mL),
satd aq NaHCO3 (50 mL), and brine (50 mL). After dry-
ing, the organic layer was concentrated, and the result-
ing residue was purified by chromatography (2:1
hexane–EtOAc) to afford 21 (815 mg, 96%) as a white
foam. Rf 0.69 (1:1 hexane–EtOAc); [a]D ꢁ23.4 (c 1.2,
CH2Cl2); 1H NMR (500 MHz, CDCl3, dH) 8.27–7.79
(m, 22H, Ar), 7.73–7.69 (m, 2H, Ar), 7.63–7.20 (m,
36H, Ar), 7.16–7.02 (m, 7H, Ar), 6.82–6.76 (m, 2H,
Ar), 6.12 (dd, 1H, J = 10.1, 10.1 Hz H-4000), 6.09 (dd,
1H, J = 9.8, 9.8 Hz, H-4), 6.01–5.90 (m, 4H, H-2, H-3000,
H-40, H-400), 5.73 (dd, 1H, J = 3.1, 1.8 Hz, H-2000), 5.71
(d, 1H, J = 1.8 Hz, H-1), 5.62 (dd, 1H, J = 10.1,
3.1 Hz, H-300), 5.49 (d, 1H, J = 2.3 Hz, H-10), 5.41 (dd,
1H, J = 2.7, 2.3 Hz, H-20), 5.27 (br s, 1H, H-100), 4.87
(dd, 1H, J = 9.7, 3.3 Hz, H-3), 4.74 (d, 1H, J = 1.8 Hz,
H-1000), 4.66–4.42 (m, 6H, H-5, H-30, H-50, H-6a, H-6b,
H-60a), 4.41–4.26 (m, 3H, H-5000, H-6b0 ; H-60a00), 4.23–4.04
(m, 4H, H-500, H-60b00; H-6a00; H-60b0), 4.00 (dd, 1H, J = 3.1,
1.9 Hz, H-200), 3.76 (s, 3H, ArOCH3); 13C NMR
(125 MHz, CDCl3, dC) 166.12 (C@O), 166.08 (C@O),
166.0 (C@O), 165.9 (C@O), 165.8 (C@O), 165.7
(C@O), 165.4 (C@O), 165.3 (C@O), 165.0 (C@O),
164.9 (2 ꢀ C@O), 164.7 (C@O), 164.6 (C@O), 155.5
(Ar), 149.7 (Ar), 133.9 (Ar), 133.8 (Ar), 133.44 (Ar),
133.38 (Ar), 133.3 (2 ꢀ Ar), 133.08 (Ar), 133.06
To a solution of a mixture of trisaccharide 18 (618 mg,
0.43 mmol) and imidate 16 (497 mg, 0.71 mmol) in dry
CH2Cl2 (20 mL) was added activated 4 A molecular
˚
sieves (1 g), and the mixture was stirred at rt for
30 min. The mixture was then cooled to 0 °C and a solu-
tion of 10% TMSOTf in CH2Cl2 (78.7 lL, 0.04 mmol)
was added. The reaction mixture was stirred for 1 h at
0 °C, and then it was neutralized by the addition of
Et3N, filtered, and concentrated to give a residue that
was purified by chromatography (4:1 hexane–EtOAc)
to afford 19 (782 mg, 92%) as a colorless oil. Rf 0.41
(3:1 hexane–EtOAc); [a]D +25.6 (c 1.2, CH2Cl2); 1H
NMR (500 MHz, CDCl3, dH) 8.11–8.05 (m, 2H, Ar),
7.59–7.53 (m, 1H, Ar), 7.42–7.07 (m, 62H, Ar), 6.99–
6.93 (m, 2H, Ar), 6.81–6.75 (m, 2H, Ar), 5.76 (dd, 1H,
J = 2.3, 2.3 Hz, H-2000), 5.44 (d, 1H, J = 1.4 Hz, H-1),
5.35 (d, 1H, J = 1.7 Hz, H-10), 5.31 (br s, 1H, H-100),
5.11 (br s, 1H, H-1000), 4.88 (d, 1H, J = 11.1 Hz, PhCH2),
4.83 (d, 1H, J = 10.9 Hz, PhCH2), 4.78–4.34 (m, 23H,
21 ꢀ PhCH2, H-3, H-300), 4.30–4.24 (m, 2H, H-30,
PhCH2), 4.17–3.87 (m, 13H, H-2, H-20, H-200, H-3000,
H-400, H-60a, H-6a, H-6b, H-5, H-4, H-40, H-4000, H-50),
3.77 (s, 3H, ArOCH3), 3.75–3.50 (m, 6H, H-500,
H-60a0; H-60b0; H-6b0 ; H-6a000; H-60b00), 3.44–3.36 (m, 1H,
H-5000); 13C NMR (125 MHz, CDCl3, dC) 165.5 (C@O),
155.2 (Ar), 150.7 (Ar), 139.0 (Ar), 138.9 (Ar), 138.7
(Ar), 138.6 (2 ꢀ Ar), 138.44 (Ar), 138.35 (2 ꢀ Ar),
138.2 (2 ꢀ Ar), 133.0 (Ar), 130.1 (Ar), 130.0 (2 ꢀ Ar),
128.44 (Ar), 128.38 (4 ꢀ Ar), 128.36 (4 ꢀ Ar), 128.33
(2 ꢀ Ar), 128.31 (2 ꢀ Ar), 128.26 (4 ꢀ Ar), 128.23
(2 ꢀ Ar), 128.19 (4 ꢀ Ar), 128.1 (2 ꢀ Ar), 128.03
(2 ꢀ Ar), 128.01 (2 ꢀ Ar), 128.00 (2 ꢀ Ar), 127.93
(Ar), 127.87 (2 ꢀ Ar), 127.73 (2 ꢀ Ar), 127.71
(2 ꢀ Ar), 127.65 (2 ꢀ Ar), 127.60 (2 ꢀ Ar), 127.56
(2 ꢀ Ar), 127.5 (4 ꢀ Ar), 127.44 (2 ꢀ Ar), 127.41
(2 ꢀ Ar), 127.37 (2 ꢀ Ar), 127.31 (4 ꢀ Ar), 127.26

D. Hou et al. / Carbohydrate Research 343 (2008) 1778–1789
1787
(2 ꢀ Ar), 133.0 (2 ꢀ Ar), 132.94 (2 ꢀ Ar), 132.90 (Ar),
132.7 (Ar), 130.1 (Ar), 130.0 (2 ꢀ Ar), 129.9 (4 ꢀ Ar),
129.81 (4 ꢀ Ar), 129.79 (4 ꢀ Ar), 129.76 (4 ꢀ Ar),
129.7 (4 ꢀ Ar), 129.6 (2 ꢀ Ar), 129.3 (Ar), 129.24
(2 ꢀ Ar), 129.22 (2 ꢀ Ar), 129.1 (4 ꢀ Ar), 129.0 (Ar),
128.92 (2 ꢀ Ar), 128.87 (Ar), 128.8 (Ar), 128.74
(2 ꢀ Ar), 128.70 (2 ꢀ Ar), 128.66 (2 ꢀ Ar), 128.52
(4 ꢀ Ar), 128.46 (2 ꢀ Ar), 128.44 (2 ꢀ Ar), 128.38
(2 ꢀ Ar), 128.34 (4 ꢀ Ar), 128.31 (4 ꢀ Ar), 128.1
(2 ꢀ Ar), 118.0 (Ar), 114.7 (Ar), 100.5 (C-100), 99.7
(C-10), 99.4 (C-1000), 96.5 (C-1), 77.0 (C-200), 76.9 (C-3),
75.8 (C-30), 71.8 (C-2), 71.6 (C-20), 70.3 (C-2000), 70.0
(C-300), 69.9 (C-50), 69.73 (C-500), 69.70 (C-5000), 69.4
(C-5), 69.2 (C-3000), 68.2 (C-40), 68.0 (C-4000), 66.7 (C-
400), 66.2 (C-4), 63.0 (C-6), 62.9 (C-6000), 62.5 (C-60),
61.8 (C-600), 55.6 (ArOCH3). MALDI MS: m/z calcd
for [C122H100O35]Na+: 2147.5937. Found: 2147.5951.
5.32 (dd, 1H, J = 3.1, 2.2 Hz, H-2), 5.19 (br s, 1H,
H-100), 5.07 (d, 1H, J = 1.5 Hz, H-10), 4.70 (d, 1H,
J = 1.7 Hz, H-1000), 4.65 (dd, 1H, J = 12.2, 2.6 Hz,
H-60a00Þ, 4.60 (dd, 1H, J = 9.8, 3.1 Hz, H-3), 4.56 (dd,
1H, J = 12.5, 2.4 Hz, H-60a), 4.51–4.44 (m, 2H, H-6b000,
H-30), 4.37–4.27 (m, 5H, H-50, H-5, H-5000, H-6a, H-6b),
4.18 (dd, 1H, J = 12.5, 3.0 Hz, H-60b), 4.15–4.09 (m,
2H, H-60a0, H-500), 4.05–3.92 (m, 2H, H-60b0, H-200), 3.79–
3.72 (ddd, 1H, J = 9.7, 6.9, 6.9 Hz, octyl OCH2), 3.54–
3.46 (ddd, 1H, J = 9.7, 6.9, 6.9 Hz, octyl OCH2), 3.25
(dd, 2H, J = 7.0, 7.0 Hz, CH2N3), 1.67–1.56 (m, 5H,
octyl CH2), 1.41–1.30 (m, 7H, octyl CH2); 13C NMR
(125 MHz, CDCl3, dC) 166.07 (C@O), 166.05 (C@O),
166.0 (C@O), 165.9 (C@O), 165.8 (C@O), 165.7
(C@O), 165.34 (C@O), 165.31 (C@O), 164.93 (C@O),
164.87 (C@O), 164.8 (C@O), 164.64 (C@O), 164.57
(C@O), 133.7 (2 ꢀ Ar), 133.4 (Ar), 133.31 (Ar), 133.26
(2 ꢀ Ar), 133.01 (2 ꢀ Ar), 132.97 (Ar), 132.92 (Ar),
132.86 (Ar), 132.8 (Ar), 132.7 (Ar), 130.1 (Ar), 130.00
(Ar), 129.96 (Ar), 129.93 (Ar), 129.90 (2 ꢀ Ar), 129.86
(4 ꢀ Ar), 129.83 (4 ꢀ Ar), 129.78 (4 ꢀ Ar), 129.7
(4 ꢀ Ar), 129.64 (2 ꢀ Ar), 129.60 (2 ꢀ Ar), 129.3 (Ar),
129.22 (Ar), 129.21 (2 ꢀ Ar), 129.03 (Ar), 129.02 (Ar),
128.98 (2 ꢀ Ar), 128.9 (Ar), 128.85 (Ar), 128.78 (Ar),
128.7 (2 ꢀ Ar), 128.61 (2 ꢀ Ar), 128.56 (4 ꢀ Ar), 128.5
(4 ꢀ Ar), 128.38 (4 ꢀ Ar), 128.36 (4 ꢀ Ar), 128.33
(4 ꢀ Ar), 128.29 (Ar), 128.26 (Ar), 128.1 (2 ꢀ Ar),
100.5 (C-10), 99.6 (C-1), 99.3 (C-1000), 97.4 (C-100), 77.6
(C-200), 75.9 (C-20), 72.0 (C-020 ), 71.6 (C-30), 70.2 (C-2000),
70.0 (C-4), 69.7 (2C, C-4 , C-3000), 69.6 (2C, C-4000,
C-3), 69.2 (C-40), 68.8 (C-300), 68.7 (octyl OCH2), 68.3
(C-5), 67.8 (C-50), 66.7 (C-5000), 66.2 (C-500), 63.0 (C-6),
62.8 (C-600), 62.2 (C-60), 61.8 (C-6000), 51.4 (CH2N3),
29.3 (octyl CH2), 29.3 (octyl CH2), 29.1 (octyl CH2),
28.8 (octyl CH2), 26.7 (octyl CH2), 26.0 (octyl CH2).
MALDI MS: m/z calcd for [C123H109O34N3]Na+:
2194.6785. Found: 2194.6800.
3.16. 8-Azidooctyl 2,3,4,6-tetra-O-benzoyl-a-D-manno-
pyranosyl-(1?2)-3,4,6-tri-O-benzoyl-a-D-mannopyran-
osyl-(1?3)-2,4,6-tri-O-benzoyl-D-mannopyranosyl-
(1?3)-2,4,6-tri-O-benzoyl-a-D-mannopyranoside (24)
A solution of compound 21 (196 mg, 0.09 mmol) and
ceric ammonium nitrate (506 mg, 0.9 mmol) in 8:1
CH3CN–H2O (18 mL) was stirred at rt for 1 h, diluted
with EtOAc (40 mL), and washed with water (40 mL),
satd aq NaHCO3 (40 mL), and brine (40 mL). The
organic layer was then dried and concentrated, and the
residue was purified by chromatography (1:1 hexane–
EtOAc) to afford 22 (180 mg, 97%) as a colorless oil.
Compound 22 (174 mg, 0.09 mmol) was then dissolved
in dry CH2Cl2 (20 mL) and then CCl3CN (0.18 mL,
1.7 mmol) and DBU (4 lL) were added in succession
at 0 °C. The solution was stirred at rt for 2 h and then
concentrated. The resulting oil was purified by chroma-
tography (1:1 hexane–EtOAc and 1% Et3N) to afford 23
(187 mg, 100%) as a colorless oil, which, following dry-
ing under vacuum overnight, was used immediately in
the next step. To a solution of a mixture of 8-azido-1-
octanol9 (8 mg, 0.06 mmol) and 23 (35 mg, 0.02 mmol)
3.17. 2,4,6-tri-O-benzoyl-3-O-methyl-a-D-mannopyrano-
syl trichloroacetimidate (25)
˚
in dry CH2Cl2 (10 mL) was added activated 4 A molec-
A solution of compound 8 (187 mg, 0.31 mmol) and
ceric ammonium nitrate (502 mg, 0.92 mmol) in 4:1
CH3CN–H2O (25 mL) was stirred at rt for 1 h, diluted
with EtOAc (40 mL), and washed with water (40 mL),
satd aq NaHCO3 (40 mL), and brine (40 mL). The
organic layer was dried and concentrated, and the resi-
due was purified by chromatography (3:1 hexane–
EtOAc) to afford reducing monosaccharide (154 mg,
100%) as a colorless oil. The resulting oil (154 mg,
0.30 mmol) was then dissolved in dry CH2Cl2 (20 mL),
and then CCl3CN (0.66 mL, 6.60 mmol) and DBU
(13.7 lL, 0.09 mmol) were sequentially added at 0 °C.
The solution was stirred at rt for 2 h and then concen-
trated. The resulting oil was purified by chromatogra-
phy (5:1 hexane–EtOAc and 1% Et3N) to afford 25
ular sieves (50 mg). After stirring at rt for 30 min, a solu-
tion of 10% TMSOTf in CH2Cl2 (8.7 lL) was added.
The reaction mixture was then stirred for 16 h at rt,
and then it was neutralized with Et3N; concentration
gave a residue that was purified by chromatography
(4:1 toluene–EtOAc) to afford compound 24 (35 mg,
79%) as a colorless oil. Rf 0.79 (4:1 toluene–EtOAc);
[a]D ꢁ27.2 (c 1.8, CH2Cl2); 1H NMR (500 MHz, CDCl3,
dH) 8.20–7.75 (m, 24H, Ar), 7.69–7.65 (m, 2H, Ar),
7.60–7.20 (m, 35H, Ar), 7.12–7.00 (m, 4H, Ar), 6.08
(dd, 1H, J = 10.1, 10.1 Hz, H-4000), 6.01 (dd, 1H,
J = 10.0, 10.0 Hz, H-40), 5.95–5.88 (m, 3H, H-4, H-400,
H-3000), 5.70–5.65 (m, 2H, H-2000, H-20), 5.57 (dd, 1H,
J = 10.0, 3.1 Hz, H-300), 5.38 (d, 1H, J = 2.2 Hz, H-1),

1788
D. Hou et al. / Carbohydrate Research 343 (2008) 1778–1789
(155 mg, 78%) as a colorless oil, which, following drying
under vacuum overnight, was used immediately in the
next step.
3.19. p-Methoxyphenyl 2,4,6-tri-O-benzoyl-3-O-methyl-
a-D-mannopyranosyl-(1?2)-3,4,6-tri-O-benzoyl-a-D-
mannopyranosyl-(1?3)-2,4,6-tri-O-benzoyl-D-mannopyr-
anosyl-(1?3)-2,4,6-tri-O-benzoyl-a-D-mannopyranoside
(28)
3.18. p-Methoxyphenyl 2,4,6-tri-O-benzoyl-3-O-methyl-
a-D-mannopyranosyl-(1?2)-3,4,6-tri-O-benzyl-a-D-man-
nopyranosyl-(1?3)-2,4,6-tri-O-benzyl-a-D-mannopyrano-
syl-(1?3)-2,4,6-tri-O-benzyl-a-D-mannopyranoside (26)
To a solution of compound 26 (229 mg, 0.12 mmol) in
3:1 CH3OH–EtOAc (12 mL) was added 20% Pd(OH)2-
on-carbon (15 mg), and the reaction mixture was stirred
for 24 h under a hydrogen atmosphere. The mixture was
filtered through Celite and then concentrated; a 1H
NMR spectrum of the crude product revealed that all
of the benzyl groups had been removed. The resulting
oil (27) was dissolved in pyridine (8 mL), and to this solu-
tion were added DMAP (2 mg) and benzoyl chloride
(0.15 mL, 1.32 mmol) dropwise at rt. The reaction mix-
ture was stirred at 50 °C for 15 h, and it was then cooled
to rt and diluted with CH2Cl2 (30 mL). The organic layer
was washed with 1N HCl (2 ꢀ 30 mL), satd aq NaHCO3
(30 mL), and brine (30 mL). After drying, the organic
layer was concentrated and the residue was purified by
chromatography (2:1 hexane–EtOAc) to afford 28
(227 mg, 93%) as an oil. Rf 0.65 (1:1 hexane–EtOAc);
[a]D ꢁ11.8 (c 0.1, CH2Cl2); 1H NMR (500 MHz, CDCl3,
dH) 8.22–7.91 (m, 22H, Ar), 7.87–7.78 (m, 4H, Ar), 7.72–
7.66 (m, 2H, Ar), 7.63–7.24 (m, 28H, Ar), 7.11–7.00 (m,
6H, Ar), 6.80–6.74 (m, 2H, Ar), 6.05 (dd, 1H, J = 9.8, 9.8
Hz, H-4), 5.93–5.80 (m, 4H, H-2, H-40, H-400, H-4000), 5.68
(d, 1H, J = 1.8 Hz, H-1), 5.56–5.50 (m, 2H, H-2000, H-300),
5.46 (d, 1H, J = 2.2 Hz, H-10), 5.39 (dd, 1H, J = 2.7,
2.2 Hz, H-20), 5.23 (br s, 1H, H-100), 4.84 (dd, 1H,
J = 9.7, 3.3 Hz, H-3), 40.64–4.41 (m, 7H, H-1000, H-5,
H-6a, H-6b, H-50, H-6a, H-30), 4.34–4.27 (m, 2H,
H-60b; H-60a0), 4.23–4.03 (m, 5H, H-5000, H-60a00; H-6b000,
H-500, H-60b0Þ, 3.96–3.74 (m, 2H, H-3000, H-200), 3.75
(s, 3H, ArOCH3), 3.24 (s, 3H, OCH3); 13C NMR
(125 MHz, CDCl3, dC) 166.1 (C@O), 166.0 (C@O),
165.84 (C@O), 165.79 (2 ꢀ C@O), 165.7 (C@O), 165.30
(C@O), 165.27 (C@O), 164.99 (C@O), 164.96 (C@O),
164.9 (C@O), 164.8 (C@O), 155.4 (Ar), 149.6 (Ar),
134.5 (Ar), 133.8 (Ar), 133.6 (Ar), 133.54 (Ar), 133.53
(Ar), 133.49 (Ar), 133.43 (Ar), 133.37 (Ar), 133.20 (Ar),
133.18 (Ar), 133.1 (Ar), 133.0 (Ar), 132.9 (Ar), 130.6
(Ar), 130.2 (2 ꢀ Ar), 130.02 (Ar), 130.00 (Ar), 129.94
(4 ꢀ Ar), 129.91 (4 ꢀ Ar), 129.84 (4 ꢀ Ar), 129.76
(4 ꢀ Ar), 129.7 (Ar), 129.6 (Ar), 129.49 (Ar), 129.48
(Ar), 129.13 (2 ꢀ Ar), 129.09 (2 ꢀ Ar), 129.04 (2 ꢀ Ar),
129.00 (Ar), 128.96 (Ar), 128.9 (Ar), 128.84 (2 ꢀ Ar),
128.77 (Ar), 128.73 (Ar), 128.69 (Ar), 128.63 (2 ꢀ Ar),
128.61 (2 ꢀ Ar), 128.5 (4 ꢀ Ar), 128.44 (4 ꢀ Ar),
128.36 (4 ꢀ Ar), 128.3 (4 ꢀ Ar), 128.2 (2 ꢀ Ar), 117.9
(Ar), 114.7 (Ar), 100.5 (C-100), 99.8 (C-10), 99.7 (C-1000),
96.4 (C-1), 77.1 (C-3000), 76.8 (C-3), 75.7 (C-30), 71.8
(C-2), 71.5 (C-20), 70.4 (C-300), 69.8 (C-5), 69.7 (C-50),
69.5 (C-500), 69.4 (C-5000), 69.2 (C-2000), 68.3 (C-4), 68.1
(C-400), 67.8 (C-4000), 67.6 (C-40), 66.6 (C-200), 63.0 (C-6),
To a solution of a mixture of trisaccharide 18 (246 mg,
0.17 mmol) and imidate 25 (177 mg, 0.27 mmol) in dry
CH2Cl2 (15 mL) was added activated 4 A molecular
˚
sieves (500 mg). The reaction mixture was stirred at rt
for 30 min and then cooled to 0 °C before a solution
of 10% TMSOTf in CH2Cl2 (31.3 lL, 0.04 mmol) was
added. The reaction mixture was stirred for 1 h at
0 °C, and then it was neutralized by the addition of
Et3N and concentrated to give a residue that was puri-
fied by chromatography (4:1 hexane–EtOAc) to afford
26 (262 mg, 79%) as a colorless oil. Rf 0.61 (2:1 hex-
ane–EtOAc); [a]D +25.7 (c 2.3, CH2Cl2); 1H NMR
(500 MHz, CDCl3, dH) 8.13–8.03 (m, 4H, Ar), 7.99–
7.94 (m, 2H, Ar), 7.60–7.49 (m, 3H, Ar), 7.42–7.49
(m, 51H, Ar), 7.00–6.95 (m, 2H, Ar), 6.81–6.76 (m,
2H, Ar), 5.85 (dd, 1H, J = 9.9, 9.9 Hz, H-4000), 5.77
(dd, 1H, J = 2.5, 2.5 Hz, H-2000), 5.45 (d, 1H,
J = 1.5 Hz, H-1), 5.37 (s, 2H, H-10, H-100), 5.16 (br s,
1H, H-1000), 4.90 (d, 1H, J = 11.2 Hz, PhCH2), 4.77–
4.22 (m, 23H, 17 ꢀ PhCH2, H-3, H-4, H-5, H-6a,
H-6b, H-3000), 4.14–3.86 (m, 11H, H-6a000; H-60a, H-2,
H-200, H-20, H-300, H-400, H-500, H-5000, H-60a0; H-6b00), 3.77
(s, 3H, ArOCH3), 3.76–3.49 (m, 5H, H-60b00, H-30, H-40,
H-50, H-60 ), 3.30 (s, 3H, OCH3); 13C NMR (125 MHz,
b
CDCl3, dC) 166.0 (C@O), 165.3 (2 ꢀ C@O), 154.9
(Ar), 150.4 (Ar), 138.7 (2 ꢀ Ar), 138.3 (2 ꢀ Ar), 138.2
(2 ꢀ Ar), 138.1 (2 ꢀ Ar), 138.0 (2 ꢀ Ar), 133.1
(2 ꢀ Ar), 132.8 (Ar), 130.2 (Ar), 129.95 (2 ꢀ Ar),
129.85 (2 ꢀ Ar), 129.8 (2 ꢀ Ar), 129.7 (4 ꢀ Ar), 128.50
(4 ꢀ Ar), 128.48 (2 ꢀ Ar), 128.40 (2 ꢀ Ar), 128.37
(2 ꢀ Ar), 128.35 (2 ꢀ Ar), 128.33 (2 ꢀ Ar), 128.24
(2 ꢀ Ar), 128.23 (2 ꢀ Ar), 128.18 (2 ꢀ Ar), 127.8
(2 ꢀ Ar), 127.72 (2 ꢀ Ar), 127.70 (2 ꢀ Ar), 127.66
(4 ꢀ Ar), 127.61 (Ar), 127.59 (Ar), 127.57 (2 ꢀ Ar),
127.54 (2 ꢀ Ar), 127.49 (Ar), 127.45 (2 ꢀ Ar), 127.4
(Ar), 127.33 (Ar), 127.29 (2 ꢀ Ar), 127.19 (2 ꢀ Ar),
127.16 (Ar), 127.1 (2 ꢀ Ar), 117.9 (2 ꢀ Ar), 114.5
(2 ꢀ Ar), 99.9 (2C, C-10, C-100), 99.1 (C-1000), 96.5
(C-1), 79.5 (C-200), 77.7 (C-3), 77.2 (C-2, C-2000), 75.8
(C-300), 75.4 (C-4), 74.9 (C-5, C-3000), 74.8 (PhCH2),
74.7 (PhCH2), 74.3 (C-500), 73.5 (PhCH2), 73.34
(PhCH2), 73.29 (2 ꢀ PhCH2), 72.6 (C-40), 72.5 (PhCH2),
72.4 (C-400, C-20), 72.3 (PhCH2), 72.1 (PhCH2), 69.3
(C-30), 69.1 (C-60, C-600), 68.9 (C-6), 68.4 (2C, C-5000,
C-50), 68.1 (C-4000), 62.5 (C-6000), 57.8 (OCH3), 55.6 (Ar-
OCH3). MALDI MS: m/z calcd for [C116H116O25]Na+:
1931.7698. Found: 1931.7699.

D. Hou et al. / Carbohydrate Research 343 (2008) 1778–1789
1789
62.6 (C-60), 62.5 (C-600), 62.0 (C-6000), 57.7 (OCH3), 55.6
(ArOCH3). MALDI MS: m/z calcd for [C116H98O34]-
Na+: 2057.5832. Found: 2057.5834.
(C@O), 164.87 (C@O), 164.85 (C@O), 164.8 (C@O),
134.5 (Ar), 133.64 (Ar), 133.58 (Ar), 133.55 (Ar), 133.4
(Ar), 133.3 (Ar), 133.2 (Ar), 133.01 (Ar), 133.97 (Ar),
133.9 (Ar), 132.83 (Ar), 132.81 (Ar), 130.6 (Ar), 130.17
(Ar), 130.15 (Ar), 130.0 (Ar), 129.91 (2 ꢀ Ar), 129.88
(2 ꢀ Ar), 129.86 (2 ꢀ Ar), 129.82 (2 ꢀ Ar), 129.79
(4 ꢀ Ar), 129.74 (4 ꢀ Ar), 129.70 (4 ꢀ Ar), 129.68
(4 ꢀ Ar), 129.6 (Ar), 129.48 (2 ꢀ Ar), 129.46 (Ar),
129.2 (Ar), 129.14 (Ar), 129.11 (2 ꢀ Ar), 129.09
(2 ꢀ Ar), 129.0 (Ar), 128.9 (Ar), 128.8 (Ar), 128.7 (Ar),
128.61 (Ar), 128.58 (4 ꢀ Ar), 128.5 (4 ꢀ Ar), 128.43
(4 ꢀ Ar), 128.38 (2 ꢀ Ar), 128.36 (Ar), 128.33 (Ar),
128.30 (Ar), 128.1 (Ar), 100.5 (C-10), 99.8 (C-1), 99.7
(C-1000), 97.4 (C-100), 77.4 (C-2), 77.1 (C-3000), 75.8 (C-200),
72.1 (C-2000), 71.5 (C-4), 70.4 (C-20), 69.7 (2C, C-30,
C-400), 69.6 (C-300), 69.1 (C-3), 68.8 (C-40), 68.7 (octyl
OCH2), 68.4 (C-4000), 67.9 (C-500), 67.7 (C-5), 67.5 (C-50),
66.6 (C-5000), 63.1 (C-6), 62.5 (C-600), 62.3 (C-60), 62.0
(C-6000), 57.6 (OCH3), 51.4 (CH2N3), 29.3 (octyl CH2),
29.2 (octyl CH2), 29.0 (octyl CH2), 28.8 (octyl CH2),
26.6 (octyl CH2), 26.0 (octyl CH2). MALDI MS: m/z
calcd for [C117H107O33N3]Na+: 2104.6679. Found:
2104.6689.
3.20. 8-Azidooctyl 2,4,6-tri-O-benzoyl-3-O-methyl-a-D-
mannopyranosyl-(1?2)-3,4,6-tri-O-benzoyl-a-D-manno-
pyranosyl-(1?3)-2,4,6-tri-O-benzoyl-D-mannopyranosyl-
(1?3)-2,4,6-tri-O-benzoyl-a-D-mannopyranoside (31)
A solution of tetrasaccharide 28 (146 mg, 0.07 mmol)
and ceric ammonium nitrate (393 mg, 0.7 mmol) in 8:1
CH3CN–H2O (18 mL) was stirred at rt for 1 h, diluted
with EtOAc (40 mL), and washed with water (40 mL),
satd aq NaHCO3 (40 mL), and brine (40 mL). The or-
ganic layer was dried and concentrated, and the residue
was purified by chromatography (1:1 hexane–EtOAc)
to afford 29 (126 mg, 91%) as a colorless oil. Compound
29 (126 mg, 0.07 mmol) was then dissolved in dry CH2Cl2
(10 mL), and then CCl3CN (0.13 mL, 1.31 mmoL) and
DBU (2.9 lL) were sequentially added at 0 °C. The solu-
tion was stirred at rt for 2 h and then concentrated. The
resulting oil was purified by chromatography (1:1 hex-
ane–EtOAc and 1% Et3N) to afford 30 (124 mg, 91%)
as a colorless oil, which, following drying under vacuum
overnight, was used immediately in the next step. To a
solution of a 8-azido-1-octanol9 (10 mg, 0.06 mmol)
and 30 (41 mg, 0.02 mmol) in dry CH2Cl2 (5 mL) was
Acknowledgments
This work was supported by the Alberta Ingenuity
Centre for Carbohydrate Science, The University of
Alberta and The Natural Sciences and Engineering
Research Council of Canada.
˚
added freshly activated 4 A molecular sieves (50 mg),
and the mixture was stirred at rt for 30 min. A 10% solu-
tion of TMSOTf in CH2Cl2 (11 lL) was added at rt. The
reaction mixture was then stirred for 16 h, and it was then
neutralized by adding Et3N. The solution was then
filtered and concentrated to a residue that was purified
by chromatography (4:1 toluene–EtOAc) to afford 31
(30 mg, 72%) as a colorless oil. Rf 0.80 (4:1 toluene–
EtOAc); [a]D ꢁ9.7 (c 1.2, CH2Cl2); 1H NMR
(500 MHz, CDCl3, dH) 8.18–7.89 (m, 20H, Ar), 7.85–
7.82 (m, 2H, Ar), 7.78–7.74 (m, 2H, Ar), 7.70–7.66 (m,
2H, Ar), 7.60–7.23 (m, 32H, Ar), 7.08–7.01 (m, 2H,
Ar), 5.99 (dd, 1H, J = 10.0, 10.0 Hz, H-40), 5.91 (dd,
1H, J = 9.8, 9.8 Hz, H-4), 5.84–5.77 (m, 2H, H-4000,
H-400), 5.66 (dd, 1H, J = 3.4, 1.8 Hz, H-20), 5.53 (dd,
1H, J = 2.5, 2.5 Hz, H-2000), 5.49 (dd, 1H, J = 10.1,
3.1 Hz, H-300), 5.37 (d, 1H, J = 2.1 Hz, H-1), 5.33 (dd,
1H, J = 3.1, 2.1 Hz, H-2), 5.18 (s, 1H, H-100), 5.06 (d,
1H, J = 1.8 Hz, H-10), 4.68–4.54 (m, 4H, H-1000, H-30,
H-6a0 , H-6a000), 4.51–4.44 (m, 2H, H-60b, H-3), 4.36–4.26
(m, 3H, H-50, H-5, H-6a), 4.25–4.17 (m, 2H, H-5000,
H-60b00), 4.15–3.99 (m, 4H, H-500, H-60a0; H-6b00, H-6b),
3.92–3.86 (m, 2H, H-3000, H-200), 3.74 (ddd, 1H, J = 9.6,
6.7, 6.7 Hz, octyl OCH2), 3.49 (ddd, 1H, J = 9.6, 6.7,
6.7 Hz, octyl OCH2), 3.27–3.21 (m, 5H, OCH3,
CH2N3), 2.06–1.55 (m, 5H, octyl CH2), 1.41–1.31 (m,
7H, octyl CH2); 13C NMR (125 MHz, CD3Cl, dC)
166.1 (C@O), 166.0 (C@O), 165.9 (C@O), 165.8
(3 ꢀ C@O), 165.29 (C@O), 165.27 (C@O), 165.0
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2008.02.025.
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