Synthesis of the β-1,3-glucan, laminarahexaose: NMR and conformational studies

Article abstract of DOI:10.1016/j.carres.2008.12.014

The synthesis of laminarahexaose is described. NMR studies of several of the intermediates leading to the β-1,3-glucan show anomalously small coupling constants for some of the C-1 hydrogens. An X-ray structure for the protected hexasaccharide shows that

Full text of DOI:10.1016/j.carres.2008.12.014

Carbohydrate Research 344 (2009) 439–447
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of the b-1,3-glucan, laminarahexaose: NMR
and conformational studies
*
Kai-For Mo, Huiqing Li, Joel T. Mague, Harry E. Ensley
Department of Chemistry, Tulane University, New Orleans, LA 70118, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 October 2008
Received in revised form 8 December 2008
Accepted 16 December 2008
Available online 25 December 2008
The synthesis of laminarahexaose is described. NMR studies of several of the intermediates leading to the
b-1,3-glucan show anomalously small coupling constants for some of the C-1 hydrogens. An X-ray struc-
ture for the protected hexasaccharide shows that the small coupling constants are due to some of the glu-
copyranose rings adopting a twist-boat conformation. The X-ray studies also explain other unexpected
NMR observations.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Laminarahexaose
b-1,3-Glucan
Oligosaccharide synthesis
1. Introduction
A complicating component in the preparation of b-(1?3)-glu-
cans is that of determining when the unexpected formation of an
The construction of structurally well-defined products in gly-
cosylation reactions is one of the most important objectives in
a anomer has occurred. In relatively simple carbohydrate systems,
a small (2–6 Hz) H1–H2 coupling constant can be indicative; how-
ever, as shown below, this is not always a reliable diagnostic tool.
carbohydrate chemistry.1 The production of a mixture of
a and
b anomers not only lowers the yield of the desired product,
but also increases the workload in separation and purification
processes. It is generally accepted that b-glycosidic bonds will
be formed if an acyl group is present at the C-2 position of
2. Results and discussion
2.1. Preparation of the protected hexasaccharide 16 and
laminarahexaose
the
D-glucose donor due to its neighboring group participation
via the oxo-carbenium intermediate.2 Because of the neighboring
group effect, the nucleophilic acceptor attacks the upper face of
the anomeric carbon of the donor to form the desired b-linkage.
Recently, our group has utilized a new type of C-2 acyl protect-
ing group, 4-acetoxy-2,2-dimethylbutanoate (ADMB),6 as the
neighboring participation group in b-glucosylations with high
yield and good diastereoselectivity. We have used this C-2 acyl
protecting group in the synthesis of b-(1?3)-glucans,7 and our
group is constructing a small library of linear b-(1?3)-glucans
and branched glucans (having b-(1?6)-glycosidic linkages with a
side chain of varying length along the linear glucan backbone)
for physiological studies.8,9 During the course of this work we pre-
pared the linear b-(1?3)-hexaglucoside (1) and we noted a num-
ber of interesting spectroscopic anomalies along the way.
However, in some cases,
a anomers are unexpectedly formed.
For example, Du and co-workers3 reported the synthesis of a
branching hexasaccharide via 3+3 coupling yielding only the
unexpected
a anomer even though C-2 acetate protection was
used in the trisaccharide donor. Du and co-workers concluded
that the steric bulk of the C-3 hydroxyl acceptor was responsible
for this unexpected result. Kong and co-workers4 have also re-
ported unexpected
a anomer formation in the attempted synthe-
sis of b-(1?3)-glucans. These workers proposed that the by-
The synthesis started from the known 4,6-O-benzylidene-1-
product of glucosylation reactions, the orthoester, rearranged in
the presence of acid to give the unexpected
thio-b-D
-glucopyranoside10 (Scheme 1). Selective silylation at the
a
anomer.5 There-
C-3 hydroxyl (to give 2), followed by benzoylation at the C-2 hy-
droxyl group gave the monosaccharide donor 3. Hydrolysis of 3
was conducted according to the method previously developed in
our group.11 The reaction resulted in not only the hydrolysis of
the thioethyl group but also the migration of C-2 benzoate to the
anomeric carbon to give 4. Treatment of the crude 4 with NEt3/
CH2Cl2, which effected quantitative rearrangement of 4 to 5,
fore, methodology for increasing stereoselectivity in b-glucosyla-
tions and, more importantly, reliability in b-glycosylations is still
in great need.
* Corresponding author. Tel.: +1 504 865 5573.
E-mail address: hensley@tulane.edu (H.E. Ensley).
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.12.014

440
K.-F. Mo et al. / Carbohydrate Research 344 (2009) 439–447
followed by standard Schmidt conditions,12 provided the trichloro-
acetimidate donor 6 as the second building block.
Secondly, the chemical shift of one of the benzylidene protons shifted
far upfield to 3.94 ppm from its usual 5 to 6 ppm range (Table 1).
HO
HO
HO
HO
HO
HO
HO
HO
HO
VI
V
IV
III
II
I
O
O
O
O
O
O
HO
HO
HO
O
O
O
O
O
HO
OH
OH
OH
OH
OH
OH
OH
1
Removal of the C-3 TBDMS group of 14 yielded the corre-
Benzoylation of compound 4, followed by desilylation with HF/
pyridine gave 8, which would function as the reducing terminus
sponding tetrasaccharide acceptor 15 in 93% yield, in which the
coupling constant of H-1III (JH-1,H-2 = 2 Hz) and the chemical shift
of the benzylidene proton (4.14 ppm) remained almost the same,
indicating that these two phenomena were not related to the
bulky TBDMS protecting group. Coupling of the donor 13 and
acceptor 15 using NIS/AgOTf gave the hexasaccharide 16 in 92%
yield. The NMR spectral data for hexasaccharide 16 were similar
to that presented by tetrasaccharides 14 and 15. However, in
the case of 16, both H-1III and H-1V have JH-1,H-2 < 1 Hz. The small
coupling constants for the H-1 protons suggested that the newly
acceptor (Scheme 2). Selective acylation of the
a-thioglucoside
913 at the C-2 hydroxyl by ADMB chloride in pyridine afforded
10 in 87% yield.
As shown in Scheme 3, coupling of the monosaccharide acceptor
8 and thioglucoside 3 using NIS and AgOTf as the promoter afforded
the disaccharide 11 in 95% yield. The coupling constant of H-1II
(4.96 ppm) is 7.6 Hz, and C-1II appears at 101.3 ppm, indicating that
only the b isomer was formed. Removal of the TBDMS group using
HFÁPy provided the corresponding disaccharide acceptor 12. Upon
standard activation by catalytic TMSOTf, the glucosyl imidate 6 cou-
pled with the acceptor 10 to give the disaccharide donor 13 in 92%
yield. The coupling constant of H-1II (5.00 ppm) is 6.8 Hz, and the
C-1II was at 99.4 ppm, again indicating that the expected b-linkage
was formed.
formed linkage had the
a
configuration; however, again, the 13C
NMR chemical shift suggested the b configuration (C-1III 95.8
and C-1V 95.9 ppm).14 In addition, two of the six benzylidine pro-
tons were now dramatically shifted upfield in the 1H NMR
spectrum.
There are two examples in the literature where careful NMR
analysis of oligoglucosides suggested that small coupling constants
can sometimes be observed for b-glucopyranoside linkages. Collins
With both disaccharide donor and acceptor in hand, the stage was
set for the iterative synthesis. Condensation of the b-(1?3)-disaccha-
ride acceptor 12 and the b-(1?3)-disaccharide donor 13 (Scheme 4)
with NIS and AgOTf as the promoter provided tetrasaccharide 14.
The 1H NMR spectrum of the crude reaction mixture showed only
one isomer formed, which was thought to be the desired tetrasaccha-
ride with a new b-glycosidic bond. The coupling of the disaccharide
donor analogous to 13 (but where the ADMBprotecting group was re-
placed with a benzoyl group) with acceptor 12 gave a 4:3 mixture of
and Ali15 reported that the coupling constant of the third sugar res-
III
1;2
idue (J ) of the trisaccharide 18 is only 5.2 Hz. He attributed this
anomaly to an unexplained conformational change in the trisac-
charide. Also, Vetvicka and co-workers16 noticed an exceptionally
II
1;2
small coupling constant (J 4.3 Hz) for the second sugar residue
of trisaccharide 19. Removal of the bulky C-3 protecting group to
give 20 did not alter this value. Nuclear Overhauser effect (NOE)
spectroscopy led Vetvicka and co-workers to propose that the
4,6-O-benzylidene acetal protection had distorted the ring confor-
mation of the central glucose unit from the normal chair (4C1) to a
boat (1,4B or B2,5), thus lowering the coupling constant of H-1 in the
second sugar residue.17
the
a
and b glycosides, respectively, in 86% yield. Analysis of 14 via 1H
NMR, 13C NMR, 1H–1H COSY, and HMQC experiments revealed two
interesting results. First, the signal of H-1III (4.66 ppm, see Table 1)
had JH-1,H-2 < 1 Hz, which indicated that the a-glycosidic linkage had
been formed. However, 13C NMR showed that the chemical shift of
C-1III was 95.9 ppm, which supported formation of the b anomer.14
Ph
O
Ph
O
Ph
O
(a)
(b)
(c)
O
O
O
O
O
O
SEt
SEt
SEt
TBDMSO
HO
TBDMSO
HO
BzO
HO
2
3
Ph
O
Ph
O
5
O
R = H
(d)
O
O
O
(e)
TBDMSO
TBDMSO
6 R = C(NH)CCl3
OR
HO
BzO
OBz
4
Scheme 1. Reagents and conditions: (a) TBDMSCl, Imidazole, DMF, 86%; (b) BzCl, DMAP, Py/CH2Cl2, 80 °C, 92%; (c) NBS, TMSOTf, CH2Cl2, 0 °C; (d) NEt3/CH2Cl2, 85% for two
steps; (e) Cl3CCN, DBU, CH2Cl2, 0 °C , 92% (a/b = 8:1).
Ph
O
O
O
O
HO
Ph
O
Ph
O
O
O
(a)
(c)
O
O
RO
4
HO
SEt
BzO
HO
OBz
SEt
O
9
7
R = TBDMS
AcO
(b)
10
8 R = H
Scheme 2. Reagents and conditions: (a) BzCl, DMAP, Py, 99%; (b) HFÁPy, Py/THF, 92%; (c) ADMB chloride, Py, 86%.

K.-F. Mo et al. / Carbohydrate Research 344 (2009) 439–447
441
Ph
O
Ph
O
O
Ph
O
O
III
II
I
O
O
O
O
O
O
III
II
I
O
O
O
O
O
O
O
O
O
O
O
O
O
OBn
RO
BzO
BzO
BzO
BzO
BzO
O
Cl
O
H
Ph
18
19 R=2-naphthylmethyl
20 R=H
Complete deprotection of 16 was accomplished in a three-
step process. The TBDMS group was removed by treatment with
HFÁPy in THF, and the benzylidene groups were removed by
catalytic hydrogenolysis using 10% Degussa Pd/C in methanol/
tetrahydrofuran/acetic acid (8:4:2.4). Removal of the acyl pro-
tecting groups (NaOMe–MeOH), followed by Sephadex LH-20
chromatography gave the hexasaccharide as a 1:1.2 mixture of
deed b-linkages. Also, the third and fifth (from the reducing
end) glucose rings are in a twist-boat conformation, while the
remaining four glucopyranose rings are chairs. The H1–H2 dihe-
dral angles of rings III and V of 16 are 63.6° and 59.3°, respec-
tively, resulting in J1,2 coupling constant of less than 2 Hz as
predicted by the Karplus correlation.19 Additionally, the X-ray
structure shows that the benzylidene acetal proton of the third
and fifth glucose units is pointed directly toward the face of the
aromatic ring of the benzylidene group on the second and fourth
glucose units, respectively (Figs. 2 and 3). The distance from the
III-ring benzylidene proton to the C-1 carbon of the II-ring ben-
zylidene group is 3.12 Å, and the distance from the V-ring benzyl-
idene proton to the C-1 carbon of the IV-ring benzylidene group
is 3.13 Å. The locations of these two protons near the face of
the benzylidene acetal aromatic rings explain the >1 ppm upfield
shift in the NMR for these two protons.20 Although it is true that
the solid-state crystal structure of 16 is not necessarily indicative
of its solution conformation, the fact that the X-ray structure ex-
plains both of the unusual features of the NMR spectrum of 16
supports the fact that the conformation of 16 is the same in the
solution and solid state.
a
and b anomers. The laminarahexaose 1H NMR, 13C NMR,
and HMQC spectra were identical (except for integration) to
those reported by Lim and co-workers18 for laminaraheptaose,
which was prepared by partial hydrolysis of laminarin. In par-
ticular, the JH-1,H-2 coupling constants returned to the expected
7.6–8 Hz range.
2.2. X-ray crystallography
Fortunately, after a great deal of effort, we were able to obtain
crystals of protected hexasaccharide 16 (containing one cyclohex-
ane molecule of crystallization), which were suitable for X-ray
crystallography.18 Crystal data are given in Table 2. The X-ray
structure (Fig. 1) showed that all the glycosidic linkages are in-
Ph
O
Ph
O
Ph
O
O
II
I
Ph
O
O
O
O
(a)
O
O
O
O
O
+
HO
RO
SEt
TBDMSO
BzO
BzO
BzO
BzO
OBz
OBz
11 R = TBDMS
12 R = H
3
8
(b)
Ph
O
Ph
O
Ph
O
O
II
I
O
O
O
O
O
O
O
Ph
O
O
HO
TBDMSO
O
(c)
TBDMSO
O
O
BzO
O
CCl3
SEt
SEt
BzO
O
NH
6
AcO
AcO
10
13
Scheme 3. Reagents and conditions: (a) NIS, AgOTf/toluene, 4 Å MS, CH2Cl2, À20 °C, 95%; (b) HFÁPy, Py/THF, 92%; (c) TMSOTf, 4 Å MS, CH2Cl2, À70 °C, 92%.
Ph
O
Ph
O
O
Ph
O
O
Ph
O
O
IV
III
II
I
O
O
O
O
O
O
O
O
O
RO
(a)
BzO
BzO
BzO
12 + 13
OBz
O
14
R = TBDMS
(b)
AcO
15 R = H
Ph
O
Ph
O
Ph
O
Ph
O
O
Ph
O
O
O
Ph
O
O
VI
V
IV
III
II
I
O
O
O
O
O
O
O
O
O
O
O
O
O
RO
(c)
BzO
O
O
BzO
O
O
BzO
BzO
13 + 15
OBz
AcO
AcO
16 R = TBDMS
17
(b)
R = H
Scheme 4. Reagents and conditions: (a) NIS, AgOTf/toluene, 4 Å MS, CH2Cl2, À20 °C, 91%; (b) HFÁPy, Py/THF, 93%; (c) NIS, AgOTf/toluene, 4 Å MS, CH2Cl2, À20 °C, 92%.

442
K.-F. Mo et al. / Carbohydrate Research 344 (2009) 439–447
Table 1
Selected proton NMR data for compounds 1, 14, 15, 16, and 17
Compound
d H-1I
JIH-1;H-2
d H-BI
d H-1II
d H-1III
d H-1IV
d H-1V
JVH-1;H-2
d H-BV
d H-1VI
II
III
IV
VI
J
J
J
J
H-1;H-2
H-1;H-2
H-1;H-2
H-1;H-2
d H-BII
d H-BIII
d H-BIV
d H-BVI
14
15
16
17
1
6.56
4
5.76
4.75
7.6
5.22
4.66
<1.0
3.94
5.1
8
5.48
NA
NA
6.56
3.6
5.74
4.81
6.8
5.15
4.7
2
4.14
5.15
7.6
5.47
NA
NA
6.54
4
5.72
4.71–4.76
ND
5.23
4.62
0.8
4.09
5.19
8.4
5.33
4.71–4.76
<1.0
3.94
4.9
8
5.51
6.54
3.6
5.72
4.73
7.6
5.18
4.63
1.2
4.12
5.24
8
5.31
4.79
<1.0
4.1
4.90–4.97
ND
5.51
4.35 (b), 4.95 (
7.6 (b), 3.6 (a)
a
)
4.42–4.45
NA
4.51
8
4.51
8
4.51
8
4.38
7.6
NA
NA
NA
NA
NA
NA
d H-1I is the chemical shift of the anomeric proton on the reducing terminus pyranose; JIH-1;H-2 is the coupling constant (in Hz) of the anomeric proton on the reducing
terminus pyranose; d H-BI is the chemical shift for the benzylidene proton on the reducing terminus pyranose.
Table 2
Crystal data for 16
the d-scale in parts per million (ppm). Residual solvent signals
were used as an internal reference. Low- and high-resolution
Empirical formula
Formula weight
Temperature
C141H156O42Si
2550.75
100(2) K
Mo Ka (graphite monochromated,
0.71073 Å)
(HRMS) electrospray (ESI) mass spectra were recorded using a Bru-
ker Microtof instrument.
Thin-layer chromatography (TLC) was carried out on Sorbent
Technologies UV254 Silica Gel plates. Flash column chromatogra-
Radiation (wavelength)
phy was carried out on silica gel (40–63 lm, Sorbent Technolo-
Crystal size
Crystal habit
Crystal system
Space group
Unit cell dimensions
a (Å)
0.26 Â 0.24 Â 0.24 mm
Colorless block
Orthorhombic
C2221
gies). All solvents were dried and freshly distilled using standard
procedures. Organic solutions were concentrated under dimin-
ished pressure with bath temperatures <40 °C.
17.3435(5)
22.9997(7)
73.405(2)
90
90
b (Å)
c (Å)
3.2. Ethyl 4,6-O-benzylidene-3-O-(tert-butyldimethylsilyl)-1-
thio-b-D-glucopyranoside (2)
a
(°)
b (°)
(°)
c
90
To a stirred solution of ethyl 4,6-O-benzylidene-1-thio-b-D-
Volume
Z
29280.9(15) Å3
8
glucopyranoside10 (10.0 g, 32.0 mmol) and imidazole (3.28 g,
48.0 mmol) in anhyd DMF (50 mL) was added tert-butyldimethyl-
silyl chloride (5.23 g, 35.2 mmol) in small portions at 0 °C. Upon
being stirred at room temperature for 24 h, the reaction was
quenched by addition of EtOAc (300 mL) and satd NH4Cl
(100 mL). The organic layer was washed with satd NaHCO3
(2 Â 250 mL) and brine (150 mL), and dried over Na2SO4. Removal
of the solvent under reduced pressure afforded a yellow oil that
was purified by flash chromatography over silica gel (1:12
EtOAc–hexanes) to give pure 2 (5.87 g, 86%) as a colorless oil: Rf
0.23 (1:10 EtOAc–hexanes); 1H NMR d 0.02 (s, 3H), 0.09 (s, 3H),
0.85 (s, 9H), 1.30 (t, J = 7.4 Hz, 3H), 2.65–2.80 (m, 2H), 3.41–3.51
(m, 3H, H-2, H-4 and H-5), 3.71–3.76 (m, 2H H-3 and H-6a), 4.31
(dd. J = 4.8 Hz and 10.4 Hz, 1H, H-6e), 4.44 (d, J = 9.6 Hz, 1H, H-1),
5.50 (s, 1H), 7.32–7.47 (m, 5H); 13C NMR d À4.6, À4.2, 15.5, 18.5,
24.8, 26.0, 68.8 (C-6), 70.9 (C-5), 74.3 (C-2), 76.0 (C-3), 81.3 (C-
4), 86.7 (C-1), 101.8, 126.3, 128.3, 129.1, 137.3; ESI-TOF-HRMS:
calcd for C21H34O5SSiNa [M+Na]+ m/z 449.1794; found m/z
449.1790.
Density (calculated)
Absorption coefficient
F(000)
h Range for data collection
Index ranges
1.157 Mg/m3
0.093 mmÀ1
10,816
1.84–22.50°
À18 6 h 6 5,
À23 6 k 6 14,
À78 6 l 6 23
164,523
15,340 (0.052)
14,354/1320/1579
R1 = 0.094; wR2 = 0.2495
R1 = 0.097; wR2 = 0.2528
1.08
Total reflections
Unique reflections (Rint
Data (I P 2 (I))/restraints/parameters
Final R indices (Fo P 4 (Fo))
)
r
r
Final R indices (all data)
GOF (on F2)
Largest final difference peak/hole (e ÅÀ3
)
0.68/À0.53
3. Experimental
3.1. General methods
1H NMR spectra were recorded on a Varian Unity Inova 400
(400 MHz) spectrometer using CDCl3 as solvent (except for com-
pound 1); multiplicities are quoted as singlet (s), doublet (d), dou-
blet of doublets (dd), triplet (t), doublet of triplets (dt), or multiplet
(m). Carbon nuclear magnetic resonance (13C) spectra were re-
corded on Varian Unity Inova 400 (100 MHz) spectrometers using
CDCl3 unless otherwise noted. Spectra were assigned using COSY,
DEPT, and HMQC experiments. All chemical shifts are quoted on
3.3. Ethyl 2-O-benzoyl-4,6-benzylidene-3-O-(tert-butyldimeth-
ylsilyl)-1-thio-b-D-glucopyranoside (3)
To a stirred solution of 2 (4.50 g, 10.6 mmol) and DMAP
(645 mg, 5.28 mmol) in anhyd pyridine (20 mL) and anhyd CH2Cl2
(30 mL) was added benzoyl chloride (3.07 mL, 26.4 mmol) at 0 °C.
The reaction mixture was heated to 80 °C for 24 h under N2. Upon

K.-F. Mo et al. / Carbohydrate Research 344 (2009) 439–447
443
Figure 1. ORTEP of hexasaccharide 16. Protecting groups and most hydrogens not shown for clarity. The reducing terminus is on the left.
Figure 3. Partial ORTEP of hexasaccharide 16 showing the fourth and fifth glucose
units from the reducing end (to the left). R3 designates the third glucose residue
and R6 designates the sixth glucose residue. Notice the twist-boat conformation of
the fifth glucose unit and the proximity of the benzylidene hydrogen (H29) on the
fifth glucose unit protecting group and the aromatic ring of the benzylidene group
on the fourth glucose unit.
Figure 2. Partial ORTEP of hexasaccharide 16 showing the second and third glucose
units from the reducing end (to the left). R1 designates the first glucose residue and
R4 designates the fourth glucose residue. Notice the twist-boat conformation of the
third glucose unit and the proximity of the benzylidene hydrogen (H70) on the third
glucose unit protecting group and the aromatic ring of the benzylidene group on the
second glucose unit.
1H, H-1), 5.29 (dd, J = 8.6 Hz and 9.8 Hz, 1H, H-2), 5.54 (s, 1H),
7.33–8.08 (m, 10H); 13C NMR d À4.9, À4.1, 14.8, 17.9, 23.9, 25.6,
68.6 (C-6), 70.8 (C-5), 73.3 (C-2), 74.2 (C-3), 81.4 (C-4), 84.1 (C-
1), 101.7, 126.3, 128.1, 128.4, 129.0, 129.8, 130.0, 133.1, 137.1,
165.2; ESI-TOF-HRMS: calcd for C28H38O6SSiNa [M+Na]+ m/z
553.2056; found m/z 553.2128.
removal of solvent under reduced pressure, the residue was dis-
solved in EtOAc (250 mL) and extracted with satd NH4Cl
(100 mL). The organic layer was washed with satd NaHCO3
(2 Â 250 mL), and brine (150 mL), and dried over Na2SO4. Removal
of the solvent under reduced pressure afforded a yellow oil that
was purified by flash chromatography over silica gel (1:15
EtOAc–hexanes) to give pure 3 (5.15 g, 92%) as a white solid: mp
38–41 °C; Rf 0.37 (1:10 EtOAc–hexanes); 1H NMR d À0.13 (s, 3H),
À0.04 (s, 3H), 0.69 (s, 9H), 1.21 (t, J = 7.4 Hz, 3H), 2.67–2.77 (m,
2H), 3.55 (dt, J = 4.8 Hz and 9.6 Hz, 1H, H-5), 3.63 (t, J = 9.2 Hz,
1H, H-4), 3.79 (t, J = 10.0 Hz, 1H, H-6a), 4.05 (t, J = 8.8 Hz, 1H, H-
3), 4.38 (dd. J = 4.8 Hz and 10.4 Hz, 1H, H-6e), 4.63 (d, J = 10.0 Hz,
3.4. Benzoyl 4,6-O-benzylidene-3-O-(tert-butyldimethylsilyl)-
a-D-glucopyranose (4)
To a stirred solution of 3 (300 mg, 0.57 mmol) in wet CH2Cl2 (1:200
H2O–CH2Cl2) (5 mL) at 0 °C was added NBS (101 mg, 0.57 mmol).
TMSOTf solution (10 L in 1.0 mL CH2Cl2, 0.057 mmol) was added in
one portion, and the reaction mixture was stirred at the same
l

444
K.-F. Mo et al. / Carbohydrate Research 344 (2009) 439–447
temperature under N2. After the reaction was completed (ꢀ10 min,
monitored by TLC), satd NaHCO3 (10 mL) was added, and the mixture
solution wasextractedwith CH2Cl2 (3 Â 15 mL). Thecombinedorganic
extract was washed with brine (2 Â 50 mL) and dried over Na2SO4. Re-
moval of solvent under reduced pressure afforded a yellow oil that was
purified by flash chromatography over silica gel (1:8 EtOAc–hexanes)
to give pure 4 (206 mg, 75%) as a white solid: mp 61–64 °C: Rf 0.23 (1:6
EtOAc–hexanes); 1HNMRd0.10(s, 3H), 0.15 (s, 3H),0.91 (s, 9H), 3.57(t,
J = 9.4 Hz, 1H, H-4), 3.73 (t, J = 10.4 Hz, 1H, H-6a), 3.86–3.90 (m, 1H, H-
2), 4.00 (dt, J = 4.9 Hz and 9.9 Hz, 1H, H-5), 4.12 (t, J = 9.2 Hz, 1H, H-3),
4.29 (dd, J = 4.8 and 10.4 Hz, 1H, H-6e), 5.55 (s, 1H), 6.50 (d, J = 3.6 Hz,
1H, H-1), 7.35–8.10 (m, 10H); 13C NMR (CDCl3) d -4.6, -4.0, 18.5, 26.0,
65.4 (C-5), 68.9 (C-6), 72.9 (C-2), 72.9 (C-3), 81.4 (C-4), 92.7 (C-1),
101.9, 126.3, 128.3, 128.8, 129.2, 129.5, 130.1, 133.8, 137.2, 165.3;
ESI-TOF-HRMS: calcd for C26H34O7SiNa [M+Na]+ m/z 509.1971; found
m/z 509.1992.
zoyl chloride (1.6 mL, 18 mmol) in one portion. After stirring
overnight at room temperature, the reaction mixture was diluted
with CH2Cl2 (140 mL). The organic solution was washed with satd
NaHCO3 (2 Â 100 mL), and brine (90 mL), and dried over Na2SO4.
Removal of solvent under reduced pressure afforded a yellow oil.
The crude product was purified by flash chromatography over sil-
ica gel (1:15 EtOAc–hexanes) to give pure 7 (4.80 g, 99%) as a
white solid: mp 49–52 °C; Rf 0.27 (1:10 EtOAc–hexanes); 1H
NMR d À0.07 (s, 3H), 0.03 (s, 3H), 0.71, (s, 9H), 3.73–3.83 (m,
2H, H-4 and H-6a), 4.10 (dt, J = 4.9 Hz and 9.8 Hz, 1H, H-5), 4.34
(dd. J = 4.8 Hz and 10.4 Hz, 1H, H-6e), 4.42 (t, J = 9.2 Hz, 1H, H-
3), 5.42 (dd, J = 3.8 Hz and 9.4 Hz, 1H, H-2), 5.60 (s, 1H), 6.60
(d, J = 3.6 Hz, 1H, H-1), 7.31–8.07 (m, 15H); 13C NMR d À4.7,
À4.0, 18.2, 25.7, 65.5 (C-5), 68.9 (C-6), 70.3 (C-3), 73.1 (C-2),
81.8 (C-4), 91.1 (C-1), 102.1, 126.4, 128.4, 128.5, 128.9, 129.3,
129.5, 129.5, 129.9, 130.1, 133.5, 134.0, 137.1, 164.8, 165.7; ESI-
TOF-HRMS: calcd for C33H38O8SiNa [M+Na]+ m/z 613.2234; found
m/z 613.2294.
3.5. 2-O-Benzoyl-4,6-O-benzylidene-3-O-(tert-butyldimethyl-
silyl)-a-D-glucopyranose (5)
3.8. 1,2-Di-O-benzoyl-4,6-O-benzylidene-a-D-glucopyranose (8)
Crude product from the hydrolysis of 3 (300 mg, 0.566 mmol)
was dissolved in a solution of 4:1 CH2Cl2–Et3N (5 mL). The result-
ing reaction mixture was stirred at room temperature for 24 h. Re-
moval of solvent under reduced pressure afforded a yellow oil that
was purified by flash chromatography over silica gel (1:6?1:4
To a stirred solution of 7 (2.5 g, 4.24 mmol) in anhyd THF
(10 mL) and anhyd pyridine (1.5 mL) was added HFÁPy (2.0 mL,
excess) in one portion at 0 °C under N2. After stirring at room
temperature for 48 h, the reaction mixture was diluted with
Et2O (200 mL). The organic solution was washed with satd
NaHCO3 (2 Â 150 mL), and brine (100 mL), and dried over
Na2SO4. Removal of solvent under reduced pressure afforded a
yellow oil. The crude product was purified by flash chromatogra-
phy over silica gel (1:3 EtOAc–hexanes) to give pure 8 (1.82 g,
90%) as a white solid: mp 152–154 °C; Rf 0.22 (1:3 EtOAc–hex-
anes); 1H NMR d 3.76–3.84 (m, 2H, H-4 and H-6a), 4.08–4.14
(m, 1H, H-5), 4.35 (dd, J = 5.2 Hz and 10.4 Hz, 1H, H-6e), 4.51
(t, J = 9.4 Hz, 1H, H-3), 5.39 (dd, J = 3.8 Hz and 9.8 Hz, 1H, H-2),
5.62 (s, 1H), 6.66 (d, J = 4.0 Hz, 1H, H-1), 7.24–8.09 (m, 15H);
13C NMR d 65.0 (C-5), 68.7 (C-6), 69.4 (C-3), 72.6 (C-2), 81.0
(C-4), 90.6 (C-1), 102.2, 126.4, 128.6, 128.9, 129.1, 129.2, 129.6,
130.0, 130.1, 133.6, 134.0, 136.9, 164.7, 166.0; ESI-TOF-HRMS:
calcd for C27H24O8Na [M+Na]+ m/z 499.1369; found m/z
499.1435.
EtOAc–hexanes) to give an inseparable mixture of
a
and b 5
ano-
(234 mg, 85%, :b = 3.2:1) as a white solid: mp 62–69 °C; (
a
a
mer) Rf 0.25 (1:3 EtOAc–hexanes); 1H NMR d À0.05 (s, 3H),
À0.02 (s, 3H), 0.68 (s, 9H), 3.59 (t, J = 9.4 Hz, 1H, H-4), 3.75 (t,
J = 10.2 Hz, 1H, H-6a), 4.13 (dt, J = 4.8 Hz and 10.0 Hz, 1H, H-5),
4.27 (dd, J = 5.0 Hz and 10.2 Hz, 1H, H-6e), 4.35 (t, J = 9.2 Hz, 1H,
H-3), 5.11 (dd, J = 3.8 Hz and 9.4 Hz, 1H, H-2), 5.45 (t, J = 3.8 Hz,
1H, H-1), 5.54 (s, 1H), 7.32–8.10 (m, 10H); 13C NMR d À4.7, À4.1,
18.1, 25.7, 62.5 (C-5), 69.0 (C-6), 69.5 (C-3), 74.8 (C-2), 82.3 (C-
4), 91.3 (C-1), 102.0, 126.4, 128.2, 128.5, 129.1, 129.7, 130.1,
133.4, 137.2, 166.3; ESI-TOF-HRMS: calcd for C26H34O7SiNa
[M+Na]+ m/z 509.1971; found m/z 509.1998.
3.6. 2-O-Benzoyl-4,6-O-benzylidene-3-O-(tert-butyldimethyl-
silyl)-a-D-glucopyranosyl trichloroacetimidate (6)
To a stirred solution of 5 (2.3 g, 4.73 mmol) in anhyd CH2Cl2
3.9. Ethyl 2-O-(4-acetoxy-2,2-dimethylbutanoyl)-4,6-O-ben-
(20 mL) was added 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU,
zylidene-1-thio-a-D-glucopyranoside (10)
50 lL). The resulting solution was cooled to 0 °C and trichloroace-
tonitrile (2.37 mL, 23.67 mmol) was added in one portion. After
stirring at room temperature for 5 h, the reaction mixture was con-
centrated under reduced pressure to afford a yellow oil that was
purified by flash chromatography over silica gel (1:8 EtOAc–hex-
To a stirred solution of 913 (1.71 g, 5.45 mmol) in anhyd pyri-
dine (15 mL) at 0 °C was slowly added 4-acetoxy-2,2-dimethylbu-
tyryl chloride (1.2 g, 6.2 mmol). The reaction mixture was stirred
at room temperature for 24 h. Upon removal of solvent under re-
duced pressure, the residue was dissolved in EtOAc (150 mL) and
water (30 mL). The organic layer was washed with satd NaHCO3
(2 Â 130 mL), and brine (100 mL), and dried over Na2SO4. Re-
moval of solvent under reduced pressure afforded a yellow oil.
The crude product was purified by flash chromatography over sil-
ica gel (1:3 EtOAc–hexanes) to give pure 10 (2.20 g, 86%) as a
white solid: mp 75–77 °C; Rf 0.27 (1:3 EtOAc–hexanes); 1H
NMR d 1.21 (s, 3H), 1.23 (t, J = 7.4 Hz, 3H), 1.27 (s, 3H), 1.75–
1.82 (m, 1H), 2.00 (s, 3H), 2.02–2.10 (m, 1H), 2.47–2.58 (m, 2H),
3.19 (d, J = 2.8 Hz, 1H, hydroxyl proton) 3.57–3.81 (m, 1H, H-4),
3.75–3.81 (m, 1H, H-6a), 3.98–4.05 (m, 1H, one of methylene pro-
tons in AcO-CH2), 4.11 (dt, J = 2.7 Hz and 10.4 Hz, 1H, H-3), 4.18–
4.30 (m, 3H, H-5, H-6e and one of methylene protons in AcO-
CH2), 4.90 (dd, J = 5.8 Hz and 9.8 Hz, 1H, H-2), 5.55 (s, 1H), 5.61
(d, J = 6.0 Hz, 1H, H-1), 7.33–7.37 (m, 3H), 7.48–7.50 (m, 2H);
13C NMR d 14.9, 21.0, 24.2, 24.6, 26.1, 38.2, 40.7, 61.6, 62.6 (C-
5), 68.6 (C-6), 68.9 (C-3), 73.7 (C-2), 81.1 (C-4), 82.4 (C-1),
anes) to give a mixture of
a and b 6 (2.75 g, 92%, a/b = 8:1) as a
white solid: ( anomer) mp 55–58 °C; Rf 0.37 (1:6 EtOAc–hex-
a
anes); 1H NMR d À0.07 (s, 3H), 0.02 (s, 3H), 0.70 (s, 9H), 3.72 (t,
J = 9.4 Hz, 1H, H-4), 3.80 (t, J = 10.4 Hz, 1H, H-6a), 4.08 (dt,
J = 4.9 Hz and 9.9 Hz, 1H, H-5), 4.36 (dd. J = 4.8 Hz and 10.4 Hz,
1H, H-6e), 4.41 (t , J = 9.4 Hz, 1H, H-3), 5.35 (dd, J = 3.6 Hz and
9.2 Hz, 1H, H-2), 5.59 (s, 1H), 6.56 (d, J = 4.0 Hz, 1H, H-1), 7.34–
8.03 (m, 10H), 8.51 (s, 1H); 13C NMR d À4.7, À4.1, 18.2, 25.7,
65.5 (C-5), 68.8 (C-6), 69.9 (C-3), 73.4 (C-2), 81.6 (C-4), 91.1, 94.3
(C-1), 102.0, 126.4, 128.3, 128.5, 129.3, 129.4, 130.1, 133.6, 137.1,
160.9, 165.8; ESI-TOF-HRMS: calcd for C28H34Cl3NO7SiNa
[M+Na]+ m/z 652.1068; found m/z 652.1121.
3.7. 1,2-Di-O-benzoyl-4,6-O-benzylidene-3-O-(tert-butyldimeth-
ylsilyl)-a-D-glucopyranose (7)
To a stirred solution of 4 (4.03 g, 8.24 mmol) and DMAP
(215 mg, 1.77 mmol) in anhyd pyridine (20 mL) was added ben-

K.-F. Mo et al. / Carbohydrate Research 344 (2009) 439–447
445
101.9, 126.4, 128.3, 129.2, 137.1, 171.3, 176.3; ESI-TOF-HRMS:
calcd for C23H32O8SNa [M+Na]+ m/z 491.1716; found m/z
491.1783.
3.12. Ethyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(tert-butyldi-
methylsilyl)-b- -glucopyranosyl-(1?3)-2-O-(4-acetoxy-2,
2-dimethylbutanoyl)-4,6-O-benzylidene-1-thio–D-glucopy-
D
ranoside (13)
3.10. 2-O-Benzoyl-4,6-O-benzylidene-3-O-(tert-butyldimethyl-
A mixture of the glucosyl acceptor 10 (912 mg, 1.95 mmol)
and glucosyl donor 6 (1.36 g, 2.16 mmol) and 4 Å molecular
sieves (1.5 g) in CH2Cl2 (20 mL) was stirred at room temperature
under N2 for 1 h. Then it was cooled to À70 °C and a TMSOTf
silyl)-b-
D-glucopyranosyl]-(1?3)-1,2-di-O-benzoyl-4,6-O-ben-
zylidene-
a
-D
-glucopyranose (11)
A mixture of the glucosyl acceptor 8 (840 mg, 1.76 mmol) and
glucosyl donor 3 (1.13 g, 2.13 mmol), 4 Å molecular sieves (1.5 g)
and NIS (660 mg, 2.93 mmol) in CH2Cl2 (20 mL) was stirred at
room temperature under N2 for 1 h. The reaction mixture was
cooled to À20 °C and AgOTf solution (100 mg in 1 mL toluene,
0.389 mmol) was added. After being stirred at the same temper-
ature under N2 for 2 h, the reaction was quenched by addition of
Et3N (2 mL). The reaction mixture was filtered, and the filtrate
was concentrated under reduced pressure to afford a yellow oil
that was purified by flash chromatography over silica gel (1:4
EtOAc–hexanes) to give pure 11 (1.58 g, 95%) as a white solid:
mp 124–126 °C; Rf 0.27 (1:4 EtOAc–hexanes); 1H NMR d À0.29
(s, 3H), À0.15 (s, 3H), 0.60 (s, 9H), 3.49 (dt, J = 4.8 Hz and
9.8 Hz, 1H, H-5II), 3.65 (t, J = 9.2 Hz, 1H, H-4II), 3.76 (t,
J = 10.2 Hz, 1H, H-6aII), 3.80 (t, J = 10.2 Hz, 1H, H-6aI), 3.85–3.91
(m, 2H, H-3II and H-4I), 4.11 (dt, J = 4.5 Hz and 9.9 Hz, 1H, H-
5I), 4.30–4.36 (m, 2H, H-6eI and H-6eII), 4.49 (t, J = 9.4 Hz, 1H,
H-3I), 4.96 (d, J = 7.6 Hz, 1H, H-1IIHH), 5.22 (t, J = 8.0 Hz, 1H, H-
2II), 5.27 (dd, J = 3.8 Hz and 9.4 Hz, 1H, H-2I), 5.42 (s, 1H), 5.62
(s, 1H), 6.57 (d, J = 4.0 Hz, 1H, H-1I), 7.10–7.98 (m, 25H); 13C
NMR d À5.0, À4.1, 18.0, 25.6, 65.4 (C-5I), 66.5 (C-5II), 68.7 (C-
solution (39 lL in 2.16 mL CH2Cl2, 0.1 M, 0.216 mmol) was
added. After being stirred at À70 °C under N2 for 6 h, the reac-
tion was quenched by addition of Et3N (2 mL). The reaction mix-
ture was filtered, and the filtrate was concentrated under
reduced pressure to afford a yellow oil that was purified by flash
chromatography over silica gel (1:5 EtOAc–hexanes) to give pure
13 (1.67 g, 92%) as a white solid: mp 68–70 °C; Rf 0.30 (1:4
EtOAc–hexanes); 1H NMR d À0.16 (s, 3H), À0.10, (s, 3H), 0.69
(s, 9H), 1.14 (s, 3H), 1.14 (s, 3H), 1.18 (t, J = 7.4 Hz, 3H), 1.84
(dd, J = 6.6 Hz and 8.2 Hz, 2H), 2.01 (s, 3H), 2.42–2.52 (m, 2H),
3.40 (dt, J = 4.8 Hz and 9.8 Hz, 1H, H-5II), 3.66–3.71 (m, 3H, H-
4I, H-4II and H-6aII), 3.77 (t, J = 10.0 Hz, 1H, H-6aI), 3.88 (dd,
J = 7.4 Hz and 8.6 Hz, 1H, H-3II), 4.04–4.10 (m, 2H, AcO-CH2),
4.20–4.31 (m, 4H, H-3I, H-5I, H-6eI and H-6eII), 4.88 (dd,
J = 6.0 Hz and 9.6 Hz, 1H, H-2I), 5.00 (d, J = 6.8 Hz, 1H, H-1II),
5.17 (t, J = 7.0 Hz, 1H, H-2II), 5.18 (s, 1H), 5.53 (s, 1H), 5.60 (d,
J = 5.6 Hz, 1H, H-1I), 7.29–7.95 (m, 15H); 13C NMR d -4.9, -4.2,
14.9, 18.0, 21.2, 24.2, 25.08, 25.14, 25.7, 38.0, 40.8, 61.5, 62.9
(C-5I), 66.2 (C-5II), 68.8 (C-6I and C-6II), 73.3 (C-3II), 73.7 (C-3I),
74.0 (C-2I), 75.5 (C-2II), 79.7 (C-4I), 81.1 (C-4II), 82.1 (C-1I), 99.4
(C-1II), 101.5, 101.9, 126.3, 126.4, 128.1, 128.4, 128.5, 129.0,
129.4, 129.8, 130.0, 133.2, 137.27, 137.3, 165.1, 171.0, 176.2;
ESI-TOF-HRMS: calcd for C49H64O14SSiNa [M+Na]+ m/z
959.3684; found m/z 959.3654.
or
or
6I
II), 68.9 (C-6I
II), 72.5 (C-2I), 73.1 (C-3II), 75.4 (C-2II and
C-3I), 79.1 (C-4I), 81.4 (C-4II), 90.4 (C-1I), 101.3 (C-1II), 101.4,
101.9, 126.2, 126.4, 128.2, 128.3, 128.4, 128.5, 128.7, 129.0,
129.15, 129.19, 129.3, 129.5, 129.6, 129.0, 130.0, 132.9, 133.4,
133.9, 137.2, 137.2, 164.5, 164.9, 165.0; ESI-TOF-HRMS:
calcd for C53H56O14SiNa [M+Na]+ m/z 967.3337; found m/z
967.3327.
3.13. 2-O-Benzoyl-4,6-O-benzylidene-3-O-(tert-butyldimethyl-
silyl)-b-
tanoyl)-4,6-O-benzylidene-b-
benzoyl-4,6-O-benzylidene-b-
di-O-benzoyl-4,6-O-benzylidene-
D
-glucopyranosyl-(1?3)-2-O-(4-acetoxy-2,2-dimethylbu-
-glucopyranosyl-(1?3)-2-O-
-glucopyranosyl-(1?3)-1,2
-glucopyranose (14)
D
D
3.11. 2-O-Benzoyl-4,6-O-benzylidene-b- -glucopyranosyl-(1?3)-
D
a
-D
1,2-di-O-benzoyl-4,6-O-benzylidene- -D-glucopyranose (12)
a
A
mixture of the disaccharide acceptor 12 (370 mg,
To a stirred solution of 11 (2.11 g, 2.22 mmol) in anhyd THF
(20 mL) and anhyd pyridine (1.8 mL) was added HFÁPy (1.8 mL,
excess) in one portion at 0 °C under N2. After stirring at room
temperature for 48 h, the reaction mixture was diluted with
diethyl ether (180 mL). The organic solution was washed with
satd NaHCO3 (2 Â 150 mL), and brine (120 mL), and dried over
Na2SO4. Removal of solvent under reduced pressure afforded a
yellow oil. The crude product was purified by flash chromatogra-
phy over silica gel (1:2 EtOAc–hexanes) to give pure 12 (1.69 g,
92%) as a white solid: mp 134–137 °C; Rf 0.30 (1:2 EtOAc–hex-
anes); 1H NMR d 3.52 (dt, J = 4.8 Hz and 9.5 Hz, 1H, H-5II),
3.71–3.78 (m, 2H, H-4II and H-6aII), 3.81 (t, J = 10.6 Hz, 1H, H-
6aI), 3.86–3.93 (m, 2H, H-3II and H-4I), 4.12 (dt, J = 5.2 Hz and
10.0 Hz, 1H, H-5I), 4.30–4.35 (m, 2H, H-6eI and H-6eII), 4.53 (t,
J = 9.4 Hz, 1H, H-3I), 5.04 (d, J = 7.2 Hz, 1H, H-1IIHH), 5.15 (dd,
J = 7.4 Hz and 8.2 Hz, 1H, H-2II), 5.33 (dd, J = 4.0 Hz and 9.6 Hz,
1H, H-2I), 5.40 (s, 1H), 5.63 (s, 1H), 6.60 (d, J = 4.0 Hz, 1H, H-
1I), 7.15–7.99 (m, 20H); 13C NMR d 65.2 (C-5I), 66.2 (C-5II),
0.446 mmol) and disaccharide donor 13 (560 mg, 0.598 mmol),
4 Å molecular sieves (1.0 g) and NIS (185 mg, 0.882 mmol) in
CH2Cl2 (10 mL) was stirred at room temperature under N2 for
1 h. The reaction mixture was cooled to À20 °C and a AgOTf solu-
tion (27 mg in 1.0 mL toluene, 0.105 mmol) was added. After being
stirred at À20 °C under N2 for 4 h, the reaction was quenched by
addition of Et3N (1 mL). The reaction mixture was filtered, and
the filtrate was concentrated under reduced pressure to afford a
yellow oil that was purified by flash chromatography over silica
gel (2:5 EtOAc–hexanes) to give pure 14 (694 mg, 91%) as a white
solid: mp 158–161 °C; Rf 0.37 (1:2 EtOAc–hexanes); 1H NMR d
À0.17 (s, 3H), À0.07 (s, 3H), 0.67 (s, 9H), 0.83 (s, 3H), 0.87 (s,
3H), 1.55–1.61 (m, 2H), 1.90 (s, 3H), 2.58 (t, J = 9.6 Hz, 1H, H-4III),
3.27 (t, J = 10.0 Hz, 1H), 3.32–3.52 (m, 4H), 3.58–3.67 (m, 2H),
3.71–3.76 (m, 2H), 3.79–4.37 (m, 12H), 4.41 (t, J = 9.4 Hz, 1H, H-
3I), 4.56–4.60 (m, 2H, H-2II and H-2III), 4.66 (br s: J < 1.0 Hz, 1H,
H-1III), 4.75 (d, J = 7.6 Hz, 1H, H-1II), 5.10 (d, J = 8.0 Hz, 1H, H-1IV),
5.22 (s, 1H), 5.29 (t, J = 8.0 Hz, 1H, H-2IV), 5.33 (dd, J = 4.0 Hz and
9.6 Hz, 1H, H-2I), 5.48 (s, 1H), 5.76 (s, 1H), 6.56 (d, J = 4.0 Hz, 1H,
H-1I), 6.95–8.13 (m, 40H); 13C NMR d À4.9, À4.2, 18.0, 21.0,
24.77, 24.79, 25.7, 37.9, 40.5, 60.9, 64.7, 65.5, 66.3, 66.5, 68.8,
71.6, 71.8, 72.2, 73.0, 74.6, 74.95, 74.99, 75.4, 76.7, 77.9, 79.1,
81.7, 90.4 (C-1I), 95.9 (C-1III), 97.1 (C-1IV), 99.9, 100.8 (C-1II),
101.7, 101.8, 102.5, 126.1, 126.35, 126.39, 126.8, 127.8, 128.2,
128.4, 128.49, 128.51, 128.6, 128.8, 128.9, 129.1, 129.1, 129.2,
or
or
68.5 (C-6I
II), 68.6 (C-6I
II), 72.25 (C-2I), 72.29 (C-3II), 75.1
(C-2II), 75.5 (C-3I), 78.9 (C-4I), 80.5 (C-4II), 90.3 (C-1I), 100.9 (C-
1II), 101.3, 101.7, 126.1, 126.4, 128.2, 128.29, 128.34, 128.4,
128.67, 128.7, 129.0, 129.1, 129.3, 129.6, 129.9, 133.0, 133.4,
133.9, 137.0, 137.0, 164.5, 164.9, 165.6; ESI-TOF-HRMS:
calcd for C47H42O14Na [M+Na]+ m/z 853.2472; found m/z
853.2445.

446
K.-F. Mo et al. / Carbohydrate Research 344 (2009) 439–447
129.5, 129.59, 129.6, 129.7, 129.9, 130.0, 130.3, 133.1, 133.5, 133.9,
134.1, 137.1, 137.2, 137.3, 137.5, 164.5, 164.6, 164.8, 165.4, 170.9,
175.7; ESI-TOF-HRMS: calcd for C94H100O28SiNa [M+Na]+ m/z
1727.6068; found m/z 1727.6066.
H-2IV and benzylidene proton), 5.51 (s, 1H), 5.72, (s, 1H), 6.54 (d,
J = 4.0 Hz, 1H, H-1I), 6.94–8.21 (m, 55H); 13C NMR d À4.9, À4.3,
17.9, 20.9, 21.0, 24.5, 24.7, 25.6, 37.8, 37.9, 40.3, 40.4, 60.7, 60.8,
64.5, 65.3, 66.0, 66.2, 66.4, 68.4, 68.6, 68.7, 71.1, 71.2, 71.6, 71.8,
71.9, 73.0, 74.2, 74.35, 74.4, 74.5, 74.8, 75.6, 76.7, 76.8, 77.9,
78.1, 79.1, 81.5, 90.3 (C-1I), 95.8 (C-1V), 95.9 (C-1III and C-1VI),
96.5 (C-1IV), 99.9, 100.0, 100.8 (C-1II), 101.5, 101.6, 102.4, 102.7,
126.1, 126.2, 126.3, 126.7, 126.8, 127.7, 127.8, 128.1, 128.4, 128.5,
128.6, 128.7, 128.8, 128.9, 129.1, 129.2, 129.4, 129.5, 129.6, 129.8,
130.3, 133.0, 133.3, 133.8, 134.2, 137.1, 137.16, 137.2, 137.3, 137.33,
137.5, 164.3, 164.4, 164.7, 165.0, 165.3, 170.6, 170.8, 175.6, 175.9;
ESI-TOF-HRMS: calcd for C135H144O42SiNa [M+Na]+ m/z 2487.8799;
found m/z 2487.8750.
3.14. 2-O-Benzoyl-4,6-O-benzylidene-b-
2-O-(4-acetoxy-2,2-dimethylbutanoyl)-4,6-O-benzylidene-
b- -glucopyranosyl-(1?3)-2-O-benzoyl-4,6-O-benzylidene-b-
glucopyranosyl-(1?3)-1,2-di-O-benzoyl-4,6-O-benzylidene-
-glucopyranose (15)
D-glucopyranosyl-(1?3)-
D
D-
a
-D
To a stirred solution of 14 (700 mg, 0.411 mmol) in anhyd THF
(10 mL) and anhyd pyridine (1 mL) was added HFÁPy (1 mL, excess)
in one portion at 0 °C under N2. After stirring at room temperature
for 48 h, the reaction mixture was diluted with Et2O (150 mL). The
organic solution was washed with satd NaHCO3 (2 Â 90 mL), and
brine (80 mL), and dried over Na2SO4. Removal of solvent under re-
duced pressure afforded a yellow oil. The crude product was puri-
fied by flash chromatography over silica gel (4:7 EtOAc–hexanes)
to give pure 15 (607 mg, 93%) as a white solid: mp 166–170 °C;
Rf 0.26 (5:8 EtOAc–hexanes); 1H NMR d 0.86 (s, 3H), 0.90 (s, 3H),
1.57–1.61 (m, 2H), 1.90 (s, 3H), 2.82 (t, J = 9.4 Hz, 1H, H-4III), 3.31
(t, J = 10.2 Hz, 1H), 3.37–3.56 (m, 4H), 3.64–3.77 (m, 4H), 3.81–
4.37 (m, 12H), 4.44 (t, J = 9.6 Hz, 1H, H-3I), 4.64 (broad s, 1H, H-
2III), 4.70 (d, J = 2.0 Hz, 1H, H-1III), 4.73 (t, J = 7.6 Hz, 1H, H-2II),
4.81 (d, J = 6.8 Hz, 1H, H-1II), 5.15 (d, J = 7.6 Hz, 1H, H-1IV), 5.15
(s, 1H), 5.25 (t, J = 8.2 Hz, 1H, H-2IV), 5.31 (dd, J = 4.0 Hz and
9.6 Hz, 1H, H-2I), 5.47 (s, 1H), 5.74 (s, 1H), 6.56 (d, J = 3.6 Hz, 1H,
H-1I), 7.04–8.14 (m, 40H); 13C NMR d 20.9, 24.6, 24.7, 37.8, 40.3,
60.8, 64.6, 65.4, 66.1, 66.2, 68.6, 71.7, 72.2, 72.4, 74.3, 74.8, 75.1,
75.2, 76.7, 77.8, 79.0, 80.9, 90.3 (C-1I), 96.1 (C-1III), 97.0 (C-1IV),
100.0, 100.5 (C-1II), 101.6, 101.8, 102.3, 126.1, 126.3, 126.4,
126.7, 127.8, 128.35, 128.38, 128.4, 128.7, 128.8, 128.97, 129.0,
129.3, 129.4, 129.6, 129.8, 130.0, 133.1, 133.4, 133.8, 134.1,
137.0, 137.1, 137.3, 164.4, 164.5, 164.8, 165.8, 170.9, 175.5; ESI-
TOF-HRMS: calcd for C88H86O28Na [M+Na]+ m/z 1613.5203; found
m/z 1613.5166.
3.16. X-ray crystallography of 16
A colorless, crystalline mass obtained by the slow diffusion of
cyclohexane into a solution of 16 in toluene was separated under
a microscope to give a single crystal which was coated with Par-
atone oil, mounted on a thin Nylon loop and placed in the cold
nitrogen stream of a Kryoflex attachment on a Bruker-AXS APEX I
diffractometer. A crystal-to-detector distance of 120 mm was used
because of the length of the c-axis, and a data collection scheme
comprising 14 runs with a combination of
x and u scans, an 0.5°
width per frame, and a scan time of 30 s/frame was prepared by
COSMO.21 Data collection was controlled by the APEX2 program
suite.22 The raw data were processed with SAINT to a resolution
23
limit of 0.93 Å, which also provided a final unit cell based on a
least-squares refinement of 9514 reflections chosen from diverse
regions of reciprocal space, while corrections for possible crystals
decay and merging of symmetry equivalent reflections were per-
formed with SADABS
in Table 2. Approximately 60% of the structure was revealed by the
direct methods program SHELXM
25 and the remainder slowly devel-
.
24 Further crystallographic details are presented
,
oped by a combination of full-matrix, least-squares refinement, fol-
lowed by computation of a difference Fourier synthesis (SHELXL26).
Because of the limited resolution of the data set (very little signif-
icant measurable intensity beyond a 0.93 Å resolution shell and
none at all beyond a 0.84 Å shell), all phenyl rings were refined
as rigid groups of ideal geometry. Those carbon atoms showing ex-
tremely elongated displacement ellipsoids were restrained to
approximate more isotropic behavior. Hydrogen atoms were in-
cluded as riding contributions with isotropic displacement param-
eters tied to those of the attached carbon atoms using appropriate
SHELXL HFIX instructions. Residual diffuse electron density appeared
in a final difference map (following inclusion of two molecules of
solvent cyclohexane, each of half occupancy). More highly disor-
dered solvent was removed with the SQUEEZE option of the PLATON
program suite.27 Calculations other than those noted above were
performed with the SHELXLS software package.28
3.15. 2-O-Benzoyl-4,6-O-benzylidene-3-O-(tert-butyldimethyl-
silyl)-b-
2-dimethylbutanoyl)-4,6-O-benzylidene-b-
(1?3)-2-O-benzoyl-4,6-O-benzylidene-b- -glucopyranosyl-
(1?3)-2-O-(4-acetoxy-2,2-dimethylbutanoyl)-4,
D-glucopyranosyl-(1?3)-2-O-(4-acetoxy-2,
D-glucopyranosyl-
D
6-O-benzylidene-b-
6-O-benzylidene-b-
D
-glucopyranosyl-(1?3)-2-O-benzoyl-4,
-glucopyranosyl-(1?3)-1,2-di-O-benzoyl-4,
D
6-O-benzylidene-a-D-glucopyranose (16)
A
mixture of the tetrasaccharide acceptor 15 (370 mg,
0.223 mmol) and disaccharide donor 13 (350 mg, 0.374 mmol),
4 Å molecular sieves (800 mg) and NIS (129 mg, 0.573 mmol) in
CH2Cl2 (10 mL) was stirred at room temperature under N2 for
1 h. The reaction mixture was cooled to À20 °C and a AgOTf solu-
tion (20 mg in 0.7 mL toluene, 0.778 mmol) was added. After being
stirred at À20 °C under N2 for 6 h, the reaction was quenched by
addition of Et3N (1 mL). The reaction mixture was filtered, and
the filtrate was concentrated under reduced pressure to afford a
yellow oil that was purified by flash chromatography over silica
gel (4:7 EtOAc–hexanes) to give pure 16 (527 mg, 92%) as a white
solid: mp 169–172 °C; Rf 0.21 (1:2 EtOAc–hexanes); 1H NMR d
À0.13 (s, 3H), À0.04 (s, 3H), 0.71 (s, 9H), 0.81 (s, 3H), 0.86 (s,
3H), 0.89 (s, 3H), 0.93 (s, 3H), 1.53–1.67 (m, 4H), 1.90 (s, 3H),
1.96 (s, 3H), 2.32 (t, J = 9.2 Hz, 1H), 2.54 (t, J = 9.4 Hz, 1H) , 3.28–
3.67 (m, 12H), 3.71–4.15 (m, 18H), 4.22–4.41 (m, 4H), 4.49–4.53
(m, 2H, two H-2), 4.62 (d, J = 0.8 Hz, 1H, H-1III), 4.71–4.76 (m, 4H,
H-1II and H-1V and two H-2), 4.90 (d, J = 8.0 Hz, 1H, H-1VI), 5.19
(d, J = 8.4 Hz, 1H, H-1IV), 5.53 (s, 1H), 5.28–5.36 (m, 3H, H-2I,
3.17. 2-O-Benzoyl-4,6-O-benzylidene-b-
2-O-(4-acetoxy-2,2-dimethylbutanoyl)-4,6-O-benzylidene-b-
glucopyranosyl-(1?3)-2-O-benzoyl-4,6-O-benzylidene-b-
glucopyranosyl-(1?3)-2-O-(4-acetoxy-2,2-dimethylbutanoyl)-
4,6-O-benzylidene-b- -glucopyranosyl-(1?3)-2-O-benzoyl-4,
6-O-benzylidene-b- -glucopyranosyl-(1?3)-1,2-di-O-benzoyl-4,
6-O-benzylidene- -glucopyranose (17)
D-glucopyranosyl-(1?3)-
D-
D-
D
D
a-D
To a stirred solution of 16 (700 mg, 0.284 mmol) in anhyd THF
(10 mL) and anhyd pyridine (1 mL) was added HFÁPy (1 mL, excess)
in one portion at 0 °C under N2. After stirring at room temperature
for 48 h, the reaction mixture was diluted with Et2O (150 mL). The
organic solution was washed with satd NaHCO3 (2 Â 90 mL), brine
(80 mL), and dried over Na2SO4. Removal of solvent under reduced
pressure afforded a yellow oil. The crude product was purified by

K.-F. Mo et al. / Carbohydrate Research 344 (2009) 439–447
447
III, IV, and
flash chromatography over silica gel (2:3 EtOAc–hexanes) to give
(C-1II,
V), 103.4 (C-1VI); ESI-TOF-HRMS: calcd for
pure 17 (621 mg, 93%) as a white solid: mp 172–175 °C; Rf 0.36
(2:3 EtOAc–hexanes); 1H NMR d 0.81 (s, 3H), 0.86 (s, 3H), 0.89 (s,
3H), 0.93 (s, 3H), 1.53–1.67 (m, 4H), 1.89 (s, 3H), 1.96 (s, 3H),
2.49 (t, J = 9.4 Hz, 1H, H-4V), 2.55 (t, J = 9.4 Hz, 1H, H-4III) , 2.61
(d, J = 3.6 Hz, 1H, hydroxyl proton), 3.30–3.62 (m, 10H), 3.67–
4.15 (m, 20H), 4.22–4.41 (m, 4H), 4.51–4.55 (m, 2H, H-2II and H-
2III), 4.63 (d, J = 1.2 Hz, 1H, H-1III), 4.73 (d, J = 7.6 Hz, 1H, H-1II)
4.79 (s, 2H, H-1V and H-2V), 4.90–4.97 (m, 2H, H-1VI and H-2VI),
5.18 (s, 1H), 5.24 (d, J = 8.0 Hz, 1H, H-1IV), 5.27–5.32 (m, 3H, H-2I,
H-2IV and benzylidene proton), 5.51 (s, 1H), 5.72, (s, 1H), 6.54 (d,
J = 3.6 Hz, 1H, H-1I), 7.04–8.22 (m, 55H); 13C NMR d 21.0, 21.1,
24.7, 24.9, 37.9, 37.94, 40.4, 40.6, 60.9, 61.0, 64.6, 64.7, 65.4, 66.1,
66.3, 68.5, 68.7, 68.76, 68.8, 71.3, 71.35, 72.0, 72.1, 72.5, 72.6, 74.35,
74.4, 74.9, 75.0, 75.7, 78.1, 78.4, 79.2, 81.1, 90.4 (C-1I), 96.05 (C-1VI),
96.1 (C-1V), 96.2 (C-1III), 96.6 (C-1IV), 100.3, 100.9 (C-1II), 101.7,
102.0, 102.5, 102.8, 126.3, 126.3, 126.3, 126.5, 126.78, 126.8, 127.9,
127.94, 128.46, 128.49, 128.6, 128.7, 128.75, 128.8, 129.0, 129.1,
129.2, 129.3, 129.4, 129.5, 129.6, 129.69, 129.7, 130.0, 130.1, 133.1,
133.5, 133.9, 134.4, 134.6, 137.1, 137.16, 137.2, 137.3, 137.4, 137.5,
164.48, 164.5, 164.8, 165.2, 166.0, 170.8, 171.0, 175.7, 175.9; ESI-
TOF-HRMS: calcd for C129H130O42Na [M+Na]+ m/z 2373.7934; found
m/z 2373.7959.
C36H62O31Na [M+Na]+ m/z 1013.3173; found m/z 1013.3160.
Supplementary data
Included in the Supplementary data are X-ray tables for com-
pound 16 including atomic coordinates, bond lengths, and bond
angles. Complete crystallographic data for the structural analysis
have been deposited with the Cambridge Crystallographic Data
Center, CCDC no. 701670. Copies of this information may be
obtained free of charge from the Director, Cambridge Crystallo-
graphic Data Centre, 12, Union Road, Cambridge, CB2 1EZ, UK.
(fax: +44-1223-336033, e-mail: deposit@ccdc.cam.ac.uk or via:
www.ccdc.cam.ac.uk). Supplementary data associated with this
article can be found, in the online version, at doi:10.1016/
j.carres.2008.12.014.
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3.18. Laminarahexaose (1)
A solution of 17 (50 mg, 0.021 mmol) in MeOH (8 mL), THF
(4 mL), and HOAc (2.4 mL) was treated with Pd/C (220 mg, 10 wt %,
Degussa type), and the mixture was hydrogenated (60 psi) for
4.5 h. The reaction mixture was filtered, and the filtrate was concen-
trated under reduced pressure to afford a yellow oil that was co-
evaporated with toluene (3 Â 50 mL) to remove traces of HOAc.
The crude product was purified by flash chromatography over silica
gel using a gradient of 1:15?1:5 MeOH–CH2Cl2 to give the hydrog-
enolysisproduct(32 mg, 83%)asa whitesolid. Toa stirredsolutionof
the purified hydrogenolysis product (32 mg, 0.018 mmol) in MeOH
(3 mL) was added NaOMe–MeOH solution (9 lL, 1.0 M, 0.009 mmol)
at room temperature, and the reaction mixture was stirred for 24 h.
The reaction mixture was then neutralized with Amberlite IRPÒ-64
ion-exchange resin (H+ form, 40 mg). After removal of the resin by
filtration, the filtrate was concentrated under reduced pressure,
and the crude product was purified by size-exclusion chromatogra-
phy over lipophilic Sephadex LH-20 (1:1 MeOH–H2O) and lyophi-
17. Bentley, R. Ann. Rev. Biochem. 1972, 41, 953–996.
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21. COSMO, Version 1.40; Bruker-AXS: Madison, WI, 2008.
22. APEX2, Version 2008.2-0; Bruker-AXS: Madison, WI, 2008.
23. SAINT+, Version 7.53A; Bruker-AXS: Madison, WI, 2008.
24. Sheldrick, G. M. SADABS, Version 2008/2; University of Göttingen: Germany,
2008.
25. Sheldrick, G, M. SHELXM, Version 2004/1; University of Göttingen: Germany,
2004.
26. Sheldrick, G. M. SHELXL-97; University of Göttingen: Germany, 2008.
27. Spek, A. L. PLATON, A Multipurpose Crystallographic Tool; Utrecht University:
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lized to give an inseparable mixture of
a- and b-1 (18 mg, 100%
a
:b = 1:1.2) as a white solid. 1H NMR (DMSO/D2O/TFAA: 95/5/trace)
d 3.00–3.69 (m, 36H), 4.35 (d, J = 7.6 Hz, b H-1I), 4.38 (d, J = 7.6 Hz,
1HVI), 4.42–4.45 (m, 1H, H-1II), 4.51 (d, J = 8.0 Hz, 3H, H-1III, IV, and V),
4.95 (d, J = 3.6 Hz,
d 61.2, 68.6, 70.1, 71.6, 71.8, 73.8, 73.9, 74.0, 74.4, 76.1, 76.2,
76.5, 82.8, 84.5, 84.7, 85.0, 92.6 (
C-1I), 96.2 (b C-1I), 103.1
a
H-1); 13C NMR (D2O, acetone as standard)
28. SHELXLS, Version 2008/3; Bruker-AXS: Madison, WI, 2008.
a