Synthesis of the starfish ganglioside LLG-3 tetrasaccharide

Article abstract of DOI:10.1016/j.carres.2009.02.003

The first synthesis of the ganglioside LLG-3 tetrasaccharide, which has attractive biological activities as well as a unique structure, is described. A C8-methoxy decorated sialic acid building block was initially prepared and a glycolic acid moiety was t

Full text of DOI:10.1016/j.carres.2009.02.003

Carbohydrate Research 344 (2009) 747–752
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of the starfish ganglioside LLG-3 tetrasaccharide
Shinya Hanashima a,b, Daichi Ishikawa a, Shoji Akai a, Ken-ichi Sato a,
*
a Material and Life Chemistry, Faculty of Engineering, Kanagawa University, 3-27-1 Rokkakubashi, Kanagawa-ku, Yokohama, Kanagawa 221-8686, Japan
b Structural Glycobiology Team, Systems Glycobiology Research Group, Chemical Biology Department, Advanced Science Institutes, RIKEN, 2-1 Hirosawa, Wako-shi,
Saitama 351-0198, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The first synthesis of the ganglioside LLG-3 tetrasaccharide, which has attractive biological activities as
well as a unique structure, is described. A C8-methoxy decorated sialic acid building block was initially
prepared and a glycolic acid moiety was then introduced by sialylation. Amide condensation between the
sialyl glycolic acid and an amino group at C5 on the sialyllactoside unit afforded the fully protected LLG-3
tetrasaccharide. Finally, the desired tetrasaccharide part of LLG-3 was obtained after careful global depro-
tection.
Received 10 December 2008
Received in revised form 30 January 2009
Accepted 4 February 2009
Available online 8 February 2009
Keywords:
HO
OH
Ganglioside
Sialic acid
N-Glycolylneuraminic acid
Glycosylation
COOH
HO
OMe
OH
HO
OH
O
COOH
O
O
HO
HN
O
O
OSE
O
HO
AcHN
HO
O
OH
OH
HO
O
HO
LLG-3 tetrasaccharide
BnO
OMe
AcO
OAc
COOMe
AcO
COOMe
O
OBn
OBn
O
+
OH
BnO
AcHN
O
O
AcO
H2N
O
O
OSE
O
BnO
O
BnO
OBn
OBn
AcO
.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
Another remarkable characteristic of this class of glycoconju-
gates is the connecting mode around the sialic acid residues.4
One class of such gangliosides having di/tri-sialic acid residues
The structural diversity found in sialic acid and sialyl-glycolip-
ids has great significance in biological systems and metabolic path-
ways of organisms.1,2 Sialic acids identified in mammalian
gangliosides mainly possess N-acetylneuraminic acid (Neu5Ac)
with an
a-(2?11) linkage has been found to possess nerve cell-
stimulating and -protecting activities.4,5 Due to the significant bio-
activities of these unique sialic acid linkages, some pioneering
studies toward the synthesis of these echinoderm gangliosides
have been reported.6
and N-glycolylneuraminic acid (Neu5Gc) residues with
a-(2?3/
6) linkages to adjacent galactose, glucosamine, and galactosamine
residues. Echinoderm gangliosides have more modifications of the
sialic acid residues and exhibit various attachment linkages be-
tween the sialic acids and neighboring sugar residues. In some
cases, distinctive sialic acids modified by acetylation and sulfation
are known to be significant gateways for viral infections.3 How-
ever, the actions of methylated sialic acids have remained unclear
because of their presence in natural sources in small amount.
The unique ganglioside LLG-3 (Scheme 1), isolated from the
starfish Linckia laevigata,5a has potent nerve cell-stimulating activ-
ity for the neuron-like rat adrenal pheochromocytoma (PC-12) cell
line through nerve cell growth factors (NGFs), which is of compa-
rable magnitude to the mammalian ganglioside GM1.5c,7 The dis-
tinctive tetrasaccharide sequence of LLG-3 contains
a C8-
methylated Neu5Ac (Neu8Me5Ac) terminus, which is connected
to the N-glycolyl hydroxyl group (C11-OH) of the neighboring
Neu5Gc residue. Because of its intriguing potent nerve cell-stimu-
* Corresponding author. Tel.: +81 45 481 5661x3853; fax: +81 45 413 9770.
E-mail address: satouk01@kanagawa-u.ac.jp (K. Sato).
lating activities and its unique a-(2?11)-disialyl connection with
0008-6215/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2009.02.003

748
S. Hanashima et al. / Carbohydrate Research 344 (2009) 747–752
HO
COOH
OH
COOH
HO
HO
OMe
OH
OH
O
O
HO
HN
O
O
OR
O
HO
AcHN
HO
O
O
OH
OH
HO
O
HO
LLG-3: R = Ceramides
1
:
R = SE
BnO
OMe
AcO
OAc
COOMe
COOMe
O
OBn
AcO
OBn
O
+
OH
BnO
AcHN
O
O
AcO
H2N
O
O
OSE
O
BnO
O
BnO
OBn
OBn
AcO
2
3
Scheme 1.
Ph
HO
OH
BnO
OR
O
O
SPh
BH3-Et3N, AlCl3,
MS4A, THF, 0 oC, 72%
PhCH(OMe)2, CSA,
CH3CN, quant.
SPh
SPh
HO
AcHN
O
COOMe
BnO
AcHN
RO
AcHN
O
O
COOMe
COOMe
HO
BnO
RO
4
7
: R = H
5: R = H
BnBr, Ba(OH)2, BaO, DMF,
then MeI, KHCO3, DMF, 59%
MeI, Ba(OH)2, BaO, MS4A, DMF,
then MeI, KHCO3, DMF, 72%
8: R = Me
6
: R = Bn
OBn
HO
BnO
OMe
O
COOMe
O
OR
9: R = Bn
BnO
AcHN
O
H2, 10% Pd-C, NH4OAc,
MeOH, 98%
NIS, TfOH, EtCN
MS 4A, −78 ºC to 0 ºC
2
: R = H
O
BnO
Scheme 2.
Neu8Me5Ac capping, we carried out the first synthesis of the LLG-3
tetrasaccharide, 1.
protection of the hydrolyzed carboxylic acid with a methyl group
produced 6 in 59% yield. Reductive benzylidene ring opening with
BH3ꢀMe3N and AlCl3 produced 7 in 72% yield.12 To introduce a
methyl group on the liberated C8-hydroxyl group, 7 was treated
with MeI, Ba(OH)2, and BaO in the presence of 4 Å molecular
sieves; re-esterification produced the desired 8 in 72% yield. The
obtained Neu8Me5Ac building block 8 was subjected to sialylation
reactions with commercial benzyl glycolate in the presence of
2. Results and discussion
2.1. Synthetic plan
In our synthetic plan (Scheme 1), tetrasaccharide 1 is con-
structed by a [1+3] amide condensation between carboxylic acid
2 and the sialyllactoside 3, which contains an amino group at C5.
This approach avoids a poorly selective sialylation reaction later
in the synthetic route.8 To synthesize carboxylic acid unit 2, the
glycolic acid is liberated after introduction of the 8-methoxy sialic
acid building block 8 by a glycosylation. The GM3-type trisaccha-
ride 3 is synthesized by sialylation between the reactive N-Troc
protected sialic acid building block 10 and lactose acceptor 11,9 fol-
lowed by removal of the N-Troc group.
NIS and TfOH to afford 9 in 81% yield (
a/b = 77/23, a-anomer:
3JC1–H3ax = 4.9 Hz).13 The desired carboxylic acid 2 was produced
by catalytic hydrogenation for 9
a with 10% Pd–C and excess
NH4OAc in 98% yield.14
2.3. Synthesis of LLG-3 tetrasaccharide (1)
The synthesis of the target LLG-3 tetrasaccharide 1, involving
formation of the full sequence through stepwise glycan chain elon-
gations as well as subsequent global deprotections, is depicted in
Scheme 3. To form the sialyllactoside 12 on a gram scale, initial
sialylation between the 1-O-trimethylsilylethyl (SE) protected lac-
tose acceptor 1115 and the potent N-Troc sialic acid building donor
10,16 which was activated by NIS–TfOH in CH3CN, produced an
2.2. Synthesis of building block (2)
Synthesis of the Neu8Me5Ac carboxylic acid building block 2
began with b-phenyl thiosialoside 410 (Scheme 2). The C8- and
C9-hydroxyl groups were first protected as a benzylidene acetal
by treatment with benzaldehyde dimethylacetal and camphorsul-
fonic acid to give 5 quantitatively. The C4/C7 di-O-benzyl interme-
diate was successfully produced by subsequent treatment with a
combination of benzyl bromide, Ba(OH)2 and BaO, conditions that
were able to avoid undesirable N-benzylation.11 Subsequent re-
inseparable crude mixture of trisaccharide. This mixture was acet-
3
ylated17 to give 12
a
(a/b = 6:1,
a
-anomer: JC1–H3ax = 4.1 Hz)18 in
67% yield. The desired trisaccharide was readily separated by silica
gel chromatography after acetylation.
The N-Troc group of 12 was reductively removed using Zn pow-
der in AcOH to afford the desired amine 3 in 85% yield. The liber-

S. Hanashima et al. / Carbohydrate Research 344 (2009) 747–752
749
NIS, TfOH, MS 4A,
MeCN, −40 ºC, then
AcO
OAc
AcO
OAc
COOMe
AcO
Ac2O, pyridine, 67%
OBn
O
SPh
HO
HO
OBn
O
OBn
O
OBn
O
AcO
TrocHN
O
O
COOMe +
OSE
AcO
RHN
O
BnO
O
OSE
O
BnO
OBn
AcO
OBn
OBn
OBn
AcO
10
11
12: R = Troc
Zn, AcOH, 85%
3
:
R = H
AcO
OAc
COOMe
AcO
BnO
2, EDCI, HOBt,
NaHCO3, MeCN, 57%
OMe
OBn
O
i) LiCl, pyridine, 120 ºC
ii) H2, 10% Pd-C, MeOH
OBn
O
COOMe
O
1
AcO
HN
O
O
OSE
O
BnO
AcHN
BnO
O
OBn
OBn
AcO
iii) 0.1 M NaOH, 91% (3 steps)
O
BnO
13
Scheme 3.
ated amine 3 was coupled with the carboxylic acid 2 using EDCI,
HOBt, and NaHCO3 to provide the fully protected LLG-3 tetrasac-
charide 13 in 57% yield.19 In addition, acetyl migration to liberated
amino group of the compound 3 was observed. As a final task, glo-
bal deprotection of 13 was carefully undertaken to avoid undesired
side reactions at the labile glycolyl moiety, which could involve
amide migration19b and elimination.
To remove the methyl esters of 13 selectively, the tetrasaccha-
ride 13 was treated with excess LiCl in pyridine under reflux con-
ditions. Following acidic treatment readily provided biscarboxylic
acid intermediate.19b,20 All the benzyl protecting groups were
reductively removed by hydrogenation with 10% Pd–C. Finally,
careful basic treatment with 0.1 M aqueous NaOH for 2 h yielded
the fully deprotected tetrasaccharide 1 (91% 3 steps). Purification
(desalting) was achieved by direct application of the reaction mix-
ture to a Sephadex G-15 size exclusion column with elution with
water.
4. Experimental procedures
4.1. General procedures
Optical rotations were measured in a 0.5 dm tube with a JASCO
P-1020 polarimeter. IR spectra were recorded with Shimadzu Pres-
tege-21 and JASCO FT/IR-4200 spectrometers. 1H and 13C NMR
spectra were measured with JEOL ECX-400, ECA-500, and ECA-
600 spectrometers and Bruker DRX-600 spectrometer referenced
to tetramethylsilane (0.00 ppm). Column chromatography was
performed on silica gel (Silica gel 60 N, spherical, neutral, 70–230
mesh, Kanto Kagaku Co.). Thin-layer chromatography (TLC) on Sil-
ica gel 60F254 (Merck) was used to monitor the reactions. High-res-
olution ESI-TOF mass spectra were measured with JEOL JMS-
T100LC AccuTOF mass spectrometer.
4.2. Methyl (phenyl 5-acetamido-8,9-O-benzylidene-3,5-
The structure of tetrasaccharide 1 was totally supported by HR-
ESI-MS data and 2D NMR spectra. Direct comparison of the 13C
NMR spectra with natural LLG-3 ganglioside in pyridine-d5, which
was reported by Higuchi et al.,5a failed because the synthesized tet-
rasaccharide 1 was completely insoluble in pyridine-d5. Addition-
ally, a set of 1H and 13C NMR data derived from the terminal
disialyl structure showed good agreement with the corresponding
dideoxy-2-thio-
(5)
D-glycero-b-D-galacto-2-nonulopyranosid)onate
A solution of 4 (2.13 g, 5.14 mmol) in CH3CN (50 mL) containing
benzaldehyde dimethyl acetal (1.0 mL, 11 mmol), and camphorsul-
fonic acid (100 mg, 0.43 mmol) was stirred for 1 h. Then, the reac-
tion mixture was neutralized with Et3N, and concentrated. The
resulting crude product was crystallized from diethyl ether to give
5 (2.58 g, 5.14 mmol, quant.). 1H NMR (600 MHz, CDCl3) d 7.51–
7.20 (m, 10H, Ar), 4.21 (m, 1H, J7,8 = 7.0 Hz, J8,9 = 7.0 Hz,
J8,9 = 5.1 Hz, H-8), 4.12 (dd, 1H, J = 8.4 Hz, H-9a), 3.91 (m, 1H, H-
9b), 3.71 (m, 1H, H-5), 3.56–3.37 (m, 2H, H-4, H-7), 3.32–3.30
(m, 4H, H-6, OMe), 2.71 (dd, 1H, Jgem = 12.5 Hz, J3eq,4 = 4.8 Hz, H-
3eq), 1.85 (s, 3H, 3 ꢁ Ac), 1.69 (t, 1H, J3ax,4 = 11.7 Hz, H-3ax); 13C
NMR (150 MHz, CDCl3) d 176.0, 175.7, 174.3, 174.1, 139.8, 139.2,
135.7, 135.5, 133.6, 130.4, 130.2, 129.5, 129.4, 129.33, 129.27,
128.94, 128.87, 127.8, 127.7, 105.6, 104.8, 93.4, 93.0, 76.22,
76.20, 74.0, 73.8, 71.8, 71.2, 69.8, 69.6, 69.2, 69.0, 54.0, 53.9,
43.6, 43.5, 23.0.
synthetic Neu5Ac-a
-(2?11)-Neu5Gc spectrum reported by Ren.20a
The precise 2D NMR spectra of compound 1 in D2O are given in
Supplementary data.
3. Conclusion
The synthesis of the LLG-3 tetrasaccharide 1, which is termi-
nated with Neu8Me5Ac and contains an a-(2?11) disialyl linkage,
was achieved. For the synthesis of the building block 2, the C8-hy-
droxyl group was selectively methylated with MeI, BaO, and
Ba(OH)2. Subsequent coupling with benzyl glyclolate and selective
removal of its benzyl ester produced carboxylic acid 2. The sialyl-
lactose building block 3 was produced via a sialylation reaction be-
tween the known N-Troc sialic acid building block 10 and lactose
derivative 11. To form the complete tetrasaccharide 13, carboxylic
acid 2 and amino trisaccharide 3 were condensed under mild basic
conditions. Finally, a sequence of global deprotection procedures
involving removal of methyl esters, hydrogenolysis of benzyl
ethers, and hydrolysis of acetyl and methyl esters provided the de-
sired LLG-3 tetrasaccharide 1 in excellent yield.
4.3. Methyl (phenyl 5-acetamido-8,9-O-benzylidene-4,7-di-O-
benzyl-3,5-dideoxy-2-thio-
D-glycero-b-D-galacto-2-
nonulopyranosid)onate (6)
To a solution of 5 (2.59 g, 5.15 mmol) in dry DMF (40 mL) were
added BaO (5.50 g, 35.9 mmol), Ba(OH)2 (1.6 g, 5.1 mmol), and
BnBr (6.0 mL, 8.6 mmol), and the mixture was stirred for 15 h at

750
S. Hanashima et al. / Carbohydrate Research 344 (2009) 747–752
room temperature. Then, the reaction mixture was filtered through
a Celite pad. The filtrate was diluted with NaHCO3 solution, and the
aqueous phase was extracted twice with CH2Cl2. Next, the com-
bined organic layers were washed with NaHCO3 solution and brine,
dried over MgSO4, filtered, and concentrated under reduced pres-
sure. Then, the residue was re-dissolved in DMF (40 mL) before
the addition of KHCO3 (5.04 g, 50.3 mmol) and MeI (1.6 mL,
26 mmol). The mixture was stirred for 2 h at room temperature,
and poured into NaHCO3 solution. The aqueous phase was ex-
tracted with CH2Cl2, and the organic layers were washed with
NaHCO3 solution and brine, dried over MgSO4, filtered, and concen-
trated under reduced pressure. The residue was purified with sil-
ica-gel flash column chromatography (hexane–EtOAc 2:1 ? 1:1)
to give 6 (1.5 g, 2.2 mmol, 59%). 1H NMR (600 MHz, CDCl3) d
7.58–7.27 (m, 10H, 2Ph), 5.45 (s, 1H, CHPh), 5.13 (d, 1H,
JNH,5 = 8.8 Hz, NH), 4.69 (dd, 1H, J6,7 = 1.6 Hz, J7,8 = 8.9 Hz, H-7),
4.68 (d, 1H, JAB = 11.3 Hz, CH2Ph), 4.58 (d, 1H, JAB = 11.3 Hz, CH2Ph),
4.54 (d, 1H, JAB = 12.0 Hz, CH2Ph), 4.23 (d, 1H, JAB = 12.0 Hz, CH2Ph),
4.36 (dd, 1H, J9a,9b = 10.6 Hz, J8,9 = 5.0 Hz, H-9), 4.23 (m, 1H, H-5),
4.04–4.01 (m, 2H, H-4, H-8), 3.82 (dd, 1H, J5,6 = 9.5 Hz, H-6), 3.60
(dd, 1H, H-9b), 3.60 (s, 3H, OMe), 2.75 (dd, 1H, J3ax,3eq = 14.0 Hz,
J3eq,4 = 4.5 Hz, H-3eq), 1.97 (m, 4H, H-3ax, Ac); 13C NMR
(150 MHz, CDCl3): d 170.2, 168.9, 138.1, 138.1, 137.6, 135.3,
135.2, 131.0, 129.2–126.2, 101.3, 89.5, 73.5, 72.1, 71.0, 70.4, 69.6,
67.8, 62.0, 52.6, 50.3, 36.6, 29.0, 23.7; HR-ESI-MS: m/z [M+Na]+:
calcd for C39H41NO8SNa, 706.2450; found, 706.2425.
was extracted twice with CH2Cl2. The combined organic layers were
washed with NaHCO3 solution and brine, dried over MgSO4, and
concentrated. The residue was purified by silica gel flash column
chromatography (hexane–EtOAc 3:2) to give 8 (570 mg, 0.81 mmol,
72%). ½a 2D6
ꢂ
ꢃ62 (c 2.1, CHCl3); 1H NMR (600 MHz, CDCl3) d 7.43–7.20
(m, 20H, Ar), 4.80 (d, 1H, JNH,5 = 8.1 Hz, HN), 4.69 (d, 1H,
J5,6 = 10.3 Hz, H-6), 4.72 (d, 1H, JA,B = 11.5 Hz, CH2Ph), 4.65 (d, 1H,
JA,B = 11.5 Hz, CH2Ph), 4.56 (d, 1H, JA,B = 12.0 Hz, CH2Ph), 4.35 (d,
1H, JA,B = 12.0 Hz, CH2Ph), 4.49 (s, 2H, CH2Ph), 4.21 (ddd, 1H,
J3ax,4 = 4.8, J3eq,4 = 4.3, J4,5 = 9.1 Hz, H-4), 4.01 (dd, 1H, J8,9a = 2.6,
J9a,9b = 10.7 Hz, H-9a), 3.83 (dd, 1H, J8,9b = 5.3 Hz, H-8), 3.70 (dd,
1H, H-9b), 3.64 (ddd, 1H, H-5), 3.56 (dd, 1H, J7,8 = 7.8 Hz, H-7),
3.48 (s, 3H, COOMe), 3.43 (s, 3H, OMe), 2.70 (dd, 1H,
J3ax,3eq = 13.7 Hz, H-3eq), 2.04 (s, 3H, HNAc), 2.00 (dd, 1H, H-3ax);
13C NMR (150 MHz, CDCl3) d 170.2, 168.9, 138.5, 138.4, 138.3,
135.6, 130.5, 129.1–127.6, 89.7, 81.1, 74.9, 73.4, 73.3, 73.2, 72.5,
71.0, 69.3, 57.9, 52.7, 52.3, 37.5, 23.6; HR-ESI-MS: m/z [M+Na]+:
calcd for C40H45NO8SNa, 722.2763; found 722.2723.
4.6. Methyl (benzyloxycarbonylmethyl 5-acetamido-4,7,9-tri-
O-benzyl-3,5-dideoxy-8-O-methyl-D-glycero-a-D-galacto-2-
nonulopyranosid)onate (9)
A solution of 8 (532 mg, 0.76 mmol) in dry EtCN (5 mL) contain-
ing benzyl glycolate (170 L, 1.20 mmol) and 4 Å molecular sieves
l
(250 mg) was stirred for 30 min under an atmosphere of argon.
Then, the reaction mixture was cooled to ꢃ78 °C, and NIS
4.4. Methyl (phenyl 5-acetamido-4,7,9-tri-O-benzyl-3,5-
(284 mg, 1.24 mmol) and TfOH (6 lL, 0.07 mmol) were added.
di-deoxy-2-thio-
(7)
D
-glycero-b-
D
-galacto-2-nonulopyranosid)onate
The reaction mixture was gradually warmed up from ꢃ78 °C to
0 °C and was then neutralized with Et3 N. The mixture was then fil-
tered through a Celite pad, and the filtrate was diluted with CH2Cl2.
The organic layer was washed with 3% Na2S2O3 solution and brine,
dried over MgSO4, and concentrated. The residue was purified by
silica gel flash column chromatography (hexane–EtOAc
To a solution of 6 (1.5 g, 2.2 mmol) in dry THF (40 mL) were
added AlCl3 (1.8 g, 14 mmol), BH3ꢀMe3 N (973 mg, 13.3 mmol),
and 4 Å molecular sieves (3.2 g) at 0 °C, and the mixture was grad-
ually warmed up to room temperature over 1 day. Then, the reac-
tion mixture was poured into 1 M aqueous H2SO4 solution and
filtered through a Celite pad. The filtrate was extracted twice with
Et2O, and the combined organic layers were washed with saturated
NaHCO3 solution and brine, dried over MgSO4, and concentrated
under reduced pressure. The residue was purified by silica gel flash
column chromatography (hexane–EtOAc 2:1) to give 7 (1.08 g,
2:1 ? 1:1) to give 9
0.150 mmol, 19%). Compound 9
a (373 mg, 0.49 mmol, 62%) and 9b (116 mg,
a
: ½a 2D5
ꢂ
ꢃ8.5 (c 1.1, CHCl3); 1H
NMR (600 MHz, CDCl3) d 7.51–7.25 (m, 20H, Ar), 5.09 (d, 1H,
J = 12.2 Hz, CO2CH2Ph), 5.03 (d, 1H, J = 12.2 Hz, CO2CH2Ph), 4.67
(d, 1H, JA,B = 12.0 Hz, CH2Ph), 4.63 (d, 1H, JA,B = 12.0 Hz, CH2Ph),
4.58 (d, 1H, JNH,5 = 8.8 Hz, HN), 4.56 (s, 2H, CH2Ph), 4.54 (d, 1H,
JA,B = 12.0 Hz, CH2Ph), 4.39 (d, 1H, JA,B = 12.0 Hz, CH2Ph), 4.37 (d,
1H, Jgem = 16.5 Hz, CH2CO2Bn), 4.28 (d, 1H, Jgem = 16.5 Hz,
CH2CO2Bn), 3.87 (m, 2H, H-6, H-9a), 3.80 (ddd, 1H, J4,5 = 8.7 Hz,
H-5), 3.68 (m, 1H, H-4), 3.66 (s, 3H, COOMe), 3.58 (m, 1H, H-8),
3.66 (m, 2H, H-9b, H-7), 3.42 (s, 3H, OMe), 2.87 (dd, 1H,
J3eq,4 = 4.5 Hz, J3ax,3eq = 12.6 Hz, H-3eq), 1.77 (s, 3H, Ac), 1.75 (dd,
1H, J3ax,4 = 7.3 Hz, H-3ax); 13C NMR (CDCl3, 150 MHz) d 170.1,
169.6, 167.9, 138.4, 138.1, 135.4, 128.9–127.7, 98.4, 79.2, 74.3,
73.7, 73.5, 73.5, 73.5, 72.8, 70.6, 67.4, 66.6, 66.6, 66.6, 61.4, 57.7,
57.7, 52.5, 52.5, 51.2, 37.0, 29.5, 23.7, 14.2; HR-ESI-MS: m/z
[M+Na]+: calcd for C43H49NO11SNa, 778.3203; found, 778.3193.
1.58 mmol, 72%). ½a D26
ꢂ
ꢃ63 (c 0.4, CHCl3); 1H NMR (600 MHz,
CDCl3) d 7.43–7.20 (m, 20H, Ar), 4.84 (d, 1H, J5,6 = 10.3 Hz, H-6),
4.68 (s, 2H, CH2Ph), 4.60 (d, 1H, JNH,5 = 8.1 Hz, HN), 4.56 (d, 1H,
JA,B = 12.0 Hz, CH2Ph), 4.34 (d, 1H, JA,B = 12.0 Hz, CH2Ph), 4.48 (d,
1H, JA,B = 11.7 Hz, CH2Ph), 4.46 (d, 1H, JA,B = 11.7 Hz, CH2Ph), 4.27
(ddd, 1H, J3ax,4 = 6.0, J3eq,4 = 4.8, J4,5 = 9.1 Hz, H-4), 3.77 (dd, 1H,
J8,9a = 4.6, J9a,9b = 9.5 Hz, H-9a), 4.08 (m, 1H, H-8), 3.75 (bs, 1H, H-
7), 3.68 (dd, 1H, J8,9a = 6.5 Hz, H-9b), 3.60 (m, 1H, H-5), 3.48 (s,
3H, COOMe), 2.90 (br d, 1H, OH), 2.77 (dd, 1H, J3ax,3eq = 13.7 Hz,
H-3eq), 1.96 (dd, 1H, H-3ax), 1.68 (s, 3H, HNAc); 13C NMR
(150 MHz, CDCl3) d 170.2, 168.6, 138.3, 138.2, 138.1, 136.1,
129.4–127.8, 125.3, 89.9, 75.0, 73.4, 72.9, 72.6, 72.3, 71.3, 71.1,
70.9, 52.8, 52.4, 37.8, 23.6; HR-ESI-MS: m/z [M+Na]+: calcd for
C39H43NO8SNa, 708.2607; found, 708.2625.
4.7. Methyl (carboxymethyl 5-acetamido-4,7,9-tri-O-benzyl-
3,5-dideoxy-8-O-methyl-D-glycero-a-D-galacto-2-
nonulopyranosid)onate (2)
4.5. Methyl (phenyl 5-acetamido-4,7,9-tri-O-benzyl-3,5-
A solution of compound 9a (357 mg, 0.472 mmol) in MeOH
dideoxy-8-O-methyl-2-thio-
nonulopyranosid)onate (8)
D
-glycero-b-
D
-galacto-2-
(3 mL) containing 10% Pd–C (382 mg) and NH4OAc (96 mg,
1.3 mmol) was stirred for 1 h at room temperature under an atmo-
sphere of hydrogen. Then, the Pd catalyst was removed by filtra-
tion through a pad of Celite, and filtrate was concentrated. The
residue was purified by silica gel flash column chromatography
To a solution of 7 (776 mg, 1.13 mmol) in dry DMF (5.0 mL) were
added 4 Å molecular sieves (500 mg), BaO (356 mg, 2.32 mmol),
Ba(OH)2 (362 mg, 1.15 mmol), and MeI (400
l
L, 5.65 mmol). The
(MeOH–chloroform 1:20 with 1% AcOH) to give 2 (308 mg,
mixture was stirred for 1.5 h at room temperature under an atmo-
sphere of argon and then filtered through a pad of Celite. Then, the
filtrate was diluted with NaHCO3 solution, and the aqueous phase
0.463 mmol, 98%). ½a D23
ꢂ
ꢃ21 (c 0.7, CHCl3); 1H NMR (CD3OD,
600 MHz) d 7.36–7.25 (m, 10H), 4.67 (d, 1H, JA,B = 11.0 Hz, CH2Ph),
4.65 (d, 1H, JA,B = 12.4 Hz, CH2Ph), 4.48 (d, 1H, JA,B = 12.4 Hz,

S. Hanashima et al. / Carbohydrate Research 344 (2009) 747–752
751
CH2Ph), 4.58 (d, 1H, JA,B = 11.7 Hz, CH2Ph), 4.54 (d, 1H,
JA,B = 11.7 Hz, CH2Ph), 4.32 (d, 1H, JA,B = 16.5 Hz, OCH2CO), 4.23 (d,
1H, JA,B = 16.5 Hz, OCH2CO), 4.16 (dd, 1H, J3,4 = J4,5 = 10.3 Hz, H-4),
3.90 (dd, 1H, J5,6 = 11.0 Hz, J6,7 = 1.2 Hz, H-6), 3.77 (d, 1H,
J9a,9b = 11.0 Hz, H-9a), 3.74 (s, 3H, OMe), 3.67–3.64 (m, 2H, H-5,
H-9b), 3.58 (m, 1H, H-7), 3.50 (m, 1H, H-8), 3.45 (s, 3H, OMe),
2.86 (dd, 1H, J3eq,4 = 4.1 Hz, J3,3ax = 12.4 Hz, H-3eq), 1.94 (s, 3H,
Ac), 1.68 (dd, 1H, J3ax,4 = 12.4 Hz, H-3ax); 13C NMR (CD3OD,
150 MHz) d 173.4, 169.3, 139.7, 139.5, 129.5–128.6, 99.7, 80.4,
76.8, 75.9, 74.5, 74.3, 72.0, 68.3, 62.1, 58.1, 53.0, 51.4, 38.1, 23.1;
HR-ESI-MS: m/z [M+Na]+: calcd for C36H43 NO11Na, 688.2734;
found, 688.2743.
(m, H-5Gal), 3.81–3.66 (m, 3H, H-5Neu, H-6abGlc), 3.59 (m, 1H,
SE), 3.51 (dd, 1H, J = 9.2, 9.2 Hz, H-3Glc), 3.48 (dd, 1H, J = 7.8,
9.3 Hz, H-2Gal), 3.43 (s, 3H, OMe), 3.41–3.32 (m, 3H, H-2Glc, H-
6abGal), 3.26 (m, 1H, H-5Glc), 2.62 (dd, 1H, J = 5.0, 13.8 Hz, H-
3eqNeu), 2.13 (s, 3H), 2.08 (s, 3H), 2.00 (s, 3H), 1.97 (s, 3H),
1.84 (s, 3H), 1.78 (dd, 1H, J = 13.8, 13.8 Hz, H-3axNeu), 1.02 (m,
2H, SE), 0.02 (s, 9H, SiMe3); 13C NMR (CDCl3, 100 MHz)
d
170.6, 171.9, 170.7, 170.5, 170.4, 169.9, 166.5, 154.6, 139.1,
138.9, 138.5, 138.2, 137.9, 129.3–127.4, 103.2, 102.1, 99.2, 95.6,
82.6, 82.0, 78.4, 75.8, 75.7, 75.3, 75.11, 75.05, 74.7, 73.3, 72.6,
72.2, 71.8, 71.7, 70.7, 69.4, 68.5, 67.4, 67.3, 62.8, 52.4, 50.8,
37.3, 21.1, 20.9, 20.8, 18.6, -1.3; HR-ESI-MS: m/z [M+Na]+: calcd
for C75H92Cl3NO25SiNa, 1562.4691; found, 1562.4652.
4.8. 2-(Trimethylsilyl)ethyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-5-trichloroethoxycarbonylamino-
galacto-2-nonulopyranosylonate)-(2?3)-4-O-acetyl-2,6-di-O-
benzyl-b- -galactopyranosyl-(1?4)-2,3,6-tri-O-benzyl-b-
glucopyranoside (12)
D
-glycero-
a
-
D
-
4.9. 2-(Trimethylsilyl)ethyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-5-amino-
onate)-(2?3)-4-O-acetyl-2,6-di-O-benzyl-b-
(1?4)-2,3,6-tri-O-benzyl-b- -glucopyranoside (3)
D-glycero-a-
D-galacto-2-nonulopyranosyl-
D
D
-
D
-galactopyranosyl-
D
A
solution of 10 (1.20 g, 1.67 mmol) and 11 (1.06 g,
A solution of 12 (208 mg, 0.135 mmol) in acetic acid (5 mL) con-
taining zinc powder (2.13 g) was stirred for 1 h. Then, the mixture
was filtered through a pad of Celite, and filtrate was concentrated
under reduced pressure at 30 °C. The residue was purified with sil-
ica gel flash column chromatography (toluene–acetone 4:1) to give
1.13 mmol) in dry CH3CN (30 mL) containing 4 Å molecular
sieves (2.5 g) was stirred for 20 min at room temperature under
an atmosphere of argon. Then, the mixture was cooled to ꢃ40 °C,
and NIS (578 mg, 2.57 mmol) and TfOH (35 lL, 0.40 mmol) were
added. After stirring for 2 h at ꢃ40 °C, the mixture was neutral-
ized with Et3N, filtered through a pad of Celite, and the filtrate
was diluted with EtOAc. The organic layer was then washed with
3% Na2S2O3 solution and brine, dried over MgSO4, and concen-
trated. The residue was re-dissolved into dry pyridine (15 mL),
and Ac2O (7.5 mL) was added at 0 °C. After stirring overnight
at room temperature, the mixture was concentrated and re-dis-
solved in EtOAc. Purification by silica gel flash column chroma-
3 (157 mg, 0.115 mmol, 85%). ½a D23
ꢂ
ꢃ3.5 (c 1.1, CHCl3); 1H NMR
(CDCl3, 600 MHz) d 7.37–7.18 (m, 25H), 5.66 (m, 1H), 5.42 (dd,
1H, J = 1.4, 8.9 Hz), 5.09 (d, 1H, J = 3.4 Hz), 4.97 (d, 1H,
J = 10.3 Hz), 4.94 (d, 1H, J = 12.4 Hz), 4.88 (d, 1H, J = 11.0 Hz), 4.75
(d, 1H, J = 12.4 Hz), 4.74 (d, 1H, J = 9.6 Hz), 4.71 (m, 1H), 4.69 (d,
1H, J = 11.0 Hz), 4.62 (d, 1H, J = 12.4 Hz), 4.50 (d, 1H, J = 11.7 Hz),
4.41 (d, 1H, J = 11.7 Hz), 4.37–4.33 (m, 3H), 4.30 (dd, 1H, J = 2.1,
13.1 Hz), 4.20 (d, 1H, J = 11.7 Hz), 4.17 (dd, 1H, J = 4.1, 13.1 Hz),
3.99 (m, 1H), 3.92 (dd, 1H, J = 9.6, 9.6 Hz), 3.80 (s, 3H), 3.78 (d,
1H, J = 9.6 Hz), 3.62 (dd, 1H, J = 6.9, 6.9 Hz), 3.61–3.56 (m, 2H),
3.54 (dd, 1H, J = 8.9, 8.9 Hz), 3.49 (dd, 1H, J = 1.4, 10.3 Hz), 3.44
(dd, 1H, J = 8.2, 9.6 Hz), 3.39–3.34 (m, 2H), 3.28 (d, 2H, J = 6.2 Hz),
2.66 (dd, 1H, J = 4.8, 13.1 Hz), 2.53 (dd, 1H, J = 9.6, 10.3 Hz), 2.10
(s, 3H), 2.07 (s, 3H), 2.00 (s, 3H), 2.00 (s, 3H), 1.70 (s, 3H) 1.03
(m, 2H), 0.01 (s, 9H); 13C NMR (CDCl3, 150 MHz) d 170.8, 170.6,
170.2, 169.80, 169.78, 167.9, 139.5, 139.3, 138.8, 138.7, 138.2,
128.2–127.1, 103.0, 102.2, 97.5, 82.8, 82.0, 79.3, 76.7, 75.05,
75.02, 74.9, 74.64, 74.62, 73.8, 73.2, 72.9, 72.3, 71.7, 69.0, 67.9,
67.8, 67.3, 52.9, 51.1, 36.8, 21.2, 21.0, 20.7, 20.3, ꢃ1.4; HR-ESI-
MS: m/z [M+Na]+: calcd for C72H91NO23SiNa, 1388.5649; found,
1388.5629.
tography (hexane–EtOAc 4:1 ? 2:1) gave 12
0.760 mmol, 67%,
a
(1.17 g,
a
/b = 6:1). Compound 12
a
: ½a 2D6
ꢂ
+1.0 (c 2.3,
CHCl3); 1H NMR (600 MHz, CDCl3) d 7.40–7.20 (m, 25H, Ar),
5.59–5.57 (m, 1H, H-8Neu), 5.35 (dd, 1H, J = 1.7, 8.4 Hz, H-7Neu),
5.03 (d, 1H, J = 2.7 Hz, H-4Gal), 4.97 (d, 1H, J = 10.7 Hz, Bn), 4.75
(d, 1H, J = 10.7 Hz, Bn), 5.00 (ddd, 1H, J = 11.9, 4.6, 10.9 Hz, H-
4Neu), 4.90 (d, 1H, J = 11.7 Hz, Bn), 4.67 (d, 1H, J = 11.7 Hz, Bn),
4.80 (d, 1H, J = 9.9 Hz, NH), 4.77 (d, 1H, J = 12.0 Hz, Bn), 4.71
(d, 1H, J = 12.0 Hz, Bn), 4.74 (d, 1H, J = 7.6 Hz, H-1Gal), 4.48 (d,
1H, J = 12.9 Hz, Troc), 4.47 (d, 1H, J = 12.9 Hz, Troc), 4.41 (d,
1H, J = 11.9 Hz, Bn), 4.33 (d, 1H, J = 11.9 Hz, Bn), 4.36 (d, 1H,
J = 7.7 Hz, H-1Glc), 4.24 (dd, 1H, J = 2.3, 12.5 Hz, H-9aNeu), 4.08
(dd, 1H, J = 10.8 Hz, H-6Neu), 4.05ꢃ3.97 (m, 2H, SE, H-9bNeu),
3.91 (t, 1H, J = 9.2 Hz, H-3Glc), 3.71–3.65 (m, 3H, H-6aGlc, H-6bGlc
,
H-5Neu), 3.61–3.55 (m, 1H, SE), 3.52 (dd, 1H, J = 7.7, 7.7 Hz, H-
4Glc), 3.48–3.46 (m, 3H, H-2Gal, H-5Glc, H-6aGal), 3.39–3.37 (m, 3H,
H-2Glc, H-5Gal, H-6bGal), 2.63 (dd, 1H, J = 4.6, 11.6 Hz, H-3eqNeu),
2.08 (s, 3H, Ac), 1.99 (s, 3H, Ac), 1.97 (s, 3H, Ac), 1.75 (s, 3H,
Ac), 1.76 (dd, 1H, J = 11.6, 11.6 Hz, H-3axNeu), 1.03–0.88 (m, 2H,
SE), 0.02 (s, 9H, SiMe3); 13C NMR (CDCl3, 150 MHz) d 170.6,
170.2, 169.9, 169.8, 169.8, 167.7, 154.2, 139.5, 139.3, 138.8,
138.7, 138.2, 128.3–127.0, 103.0, 102.1, 97.2, 95.3, 82.8, 82.0,
79.4, 76.7, 75.0, 75.0, 74.7, 74.5, 73.9, 73.2, 72.8, 71.7, 71.4,
69.0, 69.0, 68.7, 68.1, 67.6, 67.3, 67.1, 62.0, 53.1, 51.4, 37.6,
21.2–20.4, 18.5, –1.4; HR-ESI-MS: m/z [M+Na]+: calcd for
C75H92Cl3NO25SiNa, 1562.4691; found, 1562.4646; Compound
4.10. 2-(Trimethylsilyl)ethyl (methyl 5-acetamido-4,7,9-tri-O-
benzyl-8-O-methyl-3,5-dideoxy-
nonulopyranosylonate)-(2?11)-(methyl 4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-5-glycolylamido- -glycero- -galacto-2-nonulo-
pyranosylonate)-(2?3)-4-O-acetyl-2,6-di-O-benzyl-b- -galacto-
pyranosyl-(1?4)-2,3,6-tri-O-benzyl-b- -glucopyranoside (13)
D-glycero-a-D-galacto-2-
D
a-D
D
D
A solution of 3 (92 mg, 0.15 mmol) and crude 2 (157 mg,
0.115 mmol) in CH3CN (2 mL) containing NaHCO3 (61 mg,
0.72 mmol), HOBt (40 mg, 0.29 mmol), and EDCI (59 mg,
0.31 mmol) was stirred at room temperature for 18 h under an
atmosphere of argon. Then, the mixture was diluted with H2O
and extracted three times with EtOAc. The combined organic layers
were washed with brine, dried over MgSO4, and filtered. The
remaining residue was purified by silica gel flash column chroma-
tography (toluene–acetone 4:1) to give 13 (132 mg, 0.065 mmol,
12b: ½a 2D4
ꢂ
ꢃ2 (c 0.2, chloroform); 1H NMR (400 MHz, CDCl3) d
7.52–7.10 (m, 25H), 5.40 (m, 1H, H-7Neu), 5.32–5.29 (m, 2H, H-
8Neu, H-4Gal), 5.16 (d, 1H, J = 11 Hz, NH), 5.07 (ddd, 1H, J = 5.0,
10.5, 11.9 Hz, H-4Neu), 4.95–4.89 (m, 4H, H-9aNeu, Bn), 4.71 (d,
1H, J = 11.0 Hz, Bn), 4.70 (d, 1H, J = 11.0 Hz, Bn), 4.67 (dd, 1H,
J = 1.8, 8.7 Hz, H-6Neu), 4.62 (s, 2H, Bn), 4.58–4.49 (m, 5H, H-
1Gal, H-3Gal, Bn, Troc), 4.33 (d, 1H, J = 7.8 Hz, H-1Glc), 4.27 (d,
1H, J = 12.4 Hz, Bn), 4.02–3.90 (m, 3H, H-9bNeu, H-4Glc, SE), 3.83
57%). ½a 2D6
ꢂ
ꢃ6.2 (c 0.9, CHCl3); 1H NMR (600 MHz, CDCl3) d 7.39–
7.18 (m, 40H), 6.23 (d, 1H, J = 10.3 Hz), 5.60 (m, 1H), 5.21 (dd,
1H, J = 2.5, 8.2 Hz), 4.98 (d, 1H, J = 11.7 Hz), 4.89 (m, 1H), 4.88 (d,

752
S. Hanashima et al. / Carbohydrate Research 344 (2009) 747–752
2H, J = 11.0 Hz), 4.77 (d, 1H, J = 7.6 Hz), 4.76 (d, 1H, J = 11.0 Hz),
4.68 (d, 1H, J = 11.0 Hz), 4.67 (d, 1H, J = 10.6 Hz), 4.64 (d, 1H,
J = 13.1 Hz), 4.61 (d, 1H, J = 11.0 Hz), 4.58–4.55 (m, 3H), 4.54 (d,
1H, J = 11.0 Hz), 4.48 (d, 1H, J = 12.4 Hz), 4.47 (dd, 1H, J = 2.7,
9.9 Hz), 4.40 (d, 1H, J = 12.4 Hz), 4.36 (d, 2H, J = 12.4 Hz), 4.34 (d,
1H, J = 8.2 Hz), 4.28 (dd, 1H, J = 2.8, 12.4 Hz), 4.20 (d, 1H,
J = 15.1 Hz), 4.19 (d, 1H, J = 11.7 Hz), 4.10 (ddd, 1H, J = 10.3, 10.3,
11.0 Hz), 4.03 (d, 1H, J = 11.0 Hz), 3.98 (m, 1H), 3.95 (dd, 1H,
J = 5.5, 12.4 Hz), 3.91 (dd, 1H, J = 9.6, 9.6 Hz), 3,86–3.75 (m, 4H),
3.82 (s, 3H), 3.79 (s, 3H), 3.69–3.62 (m, 4H), 3.59–3.54 (m, 3H),
3.54 (dd, 1H, J = 9.6, 9.6 Hz), 3.44 (dd, 1H, J = 7.6, 9.6 Hz), 3.41 (s,
3H), 3.37 (m, 1H), 3.37 (dd, 1H, J = 7.8, 8.9 Hz), 3.60 (d, 2H,
J = 6.2 Hz), 2.82 (dd, 1H, J = 4.8, 13.1 Hz), 2.63 (dd, 1H, J = 4.1,
12.4 Hz), 2.08 (s, 3H), 1.99 (s, 3H), 1.94 (s, 3H), 2.82 (dd, 2H,
J = 12.4, 12.4 Hz), 1.78 (s, 3H), 1.76 (s, 3H), 1.69 (s, 3H), 1,04 (m,
2H), 0.01 (s, 9H); 13C NMR (CDCl3, 150 MHz) d 170.5, 170.4,
170.1, 170.0, 169.9, 169.4, 168.2, 168.0, 139.5, 139.3, 138.8,
138.7, 138.2, 138.2, 138.1, 138.0, 129.1–127.0, 103.0, 102.1, 98.7,
97.4, 82.9, 82.0, 79.5, 79.0, 76.7, 75.0, 75.0, 74.9, 74.7, 74.3, 73.8,
73.7, 73.6, 73.5, 73.2, 72.9, 72.8, 72.4, 71.4, 70.6, 69.0, 68.8, 68.7,
68.3, 67.6, 67.3, 67.2, 67.1, 63.2, 62.2, 57.6, 53.2, 52.7, 51.3, 48.5,
37.7, 37.0, 29.7, 23.7, 21.2ꢃ20.5, 18.5, 1.1, –1.4; HR-ESI-MS: m/z
[M+Na]+: calcd for C108H132N2O33SiNa, 2035.8379; found,
2035.8399.
Acknowledgments
We thank Dr. Yoshiki Yamaguchi (RIKEN ASI) for his assistance
with NMR spectroscopy. This work was supported by the Science
Frontier Project of Kanagawa University and by Grants-in-Aid for
Scientific Research for Young Scientists B (No. 20710171 to S. H.)
from the Japan Society for the Promotion of Science (JSPS). We
thank Ms. I. Hara, Ms. A. Takahashi, and Ms. K. Ueno for their tech-
nical assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.carres.2009.02.003.
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4.11. 2-(Trimethylsilyl)ethyl 5-acetamido-8-O-methyl-3,5-di-
deoxy-
(2?11)-3,5-dideoxy-5-glycolylamido-
nonulopyranosylonic acid-(2?3)-b- -galactopyranosyl-(1?4)-
b- -glucopyranoside (1)
D-glycero-
a-
D-galacto-2-nonulopyranosylonic acid-
D
-glycero- -galacto-2-
a-D
D
D
A solution of 13 (63 mg, 0.032 mmol) in pyridine (2 mL) con-
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an atmosphere of argon. Then, the mixture was cooled to room
temperature and concentrated. The residue was acidified with
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M.; Petrie C.R. III; Korytnyk, W. Carbohydr. Res. 1988, 175, 25–34.
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5490–5497.
15, H2O) to give 1 (31 mg, 0.029 mmol, 91%). ½a D24
ꢃ16 (c 1.4,
ꢂ
H2O); 1H NMR (600 MHz, with CH3OH as internal standard) d
4.40 (d, 1H, J = 7.6 Hz, H-1Gal), 4.36 (d, 1H, J = 8.3 Hz, H-1Glc), 4.15
(d, 1H, J = 15.3 Hz, OCH2CO), 3.98 (m, 1H, H-3Gal), 3.97 (d, 1H,
J = 15.6 Hz, OCH2CO), 3.92–3.21 (m, 27H), 3.34 (s, 3H, OMe), 3.15
(dd, 1H, J = 8.2, 8.9 Hz, H-2Glc), 2.63 (dd, 1H, J = 4.1, 12.4 Hz,
H-3eqNeu5Ac), 2.50 (dd, 1H, J = 4.1, 12.4 Hz, H-3eqNeu5Gc), 1.89 (s,
3H), 1.67 (dd, 1H, J = 11.7, 12.4 Hz, H-3axNeu5Ac), 1.60 (dd, 1H,
11.7, 12.4 Hz, H-3axNeu5Gc), 0.94 (ddd, 1H, J = 5.3, 12.4, 13.1 Hz,
SE), 0.84 (ddd, 1H, J = 5.4, 12.4, 13.1 Hz, SE), –0.11 (s, 9H, SE); 13C
NMR (D2O with CH3OH as internal standard, 150 MHz) d 175.6
(N-Ac), 174.5 (C-1Neu5Ac), 174.0 (C-1Neu5Gc), 173.4 (N-glycolyl),
103.3 (C-1Gal), 102.0 (C-1Glc), 101.3 (C-2Neu5Ac), 100.5 (C-2Neu5Gc),
80.7 (C-8Neu5Ac), 78.8 (C-4Glc), 76.1 (C-3Gal), 75.8, 75.4, 75.2, 73.5,
73.3, 73.2, 72.6, 70.0, 69.1, 68.7, 68.6, 68.1, 67.7, 63.8, 63.3, 61.7,
60.7 (SE), 60.1 (C-9Neu5Ac), 57.2 (OMe), 52.7 (C-5Neu5Gc), 52.2
(C-5Neu5Ac), 40.3 (C-3Neu5Ac), 40.1 (C-3Neu5Gc), 22.7 (Ac), 18.2 (SE),
–1.9 (SE); HR-ESI-MS: m/z [M-H]ꢃ: calcd for C40H69N2O28Si,
1053.3806; found, 1053.3810.
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3
18. JC1–H3ax values were determined by gradient-enhanced phase-sensitive HMBC
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