Synthesis of a chito-tetrasaccharide β-1,4-GlcNAc-β-1,4-GlcN repeating unit

Article abstract of DOI:10.1007/s00706-009-0174-y

tert-Butyldimethylsilyl (4-O-acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-β- d-glucopyranosyl)-(1 → 4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-d- glucopyranoside (Kawada and Yoneda [MOCHEM-D-09-00120], 2009), designed as a repeating disaccharide unit in a β-glu

Full text of DOI:10.1007/s00706-009-0174-y

Monatsh Chem (2009) 140:1251–1256
DOI 10.1007/s00706-009-0174-y
ORIGINAL PAPER
Synthesis of a chito-tetrasaccharide b-1,4-GlcNAc-b-1,4-GlcN
repeating unit
Toshinari Kawada Æ Yuko Yoneda
Received: 28 March 2009 / Accepted: 2 August 2009 / Published online: 1 September 2009
Ó Springer-Verlag 2009
Abstract tert-Butyldimethylsilyl (4-O-acetyl-2-azido-3,
6-di-O-benzyl-2-deoxy-b-D-glucopyranosyl)-(1 ? 4)-3,6-di-
O-benzyl-2-deoxy-2-phthalimido-b-D-glucopyranoside (Ka-
wada and Yoneda [MOCHEM-D-09-00120], 2009), designed
as a repeating disaccharide unit in a b-glucan having two
different faces, was converted into a glycosyl donor and an
acceptor. The glycosyl acceptor was glycosylated with the
donor to afford a chito-tetrasaccharide derivative in good
yield. Phthalimido and azido groups in the tetrasaccharide
were successively converted into acetamido and free amino
groups, and all other protecting groups were cleaved to
obtain the chito-tetrasaccharide (2-amino-2-deoxy-b-D-glu-
copyranosyl)-(1 ? 4)-(2-acetamido-2-deoxy-b-D-glucopy-
ranosyl)-(1 ? 4)-(2-amino-2-deoxy-b-D-glucopyranosyl)
-(1 ? 4)-2-acetamido-2-deoxy-D-glucopyranose.
and reported on the preparation of the repeating disaccha-
ride unit, tert-butyldimethylsilyl (4-O-acetyl-2-azido-3,
6-di-O-benzyl-2-deoxy-b-D-glucopyranosyl)-(1 ? 4)-3,6-di-
O-benzyl-2-deoxy-2-phthalimido-b-D-glucopyranoside (1).
In this report, a conversion of the disaccharide unit 1 into a
disaccharide acceptor, tert-butyldimethylsilyl (2-azido-3,6-
di-O-benzyl-2-deoxy-b-D-glucopyranosyl)-(1 ? 4)-2-deo-
xy-3,6-di-O-benzyl-2-phthalimido-b-D-glucopyranoside (4)
as well as into a disaccharide donor precursor (4-O-acetyl-2-
azido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyranosyl)-(1 ?
4)-2-deoxy-3,6-di-O-benzyl-2-phthalimido-D-glucopyrano-
se (2) is reported and the synthesis of chito-tetrasaccharide 9
is described in detail. The glycosylation procedure for syn-
thesizing the tetrasaccharide presents a versatile reaction for
future applications in glycosylation reactions directed
towards oligosaccharides and/or polysaccharides.
Keywords Carbohydrates ꢀ Protecting groups ꢀ
Glycosides ꢀ Oligosaccharides
Results and discussion
Introduction
The repeating disaccharide unit 1 was converted into gly-
cosyl acceptor 4 and donor 3 (Scheme 1). The glycosyl
acceptor 4 was prepared by cleavage of the 40-O-acetyl
group and the corresponding glycosyl donor precursor 2 by
cleavage of the tert-butyldimethylsilyl (TBDMS) group [2]
in the disaccharide unit 1 using tetra-n-butylammonium
fluoride (TBAF) [3, 4] in 78% yield. The glycosyl donor 3
was prepared by attaching a trichloroacetimidate moiety
using trichloroacetonitrile [5] and 1,8-diazabicyclo[5.4.0]
undec-7-ene (DBU) [6] in 88% yield.
A b-1,4-glucan chain exhibiting two different faces can be
synthesized by attaching the same functional group at the
same carbon position in each sugar residue of two neigh-
boring monosaccharides. In a previous paper [1], we
proposed a stepwise method for synthesizing b-1,4-glucan
The glycosylation of the disaccharide acceptor 4 with
the disaccharide imidate donor 3 presents an efficient
precedent for elongation of the glucan chain. Thus, in
glucan chains extensions can be done either by applying
linear or convergent synthetic methods [7] as well as by
T. Kawada (&) ꢀ Y. Yoneda
Graduate School of Life and Environmental Sciences,
Kyoto Prefectural University, Shimogamo, Sakyo-ku,
Kyoto 606-8522, Japan
e-mail: kawada@kpu.ac.jp
123

1252
T. Kawada, Y. Yoneda
Scheme 1
OBn
O
OBn
O
N3
N3
TBAF
BnO
AcO
BnO
AcO
O
O
OTBDMS
BnO
THF-AcOH
BnO
O
OBn
OH
O
OBn
NPhth
NPhth
2
1
CCl3CN, DBU,CH2Cl2
NaOMe, MeOH-CH2Cl2
OBn
O
OBn
NH
N3
N3
O
BnO
HO
BnO
O
O
OTBDMS
AcO
BnO
O
OBn
BnO
CCl3
O
O
OBn
NPhth
NPhth
4
3
TMSOTf, CH2Cl2
OBn
O
OBn
O
N3
N3
O
BnO
BnO
O
OTBDMS
O
AcO
BnO
BnO
O
O
OBn
NPhth
NPhth
5
OBn
(i) H2NCH2CH2NH2, n-BuOH
(ii) Ac2O, DMAP, Py
OBn
O
OBn
O
N3
N3
BnO
BnO
O
O
OTBDMS
O
AcO
BnO
BnO
O
OBn
O
OBn
NHAc
NHAc
6
NaOMe, MeOH
N3
OBn
O
OBn
O
N3
BnO
BnO
O
O
OTBDMS
NHAc
O
HO
BnO
BnO
O
O
OBn
NHAc
7
OBn
TBAF, THF
N3
OBn
O
OBn
O
N3
BnO
BnO
HO
O
O
O
BnO
BnO
OH
O
O
OBn
NHAc
NHAc
8
OBn
10 % Pd-C, MeOH
NH2
OH
O
OH
O
NH2
HO
HO
HO
O
O
O
NHAc
HO
HO
OH
O
O
NHAc
9
OH
OH
using the polymerization method, wherein the disaccharide
imidate 3 plays a central role as glycosyl donor. At the
reducing end of donor 3 a phthalimido group at C-2
is attached and thus is expected to favor b-glycosidation
due to neighboring group participation and steric hindrance
[8].
applying a series of experiments the optimum reaction
conditions were found using 0.01 equivalents (for the im-
idate 3) of trimethylsilyl trifluoromethanesulfonate
(TMSOTf) [9] as a catalyst to afford the stereochemically
pure target b-glycoside. However, an increase of the
amount of catalyst up to 0.05 equivalents caused unex-
pected side reactions and decreased the yield of the
reaction. The produced tetrasaccharide 5 was purified by
flash column chromatography. The 1H NMR spectrum of 5
showed a doublet for the newly formed anomeric proton at
d = 5.20 ppm, having a coupling constant of 8.3 Hz. This
fact is consistent with a 1,2-trans-relationship between the
newly formed anomeric proton and the C-2 proton typical
for a b-linkage. In addition, the 13C NMR spectrum of 5
showed a signal of C-1 at d = 96.8 ppm, which is empir-
ically indicative for a b-glucoside [10, 11].
A variety of glycosylation reaction conditions, con-
ducted in dichloromethane, were investigated. The reaction
temperature at -20 °C was found to be suitable as the
starting disaccharide imidate 3 was consumed within
5 min, preventing complex side reactions. Higher reaction
temperatures than -20 °C are leading to decomposition
and consequently to a dramatic decrease in yield. Addi-
tionally, the use of boron trifluoride–diethyl etherate as
glycosylation catalyst [5] generated a large number of
byproducts including fluorides and a-glycoside. By
123

Synthesis of a chito-tetrasaccharide b-1,4-GlcNAc-b-1,4-GlcN repeating unit
Tetrasaccharide 5 was transformed to chito-tetraose 9 by
1253
(500 MHz, CDCl3): d = 1.84 (s, 3H, COMe), 3.24 (t, 1H,
J2 ,3 = 9.6 Hz, J3 ,4 = 9.6 Hz, H-30), 3.30 (ddd, 1H,
0
0
0
0
the conversion of the phthalimido groups into acetamido
groups [12], cleavage of the 40-O-acetyl group, cleavage of
J4 ,5 = 9.6 Hz, J5 ,6 a = 3.7 Hz, J5 ,6 b = 5.3 Hz, H-50),
0
0
0
0
0
0
3.35 (dd, 1H, J5 ,6 a = 3.7 Hz, J6 a,6 b = 10.6 Hz, H-60a),
3.40 (dd, 1H, J1 ,2 = 8.2 Hz, J2 ,3 = 9.6 Hz, H-20),
3.44 (d, 1H, J1,OH = 8.5 Hz, 1-OH), 3.47 (dd, 1H,
0
0
0
0
the anomeric TBDMS group [3, 4] and hydrogenolysis of
benzyl and azide groups into free hydroxyl and free amino
groups, respectively.
0
0
0
0
J5 ,6 b = 5.3 Hz, J6 a,6 b = 10.6 Hz, H-60b), 3.71 (ddd, 1H,
J4,5 = 10.1 Hz, J5,6a = 1.6 Hz, J5,6b = 3.4 Hz, H-5), 3.83
(dd, 1H, J5,6a = 1.6 Hz, J6a,6b = 11.0 Hz, H-6a), 3.97 (dd,
1H, J5,6b = 3.4 Hz, J6a,ab = 11.0 Hz, H-6b), 4.09 (dd, 1H,
J1,2 = 8.5 Hz, J2,3 = 10.8 Hz, H-2), 4.12 (dd, 1H,
J3,4 = 8.7 Hz, J4,5 = 10.1 Hz, H-4), 4.32 (d, 1H, CH2Ph),
0
0
0
0
In conclusion, the disaccharide imidate 3 having
phthalimido at the neighboring C-2 position stereospecifi-
cally afforded a b-glycosidic linkage using TMSOTf as
catalyst and the disaccharide acceptor 4. This result dem-
onstrates that the disaccharide unit 1 is a suitable building
block for constructing a b-glucan chain exhibiting two
distinguished surfaces.
4.35 (d, 1H, J1 ,2 = 8.2 Hz, H-10), 4.36 (dd, 1H,
J2,3 = 10.8 Hz, J3,4 = 8.7 Hz, H-3), 4.40–4.43 (2 9 d,
2H, CH2Ph), 4.51 (d, 1H, CH2Ph), 4.59 (d, 1H, CH2Ph),
0
0
0
0
4.73–4.82 (3 9 d, 3H, CH2Ph), 4.96 (t, 1H, J3 ,4 = 9.6 Hz,
0
Experimental
J4 ,5 = 9.6 Hz, H-40), 5.34 (t, 1H, J1,2 = 8.5 Hz,
0
J1,OH = 8.5 Hz, H-1), 6.81–6.84 (m, 3H, Ph), 6.94–6.96
(m, 2H, Ph), 7.22–7.39 (m, 15H, Ph), 7.61–7.79 (m, 4H,
Phth) ppm; 13C NMR (125 MHz, CDCl3): d = 20.8
(COMe), 57.5 (C-2), 66.5 (C-20), 68.0 (C-6), 69.4 (C-60),
71.0 (C-40), 73.3, 73.5, 73.6 (C-50, 2 9 CH2Ph), 74.6, 74.7,
74.9 (C-5, 2 9 CH2Ph), 77.3 (C-3), 78.1 (C-4), 80.6 (C-30),
92.9 (C-1), 100.9 (C-10), 123.2 (Phth), 127.0–128.5 (Ph),
131.6, 133.7 (Phth), 137.6, 137.8, 137.9, 138.5 (Ph), 168.1
(COMe) ppm.
General methods
Nuclear magnetic resonance (NMR) spectra were measured
with tetramethylsilane as an internal standard. The
assignments of the signals were determined using H–1H
correlated spectroscopy and/or 13C–1H heteronuclear mul-
tiple-quantum correlation technique. Coupling constants
(J) are given in Hz. Matrix-assisted laser desorption/ioni-
zation time-of-flight mass spectroscopy (MALDI-TOF MS)
spectra were recorded on Bruker Reflex III using 2,5-
dihydroxybenzoic acid as a matrix. Anhydrous dichloro-
1
Trichloroacetimidoyl (4-O-acetyl-2-azido-3,6-di-O-benzyl-
2-deoxy-b-D-glucopyranosyl)-(1 ? 4)-2-deoxy-3,
6-di-O-benzyl-2-phthalimido-D-glucopyranoside
methane and tetrahydrofuran were prepared by distilling
from phosphorus pentoxide and sodium/benzophenone,
respectively. Flash column chromatography was performed
on silica gel (Wakogel FC-40). Preparative thin-layer
chromatography (TLC) was done on silica gel plates
(Kieselgel 60 F254, Merck). Results of elemental analyses
agreed favorably with calculated values. Unless otherwise
indicated, the usual workup for each reaction mixture
consists of extraction with ethyl acetate, washing with
brine, drying over sodium sulfate and evaporation in vacuo.
(3, C52H50Cl3N5O12)
To a solution of 400 mg 2 (445 lmol) in 5 cm3 anhydrous
CH2Cl2 6.2 mm3 1,8-diazabicyclo[5.4.0]undec-7-ene
(45 lmol) and 318 mg trichloroacetonitrile (2.2 mmol)
were added at 0 °C. After stirring at room temperature for
2 h, another portion of 3.0 mm3 DBU (22 lmol) and
66.24 mm3 trichloroacetonitrile (890 lmol) were added.
The reaction mixture was stirred for 1 h, then purified by
flash column chromatography (EtOAc/n-hexane, 1:3, v/v)
to give 407 mg (88%) 3 as colorless crude crystals, which
were directly used in the next step.
(4-O-Acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-b-D-gluco-
pyranosyl)-(1 ? 4)-2-deoxy-3,6-di-O-benzyl-2-phthali-
mido-D-glucopyranose (2, C50H50N4O12)
tert-Butyldimethylsilyl (2-azido-3,6-di-O-benzyl-2-deoxy-
b-D-glucopyranosyl)-(1 ? 4)-2-deoxy-3,6-di-O-benzyl-2-
phthalimido-b-D-glucopyranoside (4, C54H62N4O11Si)
To a solution of 874 mg tert-butyldimethylsilyl (4-O-
acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyranosyl)-
(1 ? 4)-2-deoxy-3,6-di-O-benzyl-2-phthalimido-b-D-glu-
copyranoside (1, 0.86 mmol) in 15 cm3 MeOH and 10 cm3
CH2Cl2 52 mm3 sodium methoxide in MeOH (28%,
0.26 mmol) were added dropwise at 0 °C. The reaction
mixture was stirred at room temperature overnight, then
another portion of 52 mm3 sodium methoxide in MeOH
(28%, 0.26 mmol) was added. Stirring was continued
To a stirred solution of 1.7 g tert-butyldimethylsilyl (4-O-
acetyl-2-azido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyranosyl)
-(1 ? 4)-2-deoxy-3,6-di-O-benzyl-2-phthalimido-b-D-gluco-
pyranoside (1, 1.68 mmol) in 17 cm3 anhydrous THF
46 mm3 acetic acid (0.8 mmol) and 3.2 cm3 tetra-n-
butylammonium fluoride solution (1.0 M in THF,
3.2 mmol) were added at 0 °C. The reaction mixture was
stirred for 1 h and subsequently worked up. The residue
was purified by flash column chromatography (EtOAc/
toluene, 1:2, v/v) to give 1.17 g (78%) 2 as colorless
crystals. Rf = 0.45 (EtOAc/toluene, 1:2, v/v); 1H NMR
123

1254
T. Kawada, Y. Yoneda
overnight. Subsequently the reaction mixture was neutral-
ized with DOWEX 50W-X8 (H?) resin. The resin was
filtered off, and the filtrate was concentrated in vacuo and
purified by flash column chromatography (EtOAc/toluene,
1:9, v/v) to yield 805 mg (96%) 4 as colorless glass.
Rf = 0.43 (EtOAc/n-hexane, 1:2, v/v); 1H NMR
(500 MHz, CDCl3): d = -0.11 (s, 3H, SiMe), 0.03 (s,
3H, SiMe), 0.66 (s, 9H, SiCMe3) 2.94 (d, 1H,
J = 9.2 Hz, H-4), 4.05 (dd, 1H, J1,2 = 8.3 Hz,
J2,3 = 10.5 Hz, H-2), 4.10–4.16 (m, 3H, H-10, H-40, H-
400), 4.17–4.23 (m, 2H, H-3, H-200), 4.26–4.41 (m, 10H, H-
300, H-1000, CH2Ph), 4.53 (d, 1H, CH2Ph), 4.55 (d, 1H,
CH2Ph), 4.61 (d, 1H, CH2Ph), 4.71–4.78 (3 9 d, 3H,
CH2Ph), 4.81 (d, 1H, CH2Ph), 4.97 (t, 1H, J = 9.5 Hz, H-
4000), 5.07 (d, 1H, CH2Ph), 5.20 (d, 1H, J1 ,2 = 8.3 Hz, H-
00 00
100), 5.28 (d, 1H, J1,2 = 8.3 Hz, H-1), 6.68–7.43 (m, 40H,
Ph), 7.53–7.84 (m, 8H, Phth) ppm; 13C NMR (125 MHz,
CDCl3): d = -5.6, -4.3 (SiMe), 17.5 (SiCMe3), 20.7
(COMe), 25.3 (SiCMe3), 56.5 (C-200), 57.8 (C-2), 66.4, 66.5
(C-20, C-2000), 67.5, 67.6, 68.0 (C-6, C-60, C-600), 69.4
(C-6000), 71.0 (C-4000), 72.4 (CH2Ph), 73.0, 73.1, 73.5, 73.8,
74.2, 74.3, 74.69, 74.72, 74.77, 74.9 (C-5, C-40, C-50, C-500,
C-5000, 7 9 CH2Ph), 76.6, 77.1 (C-3, C-300), 77.7, 78.1 (C-4,
C-400), 80.6 (C-3000), 80.9 (C-30), 93.3 (C-1), 96.8 (C-100),
100.7, 100.8 (C-10, C-1000), 123.0–123.2 (Phth), 126.8–
129.0 (Ph), 131.4–131.7, 133.5–133.8 (Phth), 137.6, 137.9,
138.0, 138.1, 138.3, 138.4, 138.5, 138.7 (Ph), 167.6–168.2
(Phth), 169.6 (COMe) ppm; MALDI-TOF-MS: m/z =
1889.82 [M ? K]?, 1873.81 [M ? Na]?, 1863.76 [M
J4 ,OH = 1.8 Hz, 40-OH), 3.20 (dd, 1H, J2 ,3 = 9.6 Hz,
0
0
0
J3 ,4 = 8.7 Hz, H-30), 3.24 (ddd, 1H, J4 ,5 = 9.9 Hz,
0
0
0
0
J5 ,6 a = 5.5 Hz, J5 ,6 b = 4.4 Hz, H-50), 3.33 (dd, 1H,
0
0
0
0
J1 ,2 = 8.0 Hz, J2 ,3 = 9.6 Hz, H-20), 3.51 (dd, 1H,
0
0
0
0
J5 ,6 a = 5.5 Hz, J6 a,6 b = 9.9 Hz, H-60a), 3.62–3.67 (m,
3H, H-40, H-60b, H-5), 3.78 (dd, 1H, J5,6a = 1.4 Hz,
J6a,6b = 11.1 Hz, H-6a), 3.97 (dd, 1H, J5,6b = 3.4 Hz,
J6a,6b = 11.1 Hz, H-6b), 4.11–4.13 (m, 2H, H-2, H-4), 4.29
(dd, 1H, J2,3 = 10.8 Hz, J3,4 = 8.6 Hz, H-3), 4.41–4.47
(m, 4H, H-10, CH2Ph), 4.56 (d, 1H, CH2Ph), 4.73–4.76
(2 9 d, 2H, CH2Ph), 4.86 (s, 2H, CH2Ph), 5.32 (d, 1H,
J1,2 = 8.0 Hz, H-1), 6.81–6.85 (m, 3H, Ph), 6.96–6.98 (m,
2H, Ph), 7.24–7.42 (m, 15H, Ph), 7.60–7.78 (m, 4H, Phth)
ppm; 13C NMR (125 MHz, CDCl3): d = -5.5, -4.3
(SiMe), 17.5 (SiCMe3), 25.3 (SiCMe3), 57.8 (C-2), 66.2
(C-20), 68.1 (C-6), 70.7 (C-60), 73.0 (C-50), 73.3, 73.3, 73.6
(CH2Ph), 74.1, 74.8 (C-40, C-5), 75.1 (CH2Ph), 76.6 (C-3),
78.0 (C-4), 82.7 (C-30), 93.3 (C-1), 101.0 (C-10), 123.1
(Phth), 126.9–128.5 (Ph), 131.6, 133.6 (Phth), 137.5,
138.1, 138.1, 138.7 (Ph) ppm.
0
0
0
0
(N3 ? NH2) ? K]?, 1847.81
[M (N3 ? NH2) ?
Na]?.
tert-Butyldimethylsilyl (4-O-acetyl-2-azido-3,6-di-O-ben-
zyl-2-deoxy-b-D-glucopyranosyl)-(1 ? 4)-(2-acetamido-
3,6-di-O-benzyl-2-deoxy-b-D-glucopyranosyl)-(1 ? 4)
-(2-azido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyranosyl)-
(1 ? 4)-2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-gluco-
pyranoside (6, C92H110N8O20Si)
tert-Butyldimethylsilyl (4-O-acetyl-2-azido-3,6-di-O-ben-
zyl-2-deoxy-b-D-glucopyranosyl)-(1 ? 4)-(2-deoxy-3,6-di-
O-benzyl-2-phthalimido-b-D-glucopyranosyl)-(1 ? 4)-
(2-azido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyranosyl)-
(1 ? 4)-2-deoxy-3,6-di-O-benzyl-2-phthalimido-b-D-
glucopyranoside (5, C104H110N8O22Si)
To a solution of 515 mg 5 (0.28 mmol) in 5 cm3 n-butanol
945 mm3 ethylenediamine (14 mmol) was added at 90 °C.
After stirring at 90 °C for 5 h, another portion of 189 mm3
ethylenediamine (2.80 mmol) was added. The reaction
mixture was stirred at 90 °C for 1 h and then co-evaporated
with EtOH. The residue was re-dissolved in 7 cm3
pyridine, and 3 cm3 acetic anhydride and 6 mg 4-dimeth-
ylaminopyridine (49 lmol) were added at 0 °C. The
reaction mixture was stirred overnight at room temperature
and then worked up. The crude material was purified by
flash column chromatography (EtOAc/toluene, 2:3, v/v) to
yield 444 mg (95%) 6 as colorless glass. Rf = 0.27
To a stirred solution of 886 mg 3 (0.85 mmol) and 882 mg
4 (0.91 mmol) in 20 cm3 anhydrous CH2Cl2 1.5 mm3
trimethylsilyl trifluoromethanesulfonate (8.5 lM) was
added dropwise at -20 °C. After stirring for 10 min, the
reaction mixture was neutralized with triethylamine and
worked up. The crude material obtained was purified by
flash column chromatography (EtOAc/toluene, 1:4, v/v) to
yield 1.192 g (76%) 5 as colorless glass. Rf = 0.67
1
(EtOAc/n-hexane, 1:1, v/v); H NMR (500 MHz, CDCl3):
1
(EtOAc/toluene, 1:4, v/v); H NMR (500 MHz, CDCl3):
d = 0.06 (s, 3H, SiMe), 0.11 (s, 3H, SiMe), 0.87 (s, 9H,
SiCMe3), 1.68 (s, 3H, COMe), 1.78 (s, 3H, COMe), 1.80 (s,
3H, COMe), 3.07 (m, 1H, H-50), 3.15–3.31 (m, 6H, H-20,
H-30, H-3000, H-5000, H-6000a, H-5NHAc), 3.36–3.54 (m, 7H,
H-2, H-3, H-60a, H-2000, H-6000b, H-5NHAc, H-6NHAc), 3.61–
3.68 (m, 3H, H-60b, H-200, H-6NHAc), 3.74 (br d, 1H,
H-6NHAc), 3.87 (dd, 1H, J = 3.2 Hz, J = 10.9 Hz, H-
d = -0.14 (s, 3H, SiMe), 0.00 (s, 3H, SiMe), 0.64 (s, 9H,
SiCMe3), 1.81 (s, 3H, COMe), 2.75 (br d, 1H, H-50), 3.12
(t, 1H, J = 8.7 Hz, J = 9.6 Hz, H-30), 3.20–3.28 (m, 5H,
H-20, H-60a, H-500, H-3000, H-5000), 3.35 (dd, 1H, J = 4.6 Hz,
J = 10.6 Hz, H-6000a), 3.38 (dd, 1H, J = 8.0 Hz,
J = 9.6 Hz, H-2000), 3.45–3.48 (m, 2H, H-60b, H-6000b),
3.55–3.58 (br d, 2H, H-5, H-600a), 3.67 (dd, 1H,
J = 2.8 Hz, J = 11.0 Hz, H-600b), 3.70 (br d, 1H, H-6a),
3.89 (dd, 1H, J = 2.8 Hz, J = 11.0 Hz, H-6b), 4.00 (t, 1H,
6
NHAc), 3.91–3.95 (m, 2H, H-3, H-40), 4.01–4.05 (m, 2H,
H-4, H-400), 4.21–4.37 (m, 6H, H-10 (d = 4.31), H-1000
(d = 4.34), CH2Ph), 4.43 (d, 1H, CH2Ph), 4.49–4.54
123

Synthesis of a chito-tetrasaccharide b-1,4-GlcNAc-b-1,4-GlcN repeating unit
1255
(m, 4H, H-100, CH2Ph), 4.56–4.60 (2 9 d, 2H, CH2Ph),
(SiCMe), 23.3, 23.4 (COMe), 25.6 (SiCMe3), 55.8 (C-200),
58.2 (C-2), 66.2, 66.5 (C-20, C-2000), 67.9, 68.2, 68.4, 70.7
(C-6, C-60, C-600, C-6000), 72.9, 73.0, 73.2, 73.3, 73.6, 73.8,
74.3, 74.6, 74.7, 75.0 (C-4azido, C-5, C-50, C-500, C-5000,
8 9 CH2Ph), 76.1, 76.5, 77.1, 78.4 (C-3, C-4, C-400, C-
00
4.64–4.67 (2 9 d, 2H, CH2Ph), 4.71 (d, 1H, J2 ,NHAc
=
8.7 Hz, 200-NHAc), 4.76 (d, 1H, CH2Ph), 4.86 (d, 1H,
CH2Ph), 4.92–4.95 (m, 2H, H-1, CH2Ph), 4.97 (t,
J = 9.7 Hz, H-4000), 5.08 (d, 1H, CH2Ph), 5.39 (d, 1H,
J = 7.8 Hz, 2-NHAc), 7.18–7.37 (m, 40H, Ph) ppm; 13C
NMR (125 MHz, CDCl3): d = -5.4, -4.3 (SiMe), 17.8
(SiCMe3), 20.7, 23.3, 23.4 (COMe), 25.5 (SiCMe3), 57.8
(C-200), 58.1 (C-2), 66.4, 66.5 (C-20, C-200), 67.8, 68.1, 68.3
(C-6, C-60, C-600), 69.4 (C-600), 70.9 (C-4000), 72.9, 73.0,
73.2, 73.5, 73.7, 73.9, 74.2, 74.5, 74.6, 74.8, 75.0 (C-5,
C-50, C-500, C-5000, 8 9 CH2Ph), 75.9 (C-3 or C-40), 76.7,
77.3 (C-4, C-400), 78.3 (C-3 or C-40), 79.8 (C-300), 80.3,
81.3 (C-30, C-3000), 95.1 (C-1), 100.0 (C-100), 100.7, 100.9
(C-10, C-1000), 127.1–128.6 (Ph), 137.6, 137.8, 137.8,
138.0, 138.1, 138.6, 138.9, 139.2 (Ph), 169.6, 169.7,
169.8 (COMe) ppm; MALDI-TOF-MS: m/z = 1713.77
[M ? K]?, 1697.78 [M ? Na]?, 1687.75 [M (N3 ?
NH2) ? K]?, 1671.76 [M (N3 ? NH2) ? Na]?.
4
azido), 79.8 (C-300), 81.4, 82.3 (C-30, C-3000), 95.1 (C-1),
100.0 (C-100), 100.7, 100.9 (C-10, C-1000), 127.1–128.9 (Ph),
137.4, 137.8, 138.1, 138.7, 139.0, 139.2 (Ph), 169.7, 169.8
(COMe)
ppm;
MALDI-TOF-MS:
m/z = 1671.65
[M ? K]?, 1655.66 [M ? Na]?, 1645.67 [M (N3 ?
NH2) ? K]?, 1629.67 [M (N3 ? NH2) ? Na]?.
(2-Azido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyranosyl)-
(1 ? 4)-(2-acetamido-3,6-di-O-benzyl-2-deoxy-b-D-gluco-
pyranosyl)-(1 ? 4)-(2-azido-3,6-di-O-benzyl-2-deoxy-b-D-
glucopyranosyl)-(1 ? 4)-2-acetamido-3,6-di-O-benzyl-2-
deoxy-D-glucopyranose (8, C84H94N8O19)
To a stirred solution of 458 mg 7 (0.28 mmol) in 5 cm3
anhydrous THF 8 mm3 acetic acid (0.14 mmol) and
0.56 cm3 TBAF solution (0.56 mmol) were added at
0 °C. After stirring at room temperature for 24 h, another
portion of 0.56 cm3 TBAF (0.56 mmol) was added. The
reaction mixture was stirred for 8 h and then worked up.
The residue was purified by flash column chromatography
(MeOH/CH2Cl2, 1:19, v/v) to yield 396 mg (93%) 8 as
tert-Butyldimethylsilyl (2-azido-3,6-di-O-benzyl-2-deoxy-
b-D-glucopyranosyl)-(1 ? 4)-(2-acetamido-3,6-di-O-ben-
zyl-2-deoxy-b-D-glucopyranosyl)-(1 ? 4)-(2-azido-3,6-di-
O-benzyl-2-deoxy-b-D-glucopyranosyl)-(1 ? 4)-2-acetam-
ido-3,6-di-O-benzyl-2-deoxy-b-D-glucopyranoisde
1
(7, C90H108N8O19Si)
colorless glass. Rf = 0.24 (MeOH/CH2Cl2, 1:19, v/v); H
NMR (500 MHz, CDCl3): d = 1.68 (s, 3H, COMe), 1.73
To a solution of 426 mg 6 (0.25 mmol) in 5 cm3 MeOH
15 mm3 sodium methoxide in MeOH (28%, 0.07 mmol)
was added dropwise at 0 °C. The reaction mixture was
stirred at room temperature overnight, then neutralized
with DOWEX 50W-X8 (H?) resin. The resin was filtered
off, and the filtrate was concentrated in vacuo and purified
by flash column chromatography (EtOAc/toluene, 2:3, v/v)
to yield 404 mg (97%) 7 as colorless glass. Rf = 0.29
000
(s, 3H, COMe), 2.97 (d, 1H, J4
= 1.8 Hz, 4000-OH),
,OH
2.98 (m, 1H, H-50), 3.04–3.09 (m, 2H, H-3000, H-5000), 3.15
(t, 1H, J = 9.4 Hz, H-30), 3.25–3.30 (m, 3H, H-20, H-500,
H-2000), 3.37 (t, 1H, J = 9.2 Hz, H-300), 3.42 (dd, 1H,
J = 6.0 Hz, J = 10.1 Hz, H-6), 3.48 (br s, 2H, 2 9 H-6),
3.52 (dd, 1H, J = 4.6 Hz, J = 10.1 Hz, H-6), 3.60 (dt, 1H,
000
J4
= 1.8 Hz, J = 9.1 Hz, H-4000), 3.64–3.69 (m, 5H,
,OH
1
(MeOH/CH2Cl2, 1:19, v/v); H NMR (500 MHz, CDCl3):
H-3, H-200, 3 9 H-6), 3.88 (dd, 1H, J = 2.8 Hz,
J = 10.1 Hz, H-6), 3.90 (t, 1H, J = 9.2 Hz, H-40), 3.95–
4.08 (m, 4H, H-2, H-4, H-5, H-400), 4.12 (d, 1H,
J = 8.3 Hz, H-1azido), 4.19 (d, 1H, CH2Ph), 4.24 (br s,
1H, 1-OH), 4.28 (d, 1H, CH2Ph), 4.30 (d, 1H, J = 8.3 Hz,
H-1azido), 4.37–4.49 (m, 7H, CH2Ph), 4.45 (d, 1H,
J = 8.3 Hz, H-100), 4.62–4.65 (2 9 d, 2H, CH2Ph), 4.68
d = 0.06 (s, 3H, SiMe), 0.10 (s, 3H, SiMe), 0.87 (s, 9H,
SiCMe3), 1.67 (s, 3H, COMe), 1.77 (s, 3H, COMe), 2.93
(br s, 1H, 4000-OH), 3.05–3.09 (m, 3H, H-3azido, H-50, H-
5000), 3.23 (t, 1H, J = 9.2 Hz, H-3azido), 3.25–3.30 (m, 3H,
H-20, H-2000, H-5NHAc), 3.38–3.45 (m, 3H, H-2, H-300, H-6),
3.48 (br s, 2H, 2 9 H-6), 3.51–3.54 (m, 2H, H-5NHAc, H-
6), 3.59–3.68 (m, 4H, H-200, H-4azido, 2 9 H-6), 3.74 (br d,
1H, H-6), 3.87 (dd, 1H, J = 3.2 Hz, J = 11.0 Hz, H-6),
3.90–3.95 (m, 2H, H-3, H-4azido), 3.99–4.04 (m, 2H, H-4,
H-400), 4.23 (d, 1H, CH2Ph), 4.28 (d, 1H, CH2Ph), 4.30 (d,
1H, J = 8.2 Hz, H-1azido), 4.31 (d, 1H, J = 7.8 Hz, H-
(d, 1H, J2 ,NHAc = 8.8 Hz, 200-NHAc), 4.81–4.85 (m, 3H,
00
CH2Ph), 4.94 (d, 1H, CH2Ph), 5.08 (d, 1H, CH2Ph), 5.19
00
(br s, 1H, H-100), 4.68 (d, 1H, J2 ,NHAc = 8.2 Hz, 200-
NHAc), 7.19–7.41 (m, 40H, Ph) ppm; 13C NMR
(125 MHz, CDCl3): d = 23.2, 23.3 (COMe), 52.8 (C-2),
55.5 (C-200), 66.1, 66.4 (C-20, C-2000), 67.0, 67.8, 68.2
(3 9 C-6), 70.2 (C-5), 70.6 (C-6), 72.9, 73.0, 73.2, 73.3,
73.6, 73.7, 74.0, 74.1, 74.6, 74.9, 75.0 (C-50, C-500, C-4000,
C-5000, 8 9 CH2Ph), 76.1 (C-40), 76.4, 77.1, 77.5 (C-3, C-4,
C-400), 80.0 (C-300), 81.2 (C-30), 82.3 (C-3000), 91.5 (C-1),
100.1 (C-100), 100.7, 101.0 (C-10, C-1000), 127.3–128.8
(Ph), 137.4, 137.7, 138.0, 138.6, 138.9, 139.1 (Ph), 169.8,
1
azido), 4.37–4.43 (m, 3H, CH2Ph), 4.47–4.53 (m, 4H, H-
100, CH2Ph), 4.59 (d, 1H, CH2Ph), 4.63–4.66 (2 9 d, 2H,
CH2Ph), 4.71 (d, 1H, J2 , NHAc = 8.7 Hz, 200-NHAc), 4.81–
00
4.83 (m, 3H, CH2Ph), 4.91–4.95 (2 9 d, 2H, H-1, CH2Ph),
5.08 (d, 1H, CH2Ph), 5.34 (d, 1H, J2,NHAc = 7.8 Hz, 2-
NHAc), 7.15–7.40 (m, 40H, Ph) ppm; 13C NMR
(125 MHz, CDCl3): d = -5.3, -4.2 (SiMe), 17.9
123

1256
T. Kawada, Y. Yoneda
the support of a Grant-in-Aid for Scientific Research (18658133) from
Japan Society for the Promotion of Science (JSPS).
170.3 (COMe) ppm; MALDI-TOF-MS: m/z = 1557.53
[M ? K]?, 1541.55 [M ? Na]?, 1515.54 [M (N3 ?
NH2) ? Na]?.
(2-Amino-2-deoxy-b-D-glucopyranosyl)-(1 ? 4)-(2-acet-
amido-2-deoxy-b-D-glucopyranosyl)-(1 ? 4)-(2-amino-2-
deoxy-b-D-glucopyranosyl)-(1 ? 4)-2-acetamido-2-deoxy-
D-glucopyranose (9, C28H50N4O19)
References
1. Kawada T, Yoneda Y (2009) Monatsh Chem 140(10). doi:
10.1007/s00706-009-0172-0
Compound 8 (208 mg, 0.136 mmol) was dissolved in
5 cm3 MeOH containing 400 mg 10% palladium on
carbon, and the resulting mixture was stirred under
hydrogen at normal pressure for 30 min at 50 °C. The
catalyst was removed by filtration, and the filtrate was
evaporated to dryness. The residue was purified by gel
filtration (Sephadex LH-20) using MeOH to yield 72.9 mg
(72%) 9 as colorless crystals. Rf = 0.20 (AcOH/MeOH,
1:2, v/v); MALDI-TOF-MS: m/z = 785.26 [M ? K]?,
769.30 [M ? Na]?, 747.28 [M ? H]?.
2. Cory EJ, Venkateswarlu A (1972) J Am Chem Soc 94(17):6190
3. Clark JH (1980) Chem Rev 80(5):429
4. Smith AB, Ott GR (1996) J Am Chem Soc 118(51):13095
5. Schmidt TT, Michel J (1980) Angew Chem Int Ed Engl
19(9):731
6. Sugimoto M, Hirosakai T, Ogawa T (1985) Glycoconj J 2:11
7. Velluz L, Valls J, Mathieu J (1967) Angew Chem Int Ed Engl
6(9):778
8. Banoub J, Boullanger P, Lafont D (1992) Chem Rev 92:1167
9. Schmidt RR (1986) Angew Chem Int Ed Engl 25(3):212
10. Bock K, Pedersen C (1983) Advances in carbohydrate chemistry
and biochemistry, vol 41. Academic Press, Inc, London, p 27
11. Bock K, Pedersen C, Pedersen H (1984) Advances in carbohy-
drate chemistry and biochemistry, vol 42. Academic Press, Inc,
London, p 193
Acknowledgments We are grateful to Dr. Hiroshi Kamitakahara,
Graduate School of Agriculture, Kyoto University, Japan, for
MALDI-TOF-MS measurements. This research was conducted with
12. Demchenko AV, Boons GJ (2001) J Org Chem 66(8):2547
123