An efficient synthesis of a biantennary sialooligosaccharide analog using a 1,6-anhydro-β-lactose derivative as a key synthetic block

Article abstract of DOI:10.1016/S0040-4020(03)00711-7

An efficient and versatile method for the synthesis of a biantennary octasaccharide derivative was established by combined chemical and enzymatic manipulations of 1,6-anhydro-β-lactose as a key starting material. A key 1,6-anhydro-β-lactose derivative having two unprotected hydroxyl groups at C-3′ and C-6′ positions was prepared and employed for the chemical coupling reaction with a known 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl imidate to afford a tetrasaccharide derivative with two GlcNAc branches in 69% yield. Enzymatic galactosylation using UDP-Gal with a bovine milk β1,4-galactosyltransferase and subsequent sialylation with a recombinant α2,3-sialyltransferase in the presence of CMP-Neu5Ac proceeded smoothly and gave a desired model compound, a bivalent sialooctasaccharide (1), in 73% overall yield from the tetrasaccharide intermediate.

Full text of DOI:10.1016/S0040-4020(03)00711-7

Tetrahedron 59 (2003) 5105–5113
An efficient synthesis of a biantennary sialooligosaccharide analog
using a 1,6-anhydro-b-lactose derivative as a key synthetic block
Tetsuya Furuike,a Kuriko Yamada,a Takashi Ohta,b Kenji Mondea,b
and Shin-Ichiro Nishimuraa,b,c
,
*
aSapporo Laboratory for Glycocluster Project, Japan Bioindustry Association, Hokkaido University, Sapporo 060-0810 Japan
bLaboratory for Bio-Macromolecular Chemistry, Division of Biological Sciences, Graduate School of Science, Hokkaido University,
Kita-ku, Sapporo 060-0810 Japan
cGlycochemosynthesis Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST),
Sapporo 062-8517, Japan
Received 30 September 2002; accepted 30 April 2003
Abstract—An efficient and versatile method for the synthesis of a biantennary octasaccharide derivative was established by combined
chemical and enzymatic manipulations of 1,6-anhydro-b-lactose as a key starting material. A key 1,6-anhydro-b-lactose derivative having
two unprotected hydroxyl groups at C-30 and C-60 positions was prepared and employed for the chemical coupling reaction with a known
3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl imidate to afford a tetrasaccharide derivative with two GlcNAc branches in
69% yield. Enzymatic galactosylation using UDP-Gal with a bovine milk b1,4-galactosyltransferase and subsequent sialylation with a
recombinant a2,3-sialyltransferase in the presence of CMP-Neu5Ac proceeded smoothly and gave a desired model compound, a bivalent
sialooctasaccharide (1), in 73% overall yield from the tetrasaccharide intermediate. q 2003 Elsevier Science Ltd. All rights reserved.
1. Introduction
degree of branching.4–6 The branching of the linear
lactosaminoglycans has also been known as a crucial on/
off switch for regulating the functional roles in Ii-antigenic
structures. Thus, I-antigens produced from the linear
lactosaminoglycans (i-antigens) are key antigenic structures
as cell surface signals to control cell differentiation, aging,
and malignant transformation of cells.7–14
Lactosaminoglycans are high molecular weight carbo-
hydrates bound to proteins or lipids and composed of
unique N-acetyllactosamine repeating units [Galb(1!4)
GlcNAcb(1!3)]n.1 It was also reported that the linear
lactosaminoglycan chains sometimes involve branching
structures at C-6 position of the D-galactose residues of
this repeating disaccharide unit. In addition, it has also
known that introduction of sialic acid and/or fucose residues
to the lactosaminoglycans may lead to further complicated
structures showing significant biological functions.2,3 In
fact, a variety of lactosaminoglycans carrying modified
oligosaccharide branches play important roles as cell
surface antigens and as specific ligands (markers) for the
carbohydrate-binding proteins in cellular recognition pro-
cesses. For example, it has been suggested that the structure
of each lactosaminoglycan isolated from teratocarcinoma,
Ehrlich’s ascites carcinoma or human granulocyte remark-
ably differs in terms of the average molecular weight and the
The synthetic study of lactosaminoglycans and related
compounds will apparently accelerate the investigation for
the biological significance of these attractive carbohydrates
and this might contribute to the development of the effective
therapeutic reagents to inhibit cancer metastasis or prevent
cells from malignant alterations and viral infections.
Moreover, multivalency of carbohydrate chains is of
growing interest from medicinal chemistry for the synthesis
of carbohydrate-based therapeutic reagents as well as
biological importance of naturally occurring glycoproteins
with multiple sugar branches such as biantennary,
triantennary, and tetraantennary structures.15 Our interest
has been, therefore, focused on the efficient and systematic
synthesis of branched-type lactosaminoglycans as a key and
specific carbohydrate signals in biomolecular recognitions.
Although chemical syntheses of some lactosaminoglycan-
related compounds were carried out by means of a
conventional strategy, these syntheses often involve com-
plicated synthetic scheme with tedious multistep protection/
deprotection processes and purification of anomeric mixture
Keywords: lactosaminoglycan; biantennary oligosaccharide; 1,6-anhydro-
b-lactose; b1,4-galactosyltransferase; a2,3-sialyltransferase; glycoprotein;
glycolipid.
*
Corresponding author. Address: Graduate School of Science, Division of
Biological Sciences, Hokkaido University, Building of Next Generation
Post-Genome, Kita-21 Nishi-l, Kitaku, Sapporo 001-0021, Japan. Tel.:
þ81-11-706-9043; fax: þ81-11-706-9042;
e-mail: shin@glyco.sci.hokudai.ac.jp
0040–4020/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4020(03)00711-7

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T. Furuike et al. / Tetrahedron 59 (2003) 5105–5113
Figure 1. A strategy for chemo-enzymatic synthesis of a biantennary sialyllactosaminoglycan model 1.
in glycosylation reactions.16–23 Advent of an efficient and
versatile strategy for rapid synthesis of the lactosamino-
glycans and related compounds is strongly required. In the
present study, we would like to report an efficient synthesis
for these unique oligosaccharide analogs based on the
combined chemical and enzymatic strategy. The method
described herein involves the use of a novel key
disaccharide intermediate derived from naturally abundant
lactose and recombinant glycosyltransferases as bio-
catalysts for the sugar elongation reactions. Merits of the
novel synthetic concept will be demonstrated by construct-
ing a bivalent octasaccharide derivative 1 having two sialic
acid residues as a potential candidate of the sugar-based
therapeutic reagent. Some synthetic intermediates prepared
herein will become versatile materials for the construction
of a variety of synthetic multivalent glycoligands as stable
and standard compounds. These standard glycoligands
prepared in a large scale manner will become valuable
tools for the investigation of the functional roles of
‘multivalency’ in carbohydrate–protein interactions. The
synthesis of 1 was basically achieved by (i) preparation of a
key disaccharide intermediate 10 from lactose, (ii) intro-
duction of two glucosamine residues to C-30 and C-60
positions of compound 10, and (iii) enzymatic galactosyl-
ation and sialylation of the tetrasaccharide intermediate as
outlined in Figure 1.
ide resource produced from bovine milk and one of the most
important starting materials that can be used for the large
scale syntheses of bioactive glycoconjugates. It has been
widely accepted that 1,6-anhydro-b-lactose obtained readily
from lactose monohydrate can be efficiently converted to
some potential intermediates for the synthesis of N-
acetyllactosamine and its derivatives as reported previously
by Kuzuhara et al.,24 Tejima et al.,25,26 and our group.27–29
Feasibility of an intermediate derived from 1,6-anhydro-b-
lactose in the synthetic study of a complicated oligo-
saccharide derivative has been preliminarily demonstrated
by an efficient synthesis of biologically important Lewis X
(LeX) trisaccharide derivative and the preparation of a
macromolecular ligand carrying this LeX antigenic
trisaccharide structure.28
In the present synthesis, we selected a 1,6-anhydro-b-
lactose derivative 10 having two unprotected hydroxyl
groups at C-30 and C-60 positions as a key intermediate for
the efficient syntheses of a bivalent sialooligosaccharide
derivative. Although compound 10 could be converted from
a known O-(3-O-allyl-2,4-di-O-benzyl-6-O-triphenyl-
methyl-b-D-galactopyranosyl)-(1!4)-1,6-anhydro-2-
azido-2-deoxy-b-D-glucopyranose,29 we prepared herein a
new intermediate 9 in order to achieve an efficient and direct
synthesis of the compound 10 as indicated in Scheme 1.
First, 1,6-anhydro-b-lactose 2 was subjected to stannyl-
ation30–32 by using bis(tri-n-butyltin)oxide, followed by the
introduction of two O-allyl groups with 3-bromo-1-propene
and acetylation to afford 30,60-di-O-allylated derivative 3 in
68% yield. After de-O-acetylation of 3, the tetraol 4 was
converted into 2-O-(2,4,6-triisopropylbenzenesulfonyl)
(TIPBS) derivative 5 in 78% yield. Intramolecular nucleo-
philic substitution of compound 5 with an aqueous solution
2. Results and discussion
2.1. Synthesis of a key intermediate 10 from 1,6-
anhydro-b-lactose as a starting material
Lactose, Galb(1!4)Glc, is a naturally abundant disacchar-
Scheme 1. Reagents and conditions: (i) (a) (n-Bu3Sn)2O/toluene, azeotrope; (b) allyl bromide, 808C; (c) Ac2O/pyridine, room temperature, 68%.
(ii) NaOMe/MeOH–THF, room temperature, q.y. (iii) TIPBS-Cl, DMAP/pyridine, room temperature, 78%. (iv) 1N NaOHaq./MeOH–THF, room
temperature, 78%. (v) BnBr, NaH/DMF, 92%. (vi) NaN3, CsF/DMF, 1108C, 65%. (vii) BnBr, NaH/DMF, 82%. (viii) PdCl2, NaOAc/95%AcOHaq.,
ultrasonication, room temperature, 81%.

T. Furuike et al. / Tetrahedron 59 (2003) 5105–5113
5107
˚
Scheme 2. Reagents and conditions: (i) TMSOTf, MS4A/CH2Cl2, 2158C, 69% for 12, 26% for 13.
of 1N sodium hydroxide efficiently gave an epoxide 6 in
78% yield. After O-benzylation at C-20 and C-40 positions of
6, nucleophilic attack by an azide ion in the presence of
cesium salt as accelerator to the epoxide 7 gave 2-azido
derivative 8 in 65% yield. O-Benzylation at C-3 position of
8 and subsequent de-O-allylation of 9 under standard
conditions30,31 afforded a desired diol 10 in 81% yield. The
standardized intermediate 10 will become a key synthetic
block to construct longer and more complicated branched-
type lactosaminoglycan chains.
tetrasaccharide 15, hydrogenation over 5% palladium on
charcoal removed benzyl protections at C-3, 20, and 40
positions to give nonaol 16 in 81% yield (2 steps).
Galactosylation of tetrasaccharide 16 was carried out by
using b1,4-galactosyltransferase with UDP-Gal in 50 mM
HEPES buffer (pH 6.0) for 24 h according to the condition
reported previously (Scheme 3).35–37 Hexasaccharide 17
was isolated by subjecting a reaction mixture to the simple
gel filtration by means of a column of Sephadex G-15 with
water as an eluent in 87% yield. No pentasaccharide
derivative (by-product) was detected and obtained by this
procedure, suggesting that the terminal GlcNAc residues in
lactosaminoglycans could be galactosylated equally by this
enzyme. Finally, compound 17 was subjected to the
following sialylation by means of rat liver a2,3-sialyltrans-
ferase in the presence of CMP-Neu5Ac in 50 mM sodium
cacodylate buffer (pH 7.3) to give octasaccharide 1 in 84%
yield. Chemical structure of the final product was confirmed
by a matrix-assisted laser desorption ionization time-of-
flight (MALDI-TOF), and electrospray ionization high
resolution (ESI-HRMS) mass spectrometry, and 1H and
13C NMR spectroscopy. The MALDI-TOF mass spectra of
hexasaccharide 17 and octasaccharide 1 are shown in
Figure 2. It was suggested that the molecular weight
observed at 1700.8 was found to be in good agreement with
2.2. Chemical and enzymatic synthesis of an
octasaccharide 1 from compound 10
A disaccharide synthetic block 10 was allowed to react with
a known glycosyl donor 1133,34 readily obtained from the
peracetate of 2-phthalimido-2-deoxy-D-glucopyranose
(Scheme 2). Coupling reaction of 10 with 11 proceeded
smoothly in the presence of trimethylsilyl trifluoromethane-
sulfonate (TMSOTf) as promoter and gave tetrasaccharide
12 in 69% yield together with trisaccharide 13 (26%) as a
by-product. De-N-phthaloylation with hydradine and sub-
sequent N-acetylation afforded compound 14 in 77% yield.
Next, azide group at C-2 position of 14 was converted into
acetamido group by treating with thioacetic acid–pyridine
to yield 15 quantitatively. After de-O-acetylation of
Scheme 3. Reagents and conditions: (i) (a) NH2NH2·H2O/EtOH, reflux; (b) Ac2O/Pyr, room temperature, 77%. (ii) AcSH/Pyr, room temperature, 96%. (iii) (a)
NaOMe/MeOH, room temperature; (b) H2, 5% Pd–C/MeOH–H2O, room temperature, 81%. (iv) UDP-galactose, b-1,4-galactosyltransferase, a-lactalbumin,
MnCl2·4H2O/50 mM HEPES buffer (pH6.0), 378C, 87%. (v) CMP-NANA, a-2,3-sialyltransferase, BSA, MnCl2·4H2O, Triton CF-54/50 mM sodium
cacodylate buffer (pH 7.4), 378C, 84%.

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T. Furuike et al. / Tetrahedron 59 (2003) 5105–5113
Figure 2. MALDI-TOF mass spectra of hexasaccharide 17 and octasaccharide 1.
its [MþNa]þ adduct of compound 1. Fully assigned 13C
NMR spectrum of octasaccharide 1 is also shown in
Figure 3. The data obtained by 1H and 13C NMR
measurements were described in Section 3. These results
clearly demonstrate that both galactosyl and sialyltrans-
ferases recognize non-reducing two GlcNAc moieties
bound to the 1,6-anhydro-b-lactose and an unusual 1C
type conformation at the reducing end of 16 does not reduce
the efficiency of the sugar elongation reaction catalyzed by
this enzyme at all. These findings mean obviously the wide
applicability of 1,6-anhydro-b-lactose derivatives as versa-
tile scaffolds in the combined chemical and enzymatic
synthesis of a variety of complicated glycoconjugates
involving non-natural oligosaccharides. A series of oligo-
saccharide derivatives might become valuable tools for the
preparation of carbohydrate-based therapeutic reagents,
since octasaccharide derivative 1 seems to be one of the
bivalent sialooligosaccharide ligands with potent biological
Figure 3. 13C NMR spectrum of octasaccharide 1.

T. Furuike et al. / Tetrahedron 59 (2003) 5105–5113
5109
activity such as antiinfluenza and antitumor activities.
Application of these synthetic multivalent glycoligands in
medical field is under investigation and the result will be
reported in the nearest future.
H-40), 5.44 (s, 1H, H-1), 5.21–5.11 (m, 5H, CH2vCH and
H-20), 5.10 (s, 1H, H-2), 4.71 (d, 1H, J1 ,2 ¼7.9 Hz, H-1 ),
4.61 (d, 1H, J5,6a¼5.0 Hz, H-5), 4.53 (s, 1H, H-3), 4.17–
4.10 (br, 2H, H-60a and H-60b), 4.19–3.86 (m, 6H,
CH2vCH–CH2, H-6a, and H-6b), 3.59 (s, 1H, H-4),
3.55–3.45 (br, 2H, H-30 and H-50), 2.13–2.08 (all s, 12H,
4COCH3). Anal. calcd for C26H36O14·0.5H2O: C, 53.70; H,
6.41%. Found: C, 53.77; H, 6.26%.
0
0
0
In conclusion, 1,6-anhydro-b-lactose was efficiently con-
verted into a key lactosamine intermediate having
unprotected hydroxyl groups at C-30 and C-60 positions. It
was clearly demonstrated that combined chemical and
enzymatic modifications of this key compound greatly
accelerated the large-scale synthesis of a bivalent sialo-
octasaccharide derivative as a potent glycoligand.
3.1.2. 3,6-Di-O-allyl-b-D-galactopyranosyl-(1!4)-1,6-
anhydro-2-O-triisopropylbenzenesulfonyl-b-D-gluco-
pyranose (5). A solution of compound 3 (1.10 g,
1.92 mmol) and sodium methoxide (60 mg) in 1:1 (v/v)
methanol–THF (20 mL) were stirred for 2 h at room
temperature. The mixture was made neutral with the
Dowex 50W-X8(Hþ) resin, and the suspension was filtered.
The filtrate was evaporated to give a quantitative yield of 4
(0.77 g). To a solution of 4 in pyridine (10 mL) were added
4-dimethylaminopyridine (DMAP) (560 mg, 4.61 mmol)
and TIPBS-Cl (1.56 g, 4.61 mmol) at 08C, and the mixture
was stirred for 72 h at room temperature under nitrogen
atmosphere. After concentration, the obtained residue was
dissolved in chloroform and washed successively with 1N
sulfuric acid, saturated sodium hydrogen carbonate and
brine, dried and evaporated. The residue was chromato-
graphed on silica gel with chloroform–methanol (100:1,
v/v) to give 5 (1.0 g, 78%) as a crystalline mass: mp 808C;
[a]2D5¼27.68 (c 0.240, CHCl3); 1H NMR (CDCl3) d7.17 (s,
2H, aromatic), 5.88 (m, 2H, CH2vCH), 5.43 (br s, 1H,
3. Experimental
3.1. General methods
Unless otherwise stated, all commercially available solvents
and reagents were used without further purification. Optical
rotations were determined with a Horiba SEPA-200 digital
polarimeter at 258C. 1H and proton decoupled carbon NMR
spectra were recorded at 400 and 100.4 MHz, respectively,
on a JEOL ALPHA-400 spectrometer. Ring-proton assign-
ments in NMR were made by first-order analysis of the
spectra and supported by HH-COSY experiments. Elemen-
tal analyses were performed with a Sartorius 4503 Micro,
7079 printer. Bovine milk b1,4-galactosyltransferase (EC
2.4.1.22) was purchased from Sigma Co. Ltd.. Rat liver
a2,3-sialyltransferase was purchased from Calbiochem Co.
Ltd. Reactions were monitored by thin-layer chromato-
graphy (t.l.c.) on a precoated plate of silica gel 60F254 (layer
thickness, 0.25 mm; E. Merck, Darmstadt, Germany).
Column chromatography was performed on silica gel
(Wakogel C-200; 100–200 mesh, Wako Pure Chemical
Industries Co. Ltd. Japan). MALDI-TOF mass spectrometry
was carried out on a Finnigan LASERMAT 2000 instru-
mental using 2,5-dihydroxybenzoic acid. The instrument
was operated in the positive ion reflection mode with an
accelerating potential of 20 kV. Solvent extracts were dried
with anhydrous magnesium sulfate and concentrated below
408C under diminished pressure.
H-1), 5.35–5.15 (m, 4H, CH2vCH), 4.60 (d, 1H,
0
0
0
J5,6a¼6.8 Hz, H-5), 4.38 (d, 1H, J1 ,2 ¼7.8 Hz, H-1 ), 4.36
(d, 1H, J1,2¼5.6 Hz, H-2), 4.20 (m, 2H, CH2vCH–CH2),
4.14 [m, 3H, (CH3)2–CH], 4.02–3.93 (m, 2H, H-3, and
H-40), 3.81 (t, 2H, H-20), 3.70–3.63 (m, 05H, H-4, H-6a,
H-6b, H-60a, and H-60b), 3.55 (d, 1H, H-5 ), 3.370 (dd, 1H,
0
0
0
0
0
J3 ,4 ¼3.2 Hz, J2 ,3 ¼10.4 Hz, H-3 ), 2.90 (br, 2H, 2 -OH and
40-OH), 2.50 (br, 1H, 3-OH), 1.27–1.25 [each s, each 6H,
(CH3)2CH]. Anal. calcd for C33H50O12S: C, 59.08; H, 7.51;
S, 4.78%. Found: C, 59.05; H, 7.57; S, 5.07%.
3.1.3. 3,6-Di-O-allyl-b-D-galactopyranosyl-(1!4)-
1,6:2,3-dianhydro-b-D-mannopyranose (6). To a solution
of 5 (650 mg, 0.969 mmol) in 1:1 (v/v) methanol–THF
(10 mL) was added an aqueous solution of 1N sodium
hydroxide (2.90 mL) and the mixture was stirred for 24 h at
room temperature. After concentration, the residual oil was
dissolved in chloroform, washed with brine, dried and
evaporated in vacuo. The residue was purified by chroma-
tography on silica gel with chloroform–methanol (80:1,
v/v) as an eluent to give a syrup 6 (290 mg, 78%):
[a]2D5¼216.48 (c 0.251, CHCl3); 1H NMR (CDCl3) d
6.00–5.84 (m, 2H, CH2vCH) 5.71 (d, 1H, J1,2¼3.0 Hz, H-
3.1.1. 2,4-Di-O-acetyl-3,6-di-O-allyl-b-D-galactopyrano-
syl-(1!4)-2,3-di-O-acetyl-1,6-anhydro-b-D-glucopyra-
nose (3). To a solution of 2 (2.5 g, 6.18 mmol) in toluene
(300 mL) was added bis(tri-n-butyltin)oxide (4.27 mL,
8.03 mmol), and the mixture was stirred for 4 h at 1408C
with continuous azeotropic removal of water. After
evaporation to the half volume, 3-bromo-1-propene
(20 mL) was added to the solution and the reaction mixture
was stirred for 12 h at 808C. After concentration, the
residual syrup was dissolved in pyridine (50 mL) and to the
mixture were added DMAP (0.76 g, 6.18 mmol) and acetic
anhydride (50 mL). After being stirred for 15 h at room
temperature, the mixture was evaporated and diluted with
chloroform. The solution was washed successively with 1N
sulfuric acid, saturated sodium hydrogen carbonate and
brine, dried and evaporated. The residue was chromato-
graphed on silica gel with toluene–ethyl acetate (5:1, v/v) as
an eluent to afford compound 3 (2.4 g, 68%): mp 1168C;
[a]D25¼237.48 (c 0.149, CHCl3); 1H NMR (CDCl3) d
1), 5.36–5.19 (m, 4H, CH2vCH), 4.54 (t, 1H, H-5), 4.45 (d,
0
0
0
1H, J1 ,2 ¼7.7 Hz, H-1 ), 4.21 (m, 2H, CH2vCH–CH2),
4.06–4.04 (m, 3H, H-40 and CH2vCH–CH2), 3.98 (br s,
1H, H-3), 3.84–3.79 (m, 2H, H-20 and H-6b), 3.78–3.68 (m,
0
0
0
0
1H, H-6a), 3.63 (dd, 1H, J5 ,6 a¼5.7, J6 a,6 b¼11.6 Hz,
H-60b), 3.47–3.42 (m, 4H, H-2, H-4,0H-50 and H-60a),03.38
0
0
0
0
(dd, 1H, J3 ,4 ¼3.3, J2 ,3 ¼9.4 Hz, H-3 ), 2.77 (br, 2H, 2 -OH
and 40-OH). Anal. calcd for C18H26O9·0.5H2O: C, 54.67; H,
6.88%. Found: C, 54.50; H, 6.58%.
3.1.4. 3,6-Di-O-allyl-2,4-di-O-benzyl-b-D-galactopyrano-
0
0
5.90–5.70 (m, 2H, CH2vCH), 5.47 (d, 1H, J3 ,4 ¼3.3 Hz,

5110
T. Furuike et al. / Tetrahedron 59 (2003) 5105–5113
syl-(1!4)-1,6:2,3-dianhydro-b-D-mannopyranose (7).
To a solution of 6 (200 mg, 0.518 mmol) dissolved in
DMF (10 mL) were added sodium hydride (60% oil
suspension; 62 mg, 1.55 mmol) and benzyl bromide
(0.32 mL, 2.59 mmol), and the mixture was stirred at
room temperature. After 24 h, excess of sodium hydride was
carefully quenched by addition of methanol. The resulting
solution was dissolved in ethyl acetate, washed with water,
brine and dried. After evaporation, the residual oil was
purified by silica gel column chromatography with toluene–
ethyl acetate (5:1, v/v) as an eluent, and recrystallization
from ethanol gave 7 (270 mg, 92 %): mp 1388C;
[a]2D5¼223.38 (c 0.227, CHCl3); 1H NMR (CDCl3) d
7.42–7.25 (m, 10H, aromatic), 5.90–5.78 (m, 2H,
CH2vCH), 5.72 (d, 1H, J1,2¼2.8 Hz, H-1), 5.37–5.15 (m,
CH2vCH–CH2), 3.94 (d, 1H, J6a,6b¼7.1 Hz, H-6b), 3.85
(s, 1H, H-3), 3.82–3.70 (m, 5H, H-4, H-20, H-40 and
CH2vCH–CH2), 3.63 (t, 1H, J5,6a¼6.1 Hz and
J6a,6b¼7.2 Hz, H-6a), 3.43–3.32 (m, 4H, H-30, H-50 H-60a
and H-60b), 3.13 (s, 1H, H-2). Anal. calcd for
C39H45N3O9·0.5H2O: C, 66.09; H, 6.54; N, 5.92%. Found:
C, 65.80; H, 6.53; N, 5.70%.
3.1.7. 2,4-Di-O-benzyl-b-D-galactopyranosyl-(1!4)-1,6-
anhydro-2-azido-3-O-benzyl-2-deoxy-b-D-glucopyra-
nose (10). To a solution of 9 (80 mg, 0.114 mmol) in acetic
acid (1.9 mL) and H2O (0.1 mL) were added sodium acetate
(93 mg, 1.14 mmol) and palladium (II) chloride (81 mg,
0.456 mmol), and the mixture was kept for 2 h under
ultrasonication. After filtration off through a celite bed, the
filtrate was extracted with chloroform. The organic layer
was washed with saturated sodium hydrogen carbonate,
brine, dried and evaporated in vacuo. The residual oil was
chromatographed on a silica gel column with chloroform:
methanol (60:1, v/v) as an eluent to afford 10 (57 mg, 81%)
4H, CH2vCH), 4.97–4.64 (m, 4H, PhCH2), 4.50 (d, 1H,
0
CH2vCH–CH2), 3.91–3.86 (m, 4H, H-3, H-6b and
0
0
J1 ,2 ¼7.7 Hz, H-1 ), 4.46 (m, 1H, H-5), 4.22–4.20 (m, 2H,
CH2vCH–CH2), 3.89 (d, 1H, H-40), 3.82 (dd, 1H,
0
0
0
0
0
J1 ,2 ¼7.6 Hz, J2 ,3 ¼9.7 Hz, H-2 ), 3.67 (d, 1H, H-6a), 3.52
(m, 2H, H-60a and H-60b), 3.45–3.41 (m, 4H, H-2, H-4, H-
30, and H-50). Anal. calcd for C32H38O9: C, 67.83; H, 6.76%.
Found: C, 67.71; H, 6.89%.
1
as a syrup foam: [a]D25¼þ2.08 (c 0.19, CHCl3); H NMR
(CDCl3) d7.40–7.30 (m, 15H, aromatic), 5.51 (brs, 1H,
H-1), 5.10–4.64 (m, 6H, PhCH2), 4.61 (d, 01H, J5,6a¼6.1 Hz,
0
0
H-5), 4.44 (d, 1H, J1 ,2 ¼7.0 Hz, H-1 ), 4.07 (d, 1H,
J6a,6b¼7.4 Hz, H-6b), 3.91 (br s, 1H, H-3), 3.78 (m, 2H,
H-20 and H-40), 3.75 (br s, 1H, H-4), 3.72 (d, 1H,
3.1.5. 3,6-Di-O-allyl-2,4-di-O-benzyl-b-D-galactopyrano-
syl-(1!4)-1,6-anhydro-2-azido-2-deoxy-b-D-glucopyra-
nose (8). A mixture of 7 (100 mg, 0.176 mmol), cesium
fluoride (270 mg, 1.76 mmol) and sodium azide (114 mg,
1.76 mmol) dissolved in DMF (2.0 mL) were stirred for
24 h at 1108C under nitrogen atmosphere. After cooling to
room temperature, the mixture was diluted with water,
extracted with ethyl acetate and washed with brine. The
mixture was dried, evaporated and subjected to chromato-
graphy on silica gel column with n-hexane–ethyl acetate
(3:1, v/v) as an eluent to give 8 (70 mg, 65%) as a crystalline
powder: mp 878C; [a]2D5¼240.78 (c 0.229, CHCl3); 1H
NMR (CDCl3) d 7.35–7.30 (m, 10H, aromatic), 6.00–5.80
(m, 2H, CH2vCH), 5.38–5.16 (m, 4H, CH2vCH), 5.20 (d,
J5,6a¼6.1 Hz, J6a,6b¼7.4 Hz, H-6a), 3.68 (m, 1H, H-60b),
0
0
0
0
0
3.68 (dd, 1H, J3 ,4 ¼3.2 Hz, J2 ,3 ¼9.9 Hz, H-3 ), 3.56 (dd,
1H, J5 ,6 a¼5.6 Hz, J6 a,6 b¼10.1 Hz, H-60a), 3.41 (t, 1H,
0
0
0
0
J5 ,6 a¼5.6 Hz, J5 ,6 b¼6.5 Hz, H-50), 3.27 (br s, 1H, H-2),
3.09 (br, 2H, 30-OH and 60-OH); 13C NMR (CDCl3) d 138.3,
138.0, 137.3, 128.7–127.5 (Bn), 103.1 (C-10), 100.5 (C-1),
79.0 (C-20), 77.4 (C-3), 76.1 (C-4), 75.0, 74.8, 74.6
(PhCH2£3), 74.4 (C-50), 74.1 (C-5,40), 72.3 (C-30), 64.9 (C-
6), 61.7 (C-2), 59.6 (C-60). Anal. calcd for C33H37N3O9: C,
63.96; H, 6.02; N, 6.78%. Found: C, 63.78; H, 6.20; N, 6.80%.
0
0
0
0
3.1.8. 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-b-D-
glucopyranosyl-(1!3)-[3,4,6-tri-O-acetyl-2-deoxy-2-
phthalimido-b-D-glucopyranosyl-(1!6)]-2,4-di-O-ben-
zyl-b-D-galactopyranosyl-(1!4)-1,6-anhydro-2-azido-3-
O-benzyl-2-deoxy-b-D-glucopyranose (12) and 3,4,6-tri-
O-acetyl-2-deoxy-2-phthalimido-b-D-glucopyranosyl-
(1!6)-2,4-di-O-benzyl-b-D-galactopyranosyl-(1!4)-1,6-
anhydro-2-azido-3-O-benzyl-2-deoxy-b-D-glucopyra-
nose (13). To a mixture of 1,6-anhydrolactose derivative 10
(160 mg, 0.259 mmol), glucosaminyl imidate 11 (360 mg,
1H, H-1), 4.97–4.51 (m, 4H, PhCH2), 4.38 (d, 1H,
0
0
CH2vCH–CH2), 3.91 (m, 2H, CH2vCH–CH2), 3.84 (d,
0
J1 ,2 ¼7.8 Hz, H-1 ), 4.34 (br t, 1H, H-5), 4.23 (m, 2H,
0
0
0
1H, J1 ,2 ¼7.7 Hz, H-2 ), 3.88–3.79 (m, 1H, H-3), 3.75 (d,
0
0
0
1H, J3 ,4 ¼3.3 Hz, H-4 ), 3.64–3.53 (m, 4H, H-6a, H-6b, H-
60a and H-60b), 3.43 (dd, 1H, J3 4 ¼2.9 Hz and J2 ,3 ¼9.8 Hz,
0
0
0
0
H-30), 3.36 (d, 1H, J3,4¼6.3 Hz, H-4), 3.26 (d, 1H,
J5 ,6 a¼6.3 Hz, H-50), 3.22 (d, 1H, J2,3¼8.6 Hz, H-2), 1.64
(br s, 1H, 3-OH). Anal. calcd for C32H39N3O9: C, 63.04; H,
6.45; N, 6.89%. Found: C, 63.08; H, 6.45; N, 6.74%.
0
0
˚
0.622 mmol) and MS4A (300 mg) in dry dichloromethane
(4.0 mL) was added trimethylsilyl trifluoromethane-
sulfonate (TMSOTf) (40 mL, 0.207 mmol) at 2158C
under nitrogen atmosphere. After stirring for 1 h at room
temperature, the mixture was neutralized with triethylamine
and concentrated. The resulting residue was purified by
chromatography on a silica gel first with 3:1 and then n-
hexane–ethyl acetate (1:1, v/v) to give tetrasaccharide 12
(260 mg, 69%) and trisaccharide 13 (70 mg, 26%).
3.1.6. 3,6-Di-O-allyl-2,4-di-O-benzyl-b-D-galactopyrano-
syl-(1!4)-1,6-anhydro-2-azido-3-O-benzyl-2-deoxy-b-
D-glucopyranose (9). To a solution of 8 (180 mg,
0.295 mmol) in DMF (10 mL) were added sodium hydride
(60% oil suspension; 24 mg, 0.590 mmol) and benzyl
bromide (0.11 mL, 0.885 mmol), and stirred for 14 h at
room temperature. The same manner as described for 7 was
carried out, and the obtained syrup was purified on silica gel
chromatography with n-hexane–ethyl acetate (5:1, v/v) as
an eluent to give 9 (170 mg, 82%): [a]2D5¼26.68 (c 0.290,
CHCl3); 1H NMR (CDCl3) d 7.38–7.18 (m, 15H, aromatic),
5.97–5.65 (m, 2H, CH2vCH), 5.42 (s, 1H, H-1), 5.30–5.02
1
Compound 12. [a]2D5¼þ13.08 (c 0.258, CHCl3); H NMR
(CDCl3) d 7.81–7.57 [4m, 8H, aromatic(Phth)], 7.31–7.20
[m, 15H, aromatic(Bn)], 5.70 (dd, 1H, J¼10.6, 9.1 Hz,
H-3c), 5.70 (d, 1H, J¼8.3 Hz, H-1c), 5.62 (dd, 1H, J¼10.6,
9.2 Hz, H-3c), 5.48 (d, 1H, J¼8.4 Hz, H-1c), 5.32 (s, 1H,
H-1a), 5.18 (t, 1H, J¼9.2, 9.5 Hz, H-4c), 5.11 (t, 1H, J¼9.4,
(m, 4H, CH2vCH), 4.95–4.50 (m, 6H, PhCH2), 4.55 (d,
0
0
0
1H, H-5), 4.38 (d, 1H, J1 ,2 ¼7.6 Hz, H-1 ), 4.14 (m, 2H,

T. Furuike et al. / Tetrahedron 59 (2003) 5105–5113
5111
9.6 Hz, H-4c), 4.85–4.30 (6d, 6H, PhCH2), 4.36 (d, 1H,
J¼7.3 Hz, H-1b), 4.35 (dd, 1H, J¼8.4, 10.9 Hz, H-2c), 4.22
(d, 1H, J¼8.6 Hz, H-2c), 3.96 (d, 1H, J¼7.4 Hz, H-2b), 3.73
(d, 1H, H-4a), 3.62 (s, 1H, H-3a), 3.36 (br t, 1H, H-5b), 3.15
(d, 1H, H-2a), 2.04–1.82 (all s, 18H, 6COCH3); 13C NMR
(CDCl3) d 170.6–169.5 (COCH3), 138.4, 138.3, 137.5,
(Bn), 134.4, 134.1, (Phth 4/5), 131.2, 131.1 (Phth 1/2),
128.4–127.3 (Bn), 123.6, 123.5 (Phth 3/6), 102.2 (C-1b),
100.7 (C-1a), 98.5, 97.5 (C-1c£2), 80.3 (C-3b), 78.1, 77.7
(C-2b, C-3a), 76.1 (C-4a), 74.7, 74.5, 74.2 (PhCH2£3, C-4b,
C-5a), 72.8 (C-5b), 71.8, 71.7 (C-5c£2), 70.8, 70.4
(C-4c£2), 68.8, 68.5, 68.0 (C-3c£2, C-6b), 65.5 (C-6a),
61.6 (C-2a, C-6c£2), 55.1, 54.4 (C-2c£2), 20.7–20.4
(COCH3). Anal. calcd for C73H75N5O27·H2O: C, 59.55; H,
5.27; N, 4.76%. Found: C, 59.64; H, 5.07; N, 4.78%.
(C-1c), 80.1, 79.2, 78.1, 76.2, 75.5, 74.6, 74.6, 74.2, 73.8,
72.7, 72.6, 71.9, 71.9, 71.7, 68.5, 68.4, 68.3, 65.4, 61.9,
61.7, 60.8, 55.1, 54.2, 22.8, 23.2 (NHCOCH3£2), 20.8–
20.6 (OCOCH3). Anal. calcd for C61H75N5O25·CH3OH: C,
56.84; H, 6.08; N, 5.34%. Found: C, 56.74; H, 6.04; N,
5.21%.
3.1.10. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-
glucopyranosyl-(1!3)-[2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-b-D-glucopyranosyl-(1!6)]-O-2,4-di-O-benzyl-
b-D-galactopyranosyl-(1!4)-2-acetamido-1,6-anhydro-
3-O-benzyl-2-deoxy-b-D-glucopyranose (15). To
a
solution of tetrasaccharide 14 (100 mg, 78.2 mmol) in
pyridine (1.5 mL) was added thioacetic acid (3.0 mL) and
the mixture was stirred for 24 h at room temperature. After
concentration, the residual oil was purified by silica gel
column chromatography with chloroform–methanol (40:1,
v/v) as an eluent to give 15 (97 mg, 96%): [a]2D5¼247.88 (c
Compound 13. [a]2D5¼þ14.08 (c 0.253, CHCl3); H NMR
1
(CDCl3) d 7.82–7.69 [2m, 4H, aromatic(Phth)], 7.36–7.32
[m, 15H, aromatic(Bn)], 5.70 (dd, 1H, J¼10.5, 9.1 Hz,
H-3c), 5.47 (d, 1H, J¼8.5 Hz, H-1c), 5.46 (s, 1H, H-1a),
5.12 (t, 1H, J¼9.1, 9.9 Hz, H-4c), 5.03–4.57 (6d, 6H,
PhCH2), 4.62 (m, 1H, H-5a), 4.39 (d, 1H, J¼7.4 Hz, H-1b),
4.27 (dd, 1H, J¼8.5, 10.4 Hz, H-2c), 4.22 (dd, 1H, J¼4.4,
12.5 Hz, H-6c), 4.08 (dd, 1H, J¼7.4 Hz, H-6a), 3.97 (dd,
1H, J¼12.5 Hz, H-6c), 3.86 (dd, 1H, J¼10.2 Hz, H-5c),
3.82 (s, 1H, H-3a), 3.79 (s, 1H, H-4a), 3.44 (dd, 1H, J¼7.5,
9.7 Hz, H-2b), 3.39 (m, 1H, H-3b), 3.24 (s, 1H, H-2a), 2.16
1
0.524, CHCl3); H NMR (CDCl3) d 7.30–7.40 [m, 15H,
aromatic(Bn)], 6.72 (d, 1H, NH), 6.05 (d, 1H, NH), 5.53 (t,
1H, J¼9.6, 10.0 Hz, H-3c), 5.34 (s, 1H, H-1a), 5.10 (dd, 1H,
J¼9.4, 9.8 Hz, H-3c), 5.01 (t, 1H, H-4c), 5.00 (t, 1H, H-4c),
4.98 (d, 1H, J¼7.7 Hz, H-1c), 4.97–4.41 (6d, 6H, PhCH2),
4.82 (d, 1H, J¼8.7 Hz, H-1c), 4.28 (d, 1H, J¼8.7 Hz, H-1b),
2.07–1.59 (all s, 27H, 9COCH3); 13C NMR (CDCl3) d
170.9, 170.9, 170.6, 170.6, 170.5, 170.2, 169.8, 169.5, 169.3
(COCH3£9), 138.8, 138.4, 137.9 (Bn£3), 128.8–127.7
(Bn), 103.6 (C-1b), 101.9 (C-1c), 100.6 (C-1a), 100.2
(C-1c), 81.0, 79.6, 78.1, 75.8, 75.7, 74.7, 74.6, 74.4, 73.7,
72.6, 72.3, 72.0, 71.9, 71.8, 68.8, 68.6, 68.4, 64.9, 61.9,
61.8, 55.9, 54.3, 48.2, 23.2, 22.9, 22.6 (NHCOCH3£3),
20.8–20.6 (OCOCH3). Anal. calcd for C63H79N3O26·2H2O:
C, 56.88; H, 6.28; N, 3.15%. Found: C, 56.91; H, 6.04; N,
3.18%.
(br s, 1H, 3b-OH), 2.00, 1.99, 1.84 (all s, 9H, 3COCH3); 13
C
NMR (CDCl3) d 170.5, 170.0, 169.3 (COCH3), 138.3, 138.2,
137.5 (Bn), 134.4, (Phth 4/5), 131.0 (Phth 1/2), 128.4–127.7
(Bn), 123.6 (Phth 3/6), 102.2 (C-1b), 100.6 (C-1a), 97.7
(C-1c), 79.0 (C-2b), 77.4 (C-3a), 76.1 (C-4a), 75.1, 74.9, 74.8
(PhCH2£3), 74.1, 73.8 (C-5a, C-5b, C-4b), 72.4 (C-3b), 71.9
(C-5c),70.7(C-4c), 68.7(C-3c), 67.6(C-6b), 65.2(C-6a), 61.7
(C-2a), 60.4 (C-6c), 54.5 (C-2c), 20.7–20.4 (COCH3). Anal.
calcd for C53H56N4O18·H2O: C,60.34; H, 5.54; N, 5.31%.
Found: C, 60.34; H, 5.39; N, 5.34%.
3.1.11. 2-Acetamido-2-deoxy-b-D-glucopyranosyl-
(1!3)-[2-acetamido-2-deoxy-b-D-glucopyranosyl-
(1!6)]-O-b-D-galactopyranosyl-(1!4)-2-acetamido-1,6-
anhydro-2-deoxy-b-D-glucopyranose (16). To a solution
of tetrasaccharide 15 (60 mg, 46.4 mmol) in 1:1 (v/v)
methanol–THF (2.0 mL) was added sodium methoxide
(1 mg). After being stirred for 2 h at room temperature, the
mixture was neutralized with the Dowex 50W-X8(Hþ)
resin, filtered, and concentrated. 5% Pd/C (30 mg) was
added to a solution of the residual syrup dissolved in
methanol (2.0 mL) and the reaction mixture was stirred for
48 h at room temperature under hydrogen atmosphere. After
filtration, the filtrate was evaporated to give 16 (29 mg,
81%): [a]2D5¼228.78 (c 0.483, H2O); 1H NMR (D2O) d 5.37
(s, 1H, H-1a), 4.61 (d, 1H, J¼8.7 Hz, H-1c), 4.46 (d, 2H,
J¼8.3 Hz, H-1b and H-1c), and 1.95, 1.95, 1.93 (3s, 9H,
3COCH3); 13C NMR (D2O) d 175.8, 175.3, 174.5
(COCH3£3), 103.6 (C-1c), 103.2 (C-1b), 102.1 (C-1c),
100.8 (C-1a), 82.6, 78.9, 76.6, 76.4, 75.0, 74.6, 74.3, 74.2,
70.7, 70.6, 70.5, 70.1, 69.7, 69.2, 65.9, 61.5, 61.3, 56.5,
56.3, 52.7, 23.1, 23.1, 22.7 (NHCOCH3£3); MALDI-TOF
MS calcd for C30H49N3O20: 771.7. Found: m/z 793.7
[MþNa]þ. Anal. calcd for C30H49N3O20·3H2O: C, 43.63;
H, 6.71; N, 5.09%. Found: C, 43.27; H, 6.38; N, 4.87%.
3.1.9. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-D-
glucopyranosyl-(1!3)-[(2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-b-D-glucopyranosyl-(1!6)]-2,4-di-O-benzyl-b-
D-galactopyranosyl-(1!4)-1,6-anhydro-2-azido-3-O-
benzyl-2-deoxy-b-D-glucopyranose (14). To a solution of
tetrasaccharide 12 (150 mg, 0.103 mmol) in ethanol
(5.0 mL) was added hydrazine monohydrate (0.50 mL).
After refluxing for 5 h, the mixture was evaporated in vacuo,
and the residual syrup was dissolved in pyridine (10 mL).
DMAP (13 mg) and acetic anhydride (10 mL) were added to
the solution at 08C and the mixture was stirred for 14 h at
room temperature. After evaporation, the residue was
purified by silica gel column chromatography with chloro-
form–methanol (40:1, v/v) as an eluent to give 14 (102 mg,
77%): [a]2D5¼217.78 (c 0.579, CHCl3); 1H NMR (CDCl3) d
7.45–7.22 [m, 15H, aromatic(Bn)], 5.47 (t, 1H, H-3c), 5.44
(s, 1H, H-1a), 5.25 (dd, 1H, J¼9.5, 10.4 Hz, H-3c), 5.13 (d,
1H, NH), 5.09 (t, 1H, J¼8.3, 9.7 Hz, H-4c), 5.00 (d, 1H,
NH), 4.96 (t, 1H, J¼9.5, 10.7 Hz, H-4c), 4.92–4.56 (6d, 6H,
PhCH2), 4.67 (d, 1H, J¼8.2 Hz, H-1c), 4.62 (d, 1H,
J¼7.2 Hz, H-1c), 4.49 (d, 1H, J¼7.6 Hz, H-1b), 3.27 (br,
1H, H-2a), 2.08–1.62 (all s, 24H, 8COCH3); 13C NMR
(CDCl3) d 170.8, 170.7, 170.6, 170.6, 170.2, 169.8, 169.4,
169.3, (COCH3£8), 139.0, 138.6, 137.7 (Bn£3), 128.8–
126.8 (Bn), 102.2 (C-1b), 101.5 (C-1c), 100.7 (C-1a), 100.3
3.1.12. b-D-Galactopyranosyl-(1!4)-2-acetamido-2-
deoxy-b-D-glucopyranosyl-(1!3)-[b-D-galactopyrano-
syl-(1!4)-2-acetamido-2-deoxy-b-D-glucopyranosyl-

5112
T. Furuike et al. / Tetrahedron 59 (2003) 5105–5113
2. Fukuda, M. N.; Dell, A.; Oates, J. E.; Fukuda, M. J. Biol.
Chem. 1985, 260, 6623–6631.
(1!6)]-b-D-galactopyranosyl-(1!4)-2-acetamido-1,6-
anhydro-2-deoxy-b-D-glucopyranose (17). Tetrasaccharide
16 (10.5 mg, 13.6 mmol), UDP-galactose (25 mg, 41 mmol),
a-lactalbumin (241.2 mg), MnCl2·4H2O (990 mg) and
b-1,4-galactosyltransferase (3 U) in a HEPES buffer
(50 mM, pH 6.0, 0.50 mL) were incubated for 48 h at
378C. The mixture was purified by a column of Sephadex
G-15 with water as an eluent and afford hexasaccharide 17
(13 mg, 87%); [a]D25¼221.28 (c 0.156, H2O); 1H NMR
(D2O, 508C) d 5.48 (s, 1H, H-1a), 4.71 (d, 1H, J¼8.0 Hz,
H-1c), 4.56 (d, 1H, J¼7.8 Hz, H-1b), 4.54 (d, 1H, J¼7.8 Hz,
H-1c), 4.49 (d, 1H, J¼7.5 Hz, H-1d), 4.47 (d, 1H, J¼7.6 Hz,
H-1d), 2.04, 2.04, 2.03 (3s, 9H, 3COCH3); 13C NMR (D2O,
508C) d177.7, 177.2, 176.4 (COCH3£3), 105.7 (C-1d),
105.7 (C-1d), 105.4 (C-1c), 105.3 (C-1c), 104.0 (C-1b),
102.9 (C-1a), 84.7, 81.5, 81.3, 81.2, 78.1, 78.1, 77.6, 77.4,
77.1, 76.3, 75.4, 75.4, 75.2, 75.0, 73.8, 73.8, 72.6, 72.2,
71.6, 71.4, 71.4, 71.2, 67.9, 63.8, 63.8, 63.0, 62.9, 58.1,
57.9, 54.9, 25.0, 25.0, 24.7 (NHCOCH3£3); MALDI-TOF
MS calcd for C42H69N3O30 [MþNa]þ: 1096.0.
Found: 1118.6. Anal. calcd for C42H69N3O30·3H2O: C,
43.86; H, 6.57; N, 3.65%. Found: C, 43.67; H, 6.38; N,
3.92%.
3. Carlsson, S. R.; Roth, J.; Piller, F.; Fukuda, M. J. Biol. Chem.
1988, 263, 18911–18919.
4. Muramatsu, H.; Muramatsu, T.; Avner, P. Cancer Res. 1982,
42, 1749–1752.
5. Feizi, T. Nature 1985, 314, 53–57.
6. Phillips, M. L.; Nudelman, E.; Gaeta, F. C.; Perez, M.;
Singhal, A. K.; Hakomori, S.; Paulson, J. C. Science 1990,
250, 1130–1132.
7. Muramatsu, T.; Gachelin, G.; Dammonneville, M.; Delarbre,
C.; Jacob, F. Cell 1979, 18, 183–191.
8. Fukuda, M.; Koeffler, H. P.; Minowada, J. Proc. Natl Acad.
Sci. USA 1981, 78, 6299–6303.
9. Muramatsu, H.; Ishihara, H.; Miyauchi, T.; Gachelin, G.;
Fjisaki, T.; Tejima, S.; Muramatsu, T. J. Biochem. 1983, 94,
799–810.
10. Fukuda, M.; Dell, A.; Fukuda, M. N. J. Biol. Chem. 1984, 259,
4782–4791.
11. Ozawa, M.; Muramatsu, T.; Solter, D. Cell Differ. 1985, 16,
169–173.
12. Ozawa, M.; Muramatsu, T.; Kemler, R. J. Biochem. 1985, 97,
307–315.
13. Crandall, J. E.; Dibble, C.; Butler, D.; Pays, L.; Ahmad, N.;
Kostek, C.; Puschel, A. W.; Schwarting, G. A. J. Neurobiol.
2000, 45, 195–206.
3.1.13. 5-Acetamido-3,5-dideoxy-D-glycero-a-D-galacto-
non-2-ulopyranosylonic acid-(2!3)-b-D-galactopyrano-
syl-(1!4)-2-acetamido-2-deoxy-b-D-glucopyranosyl-
(1!3)-[5-acetamido-3,5-dideoxy-D-glycero-a-D-galacto-
non-2-ulopyranosylonic acid-(2!3)-b-D-galactopyrano-
syl-(1!4)-2-acetamido-2-deoxy-b-D-glucopyranosyl-
(1!6)]-b-D-galactopyranosyl-(1!4)-2-acetamido-1,6-
anhydro-2-deoxy-b-D-glucopyranose (1). Hexasaccharide
17 (8.0 mg, 7.30 mmol), CMP-NANA (13.0 mg, 14.6
mmol), BSA (400 mg), MnCl2·4H2O (400 mg) and a-2,3-
sialyltransferase (50 mU) in a sodium cacodylate buffer
(50 mM, pH 7.3, 0.40 mL) were incubated for 48 h at 378C.
The mixture was purified by DEAE-Sephacel chromato-
graphy using 0.01–0.07 M NH4HCO3 solution as eluent.
Ion-exchange chromatography (Dowex50W-X8(Naþ)) and
subsequent desalting on Sephadex G-25 column with
deionized water as an eluent afforded the desired octa-
saccharide 1 (10.5 mg, 84%): [a]2D5¼222.48 (c 0.324, H2O);
1H NMR (D2O) d 5.47 (s, 1H, H-1a), 4.70 (d, 1H, J¼8.3 Hz,
H-1c), 4.58–4.51 (m, 4H, H-1b, H-1c, H-1d, H-1d), 2.75
(dd, 1H, J¼4.1, 12.1 Hz, H-3eq(e)), 2.03 (brs, 15H,
5COCH3), 1.80 (t, 1H, H-3ax(e)); 13C NMR (D2O, 408C)
d 177.8, 177.8, 177.6, 177.2, 176.5, 176.5, 176.4 (CvO£7),
105.4, 105.4, 105.4, 105.2, 104.0, 102.8, 102.6, 102.6, 84.7,
81.2, 81.0, 80.9, 78.3, 78.3, 77.9, 77.9, 77.5, 77.4, 77.0,
76.3, 75.7, 75.7, 75.1, 74.9, 74.5, 74.5, 72.5, 72.1, 72.1,
72.1, 71.8, 71.2, 71.0, 71.0, 70.9, 70.9, 70.3, 70.3, 67.8,
65.4, 65.4, 63.7, 63.7, 62.9, 62.7, 58.0, 57.8, 54.7, 54.5,
54.5, 42.4, 42.4, 25.0, 25.0, 24.8, 24.8, 24.7
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(NHCOCH3£5);
MALDI-TOF
MS
calcd
for
C64H103N5O46 [MþNa]þ, 1700.58 Found: 1700.8; ESI-
HRMS calcd for C64H101N5O46Na [M2Na]2,1698.5616
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