Synthesis of Branched Tetrasaccharide Derivatives of Schizophyllan-like β-Glucan

Article abstract of DOI:10.1080/07328303.2015.1044754

Branched tetrasaccharide derivatives as the repeating units of schizophyllan were synthesized using a common intermediate that had different protecting groups on the hydroxyl groups. Accordingly, β-1,3-disaccharide acceptors were efficiently glycosylated

Full text of DOI:10.1080/07328303.2015.1044754

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Synthesis of Branched Tetrasaccharide
Derivatives of Schizophyllan-like β-
Glucan
Atsushi Miyagawaa, Tomoka Matsudaa & Hatsuo Yamamuraa
a Department of Life and Materials Engineering, Faculty of
Engineering, Nagoya Institute of Technology,, Nagoya, Aichi,
466-8555, Japan
Published online: 01 Jul 2015.
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To cite this article: Atsushi Miyagawa, Tomoka Matsuda & Hatsuo Yamamura (2015): Synthesis of
Branched Tetrasaccharide Derivatives of Schizophyllan-like β-Glucan, Journal of Carbohydrate
Chemistry, DOI: 10.1080/07328303.2015.1044754
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Journal of Carbohydrate Chemistry, 00:1–33, 2015
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Copyright Taylor & Francis Group, LLC
ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2015.1044754
Synthesis of Branched
Tetrasaccharide Derivatives of
Schizophyllan-like β-Glucan
Atsushi Miyagawa, Tomoka Matsuda, and Hatsuo Yamamura
Department of Life and Materials Engineering, Faculty of Engineering, Nagoya
Institute of Technology, Gokiso-cho, Showa-ku, Nagoya, Aichi, 466-8555, Japan
GRAPHICAL ABSTRACT
Branched tetrasaccharide derivatives as the repeating units of schizophyllan were syn-
thesized using a common intermediate that had different protecting groups on the hy-
droxyl groups. Accordingly, β-1,3-disaccharide acceptors were efficiently glycosylated in
a stepwise manner using monosaccharide donors to construct the branched tetrasac-
charides. As a result, oligosaccharides with an internal branch rather than a branch on
the terminal residue suitable for the synthesis of branched β-glucan were obtained.
Keywords β-Glucan; Glycosylation; Aglycon transfer; Schizophyllan
INTRODUCTION
Polysaccharides are very important biomolecules. They have unique biological
functions, such as serving as structural components of cell walls and function-
ing in energy storage, cell recognition, regulation of signaling, and immune
Received February 23, 2015; accepted April 21, 2015.
Address correspondence to Atsushi Miyagawa, Department of Life and Materials Engi-
neering, Faculty of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku,
Nagoya, Aichi 466-8555, Japan. E-mail: miyagawa.atsushi@nitech.ac.jp
1

A. Miyagawa et al.
2
responses.[1–3] Consequently, polysaccharides have attracted considerable at-
tention in the study of the mechanisms of biological events.[4,5] The structures
of polysaccharides are highly diverse. It is difficult to obtain polysaccharides by
purification from natural sources containing heterogeneous compounds, such
as proteins and lipids, and structurally homogenous polysaccharides cannot
be isolated from the polysaccharides of various structural isomers.[6–8] There-
fore, it is difficult to evaluate the biological activity of polysaccharides quan-
titatively, and a method for the preparation of pure and structurally defined
polysaccharides is an important undertaking.
Recently, Brown et al.[9] identified dectin-1 as a β-1,3-glucan receptor.
Dectin-1 is expressed predominantly on myeloid tissue cells.[10] Dectin-1 me-
diates cellular responses to β-glucans as immune cell activators and induces
the production of cytokines and chemokines.[11,12] Consequently, the use of
β-glucan to elucidate this immune mechanism has been intensively investi-
gated. Palma et al.[13] reported that dectin-1 did not recognize mammalian
oligosaccharides linked on microarray, and when using oligosaccharides frac-
tionated from curdlan hydrolysate, the minimum length required for de-
tectable binding by dectin-1 was an 11-mer. Tanaka et al.[8] evaluated the
binding activity of synthetic β-1,3-glucan 10−17-mer for dectin-1 and found
that synthetic glucan ligands promoted the activation of NF-κB by binding to
dectin-1.
When higher plants are attacked by pathogens, they initiate immune re-
sponses against penetration and infection.[14] The interaction and signaling
between plants and pathogens have been investigated to identify binding
molecules and receptors. Elicitor molecules induce synthesis and accumulation
of antimicrobial compounds in plant cells.[15,16] It has been found that β-glucan
fragments, characterized as an elicitor, from the cell wall of fungi and plants
have the ability to elicit biosynthesis of phytoalexins, which act as natural an-
tibiotics in plants.[17,18] The structure of the fragment with high elicitor activity
was elucidated, and it was determined that the smallest active structure was a
branched hepta-β-glucan. This result was achieved by analysis of phytoalexin
accumulation activity using synthetic β-glucans.[19] However, the biological ac-
tivities of β-glucans are not completely clear, and interesting challenges remain
in the synthesis of branched β-glucans.
In 1982, Ogawa and Kaburagi[20] reported the first synthesis of tetra-β-
glucan having β-1,6-branched glucose on the reducing end of the main chain.
Takeo and Saimei[21] synthesized three types of repeating units of schizophyl-
lan. In these reports, the syntheses of β-glucans were achieved in high yields
and excellent stereoselectivity using glucosyl bromides as donors. However, the
strong acidic and oxidative conditions involved in the preparation of glucosyl
bromides were not suitable for continuous glycosylations, which need selective
deprotection and activation. Some studies utilized thioglucosides and glucosyl

Synthesis of Branched Oligo-β-Glucan Derivatives
3
imidates as donors and found that these glycosylations formed β-1,3-linkages
with anomalous stereoselectivity despite having an acyl group that assists β-
selectivity by neighboring group participation at the 2-position on the gluco-
syl donor.[22–25] Therefore, a novel protecting group for β-1,3-glycosylation was
developed by adding a steric hindering group, which provided β-selective gly-
cosylation with thioglucosyl and glucosyl imidate donors.[25–27] However, NMR
study showed that the synthesized intermediates of β-glucans, which were pro-
tected by the novel protecting group, showed a small coupling constant for J1,2
and the peak of the C-1 appeared in a higher magnetic field like α-anomer.[26]
Consequently, it was not easy to identify the anomeric configuration of the
synthetic compounds, and deprotection was necessary to confirm the structure,
indicating that a more practical method for the synthesis of β-glucans should
be developed. Recently, Tanaka et al.[8,28] achieved the synthesis of hexadeca-β-
glucan using a thioglucosyl donor and glucosyl acceptor protected with a ben-
zylidene group. On the glucosyl acceptor, the benzylidene group is effective
for less steric hindrance and the 2-free hydroxyl group enhances the reactiv-
ity of the 3-hydroxyl group for the glucosyl donor. However, it is difficult to
synthesize branched β-glucan and to completely avoid 2-O-glycosylation using
this method because the benzylidene group only protected the 4,6-O-positions.
Branches are known to be important for β-glucans in terms of solubility, he-
lix conformation, and biological activity and consequently have attracted the
attention of researchers in both biology and medicine.[29–32] Therefore, a more
reliable method to synthesize branched β-glucans is desirable. Herein, the syn-
thesis of repeating units of schizophyllan with branches is reported. The re-
sults suggest a strategy for the introduction of branches in the construction of
β-glucans.
RESULTS AND DISCUSSION
Synthesis of tetrasaccharide derivatives 1–3 (Fig. 1) of the repeating unit of
schizophyllan was attempted by glycosylation using thioglucosides as glucosyl
acceptors and glucosyl imidates as glucosyl donors. The acceptors and donors
were derived from a common intermediate 12 that had tert-butyldimethylsilyl
(TBDMS) and a chloroacetyl group (ClAc) at the 3- and 6-O-positions. An acetyl
group with less steric hindrance at the 2-O-position was selected for β-selective
glycosylation utilizing neighboring group participation. The intermediate was
converted to glucosyl donors and glucosyl acceptors that were then utilized to
prepare disaccharides as main chain units and branch units. Repeating units
of schizophyllan were synthesized using these disaccharides to investigate a
suitable route for the synthesis of β-glucans.

A. Miyagawa et al.
4
Figure 1: Branched tetrasaccharide derivatives 1–3 as repeating units of schizophyllan.
Methyl thioglucoside 4 was used as a starting material (Sch. 1). A benzyli-
dene group was introduced to protect the 4,6-O-positions, followed by treat-
ment with TBDMS chloride to protect the 3-O-position and then protection of
the 2-O-position with the acetyl group to afford 5 in a 90% yield. Finally, the
[33]
benzylidene group in 5 was selectively cleaved by CoCl2 and BH3
to give 6
in a 98% yield, which was used as the glucosyl acceptor for the introduction of
a branch.
Scheme 1: Synthesis of thioglucoside acceptor 6. Reagents and conditions: (a) i.
C6H5CH(OCH3)2, CSA, DMF, 60◦C, 2.5 h, under reduced pressure; ii. TBDMSCl, imidazole, DMF,
0◦C, 14.5 h, 90% (2 steps); iii. Ac2O, pyridine, rt, 22 h, quant. (b) BH3-THF, CoCl2, rt, 22.5 h,
quant.
Initially, we planned to directly use thioglycoside 6 as a glycosyl donor.
However, when it reacted with glucosyl imidate 7, instead of getting the de-
sired disaccharide 8, an aglycon transfer reaction[34–38] occurred to give only
methyl thioglucoside 9 (Fig. 2).
Therefore, in the synthesis, the aglycon portion of the glucosyl acceptor
6 was changed to a p-methoxyphenyl (MP) group (Sch. 2).[34] The 6-hydroxyl
group of 6 was chloroacetylated first, and then the methyl thioglucoside 10 was
hydrolyzed with N-bromosuccinimide[38] and converted to glucosyl imidate 11
efficiently. The imidate was glycosidated with p-methoxyphenol to afford 12
in a 75% yield. The common intermediate 12 was treated with DABCO[39] to
give glucosyl acceptor 13, which had a free hydroxyl group at the 6-position.

Synthesis of Branched Oligo-β-Glucan Derivatives
5
Figure 2: Aglycon transfer reaction of methyl thioglucoside 6.
Scheme 2: Synthesis of glucosyl donor 11 and acceptors 13 and 14. Reagents and
conditions: (a) ClCH2COCl, pyridine, 0◦C, 12 h, quant. (b) i. NBS, acetone, H2O, rt, 10 min,
97%; ii. Cl3CCN, DBU, CH2Cl2, rt, 12 h, 70%. (c) p-methoxyphenol, MeOTf, CH2Cl2, MS4Å,
−20◦C, 1 h, 0◦C, 3.5 h, 75%. (d) DABCO, EtOH, 50◦C, 30 min, 89%. (e) i. Ac2O, pyridine, rt,
16.5 h, 96%; ii. BF3·Et2O, CH3CN, 0◦C, 15 min, 71%.
Subsequently, acetylation of 13 and subsequent removal of the TBDMS group
using BF3-Et2O[40] provided glucosyl acceptor 14 with a free 3-hydroxyl
group.
Acceptors 13 and 14 were utilized to construct the β-1,6-linked branch
units and the β-1,3-linked main chain units, respectively (Sch. 3). The gly-
cosylation of glucosyl acceptor 13 with glucosyl imidate 7 in the presence of
BF3-Et2O as a catalytic promoter proceeded smoothly to afford only β-1,6-
disaccharide 15 in an 87% yield. Glycosylation of 14 with 11 in an accep-
tor/donor ratio of 2/1 formed β-1,3-disaccharide 16 in a 68% yield with 65%
recovery of the acceptor 14.
Disaccharides 15 and 16 were converted to acceptors and donors (Sch. 4),
respectively, to construct the repeating units of schizophyllan. Disaccharide 15
was treated with BF3-Et2O and was subsequently acetylated to give disaccha-
ride 17 in an 80% yield. The MP group in 15 and 17 was selectively removed

A. Miyagawa et al.
6
Scheme 3: Synthesis of disaccharides 15 and 16. Reagents and conditions: (a) BF3-Et2O,
CH2Cl2, MS4Å, −40◦C, 2 h, 87%. (b) BF3-Et2O, CH2Cl2, MS4Å, −40◦C, 4.5 h, 68%.
Scheme 4: Synthesis of disaccharide donors 18, 19, 22, and 23 and acceptors 20 and 24.
Reagents and conditions: (a) i. BF3-Et2O, CH3CN, −20◦C, 13 min, quant.; ii. Ac2O, pyridine, rt,
16 h, 89%. (b) i. CAN, CH3CN, H2O, 0◦C, 7 min, 92%; ii. Cl3CCN, DBU, CH2Cl2, rt, 31 h, 75% (α
only) for 18; i. CAN, CH3CN, H2O, 0◦C, 17 min, 94%; ii. Cl3CCN, DBU, CH2Cl2, rt, 8.5 h, 94% (α:β
= 10:1) for 19. (c) BF3-Et2O, CH3CN, −20◦C, 17 min, 98%. (d) i. BF3-Et2O, CH3CN, 0◦C, 30 min,
quant.; ii. Ac2O, pyridine, rt, 17 h, 89%. (e) i. CAN, CH3CN, H2O, 0◦C, 20 min, 98%; ii. Cl3CCN,
DBU, CH2Cl2, rt, 25 h, 74% (α only) for 22; i. CAN, CH3CN, H2O, 0◦C, 15 min, 85%; ii. Cl3CCN,
DBU, CH2Cl2, rt, 12.5 h, 84% (α:β = 25:1) for 23. (f) BF3-Et2O, CH3CN, 0◦C, 30 min, 91%.

Synthesis of Branched Oligo-β-Glucan Derivatives
7
by oxidation and then treated with trichloroacetonitrile and DBU to afford dis-
accharide donors 18 and 19 in a 68% and 89% yield, respectively. On the other
hand, 15 was treated with BF3-Et2O to give disaccharide acceptor 20 in a 91%
yield. By similar procedures, disaccharide donors 22 and 23 and disaccharide
acceptor 24 for the branch units were prepared efficiently from disaccharide
16.
Next, an attempt was made to prepare tetrasaccharides with a branch at
terminal positions (Sch. 5) by combining the donors and the acceptors obtained
above. First, acceptor 24 was glycosylated with donor 18 protected with the
TBDMS group to provide tetrasaccharide 25 in a 26% yield. Similarly, the gly-
cosylation of acceptor 20 with donor 22 having the TBDMS protection gave
tetrasaccharide 27 in a 27% yield. Glycosylation of acceptor 24 with donor 19
having an acetyl protection at the 3-O-position afforded a moderate yield (56%)
of tetrasaccharide 26. Glycosylation of acceptor 20 with donor 23 gave tetrasac-
charide 28 in a 15% yield only. These results showed that the glycosylation
of the glucose 3-OH using β-1,6-linked disaccharide donors was hampered by
the steric hindrance at the 6-O-position. Therefore, tetrasaccharides having a
branch at the reducing or nonreducing end were not suitable for use as the
repeating unit toward the construction of β-glucans.
Scheme 5: Synthesis of branched tetrasaccharides 25–28 at the terminal position. Reagents
and conditions: (a) BF3-Et2O, CH2Cl2, MS4Å, −40◦C, 2 h, −20◦C, 2 h, then TMSOTf, 0◦C, 3 h,
26% for 25; BF3-Et2O, CH2Cl2, MS4Å, −40◦C, 1 h, 0◦C, 1.5 h, rt, 14 h, 56% for 26. (b) TMSOTf,
CH2Cl2, MS4Å, −40◦C, 0.5 h, rt, 5.5 h, 27% for 27; BF3-Et2O, CH2Cl2, MS4Å, −40◦C, 1 h, rt,
45 min, 15% for 28.
Therefore, disaccharide acceptor 24 was sequentially glycosylated to intro-
duce a main chain sugar unit and a branch to form tetrasaccharide repeating

A. Miyagawa et al.
8
Scheme 6: Synthesis of branched tetrasaccharides 34 and 35 at the central position.
Reagents and conditions: (a) TMSOTf, CH2Cl2, MS4Å, −40◦C, 50 min, 87% for 30; BF3·Et2O,
CH2Cl2, MS4Å, −40◦C, 2 h, rt, 15 h, then BF3·Et2O, rt, 1 h, 60% for 31. (b) DABCO, EtOH, 50◦C,
7.5 h, quant. for 32; DABCO, EtOH, 50◦C, 13 h, quant. for 33. (c) 7, TMSOTf, CH2Cl2, MS4Å,
−40◦C, 3 h, 75% for 34; 7, TMSOTf, CH2Cl2, MS4Å, −40◦C, 35 min, 83% for 35.
units with a branch at the central sugar unit (Sch. 6). First, we attempted
a procedure to remove the 6-O-ClAc group in 24 and glycosylate both the 3-
and 6-O-positions with imidate 7 in one step to obtain tetrasaccharide 35.
However, only a trisaccharide was given, owing to the steric hindrance re-
sulting from 6-O-glycosylation that proceeded first. Therefore, the tetrasac-
charides were constructed by introducing two glucose units stepwise. Initially,
24 was glycosylated with glucosyl donor 29, which was prepared readily from
6 by a procedure similar to that used to prepare 11 or with imidate 7 to af-
ford the β-linked trisaccharide 30 or 31 in a 87% or 56% yield, respectively.
The result that 29 gave better yields than 11 showed that the TBDMS and
benzyl groups enhanced the reactivity of the glucosyl donor in glycosylation.
Then, trisaccharides 30 and 31 were treated with DABCO to remove the ClAc
group, and glycosylation of the resulting trisaccharide acceptors 32 and 33
with 7 provided tetrasaccharides 34 and 35 efficiently in 75% and 83% yields,
respectively.
CONCLUSION
Branched tetrasaccharide derivatives as the repeating units of schizophyl-
lan were synthesized, and in the process, the influence of a branch on β-1,3-
glycosylation was investigated. The tetrasaccharide derivatives were synthe-
sized using 12 as the common intermediate, and the intermediate was effi-
ciently converted to glucosyl acceptors and donors by regioselective modifica-
tions of the hydroxyl groups. It is noteworthy that glucosyl donors with an

Synthesis of Branched Oligo-β-Glucan Derivatives
9
acetyl group at the 2-O-position provided exclusively β-glycosylation without
orthoester formation. In the construction of tetrasaccharide derivatives, the
benzyl group at the 4-O-position of disaccharide acceptors sterically hindered
β-1,3-glycosylation. Furthermore, glycosylation of β-1,6-linked disaccharide ac-
ceptor 20 with donors 22 and 23 did not provide 3-O-glycosylation owing to
the steric hindrance of the branch. Thus, unlike previous reports using glu-
cosyl bromide,[20,21] the disaccharide imidates 22 and 23 cannot be used to
achieve double glycosylation in a one-pot manner. On the other hand, step-
wise glycosylation of 1,3-disacchride acceptor 24 with monosaccharide donors
proceeded efficiently to afford tetrasaccharides 34 and 35 with a branch at
the central sugar unit. The results suggest that the branch should be intro-
duced as late as possible to avoid steric hindrance, and the repeating unit
with a branch at the central position rather than at the terminal position
is more suitable for continuous glycosylation, which may enable the con-
struction of schizophyllan-like β-glucans to develop artificial immunological
agents.
EXPERIMENTAL SECTION
General Methods
1H NMR spectra were recorded at 400, 500, or 600 MHz using a Bruker
AVANCE 400 Plus Nanobay, AVANCE 500US with a cryoprobe, or AVANCE
600 spectrometer in chloroform-d. 13C NMR spectra were recorded at 101, 126,
or 151 MHz with the same instruments. Chemical shifts are given in ppm (δ)
and referenced to tetramethylsilane or to the internal solvent signal used as an
internal standard. Assignments in the NMR spectra were made by first-order
analysis of spectra and supported by correlation spectroscopy and heteronu-
clear chemical shift correlation. Matrix-assisted laser desorption ionization
time-of-flight high-resolution mass spectrometry (MALDI-TOF HRMS) spec-
tra were recorded on a Jeol JMS-S3000 using 2,5-dihydroxylbenzoic acid as
matrix.
Unless otherwise stated, all commercially available solvents and reagents
were used without further purification. Dry solvents were prepared by storage
over molecular sieves, which were activated in vacuum at 200◦C. Reactions
were monitored by thin-layer chromatography (TLC) on a precoated plate of
silica gel 60 F254 (layer thickness, 0.25 mm; E. Merk, Darmstadt, Germany).
For detection of intermediates, TLC sheets were dipped in a solution of 85:10:5
(v/v/v) methanol–resorcinol–concentrated sulfuric acid and heated for a few
minutes or irradiated with UV lamp.

A. Miyagawa et al.
10
Methyl 2-O-acetyl-4,6-O-benzylidene-3-O-tert-butyldimethylsi-
lyl-β-D- thioglucopyranoside (5)
To a solution of 4 (500 mg, 951 μmol) in DMF (2.4 mL) was added benzalde-
hyde dimethylacetal (213 μL, 1.43 mmol) and para-toluenesulfonic acid mono-
hydrate (16.4 mg, 95.1 μmol), and the mixture was stirred under reduced pres-
sure at 60◦C for 2.5 h. The solution was cooled on an ice bath and triethylamine
(26 μL, 190 μmol) added to neutralize. The solution was then evaporated un-
der vacuum to give the mixture. The mixture was dissolved in DMF (951 μL)
and cooled at 0◦C. To the solution were added imidazole (97.2 mg, 1.43 mmol)
and tert-butyldimethylsilyl chloride (172 mg, 1.14 mmol) and the solution was
stirred for 14.5 h. Then, the mixture was diluted with dichloromethane and
washed with aqueous sodium hydrogen carbonate and brine, dried over an-
hydrous sodium sulfate, filtered, and evaporated. The residue was purified by
flash silica gel chromatography with 25:1 (v/v) hexane–ethyl acetate to give
3-O-silylate (354 mg, 90%). The silylate (220 mg, 533 μmol) was subsequently
stirred with pyridine (1.2 mL) and acetic anhydride (1.0 mL) at rt for 22 h
and the mixture was coevaporated with toluene to give 5 (242 mg, quantita-
1
tive) as a white powder: H NMR δ (CDCl3, 400 MHz) 7.49–7.47 (m, 2 H, Ph),
7.36–7.35 (m, 3 H, Ph), 5.52 (s, 1 H, Ph-CH-O2-), 5.02 (dd, 1 H, J2,3 = 8.4 Hz,
H-2), 4.38–4.34 (m, 1 H, H-6a), 4.36 (d, 1 H, J1, 2 = 9.6 Hz, H-1), 3.89 (t, 1 H,
H-3), 3.75 (br-t, 1 H, H-6b), 3.56–3.47 (m, 2 H, H-4, 5), 2.16 (s, 3 H, S-CH3),
2.11 (s, 3 H, Ac), 0.82 (s, 9 H, t-Bu), 0.03 (s, 3 H, Si-CH3), 0.00 (s, 3 H, Si-
CH3); 13C NMR (CDCl3,101 MHz): 169.6, 137.0, 129.1, 128.2, 126.2, 101.8 (C-
1), 83.5 (C-4), 81.4 (C-3), 74.0 (C-2), 72.0 (C-5), 68.6 (C-6), 25.6, 21.2, 18.0, 11.2,
−4.2, −5.0; MALDI-TOF HRMS (positive ion): calcd for (C22H34O6SSi+Na+):
477.1743; found m/z: 477.1708.
Methyl 2-O-acetyl-4-O-benzyl-3-O-tert-butyldimethylsilyl-β-D-thi-
oglucopyranoside (6)
To the compound 5 were added 1M borane-tetrahydrofuran complex solu-
tion (22.3 mL, 22.3 mmol) and cobalt (II) chloride (2.90 g, 22.3 mmol) on an ice
bath and the mixture was stirred at rt for 22.5 h. The suspension was filtered
and diluted with dichloromethane and washed with water and brine. The so-
lution was dried over anhydrous sodium sulfate, filtered, and evaporated. The
residue was purified by silica gel chromatography with 10:1 (v/v) hexane–ethyl
acetate to give 6 (3.34 g, 99%) as a white powder: 1H NMR δ (CDCl3, 400 MHz)
7.34–7.26 (m, 5 H, Ph), 5.02 (t, 1 H, J2, 3 = 9.4 Hz, H-2), 4.85 (d, 1 H, J =
11.4 Hz, Ph-CH-), 4.63 (d, 1 H, J = 11.6 Hz, Ph-CH-), 4.27 (d, 1 H, J1, 2
=
10.0 Hz, H-1), 3.87–3.79 (m, 2 H, H-3, 6a), 3.67–3.61 (m, 1 H, H-6b), 3.48 (t, 1
H, J3,4 = 9.2 Hz, H-4), 3.41–3.39 (m, 1 H, H-5), 2.14 (s, 3 H, S-CH3), 2.11 (s,
3 H, Ac), 0.89 (s, 9 H, t-Bu), 0.07 (s, 6 H, Si-CH3); 13C NMR (CDCl3,101 MHz):

Synthesis of Branched Oligo-β-Glucan Derivatives
11
169.7, 137.8, 128.4, 127.7, 127.6, 83.1 (C-1), 79.7(C-5), 78.3(C-4), 75.1 (C-
3), 71.8 (C-2), 61.9 (C-6), 25.7, 21.4, 17.8, 11.4, −4.1, −4.4; MALDI-TOF
HRMS (positive ion): calcd for (C22H36O6SSi+Na+) 479.1900; found m/z: 479.
1921.
Methyl 2,3,4,6-tetra-O-acetyl-β-D-thioglucopyranoside (9) from
aglycon transfer reaction
To a solution of 6 (100 mg, 219 μmol) and 7 (162 mg, 329 μmol) in
˚
dichloromethane (2.0 mL) was added molecular sieves 4A powder (200 mg),
and the mixture was stirred at rt for 1 h and then cooled at –40◦C.
To the mixture was added trimethylsilyl trifluoromethansulfonate (6.0 mL,
21.9 mmol) and it was stirred at –40◦C for 4.0 h. The solution was diluted
with dichloromethane and filtered through Celite, and the filtrate was washed
successively with aqueous sodium hydrogen carbonate and brine, dried over
anhydrous sodium sulfate, filtered, and evaporated. The residue was purified
by silica gel chromatography with 10:1 (v/v) hexane–ethyl acetate to give 9
1
(75.7 mg, 91%) as a syrup: H NMR δ (CDCl3, 400 MHz) 5.24 (t, 1 H, J3,4
=
9.4 Hz, H-3), 5.11–5.05 (m, 2 H, H-2, 4), 4.40 (d, 1 H, J1, 2 = 10.0 Hz, H-1), 4.26
(dd, 1 H, J5,6a = 4.8 Hz, J6a,6b = 12.4 Hz, H-6a), 4.15 (dd, 1 H, J5,6b = 2.4 Hz,
H-6b), 3.74 (ddd, 1 H, J4,5 = 4.8 Hz, H-5), 2.17 (s, 3 H, S-CH3), 2.09–2.02 (m,
12 H, Ac).
Methyl 2-O-acetyl-4-O-benzyl-3-O-tert-butyldimethylsilyl-6-
O-chloroacetyl-β- D-thioglucopyranoside(10)
Compound 6 (811 mg, 1.78 mmol) was dissolved in dichloromethane
(8.9 mL) and pyridine (0.72 mL, 8.90 mmol), and the solution was cooled on
an ice bath. Then to the solution was added chloroacetyl chloride (170 μL,
2.14 mmol) dropwise and the mixture was stirred at rt for 12 h. The solution
was diluted with dichloromethane and washed with aqueous sodium hydro-
gen carbonate and brine, dried over anhydrous sodium sulfate, filtered, and
evaporated. The residue was purified by silica gel chromatography with 10:1
1
(v/v) hexane–ethyl acetate to give 10 (994 mg, 100%) as a syrup: H NMR δ
(CDCl3, 400 MHz) 7.35–7.27 (m, 5 H, Ph), 4.92 (t, 1 H, J2, 3 = 9.4 Hz, H-
2), 4.87 (d, 1 H, J = 11.6 Hz, Ph-CH-), 4.54 (d, 1 H, Ph-CH-), 4.23 (d, 1 H,
J1, 2 = 10.0 Hz, H-1), 4.15–4.09 (m, 1 H, 6a), 3.98 (d, 2 H, J = 4.4 Hz, ClAc),
3.82 (t, 1 H, J3,4 = 8.8 Hz, H-3), 3.58–3.54 (m, 1 H, H-5), 3.46 (t, 1 H, H-
4), 2.12 (s, 3 H, S-CH3), 2.11 (s, 3 H, Ac), 0.91 (s, 9 H, t-Bu), 0.09 (s, 6 H,
Si-CH3); 13C NMR (CDCl3,101 MHz): 169.7, 166.9, 137.4, 128.7, 128.5, 128.3,
128.2, 128.0, 127.8, 82.9 (C-1), 77.9 (C-4), 76.7 (C-5), 76.6 (C-3), 75.2, 71.4 (C-
2), 64.4 (C-6), 40.6, 25.7, 25.6, 21.4, 17.8, 11.1, −4.0, −4.3; MALDI-TOF HRMS

A. Miyagawa et al.
12
(positive ion): calcd for (C24H37ClO7SSi+Na+): 555.1615; found m/z: 555.
1641.
2-O-Acetyl-4-O-benzyl-3-O-tert-butyldimethylsilyl-6-O-chloro-
acetyl-α-D- glucopyranosyl 2,2,2-trichloroacetimidate (11)
Compound 9 (822 mg, 1.54 mmol) was dissolved in acetone/water (36.7 mL,
4.8/1, v/v). To the solution was added N-bromosuccinimide (1.37 g, 7.70 mmol)
and the mixture was stirred vigorously at rt for 10 min. The solution was
diluted with dichloromethane and washed with aqueous sodium sulfite and
brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The
residue was purified by silica gel chromatography with 10:1 to 4:1 (v/v)
hexane–ethyl acetate to give an intermediate with a free 1-hydroxyl group
(754 mg, 97%). The intermediate (710 mg, 1.41 mmol) was dissolved in
dichloromethane (14.1 mL) and trichloroacetonitrile (713 μL, 7.05 mmol),
and the solution was cooled on an ice bath. Then to the solution was added
1,8-diazabicyclo[5.4.0]-7-undecene (31.6 μL, 212 μmol) and the mixture was
stirred at rt for 12.0 h. The solution was concentrated and the residue was
purified by silica gel chromatography with 10:1 (v/v) hexane–ethyl acetate to
1
give 11 (640 mg, 70%) as a syrup: H NMR δ (CDCl3, 400 MHz) 8.57 (s, 1 H,
NH), 7.37–7.31 (m, 5 H, Ph), 6.45 (d, 1 H, J1, 2 = 2.0 Hz, H-1), 4.92–4.89 (m,
2 H, H-2, Ph-CH-), 4.58 (d, 1 H, J = 11.6 Hz, Ph-CH-), 4.40 (dd, 1 H, J6a,6b
=
8.0 Hz, H-6a), 4.26 (t, 1 H, J3,4 = 6.2 Hz, H-3), 4.17 (dd, 1 H, J5,6b = 2.8 Hz,
H-6b), 4.04–4.02 (m, 1 H, H-5), 3.95 (d, 2 H, J = 10.0 Hz, ClAc), 3.56 (t, 1 H,
H-4), 2.05 (s, 3 H, Ac), 0.93 (s, 9 H, t-Bu), 0.15 (s, 3 H, Si-CH3), 0.13 (s, 3 H,
Si-CH3); 13C NMR (CDCl3,151 MHz): 166.7, 160.9, 137.2, 128.5, 128.2, 128.1,
93.5 (C-1), 75.5, 72.7 (C-2), 72.0 (C-3), 71.4 (C-5), 63.8 (C-6), 40.5, 25.7, 20.8,
18.0, −4.0, −4.5.
para-Methoxyphenyl 2-O-acetyl-4-O-benzyl-3-O-tert-butyldime-
thylsilyl-6-O-chloroacetyl-β-D-glucopyranoside (12)
To a solution of compound 10 (550 mg, 1.03 mmol) and para-methoxy phe-
nol (639 mg, 5.15 mmol) in dichloromethane (5.15 mL) was added molecular
◦
˚
sieves 4A powder (2.0 g) and it was stirred at rt for 1.5 h and cooled at –20 C. To
the mixture was added methyl trifluoromethansulfonate (451 μL, 4.12 μmol)
and it was stirred at –20◦C for 1.0 h, and then at 0◦C for 3.5 h. The solution was
diluted with dichloromethane and filtered through Celite, and the filtrate was
washed successively with aqueous sodium hydrogen carbonate and brine, dried
over anhydrous sodium sulfate, filtered, and evaporated. The residue was pu-
rified by flash silica gel chromatography with 8:1 to 2:1 (v/v) hexane–ethyl ac-
etate to give 12 (380 mg, 75%) as a white powder: 1H NMR δ (CDCl3, 600 MHz)

Synthesis of Branched Oligo-β-Glucan Derivatives
13
7.38–7.29 (m, 5 H, Ph-CH2-), 6.93–6.89 (m, 2 H, Ph), 6.81–6.77 (m, 2 H, Ph),
5.11 (dd, 1 H, J2, 3 = 9.2 Hz, H-2), 4.88 (d, 1 H, J = 11.6 Hz, Ph-CH-), 4.78 (d, 1
H, J1, 2 = 7.6 Hz, H-1), 4.56 (d, 1 H, Ph-CH-), 4.44 (dd, 1 H, J5,6a = 2.4 Hz, J6a,6b
= 12.0 Hz, H-6a), 4.15 (dd, 1 H, J5,6b = 5.2 Hz, H-6b), 3.95 (d, 2 H, J = 8.4 Hz,
ClAc), 3.86 (t, 1 H, J3,4 = 9.2 Hz, H-3), 3.75 (s, 3 H, OCH3), 3.66–3.62 (m, 1 H,
H-5), 3.54 (t, 1 H, J4,5 = 9.6 Hz, H-4), 2.12 (s, 3 H, Ac), 0.92 (s, 9 H, t-Bu), 0.10
(s, 6 H, Si-CH3); 13C NMR (CDCl3,151 MHz): 169.4, 166.8, 155.5, 151.28, 137.3,
128.5, 128.1, 127.9, 118.5, 114.5, 100.6 (C-1), 78.0 (C-4), 75.2 (Ph-CH2-), 75.1
(C-3), 73.5 (C-2), 72.8 (C-5), 64.4 (C-6), 55.6, 40.6, 25.7, 21.2, 17.9, −4.1, −4.4;
MALDI-TOF HRMS (positive ion): calcd for (C30H41ClO9Si+Na+): 631.2106;
found m/z: 631.2126.
para-Methoxyphenyl 2-O-acetyl-4-O-benzyl-3-O-tert-butyl-
dimethylsilyl-β-D- glucopyranoside (13)
To a solution of 12 (50.0 mg, 82.1 μmol) in ethanol (4.0 mL) was added
1,4-diazabicyclo[2.2.2]octane (58 mg, 517 μmol), and the mixture was stirred
at 50◦C for 30 min. The solution was evaporated and the residue was purified
by silica gel chromatography with 6:1 (v/v) hexane–ethyl acetate to give 13
(39.0 mg, 89%) as a white powder: 1H NMR δ (CDCl3, 400 MHz) 7.38–7.26 (m,
5 H, Ph-CH2), 6.92–6.88 (m, 2 H, Ph), 6.83–6.77 (m, 2 H, Ph), 5.11 (dd, 1 H,
J 2, 3 = 9.2 Hz, H-2), 4.87 (d, 1 H, J = 11.6 Hz, Ph-CH-), 4.84 (d, 1 H, J1, 2
=
8.0 Hz, H-1), 4.65 (d, 1 H, Ph-CH-), 3.86 (m, 2 H, H-3, 6a), 3.70 (s, 3 H, OCH3),
3.68 (dd, 1 H, J5,6b = 4.4 Hz, J6a,6b = 12.4 Hz, H-6b), 3.59 (t, 1 H, J4,5 = 9.6 Hz,
H-4), 3.49–3.45 (m, 1 H, J5,6a = 2.8 Hz, H-5), 2.11 (s, 3 H, Ac), 0.91 (s, 9 H,
t-Bu), 0.08 (s, 6 H, Si-CH3); 13C NMR (CDCl3,101 MHz): 169.4, 155.4, 151.3,
137.8, 128.4, 127.8, 127.6, 118.1, 114.6, 100.5 (C-1), 78.2 (C-4), 75.6 (C-5), 75.1,
74.9 (C-3), 73.7 (C-2), 61.8 (C-6), 55.6, 25.7, 21.3, 17.9, −4.1, −4.4; MALDI-
TOF HRMS (positive ion): calcd for (C28H40O8Si+Na+): 555.2390; found m/z:
555.2387.
para-Methoxyphenyl 2,6-di-O-acetyl-4-O-benzyl-β-D-glucop-
yranoside (14)
Compound 13 (5.10 g, 9.57 mmol) was dissolved in dichloromethane
(19.1 mL), and pyridine (3.87 mL, 47.8 mmol) and acetic anhydride (2.71 mL,
28.7 mmol) were added. The mixture was stirred at rt for 16.5 h. The solu-
tion was evaporated and the residue was purified by silica gel chromatogra-
phy with 100:1 (v/v) toluene–acetone to give acetate (5.25 g, 96%). The acetate
(4.98g, 8.66 mmol) was dissolved in acetonitrile (155 mL) and cooled at –40◦C.
To the solution was added boron trifluoride–ethyl ether complex (2.18 mL,
17.4 mmol) diluted with acetonitrile (18.2 mL), and the mixture was stirred

A. Miyagawa et al.
14
at 0◦C for 15 min. The solution was diluted with dichloromethane and washed
successively with aqueous sodium hydrogen carbonate and brine, dried over
anhydrous sodium sulfate, filtered, and evaporated. The residue was purified
by silica gel chromatography with 6:1 (v/v) toluene–ethyl acetate to give 14
(3.60 g, 71%) as a white powder: 1H NMR δ (CDCl3, 400 MHz) 7.38–7.26 (m, 5
H, Ph-CH2), 6.96–6.92 (m, 2 H, Ph), 6.82–6.78 (m, 2 H, Ph), 5.11 (dd, 1 H, J 2, 3
= 9.6 Hz, H-2), 4.87 (d, 1 H, J = 11.2 Hz, Ph-CH-), 4.84 (d, 1 H, J 1, 2 = 7.6 Hz,
H-1), 4.69 (d, 1 H, Ph-CH-), 4.38 (dd, 1 H, J 5,6a = 2.0 Hz, J 6a,6b = 12.0 Hz, H-
6a), 4.25 (dd, 1 H, J 5,6b = 5.0 Hz, H-6b), 3.86 (m, 1 H, J 3,4 = 9.6 Hz, H-3), 3.76
(s, 3 H, OCH3), 3.64–3.55 (m, 2 H, H-4, 5), 2.86 (d, 1 H, J 3,OH = 4.8 Hz, 3-OH),
2.14 (s, 3 H, Ac), 2.04 (s, 3 H, Ac); 13C NMR (CDCl3,101 MHz): 170.9, 170.7,
155.6, 151.2, 137.6, 128.7, 128.3, 128.2, 118.6, 114.5, 100.1 (C-1), 77.7 (C-4),
76.1 (C-3), 74.9, 74.2 (C-2), 73.0 (C-5), 63.0 (C-6), 55.7, 20.9, 20.8; MALDI-TOF
HRMS (positive ion): calcd for (C24H28O9+Na+): 483.1631; found m/z: 483.
1632.
para-Methoxyphenyl 2-O-acetyl-4-O-benzyl-3-O-tert-butyldim-
ethylsilyl-6-O-(2,3,4,6- tetra-O-acetyl-β-D-glucosyl)-β-D-gluc-
opyranoside (15)
To a solution of 7 (60.1 mg, 122 μmol) and 13 (50.0 mg, 93.9 μmol) in
˚
dichloromethane (470 μL) was added molecular sieves 4A powder (4.14 g), and
the mixture was stirred at rt for 1.0 h and cooled at –40◦C. To the mixture was
added boron trifluoride–ethyl ether complex (3.1 μL, 24.6 μmol) and it was
stirred at –40◦C for 2.0 h. The solution was diluted with dichloromethane and
filtered through Celite, and the filtrate was washed successively with aqueous
sodium hydrogen carbonate and brine, dried over anhydrous sodium sulfate,
filtered, and evaporated. The residue was purified by silica gel chromatogra-
phy with 3:1 (v/v) hexane–ethyl acetate to give 15 (70.6 mg, 87%) as a white
1
powder: H NMR δ (CDCl3, 600 MHz) 7.35–7.30 (m, 5 H, Ph-CH2-), 6.93–6.92
(m, 2 H, Ph), 6.88–6.85 (m, 2 H, Ph), 5.16–5.05 (m, 3 H, H-2, 3ꢁ, 4ꢁ), 5.01 (t, 1 H,
J 1, 2 = 8.4 Hz, H-2ꢁ), 4.85 (d, 1 H, J = 11.2 Hz, Ph-CH-), 4.76 (d, 1 H, J 1, 2
=
8.0 Hz, H-1), 4.56 (d, 1 H, J 1, 2 = 8.4 Hz, H-1ꢁ), 4.53 (d, 1 H, Ph-CH-), 4.22 (dd,
1 H, J 5 ,6a = 4.4 Hz, J 6a ,6b = 12.4 Hz, H-6aꢁ), 4.09 (dd, 1 H, J 5 ,6b = 2.4 Hz,
H-6bꢁ), 4.04 (br-d, 1 H, J 6a,6b = 10.0 Hz, H-6a), 3.84 (t, 1 H, J 3,4 = 9.0 Hz, H-
3), 3.78 (s, 3 H, OCH3), 3.67–3.54 (m, 3 H, H-5, 5ꢁ, 6b), 3.35 (t, 1 H, H-4), 2.11
(s, 3 H, Ac), 2.02 (s, 3 H, Ac), 2.01 (s, 3 H, Ac), 2.00 (s, 3 H, Ac), 0.90 (s, 9 H,
t-Bu), 0.07 (s, 3 H, Si-CH3), 0.06 (s, 3 H, Si-CH3); 13C NMR (CDCl3,151 MHz):
170.6, 170.2, 169.3, 169.3, 169.2, 155.4, 151.5, 137.5, 128.4, 128.3, 127.9, 127.7,
117.9, 114.7, 100.4 (C-1ꢁ), 100.4 (C-1), 78.8 (C-4), 75.5 (C-5), 75.2, 74.9 (C-
3), 73.6 (C-2), 72.9 (C-3ꢁ), 71.8 (C-5ꢁ), 71.1 (C-2ꢁ), 68.3 (C-4ꢁ), 68.0 (C-6ꢁ), 61.9
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ

Synthesis of Branched Oligo-β-Glucan Derivatives
15
(C-6), 55.6, 25.7, 21.2, 20.6, 20.6, 20.5, 20.5, 17.9, −4.1, −4.4; MALDI-TOF
HRMS (positive ion): calcd for (C42H58O17Si+Na+): 885.3341; found m/z: 885.
3364.
para-Methoxyphenyl 3-O-(2-O-acetyl-4-O-benzyl-3-O-tert-buty-
ldimethylsilyl-6-O- chloroacetyl-β-D-glucopyranosyl)-2,6-
di-O-acetyl-4-O-benzyl-β-D-glucopyranoside (16)
To a solution of 11 (1.05 g, 1.72 mmol) and 14 (1.70 g, 3.43 mmol) in
˚
dichloromethane (8.58 mL) was added molecular sieves 4A powder (4.3 g),
and the mixture was stirred at rt for 2.0 h and cooled at –40◦C. To the mix-
ture was added boron trifluoride–ethyl ether complex (86.8 μL, 860 μmol) di-
luted in dichloromethane (1.72 mL) and stirred at –40◦C for 4.5 h. The solution
was diluted with dichloromethane and filtered through Celite, and the filtrate
was washed successively with aqueous sodium hydrogen carbonate and brine,
dried over anhydrous sodium sulfate, filtered, and evaporated. The residue
was purified by flash silica gel chromatography with 4:1 (v/v) hexane-ethyl
acetate and reverse phase silica gel chromatography with 1:9 to 17:3 (v/v)
acetonitrile–water to give 16 (1.10 g, 68%) and the recovery of acceptor 14
(1.11 g, 65%) as a white powder: 1H NMR δ (CDCl3, 400 MHz) 7.34–7.26 (m, 10
H, Ph-CH2), 6.90–6.87 (m, 2 H, Ph), 6.80–6.78 (m, 2 H, Ph), 5.12 (dd, 1 H, J1, 2
= 7.6 Hz, H-2), 5.00–4.94 (m, 2 H, H-2ꢁ, Ph-CH-), 4.84 (d, 1 H, J = 11.6 Hz,
ꢁ
ꢁ
Ph-CH-), 4.80 (d, 1 H, J1, 2 = 7.6 Hz, H-1), 4.62 (d, 1 H, J1 , 2 = 8.0 Hz, H-
1ꢁ), 4.52 (d, 1 H, J = 11.2 Hz, Ph-CH2-), 4.50 (d, 1 H, Ph-CH2-), 4.36 (dd, 1
H, J5 ,6a = 2.2 Hz, J6a ,6b = 11.6 Hz, H-6aꢁ), 4.27 (br-d, 1 H, J6a,6b = 10.8 Hz,
H-6a), 4.16–4.09 (m, 2 H, H-6b, 6bꢁ), 4.01 (m, 1 H, H-3), 3.77 (m, 1 H, H-3ꢁ),
3.76 (s, 3 H, OCH3), 3.66 (d, 2 H, J = 3.6 Hz, ClAc), 3.61–3.47 (m, 4 H, H-
4, 4ꢁ, 5, 5ꢁ), 2.15 (s, 6 H, Ac), 1.93 (s, 3 H, Ac), 0.91 (s, 9 H, t-Bu), 0.09 (s,
6 H, Si-CH3); 13C NMR (CDCl3,101 MHz): 170.6, 169.9, 168.9, 166.7, 155.4,
150.9, 137.9, 137.2, 128.5, 128.2, 128.0, 128.0, 127.8, 118.3, 114.4, 100.8 (C-1ꢁ),
99.6 (C-1), 80.5 (C-3), 78.1 (C-3ꢁ), 77.2 (C-4ꢁ), 75.3 (C-4), 75.1, 74.3, 74.2 (C-2ꢁ),
73.1 (C-2), 73.1 (C-5ꢁ), 72.9 (C-5), 72.6, 64.3 (C-6ꢁ), 62.6 (C-6), 55.6, 40.4, 25.6,
21.2, 20.9, 20.7, 17.8, −4.0, −4.5; MALDI-TOF HRMS (positive ion): calcd for
(C47H61ClO16Si+Na+): 967.3315; found m/z: 967.3362.
ꢁ
ꢁ
ꢁ
ꢁ
para-Methoxyphenyl 2,3-di-O-acetyl-4-O-benzyl-6-O-(2,3,4,6-
tetra-O-acetyl-β-D- glucosyl)-β-D-glucopyranoside (17)
Compound 15 (80 mg, 92.7 μmol) was dissolved in acetonitrile (1.60 mL),
and the solution was cooled at –20◦C. To the solution was added boron
trifluoride–ethyl ether complex (23.3 μL, 185 μmol) diluted with acetonitrile
(0.24 mL) and the mixture was stirred at –20◦C for 13 min. The solution was

A. Miyagawa et al.
16
diluted with dichloromethane and washed successively with aqueous sodium
hydrogen carbonate and brine, dried over anhydrous sodium sulfate, filtered,
and evaporated. The residue was purified by silica gel chromatography with
5:1 to 1:1 (v/v) toluene–ethyl acetate to give 20 (69 mg, 100%). To a solution
of 20 (20.0 mg, 26.7 μmol) in dichloromethane (350 μL) was added pyridine
(108 μg, 1.34 mmol) and acetic anhydride (76 μg, 0.801 mmol), and the mix-
ture was stirred at rt for 4.5 h. The solution was evaporated under vacuum
1
to give 17 (39.0 mg, 89%) as a white powder: H NMR δ (CDCl3, 600 MHz)
7.34–7.25 (m, 5 H, Ph-CH2), 6.95–6.93 (m, 2 H, Ph), 6.88–6.86 (m, 2 H, Ph),
5.30 (t, 1 H, J2, 3 = 9.6 Hz, H-3), 5.17–5.07 (m, 3 H, H-2, 3ꢁ, 4ꢁ), 5.03 (br-t, 1
H, H-2ꢁ), 4.94 (d, 1 H, J1, 2 = 7.8 Hz, H-1), 4.63 (d, 1 H, J1 , 2 = 8.0 Hz, H-1ꢁ),
ꢁ
ꢁ
ꢁ
ꢁ
4.61 (d, 1 H, J = 11.4 Hz, Ph-CH2-), 4.57 (d, 1 H, Ph-CH2-), 4.22 (dd, 1 H, J5 ,6a
= 4.8 Hz, J6a ,6b = 12.6 Hz, H-6aꢁ), 4.12 (dd, 1 H, J5 ,6b = 2.4 Hz, H-6bꢁ), 4.09
(br-d, 1 H, J6a,6b = 10.8 Hz, H-6a), 3.79 (s, 3 H, OCH3), 3.79–3.70 (m, 2 H, H-5,
6b), 3.59 (t, 1 H, J3, 4 = 9.0 Hz, H-4), 3.58–3.55 (m, 1 H, H-5ꢁ), 2.06 (s, 3 H, Ac),
2.05 (s, 3 H, Ac), 2.02 (s, 3 H, Ac), 2.01 (s, 3 H, Ac), 1.99 (s, 3 H, Ac), 1.90 (s,
3 H, Ac); 13C NMR (CDCl3,151 MHz): 171.5, 170.6, 168.9, 166.9, 155.5, 151.0,
137.9, 137.4, 128.6, 128.3, 128.3, 128.2, 127.9, 127.8, 118.4, 114.5, 100.6 (C-1ꢁ),
99.9 (C-1), 81.1 (C-3), 77.2 (C-4ꢁ), 76.6 (C-3ꢁ), 74.8 (C-4), 74.7, 74.5, 74.0 (C-2ꢁ),
73.0 (C-2), 73.0 (C-5ꢁ), 72.5 (C-5), 64.3 (C-6ꢁ), 62.7 (C-6), 55.6, 40.4, 21.0, 20.7,
20.7; MALDI-TOF HRMS (positive ion): calcd for (C38H46O18+Na+): 813.2582;
found m/z: 813.2546.
ꢁ
ꢁ
ꢁ
ꢁ
2-O-Acetyl-4-O-benzyl-3-O-tert-butyldimethylsilyl-6-O-(2,3,4,6-
tetra-O-acetyl-β-D- glucosyl)-α-D-glucopyranosyl 2,2,2-tri-
chloroacetimidate (18)
Compound 15 (150 mg, 174 μmol) was dissolved in acetonitrile/water
(6.1 mL, 4.1/1, v/v), and the solution was cooled at 0◦C. To the solution was
added cerium (IV) ammonium nitrate (476 mg, 868 μmol) diluted with ace-
tonitrile/water (2.5 mL, 4/1, v/v), and the mixture was stirred at 0◦C for 7 min.
The solution was poured into aqueous sodium hydrogen carbonate, extracted
with dichloromethane, washed with brine, dried over anhydrous sodium sul-
fate, filtered, and evaporated. The residue was purified by silica gel chromatog-
raphy with 3:1 (v/v) toluene–ethyl acetate to give an intermediate with a free 1-
hydroxyl group (121 mg, 92%). The intermediate (120 mg, 159 μmol) dissolved
in dichloromethane (1.6 mL) and trichloroacetonitrile (79.7 μL, 795 mmol) was
cooled on an ice bath. Then to the solution was added 1,8-diazabicyclo[5.4.0]-
7-undecene (3.6 μL, 23.9 μmol) and the mixture was stirred at rt for 31 h.
The solution was concentrated and the residue was purified by silica gel chro-
matography with 10:1 to 3:1 (v/v) toluene–ethyl acetate to give 18 (107 mg,
1
75%) as a white powder: H NMR δ (CDCl3, 400 MHz) 8.55 (s, 1 H, NHα),

Synthesis of Branched Oligo-β-Glucan Derivatives
17
7.38–7.29 (m, 5 H, Ph-CH2), 6.50 (d, 1 H, J1, 2 = 3.2 Hz, H-1α), 5.19–5.02 (m,
3 H, H-2ꢁ, 3ꢁ, 4ꢁ), 4.88–4.85 (m, 2 H, H-2, Ph-CH-), 4.59–4.54 (m, 2 H, J1 , 2
ꢁ
ꢁ
= 8.0 Hz, H-1ꢁ, Ph-CH-), 4.26–4.20 (m, 2 H, H-3, 6aꢁ), 4.11 (dd, 1 H, J5 ,6b
=
ꢁ
ꢁ
2.4 Hz, J6a ,6b = 10.4 Hz, H-6bꢁ), 4.06 (br-d, 1 H, J5,6a = 1.6 Hz, J6a,6b = 11.2 Hz,
H-6a), 4.00 (m, 1 H, H-5), 3.76 (dd, 1 H, J5,6b = 4.4 Hz, H-6b), 3.65 (m, 1 H,
H-5ꢁ), 3.53 (t, 1 H, J3,4 = 9.2 Hz, H-4), 2.06 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.02
(s, 3 H, Ac), 2.00 (s, 3 H, Ac), 1.96 (s, 3 H, Ac), 0.90 (s, 9 H, t-Bu), 0.09 (s, 3
H, Si-CH3), 0.08 (s, 3 H, Si-CH3); 13C NMR (CDCl3,101 MHz): 170.7, 170.3,
170.0, 169.3, 169.1, 161.0, 137.7, 128.5, 127.9, 127.5, 100.4 (C-1ꢁ), 93.8 (C-1),
78.1 (C-4), 75.2, 73.2 (C-2), 73.0 (C-5), 73.0 (C-3ꢁ), 71.9 (C-5ꢁ), 71.8 (C-3), 71.2
(C-4ꢁ), 68.3 (C-2ꢁ), 67.6 (C-6), 61.8 (C-6ꢁ), 25.7, 20.8, 20.7, 20.6, 20.6, 18.0, −4.1,
−4.5.
ꢁ
ꢁ
2,3-Di-O-acetyl-4-O-benzyl-6-O-(2,3,4,6-tetra-O-acetyl-β-D-glu-
cosyl)-D- glucopyranosyl 2,2,2-trichloroacetimidate (19)
Compound 17 (1.10 g, 1.39 mmol) was dissolved in acetonitrile/water
(27.5 mL, 4/1, v/v), and the solution was cooled at 0◦C. To the solution was
added cerium (IV) ammonium nitrate (3.81 g, 6.95 mmol) dissolved with ace-
tonitrile/water (10.4 mL, 4.2/1, v/v), and the mixture was stirred at 0◦C for
17 min. The solution was poured into aqueous sodium hydrogen carbonate, ex-
tracted with dichloromethane and washed with brine, dried over anhydrous
sodium sulfate, filtered, and evaporated. The residue was purified by silica
gel chromatography with 2:1 (v/v) toluene–ethyl acetate to give an interme-
diate with a free 1-hydroxyl group (896 mg, 94%). The intermediate (813 mg,
1.19 mmol) dissolved in dichloromethane (1.19 mL) and trichloroacetonitrile
(597 μL, 5.95 mmol) was cooled on an ice bath. To the solution was added
1,8-diazabicyclo[5.4.0]-7-undecene (26.6 μL, 179 μmol) and the mixture was
stirred at rt for 8.5 h. The solution was concentrated and the residue was
purified by silica gel chromatography with 3:1 to 2:1 (v/v) toluene–ethyl ac-
1
etate to give 19 (928 mg, 94%, α:β = 10:1) as a white powder: H NMR δ
(CDCl3, 600 MHz) 8.76 (s, 1 H, NHβ), 8.63 (s, 1 H, NHα), 6.50 (d, 1 H, J1α, 2
=
1.8 Hz, H-1α), 5.84 (d, 1 H, J1β, 2 = 8.0 Hz, H-1β), 5.63 (t, 1 H, J3,4 = 9.0 Hz,
H-3), 5.20 (t, 1 H, J3 ,4 = 9.0 Hz, H-3ꢁ), 5.11 (t, 1 H, H-4ꢁ), 5.08–5.03 (m, 2
ꢁ
ꢁ
H, H-2, 2ꢁ), 4.63 (d, 1 H, J1 , 2 = 7.2 Hz, H-1ꢁ), 4.59 (s, 2 H, H-2, Ph-CH-),
ꢁ
ꢁ
4.26–4.18 (dd, 1 H, J5 ,6a = 3.9 Hz, J6a ,6b = 12.6 Hz, H-6aꢁ), 4.11 (br-d, 1
H, H-6bꢁ), 4.09 (br-d, 2 H, H-5, 6a), 4.00 (m, 1 H, H-5), 3.86 (dd, 1 H, J5,6b
= 3.3 Hz, J6a,6b = 11.2 Hz, H-6b), 3.77 (t, 1 H, H-4), 3.69 (m, 1 H, H-5ꢁ),
2.07 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.01 (s, 3 H, Ac), 1.98 (br-s, 9 H, Ac);
13C NMR (CDCl3, 151 MHz): 170.6, 170.3, 170.0, 169.6, 169.3, 169.0, 161.0,
137.2, 129.0, 128.6, 128.2, 127.9, 125.2, 100.4 (C-1ꢁ), 93.3 (C-1α), 90.8 (C-
1β), 75.6, 74.8, 72.9, 72.6, 71.9, 71.5, 71.3, 68.2, 67.3, 61.8, 20.8, 20.7, 20.5,
20.4.
ꢁ
ꢁ
ꢁ
ꢁ

A. Miyagawa et al.
18
para-Methoxyphenyl 2-O-acetyl-4-O-benzyl-6-O-(2,3,4,6-tet-
ra-O-acetyl-β-D-glucosyl)- β-D-glucopyranoside (20)
Compound 15 (150 mg, 174 μmol) was dissolved in acetonitrile (3.00 mL)
and cooled at –20◦C. To the solution was added boron trifluoride–ethyl ether
complex (43.7 μL, 358 μmol) diluted with acetonitrile (0.48 mL) and the
mixture was stirred at –20◦C for 17 min. The solution was diluted with
dichloromethane and washed successively with aqueous sodium hydrogen car-
bonate and brine, dried over anhydrous sodium sulfate, filtered, and evapo-
rated. The residue was purified by silica gel chromatography with 5:1 to 1:1
1
(v/v) toluene–ethyl acetate to give 20 (127 mg, 98%) as a white powder: H
NMR δ (CDCl3, 600 MHz) 7.34 (m, 5 H, Ph-CH2), 6.95 (br-d, 2 H, Ph), 6.87 (br-
d, 2 H, Ph), 5.17–4.99 (m, 4 H, H-2, 2ꢁ, 3ꢁ, 4ꢁ), 4.88 (d, 1 H, J = 12.0 Hz, Ph-CH-),
4.85 (d, 1 H, J1, 2 = 9.2 Hz, H-1), 4.67 (d, 1 H, Ph-CH-), 4.63 (d, 1 H, J1, 2
=
8.0 Hz, H-1ꢁ), 4.24–4.15 (m, 2 H, J5 ,6a = 4.4 Hz, J6a ,6b = 12.4 Hz, H-6aꢁ, 6bꢁ),
4.10 (br-d, 1 H, J6a,6b = 11.2 Hz, H-6a), 3.86–3.79 (m, 2 H, H-3, 6b), 3.79 (s, 3 H,
OCH3), 3.64 (br-t, 1 H, H-5), 3.56 (m, 1 H, H-5ꢁ), 3.47 (br-t, 1 H, H-4), 2.14 (s, 3
ꢁ
ꢁ
ꢁ
ꢁ
H, Ac), 2.05 (s, 3 H, Ac), 2.02 (s, 3 H, Ac), 2.00 (s, 3 H, Ac), 1.90 (s, 3 H, Ac); 13
C
NMR (CDCl3,151 MHz): 170.8, 170.7, 170.2, 169.4, 169.2, 155.6, 151.2, 137.7,
128.6, 128.1, 128.1, 118.1, 114.7, 100.6 (C-1ꢁ), 99.8 (C-1), 78.1, 76.0, 75.3, 75.0,
74.1, 72.9, 71.8, 71.3, 68.3, 68.2, 61.7, 55.6, 20.9, 20.7, 20.6, 20.5; MALDI-TOF
HRMS (positive ion): calcd for (C36H44O17+Na+): 771.2476; found m/z: 771.
2467.
para-Methoxyphenyl 2,6-di-O-acetyl-4-O-benzyl-3-O-(2,3-di-O-
acetyl-4-O-benzyl-6-O- chloroacethyl-β-D-glucopyranosyl)-
β-D-glucopyranoside (21)
Compound 16 (1.60 g, 1.69 mmol) was dissolved in acetonitrile (30.4 mL)
and the solution was cooled at –20◦C. To the solution was added boron
trifluoride–ethyl ether complex (425 μL, 3.38 mmol) diluted with acetonitrile
(3.52 mL) and the mixture was stirred at 0◦C for 30 min. The solution was
diluted with dichloromethane and washed successively with aqueous sodium
hydrogen carbonate and brine, dried over anhydrous sodium sulfate, filtered,
and evaporated. The residue was purified by silica gel chromatography with
3:1 to 1:1 (v/v) hexane–ethyl acetate to give 24 (1.40 g, 99%) as a white pow-
der. To a solution of 24 (292 mg, 351 μmol) in dichloromethane (3.51 mL) was
added pyridine (1.0 mL, 12.4 mmol) and acetic anhydride (1.0 mL, 10.6 mmol),
and the mixture was stirred at rt for 17 h. The solution was diluted with
dichloromethane and washed successively with aqueous 1M hydrochloric acid
and brine, dried over anhydrous sodium sulfate, filtered, and evaporated. The

Synthesis of Branched Oligo-β-Glucan Derivatives
19
solution was concentrated and the residue was purified by silica gel chro-
matography with 6:1 to 3:1 (v/v) toluene–ethyl acetate to give 19 (274 mg,
1
89%) as a white powder: H NMR δ (CDCl3, 600 MHz) 7.37–7.22 (m, 10 H,
ꢁ
ꢁ
Ph-CH2), 6.89–6.87 (m, 2 H, Ph), 6.80–6.77 (m, 2 H, Ph), 5.25 (t, 1 H, J3 ,4
= 9.6 Hz, H-3ꢁ), 5.16 (dd, 1 H, J1, 2 = 7.8 Hz, J2,3 = 9.0 Hz, H-2), 5.00 (d, 1
ꢁ
ꢁ
ꢁ
ꢁ
H, J = 11.4 Hz, Ph-CH-), 5.16 (dd, 1 H, J1 ,2 = 8.4 Hz, J2 ,3 = 9.6 Hz, H-
2ꢁ), 4.77 (d, 2 H, H-1, H-1ꢁ), 4.60 (d, 1 H, J = 11.4 Hz, Ph-CH-), 4.51 (d, 1
ꢁ
ꢁ
ꢁ
ꢁ
H, Ph-CH-), 4.50 (d, 1 H, Ph-CH-), 4.36 (dd, 1 H, J5 ,6a = 1.8 Hz, J6a ,6b
=
12.0 Hz, H-6aꢁ), 4.29–4.25 (m, 2 H, J6a,6b = 4.2 Hz, J5,6a = 6.6 Hz, H-6a, 6bꢁ),
4.13 (m, 1 H, J5,6b = 1.8 Hz, H-6b), 4.02 (m, 1 H, H-3), 3.74 (s, 3 H, OCH3),
3.68 (t, 1 H, H-4ꢁ), 3.62 (s, 1 H, ClAc), 3.58 (br-d, 1 H, H-4, 5), 2.17 (s, 3 H,
Ac), 2.09 (s, 3 H, Ac), 1.99 (s, 3 H, Ac), 1.95 (s, 3 H, Ac); 13C NMR (CDCl3,
151 MHz): 170.5, 170.1, 169.9, 169.0, 166.7, 155.5, 151.0, 137.9, 136.9, 128.6,
128.2, 128.1, 128.0, 127.7, 118.3, 114.4, 100.7 (C-1ꢁ), 99.8 (C-1), 81.2 (C-3), 77.2
(C-4ꢁ), 76.8 (C-3ꢁ), 75.1 (C-4), 74.8, 74.6, 74.4 (C-2ꢁ), 72.9 (C-2), 72.8 (C-5ꢁ), 72.5
(C-5), 71.5, 63.9 (C-6ꢁ), 62.6 (C-6), 55.5, 40.3, 20.9, 20.7, 20.6, 20.4; MALDI-TOF
HRMS (positive ion): calcd for (C43H49ClO17+Na+): 895.2556; found m/z: 895.
2532.
3-O-(2-O-Acetyl-4-O-benzyl-3-O-tert-butyldimethylsilyl-6-O-chl-
oroacethyl-β-D- glucopyranosyl)-2,6-di-O-acetyl-4-O-benz-
yl-α-D-glucopyranosyl 2,2,2-trichloroacetimidate (22)
Compound 16 (1.10 g, 1.16 mmol) was dissolved in acetonitrile/water
(29.0 mL, 4/1, v/v), and the solution was cooled at 0◦C. To the solution was
added cerium (IV) ammonium nitrate (3.18 g, 5.80 mmol) dissolved in ace-
tonitrile/water (14.8 mL, 6/1, v/v), and the mixture was stirred at 0◦C for
20 min. The solution was poured into aqueous sodium hydrogen carbonate,
extracted with dichloromethane and washed with brine, dried over anhydrous
sodium sulfate, filtered, and evaporated. The residue was purified by silica
gel chromatography with 9:1 to 1:4 (v/v) hexane–ethyl acetate to give an in-
termediate with a free 1-hydroxyl group (958 mg, 98%). The intermediate
(242 mg, 289 μmol) dissolved in dichloromethane (1.4 mL) and trichloroace-
tonitrile (146 μL, 1.45 mmol) was cooled on an ice bath. Then to the solution
was added 1,8-diazabicyclo[5.4.0]-7-undecene (6.5 μL, 43.4 μmol) and the mix-
ture was stirred at rt for 25 h. The solution was concentrated and the residue
was purified by silica gel chromatography with 20:1 to 10:1 (v/v) toluene–ethyl
1
acetate to give 22 (211 mg, 74%) as a syrup: H NMR δ (CDCl3, 400 MHz)
8.62 (s, 1 H, NHα), 7.37–7.28 (m, 10 H, Ph), 6.39 (d, 1 H, J1α,2 = 3.6 Hz, H-
1α), 5.00–4.93 (m, 3 H, H-2, 2ꢁ, Ph-CH-), 4.84 (d, 1 H, J = 11.6 Hz, Ph-CH-
), 4.67 (d, 1 H, J 1 β, 2 = 8.4 Hz, H-1ꢁβ), 4.54 (d, 1 H, J = 11.2 Hz, Ph-CH-),
ꢁ
ꢁ
4.51 (d, 1 H, J = 11.6 Hz, Ph-CH-), 4.38 (dd, 1 H, J5,6b = 2.0 Hz, J6a,6b
=
12.0 Hz, H-6a), 4.28–4.13 (m, 4 H, H-3, 6b, 6aꢁ, 6bꢁ), 3.99 (m, 4 H, H-5), 3.79

A. Miyagawa et al.
20
(t, 4 H, J3 , 4 = 9.0 Hz, H-3ꢁ), 3.71 (d, 2 H, J = 4.4 Hz, ClAcα), 3.59 (m, 2 H,
H-4, 5ꢁ), 3.49 (t, 1 H, H-4ꢁ), 2.13 (s, 3 H, Ac), 2.13 (s, 3 H, Ac), 1.98 (s, 3 H,
Ac), 0.90 (s, 9 H, t-Bu), 0.08 (s, 3 H, Si-CH3), 0.07 (s, 3 H, Si-CH3); 13C NMR
(CDCl3,101 MHz): 170.6, 169.8, 169.6, 166.8, 160.6, 137.7, 137.3, 128.5, 128.3,
128.0, 127.9, 127.7, 101.3 (C-1ꢁ), 93.3 (C-1), 90.8, 78.4, 78.2, 77.3, 75.3, 75.1,
74.6, 73.6, 73.4, 72.8, 72.6, 64.4, 62.1, 40.4, 25.6, 21.2, 20.8, 20.7, 17.8, −4.0,
−4.4.
ꢁ
ꢁ
2,6-Di-O-acetyl-4-O-benzyl-3-O-(2,3-di-O-acetyl-4-O-benzyl-6-O-
chloroacethyl-β-D-glucopyranosyl)-D-glucopyranosyl 2,2,
2-trichloroacetimidate (23)
Compound 21 (380 mg, 435 μmol) was dissolved in acetonitrile/water
(8.6 mL, 4.1/1, v/v), and the solution was cooled at 0◦C. To the solution was
added cerium (IV) ammonium nitrate (1.19 g, 2.18 mmol) dissolved in ace-
tonitrile/water (3.6 mL, 4.2/1, v/v), and the mixture was stirred at 0◦C for
15 min. The solution was poured into aqueous sodium hydrogen carbonate,
extracted with dichloromethane and washed with brine, dried over anhydrous
sodium sulfate, filtered, and evaporated. The residue was purified by silica
gel chromatography with 2:1 to 1:1 (v/v) toluene–ethyl acetate to give an in-
termediate with a free 1-hydroxyl group (305 mg, 85%). The intermediate
(290 mg, 378 μmol) dissolved in dichloromethane (1.9 mL) and trichloroace-
tonitrile (190 μL, 1.89 mmol) was cooled on an ice bath. Then to the solution
was added 1,8-diazabicyclo[5.4.0]-7-undecene (8.5 μL, 56.9 μmol) and the mix-
ture was stirred at rt for 12.5 h. The solution was concentrated and the residue
was purified by silica gel chromatography with 6:1 to 3:1 (v/v) toluene–ethyl
1
acetate to give 23 (343 mg, 99%; α:β = 25:1) as a syrup: H NMR δ (CDCl3,
600 MHz) 8.64 (s, 1 H, NH), 7.35–7.23 (m, 10 H, Ph), 6.39 (d, 1 H, J1α, 2
=
ꢁ
ꢁ
3.6 Hz, H-1α), 5.69 (d, 1 H, J1β, 2 = 7.2 Hz, H-1β), 5.25 (t, 1 H, J2 , 3 = 9.6 Hz,
H-3ꢁ), 5.01–4.94 (m, 3 H, H-2, 2ꢁ, Ph-CH-), 4.84 (d, 1 H, J1 , 2 = 8.4 Hz, H-
1ꢁ), 4.61 (d, 1 H, J = 11.6 Hz, Ph-CH-), 4.54 (d, 1 H, J = 11.6 Hz, Ph-CH-),
ꢁ
ꢁ
ꢁ
ꢁ
4.53 (d, 1 H, J = 11.6 Hz, Ph-CH-), 4.40 (br-d, 1 H, J6a , 6b = 12.0 Hz, H-
6aꢁ), 4.31–4.23 (m, 3 H, H-3, 6a, 6bꢁ), 4.17 (dd, 1 H, J5, 6b = 3.6 Hz, J6a, 6b
=
12.0 Hz, H-6b), 4.00 (m, 1 H, H-5), 3.75 (s, 2 H, ClAc), 3.71–3.65 (m, 2 H,
H-4ꢁ, 5ꢁ), 3.61 (t, 1 H, J3, 4 = 9.6 Hz, H-4), 2.15–1.95 (s, 12 H, Ac); 13C NMR
(CDCl3,151 MHz): 170.5, 170.0, 169.9, 167.3, 166.8, 160.8, 137.7, 137.0, 128.6,
128.3, 128.2, 128.1, 128.0, 127.9, 101.0 (C-1ꢁ), 93.3 (C-1), 90.8, 78.8, 78.15, 77.2,
75.3, 75.1, 74.7, 74.6, 74.1, 72.8, 72.2, 71.9, 71.2, 64.0, 62.1, 40.4, 20.8, 20.7,
20.5.

Synthesis of Branched Oligo-β-Glucan Derivatives
21
para-Methoxyphenyl 3-O-(2-O-acetyl-4-O-benzyl-6-O-chloroac-
ethyl-β-D- glucopyranosyl)-2,6-di-O-acetyl-4-O-benzyl-β-
D-glucopyranoside (24)
Compound 16 (200 mg, 212 μmol) was dissolved in acetonitrile (3.80 mL),
and the solution was cooled at –20◦C. To the solution was added boron
trifluoride–ethyl ether complex (53.3 μL, 424 μmol) diluted with acetonitrile
(0.44 mL) and the mixture was stirred at 0◦C for 30 min. The solution was
diluted with dichloromethane and washed successively with aqueous sodium
hydrogen carbonate and brine, dried over anhydrous sodium sulfate, filtered,
and evaporated. The residue was purified by silica gel chromatography with
3:1 to 2:1 (v/v) toluene–ethyl acetate to give 24 (161 mg, 91%) as a white pow-
der: 1H NMR δ (CDCl3, 400 MHz) 7.37–7.26 (m, 10 H, Ph-CH2), 6.89 (d, 2 H, J
= 9.0 Hz, Ph), 6.79 (d, 2 H, J = 9.0 Hz, Ph), 5.18 (t, 1 H, J1, 2 = 7.8 Hz, H-2),
5.00 (d, 1 H, J = 11.4 Hz, Ph-CH-), 4.85–4.82 (m, 2 H, H-2ꢁ, Ph-CH-), 4.78 (d,
ꢁ
ꢁ
1 H, J1,2 = 7.8 Hz, H-1), 4.70 (d, 1 H, J = 12.0 Hz, Ph-CH-), 4.69 (d, 2 H, J1 ,2
= 7.8 Hz, H-1ꢁ), 4.49 (d, 1 H, J = 11.4 Hz, Ph-CH-), 4.36 (br-d, 1 H, J6a ,6b
=
ꢁ
ꢁ
12.0 Hz, H-6aꢁ), 4.30–4.78 (m, 2 H, H-6a, 6bꢁ), 4.13 (m, 1 H, J5,6b = 3.0 Hz,
J6a,6b = 11.4 Hz, H-6b), 4.02 (br-t, 1 H, J3,4 = 8.4 Hz, H-3), 3.78 (m, 1 H, H-3ꢁ),
3.76 (s, 3 H, OCH3), 3.68 (s, 2 H, ClAc), 3.60 (br-d, 2 H, H-4, 5), 3.57–3.50 (m, 2
H, H-4ꢁ, 5ꢁ), 2.55 (d, 1 H, J = 4.2 Hz, 3-OH), 2.19 (s, 3 H, Ac), 2.15 (s, 3 H, Ac),
1.96 (s, 3 H, Ac); 13C NMR (CDCl3,101 MHz): 171.5, 170.6, 168.9, 166.9, 155.5,
151.0, 137.9, 137.5, 128.6, 128.4, 128.3, 128.2, 127.9, 127.8, 118.4, 114.5, 100.6
(C-1ꢁ), 99.9 (C-1), 81.1 (C-3), 77.2 (C-4ꢁ), 76.6 (C-3ꢁ), 74.8 (C-4), 74.7, 74.5, 74.0
(C-2ꢁ), 73.0 (C-2), 73.0 (C-5ꢁ), 72.5 (C-5), 64.3 (C-6ꢁ), 62.7 (C-6), 55.6, 40.4, 21.0,
20.7, 20.7; MALDI-TOF HRMS (positive ion): calcd for (C41H47ClO16+Na+):
853.2450; found m/z: 853.2463.
para-Methoxyphenyl 2,6-di-O-acetyl-4-O-benzyl-3-O-(2-O-acet-
yl-4-O-benzyl-3-O-((2- O-acetyl-4-O-benzyl-3-O-tert-butyld-
imethylsilyl-6-O-(2,3,4,6-tetra-O-acetyl-β-D- glucopyranosyl)-
6-O-chloroacetyl-β-D-glucopyranosyl)-β-D-glucopyranos-
yl))-β-D-glucopyranoside (25)
To a solution of 18 (105 mg, 117 μmol) and 24 (80.0 mg, 97.1 μmol) in
˚
dichloromethane (900 μL) was added molecular sieves 4A powder (100 mg),
and the mixture was stirred for 30 min and cooled at –40◦C. To the mixture
was added boron trifluoride–ethyl ether complex (7.3 μL, 58.1 μmol) diluted
in dichloromethane (70 μL) and stirred at –40◦C for 2.0 h and then at –20◦C
for 2.0 h. To the mixture was added trimethylsilyl trifluoromethansulfonate
(10.1 μL, 58.1 μmol) and it was stirred at 0◦C for 3.0 h. The solution was di-
luted with dichloromethane and filtered through Celite, and the filtrate was

A. Miyagawa et al.
22
washed with brine, dried over anhydrous sodium sulfate, filtered, and evap-
orated. The residue was purified by flash silica gel chromatography with 6:1
(v/v) toluene–ethyl acetate and reverse-phase silica gel chromatography with
7:3 to 9:1 (v/v) methanol–water to give 25 (39.0 mg, 26%) as a white powder
1
and the recovery of acceptor 14 (48.0 mg, 60%): H NMR δ (CDCl3, 400 MHz)
7.37–7.24 (m, 15 H, Ph-CH2), 6.89–6.87 (m, 2ꢁꢁH,ꢁꢁPꢁ h), 6.80–6.79 (m, 2 H, Ph),
ꢁꢁ
ꢁ
ꢁꢁꢁ
ꢁꢁꢁ
ꢁꢁ
ꢁꢁ
5.05–4.93 (m, 8 H, J1 , 2 = 8.4 Hz, H-1 , 2, 2 , 2 , 2 , 3 , 4 , Ph-CH-), 4.89–4.83
(m, 3 H, H-1ꢁ, Ph-CH- ×2), 4.84 (d, 1 H, J1, 2 = 7.8 Hz, H-1), 4.58–4.45 (m, 5
ꢁꢁꢁ, 2ꢁꢁꢁ
H, J1
= 7.8 Hz, H-1ꢁꢁꢁ, 6a, Ph-CH- ×3), 4.35 (dd, 1 H, J5,6b = 3.9 Hz, J6a,6b
= 12.0 Hz, H-6b), 4.30 (dd, 1 H, J5 ,6b = 1.2 Hz, J6a ,6b = 12.0 Hz, H-6aꢁ), 4.15
ꢁ
ꢁ
ꢁ
ꢁ
(t, 1 H, J3,4 = 9.0 Hz, H-3), 4.10–4.06 (m, 2 H, H-3ꢁ, 6bꢁ), 4.01 (m, 1 H, H-6aꢁꢁ),
= 12.0 Hz, H-6aꢁꢁꢁ), 3.86 (m, 1 H, H-5),
ꢁꢁꢁ,6aꢁꢁꢁ
ꢁꢁꢁ,6bꢁꢁꢁ
3.91 (dd, 1 H, J5
= 3.0 Hz, J6a
3.76 (m, 4 H, H-3, OCH3), 3.71–3.68 (m, 3 H, H-4ꢁ, 5ꢁ, 6bꢁꢁꢁ), 3.60–3.56 (m, 3 H,
H-4, ClAc), 3.54–3.50 (m, 2 H, H-5ꢁꢁ, 6bꢁꢁ), 3.24 (t, 1 H, J3ꢁꢁ, 4ꢁꢁ = 8.4 Hz, H-4ꢁꢁ),
2.83 (m, 1 H, H-5ꢁꢁꢁ), 2.36 (s, 3 H, Ac), 2.17 (s, 3 H, Ac), 2.12 (s, 3 H, Ac), 2.10
(s, 3 H, Ac), 2.08 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.02 (s, 3 H, Ac), 2.00 (s, 3 H,
Ac), 1.97 (s, 3 H, Ac), 1.86 (s, 3 H, Ac), 0.90 (s, 9 H, t-Bu), 0.08 (s, 3 H, Si-CH3),
0.07 (s, 3 H, Si-CH3); 13C NMR (CDCl3,151 MHz): 170.6, 170.2, 170.0, 169.4,
169.1, 169.0, 169.0, 167.0, 155.4, 150.8, 138.0, 137.6, 137.1, 129.1, 129.0, 128.5,
128.3, 128.2, 127.9, 127.7, 118.0, 114.5, 101.1 (C-1ꢁꢁ), 100.0 (C-1ꢁ), 99.8 (C-1),
98.8 (C-1ꢁꢁꢁ), 80.1, 79.9, 79.1, 75.5, 75.4, 75.2, 74.9, 74.2, 73.7, 73.4, 73.2, 72.9,
72.7, 72.1, 71.8, 70.9, 68.0, 67.5, 64.1, 62.6, 61.5, 55.6, 40.5, 25.6, 22.7, 21.4,
21.3, 21.0, 20.8, 20.7, 20.6, 17.8, −4.0, −4.4; MALDI-TOF HRMS (positive ion):
calcd for (C76H97ClO31Si+Na+): 1591.5369; found m/z: 1591.5359.
2,6-Di-O-acetyl-4-O-benzyl-3-O-(2-O-acetyl-4-O-benzyl-3-O-((4-
O-benzyl-2,6-di-O-acetyl-6-O-(2,3,4,6-tetra-O-acetyl-β-D-
glucopyranosyl)-6-O-chloroacetyl-β-D-glucopyranosyl)-
β-D-glucopyranosyl))-β-D-glucopyranoside (26)
To a solution of 19 (673 mg, 812 μmol) in dichloromethane (2.71 mL) was
˚
added molecular sieves 4A powder (100 mg), and the mixture was stirred for
1 h and cooled at –40◦C. To the solution was added boron trifluoride–ethyl
ether complex (41 μL, 406 μmol), then added 24 (450 mg, 541 μmol) dissolved
in dichloromethane (1.62 mL) dropwise, and the mixture was stirred at –40◦C
for 1.0 h, at 0◦C for 1.5 h, and then at rt for 14 h. The mixture was diluted
with dichloromethane and filtered through Celite. The filtrate was washed suc-
cessively with aqueous sodium hydrogen carbonate and brine, dried over an-
hydrous sodium sulfate, filtered, and evaporated. The residue was purified by
reverse-phase silica gel chromatography with 4:6 to 7:3 (v/v) acetonitrile–water
1
to give 26 (450 mg, 56%) as a white powder: H NMR δ (CDCl3, 600 MHz)
7.37–7.23 (m, 15 H, Ph-CH2), 6.89–6.88 (m, 2 H, Ph), 6.81–6.79 (m, 2 H, Ph),

Synthesis of Branched Oligo-β-Glucan Derivatives
23
5.25 (t, 1 H, J3ꢁꢁ,4ꢁꢁ = 9.3 Hz, H-3ꢁꢁ), 5.08–4.93 (m, 9 H, J1 , 2 = 7.8 Hz, J1ꢁꢁ, 2ꢁꢁ
ꢁ
ꢁ
= 8.4 Hz, H-1ꢁ, 1ꢁꢁ, 2, 2ꢁ, 2ꢁꢁ, 2ꢁꢁꢁ, 4ꢁꢁꢁ, Ph-CH- ×2), 4.87 (t, 1 H, J3
= 9.0 Hz,
ꢁꢁꢁ,4ꢁꢁꢁ
H-3ꢁꢁꢁ), 4.77 (d, 1 H, J1, 2 = 8.4 Hz, H-1), 4.70 (d, 1 H, J1
= 8.4 Hz, H-1ꢁꢁꢁ),
ꢁꢁꢁ, 2ꢁꢁꢁ
4.61 (d, 1 H, J = 11.4 Hz, Ph-CH-), 4.56 (d, 1 H, J = 11.4 Hz, Ph-CH-), 4.52
(d, 1 H, J = 11.4 Hz, Ph-CH-), 4.51 (d, 1 H, J = 11.4 Hz, Ph-CH-), 4.47 (dd, 1
H, J5ꢁꢁ,6aꢁꢁ = 1.8 Hz, J6aꢁꢁ,6bꢁꢁ = 12.0 Hz, H-6aꢁꢁ), 4.37 (dd, 1 H, J5ꢁꢁ,6bꢁꢁ = 3.6 Hz,
H-6bꢁꢁ), 4.30 (dd, 1 H, J5 ,6a = 2.1 Hz, J6a ,6b = 11.7 Hz, H-6aꢁ), 4.18 (t, 1 H,
J3,4 = 9.3 Hz, H-3), 4.10–4.07 (m, 3 H, H-3, 6a, 6bꢁ), 3.92–3.88 (m, 2 H, H-5ꢁ,
6aꢁꢁꢁ), 3.76 (s, 3 H, OCH3), 3.72–3.56 (m, 8 H, H-4, 4ꢁ, 5, 5ꢁꢁ, 6b, 6bꢁꢁꢁ, ClAc), 3.45
(t, 1 H, H-4ꢁꢁꢁ), 2.81 (m, 1 H, H-5ꢁꢁꢁ), 2.14 (s, 3 H, Ac), 2.11 (s, 3 H, Ac ×3), 2.04
(s, 3 H, Ac), 2.01 (s, 3 H, Ac), 2.00 (s, 3 H, Ac), 1.98 (s, 3 H, Ac), 1.86 (s, 3 H,
Ac); 13C NMR (CDCl3,151 MHz): 170.6, 170.2, 170.0, 169.2, 169.1, 169.0, 169.0,
168.8, 167.0, 155.3, 150.7, 138.0, 137.4, 136.7, 129.0, 128.6, 128.5, 128.4, 128.3,
128.3, 127.9, 127.8, 127.7, 117.9, 114.5, 100.7 (C-1ꢁꢁ), 100.0 (C-1ꢁ), 99.7 (C-1),
98.7 (C-1ꢁꢁꢁ), 80.5, 80.0, 79.1, 76.2, 75.7, 75.7, 75.1, 74.8, 74.8, 74.2, 73.4, 73.1,
72.8, 72.6, 71.9, 71.8, 71.7, 70.9, 67.8, 67.5, 64.0, 62.6, 61.3, 55.6, 40.4, 20.9,
20.9, 20.7, 20.6, 20.6, 20.5, 20.5; MALDI-TOF HRMS (positive ion): calcd for
(C72H85ClO32+Na+): 1519.4610; found m/z: 1519.4638.
ꢁ
ꢁ
ꢁ
ꢁ
para-Methoxyphenyl2-O-acetyl-4-O-benzyl-6-O-(2,3,4,6-tetra-O-
acetyl-β-D- glucopyranosyl)-3-O-(2,6-di-O-acetyl-4-O-benz-
yl-3-O-(2-O-acetyl-4-O-benzyl-3-O- tert-butyldimethylsilyl-6-
O-chloroacetyl-β-D-glucopyranosyl)-β-D- glucopyranosyl)-
β-D-glucopyranoside (27)
To a solution of 20 (50 mg, 66.8 μmol) and 22 (197 mg, 200 μmol) in
˚
dichloromethane (628 μL) was added molecular sieves 4A powder (200 mg),
and the mixture was stirred for 30 min and cooled at –40◦C. To the mixture
was added trimethylsilyl trifluoromethansulfonate (3.6 μL, 20.0 μmol) diluted
in dichloromethane (40 μL), and the mixture was stirred at –40◦C for 0.5 h and
then at rt for 5.5 h. The solution was diluted with dichloromethane and filtered
through Celite, and the filtrate was washed with brine, dried over anhydrous
sodium sulfate, filtered, and evaporated. The residue was purified by flash sil-
ica gel chromatography with 5:1 to 4:1 (v/v) toluene–ethyl acetate to give 27
(34.2 mg, 27%) as a white powder: 1H NMR δ (CDCl3, 400 MHz) 7.40–7.17 (m,
15 H, Ph-CH2), 6.91–6.84 (m, 4 H, Ph), 5.13–4.89 (m, 8 H, H-2, 2ꢁ, 2ꢁꢁ, 2ꢁꢁꢁ, 3ꢁꢁꢁ,
4
ꢁꢁꢁ, Ph-CH- ×3), 4.84 (d, 1 H, J = 11.6 Hz, Ph-CH-), 4.58 (d, 1 H, J1ꢁꢁ, 2ꢁꢁ
=
8.0 Hz, H-1ꢁꢁ), 4.56 (d, 1 H, J1 , 2 = 8.0 Hz, H-1ꢁ), 4.50 (d, 1 H, J1
= 7.6 Hz,
ꢁ
ꢁ
ꢁꢁꢁ, 2ꢁꢁꢁ
H-1ꢁꢁꢁ), 4.47 (d, 1 H, J = 11.6 Hz, Ph-CH-), 4.44 (d, 1 H, J = 11.6 Hz, Ph-CH-),
4.34 (dd, 1 H, J5ꢁꢁ,6aꢁꢁ = 2.0 Hz, J6aꢁꢁ,6bꢁꢁ = 12.0 Hz, H-6aꢁꢁ), 4.22–3.99 (m, 7 H,
H-3, 3ꢁꢁ, 6a, 6aꢁ, 6bꢁꢁ, 6aꢁꢁꢁ, 6bꢁꢁꢁ), 3.87 (t, 1 H, J3 ,4 = 8.0 Hz, H-3ꢁ), 3.78 (s, 3 H,
OCH3), 3.72 (m, 1 H, H-5ꢁꢁ), 3.65–3.46 (m, 5 H, H-4ꢁꢁ, 5, 6b, ClAc), 3.52–3.46
ꢁ
ꢁ

A. Miyagawa et al.
24
(m, 3 H, H-4ꢁ, 5ꢁ, 5ꢁꢁꢁ), 3.39 (t, 1 H, J3,4 = 8.6 Hz, H-4), 2.17 (s, 3 H, Ac), 2.15
(s, 6 H, Ac ×2), 2.05 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.00 (s, 3 H, Ac), 1.99 (s,
3 H, Ac), 1.88 (s, 3 H, Ac), 1.63 (s, 3 H, Ac), 0.90 (s, 9 H, t-Bu), 0.09 (s, 3 H,
Si-CH3), 0.07 (s, 3 H, Si-CH3); 13C NMR (CDCl3,151 MHz): 170.6, 170.6, 170.2,
169.9, 169.3, 169.3, 169.2, 169.0, 167.7, 166.7, 155.5, 151.1, 138.0, 137.9, 137.3,
132.5, 130.9, 128.8, 128.5, 128.3, 128.3, 128.1, 128.0, 127.8, 127.8, 117.9, 114.7,
101.3 (C-1ꢁꢁ), 100.6 (C-1ꢁꢁꢁ), 100.4 (C-1ꢁ), 99.6 (C-1), 80.9, 80.3, 78.3, 75.5, 75.3,
75.2, 74.6, 74.5, 74.3, 73.3, 73.2, 73.1, 72.9, 72.6, 71.7, 71.3, 68.4, 68.2, 68.1,
64.4, 61.8, 60.4, 55.6, 40.4, 38.7, 30.4, 29.7, 28.9, 25.6, 23.7, 23.0, 21.3, 21.1,
20.9, 20.7, 20.6, 20.6, 20.5, 20.5, 17.8, 14.2, 14.0, 10.9, −4.0, −4.4; MALDI-
TOF HRMS (positive ion): calcd for (C76H97ClO31Si+Na+): 1591.5369; found
m/z: 1591.5344.
para-Methoxyphenyl p-Methoxy phenyl 2-O-acetyl-4-O-benzyl-
6-O-(2,3,4,6-tetra-O- acetyl-β-D-glucopyranosyl)-3-O-(2,6-di-
O-acetyl-4-O-benzyl-3-O-(4-O-benzyl-2,3-di-O-acetyl-6-O-
chloroacetyl-β-D-glucopyranosyl)-β-D-glucopyranosyl)-β-D-
glucopyranoside (28)
To a solution of 20 (279 mg, 373 μmol) in dichloromethane (1.86 mL) was
˚
added molecular sieves 4A powder (1.0 g), and the mixture was stirred for 1 h
and cooled at –40◦C. To the mixture was added boron trifluoride–ethyl ether
complex (18.8 μL, 186 μmol) and then 24 (340 mg, 373 μmol) dissolved in
dichloromethane (1.24 mL) dropwise, and the mixture was stirred at –40◦C for
1.0 h and at rt for 45 min. The mixture was diluted with dichloromethane and
filtered through Celite, and the filtrate was washed successively with aqueous
sodium hydrogen carbonate and brine, dried over anhydrous sodium sulfate,
filtered, and evaporated. The residue was purified by reverse-phase silica gel
chromatography with 4:6 to 3:1 (v/v) acetonitrile–water and silica gel chro-
matography with 9:1 to 17:3 (v/v) toluene–acetone to give 28 (82.2 mg, 15%)
1
as a white powder: H NMR δ (CDCl3, 600 MHz) 7.34–7.22 (m, 15 H, Ph-CH2
×3), 6.90–6.85 (m, 4 H, Ph), 5.24 (t, 1 H, J3 ,4 = 9.3 Hz, H-3ꢁꢁ), 5.11–4.93 (m, 8
H, H-2, 2ꢁ, 2ꢁꢁ, 2ꢁꢁꢁ, 3ꢁꢁꢁ, 4ꢁꢁꢁ, Ph-CH- ×2), 4.79 (d, 1 H, J1, 2 = 7.8 Hz, H-1), 4.71
(d, 1 H, J1ꢁꢁ, 2ꢁꢁ = 7.8 Hz, H-1ꢁꢁ), 4.60 (d, 1 H, J = 10.8 Hz, Ph-CH-), 4.51 (d, 1
ꢁ
ꢁ
H, J = 11.4 Hz, Ph-CH-), 4.50 (d, 1 H, J1
= 7.8 Hz, H-1ꢁꢁꢁ), 4.45 (d, 1 H, J
ꢁꢁꢁ, 2ꢁꢁꢁ
= 11.4 Hz, Ph-CH-), 4.44 (d, 1 H, J = 11.4 Hz, Ph-CH-), 4.35 (dd, 1 H, J5ꢁꢁ,6aꢁꢁ
= 2.4 Hz, J6aꢁꢁ,6bꢁꢁ = 12.0 Hz, H-6aꢁꢁ), 4.26 (dd, 1 H, J5ꢁꢁ,6bꢁꢁ = 4.2 Hz, H-6bꢁꢁ),
4.21–4.18 (m, 2 H, H-6aꢁ,6aꢁꢁꢁ), 4.11 (dd, 1 H, J5 ,6b = 3.6 Hz, J5 ,6b = 12.0 Hz,
ꢁ
ꢁ
ꢁ
ꢁ
H-6bꢁ), 4.07–4.01 (m, 3 H, H-3, 6b, 6bꢁꢁꢁ), 3.89 (t, 1 H, J3 ,4 = 9.0 Hz, H-3ꢁ), 3.77
(s, 3 H, OCH3), 3.70–3.46 (m, 9 H, H-4ꢁ, 4ꢁꢁ, 5ꢁ, 5ꢁ, 5ꢁꢁ, 5ꢁꢁꢁ, 6a), 2.19 (s, 3 H, Ac),
2.16 (s, 3 H, Ac), 2.08 (s, 3 H, Ac), 2.03 (s, 3 H, Ac), 2.01 (s, 3 H, Ac), 1.99 (s, 3 H,
Ac ×2), 1.87 (s, 3 H, Ac), 1.76 (s, 3 H, Ac); 13C NMR (CDCl3,151 MHz): 170.6,
ꢁ
ꢁ

Synthesis of Branched Oligo-β-Glucan Derivatives
25
170.5, 170.1, 170.1, 169.8, 169.4, 169.3, 169.1, 169.0, 166.6, 155.4, 151.0, 138.0,
137.9, 136.9, 128.9, 128.8, 128.6, 128.5, 128.4, 128.2, 128.2, 128.1, 128.0, 127.9,
125.2, 117.8, 114.6, 100.9 (C-1ꢁꢁ), 100.5 (C-1ꢁꢁꢁ), 100.2 (C-1ꢁ), 99.4 (C-1), 81.5,
80.0, 75.5, 75.3, 75.1, 74.8, 74.5, 74.5, 74.4, 73.2, 72.9, 72.8, 72.8, 71.6, 71.5,
71.1, 68.3, 68.1, 63.9, 62.6, 61.7, 55.6, 40.3, 21.0, 20.9, 20.7, 20.6, 20.5, 20.5,
20.4, 20.4; MALDI-TOF HRMS (positive ion): calcd for (C72H85ClO32+Na+):
1519.4610; found m/z: 1519.4663.
2,6-Di-O-Acetyl-4-O-benzyl-3-O-tert-butyldimethylsilyl-α-D-gluc-
opyranosyl 2,2,2-trichloroacetimidate (29)
Compound 6 (2.51 g, 5.50 mmol) was dissolved in dichloromethane
(20.0 mL), pyridine (4.00 mL, 49.5 mmol), and acetic anhydride (3.00 mL,
27.2 mmol), and the mixture was stirred at rt for 23.5 h. Then, the mixture
was coevaporated with toluene. The residue was dissolved in acetone/water
(139 mL, 4/1, v/v). To the solution was added N-bromosuccinimide (4.93 g,
27.7 mmol) and the mixture was stirred vigorously at rt for 100 sec. The so-
lution was diluted with ethyl acetate and washed with aqueous sodium hy-
drogen carbonate and brine, dried over anhydrous sodium sulfate, filtered,
and evaporated. The residue was dissolved in dichloromethane (18.4 mL) and
trichloroacetonitrile (2.77 mL, 27.6 mmol) cooled on an ice bath. Then to the so-
lution was added 1,8-diazabicyclo[5.4.0]-7-undecene (165 μL, 1.11 mmol) and
the mixture was stirred at rt for 17 h. The solution was concentrated and the
residue was purified by silica gel chromatography with 9:1 (v/v) hexane–ethyl
acetate to give 29 (2.32 g, 68%) as a white powder: 1H NMR δ (CDCl3, 400 MHz)
8.56 (s, 1 H, NH), 7.37–7.29 (m, 5H, Ph), 6.48 (d, 1 H, J1, 2 = 3.2 Hz, H-1),
4.93–4.88 (m, 2 H, H-2, Ph-CH-), 4.56 (d, 1 H, J = 11.2 Hz, Ph-CH-), 4.34 (dd,
1 H, J5,6a = 2.0 Hz, J6a,6b = 12.0 Hz, H-6a), 4.26 (t, 1H, J3,4 = 9.2 Hz, H-3),
4.13 (dd, 1 H, J5,6b = 3.6 Hz, H-6b), 4.02 (br-d, 1 H, H-5), 3.58 (t, 1 H, H-4), 2.05
(s, 3 H, Ac), 2.01 (s, 3 H, Ac), 0.91 (s, 9 H, t-Bu), 0.13 (s, 3 H, CH3), 0.11 (s, 3
H, CH3); 13C NMR (CDCl3,101 MHz): 170.5, 170.1, 161.0, 137.3, 128.6, 128.1,
127.9, 93.7 (C-1α), 77.7, 75.6, 72.9, 72.0, 71.8, 62.4, 25.7, 20.8, 20.8, 18.0, −4.0,
−4.5.
para-Methoxyphenyl 2,6-di-O-acetyl-4-O-benzyl-3-O-(2-O-acet-
yl-4-O-benzyl-3-O-(2,6-di-O-acetyl-4-O- benzyl-3-O-tert-butyl-
dimethylsilyl-β-D-glucopyranosyl)-6-O-chloroacetyl-β-D- glu-
copyranosyl)-β-D-glucopyranoside (30)
To a solution of 29 (2.32 g, 3.78 mmol) and 24 (6.29 g, 7.57 mmol) in
˚
dichloromethane (34.2 mL) was added molecular sieves 4A powder (8.6 g), and
the mixture was stirred at rt for 1.0 h and cooled at –40◦C for 30 min. To the
mixture was added trimethylsilyl trifluoromethansulfonate (137 μL, 758 μmol)

A. Miyagawa et al.
26
diluted in dichloromethane (3.64 mL) and the mixture was stirred at –40◦C
for 50 min. Then the solution was diluted with dichloromethane and filtered
through Celite, and the filtrate was washed successively with aqueous sodium
hydrogen carbonate and brine, dried over anhydrous sodium sulfate, filtered,
and evaporated. The residue was purified by flash silica gel chromatography
with 19:1 to 12:1 (v/v) toluene–acetone to give 30 (4.23g, 87%) as a white pow-
1
der: H NMR δ (CDCl3, 600 MHz) 7.35–7.24 (m, 15 H, Ph-CH2), 6.89–6.87 (m,
2 H, Ph), 6.80–6.78 (m, 2 H Ph), 5.11 (d, 1 H, J 2, 3 = 8.4 Hz, H-2), 4.98–4.91
(m, 4 H, H-2ꢁ, 2ꢁꢁ, Ph-CH- ×2), 4.89 (d, 1 H, J = 11.4 Hz, Ph-CH-), 4.84 (d, 1 H,
J = 12.0 Hz, Ph-CH-), 4.80 (d, 1 H, J 1, 2 = 7.8 Hz, H-1), 4.61 (d, 1 H, J 1ꢁꢁ, 2ꢁꢁ
=
7.8 Hz, H-1ꢁꢁ), 4.59 (d, 1 H, J 1 , 2 = 7.8 Hz, H-1ꢁ), 4.57 (d, 1 H, J = 12.0 Hz, Ph-
CH-), 4.51 (d, 1 H, J = 11.4 Hz, Ph-CH-), 4.48 (d, 1 H, J = 10.8 Hz, Ph-CH-),
4.36 (br-d, 1 H, J 6a,6b = 12.0 Hz, H-6a), 4.26 (br-d, 1 H, H-6aꢁꢁ), 4.22 (dd, 1 H,
ꢁ
ꢁ
J 5 ,6b = 2.4 Hz, J 6a ,6b = 12.0 Hz, H-6aꢁ), 4.17–4.11 (m, 3 H, H-6b, 6bꢁ, 6bꢁꢁ),
ꢁ
ꢁ
ꢁ
ꢁ
3.99 (m, 1 H, H-3), 3.91 (dd, 1 H, J 2 ,3 = 8.4 Hz, J 3 ,4 = 9.6 Hz, H-3ꢁ), 3.77 (m,
1 H, H-3ꢁꢁ), 3.75 (s, 3 H, OCH3), 3.59–3.47 (m, 8 H, H-4, 4ꢁ, 4ꢁꢁ, 5, 5ꢁ, 5ꢁꢁ, ClAc),
2.17 (s, 3 H, Ac), 2.17 (s, 3 H, Ac), 2.16 (s, 3 H, Ac), 1.93 (s, 3 H, Ac), 1.84 (s,
3 H, Ac), 0.91 (s, 9 H, t-Bu), 0.09 (s, 3 H, Si-CH3), 0.07 (s, 3 H, Si-CH3); 13C
NMR (CDCl3,151 MHz): 170.6, 170.5, 170.0, 169.3, 169.1, 166.8, 155.5, 151.0,
137.8, 137.7, 137.4, 128.6, 128.4, 128.4, 128.3, 128.0, 127.9, 127.9, 127.8, 127.6,
118.3, 114.5, 101.2 (C-1ꢁꢁ), 100.4 (C-1ꢁ), 99.6 (C-1), 80.8, 80.7, 75.2, 74.7, 74.4,
74.1, 73.2, 73.2, 73.1, 72.9, 72.6, 64.2, 62.7, 62.6, 55.6, 40.4, 25.6, 21.3, 21.1,
20.9, 20.7, 20.6, 17.8, −4.0, −4.4; MALDI-TOF HRMS (positive ion): calcd for
(C64H81ClO23Si+Na+): 1303.4524; found m/z: 1303.4525.
ꢁ
ꢁ
ꢁ
ꢁ
para-Methoxyphenyl 2,6-di-O-acetyl-4-O-benzyl-3-O-(2-O-ace-
tyl-4-O-benzyl-6-O-chloroacetyl-3-O-(2,3,4,6-tetra-O-acetyl-
β-D-glucopyranosyl)-β-D-glucopyranosyl)-β-D-glucopyrano-
side (31)
To a solution of 7 (474 mg, 962 μmol) and 24 (400 mg, 481 μmol) in
˚
dichloromethane (1.6 mL) was added molecular sieves 4A powder (1.6 g), and
the mixture was stirred at rt for 30 min and cooled at –40◦C for 30 min. To the
mixture was added boron trifluoride–ethyl ether complex (60.4 μL, 481 μmol)
diluted in dichloromethane (962 μL) and the mixture was stirred at –40◦C
for 2.0 h and then at rt for 15 h. Moreover, to the mixture was added boron
trifluoride–ethyl ether complex (60.4 μL, 481 μmol) diluted in dichloromethane
(962 μL), and it was stirred at rt for 1.0 h. The solution was diluted with
dichloromethane and filtered through Celite, and the filtrate was washed suc-
cessively with aqueous sodium hydrogen carbonate and brine, dried over an-
hydrous sodium sulfate, filtered, and evaporated. The residue was purified by

Synthesis of Branched Oligo-β-Glucan Derivatives
27
reverse-phase silica gel chromatography with 5:5 to 7:3 (v/v) acetonitrile–water
1
to give 31 (336 mg, 60%) as a white powder: H NMR δ (CDCl3, 600 MHz)
7.31–7.25 (m, 10 H, Ph-CH2), 6.89–6.88 (d, J = 9.1 Hz, 2 H, Ph), 6.80–6.79 (d,
J = 9.1 Hz, 2 H Ph), 5.20–5.06 (m, 4 H, H-2, 2ꢁꢁ, 3ꢁꢁ, 4 ꢁꢁ), 5.01–4.95 (m, 4 H, H-
2ꢁ, Ph-CH- ×2), 4.82 (d, 1 H, J1, 2 = 7.8 Hz, H-1), 4.75 (d, 1 H, J1ꢁꢁ, 2ꢁꢁ = 7.8 Hz,
H-1ꢁꢁ), 4.61 (d, 1 H, J1 , 2 = 8.4 Hz, H-1ꢁ), 4.52 (d, 1 H, J = 11.6 Hz, Ph-CH-),
ꢁ
ꢁ
4.49 (d, 1 H, J = 11.2 Hz, Ph-CH-), 4.33 (dd, 1 H, J5ꢁꢁ,6aꢁꢁ = 3.9 Hz, J6aꢁꢁ,6bꢁꢁ
=
12.0 Hz, H-6aꢁꢁ), 4.29–4.26 (m, 2 H, H-6a, 6aꢁ), 4.21 (m, 1 H, H-6b), 4.10 (m, 2
H, H-6bꢁ, 6bꢁꢁ), 4.01 (t, 1 H, J3,4 = 8.2 Hz, H-3), 3.94 (m, 1 H, H-3ꢁ), 3.76 (s, 3 H,
OCH3), 3.74 (m, 1 H, H-5ꢁꢁ), 3.64–3.58 (m, 4 H, H-4, 5ꢁ, ClAc), 3.56–3.54 (m, 2
H, H-4ꢁ, 5), 2.20 (s, 3 H, Ac), 2.19 (s, 3 H, Ac), 2.11 (s, 3 H, Ac), 2.02 (s, 3 H, Ac),
2.01 (s, 3 H, Ac), 1.96 (s, 3 H, Ac), 1.92 (s, 3 H, Ac); 13C NMR (CDCl3,151 MHz):
170.5, 170.1, 169.7, 169.4, 169.3, 169.0, 166.8, 155.4, 150.8, 137.8, 137.5, 128.4,
128.2, 128.0, 127.9, 127.8, 118.2, 114.4, 101.0 (C-1ꢁꢁ), 100.2 (C-1ꢁ), 99.4 (C-1),
81.6, 80.5, 74.6, 74.6, 74.3, 73.2, 72.8, 72.5, 71.7, 71.0, 68.2, 64.1, 62.6, 61.6,
55.6, 40.4, 21.1, 20.9, 20.6, 20.5, 20.5, 20.4; MALDI-TOF HRMS (positive ion):
calcd for (C55H65ClO25+Na+): 1183.3401; found m/z: 1183.3422.
para-Methoxyphenyl 2,6-di-O-acetyl-4-O-benzyl-3-O-(2-O-ace-
tyl-4-O-benzyl-3-O-(2,6-di-O-acetyl-4-O-benzyl-3-O-tert-but-
yldimethylsilyl-β-D-glucopyranosyl)-β-D-glucopyranosyl)-
β-D-glucopyranoside (32)
To a solution of 30 (2.00 g, 1.56 mmol) in ethanol (78.0 ml) was added 1,4-
diazabicyclo[2.2.2]octane (1.10 g, 9.81 mmol), and the mixture was stirred at
50◦C for 7.5 h. The solution was diluted with dichloromethane and washed suc-
cessively with aqueous 1M hydrochloric acid and brine, dried over anhydrous
sodium sulfate, filtered, and evaporated. The residue was purified by silica gel
chromatography with 9:1 to 5:1 (v/v) toluene–acetone to give 32 (1.88g, quan-
1
titative) as a white powder: H NMR δ (CDCl3, 500 MHz) 7.35–7.24 (m, 15 H,
Ph-CH2), 6.89 (d, J = 8.5 Hz, 2 H, Ph), 6.79 (d, J = 9.1 Hz, 2 H, Ph), 5.16 (t,
1 H, J2ꢁꢁ, 3ꢁꢁ = 3.5 Hz, H-2ꢁꢁ), 5.01–4.89 (m, 4 H, H-2, 2ꢁ, Ph-CH- ×2), 4.83 (d,
1 H, J = 11.5 Hz, Ph-CH-), 4.78 (d, 1 H, J1ꢁꢁ, 2 ꢁꢁ = 8.0 Hz, H-1ꢁꢁ), 4.58 (d, 2 H,
J = 9.5 Hz, H-1, Ph-CH-), 4.53 (d, 1 H, J1 , 2 = 8.0 Hz, H-1ꢁ), 4.50–4.46 (d, 2
H, J = 11.5 Hz, Ph-CH- ×2), 4.36–4.30 (m, 2 H, H-6a, 6aꢁꢁ), 4.22–4.15 (m, 2
ꢁ
ꢁ
ꢁ
ꢁ
H, H-6b, 6bꢁꢁ), 3.96 (t, 1 H, J2ꢁꢁ,3ꢁꢁ = 8.3 Hz, H-3ꢁꢁ), 3.90 (t, 1 H, J2 ,3 = 9.0 Hz,
H-3ꢁ), 3.75 (m, 4 H, H-3, OCH3), 3.64–3.60 (m, 3 H, H-4ꢁꢁ, 5ꢁꢁ, 6aꢁ), 3.54–3.53
(m, 2 H, H-4, 5), 3.42 (t, 1 H, J3 ,4 = 8.8 Hz, H-4ꢁ), 3.31–3.29 (m, 2 H, H-5ꢁ, 6bꢁ),
2.18 (s, 3 H, Ac), 2.16 (s, 3 H, Ac), 2.15 (s, 3 H, Ac), 2.00 (s, 3 H, Ac), 1.80 (s, 3
H, Ac), 0.90 (s, 9 H, t-Bu), 0.08 (s, 3 H, Si-CH3), 0.07 (s, 3 H, Si-CH3); 13C NMR
(CDCl3,126 MHz): 170.6, 170.6, 170.0, 169.3, 169.1, 155.5, 151.0, 138.2, 137.9,
137.7, 129.0, 128.5, 128.5, 128.3, 128.2, 128.1, 128.0, 127.9, 127.7, 127.7, 127.6,
ꢁ
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A. Miyagawa et al.
28
125.3, 118.4, 114.5, 101.2 (C-1), 100.6 (C-1ꢁ), 99.9 (C-1ꢁꢁ), 81.0, 80.7, 78.4, 75.7,
75.2, 75.2, 74.9, 74.7, 74.6, 73.4, 73.2, 73.1, 73.1, 73.0, 62.9, 62.6, 62.1, 55.6,
25.6, 21.3, 21.0, 20.9, 20.8, 20.6, 17.8, −4.0, −4.4; MALDI-TOF HRMS (posi-
tive ion): calcd for (C62H80O22Si +Na+): 1227.4808; found m/z: 1227.4826.
para-Methoxyphenyl 2,6-di-O-acetyl-4-O-benzyl-3-O-(2-O-acet-
yl-4-O-benzyl-3-O-(2,3,4,6-tetra-O-acetyl-β-D-glucopyrano-
syl)-β-D-glucopyranosyl)-β-D-glucopyranoside (33)
To a solution of 31 (280 mg, 241 μmol) in ethanol (12.0 mL) was added 1,4-
diazabicyclo[2.2.2]octane (170 mg, 1.52 mmol), and the mixture was stirred at
50◦C for 13 h. The solution was diluted with dichloromethane and washed suc-
cessively with aqueous 1M hydrochloric acid and brine, dried over anhydrous
sodium sulfate, filtered, and evaporated. The residue was purified by silica gel
chromatography with 3:1 to 1:1 (v/v) toluene–ethyl acetate to give 33 (258 mg,
99%) as a white powder: 1H NMR δ (CDCl3, 400 MHz) 7.36–7.23 (m, 10 H, Ph-
CH2), 6.89 (d, J = 9.1 Hz, 2 H, Ph), 6.78 (d, J = 9.1 Hz, 2 H Ph), 5.20–4.89
(m, 7 H, H-2, 2ꢁ,2ꢁꢁ, 3ꢁꢁ, 4ꢁꢁ, Ph-CH- ×2), 4.78 (d, 1 H, J1 , 2 = 7.8 Hz, H-1ꢁ),
4.74 (d, 1 H, J1ꢁꢁ, 2ꢁꢁ = 8.0 Hz, H-1ꢁꢁ), 4.58 (d, 1 H, J = 9.6 Hz, Ph-CH-), 4.55
(d, 1 H, J1, 2 = 7.7 Hz, H-1), 4.47 (d, 1 H, J = 11.0 Hz, Ph-CH-), 4.33–4.28
ꢁ
ꢁ
(m, 2 H, H-6aꢁ, 6aꢁꢁ), 4.18 (dd, 1 H, J5 ,6a = 3.8 Hz, J6a ,6b = 11.8 Hz, H-6bꢁ),
4.04-4.00 (m, 2 H, H-3ꢁ, 6bꢁꢁ), 3.92 (t, 1 H, J2,3 = 9.0 Hz, H-3), 3.71 (s, 3 H,
OCH3), 3.71–3.54 (m, 4 H, H-4ꢁ, 5ꢁ, 5ꢁꢁ, 6a), 3.46 (t, 1 H, J 3,4 = 9.0 Hz, H-4),
3.40–3.30 (m, 2 H, H-5, 6b), 2.21 (s, 3 H, Ac), 2.14 (s, 3 H, Ac), 2.09 (s, 3 H, Ac),
1.98 (s, 3 H, Ac), 1.98 (s, 3 H, Ac), 1.96 (s, 3 H, Ac), 1.92 (s, 3 H, Ac); 13C NMR
(CDCl3,101 MHz): 170.3, 169.9, 169.5, 169.2, 169.0, 168.8, 155.3, 150.7, 137.8,
137.5, 137.4, 128.8, 128.6, 128.2, 128.2, 128.1, 128.0, 128.0, 127.8, 127.6, 127.6,
127.5, 127.1, 118.1, 114.3, 100.9 (C-1ꢁꢁ), 100.2 (C-1ꢁ), 99.5 (C-1), 81.5, 80.0, 75.6,
75.2, 74.7, 74.5, 73.0, 72.8, 72.7, 72.6, 72.4, 70.9, 68.0, 62.3, 61.6, 61.5, 55.3,
21.2, 20.8, 20.7, 20.4, 20.3, 20.2, 20.2; MALDI-TOF HRMS (positive ion): calcd
for (C53H64O24+Na+): 1107.3685; found: m/z: 1107.3624.
ꢁ
ꢁ
ꢁ
ꢁ
para-Methoxyphenyl 2,6-di-O-acetyl-4-O-benzyl-3-O-(2-O-acet-
yl-4-O-benzyl-3-O-(2,6-di-O-acetyl-4-O-benzyl-3-O-tert-butyl-
dimethylsilyl-β-D-glucopyranosyl)-6-O-(2,3,4,6-tetra-O-acetyl-
β-D-glucopyranosyl)-β-D-glucopyranosyl)-β-D-glucopyranos-
ide (34)
To a solution of 7 (1.56 g, 3.17 mmol) and 32 (1.85 g, 1.53 mmol) in
˚
dichloromethane (13.8 mL) was added molecular sieves 4A powder (5.00 g),
and the mixture was stirred at rt for 1.0 h and then at –40◦C for 30 min.
To the mixture was added trimethylsilyl trifluoromethansulfonate (166 μL,