Synthesis of type 2 Lewis antigens via novel regioselective glycosylation of an orthogonally protected lactosamine diol derivative

Article abstract of DOI:10.1016/j.carres.2016.01.003

The novel and efficient synthesis of type 2 Lewis antigens is reported in this study. The rationally designed lactosamine-3,2′-diol derivative with an orthogonal set of protecting groups is efficiently glycosylated with a benzyl protected 1-thio-L-fucosid

Full text of DOI:10.1016/j.carres.2016.01.003

Carbohydrate Research 422 (2016) 34–44
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis of type 2 Lewis antigens via novel regioselective
glycosylation of an orthogonally protected lactosamine diol derivative
Yuji Yamazaki, Kyohei Sezukuri, Junko Takada, Hiroaki Obata, Shunsaku Kimura,
Masashi Ohmae *
Department of Material Chemistry, Graduate School of Engineering, Kyoto University, Kyoto-daigaku-katsura, Nishikyo-ku, Kyoto 615-8510, Japan
A R T I C L E
I N F O
A B S T R A C T
Article history:
Received 26 November 2015
Received in revised form 12 January 2016
Accepted 13 January 2016
Available online 19 January 2016
The novel and efficient synthesis of type 2 Lewis antigens is reported in this study. The rationally de-
signed lactosamine-3,2′-diol derivative with an orthogonal set of protecting groups is efficiently glycosylated
with a benzyl protected 1-thio-l-fucoside donor in a unique regioselective manner to produce Lewis x
(Lex) and Lewis y (Ley) derivatives in good yields. These derivatives can be prepared not only exclusively
but also synchronously by choosing the appropriate reaction temperature and donor–acceptor molar ratio.
The Lex derivatives are easily converted into sulfated or non-sulfated Lex bearing a terminal azido
functionalized oligo-(ethyleneoxide) linker; the Ley derivative having the same linker can also be pre-
pared, all of which can be further used for the chemical modification of other compounds and materials.
© 2016 Elsevier Ltd. All rights reserved.
Keywords:
Type 2 Lewis antigens
Sulfated Lewis x
Lewis y
Regioselective glycosylation
The Heyns rearrangement
1. Introduction
synthetic methods. A large number of reports on the synthesis of
Lewis antigens have been published to date;5 however, none of them
Lewis antigens are well-known as glycan-based blood group
antigens,1 which are classified as type 1 and type 2, depending on
the disaccharide core structure. Type 1 Lewis antigens, Lewis a and
Lewis b, have a common backbone [→3Galβ(1→3) GlcNAcβ1→] and
exist widely in the membrane of erythrocytes. The type 2 core struc-
ture [→3Galβ(1→4) GlcNAcβ1→] is also found in numerous
glycoconjugates; however, the distribution of its fucosylated de-
rivatives, Lewis x (Lex) and Lewis y (Ley), classified as type 2 Lewis
antigens (T2-LAs), is limited to some epithelial cells and leuko-
cytes. Notably, T2-LAs are overexpressed in various tumor cells,2 and
are thus frequently used as biomarkers for the diagnosis of cancer.
Furthermore, these T2-LAs are sometimes found in their sulfated
form, which includes a 6-O-sulfo-GlcNAc residue.3 The Lex deter-
minant also plays a critical role in various biological events such
as inflammation, lymphocyte homing, and infection of pathogens.4
Thus, T2-LAs have attracted much attention as promising target com-
pounds for cancer therapy as well as for the treatment of
inflammation, infectious diseases, etc.
have reported the use of common key intermediates to construct
T2-LAs, including their sulfated form. In the present study, we report
the successful and rapid assembly of T2-LAs (3–5) via a refined di-
saccharide key intermediate 2, which can be readily prepared from
lactulose 1 through the Heyns rearrangement method6 (Fig. 1). This
method is very convenient to obtain useful 2-amino-2-deoxy sugars,
particularly lactosamine derivatives.7
2. Results and discussion
2.1. Refinement of the molecular design of key intermediate 2
In a previous paper,8 we reported the synthesis of T2-LAs having
a set of orthogonal protecting groups via the useful disaccharide in-
termediate 2′ (Fig. 2). Compound 2′ was found to be an excellent
intermediate for the synthesis of T2-LAs, but had a major draw-
back concerning the removal of the anomeric 4-methoxyphenyl
(PMP) group: the 6-O-tert-butyldimethylsilyl (TBDMS) group was
found to be labile under oxidation conditions by cerium(IV) am-
monium nitrate (CAN). It is acceptable to produce neutral, non-
sulfated T2-LAs; however, in order to synthesize structurally
complicated and highly bioactive sulfated T2-LAs, the protecting
group at glucosamine C6 must be stable under oxidation condi-
tions. Thus, we refined the molecular design from 2′ to 2: the stability
of the tert-butyldiphenylsilyl (TBDPS) group is several hundred times
higher than that of TBDMS under acidic conditions, whereas both
In order to utilize T2-LAs and their derivatives as bioactive com-
pounds, it is essential to establish versatile, widely applicable
*
Corresponding author. Department of Material Chemistry, Graduate School of
Engineering, Kyoto University, Kyoto-daigaku-katsura, Nishikyo-ku, Kyoto 615-8510,
Japan. Tel.: +81 75 383 2403; fax: +81 75 383 2401.
E-mail address: ohmae@peptide.polym.kyoto-u.ac.jp (M. Ohmae).
http://dx.doi.org/10.1016/j.carres.2016.01.003
0008-6215/© 2016 Elsevier Ltd. All rights reserved.

Y. Yamazaki et al./Carbohydrate Research 422 (2016) 34–44
35
OSO3Na
OH
O
O
HO
HO
O
O
N3
O
HO
HO
OH
O
OH
OH
NHAc
O
3
OH
OH
HO
O
H3C
O
OH
O
OH
OH
HO
1
3: sulfo-Lex
OH
OH
O
O
HO
HO
O
O
OTBDPS
O
O
N3
OH
O
NHAc
3
BzO
O
OH
OPMP
NPhth
HO
O
H3C
O
O
OH
O
OH
HO
4: Lex
2
PMP
HO
OH
OH
O
H3C
HO
OH
O
O
O
O
N3
O
O
O
NHAc
3
OH
H3C
HO
O
OH
OH
HO
5: Ley
Fig. 1. Efficient synthesis of T2-LAs (3–5) via the refined orthogonally protected T2 disaccharide derivative 2 derived from lactulose 1.
show similar susceptibility to tetra-n-butylammonium fluoride
(TBAF).9 Therefore, TBDPS was selected as a more suitable protect-
ing group for the C6 position of the glucosamine residue. Further,
the 4,6-O-benzylidene protecting group for the Gal residue was re-
placed with the p-methoxybenzylidene group, which is more rapidly
removed by hydrogenolysis.
2.2. On demand synthesis of Lex and Ley derivatives by regioselective
α-fucosylation of 2
Glycosylation of 2 with benzyl-protected phenyl 1-thio-l-
fucopyranoside (9)11 is the extremely unique reaction throughout the
synthesis of T2-LAs (Scheme 2). In order to obtain the Lex deriva-
tive 10, the glycosylation was carried out at lower temperature (−78 °C)
with a slight excess of 9 over 2; under these conditions, 10 was iso-
lated as the sole product in 76% yield (Table 1, entry 1). Ley derivative
11 was exclusively formed in 83% yield when more than twice the
amount of 9 (2.4 eq) relative to 2 was used at a higher temperature
of −40 °C (entry 2). The synchronous synthesis of 10 and 11 using 9
and 2 (entries 3 and 4) is worth mentioning. Compound 11 ap-
peared at higher temperatures (−40 °C or −50 °C) than that in entry
1. Furthermore, both 10 and 11 were obtained efficiently in 49% and
45% yields, respectively, using an excess amount of 9 (1.8 eq) at −40 °C
(entry 4). These results indicate that the synthesis of 10 and 11 can
be finely controlled by varying the reaction temperature and the feed
ratio. Notably, compounds 10 and 11 can be easily separated by con-
ventional silica gel column chromatography: the Rf values for 10 and
11 in an n-hexane–EtOAc 2:1 mixture are 0.28 and 0.44, respectively.
According to our previous report,8 the lactosamine derivative of
6 was readily prepared from lactulose 1 via the Heyns rearrange-
ment (Scheme 1). After removal of the acetyl protecting groups in
6, the 4′- and 6′-hydroxy moieties were protected with a
p-methoxybenzylidene group to afford 7 in 69% yield via a two-
step procedure. The 6-OH moiety in 7 reacted selectively with TBDPS-
Cl in pyridine, giving 8 in moderate yield (59%). We originally
controlled the selectivity in the regioselective 3′-O-benzoylation by
lowering the reaction temperature (−50 °C). However, the unfavor-
able 3,3′-di-O-benzoyl product was formed even under the optimized
conditions designed to obtain the target 3′-mono-O-benzoyl product.
In the present study, we exclusively obtained the target product 2
at ambient temperature in 73% yield by employing a metal-
coordinated regio- and chemoselective nucleophilic substitution
method.10
OTBDMS
OH
OTBDPS
OH
O
O
BzO
O
BzO
O
OPMP
NPhth
HO
OPMP
NPhth
HO
O
O
O
O
O
O
Ph
2'
2
PMP
Fig. 2. Refinement of the molecular design of key intermediate 2.

36
Y. Yamazaki et al./Carbohydrate Research 422 (2016) 34–44
HO
O
HO
OH
OH
OH
OH
HO
O
O
OH
1
OAc
OH
O
OH
OAc
O
AcO
OAc
a
HO
O
O
OPMP
NPhth
AcO
OPMP
HO
O
O
NPhth
OAc
O
O
6
7
PMP
b
OTBDPS
O
OTBDPS
O
OH
O
OH
O
c
HO
O
BzO
O
O
OPMP
NPhth
HO
OPMP
NPhth
HO
O
O
O
2
8
PMP
PMP
Scheme 1. Reagents and conditions: (a) (1) MeONa/MeOH, (2) p-anisaldehyde dimethylacetal, ( )-10-camphorsulfonic acid/DMF, 30 °C, 24 h, 69% (2 steps); (b) TBDPS-Cl/
pyridine, rt, 64 h, 59%; (c) Bu2SnCl2, PEMP, BzCl/THF, rt, 48 h, 73%. NPhth: phthalimido, PMP: 4-methoxyphenyl, PEMP: 1,2,2,6,6-pentamethylpiperidine.
It is very intriguing that the reactivity of the two hydroxy groups
in 2 is strictly fixed as 3-OH > 2′-OH: a mono-fucosylated product
at 2′-OH, that is, a type 2H [Fucα(1→2)Galβ(1→4)GlcNAcβ1→]
derivative is not formed at all in this series of reactions. These results
are consistent with our previous report,8 although the molecular
design of acceptor 2 is slightly different from that of 2′. Thus, the
combination of the newly designed diol acceptor 2 and donor 9 has
proved to be highly effective for not only the selective synthesis of
Lex or Ley derivatives but also the synchronous synthesis of both
derivatives in a one-pot reaction. Although there are a few reports
on lactosamine diol derivatives for the synthesis of T2-LAs,12 the order
of reactivity of the two hydroxy groups in these derivatives is
OTBDPS
OH
O
BzO
H3C
BnO
SPh
OBn
O
O
a
OPMP
O
2
O
+
NPhth
OBn
O
O
9
H3C
O
OBn
PMP
OBn
BnO
10
BnO
H3C
OBn
OBn
O
OTBDPS
O
O
BzO
O
O
OPMP
NPhth
b
O
O
O
H3C
PMP
O
OBn
OBn
BnO
11
c
10
11
+
Scheme 2. On demand and synchronous syntheses of Lex and Ley derivatives via glycosylation of 2 with 9 under the reaction conditions summarized in Table 1.

Y. Yamazaki et al./Carbohydrate Research 422 (2016) 34–44
37
2.3. Synthesis of 6-O-sulfo-Lex 3 and non-sulfated Lex 4 bearing a
terminal azido functionalized oligo-ethyleneoxide linker
Table 1
One-pot synthesis of 10 and 11 under different reaction conditionsa
Entry Path 2/eq 9/eq NIS/eq TfOH/eq T/°C Time/hb Yield/%
For future applications of T2-LAs, we introduced a terminal azido
functionalized aglycon moiety. A highly hydrophilic oligo-
(ethyleneoxide) structure is advantageous for conjugation with all
kinds of bioactive compounds such as proteins, lipids, polysaccha-
rides, and synthetic polymers. Furthermore, azides are the first choice
in current biochemical and materials sciences as they allow con-
jugation with a range of substances having alkyne groups via a
Huisgen cycloaddition (“click chemistry”).13,14 Hence, we selected
a 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl group as an effi-
cient linker for T2-LAs, as shown in Fig. 1.
10
11
1
2
3
4
a
b
c
c
1.0
1.0
1.0
1.0
1.2
2.4
1.2
1.8
2.5
5.0
2.5
2.5
0.2
0.4
0.2
0.2
−78
−40
−50
−40
1.0
3.0
2.0
1.0
76
n.d.c
83
16
n.d.c
25
49
45
a
Reaction was carried out under the indicated conditions in CH2Cl2–Et2O using the
NIS–TfOH activation system. b Time for complete consumption of 9. c Not detected.
2′-OH > 3-OH without exception, which is opposite to that for 2 and
renders the preparation of Lex derivatives difficult. Therefore, com-
pound 2 is the most effective acceptor capable of providing both
Lex and Ley derivatives when using 9 as the donor.
Two types of Lex derivatives (3 and 4) were synthesized follow-
ing the reactions outlined in Scheme 3. In order to avoid damaging
the α-l-fucoside linkage and azido group, hydrogenation of 10 was
OR3
OR2
R1O
O
O
a
OR4
NPhth
O
10
O
OR2
OR2
H3C
O
OR2
OR2
R2O
12: R1=Bz, R2=Ac, R3=TBDPS, R4=PMP
b
OR3
O
OR3
OR2
OR2
R1O
R1O
c
O
O
O
NH
O
O
O
O
O
OH
OR2
NPhth
OR2
NPhth
OR2
H3C
CCl3
OR2
H3C
O
O
OR2
OR2
OR2
OR2
R2O
R2O
14: R1=Bz, R2=Ac, R3=TBDPS
13: R1=Bz, R2=Ac, R3=TBDPS
d
OR3
OR3
OR2
OR2
e
R1O
R1O
O
O
O
O
OR4
NPhth
OR4
O
O
O
O
OR2
OR2
NHAc
OR2
H3C
OR2
H3C
O
O
OR2
OR2
OR2
OR2
R2O
R2O
16: R1=R2=Ac, R3=H,
15: R1=Bz, R2=Ac, R3=TBDPS,
R4=
R4=
N3
N3
f
O 3
O 3
17: R1=R2=Ac, R3=SO3HNEt3,
R4=
N3
O 3
g
h
3: R1=R2=H, R3=SO3Na,
R4=
N3
N3
O 3
4: R1=R2=R3=H,
R4=
O 3
Scheme 3. Reagents and conditions: (a) (1) Pd(OH)2-C, H2/MeOH, rt, 10 h, (2) Ac2O/pyridine, rt, overnight, 39% (2 steps); (b) CAN/CH3CN–H2O, 0 °C, 6 h, 61%; (c) CCl3CN,
DBU/CH2Cl2, 0 °C, 5 h, 83%; (d) HO(CH2CH2O)3C2H4N3, TMSOTf, MS4A/CH2Cl2, −50 °C, 8 h, 48%; (e) (1) MeONa/MeOH, rt, overnight, (2) NH2NH2·H2O/EtOH, 90 °C, 7 h, (3) Ac2O/
pyridine, rt, 48 h, (4) TBAF–AcOH/THF, rt, 72 h, 56% (4 steps); (f) SO3·NMe3/DMF, 55 °C, 72 h, 91%; (g) MeONa/MeOH, rt, overnight, 63%; (h) MeONa/MeOH, rt, overnight,
15% (5 steps from 15).

38
Y. Yamazaki et al./Carbohydrate Research 422 (2016) 34–44
first carried out with Pd(OH)2 on activated carbon (Pd(OH)2-C) under
H2 atmosphere. This reaction normally proceeds to completion within
10 h, and the reaction mixture must be immediately worked-up,
as prolonged reaction accelerates the undesirable cleavage of the
α-l-fucoside linkage due to the acidity of the reagents. After acety-
lation, compound 12 was obtained in 39% yield, in two steps.
Considering the low yield and our observations by TLC monitor-
ing during the hydrogenation, removal of the benzyl protection in
11 without cleaving the α-l-fucoside linkage seems difficult. The
anomeric PMP group of 12 was smoothly removed by CAN oxida-
tion to produce 13 in 61% yield. Compound 13 was converted into
the activated glycosyl donor of trichloroacetimidate 14 through the
reaction of trichloroacetonitrile with DBU in 83% yield. The linker
moiety was introduced through the glycosylation of 2-(2-(2-(2-
azidoethoxy)ethoxy)ethoxy)-1-ethanol with 14, promoted by the
addition of TMSOTf at −50 °C, affording 15 in a modest yield of 48%.
The acyl groups in 15 were removed successively by treatment with
MeONa and hydrazine monohydrate, followed by acetylation in pyri-
dine and the removal of the TBDPS group with TBAF, affording 16
in 56% yield (4 steps). Sulfation at the 6-OH group in 16 was carried
out by the addition of SO3·NMe3 to produce 17 in excellent yield
(91%). Finally, all of the O-acetyl groups in 17 were removed with
MeONa, which resulted in the target 6-O-sulfo-Lex 3 in 63% yield.
The non-sulfated Lex derivative 4 was obtained from 15 through the
reactions in steps e and h in 15% yield (5 steps). Thus, the sulfated
and non-sulfated forms of the Lex derivatives were efficiently syn-
thesized from a common intermediate, 10, which could be readily
prepared through the regioselective glycosylation described above.
reaction, i.e., removal of the acetyl and benzoyl groups by MeONa,
removal of the phthaloyl group by hydrazine monohydrate and acety-
lation, which gave 21 in 64% yield. All the protecting groups in 21
were removed by successive treatment with TBAF in THF and MeONa
in MeOH, which led to the target Ley derivative 5 in 57% yield via two
steps. Thus, the Ley derivative can also be synthesized easily from 11,
which in turn can be obtained by the glycosylation described above.
3. Conclusion
In the present study, we have demonstrated for the first time
the feasibility of the on-demand synthesis of Lex and Ley deriva-
tives, in addition to their synchronous synthesis in one-pot through
the combined use of diol acceptor 2 and benzyl-protected thiophenyl
fucoside donor 9. The selectivity was easily controlled by varying
the reaction temperature and the ratio of 2 and 9. The derivatives
of Lex and Ley were further functionalized by introducing an oligo-
ethyleneoxide-azide linker through glycosylation. The versatile set
of orthogonal protecting groups of the obtained Lex derivative 15
enabled the facile and regioselective synthesis of both sulfated Lex
3 and non-sulfated Lex 4. Ley derivative 5 was also easily prepared
from 11. Thus, our method is highly efficient for the synthesis of
T2-LAs and will be further applied to the preparation of a variety
of bioactive materials.
4. Experimental design
4.1. General methods
2.4. Synthesis of Ley bearing a terminal azido functionalized
oligo-ethyleneoxide linker 5
Anhydrous solvents were purchased from Wako Pure Chemical
Industries, Ltd., and stored under Ar atmosphere prior to use. Other
chemicals were used without further purification unless other-
wise stated. Molecular sieves (MS) AW300 and 4A were powdered
and activated over 100 °C under reduced pressure with P2O5 as des-
iccant prior to use. Silica gel flash column chromatography was
performed on Silica Gel 60, spherical, neutrality (Nacalai Tesque),
or with a CombiFlash Rf 75 Var (Teledyne Isco) on RediSep Rf Gold
Normal Phase Silica columns. The reactions were monitored by TLC
(silica gel 60 F254, Merck) visualized by sprayed with a mixture of
H3(PMo12O40)·n H2O (12.5 g) and Ce(SO4)2·nH2O (5 g) in 10% H2SO4
(500 mL) and colored by heating at 140 °C. Glycosylation reac-
tions at −40 °C or lower were performed on UCR-150 (Techno-
sigma). Optical rotations were measured with a P-1010 polarimeter
(Jasco). 1H and 13C NMR were recorded on a DPX-400 spectrometer
The Ley derivative bearing a terminal azido functionalized oligo-
ethyleneoxide linker 5 was also prepared according to the reactions
outlined in Scheme 4. Compound 11 was treated with Pd(OH)2-C in
THF–MeOH (1:1) mixture under H2 atmosphere as described for the
synthesis of 12. The obtained mixture was subjected to acetylation
to provide pure 18. The anomeric PMP group in 18 was removed by
CAN oxidation, followed by trichloroacetimidation to give 19.
Glycosidation of 19 with 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)-1-
ethanol, which is the same acceptor employed in the synthesis of 15,
proceeded very smoothly with the addition of a catalytic amount of
TMSOTf at −50 °C and gave 20 within 30 min in a very good yield of
80%. Compound 20 was converted into 21 through a three-step
R2O
OR2
R2O
OR2
OR2
OR2
O
O
H3C
H3C
OR3
O
OR3
O
O
O
R1O
R1O
a
O
d
O
OR4
NPhth
O
OR4
11
O
O
O
NHAc
OR2
OR2
OR2
H3C
OR2
H3C
O
O
OR2
OR2
OR2
OR2
R2O
R2O
18: R1=Bz, R2=Ac, R3=TBDPS, R4=PMP
21: R1=R2=Ac, R3=TBDPS,
b
19: R1=Bz, R2=Ac, R3=TBDPS, R4=C(=NH)CCl3
R4=
N3
O 3
e
c
20: R1=Bz, R2=Ac, R3=TBDPS, R4=
N3
O 3
5: R1=R2=R3=H,
R4=
N3
O 3
Scheme 4. Reagents and conditions: (a) (1) Pd(OH)2-C, H2/THF–MeOH, rt, 10 h, (2) Ac2O, DMAP/pyridine, rt, 24 h, 68% (2 steps); (b) (1) CAN/CH3CN–H2O, rt, 2 h, (2) CCl3CN,
DBU/CH2Cl2, 0 °C, 4 h, 56% (2 steps); (c) HO(CH2CH2O)3C2H4N3, TMSOTf, MS4A/CH2Cl2, −50 °C, 0.5 h, 80%; (d) (1) MeONa/MeOH, rt, 2 h, (2) NH2NH2·H2O/EtOH, 90 °C, 13 h,
(3) Ac2O/pyridine, rt, overnight, 64% (3 steps); (e) (1) TBAF/THF, rt, 72 h, (2) MeONa/MeOH, rt, overnight, 57% (2 steps).

Y. Yamazaki et al./Carbohydrate Research 422 (2016) 34–44
39
(Bruker). Assignments were based on homo- and heteronuclear cor-
relation measurements, and DEPT measurements. High resolution
mass spectrometry was carried out with JMS-HX110A spectrom-
eter (Jeol, for FAB-MS) or Exactive spectrometer (Thermo Fisher
Scientific, for ESI-MS). Melting points were determined with an MP-
500P (Yanaco).
H-2II, OMe), 3.59 (ddd, 1H, J2,3 9.5, J3,OH 9.5, J3,4 3.5 Hz, H-3II), 3.49 (bs,
1H, H-5II), 2.44 (d, 1H, J3,OH 9.5 Hz, 3II-OH), 2.34 (d, 1H, J2,OH 2.5 Hz,
2II-OH), 1.08 (s, 9H, Me3 of tert-Bu); 13C NMR (100 MHz, CDCl3): δ
168.51, 168.04 (C = O), 160.11, 155.33, 150.98, 149.54, 136.20, 135.84,
135.65, 134.09, 133.49, 132.78, 131.70, 130.04, 129.68, 127.76, 127.66,
123.81, 118.80, 114.39, 113.49 (aromatic), 103.73 (C1II), 101.17 (CH
of 4-methoxybenzylidene), 97.42 (C1I), 81.06 (C4I), 75.32 (C3II), 75.09
(C4II), 72.81 (C5I), 71.19 (C2II), 69.75 (C3I), 68.67 (C6II), 66.93 (C5II),
62.41 (C6I), 56.37 (C2I), 55.61, 55.22 (OMe), 26.81 (CMe3 of tert-
Bu), 19.36 (CMe3 of tert-Bu); HRMS (FAB, positive ion mode, NBA)
m/z = 956.3308 [M + Na]+, calcd for C51H55NO14SiNa, 956.3290.
4.2. 4-Methoxyphenyl 4,6-O-(4-methoxybenzylidene)-β-D-
galactopyranosyl-(1→4)-2-deoxy-2-phthalimido-β-D-
glucopyranoside (7)
Compound 68 (13.5 g, 16.2 mmol) in dry MeOH (250 mL) was
treated with MeONa in MeOH (ca. 28wt%, 2.4 mL) at 0 °C under dry
atmosphere for 23 h. The formed precipitate was filtered through
filter paper, and washed with MeOH. The filtrate was neutralized
by addition of Dowex 50W-X8 (H+ form), filtered through a cotton
bed, and concentrated to dryness by vacuum pump overnight. The
former precipitate and the latter residue were combined and dis-
solved in dry DMF (120 mL). To a solution of the mixture was added
p-anisaldehyde dimethylacetal (3.16 mL, 18.6 mmol) under acidic
conditions in the presence of catalytic amount of ( )-10-
camphorsulfonic acid. After stirring at rt for 7 h, excess amount of
Et3N was added to neutralize the reaction system. The mixture was
concentrated under diminished pressure, and coevaporated with
toluene. The residue was purified by silica gel column chromatog-
raphy (CHCl3/MeOH, 20:1, v/v, containing 0.5% Et3N) to provide 7
(7.75 g, 11.1 mmol, 69 %) as a white solid.
4.4. 4-Methoxyphenyl 3-O-benzoyl-4,6-O-(4-methoxybenzylidene)-
β-D-galactopyranosyl-(1→4)-6-O-tert-butyldiphenylsilyl-2-deoxy-
2-phthalimido-β-D-glucopyranoside (2).
To a solution of compound 8 (2.40 g, 2.57 mmol) in dry THF
(50 mL) was added Bu2SnCl2 (78.1 mg, 0.26 mmol) and 1,2,2,6,6-
pentamethylpiperidine (0.92 mL, 5.14 mmol), followed by stirring
for 10 min at rt under Ar atmosphere. Benzoyl chloride (0.36 mL,
2.57 mmol) was added dropwise to the mixture at rt under Ar at-
mosphere. After stirring for 48 h, MeOH was added to quench excess
reagent, and the mixture was evaporated under reduced pressure.
The residue was diluted with CHCl3, and washed with satd aq
NaHCO3 and brine. The organic layer was dried over MgSO4, fil-
tered through a Celite bed, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography (CHCl3/
EtOAc, 1:0 to 0:1, v/v, linear gradient) to afford 2 (1.95 g, 1.88 mmol,
73%) as colorless amorphous.
23
[α]D −20.8 (c 0.64, CHCl3); mp 128–130 °C; Rf 0.30 (CHCl3/
MeOH, 10:1); 1H NMR (400 MHz, CD3OD): δ (ppm) 7.96–7.78 (m,
4H, NPhth), 7.47–6.72 (m, 8H, -C6H4-OMe × 2), 5.70 (d, 1H, J1,2 8.5 Hz,
H-1I), 5.56 (s, 1H, CH of p-methoxybenzylidene), 4.55 (d, 1H, J1,2
7.5 Hz, H-1II), 4.51 (dd, 1H, J2,3 11.0, J3,4 8.5 Hz, H-3I), 4.28 (dd, 1H,
J1,2 8.6, J2,3 11.0 Hz, H-2I), 4.24–4.06 (m, 3H, H-4II, H-6IIa, H-6Ia), 4.06–
3.93 (m, 2H, H-6IIb, H-6Ib), 3.84 (t, 1H, J3,4 = J4,5 = 9.6 Hz, H-4I), 3.77
(s, 3H, OMe), 3.73–3.63 (m, 7H, H-5I, H-2II, H-3II, H-5II, OMe); 13C NMR
(100 MHz, CDCl3): δ 168.23, 167.93 (C = O), 159.89, 155.21, 150.79,
134.03, 131.58, 130.38, 127.76, 123.32, 118.32, 114.32, 113.30 (ar-
omatic), 103.99 (C1II), 100.92 (CH of p-methoxybenzylidene), 97.45
(C1I), 81.92 (C4I), 75.53 (C4II), 75.05 (C5I), 72.08 (C3II), 70.58 (C2II),
69.70 (C3I), 68.73 (C6II), 66.82 (C5II), 61.54 (C6I), 56.07 (C2I), 55.47,
55.17 (OMe); HRMS (FAB, positive ion mode, NBA) m/z = 718.2103
[M + Na]+, calcd for C35H37NO14Na, 718.2112.
[α]D23 +23.9 (c 1.0, CHCl3); Rf 0.41 (n-hexane/EtOAc, 1:1); 1H NMR
(400 MHz, CDCl3, TMS): δ 8.13–6.69 (m, 27H, aromatic), 5.75 (d,1H,
J1,2 8.0 Hz, H-1I), 5.41 (s, 1H, CH of 4-methoxybenzylidene), 5.01 (dd,
1H, J2,3 10.0, J3,4 3.5 Hz, H-3II), 4.60 (d, 1H, J1,2 7.6 Hz, H-1II), 4.54 (t,
1H, J2,3 = J3,4 = 8.6 Hz, H-3I), 4.50–4.41 (m, 2H, H-2I, H-4II), 4.27 (m,
1H, H-6IIa), 4.23 (s, 1H, 3I-OH), 4.14–3.98 (m, 4H, H-2II, H-6Ia, H-6Ib,
H-6IIb), 3.86 (t, 1H, J3,4 = J4,5 = 8.5 Hz, H-4I) 3.82–3.70 (m, 7H, H-5I,
OMe × 2), 3.59 (s, 1H, H-5II), 2.17 (d, 1H, J2,OH 4.0 Hz, 2II-OH), 1.09 (s,
9H, Me3 of tert-Bu); 13C NMR (100 MHz, CDCl3): δ 168.55, 168.13,
166.45 (C = O), 160.03, 155.39, 151.03, 135.94, 135.69, 134.16, 133.48,
133.35, 132.85, 131.79, 130.16, 129.98, 129.83, 129.77, 129.65, 128.53,
127.87, 127.73, 127.53, 123.67, 123.41, 118.80, 114.44, 113.49 (ar-
omatic), 104.03 (C1II), 100.73 (CH of 4-methoxybenzylidene), 97.48
(C1I), 81.94 (C4I), 75.40 (C5I), 74.25 (C3II), 73.31 (C4II), 69.87 (C3I),
68.69 (C2II), 68.56 (C6II), 66.81 (C5II), 62.79 (C6I), 56.24 (C2I), 55.66,
55.29 (OMe), 26.88 (CMe3 of tert-Bu), 19.38 (CMe3 of tert-Bu); HRMS
(FAB, positive ion mode, NBA) m/z = 1037.3693 [M]+, calcd for
C58H59NO15Si, 1037.3654.
4.3. 4-Methoxyphenyl 4,6-O-(4-methoxybenzylidene)-β-D-
galactopyranosyl-(1→4)-6-O-tert-butyldipheylsilyl-2-deoxy-2-
phthalimido-β-D-glucopyranoside (8)
To a solution of 7 (7.75 g, 11.1 mmol) in anhydrous pyridine
(120 mL) was added tert-butyldiphenylchlorosilane (5.36 mL,
20.9 mmol) at rt under dry atmosphere. After stirring for 64 h, MeOH
was added to quench excess reagent, and then the mixture was con-
centrated under reduced pressure. The residue was coevaporated
with toluene and extracted with CHCl3, washed successively with
satd aq NaHCO3 and brine. The organic layer was dried over MgSO4,
filtered through a Celite bed and concentrated under diminished
pressure. The residue was subjected to silica gel column chroma-
tography eluting with CHCl3/EtOAc (2:1, v/v, containing 0.1% Et3N)
to provide pure 8 (6.12 g, 6.55 mmol, 59 %) as an amorphous powder.
4.5. 4-Methoxyphenyl 3-O-benzoyl-4,6-O-(4-methoxybenzylidene)-
β-D-galactopyranosyl-(1→4)-[2,3,4-tri-O-benzyl-α-L-fucopyranosyl-
(1→3)]-6-O-tert-butyldiphenylsilyl-2-deoxy-2-phthalimido-β-D-
glucopyranoside (10)
Compound 2 (817 mg, 787 μmol) was added to a solution of 9
(497 mg, 944 μmol) in anhydrous CH2Cl2 (10 mL), and then diluted
with anhydrous Et2O (20 mL). The mixture was stirred at rt for 30 min
under Ar atmosphere in the presence of activated powdered mo-
lecular sieves (MS) AW 300 (1.00 g). N-Iodosuccinimide (443 mg,
1.97 mmol) was added to the mixture, followed by cooling down
to −78 °C under Ar atmosphere. Triflic acid (13.8 μL, 141 μmol) in
anhydrous Et2O (124 μL) was added dropwise to the mixture. After
stirring for 1 h, excess amount of Et3N was added to terminate the
reaction. After kept stirring for 15 min, the mixture was filtered
through a bed of Celite, diluted with CHCl3, washed successively with
5 wt% aq Na2S2O3, satd aq NaHCO3 and brine. The organic layer was
dried over MgSO4, filtered through a bed of Celite, and concentrated
23
[α]D −20.6 (c 1.0, CHCl3); Rf 0.33 (CHCl3/EtOAc, 1:1), 1H NMR
(400 MHz, CDCl3, TMS): δ 7.96–6.68 (m, 22H, NPhth, -OSiPh2CMe3,
-C6H4OMe × 2), 5.75 (d, 1H, J1,2 8.0 Hz, H-1I), 5.45 (s, 1H, CH of
4-methoxybenzylidene), 4.55 (dd, 1H, J2,3 10.5, J3,4 8.04 Hz, H-3I), 4.50
(d, 1H, J1,2 8.0 Hz, H-1II), 4.44 (dd, 1H, J1,2 8.5, J2,3 11.0 Hz, H-2I),
4.30–4.22 (m, 2H, H-6IIa, 3I-OH), 4.17 (d, 1H, J3,4 3.0 Hz, H-4II),
4.14–4.08 (m, 1H, H-6Ia), 4.06–3.97 (m, 2H, H-6Ib, H-6IIb), 3.86 (t,
1H, J3,4 = J4,5 = 9.5 Hz, H-4I), 3.79 (s, 3H, OMe), 3.76–3.67 (m, 5H, H-5I,

40
Y. Yamazaki et al./Carbohydrate Research 422 (2016) 34–44
under reduced pressure. The residue was subjected to silica gel
column chromatography (n-hexane/EtOAc, 2:1, v/v, containing 0.1%
Et3N), providing pure 10 (867 mg, 596 μmol, 76%) as a white solid.
[α]D23 −10.2 (c 0.64, CHCl3); mp 106–107 °C; Rf 0.28 (n-hexane/
EtOAc, 2:1); 1H NMR (400 MHz, CDCl3, TMS): δ 8.20–6.64 (m, 42H,
aromatic), 5.55 (d, 1H, J1,2 8.5 Hz, H-1I), 5.52 (s, 1H, CH of
4-methoxybenzylidene), 5.13–5.07 (m, 2H, H-1III, H-3III), 4.91–4.84
(m, 2H, H-3I, H-5II), 4.76–4.69 (m, 2H, H-2I, H-1II), 4.61 (s, 2H, -CH2Ph),
4.53–4.25 (m, 6H, H-4I, H-6Ia, H-4III, H-6IIIa, -CH2Ph), 4.19–4.10 (m,
2H, H-2III, -CH2Ph), 4.10–3.96 (m, 3H, H-6Ib, H-6IIIb, H-3II), 3.71 (s,
3H, OMe), 3.69–3.62 (m, 2H, H-5I, H-2II), 3.59–3.48 (m, 4H, OMe,
-CH2Ph), 3.42 (s, 1H, H-5III), 3.21 (s, 1H, H-4II), 2.38 (d, 1H, J2,OH 3.0 Hz,
2III-OH), 1.13 (s, 9H, Me3 of tert-Bu), 1.08 (d, 3H, H-6II, J5,6 6.5 Hz);
13C NMR (100 MHz, CDCl3): δ 171.28, 166.35 (C = O), 159.96, 155.37,
151.18, 139.56, 139.48, 138.16, 136.11, 135.44, 134.29, 133.88, 133.53,
132.29, 130.28, 130.00, 129.96, 129.80, 128.63, 128.57, 128.39, 128.29,
128.23, 128.17, 128.05, 127.98, 127.93, 127.85, 127.78, 127.66, 127.46,
127.39, 127.25, 127.12, 127.07, 127.02, 126.78, 123.76, 118.83, 114.44,
113.39 (aromatic), 101.78 (C1III), 99.72 (CH of 4-methoxybenzylidene),
98.19 (C1II), 97.89 (C1I), 79.14 (C3II), 78.77 (C4II), 76.00 (C2II), 74.90
(CH2Ph), 74.61 (C4I), 74.36 (C3III), 73.64 (C4III), 73.42 (C5I), 73.01
(CH2Ph), 72.41 (C3I), 71.47 (CH2Ph), 69.63 (C2III), 69.08 (C6III), 66.63
(C5III), 66.45 (C5II), 61.79 (C6I), 56.81 (C2I), 55.70, 55.06 (OMe), 26.99
(Me3 of tert-Bu), 19.74 (CMe3 of tert-Bu), 16.63 (C6II); HRMS (FAB,
positive ion mode, NBA) m/z = 1476.5502 [M + Na]+, calcd for
C85H87NO19SiNa, 1476.5539.
126.97, 126.65, 119.18, 114.45, 113.43 (C = O, aromatic), 99.86 (C1II),
99.83 (CH of p-methoxybenzylidene), 98.75 (C1IV), 98.41 (C1I), 98.36
(C1III), 79.48 (C4III), 79.38 (C3IV), 78.73 (C4IV), 77.96 (C3III), 77.55 (C4I),
76.61 (C2III), 76.16 (C3II), 75.99 (C5I), 75.07, 74.73 (CH2Ph), 73.30 (C4II),
73.28 (CH2Ph), 73.09 (CH2Ph), 72.94 (C3I, CH2Ph), 72.83 (C2IV), 72.31
(C2II), 71.38 (CH2Ph), 69.14 (C6II), 67.51 (C5III), 66.84 (C5IV), 66.33 (C5II),
61.47 (C6I), 56.92 (C2I), 55.84, 55.10 (OMe), 26.87 (Me3 of tert-Bu),
19.74 (CMe3 of tert-Bu), 16.39 (C6III), 16.31 (C6IV); HRMS (FAB, pos-
itive ion mode, NBA) m/z = 1892.7506 [M + Na]+, calcd for
C112H115NO23SiNa, 1892.7527.
4.7. 4-Methoxyphenyl 2,4,6-tri-O-acetyl-3-O-benzoyl-β-D-
galactopyranosyl-(1→4)-[2,3,4-tri-O-acetyl-α-L-fucopyranosyl-
(1→3)]-6-O-tert-butyldiphenylsilyl-2-deoxy-2-phthalimido-β-D-
glucopyranoside (12)
Compound 10 (0.43 g, 294 μmol) was dissolved in the mixture
of dry MeOH (4.0 mL) and dry THF (4.0 mL) followed by addition
of Pd(OH)2 on activated carbon (20%, 200 mg). After stirring at rt
under H2 atmosphere for 10 h, the mixture was filtered through filter
paper, followed by concentration under reduced pressure. To a so-
lution of the residue in pyridine (8 mL) was added Ac2O (350 μL,
3.70 mmol) under dry atmosphere at rt overnight, and methanol
was added to quench excess reagents. The mixture was concen-
trated and coevaporated with toluene under reduced pressure. The
residue was dissolved in CHCl3, and washed with satd aq NaHCO3
and brine. The organic layer was dried over MgSO4, filtered through
a Celite bed, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (Rf 75 system,
n-hexane/EtOAc, 1:0 to 0:1, v/v, linear gradient) to afford 12 (153 mg,
116 μmol, 39%) as colorless amorphous.
4.6. 4-Methoxyphenyl 2,3,4-tri-O-benzyl-α-L-fucopyranosyl-(1→2)-
3-O-benzoyl-4,6-O-benzylidene-β-D-galactopyranosyl-(1→4)-[2,3,4-
tri-O-benzyl-α-L-fucopyranosyl-(1→3)]-6-O-tert-butyldiphenylsilyl-
2-deoxy-2-phthalimido-β-D-glucopyranoside (11)
[α]D30 −42.7 (c 0.05, CHCl3); Rf 0.32 (n-hexane/EtOAc, 1:1); 1H NMR
(CDCl3, 400 MHz, TMS): δ 7.95–6.69 (23H, m, aromatic), 5.60 (d, 1H,
J3,4 3.2 Hz, H-4III), 5.55 (d, 1H, J1,2 8.4 Hz, H-1I), 5.42 (s, 1H, H-4III),
5.29–5.02 (m, 5H, H-3II, H-5II, H-1III, H-2III, H-3III), 4.98 (d, 1H, J1,2 4.0 Hz,
H-1II), 4.87–4.82 (m, 2H, H-3I, H-2II), 4.58–4.51 (m, 2H, H-2I, H-6IIIa),
4.37–4.30 (m, 2H, H-4I, H-6IIIb), 3.91–3.84 (m, 1H, H-5I), 3.73 (s, 3H,
OMe), 3.53 (bd, 1H, J5,6 10.0 Hz, H-5III), 2.14–1.80 (m, 18H, Ac), 1.27
(d, 3H, J5,6 6.0 Hz, H-6II), 1.12 (s, 9H, Me3 of tert-Bu); 13C NMR (CDCl3,
100 MHz): δ 170.87, 170.73, 170.48, 170.42, 169.80, 169.05, 165.32
(C = O), 155.57, 150.98, 136.13, 135.89, 134.56, 133.60, 130.25, 130.04,
129.94, 128.70, 128.23, 128.15, 127.84, 123.81, 118.83, 114.54 (ar-
omatic), 99.92 (C1III), 97.74 (C1I), 95.45 (C1II), 75.56 (C5I), 74.17 (C4I),
72.00 (C3III), 71.60 (C4II), 71.48 (C3I), 71.36 (C5III), 69.35 (C2III), 68.30
(C3II), 67.94 (C2II), 67.07 (C4III), 64.37 (C5II), 61.19 (C6I), 61.10 (C6III),
56.60 (C2I), 55.74 (OMe), 27.01 (Me3 of tert-Bu), 20.95, 20.87, 20.81,
20.67, 20.62 (Ac), 19.46 (CMe3 of tert-Bu), 16.10 (C6II); HRMS (ESI,
positive ion mode) m/z = 1340.4334 [M + Na]+, calcd for
C68H75NO24SiNa, 1340.4360.
Compound 2 (2.01 g, 1.94 mmol) was added to a solution of 9
(2.45 g, 4.66 mmol) in anhydrous CH2Cl2 (15 mL), and then diluted
with anhydrous Et2O (30 mL). The mixture was kept stirring at rt
for 30 min under Ar atmosphere in the presence of activated pow-
dered MS AW 300 (2.0 g). N-Iodosuccinimide (2.18 g, 9.70 mmol)
was added to the mixture, and then it was cooled down to −40 °C
under Ar atmosphere. Triflic acid (68 μL, 776 μmol) in anhydrous
Et2O was injected to the mixture. After stirring for 3 h, excess amount
of Et3N was added to terminate the reaction. The mixture was fil-
tered through a bed of Celite, diluted with CHCl3, washed successively
with 5 wt% aq Na2S2O3, satd aq NaHCO3 and brine. The organic layer
was dried over MgSO4, filtered through a Celite bed, and concen-
trated under reduced pressure. The residue was subjected to silica
gel column chromatography (n-hexane/EtOAc, 1:0 to 0:1, v/v, linear
gradient), providing pure 11 (3.03 g, 1.61 mmol, 83%) as a white solid.
[α]D −28.2 (c 0.54, CHCl3); mp 90–91 °C; Rf 0.79 (n-hexane/
EtOAc, 1:1); 1H NMR (400 MHz, CDCl3, TMS): δ 8.10–6.68 (57H, m,
aromatic), 5.57 (1H, d, J1,2 3.6 Hz, H-1III), 5.46 (1H, s, -CH of
p-methoxybenzylidene), 5.42 (1H, d, J1,2 8.0 Hz, H-1I), 5.18 (1H, d,
J1,2 8.0 Hz, H-1II), 5.13 (1H, dd, J2,3 10.0, J3,4 3.8 Hz, H-3II), 4.98 (1H,
d, Jgem 11.8 Hz, -CH2Ph), 4.91 (1H, m, H-5IV), 4.75–4.73 (2H, m, H-2I,
H-3I), 4.67 (1H, d, J1,2 4.0 Hz, H-1IV), 4.62 (1H, d, Jgem 11.6 Hz, -CH2Ph),
4.57–4.49 (5H, m, -CH2Ph × 4, H-4I), 4.45–4.29 (7H, m, H-4II, H-2II,
H-6IIb, -CH2Ph × 4), 4.21 (1H, m, H-5III), 4.16 (1H, m, H-6Ia), 4.05–
3.98 (4H, m, H-6IIa, H-2III, H-3IV, -CH2Ph), 3.76 (3H, s, OMe), 3.71–
3.65 (2H, m, H-6Ib, H-2IV), 3.55–3.52 (4H, m, OMe, H-3III), 3.45 (1H,
d, J3,4 3.2 Hz, H-4III), 3.43 (1H, d, Jgem 12.4 Hz, -CH2Ph), 3.33 (1H, m,
H-5II), 3.20 (1H, m, H-5I), 3.09 (1H, s, H-4IV), 1.30 (1H, d, J5,6 7.2 Hz,
4.8. 2,4,6-Tri-O-acetyl-3-O-benzoyl-β-D-galactopyranosyl-
(1→4)-[2,3,4-tri-O-acetyl-α-L-fucopyranosyl-(1→3)]-6-O-tert-
butyldiphenylsilyl-2-deoxy-2-phthalimido-D-glucopyranose (13)
Compound 12 (153 mg, 116 μmol) was dissolved in a mixed so-
lution of CH3CN-H2O (8.0 mL – 2.0 mL) followed by addition of
cerium(IV) ammonium nitrate (CAN) (190 mg, 348 μmol). After stir-
ring at rt for 6 h, the mixture was concentrated under reduced
pressure to remove CH3CN. The residue was dissolved in CHCl3,
washed successively with satd aq NaHCO3 and brine. The organic
layer was dried over MgSO4, filtered through a Celite bed, and con-
centrated under reduced pressure. The residue was purified by silica
gel column chromatography eluting with CH3Cl/EtOAc (1:0 to 0:1,
v/v, linear gradient, Rf 75 system) to afford 13 (86 mg, 70.9 μmol,
61%) as a yellowish amorphous powder.
H-6III), 1.19 (1H, d, J5,6 6.4 Hz, H-6IV), 1.11 (9H, s, Me3 of tert-Bu); 13
C
NMR (100 MHz, CDCl3): δ 165.61, 159.97, 155.59, 151.32, 139.74,
139.57, 138.95, 138.78, 138.45, 138.19, 136.06, 135.15, 133.093,
133.71, 132.32, 130.18, 130.09, 129.94, 129.89, 129.70, 128.88, 128.46,
128.39, 128.33, 128.30, 128.27, 128.21, 128.18, 128.11, 128.04, 127.93,
127.82, 127.71, 127.57, 127.44, 127.37, 127.34, 127.10, 127.08, 127.05,

Y. Yamazaki et al./Carbohydrate Research 422 (2016) 34–44
41
28
[α]D −22.2 (c 0.1, CHCl3); Rf 0.56 (CH3Cl/EtOAc, 2:1); 1H NMR
completed, the reaction mixture was neutralized by addition of Et3N,
filtered through a Celite bed, and the filtrate was concentrated under
reduced pressure. The residue was dissolved in CHCl3, and washed
with satd aq NaHCO3 and brine. The organic layer was dried over
MgSO4, filtered through a Celite bed, and concentrated under reduced
pressure. The residue was purified by silica gel column chroma-
tography (n-hexane/EtOAc 1:0 to 0:1, v/v, Rf 75 system, linear
gradient) to afford 15 (20 mg, 14.2 μmol, 48%) as colorless
amorphous.
(CDCl3, 400 MHz, TMS): δ 7.94–7.41 (19H, m, aromatic), 5.58 (d, 1H,
J3,4 3.2 Hz, H-4III), 5.39 (d, 1H, J3,4 3.2 Hz, H-4 II), 5.29–5.16 (m, 3H,
H-1I, H-3II, H-2III), 5.13–5.00 (m, 3H, H-1III, H-5II,), 5.03 (dd, 1H, J2,3
10.0, J3,4 10.0 Hz, H-3III), 4.95 (d, 1H, J1,2 4.0 Hz, H-1II), 4.84–4.77 (m,
2H, H-3I, H-2II), 4.54 (dd, 1H, J5,6a 6.5, J6a,6b 11.2 Hz, H-6IIIa), 4.33–4.10
(m, 3H, H-4I, H-6IIIb, H-2I), 4.04 (m, 2H, H-6Ia, H-6Ib), 3.84–3.79 (m,
1H, H-5III), 3.47 (m, 1H, H-5I), 2.66 (d, 1H, J1,OH 8.2 Hz, 1-OH), 2.13–1.85
(m, 18H, Ac), 1.25 (d, 3H, J5,6 6.0 Hz, H-6II), 11.5 (s, 9H, CMe3 of tert-
Bu); 13C NMR (CDCl3, 100 MHz): δ 170.89, 170.77, 170.48, 170.27,
169.82, 169.12, 165.35 (C = O), 136.26, 135.61, 134.55, 133.62, 133.45,
132.46, 130.34, 130.12, 129.77, 129.15, 128.73, 128.21, 127.89, 123.77
(aromatic), 99.86 (C1III), 95.30 (C1II), 92.86 (C1I), 75.79 (C5I), 74.19
(C4I), 72.04 (C3 III), 71.64 (C4II), 71.26 (C5III, C3I), 69.36 (C2III), 68.28
(C3II), 68.05 (C2II), 67.02 (C4III), 64.37 (C5II), 61.37 (C6I), 61.09 (C6III),
58.58 (C2I), 27.12 (CMe3 of tert-Bu), 20.99, 20.92, 20.84, 20.73, 20.71,
20.65 (Ac), 19.55 (CMe3 of tert-Bu), 16.12 (C6II); HRMS (ESI, posi-
tive ion mode) m/z = 1234.3911 [M + Na]+, calcd for C61H69NO23SiNa,
1234.3927.
[α]D29 −44.4 (c 0.1, CHCl3); Rf 0.31 (n-hexane/EtOAc 2:3); 1H NMR
(CDCl3, 400 MHz, TMS): δ 7.95–7.36 (m,19H, aromatic), 5.58 (d, 1H,
J3,4 3.2 Hz, H-4III), 5.40 (d, 1H, J3,4 2.4 Hz, H-4II), 5.28 (dd, 1H, J1,2 8.4,
J2,3 10.0 Hz, H-2III), 5.22–5.17 (m, 2H, H-1III, H-3II), 5.13–5.07 (m, 3H,
H-1I, H-3III, H-5II), 4.95 (d, 1H, J1,2 4.0 Hz, H-1II), 4.82 (dd, 1H, J1,2 4.0,
J2,3 10.9 Hz, H-2II), 4.77 (t, 1H, J2,3 = J3,4 = 9.9 Hz, H-3I), 4.55 (dd, 1H,
J5,6a 6.7, J6a,6b 11.6 Hz, H-6IIIa), 4.35–4.22 (m, 3H, H-2I, H-4I, H-6IIIb),
4.07–3.98 (m, 2H, H-6Ia, H-6Ib), 3.89–3.83 (m, 2H, H-5III, PEG),
3.65–3.25 (m, 16H, H-5I, PEG), 2.12, 2.10, 2.06, 1.91, 1.82 (s × 6, 18H,
Ac), 1.25 (d, 3H, J5,6 6.4 Hz, H-6II), 1.15 (s, 9H, Me3 of tert-Bu); 13C NMR
(CDCl3, 100 MHz): δ 170.91, 170.89, 170.51, 170.29, 169.83, 169.12,
165.36 (C = O), 136.23, 135.50, 134.48, 133.62, 133.46, 132.25, 130.29,
130.07, 129.79, 129.16, 128.73, 128.24, 127.85, 123.68 (aromatic),
99.93 (C1III), 98.18(C1I), 95.36 (C1II), 75.44 (C5I), 74.38 (C4I), 72.06
(C3III), 71.68 (C4II), 71.54 (C3I), 71.31 (C5III), 70.72, 70.64, 70.62, 70.54,
70.20, 70.16 (PEG), 69.45 (C2III), 68.53 (PEG), 68.32 (C3II), 68.04 (C2II),
67.11 (C4III), 64.33 (C5II), 61.18 (C6I, C6III), 56.71 (C2I), 50.79 (PEG),
27.07 (CMe3 of tert-Bu), 20.98, 20.95, 20.85, 20.71, 20.67 (Ac), 19.54
(CMe3 of tert-Bu), 16.14 (C6II); HRMS (ESI, positive ion mode)
m/z = 1435.5026 [M + Na]+, calcd for C69H84N4O26SiNa, 1435.5041.
4.9. 2,4,6-Tri-O-acetyl-3-O-benzoyl-β-D-galactopyranosyl-
(1→4)-[2,3,4-tri-O-acetyl-α-L-fucopyranosyl-(1→3)]-6-O-tert-
butyldiphenylsilyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl
trichloroacetimidate (14)
To a solution of compound 13 (32 mg, 26.4 μmol) in dry CH2Cl2
(5.0 mL) was added trichloroacetonitrile (53 μL, 527 μmol). After stir-
ring at 0 °C under Ar atmosphere for 30 min, DBU (1 μL, 7.9 μmol)
was added, then the reaction mixture was kept stirring at 0 °C under
Ar atmosphere for 5 h. The mixture was evaporated under reduced
pressure. The residue was purified by silica gel column chroma-
tography (n-hexane-ethyl acetate containing 0.5% Et3N, 1:0 to 0:1,
v/v, linear gradient, Rf 75 system) to afford 14 (30 mg, 22.1 μmol,
83%) as colorless amorphous.
4.11. 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl 2,3,4,6-tetra-O-
acetyl-β-D-galactopyranosyl-(1→4)-[2,3,4-tri-O-acetyl-α-L-
fucopyranosyl-(1→3)]-2-acetamido-2-deoxy-β-D-glucopyranoside
(16)
[α]D27 −42.7 (C 0.1, CHCl3); Rf 0.41 (n-hexane/EtOAc 1:1 contain-
ing with Et3N); 1H NMR (CDCl3, 400 MHz, TMS): δ 8.55 (s, 1H,
-CNHCCl3), 7.96–7.30 (m,19H, aromatic), 6.36 (d, 1H, J1,2 8.8 Hz, H-1I),
5.60 (d, 1H, J3,4 3.2 Hz, H-4III), 5.42 (d, 1H, J3,4 2.8 Hz, H-4 II), 5.31 (dd,
1H, J1,2 10, J2,3 10 Hz, H-2III), 5.24–5.19 (m, 2H, H-1III, H-3II), 5.16–5.12
(m, 2H, H-3III, H-5II), 5.01 (d, 1H, J1,2 4.0 Hz, H-1II), 4.91 (t, 1H,
J2,3 = J3,4 = 8.7 Hz, H-3I), 4.82 (dd, 1H, J1,2 3.9, J2,3 10.9 Hz, H-2II), 4.65–4.52
(m, 2H, H-2I, H-6IIIa), 4.40–4.31 (m, 2H, H-4I, H-6IIIb), 4.08 (bs, 2H,
H-6Ia, H-6Ib), 3.86 (m, 1H, H-5III), 3.66–3.60 (m, 1H, H-5I), 2.13, 2.12,
2.09, 2.08, 1.93, 1.84 (s × 6, 18H, Ac), 1.28 (d, 3H J5,6 6.4 Hz, H-6II),
1.15 (s, 9H, Me3 of tert-Bu); 13C NMR (CDCl3, 100 MHz): δ 170.86,
170.82, 170.42, 170.21, 169.77, 169.03, 165.30, 160.73 (C = O,
-CNHCCl3), 136.14, 135.38, 134.59, 133.60, 133.47, 131.95, 130.24,
130.02, 129.75, 128.69, 128.20, 127.80, 123.71 (aromatic), 99.95 (C1III),
95.39 (C1II), 93.62 (C1I), 90.47 (-CCl3), 76.06 (C5I), 73.95 (C4I), 71.98
(C3III), 71.56 (C4II), 71.43 (C5III), 71.20 (C3I), 69.31 (C2III), 68.22 (C3II),
68.09 (C2II), 67.10 (C4III), 64.38 (C5II), 61.18 (C6III), 60.09 (C6I), 55.58
(C2I), 26.98 (C(CH3)3 of tert-Bu), 20.95, 20.89, 20.81, 20.65, 20.61 (Ac),
19.55 (C(CH3)3 of tert-Bu), 16.09 (C6II); HRMS (ESI, positive ion mode)
m/z = 1377.2974 [M + Na]+, calcd for C63H69N2O23SiCl3Na, 1377.3024.
Compound 15 (51 mg, 36.8 μmol) was suspended in dry MeOH
(5.0 mL) followed by the addition of ca. 28 wt-% MeONa in MeOH
(3 μL, 19.7 μmol). After stirring at rt under dry atmosphere over-
night, the reaction mixture was neutralized by the addition of Dowex
50W-X4 (H+ form), filtered through cotton, and concentrated under
reduced pressure. The residue dissolved in EtOH (5.0 mL) was added
NH2NH2·H2O (10 μL, 205.8 μmol). After stirring at 90 °C for 7 h, re-
action mixture was concentrated to dryness under reduced pressure.
The residue was dissolved in pyridine (5.0 mL) followed by addi-
tion of Ac2O (65 μL, 687.6 μmol) and DMAP (3 mg, 24.6 μmol). After
stirring at rt under dry atmosphere for 36 h, MeOH was added to
quench excess reagents, followed by concentration under reduced
pressure. The residue was dissolved in CHCl3, and washed with satd
aq NaHCO3 and brine. The organic layer was dried over MgSO4, fil-
tered through a Celite bed, and concentrated under reduced pressure.
The residue was roughly purified by silica gel column chromatog-
raphy eluting with CH3Cl/MeOH (1:0 to 6:1, v/v, Rf 75 system, linear
gradient). The obtained compound was dissolved in dry THF (3.0 mL)
followed by the addition of AcOH (5 μl, 91.8 μmol) and 1M TBAF
in THF (207 μl, 207 μmol). After stirring at rt under Ar atmo-
sphere for 72 h, the reaction mixture was concentrated under
reduced pressure and extracted with CHCl3, washed with satd aq
NaHCO3 and brine. The organic layer was dried over MgSO4, fil-
tered through a Celite bed, and concentrated under reduced pressure.
The residue was purified by silica gel column chromatography eluting
with CHCl3/MeOH (1:0 to 6:1, v/v, linear gradient, Rf 75 system) to
afford the compound 16 (21 mg, 20.5 μmol, 56%, 4 steps) as color-
less amorphous.
4.10. 2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl 2,4,6-tri-O-
acetyl-3-O-benzoyl-β-D-galactopyranosyl-(1→4)-[2,3,4-tri-O-acetyl-
α-L-fucopyranosyl-(1→3)]-6-O-tert-butyldiphenylsilyl-2-deoxy-2-
phthalimido-β-D-glucopyranoside (15)
To a solution of compound 14 (40 mg, 29.8 μmol) in dry CH2Cl2
(5.0 mL) was added 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethanol
(19 mg, 88.5 μmol) and activated 4Å molecular sieves (MS4A, 80 mg).
After stirring at −50 °C under Ar atmosphere for 30 min, TMSOTf
(2 μL, 8.85 μmol) was added to the mixture, followed by stirring at
−50 °C under Ar atmosphere for 8 h. After the reaction was
[α]D26 −71.4 (c 0.2, CHCl3); Rf 0.24 (CHCl3/MeOH, 10:1); 1H NMR
(CDCl3, 400 MHz, TMS): δ 6.31 (d, 1H, J2,NHAc 9.4 Hz, NHAc), 5.42–5.38
(m, 3H, H-1II, H-4II, H-4III), 5.22 (dd, 1H J2,3 11.0, J3,4 3.4 Hz, H-3II),

42
Y. Yamazaki et al./Carbohydrate Research 422 (2016) 34–44
5.10–4.98 (m, 4H, H-2II, H-5II, H-2III, H-3III), 4.73 (d, 1H, J1,2 7.28 Hz,
H-1III), 4.67 (d, 1H, J1,2 8.40 Hz, H-1I), 4.50 (dd, 1H, J5,6a 6.2, J6a,6b 11.4 Hz,
H-6aIII), 4.32 (dd, 1H, J5,6b 7.9, J6a,6b 11.4 Hz, H-6bIII), 4.03–3.90 (m,
4H, H-2I, H-4I, H-6Ia, H-5III), 3.85–3.54 (m, 16H, H-3I, H-6Ib, PEG),
3.45–3.39 (m, 2H, PEG), 3.28–3.24 (m, 1H, H-5I), 2.30 (dd, 1H, J6a,OH
3.96, J6Ib,OH 9.6 Hz, I6-OH), 2.19, 2.14, 2.13, 2.08, 2.05, 1.98, 1.97, 1.95
(s × 8, 24H, Ac), 1.19 (d, 3H J5,6 6.5 Hz, H-6II); 13C NMR (CDCl3,
100 MHz): δ 171.70, 171.15, 170.87, 170.67, 170.64, 170.14, 169.79,
169.26 (C = O), 102.11 (C1I), 100.40 (C1III), 95.58(C1II), 75.45 (C5I),
74.55 (C4I), 73.76 (C5III), 71.73 (C4III, PEG), 71.09, 71.05 (C3I, C3III),
70.99, 70.69, 70.67, 70.52, 70.10 (PEG), 69.47 (C2III), 68.59 (PEG), 68.11
(C2II, C3II), 67.05 (C4II), 64.14 (C5II), 60.87 (C6I), 60.59 (C6III), 55.97
(C2I), 50.76 (PEG), 23.35, 21.26, 20.96, 20.86, 20.83, 20.81, 20.76, 20.70
(Ac), 15.97 (C6II); HRMS (ESI, positive ion mode) m/z = 1047.3740
[M + Na]+, calcd for C42H64N4O25Na, 1047.3757.
filtered through a Celite bed, and concentrated under reduced pres-
sure. The residue was subjected to silica gel column chromatography
eluting with n-hexane/EtOAc (1:2, v/v, containing 0.5% Et3N) to afford
18 (562 mg, 0.36 mmol, 68%) as colorless amorphous.
27
[α]D −110.1 (c 0.03, CHCl3); Rf 0.63 (n-hexane/EtOAc, 1:2); 1H
NMR (400 MHz, CDCl3, TMS): δ 7.89–7.32, 7.23–6.74 (23H, m, aro-
matic), 5.51 (bd, 1H, J3,4 3.4 Hz, H-4II), 5.45 (d, 1H, J1,2 8.5 Hz, H-1I),
5.40 (bd, 1H, J3,4 2.7 Hz, H-4III), 5.26–5.09 (7H, m, H-1IV, H-4IV, H-3III,
H-3II, H-1II, H-2IV, H-5II), 4.97–4.81 (m, 4H, H-1III, H-3IV, H-2III, H-3I),
4.59 (dd, 1H, J1,2 8.6, J2,3 10.0 Hz, H-2I), 4.53–4.40 (m, 3H, H-4I, H-6IIa,
H-5IV), 4.30 (dd, 1H, J5,6 7.4, J6a,6b 11.4 Hz, H-6IIb), 4.23–4.15 (m, 2H,
H-6Ia, H-6Ib), 3.92 (bt, 1H, J1,2 = J2,3 = 9.0 Hz, H-2II), 3.85–3.80 (m, 1H,
H-5II), 3.75 (s, 3H, OMe), 3.47–3.43 (m, 1H, H-5I), 2.15, 2.11, 2.09,
2.08, 2.07, 1.92, 1.86, 1.72 (s × 8, 24H, Ac), 1.27–1,24 (m, 6H, H-6III,
H-6IV), 1.16 (s, 9H, Me3 of tert-Bu); 13C NMR (CDCl3, 100 MHz): δ
171.00, 170.88, 170.67, 170.40, 170.15, 169.88, 169.85, 169.61, 169.15
(C = O), 155.69, 1150.94, 135.99, 135.41, 134.55, 133.90, 133.46,
132.04, 130.04, 129.99, 129.93, 129.96, 128.90, 128.27, 128.15, 127.89,
127.86 (aromatic), 99.86 (C1II), 98.16 (C1I), 97.72 (C1IV), 95.92 (C1III),
75.42 (C5I), 74.21 (C2II), 74.07 (C3II), 73.04 (C4I), 72.47 (C3I), 71.70
(C4III), 71.20 (C5II), 71.06 (C4IV), 68.23 (C3III), 67.98 (C3IV), 67.67 (C2III),
67.27(C4II), 66.88 (C2IV), 65.41 (C5IV), 64.41 (C5III), 61.14, 61.08 (C6I,
C6II), 56.83 (C2I), 55.80 (OMe), 27.16 (Me3 of tert-Bu), 21.06, 20.97,
20.82, 20.73, 20.69 × 2, 20.67, 20.34 (Ac), 19.43 (CMe3 of tert-Bu),
4.12. Triethylammonium {2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)
ethyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→4)-[2,3,4-tri-
O-acetyl-α-L-fucopyranosyl-(1→3)]-2-acetamido-2-deoxy-6-O-
sulfonato-β-D-glucopyranoside} (17)
Compound 16 (28 mg, 27.3 μmol) was dissolved in dry DMF
(3.0 mL) and Et3N (600 μL). After stirring at 55 °C under Ar atmo-
sphere for 20 min, the reaction mixture was added to SO3·NMe3
(145 mg, 934 μmol). After stirring at 55 °C for 72 h, MeOH (1 mL)
was added to quench excess reagents, and evaporated under reduced
pressure. The residue was purified by LH-20 size exclusion column
chromatography eluting with MeOH to afford 17 (30 mg, 24.9 μmol,
91%) as colorless amorphous.
15.59, 15.52 (C6III
,
C6IV); HRMS (ESI, positive ion mode)
m/z = 1570.5151 [M + Na]+, calcd for C78H89NO30SiNa, 1570.5136.
4.14. 2,3,4-Tri-O-acetyl-α-L-fucopyranosyl-(1→2)-4,6-di-O-acetyl-
3-O-benzoyl-β-D-galactopyranosyl-(1→4)-[2,3,4-tri-O-acetyl-α-L-
fucopyranosyl-(1→3)]-6-O-tert-butyldiphenylsilyl-2-deoxy-2-
phthalimido-β-D-glucopyranosyl trichloroacetimidate (19)
[α]D23 −83.8 (c 0.07, MeOH); Rf 0.17 (CHCl3/MeOH 10:1); 1H NMR
(400 MHz, CDCl3, TMS): δ 9.46 (1H, bs, SO3H), 6.43 (d, 1H, J2,NHAc
9.6 Hz, NHAc), 5.45 (bs, 1H, H-4III), 5.39 (d, 1H, J1,2 3.8 Hz, H-1II), 5.36
(bd, 1H, J3,4 3.0 Hz, H-4II), 5.26 (dd, 1H, J2,3 10.9, J3,4 3.3 Hz, H-3II),
5.10–4.97 (m, 5H, H-2II, H-3III, H-5II, H-1III, H-2III), 4.60 (d, 1H, J1,2
8.40 Hz, H-1I), 4.44 (dd, 1H, J5,6a 5.8, J6a,6b 11.2 Hz, H-6IIIa), 4.32 (bs,
2H, H-6Ia, H-6Ib), 4.26 (dd, 1H, J5,6b 8.7, J6a,6b 11.1 Hz, H-6IIIb), 4.05–3.93
(m, 3H, H-2I, H-5III, PEG), 3.80–3.54 (m, 14H, PEG), 3.47–3.42 (m,
4H, H-3I, H-4I, H-5I, PEG), 3.20 (6H, m, N(CH2CH3)3), 2.17, 2.14, 2.12,
2.08, 2.06, 1.96, 1.95, 1.94 (s × 8, 24H, Ac), 1.40 (t, 9H, J 7.3 Hz,
N(CH2CH3)3), 1.20 (d, 3H, J5,6 6.5 Hz, H-6II); 13C NMR (CDCl3, 100 MHz):
δ 171.60, 171.26, 170.83, 170.61, 169.83, 169.80, 169.79, 169.76
(C = O), 102.04 (C1I), 99.66 (C1III), 95.53 (C1II), 74.63 (PEG), 73.79 (C3I,
C5I), 73.43 (C5III), 71.83 (C4II), 71.65 (PEG), 71.29 (C3III), 70.88 (PEG),
70.72 (C4I), 70.65, 70.61, 70.46, 70.06 (PEG), 69.44 (C2III), 68.47 (PEG),
68.20 (C2II), 67.94 (C3II), 67.25 (C4III), 64.91 (C6I), 64.30 (C5II), 60.64
(C6III), 55.46 (C2I), 50.78 (PEG), 46.71 (N(CH2CH3)3), 23.30, 21.22,
21.02 × 2, 20.88, 20.83, 20.77, 20.69 (Ac), 15.97 (C6II), 8.83
(N(CH2CH3)3): HRMS (ESI, negative ion mode) m/z = 1103.3360
[M − HNEt3]−, calcd for C48H63N4O28S, 1103.3350.
Compound 18 (562 mg, 0.36 mmol) was dissolved in a mixed so-
lution of CH3CN (8.0 mL)–H2O (2.0 mL) followed by addition of CAN
(592 mg, 1.08 mmol). After stirring at rt for 2 h, the reaction mixture
was extracted with CHCl3, washed successively with satd aq NaHCO3
and brine. The organic layer was dried over MgSO4, filtered through
a Celite bed, and concentrated under reduced pressure. The residue
was purified by silica gel column chromatography (CHCl3/MeOH,
30:1, containing 0.5% Et3N) to afford the corresponding anomer-
free compound (440 mg). To a solution of this compound (440 mg)
in dry CH2Cl2 (10 mL) was added CCl3CN (290 μL, 2.90 mmol). After
stirring at 0 °C under Ar for 15 min, DBU (13 μL, 87 μmol) was added
to the mixture. After kept stirring for 4 h, the mixture was concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography (CHCl3/EtOAc, 3:1, containing 0.5% Et3N)
to afford 19 (314 mg, 0.20 mmol, 2 steps, 56%) as colorless
amorphous.
[α]D −77.4 (c 0.5, CHCl3); Rf 0.57 (CHCl3/EtOAc 2:1); 1H NMR
27
(400 MHz, CDCl3, TMS): δ 8.58 (s, 1H, NH), 7.90–7.32 (19H, m, ar-
omatic), 6.32 (d, 1H, J1,2 8.8 Hz, H-1I), 5.51 (bd, 1H, J3,4 3.6 Hz, H-4II),
5.39 (bd, 1H, J3,4 2.8 Hz, H-4III), 5.26–5.24 (m, 2H, H-1IV, H-4IV),
5.23–5.16 (m, 2H, H-3II, H-3III), 5.14-5.07 (m, 3H, H-5III, H-1II, H-2IV),
5.02–4.95 (m, 2H, H-3IV, H-1III), 4.94–4.84 (m, 2H, H-3I, H-2III), 4.63
(dd, 1H, J1,2 8.9, J2,3 10.2 Hz, H-2I), 4.53–4.44 (m, 2H, H-4I, H-6IIa),
4.42–4.36 (m, 1H, H-5IV), 4.33-4.18 (m, 3H, H-6Ia, H-6Ib, H-6IIb), 3.92
(dd, 1H, J1,2 8.1, J2,3 9.9 Hz, H-2II), 3.80–3.75 (m,1H, H-5II), 3.62–3.58
(m, 1H, H-5I), 2.12, 2.11, 2.09, 2.08, 2.07, 1.93, 1.86, 1.71 (s × 8, 24H,
4.13. 4-Methoxyphenyl 2,3,4-tri-O-acetyl-α-L-fucopyranosyl-(1→2)-
4,6-di-O-acetyl-3-O-benzoyl-β-D-galactopyranosyl-(1→4)-[2,3,4-tri-
O-acetyl-α-L-fucopyranosyl-(1→3)]-6-O-tert-butyldiphenylsilyl-2-
deoxy-2-phthalimido-β-D-glucopyranoside (18)
To a solution of compound 11 (1.00 g, 0.53 mmol) in 20.0 mL of
THF–MeOH (1:1,v/v) was added Pd(OH)2-C (20%, 0.25 g, 1.78 mmol).
After stirring at rt under H2 for 10 h, the reaction mixture was fil-
tered through a Celite bed, and the filtrate was concentrated under
reduced pressure. The residue was dissolved in dry pyridine
(10.0 mL), followed by addition of DMAP (64 mg 0.53 mmol) and
Ac2O (1.0 mL, 10.6 mmol). After stirring at rt under dry atmo-
sphere for 24 h, MeOH was added to quench excess reagents, and
then concentrated and coevaporated with toluene under reduced
pressure. The residue was dissolved in CHCl3, and washed with satd
aq NaHCO3 and brine. The organic layer was dried over MgSO4,
Ac), 1.28–1.24 (m, 6H, H-6III, H-6IV), 1.16 (s, 9H, CMe3 of tert-Bu); 13
C
NMR (CDCl3, 100 MHz): δ 170.97, 170.86, 170.53, 170.38, 170.13,
169.86, 169.61, 168.05, 165.23, 160.74 (C = O), 136.03, 135.40, 134.65,
133.87, 133.57, 132.19, 131.45, 130.09, 130.01, 129.57, 128.95, 128.72,
128.20, 127.88, 123.72 (aromatic, C = NH), 100.01 (C1II), 97.80 (C1IV),
95.89 (C1III), 94.08 (C1I), 90.48 (CCl3), 76.03 (C5I), 74.39 (C2II), 73.97
(C3II), 72.99 (C4I), 72.12 (C3I), 71.65 (C4III), 71.25 (C5II), 71.15 (C4IV),
68.14 (C3III), 67.86 (C3IV), 67.83 (C2III), 67.33 (C4II), 67.02 (C2IV), 65.57

Y. Yamazaki et al./Carbohydrate Research 422 (2016) 34–44
43
(C5IV), 64.42 (C5III), 61.14, 61.08 (C6I, C6III), 55.73 (C2I), 27.14 (CMe3
of tert-Bu), 21.00, 20.98, 20.82, 20.70, 20.68, 20.65, 20.64, 20.34 (Ac),
19.66 (CMe3 of tert-Bu), 16.14, 15.73 (C6III, C6IV); HRMS (ESI)
m/z = 1340.4334 [M + Na]+, calcd for C68H75NO24SiNa, 1340.4360.
brine. The organic layer was dried over MgSO4, filtered through a
Celite bed, and concentrated under reduced pressure. The residue
was purified by silica gel chromatography (MeOH/EtOAc, 1:30, v/v,
containing 1% Et3N) to afford 21 (151 mg, 101 μmol, 64%) as color-
less amorphous.
4.15. 2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl 2,3,4-tri-O-
acetyl-α-L-fucopyranosyl-(1→2)-4,6-di-O-acetyl-3-O-benzoyl-β-D-
galactopyranosyl-(1→4)-[2,3,4-tri-O-acetyl-α-L-fucopyranosyl-
(1→3)]-6-O-tert-butyldiphenylsilyl-2-deoxy-2-phthalimido-β-D-
glucopyranoside (20)
[α]D25 −115.5 (c 0.24, CHCl3); Rf 0.51 (CHCl3/MeOH, 10:1); 1H NMR
(400 MHz, CDCl3, TMS): δ 7.76–7.70, 7.42–7.32 (m, 10H, aromatic),
6.11 (d,1H, J2,NH 8.6 Hz, NH), 5.39–5.37 (m, 2H, H-1IV, H-4III), 5.33–5.28
(m, 2H, H-4I, H-1III), 5.24 (dd, 1H, J2,3 11.0, J3,4 3.0 Hz, H-3III), 5.17 (bs,
1H, H-4IV), 5.07–4.95 (m, 5H, H-1II, H-2III, H-3IV, H-2IV, H-5III), 4.92
(dd, 1H, J2,3 10.0, J3,4 3.5 Hz, H-3II),4.61 (d, 1H, J1,2 7.4 Hz, H-1I), 4.45
(dd, 1H, J5,6a 6.4, J6a,6b 11.5 Hz, H-6IIa), 4.32–4.20 (m, 3H, H-4I, H-6IIb,
H-5IV), 4.15–4.04 (m, 2H, H-6Ia, H-6Ib), 3.93-3.86 (m, 4H, H-2I, H-3I,
PEG × 1), 3.78-3.59 (m, 14H, H-2II, H-5II, PEG × 6), 3.42–3.39 (m, 2H,
PEG × 1), 3.16–3.12 (m, 1H, H-5I), 2.18, 2.14, 2.13, 2.11, 2.05 × 2, 1.99,
1.96, 1.95, 1.91 (s × 10, 3H × 10, Ac × 10), 1.17–1.06 (m, 15H, H-6IV,
H-6III, Me3 of tert-Bu); 13C NMR (100 MHz, CDCl3): δ 171.64, 171.37,
170.90, 170.66, 170.60 × 2, 170.39, 170.24, 169.86, 169.80 (C = O),
136.05, 135.46, 133.61, 132.47, 129.89, 128.07, 127.81 (aromatic),
101.48 (C1I), 100.56 (C1II), 96.64 (C1III), 96.09 (C1IV), 75.37 (C5I), 73.61
(C2II), 73.40 (C3II), 73.08 (C4I), 71.79 (C4III), 71.36, 71.07 (PEG), 70.97
(C4IV), 70.94 (C5II), 70.68, 70.67, 70.55, 70.07 (PEG), 68.19 (C3III), 68.10
(C3I, C2IV), 68.04 (C2I), 67.96 (C2III), 67.72 (C3IV), 67.33 (C4II), 65.03
(C5IV), 64.06 (C5III), 61.36 (C6I), 61.04 (C6II), 50.78 (PEG), 27.10 (Me3
of tert-Bu), 23.48, 21.33, 20.96, 20.84 × 2, 20.83 × 3, 20.76, 20.71, (Ac),
19.57 (CMe3 of tert-Bu) 16.14, 15.71 (C6III, C6IV); HRMS (ESI, posi-
tive ion mode) m/z = 1510.6196 [M + NH4]+, calcd for C68H100N5O31Si1,
1510.6172.
To a solution of compound 19 (314 mg, 200 μmol) and 2-(2-(2-
(2-azidoethoxy)ethoxy)ethoxy)ethanol (65 mg, 0.30 mmol) in dry
CH2Cl2 (5.0 mL) was added activated MS4A. The mixture was kept
stirring at −50 °C for 15 min under Ar atmosphere, followed by ad-
dition of TMSOTf (11 μL, 59 μmol). After stirring at −50 °C for 30 min,
Et3N was added to terminate the reaction. The mixture was fil-
tered through a Celite bed, and the filtrate was washed successively
with satd aq NaHCO3 and brine. The organic layer was dried over
MgSO4, filtered through a Celite bed, and concentrated under reduced
pressure. The residue was purified by silica gel chromatography (n-
hexane/EtOAc 1:2, v/v, containing 0.5% Et3N) to afford 20 (262 mg,
159 μmol, 80%) as colorless amorphous.
[α]D25 −73.6 (c 0.1, CHCl3); Rf 0.39 (n-hexane/EtOAc 2:3); 1H NMR
(400 MHz, CDCl3, TMS): δ 7.90–7.36 (19H, m, aromatic), 5.50 (bd,
1H, J3,4 3.8 Hz, H-4II), 5.38 (bd, 1H, J3,4 3.0 Hz, H-4III), 5.26–5.08 (m,
7H, H-1IV, H-4IV, H-3III, H-3II, H-1I, H-5III, H-3IV), 5.02 (d, 1H, J1,2 8.5 Hz,
H-1I), 4.98–4.93 (m, 2H, H-2IV, H-1III), 4.87 (dd, 1H, J1,2 4.0, J2,3 11.0 Hz,
H-2III), 4.78 (t, 1H, J2,3 = J3,4 = 9.7 Hz, H-3I), 4.50–4.38 (m, 3H, H-5IV,
H-6IIa, H-4I), 4.36–4.26 (m, 2H, H-2I, H-6IIb), 4.20 (bs, 2H, H-6Ia, H-6Ib),
3.95–3.88 (m, 2H, H-2II, PEG), 3.82–3.78 (m, 1H, H-5II), 3.70–3.23
(m, 16H, H-5I, PEG), 2.11, 2.10, 2.08 × 2, 2.07, 1.92, 1.87, 1.73 (s × 8,
24H, Ac), 1.27–1.24 (m, 6H, C6III, C6IV), 1.16 (s, 9H, Me3 of tert-Bu);
13C NMR (CDCl3, 100 MHz): δ 170.96 × 2, 170.88, 170.67, 170.38,
170.13, 169.86 × 2, 169.63, 165.22 × 2 (C = O), 136.06, 135.47, 134.46,
133.88, 133.52, 132.40, 130.06, 130.01, 129.56, 128.94, 128.73, 128.22,
127.87, 123.61 (aromatic), 99.88 (C1II), 98.50 (C1I), 97.63 (C1IV), 95.81
(C1III), 75.30 (C5I), 74.07 (C2II, C3II), 73.28 (C4I), 72.51 (C3I), 71.72 (C4III),
71.14 (C5II), 71.07(C4IV), 770.87, 70.83, 70.76, 70.72, 70.66, 70.61, 70.55,
70.15, 70.10, 68.88 (PEG), 68.19 (C3III), 68.03 (C2IV), 67.75 (C2III), 67.30
(C4II), 66.93 (C3IV), 65.41 (C5IV), 64.34 (C5III), 61.27 (C6I), 61.04 (C6II),
56.87 (C2I), 50.80 (PEG), 27.15 (CMe3 of tert-Bu), 21.01, 20.96, 20.83,
20.70 × 2, 20.68 × 2, 20.37 (Ac), 19.61 (CMe3 of tert-Bu), 16.14, 15.65
(C6III,C6IV); HRMS (ESI) m/z = 1665.5807 [M + Na]+, calcd for
C79H98N4O32SiNa, 1665.5831.
4.17. Sodium {2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethyl β-D-
galactopyranosyl-(1→4)-[α-L-fucopyranosyl-(1→3)]-2-acetamido-2-
deoxy-6-O-sulfonato-β-D-glucopyranoside} (3)
To a solution of the compound 17 (30 mg, 24.9 μmol) in MeOH
(3.0 mL) was added MeONa (ca. 28 wt-%) in MeOH (2 μL, 12.4 μmol).
After stirring at rt overnight, the reaction mixture was concen-
trated under reduced pressure. The residue was purified by Biogel
P-2 gel column eluting with H2O to afford 3 (13 mg, 15.6 μmol, 63%)
as colorless amorphous.
[α]D25 −37.5 (c 0.2, MeOH); Rf 0.51(CHCl3/MeOH/H2O, 5:4:1); 1H
NMR (400 MHz, CD3OD): δ 5.04 (d, 1H, J1,2 3.9 Hz, H-1II), 4.83–4.76
(m, 1H, H-5II), 4.59 (d, 1H, J1,2 7.6 Hz, H-1III), 4.55 (d, 1H, J1,2 7.7 Hz,
H-1I), 4.44 (dd, 1H, J5,6a 3.4, J6a,6b 10.8 Hz, H-6IIIa), 4.31 (dd, 1H, J5,6b
2.5, J6a,6b 10.9 Hz, H-6IIIb), 3.98–3.84 (m, 5H, H-2I, H-3II, H-4I, PEG × 1),
3.82 (d, 1H, J3,4 2.9 Hz, H-4III), 3.79–3.61 (m, 18H, H-6Ia, H-6Ib, H-2II,
H-3I, H-4II, H-5III, PEG × 6), 3.56–3.1 (m, 2H, H-3 III, H-5I), 3.47 (dd,
1H, J1,2 7.6, J2,3 9.6 Hz, H-2III), 3.42–3.38 (m, 2H, PEG × 1), 1.97 (s, 3H,
Ac), 1.17 (d, 3H, J5,6 6.5 Hz, H-6II); 13C NMR (CD3OD, 100 MHz) δ 173.83
(C = O),103.61 (C1III), 102.40 (C1I), 100.15 (C1II), 76.40 (C5I), 76.27,
75.17, 75.07, 74.80, 73.72 (C4II, C3III, C5III, C3I, C4I), 72.97 (C2III), 71.51,
71.48, 71.44, 71.40, 71.35 (PEG), 71.15 (C3II), 71.04 (PEG), 70.11 (C4III),
69.98 (C2II), 69.94 (PEG), 67.72 (C5II), 66.96 (C6III), 62.66 (C6I), 56.71
(C2I), 51.77 (PEG), 23.08 (Ac), 16.61 (C6II); HRMS (ESI, negative ion
mode) m/z = 809.2626 [M − Na]−, calcd for C28H49N4O21S, 809.2610.
4.16. 2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl 2,3,4-tri-O-
acetyl-α-L-fucopyranosyl-(1→2)-3,4,6-tri-O-acetyl-β-D-
galactopyranosyl-(1→4)-[2,3,4-tri-O-acetyl-α-L-fucopyranosyl-
(1→3)]-6-O-tert-butyldiphenylsilyl-2-acetamido-2-deoxy-β-D-
glucopyranoside (21)
To a solution of compound 20 (262 mg, 159 μmol) in MeOH
(5.0 mL) was added MeONa in MeOH solution (ca. 28 wt%; 32 μL,
159 μmol). After stirring at rt for 2 h, DOWEX 50W-X8 (H+ form)
was added to neutralize the reaction system, and then filtered
through cotton, concentrated under reduced pressure to obtain the
crude mixture (203 mg). To a solution of the mixture (164 mg, 136
μmol) in EtOH (4.0 ml) was added NH2NH2·H2O (33 μL, 681 μmol).
After stirring at 90 °C for 13 h, the reaction mixture was concen-
trated under reduced pressure. The residue was dissolved in pyridine
(5.0 mL), followed by addition of Ac2O (192 μL, 2.04 mmol) and DMAP
(8 mg, 68.0 μmol). After stirring at rt under dry atmosphere over-
night, MeOH (1 mL) was added to quench excess reagents, and the
mixture was concentrated with toluene under reduced pressure. The
residue was dissolved in CHCl3, washed with satd aq NaHCO3 and
4.18. 2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl β-D-
galactopyranosyl-(1→4)-[α-L-fucopyranosyl-(1→3)]-2-acetamido-2-
deoxy-β-D-glucopyranoside (4)
Compound 15 (51 mg, 36.1 μmol) was suspended in dry MeOH
(3.0 mL) followed by the addition of ca. 28 wt-% MeONa in MeOH
(5 μL, 32.9 μmol). After stirring at rt under dry atmosphere over-
night, the reaction mixture was neutralized by the addition of Dowex
50W-X4 (H+ form), filtered through cotton, and concentrated under
reduced pressure. The residue dissolved in EtOH (4.0 mL) was added
NH2NH2·H2O (10 μL, 212.8 μmol). After stirring at 90 °C overnight,

44
Y. Yamazaki et al./Carbohydrate Research 422 (2016) 34–44
the reaction mixture was concentrated under reduced pressure. To
a solution of the residue in pyridine (3.0 mL) was added Ac2O (50 μL,
530.7 μmol). After stirring at rt under dry atmosphere for 36 h, MeOH
(1 mL) was added to quench excess reagents, then concentrated with
toluene under reduced pressure. The residue was purified by silica
gel column chromatography (CH3Cl/MeOH, 1:0 to 6:1, v/v, linear gra-
dient, Rf 75 system). The obtained compound was dissolved in dry
THF (3.0 mL) followed by the addition of AcOH (3 μL, 58.6 μmol)
and 1M TBAF in THF (550 μl, 550 μmol). After stirring at rt under
Ar atmosphere for 3 days, the reaction mixture was concentrated
under reduced pressure and extracted with CHCl3, washed with satd
aq NaHCO3 and brine. The organic layer was dried over MgSO4, fil-
tered through a Celite bed, and concentrated under reduced pressure.
The residue was roughly purified by silica gel column chromatog-
raphy (CHCl3/Methnol, 1:0 to 6:1, v/v, linear gradient, Rf 75 system)
to afford 16. To a solution of 16 in MeOH (4.0 mL) was added MeONa
in MeOH (ca. 28 wt-%, 6 μL, 40.9 μmol). After stirring at rt over-
night, the reaction mixture was concentrated under reduced
pressure. The residue was subjected to LH-20 size-exclusion column
chromatography eluting with H2O, and then to reversed phase
column chromatography eluting with H2O/MeOH (1:0 to 0:100, v/v,
linear gradient, Rf 75 system) to afford 4 (4 mg, 5.47 μmol, 15%, 5
steps) as colorless amorphous.
(m, 2H, PEG × 1), 1.97 (s, 3H, Ac), 1.26–1.21 (m, 6H, H-6III, H-6IV); 13
C
NMR (CDCl3, 100 MHz): δ 173.89 (C = O), 102.53, 102.21 (C1I, C1II),
102.14 (C1IV), 100.36 (C1III), 79.45, 77.45, 76.72, 76.64, 76.52, 75.30,
74.46, 73.71, 73.67, 71.87, 71.68, 71.60, 71.53, 71.25, 71.13, 70.79, 70.13,
69.96, 69.93 (C5I, C4I, C5II, C3I, C4II, C2II, C3II, C4II, C2III, C3III, C2IV, C3IV,
C4IV, PEG), 68.28 (C5III), 67.65 (C5IV), 62.94, 62.71 (C6I, C6II), 57.32
(C2I), 51.78 (PEG), 23.12 (Ac), 16.86, 16.80 (C6III, C6IV); HRMS (ESI,
positive ion mode) m/z = 899.3592 [M + Na]+, calcd for C34H60N4O22Na,
899.3597.
4.20. A typical procedure for synchronous synthesis of 10 and 11
Compound 2 (100 mg, 96.3 μmol) was added to a solution of 9
(90 mg, 170.9 μmol) in anhydrous CH2Cl2 (1.0 mL), and then diluted
with anhydrous Et2O (2.0 mL). The mixture was kept stirring at rt
for 30 min under Ar atmosphere in the presence of activated MSAW
300 (100 mg). N-Iodosuccinimide (55 mg, 244.5 μmol) was added
to the mixture, and then it was cooled down to −40 °C under Ar at-
mosphere. Triflic acid (1.7 μL, 19.3 μmol) in anhydrous Et2O (100 μL)
was injected to the mixture. After stirring for 1 h, excess amount
of Et3N was added to terminate the reaction. The mixture was fil-
tered through a Celite bed, diluted with CHCl3, washed successively
with 5% aq Na2S2O3, satd aq NaHCO3 and brine. The organic layer
was dried over MgSO4, filtered through a Celite bed, and concen-
trated under reduced pressure. The residue was subjected to silica
gel column chromatography (n-hexane/EtOAc, 1:0 to 0:1, v/v, linear
gradient, Rf75 system), providing 10 (69 mg, 48.4 μmol, 49%) and
11 (73 mg, 43.3 μmol, 45%).
24
[α]D −56.0 (c 0.04, MeOH); Rf 0.58 (CHCl3/MeOH/H2O, 5:4:1);
1H NMR (400 MHz, CD3OD): δ 5.03 (d, 1H, J1,2 3.9 Hz, H-1II), 4.86–4.81
(m, 1H, H-5II), 4.54 (d, 1H, J1,2 7.5 Hz, H-1I), 4.44 (d, 1H, J1,2 7.3 Hz,
H-1III), 3.95–3.84 (m, 6H, H-6IIIa, H-4I, H-2I, H-3II, PEG × 1), 3.82–3.60
(m, 19H, H-4III, H-6Ia, H-4II, H-6IIIb, H-6Ib, H-5III, H-2II, PEG × 6),
3.54–3.35 (m, 6H, H-2III, H-3III, H-3I, H-5I, PEG × 1), 1.97 (s, 3H, Ac),
1.18 (d, 3H, J5,6 6.6 Hz, H-6II); 13C NMR (CD3OD, 100 MHz): δ 173.90
(C = O), 103.89 (C1III), 102.40 (C1I), 100.35 (C1II), 77.42 (C5I), 76.66
(C3I, C5III), 75.17 (C3II), 74.90 (C3III), 73.69 (C4II), 72.75 (C2III), 71.69,
71.62, 71.54, 71.51 (PEG), 71.22 (C4I), 71.13 (PEG), 70.00 (C4III), 69.94
(C2II), 69.90 (PEG), 67.67 (C5II), 62.77 (C6I), 61.39 (C6III), 57.38 (C2I),
51.77 (PEG), 23.12 (Ac), 16.61 (C6II); HRMS (ESI, positive ion mode)
m/z = 753.3000 [M + Na]+, calcd for C28H50N4O18Na, 753.3018.
Acknowledgment
This work was partially supported by JSPS KAKENHI (Grant
Number 24550134).
Appendix: Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.carres.2016.01.003.
4.19. 2-(2-(2-(2-Azidoethoxy)ethoxy)ethoxy)ethyl α-L-
fucopyranosyl-(1→2)-β-D-galactopyranosyl-(1→4)-[α-L-
fucopyranosyl-(1→3)]-2-acetamido-2-deoxy-β-D-glucopyranoside
(5)
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