Chemical approach for the syntheses of GM4 isomers with sialic acid to non-natural linkage positions on galactose

Article abstract of DOI:10.1016/j.carres.2014.10.018

Cell-surface glycans containing sialic acid are involved in various biological phenomena. However, the syntheses of GM4 derivatives with (2→2) and (2→4) linkages have not been investigated to date. In this study, sialylation of all of the hydroxyl groups on galactose were investigated for the syntheses of GM4 isomers. Regioselective sialylation was achieved via protection of galactosyl acceptors using electron-rich benzyl groups. These synthetic sialylated glycans will prove to be useful tools for studying unidentified carbohydrate-mediated biological roles.

Full text of DOI:10.1016/j.carres.2014.10.018

Carbohydrate Research 401 (2015) 39–50
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Chemical approach for the syntheses of GM4 isomers with sialic acid
to non-natural linkage positions on galactose
⇑
Kenta Kurimoto, Hatsuo Yamamura, Atsushi Miyagawa
Graduate School of Engineering, Nagoya Institute of Technology, Gokiso-cho, Showa-ku, Nagoya 466-8555, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 26 August 2014
Received in revised form 21 October 2014
Accepted 21 October 2014
Available online 29 October 2014
Cell-surface glycans containing sialic acid are involved in various biological phenomena. However, the
syntheses of GM4 derivatives with (2?2) and (2?4) linkages have not been investigated to date. In this
study, sialylation of all of the hydroxyl groups on galactose were investigated for the syntheses of GM4
isomers. Regioselective sialylation was achieved via protection of galactosyl acceptors using electron-rich
benzyl groups. These synthetic sialylated glycans will prove to be useful tools for studying unidentified
carbohydrate-mediated biological roles.
Keywords:
Sialylation
Ó 2014 Elsevier Ltd. All rights reserved.
Oxazolidinone
Anomeric configuration
GM4
1. Introduction
many important biological events.1,4,5 Therefore, sialic acid-
containing glycans have attracted attention in the fields of
Cell-surface glycans, including N- and O-linked glycans, glyco-
sphingolipids, glycosaminoglycans, glycophospholipid anchors,
and lipo–oligo/polysaccharides, participate in a wide range of bio-
logical processes.1,2 Unlike nucleic acids and proteins, oligosaccha-
rides, which often have branched units, are rich in diversity. The
combination of three different nucleotides or amino acids can only
generate six trimers, while three different hexoses could theoreti-
cally generate more than 1000 trisaccharide regioisomers. Thus, by
selecting a limited number of combinations from the many theo-
retical saccharide isomers, organisms construct sugar chain struc-
tures with specific functions.
Sialic acids are a family of naturally occurring 2-keto-3-deoxy-
nononic acids that are involved in various functions such as cell–
cell interactions, binding for bacterial toxins and viruses, and signal
transduction.3 The structural basis for the diversity of sialic acids
arises from substitutions of one or more of the hydroxyl groups
of N-acetylneuraminic acid (Neu5Ac), Neu5Gc, or KDN with acetyl,
methyl, lactyl, phosphate, or sulfate groups.4 Among the more than
50 derivatives of sialic acid, Neu5Ac is the most common and nor-
mally appears at the non-reducing ends of glycoproteins and gly-
colipids. Because they are generally present at the terminal ends
of glycoconjugates located on cell surfaces, sialic acids act either
as masks or recognition sites for ligand–receptor interactions in
immunology and medicinal chemistry. In naturally occurring
sialosides, Neu5Ac is observed on galactosides linked through the
a
difference in the linkage to the sialic acid modulates its interaction
with receptor proteins.
Influenza A viruses can be classified into subtypes depending on
the types of surface glycoprotein present: hemagglutinin (HA) and
neuraminidase (NA). There are 16 known HA subtypes, and 9
known NA subtypes of a total of 144 theoretically possible sub-
types. Influenza virus infection is initiated by the attachment of
the HA to sialic acid-containing ligands such as gangliosides and
glycans on glycoproteins, which are located on host cell surfaces.
(2?3) or a
(2?6) bonds in glycoproteins and glycolipids.6 The
Avian influenza virus HAs bind to sialic acid linked via
a(2?3) gly-
cosidic bonds, while human influenza virus HAs bind to
a(2?6)
receptors. Thus, the difference in the binding position of the sialic
acid to the galactose is very important for species recognition. The
ability of synthetic chemists to construct desired glycoside bonds
has progressed and has contributed to our understanding of the
recognition mechanisms for receptor proteins and the develop-
ment of enzyme inhibitors.7–10
Sialylation is one of the most challenging reactions in carbohy-
drate chemistry and often proceeds with low yield and poor
stereoselectivity.11–13 In particular, the stereoselective synthesis
of a-sialosides is complicated because sialic acid does not contain
a neighboring C-3 functional group to direct the stereochemical
outcome of the glycosylation. Moreover, the electron-withdrawing
property of the carboxylic acid group at the anomeric position
⇑
Corresponding author at present address: 19-524 Gokiso-cho, Showa-ku,
Nagoya, Aichi 466-8555, Japan. Tel./fax: +81 52 735 5239.
E-mail address: miyagawa.atsushi@nitech.ac.jp (A. Miyagawa).
http://dx.doi.org/10.1016/j.carres.2014.10.018
0008-6215/Ó 2014 Elsevier Ltd. All rights reserved.

40
K. Kurimoto et al. / Carbohydrate Research 401 (2015) 39–50
OAc
and the lack of a hydroxyl group at C-3 makes sialyl donors
prone to undesired 2,3-elimination due to destabilization of the
oxocarbenium ion intermediate generated from the sialyl donor.
Consequently, many synthetic sialylation methods have been
developed to improve the stereoselectivity and coupling efficiency
of the reaction.11 However, these methodologies are focused on
AcO
SPh
R3O
R2O
OR4
O
O
COOMe
AcN
AcO
O
R1O
O
O
2-OH acceptor 1 : R1 = H, R2 = Bn, R3 = Bn, R4 = Bn
3-OH acceptor 2 : R1 = Bn, R2 = H, R3 = H, R4 = Bn
3-OH acceptor 3 : R1 = Bz, R2 = H, R3 = H, R4 = Bz
4-OH acceptor 4 : R1 = Bn, R2 = Bn, R3 = H, R4 = Bn
6-OH acceptor 5 : R1 = Bn, R2 = Bn, R3 = Bn, R4 = H
6-OH acceptor 6 : R1 = Bz, R2 = Bz, R3 = Bz, R4 = H
sialyl donor 8
efficient a-sialoside installation with a(2?3) or a(2?6) linkages;
thus, the strategies for the synthesis of non-natural sialosides with
bonds to the 2- or 4-position of galactose remain undeveloped.
Therefore, a synthetic method that enables the introduction of sia-
lic acid groups at the sites of the poor reactive hydroxyl groups in
Figure 1. Six galactosyl acceptors and sialyl donor.
galactose is required. Recently, Mine et al.14 reported that
a2,3-
sialyltransferase from Photobacterium sp. JT-ISH-224 produced a
regio-mistaken sialyl-transferred sialoside which had sialic acid
at the C2 hydroxyl group of lactose. This results may indicate that
some bacterial sialyltransferases have a potency to synthesize unu-
sual sialosides in vivo. The development of chemical synthesis
method for non-natural sialosides enables supply of an adequate
amount of the sialoside to explore unknown biological function.
Here, we describe the comprehensive synthesis of sialyl galac-
toside isomers with both natural and non-natural sialic acid bind-
ing positions to galactose (Scheme 1). Access to such a range of
gangliosides, including those with non-natural linkages, should
lead to the discovery of novel biological functions and new aspects
of the enzymatic mechanisms related to gangliosides. In this study,
natural sialosides were synthesized to investigate the reactivity of
galactosyl acceptors with hydroxyl group protected by acyl or alkyl
groups. Based on these results, non-natural gangliosides were then
efficiently synthesized by modulating the poor reactive hydroxy
groups to more reactive ones.
The nature of the 5-N-protecting group can greatly influence
the efficiency and stereoselectivity of the sialylation.12,13,15 Crich
and Li achieved an elegant synthesis of (2?3) and (2?6)-linked
sialosides using an N-acetyl-5-N,4-O-oxazolidinone-protected sial-
yl donor.16 In addition, it was observed that the sialyl donor with-
out oxazolidinone ring was low reactivity to 6-OH acceptor,
whereas oxazolidinone-protected sialyl donor gave higher reactiv-
ity to the acceptor. For synthesis of non-natural linkages to axial
and sterically hindered hydroxyl groups, the donor should have
the high reactivity. Moreover, if b-linked sialosides are produced
by sialylation, b-linked sialosides will also be attractive to investi-
gate biological function. Thus, sialyl donor 8 (Fig. 1) bearing the
N-acetyl-5-N,4-O-oxazolidinone moiety was employed as a donor
in this study.
2.2. Preparation of the 6-OH acceptor
To obtain the 6-OH glycosyl acceptor, the trityl group was
selected as a temporary and orthogonally functional protecting
group (Scheme 2).17 The allyl b-
D-galactoside 7 was regioselec-
2. Results and discussion
tively tritylated at the 6-position, and then the remaining hydroxyl
groups were protected with benzyl groups to afford 9 (74% yield, in
two steps). Removal of the trityl group under acidic conditions and
hydrogenolysis with Lindlar’s catalyst afforded the 6-OH glycosyl
acceptor 5 (77% yield, in two steps). Similarly, the 6-OH glycosyl
acceptor 6 was prepared from 7 via benzoylation instead of
benzylation.
2.1. Design of the glycosyl acceptors and sialyl donor
Regioselective glycosylation is generally achieved when the gly-
cosyl acceptor possesses protected hydroxyl groups with only one
free hydroxyl group for reaction with the glycosyl donor. The out-
comes of sialylations are known to depend primarily on the nature
of the protecting groups introduced on the glycosyl donor and
acceptor, which differ with respect to their electronic and steric
properties. In this study, benzyl and benzoyl groups were selected
as the protecting groups for the donor to investigate the reactivity
of various glycosyl acceptors with different protecting groups. The
allyl group was selected as a temporary protecting group for the
aglycones of the glycosyl acceptors. This aglycone can be removed
selectively or applied to labeling or immobilization, and so allyl
glycoside was used as starting material. However, sialylation to
allyl acceptor with thiophenyl sialoside as a donor decreased sig-
nificantly in the yield under the NIS/TfOH activation method.
Therefore, allyl group was hydrogenated using Lindlar catalyst to
the corresponding propyl group. The propyl glycosides were
used as acceptors to investigate sialylations. A set of selectively
protected glycosyl acceptors (1–6, Fig. 1) prepared from allyl
2.3. Preparation of the 3,4-OH acceptor
Regioselective sialylation of the 3,4-diol groups of galactose to
synthesize naturally occurring
a
(2?3) linkages has been success-
fully achieved.18–21 In general, glycosyl acceptors for
a
(2?3) sialy-
lation have 3,4-diol structures designed for regioselective
glycosylation at the more reactive C3 hydroxyl group.
To construct the 3,4-diol structure, an isopropylidene acetal
group was introduced to the 3- and 4-positions of galactose. Thus,
compound 7 was treated with acetone and a catalytic amount of
CSA in DMF to afford 3,4-O-isopropylidene protected derivative
11 in 64% yield (Scheme 3). The free OH group in 11 was then con-
verted to the benzyl ether 12, and the isopropylidene acetal group
of 12 was selectively removed using 0.5 M aqueous HCl. Finally, the
deprotected compound was hydrogenated with Lindlar’s catalyst
under H2 gas in EtOAc and EtOH to afford the 3,4-OH acceptor 2
b-
D-galactopyranoside 7 was then synthesized as described in
Section 2.2–2.5.
OAc
OAc
COOMe
O
AcO
SPh
AcO
O
Acid
O
O
O
COOMe
HO
O
AcN
AcO
AcN
AcO
+
O
O
O
O
O
Donor
Acceptor
Scheme 1. Plan for the synthesis of sialyl galactoside isomers.

K. Kurimoto et al. / Carbohydrate Research 401 (2015) 39–50
41
BnO
BnO
HO
HO
BnO
BnO
OH
O
OH
O
OTr
O
a
b
O
O
O
O
O
BnO
5
HO
7
BnO
9
6-OH Bn acceptor
BzO
HO
HO
BzO
BzO
OH
OH
O
OH
O
c
O
d
O
BzO
BzO
6
HO
7
BzO
10
6-OH Bz acceptor
Scheme 2. Synthesis of 6-OH acceptors 5 and 6. (a) (i) TrCl, pyridine, 70 °C, 10 h; (ii) BnBr, NaH, DMF, rt, 13 h, 74% (2 steps); (b) (i) 4 M HCl/Dox, rt, 20 min, 79%; (ii) Lindlar’s
catalyst, H2 gas, EtOAc, EtOH, rt, 6 h, 98%; (c) (i) TrCl, pyridine, 70 °C, 16 h; (ii) BzCl, pyridine, rt, 2.5 h; (iii) 4 M HCl/THF, rt, 1 h, 64% (3 steps); (d) Pd(OH)2, H2 gas, EtOAc, rt, 5 h,
98%.
O
HO
HO
OBn
O
HO
HO
OR
O
OH
O
a
c
O
O
O
O
BnO
2
RO
11
12
HO
7
: R = H
: R = Bn
b
d
3,4-OH Bn acceptor
HO
OBz
O
e
13
: R = Bz
O
HO
BzO
3
3,4-OH Bz acceptor
Scheme 3. Synthesis of 3,4-OH acceptors 2 and 3. (a) acetone, CSA, DMF, rt, 72 h, 64%; (b) BnBr, NaH, DMF, rt, 14 h, quant.; (c) (i) 0.5 M HCl/THF, 40 °C, 8 h, 97%; (ii) Lindlar’s
catalyst, H2 gas, EtOAc, EtOH, rt, 6.5 h, 99%; (d) BzCl, pyridine, CH2Cl2, 0 °C, 3.5 h, quant.; (e) (i) 0.5 M HCl/THF, 40 °C, 8 h, 96%; (ii) Pd(OH)2, H2 gas, EtOAc, rt, 4 h, 96%.
(96% yield, in three steps). Similarly, treatment of compound 11
with benzoyl chloride and pyridine in CH2Cl2 afforded the benzoy-
lated compound 13; isopropylidene group removal and palladium-
mediated hydrogenolysis afforded the corresponding 3,4-OH
acceptor 3 (92% yield, in three steps).
it reacted with the BH3. Subsequently, regioselective reduction of
the 4,6-O-benzylidene acetal derivative using CoCl2 and BH3ꢀTHF23
afforded the corresponding 4-O-benzyl derivative 16 in 83% yield.
Next, compound 16 was benzylated with benzyl imidate under
acidic conditions, in which the acetate esters were stable and not
hydrolyzed.24,25 Finally, the acetyl group was removed using
NaOMe in MeOH and aqueous NH3 to afford 2-OH derivative 1,
which served as the glycosyl acceptor in the synthesis of
Neu(2?2)Gal.
2.4. Preparation of the 2-OH acceptor
Synthetic route via an orthoester22 was initially tried to synthe-
size compound 1. However, the method gave low yield in benzyla-
tion. Therefore, the synthetic route via the benzylidene after
introducing aglycone was planned. Compound 7 was protected
with benzylidene acetal to afford 4,6-O-benzylidenated glalacto-
sides (Scheme 4). Unfortunately, the benzylidene-protected prod-
uct was not soluble in many organic solvents such as EtOAc,
MeOH, CH2Cl2, and toluene. Thus, to purify the reaction mixture
using normal phase silica gel chromatography, acetylation of the
2,3-diol moiety on the benzylidene-protected product was per-
formed to afford 14 in 83% yield (2 steps). Acetyl groups of com-
pound 14 were removed by NaOMe in MeOH, followed by
monobenzylation of the C3 hydroxyl group and acetylation of the
C2 hydroxyl group to afford 15 (22%). The low yield of the desired
monobenzylated product was due to the formation of byproducts,
that is, the 2,3-di-O-benzyl derivative (29%) and the 2-O-benzyl
derivative (2%). Prior to reduction, the allyl group of compound
15 was converted to the propyl group via hydrogenolysis because
2.5. Preparation of the 4-OH acceptor
Compound 7 was first protected with benzylidene acetal at the
4- and 6-positions, and then the remaining hydroxyl group was
benzylated to afford 17 (74% yield, in two steps) (Scheme 5). Reg-
ioselective ring-opening of the benzylidene acetal of 17 using
NaBH3CN and 4 M HCl in dioxane afforded 18 almost exclusively
in 82% yield.26,27 Treatment of compound 18 with Lindlar’s catalyst
under H2 gas in EtOAc and EtOH afforded the 4-OH propyl acceptor
4 in 98% yield.
2.6. Sialylation with galactosyl acceptors
To investigate the solvent effect on the synthesis of the a(2?6)
linkage, coupling reactions of donor 8 with acceptor 5 were exam-
ined in various mixtures of MeCN and CH2Cl2 at ꢁ40 °C using the
Ph
O
O
R2O
BnO
BnO
HO
HO
BnO
BnO
OBn
O
OH
O
OH
O
a
O
c
d
O
O
O
O
R1O
HO
1
HO
7
AcO
16
14 : R1, R2 = Ac
b
15 : R1 = Ac, R2 = Bn
2-OH Bn acceptor
Scheme 4. Synthesis of 2-OH Bn acceptor 1. (a) (i) PhCH(OCH3)2, CSA, DMF, rt, 17 h; (ii) Ac2O, pyridine, 50 °C, 2 h, 83% (2 steps); (b) (i) NaOMe, MeOH, DMF, rt, 48 h; (ii) BnBr,
NaH, DMF, 0 °C, 23 h; (iii) Ac2O, pyridine, rt, 2.5 h, 22% (3 steps); (c) (i) Lindlar’s catalyst, H2 gas, EtOAc, EtOH, THF, rt, 4 h, quant.; (ii) CoCl2, BH3ꢀTHF, from 0 °C to rt, 55 min,
83%; (d) (i) benzyl imidate, TfOH, CH2Cl2, cHex, MS4A, from 0 °C to rt, 1.5 h; (ii) NaOMe, MeOH, NH3 aq, 50 °C, 28 h, 44% (2 steps).

42
K. Kurimoto et al. / Carbohydrate Research 401 (2015) 39–50
Ph
O
O
HO
BnO
HO
HO
HO
BnO
OBn
O
OH
O
OBn
O
a
O
b
c
O
O
O
O
BnO
BnO
17
BnO
HO
7
BnO
18
4
4-OH Bn acceptor
Scheme 5. Synthesis of 4-OH Bn acceptor 4. (a) (i) PhCH(OCH3)2, CSA, DMF, rt, 24 h; (ii) BnBr, NaH, DMF, rt, 19 h, 74% (2 steps); (b) NaBH3CN, 4 M HCl/Dox, THF, 0 °C, 2 h, 82%;
(c) Lindlar’s catalyst, H2 gas, EtOAc, EtOH, rt, 3 h, 98%.
Table 1
Effect of solvent on sialylation
OAc
COOMe
AcO
OAc
AcO
SPh
BnO
BnO
OH
O
O
O
NIS, TfOH
AcN
AcO
BnO
O
COOMe
O
AcN
AcO
᧧
O
O
Solvent, MS3A,
-40 ºC, 30 min
O
BnO
5
BnO
O
O
BnO
8
19
O
Entry
Solvent
Yield (%)
Ratio (a:b)
1
2
3
CH2Cl2
MeCN
MeCN/CH2Cl2
(1/1)
91.3
94.2
98.3
7.1:1
6.0:1
10.0:1
Anomeric ratios were determined by 1H NMR analysis.
Donor:acceptor = 1.5:1.
Table 2
Effect of the acceptor protecting groups on sialylation
OAc
OAc
COOMe
AcO
AcO
SPh
NIS, TfOH
O
O
O
O
8
AcN
AcO
COOMe
O
O
AcN
AcO
HO
O
᧧
MeCN / CH2Cl2
( 1 / 1 ), MS3A,
-40 ºC, 30 min
O
O
O
19-22
Acceptor
O
Entry
1
Acceptor
Product
Yield (%)
Ratio (
10.1:1
a:b)
BnO
BnO
OAc
OH
O
COOMe
O
AcO
O
O
AcN
AcO
BnO
BnO
O
98.3
O
5
O
BnO
O
BnO
19
BzO
BzO
OAc
OH
O
COOMe
AcO
AcO
AcO
O
O
O
AcN
AcO
BzO
BzO
2
3
4
96.8
76.7
56.6
a only
O
O
6
O
BzO
O
BzO
20
HO
HO
OAc
OBn
O
COOMe
HO
O
OBn
O
O
O
AcN
AcO
BnO
O
O
1:1
O
BnO
2
O
21
HO
HO
OAc
OBz
COOMe
HO
O
O
OBz
O
O
O
AcN
AcO
BzO
1:1.9
O
BzO
3
O
22
Anomeric ratios were determined by 1H NMR analysis.
Donor:acceptor = 1.5:1.
promoter NIS/TfOH (Table 1).16 As shown in Table 1 entry 3, the
highest yield (98%) and stereoselectivity ( :b = 10:1) for the
(2?6) sialylation was obtained in a 1:1 (v/v) mixture of MeCN
tion of the oxocarbenium ion due to the nitrile effect.28–30 This
same solvent-system was applied to following sialylations.
Next, the effect of acceptors with acyl and alkyl protecting
groups on the sialylation was investigated using synthesized
a
a
and CH2Cl2. The influence of the solvent on the sialylation was
probably due to both the moderate solvent polarity and stabiliza-
a(2?6) and a(2?3) linked GM4 derivatives (Table 2). The ano-

K. Kurimoto et al. / Carbohydrate Research 401 (2015) 39–50
43
Table 3
Synthesis of sialyl galactose regioisomers
OAc
AcO
OAc
COOMe
O
SPh
AcO
NIS, TfOH
O
O
O
O
8
COOMe
AcN
AcO
AcN
O
HO
O
MeCN / CH2Cl2
( 1 / 1 ), MS3A,
-40 ºC, 30 min
᧧
AcO
O
O
O
23-24
Acceptor
O
Entry
Acceptor
Product
BnO
BnO
MeOOC
Yield (%)
Ratio (a:b)
BnO
BnO
OBn
O
OBn
O
O
O
O
HO
1
O
1
>99
2.4:1
O
AcO
O
AcO
NAc
AcO
23
HO
OBn
O
BnO
4
O
BnO
O
OBn
O
BnO
2
OAc
57.7*
b only
O
AcO
OBn
COOMe
O
AcN
AcO
O
O
24
Anomeric ratios were determined by 1H NMR analysis.
Donor:acceptor = 1.5:1.
*
Acceptor recovery = 11.9%.
meric configurations of all sialylated products were assigned on
the basis of both the chemical shifts of H-3eq and the 3JC1,H-3ax cou-
pling constants.31 Sialylation with the 6-OH derivatives 5 and 6
afforded the corresponding sialosides in high yields (98% and
reactive than the C2 hydroxyl group. Nevertheless, in our case,
sialylation with the 2-OH acceptor 1 afforded sialylation product
in higher yield than that of the 3,4-OH acceptor 2, which afforded
the desired product in a modest yield along with sialyl lactone and
disialylated byproducts. Moreover, reaction of the 2-OH acceptor 1
97%) with excellent stereoselectivity (10:1 and
and 2, respectively). When compared to their stereoselectivity,
the benzoyl acceptor 6 was suited to afford -isomer in 6-O-sialy-
a only, entries 1
proceeded with higher
a-stereoselectivity (a:b = 2.4:1) than 3,4-
a
OH acceptor 2. Based on these results, it can be concluded that
sialylation of the C2 hydroxyl group can be performed using accep-
tor 1, which is protected with an electron-rich benzyl group.
Sialylation with 4-OH glycosyl acceptor 4 afforded the corre-
sponding disaccharide 24 in 58% yield. In contrast to sialylation with
2-OH acceptor, the sialylation gave lower yields of the disaccharide
due to poor reactivity on axial hydroxyl group, along with only 12%
recovery of the unreacted acceptor 4. (Sialylation with other posi-
tion of galactose hydroxyl groups were not isolated recovery of the
unreacted acceptor.) Moreover, surprisingly, the (2?4) linked prod-
lation. In the case of the 3,4-OH acceptors 2 and 3, 3-O-sialylated
products 21 and 22 were obtained with moderate yields (77%
and 57%) and low stereoselectivity (1:1 and 1:1.9), respectively,
(entries 3 and 4). Crich and Li16 and Farris and Meo32 observed sim-
ilar b-stereoselectivity in the coupling of donor 8 with 3,4-diol
acceptors. Note that, in the sialylation using 3,4-OH acceptors 2
and 3, disialylates at both the 3 and 4-positions were obtained in
9% and 7% yield, respectively, and a small amount of sialyl lactone
was also obtained. In both reactions, the (2?4)-monosialylate was
not detected in the crude mixture. Due to these side reactions, the
yields of the target compounds were decreased. As Table 2, the
sialylation with the 6-OH acceptors 5 and 6 afforded higher yield
with excellent stereoselectivity than the sialylation with the 3,4-
OH acceptors 2 and 3. Moreover, the glucosyl acceptors protected
with the electron-donating benzyl group exhibited increased reac-
tivity for sialylation.
uct was detected only b-isomer (3JC-1 ,H-3 ax = 0 Hz). To investigate
the b-selectivity, additional experiments were performed in differ-
0
0
ent conditions of temperature and solvent. However, the a-isomer
was not detected in the conditions, and there was no noticeable
effect on the stereoselectivity. This anomeric configuration results
showed that the reactivity of 4-OH on galactose with sialyl donor
is clearly different from hydroxyl groups of the other position. How-
ever, the mechanism for this unique b-sialylation is not explained
except for dependency on thermodynamic stability of product.
Therefore, it remains interesting to investigate the mechanism and
Based on the results for the a(2?6) and a(2?3) sialylation, the
benzyl group used to protect acceptors designed for the synthesis
of GM4 isomers with non-natural linkages. As presented in Table 3,
the synthesis of (2?2) linked GM4 derivative 23 was realized in
a-selective synthetic method.
99% yield with moderate selectivity (entry 1,
a:b = 2.4:1). Previ-
ously, a galactoside blocked at only the C6 hydroxyl group was
reported to react with sialyl donors specifically at the C3
position,33 and regioselective sialylation with a 2,3-diol galactosyl
3. Conclusion
In this study, comprehensive GM4 isomers with Neu5Ac resi-
dues at each hydroxyl group of galactose were synthesized for
the first time. A set of selectively protected glycosyl acceptors were
acceptor led to the formation of only the
a(2?3) linkage prod-
uct.21,34 Thus, the C3 hydroxyl group is assumed to be more

44
K. Kurimoto et al. / Carbohydrate Research 401 (2015) 39–50
prepared and their sialylations with an N-acetyl-5-N,4-O-oxazolid-
inone-protected sialyl donor were investigated. A mixture of MeCN
and CH2Cl2 (1:1, v/v) was determined to be effective for achieving
bromide (4.0 mL, 33.6 mmol) was added dropwise to the solution
in an ice bath, and the mixture was warmed to room temperature,
and stirred for 13 h. The reaction was quenched by addition of
methanol (10 mL). The mixture was then diluted with H2O and
extracted with EtOAc. The organic phase was dried over Na2SO4,
filtered, and evaporated. The residue was purified by silica gel col-
umn chromatography, eluting with Hex/EtOAc (15:1) to give 9
(2.94 g, 4.01 mmol, 73.6%). 1H NMR (400 MHz, CDCl3): d 7.41–
7.11 (m, 30H, 6ꢂPh), 5.94 (m, 1H, OCH2CH@CH2), 5.31 (ddd, 1H,
Jallylic = 1.6 Hz, Jgem = 3.0 Hz, Jtrans = 17.6 Hz, OCH2CH@CHH), 5.17
(ddd, 1H, Jallylic = 1.6 Hz, Jcis = 10.4 Hz, OCH2CH@CHH), 4.83 (d, 2H,
J = 10.8 Hz, PhCH2), 4.72 (d, 2H, J = 12.0 Hz, PhCH2), 4.65 (d, 2H,
J = 11.6 Hz, PhCH2), 4.40 (ddt, 1H, J = 5.2 Hz, J = 12.8 Hz,
OCHHCH@CH2), 4.36 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.12 (ddt, 1H,
J = 6.0 Hz, J = 12.8 Hz, OCHHCH@CH2), 3.84 (d, 1H, J3,4 = 2.8 Hz, H-
4), 3.80 (dd, 1H, J2,3 = 9.6 Hz, H-2), 3.51–3.45 (m, 2H, H-6b, H-3),
3.34 (t, 1H, J5,6 = 6.4 Hz, H-5), 3.20 (dd, 1H, J6a,6b = 9.2 Hz, H-6a).
stereoselective sialylation in Neua(2?6)Gal derivative, and GM4
isomers could be synthesized efficiently using this solvent system.
Moreover, it was demonstrated that the oxazolidinone-protected
sialyl donor could be utilized to efficiently sialylate the 2 and 4
positions of galactoside. All of the hydroxyl groups of galactoside
were linked to sialic acid to form Neua(2?2)Gal, Neub(2?2)Gal,
Neu (2?3)Gal, Neub(2?3)Gal, Neub(2?4)Gal, Neu
a
a(2?6)Gal
and Neub(2?6)Gal derivatives. Most importantly, sialylation was
performed at the less reactive C2 and C4 hydroxyl groups of galact-
ose acceptors to afford non-natural GM4 derivatives. Thus, this
synthetic method is effective for the preparation of GM4 deriva-
tives with Neu5Ac residues of galactoside at various linkage
positions. When the glycosyl acceptors were protected by benzyl
group, the reactivity of each hydroxyl group on the acceptor in
sialylation got to the following order: 6-OH ; 2-OH > 3-OH > 4-OH.
Meanwhile, (2?4) linked sialoside was given only b-anomer and
4.2.2. Propyl 2,3,4-tri-O-benzyl-b-D-galactopyranoside (5)
further studies on the synthesis of Neu
improved the stereoselectivity and coupling efficiency.
a
(2?4)Gal needs to be
Compound 9 (1.14 g, 1.56 mmol) was dissolved in hydrogen
chloride-dioxane (4 M, 10 mL) at 0 °C. The mixture was warmed
to room temperature and stirred for 20 min. Subsequently, the sol-
vent was evaporated under reduced pressure. The residue was
purified by silica gel column chromatography, eluting with Hex/
4. Experimental section
4.1. General
EtOAc (4:1 to 2:1) to give allyl 2,3,4-tri-O-benzyl-b-
D
-galactopy-
ranoside (604 mg, 1.23 mmol, 79.2%).
A solution of compound allyl 2,3,4-tri-O-benzyl-b-
ranoside (413 mg, 841 lmol) in EtOAc, EtOH (5:2, v/v, 14 mL)
D
-galactopy-
All reagents were purchased from commercial sources and used
without further purification whereas NIS was recrystallized by 1,4-
dioxane and tetrachloromethane before use.35 Dry solvents were
dried by molecular sieves (3 or 4 Å) that activated in vacuo at
200 °C and stored over molecular sieves. TLC was performed on
Merck TLC plates silica gel 60 F254 0.25 mm. Compounds were
detected by UV and/or by treatment with EtOH/H2O/H2SO4/orcinol
and subsequent heating. Column chromatography was performed
with Kanto kagaku silica gel 60 N, spherical neutral, particle size
was stirred vigorously with Lindlar’s catalyst (57 mg) at room
temperature under H2 atmosphere for 6 h. The solid material was
filtered off, and the filtrate was concentrated under reduced
pressure. The residue was purified by silica gel column chromatog-
raphy, eluting with Hex/EtOAc (3:1) to give 5 (404 mg, 821 mmol,
97.6%). 1H NMR (400 MHz, CDCl3): d 7.38–7.25 (m, 15H, 3ꢂPh),
4.96 (d, 2H, J = 11.6 Hz, PhCH2), 4.83–4.65 (m, 4H, 2ꢂPhCH2),
4.36 (d, 1H, J1,2 = 8.0 Hz, H-1), 3.93–3.88 (m, 1H, OCHHCH2CH3),
3.84 (dd, 1H, J2,3 = 9.6 Hz, H-2), 3.79–3.73 (m, 2H, H-4, H-6b),
3.52 (dd, 1H, J3,4 = 3.2 Hz, H-3), 3.49–3.43 (m, 2H, H-6a, OCHHCH2
CH3), 3.36 (t, 1H, J5,6 = 6.0 Hz, H-5), 1.70–1.64 (m, 2H, OCH2CH2
CH3), 1.51 (dd, 1H, OH-6), 0.96 (t, 3H, OCH2CH2CH3). 13C NMR
(100.6 MHz, CDCl3): d 138.75, 138.48, 138.27, 128.72, 128.45,
128.31, 128.20, 127.98, 127.70, 127.64, 127.59 (arom), 104.11
(C-1), 82.31 (C-3), 79.71 (C-2), 75.24 (PhCH2), 74.49 (C-5), 74.12
(PhCH2), 73.45 (PhCH2), 72.87 (C-4), 71.73 (OCH2CH2CH3), 62.06
(C-6), 23.03 (OCH2CH2CH3), 10.71 (OCH2CH2CH3). HRMS (m/z)
Calcd for C30H36O6Na (M+Na+): 515.2410, Found: 515.2446.
40–50 lm or 63–210 lm. Solvents for column chromatography
were purchased from commercial sources and used as received.
1H and 13C NMR spectra were recorded with Bruker AV-400 spec-
trometers (400 MHz for 1H, 100.6 MHz for 13C) or Bruker AV-600
spectrometers (600 MHz for 1H, 150.9 MHz for 13C). Chemical
shifts (in ppm) were referenced to tetramethylsilane (d = 0 ppm)
in deuterated chloroform. 13C NMR spectra were also recorded on
the same NMR spectrometers and were calibrated with CDCl3 peak
3
(d = 77.00 ppm). The JC1,H-3ax coupling constants were measured
by the phase-sensitive gradient-enhanced HMBC spectra.30,36 Mul-
tiplicities are reported by using the following abbreviations:
s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet,
br = broad; J = coupling constant values in Hertz. Mass spectra
(MS) and high-resolution mass spectra (HRMS) were recorded with
a JEOL JMS-S3000 mass spectrometer using 2,5-dihydroxybenzoic
acid (DHBA) as the matrix.
4.3. Synthesis of 6-OH Bz acceptor
4.3.1. Allyl 2,3,4-tri-O-benzoyl-b-
D
-galactopyranoside (10)
-galactopyranoside 7 (4.00 g,
A stirred solution of allyl b-
D
18.1 mmol) in dry pyridine (80 mL) was added triphenylmethyl
chloride (6.60 g, 23.6 mmol) and heated to 70 °C. After 16 h, the
reaction mixture was cooled down to 0 °C, and benzoyl chloride
(2.40 mL, 20.7 mmol) was added dropwise. After stirring for 2.5 h
at room temperature, the reaction mixture was diluted with CH2-
Cl2, then washed with 1 M HCl aqueous, saturated aqueous
NaHCO3, and brine, dried over Na2SO4, filtered, and evaporated.
The residue was dissolved in hydrogen chloride–THF (4 M,
150 mL) at 0 °C. The mixture was warmed to room temperature
and stirred for 1 h. The reaction mixture was diluted with CH2Cl2,
then washed with saturated aqueous NaHCO3, and brine, dried
over Na2SO4, filtered, and evaporated. The residue was purified
by silica gel column chromatography, eluting with Hex/EtOAc
(3:1) to give 10 (6.16 g, 11.6 mmol, 63.7%). 1H NMR (400 MHz,
4.2. Synthesis of 6-OH Bn acceptor
4.2.1. Allyl 2,3,4-tri-O-benzyl-6-O-triphenylmethyl-b-D-
galactopyranoside (9)
A stirred solution of allyl b-D-galactopyranoside 7 (1.20 g,
5.45 mmol) in dry pyridine (20 mL) was added triphenylmethyl
chloride (1.67 g, 5.99 mmol) and heated to 70 °C. After 10 h, the
reaction mixture was cooled down to room temperature and
quenched by addition of methanol (4 mL). Subsequently, the sol-
vent was evaporated under reduced pressure. The residue was dis-
solved in dry DMF (30 mL) and added sodium hydride (60%
dispersion in oil, 1.30 g, 32.5 mmol) in an ice bath. After stirring
for 1 h at room temperature under reduced pressure, benzyl

K. Kurimoto et al. / Carbohydrate Research 401 (2015) 39–50
45
CDCl3): d 8.13–7.23 (m, 15H, 3ꢂPh), 5.90–5.77 (m, 3H, H-2, OCH2-
CH@CH2, H-4), 5.59 (dd, 1H, J2,3 = 10.6 Hz, J3,4 = 3.2 Hz, H-3), 5.27
(ddd, 1H, Jallylic = 1.6 Hz, Jgem = 3.0 Hz, Jtrans = 17.2 Hz, OCH2-
CH@CHH), 5.16 (ddd, 1H, Jallylic = 1.6 Hz, Jcis = 10.4 Hz, OCH2-
CH@CHH), 4.86 (d, 1H, J1,2 = 7.6 Hz, H-1), 4.41 (ddt, 1H, J = 4.8 Hz,
J = 13.2 Hz, OCHHCH@CH2), 4.20 (ddt, 1H, J = 6.0 Hz, J = 13.2 Hz,
OCHHCH@CH2), 4.03 (t, 1H, J5,6 = 6.8 Hz, H-5), 3.87–3.63 (m, 2H,
H-6), 2.66 (t, 1H, OH-6).
CH@CHH), 5.20 (ddd, 1H, Jallylic = 1.6 Hz, Jcis = 10.8 Hz, OCH2-
CH@CHH), 4.82 (dd, 2H, J = 12.0 Hz, PhCH2), 4.60 (dd, 2H,
J = 12.0 Hz, PhCH2), 4.42 (ddt, 1H, J = 5.2 Hz, J = 12.8 Hz,
OCHHCH@CH2), 4.37 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.16–4.11 (m, 3H,
H-3, H-4, OCHHCH@CH2), 3.90 (dt, 1H, J4,5 = 1.6 Hz, J5,6 = 6.8 Hz,
H-5), 3.79 (m, 2H, H-6), 3.42 (m, 1H, H-2), 1.35 (s, 3H, OCH3),
1.33 (s, 3H, OCH3).
4.4.3. Propyl 2,6-di-O-benzyl-b-D-galactopyranoside (2)
4.3.2. Propyl 2,3,4-tri-O-benzoyl-b-
D
-galactopyranoside (6)
mol) in EtOAc (6 mL)
Compound 12 (494 mg, 1.12 mmol) was dissolved in THF
(24 mL) and added 1 M HCl aqueous (40 mL) in an ice bath. Subse-
quently, the mixture was warmed to 40 °C and stirred for 8 h. The
reaction mixture was then cooled down to room temperature and
diluted with H2O and extracted with CH2Cl2. The organic phase
was washed with saturated aqueous NaHCO3, and brine, dried over
Na2SO4, filtered, and evaporated. The residue was purified by silica
gel column chromatography, eluting with Hex/EtOAc (4:1 to 2:1)
A solution of compound 10 (203 mg, 380
l
was stirred vigorously with palladium hydroxide (100 mg) at room
temperature under H2 atmosphere for 5 h. The solid material was
filtered off, and the filtrate was concentrated under reduced pres-
sure. The residue was purified by silica gel column chromatogra-
phy, eluting with Hex/EtOAc (3:1 to 2:1) to give 6 (199 mg,
373 mmol, 98.0%). 1H NMR (400 MHz, CDCl3): d 8.13–7.23 (m,
15H, 3ꢂPh), 5.87–5.81 (m, 2H, H-2, H-4), 5.58 (dd, 1H,
J2,3 = 10.4 Hz, J3,4 = 3.6 Hz, H-3), 4.80 (d, 1H, J1,2 = 8.0 Hz, H-1),
4.03 (m, 1H, H-5), 3.96–3.90 (m, 1H, OCHHCH2CH3), 3.88–3.81
(m, 1H, H-6b), 3.69–3.63 (m, 1H, H-6a), 3.56–3.50 (m, 1H,
OCHHCH2CH3), 2.65 (t, 1H, OH-6), 1.63–1.53 (m, 2H, OCH2CH2CH3),
0.80 (t, 3H, OCH2CH2CH3). 13C NMR (100.6 MHz, CDCl3): d 133.83,
133.34, 133.20, 130.17, 129.77, 129.68, 129.49, 128.79, 128.75,
128.65, 128.39, 128.34 (arom), 101.74 (C-1), 74.00 (C-5), 72.13
(OCH2CH2CH3), 71.90 (C-3), 70.07 (C-2), 69.08 (C-4), 60.63 (C-6),
22.72 (OCH2CH2CH3), 10.23 (OCH2CH2CH3). HRMS (m/z) Calcd for
to give allyl 2,6-di-O-benzyl-b-
1.09 mmol, 96.8%). A solution of compound allyl 2,6-di-O-benzyl-
b- -galactopyranoside (426 mg, 1.06 mmol) in EtOAc, EtOH (10:3,
D-galactopyranoside (435 mg,
D
v/v, 13 mL) was stirred vigorously with Lindlar’s catalyst
(57.2 mg) at room temperature under H2 atmosphere for 6.5 h.
The solid material was filtered off, and the filtrate was concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography, eluting with Hex/EtOAc (4:1 to 2:1)
to give 2 (424 mg, 1.05 mmol, 99.1%). 1H NMR (400 MHz, CDCl3):
d 7.36–7.26 (m, 10H, 2ꢂPh), 4.82 (d, 2H, J = 11.2 Hz, PhCH2), 4.59
(s, 2H, PhCH2), 4.37 (d, 1H, J1,2 = 7.6 Hz, H-1), 3.99 (t, 1H,
J3,4 = 3.0 Hz, H-4), 3.96–3.91 (m, 1H, OCHHCH2CH3), 3.81–3.72
(m, 2H, H-6), 3.63–3.57 (m, 2H, H-3, H-5), 3.52–3.46 (m, 2H, H-2,
OCHHCH2CH3), 2.56 (dd, 1H, OH-4), 2.50 (dd, 1H, OH-3), 1.71–
1.63 (m, 2H, OCH2CH2CH3), 0.97 (t, 3H, OCH2CH2CH3). 13C NMR
(100.6 MHz, CDCl3): d 138.38, 137.87, 128.49, 128.41, 128.10,
127.84, 127.74, 127.70 (arom), 103.63 (C-1), 79.04 (C-2), 74.53
(PhCH2), 73.63 (PhCH2), 73.25, 73.12 (C-3, C-5), 71.55 (OCH2CH2-
CH3), 69.32 (C-6), 68.91 (C-4), 22.96 (OCH2CH2CH3), 10.67 (OCH2-
CH2CH3). HRMS (m/z) Calcd for C23H30O6Na (M+Na+): 425.1940,
Found: 425.1951.
C
30H30O9Na (M+Na+): 557.1788, Found: 557.1788.
4.4. Synthesis of 3-OH Bn acceptor
4.4.1. Allyl 3,4-O-isopropylidene-b-
D
-galactopyranoside (11)
-galactopyranoside 7 (201 mg,
mol) in dry DMF (1.80 mL) was added acetone (1.34 mL,
18.2 mmol) and CSA (44.0 mg, 189 mol) at room temperature
A stirred solution of allyl b-
D
913
l
l
and stirred for 72 h. Subsequently, the reaction mixture was neu-
tralized with triethylamine (0.05 mL, excess), and the solvent
was evaporated under reduced pressure. The residue was purified
by flash silica gel column chromatography, eluting with Hex/EtOAc
(1:1) to give 11 (150 mg, 577 mmol, 63.7%). 1H NMR (400 MHz,
CDCl3): d 5.93 (m, 1H, OCH2CH@CH2), 5.32 (ddd, 1H, Jallylic = 1.6 Hz,
Jgem = 3.2 Hz, Jtrans = 17.6 Hz, OCH2CH@CHH), 5.23 (ddd, 1H,
Jallylic = 1.2 Hz, Jcis = 10.4 Hz, OCH2CH@CHH), 4.39 (ddt, 1H,
J = 5.6 Hz, J = 12.8 Hz, OCHHCH@CH2), 4.26 (d, 1H, J1,2 = 8.4 Hz, H-
1), 4.18–4.09 (m, 3H, H-3, H-4, OCHHCH@CH2), 3.99 (m, 1H, H-
5), 3.85 (m, 2H, H-6), 3.59 (dt, 1H, H-2), 2.54 (s, 1H, OH-2), 2.19
(br d, 1H, OH-6), 1.53 (s, 3H, OCH3), 1.35 (s, 3H, OCH3).
4.5. Synthesis of 3-OH Bz acceptor
4.5.1. Allyl 2,6-di-O-benzoyl-3,4-O-isopropylidene-b-D-
galactopyranoside (13)
Compound 11 (503 mg, 1.93 mmol) was dissolved in dry CH2Cl2
(8 mL) and added dry pyridine (1 mL, 12.4 mmol) and cooled to
0 °C, after which benzoyl chloride (670 lL 5.77 mmol) was added
dropwise. After 3.5 h, the reaction mixture was diluted with CH2-
Cl2, then washed with 1 M HCl aqueous, saturated aqueous
NaHCO3, and brine, dried over Na2SO4, filtered, and evaporated.
The residue was purified by silica gel column chromatography,
eluting with Hex/EtOAc (10:1) to give 13 (940 mg, 2.00 mmol,
quant). 1H NMR (400 MHz, CDCl3): d 8.07, 7.57, 7.45 (m, 10H,
2ꢂPh), 5.79 (m, 1H, OCH2CH@CH2), 5.30 (dd, 1H, J2,3 = 7.2 Hz,
H-2), 5.17 (ddd, 1H, Jallylic = 1.6 Hz, Jgem = 3.2 Hz, Jtrans = 17.2 Hz,
OCH2CH@CHH), 5.07 (ddd, 1H, Jallylic = 1.6 Hz, Jcis = 10.8 Hz, OCH2
CH@CHH), 4.67 (m, 2H, H-6), 4.57 (d, 1H, J1,2 = 8.2 Hz, H-1), 4.38
(m, 1H, J3,4 = 5.6 Hz, H-3), 4.33–4.28 (m, 2H, H-4, OCHHCH@CH2),
4.20 (m, 1H, H-5), 4.11 (ddt, 1H, J = 13.6 Hz, OCHHCH@CH2), 1.64
(s, 3H, OCH3), 1.37 (s, 3H, OCH3).
4.4.2. Allyl 3,4-O-isopropylidene-2,6-di-O-benzyl-b-D-
galactopyranoside (12)
Sodium hydride (60% dispersion in oil, 2.00 g, 50.0 mmol)
washed thrice with hexane was placed in dry DMF (5 mL). The
slurry was cooled in an ice bath and compound 11 (3.21 g,
12.3 mmol) dissolved in dry DMF (60 mL) was added dropwise
with stirring. When the addition was completed, the stirring was
continued for 1 h at room temperature under reduced pressure.
Bnzyl bromide (4.4 mL, 37.0 mmol) was added dropwise to the
solution in an ice bath, and the mixture was warmed to room tem-
perature and stirred for 14 h. The reaction was quenched by addi-
tion of methanol (2 mL). The mixture was then diluted with H2O
and extracted with EtOAc. The organic phase was dried over
Na2SO4, filtered, and evaporated. The residue was purified by silica
gel column chromatography, eluting with Hex/EtOAc (15:1 to
13:1) to give 12 (5.48 g, 12.4 mmol, quant). 1H NMR (400 MHz,
CDCl3): d 7.41–7.23 (m, 10H, 2ꢂPh), 5.95 (m, 1H, OCH2CH@CH2),
5.34 (ddd, 1H, Jallylic = 1.6 Hz, Jgem = 3.2 Hz, Jtrans = 17.2 Hz, OCH2-
4.5.2. Propyl 2,6-di-O-benzoyl-b-D-galactopyranoside (3)
Compound 13 (500 mg, 1.07 mmol) was dissolved in THF
(24 mL) and added 1 M HCl aqueous (40 mL) in an ice bath. Subse-
quently, the mixture was warmed to 40 °C and stirred for 8 h. The
reaction mixture was then cooled down to room temperature,
diluted with CH2Cl2, washed with saturated aqueous NaHCO3,

46
K. Kurimoto et al. / Carbohydrate Research 401 (2015) 39–50
and brine, dried over Na2SO4, filtered, and evaporated. The residue
was purified by silica gel column chromatography, eluting with
Na2SO4, filtered, and evaporated. The residue was purified by silica
gel column chromatography, eluting with Hex/EtOAc (8:1 to 2:1)
to give mono-O-benzylated derivative (646 mg) and 2,3-di-O-ben-
zyl derivative (820 mg, 29%).
Hex/EtOAc (4:1 to 2:1) to give allyl 2,6-di-O-benzoyl-b-
D-galacto-
pyranoside (438 mg, 1.02 mmol, 95.7%).
A solution of compound allyl 2,6-di-O-benzoyl-b-
D
-galactopy-
A solution of mono-O-benzylated derivative (646 mg) in dry
pyridine (4.5 mL) was treated with acetic anhydride (3 mL) and
stirred for 2.5 h at room temperature. Then, the solvent was con-
centrated under reduced pressure. The residue was purified by sil-
ica gel column chromatography, eluting with Hex/EtOAc (4:1 to
2:1) to give 2-O-acetyl-3-O-benzyl derivative 15 (547 mg,
1.24 mmol, 21.5%) and 2-O-benzyl-3-O-acetyl derivative (52 mg,
0.12 mmol, 2%). 1H NMR (400 MHz, CDCl3): d 7.56–7.29 (m, 10H,
2ꢂPh), 5.86 (m, 1H, OCH2CH@CH2), 5.48 (s, 1H, PhCH), 5.39 (dd,
1H, J2,3 = 10.2 Hz, H-2), 5.26 (ddd, 1H, Jallylic = 1.6 Hz, Jgem = 3.0 Hz,
Jtrans = 17.2 Hz, OCH2CH@CHH), 5.15 (ddd, 1H, Jallylic = 1.4 Hz,
Jcis = 10.4 Hz, OCH2CH@CHH), 4.67 (d, 2H, J = 12.6 Hz, PhCH2),
4.47 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.39–4.30 (m, 2H, OCHHCH@CH2,
H-6b), 4.17 (d, 1H, J3,4 = 3.2 Hz, H-4), 4.10 (ddt, 1H, J = 6.0 Hz,
J = 13.2 Hz, OCHHCH@CH2), 4.03 (dd, 1H, H-6a), 3.60 (dd, 1H, H-
3), 3.35 (d, 1H, H-5), 2.07 (s, 3H, OCOCH3).
ranoside (432 mg, 1.01 mmol) in EtOAc (13 mL) was stirred vigor-
ously with palladium hydroxide (40 mg) at room temperature
under H2 atmosphere for 4 h. The solid material was filtered off,
and the filtrate was concentrated under reduced pressure. The res-
idue was purified by silica gel column chromatography, eluting
with Hex/EtOAc (4:1 to 2:1) to give 3 (417 mg, 0.970 mmol,
96.2%). 1H NMR (400 MHz, CDCl3): d 8.45, 7.57, 7.44 (m, 10H,
2ꢂPh), 5.22 (dd, 1H, J1,2 = 8.0 Hz, J2,3 = 9.6 Hz, H-2), 4.70 (dd, 1H,
J5,6b = 6.4 Hz, J6a,6b = 11.6 Hz, H-6b), 4.61–4.57 (m, 2H, H-6a, H-1),
4.06 (br s, 1H, H-4), 3.91–3.82 (m, 3H, H-5, H-3, OCHHCH2CH3),
3.55 (br d, 1H, OH-3), 3.50–3.45 (m, 1H, OCHHCH2CH3), 3.21 (br
d, 1H, OH-4), 1.60–1.50 (m, 2H, OCH2CH2CH3), 0.80 (t, 3H, OCH2-
CH2CH3). 13C NMR (100.6 MHz, CDCl3): d 167.22, 166.56, 133.32,
133.29, 129.87, 129.71, 129.62, 129.57, 128.44, 128.35 (arom),
101.00 (C-1), 74.28 (C-2), 72.80 (C-3), 72.14 (C-5), 71.63 (OCH2CH2-
CH3), 68.72 (C-4), 62.91 (C-6), 22.71 (OCH2CH2CH3), 10.29 (OCH2-
CH2CH3). HRMS (m/z) Calcd for C23H26O8Na (M+Na+): 453.1525,
Found: 453.1523.
4.6.3. Propyl 2-O-acetyl-3,4-di-O-benzyl-b-
D
-galactopyranoside
mol) in EtOAc–
(16)
A solution of compound 15 (393 mg, 893
l
4.6. Synthesis of 2-OH Bn acceptor
EtOH–THF (20:4:1, v/v, 25 mL) was stirred vigorously with Lind-
lar’s catalyst (80 mg) at room temperature under H2 atmosphere
for 6 h. The solid material was filtered off, and the filtrate was con-
centrated under reduced pressure to give propyl 2-O-acetyl-3,4-di-
4.6.1. Allyl 2,3-di-O-acetyl-4,6-O-benzylidene-b-D-
galactopyranoside (14)
A stirred solution of allyl b-
D-galactopyranoside 7 (3.02 g,
O-benzyl-b-D-galactopyranoside (394 mg, 890 lmol, 99.7%).
13.7 mmol) in dry DMF (30 mL) was added benzaldehyde dimethyl
acetal (3.1 mL, 20.6 mmol) and CSA (316 mg, 1.36 mmol) in an ice
bath and then was warmed to room temperature. After 17 h, the
reaction mixture was neutralized with triethylamine (1.2 mL),
and the solvent was evaporated under reduced pressure. The resi-
due was acetylated with acetic anhydride (6 mL) in dry pyridine
(9 mL) at 50 °C. After 2 h, the solvent was concentrated under
reduced pressure. The residue was purified by silica gel column
chromatography, eluting with Hex/EtOAc (4:1) to give 14 (4.45 g,
11.3 mmol, 82.7%). 1H NMR (400 MHz, CDCl3): d 7.52, 7.37 (m,
5H, Ph), 5.88 (m, 1H, OCH2CH@CH2), 5.51 (s, 1H, PhCH), 5.42 (dd,
1H, J2,3 = 10.4 Hz, H-2), 5.29–5.16 (m, 2H, OCH2CH@CH2), 4.97
(dd, 1H, J3,4 = 3.6 Hz, H-3), 4.56 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.41–
4.32 (m, 3H, H-4, OCHHCH@CH2, H-6b), 4.15–4.05 (m, 2H,
OCHHCH@CH2, H-6a), 3.51 (d, 1H, H-5), 2.07 (s, 3H, OCOCH3),
2.06 (s, 3H, OCOCH3).
To a stirred solution of propyl 2-O-acetyl-3,4-di-O-benzyl-b-D-
galactopyranoside (567 mg, 1.28 mmol) in THF solution (1 M BH3
solution in THF, 15 mL), anhydrous CoCl2 (500 mg, 3.85 mmol)
was added at 0 °C. After stirring for 15 min at 0 °C, 40 min at room
temperature, the blue reaction mixture was diluted with an excess
volume of CH2Cl2, and filtered through Celite. The organic layer
was washed with H2O, dried over Na2SO4, filtered, and concen-
trated under reduced pressure. The residue was purified by silica
gel column chromatography, eluting with Hex/EtOAc (3:1) to give
16 (469 mg, 1.06 mmol, 82.5%). 1H NMR (400 MHz, CDCl3): d 7.37,
7.29 (m, 10H, 2ꢂPh), 5.38 (dd, 1H, J2,3 = 10.0 Hz, H-2), 4.80 (d, 2H,
J = 12.0 Hz, PhCH2), 4.64 (d, 2H, J = 12.2 Hz, PhCH2), 4.34 (d, 1H,
J1,2 = 8.0 Hz, H-1), 3.85–3.78 (m, 3H, H-4, H-6b, OCHHCH2CH3),
3.55–3.52 (m, 2H, H-3, H-6a), 3.43–3.36 (m, 2H, H-5, OCHHCH2-
CH3), 2.04 (s, 3H, OCOCH3), 1.63–1.51 (m, 3H, OCH2CH2CH3), 0.88
(t, 3H, OCH2CH2CH3).
4.6.2. Allyl 2-O-acetyl-3-O-benzyl-4,6-O-benzylidene-b-
D
-
4.6.4. Propyl 3,4,6-tri-O-benzyl-b-
D-galactopyranoside (1)
galactopyranoside (15)
After a mixture of 16 (239 mg, 538
lmol) and activated molec-
Sodium methoxide (110 mg, 2.04 mmol) was added to the solu-
tion of compound 14 (2.27 g, 5.78 mmol) in methanol (35 mL) at
room temperature. After stirring for 1 h, the reaction product
was crystallized and the reaction mixture became suspension.
Then DMF (5 mL) was added to the reaction mixture. After stirring
for 48 h at room temperature, the reaction mixture was neutral-
ized with Diaion SK 1B, which was removed by filtration. The fil-
trate was concentrated under reduced pressure.
Sodium hydride (60% dispersion in oil, 695 mg, 17 mmol)
washed thrice with hexane was placed in dry DMF (1 mL). The
slurry was cooled in an ice bath and the residue dissolved in dry
DMF (50 mL) was added dropwise with stirring. When the addition
was completed, the stirring was continued for 1 h at room temper-
ature under reduced pressure. The mixture was then cooled down
ular sieves (4 Å, 1.0 g) in dry CH2Cl2 (2.0 mL) and cyclohexane
(1.0 mL) was stirred at room temperature for 1 h, the mixture
was cooled to 0 °C, and added benzyl trichloroacetimidate
(500 lL, 2.69 mmol) and TfOH (50 lL, 563 lmol). After stirring
for 1.5 h, the mixture was warmed to room temperature and stir-
red for 10 min. The reaction mixture was diluted with an excess
volume of CH2Cl2, washed with saturated aqueous NaHCO3, dried
over Na2SO4, filtered, and evaporated. The residue was purified
by silica gel column chromatography, eluting with Hex/EtOAc
(15:1 to 10:1) to give the mixture of propyl 2-O-acetyl-3,4,6-tri-
O-benzyl-b-
The mixture dissolved in MeOH (2.7 mL) was added NaOMe
(6 mg, 110 mol) at room temperature. After 3 h, an additional
amount of NaOMe (6 mg, 110 mol) was added, and the stirring
D-galactopyranoside and by-product (273 mg).
l
l
at 0 °C, benzyl bromide (800
l
L, 6.8 mmol) was added dropwise to
was continued for 17 h at 50 °C. Then, 28% aqueous NH3 (4 mL)
was added. After 6 h, an additional amount of 28% aqueous NH3
(10 mL) and MeOH (10 mL) was added and the stirring was contin-
ued for 2 h at 65 °C. The reaction mixture was diluted with an
the solution and stirred for 23 h. The reaction was quenched by
addition of methanol (7 mL). The mixture was then diluted with
H2O and extracted with EtOAc. The organic phase was dried over

K. Kurimoto et al. / Carbohydrate Research 401 (2015) 39–50
47
excess volume of CH2Cl2, washed with H2O, dried over Na2SO4, fil-
tered, and evaporated. The residue was purified by silica gel col-
umn chromatography, eluting with Hex/EtOAc (8:1 to 6:1) to
J = 13.6 Hz, OCHHCH@CH2), 4.02 (br t, 1H, H-4), 3.82–3.71 (m,
2H, H-6), 3.67 (dd, 1H, J2,3 = 9.6 Hz, H-2), 3.55 (t, 1H, J5,6 = 5.8 Hz,
H-5), 3.49 (dd, 1H, J3,4 = 3.4 Hz, H-3), 2.49 (s, 1H, OH-4).
give 1 (116 mg, 235 l
mol, 43.6%). 1H NMR (400 MHz, CDCl3): d
7.38–7.24 (m, 15H, 3ꢂPh), 4.75 (d, 2H, J = 11.6 Hz, PhCH2), 4.70
(d, 2H, J = 12.0 Hz, PhCH2), 4.45 (d, 2H, J = 11.8 Hz, PhCH2), 4.23
(d, 1H, J1,2 = 7.6 Hz, H-1), 3.97–3.92 (m, 2H, H-2, H-4), 3.87–3.81
(m, 1H, OCHHCH2CH3), 3.65–3.55 (m, 3H, H-5, H-6), 3.48–3.42
(m, 2H, H-3, OCHHCH2CH3), 2.35 (s, 1H, OH-2), 1.67–1.60 (m, 2H,
OCH2CH2CH3), 0.91 (t, 3H, OCH2CH2CH3). 13C NMR (100.6 MHz,
CDCl3): d 138.53, 138.14, 137.89, 128.48, 128.42, 128.23, 128.15,
127.88, 127.79, 127.75, 127.62, 127.53 (arom), 103.21 (C-1),
81.94 (C-3), 74.51 (PhCH2), 73.73 (C-5), 73.55 (PhCH2), 72.95 (C-
4), 72.39 (PhCH2), 71.47 (C-2), 71.43 (OCH2CH2CH3), 68.75 (C-6),
22.78 (OCH2CH2CH3), 10.39 (OCH2CH2CH3). HRMS (m/z) Calcd for
4.7.3. Propyl 2,3,6-tri-O-benzyl-b-
D
-galactopyranoside (4)
A solution of compound 18 (268 mg, 546
lmol) in EtOAc, EtOH
(1:1, v/v, 12 mL) was stirred vigorously with Lindlar’s catalyst
(60 mg) at room temperature under H2 atmosphere for 3 h. The
solid material was filtered off, and the filtrate was concentrated
under reduced pressure. The residue was purified by silica gel col-
umn chromatography, eluting with Hex/EtOAc (6:1) to give 4
(263 mg, 533 mmol, 97.5%). 1H NMR (400 MHz, CDCl3): d 7.38–
7.26 (m, 15H, 3ꢂPh), 4.83 (d, 2H, J = 10.8 Hz, PhCH2), 4.72 (s, 2H,
PhCH2), 4.59 (s, 2H, PhCH2), 4.36 (d, 1H, J1,2 = 7.6 Hz, H-1), 4.02
(brt, 1H, H-4), 3.95–3.89 (m, 1H, OCHHCH2CH3), 3.80 (dd, 1H,
J5,6 = 6.0 Hz, J6a,6b = 10.0 Hz, H-6b), 3.72 (dd, 1H, H-6a), 3.64 (dd,
1H, J2,3 = 9.2 Hz, H-2), 3.55 (t, 1H, H-5), 3.51–3.45 (m, 2H, H-3,
OCHHCH2CH3), 2.48 (d, 1H, OH-4), 1.70–1.65 (m, 2H, OCH2CH2-
CH3), 0.97 (t, 3H, OCH2CH2CH3). 13C NMR (100.6 MHz, CDCl3): d
138.60, 138.00, 137.90, 128.42, 128.40, 128.28, 128.12, 127.84,
127.80, 127.76, 127.72, 127.59 (arom), 103.69 (C-1), 80.55 (C-3),
78.96 (C-2), 75.15 (PhCH2), 73.69 (PhCH2), 73.11 (C-5), 72.39
(PhCH2), 71.56 (OCH2CH2CH3), 69.19 (C-6), 66.89 (C-4), 22.99
(OCH2CH2CH3), 10.67 (OCH2CH2CH3). HRMS (m/z) Calcd for C30H36-
O6Na (M+Na+): 515.2410, Found: 515.2417.
C
30H36O6Na (M+Na+): 515.2410, Found: 515.2448.
4.7. Synthesis of 4-OH acceptor
4.7.1. Allyl 2,3-di-O-benzyl-4,6-O-benzylidene-b-D-
galactopyranoside (17)
A stirred solution of allyl b-
4.54 mmol) in dry DMF (10 mL) was added benzaldehyde dimethyl
acetal (1.05 mL, 7.01 mmol) and CSA (105 mg, 452 mol) at room
D-galactopyranoside 7 (1.00 g,
l
temperature and stirred for 24 h. Subsequently, the reaction mix-
ture was neutralized with triethylamine (0.4 mL, excess), and the
solvent was evaporated under reduced pressure.
4.8. Synthesis of a(2?6) GM4 derivatives
Sodium hydride (60% dispersion in oil, 730 mg, 18.3 mmol)
washed thrice with hexane was placed in dry DMF (6 mL). The
slurry was cooled in an ice bath and the residue dissolved in dry
DMF (14 mL) was added dropwise with stirring. When the addition
is completed, the stirring was continued for 2 h at room tempera-
ture under reduced pressure. Benzyl bromide (1.70 mL, 14.3 mmol)
was added dropwise to the solution in an ice bath, and the mixture
was warmed to room temperature and stirred for 19 h. The reac-
tion was quenched by addition of methanol (5 mL). The mixture
was then diluted with H2O and extracted with EtOAc. The organic
phase was dried over Na2SO4, filtered, and evaporated. The residue
was purified by silica gel column chromatography, eluting with
Hex/EtOAc (8:1) to give 17 (1.64 g, 3.36 mmol, 73.9%). 1H NMR
(400 MHz, CDCl3): d 7.57–7.26 (m, 15H, 3ꢂPh), 5.97 (m, 1H, OCH2-
CH@CH2), 5.50 (s, 1H, PhCH), 5.34 (ddd, 1H, Jallylic = 1.6 Hz,
Jgem = 3.0 Hz, Jtrans = 17.2 Hz, OCH2CH@CHH), 5.19 (ddd, 1H,
Jallylic = 1.4 Hz, Jcis = 10.4 Hz, OCH2CH@CHH), 4.86 (d, 2H,
J = 10.8 Hz, PhCH2), 4.77 (m, 2H, H-6), 4.48–4.36 (m, 2H,
OCHHCH@CH2, H-1), 4.30, 4.01 (m, 2H, PhCH2), 4.17–4.10 (m, 2H,
OCHHCH@CH2, H-4), 3.88 (dd, 1H, J1,2 = 8.0 Hz, J2,3 = 9.8 Hz, H-2),
3.56 (dd, 1H, J3,4 = 3.6 Hz, H-3), 3.31 (d, 1H, H-5).
4.8.1. Propyl-O-(Methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-
carbonyl-3,5-dideoxy- -glycero- -galacto-non-2-
ulopyranosylonate)-(2?6)-2,3,4-tri-O-benzyl-b-D-
D
a-D
galactopyranoside (19)
A mixture of sialyl donor 8 (156 mg, 274
lmol, 1.5 equiv),
galactosyl acceptor 5 (90 mg, 183 mol, 1.0 equiv) and activated
l
3 Å molecular sieves (1 g) in dry CH2Cl2/MeCN (3.2 mL, 1:1, v/v)
was stirred under argon atmosphere at room temperature for 1 h
to remove any trace amounts of water. The reaction mixture was
then cooled to ꢁ40 °C. N-Iodosuccinimide (148 mg, 658
3.6 equiv) was added followed by a catalytic amount of triflic acid
(24 L, 271 mol, 1.5 equiv) in 100 L of dry MeCN. After being
stirred at ꢁ40 °C for 30 min, the mixture was quenched with tri-
ethylamine (100 L), diluted with CH2Cl2, and filtered through a
lmol,
l
l
l
l
pad of Celite. The resulting filtrate was washed with 20% aqueous
Na2S2O3 solution, dried over anhydrous Na2SO4, filtered, and con-
centrated under reduced pressure. The residue was purified by sil-
ica gel column chromatography, eluting with Hex/EtOAc (3:1 to
2:1) to give 19 (171 mg, 180 lmol, 98.3%, a
:b = 10:1). 1H NMR
(600 MHz, CDCl3): d 7.36–7.23 (m, 15H, 3ꢂPh), 5.53 (dd, 1H,
J7 ,8 = 6.9 Hz, H-70), 5.40 (dt, 1H, J8 ,9a = 3.0 Hz, J8 ,9b = 7.2 Hz, H-
80), 4.84 (d, 2H, J = 10.8 Hz, PhCH2), 4.83 (d, 2H, J = 11.4 Hz, PhCH2),
0
0
0
0
0
0
4.7.2. Allyl 2,3,6-tri-O-benzyl-b-D-galactopyranoside (18)
4.75 (d, 2H, J = 12.0 Hz PhCH2), 4.55 (dd, 1H, J6 ,7 = 1.8 Hz, H-60),
0
0
To a solution of compound 17 (2.22 g, 4.54 mmol) and sodium
cyanoborohydride (2.43 g, 38.7 mmol) in dry THF (45 mL) was
added powdered 4 Å molecular sieves (22 g), and the mixture
was stirred for 30 min at room temperature, then cooled to 0 °C.
Hydrogen chloride-dioxane (4 M, 10.5 mL) was added dropwise
until the evolution of gas ceased. After 2 h, the reaction mixture
was diluted with toluene, filtered through Celite, washed with sat-
urated aqueous NaHCO3, dried over Na2SO4, and concentrated
under reduced pressure. The residue was purified by silica gel col-
umn chromatography, eluting with Hex/EtOAc (6:1) to give 18
(1.82 g, 3.71 mmol, 81.6%). 1H NMR (400 MHz, CDCl3): d 7.36–
7.28 (m, 15H, 3ꢂPh), 5.95 (m, 1H, OCH2CH@CH2), 5.30 (ddd, 1H,
Jallylic = 1.6 Hz, Jgem = 3.0 Hz, Jtrans = 17.2 Hz, OCH2CH@CHH), 5.19
(ddd, 1H, Jallylic = 1.6 Hz, Jcis = 10.4 Hz, OCH2CH@CHH), 4.83 (d, 2H,
J = 10.8 Hz, PhCH2), 4.72 (s, 2H, PhCH2), 4.59 (s, 2H, PhCH2), 4.45–
4.40 (m, 2H, OCHHCH@CH2, H-1), 4.14 (ddt, 1H, J = 6.0 Hz,
0
0
4.38 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.35 (dd, 1H, J9a ,9b = 12.0 Hz, H-
9a0), 4.07 (dd, 1H, H-9b0), 4.00–3.96 (m, 1H, H-40), 3.93–3.88 (m,
3H, H-4, H-6b, OCHHCH2CH3), 3.81 (dd, 1H, J2,3 = 9.6 Hz, H-2),
3.70 (dd, 1H, J5 ,6 = 9.6 Hz, H-50), 3.64 (s, 3H, COOCH3), 3.60–3.55
(m, 2H, H-5, H-6a), 3.53 (dd, 1H, J3,4 = 3.0 Hz, H-3), 3.49–3.46 (m,
0
0
0
0
0
0
1H, OCHHCH2CH3), 2.87 (dd, 1H, J3eq ,4 = 3.6 Hz, J3eq ,3ax = 12.0 Hz,
H-3eq0), 2.49 (s, 3H, NCOCH3), 2.12 (s, 3H, OCOCH3), 2.10–2.08
(m, 4H, H-3ax0, OCOCH3), 2.00 (s, 3H, OCOCH3), 1.69–1.63 (m, 2H,
OCH2CH2CH3), 0.96 (t, 3H, OCH2CH2CH3). 13C NMR (150.9 MHz,
3
CDCl3): d 172.03, 170.54, 170.09, 169.92, 168.24 (C-10, JC-1 ,H-
0
0
3 ax = 6.46 Hz), 153.65, 138.84, 138.71, 138.55, 128.29, 128.23,
128.18, 128.05, 127.70, 127.51, 127.47, 127.35, 127.26, 103.96
(C-1), 99.17, 82.09 (C-3), 79.46 (C-2), 75.80 (C-60), 75.11 (PhCH2),
74.92 (C-40), 74.22 (PhCH2), 73.42 (C-4), 73.02 (PhCH2), 72.64 (C-
5), 72.02 (C-70), 71.64 (OCH2CH2CH3), 69.56 (C-80), 63.54 (C-6),

48
K. Kurimoto et al. / Carbohydrate Research 401 (2015) 39–50
62.82 (C-90), 59.06 (C-50), 53.05 (COOCH3), 36.39 (C-30), 24.69
(NCOCH3), 22.96 (OCH2CH2CH3), 21.05, 20.86, 20.70 (3ꢂOCOCH3),
10.69 (OCH2CH2CH3). HRMS (m/z) Calcd for C49H59NO18Na
(M+Na+): 972.3630, Found: 972.3639.
3:2) to give 21 (148 mg, 172 lmol, 76.7%, a:b = 1:1). (21a)
1H
NMR (600 MHz, CDCl3): d 7.38–7.27 (m, 10H, 2ꢂPh), 5.57 (dd,
1H, J7 ,8 = 7.2 Hz, H-70), 5.44 (dt, 1H, J8 ,9a = 2.4 Hz, J8 ,9b = 7.2 Hz,
H-80), 4.78 (d, 2H, J = 12.0 Hz, PhCH2), 4.57 (s, 2H, PhCH2), 4.48
0
0
0
0
0
0
(dd, 1H, J6 ,7 = 1.8 Hz, H-60), 4.44–4.41 (m, 2H, J1,2 = 7.8 Hz, H-1,
H-9a0), 4.03 (dd, 1H, J3,4 = 3.0 Hz, H-3), 3.99–3.93 (m, 2H, H-40, H-
9b0), 3.92–3.89 (m, 1H, OCHHCH2CH3), 3.88 (br s, 1H, H-4), 3.82–
3.78 (m, 4H, H-6b, COOCH3), 3.72–3.70 (m, 1H, H-6a), 3.66 (dd,
0
0
4.8.2. Propyl-O-(Methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-
carbonyl-3,5-dideoxy-D-glycero-a-D-galacto-non-2-
ulopyranosylonate)-(2?6)-2,3,4-tri-O-benzoyl-b-D-
galactopyranoside (20)
1H, J5 ,6 = 9.6 Hz, H-50), 3.61 (t, 1H, H-5), 3.56 (dd, 1H,
J2,3 = 9.6 Hz, H-2), 3.52–3.49 (m, 1H, OCHHCH2CH3), 2.81–2.78
0
0
A mixture of sialyl donor 8 (144 mg, 253
lmol, 1.5 equiv),
(m, 2H, J3eq ,4 = 3.6 Hz, J3eq ,3ax = 12.0 Hz, H-3eq0, OH-4), 2.48 (s,
3H, NCOCH3), 2.22 (t, 1H, H-3ax0), 2.11 (s, 3H, OCOCH3), 2.03 (s,
3H, OCOCH3), 1.89 (s, 3H, OCOCH3), 1.69–1.65 (m, 2H, OCH2CH2-
0
0
0
0
galactosyl acceptor 6 (90 mg, 168 mol, 1.0 equiv) and activated
l
3 Å molecular sieves (1 g) in dry CH2Cl2/MeCN (3.2 mL, 1:1, v/v)
was stirred under argon atmosphere at room temperature for 1 h
to remove any trace amounts of water. The reaction mixture was
CH3), 0.95 (t, 3H, OCH2CH2CH3). 13C NMR (150.9 MHz, CDCl3): d
3
172.00, 170.63, 170.24, 169.69, 168.35 (C-10, JC-1 ,H-3 ax = 6.41 Hz),
153.56, 138.80, 138.06, 128.35, 128.13, 127.93, 127.68, 127.51,
103.76 (C-1), 98.78, 77.09 (C-2), 76.21 (C-3), 75.65 (C-60), 75.04
(C-40), 74.73 (PhCH2), 73.54 (PhCH2), 72.75 (C-5), 71.88 (C-70),
71.59 (OCH2CH2CH3), 69.62 (C-80), 69.02 (C-6), 68.39 (C-4), 62.97
(C-90), 58.85 (C-50), 53.23 (COOCH3), 34.86 (C-30), 24.61 (NCOCH3),
22.95 (OCH2CH2CH3), 21.14, 20.77, 20.54 (3ꢂOCOCH3), 10.63
(OCH2CH2CH3). HRMS (m/z) Calcd for C42H53NO18Na (M+Na+):
882.3160, Found: 882.3127.
0
0
then cooled to ꢁ40 °C. N-Iodosuccinimide (136 mg, 604
3.6 equiv) was added followed by a catalytic amount of triflic acid
(22.4 L, 253 mol, 1.5 equiv) in 100 L of dry MeCN. After being
stirred at ꢁ40 °C for 30 min, the mixture was quenched with tri-
ethylamine (100 L), diluted with CH2Cl2, and filtered through a
lmol,
l
l
l
l
pad of Celite. The resulting filtrate was washed with 20% aqueous
Na2S2O3 solution, dried over anhydrous Na2SO4, filtered, and con-
centrated under reduced pressure. The residue was purified by sil-
ica gel column chromatography, eluting with Hex/EtOAc (3:1 to
2:1) to give 20 (162 mg, 163
lmol, 96.8%,
a
only). 1H NMR
(21b) 1H NMR (600 MHz, CDCl3): d 7.37–7.28 (m, 10H, 2ꢂPh),
5.50 (br t, 1H, H-70), 5.40 (bs, 1H, H-80), 4.75 (d, 2H, J = 11.4 Hz,
PhCH2), 4.65–4.62 (m, 1H, H-9a0), 4.60 (s, 2H, PhCH2), 4.55 (dt,
(600 MHz, CDCl3): d 8.08–7.23 (m, 15H, 3ꢂPh), 6.01 (d, 1H,
J3,4 = 3.6 Hz, H-4), 5.71 (dd, 1H, J2,3 = 10.2 Hz, H-2), 5.64 (dd, 1H,
H-3), 5.58 (dd, 1H, J7 ,8 = 8.4 Hz, H-70), 5.48 (dt, 1H, J8 ,9a = 2.4 Hz,
1H, H-40), 4.48 (dd, 1H, J6 ,7 = 2.4 Hz, H-60), 4.37 (d, 1H,
J1,2 = 7.8 Hz, H-1), 4.03–3.99 (m, 2H, H-4, H-9b0), 3.95–3.92 (m,
1H, OCHHCH2CH3), 3.85–3.77 (m, 3H, H-3, H-6), 3.69 (t, 1H,
0
0
0
0
0
0
J8 ,9b = 7.8 Hz, H-80), 4.89 (d, 1H, J1,2 = 7.8 Hz, H-1), 4.62 (dd, 1H,
0
0
J6 ,7 = 1.8 Hz, H-60), 4.49 (dd, 1H, J9a ,9b = 12.6 Hz, H-9a0), 4.27 (m,
0
0
0
0
1H, H-5), 4.06 (dd, 1H, H-9b0), 3.97–3.89 (m, 3H, H-40, H-6b,
J5 ,6 = 9.6 Hz, H-50), 3.65–3.56 (m, 2H, H-2, H-5), 3.52 (s, 3H,
COOCH3), 3.51–3.47 (m, 1H, OCHHCH2CH3), 2.84 (dd, 1H,
0
0
OCHHCH2CH3), 3.73 (dd, 1H, J5 ,6 = 9.6 Hz, H-50), 3.64–3.61 (m,
1H, H-6a), 3.57–3.53 (m, 1H, OCHHCH2CH3), 3.46 (s, 3H, COOCH3),
0
0
J3eq ,4 = 3.6 Hz, J3eq ,3ax = 12.6 Hz, H-3eq0), 2.48 (s, 3H, NCOCH3),
2.18–2.06 (m, 7H, H-3ax0, 2ꢂOCOCH3), 2.03 (s, 3H, OCOCH3),
1.72–1.64 (m, 2H, OCH2CH2CH3), 0.95 (t, 3H, OCH2CH2CH3). 13C
0
0
0
0
2.73 (dd, 1H, J3eq ,4 = 3.6 Hz, J3eq ,3ax = 12.0 Hz, H-3eq0), 2.48 (s, 3H,
NCOCH3), 2.22 (s, 3H, OCOCH3), 2.12 (s, 3H, OCOCH3), 2.08 (t, 1H,
H-3ax0), 2.04 (s, 3H, OCOCH3), 1.61–1.53 (m, 2H, OCH2CH2CH3),
0
0
0
0
NMR (150.9 MHz, CDCl3): d 172.59, 170.78, 170.61, 169.68 (C-10,
0.79 (t, 3H, OCH2CH2CH3). 13C NMR (150.9 MHz, CDCl3): d 172.04,
JC-1 ,H-3 ax = 0 Hz), 166.49, 153.73, 138.32, 137.86, 128.38, 128.37,
128.24, 127.75, 127.72, 127.68 103.99 (C-1), 99.06, 76.64 (C-3),
75.32 (C-60), 74.88 (PhCH2), 74.35 (C-40), 73.71 (PhCH2), 72.81 (C-
5), 72.78 (C-70), 71.68 (OCH2CH2CH3), 70.95 (C-80), 69.92 (C-6),
69.24 (C-4), 63.29 (C-90), 59.30 (C-50), 52.77 (COOCH3), 35.63 (C-
30), 24.68 (NCOCH3), 22.97 (OCH2CH2CH3), 21.07, 20.78, 20.73
(3ꢂOCOCH3), 10.66 (OCH2CH2CH3). HRMS (m/z) Calcd for C42H53-
NO18Na (M+Na+): 882.3160, Found: 882.3186.
3
0
0
3
170.70, 170.42, 170.13, 168.20 (C-10, JC-1 ,H-3 ax = 6.31 Hz), 165.55,
165.30, 165.29, 153.60, 133.30, 133.09, 133.04, 129.93, 129.73,
129.66, 129.60, 129.55, 128.99, 128.52, 128.28, 128.20, 101.43
(C-1), 99.51, 75.76 (C-60), 74.74 (C-40), 71.96 (OCH2CH2CH3),
71.92 (C-3), 71.62 (C-5, C-70), 70.00 (C-2), 68.84 (C-80), 67.79 (C-
4), 63.50 (C-90), 63.21 (C-6), 58.93 (C-50), 52.87 (COOCH3), 36.43
(C-30), 24.70 (NCOCH3), 22.67 (OCH2CH2CH3), 21.05, 20.96, 20.76
(3ꢂOCOCH3), 10.21 (OCH2CH2CH3). HRMS (m/z) Calcd for C49H53-
NO21Na (M+Na+): 1014.3008, Found: 1014.3005.
0
0
4.9.2. Propyl-O-(methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-
carbonyl-3,5-dideoxy-D-glycero-a-D-galacto-non-2-
4.9. Synthesis of
a(2?3) GM4 derivatives
ulopyranosylonate)-(2?3)-2,6-di-O-benzoyl-b-
D-
galactopyranoside (22)
4.9.1. Propyl-O-(methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-O-
carbonyl-3,5-dideoxy- -glycero- -galacto-non-2-
A mixture of sialyl donor 8 (178 mg, 314 lmol, 1.5 equiv),
galactosyl acceptor 3 (90 mg, 209 lmol, 1.0 equiv) and activated
D
a-
D
ulopyranosylonate)-(2?3)-2,6-di-O-benzyl-b-
galactopyranoside (21)
D
-
3 Å molecular sieves (1 g) in dry CH2Cl2/MeCN (3.2 mL, 1:1, v/v)
was stirred under argon atmosphere at room temperature for 1 h
to remove any trace amounts of water. The reaction mixture was
A mixture of sialyl donor 8 (190 mg, 335
lmol, 1.5 equiv),
galactosyl acceptor 2 (90 mg, 224 mol, 1.0 equiv) and activated
l
then cooled to ꢁ40 °C. N-iodosuccinimide (169 mg, 751
3.6 equiv) was added followed by a catalytic amount of triflic acid
(28 L, 316 mol, 1.5 equiv) in 100 L of dry MeCN. After being
stirred at ꢁ40 °C for 30 min, the mixture was quenched with tri-
ethylamine (100 L), diluted with CH2Cl2, and filtered through a
pad of Celite. The resulting filtrate was washed with 20% aqueous
Na2S2O3 solution, dried over anhydrous Na2SO4, filtered, and con-
centrated under reduced pressure. The residue was purified by sil-
ica gel column chromatography, eluting with Hex/EtOAc (3:1 to
lmol,
3 Å molecular sieves (1 g) in dry CH2Cl2/MeCN (3.2 mL, 1:1, v/v)
was stirred under argon atmosphere at room temperature for 1 h
to remove any trace amounts of water. The reaction mixture was
l
l
l
then cooled to ꢁ40 °C. N-Iodosuccinimide (181 mg, 805
3.6 equiv) was added followed by a catalytic amount of triflic acid
(30 L, 339 mol, 1.5 equiv) in 100 L of dry MeCN. After being
stirred at ꢁ40 °C for 30 min, the mixture was quenched with tri-
ethylamine (100 L), diluted with CH2Cl2, and filtered through a
lmol,
l
l
l
l
l
pad of Celite. The resulting filtrate was washed with 20% aqueous
Na2S2O3 solution, dried over anhydrous Na2SO4, filtered, and con-
centrated under reduced pressure. The residue was purified by sil-
ica gel column chromatography, eluting with Hex/EtOAc (3:1 to
3:2) to give 22 (105 mg, 118 lmol, 56.6%, a:b = 1:1.9). (22ab mix-
ture) 1H NMR (400 MHz, CDCl3): d 8.15–7.43 (m, 10H, 2ꢂPh), 5.60–
5.55 (m, 1H, H-80 a), 5.49 (dd, 1H, H-70 a), 5.41 (dd, 1H, J2,3 = 9.6 Hz,
H-2 b), 5.38 (dd, 1H, J2,3 = 10.0 Hz, H-2 a), 4.68 (d, 1H, J1,2 = 7.6 Hz,

K. Kurimoto et al. / Carbohydrate Research 401 (2015) 39–50
49
H-1
a
), 4.58 (d, 1H, J1,2 = 8.0 Hz, H-1 b), 3.72 (m, 3H, COOCH3
a
),
70.87 (OCH2CH2CH3), 68.47 (C-6), 63.40 (C-90), 59.27 (C-50), 52.53
(COOCH3), 34.96 (C-30), 24.65 (NCOCH3), 22.34 (OCH2CH2CH3),
21.09, 20.83, 20.67 (3ꢂOCOCH3), 10.32 (OCH2CH2CH3). HRMS
0
0
3.58 (m, 3H, COOCH3 b), 2.88 (dd, 1H, J3eq ,4 = 3.2 Hz,
J3eq ,3ax = 12.0 Hz, H-3eq0
a
), 2.70 (dd, 1H, J3eq ,4 = 3.6 Hz,
0
0
0
0
J3eq ,3ax = 12.8 Hz, H-3eq0 b), 2.01 (H-3ax0
a, b).
(m/z) Calcd for C
49H59NO18Na (M+Na+): 972.3630, Found:
0
0
972.3669.
4.10. Synthesis of a(2?2) GM4 derivatives
4.11. Synthesis of b(2?4) GM4 derivatives
4.10.1. Propyl-O-(methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-
O-carbonyl-3,5-dideoxy- -glycero- -galacto-non-2-
D
a-D
4.11.1. Propyl-O-(methyl 5-acetamido-7,8,9-tri-O-acetyl-5-N,4-
ulopyranosylonate)-(2?2)-3,4,6-tri-O-benzyl-b-
D
-
O-carbonyl-3,5-dideoxy-D-glycero-b-D-galacto-non-2-
galactopyranoside (23)
ulopyranosylonate)-(2?4)-2,3,6-tri-O-benzyl-b-D-
A mixture of sialyl donor 8 (156 mg, 274
lmol, 1.5 equiv),
galactopyranoside (24)
galactosyl acceptor 1 (90 mg, 183 mol, 1.0 equiv) and activated
l
A mixture of sialyl donor 8 (157 mg, 277
lmol, 1.5 equiv),
3 Å molecular sieves (1 g) in dry CH2Cl2/MeCN (3.2 mL, 1:1, v/v)
was stirred under argon atmosphere at room temperature for 1 h
to remove any trace amounts of water. The reaction mixture was
galactosyl acceptor 4 (90 mg, 183 mol, 1.0 equiv) and activated
l
3 Å molecular sieves (1 g) in dry CH2Cl2/MeCN (3.2 mL, 1:1, v/v)
was stirred under argon atmosphere at room temperature for 1 h
to remove any trace amounts of water. The reaction mixture was
then cooled to ꢁ40 °C. N-iodosuccinimide (148 mg, 658
3.6 equiv) was added followed by a catalytic amount of triflic acid
(24 L, 271 mol, 1.5 equiv) in 100 L of dry MeCN. After being
stirred at ꢁ40 °C for 30 min, the mixture was quenched with tri-
ethylamine (100 L), diluted with CH2Cl2, and filtered through a
lmol,
then cooled to ꢁ40 °C. N-Iodosuccinimide (148 mg, 658
3.6 equiv) was added followed by a catalytic amount of triflic acid
(24 L, 271 mol, 1.5 equiv) in 100 L of dry MeCN. After being
stirred at ꢁ40 °C for 30 min, the mixture was quenched with tri-
ethylamine (100 L), diluted with CH2Cl2, and filtered through a
lmol,
l
l
l
l
l
l
l
pad of Celite. The resulting filtrate was washed with 20% aqueous
Na2S2O3 solution, dried over anhydrous Na2SO4, filtered, and con-
centrated under reduced pressure. The residue was purified by sil-
ica gel column chromatography, eluting with Hex/EtOAc (4:1 to
l
pad of Celite. The resulting filtrate was washed with 20% aqueous
Na2S2O3 solution, dried over anhydrous Na2SO4, filtered, and con-
centrated under reduced pressure. The residue was purified by sil-
ica gel column chromatography, eluting with Hex/EtOAc (6:1 to
2:1) to give 23 (174 mg, 183 lmol, >99%, a:b = 2.4:1). (23a)
1H
NMR (600 MHz, CDCl3): d 7.44–7.20 (m, 15H, 3ꢂPh), 5.55 (dd,
2:1) to give 24 (100 mg, 105 lmol, 58%, b only).
1H, J7 ,8 = 6.0 Hz, H-70), 5.54–5.52 (m, 1H, H-80), 4.76 (d, 2H,
J = 12.0 Hz, PhCH2), 4.69 (d, 2H, J = 13.2 Hz, PhCH2), 4.57 (dd, 1H,
(24b) 1H NMR (600 MHz, CDCl3): d 7.37–7.25 (m, 15H, 3ꢂPh),
0
0
5.75 (d, 1H, J7 ,8 = 6.0 Hz, H-70), 5.68 (bs, 1H, H-80), 4.75–4.73 (m,
1H, H-60), 4.73 (d, 2H, J = 10.8 Hz, PhCH2), 4.65 (d, 2H, J = 12.3 Hz,
PhCH2), 4.52 (d, 2H, J = 12.6 Hz PhCH2), 4.52–4.47 (m, 2H, H-40,
H-9a0), 4.31–4.30 (m, 2H, J1,2 = 7.8 Hz, H-1, H-4), 4.27–4.24 (m,
1H, H-9b0), 3.88–3.85 (m, 1H, OCHHCH2CH3), 3.78 (bt, 1H,
0
0
J8 ,9a = 3.0 Hz, J9a ,9b = 12.0 Hz, H-9a0), 4.40 (m, 1H, H-9b0), 4.37
(dd, 1H, J2,3 = 9.6 Hz, H-2), 4.33 (m, 2H, PhCH2), 4.23 (dd, 1H,
0
0
0
0
J6 ,7 = 1.8 Hz, H-60), 4.16 (d, 1H, J1,2 = 7.2 Hz, H-1), 4.01–3.96 (m,
0
0
1H, H-40), 3.77–3.75 (m, 4H, H-4, COOCH3), 3.71 (dd, 1H, J5 ,6 = 9.6
Hz, H-50), 3.69–3.63 (m, 1H, OCHHCH2CH3), 3.52–3.50 (m, 1H,
H-6b), 3.45–3.39 (m, 3H, H-5, H-6a, OCHHCH2CH3), 3.34 (dd, 1H,
0
0
J2,3 = 9.6 Hz, H-2), 3.71 (t, 1H, J5 ,6 = 10.6 Hz, H-50), 3.66 (t, 1H,
0
0
H-6b), 3.59–3.53 (m, 2H, H-5, H-6a), 3.50 (s, 3H, COOCH3), 3.46–
0
0
0
0
0
0
J3,4 = 2.4 Hz, H-3), 3.14 (dd, 1H, J3eq ,4 = 3.6 Hz, J3eq ,3ax = 12.0 Hz,
H-3eq0), 2.48 (s, 3H, NCOCH3), 2.18–2.08 (m, 4H, H-3ax0, OCOCH3),
1.93 (s, 3H, OCOCH3), 1.88 (s, 3H, OCOCH3), 1.64–1.58 (m, 2H,
3.39 (m, 2H, H-3, OCHHCH2CH3), 2.93 (dd, 1H, J3eq ,4 = 3.6 Hz,
J3eq ,3ax = 12.0 Hz, H-3eq0), 2.49 (s, 3H, NCOCH3), 2.13 (s, 3H,
OCOCH3), 2.06 (s, 3H, OCOCH3), 1.95–1.91 (m, 4H, H-3ax0, OCOCH3),
1.68–1.62 (m, 2H, OCH2CH2CH3), 0.93 (t, 3H, OCH2CH2CH3). 13C NMR
0
0
OCH2CH2CH3), 0.86 (t, 3H, OCH2CH2CH3). 13C NMR (150.9 MHz,
3
CDCl3): d 171.93, 170.67, 170.35, 169.91, 167.07 (C-10, JC-1 ,H-3 ax
= 6.58 Hz), 153.90, 138.45, 138.32, 137.81, 128.47, 128.35,
128.33, 128.18, 127.94, 127.79, 127.72, 127.62, 102.40 (C-1),
99.93, 80.69 (C-3), 75.86 (C-60), 75.84 (C-40), 74.34 (C-2), 74.15
(PhCH2), 73.57 (C-5), 73.40 (PhCH2), 73.21 (C-4), 72.77 (C-70),
72.43 (PhCH2), 72.03 (OCH2CH2CH3), 70.97 (C-80), 68.89 (C-6),
62.13 (C-90), 59.24 (C-50), 53.02 (COOCH3), 36.62 (C-30), 24.70
(NCOCH3), 22.51 (OCH2CH2CH3), 21.09, 20.70, 20.57 (3ꢂOCOCH3),
10.23 (OCH2CH2CH3). HRMS (m/z) Calcd for C49H59NO18Na
(M+Na+): 972.3630, Found: 972.3628.
(150.9 MHz, CDCl3): d 171.86, 170.65, 170.53, 170.08, 167.11 (C-10,
0
0
3
0
0
JC-1 ,H-3 ax = 0 Hz), 153.80, 138.49, 137.84, 137.28, 128.59, 128.50,
128.20, 128.12, 128.07, 127.99, 127.70, 127.61, 127.51, 104.19
(C-1), 98.36, 81.31 (C-3), 78.46 (C-2), 74.97 (PhCH2), 74.82 (C-60),
74.72 (C-40), 73.43 (PhCH2), 73.28 (PhCH2), 72.55 (C-5), 72.21
(C-70), 71.96 (OCH2CH2CH3), 70.22 (C-80), 68.17 (C-4), 67.78 (C-6),
62.90 (C-90), 59.33 (C-50), 52.25 (COOCH3), 37.62 (C-30), 24.73
(NCOCH3), 22.88 (OCH2CH2CH3), 21.10, 20.81, 20.62 (3ꢂOCOCH3),
10.58 (OCH2CH2CH3). HRMS (m/z) Calcd for C49H59NO18Na
(M+Na+): 972.3630, Found: 972.3632.
(23b) 1H NMR (600 MHz, CDCl3): d 7.35–7.26 (m, 15H, 3ꢂPh),
5.59 (s, 1H, H-70), 5.30 (bt, 1H, H-80), 5.00 (d, 1H, J5 ,6 = 9.6 Hz,
0
0
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0
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J9a ,9b = 12.0 Hz, H-9a0), 4.56–4.51 (m, 3H, H-40, PhCH2), 4.43 (m,
2H, J = 11.4 Hz, PhCH2), 4.22 (d, 1H, J1,2 = 7.2 Hz, H-1), 4.16 (t, 1H,
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