Use of a recycle-type SEC method as a powerful tool for purification of thiosialoside derivatives

Article abstract of DOI:10.1016/j.carres.2008.05.014

An efficient separation between fully acetylated thiosialoside methyl esters and fully acetylated Neu5Ac2en methyl esters was accomplished by means of a size-exclusion chromatography (SEC) method. Purity determinations and structural elucidation of the isolated compounds were performed by a combination of elemental analyses and spectroscopic analyses, including IR, ¹H, and ¹³C NMR, and mass spectroscopic analyses.

Full text of DOI:10.1016/j.carres.2008.05.014

Carbohydrate Research 343 (2008) 2735–2739
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Use of a recycle-type SEC method as a powerful tool for purification
of thiosialoside derivatives
*
Jun-Ichi Sakamoto, Chiharu Takita, Tetsuo Koyama, Ken Hatano, Daiyo Terunuma, Koji Matsuoka
Division of Material Science, Graduate School of Science and Engineering, Saitama University, Sakura, Saitama 338-8570, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 25 April 2008
Received in revised form 12 May 2008
Accepted 14 May 2008
Available online 22 May 2008
An efficient separation between fully acetylated thiosialoside methyl esters and fully acetylated Neu5A-
c2en methyl esters was accomplished by means of a size-exclusion chromatography (SEC) method. Purity
determinations and structural elucidation of the isolated compounds were performed by a combination
of elemental analyses and spectroscopic analyses, including IR, 1H, and 13C NMR, and mass spectroscopic
analyses.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Thioglycosides
Sialic acid
SEC purification
Sialooligosaccharides
Glycals
1. Introduction
2. Results and discussion
Much interest has been shown in the synthesis of sialyl oligo-
saccharides because of their importance in biological systems.1
Therefore, much effort has been devoted to the development of sia-
lic acid chemistry.2 We have also been synthesizing sialyl oligosac-
charides and their glycoclusters by chemical and enzymatic
methods, and the glycoclusters have been used in the biochemical
and biomedical fields.3 In our ongoing study on the synthesis of the
sialyl oligosaccharides having thioglycosidic linkages, we have
encountered a problem in that an inseparable mixture of a desired
sialyl derivative and the 2,3-didehydro sialic acid byproduct was
obtained in some cases. A similar phenomenon has been reported
by von Itzstein’s group, and the separation of products was
achieved by using HPLC purification methodology with a re-
versed-phase C18 column after deprotection of the product mix-
ture.4 An alternative and a highly convenient purification method
for isolation of the sialyl oligosaccharides without the 2,3-didehy-
dro sialic acid byproduct is required. Differences in the molecular
size of the products prompted us to consider size-exclusion chro-
matography (SEC), which separates molecules based on differences
in molecular size. Consequently, SEC was chosen for separation of
the products. This paper deals with a convenient method for re-
moval of the 2,3-didehydro sialic acid byproduct in the reaction
mixture by the SEC method.
Scheme 1 summarizes production of the 2,3-didehydro sialic
acid byproduct 35 accompanied by thioacetate 2 as major product
by simple SN2 reaction of known anomeric chloride 1 and KSAc.6
The reaction proceeds smoothly, but simultaneous elimination of
HCl from 2 giving a side product 3 was often observed. After the
usual workup, we tried to separate these mixtures by well-known
procedures, including crystallization and normal-phase chromato-
graphy, but separation unfortunately failed. Both compounds had
the same Rf on TLC, even when a variety of solvent systems as elu-
ents were tested for TLC. In our previous study, a 5-azido sialic acid
derivative did not have an amide proton at C-5 as a highly hydro-
philic group, and it indeed showed excellent separation from side
products after the O-glycosidation reaction.7 However, in the pres-
ent study, another direct method for the isolation of thiosialoside
having both an N-acetamido group and an amide proton was
needed.
Therefore, we next turned our attention to purification of sia-
looligosaccharide after forming their respective thioglycosides. Al-
kyl halide-type glycosides
4 and 6 as candidates for the
thioglycosidation were synthesized from the corresponding 4-pen-
tenyl glycosides in good yields by the previously reported method.8
Scheme 2 shows the synthetic conversion of thioacetate 2 into the
corresponding disaccharides having a newly formed interglycosi-
dic sulfide linkage. Condensation of 6-bromo-6-deoxy-D-glucoside
4 with thioacetate 2, after producing a thiolate anion by treatment
of 2 with diethylamine,9 followed by chromatographic purification
using silica gel, gave a mixture of disaccharide 5 having an
* Corresponding author. Tel./fax: +81 48 858 3099.
E-mail address: koji@fms.saitama-u.ac.jp (K. Matsuoka).
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.05.014

2736
J.-I. Sakamoto et al. / Carbohydrate Research 343 (2008) 2735–2739
OAc
OAc
OAc
OAc
OAc
Cl
COOMe
OAc
COOMe
O
AcO
i)
AcO
AcO
O
O
COOMe
AcHN
SAc
AcHN
AcHN
+
OAc
OAc
OAc
3
1
2
Scheme 1. Reagents and conditions: (i) KSAc (5 M excess), CH2Cl2, 0 °C?rt, 2 d.
OAc
COOMe
S
OAc
O
Br
O
AcO
ii)
i)
AcHN
AcO
O
5
O
(+ 3)
(+ 3)
2 (+ 3) +
2 (+ 3) +
AcO
OAc AcO
O
OAc
AcO
OAc
4
5
OAc
OAc
COOMe
AcO
S
AcO
AcO
Br
O
AcO
O
ii)
iii)
AcHN
O
O
7
OAc
O
OAc
6
AcO
OAc
7
Scheme 2. Reagents and conditions: (i) diethylamine (10 M excess vs Br), DMF, 0 °C?rt, overnight; (ii) purified by SEC; (iii) K2CO3, MeOH–DMF, 0 °C?rt, 2 d, then
Ac2O–pyridine, rt, overnight.
endo-thioglycosidic linkage and glycal 3. Figure 1A shows the 1H
NMR spectrum of the mixture of 5 and 3. Characteristic signals
due to both compounds were observed. Since chromatographic
purification of the products by silica gel as a solid phase adsorbent
was unfortunately unsuccessful, another separation method was
needed. Therefore, we turned to size-exclusion chromatography
(SEC) for separation of the inseparable mixture, a process that is
based on different molecular sizes of the compounds and does
not involve physical adsorption. The inseparable mixture was sub-
jected to a recycle-type SEC system using chloroform as the eluent.
OMe
H-9b
H-9a H-5
A
CH=
(aglycon)
SCH2
H-3eq
SAc
CH=
(glycal)
B
C
D
Figure 1. 1H NMR spectra of (A) mixture of disaccharide 5 and glycal 3, (B) thioacetate 2, (C) glycal 3, and (D) pure disaccharide 5.

J.-I. Sakamoto et al. / Carbohydrate Research 343 (2008) 2735–2739
2737
Table 1
100
80
60
40
20
0
1H NMR chemical shifts and multiplicities of Neu5Ac moieties of compounds 1–10a
I
Compounds
Chemical shifts (d), multiplicity
II
1
2
3b
5
7
10
H-9b
H-9a
H-8
H-7
H-6
H-5
H-4
H-3eq
H-3ax
Me (ester)
NH
4.45, dd
4.08, dd
4.41, dd
4.03, dd
4.60, dd
4.20, dd
4.29, dd
4.10, dd
4.29, dd
4.14, dd
4.30, dd
4.09, dd
5.36, ddd
5.31, br d
3.81, br d
4.04, q
5.18, ddd 5.23, ddd 5.37, ddd 5.34, ddd 5.3, mc
5.49, dd
4.38, dd
4.23, q
5.37, dd
4.66, dd
4.12, q
5.51, dd
4.41, dd
4.38, ddd 4.01, q
5.29, dd
3.82, dd
5.3, mc
3.9, mc
3.9, mc
5.24, ddd 4.91, ddd 5.52, dd
4.87, ddd 4.92, ddd 4.87, dt
2.79, dd
2.27, dd
3.88, s
5.81, d
1.91, s
2.13, s
2.09, s
2.06, s
2.06, s
2.62, dd
1.89, m
3.80, s
5.44, s
1.88, s
2.15, s
2.13, s
2.04, s
2.02, s
2.72, dd
2.72, dd
2.73, dd
2.00, t
3.80, s
5.17, d
1.88, s
2.16, s
2.14, s
2.04, s
2.03, s
6.00, d
NDc
NDc
3.81, s
5.55, d
1.94, s
2.13, s
2.08, s
2.07, s
2.06, s
3.81, s
5.19, d
1.87, s
NDc
3.82, s
5.22, d
1.80, s
NDc
NAc
OAc
a
In chloroform-d with TMS as the internal standard.
Ref. 5.
Overlapped and could not be determined.
Impurity
III
b
c
Mixture load
II
III
2nd cycle
Cut off
3rd cycle
Recycle
1
4th cycle
Table 2
1H NMR first-order coupling constants of the Neu5Ac moieties of compounds 1–10a
0
2
3
Retention Time (hours)
Compounds
First-order coupling constants (Hz)
1
2
3b
5
7
10
Figure 2. SEC profile of the separation of disaccharide 5 and glycal 3. Elution
conditions are described in Section 3.
J9a,9b
J8,9b
J8,9a
J7,8
J6,7
J5,6
12.5
6.3
2.6
6.7
2.4
10.8
10.7
4.3
10.7
13.9
10.1
12.3
4.9
2.1
6.4
2.1
11.0
11.0
4.6
11.0
13.1
10.2
12.5
7.0
3.2
4.3
3.5
8.9
7.0
12.4
5.2
2.6
8.2
1.8
12.7
4.0
2.2
12.3
4.8
1.5
NDc
NDc
NDc
11.4
4.6
9.3
The chromatographic profile of the separation of 5 and 3 is shown
in Figure 2. The peak of the inseparable mixture by silica gel chro-
matography was initially one peak (I), which was gradually split
into two peaks (II and III) after several recyclings. An impurity
was eluted later than peak (I) and was cut off at the first stage.
When the complete separation was monitored (4th cycle), peak
(II) and peak (III) were independently fractionated. Structural elu-
cidation and a purity check of thioacetate 2, glycal 3, and disaccha-
ride 5 were carried out by means of 1H NMR spectroscopic
analyses, the results of which are shown in Figure 1B–D. Since Fig-
ure 1D clearly shows disaccharide 5 without impurities, attempts
to purify the inseparable mixture by using the SEC method were
deemed likely to succeed. The final yield of the isolated product
5 was 78.8%. Furthermore, a galactosyl bromide 6 was also used
ꢀ1
10.3
10.3
4.6
10.3
10.7
4.5
J4,5
J3eq,4
J3ax,4
J3ax,3eq
J5,NH
NDc
12.7
10.3
NDc
12.8
9.5
12.4
12.7
10.0
3.4
—
8.8
a
In chloroform-d with TMS as the internal standard.
Ref. 5.
Overlapped and could not be determined.
b
c
96.9% yield. Thus, the thioacetate 2, including a small amount of 3,
was treated with a mesylate 9 having an azide at the -position in
x
the presence of potassium carbonate to give crude products, which
were first passed through a column of silica gel to remove unde-
sired byproducts and decomposed compounds. The mixture of 9
and 3 was then applied to the recycle-type SEC apparatus to give
a pure thiosialoside 9 in 72.0% yield. 1H NMR spectral data for pure
9 are summarized in Tables 1 and 2.
In conclusion, the purification of thiosialosides has been effi-
ciently demonstrated by means of the SEC method as a convenient
and a useful tool. Therefore, this methodology is applicable for the
separation of other thiosialoside mixtures, and synthetic studies
that make use of this separation system are now underway. Results
of further application of this methodology will be reported
elsewhere.
for the preparation of sialyl
a-(2?6)-D-galactose analogue. The
coupling reaction proceeded smoothly, and isolation by using the
same procedure as that described for the preparation of 5 afforded
pure 6 in 42.5% yield, the purity of which was determined by 1H
NMR spectroscopy. The results of the 1H NMR analyses are summa-
rized in Tables 1 and 2. The low yield of this condensation reaction
was apparently caused by a b-elimination of the galactose residue
under the basic reaction conditions, as well as by the relatively low
reactivity of the galactosyl bromide due to steric hindrance of the
4-acetoxyl group that has an axial orientation.
Since separation of the sialyl disaccharide and glycal was suc-
cessfully accomplished, our attention was then turned to the sep-
aration of the monosaccharidic thiosialoside from the product
mixture after thioglycosidation, including the side product 3. We
previously reported glycopolymers having thiosialoside moieties
as pendant-type epitopes, and the glycopolymers had inhibitory
activities against sialidases of human influenza virus.10 Therefore,
the convenient separation method by SEC was applied to the prep-
aration of the glycomonomers, and the synthetic scheme is shown
in Scheme 3. Before the coupling reaction of 2, bifunctional com-
pound 9, which has a leaving group and an azide moiety as a pre-
cursor of an amine, was prepared from a known azidoalcohol 811 in
3. Experimental
3.1. General methods
Unless otherwise stated, all commercially available solvents
and reagents were used without further purification. Pyridine
(Pyr) and N,N-dimethylformamide (DMF) were stored over mole-
cular sieves (4 Å MS), and methanol (MeOH) was stored over 3 Å

2738
J.-I. Sakamoto et al. / Carbohydrate Research 343 (2008) 2735–2739
i)
N
O
N
O
3
3
O
OH
O
OMs
8
9
OAc
OAc
COOMe
S
ii)
iii)
O
N
3
AcO
O
(+ 3)
2 (+ 3)
O
10
AcHN
OAc
10
Scheme 3. Reagents and conditions: (i) MsCl, pyridine, 0 °C, 4 h; (ii) K2CO3, MeOH–DMF, 0 °C?rt, overnight, then Ac2O–pyridine, DMAP, rt, overnight.
MS before use. The optical rotations were determined with a JASCO
DIP-1000 digital polarimeter. The IR spectra were obtained using a
Shimadzu IR Prestage-21 spectrometer. The 1H NMR spectra were
recorded at 400 MHz with a Bruker DPX-400 or a Bruker DRX-400
spectrometer or at 200 MHz with a Varian Gemini-2000 spectro-
meter in chloroform-d (CDCl3), including tetramethylsilane (TMS)
as the internal standard. The internal standards used for 13C NMR
spectra were CDCl3 (77.0 ppm) in CDCl3. Ring-proton assignments
in the 1H NMR spectra were made by first-order analysis of the
spectra and are supported by the results of homonuclear decou-
pling experiments and H–H or C–H COSY experiments. Elemental
analyses were performed with a Fisons EA1108 on samples exten-
sively dried at 50–60 °C over phosphorus pentoxide for 4–5 h. Fast-
atom bombardment mass (FABMS) spectra were recorded with a
JEOL DX-303 spectrometer. Reactions were monitored by thin-
layer chromatography (TLC) on a precoated plate of Silica Gel
60F254 (layer thickness, 0.25 mm; E. Merck, Darmstadt, Germany).
For detection of the intermediates, TLC sheets were sprayed with
(a) a solution of 85:10:5 (v/v/v) MeOH–p-anisaldehyde–concd
H2SO4 and heated for a few minutes (for carbohydrate) or (b) an
aq solution of 5 wt % KMnO4 and heated similarly (for detection
of C@C double bonds). Column chromatography was performed
the inseparable mixture of 5 and 3 (ca. 500 mg) was applied to
the SEC apparatus to afford pure 5 as a white foam. Total yield of
pure 5 was 8.44 g (78.8%): Rf 0.56 [5:4:1 (v/v/v) CHCl3–EtOAc–
MeOH]; ½a 2D4
ꢂ
ꢃ31.3° (c 1.07, CHCl3); IR (KBr) 2955 (
C@O, amide I), 1541 (dN–H, amide II), 1223 (
1038 (m ;
CꢃOꢃC) cmꢃ1 1H NMR (400 MHz, CDCl3): d 1.67 (m, 2H,
m
C–H), 1748
(m
C@O), 1655 (
m
m
CꢃO),
CH2), 1.99, 2.03, 2.03, 2.05, 2.07, 2.13, and 2.13 (each s, 21H, 7
COCH3), 2.91 (dd 1H, J5,6b = 7.1 Hz and J6a,6b = 14.1 Hz, H-6b), 2.97
(dd 1H, J5,6a = 3.8 Hz, H-6a), 3.57 (m, 1H, H-5), 3.67 (m, 2H,
OCH2), 4.45 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.98 (m, 4 H, H-2, H-4, and
@CH2), 5.16 (t, 1H, J2,3 = J3,4 = 9.4 Hz, H-3), and 5.79 (m, 1H,
CH@), and other signals are summarized in Tables 1 and 2; 13C
NMR (100 MHz, CDCl3): d 20.60, 20.63, 20.70, 20.72, 20.78, 20.80,
21.09, 23.16, 28.51, 29.81, 30.24, 37.99, 49.37, 52.96, 62.10,
67.30, 68.50, 69.14, 69.43, 71.41, 72.60, 72.91, 74.10, 77.21,
82.66, 100.62 (C-1), 115.02 (CH2@), 137.81 (CH@), 169.32 (C@O),
169.26 (C@O), 169.50 (C@O), 169.79 (C@O), 170.08 (C@O),
170.12 (C@O), 170.32 (C@O), 170.60 (C@O), and 170.87 (C@O);
FABMS calcd for [M+H+]: 864.3. Found: m/z 864.6, [M+Na+]:
886.3. Found: m/z 886.4.
Anal. Calcd for C37H53NO20S: C, 51.44; H, 6.18; N, 1.62. Found: C,
51.26; H, 6.11; N, 1.52.
on silica gel (Silica Gel 60; 63–200
chromatography was performed on silica gel (Silica Gel 60, spher-
ical neutral; 40–100 m, E. Merck). Preparative SEC was performed
lm, E. Merck). Flush column
3.2.2. 4-Pentenyl S-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
l
3,5-dideoxy-
D
-glycero-
a-
D-galacto-2-nonulopyranosylonate)-
by an SEC recycling apparatus [HLC-50G system (Shimamura
Instruments Works, Co., Tokyo, Japan)] using tandem-bonded Sho-
dex H-2001L (I.D., 20.0 mm ꢁ 600 mm) and H-2002 columns (I.D.,
20.0 mm ꢁ 500 mm).12 The columns were equilibrated with CHCl3
at ambient temperature and the flow rate of the apparatus was
3.8 mL/min. A refractive index (RI) detector was used for monitor-
ing the chromatograms. All extractions were concentrated below
45 °C under diminished pressure.
(2?6)-3,4,6-tri-O-acetyl-6-thio-b-
D
-galactopyranoside (7)
To
deoxy-b-
a
solution of 4-pentenyl 2,3,4-tri-O-acetyl-6-bromo-6-
-galactopyranoside (6) (145 mg, 0.322 mmol) and sialyl
D
derivative 26 (364 mg, 0.662 mmol) in 1:1 DMF–MeOH (1.4 mL)
was added K2CO3 (91 mg, 0.658 mmol) portionwise at 0 °C under
an Ar atmosphere, and the reaction mixture was stirred at room
temperature for 45 h. When TLC indicated the end of the reaction,
AcOH (76 lL, 1.24 mmol) was added to the reaction mixture at
0 °C. After concentration in vacuo, the resulting mixture was then
treated with pyridine (4 mL) and Ac2O (3 mL) at 0 °C, and the mix-
ture was stirred at room temperature for 21 h. The reaction mix-
ture was concentrated in vacuo, and the resulting residue was
diluted with CHCl3. The organic solution was successively washed
with 1 M aq H2SO4, satd aq NaHCO3, and brine, dried over anhyd
MgSO4, filtered, and the solvents were evaporated to yield a yellow
foam (394 mg). Chromatographic purification of the residue on sil-
ica gel (20 g) with 15:14:1 (v/v/v) CHCl3–EtOAc–MeOH as the elu-
ent gave an inseparable mixture of 7 and 3 (264 mg), which was
further purified by the SEC apparatus to give pure 7 (122 mg,
42.5%) as a white foam: Rf 0.55 [5:4:1 (v/v/v) CHCl3–EtOAc–
3.2. Synthesis
3.2.1. 4-Pentenyl S-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-
(2?6)-3,4,6-tri-O-acetyl-6-thio-b-
Diethylamine (12.9 mL, 124 mmol) was added dropwise to a
mixture of 4-pentenyl 2,3,4-tri-O-acetyl-6-bromo-6-deoxy-b-
glucopyranoside 4 (5.42 g, 12.4 mmol) and methyl 5-acetamido-
4,7,8,9-tetra-O-acetyl-2-S-acetyl-3,5-dideoxy-2-thio- -glycero-
-galacto-2-nonulopyranosonate (2)6 (10.2 g, 18.6 mmol) in DMF
D-glycero-
a-
D
-galacto-2-nonulopyranosylonate)-
D
-glucopyranoside (5)
D
-
D
a-
D
(25 mL) at 0 °C under Ar atmosphere, and the reaction mixture
was stirred at room temperature for 10 h. The reaction mixture
was concentrated in vacuo and the resulting residue was diluted
with CHCl3. The organic solution was successively washed with
water and brine, dried over anhyd MgSO4, filtered, and evaporated.
The residue was chromatographed on silica gel (450 g) with
2:3?0:1 (v/v) toluene–EtOAc as the eluent to give an inseparable
mixture 5 including a small amount of 3 (9.2 g) as a yellow foam,
in which each compound has the same Rf on TLC. A portion of
MeOH]; ½a 2D5
ꢂ
ꢃ10.4° (c 1.09, CHCl3); IR (KBr) 2957 (
C@O, amide I), 1549 (dN–H, amide II), 1225 (
1057 (m ;
CꢃOꢃC) cmꢃ1 1H NMR (400 MHz, CDCl3): d 1.69 (m, 2H,
m
C–H), 1748
(m
C@O), 1668 (
m
m
CꢃO),
CH2), 1.97, 2.03, 2.04, 2.05, 2.14, 2.15, and 2.15 (each s, 21H, 7
COCH3), 2.66 (dd 1H, J5,6b = 7.1 Hz and J6a,6b = 14.3 Hz, H-6b), 2.90
(dd 1H, J5,6a = 7.2 Hz, H-6a), 3.75 (m, 2H, OCH2), 3.90 (m, 1H, H-
5), 4.61 (d, 1H, J1,2 = 7.9 Hz, H-1), 5.00 (m, 2H, @CH2), 5.07 (dd,
1H, J2,3 = 10.4 Hz and J3,4 = 2.7 Hz, H-3), 5.17 (dd, 1H, H-2), 5.53

J.-I. Sakamoto et al. / Carbohydrate Research 343 (2008) 2735–2739
2739
(d, 1H, H-4), and 5.80 (m, 1H, CH@), and other signals are summa-
rized in Tables 1 and 2; 13C NMR (100 MHz, CDCl3): d 20.59, 20.68,
20.75, 20.78, 21.03, 23.19, 28.61, 29.71, 29.85, 38.04, 49.51, 53.10,
62.22, 66.78, 67.82, 68.29, 69.01, 69.17, 71.17, 72.24, 73.82, 83.72,
100.78 (C-1), 114.81 (CH2@), 138.01 (CH@), 169.15 (C@O), 169.48
(C@O), 169.64 (C@O), 169.92 (C@O), 170.09 (C@O), 170.19 (C@O),
170.40 (C@O), 170.60 (C@O), and 170.81 (C@O); FABMS calcd for
[M+H+]: 864.3. Found: m/z 864.5, [M+Na+]: 886.3. Found: m/z
886.4. Anal. Calcd for C37H53NO20Sꢄ0.5 H2O: C, 50.91; H, 6.24; N,
1.61. Found: C, 50.96; H, 6.09; N, 1.52.
give corresponding 10 (520 mg, 71.8%): Rf 0.40 [5:4:1 (v/v/v)
CHCl3–EtOAc–MeOH]; ½a D27
ꢂ
+17.8° (c 1.00, CHCl3); IR (KBr) 2959
N@N@N), 1742 ( C@O), 1655 ( C@O, amide
CꢃO), 1037 (
CꢃOꢃC) cmꢃ1; 1H NMR
(m
C–H), 2870 (
m
C–H), 2104 (
m
m
m
I), 1560 (dN–H, amide II), 1223 (
m
m
(400 MHz, CDCl3): d 2.81 (dt, 1H, Jvic = 4.5, Jgem = 13.5 Hz, SCHb),
2.95 (dt, 1H, Jvic = 5.7 Hz, SCHa), 3.39 (t, 2H, J = 4.9 Hz, CH2N3),
3.67 (m, 8H, 4CH2), and other signals are summarized in Tables 1
and 2; FABMS calcd for [M+H+]: 665.7. Found: m/z 665.2,
[M+Na+]: 687.7. Found: m/z 687.1. Anal. Calcd for C26H40NO14S:
C, 46.98; H, 6.07; N, 8.43. Found: C, 47.10; H, 6.06; N, 8.38.
3.2.3. 2-[2-(2-Azidoethoxy)ethoxy]ethyl methanesulfonate (9)
Known 2-[2-(2-azidoethoxy)ethoxy]ethanol (8)11 (1.32 g, 7.53
mmol) in pyridine (10 mL) was treated with methanesulfonyl chlo-
ride (1.17 mL, 15.1 mmol) at 0 °C under an Ar atmosphere, and the
reaction mixture was stirred at the same temperature for 4 h.
To the mixture were added CHCl3 and water, and the mixture
was partitioned. The organic solution was successively washed
with 1 M aq H2SO4, satd aq NaHCO3, and brine, dried over anhyd
MgSO4, filtered, and the solvents were evaporated to yield pure 9
as a light-yellow syrup (1.85 g, 96.9%), which was used for the next
reaction without further purification: Rf 0.65 (EtOAc); 1H NMR
(200 MHz, CDCl3): d 3.39 (t, 2H, J = 4.9 Hz, CH2N3), 3.67 (m, 6H,
3CH2), 3.79 (m, 2H, CH2CH2OMs), and 4.39 (m, 2H, CH2OMs).
Acknowledgments
We are grateful to Snow Brand Milk Products Co., Ltd for provid-
ing the sialic acid used in this study. This work was supported in
part by a grant for ‘Development of Novel Diagnostic and Medical
Applications through Elucidation of Sugar Chain Functions’ from
New Energy and Industrial Technology Development Organization
(NEDO).
References
1. Varki, A. Glycobiology 1992, 2, 25–40.
2. (a) Boons, G.-J.; Demchenko, A. V. Chem. Rev. 2000, 100, 4539–4565; (b) Kiefel,
M. J.; von Itzstein, M. Chem. Rev. 2002, 102, 471–490.
3. (a) Matsuoka, K.; Oka, H.; Koyama, T.; Esumi, Y.; Terunuma, D. Tetrahedron Lett.
2001, 42, 3327–3330; (b) Matsuoka, K.; Onaga, T.; Mori, T.; Sakamoto, J.-I.;
Koyama, T.; Sakairi, N.; Hatano, K.; Terunuma, D. Tetrahedron Lett. 2004, 45,
9383–9386; (c) Sakamoto, J.-I.; Koyama, T.; Miyamoto, D.; Yingsakmongkon, S.;
Hidari, K. I. P. J.; Jampangern, W.; Suzuki, T.; Suzuki, Y.; Esumi, Y.; Hatano, K.;
Terunuma, D.; Matsuoka, K. Bioorg. Med. Chem. Lett. 2007, 17, 717–721.
4. Groves, D. R.; Bradley, S. J.; Rose, F. J.; Kiefel, M. J.; von Itzstein, M.
Glycoconjugate J. 1999, 16, 13–17.
5. Okamoto, K.; Kondo, T.; Goto, T. Bull. Chem. Soc. Jpn. 1987, 60, 631–636.
6. Hasegawa, A.; Nakamura, J.; Kiso, M. J. Carbohydr. Chem. 1986, 5, 11–19.
7. Matsuoka, K.; Oka, H.; Terunuma, D.; Kuzuhara, H. Carbohydr. Lett. 2001, 4,
123–130.
8. Hasegawa, A.; Morita, M.; Ito, Y.; Ishida, H.; Kiso, M. J. Carbohydr. Chem. 1990, 9,
369–392.
9. (a) Kiefel, M. J.; Beisner, B.; Bennett, S.; Holmes, I. D.; von Itzstein, M. J. Med.
Chem. 1996, 39, 1314–1320; (b) Bennett, S.; von Itzstein, M.; Kiefel, M. J.
Carbohydr. Res. 1994, 259, 293–299.
3.2.4. Methyl {2-[2-(2-Azidoethoxy)ethoxy]ethyl 5-acetamido-
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-a-D-galacto-
2-nonulopyranosid}onate (10)
To a solution of a known thioacetate 26 (600 mg, 1.09 mmol)
and a mesylate 9 (550 mg, 2.18 mmol) in MeOH (6.0 mL) was
added K2CO3 (150 mg, 1.09 mmol) at 0 °C under an Ar atmosphere,
and the reaction mixture was stirred at room temperature over-
night. After consuming starting materials judged by TLC, the reac-
tion mixture was treated with AcOH (0.125 mL, 2.18 mmol), and
the mixture was concentrated. The residue was allowed to react
with Ac2O (2.06 mL, 21.8 mmol) in pyridine (6.0 mL) in the pres-
ence of 4-dimethylaminopyridine (DMAP) at 0 °C under Ar atmo-
sphere, and the reaction mixture was stirred at room
temperature overnight. The mixture was diluted with CHCl3 and
then poured into ice-cold water. The mixture was partitioned,
and the organic layer was successively washed with 1 M aq
H2SO4, satd aq NaHCO3, and brine, dried over anhyd MgSO4, fil-
tered, and concentrated in vacuo. Chromatographic purification
of the residue on silica gel with 1:3 (v/v) toluene–EtOAc as the elu-
ent was followed by further purification using the SEC apparatus to
10. Matsuoka, K.; Takita, C.; Koyama, T.; Miyamoto, D.; Yingsakmongkon, S.;
Hidari, K. I. P. J.; Jampangern, W.; Suzuki, T.; Suzuki, Y.; Esumi, Y.; Hatano, K.;
Terunuma, D. Bioorg. Med. Chem. Lett. 2007, 17, 3826–3830.
11. (a) Amvam-Zollo, P.-H.; Sinaÿ, P. Carbohydr. Res. 1986, 150, 199–212; (b) Sasaki,
A.; Murahashi, N.; Yamada, H.; Morikawa, A. Biol. Pharm. Bull. 1994, 17, 680–
685.
12. Kimura, T.; Xue, F.; Kobayashi, T.; Onoda, T.; Yamamoto, H. Polym. J. 2004, 36,
205–208.