One-pot synthesis of sialo-containing glycosyl amino acids by use of an N-trichloroethoxycarbonyl-β-thiophenyl sialoside

Article abstract of DOI:10.1002/chem.200400840

We describe an efficient synthesis of 2,6- and 2,3-sialyl T antigens linked to serine in a one-pot glycosylation. We first investigated the glycosidation of thiosialosides by varying the N-protecting group. Modification of the C-5 amino group of β-thiosialosides into the N-9-fluorenylmethoxycarbonyl, N-2,2,2-trichloroethoxycarbonyl (N-Troc), and N-trichloroacetyl derivatives enhanced the reactivity of these compounds towards glycosidation. Addition of a minimum amount of 3 A molecular sieves was also effective in improving the yield of α-linked sialosides. Next, we conducted one-pot syntheses of the glycosyl amino acids by using the N-Troc sialyl donor. The N-Troc derivative can be converted into the N-acetyl derivative without racemization of the amino acids. Branched-type one-pot glycosylation, initiated by regioselective glycosylation of the 3,6-dihydroxy galactoside with the N-Troc-β-thiophenyl sialoside, provided the protected 2,6-sialyl T antigen in good yield. Linear-type one-pot glycosylation, initiated by chemoselective glycosylation of galactosyl fluoride with the N-Troc-β-thiophenyl sialoside, afforded the protected 2,3-sialyl T antigen in excellent yield. Both protected glycosyl amino acids were converted into the fully deprotected 2,6- and 2,3-sialyl T antigens linked to serine in good yields.

Full text of DOI:10.1002/chem.200400840

FULL PAPER
One-Pot Synthesis of Sialo-Containing Glycosyl Amino Acids by Use of an
N-Trichloroethoxycarbonyl-b-thiophenyl Sialoside
Hiroshi Tanaka, Masaatsu Adachi, and Takashi Takahashi*[a]
Abstract: We describe an efficient syn-
thesis of 2,6- and 2,3-sialyl T antigens
linked to serine in a one-pot glycosyla-
tion. We first investigated the glycosi-
dation of thiosialosides by varying the
N-protecting group. Modification of
the C-5 amino group of b-thiosialosides
into the N-9-fluorenylmethoxycarbon-
yl, N-2,2,2-trichloroethoxycarbonyl (N-
Troc), and N-trichloroacetyl derivatives
enhanced the reactivity of these com-
pounds towards glycosidation. Addi-
tion of a minimum amount of 3 ꢀ mo-
lecular sieves was also effective in im-
proving the yield of a-linked sialosides.
Next, we conducted one-pot syntheses
of the glycosyl amino acids by using
the N-Troc sialyl donor. The N-Troc
derivative can be converted into the N-
acetyl derivative without racemization
of the amino acids. Branched-type one-
pot glycosylation, initiated by regiose-
lective glycosylation of the 3,6-dihy-
droxy galactoside with the N-Troc-b-
thiophenyl sialoside, provided the pro-
tected 2,6-sialyl T antigen in good
yield. Linear-type one-pot glycosyla-
tion, initiated by chemoselective glyco-
sylation of galactosyl fluoride with the
N-Troc-b-thiophenyl sialoside, afforded
the protected 2,3-sialyl T antigen in ex-
cellent yield. Both protected glycosyl
amino acids were converted into the
fully deprotected 2,6- and 2,3-sialyl
T antigens linked to serine in good
yields.
Keywords: glycosyl amino acids ·
glycosylation
· oligosaccharides ·
sialic acids · thioglycosides
Introduction
Sialic acids such as N-acetylneuraminic acid (Neu5Ac), N-
glycolylneuraminic acid (Neu5Glc), and 3-deoxy-d-glycero-
d-galacto-non-2-ulopyranosonic acid (KDN) are often at-
tached at the nonreducing end of glycoconjugates on the
cell surface through a-glycosidic bonds and they play a cen-
tral role in cell-surface recognition phenomena.[1] For exam-
ple,
Galb(1!3)–[Neu5Aca(2!6)]–GalNAca(1!3)–Ser
(1a) or -Thr (1b) and Neu5Aca(2!3)–Galb(1!3)–GalNA-
ca(1!3)–Ser (2a) or -Thr (2b) are important tumor-associ-
ated antigens carried on the MUC1 and MUC4 proteins
(Scheme 1).[2–4] Additionally, various related glycoforms con-
taining a-sialosides are found in related antigens.[5] There-
fore, sialo-containing glycosyl amino acids and peptides
[a] Dr. H. Tanaka, Dr. M. Adachi, Prof. Dr. T. Takahashi
Department of Applied Chemistry
Scheme 1. Structures of 2,6- and 2,3-sialyl T antigens 1 and 2.
Graduate School of Science and Engineering
Tokyo Institute of Technology
2-12-1 Ookayama, Meguro, Tokyo 152–8552 (Japan)
Fax : (+81)3-5734-2884
E-mail: ttak@apc.titech.ac.jp
have been synthesized to generate tumor-selective immuno-
stimulating antigens.[6]
One-pot glycosylation[7–11] to form two or more glycosidic
bonds by chemoselective activation of glycosyl donors is an
Supporting information for this article is available on the WWW
under http://www.chemeurj.org/ or from the author.
Chem. Eur. J. 2005, 11, 849 – 862
DOI: 10.1002/chem.200400840
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
849

effective solution-phase method, not only for the high-speed
synthesis of a target oligosaccharide but also for the parallel
synthesis of oligosaccharide libraries[11] because it circum-
vents the need for purification of the synthetic intermedi-
ates. Wong and co-workers have reported the Optimer
method, involving sequential activation of thioglycosides
whose reactivity was tuned by the protecting groups on the
basis of the armed and disarmed concept.[8j,l] We have inves-
tigated one-pot glycosylation based on chemoselective acti-
vation of glycosyl donors with different leaving group-
s[8a,c,k,9–11] by appropriate activators. The order of activation
of glycosyl donors can be tuned, not only by their protecting
groups but also by the combination of their leaving groups
and activators. If sialylation could be adapted to one-pot
glycosylation, this would be an effective and attractive way
for the synthesis of sialo-containing oligosaccharides. How-
ever, a-selective sialylation is a problematic step in the
chemical synthesis of oligosaccharides.[12] Therefore, the suc-
cessful synthesis of glycosyl amino acids by one-pot glycosy-
lations including sialylation requires the development of a
new effective sialylation method. Herein we report an effec-
tive sialylation method that uses N-Troc-, N-Fmoc-, and N-
trichloroacetyl-b-thiophenyl sialosides and the application of
the N-Troc sialyl donor to the synthesis of two sialo-contain-
ing glycosyl amino acids, 1 and 2, by using branched and
linear one-pot glycosylations.[13]
Scheme 2. Synthesis and glycosidation of b-thiophenyl sialosides 3a–i.
Reagents and conditions: a) MsOH, MeOH, 608C, 12 h; b) Z-OSu,
Alloc-OSu, Fmoc-OSu, Troc-OSu, or (Boc)2O, NEt3, CH3CN/H2O, RT,
5.5 h; c) CF3CO2Me or CCl3CO2Me, NEt3, MeOH, RT, 1 h; d) Ac2O, Py,
DMAP, 63% for 3b, 76% for 3c, 68% for 3d, 27% for 3e, 89% for 3 f,
77% for 3 g, and 70% for 3h in three steps from 3a; e) 3a–i
(1.00 equiv), 4 (1.50 equiv), NIS (1.20 equiv), TfOH (0.20 equiv), CH3CN,
3 ꢀ MS (10 gmmolꢀ1), ꢀ358C, 1 h; f) 3a–i (1.00 equiv), 4 (1.50 equiv),
DMTST (1.50 equiv), CH3CN, 3 ꢀ MS (10 gmmolꢀ1), ꢀ108C, 8 h.
Alloc=allyloxycarbonyl,
Bn=benzyl,
Boc=tert-butoxycarbonyl,
DMAP=4-dimethylaminopyridine, DMTST=(dimethylthio)methylsulfo-
nium trifluoromethanesulfonate, Fmoc=9-fluorenylmethoxycarbonyl,
MsOH=methanesulfonic acid, NIS=N-iodosuccinimide, Py=pyridine,
Su=succinimidyl, Tf=trifluoromethanesulfonyl, TFA=trifluoroacetyl,
Troc=2,2,2-trichloroethoxycarbonyl, Z=benzyloxycarbonyl.
Results and Discussion
In 1998, Demchenko and Boons reported that di-N-acetyl-
thiomethyl sialoside was effective for glycosidation in terms
of reactivity in comparison with N-acetyl-thiomethyl sialo-
side.[14] Recently, modification of the C-5 amino group of
the sialyl donor into an azido group[15] or an N-TFA
group[16] has been reported to improve reactivity toward gly-
cosidation. Based on this information, we first investigated
glycosidation of b-thiophenyl sialosides 3a–i with various N-
protecting groups (Scheme 2). Although b-thiophenyl sialo-
sides are less reactive to glycosidation than a-thiophenyl sia-
losides, they can be simply prepared from N-acetylneura-
minic acid (Neu5Ac).[17] Thiosialosides 3b–h were synthe-
sized by removal of all acetyl groups from the N-acetyl thio-
sialoside 3a under acidic conditions,[18] followed by selective
acylation of the amino group with various acid deriva-
tives[19,20] and acetylation of the remaining hydroxy groups.
All compounds except for N-Fmoc derivative 3e were ob-
tained in good yields (63–89% in three steps).
Glycosylation of the primary alcohol of glucose 4 with the
sialyl donors 3a–i was examined. Acetonitrile was used as
the solvent because it is known to improve a selectivity in
sialylation.[21] We first examined activation of thiosialosides
3a–i by DMTST,[22] which is a mild activator for thioglyco-
sides. Treatment of donors 3b–i with 1.5 equivalents of ac-
ceptor 4 and DMTST in the presence of 3 ꢀ molecular
sieves (MS; 10 gmmolꢀ1) in CH3CN at ꢀ108C resulted in no
significant improvement in the yields of 5b–i in comparison
with the result of glycosidation of N-acetyl derivative 3a
(Table 1). We next applied NIS and TfOH to the glycosida-
tion; these are the most common and strongest reagents for
activation of thioglycosides, and the N-Boc and N-Alloc pro-
tecting groups, unfortunately, might not survive these condi-
tions. The sialyl donors 3a–i were treated with acceptor 4
(1.5 equiv) and NIS (1.20 equiv)/TfOH (0.20 equiv)[23] in the
presence of 3 ꢀ MS (10 gmmolꢀ1) in CH3CN at ꢀ358C
(Table 2). Glycosidation of N-TFA derivative 3g provided
Table 1. Glycosylation of 4 (1.50 equiv) with b-thiophenyl sialosides 3a–i
(1.00 equiv) by DMTST.
Entry Donor Disaccharide Yield
of 5 [%]
a:b[a]
Glycal Yield
of 6 [%]
1
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3 f
3g
3h
3i
5a
5b
5c
5d
5e
5 f
5g
5h
5i
40
24
–
56
55
40
35
41
36
85:15 6a
84:16 6b
41
–
–
39
40
35
42
38
47
–
6c
84:16 6d
86:14 6e
85:15 6 f
87:13 6g
76:24 6h
75:25 6i
[a] The a:b ratio was determined by HPLC analysis based on refractive-
index detection.
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Sialo-Containing Glycosyl Amino Acids
FULL PAPER
Table 2. Glycosylation of 4 (1.50 equiv) with b-thiophenyl sialosides 3a–i
(1.00 equiv) by NIS/TfOH.
would be suitable for the synthesis of glycosyl amino acids
and glycopeptides. Recently, Kiso, Ando, and co-workers
have reported the effectiveness of an N-Troc-a-thiophenyl
sialoside for the synthesis of sialo-containing glycosides.[26]
To demonstrate the feasibility of the sialylation method,
we planned the one-pot syntheses of 1 and 2. We first exam-
ined a one-pot two-step synthesis of 2,6-sialyl T antigen
1a.[27] Our strategy for the synthesis of 1a involved a
branched-type one-pot glycosylation initiated by regioselec-
tive glycosylation (Scheme 3). 3,6-Dihydroxy galactoside 7
linked to serine was chosen as the glycosyl acceptor.[27d,e,28,29]
Entry Donor Disaccharide Yield
of 5 [%]
a:b[a]
Glycal Yield
of 6 [%]
1
2
3
4
5
6
7
8
9
3a
3b
3c
3d
3e
3 f
3g
3h
3i
5a
5b
5c
5d
5e
5 f
5g
5h
5i
47
68
–
44
91
91
92
83
65
85:15 6a
84:16 6b
28
8
–
20
7
6
5
4
23
–
6c
88:12 6d
86:14 6e
89:11 6 f
92:8 6g
91:9 6h
62:38 6i
[a] The a:b ratio was determined by HPLC analysis based on refractive-
index detection.
disaccharide 5g in excellent yield (92%, a:b=92:8) along
with a small amount of glycal 6g in 5% yield. The N-Fmoc
and N-Troc derivatives 3e and 3 f[24] served as effective gly-
cosyl donors to afford the corresponding disaccharides 5e
and 5 f in excellent yields and with acceptable a selectivity
(91%, a:b=86:14 for 5e; 91%, a:b=89:11 for 5 f). The N-
trichloroacetyl derivative 3h was converted into disacchar-
ide 5h in good yield. The anomeric configurations of the re-
1
sulting sialosides were determined by analysis of H NMR
spectra based on the chemical shift values of the H-3eq and
H-4 signals, the coupling constant J7,8, and the value of
Dd(H-9’ꢀH-9), which were in accordance with the empirical
rules for defining the anomeric configuration of N-acetyl
sialic acid glycosides (Table 3).[25] Additionally, the structural
confirmation for the a- and b-N-Troc sialosides 5 f was ac-
complished by transformation of these compounds into the
corresponding a- and b-N-acetyl sialosides 5a.
Scheme 3. Strategy for the one-pot two-step synthesis of 1a. Bz=benzo-
yl.
The N-Troc-b-thiophenyl sialoside 3 f was selected as the
glycosyl donor because the availability of 3 f from N-acetyl-
neuraminic acid was better than that of the N-Fmoc deriva-
tive 3e. Regioselective glycosylation of the primary alcohol
on position 6 of 7 with the N-Troc derivative 3 f, followed by
glycosylation of the alcohol on position 3 with galactoside 8
would provide the protected 2,6-sialyl T antigen. Transfor-
mation of the azido and N-Troc groups into N-acetyl groups
followed by deprotection of all protecting groups should be
achieved without racemization of the amino acid to provide
the fully deprotected 2,6-sialyl T antigen 1a.
Stepwise synthesis of the protected trisaccharide 11 was
undertaken (Scheme 4). We first examined the glycosylation
of the primary alcohol of 7 with an equivalent of N-Troc-b-
thiophenyl sialoside 3 f (Table 4). Treatment of diol 7 with
the sialyl donor 3 f (1 equiv) in the presence of NIS
(1.20 equiv), TfOH (0.20 equiv), and 3 ꢀ MS (10 gmmolꢀ1)
in CH3CN (10 mLmmolꢀ1) at ꢀ358C for 1 h provided disac-
charide 9 in moderate yield (57%, a:b=84:16), along with
disialyl trisaccharide 10 in 4% yield and glycal 6 f in 20%
yield (Table 4, entry 1).[30,31] Further optimization of the re-
action revealed that the quantity of 3 ꢀ MS and the concen-
tration of the substrates influenced the efficiency of the gly-
cosidation. Glycosylation of 7 with 3 f (1 equiv) in the pres-
ence of TfOH (0.20 equiv) and 3 ꢀ MS (0.50 gmmolꢀ1) in
CH3CN (5 mLmmolꢀ1) provided disaccharide 9 in 75%
yield (a:b=80:20), along with disialyl trisaccharide 10 in
4% yield and glycal 6 f in 8% yield (entry 7). Use of propio-
1
Table 3. Partial H NMR signal assignment for disaccharides 5e–g.
R3R2N
dH-3eq
dH-4
J7,8
[Hz]
Dd(H-9’ꢀH-9)
[ppm]
[ppm]
[ppm]
AcHN
5a-a
5a-b
5e-a
5e-b
5f-a
5f-b
5g-a
5g-b
2.65
2.48
2.70
2.54
2.72
2.54
2.70
2.45
4.84
5.17
4.90
5.21
4.98
5.25
4.99
5.21
9.3
1.9
9.2
8.7
–
4.8
8.8
–
0.29
0.94
0.31
0.93
0.27
0.91
0.30
0.91
FmocHN
TrocHN
TFAHN
To adapt the sialylation to the synthesis of glycosyl amino
acids and peptides, removal of the C-5 protecting group
without racemization of the amino acid is required. Unfortu-
nately, deprotection of the N-TFA group requires relatively
strongly basic conditions, in which racemization of the
amino acid would occur. On the other hand, the N-Fmoc
and N-Troc groups can be converted into various N-acyl
groups under mildly acidic or basic conditions. Therefore,
the N-Fmoc and N-Troc-b-thiophenyl sialosides 3e and 3 f
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T. Takahashi et al.
nitrile as the solvent requires a higher reaction temperature
(ꢀ258C) and longer reaction time (3 h) and resulted in a re-
duced yield of 9 (58%, a:b=82:18; entry 8). Finally, mini-
mization of the amount of remaining acceptor 7 and the
side products 10 and 6 f was accomplished by glycosidation
of sialyl donor 3 f (1.2 equiv) with NIS (1.44 equiv), TfOH
(0.10 equiv), and 3 ꢀ MS (0.50 gmmolꢀ1) in CH3CN
(5 mLmmolꢀ1) at ꢀ358C to provide disaccharide 9 in 93%
yield (a:b=78:22) along with trisaccharide 10 (6%) and
glycal 6 f (entry 9). The yield of glycal 6 f was 12%, based
on donor 3 f.
Next, glycosylation of the remaining secondary alcohol of
9 with galactoside 8 was examined. In the one-pot glycosyla-
tion, galactosylation should be achieved after the sialylation.
Therefore, we first examined glycosylation of 9a with 8 in
CH3CN. Galactosyl fluoride 8b was not completely activat-
ed by ZrCp2Cl2/AgOTf[32] in CH3CN. On the other hand,
treatment of disaccharide 9a with thiogalactoside 8a in the
presence of NIS/TfOH in CH3CN at room temperature pro-
vided trisaccharide 11 in 60% yield and with b selectivity
(a:b=19:81).[33,34] The anchimeric effect of the C-2 acyl pro-
tecting group to provide 1,2-trans glycosidic linkage did not
work well under these reaction conditions. After optimiza-
tion of the reaction conditions, we found that treatment of
disaccharide 9a with 8a (1.50 equiv) in CH2Cl2/CH3CN
(9:1) at ꢀ308C afforded trisaccharide 11 in 82% yield and
with acceptable selectivity (a:b=6:94).
One-pot glycosylation with 3 f, 7, and 8a was examined.
Treatment of diol 7 with the N-Troc-b-thiophenyl sialoside
3 f under the above-described conditions provided disacchar-
ide 9. Dilution of the reaction mixture with CH2Cl2 followed
by addition of thioglactoside 8a and additional NIS/TfOH
afforded the protected trisaccharide 11. Purification of the
crude mixture was achieved by silica-gel column chromatog-
raphy and gel-permeation chromatography (GPC) to pro-
vide trisaccharide 11 in 77% overall yield, based on 7, and
with good selectivity (a:b=78:22). The analytical data of
the trisaccharides 11a and 11b were identical to those of
authentic sample synthesized by the stepwise synthesis.
Transformation of 11a into 1a proceeded as follows. The
simultaneous reduction of the azido and N-Troc groups of
11a,[35] followed by acetylation,
Scheme 4. Synthesis of 2,6-sialyl T antigen 1a by a a stepwise process and
by a branched-type one-pot glycosylation. Reagents and conditions: a) 3 f
(1.20 equiv),
NIS
(1.44 equiv),
TfOH
(0.10 equiv),
CH3CN
(5 mLmmolꢀ1), 3 ꢀ MS (0.50 gmmolꢀ1), ꢀ358C, 93%, a:b=78:22; b) 8a
(1.50 equiv), NIS (2.00 equiv), TfOH (0.20 equiv), CH2Cl2/CH3CN (9:1,
5 mLmmolꢀ1), 3 ꢀ MS (0.50 gmmolꢀ1), ꢀ308C, 82%, a:b=6:94; c) 3 f
(1.20 equiv), 7 (1.00 equiv), NIS (1.44 equiv), TfOH (0.10 equiv), CH3CN
(5 mLmmolꢀ1), 3 ꢀ MS (0.50 gmmolꢀ1), ꢀ358C; then 8a (1.80 equiv),
NIS (2.70 equiv), TfOH (0.20 equiv), CH2Cl2 (45 mLmmolꢀ1), ꢀ308C,
77%, a:b=78:22; d) Zn dust, THF, AcOH, Ac2O, 0 8C, 1 h, 92%;
e) Pd(OH)2, THF, MeOH, AcOH, H2O, 2 h; f) 0.1m aq. NaOH, MeOH;
then H2O, 85% over two steps. THF=tetrahydrofuran.
provided the corresponding N-
acetyl derivative 12 in 92%
Table 4. Optimization of regioselective sialylation of diol 7 with the N-Troc-b-thiophenyl sialoside 3 f.[a]
yield. Complete deprotection of
12 was accomplished in two
steps, involving hydrogenolysis
of the benzyl ethers and subse-
quent hydrolysis of the ben-
zoates, acetyls, and the methyl
ester, to provide the fully de-
protected 2,6-sialyl T antigen 1a
in 85% overall yield. The ana-
lytical data (1H NMR spec-
troscopy, optical rotation) were
identical to those previously re-
ported.[27a]
Entry Donor TfOH
Concentration Solvent 3 ꢀ MS
[gmmolꢀ1
Yield
of 9 [%]
a:b[b] Yield
of 10 [%] of 6 f [%][c]
Yield
[equiv] [equiv] [mLmmolꢀ1
]
]
1
2
3
4
5
6
7
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.2
0.2
0.2
0.2
0.2
0.2
0.2
0.1
0.1
0.1
10
10
10
10
10
5
CH3CN 10
57
65
55
71
69
70
75
58
93
84:16
78:22
79:21
80:20
81:19
79:21
80:20
82:18
78:22
4
5
4
4
5
5
4
6
6
20
12
17
12
10
17
8
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
EtCN
5.0
2.5
0.50
0.25
0.50
0.50
0.50
0.50
5
5
8[d]
9[e]
25
12
5
CH3CN
[a] Conditions: NIS (1.2 equiv based on 3 f) and TfOH at ꢀ358C for 1 h. [b] The a:b ratio was determined by
HPLC analysis based on refractive-index detection. [c] Yields are based on donor 3 f. [d] The reaction was car-
ried out at ꢀ258C for 3 h. [e] 1.44 equivalents of NIS were used.
852
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Sialo-Containing Glycosyl Amino Acids
FULL PAPER
Next, we planned to conduct the one-pot synthesis of 2a
by a method involving chemoselective glycosylation of gal-
actoside 13 with thiosialoside 3 f (Scheme 5).[36] Subsequent
Scheme 5. Strategy for the one-pot two-step synthesis of 2a.
coupling of the resulting disaccharide and the galactosyl
serine 14[29,37] would provide the protected 2,3-sialyl-T anti-
gen. The glycosyl acceptor 13 must survive under the activa-
tion conditions for 3 f. Additionally, the resulting sialo-con-
taining disaccharide should smoothly undergo glycosidation
by the secondary hydroxy group. The glycosyl fluoride 13a
and thioglycoside 13b were candidate acceptors in the gly-
cosylation. We selected the glycosyl fluoride 13a as the gly-
cosyl acceptor in the chemoselective glycosidation with thio-
sialoside 3 f[38,39] and avoided the use of thioglycoside 13b as
it is tuned to have lower reactivity than the sialoside. Deac-
tivation of the acceptor by the protecting groups in compari-
son with the sialoside 3 f would result in a reduced yield of
the following glycosylation because sialosides are known to
be one of the most unreactive glycosyl donors.
Stepwise synthesis of the protected trisaccharide 16 was
examined (Scheme 6). We first conducted the chemoselec-
tive glycosylation of galactosyl fluoride 13a with the N-
Troc-b-thiophenyl sialoside 3 f (Table 5). Treatment of the
galactosyl fluoride 13a with the sialyl donor 3 f (1 equiv) in
the presence of NIS (1.35 equiv), TfOH (0.30 equiv), and
3 ꢀ MS (0.50 gmmolꢀ1) in CH3CN (5 mLmmolꢀ1) at ꢀ308C
for 15 min provided disaccharide 15 in 56% yield with rela-
tively low anomeric selectivity (a:b=70:30), along with
glycal 6 f in 35% yield (Table 5, entry 1).[40,41] Further opti-
mization of the reaction revealed that control of the reac-
tion temperature was critical for improving the a selectivity
of the glycosylation. Finally, we found that glycosylation of
13a with an equivalent of 3 f in CH3CN/CH2Cl2 (2:3) at
ꢀ788C provided disaccharide 15 in moderate yield (65%)
with excellent selectivity (a:b=93:7), along with glycal 6 f
in 31% (entry 11). Use of CH2Cl2 as the solvent resulted in
a reduced yield (17%) of disaccharide 15 but provided
products with moderately a-selective glycosylation
(entry 12). These results indicated that acetonitrile would be
effective for improving the coupling yield as well as the a
selectivity. Minimization of the remaining acceptor 13a was
accomplished by glycosidation of sialyl donor 3 f (1.5 equiv)
Scheme 6. Synthesis of 2,3-sialyl T antigen 2a by a stepwise process and
by a linear-type one-pot glycosylation. Reagents and conditions: a) 3 f
(1.50 equiv), NIS (2.00 equiv), TfOH (0.30 equiv), CH3CN/CH2Cl2 (2:3,
5 mLmmolꢀ1), 3 ꢀ MS (0.50 gmmolꢀ1), ꢀ788C, 86%, a:b=93:7; b) 14
(1.30 equiv), ZrCp2Cl2 (1.50 equiv), AgOTf (3.00 equiv), CH3CN/CH2Cl2
(2:3, 5 mLmmolꢀ1), 3 ꢀ MS (0.50 gmmolꢀ1), ꢀ58C, 96%; c) 3 f
(1.50 equiv), 13a (1.00 equiv), NIS (2.00 equiv), TfOH (0.30 equiv),
CH3CN/CH2Cl2 (2:3, 5 mLmmolꢀ1), 3 ꢀ MS (0.50 gmmolꢀ1), ꢀ788C;
then 14 (1.50 equiv), ZrCp2Cl2 (2.00 equiv), AgOTf (4.00 equiv), ꢀ58C,
88%, a:b=93:7; d) Zn dust, THF, AcOH, Ac2O, 0 8C, 1 h, 93%;
e) Pd(OH)2, THF, MeOH, AcOH, H2O, 2 h; f) 0.1n NaOH, MeOH; then
H2O, 90% over two steps.
with NIS (2.00 equiv), TfOH (0.30 equiv), and 3 ꢀ MS
(0.50 gmmolꢀ1) in CH3CN/CH2Cl2 (2:3; 5 mLmmolꢀ1) at
ꢀ788C to provide disaccharide 15 in 86% yield (a:b=93:7),
along with glycal 6 f (entry 13). The yield of glycal 6 f was
44%, based on donor 3 f. This chemoselective glycosylation
method should be effective for the preparation of various
glycosyl fluorides attached to a-sialosides.
Next, glycosylation of the glycosyl amino acid 14 with dis-
accharide 15 was examined. In the one-pot glycosylation,
the galactosylation should be achieved after the sialylation.
Therefore, we examined glycosylation of 14 with disacchar-
ide 15a in CH3CN/CH2Cl2 (2:3). Treatment of the glycosyl
fluoride 15a with the glycosyl amino acid 14 in the presence
of [ZrCp2Cl2]/AgOTf in CH3CN/CH2Cl2 (2:3) at ꢀ58C pro-
Chem. Eur. J. 2005, 11, 849 – 862
www.chemeurj.org
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
853

T. Takahashi et al.
Table 5. Optimization of regio-selective sialylation of galactosyl fluoride 13a with the N-Troc b-thiophenyl
sialoside 3 f.[a]
good yield. A linear-type one-
pot glycosylation initiated by
chemoselective glycosylation of
glycosyl fluoride 13a with the
sialyl donor 3 f afforded the
protected 2,3-sialyl T antigen 16
in excellent yield. Modification
of the N-Troc and azido groups
into N-acetyl groups followed
by removal of all protecting
groups without racemization of
the a position of the amino
acid was accomplished to pro-
vide sialo-containing glycosyl
amino acids 1a and 2a. Appli-
cation of the glycosylation
method to the synthesis of an
oligosaccharide library is in
progress.
Entry
Donor
[equiv]
Acid
[equiv]
Solvent
T
[8C]
Yield
of 15 [%]
a:b[b]
Yield
of 6 f [%][c]
1
2
3
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.5
TfOH (0.3)
TfOH (0.3)
TMSOTf[d] (0.3)
TfOH (0.5)
TfOH (0.3)
TfOH (0.3)
TfOH (0.3)
TfOH (0.3)
TfOH (0.3)
TfOH (0.3)
TfOH (0.3)
TfOH (0.3)
TfOH (0.3)
CH3CN
CH3CN
CH3CN
EtCN
ꢀ30
ꢀ35
ꢀ35
ꢀ30
ꢀ35
ꢀ50
ꢀ55
ꢀ60
ꢀ65
ꢀ70
ꢀ78
ꢀ78
ꢀ78
56
61
55
45
45
45
63
64
65
66
65
17
86
70:30
75:25
75:25
72:28
75:25
84:16
85:15
87:13
91:9
91:9
93:7
62:38
93:7
35
31
35
47
45
46
31
28
25
30
31
82
44
4[e]
5
CH3CN/CH2Cl2 5:1
CH3CN/CH2Cl2 1:1
CH3CN/CH2Cl2 1:1
CH3CN/CH2Cl2 1:1
CH3CN/CH2Cl2 1:1
CH3CN/CH2Cl2 2:3
CH3CN/CH2Cl2 2:3
CH2Cl2
6
7
8
9
10
11
12[f]
13[g]
CH3CN/CH2Cl2 2:3
[a] Conditions: NIS (1.35 equiv based on 3 f) for 15 min. [b] The a:b ratio was determined by HPLC analysis
based on refractive-index detection. [c] Yields were based on donor 3 f. [d] TMS=trimethylsilyl. [e] The reac-
tion time was 3 h. [f] The reaction time was 5 h. [g] 2.00 equivalents of NIS were used.
vided trisaccharide 16 in 96% yield and with excellent b se-
lectivity (a:b=< 1:99).
Experimental Section
One-pot glycosylation with 3 f, 13a, and 14 was examined
next. Chemoselective glycosylation of the glycosyl fluoride
13a with the N-Troc-b-thiophenyl sialoside 3 f under the
above-described conditions provided disaccharide 15. Subse-
quent addition of acceptor 14, [ZrCp2Cl2], and AgOTf af-
forded the protected trisaccharide 16 as two diastereomers
in 88% overall yield, based on 13a, and with excellent selec-
tivity (a:b=93:7). The analytical data of the trisaccharides
16a and 16b were identical to those of authentic samples
synthesized by the stepwise synthesis.
Transformation of 16a into 2a proceeded as follows. Si-
multaneous reduction of the azido and N-Troc groups of
16a followed by acetylation provided the corresponding N-
acetyl derivative 17 in 93% yield. Complete deprotection of
17 was accomplished in two steps, involving hydrogenolysis
of the benzyl ethers followed by hydrolysis of the benzoates,
acetates, and the methyl ester, to provide the fully depro-
tected 2,3-sialyl T antigen 2a in 90% overall yield. The ana-
lytical data (1H NMR spectroscopy) were identical to those
previously reported.[42]
General: NMR spectra were obtained on
a JEOL model ECP-400
(400 MHz for 1H, 100 MHz for 13C) instrument in the indicated solvent.
Chemical shifts are reported in parts per million (ppm) relative to the
signal (0 ppm) for internal tetramethylsilane for solutions in CDCl3.
1H NMR spectral data are reported as follows: CDCl3 (7.26 ppm) or D2O
(4.7015 ppm at 303 K; as an internal standard with 3-(trimethylsilyl)-1-
propanesulfonic acid sodium salt as anexternal standard). 13C NMR spec-
tral data are reported as follows: CDCl3 (77.0 ppm) or [D6]acetone
(30.3 ppm; as an internal standard for D2O). Multiplicities 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. FT-IR spectra were recorded on a Perkin–Elmer Spectrum One
spectrometer. Data are given in cmꢀ1 with only significant diagnostic
bands reported. Optical rotations were measured with a JASCO P-1020
polarimeter. Column chromatography was performed with silica gel
(Merck). Gel-permeation chromatography (GPC) for qualitative analyses
was performed on a Japan Analytical Industry model LC908 (recycling
preparative HPLC) instrument, with a Japan Analytical Industry model -
RI-5 refractive-index detector and a Japan Analytical Industry model 310
UV detector, on a polystylene gel column (JAIGEL-1H, 20ꢂ600 mm),
with chloroform as the solvent (Flow rate: 3.5 mLminꢀ1). HPLC was per-
formed on a Waters apparatus with a Senshu Pak Silica-3301-N column
and a Waters 2414 refractive-index detector. Dry tetrahydrofuran (THF),
hexane, benzene, toluene, diethyl ether, and 1,2-dimethoxyethane
(DME) were distilled from sodium wire contained with
a catalytic
amount of benzophenone. Dry dichloromethane was distilled from P2O5.
Dry N,N-dimethylformamide (DMF), triethylamine, and pyridine were
distilled from CaH2. Dry methanol and ethanol were distilled from mag-
nesium contained with a catalytic amount of iodine.
Conclusion
We have described an effective sialylation method that uti-
lizes the N-Fmoc, N-Troc, and N-trichloroacetyl-b-thiophen-
yl sialosides 3e, 3 f, and 3h. Additionally, use of a minimum
amount of 3 ꢀ molecular sieves improved the yield of the
coupled products. An application of this sialylation with N-
Troc-b-thiosialoside 3 f to the synthesis of glycosyl amino
Methyl (phenyl 4,7,8,9-tetra-O-acetyl-5-(benzyloxycarbonylamino)-3,5-di-
deoxy-2-thio-d-glycero-b-d-galacto-2-nonulopyranosid)onate (3b): Meth-
anesulfonic acid (0.66 mL, 10.2 mmol) was added to a stirred solution of
methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-d-
glycero-b-d-galacto-2-nonulopyranosid)onate (3a; 595 mg, 1.02 mmol) in
methanol (20 mL) at room temperature. After being stirred at 608C for
24 h, the reaction mixture was neutralized with triethylamine and concen-
trated in vacuo. The residue was used for the next reaction without fur-
ther purification. Benzyl succinimidyl carbonate (254 mg, 1.02 mmol) and
triethylamine (0.21 mL, 1.53 mmol) were added to a stirred solution of
the residue in CH3CN/H2O (17:1; 9.00 mL) at 08C. After being stirred at
room temperature for 2.5 h, the reaction mixture was concentrated in
acids by one-pot glycosylation was demonstrated.
A
branched-type one-pot two-step glycosylation initiated by
regioselective glycosidation of 3 f on the primary alcohol of
acceptor 7 provided the protected 2,6-sialyl T antigen 11 in
854
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 849 – 862

Sialo-Containing Glycosyl Amino Acids
FULL PAPER
vacuo. The residue was used for the next reaction without further purifi-
cation. Acetic anhydride (0.86 mL, 6.12 mmol) and a catalytic amount of
DMAP were added to a stirred solution of the residue in pyridine
(0.99 mL, 12.2 mmol) at 08C. After being stirred at room temperature for
3 h, the reaction mixture was poured into ice-cooled water. The aqueous
layer was extracted with two portions of ethyl acetate. The combined ex-
tracts were washed with 1m HCl, saturated aq. NaHCO3, and brine, dried
over MgSO4, filtered, and concentrated in vacuo. The residue was puri-
fied by chromatography on silica gel with chloroform/methanol (98:2) to
give 3b (426 mg, 0.630 mmol, 63% over three steps); 1H NMR
(400 MHz, CDCl3): d=7.27–7.45 (m, 10H, aromatic), 5.55 (brs, 1H, H-
7), 5.36 (ddd, 1H, H-4, J3ax,4 =11.7, J3eq,4 =4.4, J4,5 =10.3 Hz), 5.17 (d, 1H,
being stirred at room temperature for 3 h, the reaction mixture was
poured into ice-cooled water. The aqueous layer was extracted with two
portions of ethyl acetate. The combined extracts were washed with 1m
HCl, saturated aq. NaHCO3, and brine, dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by chromatography on
silica gel with chloroform/methanol (98:2) to give 3d (319 mg,
0.510 mmol, 67% over three steps); 1H NMR (400 MHz, CDCl3): d=
7.33–7.45 (m, 5H, aromatic), 5.88 (dddd, 1H, J=5.8, J=5.8, J=10.3, J=
10.3 Hz), 5.54 (brs, 1H, H-7), 5.39 (ddd, 1H, H-4, J3ax,4 =J3eq,4 =J4,5
10.2 Hz), 5.26 (dd, 1H, J=10.3, Jgem =1.0 Hz), 5.20 (dd, 1H, J=10.3,
gem =1.0 Hz), 5.00 (brd, 1H, H-8, J=8.3 Hz), 4.87 (d, 1H, NH, J=
10.2 Hz), 4.62 (dd, 1H, H-6, J5,6 =10.7, J6,7 =2.4 Hz), 4.57 (dd, 1H, J=5.8,
gem =13.6 Hz), 4.48 (dd, 1H, H-9’, J8,9’ =2.0, Jgem =12.2 Hz), 4.44 (dd, 1H,
=
J
J
gem =12.2 Hz), 5.01 (brd, 1H, H-8, J=8.3 Hz), 4.93 (d, 1H, Jgem
=
J
12.2 Hz), 4.86 (d, 1H, NH, J=10.6 Hz), 4.61 (dd, 1H, H-6, J5,6 =11.0,
J=5.8, Jgem =13.6 Hz), 4.02 (ddd, 1H, H-5, J4,5 =J5,6 =J5,NH =10.2 Hz),
3.60 (s, 3H, OMe), 2.72 (dd, 1H, H-3eq, J3eq,4 =4.9, Jgem =14.2 Hz), 2.11
(dd, 1H, H-3ax, J3ax,4 =11.2 Hz), 1.97, 2.03, 2.06, 2.11 (4s, 12H, Ac) ppm;
13C NMR (100 MHz, CDCl3): d=170.9, 170.5, 170.3, 170.0, 168.2, 155.8,
136.1, 132.6, 129.7, 129.1, 128.8, 117.6, 88.8, 73.1, 72.8, 69.0, 65.9, 62.6,
52.5, 51.5, 37.5, 21.0, 20.8, 20.7, 20.7 ppm; IR (KBr): n˜ =3454, 2998, 2911,
J
J
6,7 =2.0 Hz), 4.48 (brd, 1H, H-9’, Jgem =12.2 Hz), 4.03 (dd, 1H, H-9,
8,9 =8.8, Jgem =12.2 Hz), 3.81 (ddd, 1H, H-5, J4,5 =J5,6 =J5,NH =10.3 Hz),
3.59 (s, 3H, OMe), 2,69 (dd, 1H, H-3eq, J3eq,4 =4.4, Jgem =13.7 Hz), 2.08
(dd, 1H, H-3ax, J3ax,4 =11.7 Hz), 1.84, 1.98, 2.07, 2.13 (4s, 12H, Ac) ppm;
IR (KBr): n˜ =3480, 2801, 1703, 1489, 1460, 1045, 861 cmꢀ1
.
1730, 1487, 1123, 790 cmꢀ1
.
Methyl (phenyl 4,7,8,9-tetra-O-acetyl-5-(tert-butoxycarbonylamino)-3,5-
dideoxy-2-thio-d-glycero-b-d-galacto-2-nonulopyranosid)onate (3c):
Methanesulfonic acid (1.32 mL, 20.3 mmol) was added to a stirred so-
Methyl (phenyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(9-fluorenylmethoxy-
carbonylamino)-2-thio-d-glycero-b-d-galacto-2-nonulopyranosid)onate
(3e): Methanesulfonic acid (0.86 mL, 13.2 mmol) was added to a stirred
solution of methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-di-
lution of methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
2-thio-d-glycero-b-d-galacto-2-nonulopyranosid)onate
(3a;
1.12 g,
2.03 mmol) in methanol (40 mL) at room temperature. After being stir-
red at 608C for 24 h, the reaction mixture was neutralized with triethyla-
mine and concentrated in vacuo. The residue was used for the next reac-
tion without further purification. Di-tert-butyldicarbonate (2.20 g,
10.2 mmol) and triethylamine (0.85 mL, 6.09 mmol) were added to a stir-
red solution of the residue in dioxane/MeOH/H2O (1:1:1; 9.00 mL) at
08C. After being stirred at room temperature for 2.5 h, the reaction mix-
ture was concentrated in vacuo. The residue was used for the next reac-
tion without further purification. Acetic anhydride (1.71 mL, 12.1 mmol)
and a catalytic amount of DMAP were added to a stirred solution of the
residue in pyridine (1.97 mL, 24.4 mmol) at 08C. After being stirred at
room temperature for 3 h, the reaction mixture was poured into ice-
cooled water. The aqueous layer was extracted with two portions of ethyl
acetate. The combined extracts were washed with 1m HCl, saturated aq.
NaHCO3, and brine, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by chromatography on silica gel with
chloroform/methanol (98:2) to give 3c (889 mg, 1.38 mmol, 68% over
three steps); 1H NMR (400 MHz, CDCl3): d=7.30–7.45 (m, 5H, aromat-
deoxy-2-thio-d-glycero-b-d-galacto-2-nonulopyranosid)onate
(3a;
770 mg, 1.32 mmol) in methanol (26 mL) at room temperature. After
being stirred at 608C for 24 h, the reaction mixture was neutralized with
triethylamine and concentrated in vacuo. The residue was used for the
next reaction without further purification. 9-Fluorenylmethyl succinimid-
yl carbonate (445 mg, 1.32 mmol) was added to a stirred solution of the
residue in CH3CN/H20 (11:1; 12.0 mL) at 08C. After being stirred at
room temperature for 2.5 h, the reaction mixture was concentrated in
vacuo. The residue was used for the next reaction without further purifi-
cation. Acetic anhydride (1.10 mL, 7.92 mmol) and a catalytic amount of
DMAP were added to a stirred solution of the residue in pyridine
(1.30 mL, 15.8 mmol) at 08C. After being stirred at room temperature for
3 h, the reaction mixture was poured into ice-cooled water. The aqueous
layer was extracted with two portions of ethyl acetate. The combined ex-
tracts were washed with 1m HCl, saturated aq. NaHCO3, and brine, dried
over MgSO4, filtered, and concentrated in vacuo. The residue was puri-
fied by chromatography on silica gel with chloroform/methanol (98:2) to
give 3e (296 mg, 0.387 mmol, 27% over three steps); 1H NMR
(400 MHz, CDCl3): d=7.30–7.77 (m, 13H, aromatic), 5.61 (brs, 1H, H-
7), 5.45 (ddd, 1H, H-4, J3ax,4 =10.7, J3eq,4 =4.8, J4,5 =10.7 Hz), 5.00 (brd,
1H, H-8, J=8.3 Hz), 4.89 (d, 1H, NH, J=10.2 Hz), 4.67 (dd, 1H, H-6,
ic), 5.57 (brs, 1H, H-7), 5.33 (ddd, 1H, H-4, J3ax,4 =10.8, J3eq,4 =4.9, J4,5
10.8 Hz), 5.00 (brd, 1H, H-8, J=8.8 Hz), 4.67 (m, 1H, NH), 4.59 (dd,
1H, H-6, J5,6 =10.8, J6,7 =2.4 Hz), 4.53 (dd, 1H, H-9’, J8,9’ =1.5, Jgem
12.2 Hz), 4.00 (dd, 1H, H-9, J8,9 =8.8, Jgem =12.2 Hz), 3.81 (ddd, 1H, H-5,
=
=
J
5,6 =10.2, J6,7 =1.9 Hz), 4.49 (dd, 1H, H-9’, J8,9’ =2.0, Jgem =12.2 Hz), 4.35
J
J
4,5 =J5,6 =J5,NH =10.7 Hz), 3.59 (s, 3H, OMe), 2.70 (dd, 1H, H-3eq,
3eq,4 =4.9, Jgem =13.7 Hz), 2.11 (dd, 1H, H-3ax, J3ax,4 =10.8 Hz), 1.84, 1.97,
(m, 1H), 4.17–4.25 (m, 2H), 4.03 (dd, 1H, H-9, J8,9 =8.8, Jgem =12.2 Hz),
3.84 (ddd, 1H, H-5, J4,5 =J5,6 =J5,NH =10.2 Hz), 3.60 (s, 3H, OMe), 2.74
(dd, 1H, H-3eq, J3eq,4 =4.9, Jgem =13.7 Hz), 2.13 (dd, 1H, H-3ax, J3ax,4 =
2.05, 2.17 (4s, 12H, Ac), 1.40 (s, 9H, Me) ppm; 13C NMR (100 MHz,
CDCl3): d=170.9, 170.4, 170.2, 169.7, 168.2, 155.2, 136.1, 129.6, 129.0,
128.8, 88.8, 80.0, 73.2, 72.8, 69.3, 68.9, 62.7, 54.5, 50.7, 37.6, 28.6, 21.0,
20.7, 20.7, 20.6 ppm; IR (KBr): n˜ =3373, 2979, 1741, 1524, 1370, 1235,
10.7 Hz), 1.96ꢂ2, 2.05, 2.13 (3s, 12H, Ac) ppm; 13C NMR (100 MHz,
CDCl3): d=170.9, 170.6, 170.2, 170.0, 168.2, 155.9, 144.3, 143.5, 141.3,
141.1, 136.1, 129.7, 129.1, 128.8, 127.6, 127.6, 127.0, 126.4, 125.0, 119.9,
119.8, 88.8, 77.2, 73.2, 72.9, 68.9, 67.5, 62.6, 52.5, 51.5, 46.9, 37.5, 21.0,
20.8, 20.3ꢂ2 ppm; IR (KBr): n˜ =3354, 3022, 2952, 1740, 1534, 1234,
1037, 752 cmꢀ1
.
Methyl (phenyl 4,7,8,9-tetra-O-acetyl-5-(allyloxycarbonylamino)-3,5-di-
deoxy-2-thio-d-glycero-b-d-galacto-2-nonulopyranosid)onate (3d): Meth-
anesulfonic acid (0.434 mL, 6.70 mmol) was added to a stirred solution of
methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-d-
glycero-b-d-galacto-2-nonulopyranosid)onate (3a; 391 mg, 0.670 mmol)
in methanol (13 mL) at room temperature. After being stirred at 608C
for 24 h, the reaction mixture was neutralized with triethylamine and
concentrated in vacuo. The residue was used for the next reaction with-
out further purification. A 0.5m dioxane solution of allyl succinimidyl
carbonate (1.35 mL, 0.670 mmol) and triethylamine (0.14 mL, 1.01 mmol)
were added to a stirred solution of the residue in CH3CN/H2O (9:1;
6.00 mL) at 08C. After being stirred at room temperature for 2.5 h, the
reaction mixture was concentrated in vacuo. The residue was used for the
next reaction without further purification. Acetic anhydride (0.65 mL,
4.20 mmol) and a catalytic amount of DMAP were added to a stirred so-
lution of the residue in pyridine (0.65 mL, 8.04 mmol) at 08C. After
741 cmꢀ1
.
Methyl
(phenyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-5-(2,2,2-
trichloroethoxycarbonylamino)-d-glycero-b-d-galacto-2-nonulopyrano-
sid)onate (3 f): Methanesulfonic acid (0.46 mL, 7.07 mmol) was added to
a stirred solution of methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-
3,5-dideoxy-2-thio-d-glycero-b-d-galacto-2-nonulopyranosid)onate (3a;
421.8 mg, 0.707 mmol) in methanol (14 mL) at room temperature. After
being stirred at 608C for 24 h, the reaction mixture was neutralized with
triethylamine and concentrated in vacuo. The residue was used for the
next reaction without further purification. Succinimidyl 2,2,2-trichloro-
ethyl carbonate (205 mg, 0.707 mmol) and triethylamine (0.15 mL,
1.06 mmol) were added to a stirred solution of the residue in CH3CN/H20
(15:1; 6.40 mL) at 08C. After being stirred at room temperature for 2.5 h,
the reaction mixture was concentrated in vacuo. The residue was used for
the next reaction without further purification. Acetic anhydride
Chem. Eur. J. 2005, 11, 849 – 862
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ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
855

T. Takahashi et al.
(0.40 mL, 4.24 mmol) and a catalytic amount of DMAP were added to a
stirred solution of the residue in pyridine (0.69 mL, 4.84 mmol) at 08C.
After being stirred at room temperature for 3 h, the reaction mixture was
poured into ice-cooled water. The aqueous layer was extracted with two
portions of ethyl acetate. The combined extracts were washed with 1m
HCl, saturated aq. NaHCO3, and brine, dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by chromatography on
silica gel with chloroform/methanol (98:2) and recrystallized from ethyl
acetate/hexane to give 3 f (450 mg, 0.64 mmol, 89% over three steps);
1H NMR (400 MHz, CDCl3): d=7.31–7.46 (m, 5H, aromatic), 5.51 (dd,
The residue was used for the next reaction without further purification.
Acetic anhydride (1.70 mL, 18.0 mmol) and a catalytic amount of DMAP
were added to a stirred solution of the residue in pyridine (2.90 mL,
36.0 mmol) at 08C. After being stirred at room temperature for 3 h, the
reaction mixture was poured into ice-cooled water. The aqueous layer
was extracted with two portions of ethyl acetate. The combined extracts
were washed with 1m HCl, saturated aq. NaHCO3, and brine, dried over
MgSO4, filtered, and concentrated in vacuo. The residue was purified by
chromatography on silica gel with chloroform/methanol (98:2) and re-
crystallized from ethyl acetate/hexane to give 3h (1.44 g, 2.10 mmol, 70%
over three steps); 1H NMR (400 MHz, CDCl3): d=7.31–7.47 (m, 5H, ar-
omatic), 7.19 (d, 1H, NH, J=10.3 Hz), 5.57 (ddd, 1H, H-4, J3ax,4 =10.8,
1H, H-7, J6,7 =1.9, J7,8 =2.4 Hz), 5.45 (ddd, 1H, H-4, J3ax,4 =11.2, J3eq,4
4.9, J4,5 =10.3 Hz), 5.09 (d, 1H, NH, J=10.2 Hz), 5.01 (d, 1H, Jgem
12.2 Hz), 4.67 (dd, 1H, H-6, J5,6 =10.8, J6,7 =1.9 Hz), 4.49 (d, 1H, Jgem
=
=
=
J
3eq,4 =4.9 Hz), 5.53 (dd, 1H, H-7, J6,7 =2.4, J7,8 =2.4 Hz), 5.07 (ddd, 1H,
H-8, J7,8 =2.4, J8,9’ =2.0, J8,9 =8.8 Hz), 4.89 (dd, 1H, H-6, J5,6 =10.7, J6,7
2.4 Hz), 4.55 (dd, 1H, H-9’, J8,9’ =2.0, Jgem =12.7 Hz), 4.08 (dd, 1H, H-9,
8,9 =8.3, Jgem =12.7 Hz), 4.06 (ddd, 1H, H-5), 3.58 (s, 3H, OMe), 2.72
12.2 Hz), 4.46 (dd, 1H, H-9’, J8,9’ =2.0, Jgem =12.7 Hz), 4.03 (dd, 1H, H-9,
=
J
8,9 =8.3, Jgem =12.7 Hz), 3.76 (ddd, 1H, H-5, J4,5 =10.3, J5,6 =10.8, J5,NH
=
10.3 Hz), 3.60 (s, 3H, OMe), 2.73 (dd, 1H, H-3eq, J3eq,4 =4.9, Jgem
=
J
13.6 Hz), 2.07 (dd, 1H, H-3ax, J3ax,4 =11.2 Hz), 1.83, 2.01, 2.05, 2.12 (4s,
12H, Ac) ppm; 13C NMR (100 MHz, CDCl3): d=171.0, 170.4, 170.3,
170.0, 168.1, 154.3, 136.1, 129.7, 129.1, 128.7, 95.4, 88.8, 77.2, 74.5, 72.8,
69.0, 68.6, 62.5, 51.7, 37.5, 21.0, 20.8, 20.7, 20.7 ppm; IR (KBr): n˜ =3329,
2954, 1743, 1540, 1371, 1253, 1039, 755, 694 cmꢀ1; elemental analysis
calcd (%) for C27H32Cl3NO13S: C 45.23, H 4.50, N 1.95; found: C 45.04,
H 4.48, N 1.95.
(dd, 1H, H-3eq, J3eq,4 =4.9, Jgem =12.7 Hz), 2.12 (dd, 1H, H-3ax, J3ax,4
=
10.8 Hz), 1.92, 2.04, 2.11, 2.14 (4s, 12H, Ac) ppm; 13C NMR (100 MHz,
CDCl3): d=172.0, 170.5, 170.2, 170.0, 167.8, 162.4, 136.2, 129.8, 129.1,
128.9, 128.8, 128.1, 92.0, 89.2, 73.6, 72.3, 68.5, 68.1, 62.5, 52.6, 51.4, 37.5,
21.1, 20.8, 20.6, 20.5 ppm; IR (KBr): n˜ =3319, 2953, 1744, 1526, 1232,
1038, 941, 751 cmꢀ1
.
General procedure for sialylation of methyl 2,3,4-tri-O-benzyl-a-d-gluco-
pyranoside (4) with thiosialosides 3a–i:
Methyl
(phenyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-5-trifluoro-
acetamido-d-glycero-b-d-galacto-2-nonulopyranosid)onate (3g): Meth-
anesulfonic acid (0.86 mL, 13.3 mmol) was added to a stirred solution of
methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-d-
glycero-b-d-galacto-2-nonulopyranosid)onate (3a; 775 mg, 1.33 mmol) in
methanol (26 mL) at room temperature. After being stirred at 608C for
24 h, the reaction mixture was neutralized with triethylamine and concen-
trated in vacuo. The residue was used for the next reaction without fur-
ther purification. Trifluoroacetic acid methyl ester (1.34 mL, 13.3 mmol)
and triethylamine (0.37 mL, 2.66 mmol) were added to a stirred solution
of the residue in methanol (13 mL) at 08C. After being stirred at room
temperature for 2.5 h, the reaction mixture was concentrated in vacuo.
The residue was used for the next reaction without further purification.
Acetic anhydride (0.75 mL, 7.98 mmol) and a catalytic amount of DMAP
were added to a stirred solution of the residue in pyridine (1.30 mL,
16.0 mmol) at 08C. After being stirred at room temperature for 3 h, the
reaction mixture was poured into ice-cooled water. The aqueous layer
was extracted with two portions of ethyl acetate. The combined extracts
were washed with 1m HCl, saturated aq. NaHCO3, and brine, dried over
MgSO4, filtered, and concentrated in vacuo. The residue was purified by
chromatography on silica gel with chloroform/methanol (98:2) and re-
crystallized from ethyl acetate/hexane to give 3g (656 mg, 1.03 mmol,
77% over three steps); 1H NMR (400 MHz, CDCl3): d=7.32–7.43 (m,
Method A (NIS/TfOH, CH3CN): A mixture of the appropriate thiosialo-
side (3a–i; 1.00 equiv), methyl 2,3,4-tri-O-benzyl-a-d-glucopyranoside (4;
1.50 equiv; azeotroped three times with toluene), and pulverized activat-
ed 3 ꢀ MS (10 gmmolꢀ1) in dry CH3CN was stirred at room temperature
for 10 min under argon to remove any trace amounts of water. The reac-
tion mixture was then cooled to ꢀ358C. N-Iodosuccinimide (1.20 equiv)
and a catalytic amount of trifluoromethanesulfonic acid (0.20 equiv) was
added to the reaction mixture at ꢀ358C. After being stirred at the same
temperature for 1.0 h, the reaction mixture was neutralized with triethyl-
amine and filtered through a pad of celite. The filtrate was poured into a
mixture of saturated aq. NaHCO3 and saturated aq. Na2S2O3 with cool-
ing. The aqueous layer was extracted with two portions of ethyl acetate.
The combined extracts were washed with saturated aq. NaHCO3/Na2S2O3
and brine, dried over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by chromatography on silica gel with chloroform/
methanol (97:3) to give the respective disaccharide (5a–i). The a:b ratio
was determined by HPLC analysis (Senshu Pak Silica-3301-N column;
eluent: hexane/2-propanol 94:6; flow rate: 3.0 mLminꢀ1).
Method B (DMTST, CH3CN): A mixture of the appropraite thiosialoside
(3a–i; 1.00 equiv), methyl 2,3,4-tri-O-benzyl-a-d-glucopyranoside (4;
1.50 equiv; azeotroped three times with toluene), and pulverized activat-
ed 3 ꢀ MS (10 gmmolꢀ1) in dry CH3CN was stirred at room temperature
for 10 min under argon to remove any trace amounts of water. The reac-
tion mixture was then cooled to ꢀ108C. A 1.5m CH3CN solution of di-
methyl(methylthio)sulfonium trifluoromethanesulfonate (1.50 equiv) was
added to the reaction mixture at ꢀ108C. After being stirred at the same
temperature for 8 h, the reaction mixture was neutralized with triethyl-
amine and filtered through a pad of celite. The filtrate was poured into
saturated aq. NaHCO3 with cooling. The aqueous layer was extracted
with two portions of ethyl acetate. The combined extracts were washed
with saturated aq. NaHCO3 and brine, dried over MgSO4, filtered, and
concentrated in vacuo. The residue was purified by chromatography on
silica gel with chloroform/methanol (97:3) to give the respective disac-
charide (5a–i). The a:b ratio was determined by HPLC analysis (condi-
tions as for method A).
6H, NH, aromatic), 5.55 (ddd, 1H, H-4, J3ax,4 =13.7, J3eq,4 =4.9, J4,5
10.0 Hz), 5.51 (brs, 1H, H-7), 5.02 (ddd, 1H, H-8, J7,8 =2.0, J8,9’ =2.4,
8,9 =8.0 Hz), 4.86 (dd, 1H, H-6, J5,6 =10.8, J6,7 =2.4 Hz), 4.53 (dd, 1H, H-
9’, 8,9’ =2.0, gem =10.2 Hz), 4.13 (ddd, 1H, H-5, 4,5 =J5,6 =J5,NH
=
J
J
J
J
=
10.2 Hz), 4.05 (dd, 1H, H-9, J8,9 =8.8, Jgem =10.2 Hz), 3.57 (s, 3H, OMe),
2.73 (dd, 1H, H-3eq, J3eq,4 =4.9, Jgem =12.9 Hz), 2.16 (dd, 1H, H-3ax,
J
3ax,4 =13.7 Hz), 1.95, 2.07, 2.11, 2.12 (4s, 12H, Ac) ppm; 13C NMR
(100 MHz, CDCl3): d=171.7, 171.1, 170.3ꢂ2, 169.9, 167.9, 136.5, 136.0,
130.0, 129.8, 129.1, 128.9, 128.6, 89.1, 73.2, 72.1, 68.5, 68.4, 62.5, 52.6, 50.0,
37.4, 21.0, 20.7, 20.6, 20.5 ppm; IR (KBr): n˜ =3056, 2971, 1708, 1410,
1205, 760 cmꢀ1
Methyl (phenyl
.
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-5-trichloro-
acetamido-d-glycero-b-d-galacto-2-nonulopyranosid)onate (3h): Meth-
anesulfonic acid (1.95 mL, 30.0 mmol) was added to a stirred solution of
methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-d-
glycero-b-d-galacto-2-nonulopyranosid)onate (3a; 1.75 g, 3.00 mmol) in
methanol (30 mL) at room temperature. After being stirred at 608C for
24 h, the reaction mixture was neutralized with triethylamine and concen-
trated in vacuo. The residue was used for the next reaction without fur-
ther purification. Trichloroacetic acid methyl ester (3.57 mL, 30.0 mmol)
and triethylamine (0.84 mL, 6.00 mmol) were added to a stirred solution
of the residue in methanol (30 mL) at 08C. After being stirred at room
temperature for 2.5 h, the reaction mixture was concentrated in vacuo.
Methyl 2,3,4-tri-O-benzyl-6-O-(methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
5-(2,2,2-trichloroethoxycarbonylamino)-d-glycero-d-galacto-2-nonulopyr-
anosylonate)-a-d-glucopyranoside (5 f): In accordance with method A,
thiosialoside 3 f (17.2 mg, 0.0239 mmol) was treated with methyl 2,3,4-tri-
O-benzyl-a-d-glucopyranoside (4; 16.7 mg, 0.0360 mmol) and N-iodosuc-
cinimide (10.8 mg, 0.0478 mmol) in CH3CN (0.25 mL) to provide disac-
charide 5 f (23.2 mg, 0.0217 mmol, 91%, a:b=89:11). In accordance with
method B, thiosialoside 3 f (17.7 mg, 0.0247 mmol) was treated with
methyl 2,3,4-tri-O-benzyl-a-d-glucopyranoside (4; 17.2 mg, 0.037 mmol)
856
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 849 – 862

Sialo-Containing Glycosyl Amino Acids
FULL PAPER
and DMTST (25 mL, 0.037 mmol, 1.50 equiv) in CH3CN (0.25 mL) to pro-
vide disaccharide 5 f (55.0 mg, 0.0136 mmol, 55%, a:b=86:14).
5.54 (brs, 1H, Neu-H-7), 5,32 (brd, 1H, Neu-H-8, J=8.8 Hz), 5.21 (ddd,
1H, Neu-H-4, J3ax,4 =11.7, J3eq,4 =4.9 Hz), 5.09 (dd, 1H, Neu-H-9’, J8,9’
=
5 f a isomer: Rt =19.2 min; 1H NMR (400 MHz, CDCl3): d=7.30–3.74
(m, 15H, aromatic), 5.31 (brs, 2H, Neu-H-7, H-8), 4.98 (ddd, 1H, Neu-
H-4, J3ax,4 =12.7, J3eq,4 =4.9 Hz), 4.91 (d, 1H, Jgem =10.8 Hz), 4.89 (d, 1H,
1.5, Jgem =12.2 Hz), 4.98 (d, 1H, Jgem =11.2 Hz), 4.92 (d, 1H, Glc-H-1,
J
1,2 =3.4 Hz), 4.90 (d, 1H, Neu-NH, J=10.2 Hz), 4.89 (d, 1H, Jgem
=
=
11.2 Hz), 4.83 (d, 1H, Jgem =10.7 Hz), 4.79 (2d, 2H), 4.71 (d, 1H, Jgem
11.2 Hz), 4.20–4.38 (m, 4H, Neu-H-6), 4.15 (dd, 1H, Neu-H-9, J8,9 =9.3,
gem =12.2 Hz), 3.98 (dd, 1H, Glc-H-3, J2,3 =9.3, J3,4 =9.3 Hz), 3.71–3,86
J
gem =12.2 Hz), 4.85 (d, 1H, Jgem =10.8 Hz), 4.84 (d, 1H, Neu-NH, J=
J
10.7 Hz), 4.79 (d, 1H, Jgem =12.7 Hz), 4.78 (d, 1H, Jgem =10.7 Hz), 4.74 (d,
1H, Jgem =11.2 Hz), 4.66 (d, 1H, Jgem =12.2 Hz), 4.61 (d, 1H, Glc-H-1,
(m, 8H, Neu-H-5, OMe, Glc-H-4, H-5, H-6’, H-6), 3.65 (dd, 1H, Glc-H-
2, J1,2 =3.4, J2,3 =9.3 Hz), 3.36 (s, 3H, Glc-OMe), 2.54 (dd, 1H, Neu-H-
3eq, J3eq,4 =4.9, Jgem =12.7 Hz), 1.93, 1.97, 2.02, 2.17 (4s, 12H, Ac), 1.86
(dd, 1H, Neu-H-3ax, J3ax,4 =11.7 Hz) ppm; 13C NMR (100 MHz, CDCl3):
d=170.7, 170.6, 170.4, 170.3, 167.2, 155.9, 144.3, 143.5, 141.3, 138.7, 138.5,
138.2, 128.6, 128.4, 128.3, 128.0, 128.0, 127.8, 127.6, 127.5, 127.0, 125.4,
125.0, 119.9, 97.9, 97.8, 82.0, 80.1, 77.3, 75.8, 75.1, 73.6, 73.2, 71.7, 69.2,
69.1, 68.8, 67.4, 62.9, 62.1, 54.9, 52.7, 51.4, 47.0, 37.6, 29.7, 21.0, 20.9, 20.8,
20.7 ppm; IR (KBr): n˜ =3387, 2928, 11742, 1518, 1452, 1370, 1224, 1074,
J
1,2 =3.9 Hz), 4.44 (d, 1H, Jgem =12.2 Hz), 4.21 (dd, 1H, Glc-H-6’, J5,6’
3.9, Jgem =11.2 Hz), 4.18 (brd, 1H, Neu-H-6, J5,6 =10.2 Hz), 4.00 (brd,
1H, Neu-H-9’, Jgem =10.2 Hz), 3.95 (dd, 1H, Glc-H-3, J2,3 =9.3, J3,4
9.3 Hz), 3.73–3.80 (m, 5H, Neu-OMe, H-5, H-9), 3.60 (dd, 1H, Glc-H-4,
3,4 =9.3, J4,5 =9.3 Hz), 3.56 (ddd, 1H, Glc-H-5, J4,5 =9.3, J5,6’ =3.9, J5,6
=
=
J
=
2.0 Hz), 3.52 (dd, 1H, Glc-H-2, J1,2 =3.9, J2,3 =9.3 Hz), 3.42 (dd, 1H, Glc-
H-6, J5,6 =2.0, Jgem =11.2 Hz), 3.36 (s, 3H, Glc-OMe), 2.72 (dd, 1H, Neu-
H-3eq,
J3eq,4 =4.9, Jgem =12.7 Hz), 1.89 (dd, 1H, Neu-H-3ax, J3ax,4 =
741, 698 cmꢀ1
.
12.7 Hz), 1.82, 2.01ꢂ2, 2.12 (3 s, 12H, Ac) ppm; 13C NMR (100 MHz,
CDCl3): d=170.6, 170.4, 169.9, 169.7, 167.8, 154.0, 138.8, 138.6, 138.2,
128.4, 128.3, 128.1, 128.0, 127.9, 127.7, 127.6, 98.4, 98.2, 95.3, 82.0, 82.0,
79.4, 77.7, 75.8, 74.7, 74.5, 73.4, 71.7, 69.5, 68.4, 67.4, 66.9, 63.5, 61.7, 55.2,
52.8, 51.6, 38.3, 21.1, 20.8, 20.4 ppm; IR (KBr): n˜ =3333, 2927, 1744, 1535,
Methyl 2,3,4-tri-O-benzyl-6-O-(methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
5-trifluoroacetamido-d-glycero-d-galacto-2-nonulopyranosylonate)-a-d-
glucopyranoside (5g): In accordance with method A, thiosialoside 3g
(22.5 mg, 0.0353 mmol) was treated with methyl 2,3,4-tri-O-benzyl-a-d-
glucopyranoside (4; 24.6 mg, 0.0529 mmol) and N-iodosuccinimide
(9.50 mg, 0.0424 mmo) in CH3CN (0.35 mL) to provide disaccharide 5g
(32.1 mg, 0.0323 mmol, 92%, a:b=92:8). In accordance with method B,
thiosialoside 3g (17.7 mg, 0.0277 mmol) was treated with methyl 2,3,4-tri-
O-benzyl-a-d-glucopyranoside (4; 19.3 mg, 0.0416 mmol) and DMTST
(28 mL, 0.042 mmol) in CH3CN (0.28 mL) to provide disaccharide 5g
(10.9 mg, 0.011 mmol, 40%, a:b=85:15).
1454, 1369, 1218, 1042, 735, 699 cmꢀ1
.
5 f b isomer: Rt =24.9 min; 1H NMR (400 MHz, CDCl3): d=7.29–7.42
(m, 15H, aromatic), 5.44 (brs, 1H, Neu-H-7), 5.31 (brd, 1H, Neu-H-8,
J=9.3 Hz), 5.25 (ddd, 1H, Neu-H-4,
J3ax,4 =11.7, J3eq,4 =5.4 Hz), 5.06
(brd, 2H, Neu-NH, H-9’), 4.97 (d, 1H, Jgem =10.8 Hz), 4.95 (d, 1H, Jgem
=
12.2 Hz), 4.89 (d, 1H, Glc-H-1, J1,2 =3.4 Hz), 4.87 (d, 1H, Jgem =10.8 Hz),
4.83 (d, 1H, Jgem =11.2 Hz), 4.80 (d, 1H, Jgem =11.7 Hz), 4.76 (d, 1H,
J
gem =11.2 Hz), 4.70 (d, 1H, Jgem =10.8 Hz), 4.43 (d, 1H, Jgem =12.2 Hz),
4.39 (dd, 1H, Neu-H-6, J5,6 =10.8, J6,7 =2.0 Hz), 4.13 (dd, 1H, Neu-H-9,
8,9 =9.3, Jgem =12.2 Hz), 3.97 (dd, 1H, Glc-H-3, J2,3 =8.8, J3,4 =8.8 Hz),
5g a isomer: Rt =22.6 min; 1H NMR (400 MHz, CDCl3): d=7.18–7.39
(m, 15H, aromatic), 6.50 (d, 1H, Neu-NH, J=10.3 Hz), 5.31 (ddd, 1H,
J
Neu-H-8, J7,8 =8.8, J8,9’ =2.0, J8,9 =4.9 Hz), 5.22 (dd, 1H, Neu-H-7, J6,7
2.0, J7,8 =8.8 Hz), 4.99 (m, 1H, Neu-H-4), 4.92 (d, 1H, Jgem =11.2 Hz),
4.80–4.84 (2d, 2H), 4.79 (d, 1H, Jgem =11.7 Hz), 4.75 (d, 1H, Jgem
=
3.71–3.84 (m, 8H, Neu-OMe, H-5, Glc-H-4, H-5, H-6’, H-6), 3.63 (dd,
1H, Glc-H-2, J1,2 =3.4, J2,3 =9.8 Hz), 3.35 (s, 3H, Glc-OMe), 2.54 (dd,
1H, Neu-H-3eq, J3eq,4 =5.4, Jgem =13.2 Hz), 1.98, 2.00, 2.04, 2.15 (4s, 12H,
Ac), 1.83 (dd, 1H, Neu-H-3ax, J3ax,4 =11.7 Hz) ppm; IR (KBr): n˜ =3428,
=
11.2 Hz), 4.66 (d, 1H, Jgem =12.2 Hz), 4.60 (d, 1H, Glc-H-1, J1,2 =3.4 Hz),
4.25 (dd, 1H, Neu-H-6, J5,6 =10.7, J6,7 =2.0 Hz), 4.20 (dd, 1H, Glc-H-6’,
J5,6’ =3.9, Jgem =10.8 Hz), 4.06 (dd, 1H, Neu-H-9’, J8,9’ =2.0, Jgem =
2927, 1745, 1568, 1454, 1229, 1215, 1005, 733 cmꢀ1
.
10.2 Hz), 3.95 (dd, 1H, Glc-H-3, J2,3 =9.8, J3,4 =9.8 Hz), 3.90 (ddd, 1H,
Neu-H-5, J5,6 =10.7, J5,NH =10.3 Hz), 3.76 (dd, 1H, Neu-H-9, J8,9 =4.9,
Methyl 2,3,4-tri-O-benzyl-6-O-(methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-
5-(9-fluorenylmethoxycarbonylamino)-d-glycero-d-galacto-2-nonulopyra-
nosylonate)-a-d-glucopyranoside (5e): In accordance with method A, thi-
osialoside 3e (23.0 mg, 0.030 mmol) was treated with methyl 2,3,4-tri-O-
benzyl-a-d-glucopyranoside (4; 21.0 mg, 0.0452 mmol) and N-iodosucci-
nimide (8.12 mg, 0.0361 mmol) in CH3CN (0.30 mL) to provide disacchar-
ide 5e (30.7 mg, 0.0275 mmol, 91%, a:b=86:14). In accordance with
method B, thiosialoside 3e (20.0 mg, 0.0262 mmol) was treated with
methyl 2,3,4-tri-O-benzyl-a-d-glucopyranoside (4; 18.2 mg, 0.0393 mmol)
and DMTST (26 mL, 0.039 mmol) in CH3CN (0.25 mL) to provide disac-
charide 5e (16.4 mg, 0.0147 mmol, 56%, a:b=86:14).
5e a isomer: Rt =19.3 min; 1H NMR (400 MHz, CDCl3): d=7.76 (d, 2H,
aromatic), 7.53–7.60 (m, 2H, aromatic), 7.30–7.40 (m, 19H, aromatic),
5.31–5.41 (m, 2H, Neu-H-7, H-8), 4.91 (d, 1H, Jgem =11.2 Hz), 4.90 (m,
1H, Neu-H-4), 4.87 (d, 1H, Jgem =10.8 Hz), 4.80 (d, 1H, Jgem =12.2 Hz),
4.79 (d, 1H, Jgem =11.2 Hz), 4.75 (d, 1H, Jgem =10.7 Hz), 4.66 (d, 1H,
J
J
gem =10.2 Hz), 3.74 (s, 3H, Neu-OMe), 3.58 (dd, 1H, Glc-H-4, J3,4 =9.8,
4,5 =9.3 Hz), 3.51 (dd, 1H, Glc-H-2, J1,2 =3.4, J2,3 =9.8 Hz), 3.45 (dd, 1H,
Glc-H-6, J5,6 =1.4, Jgem =10.8 Hz), 3.36 (s, 3H, Glc-OMe), 2.70 (dd, 1H,
Neu-H-3eq, J3eq,4 =4.9, Jgem =13.2 Hz), 1.84, 1.99, 2.00, 2.13 (4s, 12H, Ac),
1.96 (dd, 1H, Neu-H-3ax,
J
3ax,4 =12.7 Hz) ppm; 13C NMR (100 MHz,
CDCl3): d=170.8, 170.6, 169.9, 167.7, 157.7, 157.3, 138.7, 138.5, 138.1,
128.9, 128.6, 128.4, 128.3, 128.2, 128.1, 127.9, 127.9, 127.7, 127.7, 127.6,
98.6, 98.2, 81.9, 79.4, 79.1, 75.8, 74.7, 73.3, 71.4, 69.4, 68.2, 67.8, 66.6, 63.6,
61.7, 55.2, 52.9, 50.2, 38.0, 21.0, 20.6, 20.5, 20.3 ppm; IR (KBr): n˜ =3318,
2929, 1751, 1562, 1456, 1371, 1216, 1056, 752 cmꢀ1
.
5g b isomer: Rt =27.0 min; 1H NMR (400 MHz, CDCl3): d=7.10–7.33
(m, 15H, aromatic), 7.02 (d, 1H, Neu-NH, J=10.3 Hz), 5.27 (brs, 1H,
Neu-H-7), 5.15–5.24 (m, 2H, Neu-H-4, H-8), 4.97 (dd, 1H, Neu-H-9’,
J
8,9’ =2.4, Jgem =12.7 Hz), 4.90 (d, 1H, Jgem =11.2 Hz), 4.79 (d, 1H, Jgem
12.7 Hz), 4.78 (d, 1H, Glc-H-1, J1,2 =3.9 Hz), 4.77 (d, 1H, Jgem =10.7 Hz),
4.74 (2d, 2H), 4.61 (d, 1H, Jgem =12.7 Hz), 4.39 (dd, 1H, Neu-H-6, J5,6
=
J
gem =12.2 Hz), 4.60 (d, 1H, Glc-H-1, J1,2 =3.4 Hz), 4.59 (d, 1H, Neu-NH,
J=11.2 Hz), 4.10–4.34 (m, 5H, Neu-H-6, Glc-H-6’), 4.03 (dd, 1H, Neu-
H-9’, J8,9’ =2.0, Jgem =12.7 Hz), 3.95 (dd, 1H, Glc-H-3, J2,3 =9.8, J3,4
=
10.7, J6,7 =2.0 Hz), 4.04 (dd, 1H, Neu-H-9, J8,9 =9.3, Jgem =12.7 Hz), 3.88–
4.01 (m, 2H, Neu-H-5, Glc-H-3), 3.64–3.75 (m, 7H, Neu-OMe, Glc-H-4,
=
9.8 Hz), 3.73–3.77 (m, 5H, Neu-H-9, OMe, Glc-H-5), 3.71 (m, 1H, Neu-
H-5), 3.60 (dd, 1H, Glc-H-4, J3,4 =9.8, J4,5 =3.4 Hz), 3.51 (dd, 1H, Glc-H-
2, J1,2 =3.4, J2,3 =9.8 Hz), 3.43 (brd, 1H, Glc-H-6, J=9.8 Hz), 3.36 (s, 3H,
Glc-OMe), 2.70 (dd, 1H, Neu-H-3eq, J3eq,4 =4.4, Jgem =12.7 Hz), 1.83,
1.93, 1.97, 2.13 (4s, 12H, Ac), 1.92 (dd, 1H, Neu-H-3ax) ppm; 13C NMR
(100 MHz, CDCl3): d=170.6ꢂ2, 169.8, 167.9, 155.7, 155.7, 144.2, 143.4,
141.3, 141.1, 138.8, 138.6, 138.2, 128.6, 128.4, 128.3, 128.1, 127.9, 127.9,
127.7, 127.5, 127.0, 125.2, 124.9, 119.9, 119.9, 98.5, 98.2, 82.0, 75.8, 74.8,
73.3, 72.3, 69.5, 68.8, 67.6, 67.4, 66.8, 63.5, 61.8, 55.2, 52.8, 51.4, 46.9, 38.3,
21.0, 20.8, 20.7, 20.5 ppm; IR (KBr): n˜ =3385, 2928, 1745, 1532, 1452,
H-5, H-6’, H-6), 3.27 (s, 3H, Glc-OMe), 2.45 (dd, 1H, Neu-H-3eq, J3eq,4
=
5.4, Jgem =12.7 Hz), 1.90, 1.92, 1.98, 2.07 (4s, 12H, Ac), 1.80 (dd, 1H,
Neu-H-3ax, J3ax,4 =12.2 Hz) ppm; IR (KBr): n˜ =3374, 2926, 2855, 2749,
1456, 1372, 1229, 1029, 803, 745, 699 cmꢀ1
.
N-(Benzyloxycarbonyl)
3-O-(2-azido-4-O-benzyl-2-deoxy-6-O-(methyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(2,2,2-trichloroethoxycarbonylami-
no)-d-glycero-d-galacto-2-nonulopyranosylonate)-a-d-galactopyranosyl)-
l-serine benzyl ester (9):
A mixture of thiosialoside 3 f (20.0 mg,
0.0279 mmol), N-(benzyloxycarbonyl)-3-O-(2-azido-4-O-benzyl-2-deoxy-
a-d-galactopyranosyl)-l-serine benzyl ester (7; 14.1 mg, 0.0232 mmol;
azeotroped three times with toluene), and pulverized activated 3 ꢀ MS
(11.6 mg) in dry CH3CN (0.116 mL) was stirred at room temperature for
1217, 1040, 740 cmꢀ1
.
5e b isomer: Rt =23.9 min; 1H NMR (400 MHz, CDCl3): d=7.75 (d, 2H,
aromatic), 7.56–7.63 (m, 2H, aromatic), 7.26–7.47 (m, 19H, aromatic),
Chem. Eur. J. 2005, 11, 849 – 862
www.chemeurj.org
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
857

T. Takahashi et al.
10 min under argon to remove any trace amounts of water. The reaction
mixture was then cooled to ꢀ358C. N-iodosuccinimide (7.52 mg,
0.0334 mmol) and a catalytic amount of trifluoromethanesulfonic acid
(0.20 mL, 4.64 mmol, 0.10 equiv) were added to the reaction mixture at
ꢀ358C. After being stirred at the same temperature for 1 h, the reaction
mixture was neutralized with triethylamine and filtered through a pad of
celite. The filtrate was poured into a mixture of saturated aq. NaHCO3
and saturated aq. Na2S2O3 with cooling. The aqueous layer was extracted
with two portions of ethyl acetate. The combined extracts were washed
with saturated aq. NaHCO3/Na2S2O3 and brine, dried over MgSO4, fil-
tered, and concentrated in vacuo. The residue was purified by chromatog-
raphy on silica gel with chloroform/methanol (97:3) and further purified
by gel-permeation chromatography to give 9 (26.2 mg, 2.16 mmol, 93%,
a:b=78:22). The a:b ratio was determined by HPLC analysis (Senshu
Pak Silica-3301-N column; eluent: hexane/2-propanol 90:10; flow rate:
3.0 mLminꢀ1; retention times: a isomer=10.7 min, b isomer=11.7 min).
The a,b isomers were separated by chromatography on silica gel with tol-
uene/acetone (86:14) to give the a isomer and with toluene/acetone
(88:12) to give the b isomer.
The reaction mixture was then cooled to ꢀ308C. N-iodosuccinimide
(7.90 mg, 0.0352 mmol) and a catalytic amount of trifluoromethanesul-
fonic acid (0.50 mL, 8.80 mmol) were added to the reaction mixture at
ꢀ308C. After being stirred at the same temperature for 1 h, the reaction
mixture was neutralized with triethylamine and filtered through a pad of
celite. The filtrate was poured into a mixture of saturated aq. NaHCO3
and saturated aq. Na2S2O3 with cooling. The aqueous layer was extracted
with two portions of ethyl acetate. The combined extracts were washed
with saturated aq. NaHCO3/Na2S2O3 and brine, dried over MgSO4, fil-
tered, and concentrated in vacuo. The residue was purified by chromatog-
raphy on silica gel with chloroform/methanol (97:3) and further purified
by gel-permeation chromatography to give 11 (25.3 mg, 0.0145 mmol,
82%, a:b=6:94). The a–a:a–b ratio was determined by HPLC analysis
(Senshu Pak Silica-3301-N column; eluent: hexane/2-propanol 95:5; flow
rate: 3.0 mLminꢀ1; retention times: a–a isomer=12.9 min, a–b isomer=
16.1 min). The a–a,a–b isomers were separated by chromatography on
silica gel with toluene/acetone (91:9) to give the a–a isomer and with tol-
uene/acetone (90:10) to give the a–b isomer.
1
11a–b isomer: [a]2D1 +23.8 (c 1.36, CHCl3); H NMR (400 MHz, CDCl3):
1
9a isomer: [a]2D1 +43.4 (c 1.00, CHCl3); H NMR (400 MHz, CDCl3): d=
d=8.05 (d, 2H, aromatic), 7.13–7.57 (m, 33H, aromatic), 5.74 (dd, 1H,
Gal-H-2, J1,2 =7.3, J2,3 =8.2 Hz), 5.74 (d, 1H, Ser-NH, J=6.8 Hz), 5.39
(m, 1H, Neu-H-8), 5.35 (dd, 1H, Neu-H-7, J6,7 <1, J7,8 =8.3 Hz), 5.09–
7.30–7.60 (m, 15H, aromatic), 5.89 (d, 1H, Ser-NH, J=8.3 Hz), 5.40
(ddd, 1H, Neu-H-8), 5.36 (dd, 1H, Neu-H-7, J6,7 =1.4, J7,8 =8.8 Hz), 5.35
(d, 1H, Jgem =12.2 Hz), 5.14 (d, 1H, Jgem =12.6 Hz), 5.11 (2d, 2H), 5.00
(brdd, 1H, Neu-H-4, J3ax,4 =4.9, J3eq,4 =10.8 Hz), 4.94 (d, 1H, Neu-Troc-
5.17 (m, 4H), 5.05 (d, 1H, J=11.2 Hz), 4.97 (ddd, 1H, Neu-H-4, J3ax,4
=
12.2, J3aq,4 =4.9, J4,5 =9.6 Hz), 4.94 (d, 1H, J=11.7 Hz), 4.90 (d, 1H, J=
12.7 Hz), 4.87 (d, 1H, J=10.8 Hz), 4.77 (d, 1H, Gal-H-1, J1,2 =7.8 Hz),
4.69 (d, 1H, GalNAc-H-1, J1,2 =3.4 Hz), 4.67 (d, 1H, J=12.2 Hz), 4.61 (d,
1H, J=11.2 Hz), 4.58 (d, 1H, J=11.7 Hz), 4.56 (m, 1H, Ser-a), 4.53 (d,
1H, J=12.7 Hz), 4.46 (d, 1H, J=12.2 Hz), 4.45 (d, 1H, J=12.2 Hz), 4.29
(dd, 1H, Neu-H-9’, Jgem =11.2 Hz), 4.14 (dd, 1H, Neu-H-9, J8,9 =4.9,
NH, J=10.3 Hz), 4.90 (d, 1H,
11.7 Hz), 4.78 (d, 1H, GalNAc-H-1, J1,2 =3.4 Hz), 4.65 (d, 1H, Jgem
J
gem =12.2 Hz), 4.79 (d, 1H, Jgem
=
=
11.2 Hz), 4.60 (m, 1H, Ser-a), 4.36 (d, 1H, Jgem =12.2 Hz), 4.24 (d, 1H,
Neu-H-9’, J8,9’ =1.9, Jgem =12.7 Hz), 4.16 (brd, 1H, Neu-H-6, J=10.8 Hz),
4.06 (dd, 1H, Neu-H-9,
J8,9 =4.9, Jgem =12.7 Hz), 3.91–4.01 (m, 3H,
GalNAc-H-6’, Ser-b), 3.79–3.85 (m, 3H, GalNAc-H-3, H-4, H-6), 3.65–
3.69 (m, 4H, Neu-H-5, OMe), 3.52 (brdd, 1H, GalNAc-H-5), 3.36 (dd,
1H, GalNAc-H-2, J1,2 =3.0, J2,3 =10.3 Hz), 2.66 (dd, 1H, Neu-H-3eq,
Jgem =11.2 Hz), 4.13 (dd, 1H, Neu-H-6, J5,6 =9.8, J6,7 <1 Hz), 4.05 (brdd,
1H, Gal-H-4), 3.89–3.99 (m, 4H, Ser-b, GalNAc-H-3, H-6’), 3.67–3.69 (m,
6H, GalNAc-H-4, H-6, Gal-H-3, H-5, H-6, H-6’), 3.59–3.64 (m, 4H, Neu-
H-5, OMe), 3.50 (dd, 1H, GalNAc-H-2, J1,2 =3.0, J2,3 =11.2 Hz), 3.20
(ddd, 1H, GalNAc-H-5, J=4.9, J=9.3 Hz), 2.56 (dd, 1H, Neu-H-3eq,
J
3eq,4 =4.9, Jgem =13.2 Hz), 2.25 (d, 1H, GalNAc-3OH, J=8.8 Hz), 1.97,
2.00, 2.09, 2.11 (4s, 12H, Ac), 1.92 (dd, 1H, Neu-H-3ax, J3ax.4
=
10.8 Hz) ppm; 13C NMR (100 MHz, CDCl3): d=172.0, 170.7, 170.5, 169.8,
166.6, 156.0, 154.5, 138.0, 136.0, 134.9, 128.6, 128.6, 128.5, 128.4, 128.1,
127.9, 127.8, 98.7, 98.2, 95.5, 77.9, 77.2, 75.4, 74.4, 72.3, 71.8, 70.7, 69.2,
68.4, 68.2, 68.1, 67.9, 67.2, 63.3, 62.7, 60.4, 53.8, 52.7, 50.9, 37.0, 20.9, 20.8,
20.7, 20.6 ppm; IR (KBr): n˜ =3347, 2534, 2109, 1745, 1523, 1369, 1218,
J3eq,4 =9.3, Jgem =12.7 Hz), 1.97, 1.99, 2.07, 2.11 (4s, 12H, Ac), 1.84 (dd,
1H, Neu-H-3ax, J3ax,4 =12.2 Hz) ppm; 13C NMR (100 MHz, CDCl3): d=
170.6, 170.3, 170.1, 169.8, 169.6, 169.6, 167.7, 165.3, 156.0, 154.0, 138.6,
138.5, 137.7, 137.6, 136.2, 135.1, 132.8, 132.8, 130.2, 129.8, 128.7, 128.6,
128.5, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6,
127.4, 127.3, 102.6, 99.3, 98.4, 95.4, 79.7, 77.2, 76.8, 75.3, 74.5, 74.4, 74.3,
73.6, 73.4, 72.6, 72.1, 72.0, 71.8, 69.8, 68.7, 68.6, 68.4, 68.3, 67.5, 67.1, 63.8,
62.1, 59.4, 54.5, 52.7, 51.5, 37.7, 21.0, 20.7, 20.7 ppm; IR (KBr): n˜ =3347,
1038, 738, 738, 698 cmꢀ1
;
elemental analysis calcd (%) for
C52H60Cl3N5O22: C 41.47, H 4.98, N 5.77; found: C 51.03, H 5.02, N 5.47.
1
9b isomer: [a]2D2 +37.5 (c 1.00, CHCl3); H NMR (400 MHz, CDCl3): d=
2952, 2109, 1745, 1520, 1454, 1218, 1040, 737, 698 cmꢀ1
.
7.30–7.39 (m, 15H, aromatic), 6.36 (d, 1H, Ser-NH, J=8.8 Hz), 6.22 (d,
1H, Neu-Troc-NH, J=10.3 Hz), 5.54 (ddd, 1H, Neu-H-4, J3ax,4 =12.2,
11a–a isomer: 1H NMR (400 MHz, CDCl3): d=6.88–7.91 (m, 35H, aro-
J
3eq,4 =4.4 Hz), 5.47 (brs, 1H, Neu-H-7), 5.37 (d, 1H, Jgem =13.9 Hz), 5.34
matic), 5.89 (d, 1H, Ser-NH, J=7.9 Hz), 5.61 (dd, 1H, Gal-H-2, J1,2 =3.4,
(m, 1H, Neu-H-8), 5.12 (d, 1H, Jgem =13.9 Hz), 5.11 (2d, 2H), 4.96 (brd,
1H, Neu-H-9’, J=11.2 Hz), 4.89 (d, 1H, GalNAc-H-1, J1,2 =2.9 Hz), 4.76
(m, 1H, Ser-a), 4.75 (d, 1H, Jgem =12.2 Hz), 4.69 (2d, 2H), 4.53 (d, 1H,
J2,3 =10.3 Hz), 5.55 (d, 1H, Gal-H-1, J1,2 =3.4 Hz), 5.36 (brs, 2H, Neu-H-
7, H-8), 5.20 (d, 1H, Jgem =12.2 Hz), 5.10 (d, 1H, Jgem =12.3 Hz), 5.09
(2d, 2H), 4.91 (m, 3H, Neu-H-4), 4.81 (d, 1H, Neu-Troc-NH, J=
J
J
gem =11.7 Hz), 4.10 (m, 1H, Ser-b’), 4.03 (dd, 1H, Neu-H-9, J8,9 =8.8,
gem =12.7 Hz), 3.94 (m, 2H, Neu-H-6, H-5), 3.62 (m, 9H, Neu-H-5,
10.3 Hz), 4.79 (d, 1H, GalNAc-H-1, J1,2 =3.4 Hz), 4.63 (d, 1H, Jgem
12.3 Hz), 4.62 (2d, 2H), 4.57 (d, 1H, Jgem =12.0 Hz), 4.52 (d, 1H, Jgem
=
=
OMe, GalNAc-H-3, H-4, H-6’, H-6), 3.36 (dd, 1H, GalNAc-H-2, J1,2
=
11.7 Hz), 4.50 (m, 1H, Ser-a), 4.46 (d, 1H, Jgem =12.2 Hz), 4.42 (d, 1H,
gem =12.2 Hz), 3.98–4.26 (m, 8H, Neu-H-6, H-9’, H-9, GalNAc-H-3, H-4,
Gal-H-3, H-4, Ser-b’), 3.88 (dd, 1H, Ser-b, Ja,b =2.9, Jgem =12.2 Hz), 3.54–
3.81 (m, 7H, Neu-H-5, GalNAc-H-2, H-6’, H-6, Gal-H-5, H-6’, H-6), 3.25
(dd, 1H, GalNAc-H-5, J=8.3 Hz), 2.51 (dd, 1H, Neu-H-3eq, J3eq,4 =4.9,
2.9, J2,3 =10.8 Hz), 2.51 (dd, 1H, Neu-H-3eq, J3eq,4 =4.4, Jgem =13.9 Hz),
1.96 (d, 1H, GalNAc-3OH), 1.91, 1.97ꢂ2, 2.01 (3 s, 12H, Ac), 1.83 (dd,
1H, Neu-H-3ax, J3ax,4 =12.2 Hz) ppm; 13C NMR (100 MHz, CDCl3): d=
170.7, 170.3, 170.0, 169.8, 169.7, 167.7, 156.0, 154.0, 138.0, 156.0, 138.0,
136.1, 135.1, 128.5, 128.5, 128.1, 128.0, 128.0, 127.8, 99.3, 98.4, 95.3, 76.6,
75.1, 74.4, 71.9, 69.6, 69.1, 68.3, 68.0, 67.9, 67.5, 67.3, 67.0, 63.0, 62.2, 60.8,
54.4, 52.8, 51.4, 37.7, 21.0, 20.8, 20.7, 20.6 ppm; IR (KBr): n˜ =3368, 2955,
J
J
gem =12.2 Hz), 1.97, 2.03, 2.06ꢂ2 (3s, 12H, Ac), 1.76 (dd, 1H, Neu-H-
3ax, J3ax,4 =11.2 Hz) ppm; 13C NMR (100 MHz, CDCl3): d=170.6, 170.3,
170.1, 169.6, 169.6, 167.5, 166.4, 156.1, 154.0, 138.5, 138.1, 138.0, 136.3,
135.3, 133.1, 130.9, 129.9, 129.5, 128.8, 128.6, 128.5, 128.4, 128.3, 128.2,
128.1, 128.1, 127.9, 127.9, 127.6, 127.1, 126.7, 99.3, 98.3, 95.4, 94.7, 77.2,
74.9, 74.5, 74.5, 74.1, 73.9, 73.5, 73.0, 72.8, 71.9, 71.7, 69.7, 69.1, 68.4, 68.3,
68.1, 68.1, 67.3, 67.3, 67.0, 62.5, 62.0, 59.4, 54.5, 52.7, 51.5, 38.0, 21.0, 20.8,
20.8, 20.7 ppm; IR (KBr): n˜ =3473, 2850, 2115, 1707, 1554, 1493, 1212,
2110, 1744, 1530, 1370, 1223, 1036, 944, 736, 696 cmꢀ1
.
N-(Benzyloxycarbonyl) 3-O-(2-azido-4-O-benzyl-2-deoxy-3-O-(2-O-ben-
zoyl-3,4,6-tri-O-benzyl-d-galactopyranosyl)-6-O-(methyl 4,7,8,9-tetra-O-
acetyl-3,5-dideoxy-5-(2,2,2-trichloroethoxycarbonylamino)-d-glycero-a-d-
galacto-2-nonulopyranosylonate)-a-d-galactopyranosyl)-l-serine benzyl
ester (11): A mixture of 9a (21.4 mg, 0.0176 mmol), phenylthio 2-O-ben-
zoyl-3,4,6-tri-O-benzyl-b-d-galactopyranoside (8a; 17.1 mg, 0.0265 mmol;
azeotroped three times with toluene), and pulverized activated 3 ꢀ MS
(8.80 mg) in dry CH3CN/CH2Cl2 (1:9; 2.00 mL) was stirred at room tem-
perature for 10 min under argon to remove any trace amounts of water.
1059, 594, 690 cmꢀ1
One-pot glycosylation procedure for the synthesis of N-(benzyloxycar-
bonyl) 3-O-(2-azido-4-O-benzyl-2-deoxy-3-O-(2-O-benzoyl-3,4,6-tri-O-
benzyl-b-d-galactopyranosyl)-6-O-(methyl 4,7,8,9-tetra-O-acetyl-3,5-di-
deoxy-5-(2,2,2-trichloroethoxycarbonylamino)-d-glycero-d-galacto-2-non-
.
858
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 849 – 862

Sialo-Containing Glycosyl Amino Acids
FULL PAPER
ulopyranosylonate)-a-d-galactopyranosyl)-l-serine benzyl ester (11): A
mixture of thiosialoside 3 f (24.0 mg, 0.0335 mmol), N-(benzyloxycar-
bonyl)-3-O-(2-azido-4-O-benzyl-2-deoxy-a-d-galactopyranosyl)-d-serine
benzyl ester (7; 16.9 mg, 0.0279 mmol; azeotroped three times with tolu-
ene), and pulverized activated 3 ꢀ MS (14.0 mg) in dry CH3CN
(0.140 mL) was stirred at room temperature for 30 min under argon to
remove any trace amounts of water. The reaction mixture was then
cooled to ꢀ358C. N-iodosuccinimide (9.40 mg, 0.0419 mmol) and a cata-
lytic amount of trifluoromethanesulfonic acid (0.50 mL) were added to
the reaction mixture at ꢀ358C. After being stirred at the same tempera-
ture for 1 h, the reaction mixture was warmed to ꢀ308C. A solution of
phenylthio 2-O-benzoyl-3,4,6-tri-O-benzyl-b-d-galactopyranoside (8a;
32.5 mg, 0.0502 mmol; azeotroped three times with toluene) in dry
CH2Cl2 (1.25 mL) was added to the reaction mixture. After 10 min, N-io-
dosuccinimide (17.0 mg, 0.0753 mmol) and a catalytic amount of trifluor-
omethanesulfonic acid (0.50 mL, 5.58 mmol) were added to the reaction
mixture at ꢀ308C. After being stirred at the same temperature, the reac-
tion mixture was neutralized with triethylamine and filtered through a
pad of celite. The filtrate was poured into a mixture of saturated aq.
NaHCO3 and saturated aq. Na2S2O3 with cooling. The aqueous layer was
extracted with two portions of ethyl acetate. The combined extracts were
washed with saturated aq. NaHCO3/Na2S2O3 and brine, dried over
MgSO4, filtered, and concentrated in vacuo. The residue was purified by
chromatography on silica gel with chloroform/methanol (97:3) and fur-
ther purified by gel-permeation chromatography to give 11 (37.3 mg,
0.0214 mmol, 77%, a:b=78:22). The a:b ratio was determined by HPLC
analysis (Eluent: hexane/2-propanol 95:5; flow rate: 3.0 mLminꢀ1; reten-
tion times: a isomer=15.2 min, b isomer=12.4 min). The a isomer was
separated by chromatography on silica gel with toluene/acetone (92:8).
being stirred at the same temperature for 4 h, H2O (0.30 mL) was added
and the reaction mixture was neutralized with Dowex 50W-4X. After fil-
tration and evaporation of the solvent, the residue was purified by re-
versed-phase column chromatography (Bond Elut-C18 column) to give
1a (5.28 mg, 6.94 mmol, 85% over two steps); [a]2D6 +67.0 (c 0.16, H2O);
1H NMR (400 MHz, D2O, 303 K): d=4.88 (d, 1H, GalNAc-H-1, J1,2
3.9 Hz), 4.43 (d, 1H, Gal-H-1, J1,2 =7.8 Hz), 4.31 (dd, 1H, GalNAc-H-2,
1,2 =3.4, J2,3 =11.2 Hz), 4.22 (brd, GalNAc-H-4, J=2.9 Hz), 4.10 (dd,
=
J
1H, Ser-b’, Ja,b’ =2.4, Jgem =10.7 Hz), 4.04 (dd, 1H, GalNAc-H-3, J2,3
11.2, J3,4 =2.9 Hz), 4.01 (m, 1H, GalNAc-H-5), 3.97 (dd, 1H, Ser-a, Ja,b’
=
=
2.4, Ja,b =4.9 Hz), 3.90 (dd, 1H, Ser-b, Ja,b =4.9, Jgem =10.7 Hz), 3.61–3.88
(12H, Neu5Ac-H-4, H-5, H-6, H-7, H-8, H-9’, H-9, GalNAc-H-6’, H-6,
Gal-H-5, H-6’), 3.58 (dd, 1H, Gal-H-3, J2,3 =10.3, J3,4 =3.9 Hz), 3.55 (dd,
1H, Gal-H-6, J5,6 =1.0, Jgem =10.3 Hz), 3.48 (dd, 1H, Gal-H-2, J1,2 =7.8,
J
2,3 =10.3 Hz), 2.70 (dd, 1H, Neu5Ac-H-3eq, J3eq,4 =4.9, Jgem =12.7 Hz),
2.00, 2.01 (2s, 6H, Ac), 1.65 (dd, 1H, Neu5Ac-H-3ax, J3ax,4
=
12.2 Hz) ppm; 13C NMR (100 MHz, D2O, acetone-d6): d=175.8, 175.4,
173.9, 172.0, 105.5, 100.9, 99.1, 77.4, 75.8, 73.5, 73.4, 72.4, 71.4, 70.3, 69.4,
69.3, 69.7, 68.9, 67.3, 64.6, 63.5, 61.8, 54.8, 52.6, 49.2, 40.9, 22.9ꢂ2 ppm;
IR (KBr): n˜ =3330, 1641, 1572, 1393, 1121, 1055, 930, 669 cmꢀ1; HRMS
(ESI-TOF): calcd for C28H48N3O21 [M+H]+: 762.2775; found: 762.2772.
2-O-Benzoyl-4,6-O-benzylidene-3-O-(methyl 4,7,8,9-tetra-O-acetyl-3,5-di-
deoxy-5-(2,2,2-trichloroethoxycarbonylamino)-d-glycero-d-galacto-2-non-
ulopyranosylonate)-b-d-galactopyranosyl fluoride (15): A mixture of thi-
osialoside 3 f (45.0 mg, 0.0627 mmol), 2-O-benzoyl-4,6-O-benzylidene-b-
d-galactopyranosyl fluoride (13a; 15.6 mg, 0.0418 mmol; azeotroped
three times with toluene), and pulverized activated 3 ꢀ MS (21.0 mg) in
dry CH3CN/CH2Cl2 (9:13; 0.22 mL) was stirred at room temperature for
10 min under argon to remove any trace amounts of water. The reaction
mixture was then cooled to ꢀ788C. N-iodosuccinimide (18.8 mg,
0.0836 mmol) and a catalytic amount of trifluoromethanesulfonic acid
(1.10 mL, 0.0125 mmol) were added to the reaction mixture at ꢀ788C.
After being stirred at the same temperature for 1.5 min, the reaction mix-
ture was neutralized with triethylamine and filtered through a pad of
celite. The filtrate was poured into a mixture of saturated aq. NaHCO3
and saturated aq. Na2S2O3 with cooling. The aqueous layer was extracted
with two portions of ethyl acetate. The combined extracts were washed
with saturated aq. NaHCO3/Na2S2O3 and brine, dried over MgSO4, fil-
tered, and concentrated in vacuo. The residue was purified by chromatog-
raphy on silica gel with chloroform/methanol (97:3) and further purified
by gel-permeation chromatography to give 15 (35.2 mg, 0.0360 mmol,
86%, a:b=93:7). The a:b ratio was determined by HPLC analysis
(Senshu Pak Silica-3301-N column; eluent: hexane/2-propanol 89:11;
flow rate: 3.0 mLminꢀ1; retention times: a isomer=20.6 min, b isomer=
22.7 min). The a,b isomers were separated by chromatography on silica
gel with toluene/acetone (86:14) to give the a isomer and with toluene/
acetone (88:12) to give the b isomer.
N-(Benzyloxycarbonyl) 3-O-(2-acetamido-4-O-benzyl-2-deoxy-3-O-(2-O-
benzoyl-3,4,6-tri-O-benzyl-b-d-galactopyranosyl)-6-O-(methyl 5-acetami-
do-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-d-glycero-a-d-galacto-2-nonulopyra-
nosylonate)-a-d-galactopyranosyl)-l-serine benzyl ester (12): Activated
zinc dust (30 mg) was added to a solution of trisaccharide 11a (15.0 mg,
8.57 mmol) in dry THF/AcOH/Ac2O (3:2:1; 0.60 mL) at 08C. After being
stirred at the same temperature for 1 h, the reaction mixture was filtered
through a pad of celite. The filtrate was concentrated in vacuo. The resi-
due was purified by chromatography on silica gel with chloroform/metha-
nol (98:2) to give 12 (12.8 mg, 7.85 mmol, 92%); [a]2D7 +24.8 (c 0.985,
CHCl3); 1H NMR (400 MHz, CDCl3): d=8.00 (d, 2H, aromatic), 7.14–
7.57 (m, 33H, aromatic), 5.74 (d, 1H, Ser-NH, J=11.2 Hz), 5.73 (d, 1H,
GalNAc-NH, J=7.3 Hz), 5.68 (dd, 1H, Gal-H-2, J1,2 =7.8, J2,3 =9.8 Hz),
5.38 (m, 1H, Neu5Ac-H-8), 5.30 (dd, 1H, Neu5Ac-H-7, J6,7 =2.0, J7,8
7.8 Hz), 5.16 (m, 2H, Neu5Ac-NH), 5.09 (m, 2H), 5.03 (d, 1H, Jgem
=
=
12.2 Hz), 4.99 (d, 1H,
Jgem =12.2 Hz), 4.84 (m, 2H, Neu5Ac-H-4,
GalNAc-H-1), 4.76 (d, 1H, Gal-H-1, J1,2 =7.8 Hz), 4.38–4.68 (m, 6H,
GalNAc-H-2, Gal-H-4, H-5, H-6’, H-6, Ser-a), 4.31 (dd, 1H, Neu5Ac-H-
9’, J8,9’ =2.4, Jgem =12.7 Hz), 4.11 (m, 1H, Neu5Ac-H-9, J8,9 =5.8, Jgem
=
15a isomer: [a]2D3 +59.6 (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3):
d=8.13 (d, 2H, aromatic), 7.34–7.62 (m, 8H, aromatic), 5.51–5.63 (m,
3.5H, Neu-H-8, Gal-H-1’, Gal-H-2), 5.45 (d, 0.5H, Gal-H-1, J1,2 =7.3 Hz),
5.40 (s, 1H, benzylic-H), 5.31 (dd, 1H, Neu-H-7, J6,7 =1.4, J7,8 =9.8 Hz),
4.85 (d, 1H, Jgem =12.2 Hz), 4.83 (m, 1H, Neu-H-4), 4.75 (d, 1H, Neu-
Troc-NH, J=9.8 Hz), 4.61 (dd, 1H, Gal-H-3, J2,3 =10.3, J3,4 =3.4 Hz), 4.44
(d, 1H, Jgem =12.2 Hz), 4.37 (brd, 1H, Gal-H-6’, J=12.7 Hz), 4.32 (dd,
1H, Neu-H-9’, J8,9 =2.4, Jgem =12.7 Hz), 4.17 (brd, 1H, Gal-H-6, J=
12.2 Hz), 3.51–4.05 (m, 9H, Neu5Ac-H-5, H-6, GalNAc-H-3, H-4, H-6’,
H-6, Gal-H-3, Ser-b’, Ser-b), 3.48 (s, 3H, Neu5Ac-OMe), 3.27 (dd, 1H,
GalNAc-H-5), 2.52 (dd, 1H, Neu5Ac-H-3eq, J3eq,4 =4.9, Jgem =12.2 Hz),
2.00 (m, 1H, Neu5Ac-H-3ax), 1.87, 1.98ꢂ2, 2.02, 2.07, 2.10 (6s, 18H,
Ac) ppm; 13C NMR (100 MHz, CDCl3): d=170.9, 170.6, 170.2ꢂ2, 170.0,
169.9, 167.8, 165.1, 155.9, 138.9, 138.2, 137.5, 137.4, 136.1, 134.9, 133.1,
130.1, 130.0, 129.9, 128.7, 128.6, 128.5, 128.4, 128.4, 128.3, 128.2, 128.2,
128.1, 128.1, 128.0, 128.0, 127.9, 127.8, 127.7, 127.6, 127.6, 126.9, 100.9,
98.8, 98.6, 79.7, 72.2, 75.1, 74.8, 74.2, 74.0, 73.5, 72.6, 72.1, 71.7, 69.5, 69.1,
69.0, 68.8, 68.3, 67.5, 67.4, 67.2, 63.2, 62.3, 54.6, 52.6, 49.4, 37.7, 23.2, 22.6,
21.0, 20.8, 20.8, 20.7 ppm; IR (KBr): n˜ =3358, 2923, 2856, 1732, 1660,
12.7 Hz), 4.05 (brs, 1H, Gal-H-4), 4.03 (dd, 1H, Neu-H-9, J8,9 =6.3, Jgem
=
12.2 Hz), 3.97 (dd, 1H, Neu-H-6, J5,6 =10.8, J6,7 =2.0 Hz), 3.71 (brs, 1H,
Gal-H-5), 3.59 (s, 3H, Neu-OMe), 3.52 (ddd, 1H, Neu-H-5, J5,6 =10.8,
1530, 1455, 1368, 1213, 1036, 736, 697 cmꢀ1
.
J
5,NH =9.8 Hz), 2.68 (dd, 1H, Neu-H-3eq, J3eq,4 =4.4, Jgem =12.2 Hz), 1.83,
1.94, 2.07, 2.22 (4s, 12H, Ac), 1.66 (dd, 1H, Neu-H-3ax, J3ax,4
=
3-O-(2-Acetamido-2-deoxy-3-O-(b-d-galactopyranosyl)-6-O-(5-acetami-
do-3,5-dideoxy-d-glycero-a-d-galacto-2-nonulopyranosylonic acid)-a-d-
galactopyranosyl)-l-serine (1a): Pd(OH)2 (30 mg) was added to a so-
lution of 12 (13.3 mg, 8.15 mmol) in THF/MeOH/AcOH/H2O
(100:80:10:7; 1.97 mL). The reaction mixture was hydrogenolyzed for 2 h
under an H2 atmosphere. The reaction mixture was filtered and the fil-
trate was concentrated in vacuo. The residue was used for the next reac-
tion without further purification. 0.1m aq. NaOH (350 mL) was added to
a stirred solution of the residue in methanol (1.20 mL) at 08C. After
12.7 Hz) ppm; 13C NMR (100 MHz, CDCl3): d=170.8, 170.4, 170.2, 169.9,
168.5, 165.0, 154.0, 137.4, 133.2, 129.9, 129.8, 129.0, 128.3, 128.1, 126.3,
(108.6, 106.4 (JC-F =217 Hz)), 100.9, 96.5, 95.2, 74.4, 72.7, 71.9, 71.3, 71.2,
70.3, 70.0, 68.6, 68.2, 67.3, 67.0, 66.5, 66.4, 62.5, 52.8, 51.2, 38.3, 21.4, 20.7,
20.7, 20.7 ppm; 19F NMR (376 MHz, CDCl3): d=ꢀ144.2 (dd, J1,F =55.0,
J
2,F =14.7 Hz) ppm; IR (KBr): n˜ =3319, 2959, 1743, 1715, 1549, 1337,
1265, 1097, 821, 737, 716 cmꢀ1
C41H45Cl3FNO19: C 50.19, H 4.62, N 1.43; found: C 49.99, H 4.71, N 1.39;
; elemental analysis calcd (%) for
Chem. Eur. J. 2005, 11, 849 – 862
www.chemeurj.org
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
859

T. Takahashi et al.
HRMS (ESI-TOF): calcd for C41H45Cl3NO19Na [M+Na]+: 1004.1533;
5.00,
N
4.35; MS (ESI-TOF): calcd for C72H76Cl3N5O28 [M+NH4]+:
found: 1004.1528.
1583.4; found: 1583.4.
15b isomer: [a]2D4 +81.2 (c 1.00, CHCl3); 1H NMR (400 MHz, CDCl3):
d=8.03 (d, 2H, aromatic), 7.39–7.65 (m, 8H, aromatic), 5.68 (s, 1H, ben-
One-pot glycosylation procedure for the synthesis of N-(benzyloxycar-
bonyl)-3-O-(2-azido-4,6-O-benzylidene-2-deoxy-3-O-(2-O-benzoyl-4,6-O-
zylic-H), 5.65 (m, 1H, Gal-H-2), 5.45 (dd, 1H, Gal-H-1, J1,2 =7.8, JH-F
53.5 Hz), 5.34 (ddd, 1H, Neu-H-8, J7,8 =2.9, J8,9’ =2.9, J8,9 =8.8 Hz), 5.27
(brs, 1H, Neu-H-7), 5.10 (ddd, 1H, Neu-H-4, J3ax,4 =12.7, J3eq,4 =4.4, J4,5
10.8 Hz), 5.06 (dd, 1H, Neu-H-9’, J8,9’ =2.9, Jgem =12.6 Hz), 4.83 (d, 1H,
gem =12.2 Hz), 4.49 (brs, 1H, Gal-H-4), 4.41 (brd, 1H, Gal-H-6’, J=
11.7 Hz), 4.38 (d, 1H, Jgem =12.2 Hz), 4.29 (dd, 1H, Gal-H-3, J2,3 =10.3,
3,4 =3.9 Hz), 4.17 (brd, 1H, Gal-H-6, J=11.7 Hz), 4.03 (dd, 1H, Neu-H-
6, 5,6 =12.7, 6,7 =2.4 Hz), 3.90 (dd, 1H, Neu-H-9, 8,9 =8.8, Jgem =
=
benzylidene-3-O-(methyl
4,7,8,9-tetra-O-acetyl-3,5-dideoxy-5-(2,2,2-tri-
chloroethoxycarbonylamino)-d-glycero-d-galacto-2-nonulopyranosylo-
nate)-b-d-galactopyranosyl)-a-d-galactopyranosyl)-l-serine benzyl ester
(16): A mixture of thiosialoside 3 f (43.1 mg, 0.0602 mmol), 2-O-benzoyl-
=
J
4,6-O-benzylidene-b-d-galactopyranosyl
fluoride
(13a;
15.0 mg,
0.0401 mmol; azeotroped three times with toluene), and pulverized acti-
vated 3 ꢀ MS (20.0 mg) in dry CH3CN/CH2Cl2 (2:3; 0.50 mL) was stirred
at room temperature for 30 min under argon to remove any trace
amounts of water. The reaction mixture was then cooled to ꢀ788C. N-io-
dosuccinimide (18.3 mg, 0.0812 mmol) and a catalytic amount of trifluor-
omethanesulfonic acid (1.10 mL, 0.0120 mmol, 0.30 equiv) were added to
the reaction mixture at ꢀ788C. After being stirred at the same tempera-
ture for 1.5 min, the reaction mixture was warmed to ꢀ58C. N-(Benzyl-
oxycarbonyl)-3-O-(2-azido-4,6-O-benzylidene-2-deoxy-a-d-galactopyra-
nosyl)-l-serine benzyl ester (14; 36.4 mg, 0.0602 mmol) was added to the
reaction mixture. After 10 min, ZrCp2Cl2 (23.4 mg, 0.0802 mmol) and
silver trifluoromethanesulfonate (41.2 mg, 0.1604 mmol) were added to
the reaction mixture at ꢀ58C. After being stirred at the same tempera-
ture for 2 h, the reaction mixture was neutralized with triethylamine and
filtered through a pad of celite. The filtrate was poured into a mixture of
saturated aq. NaHCO3 and saturated aq. Na2S2O3 with cooling. The aque-
ous layer was extracted with two portions of ethyl acetate. The combined
extracts were washed with saturated aq. NaHCO3/Na2S2O3 and brine,
dried over MgSO4, filtered, and concentrated in vacuo. The residue was
purified by chromatography on silica gel with chloroform/methanol
(97:3) and further purified by gel-permeation chromatography to give 16
(55.5 mg, 0.0355 mmol, 88%, a:b=93:7). The a,b isomers were separated
by chromatography on silica gel with toluene/acetone (90:10) to give the
a isomer and with toluene/acetone (92:8) to give the b isomer.
J
J
J
J
12.6 Hz), 3.78 (brs, 1H, Gal-H-5), 3.71 (ddd, 1H, Neu-H-5), 3.36 (s, 3H,
Neu-OMe), 2.58 (dd, 1H, Neu-H-3eq, J3eq,4 =4.4, Jgem =13.7 Hz), 1.97,
2.05, 2.09ꢂ2 (3s, 12H, Ac), 1.77 (dd, 1H, Neu-H-3ax, J3ax,4
=
12.7 Hz) ppm; 13C NMR (100 MHz, CDCl3): d=171.1, 170.8, 170.3, 169.7,
166.9, 164.7, 154.3, 137.3, 133.8, 129.8, 129.7, 129.5, 128.6, 126.1, (108.1,
105.1 (JC-F =217 Hz)), 99.2, 95.3, 74.5, 73.8, 73.7, 73.5, 73.0, 71.5, 70.6,
70.3, 68.9, 68.7, 68.1, 66.7, 66.7, 62.6, 52.7, 51.2, 36.5, 20.9, 20.8, 20.8,
20.7 ppm; 19F NMR (376 MHz, CDCl3): d=ꢀ144.4 (dd, J1,F =57.4, J2,F
=
13.4 Hz) ppm; IR (KBr): n˜ =3397, 2952, 2896, 1742, 1516, 1369, 1238,
1218, 1095, 1033, 709 cmꢀ1
;
elemental analysis calcd (%) for
C41H45Cl3FNO19: C 50.19, H 4.62, N 1.43; found: C 49.83, H 4.62, N 1.46;
HRMS (ESI-TOF): calcd for C41H45Cl3NO19Na [M+Na]+: 1004.1533;
found: 1002.1528.
N-(Benzyloxycarbonyl)-3-O-(2-azido-4,6-O-benzylidene-2-deoxy-3-O-(2-
O-benzoyl-4,6-O-benzylidene-3-O-(methyl 4,7,8,9-tetra-O-acetyl-3,5-di-
deoxy-5-(2,2,2-trichloroethoxycarbonylamino)-d-glycero-a-d-galacto-2-
nonulopyranosylonate)-b-d-galactopyranosyl)-a-d-galactopyranosyl)-l-
serine benzyl ester (16):
A mixture of disaccharide 15a (20.0 mg,
0.0204 mmol), N-(benzyloxycarbonyl)-3-O-(2-azido-4,6-O-benzylidene-2-
deoxy-a-d-galactopyranosyl)-l-serine benzyl ester (14; 16.0 mg,
0.0265 mmol; azeotroped three times with toluene), and pulverized acti-
vated 3 ꢀ MS (60.0 mg) in dry CH3CN/CH2Cl2 (2:3; 0.25 mL) was stirred
at room temperature for 10 min under argon to remove any trace
amounts of water. The reaction mixture was then cooled to ꢀ58C.
ZrCp2Cl2 (9.00 mg, 0.0309 mmol) and silver trifluoromethanesulfonate
(15.7 mg, 0.0612 mmol) were added to the reaction mixture. After being
stirred at the same temperature for 2 h, the reaction mixture was neutral-
ized with triethylamine and filtered through a pad of celite. The filtrate
was poured into saturated aq. NaHCO3 with cooling. The aqueous layer
was extracted with two portions of ethyl acetate. The combined extracts
were washed with saturated aq. NaHCO3 and brine, dried over MgSO4,
filtered, and concentrated in vacuo. The residue was purified by chroma-
tography on silica gel with toluene/acetone (90:10) to give 16 (30.6 mg,
0.0195 mmol, 96%); [a]D24 +63.4 (c 1.00, CHCl3); 1H NMR (400 MHz,
CDCl3): d=8.11 (d, 2H, aromatic), 7.18–7.54 (m, 23H, aromatic), 5.86
(d, 1H, Ser-NH, J=8.3 Hz), 5.52 (m, 2H, Neu-H-8, Gal-H-2), 5,46 (s,
N-(Benzyloxycarbonyl)-3-O-(2-acetamido-4,6-O-benzylidene-2-deoxy-3-
O-(2-O-benzoyl-4,6-O-benzylidene-3-O-(methyl
5-acetamido-4,7,8,9-
tetra-O-acetyl-3,5-dideoxy-d-glycero-a-d-galacto-2-nonulopyranosylo-
nate)-b-d-galactopyranosyl)-a-d-galactopyranosyl)-l-serine benzyl ester
(17): Activated zinc dust (100 mg) was added to a solution of trisacchar-
ide 16a (113 mg, 0.0722 mmol) in dry THF/AcOH/Ac2O (3:2:1)
(1.20 mL) at 08C. After being stirred at the same temperature for 1 h,
the reaction mixture was filtered through a pad of celite. The filtrate was
concentrated in vacuo. The residue was purified by chromatography on
silica gel with chloroform/methanol (97:3) to give 17 (97.4 mg,
0.0672 mmol, 93%); [a]D27 +68.3 (c 1.00, CHCl3); 1H NMR (400 MHz,
CDCl3): d=8.12 (d, 2H, aromatic), 7.11–7.60 (m, 23H, aromatic), 5.96
(d, 1H, GalNAc-NH, J=6.4 Hz), 5.91 (d, 1H, Ser-NH, J=8.8 Hz), 5.52
(m, 2H, Neu5Ac-H-8, Gal-H-2), 5.39 (s, 1H, benzylic-H), 5.26 (s, 1H,
benzylic-H), 5.21 (dd, 1H, Neu5Ac-H-7), 5.17 (d, 1H, Gal-H-1, J1,2
=
1H, benzylic-H), 5.38 (s, 1H, benzylic-H), 5.32 (dd, 1H, Neu-H-7, J6,7
1.4, J7,8 =9.3 Hz), 5.16 (2d, 2H), 5.11 (2d, 2H), 5.07 (d, 1H, Gal-H-1,
1,2 =7.8 Hz), 4.85 (d, 1H, Jgem =9.3 Hz), 4.83 (d, 1H, GalNAc-H-1, J1,2
=
8.2 Hz), 5.13 (d, 1H, Jgem =11.2 Hz), 5.12 (brs, 1H, GalNAc-H-1), 5.07
(d, 1H, Neu5Ac-NH, J=10.2 Hz), 5.06 (d, 1H, Jgem =11.2 Hz), 5.05 (d,
1H, Jgem =12.2 Hz), 5.00 (d, 1H, Jgem =11.2 Hz), 4.74 (m, 1H, Neu5Ac-H-
4), 4.58 (dd, 1H, Gal-H-3, J2,3 =9.8, J3,4 =2.9 Hz), 4.23–4.38 (m, 6H,
Neu5Ac-H-9’, Gal-H-6’, GalNAc-H-2, H-3, H-4, Ser-a), 4.14 (2brd, 2H),
3.81–4.05 (m, 6H, Neu5Ac-H-5, H-6, H-9, Gal-H-4, GalNAc-H-6, Ser-b’),
3.72 (brd, 1H, Ser-b, J=11.7 Hz), 3.70 (brs, 1H, Gal-H-5), 3.62 (s, 3H,
Neu-OMe), 3.42 (brs, 1H, GalNAc-H-5), 2.60 (dd, 1H, Neu5Ac-H-3eq,
J
=
2.9 Hz), 4.80 (m, 1H, Neu-H-4), 4.72 (d, 1H, Neu-Troc-NH, J=10.2 Hz),
4.57 (dd, 1H, Gal-H-3, J2,3 =10.2, J3,4 =3.4 Hz), 4.52 (m, 1H, Ser-a), 4.43
(d, 1H, Jgem =12.7 Hz), 4.40 (brs, 1H, GalNAc-H-4), 4.31 (2brdd, 2H,
Gal-H-6’, Neu-H-9’), 4.16 (brd, 1H, Gal-H-6), 4.00–4.11 (m, 5H, Neu-H-
9, Gal-H-4, GalNAc-H-3, H-6’, Ser-b’), 3.99 (brd, 1H, Neu-H-6, J=
10.7 Hz), 3.93 (dd, 1H, Ser-b, Ja,b =2.4, Jgem =10.8 Hz), 3.84 (brd, 1H,
GalNAc-H-6, J=12.2 Hz), 3.66 (brs, 1H, Gal-H-5), 3.62 (dd, 1H,
GalNAc-H-2, J1,2 =3.4, J2,3 =10.8 Hz), 3.55 (s, 3H, Neu-OMe), 3.44–3.51
(m, 2H, Neu-H-5, GalNAc-H-5), 2.65 (dd, 1H, Neu-H-3eq, J3eq,4 =4.4,
J
3eq,4 =4.4, Jgem =12.7 Hz), 1.71, 1.81ꢂ2, 1.94, 2.04, 2.22 (6 s, 18H, Ac),
1.72 (dd, 1H, Neu5Ac-H-3ax) ppm; 13C NMR (100 MHz, CDCl3): d=
170.8, 170.8, 170.4, 170.2, 170.1ꢂ2, 169.8, 168.7, 165.3, 156.1, 137.5, 137.5,
136.3, 135.0, 133.3, 130.0, 129.8, 129.2, 128.6, 128.6, 128.5, 128.4, 128.2,
128.1, 128.0, 127.8, 126.4, 126.1, 101.3, 100.7, 99.7, 99.1, 96.8, 77.2, 75.0,
73.5, 72.2, 72.0, 69.9, 69.5, 69.4, 69.2, 68.7, 67.5, 66.9, 66.9, 66.4, 63.4, 62.7,
54.8, 52.8, 49.0, 48.7, 38.1, 23.1, 22.9, 21.4, 20.8, 20.7, 20.4 ppm; IR (KBr):
n˜ =3356, 2955, 1723, 1661, 1518, 1453, 1369, 1269, 1031, 696 cmꢀ1; HRMS
(ESI-TOF): calcd for C73H81N3O28Na [M+Na]+: 1470.4899; found:
1470.4899.
J
gem =12.7 Hz), 1.83, 1.92, 2.06, 2.22 (4s, 12H, Ac), 1.62 (dd, 1H, Neu-H-
3ax, J3ax,4 =12.7 Hz) ppm; 13C NMR (100 MHz, CDCl3): d=170.8, 170.3,
170.2, 170.0, 169.6, 168.7, 165.1, 155.9, 154.0, 137.8, 137.5, 136.1, 135.0,
132.8, 130.4, 129.8, 129.0, 128.6, 128.6, 128.4, 128.3, 128.2, 127.9, 126.4,
126.0, 101.4, 100.9, 100.4, 100.2, 96.5, 95.3, 77.2, 75.6, 74.5, 73.1, 72.9, 72.2,
71.9, 70.2, 69.8, 69.1, 68.8, 68.2, 67.7, 67.5, 67.1, 67.0, 66.2, 63.6, 62.3, 58.6,
54.6, 52.7, 51.3, 38.4, 21.4, 20.8, 20.7, 20.5 ppm; IR (KBr): n˜ =3353, 2951,
2110, 1736, 1521, 1367, 1211, 1088, 1037, 741, 698 cmꢀ1; elemental analysis
calcd (%) for C72H76Cl3N5O28: C 55.23, H 4.89, N 4.47; found: C 55.01, H
3-O-(2-Acetamido-2-deoxy-3-O-(3-O-(5-acetamido-3,5-dideoxy-d-glyc-
ero-a-d-galacto-2-nonulopyranosylonic acid)-b-d-galactopyranosyl)-a-d-
860
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Chem. Eur. J. 2005, 11, 849 – 862

Sialo-Containing Glycosyl Amino Acids
FULL PAPER
galactopyranosyl)-l-serine (2a): Pd(OH)2 (30 mg) was added to a so-
lution of trisaccharide 17 (26.0 mg, 0.0180 mmol) in THF/MeOH/AcOH/
H2O (100:80:7:10; 1.97 mL). The reaction mixture was hydrogenolyzed
for 2 h under an H2 atmosphere. The reaction mixture was filtered and
the filtrate was concentrated in vacuo. The residue was used for the next
reaction without further purification. 0.1m aq. NaOH (120 mL) was added
to a stirred solution of the residue in methanol (1.00 mL) at 08C. After
being stirred at the same temperature for 4 h, H2O (0.35 mL) was added
and the reaction mixture was neutralized with Dowex 50W-4X. After fil-
tration and evaporation of the solvent, the residue was purified by re-
versed-phase column chromatography (Bond Elut-C18 column) to give
2a (12.3 mg, 0.0162 mmol, 90% over two steps); 1H NMR (400 MHz,
D2O, 303 K): d=4.90 (d, 1H, GalNAc-H-1, J1,2 =2.4 Hz), 4.50 (d, 1H,
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Gal-H-1,
J1,2 =7.8 Hz), 4.32 (dd, 1H, GalNAc-H-2, J1,2 =2.4, J2,3 =
10.3 Hz), 4.20 (brs, 1H, GalNAc-H-4), 4.11 (brd, 1H, Ser-b’, J=11.2 Hz),
4.02–4.05 (m, 2H, Gal-H-3, GalNAc-H-3), 3.57–4.01 (m, 16H), 3.50 (dd,
1H, Gal-H-2,
J
J
J
1,2 =7.8,
3eq,4 =4.9, Jgem =12.7 Hz), 2.00 (s, 6H, Ac), 1.75 (dd, 1H, Neu5Ac-H-3ax,
3ax,4 =12.7 Hz) ppm; 13C NMR (100 MHz, D2O, acetone-d6): d=175.8,
J2,3 =8.8 Hz), 2.73 (dd, 1H, Neu5Ac-H-3eq,
175.5, 174.7, 172.4, 105.3, 100.5, 99.0, 77.0, 76.5, 75.6, 73.6, 72.7, 72.0, 71.7,
69.8, 69.4, 69.1, 68.9, 68.2, 67.3, 63.4, 62.0, 61.8, 55.4, 52.5, 49.1, 40.6, 23.0,
22.9 ppm; IR (KBr): n˜ =3332, 1614, 1401, 1031, 932, 823, 668 cmꢀ1
;
HRMS (ESI-TOF): calcd for C28H48N3O21 [M+H]+: 762.2775; found:
762.2767.
Acknowledgements
This work was supported by a Grant-in-Aid for Scientific Research on
Priority Area (S) from the Ministry of Education, Culture, Sports, Sci-
ence, and Technology (Grant-in-Aid No.: 14103013).
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propanol 95:5; flow rate: 3.0 mLminꢀ1; retention time: a isomer:
12.9 min, b isomer: 16.1 min).
[24] Glycosidation of an N-Troc-b-thiophenyl sialoside with a primary al-
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[28] The acceptor 7 was prepared according to the following procedure.
Glycosylation of N-(benzyloxycarbonyl)-l-serine benzyl ester with
2-azido-4,6-O-benzylidene-3-O-chloroacetyl-2-deoxy-b-d-galactopyr-
anoside in the presence of NIS/TfOH in CH2Cl2 at 08C provided N-
(benzyloxycarbonyl)-3-O-(2-azido-4,6-O-benzylidene-3-O-chloroace-
tyl-2-deoxy-d-galactopyranosyl)-l-serine benzyl ester in 94% yield
(a:b=66:34). Each isomer was purified by column chromatography
on silica gel. Reductive opening of the benzylidene acetal on the a
isomer with 1m borane/tetrahydrofuran solution and 1m dibutylbor-
on triflate solution, followed by removal of the chloroacetyl group
with thiourea in N,N-dimethylformamide (DMF) at 708C gave N-
(benzyloxycarbonyl)-3-O-(2-azido-4-O-benzyl-2-deoxy-a-d-galacto-
pyranosyl)-l-serine benzyl ester (7) in 92% yield over two steps.
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propanol 90:10; flow rate: 3.0 mLminꢀ1; retention time: a isomer:
10.7 min, b isomer: 11.7 min). The anomeric configuration of disac-
charide 9 was determined by NMR spectroscopy measurements ac-
cording to the empirical rule.
[34] Selected analytical date for sialoside 11: For the 11a isomer:
1H NMR (400 MHz, CDCl3): d=5.61 (dd, 1H, Gal-H-2, J1,2 =3.4,
J
2,3 =10.3 Hz), 5.55 (d, 1H, Gal-H-1, J1,2 =3.4 Hz) ppm. For the 11b
isomer: 1H NMR (400 MHz, CDCl3): d=5.74 (dd, 1H, Gal-H-2,
1,2 =7.3, J2,3 =8.2 Hz), 4.77 (d, 1H, Gal-H-1, J1,2 =7.8 Hz) ppm.
J
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[37] The acceptor 14 was prepared according to the following procedure.
Treatment of N-(benzyloxycarbonyl)-3-O-(2-azido-4,6-O-benzyl-
idene-3-O-chloroacetyl-2-deoxy-a-d-galactopyranosyl)-l-serine
benzyl ester with thiourea in DMF at 708C provided N-(benzyloxy-
carbonyl)-3-O-(2-azido-4,6-O-benzylidene-2-deoxy-a-d-galactopyra-
nosyl)-l-serine benzyl ester (14) in 96% yield.
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[40] The a:b ratio was determined by HPLC analysis (eluent: hexane/2-
propanol 89:11; flow rate: 3.0 mLminꢀ1; retention time: a isomer:
20.6 min, b isomer: 22.7 min). The anomeric configuration of disac-
charide 15 was determined by NMR spectroscopy measurements ac-
cording to the empirical rule.
[41] Partial 1H NMR signal assignment for disaccharide 15: For the 15a
isomer: dH-3eq =2.69 ppm, dH-4 =4.83 ppm, J7,8 =9.8 Hz, Dd(H-9’ꢀH-
9)=0.30 ppm. For the 15b isomer: dH-3eq =2.58 ppm, dH-4 =5.10 ppm,
J
7,8 =2.9 Hz, Dd(H-9’ꢀH-9)=1.15 ppm.
[42] Y. Hirabayashi, Y. Matsumoto, Matsumoto, T. Toida, N. Iida, T.
Matsubara, T. Kanzaki, M. Yokota, I. Ishizuka, J. Biol. Chem. 1990,
265, 1693–1701.
[31] Partial 1H NMR signal assignment for disaccharide 9: For the 9a
isomer: dH-3eq =2.67 ppm, dH-4 =5.00 ppm, J7,8 =8.8 Hz, Dd(H-9’ꢀH-
9)=0.17 ppm. For the 9b isomer: dH-3eq =2.52 ppm, dH-4 =5.55 ppm,
Dd(H-9’ꢀH-9)=0.94 ppm.
Received: August 13, 2004
Published online: December 6, 2004
862
ꢁ 2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
Chem. Eur. J. 2005, 11, 849 – 862