Synthesis of cancer-associated glycoantigens: stage-specific embryonic antigen 3 (SSEA-3)

Article abstract of DOI:10.1016/j.carres.2006.03.041

The synthesis of the tumor-associated carbohydrate antigens SSEA-3 and Gb3 in a semi-convergent fashion using building blocks bearing a S-thiazolinyl (STaz) moiety is reported. Complete stereoselective control of a difficult α-(1→4)-galactosylation and hi

Full text of DOI:10.1016/j.carres.2006.03.041

Carbohydrate Research 341 (2006) 1458–1466
Synthesis of cancer-associated glycoantigens: stage-specific
embryonic antigen 3 (SSEA-3)
Papapida Pornsuriyasak and Alexei V. Demchenko*
Department of Chemistry and Biochemistry, University of Missouri—St. Louis, One University Blvd., St. Louis, MO 63121, USA
Received 15 February 2006; received in revised form 16 March 2006; accepted 29 March 2006
Available online 27 April 2006
Abstract—The synthesis of the tumor-associated carbohydrate antigens SSEA-3 and Gb3 in a semi-convergent fashion using build-
ing blocks bearing a S-thiazolinyl (STaz) moiety is reported. Complete stereoselective control of a difficult a-(1!4)-galactosylation
and high overall yields were achieved.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Convergent strategy; Glycosylation; Stereoselective synthesis; Selective activation
1. Introduction
monosialosyl galactosyl globoside (MSGG), bearing a
(2!3)-linked terminal sialic acid unit; and disialosyl
galactosyl globoside (DSGG), bearing an additional sia-
lic acid unit at C-6 of the subterminal galactosamine
residue.
Glycosphingolipids of the globo series (globosides) are
commonly associated with different types of human car-
cinomas including lung, breast, kidney, and ovarian.1
Over the last few years, anti-cancer immunotherapy
has emerged as a new promising direction for control-
ling tumor development and growth. It has been demon-
strated that carbohydrate antigens in the form of their
protein conjugates can be used as suitable candidates
for highly immunogenic anti-cancer vaccines.2 The high
demand for the globosides for quantitative studies and
the difficulty in their isolation in pure form have under-
scored the need for their chemical synthesis.
The stage-specific embryonic antigen-3 (SSEA-3)
represents the core pentasaccharide sequence b-D-
Galp-(1!3)-b-D-GalpNAc-(1!3)-a-D-Galp-(1!4)-b-D-
Galp-(1!4)-D-Glcp, which is common to a number of
structurally related glycosphingolipids (Fig. 1). The tri-
saccharide composing the reducing end of the molecule
is commonly referred to as globotriaosyl ceramide (Gb3,
Fig. 1). Other related structures include glycosphingo-
lipids of the globo series: Globo-H bearing an addi-
tional L-fucose moiety at C-2 of the terminal galactose
unit; SSEA-4, which is commonly referred to as
In spite of the considerable progress that has been
made in the area of convergent,3,4 polymer-supported,5
one-pot,6 and automated7 oligosaccharide synthesis,
the availability of synthetic glycostructures remains
low. Indeed, in the current state of knowledge, success-
ful total syntheses of complex carbohydrate structures
demand significant resources. As a consequence, syn-
thetic glycoconjugates of the globo series had been
mainly accessible to an elite circle of chemists: SSEA-
3—Ogawa,8 Magnusson,9 Danishefsky,10 and Seeber-
ger;11 Globo-H—Danishefsky,12 Schmidt,13 Boons,14
Wong,15 and Seeberger,11 or SSEA-4—Hasegawa16
and Schmidt/Garegg.17
As a part of a program to develop convergent strate-
gies for rapid oligosaccharide synthesis, we describe
herein the synthesis of the spacer-containing carbo-
hydrate antigens Gb3 and SSEA-3 using S-thiazolinyl
(STaz) glycosides, an approach that has been recently
developed in our laboratory.18 The methodological
importance of synthesizing the SSEA-3 pentasaccharide
derives from the fact that its backbone structure is struc-
turally related to other complex glycosphingolipids of
*
Corresponding author. E-mail: demchenkoa@umsl.edu
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.03.041

P. Pornsuriyasak, A. V. Demchenko / Carbohydrate Research 341 (2006) 1458–1466
1459
HO
HO
HO
HO
R2O
HO
O
OR1
O
OH
O
OH
O
OH
O
O
NHAc
HO
HO
OR3
O
O
OH
O
OH
O
OH
O
OH
O
HO
O
HO
O
OR'
OR'
HO
HO
OH
OH
OH
OH
SSEA-3: R1=R2=R3=H
Globo-H: R1=R2=H, R3=Fuc
SSEA-4: R1=R3=H, R2=Neu
DSGG: R1=R2=Neu, R3=H
Gb3
R' = Ceramide, Fuc = L-fucose residue, Neu = sialic acid residue
Figure 1. Major tumor-associated antigens of the globo series and Gb3.
the globo series (see Fig. 1). Therefore, it is anticipated
that the discoveries made during this synthesis could
be translated to future syntheses of other compounds
shown in Figure 1. The spacer unit, introduced in place
of the ceramide arm, should enable conjugation to an
immunogenic carrier protein to alter the poor immuno-
genicity of the bare oligosaccharide or glycosphingolipid
molecules.
group can serve as a suitable masking functionality
for the amino functionality, which can be removed by
simple reduction.20 Compound 1 was deacetylated
(NaOCH3 in CH3OH) and positions 40 and 60 were
protected as benzylidene acetal with dimethoxytoluene
(DMT) in the presence of camphorsulfonic acid
(CSA) in CH3CN to afford selectively protected com-
pound 3 (Scheme 1).21 The remaining hydroxyl groups
of 3 were protected under standard benzylation condi-
tions (BnBr, NaH, DMF), and the benzylidene acetal
was reductively opened with NaCNBH3 in an acidic
medium22 to afford 5 with complete regioselectivity in
a high overall yield of 60% over four synthetic steps
starting from 1.
The introduction of the a-(1!4)-linked galactose res-
idue at the terminal end of the GB3 trisaccharide is
arguably a major challenge in the synthesis of the tumor
antigens of the globo series.23 A variety of glycosyl
donors have been previously tested in this synthetic step:
fluoride,12 trichloroacetamidate,13 and glycosyl phos-
phate.11 In many cases, the stereoselectivity could be
improved by elaborate optimizing of reaction conditions,
in turn influencing the stereoselectivity of glycosyl-
ation.11,12,17 Typically, only average yields, rarely exceed-
ing 60%, could be achieved at this stage of the synthesis.
As recently demonstrated in our laboratory, glycosyl
thioimidates often provide excellent yields and elevated
1,2-cis stereoselectivity in a variety of applications.24
Therefore, we decided to investigate whether the use of
2. Results and discussion
The carbohydrate part of SSEA-3 is a core pentasaccha-
ride consisting of three galactose units, one galactos-
amine, and one glucose unit, all of the D-pyranose
series. It was our intention to execute the synthesis in
a
2+3 fashion from the terminal disaccharide
b-D-Galp-(1!3)-D-GalpNAc and the reducing end tri-
saccharide
a-D-Galp-(1!4)-b-D-Galp-(1!4)-D-Glcp
building blocks. The latter oligosaccharide represents
the carbohydrate sequence of Gb3 (Fig. 1).
2.1. Synthesis of the Gb3 trisaccharide
The construction of the Gb3 target molecule began
with the preparation of a suitably protected lactose
building block. For this purpose, we selected known
lactose derivative 1 bearing a 3-azidopropyl spacer at
the anomeric position.19 In this context, the azido
Ph
O
AcO
O
AcO
OAc
O
RO
ii
AcO
O
O
RO
iii
RO
O
O
N3
O
N3
AcO
O
RO
O
OAc
AcO
OR
1: R=Ac
2: R=H
3: R=H
i
4: R=Bn
HO
BnO
OBn
iv
O
BnO
O
O
N3
BnO
O
OBn
BnO
5
Scheme 1. Synthesis of the lactose acceptor 5. Reagents and conditions: (i) NaOCH3, CH3OH, 89%; (ii) PhCH(OCH3)2, CSA, CH3CN, 86%; (iii)
˚
BnBr, NaH, DMF, 93%; (iv) NaCNBH3, HCl/Et2O, THF, 3 A molecular sieves, 84%.

1460
P. Pornsuriyasak, A. V. Demchenko / Carbohydrate Research 341 (2006) 1458–1466
the STaz glycosyl donor 618 bearing a non-participating
benzyl group at C-2 would prove advantageous for this
key step of the synthesis. Initially, the standard reaction
conditions for activation of the STaz moiety with the use
of AgOTf or CH3OTf as promoters in 1,2-dichloro-
ethane at room temperature were investigated.23 In both
cases, the glycosylation proceeded smoothly leading to
the formation of target disaccharide 7 in yields of 87%
or 80%, respectively (Scheme 2). Very importantly, com-
plete a-stereoselectivity was obtained in each case, which
was sufficient to halt cumbersome experimenting with
the reaction conditions.
Ph
O
O
O
HO
BnO
O
BnO
OBn
O
i,ii
7
BnO
O
O
N3
BnO
O
OBn
BnO
8
Scheme 3. Synthesis of the trisaccharide acceptor 8. Reagents and
conditions: (i) NaOCH3, CH3OH, 99%; (ii) PhCH(OCH3)2, CSA,
CH3CN, 73%.
2.2. Preparation of the SSEA-3 pentasaccharide
functionality, including N-phthalimido, N-acetyl, N-tri-
chloroethoxycarbonyl (N-Troc), and azido. Amongst
these, the 2-N-Troc substituent,26,27 in combination with
the O-pentenyl moiety,28 seemed to be the most attrac-
tive. Considerations such as the ease of the synthesis,
regioselective modification, and glycosylation have con-
tributed to this selection. The synthesis of the building
block was started from known acetate 9,27 which was
converted into O-pentenyl glycoside 11 via the two-step
procedure, involving anomeric bromination (AcBr,
AcOH) and O-glycosylation of pent-4-enol under Helfe-
rich conditions.29 Compound 11 was then deacetylated
in the presence of guanidine and NaOCH3,30 and the
resulting triol was protected as 4,6-benzylidene acetal
13. This synthesis was accomplished in 58% yield over
four steps starting from 9 (Scheme 4).
The last building block required for the introduction
of the terminal galactose residue of SSEA-3, 14, was
obtained from known ethyl 2,6-di-O-benzoyl-3,4-O-
isopropylidene-1-thio-b-D-galactopyranoside31 by the
previously reported two-step procedure involving ano-
meric bromination followed by the introduction of the
STaz moiety.25,32 This two-stage procedure afforded
the requisite compound 14 in the total yield of 78%.
Having obtained building blocks 8, 13, and 14, we
refocused our attention on the final assembly of the
pentasaccharide of SSEA-3. To achieve this goal, we
With the desired Gb3 trisaccharide 7 in hand, we turned
our attention to the assembly of the remaining sequence
of the title pentasaccharide. Trisaccharide 7 needed to be
modified to be able to act as the glycosyl acceptor. This
was achieved by simple protecting group chemistry
involving the two-step deacetylation-benzylidene acetal
installation sequence. These manipulations were
performed under similar reaction conditions to those
described for the synthesis of the building block 5. As
a result, the 300-OH acceptor 8 was obtained in 73% yield
over two synthetic steps (Scheme 3).
In addition, the synthesis of two new building blocks
of the D-galacto and D-galactosamino series for the
introduction of the terminal and subterminal units,
respectively, was required. Our intention to assemble
the final sequence in a highly convergent manner
required the use of two leaving groups, one of which
could have been activated over another under a suitable
set of reaction conditions (selective activation).4 We
have already demonstrated that the STaz moiety can
be selectively activated over the O-pentenyl moiety using
AgOTf as a promoter.25 Therefore, the O-pentenyl pro-
tection was chosen for the synthesis of the D-galactos-
amino building block.
Throughout the course of our studies, we investigated
a variety of temporary protecting groups for the 2-amino
AcO
OAc
O
AcO
OAc
O
AcO
STaz
AcO
BnO
O
BnO
BnO
OBn
O
A or B
BnO
O
6
O
N3
BnO
O
OBn
BnO
7
HO
BnO
OBn
O
BnO
O
O
N3
BnO
O
OBn
BnO
5
Scheme 2. Synthesis of Gb3 trisaccharide 7. Reagents and conditions: (A) AgOTf, 1,2-dichloroethane, 25 h, 87% (a only); (B) CH3OTf, 1,2-
dichloroethane, 16 h, 80% (a only).

P. Pornsuriyasak, A. V. Demchenko / Carbohydrate Research 341 (2006) 1458–1466
1461
performed two sequential glycosylations in a highly con-
vergent manner.4 First, the STaz moiety of the glycosyl
donor 14 was activated over the O-pentenyl moiety of
the glycosyl acceptor 13. This selective glycosylation
was achieved in the presence of AgOTf, conditions
under which the O-pentenyl moiety remained entirely
inert. As a result, the disaccharide derivative 15 was
obtained in 79% yield with complete b-stereoselectivity
(Scheme 5). In this context, the reported experimental
evidence implies the difficulty of glycosylations at the
C-3 position of glycosamines. These examples include
a relatively low reactivity of the 3-OH that has been
observed with N-acetamido derivatives33 or steric fac-
tors caused by bulky N-phthalimido group.34
It has to be also noted that the use of S-ethyl glycoside
as glycosyl donor did not seem to be feasible. Although
we have already demonstrated that the S-ethyl moiety
can be selectively activated in the presence of O-pentenyl
glycosides,35 the necessity to use CH3OTf as a promoter
for such activation could also cause undesirable (and
RO
OR
AcO
AcO
OAc
O
OAc
O
O
i
ii
O
RO
TrocHN
OAc
AcO
AcO
TrocHN
TrocHN
Br
9
11: R=Ac
12: R=H
10
Ph
iii
O
O
iv
O
O
HO
TrocHN
13
Scheme 4. Synthesis of the glycosyl acceptor 13. Reagents and conditions: (i) 30% HBr/AcOH, CH2Cl2, 95%; (ii) 4-pentenol, HgO/HgBr2, CH2Cl2,
˚
4 A molecular sieves, 74%; (iii) guanidineÆHCl, CH3OH/CH2Cl2, Na, 99%; (iv) PhCH(OCH3)2, CSA, CH3CN, 83%.
Ph
O
O
O
OBz
O
O
O
STaz
Ph
HO
TrocHN
O
OBz
14
13
i
Ph
O
O
O
O
O
OBz
O
O
O
O
O
HO
O
TrocHN
BnO
OBz
O
BnO
OBn
O
BnO
O
15
O
N3
Bn
O
O
OBn
BnO
ii
8
Ph
Ph
O
O
O
O
O
OBz
O
O
O
O
O
O
TrocHN
BnO
OBz
O
BnO
OBn
O
BnO
O
N3
O
16
BnO
O
OBn
BnO
˚
Scheme 5. The final assembly of the SSEA-3 pentasaccharide 16. Reagents and conditions: (i) AgOTf, 1,2-dichloroethane, 3 A molecular sieves, 79%;
˚
(ii) NIS/TMSOTf, 1,2-dichloroethane, 4 A molecular sieves, 73%.

1462
P. Pornsuriyasak, A. V. Demchenko / Carbohydrate Research 341 (2006) 1458–1466
irreversible) 2-N-methylation36 of the glycosyl acceptor
13 and/or the disaccharide 15.
in dry CH3OH (10 mL) and the reaction mixture was
stirred for 1 h at rt. The reaction was then neutralized
with Dowex (H+), filtered, and concentrated in vacuo
to yield crude 2 as white foam (163 mg, 89% yield).
The product 2 was dissolved in dry CH3CN (7 mL) con-
taining PhCH(OCH3)2 (0.11 mL, 0.74 mmol) and CSA
(5 mg, 0.02 mmol). The reaction mixture was kept for
2 h at rt, then neutralized with Et3N and concentrated
in vacuo. The residue was purified by column chroma-
tography on silica gel (CH2Cl2/CH3OH gradient elu-
tion) to allow 3 as a colorless syrup (168 mg, 86%
yield). The crude 3 was dissolved in dry DMF (5 mL)
and benzyl bromide (0.27 mL, 2.29 mmol) followed by
portionwise addition of NaH (60% in mineral oil,
137 mg, 3.44 mmol) at rt over 15 min. The stirred reac-
tion mixture was kept for additional 1 h, then quenched
by adding crushed ice and stirred until cessation of H2
evolution. The mixture was then extracted with
EtOAc/Et2O (3 · 20 mL, 1:1, v/v) and the combined
organic layer was washed with water (3 · 10 mL). The
organic extract was separated, dried (MgSO4), and
evaporated in vacuo. The residue was purified by
column chromatography on silica gel (EtOAc/hexane
gradient elution) to afford 4 as white solid (293 mg,
The final step of the oligosaccharide assembly
involved coupling of the disaccharide 15 with the trisac-
charide acceptor 8. This reaction was performed in the
presence of NIS and catalytic TMSOTf, conventional
reaction conditions for the O-pentenyl moiety activa-
tion.28 As a result, the target pentasaccharide 16 was iso-
lated in 73% yield.
3. Conclusions
In summary, we have synthesized SSEA-3, the tumor-
associated antigen of the globo series using STaz glyco-
sides for the installation of two critical glycosidic bonds
of the sequence. In parallel, the synthesis of Gb3 was also
accomplished. This strategy can be applied for the synthe-
sis of the other tumor-associated antigens, the syntheses
of which are currently under pursuit in our laboratory.
4. Experimental
1
4.1. General methods
93% yield). H NMR: d 1.89 (m, 2H, CH2CH2CH2),
2.95 (s, 1H, H-50), 3.37–3.45 (m, 5H, H-20, H-3, H-5,
OCH2), 3.60–3.70 (m, 3H, H-30, CH2N3), 3.70–3.80
(m, 2H, J2,3 = 7.9 Hz, H-2, H-6b), 3.80–3.92 (m, 2H,
Column chromatography was performed on Silica Gel
60 (EM Science, 70–230 mesh). Reactions were moni-
tored by TLC on Kieselgel 60 F254 (EM Science). The
compounds were detected by examination under UV
light and by charring with 10% sulfuric acid in CH3OH.
Solvents were removed under reduced pressure at
<40 ꢁC. CH2Cl2, ClCH2CH2Cl, and CH3CN were dis-
tilled from CaH2 directly prior to use. CH3OH was dried
by heating at reflux over magnesium methoxide, dis-
tilled, and stored under argon. Anhydrous DMF (EM
Science) and THF were used as received. Molecular
H-4, H-6b0), 3.92–4.05 (m, 3H, J4 ;5 ¼ 3:6 Hz, H-40,
0
0
H-6a, CH2N3), 4.20–4.25 (dd, 1H, H-6a0), 4.33 (d, 1H,
CH2Ph), 4.38 (d, 1H, J1 ;2 ¼ 7:8 Hz, H-10), 4.46 (d,
1H, J1,2 = 6.7 Hz, H-1), 4.56 (d, 1H, CH2Ph), 4.73–
4.88 (m, 7H, CH2Ph), 5.20 (d, 1H, CH2Ph), 5.48 (s,
1H, CHPh), 7.17–7.55 (m, 30H, aromatic); FABMS
calcd for C57H61N3NaO11 [M+H]+: 986.4204. Found:
986.4204.
0
0
˚
sieves (3 or 4 A) used for reactions, were crushed and
4.3. 3-Azidopropyl 2,3,6-tri-O-benzyl-b-D-galactopyranos-
yl-(1!4)-2,3,6-tri-O-benzyl-4-O-b-D-glucopyranoside (5)
activated in vacuo at 390 ꢁC for 8 h in the first instance
and then for 2–3 h at 390 ꢁC directly prior to use.
AgOTf (Acros) was co-evaporated with toluene
(3 · 10 mL) and dried in vacuo for 2–3 h directly prior
to use. Optical rotations were measured on a Jasco
A mixture of the acetal 4 (900 mg, 0.93 mmol) and
˚
molecular sieves (3 A, 4 g) in THF (36 mL) was stirred
under argon for 1 h. NaCNBH3 (0.78 g, 12.4 mmol)
was then added, followed by the dropwise addition of
2 M HCl/Et2O (6.2 mL, 12.4 mmol) until cessation of
H2 evolution. The reaction mixture was kept for an
additional 30 min at rt and the solid was filtered-off
and washed with CH2Cl2. The combined filtrate
(200 mL) was washed with water (50 mL), 20% aq NaH-
CO3 (50 mL), and water (2 · 50 mL). The organic
extract was separated, dried (MgSO4), and evaporated
in vacuo. The residue was purified by column chroma-
tography on silica gel (EtOAc/hexane gradient elution)
to afford acceptor 5 as colorless syrup (757 mg, 84%
1
P-1020 polarimeter. H NMR spectra were recorded in
CDCl3 at 300 MHz (Bruker Advance), 13C NMR
spectra were recorded in CDCl3 at 75 MHz (Bruker
Advance) or at 125 MHz (Bruker ARX-500). HRMS
determinations were made with the use of JEOL
MStation (JMS-700) Mass Spectrometer.
4.2. 3-Azidopropyl 2,3-di-O-benzyl-4,6-O-benzylidene-b-
D-galactopyranosyl-(1!4)-2,3,6-tri-O-benzyl-4-O-b-D-
glucopyranoside (4)
28
A 1.0 M solution of NaOCH3 in CH3OH (1 mL) was
yield): Rf = 0.55 (2:3, EtOAc/hexanes); ½aꢀD +16.8 (c
added to a stirred suspension of 119 (310 mg, 0.43 mmol)
1.0, CHCl3); 1H NMR: 1.81 (m, 2H, CH2CH2CH2),

P. Pornsuriyasak, A. V. Demchenko / Carbohydrate Research 341 (2006) 1458–1466
1463
3.20–3.34 (m, 6H, H-20, H-3, H-5, H-50, OCH2), 3.34–
3.42 (dd, 1H, J5,6a = 5.2 Hz, J6a,6b = 9.7 Hz, H-6a),
3.45–3.63 (m, 5H, H-2, H-30, H-6a0, H-6b, CH2N3),
filtrate (30 mL) was washed with 20% aq NaHCO3
(10 mL) and water (3 · 10 mL), the organic phase was
separated, dried (MgSO4), and concentrated in vacuo.
The residue was purified by column chromatography on
silica gel (EtOAc/hexane gradient elution) to afford tri-
saccharide 7 as white solid (47 mg, 87% yield) with com-
plete a-stereoselectivity. Method B. A mixture of
glycosyl donor 6 (20 mg, 0.04 mmol), glycosyl acceptor
5 (25.8 mg, 0.027 mmol), and freshly activated molecular
3.72 (dd, 1H, J5,6a = 4.2 Hz, J6a0;6b ¼ 10:9 Hz, H-6b0),
0
3.85–3.95 (m, 3H, H-4, H-40, CH2N3), 4.27–4.36 (m,
5H, J1,2 = 7.4 Hz, J1 ;2 ¼ 7:8 Hz, H-1, H-10, CH2Ph),
4.47 (d, 1H, CH2Ph), 4.55–4.70 (m, 6H, CH2Ph), 4.75
(d, 1H, CH2Ph), 4.91 (d, 1H, CH2Ph), 7.10–7.30 (m,
30H, aromatic); 13C NMR: d 29.5, 48.5, 66.3, 66.7,
68.6, 72.2, 73.0, 73.4, 73.7, 75.2, 75.3, 75.5, 75.57, 76.8,
77.4, 79.6, 81.3, 82.0, 83.1, 102.7, 103.8, 127.5, 127.7
(·2), 127.8 (·2), 127.9 (·4), 128.0 (·4), 128.1 (·4),
128.3 (·4), 128.5 (·6), 128.6 (·2), 128.7 (·2), 138.1,
130.4, 138.5, 138.8 (·2), 139.3; FABMS calcd for
C57H63N3NaO11 [M+Na]+: 988.4360. Found 988.4360.
0
0
˚
sieves (3 A, 60 mg) in ClCH2CH2Cl (0.5 mL) was stirred
under argon for 1.5 h. CH3OTf (15 lL, 0.12 mmol) was
added and the reaction mixture was stirred for 16 h at
rt, then diluted with CH2Cl2, the solid was filtered-off
and the residue was washed with CH2Cl2. The combined
filtrate (30 mL) was washed with 20% aq NaHCO3
(10 mL) and water (3 · 10 mL), the organic phase was
separated, dried (MgSO4), and concentrated in vacuo.
The residue was purified by column chromatography on
silica gel (EtOAc/hexane gradient elution) to afford tri-
saccharide 7 (43.2 mg, 80% yield) with complete a-stereo-
4.4. 2-Thiazolinyl 3,4,6-tri-O-acetyl-2-O-benzyl-1-thio-b-
D-galactopyranoside (6)
NaSTaz (1.16 g, 8.22 mmol) was added to a stirred solu-
28
tion
of
3,4,6-tri-O-acetyl-2-O-benzyl-a-D-galacto-
selectivity: Rf = 0.45 (2:3, EtOAc/hexanes); ½aꢀD +38.9
pyranosyl bromide37 (2.5 g, 5.48 mmol) in dry CH3CN
(15 mL) under argon. The reaction mixture was stirred
for 30 min at rt. Upon completion, the mixture was di-
luted with toluene (200 mL) and washed with 1% aq
NaOH (50 mL) and water (3 · 50 mL), the organic
phase was separated, dried (MgSO4), and concentrated
in vacuo. The residue was purified by column chromato-
graphy on silica gel (EtOAc/hexane gradient elution)
to afford the STaz glycoside 6 (2 g, 73%) as white solid:
(c 1.0, CHCl3); H NMR: d 1.84, 1.85, 2.02 (3s, 9H,
1
3 · COCH3), 1.87 (m, 2H, CH2CH2CH2), 3.25–3.46 (m,
7H, H-20, H-3, H-5, H-50, H-6b, CH2N3), 3.49–3.70 (m,
5H, H-2, H-30, H-6a, H-6a0, CH2 O), 3.79–4.07 (m, 6H,
a
H-200, H-4, H-40, H-500, H-6b0, CH2 O), 4.28 (s, 2H,
b
0
0
CH2Ph), 4.33 (d, 1H, CH2Ph), 4.34 (d, 1H, J1 ;2
¼
9:4 Hz, H-10), 4.43 (d, 1H, J1,2 = 7.7 Hz, H-1), 4.47–4.93
(m, 12H, H-6a00, H-6b00, CH2Ph), 5.04 (d, 1H, CH2Ph),
5.14 (d, 1H, J1 ;2 ¼ 3:4 Hz, H-100), 5.32–5.37 (dd, 1H,
00 00
23
J3 ;4 ¼ 3:2 Hz, H-300), 5.40 (dd, 1H, J4 ;5 ¼ 1:1 Hz, H-
400), 7.20–7.40 (m, 35H, aromatic); 13C NMR: d 20.8,
20.9, 29.4, 48.5, 61.0, 66.4, 66.7, 67.4, 68.4, 68.7, 70.3,
72.9, 73.3, 73.6, 74.0, 75.1, 75.2, 75.4, 77.4, 79.6, 80.9,
81.9, 82.9, 99.9, 103.2, 103.7, 127.2, 127.7, 127.7, 127.8,
127.9, 128.0, 128.1 (·2), 128.2, 128.3, 128.4, 128.5,
128.5, 128.5, 128.6, 128.6, 138.2, 138.3, 138.5, 138.6,
138.8, 139.6; HR-FAB MS calcd for C76H85N3NaO19
[M+Na]+: 1366.5675. Found: 1366.5682.
00 00
00 00
Rf = 0.48 (1:4, EtOAc/CH2Cl2); ½aꢀD +28.2 (c 1.0,
CHCl3); 1H NMR: d 1.95, 2.05, 2.15 (3s, 9H,
3 · COCH3), 3.40 (dd, 2H, CH2N), 3.79 (dd, 1H,
J2,3 = 9.8 Hz, H-2), 4.02 (dd, 1H, J5,6 = 6.8 Hz, H-5),
4.09–4.19 (m, 3H, H-6a, H-6b, CH2S), 4.22–4.35 (m,
1H, JCH S;CH N ¼ 7:5 Hz, CH2S), 4.62 (d, 1H, CH2Ph),
2
2
4.80 (d, 1H, CH2Ph), 5.07 (dd, 1H, J3,4 = 3.4 Hz,
H-3), 5.39–5.44 (dd, 2H, H-1, H-4), 7.27–7.34 (m, 5H,
aromatic); 13C NMR: d 20.9 (·3), 61.5, 64.3, 67.8,
74.3, 74.7, 75.5, 75.8, 77.4, 85.1, 128.2 (·2), 128.6 (·2),
137.6, 170.0, 170.3, 170.6; FABMS calcd for
C22H28NO8S2 [M+H]+: 498.1256. Found: 498.1254.
4.6. 3-Azidopropyl 2-O-benzyl-4,6-O-benzylidene-a-D-
galactopyranosyl-(1!4)-2,3,6-tri-O-benzyl-b-D-galacto-
pyranosyl-(1!4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside
(8)
4.5. 3-Azidopropyl 3,4,6-tri-O-acetyl-2-O-benzyl-a-D-
galactopyranosyl-(1!4)-2,3,6-tri-O-benzyl-b-D-galacto-
pyranosyl-(1!4)-2,3,6-tri-O-benzyl-b-D-glucopyranoside
(7)
The title compound was obtained from 7 by the deacet-
ylation-benzylidene acetal formation sequence as de-
scribed in the synthesis of 4 in 72% yield as colorless
28
Method A. A mixture of glycosyl donor 6 (20 mg,
0.04 mmol), glycosyl acceptor 5 (25.8 mg, 0.027 mmol),
syrup: Rf = 0.52 (2:3, EtOAc/hexanes); ½aꢀD +51.6 (c
1
1.0, CHCl3); H NMR: d 1.90 (m, 2H, CH2CH2CH2),
and freshly activated molecular sieves (3 A, 60 mg) in
3.29–3.50 (m, 7H, H-20, H-3, H-5, H-50, H-6b, OCH2),
3.55–3.68 (m, 4H, H-2, H-30, H-6a, CH2N3), 3.75 (dd,
˚
ClCH2CH2Cl (0.5 mL) was stirred under argon for
1.5 h. Freshly conditioned AgOTf (20.6 mg, 0.08 mmol)
was added and the reaction mixture was stirred for 25 h
at rt, then diluted with CH2Cl2, the solid was filtered-off
and the residue was washed with CH2Cl2. The combined
1H, H-6b0), 3.80–3.90 (m, 2H, J2 ;3 ¼ 3:0 Hz, H-200,
00 00
H-6a0), 3.95–4.03 (m, 2H, H-40, CH2N3), 4.07–4.14 (m,
4H, H-400, H-500, H-6a00, H-6b00), 4.18 (dd, 1H, J3 ;4
¼
00 00
10:2 Hz), 4.23–4.43 (m, 4H, J1 ;2 ¼ 8:1 Hz, H-10,
0
0

1464
P. Pornsuriyasak, A. V. Demchenko / Carbohydrate Research 341 (2006) 1458–1466
CH2Ph), 4.50 (d, 1H, J1,2 = 7.6 Hz, H-1), 4.55–4.90 (m,
4.95–5.05 (m, 2H, CH2@), 5.30 (d, 1H, JNH,2 = 6.8 Hz,
NH), 5.56 (s, 1H, CHPh), 5.79 (m, 1H, CH@), 7.35–
7.55 (m, 5H, aromatic); 13C NMR: d 29.0, 30.5, 56.1,
66.9, 69.3, 69.6, 75.0, 75.5, 77.7, 95.9, 101.0, 101.7,
115.4, 126.8 (·2), 128.7 (·2), 129.7, 137.9, 138.5, 155.2;
FABMS calcd for C21H27Cl3NO7 [M+H]+: 510.0853.
Found: 510.0854.
00 00
10H, CH2Ph), 5.08 (d, 1H, CH2Ph), 5.20 (d, 1H, J1 ;2
¼
3:1 Hz, H-100), 5.40 (s, 1H, CHPh), 7.20–7.40 (m, 40H,
aromatic); 13C NMR: d 29.5, 48.6, 63.0, 66.7, 67.4,
68.6, 68.9, 69.4, 72.4, 73.1, 73.3, 73.4, 73.7, 74.3, 75.1,
75.2, 75.3, 75.3, 76.6, 77.0, 77.5, 78.9, 81.5, 81.9, 82.9,
100.5, 101.1, 103.1, 103.7, 126.5, 127.3, 127.4, 127.7,
127.8, 127.8, 127.9, 128.2, 128.2, 128.3, 128.4, 128.5,
128.5, 128.5, 128.6, 128.6, 128.7, 129.2, 137.9, 138.4,
138.6, 138.6, 138.8, 139.5; FABMS calcd for
C77H83N3NaO16 [M+Na]+: 1328.5671. Found 1328.5681.
4.8. 2-Thiazolinyl 2,6-di-O-benzoyl-3,4-O-isopropylidene-
1-thio-b-D-galactopyranoside (14)
The solution of ethyl 2,6-di-O-benzoyl-3,4-O-isopropylid-
ene-1-thio-b-D-galactopyranoside31 (1.6 g, 3.39 mmol)
and activated molecular sieves (3 A, 1.7 g) in CH2Cl2
4.7. 4-Pentenyl 4,6-O-benzylidene-2-deoxy-2-(2,2,2-tri-
chloroethoxycarbonylamino)-b-D-galactopyranoside (13)
˚
(51 mL) was stirred under argon for 1 h. A freshly pre-
pared solution of Br2 in CH2Cl2 (32 mL, 1/165, v/v)
was then added and the reaction mixture was kept for
5 min at rt, then concentrated under reduced pressure
at rt. The crude residue was then treated with NaSTaz
(2.39 g, 16.9 mmol, obtained by mixing stoichiometric
amounts of NaOCH3 and 2-mercaptothiazoline in
distilled CH3OH at rt)18 in dry CH3CN (20 mL) under
argon for 20 min at rt. Upon completion, the mixture
was diluted with toluene, then filtered. The solid was
washed with toluene. The combined filtrate (200 mL)
was washed with 1% aq NaOH (50 mL) and H2O
(3 · 50 mL), the organic layer was separated, dried
(MgSO4), and concentrated in vacuo. The residue was
purified by column chromatography on silica gel
(EtOAc/hexane gradient elution) to afford STaz glyco-
side 14 as white foam (1.4 g, 78% yield): Rf = 0.43
To a stirred solution of 927 (3.2 g, 6.13 mmol) in CH2Cl2
(25 mL), 30% HBr in acetic acid (20 mL) was added. The
solution was stirred at rt for 1 h, then diluted with
CH2Cl2 (250 mL) and washed with cold water (60 mL),
20% aq NaHCO3 (2 · 60 mL), and cold water
(3 · 60 mL). The organic phase was separated, dried
(MgSO4), and concentrated in vacuo to obtain com-
pound 10 as white foam. To a stirred solution of the
crude bromide 10 in CH2Cl2 (20 mL), 4-pentenol
(0.91 mL, 8.84 mmol), freshly activated molecular sieves
˚
(4 A, 9.6 g), HgO (1.27 g, 5.89 mmol), and HgBr2
(106 mg, 0.29 mmol) were added and the reaction mix-
ture was stirred under argon for 2 h. Upon completion,
the solid was filtered-off and the combined filtrate was
separated and concentrated in vacuo. The residue was
purified by column chromatography on silica gel
(EtOAc/hexane gradient elution) to afford O-pentenyl
glycoside 11 as white foam (2.4 g, 74% yield). Compound
11 was then dissolved in the solution containing guani-
dine hydrochloride (2.5 g, 26.2 mmol), Na metal
(280 mg, 12.2 mmol) in CH3OH/CH2Cl2 (260 mL, 9:1,
v/v). The reaction was stirred for 20 min at rt to allow
12 as white solid (1.82 g, 99% yield). Crude compound
12 (1 g, 2.37 mmol) was dissolved in dry CH3CN
(15 mL) containing dimethoxytoluene (0.71 mL,
4.75 mmol) and CSA (28 mg, 0.12 mmol). The reaction
mixture was kept for 3 h at rt, then neutralized with
Et3N. The reaction mixture was diluted with CH2Cl2
(100 mL) and washed with H2O (20 mL), 20% aq NaH-
CO3 (20 mL), and H2O (3 · 20 mL). The organic phase
was separated, dried (MgSO4), and concentrated in
vacuo. The residue was purified by column chromato-
graphy on silica gel (acetone/toluene gradient elution)
to afford 13 as white solid (1 g, 83% yield): Rf = 0.16
28
(1:1, EtOAc/hexanes); ½aꢀD +39.2 (c 1.0, CHCl3); 1H
NMR: d 1.36 (s, 3H, CH3), 1.60 (s, 3H, CH3), 3.14–
3.20 (m, 2H, CH2N), 4.01 (dd, 2H, CH2S), 4.35–4.41
(m, 2H, H-4,5), 4.47 (dd, 1H, J3,4 = 6.2 Hz, H-3),
4.55–4.61 (m, 1H, H-6a), 4.65 (dd, 1H, H-6b), 5.41
(dd, 1H, J2,3 = 6.5 Hz, H-2), 5.57 (d, 1H, J1,2
=
9.3 Hz, H-1), 7.38–7.55 (m, 6H, aromatic), 8.01–8.08
(m, 4H, aromatic); 13C NMR: d 26.2, 27.5, 35.1, 63.8,
64.1, 71.3, 73.6, 74.4, 76.5, 77.4, 82.2, 111.1, 128.4
(·4), 129.3, 129.8 (·2), 130.0 (·2), 133.2, 133.5, 163.3,
165.4, 166.3; FABMS calcd for C26H27S2NaNO7
[M+Na]+: 552.1127. Found: 552.1129.
4.9. 4-Pentenyl 2,6-di-O-benzoyl-3,4-O-isopropylidene-b-
D-galactopyranosyl-(1!3)-4,6-O-benzylidene-2-deoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-b-D-galactopyran-
oside (15)
28
(1:1, EtOAc/hexanes); ½aꢀD ꢁ3.14 (c 1.0, CHCl3); 1H
The title compound was obtained by Method A as
NMR: d 1.71 (m, 2H, CH2CH2CH2), 2.11 (m, 2H,
CH2CH@), 3.45–3.49 (m, 2H, H-5, OCH2), 3.56 (m,
1H, H-2), 3.90–3.97 (m, 2H, H-3, OCH2), 4.04–4.09
described for the synthesis of 7 (AgOTf) as white solid,
29
79% yield: Rf = 0.32 (1:9, acetone/toluene); ½aꢀD +25.6
1
(c 1.0, CHCl3); H NMR: d 1.36 (s, 3H, CH3), 1.62 (s,
(dd, 1H, J5,6a = 1.6 Hz, H-6a), 4.18 (d, 1H, J4,5
=
3H, CH3), 1.60 (m, 2H, CH2CH2CH2), 2.03 (m, 2H,
CH2CH@), 3.17 (s, 1H, H-50), 3.32–3.42 (m, 2H, H-20,
3.2 Hz, H-4), 4.30–4.35 (dd, 1H, J5,6b = 1.1 Hz, H-6b),
4.55 (d, 1H, J1,2 = 8.0 Hz, H-1), 4.72 (s, 2H, CH2CCl3),

P. Pornsuriyasak, A. V. Demchenko / Carbohydrate Research 341 (2006) 1458–1466
1465
OCH2), 3.71 (d, 1H, H-6b0), 3.81–3.93 (m, 1H, OCH2),
4.10–4.25 (m, 3H, H-4, H-6a0, H-6b), 4.28–4.50 (m, 4H,
(CHE-9708640) used in this work; Dr. R. E. K. Winter
and Mr. J. Kramer for HRMS determinations.
H-10, H-3, H-30, H-5), 4.58 (dd, 1H, J4 ;5 ¼ 3:8 Hz, H-
40), 4.69 (d, 1H, H-6a), 4.76–4.89 (m, 3H, J1,2 = 8.1 Hz,
H-1, CH2CCl3), 4.90–5.05 (m, 2H, CH2@), 5.20 (m, 1H,
NH), 5.25 (dd, 1H, J2,3 = 7.1 Hz, H-2), 5.46 (s, 1H,
CHPh), 5.74 (m, 1H, CH@), 7.32–8.11 (m, 15H, aro-
matic); 13C NMR: d 26.5, 27.8, 28.8, 30.2, 54.2, 64.0,
65.5, 66.7, 69.2, 69.2, 71.4, 73.6, 73.7, 76.2, 77.4, 95.9,
100.9, 101.7, 111.3, 115.0, 126.5 (·3), 128.2 (·3), 128.7
(·2), 128.8 (·2), 129.0, 129.9 (·2), 130.0 (·2), 133.5,
133.6, 138.0, 138.3, 154.0, 165.3, 166.4; FABMS calcd
for C44H48Cl3NaNO14 [M+Na]+: 942.2038. Found:
942.2023.
0
0
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28
(2:3, EtOAc/hexane); ½aꢀD +42.2 (c 0.2, CHCl3); 1H
NMR: d 1.34 (s, 3H), 1.57 (s, 3H), 1.88 (m, 2H), 3.25–
3.30 (m, 2H), 3.30–3.45 (m, 5H), 3.50–3.65 (m, 8H),
3.65–3.75 (dd, 1H), 3.75–3.85 (dd, 1H), 3.85–4.04 (m,
6H), 4.05–4.23 (m, 6H), 4.40–4.68 (m, 7H), 4.70–4.90
(m, 10H), 5.01 (d, 1H), 5.12 (d, 1H), 5.20 (dd, 1H),
5.36 (s, 1H), 5.39 (s, 1H), 7.20–8.20 (m, 55H, aromatic);
13C NMR: d 100.7, 100.78, 101.5, 103.3, 103.7; FABMS
calcd for C116H121Cl3N4NaO29 [M+Na]+: 2163.7113.
Found: 2163.6946.
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The authors thank ACS PRF (42397-G1) and NSF
(CAREER CHE-0547566) for financial support of this
research; NSF for grants to purchase the NMR spec-
trometer (CHE-9974801) and the mass spectrometer

1466
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