Synthesis and biological evaluation of the Forssman antigen pentasaccharide and derivatives by a one-pot glycosylation procedure

Article abstract of DOI:10.1002/chem.201203865

The synthesis and biological evaluation of the Forssman antigen pentasaccharide and derivatives thereof by using a one-pot glycosylation and polymer-assisted deprotection is described. The Forssman antigen pentasaccharide, composed of GalNAcα(1,3)GalNAcβ(

Full text of DOI:10.1002/chem.201203865

FULL PAPER
DOI: 10.1002/chem.201203865
Synthesis and Biological Evaluation of the Forssman Antigen
Pentasaccharide and Derivatives by a One-Pot Glycosylation Procedure
A
A
Hiroshi Tanaka,*[a] Ryota Takeuchi,[a] Mitsuru Jimbo,[b] Nami Kuniya,[b] and
Takashi Takahashi*[a]
Abstract: The synthesis and biological
evaluation of the Forssman antigen
pentasaccharide and derivatives thereof
by using a one-pot glycosylation and
polymer-assisted deprotection is de-
Troc-protected (Troc=2,2,2-trichloro-
AHCTUNGTRENNeGUN thoxycarbonyl) thioglycoside with a 2-
azide-1-hydroxyl glycosyl donor, fol-
lowed by glycosidation of the resulting
disaccharide at the C3 hydroxyl group
of the trisaccharide acceptor in a one-
An assay of the binding of the synthet-
ic oligosaccharides to a fluorescent-la-
beled SLL-2 revealed that the NHAc
substituents and the length of the oli-
gosaccharide chain were both impor-
tant for the binding of the oligosacchar-
ide to SLL-2. The inhibition effect of
the oligosaccharide relative to the mor-
phological changes of Symbiodinium
by SLL-2, was comparable to their
binding affinity to SLL-2. In addition,
we fortuitously found that the synthetic
Forssman antigen pentasaccharide di-
A
ACHTUNGTRENNUNGed. The Forssman antigen penta-
pot pro
ACHTUNGTRENNUNG
A
E
U
pot glycosACTHUNGTRENNUNG
N
G
sis of pentasaccharides in which the
galactosamine units were partially and
fully replaced by galactose units.
Among the three possible pentasac-
ligand of the lectin SLL-2 isolated
from an octocoral Sinularia lochmodes.
The chemo- and a-selective glycosyla-
tion of a thiogalactoside with a hemi-
charides, GalaACHTUNGTRNE(NUNG 1,3)GalNAc and Gala-
A
E
rectly promotes
a
morphological
Tf2O, TTBP and Ph2SO, followed by
activation of the remaining thioglyco-
side, provided the trisaccharide at the
reducing end in a one-pot procedure.
The pentasaccharide was prepared by
the a-selective glycosylation of the N-
prepared by the established method.
change in Symbiodinium. These results
strongly indicate that the Forssman an-
tigen also functions as a chemical me-
diator of Symbiodinium.
Keywords: chemical biology · gly-
cosylation · lectin · oligosacchar-
ides · one-pot
Introduction
into a non-flagellated coccoid form from a flagellated-swim-
ming form. Frontal affinity chromatography analyses[3] of in-
teractions between SLL-2 and various pyridylaminated oli-
gosaccharides from various glycolipids and glycoproteins re-
Zooxanthellae symbiosis is nearly a standard relationship
that corals depend on for energy and nutrients. Recent envi-
ronmental changes have influenced these relationships and
have resulted in the bleaching of coral and a subsequent de-
terioration in the extent of symbiosis.[1] A lectin SLL-2 iso-
lated from an octocoral Sinularia lochmodes is an important
mediator of the symbiotic relationship between corals and
symbiotic microalgae (Symbiodinium).[2] Treatment of Sym-
biodinium cells with SLL-2 promotes their transformation
vealed that
a
pyridylaminated GalNAca
ACHTUNGTREN(NUNG 1,3)GalNAcb-
A
R
(1,4)Glc, namely the Forssman antigen[4]
1 acts as a high affinity ligand to SLL-2.[5] Therefore, the an-
tigen and structurally related oligosaccharides would be ex-
pected to function as effective chemical probes for the eluci-
dation, not only of the biological roles of SLL-2, but also
the mechanism associated with the symbiotic relationship.
However, oligosaccharides from natural sources are limited
in terms of structural diversity and can be produced in only
limited qualities. In addition, the effect of a glucose unit at
the reducing end on their binding affinity is not clear be-
cause the pyridiylamination of oligosaccharides results in
the formation of an acyclic form in the case of the sugar
unit at the reducing end.[6] Therefore, the chemical synthesis
of a Forssman antigen pentasaccharide and related oligosac-
charides would be highly desirable in terms of structure–ac-
tivity relationship studies. Several reports have appeared on
the synthesis of the Forssman antigen oligosaccharide, based
on a target-oriented strategy.[7] Therefore, an effective
method for the synthesis of the Forssman antigen and relat-
[a] Prof. Dr. H. Tanaka, R. Takeuchi, Prof. Dr. T. Takahashi
Department of Applied Chemistry
Tokyo Institute of Technology (Japan)
Fax: (+81)3-5734-24884
E-mail: thiroshi@apc.titech.ac.jp
ttak@apc.titech.ac.jp
[b] Prof. Dr. M. Jimbo, N. Kuniya
School of Marine Biosciences, Kitasato Universty
1-15-1 kitasato minami-ku Sagamihara-shi
Kanagawa, 252-0373 (Japan)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201203865.
Chem. Eur. J. 2013, 19, 3177 – 3187
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3177

ed oligosaccharides based on a diverse-orientated strategy
continues to remain highly desirable.
In a previous study, we reported on an efficient methodol-
ogy for the synthesis of oligosaccharides based on a one-pot
glycosylation procedure and polymer-assisted deprotec-
tion.[8] The one-pot glycosylation procedure involves the se-
quential chemo- and regioselective glycosylation to directly
provide oligosaccharides from several simple building blocks
without the need for purifying intermediates and is effective
for the synthesis of a single target oligosaccharide as well as
oligosaccharide libraries.[9] Polymer-assisted deprotection in-
volves the deprotection of solid-supported oligosaccharides
followed by their release from the solid. This procedure sim-
plifies the manipulation of the highly polar, fully deprotect-
ed oligosaccharides and their synthetic intermediates.[10]
Herein we report on the synthesis of the pentasaccharide 2
and derivatives that contain 2-trimethylsilylethyl group at
the reducing end by two one-pot glycosylation reactions and
polymer-assisted deprotection, and the biological evaluation
of the final products.
Results and Discussion
We first planned to prepare the Forssman antigen pentasac-
charide 2a with a 2-trimethysilylethyl group at the reducing
end and derivatives 2b–d, in which one and/or two galacto-
sides are attached in place of the galactosamine units. The
2-trimethysilylethyl group at the non-reducing end is a lipo-
philic tag that allows for easy purification and can be che-
moselectively removed by treatment with trifluoroacetic
[12]
acid (TFA)[11] or LiBF4
for further derivatization. Our
strategy for the synthesis of the pentasaccharide derivatives
2a–d involved two one-pot glycosylations combined with a
polymer-assisted deprotection (Scheme 1). The first one-pot
glycosylation involved the chemo- and a-stereoselective gly-
cosylation of the thiogalactoside 10 with the galactoside 6
and the subsequent b-selective glycosyation of 11 with a di-
ACHTUNGTRENNUNGsaccharide to prepare the common trisaccharide 9 at the re-
ducing end. The methyl substituents on the phenyl group of
10 prevent the thiotransfer of the thioglycoside 10 in this
Scheme 1. Strategy for the synthesis of the pentasaccharides 2.
chemoselective glycosylation.[13] The second one-pot gly
ACHTUNGTRENNUNG
ACHTUNGTRENNUNG
different leaving group and 2,6-dimethylphenylthioglycoside
10 with the corresponding requisite activating reagents pro-
vided the disaccharide 12 with good a-selectivity (Table 1).
Glycosidation of the activated hemiacetal 6a with Tf2O,
2,4,6-tri-tert-butylpyrimidine (TTBP), and Ph2SO resulted in
ACHTUNGTRENNUNG
tion of the resulting disaccharide donors at the C3 hydroxyl
group of the trisaccharide acceptor 9 provides the protected
Forssman pentasaccharides. Deprotection of the pentasac-
charides involving modification of the N-Troc amino and
azide groups to N-acetamides and the complete removal of
all O-protecting groups was achieved by polymer-assisted
deprotection by using the prelinker 3 and the amino func-
tionalized ArgoPore resin 4 (Scheme 1).
Synthesis of the trisaccharide 9 at the reducing end is
shown in Scheme 2. We first examined the chemo- and a-se-
lective glycosylation of thioglycoside 10 with the 2O-benzyl-
protected donors 6a–d. Treatment of donors 6a–d with a
Table 1. a-Selective galactosylation at C4 position of glucose 10.
Entry Donor Conditions
Yield a/b[a]
[%]
1
2
3
4
6a
6b
6c
6d
Tf2O, TTBP, Ph2SO ꢀ78 to ꢀ308C, 4.0 h 92
95:5
95:5
95:5
95:5
MeOTf, RT, 4.0 h
AgOTf, ꢀ78 to ꢀ408C, 2.5 h
TMSOTf, ꢀ788C, 2.0 h
1
85
43
74
[a] The ratio was determined based on the H NMR spectra.
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Forssman Antigen Pentasaccharide and Derivatives
FULL PAPER
Scheme 2. Stepwise, one-pot synthesis of trisaccharide 13.
the highest yield of the disaccharide 12 among the com-
pounds tested (Table 1, entry 1).[14] The resulting disacchar-
ide 12 was activated with N-iodosuccinimide (NIS) and a
catalytic amount of TfOH in the presence of 1.5 equivalents
of acceptor 11 to give the trisaccharide 13 in 75% yield with
good b-selectivity. The one-pot synthesis of trisaccharide 13
from the three building blocks 6a, 10, and 11 was next ex-
amined. Treatment of the hemiacetal 10a and thioglycoside
11 with Tf2O, TTBP, and Ph2SO, followed by the addition of
2.0 equivalents of acceptor 11 and NIS resulted in the
Scheme 3. Stepwise, one-pot synthesis of pentasaccharide 16.
and imidate 5b or 5c provided an anomeric mixture of the
disaccharide 15 (Table 2, entries 2 and 3). On the other
hand, activation of the hemiacetal 5a with Tf2O, Ph2SO, and
TTBP in the presence of thioglycoside 7 provided the disac-
charide 15 in 92% yield with excellent a-selectivity (a/b=
92:8; Table 2, entry 1). The disaccharide 15 was activated by
reaction with NIS and a catalytic amount of TfOH in the
presence of trisaccharide 9 to give the pentasaccharide 16 in
92% yield. The one-pot synthesis of pentasaccharide 16
from the three building blocks 5a, 7, and 9 was then exam-
ined. Treatment of the hemiacetal 5a and thioglycoside 7
with Tf2O, TTBP, and Ph2SO, followed by the addition of
1.2 equivalents of acceptor 9 and NIS resulted in the stereo-
selective formation of the trisaccharide 16 in 52% yield
based on 7. The chloroacetyl group of the trisaccharide was
removed by treatment with thiourea to give the alcohol 17
in 96% yield.
stereoACHTUNGTRENNUNGselective formation of the trisaccharide 13 in 54%
yield based on 10. The chloroacetyl group of the trisacchar-
ide 13 was removed by treatment with thiourea to give the
alcohol 9 in 94% yield, which was used as the acceptor in
the next one-pot glycosylation.
The synthesis of the pentasaccharide 17 was examined
next (Scheme 3). We first examined the chemo- and a-selec-
tive glycosylation of thioglycoside 7[15] with the 2-azido-gly-
cosyl donors 5a–c with a different leaving group (Table 2).
Glycosylation of the thioglycoside 7 with glycosyl bromide
The one-pot synthesis of pentasaccharides 2c–d following
the established method was examined (Scheme 4). Treat-
ment of the hemiacetal 6a and thioglycosides 7 and 8 with
Tf2O, TTBP, and Ph2SO provided the disaccharides 18 and
19, respectively. The successive addition of 1.2 equivalents
of acceptor 9 and NIS to the reaction mixture provided the
trisaccharide 20 and 22 in 46 and 41% overall yields based
Table 2. a-Selective galactosylation at the C3 position of glucose 12.
Entry Donor Conditions
Yield a/b[a]
[%]
1
2
3
5a
5b
5c
Tf2O, TTBP, Ph2SO ꢀ78 to ꢀ308C, 4.0 h 77
92:8
63:37
74:26
AgOTf, ꢀ78 to ꢀ408C, 3.0 h
57
66
TMSOTf, ꢀ788C, 2.0 h
1
[a] The ratio was determined based on the H NMR spectra.
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3179

H. Tanaka, T. Takahashi et al.
Scheme 5. Chemo- and a-stereoselective glycosylation of 8 with hemiace-
tal 6a.
supported pentasaccharides to a mixed solution (1m HCl aq.
CH2Cl2/MeOH) provided the fully deprotected pentasac-
charides 2a, 2c, and 2d in 43, 34 and 43% overall yields
based on 17, 21, and 23, respectively. Partial hydrolysis of
the 2-trimethylsilylethyl glycosides occurred when TFA was
used as an acid instead of HCl.
A biological evaluation of the pentasaccharides 2a, 2c
and 2d, and the related oligosaccharides 27–31 was carried
out (Figure 1).[15] We first examined the use of a fluores-
Scheme 4. One-pot synthesis of pentasaccharides 20 and 22.
on 7 and 8, respectively. The chloroacetyl group of the tri-
saccharides 20 and 22 was removed by treatment with thio-
urea to afford the alcohol 21 and 23 in 86 and 88% yields.
However, the synthesis of pentasaccharide 2b through a
one-pot glycosylation failed because the glycosylation of the
benzoate acceptor 8 with the azide donor 6a did not pro-
ceed under these reaction conditions (Scheme 5). Increasing
the reaction temperature and prolonging the reaction time
resulted in the decomposition of the substrates 5a and 8.
The procedure used for the polymer-assisted deprotection
of the protected oligosaccharides 17, 21, and 23 is shown in
Scheme 6. Acetalization of pentasaccharides 17, 21, and 23
with the prelinker 3 under acidic conditions, followed by
amidation of the remaining activated ester with amine 4 at-
tached to an ArgoPore resin provided the solid-supported
protected oligosaccharides 24–26. The esters and carbamate
were removal under basic conditions. The azide group was
reduced to an amine by treatment with trimethylphosphine.
The amino groups were converted into acetamides. The
benzyl ethers and benzylidene acetals were removed under
Birch reduction conditions. Finally, exposure of the solid-
Figure 1. Structure of Forssman antigen-related oligosaccharides 27–31.
cence enhancement assay to analyze the interaction between
the synthetic oligosaccharides and FITC-labeled SLL-2.
Figure 2 shows the relative fluorescent intensity of a mixture
of FITC-labeled SLL-2 and the oligosaccharide based on
the FITC-labeled SLL-2. The affinity of the oligosaccharides
for SLL-2 was dependent on the number of sugar units in
the oligosaccharide. The pentasaccharide 5 exhibited the
strongest binding affinity. Modification of the GalNAc unit
to a Gal unit reduced the binding affinity to SLL-2. These
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Forssman Antigen Pentasaccharide and Derivatives
FULL PAPER
Figure 2. A fluorescence enhancement assay for analysis of the interac-
tion between the synthetic oligosaccharides 2a, 2c, 2d, and 27–31 and
FITC-labeled SLL-2. The error bar indicates standard deviation (n=3).
Figure 3. Inhibitory effect of the oligosaccharides on the SLL-2-induced
morphological change of CS-156. The black bars and the white bars indi-
cate CS-156 that was cultured with or without SLL-2, respectively. The
error bar represents the standard deviation (n>3).
form. Figure 3 provides information on the inhibitory effect
of the oligosaccharides on the SLL-2-induced morphological
change in CS-156. The inhibitory effects of the oligosacchar-
ides were dependent on their binding affinity for SLL-2,
except for the original pentasaccharide 2a. These results
clearly indicate that the binding site of the oligosaccharides
to SLL-2 is comparable to that of the native ligand on Sym-
biodinium. On the other hand, it should be noted that the
Forssman pentasaccharide 2a directly initiates a morpholog-
ical change in CS-156. These unexpected results suggest that
CS-156 might have receptors for the Forssman pentasacchar-
ide, which promote this morphological change. However,
the mechanism of this phenomenon is not currently clear.
Additional biological studies are required to clarify this
aspect of the process.
Scheme 6. Polymer-assisted deprotection of 17, 21, and 23.
results indicate that SLL-2 not only recognized the acet-
amide group at the non-reducing end of the Forsmann anti-
gen, but also the sugar units at the reducing end. In addi-
tion, a-GalNAc 31 showed a stronger affinity than b-
GalNAc 30, comparable to that of tetrasaccharide 27. A hy-
drophobic interaction of the 2-trimethysilylethyl group with
the SLL-2 binding pocket might be responsible for the en-
hanced binding affinity of the monosaccharide unit.
We next examined the inhibition of the SLL-2-induced
morphological change caused by oligosaccharides 2a, 2c,
2d, and 27–31. SLL-2 promotes a morphological change in
symbiotic microalgae from the motile form to a coccoid
Conclusion
We have described the synthesis of the Forssman antigen
pentasaccharide 2a and its derivatives 2c and 2d by two
one-pot glycosylation reactions and polymer-assisted depro-
tection and their biological evaluation. The chemo-and a-
stereoselective glycosylation of thioglycosides with the hemi-
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H. Tanaka, T. Takahashi et al.
remove a trace amount of H2O. Then, the reaction mixture was cooled to
ꢀ788C. After 5 min, Tf2O (8.30 mL, 49.5 mmol) was added to the reaction
mixture at the same temperature. After being stirred at ꢀ408C for 1 h, a
solution of 10 (16.7 mg, 27.5 mmol; azeotroped twice with dry toluene) in
dry CH2Cl2 (1.00 mL) was added at the same temperature. After being
stirred for 2 h, the reaction mixture was neutralized with NEt3 and fil-
tered through a pad of Celite. Then, the filtrate was concentrated in
vacuo. The residue was purified by column chromatography on silica gel
(elution with 99:1 CHCl3/MeOH) and by using gel permeation chroma-
tography (GPC) to give 12 (25.2 mg, 25.2 mmol, 92%). [a]2D6 = +129 (c=
1.22, CHCl3); 1H NMR (400 MHz, CDCl3): d=8.08 (d, 2H, J=7.3 Hz),
7.60 (dd, 1H, J=7.3, 7.7 Hz), 7.50–7.04 (m, 25H), 5.56 (dd, 1H, J=9.7,
10.2 Hz), 5.50 (dd, 1H, J=3.4, 10.6 Hz), 5.41 (s, 1H), 5.23 (d, 1H, J=
3.9 Hz), 4.84 (d, 1H, J=12.1 Hz), 4.72 (d, 1H, J=12.1 Hz), 4.70 (d, 1H,
J=12.1 Hz), 4.59 (brd, 1H, J=3.4 Hz), 4.53 (d, 1H, J=9.7 Hz), 4.46 (d,
1H, J=12.1 Hz), 4.34 (brs, 1H), 4.27 (brd, 1H, J=2.9 Hz), 4.26 (d, 1H,
J=11.6 Hz), 4.24 (dd, 1H, J=3.9, 10.6 Hz), 4.18 (d, 1H, J=11.6 Hz),
4.03 (d, 1H, J=15.0 Hz), 4.02 (dd, 1H, J=7.7, 8.7 Hz), 3.95 (d, 1H, J=
15.0 Hz), 3.63 (brs, 2H), 3.57 (dd, 1H, J2,3 =2.9, 10.2 Hz), 3.49 (dd, 1H,
J=5.8, 8.7 Hz), 2.44 ppm (s, 6H); 13C NMR (100 MHz, CDCl3): d=166.6,
165,2, 144.2, 138.4, 137.9, 137.8, 137.3, 133.1, 131.5, 130.0, 129.9, 129.8,
129.0, 128.4, 128.3, 128.0, 127.7, 127.6, 127.5, 127.4, 127.1, 126.1, 100.7,
100.4, 88.6, 80.4, 74.3, 74.2, 73.8, 73.7, 73.6, 73.2, 71.5, 70.4, 69.0, 67.5,
62.4, 40.8, 22.5 ppm; FTIR (KBr): n˜ =3421, 2927, 1732, 1636, 1384, 1265,
acetal donors 6a and 5a by using a mixture of Tf2O, TTBP,
and Ph2SO was effective for formation of a-glycosidic link-
age in one-pot glycosylation. However, the chemoselective
and a-selective glycosylation of thioglycoside 8 containing a
benzoate at the C2 position with the 2-azide glycoside 6a
did not proceed due to the low reactivity the 2-azide glyco-
syl donor 6a. An analysis of the interaction between the
synthetic oligosaccharides 2a, 2c, 2d, and 27–31 and the
FITC-labeled SLL-2 by a fluorescence assay revealed that
the NHAc substituents and the length of the oligosaccharide
chains were important for the binding of the oligosaccharide
to SLL-2. The inhibition effect of the oligosaccharide, rela-
tive to the morphological changes of Symbiodinium by SLL-
2, was comparable to their binding affinity to SLL-2. In ad-
dition, we fortuitously discovered that the synthetic penta-
saccharide 2a directly promotes the morphological change
in Symbiodinium. These results strongly indicate that the
Forssman antigen also functions as a chemical mediator of
Symbiodinium. A biological study to elucidate the mecha-
nism of the morphological change derived from the penta-
saccharide 2a is currently in progress.
1096, 756 cmꢀ1
; HRMS (ESI-TOF): m/z calcd for C57H61NO12SCl
1018.3603 [M+NH4]+; found: 1018.3598.
2-(Trimethylsilyl)ethyl (2-O-benzyl-4,6-di-O-benzylidene-3-O-chloroace-
tyl-a-d-galactopyranosyl)-(1!4)-(2-O-benzoyl-3,6-di-O-benzyl-4-O-b-d-
galactopyranosyl)-(1!4)-2,3,6-tri-O-benzyl-b-d-glucopyranoside (13): A
mixture of 12 (45.0 mg, 44.9 mmol), compound 11 (37.1 mg, 67.4 mmol;
azeotroped twice with dry toluene) and pulverized activated MS 4ꢂ
(120 mg) in dry CH2Cl2 (1.20 mL) was stirred at room temperature for
1 h under argon to remove a trace amount of H2O. Then, the reaction
mixture was cooled to ꢀ788C. After 5 min, NIS (15.2 mg, 67.4 mmol) and
a catalytic amount of TfOH (2.00 mL, 22.5 mmol) was added to the reac-
tion mixture at the same temperature. After being stirred for 3.5 h,
during which time the solution was allowed to warm to ꢀ608C, the reac-
tion mixture was neutralized with NEt3 and filtered through a pad of
Celite. Then, the filtrate was concentrated in vacuo and the residue was
purified by column chromatography on silica gel (elution with CHCl3/
MeOH=99:1) and gel permeation chromatography (GPC) to give 13
(47.8 mg, 33.8 mmol, 75%). [a]2D5 =+88.3 (c=0.890, CHCl3); 1H NMR
(400 MHz, CDCl3): d=8.00 (d, 2H, J=8.2 Hz), 7.61–7.07 (m, 38H,), 5.46
(dd, 1H, J=8.2, 10.2 Hz), 5.41 (dd, 1H, J=3.4, 10.6 Hz), 5.38 (s, 1H),
5.20 (d, 1H, J=2.9 Hz), 5.05 (d, 1H, J=11.6 Hz), 4.87 (d, 1H, J=
10.6 Hz), 4.84 (d, 1H, J=11.6 Hz), 4.82 (d, 1H, J=8.2 Hz), 4.74 (d, 1H,
J=12.6 Hz), 4.68 (brs, 2H), 4.65 (d, 1H, J=10.6 Hz), 4.54 (brd, 1H, J=
3.4 Hz), 4.52 (d, 1H, J=12.1 Hz), 4.38 (d, 1H, J=12.1 Hz), 4.33 (brs,
1H), 4.31 (d, 1H, J=8.2 Hz), 4.26 (d, 1H, J=12.1 Hz), 4.24 (d, 1H, J=
12.1 Hz), 4.23 (brs, 1H), 4.22 (dd, 1H, J=2.9, 10.2 Hz), 4.21 (d, 1H, J=
12.1 Hz), 4.18 (dd, 1H, J=7.3, 8.7 Hz), 3.93 (dt, 1H, J=7.3, 9.2 Hz), 3.88
(d, 1H, J=15.0 Hz), 3.86 (dd, 1H, J=9.2, 9.7 Hz), 3.80 (d, 1H, J=
15.0 Hz), 3.66 (brd, 1H, J=12.6 Hz), 3.63 (dd, 1H, J=8.7, 9.2 Hz), 3.61
(dd, 1H, J=9.2, 9.7 Hz), 3.58 (brd, 1H, J=12.6 Hz), 3.54 (dd, 1H, J=
6.8, 9.7 Hz), 3.53 (dt, 1H, J=7.3, 9.2 Hz), 3.44 (dd, 1H, J=2.9, 10.2 Hz),
3.43–3.31 (m, 4H), 0.99 (dt, 2H, J=7.3, 9.2 Hz), 0.00 ppm (s, 9H, Si-
Experimental Section
General procedure: NMR spectra were recorded on a JEOL Model EX-
270 (270 MHz for 1H, 67.8 MHz for 13C) or a JEOL Model ECP-400
(400 MHz for 1H, 100 MHz for 13C) in the indicated solvent. Chemical
shifts were reported in part per million (ppm) relative to the signal
(0.00 ppm) for internal tetramethylsilane solutions in CDCl3. 1H NMR
spectral data are reported as follows: CDCl3 (7.26 ppm), CD2Cl2
(5.32 ppm), [D6]acetone (2.04 ppm), [D6]DMSO (2.50 ppm), CD3OD
(3.30 ppm) or D2O (HOD (4.7015 ppm at 303 K, 4.5977 ppm at 313 K,
4.5541 ppm at 318 K, 4.3168 ppm at 343 K)). 13C NMR spectral data are
reported as follows: CDCl3 (77.0 ppm), CD2Cl2 (53.8 ppm), [D6]acetone
(29.8 ppm), [D6]DMSO (39.5 ppm), CD3OD (49.8 ppm) or D2O
([D6]acetone (206.0 ppm) as an internal standard). Infrared spectra (IR)
were recorded on a Perkin–Elmer Spectrum 1. Only the strongest and/or
structurally important absorbances are reported as the IR data given in
cmꢀ1. Optical rotations were measured on a JASCO model P-1020 polar-
imeter. All reactions were monitored by using thin layer chromatography
carried out on Merck precoated TLC plates (60F-254) by using UV light
and p-anisaldehyde H2SO4 ethanol solution or 10% ethanolic phospho-
molybdic acid. Column chromatography separations were performed by
using silica gel (Merck, silica gel). Flash column chromatography separa-
tions were performed by using silica gel (KANTO, silica gel 60N, spheri-
cal, neutral, 40–100 mm). ESI-TOF Mass spectra were measured with Ap-
pliedBioSystems Mariner TK-3500 Biospectrometry Workstation mass
spectrometers and Waters LCT Premier XE. HRMS (ESI-TOF) were
calibrated with angiotensin I (SIGMA), bradykinin (SIGMA), and neu-
rotensin (SIGMA) as an internal standard. Gel permeation chromatogra-
phy (GPC) for qualitative analysis were performed on Japan Analytical
Industry Model LC908 (recycling preparative HPLC) by using a polysty-
lene gel column (JAIGEL-1H, 20 mmꢁ600 mm). The detection of prod-
ucts was achieved by using a UV detector (Japan Analytical Industry
Model 310) and refractive index detector (Japan Analytical Industry
Model RI-5)
AHCTUNGTRENNUNG
(CH3)3); 13C NMR (100 MHz, CDCl3): d=166.4, 160.1, 139.5, 138.7,
138.4, 138.2, 138.1, 137.8, 137.4, 133.1, 130.0, 129.8, 128.7, 128.4, 128.3,
128.2, 128.1, 128.0, 127.9, 127.7, 127.6, 127.5, 127.0, 126.9, 126.1, 102.9,
101.1, 101.0, 100.3, 83.1, 82.0, 79.3, 77.6, 76.5, 75.1, 74.7, 74.3, 73.9, 73.8,
73.5, 73.2, 73.1, 72.2, 71.2, 69.0, 68.5, 67.3, 67.1, 62.6, 60.3, 40.6, 18.4, 14.2,
ꢀ1.49 ppm; FTIR (neat): n˜ =2922, 1732, 1453, 1267, 1096, 736, 698 cmꢀ1
;
HRMS (ESI-TOF): m/z calcd for C81H93NO18ClSi: 1430.5851 ACHTNUGRTNEUNG
[M+NH4]+;
found 1430.5850.
2,6-Dimethylphenylthio (2-O-benzyl-4,6-O-benzylidene-3-O-chloroacetyl-
a-d-galactopyranoyl)-(1!4)-2-O-benzoyl-3,6-di-O-benzyl-4-O-b-d-galac-
topyranoside (12): A mixture of 6a (17.9 mg, 41.2 mmol), Ph2SO (13.3 mg,
66.0 mmol), TTBP (17.7 mg, 71.4 mmol; azeotroped twice with dry tol-
uene), and pulverized activated MS 4ꢂ (200 mg) in dry CH2Cl2
(1.00 mL) was stirred at room temperature for 1 h under argon to
One-pot synthesis of 13: A mixture of 6a (17.5 mg, 40.3 mmol), Ph2SO
(11.3 mg, 55.7 mmol), TTBP (16.1 mg, 65.0 mmol) was azeotroped twice
with dry toluene and was diluted with dry CH2Cl2 (500 mL). The solution
was stirred at room temperature for 1 h under argon in the presence of
activated MS 4ꢂ (200 mg) to remove a trace amount of H2O. Then, Tf2O
3182
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 3177 – 3187

Forssman Antigen Pentasaccharide and Derivatives
FULL PAPER
(7.80 mL, 46.4 mmol) was added to the reaction mixture at ꢀ788C and stir-
red at ꢀ408C for 1 h. A solution of 10 (18.1 mg, 31.0 mmol) was azeotrop-
ed twice with dry toluene, diluted with dry CH2Cl2 (500 mL), and the sol-
ution was added to the reaction mixture at ꢀ788C. After additionally
being stirred for 2.5 h at the same temperature, the reaction mixture was
warmed to 08C. After being stirred for 30 min, the reaction mixture was
cooled to ꢀ788C. After 5 min, a solution of 11 (34.1 mg, 61.9 mmol) in
dry CH2Cl2 (800 mL), NIS (17.4 mg, 77.3 mmol, 2.50 equiv) and TfOH
(2.80 mL, 30.1 mmol) was added to the reaction mixture. After being stir-
red for 2.5 h at the same temperature, the reaction mixture was neutral-
ized with NEt3 and filtered through a pad of Celite. Then, the filtrate was
concentrated in vacuo. The residue was purified by column chromatogra-
phy on silica gel (elution with CHCl3/MeOH=99:1) and further purified
by gel permeation chromatography (GPC) to give 13 (23.8 mg, 15.7 mmol,
54% based on 10).
CHCl3/MeOH=99:1) and gel permeation chromatography (GPC) to
give 15 (102 mg, 116 mmol, 77%, a/b=92:8). The a/b ratio was deter-
mined by 1H NMR spectroscopic analysis. The a isomer was purified by
an additional column chromatography on silica gel. [a]2D5 =+127 (c=1.15,
CH2Cl2); 1H NMR (400 MHz, [D6]acetone): d=7.61 (dd, 2H, J=1.4,
7.7 Hz), 7.61 (dd, 2H, J=5.3, 7.7 Hz), 7.39–7.33 (m, 6H), 7.24 (brd, 1H,
J=9.2 Hz), 7.16–7.10 (m, 3H,), 5.71 (s, 1H), 5.63 (s, 1H), 5.48 (d, 1H, J=
3.4 Hz), 5.31 (dd, 1H, J=3.4, 11.1 Hz), 4.96 (d, 1H, J=12.1 Hz), 4.78 (d,
1H, J=12.1 Hz), 4.72 (d, 1H, J=10.2 Hz), 4.66 (brd, 1H, J=3.4 Hz),
4.57 (brd, 1H, J=3.4 Hz), 4.37 (d, 1H, J=15.0 Hz), 4.31 (d, 1H, J=
15.0 Hz), 4.25 (brdd, 1H, J=3.4, 10.6 Hz), 4.18 (dd, 1H, J=10.2,
10.6 Hz), 4.16–4.12 (m, 4H), 4.05 (brs, 1H), 3.94 (dd, 1H, J=3.4,
11.1 Hz), 3.53 (brs, 1H), 2.55 ppm (s, 6H); 13C NMR (67.8 MHz,
[D6]acetone): d=167.6, 155.2, 147.6, 144.9, 140.0, 139.3, 132.3, 131.7,
130.2, 129.6, 129.5, 129.2, 128.8, 127.1, 127.0, 125.0, 101.1ꢁ2, 96.8, 96.2,
89.5, 76.4, 75.1, 74.0, 72.2, 71.5, 70.4, 69.5, 63.8, 57.9, 53.4, 41.4, 22.7 ppm;
2-(Trimethylsilyl)ethyl (2-O-benzyl-4,6-di-O-benzylidene-a-d-galactopy-
FTIR (KBr): n˜ =2115, 1763, 1708, 1443, 1090, 1044, 756, 537 cmꢀ1
;
A
ACHTUNGTRENNUNG
HRMS (ESI-TOF): m/z calcd for C39H44N5O11SiCl4: 930.1512 ACHTNUGRTNEUNG
[M+NH4]+;
found: 930.1531.
149 mmol) and 2,6-lutidine (14.0 mL, 120 mmol) were added to a stirred
solution of 13 (35.2 mg, 24.9 mmol) in dry DMF (600 mL) at room temper-
ature. After being stirred at 508C for 14 h, the reaction mixture was
poured into 1m HCl with cooling. The aqueous layer was extracted with
two portions of ethyl acetate. The combined extract was washed with 1m
HCl, saturated aq. NaHCO3 and brine, dried over MgSO4, filtered and
concentrated in vacuo. The residue was purified by chromatography on
2-(Trimethylsilyl)ethyl
(2-azido-4,6-O-benzylidene-3-O-chloroacetyl-2-
deoxy-a-d-galactopyranosyl)-(1!3)-[4,6-O-benzylidene-2-deoxy-(2,2,2-
trichloroethoxycarbonylamino)-b-d-galactopyranosyl]-(1!3)-(2-O-
benzyl-4,6-di-O-benzylidene-a-d-galactopyranosyl)-(1!4)-(2-O-benzoyl-
3,6-di-O-benzyl-a-d-galactopyranosyl)-(1!4)-2,3,6-tri-O-benzyl-b-d-glu-
copyranoside (16): A mixture of 15 (45.5 mg, 45.6 mmol), compound 9
(30.5 mg, 22.8 mmol; azeotroped twice with dry toluene) and pulverized
activated MS 4ꢂ (50.0 mg) in dry CH2Cl2 (1.20 mL) was stirred at room
temperature for 1 h under argon to remove a trace amount of H2O.
Then, the reaction mixture was cooled to ꢀ208C. After 5 min, NIS
silica gel (elution with toluene/acetone=96:4) to give
5 (31.2 mg,
23.3 mmol, 94%). [a]2D0 =+89.3 (c=1.06, CHCl3); 1H NMR (400 MHz,
[D6]acetone): d=8.11 (dd, 2H, J=1.0, 7.7 Hz), 7.99–7.10 (m, 48H), 5.71
(s, 1H), 5.64 (s, 1H), 5.55 (dd, 1H, J=7.7, 10.2 Hz), 5.47 (s, 1H), 5.30 (d,
1H, J=3.4 Hz), 5.08 (d, 1H, J=3.4 Hz), 5.04 (brs, 2H), 5.03 (d, 1H, J=
7.7 Hz), 4.92 (d, 1H, J=12.1 Hz), 4.90 (d, 1H, J=11.1 Hz), 4.77 (d, 1H,
J=12.6 Hz), 4.72 (d, 1H, J=8.7 Hz), 4.67 (d, 1H, J=12.1 Hz), 4.62 (brd,
1H, J=2.9 Hz), 4.61 (d, 1H, J=11.1 Hz), 4.52 (brs, 1H), 4.51 (d, 1H, J=
12.6 Hz), 4.43 (d, 1H, J=11.1 Hz), 4.42 (d, 1H, J=8.2 Hz), 4.34 (d, 1H,
J=10.2 Hz), 4.33 (dd, 1H, J=2.9, 10.2 Hz), 4.32 (brd, 1H, J=2.9 Hz),
4.31 (brd, 1H, J=10.2 Hz), 4.30 (brs, 2H), 4.25 (d, 1H, J=12.1 Hz), 4.23
(brd, 1H, J=12.1 Hz), 4.18 (dd, 1H, J=6.3, 8.7 Hz), 4.17 (d, 1H, J=
10.2 Hz), 4.15 (brd, 1H, J=10.2 Hz), 4.11 (brdd, 1H, J=8.7, 10.6 Hz),
4.10 (d, 1H, J=12.1 Hz), 4.09 (dd, 1H, J=3.4, 11.6 Hz), 4.09 (dd, 1H,
J=1.4, 12.1 Hz), 3.95 (dd, 1H, J=3.4, 10.2 Hz), 3.94 (dd, 1H, J=3.4,
10.6 Hz), 3.93 (brs, 1H), 3.90 (dd, 1H, J=8.7, 9.7 Hz), 3.92 (dt, 1H, J=
7.7, 8.7 Hz), 3.80 (dd, 1H, J=2.9, 10.2 Hz), 3.76 (d, 1H, J=12.1 Hz),
3.68–3.62 (m, 6H), 3.57 (dd, 1H, J=4.8, 9.2 Hz), 3.56 (dd, 1H, J=1.9,
10.6 Hz), 3.45 (ddd, 1H, J=3.4, 4.8, 9.7 Hz), 3.38 (dd, 1H, J=5.8,
9.2 Hz), 3.31 (dd, 1H, J=8.2, 9.2 Hz), 0.98 (t, 2H, J=7.7 Hz), 0.00 ppm
(s, 9H); 13C NMR (100 MHz, [D6]acetone): d=165.6, 155.4, 141.2, 140.8,
140.5, 140.3, 140.2, 140.1, 140.0, 139.7, 134.6, 131.7, 131.0, 130.0, 129.9,
129.6, 129.5, 129.4, 129.3, 129.1, 129.0, 128.9, 128.8, 128.7, 128.6, 128.5,
128.2, 127.8, 127.6, 104.2, 104.0, 102.7, 101.9ꢁ2, 101.6, 101.5, 97.4, 96.3,
83.7, 83.6, 80.8, 80.1, 79.1, 78.1, 77.8, 76.2, 75.7, 75.5, 75.4, 75.0, 74.8, 74.6,
74.3, 74.1, 74.0, 73.7, 72.3, 72.2, 70.6, 70.4, 70.3, 70.1, 68.7, 67.8. 67.6, 67.5,
64.8, 64.4, 61.2, 54.3, 19.4, ꢀ0.79 ppm; FTIR (KBr): n˜ =3420, 2923, 2111,
1730, 1453, 1268, 1219, 1097, 1023, 772 cmꢀ1; HRMS (ESI-TOF): m/z
calcd for C108H121N5O27SiCl3: 2052.7084 [M+NH4]+; found: 2052.7026.
(12.3 mg, 54.7 mmol) and
a catalytic amount of TfOH (1.00 mL,
11.4 mmol) was added to the reaction mixture at the same temperature.
After being stirred for 2 h, during which time the solution was allowed to
warm to 08C, the reaction mixture was neutralized with NEt3 and filtered
through a pad of Celite. Then, the filtrate was concentrated in vacuo and
the residue was purified by column chromatography on silica gel (elution
with CHCl3/MeOH=99:1) and gel permeation chromatography (GPC)
to give 16 (41.3 mg, 19.5 mmol, 92%). [a]2D7 = +110 (c=1.28, CHCl3);
1H NMR (400 MHz, [D6]acetone): d=8.10 (dd, 2H, J=1.0, 7.3 Hz), 7.99–
7.13 (m, 48H), 6.56 (brd, 1H, J=9.7 Hz), 5.70 (s, 1H), 5.65 (s, 1H), 5.55
(dd, 1H, J=7.7, 10.2 Hz), 5.47 (s, 1H), 5.46 (d, 1H, J=3.9 Hz), 5.23
(brdd, 1H, J=3.4, 11.1 Hz), 5.08 (d, 1H, J=3.4 Hz), 5.04 (brs, 2H), 5.04
(d, 1H, J=7.7 Hz), 4.92 (d, 1H, J=12.6 Hz), 4.90 (d, 1H, J=11.6 Hz),
4.77 (d, 1H, J=12.1 Hz), 4.74 (d, 1H, J=7.7 Hz), 4.68 (d, 1H, J=
12.6 Hz), 4.64 (d, 1H, J=11.6 Hz), 4.63 (brs, 1H), 4.56 (brs, 1H), 4.52
(d, 1H, J=12.1 Hz), 4.43 (d, 1H, J=7.7 Hz), 4.42 (d, 1H, J=11.6 Hz),
4.35–4.30 (m, 6H), 4.28 (d, 1H, J=13.1 Hz), 4.24 (brd, 1H, J=12.1 Hz),
4.21 (d, 1H, J=11.6 Hz), 4.19 (dd, 1H, J=5.3, 9.2 Hz), 4.17 (d, 1H, J=
11.6 Hz), 4.17 (brdd, 1H, J=7.7, 10.6 Hz), 4.16 (d, 1H, J=12.1 Hz), 4.16
(brd, 1H, J=11.1 Hz), 4.15 (brs, 1H), 4.12 (d, 1H, J=12.1 Hz), 4.12
(brd, 1H, J=12.1 Hz), 4.02 (brs, 1H), 3.95 (dd, 1H, J=3.4, 10.6 Hz),
3.94 (dt, 1H, J=7.7, 9.7 Hz), 3.94 (dd, 1H, J=3.9, 11.1 Hz), 3.90 (dd,
1H, J=9.2 Hz, 9.7 Hz), 3.85 (d, 1H, J=12.6 Hz), 3.81 (dd, 1H, J=2.4,
10.6 Hz), 3.68 (dd, 1H, J=4.8, 5.3 Hz), 3.66 (dd, 1H, J=9.7, 9.7 Hz), 3.66
(dd, 1H, J=4.8, 8.7 Hz), 3.62 (dt, 1H, J=7.7, 9.7 Hz), 3.59 (dd, 1H, J=
4.8, 8.7 Hz), 3.58 (brd, 1H, J=11.6 Hz), 3.56 (brd, 1H„ J=11.6 Hz), 3.46
(brdt, 1H, J=4.8, 9.7 Hz), 3.45 (brs, 1H), 3.38 (dd, 1H, J=4.8, 9.2 Hz),
3.31 (dd, 1H, J=7.7, 9.7 Hz), 0.98 (t, 2H, J=7.7 Hz), 0.00 ppm (s, 9H);
13C NMR (67.8 MHz, [D6]acetone): d=168.0, 166.6, 155.5, 141.2, 140.8,
140.5, 140.4, 140.2, 139.9, 139.8, 139.6, 134.6, 131.2, 131.0, 130.0, 129.7,
129.6, 129.5, 129.4, 129.3, 129.1, 129.0, 128.9, 128.8, 128.7, 128.6, 128.5,
128.4, 128.2, 127.7, 127.6, 104.2, 103.9, 102.7, 101.9, 101.7, 101.6, 101.5,
97.2, 95.8), 83.4, 80.8, 80.2, 79.1, 78.1, 76.1, 75.7, 75.4, 75.0, 74.8, 74.5,
74.1, 74.0, 73.7, 72.3, 72.0, 71.8, 70.6, 70.3, 70.1, 68.7, 67.8, 67.5, 64.4, 64.1,
58.0, 54.1, 41.8, 19.4, ꢀ0.79 ppm; FTIR (KBr): n˜ =2921, 2112, 1731, 1454,
2,6-Dimethylphenylthio (2-azido-4,6-O-benzylidene-3-O-chloroacetyl-2-
deoxy-a-d-galactopyranosyl)-(1!3)-4,6-O-benzylidene-2-deoxy-(2,2,2-tri-
chloroethoxycarbonylamino)-b-d-galactopyranoside (15): A mixture of
6a (84.0 mg, 227 mmol), Ph2SO (85.6 mg, 423 mmol), TTBP (113 mg,
454 mmol; azeotroped twice with dry toluene) and pulverized activated
MS 4ꢂ (150 mg) in dry CH2Cl2 (1.00 mL) were stirred at room tempera-
ture for 1 h under argon to remove a trace amount of H2O. Then, the re-
action mixture was cooled to ꢀ788C. After 5 min, Tf2O (53.4 mL,
318 mmol) was added to the reaction mixture at the same temperature.
After being stirred at ꢀ408C for 1 h, a solution of 7 (81.9 mg, 151 mmol;
azeotroped twice with dry toluene) in dry CH2Cl2 (5.00 mL) was added
at the same temperature. After being stirred for 1 h at the same tempera-
ture, the reaction mixture was neutralized with NEt3 and filtered through
a pad of Celite. Then, the filtrate was concentrated in vacuo and the resi-
due was purified by column chromatography on silica gel (elution with
1268, 1219, 1095, 772, 698 cmꢀ1
; HRMS (ESI-TOF): m/z calcd for
C110H122N5O28SiCl4: 2128.6800 [M+NH4]+; found: 2128.6799.
One-pot synthesis of 16: A mixture of 5a (28.0 mg, 64.7 mmol), Ph2SO
(20.1 mg, 99.6 mmol), TTBP (27.5 mg, 111 mmol) was azeotroped twice
with dry toluene and was diluted with dry CH2Cl2 (1.00 mL) in the pres-
Chem. Eur. J. 2013, 19, 3177 – 3187
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
3183

H. Tanaka, T. Takahashi et al.
ence of pulverized activated MS 4ꢂ (110 mg). The mixture was stirred at
room temperature for 1 h under argon to remove a trace amount of H2O.
Tf2O (13.0 mL, 77.5 mmol) was then added to the reaction mixture at
ꢀ788C. The reaction mixture was warmed at ꢀ408C and then stirred at
the same temperature for 1 h. Compound 7 (30.0 mg, 55.3 mmol) was
then azeotroped twice with dry toluene and diluted with dry CH2Cl2
(2.50 mL). After being stirred for 1 h, the solution of 7 was added to the
reaction mixture at the same temperature. After being stirred for 5 h at
the same temperature the reaction mixture was warmed to 08C and stir-
red for 30 min. The reaction mixture was then cooled to ꢀ788C. A solu-
tion of 9 (88.8 mg, 66.4 mmol) in dry CH2Cl2 (1.00 mL) and NIS (14.9 mg,
66.4 mmol) was then added to the reaction mixture at the same tempera-
ture. After being stirred for 1 h, the reaction mixture was neutralized
with NEt3 at ꢀ208C and filtered through a pad of Celite. Then, the fil-
trate was concentrated in vacuo. The residue was purified by column
chromatography on silica gel (elution with CHCl3/MeOH=99:1) and fur-
ther purified by gel permeation chromatography (GPC) to give 16
(58.6 mg, 28.9 mmol, 52% based on 7). [a]2D7 = +110 (c=1.28, CHCl3);
1H NMR (400 MHz, [D6]acetone): d=8.10 (dd, 2H, J=1.0, 7.3 Hz), 7.99–
7.13 (m, 48H), 6.56 (brd, 1H, J=9.7 Hz), 5.70 (s, 1H), 5.65 (s, 1H), 5.55
(dd, 1H, J=7.7, 10.2 Hz), 5.47 (s, 1H), 5.46 (d, 1H, J=3.9 Hz), 5.23
(brdd, 1H, J2,3 =3.4, 11.1 Hz), 5.08 (d, 1H, J=3.4 Hz), 5.04 (brs, 2H),
5.04 (d, 1H, J=7.7 Hz), 4.92 (d, 1H, J=12.6 Hz), 4.90 (d, 1H, J=
11.6 Hz), 4.77 (d, 1H, J=12.1 Hz), 4.74 (d, 1H, J=7.7 Hz), 4.68 (d, 1H,
J=12.6 Hz), 4.64 (d, 1H, J=11.6 Hz), 4.63 (brs, C-4), 4.56 (brs, 1H),
4.52 (d, 1H, J=12.1 Hz), 4.43 (d, 1H, J=7.7 Hz), 4.42 (d, 1H, J=
11.6 Hz), 4.35–4.30 (m, 6H), 4.28 (d, 1H, J=13.1 Hz), 4.24 (brd, 1H, J=
12.1 Hz), 4.21 (d, 1H, J=11.6 Hz), 4.19 (dd, 1H, J=5.3, 9.2 Hz), 4.17 (d,
1H, J=11.6 Hz), 4.17 (brdd, 1H, J=7.7, 10.6 Hz), 4.16 (d, 1H, J=
12.1 Hz), 4.16 (brd, 1H, J=11.1 Hz), 4.15 (brs, 1H), 4.12 (d, 1H, J=
11.1 Hz), 4.42 (d, 1H, J=8.2 Hz), 4.34 (d, 1H, J=10.2 Hz), 4.33 (dd, 1H,
J=2.9, 10.2 Hz), 4.32 (brd, 1H, J=2.9 Hz), 4.31 (brd, 1H, J=10.2 Hz),
4.30 (brs, 2H), 4.25 (d, 1H, J=12.1 Hz), 4.23 (brd, 1H, J=12.1 Hz), 4.18
(dd, 1H, J=6.3, 8.7 Hz), 4.17 (d, 1H, J=10.2 Hz), 4.15 (brd, 1H, J=
10.2 Hz), 4.11 (brdd, 1H, J=8.7, 10.6 Hz), 4.10 (d, 1H, J=12.1 Hz), 4.09
(dd, 1H, J=3.4, 11.6 Hz), 4.09 (dd, 1H, J=1.4, 12.1 Hz), 3.95 (dd, 1H,
J=3.4, 10.2 Hz), 3.94 (dd, 1H, J=3.4, 10.6 Hz), 3.93 (brs, 1H), 3.90 (dd,
1H, J=8.7, 9.7 Hz), 3.92 (dt, 1H, J=7.7, 8.7 Hz), 3.80 (dd, 1H, J=2.9,
10.2 Hz), 3.76 (d, 1H, J=12.1 Hz), 3.68–3.62 (m, 6H), 3.57 (dd, 1H, J=
4.8, 9.2 Hz), 3.56 (dd, 1H, J5,6a =1.9, 10.6 Hz), 3.45 (ddd, 1H, J=3.4, 4.8,
9.7 Hz), 3.38 (dd, 1H, J=5.8, 9.2 Hz), 3.31 (dd, 1H, J=8.2, 9.2 Hz), 0.98
(t, 2H, J=7.7 Hz), 0.00 ppm (s, 9H, SiACTHNUTRGNEUNG
(CH3)3); 13C NMR (100 MHz,
[D6]acetone): d=165.6, 155.4, 141.2, 140.8, 140.5, 140.3, 140.2, 140.1,
140.0, 139.7, 134.6, 131.7, 131.0, 130.0, 129.9, 129.6, 129.5, 129.4, 129.3,
129.1, 129.0, 128.9, 128.8, 128.7, 128.6, 128.5, 128.2, 127.8, 127.6, 104.2,
104.0, 102.7, 101.9ꢁ2, 101.6, 101.5, 97.4, 96.3, 83.7, 83.6, 80.8, 80.1, 79.1,
78.1, 77.8, 76.2, 75.7, 75.5, 75.4, 75.0, 74.8, 74.6, 74.3, 74.1, 74.0, 73.7, 72.3,
72.2, 70.6, 70.4, 70.3, 70.1, 68.7, 67.8. 67.6, 67.5, 64.8, 64.4, 61.2, 54.3, 19.4,
ꢀ0.79 ppm; FTIR (KBr): n˜ =3420, 2923, 2111, 1730, 1453, 1268, 1219,
1097, 1023, 772 cmꢀ1
;
HRMS (ESI-TOF): m/z calcd for
C108H121N5O27SiCl3: 2052.7084 [M+NH4]+; found: 2052.7026.
2-(Trimethylsilyl)ethyl (2-O-benzyl-4,6-O-benzylidene-3-O-chloroacetyl-
a-d-galactopyranosyl)-(1!3)-[4,6-O-benzylidene-2-deoxy-2-(2,2,2-tri-
chloroethoxycarbonylamino)-b-d-galactopyranosyl]-(1!3)-(2-O-benzyl-
4,6-O-benzylidene-a-d-galactopyranosyl)-(1!4)-(2-O-benzoyl-3,6-di-O-
benzyl-b-d-galactopyranosyl)-(1!4)-2,3,6-tri-O-benzyl-b-d-glucopyrano-
side (20):
A mixture of 6a (15.5 mg, 35.7 mmol), Ph2SO (9.30 mg,
45.9 mmol), TTBP (12.7 mg, 51.0 mmol; azeotroped twice with dry tol-
uene) and pulverized activated MS 4ꢂ (200 mg) in dry CH2Cl2 (1.00 mL)
was stirred at room temperature for 1 h under argon to remove a trace
amount of H2O. Then, the reaction mixture was cooled to ꢀ788C. After
5 min, Tf2O (6.00 mL, 35.7 mmol) was added to the reaction mixture at
the same temperature. After being stirred at ꢀ408C for 1 h, a solution of
7 (13.9 mg, 25.5 mmol; azeotroped twice with dry toluene) in dry CH2Cl2
(2.00 mL) was added at the same temperature. After being stirred for
1.5 h at the same temperature, the reaction mixture was warmed to 08C
and stirred for 30 min. The reaction mixture was then cooled to ꢀ258C.
After 5 min, a solution of 9 (40.9 mg, 30.5 mmol) in dry CH2Cl2 (750 mL)
and NIS (8.60 mg, 38.3 mmol) was added to the reaction mixture. After
being stirred for 2 h at the same temperature, the reaction mixture was
neutralized with NEt3 and filtered through a pad of Celite. Then, the fil-
trate was concentrated in vacuo and the residue was purified by column
chromatography on silica gel (elution with CHCl3/MeOH=99:1) and gel
permeation chromatography (GPC) to give 20 (22.3 mg, 10.5 mmol, 41%
based on 7). [a]2D3 = +106 (c=0.885, CHCl3); 1H NMR (400 MHz,
[D6]acetone): d=8.11 (dd, 2H, J=1.5, 7.3 Hz), 7.68 (t, 1H, J=7.3 Hz),
7.57–7.10 (m, 52H), 6.64 (brd, 1H, J=9.7 Hz), 5.66 (s, 1H), 5.60 (s, 1H),
5.55 (dd, 1H, J=7.7, 10.2 Hz), 5.48 (d, 1H, J=2.9 Hz), 5.47 (s, 1H), 5.25
(dd, 1H, J=3.4, 10.2 Hz), 5.08 (d, 1H, J=3.4 Hz), 5.04 (d, 1H, J=
7.7 Hz), 5.03 (brs, 2H), 4.95 (d, 1H, J=13.1 Hz), 4.90 (d, 1H, J=
11.1 Hz), 4.77 (d, 1H, J=12.1 Hz), 4.76 (d, 1H, J=7.7 Hz), 4.70 (d, 1H,
J=13.1 Hz), 4.65 (d, 1H, J=11.1 Hz), 4.64 (d, 1H, J=11.1 Hz), 4.63
(brd, 1H, J=2.4 Hz), 4.62 (d, 1H, J=12.1 Hz), 4.56 (brs, 1H), 4.53 (d,
1H, J=12.1 Hz), 4.51 (d, 1H, J=12.1 Hz), 4.48 (brd, 1H, J=3.4 Hz),
4.44 (d, 1H, J=7.7 Hz), 4.41 (d, 1H, J=11.6 Hz), 4.33 (brd, 1H, J=
2.9 Hz), 4.32 (dd, 1H, J=2.4, 9.7 Hz), 4.28 (d, 1H, J=12.1 Hz), 4.28
(brd, 1H, J=11.1 Hz), 4.25 (brs, 1H), 4.24 (d, 1H, J=12.6 Hz), 4.23 (d,
1H, J=14.1 Hz), 4.23 (brd, 1H, J=11.6 Hz), 4.18 (dd, 1H, J=8.7,
8.7 Hz), 4.16 (brdd, 1H, J=9.2, 10.2 Hz), 4.13–4.07 (m, 6H), 4.02 (dd,
1H, J=2.4, 10.2 Hz), 4.01 (brs, 1H), 3.96 (dd, 1H, J=3.4, 9.7 Hz), 3.95
(dt, 1H, J=6.8, 8.7 Hz), 3.89 (dd, 1H, J=8.7, 9.2 Hz), 3.83 (d, 1H, J=
12.1 Hz), 3.81 (dd, 1H, J=2.9, 10.2 Hz), 3.71–3.54 (m, 7H), 3.47 (dt, 1H,
J=4.8, 8.7 Hz), 3.41 (brs, 1H), 3.36 (dd, 1H, J=4.8, 8.7 Hz), 3.12 (dd,
1H, J=7.7, 8.7 Hz), 0.98 (t, 2H, J=8.7 Hz), 0.00 ppm (s, 9H); 13C NMR
(67.8 MHz, [D6]acetone): d=167.5, 166.0, 155.0, 140.6, 140.3, 139.9, 139.8,
139.6, 139.5, 139.4, 139.1, 134.0, 131.1, 130.5, 129.5, 129.4, 129.1, 129.0,
128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9,
127.8, 127.6, 127.3, 127.0, 103.8, 103.4, 102.2, 101.4, 101.3, 101.0, 100.9,
96.7, 94.9, 83.1, 83.0, 79.6, 77.6, 75.5, 75.2, 74.8, 74.4, 74.3, 74.2, 74.1, 74.0,
12.1 Hz), 4.12 (brd, 1H, J=12.1 Hz), 4.02 (brs, 1H), 3.95 (dd, 1H, J1,2
=
3.4 Hz, 10.6 Hz), 3.94 (dt, 1H, J=7.7, 9.7 Hz), 3.94 (dd, 1H, J1,2 =3.9,
11.1 Hz), 3.90 (dd, 1H, J=9.2, 9.7 Hz), 3.85 (d, 1H, J=12.6 Hz), 3.81
(dd, 1H, J=2.4, 10.6 Hz), 3.68 (dd, 1H, J=4.8, 5.3 Hz), 3.66 (t, 1H, J
9.7 Hz), 3.66 (dd, 1H, J=4.8, 8.7 Hz), 3.62 (dt, 1H, J=7.7, 9.7 Hz), 3.59
(dd, 1H, J=4.8, 8.7 Hz), 3.58 (brd, 1H, J=11.6 Hz), 3.56 (brd, 1H, J=
11.6 Hz), 3.46 (brdt, 1H, J=4.8, 9.7 Hz), 3.45 (brs, 1H), 3.38 (dd, 1H,
J=4.8, 9.2 Hz), 3.31 (dd, 1H, J=7.7, 9.7 Hz), 0.98 (t, 2H, J=7.7 Hz),
0.00 ppm (s, 9H, SiACHTUNGTRENNUNG
(CH3)3); 13C NMR (67.8 MHz, [D6]acetone): d=168.0,
166.6, 155.5, 141.2, 140.8, 140.5, 140.4, 140.2, 139.9, 139.8, 139.6, 134.6,
131.2, 131.0, 130.0, 129.7, 129.6, 129.5, 129.4, 129.3, 129.1, 129.0, 128.9,
128.8, 128.7, 128.6, 128.5, 128.4, 128.2, 127.7, 127.6, 104.2, 103.9, 102.7,
101.9, 101.7, 101.6, 101.5, 97.2, 95.8, 83.4, 80.8, 80.2, 79.1, 78.1, 76.1, 75.7,
75.4, 75.0, 74.8, 74.5, 74.1, 74.0, 73.7, 72.3, 72.0, 71.8, 70.6, 70.3, 70.1, 68.7,
67.8, 67.5, 64.4, 64.1, 58.0, 54.1, 41.8, 19.4, ꢀ0.79 ppm; FTIR (KBr): n˜ =
2921, 2112, 1731, 1454, 1268, 1219, 1095, 772, 698 cmꢀ1; HRMS (ESI-
TOF): m/z calcd for C110H122N5O28SiCl4: 2128.6800 [M+NH4]+; found:
2128.6799.
2-(Trimethylsilyl)ethyl (2-azido-4,6-O-benzylidene-2-deoxy-a-d-galacto-
pyranosyl)-(1!3)-[4,6-O-benzylidene-2-deoxy-(2,2,2-trichloroethoxycar-
bonylamino)-b-d-galactopyranosyl]-(1!3)-(2-O-benzyl-4,6-di-O-benzyli-
dene-a-d-galactopyranosyl)-(1!4)-(2-O-benzoyl-3,6-di-O-benzyl-a-d-gal-
actopyranosyl)-(1!4)-2,3,6-tri-O-benzyl-b-d-glucopyranoside (17): Thio-
urea (8.70 mg, 114 mmol) and 2,6-lutidine (10.0 mL, 91.5 mmol) were
added to a stirred solution of 16 (35.2 mg, 24.9 mmol) in dry DMF
(1.00 mL) at room temperature. After being stirred at the same tempera-
ture for 14 h, the reaction mixture was poured into 1m HCl with cooling.
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 column chromatography on silica gel (elution with 92:8
toluene/acetone) to give 17 (37.1 mg, 18.4 mmol, 96%). [a]2D0 = +89.3 (c=
1
1.06, CHCl3); H NMR (400 MHz, [D6]acetone): d=8.11 (dd, 2H, J=1.0,
7.7 Hz), 7.99–7.10 (m, 48H), 5.71 (s, 1H), 5.64 (s, 1H), 5.55 (dd, 1H, J1 =
7.7, 10.2 Hz), 5.47 (s, 1H), 5.30 (d, 1H, J=3.4 Hz), 5.08 (d, 1H, J=
3.4 Hz), 5.04 (brs, 2H), 5.03 (d, 1H, J=7.7 Hz), 4.92 (d, 1H, J=12.1 Hz),
4.90 (d, 1H, J=11.1 Hz), 4.77 (d, 1H, J=12.6 Hz), 4.72 (d, 1H, J=
8.7 Hz), 4.67 (d, 1H, J=12.1 Hz), 4.62 (brd, 1H, J=2.9 Hz), 4.61 (d, 1H,
J=11.1 Hz), 4.52 (brs, 1H), 4.51 (d, 1H, J=12.6 Hz), 4.43 (d, 1H, J=
3184
www.chemeurj.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2013, 19, 3177 – 3187

Forssman Antigen Pentasaccharide and Derivatives
FULL PAPER
73.7, 73.6, 73.5, 73.4, 73.1, 72.8, 72.0, 71.8, 71.7, 70.0, 69.9, 69.6, 68.1, 67.2,
67.1, 63.8, 63.3, 53.7, 51.5, 41.4, 18.8, ꢀ1.40 ppm; FTIR (KBr): n˜ =2954,
1732, 1453, 1366, 1269, 1097, 753, 698 cmꢀ1; HRMS (ESI-TOF): m/z calcd
for C117H129N2O29SiCl4: 2193.7204 [M+NH4]+; found: 2193.7200.
7.51–7.03 (m, 55H), 5.68 (dd, 1H, J=7.7, 9.7 Hz), 5.49 (dd, 1H, J=7.7,
10.2 Hz), 5.45 (s, 1H), 5.26 (s, 2H), 5.19 (dd, 1H, J=3.4, 10.2 Hz), 5.08
(d, 1H, J=3.4 Hz), 5.08 (d, 1H, J=3.4 Hz), 5.00 (d, 1H, J=11.1 Hz),
4.90 (d, 1H, J=11.1 Hz), 4.89 (d, 1H, J=13.1 Hz), 4.86 (d, 1H, J=
7.7 Hz), 4.79 (d, 1H, J=7.7 Hz), 4.73 (d, 1H, J=12.6 Hz), 4.64 (d, 1H,
J=11.1 Hz), 4.62 (d, 1H, J=11.1 Hz), 4.62 (d, 1H, J=12.6 Hz), 4.54 (d,
1H, J=12.1 Hz), 4.52 (d, 1H, J=12.1 Hz), 4.50 (brs, 1H), 4.43 (d, 1H,
J=12.6 Hz), 4.37 (d, 2H, J=12.6 Hz), 4.37 (dd, 1H, J=3.4, 10.6 Hz),
4.36 (brd, 1H, J=10.6 Hz), 4.32 (d, 1H, J=7.7 Hz), 4.26 (d, 1H, J=
12.1 Hz), 4.22 (brs, 1H), 4.14 (brd, 1H, J=2.4 Hz), 4.13 (dd, 1H, J=8.2,
8.7 Hz), 4.10 (brd, 1H, J=3.4 Hz), 4.08 (brd, 1H, J=2.4 Hz), 4.07 (dd,
1H, J=3.4, 10.2 Hz), 4.06 (d, 1H, J=15.0 Hz), 4.02 (dd, 1H, J=3.4,
10.6 Hz), 3.98 (d, 1H, J=15.0 Hz), 3.96 (dt, 1H, J=6.8, 9.2 Hz), 3.87 (dd,
1H, J=9.2, 9.7 Hz), 3.87 (d, 1H, J=12.1 Hz), 3.86 (brd, 1H, J=10.6 Hz),
3.65 (dd, 1H, J=2.4, 9.7 Hz), 3.61–3.41 (m, 9H), 3.39 (dd, 1H, J=2.4,
10.2 Hz), 3.37 (dd, 1H, J=7.7, 9.2 Hz), 3.33 (dd, 1H, J=4.8, 8.2 Hz), 3.32
(dd, 1H, J=4.8, 8.7 Hz), 3.30 (dt, 1H, J=4.8, 9.2 Hz), 3.05 (brs, 1H),
1.02–0.98 (m, 2H), 0.01 ppm (s, 9H); 13C NMR (100 MHz, CDCl3): d=
166.4, 165.3, 164.9, 139.4, 138.7, 138.4, 138.3, 138.2, 138.1, 138.0, 137.8,
137.5, 137.4, 133.2, 132.9, 129.9, 129.8, 129.7, 129.6, 128.9, 128.7, 128.5,
128.3, 128.2, 128.1, 128.0, 127.8, 127.7, 127.6, 127.4, 127.3, 127.2, 126.9,
126.3, 126.2, 126.0, 103.0, 101.8, 101.4, 101.2, 100.7, 100.4, 100.2, 98.4,
82.5, 82.0, 79.2, 78.5, 76.2, 75.3, 74.8, 74.6, 74.4, 73.7, 73.5, 73.4, 73.3, 73.1,
72.9, 72.1, 71.1, 70.8, 69.0, 68.7, 68.5, 67.3, 67.0, 66.2, 63.3, 62.6, 40.7, 18.4,
2-(Trimethylsilyl)ethyl (2-O-benzyl-4,6-O-benzylidene-a-d-galactopyran-
ACHTUNGTRENNUNGoACHTUNGTRENNUNG
ACHTUNGTRENNUNG
A
ACHTUNGTRENNUNG
(5.80 mg, 76.3 mmol, 6.00 equiv) and 2,6-lutidine (6.80 mL, 61.0 mmol,
4.80 equiv) was added to a stirred solution of 20 (27.7 mg, 12.7 mmol) in
dry DMF (1.00 mL) was added at room temperature. After being stirred
at 508C for 14 h, the reaction mixture was poured into 1m HCl with cool-
ing. The aqueous layer was extracted with two portions of ethyl acetate.
The combined extracts were washed with 1m HCl, saturated NaHCO3
(aq.) and brine, dried over MgSO4, filtered, and concentrated in vacuo.
The residue was purified by column chromatography on silica gel (elution
with 94:6 toluene/acetone) to give 21 (23.0 mg, 10.9 mmol, 86%). [a]D23
=
+92.7 (c=0.335, CHCl3); 1H NMR (400 MHz, [D6]acetone): d=8.12 (d,
2H, J=7.3 Hz), 7.68 (dd, 1H, J=7.3, 7.7 Hz), 7.57–7.11 (m, 52H), 6.57
(brd, 1H, J=9.7 Hz), 5.68 (s, 1H), 5.59 (s, 1H), 5.55 (dd, 1H, J=7.7,
10.2 Hz), 5.47 (s, 1H), 5.39 (d, 1H, J=3.4 Hz), 5.09 (d, 1H, J=3.4 Hz),
5.05 (d, 1H, J=7.7 Hz), 5.03 (brs, 2H), 4.95 (d, 1H, J=13.1 Hz), 4.90 (d,
1H, J=11.6 Hz), 4.77 (d, 1H, J=12.6 Hz), 4.75 (d, 1H, J=7.7 Hz), 4.67
(d, 1H, J=13.1 Hz), 4.66 (d, 1H, J=11.6 Hz), 4.64 (d, 1H, J=11.6 Hz),
4.64 (brs, 1H), 4.63 (d, 1H, J=12.1 Hz), 4.59 (d, 1H, J=12.6 Hz), 4.55
(brs, 1H), 4.51 (d, 1H, J=12.6 Hz), 4.44 (d, 1H, J=8.2 Hz), 4.41 (d, 1H,
J=12.1 Hz), 4.33 (brd, 1H, J=2.4 Hz), 4.31 (dd, 1H, J=3.4, 10.6 Hz),
4.30–4.26 (m, 2H), 4.24 (brs, 1H), 4.22 (d, 1H, J=12.1 Hz), 4.21 (brd,
1H, J=3.4 Hz), 4.20 (brd, 1H, J=10.2 Hz), 4.19 (brd, 1H, J=9.2 Hz),
4.16 (dd, 1H, J=8.4, 9.2 Hz), 4.14 (brdd, 1H, J=7.7, 10.2 Hz), 4.12 (brd,
1H, J=10.2 Hz), 4.10 (d, 1H, J=11.6 Hz), 4.07 (brd, 1H, J=10.2 Hz),
4.06 (brdd, 1H, J=3.4, 10.2 Hz), 3.98–3.83 (m, 6H), 3.81 (dd, 1H, J=2.4,
10.2 Hz), 3.70–3.53 (m, 8H), 3.47 (ddd, 1H, J=4.4, 4.8, 9.2 Hz), 3.42 (brs,
1H), 3.35 (dd, 1H, J=5.8, 8.7 Hz), 3.31 (dd, 1H, J=8.2, 8.7 Hz), 0.98 (t,
2H, J=8.7 Hz), 0.00 ppm (s, 9H); 13C NMR (67.8 MHz, [D6]acetone):
d=166.0, 154.9, 140.6, 140.3, 139.9, 139.7, 139.5, 139.0, 134.0, 131.1, 130.5,
129.5, 129.0, 128.9, 128.7, 128.6, 128.5, 128.4, 128.2, 128.1, 128.0, 127.6,
127.3, 127.2, 103.4, 102.3, 101.3, 101.2, 101.1, 100.9, 96.8, 94.9, 94.8, 83.2,
83.1, 80.2, 79.8, 78.7, 77.8, 76.8, 76.2, 75.6, 74.8, 74.2, 73.8, 73.7, 73.4, 73.3,
71.8, 71.7, 69.4, 68.3, 68.1, 67.2, 67.0, 63.8, 53.6, 18.8, ꢀ1.37 ppm; FTIR
(KBr): n˜ =3569, 2869, 1730, 1454, 1366, 1269, 1097, 753, 698 cmꢀ1; HRMS
(ESI-TOF): m/z calcd for C115H128N2O28SiCl3: 2117.7488 [M+NH4]+;
found: 2117.7485.
ꢀ1.50 ppm; FTIR (KBr): n˜ =2861, 1734, 1267, 1097, 1000, 750, 698 cmꢀ1
;
HRMS (ESI-TOF): m/z calcd for
NH4]+; found: 2124.8240.
C121H131NO29SiCl: 2124.8265 [M+
2-(Trimethylsilyl)ethyl (2-O-benzyl-4,6-O-benzylidene-a-d-galactopyran-
syl)-(1!3)-(2-O-benzoyl-4,6-O-benzylidene-b-d-galactopyranosyl)-(1!
ACTHUNGTRENNUGoAHCTUNGTRENNNUG
3)-(2-O-benzyl-4,6-O-benzylidene-a-d-galactopyranosyl)-(1!4)-(2-O-
benzoyl-3,6-di-O-benzyl-b-d-galactopyranosyl)-(1!4)-2,3,6-tri-O-benzyl-
b-d-glucopyranoside (23): Thiourea (8.70 mg, 114 mmol) and 2,6-lutidine
(10.6 mL, 91.2 mmol) to a stirred solution of 22 (40.0 mg, 19.0 mmol) in dry
DMF (1.00 mL) was added at room temperature. After being stirred at
508C for 12 h, the reaction mixture was poured into 1m HCl with cooling.
The aqueous layer was extracted with two portions of ethyl acetate. The
combined extracts were washed with 1m HCl, saturated NaHCO3 (aq.)
and brine, dried over MgSO4, filtered and concentrated in vacuo. The res-
idue was purified by column chromatography on silica gel (elution with
95:5 toluene/acetone) to give 23 (33.9 mg, 16.7 mmol, 88%). [a]2D2 =+80.2
(c=0.935, CHCl3); 1H NMR (400 MHz, CDCl3): d=7.98 (d, 2H, J=
7.3 Hz), 7.86 (d, 2H, J=7.7 Hz), 7.60 (dd, 1H, J=7.3 Hz, J=7.7 Hz),
7.48–7.03 (m, 55H), 5.70 (dd, 1H, J=8.2, 10.2 Hz), 5.49 (dd, 1H, J=7.7,
10.2 Hz), 5.45 (s, 1H), 5.31 (s, 1H), 5.30 (s, 1H), 5.13 (d, 1H, J=2.9 Hz),
5.03 (d, 1H, J=3.4 Hz), 4.99 (d, 1H, J=11.1 Hz), 4.90 (d, 2H, J=
11.1 Hz), 4.89 (d, 1H, J=8.2 Hz), 4.78 (d, 1H, J=7.7 Hz), 4.72 (d, 1H,
J=11.6 Hz), 4.65 (d, 1H, J=11.1 Hz), 4.62 (d, 1H, J=11.6 Hz), 4.53 (d,
1H, J=13.1 Hz), 4.51 (brd, 1H, J=3.9 Hz), 4.49 (d, 1H, J=12.1 Hz),
4.42 (d, 1H, J=12.6 Hz), 4.37 (d, 1H, J=13.1 Hz), 4.36 (dd, 1H, J=3.9,
10.2 Hz), 4.35 (d, 1H, J=12.6 Hz), 4.31 (d, 1H, J=7.7 Hz), 4.22 (brs,
1H), 4.25 (d, 2H, J=12.1 Hz), 4.12 (brd, 1H, J=3.4 Hz), 4.12 (brd, 1H,
J=3.4 Hz), 4.11 (dd, 1H, J=7.7, 8.7 Hz), 4.03 (brd, 1H, J=11.6 Hz),
4.02 (dd, 1H, J=3.4, 10.2 Hz), 3.98–3.83 (m, 4H), 3.79 (dd, 1H, J=2.9,
9.7 Hz), 3.78 (brs, 1H), 3.68 (dd, 1H, J=3.4, 10.2 Hz, J=3.4 Hz), 3.67
(brd, 1H, J=9.7 Hz), 3.62–3.52 (m, 6H), 3.51 (brs, 1H), 3.40–3.29 (m,
7H,), 3.07 (brs, 1H), 2.33 (brd, 1H, J=6.8 Hz), 0.99 (dt, 2H, J=6.3,
10.2 Hz), 0.00 ppm (s, 9H); 13C NMR (67.8 MHz, CDCl3): d=165.3,
164.8, 139.4, 138.6, 138.4, 138.3, 138.2, 138.1, 138.0, 137.8, 137.5, 137.4,
133.2, 133.0, 129.9, 129.7, 129.5, 129.0, 128.8, 128.5, 128.5, 128.3, 128.2,
128.1, 128.0, 127.8, 127.6, 127.5, 127.3, 127.2, 126.9, 126.4, 126.2, 126.1,
102.9, 101.9 x2, 101.4, 101.2, 100.9, 100.2, 97.0, 82.4, 82.0, 79.2, 76.3, 76.2,
75.8, 75.3, 74.8, 74.6, 74.4, 73.7, 73.5, 73.2, 72.8, 72.6, 72.2, 72.1, 71.1, 70.8,
69.1, 69.0, 68.9, 68.5, 67.9, 67.3, 67.0, 66.1, 62.9, 29.7, 18.5, ꢀ1.50 ppm;
FTIR (KBr): n˜ =3572, 2861, 1732, 1268, 1097, 752, 698 cmꢀ1; HRMS
(ESI-TOF): m/z calcd for C119H130NO28Si: 2048.8549 [M+NH4]+; found:
2048.8552.
2-(Trimethylsilyl)ethyl (2-O-benzyl-4,6-O-benzylidene-3-O-chloroacetyl-
a-d-galactopyranosyl)-(1!3)-(2-O-benzoyl-4,6-O-benzylidene-b-d-galac-
topyranosyl)-(1!3)-(2-O-benzyl-4,6-O-benzylidene-a-d-galactopyrano-
syl)-(1!4)-(2-O-benzoyl-3,6-di-O-benzyl-b-d-galactopyranosyl)-(1!4)-
2,3,6-tri-O-benzyl-b-d-glucopyranoside (22): A mixture of 6a (21.4 mg,
49.3 mmol), Ph2SO (18.3 mg, 90.3 mmol), TTBP (26.9 mg, 108 mmol; azeo-
troped twice with dry toluene) and pulverized activated MS 4ꢂ (300 mg)
in dry CH2Cl2 (1.20 mL) was stirred at room temperature for 1 h under
argon to remove a trace amount of H2O. Then, the reaction mixture was
cooled to ꢀ788C. After 5 min, Tf2O (11.0 mL, 65.0 mmol) was added to
the reaction mixture at the same temperature. After being stirred at
ꢀ408C for 1 h, a solution of 8 (15.1 mg, 36.3 mmol; azeotroped twice with
dry toluene) in dry CH2Cl2 (1.00 mL) was added at the same tempera-
ture. After being stirred for 3.5 h at the same temperature, the reaction
mixture was warmed to 08C and stirred for 30 min. The reaction mixture
was then cooled to ꢀ208C. After 5 min,
a solution of 9 (58.0 mg,
43.4 mmol) in dry CH2Cl2 (1.00 mL) and NIS (12.2 mg, 54.1 mmol,
1.50 equiv) was added to the reaction mixture. After being stirred for 6 h
at the same temperature, the reaction mixture was neutralized with NEt3
and filtered through a pad of Celite. Then, the filtrate was concentrated
in vacuo. The residue was purified by column chromatography on silica
gel (elution with CHCl3/MeOH=99:10) and by gel permeation chroma-
tography (GPC) to give 22 (29.7 mg, 16.6 mmol, 46% based on 8). [a]D30
=
+90.0 (c=1.12, CHCl3); 1H NMR (400 MHz, CDCl3): d=7.98 (dd, 2H,
J=1.5, 7.3 Hz), 7.90 (d, 2H, J=7.3 Hz), 7.61 (dd, 1H, J=7.3, 7.7 Hz),
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H. Tanaka, T. Takahashi et al.
Polymer-assisted deprotection
58.5, 58.4, 54.5, 51.8, 51.7, 51.2, 51.1, 41.7, 40.2, 13.1, 12.8, 8.37,
ꢀ11.7 ppm; FTIR (KBr): n˜ =3254, 1629, 1557, 1417, 1248, 1039, 713 cmꢀ1
;
2-(Trimethylsilyl)ethyl (2-acetamide-2-deoxy-a-d-galactopyranosyl)-(1!
3)-(2-acetamide-2-deoxy-a-d-galactopyranosyl)-(1!3)-a-d-galactopyrano-
syl-(1!4)-a-d-galactopyranosyl-b-d-glucopyranoside (2a): A mixture of
17 (8.60 mg, 4.20 mmol), prelinker 3 (5.90 mg, 21.0 mmol; azeotroped
twice with dry toluene) and pulverized activated MS 4ꢂ (100 mg) in dry
CH2Cl2 (1.00 mL) was stirred at room temperature for 1 h under argon to
remove a trace amount of H2O. Then, CSA (1.00 mg, 4.20 mmol) was
added to the reaction mixture at 08C. After being stirred at the same
temperature for 4 h, the reaction mixture was filtered through a pad of
Celite. Then, the filtrate was concentrated in vacuo. The residue was pu-
rified by column chromatography on silica gel (elution with CHCl3) and
further purified by gel permeation chromatography (GPC). To a solution
of the above activated ester in dry CH2Cl2 (150 mL) and dry DMF
(150 mL) was added ArgoPore-NH2 resin (4) (56.3 mg, 42.0 mmol), a cata-
lytic amount of DIEA and DMAP at room temperature. After being
shaken at room temperature for 18 h, the reaction mixture was filtered
and the resin was washed three times each with THF/H2O (1:1)
(3.00 mL), MeOH (3.00 mL) and dry CH2Cl2 (3.00 mL), respectively. The
resin was dried in vacuo to give 24 (FTIR (KBr): n˜ =3327, 2924, 2116,
1717, 1665, 1493, 1219, 1095, 772 cmꢀ1). To a suspension of 24 packed
into MacroKans microreactor in 1,4-dioxane (20.0 mL) and H2O
(10.0 mL) was added LiOH·H2O (400 mg) at room temperature. After
being stirred at 808C for 18 h, the reaction mixture was filtered and the
resin was washed three times each with THF/H2O (1:1) (3.00 mL),
MeOH (3.00 mL), and dry CH2Cl2 (3.00 mL). The resin was dried in
vacuo (FTIR (KBr): n˜ =3313, 2927, 1666, 1602, 1451, 1173 1026,
700 cmꢀ1). LiOH·H2O (100 mg) and PMe3 (200 mL, 1.0m in THF solution)
was added to a suspension of the resultant resin packed into MacroKans
microreactor in toluene (5.00 mL) and H2O (5.00 mL) was added at
room temperature. After being stirred at 1408C for 14 h, the reaction
mixture was filtered and the resin was washed three times each with
THF/H2O (1:1) (3.00 mL), MeOH (3.00 mL) and dry CH2Cl2 (3.00 mL),
respectively. The resin was dried in vacuo (FTIR (KBr): n˜ =3325, 2916,
1664, 1602, 1493, 1451, 984, 826 cmꢀ1). AcOH (70.0 mL), DIC (150 mL)
and DIEA (330 mL) was added at room temperature to a suspension of
the resultant resin packed into MacroKans microreactor in dry CH2Cl2
(10.0 mL). After being stirred at room temperature for 12 h, the reaction
mixture was filtered and the resin was washed three times each with
THF/H2O (1:1) (3.00 mL), MeOH (3.00 mL) and dry CH2Cl2 (3.00 mL),
respectively. The resin was dried in vacuo. Liquid NH3 (36.0 mL) was
added to a suspension of the resultant resin packed into MacroKans mi-
croreactor in dry THF (4.00 mL). Sodium granules (40.0 mg) were subse-
quently added to the reaction mixture at ꢀ788C. After being stirred at
the same temperature for 1.5 h, the reaction mixture was allowed to
warm to ꢀ358C. After heating at reflux for 1 h, the reaction mixture was
quenched with MeOH. The reaction mixture was filtered and the resin
was washed three times each with THF/H2O (1:1) (3.00 mL), MeOH
(3.00 mL) and dry CH2Cl2 (3.00 mL), respectively. Then, the resin was
dried in vacuo. The resultant resin was treated with 1m HCl (300 mL),
dry CH2Cl2 (2.40 mL) and MeOH (200 mL) at room temperature for
20 min. Then, the reaction mixture was filtered and rinsed with dry
CH2Cl2 (3.00 mL), MeOH (3.00 mL) and H2O (3.00 mL). The residue
was dried in vacuo and purified by reverse-phase column chromatogra-
phy (Bond Elut-C18) to give 2a (1.70 mg, 1.50 mmol, 43% in 7 steps
based on 17). [a]2D3 = +97.8 (c 0.250, H2O); 1H NMR (400 MHz, D2O):
d=5.05 (d, 1H, J=3.9 Hz), 4.94 (d, 1H, J=2.9 Hz), 4.72 (d, 1H, J=
8.2 Hz), 4.49 (d, 1H, J=7.7 Hz), 4.45 (d, 1H, J=7.7 Hz), 4.29 (dd, 1H,
J=6.3, 5.8 Hz), 4.23 (brs, 1H), 4.21 (dd, 1H, J=3.9, 11.1 Hz), 4.09 (dd,
1H, J=8.2, 11.1 Hz), 4.07 (brd, 1H, J=3.4 Hz), 4.03 (brd, 1H, J=
3.4 Hz), 3.99 (brd, 1H, J=2.9 Hz), 3.97 (dt, 1H, J=6.3, 8.7 Hz), 3.96 (dd,
1H, J=3.9, 10.2 Hz), 3.95 (brddd, 1H, J=3.9, 4.4, 9.7 Hz), 3.94 (dd, 1H,
J=2.9, 10.2 Hz), 3.91–3.74 (m, 13H), 3.71 (dd, 1H, J=3.4, 10.2 Hz), 3.70
(brd, 1H, J=5.3 Hz), 3.70 (brd, 1H, J=5.3 Hz), 3.61 (dd, 1H, J=5.3,
6.3 Hz), 3.61 (t, 1H, J=8.7, 9.2 Hz), 3.56 (dd, 1H, J=7.7, 10.2 Hz), 3.54
(dd, 1H, J=8.7, 9.7 Hz), 3.28 (dd, 1H, J=7.7, 9.2 Hz), 2.03 (s, 3H), 2.02
(s, 3H, 1.07–0.91 (m, 2H), 0.00 ppm (s, 9H); 13C NMR (67.8 MHz, D2O,
[D6]acetone): d=165.4, 165.2, (94.1, 93.4, 92.2, 91.2, 84.4 anomeric), 69.7,
69.6, 68.1, 66.2, 65.6, 65.5, 65.4, 63.7, 63.0, 62.1, 61.7, 61.1, 59.7, 59.1, 58.9,
HRMS (ESI-TOF): m/z calcd for C39H71N2O26Si: 1011.4064 [M+H]+;
found: 1011.4064.
(a-d-Galactopyranosyl)-(1!3)-(2-acetamide-2-deoxy-a-d-galactopyrano-
syl)-(1!3)-a-d-galactopyranosyl-(1!4)-a-d-galactopyranosyl-b-d-gluco-
pyranoside (2c): According to the polymer-assisted deprotection of 17,
compound 2c was prepared from 21 (1.80 mg, 1.86 mmol, 34% in 6 steps
based on 21). [a]2D6 = +107 (c=0.240, H2O); 1H NMR (400 MHz, D2O):
d=5.08 (d, 1H, J=3.4 Hz), 4.93 (d, 1H, J=2.9 Hz), 4.72 (d, 1H, J=
8.2 Hz), 4.48 (d, 1H, J=7.7 Hz), 4.44 (d, 1H, J=7.7 Hz), 4.28 (t, 1H, C5,
J=6.3 Hz), 4.26 (brd, 1H, J=2.9 Hz), 4.13 (brd, 1H, J=1.9 Hz), 4.06
(dd, 1H, J=8.2, 10.6 Hz), 4.02 (brd, 1H, J=1.9 Hz), 3.97 (brd, 1H, J=
1.9 Hz), 3.95 (brdt, 1H, J=4.8, 9.7 Hz), 3.94 (dt, 1H, J=6.8, 10.2 Hz),
3.93 (dd, 1H, J2,3 =2.9, 11.6 Hz), 3.89 (dd, 1H, J=2.9, 11.6 Hz), 3.85–3.68
(m, 17H), 3.64 (dd, 1H, J5,6a =5.3, 5.8 Hz), 3.60 (dd, 1H, J=8.7 Hz,
9.2 Hz), 3.55 (dd, 1H, J=7.7, 10.6 Hz), 3.51 (dd, 1H, J=9.2, 9.7 Hz), 3.27
(dd, 1H, J=7.7, 8.7 Hz), 2.01 (s, 3H), 1.06–0.90 (m, 2H), ꢀ0.01 (s, 9H);
13C NMR (67.8 MHz, D2O): d=162.8, 94.0, 93.2, 92.2, 91.1, 86.2, 69.9,
68.4, 66.5, 66.1, 65.5, 63.7, 63.2, 61.9, 61.8, 61.4, 60.2, 60.0, 59.9, 59.4, 58.8,
58.7, 58.4, 56.9, 54.9, 51.8, 51.7, 51.6, 51.5, 51.2, 51.1, 32.2, 13.1, 8.33,
ꢀ11.8 ppm; FTIR (KBr): n˜ =3371, 1642, 1437, 1250, 1035, 805, 591 cmꢀ1
;
HRMS (ESI-TOF): m/z calcd for C37H68NO26Si: 970.3799 [M+H]+;
found: 970.3797.
(a-d-Galactopyranosyl)-(1!3)-(a-d-galactopyranosyl)-(1!3)-a-d-galac-
topyranosyl-(1!4)-a-d-galactopyranosyl-b-d-glucopyranoside (2d): Ac-
cording to the polymer-assisted deprotection of 17, compound 2d was
prepared from 21 (2.60 mg, 2.84 mmol, 43% in 5 steps based on 23).
[a]2D7 = +73.8 (c=0.195, H2O); 1H NMR (400 MHz, D2O): d=5.12 (d,
1H, J=3.9 Hz), 4.98 (d, 1H, J=2.4 Hz), 4.66 (d, 1H, J=7.3 Hz), 4.48 (d,
1H, J=7.7 Hz), 4.44 (d, 1H, J=7.7 Hz), 4.30 (t, 1H, J=6.3 Hz), 4.24
(brs, 1H, C-4), 4.17 (t, 1H, J=6.8 Hz), 4.13 (brd, 1H, J=2.4 Hz), 4.02–
3.83 (m, 8H), 3.81–3.68 (m, 15H), 3.66 (dd, 1H, J=5.8, 6.3 Hz), 3.60 (dd,
1H, J=8.7, 9.2 Hz), 3.56 (dd, 1H, J=7.7, 10.2 Hz), 3.54 (dd, 1H, J=8.7,
9.7 Hz), 3.28 (dd, 1H, J=7.7, 9.2 Hz), 1.07–0.91 (m, 2H), 0.00 ppm (s,
9H); 13C NMR (67.8 MHz, D2O): d=94.8, 94.1, 92.3, 91.1, 86.5, 70.1,
69.7, 68.5, 66.1, 65.6, 65.5, 63.8, 63.4, 62.0, 61.8, 61.6, 60.6, 60.3, 60.2, 59.8,
59.1, 58.8, 58.6, 56.0, 51.9, 51.8, 51.6, 51.3, 51.2, 8.45, ꢀ11.7 ppm; FTIR
(KBr): n˜ =3371, 1649, 1421, 1250, 1033, 804, 580 cmꢀ1; HRMS (ESI-
TOF): m/z calcd for C35H65O26Si: 923.3533 [M+H]+; found: 929.3527.
Biological evaluation
SLL-2 labeling: SLL-2 (1 mg) was dissolved in NaHCO3 (pH 9.0, 500 mL
of 0.1m). After the addition of fluorescein isothiocyanate (FITC; 100 mL
of 10 mgmLꢀ1) in dimethylsulfoxide, the solution was mixed and incubat-
ed at room temperature for 1 h in the dark. The solution was dialyzed
against 1 L of phosphate buffered saline at 48C overnight.
Binding of the Forssman-antigen-related oligosaccharides to SLL-2: The
relative affinity of oligosaccharides to SLL-2 was tested by the change of
fluorescence intensity using FP-715 (JASCO, Tokyo, Japan). Assay solu-
tion (400 mL of a solution of 150 mm NaCl, 0.1% Triton X-100, 20 mm
Tris-HCl, pH 8.0) and FITC-labeled SLL-2 (3 mm) was added to a cuvette
(ø=7.0 mm). The fluorescence (excitation=480 nm, emission=530 nm)
was measured every 15 s. After 1 min, an oligosaccharide was added and
the fluorescence was measured after 5 min. The all measurement was car-
ried out at 25.08C with stirring. The oligosaccharides used were
AHCTUNGTERGFNNUN orssman-related saccharides. The specimens were analyzed in triplicate.
Inhibition of SLL-2 activity by Forssman-related oligosaccharides: The in-
hibition of SLL-2 effect to the morphology of CS-156 by the Forssman-
related oligosaccharides was evaluated. The CS-156 was cultured to more
than 10000 cellsmLꢀ1. CS-156 (100 mL), adjusted to 3000 cellsmLꢀ1 by
IMK medium, was dispensed into a well of 96-well titer plate (Corning).
The culture was incubated at 258C for 24 h. Forssman-related oligosac-
charides (150 mL), at a concentration of 0.3 mm in IMK medium, were
mixed with the filtrated SLL-2 (150 mL) at
a concentration of
200 mgmLꢀ1 in IMK medium. This solution (100 mL) was added to each
CS-156 (100 mL) culture, and incubated at 258C and cultured for 5 days.
Photographs were taken at ten different fields for each well. A blurred
3186
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Chem. Eur. J. 2013, 19, 3177 – 3187

Forssman Antigen Pentasaccharide and Derivatives
FULL PAPER
shape and a clear shape were identified as the motile form and a coccoid
form, respectively. The motile cells and coccoid cells were counted, and
then the motile percentage was determined. The specimens were ana-
lyzed in triplicate.
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This work was supported by a Grant-in-Aid for Scientific Research (B;
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porting Information.
Received: October 29, 2012
Revised: December 4, 2012
Published online: January 16, 2013
Chem. Eur. J. 2013, 19, 3177 – 3187
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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3187