Synthesis of Lacto- and Neolacto-series Ganglioside Analogs Containing N-Glycolylneuraminic Acid: Probes for Investigation of Specific Receptor Structures Recognized by Influenza a Viruses

Article abstract of DOI:10.1081/CAR-120026602

Sialic acids are essential components of host-cell surface receptors for infection of influenza virus. To investigate the specific receptor structures recognized by various influenza A viruses, a series of lacto- and neolacto-series ganglioside analogs co

Full text of DOI:10.1081/CAR-120026602

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Synthesis of Lacto‐ and Neolacto‐series Ganglioside
Analogs Containing N‐Glycolylneuraminic Acid: Probes
for Investigation of Specific Receptor Structures
Recognized by Influenza A Viruses
Kyoko Fukunaga a b , Tsuyoshi Toyoda a , Hideharu Ishida a & Makoto Kiso a
a Department of Applied Bioorganic Chemistry , Gifu University , Gifu, 501‐1193, Japan
b Basic Research Laboratory , Kanebo Ltd. , Kotobuki‐cho, Odawara‐shi, Kanagawa, Japan
Published online: 02 Feb 2007.
To cite this article: Kyoko Fukunaga , Tsuyoshi Toyoda , Hideharu Ishida & Makoto Kiso (2003) Synthesis of Lacto‐
and Neolacto‐series Ganglioside Analogs Containing N‐Glycolylneuraminic Acid: Probes for Investigation of Specific
Receptor Structures Recognized by Influenza A Viruses , Journal of Carbohydrate Chemistry, 22:9, 919-937, DOI: 10.1081/
CAR-120026602
To link to this article: http://dx.doi.org/10.1081/CAR-120026602
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JOURNAL OF CARBOHYDRATE CHEMISTRY
Vol. 22, No. 9, pp. 919–937, 2003
Synthesis of Lacto- and Neolacto-series Ganglioside
Analogs Containing N-Glycolylneuraminic Acid:
Probes for Investigation of Specific Receptor
Structures Recognized by Influenza A Viruses#
z
*
Kyoko Fukunaga, Tsuyoshi Toyoda, Hideharu Ishida,
and Makoto Kiso*
Department of Applied Bioorganic Chemistry, Gifu University, Gifu, Japan
ABSTRACT
Sialic acids are essential components of host-cell surface receptors for infection of
influenza virus. To investigate the specific receptor structures recognized by various
influenza A viruses, a series of lacto- and neolacto-series ganglioside analogs con-
taining N-glycolylneuraminic acid (Neu5Gc) have been synthesized. The penta-
saccharide structures of Neu5Gc-a-(2!3)/(2!6)-lactotetraose (IV3(6)Neu5GcLcOse)
and Neu5Gc-a-(2!3)/(2!6)-neolactotetraose (IV3(6)Neu5GcnLcOse) were con-
structed by glycosylation of the suitably protected trisaccharide acceptors (2A and 2B)
with the Neu5Gc-a-(2!3)/(2!6)-Gal trichloroacetimidate donors (1 and 21), re-
spectively. Transformation of the 2-(trimethylsilyl)ethyl group at the reducing end in
4, 11, 23, and 30 into the trichloroacetimidate group gave a series of Neu5Gc-a-
(2!3)/(2!6)-lacto- and neolactotetraose donors (7, 13, 26, and 33), which were
#Synthetic studies on sialoglycoconjugates, Part 129. For Part 128, see Ando, T.; Ishida, H.; Kiso,
M. Carbohydr. Res., 2003, 338, 503–514.
zCurrent address: Basic Research Laboratory, Kanebo Ltd., Kotobuki-cho, Odawara-shi,
Kanagawa, Japan.
*
Correspondence: Hideharu Ishida, Department of Applied Bioorganic Chemistry, Gifu Univer-
sity, Gifu 501-1193, Japan; E-mail: ishida@cc.gifu-u.ac.jp. Makoto Kiso, Department of Applied
Bioorganic Chemistry, Gifu University, Gifu 501-1193, Japan; E-mail: kiso@cc.gifu-u.ac.jp.
919
DOI: 10.1081/CAR-120026602
0732-8303 (Print); 1532-2327 (Online)
www.dekker.com
Copyright D 2003 by Marcel Dekker, Inc.

920
Fukunaga et al.
coupled with 2-(tetradecyl)hexadecanol (8), to give the corresponding glycolipids (9,
14, 27, and 34). Finally, the complete removal of the O-acyl groups and saponification
of the methyl ester group gave the desired ganglioside analogs (10, 15, 28, and 35).
Key Words: Sialic acid; Ganglioside; Influenza; Glycosylation; Carbohydrate.
INTRODUCTION
Influenza virus possesses both the receptor-binding protein (hemagglutinin: HA)
and the receptor-destroying enzyme (neuraminidase: NA) on the cell surface, which are
responsible for viral infection and budding from the host cells. It has been reported that
the sialyl-lacto- and neolacto-series sugar chains, such as sialyl-a-(2!3)/(2!6)-Gal-b-
(1!3)/(1!4)-GlcNAc in both glycolipids and glycoproteins, are the functional
receptors for HA of influenza A virus.[1–4] It has also been suggested that HA dis-
criminates the species of sialic acid molecules as well as the linkage form of sialyl-
galactose on these sialoglycoconjugates.[1–3,5] These receptors differ among humans
and the other animals,[3,4] so that it has been speculated that HA might continue some
mutations for succession of the binding ability against these receptors on the occasion
of infection between different animal species.[3–6]
We have achieved a systematic synthesis of gangliosides to elucidate the structure
and functions of sialoglycoconjugates.[7,8] The sialyl lacto-(type I) and sialyl neolacto-
(type II) tetraosyl ceramides containing N-acetylneuraminic acid[9,10] or KDN[11] have
successfully been synthesized by our established method.
In this paper, we describe the synthesis of four kinds of Neu5Gc-a-(2!3)/(2!6)-
Gal-b-(1!3)/(1!4)-GlcNAc-b-(1!3)-Gal-b-(1!4)-Glc-b-(1!1)-OR probes contain-
ing N-glycolylneuraminic acid (Neu5Gc), which are widely found in many animals but
have not yet been detected in normal human tissues,[12,13] to investigate the receptor
specificity of influenza A virus HA at the molecular level.
RESULTS AND DISCUSSION
For an efficient construction of a series of pentasaccharide structures, we employed
the Neu5Gc-a-(2!3)/(2!6)-Gal trichloroacetimidate derivatives (1[14] and 21) as the
common glycosyl donors and two kinds of the suitably protected trisaccharide accep-
tors (2A[9] for lacto-series and 2B[11] for neolacto-series).
As shown in Scheme 1, Neu5Gc-a-(2!3)-Gal trichloroacetimidate donor 1, pre-
viously reported by Tanahashi et al.[14] was coupled with the lacto-series acceptor 2A in
the presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) in CH2Cl2 to give
1
the desired pentasaccharide 3 in 95% yield. In the H NMR spectrum of 3, one-proton
doublet (J = 8.1 Hz) appeared at d 5.11 indicating the newly formed glycosidic linkage
to be b. Hydrogenolytic removal of the benzyl and benzylidene groups in 3 over 20%
Pd(OH)2 on carbon in EtOH, followed by complete acetylation of the resulting free
hydroxyl groups with Ac2O-pyridine, afforded the fully acylated pentasaccharide 4 in
86% yield. Removal of the O-acyl groups and saponification of the methyl ester group
1
in 4 afforded 5 in a quantitative yield. Significant signals in the H NMR spectrum of 5

Investigation of Specific Receptor Structures
921
Scheme 1. Synthesis of Neu5Gc-a-(2-3)lactotetraosyl lipid.
were two-proton multiplets (d 1.00, TMS CH2–), one-proton triplet (d 1.77, H-3ax of
NeuGc), three-proton singlet (d 2.00, AcN), one-proton doublet of doublets (d 2.75, H-
3eq of NeuGc), and four one-proton doublets (d 4.41 ꢀ 4.70, J = 7.7 ꢀ 8.4 Hz) com-
prised of four b-glycosidic linkages, clearly showing the desired structure.
The 2-(trimethylsilyl)ethyl group in 4 was selectively cleaved by treatment[15,16]
with trifluoroacetic acid (TfOH) in CH2Cl2 to give the 1-hydroxy derivative 6, which

922
Fukunaga et al.
upon further treatment[17] with trichloroacetonitrile under 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU) in CH2Cl2 afforded the trichloroacetimidate donor 7 in 86% yield. In the
same way, the neolacto-series pentasaccharide 11,[14] which was prepared by coupling
1
of 1 with 2B (Scheme 2), was also converted to 13 in 85% yield. In the H NMR
spectra of 7 and 11, a significant one-proton doublet was observed at d 6.48 (J = 3.7
Hz) and d 6.47 (J = 3.6 Hz), respectively, showing the anomeric configuration of the
imidate to be a. Coupling of 7 with 2-(tetradecyl)hexadecanol 8,[18] a mimic of cer-
amide, was performed in the presence of TMSOTf in CH2Cl2 at about 20°C to give the
desired glycolipid derivative 9 in 54% yield. The coupling of 13 with 8 in the same
manner afforded the desired glycolipid 14 in 50% yield. Finally, removal of the O-acyl
groups and saponification of the methyl ester group in 9 and 14 gave the target
Neu5Gc-a-(2!3)-lacto- and -neolacto-series ganglioside analogs (10 and 15) in high
yields after column chromatography on Sephadex LH-20.
Scheme 2. Synthesis of Neu5Gc-a-(2-3)neolactotetraosyl lipid.

Investigation of Specific Receptor Structures
923
Scheme 3. Synthesis of Neu5Gc-a-(2-6)lactotetraose and its glycolipid.

924
Fukunaga et al.
Scheme 4. Synthesis of Neu5Gc-a-(2-6)neolactotetraose and its glycolipid.
The synthetic routes of Neu5Gc-a-(2!6)-lactotetraose and neolactotetraose deri-
vatives are shown in Schemes 3 and 4.
For the preparation of Neu5Gc-a-(2!6)-Gal donor, methyl (phenyl 5-acetoxy-
acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-a-D-galacto-2-nonulopy-
ranosid)onate 16[19] was coupled with 2-(trimethylsilyl)ethyl 3-O-benzyl-b-D-galacto-
pyranoside 17[20] in the presence of N-iodosuccinimide (NIS) and TfOH in CH3CN at
ꢁ 35°C to give Neu5Gc-a-(2!6)-Gal derivative 18 in 67% yield (Scheme 3). Ben-
zoylation of 18 with benzoic anhydride (Bz2O) and 4-dimethylaminopyridine (DMAP)
afforded 19 which was converted, by cleavage of the 2-(trimethylsilyl)ethyl group and
trichloroacetimidate formation, to the desired Neu5Gc-a-(2!6)-Gal donor 21 in good

Investigation of Specific Receptor Structures
925
yield. Couplings of trichloroacetimidate donor 21 with 2A and 2B were carried out
in the presence of TMSOTf in CH2Cl2 to afford the corresponding pentasaccharides
22 (51%) and 29 (82%), respectively. Hydrogenolytic removal of the benzyl and
benzylidene groups from 22 and 29, and complete acetylation of the resulting free
hydroxyl groups afforded the fully acylated pentasaccharides 23 and 30. Removal of
the O-acyl groups and saponification of the methyl ester group in 23 and 30
afforded the Neu5Gc-a-(2!6)-lactotetraose and neolactotetraose derivatives (24 and
1
31), quantitatively. In the H NMR spectra of 24 and 31, four one-proton doublets
(J = 8.0–8.2 Hz), each corresponding to the b-glycosidic linkages, were clearly
observed at d 4.36–4.71, indicating the desired structures. The pentasaccharide
donors 26 and 33 were prepared from 23 and 30 as described for 7 and 13, and
coupled with 8 to give the desired glycolipid derivatives 27 and 34 in 55% and 51%
yields, respectively. Removal of the O-acyl groups and saponification of the methyl
ester group in 27 and 34 gave the target Neu5Gc-a-(2!6)-lactotetraosyl and neo-
lactotetraosyl glycolipids (28 and 35), quantitatively.
The synthetic ganglioside analogs (10, 15, 28, 35) have successfully been utilized
as the molecular probes for analyzing the recognition specificity of influenza A virus
hemagglutinin,[21] demonstrating that a few amino acid residues in hemagglutinin affect
binding reactivity to the molecular species of sialic acid (Neu5Ac/Neu5Gc).
It has also been shown that both sialic acid species (Neu5Ac/Neu5Gc) and the
sialoside linkage to galactose (a2!3/a2!6) are critically associated with intestinal
replication of influenza A virus in ducks[22] as well as the host range restriction in viral
infection among different animals.[23]
EXPERIMENTAL
General procedures. Specific rotations were determined with a Union PM-201
polarimeter at 25°C, and IR spectra were recorded with a Jasco IRA-100 spectropho-
tometer. H NMR spectra were recorded at 400 MHz with a Varian Inova 400, or 200
1
MHz with a Varian Gemini-2000 spectrometer. TLC was performed on Silica Gel 60
(E. Merck), and column chromatography on silica gel (Fuji Silysia Co., 300 mesh) was
accomplished with the solvent systems (v/v) specified. Concentrations and evaporations
were conducted in vacuo.
2-(Trimethylsilyl)ethyl (methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonate)-(2!3)-(2,4,6-tri-O-benzoyl-
-D-galactopyranosyl)-(1!3)-(2-acetamido-4,6-O-benzylidene-2-deoxy- -D-gluco-
pyranosyl)-(1!3)-(2,4,6-tri-O-benzyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-O-ben-
zyl- -D-glucopyranoside (3). To a solution of 1 (408 mg, 0.36 mmol) and 2A (365
˚
mg, 0.29 mmol) in CH2Cl2 (4 mL) was added MS4A (600 mg), the reaction mixture
was stirred for 3 h at room temperature and then cooled to 0°C. To the stirred mixture
TMSOTf (11 mL, 58 mmol) was added, and the stirring was continued for 48 h at 0°C,
being monitored by TLC. The solids were collected and washed with CHCl3, and the
combined filtrate and washings were washed with sat. Na2CO3 and water, dried
(Na2SO4), and concentrated. Column chromatography (CHCl3:MeOH = 80:1) of the

926
Fukunaga et al.
residue on silica gel gave 3 (610 mg, 95%) as an amorphous mass; [a]D + 2.9 (c 0.34,
CHCl3); IR (film) 3400, 2950, 1750, 1660, 1550, 860, 840, 700cmꢁ 1 1H NMR
;
(CDCl3): d 1.00 (m, 2H, Me3SiCH2CH2), 1.44 (s, 3H, AcN), 1.59 (t, 1H, J = 12.8 Hz,
H-3e(ax)), 1.79, 1.88, 2.03, 2.13, 2.16 (5s, 15H, 5AcO), 2.44 (dd, 1H, J = 12.8, 4.4 Hz,
H-3e(eq)), 3.81 (s, 3H, MeO), 4.78 (dd, 1H, J = 9.9, 2.9 Hz, H-3d), 5.11 (d, 1H, J = 8.1
Hz, H-1d), 5.16 (dd, 1H, J = 2.6, 9.5 Hz, H-7e), 5.29 (d, 1H, J = 2.9 Hz, H-4d), 5.37
(dd, 1H, J = 8.1, 9.9 Hz, H-2d), 5.57 (s, 1H, PhCH), 5.61 (m, 1H, H-8e), 5.66 (d, 1H,
J = 10.3 Hz, NH), 7.10–8.18 (m, 50H, 10Ph).
Anal. Calcd for C123H138N2O38Si: C, 64.78; H, 6.10; N, 1.23. Found: C, 64.70; H,
5.88; N, 1.21.
2-(Trimethylsilyl)ethyl (methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonate)-(2!3)-(2,4,6-tri-O-benzoyl-
-D-galactopyranosyl)-(1!3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy- -D-glucopyr-
anosyl)-(1!3)-(2,4,6-tri-O-acetyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-O-acetyl- -
D-glucopyranoside (4). A solution of 3 (610 mg, 0.27 mmol) in acetic acid (1 mL)
and EtOH (10 mL) was treated with hydrogen over Pd(OH)2 (600 mg) overnight. The
solids were filtered off and the filtrate was concentrated. The residue was treated with
acetic anhydride (0.1 mL) in pyridine (0.5 mL) for 12 h at room temperature and
worked up. The residue was taken up in CHCl3, washed with 2N HCl and water, dried
(Na2SO4) and concentrated. Column chromatography (CHCl3:MeOH = 50:1) of the
residue on silica gel gave 4 (457 mg, 86%) as an amorphous mass; [a]D + 14.5 (c 1.2,
CHCl3); IR (film) 3400, 2950, 1750, 1660, 1550, 860, 840, 700cmꢁ 1 1H NMR
;
(CDCl3): d 0.93 (m, 2H, Me3SiCH2CH2), 1.51 (s, 3H, AcN), 1.60 (t, 1H, J = 12.8 Hz,
H-3e(ax)), 1.87, 1.91, 1.97, 1.98, 2.00, 2.03, 2.04, 2.057, 2.058, 2.06, 2.10, 2.12, 2.16
(13s, 39H, 13AcO), 2.44 (dd, 1H, J = 12.8, 4.6 Hz, H-3e(eq)), 3.81 (s, 3H, MeO), 4.19,
4.45 (2d, 2H, J = 15.3 Hz, NHC(O)CH2OAc), 5.18 (dd, 1H, J = 2.8, 9.8 Hz, H-7e),
5.30 (dd, 1H, J = 8.0, 10.1 Hz, H-2d), 5.33 (d, 1H, J = 3.2 Hz, H-4d), 5.60 (m, 1H, H-
8e), 5.75 (d, 1H, NH), 7.40–8.17 (m, 15H, 3Ph).
Anal. Calcd for C90H114N2O46Si: C, 54.38; H, 5.78; N, 1.41. Found: C, 54.22; H,
5.73; N, 1.21.
2-(Trimethylsilyl)ethyl (3,5-dideoxy-5-glycolylamino-D-glycero- -D-galacto-2-
nonulopyranosylonic acid)-(2!3)-( -D-galactopyranosyl)-(1!3)-(2-acetamido-2-de-
oxy- -D-glucopyranosyl)-(1!3)-( -D-galactopyranosyl)-(1!4)- -D-glucopyranoside
(5). To a solution of 4 (70 mg, 36 mmol) in MeOH (1 mL) was added a catalytic amount
of 28% sodium methoxide in MeOH, the reaction mixture was stirred for 24 h at room
temperature, and then water (0.1 mL) was added. After completion of the reaction, the
solution was neutralized with Amberlite IR-120 (H+) resin. The resin was filtered off and
washed with MeOH, and the combined filtrate and washings was concentrated. Column
chromatography (MeOH:H2O = 1:1) of the residue on Sephadex LH-20 gave 5 (34 mg,
95%) as an amorphous mass; [a]D ꢁ 15 (c 0.8, MeOH); IR (KBr) 3400, 2950, 1660,
1
1550, 860, 840cmꢁ 1; H NMR (D2O): d 1.00 (m, 2H, Me3SiCH2CH2), 1.77 (t, 1H,
J = 12.1 Hz, H-3e(ax)), 2.00 (s, 3H, AcN), 2.75 (dd, 1H, J = 12.1, 4.4 Hz, H-3e(eq)), 4.41
(J = 8.1 Hz), 4.47 (J = 8.1 Hz), 4.48 (J = 7.7 Hz), 4.70 (J = 8.4 Hz) (4d, 4H, four
anomeric protons).

Investigation of Specific Receptor Structures
927
Anal. Calcd for C42H74N2O30Si: C, 45.24; H, 6.69; N, 2.51. Found: C, 44.97; H,
6.48; N, 2.39.
(Methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- -D-
galacto-2-nonulopyranosylonate)-(2!3)-(2,4,6-tri-O-benzoyl- -D-galactopyranosyl)-
(1!3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy- -D-glucopyranosyl)-(1!3)-(2,4,6-tri-
O-acetyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-O-acetyl- -D-glucopyranosyl trichlo-
roacetimidate (7). A solution of 4 (150 mg, 78 mmol) in CH2Cl2 (1.0 mL) was
cooled to 0°C. TFA (1.0 mL) was added to the solution, and the mixture was stirred at
room temperature. After 1 h, the mixture was concentrated at 35°C. Column chroma-
tography (CHCl3:MeOH = 45:1) of the residue on silica gel gave 6 (129 mg, 91%) as an
amorphous mass; IR (film) 3400, 2950, 1750, 1660, 1550, 700cmꢁ 1. 1H NMR (CDCl3):
d 1.53 (s, 3H, AcN), 1.62 (t, 1H, J = 12.6 Hz, H-3e(ax)), 1.89–2.18 (13s, 39H, 13AcO),
2.46 (t, 1H, J = 12.6, 4.3 Hz, H-3e(eq)), 3.83 (s, 3H, MeO), complete loss of the TMS
ethyl group. To a solution of 6 (116 mg, 63 mmol) in CH2Cl2 (1 mL) were added
trichloroacetonitrile (0.19 mL, 1.8 mmol) and DBU (10 mL, 69 mmol) at 0°C. The
reaction mixture was stirred at 0°C for 45 min. After completion of the reaction, the
mixture was chromatographed (CHCl3:MeOH = 50:1) on a column of silica gel gave 7
(108 mg, 86%) as an amorphous mass; [a]D + 38.6 (c 0.76, CHCl3); IR (film) 3400,
2950, 1750, 1660, 1540, 700cmꢁ 1; 1H NMR (CDCl3): d 1.52 (s, 3H, AcN), 1.62 (t, 1H,
J = 12.8 Hz, H-3e(ax)), 1.89, 1.93, 1.94, 1.99, 2.00, 2.02, 2.05, 2.06, 2.080, 2.083, 2.11,
2.14, 2.18 (13s, 39H, 13AcO), 2.46 (dd, 1H, J = 12.8, 4.6 Hz, H-3e(eq)), 3.83 (s, 3H,
MeO), 4.21, 4.47 (2d, 2H, J = 15.3 Hz, NHC(O)CH2OAc), 5.16 (d, 1H, J = 8.0 Hz, H-
1d), 5.18 (dd, 1H, J = 2.5, 9.8 Hz, H-7e), 5.32 (dd, 1H, J = 8.0, 10.0 Hz, H-2d), 5.35 (d,
1H, J = 3.2 Hz, H-4d), 5.62 (m, 1H, H-8e), 5.70 (d, 1H, J = 9.8 Hz, NH), 6.48 (d, 1H
J=3.7 Hz, H-1a), 7.42–8.19 (m, 15H, 3Ph), 8.66 (s, 1H, C NH).
Anal. Calcd for C87H102Cl3N3O46: C, 51.42; H, 5.06; N, 2.07. Found: C, 51.23; H,
4.76; N, 2.00.
2-(Tetradecyl)hexadecyl (methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonate)-(2!3)-(2,4,6-tri-O-benzoyl-
-D-galactopyranosyl)-(1!3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy- -D-glucopyr-
anosyl)-(1!3)-(2,4,6-tri-O-acetyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-O-acetyl- -
D-glucopyranoside (9). To a solution of 7 (108 mg, 54 mmol) and 2-(tetrade-
˚
cyl)hexadecanol 8 (59 mg, 135 mmol) in CH2Cl2 (0.7 mL) was added MS4A (160 mg), the
reaction mixture was stirred for 3 h at room temperature and then cooled to 0°C. To the
mixture was added TMSOTf (0.62 mL, 3.3 mmol), and the reaction mixture was stirred for
12 h at 20°C. Work-up as described for 3 gave 9 (66 mg, 54%) as an amorphous mass;
[a]D + 19.4 (c 1.3, CHCl3); IR (film) 3400, 2950, 1750, 1660, 1540, 700cmꢁ 1; 1H NMR
(CDCl3): d 0.88 (t, 6H, 2MeCH2), 1.25 (s, 53H, 26CH2, CH), 1.52 (s, 3H, AcN), 1.62 (t, 1H,
J = 12.6 Hz, H-3e(ax)), 1.69–2.17 (13s, 39H, 13AcO), 2.46 (dd, 1H, J = 12.8, 4.4 Hz,
H-3e(eq)), 3.83 (s, 3H, MeO), 4.21, 4.48 (2d, 2H, J = 15.3 Hz, NHC(O)CH2OAc), 5.32
(dd, 1H, J = 8.0, 9.6 Hz, H-2d), 5.63 (m, 1H, H-8e), 5.67 (d, 1H, NH), 7.42–8.19 (m,
15H, 3Ph).
Anal. Calcd for C115H162N2O46: C, 59.83; H, 7.07; N, 1.21. Found: C, 59.57; H,
6.95; N, 0.98.

928
Fukunaga et al.
2-(Tetradecyl)hexadecyl (3,5-dideoxy-5-glycolylamino-D-glycero- -D-galacto-2-
nonulopyranosylonic acid)-(2!3)-( -D-galactopyranosyl)-(1!3)-(2-acetamido-2-de-
oxy- -D-glucopyranosyl)-(1!3)-( -D-galactopyranosyl)-(1!4)- -D-glucopyranoside
(10). To a solution of 9 (66 mg, 33 mmol) in MeOH (3 mL) was added a catalytic
amount of 28% sodium methoxide in MeOH, the reaction mixture was stirred for 24
h at room temperature, and then water (0.1 mL) was added. After completion of the
reaction, the solution was neutralized with Amberlite IR-120 (H+) resin and filtered.
The resin was washed with MeOH, and combined filtrate and washings was concen-
trated. Column chromatography (CHCl3:MeOH:H2O = 4:1:0.1) of the residue on
Sephadex LH-20 gave 10 (35 mg, 96%) as an amorphous mass; [a]D + 7.7 (c 0.39,
CHCl3:MeOH:H2O = 4:1:0.1); IR (KBr) 3400, 2950, 1650, 1550cmꢁ 1 1H NMR
;
(DMSO–d6): d 0.86 (t, 6H, 2MeCH2), 1.24 (s, 53H, 26CH2, CH), 1.52 (t, 1H, J = 12.6
Hz, H-3e(ax)), 1.79 (s, 3H, AcN), 2.79 (dd, 1H, J = 12.6, 4.4 Hz, H-3e(eq)).
Anal. Calcd for C67H122N2O30: C, 56.05; H, 8.57; N, 1.95. Found: C, 56.00; H,
8.53; N, 1.89.
(Methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- -D-
galacto-2-nonulopyranosylonate)-(2!3)-(2,4,6-tri-O-benzoyl- -D-galactopyranosyl)-
(1!4)-(2-acetamido-3,6-di-O-acetyl-2-deoxy- -D-glucopyranosyl)-(1!3)-(2,4,6-tri-
O-acetyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-O-acetyl- -D-glucopyranosyl trichlo-
roacetimidate (13). A solution of 11[14] (240 mg, 0.12 mmol) in CH2Cl2 (2 mL) was
cooled to 0°C. TFA (2 mL) was added to the solution, and the mixture was stirred at
room temperature. After 1 h, the mixture was concentrated at 35°C. Column chro-
matography (CHCl3:MeOH = 35:1) of the residue on silica gel gave 12 (220 mg, 98%)
as an amorphous mass; IR (film) 3400, 2950, 1750, 1650, 1550, 700cmꢁ 1. To a
solution of 12 (220 mg, 0.12 mmol) in CH2Cl2 (1 mL) were added trichloroacetonitrile
(0.1 mL, 3.6 mmol) and DBU (21 mL, 0.14 mmol) at 0°C. The mixture was stirred at
0°C for 45 min. After completion of the reaction, the mixture was chromatographed
(CHCl3:MeOH = 50:1) on a column of silica gel gave 13 (201 mg, 85%) as an
amorphous mass; [a]D + 38.9 (c 0.38, CHCl3); IR (film) 3400, 2950, 1750, 1660, 1540,
1
700cmꢁ 1; H NMR (CDCl3): d 1.89 (s, 3H, AcN), 1.94–2.14 (13s, 39H, 13AcO), 2.48
(dd, 1H, J = 12.6, 4.2 Hz, H-3e(eq)), 3.70 (s, 3H, MeO), 5.01 (dd, 1H, J = 3.6, 10.3 Hz,
H-2a), 6.47 (d, 1H J = 3.6 Hz, H-1a), 7.43–8.19 (m, 15H, 3Ph), 8.65 (s, 1H, C NH).
Anal. Calcd for C87H102Cl3N3O46: C, 51.42; H, 5.06; N, 2.07. Found: C, 51.22; H,
4.90; N, 1.97.
2-(Tetradecyl)hexadecyl (methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonate)-(2!3)-(2,4,6-tri-O-benzoyl-
-D-galactopyranosyl)-(1!4)-(2-acetamido-3,6-di-O-acetyl-2-deoxy- -D-glucopyr-
anosyl)-(1!3)-(2,4,6-tri-O-acetyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-O-acetyl- -
D-glucopyranoside (14). To a solution of 13 (195 mg, 97 mmol) and 8 (85 mg, 190
˚
mmol) in CH2Cl2 (1 mL) was added MS4A (130 mg), the reaction mixture was stirred
for 3 h at room temperature and then cooled to 0°C. To the mixture was added
TMSOTf (1.1 mL, 5.8 mmol), and the reaction mixture was stirred for 12 h at 20°C.
Work-up as described for 3 gave 14 (110 mg, 50%) as an amorphous mass; [a]D + 35.2
1
(c 1.3, CHCl3); IR (film) 3400, 2950, 1750, 1660, 1540, 700cmꢁ 1; H NMR (CDCl3):
d 0.88 (t, 6H, 2MeCH2), 1.25 (s, 53H, 26CH2, CH), 1.52 (s, 3H, AcN), 1.62 (t, 1H,

Investigation of Specific Receptor Structures
929
J = 12.6 Hz, H-3e(ax)), 1.69–2.17 (13s, 39H, 13AcO), 2.46 (dd, 1H, J = 12.8, 4.4 Hz,
H-3e(eq)), 3.83 (s, 3H, MeO), 4.21, 4.48 (2d, 2H, J = 15.3 Hz, NHC(O)CH2OAc), 5.32
(dd, 1H, J = 8.0, 9.6 Hz, H-2d), 5.63 (m, 1H, H-8e), 5.67 (d, 1H, NH), 7.42–8.19 (m,
15H, 3Ph).
Anal. Calcd for C115H162N2O46: C, 59.83; H, 7.07; N, 1.21. Found: C, 59.76; H,
7.03; N, 0.92.
2-(Tetradecyl)hexadecyl (3,5-dideoxy-5-glycolylamino-D-glycero- -D-galacto-2-
nonulopyranosylonic acid)-(2!3)-( -D-galactopyranosyl)-(1!4)-(2-acetamido-2-
deoxy- -D-glucopyranosyl)-(1!3)-( -D-galactopyranosyl)-(1!4)- -D-glucopyrano-
side (15). Complete O-deacylation and saponification of the methyl ester group in 14
(60 mg) were carried out as described for 10 to give 15 (33 mg, 90%) as an amorphous
mass; [a]D + 9.8 (c 0.51, CHCl3:MeOH:H2O = 4:1:0.1); IR (KBr) 3400, 2950, 1650,
1
1550cmꢁ 1; H NMR (DMSO–d6): d 0.86 (t, 6H, 2MeCH2), 1.24 (s, 53H, 26CH2, CH),
1.52 (t, 1H, J = 12.4 Hz, H-3e(ax)), 1.79 (s, 3H, AcN), 2.79 (dd, 1H, J = 12.4, 4.6 Hz,
H-3e(eq)).
Anal. Calcd for C67H122N2O30: C, 56.05; H, 8.57; N, 1.95. Found: C, 55.92; H,
8.46; N, 1.76.
2-(Trimethylsilyl)ethyl (methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonate)-(2!6)-(3-O-benzyl- -D-
galactopyranoside (18). To a solution of 16[19] (7.8 g, 12.2 mmol) and 17[20] (3.0 g,
˚
8.1 mmol) in CH3CN (70 mL) was added MS3A (11 g), the reaction mixture was
stirred for 6 h at room temperature and then cooled to ꢁ 35°C. To the mixture were
added NIS (4.1 g, 18.2 mmol) and TfOH (0.11 mL, 1.2 mmol), and the reaction
mixture was stirred for 12 h at ꢁ 35°C, being monitored by TLC. The solids were
collected and washed with CHCl3, and the combined filtrate and washings was washed
with sat. Na2CO3 and sat. Na2S2O3, dried (Na2SO4), and concentrated. Column chro-
matography (CHCl3:MeOH = 90:1) of the residue on silica gel gave 18 (4.9 g, 67%) as
an amorphous mass; [a]D ꢁ 18.6 (c 0.97, CHCl3); IR (film) 3500, 3300, 2950, 1750,
1
1650, 860, 840, 700cmꢁ 1; H NMR (CDCl3): d 1.01 (m, 2H, Me3SiCH2CH2), 2.01–
2.18 (5s, 15H, 5AcO), 2.61 (dd, 1H, J = 12.7, 4.4 Hz, H-3b(eq)), 3.81 (s, 3H, MeO),
4.30, 4.59 (2d, 2H, J = 15.0, Hz, NHC(O)CH2OAc), 4.72 (dd, 2H, J = 11.7 Hz,
PhCH2O), 4.93 (m, 1H, H-4b), 5.27 (dd, 1H, J = 1.5, 7.7 Hz, H-7e), 5.36 (m, 1H, H-
8b), 5.94 (d, 1H, J = 9.7 Hz, NH), 7.28–7.47 (m, 5H, Ph).
Anal. Calcd for C40H59NO20Si: C, 53.26; H, 6.59; N, 1.55. Found: C, 53.08; H,
6.31; N, 1.53.
2-(Trimethylsilyl)ethyl (methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonate)-(2!6)-2,4-di-O-benzoyl-3-
O-benzyl- -D-galactopyranoside (19). To a solution of 18 (3.9 g, 4.3 mmol) in
pyridine (40 mL) were added Bz2O (3.9 g, 17.2 mmol) and DMAP (0.52 g, 4.3 mmol),
and the reaction mixture was stirred at room temperature overnight. The mixture was
diluted with CHCl3 and washed with 2N HCl and water. The organic layer was dried
over Na2SO4 and concentrated. Column chromatography (CHCl3:MeOH = 100:1) of the
residue on silica gel gave 19 (4.3 g, 91%) as an amorphous mass; [a]D + 31 (c 0.97,
CHCl3); IR (film) 3300, 2950, 1750, 1660, 1550, 860, 840, 700cmꢁ 1 1H NMR
;

930
Fukunaga et al.
(CDCl3): d 0.89 (m, 2H, Me3SiCH2CH2), 2.10, 2.11, 2.19, 2.21, 2.26 (5s, 15H, 5AcO),
2.60 (dd, 1H, J = 12.6, 4.4 Hz, H-3b(eq)), 3.29 (s, 3H, MeO), 3.89 (dd, 1H, J = 10.1, 3.4
Hz, H-3a), 4.29, 4.53 (2d, 2H, J = 15.4 Hz, NHC(O)CH2OAc), 4.59, 4.75 (dd, 2H,
J = 12.5 Hz, PhCH2O), 4.70 (d, 1H, J = 8.2 Hz, H-1a), 4.85 (m, 1H, H-4b), 5.24 (dd,
1H, J = 1.5, 7.7 Hz, H-7b), 5.38 (m, 1H, H-8b), 5.46 (dd, 1H, J = 8.2, 10.1 Hz, H-2a),
6.00 (d, 1H, J = 9.8 Hz, NH), 6.02 (d, 1H, J = 3.4 Hz, H-4a), 7.13–8.25 (m, 15H, 3Ph).
Anal. Calcd for C54H67NO22Si: C, 58.42; H, 6.08; N, 1.26. Found: C, 58.29; H,
5.83; N, 1.15.
(Methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- -D-
galacto-2-nonulopyranosylonate)-(2!6)-2,4-di-O-benzoyl-3-O-benzyl- -D-galacto-
pyranosyl trichloroacetimidate (21). A solution of 19 (825 mg, 0.7 mmol) in
CH2Cl2 (5 mL) was cooled to 0°C. TFA (5 mL) was added to the solution, and the
reaction mixture was stirred at room temperature. After 2 h, the mixture was con-
centrated at 35°C. Column chromatography (CHCl3:MeOH = 50:1) of the residue on
silica gel gave 20 (749 mg, 97%) as an amorphous mass; IR (film) 3400, 2950, 1750,
1660, 1550, 700cmꢁ 1. To a solution of 20 (100 mg, 92 mmol) in CH2Cl2 (0.8 mL)
were added trichloroacetonitrile (279 mL, 2.8 mmol) and DBU (15 mL, 102 mmol) at
0°C. The reaction mixture was stirred at 0°C for 45 min. After completion of the
reaction, the mixture was chromatographed (CHCl3:MeOH = 70:1) on a column of
silica gel gave 21 (111 mg, 96%) as an amorphous mass; [a]D + 55.7 (c 1.1, CHCl3);
1
IR (film) 3400, 2950, 1750, 1660, 1550, 700cmꢁ 1; H NMR (CDCl3): d 2.01, 2.02,
2.06, 2.12, 2.17 (5s, 15H, 5AcO), 2.39 (dd, 1H, J = 12.8, 4.4 Hz, H-3e(eq)), 3.26 (s,
3H, MeO), 4.28, 4.57 (2d, 2H, J = 12.5 Hz, NHC(O)CH2OAc), 4.62, 4.79 (2d, 2H,
J = 12.5 Hz, PhCH2), 4.85 (m, 1H, H-4b), 5.25 (dd, 1H, J = 2.2, 8.4 Hz, H-7b), 5.37
(m, 1H, H-8b), 5.63 (dd, 1H, J = 3.3, 10.3 Hz, H-2a), 5.86 (d, 1H, NH), 6.06 (d, 1H,
J = 2.9 Hz, H-4a), 6.77 (d, 1H, J = 3.3 Hz, H-1a), 7.14–8.17 (m, 15H, 3Ph), 8.46 (s,
1H, C NH).
Anal. Calcd for C51H55Cl3N2O22: C, 53.07; H, 4.80; N, 2.43. Found: C, 52.77; H,
4.58; N, 2.42.
2-(Trimethylsilyl)ethyl (methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonate)-(2!6)-(2,4-di-O-benzoyl-3-
O-benzyl- -D-galactopyranosyl)-(1!3)-(2-acetamido-4,6-O-benzylidene-2-deoxy- -
D-glucopyranosyl)-(1!3)-(2,4,6-tri-O-benzyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-
O-benzyl- -D-glucopyranoside (22). To a solution of 21 (111 mg, 90 mmol) and 2A
˚
(138 mg, 108 mmol) in CH2Cl2 (0.7 mL) was added MS4A (250 mg), the reaction
mixture was stirred for 3 h at room temperature and then cooled to 0°C. To the mixture
was added TMSOTf (1.4 mL, 7.2 mmol), and the reaction mixture was stirred for 12 h at
0°C, being monitored by TLC. Work-up as described for 3 gave 22 (108 mg, 51%)
as an amorphous mass; [a]D + 21 (c 1.6, CHCl3); IR (film) 3400, 2950, 1750, 1660,
1
1550, 860, 840, 700cmꢁ 1; H NMR (CDCl3): d 1.00 (m, 2H, Me3SiCH2CH2), 1.70 (s,
3H, AcN), 1.89, 2.00, 2.02, 2.10, 2.17 (5s, 15H, 5AcO), 2.58 (dd, 1H, J = 12.8, 4.4 Hz,
H-3e(eq)), 3.06 (s, 3H, MeO), 4.28, 4.59 (2d, 2H, J = 15.7, Hz, NHC(O)CH2OAc), 4.80
(m, 1H, H-4e), 5.27 (dd, 1H, J = 2.8, 9.2 Hz, H-7e), 5.34 (dd, 1H, J = 8.2, 10.1 Hz,
H-2d), 5.42 (m, 1H, H-8e), 5.75 (d, 1H, J = 3.3 Hz, H-4d), 5.78 (s, 1H, PhCH), 5.80 (d,
1H, NH), 6.94–8.16 (m, 50H, 10Ph).

Investigation of Specific Receptor Structures
931
Anal. Calcd for C123H140N2O37Si: C, 65.18; H, 6.23; N, 1.24. Found: C, 64.88; H,
6.06; N, 1.23.
2-(Trimethylsilyl)ethyl (methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonate)-(2!6)-(3-O-acetyl-2,4-di-O-
benzoyl- -D-galactopyranosyl)-(1!3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy- -D-
glucopyranosyl)-(1!3)-(2,4,6-tri-O-acetyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-O-
acetyl- -D-glucopyranoside (23). A solution of 22 (100 mg, 42.7 mmol) in EtOH (5
mL) and AcOH (0.1 mL) was treated with hydrogen over Pd(OH)2 (100 mg) overnight.
Work-up and acetylation as described for 4 gave 23 (55 mg, 65%) as an amorphous
mass; [a]D + 9.3 (c 1.1, CHCl3); IR (film) 3400, 3100–2900, 1750, 1660, 1540, 860,
1
840, 700cmꢁ 1; H NMR (CDCl3): d 0.95 (m, 2H, Me3SiCH2CH2), 1.84 (s, 3H, AcN),
1.85, 2.00, 2.01 ꢂ 2, 2.02, 2.029 ꢂ 2, 2.034, 2.05, 2.09, 2.11 ꢂ 2, 2.175, 2.181 (14s,
42H, 14AcO), 2.52 (dd, 1H, J = 12.8, 4.4 Hz, H-3e(eq)), 3.30 (s, 3H, MeO), 4.30, 4.60
(2d, 2H, J = 15.6 Hz, NHC(O)CH2OAc), 5.71 (d, 1H, J = 3.3 Hz, H-4d), 5.86 (d, 1H,
NH), 7.45–8.12 (m, 10H, 2Ph).
Anal. Calcd for C85H112N2O46Si: C, 53.01; H, 5.86; N, 1.45. Found: C, 52.94; H,
5.58; N, 1.28.
2-(Trimethylsilyl)ethyl (3,5-dideoxy-5-glycolylamino-D-glycero- -D-galacto-2-
nonulopyranosylonic acid)-(2!6)-( -D-galactopyranosyl)-(1!3)-(2-acetamido-2-
deoxy- -D-glucopyranosyl)-(1!3)-( -D-galactopyranosyl)-(1!4)- -D-glucopyrano-
side (24). To a solution of 23 (30 mg, 20 mmol) in MeOH (2 mL) was added a
catalytic amount of 28% sodium methoxide in MeOH, the reaction mixture was stirred
for 24 h at room temperature, and then water (0.1 mL) was added. Work-up as de-
scribed for 5 gave 24 (18 mg, 98%) as an amorphous mass; [a]D ꢁ 17 (c 0.4, MeOH);
1
IR (KBr) 3400, 2950, 1660, 1550, 860, 840cmꢁ 1; H NMR (D2O) : d 1.00 (m, 2H,
Me3SiCH2CH2), 1.69 (t, 1H, J = 12.4 Hz, H-3e(ax)), 2.00 (s, 3H, AcN), 2.69 (dd, 1H,
J = 12.4, 4.6 Hz, H-3e(eq)), 4.36 (J = 8.0 Hz), 4.42 (J = 8.0 Hz), 4.47 (J = 8.0 Hz),
4.71 (J = 8.2 Hz) (4d, 4H, four anomeric protons).
Anal. Calcd for C42H74N2O30Si: C, 45.24; H, 6.69; N, 2.51. Found: C, 45.01; H,
6.48; N, 2.30.
(Methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- -D-
galacto-2-nonulopyranosylonate)-(2!6)-(3-O-acetyl-2,4-di-O-benzoyl- -D-galacto-
pyranosyl)-(1!3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy- -D-glucopyranosyl)-
(1!3)-(2,4,6-tri-O-acetyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-O-acetyl- -D-gluco-
pyranosyl trichloroacetimidate (26). A solution of 23 (100 mg, 50 mmol) in
CH2Cl2 (1 mL) was cooled to 0°C. TFA (1 mL) was added to the solution, and the
reaction mixture was stirred at room temperature. After 1 h, the mixture was con-
centrated at 35°C. Column chromatography (CHCl3:MeOH = 35:1) of the residue on
silica gel gave 25 (83 mg, 88%) as an amorphous mass; IR (film) 3400, 2950, 1750,
1
1660, 1540, 700cmꢁ 1; H NMR (CDCl3): d 1.85 (s, 3H, AcN), 2.01–2.18 (14s, 42H,
14AcO), 2.52 (dd, 1H, J = 12.8, 4.4 Hz, H-3e(eq)), 3.29 (s, 3H, MeO), 4.30, 4.58
(2d, 2H, J = 15.4 Hz, NHC(O)CH2OAc), 5.70 (d, 1H, J = 3.3 Hz, H-4d), 5.85 (d, 1H,
J = 9.9 Hz, NH), 7.47–8.12 (m, 10H, 2Ph). To a solution of 25 (83 mg, 44 mmol) in
CH2Cl2 (0.3 mL) were added trichloroacetonitrile (131 mL, 1.3 mmol) and DBU (7.2

932
Fukunaga et al.
mL, 48 mmol) at 0°C. The reaction mixture was stirred at 0°C for 1 h. Work-up as
described for 7 gave 26 (86 mg, 97%) as an amorphous mass; [a]D + 33.2 (c 1.7,
CHCl3); IR (film) 3400, 2950, 1750, 1660, 1540, 700cmꢁ 1 1H NMR (CDCl3): d
;
1.85 (s, 3H, AcN), 2.00–2.18 (14s, 42H, 14AcO), 2.53 (dd, 1H, J = 13.0, 4.6 Hz, H-
3e(eq)), 3.31 (s, 3H, MeO), 4.28, 4.57 (2d, 2H, J = 15.3 Hz, NHC(O)CH2OAc), 4.91
(m, 1H, H-4e), 5.12 (d, 1H, J = 8.0 Hz, H-1d), 5.31 (dd, 1H, J = 3.2, 10.5 Hz, H-3d),
5.34 (m, 1H, H-8e), 5.38 (dd, 1H, J = 8.0, 10.5 Hz, H-2d), 5.71 (d, 1H, J = 3.2 Hz,
H-4d), 5.83 (d, 1H, NH), 6.47 (d, 1H, J = 3.7 Hz, H-1a), 7.50–8.12 (m, 10H, 2Ph),
8.66 (s, 1H, C NH).
Anal. Calcd for C82H100Cl3N3O46: C, 49.99; H, 5.12; N, 2.13. Found: C, 49.83; H,
4.92; N, 1.99.
2-(Tetradecyl)hexadecyl (methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonate)-(2!6)-(3-O-acetyl-2,4-di-O-
benzoyl- -D-galactopyranosyl)-(1!3)-(2-acetamido-4,6-di-O-acetyl-2-deoxy- -D-
glucopyranosyl)-(1!3)-(2,4,6-tri-O-acetyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-O-
acetyl- -D-glucopyranoside (27). To a solution of 26 (86 mg, 42 mmol) and 8 (42 mg,
˚
97 mmol) in CH2Cl2 (0.3 mL) was added MS4A (140 mg), the reaction mixture was stirred
for 3 h at room temperature and then cooled to 0°C. To the mixture was added TMSOTf
(0.45 mL, 2.3 mmol), and the reaction mixture was stirred for 12 h at 0°C, being monitored
by TLC. Work-up as described for 3 gave 27 (53 mg, 55%) as an amorphous mass;
[a]D + 26.3 (c 0.75, CHCl3); IR (film) 3400, 2950, 1750, 1660, 1540, 700cmꢁ 1; 1H NMR
(CDCl3): d 0.88 (t, 6H, 2MeCH2), 1.26 (s, 53H, 26CH2, CH), 1.84 (s, 3H, AcN), 1.88 (t,
1H, H-3e(ax)), 1.85–2.18 (14s, 42H, 14AcO), 2.52 (dd, 1H, J = 13.0, 4.6 Hz, H-3e(eq)),
3.29 (s, 3H, MeO), 4.30, 4.59 (2d, 2H, J = 15.3 Hz, NHC(O)CH2OAc), 5.12 (d, 1H,
J = 8.2 Hz, H-1d), 5.31 (dd, 1H, J = 3.2, 10.3 Hz, H-3d), 5.35 (m, 1H, H-8e), 5.71 (d, 1H,
J = 3.2 Hz, H-4d), 5.81 (d, 1H, NH), 7.47–8.12 (m, 10H, 2Ph).
Anal. Calcd for C110H160N2O46: C, 58.81; H, 7.18; N, 1.25. Found: C, 58.59; H,
6.95; N, 1.04.
2-(Tetradecyl)hexadecyl (3,5-dideoxy-5-glycolylamino-D-glycero- -D-galacto-2-
nonulopyranosylonic acid)-(2!6)-( -D-galactopyranosyl)-(1!3)-(2-acetamido-2-de-
oxy- -D-glucopyranosyl)-(1!3)-( -D-galactopyranosyl)-(1!4)- -D-glucopyranoside
(28). To a solution of 27 (37 mg, 16 mmol) in MeOH (2 mL) and THF (1.5 mL) was added
a catalytic amount of 28% sodium methoxide in MeOH, the reaction mixture was stirred
for 24 h at room temperature, and then water (0.1 mL) was added. Work-up as described
for 5 gave 28 (21 mg, 98%); [a]D + 10.8 (c 0.61, CHCl3:MeOH:H2O = 4:1:0.1); IR (KBr)
1
3400, 2950, 1650, 1550cmꢁ 1; H NMR (DMSO–d6): d 0.86 (t, 6H, 2MeCH2), 1.24 (s,
53H, 26CH2, CH), 1.82 (s, 3H, AcN), 2.64 (dd, 1H, J = 11.7, 4.4 Hz, H-3e(eq)).
Anal. Calcd for C67H122N2O30: C, 56.05; H, 8.57; N, 1.95. Found: C, 56.05; H,
8.46; N, 1.78.
2-(Trimethylsilyl)ethyl (methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonate)-(2!6)-(2,4-di-O-benzoyl-3-
O-benzyl- -D-galactopyranosyl)-(1!4)-(2-acetamido-3,6-di-O-benzyl-2-deoxy- -D-
glucopyranosyl)-(1!3)-(2,4,6-tri-O-benzyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-
O-benzyl- -D-glucopyranoside (29). To a solution of 21 (334 mg, 271 mmol) and
˚
2B (204 mg, 149 mmol) in CH2Cl2 (1.5 mL) was added MS4A (200 mg), the reaction

Investigation of Specific Receptor Structures
933
mixture was added TMSOTf (5.3 mL, 27 mmol), and the reaction mixture was stirred
for 12 h at 0°C, being monitored by TLC. Work-up as described for 3, and column
chromatography (n-hexane:AcOEt = 1:2) of the residue on silica gel gave 29 (296 mg,
82%) as an amorphous mass; [a]D + 6.2 (c 0.45, CHCl3); IR (film) 3400, 2950, 1750,
1
1660, 1540, 860, 840, 700cmꢁ 1; H NMR (CDCl3): d 1.00 (m, 2H, Me3SiCH2CH2),
1.50 (s, 3H, AcN), 1.94, 2.00, 2.05, 2.07, 2.17 (5s, 15H, 5AcO), 2.52 (dd, 1H,
J = 12.8, 4.4 Hz, H-3e(eq)), 3.25 (s, 3H, MeO), 4.28, 4.58 (2d, 2H, J = 15.4 Hz,
NHC(O)CH2OAc), 5.26 (dd, 1H, J = 1.8, 8.4 Hz, H-7e), 5.37 (m, 1H, H-8e), 5.40 (dd,
1H, J=8.1, 10.9 Hz, H-2d), 5.83 (d, 1H, NH), 5.84 (d, 1H, J = 3.3 Hz, H-4d), 7.07–
8.07 (m, 55H, 11Ph).
Anal. Calcd for C130H148N2O37Si: C, 66.20; H, 6.32; N, 1.19. Found: C, 65.98; H,
6.24; N, 1.05.
2-(Trimethylsilyl)ethyl (methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonate)-(2!6)-(3-O-acetyl-2,4-di-O-
benzoyl- -D-galactopyranosyl)-(1!4)-(2-acetamido-3,6-di-O-acetyl-2-deoxy- -D-
glucopyranosyl)-(1!3)-(2,4,6-tri-O-acetyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-O-
acetyl- -D-glucopyranoside (30). A solution of 29 (220 mg, 90 mmol) in EtOH (10
mL) was hydrogenated over Pd(OH)2 (220 mg) overnight. The solid was filtered off
and the filtrate was concentrated. The residue was acetylated with acetic anhydride (0.1
mL) in pyridine (0.5 mL) for 12 h at room temperature. Work-up as described for 3
gave 30 (145 mg, 82%) as an amorphous mass; [a]D + 1.0 (c 1.2, CHCl3); IR (film)
1
3400, 2950, 1750, 1660, 1550, 860, 840, 700cmꢁ 1; H NMR (CDCl3): d 0.95 (m, 2H,
Me3SiCH2CH2), 1.84 (s, 3H, AcN), 1.91–2.18 (14s, 42H, 14AcO), 2.53 (dd, 1H,
J = 12.8, 4.4 Hz, H-3e(eq)), 3.43 (s, 3H, MeO), 4.30, 4.59 (2d, 2H, J = 15.4 Hz,
NHC(O)CH2OAc), 5.33 (dd, 1H, J = 3.1, 10.6 Hz, H-3d), 5.38 (m, 1H, H-8e), 5.46 (dd,
1H, J = 8.1, 10.6 Hz, H-2d), 5.59 (d, 1H, NH), 5.78 (d, 1H, J = 3.1 Hz, H-4d), 5.90 (d,
1H, NH), 7.43–8.11 (m, 10H, 2Ph).
Anal. Calcd for C85H112N2O46Si: C, 53.01; H, 5.86; N, 1.45. Found: C, 52.77; H,
5.69; N, 1.19.
2-(Trimethylsilyl)ethyl (3,5-dideoxy-5-glycolylamino-D-glycero- -D-galacto-2-
nonulopyranosylonic acid)-(2!6)-( -D-galactopyranosyl)-(1!4)-(2-acetamido-2-
deoxy- -D-glucopyranosyl)-(1!3)-( -D-galactopyranosyl)-(1!4)- -D-glucopyrano-
side (31). To a solution of 30 (35 mg, 18 mmol) in MeOH (2 mL) was added a
catalytic amount of 28% sodium methoxide in MeOH, the reaction mixture was stirred
for 24 h at room temperature, and then water (0.1 mL) was added. Work-up as de-
scribed for 5 gave 31 (20 mg, 98%) as an amorphous mass; [a]D ꢁ 12.8 (c 0.4,
1
MeOH); IR (KBr) 3400, 2950, 1660, 1550, 860, 840cmꢁ 1; H NMR (D2O) : d 1.00
(m, 2H, Me3SiCH2CH2), 1.71 (t, 1H, J = 12.4 Hz, H-3e(ax)), 2.03 (s, 3H, AcN), 2.66
(dd, 1H, J = 12.4, 4.6 Hz, H-3e(eq)), 4.41 (J = 8.2 Hz), 4.43 (J = 8.0 Hz), 4.47 (J = 8.0
Hz), 4.70 (J = 8.0 Hz) (4d, 4H, four anomeric protons).
Anal. Calcd for C42H74N2O30Si: C, 45.24; H, 6.69; N, 2.51. Found: C, 45.04; H,
6.67; N, 2.34.
(Methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero- -D-
galacto-2-nonulopyranosylonate)-(2!6)-(3-O-acetyl-2,4-di-O-benzoyl- -D-galacto-
mixture was stirred for 3 h at room temperature and then cooled to 0°C. To the

934
Fukunaga et al.
pyranosyl)-(1!4)-(2-acetamido-3,6-di-O-acetyl-2-deoxy- -D-glucopyranosyl)-
(1!3)-(2,4,6-tri-O-acetyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-O-acetyl- -D-gluco-
pyranosyl trichloroacetimidate (33). A solution of 30 (80 mg, 40 mmol) in CH2Cl2
(1 mL) was cooled to 0°C. TFA (1 mL) was added to the solution, and the reaction
mixture was stirred at room temperature. After 1 h, the mixture was concentrated at
35°C. Column chromatography (CHCl3:MeOH = 35:1) of the residue on silica gel gave
32 (73 mg, 96%) as an amorphous mass; IR (film) 3400, 2950, 1750, 1660, 1550,
700cmꢁ 1. To a solution of 32 (73 mg, 38 mmol) in CH2Cl2 (0.7 mL) were added
trichloroacetonitrile (115 mL, 1.1mmol) and DBU (6.3 mL, 42 mmol) at 0°C. The
reaction mixture was stirred at 0°C for 1 h. Work-up as described for 7 gave 33
(69 mg, 88%) as an amorphous mass; [a]D + 25.5 (c 1.2, CHCl3); IR (film) 3400,
2950, 1750, 1680, 1550, 700cmꢁ 1; 1H NMR (CDCl3): d 1.84 (s, 3H, AcN), 1.91–2.18 (14s,
42H, 14AcO), 2.52 (dd, 1H, J = 12.8, 4.6 Hz, H-3e(eq)), 3.43 (s, 3H, MeO), 4.29, 4.60 (2d,
2H, J = 15.3 Hz, NHC(O)CH2OAc), 4.82 (d, 1H, J = 7.8 Hz, H-1d), 4.89 (m, 1H, H-4e),
5.33 (dd, 1H, J = 3.2, 10.5 Hz, H-3d), 5.37 (m, 1H, H-8e), 5.45 (dd, 1H, J = 7.8, 10.5 Hz, H-
2d), 5.78 (d, 1H, J = 3.2 Hz, H-4d), 5.86 (d, 1H, NH), 6.46 (d, 1H, J = 3.7 Hz, H-1a), 7.43–
8.11 (m, 10H, 2Ph), 8.66 (s, 1H, C NH).
Anal. Calcd for C82H100Cl3N3O46: C, 49.99; H, 5.12; N, 2.13. Found: C, 49.76; H,
4.88; N, 2.06.
2-(Tetradecyl)hexadecyl (methyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-
dideoxy-D-glycero- -D-galacto-2-nonulopyranosylonate)-(2!6)-(3-O-acetyl-2,4-di-O-
benzoyl- -D-galactopyranosyl)-(1!4)-(2-acetamido-3,6-di-O-acetyl-2-deoxy- -D-
glucopyranosyl)-(1!3)-(2,4,6-tri-O-acetyl- -D-galactopyranosyl)-(1!4)-2,3,6-tri-O-
acetyl- -D-glucopyranoside (34). To a solution of 33 (61 mg, 30 mmol) and 2-
˚
(tetradecyl)hexadecanol 8 (30 mg, 68 mmol) in CH2Cl2 (0.3 mL) was added MS4A
(100 mg), the reaction mixture was stirred for 3 h at room temperature and then
cooled to 0°C. To the mixture was added TMSOTf (0.29 mL, 1.5 mmol), and the
reaction mixture was stirred for 12 h at 0°C, being monitored by TLC. Work-up as
described for 3 gave 34 (36 mg, 51%) as an amorphous mass; [a]D + 16.4 (c 0.71,
CHCl3); IR (film) 3400, 2950, 1750, 1660, 1540, 700cmꢁ 1 1H NMR (CDCl3): d
;
0.89 (t, 6H, 2MeCH2), 1.25 (s, 53H, 26CH2, CH), 1.84 (s, 3H, AcN), 1.91–2.18 (14s,
42H, 14AcO), 2.52 (dd, 1H, J = 12.8, 4.4 Hz, H-3e(eq)), 3.43 (s, 3H, MeO), 4.30, 4.6
(2d, 2H, J = 15.4 Hz, NHC(O)CH2OAc), 4.82 (d, 1H, J = 7.7 Hz, H-1d), 4.94 (m, 1H,
H-4e), 5.32 (dd, 1H, J = 3.3, 10.3 Hz, H-3d), 5.37 (m, 1H, H-8e), 5.46 (dd, 1H,
J = 7.7, 10.3 Hz, H-2d), 5.77 (d, 1H, J = 3.3 Hz, H-4d), 5.83 (d, 1H, NH), 7.43–8.11
(m, 10H, 2Ph).
Anal. Calcd for C110H160N2O46: C, 58.81; H, 7.18; N, 1.25. Found: C, 58.67; H,
6.97; N, 1.16.
2-(Tetradecyl)hexadecyl (3,5-dideoxy-5-glycolylamino-D-glycero- -D-galacto-2-
nonulopyranosylonic acid)-(2!6)-( -D-galactopyranosyl)-(1!4)-(2-acetamido-2-
deoxy- -D-glucopyranosyl)-(1!3)-( -D-galactopyranosyl)-(1!4)- -D-glucopyrano-
side (35). To a solution of 34 (36 mg, 15 mmol) in MeOH (2 mL) and THF (3 mL) was
added a catalytic amount of 28% sodium methoxide in MeOH, the reaction mixture was
stirred for 24 h at room temperature, and then water (0.1 mL) was added. Work-up as

Investigation of Specific Receptor Structures
935
described for 5 gave 36 (20 mg, 97%); [a]D + 8.1 (c 0.57, CHCl3:MeOH:H2O = 4:1:0.1);
IR (KBr) 3400, 2950, 1660, 1550cmꢁ 1; 1H NMR (DMSO–d6): d 0.82 (t, 6H, 2MeCH2),
1.21 (s, 53H, 26CH2, CH), 1.81 (s, 3H, AcN), 2.62 (dd, 1H, J = 12.8, 4.4 Hz, H-3e(eq)).
Anal. Calcd for C67H122N2O30: C, 56.05; H, 8.57; N, 1.95. Found: C, 56.02; H,
8.38; N, 1.94.
ACKNOWLEDGMENT
This work was supported in part by a Grant-in-Aid (No. 12306007) for Scientific
Research from Japan Society for the Promotion of Science.
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Received April 1, 2003
Accepted August 26, 2003