Syntheses of new peptidic glycoclusters derived from β-alanine: di- and trimerized glycoclusters and glycocluster-clusters

Article abstract of DOI:10.1016/j.carres.2006.02.015

Fluorescence-labeled glycoclusters 1 and 2 have been synthesized to elongate glycocluster units that contain β-alanine derivative 6 and sugar unit 7. Similarly, di- and trimerized glycoclusters 3 and 4 have been obtained by coupling glycocluster 17 with s

Full text of DOI:10.1016/j.carres.2006.02.015

Carbohydrate Research 341 (2006) 836–845
Syntheses of new peptidic glycoclusters derived from b-alanine:
di- and trimerized glycoclusters and glycocluster–clusters I
Koji Sato, Noriyasu Hada and Tadahiro Takeda*
Kyoritsu University of Pharmacy, 1-5-30 Shibakoen, Minato-ku, Tokyo 105-8512, Japan
Received 8 August 2005; accepted 16 February 2006
Available online 13 March 2006
Abstract—Fluorescence-labeled glycoclusters 1 and 2 have been synthesized to elongate glycocluster units that contain b-alanine
derivative 6 and sugar unit 7. Similarly, di- and trimerized glycoclusters 3 and 4 have been obtained by coupling glycocluster 17
with succinyl chloride and/or trimesoyl chloride, respectively. Furthermore, glycocluster–cluster 5 was also synthesized by a conver-
gent growth approach.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Glycocluster; Glycocluster–cluster; Elongation cycle; D-Galactose; Fluorescence label
1. Introduction
be developed with high affinity to the specific receptor
due to the entropic gain. Recently, artificial carbo-
hydrate ligands to the receptors of AB5 heat-labile
enterotoxin and also Shiga-like toxin based on the
molecular design described above have been developed
by two independent groups,31,32 but still, there are few
other such examples. When structural information on
protein receptors is poor, it is necessary to give diversity
to the structure for searching out suitable ligands. For
this purpose, we synthesized new peptidic glycoclusters
and a glycodendron consisting of a b-alanine derivative,
a sugar unit and an x-amino acid.33,34 By this method, it
is possible to create diverse ‘glycocluster units’ by only
replacing the x-amino acid with another one as spacer,
which is advantageous for the synthesis of diverse
glycoclusters in which the distance from the main chain
to the carbohydrate residue and/or the distance between
the side chain branch points can be readily modulated.
By giving diversity to glycoclusters, the construction of
a glycocluster library may be possible, and such a library
can be expected to aid in determining the elucidation of
the higher-order structure of the receptors.
Carbohydrate chains are known to interact with protein
receptors as natural clusters (molecular assemblies) to
form rafts (glycolipids),1 modified proteins (glycopro-
teins)2 and repeating structures (polysaccharides),3 and
these express strong molecular recognition properties.
This has been called the multivalent or cluster effect.
On the other hand, synthetic or isolated carbohydrate
chains have low affinities towards the receptors in many
cases.4 Therefore, the elucidation of their bioactivity has
been complicated in glycoscience. For resolution of this
problem, various multivalent carbohydrates and glyco-
conjugates including glycoclusters,5–16 glycodendri-
mers17–26 and glycopolymers27–30 have been synthesized
with the aim of creating ‘glycoside cluster effects’. How-
ever, almost all of these cluster molecules have been
randomly functionalized, and these have symmetric or
repeating structures arising from their synthetic strate-
gies. If the steric structure and geometry of the binding
site on the targeted carbohydrate-binding protein could
be analyzed and designed, a carbohydrate ligand might
In this paper, first, we report on the conventional
tri- and pentavalent fluorescence-labeled glycoclusters
(1 and 2), which do not incorporate an x-amino acid
as a spacer, then we describe the synthesis of di- and
q Part 3 of the series.
*
Corresponding author. Tel.: +81 3 54002696; fax: +81 3 54002556;
e-mail: takeda-td@kyoritsu-ph.ac.jp
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2006.02.015

K. Sato et al. / Carbohydrate Research 341 (2006) 836–845
837
di-carboxylic acid
(A)
(B)
tri-carboxylic acid
cluster-chain unit
(C)
glycocluster-cluster unit
tri-carboxylic acid
ω-amino acid di-carboxylic acid
sugar unit
cluster chain unit
Figure 1. Polymerization of glycocluster: (A) dimerization with a dicarboxylic acid as the dendrimer core; (B) trimerization with a tricarboxylic acid
as the dendrimer core; (C) clusterization of glycocluster–cluster unit.
trimerized glycoclusters (3 and 4) by coupling with the
precursor trivalent glycocluster 17 and a di- or tricarb-
oxylic acid in anticipation of higher affinity as an appli-
cation of our method (Fig. 1A and B). Finally, the
conversion of a simple sugar unit to a glycocluster unit
in order to synthesize a novel ‘glycocluster–cluster (5)’
is carried out using a glycocluster–cluster unit in which
a condensed glycocluster unit is condensed with a cluster
chain unit (Fig. 1C). These target compounds 1–5 are
depicted in Figure 2.
have formed the conventional glycocluster unit 8 by
using only cluster–chain unit 6 and sugar unit 7 to sim-
plify the process. Cluster–chain unit 6 and sugar unit 7
were prepared according to the previous paper.33 Elon-
gation of the glycocluster is composed of three reactions:
peptide coupling, deprotection of the tert-butyloxycar-
bonyl (Boc) group and deprotection of the trichloro-
ethyl ester (Tce) group. Coupling of cluster–chain unit
6 with the sugar unit 7 in the presence of diethyl phos-
phorocyanidate (DEPC) in dry DMF yielded 80% of
the glycocluster 8. Subsequent removal of the Boc group
with 50% TFA in CH2Cl2 or the Tce group with Zn–
AcOH afforded glycocluster 9 (quant) with an amino
group or glycocluster 10 (90%) with a carboxylic acid,
respectively. Coupling of 9 with 10 as described for 8
gave a dimer derivative 11 in 80% yield. By repeating
the coupling and deprotection, the ‘elongation cycle’
2. Results and discussion
In our previous work,33,34 we exploited glycoclusters
either attached to or inserted into an x-amino acid for
modulating steric distribution. However, this time we

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K. Sato et al. / Carbohydrate Research 341 (2006) 836–845
O
OH
HO
O
O
N
H
OH
O
HO
HO
N
O
N
OH
O
O
N
O
O
OH
OH
HO
O
H
O
N
H
OH
OH
HO
O
O
OH
O
O
HO
HO
HO
HO
N
O
N
OH
OH
HO
O
N
O
O
N
O
N
H
O
O
H
O
H
OH
OH
OH
N
O
N
O
HO
HO
OH
OH
HO
HO
O
N
HO
O
N
O
N
O
O
O
OH
O
N
H
HO
HO
O
O
O
N
H
H
OH
OH
O
H
O
O
N
OH
OH
O
HO
O
OH
O
HO
HO
HO
OH
HO
O
N
O
O
N
HO
HO
O
N
OH
H
NH
O
H
H
NH
OH
N
O
OH
O
S
O
3
O
N
O
S
O
N
HO
HO
OH
OH
O
OH
2
OH
NH
O
O
1
O
HO
OH
O
O
HO
N
C
CO
HO
OH
N
H3
CH3
CO
OH
H
H3
C
CH3
OH
O
O
C
OH
HO
O
N
HO
OH
N
CO
OH
O
OH
OH
H
HO
O
O
HO
OH
O
OH
O
OH
OH
O
C
OH
O
N
H
NH
OH
O
HO
N
OH
O
HO
N
H
OH
O
OH
O
HO
O
O
O
O
O
HO
OH
O
HO
N
O
C
N
O
O
O
N
H
OH
N
OH
CO
HN
OH
OH
CH3
HO
H
OH
OH
OH
O
HO
N
OH
O
NH
O
O
CH3
O
N
O
O
C
N
HO
N
O
N
O
HO
OH
O
O
O
N
H
O
N
OH
OH
N
H
H
OH
N
O
N
H
O
N
H
H
N
H
N
O
O
O
O
O
N
O
N
HO
S
N
O
O2
O
OH
H
O
O
N
NH
NH
O
O
NH
N
H
N
H
N
OH
O
OH
O
O
NH
NH
OH
O
HO
O
O
C
N
HO
OH
OH
N
O
C
O
O
O
O
OH
OH
OH
O
H
HO
OH
H
OH
CO
N
OH
CO
N
O
O
HO
OH
HO
OH
C
N
O
O
HO
OH
HO
OH
C
N
O
O
4
O
O
O
HO
O
O
OH
OH
OH
OH
HO
H
OH
H
N
N
OH
CO
CO
O
O
5
HO
OH
OH
OH
HO
HO
Figure 2. Structures of synthetic glycoclusters.
was completed and the sugar residues on the glycoclus-
ter were increased exponentially. The C-terminal ends of
the glycoclusters were closed by coupling with sugar unit
7. When the elongation cycle was terminated, the free
acid glycoclusters 13 and 15 gave 16 (78%) and 18
(79%), respectively. Subsequent treatment with 50%
TFA to remove the Boc groups gave 17 (79%) and 19
(86%), respectively. Alternatively, the N-terminal end
of the glycocluster could be elaborated by modifying
the free amine. The free amine trimer 17 and pentamer
19 were treated with dansyl glycine in the presence of
DEPC, and complete removal of the O-benzoyl groups
provided the target compound 1 (quant) and 2 (96%)
with free hydroxyl groups on all the fluorescence-labeled
glycoclusters (Scheme 1).
Glycocluster compound 17 with a free amine can be
coupled with poly(carboxylic) acid derivatives as the
dendrimer cores to increase valency and to enhance affin-
ity. If the higher valency glycoclusters are needed in the
glycocluster library, the glycoclusters with deprotected
amines can be used. Therefore, the trimer derivative 17
was coupled with succinyl chloride and trimesoyl chlo-
ride as the dendrimer cores to give the di- and trimerized
trimer derivatives 22 (85%) and 23 (81%), respectively.
Subsequent removal of the acyl groups from 22 and 23
afforded the di- and trimerized trimer glycocluster 3
(95%) and 4 (quant), respectively (Scheme 2).
The sugar unit with a free amine and a cluster–chain
unit with a free carboxylate of the glycocluster unit
made the additional clustering of the glycoclusters pos-
sible leading to the synthesis of a ‘glycocluster–cluster
unit’. Compound 17 was used as the glycocluster deriv-
ative to synthesize a glycocluster–cluster unit. Since the
unit seemed to be too bulky, a cluster–chain unit was
attached by means of a spacer. In brief, compound 6
was coupled with benzyl 6-aminohexanoate to give
compound 24 in 77% yield. Removal of the Tce group
as described for 10 subsequently afforded compound
25 (80%), which was then converted to 26 by coupling
with 2,2,2-trichloroethyl 6-aminohexanoate in 64%
yield. Finally, removal of the benzyl group in 26 under
neutral conditions by hydrogenation over 10% Pd–C
afforded compound 27 (39%) as a spacer-modulated
cluster–chain unit. Glycocluster–cluster unit 28 was
synthesized by coupling 27 with 17. Subsequent removal
of the Tce group as described for 10 gave compound 29
(85%), the C-terminal free acid of which was coupled
with 17 to give compound 30 (89%). Removal of the
Boc group as described for 9 afforded compound 31 in
73% yield, and it was subsequently coupled with 29 to
give compound 32 (66%), which is elongated by one unit
relative to 30. Removal of the Boc group and coupling
with 29 were carried out repeatedly, and the resulting
compound was again coupled with dansyl glycine as

K. Sato et al. / Carbohydrate Research 341 (2006) 836–845
839
COOTce
OBz
OBz
O
+
HOOC
N
O
BzO
Boc
NH2
6
7OBz
a
OTce
O
O
OBz
OBz
O
N
O
BzO
Boc
2n
N
OBz
H
n = 0
8
n = 1 11
n = 2 14
c
b
a
OTce
OH
O
O
O
OBz
OBz
OBz
OBz
O
O
O
O
N
O
N
BzO
BzO
H
2n
N
Boc
2n
N
OBz
OBz
H
H
n = 0 10
n = 1 13
n = 2 15
n = 0 9
n = 1 12
elongation clycle
OBz
OR
d
OBz
OR
O
O
O
O
BzO
OBz
RO
OR
OBz
OR
O
O
N
HN
HN
OBz
OR
e
O
O
O
O
N
O
O
O
BzO
RO
R
2n
N
H
N
OBz
n =
OR
n =
2n
1
2H
1
2
NH
R = Bz 20 21
R = H
R = Boc 16 18
R = H 17 19
Dansyl
f
b
1
2
Scheme 1. Reagents: (a) DEPC, Et3N, DMF; (b) 50% TFA, CH2Cl2; (c) Zn–AcOH; (d) 7, DEPC, Et3N, DMF; (e) dansyl Gly, DEPC, Et3N, DMF;
(f) NaOMe, MeOH-1,4-dioxane.
described for 20 to give compound 36 (51%), similar to
the fluorescence-labeled compound 17. Finally,
complete removal of the acyl groups afforded the
fluorescence-labeled glycocluster–cluster 5 in 36% yield
(Scheme 3).
In conclusion, construction of the bioactive glycoclus-
ter and glycoclusters with the higher affinities for recep-
tor binding could be expected using this method.
Moreover, it is worthwhile to synthesize various kinds
of glycoclusters using common synthetic routes and
inexpensive materials.
recorded with a JNM A 500 FT NMR spectrometer
with Me4Si as the internal standard for solutions in
CDCl3 or CD3OD. MALDI-TOF MS was recorded
on a Perceptive Voyager RP mass spectrometer. TLC
was performed by Silica Gel 60-F254 (E. Merck) with
detection by quenching of UV fluorescence and by
spraying with 5% ninhydrin and 10% H2SO4. Column
chromatography was carried out on Silica Gel 60
(E. Merck).
3.2. Typical procedure for the peptide condensation
reaction
3. Experimental
3.1. General
To a solution of cluster–chain unit 6 (202 mg,
0.53 mmol) and sugar unit 7 (434 mg, 0.64 mmol) in
DMF (2 mL) were added diethyl phosphorocyanidate
(DEPC, 90 lL, 0.59 mmol) and Et3N (110 lL,
0.79 mmol). The mixture was stirred for 16 h at room
temperature. After completion of the reaction (TLC
Optical rotations were determined with a Jasco digital
polarimeter. 1H NMR and 13C NMR spectra were

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K. Sato et al. / Carbohydrate Research 341 (2006) 836–845
OR
RO
RO
O
O
OR
O
OR
OR
NH
OR
RO
O
O
RO
O
O
O
RO
N
O
O
N
OR
O
N
H
OR
O
OR
HN
O
OR
OR
RO
OR
OR
RO
O
O
O
O
O
RO
N
O
N
O
N
OR
O
N
H
OR
OR
RO
OR
H
OR
OR
O
HN
O
OR
O
O
O
O
RO
N
H
OR
RO
O
N
O
N
N
O
N
H
OR
OR
O
O
a
c
OR
17
O
O
O
NH
O
O
RO
N
O
N
H
OR
O
NH
H
N
OR
OR
O
O
RO
O
N
O
NH
O
O
N
OR
O
OR
H
N
OR
RO
OR
O
O
RO
O
OR
O
O
OR
RO
OR
OR
O
RO
NH
R = Bz 22
R = H
OR
O
OR
b
O
O
3
RO
RO
OR
RO
R = Bz 23
R = H
OR
b
RO
OR
4
Scheme 2. Reagents: (a) succinyl chloride, Et3N, CH2Cl2; (b) NaOMe, MeOH-1,4-dioxane; (c) trimesoyl chloride, Et3N, CH2Cl2.
Scheme 3. Reagents: (a) benyl 6-aminohexanoate tosylate, Et3N, DEPC, DMF; (b) Zn–AcOH; (c) 2,2,2-trichloroethyl 6-aminohexanoate tosylate,
Et3N, DEPC, DMF; (d) Pd–C, H2, THF; (e) 17, Et3N, DEPC, DMF; (f) 50% TFA; (g) 29, Et3N, DEPC, DMF; (h) Dansyl Gly, DEPC, Et3N, DMF;
(i) NaOMe, MeOH-1,4-dioxane.
23
1
monitoring), the mixture was extracted with EtOAc,
washed with H2O, dried (Na2SO4), and concentrated.
The product was purified by silica gel column chromato-
graphy (5:1 benzene–acetone as eluent) to give 8
(157 mg, 79.6%): ½aꢀD +59.0 (c 1.9, CHCl3); H NMR
(500 MHz, CDCl3): d 8.10–7.23 (m, 20H, Ar-H), 6.25
(br t, 1H, NH), 6.00 (br d, 1H, H-4), 5.77 (br dd, 1H,
H-2), 5.63 (br dd, 1H, H-3), 4.87 (br d, 1H, H-1), 4.72

K. Sato et al. / Carbohydrate Research 341 (2006) 836–845
841
(br s, 2H, CH2CCl3), 4.67 (br dd, 1H, H-6a), 4.45 (br dd,
1H, H-6b), 4.36 (m, 1H, H-5), 3.98 and 3.76 (m, 2H,
OCH2 of sugar unit), 3.65 (br s, 2H, NCH2CO), 3.55–
3.49 (m, 4H, NCH2 of sugar unit, NCH2 of b-Ala),
2.72 (br t, 2H, COCH2 of b-Ala), 1.43 (9H, br s,
t-Bu); 13C NMR (125 MHz, CDCl3): d 169.3, 166.0,
165.5, 165.3, 133.7, 133.5, 133.4, 133.3, 130.0, 129.7,
129.3, 129.2, 129.0, 128.7, 128.6, 128.5, 128.3, 101.7,
94.8, 80.9, 74.1, 71.6, 71.5, 69.8, 68.9, 68.1, 62.1, 51.7,
44.9, 39.2, 33.0, 28.3. MALDI-TOF MS: Calcd for
C48H49Cl3N2NaO15: m/z 1021.2. Found: m/z 1021.9
[M+Na]+.
Ar-H), 6.00 (br d, 5H, H-4), 5.76 (br dd, 5H, H-2),
5.66 (br dd, 5H, H-3), 4.91 (br d, 5H, H-1), 4.65 (m,
5H, H-6a), 4.38 (m, 10H, H-5, H-6b), 3.94–3.36 (m,
36H, NCH2,OCH2), 2.53–2.08 (m, 8H, CCH2), 1.37,
1.31 (2s, 9H, t-Bu). MALDI-TOF MS: Calcd for
C
205H193N9NaO60: m/z 3763.2. Found: m/z 3763.2
[M+Na]+.
23
3.2.6. Data for 20. Yield: 15 mg (98%); ½aꢀD +61.1 (c
1
0.4, CHCl3); H NMR (CDCl3): d 8.26–7.21 (m, 66H,
Ar-H), 5.99 (br d, 3H, H-4), 5.76 (br dd, 3H, H-2),
5.65 (br dd, 3H, H-3), 4.89 (br d, 3H, H-1), 4.66 (m,
3H, H-6a), 4.41 (m, 6H, H-5, H-6b), 3.95–2.04 (m,
32H, CH2, NCH3). MALDI-TOF MS: Calcd for
3.2.1. Compounds 11, 14, 16, 18, 20, 21, 24, 26, 28, 30,
32, 34 and 36. These compounds were obtained in a
similar manner by purification by column chromato-
graphy using a gradient of 40:1!20:1 CHCl3–MeOH
as eluent.
C
132H123N7NaO37S: m/z 2452.8. Found: m/z 2452.5
[M+Na]+.
3.2.7. Data for 21. Yield, 9 mg (83%); ½aꢀ2D3 +75.6 (c 0.2,
1
CHCl3); H NMR (CDCl3): d 8.38–7.22 (m, 106H, Ar-
23
3.2.2. Data for 11. Yield: 112 mg (80%); ½aꢀD +63.9
H), 5.99 (br d, 5H, H-4), 5.75 (br dd, 5H, H-2), 5.66 (br
dd, 5H, H-3), 4.91 (br d, 5H, H-1), 4.66 (m, 5H, H-6a),
4.37 (m, 10H, H-5, H-6b), 3.94–2.04 (m, 52H, CH2).
MALDI-TOF MS: Calcd for C214H199N11NaO61S: m/z
3953.3. Found: m/z 3953.8 [M+Na]+.
(c 0.9, CHCl3); 1H NMR (CDCl3): d 8.08–7.24 (m,
40H, Ar-H), 6.00 (br d, 2H, H-4), 5.76 (br dd, 2H, H-
2), 5.63 (br dd, 2H, H-3), 4.89 (br d, 2H, H-1), 4.73 (s,
2H, CH2CCl3), 4.68 (m, 2H, H-6a), 4.42–4.34 (m, 4H,
H-5, H-6b), 3.98–3.31 (m, 16H, OCH2 of sugar unit,
NCH2 of sugar unit, NCH2CO, NCH2 of b-Ala),
2.72–2.05 (m, 4H, COCH2 of b-Ala), 1.39 and 1.32
(2s, 9H, t-Bu); 13C NMR (CDCl3): d 170.2, 166.0,
165.5, 165.3, 165.2, 133.6, 133.5, 133.3, 130.0, 129.7,
129.3, 129.2, 129.0, 128.6, 128.5, 128.3, 101.0, 94.8,
74.1, 71.6, 71.4, 69.84, 69.76, 68.1, 67.6, 61.8, 45.8,
43.8, 39.2, 38.9, 31.7, 28.2. MALDI-TOF MS: Calcd
for C89H87Cl3N4NaO27: m/z 1771.5. Found: m/z
1771.8 [M+Na]+.
3.2.8. Data for 24. Yield: 963 mg (77%); 1H NMR
(CDCl3): d 7.37–7.17 (m, 5H, Ar-H), 6.27 (br t, 1H,
NH), 5.11 (s, 2H, CH2Ph), 4.74 (s, 2H, CH2CCl3),
3.85 (s, 2H, NCH2CO), 3.63 (t, 2H, J 6.7 Hz, NCH2
of b-Ala), 3.25 (m, 2H, NCH2 of 6-aminohexanoic acid),
2.76 (t, 2H, CH2CO of b-Ala), 2.36 (t, 2H, J 6.1 Hz,
CH2CO of 6-aminohexanoic acid), 1.69–1.34 (m, 6H,
CH2), 1.45 (s, 9H, t-Bu); 13C NMR (CDCl3): d 173.3,
169.2, 136.0, 128.5, 128.2, 94.7, 81.2, 74.1, 66.1, 52.6,
45.0, 39.1, 34.0, 33.2, 29.2, 28.3, 26.3, 24.4. MALDI-
TOF MS: Calcd for C25H35Cl3N2NaO7: m/z 603.2.
Found: m/z 603.7 [M+Na]+.
23
3.2.3. Data for 14. Yield: 45 mg (41%); ½aꢀD +55.4 (c
1
0.4, CHCl3); H NMR (CDCl3): d 8.07–7.05 (m, 80H,
Ar-H), 6.00 (br d, 4H, H-4), 5.76 (br dd, 4H, H-2),
5.66 (br dd, 4H, H-3), 4.91 (br d, 4H, H-1), 4.73–4.65
(m, 6H, CH2CCl3, H-6a), 4.43–4.38 (m, 8H, H-5,
H-6b), 3.95–3.41 (m, 32H, OCH2, NCH2), 2.69–2.02
(m, 8H, CCH2), 1.46 1.38, 1.36 1.34, 1.33, 1.31 (6s, 9H,
t-Bu). MALDI-TOF MS: Calcd for C171H163Cl3N8NaO51:
m/z 3272.9. Found: m/z 3272.1 [M+Na]+.
3.2.9. Data for 26. Yield: 547 mg (64%); 1H NMR
(CDCl3): d 7.35–7.27 (m, 5H, Ar-H), 6.95 (br t, 1H,
NH), 5.11 (s, 2H, CH2Ph), 4.74 (s, 2H, CH2CCl3),
4.05 (s, 2H, NCH2CO), 3.79 (br t, 2H, NCH2), 3.25–
3.21 (m, 4H, NCH2), 2.47–2.36 (m, 6H, CH2CO),
1.78–1.33 (m, 12H, CH2), 1.43 (s, 9H, t-Bu); 13C
NMR (CDCl3): d 173.4, 171.8, 136.0, 128.5, 128.2,
128.1, 95.0, 80.8, 73.8, 66.1, 62.2, 52.6, 41.1, 39.4, 39.2,
34.1, 33.7, 29.1, 29.0, 28.3, 26.3, 26.0, 24.4, 24.3. MAL-
DI-TOF MS: Calcd for C31H46Cl3N3NaO8: m/z 716.2.
Found: m/z 716.6 [M+Na]+.
23
3.2.4. Data for 16. Yield: 53 mg (78%); ½aꢀD +75.4 (c
1
0.9, CHCl3); H NMR (CDCl3): d 8.08–7.21 (m, 60H,
Ar-H), 6.00 (br d, 3H, H-4), 5.76 (br dd, 3H, H-2), 5.64
(br dd, 3H, H-3), 4.89 (br d, 3H, H-1), 4.67 (m, 3H, H-
6a), 4.41–4.35 (m, 6H, H-5, H-6b), 3.97–3.43 (m, 20H,
OCH2, NCH2), 2.48–2.09 (m, 4H, CCH2), 1.31 (s, 9H,
t-Bu). MALDI-TOF MS: Calcd for C123H117N5NaO36:
m/z 2262.7. Found: m/z 2262.9 [M+Na]+.
23
3.2.10. Data for 28. Yield: 239 mg (83%); ½aꢀD +59.6 (c
1
2.3, CHCl3); H NMR (CDCl3): d 8.09–7.21 (m, 60H,
Ar-H), 5.99 (br d, 3H, H-4), 5.75 (br dd, 3H, H-2),
5.65 (br dd, 3H, H-3), 4.89 (br d, 3H, H-1), 4.73 (s, 2H,
CH2CCl3), 4.66 (m, 3H, H-6a), 4.43–4.36 (m, 6H, H-5,
H-6b), 3.95–3.18 (m, 28H, OCH2, NCH2), 2.45–2.03
23
3.2.5. Data for 18. Yield: 31 mg (79%); ½aꢀD +75.3 (c
1
0.8, CHCl3); H NMR (CDCl3): d 8.06–7.20 (m, 100H,

842
K. Sato et al. / Carbohydrate Research 341 (2006) 836–845
(m, 10H, CCH2), 1.70–1.36 (m, 12H, CH2), 1.40 (s, 9H, t-
Bu). MALDI-TOF MS: Calcd for C142H147Cl3N8NaO41:
m/z 2747.9. Found: m/z 2748.1 [M+Na]+.
unit), 3.55 and 3.46 (m, 2H, NCH2 of sugar unit), 3.10
(br t, 2H, NHCH2CO), 2.81 (m, 2H, NCH2 of b-Ala),
2.60 (br t, 2H, COCH2 of b-Ala); 13C NMR (CDCl3):
d 170.5, 170.3, 166.0, 165.5, 165.2, 133.6, 133.4,
133.32, 133.28, 130.0, 129.7, 129.3, 129.2, 129.0, 128.6,
128.5, 128.3, 101.4, 94.7, 74.0, 71.54, 71.46, 69.8, 68.7,
68.1, 62.0, 51.4, 44.4, 38.8, 33.5. MALDI-TOF MS:
Calcd for C46H48N2NaO15: m/z 921.2. Found: m/z
921.4 [M+Na]+.
23
3.2.11. Data for 30. Yield: 83 mg (89%); ½aꢀD +67.0 (c
1
1.7, CHCl3); H NMR (CDCl3): d 8.08–7.20 (m, 120H,
Ar-H), 6.00 (br d, 6H, H-4), 5.75 (br dd, 6H, H-2),
5.66 (br dd, 6H, H-3), 4.90 (br d, 6H, H-1), 4.67 (m,
6H, H-6a), 4.41–4.37 (m, 12H, H-5, H-6b), 3.96–3.20
(m, 48H, OCH2, NCH2), 2.58–2.04 (m, 14H, CH2CO),
1.58–1.34 (m, 12H, CH2), 1.37 (s, 9H, t-Bu). MALDI-
TOF MS: Calcd for C258H253N13NaO74: m/z 4739.6.
Found: m/z 4740.4 [M+Na]+.
3.3.1. Deprotection of compounds 12, 17, 19, 31, 33 and
35. These compounds were deprotected using the pro-
cedure outlined in Section 3.3, above.
23
23
3.2.12. Data for 32. Yield: 61 mg (66%); ½aꢀD +63.3 (c
3.3.2. Data for 12. Yield: 56 mg (quant); ½aꢀD +65.2 (c
1
1
1.2, CHCl3); H NMR (CDCl3): d 8.11–7.18 (m, 180H,
1.4, CHCl3); H NMR (CDCl3): d 8.10–7.14 (m, 40H,
Ar-H), 5.99 (br d, 9H, H-4), 5.75 (br dd, 9H, H-2),
5.66 (br dd, 9H, H-3), 4.91 (br d, 9H, H-1), 4.66 (m,
9H, H-6a), 4.40–4.37 (m, 18H, H-5, H-6b), 3.96–3.15
(m, 76H, OCH2, NCH2), 2.53–2.06 (m, 24H, CH2CO),
1.56–1.35 (m, 33H, CH2 and t-Bu).
Ar-H), 6.00 (br d, 2H, H-4), 5.75 (br dd, 2H, H-2),
5.65 (br dd, 2H, H-3), 4.88 (br d, 2H, H-1), 4.72–4.66
(m, 4H, CH2CCl3, H-6a), 4.47–4.33 (m, 4H, H-5, H-
6b), 3.99–3.09 (m, 16H, OCH2 of sugar unit, NCH2 of
sugar unit, NCH2CO, NCH2 of b-Ala), 2.86–2.63 (m,
4H, COCH2 of b-Ala); 13C NMR (CDCl3): d 170.7,
169.6, 168.3, 166.1, 165.5, 165.4, 133.6, 133.3, 130.0,
129.7, 129.2, 129.0, 128.6, 128.5, 128.3, 101.7, 101.5,
74.1, 71.6, 71.4, 70.0, 69.9, 69.5, 68.8, 68.6, 68.3, 68.2,
68.1, 62.1, 61.9, 52.1, 49.7, 44.4, 39.4, 39.1, 32.6. MAL-
DI-TOF MS: Calcd for C84H79Cl3N4NaO25: m/z 1671.4.
Found: m/z 1671.9 [M+Na]+.
23
3.2.13. Data for 34. Yield: 24 mg (54%); ½aꢀD +51.9 (c
1
1.2, CHCl3); H NMR (CDCl3): d 8.05–7.21 (240H, m,
Ar-H), 5.99 (12H, br d, H-4), 5.75 (12H, br dd, H-2),
5.65 (12H, br dd, H-3), 4.90 (12H, br d, H-1), 4.65
(12H, m, H-6a), 4.41–4.25 (24H, m, H-5, H-6b), 3.96–
3.13 (104H, m, OCH2, NCH2), 2.57–2.00 (34H, m,
CH2CO), 1.53–1.25 (45H, m, CH2 and t-Bu). MALDI-
TOF MS: Calcd for C528H525N29NaO150: m/z 9693.4.
Found: m/z 9693.6 [M+Na]+.
23
3.3.3. Data for 17. Yield: 94 mg (99%); ½aꢀD +85.9 (c
1
1.1, CHCl3); H NMR (CDCl3): d 8.08–7.23 (m, 60H,
Ar-H), 6.01 (br d, 3H, H-4), 5.78 (br dd, 3H, H-2),
5.66 (br dd, 3H, H-3), 4.89 (br d, 3H, H-1), 4.68 (m,
3H, H-6a), 4.43–4.37 (m, 6H, H-5, H-6b), 3.67–3.08
(m, 20H, OCH2, NCH2), 2.74–2.16 (m, 4H, CCH2);
MALDI-TOF MS: Calcd for C118H109N5NaO34: m/z
2162.7. Found: m/z 2163.2 [M+Na]+.
23
3.2.14. Data for 36. Yield: 6 mg (51%); ½aꢀD +34.5 (c
1
0.6, CHCl3); H NMR (CDCl3): d 8.04–7.20 (m, 246H,
Ar-H), 5.99 (br d, 12H, H-4), 5.74 (br dd, 12H, H-2),
5.65 (br dd, 12H, H-3), 4.90 (br d, 12H, H-1), 4.65 (m,
12H, H-6a), 4.40–4.38 (m, 24H, H-5, H-6b), 3.95–3.12
(m, 112H, OCH2, NCH2, NCH3), 2.36–2.00 (m, 34H,
CH2CO), 1.60–1.25 (m, 36H, CH2). MALDI-TOF
23
3.3.4. Data for 19. Yield: 26 mg (86%); ½aꢀD +78.1
MS: Calcd for
C
537H531N31NaO151S: m/z 9883.5.
(c 0.6, CHCl3); 1H NMR (CDCl3): d 8.06–7.22 (m,
100H, Ar-H), 6.00 (br d, 5H, H-4), 5.75 (br dd, 5H,
H-2), 5.66 (br dd, 5H, H-3), 4.90 (br d, 5H, H-1), 4.67
(m, 5H, H-6a), 4.39 (m, 10H, H-5, H-6b), 3.93–
2.06 (m, 44H, CH2). MALDI-TOF MS: Calcd for
Found: m/z 9884.4 [M+Na]+.
3.3. Typical procedure for removal of the Boc group
To a solution of compound 8 (116 mg, 0.12 mmol) in
CH2Cl2 (1 mL) was added 50% aq TFA (1 mL). The
reaction mixture was stirred for 1 h at room tempera-
ture. After completion of the reaction (TLC monitor-
ing), the mixture was concentrated and purified by
silica gel column chromatography (1:1 benzene–acetone
C
200H185N9NaO58: m/z 3663.2. Found: m/z 3663.9
[M+Na]+.
23
3.3.5. Data for 31. Yield: 59 mg (73%); ½aꢀD +70.3 (c
1
1.5, CHCl3); H NMR (CDCl3): d 8.06–7.20 (m, 120H,
Ar-H), 6.00 (br d, 6H, H-4), 5.75 (br dd, 6H, H-2),
5.66 (br dd, 6H, H-3), 4.94 (br d, 6H, H-1), 4.67 (m,
6H, H-6a), 4.42–4.39 (m, 12H, H-5, H-6b), 3.96–3.18
(m, 48H, OCH2, NCH2), 2.80–2.03 (m, 14H, CH2CO),
1.54–1.26 (m, 12H, CH2). MALDI-TOF MS: Calcd
for C253H245N13NaO72: m/z 4639.6. Found: m/z 4640.1
[M+Na]+.
23
as eluant) to give 9 (109 mg, quant): ½aꢀD +68.1 (c 1.1,
1
CHCl3); H NMR (CDCl3): d 8.09–7.23 (m, 20H, Ar-
H), 6.00 (br d, 1H, H-4), 5.78 (br dd, 1H, H-2), 5.62
(br dd, 1H, H-3), 4.87 (br d, 1H, H-1), 4.75 (s, 2H,
CH2CCl3), 4.68 (m, 1H, H-6a), 4.43 (m, 1H, H-6b),
4.35 (m, 1H, H-5), 4.00 and 3.77 (m, 2H, OCH2 of sugar

K. Sato et al. / Carbohydrate Research 341 (2006) 836–845
843
23
3.3.6. Data for 33. Yield: 33 mg (55%); ½aꢀD +64.6 (c
NCH2CO, NCH2 of b-Ala), 2.59–2.35 (m, 4H, COCH2
of b-Ala), 1.44, 1.38 and 1.32 (3s, 9H, t-Bu); 13C
NMR (CDCl3): d 173.7, 172.7, 168.7, 166.0, 165.5,
133.6, 133.4, 133.3, 129.9, 129.7, 129.2, 129.1, 129.0,
128.6, 128.5, 128.2, 101.4, 80.9, 71.6, 71.3, 69.8, 69.7,
68.1, 62.0, 52.8, 39.1, 32.4, 31.7, 29.6, 28.2. MALDI-
TOF MS: Calcd for C87H86N4NaO27: m/z 1641.6.
Found: m/z 1642.3 [M+Na]+.
1
0.8, CHCl3); H NMR (CDCl3): d 8.06–7.20 (m, 180H,
Ar-H), 5.99 (br d, 9H, H-4), 5.75 (br dd, 9H, H-2),
5.66 (br dd, 9H, H-3), 4.90 (br d, 9H, H-1), 4.66 (m,
9H, H-6a), 4.41–4.37 (m, 18H, H-5, H-6b), 3.96–3.17
(m, 76H, OCH2, NCH2), 2.57–2.01 (m, 24H, CH2CO),
1.54–1.25 (m, 24H, CH2). MALDI-TOF MS: Calcd
for
C
388H381N21NaO110
:
m/z 7116.5. Found: m/z
7117.8 [M+Na]+.
23
3.4.3. Data for 15. Yield: 35 mg (81%); ½aꢀD +65.1 (c
23
1
3.3.7. Data for 35. Yield: 12 mg (49%); ½aꢀD +42.7 (c
1.1, CHCl3); H NMR (CDCl3): d 8.07–7.22 (m, 80H,
1
0.8, CHCl3); H NMR (CDCl3): d 8.05–7.21 (240H, m,
Ar-H), 6.00 (br d, 4H, H-4), 5.76 (br dd, 4H, H-2),
5.65 (br dd, 4H, H-3), 4.90 (br d, 4H, H-1), 4.66 (m,
4H, H-6a), 4.42–4.37 (m, 8H, H-5, H-6b), 3.96–3.41
(m, 32H, OCH2, NCH2), 2.45–2.05 (m, 8H, CCH2),
1.36, 1.34, 1.33, 1.30 (4s, 9H, t-Bu). MALDI-TOF
MS: Calcd for C169H162N8NaO51: m/z 3142.0. Found:
m/z 3142.9 [M+Na]+.
Ar-H), 5.99 (12H, br d, H-4), 5.75 (12H, br dd, H-2),
5.66 (12H, br dd, H-3), 4.90 (12H, br d, H-1), 4.65
(12H, m, H-6a), 4.40–4.38 (24H, m, H-5, H-6b), 3.95–
3.12 (104H, m, OCH2, NCH2), 2.57–2.00 (34H, m,
CH2CO), 1.75–1.25 (36H, m, CH2). MALDI-TOF
MS: Calcd for C523H518N29O148: m/z 9571.5. Found:
m/z 9572.3 [M+H]+.
3.4.4. Data for 25. Yield: 568 mg (80%); 1H NMR
(CDCl3): d 7.34–7.25 (m, 5H, Ar-H), 7.01 (br t, 1H,
NH), 5.11 (s, 2H, CH2Ph), 4.01 (s, 2H, NCH2CO),
3.62 (br t, 2H, NCH2 of b-Ala), 3.29 (m, 2H, NCH2
of 6-aminohexanoic acid), 2.61 (br t, 2H, CH2CO of
b-Ala), 2.35 (br t, 2H, CH2CO of 6-aminohexanoic
acid), 1.64–1.34 (m, 6H, CH2 · 3), 1.44 (s, 9H, t-Bu);
13C NMR (CDCl3): d 173.9, 171.8, 135.9, 128.6, 128.2,
82.2, 66.4, 52.5, 45.8, 39.7, 34.0, 28.6, 28.2, 26.1, 24.3.
MALDI-TOF MS: Calcd for C23H34N2NaO7: m/z
473.2. Found: m/z 473.7 [M+Na]+.
3.4. Typical procedure for the removal of the 2,2,2-
trichloroethyl (Tce) group
To a solution of compound 8 (189 mg, 0.19 mmol) in
HOAc (2 mL) was added Zn powder (104 mg), and the
reaction mixture was stirred for 1 h at room temper-
ature. After completion of the reaction (TLC monitor-
ing), the mixture was filtered through Celite. The
filtrate was concentrated and purified by silica gel col-
umn chromatography (30:1:0.1 CHCl3–MeOH–HOAc)
23
to give 10 (147 mg, 90%): ½aꢀD +67.9 (c 1.4, CHCl3);
23
1H NMR (CDCl3): d 8.11–7.22 (m, 20H, Ar-H), 6.00
(d, 1H, H-4), 5.78 (dd, 1H, J2,3 9.8 Hz, H-2), 5.62 (dd,
1H, J3,4 3.7 Hz, H-3), 4.87 (d, 1H, J1,2 7.9 Hz, H-1),
4.67 (m, 1H, H-6a), 4.46 (m, 1H, H-6b), 4.38 (m, 1H,
H-5), 3.99–3.50 (m, 8H, OCH2 of sugar unit, NCH2 of
sugar unit, NCH2CO, NCH2 of b-Ala), 2.49 (br t, 2H,
COCH2 of b-Ala), 1.43 (s, 9H, t-Bu); 13C NMR
(CDCl3): d 166.1, 165.51, 165.46, 155.2, 133.7, 133.6,
133.4, 133.3, 130.8, 130.0, 129.7, 129.2, 129.0, 128.9,
128.64, 128.56, 128.5, 128.3, 101.7, 81.3, 71.6, 71.4,
70.0, 68.8, 68.1, 62.1, 51.4, 45.9, 39.6, 35.1, 28.9. MAL-
DI-TOF MS: Calcd for C46H48N2NaO15: m/z 891.3.
Found: m/z 891.7 [M+Na]+.
3.4.5. Data for 29. Yield: 140 mg (85%); ½aꢀD +62.1 (c
1
1.2, CHCl3); H NMR (CDCl3): d 8.09–7.21 (m, 60H,
Ar-H), 6.00 (br d, 3H, H-4), 5.75 (br dd, 3H, H-2), 5.66
(br dd, 3H, H-3), 4.90 (br d, 3H, H-1), 4.66 (m, 3H,
H-6a), 4.42–4.36 (m, 6H, H-5, H-6b), 3.94–3.20 (m,
28H, OCH2, NCH2), 2.40–2.05 (m, 10H, CH2CO),
1.60–1.34 (m, 12H, CH2), 1.41 (s, 9H, t-Bu); 13C NMR
(CDCl3): d 166.0, 165.5, 165.4, 133.6, 133.53, 133.47,
133.34, 133.3, 130.0, 129.7, 129.3, 129.22, 129.17, 129.0,
128.6, 128.5, 128.3, 101.7, 101.6, 101.4, 80.8, 71.6, 71.54,
71.46, 71.4, 69.9, 68.9, 68.6, 68.1, 61.9, 39.2, 29.0, 28.3,
26.3, 26.0, 24.2. MALDI-TOF MS: Calcd for C140H146
-
N8NaO41: m/z 2618.0. Found: m/z 2618.4 [M+Na]+.
3.4.1. Removal of the Tce group to give compounds 13, 15,
25 and 29. Removal of the Tce group was carried out
to give these compounds according to the procedure in
Section 3.4.
3.5. Typical procedure for the preparation of fully
deprotected peptidic glycoclusters
To a solution of 20 (15 mg, 6.17 l mol) in 1:1 1,4-diox-
ane–MeOH (2 mL) was added NaOMe (24 mg), and the
mixture was stirred for 4 h at room temperature and
then neutralized with Amberlite IR-120 (H+) resin.
The resin was filtered off and washed with MeOH and
water. The filtrate and washings were combined and
concentrated. Column chromatography (1:1 MeOH–
H2O) of the residue on Sephadex LH-20 gave 1
23
3.4.2. Data for 13. Yield: 53 mg (quant); ½aꢀD +61.6 (c
1
1.4, CHCl3); H NMR (CDCl3): d 8.09–7.14 (m, 40H,
Ar-H), 6.00 (br d, 2H, H-4), 5.77 (br dd, 2H, H-2),
5.65 (br dd, 2H, H-3), 4.90 (br d, 2H, H-1), 4.66 (m,
2H, H-6a), 4.42–4.35 (m, 4H, H-5, H-6b), 3.98–3.44
(m, 16H, OCH2 of sugar unit, NCH2 of sugar unit,

844
K. Sato et al. / Carbohydrate Research 341 (2006) 836–845
(8.5 mg, quant): 13C NMR (D2O): d 128.5, 128.4, 123.6,
102.5, 74.7, 72.3, 70.4, 68.2, 67.9, 67.8, 60.6, 48.4, 44.6,
39.01, 38.96, 39.9, 38.8. MALDI-TOF MS: Calcd for
C48H75N7NaO25S: m/z 1204.5. Found: m/z 1204.5
[M+Na]+.
(m, 4H, NCH2), 2.49–2.33 (m, 6H, CH2CO), 1.75–1.37
(m, 12H, CH2), 1.44 (s, 9H, t-Bu); 13C NMR (CDCl3):
d 176.2, 171.9, 81.0, 73.9, 68.4, 52.2, 39.4, 38.9, 33.7,
33.5, 28.9, 28.7, 28.2, 26.2, 25.8, 24.3, 24.1. MALDI-
TOF MS: Calcd for C24H40Cl3N3NaO8: m/z 626.2.
Found: m/z 626.6 [M+Na]+.
3.5.1. Deacylated compounds 2, 3, 4 and 5. These com-
pounds were obtained using the procedure in Section
3.5.
Acknowledgements
3.5.2. Data for 2. Yield: 4 mg (96%); MALDI-TOF
MS: Calcd for C74H119N11NaO41S: m/z 1872.7. Found:
m/z 1872.7 [M+Na]+.
This work was supported by a Grant-in-Aid for Scien-
tific Research (No. 14771250) from the Ministry of Edu-
cation, Culture, Sports, Science and Technology of
Japan and also the ‘High-Tech Research Center’ project
of the same ministry. We gratefully acknowledge finan-
cial support from Sasagawa Scientific Research.
3.5.3. Data for 3. Yield: 4 mg (95%); 13C NMR (D2O):
d 170.6, 102.5, 74.7, 72.3, 70.4, 68.2, 67.9, 67.8, 60.6,
48.4, 39.1, 39.0, 38.9. MALDI-TOF MS: Calcd for
C72H124N10NaO46: m/z 1887.8. Found: m/z 1888.2
[M+Na]+.
References
3.5.4. Data for 4. Yield: 7 mg (quant); MALDI-TOF
MS: Calcd for C111H183N15NaO69: m/z 2853.1. Found:
m/z 2853.5 [M+Na]+.
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3.5.5. Data for 5. Yield: 1.1 mg (36%); 13C NMR (1:1
CD3OD-D2O): d 171.7, 171.6, 171.4, 171.3, 104.4,
76.3, 74.1, 72.9, 72.1, 69.8, 62.1, 61.7, 49.1, 40.5, 40.4.
MALDI-TOF MS: Calcd for C202H343N31NaO103S:
m/z 4906.3. Found: m/z 4907.2 [M+Na]+.
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23
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23
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