Oligomers of glycamino acid.

Article abstract of DOI:10.1016/S0968-0896(02)00020-2

Glycamino acids, a family of sugar amino acids, are derivatives of C-glycosides that possesses a carboxyl group at the C-1 position and an amino group replacing one of the hydroxyl groups at either the C-2, 3, 4, or 6 position. We have prepared a series of glucose-type glycamino acids as monomeric building blocks: these are derivatives of 2-NH(2)-Glc-beta-CO(2)H 1, 3-NH(2)-Glc-beta-CO(2)H 2, 4-NH(2)-Glc-beta-CO(2)H 3, and 6-NH(2)-Glc-beta-CO(2)H 4 and constructed four types of homo-oligomers, beta(1-->2)-linked I, beta(1-->3)-linked II, beta(1-->4)-linked III, and beta(1-->6)-linked IV, employing the well-established N-Boc and BOP strategy. CD and NMR spectral studies of these oligomers suggested that only the beta(1-->2)-linked homo-oligomer possessed a helical structure that seems to be predetermined by the linkage position. Homo-oligomers with beta(1-->2)-linkages I and beta(1-->6)-linkages IV were also subjected to O-sulfation, and these O-sulfated oligomers were found to be able, in a linkage-specific manner, to effectively inhibit L-selectin-mediated cell adhesion, HIV infection, and heparanase activity without the anticoagulant activity associated with naturally occurring sulfated polysaccharides such as heparin.

Full text of DOI:10.1016/S0968-0896(02)00020-2

Bioorganic & Medicinal Chemistry 10 (2002) 1999–2013
Oligomers of Glycamino Acid
Yoshitomo Suhara,a,y Yoshiki Yamaguchi,c Brian Collins,a
Ronald L. Schnaar,a Masaki Yanagishita,b James E.K. Hildreth,a
Ichio Shimadac and Yoshitaka Ichikawaa,*
aDepartment of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine,
725 North Wolfe Street, Baltimore, MD 21205, USA
bDepartment of Biochemistry, School of Dentistry, Tokyo Medical and Dental University, 1-5-45 Yushima,
Bunkyo-ku, Tokyo 113-8549, Japan
cGraduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku,
Tokyo 113-0033, Japan
Received 21 May 2001; accepted 5 December 2001
Abstract—Glycamino acids, a family of sugar amino acids, are derivatives of C-glycosides that possesses a carboxyl group at the
C-1 position and an amino group replacing one of the hydroxyl groups at either the C-2, 3, 4, or 6 position. We have prepared a
series of glucose-type glycamino acids as monomeric building blocks: these are derivatives of 2-NH2-Glc-b-CO2H 1, 3-NH2-Glc-b-
CO2H 2, 4-NH2-Glc-b-CO2H 3, and 6-NH2-Glc-b-CO2H 4 and constructed four types of homo-oligomers, b(1!2)-linked I,
b(1!3)-linked II, b(1!4)-linked III, and b(1!6)-linked IV, employing the well-established N-Boc and BOP strategy. CD and
NMR spectral studies of these oligomers suggested that only the b(1!2)-linked homo-oligomer possessed a helical structure that
seems to be predetermined by the linkage position. Homo-oligomers with b(1!2)-linkages I and b(1!6)-linkages IV were also
subjected to O-sulfation, and these O-sulfated oligomers were found to be able, in a linkage-specific manner, to effectively inhibit
L-selectin-mediated cell adhesion, HIV infection, and heparanase activity without the anticoagulant activity associated with natu-
rally occurring sulfated polysaccharides such as heparin. # 2002 Elsevier Science Ltd. All rights reserved.
cyclo-hexane and -pentane, and 3-carboxy piperidine;4,5,7
the biological activities of some of these oligomers have
already been evaluated.4j,5g,7
Introduction
Considerable interest has been devoted to designing and
constructing a newclass of peptide molecules with the
expectation that these molecules may provide stable
alternatives to the biologically important natural
a-peptides,1 and a variety of oligomers have been pre-
pared and studied with respect to their con-
formations.2À8 Among them, b-peptides are composed
of b-amino acids, in which an amino and carboxyl
groups are separated by an additional methylene group,
and their oligomers have been found to form stable
helical conformations as comparable to those in natu-
rally occurring peptides.2 b-Amino acid derivatives that
have been designed and prepared are counterparts of the
natural a-amino acids, derivatives of 2-amino-carboxyl
Carbohydrate-derived amino acids, or sugar amino
acids (also called glycamino acids)9,10 have also been
developed as non-natural amino acid analogues with
additional functionality on the molecules, that is a
hydroxyl group. They possess both a carboxylate group
at the anomeric position and an amino group replacing
one of the OH groups of the sugar. Several oligomers
composed of glycamino acid analogues have been pre-
pared and characterized their conformation, and some
have been found to assume a rigid conformation by
NMR and CD analysis.11À20
As a part of our ongoing program for developing a
newclass of molecules that possess the structural and
functional features of both carbohydrates and pep-
tides, we have reported the first homo-oligomers of
glucose-based glycamino acids in which the glucose-
based glycamino acid residues are connected via
*Corresponding author. Tel.: +1-410-614-2892; fax: +1-410-955-
3023; e-mail: ichikawa@jhmi.edu
yCurrent address: Faculty of Pharmaceutical Sciences, Teikyo Uni-
versity, Sagamiko, Kanagawa 199-0195, Japan.
0968-0896/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(02)00020-2

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Y. Suhara et al. / Bioorg. Med. Chem. 10 (2002) 1999–2013
b(1!2)-* (I)21 and b(1!6)-linkages (IV).22 We found
that upon O-sulfation the amido-linked glycamino acid
oligomers were able to inhibit the replication of HIV-1
and sialyl Lewis x-dependent cell adhesion. Because the
b(1!2)-type glycamino acid can be recognized as a
b-amino acid,2a we expected that the oligomer would
showa rigid secondary conformation. We also won-
dered whether other type of linkages, such as b(1!3)-
(II), b(1!4)- (III), and b(1!6)-linked (IV) oligomers
might form a particular rigid conformation, and we
therefore synthesized all the four types of oligomers of
glucose-type glycamino acids (I–IV). We describe
herein the detailed synthesis of monomers of the
b(1!3)- and b(1!4)-glycamino acids and their oligo-
mers and present the result of our biological evaluation
and our conformational studies using CD and NMR
spectroscopy.
Results
Synthesis of ꢀ(1!3)-linked homooligomer 14 (Scheme 1)
The b(1!3)-monomer 2 was synthesized from a known
3-azido glucose derivative 5.23 Bromination24 of 5 with
TiBr4 gave the glycosyl bromide, which was purified by
silica gel chromatography (hexane–EtOAc 3:1) and
y
immediately used for glycosylation with Hg(CN)2 to
afford the b-glycosyl cyanide 6 in 57% yield. Sequential
treatment of 6 with: (i) NaOMe in MeOH and (ii)
NaOH in water gave the C-1 carboxylate derivative,25
which was purified with Dowex 1 [OHÀ] resin and
eluted with 2 N AcOH. Esterification of the free car-
boxylate was accomplished by dissolving the product in
refluxing MeOH,26 to afford the methyl ester 7 in 53%
overall yield. The azido group of 7 was reduced with
H2S and the resulting amino group was protected with a
Boc group (BocON27 in MeOH) to give the N-Boc
derivative 8. The free OH groups of 8 were benzylated
with NaH and BnBr, and the reaction products were
treated with NaOMe (in order to convert the con-
comitant benzyl ester to the methyl ester) to give 9 in
49% overall yield. The methyl ester group of 9 was
hydrolyzed with LiOH28 to afford the b(1!3)-monomer
2 in 97% yield.
The oligomers were assembled in a manner similar to that
used for the synthesis of the b(1!2)-linked oligomers:21
The C-terminus of 2 was blocked with l-phenylalanine
Scheme 1. (a) (i) TiBr4, CH2Cl2/EtOAc, 12 h, 0 ꢀC to rt, 62%; (ii) Hg
(CN)2, MeNO2, 1 h, 80 ꢀC, 57%; (b) (i) 25% NaOMe, MeOH, 1 h, rt;
(ii) NaOH, H2O, 12 h, reflux; (iii) MeOH, 12 h, reflux, 53% overall; (c)
(i) H2S gas, pyridine/H2O (1:1), 2 h, 0 ꢀC then 12 h, rt; (ii) 5 equiv of
Boc2O, 5 equiv of Et3N, MeOH, 12 h, rt, 77% overall yield; (d) (i) 6
equiv of BnBr, 4.5 equiv of 60% NaH, DMF, 16 h, rt; (ii) 25%
.
NaOMe, MeOH, 30 min, rt, 49% overall; (e) 2 equiv of LiOH H2O,
MeOH/THF/H2O (3:3:1), 20 min, rt, 90%; (f) 1.2 equiv of l-phenyl-
alanine methyl ester, 1.5 equiv of BOP, 3 equiv of DIEA, 16 h, 0 ꢀC to
rt, 55%; (g) (i) 2 N HCl in EtOAc, 3 h, 0 ꢀC to rt; (ii) 1.2 equiv of 2, 1.5
equiv of BOP, 3 equiv of BOP, 3 equiv of DIEA, 16 h, 0 ꢀC to rt; (h)
20% Pd(OH)2, H2, MeOH, 16 h, rt, 85%.
*In this text, we have used a nomenclature convention in which gly-
camino acids and their synthetic precursors are named as derivatives of
the parent sugar [i.e., (2,4,6-tri-O-benzyl-3-tert-butoxycarbonylamido-3-
deoxy-b-d-glucopyranosyl)-C-carboxylic acid for compound 2), and
accordingly the numbering of the skeletal carbons is based on that of
the parent sugar. This convention is recommended for our present
purposes because higher-carbon sugar names are not easily applied to
describe the relationship between the structure of the compound and
its IUPAC names provided in the Experimental. Compound 2 would
be named, in another proper convention, as 2,6-anhydro-3,5,7-tri-O-
benzyl-4-tert-butoxycarbonylamido-4-deoxy-d-glycero-d-gulo-hep-
turonic acid.
yA commercially available Hg(CN)2 was pretreated for the glycosyl
cyanation by: (1) co-evaporation from a suspension of Hg(CN)2 in
toluene three times and (2) drying under vacuum for 2 h. Without this
pretreatment, it was difficult to obtain the product.

Y. Suhara et al. / Bioorg. Med. Chem. 10 (2002) 1999–2013
2001
methyl ester in the presence of benzotriazol-1-yloxy-
tris(dimethylamino)phosphonium hexafluorophosphate
(BOP reagent)29 to give 10 in 55% yield. Removal of the
Boc group and subsequent coupling with the mono-
meric 2 gave, sequentially, the protected forms of the
dimer 11, the trimer 12, and the tetramer 13 in good
yields. O-Benzyl groups of 13 were removed by hydro-
genolysis to afford the OH-free tetramer 14 in 85% yield
after purification by a Sephadex column chromato-
graphy, with a water elution.
coupling O-benzylated monomer 21, we decided to per-
form the assembly of the oligomer using the monomeric
derivative 3 with the non-protected OH groups, which
was obtained from 20 by hydrogenolysis.
The first coupling with l-phenylalanine methyl ester
was carried out with the benzylated monomer 21, pre-
pared by alkaline hydrolysis of the methyl ester of 20, to
give 22, which was then hydrogenolyzed to give 23.
Removal of the Boc group and coupling with the OH
free monomer 3 gave 24, after purification, as the
O-acetyl derivative. This sequence was repeated to give
the O-acetylated derivatives of a trimer 25 and a tetra-
mer 26 in good yields. O-Deacetylation of 26 with
NaOMe in MeOH afforded the tetramer 27.
Synthesis of ꢀ(1!4)-linked homooligomer 2730 (Scheme 2)
The b(1!4)-monomer 3 was prepared from a known
(b-d-galactopyranosyl)-C-carboxylic acid methyl ester
(methyl 2,6-anhydro-d-glycero-l-manno-hepturonate)
15.31 Benzylidenation of 15 with benzaldehyde and for-
mic acid32 gave a benzylidene derivative 1633 (65%
yield) which was then benzylated to give 17. Reductive
opening of the benzylidene group34 with HCl–
Na(CN)BH3 gave the 2,3,6-tri-O-benzyl derivative 18 in
80% yield. Sequential treatment of 18 with: (i) tri-
fluoromethanesulfonic anhydride (Tf2O) and (ii) NaN3
afforded the 4-azido derivative with a gluco-configura-
tion 19. The azido group of 19 was reduced with H2S
and protected with a Boc group to give 20 in 97%
overall yield. Since we have experienced difficulty in
Conformational analysis of the glycamino acid homo-
oligomers
Circular dichroism (CD) techniques are extensively used
to analyze the secondary structure of a-amino acid
polypeptides.35 In the case of non-a-peptides, efforts
have been made to correlate CD spectra with types of
secondary structure36 using other structural data
obtained by NMR or X-ray crystallography. We first
examined CD spectra of all the oligomers with different
linkages to see whether any of them had a rigid second-
ary structure and then used NMR measurement to fur-
ther study their conformation.
Circular dichroism (CD). Figure 1a shows CD spectra
for a series of b(1!2)-homo-oligomers. All of the
b(1!2)-oligomers except the dimer displayed minima
([y]=À8Â103 ꢁÀ3Â103) between 215 and 225 nm and
maxima ([y]=À2Â103 ꢁ5Â103) at 200 nm. For the
b(1!2)-hexamer, molar ellipticity is 4Â103 at 200 nm
and À7Â103 at 220 nm. The observed CD pattern of the
b(1!2)-oligomers was quite similar to that of the
14-helical b-peptides reported by Seebach et al.,5a,b,d
suggesting that the b(1!2)-oligomers do indeed adopt a
14-helical structure. However, the magnitudes of the
molar ellipticity of the b(1!2)-oligomers were rather
small when compared to the reported values; this find-
ing suggests that b(1!2)-oligomers may equilibrate
between 14-helix and unfolded conformation.
The b(1!3)-oligomers produced CD spectra with a
negative band at about 205 nm (Fig. 1b). The CD spec-
tra of the b(1!4)-oligomers had a positive band at
about 218 nm and a negative band at 195 nm (Fig. 1c).
The b(1!6)-oligomers had CD spectra with positive
bands at about 198 and 218 nm and a negative band at
190 nm (Fig. 1d). From these CD studies, we concluded
that among the glycamino acid oligomers, only the
b(1!2)-oligomers likely possess a 14-helical structure.
Scheme 2. (a) PhCHO, HCO2H, 1 h, rt, 65%; (b) (i) 2.2 equiv of 60%
NaH, 3 equiv of BnBr, DMF, 12 h, rt; (ii) 25% NaOMe, MeOH, 1 h,
rt, 93% overall; (c) 6 equiv of Na(CN)BH3, MS3A, HCl satd EtOAc,
THF, 20 min, rt, 80%; (d) (i) Tf2O, pyridine, CH2Cl2, 2 h, À5 ꢀC; (ii)
NaN3, DMF, 12 h, rt, 60% overall; (e) (i) H2S gas, pyridine/H2O (1:1),
2 h, 0 ꢀC then 12 h, rt; (ii) 10 equiv of Boc2O, 9 equiv of Et3N, MeOH,
12 h, rt, 97% overall; (f) (i) 20% Pd(OH)2, H2, THF/MeOH/H2O
(5:5:1), rt, 12 h; (ii) 2 equiv of LiOH, THF/MeOH/H2O (3:3:1), 20
min, rt, 90% overall; (g) 2 equiv of LiOH, THF/MeOH/H2O (3:3:1),
20 min, rt, 93%; (h) (i) 1.2 equiv of l-phenylalanine methyl ester, 2
equiv of BOP, 3 equiv of DIEA, DMF, 16 h, 0 ꢀC to rt, 65%; (ii) 20%
Pd(OH2), H2, THF/MeOH/H2O (5:5:1), rt, 12 h, 90%; (j) (i) 2 N HCl
in EtOAc, 3 h, 0 ꢀC to rt; (ii) 1.2 equiv of 3, 2 equiv of BOP, 3 equiv of
DIEA, DMF, 16 h, rt; (iii) Ac2O, pyridine, 12 h, rt, 73% overall; (k)
25% NaOMe, MeOH (pH 11), 2 h, rt.
Nuclear magnetic resonance (NMR). Further conforma-
tional studies by NMR were carried out for the b(1!2)-
oligomers. The b(1!2)-oligomers were dissolved in pyri-
dine-d5 or H2O/D2O, with the former giving a better dis-
persion of the NMR signals. NMR signal assignments
were established by DOF-COSY, TOCSY, ROESY,
1H–13C HSQC, and 1H–13C HSQC-TOCSY experiments.

2002
Y. Suhara et al. / Bioorg. Med. Chem. 10 (2002) 1999–2013
Figure 1. (a) CD spectra of b(1!2)-dimer, trimer, tetramer, and hexamer; (b) CD spectra of b(1!3)-dimer, trimer, and tetramer; (c) CD spectra of
b(1!4)-dimer and trimer; (d) CD spectra of b (1!6)-trimer and tetramer.
3
We first observed J values for the coupling between
NH and H2 protons in the b(1!2)-oligomers in H2O
because 3J coupling constants between NH and H2
protons provide information about the dihedral angles
(1) (Fig. 2). NH protons of the b(1!2)-oligomers
exhibited large J values ranging from 8.5 to 9.6 Hz,
which corresponds to a nearly anti-periplanar arrange-
3
ment of N–H and Cb–H (H2). Large J(NH,H2) values
were also reported for the 14-helical b-peptide.5b,d
We next examined long-rang ROE cross-peaks to define
a 14-helical structure. Figure 2 shows the ROESY spec-
trum observed for the b(1!2)-trimer. In this spectrum,
the inter-residue [NH(i)–H1(i+1)] and intra-residue
[NH(i)-H3(i)/H1(i)] cross-peaks were observed. The
intensities of the inter- and intra-residue ROE cross-
peaks were almost same in both solvents (H2O and
pyridine-d5), suggesting that the b(1!2)-trimer assumes
a 14-helical conformation (see Fig. 2 and Table 1).
However, no long-range ROE cross-peaks, which are
used to identify the 14-helix in b-peptides,4f,5a,b,d could
be observed, probably because an equilibrium between
the 14-helix and unfolded conformations. For the
b(1!2)-tetramer, similar ROE data were obtained
(Table 1). Therefore, these ROE data revealed that the
b(1!2)-trimer and tetramer in solution tends to assume
Figure 2. A part of ROESY spectrum of b(1!2) trimer in H2O at 303 K.
a 14-helical conformation.37

Y. Suhara et al. / Bioorg. Med. Chem. 10 (2002) 1999–2013
2003
Table 1. ROE cross-peak intensities of intra-residue ROE (NH(i)
H1(i)H3(i) and inter-residue ROE (NH(i) $ H2(i+1$) for b(1!2)
trimer and tetramera
the glycamino acid-based oligomers are derivatives of
C-glycosides and are not cleaved by glycosidases, we
examined the effect of O-sulfation and evaluated the bio-
logical activities of glycamino acid-based oligomers in
three biological assays. The b(1!2)- and b(1!6)-linked
homo-oligomers were O-sulfated according to a pub-
lished procedure,39 and the degree of sulfation was esti-
mated from the elemental analytical data.
Trimer
Tetramer
(in pyridine)
1NH $ 1H1
1NH $ 1H3
1NH $ 2H1
20
42
37
12
30
30
Anti-HIV activity. It has been suggested that the anti-
HIV activity of the O-sulfated oligosaccharides is highly
dependent upon its degree of sulfation.39 Sulfated poly-
saccharides such as heparin and dextran sulfate are
known to inhibit HIV infection of T-cells,40 but these
compounds also showundesirable properties such as
short half-life in circulation, poor bioavailability, and
toxicity.41 Therefore, a variety of O-sulfated oligo-
saccharides or anionic compounds have been prepared
as a way of overcoming these drawbacks.39À41
2NH $ 2H1
2NH $ 2H3
2NH $ 3H1
3NH $ 3H1
3NH $ 3H3
3NH $ 4H1
24
18
16
16
42
32
12
—
10
(in H2O)
b
1NH $ 1H1
1NH $ 1H3
1NH $ 2H1
18
24
16
—
—
—
2NH $ 2H1
2NH $ 2H3
2NH $ 3H1
3NH $ 3H1
3NH $ 3H3
3NH $ 4H1
31
38
26
—
—
—
Determination of the antiviral activity of the com-
pounds was based on the inhibition of the expression of
the HIV p24 protein in supernatant using a standard
core ELISA assay, as described previously.42 MT-2 cells
are exposed to virus (HTLVRF) in the presence or
absence of various concentrations of each of the syn-
thesized analogues for 5 days at 37 ꢀC. Viral production
was then determined by ELISA. We observed that
whereas the O-sulfated b(1!6)-linked tetramer was
highly active in inhibiting HIV-1 infectivity, with an
IC50 of 1 mM, the corresponding trimer showed no
inhibitory activity up to 500 mM. In addition, the
O-sulfated b(1!6)-linked tetramer was more potent
than the b(1!2)-linked tetramer. It should be mentioned
that because of their limited size, none of these com-
pounds showed any heparin-like anticoagulant activity.
—
—
—
aValues are in arbitary unit.
bAnalysis of b(1!2) tetramer in H2O was hampered due to signal
overlap in ROESY spectrum.
In order to gain insight into intramolecular hydrogen-
bonding patterns, we examined the temperature depen-
dence of amide proton chemical shifts. Data obtained for
the b(1!2)-tetramer are shown in Table 2. These data
suggest the existence of an intramolecular hydrogen bond
for the NH of residues 2 and 3. In contrast, NH tempera-
ture coefficients for the other b(1!3)-oligomers ranged
from 14.8 to 22.8, suggesting that the b(1!3)-oligomers
did not form intramolecular hydrogen bonds (data not
shown). Thus the b(1!3)-oligomers did not have well-
defined conformation since the long range ROE was not
found by NMR measurement, therefore, the conforma-
tion of b(1!3)-oligomers could be corresponding to the
CD data. On the basis of the NMR and CD data, we
concluded that b(1!2)-oligomers, except the dimer,
assume a 14-helical conformation in solution.
Inhibition of sialyl Lewis x selectin-mediated cell adhe-
sion (Fig. 3). Because the interaction between sialyl
Lewis x and selectins is now considered to be the first
step in the intercellular adhesion leading to inflamma-
tion,43 considerable effort has been devoted to develop-
ing an effective inhibitor of this process.
The inhibitory activity of these compounds against sia-
lyl Lewis x-mediated cell adhesion was determined using
an assay described previously:44 COS cells expressing
human E- or P-selectins were incubated with our syn-
thesized compounds, and then incubated on surfaces
bearing immobilized sialyl Lewis x-containing glycoli-
pid. COS cells that bound to sialyl Lewis x were lysed
and quantified spectrophotometrically. We observed
that the O-sulfated b(1!2)- and b(1!6)-linked oligo-
mers (up to tetramers) were weak inhibitors of E-selec-
tin-dependent sialyl Lewis x adhesion; however, the
O-sulfated b(1!2)-linked tetramer was an effective
inhibitor of P-selectin-dependent adhesion, with an IC50
of 100 mM (Fig. 3). This observation can be explained
by the fact that two binding sites have been suggested
for P- and L-selectins: one site for sialyl Lewis x and the
other for negatively charged carbohydrates.45 In fact,
sulfated oligosaccharides and tyrosine residues have
been demonstrated to be ligands for P-selectin.46
Table 2. Temperature coefficient of amide protons of b(12) tetramer
in pyridine-d5
a
NH (Phe)
1NH
2NH
12.0
12.8
7.1
3NH
4NH
6.3
14.3
aValues are in -ppb/K.
Biological evaluation of sulfated homo-oligomers as a
stable analogue of the O-sulfated oligosaccharides in sev-
eral assay systems. Sulfated oligosaccharides and poly-
saccharides are known to show a variety of biological
activities in important biological systems.38 However,
these compounds are highly susceptible to glycosidase
digestion and therefore have a very short half-lives. Since

2004
Y. Suhara et al. / Bioorg. Med. Chem. 10 (2002) 1999–2013
Our biological evaluation of oligomers of the glycamino
acids as amide-linked oligosaccharide analogues has
shown that the O-sulfated derivatives are equally or
more potent anti-HIV agents than are the currently
available anti-HIV agents that are sulfated derivatives
of naturally occurring oligosaccharides. Furthermore,
our compounds were found to be very potent inhibitors
of heparanase, which is a key enzyme in cell migration,
including tumor metastasis. In addition, we have shown
that our designed b(1!2)-amide-linked analogues with
O-sulfate groups are potent inhibitors of sialyl Lewis
x-P-selectin-mediated cell adhesion. These biological
activities were found to be dependent on the linkage
position of the analogues. The O-sulfated derivatives of
b(1!2)- and b(1!6)- linked oligomers that we have
tested did not showany anticoagulant activity, which
has been a major side effect of the O-sulfated natural-
type oligosaccharide derivatives. Although we have not
yet fully optimized the structure–activity relationships
of these glycamino acid derivatives, our preliminary
observations strengthen our hypothesis that these gly-
camino acid-based molecules have great potential for
producing newand biologically effective compounds.
These findings may also lead to the design of a newclass
of designed molecules: oligosaccharide and oligopeptide
analogues with significant biological activities.
Figure 3. Inhibition of sialyl Lewis x and P-selectin-mediated cell
adhesion with O-sulfated glycamino acid oligomers.
Inhibition of heparanase activity (Fig. 4). Since heparan
sulfate proteoglycans are ubiquitous macromolecules
and components of extracellular matrices and cell sur-
faces,47 migration of T-cells and tumor cells is asso-
ciated with the secretion of heparanase, which degrades
heparan sulfate chains in the extracellular matrix.48
Inhibition of heparanase activity by heparin fragments
has been shown to decrease the heparanase-mediated
infiltration of T-cells into inflammatory lesions49 or of
tumor cells into metastatic sites.50 However, use of
heparin fragments causes the unwanted side effect of
blood anticoagulation.41 It is therefore highly desirable
to develop a compound that inhibits heparanase with-
out producing such side effects.
Conclusion
We have described the synthesis of a newclass of oli-
gomers composed of glycamino acids, which bear a
carboxylate at the anomeric position and an amino
group replacing one of the hydroxyl groups of the
monosaccharide. On the basis of the CD and NMR
spectral analyses, they were found to form rigid sec-
ondary structures, in particular the b(1!2)-linked oli-
gomers, which were very likely to form a 14-helical
structure. In addition, since some divalent metal cations
such as Ca2+ are known to form a complex with the
OH groups of carbohydrates and such complexes play
an important role in carbohydrate-receptor interactions,
it would be of considerable interest to study the solution
conformation of these glycamino acid-based oligomers
in the presence of divalent metals. Though it may be too
early to discuss the usefulness of these glycamino acid-
based oligomers as alternate analogues of biologically
active peptides or oligosaccharides, we have observed
that, upon O-sulfation, these oligomers effectively
inhibited HIV infection, sialyl Lewis x-mediated cell
adhesion, and heparanase activity in a linkage-specific
manner without evidence of the anticoagulant activity
that is characteristic of the sulfated derivatives of natu-
rally occurring oligosaccharides.
Determination of the inhibitory activity of the com-
pounds against heparanase was based on the inhibition
of its hydrolase activity. 35SO4-radiolabeled heparan
sulfate proteoglycans51 are incubated with a partially
purified heparanase52 and various concentrations of the
O-sulfated amide-linked carbohydrate analogues or
peptide–sugar hybrid analogues.53 The incubation mix-
tures were then centrifuged through an Amicon1 mem-
brane filter (molecular weight cut-off 30,000), and the
radioactivity in the filtrate (cleaved material) was deter-
mined to assess the inhibitory potency of the com-
pounds. Among the O-sulfated carbohydrate analogues
tested, the b(1!6)-linked tetramer was the most potent
inhibitor of heparanase, with an IC50 of 0.6 mM; the
b(1!6)-linked trimer (IC50, 20 mM) and the b(1!2)-
linked tetramer (IC50, 10 mM) were less potent (Fig. 4).
Experimental
Melting points were determined with a Fisher–Johns
melting point apparatus and are uncorrected. Elemental
analyses were performed by Galbraith Laboratories Inc.
(Knoxville, TN, USA). Mass spectra were measured by
Mass Spectrometry Laboratory, University of Illinois at
Figure. 4. Inhibition of heparanase activity with O-sulfated glycamino
acid oligomers.

Y. Suhara et al. / Bioorg. Med. Chem. 10 (2002) 1999–2013
2005
Urbana-Champaign (Urbana, IL, USA). Organic
extracts were dried over anhydrous MgSO4, and were
concentrated under diminished pressure. Chromato-
graphy was performed on a column of silica gel (60–200
mesh, Fisher Scientific), and thin-layer chromatography
was conducted on precoated plates silica gel 60 F254
(Merck). Gel filtration was performed on a column of
room temperature and concentrated. Solid NaOH (336
mg, 8.40 mmol) was added to a solution of the residue
in water (100 mL), and the mixture was refluxed for 12
h. After cooled, the mixture was neutralized with
Dowex 50W-X8 [H+] resin, and the resin was filtered
off. The filtrate was diluted with water (1 L) and was
applied onto a column of Dowex 1 [OH-]-form resin.
The column was washed with water and the product
was eluted out with 2 N AcOH. The fractions contain-
ing the product were pooled and concentrated. A solu-
tion of the residue in MeOH (100 mL) was refluxed for
12 h and concentrated. The residue was chromato-
graphed on SiO2, with hexanes–EtOAc 1:2 to EtOAc
only, to give 7 (1.30 g, 53%) as a colorless oil; 1H NMR
(300 MHz, CD3OD) d 4.10 (1H, q, J=1.2, 13.2 Hz, H-
5), 3.88 (1H, d, J=9.5 Hz, H-2), 3.82 (1H, dd, J=1.3,
14.4 Hz, H-7a), 3.77 (3H, s, CO2CH3), 3.65 (1H, dd,
J=1.0, 11.1 Hz, H-7b), 3.49 (1H, t, J=9.6 Hz, H-4),
3.61–3.33 (1H, m, H-3); 13C NMR (72.5 MHz, CD3OD)
d 171.6 (CO2CH3), 82.2, 81.1, 72.2, 72.0, 69.7, 62.3 (C-
1,2,3,4,5,6), 52.8 (CO2CH3); mass spectrum (FAB) m/e
248 ((M+H+), calcd for C8H14N3O6 248).
1
Sephadex G-25 (Pharmacia). H and 13C NMR spectra
were recorded with a Bruker AMX 300 or a JEOL
GX-400 spectrometer.
1,2,4,6-Tetra-O-acetyl-3-azido-3-deoxy-D-glucopyranose
(5). Compound 5 was prepared from 3-azido-3-deoxy-
1,2:5,6-di-O-isopropylidene-b-d-glucofuranose23 by acid
hydrolysis and acetylation; 1H NMR (300 MHz,
CDCl3) d 6.25 (1H, d, J=3.5 Hz, H-1a), 5.68 (1H, d,
J=8.2 Hz, H-1b), 5.07–4.93 (4H, m), 4.27–4.19 (2H, m),
4.11–3.93 (4H, m), 3.82–3.76 (1H, m, H-5), 3.70 (1H, t,
J=10.0 Hz, H-4); mass spectrum (FAB) m/e 396
((M+Na+), calcd for C14H19N3O9Na 396), 314
((MÀOAc), calcd for C12H16N3O7 314).
Methyl 2,6-anhydro-4-tert-butoxycarbonylamido-4-deoxy-
D-glycero-D-gulo-hepturonate (8). H2S gas was bubbled
through a solution of 7 (1.40 g, 5.66 mmol) in pyridine
(50 mL)–water (50 mL) for 2 h at room temperature.
The mixture was stirred for another 12 h at room tem-
perature and concentrated. BOC-ON (2.82 g, 11.3
mmol) was added to a solution of the residue and Et3N
(1.58 mL, 11.3 mmol) in MeOH (100 mL), and the
mixture was stirred for 12 h at room temperature and
concentrated. The residue was chromatographed on
SiO2, with CHCl3–MeOH, to give 8 (1.02 g, 77%) as a
white powder, mp 148–151 ꢀC; 1H NMR (300 MHz,
CD3OD) d 4.10 (1H, dd, J=7.1, 14.2 Hz, H-5), 3.88
(1H, d, J=9.3 Hz, H-2), 3.84 (1H, m, H-7a), 3.76 (3H,
s, CO2CH3), 3.65 (1H, dd, J=4.5, 12.2 Hz, H-7b), 3.57–
3.44 (2H, m, H-3,4); 13C NMR (72.5 MHz, CD3OD) d
171.9 (C-1), 159.1 (NHOCOtBu), 82.9, 81.1, 71.7, 69.5,
62.6, 61.5 (C-1,2,3,4,5,6), 52.8 (CO2CH3), 28.8 (tBu);
mass spectrum (FAB) m/e 322 ((M+H+), calcd for
C13H24NO8 322), 222 ((M–Boc+2H+), calcd for
C8H16NO6 222).
2,6-Anhydro-3,5,7-tri-O-acetyl-2,6-anhydro-4-azido-4-
deoxy-D-glycero-D-gulo-heptononitrile (6). TiBr4 (22.6 g,
616 mmol) was added portionwise to a cooled solution
of 5 (11.5 g, 30.8 mmol) in CH2Cl2 (150 mL)–EtOAc (50
mL) at 0 ꢀC, and the mixture was stirred for 36 h at
room temperature. The reaction mixture was cooled and
NaOAc (22.6 g) was added to the mixture, and the
resulting mixture was stirred for 1 h at room tempera-
ture. The mixture was diluted with CH2Cl2 and was
successively washed with water, satd NaHCO3, and
brine, dried, and concentrated. The residue was roughly
chromatographed on SiO2, with hexanes–EtOAc 3:1,
to give the glycosyl bromide (7.21 g, 62%) as a yellow
oil, which was used for the next step without further
purification.
The bromide (7.21 g, 190 mmol) and Hg(CN)2 (9.60 g,
380 mmol) were suspended in CH3NO2 (10 mL), and
the resulting reaction mixture was heated for 1 h at
80 ꢀC. The reaction mixture was diluted with CHCl3
and the precipitate was filtered off. The filtrate was
washed with N KBr and brine, dried, and concentrated.
The residue was chromatographed on SiO2, w ith tolu-
ene–EtOAc 10:1, to ꢀgive 6 (3.70 g, 57%) as a white
powder, mp 120–124 C; 1H NMR (300 MHz, CDCl3) d
5.26 (1H, t, J=10.2 Hz, H-4), 5.00 (1H, t, J=10.0 Hz,
H-3), 4.30 (1H, d, J=10.2 Hz, H-1), 4.21 (1H, dd,
J=4.9, 12.7 Hz, H-6a), 4.12 (1H, dd, J=2.4, 10.2 Hz,
H-6b), 3.72–3.67 (1H, m, H-5), 3.66 (1H, t, J=9.9 Hz,
H-2), 2.21, 2.13, 2.10 (3H each, 3s, 3ÂCH3CO); 13C
Methyl 2,6-anhydro-3,5,7-tri-O-benzyl-4-tert-butoxycar-
bonylamido-4-deoxy-D-glycero-D-gulo-hepturonate (9).
NaH (60% mineral oil dispersion; 359 mg, 8.96 mmol)
was added to a cooled solution of 8 (800 mg, 2.49
mmol) in DMF (30 mL), and the mixture was stirred for
10 min at 0 ꢀC. BnBr (1.33 mL, 11.2 mmol) was added
to the mixture, and the mixture was stirred for 12 h at
room temperature. The reaction mixture was carefully
poured into ice water, and extracted with EtOAc. The
extracts were successively washed with water and brine,
dried, and concentrated. The residue, a mixture of the
methyl ester and the corresponding benzyl ester, was
treated with 25% NaOMe–MeOH (200 mL) in MeOH
(20 mL) for 2 h at room temperature. The reaction
mixture was neutralized with Dowex 50W-X8 [H+]
resin, and the resin was filtered off and the filtrate was
concentrated. The residue was chromatographed on
SiO2, with hexanes only to hexanes–EtOAc 5:1, to give
NMR (72.5 MHz, CDCl3)
d 170.6, 169.1, 168.6
(3ÂCH3CO), 114.2 (CN), 68.9, 67.5, 66.8, 64.7, 61.6,
20.8, 20.6, 20.5 (3ÂCH3CO); mass spectrum (FAB) m/e
341 ((M+H+), calcd for C13H17N4O7 341).
Methyl 2,6-anhydro-4-azido-4-deoxy-D-glycero-D-gulo-
hepturonate (7). The pH of a solution of 6 (3.40 g, 9.99
mmol) in MeOH (100 mL) was adjusted to 11 with 25%
NaOMe–MeOH, and the mixture was stirred for 1 h at

2006
Y. Suhara et al. / Bioorg. Med. Chem. 10 (2002) 1999–2013
9 (920 mg, 64%), mp 150–153 ꢀC; H NMR (300 MHz,
CDCl3) d 7.35–7.18 (15H, m, Ph), 4.72–4.46 (6H, m,
3ÂCH2Ph), 3.95–3.84 (2H, m), 3.72 (3H, s, CO2CH3),
3.77–3.59 (4H, m), 3.48–3.44 (1H, m); 13C NMR
(72.5 MHz, CDCl3) d 169.5 (C-1), 155.3 (NHOCOtBu),
137.9, 137.7 (CPh), 128.30, 128.28, 128.11, 128.02,
127.91, 127.88, 127.77, 127.61 (Ph), 80.4, 79.6, 78.9,
73.4, 68.9, 73.97, 73.92, 73.89 (CH2Ph), 52.3 (CO2CH3),
28.3 (tBu); mass spectrum (FAB) m/e 592 ((M+H+),
calcd for C34H42NO8 592), 492 ((M–Boc+2H+), calcd
for C29H35NO6 492).
128.17, 128.14, 128.0, 127.8, 127.7, 127.5, 127.0 (Ph),
79.3, 78.0, 77.1, 73.43, 73.36, 52.8 (CHCO2CH3 of l-
Phe), 52.2 (CO2CH3 of l-Phe), 37.3 (CH2Ph of l-Phe),
28.4 (tBu); mass spectrum (FAB) m/e 739 ((M+H+),
calcd for C43H51N2O9 739), 639 ((MÀBoc+2H+),
calcd for C38H43N2O7 639).
1
(2,4,6-Tri-O-benzyl-3-tert-butoxycarbonylamido-3-deoxy-
ꢀ-D-glucopyranosyl)-C-carboxamido-3-N-(2,4,6-tri-O-ben-
zyl-3-deoxy-ꢀ-D-glucopyranosyl)-C-carboxamido-N-L-phe-
nylalanine methyl ester (11). To a cooled solution of 10
(147 mg, 199 mmol) in EtOAc (10 mL) was added 4 N
HCl/EtOAc (10 mL), and the mixture was stirred for 1 h
at 0 ꢀC. The reaction mixture was concentrated and the
residue was washed with Et2O to give the amino deri-
vative, which was used for the next step without further
purification. To a solution 2 (172 mg, 298 mmol) in
DMF (20 mL) were added the amino derivative, BOP
reagent (176 mg, 398 mmol) and DIEA (104 mL, 597
mmol), and the mixture was stirred for 16 h at room
temperature. The reaction mixture was diluted with
EtOAc and was successively washed with water, satd
NaHCO3, and brine, dried, and concentrated. The residue
was chromatographed on SiO2, with toluene–EtOAc 5:1
to 1:1, to give 11 (138 mg, 58%) as a white powder, mp
2,6-Anhydro-3,5,7-tri-O-benzyl-4-tert-butoxycarbonyl-
amido-4-deoxy-D-glycero-D-gulo-hepturonic acid (2). A
.
mixture of 9 (900 mg, 1.52 mmol) and LiOH H2O (128
mg, 3.05 mmol) in THF (15 mL)–MeOH (15 mL)–water
(5 mL) was stirred for 20 min at room temperature. The
reaction mixture was neutralized with Dowex 50W-X8
[H+] resin, and the resin was filtered off. The filtrate was
concentrated, and the residue was chromatographed on
SiO2, with CHCl3–MeOH (10:1 to 5:1), to give 2 as a
colorless amorphous (880 mg, 97%), mp 118–120 ꢀC; 1H
NMR (300 MHz, CD3Cl/CD3OD 3:1) d 7.33–7.10 (15H,
m, Ph), 4.78–4.42 (6H, m, CH2Ph), 4.12–3.43 (7H, m),
1.42 (9H, s, NHCO2tBu); 13C NMR (72.5 MHz, CD3OD/
CDCl3 3:1) d 155.6 (NHOCOtBu), 137.8 (CPh), 129.9,
128.5, 128.4, 128.2, 127.9, 127.7, 127.0, 126.6 (Ph), 80.2,
79.4, 79.0, 78.7, 73.9, 73.5, 73.3 (CH2Ph), 50.8
(CO2CH3), 28.4 (tBu); mass spectrum (FAB) m/e 600
((M+Na+), calcd for C33H39NO8Na 600).
ꢀ
1
219–221 C; H NMR (300 MHz, CDCl3) d 7.30–7.07
(35H, m, Ph), 5.22 (1H, d, J=8.2 Hz, NH), 4.78 (1H, t,
J=6.5 Hz, CHCO2CH3), 4.65–4.35 (12H, m,
6ÂCH2Ph), 4.24 (1H, t, J=7.9 Hz), 3.96 (1H, d, J=7.8
Hz), 3.86–3.73 (4H, m), 3.69–3.54 (7H, m), 3.63 (3H, s,
CO2CH3), 3.46–3.38 (1H, m), 3.05 (1H, dd, J=6.0, 13.9
Hz, CH2Ph of l-Phe), 2.94 (1H, dd, J=7.0, 13.8 Hz,
CH2Ph of l-Phe), 1.42 (9H, s, NHCO2tBu); 13C NMR
(72.5 MHz, CDCl3) d 171.8, 169.1, 155.5, 153.1 (CO),
137.94, 137.87, 137.7, 137.4, 135.8 (CPh), 129.0, 128.4,
128.2, 128.0, 127.94, 127.89, 127.81, 127.7 (Ph), 73.5,
28.2 (tBu); mass spectrum (FAB) m/e 1099
((MÀBoc+2H+), calcd for C66H72N3O12 1099).
(2,4,6-Tri-O-benzyl-3-tert-butoxycarbonylamido-3-deoxy-
ꢀ-D-glucopyranosyl)-C-carboxamido-N-L-phenylalanine
methyl ester (10). Method A. DEPC (67.4 mL, 413
mmol) was added to a cooled solution of 2 (159 mg, 275
mmol), l-phenylalanine methyl ester (71.2 mg, 330
mmol), and Et3N (115 mL, 826 mmol) in DMF (10 mL)
at 0 ꢀC, and the mixture was stirred for 12 h at room
temperature. The reaction mixture was diluted with
EtOAc, and was successively washed with water, sat.
NaHCO3, and brine, dried, and concentrated. The resi-
due was chromatographed on SiO2, with toluene–
EtOAc 5:1, to give 10 (75.2 mg, 37%) as a white pow-
der.
(2,4,6-Tri-O-benzyl-3-tert-butoxycarbonylamido-3-deoxy-
ꢀ-D-glucopyranosyl)-C-carboxamido-3-N-(2,4,6-tri-O-ben-
zyl-3-deoxy-ꢀ-D-glucopyranosyl)-C-carboxamido-3-N-
(2,4,6-tri-O-benzyl-3-deoxy-ꢀ-D-glucopyranosyl)-C-car-
boxamido-N-L-phenylalanine methyl ester (12). The
dimeric derivative 11 (113 mg, 94.2 mmol) in EtOAc (10
mL) was treated with 4 N HCl/EtOAc (10 mL) for 1 h at
0 ꢀC, and the mixture was concentrated. The residue was
washed with Et2O to give the amino derivative, which
was used for the next step without further purification.
To a solution of 2 (81.7 mg, 114 mmol) in DMF (20 mL)
were added the amino derivative, BOP reagent (83.4 mg,
189 mmol), and DIEA (49.3 mL, 283 mmol), and the
mixture was stirred for 12 h at room temperature. The
reaction mixture was diluted with EtOAc and was suc-
cessively washed with water, satd NaHCO3, and brine,
dried, and concentrated. The residue was chromato-
graphed on SiO2, with toluene–EtOAc 1:1, to give 12
Method B. To a cooled solution of 2 (166 mg, 287
mmol) in DMF (20 mL) were added BOP reagent (74.4
mg, 345 mmol) and DIEA (150 mL, 863 mmol), and the
mixture was stirred for 12 h at room temperature. The
reaction mixture was diluted with EtOAc, and was suc-
cessively washed with water, satd NaHCO3, and brine,
dried, and concentrated. The residue was chromato-
graphed on SiO2, with toluene–EtOAc 5:1, to give 10
(116 mg, 55%) as a white powder, mp 183–186 ꢀC; H
1
NMR 300 MHz, CDCl3) d 7.36–7.05 (20H, m, Ph), 5.05
(1H, br s, NH), 4.80 (1H, dd, J=6.3, 13.4 Hz,
CHCO2CH3), 4.71–4.46 (6H, m, 3ÂCH2Ph), 4.02 (1H,
d, J=5.9 Hz, H-2), 3.91–3.83 (3H, m), 3.69–3.64 (3H,
m), 3.68 (3H, s, CO2CH3), 3.03 (1H, dd, J=5.5, 13.9
Hz, CH2Ph of l-Phe), 2.84 (1H, dd, J=7.0, 13.6 Hz,
CH2Ph of l-Phe), 1.44 (9H, s, NHCO2tBu); 13C NMR
(72.5 MHz, CDCl3) d 171.9, 169.3, 155.2 (C-1, CO),
137.80, 137.75, 135.7 (CPh), 129.0, 128.5, 128.3, 128.23,
(63 mg, 40%) as a white powder, mp 177–178 ꢀC; H
1
NMR (300 MHz, CDCl3) d 7.31–7.04 (50H, m, Ph), 6.86
(1H, d, J=7.9 Hz, NH), 6.76 (1H, d, J=8.7 Hz, NH),
4.80 (1H, dd, J=6.9, 13.2 Hz, CHCO2CH3), 4.67–4.38
(18H, m, 9ÂCH2Ph), 4.33–4.23 (2H, m), 4.15 (1H, m),
4.05 (1H, d, J=6.4 Hz), 3.89 (1H, t, J=6.3 Hz), 3.83–3.52

Y. Suhara et al. / Bioorg. Med. Chem. 10 (2002) 1999–2013
2007
(12H, m), 3.65 (3H, s, CO2CH3), 3.40 (1H, m), 3.03 (1H,
dd, J=5.7, 13.8 Hz, CH2Ph of l-Phe), 2.82 (1H, dd,
J=6.7, 13.6 Hz, CH2Ph of l-Phe), 1.41 (9H, s,
NHCO2tBu); 13C NMR (72.5 MHz, CDCl3) d171.8,
168.2 (CO), 137.9, 137.8, 135.8 (CPh), 129.0, 128.5,
128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.7 (Ph), 73.5,
73.1, 72.8, 28.4 (tBu); mass spectrum (FAB) m/e 1557
((MÀBoc+2H+), calcd for C94H101N4O17 1557).
Methyl 2,6-anhydro-5,7-O-benzylidene-D-glycero-L-manno-
hepturonate (16). A mixture of (b-d-galactopyranosyl)-
C-carboxylic acid methyl ester 15 (7.0 g, 31.5 mmol) and
HCO2H (50 mL) in benzaldehyde (50 mL) was stirred
for 1 h at room temperature. The reaction mixture was
diluted with EtOAc and was carefully neutralized with
solid anhydrous NaHCO3 (30 g). The suspension was
filtered through a Celite pad, and the filtrate was suc-
cessively washed with satd NaHCO3, water, and brine,
dried, and concentrated. The residue was chromato-
graphed on SiO2, with hexanes–EtOAc 1:1, to give 16
(6.1 g, 61%), mp 54–57 ꢀC; 1H NMR (300 MHz,
CD3OD) d 7.32–7.30 (2H, m, Ph), 7.14–7.11 (3H, m,
Ph), 5.35 (1H, s, CHPh), 4.00–3.49 (6H, m), 3.55 (3H, s,
CO2CH3), 3.39 (1H, dd, J=3.5, 9.5 Hz); 13C NMR
(72.5 MHz, CD3OD) d 171.3 (CO2CH3), 139.7 (CPh),
130.0, 129.0, 127.5 (Ph), 102.3 (CHPh), 80.7, 77.5, 74.5,
71.6, 70.1, 69.6 (C-1,2,3,4,5,6), 52.7 (CO2CH3); mass spec-
trum (FAB) m/e 311 ((M+H+), calcd for C15H19O8 311).
(2,4,6-Tri-O-benzyl-3-tert-butoxycarbonylamido-3-deoxy-
ꢀ-D-glucopyranosyl)-C-carboxamido-3-N-(2,4,6-tri-O-
benzyl-3-deoxy-ꢀ-D-glucopyranosyl)-C-carboxamido-3-N-
(2,4,6-tri-O-benzyl-3-deoxy-ꢀ-D-glucopyranosyl)-C-car-
boxamido-3-N-(2,4,6-tri-O-benzyl-3-deoxy-ꢀ-D-glucopyr-
anosyl)-C-carboxamido-N-D-phenylalanine methyl ester
(13). The trimetic derivative 12 (45 mg, 27.1 mmol) in
EtOAc (10 mL) was treated with 4 N HCl/EtOAc (10
mL) for 1 h at 0 ꢀC, and the mixture was concentrated.
The residue was washed with Et2O to give an amino
derivative which was used for the next step without
further purification. To a solution of 2 (23.5 mg, 40.7
mmol) in DMF (10 mL) were added the amino deriva-
tive, BOP reagent (24.0 mg, 54.3 mmol) and DIEA (10.4
mL, 59.7 mmol), and the mixture was stirred for 12 h at
room temperature. The reaction mixture was diluted
with EtOAc and was successively washed with water,
satd NaHCO3, and brine, dried, and concentrated. The
residue was chromatographed on SiO2, with toluene–
EtOAc 1:1, to give 13 (27.1 mg, 47%) as a white pow-
der, mp 162–165 ꢀC; 1H NMR (300 MHz, CDCl3) d
7.28–7.06 (65H, m, Ph), 6.87 (1H, d, J=8.7 Hz, NH),
6.84 (1H, d, J=8.8 Hz, NH), 6.61 (1H, d, J=8.7 Hz,
NH), 4.80 (1H, dd, J=7.0, 13.1 Hz, CHCO2CH3),
4.68–4.38 (24H, m, 9ÂCH2Ph), 4.33–4.16 (2H, m),
4.15–4.05 (3H, m), 3.91 (1H, t, J=6.2 Hz), 3.81–3.50
(21H, m), 3.65 (3H, s, CO2CH3), 3.40 (1H, m), 3.04
(1H, dd, J=5.6, 13.6 Hz, CH2Ph of l-Phe), 2.82 (1H,
dd, J=6.6, 13.6 Hz, CH2Ph of l-Phe), 1.42 (9H, s,
NHCO2tBu); 13C NMR (72.5 MHz, CDCl3) d 171.4,
168.8 (CO), 138.8, 138.3 (CPh), 128.48, 128.38, 128.33,
128.1, 127.9 (Ph), 77.8, 28.2 (tBu); mass spectrum (FAB)
m/e 2018 ((MÀBoc+2H+), calcd for C122H131N5O22
2018).
Methyl 2,6-anhydro-3,4-di-O-benzyl-5,7-O-benzylidene-
D-glycero-L-manno-hepturonate (17). NaH (60% mineral
oil dispersion; 1.09 g, 27.2 mmol) was added portion-
wise to a cooled solution of 16 (3.80 g, 11.4 mmol) and
benzyl bromide (6.08 mL, 51.1 mmol) in DMF (50 mL)
at 0 ꢀC, and the mixture was stirred for 12 h at room
temperature. The excess NaH was destroyed by the
addition of MeOH with cooling, and the mixture was
poured into ice-water and extracted with EtOAc. The
extracts were washed with water and brine, dried, and
concentrated. The residue was treated with NaOMe in
MeOH (100 mL) at pH ꢁ11 for 2 h at room tempera-
ture. The reaction mixture was neutralized with Dowex
50W-X8 [H+] resin, and the resin was filtered off and
the filtrate was concentrated. The residue was chroma-
tographed on SiO2, with hexanes only to hexanes–EtOAc
3:1, to give 17 (5.20 g, 93%) as a colorless oil; 1H NMR
(300 MHz, CDCl3) d 7.56–7.54 (2H, m, Ph), 7.36–7.25
(13H, m, Ph), 5.44 (1H, s, CHPh), 4.90 (1H, d, J=10.9
Hz, CH2Ph), 4.72–4.53 (3H, m, CH2Ph), 4.28–4.20 (2H,
m), 4.12 (1H, d, J=3.1 Hz), 3.91–3.84 (2H, m), 3.73–3.67
(4H, m, H-7a and CO2CH3), 3.61 (1H, dd, J=3.4, 9.4
Hz, H-7b); 13C NMR (72.5 MHz, CDCl3) d 168.7
(CO2CH3), 138.3, 138.1, 137.8 (CPh), 128.9, 128.3,
128.2, 128.1, 127.9, 127.7, 127.6, 126.4 (Ph), 101.3
(CHPh), 80.7, 78.4, 73.4, 71.2, 70.2, 69.0 (C-1,2,3,4,5,6),
75.3, 75.2 (CH2Ph), 52.3 (CO2CH3); mass spectrum
(FAB) m/e 491 ((M+H+), calcd for C29H31O7 491).
(3-tert-Butoxycarbonylamido-3-deoxy-ꢀ-D-glucopyranosyl)-
C-carboxamido-3-N-(3-deoxy-ꢀ-D-glucopyranosyl)-C-car-
boxamido-3-N-(3-deoxy-ꢀ-D-glucopyranosyl)-C-carboxa-
mido-3-N-(3-deoxy-ꢀ-D-glucopyranosyl)-C-carboxamido-
N-L-phenylalanine methyl ester (14). The tetrametic
derivative 13 (27.1 mg, 12.8 mmol) was hydrogenated
over 20% Pd(OH)2 (20 mg) in THF (10 mL)–MeOH (10
mL)–water (2 mL) for 12 h at room temperature. The
reaction mixture was filtered through a Celite pad and
the filtrate was concentrated. The residue was purified
on Sephadex G-25, with water, and the fractions con-
taining the product were pooled and concentrated. The
product was lyophilized from water to give 14 (12 mg,
Methyl 2,6-anhydro-3,4,7-tri-O-benzyl-D-glycero-L-manno-
hepturonate (18). Saturated HCl/EtOAc was added
dropwise to a suspension of 17 (1.80 g, 3.67 mmol),
NaBH3CN (1.38 g, 22.0 mmol), and activated molecular
sieves 3A (3 g) in THF (100 mL) until the pH of the
mixture became under 1.0, and the resulting mixture
was stirred for 20 min at room temperature. The reac-
tion mixture was filtered through a Celite pad, and the
filtrate was concentrated. The residue was chromato-
graphed on SiO2, with toluene only to toluene–EtOAc
5:1, to give 18 (1.71 g, 94%); 1H NMR (300 MHz,
CDCl3) d 7.34–7.25 (15H, m, Ph), 4.80–4.53 (6H, m,
3ÂCH2Ph), 4.14–4.03 (2H, m), 3.83 (1H, d, J=9.8 Hz,
H-1), 3.78–3.71 (2H, m), 3.67 (3H, s, CO2CH3), 3.61–3.55
1
90%); H NMR (300 MHz, D2O) d 7.28–7.12 (5H, m,
Ph), 3.87–3.73 (15H, m), 3.65–3.48 (4H, m), 3.62 (3H, s,
CO2CH3), 3.45–3.34 (10H, m), 3.10–2.98 (1H, m,
CH2Ph of l-Phe), 1.31 (9H, s, NHCO2tBu); mass spec-
trum (FAB) m/e 1037 ((M+2H+), calcd for
C43H67N5O24 1037.

2008
Y. Suhara et al. / Bioorg. Med. Chem. 10 (2002) 1999–2013
(2H, m, H-7a, 7b); 13C NMR (72.5 MHz, CDCl3) d
168.1 (CO2CH3), 137.7, 137.5, 137.4 (CPh), 128.2,
128.1, 128.0, 127.7, 127.6, 127.53, 127.45 (Ph), 81.9,
78.2, 77.3, 73.3, 71.2, 68.9 (C-1,2,3,4,5,6), 75.5, 75.4,
74.9 (CH2Ph), 51.4 (CO2CH3);. mass spectrum (FAB)
m/e 493 ((M+H+), calcd for C29H33O7 493).
(5 mL) at 0 ꢀC, and the mixture was stirred for 20 min at
0 ꢀC. The reaction mixture was neutralized with Dowex
50W-X8 [H+] resin, and the resin was filtered off. The
filtrate was concentrated and the residue was purified on
Sephadex G-25, with water. The fractions containing the
product were pooled and concentrated, and the residue
was lyophilized from water to give 3 (212 mg, 82%
overall), mp 204–208 ꢀC. H NMR (300 MHz, D2O) d
1
Methyl 2,6-anhydro-5-azido-3,4,7-tri-O-benzyl-5-deoxy-
D-glycero-D-gulo-hepturonate (19). Trifluoromethane-
sulfonic anhydride (973 mL, 5.76 mmol) was added
dropwise to a cooled solution of 18 (1.90 g, 3.86 mmol)
and pyridine (2.15 mL, 15.4 mmol) in CH2Cl2 at
À15 ꢀC, and the mixture was stirred for 2 h at À15 ꢀC.
The reaction mixture was diluted with CH2Cl2, and was
successively washed with dil HCl, water, satd NaHCO3,
and water, dried, and concentrated to give the triflate
derivative, which was used for the next step without
further purification. The triflate derivative was treated
with NaN3 (1.27 g, 19.3 mmol) in DMF (50 mL) for 12
h at room temperature. The reaction mixture was dilu-
ted with EtOAc, and washed with water and brine,
dried, and concentrated. The residue was chromato-
graphed on SiO2, with toluene–EtOAc 20:1, to give 19
3.72–3.49 (3H, m), 3.43–3.25 (4H, m), 1.33 (9H, s, tBu);
13C NMR (72.5 MHz, D2O) d 159.0 (CO), 81.9, 79.5,
79.1, 75.6, 72.7, 61.9 (C-1,2,3,4,5,6), 28.2 (tBu); mass
spectrum (FAB) m/e 307 (M+, calcd for C12H21NO8 307).
2,6-Anhydro-3,4,7-tri-O-benzyl-5-tert-butoxycarbonyl-
amido-5-deoxy-D-glycero-D-gulo-hepturonic acid lithium
.
salt (21). LiOH H2O (213 mg, 5.07 mmol) was added
portionwise to a cooled solution of 20 (1.50 g, 2.53
mmol) in THF (30 mL)ÀMeOH (30 mL)Àwater (10
mL) at 0 ꢀC, and the mixture was stirred for 20 min
at 0 ꢀC. The reaction mixture was neutralized with
Dowex 50W-X8 [H=] resin, and the resin was filtered
off. The filtrate was concentrated and the residue was
chromatographed on SiO2, with CHCl3–MeOH 10:1
(1.20 g, 60%) as a colorless oil; H NMR (300 MHz,
to 5:1, to give 21 (1.31 g, 90%), mp 215–217 ꢀC. H
1
1
CDCl3) d 7.36–7.24 (15H, m, Ph), 4.91–4.51 (6H, m,
3ÂCH2Ph), 3.87–3.53 (6H, m), 3.71 (3H, s, CO2CH3),
3.34–3.29 (1H, m, H-6); 13C NMR (72.5 MHz, CDCl3) d
168.1 (CO2CH3), 162.0 (CO), 137.6, 137.1 (CPh), 128.4,
128.2, 127.9, 127.8, 127.7, 127.6, 77.2, 75.1, 74.6, 73.4,
68.9, 52.3 (CO2CH3); mass spectrum (FAB) m/e 518
((M+H+), calcd for C29H32N3O6 518).
NMR (300 MHz, CD3OD) d 7.34–7.24 (15H, m, Ph),
4.82–4.56 (6H, m, 3ÂCH2Ph), 3.79–3.51 (7H, m), 3.37
(3H, s, CO2CH3), 1.41 (9H, s, tBu); 13C NMR
(72.5 MHz, CD3OD) d 159.1 (CO), 140.8, 139.5, 138.5
(CPh), 129.1, 128.8, 128.5, 128.4, 128.2, 128.1, 127.9,
127.8, 127.5 (Ph), 84.7, 82.1, 79.9, 79.1, 78.6, 28.8 (tBu);
mass spectrum (FAB) m/e 585 (M+, calcd for
C33H38NO8Li 585).
Methyl 2,6-anhydro-3,4,7-tri-O-benzyl-5-tert-butoxycarb-
onylamido-5-deoxy-D-glycero-gulo-hepturonate (20). H2S
gas was bubbled through a solution of 19 (1.20 g, 2.31
mmol) in pyridine (50 mL)–water (50 mL) for 1 h at
room temperature. The mixture was concentrated to
give the crude amino derivative which was dried
azeotropically by toluene twice. BOC-ON (5.77 g,
23.2 mmol) was added portionwise to a solution of the
residue and Et3N (3.23 g, 23.2 mmol) in MeOH (50
mL), and the resulting mixture was stirred for 12 h at
room temperature. The reaction mixture was con-
centrated and the residue was chromatographed on
SiO2, with hexanes–EtOAc 5:1, to give 20 (1.31 g,
95%), mp 137–139 C. H NMR (300 MHz, CDCl3) d
7.33–7.25 (15H, m, Ph), 4.85–4.50 (6H, m, 3ÂCH2Ph),
3.92 (1H, d, J=9.2 Hz), 3.82–3.58 (4H, m), 3.72 (3H, s,
CO2CH3), 3.48 (1H, t, J=9.4 Hz), 1.40 (9H, s, tBu);
13C NMR (72.5 MHz, CDCl3) d 169.4 (CO), 128.44,
128.40, 128.3, 128.1, 127.92, 127.86, 127.6 (Ph), 80.3, 78.1,
74.8, 73.5, 69.8, 52.3 (CO2CH3), 28.3 (tBu); mass spectrum
(FAB) m/e 592 ((M+H+), calcd for C34H42NO8 592), 492
((M–Boc+2H+), calcd for C29H34NO6 492).
(2,3,6-Tri-O-benzyl-4-tert-butoxycarbonylamido-4-deoxy-
ꢀ-D-glucopyranosyl)-C-carboxamido-N-L-phenylalanine
methyl ester (22). BOP reagent (919 mg, 2.08 mmol)
was added to a cooled solution of 21 (600 mg, 1.03
mmol), d-phenylalanine methyl ester (269 mg, 1.25
mmol), and DIEA (543 mL, 3.15 mmol) in DMF (30 mL)
at 0 ꢀC, and the mixture was stirred for 12 h at room
temperature. The reaction mixture was diluted with
EtOAc, and was successively washed with water, satd
NaHCO3, and brine, dried, and concentrated. The resi-
due was chromatographed on SiO2, with toluene–EtOAc
5:1, to give 22 (631 mg, 82%), mp 184–186 ꢀC. 1H NMR
(300 MHz, CDCl3) d 7.32–7.19 (18H, m, Ph), 7.11–7.07
(2H, m, Ph), 6.90 (1H, d, J=7.7 Hz, NH), 4.81 (1H, dd,
J=6.1, 13.7 Hz, CHCO2CH3), 4.73–4.52 (6H, m,
3ÂCH2Ph), 4.00 (1H, d, J=9.2 Hz), 3.79 (1H, t, J=7.0
Hz), 3.69–3.52 (4H, m), 3.61 (3H, s, CO2CH3), 3.11 (1H,
q, J=6.0, 14.6 Hz, CH2Ph of d-Phe), 3.07 (1H, q,
ꢀ
1
J=6.0, 14.1 Hz, CH2Ph of l-Phe), 1.40 (9H, s, tBu); 13
C
NMR (72.5 MHz, CDCl3) d 171.3, 168.7 (CO), 138.0,
135.7 (CPh), 129.2, 128.5, 128.4, 128.3, 127.9, 127.8,
127.7, 127.6, 127.0 (Ph), 80.4, 79.6, 78.2, 77.9, 74.0, 73.6,
70.5, 52.8 (CHCO3CH3 of Phe), 52.1 (CO2CH3 of Phe),
37.7 (C(CH3)3) 28.3 (tBu); mass spectrum (FAB) m/e
739 ((M+H+), calcd for C43H51N2O9 739), 638
((MÀBoc+2H+), calcd for C38H42N2O7 638).
2,6-Anhydro-5-tert-butoxycarbonylamido-5-deoxy-D-gly-
cero-D-gulo-hepturonic acid (3). Compound 20 (500 mg,
845 mmol) was hydrogenated over 20% Pd(OH)2 (400
mg) in MeOH (20 mL) for 16 h at room temperature.
The reaction mixture was filtered through a Celite pad,
.
and the filtered was concentrated. LiOH H2O (70.9 mg,
1.69 mmol) was added portionwise to a cooled solution
of the residue in THF (20 mL)–MeOH (20 mL)–water
(4-tert-Butoxycarbonylamido-4-deoxy-ꢀ-D-glucopyrano-
syl)-C-carboxamido-N-L-phenylalanine methyl ester (23).
Compound 22 (200 mg, 271 mmol) was hydrogenated

Y. Suhara et al. / Bioorg. Med. Chem. 10 (2002) 1999–2013
2009
over 20% Pd(OH)2 (150 mg) in THF (20 mL)–MeOH
(20 mL)–water (4 mL) for 12 h at room temperature.
The reaction mixture was filtered through a Celite pad,
and the filtrate was concentrated to give 23 (120 mg,
95%) as a colorless oil; 1H NMR (300 MHz, CD3OD) d
7.21–7.09 (5H, m, Ph), 4.62 (1H, dd, J=5.9, 8.2 Hz,
CHCO2CH3), 3.62–3.45 (3H, m), 3.59 (3H, s, CO2CH3),
3.35–3.21 (4H, m), 3.08 (1H, dd, J=5.8, 13.8 Hz,
CH2Ph of l-Phe), 2.96 (1H, dd, J=8.3, 13.7 Hz, CH2Ph
of l-Phe), 1.35 (9H, s, tBu); 13C NMR (72.5 MHz,
CD3OD) d 173.1, 172.3, 158.7 (CO), 138.0, (CPh),
130.2, 129.5, 127.9 (Ph), 81.3, 80.5, 78.8, 76.3, 74.0, 63.0,
54.9, 53.8, 52.8 (CO2CH3 of Phe), 38.1 (C(CH3)3), 28.7
(tBu); mass spectrum (FAB) m/e 469 ((M+H+), calcd
for C22H33N2O9 469).
stirred for 1 h at room temperature. The reaction mix-
ture was neutralized with Dowex 50W-X8 [H+], and the
resin was filtered off. The filtrate was concentrated to
give the O-deacetylated derivative, which, in EtOAc (30
mL), was then treated with 4 N HCl/EtOAc (30 mL) for
1 h at 0 ꢀC, and the mixture was concentrated to afford
the amino derivative, which was used for the next step
without further purification. To a solution of 3 (65 mg,
211 mmol) in DMF (20 mL) were added the amino
derivative, BOP reagent (156 mg, 352 mmol), and DIEA
(92 mL, 528 mmol), and the mixture was stirred for 12 h
at room temperature. Pyridine (20 mL) and acetic
anhydride (10 mL) were added to the reaction mixture
at 0 ꢀC, and the mixture was stirred for 12 h at room
temperature. The reaction mixture was diluted with
EtOAc, and was successively washed with dil. HCl,
water, satd NaHCO3, and brine, dried, and con-
centrated. The residue was chromatographed on SiO2,
with toluene–EtOAc 5:1 to 1:1, to give 25 (152 mg, 71%
(2,3,6-Tri-O-acetyl-4-tert-butoxycarbonylamido-4-deoxy-
ꢀ-D-glucopyranosyl)-C-carboxamido-4-N-(2,3,6-tri-O-
acetyl-4-deoxy-ꢀ-D-glucopyranosyl)-C-carboxamido-N-
L-phenylalanine methyl ester (24). To a cooled solution
of 23 (150 mg, 320 mmol) in EtOAc (30 mL) was added
4 N HCl/EtOAc (30 mL) at 0 ꢀC, and the mixture was
stirred for 1 h at room temperature. The reaction mix-
ture was concentrated and the residue was washed with
Et2O to give the amino derivative, which was used for
the next step without further purification. To a solution
of 3 (118 mg, 384 mmol) in DMF (20 mL) were added
the amino derivative, BOP reagent (283 mg, 640 mmol),
and DIEA (167 mL, 960 mmol), and the mixture was
stirred for 16 h at room temperature. Pyridine (30 mL)
and acetic anhydride (15 mL) were then added to the
mixture, and the resulting mixture was stirred for 16 h
at room temperature. The reaction mixture was diluted
with EtOAc, and was successively washed with dil HCl,
water, satd NaHCO3, and brine, dried, and con-
centrated. The residue was chromatographed on SiO2,
with toluene–EtOAc 5:1 to 1:1, to give 24 (160 mg,
overall), mp 234 ꢀC–dec. H NMR (300 MHz, CDCl3)
1
d 7.31–7.23 (3H, m, Ph), 7.18–7.10 (2H, m, Ph), 6.90
(1H, d, J=8.0 Hz, NH), 6.74 (1H, d, J=8.1 Hz, NH),
5.16–5.03 (6H, m), 4.76 (1H, dd, J=7.4, 13.7 Hz,
CHCO2CH3), 4.65 (1H, br s, NH), 4.33–4.04 (8H, m),
3.91 (1H, d, J=9.2 Hz, H-1), 3.81 (1H, d, J=8.5 Hz, H-1),
3.75–3.69 (8H, m), 3.71 (3H, s, CO2CH3), 3.13 (2H, m,
CH2Ph of l-Phe), 2.14, 2.04, 1.99 (3H each, s,
9ÂCH3CO), 1.41 (9H, s, tBu); 13C NMR (72.5 MHz,
CDCl3) d 171.2, 171.0, 170.6, 170.48, 170.45, 169.7,
169.6, 169.4, 167.7, 167.6, 166.5 (CO), 135.6 (CPh),
129.1, 128.5 (Ph), 80.5, 80.4, 75.63, 75.59, 72.8, 69.74,
69.70, 68.7, 63.3, 63.0, 53.1, 52.2 (CO2CH3 of Phe), 28.1
(tBu), 21.0, 20.9, 20.7, 20.6, 20.5, 20.4, 20.2 (CH3CO);
mass spectrum (FAB) m/e 1225 ((M+H+), calcd for
C54H73N4O28 1225).
(2,3,6-Tri-O-acetyl-4-tert-butoxycarbonylamido-4-deoxy-
ꢀ-D-glucopyranosyl)-C-carboxamido-4-N-(2,3,6-tri-O-
acetyl-4-deoxy-ꢀ-D-glucopyranosyl)-C-carboxamido-4-N-
(2,3,6-tri-O-acetyl-4-deoxy-ꢀ-D-glucopyranosyl)-C-car-
boxamido-4-N-(2,3,6-tri-O-acetyl-4-deoxy-ꢀ-D-glucopyr-
anosyl)-C-carboxamido-N-L-phenylalanine methyl ester
(26). The pH of a solution of the trimeric derivative
(25) (57 mg, 46.6 mmol) in MeOH (10 mL) was adjusted
to 10ꢁ11 by the addition of 25% NaOMe–MeOH, and
the mixture was stirred for 1 h at room temperature.
The reaction mixture was neutralized with Dowex 50W-
X8 [H+], and the resin was filtered off. The filtrate was
concentrated to give the O-deacetylated derivative,
which, in EtOAc (10 mL), was then treated with 4 N
HCl/EtOAc (10 mL) for 1 h at 0 ꢀC, and the mixture
was concentrated to afford the amino derivative, which
was used for the next step without further purification.
To a solution of 3 (17 mg, 56 mmol) in DMF (10 mL)
were added the amino derivative, BOP reagent (41 mg,
93 mmol), and DIEA (73 mL, 140 mmol), and the mixture
was stirred for 12 h at room temperature. Pyridine (10
mL) and acetic anhydride (5 mL) were added to the
reaction mixture at 0 ꢀC, and the mixture was stirred for
12 h at room temperature. The reaction mixture was
diluted with EtOAc, and was successively washed with
dil HCl, water, sat. NaHCO3, and brine, dried, and
concentrated. The residue was chromatographed on
58%), mp 254–257 ꢀC. H NMR (300 MHz, CDCl3) d
1
7.31–7.23 (3H, m, Ph), 7.13–7.10 (2H, m, Ph), 6.89
(1H, d, J=8.1 Hz, NH), 6.69 (1H, d, J=9.0 Hz, NH),
5.15–5.03 (4H, m), 4.78 (1H, dd, J=6.5, 14.4 Hz,
CHCO2CH3), 4.69 (1H, d, J=9.3 Hz, NH), 4.38–4.00
(5H, m), 3.88 (1H, d, J=9.5 Hz, H-1a), 3.80 (1H, d,
J=9.7 Hz, H-1b), 3.73–3.63 (6H, m), 3.72 (3H, s,
CO2CH3), 3.14 (1H, dd, J=1.4, 14.1 Hz, CH2Ph of
l-Phe), 3.08 (1H, dd, J=2.1, 16.3 Hz, CH2Ph of l-Phe),
2.14, 2.06, 2.042, 2.040, 2.03, 1.98 (3H each, s,
6CH3CO), 1.41 (9H, s, tBu); 13C NMR (72.5 MHz,
CDCl3) d 171.1, 171.0, 170.7, 170.5, 169.7, 169.3, 167.6,
166.5, 155.0 (CO), 135.6 (CPh), 129.1, 128.5, 127.0 (Ph),
80.4, 77.4, 75.7, 75.6, 72.9, 69.3, 69.1, 63.0, 52.7, 52.3,
49.7 (CO2CH3 of Phe), 37.6 (C(CH3)3), 28.1 (tBu), 20.9,
20.8, 20.6, 20.5, 20.4 (CH3CO) ; mass spectrum (FAB)
m/e 910 ((M+H+), calcd for C41H56N3O20 910).
(2,3,6-Tri-O-acetyl-4-tert-butoxycarbonylamido-4-deoxy-
ꢀ-D-glucopyranosyl)-C-carboxamido-4-N-(2,3,6-tri-O-
acetyl-4-deoxy-ꢀ-D-glucopyranosyl)-C-carboxamido-4-N-
(2,3,6-tri-O-acetyl-4-deoxy-ꢀ-D-glucopyranosyl)-C-car-
boxamido-N-L-phenylalanine methyl ester (25). The pH
of a solution of the dimeric derivative (24) (160 mg, 176
mmol) in MeOH (30 mL) was adjusted to 10ꢁ11 by the
addition of 25% NaOMe–MeOH, and the mixture was

2010
Y. Suhara et al. / Bioorg. Med. Chem. 10 (2002) 1999–2013
SiO2, with toluene–EtOAc 5:1 to 1:1, to give 26 (31 mg,
43% overall), mp 225 ꢀC–dec. 1H NMR (300 MHz,
CDCl3) d 7.31–7.25 (3H, m, Ph), 7.17–7.10 (2H, m, Ph),
6.90 (1H, s, NH), 6.78 (2H, br s, NH), 5.23–5.01 (8H,
m), 4.76 (1H, m, CHCO2CH3), 4.65 (1H, br s, NH),
4.26–4.01 (10H, m), 3.93–3.62 (10H, m), 3.73 (3H, s,
CO2CH3), 3.10 (2H, m, CH2Ph of l-Phe), 2.13, 2.07,
2.02 (3H each, s, 9ÂCH3CO), 1.42 (9H, s, tBu); 13C
NMR (72.5 MHz, CDCl3) d 171.2, 170.5, 169.6, 169.4,
167.7, 166.5 (CO), 135.6 (CPh), 129.1, 128.5, 127.0 (Ph),
80.5, 75.6, 72.9, 69.0, 63.0, 53.1, 52.8 (CO2CH3 of Phe),
28.1 (tBu), 20.9, 20.8, 20.6, 20.5, 20.4 (CH3CO); mass
spectrum (FAB) m/e 1540 ((M+H+), calcd for
C67H90N5O36 1540), 1440 ((MÀBoc+2H+), calcd for
C62H82N5O34 1440).
those for C, H, and N. Under these conditions, the
average number of the sulfate groups was estimated to
be two per each glycamino acid residue.
Inhibition of HIV infection to MT-2 cells
Determination of the antiviral activity of the com-
pounds against HIV infection was based on the inhibi-
tion of the expression of p24 in supernatant using the
virus capture assay method as described previously.42
Briefly, MT-2 cells are exposed to virus (HTLVRF) in
the presence or absence of various concentrations of
each of the synthesized analogues for 5 days at 37 ꢀC in
c-RPMI [RPMI-1640 (Bio-Whittaker, Walkersville,
MD, USA)] supplemented with 10% fetal bovine serum
(HyClone, Logan, UT, USA), 2 mM l-glutamine
(GIBCO, Grand Island, NY, USA), and 10 mM
HEPES. The number of MT-2 cells was adjusted to
40,000 cells/mL. Supernatants were collected and lysed
with 1% Triton X-100. The wells of an enzyme-linked
immunosorbent assay (ELISA) place (Costar, Cam-
bridge, MA, USA) were coated overnight with purified
anti-p24 monoclonal antibody, 1 mg/well in 50 mM Tris,
pH 9.5, and blocked with 3% bovine serum albumin
(BSA) in phosphate-buffered saline (PBA) for 1 h at
37 ꢀC. The HIV-containing lysed culture supernatants
were added to the wells followed by incubation for 1 h
at 37 ꢀC. After washing the wells six times with 200 mL
of PBS containing 0.05% Tween 20, biotinylated poly-
clonal HIV IgG was added to the wells, and incubated
for 1 h at 37 ꢀC. The wells were again washed six times
with 200 mL of PBS containing 0.05% Tween 20 and
horse radish peroxidase (HRP)–streptavidin conjugate
was added to the wells and incubated for 20 min at room
temperature. Substrate (TMB) was added in acetate
buffer and the reaction stopped after 15 min by adding
0.5 M H2SO4. The plates were read at 450 nm with a
spectrophotometric plate reader.
(4-tert-Butoxycarbonylamido-4-deoxy-ꢀ-D-glucopyrano-
syl)-C-carboxamido-4-N-(4-deoxy-ꢀ-D-glucopyranosyl)-
C-carboxamido-4-N-(4-deoxy-ꢀ-D-glucopyranosyl)-C-
carboxamido-4-N-(4-deoxy-ꢀ-D-glucopyranosyl)-C-car-
boxamido-N-L-phenylalanine methyl ester (27). Tetra-
meric derivative (26) (31 mg, 20.2 mmol) was O-
deacetylated with NaOMeÀMeOH for 1 h at room
temperature. The reaction mixture was neutralized with
Dowex 50W-X8 [H+], and the resin was filtreated off.
The filtrate was concentrated and the residue was pur-
ified on Sephadex G-25, with water, to give 27 (14 mg,
1
67%) as a white powder. H NMR (300 MHz, DMSO-
d6) d 7.21–7.10 (5H, m, Ph), 4.61 (1H, m, CHCO2CH3),
3.63–3.52 (19H, m), 3.34–3.25 (12H, m), 3.02 (2H, m,
CH2Ph of l-Phe), 1.31 (9H, s, tBu); 13C NMR
(72.5 MHz, DMSO-d6) d 173.1, 166.0 (CO), 138.0
(CPh), 129.5, 128.6 (Ph), 28.1 (tBu); mass spectrum
(FAB) m/e 1036 ((M+H+), calcd for C43H66N5O24
1036), 936 ((M–Boc+2H+), calcd for C38H58N5O22 936).
General experimental procedure for O-sulfation of
oligomers
O-Sulfation of the oligomers was performed based on
the published procedure by Uryu et al.38c A mixture of
tetramer (such as 25 or 59) (45 mg, 0.043 mmom) and
sulfur trioxide pyridine complex (2 equiv per free OH
groups; 178 mg, 1.12 mmol) in pyridine (8 mL) was
stirred for 2 h at 80 ꢀC, and cooled. Saturated aqueous
Ba(OH)2 solution was added to the reaction mixture
until it became slightly alkaline (pH 8.0 judged by a pH
paper). The formed white precipitate was removed by
centrifugation with a microcentrifuge (Eppendorf Cen-
trifuge 5415C) at 13Â1000 cpm for 10 min. The clear
supernatant was collected and was passed through a
column of Dowex 50W-X8[Na]+ (1Â5 cm), eluted with
water. The eluant was collected and concentrated in
vacuo. The residue was chromatographed, for desalting,
on a column of Sephadex G-15 (1Â65 cm), eluted with
water. Each fraction was examined with an Azure assay54
and the positive fractions were pooled and concentrated in
vacuo. The residue was lyophilyzed from water (10 mL) to
give the O-sulfated oligomers.
Inhibition of sialyl Lewis x-L-selectin mediated cell ad-
hesion. Determination of the inhibitory activity of the
compounds against sialyl Lewis x-mediated cell adhe-
sion was based on the inhibition of the selectin expres-
sing COS cell adhesion to immobilized sialyl Lewis
x-containing glycolipid derivative as described pre-
viously.44 Briefly, aliquots (50 mL) of ethanol/water (1:1)
containing phosphatidylcholine (0.5 mM), cholesterol
(1.0 mM) and sialyl Lewis x-containing glycolipid deri-
vative (0.1 or 0.8 mM) were added to microwells, and
incubated uncovered at ambient temperature for 90 min
to allow lipid absorption. Wells were preblocked with
HEPES-buffered DMEM supplemented with 25 mg/mL
BSA for 10 min at 37 ꢀC. COS-1 cells which had been
transfected 48 h earlier with plasmids encoding E- or
P-selectin were harvested with hypertonic PBS contain-
ing 1 mM EDTA, collected by centrifugation and
resuspended in the BSA/DMEM at 1.7Â105 cells/mL.
An aliquot (300 mL) of the cell suspension was pre-
incubated with the compounds at the indicated con-
centrations for 30 min at 37 ꢀC with end-over-end
rotation, and then added to the preblocked wells.
Adhesion was allowed to proceed for 45 min at 37 ꢀC.
To remove nonadherent cells, the plates were immersed
The number of sulfate groups was determined by com-
bution analysis (performed by Galbraith Laboratories,
Knoxville, TN, USA) based on the sulfur content versus

Y. Suhara et al. / Bioorg. Med. Chem. 10 (2002) 1999–2013
2011
in PBS, inverted, and placed in a Plexiglas box with was
Acknowledgements
sealed to exclude air. The box was centrifuged at 110g,
and then resubmerged in PBS. The plate was then
removed, righted (while still immersed); removed from
the PBS, and excess surface buffer was removed by
aspiration, leaving 300 mL/well. Adherent cells were lysed
by addition of 20 mL of 10% Triton X-100 to each well,
and 80 mL were removed to a fresh 96-well plate for
quantitation. Cell adhesion was quantitated by measuring
lactate dehydrogenase activity in the cell lysate after
addition of 120 mL of 0.1 M potassium phosphate pH 7.0
containing 0.7 mM NADH and 4.7 mM pyruvate. The
decrease in absorbance at 340 nm as a function of time
was measured simultaneously in each well using a Molec-
ular Devices UV multiwell kinetics plate reader.
We are grateful to Dr. K. Ikeda (University of Shi-
zuoka, Japan) for performing the NMR experiments.
Some of the NMR studies were performed in the Bio-
chemistry NMR Facility at Johns Hopkins University,
which was established by a grant from the National
Institutes of Health (GM 27512) and a Biomedical
Shared Instrumentation Grant (1S10-RR06262–0). A
fellowship from Uehara Memorial Foundation (to Y.S.)
is gratefully acknowledged. This research was partly
supported by NIH grant GM52324 (to Y.I.).
References and Notes
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Inhibition of heparanase activity
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sulfate solution (typically 1–5 mL; containing approxi-
mately 2500 cpm of 35S-labeled HS)51 were mixed in
with a buffer into a final reaction mixture volume of 100
mL, consisting of 25 mM KCl, 50 mM NaCl, 5 mM
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Circular dichroism (CD)spectra
Samples (2 mg) were dissolved in 500 mL of H2O and
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at 20 ꢀC in a CD cell with a 1.0 mm optical path
length.
NMR spectroscopy
NMR spectra were measures on a Bruker DRX-600 and
DRX-400 spectrometers. Samples (5–6 mg) were dis-
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ROESY, H–13C HSQC, and H–13C HSQC-TOCSY
were recorded using standard pulse sequences.
3J(NH,H2) coupling constants were obtained from 1D
spectra. ROESY data for integration of peak volumes
were collected with mixing times of 300 ms at 303 K.
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