Heterobifunctional ligands: Practical chemoenzymatic synthesis of a cell adhesive glycopeptide that interacts with both selectins and integrins

Article abstract of DOI:10.1021/jm000295r

An efficient and practical synthesis of cell adhesive glycopeptides exhibiting unique properties as a novel type of modulator of cellular recognition is described. A nonnatural glycopeptide 1 composed of sialyl Lewis x and Lys-Gly-Arg-Gly-Asp-Ser that int

Full text of DOI:10.1021/jm000295r

J . Med. Chem. 2001, 44, 715-724
715
Heter obifu n ction a l Liga n d s: P r a ctica l Ch em oen zym a tic Syn th esis of a Cell
Ad h esive Glycop ep tid e Th a t In ter a cts w ith Both Selectin s a n d In tegr in s
Masao Matsuda and Shin-Ichiro Nishimura*
Division of Biological Sciences, Graduate School of Science, Hokkaido University, Sapporo 060-0810, J apan
Fumio Nakajima
Sapporo Laboratory for Glycocluster Project, J apan Bioindustry Association, Sapporo 060-0810, J apan
Takashi Nishimura
Division of Immunoregulation, Research Section of Disease Control, Institute for Genetic Medicine, Hokkaido University,
Sapporo 060-0815, J apan
Received J uly 12, 2000
An efficient and practical synthesis of cell adhesive glycopeptides exhibiting unique properties
as a novel type of modulator of cellular recognition is described. A nonnatural glycopeptide 1
composed of sialyl Lewis x and Lys-Gly-Arg-Gly-Asp-Ser that interacts with both selectins and
integrins has been systematically synthesized by combined chemical and enzymatic strategy.
It is suggested that glycopeptide 1 showed much higher affinity with P-selectin (Ka ) 6.6 ×
107 M-1) and E-selectin (Ka ) 4.5 × 105 M-1) than sialyl Lewis x. This compound also inhibited
a specific interaction between human integrin â1 and its monoclonal antibody more effectively
than the tetrapeptide Arg-Gly-Asp-Ser. Interestingly, it was demonstrated by surface plasmon
resonance analysis that this heterobifunctional glycopeptide exhibited a capacity to form stable
complexes with P-selectin and integrin â1 concurrently. It is also suggested that this activity
can be used for the inhibition of integrin-mediated adhesion of activated helper T cells onto
collagen-coated plates as a cell migration model. These results indicate that the chemoenzymatic
hybridization strategy of different biological functions of carbohydrates and peptides is a new
concept for designing potent glycoconjugates as antiinflammatory and anticancer metastasis
reagents.
In tr od u ction
tional ligands designed by combining some different
biofunctional motifs are also potential and attractive
concepts for creating new types of multiple reagents.5
Ligands that interact with both selectins and integrins
are novel and potent candidates for therapeutic reagents
having antiinflammatory and anticancer metastasis
activities. As shown in Figure 1, we considered that the
hybridization of Arg-Gly-Asp-Ser (RGDS) and sialyl
Lewis x (SLex) with an appropriate flexible linker would
provide us with a nonnatural glycopeptide having
interesting properties as a heterobifunctional ligand.
The tetrapeptide RGDS motif corresponds to natural
and core sequences derived from fibronectin which has
been reported to contribute to the molecular recognition
in cell adhesion, spreading, and migration of cells
through specific interaction with integrins, one of the
most important protein families of the cell surface
receptors.2a,b It was also suggested that the pentapep-
tide Gly-Arg-Gly-Asp-Ser (GRGDS) showed strong in-
hibitory effect on experimental metastasis of murine
melanoma cells.6 On the other hand, the tetrasaccharide
SLex, Neu5AcR(2f3)Galâ(1f4)[FucR(1f3)]GlcNAc, has
been known as a common oligosaccharide ligand for the
selectins that mediate adhesion of leukocytes to the
endothelium cells in the early phase of an inflammatory
response.1c Unfortunately, because the connectivity of
SLex with selectins under equilibrium conditions is
weak, recent efforts have been centered on the increase
Cell adhesive molecules (CAMs) such as fibronectin,
laminin, vitronectin, collagen, integrin, immunoglobulin,
and some animal lectins participate in a variety of cell-
cell interactions and cell-extracellular matrix (ECM)
interactions.1 Molecular recognition by CAMs on the cell
surfaces is one of the most important biological pro-
cesses not only in cell adhesion but also in fertilization,
organ formation, cell migration, lymphocyte trafficking,
immune response, and cancer metastasis.2 It should be
noted that multiple and complex mechanisms of the
bindings between a variety of cell surface receptors with
their ligands are supposed to contribute to the construc-
tion of stable and specific cellular networks. Therefore,
artificial hybridization of some different functional
structures of CAMs is expected to become a novel and
efficient method to create useful reagents for the
regulation of specific cellular recognition and adhesion.
In the present study, we selected integrins and
selectins as a set of target receptors of the synthetic
heterobifunctional ligand, because they play crucial
roles in cellular recognition related to lymphocyte
trafficking. Although individual synthetic blockers of
selectin- or integrin-mediated cellular recognition have
been extensively investigated,3,4 the heteromultifunc-
* To whom correspondence should be addressed. Tel: +81-11-706-
3807. Fax: +81-11-706-3435. E-mail: shin@glyco.sci.hokudai.ac.jp.
10.1021/jm000295r CCC: $20.00 © 2001 American Chemical Society
Published on Web 01/09/2001

716 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Matsuda et al.
F igu r e 1. Chemical structure of glycopeptide 1.
F igu r e 2. Cell adhesion model represented by the multiple
receptor-ligand interactions.
of the affinity by designing mimics of SLex and through
multivalency.7 In the present work, these two functional
ligands are combined by the flexible and hydrophobic
dipeptide linker in order to obtain a novel class of
biological response modifiers with highly improved and
unique binding properties. Glycopeptide 1 that interacts
with two different types of families of cell adhesive
proteins such as selectins and integrins can be used for
the regulation of cell adhesion and related diseases.
The basic idea has arisen from the realization that
each cell adhesive protein might involve some different
binding sites (domains) for their ligands as represented
in Figure 2 and this must also contribute to the stable
and successful cellular recognition as well as the mul-
tivalent interactions.8 For example, although the RGDS
motif has been known as a crucial ligand of integrins,
it has also been reported that integrins interact with
ganglioside GM3 which has a sialyl lactose moiety,
Neu5AcR(2f3)Galâ(1f4)Glc. In fact, this interaction
seems to enhance the affinity of integrin with fibronectin
dramatically.9 On the other hand, it was suggested that
the affinity of P-selectin with SLex is strongly affected
by the structure and properties of peptide segments of
SLex-containing glycoproteins. For example, the peptide
sequence involving sulfated tyrosine residues found in
PSGL-1 (P-selectin glycoprotein ligand-1) seems to be
a critical structure for the specific binding with P-
selectin.10 This clearly indicates the need of the associa-
tion by peptidic binding sites in addition to the binding
by the carbohydrate-based ligands. All these reports
strongly suggest that the conjugation of some different
CAMs or functional motifs might become a promising
method to control the biological activity, specificity
F igu r e 3. Some plausible mechanisms of binding of 1 with
receptor molecules: (A, B) cooperative-type cis-interaction of
1 with selectin or integrin; (C, D) cross-linking of two different
protein receptors by glycopeptide (trans-interaction).
(targeting), and stability of each simple ligand molecule.
Figure 3 summarizes plausible models for the interac-
tion of 1 with cell adhesive proteins displayed on the
cell surfaces. As illustrated in Figure 3A,B, cooperative
effects (cis-type interactions) by artificial glycopeptide
may lead to more successful and stable recognition with
each receptor molecule than that by peptide (RGDS) or
carbohydrate (SLex) itself. Moreover, specific cross-
linking of different receptors shown in Figure 3C,D is
also expected to be a novel and efficient pathway to
modulate cell adhesion (trans-type interactions).
We report herein the full details of an efficient
chemoenzymatic synthesis of the nonnatural glycopep-
tide 1 exhibiting specific cell adhesion activities. The
concept of heteromultifunctional ligands described here
is of growing importance for the synthesis of a novel
class of bioactive glycoconjugates.
Resu lts a n d Discu ssion
Syn th esis. Although some excellent examples of the
synthetic studies on the partial structures of the natu-
rally occurring glycoproteins based on chemoenzymatic
manner have been reported previously by Wong and co-
workers,11 versatility of this synthetic strategy toward
nonnatural or related glycopeptide mimetics with potent
biological activities has not been discussed well. We
considered that an efficient and facile glycosylation
might become a key step for the creation of the carbo-

Heterobifunctional Ligands
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 717
F igu r e 4. Stereorepresentation of the possible low-energy conformations predicted by a random conformational searching
method: (A-D) four major types of the most stable conformers; (E) extended model of compound 1.
hydrate-mediated specific sorting of bioactive peptides
and proteins as well as for the enhancement of the
stability against the thermal denaturation or the deg-
radation by peptidases.12 Here, we designed an artificial
heterobifunctional glycopeptide 1 as a tentative target
molecule for evaluating the feasibility of the synthesis
using enzyme-assisted strategy. To discuss the propriety
of this compound, possible low-energy conformations
were predicted by a random conformational searching
method,13 and Figure 4 shows four major types of the
most stable conformers (A-D) in addition to a plausible
model of the extended conformation (E). These results
suggest that the flexible linker would permit the forma-
tion of hydrogen bonds between the tetrasaccharide and
hexapeptide moieties and these stable conformations of
the glycopeptide seem to be more suited for the suc-
cessful cis-interactions with the receptor proteins rather
than trans-interactions as illustrated in Figure 3.
Scheme 1 indicates a retrosynthetic pathway for the
synthesis of glycopeptide 1. The synthetic strategy is
composed of chemical synthesis of a key intermediate
214 and its enzymatic modifications. Since the acid
lability of the L-fucose residue and the difficulty in the
stereoselective glycosylation of sialic acid were crucial
problems for the chemical manipulations of SLex-
containing glycopeptides, the chemoenzymatic strategy
greatly facilitated the synthetic scheme of the complex
glycopeptide 1. First, a key intermediate 2 was prepared
by chemical synthesis from a simple n-pentenyl glyco-
side of N-acetyllactosamine 515 and a tetrapeptide
derivative 4 as starting materials (Scheme 2). The
terminal double bond of compound 5 was converted into
a carboxyl group by treatment with potassium perman-
ganate in 87% yield, and the active ester of 6 was
employed for the coupling reaction with the ꢀ-amino
group of Z-lysine to afford 7 in 71% yield. To achieve
the satisfactory flexibility of the spacer arm moiety
between SLex and RGDS, and the versatility in further
chemical manipulation, H-Gly-OBn was combined with
7 to give N-acetyllactosamine dipeptide derivative 8 in
83% yield. Consequently, deprotection of the benzyl
ester and O-acetyl groups of 8 under alkaline condition
was allowed to proceed smoothly and gave compound 3
in 87% yield without racemization. Coupling reaction
of the glycopeptide 3 having an unprotected disaccha-
ride moiety with an N-terminus generated from RGDS
derivative 4 was carried out by employing diphenyl
phosphorazidate16 as a selective promoter of the car-

718 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Sch em e 1. Retrosynthetic Route of CAGP 1
Matsuda et al.
Sch em e 2 Synthesis of a Key Intermediate 2a
a
Reagents and conditions: (i) KMnO4, AcOH(aq), rt, 87%; (ii) HONSu, DCC, DMF, 0 °C f rt, then Z-Lys, TEA, DMF-H2O (9:1), rt,
71%; (iii) DPPA, Gly-OBn, TEA, DMF, 0 °C f rt, 83%; (iv) NaOMe, dry MeOH, rt, then 1 N NaOH(aq), rt, ion-exchange resin (Dowex
50W-X8), 87%; (v) 2 N HCl/dioxane, 0 °C f rt; (vi) DPPA, TEA, DMF, 0 °C f rt, 77% (over all yield from 4); (vii) Pd-C, H2(g), MeOH-
H2O, (1:1) rt, 99%.
boxyl group of glycopeptide and afforded a hexapeptide
derivative 9 in 77% yield with no side reaction. Removal
of all protecting groups of the peptide moiety of 9 such
as benzyl, nitro, and benzyloxycarbonyl groups was
performed by usual hydrogenation in the presence of
palladium on charcoal to afford the intermediate 2 in
92% yield.
Subsequently, the glycopeptide 2 was used as sub-
strate for the corresponding glycosyltransferases with-
out any protection/deprotection procedure. It is of
interest to examine the substrate specificity of glyco-
syltransferases against the nonnatural glycopeptide 2,
since the acceptability of synthetic glycoconjugates to
the glycosyltransferases seemed to be seriously affected
by the nature of aglycons or supporting backbones.
Actually, our previous studies have also shown that the
substrate specificity of the glycosyltransferases is strongly
dependent on the structure of backbones or scaffoldings
in addition to the length and hydrophobicity of the
linker moiety between the sugar and main chain.17 For

Heterobifunctional Ligands
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 719
Sch em e 3. Enzymatic Sugar Elongation Reactionsa
a
Reagents and conditions: (i) CMP-Neu5Ac, R-2,3-sialyltransferase, calf intestine alkaline phosphatase, BSA, MnCl2‚4H2O, Triton
CF-54, 50 mM sodium cacodylate buffer (pH 7.4), 37 °C, 3 days, 80%; (ii) GDP-Fuc, R-1,3-fucosyltranseferase, calf intestine alkaline
phosphatase, NaN3, MnCl2‚4H2O, 100 mM HEPES buffer (pH 7.5), 37 °C, 3 days, 56%.
F igu r e 5. Binding curves for compound 1 or SLex to immobilized P-selectin at the same concentration ([ligand] ) 0.66 mM). It
was observed that most of compound 1 bound on the surface of the cuvette could not be replaced with 10 mM phosphate buffer
solution containing 1 mM CaCl2 (PBS/C), although SLex was completely removed by washing with PBS/C.
Ta ble 1. Kinetic and Equilibrium Constants of Synthesized
Glycopeptides Against Selectins
instance, the hydrophilic linkers prepared from ethyl-
eneglycol derivatives as well as the insufficient spacing
inevitably rendered unsatisfactory results of the sugar
elongation reactions toward synthetic glycopolymers by
some glycosyltransferases.17c Thus, the hexapeptide
derivative bearing the N-acetyllactosamine side chain
through an appropriate hydrophobic linker (2) was
proven to become an excellent substrate for both re-
combinant rat R(2f3) sialyltransferase and recombi-
nant human R(1f3) fucosyltransferase V. The sialyla-
tion and fucosylation of compound 2 proceeded smoothly,
and the targeted glycopeptide 1 was isolated by purifi-
cation with preparative HPLC in 45% overall yield from
2 (Scheme 3).
Biologica l Asp ects. Association constants of 1 or
related compounds with selectins were determined by
the surface plasmon resonance (SPR) method. Since
selectins are the receptors displayed on the surface of
cell membranes, the interaction of the selectins im-
mobilized on the surface of biosensor cuvettes with these
synthetic ligands can be regarded as an experimental
model of cell adhesion mediated by selectins and their
ligands. Figure 5 is a typical binding profile between
P-selectin with compound 1 or with SLex in the same
SLex-
KGRGDS
(1)
LacNAc-
KGRGDS
(2)
selectin
P
constant
SLex
229.6
RGDS
89.1
kass (M-1 s-1
)
978.1
114.5
k
diss (s-1
)
1.5 × 10-5 2.6 × 10-4 4.5 × 10-4 9.2 × 10-4
Ka (M-1
kass (M-1 s-1
diss (s-1
Ka (M-1
kass (M-1 s-1
diss (s-1
Ka (M-1
)
6.6 × 107 8.8 × 105 2.5 × 105 9.7 × 104
E
L
)
451.2
21.1
9.9
4.0
k
)
)
1.1 × 10-3 1.7 × 10-3 1.3 × 10-3 6.6 × 10-4
4.1 × 105 1.2 × 104 7.6 × 103 6.1 × 103
)
180.1
312.7
29.8
4.1
k
)
)
1.9 × 10-1 3.6 × 10-1 2.9 × 10-2 7.7 × 10-4
9.5 × 102 8.7 × 102 1.0 × 103 5.4
ligand concentration (6.63 × 10-4 M). It was clearly
suggested that compound 1 showed much higher affinity
with the P-selectin immobilized cuvette than SLex.
Kinetic and equilibrium constants of the interaction
between selectins and all ligands used in this study are
listed in Table 1. The results indicate that compound 1
binds P- and E-selectins more strongly than native
SLex, and the association constants of 1 with P- and
E-selectins were assumed to be 6.6 × 107 and 4.5 × 105
M, respectively. Since the SLex and simple derivatives
such as glycosides and mimetics have been known to

720 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Matsuda et al.
F igu r e 6. Inhibitory effect of glycopeptide 1 or RGDS on the interaction between integrin â1 and immobilized monoclonal antibody
to human integrin â1.
F igu r e 7. Trans-type interaction (cross-linking of selectin and integrin) by cell adhesive glycopeptide. The first line between “a”
and “b” indicates the time point of the beginning of the injection of compound 1 (0.2 mL, 1.3 mg/mL) to the P-selectin- immobilized
cuvette. The second line between “b” and “c” indicates the time point of the injection of integrin â1 (0.2 mL, 9.1 µg/mL) to this
cuvette. Integrin â1 was found to bind to the glycopeptide-P-selectin complex as indicated by a significant increase in the response
unit.
show the highest affinity with E-selectin among three
selectins,7 this result demonstrates that the specificity
and the strength in the carbohydrate-protein interac-
tions can be controlled by conjugating simple sugar
ligands with some functional oligopeptides. Very low
binding affinity with L-selectin was detected for com-
pounds 1 and 2 as well as SLex. Next, the binding of
compound 1 to integrin â1 was discussed by the com-
petition binding assays in comparison with the effect
of RGDS. The inhibitory effect by the synthetic ligands
on the binding of integrin â1 with immobilized mono-
clonal antibody against integrin â1 was measured and
presented as a decrease in the binding activity, and the
effect was dependent on the concentration of the ligands
(Figure 6). It was found that 1 inhibited the interaction
of human integrin â1 with its monoclonal antibody more
effectively than RGDS (IC50 ) 0.55 mM). These experi-
ments clearly demonstrate that the synthetic glycopep-
tide 1 exhibited an amplified affinity with selectins or
integrins on the basis of the cis-type interactions as
proposed in Figure 3A,B. Furthermore, our interest was
focused on the cross-linking of these two different
protein receptors by the glycopeptide 1. Figure 7 shows
a double-phase measurement of the P-selectin-com-
pound 1-integrin â1 interaction carried out by using
the P-selectin-immobilized optical biosensor cuvette. As
shown in this binding profile, the surface of the im-
mobilized P-selectin covered with compound 1 interacted
tightly with integrin â1, indicating that this heterobi-
functional glycopeptide can combine P-selectin and
integrin â1 at the same time. Although the compound 1
has been supposed to form some stable compact struc-
tures (Figure 4A-D) rather than an extended confor-
mation (Figure 4E) as predicted by the random confor-
mational searching analysis, this result suggests that
the spacer moiety of compound 1 is flexible enough to
function as a ligand for trans-interaction.
As a preliminary biological application of the syn-
thetic glycopeptide, we also examined the inhibitory
effect of the glycopeptide 1 on the integrin-mediated

Heterobifunctional Ligands
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 721
trated under reduced pressure to give 6 (479 mg, 87.3%): δH
(400 MHz; CDCl3) 1.90 (m, 2 H, OCH2CH2), 1.95-2.18 (all s,
21 H, 7 × COCH3), 2.41 (m, 2 H, OCH2CH2CH2), 3.51 (m, 2 H,
OCH2), 3.62 (m, 1 H, H-5), 3.79 (t, 1 H, J 7.21 and 8.79, H-4),
3.88-3.91 (m, 2 H, H-6′a and H-6′b), 4.02-4.16 (m, 3 H, H-2,
H-5′ and H-6a), 4.41(d, 1 H, J 7.70, H-1), 4.50 (d, 1 H, J 8.06,
H-1′), 4.48-4.51 (m, 1 H, H-6b), 4.98 (dd, 1 H, J 3.67 and 10.63,
H-3′), 5.06 (dd, 1 H, J 8.25 and 9.69, H-3), 5.11 (dd, 1H, J 8.06
and 10.62, H-2′), 5.36 (d, 1 H, J 3.66, H-4′), 6.03 (d, 1 H, J
9.53, NH). Anal. Calcd for C30H43NO19: C, 49.93; H, 6.00; N,
1.94. Found: C, 49.90; H, 5.98; N, 1.83.
adhesion of activated T cells to collagen-coated plates
as a cell migration model. It was found that 1 could
block this interaction at the same level (52.8% inhibi-
tion) as the inhibition by a monoclonal antibody to
integrin (60.7% inhibition). The SLex or RGDS did not
show any significant inhibition under the same condi-
tion. Since this adhesion pathway is also very important
in the regulation of lymphocyte migration into inflam-
matory tissues, it is strongly suggested that synthetic
glycopeptide 1 will become a nice reagent, “cell adhesive
glycopeptide”, to modulate immune responses through
its inhibitory effect on lymphocyte-endothelial cell
interactions. Further biological, immunological, and
clinical trials for compound 1 and analogues will be
reported in the near future.
In conclusion, we succeeded in a practical chemoen-
zymatic synthesis of a novel class of heterobifunctional
glycopeptide ligand that interacts with both selectins
and integrins. The synthetic nonnatural glycopeptide
showed extremely interesting biological properties as a
potential candidate for a reagent to control cell adhesion.
It should be noted that the concept described here is
widely applicable for the synthesis of a variety of
bioactive glycopeptides from the viewpoint of modula-
tion and/or targeting of functional carbohydrates or
peptides complementarily.
Z-Lys[3-Ca r ba m id op r op yl O-(2,3,4,6-tetr a -O-a cetyl-â-
D-galactopyr an osyl)-(1f4)-O-2-acetam ido-3,6-di-O-acetyl-
2-d eoxy-â-D-glu cop yr a n osid e]-OH (7). To a solution of 6
(530 mg, 0.734 mmol) in dry DMF (7 mL) at 0 °C were added
N-hydroxysuccinimide (HONSu) (85 mg, 0.734 mmol) and
N,N′-dicyclohexylcarbodiimide (DCC) (167 mg, 0.807 mmol).
The mixture was stirred at room temperature for 12 h. After
filtration, the solution was evaporated under reduced pressure
to give the crude active ester. To the solution of Z-Lys-OH (268
mg, 0.954 mmol) in dry DMF (4 mL) and water (1 mL) were
added triethylamine (TEA) (260 µL, 1.84 mmol) and a solution
of the active ester in DMF (5 mL), and the mixture was stirred
at room temperature for 12 h. Next, pH of the mixture was
adjusted by addition of 5% aqueous citric acid to pH 3 and the
reaction mixture was extracted with ethyl acetate (3 × 10 mL).
The organic layer was washed with 5% aqueous citric acid (2
× 20 mL) and brine (2 × 20 mL) and dried over MgSO4. After
filtration with Celite, the solution was evaporated under
reduced pressure and the residue was purified by flash silica
gel chromatography (CHCl3-MeOH, 50:1 to 5:1, v/v) to give 7
(514 mg, 71.2%): δH (400 MHz; CD3OD) 1.37 (m, 2 H, Lys-γ),
1.48 (m, 2 H, Lys-δ), 1.65 (m, 2 H, Lys-â), 1.81 (m, 2 H,
OCH2CH2), 1.89-2.12 (all s, 21 H, 7 × COCH3), 2.20 (t, 2 H,
J 7.17 and 15.11, OCH2CH2CH2), 3.13 (m, 2 H, Lys-ꢀ), 3.51
(m, 2 H, OCH2), 3.65 (m, 1 H, H-5), 3.75-3.85 (m, 3 H, H-4,
H-6′a and H-6′b), 4.02 (dd, 1 H, J 4.89 and 7.94, Lys-R), 4.09-
4.15 (m, 4 H, H-2, H-5′ and H-6a), 4.47-4.52 (m, 1 H, H-6b),
4.50 (d, 1 H, J 8.55, H-1), 4.69 (d, 1 H, J 7.94, H-1′), 4.99 (dd,
1 H, J 7.78 and 10.38, H-3′), 5.06-5.12 (m, 4 H, H-2′, H-3 and
CH2C6H5), 5.34 (d, 1 H, J 3.36, H-4′), 7.27-7.36 (m, 5 H, C6H5).
Exp er im en ta l Section
Gen er a l P r oced u r e. Recombinant rat R-2,3-sialyltrans-
ferase, recombinant human R-1,3-fucosyltranseferase, CMP-
Neu5Ac and GDP-fucose were purchased from Calbiochem Co.
Ltd. Calf intestine alkaline phosphatase and protein A (soluble)
were obtained from Sigma Co. Ltd. Recombinant human E-,
P-, and L-selectins (soluble) were from R&D Systems Europe
Ltd., U.K. Monoclonal antibodies to human integrin â1/
fibronectin receptor and integrin â1 were obtained from
TaKaRa Co. Ltd. 1H and 13C NMR spectra were recorded at
400 and 100.4 MHz, respectively, on a J EOL lambda 400
spectrometer; J values are given in Hz. Preparative HPLC was
performed on a Hitachi HPLC system equipped with an L-6250
intelligent pump, L-7400 UV detector and reversed-phase C18
column, Inertsil ODS-3 (30 × 250 mm), at a flow rate of 30
mL min-1. Analytical HPLC was performed on a Shimadzu
HPLC system equipped with a LC-6A pump, SPV-6AV UV
detector and reversed-phase C18 column, Inertsil ODS-3 (4.6
× 250 mm), at a flow rate of 0.5 mL min-1. Matrix-associated
laser-desorption ionization/time-of-flight/mass spectrometry
(MALDI-TOF-MS) was performed on a Laser mat 2000 mass
spectrometer. FAB-MS spectra were recorded on a J EOL J MS-
SX102A spectrometer. The optical biosensor IAsys cuvette
system with IAsys software and IAsys cuvettes coated with
aminosilane were from Affinity Sensors (Cambridge, U.K.).
Reactions were monitored by thin-layer chromatography (TLC)
on precoated plates of silica gel 60F254 (layer thickness, 0.25
mm; E. Merck, Darmmstadt). Column chromatography was
performed on silica gel (silica gel 60N, 100-210 µm; Kanto
Chemical Co., Inc.) and flash column chromatography was
performed on silica gel (silica gel 60N, 40-100 µm; Kanto
Chemical Co., Inc.).
Anal. Calcd for C44H61N3O22
: C, 53.71; H, 6.25; N, 4.27.
Found: C, 53.87; H, 6.13; N, 4.46.
Z-Lys[3-Ca r ba m id op r op yl O-(2,3,4,6-tetr a -O-a cetyl-â-
D-galactopyr an osyl)-(1f4)-O-2-acetam ido-3,6-di-O-acetyl-
2-d eoxy-â-D-glu cop yr a n osid e]-Gly-OBzl (8). To the solution
of 7 (921 mg, 0.936 mmol) and Gly-OBzl p-tosylate (379 mg,
1.12 mmol) in dry DMF (10 mL) were added diphenyl phos-
phorazidate (DPPA) (240 µL, 1.12 mmol) and TEA (290 µL,
2.06 mmol) and the mixture was stirred at 0 °C for 2 h and at
room temperature for 22 h. The solution was filtrated and
evaporated under reduced pressure. The residue was dissolved
in ethyl acetate (10 mL) and the solution was washed with
5% aqueous citric acid (2 × 10 mL), brine (2 × 10 mL), 5%
aqueous NaHCO3 (2 × 10 mL) and brine (3 × 10 mL) and dried
over MgSO4. After filtration with Celite, the solution was
evaporated under reduced pressure and the residue was
purified by column chromatography (CHCl3-MeOH, 30:1, v/v)
to give 8 (881 mg, 83.2%): δH (400 MHz, CDCl3) 1.10-1.36
(m, 2 H, Lys-γ), 1.38-1.51 (m, 2 H, Lys-δ), 1.58-1.72 (m, 2 H,
OCH2CH2), 1.93-2.14 (all s, 21 H, 7 × COCH3), 2.13-2.30 (m,
2 H, OCH2CH2CH2), 3.21 (m, 2 H, Lys-ꢀ), 3.47 (m, 2 H, OCH2),
3.59 (m, 1 H, H-5), 3.78 (t, 1 H, J 6.84 and 13.67, H-4), 3.88 (t,
1 H, J 8.41 and 17.09, H-5′), 4.00-4.15 (m, 6 H, H-2, H-6a,
H-6′a, H-6′b and Gly-R), 4.24 (m, 1 H, Lys-R), 4.43-4.52 (m, 2
H, H-1 and H-6b), 4.51 (d, 1 H, J 7.81, H-1), 4.97 (dd, 1 H, J
3.41 and 10.25, H-3′), 5.06-516 (m, 6 H, H-2′, H-3 and 2 ×
CH2C6H5), 5.35 (d, 1 H, J 2.93, H-4′), 5.87 (d, 1 H, J 7.81,
NHCOCH3), 6.47 (br, 2 H, Gly-NH and Lys-NH), 7.28-7.54
(m, 10 H, 2 × C6H5). Anal. Calcd for C53H70N4O23: C, 56.28;
H, 6.24; N, 4.95. Found: C, 56.55; H, 6.37; N, 5.25.
3-Ca r boxyp r op yl O-(2,3,4,6-Tetr a -O-a cetyl-â-D-ga la c-
topyr an osyl)-(1f4)-O-2-acetam ido-3,6-di-O-acetyl-2-deoxy-
â-D-glu cop yr a n osid e (6). A solution of potassium perman-
ganate (384 mg, 2.43 mmol) in aqueous acetic acid (7 mL, H2O:
glacial acetic acid, 5:1) was cooled to 0 °C. Pentenyl glycoside
5 (533 mg, 0.76 mmol) in acetic acid (7 mL) was added to this
solution in one portion and the mixture was stirred at 0 °C
for 3 h. Then, the reaction mixture was diluted with ethyl
acetate and to this solution were added solid sodium sulfite
(623 mg, 4.94 mmol) and 1 M aqueous HCl (7 mL). The organic
layer was washed with brine, dried over MgSO4 and concen-
Z-Lys[3-Ca r ba m id op r op yl O-(â-D-ga la ct op yr a n osyl)-
(1f4)-O-2-a ceta m id o-2-d eoxy-â-D-glu cop yr a n osid e]-Gly-
OH (3). To the solution of 8 (500 mg, 0.442 mmol) in methanol

722 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Matsuda et al.
(5 mL) was added sodium methoxide (20 mg). The mixture was
stirred at room temperature for 5 h and 1 M aqueous NaOH
(5 mL) was added to the solution. The mixture was stirred at
room temperature for 5 h, neutralized with ion-exchange resin
(Dowex 50W-X8, H+-form), filtered and evaporated under
reduced pressure to give the product 3 (304 mg, 87.3%): δH
(400 MHz, CD3OD) 1.19-1.23 (m, 2 H, Lys-γ), 1.30-1.42 (m,
2 H, Lys-δ), 1.58 (m, 1 H, Lys-â), 1.70-1.73 (m, 3 H, OCH2CH2
and Lys-â), 1.87 (s, 3 H, COCH3), 2.13 (m, 2 H, OCH2CH2CH2),
3.06 (m, 2 H, Lys-ꢀ), 3.27-3.89 (m, 16 H, H-2, H-2′, H-3, H-3′,
H-4, H-4′, H-5, H-5′, H-6a, H-6b, H-6′a, H-6′b, OCH2 and Gly-
R), 4.03 (dd, 1 H, J 5.38 and 8.79, Lys-R), 4.28 (d, 1 H, J 7.32,
H-1), 4.28 (d, 1 H, J 8.30, H-1′), 4.99 (q, 2 H, J 12.70 and 17.58,
1 H, J 14.34, Gly-R), 3.89 (dd, 1 H, J 3.96 and 9.61, Ser-â),
4.04 (d, 1 H, H-2, J 14.34, Gly-R), 4.34-4.50 (m, 3 H, Arg-R
and OCH2C6H5), 4.70 (t, 1 H, J 3.90 and 7.78, Ser-R), 4.97 (dd,
1 H, J 6.72 and 12.82, Asp-R), 5.04-5.18 (m, 4 H, COCH2C6H5
× 2), 7.20-7.30 (m, 15 H, C6H5 × 3). δC (100 MHz; CDCl3)
24.49 (Arg-γ), 28.29 (CH3), 30.22 (Arg-â), 36.49 (Asp-â), 40.43
(Arg-δ), 42.98 (Gly-R), 49.17 (Asp-R), 53.14 (Ser-R), 53.41 (Arg-
R), 66.96, 67.31 (2 × COCH2C6H5), 69.13 (Ser-â), 73.18
(OCH2C6H5), 80.27 (t-Bu), 127.63, 127.80, 128.04, 128.37,
128.52, 135.12, 135.26, 137.31 (C6H5), 156.01 (Arg-ú), 159.02,
169.18, 169.71, 170.62, 171.43, and 172.89 (CO). Anal. Calcd
for C41H52N8O12‚0.5H2O: C, 57.40; H, 6.23; N, 13.06. Found:
C, 57.30; H, 6.14; N, 12.99.
CH2C6H5), 7.18-7.28 (m,
34H52N4O17: C, 51.77; H, 6.65; N, 7.10. Found: C, 51.81; H,
6.67; N, 7.10.
5 H, C6H5). Anal. Calcd for
Z-Lys[3-Ca r ba m id op r op yl O-(â-D-ga la ct op yr a n osyl)-
(1f4)-O-2-a ceta m id o-2-d eoxy-â-D-glu cop yr a n osid e]-Gly-
Ar g(NgNO2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl (9). Protected
RGDS derivative 4 (115 mg, 0.135 mmol) was dissolved in 2
M HCl/dioxane (2 mL) and the solution was stirred at room
temperature for 3 h. The mixture was evaporated under
reduced pressure and the residue was used for the next step
without further purification. To a solution of H-Gly-Arg(Ng-
NO2)-Gly-Asp(OBzl)-Ser(Bzl)-OBzl prepared from 4 and 3 (117
mg, 0.149 mmol) in DMF (5 mL) were added DPPA (32 µL,
0.149 mmol) and TEA (41.4 µL, 0.297 mmol), and the mixture
was stirred at 0 °C for 2 h and at room temperature for 22 h.
The solution was evaporated under reduced pressure. The
residue was dissolved in ethyl acetate (5 mL) and the solution
was washed with 5% aqueous citric acid 2 × 5 mL), brine (2 ×
5 mL), 5% aqueous NaHCO3 (2 × 5 mL) and brine (3 × 5 mL)
and dried over MgSO4. After filtration with Celite, the solution
was evaporated under reduced pressure and the residue was
purified by column chromatography (30:1) CHCl3-MeOH (v/
v) as an eluant to give 9 (155 mg, 76.5%): δH (400 MHz;
DMSO-d6) 1.30-1.44 (m, 4 H, Lys-γ and Lys-δ), 1.53-1.63 (m,
3 H, Arg-γ and Lys-â), 1.68-1.78 (m, 5 H, Arg-â, Lys-â,
OCH2CH2), 1.87 (s, 3 H, COCH3), 2.12 (t, 2 H, J 7.57 and 15.14,
OCH2CH2), 2.66 (dd, 1 H, J 4.88 and 16.11, Asp-â), 2.83 (dd,
1 H, J 8.30 and 16.11, Asp-â), 3.05 (m, 2 H, Lys-ꢀ), 3.19 (m, 2
H, Arg-δ), 3.30-3.86 (m, 20 H, H-2, 2′, 3, 3′, 4, 4′, 5, 5′, 6a, 6b,
6a′ and 6b′, Gly-R × 2, Ser-â, OCH2), 4.00-4.11(m, 2 H, Arg-R
and Lys-R), 4.27 (d, 1 H, J 7.32, H-1), 4.37 (d, 1 H, J 7.81,
H-1′), 4.48-4.67 (m, 3 H, Ser-R, OCH2C6H5), 4.86 (m, 1 H, Asp-
R), 5.04-5.22 (m, 6 H, COCH2C6H5 × 3), 7.30-7.44 (m, 20 H,
C6H5 × 4). Anal. Calcd for C69H92N12O26: C, 55.05; H, 6.16; N,
11.16. Found: C, 55.13; H, 6.35; N, 11.36.
H -Lys[3-Ca r b a m id op r op yl O-(â-D-ga la ct op yr a n osyl)-
(1f4)-O-2-a ceta m id o-2-d eoxy-â-D-glu cop yr a n osid e]-Gly-
Ar g-Gly-Asp -Ser -OH (2). To a solution of 9 (170 mg, 0.113
mmol) in (2:1) methanol-water (3 mL) was added palladium-
activated carbon (Pd 10%) (10 mg). The mixture was stirred
at room temperature under an atmosphere of H2 gas for 2 days.
After filtration with Celite, the solution was concentrated
under reduced pressure and the residue was subjected to
Sephadex G25 column chromatography using water as an
eluant. Fractions containing the desired product were collected
and combined, concentrated and freeze-dried to give powdery
2 (119 mg, 99.2%): δH (400 MHz; D2O) 1.41-1.50 (m, 2 H,
Lys-γ × 2), 1.53-1.61 (m, 2 H, Lys-δ × 2), 1.62-1.76 (m, 2 H,
Arg-γ), 1.80-1.98 (m, 6 H, Arg-â, Lys-â, OCH2CH2), 2.03 (s, 3
H, COCH3), 2.25-2.37 (m, 2 H, OCH2CH2CH2), 2.84 (dd, 1 H,
J 8.09 and 16.68, Asp-â), 2.97 (dd, 1 H, J 5.34 and 16.70, Asp-
â), 3.13-3.26 (m, 2 H, Lys-ꢀ), 3.55-4.11 (m, 22 H, H-2, 2′, 3,
3′, 4, 4′, 5, 5′, 6a, 6b, 6a′, 6b′, Arg-δ, Gly × 2, Ser-â, OCH2),
4.34-4.45 (m, 2 H, Arg-R and Lys-R), 4.44-4.78 (m, 3 H, H-1,
H-1′ and Ser-R), 4.86 (t, 1 H, J 5.39 and 7.99, Asp-R). Anal.
Calcd for C41H71N11O22‚H2O: C, 45.26; H, 6.76; N, 14.16.
Found: C, 45.55; H, 6.55; N, 14.52.
C
Boc-Ar g(NgNO2)-Gly-Asp (OBzl)-Ser (Bzl)-OBzl (4). To a
solution of Boc-Asp(OBzl) (1.26 g, 3.89 mmol) and Ser(Bzl)-
OBzl hydrochloride (1.50 g, 4.67 mmol) in dry DMF (30 mL)
were added DPPA (1 mL, 4.67 mmol) and TEA (1.3 mL, 9.34
mmol). The mixture was stirred at 0 °C for 2 h and at room
temperature for 22 h. The solution was evaporated under
reduced pressure. The residue was dissolved in ethyl acetate
(30 mL) and the solution was washed with 5% aqueous citric
acid (2 × 30 mL), brine (2 × 30 mL), 5% aqueous NaHCO3 (2
× 30 mL) and brine (3 × 30 mL) and dried over with MgSO4.
After filtration with Celite, the solution was evaporated under
reduced pressure and the residue was purified by column
chromatography with 3:1 hexane-ethyl acetate (v/v) as an
eluant to give Boc-Asp-Ser(Bzl)-OBzl (2.06 g, 89.5%).
N-Protected dipeptide [Boc-Asp-Ser(Bzl)-OBzl, 1.91 g, 3.23
mmol] was dissolved in 2 M HCl/dioxane (50 mL) and the
solution was stirred at room temperature for 3 h. The mixture
was evaporated under reduced pressure and the residual syrup
was employed for the next reaction without further purifica-
tion.
To a solution of this amino component [H-Asp-Ser(Bzl)-
OBzl] and Boc-Gly (622 mg, 3.55 mmol) in dry DMF (30 mL)
were added DPPA (0.84 mL, 3.88 mmol) and TEA (0.99 mL,
7.11 mmol), and the mixture was stirred at 0 °C for 2 h and
at room temperature for 22 h. The solution was evaporated
under reduced pressure. The residue was dissolved in ethyl
acetate (30 mL) and the solution was washed with 5% aqueous
citric acid (2 × 30 mL), brine (2 × 30 mL), 5% aqueous
NaHCO3 (2 × 30 mL) and brine (3 × 30 mL) and dried over
MgSO4. After filtration with Celite, the solution was evapo-
rated under reduced pressure and the residue was purified
by column chromatography with 3:1 hexane-ethyl acetate (v/
v) as an eluant [Boc-Gly-Asp-Ser(Bzl)-OBzl, 1.89 g, 90.2%].
Boc-Gly-Asp-Ser(Bzl)-OBzl (2.18 g, 3.37 mmol) was dissolved
in 2 M HCl/dioxane (50 mL) and the solution was stirred at
room temperature for 3 h. The mixture was evaporated under
reduced pressure and the residual syrup was used without
further purification. To a solution of Boc-Arg(NgNO2) (1.29 g,
4.04 mmol) in dry tetrahydrofuran (THF) (30 mL) were added
TEA (0.61 mL, 4.38 mmol) at room temperature and isobutyl
chloroformate (IBCF) (0.631 mL, 4.85 mmol) at -15 °C, and
the mixture was stirred at -15 °C for 10 min to give a mixed
acid anhydride. The solution of the tripeptide [H-Gly-Asp-Ser-
(Bzl)-OBzl] prepared above and TEA (0.52 mL, 3.71 mmol) in
dioxane was mixed with the solution of a mixed acid anhydride
and the reaction mixture was stirred at 0 °C for 2 h and room
temperature for 22 h. The solution was evaporated under
reduced pressure. The residue was dissolved in ethyl acetate
(30 mL) and the solution was washed with 5% aqueous citric
acid (2 × 30 mL), brine (2 × 30 mL), 5% aqueous NaHCO3 (2
× 30 mL), brine (3 × 30 mL) and dried over MgSO4. After
filtration with Celite, the solution was evaporated under
reduced pressure and the residue was purified by column
chromatography with 40:1 CHCl3-methanol (v/v) as an eluant
to give 4 (2.04 g, 71.3%): δH (400 MHz; CDCl3) 1.90 (s, 9 H,
t-Bu), 1.58-1.68 (m, 4 H, Arg-â, Arg-γ), 2.81 (dd, 1 H, J 4.58
and 16.63, Asp-â), 2.93 (dd, 1 H, J 5.90 and 16.63, Asp-â), 3.18
(m, 2 H, Arg-δ), 3.67 (dd, 1 H, J 3.21 and 9.61, Ser-â), 3.79 (d,
H-Lys[3-Car bam idopr opyl O-(5-acetam ido-3,5-dideoxy-
D-glycer o-r-D-galacto-2-n on u lopyr an osylon ic acid)-(2f3)-
O-(â-D-ga la ctop yr a n osyl)-(1f4)-O-2-a ceta m id o-2-d eoxy-
â-D-glu copyr an oside]-Gly-Ar g-Gly-Asp-Ser -OH (10). Bovine
serum albumin (BAS) (10.0 mg) and manganese(II) chloride
tetrahydrate (1.56 mg, 7.9 µmol) were dissolved in sodium
cacodylate buffer (50 mM; 5.00 mL) and the pH of this solution

Heterobifunctional Ligands
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5 723
was adjusted by 0.25 M aqueous HCl to pH 7.40. To a solution
of 2 (20 mg, 18.7 µmol) in the buffer solution (2 mL) were added
Triton CF-54 (10 µL), cytidine-5′-monophospho-N-acetyl-
neuraminic acid (CMP-NANA) (25 mg, 40.7 µmol), carf intes-
tinal alkaline phosphate (CIAP) (20 units) and R-2,3-sialyl-
transferase (0.3 unit), and the mixture was incubated at 37
°C for 3 days. The solution was directly applied to a Sephadex
G25 column (3.0 cm × 40 cm) and the fractions containing the
desired product were collected, combined, concentrated and
lyophilized to give crude amorphous powder. Next, the crude
product was dissolved in water (1 mL) and the solution was
subjected to preparative HPLC column and eluted with 65:35
acetonitrile-water (v/v) to afford 10 (20.2 mg, 79.5%): δH (400
MHz; D2O) 1.12-1.24 (m, 2 H, Lys-γ × 2), 1.24-1.38 (m, 2 H,
Lys-δ × 2), 1.38-1.48 (m, 3 H, Arg-γ × 2, Lys-â), 1.54-1.70
(m, 6 H, 3′′ax, Arg-â × 2, Lys-â, OCH2CH2), 1.77 (s, 3 H,
COCH3), 1.82 (s, 3 H, COCH3), 1.99-2.05 (m, 2 H, OCH2-
CH2CH2), 2.55 (dd, 1 H, J 4.73 and 12.52, 3′′eq), 2.63 (dd, 1 H,
J 8.09 and 16.63, Asp-â), 2.76 (dd, 1 H, J 5.34 and 16.63, Asp-
â), 3.33-3.92 (m, 29 H, H-2, 2′, 3, 3′, 4, 4′, 4′′, 5, 5′, 5′′, 6a, 6b,
6a′, 6b′, 6′′, 7′′, 8′′, 9′′a and 9′′b, Arg-δ × 2, Gly × 4, Ser-â × 2,
OCH2), 4.06-4.20 (m, 2 H, Arg-R and Lys-R), 4.29-4.37 (m, 3
H, H-1, H-1′ and Ser-R), 4.58-4.78 (HDO, Asp-R); FAB-MS
(pos) 1361.582 (M + H+), calcd 1361.581.
H-Lys{3-Car bam idopr opyl O-(5-acetam ido-3,5-dideoxy-
D-glycer o-r-D-galacto-2-n on u lopyr an osylon ic acid)-(2f3)-
O-(â-D-ga la ct op yr a n osyl)-(1f4)-O-[r-L-fu cop yr a n osyl-
(1f3)]-O-2-a ceta m id o-2-d eoxy-â-D-glu cop yr a n osid e}-Gly-
Ar g-Gly-Asp -Ser -OH (1). To a solution of 10 (15 mg, 10.9
µmol) in HEPES buffer (100 mM, pH 7.5; 1 mL) containing
10 mM manganese(II) chloride tetrahydrate and 1 mM sodium
azide were added guanosine diphospho-fucose (GDP-Fuc) (10.3
mg, 16.4 µmol) and fucosyltransferase V (10 munit) and the
solution was incubated at 37 °C. After 2 days, phosphatase
alkaline (1 unit) was added to the solution and the mixture
was incubated again at 37 °C for 1 day. The solution was
applied to a Sephadex G25 column (3.0 cm × 40 cm) directly,
and fractions containing the desired product were combined,
concentrated and freeze-dried. The crude product was dis-
solved in water (1 mL) and purified by preparative HPLC
column with 65:35 acetonitrile-water (v/v) to afford cell
adhesive glycopeptide 1 (9.1 mg, 55.6%). The purity of the
compound 1 was assumed to be more than 99% by using a
Shimadzu HPLC system equipped with a LC-6A pump, SPV-
6AV UV detector and reversed-phase C18 column, Inertsil
ODS-3 (4.6 × 250 mm), at a flow rate of 0.5 mL min-1 with
65:35 acetonitrile-water (v/v): δH (400 MHz; D2O) 1.00 (d, 3
H, J 6.52, Fuc-6), 1.12-1.25 (m, 2 H, Lys-γ × 2), 1.28-1.38
(m, 2 H, Lys-δ × 2), 1.38-1.48 (m, 2 H, Arg-γ), 1.60-1.73 (m,
7 H, NeuNAc-3ax, Arg-â, Lys-â, OCH2CH2), 1.85 (s, 3 H,
COCH3), 1.86 (s, 3 H, COCH3), 2.25-2.12 (m, 2 H, OCH2-
CH2CH2), 2.60 (dd, 1 H, J 4.73 and 12.52, NeuNAc-3eq), 2.67
(dd, 1 H, J 7.92 and 16.76, Asp-â), 2.79 (dd, 1 H, J 5.36 and
16.76, Asp-â), 2.94-3.05 (m, 2 H, Lys-ꢀ), 3.34-3.92 (m, 33 H,
GlcNAc-2, 3, 4, 5, 5, 6a, 6b, Gal-2, 3, 4, 5, 6a, 6b, NueNAc-4,
5, 6, 7, 8, 9a, 9b, Fuc-2, 3, 4, 5, Arg-δ, Gly × 2, Ser-â, OCH2),
4.13 (dd, 1 H, J 6.26 and 7.68, Lys-R), 4.24 (t, 1 H, J 4.58 and
5.34, Arg-R), 4.33-4.41 (m, 3 H, GlcNAc-1, Gal-1 and Ser-R),
4.63-4.71 (m, 1 H, Asp-R), 4.93 (d, 1 H, J 3.97, Fuc-R); δC (100
MHz; D2O) 15.98 (Fuc-6), 22.34 (Lys-γ), 22.74 and 22.93
(NHAc), 24.93 (Arg-γ), 26.10 (Lys-δ), 28.69 (Arg-â), 32.02 (Lys-
â), 32.03 (OCH2CH2), 32.89 (OCH2CH2CH2), 36.35 (Asp-â),
40.50 (Lys-ꢀ), 41.20 (Arg-δ), 42.88 and 43.15 (Gly-R × 2), 50.72
(Asp-R), 53.27 (Ser-R), 99.32 (Fuc-1), 100.37 (NeuNAc-1),
101.70 (GlcNAc-1), 102.34 (Gal-1); FAB-MS (pos) 1507.637 (M
+ H+), calcd 1507.638.
cycles of minimization calculation 79 conformations were
generated as conformers with different energy minima.
P r ep a r a tion of Selectin -Im m obilized Op tica l Biosen -
sor Cu vettes for SP R Mea su r em en ts. Immobilization of
recombinant P-, E-, and L-selectins onto activated aminosilane
sufaces was performed according to the manufacturer’s speci-
fications.18 Briefly, after equilibration and obtaining a stable
baseline with phosphate buffer solution (PBS; 10 mM, pH 7.7),
the optical biosensor cuvette coated with aminosilane was
activated three times with 0.2 mL of bis(sulfosuccinimidyl)-
suberate (BS3) in PBS (0.56 mg/mL) to activate the cuvette
surface for 10 min. The activation solution was removed by
washing with PBS, and 0.2 mL of E-, P- or L-selectin solution
(100 µg/cm-3 in PBS) was added for 15 min. The remaining
active sites were blocked with 0.2 mL of BSA solution (2 mg/
cm-3 in PBS) for 15 min. Finally, selectin-immobilized cevettes
were washed three times with PBS (10 mM, pH 7.2) containing
1 mM CaCl2 (PBS/C) to establish a stable baseline.
Bin d in g Assa y of In ter a ction s betw een Selectin s a n d
Glycoliga n d s. All binding experiments in the IAsys instru-
ment were carried out using 10 mM PBS/C at 25 °C. Binding
of ligand onto the cuvette was monitored for 20 min using
0.200 mL of sample solution. First, 0.19 mL of PBS was added
to the cuvette to obtain a stable baseline. Next, 0.01 mL of
ligand solution was added to this cuvette. The association was
ended by aspiration of the sample and replacing it with the
same volume of PBS/C. Monitoring was continued for a couple
of minutes. For displacement studies, the same protocol was
used by using a PBS containing 0.05% TRITON X-100 (PBS/
T) instead of PBS/C.
Im m obiliza tion of Mon oclon a l An tibod y to Hu m a n
In tegr in â1 on th e Biosen sor Su r fa ce (m Ab-p r otein A
cu vette). Prior to the immobilization of monoclonal antibody,
optical biosensor cuvette was coated with protein A in order
to achieve successful orientation of Fab domain of antibody.
Protein A was immobilized according to the same manner as
that of immobilization of selectins described above. Aminosi-
lane-containing cuvette was washed three times with 0.2 mL
of BS3 in PBS (10 mM, pH 7.7) (0.56 mg/mL) to activate the
surface. Next, 0.2 mL of protein A solution (100 µg/mL) was
added to the activated cuvette and incuvated for 15 min. The
cuvette was washed three times with PBS and followed by
blocking with BSA solution (2 mg/mL). Protein A-immobilized
cuvette was reactivated with BS3 in the same way and followed
by treating with 0.190 mL of PBS (pH 7.4) and 0.010 mL of
mouse monoclonal antibody to human integrin â1/fibronectin
receptor (2.0 mg/mL in 10 mM PBS containing 1.0% BSA and
0.1% NaN3, pH 7.4). After blocking with BSA solution (2 mg/
mL), cuvette was washed 10 times with PBS/T. The binding
activity of this cuvette with integrin â1 was evaluated by using
a solution of integrin â1 (3.2 µg/mL) as a standard.
In h ibitor y Effect of Glycop ep tid e 1 or RGDS on th e
In ter a ction of Hu m a n In tegr in â1 w ith Its Mon oclon a l
An tibod y. All measurements were carried out in 10 mM PBS
containing 1 mM CaCl2 (PBS/C, pH 7.2). First, 0.18 mL of
PBS/C was added to mAb-protein A cuvette and baseline was
stabilized. Then, to this cuvette were added 0.01 mL of ligand
solution and 0.01 mL of integrin â1 solution (3.2 µg/mL). The
decrease of the binding activity of integrin â1 to the cuvette
were determined and used for evaluating the inhibition effect
by ligands. Measurements were performed in the concentration
range of ligands from 0 to 10 mM, respectively.
In h ibitor y Effect on th e Ad h esion of Activa ted T Cells
w it h Colla gen -Coa t ed P la t e. (A) P r ep a r a t ion of Th 2
Cells. CD4+CD45RB+ native T cells obtained from DO11.10
OVA323-339-specific T cell receptor transgenic mice were stimu-
lated with 10 µg/mL OVA323-339 peptide in the presence of 1
ng/mL IL-4, 50 µg/mL anti-IFN-γ mAb, 50 µg/mL anti-IL-12
mAbs and 20 U/mL IL-2. Cells were cultured with RPMI1640
supplemented with 10% fetal calf serum (FCS).
(B) Cell Ad h esion Assa y. Th2 cells were suspended at 5
× 106 cells/mL and 1 mL of this solution was cultured using
biocoat culture ware, mouse collagen IV 35 mm dish (Col-
laborative Biomedical Products, MA). To facilitate cell adhe-
Con for m ation al An alysis. Random conformational search-
ing method of the SYBYL/advanced computation module
developed by Chang et al.13 was used for conformational
prediction of the glycopeptide 1. One cycle of conformatinal
searching was composed of random adjustment of selected
torsions in this molecule and minimization of the structure
according to the procedure reported previously.18 After 1000

724 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 5
Matsuda et al.
selectin inhibitors. J . Am. Chem. Soc. 1997, 119, 3161-3162.
(c) Sprengard, U.; Schudok, M.; Schmidt, W.; Kretzshmar, G.;
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sion, 20 ng/mL phorbol myristate acetate (PMA) was added
to the culture system. After 30 min, nonadherent cells were
harvested and counted for the estimation of the ratio of
adherent cells/total cells cultured. This assay was carried out
in the presence of synthetic glycopeptide or anti-integrin mAb.
Ack n ow led gm en t. This work was supported by a
grant from NEDO for “Research and Development on
Glycocluster Controlling Biomolecules” and by a grant-
in-aid for scientific research from the Ministry of
Education, Science, and Culture (Grants 11480157 and
12555256). We also thank Ms. Akiko Maeda, Ms. Hiroko
Matsumoto, and Ms. Seiko Oka of the Center of Instru-
mental Analysis, Hokkaido University, for measuring
elemental analyses and mass spectroscopy.
(8) Nishimura, S.-I.; Lee, Y. C. Synthetic glycopolymers: New tools
for glycobiology. In Polysaccharides, Structural Diversity and
Functional Versatility; Dumitriu, S., Ed.; Marcel Dekker: New
York, 1998; pp 523-537.
(9) Zheng, M.; Fang, H.; Tsuruoka, T.; Tsuji, T.; Sasaki, T.; Hako-
mori, S.-I. Regulatory role of GM3 ganglioside in alpha 5 beta 1
integrin receptor for fibronectin-mediated adhesion of FUA169
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(10) Sako, D.; Comess, K. M.; Barone, K. M.; Camphausen, R. T.;
Cumming, D. A.; Shaw, G. D. A sulfated peptide segment at the
amino terminus of PSGL-1 is critical for P-selectin binding. Cell
1995, 83, 323-331.
(11) (a) Seitz, O.; Wong, C.-H. Chemoenzymatic solution- and solid-
phase synthesis of O-glycopeptides of the mucin domain of
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O-Sialyl-Lewis-X peptides. J . Am. Chem. Soc. 1997, 119, 8766-
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(14) We first designed and synthesized some glycoside intermediates
having an ethyleneglycol-based linker as hydrophilic substrates
for the glycosyltransferases. However, these derivatives were
found to be poor substrates for further enzymatic manipulation
studies by the preliminary assay. The relationship between the
structures of the linkers and the enzymatic accessibility as well
as the biological activity is under investigation, and the results
will be reported in due course.
(15) Nishimura, S.-I.; Matsuoka, K.; Furuike, T.; Ishii, S.; Kurita,
K.; Nishimura, K. M. Synthetic glycoconjugates. 2. n-pentenyl
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Su p p or tin g In for m a tion Ava ila ble: Spectral data for all
new compounds and details for biological evaluations. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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