First successful molecular design of an artificial Lewis oligosaccharide binding system utilizing positive homotropic allosterism

Article abstract of DOI:10.1021/ja010806e

We have designed phenylboronic acid group appended Ce(IV) bis(porphyrinate) double decker 1 and meso - meso linked porphyrin 2, useful for the allosteric binding of biologically important saccharides, Lewis oligosaccharides. Compound 1 binds Lewis oligosa

Full text of DOI:10.1021/ja010806e

J. Am. Chem. Soc. 2001, 123, 10239-10244
10239
First Successful Molecular Design of an Artificial Lewis
Oligosaccharide Binding System Utilizing Positive Homotropic
Allosterism
Atsushi Sugasaki, Kazunori Sugiyasu, Masato Ikeda, Masayuki Takeuchi, and
Seiji Shinkai*
Contribution from the Department of Chemistry and Biochemistry, Graduate School of Engineering,
Kyushu UniVersity, Fukuoka 812-8581, Japan
ReceiVed March 28, 2001. ReVised Manuscript ReceiVed August 14, 2001
Abstract: We have designed phenylboronic acid group appended Ce(IV) bis(porphyrinate) double decker 1
and meso-meso linked porphyrin 2, useful for the allosteric binding of biologically important saccharides,
Lewis oligosaccharides. Compound 1 binds Lewis oligosaccharides in aqueous media because of the boronic
acid-diol interaction, but the complexation event can occur only above the critical concentrations because of
the sigmoidal [oligosaccharide] versus [complex] isotherm. Compound 1 has a sufficiently high affinity with
Lewis oligosaccharides (K ) 105-106 M-2) with Hill coefficients n of 1.8-2.0, and LewisX series and Lewisa
series give opposite, symmetrical CD spectra. This is the first example of efficient binding of Lewis
oligosaccharides to the artificial receptor, which has become possible by positive homotropic allosterism.
Introduction
approach to these oligosaccharides to develop inhibitors for
selectins.
It is known that oligosaccharides play crucial roles in
molecular recognition events in biological systems. Particularly
noteworthy are Lewis oligosaccharides, such as LewisX (LeX)
and Lewisa (Lea), which are involved in the adhesion of
leukocytes and neutrophils to vesicular endothelical cells during
normal and pathogenic inflammatory responses.1,2 Appearance
of Lewis oligosaccharides on the cell surface and in the blood
serum above the critical concentrations is often a sign associated
with tumor progression and malignancy. For example, overex-
pression of sialyl LewisX (sLeX) containing mucins has been
found in the sera of gastrointestinal, pancreatic, and breast cancer
patients.3 Thus, it is undoubted that sensitive detection of Lewis
oligosaccharides is one of the most urgent subjects to be
developed in the chemical therapeutic field. So far, however,
the investigation has been limited to the approach from the
biochemical field;4,5 the typical examples are (i) the structural
analysis of Lewis oligosaccharides/E-, P-, and L-selectin
complexes as a model of cell adhesion and (ii) the synthetic
Our research purpose is, in contrast to these preceding
biochemical approaches, to develop rationally designed artificial
synthetic receptors for these oligosaccharides, which will lead
to generation of small molecular antagonists and design of
specific sensory systems. It is undoubted that the development
of such artificial receptors is very useful for the sensitive and
convenient detection of these oligosaccharide antigens in the
blood serum.6
(1) (a) Kobata, A. In Biology of Carbohydrates; Ginsburg, V., Robins,
P. W., Eds.; John Wiley and Sons: New York, 1984; Vol. 2, p 87. (b)
Feizi, T. Nature 1985, 314, 53. (c) Hakomori, S. AdV. Cancer Res. 1989,
52, 257. (d) Schauer, R. AdV. Carbohydr. Chem. Biochem. 1982, 40, 131.
(f) Looms, L. M.; Vemura, K.; Childs, R. A.; Paulson, J. C.; Rogers, G.
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Feizi, T. Nature 1984, 307, 560.
(2) (a) Lorant, D. E.; Topham, M. K.; Whatley, R. E.; McEver, R. P.;
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46, 2619.
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Smith, M. E. B.; Haung, R.-F.; Wong, C.-H. J. Am. Chem. Soc. 2000, 122,
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O.; Wong, C.-H. J. Am. Chem. Soc. 1997, 119, 8152.
Then, what is the potential strategy for the molecular design?
There are two basic requirements: that is, (i) to sensitively detect
Lewis oligosaccharides, the receptors must have the large
(5) (a) Ichikawa, Y.; Lin, Y.-C.; Dumas, D. P.; Shen, G.-J.; Garcia-
Junceda, E.; Williams, M. A.; Bayer, R.; Ketcham, C.; Walker, L. E.;
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T.; Inoue, Y.; Kondo, H. J. Med. Chem. 1998, 41, 2302. (e) DeFrees, S.
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10.1021/ja010806e CCC: $20.00 © 2001 American Chemical Society
Published on Web 09/29/2001

10240 J. Am. Chem. Soc., Vol. 123, No. 42, 2001
Sugasaki et al.
association constants and (ii) because they are expressed even
in normal tissues and sera,7 the receptor’s function must change
from a tense (T) state to a rest (R) state only above the critical
concentrations as alllosteric proteins seen in nature.
aqueous media to form 1:2 1/Lewis oligosaccharide complexes
with Hill coefficients 1.8-2.0. To the best of our knowledge,
It thus occurred to us that the concept of “positive homotropic
allosterism”8,9 might be successfully applied to the molecular
design of a desired Lewis oligosaccharide binding system,
because (i) the binding isotherm is characterized by a sigmoid-
shaped curve with a steep threshold region and (ii) the
association constants and binding signal are amplified by the
cooperative action in a positive homotropic allosteric binding
process.9h,i Provided that positive homotropic allosterism oper-
ates, as expected, in the Lewis oligosaccharide binding process,
it would become possible to detect these essential oligosaccha-
rides selectively and sensitively only above the critical concen-
trations.
Recently, we designed phenylboronic acid group appended
cerium(IV) bis(porphyrinate) double decker 110 and meso-meso
linked porphyrin dimer 211 as artificial oligosaccharide recep-
tors.12 In these studies, we have demonstrated that (i) compounds
1 and 2 can bind malto- or laminari-oligosaccharides coopera-
tively and effectively in aqueous solution, (ii) the positive
homotropic allosterism is indispensable for highly efficient
oligosaccharide binding, and (iii) the binding signal is amplified
according to a sigmoidal isotherm through positive homotropic
allosterism. These findings clearly show that these compounds
would act as candidates for Lewis oligosaccharide binding
receptors, which are expected to work even in aqueous solution.
In this contribution, we report that 1 can bind Lewis
oligosaccharides because of positive homotropic allosterism in
this is the first totally synthetic artificial receptor which can
touch Lewis oligosaccharides in aqueous media sensitiVely only
aboVe the critical concentrations. The rational amalgamation
of artificial saccharide binding moieties, “boronic acids,”13 and
the concept of “allosterism” now paves the way to construct a
biologically important oligosaccharide sensing system useful in
aqueous solution.
(6) Monoclonal antibody has been used to determine the concentrations
of sLex antigen and sialyl Tn antigen by immunoradiometric assay and
radioimmunoassay, respectively, see: Springer, G. F. Immunol. Ser. 1990,
53, 587.
(7) Quantitative and qualitative characterization of cancer-associated
serum glycoprotein antigens, see: (a) Kannagi, R.; Fukushi, Y.; Tachikawa,
T.; Noda, A.; Shin, S.; Shigeta, K.; Hiraiwa, N.; Fukuda, Y.; Inamoto, T.;
Hakomori, S.; Imura, T. Cancer Res. 1986, 46, 2619. (b) Kannagi, R.;
Fukushi, Y.; Tachikawa, T.; Noda, A.; Shin, S.; Kitahara, A.; Itai, S.; Arii,
S.; Shigeta, K.; Hiraiwa, N.; Fukuda, Y.; Hakomori, S.; Imura, T. Cancer
Res. 1988, 48, 3856.
(8) (a) Blanc, S.; Yakirevitch, P.; Leize, E.; Meyer, M.; Libman, J.; Van
Dorsselaer, A.; Albrecht-Gray, A. M.; Shanzer, A. J. Am. Chem. Soc. 1997,
119, 4934. (b) Kobayashi, K.; Asakawa, Y.; Kato, Y.; Aoyama, Y. J. Am.
Chem. Soc. 1992, 114, 10307. (c) Rebek, J., Jr. Acc. Chem. Res. 1984, 17,
258. (d) Rebek, J., Jr.; Costello, T.; Marshall, L.; Wattley, R.; Gadwood,
R. C.; Onan, K. J. Am. Chem. Soc. 1985, 107, 7481. (e) Ebmeyer, E.; Rebek,
J., Jr. Angew. Chem., Int. Ed. Engl. 1990, 29, 1148. (f) Glass, T. E. J. Am.
Chem. Soc. 2000, 122, 4522. (g) Takeuchi, M.; Shioya, T.; Swager, T. M.
Angew. Chem., Int. Ed. Engl. 2001, 40, 3372.
(9) (a) Takeuchi, M.; Imada, T.; Shinkai, S. Angew. Chem., Int. Ed. Engl.
1998, 37, 2096. (b) Sugasaki, A.; Ikeda, M.; Takeuchi, M.; Robertson, A.;
Shinkai, S. J. Chem. Soc., Perkin Trans. 1, 1999, 3259. (c) Ikeda, M.;
Tanida, T.; Takeuchi, M.; Shinkai, S. Org. Lett. 2000, 2, 1803. (d) Ikeda,
M.; Takeuchi, M.; Sugasaki, A.; Robertson, A.; Imada, T.; Shinkai, S.
Supramol. Chem. 2000, 12, 321. (e) Sugasaki, A.; Ikeda, M.; Koumoto,
K.; Takeuchi, M.; Shinkai, S. Tetrahedron 2000, 56, 4717. (f) Yamamoto,
M.; Sugasaki, A.; Ikeda, M.; Takeuchi, M.; Frimat, K.; James, T. D.;
Shinkai, S. Chem. Lett. 2001, 520. (g) Robertson, A.; Ikeda, M.; Takeuchi,
M.; Shinkai, S. Bull. Chem. Soc. Jpn. 2001, 74, 883. (h) Shinkai, S.; Ikeda,
M.; Sugasaki, A.; Takeuchi, M. Acc. Chem. Res. 2001, 34, 494. (i) Takeuchi,
M.; Ikeda, M.; Sugasaki, A.; Shinkai, S. Acc. Chem. Res., in press.
(10) Sugasaki, A.; Ikeda, M.; Takeuchi, M.; Shinkai, S. Angew. Chem.,
Int. Ed. 2000, 39, 3839. The phenyl boronic acid group appended Ce(IV)
bis(porphyrinate) double decker 1 can bind maltooligosaccharides effectively
through positive homotropic allosterism to form only the 1:2 complexes.
In this system, the binding of the first oligosaccharide to a pair of boronic
acid groups, although very weak, can suppress the rotation of the two
porphyrin planes; as a result, the subsequent binding of the oligosaccharide
to the residual pair of aligned boronic acid groups can occur cooperatively.
(11) Ikeda, M.; Shinkai, S.; Osuka, A. Chem. Commun. 2000, 1047.
(12) (a) Kral, V.; Rusin, O.; Schmidtchen, F. P. Org. Lett. 2001, 3, 873.
(b) Nagai, Y.; Kobayashi, K.; Toi, H.; Aoyama, Y. Bull. Chem. Soc. Jpn.
1993, 66, 2965.
Results and Discussion
Molecular Design. Diboronic acid derivatives, which can
react with four of the five OH groups of saccharide to form
intramolecular 1:1 complexes, show a different stability order,
which is related to the specific spatial position of two boronic
acid groups.13 This implies that one can recognize a specific
saccharide guest by appropriate manipulation of two boronic
acids in a same host molecule. The present research aim is to
extend this concept to the selective binding of biologically
important Lewis oligosaccharides. This idea has been tested with
a few diboronic acid systems bearing a “long” and “rigid”
spacer: for example, diphenyl-3,3′-diboronic acid, stilbene-3,3′-
diboronic acid,14 and cis-5,15-bis[2-(dihydroxyboronyl)phenyl]-
10,20-diphenylporphirin15 show some selectivity for certain
disaccharides, but the selectivity and the affinity observed so
far are not so high. To improve the affinity and the selectivity
toward oligosaccharides, one should look for a new conceptual
design scheme by which one might be able to finely tune the
distance between two boronic acid groups. From recent research
on artificial cooperative recognition systems, it has been
suggested that positive homotropic allosterism can be utilized
as a new concept to achieve both high guest selectivity and
high guest affinity which cannot be attained by the conventional
1:1-type guest binding.9 The scaffolds that show positive
(13) Recent review see: (a) James, T. D.; Sandanayake, K. R. A. S.;
Shinkai, S. Angew. Chem., Int. Ed. Engl. 1996, 35, 1910. (b) James, T. D.;
Linnane, P.; Shinkai, S. Chem. Commun. 1996, 281. (c) Shinkai, S.;
Takeuchi, M. Trends Anal. Chem. 1996, 15, 418. (d) Norrild, J. C.; Eggert,
H. J. Am. Chem. Soc. 1995, 117, 1479.
(14) Sandanayake, K. R. A. S.; Nakashima, K.; Shinkai, S. J. Chem.
Soc., Chem. Commun. 1994, 1621.
(15) Kijima, H.; Takeuchi, M.; Shinkai, S. Chem. Lett. 1998, 781.

Artificial Lewis Oligosaccharide Binding System
J. Am. Chem. Soc., Vol. 123, No. 42, 2001 10241
oligosaccharides did not yield any perceptible CD bands. In
general, when a saccharide guest is bound to a boronic acid
group in a host molecule, the resultant complex becomes
optically CD-active.13 It has been established, however, that the
complex can yield a strongly CD-active species only when the
saccharide is bound intramolecularly to two boronic acid groups
to form a macrocyclic structure.17 The results indicate that two
diol moieties in saccharides are bound to two boronic acid
groups in 1 to bridge two porphyrin planes chirally. It is seen
from Figure 1 that the CD sign (positive exciton-coupling bands)
obtained from the 1/LeX family system is opposite to that
(negative exciton-coupling bands) obtained from the 1/Lea
family system. Judging from the fact that the boronic acid group
can interact only with the cis-1,2-diol or 1,3-diol moiety in
saccharides,13 it is undoubted that phenylboronic acid groups
in 1 interact with the 4,6-diol group of the galactose moiety
and the 3,4-diol group of the fucose moiety in the LeX (Galâ1,4-
[FucR1,3]GlcNAc) or Lea (Galâ1,3[FucR1,4]GlcNAc) back-
bone. In fact, when 1 (0.50 mM) and LeX (2.0 mM) were
allowed to react in CD3OD, the 6-methyl protons adjacent to
the 3,4-diol group of the fucose moiety shifted from 1.17 to
1.71 ppm (∆δ ) 0.54 ppm) owing to the deshielding effect of
the complexed phenylboronic acid group.18 Meanwhile, we
found that compound 2 acts as an excellent maltotetraose
(tetrasaccharide) receptor.11 The distance between two boronic
acids in 2 is estimated to be about 15.8 Å, which is too long to
bridge between the galactose moiety and the fucose moiety in
Lewis oligosaccharides. Furthermore, the rotation of porphyrin
rings around the bridging C-C bond, by which the distance
between two boronic acid groups becomes variable, is relatively
limited compared with that of 1. These should be the possible
reasons why compound 2 cannot bind any Lewis oligosccha-
rides.
Figure 1. CD spectra of 1 (1.00 × 10-4 Μ) in the presence of Lewis
oligosaccharides (2.10 × 10-3 M) at 25 °C.
homotropic allosterism are mostly with dynamic motion and
are skillfully combined with the molecular recognition systems
so that the subsequent guest binding can occur more favorably
than the first guest binding. We thus amalgamate two strategies,
diboronic acid receptor and positive homotoropic allosterism,
in designing compounds 1 and 2 that are expected to work as
a robust oligosaccharide binding system. Compounds 1 and 2
are a phenylboronic acid appended bis[porphyrinato]cerium-
(IV) double decker porphyrin and a meso-meso linked por-
phyrin, respectively. In these compounds, two porphyrins can
rotate (or oscillate) relative to each other like two wheels with
the central metal ion or bridging C-C bond acting as an axle.16
One can thus expect a cooperative allosteric binding mode to
form 1:2 host-guest complexes.
To obtain further insights into the 1‚LeX and 1‚Lea complex
structures, the most stable conformations were evaluated by
computational methods (Discover 3/Insight II).19 In the initial
structures, the boronic acid complexation sites are the 4,6-diol
group of the galactose moiety and the 3,4-diol group of the
fucose moiety. The resultant structures are illustrated in Figure
2. It is seen from Figure 2 that the 1‚LeX complex has a right-
handed helical twist, whereas the 1‚Lea complex has a left-
handed helical twist. These results clearly show that the
structural difference in C-3 and C-4 of the N-acetylglucosamine
residue between LeX and Lea is the origin of the opposite CD
signs observed for the two families in Figure 1.
1
Compounds 1 and 2 were identified by IR and H NMR
spectral evidence and elemental analyses. These products were
used for further spectral measurements without removal of 1,3-
propandiol groups. 1H NMR spectral measurements have
established that the protecting groups are readily eliminated in
aqueous media. In fact, addition of 1,3-propanediol (∼10-3 M)
scarcely affected the CD intensity versus [saccharide] plot.
Hence, one can use this compound without deprotection
treatment.
Lewis Oligosaccharide Binding Studies. Binding affinities
of 1 and 2 toward Lewis oligosaccharides were evaluated by
UV-vis and CD spectroscopic methods. Addition of Lewis
oligosaccharide to a solution of 1 (1.00 × 10-5 M) or 2 (1.00
× 10-5 M) adjusted with a mixture of 50 mM carbonate buffer/
MeOH to pH 10.5 resulted in virtually no change in the
absorption and fluorescent spectra. In contrast, exciton-coupling-
type CD bands, which have a spectral pattern inherent to each
oligosaccharide structures, were clearly observed upon addition
of oligosaccharides only when we used compound 1 (Figure
1), whereas CD spectral measurements for 2 with Lewis
The CD spectral changes induced by Lewis oligosaccharides
were examined more in detail. The CD spectra measured as a
function of the saccharide concentration provided an isosbestic
point, indicating that the reaction consists of only two species
(17) (a) Takeuchi, M.; Mizuno, T.; Shinmori, H.; Nakashima, M.;
Shinkai, S. Tetrahedron 1996, 52, 1195. (b) Takeuchi, M.; Imada, T.;
Shinkai, S. J. Am. Chem. Soc. 1996, 118, 10658. (c) Takeuchi, M.; Kijima,
H.; Shinkai, S. Bull. Chem. Soc. Jpn. 1997, 70, 699. (d) James, T. D.;
Shinkai, S. J. Chem. Soc., Chem. Commun. 1995, 1483.
1
(18) The H NMR spectrum (600 MHz, -40 °C, CD3OD) of the 1‚Lea
complex was so broadened in aqueous solution and at room temperature
that the signal assignment was nearly impossible.
(16) It is known that the rate of the porphyrin ring rotation in the Ce‚
bis(porphyrinate) double decker is comparable with or slower than the NMR
time scale. However, the allosteric behavior is basically observable for the
present “static” equilibrium system as long as porphyrin rings are able to
rotate. See references: (a) Takeuchi, M.; Imada, T.; Ikeda, M.; Shinkai, S.
Tetrahedron Lett. 1998, 39, 7897. (b) Ikeda, M.; Takeuchi, M.; Shinkai,
S.; Tani, F.; Naruta, Y. Bull. Chem. Soc. Jpn. 2001, 74, 739. (c) Tashiro,
K.; Konishi, K.; Aida, T. Angew. Chem., Int. Ed. Engl. 1997, 36, 856. (d)
Tashiro, K.; Fujiwara, K.; Konishi, K.; Aida, T. Chem. Commun. 1998,
1121. (e) Tashiro, K.; Konishi, K.; Aida, T. J. Am. Chem. Soc. 2000, 122,
7921.
(19) Conformations with low potential energy encountered during 100
ps MD simulation at 500 K were selected. The system was minimized using
the conjugate gradient and Newton-Raphson methods until convergence
was attained for a gradient of 0.01 kcal mol-1 Å-1. The force field used in
this study was the ESFF. In our calculation system, C2 symmetrical
structures were selected as the initial structures. The resultant energy-
minimized structures feature D2 symmetry, and two fucose rings exist at
opposite sides of the porphyrin plane. When the calculation was started
from CS symmetrical structures, the resultant structures again feature D2
symmetry, although two fucose rings existing at opposite sides of the
porphyrin plane are a little closer to the pophyrin plane.

10242 J. Am. Chem. Soc., Vol. 123, No. 42, 2001
Sugasaki et al.
Figure 2. Energy-minimized structures of the 1‚LeX (left) and 1‚Lea (right) complexes.
Figure 3. CD spectral changes of 1 (1.00 × 10-4 M) with an increase
Figure 4. Job plot for 1‚sLeX complex formation.
in Lewis oligosaccharide concentration ([sLex] ) 0.00 to ∼2.10 × 10-3
M) at 25 °C in a mixture of water (pH 10.5 with 50 mM carbonate
buffer)/MeOH ) 1:1 (v/v).
Hill coefficients n of 1.8-2.0 for Lewis oligosaccharides are
consistent with a highly cooperative binding mechanism forming
the 1:2 complexes, (iii) the saturated CDmax values increase with
an increase in the K values in each Lewis oligosaccharide family,
and (iv) selectivity among Lewis saccharides is not so high. In
Scatchard plots,22 the positive and negative allosterisms are
expressed by the upward and downward curvatures, respectively.
The maximum values (ym) are correlated with Hill coefficients
(n) with n ) 1/(1 - ym).22 Scatchard plots of the Lewis
oligosaccharides always result in the upward curvature (see
Figure S2), which indicates the operation of the positive
allosterism in the binding to 1.
We next investigated the effect of pH on the CD intensity of
1/oligosaccharide complexes. First, the CD intensity of
1‚(maltose)2 complex was measured as a function of pH ([1] )
5.00 × 10-6 M, [maltose] ) 3.00 × 10-3 Μ), because we have
already shown that 1 possesses almost the same affinity for both
maltose and sLex. The CD intensity of 1‚(maltose)2 complex at
446 nm decreased with lowering medium pH and changed from
28 at pH 10.5 to 12 at pH 9.5 and 1.5 at pH 7.5 (Figure 6).
in a single equilibrium (Figures 3 and S1 in the Supporting
Information). The stoichiometry of the CD-active complexes
was further confirmed by a Job plot.20 The plot of the CD
intensity at 447 nm against [1]/([1] + [saccharide]) has a
maximum at 0.33. A typical example for the 1/sLex system is
shown in Figure 4. This finding supports the view that the
complex consists of one host (1) and two Lewis oligosaccha-
rides. Plots of the CD intensity at 447 nm versus [saccharide]
are shown in Figure 5. Compound 1 shows a sigmoidal binding
isotherm for Lewis oligosaccharides, indicating that the binding
of two equivalents of Lewis oligosaccharides to 1 occurs
“cooperatively”. This cooperative saccharides-binding profile
can be analyzed with the Hill equation: log(y/(1 - y)) ) n
log[saccharide] + log K, where n, K, and y are the Hill
coefficient, the association constant, and the extent of com-
plexation, respectively, and y ) K/([saccharide]-n + K)21 (Table
1). It is seen from Table 1 that (i) 1 has a sufficiently high
affinity with Lewis oligosaccharides (K ) 105-106 M-2), (ii)
(20) Job, A. Ann. Chim. 1928, 9, 113.
(21) Conners, K. A. Binding Constants; John Wiley: New York, 1987.
(22) (a) Permutter-Hyman, B. Acc. Chem. Res. 1986, 19, 90. (b) Pfeil,
A.; Lehn, J.-M. Chem. Commun. 1992, 838.

Artificial Lewis Oligosaccharide Binding System
J. Am. Chem. Soc., Vol. 123, No. 42, 2001 10243
diol and 1,3-diol in a saccharide. It is known that Lewis
oligosaccharides on the cell or protein surface are mostly
connected via 1-OH in GalNac,4,5 which is not included in the
binding event with 1. Thus, cell- or protein-linked Lewis
oligosaccharides should be also bound to 1. Second, electrostatic
interaction should not affect the binding event in aqueous
solution. In fact, we previously tested the allosteric binding of
tartarate anion to a cationic double decker porphyrin.9f The
significant binding due to electrostatic interaction was observed
only in less polar solvents (THF, methanol, etc.) but not at all
in aqueous solution.9f Hence, the binding event should not be
affected by charges present on the cell or protein surface. Third,
we tested whether coexisting D-glucose affects the binding
event: D-glucose was selected as a competing guest because
its concentration in the blood serum is considerably high
(physiological glucose level: 0.3 ∼ 1.0 mM) and the complex
gives the opposite CD sign.9e The CD spectra of the 1‚(sLex)2
complex ([1] ) 5.00 × 10-6 M, [sLex] ) 2.00 × 10-3 M) were
only weakly affected by added D-glucose: for example, the CD
intensity at 446 nm decreased 33% at [D-glucose] ) 1.00 ×
10-3 M and 50% at [D-glucose] ) 2.00 × 10-3 M.
Figure 5. Plots of CD intensity of 1 (1.00 × 10-4 Μ) versus the
concentration of oligosaccharide guests. The solid lines represent the
theoretical curves for the formation of the [1‚saccharide] complexes.
Conclusion
In summary, we have demonstrated that 1 can effectively bind
Lewis oligosaccharides in aqueous media with the aid of positive
homotropic allosterism and discriminate between LeX and Lea
structures as the opposite CD signs. As far as we are aware,
this is the first totally synthetic artificial receptor that binds
Lewis oligosaccharides in aqueous solution. Our findings
obtained here clearly show that the higher affinity of 1 toward
Lewis oligosaccharides stems from the action of positive
homotropic allosterism: that is, even though the complexation
of Lewis oligosaccharides is usually very difficult, the presence
of the cooperative action of two pairs of boronic acid groups
has enabled us to detect them by a CD spectroscopic method.
It is expected, therefore, that the utilization of positive homo-
tropic allosterism in Lewis oligosaccharide recognition repre-
sents a new paradigm in biotechnology for selective detection
of tumor-associated antigens only above the critical concentra-
tions and promises to provide a general strategy for increasing
the affinity of oligosaccharide receptors with their specific
substrates.
Table 1. Hill Coefficient (n), Association Constant (K/M-2) and
Saturated CD Intensity (CD446
)
Lewis oligosaccharide
n
K/M-2
CD446
LewisX
2.0 ( 0.13
2.0 ( 0.07
2.0 ( 0.19
1.8 ( 0.05
2.0 ( 0.08
2.0 ( 0.15
9.0 × 105
1.1 × 106
1.8 × 106
4.2 × 105
5.7 × 105
1.3 × 106
3.1
6.6
8.1
-4.6
-5.1
-7.3
sulpho LewisX
sialyl LewisX
Lewisa
sulpho Lewisa
sialyl Lewisa
Experimental Section
General. All starting materials and solvents were purchased form
Tokyo Kasei Organic Chemicals or Wako Organic Chemicals and used
as supplied. Lewis oligosaccharides were purchased from Funakoshi
and were used without further purification. 1H NMR spectra were
recorded either on a Bruker AC 250 (250 MHz) or Bruker DRX 600
(600 MHz) spectrometer. Chemical shifts are reported in ppm downfield
from tetramethylsilane as an internal standard. Mass spectral data were
obtained using a Perseptive Voyager RP MALDI TOF mass spectrom-
eter. UV-vis and CD spectra were recorded with a Shimadzu UV-
2500 PC and a JASCO J-720WI spectrophotometer, respectively.
Synthesis of 1. Compound 1 was synthesized by treatment of bis-
[5,15-bis(4-methoxyphenyl)-10,20-di(4-pyridyl)porphyrinato]cerium-
(IV)9d with 2-(4-bromomethylphenyl)-1,3-dioxa-2-borinan24 in DMF.
Detailed synthetic procedure and characterization of 1 were reported
previously.10
Figure 6. Effect of pH on the CD intensity of 1/oligosaccharide
complex; [1] ) 5.00 × 10-6 M, [maltose] ) 3.00 × 10-3 Μ (b), [sLex]
) 2.00 × 10-3 Μ (O).
This decrease is probably due to the pKa value of the phenyl-
boronic acid groups in 1, which is supposed to be ∼9.23 The
similar pH dependence was also observed for 1‚(sLex)2 complex
([sLex] ) 2.00 × 10-3 Μ).
In detecting Lewis saccharides under physiological conditions,
one must consider that a lot of chiral compounds would coexist
and may interfere with Lewis oligosaccharide binding to 1. We
believe, however, that 1 is useful also under physiological
conditions because of the following reasons. First, as mentioned
above, the boronic acid group can interact with only cis-1,2-
Synthesis of 2. 4-(1,3-dioxaborinan-2-yl)benzaldehyde (115 mg,
0.605 mmol), 2,2′-dipyrromethane (80 mg, 0.55 mmol), and two drops
of trifluoroacetic acid were dissolved in 60 mL of dry dichloromethane,
and the mixture was stirred for 15 h at room temperature. To this
reaction mixture was added chloranil (560 mg, 0.605 mmol). After
(23) Arimori, S.; Murakami, H.; Takeuchi, M.; Shinkai, S. Chem.
Commun. 1995, 961.
(24) Shinmori, H.; Takeuchi, M.; Shinkai, S. Tetrahedron 1995, 51, 1893.

10244 J. Am. Chem. Soc., Vol. 123, No. 42, 2001
Sugasaki et al.
evaporation of the solvent, the red residue was chromatographed (silica
gel, CHCl3 to CHCl3-methanol ) 100:1) to produce 50 mg of 5,15-
bis(4-[1,3,2]-dioxaborinan-2-yl-phenyl)porphyrin. Yield 30%. Mp >
the organic layer was separated. After evaporation of the solvent, the
red residue was chromatographed (silica gel, CHCl3-methanol) to
produce 2 mg of 2 in 4% isolated yield. Mp (dec) > 300 °C; 1H NMR
(250 MHz, CDCl3-CD3OD, δ ) ppm, J ) Hz): 2.15 (m, 8H), 4.26
(m, 16H), 8.05-8.06 (m, 12H), 8.22 (d, 8H), 8.59 (m, 4H), 9.06 (m,
4H), 9.41 (m, 4H), 10.27 (s, 2H). MALDI TOF-MS (CHCA): calcd.
(found) for [M+H]+: 1386.35 (1386.92).
1
300 °C; H NMR (250 MHz, CDCl3-CD3OD, δ ) ppm, J ) Hz):
2.25 (m, 4H), 4.37 (m, 8H), 8.21 (d, J ) 7.8, 4H), 8.28 (d, J ) 7.8,
4H), 9.08 (d, J ) 4.6, 4H), 9.40 (d, J ) 4.6, 4H), 10.3 (s, 2H). MALDI
TOF-MS (CHCA): calcd. (found) for [M+H]+: 631.80 (631.26). Anal.
Calcd for C38H32B2N4O4‚0.5CHCl3: C, 67.01; H, 4.75; N, 8.12%.
Found: C, 67.45; H, 4.61; N, 8.89%.
CD Spectroscopy. To a solution of 1.00 × 10-4 M of 1 or 2 in
H2O (pH 10.5, 50 mM carbonate buffer)/methanol ) 1:1 (v/v) was
added a stock solution of saccharide prepared in H2O. The CD spectra
from 250 to 500 nm were recorded with JASCO J-720WI at 15 different
concentrations of guest saccharides. The measurement temperature was
25 °C, and the cell length is 1 mm.
To a solution of 5,15-bis(4-[1,3,2]-dioxaborinan-2-yl-phenyl)por-
phyrin (40 mg, 0.063 mmol) obtained above in 10 mL of CHCl3 was
added 5 mL methanol solution of zinc acetate (680 mg, large excess),
and the mixture was stirred for 24 h at room temperature. After
evaporation of the solvent, the red residue was chromatographed (silica
gel, CHCl3) to yield 39 mg of 5,15-bis(4-dihydroxyboronophenyl)-
pH Dependence on CD Intensity. To a solution of 5.00 × 10-6
M
of 1 in H2O/methanol ) 1:1 (v/v) (pH 9.0-10.5 adjusted with 10 mM
carbonate buffer and pH 7.5-8.5 with 10 mM phosphate buffer) was
added a stock solution of saccharide prepared in H2O. The CD spectra
from 250 to 500 nm were recorded with JASCO J-720WI. The
measurement temperature was 25 °C, and the cell length is 1 cm.
1
porphyrinatozinc(II). Yield 86%. Mp (dec) > 300 °C; H NMR (250
MHz, DMSO-d6, δ ) ppm, J ) Hz): 8.22 (d, 4H), 8.24 (d, 4H), 8.38
(br s, 4H), 8.95 (d, J ) 4.2, 4H), 9.51 (d, J ) 4.2, 4H), 10.38 (s, 2H).
MALDI TOF-MS (CHCA): calcd. (found) for [M+H]+: 614.81
(614.55). Anal. Calcd for C32H22B2N4O4‚2H2O: C, 59.16; H, 4.03; N,
8.62%. Found: C, 60.68; H, 4.29; N, 8.08%.
Competing Binding Experiment. To a mixture of 5.00 × 10-6
M
of 1 and 2.00 × 10-3 M of sLex in H2O/methanol ) 1:1 (v/v) (pH 9.5
adjusted with 10 mM carbonate) was added a stock solution of
D-glucose prepared in H2O. The CD spectra from 250 to 500 nm were
recorded with JASCO J-720WI. The measurement temperature was 25
°C, and the cell length is 1 cm.
5,15-Bis(4-dihydroxyboronophenyl)porphyrinatozinc(II) (170 mg,
0.217 mmol) and propane-1,3-diol (35 µL, 2.2 eq) were dissolved in
benzene, and the mixture was refluxed overnight. The residue was
washed with water, and the organic layer was dried over anhydrous
Na2SO4. After evaporation of the solvent, 5,15-bis(4-[1,3,2]-dioxabori-
nan-2-yl-phenyl)porphyrinatozinc(II) was obtained as a purple solid in
98% isolated yield. This compound was used for the next reaction
without further purification.
Binding Isotherm Analysis. In the analysis of the binding isotherm
by Hill plot and Scatchard plot, we have evaluated the concentration
of unbound saccharide, [saccharide], by assuming that 100% 1:2
1/oligosaccharide complex is formed when the CD intensity is saturated.
To a solution of 5,15-bis(4-[1,3,2]-dioxaborinan-2-yl-phenyl)por-
phyrinatozinc(II) (50 mg, 0.072 mmol) in 50 mL of CHCl3 was added
5 mL of acetonitrile solution of AgPF6 (9 mg, 0.5 equiv), and the
mixture was stirred for 5 h at room temperature in the dark.25 During
the reaction, the color of the reaction mixture was changed from purple-
red to orange-red. The reaction was quenched by 5 mL of water, and
Acknowledgment. This work was supported by a Grant-
in-Aid for COE Research, “Design and Control of Advanced
Molecular Assembly Systems”, from the Ministry of Education,
Science and Culture, Japan (Grant #08CE2005).
Supporting Information Available: CD data and Scatchard
plots (PDF). This material is available free of charge via the
Internet at http://pubs.acs.org.
(25) (a) Nakano, A.; Osuka, A.; Yamazaki, I.; Nishimura, Y. Angew.
Chem., Int. Ed. Engl. 1998, 37, 3023. (b) Ogawa, T.; Nishimoto, Y.;
Yoshida, N.; Ono, N.; Osuka, A. Angew. Chem., Int. Ed. Engl. 1999, 38,
176 and references therein.
JA010806E