Supramolecular control of oligosaccharide-protein interactions: Switchable and tunable ligands for concanavalin A based on β-cyclodextrin

Article abstract of DOI:10.1002/anie.200601123

The ins and outs of binding: Supramolecular control of carbohydrate-protein interactions has been achieved through the design of β-cyclodextrin (βCD) based conjugates whose conformation is dependent on a reversible self-inclusion process. The accessibilit

Full text of DOI:10.1002/anie.200601123

Angewandte
Chemie
Noncovalent Interactions
mechanisms involved in the glycoside cluster effect.[5,6]
However, none of the reported examples can fully mimic
the switching between the “on” and “off” states and the
regulation of the binding intensity after an external stimulus.
We have now developed a molecular system based on b-
cyclodextrin (bCD)[7] that can reproduce this behavior by
virtue of allosteric supramolecular interactions with effector/
antagonist-like molecules.
DOI: 10.1002/anie.200601123
Supramolecular Control of Oligosaccharide–
Protein Interactions: Switchable and Tunable
Ligands for Concanavalin A Based on b-
Cyclodextrin**
Switching of carbohydrate–protein interactions has pre-
viously been achieved through chemical modification of the
protein with photoisomerizable groups that induce structural
perturbations at the protein binding site. In seminal work,
Willner prepared chemically modified lectins with reversible
photochromic properties and demonstrated the reversible
photostimulated “on/off” association and dissociation of
monosaccharides.[8] The present study aims at implementing
the “reversible structure-dependent binding” strategy by
designing conformationally switchable glycoligands that are
sensitive to chemical inputs.
Our molecular design is based on the observation that the
ability of bCD-centered glycoligands to bind lectins is
strongly influenced by the distance between the recognition
motif and the bCD scaffold.[9] We conceived that this distance
could be modulated by inserting a segment that was able to
undergo intramolecular inclusion into the bCD cavity in a
reversible manner. Previous results showed that some amino
acids grafted on the bCD core form self-inclusion complexes
in aqueous solution.[10] Interestingly, this process can be
shifted in the presence of a competing guest for bCDs bearing
a tyrosinyl residue,[11] thus suggesting that this structural motif
could act as a distance-regulating element.
To prove the above concept we have focused on con-
canavalin A (Con A), a mannose-specific lectin commonly
used to probe protein–carbohydrate interactions,[12] and the
branched trisaccharide 3,6-di-O-(a-d-mannopyranosyl)-a-d-
mannopyaranose (Man-tri) as the protein receptor and the
sugar ligand, respectively. Con A is known to possess an
extended binding site for this trisaccharide, and results in a
binding affinity (logK = 5.4) much higher than that encoun-
tered for trivalent or even higher-valency mannopyranosi-
des.[1b]
Nicolas Smiljanic, Vincent Moreau, Duplex Yockot,
Juan M. Benito, JosØ M. García Fernµndez,* and
Florence Djedaïni-Pilard*
Interactions between carbohydrate-binding proteins (lectins)
and cell-surface carbohydrate ligands have been shown to be
critical for a wide variety of intercellular recognition process-
es, including cell proliferation, signaling, and recognition.[1]
The interaction of a single carbohydrate ligand and a protein
molecule is, however, usually too weakto induce a useful
biological response. Biological systems commonly engage in
multivalent carbohydrate–protein interactions to enhance the
affinity. Remarkably, the advantage gained by multiple copies
of a ligand is generally more than that predicted for the sum of
the individual interactions.[2] This observation was first
reported by Lee and Lee and is known as the glycoside
cluster effect.[3] Since then it has been speculated that nature
uses carbohydrate surface density as an “on/off” switch to
regulate biological events involving carbohydrate–protein
interactions. However, recent results have indicated that the
situation is far more complicated. Architectural and orienta-
tional factors as well as secondary interactions in a hetero-
geneous environment may play a decisive role in the
regulation and fine tuning of the binding strength and
selectivity of the interaction between a lectin and its putative
ligand.[4]
A number of multivalent systems have been reported in
which the loading of the saccharide binding epitopes or their
overall geometry were varied to get information on the
[*] Dr. J. M. Benito, Dr. J. M. García Fernµndez
Instituto de Investigaciones Químicas
CSIC – Universidad de Sevilla
According to our previous work, we attached a succiny-
lamido substituent to the bCD prior to the incorporation of
the tyrosine segment or the mannosyl ligand.[13] It was
anticipated that this spacer would provide enough flexibility
to allow both the self-inclusion process and glycoligand–lectin
recognition events in an extended conformation. To confirm
this point, compounds 1 and 2, which differed in the absence
or presence of the aromatic amino acid, were prepared.
Additionally, conjugate 3, which possesses two Man-tri units,
was synthesized to checkhow polyvalency could affect both
the lectin binding and the self-inclusion phenomena in such a
system (Scheme 1).
The synthetic strategy to access compounds 1–3 follows a
convergent approach and makes use of standard peptide-
coupling techniques. The synthesis of the per-O-esterified
Man-tri glycosylamine 4 was achieved by using a simulta-
neous “double-glycosylation” of 2,4-di-O-benzoyl-a-d-man-
nopyranosyl azide with 2,3,4,6-tetra-O-acetyl-a-d-mannopyr-
AmØrico Vespucio 49, Isla de la Cartuja, 41092 Sevilla (Spain)
Fax: (+34)954-460-565
E-mail: jogarcia@iiq.csic.es
Dr. N. Smiljanic, Dr. V. Moreau, Dr. D. Yockot, Prof. F. Djedaïni-Pilard
Laboratoire des Glucides UMR6219
UniversitØ Picardie Jules Verne
33, rue St. Leu, 80039 Amiens (France)
Fax: (+33)322-827-562
E-mail: florence.pilard@u-picardie.fr
[**] This work was supported by the Conseil RØgional de Picardie under
the scientific IBFBio program and by the Spanish Ministerio de
Educación y Ciencia (contract number BQU2003-00937). D.Y. is
grateful to the Minist›re Gabonais des Finances et des Bourses et
Stages for a doctoral fellowship. N.S. is grateful to the Conseil
RØgional de Picardie for a doctoral fellowship.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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sponding hemiacylated adduct, the succinyl-tethered conju-
gate 1. An analogous coupling-deprotection sequence involv-
ing 4 and the tyrosinyl-armed precursor 7[11] provided the
elongated analogue 2. Replacing the trisaccharide precursor
by the amine-armed glycodendron 5, accessible by double
peptide coupling of 4 with N-Fmoc-protected glutamic acid
and subsequent removal of the carbamate protecting group,
afforded the target (Man-tri)2 conjugate 3 (Scheme 2).
Comparative 1H NMR and T-ROESYexperiments on 1–3
in D2O at different concentrations confirmed the formation of
intramolecular self-inclusion complexes with the tyrosine-
bearing conjugates 2 and 3, with the aromatic ring placed
inside the bCD cavity. These complexes were disrupted in
[D6]DMSO solution, thus indicating the reversible character
of the self-inclusion process. Addition of adamantane-1-
Scheme 1. Structure of the Man-tri–bCD conjugates evaluated.
anosyl trichloroacetimidate as described pre-
viously.[11,14] Reduction of the anomeric azido
group to a b-amine was significantly improved
by using the “catalytic transfer hydrogena-
tion” method developed by Anwer and Spa-
tola[15] (see the Supporting Information).
Amide coupling of the known monofunction-
alized bCD derivative 6[13] and glycosylamine
4 afforded, after deprotection of the corre-
Scheme 2. Synthesis of the bCD conjugates 1–3. a) DIC, HOBt, iPr2NEt, DMF, 96 h then
DMF-MeOH, MeONa (1m in MeOH), 7 days, (23%); b) Ref. [8]; c) DIC, HOBt, iPr2NEt,
DMF, 72 h, then piperidine, DMF, 90 min (76%); d) DIC, HOBt, iPr2NEt, DMF, 72 h then
MeOH, NH3 (7n in MeOH), 10 days (40%). DIC=diisopropylcarbodiimide, HOBt=1-
hydroxy-1H-benzotriazole, Fmoc=9-fluorenylmethoxycarbonyl, Bz=benzyl.
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carboxylate (AC), known to be an excellent guest for bCD
(association constant (Kass) 3.9 ” 104 mÀ1),[16] to solutions of 2
or 3 in D2O also caused departure of the tyrosine residue from
the cavity, with formation of the 1:1 intermolecular inclusion
complexes 2:AC or 3:AC (Kass = 1.6 ” 104 mÀ1), as deduced
from NMR titration experiments.[17]
inclusion complex. Accordingly, a gradual increase in activity,
up to a factor of three (IC50 1.0 mm), was observed in the
presence of increasing proportions of AC (Figure 2A). From
this data, a binding isotherm consistent with formation of a 1:1
For evaluation of Con A binding, the enzyme-linked
lectin assay (ELLA), a variation on the enzyme-linked
immunosorbent assay (ELISA), was implemented.[2b] In
agreement with our initial hypothesis, 2 was virtually unrec-
ognized by the lectin at concentrations up to 1 mm, a result
which is in starkcontrast with data for 1 that lacks the tyrosine
segment (IC50 21 mm). Interestingly, a dramatic increase in
Con A binding affinity was observed for 2 (IC50 22 mm) in the
presence of a threefold molar excess of AC, while that of 1
remained unaffected (Figure 1A). This result strongly sup-
ports the scenario depicted in Figure 1B (left-to-right). In the
“off” state the epitope is not accessible to the protein binding
site because of its conformation, while in the “on” state, the
oligosaccharide is fully exposed to interact with the lectin.
Switching between these two modes can be controlled by an
allosteric supramolecular process at the CD cavity involving
AC as a competing guest molecule.
Evaluation of the (Man-tri)2 conjugate 3 by ELLA
indicated that it had an affinity towards the protein receptor
(IC50 = 3.2 mm) that was more than sixfold higher than that of
1 or the 2:AC complex. The Man-tri unit located at the a-
substituent of the glutamic acid segment in 3 is probably now
fully accessible for binding to Con A, the second one being
only partially available to contribute to the glycoside cluster
effect because of the energetic cost of disrupting the self-
Figure 2. ELLA analysis for 3 in the presence of various concentrations
of AC (A) and binding isotherm for the 1:1 3:AC complex (B).
host/guest ligand was derived. Nonlinear regression analysis
(Figure 2B) provided a Kass value of 1.4 ” 104 mÀ1 for the 3:AC
complex, which is about 2.5-fold lower than the value
reported for the corresponding complex with bCD, probably
because of the presence of
the tyrosinyl self-inclusion
element.
It is worth mentioning
that the presence of a bulky
horseradish
peroxidase
(HRP) label on Con A, to
produce a spectrophotomet-
ric readout in the ELLA, is
supposed to prevent cross-
linking processes. These
macromolecules likely inter-
act with a single binding site
per lectin, with proximity/
statistical affinity enhance-
ment mechanisms operating.
The above result strongly
suggests that both Man-tri
groups become fully disposi-
ble for protein recruitment
after AC stimulation, even
under
(microcluster
such
conditions
effect[18,19]),
which result in an increased
biological response, by a bio-
mimetic mechanism.
We hypothesized that the
lectin affinity should be
Figure 1. ELLA analysis (% inhibition of the Con A/yeast mannan binding) for 1, 2, and 2 + AC (A) and
schematic representation of the observed “off/on” switching of carbohydrate–lectin binding through allosteric
supramolecular interactions (B).
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Communications
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receptor CT3, which exhibits a perfect size match and
[20]
symmetry complementarity with AC (Kass = 4.6 ” 104 mÀ1
)
was considered a good candidate for this purpose. Addition of
1.5 equivalents of CT3 per mol of effector molecule to AC-
activated samples of 2 or 3 led the system backto the initial
state—switch “off”—in agreement with the reversible char-
acter of the equilibria involved both at the lectin binding site
and in the CD cavity.[21]
In summary, our results indicate that reversible tuning and
switching of the binding affinity in a model carbohydrate–
lectin system is possible through logical design. This workhas
included the first examples of allosteric activation/deactiva-
tion of binding and of the multivalent effect. This switchable,
environment-sensitive, supramolecularly mediated carbohy-
drate–protein recognition offers exciting possibilities to
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ecules. Indeed, specific drug delivery using CD conjugates is
hampered by the blocking of the protein receptors by
unoccupied carriers, which might be avoided by designing
switchable systems for which the drug will act as the effector
(input “on”). We aim to investigate this possibility, as well as
the further supramolecular modulation of multivalent inter-
actions through variations in chemical modification in this
and other systems.
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Received: March 22, 2006
Revised: June 7, 2006
Published online: July 20, 2006
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Keywords: carbohydrates · cyclodextrins · lectins ·
noncovalent interactions · receptors
.
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[21] Control ELLA experiments demonstrated that CT3 does not
interfere in the binding of the mannosyl ligands 1–3 with Con A
in the absence of AC. Binding of 1 to Con A was also unaffected
by the presence of AC and AC:CT3 in up to threefold excesses.
These results strongly support that CT3 triggers the “off” state
by a mechanism involving exclusively AC trapping. Conse-
quently, further addition of AC to a CT3-switched-off sample of
2:AC restored the “on” state in a new cycle, thus confirming the
reversibility of both input processes. NMR experiments addi-
tionally supported the absence of supramolecular interactions
between CT3 and the bCD-centred glycoligands 1–3.
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