Mannose-decorated cyclodextrin vesicles: The interplay of multivalency and surface density in lectin-carbohydrate recognition

Article abstract of DOI:10.3762/bjoc.8.175

Cyclodextrin vesicles are versatile models for biological cell membranes since they provide a bilayer membrane that can easily be modified by host-guest interactions with functional guest molecules. In this article, we investigate the multivalent interact

Full text of DOI:10.3762/bjoc.8.175

Mannose-decorated cyclodextrin vesicles:
The interplay of multivalency and surface density
in lectin–carbohydrate recognition
Ulrike Kauscher and Bart Jan Ravoo*
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 1543–1551.
Organic Chemistry Institute, Westfälische Wilhelms-Universität
Münster, Correnstraße 40, 48149 Münster, Germany
doi:10.3762/bjoc.8.175
Received: 25 May 2012
Email:
Accepted: 17 August 2012
Published: 17 September 2012
Bart Jan Ravoo* - b.j.ravoo@uni-muenster.de
* Corresponding author
This article is part of the Thematic Series "Superstructures with
cyclodextrins: Chemistry and applications".
Keywords:
carbohydrates; cyclodextrins; lectins; molecular recognition;
multivalency; vesicles
Guest Editor: H. Ritter
© 2012 Kauscher and Ravoo; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Cyclodextrin vesicles are versatile models for biological cell membranes since they provide a bilayer membrane that can easily be
modified by host–guest interactions with functional guest molecules. In this article, we investigate the multivalent interaction of the
lectin concanavalin A (ConA) with cyclodextrin vesicles decorated with mannose–adamantane conjugates with one, two or three
adamantane units as well as one or two mannose units. The carbohydrate–lectin interaction in this artificial, self-assembled glyco-
calyx was monitored in an agglutination assay by the increase of optical density at 400 nm. It was found that there is a close rela-
tion between the carbohydrate density at the cyclodextrin vesicle surface and the multivalent interaction with ConA, and the most
efficient interaction (i.e., fastest agglutination at lowest concentration) was observed for mannose–adamantane conjugates, in which
both the cyclodextrin–adamantane and the lectin–mannose interaction is inherently multivalent.
Introduction
The surface modification of materials with carbohydrates has various numbers and arrangements of polysaccharides and is
attracted much attention due to the fact that such materials can found in eukaryotic as well as in prokaryotic cells. A well-
be compared to and compatible with the cell surface [1]. The known example of the pivotal role of oligosaccharides on cell
“glycocalyx” is a dense layer on the surface of the cell, which surfaces is the fact that human blood types (A, B, AB and 0) are
serves as a responsive interface with its environment and also solely determined by minor changes in the composition of the
serves as a natural protective shield. The glycocalyx consists of erythrocyte glycocalyx. Additionally, many biological mecha-
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nisms are mediated by multivalent recognition of carbohydrates. Additionally, all guest molecules possess α-mannose units,
For example, lectins are proteins that bind to specific carbohy- which bind to lectins such as concanavalin A (ConA, Figure 1).
drates on the cell surface and activate biochemical responses Guest 1 contains a single mannose and a single adamantane
[2]. In this way, protein–carbohydrate interactions regulate cell unit. Guest 2 and guest 3 contain a single mannose and two or
division, protein synthesis, the immune system, and the adhe- three adamantane units, respectively. Guest 4 contains two
sion of cells. A well-known lectin is concanavalin A (ConA), mannose as well as two adamantane units. The synthesis of 1–4
which can be readily obtained from jack-beans. It has four iden- is described in Supporting Information File 1. The analytical
tical binding sides and binds α-mannose, α-glucose and their data for 1–4 are fully consistent with their molecular structure.
derivatives. Because of the importance of carbohydrates and The synthesis of amphiphilic β-cyclodextrin 5 has been reported
their multivalent recognition by lectins in physiological previously [30]. Unilamellar vesicles with a diameter of
processes, they are also considered a promising tool for the 100–150 nm are obtained by extrusion [27,30].
development of drug-delivery systems [3].
To investigate the ability of adamantane functions to bind into
Synthetic bilayer vesicles are a versatile model for biological the cavity of cyclodextrins, the synthesized guest molecules
cell membranes, and there are a substantial number of reports were investigated regarding their 1:1 complexation behavior
on synthetic glycolipids that mimic the glycocalyx [4-23]. towards β-cyclodextrin. Isothermal titration calorimetry (ITC)
Multivalent guest interaction with the surface of the vesicles has was carried out with β-cyclodextrin and each of the synthesized
become a useful system to investigate recognition, adhesion and guest molecules 1–4. The concentrations were chosen to
fusion of biological cell membranes [24-26]. In this context, provide one cyclodextrin cavity for each adamantane unit and
amphiphilic cyclodextrins are a promising platform due to their are displayed in Table 1. The effective adamantane concentra-
ability to form stable bilayer vesicles that can be functionalized tion describes the concentration of adamantane units. A guest
by self-assembly [27]. To this end, cyclodextrins are modified with two adamantane units (2 or 4) results in an effective
with long alkyl chains (“tails”) and short oligo(ethylene glycol) adamantane concentration that is twice the concentration of the
head groups. These macrocyclic amphiphiles form unilamellar divalent guest molecule. For guest 3, the effective adamantane
bilayer vesicles in aqueous solution upon hydration of a concentration is three times the concentration of the trivalent
thin film cast by evaporation from organic solution and extru- guest molecule. The results of these titrations can be seen in
sion through a 0.1 μm polycarbonate membrane [27]. The cavi- Table 1 and Figure 2.
ties of each cyclodextrin are available to form inclusion
complexes with hydrophobic guest molecules. Adamantane is The thermodynamic parameters of guests 1–4 are characteristic
known to be an excellent guest for β-cyclodextrin cavities of the formation of a 1:1 inclusion complex of each adaman-
(Ka = (2–3) × 104 M−1). We were able to recently demonstrate tane unit with β-cyclodextrin. Based on the effective adaman-
the interaction of monovalent bifunctional guest molecules, tane concentration, the stoichiometry, the binding constants, and
containing a maltose or lactose unit and an adamantane unit, the thermodynamic parameters (negative ΔH and positive ΔS)
with cyclodextrin vesicles, and their ability to agglutinate with are very similar for each guest, with the exception of guest 3.
lectins [28]. We also showed that agglutination requires a criti- This implies that in guests 1, 2 and 4, each and every adaman-
cal density of carbohydrate ligand on the cyclodextrin vesicle tane unit is able to complex a β-cyclodextrin host molecule
surface [29]. In this work we investigate the influence of multi- independent of the other adamantanes on the guest molecule. A
valent recognition by guest molecules with an increasing significant deviation of this behavior is observed only for guest
number of adamantane and mannose units. It is our hypothesis 3, which carries three adamantane units. In this case, the stoi-
that more adamantane units in the guest molecule lead to higher chiometry appears to be less than 1:1, the binding constant is
affinity for the cyclodextrin vesicles due to multivalent inter- somewhat lower, and the thermodynamic parameters are
action at the vesicle surface. In addition, we increased the different (notably, ΔS is negative). This observation can be
number of mannose units in the guest molecule, assuming that a explained by steric hindrance in the trivalent host–guest com-
high density of carbohydrate is essential for multivalent lectin plex: apparently, three β-cyclodextrins are too large to interact
binding at the vesicle surface.
efficiently with each of the three adamantane units. The steric
bulk of the cyclodextrins hinders the deep intrusion of adaman-
tane functions into the cavity (Figure 3).
Results and Discussion
Four different guest molecules were synthesized to study the
effect of multivalency, each with a distinct number of adaman- The results of the titration calorimetry show that each adaman-
tane or mannose functions. The adamantane unit can bind into tane on guests 1–4 is able to bind to β-cyclodextrin. Accord-
the cavity of cyclodextrins embedded at the vesicle surface. ingly, the guest molecules are expected to form inclusion
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Figure 1: Mannose–adamantane conjugates 1–4 and amphiphilic cyclodextrin 5.
Table 1: Thermodynamic parameters measured with isothermal titration calorimetry.
ΔH
kJ/mol
ΔG
kJ/mol
ΔS
J/(K·mol)
Ka
M−1
compound
host
[guest]a mM
[host] mM
1
2b
3b
4
β-CD
β-CD
β-CD
β-CD
5.00
0.38
0.29
20.0
0.41
5.0
−18.93
−16.96
−28.78
−12.62
−27.71
−25.98
−23.47
−24.59
29.47
30.28
−17.82
40.19
7.2 × 104
3.6 × 104
1.3 × 104
2.1 × 104
3.6
2.4
aEffective adamantane concentration. bDue to the low solubility of guest molecules the titration was carried out in reverse mode (host added to guest).
complexes at the surface of vesicles of amphiphilic cyclodex- In view of the high affinity binding of the (multivalent) guest
trin 5. More importantly, guest molecules 2–4 are expected to molecules 1–4 and assuming that these polar molecules are not
form multivalent host–guest complexes with a much higher able to permeate through the membrane, it is our hypothesis that
effective binding constant than the monovalent binding constant even at submillimolar concentrations of guest 1–4 and host 5,
reported in Table 1. According to a quantitative treatment of most guest molecules are confined to the outer vesicle surface.
multivalent host–guest interactions at surfaces, it may be Moreover, since in all experiments the effective guest concen-
expected that for a 1:1 monovalent interaction with a binding tration is only half of the host concentration, it can be assumed
constant of ≈104 M−1, a divalent interaction can have an that most cyclodextrin cavities at the vesicle surface are occu-
apparent binding constant of ≈107 M−1 and a trivalent inter- pied by an adamantane unit. As a consequence, the density of
action can lead to an apparent binding constant of ≈1010 M−1 mannose molecules on the surface of the vesicles is expected to
[31].
be relatively high, resulting in the formation of an artificial
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Figure 2: Integrated peak area (left) and raw titration curves (right) for the ITC measurements of 1–4 with β-CD. The concentrations used are listed in
Table 1.
Figure 3: Schematic presentation of the binding between β-cyclodextrin and (A) monovalent guest 1, (B) divalent guest 2, (C) trivalent guest 3, and
(D) divalent guest 4.
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glycocalyx by self-assembly. Therefore, the addition of the
lectin ConA should lead to agglutination of the vesicles due to
the specific interaction of mannose and ConA at the vesicle
surface. Optical density measurements were carried out at a
wavelength of 400 nm to investigate the agglutination behavior
of each guest molecule.
In the case of monovalent guest 1, a typical time-dependent
agglutination was found due to the specific interaction of ConA
with mannose at the cyclodextrin vesicle surface (Figure 4). In
these experiments, the concentration of cyclodextrin 5 was
0.2 mM and the concentration of guest 1 was 0.1 mM. It was
also found that agglutination can be gradually suppressed by the
substitution of guest 1 by an “inert” guest (adamantyl tetraethyl-
eneglycol, 6) that binds cyclodextrin but not ConA (Figure 4). It
is evident that as the surface density of mannose is decreased in
the glycocalyx, the effective separation of mannose ligands
increases, and hence the multivalent interaction of mannose and
ConA is suppressed. In fact, a critical threshold is observed
around a mannose surface density of 50%: if the mannose
density is reduced even further, the average distance of the
mannose units on the vesicle surface is larger than the binding
site separation of ConA, and hence multivalent interaction is no
longer observed. The average distance between two cyclodex-
trins at the vesicle surface is approximately 2.2 nm [30]. The
distance between two mannose molecules is expected to be the
same when using a maximum surface coverage of the cyclodex-
trin host surface with guest 1. The binding site separation for
ConA is 3.6 to 4.9 nm [21,22], which roughly corresponds to
the average spacing of mannose at 50% surface coverage of
Figure 4: (A) Agglutination of β-cyclodextrin vesicles in the presence
of monovalent guest 1 and ConA. The surface coverage of guest 1 is
gradually reduced by substitution with “inert” guest 6. Concentrations:
[5] = 0.2 mM, [1] = 0–0.1 mM, [6] = 0–0.1 mM, [ConA] = 0.1 mg/mL.
(B) Maximum agglutination depending on surface coverage of guest 1.
guest 1. These observations are entirely consistent with our guest 3. We propose that due to the adjacency in the case of
previous investigation of an artificial glycocalyx of lactose and guest 3, the binding of one adamantane unit directs the other
maltose and its interaction with ConA and peanut agglutinin two units to bind to the same vesicle (intravesicular binding),
(PNA) [28,29].
whereas the larger distance between the adamantane units in
guest 2 and 4 allows the adamantane functions to bind to
Interestingly, if guests 2–4 (0.1 mM adamantane) were added to different vesicles (intervesicular binding). As discussed above
cyclodextrin vesicles (0.2 mM) an aggregation effect was for guest 1, the surface density of guests 2–4 can be reduced by
detected even in the absence of ConA (Figure 5). This effect is replacement with “inert” guest 6. It can be seen from Figure 5
due to noncovalent cross-linking of the vesicles by guest 2–4. that addition of a substantial amount of this inert competitor
Each of the adamantane units on guest 2–4 can bind to a effectively reduces the tendency to cross-link the vesicles. As
different cyclodextrin vesicle (intervesicular binding) and hence can be seen in the plot of the maximum agglutination induced
cause vesicle aggregation. We have previously observed this by guest 2, a linear dependence (rather than a threshold)
effect for homobifunctional guest molecules equipped with two between the guest surface coverage and the extent of aggrega-
azobenzene, methylbenzoyl, or tert-butylbenzyl groups [32-34]. tion is observed. If a higher percentage of cross-linker is present
It should be noted that in the case of guest 3 the aggregation due on the vesicle surface, more noncovalent cross-links between
to cross-linking is much smaller, almost invisible, compared to vesicles are formed and more extensive aggregation is
the effect found for guests 2 and 4. This can possibly be observed.
explained by the orientation of the adamantane units in the
guest molecules. Guests 2 and 4 have two adamantane units and In line with the observations discussed above, a tenfold dilution
the average distance between these units is significantly larger of the concentration of host ([5] = 20 μM) and guest ([2] = [4] =
than the average distance of the three adamantane units in 5.0 μM and [3] = 3.3 μM) results in a complete suppression of
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Figure 6: (A) Agglutination of β-cyclodextrin vesicles in the presence
of guest 2 or 3 and ConA. Legend: red = guest 2, black = guest 3. The
surface coverage of guests 2 and 3 is gradually reduced by substitu-
tion with “inert” guest 6. Concentrations: [5] = 20 μM, [2] = 0–5.0 μM,
[3] = 0–3.3 μM, [6] = 0–10 μM; [ConA] = 0.1 mg/mL. (B) Maximal
aggregation depending on surface coverage of guest 2 or 3.
Figure 5: (A) Agglutination dependence of β-cyclodextrin vesicles in
the presence of guests 2–4. Legend: red = guest 2, black = guest 3,
blue = guest 4. The surface coverage of guests 2–4 is gradually
reduced by substitution with “inert” guest 6. Concentrations: [5] =
0.2 mM, [2] = 0–0.05 mM, [3] = 0–0.033 mM, [4] = 0–0.05 mM, [6] =
0–0.075 mM. (B) Maximum agglutination depending on surface
coverage of guest 2.
surface of the vesicles. The average distance between two
cross-linking of the vesicles. Apparently, at this concentration cyclodextrins at the vesicle surface is approximately 2.2 nm
intravesicular binding is favored over intervesicular binding, [30]. The distance between two mannose molecules dCH is
and vesicle aggregation is negligible. These findings are again expected to be the same when using a 100% surface coverage of
consistent with earlier observations [32]. Hence, in this concen- cyclodextrin vesicles with monovalent guest 1. A decrease in
tration window the agglutination in the presence of ConA can the surface density of mannose due to replacement of guest 1 by
be investigated. Indeed, cyclodextrin vesicles functionalized “inert” guest 6 decreases the agglutination by ConA (see
with guests 2–4 aggregate in the presence of ConA. However, Figure 4 and Figure 7) since eventually the average spacing of
the extent and rate of agglutination is very different for each mannose on the vesicle surface (dCH) exceeds the binding-site
guest. The observations for divalent guest 2 and trivalent guest separation of ConA (dBS) [21]. However, the surface density of
3 are shown in Figure 6.
mannose also decreases if the guest molecule occupies two or
even three cyclodextrin cavities but yet carries only one
Perhaps counter intuitively, the extent of agglutination mannose unit (Figure 7).
decreases with an increasing number of adamantane functions
present in the guest molecule. In other words, agglutination is In the case of divalent guest 2 (with two adamantane units and a
less efficient for 3 compared to 2 in spite of the higher surface single mannose) the maximum surface density of mannose is
affinity of trivalent guest 3 compared to divalent guest 2. This only half of the surface density that can be achieved with mono-
observation can be explained by the density of mannose on the valent guest 1. As a consequence, the average distance dCH of
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Figure 7: Schematic presentation of the binding of (A) a monovalent, (B) a divalent, or (C) a trivalent guest onto the surface of cyclodextrin vesicles
and their interaction with ConA. dCH: average spacing of carbohydrates, dBS: effective binding-site separation of ConA.
the mannose residues is similar to the effective binding-site sep- but the extent of agglutination is similar. These observations
aration of ConA, and agglutination is quite efficient. However, can be rationalized as illustrated in Figure 9. Since guest 4 has
it can be seen that although agglutination can be completely two mannose units, it can bind in a divalent fashion to ConA
suppressed by replacement with guest 6, the concentration
dependence is completely different from that observed for guest
1: rather than showing a threshold value, agglutination persists
even at a rather low percentage of guest 2 compared to inert
guest 6. In the case of trivalent guest 3 (with three adamantane
units and a single mannose) the maximum surface density of
mannose on the cyclodexrin vesicle is only one third of the
maximum surface density for guest 1. As a consequence, the
average distance of the mannose residues exceeds the effective
binding-site separation of ConA, and very little agglutination is
observed upon addition of ConA. Thus, it could be said that
more adamantane units in the guest molecule in fact diminish
the agglutination by ConA, since they result in a substantially
lower surface coverage of mannose.
Finally, we investigated the agglutination behavior of cyclodex-
trin vesicles decorated with guest 4. Guest 4 contains two
adamantane units as well as two mannose residues. A 100%
surface coverage of the cyclodextrin vesicles with guest 4 is
therefore expected to give an average distance of 2.2 nm
between the mannose functions, similar to the average spacing
obtained for guest 1. However, due to the inherent multiva-
lency of guest 4, it should have a much higher affinity both for
the cyclodextrin vesicle surface as well as for ConA. Indeed it
was found that guest 4 ([4] = 10 μM) can induce the agglutina-
tion of cyclodextrin vesicles ([5] = 20 μM) in the presence of
ConA. Figure 8 shows the results of the optical density
measurements for guest 4 compared to guest 2. It can be seen
that the extent and rate of agglutination of the vesicles induced
by guest 4 is substantially higher than for guest 2, in particular
at low surface coverage (i.e., below 50 %). At higher surface
coverage, the rate of agglutination is much higher for guest 4,
Figure 8: (A) Agglutination of β-cyclodextrin vesicles in the presence
of guest 2 or 4 and ConA. Legend: red = guest 2, blue = guest 4. The
surface coverage of guests 2 and 4 is gradually reduced by substitu-
tion with “inert” guest 6. Concentrations: [5] = 20 μM, [2] = 0–5.0 μM:
[4] = 0–5.0 μM, [6] = 0–10 μM; [ConA] = 0.1 mg/mL. (B) Maximal
aggregation depending on surface coverage of guest 2 or 4.
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Beilstein J. Org. Chem. 2012, 8, 1543–1551.
a divalent guest molecule that carries two mannose residues:
this molecule binds inherently divalent to the cyclodextrin
vesicle surface as well as to ConA and, hence, is able to mediate
fast agglutination at low overall concentration as well as low
surface coverage. These findings should further the under-
standing of the complex and dynamic interactions of oligo-
saccharides on cell surfaces.
Experimental
Materials: Throughout this work, chemicals were used as
received from Acros Organics (Schwerte, Germany) or Sigma-
Aldrich Chemie (Taufkirchen, Germany) without further purifi-
cation. The synthesis and analysis of guest molecule 1–4 is
reported in Supporting Information File 1. Amphiphilic
β-cyclodextrin 5 was synthesized as described in the literature
[30].
Figure 9: Schematic presentation of the binding of guest molecules
with (A) one or (B) two mannose functions onto the surface of
cyclodextrin vesicles and their interaction with ConA. dCH: average
spacing of carbohydrates, dBS: effective binding site separation of
ConA.
irrespective of the surface coverage. Nevertheless, the aggluti- Methods: Isothermal titration calorimetry (ITC) measurements
nation should still be surface coverage dependent, since multi- were performed on a Nano-Isothermal Titration calorimeter III
valent intervesicular binding is more likely to occur when more (model CSC 5300; Calorimetry Sciences Corporation, London,
mannose is presented on the cyclodextrin vesicles. Since guest 2 Utah, USA). All samples were measured in distilled water at
has only one mannose unit, one would expect that ConA can 23 °C by using a stirring rate of 250 rpm. For each experiment
only bind in a divalent fashion to the cyclodextrin vesicle if the 20 injections with 10 µL volume were carried out with a 250 µL
surface coverage is rather high. However, as can be seen from syringe, into the measurement cell (V = 980.5 µL). Concentra-
Figure 6 and Figure 8, the extent of agglutination can be very tions of host and guest molecules are reported in Table 1.
high (as high as for guest 4) even at rather low surface β-Cyclodextrin vesicles are formed by extrusion of cyclodex-
coverage, albeit with a substantially lower rate of agglutination. trin 5 in a HEPES-buffer solution with a Liposofast manual
These observations can be explained on the basis of a clus- extruder through a polycarbonate membrane with a pore size of
tering of mannose residues in the mixed glycocalyx of guest 2 100 nm. Unilamellar vesicles with an average diameter of
and “inert” guest 6: clusters of guest 2 could offer multivalent 100–140 nm are obtained [30,35]. Optical density measure-
“adhesive patches” for ConA even if the average surface ments were performed in 1 mL small-volume disposable
coverage of guest 2 is far below 50%. The low rate of aggluti- PMMA cuvettes at 400 nm by using an Uvikon 923 double-
nation could be a consequence of a slow rearrangement of the beam photospectrometer. All measurements were recorded at
glycocalyx, i.e., a “receptor-induced clustering” of mannose in 23 °C in HEPES buffer (20 mM, pH 7.45) containing 1 mM
the presence of ConA.
CaCl2 and 1 mM MnCl2. Reagents were added in the following
order: To 1 mL vesicle solution, 10 μL of guest molecule stock
solution is added after 1 min, and 10 μL ConA stock solution
Conclusion
In this study we described a biomimetic model for the after 2 min.
glycocalyx of a cell membrane based on multivalent adaman-
tane–mannose conjugates that bind to cyclodextrin vesicles. In
Supporting Information
this dynamic supramolecular system the guest molecules bind
with their adamantane units to the cyclodextrin cavity, which
acts as a receptor, and as a consequence the vesicle surface is
covered by mannose. In turn, the mannose units are ligands for
the lectin ConA, which induces agglutination due to multiva-
lent cross-linking of the vesicles. Strikingly, the multivalency of
the guest molecules was found to have a detrimental effect on
the glycocalyx: divalent and trivalent guest molecules can in-
duce cross-linking of the vesicles even in the absence of ConA,
and the interaction with ConA is reduced due to the lower
surface coverage with mannose. The optimal binder therefore is
Supporting Information File 1
Synthesis, NMR and mass spectra of guest molecules 1–4.
[http://www.beilstein-journals.org/bjoc/content/
supplementary/1860-5397-8-175-S1.pdf]
Acknowledgements
We acknowledge the synthesis of starting materials by Christian
Wendeln. β-Cyclodextrin was kindly donated by Wacker
Chemie AG.
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;2-O
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