Spatially well-defined carbohydrate nanoplatforms: Synthesis, characterization and lectin interaction study

Article abstract of DOI:10.1039/c6cc06737a

Two novel dodecasubstituted carbohydrate nanoplatforms based on molecular Borromean rings and dodecaamine cages have been prepared for use in evaluating the importance of the spatial distribution of carbohydrates in their interaction with lectins. The binding affinities of the glyconanoplatforms were characterized using quartz crystal microbalance technology and compared with a monovalent reference and dodecaglycosylated fullerenes.

Full text of DOI:10.1039/c6cc06737a

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Spatially well-defined carbohydrate nanoplatforms:
synthesis, characterization and lectin interaction
study†
Cite this:DOI: 10.1039/c6cc06737a
Received 16th August 2016,
Accepted 20th September 2016
a
b
c
c
B. J. J. Timmer,‡ M. Abellan Flos,‡ L. Mønster Jørgensen, D. Proverbio,
´
c
a
c
b
S. Altun, O. Ramstrom,* T. Aastrup* and S. P. Vincent*
¨
DOI: 10.1039/c6cc06737a
www.rsc.org/chemcomm
Two novel dodecasubstituted carbohydrate nanoplatforms based yields dodecavalent carbohydrate nanoplatforms, which have
on molecular Borromean rings and dodecaamine cages have been been widely studied with varying carbohydrate types, linker
5
prepared for use in evaluating the importance of the spatial types and linker lengths.
distribution of carbohydrates in their interaction with lectins. The
To further investigate the influence of the spatial arrange-
binding affinities of the glyconanoplatforms were characterized ment of carbohydrates on multivalent recognition, new nano-
using quartz crystal microbalance technology and compared with platforms with a high degree of symmetry in ligand presentation
a monovalent reference and dodecaglycosylated fullerenes.
are desirable. A field offering access to such platforms is the field of
constitutional dynamic chemistry (CDC). The thermodynamic and
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Carbohydrates act as carriers of information in living systems self-sorting nature of CDC allows for the high-yielding synthesis
and play critical roles in regulation, modulation and adhesion of complex multi-dimensional architectures from simple starting
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processes. For efficient information transfer to occur, relatively materials. Two interesting nanoplatform architectures that can be
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strong interactions between carbohydrates and their cognate assembled using CDC are molecular Borromean rings (BRs) and
receptors are required; however, they are usually weak for dodecaamine cages. Derivatization of these core structures
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monovalent saccharides. Nature addresses this effect by multi- results in nanoplatforms with a high degree of symmetry in
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0
valent presentation of carbohydrates to enhance the affinity ligand presentation. Derivatization of Borromean rings yields
and selectivity in vivo, an effect referred to as the ‘cluster ‘globular’ S -symmetrical core structures with a diameter of 24 Å
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2
glycoside effect’. To unravel the processes in which carbo- and an octahedral addition pattern of the ligands, whereas the
hydrates are involved, it is essential to better understand this derivatized dodecaamide cages have a T -symmetrical core
d
effect. For this purpose, a wide variety of multivalent glyco- structure with a diameter of 20 Å and an icosahedral addition
sylated architectures have previously been synthesized and pattern of the ligands (Fig. 1).
assessed, e.g., glycoclusters, glycodendrimers, glycopolymers and
Herein, we report the synthesis of two novel carbohydrate
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glyconanoparticles. However, in most of these platforms it is nanoplatforms by carbohydrate functionalization of molecular
challenging to control the local density and spatial arrangement Borromean rings and dodecaamide cages. The binding kinetics
of the carbohydrate ligands. An intriguing nanoplatform which and thermodynamics of the carbohydrate nanoplatforms were
enables full control of spatial presentation is the fullerene: a C60 assessed in real-time against the lectin concanavalin A (ConA)
1
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truncated icosahedral core which can be hexafunctionalized to give using quartz crystal microbalance (QCM) technology, and
a globular T -symmetrical core with an octahedral substitution compared with previously known dodecaglycosylated fullerenes.
h
4
pattern. Carbohydrate functionalization of this core structure
a
KTH – Royal Institute of Technology, Department of Chemistry, Organic Chemistry,
Teknikringen 36, S-100 44 Stockholm, Sweden. E-mail: ramstrom@kth.se
b
University of Namur, D ´e partment de Chimie, Laboratoire de Chimie Bio-Organique,
Rue de Bruxelles 61, B-5000 Namur, Belgium. E-mail: stephane.vincent@unamur.be
c
Attana AB, Bj ¨o rnn ¨a sv a¨ gen 21, SE-114 19 Stockholm, Sweden.
E-mail: teodor.aastrup@attana.com
†
Electronic supplementary information (ESI) available: Experimental details,
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characterization data and copies of NMR ( H, C and F) and IR spectra. See
DOI: 10.1039/c6cc06737a
‡
These authors contributed equally.
Fig. 1 Symmetry and size of nanoscaffolds.
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Fig. 2 Synthesis of dodecavalent glyconanoplatforms based on (A) molecular Borromean rings, (B) dodecaamine cages and (C) fullerenes.
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In addition to the main objective of evaluating the influence of
The obtained carbohydrate nanoplatforms BRÁ(R )
6
, CÁ(R )12
,
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the spatial arrangement of carbohydrates on protein binding and FÁ(R )12 and monovalent reference 8 (see Fig. 4) were evaluated
affinity, this is the first application of the molecular Borromeate using QCM technology. This technology allowed us to monitor the
scaffold in biomolecular recognition.
binding event between ConA and the carbohydrate ligands in real
The preparation of the different carbohydrate nanoplat- time, providing additional details with respect to the binding
forms used in this study is illustrated in Fig. 2 (cf. ESI†). The kinetics and thermodynamics. ConA was selected owing to its
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synthesis of molecular Borromeate BRÁ(R )
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was achieved by established adoption in multivalency studies. QCM sensors were
polycondensation of diamine 1 with alkyne-functionalized prepared from activated, carboxyl-terminated, self-assembled
II
building block 2 in the presence of templating Zn ions. monolayers, to which ConA was attached by amide coupling.
I
1
Cu -catalyzed azide alkyne cycloaddition (CuAAC) between BRÁ(R )
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Unreacted groups were subsequently blocked by treatment with
and divalent azido-functionalized mannoside 3 yielded the ethanolamine. Immobilization of ConA on the sensor surfaces
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desired dodecaglycosylated BRÁ(R ) . The structure of the central resulted in frequency shifts (DF) of E50 Hz, indicating a successful
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Borromeate was ascertained by NMR and mass spectrometry. conjugation process (Fig. S1, ESI†).
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The dodecaamine cage CÁ(R )12 was synthesized by in situ
The binding event was monitored by injecting eight different
reduction of the spontaneously self-organized imine assembly concentrations of the respective samples, ranging from 1.56 to
À1
of trialdehyde 4 and ethylene diamine. Acylation of the formed 200 mg mL , over the ConA-functionalized sensors. This resulted
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cage with compound 5 yielded dodecaamide CÁ(R )12, from in DFs ranging from 0 Hz for the lowest concentrations to 50 Hz
5
which the glycosylated CÁ(R )12 was obtained by CuAAC with for the highest concentrations. In parallel, the samples were
compound 6. The fullerene was functionalized using a Bingel injected over unfunctionalized sensors for correction for non-
reaction between unfunctionalized fullerene and malonic specific binding. Regeneration of both sensors was performed
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ester 7 to obtain dodecasubstituted fullerene FÁ(R )12. Carbo- after each sample analysis using a 10 mM solution of glycine
hydrate functionalization was performed through CuAAC at pH 2.5. Sensor regeneration proved to be highly successful
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between FÁ(R )12 and compound 6 to obtain dodecaglyco- for nanoplatforms BRÁ(R )
6
and CÁ(R )12, whereas the surfaces
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sylated FÁ(R )12
.
after injection of fullerene FÁ(R )12 did not fully regenerate
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(
Fig. S3, ESI†). This can be explained by the increased hydrophobic other two nanoplatforms. Another observation is that the sensor-
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interactions of the fullerene core with the self-assembled monolayer gram for cage CÁ(R )12 clearly shows two binding events, indicating
present on the sensors. However, since the surfaces remained monovalent and multivalent binding interactions. For Borromeate
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unsaturated throughout the experiments, accurate binding BRÁ(R ) and fullerene FÁ(R ) , the overall stronger binding
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data were still obtained.
From the obtained sensorgrams, a selection was made to stronger multivalent binding event was mainly observed. Overall,
eliminate sample injections that showed spikes and other the estimated binding data show K -values as low as 147 nM,
obscures the visibility of two separate binding events and the
D
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factors that could interfere with proper fitting of the model. 1.21 mM and 1.02 mM for Borromeate BRÁ(R ) , cage CÁ(R )12 and
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The selected datasets were then fitted with a 1 : 2 interaction fullerene FÁ(R )12, respectively. The weakest binding to ConA, as
model to accommodate for both possible binding modes: already apparent from the sensorgrams, was estimated for cage
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monovalent and multivalent binding. The fitted datasets were CÁ(R ) . However, the lower association rate combined with a lower
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used to extract estimates of the kinetic and thermodynamic dissociation rate resulted in a very similar affinity for fullerene
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data including the association rate (k ), the dissociation rate FÁ(R ) . In contrast, the larger Borromeate BRÁ(R ) nanoplatform
a
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6
(k
d
) and the dissociation constant (K
D
). The obtained sensorgrams showed higher affinity towards ConA in consequence of a relatively
as well as the fitted datasets and estimated binding data are shown high association rate and low dissociation rate. In terms of multi-
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in Fig. 3. From the sensorgrams, it can clearly be seen that the valency and cooperativity, regular chelation is inaccessible, and
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dissociation rate of the icosahedrally glycosylated cage CÁ(R )12 was subsite chelation in combination with statistical and polyelectrolyte
significantly higher than those of the octahedrally substituted effects are expected to be the major contributors to the increased
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5
Borromeate BRÁ(R ) and fullerene FÁ(R ) . As a result, the surface affinity observed in the nanoplatforms. In CÁ(R ) , potentially
6
12
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was almost fully regenerated after a 500 s dissociation time when weaker subsite interactions may lead to the observed saturation,
treated with cage CÁ(R ) , whereas this was not the case for the whereas the difference in the K -value between the two nano-
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D
platforms with octahedral ligand presentation may be a result
of their difference in size and charge. Another explanation for
this phenomenon can be the forced higher spatial distribution
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of carbohydrate ligands in fullerene FÁ(R )12 due to substitution
of the carbohydrate ligands on a cyclopropane motif.
To be able to assess the multivalency effect, the same
experiment was performed for monovalent reference 8, as shown
in Fig. 4. As the molecular weight of compound 8 is significantly
lower in comparison to the nanoplatforms, the observed DF was
considerably lower as reflected in the reduced accuracy of the fitted
data. As clear saturation could be observed in the sensorgrams,
saturation binding was determined instead of fitting model data.
The obtained K
D
-value for compound 8 was estimated to be
109 mM. Due to the multivalency effect, the relative potency,
i.e. the binding affinity per carbohydrate unit, increased by
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approximately 66-, 8- and 10-fold for Borromeate BRÁ(R ) , cage
6
5
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CÁ(R )12 and fullerene FÁ(R )12, respectively.
In summary, using the toolbox provided by CDC we have
demonstrated the synthesis of two novel highly ordered carbo-
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hydrate nanoplatforms BRÁ(R )
6
and CÁ(R )12. Using QCM technology,
the binding affinity towards ConA could be elucidated and
Fig. 3 Selected obtained sensorgrams (black curves) and fitted datasets
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5
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(
red curves) for nanoplatforms BRÁ(R )
6
, CÁ(R )12 and FÁ(R )12 together with
Fig. 4 Obtained sensorgrams for compound 8. Inset: Saturation binding
estimated binding data.
isotherm; estimated K
D
= 109 Æ 19 mM.
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This project received funding from the European Union’s Seventh
Framework Programme for research, technological development
and demonstration under grant agreement no. 289033.
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