Hexameric supramolecular scaffold orients carbohydrates to sense bacteria

Article abstract of DOI:10.1021/ja2036767

Carbohydrates are integral to biological signaling networks and cell-cell interactions, yet the detection of discrete carbohydrate-lectin interactions remains difficult since binding is generally weak. A strategy to overcome this problem is to create multivalent sensors, where the avidity rather than the affinity of the interaction is important. Here we describe the development of a series of multivalent sensors that self-assemble via hydrophobic supramolecular interactions. The multivalent sensors are comprised of a fluorescent ruthenium(II) core surrounded by a heptamannosylated β-cyclodextrin scaffold. Two additional series of complexes were synthesized as proof-of-principle for supramolecular self-assembly, the fluorescent core alone and the core plus β-cyclodextrin. Spectroscopic analyses confirmed that the three mannosylated sensors displayed 14, 28, and 42 sugar units, respectively. Each complex adopted original and unique spatial arrangements. The sensors were used to investigate the influence of carbohydrate spatial arrangement and clustering on the mechanistic and qualitative properties of lectin binding. Simple visualization of binding between a fluorescent, multivalent mannose complex and the Escherichia coli strain ORN178 that possesses mannose-specific receptor sites illustrates the potential for these complexes as biosensors.

Full text of DOI:10.1021/ja2036767

ARTICLE
pubs.acs.org/JACS
Hexameric Supramolecular Scaffold Orients Carbohydrates
To Sense Bacteria
Dan Gr€unstein,† Maha Maglinao,† Raghavendra Kikkeri,† Mayeul Collot,† Konstantin Barylyuk,‡
Bernd Lepenies,† Faustin Kamena,† Renato Zenobi,‡ and Peter H. Seeberger*,†
†Department of Biomolecular Systems, Max Planck Institute of Colloids and Interfaces, Am M€uhlenberg 1, 14476 Potsdam, Germany,
and Institute of Chemistry and Biochemistry, Free University Berlin, Arnimallee 22, 14195 Berlin, Germany
‡Department of Chemistry and Applied Biosciences, ETH Zurich, Wolfgang-Pauli-Strasse 10, 8093 Zurich, Switzerland
S Supporting Information
b
ABSTRACT: Carbohydrates are integral to biological signaling networks and cellÀcell
interactions, yet the detection of discrete carbohydrateÀlectin interactions remains difficult
since binding is generally weak. A strategy to overcome this problem is to create multivalent
sensors, where the avidity rather than the affinity of the interaction is important. Here we
describe the development of a series of multivalent sensors that self-assemble via hydro-
phobic supramolecular interactions. The multivalent sensors are comprised of a fluorescent
ruthenium(II) core surrounded by a heptamannosylated β-cyclodextrin scaffold. Two
additional series of complexes were synthesized as proof-of-principle for supramolecular
self-assembly, the fluorescent core alone and the core plus β-cyclodextrin. Spectroscopic
analyses confirmed that the three mannosylated sensors displayed 14, 28, and 42 sugar units,
respectively. Each complex adopted original and unique spatial arrangements. The sensors
were used to investigate the influence of carbohydrate spatial arrangement and clustering on
the mechanistic and qualitative properties of lectin binding. Simple visualization of binding
between a fluorescent, multivalent mannose complex and the Escherichia coli strain ORN178 that possesses mannose-specific
receptor sites illustrates the potential for these complexes as biosensors.
’ INTRODUCTION
logical or chemical triggering by carbohydrateÀlectin binding.9
No ideal scaffold for multivalent carbohydrate presentation
exists, even though a myriad of systems have been described.
For instance, C5-symmetrical glycoconjugates that orient five
ganglioside GM1 pentasaccharides can neutralize the pentameric
cholera toxin via a specific, multivalent interaction.10 Similarly, a
multivalent C3-symmetrical ganglioside GM3 trisaccharide is a
potent inhibitor of influenza virus hemagglutination.11
With the goal of creating a new architecture for multivalent
sugar presentation using the “building-on-a-template” strategy,
we have developed a small-molecule scaffold that is synthetically
facile and robust, has tunable symmetry, can accommodate
different means of carbohydrate ligand functionalization, and
exhibits distinct optical and electrochemical properties. This
platform is based on hydrophobic supramolecular interac-
tions that allow for the self-assembly of a fluorescent scaffold
with a multivalent system, generating fluorescent and multivalent
sensors. The fluorescent core—the template—is derived from
[Ru(bipy)2]Cl2 to afford a di-adamantyl- (Ru2 2), tetra-adaman-
tyl- (Ru4 3), or hexa-adamantyl-ruthenium(II) complex (Ru6 4).
Around this core, β-cyclodextrins (βCD), each equipped with
seven mannose units to form heptamannosylated βCD12
(βCDMan), self-assemble around the template, as the adamantyl
CarbohydrateÀlectin interactions are critical to many biolo-
gical systems.1 Consequently, a better understanding of their
interplay will provide important insights into specific biological
processes. The weak affinities of carbohydrateÀprotein binding
require improved tools to analyze carbohydrateÀlectin com-
plexes. Detection and read-out of binding can be improved by
multivalent probes since they amplify the weak interactions and
enhance the potency of carbohydrate ligands.
Many multivalent carbohydrate-based tools such as dendrimers,2
polymers,3 micelles,4 and nanoparticles,5 as well as carbohydrates
built on templates of cyclodextrins (CDs)6 and calixarenes,7 have
been synthesized to monitor glycanÀlectin recognition and bind-
ing events by electronic, optical, or microgravimetric methods.8
These multivalent probes can be functionalized with carbohy-
drates either through linear synthesis, where the sugar building
blocks are incorporated during the synthetic process (“building-
with-a-template”), or via convergent synthesis, where sugar
functionalization takes place as a discrete, final step (“building-
on-a-template”).3f,5a “Building-on-a-template” is advantageous for
generating libraries of multivalent carbohydrate-based probes, as
different carbohydrate groups can be displayed by a variety of
synthetic methods on a “fixed” core structure.
Important considerations for designing multivalent probes are
valency, the spatial arrangement of the lectin target’s multiple
binding sites, and the response to external stimuli, such as bio-
Received: April 21, 2011
Published: July 26, 2011
r
2011 American Chemical Society
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Scheme 1. Overview of the Synthesis Procedure for the Ru Series 2À4
“hosts” the CD “guests” around the template. Two different
To demonstrate the supramolecular interaction between CD
series of fluorescent compounds were produced by the hostÀ
guest interactions. One group of molecules contains only un-
modified βCD (RuCD2 5, RuCD4 6, and RuCD6 7). The
second group of molecules consists of RuCDMan2 8, RuCDMan4
9, and RuCDMan6 10, bearing 14, 28, or 42 mannose units,
respectively. Multivalent carbohydrate presentation and lectin
binding were examined using the RuCDMan complexes 8À10
and the lectin concanavalin A (ConA). Surface plasmon reso-
nance (SPR) measurements provided mechanistic, qualitative,
and quantitative insights into the role of cluster arrangement.
The fluorescence emission of the Ru(II) core was employed
to visualize specific and shape-dependent interactions of 10
with Escherichia coli (E. coli) strain ORN178, which exhibits
mannose-specific receptor sites for recognition and binding to
host cells.
derivatives and the adamantyl Ru(II) cores, complexes 2À4 were
mixed with stoichiometric amounts (2, 4, and 6 equiv) of native
βCD to furnish compounds with two (RuCD2 5), four (RuCD46),
and six (RuCD6 7) CD units (Scheme 2).
Finally, a series of multivalent mannosylated structures was
generated by mixing complexes 2À4 with βCDMan in water in
stoichiometric amounts relative to the number of adamantyl
units to afford RuCDMan2 8, RuCDMan4 9, and RuCDMan6
10. These multivalent complexes expose 14, 28, or 42 mannose
units on their periphery, respectively (Scheme 3). It should be
noted that complexes 2À4 can self-assemble with βCD carrying
diverse glycosylation patterns, thereby facilitating production of a
library of new multivalent systems.
Their octahedral core symmetry and robustness as well as
strong oxidizing and reducing properties render ruthenium(II)
complexes attractive for use as sensors.13 The photophysical
properties of the Ru series 2À4 were examined and compared
with the properties of the RuCD series 5À7 to determine the
effects of covering the ruthenium core with βCD (Figure 1a,b
and Table 1). All spectra contain a first transition band (1IL),
’ RESULTS AND DISCUSSION
Three variants of a fluorescent template were synthesized
using common ligand 1 (Scheme 1) and produced three scaffolds
with distinct architectures to support the multivalent pre-
sentation of carbohydrate ligands. Ligand 1 was obtained via a
convergent synthesis (see Supporting Information (SI)). The
scaffolds were comprised of a central ruthenium(II) core that is
equipped with either two (Ru2 2), four (Ru4 3), or six (Ru6 4)
adamantyl groups (Scheme 1). The di-adamantyl complex 2
was assembled by ligand complexation of 1 with [Ru(bipy)2]Cl2.
Treatment of 1 with RuCl3, using acetic acid as a reducing agent
followed by bipyridine as a quenching agent, resulted in a mixture
of tetra-adamantyl 3 and hexa-adamantyl 4 complexes, which
were purified and separated by column chromatography.
located around 350 nm, that is assigned to metal-centered dÀd
abs
max
transitions.14 An intense absorption band at λ ≈ 475 nm and a
shoulder at 450 nm dominate the spectra and originate from the
splitting of the energy level of the first excited state caused by the
trigonal symmetry of the complex. Both bands are assigned to a
t2g(Ru) f π*(bipy) metal-to-ligand charge transfer (1MLCT).15
The emission spectra of the Ru series and the RuCD series
em
max
exhibited an emission band at λ ≈ 640 nm. An increase in
emission intensity and calculated quantum yield (Φ) directly
correlates with the increasing complexity of the structures as more
units of ligand 1 and CD are assembled around the Ru(II) core
(Figure 1c,d and Table 1).16,17 Physical shielding of the core by
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Scheme 2. Overview of the Synthesis Procedure for the RuCD Series 5À7
the surrounding scaffold prevents effective fluorescence quench-
ing by dioxygen and solvent. Comparison of the quantum yields
within the Ru series 2À4 illustrates the influence of the number of
adamantyl ligands 1 on fluorescence intensity. The consequences of
forming supramolecular assemblies, however, are highlighted by
direct comparison of a Ru series complex and its RuCD
equivalent, such as comparing 3 and 6, or 4 and 7. Interestingly,
no difference was observed when 2 and 5 were compared. This is
likely because complexes 2 and 5 differ by the addition of only
two CDs, a minor structural difference, whereas an additional
four CD molecules have been incorporated into complex 6, and
six CD molecules into complex 7. While the bulky CDs in 6 and 7
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Scheme 3. Overview of the Synthesis Procedure for the RuCDMan Series 8À10
efficiently shield the fluorescent core from the environment, the
two CDs in complex 5 are not sufficient, and consequently the
fluorescent chromophore is easily quenched. Fluorescence peak
analysis indicated a blue (hypsochromic) shift from 668.5 to
631.0 nm in the Ru series, and from 655.0 to 627.0 nm in the
RuCD series, occurring as more adamantyl ligand 1 or more CD
is added to the Ru(II) core. The observed hypsochromic shift of
luminescence is possibly a consequence of so-called rigidochro-
mism described in highly hindered complexes.18 To summarize,
an increasing number of adamantyl ligands 1 and, consequently,
CD units around the metal center strongly influenced the
photophysical and colloidal properties of the Ru(II) core.
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Figure 1. Absorption (UV) spectra of (a) the Ru series Ru2 2, Ru4 3, and Ru6 4 in MeOH and (b) the RuCD series RuCD2 5, RuCD4 6, and RuCD6 7
in H2O. Emission (fluorescence) spectra of (c) the Ru series 2À4 in MeOH upon excitation at 475 nm and (d) the RuCD series 5À7 in H2O upon
excitation at 475 nm.
Table 1. Photophysical Data for Ruthenium Complexes 2À4
(in MeOH) and Complexes 5À7 (in H2O)
keep non-covalent complexes intact during the ionization and
ion transmission processes, a native nano-ESI technique was
employed.22
(nm) ελabs (L molÀ1 cmÀ1
)
λ
(nm)
Φ
abs
max
em
max
complex Amax
λ
max
3
3
Figure 2a shows the full scan of the RuCD2 5 sample. The
positive-ion mass spectrum of fully assembled complex 5 clearly
reveals a doubly charged ion [RuCD2]2+ with m/z 1668. A broad
isotope distribution due to the presence of Ru2+ is in agreement
with the theoretically calculated isotope pattern (see Figures S3
and S4, SI). No Ru-containing ions with higher m/z values
were detected in the mass spectrum, indicating the absence of
non-specific aggregates of CD molecules and Ru-containing
ligand either in the electrospray plume or inside the mass
spectrometer.19 Peaks at m/z 1100 and 533 corresponding to
[RuCD]2+ and [Ru]2+ ions, respectively, were also detected. The
presence of these ion peaks in the mass spectrum indicated
partial dissociation of complex 5. Acceleration and collision of
the analyte ions with neutral gas molecules during the electro-
spray ionization process caused the observed dissociation of the
supramolecular complex and could even lead to covalent bond
cleavage under different conditions.23
2
3
4
5
6
7
2.38
1.73
3.15
1.76
1.61
1.99
466.0
4760
3460
6300
5189
4586
5836
668.5
0.14
0.16
0.48
0.12
0.23
0.70
471.0
460.5
477.0
478.0
468.5
644.5
631.0
655.0
641.5
627.0
Encapsulation of the ruthenium(II) core with CD led to an
increase in fluorescence intensity through protection of the core
properties.
The RuCD complexes 5À7 were subjected to mass spec-
trometry to determine their stoichiometry and to analyze
the supramolecular assemblies.19,20 Only small amounts of
the RuCDMan complexes 8À10 were available, and there-
fore these larger and heavier structures were not investigated.
ESI is recognized as a soft ionization method that allows the
generation of intact molecular ions from different species,
including large, non-volatile organic molecules and biopolymers.21
On the other hand, the detection of non-covalent assemblies
by ESI-MS remains challenging since supramolecular interac-
tions are often disrupted during the electrospray process. To
The highlighted peaks in the full-scan mass spectrum of 6
(Figure 2b) correspond to ions from fully assembled [RuCD4]2+
(m/z 3050) and the partially assembled supramolecular complexes
[RuCD3]2+ (m/z 2483), [RuCD2]2+ (m/z 1916), [RuCD]2+
(m/z 1349), and [Ru]2+ (m/z 781). No non-specific aggregates
of Ru ligand and CD were evident in the higher m/z range.
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complexes such as [RuCD5]2+ (m/z 3865), [RuCD4]2+ (m/z 3298),
[RuCD3]2+ (m/z 2731), [RuCD2]2+ (m/z 2164), [RuCD]2+
(m/z 1597), and [Ru]2+ (m/z 1030) were detected in the
mass spectrum (see Figures S7 and S8, SI). After the initial
failure to measure the fully assembled complex 7 with
conventional ESI and nano-ESI sources, different electro-
spray-based soft ionization methods, such as Electrosonic
Spray Ionization (ESSI) and cold-spray, were employed.
These measurements resulted in signals from [RuCD6]2+
above the detection limit and differences in ion yields for
assembled, partially disassembled, and fully disassembled
complexes were observed.19
Overall, nano-ESI mass spectrometry-based analysis of the
RuCD complexes (5À7) confirmed the existence of complete
supramolecular assembly. Stabilizing forces such as dispersion
interactions between adamantyl moieties of the Ru(II)-contain-
ing ligands and the cavity of the CDs were insufficient to preserve
the complexes from collision-activated dissociation, resulting in
the presence of partially assembled complexes.
To confirm that the supramolecular assemblies are an-
chored by adamantyl groups acting as hosts in the CD guests,
NMR was employed. Unfortunately, comparative measure-
ments were complicated by the fact that some complexes
(2À4) were soluble only in methanol, whereas others (5À10)
1
were soluble only in water. H NMR spectra were incon-
clusive in determining whether the inclusion complex be-
tween adamantyl moieties and native βCD or βCDMan
occurred for each adamantyl group (see SI part 3.2). Different
chemical shift patterns of the adamantyl protons were ob-
1
served when comparing the H NMR of different complexes
and may have resulted from solvent effects rather than
supramolecular assembly.
NOESY experiments24 illustrated the proximity of the
adamantyl groups to CD. Cross peaks arise from the proximity
of the adamantyl moiety and CD via NOESY analysis of
solutions of 5, 6, and 7 in D2O (Figure 3aÀc and Table 2).
The cross peaks indicate strong interactions between H3 and
H5 of βCD with all adamantyl protons (Ha, Hb, and Hc)
throughout the RuCD complexes (Figure 4). Thus, it was
confirmed that the adamantyl derivative penetrated comple-
tely into the βCD core. Since H2 and H4 of βCD are localized
outside the cavity, they do not interact with adamantyl pro-
tons. All CD protons in the 1H NMRs (complexes 5À7) were
shifted in comparison to the peaks of native βCD in D2O, and
proton signals signifying free native βCD were absent. All
βCD units observed by NMR appear to be involved in
supramolecular inclusion, indicating that all complexes were
fully assembled.
Figure 2. ESI mass spectra of (a) RuCD2 5, (b) RuCD4 6, and (c)
RuCD6 7. Peaks corresponding to doubly charged ions of assembled,
partially disassembled, and fully disassembled complexes are highlighted
with yellow bars, marked with corresponding m/z values, and magnified
(see insets).
Figure 3dÀf shows the cross peaks observed from NOESY
experiments for solutions of 8, 9, and 10 in D2O. Due to the
1
very complex H NMR between 4.2 and 3.6 ppm, it was
difficult to identify the H3 and H5 of βCDMan and assign
changes after supramolecular assembly. Instead, we focused
our attention on the protons of the adamantyl moiety and
observed a strong NOE interaction with protons from the
CDMan region. As described above for complexes 5À7, three
defined cross-coupling peaks appeared, coming from the
interaction between CD and three different protons of the
adamantyl moiety (Ha, Hb, and Hc). In comparison, for
complexes 8À10, the outermost protons of the adamantyl
group (Hc) probably split due to the larger steric environment
of βCDMan (attributable to the seven mannose residues).25
Although a peak corresponding to the doubly charged ion of fully
assembled RuCD4 complex 6 was detectable,19 the resolution
was not high enough to correctly resolve the individual isotope
peaks (see Figures S5 and S6, SI).
A full-scan mass spectrum of RuCD6 7 is shown in Figure 2c.
Since the [RuCD6]2+ ion has a m/z value of ∼4432, relatively
high acceleration voltages were applied to the ion guides to
facilitate ion transport. High acceleration of ions resulted in
collision-activated dissociation of non-covalent complexes.
Nevertheless, signals from ions of partially assembled RuCD6
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Figure 3. Significant cross peaks from the NOE spectra of (a) RuCD2 5, (b) RuCD4 6, and (c) RuCD6 7 and of (d) RuCDMan2 8, (e)
RuCDMan4 9, and (f) RuCDMan6 10. Cross peaks between H3 and H5 of βCD or βCDMan and Ha, Hb, and Hc of adamantyl are emphasized in
the red boxes.
1
Table 2. H NMR (in D2O) Chemical Shifts of CÀH Protons
from Native βCD and from Ru(II)-Complexed βCD in the
RuCD Series 5À7
proton shift (ppm)
entry
βCD
H1
H2
H3
H4
H5
H6
4.68
5.12
5.13
5.13
3.26
3.71
3.71
3.71
3.58
3.19
3.64
3.64
3.64
3.47
3.85
3.89
3.88
3.49
RuCD2 5
RuCD4 6
RuCD6 7
3.99À3.90
4.00
3.99À3.90
3.98À3.91
4.02À3.90
Figure 4. Proton designation of the relevant molecule moiety involved
in the supramolecular assembly.
4.02À3.90
has four mannose binding sites. Immobilizing the lectin at low
and high densities was intended to distinguish whether the
presence of more mannose-binding sites would stabilize
bound complexes, or whether binding site clustering would
inhibit this interaction.
Overall, the results from the NOESY experiments clearly
indicate that the inner βCD protons are in close proximity to the
adamantyl moieties, confirming the presence of fully assembled
complexes in the RuCD series 5À10. Overall, the combination of
photophysical and spectroscopic structural analyses provided a
near-complete analytical characterization of complexes 2À10.
Functional studies were conducted to determine binding beha-
vior of the multivalent RuCDMan complexes 8À10.
The SPR analyses of ConA-RuCDMan2 8 and ConA-
RuCDMan4 9 (Figure 5 and Table 3) indicated that both
complexes 8 and 9 strongly prefer to bind ConA-HD over
ConA-LD, indicating that high ConA concentration stabilized
the binding interaction. This trend correlates well with the
prior characterization of ConA binding to mannose.28 Closer
examination shows that 9 binds to ConA-HD more efficiently
than 8, and also that 8 dissociates from ConA much faster than
9 (Figure 5b,d). For the ConA-LDÀcomplex interactions, KD
for 8 is lower than for 9. The difference in the specific KD
values is most likely attributable to the generally weak binding
of low ConA concentration than to the individual binding
capacity of 8 and 9. Interestingly, complex 10 did not bind to
ConA (data not shown). The binding curves obtained for the
interactions between 10 and ConA-HD as well as ConA-LD
Surface plasmon resonance (SPR) is a valuable tool for
analyzing proteinÀcarbohydrate complex formation in real
time and for providing insights into the kinetics and mecha-
nics of binding.26 To understand the influence of mannose
density on lectin binding at the surface of the RuCDMan
complexes, the interaction of complexes 8À10 with the
mannose-specific plant lectin ConA was investigated. SPR
and kinetic analyses were based on a 1:1 interaction model.27
Two different concentrations of ConA were covalently bound
to a polycarboxylated CM5 sensor chip generating low-den-
sity (ConA-LD) and high-density (ConA-HD) surfaces. ConA
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Figure 5. SPR sensorgrams of RuCDMan2 8 (Ru-CD2-Man14) and RuCDMan4 9 (Ru-CD4-Man28): (a,b) binding of RuCDMan2 8 to ConA-LD
and ConA-HD, respectively; (c,d) binding of RuCDMan4 9 to ConA-LD and ConA-HD, respectively.
were comparable to the results obtained using only HEPES-
EP as a negative control. This outcome is not entirely
surprising, since similar results have been reported for highly
multivalent probes.29 In this case, the addition of two man-
nose-functionalized CD units (the structural difference be-
tween complexes 9 and 10) impeded binding with ConA,
despite the greater number of mannose residues available for
the interaction. One explanation for this finding is that the
inherent bulkiness of 10 may impede the formation of stable
interactions between mannose and ConA. Additionally, the
spatial arrangement of the mannose-bearing CDs is impor-
tant: in the case of 10, the mannose pattern may represent a
non-ideal setup for interacting with surface-immobilized
ConA. Together these results show that, in the context of a
multivalent platform, the strength of the binding interaction
between ConA and mannose directly correlates with the
number of participating mannose residues. Nonetheless, there
is an upper limit to that number, beyond which binding
efficiency declines.
Table 3. Equilibrium Constants, KD, of RuCDMan2 8 and
RuCDMan4 9
KD (μM)
analyte
ConA-LD
ConA-HD
RuCDMan2 8
RuCDMan4 9
RuCDMan6 10
0.8428
4.573
0.2292
0.1380
no binding
no binding
inherent red fluorescence and the bacteria by using blue DAPI
stain. Binding was evident only between mannosylated complex
10 and ORN178 (Figure 6a,b). Binding did not occur between
complex 10 and strain ORN208 as anticipated due to the
mutation in fimH31 (Figure 6d). Other controls were performed
to prove the specificity of the complex toward ORN178. As
expected, complex 7 failed to bind to both ORN178 (Figure 6c)
and ORN208 (Figure 6e).
Since the binding capacity of complex 10 was unable to be
assessed by SPR, an alternative strategy was devised using the
outer surface of bacteria as the target for lectin recognition
and binding. Two E. coli strains, ORN178 and ORN208, were
used.30 Both strains produce type 1 pili but differ in their
expression of the pilus component FimH, a mannose-specific
adhesin. While ORN178 expresses wild-type FimH, ORN208
carries a mutation in the fimH gene which renders the pili unable
to bind mannose. Each bacterial strain was incubated with either
complex 10 or the negative control, complex 7, that does not
contain mannose, and confocal microscopy was used to visualize
the interaction between the complexes and the bacterial pili. The
Ru(II)-containing complexes were observable by virtue of their
The images obtained by confocal microscopy show red
fluorescent spots (the Ru core) decorating the lateral end of
the bacteria along the pili, implying that complex 10 is
attached to the mannose-binding lectin FimH at this site.
The molecular interaction between complex 10 and E. coli
strain ORN178 was both specific and easily detectable,
giving rise to the idea that this new fluorescent and highly
multivalent system may be adapted to detect bacteria.
Star-shaped clusters of bacteria were produced during incubation
with 10 (Figure 6a,b) and may be attributable to the specific
arrangement of this octahedral ruthenium complex. This will be
the subject of future investigations.
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Figure 6. Confocal laser scanning microscopy images for the incubation of (a,b) bacteria E. coli strain ORN178 with RuCDMan6 10 and of (c)
ORN178 with RuCD6 7, (d) ORN208 with RuCDMan6 10, and (e) ORN208 with RuCD6 7 as negative controls.
’ CONCLUSIONS
’ AUTHOR INFORMATION
Here, we describe the synthesis of three different templates that
ultimately generate three new scaffolds for the presentation of
multiple multivalent carbohydrate ligands by supramolecular as-
sembly. The multivalent complexes self-assemble and display
carbohydrates with unique spatial orientation. In addition, binding
interactions between the carbohydrate epitopes and lectins could
be investigated by surface plasmon resonance. Previously, multi-
valent probes, such as supramolecular and conductive glycopoly-
mers, carbohydrate-capped gold nanoparticles, and quantum dots,
have been used as probes to identify specific E. coli strains32 or
other cell lines.33 Taking advantage of the fluorescent properties of
ruthenium(II), distinct interactions between mannosylated com-
plex 10 and E. coli strain ORN178 were observed using confocal
microscopy. The simplicity of this screenhighlights the potentialof
using these complexes as adaptable and user-friendly bacterial
sensing tools. The significance of another property of multivalent
complexes was also illustrated by incubating complex 10 with
ORN178: the bacterial clusters that formed around the complex
were distinctive and star-shaped. Therefore, it is likely that the
spatial arrangement of the ligands on the multivalent complex
ultimately restricts the orientation of the lectins. Consequently, it
may be possible to construct nanostructures that will interact with
multiple target binding sites on bacterial cells or proteins in a
shape-dependent manner, providing an easy visual read-out of a
particular molecular interaction. Application of the multivalent
probes to explore other carbohydrateÀlectin interactions will be
facilitated by the flexible architecture of this system—the manno-
sylated cyclodextrins are interchangeable with any glycosylated
β-cyclodextrin. Simple interchange of the carbohydrate epitopes will
also greatly streamline generating collections of multivalent probes.
Corresponding Author
peter.seeberger@mpikg.mpg.de
’ ACKNOWLEDGMENT
P.H.S. and D.G. thank the Max-Planck Society and the European
Union FP7 (CARMUSYS) for financial support. Generous finan-
cial support by the K€orber Foundation is gratefully acknowledged.
B.L. thanks the German Federal Ministry of Education and
Research (BMBF, Fkz. 0315446) for financial support. The authors
thank Dr. A. Sch€afer for NMR spectroscopy and Dr. A. Springer for
ESI-MS. We thank Dr. V. Mountain and Dr. R. Pragani for critically
editing the manuscript.
’ REFERENCES
(1) (a) Drickamer, K.; Taylor, M. E. Annu. Rev. Cell Biol. 1993,
9, 237–264. (b) Liu, F. T. Clin. Immunol. 2000, 97, 79–88.
(c) Konstantinov, K. N.; Robbins, B. A.; Liu, F. T. Am. J. Pathol.
1996, 148, 25–30. (d) Pilobello, K. T.; Slawek, D. E.; Mahal, L. K. Proc.
Natl. Acad. Sci. U.S.A. 2007, 104, 11534–9. (e) Bertozzi, C. R.; Kiessling,
L. L. Science 2001, 291, 2357–2364. (f) Lis, H.; Sharon, N. Chem. Rev.
1998, 98, 637–674.
(2) (a) Kikkeri, R.; Hossain, L. H.; Seeberger, P. H. Chem. Commun.
2008, 2127. (b) De Paz, J. L.; Noti, C.; Boehm, F.; Werner, S.; Seeberger,
P. H. Chem. Biol. 2007, 14, 879. (c) Wolfenden, E. K.; Cloninger, M. J.
J. Am. Chem. Soc. 2005, 127, 12168. (d) Roy, R.; Kim, J. M. Angew. Chem.,
Int. Ed. 1998, 38, 369. (e) Woller, E. K.; Walter, E. D.; Morgan, J. R.;
Singel, D. J.; Cloninger, M. J. J. Am. Chem. Soc. 2003, 125, 8820.
(f) Kikkeri, R.; Liu, X. Y.; Adibekian, A.; Tsai, Y. H.; Seeberger, P. H.
Chem. Commun. 2010, 46, 2197. (g) Lepenies, B.; Yin, J.; Seeberger,
P. H. Curr. Opin. Chem. Biol. 2010, 14, 404. (h) Bernardes, G. J. L.;
Kikkeri, R.; Maglinao, M.; Laurino, P.; Collot, M.; Hong, S. Y.; Lepenies,
B.; Seeberger, P. H. Org. Biomol. Chem. 2010, 8, 4987.
’ ASSOCIATED CONTENT
(3) (a) Disney, M. D.; Zheng, J.; Swager, T. M.; Seeberger, P. H.
J. Am. Chem. Soc. 2004, 126, 13343. (b) Yang, W.; Pan, C. Y.; Luo, M. D.;
Zhang, H. B. Biomacromolecules 2010, 11, 1840. (c) Gestwicki, J. E.;
Kiessling, L. L. Nature 2002, 415, 81. (d) Sasaki, K.; Nishida, Y.; Tsurumi,
S
Supporting Information. Details of syntheses, experi-
b
ments, analyses and NMR spectra. This material is available
free of charge via the Internet at http://pubs.acs.org.
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ARTICLE
T.; Uzawa, H.; Kondo, H.; Kobayashi, K. Angew. Chem., Int. Ed. 2002,
41, 4463. (e) Cario, C. W.; Gestwicki, J. E.; Kanai, M.; Kiessling, L. L.
J. Am. Chem. Soc. 2002, 124, 1615. (f) Laurino, P.; Kikkeri, R.; Azzouz, N.;
Seeberger, P. H. Nano Lett. 2011, 11, 73. (g) Gentsch, R.; Pippig, F.;
Nilles, K.; Theato, P.; Kikkeri, R.; Maglinao, M.; Lepenies, B.; Seeberger,
P. H.; B€orner, H. G. Macromolecules 2010, 43, 9239–9247.
(4) Ryu, J. H.; Lee, E.; Lim, Y. B.; Lee, M. J. Am. Chem. Soc. 2007,
129, 4808–4814.
(5) (a) Kikkeri, R.; Laurino, P.; Odedra, A.; Seeberger, P. H. Angew.
Chem., Int. Ed. 2010, 49, 2054. (b) Huang, C.-C.; Chen, C.-T.; Shiang,
Y.-C.; Lin, Z.-H.; Chang, H.-T. Anal. Chem. 2009, 81, 875. (c) Lin, C.-C.;
Yeh, Y.-C.; Yang, C.-Y.; Chen, C.-L.; Chen, G.-F.; Chen, C.-C.; Wu,
Y.-C. J. Am. Chem. Soc. 2002, 124, 3508.
(6) (a) Gomez-Garcia, M.; Benito, J. M.; Rodriguez-Lucena, D.; Yu,
J.-X.; Chmurski, K.; Mellet, C. O.; Gallego, R. G.; Maestre, A.; Defaye, J.;
Fernandez, J. M. G. J. Am. Chem. Soc. 2005, 127, 7970. (b) Benito, J. M.;
Gomez-Garcia, M.; Ortiz Mellet, C.; Baussanne, I.; Dafaye, J.; Fernandez, J.
M.G. J. Am. Chem. Soc. 2004, 126, 10355. (c) Mellet, C. O.; Defaye, J.;
Fernandez, J. M. G. Chem.—Eur. J. 2002, 8, 1982.
(7) (a) Fulton, D. A.; Stoddart, J. F. Bioconjugate Chem. 2001,
12, 655. (b) Baldini, L; Casnati, A.; Sansone, F.; Ungaro, R. Chem.
Soc. Rev. 2007, 36, 254. (c) Badjic, J. D.; Nelson, A.; Cantrill, S. J.;
Turnbull, W. B.; Stoddart, J. F. Acc. Chem. Res. 2005, 38, 723.
(8) (a) Kikkeri, R.; Kamena, F.; Gupta, T.; Hossain, L. H.;
Boonyarattanakalin, S.; Gorodyska, G.; Beurer, E.; Coullerez, G.; Textor,
M.; Seeberger, P. H. Langmuir 2010, 26, 1520. (b) Dai, Z.; Kawde, A.-N.;
Xiang, Y.; La Belle, J. T.; Gerlach, J.; Bhavanandan, V. P.; Joshi, L.; Wang, J.
J. Am. Chem. Soc. 2006, 128, 10018. (c) Kikkeri, R.; Gr€unstein, D.;
Seeberger, P. H. J. Am. Chem. Soc. 2010, 132, 10230. (d) Otsuka, H.;
Akiyama, Y.; Nagasaki, Y.; Kataoka, K. J. Am. Chem. Soc. 2001, 123, 8226.
(e) Kikkeri, R.; Lepenies, B.; Adibekian, A.; Laurino, P.; Seeberger, P. H.
J. Am. Chem. Soc. 2009, 131, 2110.
(25) Jing, B.; Chen, X.; Zhao, Y. R.; Wang, X. D.; Cai, J. G.; Qiu, H. Y.
J. Phys. Chem. B 2008, 112, 7191.
(26) Rich, R. L.; Myszka, D. G. J. Mol. Recognit. 2006, 19, 478.
(27) Hardman, K. D.; Ainswort., C. F. Biochemistry 1972, 11, 4910.
(28) Munoz, E. M.; Correa, J.; Fernandez-Megia, E.; Riguera, R.
J. Am. Chem. Soc. 2009, 131, 17765.
(29) (a) Dhayal, M.; Ratner, D. A. Langmuir 2009, 25, 2181.
(b) Schlick, K. H.; Cloninger, M. J. Tetrahedron 2010, 66, 5305.
(30) Kim, B. S.; Hong, D. J.; Bae, J.; Lee, M. J. Am. Chem. Soc. 2005,
127, 16333.
(31) (a) Jones, C. H.; Pinkner, J. S.; Roth, R.; Heuser, J.; Nicholes,
A. V.; Abraham, S. N.; Hultgren, S. J. Proc. Natl. Acad. Sci. U.S.A. 1995,
92, 2081. (b) Choudhury, D.; Thompson, A.; Stojanoff, V.; Langermann,
S.; Pinker, J.; Hultgren, S. J.; Knight, S. D. Science 1999, 285, 1061.
(c) Harris, S. L.; Spears, P. A.; Havell, E. A.; Hamrick, T. S.; Horton, J. R.;
Orndorff, P. E. J. Bacteriol. 2001, 183, 4099.
(32) (a) Hahan, M. A.; Tabb, J. S.; Krauss, T. D. Anal. Chem. 2005,
77, 4861. (b) Mukhopadhyay, B.; Martins, M. B.; Karamanska, R.;
Russell, D. A.; Field, R. A. Tetrahedron Lett. 2009, 50, 886. (c) Wang, C.;
Irudayaraj, J. Small 2008, 4, 2204. (d) Huang, P. J.; Tay, L. L.; Tanha, J.;
Ryan, S.; Chau, L. K. Chem.—Eur. J. 2009, 15, 9330. (e) Su, Y. L.; Li,
J. R.; Jiang, L.; Cao, J. J. Colloid Interface Sci. 2005, 284, 114. (f) Li, Y.; Ma,
B.; Fan, Y.; Kong, X.; Li, J. Anal. Chem. 2002, 74, 6349.
(33) Gottschaldt, M.; Schubert, U. S.; Rau, S.; Yano, S.; Vos, J. G.;
Kroll, T.; Clement, J.; Hilger, I. Chembiochem 2010, 11, 649.
(9) Rosenzweig, B. A.; Ross, N. T.; Tagore, D. M.; Jayawickramarajah,
J.; Saraogi, I.; Hamilton, A. D. J. Am. Chem. Soc. 2009, 131, 5020.
(10) (a) Fan, E. K.; Zhang, Z. S.; Minke, W. E.; Hou, Z.; Verlinde, C.;
Hol, W. G. J. J. Am. Chem. Soc. 2000, 122, 2663. (b) Zhang, Z. S.; Merritt,
E. A.; Ahn, M.; Roach, C.; Hou, Z.; Verlinde, C.; Hol, W. G. J.; Fan, E.
J. Am. Chem. Soc. 2002, 124, 12991.
(11) Ohta, T.; Miura, N.; Fujitani, N.; Nakajima, F.; Niikura, K.;
Sadamoto, R.; Guo, C. T.; Suzuki, T.; Suzuki, Y.; Monde, K.; Nishimura,
S. I. Angew. Chem., Int. Ed. 2003, 42, 5186.
(12) Garcia-Barrientos, A.; Garcia-Lopez, J. J.; Isac-Garcia, J.;
Ortega-Caballero, F.; Uriel, C.; Vargas-Berenguel, A.; Santoyo-Gonzalez,
F. Synthesis 2001, 1057.
(13) Kikkeri, R.; Garcia-Rubio, I.; Seeberger, P. H. Chem. Commun.
2009, 235.
(14) Hahn, U.; Vogtle, F.; De Paoli, G.; Staffilani, M.; De Cola, L.
Eur. J. Inorg. Chem. 2009, 2639.
(15) Issberner, J.; Vogtle, F.; De Cola, L.; Balzani, V. Chem.—Eur.
J. 1997, 3, 706.
(16) Faiz, J. A.; Kyllonen, L. E. P.; Contreras-Carballada, P.;
Williams, R. M.; De Cola, L.; Pikramenou, Z. Dalton Trans. 2009, 3980.
(17) Felici, M.; Contreras-Carballada, P.; Vida, Y.; Smits, J. M. M.;
Nolte, R. J. M.; De Cola, L.; Williams, R. M.; Feiters, M. C. Chem.—Eur.
J. 2009, 15, 13124.
(18) (a) Wrighton, M.; Morse, D. L. J. Am. Chem. Soc. 1974, 96, 998.
(b) Innocenzi, P.; Kozuka, H.; Yoko, T. J. Phys. Chem. B 1997, 101, 2285.
(19) Barylyuk, K.; Balabin, R. M.; Gr€unstein, D.; Kikkeri, R.;
Frankevich, V.; Seeberger, P. H.; Zenobi, R. J. Am. Soc. Mass Spectrom.
2011, 22, 1167.
(20) Hernandez, H.; Robinson, C. V. Nat. Protoc. 2007, 2, 715.
(21) (a) Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse,
C. M. Science 1989, 246, 64. (b) Kebarle, P.; Verkerk, U. H. Mass Spectrom.
Rev. 2009, 28, 898.
(22) Wilm, M.; Mann, M. Anal. Chem. 1996, 68, 1.
(23) Gabelica, V.; De Pauw, E. Mass Spectrom. Rev. 2005, 24, 566.
(24) Schneider, H. J.; Hacket, F.; Rudiger, V.; Ikeda, H. Chem. Rev.
1998, 98, 1755.
13966
dx.doi.org/10.1021/ja2036767 |J. Am. Chem. Soc. 2011, 133, 13957–13966