Detection of bacteria using glyco-dendronized polylysine prepared by continuous flow photofunctionalization

Article abstract of DOI:10.1021/nl102821f

Biocompatible glyco-dendronized poly-L-lysine (PLL) polymers carry either three or nine mannose- or galactose-bearing dendrons that selectively bind, and thus can be used to detect, bacteria. Central to the synthesis of glyco-dendronized polymers was the development of a continuous flow [2 + 2] photocycloaddition reaction to connect the dendrons and PLL. Glycodendronized polymers cluster bacteria by binding to cell-surface carbohydrate receptors and thereby result in an easy read-out using microscopic analyses.

Full text of DOI:10.1021/nl102821f

pubs.acs.org/NanoLett
Detection of Bacteria Using
Glyco-Dendronized Polylysine Prepared by
Continuous Flow Photofunctionalization
Paola Laurino,†,‡ Raghavendra Kikkeri,†,‡ Nahid Azzouz,†,‡ and Peter H. Seeberger*,†,‡
† Max Planck Institute for Colloids and Interfaces, Department of Biomolecular Systems, Research Campus Golm,
14476 Potsdam, Germany, and ‡ Freie Universita¨t Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22,
14195 Berlin, Germany
ABSTRACT Biocompatible glyco-dendronized poly-L-lysine (PLL) polymers carry either three or nine mannose- or galactose-bearing
dendrons that selectively bind, and thus can be used to detect, bacteria. Central to the synthesis of glyco-dendronized polymers was
the development of a continuous flow [2 + 2] photocycloaddition reaction to connect the dendrons and PLL. Glycodendronized
polymers cluster bacteria by binding to cell-surface carbohydrate receptors and thereby result in an easy read-out using microscopic
analyses.
KEYWORDS Glyco-polymer, continuous flow reactor, [2 + 2] photocycloaddition, protein carbohydrate interaction, bacteria detection
ood contamination with Escherichia coli, a pathogenic
bacterium, results in frequent outbreaks of infec-
tions with grave consequences.1 Rapid, reliable,
environment of the pathogen surface and optimize binding
to the bacterial carbohydrate receptors.10 The resulting
polyvalent systems can thus adjust the carbohydrate units
for the strongest and most efficient binding interaction with
the lectin. Poly-L-lysine (PLL) was chosen as the polymer
backbone for its biocompatibility and biodegradability.11 In
addition, the use of PLL is advantageous because it is
commercially available in a range of molecular weights, and
the lateral amino chain of PLL is readily functionalized to
produce various multivalent systems.12-14
Polymer functionalization often poses a severe challenge
as the coupling reaction has to be selective and high yielding
but must not contaminate the end product, which would
necessitate additional purification steps. To circumvent the
need for late stage deprotection steps, functionalization can
take place in water that allows for unprotected sugars to be
used. Widely used methods for the synthesis of glycopoly-
mers suffer from a lack of control over carbohydrate epitope
density, regioselectivity, metal contamination, and low
yields.15-17
Consequently, we explored the photochemical [2 + 2]
cycloaddition reaction, which has been previously tested as
an alternative method for polymer functionalization (Figure
1).18 [2 + 2] Cycloaddition reactions are attractive since they
can be carried out in water using inexpensive starting
materials, are not pH dependent, and circumvented the use
of heavy metals or other reagents that contaminate the
polymer product, while avoiding the formation of side
products.18 The photochemical transformation resulted in
good yields for all the glyco-dendrimers.
F
and straightforward detection of pathogenic bacteria is a
key step in avoiding the spread of contamination. Devel-
oping detection systems that meet these criteria has
proven challenging.2
The carbohydrate binding proteins3 on the surface of
pathogenic bacteria are the means by which these organ-
isms recognize and bind to hosts cells.4 Invasive strains of
E. coli express high levels of mannose binding proteins that
are displayed on the pili. The sugar-binding properties of
these proteins have been exploited in the development of
bacterial detection systems.5 Supramolecular glycopoly-
mers, conductive glycopolymers, carbohydrate-capped gold
nanoparticles, and CdS quantum dots have all been used as
probes to identify specific E. coli strains.6 These methods rely
on the multivalent presentation of carbohydrate ligands,
which is necessary because the binding affinity between an
individual monosaccharide and the lectin is weak.7,8 How-
ever, these multivalent probes have structures that do not
permit adjustment of the carbohydrate ligands to achieve
optimal multivalent binding. This structural limitation inhib-
its multimodal conjugation that would also allow for optimal
interaction with the pathogens.9
In contrast, dendronized polymers are multivalent sys-
tems that integrate the two-dimensional structure of poly-
mers and the three-dimensional structure of dendrimers to
create a flexible polymer that can bend to adapt to the
*To whom correspondence should be addressed. E-mail: peter.seeberger@
mpikg.mpg.de. Phone: +49 (0) 30-838-59300.
Received for review: 08/10/2010
Traditionally, photochemical reactions have been not or
at best poorly scalable. Utilization of a continuous-flow
photochemical reactor rendered this mode of bond forma-
Published on Web: 11/29/2010
© 2011 American Chemical Society
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DOI: 10.1021/nl102821f | Nano Lett. 2011, 11, 73–78

FIGURE 1. Schematic representation of PLL functionalization. Nonapod dendrimer (a); poly-L-lysine (b); continuous flow reactor (c); dendronized
polymer (d).
SCHEME 1. Synthesis of First- (6, 7) and Second-Generation Dendrons (10, 11)a
a (a) Acrylonitrile/NaOH, 40%, conc HCl/EtOH, 51%; 5-hexynoic acid/DIC/HOBT/DCM, 68%; (b) NaOH/MeOH; pentafluorophenol/DIC/HOBT/
DCM, 86%; (c) 2-(t-butoxycarbonylamino)ethoxy-2,3,4,6-tetra-O-acetyl-R-D-mannoside or 2-(t-butoxycarbonylamino)ethoxy-2,3,4,6-tetra-O-acetyl-
R-D-galactoside/DCM/TEA, 71%; (d) tripod-mannose (4) or galactose (5)/DCM/TEA, 67%; (e) MeOH/Na quant.; (f) MeOH/Na, quant.
tion attractive again. The high surface-to-volume ratio of the
continuous flow microdevice provides precise control over
reaction conditions, including controlled radical formation
as well as efficient and homogeneous mixing, thus enabling
facile conjugation and greatly reduced side reactions.19 Here
we report the first use of the [2 + 2] cycloaddition reaction
in a continuous flow system to generate a glyco-conjugated
polymer that successfully bound and detected E. coli bacteria.
Two sets of dendrons with three and nine carbohydrates,
respectively, were synthesized bearing either mannose or
galactose (Scheme 1).20 Starting from commercially avail-
able 2-amino-2-(hydroxymethyl)propane-1,3-diol 1, treat-
ment with acrylonitrile under basic conditions, followed by
hydrolysis and esterification, prior to coupling with 5-hex-
ynoic acid provided structure 2. Ester hydrolysis under basic
conditions, followed by coupling with pentafluorophenol
yielded the active ester 3. Reaction of 3 with peracetylated
mannose or galactose yielded first generation dendrons 4
or 5, while reaction with the peracetylated tripod mannose
or galactose second generation dendrons 8 or 9 were
obtained. Removal of the acetate groups under basic condi-
tions yielded 6 or 7 and 10 or 11.
The first- and second-generation dendrons were subse-
quently used for the functionalization of the PLL polymer
backbone 12 (Scheme 2). In this way, dendronized polymers
13-16 were created by photochemical functionalization
© 2011 American Chemical Society
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74

SCHEME 2. Functionalization of PLL with First- and Second Generation-Dendrons 5 and 7a
a (a) Succinimido 3-maleidopropanoate, quant; (b) 6 or 7, hν, 40 min, rt; (c) 10 or 11, hν, 40 min, rt.
using the [2 + 2] cycloaddition reaction in a continuous flow
reactor. Reactions were carried out at room temperature and
in water with 40 min of irradiation using a medium pressure
Hg lamp (450 W). [2 + 2] Cyclizations were analyzed by 1H
NMR (Figure 2). Two well-separated peaks of the maleimido
polymer 12 (visible at 5.8 and 6.3 ppm) disappeared after
photochemical conjugation. Peaks at 3.5 and 5 ppm show
the complete conversion of maleimido group to cyclobuten
adducts (Figure 2).
The size and shape of the dendronized polymers was
characterized by AFM analysis of samples that were im-
mobilized on a gelatin-coated mica surface.21 The nonapod-
mannose-functionalized polymer 15 adopts a globular struc-
ture with a diameter of approximate 4 Å (Figure 3). The
globular shape likely results from polymer and dendrimer
integration.
To demonstrate that we can easily detect the clustering
capability of the dendronized polymers, bacterial detection
assays were performed.22 The dendronized polymers were
incubated with either the mannose-binding, invasive strain
of E. coli, ORN178 (positive control), or with the mutant E.
coli strain, ORN208, that does not bind mannose (negative
control). Binding and any resultant clustering was detected
by confocal microscopy using DAPI (4′,6-diamidino-2-phe-
nylindole) a fluorescent dye that stains bacterial DNA. Dif-
ferent numbers of E. coli bacteria suspended in buffer and
incubated with polymer 15 and single mannose-containing
polymer (17, Supporting Information). After centrifugation
and washing, the cells were resuspended and stain with
DAPI (4′,6-diamidino-2-phenylindole). Confocal images
showed no aggregation in case of single mannose polymer
17 (see Supporting Information, Figure 1). Figure 4 shows
that incubation of the nona-mannose functionalized polymer
15 with strain ORN178 resulted in large clusters of bacteria
(Figure 4a), while strain ORN208 did not aggregate with the
polymer (Figure 4c,d).
We confirmed specific multivalent interactions between
specific E. coli strains and polymer 15 by adding ConA-FITC
(Concavanalin A-fluorescein isothiocyanate conjugate). ConA
is a lectin well-known for its strong interaction with R-man-
nose.23 Solutions containing different numbers of E. coli
bacteria (105-107) were incubated with polymer 15. The
cells were then incubated with ConA-FITC for approximately
10 min, centrifuged, and washed. ConA-FITC staining suc-
cessfully localized polymer 15 within a cluster of E. coli strain
ORN178 (Figure 4b). Neither clustering nor significant ConA-
FITC staining were observed in samples of E. coli strain
ORN208 incubated with compound 15 (Figure 4d). There-
fore, we conclude that the mannose-binding E. coli was
aggregated by a matrix of the nona-mannose dendronized
© 2011 American Chemical Society
DOI: 10.1021/nl102821f | Nano Lett. 2011, 11, 73-–78
75

FIGURE 2. 1H NMR spectra of compounds 15 (a), 10 (b), and 12 (c), respectively.
FIGURE 3. AFM images of mannose nonapod PLL 15 on gelatin-coated mica surfaces.
polymer 15, which was localized by fluorescently labeled
ConA. E. coli bacteria that do not bind to mannose were not
bound by the flexible polymer and aggregated only in a
nonspecific manner.
Finally, high-resolution atomic force microscopy (AFM)
was employed to confirm the presence of dendronized
polymers around the mannose-binding E. coli. AFM images
of a single ORN178 E. coli cell were obtained before and after
incubation with dendronized polymer 15. The surface topol-
ogy of a single cell of strain ORN178 after incubation with
polymer 15 is shown in Figure 5.
solutions of E. coli ORN178 were incubated with polymer
15 for 1 h. Samples were centrifuged, the pellets were
washed to remove unbound polymer, and the bacteria were
visualized using confocal microscopy. Polymer 15 displays
optimal binding with as little as 105 bacteria (see Supporting
Information, Figure 2). Using confocal microscopy, we found
that the detection limit of the aggregates is similar to that
observed using other multivalent biosensors.24 However,
using AFM allows for detection of an individual bacterium
that binds the glyco-dendronized polymer.
In conclusion, we have developed an efficient, fast, and
readily scalable synthetic route to synthesize dendronized
polymers in water employing a photochemical continuous
To assess the lower limit for detecting bacterial ag-
gregates in the presence of polymer 15, serially diluted
© 2011 American Chemical Society
DOI: 10.1021/nl102821f | Nano Lett. 2011, 11, 73-–78
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FIGURE 5. AFM imagines in liquid media of mannose nonapod PLL
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Acknowledgment. Generous financial support for this
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Supporting Information Available. Continuous flow re-
actor setup, general information, synthesis of dendrimers,
functionalization of polymer, cell growth and incubation of
polymer, confocal fluorescence microscopy, atomic force
microscopic (AFM) measurements, and 1H and 13C NMR data
of all the compounds. This material is available free of charge
via the Internet at http://pubs.acs.org.
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