Temperature-Switchable Glycopolymers and Their Conformation-Dependent Binding to Receptor Targets

Article abstract of DOI:10.1021/acs.biomac.0c00676

The temperature-dependent binding of copolymers from poly(N-isopropylacrylamide) (PNIPAM) and mannose ligands to Escherichia coli and concanavalin A (ConA) is determined. Through polymer analogous reactions using poly(N-acryloxysuccinimide) and amine-linked mannose residues with different linkers, glycopolymers are prepared with the variation of the mannose density. Quantitative adhesion inhibition assays show the inhibitory potential of the glycopolymers as a function of the mannose/NIPAM ratio and linker type above and below their lower critical solution temperature (LCST). Intriguingly, opposite temperature effects on the binding to E. coli and ConA are observed. While the E. coli inhibition is stronger above the LCST, the ConA inhibition is, in overall, weaker at elevated temperatures. When going beyond the LCST, the polymers undergo a coil-to-globule transition, forming microphases with surface-enriched hydrophilic sugar moieties exhibiting increased E. coli inhibition through steric shielding. However, the formation of such microphases above the LCST renders a fraction of carbohydrate ligands inaccessible,and the polymers remaining in the solution phase then have coil sizes below the minimum binding site spacing of the ConA receptor, explaining reduced ConA inhibition. Overall, these results suggest that the coil-to-globule transition of glycopolymers may induce lower or higher inhibitory potentials due to the adverse effects of steric shielding and carbohydrate ligand accessibility.

Full text of DOI:10.1021/acs.biomac.0c00676

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Article
Temperature switchable glycopolymers and their
conformation-dependent binding to receptor targets
Tanja J. Paul, Alexander K. Strzelczyk, Melina I. Feldhof, and Stephan Schmidt
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.0c00676 • Publication Date (Web): 16 Jun 2020
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Temperature switchable glycopolymers and their
conformation-dependent binding to receptor targets
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*
†#
†#
†
Tanja J. Paul , Alexander K. Strzelczyk , Melina I. Feldhof, and Stephan Schmidt
1Institute of Organic and Macromolecular Chemistry, Heinrich-Heine-University Düsseldorf,
Universitätsstraße 1, 40225 Dusseldorf, Germany
Email: Stephan Schmidt – stephan.schmidt@hhu.de
# Authors contributed equally
* Corresponding authors
Keywords
LCST, temperature stimulus, PNIPAM, carbohydrate polymer, inhibitors, E. coli, lectin
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Abstract
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The temperature-dependent binding of copolymers from poly(N-isopropylacrylamide) (PNIPAM)
and mannose ligands to E. coli and concanavalin A (ConA) is determined. Through polymer
analogous reactions using poly(N-acryloxysuccinimide) and amine-linked mannose residues with
different linkers, glycopolymers are prepared under variation of the mannose density. Quantitative
adhesion-inhibition assays show the inhibitory potential of the glycopolymers as a function of the
mannose/NIPAM ratio and linker type above and below their lower critical solution temperature
(LCST). Intriguingly, opposite temperature effects on the binding to E. coli and ConA are
observed. While the E. coli inhibition is stronger above the LCST, the ConA inhibition is overall
weaker at elevated temperature. When going beyond the LCST, the polymers undergo a coil-to-
globule transition, forming microphases with surface-enriched hydrophilic sugar moieties
explaining increased E. coli inhibition by steric shielding. However, the formation of such
microphases above the LCST renders a fraction of carbohydrate ligands inaccessibleand polymers
remaining in the solution phase then have coil sizes below the minimum binding site spacing of
the ConA receptor explaining reduced ConA inhibition. Overall these results suggest that the coil-
to-globule transition of glycopolymers may induce lower or higher inhibitory potentials due to the
adverse effects of steric shielding and carbohydrate ligand accessibly.
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1. Introduction
Interactions between carbohydrates and proteins at the surface of cells or pathogens control
numerous biological processes including infections, fertilization, recognition or signaling.1
Lectins, as a class of carbohydrate binding proteins, decorate the pathogen surface and form
complexes with glycans at the cell’s glycocalyx, which is a critical step in the development of
infectious diseases. The macromolecular glycans involved in these processes interact with the
pathogen receptors through multiple weak interactions generating sufficient adhesion across large
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interfacial areas2, 3 to drive pathogen invasion and disease progression. The ability to suppress such
lectin-glycan mediated adhesion processes by multivalent carbohydrate presenting inhibitors has
been proposed as a strategy to fight infections.4-6 Different glycoconjugates have been developed
for this purpose, e.g. nanogels,7 gold nanoparticles,8, 9 and many more, see recent reviews.10-12 To
improve the viability of such inhibitors in practice, being able to increase or decrease their affinity
toward pathogens by remote stimulus is desired. For example, to first capture pathogens at a high
affinity state of the glycoconjugate and to release the pathogen afterward for diagnosis by
switching to a low affinity state. Remote stimuli could also reduce side effects, e.g. by locally
restricting bacterial inhibition/capture to the inflamed tissues. For controlled drug release many of
such remotely switchable and site specific scaffolds were successfully established.13 Recent
studies also transferred this concept to glyco-conjugated thermo-responsive polymers in order to
control the interactions to lectins, bacteria or viruses via temperature stimulus.14-23 The involved
polymers have a lower critical solution temperature (LCST) in the physiological temperature
range. They form extended coils below the LCST, and attain a collapsed globule conformation
above the LCST. It was reasonably assumed that the temperature-controlled coil-to-globule
transition affects the presentation of carbohydrate ligands as well as the size of the scaffold and
thus controls the binding affinity of the glycoconjugate. However, the literature shows diverging
results on how temperature changes affect the affinity. One the one hand, affinities toward single
lectins or bacteria were shown to decrease above the LCST,14-16 whereas other studies showed
increasing affinities.19-23 For example, using linear copolymers composed of N-
isopropylacrylamide (NIPAM) and mannose derivatives Pasparakis et al. showed that binding to
E. coli and their clustering is preferred below the LCST.14 Using a similar pair of monomers, and
additional crosslinker to form microgels, we showed that their binding and clustering of E. coli is
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preferred above the LCST.19 Other work showed no clear effect of the temperature transition on
the binding efficiency.24-26
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This work is aimed at investigating the changes in the temperature dependent affinity of
thermosensitive glycopolymers to understand the diverging findings and interpretations in the
literature. It could be argued that carbohydrate units become inaccessible at elevated temperature
when linear LCST-polymers attain a globule conformation and aggregate to explain reduced
affinity. On the other hand, the surface presentation of hydrophilic carbohydrate units at such
globules might increase above the LCST when the polymer becomes hydrophobic. In addition the
size ratio between the glycopolymer ligands and their targets are shifted by the coil-to-globule
transition, which may affect their inhibitory potential due to steric shielding effects.27 Such steric
shielding effects describe the ability of a large inhibitor particle to block the binding between
ligand and receptor decorated surfaces due to the steric screening of binding sites.28 Furthermore,
the multivalent binding to receptor sites could be affected because of the change of the polymer
coil size when crossing the LCST, e.g. when the coil size falls below the minimum binding site of
the receptor. Therefore, here we systematically vary the compositions of linear PNIPAM/mannose
copolymers as well as the linker between mannose units and the polymer backbone. We study their
binding via inhibition assays with concanavalin A (ConA) as a well-known mannose specific lectin
with a minimum binding site distance of 7.2 nm 29 and E. coli, a bacteria binding to mannose via
monovalent FimH receptors.30 By choosing these targets with broadly different properties and
systematically varying the glycopolymer mannose density and linker type we aim to elucidate
phase transition effects in glycopolymer binding.
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2. Experimental
Materials
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α-D-mannopyranoside (99%, Acros Organics), β-D-galactose pentaacetate (95%, Fluorochem)
acetonitrile (≥99.9%, Panreac AppliChem) p-toluenesulfonic acid (98%, Alfa Aesar), sodium
methanolate (98%, Alfa Aesar), isopropylamine (99+%, Alfa Aesar), acetic anhydride (≥98%,
VWR Chemicals), n-hexane (99%, VWR Chemicals), ethyl acetate (freshly distilled), sodium
bicarbonate (100%, Fisher Chemicals), Amberlite-IR120® (Fisher Chemicals), magnesium sulfate
(62-70%, Fisher Chemicals), tetrahydrofuran (99.99%, Fisher Chemicals), chloroform (99.97%,
Fisher Chemicals), dimethylsulfoxide (99.99%, Fisher Chemicals) allyl alcohol (≥99%, Merck
KGaA), trimethylamine (>99.0%, Merck KGaA), acryloyl chloride (96%, Merck KGaA), boron
trifluoride diethyl etherate (≥98%, TCI), hydrogen (Air Liquide), N,N-dimethylformamide
(≥99.8%, Biosolve-Chemicals). All other chemicals were obtained from Sigma-Aldrich
(Germany).
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Synthesis of N-acryloxysuccinimide (NAS)
The synthesis of NAS was adapted from published protocols.16 In 200 mL of chloroform N-
Hydroxysuccinimide (14.4 g, 125 mmol) and triethylamine (22 mL, 155 mmol) were dissolved at
0 °C. Then acryloyl chloride (10 mL, 125 mmol) was added dropwise under stirring at 0 °C for
30 min. The organic phase was washed three times with saturated sodium bicarbonate solution and
dried over sodium sulfate. The volume was then reduced to 1/3 and NAS was precipitated by
addition of n-hexane. The supernatant n-hexane was removed under reduced pressure. The
remaining yellow oil was dissolved in chloroform. This cycle was repeated until the remaining oil
was not soluble in chloroform giving a yield of 18.09 g (88%).1H-NMR (300 MHz, CDCl3) δ
6.74 - 6.67 (dd, 3JHH = 17.4, 0.8 Hz, 1H, H1), δ 6.38 - 6.27 (dd, 3JHH= 16.6, 10.3 Hz, 1H, H2), δ
6.20 - 6.14 (dd, 3JHH = 10.7, 0.8 Hz, 1H, H3), δ 2.89 - 2.81 (s, 4H, H4-7) (Supporting Information
S1).
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Synthesis of Poly(N-acryloxysuccinimide) (PNAS)
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NAS (15.3 g, 90 mmol) in 140 mL N,N-dimethylformamide (DMF) was heated to 80 °C and
flushed with nitrogen for 15 min. The polymerization was initiated by addition of 4,4’-Azobis(4-
cyanovaleric acid) (255.6 mg, 0.9 mmol) in 10 mL DMF. After 20 hrs the polymer was
precipitated in cold tetrahydrofuran, filtered and dried under vacuum. A brown solid was obtained
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(yield 12.21 g, 79%, ꢀꢁ
= 15340 Da).1H-NMR (600 MHz, CDCl3) δ = 12.38 - 12.14 (s, 1H, H9)
R
3.28 - 3.00 (s, 1H, H3), 2.85 - 2.74 (s, 4H, H4-7), 2.25 - 1.90 (s, 2H, H1+2) 1.40 - 1.33 (s, 3H, H8)
(Supporting Information S2).
Synthesis of the glycopolymers
Poly(N-acryloxysuccinimide) (1.00 g, 65 µmol) was dissolved in 10 mL dimethyl sulfoxide
(DMSO) and heated to 40 °C. Depending on the Man/Gal functionalization degree, different
amounts of amine functionalized sugar (Supporting Information S4) were added. After 2 hrs of
reaction, isopropylamine (1.5 mL, 17.7 mmol) was added reacting for an additional 2 hrs. The
reaction solution was cooled down and diluted with 35 mL water and dialyzed for 48 hrs followed
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by freeze drying. The Supporting Information shows the H NMR analysis (Supporting
Information S4) and the size exclusion chromatography (Supporting Information S5) of the
polymers.
Phenol sulfuric acid (PSA) method
To determine the glycopolymer Man/Gal functionalization degree, the PSA method was used.31
At first a calibration curve was measured using a methyl α-D-mannopyranoside (MeMan) dilution
series in water (160 µM, 80 µM, 40 µM and 20 µM). To 125 µL of each MeMan solution 125 µL
of a 5wt% phenol solution in water was added and vigorously shaken. Afterward, 625 µL of
concentrated sulfuric acid was added, vigorously shaken and reacted at 30 °C for 30 min. Next,
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the absorbance at a wavelength of 490 nm was measured. For analyzing the functionalization
degree, 125 µL of the polymer solution at known concentration was used (Supporting Information
S6).
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Turbidimetry
Turbidity measurements were performed on a Tepper turbidity photometer using a 1 mW laser at
630 – 690 nm. 2 mL of a solution of 5 mg mL-1 polymer in LBB was given into a QS quartz cuvette
(Hellma Analytics, Müllheim, Germany). After reaching the starting temperature of 20 °C, 0.5 mL
of a ConA solution (1 mg mL-1) was added followed by starting the heating and cooling cycle from
20 °C up to 44 °C and back to 20 °C at a temperature rate of 1 °C min-1. To determine the cloud
point as an indication for the LCST of the polymers, the measurement was executed without
protein (supporting information S9). From the transmission-temperature traces the onset of the
transmission decrease was used to determine the cloud points.
Mannan-coating
For coating the 96-well plates mannan from Saccaromyces cerevisiae (Sigma-Aldrich) was used.
120 µL mannan solution (1.2 mg mL-1) in carbonate buffer (pH 9.5) were filled into each well.
The plates were dried at 37 °C overnight followed by washing with PBST (PBS with 0.5 wt%
Tween 20, 3 x 120 µL per well).
GFP-based bacterial adhesion inhibition assay
The adhesion assay was adapted from previous protocols.30 For blocking nonspecific binding each
well was filled with 120 µL of 1 wt% polyvinylalcohol (22000 g/mol) in PBS at 37 °C followed
by shaking at 120 rpm for 1 hrs. Next, the plates were washed three times with 120 µL PBST and
one time with PBS. Then a dilution series of the polymers was prepared and added to the well
plates. The bacteria suspension (50 µL) at a concentration of 2 mg mL-1 (OD = 0.4) was added to
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the wells, and the plates were incubated for 1 hr at 100 rpm either 20 °C or 40 °C. Afterward the
plates were washed three times with 120 µL PBS and then filled with 100 PBS µL per well. Finally,
the fluorescence intensity of the adhered E. coli was detected at 485 nm / 535nm.
FITC-ConA adhesion inhibition assay
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The mannan-coated surfaces were blocked with a 5wt% solution of bovine serum albumin in
carbonate buffer (pH 9.5) by giving 120 µL of the BSA solution into each well and shaking at 120
rpm for 1 hr at ambient temperature. After blocking, the plates were washed three times with
120µL PBST and one time with 120 µL LBB. A dilution series of the polymers was prepared on
the mannan-coated, BSA blocked well plates. A solution of FITC-ConA at a concentration of 0.1
mg mL-1 in LBB was prepared. 50 µL of the ConA solution were added to each well, and the plates
were incubated for 1 hrs at 100 rpm either at 20 °C or 40 °C. After 1 hr the plates were washed
three times with 120 µL LBB and filled with 100 µL LBB per well. The fluorescence intensity of
the adhered FITC-ConA was determined at 485 nm / 535 nm.
3. Results and discussion
3.1 Synthesis of thermo-responsive glycopolymers
The key objective of this work is to control the sugar density and linker type in thermo-responsive
glycopolymers and to test the effect of these parameters on the inhibition of ConA and E. coli
FimH receptors. Ten different polymers with varying carbohydrate densities and different linkers
were prepared. For straightforward variation of the sugar densities and linker type, a polymer
analogous reaction was chosen (Figure 1). By post-functionalization of a poly(active ester) the
NIPAM repeat units and the sugar ligands were introduced. First the succinimide-based
poly(active ester) was synthesized according to a previously published protocol.16 Via free radical
polymerization (FRP) with 4,4’-azobis(4-cyanovaleric acid) as initiator the active ester polymer
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poly(N-acryloxysuccinimide) (PNAS) was synthesized. As expected from FRP, PNAS exhibits a
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dispersity of 1.51 and a number average molecular weight (ꢀꢁ) of 15.34 kDa as determined via
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size exclusion chromatography (SEC) and 1H-NMR. As carbohydrate ligands, varying quantities
of 2-aminoethyl-α-D-mannopyranoside (ManEL) and 3-amino-2-hydroxypropyl-α-D-
mannopyranoside (ManHPL) were then reacted with PNAS. The two carbohydrates have
different linkers, where the hydroxypropyl (HPL) linker can be considered slightly more
hydrophilic due to the added hydroxy group as compared to the ethyl (EL) linker. In a second
reaction step, isopropylamine was added to quench the remaining active esters and to create the
thermo-responsive NIPAM repeating units.
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H2N
HO
O
st
∗
p
∗
n
HO
NC
m
1.
(c)
2.
CN
NH2
DMF, 80°C
O
NH
O
NH
O
O
O
N
n
N
O
O
O
N
+
OH
N
O
HO
O
HO
O
O
NC
H2N
HO
NH2
N-acryloxysuccinimide
1.
O
(b)
(a)
OH
1.
2.
NH2
2.
NH2
α
=
=
-D-mannopyranoside
st
st
m
∗
∗
NC
NC
m
p
p
β
-D-galactopyranoside
O
NH
O
NH
O
NH
O
NH
OH
HO
HO
O
O
OH
OH
OH
HO
HO
HO
NH2
HO
NH2
HO
O
HO
O
O
OH
NH2
HO
HO
HO
NH2
O
HO
OH
O
O
HO
NH2
NH2
Figure 1 Schematic of the active ester synthesis and grafting of different sugars. Grafting of (a)
ManHPL, (b) ManEL and (c) GalHPL at different carbohydrate-densities on the polymer
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backbone is followed by the addition of isopropylamine to quench the remaining active esters and
to form thermoresponsive NIPAM residues.
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The incorporation of carbohydrates into the polymers took place with an efficiency of roughly
50% (Supporting Information S4). Due to the presence of hydroxyl groups it is possible that a
fraction of sugar units was grafted to the polymer backbone via ester groups. However, as
evidenced by the absence of ester groups in the IR traces (Supporting Information S7), the esters
were likely substituted by isopropylamine groups in the second grafting step under basic
conditions. With this synthetic route five glycopolymers bearing ManHPL with carbohydrate
functionalization degrees from 1% to 97% and three glycopolymers bearing ManEL with
carbohydrate functionalization degrees from 1% to 5% were produced. As negative controls two
non-Man presenting polymers were prepared: pure PNIPAM without sugar ligands, and a
galactose decorated polymer via grafting of 3-amino-2-hydroxypropyl-β-D-galactopyranoside
(GalHPL). In Table 1 the synthesized polymers are listed and named by the grafted carbohydrate
followed by a number representing the functionalization degree, e.g. ManHPL7 signifies 7% Man
units compared to NIPAM. The polymer’s carbohydrate functionalization degree was determined
by a quantitative colorimetric test for sugars (PSA-test).32, 33 In addition, the degree of carbohydrate
functionalization was confirmed by AT-FTIR. The glycopolymer molecular weights were
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�
determined by considering the ꢀꢁ of PNAS as determined by NMR and the degree of carbohydrate
functionalization as determined by the PSA-test. Size exclusion chromatography confirmed the
�
shifts in ꢀꢁ with varying degrees of functionalization because of the higher molecular weight of
the carbohydrates compared to isopropylamine. Also, the glass transition temperature (Tg)
increased with a higher carbohydrate content (Supporting Information S8), in agreement with the
literature.34 The cloud point of the polymers, as determined by turbidimetry, increased at higher
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carbohydrate functionalization, in line with previous studies on carbohydrate functionalized
microgels.19 For ManHPL97 and ManHPL34 no temperature responsive behavior could be
observed owing to the high carbohydrate content.
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�
Table 1 The glycopolymers functionalized with different quantities of Man and Gal, ꢀꢁ, Tg, the
cloud point and the carbohydrate functionalization degree are listed. The polymers are named by
the sugar type and the functionalization degree in mol%.
Man/Gal functionalization
polymer sample
Tg [°C]b LCST [°C] c
a
����
ꢀꢁ [kDa]
degree [µmol g-1]d
0.0
PNIPAM
ManHPL1
ManHPL2
ManHPL7
ManHPL34
ManHPL97
ManEL1
10.30
10.48
10.65
11.53
16.24
27.24
10.45
10.59
11.04
10.83
117
122
126
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>45e
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5.7 ± 0.1
23 ± 0.1
99 ± 0.3
298 ± 2.2
3.5 ± 0.1
6.8 ± 0.1
14 ± 0.1
ManEL2
ManEL5
GalHPL3
40.8
8.3 ± 0.1
aThe Mn of the active ester polymer (PNAS) was determined via H-NMR, DSC measurements, turbidimetry measurements,
1
b
c
dphenol sulfuric acid assay, eno sigmoidal curve, an LCST could not be determined
3.2 Aggregation of ConA with the glycopolymers
Turbidity measurements were conducted to test the temperature dependent binding of the
glycopolymers to ConA. ConA exhibits a homotetrameric structure at neutral pH, with four Man
binding sites and a minimum spacing of 7.2 nm.35 All studies were carried out in lectin binding
buffer (LBB) containing Mn2+ and Ca2+ to activate the ConA binding sites.36-38 Since ConA
exhibits four binding sites, it typically forms clusters with multivalent carbohydrates.29, 39 The
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formation of such ConA-glycopolymer clusters can be studied via turbidimetry in real time, due
to the increase of light scattering for increased cluster sizes.40
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The temperature dependent cluster formation was tested under a heating/cooling rate of 1 °C min-1
between 20 °C and 44 °C. Without adding ConA, all polymers showed an increase in turbidity
above the phase transition temperature as indicated by the cloud point. This can be attributed to
the formation of polymer-polymer aggregates (Supporting Information S9a-h). All measurements
in the absence of ConA reach the starting turbidity value after cooling back to 20 °C. However,
when ConA is added to the measurement cell, the turbidity does not return to the starting value
upon cooling for all Man-bearing polymers. This hysteresis effect can be assigned to ConA-
glycopolymer binding and the formation of clusters that dissolve only very slowly below the cloud
point. Moreover, this hysteresis effect is stronger for the polymers bearing ManEL. This suggests
that the more hydrophobic linker leads to slower cluster dissolution. For the non-carbohydrate
bearing polymers PNIPAm and GalHPL3, the hysteresis-effect was not observed (Supporting
Information S9). This indicates that the delayed cluster dissolution for Man bearing polymers is
because of the specific binding to ConA. In addition, when inhibiting ConA binding using an
excess of MeMan the hysteresis was significantly reduced confirming specific binding. When the
cooling rate was reduced to 0.1 °C min-1 the starting turbidity value was reached indicating that
glycoclusters with Man-functionalization degrees up to 2% as tested, are not stable below the phase
transition temperature but their dissolution is merely delayed (Supporting Information S9k).
Overall, these measurements confirm a statistical/additive effect on glycopolymer binding,40 i.e.
an increased number of Man-units leads to larger and more persistent clustering with ConA.
Moreover, the linker chemistry affects the cluster dissolution, where the more hydrophilic linker
in ManHPL leads to faster dissolution than the more hydrophobic linker in ManEL.
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Figure 2 Turbidity under heating (red) and cooling (blue) at 1 °C min-1 of glycopolymer samples
in the presence of 0.2 mg mL-1 ConA. (a) Three different Man functionalized polymers are shown.
ManEL2 showed a stronger heating/cooling hysteresis compared to ManHPL2 as indicated by
the lower transmission value after cooling. (b) Negative control samples PNIPAm and GalHPL3
showed a reduced hysteresis. The inhibitor α-D-mannopyranoside (MeMan) also lead to a reduced
hysteresis upon cooling.
3.3 Temperature dependent adhesion inhibition of E. coli
The turbidity measurements confirmed the glycopolymers’ phase behavior. To understand the
phase transition effect on the glycopolymer affinity, a quantitative binding assay is required.
Therefore, we carried out adhesion inhibition assays with E. coli. These bacteria have evolved
hairy adhesive organelles, called pili or fimbriae, allowing the bacteria to adhere to cells via
carbohydrate lectin interactions and to cause infections.41 One of the best characterized adhesive
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organelles is the type 1 fimbriae, which comprises various protein subunits and the monovalent α-
D-mannopyranoside binding lectin FimH.42, 43
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To evaluate the Man-specific adhesion of the GFP-tagged type 1-fimbriated pKL1162 strain, we
use a mannan coated microtiter plate, add the bacteria, incubate/wash with the glycopolymers and
then quantify the number of adhered bacteria by fluorescent readout (Figure 3). In this manner,
glycopolymers compete with the mannan coated surface for binding to FimH. Therefore, by
increasing the concentration of the Man presenting glycopolymers, a larger inhibition of the
bacterial adhesion was achieved as measured by a reduction of the fluorescent signal.30 From the
changes in fluorescence intensity as a function of glycopolymer concentration, the inhibitory
concentration at half maximum intensity (IC50) was determined. The IC50 value represents the
concentration of glycopolymer, where 50% of the bacterial adhesion to the surface was inhibited.
If the IC50 is low, the inhibitory potency of the glycopolymer is high and vice versa.
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FimH
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Figure 3 a) The temperature dependent inhibition assay of E. coli adhesion. GFP-tagged E. coli
adhere to mannan coated surfaces. After the addition of the thermoresponsive glycopolymer the
E. coli adhesion to the mannan coated surface is hindered. The glycopolymers form aggregates
and show an increased Man surface density above the cloud point. b) A typical inhibition curve at
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At increased Man functionalization a stronger adhesion inhibition took place (Figure 4).
Furthermore, GalHPL3 showed only a small inhibition effect due to the low affinity to FimH.
Comparing ManEL and ManHPL with the same Man concentration, it can be seen that the IC50
values for ManEL are lower. This is likely due to a higher lectin binding affinity of the
hydrophobic linker, which was also observed by Lindhorst and coworkers.44 For low Man
functionalization degrees of less than 5%, an affinity increase is observed for both ManEL and
ManHPL when heating to 40 °C, where ManHPL polymers show a stronger temperature
response. At elevated Man content larger than 5%, the cloud point was above 40 °C for all
polymers, indicating that a coil-to-globule transition and glycopolymer aggregate formation did
not take place. Consequently, no clear temperature dependence on the IC50 was observed for these
polymers.
Figure 4 Left: IC50 values for the inhibition of E. coli adhesion with ManHPL glycopolymers at
20 °C (blue) and 40 °C (red). Right: IC50 values for ManEL glycopolymers. The IC50 values are
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related to the polymer concentration. All measurements were performed in triplicates, averaged
values and standard deviations are presented.
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A comparison of the Hill coefficients shows the degree of cooperative binding. Cooperative
binding is observed when ligand/receptor complex formation between multivalent structures yields
higher a binding energy as compared to the sum of energies from single ligand/receptor subunits.28
A Hill coefficient below 1 represents a negative cooperativity, a value higher than 1 stands for a
positive cooperativity effect. For all polymers incorporating less than 5 mol% Man, the Hill
coefficient increase above the cloud point, which was below 40°C for these polymers (Table 2).
This could be attributed to the formation of highly multivalent glycopolymer aggregates, which
likely show an increased surface presentation of hydrophilic Man units as these polymers
aggregate via hydrophobic polymer-polymer contacts. This results in increased sugar surface
densities, and explains the increased binding cooperativity and reduced IC50. Furthermore, the
glycopolymer aggregates can block additional areas on the bacteria besides the Man-FimH binding
sites, i.e. steric shielding by these aggregates amplifies the inhibitory potential. The fraction of
glycopolymers not incorporated into aggregates above the cloud point still attained a collapsed
coil conformation since their LCST was likely exceeded, thus showing improved affinity to FimH
due to the increased carbohydrate surface density. For polymers with larger Man functionalization
degree (ManHPL7, ManHPL34, ManHPL97, ManEL5) the Hill coefficients tend to decrease
at elevated temperature since their cloud point was not reached, i.e. a complete coil-to-globule
transition and aggregate formation did not take place.
The results overall showed that the coil-to-globule transition of glycopolymers followed by
aggregate formation and steric shielding increased the E. coli adhesion inhibition. In particular
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polymers with low Man-functionalization degrees of showed a large temperature response, most
likely due to the high fraction of thermoresponsive repeat units. The inhibitory potential of
polymers with hydrophobic linkers (ManEL) did not benefit significantly from the coil-to-globule
transition since they inhibit already strong in the coiled state below the LCST. In addition, owing
to the linker hydrophobicity, the proposed effect of an increased surface presentation of the sugars
on collapsed coils could be reduced compared to ManHPL.
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Table 2 Hill coefficients from the E. coli adhesion inhibition curves.
Sample
Hill coefficient 20 °C
0.5 ± 0.2
Hill coefficient 40 °C
3.1 ± 0.4
ManHPL1
ManHPL2
ManHPL7
ManHPL34
ManHPL97
ManEL1
0.4 ± 0.5
2.7 ± 0.2
1.1 ± 0.1
2.3 ± 0.8
0.6 ± 0.2
0.3 ± 1.0
0.4 ± 0.2
0.1 ± 1.6
1.4 ± 0.2
3.3 ± 1.1
ManEL2
1.2 ± 0.4
4.0 ± 2.7
ManEL5
0.1 ± 1.4
0.7 ± 0.1
3.4 Temperature dependent adhesion inhibition of ConA
We compared E. coli inhibition studies to inhibition studies employing ConA as carbohydrate
binding species. The microplates were again coated with mannan and the IC50 values were
determined by incubating a glycopolymer concentration series in the presence of fluorescein
labeled ConA.
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Figure 5 a) Temperature dependent inhibition of ConA binding. Below the phase transition
temperature, the extended glycopolymer coil can bind to multiple ConA binding sites resulting in
low IC50 values whereas this is not possible in the collapsed state where a large fraction of
carbohydrate units is inaccessible due to aggregate formation. b) A typical inhibition curve at
20 °C and 40 °C for ManHPL7. The full set of inhibition curves is shown in Supporting
Information S11.
As observed in the E. coli assay, when increasing the degree of Man functionalization reduced
IC50 values are observed (Figure 6). However, the results from the inhibition assay with ConA
shows two striking differences compared to the E. coli assay. First, the majority of compounds
now shows increasing IC50 values (weaker inhibition) when increasing the temperature above the
cloud point. Second, changing the linker type does not affect the IC50 values significantly.
Regarding the temperature dependence of the IC50 values, it should be noted that ConA offers four
binding sites with a minimum spacing of 7.2 nm,29, 35 whereas E. coli’s FimH receptor has only a
single binding site and is positioned at larger spacing on the bacteria.45 Dynamic light scattering
of the glycopolymers showed that their coil-size was around 8 nm at 20 °C (Supporting
Information S12). Therefore, the glycopolymers may bind to more than just one ConA binding
site, whereas this is not possible when binding to FimH. At elevated temperatures where the
polymer forms small globules, such multivalent binding to ConA is not possible leading to a
reduced affinity and increased IC50 values. In addition, an extended coil conformation at 20°C
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may increase the accessibility of Man-units for small receptors in solution phase (ConA), whereas
a large fraction of Man units was rendered inaccessible due to aggregate formation above the cloud
point temperature. The presence of microscopic aggregates thus effectively reduced the
glycopolymer inhibitory potential for ConA but not for E. coli as the bacteria are also of
microscopic size, i.e. their inhibition benefits less from an excess of low-affinity glycopolymers
in solution but more from similar sized high-affinity aggregates capable of additional sterical
shielding.27 Therefore, the temperature induced phase transition upon heating resulted in weaker
inhibition of ConA but stronger inhibition of E. coli. Nevertheless, the proposed increase in affinity
in the compact-globule state at elevated temperature due to an increased surface density of Man
units should also be present in case of binding to ConA. Therefore, the thermoresponsive binding
of the glycooligomers to ConA is mediated by adverse effects: 1) reduced multivalent binding and
lower Man accessibility above the LCST; 2) increased binding due to increased surface density of
Man in the globule state. These opposing contributions may explain the less obvious temperature
trend for inhibiting ConA as compared to inhibiting E. coli. This is also reflected in the
comparatively small changes of the Hill coefficients at 20 °C and 40 °C (Table 3).
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The reduced influence of the linker on inhibiting ConA binding when compared to E. coli binding,
could be explained by structural differences of the binding sites. The ConA binding pocket mainly
presents hydrophobic amino acids favoring hydrophobic linkers but at the binding pocket’s
entrance hydrophilic amino acids such as asparagine reside.44,46 These residues may serve as
hydrogen bond acceptors for the hydrophilic HPL linker or, alternatively, hydrogen bonding
bridged by a hydration shell may increase binding to these residues.47 In addition, such hydration
layer mediated hydrogen bonding is generally less favored at increased temperature,48 which
reduces the inhibitory potential at elevated temperature, as observed.
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Figure 6 Left: IC50 values for the inhibition of ConA with ManHPL glycopolymers at 20 °C (blue)
and 40 °C (red). Right: IC50 values for ManEL glycopolymers. All measurements were performed
in triplicates.
Table 3 Hill coefficients from the ConA inhibition curves.
Sample
Hill coefficient at 20 °C
5.2 ± 1.4
Hill coefficient at 40 °C
1.8 ± 0.4
ManHPL1
ManHPL2
ManHPL7
ManEL1
ManEL2
ManEL5
1.8 ± 0.4
3.4 ± 1.0
2.0 ± 0.7
2.6 ± 1.2
1.7 ± 0.9
3.6 ± 0.8
2.5 ± 0.1
2.7 ± 1.1
3.4 ± 1.1
4.4 ± 0.8
Conclusion
Taken together, the straightforward synthesis of a poly(active ester) followed by grafting of
carbohydrates with varying linker hydrophobicity and isopropylamine to induce
thermoresponsiveness was carried out. A set of polymers was synthesized with carbohydrate
functionalization degrees between 1% and 97% and tested by inhibition assays with ConA and
type 1-fimbriated E. coli at 20°C and 40°C. For polymers with low functionalization degrees of
1% to 2% reaching the cloud point at around 40°C a large shift in affinity was observed.
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Importantly, the inhibition of E. coli was increased under cloud point conditions, whereas the
inhibition of ConA had the tendency to decrease at elevated temperatures although the results are
not fully consistent in the case of ConA binding. This confirms the contradicting observations by
several groups studying the effect of the temperature induced coil-to-globule transition on
glycopolymer binding affinities. We propose that the enhanced inhibition of E. coli binding is
driven by an enhanced presentation of carbohydrate units in the collapsed state where the
glycopolymers form micrometer sized high-affinity aggregates capable of sterical shielding. ConA
binding is reduced above the LCST due to reduced multivalent binding and reduced accessibility
of the Man units compared to the extended coil state below the phase transition temperature. This
suggests that small receptor targets such as single lectins in the solution phase are bound stronger
by non-aggregated multivalent glycopolymers in the extended coil conformation, whereas the
inhibition of large targets with surface-anchored receptors having access only to the exterior of
polymer coils (bacteria, viruses) benefits from the increased surface density of ligands and steric
shielding of polymer aggregates above the phase transition temperature. Overall, these results shed
light on the conformation-dependent binding of glycopolymers and give the blueprint for the
design of switchable ligand presenting polymers, e.g. for biomedical applications.
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Acknowledgments
The authors thank Maria Breuer for performing the NMR measurements and Stephanie Scheelen
for performing the SEC measurements. The authors acknowledge funding by the German Research
foundation (DFG) in the project SCHM 2748/5-1.
Conflict of Interest
The authors declare no conflict of interest.
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Tanja J. Paul and Alexander K. Strzelczyk contributed equally to this paper.
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Supporting Information
Chemical analysis, polymer characterization, IC50 curves, DLS measurements, turbidity traces,
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details on instrumentation, buffers and media
References
1.
Reitsma, S.; Slaaf, D. W.; Vink, H.; van Zandvoort, M. A.; oude Egbrink, M. G., The
endothelial glycocalyx: composition, functions, and visualization. Pflugers Arch 2007, 454 (3),
345-59.
2.
Schmidt, S.; Paul, T. J.; Strzelczyk, A. K., Interactive Polymer Gels as Biomimetic
Sensors for Carbohydrate Interactions and Capture–Release Devices for Pathogens. Macromol
Chem Phys 2019, 220 (22), 1900323.
3.
Jacobi, F.; Camaleno de la Calle, A.; Boden, S.; Grafmuller, A.; Hartmann, L.; Schmidt,
S., Multivalent Binding of Precision Glycooligomers on Soft Glycocalyx Mimicking Hydrogels.
Biomacromolecules 2018, 19 (8), 3479-3488.
4.
and problems. FEMS Immunol Med Microbiol 2003, 38 (3), 181-91.
5. Kiessling, L. L.; Grim, J. C., Glycopolymer probes of signal transduction. Chem Soc Rev
2013, 42 (10), 4476-91.
Ofek, I.; Hasty, D. L.; Sharon, N., Anti-adhesion therapy of bacterial diseases: prospects
6.
Hudak, J. E.; Bertozzi, C. R., Glycotherapy: new advances inspire a reemergence of
glycans in medicine. Chem Biol 2014, 21 (1), 16-37.
7.
Steinhilber, D.; Seiffert, S.; Heyman, J. A.; Paulus, F.; Weitz, D. A.; Haag, R.,
Hyperbranched polyglycerols on the nanometer and micrometer scale. Biomaterials 2011, 32 (5),
1311-6.
8.
Papp, I.; Sieben, C.; Ludwig, K.; Roskamp, M.; Bottcher, C.; Schlecht, S.; Herrmann, A.;
Haag, R., Inhibition of influenza virus infection by multivalent sialic-acid-functionalized gold
nanoparticles. Small 2010, 6 (24), 2900-6.
9.
Li, M. H.; Choi, S. K.; Leroueil, P. R.; Baker, J. R., Jr., Evaluating binding avidities of
populations of heterogeneous multivalent ligand-functionalized nanoparticles. ACS Nano 2014, 8
(6), 5600-9.
10.
Bernardi, A.; Jimenez-Barbero, J.; Casnati, A.; De Castro, C.; Darbre, T.; Fieschi, F.;
Finne, J.; Funken, H.; Jaeger, K. E.; Lahmann, M.; Lindhorst, T. K.; Marradi, M.; Messner, P.;
Molinaro, A.; Murphy, P. V.; Nativi, C.; Oscarson, S.; Penades, S.; Peri, F.; Pieters, R. J.;
Renaudet, O.; Reymond, J. L.; Richichi, B.; Rojo, J.; Sansone, F.; Schaffer, C.; Turnbull, W. B.;
Velasco-Torrijos, T.; Vidal, S.; Vincent, S.; Wennekes, T.; Zuilhof, H.; Imberty, A., Multivalent
glycoconjugates as anti-pathogenic agents. Chem Soc Rev 2013, 42 (11), 4709-27.
11.
Architectures. J Am Chem Soc 2016, 138 (28), 8654-66.
12. Muller, C.; Despras, G.; Lindhorst, T. K., Organizing multivalency in carbohydrate
Bhatia, S.; Camacho, L. C.; Haag, R., Pathogen Inhibition by Multivalent Ligand
recognition. Chem Soc Rev 2016, 45 (11), 3275-302.
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Biomacromolecules
1
2
3
4
5
6
7
8
9
13.
Stuart, M. A.; Huck, W. T.; Genzer, J.; Muller, M.; Ober, C.; Stamm, M.; Sukhorukov,
G. B.; Szleifer, I.; Tsukruk, V. V.; Urban, M.; Winnik, F.; Zauscher, S.; Luzinov, I.; Minko, S.,
Emerging applications of stimuli-responsive polymer materials. Nat Mater 2010, 9 (2), 101-13.
14.
thermoresponsive glycopolymers. J Am Chem Soc 2007, 129 (36), 11014-5.
15. Mahon, C. S.; Wildsmith, G. C.; Haksar, D.; de Poel, E.; Beekman, J. M.; Pieters, R. J.;
Pasparakis, G.; Cockayne, A.; Alexander, C., Control of bacterial aggregation by
10
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27
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42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Webb, M. E.; Turnbull, W. B., A 'catch-and-release' receptor for the cholera toxin. Faraday
Discuss 2019, 219 (0), 112-127.
16.
Dalier, F.; Eghiaian, F.; Scheuring, S.; Marie, E.; Tribet, C., Temperature-Switchable
Control of Ligand Display on Adlayers of Mixed Poly(lysine)-g-(PEO) and Poly(lysine)-g-
(ligand-modified poly-N-isopropylacrylamide). Biomacromolecules 2016, 17 (5), 1727-36.
17.
Tang, J. S. J.; Rosencrantz, S.; Tepper, L.; Chea, S.; Klopzig, S.; Kruger-Genge, A.;
Storsberg, J.; Rosencrantz, R. R., Functional Glyco-Nanogels for Multivalent Interaction with
Lectins. Molecules 2019, 24 (10), 22.
18.
Siirila, J.; Hietala, S.; Ekholm, F. S.; Tenhu, H., Glucose and Maltose Surface-
Functionalized Thermoresponsive Poly(N-Vinylcaprolactam) Nanogels. Biomacromolecules
2020, 21 (2), 955-965.
19.
Paul, T. J.; Rubel, S.; Hildebrandt, M.; Strzelczyk, A. K.; Spormann, C.; Lindhorst, T. K.;
Schmidt, S., Thermosensitive Display of Carbohydrate Ligands on Microgels for Switchable
Binding of Proteins and Bacteria. ACS Appl Mater Interfaces 2019, 11 (30), 26674-26683.
20.
Vasani, R. B.; Janardanan, N.; Prieto-Simon, B.; Cifuentes-Rius, A.; Bradley, S. J.;
Moore, E.; Kraus, T.; Voelcker, N. H., Microwave Heating of Poly(N-isopropylacrylamide)-
Conjugated Gold Nanoparticles for Temperature-Controlled Display of Concanavalin A. ACS
Appl Mater Interfaces 2015, 7 (50), 27755-64.
21.
Wang, Y.; Kotsuchibashi, Y.; Liu, Y.; Narain, R., Study of bacterial adhesion on
biomimetic temperature responsive glycopolymer surfaces. ACS Appl Mater Interfaces 2015, 7
(3), 1652-61.
22.
Probing Carbohydrate-Carbohydrate Interactions. ACS Macro Lett. 2018, 7 (2), 178-183.
23. Won, S.; Richards, S. J.; Walker, M.; Gibson, M. I., Externally controllable glycan
Won, S.; Hindmarsh, S.; Gibson, M. I., Triggerable Multivalent Glyconanoparticles for
presentation on nanoparticle surfaces to modulate lectin recognition. Nanoscale Horiz 2017, 2
(2), 106-109.
24.
Siirilä, J.; Hietala, S.; Ekholm, F. S.; Tenhu, H., Glucose and Maltose Surface-
Functionalized Thermoresponsive Poly(N-Vinylcaprolactam) Nanogels. Biomacromolecules
2020, 21 (2), 955-965.
25.
Hoshino, Y.; Nakamoto, M.; Miura, Y., Control of protein-binding kinetics on synthetic
polymer nanoparticles by tuning flexibility and inducing conformation changes of polymer
chains. J Am Chem Soc 2012, 134 (37), 15209-12.
26.
Tang, J. S. J.; Rosencrantz, S.; Tepper, L.; Chea, S.; Klöpzig, S.; Krüger-Genge, A.;
Storsberg, J.; Rosencrantz, R. R., Functional Glyco-Nanogels for Multivalent Interaction with
Lectins. Molecules 2019, 24 (10), 1865.
27.
Vonnemann, J.; Liese, S.; Kuehne, C.; Ludwig, K.; Dernedde, J.; Bottcher, C.; Netz, R.
R.; Haag, R., Size dependence of steric shielding and multivalency effects for globular binding
inhibitors. J Am Chem Soc 2015, 137 (7), 2572-9.
23
ACS Paragon Plus Environment

Biomacromolecules
Page 24 of 26
1
2
3
4
5
6
7
8
9
28.
Mammen, M.; Choi, S. K.; Whitesides, G. M., Polyvalent interactions in biological
systems: Implications for design and use of multivalent ligands and inhibitors. Angew Chem Int
Ed 1998, 37 (20), 2755-2794.
29.
using synthetic biomolecules. Curr Opin Chem Biol 2013, 17 (6), 982-9.
30. Hartmann, M.; Horst, A. K.; Klemm, P.; Lindhorst, T. K., A kit for the investigation of
live Escherichia coli cell adhesion to glycosylated surfaces. Chem Commun 2010, 46 (2), 330-2.
31. Gerchakov, S. M.; Hatcher, P. G., Improved Technique for Analysis of Carbohydrates in
Sediments. Limnol Oceanogr 1972, 17 (6), 938-943.
32. DuBois, M.; Gilles, K. A.; Hamilton, J. K.; Rebers, P. A.; Smith, F., Colorimetric Method
for Determination of Sugars and Related Substances. Anal Chem 1956, 28 (3), 350-356.
33. Boden, S.; Wagner, K. G.; Karg, M.; Hartmann, L., Presenting Precision
Glycomacromolecules on Gold Nanoparticles for Increased Lectin Binding. Polymers 2017, 9
(12), 716.
34.
Fernández-García, M., Glycopolymers with glucosamine pendant groups: Copolymerization,
physico-chemical and interaction properties. React Funct Polym 2011, 71 (1), 1-10.
35.
Chem Soc Rev 2013, 42 (10), 4492-503.
36. Becker, J. W.; Reeke, G. N., Jr.; Cunningham, B. A.; Edelman, G. M., New evidence on
the location of the saccharide-binding site of concanavalin A. Nature 1976, 259 (5542), 406-9.
37. Bryce, R. A.; Hillier, I. H.; Naismith, J. H., Carbohydrate-protein recognition: molecular
dynamics simulations and free energy analysis of oligosaccharide binding to concanavalin A.
Biophys J 2001, 81 (3), 1373-88.
38.
Wittmann, V., Structural investigation of multivalent carbohydrate-protein interactions
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Bordegé, V.; Muñoz-Bonilla, A.; León, O.; Sánchez-Chaves, M.; Cuervo-Rodríguez, R.;
Wittmann, V.; Pieters, R. J., Bridging lectin binding sites by multivalent carbohydrates.
Dam, T. K.; Brewer, C. F., Thermodynamic studies of lectin-carbohydrate interactions by
isothermal titration calorimetry. Chem Rev 2002, 102 (2), 387-429.
39.
78.
40.
Lundquist, J. J.; Toone, E. J., The cluster glycoside effect. Chem Rev 2002, 102 (2), 555-
Gestwicki, J. E.; Cairo, C. W.; Strong, L. E.; Oetjen, K. A.; Kiessling, L. L., Influencing
receptor-ligand binding mechanisms with multivalent ligand architecture. J Am Chem Soc 2002,
124 (50), 14922-33.
41.
domain of the bacterial adhesin FimH: Design, synthesis and binding properties of mannoside
ligands. Org Biomol Chem 2006, 4 (21), 3913-22.
Sperling, O.; Fuchs, A.; Lindhorst, T. K., Evaluation of the carbohydrate recognition
42.
Knight, S. D.; Bouckaert, J., Structure, function, and assembly of type 1 fimbriae. Topics
curr chem 2009, 288, 67-107.
43.
for the design of high affinity lectin ligands. Chem Rev 2015, 115 (1), 525-61.
44.
Weber, M.; Hartmann, L., Linear Precision Glycomacromolecules with Varying Interligand
Spacing and Linker Functionalities Binding to Concanavalin A and the Bacterial Lectin FimH.
Macromol Biosci 2017, 17 (12), 1700198.
Cecioni, S.; Imberty, A.; Vidal, S., Glycomimetics versus multivalent glycoconjugates
Igde, S.; Roblitz, S.; Muller, A.; Kolbe, K.; Boden, S.; Fessele, C.; Lindhorst, T. K.;
45.
Mortezaei, N.; Singh, B.; Zakrisson, J.; Bullitt, E.; Andersson, M., Biomechanical and
structural features of CS2 fimbriae of enterotoxigenic Escherichia coli. Biophys J 2015, 109 (1),
49-56.
24
ACS Paragon Plus Environment

Page 25 of 26
Biomacromolecules
1
2
3
4
5
6
46.
Sanders, D. A.; Moothoo, D. N.; Raftery, J.; Howard, A. J.; Helliwell, J. R.; Naismith, J.
H., The 1.2 A resolution structure of the Con A-dimannose complex. J Mol Biol 2001, 310 (4),
875-84.
47.
Loris, R.; Stas, P. P.; Wyns, L., Conserved waters in legume lectin crystal structures. The
7
importance of bound water for the sequence-structure relationship within the legume lectin
family. J Biol Chem 1994, 269 (43), 26722-33.
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
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24
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
48.
Lemieux, R. U., How Water Provides the Impetus for Molecular Recognition in Aqueous
Solution. Acc Chem Res 1996, 29 (8), 373-380.
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Temperature switchable glycopolymers and their
conformation-dependent binding to receptor targets
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*
†#
†#
†
Tanja J. Paul , Alexander K. Strzelczyk , Melina I. Feldhof, and Stephan Schmidt
E. coli
20°C 40°C
ConA
20°C 40°C
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