Preparation and characterization of glycopolymers with biphenyl spacers: Via Suzuki coupling reaction

Article abstract of DOI:10.1039/d1ob00617g

Poly(vinylbiphenyl)s bearing glycoside ligands at the side chains were prepared using the Suzuku coupling reaction. Effects of glycoside reactant concentration, halide species, glycoside species, and catalyst species on the incorporation of glycoside ligand into the polymer were investigated. The obtained glycopolymers exhibited specific binding to proteins corresponding to the glycoside ligands. In addition, the biphenyl spacers formed by the Suzuki coupling reaction in the glycopolymer were fluorescent, whereas the polymer precursor was not.

Full text of DOI:10.1039/d1ob00617g

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Preparation and characterization of glycopolymers
with biphenyl spacers via Suzuki coupling
reaction†
Cite this: DOI: 10.1039/d1ob00617g
Received 31st March 2021,
Accepted 24th April 2021
Hirokazu Seto, * Takumi Tono, Akiko Nagaoka, Mai Yamamoto, Yumiko Hirohashi
and Hiroyuki Shinto
DOI: 10.1039/d1ob00617g
rsc.li/obc
Poly(vinylbiphenyl)s bearing glycoside ligands at the side chains
Two methods for the preparation of glycopolymers are con-
were prepared using the Suzuku coupling reaction. Effects of gly- sidered. One is the direct polymerization of the glycomono-
coside reactant concentration, halide species, glycoside species, mer, which has already been employed to synthesize many
and catalyst species on the incorporation of glycoside ligand into polymerizable glycosides.19 The other method involves the
the polymer were investigated. The obtained glycopolymers preparation of polymer precursors with functional groups, fol-
exhibited specific binding to proteins corresponding to the glyco- lowed by the incorporation of glycoside ligands via chemical
side ligands. In addition, the biphenyl spacers formed by the reactions. Various chemoselective reactions have been used in
Suzuki coupling reaction in the glycopolymer were fluorescent, post-polymerization functionalization.20 The glycoside density
whereas the polymer precursor was not.
in the polymer is tuned by the feed ratio of the monomer and
the concentration of the glycoside reactant in the direct
polymerization and post-incorporation methods, respectively.
The direct polymerization method allows for sequence control
of polymeric units, such as in the preparation of block copoly-
mers. In contrast, the post-incorporation method is suitable
for the preparation of uniform polymers with identical average
molecular weights and distributions. A series of different gly-
copolymers based on a single polymer backbone is easily pro-
duced, and the biological function of glycopolymers with
different glycoside species and densities can be investigated
without considering the factor of inhomogeneous structures.
Nagao et al. have successfully prepared glycopolymers with oli-
gomeric glycosides using reversible addition–fragmentation
chain transfer polymerization (RAFT) and post-click chemistry
between azide and alkyne.21–23 Living radical polymerization
techniques facilitate both methods of glycopolymer
preparation.
Cross-coupling reactions using palladium catalysts are
powerful tools for the chemical transformations of pharmaceu-
ticals, agrochemicals, liquid crystal materials, and natural pro-
ducts. The Suzuki coupling reaction between phenylboronic
acid and phenyl halide offers several advantages:24 (1) various
phenylborates are commercially and synthetically available, (2)
phenylborates are stable in air and water, (3) the coupling reac-
tions proceed under mild conditions (even in water), (4) the
boron byproducts have low toxicity and can be easily removed,
(5) the tolerability of functional groups is high, (6) regio- and
stereoselectivities are high, and (7) the reaction is less suscep-
tible to steric hindrance. A large variety of phenylborate poly-
Glycosides on cellular membranes are involved in various bio-
logical phenomena, including pathogen infection, cell
adhesion, and cancerous metastasis.1 The specificity of the gly-
coside ligands enables applications in the fields of biosensing,
bioseparation, and biotargeting. Unfortunately, glycoside-
protein interactions are relatively weak in the monomeric state.
On cellular membranes, glycosides are conjugated with lipids
and proteins, and tend to form densely packed structures (rafts
and caveolae). These multivalent states amplify the glycoside-
protein interaction, known as the glyco-cluster effect.2–4
Various models have been developed on the biological
systems, artificial glycoconjugates based on peptides,5,6 DNA,7
liposomes,8,9 cyclodextrins,10,11 calixarenes,12,13 and synthetic
polymers.14–16 Interfaces, which are densely immobilized with
glycoside ligands, also exhibit specific and robust binding to
various biomolecules, and are useful in bioapplications.
Recently, we reported a method for the formation of a glyco-
functionalized interface and its application in biosensing.17,18
Among the glycoconjugates, glycopolymers with glycoside
ligands at side chains have been the subject of conspicuous
attention.
Department of Chemical Engineering, Fukuoka University, 8-19-1 Nanakuma, Jonan-
ku, Fukuoka, 814-0180, Japan. E-mail: hirokazuseto@fukuoka-u.ac.jp
†Electronic supplementary information (ESI) available: Syntheses of polymer
precursor and p-halophenyl glycosides, process of the Suzuki coupling reaction,
and QCM and fluorescent spectra measurements. See DOI: 10.1039/
D1OB00617G
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mers have been suggested,25 which were synthesized via percentage of the glycoside ligand was estimated from nuclear
RAFT,26,27 atom transfer radical polymerization (ATRP),28,29 magnetic resonance (NMR) spectra (Fig. S2, ESI†). When
and nitroxide-mediated polymerization (NMP).30,31 To the best pBP-Man with a concentration of 20 mmol L−1 was coupled
of our knowledge, there are no reports on the post-incorpor- with poly(VPBA), a remarkable peak, corresponding to an
ation method using the Suzuki coupling reaction for the prepa- anomeric proton, appeared in the NMR spectrum, suggesting
ration of glycopolymers.
that the glycosides were incorporated into the polymer. The
Here, we report a novel method for the preparation of glyco- incorporation of the glycosides in the polymer was also con-
polymers via post-incorporation using living radical polymeriz- firmed by Fourier transform infrared spectroscopies (Fig. S3,
ation and catalytic coupling reaction with palladium catalysts. ESI†). In this Suzuki coupling reaction, DMF should function
In the post-incorporation method using the Suzuki coupling as a reductant and protectant for palladium.33 The glycoside
reaction, phenylborate polymer precursors obtained via NMP ligand was incorporated into the polymer using pBP-Man at
should be applicable rather than those obtained via RAFT, various concentrations (Fig. 2). The incorporation percentage
because the terminal thiocarbonylthio group acts as a catalyst of glycoside increased with increasing concentration of
poison during the subsequent palladium-catalyzed reaction. pBP-Man, and Man ligand could be incorporated into almost
Various glycoside ligands were incorporated into a single all phenylboronic acid units in the polymer at more than
polymer backbone with boronic acids via Suzuki coupling 100 mmol L−1. The Suzuki coupling reaction between poly
(Fig. 1). The effects of glycoside reactant concentration, halide (VPBA) and pBP-Man was also performed in a heterogenous
species, and glycoside species on the incorporation of glyco- system (see ESI†). The incorporation of the Man ligand into
side ligands were investigated.
the polymer using Pd/C was confirmed, but the incorporation
The poly(vinylphenylboronic acid) (poly(VPBA)) precursor percentage was low.
was synthesized via NMP (Fig. S1, ESI†). During the polymeriz-
The catalytic reactivities of pCP-Man, pBP-Man, and
ation, water was added to suppress the formation of cyclic pIP-Man were compared (Table 1). The Man ligand was not
borate trimers.30–32 The polydispersity index of poly(VPBA), incorporated using pCP-Man. However, the incorporation per-
determined by gel permeation chromatography analysis, was centage using pIP-Man was higher than that using pBP-Man.
narrow owing to the living radical polymerization technique. This reactivity was consistent with the typical order for the
p-Bromophenyl-α-D-mannopyranoside (pBP-Man), p-chlorophe- Suzuki coupling reaction.34 Suzuki coupling proceeds via oxi-
nyl-α-D-mannopyranoside (pCP-Man), p-iodophenyl-α-D-man- dative addition, transmetallation, and reductive elimination
nopyranoside (pIP-Man), p-bromophenyl-β-D-galactopyranoside steps. Oxidative addition is often the rate-determining step in
(pBP-Gal), and p-bromophenyl-N-acetyl-β-D-glucosaminide the catalytic cycle. The presence of electron-withdrawing
(pBP-GlcNAc) were prepared (see ESI†).
groups promotes oxidative addition, compared to that of elec-
Poly(VPBA) was dissolved in a solvent mixture of N,N-di- tron-donating groups. Therefore, the reactivity was in the order
methylformamide (DMF) and water (50 : 50) to obtain a con- of pIP-Man > pBP-Man > pCP-Man. The incorporation of glyco-
centration of 2 g L−1, which corresponded to a phenylboronic side ligands using pCP-glycoside is expected to improve by
acid unit concentration of 13.5 mmol L−1. Na2CO3 (20 mmol employing bulky phosphine-coordinated Pd catalysts, such as
L−1) and p-halophenyl glycoside were added to the polymer Pd2(dba)3/P(t-Bu)3,35 PdCl2/JohnPhos,36 XPhos Pd G2,37 and
solution, and the mixture was heated to 80 °C. The Suzuki XantPhos Pd G3,38 which have been successfully used in the
coupling reaction was initiated by the addition of Pd(OAc)2. cross-coupling reaction of aryl chlorides. Furthermore, the
The mixture was stirred at 80 °C for 24 h. After the reaction,
the palladium catalyst was removed using a scavenger. The
products were purified by dialysis (molecular weight cut-off:
3500 Da) against DMSO and then water. The solvent was evap-
orated, and the sample was dried in vacuo. The incorporation
Fig. 1 Preparation of glycopolymers with biphenyl spacer via Suzuki
coupling reaction between poly(VPBA) and p-halophenyl glycoside (X:
Cl, Br, or I).
Fig. 2 Incorporation percentage of Man ligand into poly(VPBA) at
different concentrations of pBP-Man. Incorporation percentages were
determined from two different samples.
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Table 1 Incorporation percentages of glycoside ligand to polymer
main chain using different p-halophenyl glycoside
p-Halophenyl glycoside
pBP-Man
Incorporation percentage (%)
64 10
pCP-Man
pIP-Man
<1
84
4
pBP-Gal
69
32
4
8
Fig. 3 Frequency changes in QCM for adsorption of proteins to the
poly(VBiph-Man)-immobilized surface at the different protein concen-
trations. Solid line represents curve fitting based on the Langmuir
equation (R2 = 0.986). The error bars were determined from two
different measurements.
pBP-GlcNAc
[Poly(VPBA)]: 2 g L−1, [p-halophenyl glycoside]: 20 mmol L−1, [Pd
(OAc)2]: 0.1 mmol L−1, 80 °C, 24 h. Incorporation percentages were
determined from two different samples.
catalytic reactivities of different pBP-glycosides to poly(VPBA)
were compared (Table 1). When all the pBP-glycosides were
used, incorporation of glycosides was confirmed. However, the
incorporation percentage using pBP-GlcNAc was low. The
incorporation of pBP-GlcNAc should be decreased by the steric
hindrance of the acetamide group at the C2 position. The use
of pIP-glycoside will most likely lead to an improvement in the
incorporation of GlcNAc ligands.
The protein-binding abilities of the glycopolymers obtained
by the Suzuki coupling reaction were evaluated using quartz
crystal microbalance (QCM). The Man-incorporated glycopoly-
mer with biphenyl spacers (poly(VBiph-Man)) was prepared at
a high concentration of pBP-Man, and immobilized on the
surface of sensor chips via an amine coupling reaction (see
ESI†). Concanavalin A (Con A) is a glucoside- and mannoside-
binding protein. Wheat germ agglutinin (WGA) is an N-acetyl-
glucosaminide-binding protein. The −ΔF values of the poly
Fig. 4 Fluorescent spectra of poly(VPBA) and poly(VBiph-Man) in
water.
(VBiph-Man) for protein adsorption at different concentrations while a fluorescence peak was observed at 340 nm in the spec-
are shown in Fig. 3. The −ΔF value for Con A was greater than trum of poly(VBiph-Man). The poly(VBiph-Gal) and poly
that for WGA. The specificity of poly(VBiph-Man) was relatively (VBiph-GlcNAc) were also fluorescent (Fig. S5, ESI†). Biphenyl
high. Using the Langmuir equation, the apparent dissociation spacers, which were formed by the Suzuki coupling reaction
constant (KD) between poly(VBiph-Man) and Con A was esti- between polymer main chain and glycoside, functioned as
mated to be 2.5 × 10−7 mol L−1. This KD value was of the same fluorescent groups. However, note that the excitation and
order as that of previously reported polyacrylamide- and poly- emission wavelengths of poly(VBiph-Man) were in the UV
methacrylate-based glycopolymers,39,40 and was sufficient for region, whereas operation at longer wavelengths is desired for
applications as a ligand in the field of biosensing and biose- bioapplication. Improvement in the properties of poly(VBiph-
paration. Poly(VBiph-Gal) and poly(VBiph-GlcNAc) also showed glycoside), which has binding ability and fluorescence, and its
high specificity and binding ability (Fig. S4, ESI†).
applications, such as cellular staining, are under investigation.
In summary, we proposed the preparation of glycopolymers
We discovered another interesting feature of poly(VBiph-
Man) other than its protein-binding ability. The fluorescence via post-incorporation using the Suzuki coupling reaction.
of the polymers before and after the Suzuki coupling reaction Various glycosides were incorporated into poly(VPBA). To
was determined (see ESI†). The fluorescence spectra of poly prepare poly(VBiph-glycoside) with a high incorporation per-
(VPBA) and poly(VBiph-Man) in water are shown in Fig. 4. As a centage, the use of pIP-glycoside and excess glycoside reactant
polymer precursor, poly(VPBA) did not exhibit fluorescence, was effective. Increasing the catalyst concentration may also be
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Conflicts of interest
There are no conflicts to declare.
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Acknowledgements
This study was supported by JSPS KAKENHI (grant number
19K05042). This study was funded by the Kurita Water and
Environment Foundation, Shiraishi Scientific Research Fund
of Fukuoka University (no. SK2001), and the Central Research
Institute of Fukuoka University (no. 175009 and 207103). We
are grateful for the assistance of Prof. Y. Miura and Assoc.
Prof. Y. Hoshino (Kyushu University) for providing access to
the Affinix Q8 system. We would like to thank Editage for
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