Preparation of functional monomers as precursors of bioprobes from a common styrene derivative and polymer synthesis

Article abstract of DOI:10.3390/molecules23112875

CM-Str (4-(Chloromethyl)styrene) was used as a useful starting material for the construction of a series of functional monomers. Substitution of the chlorine to the corresponding azide was performed, and the reduction of the azide proceeded smoothly to afford an aminostyrene, which was used as a common precursor for the preparation of functional monomers. Condensation of the amine with a fluorophore, biotin and carbohydrate was accomplished. Among the monomers, a carbohydrate monomer was polymerized with or without acrylamide as a model polymerization to yield the corresponding water-soluble glycopolymers, and biological evaluations of the glycopolymers for a lectin, and wheat germ agglutinin (WGA), were carried out on the basis of the fluorescence change of tryptophan in the WGA.

Full text of DOI:10.3390/molecules23112875

molecules
Article
Preparation of Functional Monomers as Precursors of
Bioprobes from a Common Styrene Derivative and
Polymer Synthesis
1
1
1,2
, Ken Hatano 1,2
Riho Hayama , Tetsuo Koyama , Takahiko Matsushita
and Koji Matsuoka 1
,2,
*
1
Division of Material Science, Graduate School of Science and Engineering, Saitama University, Sakura,
Saitama 338-8570, Japan; ri11270825@gmail.com (R.H.); koyama@fms.saitama-u.ac.jp (T.K.);
takahiko@fms.saitama-u.ac.jp (T.M.); khatano@fms.saitama-u.ac.jp (K.H.)
Medical Innovation Research Unit (MiU), Advanced Institute of Innovative Technology (AIIT),
Saitama University, Sakura, Saitama 338-8570, Japan
2
*
Correspondence: koji@fms.saitama-u.ac.jp; Tel.: +81-48-858-3099
Academic Editor: Lothar Elling
Received: 28 September 2018; Accepted: 2 November 2018; Published: 4 November 2018
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: CM-Str (4-(Chloromethyl)styrene) was used as a useful starting material for the construction
of a series of functional monomers. Substitution of the chlorine to the corresponding azide
was performed, and the reduction of the azide proceeded smoothly to afford an aminostyrene,
which was used as a common precursor for the preparation of functional monomers. Condensation
of the amine with a fluorophore, biotin and carbohydrate was accomplished. Among the
monomers, a carbohydrate monomer was polymerized with or without acrylamide as a model
polymerization to yield the corresponding water-soluble glycopolymers, and biological evaluations
of the glycopolymers for a lectin, and wheat germ agglutinin (WGA), were carried out on the basis of
the fluorescence change of tryptophan in the WGA.
Keywords: styrene; glycopolymers; radical polymerization; lectins; fluorescence spectrometry
1
. Introduction
Although oligosaccharides and their derivatives are of great interest as bioactive compounds
in various research areas, the affinities of the carbohydrate moieties are not so high [1,2]. In nature,
the oligosaccharide chains in glycoconjugates play a role and have high specificities in biological
events, and a recent report suggested that the affinities of oligosaccharide chains in glycolipids and
glycoproteins were enhanced by the self-assembly of glycoconjugates, which display multivalent-type
clusters. Much attention has been paid to the formation of synthetic glycoclusters by covalent bonds,
and glycodendrimers [3–5] and glycopolymers [6–10] have been successfully developed. In our
ongoing studies of glycopolymers, we have reported a conventional method for the preparation of a
glycopolymer by means of Huisgen cycloaddition as a key reaction for the synthesis of monomers and
glycopolymers [11]. The efficient assembly of carbohydrate moieties induced an enhancement of the
affinities for a lectin on the basis of carbohydrate-protein interactions [12–15].
The functionalization of monomers as bioprobes is highly attractive and of great importance in
order to evaluate their biological phenomena. For example, fluorogenic monomers are useful in order
to evaluate an interaction between carbohydrate molecules and a glyscosidase [16]. In addition to
the fluorogenic compounds, the biotin-avidin interaction is also an important biological event and
a useful biological tool for biochemical and biomedical uses [17
,18]. In this paper, we describe an
alternative pathway for the convenient preparation of a glycomonomer and functional monomers
Molecules 2018, 23, 2875; doi:10.3390/molecules23112875
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2875
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using a commercially available and inexpensive reagent, 4-chloromethylstyrene (CM-Str), as a starting
material. The further transformation of the glycomonomer into corresponding glycopolymers is
reported. In addition to the syntheses, the results of the biological evaluations of the glycopolymers
with N-acetyl-D-glucosamine (NAG) residues for wheat germ agglutinin (WGA) [19] as a model lectin
are also described.
2
. Results and Discussion
The utilization of 4-(chloromethyl)styrene
1 (CM-Str) as a starting material for the preparation of
a series of functionalized monomers was examined in order to construct polymeric bioprobe families.
We previously reported a convenient synthesis of glycomonomers using CM-Str as a starting material
by means of the Huisgen cycloaddition reaction [20 23] and polymer syntheses [11]. In this paper,
alternative preparations of a glycomonomer and other useful functional monomers are described.
1
–
2
.1. Conversion of 4-(Chloromethyl)styrene into an Amine as a Common Precursor
Commercially available CM-Str as a versatile precursor for an azide compound was used as a
convenient starting material, and the conversion of the azide (AZ-Str) [24] into the corresponding
amine derivative by the Staudinger reaction was performed. Scheme 1 summarizes the preparation
of aminostyrene derivative (AMN-Str) [25] from CM-Str . The S 2 replacement of the chloride of
CM-Str was performed by an azide anion in a polar aprotic solvent at a slightly high temperature
to afford the corresponding AZ-Str in 90% yield [11]. The Staudinger reaction of AZ-Str with
triphenylphosphine (TPP) afforded the corresponding CM-Str in 96% yield. Two-step-conversion
from to was reported and the yield was moderate (63% in two steps) [25]. In our case, the yield
was improved (86% in two steps) by the replacement of a solvent from benzene to THF in Scheme 1(ii).
1
2
3
1
N
1
2
2
3
1
3
i)
ii)
Cl
N3
NH2
1: CM-Str
2: AZ-Str
3: AMN-Str
◦
◦
Scheme 1. Reagents and conditions: (i) NaN , DMF, 80 C, 1 h, (ii) TPP, H O, THF, 0 C, 2.5 h.
3
2
2
.2. Synthetic Conversion of the Amine into Functionalized Monomers
Given the success of the simple preparation of AMN-Str
amino function, we next turned our attention to the applicability of AMN-Str
precursor for the synthesis of functional monomers. Scheme 2 shows the chemical conversion of
AMN-Str into useful monomers with a styrene moiety. Commercially available acid chloride
of a dansyl derivative was first used to introduce fluorescent moieties into the synthetic polymer.
Thus, the dansylation of AMN-Str with dansyl chloride in the presence of an acid scavenger
proceeded smoothly to afford the dansylated styrene derivative
3, which had a highly reactive primary
3
as a common
3
3
4
(DSL-Str) [26] as a fluorogenic
1
monomer in 76% yield. Structural elucidations were performed by a combination of IR, H-NMR and
elemental analyses. An alternative functionalization of AMN-Str
3 was performed using a biotin as an
attractive biomolecule in order to obtain a strong interaction between an avidin and the biotin [18].
Thus, the D-(+)-biotin was coupled with AMN-Str
3
using typical condensing reagents [27] such as
0
1
-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), N,N -diisopropyl carbodiimide
(
DIC), and (benzotriazol-1-yloxy)tris(dimethylamino)phosphonium hexafluorophosphate (BOP).
All of the coupling reagents unfortunately produced unsuccessful or poor results due to
a solubility problem of the biotin derivatives. The results are summarized in Table 1.
Since well-known condensing reagents did not work in this coupling reaction, we decided to
use 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), which was

Molecules 2018, 23, 2875
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introduced by Kunishima et al. [28]. The biotin was treated with AMN-Str
3
in the presence of
DMT-MM to afford the corresponding amide 5 (BTN-Str) in 91% yield.
Me
N
Me
O
S
Cl
Me
Me
O
N
O
S
i)
N
H
O
4: DSL-Str
H2N
HN
3
: AMN-Str
O
S
O
HN
NH
ii)
O
H
H
NH
O
H
H
N
H
OH
5: BTN-Str
S
Scheme 2. Reagents and conditions: (i) Dansyl chloride, Et N, dichloromethane (DCM), rt, 5.5 h,
3
(ii) biotin, DMT-MM, DMF, rt, 1.5 h.
Table 1. Results of the coupling reaction of AMN-Str 3 and biotin.
◦
Reagent
Solvent
Temp ( C)
Time (h)
Yield (%)
DIC
EDC
BOP
DMF
DMF
Rt
Rt
Rt
Rt
Rt
on
5
3
1.5
2
0
0
36
17
91
1
CH CN
3
1
BOP
DMF
DMF
DMT-MM
1
including a trace of inseparable byproducts.
2
.3. Preparation of a Carbohydrate Monomer
Although the effective conversion of the aminostyrene
3
(AMN-Str) into functional monomers
was accomplished, the further transformation of the amine was required in order to produce
carbohydrate monomers, which are bioactive compounds and are recognized by various lectin
molecules. A schematic diagram of the preparation of a glycomonomer 10 (NAG-Str) is shown in
Scheme 3. Since the hydrophilicity of the styrene moiety was low, we decided to use a polyethyleneoxy
alcohol, which has the appropriate hydrophilicities as an aglycon moiety. Thus, a known oxazoline
0
derivative
6
[
29] was treated with 2,2 -(ethylenedioxy)diethanol in the presence of camphorsulfonic
acid (CSA) [30] to yield a monoalcoholic glycoside
7
as a yellow syrup in 76% yield. An anomeric
1
configuration of glycoside
assignable to H-1 at
The primary alcohol had to be oxidized to carboxylic acid before the coupling reaction of the
glycoside with the amine . Thus, 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO)-mediated
oxidation [31] of the alcohol was carried out to produce the desired carboxylic acid , which was
7
was determined using H-NMR spectra. The results revealed a signal
δ
4.77 as a doublet with J1,2 = 8.60 Hz, which was confirmed to be a β-glycoside.
3
7
8
directly used for the next step due to the production of an inseparable byproduct in the reaction.
We therefore estimated the yield of the oxidation to be approximately 46%. Freshly prepared carboxylic
acid
proceeded smoothly to produce the corresponding styrene derivative
yield after chromatographic purification. Structural elucidation of product
8
and the amine
3
were applied for a DMT-MM-mediated coupling reaction, and the reaction
as a colorless syrup in 78%
was performed by means
9
9
1
13
of H- and C-NMR spectroscopic analyses. Since the appearance of aromatic ring proton signals
1
at 7.39 ppm and 7.27 ppm in
9
was observed in the H-NMR results, the progress of the coupling
1
13
reaction was confirmed. In addition to the H-NMR results, the combined results of the C-NMR
and HMQC experiments also supported the formation of . The removal of the acetyl protection in
9
9

Molecules 2018, 23, 2875
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was performed by using the Zemplén method [32] to afford NAG-Str 10 as a water-soluble material in
9
9% yield.
OAc
O
OAc
O
OR
O
O
AcO
AcO
i)
AcO
AcO
iii)
RO
RO
O
O
R
O
O
O
O
N
H
NHAc
: R = CH2OH
8 : R = COOH
NHAc
O
N
7
9 : R = Ac
10 : R = H (NAG-Str)
ii)
iv)
6
CH3
0
Scheme 3. Reagents and conditions: (i) 2,2 -[ethane-1,2-diylbis(oxy)]di(ethan-1-ol), CSA, ClCH CH Cl,
9
2
2
◦ ◦
0 C, 1 h, (ii) aq. NaHCO , KBr, TEMPO, trichloroisocyanuric acid, acetone, 0 C, 20 min, then rt, 4.5 h;
3
(iii) AMN-Str 3, DMT-MM, DCM, rt, 3.5 h; (iv) NaOMe, MeOH, rt, 1.5 h.
2
.4. Polymerization of a Glycomonomer by Means of Radical Polymerization
The synthetic conversion of AMN-Str into functional monomers was successfully demonstrated,
3
and our next objective was the chemical conversion of the monomers into the corresponding polymers.
In our synthetic study of glycoclusters, radical polymerization was applied for the synthetic assembly
of carbohydrate epitopes. This type of monomer had not hitherto been used in our study. Thus,
the N-acetyl-D-glucosaminyl monomer 10 (NAG-Str) was used as a model monomer for the preparation
of glycopolymers, as shown in Scheme 4. The polymerization ability of NAG-Str 10 was first evaluated
by radical polymerization without a comonomer in aqueous media at room temperature. NAG-Str 10
underwent homopolymerization to yield white powdery 11a in 71% yield after dialysis against water
followed by lyophilization. A successful polymerization protocol was applied for the preparation of
copolymers of NAG-Str 10 and acrylamide, and the results of the copolymerizations are summarized
in Table 2. The weight-average molecular weights (Mw) of the glycopolymers were estimated by
size exclusion chromatography in 0.3 M aqueous NaCl solution. From the weight-average molecular
weight (Mw) of glycopolymer 11a, the degree of polymerization was estimated to be 653. The polymer
1
compositions of glycopolymers 11b
,
11c and 11d were estimated on the basis of the H-NMR results.
All of the polymers (11a
–
11d) had water-soluble abilities and appropriate molecular weights as
estimated by size-exclusion chromatography (SEC) analyses.
OH
O
OH
O
O
HO
HO
O
O
O
HO
HO
O
O
i)
O
N
H
O
N
H
NHAc
x
y
NHAc
1
0
11a : x:y:n = 1:0:653
1
1
1
1b : x:y:n = 1:4:616
1c : x:y:n = 1:11:145
1d : x:y:n = 1:22:145
H2N
O
n
Scheme 4.
Reagents and conditions:
(i) acrylamide, ammonium persulfate (APS),
tetramethylethylenediamine (TEMED) rt.
Table 2. Results of polymerizations of NAG-Str 10 with or without acryl amide (AAm).
Monomer
Ratio
Total
Yield
(%)
Polymer
Composition
x:y:n
Sugar
Content
(wt%)
1
2
Mw 3 (kDa) D 4
Polymer
Time (h)
Solvent
1
0:AAm
2
1
1
:0
:4
11a
11b
11c
11d
69
24
24
24
H2O
H2O
H2O
H2O
71
81
99
90
1:0:6.5 × 10
1:4:6.2 × 10
1:11:1.5 × 10
1:22:1.5 × 10
100
63
38
315
472
183
297
1.13
1.01
2.52
1.34
2
2
1
1
:10
:20
2
24
1
2
Total yields were calculated on the basis of the quantities of monomers used. Polymer compositions of sugar
1
3
unit:acrylamide unit were estimated on the basis of the H-NMR results. The weight-average molecular weights
were estimated by size-exclusion chromatography in 0.3 M aq NaCl solution using a Shodex GF-510HQ column.
Calibration curves were obtained using pullulan standards (5.9, 11.8, 22.8, 47.3, 112, 212, 404, and 788 kDa, Shodex
4
P-82). Dispersity (D) were calculated on the basis of (Mw)/(Mn).

Molecules 2018, 23, 2875
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2
.5. Biological Evaluations of the Glycopolymers for Lectin
Once the synthetic assembly of sugar moieties using polymer chemistry was accomplished,
we turned our attention to the biological properties of the glycopolymers. Interactions between
glycopolymers 11a 11d and WGA, which showed high binding specificity for N-acetyl-D-glucosamine
NAG) and its oligomers [33 34], were therefore evaluated on the basis of fluorescence measurements.
–
(
,
Fluorescence measurements of WGA and polymers with NAG residues were preliminarily performed
◦
in 0.65
µ
M of WGA solution in 50 mM Tris-HCl buffer (pH 7.4) at 4 C [11]. The results of the
measurement using 11b and the analyses of the results are shown in Figure 1 as an example.
The intensity of the peaks at 348 nm gradually increased upon addition of NAG polymer solutions,
as shown in Figure 1a. The difference between the wavelengths on the maximum relative fluorescence
intensity of WGA alone and WGA with the saturated concentration of a glycopolymer is represented
as ∆λ. From the results in Figure 1a, we can see that ∆λ was
−
14.2 nm, as shown in Table 3.
These phenomena occurred due to the change in the environment around the tryptophan residue
located at or near the binding sites of WGA. The changes in fluorescence intensity were substituted
into the Hill equation [35]:
ꢀ
ꢁ
∆
F
log
= log[S] + log Ka
(1)
∆
FMAX − ∆F
where
∆
F (
∆
F = F
−
F0) is the difference in relative fluorescence intensity at 348 nm of WGA between
F, which is the fluorescence intensity of WGA with various concentrations of a glycopolymer and F ,
0
which is fluorescence intensity of a solution of WGA itself (without a glycopolymer); and ∆FMAX is the
difference between FMAX, which is the final fluorescence intensity at the saturated concentration of a
glycopolymer, and F0.
Figure 1. Biological evaluations of the WGA–carbohydrate interaction of 11b
.
(
a
) Changes in
M, 3.0 mL, 50 mM Tris-HCl buffer containing 150 mM
M). ( ) Plots of F’/F0
F’ is the change in the intensity at 348 nm of WGA with various concentrations
) Hill plots of log
fluorescence emission spectra of WGA (0.65
µ
◦
NaCl, pH 7.4, 4 C) upon addition of 2.5-µL aliquots of glycopolymer 11b (74.5
µ
b
∆
versus [S], where
∆
of 11b, F is the intensity of WGA alone, and [S] is total ligand concentration. (
c
0
0
0
0
[
∆F /(∆F
− ∆F )] versus log [S].
MAX

Molecules 2018, 23, 2875
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Table 3. Results of the binding assays for WGA on the basis of fluorescence measurements.
0
−1
)
Compounds
GlcNAc
∆λ (nm)
|∆F /F | (%)
Ka (M
Relative Potency
0
3
0
-
5
8.2 × 10
5.0 × 10
1.8 × 10
3.7 × 10
3.5 × 10
4.2 × 10
1
6
22
45
43
51
4
1
0
90
33
46
50
58
5
5
5
5
1
1
1
1
1a
1b
1c
1d
−14.2
−14.2
−13.0
−16.0
The influence of the dilution of the WGA concentration by the addition of a glycopolymer solution
was corrected using the following equation:
V
V0
0
∆
F =
∆F
(2)
where V is the initial volume of the WGA solution and V is the measured volume of the WGA solution
0
with a given glycopolymer concentration based on the sugar unit in the glycopolymer [S]; Equation (1),
therefore, is represented as
ꢀ
ꢁ
0
F
∆
log
= log[S] + logKa
(3)
0
F
MAX
− ∆F0
∆
When
fluorescence intensity by about 46% was observed, as shown in Table 3. From the results of a Hill
plot of log[ F/( was
FMAX − ∆F)] versus log[S], as shown in Figure 1c, the association constant K
estimated to be 3.7 10 M . The results for other glycopolymers against WGA are summarized
in Table 3. Association constants were estimated to be 1.8–4.2
was the largest when copolymer 11d was used. This can be explained by the suitable density
of the carbohydrate moieties on the glycopolymer, as the ligand was possibly the best fit for the
location of each binding site on WGA [36]. However, their K values were nearly in the same molar
∆F’/F was plotted versus [S], as shown in Figure 1b, an enhancement of the relative
0
∆
∆
a
5
−1
×
5
−1
×
10 M . The association constant
K
a
a
range. In addition to the fluorometric assays for the glycopolymers, biological evaluations of some
of the glycopolymers and WGA were carried out by means of surface plasmon resonance (SPR) [37].
5
−1
The results of the SPR analyses showed that the K
a
for 11b was 3.30
×
10 M ; for 11c it was
5
−1
3
.41 × 10 M . These K
a
values were in good agreement with the results from the fluorometric assay
measurements in this study. From the results of the biological evaluation of the glycopolymers for
lectin, we can see that these types of glycopolymers also have a sugar-clustering effect [38–40].
3
. Experimental
3
.1. Materials and Methods
Unless otherwise stated, all commercially available solvents and reagents were used without
further purification. N,N-Dimethylformamide (DMF) was stored over molecular sieves (4 Å MS)
and methanol (MeOH) was stored over 3 Å MS before use. Acrylamide was recrystallized from
benzene before use. Optical rotations were determined with a JASCO DIP-1000 digital polarimeter
(
(
JASCO Corp., Tokyo, Japan). IR spectra were obtained using a Shimadzu IR Prestige-21 spectrometer
1
1
Shimadzu Corp., Kyoto, Japan). H-NMR spectra were recorded at 400 MHz for H and at 100 MHz
13
1
13
for C with a Bruker DPX-400 spectrometer or at 500 MHz for H and at 125 MHz for C with a
Bruker AVANCE 500 spectrometer (Bruker BioSpin, Ettlingen, Germany) in chloroform-d (CDCl ) or
3
deuterium oxide (D O). Chemical shifts are expressed as parts per million (ppm, δ) and are relative
2
1
to an internal tetramethylsilane (TMS) in CDCl3 (δ 0.0) or HDO in D O (δ 4.78) for H and CHCl in
2 3
13
CDCl (
δ
77.0), CH in MeOD (
δ
49.0) or CH in acetone (
δ
215.0) for C. Ring-proton assignments
3
3
3
1
in the H-NMR spectra were made by first-order analysis of the spectra and are supported by the

Molecules 2018, 23, 2875
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results of homonuclear decoupling experiments and H-H or HMQC experiments. Elemental analyses
were performed with a Fisons EA1108 (Thermo Fisher Scientific Inc., Waltham, MA, USA) on samples
◦
that were extensively dried at 50–60 C over phosphorus pentoxide for 4–5 h. Matrix-assisted laser
desorption/ionization time-of-flight mass spectra (MALDI-TOF-MS) were obtained using a Bruker
AutoflexIII spectrometer (Bruker Daltonics, Bremen, Germany). Reactions were monitored by thin
layer chromatography (TLC) on a precoated plate of silica gel 60F254 (layer thickness, 0.25 mm;
E. Merck, Darmstadt, Germany). For the detection of the intermediates, TLC sheets were (a) dipped in
a solution of 85:10:5 (v/v/v) MeOH-p-anisaldehyde-concd H SO and heated for a few minutes (for
2
4
carbohydrate); or (b) dipped in an aq solution of 5wt% KMnO and heated similarly (for detection of
4
C=C double bonds). Column chromatography was performed on silica gel (Silica Gel 60; 63–200
µm,
E. Merck, Darmstadt, Germany). Flush column chromatography was also performed on silica gel
(
Silica Gel 60, spherical neutral; 40–100
µ
m, E. Merck, Darmstadt, Germany). All extractions were
◦
concentrated below 45 C under diminished pressure. Dialysis was performed against distilled water
using a dialysis tubing [molecular cutoff (MWCO): 12 k–16 kDa, Dow Chemical Co., Midland, MI,
USA]. Wheat germ agglutinin (WGA; a lectin from Triticum vulgaris) was purchased from J-Oil Mills
(Lot # 62015) (J-Oil Mills, Inc., Tokyo, Japan).
3
.2. Synthesis
3
.2.1. 4-Azidomethylstyrene (AZ-Str) (2)
To a solution of chloromethyl styrene
sodium azide (12.4 g, 190 mmol) and the mixture was stirred at 80 C for 1.0 h. After the complete
1
(CM-Str) (10.0 g, 65.5 mmol) in DMF (50 mL) was added
◦
vanishing of
1 judged by TLC, the reaction mixture was evaporated in vacuo. The residue was diluted
with CHCl and was then washed successively with water and brine and dried over anhyd MgSO .
3
4
The organic solution was filtered and evaporated in vacuo. The residual syrup was applied to a column
of silica gel with 40:1 (v/v) hexane—EtOAc as the eluent to give pure AZ-Str
yellow syrup: R 0.28 [hexane]; IR (NEAT) 3088 ( C–H, Ph), 2930 ( C-H), 2097 (
2
ν
(9.40 g, 90.1%) as a
N=N=N), 1630 ( C=C)
ν
ν
ν
f
−
1 1
cm ; H-NMR (500 MHz, CDCl ):
δ
7.43 (d, 2 H, J = 8.15 Hz, Ph), 7.28 (d, 2 H, J = 8.15 Hz, Ph), 6.72
3
(
dd, 1 H, Jcis = 10.85 Hz, Jtrans = 17.60 Hz, CH=), 5.77 [dd, 1 H, Jgem = 0.50 Hz, trans of CH =], 5.28 [dd,
2
1
H, cis of CH =], 4.32 (s, 2 H, PhCH ).
2 2
3
.2.2. Aminomethylstyrene (AMN-Str) (3)
To a solution of AZ-Str
2
(3.00 g, 18.8 mmol) in anhydr THF (10 mL) was added triphenyl
◦
phosphine (TPP) (4.94 g, 20.7 mmol). H O (30 mL) was added to the solution at 0 C and the mixture
2
was stirred at room temperature for 2.5 h. After the complete transformation of the AZ-Str
2 judged by
TLC, the reaction mixture was diluted with CHCl and the mixture was shaken with 3 M aq H SO .
3
2
4
The extraction was partitioned and the aqueous layer was treated with NaOH until ca. pH 12 to
produce precipitates. To the mixture was added CHCl and the organic solution was washed with
3
brine, dried over anhyd MgSO , and evaporated to give pure AMN-Str
3
[
24] (2.40 g, 95.6%) as a
yellow syrup: R 0.37 [5:5:1 (v/v/v) CHCl —MeOH—water]; IR (NEAT) 3281 ( N-H), 3005 ( C-H, Ph),
7.39 (d, 2 H, J = 8.10 Hz, Ph), 7.27 (d, 2 H, J = 9.15 Hz,
Ph), 6.71 (dd, 1 H, Jcis = 10.90 Hz, Jtrans = 17.60 Hz, CH=), 5.73 (dd, 1 H, Jgem = 0.83 Hz, trans of CH =)
4
ν
ν
f
3
−
628 (δN-H) cm ; H-NMR (500 MHz, CDCl ): δ
3
1 1
1
2
5
.28 (dd, 1 H, cis of CH =), 3.86 (s, 2 H, PhCH ), 1.56 (s, 2 H, NH ).
2 2 2
3
.2.3. 4-{[5-(Dimethylamino)-1-naphthalenesulfonamido]methyl}styrene (DSL-Str) (4)
To a stirred solution of AMN-Str (512 mg, 3.8 mmol) and Et N (0.53 mL, 456 mmol) in
dichloromethane (50 mL) a solution of dansyl chloride (1.23 g, 4.56 mmol) in dichloromethane (40 mL)
3
3
was added dropwise at room temperature under a N atmosphere. After stirring at room temperature
2
for 5.5 h, the reaction mixture was evaporated in vacuo. The resulting mixture was diluted with CHCl3
and was then washed successively with water and brine and dried over anhyd MgSO . The organic
4

Molecules 2018, 23, 2875
8 of 13
mixture was filtered and evaporated in vacuo. The residual syrup was applied to a column of silica
gel with 5:2 (v/v) hexane—EtOAc as the eluent to give pure DSL-Str
4
[
26] (1.052 g, 75.5%) as yellow
N-H),
S=O) cm ; H-NMR (500 MHz, CDCl ):
◦
crystals: mp 103.0-103.4 C; R 0.40 [5:2 (v/v) hexane—EtOAc]; IR (NEAT): 3286.70, 3273.20 (
ν
f
−
1 1
3084.18 (
ν
C-H, Ph), 1625.99 (
δ
N-H), 1305.81 (
ν
C-N), 1066.64 (
ν
δ
3
8
.53 (d, 2 H, J = 8.50 Hz, Naph), 8.27 (t, 2 H, J = 8.85 Hz, J = 7.45 Hz, Naph), 7.53 (dt, 2 H, J = 10.90 Hz
,
J = 17.60 Hz, Naph), 7.19 (d, 2 H, J = 7.95 Hz, Ph) 7.19 (d, 1 H, J = 6.35 Hz, Naph), 7.01 (d, 2 H,
J = 7.80 Hz, Ph), 6.60 (dd, 1 H, J =10.87 Hz, J = 17.53 Hz, Naph), 5.66 (br dd, 1 H, J = 17.61 Hz, Naph),
5
6
.20 (br dd, 1 H, J = 10.85 Hz, Naph), 4.86 (t, 2 H, J = ~6 Hz, NH), 4.06 (d, 2 H, J = 6.05 Hz, CH ), 2.89 (s,
2
1
H, NCH × 2) ( H-MNR spectrum can be found in Supplementary Materials).
3
3
.2.4. 4-({5-[(3aR,6S,6aS)-2-Oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-6-
yl]pentanamido}methyl)-styrene (BTN-Str) (5)
To a stirred solution of AMN-Str
3 (60.0 mg, 0.45 mmol) and biotin (121 mg, 495 mmol) in DMF
(
1 mL) was added DMT-MM (150 mg, 0.54 mmol) at room temperature under a N atmosphere.
2
After stirring at room temperature for 1.5 h, the white suspension was poured into ice-cold water
and extracted with EtOAc. When extraction with EtOAc was performed, an insoluble mass was also
observed. The organic solution and the insoluble mass were successively washed with ice-cold water,
satd aq NaHCO and brine, and the whole mixture was filtrated to give a white amorphous powder
3
1
BTN-Str
5
(148 mg, 91.3%): R = 0.53 [5:1 (v/v) CHCl —MeOH]; H-NMR (500 MHz, DMSO-d ):
δ
f
3
6
8
.34 (br s, 1 H, NHCH ), 7.41 (d, 2 H, J = 8.15 Hz, Ph), 7.21 (d, 2 H, J = 8.15 Hz, Ph), 6.71 (dd, 1 H,
2
Jcis = 10.95 Hz, Jtrans = 17.65 Hz, CH=CH ), 6.42 [br s, 1 H, NH (biotin)], 6.37 [s, 1 H, NH (biotin)], 5.79
2
(
dd, 1 H, Jgem = 0.70 Hz, trans of CH2=), 5.22 (d, 1 H, cis of CH =), 4.30 [dd, 1 H, J =5.15 Hz, J =7.65 Hz,
2
CHCH (biotin)], 4.24 (d, 2 H, J = 5.95 Hz, PhCH ), 4.12 (ddd, 1 H, J = 1.80 Hz, J = 4.35 Hz, J = 7.45 Hz,
2
2
NHCHCH), 3.09 (ddd, 1 H, J = 2.55 Hz, J = 4.35 Hz, J = 10.5 Hz, SCH), 2.82 (dd, 1 H, Jgem = 12.4 Hz,
1
SCH
a
), 2.58 (d, 1 H, SCH ), 2.14 (t, 2 H, J =7.45 Hz, CH CO), 1.5 (m, 4 H, CH
2
×
2) ( H-MNR spectrum
b
2
can be found in Supplementary Materials).
3
.2.5. 2-[2-(2-Hydroxyethoxy)-ethoxy]-ethyl-O-2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-
β-D-glucopyranoside (7)
The quantitative preparation of an oxazoline
6 was accomplished from 2-acetamido-1,3,4,6-tetra-
O-acetyl-
α-D-glucosamine (2.00 g, 5.14 mmol) by the method previously reported [29]. To a solution
0
of oxazoline and 2,2 -[ethane-1,2-diylbis(oxy)]di(ethan-1-ol) (3.4 mL, 25.6 mmol) in dichloroethane
(
17 mL) was added CSA (0.12 g, 0.51 mmol) under a N atmosphere and the reaction mixture was
2
◦
stirred at 90 C for 1 h. After cooling the reaction mixture, CHCl was added. The organic mixture
3
was successively washed with water, satd aq NaHCO and brine, dried over anhyd MgSO , filtered,
3
4
and evaporated to yield a dark yellow syrup, which was applied to a column of silica gel with 20:1
(v/v) CHCl —MeOH as the eluent to afford pure 7 (1.87 g, 75.7%) as a yellow syrup: R = 0.39 [10:1
3
f
(
1
v/v) CHCl —MeOH]; IR (NEAT): 3294 (
ν
), 2924 (
ν
1
), 1748 (
ν
C=O, ester), 1660 (
ν
C=O, amide I),
6.80 (d, 1 H,
3
O-H
C-H
1
−
557 (
δ
N-H, amide II), 1236 (
ν
C-O), 1043 (
ν
C-O-C) cm ; H-NMR (500 MHz, CDCl ):
δ
3
J2,NH = 9.30 Hz, NH), 5.11 (dd, 1 H, J2,3 = 10.0 Hz, J3,4 = 9.30 Hz, H-3), 5.07 (t, 1 H, J4,5 = 9.60 Hz, H-4),
.77 (d, 1 H, J1,2 = 8.60 Hz, H-1), 4.26 (dd, 1 H, J5,6b = 4.65 Hz, J6a,6b = 12.3 Hz, H-6b), 4.12 (q, 1 H,
H-2), 4.12 (dd, 1 H, J5,6a = 2.60 Hz, H-6a), 3.90 (dt, 1 H, J = 12.65 Hz, J = 2.48 Hz, OCH ), 3.86–3.60 (m,
2 H, H-5, OCH2 5, OCH ), 3.08 (br s, 1 H, OH), 2.09, 2.02, 2.01, and 1.98 (each s, 12 H, COCH 4)
(1
H-MNR spectrum can be found in Supplementary Materials); C-NMR (100 MHz, CDCl3): 171.77,
4), 102.05 (C-1), 73.88 (C-3), 72.50 (OCH ), 71.71 (C-5), 71.67 (OCH ),
4
a
1
×
×
b
3
13
δ
1
7
70.94, 170.94, and 169.48 (C=O
×
2
2
0.69 (OCH ), 70.25 (OCH ), 68.78 (C-4), 68.69 (OCH ), 62.30 (C-6), 61.39 (OCH ), 54.01 (C-2), 23.12
2
2
2
2
(
NCOCH ), 20.94, 20.89 and 20.75 (COCH × 3).
3
3

Molecules 2018, 23, 2875
9 of 13
3
.2.6. 8-O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-3,6-dioxaoctanoic Acid (8)
An aqueous 15% solution of NaHCO (19 mL) was added to a solution of alcohol
1.6 mmol) in acetone (18 mL) with stirring at 0 C. KBr (0.28 g, 2.3 mmol) and TEMPO (36.4 mg,
7 (5.58 g,
3
◦
1
0
.23 mmol) were added to the mixture. Trichloroisocyanuric acid (5.42 g, 23.2 mmol) was then added
◦
slowly over a period of 20 min at 0 C. The mixture was warmed to room temperature and stirred
for 4.5 h, and then propan-2-ol was added. The mixture was filtered on celite and treated with 15 mL
of satd aq NaHCO . The aqueous phase was washed with portions of CHCl , treated with 1 M aq
3
3
HCl and brine, and extracted twice with CHCl . The organic layers were combined and dried over
3
anhyd MgSO , and the solvent was evaporated after filtration on celite to yield the corresponding
4
carboxylic acid
8, which was used for the next step without further purification: R = 0.40 [5:1 (v/v)
f
CHCl —MeOH]; IR (NEAT) 3323 (
ν
O-H), 2931 (
N-H, Amide II), 1373 (
cm ; H-NMR (400 MHz, CDCl ): 6.59 (d, 1 H, J2,NH = 9.20 Hz, NH), 5.15 (t, 1 H, J2,3 = 10.2 Hz,
ν
C-H), 1745 (
ν
C=O, ester), 1732 (
ν
C=O, carboxy group),
3
1
643 (
ν
1 1
C=O, Amide I), 1556 (
δ
δ
O-H), 1234 (
ν
O-H, carboxy group), 1041 (
ν
C-O-C)
−
δ
3
J3,4 = 9.48 Hz, H-3), 5.07 (t, 1 H, J4,5 = 9.65 Hz, H-4), 4.87 (d, 1 H, J1,2 = 8.52 Hz, H-1), 4.27 (dd, 1 H,
J5,6b = 4.60 Hz, J6a,6b = 12.2 Hz, H-6b), 4.14 (dd, 1 H, J5,6a = 2.24 Hz, H-6a) 4.06 (q, 1H, H-2), 3.94–3.61
1
(
m, 11 H, H-5 & OCH2
be found in Supplementary Materials); C-NMR (100 MHz, CDCl3):
and 169.56 (C=O 5), 101.87 (C-1), 73.34 (C-3), 71.77 (C-5), 71.38 (OCH ), 70.40 (OCH ), 68.81 (OCH ),
×
5), 2.09, 2.02, 2.01, and 1.97 (each s, 12 H, COCH
×
4) ( H MNR spectra can
3
13
δ
172.01, 171.47, 171.47, 171.19,
×
2
2
2
6
2
5
8.55 (C-4), 68.25 (OCH ), 62.38 (C-6), 54.20 (C-2), 25.44 (OCH ), 23.10 (NCOCH ), 20.94, 20.90 and
2 2 3
+
+
0.78 (COCH3
×
3); MALDI-TOF MS calcd for [M + Na] : 515.169. Found: m/z 516.101, [M + K] :
32.143. Found: m/z 532.083.
3
.2.7. 8-O-(2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-3,6-dioxaoctanoic Acid
-vinybenzylamide (9)
4
To a stirred solution of carboxylic acid
8 (4.0 g, 8.11 mmol) and AMN-Str 3 (1.24 g, 9.31 mmol)
in dichloromethane (20 mL) was added DMT-MM (2.69 g, 9.72 mmol) and the resulting suspension
was stirred at room temperature under a N atmosphere for 3.5 h. The reaction mixture was poured
2
into ice-cold water and extracted with EtOAc. The organic layer was successively washed with satd
aq NaHCO and brine. The organic layer was dried over anhyd MgSO4 and concentrated in vacuo.
3
The residue was purified by silica gel chromatography with 50:1 (v/v) CHCl —MeOH as the eluent to
3
give styrene derivative
IR (NEAT) 3306 ( N-H), 3086, 3007 (
C=C), 1537 ( N-H, Amide II), 1234 (
7.39 (d, 2 H, Jvic =8.1 Hz, Ph), 7.27 (d, 2 H, Jvic = 8.3 Hz, Ph), 7.23 (s, 1 H, NHCH ), 6.71 (dd, 1 H,
9
(3.82 g, 77.7%) as a colorless syrup: R = 0.48 [10:1 (v/v) CHCl —MeOH];
f
3
ν
ν
C-H, Ph), 2937 (
ν
C-H), 1748 (
ν
, ester), 1667 (
ν
C=O, Amide I),
C=O
) cm 1; H-NMR (400 MHz, CDCl ):
−
1
1651 (
ν
δ
ν
C-O), 1043 (
ν
C-O-C 3
δ
2
Jcis = 10.9 Hz, Jtrans = 17.6 Hz, CH=CH ), 6.30 (d, 1 H, J
= 8.52 Hz, NHAc), 5.75 (dd, 1 H, Jgem =
2
2,NH
0
5
4
.60 Hz, trans of CH =), 5.35 (dd, 1 H, J2,3 = 10.5 Hz, J3,4 = 9.34 Hz, H-3), 5.26 (dd, 1 H, cis of CH =),
2 2
.03 (t, 1 H, J4,5 = 9.72 Hz, H-4), 4.79 (d, 1 H, J1,2 = 8.36 Hz, H-1), 4.49 (d, 2 H, Jvic = 5.92 Hz, NHCH2),
.24 (dd, 1 H, J5,6b = 4.66 Hz, J6a,6b = 12.3 Hz, H-6b), 4.19 (d, 1 H, Jgem = 15.7 Hz, NCOCH ), 4.13 (d, 1
b
H, NCOCH
a
), 4.10 (dd, 1 H, J5,6a = 2.36 Hz, H-6a), 3.88 (m, 1 H, OCH ), 3.71–3.60 (m, 9 H, H-2, H-5,
b
1
OCH , OCH2
×
3), 2.07, 2.01, 2.01, 1.89 (each s, 12 H, COCH
×
4) ( H-MNR spectrum can be found
b
3
13
in Supplementary Materials); C-NMR (100 MHz,CDCl3):
δ
170.86, 170.65, 170.42, and 169.59 (C=O
×
4
7
2
), 136.43 (CH=), 128.17 (Ph), 126.66 (Ph), 114.27 (CH =), 100.60 (C-1), 72.30 (C-3), 71.87, 71.03, 70.89,
0.70 and 70.52 (C-5, OCH2
3.29 (NCOCH ), 20.89, 20.84, and 20.77 (COCH
2
×
4), 68.86 (C-4), 68.62 (OCHa,b), 62.26 (C-6), 55.15 (C-2), 42.75 (NCH ),
2
+
×
3); MALDI-TOF MS calcd for [M + H] : 609.265.
3
3
+
+
Found: m/z 609.228, [M + Na] : 631.247. Found: m/z 631.242, [M + K] : 647.221. Found: m/z 647.225.
3
.2.8. 8-O-(2-Acetamido-2-deoxy-β-D-glucopyranosyl)-3,6-dioxaoctanoic acid 4-vinybenzylamide
(
NAG-Str) (10)
Sodium methoxide (53.2 mg, 0.98 mmol) was added to a solution of acetate
9 (2.0 g, 3.29 mmol)
in MeOH (20 mL) at room temperature under a N atmosphere and the reaction mixture was stirred
2

Molecules 2018, 23, 2875
10 of 13
+
for 1.5 h. When complete disappearance of the acetate
9 on TLC was observed, IR-120B (H ) was
added to the mixture. The suspension was filtered off and the filtrate was evaporated in vacuo to
yield the corresponding NAG-Str 10 (1.48 g, 98.9%) as a colorless syrup: R = 0.41 [65:25:4 (v/v/v)
f
CHCl —MeOH—H O]; IR (NEAT) 3312 (
ν
), 3086, 2999 (
ν
, Ph), 2922 (
ν
C-H), 1651 (
ν
C=O, Amide
7.49 (d, 2
3
2
O-H
C-H
1
−
1
I), 1556 (
δ
N-H, Amide II), 1298 (
ν
C-O), 1072 (
ν
C-O-C) cm ; H-NMR (400 MHz, D O):
δ
2
H, Jvic = 8.12 Hz, Ph), 7.32 (d, 2 H, Jvic = 8.08 Hz, Ph), 6.78 (dd, 1 H, Jcis = 11.0 Hz, Jtrans = 17.7 Hz,
CH=CH ), 5.85 (d, 1 H, trans of CH =), 5.31 (d, 1 H, cis of CH =), 4.50 (d, 1 H, J = 8.44 Hz, H-1),
2
2
2
1,2
4
.45 (s, 2 H, NHCH ), 4.13 (s, 2 H, COCH ), 3.92 (dd, 1 H, J
= 4.40 Hz, J6a,6b = 12.6 Hz, H-6b), 3.92
2
2
5,6b
(
m, 1 H, OCH ), 3.67 (m, 9 H, H-2, H-6a, OCH
a
, OCH2
×
3), 3.55 (m, 2 H, H-3 and H-4), 2.02 (s, 3 H,
b
1
13
COCH ) ( H-MNR spectrum can be found in Supplementary Materials); C-NMR (100 MHz, D O):
δ
3
2
1
74.42 (C=O, NAc), 172.47 (C=O, NCOCH ), 137.43 (Ph), 136.68 (Ph), 136.24 (CH=), 127.76 (Ph), 126.48
2
(
6
Ph), 114.34 (CH =), 101.00 (C-1), 75.86 (C-3), 73.87 (C-5), 70.53 (OCH ), 69.91 (C-4), 69.61 (OCH
2
×
2),
2
2
9.54 (OCOCH ), 68.85 (OCH ), 60.75 (C-6), 55.49 (C-2), 42.28 (NCH ), 22.16 (NCOCH ).
2
2
2
3
3
.3. Radical Polymerization
A solution of NAG-Str monomer 10 and an appropriate amount of acrylamide (AAm) in deionized
water was deaerated under reduced pressure for a few minutes, and then tetramethylethylenediamine
TEMED) (0.2 equivalent molar for 10) and ammonium persulfate (APS) (0.1 equivalent molar for 10
(
)
were added. The mixture was stirred at room temperature and diluted with 1 M aq pyridine-acetic acid
buffer (pH 5.6). The viscous solution was dialyzed against distilled water, followed by lyophilization
to provide the corresponding white powdery glycopolymers 11a–11d. The results of the radical
polymerization are summarized in Table 1.
3
.4. Biological Evaluations of Glycopolymers for WGA
Measurement of the fluorescence emission spectra and excitation spectra were carried out in
a Teflon-stoppered cuvette (12.5 mm in width 45 mm height) containing 3.0 mL of a sample.
×
The slit width of the excitation and the emission was 5.0 nm, and the scan speed was medium.
The sensitivity was high, and the interval of sampling was 3.0 nm. Emission spectra of WGA induced
by excitation at 295 nm were uncorrected and were recorded with a Shimadzu RF-5300PC fluorescence
spectrophotometer (Shimadzu Corp., Kyoto, Japan). The cuvette was mounted in a thermostated
◦
holder, and measurement was carried out at 4 C in order to eliminate the effect of nonspecific binding
on the spectra. The concentration of WGA was estimated to be 0.65
µM using the absorption coefficient
at 280 nm (E2801% = 15.0 in 50 mM Tris-HCl buffer, 0.15 M NaCl, pH 7.4) [35].
4
. Conclusions
In conclusion, chloromethylstyrene was efficiently converted into the desired functional
monomers by a combination of azide displacement, reduction and condensation with appropriate
functional substrates. The assembly of the functional monomers by means of radical polymerization
was performed using a GlcNAc monomer (NAG-Str) as a model monomer, and biological interactions
between the polymers and a protein were demonstrated. This methodology can be applied to
the preparation of various functional monomers and the corresponding polymers including the
reversible deactivation radical polymerization (RDRPs) [41
syntheses [45,46].
–
44] and microwave-assisted polymer
1
Supplementary Materials: The following are available online, Figure S1: H-NMR spectrum of DSL-Str
4
,
1
1
1
Figure S2: H-NMR spectrum of BTN-Str 5, Figure S3: H-NMR spectrum of compound 7, Figure S4: H-NMR
spectrum of compound 8 9, Figure S6: H-NMR spectrum of
, Figure S5: 1H-NMR spectrum of compound
1
NAG-Str 10.
Author Contributions: K.M. conceived and designed the experiments; R.H., T.K., T.M., K.H. and K.M. performed
the experiments and analyzed the data; R.H. and K.M. contributed to preparation of the paper; and R.H. mainly
wrote the paper. K.M. supervised the whole research.

Molecules 2018, 23, 2875
11 of 13
Funding: This work was partly supported by a grant-in-aid from Saitama Prefecture (K.M.) (Saitama Leading
Edge Project).
Acknowledgments: We are grateful to AGC Seimi Chemical Co., Ltd. for providing chloromethylstyrene (CMS)
used in this study.
Conflicts of Interest: The authors declare no conflict of interest associated with this manuscript.
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Sample Availability: Samples of the compounds 2, 4, 5 and 10 are available from the authors.
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