Preparation of Functional Monomers as Precursors of Bioprobes from a Common Styrene Derivative and Polymer Synthesis

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.


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][4][5] and glycopolymers [6][7][8][9][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][13][14][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 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.

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 1 as a starting material by means of the Huisgen cycloaddition reaction [20][21][22][23] and polymer syntheses [11]. In this paper, alternative preparations of a glycomonomer and other useful functional monomers are described.

Conversion of 4-(Chloromethyl)styrene into an Amine as a Common Precursor
Commercially available CM-Str 1 as a versatile precursor for an azide compound was used as a convenient starting material, and the conversion of the azide 2 (AZ-Str) [24] into the corresponding amine derivative by the Staudinger reaction was performed. Scheme 1 summarizes the preparation of aminostyrene derivative 3 (AMN-Str) [25] from CM-Str 1. The SN2 replacement of the chloride of CM-Str 1 was performed by an azide anion in a polar aprotic solvent at a slightly high temperature to afford the corresponding AZ-Str 2 in 90% yield [11]. The Staudinger reaction of AZ-Str 2 with triphenylphosphine (TPP) afforded the corresponding CM-Str 3 in 96% yield. Two-step-conversion from 1 to 3 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). Scheme 1. Reagents and conditions: (i) NaN3, DMF, 80 °C, 1 h, (ii) TPP, H2O, THF, 0 °C, 2.5 h.

Synthetic Conversion of the Amine into Functionalized Monomers
Given the success of the simple preparation of AMN-Str 3, which had a highly reactive primary amino function, we next turned our attention to the applicability of AMN-Str 3 as a common precursor for the synthesis of functional monomers. Scheme 2 shows the chemical conversion of AMN-Str 3 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 3 with dansyl chloride in the presence of an acid scavenger proceeded smoothly to afford the dansylated styrene derivative 4 (DSL-Str) [26] as a fluorogenic monomer in 76% yield. Structural elucidations were performed by a combination of IR, 1 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 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-2yl)-4-methylmorpholinium chloride (DMT-MM), which was introduced by Kunishima et al. [28]. The

Synthetic Conversion of the Amine into Functionalized Monomers
Given the success of the simple preparation of AMN-Str 3, which had a highly reactive primary amino function, we next turned our attention to the applicability of AMN-Str 3 as a common precursor for the synthesis of functional monomers. Scheme 2 shows the chemical conversion of AMN-Str 3 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 3 with dansyl chloride in the presence of an acid scavenger proceeded smoothly to afford the dansylated styrene derivative 4 (DSL-Str) [26] as a fluorogenic monomer in 76% yield. Structural elucidations were performed by a combination of IR, 1 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 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.

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 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 configuration of glycoside 7 was determined using 1 H-NMR spectra. The results revealed a signal assignable to H-1 at δ 4.77 as a doublet with J1,2 = 8.60 Hz, which was confirmed to be a -glycoside. The primary alcohol had to be oxidized to carboxylic acid before the coupling reaction of the glycoside with the amine 3. Thus, 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO)-mediated oxidation [31] of the alcohol 7 was carried out to produce the desired carboxylic acid 8, which was 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 8 and the amine 3 were applied for a DMT-MM-mediated coupling reaction, and the reaction proceeded smoothly to produce the corresponding styrene derivative 9 as a colorless syrup in 78% yield after chromatographic purification. Structural elucidation of product 9 was performed by means of 1 H-and 13 C-NMR spectroscopic analyses. Since the appearance of aromatic ring proton signals at 7.39 ppm and 7.27 ppm in 9 was observed in the 1 H-NMR results, the progress of the coupling reaction was confirmed. In addition to the 1 H-NMR results, the combined results of the 13 C-NMR and HMQC experiments also supported the formation of 9. The removal of the acetyl

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 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 configuration of glycoside 7 was determined using 1 H-NMR spectra. The results revealed a signal assignable to H-1 at δ 4.77 as a doublet with J 1,2 = 8.60 Hz, which was confirmed to be a β-glycoside. The primary alcohol had to be oxidized to carboxylic acid before the coupling reaction of the glycoside with the amine 3. Thus, 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO)-mediated oxidation [31] of the alcohol 7 was carried out to produce the desired carboxylic acid 8, which was 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 8 and the amine 3 were applied for a DMT-MM-mediated coupling reaction, and the reaction proceeded smoothly to produce the corresponding styrene derivative 9 as a colorless syrup in 78% yield after chromatographic purification. Structural elucidation of product 9 was performed by means of 1 H-and 13 C-NMR spectroscopic analyses. Since the appearance of aromatic ring proton signals at 7.39 ppm and 7.27 ppm in 9 was observed in the 1 H-NMR results, the progress of the coupling reaction was confirmed. In addition to the 1 H-NMR results, the combined results of the 13 C-NMR and HMQC experiments also supported the formation of 9. The removal of the acetyl protection in 9 was performed by using the Zemplén method [32] to afford NAG-Str 10 as a water-soluble material in 99% yield. protection in 9 was performed by using the Zemplén method [32] to afford NAG-Str 10 as a watersoluble material in 99% yield.

Polymerization of a Glycomonomer by Means of Radical Polymerization
The synthetic conversion of AMN-Str 3 into functional monomers was successfully demonstrated, 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 ( ) of the glycopolymers were estimated by size exclusion chromatography in 0.3 M aqueous NaCl solution. From the weight-average molecular weight ( ) of glycopolymer 11a, the degree of polymerization was estimated to be 653. The polymer compositions of glycopolymers 11b, 11c and 11d were estimated on the basis of the 1 H-NMR results. All of the polymers (11a-11d) had watersoluble abilities and appropriate molecular weights as estimated by size-exclusion chromatography (SEC) analyses.

Polymerization of a Glycomonomer by Means of Radical Polymerization
The synthetic conversion of AMN-Str 3 into functional monomers was successfully demonstrated, 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 compositions of glycopolymers 11b, 11c and 11d were estimated on the basis of the 1 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.
Molecules 2018, 23, x FOR PEER REVIEW 4 of 13 protection in 9 was performed by using the Zemplén method [32] to afford NAG-Str 10 as a watersoluble material in 99% yield.

Polymerization of a Glycomonomer by Means of Radical Polymerization
The synthetic conversion of AMN-Str 3 into functional monomers was successfully demonstrated, 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  1 Total yields were calculated on the basis of the quantities of monomers used. 2 Polymer compositions of sugar unit:acrylamide unit were estimated on the basis of the 1 H-NMR results. 3 The weightaverage molecular weights were estimated by size-exclusion chromatography in 0.3 M aq NaCl

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 µM 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]: where ∆F (∆F = F − F 0 ) 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 ∆F MAX is the difference between F MAX , which is the final fluorescence intensity at the saturated concentration of a glycopolymer, and F 0 .     The influence of the dilution of the WGA concentration by the addition of a glycopolymer solution was corrected using the following equation: where V 0 is the initial volume of the WGA solution and V is the measured volume of the WGA solution with a given glycopolymer concentration based on the sugar unit in the glycopolymer [S]; Equation (1), therefore, is represented as When ∆F'/F 0 was plotted versus [S], as shown in Figure 1b, an enhancement of the relative fluorescence intensity by about 46% was observed, as shown in Table 3. From the results of a Hill plot of log[∆F/(∆F MAX − ∆F)] versus log[S], as shown in Figure 1c, the association constant K a was estimated to be 3.7 × 10 5 M −1 . The results for other glycopolymers against WGA are summarized in Table 3. Association constants were estimated to be 1.8-4.2 × 10 5 M −1 . The association constant K a 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 a values were nearly in the same molar 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]. The results of the SPR analyses showed that the K a for 11b was 3.30 × 10 5 M −1 ; for 11c it was 3.41 × 10 5 M −1 . 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][39][40].

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 (Shimadzu Corp., Kyoto, Japan). 1 H-NMR spectra were recorded at 400 MHz for 1 H and at 100 MHz for 13 C with a Bruker DPX-400 spectrometer or at 500 MHz for 1 H and at 125 MHz for 13

4-Azidomethylstyrene (AZ-Str) (2)
To a solution of chloromethyl styrene 1 (CM-Str) (10.0 g, 65.5 mmol) in DMF (50 mL) was added sodium azide (12.4 g, 190 mmol) and the mixture was stirred at 80 • C for 1.0 h. After the complete vanishing of 1 judged by TLC, the reaction mixture was evaporated in vacuo. The residue was diluted with CHCl 3 and was then washed successively with water and brine and dried over anhyd MgSO 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 2 (9.

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 2 O (30 mL) was added to the solution at 0 • C and the mixture 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 3 and the mixture was shaken with 3 M aq H 2 SO 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 3 and the organic solution was washed with brine, dried over anhyd MgSO 4 , and evaporated to give pure AMN-Str 3 [24] (40 mL) was added dropwise at room temperature under a N 2 atmosphere. After stirring at room temperature for 5.5 h, the reaction mixture was evaporated in vacuo. The resulting mixture was diluted with CHCl 3 and was then washed successively with water and brine and dried over anhyd MgSO 4 . The organic 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] ( 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 3 and brine, and the whole mixture was filtrated to give a white amorphous powder BTN-  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 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 2 atmosphere and the reaction mixture was stirred at 90 • C for 1 h. After cooling the reaction mixture, CHCl 3 was added. The organic mixture was successively washed with water, satd aq NaHCO 3 and brine, dried over anhyd MgSO 4 , filtered, and evaporated to yield a dark yellow syrup, which was applied to a column of silica gel with 20:1 (v/v) CHCl 3 -MeOH as the eluent to afford pure 7 (1.

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.

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 (E 280 1% = 15.0 in 50 mM Tris-HCl buffer, 0.15 M NaCl, pH 7.4) [35].

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][42][43][44] and microwave-assisted polymer syntheses [45,46].