Synthesis of a New Polyanion Possessing Dense 1,2,3-Triazole Backbone

Polyanions are an important class of water-soluble polymers because polyanions are utilized in a wide range of industrial fields. It is thus a great challenge to develop polyanions with novel structures to make their applications broader. In this study, a new polyanion with a dense 1,2,3-triazole backbone, poly(4-azido-5-hexanoic acid) (poly(AH)), was synthesized by copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) polymerization of t-butyl 4-azido-5-hexanoate followed by hydrolysis of the t-butyl ester groups. Turbidimetric and potentiometric titration data indicated that poly(AH) was well soluble in water under basic conditions (pH < 7) and a weaker polyanion (apparent pKa = 5.4) than polyacrylic acid (apparent pKa = 4.5). Adsorption tests exhibited that sodium salt of poly(AH) (poly(AH)Na) adsorbed most preferably Fe3+ among the four metal ions examined, i.e., Cu2+, Pb2+, Li+, and Fe3+. 1H spin-lattice relaxation time measurements indicated that Fe3+ ions were adsorbed favorably onto the 1,2,3-triazole residues.


Introduction
Polyanions are an important class of water soluble polymers showing various effects, e.g., thickening, gelling, dispersing, cohesive, adhesive, moisturizing, and metal-ion scavenging effects. On the basis of the effects, polyanions are thus utilized in a wide range of industrial fields including detergents, cosmetics, pharmaceuticals, foods, paints, textiles, civil engineering, and construction [1][2][3][4][5][6]. There are a number of polyanions, which are categorized into natural, semi-synthetic, and synthetic polymers. Polyanions are composed of the polymer backbone, anionic residues, and linkers between the backbone and anionic residues. Polyanions are used in appropriate applications depending on their properties based on the chemical structure. Therefore, it is a great challenge to develop polyanions with novel structures to make their applications broader.
Recently, t-butyl 4-azido-5-hexanoate (tBuAH), which we designed and synthesized, was polymerized by CuAAC to yield a dense 1,2,3-triazole polymer soluble in organic solvents, including polar and halogenated ones, indicating that the t-butyl (t-Bu) ester side chains improved the solubility [32]. Since t-Bu ester is often used as a protecting group for carboxylic acid residue, a new polyanion, poly(4-azido-5-hexanoic acid) (poly(AH)), is obtained by hydrolysis of t-Bu ester, and the properties of poly(AH) are investigated preliminarily in this study.
The poly(AH) sample was then neutralized with an equimolar amount of 0.10 M NaOH, and the poly(AH) neutralized (poly(AH)Na) was then recovered by drying under reduced pressure at 45 °C for 12 h.

Turbidimetric and Potentiometric Titrations
A sample of poly(AH) not neutralized (68 μmol monomer units) was dissolved in 0.10 M NaOH (780 μL) and then diluted with water (5.4 mL). While the polymer solution was titrated with 0.10 M HCl from pH 10.9 using a microburet at 25 °C under a nitrogen atmosphere, turbidity and pH were monitored after establishing the equilibrated state, in which the pH reached a constant value after each step of titrant addition. Turbidities, reported as 100-%T, were measured with a Brinkmann PC920 probe colorimeter equipped with a 1 cm path length fiber optics probe at 620 nm. Values of pH were measured with a Horiba F-23 pH meter equipped with a Horiba 9618S-10D glass electrode. The reference electrode was calibrated with buffer solutions of pH 4.01, 6.86, and 9.18 prior to pH measurements. The degrees of neutralization (α) were calculated from the amounts of the monomer unit and the added HCl.

Adsorption Tests
Poly(AH)Na (11.5 mg, 65.7 μmol monomer units) was added to an aqueous solution of a salt (100 mg L -1 metal ion), i.e., PbCl2, CuSO4•5H2O, (FeNO3)3•9H2O, or LiCl, at pH = 7. The mixture was stirred at 35 °C for 12 h. After reaching equilibrium, 4 mL of the mixture solution was taken. The polymer-metal ion complexes were removed with a Pall Microsep TM advanced centrifugal device 1K Omega (1 kDa cut off) by centrifugation at 1000 rpm for 15 min. The filtrate obtained (1.2 mL) was analyzed by a PerkinElmer Optima 8300 ICP-Optical Emission Spectroscopy system (PerkinElmer Inc. (Waltham, MA, USA)) to evaluate the equilibrium concentration (ce) of metal ion. The concentrations of metal Scheme 1. Synthesis of poly(AH) by CuAAC polymerization of tBuAH followed by hydrolysis of the t-Bu ester.
The poly(AH) sample was then neutralized with an equimolar amount of 0.10 M NaOH, and the poly(AH) neutralized (poly(AH)Na) was then recovered by drying under reduced pressure at 45 • C for 12 h.

Turbidimetric and Potentiometric Titrations
A sample of poly(AH) not neutralized (68 µmol monomer units) was dissolved in 0.10 M NaOH (780 µL) and then diluted with water (5.4 mL). While the polymer solution was titrated with 0.10 M HCl from pH 10.9 using a microburet at 25 • C under a nitrogen atmosphere, turbidity and pH were monitored after establishing the equilibrated state, in which the pH reached a constant value after each step of titrant addition. Turbidities, reported as 100-%T, were measured with a Brinkmann PC920 probe colorimeter equipped with a 1 cm path length fiber optics probe at 620 nm. Values of pH were measured with a Horiba F-23 pH meter equipped with a Horiba 9618S-10D glass electrode. The reference electrode was calibrated with buffer solutions of pH 4.01, 6.86, and 9.18 prior to pH measurements. The degrees of neutralization (α) were calculated from the amounts of the monomer unit and the added HCl.

Adsorption Tests
Poly(AH)Na (11.5 mg, 65.7 µmol monomer units) was added to an aqueous solution of a salt (100 mg L -1 metal ion), i.e., PbCl 2 , CuSO 4 ·5H 2 O, (FeNO 3 ) 3 ·9H 2 O, or LiCl, at pH = 7. The mixture was stirred at 35 • C for 12 h. After reaching equilibrium, 4 mL of the mixture solution was taken. The polymer-metal ion complexes were removed with a Pall Microsep TM advanced centrifugal device 1K Omega (1 kDa cut off) by centrifugation at 1000 rpm for 15 min. The filtrate obtained (1.2 mL) was analyzed by a PerkinElmer Optima 8300 ICP-Optical Emission Spectroscopy system (PerkinElmer Inc. (Waltham, MA, USA)) to evaluate the equilibrium concentration (c e ) of metal ion. The concentrations of metal ions in the equilibrated state were reported as average values of three measurements. The removal ratio (r removal ) and adsorption capacity (q e ) were calculated by where c i is the initial ion concentration, m is the weight of poly(AH), and V is the volume of solution.

Synthesis and Structural Characterization of Poly(AH)
In our previous study, we synthesized tBuAH possessing t-butyl (t-Bu) ester in the side chain and conducted its CuAAC polymerization to obtain poly(tBuAH) with a dense 1,4-disubstituted 1,2,3-triazole backbone [32]. The unsubstituted dense 1,4-disubstituted 1,2,3-triazole polymer (poly(3-azido-1-propyne)) was insoluble in all the solvents studied, whereas poly(tBuAH) was soluble in many organic solvents, including polar and halogenated solvents, presumably because of the t-Bu ester moiety in the side chain. In this study, a new polyanion, poly(AH), was synthesized by hydrolysis of the t-Bu ester moieties in poly(tBuAH). According to the procedure reported previously [32], tBuAH was polymerized by CuAAC to yield poly(tBuAH) consisting of 1,4-disubstituted 1,2,3-triazole residues. The M w values for the poly(tBuAH) samples obtained were determined to be 7.9 × 10 3 and 8.8 × 10 3 , respectively, by SEC ( Table 1). The poly(tBuAH) samples were dissolved in DCM, and the t-Bu ester groups were hydrolyzed with TFA to synthesize poly(AH) samples. Since poly(AH) is a weak polyanion possessing carboxylic acid moieties, the poly(AH) samples were neutralized with an equimolar amount of NaOH to dissolve in water and then recovered as a salt-type polymer samples (poly(AH)Na) by drying under reduced pressure. Table 1. Basic characteristics of poly(tBuAH) samples prepared in this study.

Sample
M w /10 3 1 M w /M n 1 poly(tBuAH) 2 7.9 1.5 poly(tBuAH) 3 8.8 1.4 1 Determined by SEC measurements at 25 • C using DMSO containing 1.05 g L -1 LiBr as eluent. Molecular weights were calibrated with PEG and PEO standard samples. 2 The poly(AH) sample prepared from this poly(tBuAH) sample was used for the FTIR, turbidimetric and potentiometric titrations, and PGSE NMR. 3 The poly(AH) sample prepared from this poly(tBuAH) sample was used for the 1 H NMR, adsorption tests, and T 1 measurements.
Poly(AH) was characterized by 1 H NMR and FTIR. Figure 1 shows 1 H NMR spectra for poly(tBuAH) and poly(AH). As can be seen in Figure 1a, the spectrum of poly(tBuAH) in CDCl 3 contains signals ascribable to the proton in 1,4-disubstituted 1,2,3-triazole and the methine proton in the main chain at ca. 7.9 and 6.0 ppm, respectively. The signals at ca. 2.6 and 2.2 ppm are assignable to the protons of two methylenes in side chain. The signal due to the t-Bu ester was observed at ca. 1.4 ppm. This 1 H NMR spectrum is thus indicative of successful preparation of poly(tBuAH) consisting of 1,4-disubstituted 1,2,3-triazole units. In the spectrum of poly(AH) in D 2 O (Figure 1b), on the other hand, signals due to protons of the triazole, methine, and methylenes were observed, whereas the signal ascribable to the t-Bu ester was not observed. These observations indicate that poly(AH) was successfully obtained through quantitative hydrolysis of the t-Bu ester groups, as can be seen in Scheme 1.  Figure 2 shows FTIR spectra of poly(tBuAH) and poly(AH). Both the spectra indicate absorption bands ascribable to 1,2,3-triazole at ca. 1500, 1000, and 800 cm -1 . In the spectrum of poly(tBuAH), the signal attributed to the stretching vibration of the ester carbonyl was observed around 1720 cm -1 , whereas in the spectrum of poly(AH), a broad signal attributed to the carboxylic acid was observed around 1692 cm -1 . It should be noted here that the spectrum of poly(AH) contains a broad absorption band ascribable to the stretching vibration of O-H in the region of 2500-3500 cm -1 caused by intermolecular hydrogen bonding. These FTIR spectra also confirm that poly(AH) was successfully obtained by quantitative hydrolysis of the t-Bu ester groups in the side chain of poly(tBuAH) while the dense 1,2,3-triazole backbone was maintained.  Figure 2 shows FTIR spectra of poly(tBuAH) and poly(AH). Both the spectra indicate absorption bands ascribable to 1,2,3-triazole at ca. 1500, 1000, and 800 cm -1 . In the spectrum of poly(tBuAH), the signal attributed to the stretching vibration of the ester carbonyl was observed around 1720 cm -1 , whereas in the spectrum of poly(AH), a broad signal attributed to the carboxylic acid was observed around 1692 cm -1 . It should be noted here that the spectrum of poly(AH) contains a broad absorption band ascribable to the stretching vibration of O-H in the region of 2500-3500 cm -1 caused by intermolecular hydrogen bonding. These FTIR spectra also confirm that poly(AH) was successfully obtained by quantitative hydrolysis of the t-Bu ester groups in the side chain of poly(tBuAH) while the dense 1,2,3-triazole backbone was maintained.

Turbidimetric and Potentiometric Titrations
Turbidimetric and potentiometric titrations were carried out to study basic characteristics of poly(AH) as polyanion in aqueous media. The acid form of poly(AH) (10.4 mg,

Turbidimetric and Potentiometric Titrations
Turbidimetric and potentiometric titrations were carried out to study basic characteristics of poly(AH) as polyanion in aqueous media. The acid form of poly(AH) (10.4 mg, 68 µmol monomer units) and a small excess of NaOH (0.10 M, 780 µL) were dissolved in water (5.4 mL). Values of turbidity and pH were recorded while hydrochloric acid (0.10 M) was added with a microburet to the polymer aqueous solution as a titrant with stirring under a nitrogen atmosphere. The dissociation degrees (α) were calculated using the mass of poly(AH) and the amount of HCl added, and the values of turbidity and α were plotted in Figure 3 against pH. As can be seen in Figure 3, the turbidity was almost 10% at pH > 7, whereas the turbidity increased with decreasing pH from 7 to 4 and then saturated at ca. 90% in the regime of pH < 4. On the other hand, α decreased with decreasing pH and reached 0 at ca. pH = 3. The apparent pK a at which α = 0.5 was estimated to be ca. 5.4, which is larger than that of polyacrylic acid (PAA) (pK a = 4.5) [41]. This observation indicates that poly(AH) is a weaker polyanion than PAA presumably because of the more hydrophobic backbone and linker of poly(AH) [42].

Pulse-Field-Gradient Spin-Echo NMR
The molecular sizes of poly(AH) chains in aqueous solution at pH 9.0 and 12.0 (i.e., pD 9.4 and 12.4, respectively) were estimated by PGSE NMR using D2O containing 5 mM NaCl as a solvent. As can be seen in Figure 4, 2D DOSY data indicate that all the signals for poly(AH) were observed as unimodal distribution in the mutual diffusion coefficient (D) region of (4.5 − 6.5) × 10 −11 and (4 − 9) × 10 −11 m 2 s −1 at pH 9.0 and 12.0, respectively. (Here, the signals for methylene protons at ca. 2.2 ppm were not observed because the signals were very close to that for acetonitrile, the internal standard.) Using the signal attributed to the main-chain methine proton, the D values were evaluated to be 5.2 × 10 −11 and 4.7 × 10 −11 m 2 s −1 at pH 9.0 and 12.0, respectively. On the basis of these D values, the hydrodynamic radii values were calculated to be ca. 3.4 and 3.7 nm at pH 9.0 and 12.0, respectively, using the Einstein-Stokes equation. These data indicate that poly(AH) takes a conformation independent of pH under basic conditions.

Pulse-Field-Gradient Spin-Echo NMR
The molecular sizes of poly(AH) chains in aqueous solution at pH 9.0 and 12.0 (i.e., pD 9.4 and 12.4, respectively) were estimated by PGSE NMR using D 2 O containing 5 mM NaCl as a solvent. As can be seen in Figure 4, 2D DOSY data indicate that all the signals for poly(AH) were observed as unimodal distribution in the mutual diffusion coefficient (D) region of (4.5 − 6.5) × 10 −11 and (4 − 9) × 10 −11 m 2 s −1 at pH 9.0 and 12.0, respectively. (Here, the signals for methylene protons at ca. 2.2 ppm were not observed because the signals were very close to that for acetonitrile, the internal standard.) Using the signal attributed to the main-chain methine proton, the D values were evaluated to be 5.2 × 10 −11 and 4.7 × 10 −11 m 2 s −1 at pH 9.0 and 12.0, respectively. On the basis of these D values, the hydrodynamic radii values were calculated to be ca. 3.4 and 3.7 nm at pH 9.0 and 12.0, respectively, using the Einstein-Stokes equation. These data indicate that poly(AH) takes a conformation independent of pH under basic conditions. ymers 2021, 13, x FOR PEER REVIEW 8 of 13

Adsorption Tests
Poly(AH)Na possesses 1,2,3-triazole and carboxylate moieties in the monomer unit. Since 1,2,3-triazole and carboxylate moieties may act as relatively soft and hard ligands, respectively, poly(AH)Na may coordinate metal ions. Adsorption tests were thus performed using poly(AH)Na. Four salts, i.e., CuSO4•5H2O, PbCl2, LiCl, and Fe(NO3)3, were added to an aqueous solution of poly(AH)Na, stirred for 12 h to reach adsorption equilibrium, and then the polymer was removed by centrifugal filtration. The equilibrium concentrations (ce) of metal ions were determined by ICP measurements. Using the ce value of metal ion, the removal ratio (rremoval) and the amount of metal ion adsorbed on 1.0 g of poly(AH)Na (adsorption capacity, qe) were evaluated, as summarized in Table 2. The qe values were 75.12 ± 0.02, 62.0 ± 0.2, 89.22 ± 0.02, and 98.57 ± 0.03 mg g −1 for the four metal ions used, i.e., Cu 2+ , Pb 2+ , Li + , and Fe 3+ , respectively ( Figure 5). (Under the conditions in this study, the rremoval values were almost the same as the qe values; 75.10 ± 0.02, 62.0 ± 0.2, 89.20 ± 0.02, and 98.54 ± 0.03% for Cu 2+ , Pb 2+ , Li + , and Fe 3+ , respectively.) These observations indicate that poly(AH)Na can be used as an adsorbent for metal ions, especially Fe 3+ ion.

Adsorption Tests
Poly(AH)Na possesses 1,2,3-triazole and carboxylate moieties in the monomer unit. Since 1,2,3-triazole and carboxylate moieties may act as relatively soft and hard ligands, respectively, poly(AH)Na may coordinate metal ions. Adsorption tests were thus performed using poly(AH)Na. Four salts, i.e., CuSO 4 ·5H 2 O, PbCl 2 , LiCl, and Fe(NO 3 ) 3 , were added to an aqueous solution of poly(AH)Na, stirred for 12 h to reach adsorption equilibrium, and then the polymer was removed by centrifugal filtration. The equilibrium concentrations (c e ) of metal ions were determined by ICP measurements. Using the c e value of metal ion, the removal ratio (r removal ) and the amount of metal ion adsorbed on 1.0 g of poly(AH)Na (adsorption capacity, q e ) were evaluated, as summarized in Table 2.
The q e values were 75.12 ± 0.02, 62.0 ± 0.2, 89.22 ± 0.02, and 98.57 ± 0.03 mg g −1 for the four metal ions used, i.e., Cu 2+ , Pb 2+ , Li + , and Fe 3+ , respectively ( Figure 5). (Under the conditions in this study, the r removal values were almost the same as the q e values; 75.10 ± 0.02, 62.0 ± 0.2, 89.20 ± 0.02, and 98.54 ± 0.03% for Cu 2+ , Pb 2+ , Li + , and Fe 3+ , respectively.) These observations indicate that poly(AH)Na can be used as an adsorbent for metal ions, especially Fe 3+ ion.  Since Fe 3+ is a paramagnetic species, NMR relaxation may be significantly faster in the presence of Fe 3+ . Figure 6  It should be noted here that the signals due to the triazole and methine protons at ca. 8.2 and 6.0 ppm became weaker than did the signals due to the methylene protons at ca. 2.2. and 2.6 ppm. T1 values of signals were measured and compared in 1 H NMR spectra in the presence and absence of 0.6 mM Fe 3+ (Table 3). Figure 7 indicates the ratios of T1 values for the 1 H NMR signals in the presence and absence of 0.6 mM Fe 3+ . The ratio was 80.4% for the triazole proton at ca. 8.2 ppm, whereas those were 90.0-96.6% for the methine (6.0 ppm) and methylene protons (2.6 and 2.0 ppm). These observations indicate that Fe 3+ ions are adsorbed more preferably onto the 1,2,3triazole residues [43,44].  Since Fe 3+ is a paramagnetic species, NMR relaxation may be significantly faster in the presence of Fe 3+ . Figure 6 displays 1 H NMR spectra for poly(AH)Na in the presence of varying concentrations of Fe(NO 3 ) 3 . The signals due to poly(AH)Na are only slightly broader at [Fe 3+ ] = 0.1 mM. At [Fe 3+ ] = 3.1 and 9.1 mM, on the other hand, the 1 H NMR spectra show markedly broader signals. It should be noted here that the signals due to the triazole and methine protons at ca. 8.2 and 6.0 ppm became weaker than did the signals due to the methylene protons at ca. 2.2. and 2.6 ppm. T 1 values of signals were measured and compared in 1 H NMR spectra in the presence and absence of 0.6 mM Fe 3+ (Table 3). Figure 7 indicates the ratios of T 1 values for the 1 H NMR signals in the presence and absence of 0.6 mM Fe 3+ . The ratio was 80.4% for the triazole proton at ca. 8.2 ppm, whereas those were 90.0-96.6% for the methine (6.0 ppm) and methylene protons (2.6 and 2.0 ppm). These observations indicate that Fe 3+ ions are adsorbed more preferably onto the 1,2,3-triazole residues [43,44].

Conclusions
As a new polyanion with a dense 1,2,3-triazole backbone, poly(AH), was synthesized by CuAAC polymerization of tBuAH followed by hydrolysis of the t-Bu ester groups. The

Conclusions
As a new polyanion with a dense 1,2,3-triazole backbone, poly(AH), was synthesized by CuAAC polymerization of tBuAH followed by hydrolysis of the t-Bu ester groups. The 1 H NMR and FTIR data confirmed the structure of poly(AH). Turbidimetric and potentiometric titration date indicated that poly(AH) was well soluble at pH > 7, slightly soluble in the pH region of 7-4, and practically insoluble in the regime of pH < 4. The apparent pK a at which α = 0.5 was estimated to be ca. 5.4, which is larger than that of PAA (pK a = 4.5), indicating that poly(AH) is a weaker polyanion than PAA presumably because of the more hydrophobic backbone and linker of poly(AH). On the basis of PGSE NMR data, the hydrodynamic radii of poly(AH)Na were estimated to be ca. 3.4 and 3.7 nm at pH 9.0 and 12.0, respectively, indicating that that poly(AH) takes a conformation independent of pH under basic conditions. Since 1,2,3-triazole and carboxylate moieties may act as relatively soft and hard ligands, respectively, adsorption tests were thus performed using poly(AH)Na. The amounts adsorbed were 75.12 ± 0.02, 62.0 ± 0.2, 89.22 ± 0.02, and 98.57 ± 0.03 mg g −1 for Cu 2+ , Pb 2+ , Li + , and Fe 3+ , respectively. These observations indicate that poly(AH)Na can be used as an adsorbent for metal ions, especially Fe 3+ ion. The ratio of T 1 values for the 1 H NMR signals in the presence and absence of 0.6 mM Fe 3+ was calculated to be 80.4% for the triazole proton at ca. 8.2 ppm, whereas those were 90.0-96.6% for the methine (6.0 ppm) and methylene protons (2.6 and 2.0 ppm), indicating that Fe 3+ ions are adsorbed more preferably onto the 1,2,3-triazole residues. On the basis of poly(AH), synthesis of sequence-controlled amphiphilic polymers is in progress.