Poly(N-isopropylacrylamide-co-2-((diethylamino)methyl)-4-formyl-6-methoxyphenyl acrylate) Environmental Functional Copolymers: Synthesis, Characterizations, and Grafting with Amino Acids

Vanillin was used to synthesize a new derivative with an active aldehyde group and response to pH. It is named 2-((diethylamino) methyl)-4-formyl-6-methoxyphenyl acrylate, abbreviated to DEAMVA. The chemical structures were evaluated by 1H, 13C nuclear magnetic resonance (NMR), infrared (IR), and UV-Vis-spectroscopy, and all results demonstrated good statement. In order to achieve the dual responsive behavior thermo-pH with functionality, free radical polymerization of N-isopropylacrylamide with DEAMVA in different molar ratios (5, 10, 15 mol%) has been used, with azobisisobutyronitrile (AIBN) as the initiator. The chemical structure of the polymers was investigated by 1H NMR and IR. The dual responsive functional copolymer was exposed to a grafted process with tryptophan and tyrosine, both of which were also evaluated by 1HNMR and IR. Copolymers before and after grafting were physically investigated by size exclusion chromatography (SEC) for estimation of the molecular weight, the glass transition temperature by differential scanning calorimeter (DSC) and scanning electron microscope (SEM) for the surface morphology. The phase separation or lower critical solution temperature (LCST) (Tc) of the polymer solution was determined not only by a turbidity method using the change in the transmittance with temperature, but also by micro-DSC. The conversion to an amino acid-grafted polymer was detected through Beer’s law for the absorption of the –CH=N- imine group by UV-Vis-Spectroscopy.


Introduction
In nature, many living creatures can change their behavior according to the surrounding environment; examples include the Venus flytrap (closes fast enough to catch its prey), the leaves of Mimosa pudica (collapses immediately when touched), sunflowers (follow the movement of the sun), sea cucumbers (change their stiffness in the face of danger), and chameleons (change color according to the nature of the environment) [1][2][3][4].
Over the past few years, many scientists have focused their interest on a new kind of polymer material with special characterizations that can develop its behavior with the surrounding environment. Several definitions were used like environment, intelligent, smart, and responsive, according to the physical/chemical properties. These polymer materials can develop their responses to temperature, the intensity of light, humidity, pH, ionic strength, and electric/magnetic fields. Basically, they can be classified as one of single stimuli-responsive, dual stimuli-responsive, or multistimuli responsive [5][6][7].

Instrumentations and Measurements
1 H (500 MHz) and 13 C (125 MHz) NMR spectra were recorded on Bruker AV (Germany, Karlsruhe) in d 6 -DMSO or CDCl 3 . IR spectra were measured on a Vertex 70 FT-IR-4100 spectrophotometer (Germany, Karlsruhe). The samples were milled with dry potassium bromide KBr (99% Merck, Germany, Darmstadt) and pressed into pellets. The molecular weights number average molecular (Mn), weight average molecular weights (Mw), and polydispersity (Ð) were determined by gel permeation chromatography (GPC) on TSK gel α-3000, using a solution of LiBr (10 mM) in N,N-dimethylformamide (DMF) as an eluent at a flow rate of 1.0 mL/min, calibrated by polystyrene standards at 30 • C. This was used to determine the conversion of amino acid grafted polymers. UV/vis spectrometer (Perkin Elmer Lambda 45, UK) with metal covet stand and water bath (Julabo F12, UK) with thermostat for injection of the water cycle, and cooling system was used to measure the phase transition temperature (LCST) (T c ). Over manual thermostat (Temperatur-Messgerät Md 3040, Beckmann+Egle, UK) was also used to adjust the actual temperature inside the solution at 2 • C/min over a temperature range of 5 to 80 • C. The polymer solution was 1 wt % in water or pH solution. Definitional scanning calorimeter (micro-DSC) was used to determine the transition temperature (T c ) of the polymer solution; the thermograms of the polymer solutions were recorded at a cooling and heating rate of 5 • C/min. A concentration of 50 mg/mL was dissolved in deionized water, and the transition temperature was determined as the onset value in the thermogram. The glass transition temperature of solid polymers was recorded by Perkin Elmer Differential Scanning Calorimeter (DSC) Pyris 1 (USA, Waltham, MA, USA) with a heating and cooling rate of 5 • C/min. The morphological feature of the polymer before and after grafting was examined by Scanning Electron Microscopy (SEM) using a Zeiss NEON 40 instrument (USA, San Diego, CA, USA); 2 kV (30 µm aperture) and Bal-Tec SCD 500 sputter (USA, San Diego, CA, USA) coated with a film thickness monitor QSG 100. We applied approx. 4 nm of gold-palladium (Au:Pd = 80:20). The melting point was recorded by Stuart Digital Melting Point Apparatus (UK, Staffordshire, ST15 OSA), temperature range: Ambient to 300 • C, temperature resolution: 1 • C, and Ramp rates: 20 • C per min to plateau, 2 • C per min to melt. Equal amounts of (20 g, 0.13 mol) vanillin (4-hydroxy-3-methoxy benzaldehyde), (20 g, 0.66 mol) formaldehyde and (20 g, 0.27 mol) diethylamine were dissolved in 100 mL ethanol. The reaction mixture was stirred and refluxed with a water trap for 3 h in a 250 mL, single-neck, round-bottomed flask. Water was removed from the trap and the solution was allowed to cool at room temperature. The product was concentrated under reduced pressure and allowed to cool for a few hours at room temperature; it was then extracted as a solid material. It was dried overnight in a desiccator with calcium chloride, and fitted with reduced pressure. The product Yield was 97%. It comprised a yellowish white solid with a melting point (mp) of 126 • C. 1   In a 500 mL, three-neck flask fitted with an argon balloon, refluxed condenser, and dropping funnel, (20.85 g, 0.087 mol) of 3-((diethylamino)methyl)-4-hydroxy-5-methoxy-benzaldehyde (DEAMV) (I) was dissolved in dry CH 2 Cl 2 (300 mL). Pellets of sodium hydroxide (15.0 g, 0.375 mol) were added and the mixture was stirred. The overall reaction mixture was cooled in an ice bath to 0-5 • C. (8.1 g, 0.0885mol). Then, acryloyl chloride was dissolved in 50 mL dry CH 2 Cl 2 and added dropwise by dropping funnel. The solution color changed from colorless to a yellowish suspension. After 1h, the ice bath was taken off and stirring was continued at room temperature (25-27 • C) for 8 h. The precipitate of sodium chloride was filtered, and then the filtrate was taken to a rotatory evaporator to remove the solvent and concentrate the crude product. The purification process was done by dissolving the crude product in CH 2 Cl 2 and washing it three times with distilled water, once with 0.1 M Na 2 CO 3 , and again with distilled water. The pure product was dried by stirring in MgSO 4 overnight; the solvent was removed under reduced pressure to collect the product, and decanted in a dry flask injected by nitrogen. Yield was 75%, comprising an orange viscous liquid.  In a 100 mL, two neck, round bottom flask with in and outlet argon and 25 mL dropping funnel, 5, 10, and 15 mol %, 0.544 g and 1.088 g, 1.632 respectively of (II) was added to 2 g (0.0176 mol) NIPAAm in 50 mL absolute ethanol. The mixture was purged by nitrogen and stirred. After complete dissolving, AIBN was dissolved in 15 mL absolute ethanol and added as 10 −3 mol % of the total mol% of monomers. The separating funnel was removed and the mixture was stirred in an oil bath at 65 • C for 8 h under an inert atmosphere. The polymerization process was stopped and the solution cooled at room temperature and then in a refrigerator for termination. The polymer was precipitated in diethyl ether, at −40 • C (liquid nitrogen + acetone in 1.9 L Dewar flask), then dissolved in THF, and re-precipitated in diethyl ether to remove the unreacted monomers and impurities. Yield: 88%, 82%, and 76% for 10%, 15% and 20% mol of II respectively, Physical state: Yellowish solid. IR (KBr): ν (cm −1 ) 2990 (s) (6, 7-CH-Aliphatic), 1714-1743 (s) (5-C=O), 1640-1650 (s) (1-C=O), 1134 (s) (12-OCH 3 ).

Synthesis of Grafted 15 mol % Poly (NIPAAm-co-DEAMVA) with Tryptophan and Tyrosine (IV-V)
In a two-neck flask fitted with a reflux condenser with a water trap and rubber stopper to pass a syringe of inlet and outlet argon, a mixture of 1.0 g of 10 mol % Poly(NIPAAm-co-DEAMVA) and 1.0 g of (0.005 mol tryptophan, 0.055 mol tyrosine) was dissolved in 30 mL ethanol-water 50/50 V/V %. The reaction mixture was allowed to reflux and stirred for 8 h at 110-120 • C. The product was transported to a one neck flask and the solvent was evaporated under reduced pressure. The product was purified by dissolving in THF and reprecipitated in diethyl ether at −30 • C to avoid any unreacted molecules. Physical state: with VI (Tryptophan) Brownish solid, V (tyrosine) yellowish solid.

Synthesis of Grafted 15 mol % Poly (NIPAAm-co-DEAMVA)-g-tyrosine as a Function of Time
In order to study the conversion of polymer to the grafted polymer with amino acid, we repeated the previous reaction with tyrosine using the same method several times as a function of the reaction time (15,180,360, 840 min). The products were separated and purified. UV-Vis spectroscopy was used to determine the reaction conversion for each running reaction, according to the change of the absorbance intensity.

Synthesis of Monomer, Copolymers and Grafted Copolymers
Monomer, copolymers, and grafted copolymers were synthesized according to the chemical conditions described in Figure 1. In the present study, we used a vanillin compound for the synthesis of new kinds of acrylate monomers. The new monomer (II) [(diethylamino) methyl]-4-formyl-6-methoxyphenyl acrylate (DEMAVA) was synthesized in two steps, as described in Figure 1 This has been done by the reaction of vanillin with diethylamine and formaldehyde, according to Mannich reaction mechanism. In this reaction, we did not use any catalysis, especially acid catalysis, which is famously used in Mannich reaction; therefore, protonated tertiary amine has been yielded. This was done by the reaction of vanillin with diethylamine and formaldehyde according to Mannich reaction mechanism. It was chemically evaluated by 1 H NMR and 13 C and FT-IR in Figures 1 and 2. All data was in a logic state, and proved the presence of an active aldehyde group at 9.74 ppm and 196 ppm. A tertiary amine group was also detected at 1.13 ppm for 2CH 3 , at 2.66 and 3.85 ppm for 2CH 2 , N(CH 2 ) 2 and Ar-CH 2 -N respectively.  Figure 1. It was chemically evaluated by 1 H NMR and 13 C (Figures 3 and 4) and Fourier-transformed infrared spectroscopy (FT-IR). All data was in a logic state and proved the presence of an active aldehyde group at 9.95 ppm and 192.52 ppm. The chemical reactions for the preparation of dual responsive copolymer with different mole ratios DEAMVA (5, 10, 15% mol) are illustrated in Figure 1. The copolymers were prepared and evaluated by 1HNMR and FTIR, as shown in Figures 5-7. The 1 H-NMR recorded specific signals for IIIa-c at δ = 0.73-1.35 ppm for 6H dimethyl of NIPAAm, at δ 3.60-3.73 ppm for 3H methoxy group of vanillin, the aromatic protons 2H appeared at δ = 7.08-7.62 ppm, and finally, the most distinguished signal related to the aldehyde group of DEAMVA 1H at δ = 9.73-10 ppm. The FT-IR spectroscopy of polymer with dry KBr, as described in the instrumental part, exhibited signal emphasis of the results of 1 H NMR. The stretched signals were recorded at 1742 cm −1 for carbonyl ester, and at 1650 cm −1 for amide. Moreover, the 1 H NMR was used to determine the actual mole percent of each monomer in the polymer chain. The intensity of the signals at 1.21 ppm of 1H isopropyl group (CH(CH 3 ) 2 ) was a specific signal of NIPAAm, and 1H at 9.73 ppm of aldehyde (CHO) of DEAMVA, as shown in Table 1.
The grafting of tyrosine and tryptophan into the dual responsive copolymer was enhanced according to Schiff base between the aldehyde group of DEAMVA and the primary amine group of amino acids. The grafting process was done at room temperature in a mixture of ethanol and H 2 O 1:1.

Molecular Weight
Molecular weight (M n ) and molecular weight distribution (M w /M n ) of all polymers, copolymers, and terpolymers were determined by Gel-Permeation Chromatography with Knauer in dimethylacetamide DMAc.
Polymer samples (6 g/L) were prepared with 2,6-di-tert-butyl-4-methylphenol (BHT) as an internal standard. The measurements were performed at 30 • C. The molecular weight of copolymers and grafted copolymers is summarized in Table 1.
The spectra showed one peak, indicating that complete conversion of monomers to the polymer and the absence of low molecular weight and impurities [4,6,29], as shown in Figure 9A,B.

Study of the Phase Separation of Poly (NIPAAm-Co-DEAMVA) and Grafted Polymers
The lower critical solution temperature LCST at pH 1.68 and pH 7 showed an increase as the molar concentration of DEMAVA increased in the polymer chain, as shown in Table 1. This might be due to the formation of charge in the polymer chain causing electrostatic repulsion which further increases the hydrodynamic volume [39,40], as shown in Figure 9. At pH 1.68, the polymer has hydrated tertiary amine and aldehyde groups that converted to OH groups. For this reason, the hydrophilic groups are more effective than the hydrophobic groups of DEMAVA. Hence, overall, the copolymer exhibited higher hydrophilicity. At pH 7, the polymer solution shows no change because the affection of total hydrophilic groups is too weak for DEMAVA. Therefore, the polymer solutions demonstrated LCST which eas closed more to homo-poly (NIPAAm) with respect to the affection of high composition of DEMAVA for polymer IIIc with the highest (T c ) 60.6, 44 • C at pH 2 and pH 7 respectively, as shown in Figure 10A,B.
The last feature was detected at pH 11, which did not show any change in LCST starting from 10 • C to 80 • C. For the deprotonating of hydrated tertiary amine and the formation of tertiary amine with a lone pair of electrons as shown in Figure 11, this is responsible for raising LCST to 80 • C.
The post polymerization of poly (NIPAAm-co-DEAMVA) with tryptophan and tyrosine IV and V was achieved by chemical reaction with the formation of a Schiff base, which showed responsiveness to pH addition to the presence of tertiary amine group.
The conjugation of tryptophan and tyrosine with polymer molecules is based on the formation of a Schiff base through the reaction of amine group with aldehyde and the formation of imine. This kind of molecule is stable in alkaline conditions, and decomposed in stronger acidic one. For this reason, it characterizes as a stimuli-responsive linker in polymer chemistry [41]. The imine group affects the electrical charge in the polymer chain. After conjugation, the copolymers became more hydrophilic than at first, and gave a dual response character to pH and temperature. The lower critical solution temperature of the conjugated polymer solution was measured by UVVis-spectroscopy in different pH buffer solutions (pH 1.68, pH 7 and pH 12.46). For copolymer IV and V conjugated with tryptophan and tyrosine respectively, we observed a rise in T cs in pH 1.68 to more than pH 7, due to the release of amino acid and the formation of charge along the polymer chain. The other observation was the higher T c value for the conjugated copolymer IV, with tryptophan at pH 1.68 exhibiting T c = 50 • C compared to the T c value for the conjugated copolymer V with tyrosine T c = 42 • C, indicating the higher hydrophilicity of IV compared to V, according to the chemical structure of the conjugated amino acid, which proves the higher hydrophilicity of tryptophan compared to tyrosine [42]. As previously noted, the T c at stronger basic conditions, i.e., pH 12.46, could not be recorded until the highest temperature, 80 • C, due to the greater electrical charge in the polymer chain. The lower critical solution temperature T c was taken as the inflected point for all copolymers further. The cloud point was also detected at 50% transmittance. Table 1 summarizes all values. The (T c , s), Figure 10A-D showed a relationship between temperature and transmittance. All the T c values for copolymers were calculated as the inflected point, while the cloud point was taken at 50% transmittance Micro-DSC, and was also used to determine the T c of copolymer IIIa in pH 7. The different T c , svalues have been detected by UV-Vis. Spectroscopy and micro-DSC Figure 12, depended on the definitions of the T c as the inflected point or the onset respectively [43].

Determination of the Conversion of poly (NIPAAm-Co-DEAMVA) to Poly (NIPAAm-Co-DEAMVA)-g-Tryptophan
After the post polymerization and conjugation with amino acid, it was important to determine the conversion of poly (NIPAAm-co-DEAMVA) to the grafted one. For this purpose, we used poly (NIPAAm-co-DEAMVA)-g-tryptophan for the experiment. The conversion was related to time. UV.vis. Spectroscopy was used to record the conversion at the different times (15,180,360, and 840 min) by recording the absorption for each run, as shown in Figure 13. The Labert-Beer law has been used for to determine the concentrations and mol % of conversion. The absorption peaks exhibit the disappearance of the absorption of carbonyl aldehyde at 250-270 nm for n-π*, π-π* and a good appearance of the new peaks at 340-380 related to C=N imine linkage n-π*. As is clear in Figure 12, the C=N absorption increased steeply with time to get the greatest value at 840 min. Table 1 shows the conversion percentages at 840 min for poly (NIPAAm-co-DEAMVA)-g-tryptophan and poly (NIPAAm-co-DEAMVA)-g-tyrosine.

The Thermal Properties
The glass transition temperature T g is the most important thermal property. A Differential Scanning Calorimeter was used for recoding T g for all polymers. The measurements were taken for dry samples at a heating rate 5 • C/min at the midpoint inflection of the thermogram. The thermogram exhibits one T g value, proving random copolymerization [44]. A comparison of the T g , s values of PNIPAAm homopolymer, copolymer with DEAMVA and grafted copolymers with tryptophan and tyrosine, was undertaken. Table 1 summarizes the (T g , s) values. Firstly, a recent study measured the glass transition temperature of homo-PNIPAAm T g at 135 • C [44]. The incorporation of DEAMVA with its hydrophilic and hydrophobic groups in the PNIPAm main chain will shift the T g values to 122, 127, and 137 • C for IIIa, IIIb, and IIIc respectively. It has been demonstrated in Figure 14 that an increase in the T g values occurs by increasing the molar concentration of DEAMVA; this is attributed to the increase in hydrophilicity, which leads to a decrease in the spacing and lower the chain interaction lower in the flexibility [45]. After the conjugation of the copolymer with tryptophan and tyrosine, we noticed an increase in T g due to the same factors discussed previously; in spite of this, the T g of grafted copolymer with tryptophan IV has a smaller increase in T g 136 • C than tyrosine V 133 • C for the higher hydrophilicity of tryptophan than tyrosine, supporting from its chemical structure.

Morphological Studies
The morphology of solid polymers has been distinguished by a scanning electron microscope (SEM). The scanning was done for copolymer IIIb before and after grafting the copolymer with tyrosine V. Figure 15 is the SEM photograph before and after grafting at a magnification of 500×. The polymer surface before grafting seems smooth and compact, with small pores. After grafting with tyrosine, it appears as a waxy and coarse surface with some cross-links; these morphological changes were due to the grafting of tyrosine in the copolymer chains. Figure 15. SEM images of copolymer IIIb (before grafting) and after grafted copolymer with tyrosine V at 500× magnification.

Conclusions
Vanillin acrylate monomer has been prepared in two steps; first, by the preparation of vanillin with tertiary amine group which then reacted with acryloyl chloride for the preparation of the final product, DEAMVA. This monomer was distinguished by its stimuli-responsiveness to pH, in addition to its functionality. In the preparation of thermal and a pH dual responsive polymer, we used N-isopropyl acrylamide (NIPAAm) with three different molar concentrations of DEAMVA, using a free radical polymerization technique. All chemical evaluation of monomers and polymers were in a logic state, and proved the presence of aldehyde groups, which were used in the grafting of amino acid. Due to the thermal and pH-responsive behavior of the polymer, the phase separation and LCST are of great interest. The LCST of copolymers showed higher T c than PNIAAm for its higher hydrophilicity, and exhibited a regular increasing rate with the molar concentration of DEAMVA. The grafting of amino acid was facilitated by the presence of the aldehyde group in the polymer chain. The grafting process occurred with tryptophan and tyrosine; both were evaluated by chemical methods and proved the disappearance of aldehyde group and the formation of imine. The lower critical solution temperature LCST and T c was recorded and showed an increased value for tryptophan over tyrosine. The conversion was also studied as a factor with time, demonstrating the highest conversion at 840 min. DSC thermogram for copolymers and grafted copolymers showed higher T g values by increasing the concentration of DEAMVA. Moreover, the glass transition temperature of the grafted tryptophan was higher than that of the grafted tyrosine. SEM morphological photograph of the grafted copolymer showed some cross-links, which were attributed to the grafted molecule.
Funding: This research was funded by University of Tabuk, granted number S-1439-0208.