2.1. Material
2-Hydroxyethyl methacrylate, 1,2-Ethanediol mono(2-methylpropenoate), Glycol methacrylate, (HEMA, 98%, Sigma-Aldrich, Germany) was extra-purified by distillation under reduced pressure, 2,2′-Azobis(2-methylpropionitrile) (AIBN, 98%, Sigma-Aldrich), anhydrous dimethylamine (DMA, 99%, Sigma-Aldrich), formaldehyde (37 wt% in water, 10-15% methanol, Sigma-Aldrich), N-isopropylacrylamide (NIPAAm, 97%, Acrōs) has been purified by recrystallization from a mixture of benzene/n-hexane 60/40 v/v, Vanillin, 4-Hydroxy-3-methoxybenzaldehyde, (V, 99%, Sigma-Aldrich), acryloyl chloride or 2-Propenoyl chloride (98 %, Sigma-Aldrich), guanine (98%, Sigma-Aldrich). Ethanol, dichloromethane, diethyl ether, and tetrahydrofuran (THF) have been stirred overnight in potassium hydroxide at room temperature and then distilled. Other chemicals were used as purchased.
2.2. Instrumentation
Nuclear magnetic resonance (NMR) spectroscopy model Bruker AV 500 has been used for the investigation of 1H and 13 C of monomers and polymer molecules. The solid and dried material has been dissolved in deuterated CDCl3 or DMSO-d6, and then data has been measured at 500 MHz (1H) and 125 MHz (13C).
Compact ATR-FTIR spectroscopy with the Bruker Alpha has been used for the deduction of functional groups of both monomers and polymers molecules. The sample has been fixed on the surface of a ZnSe ATR crystal and then is functioning as fiber optics. By the entry of the waves of light into the sample; it was exposed to a total reflection. The total attenuated reflected beams were measured and the final results seemed as signals that converted into an infrared spectrum.
ESI/MALDI-TOF mass spectroscopy with modern soft ionization techniques such as electrospray ionization (ESI), the chemical ionization has been done under atmospheric pressure (APCI), and matrix-assisted laser desorption ionization (MALDI) has been used to investigate the ionization of monomer compounds m/z.
The vario MICRO cube elemental analyzer has been used for estimating the elemental composition of C, H, and N of the monomer compounds.
Gel permeation chromatography (GPC) is a technique that used to separate dissolved macromolecules according to their size and depending on their elution through columns filled with a porous gel. It has been used for the determination of the molecular weights including the viscosity average molecular weight (Mv), the number average molecular weight (Mn), and the weight average molecular weight (Mw); dispersity or the formal name the polydispersity index (PDI) and also known as heterogeneity index has been also measured as Mw/Mn (Ð). We used tetrahydrofuran (THF) as eluent with 2 g/L 2,6-di-tert-butyl-4-methylphenol (BHT). It was compacted with Jasco 880-PU pump and Waters RI-Detector for justification of the rate of flow at 0.75 ml/min., toluene was the internal standard at 30 ℃. Polymers have been dissolved as 6 g/L in concentration, and then the injection process was achieved by hand. Standards gel particle PSS-SDV with high-speed polymer service columns has been filled with the porosity of 106 Å (guard), 105 Å, 103 Å, and 102 Å have been respectively used; the molecular weights were detected and recorded related to a narrow polystyrene standard.
Differential scanning calorimeter-DSC Analysis-Netzsch DSC 214 PolymaIt has been used to detect the glass transition temperature of solid terpolymers Tg,s. it is characterized by the temperature range of 25 to 600 ℃, and a heating and cooling rate at 0.001 to 500 K / min. the glass transition temperature was taken It at the onset value of the thermogram.
Thermogravimetric analysis (TGA) with the Mettler Toledo TGA/SDTA851was used to elucidate the chemical decomposition as the change of mass with the temperature of polymer sample by heating from 25 ℃-600 ℃ at the rate of 5 ℃/min.
Scanning Electron Microscopy (SEM) Zeiss NEON 40; the change on the surface of the polymer due to the chemical modification has been noticed as the change in the surface morphological features of polymers. It was investigated by Scanning Electron Microscopy (SEM); Zeiss NEON 40, (USA) is the model of the instrument; it is characterized by 2 kV (30 µm aperture), a Bal-tec SCD 500 sputter coater with a film thickness monitor QSG 100. 4 nm and gold-palladium (Au: Pd = 80:20).
UV/vis spectrometer (Perkin Elmer Lambda 45)
Two experiments were tested using a UV/VIS spectrometer (Perkin Elmer Lambda 45).
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The estimation of absorption and conversion of polymer to guanine-post-polymer at different pH solutions.
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Measuring of the (LCST) (Tc,s) of terpolymers and post-polymer at different pH solutions.
By the test, the instrument was fixed by metal covet, and it has been connected with the water cycle of the water bath with a thermostat and the water pump for both heating and cooling. The manual thermostat (TEMPERATUR-MESSGERÄT MD 3040, BECKMANN+EGLE) placed inside the polymer solution via the rate of 2 ℃/min in the range of 5-75 ℃ has been used to justify the inner temperature; the polymer concentration was 1 wt. /wt.%.
Micro- Differential Scanning Calorimeter (micro-DSC) (Perkin Elmer)
This technique has been used to record the phase transition temperature and the lower critical solution temperatures (LCST) (Tc,s) of the terpolymers and post-polymer solution. 50 mg/ml of polymer sample dissolved in DI water; the samples have been exposed to cool and heat at heating rate of 5 ℃/min.; the transition temperature (Tc) was detected at the onset value.
2.1. Synthesis of pH-responsive acrylate monomer (DMAMVA)-(III) 2-((dimethylamino)methyl)-4-formyl-6-methoxyphenyl acrylate
Step 1: Synthesis of (DMAMV)-(II) 3-((dimethylamino)methyl)-4-hydroxy-5-methoxy-benzaldehyde
Vanillin was used as the start material. 14 g (0.092 mol) vanillin (4-hydroxy-3-methoxy benzaldehyde), 14 g (0.310 mol) dimethylamine, and 14 g (0.456 mol) formaldehyde were dissolved in 150 ml dry ethanol. The mixture was transferred into a 250 ml two necks round bottom flask fixed with a water condenser and water trap. It was stirred and allowed refluxing for 5 h at 130 ℃; after about 1.5 h an orange precipitate started till filling the flask. After that, the reaction was stopped and has been kept at RT. The product was concentrated by evaporation of solvent by vacuum rotatory evaporator and then was recrystallized from ethanol. 97.4 %, orange solid, melting point = 140 ℃
1 H NMR (500 MHz, CDCl 3 ): δ (ppm) = 2.37 (s, 6 H, 6-N(CH3)2), 3.75 (s, 2 H, 5-Ar-NCH2), 3.93 (s, 3 H, 3-OCH3), 6.39 (br, 1H, 1-OH), 7.15 (d, 1 H, 4J = 1.8 Hz, 9-Ar-CH), 7.33 (d, 1 H, 4J = 1.8 Hz, 7-Ar-CH), 9.76 (s, 1 H, 11-CHO).
13 C-NMR (125 MHz,CDCl 3 ): δ (ppm) = 44.32 (2 C, 6-NCH3), 56.01 (1 C, 5-NCH2), 62.21 (1 C, 3-OCH3), 109.97 (1 C, 9-Ar-CH), 123.70 (1 C, 7-Ar-CH), 125.21 (1 C, 4-Ar-C), 128.09 (1 C, 8-Ar-C), 148.68 (1 C, 2-Ar-C), 154.54 (1 C,10-Ar-C), 190.67 (1 C, 11-CHO).
IR (KBr): ν (cm −1 ) = 2450-3100 (s) (CH2, CH3), 2056-2341 (s) (C-N), 1735 (s) (C=O), 1660 (s) (Ar-C-O), 1575 (s) (Ar-C=C), 1112 (s) (-OCH3), 814 - 847 (m) (Ar-CH).
MS m/z (%)
209.11 [M+],100%
Anal. Calcd. For C11H15NO3 (209.11): Calc. (%): C, 63.14; H, 7.23; N, 6.69; Found (%): C, 62.92; H, 7.34; N, 6.23
Step 2: Synthesis of compound (III) 2-((dimethylamino)methyl)-4-formyl-6-methoxyphenyl acrylate (DMAMVA).
The final product of the new acrylate monomer (DMAMVA)-(III) can be s synthesized by the addition of 7.5 g (0.0355mol) (DMAMV)-(II) and 7.5 g (0.187mol) sodium hydroxide into 100 ml dry dichloromethane and transferred into 250 ml three-neck flask equipped with dropping funnel, water condenser, and a balloon of argon. The reaction mixture has been allowed to stir in an inert atmosphere. The reaction was cooled to 0-5 ℃ in an ice bath, and then 3.23 g (0.0355 mol) acryloyl chloride dissolved in 15 ml dichloromethane was dropped to the reaction mixture by the dropping funnel and during vigorous stirring. The suspension converted to yellow color on the edges of the flask; the stirring was continued at 0-5°C for about 1 h until the yellowish precipitate was dominated. After this step, the reaction was left at room temperature for 6 h.; it was filtered and the solvent was removed. The purification steps was carried out by dissolving in CH2Cl2 and washing several times with deionized water, and then washing with 0.1 M Na2CO3 solution, and washed two times again with DI H2O. It was separated by the separated funnel, and the organic phase was dried overnight via MgSO4. The product was concentrated by evaporation of the solvent and the final pure product was collected for further analysis and application. 75%, orange viscous liquid.
1 H NMR (500 MHz, CDCl 3 ): δ (ppm) = 2.19 (s, 6 H, 13-2CH3), 3.37 (s, 2 H, 12-NCH2), 3.84 (s, 3 H, 6-OCH3), 6.03 (dd, 2J = 1.1 Hz, 3J = 10.5 Hz,1 H, 1-Hb=CH2), 6.34 (dd, 3J = 10.5 Hz, 3J = 17.3 Hz, 1 H, 2-Hc=CH), 6.64 (dd, 2J = 1.1 Hz, 3J = 17.3 Hz, 1-Ha=CH2), 7.37 (d, 1 H, 4J = 1.7 Hz, 7-Ar-CH, 7.34 (d, 1 H, 4J = 1.7 Hz, 10-Ar-CH), 9.91 (s,1 H, 9-CHO).
13 C-NMR (125 MHz, CDCl 3 ): δ (ppm) = 45.42 (2 C, 13-2CH3), 55.72 (1 C, 12-NCH2), 62.41 (1 C, 6-OCH3), 108.75 (1 C, 7-Ar-CH), 122.37 (1 C, 11-Ar-CH), 126.25 (1 C,2=CH), 127.30 (1 C, 10-Ar-C), 132.22 (1 C, 1=CH2), 134.39 (1 C, 8-Ar-C), 143.50 (1 C, 4-Ar-C), 152.51 (1 C, 5-Ar-C), 162.81 (1 C, 3-COO), 191.64 (1 C, 9-CHO).
IR (KBr): ν (cm −1 ) = 2400-3120 (s) (CH2, CH3), 2058-2450 (s) (C-N), 1760 (s) (ester C=O), 1745(s) (aldehyde C=O),1665 (s) (Ar-C-O), 1587 (s) (Ar-C=C), 1120 (s) (-OCH3), 820 - 860 (m) (Ar-CH).
MS m/z (%)
263.12 [M+], 100%
Anal. Calcd. For C14H17NO4: Calc. (%): C, 63.87; H, 6.51; N, 5.32;; Found (%):C, 63.22; H, 5.97; N, 5.17
2.2. Synthesis of poly (N-isopropylacryamide-co-2-Hydroxyethyl methacrylate-co-2-((dimethylamino)methyl)-4-formyl-6-methoxyphenyl acrylate) poly (NIPAAm-co-HEMA-co-DMAMVA) thermo-pH stimuli-responsive terpolymer
The reaction has been done three times with different molar concentrations of DMAMVA 5, 10 and 20 mol % (0.0013 mol/0.342 g), (0.00265 mol/0.697 g) and (0.0053 mol/1.697 g) respectively, and 10 mol % of HEMA (0.00265 mol/0.345 g), and (0.0265 mol/3 g) of N-isopropylacrylamide and AIBN initiator (10−3 of the total molar concentrations of monomers); they were dissolved in 60 ml absolute ethanol. The three mixtures were reacted in 100 ml round bottom flasks fitted with rubber stoppers allowed for nitrogen injection. They allowed stirring in an oil bath for 6h at 75 ℃. After the specific time has finished the polymerization reaction has been terminated by cooling firstly at room temperature and then in the refrigerator. The terpolymers were precipitated in diethyl ether, at -20 ℃; they have been extra purified to remove the small molecules by dissolving in THF and again re-precipitated in Et2O. They were crystals varied in color from white to light yellow to yellow depending on the concentration of DMAMVA.
1 H NMR (500 MHz, CDCl 3 ): δ (ppm) = 0.95-1.22(m, 12H, 6, 12-2CH3), 1.25-1.62 (m, -CH- repeating unit), 1.83-2.22 (m, 3H, 9-CH3), 3.42-3.47(m, 2H, 5-CH2), 3.53-3.59 (m, 3H, 4-CH3), 3.60-3.68 (m, 2H, 7-CH2), 3.73-4.05 (m, 1H, 11-CH), 4.74-5.22 (m, -CH2-repeating unit), 6.72-7.84 (m, 3H, 10-NH, 2,3-Ar-H), 9.85-10.07(m, 1H, 1-CHO).
IR (KBr): ν (cm −1 ): 3254-3662 (N-H, OH), 3760-3100 (CH-Aliphatic), 1764 (s) (C=O, carbonyl), 1728 (s) (C=O, aldehyde), 1565, (s) (C=O amide), 1110 (m) (OCH3), 877 (m) (CH-Ar.).
2.3. Synthesis of poly (NIPAAm-co-HEMA-co-DMAMVA)-post-guanine
The post-polymerization of poly (NIPAAm-co-HEMA-co-DMAMVA) with guanine has been achieved according to Schiff base reaction mechanism. The terpolymer VI-10-10 was selected for implementing. The reaction between terpolymer and guanine was performed in different pH solutions pH3, pH7 and pH12. In three 50 ml round bottom flasks 1 g of the terpolymer VI-10-10 was added to (0.013 mol/2 g) of guanine were dissolved in 30 ml pH solutions. They allowed stirring for 6h at RT. The brownish precipitate start appears after 2h; the reaction continued till the limited time and then stopped and separated off. The precipitate was filtered, and then dissolved in THF and precipitated in diethyl ether at -20 ℃. The product was dark brownish crystals.
1 H NMR (500 MHz, CDCl 3 ): δ (ppm) = 0.96-1.51 (m, 12H, 9,18-2CH3), 1.53-1.66 (m, 3H, 12-1CH3), 1.65-1.95 (m, 2H, 10,14-CH repeating), 2.08-2.75 (m,6H, 11,13,15-3CH2 repeating), 3.45-3.56 (m, 3H, 7-1CH3), 3.52-3.83 (m, 2H, 20-1CH2), 3.87-3.93 (m, 2H, 19-1CH2), 4.02-4.17 (m,1H, 17-CH), 4.85-5.36 (m, 1H, 2-CH), 5.90-6.10 (m, 1H, 3-NH), 6.13-6.73 (m, 1H, 17-NH), 6.79-7.20 (m,1H, 5-Ar-H), 7.30-7.48 (m,1H, 5-Ar-H), 8.05-8.23 (m, 1H, 4-CH=N), 12.46-12.75 (m, 1H, 1-NH).
IR (KBr): ν (cm −1 ): 3430-3640 (s) (NH, OH), 2988-3185(m) (CH-Aliphatic), 1640-1647 (s) (C=O), 1556-1560 (s) (-CH=N), 1095-1113 (s) (OCH3)