Synthesis of Degradable Poly(vinyl alcohol) by Radical Ring-Opening Copolymerization and Ice Recrystallization Inhibition Activity

Transcription

1 Electronic Supporting Information for; Synthesis of Degradable Poly(vinyl alcohol) by Radical Ring-Opening Copolymerization and Ice Recrystallization Inhibition Activity Guillaume Hedir, a,c Christopher Stubbs, a Phillip Aston, a Andrew P. Dove a * and Mathew I. Gibson a,b * a Department of Chemistry, University of Warwick, Coventry, CV4 7AL, UK b Warwick Medical School, University of Warwick, Coventry, CV4 7AL, UK c Institute of Advanced Study, University of Warwick Science Park, CV4 8UW, UK Corresponding Author Information * Prof. Matthew I. Gibson m.i.gibson@warwick.ac.uk * Prof. Andrew P. Dove a.p.dove@warwick.ac.uk S1

2 Experimental Section General Phosphate-buffered saline (PBS) solutions were prepared using pre-formulated tablets (Sigma- Aldrich) in 200 ml of Milli-Q water (>18.2 Ω mean resistivity) to give [NaCl] = M, [KCl] = M, and ph 7.4. Sodium hydroxide (pellets AnalaR) and potassium carbonate (anhydrous) were purchased from Fisher. Vinyl chloroacetate (99%), Azobisisobutyronitrile (AIBN), Acetic anhydride(>99%), Pyridine(99.5%), 2,5-Dihydroxybenzoic Acid (DHB) (>99%) and Amberlite IR120 hydrogen form were purchased from Sigma Aldrich. All solvents were purchased from VWR or Sigma Aldrich and used without further purification. Physical and Analytical Methods 1 H and 13 C NMR spectra were recorded on Bruker Avance III HD 300 MHz or HD 400 MHz spectrometers using deuterated solvents obtained from Sigma-Aldrich. Chemical shifts are reported relative to residual non-deuterated solvent. Size exclusion chromatography (CHCl 3 ) analyses were performed on a system composed of a Varian 390-LC-Multi detector using a Varian Polymer Laboratories guard column (PLGel 5 µm, 50 x 7.5 mm), two mixed D Varian Polymer Laboratories columns (PLGel 5 µm, 300 x 7.5 mm) and a PLAST RT autosampler. Detection was conducted using a differential refractive index (RI). The analyses were performed in CHCl 3 containing 0.5% w/w triethylamine (Et 3 N) at a flow rate of 1.0 ml/min at 313 K. Polystyrene (PS) ( x 10 5 g/mol) standards were used to calibrate the system. Molecular weights and dispersities were determined using Cirrus v2.2 SEC software. Alternatively, samples were analysed in THF on an Agilent 390-LC MDS instrument equipped with differential refractive index (DRI) and dual wavelength UV detectors. The system was equipped with 2 x PLgel Mixed D columns (300 x 7.5 mm) and a PLgel 5 µm guard column. The eluent is THF with 2 % TEA (triethylamine) and 0.01 % BHT (butylated hydroxytoluene) additives. Samples were run at 1ml/min at 30 C. Poly(methyl methacrylate) and polystyrene standards (Agilent EasyVials) were used for calibration. For all GPC analysis, Analyte samples were filtered through a nylon membrane with 0.22 µm pore size before injection. Respectively, experimental molar mass (Mn, SEC ) and dispersity (Đ M ) values of synthesised polymers were determined by conventional calibration using Agilent GPC/SEC software. MALDI-TOF mass spectra were recorded on a Bruker Autoflex Speed MALDI-TOF mass spectrometer in a positive ion TOF detection mode performed using an acceleration voltage of 25 kv. Solutions in water using HCCA as matrix (30 mg/ml), sodium or potassium trifluoroacetate as ionization agents (2 mg/ml) and analyte (1 mg/ml) were mixed prior to being spotted on the MALDI plate and air-dried. The S2

3 samples were measured in reflector ion mode and calibrated by comparison to SpheriCal (Polymer Factory) single molecular weight standards ( g/mol). Ice wafers were annealed on a Linkam Biological Cryostage BCS196 with T95-Linkpad system controller equipped with a LNP95-Liquid nitrogen cooling pump, using liquid nitrogen as the coolant (Linkam Scientific Instruments UK, Surrey, UK). An Olympus CX41 microscope equipped with a UIS-2 20x/0.45/ /0-2/FN22 lens (Olympus Ltd, Southend on sea, UK) and a Canon EOS 500D SLR digital camera was used to obtain all images. Image processing was conducted using Image J, which is freely available from Ice Recrystallisation Inhibition Splat Assay A 10 µl droplet of polymer in PBS solution is dropped from 1.4 m onto a glass microscope coverslip, which is on top of an aluminium plate cooled to -78 C using dry ice. The droplet freezes instantly upon impact with the plate, spreading out and forming a thin wafer of ice. This wafer is then placed on a liquid nitrogen cooled cryostage held at -8 C. The wafer is then left to anneal for 30 minutes at - 8 C. Three photographs are then taken of the wafer in different locations at 20 x zoom under cross polarisers. The number of crystals in the image is counted, again using ImageJ, and the area of the field of view divided by this number of crystals to give the average crystal size per wafer, assuming equal crystal size. Synthetic and Experimental Procedures Synthesis of poly(mdo-co-vclac) using the RAFT/MADIX polymerization As a representative example, 2-methylene-1,3-dioxepane (MDO) (0.126 g, mol), vinyl chloroacetate (VClAc) (1.20 g, mol), O-hexyl-S-methyl-2-propionylxanthate (CTA) (9.7 mg, mol), AIBN (1.8 mg, mol) were placed in an ampoule and subjected to a further three freeze-pump-thaw cycles and then backfilled with nitrogen. The resulting solution was stirred and heated to 60 C for 5 hours before the polymerization was quenched by plunging the ampoule into an ice bath. An aliquot was taken prior to precipitation in order to determine the monomer conversions using 1 H NMR spectroscopy. Poly(MDO-co-VClAc) was dissolved in a small amount of CHCl 3 precipitated several times in hexane until no further monomer residue was observed. Finally, the colorless solid was dried under vacuum. 1 H NMR (400 MHz, CDCl 3, ppm) δ: (m, CH 2 CHOCO backbone, 1H), 4.58 (t, SCOCH 2 CH 2 end-group, 2H), (m, COOCH 2 CH 2 CH 2 backbone, 2H, CHOCOCH 2 Cl, 2H), 3.70 (s, CH 3 CCOCH end-group, 3H), (t, COOCH 2 CH 2 CH 2 CH 2 SC, 2H), 2.60 (m, CH 2 COOCH 2 CH 2 backbone, 2H), 2.05 (m, CHOOCCH 3 backbone, 3H), (m, CHCH 2 OCO S3

4 backbone, 1H, CH 2 CHOCOCH 2 backbone, 2H, CH 3 OCOCHCH 3 end-group, 1H, CH 3 OCOCHCH 3 end-group, 3H), (m, CH 2 CH 2 CH 2 CH 3 end-group, 2H, CH 2 CH 2 CH 2 CH 3 end-group, 2H, CH 2 COOCH 2 CH 2 CH 2, 2H), 0.90 (t, CH 2 CH 2 CH 2 CH 3 end-group, 3H). Conversion by 1 H NMR spectroscopy: VClAc conv. = 53%, MDO conv. = 41%, M n (SEC, CHCl 3 ) = 7.3 kg/mol, Ð M = 2.48, M n ( 1 H NMR) = 18.9 kg/mol. Polymer Targeted Initial feed VClAc MDO Polymer comp. DP [VClAc]:[MDO] conv. conv. [VClAc]:[MDO] (kda) (%) a (%) a b M theo n M SEC n (kda) 1 * 90: :5 * : : : : : : a conversions determined by 1 H NMR spectroscopy (CDCl 3 ), b obtained by SEC analysis in CHCl 3, c theoretical molecular weight based on monomer conversion ( 1 H NMR spectroscopy). The final composition of poly(mdo-co-vclac) was determined by comparing the characteristics signals at δ = ppm and δ = 2.60 ppm corresponding to the VClAc (CH) and MDO (CH 2 ) repeat units respectively. O-hexyl-S-methyl-2-propionylxanthate was synthesised following the procedure reported by Hedir et al. 1 2-methylene-1,3-dioxepane (MDO) was synthesised using the previously reported procedure by Bailey et al. 2 Copolymer Deprotection Ð M b As a representative example, the deprotection of the MDO-VClAc copolymer was carried out by dissolving 100 mg of copolymer in 10 ml 1:1 THF/MeOH followed by the addition of 20 mg potassium carbonate. The solution was stirred for 10 minutes at room temperature and the solvent removed under vacuum. The resulting solid was dissolved in deionised water and filtered, followed by dialysis (Spectra/Por, MWCO) for 1 hour with 3 water changes. This solution was then freeze dried to remove water, and the polymers recovered were analysed by 1 H and 13 C NMR spectroscopy. 1 H NMR (400 MHz, DMSO, ppm) δ: (m, CH 2 CHOH, 2H, COOCH 2 CH 2 CH 2 CH 2, 2H, COOCH 2 CH 2 CH 2 CH 2, 2H, COOCH 2 CH 2 CH 2 CH 2, 2H), 2.50 (m, CH 2 COO, 2H), (m, COOCH 2 CH 2 CH 2 CH 2, 2H), (m, CH 2 CHOH, 1H). 1 H NMR (400 MHz, D 2 O, ppm) δ: S4

5 1.45 (m, CH 2 CH 2 CH 2 CHOH, 2H, m, CH 2 CH 2 CH 2 CH 2 CH 2 CHOH, 2H), (m, CH 2 CH 2 CH 2 CH 2 CH 2 CHOH, 2H), 3.61 (m, OCH 2 CH 2 CH 2 CH 2 CH 2 CHOH, 2H), 3.73 (m, CH 2 CHOHCH 2 COOH, 2H), (m, CH 2 CHOHCH 2 COOCH 2, 1H). 13 C NMR (500MHz, DMSO, ppm) δ: (OCH 2 CH 2 CH 2 CH 2 CH 2 CHOH, 1C, OCH 2 CH 2 CH 2 CH 2 CH 2 CHOH, 1C), (OCH 2 CH 2 CH 2 CH 2 CH 2 CHOH, 1C), (OCH 2 CH 2 CH 2 CH 2 CH 2 CHOH, 1C), (OCH 2 CH 2 CH 2 CH 2 CH 2 CHOH, 1C), (CH 2 COOCH 2 CH 2 CH 2 CH 2, 1C), (OCH 2 CH 2 CH 2 CH 2 CH 2 CHOH, 1C), (CH 2 COOCH 2 CH 2 CH 2 CH 2, 1C) Copolymer Degradation As a representative example, the deprotection of the MDO-VClAc copolymer was carried out by dissolving 100 mg of the copolymer in 1 ml THF. 0.2 g of sodium hydroxide was dissolved in 4 ml deionised water and the solutions combined. The solution was stirred uncovered for 2 hours at room temperature, and then placed in a water bath at 60 C for 4 hours. Samples were filtered through a plug of amberlite resin to remove any residual hydroxide and dialysed ( MWCO) for 2 hours with 4 water changes. The water was removed by freeze drying and the polymers analysed by 1 H and 13 C NMR. 1 H NMR (400 MHz, D 2 O, ppm) δ: (m, HOCH 2 CH 2 CH 2 CH 2, 2H, HOCH 2 CH 2 CH 2 CH 2, 2H, HOCH 2 CH 2 CH 2 CH 2, 2H), 2.35 (m, HOCH 2 CH 2 CH 2 CH 2, 2H), 3.55 (m, CH 2 CHOHCH 2 COOH, 2H), 3.85 (m, CH 2 CHOHCH 2 COOH, 1H), (m, CH 2 CHOHCH 2 COOH, 2H). 1 H NMR (400 MHz, DMSO, ppm) δ: (m, HOCH 2 CH 2, 2H), (m, HOCH 2 CH 2, 2H), 3.85 (m, CH 2 CHOHCH 2 COOH, 1H), (m, HOCH 2 CH 2, 1H). 13 C NMR (500MHz, D 2 O, ppm) δ: (HOCH 2 CH 2 CH 2 CH 2, 1C, HOCH 2 CH 2 CH 2 CH 2, 1C), (HOCH 2 CH 2 CH 2 CH 2, 1C), (CH 2 CH 2 CH 2 CHOHCH 2, 1C, CH 2 CH 2 CH 2 CHOHCH 2, 1C), (HOCH 2 CH 2 CH 2 CH 2, 1C), (CH 2 CH 2 CH 2 CHOHCH 2, 1C), (CH 2 CHOOH 1C), (CH 2 CHOOH 1C), (CH 2 CHOOH 1C). Copolymer Reacetylation As a representative example, 5 ml of acetic anhydride was added to a subasealed vial containing 5 ml of pyridine and 10 mg of the degraded copolymer under a nitrogen atmosphere. The solution was left to stir at room temperature for 24 hours. The solvent was removed through rotary evaporation, followed by dissolution in dichloromethane. This was then washed with saturated sodium hydrogen carbonate solution and brine, and the solvent removed under reduced pressure. The resulting polymer was dissolved in THF and analysed by SEC analysis. S5

6 1 H NMR (400 MHz, CDCl 3, ppm) δ: (m, HOCH 2 CH 2 CH 2 CH 2, 2H, HOCH 2 CH 2 CH 2 CH 2, 2H, HOCH 2 CH 2 CH 2 CH 2, 2H, CH 2 CH 2 CH(OCOCH 3 )CH 2, 2H), (m, CH(OCOCH 3 )CH 2, 3H), (m, HOCH 2 CH 2 CH 2 CH 2, 2H), 4.50 (m, CH 2 CH 2 CH(OCOCH 3 )CH 2, 1H). M n (SEC, THF) = 3.8 kg/mol, Ð M = Lipase Degradation As a representative example, poly(va-co-mdo) (P2) (15 mg) was added to 1 ml deuterated H 2 O, 5 mg of Lipase immobilized from Candida Antarctica beads (200 U/g) were added and the solution incubated with stirring at 37 C. After 24 hours, the solution was filtered through a 0.22 µm filter and 1 H NMR spectroscopy and MALDI-Tof mass spectrometry were carried out. 1 H NMR (400 MHz, D 2 O, ppm) δ: (m, CH 2 CHOH 2H), (m, peaks from beads), (m, CH 2 CHOH, 1H). MALDI-TOF (Water, HCCA) below. Figures HO n OH O OH H Structure S1: The structure of the P2-MDO post degradation detected in the MALDI-TOF MS. Number of PVA units Calculated (m/z) Experimental (m/z) Table S1: MALDI-TOF of P2-MDO after degradation with lipase beads for 24 hours. MALDI carried out in water with HCCA as matrix. S6

7 Figure S1: 1 H NMR showing the PVA-MDO before (black) and after (red) degradation with lipase beads, the MDO region is indicated, this is the same region at which there is a signal caused by leaching of material from the beads themselves (blue). S7

8 Normalised dwdlogm LogM PCL 10 Minute 30 Minute 120 Minute Figure S2: Degradation of PCL under the conditions used for MDO-VAcCl deprotection, while little degradation is seen after 10 minutes, within 30 minutes the molecular weight is significantly lowered. Figure S3: 13 C NMR of polymers after deprotection (A in DMSO) and subsequent degradation (B in D 2 O). The MDO unit can be seen clearly in the initial NMR (A), however when the polymer chain is degraded (B), this peak splits into multiple carbonyl region peaks, likely due to the orientation of the carbonyl carbon to the terminal PVA unit. S8

9 Figure S4: IR showing the complete deprotection of the VClAc unit to OH Overall MDO VClAc Ln[M 0 /M] Time (h) Figure S5: Kinetic plot of the reaction between MDO and VClAc. Kinetics were tudied on a reaction with an initial monomer feed of 30 mol% MDO and 70 mol% VClAc. S9

10 Table S2: Table to show the kinetics of the reaction between MDO and VClAc. Time (h) Overall conv MDO conv. VClAc conv. Mn theo Mn SEC Mn Obs DM Figure S6: Kinetic plot of overall conversion versus molecular weight and dispersity. Molecular weight determined by NMR and SEC is shown, as well as the theoretical molecular weight and dispersity of the polymer from SEC. S10

11 120 min 30 min 10 min 0 min Chemical Shift (ppm) Figure S6: NMR of the initial P2 VClAc-MDO (t = 0, black) and the same polymer sampled after 10 minutes (red), 30 minutes (black), 2 hours (pink). NMRs were carried out in DMSO, and a methanol solvent peak can be seen at 3.16 in the 10 and 30 minute samples. As the reaction progresses the degradation becomes more prevalent ( ppm). References (1) Hedir, G. G.; Bell, C. A.; Ieong, N. S.; Chapman, E.; Collins, I. R.; O Reilly, R. K.; Dove, A. P. Macromolecules 2014, 47, (2) Bailey, W. J.; Ni, Z.; Wu, S.-R. J. Polym. Sci., Part A: Polym. Chem. 1982, 20, S11