Rapid Visible Light-Mediated Controlled Aqueous Polymerization with In Situ Monitoring

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1 Supplementary Information Rapid Visible Light-Mediated Controlled Aqueous Polymerization with In Situ Monitoring Jia Niu 1,2,4, Zachariah A. Page 2, Neil D. Dolinski 2, Athina Anastasaki 1,2, Andy T. Hsueh 3, H. Tom Soh 5,6, and Craig J. Hawker* 1,2,3 1. California NanoSystems Institute, 2. Materials Research Laboratory, 3. Department of Chemistry and Biochemistry, University of California, Santa Barbara, California 93106, United States 4. Department of Chemistry, Boston College, Chestnut Hill, Massachusetts 02467, United States 5. Department of Radiology, School of Medicine, 6. Department of Electrical Engineering, Stanford University, Stanford, CA 94305, United States I. Supplementary Methods General methods Unless noted below, all commercially available reagents were purchased from Aldrich. 5 - [(2 - Aminoethyl)amino]naphthalene sulfonic acid, sodium salt (EDANS) was purchased from Anaspec. The radical inhibitors in the commercial N, N-dimethylacrylamide (DMA) and N, N-diethylacrylamide (DEA) monomers were removed by passing through a short basic alumina column before polymerization reaction. N-Acryloylmorpholine was purified by distillation under vacuum. All the other chemicals and reagents were used without further purification. The chain transfer agent 2-(butylthiocarbonothioyl) propionic acid (BTPA) was prepared according to a previously established procedure. 1 Conventional and in situ nuclear magnetic resonance (NMR) experiments were conducted on a Varian 600 MHz instrument. 1 H NMR experiments conducted in deuterated chloroform solvent were measured relative to the signals for residual chloroform (7.26 ppm). Mass spectra (MS) were recorded on a Micromass QTOF2 Quadrupole/Time-of-Flight Tandem mass spectrometer equipped with electrospray ionization (ESI) ion sources. Gel permeation chromatography (GPC) analyses were performed on a Waters Alliance 2695 separation module with a Waters 2414 differential refractometer eluting with DMF containing 0.1% LiBr and a same instrument eluting with chloroform. Polystyrene (PS) standard was used to calibrate GPC for molecular weight measurements. Fluorescence measurement was performed Horiba FluoroMax 4 spectrometer. The LED fiber optic light source (Thorlabs, 470 nm, M470F3) was coupled into a multimode optical fiber terminated with a flat cleave and the intensity and on / off cycles were controlled through a digital-to-analog converter (National Instruments USB-6009) using LabVIEW, which was connected to a T-cube LED driver (LEDD1B) from Thorlabs. A spectrophotometer with cosine corrector and radiometric calibration (Ocean Optics, model USB 4000) was used to measure LED emission profiles and calculate the power density of emission coming out of the fiber tip. Optical fibers were used to conduct light irradiation into the NMR spectrometer and benchtop reaction vessels to induce the photopolymerization. S1

2 Experimental Procedures + DCC DMF 54% yield Synthesis of N-acryloxysulfosuccinimide (NASS) To a nitrogen flushed and dry 2-neck, 50 ml round bottom flask, equipped with a magnetic stir bar, inlet adapter, and septum was added N-hydroxysulfosuccinimide sodium salt (1.21 g, 5.6 mmol), acrylic acid (0.40 g, 5.6 mmol) and DMF (12 ml, anh). The suspension was cooled to 0 C with an ice bath and N,N - dicyclohexylcarbodiimide (1.26 g, 6.1 mmol) dissolved in DMF (12 ml, anh) was added dropwise. The reaction was allowed to warm to room temperature and stirred for 14 hours. The mixture was again cooled to 0 C with an ice bath and filtered through a plug of Celite to remove the byproduct, dicicylohexylurea (DCU). The filtrate was then precipitated into Et 2 O, filtered, and washed with Et 2 O to obtain the desired product as a white powder (1.31 g, 54% yield). 1 H NMR (600 MHz, DMSO-d6) δ 6.66 (d, J = 17.3 Hz, 1H), 6.51 (dd, J = 17.3, 10.6 Hz, 1H), 6.34 (d, J = 10.6 Hz, 1H), 3.96 (s, 1H), 3.18 (s, 1H), (m, 1H). 13 C NMR (101 MHz, DMSO-d6) δ , , , , , 56.33, IR (ATR) ν 3498, 2949, 1774, 1724, 1662, 1625, 1404, 1369, 1194, 1092, 1074, 1038, 981, 854, 777, 717 cm -1. LRMS (-ESI): m/z calcd for (M - Na) - C 7 H 6 NO 7 S , found A general procedure for the polymerization of acrylamide using the Ru(bpy) 3 Cl 2 /sodium ascorbate catalytic system without degassing procedures Various conditions have been attempted to polymerize acrylamide monomers including DMA, DEA, and NAM. The following procedure describes a reaction with the ratio of monomer : CTA: catalyst : reductant = 400 : 1 : : 2 and the irradiation time of 45 min. Other conditions typically follows a similar procedure but with different monomer ratios, or different irradiation times. For the reactions with monomer : CTA ratio 40, 20% volume of DMSO was added as the cosolvent to solubilize BTPA. Polymerization was conducted in water in a 1.5 ml transparent glass vessel equipped with a rubber septum. BTPA (0.75 mg, 3.12 µmol) was aliquoted as 7.5 µl of a 100 mg/ml solution in DMSO into the glass vial. To this container 0.25 ml water, acrylamide monomer (1.25 mmol), 12.5 µl of 100 mg/ml sodium ascorbate solution in water, and 2.4 µl of 1 mg/ml Ru(bpy) 3 Cl 2 solution in water were added sequentially. The solution was then allowed to polymerize under irradiation by a digitally controlled 470 nm LED light source conducted into the vial by the optical fiber for 45 min under constant stirring. The light intensity was separately measured as 25.8 mw/cm 2. Immediately after the light source is turned off, 25 µl of the reaction mixture was withdrawn and added into 0.5 ml D 2 O for monomer conversion analysis by NMR. The rest of the solution was then added into a dialysis tube (MWCO = 1k) and dialyzed against water for 48 h. The dialyzed product was lyophilized and redissolved in DMF for GPC analysis. S2

3 A general procedure for the polymerization of acrylamides using the Ru(bpy) 3 Cl 2 /sodium ascorbate catalytic system for the in situ NMR kinetic experiments Polymerization was conducted in D 2 O in a NMR tube with the ratio of monomer : CTA: catalyst : reductant = 400 : 1 : : 2. First, acrylamide monomer (6.3 mmol), D 2 O (2.5 ml), BTPA (3.75 mg, µmol) and Ru(bpy) 3 Cl 2 (11.75 µl of a 1 mg/ml solution in D 2 O, µmol, 2.5 ppm relative to monomer) were mixed to generate a stock solution that was used for all NMR experiments (1.6 M monomer). A fraction of the mixture was added to a 5 mm NMR tube so that the solution was ~2 mm above the measurement region. The optical fiber and a Teflon insert were placed into the NMR tube such that the fiber was ~2 mm above the solution. All NMR measurements were performed as an array and a LabVIEW program connected to a T-cube LED driver was used to automate both intensity and on / off times of a 470 nm LED. After polymerization the solution was then added into a dialysis tube (MWCO = 1k) and dialyzed against water for 48 h. The dialyzed product was lyophilized and redissolved in DMF for GPC analysis. For the degassed experiments, a gauge 27 ½ (27G, ½ ) needle was connected to the argon (Ar) source and inserted through a rubber septum to purge Ar through the stock solution for 10 min, and a gauge 27 ¼ (27G, ¼ ) needle was inserted through the septum to vent. Immediately after purging a fraction of the degassed solution was transferred to a NMR tube. A balloon filled with Ar was then used to further purge Ar through the space on top of the solution in the tube, and to keep the solution under Ar protection after purging until the sample was loaded in the NMR instrument. In situ block copolymerization Each block was polymerized with the ratio of monomer : CTA: catalyst : reductant = 40 : 1 : : 2. Polymerization was conducted in water in a 1.5 ml transparent glass vessel equipped with a rubber septum. To this container BTPA (4.82 mg, 0.02 mmol), 0.3 ml water, NAM (101.9 µl, 0.81 mmol), 80.2 µl of 100 mg/ml sodium ascorbate solution in water, and 15.2 µl of 1 mg/ml Ru(bpy) 3 Cl 2 solution in water were added sequentially. The solution was then allowed to polymerize under irradiation by a digitally controlled 470 nm LED light source conducted into the vial by the optical fiber for 15 min under constant stirring. The light intensity was separately measured as 25.8 mw/cm 2. Immediately after the light source is turned off, 25 µl of the reaction mixture was withdrawn. 20 µl of the withdrawn sample was added into 0.5 ml D 2 O for monomer conversion analysis by 1 H-NMR, and 5 µl of this sample was added into 95 µl DMF for GPC analysis. To the rest of the solution DEA (111.4 µl, 0.81 mmol) was then added. The resulting solution was irradiated for another 15 min with the same light intensity under constant stirring. Immediately after the light source is turned off, 25 µl of the reaction mixture was withdrawn. 20 µl of the withdrawn sample was added into 0.5 ml D 2 O for monomer conversion analysis by 1 H-NMR, and 5 µl of this sample was added into 95 µl DMF for GPC analysis. To the rest of the solution the third monomer DMA (83.4 µl, 0.81 mmol) was added. The resulting solution was irradiated for another 20 min with the same light intensity under constant stirring. Immediately after the light source is turned off, 25 µl of the reaction mixture was withdrawn. 20 µl of the withdrawn sample was added into 0.5 ml D 2 O for monomer conversion analysis by 1 H-NMR, and 5 µl of this sample was added into 95 µl DMF for GPC analysis. The rest of the solution was added into a dialysis tube (MWCO = 1k) and dialyzed against water for 48 h. The dialyzed product was lyophilized and redissolved in DMF for GPC analysis. S3

4 Monomer conversion analysis by NMR Determination of monomer conversions for the polymerizations of DMA was made according to the method adopted by Xu and Boyer. 2,3 Specifically, the determination of monomer conversion for the polymerization of DMA made the assumption that the integral of δ = ppm (Peak a, Figure S1) corresponds to the backbone methylene group of the polymerized DMA, and the integral of δ = ppm (Peak b and b, Figure S1) corresponds to two unreacted vinyl group. When the sum of these two integrals are normalized to 1, the integral of Peak a is the monomer conversion. Figure S1. NMR determination of monomer conversion for the polymerization of DMA. When Peak a (polymerized backbone methylene) and Peak b and b (two protons on the unreacted vinyl group) are normalized to 1, the conversion equals to the integral of Peak a as α = 89%. NMR determination of the hydrolysis kinetics of NASS The hydrolysis of NASS in the reaction condition was determined by monitoring the disappearance of the peak at δ = 4.3 ppm in the 1 H-NMR spectrum. Immediately before the experiment, NASS (31 mg, mmol) was dissolved in 0.5 ml D2O containing 18 mm sodium ascorbate. The integral of the peak at δ = 4.3 ppm in the 1 H-NMR spectrum was monitored during the course of 24 h, with the result at different time point being normalized to the time-zero integral to represent the fraction of unreacted NASS over the total amount. S4

5 Random copolymerization of NAM and NASS and subsequent in situ post-polymerization functionalization Two conditions have been attempted to polymerize NAM. The following procedure describes a reaction with the ratio of monomer : CTA: catalyst : reductant = 400 : 1 : : 2. For the other condition with with the ratio of monomer : CTA: catalyst : reductant = 40 : 1 : : 2, the reaction setup follows the same procedure, with 20% volume of DMSO being added as the cosolvent to solubilize BTPA. Both reactions were irradiated for 15 min to minimize the hydrolysis of the sulfo-nhs esters. Polymerization was conducted in water in a 1.5 ml transparent glass vessel equipped with a rubber septum. BTPA (0.75 mg, 3.12 µmol) was aliquoted as 7.5 µl of a 100 mg/ml solution in DMSO into the glass vial. To this container 0.5 ml water, NAM (143 µl, 1.14 mmol), 25 µl of 100 mg/ml sodium ascorbate solution in water, and 4.7 µl of 1 mg/ml Ru(bpy) 3 Cl 2 solution in water were added sequentially. Right before irradiation, NASS (31 mg, mmol) was added and mix to fully dissolve. The solution was then allowed to polymerize under irradiation by a digitally controlled 470 nm LED light source conducted into the vial by the optical fiber for 15 min under constant stirring. The light intensity was separately measured as 80.4 mw/cm 2. Immediately after the light source is turned off, 25 µl of the reaction mixture was withdrawn and added into 0.5 ml D 2 O for monomer conversion analysis by NMR. The rest of the solution was then split into two equal portions. One half of the solution was added into 1 ml DMF, before precipitating in 50 ml cold ether. The DMF dissolution ether precipitation cycle was repeated two more times to completely remove water and unreacted monomers, before dissolving the polymer into DCM, and then precipitating into ether : hexanes = 9 : 1. The DCM dissolution ether precipitation cycle was repeated two more times to completely remove DMF. The precipitated polymer sample was then dried under vacuum and analyzed by 1 H-NMR and GPC. For small molecule conjugation with the DP = 40 polymer, immediately after irradiation half of the reaction mixture was directly added into 3 ml 0.2 M phosphate buffer ph 8.0 containing 0.1 M L- phenylalanine or 0.1 M EDANS (5 eq. relative to sulfo-nhs). The reaction was incubated for four hours before purification by dialysis against water for 48 h using a MWCO = 1k membrane. The dialyzed polymer solution was lyophilized, and analyzed by 1 H-NMR and GPC. For protein conjugation with the DP = 400 polymer, immediately after irradiation half of the reaction mixture was directly added into 3 ml 0.2 M phosphate buffer ph 8.0 containing 0.2 mm of Bovine Serum Albumin (BSA) (0.01 eq relative to sulfo-nhs). The reaction was incubated for four hours before purification by dialysis against water for 48 h using a MWCO = 50k membrane. The dialyzed polymer- BSA conjugate was lyophilized, and analyzed by Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE). Quantitation of dye grafting ratio of the EDANS-conjugated polymer using fluorescence spectroscopy The fluorescent emission of EDANS in DMSO at 467 nm was used to quantitate the dye concentration. To establish a standard curve, 0.1 mg/ml EDANS in DMSO was titrate into 1 ml DMSO, with a rate of S5

6 10 µl per measurement, until 100 µl solution was added. The fluorescence of the polymer solution at mg/ml at 467 nm was then measured, and the value was used to calculate the dye grafting ratio. SDS-PAGE analysis of the BSA-polymer conjugate Along with unconjugated BSA and unconjugated poly(nam)-co-poly(nass), BSA-polymer conjugate was loaded onto a 4-15% Tris-Glycine SDS polyacrylamide gel, with 50 ng of each sample being loaded. The gel was run at 125 V for 60 min, and subsequently stained by SYPRO RUBY protein gel stain using the manufacturer s protocol. Probe singlet oxygen evolution by anthracene dipropionic acid (ADPA) Determination of singlet oxygen evolution using ADPA was adapted from a procedure by Boyer and coworkers. 4 A stock solution of 2.5 mm ADPA in water was prepared by dissolving into 60 mm NaOH aqueous solution. For the sample without reductant, 24 µl of 1 mg/ml Ru(bpy) 3 Cl 2 solution in water was added into 0.5 ml of ph 7.4 PBS buffer. For the sample with reductant, 24 µl of 1 mg/ml Ru(bpy) 3 Cl 2 solution in water and 125 µl 100 mg/ml sodium ascorbate solution in water was added into ml of ph 7.4 PBS buffer. Right before the experiment, 26 µl of the ADPA stock solution was added into the samples, which were subsequently irradiated by a digitally controlled 470 nm LED light source conducted into the vial by the optical fiber for a predetermined set of time. After each time point the absorbance at 400 nm was measured. The absorptivity-time relationship was established to reflect the singlet oxygen evolution measured by ADPA quenching. Mass spectrometry determination polymer chain end fidelity Polymerization with the ratio of monomer : CTA: catalyst : reductant = 40 : 1 : : 2 was irradiated at the light intensity of 25.8 mw/cm 2 for 4 min to reach 35% conversion. The resulted polymer (M n =1.6 k, M w /M n = 1.22) was analyzed by ESI-MS without further purification. References: (1) Niu, J.; Lunn, D. J.; Pusuluri, A.; Yoo, J. I.; O Malley, M. A.; Mitragotri, S.; Soh, H. T.; Hawker, C. J. Nat. Chem. 2017, 16 (6), (2) Xu, J.; Jung, K.; Atme, A.; Shanmugam, S.; Boyer, C. J. Am. Chem. Soc. 2014, 136 (14), (3) Xu, J.; Jung, K.; Corrigan, N. A.; Boyer, C. Chem. Sci. 2014, 5 (9), (4) Corrigan, N.; Rosli, D.; Jones, J. W. J.; Xu, J.; Boyer, C. Macromolecules 2016, 49 (18), S6

7 Supplementary Results Figure S2. Benchtop experimental setup. S7

8 Figure S3. Proposed mechanisms of aqueous PET-RAFT polymerization. (a) Oxidative quenching cycle in the absence of oxygen. (b) Oxidative quenching cycle in the presence of oxygen. (c) Reductive quenching cycle in the presence of reductant and oxygen. S8

9 Figure S4. Reaction kinetics of the degassed reaction and non-degassed reaction. The polymerization was conducted at a monomer:cta:catalyst:reductant ratio of 400:1: :2, under irradiation of a digitally controlled fiber optical LED light source at the intensity of 26 mw/cm 2. The degassed reaction was purged with Ar for 15 min, transferred to a sealed NMR tube, and an Ar balloon was used to flush the space above the solution prior to the in situ NMR experiment. S9

10 Figure S5. Plots of number average molecular weights (M n, black squares) and polydispersity (PDI, blue triangles) vs. conversion. The polymerization was conducted at the ratio of monomer: CTA: catalyst: reductant ratio of 400 : 1 : : 2, under irradiation of a digitally controlled fiber optical LED light source at the intensity of 26 mw/cm 2. The dotted line indicates linear fit of the molecular weight evolution over conversion. Polydispersity remains low (<1.3) over the course of the polymerization. These results indicate a good control over polymerization. S10

11 Figure S6. Reaction kinetics of the polymerization in the presence of NaAsc and NaOAc, respectively. The polymerization with NaAsc was conducted at the ratio of monomer: CTA: catalyst: reductant ratio of 400 : 1 : : 2, under irradiation of a digitally controlled fiber optical LED light source at the intensity of 26 mw/cm 2. The reaction with NaOAc replaced the NaAsc with the same molar concentration of NaOAc, while keeping the other parameters the same. S11

12 Figure S7. Singlet oxygen evolution of the photochemical transformation in the presence and absence of reductant. The photochemical reaction in the absence of the reductant generated significant amount of singlet oxygen that caused rapid APDA quenching (black filled square). In contrast, reduced singlet oxygen evolution was observed in the presence of the reductant sodium ascorbate. These results demonstrated that the reductant could reduce the singlet oxygen level in the photochemical reaction and improve reaction kinetics. S12

13 Figure S8. Inhibition period with respect to the irradiation intensity. The inhibition period was defined as the irradiation time before the polymerization takes place, which can be measured by the in situ NMR strategy. Higher intensity leads to shorter induction time. S13

14 Figure S9. SEC analysis of polymers obtained under the irradiation of different light intensities. These traces were obtained using crude reactions prior to purification. The apparent chain propagation rate (kpapp) and the dispersity (Ð) were shown in Figure 1d. Conversions as measured by NMR 8 mw/cm 2, 89% 26 mw/cm 2, 87% 43 mw/cm 2, 94% 80 mw/cm 2, 96% 112 mw/cm 2, 97% 139 mw/cm 2, 98% S14

15 Figure S10. UV-vis absorption spectra for BTPA and the corresponding molar absorptivity at absorption maximum and at 470 nm. S15

16 Figure S11. Monitoring the stability of BTPA using 1 H NMR spectroscopy under the given polymerization conditions with DMSO-d6 used in place of DMA, with and without visible light irradiation (26 mw/cm 2 ). BTPA photolysis/hydrolysis tracked via: Norm [BTPA] = ( ) ( ) + ( ) S16

17 S17

18 Figure S12. Mass spectrometry analysis of the polymer chain ends. (a) ESI-MS characterization of the crude polymer products from PET-RAFT. (b) Magnified spectrum between m/z =1,000 and 2,000. (c) Further magnified spectrum between m/z =1,000 and 1,200. Shaded areas are magnified in the next panel. Molecular ions with a single charge by proton or sodium cations correspond to the oligomer species bearing intact chain ends. In addition, doubly charged ions corresponding to oligomers with intact chain ends could also be observed with lower intensities, as shown in (b) and (c). This mass spectrum clearly demonstrated the high chain end fidelity of the fast aqueous PET-RAFT polymerization. S18

19 t 1/2 = 1 hr Figure S13. Hydrolysis kinetics of NASS. The half-life of NASS in the reaction condition is 100 min. Over 90% activated sulfo-nhs esters were still intact after 15 min. This result established that the majority of the sulfo-nhs groups can be preserved by limiting the reaction time within 15 min. S19

20 Figure S14. Aqueous SEC analysis of poly(nam-co-nass). DP400 copolymer: M n (SEC) = 10.1k, Ð = 1.33; DP40 copolymer: M n (SEC) = 1.0k, Ð = The molecular weights measured by SEC was lower than the theoretical values based on conversions. The deviation of experimental M n from the theoretical values is attributed to the difference in molecular structure of the measured copolymers and the linear polyethylene glycol standards, as well as a potential interaction of this copolymer with the stationary phase. In order to confirm the molecular weights of the copolymers, 1 H-NMR peak integration was used. Figure S14 shows an example of molecular weight and grafting ratio estimation of DP40 by 1 H-NMR. S20

21 Figure S15. Molecular weight and grafting ratio estimation of DP40 after in situ conjugation using 1 H-NMR. Poly(NAM-co-NASS) was reacted with L-Phe in situ, and the 1H-NMR spectrum of the product was collected. The peak at 0.86 ppm was assigned as the terminal -CH 3 at the chain end (a), and the broad peaks at ppm was assigned as the polymer backbone -CH 2 - (b, b, and b ). Finally, the broad peak at ppm was assigned as the phenyl protons of the grafted L-Phe (c, c, and c ). Therefore, the degree of polymerization can be derived as: The grafting ratio can be derived as: DP = Int(b + b! + b!! ) 2 g = Int(c + c! + c )/5 DP = = The molecular weight of the original poly(nam-co-nass) polymer can be calculated as 5,500 Da using the average molecular weight of the monomers and the DP value. S21

22 Figure S16. Fluorescence quantification of the dye grafting ratio of the EDANS-conjugated polymer. A standard curve was established by measuring fluorescence of a concentration series of free EDANS in DMSO. The concentrations of the free EDANS were then converted to equivalent grafting ratio (mol%) by the following equation: M!"#$% kc! C M!"# C where the M EDANS and M NAM are the molecular weights of the dye-conjugated acrylamide monomeric unit and NAM, respectively, C is the concentration of the free EDANS solution, k dilution factor of the EDANS-polymer conjugate, and C p is concentration (weight per volume) of the EDANS-polymer conjugate solution. S22

23 Figure S17. SDS-PAGE characterization of the BSA-poly(NAM-co-NASS) copolymer conjugate. Lane 1: protein ladder; lane 2: copolymer only; lane 3: BSA only; lane 4: BSA-copolymer conjugate. The yield of polymer-bsa conjugate was determined as 88% using densitometry by ImageJ software. S23