Supplemental Information for Long Tethers Binding Redox Centers to Polymer. Backbones Enhance Electron Transport in Enzyme Wiring Hydrogels by Fei

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1 Supplemental Information for Long Tethers Binding Redox Centers to Polymer Backbones Enhance Electron Transport in Enzyme Wiring Hydrogels by Fei Mao, Nicolas Mano and Adam Heller, Department of Chemical Engineering, University of Texas, Austin, TX and TheraSense Inc., 1360 South Loop Road, Alameda, CA ,2 -biimidazole. The procedure of Fieselmann, B. F.; Hendrickson, D. N.; Stucky, G. D. Inorg. Chem. 1978, 17, was modified by substituting the ammonia gas with concentrated NH 4 OH (28-30%). To a magnetically stirred solution of 40% glyoxal in H 2 O (101.2g, 1.74 mol) in a round bottom flask cooled in an ice-water bath was added drop wise 370 ml (2.76 mol) conc. NH 4 OH. The rate of addition was controlled so that the temperature did not rise above 30 C. After adding the NH 4 OH the mixture was stirred at C for 24 h, then cooled to room temperature. The dark precipitate was collected by suction filtration, dried at C under vacuum for 24 h to give 24 g of 2,2 -biimidazole. Silica gel TLC (10% MeOH/CHCl 3 ) showed that the product was pure. N,N -dimethyl-2,2 -biimidazole. Sodium hydride (60% in mineral oil) (2.7g, 67.5 mmol) was added under nitrogen to a stirred solution of 2,2 -biimidazole (4.6g, 34.3 mmol) in 100 ml anhydrous DMF cooled to 0 C. After stirring at room temperature for 1 h, methyl p-toluenesulfonate (12.8g, 68.8 mmol) in 5 ml DMF was added drop wise using a syringe over 20 min. The mixture was stirred at room temperature for 4 h, then the solvent was removed by vacuum distillation at 50 C and the remaining dark solid was triturated with hexane (50 ml) to remove the residual mineral oil. The solid was then 1

2 sublimed under high vacuum at C. The colorless crystals (80% yield) were pure. 1 H NMR (400 MHz, DMSO-d 6 ): δ 7.28 (d, 2H, J = 1.6), 7.02 (d, 2H, J = 1.6), 3.94 (s, 6H). Calculated for C 8 H 10 N 4 : C, 74.97; H, 7.55; N, Found: C, ; H, 7.51 ; N, N-methyl-2,2 -biimidazole. Sodium hydride (60% in mineral oil) (3.26g, mmol) was added in small portions under nitrogen to a stirred solution of 2,2 - biimidazole (10 g, 74.6 mmol) in 100 ml anhydrous DMF at 0 C. Stirring at 0 C was continued for 1 h, then methyl iodide (4.6 ml, 74.7 mmol) was added drop wise using a syringe. The resulting mixture was stirred at 0 C for ½ h, then at room temperature overnight. Next it was poured into 300 ml EtOAc. The precipitated NaI was removed by suction filtration, the EtOAc was evaporated in a rotary evaporator and the remaining DMF was removed by vacuum distillation. The dark tarry solid was dissolved in 40 ml boiling EtOAc and gravity filtered. The filtrate was boiled again in EtOAc and saturated with hexane ( ml). The solution was placed in a freezer overnight to allow the product to crystallize. The crystals were collected by suction filtration and recrystallized from EtOAc/hexane to give the pure product in 40% yield. 1 H NMR (400 MHz, DMSOd 6 ): δ (br s, 1H), 7.24 (s, 1H), 7.16 (br s, 1H), 7.04 (br s, 1H), 6.97 (s, 1H), 4.02 (s, 3H). Calculated for C 7 H 8 N 4 : C, 56.74; H, 5.44; N, Found: C, 56.71; H, 5.50; N, N-methyl-N -(6-phthalimidohexyl)-2,2 -biimidazole. To N-methyl-2,2 - biimidazole (1g, 6.75 mmol) dissolved in 80 ml anhydrous DMF was added under N 2 NaH (60% in mineral) ( g, 8.1 mmol). After the mixture was stirred for 1 h at room temperature, N-(6-bromohexyl)phthalimide (2.52 g, 8.1 mmol) and NaI (1.01 g,

3 mmol) were added successively. The resulting mixture was stirred under N 2 at 80 C for 24 h. The course of the reaction was followed by alumina TLC using 50%EtOAc/hexane as the solvent. The reaction mixture was cooled to room temperature and poured into ml H 2 O. The product was extracted with EtOAc (2x150 ml) and the combined extracts were washed with brine, dried with anhydrous Na 2 SO 4 and evaporated to give the crude product. The product was purified by running it through a neutral alumina column using EtOAc/hexane (10% 40%) as the eluent (yield : 98%). 1 H NMR (400 MHz, DMSO-d 6 ): δ 7.85 (m, 4H), 7.32 (d, 1H, J = 0.8), 7.25 (d, 1H, J = 1.2), 7.02 (d, 1H, J = 0.8), 6.98 (d, 1H, J = 1.2), 4.38 (t, 2H, J = 7.2), 3.89 (s, 3H), 3.51 (t, 2H, J = 7.2), 1.66 (m, 2H), 1.52 (m, 2H), 1.20 (m, 4H). Calculated for C 21 H 23 N 5 O 2 : C, 66.83; H, 6.14; N, Found: C, 66.79; H, 6.20; N, N-(6-aminohexyl)-N -methyl-2,2 -biimidazole. To a solution of N-methyl-N -(6- phthalimidohexyl)- 2,2 -biimidazole (2.5g, 6.56 mmol) in 25 ml ethanol was added hydrazine hydrate (0.387g, 6.56 mmol). The solution was refluxed for 2 h to complete the deprotection then cooled to room temperature and suction filtered to remove the phthalhydrazide precipitate. The filtrate was loaded onto a silica gel column packed in ethanol. The column was eluted first with ethanol to remove the remaining phthalhydrazide, followed by elution of the product with 10% NH 4 OH in CH 3 CN. Fractions containing pure product were pooled and evaporated to dryness to give the product as a light yellow oil in 50% yield. 1 H NMR (400 MHz, DMSO-d 6 ): δ 7.32 (d, 1H, J = 0.8), 7.27 (d, 1H, J = 0.8), 7.03 (d, 01H, J = 0.8), 7.02 (d, 1H, J = 0.8), 4.40 (t, 2H, J = 7.2), 3.90 (s, 3H), 3.37 (t, 2H, J = 6.6), 1.67 (m, 2H), (m, 6H). Calculated for C 13 H 21 N 5 : C, 63.13; H, 8.56; N, Found: C, 62.89; H, 8.71; N,

4 [Os(III)(N,N -dimethyl-2,2 -biimidazole) 2 (N-(6-aminohexyl)-N -methyl-2,2 - biimidazole)](pf 6 ) 3. A mixture of N,N -dimethyl-2,2 -biimidazole (1.05 g, 6.48 mmol) and (NH 4 ) 2 OsCl 6 (1.42 g, 3.24 mmol) 40 ml ethylene glycol was stirred under N 2 at 140 C for 24 h. N-(6-aminohexyl)-N -methyl-2,2 -biimidazole (0.8 g, 3.24 mmol) in 5 ml ethylene glycol was introduced into the reaction mixture using a syringe. Stirring under N 2 at 140 C was continued for another 24 h. The mixture was cooled to room temperature and suction filtered through a medium fritted funnel to remove a red impurity precipitate. The filtrate was diluted with 200 ml H 2 O and then mixed with 40 ml AG1X4 chloride resin. The mixture was stirred under air in an open beaker for 24 h to allow full oxidation of the Os(II) complex to the Os(III) complex. The dark green color of the Os(II) complex changed to the dark blue color of the Os(III) complex. The dark blue solution was poured slowly into a rapidly stirred NH 4 PF 6 solution (10.2 g in 150 ml H 2 O). The precipitate was collected by suction filtration through a fine fritted funnel. The crude product was redissolved in ml CH 3 CN and precipitated again from NH 4 PF 6 solution (3.4 g in 150 ml H 2 O). The precipitate was collected by suction filtration, washed with H 2 O (3x5 ml) and dried under high vacuum at C for 24 h. Yield: 84%. The product showed a clean redox wave on a glassy carbon working electrode with a E 1/2 of 0.03 V vs. Ag/AgCl in 0.2 M LiCl/MeOH. The E 1/2 shifted negatively to V vs. Ag/AgCl in ph 7 PBS buffer, showing a large solvent dependency. Calculated for C 29 H 41 F 18 N 13 OsP 3 Â+ 2 O: C, 28.23; H, 3.65; N, 14.76; F, Found: C, 27.92; H, 3.25; N, 14.55; F, Poly(4-(N-(5-carboxypentyl)pyridinium)-co-4-vinylpyridine). To a solution of poly(4-vinylpyridine) (number average molecular weight: ~160,000) (20 g) in 150 ml 4

5 DMF was added 6-bromohexanoic acid (5.6 g, 28.5 mmoles). The solution was mechanically stirred at 90 C for 24 h. The solution was cooled to room temperature and then poured slowly to a rapidly stirred EtOAc solution (1.2 L). The solvent was decanted and the solid was redissolved in MeOH (300 ml). The solution was vacuum filtered and the filtrate was concentrated by rotary evaporation to about 200 ml, then the solid was precipitated from 1 L ethyl ether, collected by vacuum filtration and dried under high vacuum at 50 C. After 24 h of drying, the solid was crushed into powder. Drying at 50 C was continued for another 48 h. Yield:90%. 1 H NMR (300 MHz, DMSO-d 6 ): δ 8.86 (br s, 6.8H), 8.23 (br s, 40.3H), 7.48 (br s, 6.9H), 6.55 (br s, 38.1H), 4.45 (br s, 5H), (br m, 99.8H). The number of protons given for each peak is relative, providing the ratios of the peak heights. The integration ratio of the pyridinium protons ( ) over the total aromatic protons (( ) + ( )) is ~15%, consistent with quantitative quaternization. Polymer I. To a suspension of poly(4-(n-(5-carboxypentyl) pyridinium)-co-4- vinylpyridine) (515 mg, 575 µmol) in 10 ml anhydrous DMF was added O-(Nsuccinimidyl)-N,N,N,N -tetramethyluronium tetrafluoroborate (TSTU) (182 mg, 604 µmol). After the suspension was stirred for 15 min, N,N-diisopropylethylamine (100 µl, 575 µmol) was added and the resulting solution was stirred for 7~8 h. [Os(III)(N,N - dimethyl-2,2 -biimidazole) 2 (N-(6-aminohexyl)-N -methyl-2,2 -biimidazole)](pf 6 ) 3 (894 mg, 748 µmol) was added to the reaction in small portions over 5 min, followed by 100 µl N,N-diisopropylethylamine. The dark blue solution was stirred at room temperature for 24 h, then poured into 200 ml EtOAc. The precipitate was collected by vacuum filtration and redissolved in ml CH 3 CN. The CH 3 CN solution was mixed with 40 5

6 ml AG1x4 chloride resin in a beaker by stirring briefly with a spatula, the 150 ml H 2 O was added. The resulting mixture was magnetically stirred for > 24 h, or until all of the polymer was dissolved, suction filtered through a medium fritted funnel and the filtrate was concentrated to ~50 ml by ultra membrane filtration under 40 psi nitrogen pressure using a Millipore polyethersulfone membrane with a molecular weight cutoff of 10,000. The concentrated polymer solution was then subjected to further ion exchange by running it through a AG1x4 resin column (2.5 cmx25 cm) with H 2 O. The collected polymer solution was dialyzed with H 2 O by ultra membrane filtration, using the same membrane. The dialysis was considered complete when the filtrate showed a conductivity of less than 50 µscm -1 as measured by a Corning CD-55 conductivity meter. The dialyzed polymer solution was lyophilized to give a light blue solid. Yield: 0.8g. Elemental analysis: C, 53.16; H, 6.16; N, 13.47; H 2 O, <0.10. The elemental analysis is indicative of the polymer structure shown in Figure 1, where the percentage numbers for the three copolymer components, pyridinium with osmium complex, pyridine and pyridinium with a carboxypentyl group, are 10.6, 85, and 4.4, respectively. The number of polymer units, 15, was based on the molecular weight (~160,000) of the starting polymer poly(4- vinylpyridine), assuming that the backbone did not break during the synthesis. 6

Crystallization & Filtration

Crystallization & Filtration Purification of Salicyclic Acid and Sodium Chloride Separation processes Liquid-liquid extraction Adsorption Filtration Solid-liquid extraction (leaching) Elution chromatography