Supporting Information for: Light Control of Protein Solubility Through Isoelectric Point Modulation. Karthik Nadendla, Simon H.

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1 Supporting Information for: Light Control of Protein Solubility Through Isoelectric Point Modulation Karthik Nadendla, Simon H. Friedman* Division of Pharmaceutical Sciences University of Missouri-Kansas City, School of Pharmacy Kansas City, MO *Telephone: Fax: TABLE OF CONTENTS MATERIALS... 2 METHODS... 3 P-INSULIN SYNTHESIS... 3 Q-INSULIN SYNTHESIS... 7 R2-INSULIN SYNTHESIS...10 P2-INSULIN SYNTHESIS...14 ISOELECTRIC POINT DETERMINATION...20 SOLUBILITY STUDIES...20 P2-INSULIN PHOTOLYSIS IN DMSO...21 P2-INSULIN PHOYOLYSIS IN 10 mm PBS RESULTS HPLC ANALYSIS...23 LCMS ANALYSIS...32 NMR SPECTROSCOPY...49 UV SPECTROSCOPY...53 ISOELECTRIC POINT DETERMINATION...55 P2-INSULIN PHOTOLYSIS IN 10 mm 7.2 PBS...59 REFERENCES S1

2 MATERIALS Acetovanillone, tert-butylbromoacetate, 70% nitric acid, hydrazine monohydrate, manganese dioxide, d6-dmso, Sigmacote, (2-aminoethyl)trimethylammonium chloride hydrochloride and human recombinant insulin were purchased from Sigma Aldrich. Acetone, acetonitrile, 1-(2- aminoethyl)pyrrolidine, isopropanol, dimethyl sulfoxide, dimethyl formamide, diethyl ether, dichloromethane, methanol, ethanol, ethyl acetate, sodium chloride, magnesium sulfate, trifluoroacetic acid, 1 N hydrochloric acid, acetic anhydride, sodium bicarbonate, 50% glutaraldehyde solution, Coomassie Brilliant Blue R-250 and Crocein Scarlet 3B were purchased from Fisher Scientific. HBTU was purchased from Advanced ChemTech. Lantus was received as a gift from Prof. Cameron Lindsey, UMKC School of Pharmacy. ChemMatrix Rink amide resin (RCM-10-HL) was purchased from Protein Technologies. Piperidine, Fmocarginine(Pbf)-OH, HATU, DIEA were purchased from Chem-Impex International Ltd. 10X IEF cathode buffer, 10X IEF anode buffer, IEF standards (pi ), IEF sample loading buffer and ph 3-10 Criterion IEF gels (18 wells, 30 µl) were purchased from Bio-Rad. Nucleosil mm, 5 µm, C18 column (Supelco Analytical) was purchased from Sigma Aldrich. Microsorb-MV mm, 5 µm, C18 column was purchased from Agilent. Luna mm, 10 µm, C18 column was purchased from Phenomenex for preparative chromatography. Dry molecular sieves were added to DMSO, DMF and NMP, allowed to stand overnight to remove water from solvents. Glass surfaces were siliconized by treating with Sigmacote for approximately 5-10 minutes. UV-visible spectroscopic analyses were performed using a USB-2000 fiber optic spectrometer (Ocean Optics, Inc.) with DT-Mini-B lamp source. HPLC analyses were performed using Agilent 1260 and 1050 systems with attached vacuum degasser, auto-sampler and diode array detector. Mass spectrometric analyses were performed using an ABI Q-Trap IEF gels were run on Bio-Rad Criterion vertical midi-format electrophoresis cell. NMR spectroscopy was performed using Varian Inova 400 MHz instrument (400 MHz for H-NMR and 100 MHz for C13- NMR). Photolysis experiments were performed using a Nichia 200 mw 365 nm LED source in 1 ml HPLC glass inserts (Fisherbrand 1 ml Shell Vial Convenience Kit, Catalog # ). S2

3 METHODS P-INSULIN SYNTHESIS Compounds 1 and 2 were synthesized as described earlier [1]. (3) Synthesis of 2-(4-acetyl-2-methoxy-5-nitro-phenoxy)-N-(2-pyrrolidin-1-yl-ethyl)- acetamide 106 mg of compound 2 (394 µmoles, 65.6 mm) was dissolved in 6 ml of anhydrous N,Ndimethylformamide. 141 mg of HATU (372 µmoles, 60 mm) was added to the solution and allowed to stand for 5 minutes. 123 µl of N,N-diisopropylethylamine (744 µmoles, 120 mm) was added, followed by addition of 47 µl of 1-(2-aminoethyl)pyrrolidine (372 µmoles, 60 mm) to the reaction mixture. Contents were stirred and allowed to react for 3 hours. The reaction mixture was then mixed with 100 ml of 0.5 N sodium hydroxide and partitioned against ethyl acetate. Ethyl acetate fraction containing compound 3 was collected. The aqueous layer was washed multiple times with ethyl acetate until the majority of he compound S3

4 was extracted into the ethyl acetate layer. Ethyl acetate fractions were pooled and dried by addition of anhydrous magnesium sulfate. Magnesium sulfate was removed by filtration. Pure compound 3 was obtained by drying ethyl acetate in vacuum at approximately 60 C for approximately 4 hours. Compound 3 was analyzed using HPLC, LCMS and NMR (See figures S1, S14, S31 and S32). Reversed phase HPLC was performed using mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes. LCMS analysis was performed using mm, 5 µm, C18 column. 100% solvent A (0.1% formic acid in water) to 100% solvent B (0.1% formic acid in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes; ESI-MS (m/z) = MH + calculated, ; found, H NMR (400 MHz, DMSO-d6) d ppm (4 H, m), 2.51 (3 H, s), (2 H, m), 3.21 (2 H, q, J=5.21 Hz), (4 H, m), 3.93 (3 H, s), 4.78 (2 H, s), 7.25 (1 H, s), 7.62 (1 H, s), 8.56 (1 H, t, J=5.27 Hz). 13C NMR (101 MHz, DMSO-d6) d ppm (s, 1 C), (s, 1 C), (s, 1 C), (s, 1 C), (s, 1 C), (s, 1 C), (s, 1 C), (s, 1 C), (s, 1 C), (s, 1 C), (s, 1 C), (s, 1 C), (s, 1 C), (s, 1 C), (s, 1 C). (4) Synthesis of 2-[4-(1-hydrazono-ethyl)-2-methoxy-5-nitro-phenoxy]-N-(2-pyrrolidin-1-ylethyl)-acetamide Purified compound 3 was dissolved in 4 ml of ethanol:acetonitrile (1:1) solution. The amount of compound 3 was quantified using its extinction coefficient [2], 4470 M -1 cm -1. A solution containing 524 µmoles of compound 3 (36.8 mm, ml) was transferred into a sealed reaction vessel. 94 µl of glacial acetic acid (524.1 µmoles) and 507 µl of hydrazine monohydrate (10.47 mmoles) were added and the reaction vessel was sealed. Contents were mixed and the reaction was allowed to proceed for 4 hours at 90 C. The reaction mixture was cooled, solvent was evaporated, and the material stored at -20 C. Note: It is not recommended to store compound 4 as it is unstable. It may be stored at lower temperatures in dried form for short time. It is recommended that the purification should be done in as minimum time as possible and the compound to be used immediately for the next reactions. Compound 4 was purified using RP-HPLC using mm, 5 µm, C18 column with a flow rate of 1 ml/min. 100% solvent A (0.1% TFA in water) to 25% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 25% solvent B to 100% solvent B over 2 minutes (28-30 minutes), 100% solvent B isocratic for 3 minutes (30-33 minutes), and 100% solvent B to 100% solvent A over 2 minutes (33-35 minutes). Compound 4 elutes from minutes. Purification on S4

5 this method was repeated multiple times to collect enough material. Collected fractions were pooled, solvent removed with rotary evaporation (without application of heat), and immediately used for the next reaction. (See figures S2 and S15). Reversed phase HPLC was performed using a mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes. Two peaks observed for E/Z isomers. LCMS analysis was performed using mm, 5 µm, C18 column. 100% solvent A (0.1% formic acid in water) to 100% solvent B (0.1% formic acid in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes; ESI-MS (m/z) = MH + calculated, 380.2; found, (5) Synthesis of 2-[4-(1-diazo-ethyl)-2-methoxy-5-nitro-phenoxy]-N-(2-pyrrolidin-1-ylethyl)-acetamide Compound 4 was dissolved in anhydrous DMSO and quantified using UV spectroscopy (extinction coefficient = 4470 M -1 cm -1 ). A solution containing 6.4 µmoles of compound 4 was diluted with anhydrous DMSO (to µl) such that the final concentration was mm. 120 mg of activated manganese (IV) oxide was added and vigorously shaken for 45 minutes. The reaction was centrifuged at RCF for 4 minutes to remove manganese oxide. DMSO containing compound 5 was immediately used for reaction with insulin. The manganese oxide pellet was washed with additional DMSO to recover more compound 5. 2 µl of reaction mixture was diluted to 200 µl and UV-visible spectrum was observed. Compound 5 has a characteristic red color and absorbance at 450 nm (Figure S38). S5

6 (6) Synthesis of P-Insulin (Compound 6) 6.4 µmoles of insulin (36.9 mg) was dissolved in µl of anhydrous DMSO. DMSO solution containing 6.4 µmoles of compound 5 (assuming 100% conversion) was added to insulin solution. The reaction was carried out for 24 hours. Compound 6 was purified by RP-HPLC using a mm, 5 µm, C18 column; with flow rate of 1 ml/min. 24% solvent B (0.1% TFA in acetonitrile) to 38% solvent B over 35 minutes, 38% solvent B to 75% solvent B over 0.5 minutes ( minutes), 75% solvent B isocratic for 2.5 minutes ( minutes), and 75% solvent B to 24% solvent B over 2 minutes (38-40 minutes) with a post time of 5 minutes. Compound 6 was collected from minutes. See figures S3, S16 and S17. Reversed phase HPLC was performed using a mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes. Multiple peaks were observed due to regioisomeric adducts at different carboxyl groups on insulin. The mass of compound 6 was confirmed by infusing a purified HPLC fraction into mass spectrometer. ESI- MS (m/z) = MH + calculated, ; found (reconstructed), S6

7 Q-INSULIN SYNTHESIS (7) Synthesis of {2-[2-(4-acetyl-2-methoxy-5-nitro-phenoxy)-acetylamino]-ethyl}-trimethylammonium 0.77 g of compound 2 (2.86 mmoles, 120 mm) was dissolved in 23.8 ml of anhydrous NMP g of HBTU (5.7 mmoles, 240 mm) was added to the solution and allowed to stand for 5 minutes. 2.4 ml of N,N-diisopropylethylamine (14.3 mmoles, 600 mm) was added, followed by addition of 0.25 g of (2-aminoethyl)trimethylammonium chloride hydrochloride (372 µmoles, 60 mm) to the reaction mixture. Contents were stirred for 12 hours. Compound 7 was purified using a mm, 10 µm, C18 reverse phase HPLC. Compound 7 was purified from 3 ml of reaction mixture in each run. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 70 minutes, 100% solvent B isocratic for 5 minutes (70-75 minutes), 100% solvent B to 100% solvent A over 5 minutes (75-80 minutes). Compound 7 was collected from minutes. See figures S4, S18, S33 and S34. S7

8 Reversed phase HPLC was performed using a mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes. LCMS analysis was performed using mm, 5 µm, C18 column. 100% solvent A (0.1% formic acid in water) to 100% solvent B (0.1% formic acid in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes; ESI-MS (m/z) = M + calculated, ; found, H NMR (400 MHz, DMSO-d 6 ) d ppm 2.53 (d, J=2.34 Hz, 3 H), 3.09 (s, 9 H), 3.40 (t, J=6.44 Hz, 2 H), 3.58 (d, J=5.86 Hz, 2 H), 3.96 (s, 3 H), 4.75 (s, 2 H), 7.28 (s, 1 H), 7.61 (s, 1 H), 8.48 (t, J=5.47 Hz, 1 H) 13 C NMR (101 MHz, DMSO-d 6 ) d ppm 1.08 (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s) (8) Synthesis of (2-{2-[4-(1-hydrazono-ethyl)-2-methoxy-5-nitro-phenoxy]-acetylamino}- ethyl)-trimethyl-ammonium Compound 7 was quantified using its extinction coefficient [2], 4470 M -1 cm -1 in DMSO. A solution containing µmoles of compound 7 was transferred into an Eppendorf tube and solvent evaporated. Compound 7 was dissolved in 772 µl of ethanol:acetonitrile solution 80:20 (38.15 mm) µl of glacial acetic acid (44.2 µmoles) and 100 µl of hydrazine monohydrate (2 mmoles) were added and reaction vessel was sealed. Contents were mixed and the reaction was allowed to proceed for 36 hours at 60 C. Note: It is not recommended to store compound 8 since it is observed to be unstable. Compound 8 was purified by precipitation. Reaction mixture was cooled, unsealed and poured into cold ether. Compound 8 precipitates out of solution. Suspension was transferred into a 50 ml falcon tube and centrifuged. The ether was discarded and the precipitate was dried in vacuum. The dried material was immediately used for the successive reaction. See figures S5 and S19. Reversed phase HPLC was performed using mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes. Two peaks observed for E/Z isomers. LCMS analysis was performed using mm, 5 µm, C18 column. 100% solvent A (0.1% formic acid in water) to 100% solvent B (0.1% formic acid in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes; ESI-MS (m/z) = MH + calculated, 368.2; found, (9) Synthesis of (2-{2-[4-(1-diazo-ethyl)-2-methoxy-5-nitro-phenoxy]-acetylamino}-ethyl)- trimethyl-ammonium S8

9 Compound 8 was dissolved in a minimal quantity of anhydrous DMSO and quantified using UV spectroscopy (extinction coefficient = 4470 M -1 cm -1 ). A solution containing µmoles of compound 8 was diluted with anhydrous DMSO (to 1880 µl) such that the final concentration was mm. To this solution, 400 mg of activated manganese (IV) oxide was added and vigorously shaken for 45 minutes. The reaction mixture was centrifuged at RCF for 4 minutes to remove manganese oxide. DMSO containing compound 9 was collected into another reaction vial for reaction with insulin. The manganese oxide pellet was washed with additional DMSO (1000 µl) to recover more compound 9. Compound 9 has a characteristic red color and an absorbance at 450 nm (Figure S39). (10) Synthesis of Q-Insulin (Compound 10) µmoles of insulin (120.5 mg) was weighed and dissolved in 500 µl of anhydrous DMSO. DMSO solution containing µmoles of compound 9 (assuming 100% conversion) was added to insulin solution. Reaction was carried out for 24 hours. Compound 10 was purified by RP-HPLC using a mm, 5 µm, C18 column; with a flow rate of 1 ml/min. 24% solvent B (0.1% TFA in acetonitrile) to 38% solvent B over 35 minutes, 38% solvent B to 75% solvent B over 0.5 minutes ( minutes), 75% solvent B isocratic for 2.5 minutes ( minutes), and 75% solvent B to 24% solvent B over 2 minutes (38-40 minutes) with a post time of 5 minutes. On this method, Q-Insulin, i.e. compound 10 was collected as a fraction from minutes. See figures S6, S20 and S21. Reversed phase HPLC was performed using a mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes. Multiple peaks observed due to regioisomeric adducts at different carboxyl groups on insulin. Mass of Q-Insulin was confirmed by infusing HPLC purified fraction into mass spectrometer. ESI-MS (m/z) = MH + calculated, ; found (reconstructed), S9

10 R2-INSULIN SYNTHESIS S10

11 (11) Synthesis of 2-{2-[2-(4-acetyl-2-methoxy-5-nitro-phenoxy)-acetylamino]-5-guanidinopentanoylamino}-5-guanidino-pentanoic acid amide Compound 11 was synthesized using solid phase peptide synthesis. Synthesis was carried out on ChemMatrix Rink amide resin using anhydrous NMP as solvent. Fmoc deprotection was carried out using 20% piperidine solution in NMP. Fmoc deprotection was repeated until the reaction mixture did not show any absorbance at 301 nm. Carboxylic acids (5 equivalents) were activated in anhydrous NMP for approximately 10 minutes, DIEA (10 equivalents) was added to activation mixture and added to the solid phase. Capping was performed with 10% acetic anhydride, 5% DIEA in NMP (anhydrous). After each step, resin was washed 5 times with anhydrous NMP. 20 µl of homogenous solid phase suspension was sampled from reaction mixture to analyze the material. Product was cleaved off the resin with 1 ml of 95% TFA 5% water mixture, for 1 hour. TFA was evaporated by blowing nitrogen gas. Crude material was washed twice with 1 ml of cold diethyl ether. Ether was discarded and compound 11 was analyzed by HPLC and LCMS. See figures S7 and S22. Reversed phase HPLC was performed using mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = 12 minutes. LCMS analysis was performed using mm, 5 µm, C18 column. 100% solvent A (0.1% formic acid in water) to 100% solvent B (0.1% formic acid in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes; ESI-MS (m/z) = M + calculated, ; found, S11

12 (12) Synthesis of 5-guanidino-2-(5-guanidino-2-{2-[4-(1-hydrazono-ethyl)-2-methoxy-5- nitro-phenoxy]-acetylamino}-pentanoylamino)-pentanoic acid amide Compound 11 was converted into compound 12 on resin. 235 µmol of ketone (assuming 100% coupling on resin, 235 µmol of resin, 0.5 g) was suspended in 7 ml of 1:1 NMP:ethanol solvent mixture in a siliconized glass reaction vial µmol (20.2 µl) glacial acetic acid was added and mixed gently. 40 times excess hydrazine monohydrate (9.4 mmol, µl) was added and reaction vessel was sealed tightly. Reaction mixture was shaken overnight using Eppendorf Thermomixer at 60 C. Resin was washed (5X) with NMP and DCM and dried. Compound 12 was cleaved off resin by treating the resin with 95% TFA 5% water solution for one hour. TFA was evaporated as quickly as possible to avoid formation of azines. Dry material was thoroughly washed with cold ether. Crude material was dried on rotovap and immediately used for the next reaction. See figures S8 and S23. NOTE: Compound 12 is unstable and may not be stored for longer time. It is recommended to synthesize the material freshly and immediately used for the next reaction. Reversed phase HPLC was performed using mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes. Two peaks observed for E/Z isomers. LCMS analysis was performed using mm, 5 µm, C18 column. 100% solvent A (0.1% formic acid in water) to 100% solvent B (0.1% formic acid in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = 9.49 minutes; ESI-MS (m/z) = MH + calculated, ; found, S12

13 (13) Synthesis of 2-(2-{2-[4-(1-diazo-ethyl)-2-methoxy-5-nitro-phenoxy]-acetylamino}-5- guanidino-pentanoylamino)-5-guanidino-pentanoic acid amide Compound 12 was dissolved in minimal amount of anhydrous DMSO and quantified using UV spectroscopy at 345 nm with its extinction coefficient 4470 M -1 cm -1 [2]. Final concentration of the solution was adjusted to mm (1.66 µmol) with anhydrous DMSO. 690 µmol (60 mg) of MnO 2 was added to compound 12 solution (1.66 µmol). The reaction mixture was shaken vigorously for 45 minutes. Reaction mixture was centrifuged at RCF for 4 minutes to remove MnO 2. The orange/red supernatant containing compound 13 was collected in another Eppendorf tube and the MnO 2 pellet was washed with additional DMSO. The diazo derivative was pooled and added to insulin solution (described in next reaction). Compound 13 has a characteristic red color and an absorbance at 450 nm (Figure S40). (14) Synthesis of R2-Insulin (Compound 14) 9.6 mg of insulin was dissolved in 150 µl of anhydrous DMSO (1.66 µmol). To this solution, compound 13 supernatant from previous reaction was added and allowed to react for 24 hours. Compound 14 was purified by RP-HPLC using a mm, 5 µm, C18 column with a flow rate of 1 ml/min. 20% solvent B (0.1% TFA in acetonitrile) to 40% solvent B over 28 minutes, 40% solvent B to 100% solvent B over 0.5 minutes ( minutes), 100% solvent B isocratic for 3.5 minutes ( minutes), and 100% solvent B to 20% solvent B over 2 minutes (38-40 minutes) with a post time of 5 minutes. Compound 14 was collected as a fraction from minutes. See figures S9, S24 and S25. Reversed phase HPLC was performed using a mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes. Multiple peaks S13

14 observed due to regioisomeric adducts at different carboxyl groups on insulin. The molecular weight of R2-Insulin was confirmed by infusing HPLC purified fraction into mass spectrometer. ESI-MS (m/z) = M + calculated, amu; found (reconstructed), P2-INSULIN SYNTHESIS S14

15 (15) Synthesis of [1,3-bis-(2-pyrrolidin-1-yl-ethylcarbamoyl)-propyl]-carbamic acid 9Hfluoren-9-ylmethyl ester 200 mg Fmoc-glutamic acid (541.8 µmoles) and mg of HATU (1.6 mmoles) were dissolved in 9 ml of NMP. Solution was allowed to stand for five minutes. DIEA (1074 µmol, 187 µl) and 1-(2-aminoethyl)pyrrolidine (1.6 mmoles, µl) were added to the solution and reacted for 3 hours. Compound 15 was purified using a mm, 10 µm, C18 reverse phase HPLC. 4 ml of the reaction mixture was injected on to the column in each purification run. The following method (5 ml/min) was used to purify compound % solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 70 minutes, 100% solvent B isocratic for 5 minutes (70-75 minutes), 100% solvent B to 100% solvent A over 5 minutes (75-80 minutes). Compound 15 was collected from minutes. See figures S10, S26, S35 and S36. Reversed phase HPLC was performed using a mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = 15.7 minutes. LCMS analysis was performed using mm, 5 µm, C18 column. 100% solvent A (0.1% formic acid in water) to 100% solvent B (0.1% formic acid in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes; ESI-MS (m/z) = M + calculated, 562.3; found, H NMR (400 MHz, DMSO-d 6 ) d ppm (m), (m), (m), (m), (m), (m), 3.58 (d, J=4.69 Hz), 3.96 (td, J=8.39, 5.47 Hz), (m), 7.34 (td, J=7.42, 1.17 Hz), (m), 7.74 (d, J=7.42 Hz), 7.90 (d, J=7.42 Hz), 8.16 (t, J=5.86 Hz), 8.29 (t, J=5.66 Hz) 13C NMR (101 MHz, DMSO-d 6 ) d ppm (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s), (s) (16, 17) Synthesis of 2-[2-(4-acetyl-2-methoxy-5-nitro-phenoxy)-acetylamino]- pentanedioic acid bis-[(2-pyrrolidin-1-yl-ethyl)-amide] S15

16 Compound 15 was dissolved in ethanol and quantified using UV-spectroscopy at 301 nm (extinction coefficient = 6800 M -1 cm -1 ). 440 µmoles of compound 15 was transferred into round bottom flask and dried. Compound 15 was dissolved in 20 ml of acetonitrile, and 20 ml of 40% dimethylamine solution was added. The flask was sealed and allowed to react at room temperature for 30 minutes. Reaction mixture was dried at approximately 60 C. The crude material was washed with 40 ml of 50% DIEA in methanol solution five times, and dried at 60 C. The resulting crude material was washed with cold ether multiple times and dried. Compound 16 was neither purified nor characterized, and used for the synthesis of compound 17. Assuming 100% Fmoc-deprotection, 440 µmoles of compound 16 was dissolved in 5 ml NMP. 660 µmoles (0.18 g) compound 2 and 990 µmoles (0.38 g) HATU were dissolved in this solution. 1.9 mmoles (345 µl) of DIEA was added and reaction was allowed to stand for 3 hours. Compound 17 was purified using a mm, 10 µm, C18 reverse phase HPLC. 4 ml of reaction mixture was injected on to the column for each purification run. The following method (5 ml/min) was used to purify compound % solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 70 minutes, 100% solvent B isocratic for 5 minutes (70-75 minutes), 100% solvent B to 100% solvent A over 5 minutes (75-80 minutes). Compound 17 was collected from minutes. See figures S11, S27 and S37. Reversed phase HPLC was performed using a mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = 13.0 minutes. LCMS analysis was performed using a mm, 5 µm, C18 column. 100% solvent A (0.1% formic acid in water) to 100% solvent B (0.1% formic acid in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes; ESI-MS (m/z) = MH + calculated, 591.3; found, H NMR (400 MHz, DMSO-d 6 ) d ppm 1.83 (br. s., 5 H), 1.99 (br. s., 5 H), 2.15 (br. s., 2 H), 2.54 (m, 3 H), 3.00 (br. s., 4 H), 3.17 (m, 4 H), (br. s., 9 H), 3.95 (s, 3 H), 4.25 (m, 1 H), 4.79 (s, 2 H), 7.28 (s, 1 H), 7.63 (s, 1 H), 8.1 (m, 1 H), 8.35 (m, 1 H), 8.45 (m, 1 H) (18) Synthesis of 2-{2-[4-(1-hydrazono-ethyl)-2-methoxy-5-nitro-phenoxy]-acetylamino}- pentanedioic acid bis-[(2-pyrrolidin-1-yl-ethyl)-amide] S16

17 Compound 17 was dissolved in minimal amount of ethanol:acetonitrile mixture (1:1), and quantified using UV spectroscopy at 345 nm from its extinction coefficient [2], 4470 M -1 cm µmoles of compound 17 was diluted to 6 ml with 1:1 ethanol:acetonitrile solution. 15 µl of glacial acetic acid and µl of hydrazine monohydrate were added and allowed to react at 90 C for 4 hours. Reaction mixture was dried and crude material was washed multiple times with cold ether. Ether was discarded and partially purified compound 18 was obtained as a yellowish-white material in the flask. Material was dried again in vacuum to remove any ether. Compound 18 was dissolved in minimal quantity of anhydrous DMSO and used for the next reaction. See figures S12 and S28. Note: Compound 18 is unstable and may not be stored for longer time. Also, drying the material completely to remove ether is crucial. Presence of any amount of ether within the material resulted in an unwanted exothermic reaction with manganese dioxide (see compound 19 synthesis) that lowered the reaction yield. Reversed phase HPLC was performed using a mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes. Two peaks observed for E/Z isomers. LCMS analysis was performed using a mm, 5 µm, C18 column. 100% solvent A (0.1% formic acid in water) to 100% solvent B (0.1% formic acid in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes; ESI-MS (m/z) = MH + calculated, ; found, (19) Synthesis of 2-{2-[4-(1-diazo-ethyl)-2-methoxy-5-nitro-phenoxy]-acetylamino}- pentanedioic acid bis-[(2-pyrrolidin-1-yl-ethyl)-amide] S17

18 Compound 18 was dissolved in minimal amount of anhydrous DMSO and quantified using UVspectroscopy at 345 nm from it s extinction coefficient, 4470 M -1 cm -1. Final concentration was adjusted to mm of compound 18 by dilution with anhydrous DMSO. 132 µmoles (16.56 mm) compound 18 was transferred into a glass reaction vial and 22.9 mmoles (2000 mg) manganese dioxide was added and shaken vigorously for 45 minutes. The reaction mixture was centrifuged at RCF for 5 minutes. Supernatant containing compound 19 was collected into another Eppendorf tube (identified by a characteristic red color). The manganese dioxide was washed with additional DMSO to recover any trapped diazo. Solutions were pooled, and added to the insulin solution (described in compound 20 synthesis). Compound 19 has a characteristic red color and an absorbance at 450 nm (Figure S41). (20) Synthesis of P2-Insulin (Compound 20) 132 µmoles of insulin (770 mg) was dissolved in 8 ml of anhydrous DMSO. Compound 19 solution (132 µmoles, assuming 100% conversion) was added to insulin solution. Reaction was carried out for 24 hours. Compound 20 was purified by RP-HPLC using mm, 5 µm, C18 column with a flow rate of 1 ml/min. 24% solvent B (0.1% TFA in acetonitrile) to 38% solvent B over 35 minutes, 38% solvent B to 75% solvent B over 0.5 minutes ( minutes), 75% solvent B isocratic for 2.5 minutes ( minutes), and 75% solvent B to 24% solvent B over 2 minutes (38-40 minutes); post time = 5 minutes. Compound 20 was collected from minutes. See figures S13, S29 and S30. S18

19 Reversed phase HPLC was performed using mm, 5 µm, C18 column. 100% solvent A (0.1% TFA in water) to 100% solvent B (0.1% TFA in acetonitrile) over 28 minutes, 100% solvent B to 100% solvent A over 2 minutes. Retention time = minutes. Multiple peaks observed due to regioisomeric adducts at different carboxyl groups on insulin. Mass of P2-Insulin was confirmed by infusing HPLC purified fraction into mass spectrometer. ESI-MS (m/z) = M + calculated, ; found (reconstructed), S19

20 Quantitation ISOELECTRIC POINT DETERMINATION 0.5 nmoles/µl solutions of insulin (quantified by weight), and other insulin derivatives (quantified by UV-spectroscopy at 345 nm, 4470 M -1 cm -1 ) were prepared in DMSO. Lantus formulation was diluted in DMSO and quantified at 280 nm with extinction coefficient 5128 M -1 cm -1. Loading samples and running IEF 1X anode buffer and 1X cathode buffers were prepared from 10X stock solutions purchased from Bio-rad. The electrophoresis unit reservoir was filled with anode buffer up to the recommended level, and the reservoir on gel cast was filled with cathode buffer. Voltage across the gel was applied as shown below, for following periods: Time Voltage 1 hour 100 V 1 hour 250 V 30 min 500 V Solutions (1) Fixing solution: 15% Glutaraldehyde in 30% methanol (2) Washing solution: 27% isopropanol, 10% acetic acid (3) Staining solution (primary stock): 0.04% w/v Coomassie Blue R-250, 0.05% w/v Crocein Scarlet in 27% isopropanol, 10% acetic acid (4) Destaining solution: First wash with 20% methanol, 5% acetic acid for approximately 120 minutes. Successive washes with 40% methanol, 10% acetic acid until the gel background becomes clear. Overnight destaining was performed with 20% methanol, 5% acetic acid. Processing the gel After the two and half hours run, gel was fixed for 2 hours with fixing solution. Gel was washed with the washing solution for 5 minutes. Gel was stained for 45 minutes with 0.1X (10 times diluted) staining solution. Gel was destained with destaining solution until the bands were clearly visible. Analysis Gel images were captured on a white background. Images were opened with Adobe Illustrator and migration distance was measured in millimeters using line segment tool, from top of the gel (bottom of well) to the band. A standard curve of Bio-Rad standards was used to determine the pi of insulin and its derivatives. See figures S42, S43, S44, and S45. SOLUBILITY STUDIES The solubility of insulin and P2-insulin were analyzed at both ph 4.0 and 7.2, using PBS (10mM phosphate, 137mM NaCl). An amount of each peptide or modified peptide was added to each Eppendorf tube such that saturation would be achieved. The amount of added P2-insulin was determined using the UV absorbance at 345nm and an extinction coefficient of 4470 M -1 cm -1. S20

21 The umodified insulin amount was determined by weight. Saturation was confirmed by observing solid in the tube at equillibrium, and by analyzing this solid. Specifically, the solid was dissolved in DMSO and analyzed by HPLC to confirm either insulin or P2-insulin presence. Solubility was determined as follows: 100 µl of the respective buffers were added (ph 7.2 and 4). The material was suspended with the help of a pipette. Small changes in ph were observed likely due to counterions in both insulin and modified insulin. Therefore the ph of the buffer was readjusted to the desired ph by addition of small quantities of 0.1 N NaOH. Tubes were shaken at 300 RPM for 2 hours to suspend the material homogenously. After 2 hours, the tubes were centrifuged at RCF for 3 minutes. This clarified the supernatant, which was then analyzed by HPLC. The amount of insulin or P2-Insulin was quantified using standard curves of insulin and compound 2 respectively. P2-INSULIN PHOTOLYSIS IN DMSO HPLC purified P2-Insulin was dissolved in DMSO and quantified using UV spectroscopy. 20 nmoles of P2-Insulin was transferred into a 1 ml glass insert (Fisherbrand) and dried. A 365 nm LED was used to photolyze the material. The absolute irradiance of the LED was measured by using a USB-2000 fiber optic spectrometer (Ocean Optics, Inc.), fitted with a cosine corrector and the SpectraSuite package. This was measured at a distance of 7cm, the same distance used for the photolysis experiments. 20 nmoles of dried P2-Insulin was dissolved in 40 µl of DMSO (0.5 nmol/µl) in a glass insert. This glass insert was placed such that the bottom was 7 cm away from the LED light source. The solution was irradiated with 365 nm light for the respective time intervals. At the end of each exposure, the LED was turned off and 2 µl of the solution were sampled from the tube for analysis. The LED was then turned on and photolysis was continued until the next time point. 2 µl of each sample was diluted with 8 µl of Bio-Rad IEF sample loading buffer and this 10 µl was loaded onto an IEF gel (ph 3-10) and run as discussed in the Isoelectric Point Determination section. The gel was photographed and analyzed using Adobe Photoshop as described earlier [3]. Briefly, the gel image was converted into grayscale and inverted. Using the marquee tool, bands and backgrounds were selected. Signal intensity in the bands (and backgrounds) was measured with the measurement tool. Band intensities were corrected for background. Photoreleased insulin was quantified from the lower band, previously shown to be insulin. The amount of insulin released was determined by normalizing the band intensity to the intensity of the 60 minute sample insulin band, which was set at 20nmoles, the starting amount of P2-insulin. The insulin photo-release profile was plotted as a function of time, and the curve was fitted in KaleidaGraph using a first order rate law, as follows: Insulin = P2-Insulin t=0 *[1-exp -kt ] KaleidaGraph format: Y=M1*(1-EXP(-M2*M0)) P2-INSULIN PHOYOLYSIS IN 10 mm PBS 7.2 HPLC purified P2-Insulin (compound 20) was dried, dissolved in DMSO and quantified using UV spectroscopy with an extinction coefficient of 4470 M -1 cm -1 at 345 nm. A volume of DMSO S21

22 equivalent to 100 nmoles of P2-Insulin was transferred into glass insert and the DMSO removed by vacuum. A 365 nm LED was used to photolyze the material. The absolute irradiance of the LED was measured by using a USB-2000 fiber optic spectrometer (Ocean Optics, Inc.), fitted with a cosine corrector and the SpectraSuite package. This was measured at a distance of 5cm, the same distance used for the photolysis experiments. 100nmoles of P2-insulin was added to 100 µl ph 7.2 PBS (10mM phosphate, 137mM NaCl). The ph of the buffer was measured and corrected to 7.2 through addition of small amounts of 0.1 N sodium hydroxide. The material was suspended with the help of a pipette. It was then centrifuged at RCF for 4 minutes to clarify the supernatant of small particle of P2-insulin. 100 µl of the supernatant was collected without disturbing the P2-Insulin pellet. This supernatant sample when analyzed was considered the zero time point, as it was unirradiated. Following this initial step, the material was resuspended in 100µl of fresh PBS, and photolyzed. Supernatant was isolated from this photolysis suspension at specific time points for analysis. These time points were 1, 2, 3, 4, 5, 10, 15, 30 and 60 minutes. Supernatant isolation was effected by centrifuging the tubes, removing the entire 100µl buffer for analysis, and replacing the removed buffer with 100µl of fresh buffer. A control reaction was also carried out, which was processed identically without exposing the sample to 365 nm LED irradiation. To 10 µl of PBS supernatant collected at respective time points, 10 µl of IEF sample buffer were added and mixed thoroughly by vortexing. This 20 µl sample was loaded onto IEF gels (ph 3-10) and run as discussed in the isoelectric point determination section. 10 µl each of 20 µm, 40 µm, 60 µm, 80 µm and 100 µm insulin solutions in DMSO were mixed with 10 µl of IEF sample buffer respectively (total 20 µl) and run as insulin standards (corresponding to 0.2 nmoles, 0.4 nmoles, 0.6 nmoles, 0.8 nmoles and 1 nmole insulin respectively). Insulin photorelease was quantified using the insulin standard curve generated from these insulin standards as run on the IEF gel. Samples were run on two gels. Gel 1 contained samples collected at 0, 1, 2, 3, 4, 5, 10 and 15 minutes (seen in the main manuscript). Gel 2 contained samples collected at 30 and 60 minutes, and the insulin standards (seen in the supporting information document). See P2-Insulin photorelease (Gel 1) in the manuscript. Gel 2 of P2-Insulin photolysis and both Gels 1 and 2 of control reaction are shown below. See figures S47, S48 and S49. The insulin photo-release profile was plotted as a function of time, and the curve was fitted in KaleidaGraph using a first order rate law, as follows: Insulin = P2-Insulin t=0 *[1-exp -kt ] KaleidaGraph format: Y=M1*(1-EXP(-M2*M0)) S22

23 RESULTS HPLC ANALYSIS (S1) Compound 3; 2-(4-Acetyl-2-methoxy-5-nitro-phenoxy)-N-(2-pyrrolidin-1-yl-ethyl)- acetamide Figure S 1 Compound 3 chromatogram. Retention time = minutes. S23

24 (S2) Compound 4; 2-[4-(1-Hydrazono-ethyl)-2-methoxy-5-nitro-phenoxy]-N-(2-pyrrolidin-1- yl-ethyl)-acetamide Figure S 2 Compound 4 chromatogram. Retention time = minutes. Two peaks observed for E/Z isomers. (S3) Compound 6; P-Insulin Figure S 3 Compound 6 (P-Insulin) chromatogram. Retention time = minutes. Multiple peaks observed due to regioisomeric adducts at different carboxyl groups on insulin. Absorbance seen at both 280 nm (for insulin + DMNPE) and 345 nm (for DMNPE moiety). S24

25 (S4) Compound 7; {2-[2-(4-Acetyl-2-methoxy-5-nitro-phenoxy)-acetylamino]-ethyl}- trimethyl-ammonium Figure S 4 Compound 7 chromatogram. Retention time = minutes. (S5) Compound 8; (2-{2-[4-(1-Hydrazono-ethyl)-2-methoxy-5-nitro-phenoxy]-acetylamino}- ethyl)-trimethyl-ammonium trifluoroacetate Figure S 5 Compound 8 chromatogram. Retention time = minutes. Two peaks observed for E/Z isomers S25

26 (S6) Compound 10; Q-Insulin Figure S 6 Compound 10 chromatogram. Retention time = minutes. Multiple peaks observed due to regioisomeric adducts at different carboxyl groups on insulin. Absorbance seen at both 280 nm (for insulin + DMNPE) and 345 nm (for DMNPE moiety). S26

27 (S7) Compound 11; 2-{2-[2-(4-Acetyl-2-methoxy-5-nitro-phenoxy)-acetylamino]-5- guanidino-pentanoylamino}-5-guanidino-pentanoic acid amide Figure S 7 Compound 11 synthesized on ChemMatrix Rink amide resin, was cleaved off using 95% TFA. Dried material, after washing with ether, was run on HPLC. Chromatogram shows compound 11 having retention time = 12 minutes. S27

28 (S8) Compound 12; 5-Guanidino-2-(5-guanidino-2-{2-[4-(1-hydrazono-ethyl)-2-methoxy-5- nitro-phenoxy]-acetylamino}-pentanoylamino)-pentanoic acid amide Figure S 8 Compound 12, synthesized on ChemMatrix Rink amide resin, was cleaved off using 95% TFA. Dried material, after washing with cold ether, was run on HPLC. Retention time = minutes. Two peaks observed for E/Z isomers. S28

29 (S9) Compound 14; R2-Insulin Figure S 9 Compound 14 (R2-Insulin) chromatogram. Retention time = minutes. Multiple peaks observed due to regioisomeric adducts at different carboxyl groups on insulin. Absorbance seen at both 280 nm (for insulin + DMNPE) and 345 nm (for DMNPE moiety). Insulin contamination (di) was observed, the ratio of absorbance at 280 nm:345 nm was not constant. S29

30 (S10) Compound 15; [1,3-Bis-(2-pyrrolidin-1-yl-ethylcarbamoyl)-propyl]-carbamic acid 9H-fluoren-9-ylmethyl ester Figure S 10. Compound 15 chromatogram. Retention time = 15.7 minutes. (S11) Compound 17; 2-[2-(4-Acetyl-2-methoxy-5-nitro-phenoxy)-acetylamino]- pentanedioic acid bis-[(2-pyrrolidin-1-yl-ethyl)-amide] Figure S 11 Compound 17 chromatogram. Retention time = 13.0 minutes S30

31 (S12) Compound 18; 2-{2-[4-(1-Hydrazono-ethyl)-2-methoxy-5-nitro-phenoxy]- acetylamino}-pentanedioic acid bis-[(2-pyrrolidin-1-yl-ethyl)-amide] Figure S 12 Compound 18 chromatogram. Retention time = minutes. Two peaks observed for E/Z isomers. (S13) Compound 20; P2-Insulin Figure S 13 Compound 20 chromatogram. Retention time = minutes. Multiple peaks observed due to regioisomeric adducts at different carboxyl groups on insulin. Absorbance seen at both 280 nm (for insulin + DMNPE) and 345 nm (for DMNPE moiety). S31

32 LCMS ANALYSIS (S14) Compound 3; 2-(4-Acetyl-2-methoxy-5-nitro-phenoxy)-N-(2-pyrrolidin-1-yl-ethyl)- acetamide Figure S 14 Compound 3 mass spectrum. Calculated MH + = ; observed = S32

33 (S15) Compound 4; 2-[4-(1-Hydrazono-ethyl)-2-methoxy-5-nitro-phenoxy]-N-(2-pyrrolidin- 1-yl-ethyl)-acetamide Figure S 15 Compound 4 mass spectrum. Calculated MH + = 380.2; observed = S33

34 (S16) Compound 6; P-Insulin Figure S 16 Compound 6 (P-Insulin) MS analysis. Insulin was infused as a standard to calculate the errors propagated in mass reconstruction. Expected mass of insulin = 5808; Observed mass = 5810; = +2. See next page for P-Insulin mass spectrum. S34

35 Figure S 17 Compound 6 (P-Insulin) MS analysis. Errors calculated from Insulin infusion (Figure S 15) were subtracted from P-Insulin observed mass to know the exact mass of compound. Expected mass of P-Insulin = ; Observed mass = 6160; = +3; of = +1 S35

36 (S18) Compound 7; {2-[2-(4-Acetyl-2-methoxy-5-nitro-phenoxy)-acetylamino]-ethyl}- trimethyl-ammonium Figure S 18 Compound 7 mass spectrum. Calculated MH+ = ; observed = S36

37 (S19) Compound 8; (2-{2-[4-(1-Hydrazono-ethyl)-2-methoxy-5-nitro-phenoxy]- acetylamino}-ethyl)-trimethyl-ammonium Figure S 19 Compound 8 mass spectrum. Calculated MH+ = 368.2; observed = S37

38 (S20) Compound 10; Q-Insulin Figure S 20 Compound 10, standard (Insulin) MS infusion. Expected mass of Insulin = Observed mass = = 5. See next page for Q-Insulin mass spectrum S38

39 Figure S 21 Compound 10 MS infusion. Expected mass of Q-Insulin = Observed mass = = 4. of = -1. S39

40 (S22) Compound 11; 2-{2-[2-(4-Acetyl-2-methoxy-5-nitro-phenoxy)-acetylamino]-5- guanidino-pentanoylamino}-5-guanidino-pentanoic acid amide Figure S 22 Compound 11 mass spectrum. Calculated mass, MH + = Observed mass = S40

41 (S23) Compound 12; 5-Guanidino-2-(5-guanidino-2-{2-[4-(1-hydrazono-ethyl)-2-methoxy- 5-nitro-phenoxy]-acetylamino}-pentanoylamino)-pentanoic acid amide Figure S 23 Compound 12 mass spectrum. Calculated mass, MH + = Observed mass = S41

42 (S24) Compound 14; R2-Insulin Figure S 24 Compound 14, Insulin standard infusion. Calculated mass = Observed mass = = 4. See R2-Insulin mass spectrum on the next page S42

43 Figure S 25 Compound 14, R2-Insulin MS infusion. Calculated mass = Observed mass = = 5. of = +1 S43

44 (S26) Compound 15; [1,3-Bis-(2-pyrrolidin-1-yl-ethylcarbamoyl)-propyl]-carbamic acid 9H-fluoren-9-ylmethyl ester Figure S 26 Compound 15 mass spectrum. Calculated mass, MH + = Observed mass = S44

45 (S27) Compound 17; 2-[2-(4-Acetyl-2-methoxy-5-nitro-phenoxy)-acetylamino]- pentanedioic acid bis-[(2-pyrrolidin-1-yl-ethyl)-amide] Figure S 27 Compound 17 mass spectrum. Calculated mass, MH+ = Observed mass = S45

46 (S28) Compound 18; 2-{2-[4-(1-Hydrazono-ethyl)-2-methoxy-5-nitro-phenoxy]- acetylamino}-pentanedioic acid bis-[(2-pyrrolidin-1-yl-ethyl)-amide] Figure S 28 Compound 18 mass spectrum. Calculated mass, MH + = Observed mass = S46

47 (S29) Compound 20; P2-Insulin Figure S 29 Compound 20 mass spectrometry analysis. Insulin (standard) infusion. Calculated mass = Observed mass = = 0. See P2-Insulin mass spectrum on the next page S47

48 Figure S 30 Compound 20 (P2-Insulin) mass spectrometry. Expected mass = Observed mass = = S48

49 NMR SPECTROSCOPY (S31) Compound 3; 2-(4-Acetyl-2-methoxy-5-nitro-phenoxy)-N-(2-pyrrolidin-1-yl-ethyl)- acetamide Figure S 31 Compound 3, H-NMR (400 MHz, d6-dmso) Figure S 32 Compound 3, C13 NMR (100 MHz, d6-dmso) S49

50 (S33) Compound 7; {2-[2-(4-Acetyl-2-methoxy-5-nitro-phenoxy)-acetylamino]-ethyl}- trimethyl-ammonium Figure S 33 Compound 7, H-NMR (400 MHz, d6-dmso) Figure S 34 Compound 7, 13C-NMR (100 MHz, d6-dmso) S50

51 (S35) Compound 15; [1,3-Bis-(2-pyrrolidin-1-yl-ethylcarbamoyl)-propyl]-carbamic acid 9H-fluoren-9-ylmethyl ester Figure S 35 Compound 15, 1H-NMR (400 MHz, d6-dmso) Figure S 36 Compound 15, 13C-NMR (100 MHz, d6-dmso) S51

52 (S37) Compound 17; 2-[2-(4-Acetyl-2-methoxy-5-nitro-phenoxy)-acetylamino]- pentanedioic acid bis-[(2-pyrrolidin-1-yl-ethyl)-amide] Figure S 37 Compound 17, 1H NMR (400 MHz, d6-dmso) S52

53 UV SPECTROSCOPY (S38) Compound 5; 2-[4-(1-Diazo-ethyl)-2-methoxy-5-nitro-phenoxy]-N-(2-pyrrolidin-1-ylethyl)-acetamide Figure S 38 Compound 5 spectrum, showing absorbance at 450 nm (S39) Compound 9; (2-{2-[4-(1-Diazo-ethyl)-2-methoxy-5-nitro-phenoxy]-acetylamino}- ethyl)-trimethyl-ammonium Figure S 39 Compound 9 spectrum, showing absorbance at 450 nm S53

54 (S40) Compound 13; 2-(2-{2-[4-(1-Diazo-ethyl)-2-methoxy-5-nitro-phenoxy]-acetylamino}- 5-guanidino-pentanoylamino)-5-guanidino-pentanoic acid amide Figure S 40 Compound 13 spectrum, showing absorbance at 450 nm (S41) Compound 19; 2-{2-[4-(1-Diazo-ethyl)-2-methoxy-5-nitro-phenoxy]-acetylamino}- pentanedioic acid bis-[(2-pyrrolidin-1-yl-ethyl)-amide] Figure S 41 Compound 19 spectrum, showing absorbance at 450 nm S54

55 (S42) Compound 6; P-Insulin IEF ISOELECTRIC POINT DETERMINATION Figure S 42 Compound 6 (P-Insulin) IEF. Observed pi = 6.62 ± 0.01 S55

56 (S43) Compound 10; Q-Insulin IEF Figure S 43 Compound 10 (Q-Insulin) IEF. Observed pi = 6.65 ± S56

57 (S44) Compound 14; R2-Insulin IEF Figure S 44 Compound 14 (R2-Insulin) IEF. Observed pi = 7.15 ± S57

58 (S45) Compound 20; P2-Insulin IEF Figure S 45 Compound 20 (P2-Insulin) IEF. Observed pi = 7.17 ± S58

59 (S46) LED wavelength P2-INSULIN PHOTOLYSIS IN 10 mm 7.2 PBS Figure S 46 Spectrum of 365 nm LED, measured using USB-2000 fiber optic spectrometer (Ocean Optics, Inc.) S59

60 (S47) P2-Insulin photolysis samples on Gel 2 Figure S 47 Gel 2 of P2-Insulin photolysis with 30 and 60 minutes samples. Insulin standards (0.2-1 nmol) were also run to determine the amount of insulin in each sample. S60

61 (S48) P2-Insulin control reaction Gel 1 Figure S 48 P2-Insulin control reaction Gel 1 not showing insulin bands. S61

62 (S49) P2-Insulin control reaction Gel 2 Figure S 49 P2-Insulin control reaction Gel 2 not showing insulin bands. Insulin standards (0.2-1 nmol) were also run on this gel. S62

63 REFERENCES [1] Holmes, C.P. J. Org. Chem. 1997, 62, [2] Jain, P.K., Karunakaran, D. and Friedman, S.H. Angew. Chem., Int.Ed , [3] Sarode, B.R., Jain, P.K. and Friedman, S.H. Macromol. Biosci , S63