Multifunctional poly[n-(2-hydroxypropyl)methacrylamide] copolymers via post-polymerization modification and sequential thiol ene chemistry

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1 Electronic Supplementary Information for: Multifunctional poly[n-(2-hydroxypropyl)methacrylamide] copolymers via post-polymerization modification and sequential thiol ene chemistry Nora Francini, Laura Purdie, Cameron Alexander,* Giuseppe Mantovani* and Sebastian G. Spain.*, School of Pharmacy, University of Nottingham, University Park, Nottingham. NG7 2RD. UK. Current address: Department of Chemistry, University of Sheffield, Dainton Building, Sheffield. S3 7HF. UK. Table of Contents Removal of dithioester end group from RAFT polymers 2 Synthesis of Allyl-pHPMA: optimization of the conditions for post-polymerization modifications of phpma with allyl isocyanate. 3 Stability of BODIPY-SH under radical thiol ene conditions 5 HPLC analysis of RGDC peptide 6 1 H NMR analysis of polymers synthesized by thiol ene chemistry 7 Thiol ene chemistry on allyl-phpma prepared by free-radical polymerization. 17

2 Removal of dithioester end group from RAFT polymers Figure S1. A. Representative UV/Vis spectra of phpma P1 before and after treatment with AIBN. B. Calibration curve for thiol determination using Ellman s assay.

3 Synthesis of Allyl-pHPMA: optimization of the conditions for post-polymerization modifications of phpma with allyl isocyanate. Scheme S1. Post-polymerization modification of phpma. A range of reaction conditions were investigated for the modification of phpma with allyl isocyanate, as reported in Table S1. As a general procedure, phpma P2 (50 mg, 0.35 mmol) was dissolved in 1.5 ml of dry solvent in a round bottomed flask equipped with a magnetic stirrer bar. The base was added under a stream of nitrogen and the resulting solution allowed to stir for 5 minutes before addition of allyl isocyanate. The reaction was allowed to proceed at room temperature or 60 C. Aliquots of the reaction mixture were taken at certain time intervals for 1 H NMR spectroscopy. Conversion was estimated by integration of the CHCH 2 signal at 6.01 and 5.82 ppm corresponding to reacted and unreacted isocyanate, respectively. Purification of the functionalized polymers was carried out by precipitation in diethyl ether and re-dissolution of the precipitate in methanol. The precipitation was performed three times. The final products were characterized by 1 H NMR spectroscopy. 1 H NMR (400 MHz, (CD 3 ) 2 SO): δ = 7.16 (broad s, 2H, CONH), 5.82 (broad s, 1H, CHCH 2 ), 5.08 (broad m, 2H, CHCH 2 ), 4.75 (broad s, 2H, CHOH and CHOCO), 3.76 (broad s, 1H, CHOH), 3.02 (broad s, 2H, CH 2 ), (broad m, 8H, CH 2 and CH 3 of polymer backbone, and CHCH 3 ).

4 Table S1. Reaction conditions for the synthesis of Allyl-pHPMA and conversions Code solvent base Reaction time (h) Temperature ( C) Equivalents of isocyanate a Conversion (%) b A DMF dry No base 24 RT B DMAc dry No base 17 RT C DMAc dry No base D DMAc dry No base E DMAc dry 0.1 eq DIPEA dry 17 RT 1 4 F DMAc dry 0.1 eq DIPEA dry G DMAc dry 5 eq DIPEA dry H DMAc dry c 5 eq TEA dry 24 RT RT 1 46 a Equivalents calculated based on hydroxyl groups of HPMA. b Determined by 1 H NMR spectroscopy in DMSO-d6. c Traces of water were removed from the solution of phpma in DMAc under high vacuum before addition of dry triethylamine and allyl isocyanate.

5 Figure S2. A. Representative 1 H NMR spectrum (reaction F), signal b was used to determined degree of modification. B. SEC chromatogram of D (2 eq, 60 C) in DMF + 0.1% LiBr. Figure S3. SEC traces of P1, P2 and P3 before and after modification with different ratios of allyl isocyanate (P1a d, P2a c, P3a c). Stability of BODIPY-SH under radical thiol ene conditions Figure S4. Aqueous SEC chromatograms for the reaction between of 6 and P1a. Fluorescence detection (λ ex 485 nm, λ em 506 nm).

6 HPLC analysis of RGDC peptide Figure S5. Reverse phase HPLC chromatogram of RGDC peptide 7 after purification.

7 1 H NMR analysis of polymers synthesized by thiol ene chemistry P5 (benzyl mercaptan) 1 H NMR (400 MHz, DMSO) δ (broad s, 7H, 2 NH and 5 CH arom ), 4.71 (broad s, 3H, CHOH, CHOCO), 3.72 (broad s, 3H, CHOH and PhCH 2 S), (broad m, 4H, NHCH 2 CH, NHCH 2 CH 2 and CH 2 CH 2 S), 2.39 (broad s, 2H, SCH 2 ), (broad m, 10H, CH 2 and CH 3 of polymer backbone, CHCH 3 and CH 2 CH 2 CH 2 ). Figure S6. 1 H NMR of P5 in DMSO-d6.

8 P6 (3-mercaptopropionic acid) 1 H NMR (400 MHz, DMSO): δ (broad s, 1H, COOH), 7.11 (broad s, 2H, NH), 4.69 (broad s, 3H, CHOH, CHOCO), 3.69 (broad s, 1H, CHOH), (broad m, 6H, NHCH 2 CH, NHCH 2 CH 2 and SCH 2 ), 2.69 (broad m, 2H, CH 2 COOH), 2.50 (broad s, 2H, SCH 2 ), (broad m, 10H, CH 2 and CH 3 of polymer backbone, CHCH 3 and CH 2 CH 2 CH 2 ). Figure S7. 1 H NMR of P6 in DMSO-d6.

9 P7 (cysteamine) 1 H NMR (400 MHz, D 2 O): δ 3.84 (broad s, 1H, CHOH), (broad m, 6H, NHCH 2 CH, NHCH 2 CH 2 and CH 2 NH 2 ) 2.81 (broad s, 2H, SCH 2 ), 2.57 (broad s, 2H, SCH 2 ), (broad m, 10H, CH 2 and CH 3 of polymer backbone, CHCH 3 and CH 2 CH 2 CH 2 ). Figure S8. 1 H NMR of P7 in D 2 O.

10 P8 (mercaptoethanol) 1 H NMR (400 MHz, D 2 O): δ 7.55 (broad s, 2H, NH), 3.84 (broad s, 1H, CHOH), 3.70 (broad s, 2H, CH 2 OH), (broad m, 4H,NHCH 2 CH and NHCH 2 CH 2 ), 2.69 (broad s, 2H, SCH 2 ), 2.58 (broad s, 2H, CH 2 S), (broad m, 10H, CH 2 and CH 3 of polymer backbone, CHCH 3 and CH 2 CH 2 CH 2 ). Figure S9. 1 H NMR of P8 in D 2 O.

11 P9 (1-thio-β-D-glucose) 1 H NMR (400 MHz, (CD 3 ) 2 SO): δ 7.14 (broad s, 2H, NH), (broad m, 7H, OH, SCH (H1),1H CH2 (H6) and CHOC), 3.68 (broad s, 2H, CHOH HPMA and CH 2(H6) OH), 3.43 (broad s, 1H, CH 2(H5) OH) (broad m, 10H, CH (H1-4), CH 2 CH HPMA,CONHCH 2 and SCH 2 ), (broad m, 10H, CH 2 and CH 3 of polymer backbone, CHCH 3 and CH 2 CH 2 CH 2 ). Figure S10. 1 H NMR of P9 in DMSO-d6.

12 P10 (BODIPY-SH) 1 H NMR (400 MHz, (CD 3 ) 2 SO): δ 7.21 (broad s, 2H, NH), 6.53 (broad s, 2H, CH arom,), 5.83 (broad s, 1H, CHCH 2 ), 5.12 (broad m, 2H, CHCH 2 ), 4.71 (broad s, 2H, CHOH and CHOCO), 3.69 (broad s, 1H, CHOH and CONHCH 2 ), 2.92 (broad m, 8H, CH 2 CH HPMA,CONHCH 2 and CH 2 S), 2.68 (broad s, 2H, SCH 2 ), 2.34 (broad s, 2H,CH 2 C), 2.09 (broad s, 6H, CH 2 alk ), (broad m, 10H, CH 2 and CH 3 of polymer backbone, CHCH 3 and CH 2 CH 2 CH 2 ). Figure S11. 1 H NMR of P10 in DMSO-d6.

13 P11 (RGDC) 1 H NMR (400 MHz,, (CD 3 ) 2 SO): δ (broad s, 5H, CONH RGD ), 7.22 (broad s, 2H, CONH), 5.82 (broad s, 1H, CHCH 2 ), 5.12 (broad m, 2H, CHCH 2 ), 4.71 (broad s, 2H, CHOH and CHOCO), 4.57 (broad s, 2H, CHCOOH RGD ), 4.17 (broad s, 8H, CH 2 S RGD, CH 2 NHCO, NH 2 CHCH 2 and CH 2 NC) 3.86 (broad s, 3H, CHOH and CONHCH 2 ) (broad m, 8H,CH 2 CH HPMA, CONHCH 2 CH 2 ), (broad m, 10H, CH 2 and CH 3 of polymer backbone, CHCH 3 and CH 2 CH 2 CH 2 ). Figure S12. 1 H NMR of P11 in DMSO-d6.

14 P12 (RGDC, 3-mercaptopropionic acid) 1 H NMR (400 MHz, (CD 3 ) 2 SO): δ (broad s, 1H, COOH), (broad m, 5H, CONH RGD ), 7.07 (broad s, 2H, CONH), 4.72 (broad s, 2H, CHOH and CHOCO), 4.57 (broad s, 2H, CHCOOH RGD ), 4.25 (broad s, 2H, CH 2 NHCO), 3.89 (broad m, 6H, CH 2 S RGD, NH 2 CHCH 2 and CH 2 NC), 3.69 (broad s, 1H, CHOH HPMA ), (broad m, 6H, CH 2 CH HPMA, CONHCH 2 and CH 2 COOH), 2.67 (brad m, 2H, SCH 2 ), 2.50 (DMSO and CH 2 S), (broad m, 10H, CH 2 and CH 3 of polymer backbone, CHCH 3 and CH 2 CH 2 CH 2 ). Figure S13. 1 H NMR of P12 in DMSO-d6.

15 P13 (BODIPY-SH, mercaptoethanol) 1 H NMR (400 MHz, (CD 3 ) 2 SO): δ 7.17 (broad s, 2H, NH), 6.24 (broad s, 2H, CH arom,) 4.70 (broad s, 3H, CHOH, CHOCO and CH 2 OH), 3.69 (broad s, 1H, CHOH), 3.52 (broad m,2h, CH 2 OH), (broad m, 4H,NHCH 2 CH and NHCH 2 CH 2 ), 2.56 (broad s, 4H, CH 2 SCH 2 ), (broad m, 10H, CH 2 and CH 3 of polymer backbone, CHCH 3 and CH 2 CH 2 CH 2 ). Figure S14. 1 H NMR of P13 in DMSO-d6.

16 P14 (RGDC, BODIPY-SH, mercaptoethanol) 1 H NMR (400 MHz, (CD 3 ) 2 SO): δ (broad m, 5H, CONH RGD ), 7.15 (broad s, 2H, CONH), 6.22 (broad s, 2H, CH arom,), 5.00 (broad s, 2H, CHCOOH RGD ), 4.78 (broad s, 1H, CH 2 OH), 4.71 (broad s, 2H, CHOH and CHOCO), 3.79 (broad s, 2H, CHCOOH RGD, CH 2 S RGD, NH 2 CHCH 2 and CH 2 NC), 3.69 (broad s, 1H, CHOH HPMA ), 3.53 (broad s, 2H, CH 2 OH), (broad m, 6H, CH 2 CH HPMA, CONHCH 2 ), 2.56 (broad m, 4H, CH 2 SCH 2 ), (broad m, 10H, CH 2 and CH 3 of polymer backbone, CHCH 3 and CH 2 CH 2 CH 2 ). Figure S15. 1 H NMR of P14 in DMSO-d6.

17 Thiol ene chemistry on allyl-phpma prepared by free-radical polymerization. Allyl-pHPMA (15 mg, 35.6 µmol of alkene) was dissolved in ~ 25 µl of DMSO-d6. DMPA (25 µl aliquot of 40 mg/ml stock solution in DMSO-d6) was then added along with benzyl mercaptan (22.1 mg, mmol). The solution was bubbled with nitrogen for 5 min and then subjected to UV irradiation at 365 nm for 20 min. The functionalized polymer was purified by precipitation in diethyl ether 4 times and dried under reduced pressure. 1 H NMR spectrum has been assigned as reported above for the analogous RAFT polymer. SEC in DMF+ 0.1% LiBr: M n =43.7 kda, Ð= Figure S16. Molecular weight distributions of phpma, allyl-phpma and benzyl-phpma prepared by free radical polymerization.