Supplementary Information for

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1 Supplementary Information for Ionic Polypeptides with Unusual Helical Stability Hua Lu 1, Jing Wang 2, Yugang Bai 1, Jason W. Lang 1, Shiyong Liu 3, Yao Lin 2,*, Jianjun Cheng 1,* 1 Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign, Urbana, IL Polymer Program, Institute of Materials Science and Department of Chemistry, University of Connecticut, Storrs, CT CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at Microscale, Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei, Anhui 2326, China * jianjunc@illinois.edu; ylin@ims.uconn.edu

2 Supplementary Methods Materials. All chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used as received unless otherwise specified. Anhydrous dimethylformamide (DMF) was dried by a column packed with 4Å molecular sieves and stored in a glove box. Tetrahydrofuran (THF) and hexane were dried by a column packed with alumina and stored in a glove box. Anhydrous CDCl 3 was prepared by treating the purchased CDCl 3 with CaSO 4 overnight, followed by distillation under nitrogen. The purified CDCl 3 was stored in the presence of 4Å MS. Dry nitrobenzene and DMSO-d 6 were prepared by treating regular nitrobenzene and DMSO-d 6 with CaH 2 followed by distillation under reduced pressure. H-Lys(Z)-OH and H-Glu(OBn)-OH were obtained from Chem-Impex International (Des Plaines, IL, USA) and used as received. Glu-NCA and Lys-NCA were prepared by following the previously reported procedures 41. Instrumentation. NMR spectra were recorded on a Varian UI4 MHz, a UI5NB MHz or a VXR-5 MHz spectrometer. Tandem gel permeation chromatography (GPC) experiments were performed on a system equipped with an isocratic pump (Model 11, Agilent Technology, Santa Clara, CA, USA), a DAWN HELEOS 18-angle laser light scattering detector (also known as multi-angle laser light scattering (MALLS) detector, Wyatt Technology, Santa Barbara, CA, USA) and an Optilab rex refractive index detector (Wyatt Technology, Santa Barbara, CA, USA). The detection wavelength of HELEOS was set at 658 nm. Separations were performed using serially connected size exclusion columns (1 Å, 5 Å, 1 3 Å and 1 4 Å Phenogel columns, 5 µm, mm, Phenomenex, Torrance, CA, USA) at 6 C using DMF containing.1 M LiBr as the mobile phase. The MALLS detector is calibrated using pure toluene with no need for calibration using polymer standards and can be used for the determination of the absolute molecular weights (MWs). The MWs of polymers were determined based on the dn/dc value of each polymer sample calculated offline by using the internal calibration system processed by the ASTRA V software (version , Wyatt Technology, Santa Barbara, CA,

3 USA). Infrared spectra were recorded on a Perkin Elmer 1 serial FTIR spectrophotometer calibrated with polystyrene film. Circular dichroism (CD) measurements were carried out on a JASCO J-7 or a JASCO 72 CD spectrometer. Ozone was produced by an OZV-8S ozone generator manufactured by Ozone Solutions Inc (Hull, IA, USA). Lyophilization was performed on a FreeZone lyophilizer (Labconco, Kansas City, MO, USA). Synthesis of poly( -(3-aminopropyl)- L -glutamate) (PAPG) PAPG 57 was synthesized through conjugation of 3-(N-Boc-amino)propanol to PLG 57 followed by deprotection, following the same protocol for the synthesis of PAHG (18 mg; 48% overall yield). The grafting efficiency was above 95%, as calculated from 1 H NMR (Supplementary Figure 1a). Synthesis of poly( -(5-aminopentanyl)- L -glutamate) (PAPTG 5 ) PAPTG 5 was synthesized through conjugation of 5-(N-Boc-amino)pentanol to PLG 57 followed by deprotection, following the same protocol for the synthesis of PAHG (5 mg, 38% yield). The grafting efficiency was above 9%, as calculated from 1 H NMR (Supplementary Fig.1b). Synthesis of poly( -((2-aminoethyl)ethyleneglycol)- L -glutamate) (PAEG 5 ) PAEG 5 was synthesized through conjugation of 2-(2 -(N-Boc)aminoethoxy)ethanol to PLG 57 followed by deprotection, following the same protocol for the synthesis of PAHG (8 mg, 46% overall yield) The grafting efficiency was above 9%, as calculated from 1 H NMR (Supplementary Fig. 1d). Synthesis of poly( -((2-aminoethyl)-heptaethyleneglycol)- L -glutamate) (PAHEG 5 ) PLG 5 (15 mg,.12 mmol) was dissolved in dry NMP (5 ml) under nitrogen. The solution was cooled in an ice bathe. O-(2-Azidoethyl)heptaethylene glycol (25 mg,.75 mmol), DMAP (15 mg, 1.23 mmol), BOP-Cl (175 mg,.7 mmol) and diisopropylethylamine (.1 ml, 1. mmol) were added to stirred solution under nitrogen. The ice bath was removed after the mixture was stirred for 2 min and the solution was stirred for an additional 6 h at room temperature followed

4 by another 4-day reaction at 45 o C. The mixture was dialysed (MWCO = 8, Da) against DI water for 24h to remove unreacted reagents. After lyophilization, the residue was dissolved in chloroform (3 ml) containing triphenylphosphine (8 mg,.3 mmol). The mixture was stirred for 2h at room temperature followed by the removal of the solvent under vacuum. DI water (1 ml) was added to the mixture and then the solution was filtered by regular filter paper. The filtrate was treated by ammonium hydroxyide (1 μl) for 4h, dialyzed in DI water and lyophilized to yield (PAHEG) 57 (18 mg, 23 % overall yield). The grafting efficiency was 9% as calculated from the 1 H NMR spectrum of (PAHEG) 57 (Supplementary Figure 1e). Synthesis of a 75mer of poly( L -Lys) (PLL) 75 Poly( -CBZ- L -lysine) (PZLL) was prepared at a monomer/initiator ratio of 75 by following literature reported procedure 41. The M n and PDI of the PZLL were 2.7 kg/mol and 1.5, respectively. The deprotection of PZLL was performed by following the standard procedure published previously 42 to give the desired PLL 75. Hydrolysis kinetics of the benzyl ester of PVBLG-1 PBS (1 L, 1 ) was lyophilized and followed by addition of D 2 O (1 ml) to prepare a PBS/D 2 O (1 ) solution. (PVBLG-1) 4 (1 mg) was dissolved in this 1 PBS/D 2 O solution and incubated at room temperature. The hydrolysis of the benzyl ester was monitored by 1 H NMR (Supplementary Figure 12). Wide-angle X-ray diffraction of PVBLG-1 Two-dimensional (2D) wide-angle x-ray diffraction (WAXD) experiments were performed on an Oxford Xcalibur diffractometer with an ONYX CCD area detector. The X-ray wavelength was.1542 nm for Cu Kα. One-dimensional (1D) profiles were obtained by integration from corresponding 2D WAXD images. The d-spacing was calibrated using silver behenate which has the first-order reflection (d 1 = Å) at q = 1.76 nm -1 (scattering vector q = (4πsin )/ with being half of the scattering angle).

5 D 2 O f&g i e&h a &d b c ppm Supplementary Figure S1. 1 H NMR of (PAPG) 57 in D 2 O

6 a d PAPTG 5 h b c e, f g (ppm) Supplementary Figure S2. 1 H NMR of (PAPTG) 5 in D 2 O

7 D 2 O a d f c b e ppm Supplementary Figure S3. 1 H NMR of (PAHG) 57 in D 2 O

8 [ ] (1-3 degrees cm 2 /dmol) (nm) ph1 ph4 ph1 ph -[ ] (cm 2 deg/dmol) * * Mean residue molar ellipticity at 222nm Supplementary Figure S4. The overlay of the CD spectra of (PAHG) 57 at ph 1, 4 and 1 in 25mM NaCl solutions. (polymer conc. =.28 mg/ml).

9 [ ] (1-3 degrees cm 2 /dmol) (nm).2mg/ml.6mg/ml.25mg/ml.5mg/ml 1.mg/ml [PAHG 6 ] (mg/ml) -[ ] (cm 2 deg/dmol) * * Mean residue molar ellipticity at 222nm Supplementary Figure S5. The overlay of the CD spectra of (PAHG) 57 at various concentrations ranging from.2 mg/ml to 1 mg/ml in aqueous solution. These experiments showed that (PAHG) 57 remains monomeric in aqueous solution.

10 [ ] (1-3 degrees cm 2 /dmol) C 15 C 25 C 35 C 45 C 55 C 7 C (nm) [Temp] ( o C) -[ ] (cm 2 deg/dmol) * * Mean residue molar ellipticity at 222nm Supplementary Figure S6. The overlay of the CD spectra of (PAHG) 57 in aqueous solution (ph=3, the concentration is.2 mg/ml.) at various temperatures ranging from 4 o C to 7 o C. These experiments showed that the helical stability of (PAHG) 57 was compromised at elevated temperature.

11 [ ] (1-3 degrees cm 2 /dmol) (nm) M.1M.3M 1.2M 3.1M 4.7M [NaCl] ([M]) -[ ] (cm 2 deg/dmol) * * Mean residue molar ellipticity at 222nm Supplementary Figure S7. The overlay of the CD spectra of (PAHG) 57 in aqueous solution (ph=3) with different NaCl concentrations ranging from to 4.7M (c =.28 mg/ml). These experiments showed that the helical conformation of (PAHG) 57 has unusual stability in salt solution.

12 [ ] (1-3 degrees cm 2 /dmol) M.5M 1.M 1.9M 2.8M 5.M 6.M (nm) [Urea] ([M]) -[ ] (cm 2 deg/dmol) * * Mean residue molar ellipticity at 222nm Supplementary Figure S8. The overlay of the CD spectra of (PAHG) 57 in aqueous solution with different urea concentrations ranging from to 6. M (c =.28 mg/ml). These experiments showed that the helical stability of (PAHG) 57 was compromised with increased concentration of urea, a helix-disrupting agent.

13 [ ] (1-3 degrees cm 2 /dmol) MSA volume fraction (nm) volume fraction MSA -[ ] (cm 2 deg/dmol) * volume fraction MSA -[ ] (cm 2 deg/dmol) * * Mean residue molar ellipticity at 222nm Supplementary Figure S9. The overlay of the CD spectra of (PAHG) 57 in aqueous solution with different MSA concentrations ranging from MSA volume fraction of % to 9% (c =.28 mg/ml). These experiments showed the two-state helix-coil transition of (PAHG) 57 with an isodichroic point around 23 nm 43 with the addition of MSA, a strong denaturing agent.

14 D 2 O (PAEG) 5 e d a f g c b pm (f1) Supplementary Figure S1. 1 H NMR of (PAEG) 5 in D 2 O

15 D 2 O e&f a&d g c b ppm Supplementary Figure S11. 1 H NMR of (PAHEG) 5 in D 2 O

16 ppm f b e d a g c i solvent solvent h N H O H N O O 2 a h g f e d c b H H H i Supplementary Figure S12. 1 H NMR of PVBLG 2 in CDCl 3 /TFA-d (v/v : 5/1)

17 1734 cm -1 ester 174 cm -1 aldehyde 1654 cm -1 amide I 1547 cm -1 amide II g f e d a c b ppm Supplementary Figure S13. The FTIR spectrum of poly( -(4-aldehydebenzyl)- L -glutamate) (PABLG) 4 in THF (top) and the 1 H NMR spectrum of (PABLG) 4 in TFA-d (bottom)

18 a b c D 2 O m j h d k e i g f e d a&f m g b&c h k i&j ppm Supplementary Figure S14. 1 H NMR of (PVBLG-1) 4 in D 2 O

19 [ ] (1-3 degrees cm 2 /dmol) ph1 ph3 ph6 ph9 ph ph -[ ] (cm 2 deg/dmol) * * Mean residue molar ellipticity at 222nm (nm) Supplementary Figure S15. The overlay of the CD spectra of (PVBLG-1) 6 at ph 3, 6, 9 and 1 in 25mM NaCl solutions (c =.28 mg/ml,). The [ ] 222 of (PVBLG-1) 6 (~ (cm 2 deg/dmol) is much larger than that of (PAHG) 57 (~ cm 2 deg/dmol, Supplementary Figure 13a), and remains unchanged between ph 1 and 1.

20 [ ] (1-3 degrees cm 2 /dmol) mg/ml.4mg/ml.45mg/ml 1.25mg/ml 2.5mg/ml Concentration (mg/ml) [ ] (cm 2 deg/dmol) * * Mean residue molar ellipticity at 222nm (nm) Supplementary Figure S16. The overlay of the CD spectra of (PVBLG-1) 6 at various concentrations ranging from.2 mg/ml to 2.5 mg/ml in aqueous solution. These experiments showed that (PVBLG-1) 6 stays monomeric in aqueous solution.

21 [ ] (1-3 degrees cm 2 /dmol) (nm) 4 o C 15 o C 25 o C 35 o C 45 o C 55 o C 7 o C Temperature ( o C) -[ ] (cm 2 deg/dmol) * * Mean residue molar ellipticity at 222nm Supplementary Figure S17. The overlay of the CD spectra of (PVBLG-1) 6 in aqueous solution (ph=3) at various temperatures ranging from 4 o C to 7 o C. These experiments showed that (PVBLG-1) 6 adopts helical structure with remarkable thermal stability. \

22 [ ] (1-3 degrees cm 2 /dmol) (nm) M.3M.5M 1.M NaCl(M) -[ ] (cm 2 deg/dmol) * * Mean residue molar ellipticity at 222nm Supplementary Figure S18. The overlay of the CD spectra of (PVBLG-1) 6 in aqueous solution with different NaCl concentrations ranging from to 1 M (c =.28 mg/ml). These experiments showed that the helical conformation of (PVBLG-1) 6 has unusual stability in salt solution.

23 [ ] (1-3 degrees cm 2 /dmol) (nm) M 1.M 1.5M 2.M 4.M 6.M Urea(M) -[ ] (cm 2 deg/dmol) * * Mean residue molar ellipticity at 222nm Supplementary Figure S19. The overlay of the CD spectra of (PVBLG-1) 6 in aqueous solution with different urea concentrations ranging from to 4 M (c =.28 mg/ml). These experiments showed that the helical stability of (PVBLG-1) 6 is remarkable, and remains nearly unchanged in 2 M urea and still has 76% of its original helical contents in a 6M urea solution

24 [ ] (1-3 degrees cm 2 /dmol) MSA volume fraction MSA Volume fraction -[ ] (cm 2 deg/dmol) * MSA Volume fraction -[ ] (cm 2 deg/dmol) * (nm) * Mean residue molar ellipticity at 222nm Supplementary Figure S2. The overlay of the CD spectra of (PVBLG-1) 6 in aqueous solution with different MSA concentrations ranging from MSA volume fraction of % to 9% (c =.28 mg/ml). These experiments showed the two-state helix-coil transition of (PVBLG-1) 6 with an isodichroic point around 23 nm 43 with the addition of MSA, a strong denaturing agent. (PVBLG-1) 6 has amazing stability against MSA and can adopt 58% of its original helical content with a MSA volume fraction of 69%.

25 [θ] ( 1-3 deg cm 2 dmol 1 ) 6 3 n Series2 =1 n Series1 =4 n Series4 =6 n Series3 =15 n Series5 =18 n Series6 = λ (nm) -[θ] 222 ( 1-3 deg cm 2 dmol 1 ) O H N O O n N H H 2 N Cl (PVBLG-1) n OH Degree of Polymerization n (n) Supplementary Figure S21. The overlay of the CD spectra of PVBLG-1 at ph =3 (left) and the plot of the [ ] 222 of (PVBLG-1) n versus of the degree of polymerization (n). We evaluated the MW dependence of the conformation of (PVBLG-1) n, where n is the degree of polymerization. As shown in Supplementary Figure 15 (left), (PVBLG-1) 1, with a -[ ] 222 value of 717 deg cm 2 dmol 1, has very low helical content. The FTIR analysis (Fig. 3i) confirmed its low helical content (1651 cm -1 and 1545 cm -1 ). The predominant conformation of (PVBLG-1) 1 is -sheet as indicated in its FTIR spectrum (1627 cm -1 and 152 cm -1, Fig. 3i), which agrees well with the conformation of short PBLG (2 mer) previously reported 44. The [ ] 222 value of PVBLG-1 s (helical contents) increases with the increase of PVBLG-1 MW until reaching a plateau of 32, to 33, deg cm 2 dmol 1 at around 6mer (Supplementary Figure S 29 (right), Table 1).

26 D 2 O e d a&f h g c&b i k ppm Supplementary Figure S22. 1 H NMR of (PVBLG-2) 45 in D 2 O

27 D 2 O e d a&f&h i g c&b ppm Supplementary Figure S23. 1 H NMR of (PVBLG-3) 45 in D 2 O

28 D 2 O i h e d a&f&g c&b ppm Supplementary Figure S24. 1 H NMR of (PVBLG-4) 4 in D 2 O

29 D 2 O g&h i e d a&f c&b ppm Supplementary Figure S25. 1 H NMR of (PVBLG-5) 4 in D 2 O

30 m, n&o e k g&i f j h d a c b ppm Supplementary Figure S26. 1 H NMR of (PVBLG-6) 2 in D 2 O

31 HO f' O e' O d HO j m k HO HO O i h N H OH g N H 2 f O Cl b O H N a e 28 c O O d b c a e N H e 12 g d a&f f i, j, m&k h c b ppm Supplementary Figure S27. 1 H NMR of (PVBLG-7) 4 in D 2 O

32 [θ] ( 1-3 deg cm 2 dmol 1 ) [θ] 222 = λ (nm) Supplementary Figure S28. CD analysis of (PVBLG-2) 45

33 [θ] ( 1-3 deg cm 2 dmol 1 ) [θ] 222 = λ (nm) Supplementary Figure S29. CD analysis of (PVBLG-3) 45

34 [θ] ( 1-3 deg cm 2 dmol 1 ) [θ] = λ (nm) Supplementary Figure S3. CD analysis of (PVBLG-4) 4

35 [θ] ( 1-3 deg cm 2 dmol 1 ) [θ] 222 = λ (nm) Supplementary Figure S31. CD analysis of (PVBLG-5) 4

36 [θ] ( 1-3 deg cm 2 dmol 1 ) [θ] = λ (nm) Supplementary Figure S32. CD analysis of (PVBLG-6) 44

37 [ ]( 1-3 deg cm dmol (nm) Supplementary Figure S33. CD analysis of (PVBLG-7) 4

38 Supplementary References 41. Lu, H. & Cheng, J. Hexamethyldisilazane-mediated controlled polymerization of alpha-amino acid N-carboxyanhydrides. J. Am. Chem. Soc. 129, (27). 42. Holowka, E.P., Sun, V.Z., Kamei, D.T. & Deming, T.J. Polyarginine segments in block copolypeptides drive both vesicular assembly and intracellular delivery. Nat. Mater. 6, (27). 43. Guerois, R. & López de la Paz, M. Protein design: methods and applications. (Humana Press, Totowa, N.J.; 26). 44. Klok, H.A., Langenwalter, J.F. & Lecommandoux, S. Self-assembly of peptide-based diblock oligomers. Macromolecules 33, (2).