L-Carnosine-Derived Fmoc-Tripeptides Forming ph- Sensitive and Proteolytically Stable Supramolecular

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1 Supporting Information: L-Carnosine-Derived Fmoc-Tripeptides Forming ph- Sensitive and Proteolytically Stable Supramolecular Hydrogels Rita Das Mahapatra, a Joykrishna Dey* a, and Richard G. Weiss b a Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur , India. b Department of Chemistry and Institute for Soft Matter Synthesis and Metrology, Georgetown University, Washington, DC , USA Synthesis of Tripeptides: An Fmoc-L-amino acid (1 eq.) was treated with N-hydroxysuccinimide (NHS) (1.1 eq.) in dry THF under a N 2 atmosphere. Then, a solution of 1,3-dicyclohexylcarbodiimide (DCC) (1.5 eq.) in dry THF was added drop wise at 0-5 o C to produce the corresponding NHS-ester. Then, the reaction was stirred at room temperature for 12 h. A white precipitate (dicyclohexyl urea (DCU)), a biproduct, was filtered off quickly from the mixture and these THF filtrate was reduced to ca. one-fourth of the initial volume. The THF solution of NHS-ester of the Fmoc-protected amino acid was added slowly to a cold (0-5 o C) aqueous solution of L- carnosine (1 eq.) in NaHCO 3 (2.2 eq.). (The ratio of water to THF was maintained at 1:9). After that, the reaction mixture was stirred for 24 h at room temperature, the THF part was evaporated leaving a white precipitate in the aqueous medium. The precipitate was filtered off and the filtrate was diluted with distilled water. Finally, 1(N) HCl was added dropwise to the filtrate to obtain a white precipitate of the tripeptide. The precipitate was collected by filtration and air dried. The product was purified by column chromatography using silica gel

2 ( mesh) and an 8:2 dichloromethane:methanol mixture as eluent. The purified product was characterized by FT-IR, NMR and high resolution mass spectrometry (HRMS). O X OH O N H O NHS, DCC THF, 0oC, 12 h O X O O N N H O O O 1. Carnosine 2. NaHCO3 3. THF/H2O, 24 h, rt O X H N O N H O O H N O OH N NH X = CH 3 CH(CH 3 ) 2 CH 2 CH(CH 3 ) 2 OH Fmoc- Ala-Car Fmoc- Val-Car Fmoc- Phe-Car Fmoc- Leu-Car Fmoc- Tyr-Car CH CH 3 CH 2 CH 3 Fmoc- Ile-Car CH 2 CH 2 SMe Fmoc- Met-Car Scheme S1. Scheme for syntheses of the hydrogelators.

3 Chemical Identification: Fmoc-Ala-Car: White solid (64 % yield), mp o C. FTIR (KBr, cm 1 ): 3402 (- OH stretching), 3297 (N-H stretching), 1683 (-C=O of COOH), 1645 (-C=O of amide), 1543 (N-H bending). ESI MS: Calculated mass [M+H] + m/z , obtained mass [M+H] + m/z , 1 H NMR (DMSO-d 6, δ ppm): (1H, d, 7.8 Hz), (1H, t, 7.2 Hz), (3H, m), (2H, t, 7.8 Hz), (1H, s), (1H, d, 7.2 Hz), (2H, m), (3H, m), (1H, s), (1H, t), (2H, t), (1H, t), (1H, m), (2H, t), (2H, t), (2H, t), (3H, d). Figure S1: HR-MS spectrum of Fmoc-Ala-Car.

4 Fmoc-Val-Car: White solid (75 % yield), mp o C, FTIR (KBr, cm 1 ): 3424 (-OH stretching), 3294 (N-H stretching), 1684 (-C=O of COOH), 1652 (-C=O of amide), 1541 (N-H bending). ESI MS: Calculated mass [M+H] + m/z , obtained mass [M+H] + m/z , 1 H NMR (DMSO-d 6, δ ppm): (1H, d, 7.8 Hz), (1H, t, 4.2 Hz), (2H, d, 7.8 Hz), (2H, t, 7.8 Hz), (1H, s), (3H, m), (2H, m), (1H, s), (1H, t), (2H, t), (1H, m), (1H, t), (2H, t), (2H, d), (2H, t), (1H, m), (6H, t). Figure S2: HR-MS spectrum of FVCAR. Figure S2: HR-MS spectrum of Fmoc-Val-Car.

5 Fmoc-Leu-Car: White solid (75 % yield), mp o C, FTIR (KBr, cm 1 ): 3405 (-OH stretching), 3297 (N-H stretching), 1691 (-C=O of COOH), 1648 (-C=O of amide), 1542 (N-H bending). ESI MS: Calculated mass [M+H]+ m/z , obtained mass [M+H]+ m/z , 1 H NMR (DMSO-d 6, δ ppm): (1H, d, 7.2 Hz), (1H, m), (2H, m), (2H, m), (1H, s), (4H, m), (1H, s), (1H, t), (2H, t), (1H, m), (1H, m), (2H, t), (6H, d). Figure S3: HR-MS spectrum of Fmoc-Leu-Car.

6 Fmoc-Phe-Car: White solid (75 % yield), mp o C, FTIR (KBr, cm 1 ): 3400 (- OH stretching), 3298 (N-H stretching), 1686 (-C=O of COOH), 1648 (-C=O of amide), 1541 (N-H bending). ESI MS: Calculated mass [M+H]+ m/z , obtained mass [M+H]+ m/z , 1 H NMR (DMSO-d 6, δ ppm): (2H, d, 7.8 Hz), (3H, m), (1H, s), (2H, m), (8H, m), (1H, s), (5H, m), (4H, t), (2H, t), (2H, m). Figure S4: HR-MS spectrum of Fmoc-Phe-Car.

7 Fmoc-Ile-Car: White solid (78 % yield), mp o C, FTIR (KBr, cm 1 ): 3405 (- OH stretching), 3293 (N-H stretching), 1684 (-C=O of COOH), 1647 (-C=O of amide), 1543 (N-H bending). ESI MS: Calculated mass [M+H]+ m/z , obtained mass [M+H]+ m/z , 1 H NMR (DMSO-d 6, δ ppm): (2H, d, 7.8 Hz), (2H, t), (1H, s), (2H, t), (2H, m), (1H, s), (2H, d), (2H, q), (1H, t), (2H, t), (2H, t), (2H, m), (3H, d), (1H, m), (2H, t), (3H, q). Figure S5: HR-MS spectrum of Fmoc-Ile-Car.

8 Fmoc-Met-Car: White solid (78 % yield), mp o C, FTIR (KBr, cm 1 ): 3405 (-OH stretching), 3293 (N-H stretching), 1684 (-C=O of COOH), 1647 (-C=O of amide), 1543 (N-H bending). ESI MS: Calculated mass [M+H]+ m/z , obtained mass [M+H]+ m/z , 1 H NMR (DMSO-d 6, δ ppm): (2H, d, 7.2 Hz), (1H, m, 7.2 Hz), (2H, m), (1H, m), (1H, d), (1H, s), (2H, m), (2H, m), (1H, s), (2H, d), (2H, m), (2H, t), (2H, m), (3H, d), (1H, m), (2H, t), (3H, q). Figure S6: HR-MS spectrum of Fmoc-Met-Car.

9 Fmoc-Tyr-Car: White solid (75 % yield), mp o C, FTIR (KBr, cm 1 ): 3400 (- OH stretching), 3317 (N-H stretching), 1685 (-C=O of COOH), 1652 (-C=O of amide), 1539 (N-H bending). ESI MS: Calculated mass [M+H]+ m/z , obtained mass [M+H]+ m/z , 1 H NMR (DMSO-d 6, δ ppm): (1H, d, 7.8 Hz), (1H, t), (2H, d), (1H, m), (1H, d), (2H, m), (2H, m), (1H, s), (1H, s), (2H, d), (1H, d), (1H, m), (3H, m), (3H, m), (3H, m), (1H, q), (2H, t).m), (3H, t), (3H, s), (2H, d). Boc-Val-Car: White solid (78 % yield), mp o C, FTIR (KBr, cm 1 ): 3400 (- OH stretching), 3322 (N-H stretching), 1681 (-C=O of COOH), 1645 (-C=O of amide), 1534 (N-H bending). ESI MS: Calculated mass [M+H]+ m/z , obtained mass [M+H]+ m/z , 1 H NMR (DMSO-d 6, δ ppm): (1H, s), (1H, s), (1H, d), (1H, t), (2H, t), (2H, d), 3.04 (1H, m), (2H, t), (9H, s).

10 Figure S7. 1 H-NMR spectrum of Fmoc-Val-Car (600 MHz) in DMSO-d 6 solvent. Figure S8. 1 H-NMR spectrum of Fmoc-Phe-Car (600 MHz) in DMSO-d 6 solvent.

11 Figure S9. Representative photographs of hydrogels of tripeptides: (A) Fmoc-Val-Car (ph 2), (B) Fmoc-Val-Car (ph 7), (C) Fmoc-Leu-Car (ph 2), (D) Fmoc-Leu-Car (ph 7), (E) Fmoc-Met-Car (ph 2), (F) Fmoc-Met-Car (ph 7), (G) Fmoc-Ile-Car, (H) Fmoc-Phe-Car (ph 2), (I) Fmoc-Phe-Car (ph 7), (j) Fmoc-Tyr-Car (ph 8). All hydrogels were prepared at a concentration equal to their respective CGC values. Determination of pk a : The acidity constants (pk a ) of the tripeptides were measured experimentally using a titration method. For this, 1 mg of the tripeptide was suspended in 1 ml Mili Q water and was dissolved by adding dilute NaOH solution. Solubilisation of the tripeptides required heating for 3-5 minutes (65-70 o C). Solutions of different peptides showed ph values > 10.5 and, therefore, it was assumed that all the peptides are ionized state at this ph. Then, dilution HCl was added dropwise to this alkaline solution, and ph values were recorded after each HCl addition. Addition of dilute HCl was continued until the solution reaches a ph < 2. After that, ph values were plotted against volume of HCl addition (Figure S10). Two flattened

12 regions were observed in each tripeptide curve, indicating the presence of two pk a values that are presented in the plots. Figure S10. ph titration curves of the tripeptides.

13 Figure S11. FESEM images of the xerogels prepared from the corresponding hydrogels of (A) Fmoc-Val-Car (ph 2), (B) Fmoc-Val-Car (ph 7), (C) Fmoc-Leu-Car (ph 7), (D) Fmoc- Ile-Car, (E) Fmoc-Phe-Car (ph 2), and (F) Fmoc-Phe-Car (ph 7). Table S1. Observed widths of fibrils from analysis of TEM images of the gelator solution (~10 4 M). Sample Width of fibrils (nm) ph 2 ph 5 ph 7 Fmoc-Val-Car 20 ± 5-7 ± 3 Fmoc-Leu-Car 10 ± ± 5 Fmoc-Met-Car 25 ± 5 25 ± ± 2 Fmoc-Ile-Car 18 ± 6 (unbuffered solution) Fmoc-Phe-Car 20 ± 3-10 ± 4 Fmoc-Tyr-Car 20 ± 2 (ph 8)

14 Figure S12. AFM images of the hydrogels of (A) Fmoc-Val-Car (ph 2), (B) Fmoc-Leu-Car (ph 7), (C) Fmoc-Met-Car (ph 7), (D) Fmoc-Ile-Car, (E) Fmoc-Phe-Car (ph 7), (F) Fmoc- Tyr-Car (ph 8); arrows indicate twisting of ribbons.

15 Figure S13. UV-vis spectra of 10 5 M hydrogelators in aqueous media. Path length of the cell was 1 cm. The gel was prepared at its CGC concentration.

16 Figure S14. Fluorescence spectra of 10 6 M tripeptide solutions and in the hydrogel ([gelator] = CGC) states; path length of the cuvette was 1 cm. Excitation wavelength was 262 nm.

17 Figure S15. Emission spectra of (a, b) Fmoc-Val-Car and (c, d) Fmoc-Phe-Car in solutions and in gels. The gel concentrations were equal to their CGC values. Path length of the cell was 1 cm; the excitation wavelength was 262 nm.

18 Figure S16. CD spectra of (a) Car (ph 7), (b) Fmoc-Val-Car (ph 2), (c) Fmoc-Leu-Car (ph 2), (d) Fmoc-Phe-Car (ph 2), (e) Fmoc-Tyr-Car (ph 8); path length of the cell was 1 mm. The spectra in solutions were measured at 10 4 M concentrations and the hydrogel concentrations were equal to CGC values.

19 Figure S17. HT data of the CD spectra for (a) Car (ph 7), (b) Fmoc-Val-Car (ph 2), (c) Fmoc-Leu-Car (ph 2), (d) Fmoc-Phe-Car (ph 2), (e) Fmoc-Tyr-Car (ph 8). The solution state spectra were measured with 10 4 M concentrations in all the cases and the hydrogel was prepared at its CGC value.

20 Figure S18. HT data of the CD spectra for (a) Fmoc-Phe-Car at ph 7 at different concentrations, (b) different gelators in the gel state (ph 7), (c) Fmoc-Phe-Car gel (ph 2) at different temperatures, (d) Fmoc-Val-Car gel (ph 2) at different temperatures.

21 Figure S19. Variation of G and G with frequency (f, Hz) for the hydrogels of (a) Fmoc- Val-Car (ph 2), (b) Fmoc-Leu-Car (ph 2), (c) Fmoc-Met-Car (ph 2), (d) Fmoc-Met-Car (ph 7), (e) Fmoc-Phe-Car (ph 2), (f) Fmoc-Phe-Car (ph 7), (g) Fmoc-Ile-Car, (h) Fmoc-Tyr-Car (ph 8). The hydrogels were prepared at their respective CGC values.

22 Figure S20. Variation of G and G with shear stress (σ) for the hydrogels of (a) Fmoc-Val- Car (ph 2), (b) Fmoc-Leu-Car (ph 2), (c) Fmoc-Met-Car (ph 2), (d) Fmoc-Met-Car (ph 7), (e) Fmoc-Phe-Car (ph 2), (f) Fmoc-Phe-Car (ph 7), (g) Fmoc-Ile-Car, (h) Fmoc-Tyr-Car (ph 8). The frequency in all the cases was 1 Hz and the hydrogels were prepared at their respective CGC values.

23 Figure S21. FTIR spectra of (a) Fmoc-Leu-Car, (b) Fmoc-Phe-Car, (c) Fmoc-Tyr-Car as neat solids and xerogels. The xerogels of Fmoc-Leu-Car and Fmoc-Phe-Car were prepared from ph 7 buffered solutions and the xerogel of Fmoc-Tyr-Car was prepared from ph 8 (20 mm).

24 Table S2. Important peaks in FTIR spectra of the neat solid and xerogel states of the peptides. Sample Fmoc-Phe-Car (solid) Fmoc-Phe-Car (xerogel prepared at ph 7) Fmoc-Leu-Car (solid) Fmoc-Leu-Car (xerogel at ph 7) Fmoc-Tyr-Car (solid) Fmoc-Tyr-Car (xerogel at ph 8) O-H stretching frequency (cm 1 ) N-H stretching frequency (cm 1 ) C=O stretching frequency (cm 1 ) N-H bending frequency (cm 1 ) (-COOH) 1648 (C=O of amide) (broad) 1639 (broad) 1538 (weak signal) (weak, -COOH) 1648 (C=O of amide) (weak signal) (weak, -COOH) (C=O of amide)

25 Figure S22. WAXD spectra of the xerogels of (a) Fmoc-Val-Car (ph 2), (b) Fmoc-Phe-Car (ph 2), (c) Fmoc-Phe-Car (ph 7), and (d) Fmoc-Leu-Car (ph 7) gelators. Table S3. Spacings (in Å) of the xerogels of Fmoc-Val-Car, Fmoc-Leu-Car, and Fmoc-Phe- Car at different ph values. Hydrogelators Peak positions Fmoc-Val-Car (ph 2) d = (2θ = 8.06 o ), d = 4.55 (2θ = o ), d = 3.54 (2θ = o ) Fmoc-Phe-Car (ph 2) d = (2θ = 7.32 o ), d = 5.13 (2θ = o ), d = 3.56 (2θ = o ) Fmoc-Phe-Car (ph 7) d = (2θ = 8.50 o ), d = 4.79 (2θ = 18.5 o ), d = 3.40 (2θ = 26.2 o ) Fmoc-Leu-Car (ph 7) d = (2θ = 8.50 o ), d = 4.52 (2θ = o ), d = 3.48 (2θ = o )

26 HPLC Measurements:

27 Figure S23. HPLC traces of the tripeptides after 0 h and 24 h with proteinase K enzyme in HEPES buffer of ph 7.4.

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