a [M+H] + b [M+H] + [M+Na] + [M+K] + 1100 1200 1300 1400 1500 1600 1700 1800 Mass (m/z) c [M+H] + 900 1000 1100 1200 1300 1400 1500 1600 Mass (m/z) d [M+H] + 1160 1180 1200 1220 1240 1260 1280 Mass (m/z) 420 440 460 480 500 Mass (m/z) e f [M+H] + [M+Na] + [M+K] + [M+H] + [M+Na] + [M+K] + 500 520 540 560 580 600 Mass (m/z) 600 640 680 720 760 800 Mass (m/z) Supplementary Figure S1 ǀ Characterization of molecular weight identity. MALDI- TOF mass spectra of molecule 2 (a), molecule 3 (b), molecule 5 (c), molecule 8 (d), molecule 9 (e), and molecule 10 (f). 1
2.0 Absorption 1.5 1.0 0.5 0.0 3480 cm-1 3480 cm-1 2600 2800 3000 3200 3400 3600 Wavenumber ( cm -1 ) Supplementary Figure S2 ǀ FT-IR spectra (2500-3700 cm -1 ). Black-solid line from dried solution of self-assembled 1 in methanol and water (2 : 3) and Red-dash line from dried solution of self-assembled 2 in water. 2
44000.8801 2 UV intensity 0.FL intensity a b 0.00350404505003c d 0.1240.0620.00040.080.0120.0161Iq -2 ( KHz nm 2 ).Intensity0Wavelength ( nm ) 1q ( nm -1 ) D app 10-7 ( cm -2 / s) 0300Diameter ( nm ) q 2 10 10 ( cm -2 ) Supplementary Figure S3 ǀ Characterization of supramolecular nanotubes of 1. (a) Absorption and emission spectra of 0.01 wt% of 1 in CHCl 3 (black and solid line) and mixed solution (H 2 O : MeOH = 7 : 3 red and dashed line). (b) Size distribution graphs of 0.01 wt% of 1 in MeOH-Water (30 % MeOH) solution and 0.01 wt% of 2 in aqueous solution. (c) Kratky plot and linear fit of 0.01 wt% of 1 in MeOH-Water (30 % MeOH) solution was confirmed the cylindrical micelle in solution. (d) Angular dependence of the apparent diffusion coefficient of 0.01 wt% of 1, Dapp ~ 0.024 for the cylindrical micelle in solution. 10001000023
7523.0050.0100.01510 11 Intensity 20 a 54321b0Iq -2 ( KHz nm 2 ) 6ln( Iq / nm -1 ) -4.5 1-5.0-5.5 0.0000 0.0001 0.0002 0.0003 q 2 ( nm -2 ) D app 10-7 ( cm -2 / s) 0.0.600.451 2 q ( nm -1 ) c d q ( nm -1 ) q 2 10 10 ( cm -2 ) Supplementary Figure S4 ǀ Characterization of supramolecular nanofibers of 2. (a) AFM image of 2 in aqueous solution (0.01 wt%) transformed on mica. (b) Small-angle X-ray diffraction pattern of 30 wt% of 2 in aqueous solution showed tetragonal columnar structure with lattice constants a = 9.4 nm implying that the cross section of a cylinder consists of 14 molecules. (c) The linear fit of Kratky plot and the Holzer plot in the inset of 0.01 wt% of 2 in aqueous solution was confirmed the cylindrical micelle in solution. (d) Angular dependence of the apparent diffusion coefficient of 0.01 wt% of 2, Dapp ~ 0.035 for the cylindrical micelle in aqueous solution. 4
a b c Viability % Supplementary Figure S5 ǀ Cell culture in gels and on tissue-culture plastic. (a) Optical images of C2C12 cells in nanofibers (Left: immediately after seeding, Middle: after 1 day, Right: after 2 days). (b) The viability of C2C12 cells using trypan blue assay in three different conditions 1 wt% (blue), 1.5 wt% (red) and 2 wt% (green) for 5 days. (c) The control image of C2C12 cells grown as a monolayer on tissue-culture plastic (2D). The scale bars in all images are 100 μm. The error bars in b mean the standard deviations of the measured cell viability from four replicate experiments. 5
Supplementary Figure S6 ǀ Optical images of grown C2C12 cells. The control image of C2C12 cells grown in 4 wt% of collagen gel (a), 0.5 wt% of 2 in the presence of 10 mol% of 3 (b), and 1 wt% of 2 in the presence of 10 mol% of 3 (c). The scale bars in all images are 100 μm. 6
Supplementary Figure S7 ǀ Cell grown in gel and released from gel. Optical images of C2C12 cells in nanofiber gel with the concentration of 2 1 wt% (a), 1.5 wt% (b), and 2 wt% (c). The grown image of C2C12 cells after release from 1 wt% (d), 1.5 wt% (e), and 2 wt% (f) of 2 in gel on culture dish for 8 hours. The scale bars in all image are 100 μm. 7
Supplementary Figure S8 ǀ Movement of cells after release from gel. Time-lapse optical images of C2C12 cells as cooling the gel with a cold cell media (scale bar in all images are 100 μm). 8
Supplementary Methods Materials and instruments Materials. 1, 2-diamino benzene, triethylene glycol monomethyl ether, diethylene glycol monomethyl ether, and 4-hydroxy benzaldehyde from Aldrich were used as received. NaH (60 %) and p-toluenesulfonyl chloride (98 %) from TCI and Tokyo Kasei were used as received. Unless otherwise indicated, all starting materials were obtained from commercial suppliers (Aldrich, TCI, Acros, etc.) and were used without purification. Hexane, dichloromethane, and ethyl acetate were distilled before use. Visualization was accomplished with UV light, iodine vapor. Flash chromatography was carried out with Silica Gel 60 (230-400 mesh) from EM Science. Dry THF was obtained by vacuum transfer from sodium and benzophenone. The synthesis of compound 1 based on diethylene oxide 2 nd generation dendrimer has been reported elsewhere. 31 2,5- dibromophenol, 32 4-biphenylboronic acid, 40 mono-tosylated tetraethylene glycol, 41 and tosylated tri ethylene 2 nd generation dendrimer 42 were prepared according to the similar procedures described previously. Instruments. 1 H NMR and 13 C NMR spectra were recorded from CDCl 3 or DMSO solutions on a Bruker AM 300 spectrometer. The purity of the products was checked by thin-layer chromatography (TLC; Merck, silica gel 60). Recycling preparative highpressure chromatography (HPLC) was performed for further purification by using HITACHI model pump L-7110, JAI model UV detector 310 and JAI model RI detector RI-7S. MALDI TOF-MS spectroscopy (MALDI-TOF-MS) was performed on a Bruker Microflex LRF20 using α-cyano-4-hydroxy cinnamic acid (CHCA) as matrix. The 9
dynamic light scattering experiment was performed by DLS-8000 from Otsuka Electronics with a 632.8 nm He-Ne laser. The Uv/vis spectra was obtained from a Hitachi U-2900 Spectrophotometer. The fluorescence spectra was obtained from a Hitachi F-7000 Fluorescence Spectrophotometer. The rheology experiment was performed by ARES-LS from TA Instrument. The transmission electron microscopy (TEM) was performed at 120 kv using JEOL-JEM 2100. Synthesis of compound 2 Reagents and conditions : (a) K 2 CO 3, CH 3 CN, reflux; (b) 4-biphenylboronic acid, 2M K 2 CO 3, tetrakistriphenylphosphine palladium(0), THF, reflux. Scheme 1. Synthesis of compound 2. 10
Compound 5. Compound 4 (0.2 g, 0.54 mmol), Tosylated tri-ethylene 2 nd generation dendrimer (0.5 g, 0.5 mmol) and K 2 CO 3 (1.1g, 7.0 mmol) were dissolved in 20 ml of CH 3 CN. The mixture was heated at reflux for 48 hours and then cooled to room temperature. The solvent was removed in a rotary evaporator, and the resulting mixture was poured into water and extracted with ethyl acetate. The ethyl acetate solution was dried over anhydrous magnesium sulfate and filtered. After the solvent was removed in a rotary evaporator, the crude products were purified by column chromatography (silica gel) using methanol : ethyl acetate (1:8 v/v) as eluent to yield 70% (0.43 g) of colorless liquid. Compound 5. 1 H-NMR (300MHz, CDCl 3, δ, ppm): 10.49 (s, 1H), 8.06 (d, J = 7.5 Hz, 2H), 7.2 (m, 2H), 6.95 (d, J = 7.5 Hz, 2H), 4.05 (d, J = 4.8 Hz, 2H), 3.62-3.35 (m, 78H), 2.39-2.04 (m, 3H). 13 C-NMR (100 MHz, CDCl 3, δ, ppm): 160.9, 135.4, 129.1, 128.5, 127.5, 123.6, 122.1, 114.9, 71.7, 70.6, 70.3, 69.8, 69.7,67.6, 54.7, 40.2. Compound 2. Compound 5 (0.4 g, 0.33 mmol) and 4-biphenylboronic acid (0.26 g, 1.31 mmol, 4 eq.) were dissolved in degassed THF (30 ml). Degassed 2M aqueous K 2 CO 3 (30 ml) was added to the solution and then tetrakis(triphenyl-phosphine) palladium (0) (0.06 g, 0.05 mmol, 0.15 eq.) was added. The mixture was heated at reflux for 48 hours with vigorous stirring under argon. Cooled to room temperature, the layers were separated, and the aqueous layer was then washed twice with methylene chloride. The combined organic layer were dried over anhydrous magnesium sulfate and filtered. The solvent was removed in a rotary evaporator, and the crude product was 11
purified by column chromatography (silica gel) using ethyl acetate : methanol (8:1 v/v) as eluent and then further purified by prep-hplc to yield 0.34 g (75 %) of colorless liquid. Compound 2. 1 H-NMR (300 MHz, CDCl 3, δ, ppm): 10.15 (s, 1H), 8.30 (d, J = 8.6 Hz, 2H), 7.79-7.38 (m, 20H), 7.06 (d, J = 8.6 Hz, 2H), 4.05 (d, J = 7.5 Hz, 2H), 3.62-3.35 (m, 78H), 2.40-2.04 (m, 3H).; 13 C-NMR (100 MHz, CDCl 3, δ, ppm): 157.3, 141.8, 136.7, 135.4, 129.1, 128.5, 127.9, 127.7, 123.4, 122.1, 114.7, 71.9, 70.6, 70.5, 69.8, 69.7,67.6, 54.7, 40.2; MALDI-TOF-MS [M+H] +, [M+Na] + and [M+K] + calcd. for C 69 H 90 N 2 O 15 : m/z 1364.67, 1387.67 and 1403.67; Found: 1364.78, 1388.06 and 1403.11. 12
Synthesis of compound 3 Reagents and conditions : (a) K 2 CO 3, ACN, reflux; (b) I TsCl, Et 3 N, DCM; II NaN 3, DMF 110 o C; (c) I Pa/C, H 2, EtOH; II Succinic anhydride, Dioxane 80 o C; (d) 4-biphenylboronic acid, 2M K 2 CO 3, tetrakistriphenylphosphine palladium(0), THF, reflux. (e) I 4-Nitrophenol, CHCl 3 ; II L-RGD, DIPEA, DCM, DMF. Scheme 2. Synthesis of compounds 3. Compound 7. Compound 6 (0.4 g, 1.6 mmol), mono-tosylated tetraethylene glycol (0.44 g, 1.4 mmol) and K 2 CO 3 (1.1g, 7.0 mmol) were dissolved in 20 ml of CH 3 CN. The mixture was heated at reflux for 12 hours and then cooled to room temperature. The 13
solvent was removed in a rotary evaporator, and the resulting mixture was poured into water and extracted with ethyl acetate. The ethyl acetate solution was dried over anhydrous magnesium sulfate and filtered. After the solvent was removed in a rotary evaporator, the crude products were purified by column chromatography (silica gel) using ethyl acetate as eluent to yield 70 % (0.37 g) of colorless liquid. Compound 7. 1 H-NMR (300MHz, CDCl 3, δ, ppm): 7.37 (d, J = 8 Hz, 1H), 7.05 (s, 1H), 6.96 (d, J = 8 Hz, 1H), 4.15 (t, J = 4.6 Hz, 2H), 3.90 (t, J = 4.6 Hz, 2H), 3.79-3.65 (m, 12H) ; 13 C-NMR (100 MHz, CDCl 3, δ, ppm): 141.9, 133.9, 132.0, 131.6, 121.8, 120.7, 71.0, 70.7, 61.5. Compound 8. P-Toluenesulfonylchloride (0.38 g, 2 mmol) was added to Compound 7 (0.57 g, 1.34 mmol) and triethylamine 4 ml in CH 2 Cl 2 20 ml at room temperature. The reaction mixture was stirred at room temperature for 6 h. Water (20 ml) was then added to the reaction mixture, the organic layer was separated and the aqueous layer was extracted with CH 2 Cl 2 (3 20 ml). The combined organic layers were dried over MgSO 4 and concentrated under vacuum. The residue was then purified by column chromatography on silica gel using ethyl acetate as eluent to yield 90 % (0.7 g) of colorless liquid. A solution of Sodium azide (86 mg, 1.32 mmol) in ethanol 20 ml was added at room temperature. The reaction mixture was stirred overnight at 70 C. The reaction was then quenched by addition of water (50 ml), and concentrated under vacuum. The aqueous layer was extracted with ethyl acetate (3 50 ml). The combined organic layers were then dried over MgSO 4 and concentrated under vacuum. The residue was then purified by column chromatography on silica gel using ethyl acetate as eluent to yield 95 % (0.52 g) of colorless liquid. 14
Compound 8. 1 H-NMR (300MHz, CDCl 3, δ, ppm): 7.38 (d, J = 8 Hz, 1H), 7.1 (s, 1H), 6.96 (d, J = 8 Hz, 1H), 4.15 (t, J = 4.6 Hz, 2H), 3.90 (t, J = 4.6 Hz, 2H), 3.79-3.65 (m, 10H), 3.37 (t, J = 4.6 Hz, 2H); 13 C-NMR (100 MHz, CDCl 3, δ, ppm): 141.6, 133.8, 132.0, 131.4, 121.6, 120.5, 71.1, 70.6, 44.7. Compound 9. To a solution of 8 (0.46 g, 1 mmol) in 12 ml of methanol, 10% Pd/C (55 mg) was added. The reaction suspension was stirred at room temperature for 12 h under hydrogen. After removing the Pd/C by filtration, the filtrate was concentrated to dryness. The residue was purified by silica-gel column chromatography using chloroform/methanol = 7/1, v/v) as eluent to give colerless liquid. The liquid was dissolved in 5 ml of dioxane and then slowly added to a solution of Succinic anhydride (1 mmol) in dioxane 10 ml. The mixture was heated to 80 o C and stirred 6 h. The reaction was then quenched by addition of water (50 ml), and concentrated under vacuum. The aqueous layer was extracted with ethyl acetate (3 50 ml). The combined organic layers were then dried over MgSO 4 and concentrated under vacuum. The residue was then purified by flash column chromatography on silica gel using chloroform/methanol = 7/1, v/v) as eluent to give 91 % (0.48 g) colerless liquid. Compound 9. 1 H-NMR (300MHz, CDCl 3, δ, ppm): 7.35 (d, J = 8 Hz, 1H), 7.04 (s, 1H), 6.90 (d, J = 8 Hz, 1H), 4.15 (t, J = 4.6 Hz, 2H), 3.88 (t, J = 4.6 Hz, 2H), 3.72-3.60 (m, 10H), 3.41 (t, J = 4.5 Hz, 2H), 2.61 (m, 2H), 2.46 (m, 2H); 13 C-NMR (100 MHz, CDCl 3, δ, ppm): 173.8, 173.1, 141.9, 133.7, 132.0, 131.6, 121.8, 120.7, 71.0, 70.7, 43.3, 30.5, 15
29.3. Compound 10. Compound 9 (0.16 g, 0.3 mmol) and 4-biphenylboronic acid (0.13 g, 1.2 mmol, 4 eq.) were dissolved in degassed THF (30 ml). Degassed 2M aqueous K 2 CO 3 (30 ml) was added to the solution and then tetrakis(triphenyl-phosphine) palladium (0) (0.06 g, 0.05 mmol, 0.15 eq.) was added. The mixture was heated and reflux for 48 hours with vigorous stirring under argon. Cooled to room temperature, the layers were separated, and the aqueous layer was then washed twice with methylene chloride. The combined organic layer were dried over anhydrous magnesium sulfate and filtered. The solvent was removed in a rotary evaporator, and the crude product was purified by column chromatography (silica gel) using chloroform/methanol = 5/1, v/v) as eluent to give 80 % (0.16 g) colerless liquid. Compound 10. 1 H-NMR (300 MHz, CDCl 3, δ, ppm): 7.73-7.45 (m, 20H), 6.85 (d, J = 8 Hz, 1H), 4.23 (t, J = 4.6 Hz, 2H), 3.82 (t, J = 4.6 Hz, 2H), 3.68-3.35 (m, 12H), 2.82-2.50 (m, 4H); 13 C-NMR (100 MHz, CDCl 3, δ, ppm): 173.8, 173.1, 156.3, 141.1, 139.7, 137.5, 131.6, 130.1, 129.7, 128.9, 128.0, 127.6, 127.4, 126.7, 120.1, 71.9, 70.6, 69.5, 43.5, 30.3, 29.1. Compounds 3. The compound 10 (50 mg, 0.063 mmol), DCC (14.3 mg) and 4- nitrophenol (8.8 mg, 0.063 mmol) was dissolved in CHCl 3 (5 ml). After stirring for 12 hours, the solvent was removed in a rotary evaporator, and the resulting mixture was 16
poured into water and extracted with ethyl acetate. The ethyl acetate solution was dried over anhydrous magnesium sulfate and filtered. After the solvent was removed in a rotary evaporator, the crude products were purified by column chromatography (silica gel) using ethyl acetate as eluent to yield colorless liquid. Then, the liquid was dissolved in anhydrous DMF (5 ml). To the solution L-RGD (38 mg, 0.063 mmol), DIPEA (25 mg) were added. After stirring for 24 hours, the solvent was removed. The residue was recrystallized by methanol gave 3 as a white solid. MALDI-TOF-MS [M+H] + calcd. for C 69 H 90 N 2 O 15 : m/z 1263; Found: 1262.62. 17
Supplementary References 40. Kim, B.-S., Hong, D.-J., Bae, J., Lee, M. Controlled Self-Assembly of Carbohydrate Conjugate Rod Amphiphiles for Supramolecular Multivalent Ligands. J. Am. Chem. Soc. 127, 16333-16337 (2005). 41. Sanders, B. C. Friscourt, F., Ledin, P. A., Mbua, N. E., Arumugam, S., Guo, J., Boltje, T. J., Popik, V. V., Boon, G.-J. J. Am. Chem. Soc. 133, 949-957 (2011). 43. Kim, J.-K., Lee, E. Huang, Z., Lee, M. Nanorings from Self-Assembly of Amphiphilic Molecular Dumbbells. J. Am. Chem. Soc. 128, 14022-14023 (2006). 18