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1 Supporting Information Self-Assembly of 1-D n-type anostructures based on aphthalene Diimide-appended Dipeptides Hui Shao, Tuan guyen, atalie C. Romano, David A. Modarelli and Jon R. Parquette*, Department of Chemistry, The hio State University, 100 W. 18 th Ave. Columbus, hio Department of Chemistry and The Center for Laser and ptical Spectroscopy, Knight Chemical Laboratory, The University of Akron, Akron, hio S1

2 Content Solid phase peptide synthesis schemes S3 Experimental section S4-8 AFM images of dipeptide A S9 AFM images of dipeptide B S10 TEM images of dipeptide C S11 Cryo-TEM images of dipeptides S12 MR spectra of dipeptides A, B and C in D 2 S13 FTIR spectra of dipeptides A, B and C in D 2 and TFE S14 UV spectra of dipeptides A, B and C in H 2 and TFE S15 CD spectra of dipeptides A, B and C at varying temperatures S16 Fluorescence spectra of dipeptides A, B and C in H 2 and TFE S17 Time-resolved fluorescence spectra of dipeptides in water (monomer) S18 Time-resolved fluorescence spectra of dipeptides in water (excimer) S19 Time-resolved fluorescence anisotropy spectra of dipeptides S20 XRD patterns of DI-dipeptides S21 A proposed model for formation of helical nanofibers by dipeptide A S22 A proposed model for formation of nanoribbons by dipeptide B S23 HPLC traces of purified DI-dipeptides S24 1 H MR, 13 C MR and ESI-MS data for dipeptides S25-34 S2

3 H 2 DMF, reflux 40% 1 Boc H Boc H Boc H H 2 Fmoc H H 4% TFA/CH 2 Cl 2 H Fmoc 1, DMF 5 min H HBTU/HBt Fmoc H H % piperidine DMF % TFA/H 2 H 2 H H 2 H Mtt H 2 Dipeptide C Boc H Boc H H % piperidine/dmf 2. Ac 2 /DIPEA 3. 4% TFA/CH 2 Cl 2, 5 min H H H 2 1, DMF HBTU/HBt H H 95 % TFA/H 2 H H H 2 Dipeptide A Mtt H H 2 Fmoc H H 1. 20% piperidine/dmf 2. Ac 2 /DIPEA 3. 4% TFA/CH 2 Cl 2, 5 min H Boc H H 1, DMF HBTU/HBt H Boc H H 95 % TFA/H 2 H Boc H H H 2 Dipeptide B H 2 Scheme 1. Synthesis of DI-dipeptides via on-resin modification of side chain method S3

4 Experimental section. General Methods. Fourier transform-infrared (FTIR) were performed on FTIR spectrometer (Thermo icolet, Madison, WI). Circular dichroism (CD) spectra were taken with an AVIV 202 CD spectrometer. Atomic force microscopy (AFM) was conducted in tapping mode. Matrix-assisted laser desorption ionization-time of flight MS (MALDI-TF MS) spectrometry was performed using 2,5-dihydroxybenzoic acid as the matrix in tetrahydrofuran (THF). All fluorescence spectroscopy were performed in a Perkin-Elmer LS-50B using a cuvette with 1 mm or 1cm pass length at 25 o C. Transmission Electron Microscopy (TEM) was performed with Technai G2 Spirit instrument operating at 80 kv. All reactions were performed under an argon or nitrogen atmosphere. 1 H MR were recorded at 400 or 500 MHz and 13 C MR spectra at 100 or 125 MHz on a Bruker DPX-400 or DPX-500 instrument as indicated. Dimethylformamide (DMF) was dried by distillation from MgS 4. Chromatographic separations were performed on silica gel 60 ( mesh, 60 Å) using the indicated solvents. All water used for sample solutions was HPLC grade and passed through membrane filter (0.02 µm) before use. Synthesis of -butyl-1,4,5,8-naphthalenetetracarboxylic acid monoanhydride (1) 1,4,5,8-aphthalenetetracarboxylic dianhydride (9.65 g, 35.9 mmol) was dissolved in 300 ml DMF and degassed with nitrogen. The reaction mixture was heated to 140 o C and A 50 ml DMF solution of n-butylamine (3.5 ml, 2.59 g, 35.3 mmol) was added dropwise over 2 h. The reaction was heated to reflux for 18 h, then cooled in the freezer for 1 h. The precipitate was filtered off and the filtrate was condensed in vacuo. The dark brown residue was purified with flash column chromatography (CHCl 3 ) to afford monoanhydride 1 as off-white solid (4.72 g, 40 %). m.p. (CHCl 3 ) o C; R f 0.25 (CHCl 3 ); 1 H MR (400 MHz, CDCl 3 ) δ 1.00 (t, J = 7.2 Hz, 3H), 1.46 (m, 2H), 1.73 (m, 2H), 4.21 (t, J = 7.6 Hz, 2H), 8.82 (s, 4H). S4

5 Peptide Preparation All DI-dipeptides were manually prepared using Fmoc/t-Bu solid-phase peptide synthesis on rink amide resin (loading 0.35mmol/g). Amide-coupling steps were accomplished with standard techniques for all amino acids: Fmoc-amino acid, 1,3- diisopropylcarbodiimide (DIC), and 1-hydroxybenzotriazole (HBt) (500 mol% each relative to resin) in 1:1 DMF/DCM for 1 h. Piperidine (20%) in DMF was used for Fmoc removal. The DI-dipeptides were cleaved from the resin by the treatment with TFA/water/triethylsilane ( 95 / 2.5 / 2.5) at room temperature for 2 h. The crude peptides were precipitated with cold dimethylether and purified by reversed-phased HPLC on preparative Varian Dynamax C18 column eluting with a linear gradient of CH 3 C/water (30/70 to 100/0 over 50 minutes, 0.1 % TFA) and stored as lyophilized powers at 0 C. Peptide purity was assessed by analytical reverse-phase HPLC and identity confirmed using MALDI-TF mass spectrometry. The attachment of DI on the solid support 1 The resin bearing peptides was treated with CH 2 Cl 2 /TFA (96/4) for 5 min to remove the lysine MTT protecting group followed by washing sequentially with CH 2 Cl 2, DMF, DMF/DIPEA (95/5), and again with DMF. 2 ml DMF solution of monoanhydride 1 (3 eq.) and DIPEA (4 eq) was added to the resin. The suspension was shaken for 30 min followed by the addition of HBTU/HBt (0.4 M DMF solution, 3 eq). The reaction mixture was shaken for 12 h at room temperature and then filtered through a fritted syringe. The resin was washed thoroughly (3 x DMF, 3 x EtH, 3 x CH 2 Cl 2 ) and submitted for the next step. Circular Dichroism (CD) Spectroscopy Measurement. CD spectra were recorded on an AVIV 202 CD spectrometer under a nitrogen atmosphere. Experiments were performed in a quartz cell with a 1 cm or 1 mm path length over the range of nm at 25 o C. S5

6 Fourier Transform Infrared (FTIR) Spectroscopy Measurement All FTIR spectra were collected on a icolet FTIR spectrometer at ambient temperature. The instrument was continuously purged with C 2 -free dry air. Spectra were recorded between 1700 and 1600 cm -1 at a resolution of 4 cm -1, and a total of 64 scans were averaged. Samples for FTIR were dissolved in D 2 or TFE (about 10 mg/ml) and analyzed in a transmission cell having CaF 2 windows and a µm path length. Atomic Force Microscopy (AFM) Measurement. The AFM images were collected on a anoscope IIIa device at ambient temperature in tapping mode using silicon tips (SC14/AIBS, MikroMasch). 10 µl of the sample solution (250 µm) was diluted 10 folds and then was place on freshly cleaved mica. After adsorption for 30 min under moist conditions, the excess solution was removed by absorption onto filter paper. The resultant substrates were rinsed with solvent (50 ul) twice to remove the loosely bound peptide, and the samples were stored in a desiccator in vacuo for 1 h before imaging. The scanning speed was at a line frequency of 1.0 Hz, and the original images were sampled at a resolution of 512 x 512 pixels. Electron Microscopy Measurement egative Stain TEM 10 µl drops of peptide solutions after incubation (at least 2 h) were applied to carboncoated copper grid (Ted Pella, Inc.) for 2 min and. After removal the excess solution with filter paper, the grid was floated on 10 µl drops of 2 % wt uranyl acetate solution for negative stain for 2 min. The excess solution was removed by filter paper. The dried specimen was observed with Technai G2 Spirit instrument operating at 80 kev. The data were analyzed with Image pro software. Electron Microscopy Measurement Cryo-TEM 10 µl drops of peptide solutions (1mM) were palced to carbon-coated copper grid (Ted Pella, Inc.), blotted with filter paper from behind, and plunged into ethane slurry maintained at liquid nitrogen temperature. The samples were transferred to a cryo-holder precooled to -175 o C before inserting into the TF30He Ploara G2 (FEI company) electron S6

7 cryo microscope operating at 120 kev. The temperature of the sample holder was maintained at -175 o C during imaging to avoid sublimation of vitreous water. X-ray Diffraction Measurement Dipeptide samples were dissolved in water at a concentration of 10 mg/ml to produce assemblies after 24 h incubation. The assemblies were isolated using Ultracentrifuge (80,000 rpm) for 2h. The bottom assemblies were collected and spread on power XRD sample holder. o assembly was found for dipeptide C sample solution. A droplet of dipeptide C solution was placed on the sample holder and air dried. Powder XRD patterns were recorded on a Riguka powder diffractometer operating at 40 kv and 25 ma, using CuK α radiation (λ = Å). Data were collected from 3 o to 50 o with a sampling interval of 0.01 o per step and a counting rate of 2 s per step. Fluorescence Spectroscopy Measurement Aqueous DI-dipeptide solutions (250 µm) were prepared by dilution from 10 mm solution and equilibrated for 24 h. The fluorescence emission spectra of DI were measured with excitation at 380 nm. Time-Resolved Fluorescence and Anisotropy Experiments Time-resolved fluorescence and time-resolved anisotropy experiments were performed using the time-correlated single-photon counting (TCSPC) technique. The instrument used in this work utilized the pulses from a frequency doubled, Coherent cavity dumped 702 dye laser pumped by the 527 nm output of a CW mode-locked d:ylf laser. The fluorescence signal was detected at 54.7 o for lifetime measurements with an emission polarizer and depolarizer, using a Hamamatsu R3809U-51 red-sensitive multichannel plate detector (MCP). Anisotropy experiments were collected with detection polarization of 0 and 90 relative to the excitation (perpendicular) laser. Data collection was accomplished with an Edinburgh Instruments data collection system, and analysis with a PicoQuant FluoFit decay analysis program. Time resolution on this instrument is estimated at ~ 7-9 ps after reconvolution. Time-resolved and time-resolved anisotropy decays were fit such that values of χ were obtained. Error limits in these S7

8 measurements are estimated at ± 10%. All TCSPC experiments were run with argonsaturated deionized water solutions with optical densities of 0.15 at the 305 nm excitation wavelength. Monomer decays were monitored at 411 nm (A), 418 nm (B) and 393 nm (C), while all excimer decays were monitored at 500 nm and at 510 nm for anisotropy data. S8

Figure S1. Self-assembly of dipeptide A in water solution (250 µm) on mica surface. (a) tapping mode AFM image showing the formation of 1-D helical nanofibers.

9 Figure S1. Self-assembly of dipeptide A in water solution (250 µm) on mica surface. (a) tapping mode AFM image showing the formation of 1-D helical nanofibers. (b) Section analysis along the line in AFM image showing the height (7 ± 1nm) of self-assembled nanofibers. (c) 3D surface plot of AFM image of dipeptide A showing regular height fluctuations. S9

Figure S2. Self-assembly of dipeptide B in water solution (250 µm) on mica surface. (a) tapping mode AFM image showing the formation of 1-D twisted nanoribbons. (b) AFM phase image of dipeptide B.

10 Figure S2. Self-assembly of dipeptide B in water solution (250 µm) on mica surface. (a) tapping mode AFM image showing the formation of 1-D twisted nanoribbons. (b) AFM phase image of dipeptide B. (c) Section analysis along the line in AFM image showing the height of the flat sheet reign (6 nm) and height of the twist (16 nm). (d) Schematic representation of the structure parameters of nanoribbons. S10

11 Figure S3. TEM image of dipeptide C in water at varying concentrations. (a) 250 µm showing no well-ordered nanostructures; (b) 25 mm, showing helical nanofibers. Scale bar: 100 nm (Carbon-coated copper grid, Uranyl acetate as negative stain). S11

12 Figure S4. Cyro-TEM images of DI-dipeptides (1mM in H 2 ) indicating the nanostructures were formed in the solution. (a) Dipeptide A showing nanofibers, (b) Dipeptide B showing twisted nanoribbons, (c) Dipeptide C showing no visible nanostructures. Scare bar: 200 nm S12

13 Figure S5. MR spectra of dipeptides A, B, and C in D 2 (10 mm). MR spectra of C showed only well-resolved peaks consistent with a minimally associated or monomeric structure, whereas the spectra of A and B exhibited highly broadened resonances typical of aggregates. S13

14 Figure S6. FTIR spectra of dipeptides in TFE (10 mg/ml) and D 2 (10 mg/ml) and secondary derivative FTIR spectra of dipeptides in D 2 (10 mg/ml) (a) (b) dipeptide A; (c) (d) dipeptide B; (e) (f) dipeptide C. S14

15 Figure S7. UV spectra of dipeptides in TFE and H 2 (1 mm). (a) dipeptide A; (b) dipeptide B; (c) dipeptide C; Both decreases in intensity and red shifts of absorption were shown in UV spectra of dipeptide A and B as the solvent changed from TFE (dissolved state) to H 2 (assembled state), indicating the formation of J-type aggregates for A and B assemblies in H 2. (d) Principal z and y polarized π-π* transitions in DI chromophore. S15

16 Figure S8. CD spectra of dipeptides at varying temperatures and comparison of CD spectra of dipeptides in water and TFE. (a) (b) Dipeptide A, (c) (d) dipeptide B, (e) (f) dipeptide C. (concentration of sample solutions: 250 µm). S16

17 Figure S9. ormalized fluorescence spectra of dipeptides in water (250 µm) and TFE (250 µm). (a) dipeptide A, (b) dipeptide B, (c) dipeptide C. (d) dipeptide A, B and C in water (250 µm). S17

Figure S10. Time-resolved fluorescence spectra of dipeptides in water (monomer). Table S1. Fluorescence lifetime data for monomeric dipeptides A, B, C, and,-di-nbutylnaphthalenediimide.

18 Figure S10. Time-resolved fluorescence spectra of dipeptides in water (monomer). Table S1. Fluorescence lifetime data for monomeric dipeptides A, B, C, and,-di-nbutylnaphthalenediimide. Sample Component Lifetimes (ps) b τ 1 τ 2 τ 3 τ avg c A 428 (0.3%) 58 (14.5%) 2 (85.2%) ~11 B 196 (1%) 3 (93 %) 54 (6%) <10 C 192 (0.5%) 59 (16.5%) 2 (83%) ~12 DI a 2 (100%) 2 a,-di-n-butylnaphthalenediimide: (Posokhov, Y.; Alp, S.; Köz, B.; Dilgin, Y.; Icli, S. Turk. J. Chem. 2004, 28, ) b Values in parenthesis represent the portion of the decay corresponding to each lifetime. It should be noted that the lifetime values <10 ps are too fast to resolve with our TCSPC system, and these values should be treated accordingly. c Average lifetimes (τ avg ) were calculated using the following equation: τ avg = a 1 τ 1 + a 2 τ 2 + a 3 τ 3. S18

Figure S11. Time-resolved fluorescence spectra of dipeptides in water (excimer). Table S2. Fluorescence lifetime data for the excimer band of dipeptides A, B, and C.

19 Figure S11. Time-resolved fluorescence spectra of dipeptides in water (excimer). Table S2. Fluorescence lifetime data for the excimer band of dipeptides A, B, and C. Sample Component Lifetimes (ps) a τ 1 τ 2 τ 3 τ avg b A 1885 (0.5%) 178 (12%) 32 (88%) 57 B 3271 (1%) 716 (9%) 66 (90%) 153 C 2560 (0.1%) 72 (0.9%) 1 (99%) <10 a Values in parenthesis represent the portion of the decay corresponding to each lifetime. It should be noted that the lifetime values <10 ps are too fast to resolve with our TCSPC system, and these values should be treated accordingly. b Average lifetimes (τ avg ) were calculated using the following equation: τ avg = a 1 τ 1 + a 2 τ 2 + a 3 τ 3. S19

20 Figure S12. Time-resolved fluorescence anisotropy spectra of dipeptides in water (λem = 500 nm). The initial anisotropy values were shown in the table. S20

21 Figure S13. XRD patterns of DI-dipeptides S21

4 Å corresponds to the plane-to-plane distance between DIs.

22 Figure S14. A proposed model for formation of helical nanofibers by dipeptide A. 4.4 Å corresponds to the interstrand distance of β-sheet along the long axis of fibers.3.4 Å corresponds to the plane-to-plane distance between DIs. 27Å is the extended length of the ends of two side chains. Two fibrils intertwine into one fiber with a diameter at ~10 nm. Double-headed arrows refer to the direction of β-sheet association. S22

Figure S15. A proposed model for formation of nanoribbons by dipeptide B. 4.5 Å corresponds to the interstrand distance of β-sheet along the long axis of ribbon.3.

23 Figure S15. A proposed model for formation of nanoribbons by dipeptide B. 4.5 Å corresponds to the interstrand distance of β-sheet along the long axis of ribbon.3.4 Å corresponds to the plane-to-plane distance between DIs. 27Å is the extended length of the ends of two side chains. Double-headed arrows refer to the direction of β-sheet association S23

24 Figure S16. HPLC traces of purified DI-dipeptides (MeC/Water 0.1% TFA: 10/90 to 100/0 over 25 min) S24

25 Dipeptide A: 1 H MR (400 MHz, DMS-d 6 ) δ 0.94 (t, J = 6.4 Hz, 3H), (m, 6H), (m, 10 H), 1.83 (s, 3H), 2.77 (m, 2H), (m, 4H), (m, 2H), 7.00 (s, 1H), 7.31 (s, 1H), 7.67 (s, 3H), 7.82 (d, J = 8 Hz, 1H), 8.00 (d, J = 8 Hz, 1H), 8.65 (s, 4H); 13 C MR (100 MHz, DMS-d 6 ) δ 13.65, 19.74, 22.18, 22.45, 22.94, 26.57, 27.21, 29.51, 31.19, 31.67, 38.69, 52.20, 52.34, 66.30, , , , , , , , , ; ESI-MS calcd for C 32 H [M+H] , found Dipeptide B: 1 H MR (500 MHz, DMS-d 6 ) δ 0.94 (t, J = 7.0 Hz, 3H), (m, 6H), (m, 10 H), 1.82 (s, 3H), 2.76 (m, 2H), (m, 4H), (m, 2H), 6.99 (s, 1H), 7.23 (s, 1H), 7.62 (s, 3H), 7.84 (d, J = 8 Hz, 1H), 8.01 (d, J = 8 Hz, 1H), 8.68 (s, 4H); 13 C MR (125 MHz, DMS-d 6 ) δ 13.65, 19.74, 22.16, 22.42, 23.02, 26.53, 27.20, 29.51, 31.22, 31.45, 38.67, 51.93, 52.86, , , , , , , , ; ESI-MS calcd for C 32 H [M+H] , found Dipeptide C: 1 H MR (500 MHz, DMS-d 6 ) δ 0.94 (t, J = 7.0 Hz, 3H), (m, 6H), (m, 10 H), 2.76 (m, 2H), 3.84 (m, 1H), 4.57 (q, J = 7.5 Hz, 4.5 Hz, 4H), 4.26 (m, 2H), 7.06 (s, 1H), 7.51 (s, 1H), 7.78 (s, 3H), 8.15 (s, 3H), 8.51 (d, J = 7.5 Hz, 1H), 8.66 (s, 4H); 13 C MR (125 MHz, DMS-d 6 ) δ 13.66, 19.75, 20.89, 23.02, 26.41, 27.27, 29.53, 30.44, 31.90, 38.46, 51.81, 52.63, , , , , , , , , , ; ESI-MS calcd for C 30 H [M+H] , found S25

26 Figure S17. ESI spectrum of Dipeptide A S26

27 Figure S18. ESI spectrum of dipeptide B S27

28 Figure S19. ESI spectra of dipeptide C. S28

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34 1 Horne, W. S.; Ashkenasy,.; Ghadiri, M. R. Chem.--Eur. J. 2005, 11, S34

Supporting Information

Supporting Information A π-conjugated ydrogel based on an Fmoc-Dipeptide aphthalene Diimide Semiconductor. By ui Shao and Jon R. Parquette* [*] Prof. J.R Parquette,. Shao Department of Chemistry The hio