Reversible, Short α-Peptide Assembly for Controlled Capture and ...
Supporting Information for
Reversible, Short α-Peptide Assembly for Controlled Capture and Selective Release of Enantiomers
Xi Chen, Ying He, Yongju Kim,* and Myongsoo Lee*
State Key Laboratory of Supramolecular Strucuture and Materials College of Chemistry, Jilin University, Changchun 130012, China
E-mail: [email protected], [email protected]
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1. General method All starting materials were obtained from commercial suppliers (Aldrich, Lancaster, Anaspec, Novabiochem, and TCI, etc.) and were used without further purification. Dichloromethane (DCM), N, N–dimethylformamide (DMF), ethyl acetate (EA), methanol (MeOH) were distilled before use. The purity of the compound 5 was checked by thin layer chromatography (TLC; Merck, silica gel 230–400). Compound 4 was prepared according to the procedures described previously.S1 Mass spectrometry was performed on a Bruker Microflex MALDITOF mass spectrometer using an α-cyano-4-hydroxy cinnamic acid as a matrix. HPLC analysis was performed with Prominence LC-20AP (SHIMADZU) equipped with Shim-pack PREP-ODS (H) KIT C18 reverse phase column (C18, 250 × 4.6 mm I.D.) for general separation and YMC CHIRALART column (250 × 4.6 mm I.D. S-5 µm) for chiral separation. The UV/vis spectrum was obtained from Hitachi U-2900. Circular Dichroism (CD) spectra were measured using a JASCO model J-810 spectropolarimeter. Spectra were recorded from 280 nm to 190 nm using a 1 mm pathlength cell. Scans were repeated for 3 times and averaged. Samples were stabilized at room temperature for 24 h before the measurement. The concentrations for the peptides used in CD analysis are as follows: Peptide 1 in KF 5 mM is 1.0 mg/mL, peptide 1 in TFE 30% is 1.0 mg/mL, peptide 2 in KF 5 mM is 1 mg/mL, peptide 3 in KF 5 mM is 1.0 mg/mL. Dynamic Light Scattering (DLS) experiments were performed by using ALV/CGS-3. The Scanning Electron Microscope (SEM) was performed at 3 kV using JEOL-JSM-6700F. Compounds were synthesized according to the procedure described in scheme 1 and then purified by silica gel column chromatography and prep HPLC. Racemic 1-(4-bromophenyl) ethanol was synthesized according to the procedure described in referenceS2 and then purified by silica gel column chromatography. The peptides were modeled and minimized with CHARMM force field using Minimization protocol in Discovery Studio 1.7. X-ray diffraction (XRD) patterns were obtained using a Rigaku D/ max 2550 diffractometer (Rigaku Co.). TEM Experiments. A drop of the sample solution was placed on a carbon-coated copper grid (Carbon Type B (15–25 nm) on 200 mesh, with Formvar; Ted Pella, Inc.) and the solution was allowed to evaporate under 50 oC conditions. These samples were stained by depositing a drop of
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sodium phosphotungstate aqueous solution (1.0–2.0 wt %) onto the surface of the sample-loaded grid. The dried specimen was observed by a JEOL–JEM HR2100 operated at 120 kV. The cryogenic transmission electron microscopy (cryo-TEM) experiments were performed with a thin film of a water solution of the peptide (5 µL) transferred to a lacey supported grid. The thin films were prepared under controlled temperature and humidity conditions (97–99 %) within a custom–built environmental chamber in order to prevent evaporation of water from sample solution. The excess liquid was blotted with filter paper for 2–3 seconds and the thin water films were rapidly vitrified by plunging them into liquid ethane (cooled by liquid nitrogen) at its freezing point. The grid was transferred, on a Gatan 626 cryoholder, using a cryo–transfer device and transferred to the JEOL– JEM HR2100 TEM. Direct imaging was carried out at a temperature of approximately –175 oC and with a 120 kV accelerating voltage, using the images acquired with a Dual vision 300 W and SC 1000 CCD camera (Gatan, Inc.; Warrendale, PA). The data were analysed using Digital Micrograph software. AFM experiments. The sample films on mica surface were prepared from evaporation of 0.005 wt % peptide 2 solution in 5 mM KF aqueous solution. The measurements were conducted on a MultiMode 8 AFM with NanoScope V controller, NanoScope software and NanoScope Analysis software (Bruker AXS Corporation, Santa Barbara, CA, USA) in air at 50 oC in the tapping mode. Images were acquired in PeakForce Tapping mode. NMR experiment. 1H-NMR (300 MHz) spectra were recorded using a solution of peptide 3 in D2O with a concentration of 0.1 wt %. At each temperature, the solution was equilibrated for 10 minutes before data acquisition. The dehydration of the ethylene oxide segments was supported by peak broadening and peak shift toward upfield. Separation of racemic mixtures. A series of racemic mixture (1-(4-bromophenyl)ethanol, Binol, and N-acetylphenylalanine) were subjected to evaluate the enantioselective permeability of the vesicular walls. Aqueous solution (1.0 mL) of racemic mixtures (5 mM) was added to 1 mg peptide 3 to give 0.1 wt % peptide 3 aqueous solution. The mixture sample was sonicated for 20 min and stabilized for 1 hour at room temperature. Then, the sample was kept at 55 oC and 0.2 mL samples
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with differenct incubation time were subjected to Sephadex G–50 (800 mg) column to remove untrapped guest molecule. Ten fractions of 7.0 mL eluate (each 0.7 mL) were collected and monitored for the residue of chiral molecules through HPLC analysis.
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2. Synthetic Method
Compound 5. Dry NaH (40 mg), compound 4 (360 mg) and freshly distilled dry THF (4.2 ml) were placed in a dry round bottomed flask under N2 atmosphere. Propargyl bromide (59 mg) was added to this mixture dropwise at room temperature. The mixture was stirred at room temperature for 15 min and then 65 oC for 12 h. After cooling to room temperature, the reaction was quenched with water and extracted with methylene chloride. The organic layer was then dried by anhydrous MgSO4 and the solvent was then removed by rotary evaporator. Purification of the residue by flash column chromatography on silica gel using a mixture of ethyl acetate and methanol (5:1 v/v) as an eluent yielded 297 mg desired product (78 %). 1
H NMR (300 MHz, CDCl3) δ 4.11 (s, 2H), 3.68 – 3.39 (m, 66H), 3.39 (s, 12H), 2.47 (t, J = 2.4 Hz,
1H), 2.16 (td, J = 11.5, 5.9 Hz, 3H).
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Compound 7. Compound 6 (375 mg) and a mixture solvent of water (4.5 ml), methanol (9 ml) and dichloromethane (7.5 ml) were placed in a round bottomed flask under N2 atmosphere. 1H-imidazole-1-sulfonyl azide·HCl (600 mg) and CuSO4·5H2O (1.5 mg) were added into this solvent and then adjust the pH to 9. After stirred
for 18 h, diluted with MC (9 ml), the aqueous
layer was isolated, then the organic layer was extracted
with saturated NaHCO3 (2 × 15 ml), The
combined aqueous extracts were washed by Et2O (2 × 15 ml), acidified to pH 2 with concentrated HCl and then extracted with Et2O (2 × 18 ml). The organic layer was then dried over anhydrous MgSO4 and the solvent was then removed in a rotary evaporator. Checking by MALDI-TOF without further Purification. Yield: 215 mg (58.7 %). MALDI-TOF-mass (m/z): [M + K]+ calcd. for C21H22N4KO4, 472.63; Found: 471.94. Peptide 1. Peptide 1 was synthesized on Rink amide MBHA resin with standard Fmoc protection process using HCTU as coupling reagent. tert-Butyloxycarbonyl (Boc) protecting group was employed for Lysine. The resin was swollen in DCM for 1h before the synthesis. Deprotection of Fmoc protecting group, a cocktail solution (20 % piperidine in DMF) was treated several times (2 × 2 min). The resin was washed with DCM and DMF several times after each step was finished. Resin was treated with a cleavage cocktail solution (TFA : anisole : Water : TIPS; 88 : 5 : 5 : 2) for 3 h after synthetic and the mixture solution was washed with ether. Peptide 1 was purified by reverse phase HPLC (water/acetonitrile with 0.1 % Formic Acid) using C18 prep scale column. Peptide 1 were confirmed by MALDI-TOF mass spectrometer. Yield of peptide 1 was calculated from the initial loading amount of the resin and after repeatative purification process, total reaction yield: 5.4 % Peptides 2 and 3. A solution of compound 8 and 9 (synthesised by the similar method with peptide 1) in H2O/t-BuOH (1/3) with catalytic amounts of sodium ascorbate (5 mol%) and CuSO4 (2 mol%) was stirred at rt for 2 h, checking by MALDI-TOF. After the solvent was removed in a rotary evaporator, diluted with water. The crude product was purified reverse phase HPLC (water/acetonitrile with 0.1% Formic Acid) using C18 prep scale column. Yields: 52 % for peptide 2 ; 45 % for peptide 3.
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3. Supporting Figures
Peptide 1. Calcd. for C108H177N29O19 [M + H]+: 2186.80; Found: 2187.33.
Peptide 2. Calcd. for C194H341N33O57 [M + H]+: 4048.49; Found: 4047.45.
Peptide 3. Calcd. for C237H423N35O76 [M + H]+: 4980.17; Found: 4979.80.
Figure S1. MALDI-TOF spectra of peptides 1, 2, and 3.
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Figure S2. HPLC Data of peptides 1, 2, and 3. Purified samples were analyzed with an analytical HPLC system consisting of a Shim-pack PREP-ODS (H) KIT column (C18, 250 × 4.6 mml.D). Solvent was changed from 5 % CH3CN in H2O (0.1 % formic acid) to 100 % CH3CN (0.1 % formic acid) for 50 min with 0.7 mL/min flow rate. Absorbance in 210 nm was detected.
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Figure S3. CD spectrum of the peptide 1 in 5 mM aqueous KF solution with various temperatures and CD spectrum of peptide 1 in 5 mM aqueous KF solution including 30 % TFE.
Figure S4. Measurement of transmittance for lower critical solution temperature (LCST) of 0.1 wt% peptide 3 in 5 mM aqueous KF solution.
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Figure S5. 1H-NMR (300 MHz) spectra of peptide 3 (0.1 wt%) in 5 mM KF solution in D2O. The spectrum showed broad proton signals at the chemical shift range of 3.2–4.2 ppm associated with oligoether backbones. Upon heating, the broad signals are upfield-shifted due to the dehydration of the oligoether dendrons.S3
Figure S6. TEM image of peptide 2 (0.1 wt%) in 5 mM aqueous KF solution at 50℃.
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Figure S7. XRD pattern on the film from evaporation of aqueous solution of peptide 3 (0.1 wt%) in 5 mM aqueous KF solution at 50℃.
Figure S8. TEM image of peptide 3 (0.002 wt%) in 5 mM aqueous KF solution at 50℃.
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Figure S9. a) Absorption spectrum of 1-(4-bromophenyl)ethanol encapsulated by vesicles after the seperation of free enantiomers. b) HPLC chromatogram of 1-(4-bromophenyl)ethanol encapsulated by vesicles just after the seperation of free enantiomers. HPLC condition: YMC CHIRALART Cellulose-C (250 *4.6 mm I.D. S-5 µm), MeOH / 20 mM NH4HCO3 / DEA : 50/50/0.1 (flow rate 0.7 mL/min).
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Figure S10. The separation experiments with (R)/(S)-Binol and a model D/L-amino acyl amide, N-acetylphenylalanine showing the maximum ee value after 3 hr to be 8 %, and with the maximum ee value to be 15 % after 2hr. HPLC condition: YMC CHIRALART Amylose-C (250 *4.6 mm I.D. S-5 µm), Hex/Isopropanol : 90/10 (flow rate 1 mL/min) for Binol; YMC CHIRALART Cellulose-C (250 *4.6 mm I.D. S-5 µm), Hex/THF/TFA : 75/25/0.1 (flow rate 1 mL/min) for N-AcPheCONH2.
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References S1) Park, I.-S.; Yoon, Y.-R.; Jung, M.; Kim, K.; Park, S. B.; Shin, S.; Lim, Y.-B.; Lee, M. Chem. Asian J. 2011, 6, 452-458. S2) Battilocchio, C.; Hawkins, J. M.; Ley, S. V. Org. Lett. 2013, 15, 2278-2281. S3) Huang, Z.; Kang, S. K.; Banno, M.; Yamaguchi, T.; Lee, D.; Seok, C.; Yashima, E.; Lee, M. Science 2012, 337, 1521-1526.
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Reversible, Short α-Peptide Assembly for Controlled Capture and Selective Release of Enantiomers
Xi Chen, Ying He, Yongju Kim,* and Myongsoo Lee*
State Key Laboratory of Supramolecular Strucuture and Materials College of Chemistry, Jilin University, Changchun 130012, China
E-mail: [email protected], [email protected]
S1
1. General method All starting materials were obtained from commercial suppliers (Aldrich, Lancaster, Anaspec, Novabiochem, and TCI, etc.) and were used without further purification. Dichloromethane (DCM), N, N–dimethylformamide (DMF), ethyl acetate (EA), methanol (MeOH) were distilled before use. The purity of the compound 5 was checked by thin layer chromatography (TLC; Merck, silica gel 230–400). Compound 4 was prepared according to the procedures described previously.S1 Mass spectrometry was performed on a Bruker Microflex MALDITOF mass spectrometer using an α-cyano-4-hydroxy cinnamic acid as a matrix. HPLC analysis was performed with Prominence LC-20AP (SHIMADZU) equipped with Shim-pack PREP-ODS (H) KIT C18 reverse phase column (C18, 250 × 4.6 mm I.D.) for general separation and YMC CHIRALART column (250 × 4.6 mm I.D. S-5 µm) for chiral separation. The UV/vis spectrum was obtained from Hitachi U-2900. Circular Dichroism (CD) spectra were measured using a JASCO model J-810 spectropolarimeter. Spectra were recorded from 280 nm to 190 nm using a 1 mm pathlength cell. Scans were repeated for 3 times and averaged. Samples were stabilized at room temperature for 24 h before the measurement. The concentrations for the peptides used in CD analysis are as follows: Peptide 1 in KF 5 mM is 1.0 mg/mL, peptide 1 in TFE 30% is 1.0 mg/mL, peptide 2 in KF 5 mM is 1 mg/mL, peptide 3 in KF 5 mM is 1.0 mg/mL. Dynamic Light Scattering (DLS) experiments were performed by using ALV/CGS-3. The Scanning Electron Microscope (SEM) was performed at 3 kV using JEOL-JSM-6700F. Compounds were synthesized according to the procedure described in scheme 1 and then purified by silica gel column chromatography and prep HPLC. Racemic 1-(4-bromophenyl) ethanol was synthesized according to the procedure described in referenceS2 and then purified by silica gel column chromatography. The peptides were modeled and minimized with CHARMM force field using Minimization protocol in Discovery Studio 1.7. X-ray diffraction (XRD) patterns were obtained using a Rigaku D/ max 2550 diffractometer (Rigaku Co.). TEM Experiments. A drop of the sample solution was placed on a carbon-coated copper grid (Carbon Type B (15–25 nm) on 200 mesh, with Formvar; Ted Pella, Inc.) and the solution was allowed to evaporate under 50 oC conditions. These samples were stained by depositing a drop of
S2
sodium phosphotungstate aqueous solution (1.0–2.0 wt %) onto the surface of the sample-loaded grid. The dried specimen was observed by a JEOL–JEM HR2100 operated at 120 kV. The cryogenic transmission electron microscopy (cryo-TEM) experiments were performed with a thin film of a water solution of the peptide (5 µL) transferred to a lacey supported grid. The thin films were prepared under controlled temperature and humidity conditions (97–99 %) within a custom–built environmental chamber in order to prevent evaporation of water from sample solution. The excess liquid was blotted with filter paper for 2–3 seconds and the thin water films were rapidly vitrified by plunging them into liquid ethane (cooled by liquid nitrogen) at its freezing point. The grid was transferred, on a Gatan 626 cryoholder, using a cryo–transfer device and transferred to the JEOL– JEM HR2100 TEM. Direct imaging was carried out at a temperature of approximately –175 oC and with a 120 kV accelerating voltage, using the images acquired with a Dual vision 300 W and SC 1000 CCD camera (Gatan, Inc.; Warrendale, PA). The data were analysed using Digital Micrograph software. AFM experiments. The sample films on mica surface were prepared from evaporation of 0.005 wt % peptide 2 solution in 5 mM KF aqueous solution. The measurements were conducted on a MultiMode 8 AFM with NanoScope V controller, NanoScope software and NanoScope Analysis software (Bruker AXS Corporation, Santa Barbara, CA, USA) in air at 50 oC in the tapping mode. Images were acquired in PeakForce Tapping mode. NMR experiment. 1H-NMR (300 MHz) spectra were recorded using a solution of peptide 3 in D2O with a concentration of 0.1 wt %. At each temperature, the solution was equilibrated for 10 minutes before data acquisition. The dehydration of the ethylene oxide segments was supported by peak broadening and peak shift toward upfield. Separation of racemic mixtures. A series of racemic mixture (1-(4-bromophenyl)ethanol, Binol, and N-acetylphenylalanine) were subjected to evaluate the enantioselective permeability of the vesicular walls. Aqueous solution (1.0 mL) of racemic mixtures (5 mM) was added to 1 mg peptide 3 to give 0.1 wt % peptide 3 aqueous solution. The mixture sample was sonicated for 20 min and stabilized for 1 hour at room temperature. Then, the sample was kept at 55 oC and 0.2 mL samples
S3
with differenct incubation time were subjected to Sephadex G–50 (800 mg) column to remove untrapped guest molecule. Ten fractions of 7.0 mL eluate (each 0.7 mL) were collected and monitored for the residue of chiral molecules through HPLC analysis.
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2. Synthetic Method
Compound 5. Dry NaH (40 mg), compound 4 (360 mg) and freshly distilled dry THF (4.2 ml) were placed in a dry round bottomed flask under N2 atmosphere. Propargyl bromide (59 mg) was added to this mixture dropwise at room temperature. The mixture was stirred at room temperature for 15 min and then 65 oC for 12 h. After cooling to room temperature, the reaction was quenched with water and extracted with methylene chloride. The organic layer was then dried by anhydrous MgSO4 and the solvent was then removed by rotary evaporator. Purification of the residue by flash column chromatography on silica gel using a mixture of ethyl acetate and methanol (5:1 v/v) as an eluent yielded 297 mg desired product (78 %). 1
H NMR (300 MHz, CDCl3) δ 4.11 (s, 2H), 3.68 – 3.39 (m, 66H), 3.39 (s, 12H), 2.47 (t, J = 2.4 Hz,
1H), 2.16 (td, J = 11.5, 5.9 Hz, 3H).
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Compound 7. Compound 6 (375 mg) and a mixture solvent of water (4.5 ml), methanol (9 ml) and dichloromethane (7.5 ml) were placed in a round bottomed flask under N2 atmosphere. 1H-imidazole-1-sulfonyl azide·HCl (600 mg) and CuSO4·5H2O (1.5 mg) were added into this solvent and then adjust the pH to 9. After stirred
for 18 h, diluted with MC (9 ml), the aqueous
layer was isolated, then the organic layer was extracted
with saturated NaHCO3 (2 × 15 ml), The
combined aqueous extracts were washed by Et2O (2 × 15 ml), acidified to pH 2 with concentrated HCl and then extracted with Et2O (2 × 18 ml). The organic layer was then dried over anhydrous MgSO4 and the solvent was then removed in a rotary evaporator. Checking by MALDI-TOF without further Purification. Yield: 215 mg (58.7 %). MALDI-TOF-mass (m/z): [M + K]+ calcd. for C21H22N4KO4, 472.63; Found: 471.94. Peptide 1. Peptide 1 was synthesized on Rink amide MBHA resin with standard Fmoc protection process using HCTU as coupling reagent. tert-Butyloxycarbonyl (Boc) protecting group was employed for Lysine. The resin was swollen in DCM for 1h before the synthesis. Deprotection of Fmoc protecting group, a cocktail solution (20 % piperidine in DMF) was treated several times (2 × 2 min). The resin was washed with DCM and DMF several times after each step was finished. Resin was treated with a cleavage cocktail solution (TFA : anisole : Water : TIPS; 88 : 5 : 5 : 2) for 3 h after synthetic and the mixture solution was washed with ether. Peptide 1 was purified by reverse phase HPLC (water/acetonitrile with 0.1 % Formic Acid) using C18 prep scale column. Peptide 1 were confirmed by MALDI-TOF mass spectrometer. Yield of peptide 1 was calculated from the initial loading amount of the resin and after repeatative purification process, total reaction yield: 5.4 % Peptides 2 and 3. A solution of compound 8 and 9 (synthesised by the similar method with peptide 1) in H2O/t-BuOH (1/3) with catalytic amounts of sodium ascorbate (5 mol%) and CuSO4 (2 mol%) was stirred at rt for 2 h, checking by MALDI-TOF. After the solvent was removed in a rotary evaporator, diluted with water. The crude product was purified reverse phase HPLC (water/acetonitrile with 0.1% Formic Acid) using C18 prep scale column. Yields: 52 % for peptide 2 ; 45 % for peptide 3.
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3. Supporting Figures
Peptide 1. Calcd. for C108H177N29O19 [M + H]+: 2186.80; Found: 2187.33.
Peptide 2. Calcd. for C194H341N33O57 [M + H]+: 4048.49; Found: 4047.45.
Peptide 3. Calcd. for C237H423N35O76 [M + H]+: 4980.17; Found: 4979.80.
Figure S1. MALDI-TOF spectra of peptides 1, 2, and 3.
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Figure S2. HPLC Data of peptides 1, 2, and 3. Purified samples were analyzed with an analytical HPLC system consisting of a Shim-pack PREP-ODS (H) KIT column (C18, 250 × 4.6 mml.D). Solvent was changed from 5 % CH3CN in H2O (0.1 % formic acid) to 100 % CH3CN (0.1 % formic acid) for 50 min with 0.7 mL/min flow rate. Absorbance in 210 nm was detected.
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Figure S3. CD spectrum of the peptide 1 in 5 mM aqueous KF solution with various temperatures and CD spectrum of peptide 1 in 5 mM aqueous KF solution including 30 % TFE.
Figure S4. Measurement of transmittance for lower critical solution temperature (LCST) of 0.1 wt% peptide 3 in 5 mM aqueous KF solution.
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Figure S5. 1H-NMR (300 MHz) spectra of peptide 3 (0.1 wt%) in 5 mM KF solution in D2O. The spectrum showed broad proton signals at the chemical shift range of 3.2–4.2 ppm associated with oligoether backbones. Upon heating, the broad signals are upfield-shifted due to the dehydration of the oligoether dendrons.S3
Figure S6. TEM image of peptide 2 (0.1 wt%) in 5 mM aqueous KF solution at 50℃.
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Figure S7. XRD pattern on the film from evaporation of aqueous solution of peptide 3 (0.1 wt%) in 5 mM aqueous KF solution at 50℃.
Figure S8. TEM image of peptide 3 (0.002 wt%) in 5 mM aqueous KF solution at 50℃.
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Figure S9. a) Absorption spectrum of 1-(4-bromophenyl)ethanol encapsulated by vesicles after the seperation of free enantiomers. b) HPLC chromatogram of 1-(4-bromophenyl)ethanol encapsulated by vesicles just after the seperation of free enantiomers. HPLC condition: YMC CHIRALART Cellulose-C (250 *4.6 mm I.D. S-5 µm), MeOH / 20 mM NH4HCO3 / DEA : 50/50/0.1 (flow rate 0.7 mL/min).
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Figure S10. The separation experiments with (R)/(S)-Binol and a model D/L-amino acyl amide, N-acetylphenylalanine showing the maximum ee value after 3 hr to be 8 %, and with the maximum ee value to be 15 % after 2hr. HPLC condition: YMC CHIRALART Amylose-C (250 *4.6 mm I.D. S-5 µm), Hex/Isopropanol : 90/10 (flow rate 1 mL/min) for Binol; YMC CHIRALART Cellulose-C (250 *4.6 mm I.D. S-5 µm), Hex/THF/TFA : 75/25/0.1 (flow rate 1 mL/min) for N-AcPheCONH2.
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References S1) Park, I.-S.; Yoon, Y.-R.; Jung, M.; Kim, K.; Park, S. B.; Shin, S.; Lim, Y.-B.; Lee, M. Chem. Asian J. 2011, 6, 452-458. S2) Battilocchio, C.; Hawkins, J. M.; Ley, S. V. Org. Lett. 2013, 15, 2278-2281. S3) Huang, Z.; Kang, S. K.; Banno, M.; Yamaguchi, T.; Lee, D.; Seok, C.; Yashima, E.; Lee, M. Science 2012, 337, 1521-1526.
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