2.4 Methods and Materials
47
2.4 Methods and Materials
2.4.1 Reagents and Equipment
All reagents including amino acids and resins were purchased from GL Biochem
(Shanghai), Shanghai Hanhong Chemical Co., J&K Scientific or Energy Chemical
and were used without further purifications. Unnatural amino acids were synthesized following reported procedures. NMP were purchased from Shenzhen Tenglong Logistics Co. and used without purification. All other solvents used were
purchased from Cantotech Chemicals, Ltd. Anhydrous solvents were purchased
from J&K Scientific. NMRs were measured on nuclear magnetic resonance (NMR)
spectroscopy (Bruker AVANCE-III 300, 400 and 500). Variable-temperature NMR
were acquired using Bruker AVANCE III 500 MHz and processed using Topspin
3.1 software package. Peptides were purified by HPLC (SHIMAZU Prominence
LC-20AT) using reverse phase C18 column Grace Vydac protein and peptide C18
250 × 10 mm, flow rate 5 ml/min or grace smart C18 250 × 4.6 mm at flow rate
1 ml/min. Deionized H 2 O (containing 0.1% TFA) and pure acetonitrile were used as
solvents in linear gradient elution. HPLC fractions containing product (screened
by ESI) were combined and lyophilized. Molecular weights were measured on
SHIMAZU-SPD2020; CD spectra were measured on Chirascan Circular Dichroism
Spectrometer.
2.4.2 Peptide Purification and Characterization
Linear peptides were synthesized, and then characterized by HPLC and LC-MS. The
photoreaction products were purified on the HPLC. In general, for peptides 1-10 and
s1-8a/b the S/R diastereomers were separable with more than 5 min retention time
differences. The purified peptides were then characterized by LC-MS. Mass spectra
were obtained by ESI in positive ion mode. All LC-MS figures were attached at the
end of supporting information.
2.4.3 CD Measurement
Peptides were dissolved in potassium phosphate solution (pH 7.0) or 20% TFE buffer
to concentrations of 10–100 μM. The spectra were obtained on an Applied Photophysics Chirascan Circular Dichroism Spectrometer at 20°C using the following
standard measurement parameters: wavelength, 185–260 nm; step resolution, 0.5 nm;
speed, 20 nm/sec; accumulations, 10; response, 1 s; bandwidth, 1 nm; path 3 length,
0.1 cm. Every sample was scanned twice and the final CD spectrum was averaged
and smoothed. The α-helical content of each peptide was calculated as reported
47
2.4 Methods and Materials
2.4.1 Reagents and Equipment
All reagents including amino acids and resins were purchased from GL Biochem
(Shanghai), Shanghai Hanhong Chemical Co., J&K Scientific or Energy Chemical
and were used without further purifications. Unnatural amino acids were synthesized following reported procedures. NMP were purchased from Shenzhen Tenglong Logistics Co. and used without purification. All other solvents used were
purchased from Cantotech Chemicals, Ltd. Anhydrous solvents were purchased
from J&K Scientific. NMRs were measured on nuclear magnetic resonance (NMR)
spectroscopy (Bruker AVANCE-III 300, 400 and 500). Variable-temperature NMR
were acquired using Bruker AVANCE III 500 MHz and processed using Topspin
3.1 software package. Peptides were purified by HPLC (SHIMAZU Prominence
LC-20AT) using reverse phase C18 column Grace Vydac protein and peptide C18
250 × 10 mm, flow rate 5 ml/min or grace smart C18 250 × 4.6 mm at flow rate
1 ml/min. Deionized H 2 O (containing 0.1% TFA) and pure acetonitrile were used as
solvents in linear gradient elution. HPLC fractions containing product (screened
by ESI) were combined and lyophilized. Molecular weights were measured on
SHIMAZU-SPD2020; CD spectra were measured on Chirascan Circular Dichroism
Spectrometer.
2.4.2 Peptide Purification and Characterization
Linear peptides were synthesized, and then characterized by HPLC and LC-MS. The
photoreaction products were purified on the HPLC. In general, for peptides 1-10 and
s1-8a/b the S/R diastereomers were separable with more than 5 min retention time
differences. The purified peptides were then characterized by LC-MS. Mass spectra
were obtained by ESI in positive ion mode. All LC-MS figures were attached at the
end of supporting information.
2.4.3 CD Measurement
Peptides were dissolved in potassium phosphate solution (pH 7.0) or 20% TFE buffer
to concentrations of 10–100 μM. The spectra were obtained on an Applied Photophysics Chirascan Circular Dichroism Spectrometer at 20°C using the following
standard measurement parameters: wavelength, 185–260 nm; step resolution, 0.5 nm;
speed, 20 nm/sec; accumulations, 10; response, 1 s; bandwidth, 1 nm; path 3 length,
0.1 cm. Every sample was scanned twice and the final CD spectrum was averaged
and smoothed. The α-helical content of each peptide was calculated as reported