Lc esi it tof ms

Prior to the purification process, the peptides were dissolved in water and a 10-fold excess of dithiothreitol (DTT) was added. The mixture was kept in an ultrasonic bath at 60 °C for 30 min. The crude peptide was purified by reverse-phase high-performance liquid chromatography (RP-HPLC) on a semi-preparative Phenomenex Luna C8 (2) (250 mm × 20 mm, 5 μm) column. The aqueous system (A) consisted of 0.1% (v/v) TFA in water, and the organic phase (B) consisted of 80% acetonitrile in water containing 0.08% (v/v) TFA. A linear gradient from 5% B to 50% B in A for 120 min was applied. Purification was monitored by UV absorption at wavelengths of 222 nm and 254 nm. The purity of the peptide was verified by liquid chromatography coupled with electrospray ionization, ion trap, and time-of-flight mass spectroscopy (LC ESI–IT–TOF MS, Shimadzu, Shimpol, Warsaw, Poland) and RP-HPLC with a Kromasil C8 analytical column (250 mm × 4.6 mm, 5 μm), using a gradient of 5% to 100% B in A for 60 min (A and B as described above).

Optical rotation was taken on the automatic polarimeter (AUTOPOL IV automatic polarimeter) in chloroform solutions at 28 °C. IR spectrum was recorded on the FTIR spectroscopy (Perkin Elmer). NMR spectra were recorded on the 600 MHz FT-NMR (Jeol) using deuterated chloroform (CDCl₃) with tetramethylsilane (TMS) as the internal standard. MS spectra were obtained using LC-ESI-IT-TOF-MS (Shimadzu). For preparative TLC, Merck Kieselgel 60 F₂₅₄ was used and Kieselgel 60 was used for column chromatography. Purification was performed using high performance liquid chromatography (LC-10 AT, Shimadzu) equipped with UV detector.

Before the purification the peptides were dissolved in H₂O, DTT was added (6-fold excess with respect to purified peptide) and the mixture was sonified for 30 min in 60°C. Purification of the crude peptide was carried out by using RP-HPLC on a semi-preparative Phenomenex Luna C8(2) (250 mm x 20 mm, 5 μm) column. A linear gradient from 5% B to 50% B in A in 150 min was used. The aqueous system (A) consisted of 0.1% (v/v) TFA solution in water, whereas the organic phase (B) was 80% acetonitrile in water, containing 0.08% (v/v) TFA. Purification was monitored by UV absorption at a wavelength of 222 and 254 nm. The purity of the peptide was verified by LC-ESI-IT-TOF/MS (Shimadzu, Shimpol, Warsaw, Poland), and by using RP-HPLC with a Kromasil C8 analytical column (250mm x 4.6 mm, 5 μm), where a gradient of 5% to 100% B in A in 60 min was employed, with A and B as described above.