Micromass lct premier

Peptides were synthesized in our laboratory by standard solid-phase peptide synthesis in N-methyl-2-pyrrolidone using Syro I and Syro II synthesizers. Amino acids and resins were used as purchased from Nova Biochem. Peptides were purified by reversed-phase HPLC using a Waters HPLC system equipped with a preparative Waters X-bridge C18 column. The mobile phase consisted of water/acetonitrile mixtures containing 0.1% TFA. Peptide purity and composition were confirmed by LC-MS using a mass spectrometer (Micromass LCT Premier; Waters) equipped with a 2795 separation module (Alliance HT) and photodiode array detector (2996; Waters Chromatography). LC-MS samples were run over a Kinetex C18 column (Phenomenex) in a water/acetonitrile gradient. Analysis was performed using MassLynx 4.1 software (Waters Chromatography). Peptides were purified twice if necessary.

Optical rotations were recorded on a Bellingham and Stanley ADP440+ polarimeter (Bellingham + Stanley Ltd., Longfield Road, Tunbridge Wells, Kent TN2 3EY, UK). NMR spectra were obtained on Bruker AVANCE III (Bruker Corporation, Billerica, MA, USA) spectrometers (400 or 500 MHz for ¹H and 100 or 125 MHz for ¹³C), using a 5 mm BBOZ probe. The spectra were referenced to residual solvent peaks, δ_H 3.31 and δ_C 49.03 for CD₃OD. Mass spectra were recorded on a TOF Waters Micromass LCT Premier mass spectrometer (Waters Corporation, 34 Maple street, Milford, CT, USA) using ESI-ionization in negative or positive modes in MS-grade acetonitrile and methanol solutions. For thin-layer chromatographic analyses, pre-coated TLC silica gel 60 F₂₅₄ (Merck) plates were used. Column chromatography was performed on Merck silica gel (230–400) mesh, and gel filtration chromatography was performed using Sephadex LH-20 (Fluka). Spots on TLC plates were observed under UV light at 254 and 365 nm, and visualised by spraying with p-anisaldehyde/H₂SO₄ spray reagent (0.5 mL of p-anisaldehyde, 10 mL of glacial acetic acid, 4 mL of concentrated H₂SO₄ acid and 85 mL of MeOH), followed by heating at 100 °C for 5 min.

Electrospray ionization-time-of-flight (ESI-TOF) mass spectra were recorded on a Waters Micromass LCT premier. The samples (40 µM) were prepared in 20 mM NH₄OAc buffer (pH 7.0) and annealed just before the measurements (from 85 to 4 °C, −1.0 °C/min).