Cms l

Melting points are uncorrected (Leica hot stage microscope), NMR spectra were recorded using the Varian spectrometers Gemini 2000 or Unity 500 (δ given in ppm, J in Hz, internal Me₄Si; typical experiments: HH-COSY, gHMBC, gHMQC, NOESY, DQFCOSY), ESI-MS spectra were taken on a Finnigan MAT LCQ 7000 (electrospray, voltage 4.1 kV, sheath gas nitrogen) instrument. ASAP-MS spectra were taken on an Advion expression CMS-L with an ASAP/APCI Ion source (capillary voltage 150 V, capillary temperature 220 °C, and voltage of the ion source: 15 V; APCI source temperature 300 °C with 5 µA). The optical rotation was measured on a PerkinElmer polarimeter at 20 °C; TLC was performed on silica gel (Merck 5554); the solvents were dried according to usual procedures. Oleanolic and ursolic acid were obtained from “betulinines” (Stribrna Skalica, Czech Republic) in bulk quantities.

HPLC/MS analyses were conducted to assess the proteolytic cleavage of STIPs by the SspA protease and catalogue peptides in the reaction mixtures. An Agilent Infinity II 1260 HPLC outfitted with a Phenomenex Lune C₅ column (4.6 by 150 mm, 5 μm) was used to separate the peptide fragments using a binary solvent system consisting of solvent A (water, 0.05% formic acid) and solvent B (acetonitrile, 0.05% formic acid) operating at 0.7 mL/min. A 5-μL volume (∼4.5 μg) of each sample was injected at 10% solvent B, followed by a two-step gradient elution from 10% to 50% solvent B in 30 min, then 50% to 100% B in 10 min. The HPLC was coupled to an Advion CMS(L) single quadrupole mass detector through an electrospray ionization source, and ions were detected between 100 and 2,000 m/z. Low temperature and fragmentation settings were used: capillary temperature 135°C, capillary voltage 120 V, source voltage offset 20 V, source gas temperature 135°C, and electrospray ionization (ESI) voltage 3,500 V.