2767 sample manager system

The TAF-Ap (1.90 g) was subjected to medium pressure chromatography (BUCHI Pump Manager, model C-615, Flawil, Switzerland) using aluminum oxide 90 (70-270 mesh ASTM, MN) and binary mixtures of hexane and methanol in an increasing polarity gradient, providing 48 fractions. The chromatographic profile of sample 8 (126 mg) was analyzed in an analytical HPLC (Infinity Series More Confident 1260 system, Agilent, Santa Clara, CA, United States). The fraction was diluted in 0.2% trifluoroacetic acid (TFA):acetonitrile (ACN) (1:1—v/v) aqueous solution at 1 mg mL⁻¹ and the volume injected into the apparatus was 50 μL. A reverse phase C-18 column was maintained at 40 °C and 0.2% TFA:ACN aqueous solution (77:23 to 65:35 v/v) was used as a mobile phase with a flow of 10 mL min⁻¹, a time of 15 min, and in gradient mode. After analysis in analytical HPLC, the sample was injected into semi-preparative HPLC (2767 Sample Manager System, Waters, with a UV-Vis photodiode detector, Milford, Massachusetts) using the same conditions as previously mentioned. At the end of the separation, a total of 10 fractions were obtained (Ap-1 to Ap-10). Fraction Ap-3 was directed to structural analysis, which allowed the identification of compounds 1 and 2 (Figure 2).

The mass spectra were obtained by injecting the samples into a high-performance liquid chromatograph with a diode array detector coupled with a low resolution electrospray ionization mass spectrometer (HPLC-DAD-ESI-MS; 2767 Sample Manager System, Waters, Milford, Massachusetts) and a quadrupolar analyzer ion trap (trap ions) operating in the positive mode. The ¹H and ¹³C NMR spectra (500 and 125 MHz, acetone-d₆) (1D and 2D) were recorded on a Varian Mercury spectrometer using TMS as the internal standard.