Bacteria were cultivated at 37 °C under aerobic conditions in tryptic soy broth supplemented with 0.6% yeast extract. The bacterial mass was harvested at the end of the logarithmic growth phase, centrifuged, washed with distilled water, and lyophilized. The LPS in a yield of 5.8% of dry bacteria mass was isolated by the phenol–water extraction (Westphal & Jann, 1965) followed by dialysis of the extract without layer separation and purification by treatment with cold aq 50% CCl3CO2H to precipitate proteins and nucleic acids; the supernatant was dialyzed and freeze-dried. A LPS sample (150 mg) was hydrolyzed with aqueous 2% HOAc at 100 °C for 4.5 h, and a lipid precipitate was removed
by centrifugation (13 000 g, 20 min). BVD-523 order The water-soluble carbohydrate portion was fractionated by gel-permeation chromatography on a column (60 × 2.5 cm) of Sephadex G-50 Superfine (Amersham Biosciences, Sweden) in 0.05 M pyridinium acetate buffer (pH 4.5) with monitoring using a differential refractometer (Knauer, Germany). The yield of the O-polysaccharide was 17% of the LPS mass. For sugar analyses, a polysaccharide
sample was subjected to hydrolysis with 10 M HCl (80 °C, 30 min) followed by reduction with an excess of NaBH4 (20 °C, 2 h) or to methanolysis (1 mL MeOH, 0.1 mL AcCl, 16 h, 100 °C). The products were acetylated with a 1 : 1 Ac2O-pyridine mixture (100 °C, 1 h) and analyzed by gas-liquid chromatography (GLC) on a Hewlett-Packard 5880 chromatograph (USA) equipped with an Ultra 2 capillary column using a temperature gradient from 160 to 290 °C at a rate of 7 °C min−1. For determination of the absolute configuration of the monosaccharides, RXDX-106 cost a polysaccharide
sample was hydrolyzed with 10 M HCl (80 °C, 30 min) and N-acetylated (400 μL NaHCO3, 60 μL Ac2O, 0 °C, 1 h) (for Qui3N) or subjected to methanolysis as above. The products were heated with (S)-2-octanol (100 μL) (Leontein & Lönngren, 1993) in the presence of CF3CO2H (15 μL) at 120 °C for 16 h, acetylated, and analyzed by GLC as above. A polysaccharide sample was deuterium-exchanged by freeze-drying twice from 99.9% D2O and then examined as a solution in 99.96% D2O using internal sodium 3-(trimethylsilyl) propanoate-d4 Thalidomide (δH 0.0) and acetone (δC 31.45) as references. 1H and 13C NMR spectra were recorded at 30 °C using a Bruker DRX-500 NMR spectrometer (Germany) and xwinnmr Bruker software. Mixing time of 100 ms and spinlock time of 150 ms were used in TOCSY and ROESY experiments, respectively. Other NMR experimental parameters were set essentially as described earlier (Hanniffy et al., 1999). Ion–cyclotron resonance Fourier transform electrospray ionization mass spectrometry (ESI MS) was performed in the negative mode using an APEX II Instrument (Bruker Daltonics) equipped with a 7-Tesla magnet and an Apollo ion source. Mass spectra were acquired using standard experimental sequences as provided by the manufacturer.