Monosaccharide compositional analysis and absolute configuration determination by GC

The monosaccharide composition of O25B O-PS (500 μg) and O25B-EPA (400 μg saccharide) was determined by GC and GC-MS after derivatization of the samples to tri-methyl-silyl ethers methyl-glycosides (methyl-glycosides TMS). The samples were prepared by methanolysis (3 M HCl for 16 h at 85 °C) and derivatized following the method described by Kim et al. [30]. After re-N-acetylation of the amino sugars with acetic anhydride, the resulting methyl glycosides were TMS-derivatized using the silylating reagent (HMDS+TMCS+Pyridine, 3:1:9 (Sylon HTP) Kit from Supelco). The samples were dried under a stream of N₂, hexane was added, followed by centrifugation to remove insoluble material. The clear supernatants were dried under N₂, the samples dissolved in hexane and analyzed either by GC on a Perkin–Elmer Autosystem XL gas chromatograph equipped with a flame ionization detector and a capillary HP-1 column, using He as carrier gas or by GC-MS on an Agilent Technologies 7890A gas chromatograph coupled to an Agilent Technologies 5975C VL MSD using the same column. The temperature program was: 150 °C for 1 min, 150–280 °C at 3 °C/min, and then held at 280 °C for 2 min. Standard retention times and response factors for the TMS methyl glycosides were determined using monosaccharides standards and the internal standard inositol. A sample of O-PS from Yersinia enterocolitica O:50 strain 3229 [31] was used to obtain a standard for L-FucNAc; identification of the peaks attributable to L-FucNAc was confirmed by GC-MS.

Determination of the absolute configuration of the monosaccharide residues in O25B-EPA was performed according to Gerwig et al. [32, 33]. 500 μg of each monosaccharide standard (all with the D configuration, except L-Rha) was subjected to butanolysis in 1 M HCl in S-(+)-2-butanol or R-(−)-2-butanol for 16 h at 80 °C. Re-N-acetylation, TMS-derivatization of the free hydroxyl groups and purification were performed as described above for the preparation of the TMS methyl-glycosides. The monosaccharide standards were used to prepare S-(+)-2-butyl-glycoside TMS as well as R-(−)-2-butyl-glycoside TMS standards for each monosaccharide. The O25B-EPA sample (400 μg) was subjected to methanolysis (3 M HCl, 16 h at 85 °C) followed by butanolysis using S-(+)-2-butanol and derivatization as described for the monosaccharide standards. Attribution to the D- or L- absolute configuration was achieved by comparing the elution time of the samples with those of the monosaccharide standards. The GC temperature program was: 50 °C for 1 min, 50–130 °C at 45 °C/min, at 130 °C for 1 min, 130–200 °C at 1 °C/min, and finally held at 200 °C for 10 min. GC-MS was used to confirm the data obtained with GC and to identify all peaks present in the chromatograms.