Application of nano-probe NMR for structure determination of low nanomole amounts of arabinoxylan oligosaccharides fractionated by analytical HPAEC-PAD.

A methodology for NMR analysis of low nanomole amounts of oligosaccharides fractionated by analytical HPAEC is presented. Arabinoxylan derived oligosaccharides purified by HPAEC-PAD on an analytical column, by single injections, were analyzed with nano-probe NMR and MALDI-TOF MS to provide full structural assignment. The NMR data were obtained with a 500 MHz NMR spectrometer equipped with a 1H-observe nano-probe. Both one- and two-dimensional experiments on arabinoxylan samples in the low nanomole range were performed, including 1H-1H DQF-COSY, 1H-1H TOCSY and 1H-1H ROESY. These experiments allowed, in combination with MALDI-TOF MS and literature NMR data, a complete structural determination of several tetra-, penta-, hexa- and heptasaccharides. Two new structures: alpha-L-Araf-(1 --> 2)-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-D-Xylp and alpha-L-Araf-(1 --> 2)[alpha-L-Araf-(1 --> 3)]-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-beta-D-Xylp-(1 --> 4)-D-Xylp) were characterized, as well as some previously published structures.