Structural analyses of mannose pentasaccharide of high mannose type oligosaccharides by 1D and 2D NMR spectroscopy.

NMR spectroscopy is a very important and useful method for the structural analysis of oligosaccharides, despite its low sensitivity. We first applied conventional measuring methods, 2D DQF COSY, (1)H-(13)C HSQC, and (1)H-(13)C HMBC, and also the Double Pulsed Field Gradient Spin Echo (DPFGSE)-TOCSY and DPFGSE-NOESY/ROESY techniques to analyze a branched mannose pentasaccharide as a model of high mannose type N-glycans in natural abundance. The NMR spectra of the model compound are very complex and difficult to analyze owing to overlapping signals. The superior selective irradiation capability of the DPFGSE technique is useful for fine structural and conformational analyses of such complex oligosaccharides. We here introduce a novel technique called DPFGSE-Double-Selective Population Transfer (SPT)-Difference and DPFGSE-NOE/ROE-SPT-Difference spectroscopy. The DPFGSE-Double-SPT-Difference method involves irradiation of two peaks from one proton and the subtraction of higher and lower peaks from each spectrum. The DPFGSE-NOE/ROE-SPT-Difference method involves the transfer of the magnetization polarized by NOE/ROE from the nuclei to the spin-coupled nuclei through scalar spin-spin interaction using the SPT method. Even if the signals in the NMR spectra overlap, each signal can be accurately assigned. In particular, DPFGSE-NOE/ROE-SPT-Difference is very useful for identifying sugar connectivity.