Abstract: A new NMR technique, two-dimensional RELAY-HOHAHA NMR spectroscopy was developed. The results obtained showed that the efficiency of magnetization transfer among sugar residues of glycoside are much better for this method than that for 2D RELAY COSY and 2D HOHAHA experiments. The subspectra corresponding to individual sugar components were extracted even from overlapping proton resonance by taking the cross sections of 2D spectra para-llel to the F2 axis at anomeric proton resonance. Thus all the sugar proton resonances can be assigned to the individual sugar components. 2D RELAY-HOHAHA experiment in combination with NOE difference spectroscopy has been successfully used for determining the glycosidation position in an aglycome moiety and the sequence of sugar moiety in a natural glycoside without chemical degradation. The sensitivity for the present procedure based on proton NMR is much better than that for glycosidation shift based on ¹³C NMR.

Key words: 2D RELAY -HOHAHA experiment, Determination of the position of linkages between sugar units, Structure analysis of glycoside