Enzymatic Synthesis and Structural Characterization of Rebaudioside D3, a Minor Steviol Glycoside of Stevia rebaudiana Bertoni

Rebaudioside D3, a novel steviol glycoside, is produced by specific UDP-glycosyltransferase of rebaudioside E, a minor steviol glycoside of Stevia rebaudiana Bertoni. The complete proton and carbon NMR spectral assignments of rebaudioside D3, 13-[(2-O-β-D-glucopyranosyl-6-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester, was achieved by the extensive 1D and 2D NMR (1H and 13C, TOCSY, HMQC, HMBC) as well as mass spectral data. Further, hydrolysis studies were performed on rebaudioside D3 using acid and enzymatic studies to identify aglycone and sugar residues in its structure. Rebaudioside D3 is detected in the commercial extract of the leaves of Stevia rebaudiana by LC-MS analysis, suggesting rebaudioside D3 is a natural steviol glycoside.