Discovery of Novel Minor Steviol Glycoside from the Stevia rebaudiana: Structural Characterization and Proposed Biosynthetic Pathway of Rebaudioside D17

A novel steviol glycoside, Rebaudioside <i>D17</i>, was identified from the leaf extract of <i>Stevia rebaudiana</i> Bertoni. This compound features a rare β-1→4 glycosidic linkage between two glucose units at the C19 position, distinguishing it from its structural isomer, Rebaudioside <i>D</i>. The aim of this study was to isolate and characterize Rebaudioside <i>D17</i> and investigate its biosynthetic origin. The compound was isolated and structurally characterized using comprehensive NMR spectroscopy including ¹H, ¹³C, COSY, NOESY, Heteronuclear Single Quantum Coherence-Distortionless Enhancement by Polarization Transfer (HSQC-DEPT), Heteronuclear Multiple Bond Correlation (HMBC), Heteronuclear Single Quantum Coherence-Total Correlated Spectroscopy (HSQC-TOCSY), along with mass spectrometry analysis. A tentative biosynthetic pathway is proposed, involving Rebaudioside <i>E19</i>, a putative intermediate bearing the same β-1→4 glycosidic linkage at C19. Rebaudioside <i>E19</i> may serve as a common precursor to both Rebaudioside <i>D17</i> and Rebaudioside <i>U3</i>, a minor steviol glycoside previously reported in <i>Stevia rebaudiana</i> leaf extract, which also contains the same β-1→4 glycosidic linkage. The discovery of Rebaudioside <i>D17</i> expands the known diversity of steviol glycosides and provides new insights into glycosylation patterns in <i>Stevia rebaudiana</i>, which may support the development and production of novel sweeteners with improved sensory and physicochemical properties.