Structural Revision of a Nornicotine Glycoside: Identification of N-[(β-D-xylopyranosyl)methyl]nornicotine from Cherry-Red Tobacco

Abstract A nornicotine glycoside─previously reported with an incorrect structure─was isolated from cherry-red tobacco and structurally re-elucidated. Its molecular formula (C15H22N2O5) was determined by high-resolution electrospray ionization mass spectrometry. Acid hydrolysis combined with gas chromatography–mass spectrometry (GC-MS) preliminarily identified the aglycone as nornicotine. Comprehensive analysis of one-dimensional (1H, 13C, DEPT) and two-dimensional (COSY, HSQC, HMBC) nuclear magnetic resonance (NMR) data unambiguously established the planar structure, confirming the sugar moiety is a six-membered pyranose linked to the pyrrolidine nitrogen of nornicotine via an N–CH2–C bond. Density functional theory (DFT)-based NMR chemical shift calculations were performed for four possible stereoisomers (β-D, β-L, α-D, α-L). Linear regression analysis showed the β-D stereoisomer had the highest fitting correlation coefficient (R2 = 0.9952 for 13C and R2 = 0.9801 for 1H), significantly outperforming the others. The mean absolute error (MAE) was 2.41 ppm for 13C NMR chemical shifts and 0.28 ppm for 1H NMR chemical shifts, indicating excellent agreement between theoretical and experimental data. Combined with the prevalent distribution of β-D configuration in natural glycosides, the sugar moiety’s stereo configuration was determined as β-D, confirming it as β-D-xylopyranose. This work corrects the long-standing misassignment of the nornicotine glycoside’s sugar moiety.