Objective As methylation affects the biological activities of secondary metabolites, novel methylated anthocyanins hold potential as new functional materials. While accurate structural determination is essential for the development of these novel derivatives, discrepancies in the nuclear magnetic resonance (NMR) assignments of anthocyanidins are observed in the literature. This study aims to achieve accurate NMR signal assignments for major natural anthocyanidins and a non-natural analog, then systematically elucidate the effects of O -methylation to establish reliable foundational data for future structural elucidation. Methods Five natural anthocyanidins (cyanidin, peonidin, delphinidin, petunidin, and malvidin) were isolated and purified from bilberry ( Vaccinium myrtillus L.) extract following acid hydrolysis. Additionally, a non-natural analog, 3,7,3′,4′-tetra- O -methylcyanidin, was synthesized from quercetin. Comprehensive structural analysis of these compounds was conducted using 1 H-, 13 C- and two-dimensional NMR spectroscopy. Results Signal assignments were achieved for all compounds, and previous literature discrepancies regarding the H-6 and H-8 signals were resolved. In 1 H-NMR, O -methylation generally induced downfield shifts for aromatic protons on the substituted rings. In 13 C-NMR, ipso and para carbons typically exhibited downfield shifts, whereas ortho methine carbons shifted upfield. While 3,7,3′,4′-tetra- O -methylcyanidin exhibited trends similar to natural anthocyanidins, O -methylation on the A- and C-rings demonstrated slightly different shift changes. Conclusion In this study, we systematically summarized the fundamental trends of 1 H- and 13 C-NMR chemical shifts for anthocyanidins. Furthermore, effects on the spectra due to O -methylation of hydroxyl groups including those on the A- and C-rings were confirmed. These findings provide an essential spectroscopic basis for identifying novel O -methylated anthocyanidins.
Shiozawa et al. (Wed,) studied this question.