The Elusive Indigo Precursors in Woad (Isatis tinctoria L.) - Identification of the Major Indigo Precursor, Isatan A, and a Structure Revision of Isatan B

A metabolite‐profiling study of shock‐frozen leaves of Isatis tinctoria L., an old indigo dye plant and medicinal herb, revealed a complex pattern of indigo‐forming compounds with higher polarities than the known indigo precursors isatan B and indican. These highly unstable compounds underwent rapid post‐harvest transformation and were not detected in air‐dried leaves. The major indigo precursor, named isatan A (4), was isolated by rapid normal‐phase and gel chromatography, along with isatan B (3). A full spectral data set of 3 showed that the previous structure assignment as ‘indoxyl‐5‐ketogluconate’ has to be revised to 1H‐indol‐3‐yl β‐D‐ribohex‐3‐ulopyranoside. Isatan A (4) was identified as 1H‐indol‐3‐yl 6′‐O‐(carboxyacetyl)‐β‐D‐ribohex‐3′‐ulopyranoside. In aqueous solution, glycosides 3 and 4 occur as hydrates and undergo rapid hydrolysis under very mild acidic or basic conditions.