Methyl Syringate Monoglucoside Is a Crucial Intermediate in Leptosperin Biosynthesis in <i>Leptospermum scoparium</i> (manuka)

Leptosperin (methyl syringate-4-<i>O</i>-β-d-gentiobioside) serves as a unique marker for ma̅nuka honey, derived from the ma̅nuka plant (<i>Leptospermum scoparium</i>). Despite its importance, the biosynthesis pathway of leptosperin remains unreported. This study investigates the molecular mechanism of leptosperin formation from its aglycone, methyl syringate (MSYR), in ma̅nuka plants. Methyl syringate-4-<i>O</i>-β-d-glucopyranoside (MSYR-glucose) was identified in ma̅nuka flower nectar but not in ma̅nuka honey. MSYR was distributed in the flowers, leaves, branches, and roots of ma̅nuka plants, while MSYR-glucose and leptosperin were only observed in the flowers. By immersing a cut flowering branch in a deuterium-labeled aqueous medium, the formation of deuterated leptosperin (leptosperin-<i>d</i>₆) and MSYR-glucose (MSYR-<i>d</i>₆-glucose) was analyzed. When MSYR-<i>d</i>₆ was added, both MSYR-<i>d</i>₆-glucose and leptosperin-<i>d</i>₆ were detected. Supplementation with synthetic MSYR-<i>d</i>₆-glucose also generated leptosperin-<i>d</i>₆, indicating that gentiobioside moiety in leptosperin forms through the conjugation of MSYR with d-glucose, followed by the addition of another d-glucose.