Methyl Syringate Monoglucoside Is a Crucial Intermediate in Leptosperin Biosynthesis in Leptospermum scoparium (ma̅nuka)

Leptosperin (methyl syringate-4-O-β-d-gentiobioside) serves as a unique marker for ma̅nuka honey, derived from the ma̅nuka plant (Leptospermum scoparium). 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-O-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-d₆) and MSYR-glucose (MSYR-d₆-glucose) was analyzed. When MSYR-d₆ was added, both MSYR-d₆-glucose and leptosperin-d₆ were detected. Supplementation with synthetic MSYR-d₆-glucose also generated leptosperin-d₆, indicating that gentiobioside moiety in leptosperin forms through the conjugation of MSYR with d-glucose, followed by the addition of another d-glucose.