Synthesis of [15,15,15‑²H₃]‑Dihydroartemisinic Acid and Isotope Studies Support a Mixed Mechanism in the Endoperoxide Formation to Artemisinin

Artemisinin
is the plant natural product used to treat malaria.
The endoperoxide bridge of artemisinin confers its antiparasitic properties.
Dihydroartemisinic acid is the biosynthetic precursor of artemisinin
that was previously shown to nonenzymatically undergo endoperoxide
formation to yield artemisinin. This report discloses the synthesis
of [15,15,15-2H3]-dihydroartemisinic acid and
its use to determine the mechanism of endoperoxide formation. Several
new observations were made: (i) Ultraviolet-C (UV-C) radiation initially
accelerates artemisinin formation and subsequently promotes homolytic
cleavage of the O–O bond and rearrangement of artemisinin to
a different product, and (ii) dideuterated and trideuterated dihydroartemisinic
acid isotopologues at C3 and C15 converted to artemisinin at a slower
rate compared to nondeuterated dihydroartemisinic acid, revealing
a kinetic isotope effect in the initial ene reaction toward endoperoxide
formation (kH/kD ∼ 2–3). (iii)
The rate of conversion from dihydroartemisinic acid to artemisinin
increased with the amount of dihydroartemisinic acid, suggesting an
intermolecular interaction to promote endoperoxide formation, and
(iv) 18O2-labeling showed incorporation of three
and four oxygen atoms from molecular oxygen into the endoperoxide
bridge of artemisinin. These results reveal new insights toward understanding
the mechanism of endoperoxide formation in artemisinin biosynthesis.