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

Abstract

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.

Autoxidation of a C2-Olefinated Dihydroartemisinic Acid Analogue to Form an Aromatic Ring: Application to Serrulatene Biosynthesis

“…Therefore, unlike the endoperoxide formation in the conversion of DHAA to artemisinin (Figure 1, 1 to 2), 1,2 there is no rate acceleration in the presence of light in the spontaneous aromatization process of 2,3-didehydro DHAA to the aromatic compound (i.e., Figure 5, 3 to 6). However, similar to transformation of DHAA to artemisinin, 2 there was a concentration dependence in the rate of autoxidation of diene 3 to compound 6 in that more of the aromatic ring was formed when there was more diene 3 in the vial (see SI, Part 10, 68 μg vs 6.8 μg).…”

“…D ihydroartemisinic acid (DHAA, 1) is the biosynthetic precursor to artemisinin (2), an endoperoxide-containing plant natural product used to treat malaria (Figure 1).…”

“…When DHAA is exposed to air, a cascade reaction involving (i) ene reaction to form an allylic hydroperoxide, (ii) C−C bond cleavage, (iii) incorporation of a second molecule of oxygen, and (iv) polycyclization to form artemisinin occurs. 1,2 Due to the important biological activity of the endoperoxide bridge, 3−7 there have been a number of previous studies 8−11 dedicated to elucidating the mechanism of endoperoxide formation in artemisinin biosynthesis. Furthermore, there have been numerous studies focused on the topic of artemisinin synthesis 12−14 and biosynthesis.…”

“…A similar effect was observed when a solid residue of DHAA was left in a vial under UV light. 2 Singlet Oxygen Promotes Aromatization of Diene 3 to Compound 6 in Solution. In order to clarify the potential role of singlet oxygen in the aromatization process of diene 3 to compound 6, diene 3 was exposed to singlet oxygen (Scheme 2).…”

“…Dihydroartemisinic acid (DHAA, 1 ) is the biosynthetic precursor to artemisinin ( 2 ), an endoperoxide-containing plant natural product used to treat malaria (Figure ). When DHAA is exposed to air, a cascade reaction involving (i) ene reaction to form an allylic hydroperoxide, (ii) C–C bond cleavage, (iii) incorporation of a second molecule of oxygen, and (iv) polycyclization to form artemisinin occurs. , Due to the important biological activity of the endoperoxide bridge, − there have been a number of previous studies − dedicated to elucidating the mechanism of endoperoxide formation in artemisinin biosynthesis. Furthermore, there have been numerous studies focused on the topic of artemisinin synthesis − and biosynthesis. , Either singlet oxygen or ozone has been reported to lead to the construction of the endoperoxide.…”