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Dihydroartemisinic acid (DHAA) is a plant natural product that undergoes a spontaneous endoperoxide-forming cascade reaction to yield artemisinin in the presence of air. The endoperoxide functional group gives artemisinin its biological activity that kills Plasmodium falciparum, the parasite that causes malaria. To enhance our understanding of the mechanism of this cascade reaction, 2,3-didehydrodihydroartemisinic acid (2,3-didehydro-DHAA), a DHAA derivative with a double bond at the C2-position, was synthesized. When 2,3-didehydro-DHAA was exposed to air over time, instead of forming an endoperoxide, this compound predominantly underwent aromatization. This olefinated DHAA analogue reveals the requirement of a monoalkene functional group to initiate the endoperoxide-forming cascade reaction to yield artemisinin from DHAA. In addition, this aromatization process was exploited to illustrate the autoxidation process of a different plant natural product, dihydroserrulatene, to form the aromatic ring in serrulatene. This spontaneous aromatization process has applications in other natural products such as leubethanol and erogorgiaene. Due to their similarity in structure to antimicrobial natural products, the synthesized compounds in this study were tested for biological activity. A group of the tested compounds had minimum inhibitory concentration (MIC) values ranging from 12.5 to 25 μg/mL against the bacterial pathogen Staphylococcus aureus and the fungal pathogen Cryptococcus neoformans.

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.

Dihydroartemisinic acid is the biosynthetic precursor to artemisinin, the endoperoxide-containing natural product used to treat malaria. The conversion of dihydroartemisinic acid to artemisinin is a cascade reaction that involves C-C bond cleavage, hydroperoxide incorporation, and polycyclization to form the endoperoxide. Whether or not this reaction is enzymatically controlled has been controversial. A method was developed to quantify the nonenzymatic conversion of dihydroartemisinic acid to artemisinin using LC-MS. A seven-step synthesis of 3,3-dideuterodihydroartemisinic acid (23) was accomplished beginning with dihydroartemisinic acid (1). The nonenzymatic rates of formation of 3,3-dideuteroartemisinin (24) from 3,3-dideuterodihydroartemisinic acid (23) were 1400 ng/day with light and 32 ng/day without light. Moreover, an unexpected formation of nondeuterated artemisinin (3) from 3,3-dideuterodihydroartemisinic acid (23) was detected in both the presence and absence of light. This formation of nondeuterated artemisinin (3) from its dideuterated precursor (23) suggests an alternative mechanistic pathway that operates independent of light to form artemisinin, involving the loss of the two C-3 deuterium atoms.

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

Dihydroartemisinic acid is the biosynthetic precursor to artemisinin, the endoperoxide-containing natural product used to treat malaria. The conversion of dihydroartemisinic acid to artemisinin is a cascade reaction that involves C–C bond cleavage, hydroperoxide incorporation, and polycyclization to form the endoperoxide. Whether or not this reaction is enzymatically controlled has been controversial. A method was developed to quantify the nonenzymatic conversion of dihydroartemisinic acid to artemisinin using LC-MS. A seven-step synthesis of 3,3-dideuterodihydroartemisinic acid (23) was accomplished beginning with dihydroartemisinic acid (1). The nonenzymatic rates of formation of 3,3-dideuteroartemisinin (24) from 3,3-dideuterodihydroartemisinic acid (23) were 1400 ng/day with light and 32 ng/day without light. Moreover, an unexpected formation of nondeuterated artemisinin (3) from 3,3-dideuterodihydroartemisinic acid (23) was detected in both the presence and absence of light. This formation of nondeuterated artemisinin (3) from its dideuterated precursor (23) suggests an alternative mechanistic pathway that operates independent of light to form artemisinin, involving the loss of the two C-3 deuterium atoms.