Synthesis of [3,3-2H2]-Dihydroartemisinic Acid to Measure the Rate of Nonenzymatic Conversion of Dihydroartemisinic Acid to Artemisinin

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.