## 3 The Biosynthesis of Artemisinin (Qinghaosu)
### Phase 2 (Amorpha-4,11-diene to Dihydroaertemisinic Acid)  
The second phase in the biosynthesis of artemisinin (**495**) involves modification to the isopropylidene group (C-11, C-12 and C-13) in amorpha-4,11-diene (**451**), yielding putative biosynthetic intermediates such as dihydroaertemisinic acid (**480**) and artemisinin acid (**473**). Phase 2 of the biosynthesis has only recently been the subject of experimental investigation and is therefore less well understood than phase 1. In 2005, artemisinin alcohol (**458**), artemisinin aldehyde (**460**), dihydroartemisinic alcohol (**482**) and dihydroartemisinic aldehyde (**483**) were all isolated as natural products from _A. annua_[135]. Collectively, these metabolites suggested the presence of two interlinked pathways for the conversion of amorpha-4,11-diene (**451**) to either dihydroaertemisinic acid (**480**) or artemisinin acid (**473**), as is shown in Scheme 11 [135]. **Scheme 11. ** Various possible routes for the oxidation of the isopropylidene group in amorpha-4,11-diene (**451**), yielding artemisinin acid (**473**) and/or dihydroartemisinic acid (**480**) in phase 2 of the biosynthesis of artemisinin. Although it has been assumed that all transformations at the 11-, 12- and 13- positions of (**451**) in phase 2 would proceed either by oxidation reactions (horizontal) or by reduction reactions (vertical), it is also possible to postulate more \"economical\" routes, such as those involving isomerases, which the author has depicted by diagonal dashed arrows in Scheme 11. Thus, isomerization of the \(\Delta^{11,13}\) bond to the \(\Delta^{11,12}\) position in (**458**) would produce an enol, which would spontaneously tautomerize to the carbonyl group in (**483**), without any necessity for either reduction at C-11/13 or oxidation at C-12. A trichome-specific cDNA for the cytochrome P\({}_{450}\) enzyme, CYP71 AV1, which catalyzes threesuccessive oxidations at the 12-position of amorpha-4,11-diene (**451**) in the \"upper\" oxidative pathway in Scheme 11, has now been isolated (see also Section 4.4). These three sequential oxidations of amorpha-4,11-diene (**451**) produce artemisininic acid (**473**) via artemisininic alcohol (**458**) and artemisininic aldehyde (**460**) [196] (note that the exocyclic 11,13-double bond remains intact in this route). Historically, it has often been assumed that artemisininic acid (**473**) is then the starting point for the third and final phase of the biosynthesis of artemisinin. However, the most recent evidence suggests that dihydroartemisinin acid (**480**), produced from the saturated \"lower\" branch in Scheme 11, is, in fact, the true precursor to artemisinin. Kim and Kim have reported that artemisininic acid is not converted to dihydroartemisinin acid in _A. annua_[297] and more recent biosynthetic studies, employing both labeled artemisininic acid [155] and dihydroartemisininic acid [185], have confirmed that there is no interconversion in either direction between dihydroartemisinin acid (**473**) and artemisininic acid (**480**), as is represented by the \"crossed\" double-headed arrow in Scheme 11. Therefore, if dihydroartemisininic acid (**480**) is the true precursor to artemisinin in phase 3 of the biosynthesis, then reduction of the exocyclic double bond in amorpha-4,11-diene (**451**) must be occurring before artemisininic acid (**473**) in Scheme 11. Interestingly, there is now some evidence for such a route to dihydroartemisininic acid (**480**), which involves two oxidations at C-12 of amorpha-4,11-diene (**451**), producing artemisinin aldehyde (**460**) _via_ artemisininic alcohol (**458**); followed by reduction of the \(\Delta^{11,13}\) double bond in (**460**) to dihydroartemisininic aldehyde (**483**); and, finally, oxidation of (**483**) at C-12 to yield dihydroartemisininic acid (**480**) [135]. A recombinant DBR2 enzyme has been purified to approximately 90% from _E.