## 3 The Biosynthesis of Artemisinin (Qinghaosu)
### Phase 3 (Dihydroartemisinin acid to Artemisinin)  
#### 3.3.1 Dihydroartemisinin Acid as a Late-Stage Precursor to Artemisinin  
In this review, it is assumed that the final steps in the biosynthetic pathway to artemisinin (**495**) proceed from dihydroartemisininic acid (**480**), rather than artemisininic acid (**473**) (or, indeed, any of the other late stage intermediates: **462**, **478**, **479**, **485**, **494** or **497** which have been proposed in the past). Discussions of phase 3 of the biosynthesis therefore commence with dihydroartemisininic acid (**480**) - although the other possibilities are also evaluated in some detail in parts b) - e) of this section. Artemisinin is a _seco_-cadinane (Table 38), and carbon-carbon cleavage at C-4/C-5 in (**495**) therefore accompanies formation of the 1,2,4-trioxane ring in this final phase of the biosynthesis. No enzymes have yet been described for any of these putative reactions in phase 3. Indeed, experiments with classical plant peroxididases, a class of enzyme with the potential for involvement in these kinds of reactions, have failed to increase the yield of artemisinin [299,300]. It is also possible that the final transformations of dihydroartemisinin acid to artemisinin might proceed via non-enzymatic processes. In this regard, it is interesting to note that artemisinin biosynthesis has recently been correlated with increased levels of singlet oxygen -although this hasbeen explained in terms of the upregulation of genes involved in artemisinin biosynthesis, rather than the operation of non-enzymatic processes [256]. A non-enzymatic mechanism involving molecular oxygen is particularly attractive in view of the variety of spontaneous autoxidation reactions which have already been suggested for the biogenesis of many other highly oxygenated terpenes from _A. annua._ For example, the formation of regular acyclic monoterpenes **263-265**, **267**, **271** and **272** in Scheme 1 (Section 2.5.1); irregular acyclic monoterpenes **279** and **280** in Scheme 3 (Section 2.5.2); eudesmane sesquiterpenes **413** and **414** in Scheme 4 (Section 2.6.3); amorphane sesquiterpenes **457**, **465-467**, **477**, **478**, **481**, **484**, **485** and **490** in Scheme 5; **479**, **481**, **485** and **493** in Scheme 6; **462**, **478** and **494** in Scheme 7 (Section 2.6.3); and diterpenes **552** and **553** in Scheme 8 (Section 2.7.1) have all been proposed to proceed by spontaneous autoxidation chemistry. The proposal that the final transformations to artemisinin (**495**) in phase 3 may be non-enzymatic also receives strong support both from _in vivo_ studies [168,185] and from experiments which have been performed _in vitro_[167,193,218], under conditions that are relevant to the living plant. Together, _in vivo_ and _in vitro_ experiments have suggested a mechanism for the conversion of dihydroartemisinin acid (**480**) to artemisinin (**495**) _via_ a spontaneous autoxidation process, involving four steps, as is shown in Scheme 12. **Scheme 12. ** A four-step mechanism for the spontaneous autoxidation of dihydroartemisinin acid (**480**) to artemisinin (**495**) in _A. annua._  
The four steps shown in Scheme 12 are: i) photo-sensitized reaction of the \(\Delta^{4,5}\)-double bond in dihydroartemisinin acid (**480**) with singlet molecular oxygen (_via_ an \"ene-type\" mechanism); ii) Hock cleavage of the resulting tertiary allylic hydroperoxide **481**; iii) oxygenation of the enol product from Hock cleavage; and iv) cyclization of the resulting vicinal hydroperxyl-aldehyde to the 1,2,4-trioxane system of artemisinin (**495**). The tertiary allylic hydroperoxide **481**, which is produced in step i), has already been shown to be a biosynthetic intermediate, linking dihydroartemisinin acid (**480**) and artemisinin (**495**), by _in vivo_ experiments with labeled dihydroartemisinin acid (**480**) [185,189]. Compound **481** has also been described independently as a natural product from _A.