## 3 The Biosynthesis of Artemisinin (Qinghaosu)
### Phase 3 (Dihydroartemisinin acid to Artemisinin)
#### 3.3.1 Dihydroartemisinin Acid as a Late-Stage Precursor to Artemisinin
annua_[167,170]; and quantitative studies, which monitored the decline of dihydroartemisinin acid (**480**) and the increase of artemisinin (**495**) during leaf development and senescence have confirmed that an intermediate, such as **481**, is probably involved in this conversion [301] [it is interesting to note that the alternative secondary allylic hydroperoxide from the spontaneous autoxidation of dihydroartemisinin acid (**480**) in step i] has also been proposed as the precursor to another amorphane sesquiterpene endoperoxide from _A. annua_, arteannuin H (**465**) [166], as is shown in Scheme 5 in Section 2.6.3). There is - as yet - no direct evidence from _in vivo_ studies to suggest how further transformation of **481** to the 1,2,4-trioxane ring in artemisinin might occur in _A. annua_. The evidence for steps ii)-iv) which are proposed in Scheme 12 comes solely from _in vitro_ studies, conducted with the tertiary allylic hydroperoxide **481**[171, 236, 237, 239, 254], which can be obtained in good yield from photosensitized oxygenation of **480**[193, 218]. These _in vitro_ experiments have shown that **481** can indeed undergo sponataneous conversion to a transient enol as proposed in step ii) of Scheme 11 [204, 238, 243, 302, 303], and it has even been possible to fully characterize this unstable intermediate by 2D-NMR at low temperature [193]. This known reaction of allylic hydroperoxides is referred to as a Hock cleavage (or a Criegee rearrangement) and it is in this step that carbon-carbon bond cleavage occurs between C-4 and C-5, thereby producing the _seco_-cadinane skeleton of artemisinin (**495**). The double bond in the enol which is produced by step ii) then reacts rapidly with a second molecule of oxygen in step iii). This reaction most probably proceeds _via_ an \"ene-type\" mechanism similar to that for the conversion of **480** to **481**, although apparently without the requirement for light (_i.e._\({}^{3}\)O\({}_{2}\) rather than \({}^{1}\)O\({}_{2}\) is perhaps the active species) [193, 236, 304, 305]. All carbons in the resultant \(\alpha\)-aldehyde hydroperoxide [238] are now at the correct oxidation level and this intermediate immediately \"zips up\" to the 1,2,4-trioxane ring of artemisinin (**495**) in step iv). It has been speculated that the proximity of the 12-carboxylic acid group to the \(\Delta^{4,5}\)-double bond in **480** and the hydroperoxide functionality in **481** might be the reason why both transformations i) and ii) proceed with such apparent ease _in vitro_[218]. Finally, it is worth reiterating that both _in vivo_ and _in vitro_ studies have shown that the tertiary allylic hydroperoxide **481** can be transformed into a wide variety of other compounds, in addition to artemisinin (**495**). Many of these compounds have been obtained previously as natural products from _A. annua_ (see Schemes 5 and 6 in Section 2.6.3). Inspection of Scheme 12 suggests that some of these metabolites, such as dihydroarteannuin B (**479**) and dihydro-_epi_-deoxyarteannuin B (**485**) may be formed by alternative reactions to the Hock cleavage of **481** in step ii), while others such as the _seco_-cadinane **493** may arise by alternative reactions of the enol from Hock cleavage in step iii).