## 3 The Biosynthesis of Artemisinin (Qinghaosu)
### Phase 3 (Dihydroartemisinin acid to Artemisinin)
#### 3.3.2 Artemisinic Acid (**473**) as a Late-Stage Precursor to Artemisinin  
[2-\({}^{14}\)C]-Mevalonic acid lactone has been shown to be incorporated into artemisinin acid (**473**) in _A. annua_ using plantlet hydroponic and stem tip feeding methods [306]. Several experiments in the older literature (1990's and earlier) have suggested that artemisinin acid (**473**) can then be converted in to both arteannuin B (**462**) and artemisinin (**495**) [215, 281, 285]. All these experiments employed radiolabelled forms of artemisinin acid that were presented to cell-free systems, which had been derived by extensive manipulations of _A. annua_ plants. However, the most recent biosynthetic study [155], which employed stable isotope-labeled artemisinin acid [15-[\({}^{2}\)H\({}_{3}\)\({}^{13}\)C]-(**473**)] administered to a whole plant system, seemed to show that artemisinin acid was not a biosynthetic precursor to artemisinin. The experimental conditions for this study were purposely chosen to allow comparison with a previous investigation, which had demonstrated that dihydroartemisinic acid [15-[\({}^{2}\)H\({}_{3}\)\({}^{13}\)C]-(**480**)] was a precursor to artemisinin (**495**)) [185] (see discussion in a) above). In addition, it should be noted that a separate study with a cell-free system has also confirmed that dihydroartemisinin acid (**480**) is a precursor to artemisinin in _A. annua_, while artemisinin acid (**473**) is not [307]. One explanation for these apparently contradictory results with artemisinin acid (**473**) may lie in the differing experimental approaches which have been adopted. The threshold for detection by NMR of a metabolite which is labeled with a stable isotope is likely to be several orders of magnitude higher than the corresponding threshold for a radioisotopic label. Thus, it is possible that the \"older\" experiments (1990s and earlier), which detected radiolabel, might - in reality - be identifying only trace quantities of metabolites, which were not derived directly from the labeled precursor. It is possible that partial degradation of a radio-labeled precursor can lead to some radioactivity appearing in a \"pool\" of small molecules, such as acetyl-CoA (see Scheme 13). These small molecules would then serve as precursors for several biosynthetic pathways, including the terpenoid pathway, and could thereby be incorporated indirectly into many metabolites from such pathways. By contrast, the stable isotope-labeled metabolites which have been detected by NMR [155] represented at least 1% (very often between 5-30%) of the label that had been supplied and therefore provide a picture only of the most significant and direct transformations which have been undergone by the precursor. In addition, it should also be pointed out that the possibilities for introducing artifacts when performing feeding studies with whole plants, which require no external manipulation, is likely to be significantly reduced as compared to cell-free extracts, which must endure many perturbations to the biological system (homogenization, addition of buffers _etc._). Although the most recent experiments with artemisinin acid (**473**) failed to observe any detectable incorporation into artemisinin, a very significant conversion was observed into artemisinin B (**462**), as well as six other highly oxygenated sesquiterpene natural products, all of which retained the 11,13-double bond, as shown in Scheme 7 in Section 2.6.3 [155] [furthermore, there was no evidence for conversion to any 11,13-dihydro metabolite, including artemisinin (**495**)]. Such transformations can most easily be accounted for by oxidation of the \(\Delta^{4,5}\)-double bond in **473** to a hydroperoxide, analogous to that postulated in Scheme 12 for dihydroartemisinin acid (**481**). There is indeed ample precedent for the formation of such hydroperoxides from **473** by photooxygenation reactions _in vitro_[243], which lead both to artemisinin B (**462**) [251] and 11,13-dehydro analogues of artemisinin [254,308].