## 3 The Biosynthesis of Artemisinin (Qinghaosu)
### Phase 1 (Isopentenyl Pyrophosphte to Amorpha-4,11-diene)  
This first phase in the biosynthesis of artemisinin is the least controversial, and most of the enzymes involved in the conversion of isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) to amorpha-4,11-diene (**451**) have now been isolated and characterized from _A. annua_. It has been proposed that the IPP used in the biosynthesis of artemisinin comes from both the mevalonate and the mevalonate-independent pathways [287], and it has recently been demonstrated that the cental isoprenoid unit in the FPP (**378**) precursor to artemisinin is predominantly biosynthesisized from the non-mevalonate pathway [102]. \({}^{14}\)C-Labelling studies have confirmed the C\({}_{5}\) terpenoid precursor IPP as a starting point for the biosynthetic pathway to artemisinin [288] and \({}^{13}\)C labeling studies have also been used to characterize the photosynthetic mechanism of _A. annua_ as C3 [289]. The first step in phase 1 involves the conversion of IPP and DMAPP (both C\({}_{5}\)) to the C\({}_{15}\) intermediate, farnesyl pyrophosphate (FPP; **378**) by the enzyme farnesyl diphosphate synthase (FPPS). A cDNA encoding this enzyme, which catalyses the \"head-to-tail\" chain extension of a DMAPP starter by two molecules of IPP, has now been cloned from _A. annua_[290]. FPP sits at a branch point in terpenoid metabolism. Further elaboration to triterpenes and plant sterols (Table 43) requires the \"head-to-head\" coupling of two molecules of FPP, which is catalysed by the enzyme squalene synthase (SQS), as discussed in Section 2.7.2. Conversion of FPP (**378**) to sesquiterpenes, on the other hand, requires the operation of ionase and cyclase enzymes, such as: (_E_)-\(\beta\)-farnesene synthase (Section 2.6.1); germacrene A synthase (Section 2.6.2); \(\beta\)-caryophyllene synthase (Secton 2.6.2); and _epi_-cedrol synthase (Section 2.6.4). The omitted intermediate in the biosynthesis of artemisinin (**495**) is the bicyclic sesquiterpene-amorpha-4,11-diene (**451**) [291], which is formed from FPP (**378**) by the action of the sesquiterpene cyclase, amorpha-4,11-diene synthase (ADS) [138,292]. **Scheme 10. ** Cyclization of FPP (**378**) to amorpha-4,11-diene (**451**), catalysed by the enzyme ADS. cDNAs encoding ADS from _A. annua_ have been isolated, sequenced, and expressed [293, 294]. The mechanism of this particular enzymatic cyclization of FPP has now been studied in detail using a recombinant amorpha-4,11-diene synthase from _A. annua_. It is proposed that FPP is first isomerized to merididyl diphosphate; ionization of merididyl pyrophosphate is followed by C-1,C-6-ring closure to generate a bisabolyl cation; next, this cation undergoes a 1,3-hydride shift [295] permitting a second ring closure between the 1- and 10-positions to generate the amorphane skeleton; and, finally, deprotonation at either C-12 or C-13 affords amorpha-4,11-diene (**451**) [136], as is shown in Scheme 10. This mechanism is supported by GC-MS analysis of several minor sesquiterpene products which are also produced by ADS. These include the known metabolites (_E_)-\(\beta\)-farnesene (**382**), \(\alpha\)-bisabolol (**385**), amorph-4-en-7-ol (**456**) and amorpha-4-7,(11)-diene (**492**). Other minor products from ADS have not yet been reported as natural products from _A. annua_, including the amorphane, amorph-4-en-11-ol (**574**); the bisabolanes: \(\beta\)-sesquiphellandrene (1,3(15),10-bisabolatriene [20307-83-9]) (**575**) [134], zingiberene (1,3,10-bisabolatriene[495-60-3]) (**576**) and zingiberenol (1,10-bisaboladien-3-ol [58334-55-7] [495-60-3]) (**577**); and the humulane: \(\gamma\)-humulene (1,3(15),6-humulatriene [26259-79-0]) (**578**) (Figure 3) [296]. **Figure 3.** Minor products from the cyclization of FPP (**378**) by ADS, which have not yet been reported as natural products from _A. annua_.