Harnessing enzyme plasticity for the synthesis of oxygenated sesquiterpenoids

8-Methoxy-γ-humulene, (E)-8-methoxy-β-farnesene, 12-methoxy-β-sesquiphellandrene and 12-methoxyzingiberene can be synthesised in amorphadiene synthase-catalysed reactions from 8- and 12-methoxyfarnesyl diphosphates due to the highly plastic yet tightly controlled carbocationic chemistry of this sesquiterpene cyclase.

Several sesquiterpene synthases including ADS accept FDP analogues containing a variety of heteroatoms and functional groups to generate unnatural sesquiterpenoids that are not easily accessible by conventional organic synthesis [10][11][12][13][14][15][16][17][18][19]. Creating novel sesquiterpenoids, not normally found in nature, is of great interest due to the important applications of terpenoids in healthcare and agriculture as well as the potential to tailor their properties to specific needs. For example, fluorinated derivatives of (E)-β-farnesene, a potent alarm pheromone for aphids [20][21][22][23], are more effective as pheromones than the parent compound, and finding high potency derivatives of (E)-β-farnesene may be of significant benefit in agriculture [24]. While (S)germacrene D is a highly volatile but unstable olfactory signal that repels invertebrate arthropod pests (insects, ticks, mites) that affect humans and livestock as well as arable crops, (S)-14,15-dimethylgermacrene D acts as an attractant of aphids [10]. β-Sesquiphellandrene and α-curcumene are both found in turmeric (Curcuma longa) and have been shown to have anti- cancer activity [25,26]. The oxygenated α-curcumene and β-sesquiphellandrene derivatives α-and β-turmerone are reported to possess anticonvulsant properties and are used to treat epilepsy [27,28]. This array of important compounds shows the potential of generating novel sesquiterpenoids with desirable bio-properties.
ADS is a high fidelity sesquiterpene synthase that produces almost exclusively a single product. Its active site plasticity nevertheless allows the conversion of 12-hydroxy-FDP (9) to dihydroartemisinic aldehyde (10), a biosynthetic intermediate and valuable precursor in the synthesis of artemisinin [29].
GC-MS analysis ( Figure 1) of the organic soluble products formed from 8-methoxy FDP (11) through ADS catalysis  revealed the formation of a major sesquiterpenoid of molecular mass 234 (85%). No organic soluble products were detected when ADS was omitted from the incubation mixture. This product was identified as 8-methoxy-γ-humulene (20) by NMR spectroscopy and comparison of its 1 H NMR spectrum ( Figure 2) with that of 8-oxo-γ-humulene, a natural sesquiterpenoid isolated from the plant Cineraria fruticulorum [36].  The ADS-catalysed 1,11-cyclisation of diphosphate 11 suggests that the 8-methoxy group prevents the formation of a conformation conducive to isomerisation to NDP (4, Scheme 1) and hence 1,6-cyclisation to generate a bisabolyl cation and the amorphane skeleton. Rather the active site conformations of 11 and cation 22 appear to enable a 1,11-cyclisation to 23.
GC-MS analysis of the organic soluble products generated from an incubation of ADS with 12-methoxy-FDP (12) revealed the formation of a 1:2.4 mixture of two sesquiterpenoids of mass m/z 234 (Figure 3). Again, no organic soluble products were detected when ADS was omitted from the incubation mixture. The 1 H NMR spectrum of this product mixture ( Figure 4) and comparison with the 1 H NMR spectra of the bisabolyl-derived sesquiterpenes β-sesquiphellandrene [38][39][40] and zingiberene [40], a hydrocarbon with antifertility, antiviral and anticancer activity [41], suggested that the major compound was 12-methoxy-β-sesquiphellandrene (26), while the minor product was identified as 12-methoxyzingiberene (27). The two doublets observed at 0.85 and 0.87 ppm correspond to the C14H 3 groups for both compounds. The 13 C-DEPT-135 spectrum (see Supporting Information File 1) showed an inverted peak at ≈109 ppm, implying the presence of an olefinic CH 2 that couples to the two overlapped doublets at 4.74 ppm. The integration of these doublets as 2 protons suggested that the exocyclic alkene was present in only one of the products. In the literature, this exocyclic alkene in β-sesquiphellandrene is observed at 4.72 ppm as a multiplet [39]. A triplet at 5.39 ppm that integrates for 2 protons can be assigned to H10 in both 26 and 27, which resonates further downfield than in β-sesquiphellandrene and zingiberene due to the methoxy group positioned two carbons away.   27 correspond to the equivalent protons (δ H = 5.61), H2 (δ H = 5.57) and H4 (δ H = 5.42) in zingiberene [40].
It is suggested that the 12-methoxy group enforces an orientation of the distal 10,11-double bond that is not conducive to the

Conclusion
In conclusion, the class I sesquiterpene cyclase amorphadiene synthase facilitates the efficient conversion of readily accessible synthetic methoxy-FDPs to sesquiterpenoids that may have applications in healthcare and agriculture. These results inform us of both the utility and limitations that non-natural functional groups have upon terpene cyclase-catalysed reaction cascades supporting the design of future biocatalytic syntheses. In particular, the presence of an ethereal oxygen atom containing π-acid functionality alongside its inductive withdrawal effect has a profound effect on the carbocationic reactivity of the intermediates. Of course a fully comprehensive interpretation of these results, regarding potentially interesting aspects such as anchimeric assistance is hampered by unknowns such as the conformation of binding to the enzyme and what effect the extra bulk of the substituents has upon the results observed, but nevertheless such empirical results will accumulate to inform future investigations. This reversal of the biosynthetic reaction order is expandable to other terpene synthases to generate libraries of unnatural sesquiterpenoids with a wide range of potential uses and applications across many areas of human activity.

Supporting Information
Supporting Information File 1 Experimental part.