Chemoenzymatic approaches to monoterpenoids

Terpenoids are the largest and structurally most diverse family of natural product. α-pinene, a monoterpene found in turpentine, is a precursor to several high-value monooxygenated terpenoids such as verbenone. It is produced naturally through the Mg2+ -dependent transformation of geranyl diphosphate (GDP) catalysed by (+)-α-pinene synthase (APS). Heterologous expression, purification and characterization of recombinant (+)-α-pinene synthase were performed. Several expression conditions and many purification methods were tried in order to get optimum results. After optimisation of His tagged α-pinene synthase (HAPS), chemoenzymatic synthesis of a simple two-step, one pot asymmetric synthesis from GDP of (+)-verbenone, an oxidation product of the monoterpene (+)-α-pinene, that is used to control attacks of pine trees by bark beetles is described. This method generates (+)-verbenone from GDP in good yield and without the need to isolate the intermediate (+)-α-pinene. Then, synthesis of some chemically modified GDP analogues to obtain (+)-α-pinene analogues was performed. These engineered (+)-α-pinene analogues may display enhanced biological activities due to the potential improvement of their physical properties. Finally, site directed mutagenesis strategy was employed to HAPS to examine the potential catalytic role of several aromatic amino acids, in stabilizing the carbocationic intermediates generated in the active site of HAPS during the natural conversion of GDP to the monoterpene (+)-α-pinene