Studies with plant cell cultures of Tripterycium wilfordii isolation of metabolites and biotransformation studies

This thesis is concerned with the use of plant cell cultures in combination with organic synthesis to provide efficient routes for the synthesis of novel diterpene analogs of triptolide (Tl) and tripdiolide (Td), the two most active principles of the Chinese medicinal plant Tripterygium wilfordii. The present study is also devoted to providing a better understanding with regard to the biosynthesis of related diterpenes in the plant cell culture line of T. wilfordii designated as TRP4a. The initial phases of the research involved isolation and identification of diterpene metabolites from TRP4a cell cultures. Twenty nine compounds (28 diterpenes) were isolated, of which 26 (including 14 new natural products) were isolated for the first time from this cell culture line. Based on the structural relationships revealed within this series, a biosynthetic pathway from dehydroabietane to Tl and Td is proposed. The synthetic studies involved initial synthesis of isodehydroabietenolide from the readily available dehydroabietic acid and subsequent ring C functionalization to provide a series of ring C "activated" analogs such as isotriptophenolide, triptophenolide and demethyl isoneotriptophenolide. A new, efficient sequence from isodehydroabietenolide to triptophenolide was developed and a synthesis of demethyl isoneotriptophenolide was completed. The later stages of the present studies were concerned with biotransformation of the various synthetic precursors in both TRP4a whole cell systems and crude enzyme preparations (cell free extract, or CFE). Incubation of isotriptophenolide in TRP4a cell cultures produced two novel epoxy dienones, (7,8)β-epoxy-19-hydroxy-12-oxo-18(4->3)afce0-abieta-3,9(ll),13- trien-18-oic acid lactone, and (7,8)α-epoxy-19-hydroxy-12-oxo-18(4->3)aZ?eo-abieta- 3,9(1 l),13-trien-18-oic acid lactone, along with additional hydroxylated compounds. This is the first example in which a "para-alkylated" phenol is enzymatically converted to the corresponding para-epoxy dienone in one step. Older cell cultures (21 day) tend to give higher yields of these epoxides. Further biotransformation results indicated a possible involvement of a quinone methide intermediate in the formation of these epoxides. Biotransformation of isotriptophenolide with CFE preparations from TRP4a cell cultures gave comparable results as the whole cell system except that the biotransformation proceeded much faster. Certain factors in relation to the yield of the epoxides were also studied. Incubation of triptophenolide in whole cell cultures and CFEs showed only a low level of biotransformation in comparison with isotriptophenolide. A large scale experiment did allow the isolation of some hydroxylated products in low yields. These compounds are structurally comparable with those obtained from biotransformation of isotriptophenolide. Biotransformation of demethyl isoneotriptophenolide in whole cell cultures showed rapid consumption of the precursor. The major product isolated was the hydroxy quinone, 12,19-Dihydroxy-l l,14-dioxo-18(4->3)aibeo-abieta-3,8,12-trien-18-oic acid lactone. Biotransformation of the above ring C "activated" precursors has demonstrated that the position and number of the hydroxy groups in ring C are critical to the fate of the biotransformation.Science, Faculty ofChemistry, Department ofGraduat