Total Synthesis and Precursor Directed Biosynthesis of Epothilone Analogues

The epothilones are a family of natural products isolated as secondary metabolites from the soil bacteria, Sorangium cellulosum. It was discovered that they inhibit tumor cell proliferation via microtubule stabilization, exhibiting the same mode of action as the famous therapeutic Paclitaxel, or Taxol, but with improved properties. This dissertation describes a precursor directed biosynthesis approach to epothilones using the enzyme machinery that produces these molecules in nature. Three precursors to epothilones were synthesized and participated in precursor directed biosynthesis using individually expressed modules of the epothilone-producing enzyme system. The completion of the synthesis of the naturally occurring epothilone D was accomplished by the appropriate enzymes expressed in host bacteria. Two analogues containing a C14-Me group were also introduced to the recombinant enzyme system. It is known that a C14-Me group can bias the conformation of the otherwise flexible polyketide in an active or inactive form based on the stereochemistry at this center. However, when intermediates including the C14-Me group were subjected to epothilone producing enzymes, the desired products were not formed in quantities comparable to the natural substrate. Studies with the isolated thioesterase domain on synthetic standards suggest this final domain's activity is diminished when a methyl substituent is present at C14, a plausible reason for lack of formation of these analogues. Precursor directed biosynthesis is aided by the availability of such synthetic standards. This thesis includes the total synthesis of one analogue, C14-methyl, C16-desmethyl-epothilone D, whose semi biosynthesis was attempted.