The epothilones are anticancer agents that stabilize microtubules by binding within the paclitaxel binding site in Ì_å_-tubulin; their mechanism of action is analogous to that of TaxolÌâå¨. The structure-activity relationships (SAR) of epothilones have been extensively investigated. Our contributions to these SAR studies have emphasized the role conformation plays in biological function. Conformational analysis predicts two major conformations for the epothilone epoxide/olefinic region. Some groups have suggested that the bioactive conformation of this region most closely resembles conformer 2. Computational methods and high-field NMR experiments on the epothilone pharmacophore have enabled us to distinguish epoxide/olefinic conformer I and epoxide/olefinic conformer II and, subsequently, to determine which most accurately describes the bioactive conformation for this region. We have confirmed through computer modeling techniques that (14S)- and (14R)-methyl epothilone D preferentially exist as epoxide/olefinic conformer I and epoxide/olefinic conformer II, correspondingly. Moreover, the C14-methyl substituent destabilizes the surrogate conformer. (14S)-methyl epothilone D exhibits biological activity against several human cancer cell lines analogous to the naturally occurring epothilones; however, (14R)-methyl epothilone D retains no significant cytotoxicity. Herein, we have employed a concise, efficient second generation synthesis of our designed, C14-methylated epothilone D analogues that shortens our synthetic route by two steps and circumvents some of the difficulties associated with our first generation synthetic route. Subsequently, we investigated their binding interactions with tubulin, including elucidating their thermodynamic binding parameters. These studies demonstrated that (14S)-methyl epothilone D incites microtubule polymerization and binds to the paclitaxel binding site (Kapp = 1.2 x 107 M-1 at 37oC). In contrast, (14R)-methyl epothilone D does not incite microtubule polymerization and does not show any appreciable affinity for the paclitaxel binding site. These data provide additional evidence towards the bioactive conformation of the epoxide/olefinic region of the epothilone class of natural products and suggests that the bioactive conformation of the epoxide/olefinic region is, indeed, epoxide/olefinic conformer I and is most similar to that adopted by (14S)-methyl epothilone D.