Interaction of a fluorescent paclitaxel analogue with tubulin

To study the mechanism of binding of the antitumor agent paclitaxel to microtubules and tubulin, we have synthesized a fluorescent analogue of the drug. A dimethylamino group was introduced onto the 3'-N-benzoyl group of paclitaxel. This compound was synthesized from N-debenzoylpaclitaxel and 3-(dimethylamino)benzoyl chloride in 67% yield. N-Debenzoyl-N-[3-(dimethylamino)benzoyl]-paclitaxel has activity similar to paclitaxel in inducing microtubule assembly and binds to tubulin at the paclitaxel-binding site. Under assembly conditions, binding of this paclitaxel analogue to tubulin occurs in a time-dependent manner and is accompanied by a large increase in fluorescence intensity, as well as a large blue shift in the emission maximum. In addition, evidence is presented to show that this compound also binds to tubulin in the dimeric state, but the binding affinity is much lower (Kd = 49 +/- 8 microM at 25 degrees C) than that reported for polymeric tubulin. The fluorescent paclitaxel analogue, with a high quantum yield, will be a useful tool in studying the mechanism of paclitaxel binding to tubulin and the environment of the paclitaxel-binding site on tubulin.