EFFECT OF 2'-OH ACETYLATION ON THE BIOACTIVITY AND CONFORMATION OF 7-O-[N-(4'-FLUORESCEINCARBONYL)-L-ALANYL]TAXOL - A NMR-FLUORESCENCE MICROSCOPY STUDY

The relationship between conformation, 2'-OH acetylation, and bioactiv ity of two fluorescent taxoids has been investigated by a combination of NMR and fluorescence microscopy techniques. These taxoids present t he structure of taxol with the 7-OH group esterified with the N-(4'-fl uoresceincarbonyl)-L-alanine group and with the 2'-OH group free (taxo id 2) or acetylated (taxoid 3). The larger water solubility of 2 and 3 compared with taxol allowed a detailed NMR study in DMSO-d(6)/D2O (3/ 7), showing that both taxoids adopt a similar collapsed conformation i n which the hydrophobic groups 2-O-benzoyl, 3'-phenyl and 4-O-acetyl a re in close proximity, with the fluorescein group displaying unrestric ted motion. On the other hand, while taxoid 2 retains essentially the ability of taxol to induce in vitro microtubule assembly and to bind t o cell microtubules, the 2'-acetylated derivative 3 does not show imme diate activity. However, when taxoid 3 is left in the cell culture, th e slow hydrolysis of the 2'-acetate group in the medium liberates the cytotoxic, microtubule-specific taxoid 2. The intense emission of this active derivative (2) allows the accurate recording of the drug-cell interaction from the very initial steps using fluorescence microscopy. These experiments show conclusively, for the first time in cell cultu res, that a free 2'-OH group in taxol is essential for the recognition of the drug by the binding site of cellular microtubules. (C) 1998 El sevier Science Ltd. All rights reserved.