INTERACTION BETWEEN THE SUBSTRATE AND THE HIGH-VALENT-IRON-OXO PORPHYRIN COFACTOR AS A POSSIBLE FACTOR INFLUENCING THE REGIOSELECTIVITY OF CYTOCHROME-P450 CATALYZED AROMATIC RING HYDROXYLATION OF 3-FLUORO(METHYL)ANILINES

In the present study the in vitro and in vivo aromatic ring hydroxylat ion of a series of amino and/or methyl containing fluorobenzenes, i.e. 3-fluoro(methyl)anilines, was investigated and compared to the calcul ated density distribution of the reactive frontier pi-electrons of the aromatic substrate, This was done (1) to study to what extent the reg ioselectivity of the aromatic ring hydroxylation of the 3-fluoro(methy l)anilines could be predicted on the basis of the calculated chemical reactivity, as was previously observed for a series of fluorinated ben zenes and monofluoroanilines, and (2) to investigate which factors con tribute to possible deviations from the predictions on the basis of th e calculated chemical reactivity. Results obtained show that the in vi tro and in vivo aromatic ring hydroxylation of the series of 3-fluoro( methyl)anilines correlates qualitatively with the calculated frontier orbital density distribution for electrophilic attack by the cytochrom e P450(FeO)(3+) species. These results indicate that the HOMO/HOMO-1 f rontier orbital densities, i.e. the chemical reactivity of the carbon centres for an electrophilic attack, predict the preferential as well as the non-reactive sites for cytochrome P450 catalysed aromatic ring hydroxylation of the tested model compounds. The absolute values, howe ver, deviated in a systematic way; C4 para hydroxylation being observe d to a higher extent than expected on the basis of chemical reactivity and C2/C6 ortho hydroxylation being observed to a lower extent than e xpected. Additional experiments were performed using different microso mal preparations and microperoxidase-8. The latter is a mini-heme prot ein of eight amino acids without a substrate binding site. In incubati ons of the model compounds with different types of microsomal preparat ions, as well as with MP-8 and purified reconstructed cytochrome P4502 B1, similar systematic deviations between the predicted and observed r egioselectivity of aromatic hydroxylation were observed. These results show that the regioselectivity of aromatic ring hydroxylation of the 3-fluoro(methyl)anilines cannot be predominantly ascribed to an intera ction between the substrate and the substrate binding site of the cyto chromes P450 dictating a specific stereoselective positioning of the s ubstrate in the active site. More likely, the systematic deviations be tween the observed and predicted regioselectivity of hydroxylation of the tested model substrates should be ascribed to an (orienting) inter action between the substrate and the activated cytochrome P450(FeO)(3) cofactor.