title: Interaction between the substrate and the high-valent-iron-oxo porphyrin cofactor as a possible factor influencing the regioselectivity of cytochrome P450 catalysed aromatic ring hydroxylation of 3-fluoro(methyl)anilines.
abstract: In the present study the in vitro and in vivo aromatic ring hydroxylation of a series of amino and/or methyl containing fluorobenzenes, i.e. 3-fluoro(methyl)anilines, was investigated and compared to the calculated density distribution of the reactive frontier pi-electrons of the aromatic substrate. This was done (1) to study to what extent the regioselectivity of the aromatic ring hydroxylation of the 3-fluoro(methyl)anilines could be predicted on the basis of the calculated chemical reactivity, as was previously observed for a series of fluorinated benzenes and monofluoroanilines, and (2) to investigate which factors contribute to possible deviations from the predictions on the basis of the calculated chemical reactivity. Results obtained show that the in vitro 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 cytochrome P450(FeO)3+ species. These results indicate that the HOMO/HOMO-1 frontier 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, however, deviated in a systematic way; C4 para hydroxylation being observed to a higher extent than expected on the basis of chemical reactivity and C2/C6 ortho hydroxylation being observed to a lower extent than expected. Additional experiments were performed using different microsomal preparations and microperoxidase-8. The latter is a mini-heme protein of eight amino acids without a substrate binding site. In incubations of the model compounds with different types of microsomal preparations, as well as with MP-8 and purified reconstructed cytochrome P4502B1, similar systematic deviations between the predicted and observed regioselectivity 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 interaction between the substrate and the substrate binding site of the cytochromes P450 dictating a specific stereoselective positioning of the substrate in the active site. More likely, the systematic deviations between the observed and predicted regioselectivity of hydroxylation of the tested model substrates should be ascribed to an (orienting) interaction between the substrate and the activated cytochrome P450(FeO)3+ cofactor.
authors.affiliation: Department of Biochemistry, Agricultural University, Wageningen, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Koerts
authors.forename: S
authors.initials: S
authors.lastname: Boeren
authors.forename: J
authors.initials: J
authors.lastname: Vervoort
authors.forename: R
authors.initials: R
authors.lastname: Weiss
authors.forename: C
authors.initials: C
authors.lastname: Veeger
authors.forename: I M
authors.initials: IM
authors.lastname: Rietjens
mesh_terms: D000814:Aniline Compounds / Q000378:metabolism*; D000815:Aniline Hydroxylase / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D005464:Fluorobenzenes / Q000378:metabolism*; D006900:Hydroxylation; D008099:Liver / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008297:Male; D008861:Microsomes / Q000201:enzymology / Q000378:metabolism; D015394:Molecular Structure; D051381:Rats; D017208:Rats, Wistar; D013379:Substrate Specificity
chemical_list: D000814:Aniline Compounds; D005464:Fluorobenzenes; D003577:Cytochrome P-450 Enzyme System; D000815:Aniline Hydroxylase