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J Vervoort

Publications and source records attributed to J Vervoort.

At least 37 records · Page 2Linked to original sources
PubMed1999

TEAC antioxidant activity of 4-hydroxybenzoates.

The influence of pH, intrinsic electron donating capacity, and intrinsic hydrogen atom donating capacity on the antioxidant potential of series of hydroxy and fluorine substituted 4-hydroxybenzoates was investigated experimentally and also on the basis of computer calculations. The pH-dependent behavior of the compounds in the TEAC assay revealed different antioxidant behavior of the nondissociated monoanionic form and the deprotonated dianionic form of the 4-hydroxybenzoates. Upon deprotonation the radical scavenging ability of the 4-hydroxybenzoates increases significantly. For mechanistic comparison a series of fluorobenzoates was synthesized and included in the studies. The fluorine substituents were shown to affect the proton and electron donating abilities of 4-hydroxybenzoate in the same way as hydroxyl substituents. In contrast, the fluorine substituents influenced the TEAC value and the hydrogen atom donating capacity of 4-hydroxybenzoate in a way different from the hydroxyl moieties. Comparison of these experimental data to computer-calculated characteristics indicates that the antioxidant behavior of the monoanionic forms of the 4-hydroxybenzoates is not determined by the tendency of the molecule to donate an electron, but by its ability to donate a hydrogen atom. Altogether, the results explain qualitatively and quantitatively how the number and position of OH moieties affect the antioxidant behavior of 4-hydroxybenzoates.

Antioxidants↗
PubMed1999

Folding and conformational analysis of AVR9 peptide elicitors of the fungal tomato pathogen Cladosporium fulvum.

The race-specific elicitor AVR9, produced by the phytopathogenic fungus Cladosporium fulvum, is a 28-residue beta-sheet peptide containing three disulfide bridges. The folding of this peptide to its native conformation was examined in the presence of oxidized (GSSG) and reduced (GSH) glutathione at concentrations resembling those present in the endoplasmic reticulum. The concentrations of GSH and GSSG, and the applied temperature strongly affected the folding efficiency. The effect of temperature appeared reversible. The conditions for in vitro folding were optimized and a maximum yield of 60-70% of correctly folded peptide was obtained. In vitro folded AVR9 is equally as active as native fungal AVR9. They both display similar NMR characteristics, indicating that they have the same 3D structure and identical disulfide bridges. Thus, AVR9 can be folded correctly in vitro. This folding can be described by disulfide bridge formation leading to scrambled three-disulfide species, followed by disulfide reshuffling to acquire the native structure. The presence of urea significantly affected the folding of AVR9, indicating that noncovalent interactions play a role in directing correct folding. Protein disulfide isomerase increased the folding rate at least 15-fold, but had no effect on the yield. The folding procedure has also been applied successfully to two mutant AVR9 peptides, (K23A)AVR9 and biotinylated AVR9. We conclude that the 28-residue sequence, without the preprosequence (as present in vivo), contains sufficient information to direct correct folding and disulfide bridge formation in vitro.

Amino Acid Sequence↗
PubMed1998

GSTP1-1 stereospecifically catalyzes glutathione conjugation of ethacrynic acid.

Using 1H NMR two diastereoisomers of the ethacrynic acid glutathione conjugate (EASG) as well as ethacrynic acid (EA) could be distinguished and quantified individually. Chemically prepared EASG consists of equal amounts of both diastereoisomers. GSTP1-1 stereospecifically catalyzes formation of one of the diastereoisomers (A). The GSTP1-1 mutant C47S and GSTA1-1 preferentially form the same diastereoisomer of EASG as GSTP1-1. Glutathione conjugation of EA by GSTA1-2 and GSTA2-2 is not stereoselective. When human melanoma cells, expressing GSTP1-1, were exposed to ethacrynic acid, diastereoisomer A was the principal conjugate formed, indicating that even at physiological pH the enzyme catalyzed reaction dominates over the chemical conjugation.

Amino Acid Substitution↗
PubMed1998

Occurrence of the NIH shift upon the cytochrome P450-catalyzed in vivo and in vitro aromatic ring hydroxylation of fluorobenzenes.

The in vivo cytochrome P450-catalyzed aromatic hydroxylation of a series of fluorobenzenes was investigated with special emphasis on the importance of the fluorine NIH shift. The results obtained demonstrate a minor role for the NIH shift in the metabolism of the fluorobenzenes to phenolic metabolites in control male Wistar rats. These in vivo results could indicate that (1) the NIH shift is an inherently minor process for fluorine substituents or (2) it is a potentially significant process but the presumed epoxide that leads to formation of the NIH-shifted metabolite is lost to an alternative metabolic pathway. In contrast to the in vivo data, in vitro experiments showed a significant amount of an NIH-shifted metabolite for 1,4-difluorobenzene. This result eliminates the explanation that the NIH shift is an inherently minor process for fluorine substituents. Results of additional experiments presented in this paper show that the reduced tendency of fluorine-substituted benzenes to undergo an NIH shift in vivo can-at least in part-be ascribed to the possible existence of alternative pathways for metabolism of the epoxide, such as, for example, GSH conjugation, being more efficient for fluorinated than chlorinated arene oxides.

Animals↗
PubMed1998

19F NMR study on the biodegradation of fluorophenols by various Rhodococcus species.

Of all NMR observable isotopes 19F is the one perhaps most convenient for studies on biodegradation of environmental pollutants. The reasons underlying this potential of 19F NMR are discussed and illustrated on the basis of a study on the biodegradation of fluorophenols by four Rhodococcus strains. The results indicate marked differences between the biodegradation pathways of fluorophenols among the various Rhodococcus species. This holds not only for the level and nature of the fluorinated biodegradation pathway intermediates that accumulate, but also for the regioselectivity of the initial hydroxylation step. Several of the Rhodococcus species contain a phenol hydroxylase that catalyses the oxidative defluorination of ortho-fluorinated di- and trifluorophenols. Furthermore, it is illustrated how the 19F NMR technique can be used as a tool in the process of identification of an accumulated unknown metabolite, in this case most likely 5-fluoromaleylacetate. Altogether, the 19F NMR technique proved valid to obtain detailed information on the microbial biodegradation pathways of fluorinated organics, but also to provide information on the specificity of enzymes generally considered unstable and, for this reason, not much studied so far.

Biodegradation, Environmental↗
PubMed1998

Solid-phase synthesis, conformational analysis, and biological activity of AVR9 elicitor peptides of the fungal tomato pathogen Cladosporium fulvum.

The race-specific peptide elicitor AVR9 of the fungal pathogen Cladosporium fulvum specifically induces a hypersensitive response in tomato genotypes carrying the complementary resistance gene Cf-9. The total chemical syntheses of this 28-residue AVR9 peptide containing three disulfide bonds, and of three mutant peptides [R8K]AVR9, [F10A]AVR9 and [F21A]AVR9, have been accomplished. The syntheses were carried out using a stepwise solid-phase approach based on tBoc chemistry. The disulfide bridges were formed by air oxidation. The correctness of the chemical structure of all folded synthetic peptides was confirmed by combined NMR and MS analyses. The biological activity and a number of physicochemical properties of folded synthetic AVR9 are identical to those of native fungal 28-residue AVR9. The overall conformations of the folded synthetic mutant peptides were comparable to that of synthetic wild-type AVR9 as demonstrated by NMR spectroscopy. Mutant [R8K]AVR9 showed a threefold higher, and mutant [F10A]AVR9 a threefold lower necrosis-inducing activity when compared to synthetic wild-type AVR9. However, mutant [F21A]AVR9 showed hardly any necrosis-inducing activity. Affinity for polyclonal antibodies raised against native fungal AVR9 is positively correlated with the necrosis-inducing activity of the synthetic AVR9 peptides ([R8K]AVR9 > wild-type AVR9 > [F10A]AVR9 > [F21A]AVR9).

Blotting, Western↗
PubMed1998

19F nuclear magnetic resonance as a tool to investigate microbial degradation of fluorophenols to fluorocatechols and fluoromuconates.

A method was developed to study the biodegradation and oxidative biodehalogenation of fluorinated phenols by 19F nuclear magnetic resonance (NMR). Characterization of the 19F NMR spectra of metabolite profiles of a series of fluorophenols, converted by purified phenol hydroxylase, catechol 1,2-dioxygenase, and/or by the yeast-like fungus Exophiala jeanselmei, provided possibilities for identification of the 19F NMR chemical shift values of fluorinated catechol and muconate metabolites. As an example, the 19F NMR method thus defined was used to characterize the time-dependent metabolite profiles of various halophenols in either cell extracts or in incubations with whole cells of E. jeanselmei. The results obtained for these two systems are similar, except for the level of muconates observed. Altogether, the results of the present study describe a 19F NMR method which provides an efficient tool for elucidating the metabolic pathways for conversion of fluorine-containing phenols by microorganisms, with special emphasis on possibilities for biodehalogenation and detection of the type of fluorocatechols and fluoromuconates involved. In addition, the method provides possibilities for studying metabolic pathways in vivo in whole cells.

Biodegradation, Environmental↗
PubMed1997

19F NMR study on the regiospecificity of hydroxylation of tetrafluoro-4-hydroxybenzoate by wild-type and Y385F p-hydroxybenzoate hydroxylase: evidence for a consecutive oxygenolytic dehalogenation mechanism.

The regiospecificity of hydroxylation of tetrafluoro-4-hydroxybenzoate (F4-POHB) by p-hydroxybenzoate hydroxylase (PHBH) and its active site mutant Y385F was investigated by 19F NMR. Evidence is provided that the hydroxylation of F4-POHB is not restricted to the C3 center of the aromatic ring but rather involves sequential oxygenation and dehalogenation steps. The catalytic efficiency of PHBH and Y385F with F4-POHB was optimal near pH 6.5. Below pH 7.0, substantial substrate inhibition occurred. Dianionic F4-POHB was a competent effector, highly stimulating upon binding the rate of flavin reduction by NADPH. Hydroxylation of F4-POHB involved the formation of quinone intermediates as primary products of oxygenolytic defluorination. Ascorbate competed favorably with NADPH for the nonenzymatic reduction of these reactive intermediates and prevented the accumulation of nonspecific oxidation products. 19F NMR showed that the initial aromatic product 2,5,6-trifluoro-3,4-dihydroxybenzoate (F3-DOHB) was further converted to 5,6-difluoro-2,3,4-trihydroxybenzoate (5,6-F2-TOHB). This reaction was most efficient with Y385F. F3-DOHB was not bound in a unique regiospecific orientation as also 2,6-difluoro-3,4, 5-trihydroxybenzoate (2,6-F2-TOHB) was formed. The oxygenolytic dehalogenation of F3-DOHB by PHBH and Y385F is consistent with the electrophilic aromatic substitution mechanism proposed for this class of flavoenzymes. Nucleophilic attack of the carbon centers of F3-DOHB onto the distal oxygen of the electrophilic flavin C(4a)-hydroperoxide occurs when the carbon center has a relatively high HOMO density and is relatively close to the distal oxygen of the flavin C(4a)-hydroperoxide.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1997

13C NMR analysis of the cysteine-sulfenic acid redox center of enterococcal NADH peroxidase.

In order to characterize the native Cys42-sulfenic acid redox center of the flavoprotein NADH peroxidase by NMR, an expression protocol has been developed which yields the [3-13C]Cys42-labeled protein in 100 mg quantities. Difference spectra of the labeled minus unlabeled oxidized enzyme (E) give a peak at 41.3 ppm (relative to dioxane) which represents the Cys42-sulfenic acid. Reduction of labeled E with 1 equiv of NADH gives the air-stable two-electron reduced (EH2) species, and oxidized minus reduced difference spectra give maxima and minima at 41.3 and 30.8 ppm, respectively, corresponding to the Cys42-sulfenic acid and -thiolate species. Peroxide inactivation of E, which has previously been attributed to oxidation of the Cys42-sulfenic acid to the Cys42-sulfinic and/or sulfonic acid states, gives rise to a new maximum in the difference spectrum of Einactive minus E at 57.0 ppm. A similar expression protocol was used to obtain the [ring-2-13C]His-labeled peroxidase HHAA mutant (His10His23Ala87Ala258); the spectral change over the pH range 5.8-7. 8 is attributed to deprotonation of the surface-exposed His23. Furthermore, replacement of Arg303, which is hydrogen bonded to His10, has no effect on the 13C spectrum. These results provide direct evidence in support of the peroxidase Cys42-sulfenic acid/thiol redox cycle and add significantly to our structure-based understanding of protein-sulfenic acid stabilization and function.

Binding Sites↗
PubMed1997

The race-specific elicitor AVR9 of the tomato pathogen Cladosporium fulvum: a cystine knot protein. Sequence-specific 1H NMR assignments, secondary structure and global fold of the protein.

The secondary structure and global fold of the AVR9 elicitor protein of Cladosporium fulvum has been determined by 2D NMR and distance-geometry protocols. The protein consists of three anti-parallel strands forming a rigid region of beta-sheet. On the basis of the NMR-derived parameters and distance geometry calculations, it is evident that the AVR9 protein is structurally very homologuous to carboxy peptidase inhibitor (CPI) of which the X-ray structure is known. The AVR9 protein reveals the presence of a cystine knot, which consists of a ring formed by two disulfide bridges and the interconnecting backbone through which the third disulfide bridge penetrates. This structural motif is found in several small proteins such as proteinase inhibitors, ion channel blockers and growth factors. The implications of the structural relationship between AVR9 and other biologically active proteins are discussed.

Amino Acid Sequence↗
PubMed1997

Three-dimensional structure in solution of the N-terminal lipoyl domain of the pyruvate dehydrogenase complex from Azotobacter vinelandii.

The three-dimensional structure of the N-terminal lipoyl domain of the acetyltransferase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been determined using heteronuclear multidimensional NMR spectroscopy and dynamical simulated annealing. The structure is compared with the solution structure of the lipoyl domain of the A. vinelandii 2-oxoglutarate dehydrogenase complex. The overall fold of the two structures, described as a beta-barrel-sandwich hybrid, is very similar. This agrees well with the high similarity of NMR-derived parameters, e.g. chemical shifts, between the two lipoyl domains. The main structural differences between the two lipoyl domains occur in a solvent-exposed loop close in space to the lipoylation site. Despite their high structural similarity, these lipoyl domains show a high preference for being reductively acylated by their parent 2-oxo acid dehydrogenase. Potential residues of the lipoyl domain involved in this process of molecular recognition are discussed.

Acylation↗
PubMed1997

Influence of substituents in fluorobenzene derivatives on the cytochrome P450-catalyzed hydroxylation at the adjacent ortho aromatic carbon center.

In a previous study, the in vivo cytochrome P450-catalyzed regioselectivity of aromatic ring hydroxylation for a series of (poly)fluorobenzenes could be quantitatively predicted by the calculated frontier orbital density distribution in the aromatic ring [Rietjens et al. (1993) Biochemistry 32, 4801-4812]. However, the relative small fluorine, its size almost comparable to a hydrogen, is not expected to influence the regioselectivity of aromatic hydroxylation due to steric hindrance. The aim of the present study was to investigate the influence of substituents larger than a hydrogen or fluorine on the possibilities for hydroxylation at the adjacent carbon center. First, the in vivo regioselectivity of aromatic ring hydroxylation of a series of C4-substituted fluorobenzenes was investigated. The results obtained demonstrate that a chlorine and cyano C4 substituent do not hamper hydroxylation at the positions ortho to the C4 carbon atom. For 4-chloro- and 4-cyanofluorobenzene, the observed regioselectivity of aromatic hydroxylation correlated with the regioselectivity predicted on the basis of the frontier orbital density distribution. In contrast, a bromine and iodine substituent affected the hydroxylation at the adjacent ortho carbon centers, reducing it to respectively 40 and 6% of the calculated intrinsic reactivity of the carbon centers. Additional experiments were performed to investigate whether the regioselectivity of the aromatic hydroxylation of the C4-substituted fluorobenzene model compounds was influenced by changes in the cytochrome P450 enzyme pattern. Results obtained demonstrate that for these relatively small substrates the regioselectivity of their hydroxylation was not significantly influenced by several cytochrome P450 inducers. This suggests that the active sites of the cytochromes P450 catalyzing the aromatic hydroxylation do not impose a stereoselective orientation of the aromatic rings with respect to the iron-oxo porphyrin reaction center. Thus, the working hypothesis for additional experiments was that the deviations for the regioselectivity of aromatic hydroxylation observed for 4-bromo- and 4-iodofluorobenzene may be ascribed to steric hindrance by the bromine and iodine substituents hampering the attack of the cytochrome P450 iron-oxo species on the adjacent carbon centers in the benzene derivative. This working hypothesis was further tested by investigating whether useful steric correction factors could be derived from the results obtained with the series C4-substituted fluorobenzenes. These correction factors should make it possible to correct calculated relative reactivities of carbon sites for steric hindrance by substituents positioned ortho with respect to the carbon to be hydroxylated. This will make it possible to better explain and predict the regioselectivities for other chlorine-, bromine-, iodine-, and cyano-containing fluorobenzenes. The in vivo regioselectivity of aromatic ring hydroxylation of a series of five chlorine-, bromine-, iodine-, or cyano-containing fluorobenzenes did not correlate with the noncorrected calculated reactivities (r = 0.49). However, upon correction of the calculated reactivity values by using the steric correction factors, a correlation between the observed and calculated regioselectivity for the substrates of the present study was obtained (r = 0.91). Together these results strongly indicate that for the fluorobenzenes studied the main factors directing the regioselectivity of their aromatic hydroxylation are (i) the nucleophilic chemical reactivity of the site to be hydroxylated and (ii) the steric influence of the substituent ortho with respect to the site of hydroxylation. This latter effect appears to be negligible for a fluorine, chlorine, and cyano substituent but significant for a bromine and iodine substituent.

Animals↗
PubMed1997

Purification and properties of 4-hydroxybenzoate 1-hydroxylase (decarboxylating), a novel flavin adenine dinucleotide-dependent monooxygenase from Candida parapsilosis CBS604.

A novel flavoprotein monooxygenase, 4-hydroxybenzoate 1-hydroxylase (decarboxylating), from Candida parapsilosis CBS604 was purified to apparent homogeneity. The enzyme is induced when the yeast is grown on either 4-hydroxybenzoate, 2,4-dihydroxybenzoate, or 3,4-dihydroxybenzoate as the sole carbon source. The purified monooxygenase is a monomer of about 50 kDa containing flavin adenine dinucleotide as weakly bound cofactor. 4-Hydroxybenzoate 1-hydroxylase from C. parapsilosis catalyzes the oxidative decarboxylation of a wide range of 4-hydroxybenzoate derivatives with the stoichiometric consumption of NAD(P)H and oxygen. Optimal catalysis is reached at pH 8, with NADH being the preferred electron donor. By using (18)O2, it was confirmed that the oxygen atom inserted into the product 1,4-dihydroxybenzene is derived from molecular oxygen. 19F nuclear magnetic resonance spectroscopy revealed that the enzyme catalyzes the conversion of fluorinated 4-hydroxybenzoates to the corresponding hydroquinones. The activity of the enzyme is strongly inhibited by 3,5-dichloro-4-hydroxybenzoate, 4-hydroxy-3,5-dinitrobenzoate, and 4-hydroxyisophthalate, which are competitors with the aromatic substrate. The same type of inhibition is exhibited by chloride ions. Molecular orbital calculations show that upon deprotonation of the 4-hydroxy group, nucleophilic reactivity is located in all substrates at the C-1 position. This, and the fact that the enzyme is highly active with tetrafluoro-4-hydroxybenzoate and 4-hydroxy-3-nitrobenzoate, suggests that the phenolate forms of the substrates play an important role in catalysis. Based on the substrate specificity, a mechanism is proposed for the flavin-mediated oxidative decarboxylation of 4-hydroxybenzoate.

Candida↗
PubMed1996

Solution structure of the lipoyl domain of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii.

The three-dimensional solution structure of the lipoyl domain of the 2-oxoglutarate dehydrogenase complex from Azotobacter vinelandii has been determined from nuclear magnetic resonance data by using distance geometry and dynamical simulated annealing refinement. The structure determination is based on a total of 580 experimentally derived distance constraints and 65 dihedral angle constraints. The solution structure is represented by an ensemble of 25 structures with an average root-mean-square deviation between the individual structures of the ensemble and the mean coordinates of 0.71 A for backbone atoms and 1.08 A for all heavy atoms. The overall fold of the lipoyl domain is that of a beta-barrel-sandwich hybrid. It consists of two almost parallel four-stranded anti-parallel beta-sheets formed around a well-defined hydrophobic core, with a central position of the single tryptophan 21. The lipoylation site, lysine 42, is found in a beta-turn at the far end of one of the sheets, and is close in space to a solvent-exposed loop comprising residues 7 to 15. The lipoyl domain displays a remarkable internal symmetry that projects one beta-sheet onto the other beta-sheet after rotation of approximately 180 degrees about a 2-fold rotational symmetry axis. There is close structural similarity between the structure of this 2-oxoglutarate dehydrogenase complex lipoyl domain and the structures of the lipoyl domains of pyruvate dehydrogenase complexes from Bacillus stearothermophilus and Escherichia coli, and conformational differences occur primarily in a solvent-exposed loop close in space to the lipoylation site. The lipoyl domain structure is discussed in relation to the process of molecular recognition of lipoyl domains by their parent 2-oxo acid dehydrogenase.

Amino Acid Sequence↗
PubMed1996

Flavin motion in p-hydroxybenzoate hydroxylase. Substrate and effector specificity of the Tyr22-->Ala mutant.

The side chain of Tyr222 in p-hydroxybenzoate hydroxylase interacts with the carboxy moiety of the substrate. Studies on the Tyr222-->Phe mutant, [F222]p-hydroxybenzoate hydroxylase, have shown that disruption of this interaction hampers the hydroxylation of 4-hydroxybenzoate. Tyr222 is possibly involved in flavin motion, which may facilitate the exchange of substrate and product during catalysis. To elucidate the function of Tyr222 in more detail, in the present study the substrate and effector specificity of the Tyr222-->Ala mutant, [A222]p-hydroxybenzoate hydroxylase, was investigated. Replacement of Tyr222 by Ala impairs the binding of the physiological substrate 4-hydroxybenzoate and the substrate analog 4-aminobenzoate. With these compounds, [A222]p-hydroxybenzoate hydroxylase mainly acts as a NADPH oxidase. [A222]p-hydroxybenzoate hydroxylase tightly interacts with 2,4-dihydroxybenzoate and 2-hydroxy-4-aminobenzoate. Crystallographic data [Schreuder, H.A., Mattevi, A., Oblomova, G., Kalk, K.H., Hol, W.G.J., van der Bolt, F.J.T. & van Berkel, W.J.H. (1994) Biochemistry 33, 10161-10170] suggest that this is due to motion of the flavin ring out of the active site, allowing hydrogen-bond interaction between the 2-hydroxy group of the substrate analogs and N3 of the flavin. [A222]p-Hydroxybenzoate hydroxylase produces about 0.6 mol 2,3,4-trihydroxybenzoate from 2,4-dihydroxybenzoate/mol NADPH oxidized. This indicates that reduction of the Tyr222-->Ala mutant shifts the equilibrium of flavin conformers towards the productive "in' position. [A222]p-Hydroxybenzoate hydroxylase converts 2-fluoro-4-hydroxybenzoate to 2-fluoro-3,4-dihydroxybenzoate. The regioselectivity of hydroxylation suggests that [A222]p-hydroxybenzoate hydroxylase binds the fluorinated substrate in the same orientation as wild-type. Spectral studies suggest that wild-type and [A222]p-hydroxybenzoate hydroxylase bind 2-fluoro-4-hydroxybenzoate in the phenolate form with the flavin ring preferring the "out' conformation. Despite activation of the fluorinated substrate and in contrast to the wild-type enzyme, [A222]p-hydroxybenzoate hydroxylase largely produces hydrogen peroxide. The effector specificity of p-hydroxybenzoate hydroxylase is not changed by the Tyr222-->Ala replacement. This supports the idea that the effector specificity is mainly dictated by the protein-substrate interactions at the re-side of the flavin ring.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1996

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.

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.

Aniline Compounds↗