Mechanism of the N-Hydroxylation of Primary and Secondary Amines

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Mechanism of the N-hydroxylation of primary and secondary amines by Cytochrome P450 Signe Teuber Seger, Patrik Rydberg, and Lars Olsen Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/tx500371a • Publication Date (Web): 04 Feb 2015 Downloaded from http://pubs.acs.org on February 11, 2015

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Chemical Research in Toxicology

Mechanism of the N-Hydroxylation of Primary and Secondary Amines by Cytochrome P450 Signe T. Seger,†* Patrik Rydberg, and Lars Olsen Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, DK-2100 Copenhagen, Denmark KEYWORDS: Cytochrome P450, N-hydroxylation, Density Functional Theory (DFT), Primary and secondary amines, Drug metabolism

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ABSTRACT: Cytochrome P450 enzymes (CYPs) metabolize alkyl- and arylamines, generating several different products. For the primary and secondary amines, some of these reactions result in hydroxylated amines, which may be toxic. Thus, when designing new drugs containing amine groups, it is important to be able to predict if a given compound will be a substrate for CYPs, in order to avoid toxic metabolites, and hence to understand the mechanism that is utilized by CYPs. Two possible mechanisms, for the N-hydroxylation of primary and secondary amines mediated by CYPs, are studied by density functional theory (DFT) for four different amines (aniline, N-methylaniline, propan-2-amine, and dimethylamine). The hydrogen abstraction and rebound mechanism is found to be preferred over a direct oxygen transfer mechanism for all four amines. However, in contrast to the same mechanism for the hydroxylation of aliphatic carbon atoms, the rebound step is shown to be rate-limiting in most cases.

Introduction In man, drugs and other xenobiotics containing amine groups (primary, secondary or tertiary) are often metabolized by the cytochromes P450 (CYP) enzyme family.1 These enzymes are promiscuous and perform a multitude of chemical reactions. For example, CYPs metabolize both alkylamines and arylamines, generating several different products. The most common product that is generated from tertiary amines results from N-dealkylation, however, for some amines the N-oxide is formed as well.2 The mechanism for the reactions leading to these two products, and the competition between them, has been investigated in several studies during the last few years.3–5 However, for primary and secondary amines, the oxidation of the nitrogen atom does not lead to the formation of an N-oxide, but to the hydroxylamine. It is important to understand the mechanism of hydroxylamine formation, as it has been shown that hydroxylamines formed from primary arylamines may be carcinogenic,6 while the ones formed from secondary


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alkylamines may lead to mechanism-based inhibition of CYP enzymes.7 Understanding the mechanisms that lead to the formation of hydroxylamines and other products is also vital for the construction of mechanistic site of metabolism prediction models.8,9 Two recent studies have investigated the mechanisms of CYP oxidation of primary amines with different results. Rydberg and Olsen10 investigated the N-hydroxylation of the primary alkylamine propan-2-amine, and came to the conclusion that two mechanisms could potentially occur: Direct oxygen addition rearrangement (OAR), and hydrogen atom transfer and rebound mechanism (HAT), shown in Scheme 1. A small preference for the HAT mechanism was observed, and the rebound step was found to have the highest transition state energy for both the doublet and quartet spin state. Ji and Schüürmann11 studied the primary arylamine aniline, and showed that the same two pathways were the most likely. However, in contrast to the previous study they found that for the OAR mechanism the rearrangement of the N-oxide to the Nhydroxyl product was the step with the highest barrier, and for the HAT pathway the rebound step was barrierless in the doublet spin state. Other mechanisms that were investigated in these two studies, and ruled out as unlikely, were: two mechanisms involving electron transfer reactions, a direct insertion of the oxygen atom into the nitrogen-hydrogen bond of the amine, and an anionic route catalyzed by the ferric peroxo dianion (FeIIIOO2-). Another study of the latter mechanism, however, has pointed out a correlation with experimental mutagenicity data for aryl amines.12 While the mentioned studies investigate different types of amines, the discrepancies between the results are significant. In this study we investigate the OAR and HAT mechanism for four different types of amines: primary and secondary aromatic and aliphatic amines (shown in Figure 1); to explore if the rate-determining step and the mechanism used by the CYPs are dependent


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upon the type of amine substrate. The results are computed using density functional theory with the B3LYP functional13–15 with an added dispersion correction. Inclusion of a dispersion correction has been shown to be extremely important when calculating reaction barriers involving highly conjugated systems such as the heme in CYPs.16

Methods All calculations have been performed with the Turbomole package,17 version 6.3. A reduced heme model without side chains has been used to describe the catalytic core of CYP. The model consists of iron porphine with CH3S- and O2- as axial ligands. The B3LYP functional13-15 has been used along with the VWN(V) correlation functional18 (unrestricted formalism for open shell systems). The geometry optimizations as well as the frequency and solvent calculations have been performed with the 6-31G(d) basis set19-21for all atoms except Fe. For Fe the double-ζ basis set of Schäfer,22 enhanced with a p function with the exponent 0.134915, was used. Subsequently, single-point calculations were performed on the optimized geometries, using the 6-311++G(2d,2p) basis set23,24 for all atoms except Fe, for which the double-ζ basis set of Schäfer,22 enhanced with s (exponent of 0.01377232), p (0.041843), d (0.1244), and two f functions (2.5 and 0.8) was employed. In all calculations the D3 dispersion correction was used.25 Calculations in implicit solvent were carried out with COSMO26 (continuum conductorlike screening model), using a dielectric constant of 4. The optimized COSMO radii in Turbomole27 have been used as the atomic radii in solvent and Fe was assigned an atomic radius of 2.0 Å. The final energies presented in this paper are the outcome of the single-point calculations (using the large basis set) and include zero point vibrational corrections and solvation effects (both calculated using the smaller basis set).


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Results and Discussion Aniline. The reaction profiles for the HAT and OAR mechanisms for aniline are shown in Figure 2, both for the solution phase (calculated with a dielectric constant of 4 to represent a protein environment) and for the gas phase. The doublet and quartet states have virtually identical energies in the reactant state and for the hydrogen abstraction transition state and intermediate in the HAT mechanism. However, the doublet state is significantly lower in energy for both the transition states involving the formation of an oxygen-nitrogen bond (the rebound step in the HAT mechanism and the direct oxygen in the OAR mechanism), and for the two product states. The results for the OAR mechanism are in agreement with earlier studies of tertiary amines and the primary alkylamine propan-2-amine,10,11,28–30 with the doublet state being favored over the quartet state. In the quartet state the electrons in the two acceptor orbitals of compound I have parallel spin (as opposed to the doublet state, where they have opposite spin), while the lone pair electrons on nitrogen have opposite spin. Thus, another higher-lying orbital of Fe is involved in the quartet state, giving rise to a product state that has higher energy than the doublet state. 10,28 As can be seen in Figure 2, the barrier for interconversion between the reactant complex and the radical intermediate is quite low compared to the barriers converting these two species to the corresponding products. Thus, we expect the reactant and the radical intermediate to be in fast equilibrium, and we do not expect diffusion of the intermediate to occur as easily. This means (according to the Curtin-Hammett principle) that the product distribution will be determined both by the transition state energies of the competing reactions, and the relative energy of the two species in equilibrium. Thus, the OAR mechanism has a reaction barrier of 71.5 kJ/mol (in solution), while the HAT rebound mechanism has a reaction barrier of 52.4 kJ/mol, making the


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latter the preferred reaction path. It can be seen that solvation lowers the energy of the ionic OAR reaction path, compared to the gas phase reaction. This is expected, as the solvent will stabilize the transition state by screening the developing charges (lowered by 13.9 kJ/mol in the doublet state), and also stabilize the charges in the zwitter-ionic product (lowered by 14.1 kJ/mol). In contrast, the rebound step of the radicaloid HAT reaction does not benefit from solvation, and the gas phase energies are slightly more favorable (by 2.4 kJ/mol for the transition state and 5.1 kJ/mol in the product). This is in accordance with the solvent effects reported for the quartet rebound transition state in a previous study of aniline.11 In the hydrogen abstraction step solvent has a stabilizing effect on the transition state. The present HAT mechanism, with a low hydrogen abstraction barrier (11 kJ/mol) and a high rebound barrier (52.4 kJ/mol), stands in contrast to the traditional mechanism for hydroxylation of aliphatic carbon atoms, for which the hydrogen abstraction barrier is the limiting step and the rebound barrier is small or nonexistent.31 The change in rate-determining step, from the hydroxylation of aliphatic carbons to the hydroxylation of aniline, can be explained by the higher stability of the formed aniline radical, than the carbon radical. This stabilization is already present in the transition state. Our results are in agreement with previous findings for propan-2-amine,10 but stand in contrast to a previous study of aniline.11 which reported an energy profile for the HAT mechanism without an intermediate or a rebound transition state in the doublet spin state both in solvent and in gas phase. However, in the quartet state we find similar energies, and the remaining transition states are also close in energy. To ensure that our findings were not an artifact of the employed method (model system, basis sets and dispersion correction), we repeated the calculations of the HAT mechanism with the model system and basis sets used in the previous study of aniline11 in the doublet spin state. We found energies for the rebound intermediate and transition state that were


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within 3 kJ/mol of the ones found with our initial setup. In addition, using B3LYP with the VWN(III) functional (employed in the previous study11) or with VWN(V) (employed in the present study) give rise to the same activation energies. Thus, the discrepancy between the rebound barriers found in this and in the previous study,11 does not seem to arise from the use of different basis sets, model systems or the use of dispersion correction. However, it may be due to different methods of searching for transition states, as some transition states can be difficult to locate, and thus might be overlooked by the program. We believe that the rate-determining step of the HAT mechanism is the rebound step and it seems unlikely that the rebound step should occur without a barrier, since the intermediate that is formed in the hydrogen abstraction step has a hydrogen bond between the amine nitrogen lone pair and the hydroxyl group on the heme (see Figure 2 and reference 11). Breaking this hydrogen bond and forming an oxygen-nitrogen bond without encountering any energetic barrier seems unlikely. Rearrangement of aniline N-oxide to hydroxylamine. The experimentally observed product from N-hydroxylation of primary and secondary amines by CYPs is not the N-oxide, but the hydroxyl amine. Thus, the N-oxide product formed in the OAR mechanism would have to rearrange into the more stable N-hydroxylamine. An enzyme mediated rearrangement has been shown to have a higher reaction barrier than the OAR transition state.11 However, the rearrangement between these two products might not be enzyme mediated, as the process could be more facile in water (previously shown for the same rearrangement of propan-2-amine10). To deduce this, we investigated if a cluster of three explicit water molecules could facilitate the movement of one proton from the nitrogen atom to the oxygen atom in the N-oxide product, and found a low barrier of 8 kJ/mol. The water mediated rearrangement takes place by a proton transfer through two water molecules (wat1 and wat2 in Figure 3a) while a third water molecule


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(wat3) stabilizes the transition state by forming hydrogen bonds to one of the other water molecules and the amine. The third water molecule was added to explicitly solvate the amine, which is usually more precise than using implicit solvent to model this interaction. The mechanism for the rearrangement is shown in Figure 3b. Thus, the fact that the N-oxide is not observed experimentally,32 does not rule out the OAR mechanism, as the subsequent rearrangement to the more stable hydroxyl amine (by 44kJ/mol) should take place instantaneously in water. However, as it appears from Figure 2 the OAR mechanism requires significantly higher activation energy than the HAT mechanism (19.1 kJ/mol), which makes the OAR mechanism unfeasible. N-methylaniline. The results for the secondary arylamine, N-methylaniline, are shown in Figure 4, and are similar to those for aniline, but with a few exceptions. The radical intermediate is 13.2 kJ/mol lower in energy than the corresponding intermediate for aniline. This is expected, as a secondary radical is more stable than a primary one. As can be seen from Figure 4, the transition state energy is lowered along with the energy of the intermediate to a degree that the hydrogen abstraction from N-methylaniline will occur spontaneously. In vacuum calculations, the transition state energy is higher than the energy of the reactant, but since both the contributions from zero-point vibrational corrections and solvent corrections lower the energy of the transition state, we end up with a total energy that is lower than the energy of the reactant state. This difference, in hydrogen abstraction barriers between a primary and a secondary arylamine, is similar to what has been found for the hydroxylation of primary and secondary aliphatic carbon atoms.33 As before, we expect the reactant complex and the radical intermediate to be in equilibrium (due to a low barrier of interconversion between these species, compared to the respective barriers of the product-forming reactions), which gives a HAT rebound barrier of


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70.1 kJ/mol and an OAR barrier of 74.5 kJ/mol. In contrast to the mechanism for aniline, the activation energies for the HAT and OAR pathways are in this case quite similar. There is a 4.4 kJ/mol preference for the HAT mechanism, but based on such a small energy difference the OAR mechanism cannot be ruled out. Propan-2-amine. Figure 5 shows the reaction profile of the primary aliphatic amine propan-2amine. In contrast to the almost planar geometry of the amino groups of the aryl amines discussed above, the amino group of propan-2-amine has a trigonal pyramidal geometry, giving rise to an additional nitrogen inversion step in the OAR mechanism. The nitrogen inversion is required in order for the substrate to reorient to the proper geometry for the direct oxidation to take place. While the arylamines also need to rotate to break the hydrogen bond, they cannot undergo an inversion since their amine groups are almost planar. The inversion required for the N-oxidation of propan-2-amine is followed by a semi-stable intermediate before the oxidation takes place with virtually no barrier (in the doublet spin state). In this case, the rebound step and the hydrogen abstraction step in the HAT mechanism have identical energies (45.3 and 45.6 kJ/mol). So in contrast to our findings for aniline and N-methylaniline, the hydrogen abstraction transition state impose a significant reaction barrier, which means that the reactant and the radical intermediate cannot be assumed to be in equilibrium. Consequently, the hydrogen abstraction step (45.3 kJ/mol), and not the rebound step (30.6 kJ/mol) will be rate determining for the hydroxylation of propan-2-amine. As before, the HAT mechanism represents the preferred reaction path, as the OAR reaction barrier is 8.5 kJ/mol higher, than the hydrogen abstraction barrier. We have previously studied the N-hydroxylation of propan-2-amine, using B3LYP without a dispersion correction.10 The energy profile we found then, is qualitatively the same as the one


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shown in Figure 5 (where a dispersion correction has been used). The major difference is that all barriers are 12-16 kJ/mol lower when the dispersion correction is applied. This difference is due to the stabilizing dispersion interaction between the heme and the substrate, which is not properly accounted for by B3LYP alone. The stabilizing effect is present both in the reactant and in the TS, but since the substrate and the heme are in closer contact in the TS, the dispersion effect is larger here; thus the lower energy barrier.16 However, the relative energies of the different barriers, calculated with and without the dispersion correction, are very similar. Dimethylamine. The reaction profile of dimethylamine is shown in Figure 6, and differs from the profile for propan-2-amine at some important points. In the OAR mechanism we find no semi-stable intermediate or oxidation transition state in the doublet spin state, which means that after the inversion transition state the formation of the N-oxide product is barrierless. In the HAT mechanism the barrier for the hydrogen abstraction step is significantly lower than the barrier for the rebound step, in contrast to our findings for propan-2-amine. The difference in the barrier height of the hydrogen abstraction step, for the two studied alkylamines, has two reasons. Firstly, dimethylamine is a secondary amine, and has two methyl groups stabilizing the radical intermediate. Secondly, the solvation effects lower the barrier by 17 kJ/mol (compared to a destabilization of 5 kJ/mol in propan-2-amine). Due to the low energy barrier for interconversion between the reactant and the radical intermediate, we assume that the reactant and the radical intermediate are in fast equilibrium, giving a total activation energy of 53.6 kJ/mol for the OAR mechanism and of 44.9 kJ/mol for the HAT mechanism. Thus, there is a preference of 8.7 kJ/mol for the HAT mechanism. Trends for N-hydroxylation of primary and secondary amines. Table 1 summarizes our findings for the four investigated amines (aniline, N-methylaniline, propan-2-amine, and


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dimethylamine). It is a general trend that the HAT mechanism is preferred over the OAR mechanism. However, in some cases the energetic preference is small; indicating that other factors, such as substrate orientation and solvation within the active site, could influence which mechanism is preferred. For all other amines than propan-2-amine, the rate-determining step of the HAT mechanism is the rebound step. Our results suggest that amines which form stable radical intermediates (arylamines and secondary alkylamines) will have a low hydrogen abstraction barrier, and thus a rate-determining rebound step, while the hydrogen abstraction step will be rate-determining for primary alkyl amines, which form less stable radical intermediates. The aromatic amines have particularly high rebound barriers, as delocalization of the radical, renders their intermediates less reactive. This is apparent from the spin distributions in the intermediate states (Table S7-10). In the case of aniline, the radical intermediate has a spin of 0.61 on nitrogen (in the doublet state), while N-methylaniline has a spin of -0.64 on nitrogen, indicating that the radical is delocalized to the aromatic ring and thus less reactive. In contrast, the aliphatic amine radicals are localized on nitrogen (spin -0.96 for propan-2-amine and -0.92 for dimethylamine) and thus more reactive. N-methylaniline has the highest reaction barrier of 70.1 kJ/mol. This is in good agreement with the fact, that hydroxylamine formation is not observed experimentally for N-alkylaniline fragments, since the competing N-dealkylation reaction has a significantly lower activation energy.34 Aniline has a substantial reaction barrier as well (52.4 kJ/mol), which might explain why aniline itself is not mutagenic.12

Conclusions In conclusion, we have made three findings regarding the CYP mediated N-hydroxylation of primary and secondary amines. Firstly, the HAT mechanism is consistently preferred to the OAR mechanism, though the energetic difference is small in some cases, indicating that the enzyme


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potentially could perform both mechanisms. Secondly, the HAT mechanism has a rebound barrier both in the doublet and quartet spin states, and finally, the rebound step is ratedetermining for the anilines and for dimethylamine, whereas the hydrogen abstraction step is rate-determining for propan-2-amine. Thus, our study shows that the rate-determining step is dependent upon the amine substrate, which means that both the hydrogen abstraction transition state and the rebound transition state of the HAT mechanism should be investigated when using computational methods to predict CYP reactivity. In a drug discovery and toxicology context, knowledge about these reaction mechanisms is important, as it can be used to avoid toxic side effects in drug candidates, by eliminating compounds that are likely to be N-hydroxylated by CYPs.

ASSOCIATED CONTENT Supporting Information: Coordinates for all computed complexes, relative energies for all states, spin distributions for all states, imaginary frequencies for all transition states, and distances for selected bonds. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected] Present Addresses


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† Department of Protein Structure and Interaction, Biopharmaceutical Research Unit, Novo Nordisk A/S, Novo Nordisk Park, DK-2760 Måløv, Denmark. Notes This manscript is dedicated to Dr. Rydberg, deceased prior to publication. The authors declare no competing financial interest. Funding Sources The authors are grateful for the financial support from the Danish Council for Independent Research and Lhasa Ltd. ABBREVIATIONS CYP, Cytochrome P450; DFT, density functional theory; OAR, oxidation addition rearrangement; HAT, hydrogen atom transfer; COSMO, continuum conductor-like screening model


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(23) Mclean, A. D. and Chandler, G. S. (1980) Contracted Gaussian basis sets for molecular calculations. I. Second row atoms, Z=11–18. J. Chem. Phys. 72, 5639–5648. (24) Krishnan, R., Binkley, J. S., Seeger, R., and Pople, J. A. (1980) Self-consistent molecular orbital methods. XX. A basis set for correlated wave functions. J. Chem. Phys. 72, 650–654. (25) Grimme, S., Antony, J., Ehrlich, S., and Krieg, H. (2010) A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements HPu. J. Chem. Phys. 132, 154104. (26) Klamt, A. and Schuurmann, G. (1993) COSMO: A new approach to dielectric screening in solvents with explicit expressions for the screening energy and its gradient. J. Chem. Soc., Perkin Trans. 2, 799–805. (27) Klamt, A., Jonas, V., Burger, T., and Lohrenz, J. C. W. (1998) Refinement and parametrization of COSMO-RS. J. Phys. Chem. A 102, 5074–5085. (28) Rydberg, P., Ryde, U., and Olsen, L. (2008) Sulfoxide, sulfur, and nitrogen oxidation and dealkylation by cytochrome P450. J. Chem. Theory Comput. 4, 1369–1377. (29) Li, C. S., Wu, W., Cho, K. B., and Shaik, S. (2009) Oxidation of tertiary amines by cytochrome P450-kinetic isotope effect as a spin-state reactivity probe. Chem.-Eur. J. 15, 8492– 8503. (30) Roberts, K. M. and Jones, J. P. (2010) Anilinic N-oxides support cytochrome P450mediated N-dealkylation through hydrogen-atom transfer. Chem.-Eur. J. 16, 8096–8107.


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(31) Shaik, S., Cohen, S., de Visser, S. P., Sharma, P. K., Kumar, D., Kozuch, S., Ogliaro, F., and Danovich, D. (2004) The "Rebound controversy": An overview and theoretical modeling of the rebound step in C-H hydroxylation by cytochrome P450. Eur. J. Inorg. Chem. 2004, 207– 226. (32) Nishida, C. R., Knudsen, G., Straub, W., and de Montellano, P. R. O. (2002) Electron supply and catalytic oxidation of nitrogen by cytochrome P450 and nitric oxide synthase. Drug Metab. Rev. 34, 479–501. (33) Shaik, S., Kumar, D., de Visser, S. P., Altun, A., and Thiel, W. (2005) Theoretical perspective on the structure and mechanism of cytochrome P450 enzymes. Chem. Rev. 105, 2279-2328. (34) Olsen, L., Rydberg, P., Rod, T. H., and Ryde, U. (2006) Prediction of activation energies for hydrogen abstraction by cytochrome P450. J. Med. Chem. 49, 6489–6499.


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TABLES Table 1. Reaction barriers, preferred mechanism, and rate-determining steps are listed for each of the four investigated amines. Amine

Type

∆G‡OARa)

∆G‡Hb)

∆G‡Rec)

(kJ/mol)

(kJ/mol)

(kJ/mol)

PMd)

RDSe)

Aniline

1o aromatic

71.5

11.6

52.4

HAT

Rebound

N-methyl aniline

2o aromatic

74.5

-7.2

70.1

HAT

Rebound

Propan-2amine

1o aliphatic

53.8

45.3

30.6

HAT

H-abstraction

Dimethyl amine

2o aliphatic

53.6

4.9

44.9

HAT

Rebound

a)

∆G‡OAR = reaction barrier for the direct oxidation reaction. b) ∆G‡H = reaction barrier for hydrogen abstraction. c) ∆G‡Re = reaction barrier for the rebound step. d) PM = Preferred mechanism. e) RDS = Rate determining step.


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FIGURE LEGENDS Figure 1. The amine substrates investigated in this study. Figure 2. Reaction profiles for aniline (black: solution phase free energy of the doublet state, gray: gas phase free energy of the doublet state, dark blue: solution phase free energy of the quartet state, light blue: gas phase free energy of the quartet state). Energies are given in kJ/mol. B3LYP energies for all species optimized without the D3 dispersion correction are given in the supporting information. For the doublet state, distances of bonds that are involved in the reaction are given in Å (gray numbers). Bond distances for the quartet state can be found in the supplementary data Table S12. Figure 3. a) Transition state structure for the water mediated rearrangement of aniline oxide to hydroxyl amine. Two water molecules (wat1 and wat2) take part in the transfer, while a third water molecule (wat3) stabilizes the transition state by forming hydrogen bonds to the amine group and wat2. b) Mechanism for the rearrangement of aniline oxide to hydroxylamine. Two water molecules are directly involved in the proton transfer. Figure 4. Reaction profiles for N-methylaniline (black: solution phase free energy of the doublet state, gray: gas phase free energy of the doublet state, dark blue: solution phase free energy of the quartet state, light blue: gas phase free energy of the quartet state). Energies are given in kJ/mol. For the doublet state, distances of bonds that are involved in the reaction are given in Å (gray numbers). Bond distances for the quartet state can be found in the supplementary data Table S13.


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Figure 5. Reaction profiles for propan-2-amine (black: solution phase free energy of the doublet state, gray: gas phase free energy of the doublet state, dark blue: solution phase free energy of the quartet state, light blue: gas phase free energy of the quartet state). Energies are given in kJ/mol. For the doublet state, distances of bonds that are involved in the reaction are given in Å (gray numbers). Bond distances for the quartet state can be found in the supplementary data Table S14.

Figure 6. Reaction profiles for dimethylamine (black: solution phase free energy of the doublet state, gray: gas phase free energy of the doublet state, dark blue: solution phase free energy of the quartet state, light blue: gas phase free energy of the quartet state). Energies are given in kJ/mol. For the doublet state, distances of bonds that are involved in the reaction are given in Å (gray numbers). Bond distances for the quartet state can be found in the supplementary data Table S15.


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FIGURES Figure 1 NH NH 2

aniline

N H

N -methylaniline

NH2

propan-2amine

dimethyl amine

Figure 2


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Figure 3


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Figure 4


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Figure 5


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Figure 6


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SCHEME LEGENDS Scheme 1. The direct oxygen addition rearrangement (OAR) and hydrogen atom transfer and rebound (HAT) mechanisms, which both are mediated by the reactive compound I intermediate. The bold lines represent the porphyrin ring surrounding the iron ion in the heme group.

SCHEMES Scheme 1


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Mechanism of the N-Hydroxylation of Primary and Secondary Amines

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