Different Geometric Requirements for Cytochrome P450-Catalyzed Aliphatic Versus Aromatic Hydroxylation Results in Chemoselective Oxidation

Abstract

The cytochrome P450 enzyme, CYP199A4 from Rhodopseudomonas palustris strain HaA2, is able to oxidize parasubstituted benzoic acids. This enzyme was used to compare aromatic versus aliphatic C−H bond oxidation, common reactions catalyzed by the P450 superfamily of heme monooxygenases. CYP199A4 was able to bind 4-phenylbenzoic acid and 4cyclohexylbenzoic acid, and the crystal structures demonstrated that both substrates are bound within the active site in a similar fashion. Despite this, while 4-cyclohexylbenzoic acid was efficiently hydroxylated, no detectable enzyme catalyzed oxidation of the aromatic 4-phenylbenzoic acid was observed. The selectivity of 4-cyclohexylbenzoic acid oxidation favored C−H bond abstraction at one of the β-sites in an enantioselective fashion (66%, 95:5 er), over C−H bond abstraction at the benzylic position (33%). In addition, unlike the oxidation of smaller alkyl-substituted benzoic acids (4-ethyl-and 4-isopropyl-), little or no desaturation of the cyclohexyl ring to give an alkene was detected (∼1%). Molecular dynamics simulations suggested that the cyclohexyl ring of 4cyclohexylbenzoic acid was able to achieve a suitable orientation to enable efficient C−H bond abstraction and oxidation by the enzyme at the expected positions. In contrast, when the distance and angle of attack were considered, the alignment of the phenyl ring of 4-phenylbenzoic acid rarely attained a productive geometry for aromatic oxidation to occur. Overall, these results illustrate the chemoselectivity that may arise due to the different geometrical requirements for efficient aromatic oxidation versus aliphatic C− H bond hydroxylation by cytochrome P450 enzymes.

Enabling Aromatic Hydroxylation in a Cytochrome P450 Monooxygenase Enzyme through Protein Engineering

“…The major metabolite was isolated by semi-prep HPLC and characterised by NMR spectroscopy (Figure S3). The NMR spectrum of the [13] 90 1.7 � 0.1 902 � 34…”

“…-[e] 4-cyclohexylBA [13] � 95 0.45 � 0.05 169 � 11 52 � 6 33 � 2 4-tert-butylBA [12] � 95 39 � 2 227 � 4 227 � 32 100 � 13 4-methoxyBA [8c] � 95 0.28 � 0.01 1340 � 30 1220 � 120 91 � 10…”

Rational Design of First-Row Transition Metal Complexes as the Catalysts for Oxidation of Arenes: A Homogeneous Approach

“…Over the years, many experimental and theoretical studies have been done to gain a clear mechanistic picture of biological aromatic oxidations. ,− In this regard, different mechanisms have been proposed for Cyt P450-mediated arene hydroxylation (Scheme ), such as (A) benzene oxide formation by the electrophilic attack of high-valent iron­(IV)-oxo porphyrin π-cation radicals (Cpd I) on the aromatic ring, (B) electrophilic attack of Cpd I on the π-system to generate the radical/cationic σ-complexes, (C) electron transfer to Cpd I prior to the formation of σ-complexes, and (D) hydrogen atom abstraction by Cpd I from the aromatic ring and the subsequent rebound of the hydroxyl group. , Further, with the help of DFT studies, pathways C and D were ruled out, and a mechanism involving the formation of radical/cationic σ-complexes (pathway B) was found to be the most feasible. ,, In addition to this, in 2016, Fuji and co-workers demonstrated a kinetic study of aromatic oxidation (benzene, anisole, and naphthalene) using a series of Cpd I model complexes having different numbers of fluorine atoms in the meso-phenyl groups of the porphyrin ligand. On the basis of the Marcus plot obtained (slope = 0.60–0.68), a new mechanism (E) was proposed in which the electron transfer process between an aromatic compound and Cpd I is in equilibrium in a solvent cage and coupled with the subsequent bond formation …”

“… , Similarly, Fe­(III) complexes of N3 ligands are also found to proceed via a hydroxyl radical mechanism, probably due to a less sterically constrained ligand environment to stabilize the iron–oxygen intermediate . With heme-iron complexes, a mechanism similar to Cyt P450 is proposed involving the formation of a high-valent iron-oxo with porphyrin π-cation radical species. − When it comes to non-heme systems, although complexes of both N4 and N5 ligands are found to form the common Fe III –OOH intermediate, the actual reactive species in both instances are understood to be different. In the case of iron complexes having N4 ligands, the high-valent iron­(V)-oxo species is identified as the reactive species, especially in the presence of a carboxylic acid as a cocatalyst resulting from the heterolytic cleavage of the O–O bond in the Fe III –OOH intermediate. , In contrast, a caged pair, [Fe IV O + • OH] produced by the homolysis of O–O bond in Fe III –OOH species is the proposed intermediate in the case of iron complexes of N5 ligands. − Besides, with diiron complexes, the formation of oxido bridged reactive species are proposed in the catalytic mechanism. ,, Our overall mechanistic understanding of heme and non-heme-iron-mediated homogeneous benzene hydroxylation is illustrated in Scheme .…”

Co-Crystal Structure-Guided Optimization of Dual-Functional Small Molecules for Improving the Peroxygenase Activity of Cytochrome P450BM3

“…In fact, there are only a few native P450s belonging to the CYP152 family that are capable of efficiently using H 2 O 2 to perform their catalytic functions [ 20 , 21 , 22 ]. Considering the significant potential of peroxygenases as practical biocatalysts [ 23 , 24 , 25 , 26 ], many efforts have been made to develop a peroxide-driven P450 system [ 27 , 28 , 29 , 30 , 31 ].…”