Carbon-fluorine bond cleavage mediated by metalloenzymes

Abstract

Fluorochemicals are a widely distributed class of compounds and have been utilized across a wide range of industries for decades. Given the environmental toxicity and adverse health threats of some fluorochemicals, the development of new methods for their decomposition is significant to public health. However, the carbon-fluorine (C-F) bond is among the most chemically robust bonds; consequently, the degradation of fluorinated hydrocarbons is exceptionally difficult. Here, metalloenzymes that catalyze the cleavage of this chemically challenging bond are reviewed. These enzymes include histidine-ligated heme-dependent dehaloperoxidase and tyrosine hydroxylase, thiolate-ligated heme-dependent cytochrome P450, and four nonheme oxygenases, namely, tetrahydrobiopterin-dependent aromatic amino acid hydroxylase, 2-oxoglutarate-dependent hydroxylase, Rieske dioxygenase, and thiol dioxygenase. While much of the literature regarding the aforementioned enzymes highlights their ability to catalyze C-H bond activation and functionalization, in many cases, the C-F bond cleavage has been shown to occur on fluorinated substrates. A copper-dependent laccase-mediated system representing an unnatural radical defluorination approach is also described. Detailed discussions on the structure-function relationships and catalytic mechanisms provide insights into biocatalytic defluorination, which may inspire drug design considerations and environmental remediation of halogenated contaminants.

Fig. 1

DHP-catalyzed reaction and its active-site architecture. (A) Oxidative C-F bond cleavage of trifluorophenol. (B) Crystal structure of DHP in a complex with TBP and oxygen. His55 interacts with oxygen via hydrogen bonding (PDB entry: 4FH6).

Fig. 2

C-F bond cleavage pathway promoted by DHP.

Fig. 3

(A) Histidine-ligated heme TyrH catalyzes the defluorination of 3-fluorotyrosine. Two products are formed with C-H and C-F bond cleavages, respectively. (B) Thiolate-ligated CYP catalyzes the defluorination of 4-fluorophenol.

Fig. 4

Proposed C-F bond cleavage mechanism promoted by heme TyrH. R: amino acid moiety.

Fig. 5

Mechanism of C-F bond cleavage promoted by CYP.

Fig. 6

Active site of nonheme TyrH and the demonstrated reactions. (A) Crystal structure of nonheme TyrH in complex with BH₄. Phe300 is self-hydroxylated to 3-OH-Phe300. PDB entry: 2TOH. (B) Natural hydroxylation of tyrosine. (C) Defluorination of 3-fluorotyrosine. (D) Dehalogenation of 4-halo-phenylalanine. 4-Fluorophenylalanine (X = F) only yields one product, namely, tyrosine. Other halogen substitutions (X = Cl, Br) afford multiple products.

Fig. 7

A plausible mechanism of C-F bond cleavage promoted by nonheme TyrH with which 4-fluorophenylalanine (4-F-Ala) is converted into tyrosine. BH₄ represents tetrahydrobiopterin. R: amino acid moiety. (Note: The product derived from fluorine substitution is unclear and requires more experimental evidence for its production).

Fig. 8

Demonstrated catalytic reactions and the crystal structure of P4H. (A) Native hydroxylation of P4H on a proline residue. (B) The catalytic active site of human P4H (white) in complex with 2OG (yellow) and a fragment of HIF peptide (green) (PDB entry: 5L9B). Catalytic iron was substituted with a manganese ion in this structure. Pro564 from HIF is the target for hydroxylation. (C) Defluorination of a fluorinated proline residue.

Fig. 9

Mechanism of C-F bond cleavage mediated by prolyl-4-hydroxylase.

Fig. 10

Architecture of Rieske oxygenase and the experimentally verified catalytic reactions. (A) Structure of enzyme in complex with carbazole (yellow) and dioxygen. The Rieske cluster and catalytic iron located in two subunits are represented by green loops and white ribbons, respectively, and they are connected by an Asp residue through H-bonding interactions. PDB entry: 3VMI. (B) Defluorination reaction catalyzed by 2HD. (C) Defluorination reaction catalyzed by toluene-1,2-dioxygenase. R: -CH₃, −CN, −X, −OCH₃, or −CF₃.

Fig. 11

C-F bond cleavage of 2-fluorobenzoate promoted by 2-halobenzoate-1,2-dioxygenase.

Fig. 12

Cofactor biogenesis of CDO and its active site. (A) Crosslink formation in wild-type ADO/CDO with C-H bond cleavage. (B) Crosslink formation in F₂-Tyr ADO/CDO with C-F bond cleavage. (C) Crystal structure of uncrosslinked CDO bound with _L-cysteine (CYS) and nitric oxide (NO^●). Cys93 exhibits two conformations. PDB entry: 6BGF.

Fig. 13

Mechanism of C-F bond cleavage promoted by CDO. Ferric superoxide is regenerated after cofactor biogenesis, and it proceeds with the dioxygenation of the ligated substrate, forming CSA as the product.

Fig. 14

Laccase-/HBT-based radical approach for defluorination. (A) A global structure of laccase from Trametes versicolor. It is composed of three-domain polypeptide and four copper ions. PDB entry: 1GYC. (B) A simplified view of the laccase active site. (C) Reaction scheme of HBT radical formation.