Halogenating Enzymes for Active Agent Synthesis: First Steps Are Done and Many Have to Follow

Abstract

Halogens can be very important for active agents as vital parts of their binding mode, on the one hand, but are on the other hand instrumental in the synthesis of most active agents. However, the primary halogenating compound is molecular chlorine which has two major drawbacks, high energy consumption and hazardous handling. Nature bypassed molecular halogens and evolved at least six halogenating enzymes: Three kind of haloperoxidases, flavin-dependent halogenases as well as α-ketoglutarate and S-adenosylmethionine (SAM)-dependent halogenases. This review shows what is known today on these enzymes in terms of biocatalytic usage. The reader may understand this review as a plea for the usage of halogenating enzymes for fine chemical syntheses, but there are many steps to take until halogenating enzymes are reliable, flexible, and sustainable catalysts for halogenation.

Keywords: active agent synthesis; biocatalysis; bromination; chlorination; halogenase; haloperoxidase; pharmaceuticals.

Figure 1

Schematic representation of electron distribution in halogens. (A): Latent polarization of a carbon-halogen bond. (B): Polarizability of large halogens (Br, I) bonded with a carbon. The external electrical field, for example, caused by an approaching electrophile/nucleophile leads to the distortion of the electron density. (C): Schematic view on the “σ-hole”. The electron density is drawn to the carbon-halogen bond, with the strength gradually increasing with the size of the halogen (I > Br > Cl >> F). This anisotropic distribution of electrons in the outer orbitals of the halogen creates an area of higher electron density around the belt of the halogen, allowing interaction with electrophiles or H-bonds. Orthogonal to the direction of the bond is an area of electron deficiency, creating a partially positively charged area in the halogen, allowing for nucleophilic attacks, commonly called “σ-hole”.

Figure 2

Examples for halogenated active agents.

Figure 3

Workflow for the provision of halogenating reagents from alkali salts. The electrolysis process thus produces molecular halogens (X₂), as well as hypohalous acids (HOX, 6) and N-halogenated succinimides (NXS, 8) in further steps.

Figure 4

Most common reactions in organic synthesis exploiting halogen moieties. [37,45,46,47,48] Besides organolithium reactions as well as Grignard/Barbier reactions all of them are Pd-based, but can in many cases be substituted by other transition metals such as nickel.

Figure 5

Scheme of the steps in cross-coupling reactions. After oxidative addition of the organo-halogen species, the transmetalation occurs. The ligands start rearrange before reductive elimination to the final product is carried out and the catalyst is regenerated.

Figure 6

Overview on the categorization of halogenating enzymes.

Figure 7

Proposed catalytic cycle of heme-iron-dependent haloperoxidases, shown on the example of CPO from C. fumago. In the resting state (3 o’ clock), water is bound to the heme-iron, which is subsequently replaced by hydrogen peroxide. After protonation of this complex by a catalytic glutamate (Glu183), water is eliminated, creating the actual active species, the Fe(IV)-oxo complex. A halide, in this case chloride, binds to the Fe(IV)-oxo species and is released as hypochloric acid, regenerating the heme-site by hydrolysis with water. Alternatively, another molecule hydrogen peroxide may attack, leading to the disproportion of the complex to molecular oxygen, water, and chloride [54,55].

Figure 8

Example reactions of Cf-CPO involved in biocatalytic conversions of organic molecules (A): Cyclization of allenes (10) to the product 11 induced by halogenation with Br⁻ by Cf-CPO. (B): Unselective chlorination of thymol (12) by Cf-CPO.

Figure 9

Proposed catalytic cycle of vanadium-dependent haloperoxidases. In its resting state (3 ‘o clock), vanadium contains four oxygen ligands, while the free coordination site is occupied by a catalytic histidine residue, resulting in a dative bond. In presence of hydrogen peroxide, a hydroxyl group is substituted by peroxide. Upon elimination of a hydroxide ion, a cycloperoxo-species is generated, which is stabilized by a catalytic lysine residue. This cyclic intermediate is opened by addition of a halide, in this case bromide, which can then be hydrolyzed by water, leading to the release of hypobromic acid, or in presence of another hydrogen peroxide molecule, be disproportioned to molecular oxygen and bromide. During catalysis, the vanadium does not alter its oxidation state (V) [55].

Figure 10

(A): (Aza-)Achmatowicz reaction transforming the furan 15 to the Michael-system [78] 14. (B): Halofunctionalization of styrene (16) [77,79].

Figure 11

Selected reactions performed by the Co-V_BrPO to illustrate the reaction spectrum [81].

Figure 12

Proposed catalytic cycle of metal-free haloperoxidases/perhydrolases. This mechanism was compiled from several sources [81,85]. The catalytic cycle is adopted from the common hydrolase catalysis encountered in lipases and esterases, for instance. In presence of a carboxylic acid, in this case acetic acid, an ester is formed with the catalytic serin residue upon elimination of water (3 ‘o clock). In presence of hydrogen peroxide, the ester is cleaved, forming a percarboxylic acid. In the following step, a halide binds to the peroxoacid, which is hydrolyzed to the hypohalous acid, while the characteristic Ser-His-Asp triad is already regenerated.

Figure 13

Halogenation of indole (28) nucleobase analogs according to Lewkowicz and co-workers [87].

Figure 14

Regioselectivity of flavin-dependent halogenases and their dependency on carrier proteins. * Natural products with halogenations are known, but so far, no enzyme is characterized. ^{^} This tryptophan halogenase is one of the few examples that is carrier protein-dependent [99].

Figure 15

Catalytic cycle of halogenation by flavin-dependent halogenases [96,115].

Figure 16

Proposed mechanism for halogenation reaction by Fe(II)/αKG-dependent halogenase via a radical C-H functionalization [142]. The highly reactive Fe(IV)-oxo (haloferryl) intermediate is produced by decarboxylation of αKG to succinate through an oxygen attack. Subsequently abstraction of a hydrogen-atom from the substrate leads to an energetically favourable rearrangement towards Fe(III). Rebound reaction with chloride was shown to depend on the distance and orientation of the substrate [143]. The catalytically cycle is re-established by the hexa-coordinated Fe(II) with water molecules, chloride and histidine.

Figure 17

Schematically example for formation of cyclopropane initiated by CmaB through halogenation.

Figure 18

(A) Schematically sequential mechanism for the F–C bond formation catalysed by the fluorinase from S. cattleya and some products of subsequently cascade reaction. Dashed lines represents hydrogen bonding contacts with amino acids in the active pocket or water. (B) Reaction scheme of fluorinase-mediated trans-halogenation. Rest R marks position of usual derivation.