Selective C-H Halogenation of Alkenes and Alkynes Using Flavin-Dependent Halogenases

Abstract

Single component flavin-dependent halogenases (FDHs) possess both flavin reductase and FDH activity in a single enzyme. We recently reported that the single component FDH AetF catalyzes site-selective bromination and iodination of a variety of aromatic substrates and enantioselective bromolactonization and iodoetherification of styrenes bearing pendant carboxylic acid or alcohol substituents. Given this inherent reactivity and selectivity, we explored the utility of AetF as catalyst for alkene and alkyne C-H halogenation. We find that AetF catalyzes halogenation of a range of 1,1-disubstituted styrenes, often with high stereoselectivity. Despite the utility of haloalkenes for cross-coupling and other applications, accessing these compounds in a stereoselective manner typically requires functional group interconversion processes, and selective halogenation of 1,1'-disubstituted olefins remains rare. We also establish that AetF and homologues of this enzyme can halogenate terminal alkynes. Mutagenesis studies and deuterium kinetic isotope effects are used to support a mechanistic proposal involving covalent catalysis for halogenation of unactivated alkynes by AetF homologues. These findings expand the scope of FDH catalysis and continue to show the unique utility of single component FDHs for biocatalysis.

Figure 1.

A) Representative substrate scope of native FDH catalysis. B) FDH-catalyzed halocyclization of substituted alkenes. C) FDH-catalyzed alkene C-H halogenation.

Figure 2.

A) Substrate scope for AetF-catalyzed halogenation of alkenes. B) AetF-catalyzed halogenation of p-methoxyphenylacetylene. Conversion is defined as % area for the product peaks relative to the % area for the starting material + product peaks in GC/MS chromatograms of crude reactions conducted on a 75 μL scale. Isolated yields were obtained from reactions conducted on a 30–40 mL scale, and the selectivity of these reactions was determined by GC/MS or NMR spectroscopy. Differences between conversions and isolated yields represent the formation of uncharacterized by-products in some cases (see GC/MS chromatograms in the Supporting Information) and product loss during protein removal and the multiple extractions required for isolation.

Figure 3.

A) Overlay of models of JamD (yellow) and MCE9 (purple) generated using AlphaFold^[50] with the crystal structure of the AetF•Trp•FAD complex (PDB ID 8CJE).^[47] B) Effects of E200A, H202A, and Y428F on JamD-catalyzed bromination of phenylacetylene and 1-decyne. Conversions determined from relative integration values of starting material and product in GC/MS or LC/MS chromatograms (see the Supporting Information). Alkyne reactions with measurable activity were conducted in triplicate; all other conversions are single measurements. See data file for complete data.

Figure 4.

Proposed mechanisms for FDH-catalyzed reactions: A) arene and alkene C-H halogenation, B) halolactonization, and C) alkyne C-H halogenation (potential role of Y428 as a proton relay between E200 and H202 omitted for clarity).