Enzymatic Halogenation of Terminal Alkynes

Abstract

The biosynthetic installation of halogen atoms is largely performed by oxidative halogenases that target a wide array of electron-rich substrates, including aromatic compounds and conjugated systems. Halogenated alkyne-containing molecules are known to occur in Nature; however, halogen atom installation on the terminus of an alkyne has not been demonstrated in enzyme catalysis. Herein, we report the discovery and characterization of an alkynyl halogenase in natural product biosynthesis. We show that the flavin-dependent halogenase from the jamaicamide biosynthetic pathway, JamD, is not only capable of terminal alkyne halogenation on a late-stage intermediate en route to the final natural product but also has broad substrate tolerance for simple to complex alkynes. Furthermore, JamD is specific for terminal alkynes over other electron-rich aromatic substrates and belongs to a newly identified family of halogenases from marine cyanobacteria, indicating its potential as a chemoselective biocatalyst for the formation of haloalkynes.

Figure 1.

(a) Dibromination reaction performed by the single-component halogenase AetF. (b) Modes of reactivity for two-component and single-component FDHs.

Figure 2.

Phylogenetic analysis reveals AetF relatives from known BGCs. (a) AetF-containing clade from a flavin-dependent enzyme phylogenetic tree. (b) Synteny plots of known BGCs highlighting AetF relatives in dark blue; percent identity indicated between relatives in gray. Partial regions of BGCs containing AetF relatives are shown due to cluster length. (c) Structures of natural products associated with highlighted BGCs with bromine atoms proposed to be installed by the corresponding AetF-like halogenases highlighted in blue.

Figure 3.

Demonstration of JamD reactivity. (a) Scheme of JamD reaction with jamaicamide B (7) to yield jamaicamide A (4). (b) LC-MS trace showing the extracted ion chromatograms (EICs) of jamaicamide B (7, [M + H]⁺ = 489.2515) and jamaicamide A (4, M + H]⁺ = 567.1620) compared to an authentic standard of jamaicamide A (4). (c) MS isotope patterns of jamaicamide B (7) and jamaicamide A (4).

Figure 4.

JamD reaction profiling. (a) Halogenase reactions for chemoselectivity assessment. (b) Heatmap of total turnover numbers (TTNs) of Co-VBPO, AetF, and JamD with phenol (8), l-tryptophan (1), and jamaicamide B (7). TTN is calculated based on the number of catalytic cycles (i.e., bromination events) to generate the indicated product relative to the molar quantity of enzyme used. n.d. = not detected.

Figure 5.

Simple and complex alkynes accepted by JamD. Reactions with substrates 10–12 and 16 were analyzed by LC-MS, products were isolated and characterized by NMR, and the reactions with substrates 13–15 were analyzed by GC-MS compared to synthetic product standards. *Percent conversion n.a. (not available) due to irregular precipitation. ^†Product isolated yield n.a. (not available) due to volatility. ^‡Dibromination observed exclusively.