Article Title: Enzymatic halogenation of terminal alkynes
Authors: Lukowski, A. L.; Hubert, F. M.; Ngo, T.-E.; Avalon, N. E.; Gerwick, W. H.; Moore, B.S.
Journal: J. Am. Chem. Soc.
Year: 2023
DOI: /10.1021/jacs.3c05750
Installation of carbon–halogen bonds (e.g., fluorine, chlorine, bromine, iodine) in molecules is a chemically challenging task as most sites are chemically inert, or unreactive. Adding these halogens onto molecules, particularly pharmaceuticals, has long been sought after as these modifications can add beneficial properties including improved solubility and stability. Enzymes have evolved multiple strategies to accomplish these difficult feats. These enzymes are known as halogenases, with one well-studied example being the flavin-dependent halogenase (FDH) AetF involved in the di-bromination of the aromatic ring of tryptophan (Figure 1A).
Unlike the vast majority of FDHs, AetF does not require a partner enzyme to recycle critical cofactors required for its chemistry. This simplicity makes AetF a prime candidate for biocatalysis ventures. One major gap in knowledge, however, is that very few single component FDHs like AetF are known. Here, Lukowski et al. report the discovery of a new FDH, JamD, which can install C–Br bonds on terminal alkyne (carbon–carbon triple bond) functional groups. Moreover, the authors demonstrate JamD has a broad substrate scope and therefore great utility in biocatalysis.
In order to discover candidate halogenases, the authors constructed a phylogenetic tree of FDHs. Sequences located most closely to AetF included JamD, VatD, and PhmJ, which are predicted to be involved in the production of the microbial products jamaicamide A, vatiamide B, and phormidolide, respectively (Figure 1B). One interesting point is that the microbe which encodes AetF is a freshwater cyanobacteria, whereas all other closely related halogenases are derived from marine cyanobacteria. As a result, there is a question of how halogenases evolved to be found in these diverse ecosystems.
Next, as is typical of most enzyme discovery workflows, the authors heterologously expressed and purified JamD from the prototypical lab strain Escherichia coli. They were able to obtain authentic standards of the two compounds jamaicamide A and B and demonstrate that JamD indeed catalyzes the conversion of jamaicamide B into A (Figure 2A). Unlike AetF, JamD does not release the reactive intermediate HO–Br into solution, which minimizes the amount of undesired side products with other nucleophiles in the reaction mixture. In these reactions, potassium bromide (KBr) is the halide source. When potassium iodide (KI) was added instead, the iodinated version of jamaicamide A was observed. However, when sodium chloride (NaCl) was substituted, no chlorinated product was observed. As a result, JamD can catalyze halogenation reactions using less electronegative (Br, I) halogens.
The authors also tested the substrate scope of JamD (Figure 2B), demonstrating that JamD can recognize and brominate a variety of short and long jamaicamide B analogs. In all cases, the bromine is installed at the terminal alkyne position. Products were validated by analysis of nuclear magnetic resonance (NMR) spectroscopy or comparison to authentic standards. One final discovery was that a different putative halogenase, PhmJ, which is involved in the production of phormidolide (Figure 1B) was also shown to catalyze the conversion of jamaicamide B into jamaicamide A. This was a surprising result as PhmJ natively installs C–Br bonds on a terminal alkene (carbon–carbon double bond) rather than an alkyne.
In this work, a variety novel, single component halogenases from marine microbes were identified and tested for activity in vitro. Notably, JamD is able to install terminal C–Br bonds on a variety of compounds, including very minimal scaffolds. As a result, further assessment of JamD and its utility for biocatalysis will be crucial. While JamD is already capable of installing C–I bonds, perhaps further mechanistic studies will lead to JamD variants capable of installing more challenging C–Cl or even C–F bonds. Continual study of JamD and related homologs will greatly expand our knowledge of how enzymes catalyze challenging chemical reactions so precisely.
