Enzymatic bromination of native peptides for late-stage structural diversification via Suzuki-Miyaura coupling

Why I chose to highlight this preprint

Biomolecule modification is a fascinating transformation that has multiple scientific and medical applications. In this preprint, the authors developed an enzymatic method for tryptophan-specific bromination. The power in the enzymatic approach lies in its orthogonality to chemical reactions: enzymes catalyse highly specific transformations to occur under mild conditions, so most functional handles for chemical reactions can be tolerated.

I particularly liked that Bridge and co-workers not just described the discovery and development of RebH and its variant 4V for tryptophan bromination, but also demonstrated the application and impact of their work. Specifically, the authors showed that their enzymatic approach could modify key substrates and that the resultant products were amenable to further diversification. The authors also demonstrated that these modified biomolecules could be applied to several areas beyond the transformation itself.

Background

Tryptophan, being one of the rarest amino acids, is an attractive target for selective modification. However, to date most chemical methods to functionalise tryptophan are not sufficiently selective for tryptophan over cysteine, histidine, and tyrosine. To overcome these limitations associated with chemical methods for tryptophan modification, the authors instead opted for an enzymatic approach involving the use of flavin-dependent halogenases (FDHs).

Key findings

Bridge and co-workers first started by establishing the bromination of RebH and its variant 4V on peptide substrates (Figure 1). The authors tested the ability of both enzymes to brominate a panel of synthesised Trp-containing peptides, noting that both RebH and 4V exhibited similar bromination activity on these substrates. Bridge and co-workers then performed further experiments using over 110 dipeptide and tripeptide substrates to investigate the impact of peptide sequence and their structural features on RebH and 4V activity, allowing them to identify hydrogen-bonding interactions at the enzymes’ active sites that are key to the enzymatic reaction.

Figure 1. Bromination of RebH and its variant 4V on various peptide substrates.

With this structural understanding in hand, Bridge and co-workers further focussed on the ability of 4V to catalyse late-stage bromination of peptides containing Trp residues. Here, the authors investigated the substrate scope (that is, the breadth of substrates that could be brominated by 4V) and showed that 4V could brominate several neuropeptides, cell-penetrating peptides, as well as antimicrobial peptides. The authors hypothesised that brominating antimicrobial peptides would increase their antimicrobial activity by improving their hydrophobicity and enhancing membrane permeability; this was confirmed using an E. coli growth inhibition assay.

Finally, Bridge and co-workers demonstrated that 4V could brominate more complicated peptides and that the resultant derivatives could undergo further functionalisation (Figure 2). Here, two specific uses were illustrated: (a) the subsequent generation of a fluorescent probe for cellular peptide uptake with a naphthyl-modified peptide, as well as (b) the diversification of endomorphin-1, a μ opioid receptor agonist, to create analogues with modified activity.

Figure 2. Bromination of peptides followed by further functionalisation of the resultant derivatives.

Future directions

The large scope of this work by Bridge and co-workers, along with the downstream applications that they have demonstrated in this preprint, lends itself to multiple potential directions for development. In this post, I offer three such ways in which I foresee that this research could be expanded.

First, the study that the authors performed on the substrates led to the identification of several interactions underlying enzymatic activity, which could be applied to rational design of enzymes and allow researchers to discover other enzyme analogues exhibiting improved activity. Next-generation enzyme analogues could become (a) more promiscuous in accepting a wider scope of substrates, (b) more selective for bromination at different positions on the tryptophan residue (some of which are more challenging than others), and (c) better at functionalising challenging substrates.

Second, subsequent studies on enzyme stability and reusability will be key to scaling up the production of these enzymes. These studies are essential for this work to be more easily adapted by potential industrial partners, and will eventually be useful in moving the enzymatic transformation from a small lab scale to an industrial one. Associated studies in this field will involve not just the enzyme itself, but also more extensive studies on its expression in different cell lines and under different growth conditions.

Third, the enzymatic bromination described in this preprint can be expanded further to potentially include other reactions. One such way forward is to introduce other forms of halogenation, such as chlorination and iodination, which would be useful in further modulating the peptides’ physicochemical properties. A more ambitious goal would be to use these enzymes to perform other fascinating reactions such as azidation.

Acknowledgements

Images created using Microsoft Powerpoint, ChemDraw, and BioRender.

Tags: biomolecule modification, bromination, enzymatic catalysis, synthetic biology, tryptophan functionalisation

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