Special boron chemistry can now also be achieved enzymatically

Enzymes come in all shapes and sizes, meaning you can carry out a vast variety of chemistry with them, including for numerous synthetic applications. Yet there are limits. Reactions based on the element boron, for instance, have been out of reach. Until now, that is.

‘Boron is a bit of a forgotten child. It offers unique chemical possibilities and it is fairly abundant on Earth, but it is mostly locked up in minerals and rocks and is therefore not very accessible. No naturally occurring enzyme contains boron’, says research leader Gerard Roelfes, professor of biomolecular chemistry and catalysis at the University of Groningen. ‘Boron chemistry and catalysis enable reaction mechanisms that you simply do not find in nature.’ To exploit these possibilities in a new way, Roelfes’s group has now created a designer enzyme into which they have genetically encoded a boronic acid.

Boron enzymes

Such a ‘boron enzyme’ can perform boron chemistry in a more sustainable manner, according to Roelfes. ‘Because of the milder reaction conditions — water as a solvent and lower reaction temperatures, for example — biocatalysis with enzymes is far greener than classical organic chemistry.’ But green chemistry is not the only reason why boron enzymes are of interest. ‘Enzymes are fantastic catalysts. The catalytic site provides greater control, faster reactions, and high stereoselectivity. Conventional boron catalysis has virtually no examples of stereoselectivity, so with our enzyme you can genuinely achieve something new.’

Although incorporating the boronic acid was relatively straightforward — ‘we’ve been doing that sort of thing for years’ — the greatest challenge lay in finding a suitable reaction. ‘Many boron reactions are possible, but the catalytic site obviously has limited space’, Roelfes explains. The reaction must also work in water at low temperature and must not interact with other functional groups on the enzyme itself. ‘On top of that, you need to find the right position within the protein. We are getting better at it all the time, but every new designer enzyme is still an adventure.’

Directed evolution

In the designer enzyme described in the Nature paper, the researchers incorporated the non-canonical amino acid para-boronophenylalanine. That boronic acid group catalyses the stereoselective condensation reaction of α-hydroxyketones with hydroxylamine to form an oxime. Roelfes: ‘The reaction proceeds via so-called kinetic resolution, which means you start with a racemic mixture of your substrate, from which one enantiomer is then selectively converted.’ Using MS and ¹¹B-NMR among other techniques — ‘a fine piece of work by postdoc Lars Longwitz’ — the researchers confirmed that it is indeed a boronic acid reaction mechanism, something you find nowhere else in nature. First authors Longwitz and Reuben Leveson-Gower then optimised the boron enzyme through directed evolution, making it highly selective.

The possibilities that enzymes offer for carrying out organic syntheses continue to astonish Roelfes. ‘I was trained as a classical organic chemist and was taught that you only use water for extraction. But once you know how to find the right parameters, you see how much chemistry you can do under such mild conditions — chemistry you would never expect to work in water. So don’t be too quick to think in absolute truths; more often than not, there turn out to be ways of doing things differently.’

A major step

This first boron enzyme is primarily a proof of principle, but it represents an important first step. ‘It shows you what the enzyme is capable of, what you need to do to get it active, and how you can ultimately translate that into other important reactions’, says Roelfes. ‘For the pharmaceutical industry, for instance, there are still entire reaction classes that cannot be performed with enzymes, such as nucleophilic aromatic substitution or cross-couplings.’

If it works with boron, there are of course many more possibilities. Roelfes: ‘Compared to the bipyridines and anilines we started out with, inserting boron into an enzyme is a truly significant step away from nature. So when you look at the periodic table, there are quite a few elements I want to explore. We are playing around with them, but I do want to ensure you can ultimately do useful chemistry with them — it mustn’t become a party trick. If you can find new reactions that can only be done with these kinds of designer enzymes, that’s when you can start thinking up genuinely interesting applications.’

Longwitz, L., Leveson-Gower, R.B. et al. (2024) Nature, DOI: 10.1038/s41586-024-07391-3