Special boron chemistry can now also be done enzymatically | News

Enzymes come in all shapes and sizes, so you can do a wide variety of chemistry with them, including for numerous synthetic applications. Yet there are limits. For example, responses based on the element boron are out of scope. At least, until now.

‘Boor is a bit of a forgotten child. It offers unique chemical possibilities and is quite common on Earth, but it is often locked up in minerals and rocks and is therefore not very accessible. No natural enzyme contains boron,” says research leader Gerard Roelfes, professor of biomolecular chemistry & catalysis at the University of Groningen. ‘Boron chemistry and catalysis make reaction mechanisms possible that are not found in nature.’ To utilize these possibilities in a new way, Roelfes’ group has now created a designer enzyme in which they have genetically coded a boric acid.

Boron enzymes

Such a ‘boron enzyme’ can do boron chemistry in a more sustainable way, according to Roelfes. ‘Because of the milder reaction conditions, such as water as a solvent and lower reaction temperatures, biocatalysis with enzymes is much greener than classical organic chemistry.’ But green chemistry is not the only reason why boron enzymes are interesting. ‘Enzymes are fantastic catalysts. The catalytic site provides greater control, faster reactions and high stereoselectivity. Conventional boron catalysis has almost no examples of stereoselectivity, so with our enzyme you can really do something new.’

Although incorporating the boric acid was relatively simple – ‘we’ve been doing that kind of thing for years’ – the biggest challenge was finding a suitable reaction. ‘Many boron reactions are possible, but the catalytic site obviously has limited space,’ Roelfes explains. The reaction must also be able to work in water at a low temperature and not interact with other functional groups on the enzyme itself. ‘You also have to find the right position in the protein. We are becoming more and more adept at it, but every new designer enzyme is an adventure.’

Directed evolution

The researchers added the designer enzyme from the Nature-paper the non-canonical amino acid para-boronophenylalanine in. This boric acid group catalyzes the stereoselective condensation reaction of α-hydroxyketones with hydroxylamine to form an oxime. Roelfes: ‘The reaction takes place with a so-called kinetic resolution, which means that you start with a racemic mixture of your substrate, of which one enantiomer is then selectively converted.’ The researchers confirmed with MS and, among other things ¹¹B-NMR – ‘a strong example of postdoc Lars Longwitz’ – that it is indeed a boric acid reaction mechanism, which is not found anywhere else in nature. Subsequently, first authors Longwitz and Reuben Leveson-Gower tested the boron enzyme with directed evolution optimized, making it now highly selective.

The possibilities that enzymes offer to carry out organic syntheses continues to amaze Roelfes. ‘I was trained as a classical organic chemist and then learned that you only use water for extraction. But if you can find the right parameters, you see how much chemistry you can do under such mild conditions. Chemistry that you wouldn’t expect to be possible in water. So don’t think too quickly in absolute truths, it often turns out that there are ways to do things differently.’

Big step

This first boron enzyme mainly offers a proof of principle, but it is an important first step. ‘This way you find out 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 example, there are still complete reaction classes that you cannot carry out with enzymes, such as nucleophilic aromatic substitution or cross-couplings.’

If it can be done with drill, there are of course many more possibilities. Roelfes: ‘Compared to the bipyridines and anilines we once started with, adding boron to an enzyme is really a big step away from nature. So if you look at the periodic table, there are still a lot of elements that I want to look at. We’re playing around with it, but I want you to eventually be able to do useful chemistry with it, not make it a trick. If you can find new reactions that can only be done with these types of designer enzymes, then you can come up with interesting applications.’

Longwitz, L., Leveson-Gower, RB et al. (2024) NatureDOI: 10.1038/s41586-024-07391-3