Simple and Enzymatic Synthesis of Amides

In the field of chemistry, finding new, efficient and clean ways to synthesise amides is an important goal. ‘Until now, practical limitations have hampered progress, including in the area of biocatalysis,’ says Francesco Mutti, Associate Professor of Biocatalysis at the University of Amsterdam. ‘Over the past decade, researchers have succeeded in simplifying the formation of amide bonds in aqueous environments, but this requires ATP-activated enzymes, which industry is reluctant to use.’

Mutti, Matteo Damian and their colleagues have, by contrast, discovered a simple alternative that requires no ATP. ‘It’s very straightforward,’ says doctoral candidate Damian. ‘You simply mix an alcohol dehydrogenase enzyme in powder or liquid form with a cofactor, an alcohol and a second reagent to produce an amide under full conversion.’ The team also uses ordinary air to supply oxygen. ‘Alcohols are inexpensive and widely used substrates, and we can play both with the nucleophile and with the promiscuity of the enzyme,’ adds Mutti. ‘There is therefore no longer any reason for industry to be reluctant to use these enzymes.’

Luck

Their publication in Angewandte Chemie International Edition on the synthesis of amides and thioesters using alcohol dehydrogenases (ADHs) was designated a Very Important Paper. ‘This work is particularly significant in the pharmaceutical industry,’ Mutti explains, ‘given that more than half of the two hundred best-selling medicines contain amide bonds of the kind we have created here.’ Damian adds: ‘It is not just the novelty of the work, but also its impact on the community. Our protocols are straightforward to implement.’

It all began with one of Mutti’s first doctoral students, Jan Vilím, who wanted to convert alcohols into amines using a combination of enzymes. ‘By chance, he discovered a new reaction involving an oxidase that was able to convert alcohols into nitriles via a hemiaminal intermediate — work that was also published in Angewandte Chemie in 2019,’ recounts Mutti. Damian was inspired by this reaction. ‘I have a poster of his work above my desk, and after reviewing the literature I realised I could do something similar with alcohol dehydrogenases via the same hemiaminal intermediate, but with the formation of amides.’ He was lucky, he says.

It appears that the reactivity of ADHs is broader and not limited to ammonia as a nucleophile. Mutti: ‘We can introduce more complex amines and sulphur donors as substrates, and in the future it may well work for other nucleophiles too. It’s work in progress!’

Unconventional project

To achieve this, however, they had to adopt an unconventional approach. ‘Anyone who works with enzymes knows they do not tolerate high pH values,’ explains Damian. ‘But when I shifted my perspective from biocatalysis to chemistry, we realised that a high pH might actually work for the reaction we had in mind. That caused our yield to shoot up.’

The project was also unusual in another respect. ‘Matteo’s position was something of a consolation prize for a larger grant application that was not awarded,’ says Mutti. ‘Initially there was insufficient funding to launch the project, so instead the university provided funding for an internal PhD studentship, which is extremely rare.’ This meant there was considerable freedom with regard to both the project details and the choice of doctoral student.

‘The current system, in which proposals are assessed by referees and committees, is generally regarded as the best possible arrangement. But sometimes freedom without oversight is precisely what leads to unexpected results. The current peer-review system is not as perfect as we tend to think. I doubt whether a project like this would ever have been approved. But if you never attempt unconventional projects, important discoveries will never come to light.’

Damian adds: ‘Freedom is important, but when I was a doctoral student, despite working hard I had not achieved many positive results by the end of my first year. Yet Francesco continued to trust me, which was enormously encouraging.’ Mutti grins broadly. ‘Matteo had his moments of self-doubt back then. But failure is part of the process and every doctoral student goes through it. That is why it is crucial to show confidence in your students, especially after the first year.’

Flow

For the time being, the scale of 500 mg remains relatively modest. There are, however, a number of promising strategies the team could implement to increase yields and turnover figures. Immobilising the enzyme in a flow system, for instance, could extend its lifespan, circumvent the limited substrate loading and improve scalability. ‘Moreover, we have not yet made any engineering modifications to further stabilise the enzyme,’ concludes Mutti. ‘By drawing on state-of-the-art engineering methods to enhance stability, the enzyme’s lifespan could be extended, enabling greater productivity. By combining all of these approaches, efficiency could in all likelihood increase by a factor of ten.’