Researchers at the Biocatalysis group led by Dr Francesco Mutti at the Van ‘t Hoff Institute for Molecular Sciences (University of Amsterdam) have developed a novel use of alcohol dehydrogenase (ADH) enzymes. In an article designated as VIP (very important paper) in Angewandte Chemie, they describe how ADHs can forge direct links between alcohols and amines or thiols. The result is a clean synthesis of a variety of amides and thioesters. These are highly valuable functional compounds in many industrial synthesis routes, from medicines to advanced materials.

Amides and thioesters are ubiquitous compounds in chemistry, used for the production of medicines, natural products, and advanced materials. Traditionally, their synthesis is a messy business, involving wasteful reagents, toxic metals, or energy-intensive conditions. The use of enzymes can offer an environmentally friendly and efficient alternative here. However, established biocatalytic methods commonly require expensive cofactors (helper molecules) like ATP that support the enzymatic conversion. In addition, the scope of the enzymatic conversion can be rather limited so that only a few structural varieties of the desired product are obtained.

In the study now published in Angewandte Chemie, the Biocatalysis researchers reveal how amides and thioesters can be produced in a relatively straightforward manner using alcohol dehydrogenase (ADH) enzymes. They were also able to extend the scope of this enzymatic conversion using enzyme engineering.

Teaching ADH enzymes a new trick

In nature, alcohol dehydrogenases catalyse the oxidation of an alcohol to a carbonyl compound, hence their name. Since this is a reversible conversion, ADHs can also catalyse the reduction of carbonyl compounds to alcohols. The “reduction direction” is in fact the most common application of ADHs in industry, in particular for producing chiral secondary alcohols starting from prochiral ketones. As a consequence, the enzymes are also referred to as carbonyl reductases or ketoreductases.

The current research by the HIMS Biocat researchers now adds to the industrial toolbox by exploiting the forward pathway of alcohol oxidation. In their Angewandte paper, the team describes how they have been able to ‘teach ADHs a new trick’: forging direct links between alcohols and amines or thiols.

Effective, clean synthesis

So what is the trick? When an ADH oxidizes an alcohol to an aldehyde, the aldehyde can react on the spot with an amine or a thiol, which acts as a nucleophile. This additional reaction creates intermediates called hemiaminal or hemithioacetal, respectively. Instead of stopping there, the enzyme goes on to carry out a second oxidation step on these intermediates. The result is the formation of an amide or a thioester, respectively, which are both highly valuable compounds in industrial synthesis.

By testing a range of ADHs, the researchers were able to reveal the novel “oxidative coupling” in about half of the cases. Yields reached up to 99% by only using 0.1 mol% of the enzyme compared to the alcohol substrate. The scalability of the reaction was also proven. As a result, this application of ADHs paves the way towards an effective, clean synthesis of amides and thioesters. Without the need for costly ATP, activated intermediates or harsh reaction conditions - just the enzyme, air, and aqueous buffer.

Broadening the scope

To broaden the scope further, the researchers used protein engineering. By mutating key residues, opening up the active site, the engineered enzyme allowed for the acceptance of bulkier amines and thiols, enabling the synthesis of even more challenging amides and thioesters. The researchers expect the scope to become much broader in future by performing more protein engineering and testing other ADHs.

This work shows how exploring and tweaking the “hidden reactivity” of known enzymes can lead to new, useful biotransformations. This green and versatile method provides a sustainable platform for synthesising building blocks central to pharmaceuticals, agrochemicals, and biomaterials, greatly contributing to cleaner industrial chemistry.

Abstract, as published with the paper:

Amide and thioester moieties are prevalent in pharmaceuticals, natural products, and functional materials, but their chemical synthesis suffers from poor atom economy and ungreen conditions, while biocatalytic methods require ATP-dependent enzymes, activated intermediates, or show limited scope and activity. Here, we report the oxidative coupling of alcohols with ammonia or amines catalyzed by alcohol dehydrogenases (ADHs) via hemiaminal intermediates to form primary and secondary amides at pH 9.5–10.5. Pf-ADH preferably converted linear aliphatic or arylaliphatic alcohols (up to 90% conversion), while Pp-ADH and Aa-ADH preferably converted branched or aromatic alcohols (up to 99% conversion). Preparative-scale synthesis of an N-methyl amide gave >99% conversion and 87% isolated yield. The method was extended to thioacid and thioester formation via hemithioacetal intermediates using hydrogen sulfide or thiols at pH 7. Pf-ADH favored linear aliphatic alcohols (up to 93% conversion), Pp-ADH branched alcohols (up to 82% conversion), and Aa-ADH aromatic alcohols (up to 98% conversion). A KPi/MTBE biphasic system enabled the reaction with poorly soluble longchain thiols. Structure-guided engineering of Aa-ADH led to the Y151A and L186A variants with expanded activity toward longer-chain amines or thiols. This work highlights how enzyme promiscuity with protein engineering can enable new-to-nature synthetic pathways for the production of valuable compounds.