Enlightening the Path to Protein Engineering: Chemoselective Turn-On Probes for High-Throughput Screening of Enzymatic Activity.

Many successful stories in enzyme engineering are based on the creation of randomized diversity in large mutant libraries, containing millions to billions of enzyme variants. Methods that enabled their evaluation with high throughput are dominated by spectroscopic techniques due to their high speed and sensitivity. A large proportion of studies relies on fluorogenic substrates that mimic the chemical properties of the target or coupled enzymatic assays with an optical read-out that assesses the desired catalytic efficiency indirectly. The most reliable hits, however, are achieved by screening for conversions of the starting material to the desired product. For this purpose, functional group assays offer a general approach to achieve a fast, optical read-out. They use the chemoselectivity, differences in electronic and steric properties of various functional groups, to reduce the number of false-positive results and the analytical noise stemming from enzymatic background activities. This review summarizes the developments and use of functional group probes for chemoselective derivatizations, with a clear focus on screening for enzymatic activity in protein engineering.

Organic Acid to Nitrile: A Chemoenzymatic Three-Step Route.

Various widely applied compounds contain cyano-groups, and this functional group serves as a chemical handle for a whole range of different reactions. We report a cyanide free chemoenzymatic cascade for nitrile synthesis. The reaction pathway starts with a reduction of carboxylic acid to aldehyde by carboxylate reductase enzymes (CARs) applied as living cell biocatalysts. The second - chemical - step includes in situ oxime formation with hydroxylamine. The final direct step from oxime to nitrile is catalyzed by aldoxime dehydratases (Oxds). With compatible combinations of a CAR and an Oxd, applied in one-pot two-step reactions, several aliphatic and aryl-aliphatic target nitriles were obtained in more than 80% conversion. Phenylacetonitrile, for example, was prepared in 78% isolated yield. This chemoenzymatic route does not require cyanide salts, toxic metals, or undesired oxidants in contrast to entirely chemical procedures.

Chemoenzymatic one-pot reaction from carboxylic acid to nitrile via oxime.

We report a new chemoenzymatic cascade starting with aldehyde synthesis by carboxylic acid reductase (CAR) followed by chemical in situ oxime formation. The final step to the nitrile is catalyzed by aldoxime dehydratase (Oxd). Full conversions of phenylacetic acid and hexanoic acid were achieved in a two-phase mode.