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...

The scarcity of practical methods for aldehyde synthesis in chemistry necessitates the development of mild, selective procedures. Carboxylic acid reductases catalyze aldehyde formation from stable carboxylic acid precursors in an aqueous solution. Carboxylic acid reductases were employed to catalyze aldehyde formation in a cell-free system with activation energy and reducing equivalents provided through auxiliary proteins for ATP and NADPH recycling. In situ product removal was used to suppress over-reduction due to background enzyme activities, and an N-protected 4-formylpiperidine pharma synthon was prepared in 61% isolated yield. This is the first report of preparative aldehyde synthesis with carboxylic acid reductases employing crude, commercially available enzyme preparations. K E Y W O R D S aldehyde, biocatalysis, carboxylic acid reductase (CAR), in vitro cofactor recycling, pharma synthon 1 INTRODUCTION Aldehydes are key intermediates in medicinal chemistry. Their reactivity is a desired feature but also creates challenges during their synthesis. Classical methods for the synthesis of aldehydes encompass numerous examples of oxidations at specific reaction centers. Benzylic positions can be oxidized by using a NaClO/TEMPO/Co(OAc) 2 [1] system or IBX. [2] Terminal double bonds can be efficiently oxidized by O 2 applying Wacker conditions.

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.