Break the template boundary: Test of different protein scaffolds by genetic code expansion resulted in NADPH-dependent secondary amine catalysis

Here, incorporation of secondary amine by genetic code expansion was used to expand the potential protein templates for artificial enzyme design. Pyrrolysine analogue containing a D-proline could be stably incorporated into proteins, including the multidrug-binding LmrR and nucleotide-binding dihydrofolate reductase (DHFR). Both modified scaffolds were catalytically active, mediating transfer hydrogenation with a relaxed substrate scope. The protein templates played a distinctive role in that, while the LmrR variants were confined to the biomimetic BNAH as the hydride source, the optimal DHFR variant favorably used the pro-R hydride from NADPH for reactions. Due to the cofactor compatibility, the DHFR secondary amine catalysis could also be coupled to an enzymatic recycling scheme. This work has illustrated the unique advantages of using proteins as hosts, and thus the presented concept is expected to find uses in enabling tailored secondary amine catalysis.

On-Demand Release of Secondary Amine Bases for the Activation of Catalysts and Crosslinkers

We demonstrate that the species present in the equilibrium of DCv ureas can be employed in reaction cascades and as triggered organocatalysts. Easily controllable stimuli like heat or addition of water shift the equilibrium towards isocyanate and free base which can function as an in situ released reagent, both catalytically and in an equimolar fashion in different reactions. While applying heat to the system leads to a reversible liberation of amine base, addition of water makes this release irreversible. We demonstrate this application of DCv ureas with two examples via ¹H-NMR spectroscopy. Firstly, we use the liberated base to activate a protected organocatalyst for acylhydrazone formation. Secondly, this base can be employed to trigger the release of nitrile-N-oxides from chlorooximes, which can react with 4-arm PEG-thiols to form a thiohydroximate polymer gel. These findings show the utility of DCv hindered ureas beyond their application in self-healing.

On-Demand Release of Secondary Amine Bases for the Activation of Catalysts and Crosslinkers

We demonstrate that the species present in the equilibrium of DCv ureas can be employed in reaction cascades and as triggered organocatalysts. Easily controllable stimuli like heat or addition of water shift the equilibrium towards isocyanate and free base which can function as an in situ released reagent, both catalytically and in an equimolar fashion in different reactions. While applying heat to the system leads to a reversible liberation of amine base, addition of water makes this release irreversible. We demonstrate this application of DCv ureas with two examples via ¹H-NMR spectroscopy. Firstly, we use the liberated base to activate a protected organocatalyst for acylhydrazone formation. Secondly, this base can be employed to trigger the release of nitrile-N-oxides from chlorooximes, which can react with 4-arm PEG-thiols to form a thiohydroximate polymer gel. These findings show the utility of DCv hindered ureas beyond their application in self-healing.