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Bridging Between Organocatalysis and Biocatalysis: The Powerful Enamine Mechanism of Organocatalysts Engineered into the Tautomerase Superfamily Scaffold

Final Report Summary - PROZYMES (Bridging Between Organocatalysis and Biocatalysis: The Powerful Enamine Mechanism of Organocatalysts Engineered into the Tautomerase Superfamily Scaffold)

Inspired by the versatile success of proline and its derivatives as organocatalysts, the Prozymes project aimed at application of the enzyme 4-oxalocrotonate tautomerase (4-OT), which carries a catalytic amino-terminal proline (Pro-1), in promiscuous catalysis of carbon–carbon bond-forming reactions. Thus the project aimed to bridge organocatalysis and biocatalysis by developing a new class of biocatalysts that use the powerful proline-based enamine mechanism of organocatalysts but that take advantage of the water solubility, stereochemical control, and relatively high catalytic rates available with enzymes. Ultimately this may lead to environmentally benign methodologies to produce pharmaceuticals and precursors.
Systematic screening of 4-OT for promiscuous carbonyl transformation activities, based on the presence of a unique nucleophilic proline residue within the active site of 4-OT, led to the discovery of promiscuous carbon-carbon bond-forming activities of this tautomerase. These activities include Michael-type additions of linear aliphatic aldehydes to various aromatic and aliphatic nitroolefins yielding valuable γ-nitroaldehydes, which are precursors for pharmaceutically active γ-aminobutyric acid (GABA) analogues. In addition, 4-OT has catalytic activity for an extensive scope of aldol reactions including homo and mixed coupling of aldehydes and ketones. Mechanistic studies have ascertained that the aldol coupling as well as the subsequent dehydration step are catalyzed by 4-OT.
The natural activity of 4-OT involves the catalysis of tautomerization reactions through its characteristic amino-terminal proline residue that acts as a catalytic base. The new promiscuous carbon-carbon bond-forming reactions proceed via another conceivable mechanism. The Pro-1 residue of 4-OT likely acts as a nucleophile, rather than a base, and forms an enamine intermediate with the carbonyl moiety of the substrate during the first catalytic step, reminiscent of organocatalysis. Rational design guided by entire protein mutability landscapes appeared to be a very powerful tool to enhance the promiscuous activities and stereoselectivities of 4-OT, and we were able to achieve considerable rate and yield improvements. Furthermore, the critical residues for the native and promiscuous activities were identified and valuable information about their specific mechanistic roles was gained.
In conclusion, by using the understanding of reaction mechanisms we have discovered new activities in a promiscuous tautomerase, and exploited this promiscuity as starting point to create tailor-made biocatalysts. Based on redesigned 4-OT variants we have developed a biocatalytic methodology that provides convenient access to both enantiomers of various γ-nitroaldehydes, with excellent enantiomeric excess, via asymmetric acetaldehyde additions to nitroalkenes. These chiral γ-nitroaldehydes are valuable precursors for GABA analogues, including the marketed pharmaceuticals Baclofen, Pregabalin, Phenibut and Rolipram. High stereoselectivity, low catalyst loading, and water as reaction medium characterize this unique biocatalytic methodology, which makes it an attractive alternative for existing organocatalytic methodologies.