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CURATOR

Postdoctoral fellow, The university of Rochester

PINBOARD SUMMARY

Non-native Biocatalysis

Enzymes and proteins are increasingly used in organic transformations due to excellent chemo-, regio- and stereo- selectivity, environmental sustainability, milder reaction conditions, improved productivity, simplified work-streams and greater economical saving potential. Use of engineered and artificial biocatalysis is an effective way to tap chemical transformations that lie beyond the realm of natural enzymes. Fasan group at the University of Rochester discovered that engineered variants of myoglobin (Mb), would support the cyclopropanation of variety of aryl-substituted olefins with excellent trans diastereoselectivity (97 to 99.9%) and enantioselectivity (96 to 99.9%, trans-1S,2S isomer) while undergoing high catalytic turnovers (up to 46,800 TON). The ability to access both enantiomeric forms of a target cyclopropane pharmacophore is critical in the context of the synthesis of bioactive molecules. We have used a systemic active-site mutagenesis approach combined with structure-reactivity-relationship (SRR) guided design for developing trans-(1R,2R)-selective Mb catalysts for 1-carboxy-2-aryl-cyclopropane synthesis. SRR strategy yielded Mb(L29T,F43W,H64V,V68F) variant that catalyzes cyclopropanation between styrene and ethyl diazoacetate (EDA) with excellent diastereoselectivity (>99%, trans) and enantioselectivity (95%, trans-1R,2R). In order to expand the scope of Mb catalyzed organic transformations, we have investigated the effect of altering the nature of the metal center and proximal ligand on the carbene transfer activity of these metalloproteins. Using this strategy, Mb catalysts capable of promoting cyclopropanation and carbene Y-H insertion reactions (Y= N, S) under aerobic conditions were identified. Aerobic enzyme reactions would further simplify the biocatalytic process. Performing hemoprotein biocatalysis under aerobic conditions is a challenge due to its greater affinity towards molecular oxygen. Replacing the porphyrin ring of the metal cofactor with other naturally occurring or artificial porphyrinoids could improve the aerobic myoglobin biocatalyst activity. To this end, we have used chlorin e6 (ce6, has 20 π electron containing tetrapyrrole ring system and a chlorophyll analog) as a porphyrin replacement in Mb heme prosthetic group and prepared various Mb variants containing Fe ce6 prosthetic group either through recombinant expression or reconstitution.

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