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The use of enzyme-catalyst interfaces is underexplored in the field of biocatalysis, particularly in studies on enabling novel reactivity of enzymes. For this thesis, the HaloTag® protein tagging platform was proposed as a bioconjugation method for a pinacol coupling reaction using lipases, as a model for novel reactivities proceeding via

The use of enzyme-catalyst interfaces is underexplored in the field of biocatalysis, particularly in studies on enabling novel reactivity of enzymes. For this thesis, the HaloTag® protein tagging platform was proposed as a bioconjugation method for a pinacol coupling reaction using lipases, as a model for novel reactivities proceeding via ketyl radical intermediates and hydrogen-bonding-facilitated redox attenuation. After an initial lipase screening of 9 lipases, one lipase (Candida rugosa) was found to perform the pinacol coupling of p-anisaldehyde under standard conditions (fluorescein and 530nm light, 3% yield). Based on a retrosynthetic analysis for the photocatalyst-incorporated HaloTag® linker, the intermediates haloamine 1 and aldehyde 6 were synthesized. Further experiments are underway or planned to complete linker synthesis and conduct pinacol coupling experiments with a bioconjugated system. This project underscores the promising biocatalytic promiscuity of lipases for performing reactions proceeding through ketyl radical intermediates, as well as the underdeveloped potential of incorporating bioengineering principles like bioconjugation into biocatalysis to overcome kinetic barriers to electron transfer and optimize biocatalytic reactions.

ContributorsMcrae, Kenna Christine (Author) / Biegasiewicz, Kyle (Thesis director) / Ghirlanda, Giovanna (Committee member) / Moore, Ana (Committee member) / Department of Physics (Contributor) / School of Human Evolution & Social Change (Contributor) / School of International Letters and Cultures (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
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In Photosystem II of plants, the proton motive force that is essential for life is generated partly by the water oxidation process where the tyrosine and histidine 190 (hydrogen bonded) amino acids play an important role. The proton-coupled electron transfer (PCET) process involving these two molecules has been replicated using

In Photosystem II of plants, the proton motive force that is essential for life is generated partly by the water oxidation process where the tyrosine and histidine 190 (hydrogen bonded) amino acids play an important role. The proton-coupled electron transfer (PCET) process involving these two molecules has been replicated using a benzimidazole-phenol (BIP) construct as an artificial model of both the intramolecular hydrogen bond interaction and the associated PCET process. BIP is a nearly planar molecule and features a strong intramolecular hydrogen bond between the phenol and the nitrogen of the benzimidazole. When the molecule is oxidized electrochemically, the phenolic proton is transferred to the nitrogen of the benzimidazole moiety in a PCET mechanism. Herein the design, synthesis, and physicochemical characterization of a new BIP derivative is described. By introducing a methyl group in the new design, we intentionally increase the dihedral angle between the benzimidazole and phenol rings. The presence of the methyl group affects the ground-state PCET and the excited-state intramolecular proton transfer processes as well. The break in the coplanarity weakens the strength of the intramolecular hydrogen bond, decreases the chemical reversibility, and quenches the emission from the excited-state intramolecular proton transfer state. The findings contribute to understanding the importance of having a nearly planar structure in bioinspired artificial photosynthetic systems.

ContributorsDipaola, Lydia (Author) / Moore, Ana (Thesis director) / Odella, Emmanuel (Thesis director) / Moore, Thomas A. (Committee member) / Barrett, The Honors College (Contributor) / School of Human Evolution & Social Change (Contributor) / School of Life Sciences (Contributor)
Created2021-12