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Structure-based design of artificial metalloenzymes and beyond

Heinisch, Max Tillmann. Structure-based design of artificial metalloenzymes and beyond. 2013, PhD Thesis, University of Basel, Faculty of Science.

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Official URL: http://edoc.unibas.ch/diss/DissB_10521

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Abstract

Optically active molecules play a fundamental role in the functions of live. The enantioselective synthesis of chiral building blocks is crucial for the production of high value compounds such as pharmaceuticals or pesticides. Besides homogenous transition metal and enzymatic catalysts, artificial metalloenzymes - that consist of metal cofactors anchored within protein scaffolds - have been developed in the past decade to access optically pure compounds. The creation and optimization of these hybrid systems requires structural information. The asymmetric transfer hydrogenation of functional carbonyl, imine or enone groups to obtain the corresponding alcohols, amines or alkanes in high optical purity is achieved by transition metal piano stool complexes as well as by a number of different enzymes. In this thesis, two artificial transfer hydrogenases based on the streptavidin-biotin system for the asymmetric reduction of cyclic imines are structurally characterized. Potential substrate binding modes are proposed and the origins of the enantioselectivities are discussed. The Sharpless osmium-catalyzed asymmetric dihydroxylation of olefins is a powerful method to obtain chiral vicinal diols from variously substituted substrate molecules. The enantioselectivity of the reaction is governed by the interactions between the substrate olefins and the bulky chiral ligands bound to the catalytic osmium tetroxide center. In this thesis, an artificial olefin dihydroxylase is structurally characterized which is based on the embedding of an osmium tetroxide catalyst within streptavidin. Although none of the four osmium-binding sites located in the crystal structures was bound to the biotin-binding pocket, the activity pattern of various streptavidin mutants suggests that the active site is located in proximity to this position. During the processing of the diffraction data of one of the streptavidin-osmium crystals, an ambiguous packing disorder was diagnosed for which a quantitative model is proposed in the final chapter of this thesis.
Human carbonic anhydrase II is a well-characterized monomeric protein and numerous arylsulfonamide inhibitors with nanomolar and subnanomolar affinities for this enzyme are described in the literature. The potential of this protein to act as a host for the creation of an artificial transfer hydrogenase is evaluated by the structural characterization of an arylsulfonamide-tethered transition metal piano stool complex bound to human carbonic anhydrase II. This study is also investigating the structural origins of the high affinities of piano stool arylsulfonamide complexes for human carbonic anhydrase II and the implications for future metallodrug design.
A number of ruthenium-arene piano stool complexes have been demonstrated to strongly inhibit the growth of human cancer cell lines by unspecific interactions with nucleobases. To introduce DNA-specificity via second coordination sphere interactions, two ruthenium arene complexes have been biotinylated and bound to streptavidin. The structural basis of the DNA-specificity governed by the streptavidin-ruthenium arene complex is investigated.
The in vivo activity of artificial metalloenzymes is a prerequisite for their genetic optimization by directed evolution. Moreover, hybrid enzymes have the potential to be used in vivo to complement metabolic pathways or to act as bioorthogonal catalyst in the activation of prodrugs. The strategy of enzyme-encapsulation into cell-penetrating nanoreactors is investigated which allows the shuttling of artificial metalloenzymes into cells.
Advisors:Ward, Thomas R.
Committee Members:Schirmer, Tilman
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie > Bioanorganische Chemie (Ward)
Item Type:Thesis
Thesis no:10521
Bibsysno:Link to catalogue
Number of Pages:168 S.
Language:English
Identification Number:
Last Modified:30 Jun 2016 10:53
Deposited On:04 Oct 2013 14:45

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