• Login
    View Item 
    •   Home
    • Theses and Dissertations
    • Dissertations
    • View Item
    •   Home
    • Theses and Dissertations
    • Dissertations
    • View Item
    JavaScript is disabled for your browser. Some features of this site may not work without it.

    Browse

    All of KAUSTCommunitiesIssue DateSubmit DateThis CollectionIssue DateSubmit Date

    My Account

    Login

    Quick Links

    Open Access PolicyORCID LibguidePlumX LibguideSubmit an Item

    Statistics

    Display statistics

    From Unnatural Amino Acid Incorporation to Artificial Metalloenzymes

    • CSV
    • RefMan
    • EndNote
    • BibTex
    • RefWorks
    Thumbnail
    Name:
    Arwa Makki - Dissertation - Final Draft.pdf
    Size:
    4.758Mb
    Format:
    PDF
    Download
    Type
    Dissertation
    Authors
    Makki, Arwa cc
    Advisors
    Eppinger, Jörg cc
    Committee members
    Peinemann, Klaus-Viktor cc
    Arold, Stefan T. cc
    Elmoslimany, Wael
    Program
    Chemical and Biological Engineering
    KAUST Department
    Physical Science and Engineering (PSE) Division
    Date
    2016-12-04
    Embargo End Date
    2017-12-11
    Permanent link to this record
    http://hdl.handle.net/10754/621994
    
    Metadata
    Show full item record
    Access Restrictions
    At the time of archiving, the student author of this dissertation opted to temporarily restrict access to it. The full text of this dissertation became available to the public after the expiration of the embargo on 2017-12-11.
    Abstract
    Studies and development of artificial metalloenzymes have developed into vibrant areas of research. It is expected that artificial metalloenzymes will be able to combine the best of enzymatic and homogenous catalysis, that is, a broad catalytic scope, high selectivity and activity under mild, aqueous conditions. Artificial metalloenzyme consist of a host protein and a newly introduced artificial metal center. The host protein merely functions as ligand controlling selectivity and augmenting reactivity, while the metal center determines the reactivity. Potential applications range from catalytic production of fine chemicals and feedstock to electron transfer utilization (e.g. fuel cells, water splitting) and medical research (e.g. metabolic screening). Particularly modern asymmetric synthesis is expected to benefit from a successful combination of the power of biocatalysis (substrate conversion via multi-step or cascade reactions, potentially immortal catalyst, unparalleled selectivity and optimization by evolutionary methods) with the versatility and mechanism based optimization methods of homogeneous catalysis. However, so far systems are either limited in structural diversity (biotin-avidin technology) or fail to deliver the selectivities expected (covalent approaches). This thesis explores a novel strategy based on the site-selective incorporation of unnatural, metal binding amino acids into a host protein. The unnatural amino acids can either serve directly as metal binding centers can be used as anchoring points for artificial metallo-cofactors. The identification expression, purification and modification of a suitable protein scaffolds is fundamental to successfully develop this field. Chapter 2 and 3 detail a rational approach leading to a highly engineered host protein. Starting with fluorescent proteins, which combine high thermal and pH stability, high expression yields, and fluorescence for ease of quantification and monitoring an efficient and fast purification protocol was developed first. The purification protocol uses a combination of heat precipitation and three-phase-partitioning (TPP). It provides high yield and purity without requiring any tag. Building on the favourable properties of fluorescent proteins, the non-metal binding, highly stable host-scaffold mTFP* was generated through rational design. The incorporation of artificial metal binding sites, the allowed the selective formation of artificial metalloenzymes, which show catalytic activity and moderat to good chiral induction in the Diels-Alder Cyclization and Friedl-Crafts Acylation Chapter 4 of the thesis describes the use of UAA incorporation to generate artificial metal binding sites. Computational studies and homology modelling successfully highlighted several positions in mTFP*, which are particularly suitable for UAA incorporation without any disruption of the protein structure. Application of a functional orthogonal aaRS/tRNA pair developed by P.G. Schultz and co-workers allowed the site-specific incorporation of UAAs in the host protein framework. Changes in fluorescence intensity revealed preferences of varieous UAAs for specific incorporations sites. The three UAAs, pIF, pAzF, and pEynF were incorporated into mTFP* in good yields, while pBF does only deliver low protein yields. A successfully established on-protein MIYAURA borylation reaction allows convert well-incorporated pIF into pBF circumventing the problem of low expression yields. Chapter 5 details the use of the azide-functionality of pAzF for the bioconjugation of artificial metal-binding cofactors through CuAAC. The triazole ring formed during this reaction serves as an additional moderate σ -donor/π –acceptor ligand of the metal binding site. We demonstrated the potential of site-specific modifications within the protein host with a versatile subset of artificial cofactors. Following transition metal binding, the newly created metal sites show catalytic activities that nature does not provide. The proof of concept study highlights the potential of the present mTFP* based catalysts in asymmetric Tsuji Trost allylation reactions and Diels-Alder cycloadditions. Dual anchoring of the cofactor lead to increased enantioselectivities, which is a direct result of a better-defined orientation of the catalytic center on the protein surface. Following the utilization of the CuAAC click reaction for the generation of artificial metalloenzymes, the last chapter of this thesis reports the development of a heterogeneous catalyst system for this reaction, which overcomes limitations of homogenous protocols. The recyclable core-shell structured Cu2O/Cu-nanowire catalyst is highly active, does not lead to protein precipitation, can be filtered off after the reaction and provides copper free bioconjugation products.
    DOI
    10.25781/KAUST-0055F
    ae974a485f413a2113503eed53cd6c53
    10.25781/KAUST-0055F
    Scopus Count
    Collections
    Dissertations; Dissertations; Physical Science and Engineering (PSE) Division; Chemical Engineering Program

    entitlement

     
    DSpace software copyright © 2002-2021  DuraSpace
    Quick Guide | Contact Us | Send Feedback
    Open Repository is a service hosted by 
    Atmire NV
     

    Export search results

    The export option will allow you to export the current search results of the entered query to a file. Different formats are available for download. To export the items, click on the button corresponding with the preferred download format.

    By default, clicking on the export buttons will result in a download of the allowed maximum amount of items. For anonymous users the allowed maximum amount is 50 search results.

    To select a subset of the search results, click "Selective Export" button and make a selection of the items you want to export. The amount of items that can be exported at once is similarly restricted as the full export.

    After making a selection, click one of the export format buttons. The amount of items that will be exported is indicated in the bubble next to export format.