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  3. Crystallographic workup of the wild type ring cleaving dioxygenase PcpA, and synthesis of associated biomimetic model complexes
Title

Crystallographic workup of the wild type ring cleaving dioxygenase PcpA, and synthesis of associated biomimetic model complexes

    Item Description
    Limited Access
    The author(s) chose to restrict access to this thesis to current Whitman students, faculty, and staff. Please log in to view it.
    Linked Agent
    Creator (cre): Porter, Noah Ritzma
    Advisor (adv): Machonkin, Tim
    Department (dpt): Whitman College. Chemistry Department
    Date
    December 9, 2016
    Graduation Year
    2017
    Abstract

    Non-heme iron enzymes are a wide class of proteins that complete a myriad of highly specified and biologically important functions. The mechanistic details of their specificity and reactivity have yet to be fully elucidated. PcpA, a ring-cleaving dioxgenase (RCDO) with a high degree of specificity for ortho-halogenated hydroquinone substrates, is an atypical enzyme of the class that offers insights into the importance of active site coordination geometry, substrate character, and proton control in the activity of RCDOs. A purification procedure was developed for the wild-type PcpA expressed in and purified from E. coli along with crystallization procedures for the purpose of structural studies of the wild-type enzyme. Synthetic models mimicking common RCDO active site moieties and substrate types were also developed. Models incorporating phenol and hydroquinone motifs in addition to a facially coordinating triad of N and O ligands were created and complexed with divalent first row transition metals. N-methylamino linked iminodiacetic acid (IDA) and histadine (His) moieties were joined with a variety of substituted phenol and hydroquinone substrates, isolated, and characterized. 2-(N-methylamino-IDA)-hydroquinone (L₁), 4-methoxy-2,6-di(N-methylamino-IDA)-phenol (L₂*), 6-tertbutyl-4-methoxy-2-(N-methylamino-IDA)-phenol (L₄), and 4-methoxy-2-(N-methylamino-His)-phenol (L₃) were synthesized and characterized via ¹H-NMR. Ni(II), Co(II) and Fe(II) metallations were attempted for all ligands and the resulting complexes characterized in part via paramagnetic ¹HNMR, Mössbauer spectroscopy, UV-Vis spectroscopy, cyclic voltammetry, and single crystal x-ray diffractometry. Crystal structures of L₂*-Co(II)₂ and L₄-Co(II) confirmed initial estimates of coordination geometry of IDA-based ligands. Structures of L₄ complexes, showing distinct 1:1 binding:non-binding repeating modes in the crystal, suggest that ortho-tertbutyl substituted linked substrates may be unsuitable for further development due to steric crowding around the coordination site compared to other complexes. Spectroscopic titrations of L₄-M(II) complexes confirmed significant pH dependence for chelation and substrate protonation state in aqueous phase complexes of this type. The dinucleating ligand, L₂*, was found to result from the preferential overreaction of 2,6-unsubstituted phenol in a Mannich-type reaction suggesting that future ligand syntheses would benefit from phenol 6-position substitutions to ensure only monosubstitution at the 2-position resulting in mononucleating products.

    Subject
    Enzymes -- Synthesis -- Research -- Testing
    Ion exchange chromatography -- Purification -- Development -- PcpA protein
    Mannich reaction -- Evolution -- Derivatives
    Ligands (Biochemistry) -- Production standards -- Study and teaching
    Science
    Academic theses
    Whitman College 2017 -- Dissertation collection -- Chemistry Department
    Genre
    Theses
    Extent
    106 pages
    Permanent URL
    http://works.whitman.edu/20170207318
    Rights
    http://rightsstatements.org/vocab/InC/1.0/
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