Browsing by Author "Ohata, Jun"

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    Convenient analysis of protein modification by chemical blotting with fluorogenic “click” reagents
    (Royal Society of Chemistry, 2015) Ohata, Jun; Farrukh, Vohidov; Ball, Zachary T.
    Direct visualization of bioorthogonal alkyne or azide handles using fluorogenic azide–alkyne cycloaddition conducted on the surface of a blot membrane. The method eliminates the need for separation steps to remove excess small molecule reagents before attachment of antigen molecules or other visualization handles, and is especially useful for the analysis of peptides and small proteins. A variety of potential fluorogenic reagents are assessed, and sensitivity (<0.1 picomole) similar to current commercially available fluorescence imaging methods is possible.
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    Copper- and rhodium-mediated approaches to site-specific bioconjugation
    (2018-02-23) Ohata, Jun; Ball, Zachary T
    Chemical functionalization of proteins has become an indispensable tool for chemical and biological studies. Recent effort has been focused on exerting a control of selectivity of the chemical reaction to connect a synthetic moiety onto a precise location of a target protein or onto a particular protein in a complicated mixture of a myriad of biomolecules. Such selective bioconjugation technologies have been increasingly studied within the past decade. This thesis focuses on development of site-specific/bioorthogonal modification of natural proteins by transition-metal catalysis without the aid of genetic incorporation of unnatural amino acids. The first two chapters review recent advances in technologies used at the interface between chemistry and biology. The first chapter covers novel site-specific conjugation technologies for antibodies mainly focusing on reports after 2014. The second chapter features application of dirhodium(II) complexes for a variety of applications such as potency enhancement as a metallodrug, identification of binding site as a transition-metal catalyst, and an indicator for decomposition of the metal complex. Development of novel selective protein modification technologies is discussed in chapter 3–5. With the rhodium(II)-metallopeptide catalysis developed in the Ball group, it is shown that site-specific modification of monoclonal antibodies are feasible through proximity-driven diazo decomposition reaction. Application of Chan-Lam cross coupling to bioconjugation method is described in chapter 4; copper(II)-catalyzed cross coupling with boronic acid reagents enabled amide backbone modification through directing effect of a neighboring histidine residue. Furthermore, unexpected reactivity of vinylboronate toward N-terminal amine group with ascorbate reagent is shown in the same chapter as well. In order for the abovementioned protein modification reactions, a facile and concise analytical technique is of great help. To that end, a new blot analysis is invented by performing click chemistry reaction on a blot membrane with luminogenic azide probes. Because of a lack of such a “turn-on” type luminescence azide probes, a series of luminogenic iridium (III) azide probes are designed, synthesized, and applied to biological experiments such as the blot membrane experiment and cellular labeling experiment.
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    Luminogenic iridium azide complexes
    (Royal Society of Chemistry, 2015) Ohata, Jun; Vohidov, Farrukh; Aliyan, Amirhossein; Huang, Kewei; Martí, Angel A.; Ball, Zachary T.
    The synthesis and characterization of luminogenic, bioorthogonal iridium probes is described. These probes exhibit long photoluminescence lifetimes amenable to time-resolved applications. A simple, modular synthesis via 5-azidophenanthroline allows structural variation and allows optimization of cell labeling.
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    Potent and selective inhibition of SH3 domains with dirhodium metalloinhibitors
    (Royal Society of Chemistry, 2015) Vohidov, Farrukh; Knudsen, Sarah E.; Leonard, Paul G.; Ohata, Jun; Wheadon, Michael J.; Popp, Brian V.; Ladbury, John E.; Ball, Zachary T.
    Src-family kinases (SFKs) play important roles in human biology and are key drug targets as well. However, achieving selective inhibition of individual Src-family kinases is challenging due to the high similarity within the protein family. We describe rhodium(II) conjugates that deliver both potent and selective inhibition of Src-family SH3 domains. Rhodium(II) conjugates offer dramatic affinity enhancements due to interactions with specific and unique Lewis-basic histidine residues near the SH3 binding interface, allowing predictable, structure-guided inhibition of SH3 targets that are recalcitrant to traditional inhibitors. In one example, a simple metallopeptide binds the Lyn SH3 domain with 6 nM affinity and exhibits functional activation of Lyn kinase under biologically relevant concentrations (EC50 200 nM).