R-3 Repository :: Browsing by Author "Ball, Zachary T"

Browsing by Author "Ball, Zachary T"

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Boronic Acid-Functionalized Macromolecules for Facile Preparation of Complex Bioconjugates
(2022-07-26) Swierczynski, Michael J; Ball, Zachary T
The conjugation of macromolecules to proteins can offer a variety of benefits from enhanced in vivo stability of protein-polymer conjugates to targeted delivery of protein-antibody conjugates. Transition-metal mediated bioconjugation with boronic acids has been shown to effectively modify natural proteins site-selectively under mild conditions with accessible reagents, though has been limited to small molecule conjugation. This thesis is on the development of boronic acid based bioconjugation methods for protein-macromolecule bioconjugation. The first two chapters review developments in protein-polymer and boronic acid bioconjugation chemistry. The first chapter details protein-polymer conjugation techniques with a focus on direct conjugation of functionalized polymers onto natural amino acids of proteins. The second chapter discusses protein bioconjugation strategies utilizing boronic acids as the warhead. The development of protein-macromolecule bioconjugates with boronic acid-functionalized macromolecules is covered in chapters 3-5. Polymer-protein conjugates are efficiently prepared via Ni(II) mediated cysteine arylation with ortho-nitroarylboronic acid functionalized polymers in chapter 3. In chapter 4, hetero-boronic acids containing an ortho-nitroarylboronic acid and a alkenylboronic acid moiety are used to conjugate cysteine containing proteins to dipeptide pyroglutamate-histidine tagged peptides and proteins. Finally, in chapter 5, phenylboronic acid-tagged proteins are reversibly conjugated to salicylhydroxamic acid-functionalized molecules including a polymer and dendrimer.
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.
Dirhodium Metallopeptides for Catalytic Protein Modification & Inhibition of Protein-Protein Interactions
(2016-06-03) Vohidov, Farrukh; Ball, Zachary T
The fundamental goal of chemical biology is to develop tools for interrogation of the activity and function of biomolecules, ultimately leading to understanding of the inner workings of biological systems. This thesis discusses three contributions towards expanding the chemical biology toolbox: i) an enzyme-like specific protein modification method, ii) a chemical blotting protocol for analyzing the products of protein modification reactions, iii) potent and selective inhibitors of protein-protein interactions. Inspired by the exquisite control and selectivity seen in enzymatic transformations, dirhodium metallopeptides were developed that catalyze selective labeling of natural proteins in their native environment. These tailored metallopeptides are highly specific for the targeted proteins, allowing quantitative modification of a target protein at micromolar concentrations even in the presence of a large number of potentially reactive biomolecules. The utility of the method for biophysical studies was demonstrated by labeling Yes protein kinase directly in cell lysate. In order to analyze products of in-lysate modification reactions, a chemical blotting protocol was established, and luminogenic probes were designed for use in chemical blotting and cell imaging applications. Design of inhibitors for protein-protein interactions is a formidable challenge due to the structural and chemical properties of protein interfaces involved in these interactions. Exploiting the potential of the dirhodium core to participate in cooperative binding, inhibitors for SH3 protein domains were developed by attaching dirhodium at different positions on a parent peptide ligand with modest SH3-affinity. The rationally designed library of dirhodium metalloinhibitors yielded the tightest known inhibitor for Lyn SH3; Lyn, a tyrosine kinase, represents an important target to further our understanding of a variety of cellular dysfunctions. Taken together, these contributions represent a set of powerful tools in chemical biology and can serve as a foundation for continued advancement of the field.
Investigations of the Specificity of Oxidosqualene Cyclization: Errors are the Rule, Not the Exception
(2014-12-05) Bodager, Paul Gregory; Matsuda, Seiichi P.T.; Ball, Zachary T; Silberg, Jonathan
This thesis describes the characterization of oxidosqualene cyclases from numerous organisms, through heterologous expression in Saccharomyces cerevisiae, and extraction from organismal tissue. Oxidosqualene cyclases are a family of proteins which catalyze the cyclization of the linear substrate oxidosqualene into cyclic compounds known as triterpene alcohols, acting in both the primary and secondary metabolism of organisms. Detailed analyses of cyclase product profiles in both primary and secondary metabolism are used herein to develop a comprehensive discussion of cyclase product specificity. First, the characterization of two oxidosqualene cyclases of secondary metabolism from the plant Arabidopsis thaliana, LUP4 and LUP5, by heterologous expression, is described. While demonstrating quite different product specificity, both cyclases make a mixture of nearly 20 triterpene alcohols. The isolation of a novel triterpene alcohol, (20S)-dammara-12,24-dienol, is reported. Next, the characterization of six oxidosqualene cyclases of primary metabolism are detailed, including lanosterol synthases from S. cerevisiae, Trypanosoma cruzi, Trypanosoma brucei, Homo sapiens, Bos taurus, and cycloartenol synthase from A. thaliana. Despite no reports of minor product generation by lanosterol synthases prior to this work, each cyclase is shown to make minor “errors”. These cyclases make different sets of minor products, and produce the major product with varying accuracy. This work demonstrates that minor product formation is characteristic of oxidosqualene cyclization, and leads to the conclusion that no cyclase produces only a single product. Finally, lanosterol synthase product profiles are extended to in vivo systems, via the analysis of triterpene alcohols present in yeast culture, as well as in mammalian tissue. This analysis demonstrates that S. cerevisiae lanosterol synthase produces at least 16 products, including three generated through B-ring-chair intermediates, the first evidence of a non-mutant cyclase accessing B-ring-boat and B-ring-chair intermediates. Analysis of bovine brain extracts led to the discovery that 18 lanosterol synthase minor products are detectable in mammalian tissue, including two novel triterpene alcohols, protosta-20(22)E-dienol and CB-thalianol A. Finally, this analysis demonstrated that one lanosterol synthase minor product, parkeol, is metabolized by enzymes in the sterol biosynthetic pathway, demonstrating that enzymatic errors generate a previously hidden level of chemical diversity in primary metabolism.
Organometallic Approaches for Selective Bioconjugation Using Boronic Acids
(2021-03-30) Miller, Mary K; Ball, Zachary T
Peptide and protein bioconjugates are important for the development of chemical tools and biopharmaceuticals. Due to the vast number of applications of bioconjugate structures, a need has arisen for diverse methodologies to create different bioconjugate structures. Organometallic methodologies have risen in prominence to meet this need due to the varying reaction types and residue selectivities that can be accessed using these strategies. The first two chapters review developments in transition metal chemistry in aqueous environments, which is important to understand when developing novel organometallic bioconjugation strategies. The first chapter details copper-mediated bioconjugation strategies with a focus on the mechanisms of the reactions described. The second chapter discusses methods for arylation of amines in aqueous conditions, including small molecule alkyl, aryl, and aromatic amines as well as larger biomolecules such as peptides and DNA. The development of selective protein/peptide bioconjugation strategies using boronic acids will be covered in chapter 3–5, focusing on the diversity of bioconjugate structures that can be accessed by judicious choice of metal mediator and boronic acid substrate. Copper(II) salts and ortho sulfonamide boronic acids were shown to arylate the N-terminus of naturally occurring peptides. This method allowed us to achieve excellent selectivity for the N-terminus over lysine side chains. Utilizing rhodium(III), tyrosine was selectively arylated in a unique three-component organometallic bioconjugation strategy wherein an η 6 -organometallic complex was formed. This method showed a very different substrate scope, with 2-carboxamideboronic acids showing increased reactivity, likely due to the chelation to the rhodium(III) center. Utilizing nickel(II), cysteine residues and pyroglutamate-histidine dipeptide sequences were found to be arylated with boronic acids π-deficient electron withdrawing groups, which is complementary to previously reported approaches. It was discovered that certain bioconjutation strategies could perhaps be used orthogonally with one another, which will be discussed in chapter 6. While nickel(II) is reactive with boronic acids with π-deficient electron withdrawing groups, copper(II) is reactive with vinyl boronic acids and arylboronic acids lacking ortho substitution. Furthermore, nickel(II) does not react with boronic acids that lack π-deficient electron withdrawing groups. By employing sequential reactions using diboronic acid reagents, protein heterodimer structures could be accessed using transition metal mediated methodologies developed in the Ball lab.
Photocleavable approaches to reversible bioconjugation
(2019-12-19) Mangubat-Medina, Alicia E; Ball, Zachary T
The folding of peptides and proteins depends on the structural conformation of the amide backbone. Historically, side-chain modifications that respond to external stimuli, such as light, were a convenient technique for controlling folding and activity. As our curiosity into complex biological systems matures, so must the techniques used to probe these systems. While thoroughly less reported than light-sensitive side-chain modifications (called photocages), light-responsive modifications to the amide backbone structure represents a direct and powerful alternative to impact structural conformation. Using a copper-mediated, histidine-directed peptide backbone modification with vinyl boronic acids, it was possible to insert a traceless photocage into the N-H bond of the amide backbone. Several obstacles required addressing in order to achieve a traceless backbone photocage: first, the bond formed required a previously unreported photocleavage between C(sp2) and N atoms. Second, the photocaging reagents must maintain a minimum level of solubility in an aqueous solution. Finally, for maximized general use in biological systems, light-induce uncaging would ideally occur using visible light and near infrared light. Here, the first peptide backbone photocaging reagents are described. These represent a new class of photocaging reagents capable of tracelessly uncaging peptides using ultraviolet, blue, and near-IR light; reversibly disrupting peptide folding; reversibly interrupting enzymatic recognition of a peptide substrate; and reversibly modifying a model protein. These results represent a jumping point to developing a vast array of backbone photocages, optimized for a wide variety of contexts.
Rhodium and copper: chemical approaches to biological problems
(2019-12-06) Martin, Samuel Cody; Ball, Zachary T
Often, the development of a tool in one field of study allows researchers in another field to address questions and solve problems previously inaccessible. Chemical biology as a field seeks to develop and use chemical tools in the study of biological processes. This body of work was intended to develop chemical tools (synthetic procedures, chemical or organometallic probes, protein-binding design strategy, bioorthogonal protein modification reactions) which might one day achieve use toward the study of biological systems. The first two chapters review the history and previous developments within the chemical biology space. The first chapter discusses techniques for the metalation of peptides and proteins with transition metals. The second chapter reviews the role of rhodium metal in biological catalysis, specifically in the alteration of nucleic acids and polypeptides. Research toward advancing scientific knowledge in this broad field is discussed in chapters 3–5. Novel hybrid organic–inorganic protein ligands, consisting of a dirhodium-metallated aminoquinoline, are designed, prepared, and shown to confer potency and selectivity in a challenging class of kinases in chapter 3. In chapter 4, selective dirhodium–diazo peptide modification is developed as a tool to enable more comprehensive analysis of protein-based systems, including purification, on-gel analysis, and exogenous ligand binding affinity determination. Finally, in chapter 5, a copper-catalyzed peptide cross-coupling reaction is applied for use in caging and photoreleasing a full-length natural protein.
Tuning Rh(II) probes for intracellular inhibition of STAT3
(2017-08-03) Minus, Matthew B; Ball, Zachary T
Biological probes play an important role in the advancement of biochemistry, chemical biology, and medicine. Molecular targeting probes can elucidate biological mechanisms by binding to a target molecule in a way that disrupts its biological function. Molecular probes can selectively label a cellular organelle or protein which assists in biological studies. The development of new molecular targeting probes is critical for the progression of biosciences. Methods for quickly developing potent molecular targeting probes are still needed. A molecular targeting probe recognizes its target through strong intermolecular binding interactions. Early small molecule probes targeted deep hydrophobic binding pockets. Unfortunately, only a small portion of the biomolecules in the human genome can be targeted this way. As an alternative, proteins and peptides have been developed as probes and therapeutics for extracellular molecular targets. However, theses alternative methods are not always amenable for intracellular molecular targeting. Therefore, there remains a class of intracellular signaling proteins, such as STAT3, that signal through shallow hydrophilic binding interfaces. These large binding interfaces usually do not bind well with hydrophobic small molecules, because these have evolved to interact with other intracellular proteins. Previous studies have shown that Rh(II) can enhance small molecule-probe binding thereby increasing the probes efficacy. However, this phenomenon had only been demonstrated in simple protein-ligand assays.

Browsing by Author "Ball, Zachary T"

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