Base-Activated Lysine Probes and their Utility in Mosquito Larvae Midguts

Abstract

Protein bioconjugation is an essential tool for the deeper understanding of protein behavior and localization. New bioconjugation tools are constantly being developed, increasing the scope of what residues can be targeted and under which conditions, ultimately reporting on the chemical environment of the system. Although many different amino acid residues can be targeted through tailored probes, the most commonly targeted residues are cysteine and lysine due to their high nucleophilic character. Lysine is much more abundant on the surface of proteins, making it an attractive target for robust protein labeling, but can also be problematic when looking to conjugate to other nucleophilic residues. We initially looked for compounds that would act as a reversible lysine probe that could be utilized as a protecting group, masking reactivity in situations where lysine is competing with another residue. In this search, we came across a Meldrum’s acid derivative, equipped with a Michael acceptor that showed reversibility when a single amine was attached. When testing this compound for our own purposes, we found an unexpected, irreversible side reaction at pH 10, which was evident by the loss of our protein band of interest in our gel analyses. Switching to small molecules to identify this side reactivity, we found the Meldrum’s acid derivative was not only capable of accepting one amine but was accepting two amines in high pH environments. This showed that the loss of our protein band was due to oligomerization by the addition of two lysine residues to the probe backbone. We were intrigued by this new reactivity and began characterizing the second amine addition further. Herein, this dissertation will discuss the characterization of the double amine addition through kinetics, water stability, and resilience against reducing agents and other nucleophiles. Our kinetic studies have shown the reaction rate of this second amine addition increases with increasing pH (fastest at pH 10), leading to use of these compounds to label proteins in basic environments within minutes. Furthermore, the single and double amine adducts have shown to be stable across a wide range of pH values, showing excellent stability of both compounds in aqueous systems. Therefore, we envisioned using this reactivity to our advantage and add a single amine equipped with our cargo of choice to the backbone and allow the second amine addition to be a pH dependent reaction with lysine residues, forming a stable, permanent covalent linkage of our cargo to the surface of the protein. We call this series of probes MaMa (Meldrum’s acid amine-reactive Michael acceptor) and these compounds have shown to be easily functionalized with different types of cargo. This dissertation will discuss the design and synthesis of many different MaMa probes, including ones equipped with fluorophores, click handles, and even a fluorogenic system, along with their pH reactivity profiles with proteins. Due to the ease of functionalization and stability, our MaMa probes overcome the limitations of currently existing lysine technologies. To test these probes in a larger system, we chose mosquito larvae, as their midguts have aunique pH gradient ranging from pH 7-11. As our MaMa probes are activated at high pH, we sought to use our probes as a tool to find new protein drug targets to help overcome the resistance mosquitoes are gaining against current control methods. This dissertation will highlight the use of a MaMa probe already equipped with a fluorophore, a fluorogenic system, and our click-ready probes in the mosquito midgut for fluorescent labeling. We have found the importance of using a bright fluorophore for optimal visualization and the need to choose fluorophores that do not cross cellular membranes. Preliminary data does show promise for the use of these probes in mosquito larvae midguts to shed light on new protein drug targets for tailored insecticide development. Finally, this dissertation will discuss attempts at altering the optimal pH necessary for the double amine addition. Since the reactivity of these probes are dependent on the pKa of both the MaMa probe and lysine, we sought to manipulate the pKa of the N-H bond on the MaMa backbone by conjugating aniline compounds equipped with varying substituents of varying degrees of electron donating and withdrawing character. This dissertation discusses the synthesis of this class of aniline probes along with small molecule kinetic data showing reactivity at various pH values. Some of these compounds have shown optimal pH conditions at lower pH values than the first class of MaMa probes, showing promise for labeling at a wider range of aqueous conditions. Additionally, the tuning of the pKa with a MaMa probe containing a propargyl linker will be discussed, as it deviates slightly from the initially determined kinetic trend. Finally, the attempt at altering pKa of the MaMa probes with other nucleophiles, such as hydrazines and hydroxylamines will be discussed. Overall, the MaMa probe series has shown promise for use in many different environments and experimental setups, including in vivo and in vitro. The story of the development and design of these probes along with their uses will be highlighted throughout this dissertation.