Molecular recognition tools for chemistry in living systems
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Molecular recognition tools for chemistry in living systems

  • Author(s): Kataki Anastasakou, Anna
  • Advisor(s): Sletten, Ellen M
  • et al.

The introduction of unnatural functionality in biological systems, coupled with detection using bioorthogonal chemical reactions, revolutionized the field of chemical biology by enabling the investigation of biological processes in live cells and simple organisms. However, the translation to complex organisms has led to less-than-optimal results with high background noise due to cross reactivity with activated reagents. This dissertation investigates the utilization of non-covalent chemistry and bioorthogonal host-guest pairs to obtain more efficient labeling of living systems. Complexation between a host and guest is diffusion-limited, hence can be efficient in dilute environments. The cucurbit[n]uril scaffold has been utilized to determine the minimum binding affinity required for efficient bioorthogonal complexation and investigate how guest size and charge affects the introduction of guests as unnatural metabolites. Carboranes, a cucurbit[7]uril guest class that can be removed “on demand” from the host cavity, were found compatible with metabolic glycoengineering and were successfully incorporated on the cell surface. The cucurbit[7]uril–carborane system serves as the first example of bioorthogonal complexation of a metabolically-incorporated guest and answers fundamental questions required for the further development of bioorthogonal host–guest pairs. Finally, work in expanding the properties of unnatural functionality that can be tolerated by biological systems led to the development of fluorescent guest molecules that could be used independently or in conjunction with molecular recognition tools to investigate living systems.Chapter One is a review on the expansion of chemical reporters beyond bioorthogonal chemistry by metabolic incorporation of alternative moieties with novel functions. These chemical reporters expand on our ability to study and manipulate biological processes with non-invasive methods. Chapters Two details the synthetic methodologies for functionalized cucurbit[7]urils. The two main synthetic approaches to obtaining mono-functionalized cucurbit[7]urils were investigated and cucurbit[7]uril-payload conjugates were synthesized that were used in the remaining work on bioorthogonal complexation described in this dissertation. Chapter Three describes the discovery of carboranes as cucurbit[7]uril guests and their derivatization to increase their binding affinity and aqueous solubility. Ortho-carborane is presented as an stimuli-responsive guest that allows recycling cucurbit[7]uril-solid support constructs through multiple payload isolation rounds in cell lysate. Chapter Four introduces bioorthogonal complexation, namely the labeling of cell-surfaces using host-guest chemistry. A variety of cucurbit[7]uril guests were labeled with a cucurbit[7]- uril-fluorescein conjugate to determine the minimum binding affinity required for bioorthogonal complexation. Metabolic incorporation of a cucurbit[7]uril guest is also presented as a sialic acid derivative on the cell surface that can be similarly labeled with cucurbit[7]uril-fluorophore conjugates. Chapters Five explores the functionality of cucurbit[7]uril guests that can be tolerated by the sialic acid biosynthetic pathway along with in vitro and in vivo methods to analyze and determine successful metabolic incorporation. Chapters Six outlines the development of fluorescent guest molecules that can serve as independent fluorophores or in conjuction with bioorthogonal complexation. The mechanism of luminescence is investigated and preliminary in cellulo data is presented that point towards potential applications.

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