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Proximity-Enhanced Reactivity for Chemical Biotechnology

  • Author(s): Robinson, Peter V.
  • Advisor(s): Bertozzi, Carolyn R
  • et al.
Abstract

Chemoselective reactions are powerful tools for the progression of basic science and biochemical technologies. Bond-forming reactions are routinely used for monitoring the status of cellular activity and in the production of precision therapeutics and diagnostics. However, the development of such reactions is a challenging feat, as one must account for the broad swathes of interfering chemical entities present within the biological milieu. One way to address this complicating factor is through the use of “bioorthogonal” chemistry. These reactions involve two reactive groups with xenochemical properties, i.e., reactivity preferences that are inert to biological moieties yet exquisitely reactive with one another. By necessity, bioorthogonal reactions are forged from deep chemical insight. A complementary strategy to achieve reaction selectivity in biological settings seeks to use proximity enhancement. Positioning two groups in close proximity to one another raises the higher effective local concentration of the desired partners, increasing the rate by which they react to afford highly selective conjugation, even amidst complex systems. In this dissertation, I explore several applications of this concept to develop a variety of tools for basic bioscience and clinical diagnostics.

In Chapter 1, I provide a brief overview of several landmark papers that employ proximity-enhanced reactions in chemical biotechnology.

In Chapter 2, I describe the use of proximity-enhanced reactivity to direct ligation and detection of protein-specific glycosylation on live cells.

In Chapter 3, I describe the application of proximity enhancement to selectively accelerate the ligation of DNA strands that have been chemically attached to different antibodies. In this particular project, we used paired antibodies that bind either to biotin or a protein of interest, driving the ligation and formation of a DNA amplicon in the presence of a biotinylated protein. In combination with chemistries that selectively attach biotin to glycans, this technology allows the use of qPCR to detect very low levels of glycosylated proteins.

In Chapter 4, I describe the development of a new technology, termed Antibody Detection by Agglutination-PCR (ADAP), which uses PCR to detect antibodies with a high degree of multiplexing and sensitivity.

In Chapter 5, I describe a method for re-purposing cyclooctynes to selectively ligate to thiols present on serum albumin as a strategy to improve the serum-half lives of therapeutic molecules.

In Chapter 6, I describe the computational modeling and synthesis of dendrimer-based glycan mimetics.

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