Development of new tools for the tetrazine ligation: installation of minimal tetrazines through silver(i) mediated cross-coupling and development of a hydrophilic trans-5-oxocene for bioorthogonal labeling
Date
2020
Authors
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Publisher
University of Delaware
Abstract
Methods to probe the biochemistry of living organisms have been greatly augmented by the relatively recent advancement of bioorthogonal chemistry. Of the many bioorthogonal reactions, the tetrazine ligation has emerged as one of the most biologically compatible due to its rapid catalyst-free kinetics, innocuous byproducts, and orthogonality toward native cellular processes. In the introductory chapter I will give a brief history of the development of the tetrazine ligation, specifically the inverse-electron demand Diels-Alder reaction between tetrazine and trans-cyclooctene. Biologically relevant applications of the tetrazine ligation include protein and oligonucleotide labeling, fluorescent and radiochemical imaging, targeted drug delivery, and the synthesis of biomimetic materials. ☐ In chapter 2 I will overview the classical and modern synthesis of tetrazines, including a new approach to minimal tetrazine conjugations to complex biologically relevant molecules. I have developed the first silver-mediated Liebeskind-Srogl cross-coupling reaction for the functionalization of aryl boronic acids with a minimal 6-methyltetrazine moiety. A new reagent for this reaction, ‘b-Tz’, has been optimized for large scale synthesis with process chemistry principles. Cross-coupling is catalyzed by PdI2(dppf) and has broad substrate scope, including an improved route toward tetrazine-fluorophore conjugates as well as the development of a tetrazine-MAGL (monoacylglycerol lipase) conjugate for the quantification of endogenous proteins in live cells. ☐ Further, the silver-mediated, palladium-catalyzed Liebeskind-Srogl reaction has recently been improved with the development of a new catalyst which will be discussed in chapter 3. Through optimization of ferrocene ligand and palladium halide pairings, I have advanced a new catalyst which promotes the cross-coupling of b-Tz with electron deficient aryl boronic acids with greatly improved yields. The efficiency of the reaction is enhanced by requiring less boronic acid, catalyst, and silver loadings while still obtaining higher yields than my first-generation protocol. ☐ Finally, chapter 4 will discuss the development of ‘oxoTCO’; a small and hydrophilic trans-cyclooctene for in vivo application. oxoTCO is synthesized in seven high yielding steps and has been experimentally tested for its aqueous solubility, stability, and overall kinetics in a tetrazine ligation. Additionally, oxoTCO has been used as an 18F-labeled probe for positron emission tomography (PET) where it displayed improved signal-to-background versus alternative trans-cyclooctenes.
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Keywords
Bioorthogonal, Cross-coupling, Biomimetic materials, Tetrazine, Trans-cyclooctene