The direct synthesis of tetrazines from carboxylic ester precursors and the development of the photocatalytic tetrazine ligation for intracellular bioorthogonal labeling and uncaging
Date
2021
Authors
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Publisher
University of Delaware
Abstract
Conventional approaches to conjugating tetrazines with bioactive molecules requires hydrophobic linkers which may have negative impact on target binding and physiochemical properties of the conjugates. The reason is the lack of mild, safe, general and practical approaches to synthesizing tetrazines. Chapter 1 described one-pot synthesis to convert carboxylic ester precursors into thiomethyl tetrazines from which monosubstituted tetrazines could be access by palladium catalyzed reduction and unsymmetrical tetrazines could be prepared through Liebeskind-Srogl coupling with boronic acids and stannane reagents. These approaches have broad functional group tolerance and avoid the direct handling of hydrazine. New tetrazine-based MAGL probes were developed and the most reactive probe was used to label MAGL protein in live cells. ☐ Chapter 2 described far-red light triggered photocatalytic turn-on of tetrazine reactivity for intracellular protein labeling with spatiotemporal control. The first DHTz/Tetrazine pair suitable for the designed intracellular application was developed. The new DHTz is highly resilient towards background oxidation in PBS and intracellular environments which minimizes uncontrolled labeling. The corresponding tetrazine was shown to be the most reactive diene in tetrazine-TCO reactions, which enables no-wash labeling of endogenous proteins in live cells. ☐ Chapter 3 introduced new photocatalytic or enzymatic drug release systems. Prodrugs consist of a DHTz core and vinyl ether linked cytotoxic drug. The release mechanism includes photocatalytic oxidation of DHTz to tetrazine, intramolecular Diels-Alder reaction between tetrazine and a tethered vinyl ether and an elimination reaction to release drugs. Gen 3 prodrugs based on amide- functionalized DHTzs with improved stability were synthesized and employed in Intracellular uncaging. The photocatalyst SiR was conjugated to Hoechst’s ligand and localized to the nucleus in order to achieve intracellular uncaging by a subcellularly localized photocatalyst. Gen4 prodrugs based on acetyl-functionalized DHTz with small size, access via mild synthetic conditions, as well as improved stability are developed and being employed for photocatalytic and enzymatic drug delivery applications.
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Keywords
Tetrazines, Bioactive molecules, Thiomethyl tetrazines, DHTz, MAGL protein, Intracellular uncaging