Dissertation/Thesis Abstract

The tetrazine ligation: Development of a novel bioorthogonal Diels-Alder reaction and its applications
by Blackman, Melissa Lynn, Ph.D., University of Delaware, 2011, 214; 3465758
Abstract (Summary)

A novel bioorthogonal reaction based on an inverse electron-demand Diels-Alder reaction has been developed. The transformation involves the cycloaddition of s-tetrazine derivatives with strained dienophiles such as cyclopropene and trans-cyclooctene.

The reaction of trans-cyclooctene and 3,6-di-(2-pyridyl)-s-tetrazine is significantly faster than conventional ‘click’ reactions and proceeds in high yield in organic solvents and aqueous media. The reaction exhibits selectivity in the presence of biological nucleophiles such as amines and thiols as well as bioorthogonal substrates such as phosphines, azides and alkynes. Experiments conducted in biological media and cell lysate do not adversely affect the reaction. In addition, the reaction has been carried out on the protein Thioredoxin (Trx) with 100% conversion within five minutes. Selectivity, fast reaction rate and aqueous compatibility make the reaction suitable for various in vivo and in vitro applications.

The scope of the reaction has been expanded by synthesizing various s-tetrazine derivatives with the purpose of identifying a relatively stable tetrazine with comparable or superior reactivity than that of 3,6-di-(2-pyridyl)-s-tetrazine. Gratifyingly, all of the derivatives demonstrated excellent stability stable and fast reactivity.

The utility of the tetrazine ligation in the fields of surface chemistry, protein engineering and radiochemistry has been explored. A variety of unnatural amino acids containing trans-cyclooctene or tetrazine functionality have been synthesized and site-specifically incorporated into proteins in vitro and in vivo. Additionally, a robust radiolabeling method based on the reaction between 3,6-diaryl-s-tetrazines and an 18F-labeled trans-cyclooctene has been developed. The fast kinetics of the tetrazine-trans-cyclooctene ligation effectively addresses the existing challenges of this application, which include reaction rate, efficiency, and selectivity.

Indexing (document details)
Advisor: Fox, Joseph M.
Commitee: Koh, John T., Thorpe, Colin, Venkatraman, Srikanth
School: University of Delaware
Department: Department of Chemistry and Biochemistry
School Location: United States -- Delaware
Source: DAI-B 72/10, Dissertation Abstracts International
Subjects: Organic chemistry
Keywords: Bioorthogonal reaction, Diels-Alder reaction, Tetrazine ligation
Publication Number: 3465758
ISBN: 978-1-124-78194-5
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