Methods for the syntheses of trans-cycloheptene, dihydrotetrazine and tetrazine compounds and their applications to bioorthogonal chemistry and drug delivery
Date
2018
Authors
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Publisher
University of Delaware
Abstract
In chapter 1, I will provide an overview of chemistry involving medium sized trans-cycloalkenes, with a focus on trans-cycloheptene and sila trans-cycloheptene. I will describe the development of a general synthetic method of trans-cycloheptenes and sila trans-cycloheptenes. These seven-membered trans-cycloalkenes are isolated and stabilized as silver nitrate complexes. trans-Cycloheptene silver complexes and sila trans-cycloheptene can engage in a range of cycloaddition reactions as well as dihydroxylation reactions. Computation was used to predict that sila trans-cycloheptene would engage in bioorthogonal reactions that are more rapid than the most reactive trans-cyclooctenes. Metal-free sila trans-cycloheptene derivatives were shown to display good stability in solution, and to engage in the fastest bioorthogonal reaction reported to date (k2 1.14 × 107 M−1 s−1 in 9: 1 H2O: MeOH). Utility in bioorthogonal protein labeling in live cells is described, including labeling of GFP with an unnatural tetrazine-containing amino acid. The reactivity and specificity of the sila trans-cycloheptene reagents with tetrazines in live mammalian cells was also evaluated using the HaloTag platform. The cell labeling experiments show that sila trans-cycloheptene derivatives are best suited as probe molecules in the cellular environment. Additionally, trans-cyclooctene silver complexes also show superior stability to metal free trans-cyclooctenes. I evaluated the stability of different trans-cyclooctene silver complex derivatives. In-gel fluorescence studies have shown trans-cyclooctene silver nitrate complexes are useful precursors to trans-cyclooctenes in bioorthogonal chemistry. ☐ Using bioorthogonal ligation reactions to trigger bond cleavage under physiological conditions has gained much attentions. Bioorthogonal cleavage reactions can serve as a spatiotemporal controllable manipulation to restore function of protected motifs such as caged-fluorophores, masked biomolecules or prodrugs. Our group have developed a NIR-triggered, methylene blue catalyzed dihydrotetrazine oxidation reaction to restore the reactivity of tetrazines. In Chapter 2, I will describe the application of this chemistry in bioorthogonal bond cleavage reaction for prodrug activation. Nirogen mustard and Combretastatin A4 dihydrotetrazine prodrugs were synthesized. These dihydrotetrazine prodrugs show good stability in PBS. The prodrug activation process is rapid, producing good yields of cytotoxic drugs. Preliminary results have shown that catalytic photodecaging can have a pronounced effect in a prostate cancer cells. ☐ The major challgene in developing new dihydrotetrazine prodrugs is the synthesis of unsymmetrical dihydrotetrazine. Dihydrotetrazine can be obstained via reduction reaction of tetrazine. However, the synthetic methodology of unsymmetrical tetrazine are limited. In Chapter 3, I will discuss the development of a novel synthetic method of unsymmetrical tetrazine. The new synthetic route involves a condensation reaction between normal orthoester or OBO orthoester and bishydrazide, followed by a palladium cross-coupling reaction with organotin reagents or boronic acids. In contrast to the widely-used nitrile/hydrazine condensation reaction, no hydrazine anhydrous or hydrazine monohydrate were used in this condensation/cross-coupling reactions. A variety of unsymmetrical tetrazines are prepared with moderate to good yield. This chemistry allows for the synthesis of useful unsymmetrical tetrazine-containing molecules in a more efficient and safe way.
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Keywords
Synthesis, Trans-cycloheptene, Dihydrotetrazine, Tetrazine, Bioorthogonal chemistry, Drug delivery