The field of bioorthogonal chemistry uses click chemistry as a tool to further understand cellular function. The tools of bioorthogonal chemistry include cell and metabolic probes, fluorescent probes with varying bioorthogonal handles that can be used for cell labelling, and gauging metabolic function. This is done by using non-toxic, orthogonal, and selective chemical probes that do not interfere with the complex environment of the cell (termed bioorthogonal). An example of a bioorthogonal reaction is the tetrazine ligation, which occurs via an Inverse Electron Demand Diels Alder (IEDDA) reaction with the 4π and 2π orbitals of a tetrazine and an alkyne/alkene, respectively. This reaction is considered bioorthogonal due to the reagents' high selectivity, quick kinetic rates of reaction, and the reaction's high yield, all of which do not disturb the native cellular environment. The key advantage of this reaction is its extremely fast rate, with second-order rate constants ranging from 10³ to 10⁶ M⁻¹s⁻¹ with the exergonic release of N₂ (g) as the sole by-product which causes the reaction to be irreversible. However, tetrazines are easily hydrolyzed and are redox-sensitive.
In this work, we describe herein an alternative bioorthogonal reagent to tetrazine in an IEDDA reaction with cyclooctynes or trans-cyclooctene (TCO). Currently, the tetrazine ligation, with an alkene/alkyne is the fastest bioorthogonal reaction known. Based on this, we investigated the synthesis of diazapyrones as an alternative heterocycle to tetrazine. We also investigated the synthesis of trans-cyclooctenes using a photochemical flow reactor developed by Dr. Fox et al. From this we aimed to develop two cycloaddition reactions, the first one using bicyclononyne (BCN) and the second using strained trans-cyclooctenes (sTCO). Both of these reagents act as the dienophiles. Evaluation of reactions between diazapyrones, cyclooctynes, and trans-cyclooctenes was done through kinetic studies using UV-Vis spectroscopy under pseudo-first-order conditions. Different diazapyrones were tested accordingly with BCN and sTCO dienophiles. The effects of varying substituents on the diazapyrones were also studied. From the results, it was determined that electron-poor diazapyrones react the fastest with BCN and sTCO. Kinetic studies also indicated that diazapyrones are unstable in aqueous environments and are prone to hydrolysis. However, this primarily poses issues for electron poor diazapyrone. The reactions between diazapyrones and cyclooctynes or trans-cyclooctenes are slower than the established tetrazine ligation (k2 ~ 1 - 10⁴ M⁻¹s⁻¹). The k2 values are 10⁻² M⁻¹s⁻¹ and 10 M⁻¹s⁻¹ respectively. However, reaction rates are comparable to other established bioorthogonal reactions such as SPACC (k~ 10⁻² - 10 M⁻¹s⁻¹) or SPANC (k~ 10 - 60 M⁻¹s⁻¹). Thus, this reaction is a useful addition to the bioorthogonal toolbox and can be applied in cell labelling experiments.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/44620 |
| Date | 15 February 2023 |
| Creators | Morrill, Kaitlyn |
| Contributors | Pezacki, John |
| Publisher | Université d'Ottawa / University of Ottawa |
| Source Sets | Université d’Ottawa |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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