Expanding the bioorthogonal chemistry toolbox: Photo-inducible Diels-Alder cycloadditions and endeavors towards a platform to study protein-protein interactions
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2024
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Doctoral Thesis
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yes
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Abstract
In the early 2000s, genetic code expansion (GCE) was developed, providing a powerful methodology for site-specific protein modification with non-canonical amino acids (ncAAs). Since then, a large variety of diverse functionalities has been introduced into proteins including post-translational modifications (PTMs), crosslinking moieties, and bioorthogonal groups. Bioorthogonal moieties are inert towards the chemistries found in biological systems and can be selectively targeted with a suitable external probe in bioorthogonal reactions. Over the past twenty years, numerous bioorthogonal reactions have been developed, with the inverse electron-demand Diels-Alder cycloaddition (iEDDAC) between tetrazines and strained dienophiles (tetrazine ligation) emerging as one of the outstanding choices. With second-order rate constants of up to 10⁷ M⁻¹ s⁻¹, the tetrazine ligation surpasses all other bioorthogonal reactions in terms of kinetics by several orders of magnitude, allowing fast and selective labeling of biomolecules within seconds at nanomolar concentrations. Various ncAAs bearing strained dienophile moieties or tetrazine functionalities have been incorporated site-specifically into proteins over the last ten years, and the combination of GCE and tetrazine ligation has provided powerful platforms to study, manipulate, and selectively target proteins in chemically complex environments such as living systems.
The first part of this thesis presents the development of a photo-inducible iEDDAC (photoiEDDAC) methodology between tetrazines and a cyclopropenone-caged dibenzoannulated bicyclo[6.1.0]nonyne probe (photo-DMBO). Recent advancements within the bioorthogonal field did not only focus on the development of more reactive moieties but also on ways to control the reactivity of these handles with external stimuli. Especially light has emerged as a convenient and non-invasive trigger for bioorthogonal reactions, enabling spatio-temporal control. Despite the popularity of the tetrazine ligation however, the selection of photo-iEDDAC moieties was limited when we initiated our investigations.
Inspired by cyclopropenone-caged dibenzocyclooctynes (DBCOs) used for photo-induced strain-promoted azide-alkyne cycloadditions (photo-SPAAC), we designed and synthesized photo-DMBO – a DBCO featuring a cyclopropane fused to its cyclooctyne core. Photo-DMBO itself exhibited no reactivity towards tetrazines, but upon irradiation with UV light, the cyclopropenone moiety was efficiently removed liberating the strained cyclooctyne (DMBO), which readily engaged in iEDDAC with tetrazines. We first established our photo-iEDDAC methodology between photo-DMBO and tetrazines on a small molecule level, characterizing important properties of the novel DMBO moiety, and then successfully expanded it to labeling experiments on tetrazine-modified proteins in vitro, in cell lysates, and on living E. coli cells.
Due to the unique reactivity of DMBO towards tetrazines, which is typically not observed for DBCOs, we further conducted kinetic experiments and computational studies employing density functional theory (DFT) calculations. These studies revealed that DMBO adopts an unusual tub-like conformation in the transition state of iEDDAC and SPAAC reactions, which allows tetrazines and azides to approach the strained alkyne from the face trajectory and greatly reduces the activation barrier of the iEDDAC, explaining DMBO’s unique reactivity. Intrigued by these findings, we expanded our studies towards other cycloalkane-fused DBCOs, where we identified the same tub-like transition state geometry as observed for DMBO, highlighting the great influence of bicyclic structures on DBCO reactivity.
In a second project, we aimed at developing a platform to study protein-protein interactions (PPIs) in living cells. PPIs are fundamental to numerous biological processes, driving essential signaling and regulatory pathways. Because of their non-covalent and typically transient character, they are however difficult to study. One possibility consists in applying GCE to sitespecifically incorporate crosslinking ncAAs, generating covalent linkages between interacting proteins in a proximity-induced manner. Nevertheless, the necessity of orthogonal tRNA/aminoacyl tRNA synthetase (tRNA/aaRS) pairs to incorporate these ncAAs into proteins limits the design space for crosslinking moieties due to active site restrictions.
We therefore aimed at establishing a platform to install several electrophilic crosslinking moieties through a combination of GCE and tetrazine ligation. In short, we introduced a bicyclo[6.1.0]nonyne-bearing ncAA (BCNK) into proteins via GCE and subsequently treated the modified protein with bifunctional tetrazine-electrophile conjugates installing the crosslinking moiety site-specifically. The scope of the electrophilic moieties that we could attach to the proteins was thus mostly limited by the synthetic accessibility of the tetrazineelectrophile conjugates. To streamline the synthesis of these conjugates, we employed a modular synthetic approach utilizing tetrazine precursors, which allowed us to attach minimal tetrazine moieties to certain functional groups within the electrophiles. We determined two suitable precursors for our crosslinking platform by thorough in vitro evaluation of several tetrazine moieties. Afterwards, we selected six different electrophiles targeting various nucleophilic amino acid side chains and synthesized the corresponding tetrazine-electrophile conjugates, which we successfully employed to label proteins in vitro and in cellulo. Furthermore, we conducted crosslinking experiments on model protein complexes such as the sfGFP homodimer and the Rab1b-DrrA complex. While these experiments faced challenges and were mostly unsuccessful, they laid the groundwork for a potential crosslinking platform to study PPIs, but warrant further investigations. Nevertheless, our in-depth evaluation of differently substituted tetrazines could constitute a valuable contribution to the scientific community as we identified several strengths and weaknesses of each tetrazine moiety.
Hence, in the third part of this thesis, we extended our investigations to tetrazines commonly used for labeling experiments to generate a comprehensive comparison between these tetrazine moieties under uniform conditions. To this end, we synthesized a total of ten differently substituted model tetrazines and evaluated them in vitro for key properties such as their kinetics in tetrazine ligation, their stability under physiological conditions, and their protein labeling efficiency. Moreover, it is our goal to extend our investigations towards fluorescent protein labeling experiments conducted in different biological settings such as in cell lysate, on living cells, and in cellulo. Ultimately, we envision that this study may serve as a guideline like a “hitchhiker`s guide to tetrazine ligation”, which allows us to recommend suitable tetrazinedienophile pairs for the intended biochemical experiment.
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Examiner: Lang, Kathrin
Examiner : Hacker, Stephan
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ETH Zurich
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09740 - Lang, Kathrin / Lang, Kathrin