The versatility of asymmetric aminoethyl-tetrazines in bioorthogonal chemistry

Sarris, A. (2025)

Doctoral Thesis

This thesis describes the synthesis, analysis of various tetrazine bearing compounds and use in bioorthogonal chemistry. Chapter 1 describes a literature survey of tetrazine chemistry developments throughout history is provided. Additionally an introduction to the emergence of click chemistry, bioorthogonal reactions, and IEDDA chemistry is provided. These developments are the foundations on which the work described in this thesis is built upon. Chapter 2 reports the synthesis of tetrazine-modified alanine building blocks as functionalized amino acids to substitute natural amino acids in activity-based probes (ABP’s) for bioorthogonal activity-based protein profiling (ABPP) of cysteine, serine and threonine peptidases. Chapter 3 describes the synthesis of a library of functionalized tetrazines, as well as the synthesis and optimization of a variety of reactive alkenes. The work includes kinetic studies to determine the reactivity of the library of tetrazines towards the synthesized variety of alkenes. Additionally, these tetrazines were attached to Bodipy-FL to obtain fluorophore-tetrazine tags for bioorthogonal live cell fluorescence microscopy of alkene-functionalized molcecules present in the cell. Chapter 4 describes the synthesis of highly water-soluble tetrazine fluorophores. These fluorophores were able to access the dense hydrophilic glycan coating (glycocalyx) around the cell surface and by doing so label the metabolically incorporated alkene-functionalized mannosamine. By using sterculic acid and Bodipy-FL tetrazine as a second bioorthogonal pair, live cells could be incubated and labeled simultaneously to achieve multicomponent labeling (“dual-labeling”) of alkene-bearing biomolecules. In Chapter 5 the library of tetrazines from chapter 3 was analyzed on their capability to perform a IEDDA-pyridazine elimination tandem reaction. The library was analyzed on their reactivity and elimination speed with AMC-coumarin, a previously employed method from the literature. The results were compared and showed that amino-ethyl functionalized tetrazines showed unprecedented elimination rates, combined with respectable elimination efficiency and some were not negatively affected by pH changes, or the lack of an acidic environment. In Chapter 6 a tool was designed and synthesized from bifunctional trans cyclooctane and the EDANS/DABCYL quencher pair to be able to correctly determine the reaction and elimination rates of tetrazines when releasing alkylic amines.

• Tetrazine
• Click chemistry
• IEDDA
• Click-to-release
• Elimination
• ABPP
• Bioorthogonal
• Reaction kinetics
• Fluorescence microscopy
• Labeling
• Live cell

• All authors: Sarris, A.
• Supervisor: Overkleeft, H.S.
• Co-supervisor: Kasteren, S.I. van
• Committee: Ubbink, M.; Codee, J.D.C..; Filippov, D.V.; Mikula, H.; Bonger, K.M
• Qualification: Doctor (dr.)
• Awarding Institution: Leiden Institute of Chemistry (LIC), Faculty of Science, Leiden University
• Date: 2025-02-20
• Sponsorship: Grant from ICI - Institute for Chemical Immunology