Biomedical Applications of Tetrazine Cycloadditions

Abstract

Conspectus Disease mechanisms are increasingly being resolved at the molecular level. Biomedical success at this scale creates synthetic opportunities for combining specifically designed orthogonal reactions in applications such as imaging, diagnostics, and therapy. For practical reasons, it would be helpful if bioorthogonal coupling reactions proceeded with extremely rapid kinetics (k > 103 M−1 sec−1) and high specificity. Improving kinetics would minimize both the time and amount of labeling agent required to maintain high coupling yields. In this Account, we discuss our recent efforts to design extremely rapid bioorthogonal coupling reactions between tetrazines and strained alkenes. These selective reactions were first used to covalently couple conjugated tetrazine near-infrared-emitting fluorophores to dienophile-modifed extracellular proteins on living cancer cells. Confocal fluorescence microscopy demonstrated efficient and selective labeling, and control experiments showed minimal background fluorescence. Multistep techniques were optimized to work with nanomolar concentrations of labeling agent over a timescale of minutes: the result was successful real-time imaging of covalent modification. We subsequently discovered fluorogenic probes that increase in fluorescence intensity after the chemical reaction, leading to an improved signal-to-background ratio. Fluorogenic probes were used for intracellular imaging of dienophiles. We further developed strategies to react and image chemotherapeutics, such as trans-cyclooctene taxol analogs, inside living cells. Because the coupling partners are small molecules (<300 daltons), they offer unique steric advantages in multistep amplification. We also describe recent success in using tetrazine reactions to label biomarkers on cells with magneto-fluorescent nanoparticles. Two-step protocols that use bioorthogonal chemistry can significantly amplify signals over both one-step labeling procedures as well as two-step procedures that use more sterically hindered biotin–avidin interactions. Nanoparticles can be detected with fluorescence or magnetic resonance techniques. These strategies are now being routinely used on clinical samples for biomarker profiling to predict malignancy and patient outcome. Finally, we discuss recent results with tetrazine reactions used for in vivo molecular imaging applications. Rapid tetrazine cycloadditions allow modular labeling of small molecules with the most commonly used positron emission tomography isotope, 18F. Additionally, in recent work we have begun to apply this reaction directly in vivo for the pre-targeted imaging of solid tumors. Future work with tetrazine cycloadditions will undoubtedly lead to optimized protocols, improved probes, and additional biomedical applications.

Selective chemical protein modification

“…Inspired by the work of Dommerholt et al 96 , it was found that trans-bicyclononene reacted at yet faster rates (while noting the caveats on rate determinations given above) 103 . In addition to allowing labelling of highly dynamic processes, such rapid and efficient reactions allow the concentrations of reactive partners to be lowered significantly, reducing background labelling particularly in cases where it is REVIEW implausible to wash away excess reagent, such as intracellularly or in animal models 104 .…”

Inverse electron demand Diels–Alder (iEDDA)-initiated conjugation: a (high) potential click chemistry scheme

“…The formed ''click'' products may be characterised, in addition to these techniques, with more sophisticated methods, such as MALDI-TOF-MS (matrixassisted laser desorption ionisation time-of-flight mass spectrometry) 20,24 or SEC (size exclusion chromatography). 12 Therefore, this section concentrates on the underlying chemistry to give an impression that features of iEDDA are responsible for its success in life sciences and could also be of interest for other fields. Herein, we focus on the three most impressive features of iEDDA: fast reaction rates, bioorthogonality and mutual orthogonality with other click reactions.…”

Single‐Vehicular Delivery of Antagomir and Small Molecules to Inhibit miR‐122 Function in Hepatocellular Carcinoma Cells by using “Smart” Mesoporous Silica Nanoparticles

“…Also,t he amino-propyl groups on the surface of MSNs were partially modified with PEG-linker-RGD peptide by the bioorthogonal tetrazine-TCO ligation reaction and copper-free DBCOazide click chemistry. [13] RGD peptide and PEG linker were previously shown to improve the nanoparticles cellular uptake and prevent their intracellular aggregation. [14] Upon cellular uptake (Steps I, II in Scheme 1) and successful endosomal/lysosomal escape (Step III), the hybridization of the complementary sequences between Ant122 and intracellular miR-122 produced arigid double helical structure and altered the conformation of Ant122, which would reduce the binding capacity of Ant122 and lead to the uncapping of the MSNs.…”