Catalytic activation of bioorthogonal chemistry without photochemistry
Date
2025
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
University of Delaware
Abstract
My research centers on developing novel strategies to activate the rapid tetrazine ligation reaction through “photochemistry-free” oxidation, enabling applications in biological systems. I demonstrated that the stable precursor dihydrotetrazine (DHTz) can be oxidized by enzymes and small molecules, via catalytic and stoichiometric pathways, to trigger subsequent tetrazine-trans-cyclooctene (TCO) ligation in the absence of light. I achieved the first intracellular enzymatic labeling reaction of DHTz in live cells using “dark” catalysis- defined as catalytic chemical reactions that occur without light. Building on this, I made progress toward development of new proximity labeling systems that leverage dark catalysis, and I have evaluated a range of dark catalysts and oxidants, comparing their efficiencies and biocompatibilities with the DHTz system. ☐ In chapter 1, I discuss my work with ascorbate peroxidase (APEX2) to turn on the bioorthogonal tetrazine ligation reaction. Kinetic studies revealed that APEX2-catalyzed oxidation of DHTz is enhanced by superoxide dismutase (SOD), a ubiquitous mammalian enzyme that regulates oxidative stress by converting superoxide into molecular oxygen (O2) and hydrogen peroxide (H2O2). The APEX2 oxidation with SOD achieved a catalytic efficiency of kcat/KM 4.90 × 103 M–1s–1 in vitro. While H2O2 is not strictly required, the addition of 10 µM H2O2 accelerated the oxidation reaction both in vitro and in live cells. Using a dual-transfection protocol expressing cytosolic APEX2 and HaloTag-DHTz conjugate, I demonstrated that APEX2 promotes DHTz oxidation and subsequent Diels- Alder chemistry in live HeLa cells. Labeling with a fluorophore-tagged TCO probe was confirmed via in-gel fluorescence, Western blot analysis, and confocal microscopy. In live PC3 cells, APEX2 also catalyzed the oxidation of a DHTz conjugated to an endogenous monoacylglycerol lipase (MAGL) through a selective covalent warhead. ☐ In chapter 2, I describe my screenings for proximity labeling using APEX2 fused to various proteins of interest (POI). Previous studies showed APEX2-biotin-phenol systems label proteins within a 20 nm radius. I wanted to compare this method for proximity labeling to a complementary approach based on enzymatically activatable bioorthogonal chemistry. I labeled lysine residues proteome-wide with a TCO-N-hydroxysuccinimide (NHS) ester, then activated a DHTz by APEX2 bearing a biological alkyne handle to assess proximity-based differences. The alkyne- labeled proteins were conjugated to biotin-azide or TAMRA-azide via a Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Among the constructs tested, cytosolic APEX2-GFP detected new proteins. While other constructs targeting cereblon (CRBN), the outer mitochondrial matrix (OMM), and a nuclear export signal (NES) did not show detectable labeling under these conditions, these results offered insight to the complexities of expression levels and accessibility for effective proximity labeling methods. To further analyze labeling targets, I optimized a small-scale streptavidin enrichment protocol. ☐ In chapter 3, I evaluate DHTz activation by small molecules for dark catalysis. Building on the heme enzyme of APEX2, I demonstrated that the iron porphyrin complex, Fe (III) tetrakis (N-methyl-4′-pyridyl) porphyrinato (FeTMPyP) activates DHTz in vitro and in extracellular environments. In addition, ferrocenium tetrafluoroborate promoted the rapid and stoichiometric oxidation of DHTz with a second-order rate constant of k2 = 1.82 x 105 M-1s-1. The low molecular weight of ferrocenium ion and the extremely rapid kinetics of DHTz oxidation make it a uniquely promising oxidant among the small molecules tested for tetrazine activation in biological applications. The compact structure of ferrocenium also offers an excellent scaffold for further functionalization. Beyond ferrocenium, I evaluated a range of other oxidants. Copper (II) sulfate (CuSO4) catalytically oxidized DHTz, while quinones acted as stoichiometric oxidants. Each class of oxidant presents unique advantages and limitations, which I critically analyzed in the context of their potential for biological compatibility, efficiency, and tunability.
Description
Keywords
Dihydrotetrazine, Ascorbate peroxidase, Nuclear export signal, Outer mitochondrial matrix