Tetrazine ligation techniques have evolved into a versatile tool set for bioconjugation. Despite their numerous applications, highly reactive tetrazines can suffer from long-term instability and off-target reactivity, and achieving cell-specific bioconjugation remains challenging. Tetrazine caging techniques intend to overcome these issues by achieving spatial and temporal control over tetrazine activation from stable dihydrotetrazine derivatives. Here we explore enzyme-initiated tetrazine uncaging, allowing for controlled release of reactive tetrazines by enzymes overexpressed in specific cell types. Using a modular synthetic strategy, we connected four distinct enzyme-cleavable motifs to caged tetrazine derivatives using a self-immolative linker unit. We validated cell-specific release using penicillin G amidase (PGA), which we genetically encoded into HEK293Ts. Cells expressing PGA were able to activate tetrazines which subsequently reacted with a dienophile-protected doxorubicin, lowering viability. In contrast, treatment of mammalian cells lacking PGA showed no reduction in cell viability. Additionally, we generated enzyme-cleavable conjugates that are hydrolyzed by enzymes commonly exploited for drug delivery in human cells, including esterases, cathepsin B, and alkaline phosphatases. We demonstrate cell-type specific activation of tetrazines based on the varying expression levels of alkaline phosphatases in different cell lines. Specifically, we compared tetrazine activation in the osteosarcoma cell line SAOS-2, known for its exceptionally high ALP expression levels, to cells that express lower levels of ALP, the colorectal adenocarcinoma cell line HT-29 and the stromal cell line HS-5. Overall, we find that tetrazine release from enzyme-cleavable tetrazine derivatives is dependent on the enzyme activity present in specific cells and therefore constitutes a promising technique for cell-type specific bioconjugation.
Materials and methods, experimental and synthetic proce-dures, NMR spectra, supplementary figures S1-S12