Late Stage Functionalization of GalNAc Tetrazine Probes for Real-time Live Cell Labeling

• Sarah Janney, Chemistry, University of Delaware

• Joseph Fox, Chemistry and Biochemistry, University of Delaware

Glycobiology probes that can perform real-time labeling are highly sought-after tools for investigating different aspects of human and bacterial biology including cancer research, immunology, and medicinal chemistry. The previous carbohydrate-based probes utilize copper-catalyzed azide-alkyne click (CuAAC) chemistry handles to undergo rapid labeling. While there is a high rate of incorporation into the intracellular and extracellular space, it suffers from modest labeling rates and the cytoxicity of copper limits the use of in vivo applications. To combat this, strain-promoted azide-alkyne click (SPAAC) chemistry was developed to monitor live systems without the need for a metal catalyst. Although the breadth of utility was increased this reaction still suffers from significantly slower kinetics leading to limited use for observing biochemical processes in real time. Both CuAAC and SPAAC reactions require lengthy incubation times for labeling and arduous washing procedures to avoid background fluorescence and over-saturation of the imaging detector. In an attempt to circumvent these limitations, a carbohydrate-based probe containing a tetrazine handle was developed to undergo the tetrazine-trans-cyclooctene (TZ-TCO) ligation. Because of its ultrafast kinetics and inert byproducts, the use of TZ-TCO ligation is an excellent candidate for real-time live cell labeling. With a second-order rate constant greater than 106M-1s-1, the TZ-TCO ligation allows for real-time monitoring of biochemical processes as it can be performed at biologically relevant concentrations which were not accessible with the CuAAC and SPAAC probes. However, initial tetrazine probes were sterically large leading to low metabolic incorporation and perturbed cell health. The work I have done utilizes recently developed methods in the Fox lab to make minimalist tetrazines that will be coupled to a carbohydrate core and then introduce different coupling conditions for late-stage functionalization in hopes of creating a library of different tetrazine probes for metabolic incorporation into bacterial and mammalian cells.