DMEDA is commonly used as a self-immolative linker or spacer in the design of cleavable and/or activatable compounds such as drug conjugates. Replacement of an N-methyl substituent with other functionalities moreover allows further modification, as very recently been applied for the development of an rTCO-caged fluorogenic resorufin derivative. For the detailed investigation of the reactions of rTCO-DMEDA-phenol conjugates with structurally different Tz we have first prepared a water-soluble release probe labeled with a boron dipyrromethene (BODIPY) fluorophore that allows detection and relative quantification of all intermediates and products by HPLC using its characteristic absorption at a wavelength of 500 nm. The active p-nitrophenyl (PNP) carbonate rTCO-PNP (1) was treated with an excess of DMEDA and (following evaporation to afford crude intermediate 2) reacted with the PNP-activated tyrosine-BODIPY conjugate 3 to obtain the rTCO-DMEDA-caged compound 4 in an overall yield of 68%. Saponification and subsequent PEGylation of the intermediate acid 5 via HBTUmediated amide coupling yielded rTCO-DMEDA-Tyr-BODIPY (6) with a yield of 76% over 2 steps (Fig. ).