Fluorogenic Imaging Agents for Enzyme Activity

Fluorogenic probes for enzymes have proven to be essential for understanding the function and regulation of enzymes in living organisms. Cellular enzymes play crucial roles in many biological and physiological processes, maintaining cellular viability, electric and ionic gradients, signal transduction, proliferation, detoxification, and many other elementary processes. One especially important and widespread class of these enzymes is the carbohydrate processing glycosidases with uncountable biological functions. The aim of this project is the development of freely cell permeable latent fluorogenic probes whose enzymatic cleavage results in the entrapment of a charged fluorogenic product in the cell. We propose to connect the enzymatic substrate and the latent fluorophore by a self-immolative linker, that excises itself after the enzymatic reaction. This linker design allows the usage of various charged leaving groups, rendering a fine tuning of the pKa of the fluorogenic products possible, securing their entrapment in the cell. Furthermore, the attachment of the fluorophore to the linker via a variety of functionalities permits the usage of a wide choice of fluorogenic dyes. The non-toxicity of the released self-immolative linker will be ensured by intramolecular deactivation of their reactive excised quinone methide-like structures by 6-endo-trig-cyclizations. This concept will be tested initially by the fluorescence-activated cell sorting (FACS)-based screening for beta-galactosidase and beta-glucosidase activity in transformed E. coli and Yeast cells. beta-Galactosidase is the most extensively used reporter gene in molecular biology and is frequently employed for the detection of cancer in mice. Furthermore these probes will be applied for histological staining of plant cells by visualizing their beta-glucuronidase activity in their endoplasmic reticulum. Successful initial studies will offer the opportunity to identify novel biocatalysts and catabolic enzymes. The synthesis of fluorogenic probes for more complex carbohydrates will enable the FACS-based screening of enzyme activity of various glycosidases involved in biomass conversion (cellulases, xylanases, hemicellulases, ligninases, ...). These enzymes have great potential for the conversion of biomass to biofuels and for the production of high value carbohydrates. The non-toxic behavior of these fluorogenic probes will set the basis for the optimization of these enzymes by directed evolution experiments in the future.

Fluorogenic probes for enzymes have proven to be essential for understanding the function and regulation of enzymes in living organisms. Cellular enzymes play crucial roles in many biological and physiological processes, maintaining cellular viability, electric and ionic gradients, signal transduction, proliferation, detoxification, and many other elementary processes. We have developed a novel fluorogenic probe for ultra-high-throughput screening of single enzyme expressing cells. We have shown by fluorescence-activated cell sorting (FACS) that our novel probes for esterases and glycosidases exhibit superior fluorescence retention in water-in-oil-in-water emulsions, providing much lower background signal and better dynamic range compared to traditional fluorescein and coumarin based imaging agents. Currently we are applying this strategy to the directed laboratory evolution of glycoside hydrolases and engineered variants thereof termed glycosynthases. These evolved glycosynthases will allow the efficient transfer of artificial carbohydrates with bioorthogonal functionalities. These enzymes are opening up the possibility for the synthesis of complex biomolecules and analogs and to study their biological functions. Furthermore we are using similar probes for ultra-high-throughput screening of bio mass degrading enzymes like glucosidases, cellobiohydrolases and cellulases, which are urgently needed for the production of biofuels.