Vesicle‐Encapsulated Chemosensing Ensembles Allow Monitoring of Transmembrane Uptake Coupled with Enzymatic Reactions

Abstract Compartmentalized models with coupled catalytic networks are considered as “protocells” in the context of research related to the origin of life. To model the kinetics of a simple cellular uptake‐metabolism process, we use a compartmentalized protocell system that combines liposome‐encapsulated intravesicular reporter pairs with co‐encapsulated enzymes to monitor the membrane transport of a substrate (analyte uptake) and its subsequent enzymatic reaction inside the vesicles (metabolism to the product). The intravesicular chemosensing ensembles consist of the macrocycles cucurbit[7]uril or p ‐sulfonatocalix[4]arene and matching fluorescent dyes to set up suitable reporter pairs. When these macrocycle/dye reporter pairs are co‐encapsulated with enzymes (trypsin, protein kinase A, or butyrylcholinesterase), it is possible to monitor first the transport of different substrates (polylysine, protamine, H‐LRRWSLG‐OH, or butyrylcholine) through added pores (outer membrane proteins F and C), with synthetic carriers (amphiphilic calixarenes), or by direct permeation (only for butyrylcholine). The subsequent enzymatic conversions of the substrates after they have entered the corresponding protocells can be monitored as consecutive reactions. The new type of in vitro assays can be applied to different enzymes and analytes, affording a comprehensive chemosensing system of high chemical complexity.