Dual-Color Real-Time Chemosensing of a Compartmentalized Reaction Network Involving Enzyme-Induced Membrane Permeation of Peptides

The design of synthetic systems with interrelated reaction sequences that model incipient biological complexity is limited by physicochemical tools that allow the direct monitoring of the individual processes in real-time. To mimic a simple digestion-resorption sequence, the authors have designed compartmentalized liposomal systems that incorporate extra- and intravesicular chemosensing ensembles. The extravesicular reporter pair consists of cucurbit[7]uril and methylene blue to monitor the enzymatic cleavage of short enkephalin-related peptides by thermolysin through a switch-off fluorescence response ("digestion"). Because the substrate is membrane-impermeable, but the dipeptide product is permeable, uptake of the latter into the pre-formed liposomes occurs as a follow-up process. The intravesicular chemosensing ensemble consists of i) cucurbit[8]uril, 2-anilinonaphthalene-6-sulfonic acid, and methyl viologen or ii) cucurbit[7]uril and berberine to monitor the uptake ("resorption") of the enzymatic products through the liposomal membranes by i) a switch-on or ii) a switch-off fluorescence response. The dyes are designed to allow selective optical excitation and read-out of the extra- and intravesicular dyes, which allow for dual-color chemosensing and, therefore, kinetic discrimination of the two sequential reactions. A dual-compartment system with two optically and spatially separated chemosensing ensembles for monitoring the enzymatic conversion and subsequent permeation of peptides is constructed. One long-wavelength fluorescent reporter pair is positioned outside the liposomes to monitor the enzymatic digestion of enkephalin substrates by thermolysin. A second shorter-wavelength chemosensing ensemble inside the liposomes signals the subsequent permeation of the enzymatic products.image