A novel three-fluorophore system as a ratiometric sensor for multiple protease detection

Abstract

A synthetic three-fluorophore system with two enzymatically cleavable linkers has been developed for the simultaneous detection of two proteases in a mixture. The probe was designed to afford single excitation/triple emission ratiometric detection through a fluorescence change during the cleavage of a peptide linker. The developed assays were verified for trypsin and chymotrypsin as the model enzymes.

Immobilized Fluorescent Probes for Simultaneous Multiple Protease Detection

“…The developed sensor for the simultaneous detection of two model proteases consists of two separate selectively cleavable peptide linkers anchored to a solid support (ITPimmobilized substrate for trypsin and ICP-immobilized substrate for chymotrypsin). Considering the recently published data [22], sequences of Ala-Lys-Ala and Ala-Phe-Ala were confirmed as suitable recognizable sites for trypsin and chymotrypsin, respectively. In addition, two poly(ethylene glycol) spacers, one between the resin and the cleavage site, and another between the cleavage site and the corresponding fluorescent dye, were introduced to both probes to improve the efficiency of the enzymatic cleavage (Figure 2A).…”

Förster resonance energy transfer (FRET)-based small-molecule sensors and imaging agents

“…36) was constructed by attaching three fluorescent dyes 7-diethylaminocoumarin-3-carboxylic acid (DEAC), fluorescein (Fl) and rhodamine B (RhB) to a short peptide. 47 Upon excitation of the donor (DEAC) at 435 nm, FRET to Fl occurs, followed by FRET from Fl to RhB. The system thus exhibited a cascadelike energy transfer process.…”

Ketone Incorporation Extends the Emission Properties of the Xanthene Scaffold Beyond 1000 nm†

“…Phthalic anhydride underwent bis-aryl addition to generate 4 which remained as a ring-opened 9,10-anthracenediyl derivative until subsequent deprotection with TFA to give ring-closed KR-2 in 27% overall yield (Scheme 1B). We also set out to prepare a water-soluble "always on" amide derivative KR dye, as related probes have been described extensively in the rhodamine literature (53)(54)(55)(56)(57)(58)(59)(60). The carboxylic acid 4 underwent amide coupling under standard conditions to give doubly protected intermediate 9.…”