Cascade Reaction-Based, Near-Infrared Multiphoton Fluorescent Probe for the Selective Detection of Cysteine.

Abstract

The ability to detect and visualize cellular events and their associated target biological analytes through use of cell-permeable profluorogenic probes is dependent on the availability of activatable probes that respond rapidly and selectively to target analytes by production of fluorescent reporting molecules whose excitation and emission energies span a broad range. Herein is described a new probe, DCM-Cys, that preferentially reacts with cysteine to form a dicyanomethylene-4H-pyran (DCM) reporter whose red-energy fluorescence can be stimulated by two-photon, near-infrared excitation so as to provide visualization of cysteine presence inside living human cells with a high signal-to-background ratio. These aforementioned characteristics and the ability of DCM-Cys to provide selective, nanomolar-level in vitro cysteine detection, as demonstrated by its lack of significant response to other thiols and potential interfering agents from biological environments, are attributed to the molecular designs of the DCM-Cys probe and DCM reporter. Attachment of an acryl moiety to the DCM reporter via a self-eliminating, electron-withdrawing benzyl alcohol-carbamate linker offers a probe having selective, sensitive reaction with cysteine to rapidly produce a reporter whose energies of excitation and emission (λabs(report) = 480 nm, λemis(report) = 640 nm) are red-shifted from those of the DCM-Cys probe (λabs(probe) = 440 nm, λemis(probe) = 550 nm), thereby leading to low background signal from abundant probe and a large signal from the resulting reporter of cysteine presence.