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Cascade Reaction-Based, Near-Infrared Multiphoton Fluorescent Probe for the Selective Detection of Cysteine Rasika R Nawimanage, Bijeta Prasai, Suraj U. Hettiarachchi, and Robin L. McCarley Anal. Chem., Just Accepted Manuscript • Publication Date (Web): 16 May 2017 Downloaded from http://pubs.acs.org on May 16, 2017
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Analytical Chemistry
Cascade Reaction-Based, Near-Infrared Multiphoton Fluorescent Probe for the Selective Detection of Cysteine Rasika R. Nawimanage, Bijeta Prasai, Suraj U. Hettiarachchi, and Robin L. McCarley* Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803-1804, United States ABSTRACT: The ability to detect and visualize cellular events and their associated target biological analytes through use of cell-permeable pro-fluorogenic 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 interferents 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 report probe probe emission (λreport abs = 480 nm, λ emis = 640 nm) are red-shifted from those of the DCM–Cys probe (λ abs = 440 nm, λ emis = 550 nm), thereby leading to low background signal from abundant probe, and a large signal from the resulting reporter of cysteine presence.
Fluorescence-based small molecule probes have recently received much attention for their use in tracking of important analytes in cellular environments, biological fluids, and in living tissues, without affecting sample integrity. By making use of the intrinsically unique reactivity of select classes of organic molecules, these probes can be tailored to detect either broad classes of analytes or specific targets as a result of analyte-induced formation of fluorescent reporters.1,2-4 The sulfur-containing small molecules glutathione (GSH), cysteine (Cys), and homocysteine (Hcy) are involved in a number of key biological processes.5 Cys and Hcy are clearly prominent among the other naturally occurring amino acids, as they are the only ones with a free thiol (–SH) moiety. Hcy and Cys differ from each other by the presence of a single methylene unit in their side chain, giving them similar structural and chemical characteristics. Owing to their near-identical chemical reactivities, the selective detection, quantification, or visualization of a single thiol analyte by discriminating against other thiol-containing small molecules remains a great challenge. Only a select few of the extant thiol-responsive probe/reporter systems have the ability to preferentially detect cysteine versus Hcy and GSH.6-10 Furthermore,
most of these cysteine-selective probes have not had reported their response to the possible cross reactant H2S,8 an important cellular gasotransmitter. And although a class-selective bio–thiol probe has been shown to be unreactive toward a prevalent thiol-containing protein,11 to date there have been no reports of the potential role of protein thiols as interferents with cysteine-selective probes. Finally, there is a great need for probes capable of selectively detecting cysteine without the requirement of multiple cysteine molecules reacting with the probe9 or probe/reporter excitation and emission energies lying solely outside the red region.6 Fluorescent dyes with excitation and emission energies in the red and NIR range (640–900 nm) possess unique advantages for tracking molecular processes in vitro and in vivo with less background interference.12 NIR dyes offer increased penetration distance of light, thereby facilitating deep-tissue imaging with minimal damage to biological components. Although long-wavelength cyanine dyes are widely used in designing NIR sensors for bioimaging, they typically suffer from poor photostability in aqueous solutions,13 thereby limiting their universal application for analyte detection, particularly those aimed at real-time assessments.
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Analytical Chemistry
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Dicyanomethylene-4H-pyrans and their derivatives (DCM dyes) have long been used for non-linear optical materials, logic gates,14 and photovoltaic sensitizers.15,16 As a donor–π–acceptor type chromophore, derivatives of DCM have attractive photophysical properties, such as tunable emission wavelength in the red or NIR region via control over electron donor ability, large intramolecular chargetransfer-based Stokes shift, and high photostability.14
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two-photon absorption cross section in the NIR region18 and emits in the red and NIR region, both key for tissue imaging applications. As a result, DCM–Cys offers quick (
