Article pubs.acs.org/ac
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 S Supporting Information *
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 redenergy fluorescence can be stimulated by two-photon, nearinfrared excitation so as to provide visualization of cysteine presence inside living human cells with a high signal-tobackground 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 (λreport abs = probe probe 480 nm, λreport emis = 640 nm) are red-shifted from those of the DCM−Cys probe (λabs = 440 nm, λemis = 550 nm), thereby leading to low background signal from abundant probe and a large signal from the resulting reporter of cysteine presence.
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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 interferences 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 near-infrared (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 deeptissue 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. Dicyanomethylene-4H-pyrans and their derivatives (DCM dyes) have long been used for nonlinear optical materials, logic
luorescence-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.1−4 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− © 2017 American Chemical Society
Received: April 12, 2017 Accepted: May 16, 2017 Published: May 16, 2017 6886
DOI: 10.1021/acs.analchem.7b01384 Anal. Chem. 2017, 89, 6886−6892
Article
Analytical Chemistry 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 charge-transfer-based Stokes shift, and high photostability.14 Furthermore, compared to traditional dyes that undergo only single-photon excitation, DCM derivatives have significant two-photon absorption cross sections.17−19 As such, use of DCM-based dyes in two-photon microscopy studies18 is predicted to allow for deeper penetration of the lower energy photons into tissues.4 The work here presents the design, synthesis, and evaluation of a fluorescent probe, DCM−Cys, for the selective detection of cysteine under physiological conditions and in living cells. The general design rationale of the cysteine-activatable DCM− Cys is described as follows. The probe contains a reporter dye attached to a cysteine-reactive acryl moiety20 via a carbamatebased, self-eliminating linker that is not prone11,21 to potential esterase-catalyzed hydrolysis within cells,22,23 Figure 1. Initially,
Stock solutions of the DCM−Cys probe were made in DMSO (Sigma-Aldrich). Water from a Barnstead NANOpure Diamond water System (18 MΩ cm) was used to prepare phosphate-buffered saline (PBS) required for those experiments requiring its use. The DCM reporter and DCM−Cys probe were synthesized as outlined in the synthetic scheme of the Supporting Information. The structures and purities of the probe and the reporter were confirmed with electrospray ionization mass spectrometry (ESI-MS), 1H NMR, and 13C NMR. The 1H NMR and 13C NMR spectra were recorded in chloroform-d at room temperature using Bruker AV-400 and AV-500 spectrometers. Spectroscopic Methods. All spectroscopic measurements were performed in 0.1 M, pH 7.4 phosphate-buffered saline solution, using 1 cm × 1 cm quartz cuvettes (1.5 mL volume, Sigma-Aldrich). Fluorescence data were collected using a PerkinElmer LS-55 spectrometer, and absorption spectra were obtained with a Varian Cary-50 spectrophotometer. Procedure for Solution-Phase Thiol Sensing. Stock solutions of 2 × 10−4 M DCM−Cys probe were prepared in 100% DMSO and were subsequently diluted to prepare preferred concentration solutions in pH 7.4 PBS/DMSO buffer. Stock solutions of thiol were freshly prepared prior to each experiment. For the calibration curve of cysteine, a fixed concentration solution of DCM−Cys was incubated with different concentrations of cysteine at 37 °C for 15 min, and spectral data were recorded. For the DCM−Cys probe (detect the release of the free dye), samples were excited at 520 nm, and emission was detected at 640 nm. The excitation and emission slit widths were 5 and 10 nm, respectively. Cell Culture. Human non-small-cell lung cancer cells H596, cell culture base media, and fetal bovine serum (FBS) were purchased from American Type Cell Culture (ATCC, Manassas, VA). All cell culture procedures were performed as suggested by ATCC. H596 cells were cultured in RPMI-1640 with 10% FBS and 10 IU mL−1 penicillin−streptomycin. Cells were incubated at 37 °C in a humidified incubator containing 5 wt %/vol CO2. Imaging of Cysteine in Cells via Scanning Laser Confocal Microscopy. H596 cells were cultured overnight on 22 mm × 22 mm glass coverslips on a treated tissue culture 6well plate purchased from Fisher Scientific. The existing growth medium was replaced with 2 mL of fresh cysteine free medium and then incubated at 37 °C. Solutions of DCM−Cys dissolved in DMSO were added to the cells to give a final DCM−Cys concentration of 3 × 10−5 M, with the DMSO concentration at
