Alexander R. Lippert

Reaction-Based Fluorescent Probes for Selective Imaging of Hydrogen Sulfide in Living Cells

Hydrogen sulfide (H(2)S) is emerging as an important mediator of human physiology and pathology but remains difficult to study, in large part because of the lack of methods for selective monitoring of this small signaling molecule in live biological specimens. We now report a pair of new reaction-based fluorescent probes for selective imaging of H(2)S in living cells that exploit the H(2)S-mediated reduction of azides to fluorescent amines. Sulfidefluor-1 (SF1) and Sulfidefluor-2 (SF2) respond to H(2)S by a turn-on fluorescence signal enhancement and display high selectivity for H(2)S over other biologically relevant reactive sulfur, oxygen, and nitrogen species. In addition, SF1 and SF2 can be used to detect H(2)S in both water and live cells, providing a potentially powerful approach for probing H(2)S chemistry in biological systems.

Cell-trappable fluorescent probes for endogenous hydrogen sulfide signaling and imaging H2O2-dependent H2S production

Hydrogen sulfide (H2S) is a reactive small molecule generated in the body that can be beneficial or toxic owing to its potent redox activity. In living systems, disentangling the pathways responsible for H2S production and their physiological and pathological consequences remains a challenge in part due to a lack of methods for monitoring changes in endogenous H2S fluxes. The development of fluorescent probes with appropriate selectivity and sensitivity for monitoring production of H2S at biologically relevant signaling levels offers opportunities to explore its roles in a variety of systems. Here we report the design, synthesis, and application of a family of azide-based fluorescent H2S indicators, Sulfidefluor-4, Sulfidefluor-5 acetoxymethyl ester, and Sulfidefluor-7 acetoxymethyl ester, which offer the unique capability to image H2S generated at physiological signaling levels. These probes are optimized for cellular imaging and feature enhanced sensitivity and cellular retention compared with our previously reported molecules. In particular, Sulfidefluor-7 acetoxymethyl ester allows for direct, real-time visualization of endogenous H2S produced in live human umbilical vein endothelial cells upon stimulation with vascular endothelial growth factor (VEGF). Moreover, we show that H2S production is dependent on NADPH oxidase–derived hydrogen peroxide (H2O2), which attenuates VEGF receptor 2 phosphorylation and establishes a link for H2S/H2O2 crosstalk.

Designing reaction-based fluorescent probes for selective hydrogen sulfide detection.

Hydrogen sulfide (H2S) is a biologically generated, gaseous signaling molecule that mediates a wide range of physiological functions and is misregulated in numerous pathologies ranging from neurodegenerative disease to hypertension to diabetes. Despite swelling interest, a deeper understanding of the biological roles played by H2S has been hindered by a lack of tools for the real-time visualization of its production in living organisms. Recently, reaction-based fluorescent probes have emerged as an ideal approach for selective H2S imaging and are attracting increasing attention with many new innovative designs being introduced. This review will highlight some of the most fruitful reaction-based strategies, including reduction-based, nucleophilic-based, and metal sulfide precipitation-based fluorescent sensors. Strategies to address the key design challenges of sensitivity, selectivity, in vivo compatibility, and quantification will be discussed using examples of recently developed molecular scaffolds for selective H2S detection.

A hydrogen peroxide-responsive hyperpolarized 13C MRI contrast agent.

We report a new reaction-based approach for the detection of hydrogen peroxide (H(2)O(2)) using hyperpolarized (13)C magnetic resonance imaging ((13)C MRI) and the H(2)O(2)-mediated oxidation of α-ketoacids to carboxylic acids. (13)C-Benzoylformic acid reacts selectively with H(2)O(2) over other reactive oxygen species to generate (13)C-benzoic acid and can be hyperpolarized using dynamic nuclear polarization, providing a method for dual-frequency detection of H(2)O(2). Phantom images collected using frequency-specific imaging sequences demonstrate the efficacy of this responsive contrast agent to monitor H(2)O(2) at pre-clinical field strengths. The combination of reaction-based detection chemistry and hyperpolarized (13)C MRI provides a potentially powerful new methodology for non-invasive multi-analyte imaging in living systems.

Chemiluminescent Probes for Imaging H2S in Living Animals

Responsive 1,2-dioxetane chemiluminescent probes have been developed that display instantaneous, sensitive, and selective responses to H2S and are capable of imaging H2S in living mice.

In Vivo Chemiluminescent Imaging Agents for Nitroreductase and Tissue Oxygenation

Tissue oxygenation is a driving parameter of the tumor microenvironment, and hypoxia can be a prognostic indicator of aggressiveness, metastasis, and poor response to therapy. Here, we report a chemiluminescence imaging (CLI) agent based on the oxygen-dependent reduction of a nitroaromatic spiroadamantane 1,2-dioxetane scaffold. Hypoxia ChemiLuminescent Probe 2 (HyCL-2) responds to nitroreductase with ∼170-fold increase in luminescence intensity and high selectivity for enzymatic reductase versus other small molecule reductants. HyCL-2 can image exogenous nitroreductase in vitro and in vivo in living mice, and total luminescent intensity is increased by ∼5-fold under low oxygen conditions. HyCL-2 is demonstrated to report on tumor oxygenation during an oxygen challenge in H1299 lung tumor xenografts grown in a murine model as independently confirmed using multispectral optoacoustic tomography (MSOT) imaging of hemoglobin oxygenation.

Synthesis of Phototrappable Shape-Shifting Molecules for Adaptive Guest Binding

We have designed and synthesized oligosubstituted bullvalenes 1 and 2 as adaptive molecules that can change their shapes in order to bind tightly to a suitable guest. By incorporation of a photolabile o-nitroveratryloxycarbonate (NVOC) group into bullvalenes 1 and 2, tightly binding species can be selectively isolated from a population of hundreds of interconverting structural isomers. Spontaneous strain-assisted Cope rearrangements allow these shape-shifting molecules to exist in a dynamic equilibrium of configurationally distinct valence isomers, as revealed by dynamic NMR and HPLC studies. When NVOC bullvalenes 1 and 2 were exposed to UV light, the cleavage of the NVOC group resulted in a mixture of static isomers of the corresponding bullvalone. Binding studies of NVOC bisporphyrin bullvalene 1 demonstrated that the dynamic isomeric equilibrium shifted in the presence of C(60), favoring configurations with more favorable binding affinities. Irradiation of a mixture of 1 and C(60) with UV light and isolation of the major static isomer yielded an isomer of bisporphyrin bullvalone with a binding affinity for C(60) that was ∼2 times larger than that of the nonadapted isomer bisporphyrin bullvalone 41.

A chemiluminescent platform for smartphone monitoring of H2O2 in human exhaled breath condensates

Noninvasive measurement of oxidative markers in clinical samples has the potential to rapidly provide information for disease management, but is limited by the need for expensive analytical instrumentation that precludes home monitoring or point-of-care applications. We have developed a simple to use diagnostic platform for airway hydrogen peroxide (H2O2) that combines optimized reaction-based chemiluminescent designs with an inexpensive home-built darkbox and readily available smartphone cameras. Specialized photography software applications and analysis of pixel intensity enables quantification of sample concentrations. Using this platform, sample H2O2 concentrations as low as 264nM can be detected. The platform has been used to measure H2O2 in the exhaled breath condensates of human subjects, showing good agreement with the standard Amplex Red assay.

Unlocking the Potential of Chemiluminescence Imaging

Improved chemiluminescence probes may help transition the technology into a widely employed tool for whole animal imaging and microscopy.

Azide-Based Fluorescent Probes: Imaging Hydrogen Sulfide in Living Systems

Hydrogen sulfide is a redox active sulfur species that is endogenously generated in mammalian systems as an antioxidant and signaling molecule to support cellular function. The fundamental and ubiquitous actions of hydrogen sulfide demand sensitive and specific methods to track this biomolecule as it is produced within living organisms with temporal and spatial regulation. In this context, the hydrogen sulfide-mediated reduction of an azide to an amine is a useful method for organic synthesis, and this reaction has successfully been exploited to yield biocompatible fluorescent probes for hydrogen sulfide detection in vitro and in cells. This chapter provides protocols and guidelines for applying azide-based fluorescence probes to detecting hydrogen sulfide in living systems, including a protocol that was used to detect endogenous hydrogen sulfide in living single cells using a confocal microscope.