Tuning Photoinduced Electron Transfer Efficiency of Fluorogenic BODIPY-α-Tocopherol Analogues.

Fluorogenic analogues of α-tocopherol developed by our group have been instrumental in monitoring reactive oxygen species (ROS) within lipid membranes. Prepared as two-segment trap-reporter (chromanol-BODIPY) probes, photoinduced electron transfer (PeT) was utilized to provide these probes with an off/on switch mechanism warranting the necessary sensitivity. Herein, we rationalize within the context of Marcus theory of electron transfer how substituents on the BODIPY core and linker length joining the trap and reporter segments, tune PeT efficiency. DFT and electrochemical studies were used to estimate the thermodynamic driving force of PeT in our constructs. By tuning the redox potential over a 400 mV range, we observed over an order of magnitude increase in PeT efficiency. Increasing the linker length between the chromanol and BODIPY by 2.8 angstroms, in turn, decreased PeT efficiency 2.7-fold. Our results illustrate how substituent and linker choice enable "darkening" the off state of fluorogenic probes based on BODIPY fluorophores, by favoring PeT over radiative emission from the singlet excited state manifold. Ultimately, our work brings light to the sensitivity ceiling one may achieve in developing fluorogenic antioxidant analogues of α-tocopherol. The work provides general guidelines applicable to those developing fluorogenic probes based on PeT.

Effect of antioxidant supplements on lipid peroxidation levels in primary cortical neuron cultures.

Oxidative stress, specifically lipid peroxidation, is a major driving force in neurodegenerative processes. However, the exact role of lipid peroxidation remains elusive as reliable real-time detection and quantification of lipid peroxyl radicals proves to be challenging in vitro and in vivo. Motivated by this methodological limitation, we have optimized conditions for real-time imaging and quantification of lipid peroxyl radical generation in primary neuron cultures using the lipophilic fluorogenic antioxidant H4BPMHC (8-((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl)-methyl)-1,5-di(3-chloropropyl)-pyrromethene fluoroborate), an α-tocopherol analog probe. By subjecting neurons to different antioxidant conditions in the presence and absence of lipid peroxidation inducing stressors (Haber-Weiss reagents), we maximized H4BPMHC sensitivity and confirmed its potential to temporally resolve subtle and marked differences in lipid peroxidation levels in real-time. Herein we report imaging and quantification of homeostatic and induced lipid peroxidation in primary neuron cultures, supporting the use of this probe for investigating healthy and diseased states. Overall these results provide the necessary foundation and impetus towards using H4BPMHC for elucidating and mapping lipid peroxyl radical contributions to ROS-associated pathological processes in neurons.

Spatio-temporal monitoring of lipid peroxyl radicals in live cell studies combining fluorogenic antioxidants and fluorescence microscopy methods.

Lipid peroxidation of polyunsaturated fatty acids in cells may occur via their catalytic autoxidation through peroxyl radicals under oxidative stress conditions. Lipid peroxidation is related to a number of pathologies, and may be invoked in new forms of regulated cell death, yet it may also have beneficial roles in cell signaling cascades. Antioxidants are a natural line of defense against lipid peroxidation, and may accordingly impact the biological outcome associated with the redox chemistry of lipid peroxidation. Critical to unraveling the physiological and pathological role of lipid peroxidation is the development of novel probes with the partition, chemical sensitivity and more importantly, molecular specificity, enabling the spatial and temporal imaging of peroxyl radicals in the lipid membranes of live cells, reporting on the redox status of the cell membrane. This review describes our recent progress to visualize lipid peroxidation in model membrane systems and in live cell studies. Our work portrays the mechanistic insight leading to the development of a highly sensitive probe to monitor lipid peroxyl radicals (LOO•). It also describes technical aspects including reagents and fluorescence microscopy methodologies to consider in order to achieve the much sought after monitoring of rates of lipid peroxyl radical production in live cell studies, be it under oxidative stress but also under cell homeostasis. This review seeks to bring attention to the study of lipid redox reactions and to lay the groundwork for the adoption of fluorogenic antioxidant probeshancement and maximum intensity recorded in turn provide a benchmark to estimate, when compared to the control BODIPY dye lacking the intramolecular PeT based switch, the overall exte and related fluorescence microscopy methods toward gaining rich spatiotemporal information on lipid peroxidation in live cells.

Cigarette smoke activates CFTR through ROS-stimulated cAMP signaling in human bronchial epithelial cells.

Air pollution stimulates airway epithelial secretion through a cholinergic reflex that is unaffected in cystic fibrosis (CF), yet a strong correlation is observed between passive smoke exposure in the home and impaired lung function in CF children. Our aim was to study the effects of low smoke concentrations on cystic fibrosis transmembrane conductance regulator (CFTR) function in vitro. Cigarette smoke extract stimulated robust anion secretion that was transient, mediated by CFTR, and dependent on cAMP-dependent protein kinase activation. Secretion was initiated by reactive oxygen species (ROS) and mediated by at least two distinct pathways: autocrine activation of EP4 prostanoid receptors and stimulation of Ca2+ store-operated cAMP signaling. The response was absent in cells expressing the most common disease-causing mutant F508del-CFTR. In addition to the initial secretion, prolonged exposure of non-CF bronchial epithelial cells to low levels of smoke also caused a gradual decline in CFTR functional expression. F508del-CFTR channels that had been rescued by the CF drug combination VX-809 (lumacaftor) + VX-770 (ivacaftor) were more sensitive to this downregulation than wild-type CFTR. The results suggest that CFTR-mediated secretion during acute cigarette smoke exposure initially protects the airway epithelium while prolonged exposure reduces CFTR functional expression and reduces the efficacy of CF drugs.

Mitochondria Alkylation and Cellular Trafficking Mapped with a Lipophilic BODIPY-Acrolein Fluorogenic Probe.

Protein and DNA alkylation by endogenously produced electrophiles is associated with the pathogenesis of neurodegenerative diseases, to epigenetic alterations and to cell signaling and redox regulation. With the goal of visualizing, in real-time, the spatiotemporal response of the cell milieu to electrophiles, we have designed a fluorogenic BODIPY-acrolein probe, AcroB, that undergoes a >350-fold fluorescence intensity enhancement concomitant with protein adduct formation. AcroB enables a direct quantification of single post-translational modifications occurring on cellular proteins via recording fluorescence bursts in live-cell imaging studies. In combination with super-resolution imaging, protein alkylation events may be registered and individually counted, yielding a map of protein-electrophile reactions within the cell lipid milieu. Alkylation is predominantly observed within mitochondria, a source, yet not a sink, of AcroB adducts, illustrating that a mitochondrial constitutive excretion mechanism ensures rapid disposal of compromised proteins. Sorting within the Golgi apparatus and trafficking along microtubules and through the cell endo- and exocytic pathways can be next visualized via live-cell imaging. Our results offer a direct visualization of cellular response to a noncanonical acrolein warhead. We envision AcroB will enable new approaches for diagnosis of pathologies associated with defective cellular trafficking. AcroB may help elucidate key aspects of mitochondria electrophile adduct excretion and cell endocytic and exocytic pathways. Conceptually, AcroB provides a new paradigm on fluorescence microscopy studies where chemical perturbation is achieved and simultaneously reported by the probe.

Rate of Lipid Peroxyl Radical Production during Cellular Homeostasis Unraveled via Fluorescence Imaging.

Reactive oxygen species (ROS) and their associated byproducts have been traditionally associated with a range of pathologies. It is now believed, however, that at basal levels these molecules also have a beneficial cellular function in the form of cell signaling and redox regulation. Critical to elucidating their physiological role is the opportunity to visualize and quantify the production of ROS with spatiotemporal accuracy. Armed with a newly developed, extremely sensitive fluorogenic α-tocopherol analogue (H4BPMHC), we report herein the observation of steady concentrations of lipid peroxyl radicals produced in live cell imaging conditions. Imaging studies with H4BPMHC indicate that the rate of production of lipid peroxyl radicals in HeLa cells under basal conditions is 33 nM/h within the cell. Our work further provides indisputable evidence on the antioxidant role of Vitamin E, as lipid peroxidation was suppressed in HeLa cells both under basal conditions and in the presence of Haber-Weiss chemistry, generated by the presence of cumyl hydroperoxide and Cu2+ in solution, when supplemented with the α-tocopherol surrogate, PMHC (2,2,5,7,8-pentamethyl-6-hydroxy-chromanol, an α-tocopherol analogue lacking the phytyl tail). H4BPMHC has the sensitivity needed to detect trace changes in oxidative status within the lipid membrane, underscoring the opportunity to illuminate the physiological relevance of lipid peroxyl radical production during cell homeostasis and disease.

Development of a Fluorogenic Reactivity Palette for the Study of Nucleophilic Addition Reactions Based on meso-Formyl BODIPY Dyes.

We describe herein a fluorescence-based assay to characterize and report on nucleophilic addition to carbonyl moieties and highlight the advantages a fluorescence-based assay and multiplex analysis can offer. The assay relies on the fluorogenic properties of meso-formyl boron-dipyrromethene (BODIPY) dyes that become emissive following nucleophilic addition. A reactivity palette is assembled based on the increasing electrophilic character of five meso-formyl BODIPY compounds tested. We show that increasing rates of emission enhancement correlate with the decreasing electrophilic character of BODIPY dyes in the presence of an acid catalyst and a nucleophile. These results are consistent with the rate-limiting step involving activation of the electrophile. Increasing product formation is shown to correlate with the increasing electrophilic character of the BODIPY dyes, as expected based on thermodynamics. In addition to providing rates of reaction, analysis of the fluorescence parameters for the reaction mixtures, including emission quantum yields and fluorescence lifetimes, enables us to determine the extent of reactant conversion at equilibrium (in our case the estimated yield of a transient species) and the presence of different products, without the need for isolation. We anticipate that our reactivity palette approach, combined with the in-depth fluorescence analysis discussed herein, will provide guidelines toward developing fluorogenic assays of reactivity offering multiplex information, beyond fluorescence intensity.

Fluorogenic Ubiquinone Analogue for Monitoring Chemical and Biological Redox Processes.

We report herein the synthesis and characterization of a fluorogenic analogue of ubiquinone designed to reversibly report on redox reactions in biological systems. The analogue, H2B-Q, consists of the redox-active quinone segment found in ubiquinone, 2,3-dimethoxy-1,4-benzoquinone, coupled to a boron-dipyrromethene (BODIPY) fluorophore segment that both imparts lipophilicity in lieu of the isoprenyl tail of ubiquinone, and reports on redox changes at the quinone/quinol segment. Redox sensing is mediated by a photoinduced electron transfer intramolecular switch. In its reduced dihydroquinone form, H2B-QH2 is highly emissive in nonpolar media (quantum yields 55-66%), while once oxidized, the resulting quinone H2B-Q emission is suppressed. Cyclic voltammetry of H2B-Q shows two reversible, 1-electron reduction peaks at -1.05 V and -1.37 V (vs ferrocene) on par with those of ubiquinone. Chemical reduction of H2B-Q by NaBH4 resulted in >200 fold emission enhancement. H2B-QH2 is shown to react with peroxyl radicals, a form of reactive oxygen species (ROS) as well as to cooperatively interact with chromanol (the active segment of α-tocopherol). Kinetic analysis further shows the antioxidant reactivity of the nonfluorescent intermediate semiquinone. We anticipate that the H2B-Q/H2B-QH2 off/on reversible couple may serve as a tool to monitor chemical redox processes in real-time and in a noninvasive manner.

Reactive Oxygen Species Mediated Activation of a Dormant Singlet Oxygen Photosensitizer: From Autocatalytic Singlet Oxygen Amplification to Chemicontrolled Photodynamic Therapy.

Here we show the design, preparation, and characterization of a dormant singlet oxygen ((1)O2) photosensitizer that is activated upon its reaction with reactive oxygen species (ROS), including (1)O2 itself, in what constitutes an autocatalytic process. The compound is based on a two segment photosensitizer-trap molecule where the photosensitizer segment consists of a Br-substituted boron-dipyrromethene (BODIPY) dye. The trap segment consists of the chromanol ring of α-tocopherol, the most potent naturally occurring lipid soluble antioxidant. Time-resolved absorption, fluorescence, and (1)O2 phosphorescence studies together with fluorescence and (1)O2 phosphorescence emission quantum yields collected on Br2B-PMHC and related bromo and iodo-substituted BODIPY dyes show that the trap segment provides a total of three layers of intramolecular suppression of (1)O2 production. Oxidation of the trap segment with ROS restores the sensitizing properties of the photosensitizer segment resulting in ∼40-fold enhancement in (1)O2 production. The juxtaposed antioxidant (chromanol) and prooxidant (Br-BODIPY) antagonistic chemical activities of the two-segment compound enable the autocatalytic, and in general ROS-mediated, activation of (1)O2 sensitization providing a chemical cue for the spatiotemporal control of (1)O2.The usefulness of this approach to selectively photoactivate the production of singlet oxygen in ROS stressed vs regular cells was successfully tested via the photodynamic inactivation of a ROS stressed Gram negative Escherichia coli strain.

When push comes to shove: unravelling the mechanism and scope of nonemissive meso-unsaturated BODIPY dyes.

We report herein spectroscopy and computational results that illustrate an efficient intramolecular deactivation pathway for meso-unsaturated boron-dipyrromethene (BODIPY) dyes in their singlet excited state. Our results show that the mechanism hinges on the structural flexibility imparted by the boron atom and on the energetic stabilization conferred by extending the conjugation into the meso substituent, which is otherwise unconjugated in the ground state. Following photoexcitation, rotation along the dihedral angle of the meso-unsaturated group results in its conjugation at the expense of shifting one pyrrole moiety in dipyrrin out of the plane. Internal conversion to an energetically hot, ground-state species efficiently competes with emission. The mechanism applies to meso-vinyl, -formyl, and -iminyl moieties. The presence of methyl groups at positions C1 and C7 exacerbates the energetic penalty toward conjugation of the meso groups leading to a small energy gap between relaxed excited state and ground state and undetected emission quantum yields. Importantly, methyls at C1 and C7 prevent nonradiative deactivation in meso-aryl moieties, illustrating that when push comes to shove, the energetic (kinetic) barrier toward reaching conjugation is too large for aryl moieties but low enough for smaller groups to effectively compete with radiative transitions. Wisely chosen meso-unsaturated BODIPY dyes may serve as richly sensitive platforms for the preparation of novel fluorogenic substrates to monitor chemical reactions or to probe the rigidity of their surrounding environment.

Electronic excited state redox properties for BODIPY dyes predicted from Hammett constants: estimating the driving force of photoinduced electron transfer.

Here we formulate equations based solely on empirical Hammett substituent constants to predict the redox potentials for the electronic excited state of boron-dipyrromethene (BODIPY) dyes. We utilized computational, spectroscopic, and electrochemical techniques toward characterizing the effect of substitution at the positions C2, C6, and C8 of the 1,3,5,7-tetramethyl BODIPY core. Working with a library of 100 BODIPY dyes, we found that highest occupied molecular orbital (HOMO) energies calculated at the B3LYP 6-31g(d) level correlated linearly with the Hammett σm value for substituents at position C8 and with Hammett σp values for substituents at positions C2 and C6. In turn, we observed that LUMO energies correlated linearly with Hammett σp at position C8 and with Hammett σm at positions C2 and C6. Focusing on a subset of 26 dyes for which reduction potentials were either previously available or measured herein and ranged from -1.84 to -0.52 V (a full 1.3 V), we found a linear relationship between redox potentials in acetonitrile and HOMO and lowest unoccupied molecule orbital (LUMO) energies determined via density functional theory (DFT). A linear correlation was thus ultimately established between redox potentials in acetonitrile and Hammett substituent constants. Combining this with equations derived for the linear relationship existing between the zero vibrational energy of the excited BODIPY and Hammett substituent constants enabled us to provide the parameters toward predicting the oxidizing/reducing power of photoexcited 1,3,5,7,-tetramethyl BODIPY dyes in their singlet excited state.

Fluorogenic α-tocopherol analogue for monitoring the antioxidant status within the inner mitochondrial membrane of live cells.

We report here the preparation of a lipophilic fluorogenic antioxidant (Mito-Bodipy-TOH) that targets the inner mitochondrial lipid membrane (IMM) and is sensitive to the presence of lipid peroxyl radicals, effective chain carriers in the lipid chain autoxidation. Mito-Bodipy-TOH enables monitoring of the antioxidant status, i.e., the antioxidant load and ability to prevent lipid chain autoxidation, within the inner mitochondrial membrane of live cells. The new probe consists of 3 segments: a receptor, a reporter, and a mitochondria-targeting element, constructed, respectively, from an α-tocopherol-like chromanol moiety, a BODIPY fluorophore, and a triphenylphosphonium cation (TPP). The chromanol moiety ensures reactivity akin to that of α-tocopherol, the most potent naturally occurring lipid soluble antioxidant, while the BODIPY fluorophore and TPP ensure partitioning within the inner mitochondrial membrane. Mechanistic studies conducted either in homogeneous solution or in liposomes and in the presence of free radical initiators show that the antioxidant activity of Mito-Bodipy-TOH is on par with that of α-tocopherol. Studies conducted on live fibroblast cells further show the antioxidant depletion in the presence of methyl viologen (paraquat), a known agent of oxidative stress and source of superoxide radical anion (and indirectly, a causative of lipid peroxidation) within the mitochondria matrix. We recorded a ca. 8-fold emission enhancement with Mito-Bodipy-TOH in cells stressed with methyl viologen, whereas no enhancement was observed in control studies with untreated cells. Our findings underscore the potential of the new fluorogenic antioxidant Mito-Bodipy-TOH to study the chemical link between antioxidant load, lipid peroxidation and mitochondrial physiology.

Correlations of ion structure with multiple fragmentation pathways arising from collision-induced dissociations of selected α-hydroxycarboxylic acid anions.

Under conditions of collision-induced dissociation (CID), anions of α-hydroxycarboxylic acids usually fragment to yield the distinctive hydroxycarbonyl anion (m/z 45) and/or the complementary product anion formed by neutral loss of formic acid (46 u). Further support for the known two-step mechanism, involving an ion-neutral complex for the formation of the hydroxycarbonyl anion from the carboxyl group, is herein provided by tandem mass spectrometric results and density functional theory computations on the glycolate, lactate and 3-phenyllactate ions. A fourth, structurally related α-hydroxycarboxylate ion, obtained by deprotonation of mandelic acid, showed only loss of carbon dioxide upon CID. Density functional theory computations on the mandelate ion indicated that similar energy inputs were required for a direct, phenyl-assisted decarboxylation and a postulated novel rearrangement to a carbonate ester, which yielded the benzyl oxide ion upon loss of CO2. Rearrangement of the glycolate ion led to expulsion of carbon monoxide, whereas the 3-phenyllactate ion showed the loss of water and formation of the benzyl anion and the benzyl radical as competing processes. The fragmentation pathways proposed for lactate and 3-phenyllactate are supported by isotopic labeling. The relative computed energies of saddle points and product ions for all proposed fragmentation pathways are consistent with the energies supplied during CID experiments and the observed relative intensities of product ions. The diverse reaction pathways characterized for this set of four α-hydroxycarboxylate ions demonstrate that it is crucial to understand the effects of structural variations when attempting to predict the gas-phase reactivity and CID spectra of carboxylate ions.