Cyanine dyes in biophysical research: the photophysics of polymethine fluorescent dyes in biomolecular environments

The breakthroughs in single molecule spectroscopy of the last decade and the recent advances in super resolution microscopy have boosted the popularity of cyanine dyes in biophysical research. These applications have motivated the investigation of the reactions and relaxation processes that cyanines undergo in their electronically excited states. Studies show that the triplet state is a key intermediate in the photochemical reactions that limit the photostability of cyanine dyes. The removal of oxygen greatly reduces photobleaching, but induces rapid intensity fluctuations (blinking). The existence of non-fluorescent states lasting from milliseconds to seconds was early identified as a limitation in single-molecule spectroscopy and a potential source of artifacts. Recent studies demonstrate that a combination of oxidizing and reducing agents is the most efficient way of guaranteeing that the ground state is recovered rapidly and efficiently. Thiol-containing reducing agents have been identified as the source of long-lived dark states in some cyanines that can be photochemically switched back to the emissive state. The mechanism of this process is the reversible addition of the thiol-containing compound to a double bond in the polymethine chain resulting in a non-fluorescent molecule. This process can be reverted by irradiation at shorter wavelengths. Another mechanism that leads to non-fluorescent states in cyanine dyes is cis-trans isomerization from the singlet-excited state. This process, which competes with fluorescence, involves the rotation of one-half of the molecule with respect to the other with an efficiency that depends strongly on steric effects. The efficiency of fluorescence of most cyanine dyes has been shown to depend dramatically on their molecular environment within the biomolecule. For example, the fluorescence quantum yield of Cy3 linked covalently to DNA depends on the type of linkage used for attachment, DNA sequence and secondary structure. Cyanines linked to the DNA termini have been shown to be mostly stacked at the end of the helix, while cyanines linked to the DNA internally are believed to partially bind to the minor or major grooves. These interactions not only affect the photophysical properties of the probes but also create a large uncertainty in their orientation.     

Ab initio multiconfiguration reference perturbation theory calculations on the energetics of low-energy spin states of iron(III) porphyrins

Although a major goal of inorganic spectroscopy is to determine the energetics of the low-lying spin states of transition metal complexes, surprisingly little has been accomplished in this respect by means of accurate ab initio calculations. Against this context, we present ab initio multiconfiguration reference perturbation theory (CASPT2) calculations with large basis sets on the low-lying spin states of Fe(III)(P)Cl and [Fe(P)Cl](+) (P(2-)=porphinato). The CASPT2 results on the energetics of various low-lying spin states studied differ significantly, sometimes even dramatically, from those obtained from density functional theory calculations.     

Biological significance of singlet oxygen

The biological significance of singlet oxygen (1O2), an electronically excited species of oxygen, has been realized only in the last two decades. This was mainly due to the lack of proper methodology to generate this reactive oxygen species (ROS) in pure form and its reactions with biological molecules. Recent studies, using newly developed detection methods, show that 1O2 being generated in many biological systems, can significantly and quite often adversely alter several crucial biomolecules including DNA, proteins and lipids with undesirable consequences including cytotoxicity and/or disesase development. The reactions of 1O2 with the biological molecules are rather specific, as compared to other ROS. There are various compounds, mainly derived from natural sources that offer protection against damage induced by 1O2. Among the antioxidants carotenoids are the most effective singlet oxygen quenchers followed by tocopherols and others. The same reactive species if generated specifically in diseased states such as cancer can lead to the cure of the disease, and this principle is utilized in the newly developing modality of cancer treatment namely photodynamic therapy. Singlet oxygen, in low concentrations can also act as signaling molecule with several biological implications. This review clearly brings out the biological significance of 1O2.     

Observations concerning light promoted electronic delocalization in covalently linked transition metal complexes

The emission spectral band shapes of several polypyridine-ligand (PP) bridged bis-ruthenium(II) complexes imply that Ru(II)/Ru(III) electronic coupling is weaker in their lowest energy metal to ligand charge transfer (MLCT) excited states than in their corresponding mixed valence ground states. In general, the amplitudes of the vibronic contributions to emission band shapes decrease markedly with the excited state-ground state energy differences, and it is expected that complexes with degenerate, or mixed valence excited states will have very weak vibronic side bands if configurational mixing of the degenerate MLCT excited states is substantial. However, the bimetallic PP-bridged ruthenium complexes emit at significantly lower energy than their monometallic analogs, but the vibronic contributions to their 77 K emission spectra are very similar to those of their monometallic complexes analogs. This indicates that the mixed valence excited states of the bimetallic complexes are electronically localized.     

One-electron transfer reactions of diquat radical to different reduction intermediates of oxygen. Formation of hydroxyl radical and electronically excited states

The one-electron transfer activation of DQ++ by microsomal fractions comprises an aerobic phase and an anaerobic phase. The aerobic phase is characterized by O2 consumption, formation of electronically excited states with main emission below 600 nm, and H2O2 formation. The anaerobic phase is characterized by H2O2 consumption, DQ+ accumulation, HO. formation, and also electronically excited state formation with main emission beyond 600 nm. Superoxide dismutase abolishes the photoemission during the aerobic phase, whereas it has no effect on the photoemission originating during the anaerobic phase. The hydroxylation products of the aromatic compound salicylate, mainly 2,3- and 2,5-dihydroxybenzoic acids--indicative of the occurrence of HO.-, were detected by h.p.l.c. with oxidative electrochemical detection during the anaerobic phase, but not during the aerobic phase. Neither H2O2 consumption nor HO. are prevented by desferrioxamine. These experimental observations are interpreted on the grounds of two main electron-transfer reactions of DQ.+: under aerobic conditions, two one-electron transfer steps to molecular O2 and O2.- to yield H2O2. Under anaerobic conditions, one-electron transfer step to contaminating iron or any ferrioxamine formed to a ferrous complex which can support a Fenton-like reduction of H2O2 with formation of HO.. The toxicological relevance for the occurrence of such reactions is also discussed in terms of the formation of electronically excited states.     

Electronic excitations of biomolecules studied by quantum chemistry

Significant methodological advances have been made over the past ten years in developing reliable quantum chemical methods for the treatment of electronically excited states. These methods can nowadays be used routinely by the experienced researcher to accurately compute excitation spectra of medium-sized organic molecules; results have been reported for several popular photobiological systems, including green fluorescent protein. First steps are currently being taken to account for the solvochromic shifts of chromophore excitations caused by particular protein environments and to dynamically simulate photochemical reactions in the excited states.     

Electronically excited state generation during the reaction of p-benzoquinone with H2O2. Relation to product formation: 2-OH- and 2,3-epoxy-p-benzoquinone. The effect of glutathione

The reaction between H2O2 and p-benzoquinone proceeds with consumption of both reactants with second order rate constants of 1.66- and 0.77 M-1S-1, respectively. The process is mainly supported by oxygen addition reactions to the quinone resulting in the formation of both 2,3-epoxy-p-benzoquinone and 2-OH-p-benzoquinone. The former product accumulates in the assay mixture without participating in further reactions. The formation of the latter product implies free radical intermediates such as 2-OH-p-benzosemiquinone anion, which supports the generation of electronically excited states upon its oxidation by H2O2, presumably as part of an organic Fenton reaction. The relaxation of the excited state is accompanied by photoemission at 485-530 nm. Glutathione was found to counteract the oxidative aspects of the reaction between p-benzoquinone and H2O2 by a series of processes involving (a) a rapid reductive addition to the quinone with formation of a substituted p-benzohydroquinone; (b) an effective quenching of photoemission, which might be attributed to the deactivation of the excited state by the p-benzohydroquinone-glutathione adduct, and (c) the decomposition of the formed 2,3-epoxy-p-benzoquinone, also by reductive cleavage of the epoxide ring.     

Sensitizer-mediated photooxidation of histidine residues: evidence for the formation of reactive side-chain peroxides

Exposure of proteins to visible light in the presence of a sensitizer results in the oxidation of Met, Trp, Tyr, Cys, and His side chains. These reactions are only partially understood, particularly with His. In this study, the oxidation of free His, His derivatives, and His-containing peptides has been examined using visible light and a range of sensitizers. It is shown that photooxidation gives rise to unstable peroxides, in a light-, illumination time-, and sensitizer-dependent manner. The yield of these materials is increased when reactions are carried out in solutions prepared with D2O, which prolongs the lifetime of 1O2, and decreased in the presence of the potent 1O2 scavenger azide, consistent with the involvement of this excited state. These peroxides have half-lives of hours, though the rate of decomposition is enhanced by elevated temperatures, reductants, and metal ions. Reducing metal ions catalyze the formation of radicals, which have been detected by EPR spin trapping. Structural analysis of His photo-products using NMR spectroscopy has provided evidence for the formation of oxygenated and cyclized compounds (e.g., 6a-hydroxy-2-oxo-octahydro-pyrollo[2,3-d]imidazole-5-carboxylic acid) and cross-linked materials. The latter materials may be partly responsible for the high yield of aggregated materials detected on photooxidation of His-containing proteins.     

Chemistries and colors of bioluminescent reactions: a review

Many different organisms, ranging from bacteria and fungi to fireflies and fish, are endowed with the ability to emit light, but the bioluminescent systems are not evolutionarily conserved: genes coding for the luciferase proteins (Lase) are not homologous, and the luciferins are also different, falling into many unrelated chemical classes. Biochemically, all known Lase are oxygenases that utilize molecular oxygen to oxidize a substrate (a luciferin; literally the ‘light-bearing’ molecule), with formation of a product molecule in an electronically excited state. The color of the light may differ, even though the same luciferin/Lase system underlies the reaction. Filters or differences in Lase structure are responsible in some cases; in others a secondary emitter associated with a second protein is involved. In the coelenterates a green fluorescent protein, whose chromophore is derived from the primary amino-acid sequence, results in a red shift of the emission. In the bacteria accessory proteins causing either blue- or red-shifts have been isolated from different species; the chromophores are noncovalently bound. Although radiationless energy transfer has been implicated in the excitation of such accessory emitters, this may not be so in all cases.     

Mechanism of chemiluminescence from the linoleate--lipoxygenase system.

Blue luminescence peaking at 420 nm arises in the early stage of lipoxygenase-catalyzed linoleate oxygenation. An excited species which involves the blue light, “excited CO₂”, is produced by the interaction of an oxidant and carbonate present in the system. An oxidant generated in a linoleate-lipoxygenase system attacks not only carbonate but also proteins and oxidizable xanthene dyes to produce their electronically excited states, which emit light in the visible region during their return to ground states. This also attacks diphenylisobenzofuran (a singlet oxygen trap) yielding o-dibenzoylbenzene identical with that obtained by a singlet oxygen-derived reaction. Neither an active form of lipoxygenase nor a linoleate peroxy radical is considered to be the oxidant. Another luminescence, which could not be characterized spectrometrically, begins to appear when most of the oxygen in the system has been consumed during the reaction. An excited species, probably involved in this luminescence, can transfer its energy to the dyes containing heavy atoms and is reasonably considered to be an excited carbonyl generated from linoleate peroxy radicals via a cyclic intermediate.     

Nonthermal decomposition of nitrous oxide in a high-current pulsed discharge

The kinetics of the nonthermal decomposition of nitrous oxide (N₂O) in a nonequilibrium plasma is investigated experimentally. A numerical model of the process is constructed and used to simulate the decomposition of N₂O in a high-current pulsed discharge. The most important channels for decomposition are revealed by analyzing the results obtained. The role of the charged, electronically excited, and vibrationally excited components is examined. It is shown that the mechanism for the thermally nonequilibrium decomposition of N₂O in a high-current pulsed discharge is governed by the reactions involving ions and electronically excited molecules.     

The effect of oxygen on the amplitude of photodriven electron transfer across the lipid bilayer-water interface.

The surprisingly small effect of oxygen on photoelectron transfer in pigmented lipid bilayers is traced to a short lifetime of the excited states. Decreasing the oxygen concentration by greater than 100-fold decreases the half saturating concentration of acceptor by only threefold and has no effect on the maximum photovoltage observed at acceptor saturation. This holds true for both magnesium octaethylporphyrin and chlorophyll with both ferricyanide and methyl viologen as acceptors. Since oxygen quenches excited states at near the encounter limit, the lifetime of reactive state must be short, less than 100 ns. About 100-fold higher concentrations of acceptor are required to quench the fluorescence (in liposomes) than to saturate the photoeffect. Thus the reactive state is most likely the triplet. The short life of the excited state is caused by concentration quenching, i.e., their reaction with ground state molecules. The increase of photovoltage with increasing pigment concentration shows that this quenching in a condensed form of the pigment produces ions that lead to the observed photovoltage by interfacial reaction of the anion with acceptor.     

Measurements in vivo of parameters pertinent to ROS/RNS using EPR spectroscopy

The technique of in vivo EPR spectroscopy can provide useful and even unique information pertinent to the study of oxygen/nitrogen radicals and related processes. The parameters that can be measured include: (a) Oxygen centered radicals (by spin trapping); (b) carbon centered radicals (by spin trapping and sometimes by direct observation); (c) sulfur centered radicals (by spin trapping and sometimes by direct observation); (d) nitric oxide (by spin trapping); (e) oxygen (using oxygen sensitive paramagnetic materials); (f) redox state (using metabolism of nitroxides); (g) thiol groups (using special nitroxides); (h) pH (using special nitroxides); (h) perfusion (using washout of paramagnetic tracers); (i) some redox active metal ions (chromium, manganese). The current state of the art for these and other measurements is discussed, especially in relationship to experiments that are likely to be useful for studies of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS).     

Visible-light-promoted degradation of the antioxidants propyl gallate and t-butylhydroquinone: Mechanistic aspects

Abstract

The kinetic and mechanistic aspects of the visible-light-mediated photodegradation of the phenolic antioxidants (PA), propyl gallate (PG), and t-butylhydroquinone (TBHQ), employing riboflavin (Rf) as photosensitizer, have been studied by time-resolved and stationary techniques. The photosensitizer Rose Bengal (RB) was used for auxiliary experiments. Results show the occurrence of chemical transformations on PA with the participation of electronically excited states of Rf and different reactive oxygen species (ROS) generated from these states. With 0.02 mM Rf and 1.0 mM PA, the electronically excited triplet state of Rf is quenched by PA, in a competitive manner with the dissolved oxygen. As a consequence, a cascade of photoprocesses produces singlet oxygen (O₂(¹Δ_g)) and H₂O₂ in the case of PG and, O₂(¹Δ_g), H₂O₂ and HO^? in the case of TBHQ. The participation of these species is supported by experiments of oxygen consumption carried out in the presence of specific ROS scavengers. TBHQ has a relatively high capacity for O₂(¹Δ_g) physical deactivation and a low photodegradation efficiency by the oxidative species. Comparatively, it can be asserted that TBHQ has a higher antioxidant capacity than PG.     

Low-lying electronic states of the ferrous high-spin (S=2) heme in deoxy-Mb and deoxy-Hb studied by highly-sensitive multi-frequency EPR

The low-lying electronic states of the ferrous high-spin heme in deoxy-myoglobin (deoxy-Mb) and deoxy-hemoglobin (deoxy-Hb) were probed by multi-frequency electron paramagnetic resonance (MFEPR) spectroscopy. An unexpected broad EPR signal was measured at the zero magnetic field using cavity resonators at 34-122 GHz that could not be simulated using any parameter sets for the S = 2 spin Hamiltonian assuming spin quintet states in the ⁵B₂ ground state. Furthermore, we have observed novel, broad EPR signals measured at 70-220 GHz and 1.5 K using a single pass transmission probe. These signals are attributed to the ferrous high-spin heme in deoxy-Mb and deoxy-Hb. The resonant peaks shifted to a higher magnetic field with increasing frequency. The energy level separation between the ground singlet and the first excited state at the zero magnetic field was directly estimated to be 3.5 cm^− 1 for deoxy-Hb. For deoxy-Mb, the first two excited singlet states are separated by 3.3 cm^− 1 and 6.5 cm^− 1, respectively, from the ground state. The energy gap at the zero magnetic field is directly derived from our MFEPR for deoxy-Mb and deoxy-Hb and strongly supports the theoretical analyses based on the Mössbauer and magnetic circular dichroism experiments.     

Model studies of the α-peroxidase system: Formation of an electronically excited product

Horseradish peroxidase—as an oxidase—converts propanaldehyde to acetaldehyde and formic acid. To some extent the enzyme also acts upon linear acids, thus mimicking even better the α-peroxidase activity of higher plants. In these reactions an electronically excited species—presumably the aldehyde—is generated, as revealed by sensitized emission. The species is long-lived; in accord with its triplet nature heavy substituents are required in the acceptor for efficient sensitization. Energy transfer occurs noncollisionally and does not appear to proceed by a long-range Förster-type T-S mechanism. A long-range triplet-triplet exciton transfer to an upper triplet state of the acceptor is proposed; then ISC occurs to the fluorescent state of the acceptor. Biological compounds which might originate from excited aldehydes are pointed out.     

Measurements in vivo of parameters pertinent to ROS/RNS using EPR spectroscopy

The technique of in vivo EPR spectroscopy can provide useful and even unique information pertinent to the study of oxygen/ nitrogen radicals and related processes. The parameters that can be measured include: (a) Oxygen centered radicals (by spin trapping); (b) carbon centered radicals (by spin trapping and sometimes by direct observation); (c) sulfur centered radicals (by spin trapping and sometimes by direct observation); (d) nitric oxide (by spin trapping); (e) oxygen (using oxygen sensitive paramagnetic materials); (f) redox state (using metabolism of nitroxides); (g) thiol groups (using special nitroxides); (h) pH (using special nitroxides); (h) perfusion (using washout of paramagnetic tracers); (i) some redox active metal ions (chromium, manganese). The current state of the art for these and other measurements is discussed, especially in relationship to experiments that are likely to be useful for studies of reactive oxygen species (ROS) and/or reactive nitrogen species (RNS).     

Factors that influence singlet oxygen formation vs. ligand substitution for light-activated ruthenium anticancer compounds

This review focuses on light-activated ruthenium anticancer compounds and the factors that influence which pathway is favored. Photodynamic therapy (PDT) is favored by π expansion and the presence of low-lying triplet excited states (e.g. ³MLCT, ³IL). Photoactivated chemotherapy (PACT) refers to light-driven ligand dissociation to give a toxic metal complex or a toxic ligand upon photo substitution. This process is driven by steric bulk near the metal center and weak metal–ligand bonds to create a low-energy ³MC state with antibonding character. With protic dihydroxybipyridine ligands, ligand charge can play a key role in these processes, with a more electron-rich deprotonated ligand favoring PDT and an electron-poor protonated ligand favoring PACT in several cases.