Paul D. Sima

Singlet-oxygen Generation from A2E¶

Abstract Singlet-oxygen generation was measured in solutions containing equilibrium mixtures of the retinal lipofuscins, 2-[2,6-dimethyl-8-(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E,7E-octatetraenyl]-1-(2-hydroxyethyl)-4-[4-methyl-6(2,6,6-trimethyl-1-cyclohexen-1-yl)-1E,3E,5E-hexatrienyl]-pyridinium (A2E) and double bond isomer of A2E (iso-A2E), using steady-state irradiation and using cholesterol as a singlet-oxygen trap. The amount of singlet oxygen generated by equilibrium mixtures of A2E and iso-A2E was compared with that generated by tetraphenylporphine (TPP) under the same conditions. Studies were carried out in ethanol-d6, acetone-d6, 80% cyclohexane-d12–20% acetone-d6 (vol/vol) and hexafluorobenzene. Using 420 nm irradiation and assuming a singlet-oxygen quantum yield of 0.60 ± 0.12 for TPP, the singlet-oxygen quantum yields were 0.8 ± 0.3 × 10−3, 1.2 ± 0.4 × 10−3, 2 ± 1 × 10−3 and 4 ± 1 × 10−3, respectively. In acetone-d6, the quantum yields were smaller at longer wavelengths, with values of 0.3 ± 0.1 × 10−3 and 0.4 ± 0.2 × 10−3 at 461 and 493 nm, respectively. Singlet-oxygen generation was greatest in solvents with the lowest dielectric constants. In view of the relatively small quantum yields, the contribution of singlet-oxygen generation to the phototoxic properties of A2E and of iso-A2E will require further study.

Structural and Environmental Requirements for Quenching of Singlet Oxygen by Cyanine Dyes

Abstract Singlet-oxygen quenching constants were measured for 19 cyanine dyes in acetonitrile. The most efficient quenchers were 1-butyl-2-[2-[3-[(1-butyl-6-chlorobenz[cd]indol-2(1H)-ylidene)ethylidene]-2-chloro-1-cyclohexen-1-yl]ethenyl]-6-chlorobenz[cd]indolium and 6-chloro-2-[2-[3-(6-chloro-1-ethylbenz[cd]indol-2(1H)-ylidene)ethylidene]-2-phenyl-1-cyclopenten-1-yl]ethenyl]-1-ethylbenz[cd]indolium, having quenching constants with diffusion-controlled values of 2.0 ± 0.1 × 1010 and 1.5 ± 0.1 × 1010 M−1 s−1, respectively. There was a trend toward increased quenching constants for cyanine dyes with the absorption band maxima at longer wavelengths. However, the quenching constants correlated better with the oxidation potentials of the cyanine dyes, suggesting that quenching proceeds by charge transfer rather than energy transfer. The quenching constants for 1,1′,3,3,3′,3′-hexamethylindotricarbocyanine perchlorate and 1,1′-diethyl-4,4′-carbocyanine iodide were measured in several solvents as well as in aqueous solutions of detergent micelles. In different solvents, the quenching constants varied by as much as a factor of 50. The quenching constants were largest in solvents with the highest values on the π* scale of Kamlet, Abboud, Abraham and Taft. This was consistent with quenching occurring by charge transfer. Within cells, cyanine dyes concentrate in membrane-bound organelles. The quenching constants were substantial within detergent micelles. To the extent that micelles are models for biological membranes, cyanine dyes may be effective biological singlet-oxygen quenchers.

Cyanine Dyes as Protectors of K562 Cells from Photosensitized Cell Damage

Abstract Several cyanine dyes were found to protect K562 leukemia cells against toxicity mediated by cis-di(4-sulfonatophenyl)diphenylporphine (TPPS2) and light. Most cyanine dyes derived from dimethylindole were better photoprotectors than cyanine dyes with other structures. This correlated with the fact that cyanine dyes derived from dimethylindole were predominately monomeric at millimolar concentrations within K562 cells, while other cyanine dyes formed aggregates. For cyanine dyes that are derived from dimethylindole and have absorption band wavelengths greater than 700 nm, fluorescence-energy transfer from TPPS2 to the cyanine dye was the most important mechanism for photoprotection. There was no spectroscopic evidence for complex formation between the cyanine dyes and TPPS2. The dimethylindole derivative, 1,1′,3,3,3′,3′-hexamethylindodicarbocyanine, was an excellent photoprotector, but a poor quencher of TPPS2 fluorescence and a relatively poor singlet-oxygen quencher. This cyanine dye may act by quenching excited triplet TPPS2. Singlet-oxygen quenching may contribute to the photoprotection provided by cyanine dyes not derived from dimethylindole. Differences in the subcellular distribution of the various cyanine dyes studied may have contributed to the different apparent mechanisms of photoprotection.

Singlet-oxygen generation at gas-liquid interfaces : a significant artifact in the measurement of singlet-oxygen yields from ozone-biomolecule reactions

Abstract— Several ozone‐biomolecule reactions have previously been shown to generate singlet oxygen in high yields. For some of these orone‐biomolecule reactions, we now show that the apparent singlet‐oxygen yields determined from measurements of 1270 nm chemiluminescence were artifactually elevated by production of gas‐phase singlet oxygen. The gas‐phase singlet oxygen results from the reaction of gas‐phase ozone with biomolecules near the surface of the solution. Through the use of a flow system that excludes air from the reaction chamber, accurate singlet‐oxygen yields can be obtained. The revised singlet‐oxygen yields (mol 1O2 per mol O3) for the reactions of ozone with cysteine, reduced glutathione, NADH, NADPH, human albumin, methionine, uric acid and oxidized glutathione are 0.23 ± 0.02, 0.26 ± 0.02, 0.48 ± 0.04, 0.41 ± 0.01, 0.53 ± 0.06, 1.11 ± 0.04, 0.73 ± 0.05 and 0.75 ± 0.01, respectively. These revised singlet‐oxygen yields are still substantial.

Singlet-Oxygen Generation from Liposomes: A Comparison of 6β-Cholesterol Hydroperoxide Formation with Predictions from a One-Dimensional Model of Singlet-Oxygen Diffusion and Quenching

Abstract— We measured 6β‐cholesterol hydroperoxide (6β‐CHP), a specific singlet‐oxygen (O2(δg)) product, during irradiation of unilamellar dimyristoyl 1‐α‐phosphatidylcholine liposomes containing cholesterol and zinc phthalocyanine (ZnPC). The effects of liposome size, the hydrophobic (O2(1δg)) quencher, β‐carotene, and hydrophilic O2(1δg) quenchers upon the amount of 6β‐CHP formed were determined and interpreted in terms of a one dimensional model of 2(1δg) quenching and diffusion. The model correctly predicted (1) that the amount of 6β‐CHP was increased with increasing liposome size, (2) that P‐carotene was more effective at reducing 6β‐CHP formation in 400 nm diameter liposomes than 100 nm diameter liposomes and (3) that the hydrophilic quencher, water, was also more effective in large liposomes than in small liposomes.

[46] Assay for singlet oxygen generation by plant leaves exposed to ozone

Publisher Summary Ozone is a well-established atmospheric toxin for plants. It is known to diffuse through the open stomata of leaves into their inner air spaces. Chameides has carried out a mathematical analysis of the interaction of ozone with plants and concluded that most of the ozone entering a leaf will react with the ascorbic acid present within the cell walls of the plant cells lining the leaf air spaces. Ascorbic acid concentrations on the order of 300 μM within the cell wall, combined with the large rate constant for the reaction of ozone with ascorbic acid, ensure that no ozone will reach the plasmalemmas of the plant cells. Because the reaction of ascorbic acid with ozone produces singlet oxygen in high yield, plants exposed to an ozone-containing atmosphere should generate singlet oxygen. The singlet oxygen generated within the cell wall should also not reach the plant cell plasmalemma, as singlet oxygen reacts rapidly with ascorbic acid and, in addition, is quenched efficiently by water. However, because most of the singlet oxygen is generated close to the outer surface of the cell wall, some singlet oxygen should diffuse back into the air spaces of the leaf.

Girard's Reagent P Derivative of β‐Apo‐8′‐carotenal: A Potent Photoprotective Agent

A cationic carotenoid derivative (GRP‐carotenal) was synthesized by the reaction of Girards reagent P and β‐apo‐8′‐carotenal. The singlet‐oxygen quenching constants for GRP‐carotenal were 1.3 ± 0.1 × 1010 and 1.0 ± 0.1 × 1010M−1 s−1 in acetonitrile and in detergent micelles, respectively. Photosensitized damage to K562 leukemia cells from cis‐di(4‐sulfonatophenyl)diphenylporphine, hypericin and protoporphyrin IX was inhibited by GRP‐carotenal under conditions where β‐apo‐8′‐carotenal, β‐carotene and crocetin were ineffective. The unique cytoprotective properties of GRP‐carotenal, relative to the other carotenoids studied, could not be explained by the differences in the cell content of the various carotenoids or by the changes in the cell content of the photosensitizers used. Photosensitizer fluorescence from labeled K562 cells was reduced by GRP‐carotenal but not by the other carotenoids studied. The novel photoprotective properties of GRP‐carotenal may be due to its subcellular distribution. In photosensitizer‐containing detergent micelles, novel properties of GRP‐carotenal were not apparent. None of the carotenoids studied reduced photosensitizer fluorescence or singlet‐oxygen generation. Singlet‐oxygen quenching by GRP‐carotenal and by β‐apo‐8′‐carotenal were roughly the same. Crocetin has a singlet‐oxygen quenching constant that is about a factor of five lower. Singlet‐oxygen quenching by β‐carotene was limited by its aggregation.

Structural Requirements for Efficient Cellular Photoprotection by Carotenoid Derivatives

Abstract We have synthesized five carotenoid derivatives: (1) Girards reagent P (GRP)-retinal from GRP and retinal; (2) GRP-carotenal from GRP and β-apo-8′-carotenal; (3) Girards reagent T (GRT)-carotenal from GRT and β-apo-8′-carotenal; (4) (GRP)2-canthaxanthin from 2 mol of GRP and 1 mol of canthaxanthin; and (5) dansyl hydrazine (DH)-carotenal from DH and β-apo-8′-carotenal. The first three derivatives are cations, whereas the fourth is a dication and the fifth is a weak base. Using K562 cells, we compared the subcellular distribution of the synthetic carotenoid derivatives with two uncharged natural carotenoids, β-carotene and β-apo-8′-carotenal. The two natural carotenoids were present mainly within the cell membranes. The synthetic carotenoid derivatives were more broadly distributed among the cell organelles. The positively charged derivatives had relatively high concentrations in mitochondria, whereas DH-carotenal had a relatively high concentration in lysosomes. We also measured the amount of photoprotection provided by the synthetic and natural carotenoids for K562 cells labeled with a photosensitizer (hypericin, protoporphyrin IX or cis-di[4-sulfonatophenyl]diphenylporphine). In this model system, only carotenoid derivatives with a permanent positive charge provided significant photoprotection. Neither the two natural carotenoids nor DH-carotenal were effective photoprotectors.

Quenching of Singlet Oxygen by a Carotenoid-Cyclodextrin Complex : The Importance of Aggregate Formation

The Girard’s reagent P derivative of canthaxanthin ((GRP)2‐canthaxanthin), a dicationic carotenoid, forms a highly water‐dispersible complex with (2‐hydroxypropyl)‐γ‐cyclodextrin. The UV–visible light spectrum of the complex is consistent with some degree of aggregation, but the spectrum is independent of concentration from 7.5 to 750 μm. Stern‐Vomer plots for singlet‐oxygen quenching by the complex are linear over a concentration range of 0–20 μm. In the presence of 1 mm (2‐hydroxypropyl)‐γ‐cyclodextrin, the singlet‐oxygen quenching constant for the complex is 7.9 ± 0.9 × 108 m−1s−1. This is about an order of magnitude lower than the singlet‐oxygen quenching constants for (GRP)2‐canthaxanthin in various organic solvents. The properties of the complex are also compared with the properties of (GRP)2‐canthaxanthin solubilized in neat water and in water containing various detergents. The singlet‐oxygen quenching constant for (GRP)2‐canthaxanthin in micelles depends strongly on the specific detergent used, varying from 9.4 × 108 m−1s−1 for hexadecyltrimethylammonium bromide (CTAB) to 1.24 ± 0.4 × 1010 m−1s−1 for sodium dodecyl sulfate. The small quenching constant in CTAB micelles correlates with spectroscopic evidence for aggregation of the (GRP)2‐canthaxanthin in this detergent.

[45] Reactive absorption of ozone: An assay for reaction rates of ozone with sulfhydryl compounds and with other biological molecules

Publisher Summary Ozone is toxic to both plants and animals. Because the reactions of ozone with many types of biological molecules are extremely fast, ozone reacts very close to the surface of tissues that are in direct contact with the atmosphere. In animals, ozone reacts with biomolecules present in the fluid lining the air passages of lungs. In plants, ozone reacts with biomolecules within the cell walls lining the air passages of leaves. As ozone is consumed within tissues through reactions with biological molecules, more ozone is drawn into the tissues in a process called reactive absorption. Mathematical models of reactive absorption are well established and provide a means of calculating reaction rate constants for gases that react with nonvolatile solutes in liquids. While most of the development of these mathematical models has occurred within the chemical engineering literature, the measurement of reactive absorption can provide some unique insights about ozone reactions with biological molecules close to the surface of a solution. Using a very simple apparatus, it is possible to obtain values for even very large reaction rate constants.