Control and selectivity of photosensitized singlet oxygen production: challenges in complex biological systems

Singlet molecular oxygen is a reactive oxygen species that plays an important role in a number of biological processes, both as a signalling agent and as an intermediate involved in oxidative degradation reactions. Singlet oxygen is commonly generated by the so-called photosensitization process wherein a light-absorbing molecule, the sensitizer, transfers its energy of excitation to ground-state oxygen to make singlet oxygen. This process forms the basis of photodynamic therapy, for example, where light, a sensitizer, and oxygen are used to initiate cell death and ultimately destroy undesired tissue. Although the photosensitized production of singlet oxygen has been studied and used in biologically pertinent systems for years, the photoinitiated behaviour is often indiscriminate and difficult to control. In this Concept, we discuss new ideas and results in which spatial and temporal control of photosensitized singlet oxygen production can be implemented through the incorporation of the sensitizer into a conjugate system that selectively responds to certain triggers or stimuli.     

Effects of quenching mechanisms of carotenoids on the photosensitized oxidation of soybean oil

The effects of 0, 1.0 × 10”⁻⁵, 2.5 × 10⁻⁵, and 5.0 × 10⁻⁵ M β-apo-8'-carotenal, β-carotene, and canthaxanthin on the photooxidation of soybean oil in methylene chloride containing 3.3 × 10⁻⁹ M chlorophyll b were studied by measuring peroxide values and conjugated diene content. β-Apo-8'-carotenal, β-carotene, and canthaxanthin contain 10,11, and 13 conjugated double bonds, respectively. The peroxide values and conjugated diene contents of oils containing the carotenoids were significantly lower (P<0.05) than those of control oil containing no carotenoid. As the number of conjugated double bonds of the carotenoids increased, the peroxide values of soybean oils decreased significantly (P<0.05). The quenching mechanisms and kinetics of the carotenoids in the photosensitized oxidation of soybean oil were studied by measuring peroxide values. The steady-state kinetics study showed that carotenoids quenched singlet oxygen to reduce chlorophyll-sensitized photooxidation of soybean oil. The singlet-oxygen quenching rate constants ofβ- apo-8'-carotenal, β-carotene, and canthaxanthin were 3.06 × 10⁹, 4.60 × 10⁹, and 1.12 × 10¹⁰ M⁻¹sec⁻¹, respectively.     

A comparative study on the susceptibilities of soybean,sunflower and peanut oils to singlet molecular oxygen photooxidation

The susceptibilities of crude soybean, sunflower and peanut oils to singlet oxygen photooxidation were determined in a kinetic study. The accumulation of photosensitized hydroperoxides, determined spectroscopically, and the quenching of singlet molecular oxygen phosphorescence by the crude oils and their fatty acid methyl esters were compared. The relative tendency to photooxidation for the oils and the methyl esters was soybean ≫ sunflower > peanut. This trend was independent of the method employed in the determination of initial photodamaging. Soybean oil was demonstrated to be the most unstable product, not only due to the presence of highly unsaturated fatty acids, but also due to the absence of natural constituents, capable of providing a protective antioxidant effect. This protection was more effective in sunflower and peanut oils.     

Christopher Foote's discovery of the role of singlet oxygen [1O2 (1Delta g)] in photosensitized oxidation reactions

The chemistry of singlet molecular oxygen [1O2 (1Delta g)], its importance in atmospheric, biological, and therapeutic processes, and its use as a reagent in organic synthesis have been of considerable interest. Many aspects of singlet oxygen chemistry have emanated from the work of Christopher S. Foote and co-workers. Singlet oxygen is a historically interesting molecule with an unusual story connected with its discovery. Foote and Wexler conducted experiments in the 1960s where evidence was obtained supporting 1O2 generation via two independent routes: (1) a photochemical reaction (dye-sensitized photooxidation) and (2) a chemical reaction (NaOCl with H2O2). An important factor in the discovery of 1O2 as the critical reaction intermediate in dye-sensitized photooxygenations was Foote's reassessment of the chemical literature of the 1930s, when 1O2 was suggested to be a viable intermediate in dye-sensitized photooxidation reactions. Experiments that used silica gel beads provided evidence for a volatile diffusible oxidant such as 1O2. However, a contemporaneous quarrel surrounded this early work, and the possible existence of solution-phase 1O2 was ignored for over 2 decades. Not long after Foote's initial studies were published in 1964, the idea of singlet oxygen as an intermediate in photooxidation chemistry gained increasing recognition and verification in organic, gas phase, and biological processes. There are many documented impacts that 1O2 has had and continues to have on biology and medicine, for example, photodynamic therapy and plant defenses.     

Participation of free fatty acids in the oxidation of purified soybean oil during microwave heating

Effects of 0 to 1.0% levels of caprylic, capric, lauric, myristic, palmitic or stearic acid on the oxidative stability of purified soybean oil were investigated under microwave heating conditions. A prooxidative effect of the fatty acids introduced into the systems was established. The extent of this effect depended on the acyl chin and levels of added fatty acids. During microwave heating, the oxidative rate of purified soybean oil by the fatty acids was rapid compared to the addition of their corresponding hydrocarbons; the shorter the chainlength and the higher the levels of fatty acids, the more accelerated was the thermal oxidation in the oil. The results are explained on the basis of the catalytic effect of the carboxylic group on the formation of free radicals by the decomposition of hydroperoxides. Therefore, particular attention should be paid to the free fatty acid content, which affects the oxidative stability of purified soybean oil.     

The impact of singlet oxygen on lipid oxidation

Oxidation plays a fundamental role in the reduction of the quality of fats, oils and many other principal ingredients in food or animal feed. A significant amount of research data is available on the impact of autoxidation caused by the most abundant and stable form of oxygen: triplet oxygen (³O₂). An alternative oxidation process occurs when lipids are exposed to a light source, even at low temperatures. Many molecules are able to absorb energy from light after which this energy is transferred to an oxygen molecule. This process leads to the conversion of the most abundant triplet oxygen into the more reactive singlet oxygen (¹O₂). Both types of oxygen react very differently with lipids and other nutrients due to the dissimilarity in their fundamental chemical structure. This allows researchers to study the different possible oxidation pathways and consequently to develop potential strategies to delay oxidation for the improvement of shelf life.     

Model systems for fluorescence and singlet oxygen quenching by metalloporphyrins

Next-generation photodynamic therapy agents will minimize extraneous phototoxicity by being active only at the target site. To this end, we have developed a model system to systematically investigate the excited-state quenching ability of a number of metalloporphyrins. Central metal ions that prefer four-coordinate, square planar orientations (Ag(II), Cu(II), Ni(II), Pd(II), and Zn(II)) were used. Porphyrin dimers based on 5-(4-aminophenyl)-10,15,20-triphenylporphyrin and comprising both a free base porphyrin and a metalloporphyrin covalently linked through a five-carbon alkyl chain were synthesized. The fluorescence and singlet oxygen quantum yields for the dimers were probed at 630 and 650 nm, respectively, resulting in the excitation of only the free base porphyrin and allowing a comparison of the quenching efficacy of each central metal ion. These results demonstrate that metalloporphyrins can serve as efficient quenchers, and may be useful in the design of novel light-activated therapeutic agents.     

Evidence for singlet oxygen in the air oxidation of surface adsorbed 2,5-diphenylfuran

Heating 2,5-diphenylfuran on a silicon or titanium dioxide surface results in its conversion tocis-dibenzoylethylene. This is considered an indication of singlet oxygen intermediacy. No significant reaction occurs with the solid or in solvents other than acetone. A singlet oxygen quencher inhibits the reaction, and a free radical scavenger has little effect. The differences between the surface reaction and lack of reactivity in solution may arise from several causes. These include local forces involved in adsorption, topographical and geometric effects, and metal catalysis. Knowledge of factors influencing singlet oxygen formation under nonphotochemical conditions has potential practical application.     

Rosmariquinone interactions in autoxidation and light-sensitized oxidation of stripped soybean oil

Two studies were conducted, using a stripped soybean oil model system, to evaluate the antioxidant interaction(s) between rosmariquinone (RQ) and oil components, including chlorophyll (CHL), β-carotene (CAR), and tocopherol(s) (TOCO), under both autoxidation and light-sensitized oxidation. In autoxidation, CHL alone had no effect on the level of oxidation, whereas CAR alone showed prooxidant (P<0.05) activity. RQ and RQ+CHL or RQ+CAR were significantly (P<0.05) better in controlling autoxidation. RQ appeared to be responsible for the activity in the test combinations. However, among the treatments containing TOCO, RQ, and RQ+TOCO, RQ+TOCO gave a level (P<0.05) of inhibition that indicated a possible synergistic relationship. When tested in a light-sensitized oxidative system, CHL and CAR had no effect on oxidation relative to the control and, again, RQ alone or in combination with CHL or CAR was responsible for the greatest (P<0.05) inhibition of oxidation. RQ+TOCO had better (P<0.05) inhibitory activity than the individual compounds. In the second study, the interaction between RQ and TOCO was studied. TOCO, added at levels found in the nonstripped soybean oil, and RQ, added at two levels (100 and 200 ppm), were tested alone or in combination in both autoxidation and light-sensitized oxidation. In autoxidation, the combination of RQ at 200 ppm and TOCO was synergistic; however, in light-sensitized oxidation, this combination was not synergistic. Monitoring the loss of both RQ and TOCO suggested that during autoxidation RQ acted as a secondary antioxidant relative to TOCO.     

Structural analysis of hydroperoxides formed by oxidation of phosphatidylcholine with singlet oxygen

Soybean phosphatidylcholine (PC) and dilinoleoyl PC (di-18∶2 PC) were oxidized with singlet molecular oxygen using methylene blue as the photosensitizer. The oxidation products, PC monohydroperoxides (PC-MHP) and PC dihydroperoxides (PC-DHP), were isolated by reverse phase liquid chromatography, and their structures were analyzed by nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). Signals for the hydroperoxy proton appeared downfield in NMR spectra of PC-MHP and PC-DHP. Soybean PC-MHP and di-18∶2 PC-MHP were converted to trimethylsilyl (TMS) derivatives of hydrogenated diglycerides when treated with phospholipase C and hydrogenated. Thetert-butyldimethylsilyl (TBDMS) derivatives of hydrogenated diglycerides were also prepared from di-18∶2 PC-MHP. Fragmentation of the TMS and TBDMS derivatives was obtained in electron impact mass spectra. The isomeric composition of hydroperoxylinoleate component in di-18∶2 PC-MHP was determined by methanolysis of the hydrogenated diglyceride and mass chromatographic analysis of the resulting isomeric hydroxy octadecanoates.     

A dimeric oxidation product of γ-tocopherol in soybean oil

A dimeric oxidation product (5-γ-tocopheroxy-γ-tocopherol) has been isolated from soybean oil and identified. The dimer content in extracted oil was increased by elevating the moisture level in raw soybeans. With moisture increase, no change in the quantity of α-tocopherol was observed, but γ- and δ-tocopherol contents were greatly decreased and two kinds of dimer were formed from γ- and δ-tocopherols. When the moisture level in moistened beans was lowered, these dimers reverted to their corresponding original tocopherols. The same results were obtained by treating pulverized soybeans with various reducing agents. γ-Tocopherol added to autoxidizing soybean oil was oxidized more easily in the presence of oxidation products derived from tocopherols and turned into the dimeric product.     

Studies in photooxidation of olive oil

Photooxidation of olive oil, bleached to remove most non-triglyceride components, was studied to elucidate the role of added chlorophyll a, pheophytin a and b,α- andβ-carotene, d-α-tocopherol and nickel dibutyldithiocarbamate. Chlorophyll functioned as a photosensitizer resulting in rapid oxidation of the oil and the added components and loss of color.α- andβ-carotene acted essentially equally as singlet oxygen quenchers.α-tocopherol had little apparent effect on the oxidation rate. Carotenes and tocopherols apparently were destroyed more rapidly when chlorophyll was present. The ratio of peroxide value to conjugated dienoic acids formed developed greater values when chlorophyll was present, thus suggesting a singlet oxygen effect in the system. Pheophytin also proved to be an oxidation promoter.     

用作植物绝缘油的精炼大豆油氧化安定性研究

采用电导率法、GB/T 12580—1990法、SH/T 0206—92法和铜催化电导率法测定了3种精炼植物油的氧化安定性,比较了3种抗氧化添加剂的作用效果。结果表明,电导率法最适合植物油氧化安定性的测定;精炼库柏FR3植物绝缘油氧化安定性最好,大豆油次之,菜籽油最差;3种抗氧化剂中,TBHQ效果最明显,BHT次之,PG最弱。对比电导率法和铜催化电导率法,试验了EDTA抑制铜的催化作用。用电导率法试验了3种抗氧化剂的适宜添加量,TBHQ的适宜添加量为0.5%,BHT的适宜添加量为0.5%～0.7%,PG的适宜添加量为0.6%～0.7%。     

Influence of oxygen and copper concentration on lipid oxidation in rapeseed oil

The influence of oxygen concentration and copper on lipid oxidation in rapeseed oil during storage at 40°C was investigated. The oil was stored in air, or with 1.1%, 0.17%, or 0.04% oxygen in the headspace, and 70 or 0.07 ppm copper was added. Volatile oxidation products and oxygen consumption were monitored. Addition of 70 ppm copper to the sample in air resulted in a 70-fold higher hexanal concentration after 35 d of storage, compared to the sample without added copper. The addition of 0.07 ppm copper to the sample stored in air gave a doubled hexanal concentration, compared to the sample without copper, after 35 d of storage. For the samples with 70 ppm copper at 0.17% and 0.04% oxygen, all oxygen was consumed after 7 d of storage. The results show the importance of minimizing the oxygen available for oxidation, especially when pro-oxidants are present. In the sample with 70 ppm added copper, in air, the hexanal increase was 65 times larger than for the same sample in 0.04% oxygen. A comparison of the effect of oxygen or copper on oxidation shows that the addition of 70 ppm copper to the 0.04% oxygen sample gave the same increase in hexanal content as an oxygen increase to 0.17%.     

Comparison of gas chromatographic methods for volatile lipid oxidation compounds in soybean oil

To develop new knowledge on undesirable flavors affecting the quality of foods containing polyunsaturated lipids, we investigated the volatiles in soybean oil oxidized at different conditions by three capillary gas chromatographic methods: (a) direct injection (5 min heating at 180 C); (b) static headspace (20 min heating at 180 C, pressurizing for one min), and (c) dynamic headspace (purging 15 min at 180 C onto a porous polymer trap, desorbing from trap for five min). A fused silica column was used with bonded polymethyl and phenyl siloxane phase. At peroxide values between 2 and 10, the major volatile products found in soybean oil by the three methods were pentane, hexanal, 2-heptenal, 2,4-heptadienal and 2,4-decadienal. The intensities of each volatile compound varied with the analytical methods used.     

Antioxidants and stabilizers. LXV. Contribution to the investigation of sensitized photooxidation of phenolic antioxidants. Quenching of singlet oxygen with stilbenequinoid compounds

The photooxidation of 2,6-di-tert-butyl-4-methylphenol (I) and 2,2′-methylene-bis(4-methyl-6-tert-butylphenol) (II) sensitized with methylene blue in dichloromethane was investigated. The oxidation of phenol I proceeds at a reduced rate, but otherwise similar to the oxidation in methanol, that is, with the formation of 2,6-di-tert-butyl-4-methyl-4-hydroperoxy-2,5-cyclohexadiene-1-one (III, R = tert-butyl). On the other hand, bisphenol II remains almost unoxidized in dichloromethane. This is due to the formation of stilbenequinoid derivatives of type VI which slow down the oxidation even at insignificant concentrations. The effect approaches that of β-carotene. Phenol I has a much weaker tendency towards the formation of an analogous derivatives, i.e. 3,5,3′,5′-tetra-tert-butylstil-benequinone (VIII), which however is also an active retarder of oxidation. A more detailed study carried out with VIII indicates that the mechanism of its effect is the same as for β-carotene, that is, the quenching of singlet oxygen. Stilbenequinone VIII used in a fourfold concentration has almost the same effect as β-carotene, but unlike the latter it is much more stable towards oxidation. The activity of VI lies between that of stilbenequinone VIII and β-carotene.     

The effects of bleached and unbleached rosemary oleoresins on light-sensitized oxidation of soybean oil

Bleached and unbleached forms of a rosemary oleoresin (RO) in stripped and nonstripped soybean oil behave both as antioxidant and prooxidant in a light-induced oxidative system. At 0.02 and 0.05% levels, RO had the greatest antioxidant activity, while at 0.01 and 0.5% levels it had the highest prooxidant activity in both stripped and nonstripped soybean oil. Treatment of both soybean oil systems with tertiary butylhydroquinone controlled light-induced oxidation of the oil better than did the oleoresin treatments. The prooxidant activity of the 0.5% RO level was probably due to an excess of prooxidant components being carried into the oil at that level, whereas the reduced antioxidant activity at 0.01% was probably due to the low initial level of active antioxidant components being added to the oil. Published as Journal Series No. 10072, Nebraska Agricultural Research Division, Department of Food Science and Technology, University of Nebraska, Lincoln, NE 68583-0919.     

Riboflavin-photosensitized singlet oxygen oxidation product of vitamin D<Subscript>2</Subscript>

The riboflavin-photosensitized singlet oxygen oxidation of vitamin D₂ in a model system of 12% water and 88% acetone was studied to understand the possible oxidation of fortified vitamin D in milk. Only the samples containing vitamin D₂ and riboflavin under light storage showed two new peaks in the HPLC chromatogram, indicating that singlet oxygen oxidation of vitamin D₂ had occurred. UV analysis indicated that a new compound was formed from the reaction of the triene of vitamin D₂ with oxygen. The mass spectrum showed that one of the two compounds had a molecular ion at m/z=412, which was an increase of the mass of vitamin D₂ by the mass of exactly one oxygen. The IR spectrum suggested the presence of a hydroxyl group and no carbonyl group in the product. The combned information from UV, MS, and FTIR spectra and the chemical mechanisms of singlet oxygen oxidation of vitamin D₂ indicated that a 5,6-epoxide of vitamin D₂ was formed from vitamin D₂ in the presence of riboflavin under light.