Recent Research Developments

Stable Dioxetane Precursors as Selective Trap-and-Trigger Chemiluminescent Probes for Singlet Oxygen

Singlet oxygen (¹O₂), the lowest excited state of molecular oxygen, is a short-lived reactive oxygen species (ROS) that is formed in both biological and aqueous environmental systems via both ?light? (photosensitized) and ?dark? processes. Singlet oxygen is a known cytotoxin, believed to be involved in DNA damage, lipid peroxidation, and the bactericidal response of certain antibodies. In sunlit surface waters, ¹O₂ reacts with several types of organic micropollutants, such as some pharmaceuticals and pesticides. An ability to detect and quantify the production of ¹O₂ in such systems allows for a better understanding of the mechanisms of oxidative damage in the body and estimations of the environmental fates of pollutants that are degraded by reaction with ¹O₂. Due to the transient nature of ¹O₂ (it has a lifetime of ~ 4 μs in water) and its very low concentrations (10^-15 – 10^-12 M steady-state concentrations are typical in sunlit surface waters), a highly sensitive detection method is needed. Since ¹O₂ is often formed simultaneously with other ROS, such as superoxide radical anion, hydroxyl radical, and peroxides, it is also imperative that the detection method be selective for ¹O₂ relative to other ROS. Graduate students Laura MacManus-Spencer, Douglas Latch, and Kim Kroncke, along with Professor Kristopher McNeill, have developed a sensitive and selective trap-and-trigger detection method based on the trapping of ¹O₂ to form a stable dioxetane intermediate that, when chemically triggered, emits a luminescent signal that is proportional to the amount of ¹O₂ trapped.

Figure 1. In this chemiluminescent detection scheme, ¹O₂ is trapped with probe 1 to form a thermally stable dioxetane (2), which emits visible light (470 nm) upon addition of a chemical trigger (F^-).

The selectivity of this detection method for ¹O₂ is afforded by the formation of a stable dioxetane intermediate upon the addition of ¹O₂ to a spiroadamantyl-substituted vinyl ether probe molecule, as a dioxetane is a characteristic product of the reaction of ¹O₂ with an electron-rich alkene and is unlikely to be formed by other ROS. The selectivity of probe 1a for ¹O₂ with respect to superoxide radical anion (O₂^-?) and hydrogen peroxide (H₂O₂), compared to another chemiluminescent probe for ROS (MCLA = methyl Cypridina luciferin analog), is illustrated in Figure 2a.The sensitivity of the detection method lies in the chemiluminescent detection of the aryloxy emitter formed in the fluoride ion-triggered decomposition of the dioxetane. The trap-and-trigger nature of this detection scheme represents a facile method to measure photochemically generated ¹O₂, as it allows for low background chemiluminescence measurements in the dark after trapping ¹O₂ in the light. It also facilitates kinetic measurements of ¹O₂ formation as a function of time.

Figure 2. (a) Comparison of the chemiluminescence (CL) signals elicited from 5 μM MCLA and 10 μM 1a when exposed to three ROS: ¹O₂, O₂^-?, and H₂O₂. Filled columns with error bars represent the average and one standard deviation for three measurements, and the unfilled columns represent the blank signal in each system. Probe 1a was used to detect ¹O₂ (b) in the thermal decomposition of a naphthalene endoperoxide, MNPO₂, and (c) in the reaction of benzoyl peroxide with O₂^-?.

The detection scheme described here has been used in the McNeill lab to quantify both photochemically and thermally generated ¹O₂ in aqueous and hydrophobic environments. Probes 1a and 1b were used to detect and quantify ¹O₂ generated photochemically using a small-molecule model photosensitizer. Using results from these experiments, it has been shown that steady-state concentrations of ¹O₂ as low as 5 ? 10^-13 M can be measured accurately with an exposure time of ten minutes. The detection scheme has been successfully applied to natural water samples (for example, Lake Superior surface water) to measure environmentally relevant ¹O₂ steady-state concentrations.

The application of probe 1a to the detection of thermally generated ¹O₂ was tested using a water-soluble naphthalene endoperoxide, which releases ¹O₂ when heated (Figure 2b). Subsequently, the selectivity of the detection scheme for ¹O₂ has been exploited to investigate ?dark? ¹O₂ generating reactions involving other ROS. Singlet oxygen was detected and quantified in the reaction of O₂^-? with benzoyl peroxide in organic solvent (Figure 2c), a model system for ?dark? biological reactions involving ROS in hydrophobic environments, such as lipid bilayers. The formation of ¹O₂ in reactions involving other ROS has important implications for understanding, for instance, the mechanisms of cytotoxicity in organisms under oxidative stress.

The trap-and-trigger chemiluminescent ¹O₂ detection scheme was described in a recent publication (L. A. MacManus-Spencer, et al.,Analytical Chemistry, 2005, http://dx.doi.org/10.1021/ac048293s ) and will be highlighted in the March issue of ?Chemistry World? (http://www.rsc.org/chemistryworld/news/free/cw005001cs.htm).

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