The effect of solvent on the lifetime of singlet oxygen, O2(a(1)Δg), particularly the pronounced H/D solvent isotope effect, has drawn the attention of chemists for almost 50 years. The currently accepted model for this phenomenon is built on a foundation in which the electronic excitation energy of O2(a(1)Δg) is transferred to vibrational modes in a solvent molecule, with oxygen returning to its ground electronic state, O2(X(3)Σg(-)). This model of electronic-to-vibrational (e-to-v) energy transfer specifically focusses on the solvent as a "sink" for the excitation energy of O2(a(1)Δg). On the basis of temperature-dependent changes in the solvent-mediated O2(a(1)Δg) lifetime, we demonstrate that this energy-sink-based model has limitations and needs to be re-formulated. We now show that the effect of solvent on the O2(a(1)Δg) lifetime is more reasonably interpreted by considering an activation barrier that reflects the extent to which a solvent molecule perturbs the forbidden O2(a(1)Δg) → O2(X(3)Σg(-)) transition. For a given solvent molecule, this barrier reflects contributions from (a) the oxygen-solvent charge transfer state that mediates nonradiative coupling between the O2(a(1)Δg) and O2(X(3)Σg(-)) states, and (b) vibrations of specific bonds in the solvent molecule. The latter establishes connectivity to the desirable features of the energy-sink-based model. Moreover, temperature-dependent H/D solvent isotope effects imply that tunneling through this barrier plays a role in the mechanism for O2(a(1)Δg) deactivation, even at room temperature. Although we focus on a long-standing problem involving O2(a(1)Δg), our results and interpretation touch fundamental issues of interest to chemists at large.

Singlet oxygen, O(2)(a(1)Δ(g)), plays a key role in many processes of cell signaling. Limitations in mechanistic studies of such processes are generally associated with the difficulty of controlling the amount and location of O(2)(a(1)Δ(g)) production in or on a cell. As such, there is great need for a system that (a) selectively produces O(2)(a(1)Δ(g)) in appreciable and accurately quantifiable yields and (b) can be localized in a specific place at the suborganelle level. A genetically encodable, protein-encased photosensitizer is one way to achieve this goal. Through a systematic and rational approach involving mutations to a LOV2 protein that binds the chromophore flavin mononucleotide (FMN), we have developed a promising photosensitizer that overcomes many of the problems that affect related systems currently in use. Specifically, by decreasing the extent of hydrogen bonding between FMN and a specific amino acid residue in the local protein environment, we decrease the susceptibility of FMN to undesired photoinitiated electron-transfer reactions that kinetically compete with O(2)(a(1)Δ(g)) production. As a consequence, our protein-encased FMN system produces O(2)(a(1)Δ(g)) with the uniquely large quantum efficiency of 0.25 ± 0.03. We have also quantified other key photophysical parameters that characterize this sensitizer system, including unprecedented H(2)O/D(2)O solvent isotope effects on the O(2)(a(1)Δ(g)) formation kinetics and yields. As such, our results facilitate future systematic developments in this field.

Singlet oxygen, O(aΔ), the lowest excited electronic state of molecular oxygen, is an omnipresent part of life on earth. It is readily formed through a variety of chemical and photochemical processes, and its unique reactions are important not just as a tool in chemical syntheses but also in processes that range from polymer degradation to signaling in biological cells. For these reasons, O(aΔ) has been the subject of intense activity in a broad distribution of scientific fields for the past ∼50 years. The characteristic reactions of O(aΔ) kinetically compete with processes that deactivate this excited state to the ground state of oxygen, O(XΣ). Moreover, O(aΔ) is ideally monitored using one of these deactivation channels: O(aΔ) → O(XΣ) phosphorescence at 1270 nm. Thus, there is ample justification to study and control these competing processes, including those mediated by solvents, and the chemistry community has likewise actively tackled this issue. In themselves, the solvent-mediated radiative and nonradiative transitions between the three lowest-lying electronic states of oxygen [O(XΣ), O(aΔ), and O(bΣ)] are relevant to issues at the core of modern chemistry. In the isolated oxygen molecule, these transitions are forbidden by quantum-mechanical selection rules. However, solvent molecules perturb oxygen in such a way as to make these transitions more probable. Most interestingly, the effect of a series of solvents on the O(XΣ)-O(bΣ) transition, for example, can be totally different from the effect of the same series of solvents on the O(XΣ)-O(aΔ) transition. Moreover, a given solvent that appreciably increases the probability of a radiative transition generally does not provide a correspondingly viable pathway for nonradiative energy loss, and vice versa. The ∼50 years of experimental work leading to these conclusions were not easy; spectroscopically monitoring such weak and low-energy transitions in time-resolved experiments is challenging. Consequently, results obtained from different laboratories often were not consistent. In turn, attempts to interpret molecular events were often simplistic and/or misguided. However, over the recent past, increasingly accurate experiments have converged on a base of credible data, finally forming a consistent picture of this system that is resonant with theoretical models. The concepts involved encompass a large fraction of chemistry's fundamental lexicon, e.g., spin-orbit coupling, state mixing, quantum tunneling, electronic-to-vibrational energy transfer, activation barriers, collision complexes, and charge-transfer interactions. In this Account, we provide an explanatory overview of the ways in which a given solvent will perturb the radiative and nonradiative transitions between the O(XΣ), O(aΔ), and O(bΣ) states.

Singlet oxygen, O2(a(1)Δg), the first excited electronic state of molecular oxygen, is an important reactive oxygen species. Its chemistry plays a role in processes ranging from polymer degradation to cell death. Although O2(a(1)Δg) is routinely produced through natural events, including photosensitized processes mediated by organic chromophores, the controlled and selective laboratory production of O2(a(1)Δg) remains a challenge, particularly in biological systems. Here we exploit the fact that ground-state oxygen, O2(X(3)Σg(-)), absorbs 765 nm light to selectively produce O2(b(1)Σg(+)) which, in turn, decays to O2(a(1)Δg). We have quantified this process in different solvents using the time-resolved 1275 nm O2(a(1)Δg) phosphorescence as an optical probe. Most importantly, 765 nm falls in the so-called "biological window", where endogenous chromophores minimally absorb. We show that femtosecond-laser-based, spatially resolved 765 nm irradiation of human tumor cells induces O2(a(1)Δg)-mediated cell death. We thus provide an accessible tool for the controlled sensitizer-free production and study of O2(a(1)Δg) in complex biological systems.

Optogenetics has been, and will continue to be, a boon to mechanistic studies of cellular processes. Genetically encodable proteins that sensitize the production of reactive oxygen species (ROS) are expected to play an increasingly important role, particularly in elucidating mechanisms of temporally and spatially dependent cell signaling. However, a substantial challenge in developing such photosensitizing proteins has been to funnel the optical excitation energy into the initial selective production of only one ROS. Singlet molecular oxygen, O(aΔ), is a ROS known to have a wide range of effects on cell function. Nevertheless, mechanistic details of singlet oxygen's behavior in a cell are lacking. On the basis of the rational optimization of a LOV-derived flavoprotein, we now report the development and photophysical characterization of a protein-encased photosensitizer that efficiently and selectively produces singlet oxygen at the expense of other ROS, especially ROS that derive from photoinduced electron transfer reactions. These results set the stage for a plethora of new experiments to elucidate ROS-mediated events in cells.