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(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.

Optogenetic sensitizers that selectively produce a given reactive oxygen species (ROS) constitute a promising tool for studying cell signaling processes with high levels of spatiotemporal control. However, to harness the full potential of this tool for live cell studies, the photophysics of currently available systems need to be explored further and optimized. Of particular interest in this regard, are the flavoproteins miniSOG and SOPP, both of which (1) contain the chromophore flavin mononucleotide, FMN, in a LOV-derived protein enclosure, and (2) photosensitize the production of singlet oxygen, O(aΔ). Here we present an extensive experimental study of the singlet and triplet state photophysics of FMN in SOPP and miniSOG over a physiologically relevant temperature range. Although changes in temperature only affect the singlet excited state photophysics slightly, the processes that influence the deactivation of the triplet excited state are more sensitive to temperature. Most notably, for both proteins, the rate constant for quenching of FMN by ground state oxygen, O(XΣ), increases ∼10-fold upon increasing the temperature from 10 to 43 °C, while the oxygen-independent channels of triplet state deactivation are less affected. As a consequence, this increase in temperature results in higher yields of O(aΔ) formation for both SOPP and miniSOG. We also show that the quantum yields of O(aΔ) production by both miniSOG and SOPP are mainly limited by the fraction of FMN triplet states quenched by O(XΣ). The results presented herein provide a much-needed quantitative framework that will facilitate the future development of optogenetic ROS sensitizers.

The effect of 16 liquid solvents on both the spectrum and molar absorption coefficient of the XΣ → bΣ transition in molecular oxygen has been examined. The ability to monitor this weak transition using air or oxygen saturated samples at atmospheric pressure was facilitated by the rapid and efficient O(bΣ) → O(aΔ) transition, which allowed the use of O(aΔ) phosphorescence as a sensitive probe of O(bΣ) production. The results of these O(aΔ) phosphorescence experiments are consistent with the results of independent experiments in which the O(aΔ) thus produced was "trapped" via a chemical reaction. The data recorded were used to calculate rate constants for the O(bΣ) → O(XΣ) radiative transition, a parameter that is otherwise difficult to directly obtain from such a wide range of solvents using O(bΣ) → O(XΣ) phosphorescence. The data show that the response of the O(bΣ) → O(XΣ) radiative transition to solvent is not the same as that of the O(bΣ) → O(aΔ) and O(aΔ) → O(XΣ) radiative transitions, both of which have been extensively examined over the years. However, our data are consistent with a theoretical model proposed by Minaev for the effect of solvent on radiative transitions in oxygen and, as such, arguably provide one of the final chapters in describing a system that has challenged the scientific community for years.

Over the last decade, we have investigated and exploited the photophysical properties of triangulenium dyes. Azadioxatriangulenium (ADOTA) and diazaoxatriangulenium (DAOTA), in particular, have features that make them useful in various fluorescence-based technologies (e.g., bioimaging). Through our work with ADOTA and DAOTA, we became aware that the reported fluorescence quantum yields (phi(fl)) for these dyes are lower than their actual values. We thus set out to further investigate the fundamental structure-property relationships in these unique conjugated cationic systems. The nonradiative processes in the systems were explored using transient absorption spectroscopy and time-resolved emission spectroscopy in combination with computational chemistry. The influence of molecular oxygen on the fluorescence properties was explored, and the singlet oxygen sensitization efficiencies of ADOTA and DAOTA were determined. We conclude that, for these dyes, the amount of nonradiative deactivation of the first excited singlet state (S-1) of the azaoxa-triangulenium fluorophores is low, that the rate of such deactivation is slower than what is observed in common cationic dyes, that there are no observable radiative transitions occurring from the first excited triplet state (T-1) of these dyes, and that the efficiency of sensitized singlet oxygen production is low (phi(Delta) <= 10%). These photophysical results provide a solid base upon which technological applications of these fluorescent dyes can be built

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