Intracellular singlet oxygen photosensitizers: on the road to solving the problems of sensitizer degradation, bleaching and relocalization

Abstract

Selected singlet oxygen photosensitizers have been examined from the perspective of obtaining a molecule that is sufficiently stable under conditions currently employed to study singlet oxygen behavior in single mammalian cells. Reasonable predictions about intracellular sensitizer stability can be made based on solution phase experiments that approximate the intracellular environment (e.g., solutions containing proteins). Nevertheless, attempts to construct a stable sensitizer based solely on the expected reactivity of a given functional group with singlet oxygen are generally not sufficient for experiments in cells; it is difficult to construct a suitable chromophore that is impervious to all of the secondary and/or competing degradative processes that are present in the intracellular environment. On the other hand, prospects are reasonably positive when one considers the use of a sensitizer encapsulated in a specific protein; the local environment of the chromophore is controlled, degradation as a consequence of bimolecular reactions can be mitigated, and genetic engineering can be used to localize the encapsulated sensitizer in a given cellular domain. Also, the option of directly exciting oxygen in sensitizer-free experiments provides a useful complementary tool. These latter systems bode well with respect to obtaining more accurate control of the "dose" of singlet oxygen used to perturb a cell; a parameter that currently limits mechanistic studies of singlet-oxygen-mediated cell signaling.

Oxygen- and pH-Dependent Photophysics of Fluorinated Fluorescein Derivatives: Non-Symmetrical vs. Symmetrical Fluorination

“…However, upon acidification, both monoanionic and neutral fluoresceins can sensitize the production of O 2 (a 1 Δ g ) in greater yield [ 12 , 14 , 37 ]. Moreover, even if produced in only small amounts, O 2 (a 1 Δ g ) can lead to the photooxidative bleaching of the fluorophore [ 6 , 7 ] which, in turn, can result in the production of an efficient O 2 (a 1 Δ g ) sensitizer [ 37 , 38 ]. It is thus incumbent upon us to quantify pH-dependent yields of O 2 (a 1 Δ g ) sensitized by these fluorescein derivatives.…”

“…For this latter exercise, it is useful to quantify not just the rate constant for total removal, k total , but to perform experiments that distinguish the rate of removal via the fluorescein-mediated physical deactivation of O 2 (a 1 Δ g ) to O 2 (X 3 Σ g − ), k phys , from the rate of O 2 (a 1 Δ g ) removal via a chemical reaction with the fluorescein, k chem . Although the photobleaching mechanisms of fluorescein derivatives depend on whether oxygen is present or not [ 3 , 5 , 6 , 7 ], the magnitudes of k phys and k chem are certainly relevant for systems in which O 2 (a 1 Δ g ) is produced, either by the fluorescein derivative itself or by another chromophore in the system.…”

“…In this regard, the light emitted is at a readily detected wavelength (~500 nm), the quantum yields of emission are large (>0.9), the molecules are generally photostable, and they do not sensitize the production of singlet molecular oxygen, O 2 (a 1 Δ g ), in appreciable yield. Although mechanisms of fluorophore photobleaching are often complicated and depend on the molecule’s structure and whether or not molecular oxygen is present in the system, it is acknowledged that O 2 (a 1 Δ g ) can be a key intermediate in this regard [ 3 , 4 , 5 , 6 , 7 ]. Thus, a low yield of sensitized O 2 (a 1 Δ g ) production not only helps mitigate photobleaching, but it also helps to minimize the extent to which the fluorescein perturbs the system in which it is used as a probe by minimizing the O 2 (a 1 Δ g )-mediated oxidative degradation of other molecules.…”

“…In this way, one can use the fluorescein derivative over a wider pH range without a protonation-dependent change in photophysical properties. Fluorination of a chromophore/fluorophore can also result in a molecule that is more stable to photooxidative degradation, partly due to a decrease in the yield of photosensitized O 2 (a 1 Δ g ) production and partly to a decreased reactivity with electrophiles such as O 2 (a 1 Δ g ) [ 7 , 12 , 14 , 19 ].…”

Engineering of relevant photodynamic processes through structural modifications of metallotetrapyrrolic photosensitizers

“…As a consequence, the intracellular localization of the PS greatly determines the site of cellular damage induced via the type I photoreactions, because oxygen-centered radicals are rather promiscuous and not selective: they react within the organelle where they are produced. In contrast, photodamage induced by singlet oxygen is not restricted to one cellular compartment and radius, 1 O 2 can explore all the cell volume and it is a selective oxidant [59,60].…”