Ashwini A. Ghogare

Using Singlet Oxygen to Synthesize Natural Products and Drugs

This Review describes singlet oxygen ((1)O2) in the organic synthesis of targets on possible (1)O2 biosynthetic routes. The visible-light sensitized production of (1)O2 is not only useful for synthesis; it is extremely common in nature. This Review is intended to draw a logical link between flow and batch reactions-a combination that leads to the current state of (1)O2 in synthesis.

Type I and Type II Photosensitized Oxidation Reactions: Guidelines and Mechanistic Pathways†

Here, 10 guidelines are presented for a standardized definition of type I and type II photosensitized oxidation reactions. Because of varied notions of reactions mediated by photosensitizers, a checklist of recommendations is provided for their definitions. Type I and type II photoreactions are oxygen‐dependent and involve unstable species such as the initial formation of radical cation or neutral radicals from the substrates and/or singlet oxygen (1O2 1∆g) by energy transfer to molecular oxygen. In addition, superoxide anion radical ( O2·− ) can be generated by a charge‐transfer reaction involving O2 or more likely indirectly as the result of O2‐mediated oxidation of the radical anion of type I photosensitizers. In subsequent reactions, O2·− may add and/or reduce a few highly oxidizing radicals that arise from the deprotonation of the radical cations of key biological targets. O2·− can also undergo dismutation into H2O2, the precursor of the highly reactive hydroxyl radical ( ·OH ) that may induce delayed oxidation reactions in cells. In the second part, several examples of type I and type II photosensitized oxidation reactions are provided to illustrate the complexity and the diversity of the degradation pathways of mostly relevant biomolecules upon one‐electron oxidation and singlet oxygen reactions.

Photosensitizer Drug Delivery via an Optical Fiber

An optical fiber has been developed with a maneuverable mini-probe tip that sparges O(2) gas and photodetaches pheophorbide (sensitizer) molecules. Singlet oxygen is produced at the probe tip surface which reacts with an alkene spacer group releasing sensitizer upon fragmentation of a dioxetane intermediate. Optimal sensitizer photorelease occurred when the probe tip was loaded with 60 nmol sensitizer, where crowding of the pheophorbide molecules and self-quenching were kept to a minimum. The fiber optic tip delivered pheophorbide molecules and singlet oxygen to discrete locations. The 60 nmol sensitizer was delivered into petrolatum; however, sensitizer release was less efficient in toluene-d(8) (3.6 nmol) where most had remained adsorbed on the probe tip, even after the covalent alkene spacer bond had been broken. The results open the door to a new area of fiber optic-guided sensitizer delivery for the potential photodynamic therapy of hypoxic structures requiring cytotoxic control.

Synthesis and Characterization of Mono-, Di-, and Tri-Poly(ethylene glycol) Chlorin e6 Conjugates for the Photokilling of Human Ovarian Cancer Cells

PEGylated chlorin e(6) photosensitizers were synthesized with tri(ethylene glycol) attached at the ester bond(s) for a 1:1 conjugate at the 17(3)-position, a 2:1 conjugate at the 15(2)- and 17(3)-positions, and a 3:1 conjugate at the 13(1)-, 15(2)-, and 17(3)-positions. These chlorin sensitizers were studied for hydrolytic stability and solubility, as well as ovarian OVCAR-5 cancer cell uptake, localization, and phototoxicity. Increasing numbers of the PEG groups in the mono-, di-, and tri-PEG chlorin conjugates increased the water solubility and sensitivity to hydrolysis and uptake into the ovarian cancer cells. The PEG chlorin conjugates accumulated in the cytoplasm and mitrochondria, but not in lysosomes. Higher phototoxicity was roughly correlated with higher numbers of PEG groups, with the tri-PEG chlorin conjugate showing the best overall ovarian cancer cell photokilling of the series. Singlet oxygen lifetimes, solvent deuteration, and the effects of additives azide ion and d-mannitol were examined to help clarify the photokilling mechanisms. A Type-II (singlet oxygen) photosensitized mechanism is suggested for the di- and tri-PEG chlorin conjugates; however, a more complicated process based in part on a Type-I (radicals or radical ions) mechanism is suggested for the parent chlorin e(6) and the mono-PEG chlorin conjugate.

A Hand-held Fiber-optic Implement for the Site-specific Delivery of Photosensitizer and Singlet Oxygen

We have constructed a fiber optic device that internally flows triplet oxygen and externally produces singlet oxygen, causing a reaction at the (Z)‐1,2‐dialkoxyethene spacer group, freeing a pheophorbide sensitizer upon the fragmentation of a reactive dioxetane intermediate. The device can be operated and sensitizer photorelease observed using absorption and fluorescence spectroscopy. We demonstrate the preference of sensitizer photorelease when the probe tip is in contact with octanol or lipophilic media. A first‐order photocleavage rate constant of 1.13 h−1 was measured in octanol where dye desorption was not accompanied by readsorption. When the probe tip contacts aqueous solution, the photorelease was inefficient because most of the dye adsorbed on the probe tip, even after the covalent ethene spacer bonds have been broken. The observed stability of the free sensitizer in lipophilic media is reasonable even though it is a pyropheophorbide‐a derivative that carries a p‐formylbenzylic alcohol substituent at the carboxylic acid group. In octanol or lipid systems, we found that the dye was not susceptible to hydrolysis to pyropheophorbide‐a, otherwise a pH effect was observed in a binary methanol‐water system (9:1) at pH below 2 or above 8.

Pointsource delivery of a photosensitizer drug and singlet oxygen: eradication of glioma cells in vitro.

We describe a pointsource sensitizer‐tipped microoptic device for the eradication of glioma U87 cells. The device has a mesoporous fluorinated silica tip which emits singlet oxygen molecules and small quantities of pheophorbide sensitizer for additional production of singlet oxygen in the immediate vicinity. The results show that the device surges in sensitizer release and photokilling with higher rates about midway through the reaction. This was attributed to a self‐amplified autocatalytic reaction where released sensitizer in the extracellular matrix provides positive feedback to assist in the release of additional sensitizer. The photokilling of the glioma cells was analyzed by global toxicity and live/dead assays, where a killing radius around the tip with ~0.3 mm precision was achieved. The implication of these results for a new PDT tool of hard‐to‐resect tumors, e.g. in the brain, is discussed.

Photoactive fluoropolymer surfaces that release sensitizer drug molecules.

We describe a physical-organic study of two fluoropolymers bearing a photoreleasable PEGylated photosensitizer that generates (1)O2((1)Δg) [chlorin e6 methoxy tri(ethylene glycol) triester]. The surfaces are Teflon/poly(vinyl alcohol) (PVA) nanocomposite and fluorinated silica. The relative efficiency of these surfaces to photorelease the PEGylated sensitizer [shown previously to be phototoxic to ovarian cancer cells (Kimani, S. et al. J. Org. Chem 2012, 77, 10638)] was slightly higher for the nanocomposite. In the presence of red light and O2, (1)O2 is formed, which cleaves an ethene linkage to liberate the sensitizer in 68-92% yield. The fluoropolymers were designed to deal with multiple problems. Namely, their success relied not only on high O2 solubility and drug repellency but also on the C-F bonds, which physically quench little (1)O2, for singlet oxygens productive use away from the surface. The results obtained here indicate that Teflon-like surfaces have potential uses in delivering sensitizer and singlet oxygen for applications in tissue repair and photodynamic therapy (PDT).

On the in vivo photochemical rate parameters for PDT reactive oxygen species modeling

Photosensitizer photochemical parameters are crucial data in accurate dosimetry for photodynamic therapy (PDT) based on photochemical modeling. Progress has been made in the last few decades in determining the photochemical properties of commonly used photosensitizers (PS), but mostly in solution or in vitro. Recent developments allow for the estimation of some of these photochemical parameters in vivo. This review will cover the currently available in vivo photochemical properties of photosensitizers as well as the techniques for measuring those parameters. Furthermore, photochemical parameters that are independent of environmental factors or are universal for different photosensitizers will be examined. Most photosensitizers discussed in this review are of the type II (singlet oxygen) photooxidation category, although type I photosensitizers that involve other reactive oxygen species (ROS) will be discussed as well. The compilation of these parameters will be essential for ROS modeling of PDT.

Fluorinated Photodynamic Therapy Device Tips and their Resistance to Fouling for In Vivo Sensitizer Release.

We describe progress on a one‐step photodynamic therapy (PDT) technique that is simple: device tip delivery of sensitizer, oxygen and light simultaneously. Control is essential for their delivery to target sites to generate singlet oxygen. One potential problem is the silica device tip may suffer from biomaterial fouling and the pace of sensitizer photorelease is slowed. Here, we have used biomaterial (e.g. proteins, cells, etc.) from SQ20B head and neck tumors and whole blood for an assessment of fouling of the silica tips by adsorption. It was shown that by exchanging the native silica tip for a fluorinated tip, a better nonstick property led to an increased sensitizer output by ~10%. The fluorinated tip gave a sigmoidal photorelease where singlet oxygen is stabilized to physical quenching, whereas the native silica tip with unprotected SiO–H groups gave a slower (pseudolinear) photorelease. A further benefit from fluorinated silica is that 15% less biomaterial adheres to its surface compared to native silica based on a bicinchoninic acid assay (BCA) and X‐ray photoelectron spectroscopy (XPS) measurements. We discuss how the fluorination of the device tip increases biofouling resistance and can contribute to a new pointsource PDT tool.

Theoretical study of the reaction formalhydrazone with singlet oxygen. Fragmentation of the C=N bond, ene reaction and other processes.

Photobiologic and synthetic versatility of hydrazones has not yet been established with 1O2 as a route to commonly encountered nitrosamines. Thus, to determine whether the “parent” reaction of formalhydrazone and 1O2 leads to facile C=N bond cleavage and resulting nitrosamine formation, we have carried out CCSD(T)//DFT calculations and analyzed the energetics of the oxidation pathways. A [2 + 2] pathway occurs via diradicals and formation of 3‐amino‐1,2,3‐dioxazetidine in a 16 kcal/mol−1 process. Reversible addition or physical quenching of 1O2 occurs either on the formalhydrazone carbon for triplet diradicals at 2–3 kcal mol−1, or on the nitrogen (N(3)) atom forming zwitterions at ~15 kcal/mol−1, although the quenching channel by charge‐transfer interaction was not computed. The computations also predict a facile conversion of formalhydrazone and 1O2 to hydroperoxymethyl diazene in a low‐barrier ‘ene’ process, but no 2‐amino‐oxaziridine‐O‐oxide (perepoxide‐like) intermediate was found. A Benson‐like analysis (group increment calculations) on the closed‐shell species are in accord with the quantum chemical results.