Dynamics of Oxygen-Independent Photocleavage of Blebbistatin as a One-Photon Blue or Two-Photon Near-Infrared Light-Gated Hydroxyl Radical Photocage

Development of versatile, chemically tunable photocages for photoactivated chemotherapy (PACT) represents an excellent opportunity to address the technical drawbacks of conventional photodynamic therapy (PDT) whose oxygen-dependent nature renders it inadequate in certain therapy contexts such as hypoxic tumors. As an alternative to PDT, oxygen free mechanisms to generate cytotoxic reactive oxygen species (ROS) by visible light cleavable photocages are in demand. Here, we report the detailed mechanisms by which the small molecule blebbistatin acts as a one-photon blue light-gated or two-photon near-infrared light-gated photocage to directly release a hydroxyl radical (•OH) in the absence of oxygen. By using femtosecond transient absorption spectroscopy and chemoselective ROS fluorescent probes, we analyze the dynamics and fate of blebbistatin during photolysis under blue light. Water-dependent photochemistry reveals a critical process of water-assisted protonation and excited state intramolecular proton transfer (ESIPT) that drives the formation of short-lived intermediates, which surprisingly culminates in the release of •OH but not superoxide or singlet oxygen from blebbistatin. CASPT2//CASSCF calculations confirm that hydrogen bonding between water and blebbistatin underpins this process. We further determine that blue light enables blebbistatin to induce mitochondria-dependent apoptosis, an attribute conducive to PACT development. Our work demonstrates blebbistatin as a controllable photocage for •OH generation and provides insight into the potential development of novel PACT agents

Reaction Mechanisms and Structural Characterization of the Reactive Intermediates Observed after the Photolysis of 3-(Hydroxymethyl)benzophenone in Acetonitrile, 2-Propanol, and Neutral and Acidic Aqueous Solutions

Nanosecond time-resolved resonance Raman (ns-TR3) spectroscopy was employed to investigate the photoinduced reactions of 3-(hydroxymethyl)benzophenone (1) in acetonitrile, 2-propanol, and neutral and acidic aqueous solutions. Density functional theory calculations were utilized to help the interpretation of the experimental spectra. In acetonitrile, the neutral triplet state 1 [denoted here as (m-BPOH)3] was observed on the nanosecond to microsecond time scale. In 2-propanol this triplet state appeared to abstract a hydrogen atom from the solvent molecules to produce the aryphenyl ketyl radical of 1 (denoted here as ArPK of 1), and then this species underwent a cross-coupling reaction with the dimethylketyl radical (also formed from the hydrogen abstraction reaction) to form a long-lived light absorbing transient species that was tentatively identified to be mainly 2-(4-(hydroxy(3-(hydroxymethyl)phenyl)methylene)cyclohexa-2,5-dienyl)propan-2-ol. In 1:1 H2O:CH3CN aqueous solution at neutral pH, (m-BPOH)3 reacted with water to produce the ArPK of 1 and then underwent further reaction to produce a long-lived light absorbing transient species. Three photochemical reactions appeared to take place after 266 nm photolysis of 1 in acidic aqueous solutions, a photoreduction reaction, an overall photohydration reaction, and a novel photoredox reaction. TR3 experiments in 1:1 H2O:CH3CN aqueous solution at pH 2 detected a new triplet biradical species, which is associated with an unusual photoredox reaction. This reaction is observed to be the predominant reaction at pH 2 and seems to face competition from the overall photohydration reaction at pH 0

Time-Resolved Spectroscopic and Density Functional Theory Study of the Photochemistry of Irgacure-2959 in an Aqueous Solution

The photocleavage reaction mechanism of 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (Irgacure-2959) was investigated using femtosecond (fs) and nanosecond (ns) transient absorption (-TA) spectroscopy and also picosecond (ps) and nanosecond (ns) time-resolved resonance Raman (-TR³) spectroscopy experiments in a water-rich (volume ratio of acetonitrile/water = 3:7) solution. TA spectroscopy was used to study the dynamics of the benzoyl radical growth and decay as well as to investigate the radical quenching process by the radical scavenger methyl acrylate. ps- and ns-TR³ spectroscopies were employed to monitor the formation of the benzoyl radical and also to characterize its electronic and structural properties. The fs-TA experiments results indicate that the Irgacure-2959 lowest lying excited singlet state S₁ underwent efficient intersystem crossing (ISC) to convert into its triplet state with a time constant of 4 ps. Subsequently, this triplet species dissociated into the benzoyl and alkyl radicals with a corresponding maximum absorption band at 415 nm. The TR³ results in conjunction with results from DFT calculations confirmed that Irgacure-2959 cleaved into the benzoyl and alkyl radicals at a fast rate on the tens of picosecond time scale

Controllable Photocleavage of Blebbistatin Derivatives as Photoremovable Protecting Groups

Blebbistatin was demonstrated as a promising two-photon near-infrared activated photoremovable protecting group of hydroxyl radicals with various potential applications. However, the photocleavage mechanism of the blebbistatin derivatives remains ambiguous. Herein, blebbistatin derivatives with various electronic characteristic leaving groups were synthesized and studied, and the photocleavage mechanism(s) and the tunable effect of the leaving groups were unveiled by combining photoproduct analysis, reactive oxygen radical species detection, femtosecond transient absorption spectroscopy, and density functional theory calculation. More substantial electron-withdrawing leaving groups facilitate heterolysis of the C–O bond, which results in a cationic intermediate and a corresponding remnant. Weaker electron-withdrawing groups lead to a higher proportion of homolysis of the C–O bond, accompanied by the generation of the reactive oxygen radical species. With this structure–property relationship, the protected groups of the molecules of interest can be rationally chosen to satisfy the different requirements needed for specific applications

Controllable Photocleavage of Blebbistatin Derivatives as Photoremovable Protecting Groups

Blebbistatin was demonstrated as a promising two-photon near-infrared activated photoremovable protecting group of hydroxyl radicals with various potential applications. However, the photocleavage mechanism of the blebbistatin derivatives remains ambiguous. Herein, blebbistatin derivatives with various electronic characteristic leaving groups were synthesized and studied, and the photocleavage mechanism(s) and the tunable effect of the leaving groups were unveiled by combining photoproduct analysis, reactive oxygen radical species detection, femtosecond transient absorption spectroscopy, and density functional theory calculation. More substantial electron-withdrawing leaving groups facilitate heterolysis of the C–O bond, which results in a cationic intermediate and a corresponding remnant. Weaker electron-withdrawing groups lead to a higher proportion of homolysis of the C–O bond, accompanied by the generation of the reactive oxygen radical species. With this structure–property relationship, the protected groups of the molecules of interest can be rationally chosen to satisfy the different requirements needed for specific applications

pH Dependent Photodeprotection of Formaldehyde: Homolytic C–C Scission in Acidic Aqueous Solution versus Heterolytic C–C Scission in Basic Aqueous Solution

The photodeprotection of formaldehyde was investigated for 3-(1-hydroxy­propan-2-yl)­benzo­phenone (3-HPBP) with ultrafast time-resolved spectroscopy. The femtosecond transient absorption results indicated the singlet excited state of 3-HPBP transformed efficiently into its triplet state by a fast intersystem crossing. In acidic (pH = 0) and basic (pH = 12.5) aqueous solutions, the triplet intermediate was a key precursor for the deprotection of formaldehyde via two different pathways. However, little photodeprotection was observed in neutral (pH = 7) aqueous solution where the triplet intermediate appeared to undergo a proton coupled electron transfer process to form a ketyl radical transient. The important benzylic biradical intermediates seen in the acidic and basic aqueous solutions were identified by time-resolved resonance Raman spectra whose vibrational frequency patterns were consistent with DFT calculation results for the benzylic biradical intermediate. The results here indicate that the β-carbon alcohol group of the triplet state 3-HPBP is deprotonated in basic aqueous solutions and this leads to a heterolytic C–C bond cleavage to deprotect formaldehyde and produce the benzylic carbanion triplet state species, whereas protonation of the carbonyl moiety of the triplet state 3-HPBP leads to direct generation of a benzylic biradical intermediate and the deprotection of formaldehyde in acidic aqueous solutions via a homolytic C–C bond cleavage

Time-Resolved Resonance Raman Study of the Effect of pH on the Photoreactions of 3-Benzoylpyridine in Aqueous Solution

A nanosecond time-resolved resonance Raman investigation of the photoreactions of 3-benzoylpyridine (3-BPy) in different pH aqueous solutions is reported. In neutral, basic, and pH = 5 aqueous solution conditions, the photoreduction reaction from the triplet 3-BPy species is observed to produce the corresponding 3-phenyl pyridyl ketyl radical that was also observed in a 2-propanol solvent. Under moderate acidic conditions (at pH = 3 for example), most of the 3-BPy triplet state species goes through two protonation steps at the nitrogen atom and the carbonyl oxygen atom after UV laser photolysis and then forms a short-lived hydration intermediate via a hydration reaction at the ortho position in the benzene ring. This new species is tentatively assigned to the o-3[3-BPyH+·H2O] hydration species. In acidic aqueous solutions with a pH ≤ 1, the protonated triplet states of 3-BPy cations at the nitrogen atom are generated from photoexcitation of the protonated ground state and are subsequently further protonated at the carbonyl oxygen atom to form a 3-BPy-dication triplet state. This dication intermediate reacts with water molecules at the ortho position of the benzene ring to produce the o-3[3-BPyH+·H2O] hydration species. The mechanisms of photoreduction observed for 3-BPy in different pH aqueous solutions were investigated using density functional theory calculations, and these results were used to help assign the intermediates observed in the experiments. The structures and properties of these species are briefly discussed, and an overall photoreaction mechanism is proposed based on the results from the time-resolved resonance Raman experiments and the density functional theory calculations

Boosting the Release of Leaving Group from Blebbistatin Derivative Photocages via Enhancing Intramolecular Charge Transfer and Stabilizing Cationic Intermediate

Blebbistatin (Bleb) derivatives are a visible light photocage platform. During the photocleavage process, intramolecular charge transfer (ICT) and cationic intermediates play a decisive role. However, slow photolysis rate and low photolysis quantum yield are the main problems for Bleb’s derivatives. Herein, by introducing a substituted OCH3 group at the para-position of the D ring, Bleb and Bleb derivatives with various leaving groups were synthesized and studied, and the photolysis performance was unveiled by steady-state spectra, photolysis rate experiments, photolysis quantum yield, and density functional theory calculations. Substituted OCH3 derivatives of Bleb may enhance the photolysis rate and increase the photolysis quantum yield because the electron-donating group can promote the ICT process and stabilize the cationic intermediate during the photolytic reaction. More generally, the insights gained from this structure–reactivity relationship may provide theoretical guidance and aid in the development of new highly efficient photoreactions

The Essence in Selectivity of Copper-Mediated Intermolecular Nucleophilic Substitution of a <i>meta</i> C–H Bond in 2‑Methyl‑<i>N</i>‑methoxyaniline: A Theoretical Study

The detailed mechanism for NHC–Cu­(I)-catalyzed intermolecular nucleophilic substitution of the C–H bonds at aniline (2-methyl-N-methoxyaniline) was studied via DFT methods to reveal the essence of the selectivity. Calculations revealed that the meta C–H functionalization proceeds via two nucleophilic attacks on the aromatic ring rather than a one-step meta C–H substitution to give the experimentally observed major product. The reaction is initiated by activation of the substrate via oxidative addition with an NHC–Cu­(I) catalyst, through which an umpolung occurs at the ring. From the activated intermediate, methoxyl group transfer to benzyl forms a resting state, while a nucleophile can attack the ortho position of benzyl to form a more stable intermediate. The nucleophile group can then transfer to the meta position by a 1,2-Wagner–Meerwein rearrangement to form the final product through a proton shuttle. In contrast, other transfer processes affording ortho- or para-substituted products encounter higher activation barriers. This work investigates the relationship of product selectivity with the umpolung of the aromatic ring, as well as the priority of a nucleophilic attack at the ortho position of the aromatic, 1,2-Wagner–Meerwein rearrangement from the ortho-substituted intermediate, and proton shuttle from the meta-substituted intermediate