Photochemistry of N-aryl and N-alkyl dibenzothiophene sulfoximines.

N-phenyl dibenzothiophene sulfoximine has been demonstrated to produce phenyl nitrene and dibenzothiophene S-oxide upon irradiation with UV-A light, and dibenzothiophene S-oxide upon further irradiation releases triplet atomic oxygen. Thus, N-phenyl dibenzothiophene sulfoximine exhibits a rare dual-release capability in its photochemistry. In this work, N-substituted dibenzothiophene sulfoximine derivatives are irradiated with UV-A light to compare their photochemistry and quantum yield of dibenzothiophene S-oxide production with that of N-phenyl dibenzothiophene sulfoximine. Both N-aryl and N-alkyl derivatives of dibenzothiophene sulfoximine are examined to observe their effects on the quantum yield of the photolysis reaction. Adding electron withdrawing N-aryl substituents is shown to increase the quantum yield of dibenzothiophene S-oxide production, while adding electron donating N-aryl substituents is shown to decrease the quantum yield. The quantum yield was slightly lowered or not increased by most N-alkyl substituents. Furthermore, the quantum yield was not augmented by branching and steric hindrance effects associated with the N-alkyl substituents. These results suggest that electronic modulation of the sulfoximine bonds affects the observed photolysis reaction.

Insights into the Mechanism of O(3P)-Mediated Oxidation of Alkenes through Kinetic Isotope Effects and Computational Modeling.

Photodeoxygenation of aryl sulfoxides, such as dibenzothiophene S-oxide (DBTO), produces atomic oxygen [O(3P)] in solution. The mechanism of alkene oxidation with O(3P) remains uncertain. To address this, the current study utilized kinetic isotope effects (KIEs) and computational approaches to study the reaction of O(3P) with styrene and its isotopologues. Notably, the 2° CH/D KIE at the internal and terminal carbons of the reactive π-bond was ∼1.00 and ∼0.87, respectively. These findings indicate a terminal addition of O(3P) to the π-bond, supporting a stepwise oxidation pathway. Both epoxide and aldehyde products go through the same rate-determining transition state and then diverge based on the intermediate conformation. The O-C-C-C dihedral angle (φ) on the triplet surface dictates the product distribution, where φ = 50° or 310° leads to epoxide formation and φ = 180° leads to aldehyde formation. Computational modeling suggests that the epoxide is formed through rapid ring closure upon intersystem crossing from the triplet to the singlet ground state. Similarly, the aldehyde is generated via a 1,2-H shift immediately following intersystem crossing. This study integrates experimental and computational methods to understand the O(3P)-mediated oxidation of alkenes, providing supporting evidence for a stepwise addition mechanism.

Comparison of Low-Density Lipoprotein Oxidation by Hydrophilic O(3 P)-Precursors and Lipid-O(3 P)-Precursor Conjugates.

Lipid oxidation by reactive oxygen species (ROS) provide several different oxidation products that have been implicated in inflammatory responses. Ground state atomic oxygen [O(3 P)] is produced by the photodeoxygenation of certain heterocyclic oxides and has a reactivity that is unique from other ROS. Due to the reactive nature of O(3 P), the site of O(3 P)-generation is expected to influence the products in heterogenous solutions or environments. In this work, the oxidation of low-density lipoprotein (LDL) by lipids with covalently bound O(3 P)-photoprecursors was compared to more hydrophilic O(3 P)-photoprecursors. Lipid oxidation products were quantified after Bligh-Dyer extraction and pentafluorobenzyl bromide (PFB) derivatization by GC-MS. Unlike the more hydrophilic O(3 P)-photoprecursors, the oxidation of LDL during the irradiation of lipid-(O3 P)-photoprecursor conjugates showed little quenching by the addition of the O(3 P)-scavenging sodium allyl sulfonate. This indicated that lipophilic O(3 P)-photoprecursors are expected to generate lipid oxidation products where other more hydrophilic O(3 P)-photoprecursors could be quenched by other reactive groups present in solution or the environment.

Effects of photodeoxygenation on cell biology using dibenzothiophene S-oxide derivatives as O(3P)-precursors.

Photodeoxygenation of dibenzothiophene S-oxide and its derivatives have been used to generate atomic oxygen [O(3P)] to examine its effect on proteins, nucleic acids, and lipids. The unique reactivity and selectivity of O(3P) have shown distinct oxidation products and outcomes in biomolecules and cell-based studies. To understand the scope of its global impact on the cell, we treated MDA-MB-231 cells with 2,8-diacetoxymethyldibenzothiophene S-oxide and UV-A light to produce O(3P) without targeting a specific cell organelle. Cellular responses to O(3P)-release were analyzed using cell viability and cell cycle phase determination assays. Cell death was observed when cells were treated with higher concentrations of sulfoxides and UV-A light. However, significant differences in cell cycle phases due to UV-A irradiation of the sulfoxide were not observed. We further performed RNA-Seq analysis to study the underlying biological processes at play, and while UV-irradiation itself influenced gene expression, there were 9 upregulated and 8 downregulated genes that could be attributed to photodeoxygenation.

Identifying cysteine residues susceptible to oxidation by photoactivatable atomic oxygen precursors using a proteome-wide analysis.

The reactivity profile of atomic oxygen [O(3P)] in the condensed phase has shown a preference for the thiol group of cysteines. In this work, water-soluble O(3P)-precursors were synthesized by adding aromatic burdens and water-soluble sulphonic acid groups to the core structure of dibenzothiophene-S-oxide (DBTO) to study O(3P) reactivity in cell lysates and live cells. The photodeoxygenation of these compounds was investigated using common intermediates, which revealed that an increase in aromatic burdens to the DBTO core structure decreases the total oxidation yield due to competitive photodeoxygenation mechanisms. These derivatives were then tested in cell lysates and live cells to profile changes in cysteine reactivity using the isoTOP-ABPP chemoproteomics platform. The results from this analysis indicated that O(3P) significantly affects cysteine reactivity in the cell. Additionally, O(3P) was found to oxidize cysteines within peptide sequences with leucine and serine conserved at the sites surrounding the oxidized cysteine. O(3P) was also found to least likely oxidize cysteines among membrane proteins.

Photochemistry of N-Phenyl Dibenzothiophene Sulfoximine.

Sulfoximines are popular scaffolds in drug discovery due to their hydrogen bonding properties and chemical stability. In recent years, the role of reactive intermediates such as nitrenes has been studied in the synthesis and degradation of sulfoximines. In this work, the photochemistry of N-phenyl dibenzothiophene sulfoximine [5-(phenylimino)-5H-5λ4 -dibenzo[b,d]thiophene S-oxide] was analyzed. The structure resembles a combination of N-phenyl iminodibenzothiophene and dibenzothiophene S-oxide, which generate nitrene and O(3 P) upon UV-A irradiation, respectively. The photochemistry of N-phenyl dibenzothiophene sulfoximine was explored by monitoring the formation of azobenzene, a photoproduct of triplet nitrene, using direct irradiation and sensitized experiments. The reactivity profile was further studied through direct irradiation experiments in the presence of diethylamine (DEA) as a nucleophile. The studies demonstrated that N-phenyl dibenzothiophene sulfoximine underwent S-N photocleavage to release singlet phenyl nitrene which formed a mixture of azepines in the presence of DEA and generated moderate amounts of azobenzene in the absence of DEA to indicate formation of triplet phenyl nitrene.

Photo-oxidation and Thermal Oxidations of Triptycene Thiols by Aryl Chalcogen Oxides.

Oxidation of thiols yield sulfenic acids, which are very unstable intermediates. As sulfenic acids are reactive, they form disulfides in the presence of thiols. However, sulfenic acids also oxidize to sulfinic acids (-SO2H) and sulfonic acids (-SO3H) at higher concentrations of oxidants. Hydrogen peroxide is a commonly used oxidant for the oxidation of thiols to yield sulfenic acids. However, hydrogen peroxide also oxidizes other reactive functional groups present in a molecule. In this work, the reaction intermediates arising from the oxidation of sterically hindered thiols by aryl chalcogen oxides, dibenzothiophene S-oxide (DBTO), dibenzoselenophene Se-oxide (DBSeO), and dibenzotellurophene Te-oxide (DBTeO), were investigated. Photodeoxygenation of DBTO produces triplet atomic oxygen [O(3P)], which has previously shown to preferentially react with thiols over other functional groups. Similarly, aryl selenoxides have also shown that they can thermally react selectively with thiols at room temperature to yield disulfides. Conversely, aryl telluroxides have been reported to oxidize thiols to disulfides thermally with no selectivity toward thiols. The results from this study demonstrate that sulfenic acids are an intermediate in the oxidation of thiols by DBTeO and by photodeoxygenation of DBTO. The results also showed that the oxidation of thiols by DBSeO yields sulfonic acids. Triptycene-9-thiol and 9-fluorotriptycene-10-thiol were for the thiols used in this oxidation reaction. This work expands the list of oxidants that can be used to oxidize thiols to obtain sulfenic acids.

Synthesis of triphenylphosphonium dibenzothiophene S-oxide derivatives and their effect on cell cycle as photodeoxygenation-based cytotoxic agents.

Photodeoxygenation of Dibenzothiophene-S-oxide (DBTO) in UV-A light produces atomic oxygen [O(3P)] and the corresponding sulfide, dibenzothiophene (DBT). Recently, DBTO has been derivatized to study the effect of UV-A light-driven photodeoxygenation in lipids, proteins, and nucleic acids. In this study, two DBTO derivatives with triphenylphosphonium groups were synthesized to promote mitochondrial accumulation. The sulfone analogs of these derivatives were also synthesized and used as fluorescent mitochondrial dyes to assess localization in mitochondria of HeLa cells. These derivatives were then used to study the effect of photodeoxygenation on MDA-MB-231 breast cancer cell line using cell viability assays, cell cycle phase determination tests, and RNA-Seq analysis. The DBTO derivatives were found to significantly decrease cell viability only after UV-A irradiation as a result of generating corresponding sulfides that were found to significantly affect gene expression and cell cycle.

Dibenzothiophene Sulfone Derivatives as Plasma Membrane Dyes.

Dibenzothiophene 5,5-dioxide (DBTOO) derivatives have recently been shown to processes utility as fluorescent cell dyes. In an effort to extend the functionality of DBTOO-based dyes to include the visualization of cellular membranes, two lipophilic DBTOO were synthesized and their ability to incorporate into the plasma membrane of HeLa cells was examined by fluorescent microscopy. The photophysical properties of the two new DBTOO derivatives were determined and both have good fluorescent quantum yields and a visible blue emission. Due to agreeable wavelengths of excitation and emission, a standard 4',6-diamindino-2-phenylindole (DAPI) filter set worked well with these dyes. After co-staining, it was confirmed that both DBTOO dyes localized in the plasma membrane. The quality of the overlap was quantified using Pearson correlation coefficient, which indicated a strong overlap between the DBTOO dyes and the standard plasma membrane dye. The novel dyes also displayed relatively low toxicity to the HeLa cells with IC50 between 10 and 100 µm. Thus, this work reports a new use of DBTOO derivatives as fluorescent microscopy stains.

In vitro oxidations of low-density lipoprotein and RAW 264.7 cells with lipophilic O(3P)-precursors.

A beneficial property of photogenerated reactive oxygen species (ROS) is the capability of oxidant generation within a specific location or organelle inside a cell. Dibenzothiophene S-oxide (DBTO), which is known to undergo a photodeoxygenation reaction to generate ground state atomic oxygen [O(3P)] upon irradiation, was functionalized to afford localization within the plasma membrane of cells. The photochemistry, as it relates to oxidant generation, was studied and demonstrated that the functionalized DBTO derivatives generated O(3P). Irradiation of these lipophilic O(3P)-precursors in the presence of LDL and within RAW 264.7 cells afforded several oxidized lipid products (oxLP) in the form of aldehydes. The generation of a 2-hexadecenal (2-HDEA) was markedly increased in irradiations where O(3P) was putatively produced. The substantial generation of 2-HDEA is not known to accompany the production of other ROS. These cellular irradiation experiments demonstrate the potential of inducing oxidation with O(3P) in cells.

Evidence for diffusing atomic oxygen uncovered by separating reactants with a semi-permeable nanocapsule barrier.

Ground-state atomic oxygen [O(3P)] is an oxidant whose formation in solution was proposed but never proven. Polymer nanocapsules were used to physically separate dibenzothiophene S-oxide (DBTO), a source of O(3P), from an O(3P)-accepting molecule. Irradiation of polymer nanocapsules loaded with DBTO resulted in oxidation of the O(3P)-acceptor placed outside nanocapsules. The results rule out a direct oxygen atom transfer mechanism and are consistent with freely diffusing O(3P) as the oxidant.

Asymmetric Dibenzothiophene Sulfones as Fluorescent Nuclear Stains.

Asymmetric dibenzothiophene S, S-dioxides (DBTOOs) were synthesized and their photophysical properties examined. Through examination, the molecules fluoresced at wavelengths between 371 and 492 nm with quantum yields of fluorescence nearing 0.59. Three of the sulfonic acid sodium salt analogues were chosen to be introduced to HeLa cells, resulting in illumination of the nucleus by fluorescent microscopy. These compounds function as nuclear stains while also affording the ability to predict the localization of the corresponding sulfoxide precursor to ground-state atomic oxygen.

Thiol Reactivity toward Atomic Oxygen Generated during the Photodeoxygenation of Dibenzothiophene S-Oxide.

Aromatic heterocyclic oxides, such as dibenzothiophene S-oxide (DBTO), have been suggested to release ground state atomic oxygen [O(3P)] upon irradiation, and as such, they have been used to create a condensed phase reactivity profile for O(3P). However, thiols, which are highly reactive with O(3P) in the gas phase, were not previously investigated. An earlier study of O(3P) with proteins in solution indicated a preference for thiols. A further investigation of the apparent thiophilicity provided the subject for this study. DBTO was employed as a putative O(3P)-precursor. However, the effective rate of O(3P) formation was found to be dependent on reactant concentrations in certain cases. All reactants were found to increase the rate of deoxygenation to some extent, but in the presence of reactants containing an alcohol linked to a reactive functional group, deoxygenation occurred substantially more rapidly. The rate enhancement was quantified and attributed to the reaction of activated O atom within the solvent cage prior to escape into the bulk solution. Through competition experiments, the relative rate constants of O(3P) with thiols and other functional groups were found. A small preference for primary thiols was observed over other thiols, sulfides, and alkenes. A much larger preference was observed for thiols, sulfides, and alkenes over aromatic groups. In summary, DBTO was successfully used as an O(3P)-precursor, and the thiophilicity of O(3P) was confirmed and quantified.

Oxidation of Plasmalogen, Low-Density Lipoprotein and RAW 264.7 Cells by Photoactivatable Atomic Oxygen Precursors.

The oxidation of lipids by endogenous or environmental reactive oxygen species (ROS) generates a myriad of different lipid oxidation products that have important roles in disease pathology. The lipid oxidation products obtained in these reactions are dependent upon the identity of the reacting ROS. The photoinduced deoxygenation of various aromatic heterocyclic oxides has been suggested to generate ground state atomic oxygen (O[3P]) as an oxidant; however, very little is known about reactions between lipids and O(3P). To identify lipid oxidation products arising from the reaction of lipids with O(3P), photoactivatable precursors of O(3P) were irradiated in the presence of lysoplasmenylcholine, low-density lipoprotein and RAW 264.7 cells under aerobic and anaerobic conditions. Four different aldehyde products consistent with the oxidation of plasmalogens were observed. The four aldehydes were: tetradecanal, pentadecanal, 2-hexadecenal and hexadecanal. Depending upon the conditions, either pentadecanal or 2-hexadecenal was the major product. Increased amounts of the aldehyde products were observed in aerobic conditions.

Redox switching of adenosine-5'-phosphosulfate kinase with photoactivatable atomic oxygen precursors.

Adenosine-5'-phosphosulfate kinase (APSK) catalyzes the phosphorylation of adenosine-5'-phospho-sulfate (APS) to 3'-phospho-APS (PAPS). In plants, this enzymatic activity is biochemically regulated through an intersubunit disulfide bond between Cys86 and Cys119 in the N-terminal loop of APSK. To examine if O((3)P) generated by the photodeoxygenation of 2,8-dihydroxymethyldibenzothiophene S-oxide could specifically oxidize APSK at its regulatory site, APSK was irradiated in the presence of 2,8-dihydroxymethyldibenzothiophene S-oxide. Near-quantitative alteration of APSK from the enzymatically active monomeric form to the inhibited dimeric form was achieved. The photoinduced increase of dimeric APSK was strongly implicated to arise from the formation of the Cys86-Cys119 disulfide bond.

Comparison of experimental and computationally predicted sulfoxide bond dissociation enthalpies.

The accurate estimation of S-O bond dissociation enthalpies (BDE) of sulfoxides by computational chemistry methods has been a significant challenge. One of the primary causes for this challenge is the well-established requirement of including high-exponent d functions in the sulfur basis set for accurate energies. Unfortunately, even when high-exponent d functions were included in Pople-style basis sets, the relative strength of experimentally determined S-O BDE was incorrectly predicted. The aug-cc-pV(n+d)Z basis sets developed by Dunning include an additional high-exponent d function on sulfur. Thus, it was expected that the aug-cc-pV(n+d)Z basis sets would improve the prediction of sulfoxide S-O BDE. This study presents the S-O BDE predicted by B3LYP, CCSD, CCSD(T), M05-2X, M06-2X, and MP2 combined with aug-cc-pV(n+d)Z, aug-cc-pVnZ, and Pople-style basis sets. The accuracy of these predictions was determined by comparing the computationally predicted values to the experimentally determined S-O BDE. Values within experimental error were obtained for dialkyl sulfoxides when the S-O BDEs were estimated using an isodesmic oxygen transfer reaction at the M06-2X/aug-cc-pV(T+d)Z level of theory. However, the S-O BDE of divinyl sulfoxide was overestimated by this method.

Ultrafast spectroscopy and computational study of the photochemistry of diphenylphosphoryl azide: direct spectroscopic observation of a singlet phosphorylnitrene.

The photochemistry of diphenylphosphoryl azide was studied by femtosecond transient absorption spectroscopy, by chemical analysis of light-induced reaction products, and by RI-CC2/TZVP and TD-B3LYP/TZVP computational methods. Theoretical methods predicted two possible mechanisms for singlet diphenylphosphorylnitrene formation from the photoexcited phosphoryl azide. (i) Energy transfer from the (π,π*) singlet excited state, localized on a phenyl ring, to the azide moiety, thereby leading to the formation of the singlet excited azide, which subsequently loses molecular nitrogen to form the singlet diphenylphosphorylnitrene. (ii) Direct irradiation of the azide moiety to form an excited singlet state of the azide, which in turn loses molecular nitrogen to form the singlet diphenylphosphorylnitrene. Two transient species were observed upon ultrafast photolysis (260 nm) of diphenylphosphoryl azide. The first transient absorption, centered at 430 nm (lifetime (τ) ∼ 28 ps), was assigned to a (π,π*) singlet S(1) excited state localized on a phenyl ring, and the second transient observed at 525 nm (τ ∼ 480 ps) was assigned to singlet diphenylphosphorylnitrene. Experimental and computational results obtained from the study of diphenyl phosphoramidate, along with the results obtained with diphenylphosphoryl azide, supported the mechanism of energy transfer from the singlet excited phenyl ring to the azide moiety, followed by nitrogen extrusion to form the singlet phosphorylnitrene. Ultrafast time-resolved studies performed on diphenylphosphoryl azide with the singlet nitrene quencher, tris(trimethylsilyl)silane, confirmed the spectroscopic assignment of singlet diphenylphosphorylnitrene to the 525 nm absorption band.

Computational investigation of the reaction mechanisms of nitroxyl and thiols.

Nitroxyl, or nitrosyl hydride, (HNO) is a pharmacologically relevant molecule whose physiological responses have been thought to result from modification of intracellular thiols. The reaction of HNO with thiols has been shown to lead to disulfides and sulfinamides. The free energies of reaction (DeltaG) and activation (DeltaG(++)) were determined for the reaction pathways of HNO and five different thiols using computational methods. The methods employed included B3LYP, MP2, and CBS-QB3, as well as IEF-PCM to approximate implicit water solvation. The five examined thiols were hydrogen sulfide, methanethiol, trifluoromethanethiol, thiophenol, and cysteine. A putative N-hydroxysulfenamide intermediate was the initial product for the reaction of HNO with a thiol. Analysis of the Wiberg bond indices indicated that the formation of the S-N bond was concerted with the proton transfers that led to the intermediate. The calculated pK(a) of protonated N-hydroxysulfenamide was approximately 13, and from the protonated N-hydroxysulfenamide intermediate, two irreversible reactions that lead to either the disulfide or sulfinamide were found. The calculated values of DeltaG(++) indicated the preferred reaction pathway would be dependent upon the hydrophobicity of the environment, the availability of a local base, and the identity of the thiol substituent. In a hydrophobic environment, the formation of the disulfide was kinetically favored. Formation of the sulfinamide product was expected to occur upon the protonation of the hydroxy group of the N-hydroxysulfenamide intermediate.

Photodeoxygenation of dibenzothiophene S-oxide derivatives in aqueous media.

The use of atomic oxygen (O((3)P)) as potent oxidant in water has suffered from the lack of a facile, efficient source. The photodeoxygenation of aromatic sulfoxides to the corresponding sulfides in organic solvents has been suggested to produce O((3)P) in low quantum yields. The photolysis of 4,6-dihydroxymethyldibenzothiophene S-oxide and 2,8-dihydroxymethyldibenzothiophene S-oxide in water results in deoxygenation at significantly higher quantum yields than in organic solvents. Depending upon conditions, a variable amount of oxidation of the hydroxymethyl substituent into an aldehyde was observed to accompany deoxygenation. Analysis of the photoproducts indicated the deoxygenation occurred by at least two different pH-sensitive mechanisms. Under basic conditions, photoinduced electron transfer yielding a hydroxysulfuranyl radical that decomposed by heterolytic S-O cleavage was thermodynamically feasible. The thermodynamics of photoinduced electron transfer were expected to become increasingly unfavorable as the pH of the solution decreased. Thus, at neutral and acidic pH, an S-O bond scission mechanism was suspected. The observed increase in the photodeoxygenation quantum yields was consistent with charge separation accompanying S-O bond scission. Oxidative cleavage of alkenes in aerobic conditions suggested O((3)P) was produced during photolysis in these conditions; however, the formation of discrete O(*-)/HO(*) may occur, particularly at low pH.

Computational study of the Curtius-like rearrangements of phosphoryl, phosphinyl, and phosphinoyl azides and their corresponding nitrenes.

The free energies of reaction (DeltaG) and activation (DeltaG) were determined for the Curtius-like rearrangement of dimethylphosphinoyl, dimethylphosphinyl, and dimethylphosphoryl azides as well as the corresponding singlet and triplet nitrenes by CBS-QB3 and B3LYP computational methods. From CASSCF calculations, it was established that the closed-shell configuration was the lower energy singlet state for each of these nitrenes. The triplet states of dimethylphosphinyl- and dimethylphosphorylnitrene are the preferred ground states. However, the closed-shell singlet state is the ground state for dimethylphosphinoylnitrene. The CBS-QB3 DeltaG values for the Curtius-like rearrangements of dimethylphosphinyl and dimethylphosphoryl azides were 45.4 and 47.0 kcal mol-1, respectively. For the closed-shell singlet dimethylphosphinyl- and dimethylphosphorylnitrene, the CBS-QB3 DeltaG values for the rate-limiting step of the Curtius-like rearrangement were 22.9 and 18.0 kcal mol-1, respectively. It is unlikely that the nitrenes will undergo a Curtius-like rearrangement because of competing bimolecular reactions that have lower activation barriers. The pharmacology of weaponized organophosphorus compounds can be investigated using phosphorylnitrenes as photoaffinity labels. Dominant bimolecular reactivity is a desirable quality for a photoaffinity label to possess, and thus, the resistance of phosphorylnitrenes to intramolecular Curtius-like rearrangements increases their usefulness as photoaffinity labels.