Dioxetane Intermediates Research Articles

Activatable Sonoafterglow Nanoprobes for T cell Imaging.

Real-time imaging of immune system benefits early diagnosis of disease and precision immunotherapy; however, most existing imaging probes either have "always-on" signals with poor correlation to immune responses, or rely on light excitation with limited imaging depth. Herein, an ultrasound-induced afterglow (sonoafterglow) nanoprobe is developed to specifically detect granzyme B for accurate imaging of T cell immunoactivation in vivo. The sonoafterglow nanoprobe (Q-SNAP) consists of sonosensitizers, afterglow substrates, and quenchers. Upon ultrasound irradiation, sonosensitizers generate singlet oxygen, which converts substrates to high-energy dioxetane intermediates that slowly release energy after ultrasound cessation. Due to the proximity, energy from substrates can be transferred to quenchers, leading to afterglow quenching. Only in the presence of granzyme B, quenchers are liberated from Q-SNAP, resulting in bright afterglow emission with an LOD (2.1nM) much lower than most existing fluorescent probes. Due to the deep tissue-penetrating ultrasound, sonoafterglow can be induced through tissue of 4cm thickness. Based on the correlation between sonoafterglow and granzyme B, Q-SNAP not only distinguishes autoimmune hepatitis from healthy liver as early as 4 hours after probe injection, but also effectively monitoring the cyclosporin A-mediated reversal of T cell hyperactivation. Q-SNAP thus offers the possibilities for dynamic monitoring of T cell dysfunction and evaluation of prophylactic immunotherapy in deep-seated lesions. This article is protected by copyright. All rights reserved.

1O2-Relevant Afterglow Luminescence of Chlorin Nanoparticles for Discriminative Detection and Isotopic Analysis of H2O and D2O

Discriminative detection between D2O and H2O is important for diverse fields but challenging due to their high similarity in chemical and physical properties. Current molecular sensors for D2O detection generally rely on the spectral change of fluorophores with suitable pKa in response to D2O and H2O with slightly different pH acidity. Herein, we report a new and facile D2O sensor by using singlet oxygen (1O2)-relevant afterglow luminescence of chlorin e4 nanoparticles (Ce4-NPs) to achieve distinguishable detection between D2O and H2O. As 1O2 is a key initiator involved in the afterglow luminescence process, it displays a 22-fold longer lifetime in D2O relative to H2O and thereafter generates more dioxetane intermediates after laser irradiation to lead to ultimate afterglow brightness of Ce4-NPs in D2O. In addition, Ce4-NPs are capable of quantitatively detecting the amount of H2O in D2O with a limit of detection (LOD) of 1.45%. Together, this study broadens the utility of afterglow materials and presents a facile strategy for isotopic purity analysis of heavy water.

Silyl Tether-Assisted Photooxygenation of Electron-Deficient Enaminoesters: Direct Access to Oxamate Formation.

Photooxygenation reactions of electron-deficient enaminoesters bearing an oxophilic silyl tether at the α-position of the nitrogen atom using methylene blue (MB) were explored to develop a mild and efficient photochemical strategy for oxidative C-C double bond cleavage reactions via singlet oxygen (1O2). Photochemically generated 1O2, through energy transfer from the triplet excited state of MB (3MB*) to molecular oxygen (3O2), was added across a C-C double bond moiety of enaminoesters to form perepoxides, which rearranged to form dioxetane intermediates. The cycloreversion of the formed dioxetane via both C-C and O-O bond cleavage processes led to the formation of oxamates. Importantly, contrary to alkyl group tether-substituted electron-deficient enaminoesters that typically disfavor photooxygenation, the silyl tether-substituted analogues undergo this photochemical transformation efficiently with the assistance of a silyl tether, which facilitates formation of the perepoxide. The observations in this study provide useful information about photosensitized oxygenation reactions of unsaturated C-C bonds, and, moreover, this photochemical strategy can be utilized as a mild and feasible method for the preparation of diversely functionalized carbonyl compounds including oxamates.

Theoretical Study of the Thermolysis Reaction and Chemiexcitation of Coelenterazine Dioxetanes.

Coelenterazine and other imidazopyrazinones are important bioluminescent substrates widespread in marine species and can be found in eight phyla of luminescent organisms. Light emission from these systems is caused by the formation and subsequent thermolysis of a dioxetanone intermediate, whose decomposition allows for efficient chemiexcitation to singlet excited states. Interestingly, some studies have also reported the involvement of unexpected dioxetane intermediates in the chemi- and bioluminescent reactions of Coelenterazine, albeit with little information on the underlying mechanisms of these new species. Herein, we have employed a theoretical approach based on density functional theory to study for the first time the thermolysis reaction and chemiexcitation profile of two Coelenterazine dioxetanes. We have found that the thermolysis reactions of these species are feasible but with relevant energetic differences. More importantly, we found that the singlet chemiexcitation profiles of these dioxetanes are significantly less efficient than the corresponding dioxetanones. Furthermore, we identified triplet chemiexcitation pathways for the Coelenterazine dioxetanes. Given this, the chemiexcitation of these dioxetanes should lead only to minimal luminescence. Thus, our theoretical investigation of these systems indicates that the thermolysis of these dioxetanes should only provide "dark" pathways for the formation of nonluminescent degradation products of the chemi- and bioluminescent reactions of Coelenterazine and other imidazopyrazinones.

Unraveling the mechanism of latex clearing protein Lcp<sub>K30</sub> catalysed oxidative cleavage of poly(<italic>cis</italic>-1,4-isoprene)

<p indent="0mm">Natural rubber or synthetic rubber materials have been extensively used in our daily life and industrial production for more than <sc>100 years</sc> due to their unique physical properties. However, a amount of rubber waste has been released into the environment, posing great challenges to the disposal of these polymers. In contrast to the disposal strategies such as combustion or stockpiling, enzyme catalyzed rubber degradation constitutes an environmentally friendly strategy. Latex clearing protein (Lcp) is an iron-heme containing endotype dioxygenase that catalyses the oxidative cleavage of the chemically inert poly(<italic>cis</italic>-1,4-isoprene) which is the main component of rubber, affording the products of aldehyde and ketone. It was found that active site E148 residue is key to the reactivity of Lcp based on the mutation experiments. However, the role of E148 and the overall mechanism are still controversial. It is generally accepted that both of dioxygen atoms are incorporated into the substrate. In previous studies, two possible mechanisms of Lcp catalyzed cleavage of poly(<italic>cis</italic>-1,4-isoprene) are proposed. In the first one, the distal oxygen atom of Fe(III)−O₂⁻ complex attacks onto the C=C double bond, leading to a three-membered epoxide intermediate. This is followed by the attack of Fe=O complex on the epoxide intermediate and subsequent C−C bond cleavage, affording the final ketone and aldehyde products. While in the second one, the proximal oxygen atom of Fe(III)−O₂⁻ complex attacks onto the C=C double bond of the substrate, which is followed by the attack of the distal oxygen atom onto the substrate radical to generate a four-membered cyclic dioxetane intermediate. The final O−O as and C−C bond cleavage in the dioxetane intermediate would afford the final products. In this study, based on the crystal structure of Lcp_K30, the combined molecular dynamic (MD) simulations and quantum mechanics/molecular mechanics (QM/MM) calculations are performed to elucidate the catalytic mechanism of Lcp catalysed cleavage of central double bond of poly(<italic>cis</italic>-1,4-isoprene). Our study shows E148 is protonated during the reaction and forms stable H bond interactions with Fe(III)−O₂⁻ species. Thus, the plausible role of E148 is related to the positioning of both Fe(III)−O₂⁻ species and the substrate. Moreover, it can be ruled out that the E148 acts as a base to abstract the proton from the substrate. The proposed mechanism of Lcp works as follows. Firstly, the distal oxygen of Fe(III)−O₂⁻ attacks onto the carbon−carbon double bond to generate the peroxide alky radical intermediate. This is followed by the rebound of the proximal oxygen to the carbon radical to generate the dioxetane intermediate, with Fe(III) being reduced to Fe(II). The following O−O bond cleavage occurs via the Fe(II)-mediated reductive cleavage of O−O bond of the dioxetane intermediate, leading to the vicinal diol radical anion intermediate. Finally, a facile C−C cleavage is undergone to yield the ketone and aldehyde products. In particular, our calculations show that the direct O−O cleavage of the dioxetane intermediate in the absence of Fe(II) catalysis requires a remarkably high barrier of <sc>160 kJ/mol,</sc> indicating the Fe(II) catalysis is vital for the further transformation of the dioxetane intermediate. For comparison, we also investigated the previously proposed mechanism involving the three-membered epoxide intermediate. However, the attack of the epoxide by Fe^IV=O complex is found to be highly unfavourable thermodynamically and thus can be ruled out. As such, this study may not only expand our understanding of the catalytic mechanism of Lcp_K30, but also provide useful information for rational design or engineering of Lcp enzyme toward the degradation of synthetic rubber materials.

How Tryptophan Oxidation Arises by "Dark" Photoreactions from Chemiexcited Triplet Acetone.

Dioxetane intermediates readily decompose to chemiluminescent triplet carbonyls, giving rise to what has been paradoxically called photochemistry in the dark. In this issue of Photochemistry and Photobiology, Bechara et al. report on mechanistic advances in such a reaction. With the use of horseradish peroxidase for isobutyraldehyde-derived triplet acetone, light emission from acetone and singlet oxygen can be quenched. The experiments reveal that the reaction depends on oxygen and the amino acid. The analysis reveals that free tryptophan is a target of this form of "carbonyl stress," with the efficient formation of mono-, bi- and tricyclic compounds (N-formylkynurenine, indoline, 1λ2 -indole and 3H-indoles).

Mechanical Insights into the Enzymatic Cleavage of Double C-C Bond in Poly(cis-1,4-isoprene) by the Latex Clearing Protein.

The b-type cytochrome LcpK30 is a latex clearing protein (Lcp), which acts as an endotype dioxygenase to catalyze the extracellular cleavage of the chemically inert aliphatic polymer poly(cis-1,4-isoprene), producing oligo-isoprenoids with different terminal carbonyl groups (aldehyde and ketone, -CH2-CHO and -CH2-COCH3). On the basis of the fact that the muteins of E148A, E148Q, and E148H have substantially reduced reactivity, and the E148-initiated reaction mechanism has been previously proposed, in which a cyclic dioxetane intermediate or an epoxide intermediate may be involved, however, open questions still remain. In this paper, on the basis of the crystal structure of LcpK30, the enzyme-substrate reactant model was constructed, and the cleavage mechanism of the central double bond of poly(cis-1,4-isoprene) was elucidated by performing quantum mechanics/molecular mechanics calculations. Our calculation results revealed that the oxidative cleavage reaction is triggered by the addition of the heme-bound dioxygen to the double bond of the polymer, and E148 does not act as the catalytic base to extract the allylic proton to assist the reaction as previously suggested. Of the two considered pathways, the pathway that involves the dioxetane intermediate was calculated to be more favorable. During the catalysis, the distal oxygen first adds to the double bond of the substrate to form a radical intermediate, and then the Fe-O1 (proximal oxygen) bond cleaves to generate the dioxetane intermediate, which can easily collapse affording the final ketone and aldehyde products. In general, the cleavage mechanism of double C-C bond catalyzed by LcpK30 is similar to those of indoleamine 2,3-dioxygenase, tryptophan 2,3-dioxygenase, and the nonheme stilbene cleavage oxygenase NOV1 that all depend on the iron-bound dioxygen to initiate the cleavage reaction.

P23 - Singlet molecular oxygen generated in dark biological process

Ultraweak chemiluminescence arising from biomolecules oxidation has been attributed to the radiative deactivation of singlet molecular oxygen [1O2] and electronically excited triplet carbonyl products involving dioxetane intermediates. As examples, we will discuss the generation of 1O2 from lipid hydroperoxides, which involves a cyclic mechanism from a linear tetraoxide intermediate. The generation of 1O2 in aqueous solution via energy transfer from the excited triplet acetone arising from the thermodecomposition of dioxetane a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source, will also be discussed. The approach used to unequivocally demonstrate the generation of 1O2 in these reactions is the use of 18O-labeled hydroperoxide / triplet dioxygen (18[3O2]), the detection of labeled compounds by HPLC coupled to tandem mass spectrometry (HPLC-MS/MS) and the direct spectroscopic detection and characterization of 1O2 light emission. Characteristic light emission at 1,270nm, corresponding to the singlet delta state monomolecular decay was observed. Using18[3O2], we observed the formation of 18O-labeled 1O2 (18[1O2]) by the chemical trapping of 18[1O2]with the anthracene-9,10-diyldiethane-2,1-diyl disulfate disodium salt (EAS) and detected the corresponding 18O-labeled EAS endoperoxide usingHPLC-MS/MS. The combined use of the thermolysis of a water-soluble naphthalene endoperoxide as a generator of 18O labeled 1O2 and the sensitivity of HPLC-MS/MS allowed the study of 1O2reactivity toward biomolecules. Photoemission properties and chemical trapping clearly demonstrate that the production of hydroperoxide and excited carbonyls generates 18[1O2], and points to the involvement of 1O2 in physiological and pathophysiological mechanism.Supported by FAPESP (2012/12663-1), CAPES, INCT Redoxoma (FAPESP/CNPq/CAPES; 573530/2008-4), NAP Redoxoma (PRPUSP; 2011.1.9352.1.8), CEPID Redoxoma (FAPESP; 2013/07937-8).

Excited singlet molecular O₂(¹Δg) is generated enzymatically from excited carbonyls in the dark.

In mammalian tissues, ultraweak chemiluminescence arising from biomolecule oxidation has been attributed to the radiative deactivation of singlet molecular oxygen [O2 (1Δg)] and electronically excited triplet carbonyl products involving dioxetane intermediates. Herein, we describe evidence of the generation of O2 (1Δg) in aqueous solution via energy transfer from excited triplet acetone. This involves thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane, a chemical source, and horseradish peroxidase-catalyzed oxidation of 2-methylpropanal, as an enzymatic source. Both sources of excited carbonyls showed characteristic light emission at 1,270 nm, directly indicative of the monomolecular decay of O2 (1Δg). Indirect analysis of O2 (1Δg) by electron paramagnetic resonance using the chemical trap 2,2,6,6-tetramethylpiperidine showed the formation of 2,2,6,6-tetramethylpiperidine-1-oxyl. Using [18O]-labeled triplet, ground state molecular oxygen [18O2 (3Σg-)], chemical trapping of 18O2 (1Δg) with disodium salt of anthracene-9,10-diyldiethane-2,1-diyl disulfate yielding the corresponding double-[18O]-labeled 9,10-endoperoxide, was detected through mass spectrometry. This corroborates formation of O2 (1Δg). Altogether, photoemission and chemical trapping studies clearly demonstrate that chemically and enzymatically nascent excited carbonyl generates 18O2 (1Δg) by triplet-triplet energy transfer to ground state oxygen O2 (3Σg−), and supports the long formulated hypothesis of O2 (1Δg) involvement in physiological and pathophysiological events that might take place in tissues in the absence of light.

Ab initio calculations on the (1)O2 quenching mechanism by trans-resveratrol.

trans-Resveratrol has shown to play an important role in a variety of biological and medical processes such as reactive oxygen species (ROS) scavenging, inhibition of apoptosis and induction of cell survival. In light of the fact that resveratrol and its oligomers were found to be selective singlet oxygen (1)O2 quenchers, we report here a systematic study on the reactivity of trans-resveratrol toward molecular oxygen in acetone simulated media. On the basis of the controversial hypotheses reported in the literature we explored, at density functional levels of theory, two different mechanisms. The first one leads to a resveratrol quinone product via an endoperoxide intermediate by attack of (1)O2 on the resorcinol ring, assisted (pathway (b)) or not (pathway (a)) by a water molecule. The second mechanism, in which the singlet oxygen reacts with the double bond connecting the two resveratrol rings leading to benzaldehyde products, involves the formation of a dioxetane intermediate. As the outcomes of our computational analysis show that the latter mechanism is kinetically more favorable than the former one, it is likely that when trans-resveratrol reacts with singlet oxygen a dioxetane intermediate is formed.

The Chemiexcitation of the Chemiluminescence of Lophine Peroxide Anions via a Partially Cyclic Transition State

AbstractIt was shown that acyclic intermediates play a role in the chemiexcitation of the chemiluminescence (CL) of lophine peroxides in addition to the dioxetane intermediates. Because the CL efficiencies of position‐isomers (R)‐9 and (R)‐10, which theoretically give the common dioxetane intermediate, were different, the different CL efficiencies are attributable to the CL mechanism involving a partially cyclic transition structure at the chemiexcitation step.

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.

Density Functional Theory Study on a Missing Piece in Understanding of Heme Chemistry: The Reaction Mechanism for Indoleamine 2,3-Dioxygenase and Tryptophan 2,3-Dioxygenase

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are heme-containing dioxygenases and catalyze oxidative cleavage of the pyrrole ring of L-tryptophan. On the basis of three recent crystal structures of these heme-containing dioxygenases, two new mechanistic pathways were proposed by several groups. Both pathways start with electrophilic addition of the Fe(II)-bound dioxygen concerted with proton transfer (oxygen ene-type reaction), followed by either formation of a dioxetane intermediate or Criegee-type rearrangement. However, density functional theory (DFT) calculations do not support the proposed concerted oxygen ene-type and Criegee-type rearrangement pathways. On the basis of DFT calculations, we propose a new mechanism for dioxygen activation in these heme systems. The mechanism involves (a) direct electrophilic addition of the Fe(II)-bound oxygen to the C2 or C3 position of the indole in a closed-shell singlet state or (b) direct radical addition of the Fe(III)-superoxide to the C2 position of the indole in a triplet (or open-shell singlet) state. Then, a radical-recombination or nearly barrierless charge-recombination step from the resultant diradical or zwitterionic intermediates, respectively, proceeds to afford metastable dioxetane intermediates, followed by ring-opening of the dioxetanes. Alternatively, homolytic O-O bond cleavage from the diradical intermediate followed by oxo attack and facile C2-C3 bond cleavage could compete with the dioxetane formation pathway. Effects of ionization of the imidazole and negatively charged oxyporphyrin complex on the key dioxygen activation process are also studied.

Peroxynitrite-Initiated Oxidation of Acetoacetate and 2-Methylacetoacetate Esters by Oxygen: Potential Sources of Reactive Intermediates in Keto Acidoses

Oxidative stress is believed to play a role in the pathogenesis of several diseases, including diabetes and inborn errors of metabolism. The types of oxidative damage observed in these pathologies have been attributed to the excessive production of reactive intermediates relating to the accumulation of toxic metabolites. The production of extremely oxidizing peroxynitrite can also be high in these pathologies. We study here the oxidation initiated by peroxynitrite of the ethyl esters of acetoacetate (EAA) and 2-methylacetoacetate (EMAA), metabolites that accumulate in diabetes and isoleucinemia, respectively. Oxygen consumption studies have confirmed that peroxynitrite promotes the aerobic oxidation of EAA and EMAA in phosphate buffer. These reactions were accompanied by ultraweak light emission, which probably arises from triplet carbonyl products formed by thermolysis of dioxetane intermediates. The kinetics of oxygen uptake and chemiluminescence by EAA and EMAA was strongly affected by the phosphate ion, known to catalyze carbonyl enolization and nucleophilic additions to carbonyls. The reaction pH profiles obtained by oxygen consumption and chemiluminescence measurements indicated that the peroxynitrite anion was the initiator of EAA and EMAA aerobic oxidation. EPR spin-trapping studies with the spin traps 3,5-dibromo-4-nitrosobenzenesulfonic acid and 2-methyl-2-nitrosopropane showed the intermediacy of methyl and a carbon-centered radical (*CH2COR) in the oxidation of EAA by peroxynitrite. In the case of EMAA, a tertiary carbon-centered radical (*EMAA) and an acyl radical were detected, the latter probably resulting from the cleavage of a triplet carbonyl product. Superstoichiometric formation of acetate from both substrates confirmed the occurrence of oxygen-dependent chain reactions, here proposed to be initiated by one-electron abstraction from the enolic form of the substrates. The free radicals and electronically excited species generated in the oxidation of EAA and EMAA may help shed further light on the molecular basis of these diseases.

Oxidative Fragmentation of Hydroxy Octadecadienoates Generates Biologically Active γ-Hydroxyalkenals

Oxidative fragmentation of polyunsaturated fatty acids (PUFAs) in vivo generates cytotoxic aldehydes. Among these, 4-hydroxynon-2-enal and analogous gamma-hydroxyalkenal phosphatidylcholines (PCs) have attracted attention because these oxidatively truncated lipids are biologically active and have been implicated in diseases. A previous study showed that hydroxydienes, generated by allylic oxygenation of linoleic acid, are unreactive toward oxidative fragmentation. We now show that, in the presence of hydroperoxides, hydroxydienes fragment as readily as the corresponding hydroperoxydienes, generating gamma-hydroxyalkenals. In a physiomimetic model study, myeloperoxidase-promoted free radical-induced fragmentation of either hydroperoxy- or hydroxyoctecadienoate esters of 2-lyso-PC in small unilamellar vesicles produced the 9-hydroxy-12-oxododec-10-enoic acid (HODA) ester HODA-PC. Therefore, hydroxydienes, that are generally more abundant in vivo than hydroperoxydienes, are plausible intermediates in the production of oxidatively truncated lipids in vivo where a constant flux of radicals and hydroperoxides is present. Our findings also show that the formation of dioxetane intermediates through peroxyradical cyclization is not required to achieve oxidative fragmentation of PUFAs.

Succinylacetone oxidation by oxygen/peroxynitrite: a possible source of reactive intermediates in hereditary tyrosinemia type I.

Hereditary tyrosinemia type I (HT1) is an inborn metabolic error characterized by hepatorenal dysfunction. Affected patients excrete large quantities of succinylacetone (SA), a tyrosine catabolite believed to be involved in the pathogenesis of HT1. A growing body of evidence relates the oxidative stress observed in metabolic disorders to free radicals generated from accumulated metabolites. In this context, oxidation of SA by peroxynitrite or cytochrome c yielding reactive intermediates and products was investigated here. Both peroxynitrite and cytochrome c were able to initiate oxygen consumption by SA, which was followed by polarimetric and chemiluminescence measurements. The light emission arises from triplet carbonyls formed by the thermolysis of dioxetane intermediates, as indicated by energy transfer experiments. EPR spin-trapping studies with 2-methyl-2-nitrosopropane revealed the intermediacy of two different carbon-centered radicals, one of them originating from cleavage of the triplet carbonyl product. The pH profiles obtained by oxygen consumption, chemiluminescence, and stopped-flow spectrophotometry point to the peroxynitrite anion as the initiator of SA aerobic oxidation. Overstoichiometric formation of organic acids based on added peroxynitrite confirms the occurrence of an oxygen-dependent chain reaction, here proposed to be initiated by one electron abstraction from the enolic form of SA. The results obtained may help shed light on the role of both SA and oxidative stress in the pathogenesis of HT1.

Substituent and solvent effects on the photosensitized oxygenation of 5,6-dihydro-1,4-oxathiins. Intramolecular oxygen transfer vs normal cleavage of the dioxetane intermediates.

The reaction of singlet oxygen with variously substituted oxathiins 1 affords dicarbonyl compounds 4 and/or ketosulfoxides 7 and 8 depending on the nature of the substituent at C3 and on the reaction conditions. The normal fragmentation of dioxetanes 2 to 4 competes with an intramolecular oxygen transfer to ring sulfur, which leads to 7 and 8, presumably via the labile epoxides 5. This new pathway is promoted by electron-withdrawing groups at C3 and, for unsubstituted and monosubstituted amide derivatives 1h and 1i, respectively, by the solvent basicity. Chemical experiments support the intermediacy of epoxides 5 for 7, whereas they are not conclusive for 8. However, the formation of the latter compounds appears to be favored by polar solvents and cation-stabilizing groups at C2 as phenyl or methyl, and these observations may be well accounted for by the suggested pathway from 5 through charged species as E. Direct oxidation by singlet oxygen to sulfur is unsignificant, except for the amide series and for 1g or when the oxygenation is carried out in methanol.

Low-temperature photosensitized oxidation of a guanosine derivative and formation of an imidazole ring-opened product.

An organic-soluble guanosine derivative, 2',3',5'-O-(tert-butyldimethylsilyl)guanosine (1), was prepared and its photosensitized oxidation was carried out in several solvents at various temperatures. Singlet oxygen is the reactive oxidizing agent responsible for this reaction. Neither an endoperoxide nor a dioxetane intermediate was detected by low-temperature NMR even at -78 degrees C. A product (A) with an oxidized imidazole ring was the only major product detected at room temperature; this compound could be isolated by low-temperature column chromatography and was characterized by (1)H and (13)C and mass spectroscopy. CO(2) was the other major product. A small amount of the corresponding 8-oxo-7,8-dihydroguanosine derivative B was detected during the initial stage of the photooxidation and was shown to be intermediate in the formation of two products of extensive degradation, C and D. Reaction of 1 with the singlet oxygen analogues 4-methyl-1,2,4-triazoline-3,5-dione (MTAD) and 4-phenyl-1,2,4-triazoline-3,5-dione (PTAD) gave products consistent with a proposed mechanism involving the rearrangement of an initially formed endoperoxide to give A and B from reaction of 1 with singlet oxygen.

Reaction of dioxygen with a cross-conjugated carbon-carbon double bond in a bis-macrocycle diiron compound

Dioxygen at atmospheric pressure attacks a cross-conjugated carbon-carbon double bond in a diiron complex to form two, like, keto macrocyclic iron(II) complexes. This reaction occurs with high yield in both solution and in the solid state. A dioxetane intermediate is, therefore, invoked. The rate of the reaction is very dependent on the nature of the axial ligands on the low-spin iron(II) ions in the bimetallic complex. The rate is at least a factor of 10 4 faster with DMF ligands than with CH 3CN axial ligands. This rate dependence is explained by stabilization of a peroxo biradical transition state en route to a dioxetane intermediate. The keto-macrocyle product has the carbonyl group conjugated with a β-diimine in a six-membered chelate ring. The conformation of this keto macrocycle is fixed on the NMR time scale and the spectra of all ten non-equivalent protons in the complex can be unambiguously assigned. The keto β-diimine ligand is an excellent π-acceptor as indicated by the high Fe(II) to Fe(III) oxidation potential of the compound and by the Mössbauer spectrum, which shows a low value for the center shift and a high value for the quadrupole splitting parameter.

Horseradish Peroxidase‐Catalyzed Generation of Acetophenone and Benzophenone in the Triplet State*

ABSTRACTHorseradish peroxidase catalyzes the aerobic oxidation of 2‐phenylpropanal and diphenylacetaldehyde at physiological pH to yield acetophenone and benzophenone partly in the triplet state, respectively. These excited products plus formic acid are expected from the thermolysis of dioxetane intermediates. The presence of acetophenone was demonstrated spectrophotometr‐ically and the chemiluminescence spectrum (δmax ‐ 430 nm) was consistent with its triplet state. Energy transfer to 9,10‐dibromoanthracene‐2‐sulfonate ion, a triplet carbonyl counter, but not to anthracene‐2‐sulfonate ion, a singlet carbonyl acceptor, occurred, confirming the triplet nature of the main emitter. In the case of the diphenylacetaldehydelperoxidase system, benzophenone could also be detected spectrophotometrically but the corresponding chemiluminescence spectrum showed only red emission (δmax ‐ 630 nm), which was tentatively attributed to singlet oxygen formed by triplet‐triplet energy transfer to ground state oxygen. Horseradish peroxidase can be replaced by other he‐meproteins such as myoglobin, hemoglobin and micro‐peroxidase as catalyst of the chemiluminescent reaction. The distinct emission spectra achieved with different hemeproteins suggest a role of the microenvi‐ronment in totally or partly protecting the excited species from oxygen collisions, resulting in emission maxima around 430 nm, 630 nm or both.