Superoxide ion reacts rapidly with nitric oxide in aqueous solutions to form the peroxonitrite (oxoperoxonitrate( 1 -), ONO2-) ion.* 1 11This anion and its conjugate acid, hydrogen oxoperoxonitrate (ONO2H), are powerful oxidants2 which are reported to rapidly oxidize sulfhydryl groups3 and thioethers4 and to nitrate and hydroxylate aromatic compounds.5-7 In

Polymer nanoparticles of 40-400 nm diameter with spiropyran-merocyanine dyes incorporated into their hydrophobic cavities have been prepared; in contrast to their virtually nonfluorescent character in most environments, the merocyanine forms of the encapsulated dyes are highly fluorescent. Spiro-mero photoisomerization is reversible, allowing the fluorescence to be switched "on" and "off" by alternating UV and visible light. Immobilizing the dye inside hydrophobic pockets of nanoparticles also improves its photostability, rendering it more resistant than the same dyes in solution to fatigue effects arising from photochemical switching. The photophysical characteristics of the encapsulated fluorophores differ dramatically from those of the same species in solution, making nanoparticle-protected hydrophobic fluorophores attractive materials for potential applications such as optical data storage and switching and biological fluorescent labeling. To evaluate the potential for biological tagging, these optically addressable nanoparticles have been delivered into living cells and imaged with a liquid nitrogen-cooled CCD.

Peroxynitrite ion (ONO2-) reacted rapidly with CO2 to form a short-lived intermediate provisionally identified as the ONO2CO2- adduct. This adduct was more reactive in tyrosine oxidation than ONO2- itself and produced 3-nitrotyrosine and 3,3'-dityrosine as the major oxidation products. With tyrosine in excess, the rate of 3-nitrotyrosine formation was independent of the tyrosine concentration and was determined by the rate of formation of the ONO2CO2- adduct. The overall yield of oxidation products was also independent of the concentration of tyrosine and medium acidity; approximately 19% of the added ONO2- was converted to products under all reaction conditions. However, the 3-nitrotyrosine/3,3'-dityrosine product ratio depended upon the pH, tyrosine concentration, and absolute reaction rate. These data are in quantitative agreement with a reaction mechanism in which the one-electron oxidation of tyrosine by ONO2CO2- generates tyrosyl and NO2 radicals as intermediary species, but are inconsistent with mechanisms that invoke direct electrophilic attack on the tyrosine aromatic ring by the adduct. Based upon its reactivity characteristics, ONO2CO2- has a lifetime shorter than 3 ms and a redox potential in excess of 1 V, and oxidizes tyrosine with a bimolecular rate constant greater than 2 x 10(5) M-1 s-1. In comparison, in CO2-free solutions, oxidation of tyrosine by peroxynitrite was much slower and gave significantly lower yields (approximately 8%) of the same products. When tyrosine was the limiting reactant, 3,5-dinitrotyrosine was found among the reaction products of the CO2-catalyzed reaction, but this compound was not detected in the uncatalyzed reaction.

Fluorescence labeling of biomacromolecules and cells has become one of the major tools to study structural organization and intra-and intermolecular processes within biological systems. 1 Indeed, both highly fluorescent dyes and semiconductive quantum dots (QDs) are widely employed as fluorescent tags. 2 Dual-color fluorescent tags, especially those that can be reversibly photoswitched, are particularly desirable because they can distinguish sites of interest from false positive signals generated by adventitious fluorescent biomolecules. 3 Herein, we report a class of polymer nanoparticles containing both fluorescent dyes and photoswitchable chromophores within its hydrophobic core. Reversible photoisomerization of the second chromophore toggles it between forms that can or cannot quench the photoexcited fluorescent dye via energy transfer. The photoexcited acceptor is also emissive, so that the energy of the emitted photon, i.e., the identity of the fluorophore, is dependent upon the isomeric state of the second photoswitchable chromophore.The specific synthetic approach uses an emulsion polymerization method to polymerize Nisopropylacrylamide (NIPAM) in the presence of other polymerizable monomers, including styrene (St), divinylbenzene (DVB), and the acrylatelinked dyes perylene diimide (PDI) and spiropyran (SP) (Scheme 1). When using a water-soluble initiator, NIPAM initially polymerizes because the other monomeric units are not appreciably water-soluble. Poly(Nisopropylacrylate) (PNIPAM), however, undergoes a hydrophilic to hydrophobic transition above its low critical transition temperature (LCST), T t = 31 °C. 4 As a result, PNIPAM collapses into hydrophobic spheres at elevated temperatures with the assistance of the surfactant Tween 20. This transition then allows the incorporation of the solubilized hydrophobic monomers into the growing polymer nanospheres.The as-prepared nanoparticles are spherical with mean diameters varying from 50 nm to 110 nm depending on the feed ratio of monomers. The particles shown in Figure 1A are nearly monodisperse and have an average size of ∼56 nm, as characterized by transmission electron microscopy (TEM). They also have negatively charged carboxyl functional groups on their surfaces; hence, they can be easily dispersed in water and are stable in many biological buffers and extracellular fluids both above and below their LCST. When the photoswitchable dye is in its ring-closed (spiro) form, the perylene chromophore located in the hydrophobic core strongly emits a distinctive green fluorescence (λ max ∼535 nm, with a strong overtone at 575 nm; quantum yield (φ≈ 0.9)). 5 However, UV-induced ring opening converts the spirocompound to its open-ring form (merocyanine) 6 whose visible absorption band (λ max ∼588 nm) nearly coincides with the perylene emission bands. Consequently, the perylene emission is strongly quenched by fluorescence resonance energy transfer (FRET). 7 Because the spiro form has no visible absorption with energy lower than the π→π* transition of perylene dy...

Oxidative degradation of biological substrates by hypochlorous acid has been examined under reaction conditions similar to those found in active phagosomes. Iron sulfur proteins are bleached extremely rapidly, followed in decreasing order by f3carotene, nucleotides, porphyrins, and heme proteins. Enzymes containing essential cysteine molecules are inactivated with an effectiveness that roughly parallels the nucleophilic reactivities of their sulfhydryl groups. Other compounds, including glucosamines, qumiones, riboflavin, and, except for N-chlorination, phospholipids, are unreactive. Rapid irreversible oxidation of cytochromes, adenine nucleotides, and carotene pigments occurs when bacterial cells are exposed to exogenous hypochlorous acid; with Escherichia coli, titrimetric oxidation of cytochrome was found to coincide with loss of aerobic respiration. The occurrence of these cellular reactions implicates hypochlorous acid as a primary microbicide in myelo.roxidase-containing leukocytes; the reactivity patterns observed are consistent with the view that bactericidal action results primarily from loss of energy-linked respiration due to destruction of cellular electron transport chains and the adenine nucleotide pool.Bacteria commonly lose their ability to divide within minutes of encountering phagocytosing leukocytes (1). Loss ofcell viability often occurs well before the onset ofcellular digestion as determined by physiological changes (2), macromolecular degradation (3, 4), or loss of macromolecular biosynthesis (5). The specific reactions giving rise to cellular death have not yet been identified, at least in part because leukocytes are capable ofinitiating a diverse set of processes which are potentially lethal (6, 7). Given the above observations, however, the microbicidal reactions must be among the first that attend interaction with the leukocyte.Reactions catalyzed by myeloperoxidase (MPOase) appear to make major contributions to the microbicidal-action of polymorphonuclear leukocytes (PMNs) (6, 7). The cell-free MPOase-H202-Cl-system is 'potentially microbicidal; chlorination of bacteria by the cell-free system (8, 9) and of macromolecular fractions during PMN digestion of bacteria (9) electrophile-nucleophile interactions involving association of electropositive chlorine with electron-rich centers on the substrate; reaction pathways are-correspondingly highly dependent upon medium-conditions, particularly H' and Cl-concentrations (14). With the exception ofamines and amino acids (11,12,15), the reactions of biological compounds with HOC1 are not understood (16).We report 'here the results of a survey of simple biological compounds which can be taken as prototypic ofvarious components of bacterial cells. :The data demonstrate that HOCI is strongly selective -toward nucleotides and compounds that are models for certain components ofrespiratory redox chains. This selectivity is shownito extend to bacterial cells. Table 1 were -determined from loss of their characteristic absorption bands when ...

Resonance Raman (RR) and optical spectroelectrochemical titrations of the cis,cis-[(bpy)2Ru(OH2)]2O4+ ion (denoted [3,3] to indicate the formal oxidation state of the Ru−O−Ru unit) were made over the range 0.8−2.0 V vs Ag/AgCl in 0.5 M trifluoromethanesulfonic acid; the results revealed sequential accumulation of three higher oxidation states. Two of these states were identified by redox titration with Os(bpy)3 2+ as one-electron ([3,4]) and four-electron oxidized species ([5,5]); spectroscopic analysis of reaction products formed upon mixing the [3,3] and [5,5] ions indicated that the third oxidation state is a two-electron oxidized species ([4,4]). The [5,5] ion underwent first-order decay to the [4,4] ion with a rate constant, k ≃ 9.5 × 10-3 s-1, that was nearly identical with the catalytic turnover rate for O2 evolution, k cat ≃ 1.3 × 10-2 s-1 measured under comparable conditions. The [4,4] ion underwent degradation more slowly to the [3,4] ion, which was stable on these time scales. An analogue bearing 4,4‘-dimethyl-2,2‘-bipyridine ligands exhibited very similar behavior, except that the oxidation steps were shifted by ∼50 mV to lower potentials. 18O isotope labeling experiments on the underivatized complex established that there was no oxygen exchange at the bridging μ-oxo position during catalytic turnover. Frozen solutions of the [5,5] ion displayed unusual low-temperature spectroscopic features, including the following:  (i) a narrow g = 2.02 axial EPR signal exhibiting an apparent six-line hyperfine interaction from a minor component; (ii) a concentration-dependent broad rhombic EPR signal in mixtures also containing the [4,4] ion; and (iii) a concentration-dependent replacement of its characteristic ruthenyl RuO stretching mode at 818 cm-1 in the RR spectrum when chemically oxidized with Ce4+ by an 18O isotope sensitive set of three bands in the 650 cm-1 region. The RR spectrum of this new species is consistent with further coordination of the terminal oxo ligands by Ce4+ to form additional ligand bridges.

The cis,cis-[(bpy)(2)Ru(III)(OH(2))](2)O(4+) micro-oxo dimeric coordination complex is an efficient catalyst for water oxidation by strong oxidants that proceeds via intermediary formation of cis,cis-[(bpy)(2)Ru(V)(O)](2)O(4+) (hereafter, [5,5]). Repetitive mass spectrometric measurement of the isotopic distribution of O(2) formed in reactions catalyzed by (18)O-labeled catalyst established the existence of two reaction pathways characterized by products containing either one atom each from a ruthenyl O and solvent H(2)O or both O atoms from solvent molecules. The apparent activation parameters for micro-oxo ion-catalyzed water oxidation by Ce(4+) and for [5,5] decay were nearly identical, with DeltaH(++) = 7.6 (+/-1.2) kcal/mol, DeltaS() = -43 (+/-4) cal/deg mol (23 degrees C) and DeltaH(++) = 7.9 (+/-1.1) kcal/mol, DeltaS(++) = -44 (+/-4) cal/deg mol, respectively, in 0.5 M CF(3)SO(3)H. An apparent solvent deuterium kinetic isotope effect (KIE) of 1.7 was measured for O(2) evolution at 23 degrees C; the corresponding KIE for [5,5] decay was 1.6. The (32)O(2)/(34)O(2) isotope distribution was also insensitive to solvent deuteration. On the basis of these results and previously established chemical properties of this class of compounds, mechanisms are proposed that feature as critical reaction steps H(2)O addition to the complex to form covalent hydrates. For the first pathway, the elements of H(2)O are added as OH and H to the adjacent terminal ruthenyl O atoms, and for the second pathway, OH is added to a bipyridine ring and H is added to one of the ruthenyl O atoms.

(18)O-isotope-labeling studies have led to the conclusion that there exist two major pathways for water oxidation catalyzed by dimeric ruthenium ions of the general type cis, cis-[L2Ru(III)(OH2)]2O(4+). We have proposed that both pathways involve concerted addition of H and OH fragments derived from H 2O to the complexes in their four-electron-oxidized states, i.e., [L2Ru(V)(O)]2O(4+), ultimately generating bound peroxy intermediates that decay with the evolution of O2. The pathways differ primarily in the site of addition of the OH fragment, which is either a ruthenyl O atom or a bipyridine ligand. In the former case, water addition is thought to give rise to a critical intermediate whose structure is L2Ru(IV)(OH)ORu(IV)(OOH)L2(4+); the structures of intermediates involved in the other pathway are less well defined but may involve bipyridine OH adducts of the type L2Ru(V)(O)ORu(IV)(OH)(L(*)OH)L(4+), which could react further to generate unstable dioxetanes or similar endoperoxides. Published experimental and theoretical support for these pathways is reviewed within the broader context of water oxidation catalysis and related reactions reported for other diruthenium and group 8 monomeric diimine-based catalysts. New experiments that are designed to probe the issue of bipyridine ligand "noninnocence" in catalysis are described. Specifically, the relative contributions of the two pathways have been shown to correlate with substituent effects in 4,4'- and 5,5'-substituted bipyridine complexes in a manner consistent with the formation of a reactive OH-adduct intermediate in one of the pathways, and the formation of OH-bipyridine adducts during catalytic turnover has been directly confirmed by optical spectroscopy. Finally, a photosensitized system for catalyzed water oxidation has been developed that allows assessment of the catalytic efficiencies of the complex ions under neutral and alkaline conditions; these studies show that the ions are far better catalysts than had previously been assumed based upon reported catalytic parameters obtained with strong oxidants in acidic media.