In Vitro Fossilization for High Spatial Resolution Quantification of Elements in Plant-Tissue Using LA-ICP-TOFMS.

Laser ablation in combination with an inductively coupled plasma time-of-flight mass spectrometer (LA-ICP-TOFMS) is an upcoming method for rapid quantitative element mapping of various samples. While widespread in geological applications, quantification of elements in biotissues remains challenging. In this study, a proof-of-concept sample preparation method is presented in which plant-tissues are fossilized in order to solidify the complex biotissue matrix into a mineral-like matrix. This process enables quantification of elements by using silicone as an internal standard for normalization while also providing consistent ablation processes similar to minerals to reduce image blurring. Furthermore, it allows us to generate a quantitative image of the element composition at high spatial resolution. The feasibility of the approach is demonstrated on leaves of sunflowers (Helianthus annuus), soy beans (Glycine max), and corn (Zea mays) as representatives for common crops, which were grown on both nonspiked and cadmium-spiked agricultural soil. The quantitative results achieved during imaging were validated with digestion of whole leaves followed by ICP-OES analysis. LA-ICP-TOFMS element mapping of conventionally dried samples can provide misleading trends due to the irregular ablation behavior of biotissue because high signals caused by high ablation rates are falsely interpreted as enrichment of elements. Fossilization provides the opportunity to correct such phenomena by standardization with Si as an internal standard. The method demonstrated here allows for quantitative image acquisition without time-consuming sample preparation steps by using comparatively safe chemicals. The diversity of tested samples suggests that this sample preparation method is well-suited to achieve reproducible and quantitative element maps of various plant samples.

Electron-Driven Nitration of Unsaturated Hydrocarbons.

Herein, we introduce an electrochemically assisted generation of nitryl radicals from ferric nitrate under mild reaction conditions using a simple setup with inexpensive graphite and stainless-steel electrodes. The mechanism of the reaction is supported by detailed spectroscopic and experimental studies. Powered by electricity and driven by electrons, the synthetic diversity of this reaction has been demonstrated through the development of highly efficient nitration protocols of various unsaturated hydrocarbons. In addition to a broad application area, these protocols are easy to scale for decagram quantities, and exhibit exceptional substrate generality and functional-group compatibility.

Impact of substitution on reactions and stability of one-electron oxidised phenyl sulfonates in aqueous solution.

Highly reactive aromatic cation radicals have been invoked lately in synthetic routes and in the degradation pathways of hydrocarbon-based polymers. Changes in the electron density of aromatic compounds are expected to alter the reaction pathway following one electron oxidation through altering the pKa of the formed intermediate cation radical. Electron-donating groups increase its stability, however, little experimental data are known. While, in theory, the cation radical can be repaired by simple electron transfer, electron transfer to or from its deprotonated form, the hydroxycyclohexadienyl radical, will cause permanent modification or degradation. Time-resolved absorption spectroscopy indicates a pKa ≈ 2-3 for the 4-(tert-butyl)-2-methoxyphenylsulfonate (BMPS) radical cation, while its parent compound 4-(tert-butyl) phenylsulfonate (BPS) is much more acidic. The stability of both compounds towards oxidation by HO˙ was evaluated under air at pH 5 and pH 0. At pH 5, both BMPS and BPS are unstable, and superstoichiometric degradation was observed. Degradation was slightly reduced for BPS at pH 0. In contrast, the more electron rich BMPS showed 80% lower degradation. We unambigously showed that in the presence of Ce(III) and H2O2 at pH 0 both BMPS and BPS could be catalytically repaired via one electron reduction, resulting in further damage moderation.

Initiation and Prevention of Biological Damage by Radiation-Generated Protein Radicals.

Ionizing radiations cause chemical damage to proteins. In aerobic aqueous solutions, the damage is commonly mediated by the hydroxyl free radicals generated from water, resulting in formation of protein radicals. Protein damage is especially significant in biological systems, because proteins are the most abundant targets of the radiation-generated radicals, the hydroxyl radical-protein reaction is fast, and the damage usually results in loss of their biological function. Under physiological conditions, proteins are initially oxidized to carbon-centered radicals, which can propagate the damage to other molecules. The most effective endogenous antioxidants, ascorbate, GSH, and urate, are unable to prevent all of the damage under the common condition of oxidative stress. In a promising development, recent work demonstrates the potential of polyphenols, their metabolites, and other aromatic compounds to repair protein radicals by the fast formation of less damaging radical adducts, thus potentially preventing the formation of a cascade of new reactive species.

Thinking Outside the Cage: A New Hypothesis That Accounts for Variable Yields of Radicals from the Reaction of CO2 with ONOO.

In biology, the reaction of ONOO- with CO2 is the main sink for ONOO-. This reaction yields CO3•-, NO2•, NO3-, and CO2. There is a long-standing debate with respect to the yield of the radicals relative to ONOO-. The reaction of ONOO- with CO2 results at first in ONOOCO2-. According to one hypothesis, ONOOCO2- is extremely short-lived and devolves into a solvent cage that contains CO3•- and NO2•. Of these solvent cages, approximately two/thirds result in NO3- and CO2, and approximately one/third release CO3•- and NO2• that oxidize the substrate. According to our hypothesis, ONOOCO2- is formed much faster, is relatively long-lived, and may also be an oxidant; the limited yield is the result of ONOOCO2- being scavenged by a second CO2 under conditions of a high CO2 concentration. We disagree with the first hypothesis for three reasons: First, it is based on an estimated K for the reaction of ONOO- with CO2 to form ONOOCO2- of ∼1 M-1, while experiments yield a value of 4.5 × 103 M-1. Second, we argue that the solvent cage as proposed is physically not realistic. Given the less than diffusion-controlled rate constant of CO3•- with NO2•, all radicals would escape from the solvent cage. Third, the reported ∼33% radical is not supported by an experiment where mass balance was established. We propose here a hybrid mechanism. After formation of ONOOCO2-, it undergoes homolysis to yield CO3•- with NO2•, or, depending on [CO2], it is scavenged by a second CO2; CO3•- oxidizes ONOO-, if present. These reactions allow us to successfully simulate the reaction of ONOO- with CO2 over a wide range of ONOO-/CO2 ratios. At lower ratios, fewer radicals are formed, while at higher ratios, radical yields between 30% and 40% are predicted. The differences in radical yields reported may thus be traced to the experimental ONOO-/CO2 ratios. Given a physiological [CO2] of 1.3 mM, the yield of CO3•- and NO2• is 19%, and lower if ONOOCO2- has a significant reactivity of its own.

Addition of carbon-centered radicals to aromatic antioxidants: mechanistic aspects.

Several recent studies have shown that the rates of formation of adduct radicals between carbon-centred radicals and aromatic molecules are virtually diffusion-controlled and reversible. This contrasts with "radical addition", the well-known multistep reaction in preparative organic chemistry where the rate-determining initial formation of radical adducts is perceived to be several orders of magnitude slower and virtually irreversible. Using pulse radiolysis and spectroscopic analysis, we have now re-examined parts of this complex mechanism. The results have significant implications for biological systems: electron-rich, aromatic structures may act like buffers for radicals, moderating their reactivity resulting in a much slower reaction determining the overall rate of oxidation. In vivo, an organism would gain time for an appropriate antioxidant reaction.

Attack of hydroxyl radicals to α-methyl-styrene sulfonate polymers and cerium-mediated repair via radical cations.

Both synthetic polymers (membranes, coatings, packaging) and natural polymers (DNA, proteins) are subject to radical-initiated degradation. In order to mitigate the deterioration of the polymer properties, antioxidant strategies need to be devised. We studied the reactions of poly(α-methylstyrene sulfonate), a model compound for fuel cell membrane materials, with different degrees of polymerization with OH˙ radicals as well as subsequent reactions. We observed the resulting OH˙-adducts to react with oxygen and eliminate H2O, the relative likelihood of which is determined by pH and molecular weight. The resulting radical cations can be reduced back to the parent molecule by cerium(iii). This 'repair' reaction is also dependent on molecular weight likely because of intramolecular stabilization. The results from this study provide a starting point for the development of new hydrocarbon-based ionomer materials for fuel cells that are more resistant to radical induced degradation through the detoxification of intermediates via damage transfer and repair pathways. Furthermore, a more fundamental understanding of the mechanisms behind conventional antioxidants in medicine, such as ceria nanoparticles, is achieved.

Thermochemical unification of molecular descriptors to predict radical hydrogen abstraction with low computational cost.

Chemistry describes transformation of matter with reaction equations and corresponding rate constants. However, accurate rate constants are not always easy to get. Here we focus on radical oxidation reactions. Analysis of over 500 published rate constants of hydroxyl radicals led us to hypothesize that a modified linear free-energy relationship (LFER) could be used to predict rate constants speedily, reliably and accurately. LFERs correlate the Gibbs activation-energy with the Gibbs energy of reaction. We calculated the latter as the sum of one-electron transfer and, if appropriate, proton transfer. We parametrized specific transition state effects to orbital delocalizability and the polarity of the reactant. The calculation time for 500 reactions is less than 8 hours on a standard desktop-PC. Rate constants were also calculated for hydrogen and methyl radicals; these controls show that the predictions are applicable to a broader set of oxidizing radicals. An accuracy of 30-40% (standard deviation) with reference to reported experimental values was found suitable for the screening of complex chemical systems for possibly relevant reactions. In particular, potentially relevant reactions can be singled out and scrutinized in detail when prioritizing chemicals for environmental risk assessment.

Synthesis, Characterization, and Reactivity of a Hypervalent-Iodine-Based Nitrooxylating Reagent.

Herein, the synthesis and characterization of a hypervalent-iodine-based reagent that enables a direct and selective nitrooxylation of enolizable C-H bonds to access a broad array of organic nitrate esters is reported. This compound is bench stable, easy-to-handle, and delivers the nitrooxy (-ONO2 ) group under mild reaction conditions. Activation of the reagent by Brønsted and Lewis acids was demonstrated in the synthesis of nitrooxylated ß-keto esters, 1,3-diketones, and malonates, while its activity under photoredox catalysis was shown in the synthesis of nitrooxylated oxindoles. Detailed mechanistic studies including pulse radiolysis, Stern-Volmer quenching studies, and UV/Vis spectroelectrochemistry reveal a unique single-electron-transfer (SET)-induced concerted mechanistic pathway not reliant upon generation of the nitrate radical.

Fast reaction of carbon free radicals with flavonoids and other aromatic compounds.

Many theoretical and experimental studies have shown that the principal initial biological targets of free radicals are nucleic acids, lipids and proteins. The reaction normally generates carbon-centered radicals which can propagate molecular damage either directly or after formation of new reactive species following reaction with oxygen. Overall damage prevention is therefore best achieved by repair of the carbon radicals before they initiate further reactions. Recent studies have shown that the repair cannot be achieved by normal levels of the endogenous antioxidants glutathione, ascorbate or urate. Since their concentrations are well regulated and cannot be enhanced by oral intake, we have investigated the effectiveness of flavonoids and other polyphenols as potential carbon radical repair agents, because their levels in vivo can be significantly enhanced by diet. Pulse radiolysis measurements of the rate constants of repair of amino acid radicals by several polyphenols showed reversible formation of radical-polyphenol adducts 100-1000 times faster than previously reported for the bimolecular stoichiometric reactions of flavonoids i.e. with rate constants in the order of 1010 M-1s-1. Adduct formation depended only on the presence of a carbon-centered radical and an aromatic moiety in the reactants, without the involvement of redox reactions at the phenolic groups. Formation of adducts lowered the reactivity of the radicals. Our results suggest that flavonoids, polyphenols and many of their metabolites can effectively reduce the damaging potential of carbon radicals at concentrations achievable in vivo by diets rich in fruits and vegetables.

Profiling the oxidative activation of DMSO-F6 by pulse radiolysis and translational potential for radical C-H trifluoromethylation.

The oxidative activation of the perfluorinated analogue of dimethyl sulfoxide, DMSO-F6, by hydroxyl radicals efficiently produces trifluoromethyl radicals based on pulse radiolysis, laboratory scale experiments, and comparison of rates of reaction for analogous radical systems. In comparison to commercially available precursors, DMSO-F6 proved to be more stable, easier to handle and overall more convenient than leading F3C-reagents and may therefore be an ideal surrogate to study F3C radicals for time-resolved kinetics studies. In addition, we present an improved protocol for the preparation of this largely unexplored reagent.

Jumpstarting the cytochrome P450 catalytic cycle with a hydrated electron.

Cytochrome P450cam (CYP101Fe3+) regioselectively hydroxylates camphor. Possible hydroxylating intermediates in the catalytic cycle of this well-characterized enzyme have been proposed on the basis of experiments carried out at very low temperatures and shunt reactions, but their presence has not yet been validated at temperatures above 0 °C during a normal catalytic cycle. Here, we demonstrate that it is possible to mimic the natural catalytic cycle of CYP101Fe3+ by using pulse radiolysis to rapidly supply the second electron of the catalytic cycle to camphor-bound CYP101[FeO2]2+ Judging by the appearance of an absorbance maximum at 440 nm, we conclude that CYP101[FeOOH]2+ (compound 0) accumulates within 5 µs and decays rapidly to CYP101Fe3+, with a k440 nm of 9.6 × 104 s-1 All processes are complete within 40 µs at 4 °C. Importantly, no transient absorbance bands could be assigned to CYP101[FeO2+por•+] (compound 1) or CYP101[FeO2+] (compound 2). However, indirect evidence for the involvement of compound 1 was obtained from the kinetics of formation and decay of a tyrosyl radical. 5-Hydroxycamphor was formed quantitatively, and the catalytic activity of the enzyme was not impaired by exposure to radiation during the pulse radiolysis experiment. The rapid decay of compound 0 enabled calculation of the limits for the Gibbs activation energies for the conversions of compound 0 → compound 1 → compound 2 → CYP101Fe3+, yielding a ΔG‡ of 45, 39, and 39 kJ/mol, respectively. At 37 °C, the steps from compound 0 to the iron(III) state would take only 4 µs. Our kinetics studies at 4 °C complement the canonical mechanism by adding the dimension of time.

Reaction rates of glutathione and ascorbate with alkyl radicals are too slow for protection against protein peroxidation in vivo.

Reaction kinetics of amino acid and peptide alkyl radicals with GSH and ascorbate, the two most abundant endogenous antioxidants, were investigated by pulse radiolysis. Rate constants in the order of 106 M-1s-1 were found. Alkyl radicals react at almost diffusion controlled rates and irreversibly with oxygen to form peroxyl radicals, and competition with this reaction is the benchmark for efficient repair in vivo. We consider repair of protein radicals and assume comparable rate constants for the reactions of GSH/ascorbate with peptide alkyl radicals and with alkyl radicals on a protein surface. Given physiological concentrations of oxygen, GSH and ascorbate, protein peroxyl radicals will always be a major product of protein alkyl radicals in vivo. Therefore, if they are formed by oxidative stress, protein alkyl radicals are a probable cause for biological damage.

Physiological Concentrations of Ascorbate Cannot Prevent the Potentially Damaging Reactions of Protein Radicals in Humans.

The principal initial biological targets of free radicals formed under conditions of oxidative stress are the proteins. The most common products of the interaction are carbon-centered alkyl radicals which react rapidly with oxygen to form peroxyl radicals and hydroperoxides. All these species are reactive, capable of propagating the free radical damage to enzymes, nucleic acids, lipids, and endogenous antioxidants, leading finally to the pathologies associated with oxidative stress. The best chance of preventing this chain of damage is in early repair of the protein radicals by antioxidants. Estimate of the effectiveness of the physiologically significant antioxidants requires knowledge of the antioxidant tissue concentrations and rate constants of their reaction with protein radicals. Previous studies by pulse radiolysis have shown that only ascorbate can repair the Trp and Tyr protein radicals before they form peroxyl radicals under physiological concentrations of oxygen. We have now extended this work to other protein C-centered radicals generated by hydroxyl radicals because these and many other free radicals formed under oxidative stress can produce secondary radicals on virtually any amino acid residue. Pulse radiolysis identified two classes of rate constants for reactions of protein radicals with ascorbate, a faster one in the range (9-60) × 107 M-1 s-1 and a slow one with a range of (0.5-2) × 107 M-1 s-1. These results show that ascorbate can prevent further reactions of protein radicals only in the few human tissues where its concentration exceeds about 2.5 mM.

An Experimental Radical Electrophilicity Index.

We present an experimental electrophilicity index (Ï») for the classification of radicals. The Ï»-scale is based on the equilibrium constant determined for the reversible addition of a radical R. to an aromatic radicophile (HisNH2 ). This experimental approach is in excellent agreement with the computed global electrophilicity index ω and serves to validate the latter.

Mechanistic insight into the thermal activation of Togni's trifluoromethylation reagents.

Herein we investigate the propensity of hypervalent iodine based electrophilic trifluoromethylating agents to undergo thermally induced fragmentation of the F3C-I-O motif. For the first time we are able to observe a dissociative electron transfer mechanism using mass spectroscopy techniques to generate and trap CF3 radicals. Consistent with this mechanism, alkyl radical elimination from these reagents is in full support of an intermediate cyclic iodanyl radical and a reagent-specific temperature of maximum radical production was found to correlate with reported solution phase reactivity.

Electrode Potentials of l-Tryptophan, l-Tyrosine, 3-Nitro-l-tyrosine, 2,3-Difluoro-l-tyrosine, and 2,3,5-Trifluoro-l-tyrosine.

Electrode potentials for aromatic amino acid radical/amino acid couples were deduced from cyclic voltammograms and pulse radiolysis experiments. The amino acids investigated were l-tryptophan, l-tyrosine, N-acetyl-l-tyrosine methyl ester, N-acetyl-3-nitro-l-tyrosine ethyl ester, N-acetyl-2,3-difluoro-l-tyrosine methyl ester, and N-acetyl-2,3,5-trifluoro-l-tyrosine methyl ester. Conditional potentials were determined at pH 7.4 for all compounds listed; furthermore, Pourbaix diagrams for l-tryptophan, l-tyrosine, and N-acetyl-3-nitro-l-tyrosine ethyl ester were obtained. Electron transfer accompanied by proton transfer is reversible, as confirmed by detailed analysis of the current waves, and because the slopes of the Pourbaix diagrams obey Nernst's law. E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH) at pH 7 are 0.99 ± 0.01 and 0.97 ± 0.01 V, respectively. Pulse radiolysis studies of two dipeptides that contain both amino acids indicate a difference in E°' of approximately 0.06 V. Thus, in small peptides, we recommend values of 1.00 and 0.96 V for E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH), respectively. The electrode potential of N-acetyl-3-nitro-l-tyrosine ethyl ester is higher, while because of mesomeric stabilization of the radical, those of N-acetyl-2,3-difluoro-l-tyrosine methyl ester and N-acetyl-2,3,5-trifluoro-l-tyrosine methyl ester are lower than that of tyrosine. Given that the electrode potentials at pH 7 of E°'(Trp(•),H(+)/TrpH) and E°'(TyrO(•),H(+)/TyrOH) are nearly equal, they would be, in principle, interchangeable. Proton-coupled electron transfer pathways in proteins that use TrpH and TyrOH are thus nearly thermoneutral.

Shielding effects in spacious macromolecules: a case study with dendronized polymers.

Dendronized polymers exhibit defined structures with bulky side chains (dendrons) on a linear polymer backbone. Upon reaction with radicals, chromophores close to the backbone were bleached. The reaction rate and yield decreased with increasing dendron size, demonstrating that the inside of dendronized polymers can be "shielded" by bulky dendrons from access by reactive species.

Why selenocysteine replaces cysteine in thioredoxin reductase: a radical hypothesis.

Thioredoxin reductases, important biological redox mediators for two-electron transfers, contain either 2 cysteines or a cysteine (Cys) and a selenocysteine (Sec) at the active site. The incorporation of Sec is metabolically costly, and therefore surprising. We provide here a rationale: in the case of an accidental one-electron transfer to a S-S or a S-Se bond during catalysis, a thiyl or a selanyl radical, respectively would be formed. The thiyl radical can abstract a hydrogen from the protein backbone, which subsequently leads to the inactivation of the protein. In contrast, a selanyl radical will not abstract a hydrogen. Therefore, formation of Sec radicals in a GlyCysSecGly active site will less likely result in the destruction of a protein compared to a GlyCysCysGly active site.

Structure and enzymatic properties of molecular dendronized polymer-enzyme conjugates and their entrapment inside giant vesicles.

Macromolecular hybrid structures were prepared in which two types of enzymes, horseradish peroxidase (HRP) and bovine erythrocytes Cu,Zn-superoxide dismutase (SOD), were linked to a fluorescently labeled, polycationic, dendronized polymer (denpol). Two homologous denpols of first and second generation were used and compared, and the activities of HRP and SOD of the conjugates were measured in aqueous solution separately and in combination. In the latter case the efficiency of the two enzymes in catalyzing a two-step cascade reaction was evaluated. Both enzymes in the two types of conjugates were highly active and comparable to free enzymes, although the efficiency of the enzymes bound to the second-generation denpol was significantly lower (up to a factor of 2) than the efficiency of HRP and SOD linked to the first-generation denpol. Both conjugates were analyzed by atomic force microscopy (AFM), confirming the expected increase in object size compared to free denpols and demonstrating the presence of enzyme molecules localized along the denpol chains. Finally, giant phospholipid vesicles with diameters of up to about 20 µm containing in their aqueous interior pool a first-generation denpol-HRP conjugate were prepared. The HRP of the entrapped conjugate was shown to remain active toward externally added, membrane-permeable substrates, an important prerequisite for the development of vesicular multienzyme reaction systems.