Reduction of Guanosyl Radicals in Reactions with Proteins Studied by TR-CIDNP

Pulsed-field-gradient nuclear magnetic resonance (NMR) combined with time-resolved chemically induced dynamic nuclear polarization (TR-CIDNP) was applied to study the reduction of guanosyl radicals in reactions with the proteins hen egg white lysozyme (HEWL) and bovine α-lactalbumin (BLA) in their native state. Guanosyl radicals were generated photochemically in the reaction of guanosine-5′-monophosphate with photosensitizer, triplet-excited 2,2′-dipyridyl. In this reaction, at pH 5 guanosyl cation radical is formed, which deprotonates to yield the neutral guanosyl radical. To minimize the contribution of the cation radical, phosphate buffer was added, which accelerates the deprotonation of guanosyl cation radical. From model simulations of CIDNP kinetics the rate constants of the reduction were found to be (3.1 ± 0.5) × 10⁷ M^?1s^?1 for HEWL and (1.6 ± 0.4) × 10⁷ M^?1s^?1 for BLA. Also, experiments were carried out at the conditions for denatured HEWL, i.e., at 50 °C in the presence of 10 M urea-d₄. The rate constant of the reduction of guanosyl radical in this case was (3.6 ± 0.5) × 10⁸ M^?1s^?1.     

The Excited Triplet State of Azoalkanes: Electron Spin Polarization and Magnetic Field Effects During Triplet-Sensitized Photolysis of trans-Azocumene in Solution

The triplet-sensitized photodecomposition of azocumene into nitrogen and cumyl radicals is investigated by time-resolved electron paramagnetic resonance and absorption spectroscopy. The radicals are found to be created spin polarized with a yield depending on the strength of the applied magnetic field. The phenomenon arises because in triplet azocumene, the decay into radicals competes with a fast triplet-sublevel selective intersystem crossing back to the azocumene ground state. The size of the initial spin polarization of the radicals and the magnetic field effect on their yield are determined in solvents of different viscosities. Data analysis yields rate constants for the intersystem crossing and the cleavage reaction of triplet azocumene as well as its zero-field splitting D _ZFS. At room temperature in nonpolar solvents, the most probable values are: k _x ?=?k _y ?=?1.2?×?10¹¹?s^?? and k _z ?=?1.9?×?10¹⁰?s^?? for the intersystem crossing from the energetically lower and upper triplet substates, respectively, k _p ?=?1.6?×?10⁹?s^?? for the cleavage reaction and for the zero-field splitting D _ZFS?=???.4?×?10^10?s^?? (0.18?cm^??).     

Time resolved CIDNP study of electron transfer reactions in proteins and model compounds

Intramolecular electron transfer (IET) from tyrosine to tryptophan cation radicals is investigated using time resolved chemically induced dynamic nuclear polarization (CIDNP) spectroscopy in combination with laser flash photolysis. In both the tryptophan-tyrosine dipeptide and the denatured state of hen lysozyme in aqueous solution, the transformation TrpH⁺ → TyrO by IET leads to an increase in the tyrosine radical concentration, growth in the tyrosine CIDNP signal, fast decay of the tryptophan CIDNP, and inversion of the phase of the CIDNP of the photosensitizing dye, 2,2′-dipyridyl. IET effects are not observed for mixtures of the amino acid or for the native state of lysozyme. The steady state CIDNP effects seen for denatured lysozyme thus depend not only on the accessibility of the amino acid residues on the surface of the protein but also on the reactivity of the radical intermediates.     

Coherent Polarization Transfer Effects Are Crucial for Interpreting Low-Field CIDNP Data

In this work we demonstrate that low-field chemically induced dynamic nuclear polarization (CIDNP) is strongly affected by re-distribution of polarization, which is formed in the course of spin evolution in transient radical pairs, in diamagnetic reaction products. This phenomenon is of importance when the spins of the reaction product are coupled strongly meaning that spin–spin interactions between them are comparable to the differences in their Zeeman interactions with the external magnetic field. In this case, polarization transfer relies on a coherent mechanism; as a consequence, spins can acquire significant polarization even when they have no hyperfine coupling to the electron spins in the radical pairs, i.e., cannot be polarized directly by CIDNP. This is demonstrated by taking CIDNP of n-butylamine as an example: in this case only the α-CH₂ protons are polarized directly, which is confirmed by high-field CIDNP, whereas the β-CH₂, γ-CH₂ and δ-CH₃ protons get polarized only indirectly due to the transfer of polarization from the α-CH₂ protons. These results show that low-field CIDNP data should be interpreted with care to discriminate between the effects of spin evolution in transient radical pairs and in diamagnetic reaction products.     

The transient EPR spectra and spin dynamics of coupled three-spin systems in photosynthetic reaction centres

The concept of introducing an additional, stable paramagnetic species into photosynthetic reaction centres to increase the information content of their spin polarized transient EPR spectra is investigated theoretically. The light-induced electron transfer in such systems generates a series of coupled three-spin states consisting of sequential photoinduced radical pairs coupled to the stable spin which acts as an “observer”. The spin polarized transient EPR spectra are investigated using the coupled three-spin system P⁺I⁻Q⁻ _A in pre-reduced bacterial reaction centres as a specific example which has been studied experimentally. The evolution of the spin system and the spin polarized EPR spectra of P⁺I⁻Q⁻ _A and Q⁻ _A following recombination of the radical pair (P = primary donor, I = primary acceptor, Q_A = quinone acceptor) are calculated numerically by solving the equations of motion for the density matrix. The net polarization of the observer spin is also calculated analytically by perturbation theory for the case of a single, short-lived, charge-separated state. The result bears a close resemblance to the chemically induced nuclear polarization (CIDNP) generated in photolysis reactions in which a nuclear spin plays the role of the observer interacting with the radical pair intermediates. However, because the Zeeman frequencies of the three electron spins involved are usually quite similar, the polarization of the electron observer spin in strong magnetic fields can reflect features of the CIDNP effect in both, high and low magnetic fields. The dependence of the quinone spin polarization on the exchange couplings in the three-spin system is investigated by numerical simulations, and it is shown that the observed emissive polarization pattern is compatible with either sign, positive or negative, for a range of exchange couplings, J_PI, in the primary pair. The microwave frequency and orientation dependence of the spectra are discussed as two of several possible criteria for determining the sign of J_PI.     

Photooxidation of Histidine by 3,3′,4,4′-Benzophenone Tetracarboxylic Acid in Aqueous Solution: Time-Resolved and Field-Dependent CIDNP Study

The photooxidation reaction between 3,3′,4,4′-benzophenone tetracarboxylic acid (TCBP) and l-histidine (His) has been investigated in neutral aqueous solution using the technique of chemically induced dynamic nuclear polarization (CIDNP). Relative values of ¹³C isotropic hyperfine couplings in the TCBP and His radicals were obtained from the ¹³C-time-resolved CIDNP spectrum, recorded during the photoreaction of TCBP with His at natural abundance of the magnetic isotope ¹³C. Good agreement was found for the hyperfine coupling constants of the TCBP ketyl radical calculated using methods of density functional theory, and those obtained from the ¹³C-time-resolved CIDNP spectrum. The mechanism of the quenching reaction of triplet-excited TCBP by His in neutral aqueous solution was established. ¹H CIDNP field dependencies for the photoreaction of TCBP with His were obtained and the g-factor of the histidyl radical was found.     

Quantitative time-resolved EPR CIDEP study of the photodecomposition oftrans-azocumene in solution

The cumyl radical system, which is created after laser flash irradiation oftrans-azocumene in benzene solution at room temperature, is investigated using time-resolved EPR spectroscopy. From the quantitative analysis of EPR time-profiles at different microwave powers the spin relaxation timesT ₁=3.5±0.3 μs andT ₂=2.5±0.1 μs are evaluated as well as the magnitude of the chemically induced electron polarization (CIDEP), which is generated by the radical pair mechanism (RPM). The geminate RPM polarization is found to be considerably smaller than the F-pair one, 32±2 and 48±5 in units of the Boltzmann polarization, respectively. This is attributed to an initial radical separation in the geminate pair, caused by the cleavage reaction. Besides cleavage, the photoexcitedtrans-azocumene also decays via isomerization to the thermally unstablecis-isomer, the lifetime of which is found to be 14±3 μs at 293 K in benzene, three times longer than in cyclohexane. The quantum yield of free radicals, escaping from the primary cage, is determined as 0.28±0.06 for the decay of the excitedtrans-azocumene and 0.18±0.04 for the thermal cleavage of thecis-isomer. The self-termination of cumyl radicals proceeds with a rate constant 2k _t=7±1)·10⁸ M^?1s^?1 in benzene at RT.     

Radical chemistry of glucosamine naphthalene acetic acid and naphthalene acetic acid: a pulse radiolysis study

Free radical‐induced oxidation reactions of glucosamine naphthalene acetic acid (GNaa) and naphthalene acetic acid (Naa) have been studied using pulse radiolysis. GNaa was synthesized by covalently attaching Naa on glucosamine. Hydroxyl adduct (from the reaction of hydroxyl radicals (^●OH) at the naphthalene ring) was identified as the major transient intermediate (suggesting that the ^●OH reaction is on the naphthalene ring) and is characterized by its absorption maxima of 340 and 400 nm. Both GNaa and Naa undergo similar reaction pattern. The bimolecular rate constants determined for the reactions are 4.8 × 10⁹ and 8.9 × 10⁹ dm³ mol^?1 s^?1 for GNaa and Naa respectively. The mechanism of reaction of ^●OH with GNaa was further confirmed using steady‐state method. Radical cation of GNaa was detected as an intermediate during the reaction of sulfate radical (SO₄^●?) with GNaa (k₂ = 4.52 × 10⁹ dm³ mol^?1 s^?1). This radical cation transforms to a ^●OH adduct at higher pH. The radical cation of GNaa is comparatively long lived, and a cyclic transition state by neighboring group participation accounts for its stability. The oxy radical anion (O^●?) reacts with GNaa (k₂ = 1.12 × 10⁹ dm³ mol^?1 s^?1) mainly by one‐electron transfer mechanism. The reduction potential values of Naa and GNaa were determined using cyclic voltammetric technique, and these are 1.39 V versus NHE for Naa and 1.60 V versus NHE for GNaa. Copyright © 2014 John Wiley & Sons, Ltd.     

On the equilibrium of peroxy radicals in the oxidation of 2-propanol by molecular oxygen

Based on kinetic data, it is demonstrated that the 1-hydroxy-1-methylethylperoxyl and hydroperoxy radicals are at equilibrium in the oxidation of 2-propanol at 323°C initiated by azobisisobutyronitrile. It is shown that the equilibrium of between the peroxy radicals is not disturbed within the covered range of alcohol concentrations. The rate constants of the reaction of the hydroperoxy radical with 2-propanol is determined: k _2.2 = (0.13 ± 0.04) L mol^?1 s^?1.     

Application of a Stopped-Flow EPR Method for the Detection of Short-Lived Flavonoid Semiquinone Radicals Produced by Oxidation Using <Superscript>15</Superscript>N-Labeled Nitrosodisulfonate Radical (Fremy’s Salt)

A stopped-flow-electron paramagnetic resonance (EPR) method was applied for the detection of short-lived radicals of flavonoids bearing a catechol moiety as the B-ring, such as flavonols (quercetin, fisetin, and rutin), flavanones (eriodictyol and taxifolin), flavanols (catechin and epicatechin), and flavone (luteolin). ¹⁵N-labeled sodium salt of nitrosodisulfonate (¹⁵NDS) was employed to obtain the highly resolved EPR hyperfine structure (hfs) of flavonoid-derived semiquinone radicals under stoichiometrically regulated reaction conditions in aqueous media (pH 10). The EPR hfs of these flavonoids radicals, except catechin and epicatechin, were recorded. Based on the g value and the proton hyperfine coupling constants (hfcc), these flavonoid-derived radicals were assigned to be semiquinone radicals of the catechol moiety (B-ring). For example, the observed EPR hyperfine structure (hfs) of the luteolin radical (Lut^?·) was composed of four sets of doublet splitting, which could be ascribed to the three protons of the B-ring (a^2′?=?0.136, a^5′?=?0.102, and a^6′?=?0.272 mT) and a vinyl proton of the C-ring (a³?=?0.099 mT). In addition, the characteristically small doublet splitting resolved for the fisetin anion radical (Fis^?·, 0.028 mT) was assigned to the aromatic proton at the C₅ carbon of the A-ring, indicating that the unpaired electron of the radials was partially delocalized onto the A-ring through the π bonds involved in the vinyl-carbonyl moiety of the C-ring. The hfcc of the methine protons at the C₂ carbon of taxifolin and eriodictyol-derived radicals (Tax^?· and Eri^?·) was, respectively, evaluated to be 0.102 and 0.230 mT. The assignment of the proton hfcc of flavonoid-derived semiquinone radicals will be discussed in relation with the molecular structure of the C-ring.     

Low field CIDNP of amino acids and proteins: characterization of transient radicals and NMR sensitivity enhancement

A new method for measuring and exploiting the magnetic field dependence of chemically induced dynamic nuclear polarization (CIDNP) is described. A solution of an amino acid or protein together with a flavin photosensitizer is irradiated with laser light at a position in the bore of a superconducting NMR magnet where the field is between 0.1 T and 7.0 T. The polarized sample is then transferred by rapid injection into an NMR tube at the centre of the magnet (at 9.4 T), where the spectrum is recorded. The observed ¹H CIDNP field dependence of tyrosine agrees well with the diffusion model of the radical pair mechanism. The field dependence of histidine, tryptophan and methionine CIDNP allows the g values of the transient radicals responsible for the polarization to be determined. Experiments in which amino acids compete for the photoexcited flavin indicate that methionine residues could be used as probes of surface accessibility, especially if the polarization is generated in low fields (～ 0.7 T) and detected in high fields (≥ 9.4 T). Possible extensions of the technique to study protein folding and the structures of partially denatured states of proteins are discussed.     

Abilities of Antioxidants to Eliminate the Peroxyl Radical Derived from 2,2′-Azobis(2-Amidinopropane) Dihydrochloride (AAPH)

Electron spin resonance (ESR) of 5-(2,2-dimethyl-1,3-propoxy cyclophosphoryl)-5-methyl-1-pyrroline N-oxide (CYPMPO) spin adducts of free radicals derived from the UV irradiation of 2,2′-azobis(2-amidinopropane) dihydrochloride (AAPH) under rapid scanning condition was examined. The ESR signal obtained was the superposition of two spectra, the spin adduct of the alkoxyl radical [CYPMPO-ORa, Ra = C(CH₃)₂-C(⁺NH₂Cl^?)NH₂] and the peroxyl radical (CYPMPO-OORa). The decay rate of CYPMPO-OORa was 8,400 times that of CYPMPO-ORa. The order of the k _SB/k _ST values (the slope of the Stern–Volmer’s plot) for the peroxyl radical (RaOO·) was l-ascorbic acid > caffeic acid > rutin ~ Trolox ~ (+)-catechin ~ glutathione (reduced), which was almost the same order as that for the alkoxyl radical (RaO·). Though the k _SB/k _ST value of each antioxidant for the peroxyl radical was about half of that for the alkoxyl radical, the ratios of the values of antioxidants to that of trolox (the relative ORAC values) were almost the same between the peroxyl and alkoxyl radicals. The relative oxygen radical absorbance capacity (ORAC) values of polyphenols estimated by the ORAC-ESR assay using both peroxyl and alkoxyl radicals were smaller than those by the ORAC-FL assay.     

Dimer radical cation of 4‐thiouracil: a pulse radiolysis and theoretical study

Pulse radiolysis with optical absorption detection has been used to study the reactions of hydroxyl radical (OH^?) with 4‐thiouracil (4TU) in aqueous medium. The transient absorption spectrum for the reaction of OH^? with 4TU is characterized by λ_max 460 nm at pH 7. A second‐order rate constant k_(4TU+OH) of 1.7 × 10¹⁰ M^?1 s^?1 is determined via competition kinetics method. The transient is envisaged as a dimer radical cation [4TU]₂^?+, formed via the reaction of an initially formed radical cation [4TU]^?+ with another 4TU. The formation constant of [4TU]₂^?+ is 1.8 × 10⁴ M^?1. The reactions of dibromine radical ion (Br₂^??) at pH 7, dichlorine radical ion (Cl₂^??) at pH 1, and azide radical (N₃^?) at pH 7 with 4TU have also produced transient with λ_max 460 nm. Density functional theory (DFT) studies at BHandHLYP/6–311 + G(d,p) level in aqueous phase showed that [4TU]₂^?+ is characterized by a two‐centerthree electron (2c‐3e) [?S∴S?] bond. The interaction energy of [?S∴S?] bond in [4TU]₂^?+ is ?13.01 kcal mol^?1. The predicted λ_max 457 nm by using the time‐dependent DFT method for [4TU]₂^?+ is in agreement with experimental λ_max. Theoretical calculations also predicted that compared with [4TU]₂^?+, 4‐thiouridine dimer is more stable, whereas 4‐thiothymine dimer is less stable. Copyright © 2013 John Wiley & Sons, Ltd.     

A Novel Ratiometric Fluorescent Mercury Probe Based on Deprotonation-ICT Mechanism

A new NBD-rhodamine dye (1) was developed as a colorimetric and ratiometric fluorescent chemosensor for Hg²⁺ with good selectivity in aqueous ethanol solutions under neutral to basic conditions. Sensor 1 showed absorption at 468 nm and a weak emission at 529 nm (? _F ?=?0.063) in ethanol/aqueous tris buffer (9:1, v/v) of pH 9.17 solution. Bathochromic shifts in both absorption (492 nm) and fluorescence spectra (569 nm, ? _F ?=?0.129), respectively upon addition of 2 equiv. of Hg²⁺ were observed. The ring-opening reaction of the spirolactam form to the corresponding xanthene form was not found. The interaction of Hg²⁺ with chemosensor 1 resulted in the deprotonation of the secondary amine conjugated to the NBD component so that the electron-donating ability of the N atom was enhanced. Deprotonation-ICT mechanism of secondary amines was suggested for the ratiometric fluorescent chemosensing for Hg²⁺.     

Evidence for a possible role of 3‐hydroxyanthranilic acid as an antioxidant

The reactions of 3‐hydroxyanthranilic acid (3‐OHAA) with N₃^?, NO₂^?, NO^?, CCl₃O₂^? , and OH^? radicals were examined using a pulse radiolysis technique mainly at pH 7. The bimolecular electron transfer from secondary one‐electron oxidants results in the formation of anilino radical (λ_max ? 380 nm). The rate constant for the reaction of N₃^? radical with 3‐OHAA at pH 7 was found to be 6.3 × 10⁹ dm³ mol^?1 s^?1. It was observed that the 3‐OHAA reacts with oxygen centered radicals. The repair rate constant for the electron transfer reaction from 3‐OHAA to guanosine radical and chlorpromazine cation radical was also examined using a pulse radiolysis technique. Kinetic studies indicate that 3‐OHAA may act as an antioxidant to repair free‐radical damage to above mentioned biologically important compounds. The rate constants of electron transfer from the 3‐OHAA to the guanosine and chlorpromazine radicals were determined. The one‐electron reduction potential for 3‐OHAA radical was found to be 0.53 ± 0.06 V versus NHE. Copyright © 2008 John Wiley & Sons, Ltd.     

Analysis of the Λb → Λ?+?? transition in the SM4 using form factors from full QCD

Using the responsible form factors calculated via full QCD, we analyze the ?? _b ?? ???⁺?^? transition in the standard model containing fourth-generation quarks (SM4). We discuss the effects of the presence of the t?? fourth-family quark on related observables like branching ratio, forward-backward asymmetry, baryon polarization as well as double-lepton polarization asymmetries. We also compare our results with those obtained in the SM as well as with predictions of the SM4 but using form factors calculated within heavy-quark effective theory. The obtained results on the branching ratio indicate that the ? transition is more probable in full QCD comparing to the heavy-quark effective theory. It is also shown that the results on all considered observables in the SM4 deviate considerably from the SM predictions when m _t?? ?? 400 GeV.     

Magnetic field effects on the hydrogen abstraction reaction of triplet 4-methoxybenzophenone with thiophenol as studied by a picosecond laser flash photolysis technique

The hydrogen abstraction reaction of triplet 4-methoxybenzophenone with thiophenol at 265 K has been studied with a newly developed picosecond laser flash photolysis apparatus under magnetic fields of 0–1.7 T. The decay rate constant of the radical pair generated was found to increase from 3.42 × 10⁹ s^?1 to 4.15 × 10⁹ s^?1 with increasing the magnetic field from 0 to 1.7 T. The observed magnetic field effects can be explained by the Δg mechanism. Using the simple kinetics model with the Δg mechanism, the rate constant of the escape process from the pair (k _esc) and two rate constants for the T-S spin conversion process (k _T-S) at 0 and 1.7 T were found to be 1.97 × 10⁹ s^?1, 1.45 × 10⁹ s^?1, and 2.12 × 10⁹ s^?1, respectively. From the magnetic field dependence on k _T-S, the difference in the g values of the 4-methoxybenzophenone ketyl and phenylthiyl radicals was estimated to be 0.0087.     

Gas‐phase oxidation of CH2 = C(CH3)CH2Cl initiated by OH radicals and Cl atoms: kinetics and fate of the alcoxy radical formed

Relative kinetics of the reactions of OH radicals and Cl atoms with 3‐chloro‐2‐methyl‐1‐propene has been studied for the first time at 298 K and 1 atm by GC‐FID. Rate coefficients are found to be (in cm³ molecule^?1 s^?1): k₁ (OH + CH₂ = C(CH₃)CH₂Cl) = (3.23 ± 0.35) × 10^?11, k₂ (Cl + CH₂ = C(CH₃)CH₂Cl) = (2.10 ± 0.78) × 10^?10 with uncertainties representing ± 2σ. Product identification under atmospheric conditions was performed by solid phase microextraction/GC‐MS for OH reaction. Chloropropanone was identified as the main degradation product in accordance with the decomposition of the 1,2‐hydroxy alcoxy radical formed. Additionally, reactivity trends and atmospheric implications are discussed. Copyright © 2015 John Wiley & Sons, Ltd.     

Electron spin polarization in consecutive spin-correlated radical Pairs: Application to short-lived and long-lived precursors in type 1 photosynthetic reaction centres

An analytical treatment of the spin dynamics in sequential photoinduced correlated coupled radical pairs is presented and applied to the spectra of the states P⁺A ₁ ^? and P⁺F _x ^? in type 1 photo-synthetic reaction centres. Expressions for the spin polarized spectra are derived for the specific limiting cases of a very short-lived and very long-lived primary radical pair which correspond to the situation found in heliobacteria and photosystem I (PSI), respectively. The inhomogeneous line-broadening due to the unresolved hyperfine couplings is taken explicitly into account. It is shown that the density matrix of the secondary pair ρ₂ can be written as the sum of two terms corresponding to (i) the part which is independent of the spin dynamics in the precursor, (ii) the additional spin polarization which is generated during the lifetime of the precursor and transferred to the secondary pair. The latter term contains two contributions which arise from the difference of the Zeeman interactions of the radicals in the primary pair and from the inhomogeneous line broadening. The predicted polarization patterns are compared to those established for chemically induced dynamic electron polarization (CIDEP) when uncoupled radicals are generated from a radical pair precursor. The expressions are then used to simulate the experimental spectra of the consecutive pairs P⁺A ₁ ^? and P⁺F _x ^? in PSI using parameters derived entirely from independent experimental data. Excellent agreement with the experimental results is obtained. The spectra of P⁺F _x ^? in heliobacteria at X- and K-band are also simulated and it is shown that the observed polarization patterns can be reproduced assuming direct electron transfer from A₀ to F_x with a time constant ofτ = 600 ps.     

Kinetics of the oxidation of 2-Propanol with molecular oxygen under conditions of selective inhibition

The specifics of the mechanism of chain propagation in the radical-chain oxidation of 2-propanol at 323 K were studied by the method of selective inhibition with the use of nitrobenzene and ionol. The rate constants for the interaction of the 1-hydroxy-1-methylethylperoxy (k _2.1 = (0.097 ± 0.004) L mol^?1 s^?1) and hydroperoxy (k _2.2 = 0.13 ± 0.05 L mol^?1 s^?1) radicals with alcohol and their relative contributions to the chain propagation reactions were determined.