Direct ESR detection of pentadienyl radicals and peroxyl radicals in lipid peroxidation: mechanistic insight into regioselective oxygenation in lipoxygenases

Well-resolved ESR spectra of free pentadienyl radicals have been observed under photoirradiation of di-tert-butylperoxide (Bu(t)OOBu(t)) and polyunsaturated fatty acids in the absence of O(2), allowing us to determine the hfc values. The hfc values of linoleyl radical indicate that the spin density is the largest at the C-11 position. The linoleyl radical is readily trapped by O(2) to produce the peroxyl radical (11-HPO.) in which O(2) is added mainly at the C-11 position of the pentadienyl radical as indicated by the comparison of the ESR spectra of peroxyl radicals derived from linoleic acid and [11,11-(2)H(2)]linoleic acid. The peroxyl radical (13-HPO.), which is initially formed by the hydrogen abstraction from 13-(S)-hydroperoxy-9(Z),11(E)-octadecadienoic acid (13-HPOD) by Bu(t)O., is found to isomerize to 11-HPO. via removal of O(2) from 13-HPO. and addition of O(2) to linoleyl radical to produce 11-HPO. . This finding supports an idea of O(2) entering via a specific protein channel, which determines the stereo- and regiochemistry of the biradical combination between O(2) and linoleyl radical in lipoxygenases.     

Separation of polyunsaturated fatty acid radicals by high-performance liquid chromatography with electron spin resonance and ultraviolet detection

In the reaction of soybean lipoxygenase (EC 1.13.11.12) with polyunsaturated fatty acids such as linoleic, linolenic and arachidonic acids, some radical species were detected using the electron spin resonance (ESR) spin-trapping technique. The radical species derived from the three polyunsaturated fatty acids were not distinguishable because the ESR spectra of the spin adducts of nitrosobenzene with their three radical species showed no difference in their hyperfine splittings. To overcome this defect of the spin-trapping technique, these spin-adducts were separated by employing high-performance liquid chromatography (HPLC) combined with ESR spectroscopy. The spin adducts were eluted from a C18 reversed-phase column in the order linolenic acid, linoleic acid and arachidonic acid. The half-lives of the spin adducts separated by HPLC-ESR were determined as linoleic acid 600 min, linolenic acid 360 min and arachidonic acid 160 min. The use of an ultraviolet detector together with the HPLC-ESR technique resulted in a 500-fold increase in sensitivity in the detection of the radical species.     

Pulsed EPR and DFT characterization of radicals produced by photo-oxidation of zeaxanthin and violaxanthin on silica-alumina

Pulsed electron nuclear double resonance (ENDOR) and two-dimensional (2D)-hyperfine sublevel correlation spectroscopy (HYSCORE) studies in combination with density functional theory (DFT) calculations revealed that photo-oxidation of natural zeaxanthin (ex Lycium halimifolium) and violaxanthin (ex Viola tricolor) on silica-alumina produces the carotenoid radical cations (Car*+) and also the neutral carotenoid radicals (#Car*) as a result of proton loss (indicated by #) from the C4(4') methylene position or one of the methyl groups at position C5(5'), C9(9'), or C13(13'), except for violaxanthin where the epoxide at positions C5(5')-C6(6') raises the energy barrier for proton loss, and the neutral radicals #Car*(4) and #Car*(5) are not observed. DFT calculations predict the largest isotropic beta-methyl proton hyperfine couplings to be 8 to 10 MHz for Car*+, in agreement with previously reported hyperfine couplings for carotenoid pi-conjugated radicals with unpaired spin density delocalized over the whole molecule. Anisotropic alpha-proton hyperfine coupling tensors determined from the HYSCORE analysis were assigned on the basis of DFT calculations with the B3LYP exchange-correlation functional and found to arise not only from the carotenoid radical cation but also from carotenoid neutral radicals, in agreement with the analysis of the pulsed ENDOR data. The formation of the neutral radical of zeaxanthin should provide another effective nonphotochemical quencher of the excited state of chlorophyll for photoprotection in the presence of excess light.     

Regio‐ and Stereospecificity in the Oxygenation of Arachidonic Acid Catalyzed by Leu597 Mutants of Rabbit 15‐Lipoxygenase: A QM/MM Study

We combined quantum mechanics/molecular mechanics calculations with molecular dynamics simulations to study the addition of O₂ to the pentadienyl radical of arachidonic acid (AA) catalyzed by the Leu597Val and Leu597Ala mutants of rabbit 15‐lipoxygenase (15‐rLO). In the Leu597Val mutant, the addition of O₂ to C15 of AA is the predominant path, although it reduces the C15/C11 product ratio by almost ten times with respect to the wildtype enzyme. The S stereochemistry is kept. Mutation to Ala causes just the opposite effect: regiospecificity favoring addition to C15 is somewhat sharper than that in the wildtype, but the stereochemistry is R. This is because the extra space created by the mutation to Ala is big enough for AA to move so that it can adopt an alternative binding mode, and this opens new feasible paths for the attack of O₂. So, we showed that the Leu597Ala mutant of 15r‐LO works as an aspirin‐acetylated cyclooxygenase‐2, which makes 15‐(R)‐ hydroperoxyeicosatetraenoic acid.     

1H and 13C hyperfine coupling constants of the tryptophanyl cation radical in aqueous solution from microsecond time-resolved CIDNP

Relative values of the 1H and 13C isotropic hyperfine couplings in the cationic oxidized tryptophan radical TrpH*+ in aqueous solution are determined. The data are obtained from the photo-CIDNP (chemically induced dynamic nuclear polarization) enhancements observed in the microsecond time-resolved NMR spectra of the diamagnetic products of photochemical reactions in which TrpH*+ is a transient intermediate. The method is validated using the tyrosyl neutral radical Tyr*, whose 1H and 13C hyperfine couplings have previously been determined by electron paramagnetic resonance spectroscopy. Good agreement is found with hyperfine coupling constants for TrpH*+ calculated using density functional theory methods but only if water molecules are explicitly included in the calculation.     

Synthesis of the enediol isofurans, endogenous oxidation products of arachidonic acid

Isofurans (IsoF's) are a new class of human arachidonic acid oxidation products. They are produced in vivo by a free radical mechanism, independent of the cyclooxygenase enzymes. These new compounds are available from natural sources only in microgram quantities as mixtures. The enantioselective preparation of two enediol isofurans, 15-epi-ent-SC-Delta13-8-IsoF and ent-SC-Delta13-8-IsoF, is described. A key transformation in the synthesis is the selective cascade cyclization of a diol epoxide benzenesulfonate to give the substituted tetrahydrofuran skeleton of the isofurans. This synthesis will make these metabolites available for physiological evaluation.     

高效液相色谱-电喷雾串联四级杆质谱法检测小鼠组织中花生四烯酸及其代谢产物

采用反相高效液相色谱梯度洗脱,电喷雾串联质谱多反应检测模式,同时分离测定小鼠组织中花生四烯酸类代谢产物.组织样品经低温超声破碎细胞,采用乙酸乙酯进行两次萃取,LC-MS/MS检测.实验用3个内标,标准溶液稀释法建立了19种花生四烯酸类物质的标准曲线.方法学确证表明: 各被测物质的线性相关系数均大于0.99;定量限(LOQ)为3～250 μg/L;相对回收率在70.7%～123.0%之间;方法重复性(RSD)均小于20%.运用本法在小鼠肺、肝、脾组织中检测到了几种花生四烯酸类代谢产物,并对肺组织里的8种代谢产物进行了含量测定,结果为11-羟基二十碳四烯酸(58.3±11.6)ng/g,15-羟基二十碳四烯酸(52.6±15.5)ng/g,前列腺素E2(210±102) ng/g,前列腺素D2(20.3±13.9)ng/g,前列腺素J2(39.2±15.9)ng/g,血栓素B2(58.3±28.4)ng/g,前列腺素F2α(3.8±6.0) ng/g,花生四烯酸(1980±544)ng/g.本方法灵敏度高、重复性良好,为研究生物组织样品中花生四烯酸类物质的代谢状况提供了分析基础.     

Fourier transform millimeter-wave spectroscopy of the ethyl radical in the electronic ground state

The pure rotational spectrum of the ethyl radical (C2H5) has been detected for the first time with the Fourier transform millimeter-wave spectrometer. The ethyl radical is produced by discharging the C2H5I gas diluted in Ar. The 1(01)-0(00) rotational transition of the ethyl radical is observed in the frequency range from 43,680 to 43,780 MHz. The observed spectrum shows a very complicated pattern of the fine and hyperfine structures of a doublet radical with the nuclear spins of five protons. The fine and hyperfine components are assigned with the aid of measurements of the Zeeman splittings. As a result, the 22 lines are ascribed to the transitions in the ground vibronic state (A2"). The rotational constant, the spin-rotation interaction constant, and hyperfine interaction constants are determined by the least-squares fit. The Fermi contact term of the alpha-proton is determined to be -64.1654 MHz in the gas phase, indicating that the structure of the -CH2 is essentially planar. The present rotational spectroscopic study further supports that the methyl group of the ethyl radical can be regarded as a nearly free internal rotor with a low energy barrier. A few unassigned lines still remain, which may be vibrational satellites of the internal rotation mode.     

Factors influencing the autoxidation of fatty acids: effect of olefin geometry of the nonconjugated diene

Autoxidations of cis,cis, cis,trans, and trans,trans nonconjugated octadecadienoates and pentadecadienes were carried out in the presence of alpha-tocopherol to investigate the effect of olefin geometry on this oxidation process and provide insight into the factors that influence the autoxidation of fatty acids. We have found that as the trans character of the diene increases, the amount of O(2) trapping at the central (bis-allylic) position of the pentadienyl radical also increases. In addition, the rate constant for beta-fragmentation (k(beta) approximately 10(6) s(-1)) of the bis-allylic peroxyl radical decreased on going from the cis,cis to the trans,trans diene. We have also found that for the cis,trans nonconjugated dienes, there is a preference for trapping of the pentadienyl radical by O(2) at the transoid end, generating the cis,trans conjugated hydroperoxide as the major product.     

13C hyperfine coupling constants in o-benzosemiquinones

The ¹³C hyperfine splitting constants of the tetrachloro-o-benzosemiquinone radical anion are measured for naturally occurring ¹³C nuclei in all the possible positions in the molecule. Their assignment is based on the linewidth analysis of the single hyperfine components and this is discussed with reference to the reported spin density distribution in o-semiquinones.     

Electron-nuclear double resonance spectroscopic evidence that S-adenosylmethionine binds in contact with the catalytically active [4Fe-4S](+) cluster of pyruvate formate-lyase activating enzyme

Pyruvate formate-lyase activating enzyme (PFL-AE) is a representative member of an emerging family of enzymes that utilize iron-sulfur clusters and S-adenosylmethionine (AdoMet) to initiate radical catalysis. Although these enzymes have diverse functions, evidence is emerging that they operate by a common mechanism in which a [4Fe-4S](+) interacts with AdoMet to generate a 5'-deoxyadenosyl radical intermediate. To date, however, it has been unclear whether the iron-sulfur cluster is a simple electron-transfer center or whether it participates directly in the radical generation chemistry. Here we utilize electron paramagnetic resonance (EPR) and pulsed 35 GHz electron-nuclear double resonance (ENDOR) spectroscopy to address this question. EPR spectroscopy reveals a dramatic effect of AdoMet on the EPR spectrum of the [4Fe-4S](+) of PFL-AE, changing it from rhombic (g = 2.02, 1.94, 1.88) to nearly axial (g = 2.01, 1.88, 1.87). (2)H and (13)C ENDOR spectroscopy was performed on [4Fe-4S](+)-PFL-AE (S = (1)/(2)) in the presence of AdoMet labeled at the methyl position with either (2)H or (13)C (denoted [1+/AdoMet]). The observation of a substantial (2)H coupling of approximately 1 MHz ( approximately 6-7 MHz for (1)H), as well as hyperfine-split signals from the (13)C, manifestly require that AdoMet lie close to the cluster. (2)H and (13)C ENDOR data were also obtained for the interaction of AdoMet with the diamagnetic [4Fe-4S](2+) state of PFL-AE, which is visualized through cryoreduction of the frozen [4Fe-4S](2+)/AdoMet complex to form the reduced state (denoted [2+/AdoMet](red)) trapped in the structure of the oxidized state. (2)H and (13)C ENDOR spectra for [2+/AdoMet](red) are essentially identical to those obtained for the [1+/AdoMet] samples, showing that the cofactor binds in the same geometry to both the 1+ and 2+ states of PFL-AE. Analysis of 2D field-frequency (13)C ENDOR data reveals an isotropic hyperfine contribution, which requires that AdoMet lie in contact with the cluster, weakly interacting with it through an incipient bond/antibond. From the anisotropic hyperfine contributions for the (2)H and (13)C ENDOR, we have estimated the distance from the closest methyl proton of AdoMet to the closest iron of the cluster to be approximately 3.0-3.8 A, while the distance from the methyl carbon to the nearest iron is approximately 4-5 A. We have used this information to construct a model for the interaction of AdoMet with the [4Fe-4S](2+/+) cluster of PFL-AE and have proposed a mechanism for radical generation that is consistent with these results.     

The Radical Ions of Dipleiadiene (Dicyclohepta[de,ij]naphthalene) and Its 12b,12c-Homo Derivative. An ESR and ENDOR study

The radical anions and the radical cations of dipleiadiene (dicyclohepta[de,ij]naphthalene; 1 ) and its 12b, 12c-homo derivative 2 were characterized by ESR and ENDOR spectroscopy. Their singly occupied orbitals are related to the degenerate nonbonding MOs of a 16-membered π-perimeter. The π-spin distribution over the perimeter is similar in the radical cations 1 ^.+ and 2 ^.+, and an analogous statement holds for the radical anions 1 ^.? and 2 ^.?. However, deviations of the π-system from planarity lead to a decrease in the absolute values of the negative coupling constants of the perimeter protons in 2 ^.+ and 2 ^.? relative to those in 1 ^.+ and 1 ^.?. The hyperfine data for the perimeter protons in the radical ions correlate with the changes in ¹³C chemical shifts on passing from the neutral compounds to the corresponding diions. It is concluded from the coupling constants of the CH₂ protons in the radical ions of 2 that the cation 2 ^.+ exists in the methano-bridged form ( A ) of the neutral 2 (and, presumably, also of the dication 2 ²⁺), whereas the anion 2 ^.? adopts the bisnorcaradiene form ( B ) of the dianion 2 ^2?.     

9-Mercaptodethiobiotin is generated as a ligand to the [2Fe-2S]+ cluster during the reaction catalyzed by biotin synthase from Escherichia coli

Biotin synthase catalyzes formation of the thiophane ring through stepwise substitution of a sulfur atom for hydrogen atoms at the C9 and C6 positions of dethiobiotin. Biotin synthase is a radical S-adenosylmethionine (SAM) enzyme that reductively cleaves S-adenosylmethionine, generating 5'-deoxyadenosyl radicals that initially abstract a hydrogen atom from the C9 position of dethiobiotin. We have proposed that the resulting dethiobiotinyl radical is quenched by the μ-sulfide of the nearby [2Fe-2S](2+) cluster, resulting in coupled formation of 9-mercaptodethiobiotin and a reduced [2Fe-2S](+) cluster. This reduced FeS cluster is observed by electron paramagnetic resonance spectroscopy as a mixture of two orthorhombic spin systems. In the present work, we use isotopically labeled 9-mercaptodethiobiotin and enzyme to probe the ligand environment of the [2Fe-2S](+) cluster in this reaction intermediate. Hyperfine sublevel correlation spectroscopy (HYSCORE) spectra exhibit strong cross-peaks demonstrating strong isotropic coupling of the nuclear spin with the paramagnetic center. The hyperfine coupling constants are consistent with a structural model for the reaction intermediate in which 9-mercaptodethiobiotin is covalently coordinated to the remnant [2Fe-2S](+) cluster.     

The effects of a thiol-containing quinazolinedione derivative (MECH) on the lipid oxidation in bilayer liposomes

In order to investigate the radical chemistry of 3-(2-mercaptoethyl)quinazoline-2,4(1H, 3H)-dione (MECH) in homogeneous and liposomal solutions experiments were performed with pulse radiolysis, γ radiolysis and the chemical radical intiator 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH). It is shown that the thiol group represents the most sensitive group to radical attack. The thiyl radical originating from MECH is detected indirectly by product analysis and by pulse radiolysis. The thiyl radical can abstract bisallylic hydrogens from polyunsaturated fatty acids. This is shown by pulse radiolysis in homogeneous and liposomal solutions via the formation of the pentadienyl radical which has a strong and characteristic absorption band at 280 nm.     

Microenvironmental and conformational structure of triblock copolymers in aqueous solution by 1H and 13C NMR spectroscopy

1H and 13C nuclear magnetic resonance (NMR) spectra of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) triblock copolymers in D2O solutions have been systematically investigated. The detailed assignments of various 1H and 13C NMR signals are presented. The hyperfine structure of PO -CH2- protons was clearly assigned, the arising reason of this hyperfine structure was attributed to the influence of the chiral center of -CHCH3- groups and the direct coupling between the PO -CH2- and -CH3 protons. The external standard 2,2-dimethyl-2-silapentane-5-sulfonate sodium salt (DSS) was firstly applied in this system. Accurate chemical shift values referenced to the external standard DSS were obtained. 1H NMR chemical shift of PO -CH2- and -CH3 signals shows a larger decrease in ppm values than that of EO -CH2- signal with the increase of PPO/PEO ratio or temperature indicating that PO segments exist in a more hydrophobic microenvironment. A new resonance signal assigned to the PO -CH2- protons appeared when the temperature is above the CMT, which is attributed to the breakdown of the intra-molecular (C-H)...O hydrogen bond between the PO -CH2- protons and the ester oxygens. The breakdown of this intra-molecular hydrogen bond may result in a decrease of gauche conformers of the PPO chain. The increase of 13C NMR chemical shift of block copolymers validates this conformational change assumption. It can be inferred that the amount of gauche conformers decreases whereas that of trans conformers increases in both PO and EO chains when elevating the PPO/PEO ratio or temperature. The observed 13C NMR chemical shifts of PO segments show a bigger increase than those of EO segments, supporting the formation of a nonpolar microenvironment around PO segments.     

Preparation of a mercaptopropyl bonded silica intermediate in supercritical carbon dioxide: synthesis, characterisation and chromatography of a quinine based chiral stationary phase

This research examines the preparation of a mercaptopropyl bonded silica intermediate in supercritical carbon dioxide (sc-CO(2)) and the subsequent conversion in sc-CO(2) to a quinine derived chiral stationary phase (CSP). The effects of reaction temperature, pressure and time on the surface coverage of the silica intermediate were investigated when porous silica particles (Exsil-Avanti, 3microm) were reacted with 3-trimethoxymercaptopropylsilane in sc-CO(2). We present results which demonstrate that a stable mercaptopropyl bonded silica intermediate can be successfully prepared under supercritical conditions of 40 degrees C, 483bar, in a substantially reduced reaction time of 1h with superior surface coverages compared to organic solvent based methods. The further utility of this supercritical fluid technology was demonstrated by the free radical addition of a quinine derived chiral selector onto a mercaptopropyl bonded silica intermediate in sc-CO(2). This supercritical fluid generated chiral stationary phase (CSP) was utilised for the direct LC enantioseparation of a series of 3,5-dinitrobenzoyl (DNB) amino acids. Bonded silica samples were characterised using elemental analysis, diffuse reflectance infrared fourier transform (DRIFT) spectroscopy, solid state (13)C and (29)Si CP-MAS NMR spectroscopy, and thermogravimetric analysis (TGA). This supercritical fluid functionalisation approach offers an efficient and cleaner alternative to existing organic solvent based approaches for the preparation of bonded silica phases.     

A study of radicals arising in the low-temperature radiolysis of thiophene and some of its derivatives

EPR was used to investigate the radicals formed by radiolysis of furan, thiophene, and some of their substitution products having the general formula C₄H₃RS, where R = Cl, Br, COOH, C(CH₃)₃, and of di-tert-butylthiophene. By analyzing the EPR spectra it is shown that radicals formed by radiolysis of furan, thiophene, their alkyl substitution products, and 2-thiophene carboxylic acid arise in primary radiation-chemical events with splitting of the C-H bond in both ring and side chain. Characteristic of the radiolysis of these compounds is formation of secondary radicals by addition of hydrogen atoms at the double bond of the heteroaromatic ring. The values of the hyperfine splittings in the spectra of the secondary radicals are, for protons of methylene group 32 e [positions 2(5)] or 40 e [positions 3(4)], and 13 e for protons of the thiophene ring. In radiolysis of 2-chlorothiophene and 3-bromothiophene the main products are radicals with the unpaired electron localized on the sulfur atom.     

Conformational disorder in the propagating radical of dimethacrylate polymers

Photopolymerization of methacrylic monomers yields samples with trapped radicals that are easily detected by electron paramagnetic resonance (EPR) spectroscopy. Despite its simplicity, there is no general agreement about the interpretation of this spectrum, in particular, about the role of methylene β protons. An extensive ENDOR study of the propagating radical in photopolymerized dimethacrylates has been carried out in order to obtain detailed information about methylene hyperfine couplings and, thus, about radical conformation. It is shown that literature models are not able to reproduce the ENDOR results and that only accurate fitting of ENDOR spectra obtained by saturating the EPR spectrum at different positions gives reliable information about radical conformation, thanks to the exploitation of conformational selectivity. It turns out that most radicals are in the minimum energy conformation, but any possible conformation is assumed by non negligible fractions of radical.     

Radical Ions in the Pentalene Series [1]. Part II. Dibenzo [b,f]pentalene and its 5, 10-dimethyl derivative

Proton hyperfine data have been determined for the radical anions and cations of dibenzo [b,f]pentalene (III) and its 5, 10-dimethyl derivative (IV) . The assignment of the coupling constants to pairs of equivalent protons follows from a simple MO model, the use of which enables one to reproduce satisfactorily the experimental values. The proton hyperfine data, a_Hμ

1 The meaning of ^aH_μis ^aH? C(x),H? C(y),whereas only x and y are given in the particular cases.

, for the radical anion III?. correlate fairly well with the π-charge populations ?_μ derived from ¹H-NMR. spectra for the carbon centres μ in the dianion III^2?. The analogous correlation is less good in the case of the radical cation III⊕. and the dication III^2⊕., presumably due to the rough approximations involved in the evaluation of the numbers ?_μ for the latter species. The coupling constants a_H5,10 for III?. and III⊕. are very close to the corresponding values a_H4,6 for the radical ions of 1,3,5-tri-t-butylpentalene (II), in accord with the prediction of the MO model. A similarity is also found between the proton hyperfine data for III?. and those for the radical anions of structurally related 1,4-diphenyl-1,3-butadiene (V). On the other hand, there are striking changes in the coupling constants of the analogous protons on passing from III?. to the radical anions of dibenzo [a,e] cyclooctene (VI) and [16] annulene (VII), as a consequence of raising the symmetry from C_2h to D_2h and D_4h, respectively.