Assignment of the EPR spectrum of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) superoxide spin adduct

[structure: see text] Spin trapping consists of using a nitrone or a nitroso compound to "trap" an unstable free radical as a long-lived nitroxide that can be characterized by electron paramagnetic resonance (EPR) spectroscopy. The formation of DMPO-OOH, the spin adduct resulting from trapping superoxide (O(2)(*)(-)) with 5,5-dimethyl-1-pyrroline N-oxide (DMPO), has been exploited to detect the generation of superoxide in a wide variety of biological and chemical systems. The 12-line EPR spectrum of DMPO-OOH has been either reported or mentioned in more than a thousand papers. It has been interpreted as resulting from the following couplings: A(N) approximately 1.42 mT, A(H)beta approximately 1.134 mT, and A(H)gamma(1H) approximately 0.125 mT. However, the DMPO-OOH EPR spectrum has an asymmetry that cannot be reproduced when the spectrum is calculated considering a single species. Recently, it was proposed that the 0.125 mT splitting was misassigned and actually results from the superimposition of two individual EPR spectra associated with different conformers of DMPO-OOH. We have prepared 5,5-dimethyl-[3,3-(2)H(2)]-1-pyrroline N-oxide (DMPO-d(2)), and we showed that the EPR spectrum of the corresponding superoxide spin adduct is composed of only six lines, in agreement with the assignment of the 0.125 mT splitting to a gamma-splitting from a hydrogen atom bonded to carbon 3 of DMPO. This result was supported by DFT calculations including water solvation, and the asymmetry of the DMPO-OOH EPR spectrum was nicely reproduced assuming a chemical exchange between two conformers.     

PROPERTIES OF THE ULTRAVIOLET-LIGHT-MEDIATED BINDING OF BOVINE SERUM ALBUMIN TO DNA*

Abstract— The binding of DNA to protein mediated by U V (254 nm) radiation has been investigated using binding of the complex to Millipore membrane filters as an assay technique. The reaction proceeds through an activated protein intermediate which then reacts with the DNA. The activated protein has a half-life of about 75 min at 0°C and about 18 min at 37°C. Short wavelengths are more efficient in forming the complex than wavelengths in the 250–280 nm range. N-ethyl maleimide treatment of protein before irradiation markedly inhibits the reaction.     

Characterization of PEDOT film functionalized with a series of automated synthesis ferrocenyl-containing oligonucleotides

In previous works we have described a fully automated synthesis of new ferrocene labelled oligonucleotides (Fc-ODNs) probes with one or more electroactive markers at different position in the chain. These Fc-ODNs have shown good properties to detect ODN target in solution. Here we describe the post-functionalization of a conducting co-polymer based on ethylenedioxythiophene (EDOT) derivatives by a series of Fc-ODNs. The grafting of the Fc-ODNs probes resulted in the appearance of the ferrocene redox couple which directly confirm the effectiveness of the ODN anchoring compared to traditional approach based on IR spectroscopy and X-ray fluorescence of the films. Moreover, the electrochemical response of the modified electrodes analysed in organic media before and after hybridization with ODN target confirm that properties obtain in solution for Fc-ODNs already exist in the film. The changes in the current intensity were found to be dependant on the structure of the grafted ODN that validate our strategy to synthesize an optimal Fc-ODNs.     

Improved solid-phase peptide synthesis of ‘difficult peptides’ by altering the microenvironment of the developing sequence

Three types of resins, related to the spacer, environmental and microenvironmental models were prepared by grafting commercial AMP polymer with 2-[2-(2-aminoethylamino)-2-oxoethoxy]acetic acid. All resins were highly loaded and functionalized with Rink-amide linker. A comparative synthesis of the classic difficult sequence ACP (65-74) on the prepared resins by Fmoc/t-Bu chemistry is presented. The ‘microenviromental’ model resin afforded the crude peptide in the highest purity (98%).     

Efficient, protection-free Suzuki-Miyaura synthesis of ortho-biphenyltetrazoles

We describe an efficient protocol for the Suzuki-Miyaura synthesis of ortho-biphenyltetrazoles from non-protected 2-bromophenyltetrazole and arylboronic acids. The optimised conditions were achieved using [1,1′-bis(diphenylphosphino)ferrocene]dichloropalladium(II) as catalyst and Na₂CO₃ as base. A panel of structurally diverse arylboronic acids was used to demonstrate the scope of the coupling procedure.     

Functional organization of the outer membrane of escherichia coli: phage and colicin receptors as components of iron uptake systems.

The functional interaction of outer membrane proteins of E. coli can be studied using phage and colicin receptors which are essential components of penetration systems. The uptake of ferric iron in the form of the ferrichrome complex requires the ton A and ton B functions in the outer membrane of E. coli. The ton A gene product is the receptor protein for phage T5 and is required together with the ton B function by the phages T1 and ?80 to infect cells and by colicin M and the antibiotic albomycin, a structural analogue of ferrichrome, to kill cells. The ton B function is necessary for the uptake of ferric iron complexed by citrate. Iron complexed by enterochelin is only transported in the presence of the ton B and feu functions. Cells which have lost the feu function are resistant to the colicins B, I or V while ton B mutants are resistant to all 3 colicins. The interaction of the ton A, ton B, and feu functions apparently permits quite different "substrates" to overcome the permeability barrier of the outer membrane. It was shown for ferrichrome dependent iron uptake that the complexing agent was not altered and could be used repeatedly. Only very low amounts of 3H-labeled ferrichrome were found in the cell. It is possible that the iron is mobilized in the membrane and that desferri-ferrichrome is released into the medium without having entered the cytoplasm. Growth on ferrichrome as the sole iron source was used to select revertants of T5 resistant ton A mutants. All revertants exhibited wild-type properties with the exception of partial revertants. In these 4 strains, as in the ton A mutants, the ton A protein was not detectable by SDS polyacrylamide gel electrophoreses of outer membranes. Albomycin resistant mutants were selected and shown to fall into 5 categories: 1) ton A; 2) ton B mutants; 3) mutants with no iron transport defects and normal ton A/ton B functions, which might be target site mutants; 4) mutants which were deficient in ferrichrome-mediated iron uptake but had normal ton A/ton B functions. We tentatively consider that the defect might be located in the active transport system of the cytoplasmic membrane; 5) a variety of mutants with the following general properties: most of them were resistant to colicin M, transported iron poorly, and, like ton B mutants, contained additional proteins in the outer membrane. The outer membrane protein patterns of wild-type and ton B mutant strains were compared by slab gel electrophoresis in an attempt to identify a ton B protein. It was observed that under most growth conditions, ton B mutants overproduced 3 proteins of molecular weights 74,000-83,000. In extracted, iron-deficient medium, both the wild-type and ton B mutant strains had similar large amounts of these proteins in their outer membranes. The appearance of these proteins was suppressed by excess iron in both wild-type and mutant. From this evidence it is apparent that the proteins appear as a response to low intracellular iron rather than being controlled by the ton B gene...     

Unusual heme-histidine bond in the active site of a chaperone

The heme chaperone CcmE is essential for the delivery of heme to c-type cytochromes. It forms an unusual transient, yet covalent, bond between an essential histidine, H130, and heme. We report on the discovery of the chemical structure of this bond solved by NMR, where the heme vinyl is cross-linked at the beta carbon to the Ndelta1 of H130. As this type of heme linkage has not been described previously in any cytochrome or hemoprotein, it represents a novel type of heme-histidine complex.     

Substituent effects on the photophysical properties of pterin derivatives in acidic and alkaline aqueous solutions

Pterins are heterocyclic compounds with important biological functions, and most of them may exist in two acid-base forms in the pH range between 3 and 13 in aqueous solution. In this work, the photophysical properties of acid and basic forms of six compounds of the pterin family (6-hydroxymethylpterin [HPT], 6-methylpterin [MPT], 6,7-dimethylpterin [DPT], rhamnopterin [RPT], N-methylfolic acid [MFA], and pteroic acid [PA]) have been studied. The effects of the chemical nature of the substituents at position 6 of the pterin moiety and the effects of the pH on the absorption and emission properties are analyzed. The fluorescence characteristics (spectra, quantum yields, lifetimes) of these compounds have been investigated using the single-photon-counting technique. Results obtained for pterin derivatives containing small substituents with 1 carbon atom (HPT, MPT, DPT) and short hydrocarbon chain (4 carbon atoms) (RPT) are different from those found for pterin derivatives containing a p-aminobenzoic acid (PABA) moiety in the substituent (MFA and PA). Fluorescence quantum yields (Phi(F)) of the first group of compounds are relatively high (>/=0.4), whereas MFA and PA exhibit very small Phi(F) values (相似文献   

Synthesis and polymerization of racemic and optically active β-substituted β-propiolactones. II: β-monosubstituted and β-disubstituted monomers and polymers with different optical purities

β-(trichloromethyl)-β-propiolactone (CCl₃-PL), β-(trifluoromethyl,methyl)-β-propiolactone (CF₃, Me-PL) and β-(trifluoromethyl,ethyl)-β-propiolactone (CF₃,Et-PL) have been obtained by the reaction of ketene with chloral, 1,1,1-trifluoroacetone and 1,1,1-trifluorobutanone, respectively. Chiral catalysis lead to optically active monomers. The enantiomeric excess of the lactones has been measured by ¹H-NMR spectroscopy, in the presence of 2,2,2-trifluoro-1-(9-anthryl)ethanol or an europium chiral shift reagent. Polymerizations have been carried out in bulk or in toluene, at 60°C or 80°C, using mainly organometallic initiators. The Polymers become insoluble and crystalline at enantiomeric excesses over 80% for CCl₃-PL and 70% for CF₃,Me-PL. Melting temperatures were recorded from 238 to 268°C for poly(CCl₃-PL) and from 78 to 100°C for poly(CF₃,Me-PL), depending upon the molecular weight and the enantiomeric excess. The ¹³C-NMR specroscopy of poly(CCL₃-PL) indicates that the polymerization of the corresponding lactone leads to polymers of increasing degrees of isotacticity with the enantiomeric excess of the monomer.