Study of the lithium thiocyanate quadrupole by ultra-violet absorption spectroscopy

A spectroscopic method for studying quadrupole formation is described, and experimental results are presented for lithium thiocyanate in a tetrahydrofuran/hexane solvent system. From molar absorptivity equations and an equation by Fuoss and Kraus, a direct relationship is derived between the quadrupole formation constant, K_Q, and the dielectric constant of the medium. Experimental results are consistent with the equation and allow an estimate of the distance between lithium and thiocyanate in the ion pair.     

Mass spectrometry in structural and stereochemical problems—CCXXIII:ortho interactions of nitro groups

The mass spectra of C¹³ labeled o- nitrobenzoic acid and o-nitrobenzaldehyde were studied. Earlier proposed fragmentations involving group migration to a charge-carrying vacant ortho position were verified. A completely unexpected interaction in o- nitroanisole was uncovered by O¹⁸labeling.     

High thermodynamic stability and extraordinary kinetic inertness of copper(II) complexes with 1,4,8,11-tetraazacyclotetradecane-1,8-bis(methylphosphonic acid): example of a rare isomerism between kinetically inert penta- and hexacoordinated copper(II) complexes

In an aqueous solution at room temperature, 1,4,8,11-tetraazacyclotetradecane-1,8-bis(methylphosphonic acid) (H(4)L(1)) and Cu(I) (I) form a pentacoordinated (pc) complex, pc-[Cu(L(1))](2-), exhibiting conformation I of the cyclam ring. At high temperature, the complex isomerises to a hexacoordinated isomer, trans-O,O-[Cu(L(1))](2-), with a trans-III conformation of the cyclam ring. In pc-[Cu(L(1))](2-), four ring nitrogen atoms and one phosphonate oxygen atom are arranged around Cu(I) (I) in a structure that is half-way between a trigonal bipyramid and a tetragonal pyramid, with one phosphonic acid group uncoordinated. In the trans-O,O-[Cu(L(1))](2-) isomer, the nitrogen atoms form a plane and the phosphonic acid groups are in a mutually trans configuration. A structurally very similar ligand, 4-methyl-1,4,8,11-tetraazacyclotetradecane-1,8-bis(methylphosphonic acid) (H(4)L(2)), forms an analogous pentacoordinated complex, pc-[Cu(L(2))](2-), at room temperature. However, the complex does not isomerise to the octahedral complex analogous to trans-O,O-[Cu(L(1))](2-). Because of the high thermodynamic stability of pc-[Cu(L(1))](2-), (logbeta=25.40(4), 25 degrees C, I=0.1 mol dm(-3) KNO(3)) and the formation of protonated species, Cu(I) (I) is fully complexed in acidic solution (-log [H(+)] approximately 3). Acid-assisted decomplexation of both of the isomers of [Cu(H(2)L(1))] takes place only after protonation of both uncoordinated oxygen atoms of each phosphonate moiety and at least one nitrogen atom of the cycle. The exceptional kinetic inertness of both isomers is illustrated by their half-lives tau(1/2)=19.7 min for pc-[Cu(H(2)L(1))] and tau(1/2) about seven months for trans-O,O-[Cu(H(2)L(1))] for decomplexation in 5 M HClO(4) at 25 degrees C. The mechanism of formation of pc-[Cu(L(1))](2-) is similar to those observed for other macrocyclic complexes.     

Mass spectrometric characterization of glycosylation of hepatitis C virus E2 envelope glycoprotein reveals extended microheterogeneity of N-glycans

Hepatitis C virus (HCV) causes acute and chronic liver disease in humans, including chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The polyprotein encoded in the HCV genome is co- and post-translationally processed by host and viral peptidases, generating the structural proteins Core, E1, E2, and p7, and five nonstructural proteins. The two envelope proteins E1 and E2 are heavily glycosylated. Studying the glycan moieties attached to the envelope E2 glycoprotein is important because the N-linked glycans on E2 envelope protein are involved in the interaction with some human neutralizing antibodies, and may also have a direct or indirect effect on protein folding. In the present study, we report the mass spectrometric characterization of the glycan moieties attached to the E2 glycoprotein. The mass spectrometric analysis clearly identified the nature, composition, and microheterogeneity of the sugars attached to the E2 glycopeptides. All 11 sites of glycosylation on E2 protein were characterized, and the majority of these sites proved to be occupied by high mannose glycans. However, complex type oligosaccharides, which have not been previously identified, were exclusively observed at two N-linked sites, and their identity and heterogeneity were determined.     

Mn2+ complexes with 12-membered pyridine based macrocycles bearing carboxylate or phosphonate pendant arm: crystallographic, thermodynamic, kinetic, redox, and 1H/17O relaxation studies

Mn(2+) complexes represent an alternative to Gd(3+) chelates which are widely used contrast agents in magnetic resonance imaging. In this perspective, we investigated the Mn(2+) complexes of two 12-membered, pyridine-containing macrocyclic ligands bearing one pendant arm with a carboxylic acid (HL(1), 6-carboxymethyl-3,6,9,15-tetraazabicyclo[9.3.1] pentadeca-1(15),11,13-triene) or a phosphonic acid function (H(2)L(2), 6-dihydroxyphosphorylmethyl-3,6,9,15-tetraazabicyclo[9.3.1]pentadeca-1(15),11,13-triene). Both ligands were synthesized using nosyl or tosyl amino-protecting groups (starting from diethylenetriamine or tosylaziridine). The X-ray crystal structures confirmed a coordination number of 6 for Mn(2+) in their complexes. In aqueous solution, these pentadentate ligands allow one free coordination site for a water molecule. Potentiometric titration data indicated a higher basicity for H(2)L(2) than that for HL(1), related to the electron-donating effect of the negatively charged phosphonate group. According to the protonation sequence determined by (1)H and (31)P pH-NMR titrations, the first two protons are attached to macrocyclic amino groups whereas the subsequent protonation steps occur on the pendant arm. Both ligands form thermodynamically stable complexes with Mn(2+), with full complexation at physiological pH and 1:1 metal to ligand ratio. The kinetic inertness was studied via reaction with excess of Zn(2+) under various pHs. The dissociation of MnL(2) is instantaneous (at pH 6). For MnL(1), the dissociation is very fast (k(obs) = 1-12 × 10(3) s(-1)), much faster than that for MnDOTA, MnNOTA, or the Mn(2+) complex of the 15-membered analogue. It proceeds exclusively via the dissociation of the monoprotonated complex, without any influence of Zn(2+). In aqueous solution, both complexes are air-sensitive leading to Mn(3+) species, as evidenced by UV-vis and (1)H NMRD measurements and X-ray crystallography. Cyclic voltammetry gave low oxidation peak potentials (E(ox) = 0.73 V for MnL(1) and E(ox) = 0.68 V for MnL(2)), in accordance with air-oxidation. The parameters governing the relaxivity of the Mn(2+) complexes were determined from variable-temperature (17)O NMR and (1)H NMRD data. The water exchange is extremely fast, k(ex) = 3.03 and 1.77 × 10(9) s(-1) for MnL(1) and MnL(2), respectively. Variable-pressure (17)O NMR measurements have been performed to assess the water exchange mechanism on MnL(1) and MnL(2) as well as on other Mn(2+) complexes. The negative activation volumes for both MnL(1) and MnL(2) complexes confirmed an associative mechanism of the water exchange as expected for a hexacoordinated Mn(2+) ion. The hydration number of q = 1 was confirmed for both complexes by (17)O chemical shifts. A relaxometric titration with phosphate, carbonate or citrate excluded the replacement of the coordinated water molecule by these small endogenous anions.     

The antigenic determinants on HIV p24 for CD4+ T cell inhibiting antibodies as determined by limited proteolysis, chemical modification, and mass spectrometry

In the last decade, mass spectrometry has been employed by more and more researchers for identifying the proteins in a macromolecular complex as well as for defining the surfaces of their binding interfaces. This characterization of protein-protein interfaces usually involves at least one of several different methodologies in addition to the actual mass spectrometry. For example, limited proteolysis is often used as a first step in defining regions of a protein that are protected from proteolysis when the protein of interest is part of a macromolecular complex. Other techniques used in conjunction with mass spectrometry for determining regions of a protein involved in protein-protein interactions include chemical modification, such as covalent cross-linking, acetylation of lysines, hydrogen-deuterium exchange, or other forms of modification. In this report, both limited proteolysis and chemical modification were combined with several mass spectrometric techniques in efforts to define the protein surface on the HIV core protein, p24, recognized by two different monoclonal human antibodies that were isolated from HIV+ patients. One of these antibodies, 1571, strongly inhibits the CD4+ T cell proliferative response to a known epitope (PEVIPMFSALSEGATP), while the other antibody, 241-D, does not inhibit as strongly. The epitopes for both of these antibodies were determined to be discontinuous and localized to the N-terminus of p24. Interestingly, the epitope recognized by the strongly inhibiting antibody, 1571, completely overlaps the T cell epitope PEVIPMFSALSEGATP, while the antibody 241-D binds to a region adjacent to the region of p24 recognized by the antibody 1571. These results suggest that, possibly due to epitope competition, antibodies produced during HIV infection can negatively affect CD4+ T cell-mediated immunity against the virus.     

Effect of montmorillonite on structure and properties of nanocomposite with PA6/PS/elastomer matrix

Polyamide nanocomposites with fair balance of mechanical properties were recently obtained by addition of finely dispersed clay-compatibilized rubber or rigid PS phase. This work deals with combination of both components, which recently led also to enhanced mechanical behaviour in an analogous reactively compatibilized ternary system.Application of clay to PA6/PS/EPR matrix leads to a decrease in particle size analogously to corresponding binary blends, but the effect of clay on toughness is predominantly contradictory, i.e., a decrease with increasing clay content was found. Also the toughening effect of formed core-shell (elastomer/clay) particles is lower in comparison with binary PA6/EPR. At the same time, in contrast to the PA/PS system, the presence of core-shell particles formed by PS/C15 preblending leads to fair mechanical behaviour including enhanced toughness. This documents a complex affecting of the system behaviour by clay and the expected synergistic cooperation of numerous clay-induced changes in both component parameters and structure. The obtained results indicate that a proper combination of rigid and elastomeric inclusions can lead to nanocomposites with balanced and enhanced mechanical behaviour.     

Radical identification by liquid chromatography/thermospray mass spectrometry

Radical adducts of 5,5-dimethyl-1-pyrroline N-oxide (DMPO) with hydroxyl, methanol-derived, and ethanol-derived radicals were detected by a combination of liquid chromatography with either electron paramagnetic resonance or thermospray mass spectrometry (LC/EPR or LC/TSP-MS) in the Fenton system (with methanol or ethanol). One radical adduct was observed in the reaction of DMPO with the hydroxyl radical or the methanol-derived radical, while two adducts were detected in the reaction of DMPO with ethanol-derived radicals. The LC/TSP-MS spectra showed quasi-molecular ions [M + H]+ at m/z 146 and m/z 160 for the methanol-derived and ethanol-derived radical adducts, respectively, and an apparent molecular ion M+ at m/z 130 for the hydroxyl radical adduct. Use of methyl-D3 alcohol (CD3OH) and ethyl-D5 alcohol (CD3CD2OH) indicated that carbon-centered radicals are formed. Experiments with partially deuterated ethanol (CD3CH2OH and CH3CD2OH) indicated that the two adducts observed in the reaction of DMPO with ethanol-derived radicals correspond to the two diastereomeric adducts of DMPO with the alpha-hydroxyethyl free radical.