Antibody immobilisation on the metal and silicon surfaces. The use of self-assembled layers and specific receptors

The use of Staphylococcal protein A and lectins as intermediate immobilising agents allows operators to orient antibodies (Ab) towards the solution due to the presence of a specific binding sites of immunoglobulin (Ig) molecules. Antibodies of different species of animals have unequal affinities to individual lectins. The effective thickness of immobilised Ab's depends on the type of substrates used and increases in the following sequence: bare gold or silicon surface, the surface treated with self-assembled polyelectrolytes (PESA) or with protein A or some lectins deposited on the preliminary formed polyelectrolyte layer. The glycolysated protein of jp51 may be selectively immobilised from the mixture of retroviral proteins (p24 and jp51), if it is necessary to distinguish infected animals from preliminarily immunised ones by means of a vaccine based on p24 protein. It was shown that the use of Staphylococcal protein A, instead of some lectins as intermediate layer for the Ab immobilisation, does not lead to a more sensitive determination of such low-weight toxins as 2,4-dichlorophenoxyacetic acid (2,4-D). The above-mentioned results were obtained with surface plasmon resonance (SPR) technique.     

Residue-specific real-time NMR diffusion experiments define the association states of proteins during folding

Characterizing the association states of proteins during folding is critical for understanding the nature of protein-folding intermediates and protein-folding pathways, protein aggregation, and disease-related aggregation. To study the association states of unfolded, folded, and intermediate species during protein folding, we have introduced a novel residue-specific real-time NMR diffusion experiment. This experiment, a combination of NMR real-time folding experiments and 3D heteronuclear pulsed field gradient NMR diffusion experiments (LED-HSQC), measures hydrodynamic properties, or molecular sizes, of kinetic species directly during the folding process. Application of the residue-specific real-time NMR diffusion experiments to characterize the folding of the collagen triple helix motif shows that this experiment can be used to determine the association states of unfolded, folded, and kinetic intermediates with transient lifetimes simultaneously. The ratio of the apparent translational diffusion coefficients of the unfolded to the folded form of the triple helix is 0.59, which correlates very well with a theoretical ratio for monomer to linear trimer. The apparent diffusion coefficients of the kinetic intermediates formed during triple helix folding indicate the formation of trimer-like associates which is consistent with previously published kinetic and relaxation data. The residue-specific time dependence of apparent diffusion coefficients of monomer and trimer peaks also illustrates the ability to use diffusion data to probe the directionality of triple helix formation. NMR diffusion experiments provide a new strategy for the investigation of protein-folding mechanisms, both to understand the role of kinetic intermediates and to determine the time-dependent aggregation processes in human diseases.     

Transition-Metal Derivatives of the Cyclopentadienylphosphine Ligands. 11. Reactivity of the Dinuclear Bridged Rhodium(II) Complexes toward Nitrogen-Containing Ligands

We report observations on the reactivity of the dinuclear bridged metal-metal-bonded carbonyl [Rh(II)(CO)(&mgr;-CpPPh(2))](2)(2+) (2(2+)) and of the bis(solvate) cations [Rh(II)(solv)(&mgr;-CpPPh(2))(2)](2)(2+) (3(2+)) with nitriles, amines and pyridine and in general with nitrosyl cation and nitrite anion. By reaction of nitriles and pyridine with 2(2+) we obtained monosubstituted [Rh(2)(CO)L(&mgr;-CpPPh(2))(2)](2+) (4(2+)) and disubstituted [Rh(L)(&mgr;-CpPPh(2))](2)(2+) (5(2+)) (L = MeCN, PhCN, pyridine). These complexes (5(2+)) were also obtained directly from 3(2+). In the reactions with 2(2+) the difference in reactivity of the two rhodium(II) sites suggests a specific role of the metal-metal bond. With secondary and primary amines, reductions of 2(2+) to [Rh(I)(CO)(&mgr;-CpPPh(2))](2) (1) were also observed, and the selectivity with respect to substitution or reduction was strongly solvent dependent. Lithium diisopropylamide induces quantitatively the reduction of 2(2+) to 1 and apparently the substitution of the solvent in 3a(2+). Finally, the new compounds [Rh(II)(THF)(&mgr;-CpPh(2))](2)(2+) (3a(2+)), [Rh(II)(&mgr;-H(2)NC(5)H(4)N)(&mgr;-CpPh(2))(2)](2+) (6(2+)) and [Rh(II)(NO(2))(&mgr;-CpPh(2))](2) (7(2+)) were obtained from 2(2+) and PhIO, from 3(2+) and aminopyridine, and from 2(2+) and nitrite anion, respectively. The compounds were characterized by elemental analysis and the usual spectroscopic methods including COSY NMR experiments. Reaction of NOBF(4) with 1 led efficiently to the compound 2(2+) again through a redox process, instead of a substitution.     

Reactivity and binding energies of transition metal halide ions with benzene

We have generated novel halogen-ligated transition metal ions MX(n)+ (M = Sc, Ti, V, and Fe, X = Cl, Br and I, n = 1-3). We have explored their reactions with benzene, a typical aromatic hydrocarbon. Attachment of one benzene molecule is usually rapid, whereas attachment of a second benzene molecule is generally much slower. The kinetics were analyzed to estimate binding energies, modeling the attachment reaction as a radiative association process. In all cases the Standard Hydrocarbon semiquantitative estimation approach was employed, and in some cases the more accurate variational transition state (VTST) kinetic modeling approach was also applied. Density functional (DFT) quantum calculations were also performed to give computed binding energies for some of the complexes. Taking previously determined binding energies for halogen-ligated alkaline-earth ions as benchmarks, it is concluded that binding of the first benzene molecule to the transition-metal species is strongly enhanced by specific chemical interactions, while binding of the second benzene molecule is more nearly electrostatic. The binding energies are not strongly dependent on the identity of the transition metal ion, and the metal-ion dependences can be rationalized in terms of valence-orbital occupations of the metals. The binding energies are nearly independent of the identity of the halogen ligands.     

A novel one-pot three-component synthesis of 3-halofurans and sequential Suzuki coupling

A novel sequence of Sonogashira coupling and electrophilic addition to an ynone, with concomitant deprotection and cyclocondensation, opens a new one-pot synthesis of 3-halofurans; the method can be readily elaborated to a new sequential Sonogashira-addition-cyclocondensation-Suzuki reaction to furnish 2,3,5-trisubstituted furans in a one-pot fashion.     

La(3+)-Catalyzed methanolysis of hydroxypropyl-p-nitrophenyl phosphate as a model for the RNA transesterification reaction

The methanolysis of hydroxypropyl-p-nitrophenyl phosphate (HPNPP, 1) promoted by La(OTf)(3) under buffered conditions was studied in methanol as a function of pH at 25 degrees C. (31)P NMR studies at -90 degrees C indicate that there are at least three La/1 complexes formed at pH approximately 5.3 of 1:1, 2:2, and 1:2 stoichiometry. Kinetic studies of the observed pseudo-first-order rate constants for the methanolysis of 1 as a function of [La(3+)] at 4.5 < pH < 10.5 indicate there are two general pH regimes. In the low pH regime between 4.5 and 7.6, the plots of k(obs) versus [La(3+)] exhibit saturation behavior with very strong 1:1 binding, with a plateau rate constant that depends on [OCH(3)(-)]. The catalytically productive species is shown to be a 2:2 complex of La(3+) and 1, where the phosphate is proposed to be doubly activated, thereby promoting the methoxide reaction by some 4.6 x 10(10)-fold. In the high pH regime from 7.9 to 10.5, 1:1, 2:2, and 2:1 La(3+)/1 complexes are formed with the La(3+) coordinated in the form of [La(3+)(OCH(3)(-))](1,2). Throughout this pH regime at high [La(3+)], a saturation complex, (La(3+)OCH(3)(-))(2)/1, is formed that spontaneously decomposes with a rate constant of (5-10) x 10(-)(3) s(-)(1), leading to an acceleration of 10(9)-fold at pH 8.0.     

One-hydrogen polarization in hydroformylation promoted by platinum-tin and iridium carbonyl complexes: a new type of parahydrogen-induced effect

The first use of parahydrogen-induced polarization (PHIP) in hydroformylation is described including a novel one-hydrogen polarization (oneH-PHIP) in the product RCHO proton. Observed in propanal formed in the reaction of trans-PtCl(COEt)(PPh3)2 + SnCl2 under parahydrogen, oneH-PHIP was examined using the model hydroformylation catalyst Ir(COEt)(CO)2(dppe) (dppe = bis(diphenylphosphino)ethane) that yields the characterized acyl dihydride IrH2(COEt)(CO)(dppe). It is found that oneH-PHIP occurs as a consequence of second-order effects in the acyl dihydride species coupled with stereospecific reductive elimination of aldehyde. Thus, even though hydrogen transfer to substrate in hydroformylation is nonpairwise, parahydrogen effects prove useful mechanistically.     

Structural Evolution of Water on ZnO(10 0): From Isolated Monomers via Anisotropic H‐Bonded 2D and 3D Structures to Isotropic Multilayers

The surface chemistry of water on zinc oxides is an important topic in catalysis and photocatalysis. Interaction of D₂O with anisotropic ZnO(10 0) surfaces was studied by IR reflection absorption spectroscopy using s‐ and p‐polarized light incident along different directions. Interpretation of the experimental data is aided using isotopologues and DFT calculations. The presence of numerous species is revealed: intact monomers, a mixed 2D D₂O/OD adlayer, an anisotropic bilayer, and H‐bonded 3D structures. The isolated water monomers are identified unambiguously at low temperatures. The thermally induced diffusion of water monomers occurs at elevated temperatures, forming dimers that undergo autocatalytic dissociation via proton transfer. Polarization‐ and azimuth‐resolved IR data provide information on the orientation and strength of H‐bonds within the 2D and 3D structures. Ab initio molecular dynamics simulations reveal strong anharmonic couplings within the H‐bond network.     

Electrostatic study of the proton pumping mechanism in bovine heart cytochrome C oxidase

Cytochrome c oxidase (CcO) is the terminal enzyme of the cell respiratory chain in mitochondria and aerobic bacteria. It catalyzes the reduction of oxygen to water and utilizes the free energy of the reduction reaction for proton pumping across the inner-mitochondrial membrane, a process that results in a membrane electrochemical proton gradient. Although the structure of the enzyme has been solved for several organisms, the molecular mechanism of proton pumping remains unknown. In the present paper, continuum electrostatic calculations were employed to evaluate the electrostatic potential, energies, and protonation state of bovine heart cytochrome c oxidase for different redox states of the enzyme along its catalytic cycle. Three different computational models of the enzyme were employed to test the stability of the results. The energetics and pH dependence of the P-->F, F-->O, and O-->E steps of the cycle have been investigated. On the basis of electrostatic calculations, two possible schemes of redox-linked proton pumping are discussed. The first scheme involves His291 as a pump element, whereas the second scheme involves a group linked to propionate D of heme a(3). In both schemes, loading of the pump site is coupled to ET between the two hemes of the enzyme, while transfer of a chemical proton is accompanied by ejection of the pumped H(+). The two models, as well as the energetics results are compared with recent experimental kinetic data. The proton pumping across the membrane is an endergonic process, which requires a sufficient amount of energy to be provided by the chemical reaction in the active site. In our calculations, the conversion of OH(-) to H(2)O provides 520 meV of energy to displace pump protons from a loading site and overall about 635 meV for each electron passing through the system. Assuming that the two charges are translocated per electron against the membrane potential of 200 meV, the model predicts an overall efficiency of 63%.     

Carbon nucleophiles in the Mitsunobu reaction. Mono- and dialkylation of bis(2,2,2-trifluoroethyl) malonates

[formula: see text] Simple dialkyl malonate esters, for example diethyl malonate, exhibit relatively limited scope as carbon nucleophiles in the Mitsunobu dehydrative alkylation reaction. In contrast, bis(2,2,2-trifluoroethyl) malonate readily undergoes dehydrative alkylation with primary alcohols, and using only a slight excess of malonate gives monoalkylated product in good yield. Some secondary alcohols can also be employed, and bis(2,2,2-trifluoroethyl) malonates can be used in a second dehydrative alkylation to give dialkylated products in good to excellent yield.