Reversible Adduct Research Articles

Nature of the parasitic chemistry during AlGaInN OMVPE

Using in situ laser light scattering, we have observed gas-phase nanoparticles formed during AlN, GaN and InN OMVPE. The response of the scattering intensity to a wide range of conditions indicates that the AlN parasitic chemistry is considerably different from the corresponding GaN and InN chemistry. A simple CVD particle-growth mechanism is introduced that can qualitatively explain the observed particle size and yields a strong residence time dependence. We also used FTIR to directly examine the reactivity of the metalorganic precursors with NH 3 in the 25–300°C range. For trimethylaluminum/NH 3 mixtures a facile CH 4 elimination reaction is observed, which also produces gas-phase aminodimethylalane, i.e. Al(CH 3) 2NH 2. For trimethylgallium and trimethylindium the dominant reaction is reversible adduct formation. All of the results indicate that the AlN particle-nucleation mechanism is predominately of a concerted nature, while the GaN and InN particle-nucleation mechanisms involve homogeneous pyrolysis and radical chemistry.

Chelerythrine stimulates GSH transport by rat Mrp2 (Abcc2) expressed in canine kidney cells.

Rat multidrug resistant protein 2 (Mrp2; Abcc2), an ATP-driven pump located on the canalicular domain of hepatocytes, exports glutathione S-conjugates (GS-X) and GSH among its wide variety of substrates. Previous studies have shown that chelerythrine (CHEL), a quaternary benzophenanthridine cation, reacts with GSH to form a reversible adduct under physiological conditions. Here we report that CHEL can strongly stimulate GSH efflux by Mrp2, when it is constitutively expressed in polarized canine kidney cells, thereby leading to the depletion of cellular GSH. Transepithelial transport experiments indicate that Mrp2 transports GSH and CHEL with a 1:1 stoichiometry, which can be readily inhibited by GS-bimane, a GS-X substrate for Mrp2. Moreover, CHEL can block Mrp2-mediated leukotriene C4 uptake by membrane vesicles with an IC50 approximately 100 microM in the presence of GSH, but not S-methyl GSH or ophthalmic acid. Thus the thiol group of GSH is required for inhibition of Mrp2 in the presence of CHEL. Our results suggest that CHEL stimulates GSH efflux by forming a reversible GS-CHEL adduct, which is transported by Mrp2 and dissociates extracellularly.

Theoretical characterization of stable eta1-N2O-, eta2-N2O-, eta1-N2-, and eta2-N2-bound species: intermediates in the addition reactions of nitrogen hydrides with the pentacyanonitrosylferrate(II) ion.

The addition of nitrogen hydrides (hydrazine, hydroxylamine, ammonia, azide) to the pentacyanonitrosylferrate(II) ion has been analyzed by means of density functional calculations, focusing on the identification of stable intermediates along the reaction paths. Initial reversible adduct formation and further decomposition lead to the eta(1)- and eta(2)-linkage isomers of N(2)O and N(2), depending on the nucleophile. The intermediates (adducts and gas-releasing precursors) have been characterized at the B3LYP/6-31G level of theory through the calculation of their structural and spectroscopic properties, modeling the solvent by means of a continuous approach. The eta(2)-N(2)O isomer is formed at an initial stage of adduct decompositions with the hydrazine and azide adducts. Further conversion to the eta(1)-N(2)O isomer is followed by Fe-N(2)O dissociation. Only the eta(1)-N(2)O isomer is predicted for the reaction with hydroxylamine, revealing a kinetically controlled N(2)O formation. eta(1)-N(2) and eta(2)-N(2) isomers are also predicted as stable species.

Molecular Mechanics Analysis of Structure and Diastereoselectivity toward Lithiation in Amido- and α-Aminoferrocene Complexes

A molecular mechanics force field has been developed for the conformational analysis of amido- and α-aminoferrocenes. Parameterization for ring-substituent rotational barriers in amidoferrocenes and other cross-conjugated derivatives have been calculated using DFT on both the free and complexed cyclopentadienyl ligand. Modeled structures of (diisopropylamido)- and (dimethylamido)ferrocene and N,N-dimethyl-α-ferrocenylethylamine are in agreement with those determined through single-crystal X-ray diffraction. The diastereoselective lithiation of N,N-dimethylferrocenylethylamine and sparteine-mediated enantioselective lithiation of (diisopropylamido)ferrocene using MeLi have been modeled through an assumed reversible adduct formation at the amine nitrogen or amide oxygen, followed by an irreversible ring lithiation. Results indicate that selectivity results from ring lithiation via the adduct conformer with the shortest C−Hring- - -H3C−Li interaction.

The electrophilic reactions of pentacyanonitrosylferrate(II) with hydrazine and substituted derivatives. Catalytic reduction of nitrite and theoretical prediction of eta(1)-, eta(2)-N(2)O bound intermediates.

The electrophilic reactivity of the pentacyanonitrosylferrate(II) ion, [Fe(CN)(5)NO](2)(-), toward hydrazine (Hz) and substituted hydrazines (MeHz, 1,1-Me(2)Hz, and 1,2-Me(2)Hz) has been studied by means of stoichiometric and kinetic experiments (pH 6-10). The reaction of Hz led to N(2)O and NH(3), with similar paths for MeHz and 1,1-Me(2)Hz, which form the corresponding amines. A parallel path has been found for MeHz, leading to N(2)O, N(2), and MeOH. The reaction of 1,2-Me(2)Hz follows a different route, characterized by azomethane formation (MeNNMe), full reduction of nitrosyl to NH(3), and intermediate detection of [Fe(CN)(5)NO](3)(-). In the above reactions, [Fe(CN)(5)H(2)O](3)(-) was always a product, allowing the system to proceed catalytically for nitrite reduction, an issue relevant in relation to the behavior of the nitrite and nitric oxide reductase enzymes. The mechanism comprises initial reversible adduct formation through the binding of the nucleophile to the N-atom of nitrosyl. The adducts decompose through OH(-) attack giving the final products, without intermediate detection. Rate constants for the adduct-formation steps (k = 0.43 M(-)(1) s(-)(1), 25 degrees C for Hz) decrease with methylation by about an order of magnitude. Among the different systems studied, one-, two-, and multielectron reductions of bound NO(+) are analyzed comparatively, with consideration of the role of NO, HNO (nitroxyl), and hydroxylamine as bound intermediates. A DFT study (B3LYP) of the reaction profile allows one to characterize intermediates in the potential hypersurface. These are the initial adducts, as well as their decomposition products, the eta(1)- and eta(2)-linkage isomers of N(2)O.

Kinetic and thermodynamic studies of reversible adduct formation between Ru5C(CO)15 and acetonitrile

Stopped-flow kinetic measurements have been made on the reversible addition of NCMe to Ru5C(CO)15 in 1,2-dichloroethane to form the adduct in which a Ru-Ru bond in the cluster has been broken and replaced by a Ru-NCMe bond. The measurements lead to the kinetic parameters kf (25°C)=24.3±1.4 M−1 s−1, ΔHf≠=31.7±3.5 kJ mol−1 and ΔSf≠=−91±12 J K−1 mol−1; kr (25°C)=296±18 s−1, ΔHr≠=51.0±1.3 kJ mol−1 and ΔSr≠=−47±4 J K−1 mol−1. Addition of NCMe requires an exceptionally low activation enthalpy and suggests a very early transition state, perhaps with attack at the carbon atom of a coordinated CO ligand. Loss of NCMe from the adduct also suggests a late transition state for the reverse process with a great deal of Ru-Ru bond making and a consequently pronounced negative entropy of activation. These data, when combined with UV–Vis measurements on reactions at equilibrium, lead to the thermodynamic parameters K (25°C)=12.6 M−1, ΔH°=−17.6±1.2 kJ mol−1 and ΔS°=−38±4 J K−1 mol−1. These novel parameters provide a quantitative measure of the release of strain within the Ru5C cluster when it is opened up by reaction with the rather weak ligand NCMe.

Genesis, Redox, and Acid−Base Relationships among WC, WC, and W⋮C Functionalities over an Oxo Surface Modeled by Calix[4]arene

This report deals with the chemistry of anionic W-alkylidynes generated over the oxo surface defined by a calix[4]arene tetraanion. The exhaustive alkylation of [cis-(Cl)2W{p-But-calix[4]−(O)4}] (1), with an excess of alkylating agent led to [{p-But-calix[4]−(O)4}W⋮C−R][M] (R = Ph, M = 1/2 Mg, 2; R = Prn, M = Li, 3; R = SiMe3, M = Li, 4). The protonation [PyHCl] of 2 and 3 led to the corresponding alkylidenes [{p-But-calix[4]−(O)4}WC(H)R] (R = Ph, 7; R = Prn, 8), which were reversibly deprotonated back to the starting akylidynes using LiBu. The reaction of 2−4 with AgNO3 led to dimetallic alkylidenes [{p-But-calix[4]−(O)4}WC(R)Ag)] (R = Ph, 12; R = Prn, 13; R = SiMe3, 14). The alkylation of 3 and 4 with MeOTf occurred at the carbon in 4 and at both carbon and oxygen in 3 leading to [{p-But-calix[4]−(O)4}WC(Me)SiMe3] (9) and to a mixture of [{p-But-calix[4]−(O)4}WC(Me)Prn], (10) and [{p-But-calix[4]−(O)3(OMe)}W⋮C−Prn] (11), respectively. The functionalization of the anionic alkylidynes was achieved by reacting 2 with electrophiles such as PhCHO leading to [{{p-But-calix[4]−(O)4}WC(Ph)-C(H)(Ph)-O}2Mg(thf)] (15) and with Ph2CCO forming [{{p-But-calix[4]−(O)4}WC(Ph)-C(CPh2)-O}2Mg] (16). An indirect, but synthetically quite versatile, functionalization is the oxidation of 2 with I2 producing [{p-But-calix[4]−(O)4}WC(Ph)-I] (20). The alkylidenes 7 and 8 were unexpectedly unreactive in the presence of olefins and aldehydes, e.g., PhCHO, which formed a reversible adduct binding inside the calix[4]arene cavity. However, the trans labilization of the alkylidene functionality was achieved by reacting 7 with ButNC, a reaction leading to cis-stilbene and the WW dimer [{μ-p-But-calix[4]−(O)4}2W2(CNBut)2] (18). The one-electron oxidation of 2 by Cp2FeBPh4 followed two different pathways via the common free-radical intermediate [{p-But-calix[4]−(O)4}WC·(Ph)], leading to either 7 (hydrogen abstraction) or [{p-But-calix[4]−(O)4}2W2(μ2-η2:η2-Ph2C2)], (19). The nature of the anionic alkylidynes, the reaction pathways with electrophiles, and the one-electron oxidation reactions were analyzed using extended Hückel calculations.

Coagulation protein function VI: augmentation of anticoagulant function by acetaldehyde-treated heparin.

Acetaldehyde (AcH) at preincubation concentrations of 447, 89.4, and 17.9 mM potentiates the effects of heparin on the clotting time of plasma. While control plasma clotted in the range of 12.6+/-0.1 to 13.8+/-0.1 sec, and heparin-treated plasma clotted in a range from 131.5+/-2.5 to 168.2+/-1.2 sec, heparin that was preincubated at room temperature for 30 min with 89.4 or 447 mM AcH did not clot plasma in 300 sec. Heparin exposed to 17.9 mM AcH clotted plasma in 193+/-1.1 sec. Ethanol at a 404 mM concentration also prolonged the clotting time of heparin-treated plasma >300 sec, while 202 mM ethanol prolonged the clotting time of heparin-treated plasma from 149.0+/-2.0 sec to 219.5+/-1.7 sec. It is suggested that AcH alters the tertiary structure of heparin by adduct formation, possibly by formation of cyclic acetals with iduronic and glucuronic acids, thereby more readily affecting binding of the glycosaminoglycan to antithrombin III and/or thrombin, prolonging clotting time. Ethanol, which does not react covalently with heparin, might affect its conformation as a consequence of an organic solvent effect. Protamine sulfate prolonged the clotting time of plasma from 13.6+/-0.1 sec to 17.9+/-0.2 sec. Protamine sulfate-treated heparin clotted plasma in 21.0+/-0.4 sec relative to heparin-treated plasma (160.4+/-1.7 sec). In subsequent experiments, AcH-treated protamine sulfate extended the clotting time of protamine sulfate from 17.9+/-0 sec to 33.7+/-0.6 sec. Prior addition of protamine sulfate to AcH-heparin mixtures or heparin to protamine sulfate-AcH mixtures before addition to plasma resulted in clotting times of 22.0+/-0.4 sec and 24.1+/-0.5 sec, respectively, relative to control clotting times of 162.3+/-2.6 sec for plasma-heparin mixtures. These results confirm both the reduction in coagulation time of heparin-treated plasma by protamine sulfate and the prolongation of clotting time of plasma by protamine sulfate. Furthermore, and importantly, they indicate that acetaldehyde-treated protamine sulfate is a more effective anticoagulant than protamine sulfate. It is suggested that reversible adduct formation between acetaldehyde, heparin, and protamine sulfate may occur as a means explaining the essentially identical coagulation time of these mixtures when added to plasma regardless of the order of premixing. Ethanol (404 mM) did not influence protamine sulfate effects. Lastly, the potentiation of the anticoagulant function of heparin by acetaldehyde suggests that a structural modification of the glycosaminoglycan may occur in alcoholics.

Catalytic Generation of Oxalate through a Coupling Reaction of Two CO(2) Molecules Activated on [(Ir(eta(5)-C(5)Me(5)))(2)(Ir(eta(4)-C(5)Me(5))CH(2)CN)(&mgr;(3)-S)(2)

Electrochemical reduction of [(Ir(eta(5)-C(5)Me(5)))(3)(&mgr;(3)-S)(2)](BPh(4))(2) ([Ir(3)S(2)](BPh(4))(2)) in CO(2)-saturated CH(3)CN at -1.30 V (vs Ag/AgCl) produced C(2)O(4)(2)(-) and [(Ir(eta(5)-C(5)Me(5)))(2)(Ir(eta(4)-C(5)Me(5))CH(2)CN)(&mgr;(3)-S)(2)](+) ([Ir(3)S(2)CH(2)CN](+)). The crystal structure of [Ir(3)S(2)CH(2)CN](BPh(4)) by X-ray analysis revealed that a linear CH(2)CN group is linked at the exo-position of a C(5)Me(5) ligand, and the C(5)Me(5)CH(2)CN ligand coordinates to an Ir atom with an eta(4)-mode. The cyclic voltammogram of [Ir(3)S(2)CH(2)CN](+) in CH(3)CN under CO(2) exhibited a strong catalytic current due to the reduction of CO(2), while that of [Ir(3)S(2)](2+) did not show an interaction with CO(2) in the same solvent. The reduced form of [Ir(3)S(2)CH(2)CN](+) works as the active species in the reduction of CO(2). The IR spectra of [Ir(3)S(2)CH(2)CN](+) in CD(3)CN showed a reversible adduct formation with CO(2) and also evidenced the oxalate generation through the reduced form of the CO(2) adduct under the controlled potential electrolysis of the solution at -1.55 V. A coupling reaction of two CO(2) molecules bonded on adjacent &mgr;(3)-S and Ir in [Ir(3)S(2)CH(2)CN](0) is proposed for the first catalytic generation of C(2)O(4)(2)(-) without accompanying CO evolution.

An agent cleaving glucose-derived protein crosslinks in vitro and in vivo.

Glucose and other reducing sugars react with proteins by a nonenzymatic, post-translational modification process called nonenzymatic glycosylation or glycation. The sugar-derived carbonyl group adds to a free amine, forming a reversible adduct which over time rearranges to produce a class of products termed advanced-glycation end-products (AGEs). These remain irreversibly bound to macromolecules and can covalently crosslink proximate amino groups. The formation of AGEs on long-lived connective tissue and matrix components accounts largely for the increase in collagen crosslinking that accompanies normal ageing and which occurs at an accelerated rate in diabetes. AGEs can activate cellular receptors and initiate a variety of pathophysiological responses. They modify an appreciable fraction of circulating low-density lipoproteins preventing uptake of these particles by their high-affinity tissue receptors. Advanced glycation has also been implicated in the pathology of Alzheimer's disease. Because AGEs may form by a pathway involving reactive alpha-dicarbonyl intermediates, we investigated a potential pharmacological strategy for selectively cleaving the resultant glucose-derived protein crosslinks. We now describe a prototypic AGE crosslink 'breaker', N-phenacylthiazolium bromide (PTB), which reacts with and cleaves covalent, AGE-derived protein crosslinks. The ability of PTB to break AGE crosslinks in vivo points to the importance of an alpha-dicarbonyl intermediate in the advanced glycation pathway and offers a potential therapeutic approach for the removal of established AGE crosslinks.

Production of α-Siloxycarbenium Ions by Protonation of Photochemically Generated α-Siloxycarbenes. Formation Mechanism and Reactivities with Nucleophiles

The acyltrimethylsilanes 4-RC6H4C(O)SiMe3 (R = H, Me, MeO) and β-naphthylC(O)SiMe3, upon photolysis in acetonitrile with 20 ns pulses of 248 nm light from an KrF* excimer laser, give rise to the corresponding α-siloxycarbenes ArC:OSiMe3, whose absorption spectra (λmax between 270 and 310 nm), lifetimes (between 130 and 260 ns), and reactivities with proton donors (ROH, mainly alcohols) are reported. With highly acidic ROH, such as 1,1,1,3,3,3-hexafluoroisopropyl alcohol (HFIP), the reaction is of simple second order, rate constants being in the 109 M-1 s-1 range and virtually independent of the nature of the aromatic moiety of the carbene. Isotopic substitution of H in ROH by D has no effect on the rate constant for reaction with carbene. For less acidic alcohols such as, e.g., methanol, the reactivity of the carbenes increases with increasing [ROH]. This behavior is interpreted in terms of reversible adduct formation between carbene and alcohol followed by reaction with further alcohol molecule(s) to giv...

Reversible adduct formation between phosphines and triarylboron compounds

The acceptor strength of a number of Lewis-acidic fluorinated triarylboron compounds has been established and shown to depend on the amount and position of fluorine substitution. The donor strength of tert-butyl-phosphine has been found to be greater than phosphine towards these acceptor compounds; two series of adducts have been characterised and reversible adduct formation has been demonstrated for some adducts. Crystal structures of the phosphine adducts of tris(pentafluorophenyl)boron and tris(2,6-difluorophenyl)boron have been compared with those of the tert-butylphosphine adducts of tris(pentafluorophenyl)boron and tris(3,4,5-trifluorophenyl)boron to show a correlation between the length and the strength of the adduct bond. Mixing aryl groups in the acceptor compounds has not produced new adducts. The strong acceptor compounds have been found to form unstable adducts with water which act as drying agents ultimately producing arylboric acids; the crystal structure of (2,6-difluoropheny)dihydroxyborane has been determined.

Oxygen Reduction at Co ( II ) 2 ‐ Disalophen Modified Carbon Electrodes

Oxygen reduction at a dinuclear Co(II)‐Schiff‐base complex, , modified carbon electrodes has been studied and results thereof are reported. The absorbed complex is shown to have a large catalytic effect for oxygen reduction. The oxygen reduction at the modified carbon electrodes is reasonably reversible in alkaline solutions with an apparent number of electrons transferred of around one and an estimated exchange rate constant of about 0.04 cm/s. The strong catalytic activity is explained by the formation of a reversible adduct with oxygen, i.e., , followed by reduction of Co(III) in the adduct. The cyclic voltammetric and chronoamperometric experimental results suggest that the first electron transfer should be a rate limiting step.

Chemical modification of human placental glutathione transferase by pyridoxal 5′-phosphate

Incubation of GST pi from human placenta with 8 mM PLP resulted in a rapid loss of activity during the first 10 min, concomitant with a Schiff base formation. This inactivation was probably due to the formation of a reversible adduct between PLP and the enzyme. After sodium borohydride treatment this adduct was reduced and stabilized. Stoichiometry and peptide isolation studies showed that three lysine residues were modified during reaction of GST and PLP. Protection of the enzyme against inactivation was achieved in the presence of 4 mM GSH suggesting that at least one lysyl residue is associated with the substrate binding site. Peptide mapping by digesting the enzyme with trypsin revealed that lysine shielded by GSH is Lys-127. Our results suggest that this residue may play an important role in enzymatic activity.

Rhodium iminophosphine complexes as efficacious oxygen carriers. Crystal structure of a representative dioxygen adduct

The synthesis, characterization and reactivity of some square-planar rhodium(I) complexes with new bidentate ligands o-Ph2PC6H4CHNR (R = Et, Prn, Pri or But) having NP donor atoms have been investigated. Depending on the steric crowding of the ligands the complexes can reversibly form dioxygen adducts, in organic solvent solutions at room temperature. The oxygenation–deoxygenation cycles, which have been monitored by 31P NMR spectroscopy, can be repeated several times with minor loss in the starting material. A reversible adduct is similarly obtained by reaction with CO. The reaction of the dioxygen adduct with SO2 allows the formation of a sulfate derivative. The molecular structure of the dioxygen complex [Rh(o-Ph2PC6H4CHNPri)2(O2)]BPh4 has been determined by a single-crystal diffraction study: triclinic, space group P, a= 17.450(7), b= 16.518(7), c= 9.624(5)A, α= 92.79(6), β= 92.52(7), γ= 92.69(7)°, Z= 2, R= 0.057. The co-ordination of the metal may be alternatively described as distorted trigonal bipyramidal or distorted octahedral, according to whether the dioxygen molecule is treated as occupying one or two equatorial sites respectively. The two oxygen atoms, which are equidistant from the metal, are 1.436(9)A apart.

Mechanism-based inactivation of alanine racemase by 3-halovinylglycines.

Alanine racemase, an enzyme important to bacterial cell wall synthesis, is irreversibly inactivated by 3-chloro- and 3-fluorovinylglycine. Using alanine racemase purified to homogeneity from Escherichia coli B, the efficient inactivation produced a lethal event for every 2.2 +/- 0.2 nonlethal turnovers, compared to 1 in 800 for fluoroalanine. The mechanism of inhibition involves enzyme-catalyzed halide elimination to form an allenic intermediate that partitions between reversible and irreversible covalent adducts, in the ratio 3:7. The reversible adduct (lambda max = 516 nm) decays to regenerate free enzyme with a half-life of 23 min. The lethal event involves irreversible alkylation of a tyrosine residue in the sequence -Val-Gly-Tyr-Gly-Gly-Arg. The second-order rate constant for this process with D-chlorovinylglycine (122 +/- 14 M-1 s-1), the most reactive analog examined, is faster than the equivalent rate constant for D-fluoroalanine (93 M-1 s-1). The high killing efficiency and fast turnover of these mechanism-based inhibitors suggest that their design, employing the haloethylene moiety to generate a reactive allene during catalysis, could be extended to provide useful inhibitors of a variety of enzymes that conduct carbanion chemistry.

Gas chromatographic study of reversible adduct formation between nucleophilic solutes and stationary phases containing lanthanide β-diketonates

Complex formation between fluorinated and methylated β-diketonates of praseodymium and europium, dissolved in a polydimethylsiloxane stationary phase, and aliphatic alcohols, methyl ketones, methyl ethers and carboxylic acid methyl esters has been studied. Investigations were carried out isothermally in the range 90 to 130°C. An increase in retention values measured on the complexing stationary phase for the test solutes was the primary observation. Equilibrium constants (K) of adduct formation were determined from retention data. It was established that fluorinated lanthanide β-diketonates enhance adduct formation more selectively than the corresponding methyl compounds. Decrease in the ionic radii from praseodymium to europium gave rise to increased K values.

Kinetics and thermochemistry of reversible adduct formation in the reaction of atomic chlorine with carbon disulfide

Reversible adduct formation in the reaction of Cl((sup 2)P(sub J)) with CS2 has been observed over the temperature range 193-258 K by use of time-resolved resonance fluorescence spectroscopy to follow the decay of pulsed-laser-generated Cl((sup 2)P(sub J)) into equilbrium with CS2Cl. Rate coefficients for CS2Cl formation and decomposition have been determined as a function of temperature and pressure; hence, the equilbrium constant has been determined as a function of temperature. A second-law analysis of the temperature dependence of Kp and heat capacity corrections calculated with use of an assumed CS2Cl structure yields the following thermodynamic parameters for the association reaction: Delta-H(sub 298) = -10.5 +/- 0.5 kcal/mol, Delta-H(sub 0) = -9.5 +/- 0.7 kcal/mol, Delta-S(sub 298) = -26.8 +/- 2.4 cal/mol.deg., and Delta-H(sub f,298)(CS2Cl) = 46.4 +/- 0.6 kcal/mol. The resonance fluorescence detection scheme has been adapted to allow detection of Cl((sup 2)P(sub J)) in the presence of large concentrations of O2, thus allowing the CS2Cl + Cl + O2 reaction to be investigated. We find that the rate coefficient for CS2Cl + O2 reaction via all channels that do not generate Cl((sup 2)P(sub J)) is less than 2.5 x 10(exp-16) cu cm/(molecule.s) at 293 K and 300-Torr total pressure and that the total rate coefficient is less than 2 x 10 (exp -15) cu cm/(molecule.s) at 230 K and 30-Torr total pressure. Evidence for reversible adduct formation in the reaction of Cl((sup 2)P(sub J)) with COS was sought but not observed, even at temperatures as low as 194 K.

Properties of D‐amino‐acid oxidase from Rhodotorula gracilis

The flavoprotein D-amino-acid oxidase was purified to homogeneity from the yeast Rhodotorula gracilis by a highly reproducible procedure. The amino acid composition of the protein was determined; the protein monomer had a molecular mass of 39 kDa and contained one molecule of FAD. The ratio between A274/A455 was about 8.2. D-Amino-acid oxidase from yeast showed typical flavin spectral perturbations on binding of the competitive inhibitor benzoate and was reduced by D-alanine under anaerobiosis. The enzyme reacted readily with sulfite to form a covalent reversible adduct and stabilized the red anionic form of the flavin semiquinone on photoreduction in the presence of 5-deazariboflavin; the 3,4-dihydro-FAD form was not detectable after reduction with sodium borohydride. Thus D-amino-acid oxidase from yeast exhibited most of the general properties of the dehydrogenase/oxidase class of flavoproteins; at the same time, the enzyme showed some peculiar features with respect to the same protein from pig kidney.

3H]Ro 19-6327: A reversible ligand and affinity labelling probe for monoamine oxidase-B

This study demonstrated the existence of specific binding sites [ 3H]Ro 19-6327 in human platelet membranes. This compound is a novel, time-dependent inhibitor of monoamine oxidase type B (MAO-B) and is structurally closely related to [ 3H]Ro 16-6491. The density of the sites labelled with high affinity by [ 3H]Ro 19-6327 was similar to that observed in previous studies with [ 3H]Ro 16-6491 as ligand. Binding was reversible at 20°C and showed a relatively slow dissociation ( t 1 2 =220 min) . THe dissociation rate was markedly decreased ( t 1 2 = > 24 h) at 0 °C. MAO-B, but not MAO-A inhibitors, effectively prevented the binding of [ 3H]Ro 19-6327. Like [ 3H]Ro 16-6491, [ 3H]Ro 19-6327 is recognized as a substrate by MAO-B, being eventually deaminated by the enzyme. Since the deaminated aldehyde derivative of Ro 19-6327 did not inhibit MAO-B, a still unidentified reversible adduct, formed at the MAO-B active site, might explain the high potency and selectivity of [ 3H]Ro 19-6327. Incubation of the radioligand-enzyme complex from platelet and brain membranes with NaBH 3CN and acetic acid (to pH 4.5) caused the irreversible incorporation of the radioactivity into a single polypeptide as shown by SDS-PAGE analysis. This polypeptide had a molecular weight identical to that of the MAO-B subunit, i.e. 58 000. The presence of unlabelled MAO-B inhibitors in the incubation mixture prevented the covalent incorporation of [ 3H]Ro 19-6327. The irreversible MAO-B inhibitor, [ 3H] pargyline, labelled a protein with a molecular weight identical to the protein labelled by [ 3H]Ro 19-6327. These data indicate that [ 3H]Ro 19-6327 in the presence of NaBH 3CN is irreversibly incorporated into one of the two polypeptide subunits of MAO-B. Therefore, [ 3H]Ro 19-6327 can also be used as an irreversible affinity-labelling probe for MAO-B. Since both Ro 19-6327 and Ro 16-6491 inhibit MAO-B irreversibly in the presence of NaBH 3CN these compounds might be very useful for the localization and identification of the active site of the enzyme.