Direct hydride transfer in the reaction mechanism of quinoprotein alcohol dehydrogenases: a quantum mechanical investigation

Oxidation of alcohols by direct hydride transfer to the pyrroloquinoline quinone (PQQ) cofactor of quinoprotein alcohol dehydrogenases has been studied using ab initio quantum mechanical methods. Energies and geometries were calculated at the 6-31G(d,p) level of theory. Comparison of the results obtained for PQQ and several derivatives with available structural and spectroscopic data served to judge the feasibility of the calculations. The role of calcium in the enzymatic reaction mechanism has been investigated. Transition state searches have been conducted at the semiempirical and STO-3G(d) level of theory. It is concluded that hydride transfer from the Calpha-position of the substrate alcohol (or aldehyde) directly to the C(5) carbon of PQQ is energetically feasible. Copyright 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1732-1749, 2001     

Oxidative deamination of benzylamine by electrogenerated quinonoid systems as mimics of amine oxidoreductases cofactors

The reactions of a new type of quinonoid system with benzylamine have been investigated in methanol in order to mimic the reactions occurring in the course of the enzymatic oxidation of amines by quinone cofactors. Under strictly anaerobic conditions, unstable quinonoid species 1(ox)()-4(ox)() have been selectively electrogenerated using anodic-controlled potential electrolysis. Thus, we have demonstrated that 3,4-quinone 1(ox)() is incapable of deaminating benzylamine, while 3,4-iminoquinone species 3(ox)() and 4(ox)() act as efficient catalysts for the autorecycling oxidation of benzylamine: the reaction efficiency reached 64 turnovers. Additional mechanistic investigations reveal that the oxidation of benzylamine by our quinonoid model cofactors proceeds unambiguously via a transamination mechanism, as suggested for many enzymatic systems.     

In silico studies of the mechanism of methanol oxidation by quinoprotein methanol dehydrogenase

The mechanism of bacterial methanol dehydrogenase involves hydride equivalent transfer from substrate to the ortho-quinone PQQ to provide a C5-reduced intermediate that subsequently rearranges to the hydroquinone PQQH(2). We have studied the PQQ reduction by molecular dynamic (MD) simulations in aqueous solution. Among the five simulated structures, either Asp297 or Glu171 or both are ionized. Reasonable structures are obtained only when both carboxyl groups are ionized. This is not unexpected since the kinetic pH optimum is 9.0. In the structure of the enzyme.PQQ.HOCH(3) complex, the hydrogen bonded Glu171-CO(2)(-).H-OCH(3) is in a position to act as a general base catalyst for hydride equivalent transfer to C5 of PQQ. We thus suggest that Glu171 plays the role of general base catalyst in PQQ reduction rather than Asp297 as previously suggested. The reduction is assisted by Arg324, which hydrogen bonds to the ortho-quinone moiety of PQQ. The rearrangement of the C5-reduced intermediate to provide hydroquinone PQQH(2) is also assisted by proton abstraction by Glu171-CO(2)(-) and the continuous hydrogen bonding of Arg324 throughout the entire reaction. These features as well as the mapping of the channel for substrate and water into the active site entrance are the observations of major importance.     

Computational studies of aliphatic amine basicity

Computational studies have been used to examine the structural and energetic effects of adding small numbers of water molecules to ammonia, methylamine, dimethylamine, and trimethylamine, and their respective ammoniums ions using the effective fragment potential method. Distinct structural effects with only a few fragment water molecules are revealed. The complexity of structures increases with the number of water fragments with the water fragments forming complex networks. Structural and energetic effects are used to probe the so-called anomalous basicity effect of ammonia and the methylamines on going from the gas phase to aqueous solution.     

Chemical, pulse radiolysis and density functional studies of a new, labile 5,6-indolequinone and its semiquinone

The chemical and spectroscopic characterization of 5,6-indolequinones and their semiquinones, key transient intermediates in the oxidative conversion of 5,6-dihydroxyindoles to eumelanin biopolymers, is a most challenging task. In the present paper, we report the characterization of a novel, relatively long-lived 5,6-indolequinone along with its semiquinone using an integrated chemical, pulse radiolytic, and computational approach. The quinone was obtained by oxidation of 5,6-dihydroxy-3-iodoindole (1a) with o-chloranil in cold ethyl acetate or aqueous buffer: it displayed electronic absorption bands around 400 and 600 nm, was reduced to 1a with Na2S2O4, and reacted with o-phenylenediamine to give small amounts of 3-iodo-1H-pyrrolo[2,3-b]phenazine (2). The semiquinone exhibited absorption maxima at 380 nm (sh) and 520 nm and was detected as the initial species produced by pulse radiolytic oxidation of 1a at pH 7.0. DFT investigations indicated the 6-phenoxyl radical and the N-protonated radical anion as the most stable tautomers for the neutral and anion forms of the semiquinone, respectively. Calculated absorption spectra in water gave bands at 350 (sh) and 500 nm for the neutral form and at 310 and 360 (sh) nm for the anion. Disproportionation of the semiquinone with fast second-order kinetics (2k = 1.1 x 1010 M-1 s-1) gave a chromophore with absorption bands resembling those of chemically generated 1a quinone. Computational analysis predicted 1a quinone to exist in vacuo as the quinone-methide tautomer, displaying low energy transitions at 380 and 710 nm, and in water as the o-quinone, with calculated absorption bands around 400 and 820 nm. A strong participation of a p orbital on the iodine atom in the 360-380 nm electronic transitions of the o-quinone and quinone-methide was highlighted. The satisfactory agreement between computational and experimental electronic absorption data would suggest partitioning of 1a quinone between the o-quinone and quinone-methide tautomers depending on the medium.     

Mechanism of glucose oxidation by quinoprotein soluble glucose dehydrogenase: insights from molecular dynamics studies

We have generated 3 ns molecular dynamic (MD) simulations, in aqueous solution, of the bacterial soluble glucose dehydrogenase enzyme.PQQ.glucose complex and intermediates formed in PQQ reduction. In the MD structure of enzyme.PQQ.glucose complex the imidazole of His144 is hydrogen bonded to the hydroxyl hydrogen of H[bond]OC1(H) of glucose. The tightly hydrogen-bonded triad Asp163-His144-glucose (2.70 and 2.91 A) is involved in proton abstraction from glucose concerted with the hydride transfer from the C1[bond]H of glucose to the >C5[double bond]O quinone carbon of PQQ. The reaction is assisted by Arg228 hydrogen bonding to the carbonyl oxygen of >C5[double bond]O. The rearrangement of [bond](H)C5(O-)[bond]C4([double bond]O)[bond] of II to [bond]C5(OH)[double bond]C4(OH)[bond] of PQQH(2) hydroquinone is assisted by general acid protonatation of the >C4[double bond]O oxygen by protonated His144 and hydrogen bonds of Arg228 to the oxyanion O5. The continuous hydrogen bonding of the amide side chain of Asn229 to >C4[double bond]O4 oxygen and that of the O5 oxygen of the cofactor to Wat89 is observed throughout the entire reaction.     

Synthesis of 2,4-substituted 6,7-phenanthreno-and 6,7-acenaphthenopteridines

A series of 6,7-phenanthreno- and 6,7-acenaphthenopteridines bearing different substituents at positions 2 and 4 are prepared. The structures of the compounds are confirmed by spectroscopic studies and elemental analyses.     

Thermogravimetric studies on some Cu(II) amine complexes

Thermogravimetric analysis of solid state bis-Cu(II) amine complexes CuL₂X₂, whereX=Cl^– or NO ₃ ^– andL=ethylenediamine (en), tetramethylethylenediamine (tmn),o-phenylenediamine (opd) and 2,2 bipyridyl (bpy), has been carried out and the kinetic parameters have been determined by fitting the observed mass losses corresponding to various steps using the Coats and Redfern equation. These studies show that the activation energies of these complexes decrease on changing the ligand in the order: bpy > opd > en > tmn. This trend can be attributed to the rigidity of the N-C-C-N skeleton in the bpy and opd ligands and steric crowding at the nitrogens in the tmn ligand. The majority of the decomposition steps were found to be composite in nature and frequently involved fractional groups expelled from the complexes. From the observation ofE _a> H, the thermal decomposition process of these complexes appears to be governed by the nucleation and growth mechanism.

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Zusammenfassung Kupfer(II)-bisaminkomplexe der Formel CuL₂X₂ mitX=Cl^– oder NO ₃ ^– undL=Äthylendiamin (en), Tetramethyläthylendiamin (tmn),o-Phenylendiamin (opd) und 2,2-Bipyridyl (bpy) wurden thermogravimetrisch untersucht und die kinetischen Parameter aus den gemessenen, zu den entsprechenden Schritten gehörenden Masseverlustwerten unter Anwendung der Gleichung von Coats und Redfern bestimmt. Es ergab sich, daß die Aktivierungsenergie dieser Komplexe entsprechend folgender Ligandenreihe abnimmt: bpy > opd > en > tmn. Dies kann bei den bpy und opd Liganden auf die Starrheit des N-C-C-N Fragmentes und im Falle der tmn Liganden auf eine räumliche Überfülltheit am Stickstoffatom zurückgeführt werden. Die Mehrzahl der Zersetzungsschritte sind komplexer Natur und haben häufig aus den Komplexen stammende Teilgruppen zum Ergebnis. Aufgrund der BeobachtungE _a>H scheint der thermische Zersetzungsvorgang dieser Komplexe durch einen Keimbildungs und -Wachstums mechanismus bestimmt zu werden.

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-() (II) CuL₂X₂, X = , aL= (en), (tmn),o- (opd) 2,2- (bpy). C - . , bpy\s>opd\s>en\s>tmn. N-C-C-N o- . , , . E _a H, , « ».

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A research grant from the University Grants Commission (India) to LSP and a JRF to JKK are thankfully acknowledged.     

Simultaneous kinetic and microdielectric studies of some epoxy–amine systems

Three reactive epoxy–amine systems based on diglycidyl ether of bisphenol A (DGEBA) with 4,4′-diaminodiphenylsulfone (DDS), 4,4′-methylenebis [3-chloro 2,6-diethylaniline] (MCDEA), and 4,4′-methylenebis [2,6-diethylaniline] (MDEA), were studied during isothermal curings at 140 and 160°C. The simultaneous kinetic and dielectric studies allow to express conductivity, σ, in terms of conversion, x, and of glass transition temperature, T_g. The conductivity, σ₀, of the initial monomer mixture and, σ_∞ of the fully cured network are measured. It is found that:

• The glass transition temperature, T_g, versus conversion, x, curves follows the equation of Di Benedetto modified by Pascault and Williams
• There exists a linear relation between log σ/log σ₀ and T_g.

So, it is possible to predict both kinetic and dielectric behaviors of these epoxy-amine systems by the knowledge of T_g0, ΔC_p0, and σ₀, respectively, glass transition temperature, heat capacity, and conductivity of initial monomer mixture, T_g∞ and ΔC_p∞, and σ_∞, respectively, glass transition temperature and heat capacity and conductivity of fully cured network. © 1998 John Wiley & Sons, Inc. J Polym Sci B: Polym Phys 36: 2911–2921, 1998     

Model compound studies

The influence of other hemicellulosic sugars (arabinose, galactose, mannose, and glucose), oxygen limitation, and initial xylose concentration on the fermentation of xylose to xylitol was in vestigated using experimental design methodology. Oxygen limitation and initial xylose concentration had strong influences on xylitol production by Candida tropicalis ATCC 96745. Under semiaerobic conditions, xylitol yield was highest (0.62 g/g), whereas under aerobic conditions volumetric productivity was highest (0.90g/[L·h]). In the presence of glucose, xylose utilization was strongly repressed and sequential sugar utilization was observed. Ethanol produced from the glucose caused a 50% reduction in xylitol yield when the ethanol con centration exceeded 30 g/L. When complex synthetic hemicellulosic sugars were fermented, glucose was initially consumed followed by a simultaneous uptake of the other sugars. The highest xylitol yield (0.84 g/g) and volumetric productivity (0.49 g/[L·h]) were obtained for substrates containing high arabinose and low glucose and mannose contents.     

Syntheses of 7-fluoro- and 6,7-difluoroserotonin and 7-fluoro- and 6,7-difluoromelatonin

The Abramovitch adaption of the Fischer indole synthesis gave low yields of 7-fluoro-5-methoxytryptamine due in part to decomposition during the required decarboxylation step. Therefore, 7-fluoro- and 6,7-difluoro-5-methoxytryptamines were prepared by reaction of aminobutyraldehyde (generated in situ from the diethyl acetal) with 2-fluoro- and 2,3-difluoro-4-methoxyphenylhydrazine, and the products converted to the corresponding serotonins. The melatonins were prepared by a one-pot reaction that involved in situ acetylation of the aminobutyraldehyde.     

Stereochemical studies of substituted 6,7- benzomorphan derivatives by 13C and 1H NMR spectroscopy

The ¹³C NMR spectra of a series of 6,7-benzomorphan derivatives variously substituted at C-5 and C-9 by methyl and at C-3 by cyano, alkyl and aralkyl groups, together with certain 3-cyano, 3-allyl or benzyl congeners, are reported and chemical shift data analysed in terms of the configuration of isomeric pairs and compounds isolated as single diastereoisomers. Special attention is given to the consequences of γ-shielding interactions, the effects of the nitrogen lone-pair orbital and anisotropic shielding by the aromatic region of the molecule. Deductions of stereochemistry are supported by ¹H NMR data and the NMR features of the corresponding methiodide salts.     

Studies on amine hydrothiocyanates. IV. Thermal decomposition studies using TG

The thermal decomposition of piperidine hydrothiocyanate, piperazine hydrothiocyanate, and the dihydrothiocyanates of ethylenediamine and 1,3-diaminopropane has been studied using TG. Piperidine hydrothiocyanate decomposes in a single step while the dihydrothiocyanates follow more complicated decomposition patterns yielding H₂S and half of the organic moiety first. The second step involves the loss of H₂S and the remainder of the organic moiety. In each case, complex polymeric materials result. Piperazine hydrothiocyanate also decomposes in two steps, the first involving the loss of half of the piperazine and the second involving the loss of piperazine and H₂S. Kinetic parameters have been determined for all these reactions.     

Model reactions of amine curing of glycidylamine epoxy resins: Homopolymerization of N-methylglycidylaniline

The kinetics of homopolymerization of the monofunctional epoxide N-methylglycidylaniline in the presence of a tertiary amine or an amino alcohol has been followed by reversed phase high performance liquid chromatography and size exclusion chromatography. The reaction products were identified by mass spectrometry using potassium ionization of desorbed species (K⁺IDS). 1,3-Di-N-methylanilino-2-propanol (P) was the main reaction product and low molecular weight oligomers with M_n > 600 were also formed. The molecular weight and fraction of oligomers decrease with increasing concentration of the initiator. The suggested complex reaction mechanism involves formation of four stable oligomeric series initiated by reaction of the epoxide with either an OH group of (a) the amino alcohol, (b) product P, (c) traces of water, or (d) the tertiary amine to form ionic species resulting in the ionic propagation. Regeneration of the initiator and formation of new initiating centers during the polymerization are the causes of low molecular weights of oligomers. © 1992 John Wiley & Sons, Inc.     

Ruthenium(II) complexes of 6,7-dicyanodipyridoquinoxaline: synthesis, luminescence studies, and DNA interaction

The hexaflurophosphate and chloride salts of a series of ruthenium(II) complexes incorporating a new dipyridophenazine-based ligand, dicnq (6,7-dicyanodipyrido[2,2-d:2',3'-f]quinoxaline), are synthesized in good-to-moderate yields. These mono ([Ru(phen)2(dicnq)]2+; phen = 1,10-phenanthroline), bis ([Ru(phen)(dicnq)2]2+), and tris ([Ru(dicnq)3]2+) complexes are fully characterized by elemental analysis, infrared, FAB-MS, 1H NMR, and cyclic voltammetric methods. Results of absorption titration and thermal denaturation studies reveal that these complexes are moderately strong binders of calf-thymus (CT) DNA, with their binding constants spanning the range (1-3) x 10(4) M-1. On the other hand, under the identical set of experimental conditions of light and drug dose, the DNA (pBR 322)-photocleavage abilities of these ruthenium(II) complexes follow the order [Ru(phen)2(dicnq)]2+ > [Ru(phen)(dicnq)2]2+ > [Ru(dicnq)3]2+, an order which is the same as that observed for their MLCT emission quantum yields. Steady-state emission studies carried out in nonaqueous solvents and in aqueous media with or without DNA reveal that while [Ru(dicnq)3]2+ is totally nonemissive under these solution conditions, both [Ru(phen)2(dicnq)]2+ and [Ru(phen)(dicnq)2]2+ are luminescent and function as "molecular light switches" for DNA. Successive addition of CT DNA to buffered aqueous solutions containing the latter two complexes results in an enhancement of the emission in each case, with the enhancement factors at saturation being approximately 16 and 8 for [Ru(phen)2(dicnq)]2+ and [Ru(phen)(dicnq)2]2+, respectively. These results are discussed in light of the relationship between the structure-specific deactivations of the MLCT excited states of these metallointercalators and the characteristic features of their DNA interactions, and attempts are made to compare and contrast their properties with those of analogous dipyridophenazine-based complexes, including the ones reported in the preceding paper.     

Model studies onRNA-replication I

A simplified mechanism ofRNA-replication by a specific polymerase is analysed by direct numerical integration and by means of the quasiequilibrium approximation. The quasiequilibrium approximation is formulated in precise mathematical terms for three simple, two step reactions which describe approach towards equilibrium, irreversible transformation and unlimited growth.Dedicated to Prof. Dr.G. Kainz on the occasion of his 60th birthday.     

Hydration of tri-n-hexyl amine hydrochloride,tri-n-octyl amine hydrochloride,tri-n-decyl amine hydrochloride and tri-n-dodecyl amine hydrochloride benzene solutions

Isopiestic equilibrations of THxA·HCl, TOA·HCl, TDA·HCl and TLA·HCl benzene solutions have been made in the concentration range from 0·02 to 0·13 mole/kg at different water activities and 25°C. The mean hydration number of the chloride ion at unit water activity was found to be 1·024±0·015 for the systems investigated. To determine the effect of water activity on the amine salt activity the results were treated on the basis of the Gibbs-Duhem equation and a simple linear relation obtained between salt and water activities. IR spectrophotometric measurements were performed to identify the nature of the hydrated species.     

(±)-6,7-丙酮缩二醇-3-酮-庚酸乙酯合成研究

用对甲苯磺酸-2,3-丙酮缩甘油酯(2)与乙酰乙酸乙酯盐、碳酸二乙酯反应, 制备β-酮酯类衍生物1. 以(±)-1,2-丙酮缩甘油为起始物, 经对甲苯磺酰化、亲核取代、脱羧等反应, 方便、高产率地合成了6,7-丙酮缩二醇-3-酮-庚酸乙酯(1). 试图通过对甲苯磺酸-2,3-丙酮缩甘油酯(2)和乙酰乙酸乙酯双阴离子反应制备6,7-丙酮缩二醇-3-酮-庚酸乙酯(1)未获成功. 所合成的化合物经元素分析, IR, ¹H NMR, ¹³C NMR和MS光谱表征.     

Syntheses of triazolo[6,7-d]phthalide and triazolo[6,7-d]dihydrocoumarin

Triazolo[6,7-d]phthalide has been synthesized in a seven step sequence from phthalide in 24% overall yield, and triazolo[6,7-d]dihydrocoumarin has been prepared in nine steps from dihydrocoumarin in 20% overall yield. The ¹H and ¹³C nmr spectra and the tautomeric equilibria of the final compounds and intermediates are discussed.     

6,7-二甲氧基-2-苯乙烯基喹啉的合成

在乙醇钠催化下,6-氨基藜芦醛与卞叉丙酮发生Friedlainder缩合反应得到新化合物6,7-二甲氧基-2-苯乙烯基喹啉,产率59％。其结构经UV,^1H NMR,IR和MS表征。