oxidation of catechol in plants iv. purification and properties of the 3,4,3',4'-tetrahydroxydiphenyl-forming enzyme system from tecoma leaves

Acetone powders prepared from leaf extracts of Tecoma stans L. were found to catalyze the oxidation of catechol to 3,4,3',4'-tetrahydroxydiphenyl. Fractionation of the acetone powders obtained from Tecoma leaves with acetone, negative adsorption of the acetone fraction with tricalcium phosphate gel, and chromatography of the gel supernatant on DEAE-Sephadex yielded a 68-fold purified enzyme with 66% recovery. The enzyme had an optimum pH around 7.2. It showed a temperature optimum of 30° and the Km for catechol was determined as 2 x 10-4 m. The purified enzyme moved as a single band on polyacrylamide gel electrophoresis. Its activity was found to be partially stimulated by Mg2+. The reaction was not inhibited by o-phenanthroline and agr,agr'-dipyridyl. The purified enzyme was highly insensitive to a range of copper-chelating agents. It was not affected appreciably by thiol inhibitors. The reaction was found to be suppressed to a considerable extent by reducing agents like GSH, cysteine, cysteamine, and ascorbic acid. The purified enzyme was remarkably specific for catechol. Catalase affected neither the enzyme activity nor the time course of the reaction. Hydrogen peroxide was not formed as a product of the reaction.

Anaerobic DNA cleavage activity in red light and photocytotoxicity of (pyridine-2-thiol)cobalt(III) complexes of phenanthroline bases

Cobalt(III) complexes [Co(pnt)(B)(2)](NO3)(2) (1-3) of pyridine-2-thiol (pnt) and phenanthroline bases (B), viz. 1,10-phenanthroline (phen in 1), dipyrido[3,2-d: 2',3'-f]quinoxaline (dpq in 2) and dipyrido[3,2-a:2',3'-c] phenazine (dppz in 3), have been prepared, characterized and their photo-induced anaerobic DNA cleavage activity studied. The crystal structure of 1a as mixed ClO4- and PF6- salt of 1 shows a (CoN5S)-N-III coordination geometry in which the pnt and phen showed N,S- and N,N-donor binding modes, respectively. The complexes exhibit Co(III)/Co(II) redox couple near -0.3 V (vs. SCE) in 20% DMF-Tris-HCl buffer having 0.1 M TBAP. The complexes show binding propensity to calf thymus DNA giving K-b values within 2.2 x 10(4)-7.3 x 10(5) M-1. Thermal melting and viscosity data suggest DNA surface and/or groove binding of the complexes. The complexes show significant anaerobic DNA cleavage activity in red light under argon atmosphere possibly involving sulfide anion radical or thiyl radical species. The DNA cleavage reaction under aerobic medium in red light is found to involve both singlet oxygen and hydroxyl radical pathways. The dppz complex 3 shows non-specific BSA and lysozyme protein cleavage activity in UV-A light of 365 nm via both hydroxyl and singlet oxygen pathways. The dppz complex 3 exhibits photocytotoxicity in HeLa cervical cancer cells giving IC50 values of 767 nM and 19.38 mu M in UV-A light of 365 nm and in the dark, respectively. A significant reduction of the dark toxicity of the dppz base (IC50 = 8.34 mu M in dark) is observed on binding to the cobalt(III) center.

Solubility of n-(4-Ethoxyphenyl)ethanamide in Supercritical Carbon Dioxide

The solubility of a drug, n-(4-ethoxyphenyl)ethanamide (phenacetin), in supercritical carbon dioxide was determined by a Saturation method at (308, 318, and 328) K from (9 to 19) MPa. The Solubilities in mole fraction of n-(4-ethoxyphenyl)ethanamide in supercritical carbon dioxide were in the range of 1.29.10(-5) to 2.88.10(-5), 1.13.10(-5) to 3.65.10(-5), and 0.91.10(-5) to 4.28.10(-5) at (308, 3 18, and 328) K, respectively. The solubility data were correlated with the Peng-Robinson equation of state models and the Mendez-Santiago and Teja model.

Hydration and distortion of peptide helices in crystals. α-Helical structure of a dodecapeptide, Boc-(Ala-Leu-Aib)4-OMe

The dodecapeptide Boc-(Ala-Leu-Aib)(4)-OMe crystallized with two independent helical molecules in a triclinic cell. The two molecules are very similar in conformation, with a 3(10)-helix turn at the N-terminus followed by an alpha-helix, except for an elongated N(7)...O(3) distance in both molecules. All the helices in the crystal pack in a parallel motif. Eleven water sites have been found in the head-to-tail region between the apolar helices that participate in peptide-water hydrogen bonds and a network of water-water hydrogen bonds. The crystal parameters are as follows: 2(C58H104N12O15)+ca. 10H(2)O, space group P1 with a = 12.946(2), b = 17.321(3), c = 20.465(4) Angstrom, alpha = 103.12(2), beta = 105.63(2), gamma = 107.50(2)degrees, Z = 2, R = 10.9% for 5152 data observed > 3 sigma(F), resolution 1.0 Angstrom. In contrast to the shorter sequences [Karle et al. (1988)Proc. Natl. Acad. Sci. USA 85, 299-303] and Boc-(Ala-Leu-Aib)(2)-OMe [Karle et al. (1989) Biopolymers 28, 773-781], no insertion of a water molecule into the helix is observed. However, the elongated N---O distance between Ala(7) NH and Aib(3) CO in both molecules (molecule A, 3.40 Angstrom; molecule B, 3.42 Angstrom) is indicative of an incipient break in the helices. (C) Munksgaard 1994.

Chain structures in alkali metal borophosphates: synthesis and characterization of K3[BP3O9(OH)3] and Rb3[B2P3O11(OH)2]

Two new alkali metal borophosphates, K-3[BP(3)o(9)(OH)(3)] and Rb-3[B2P3O11(OH)(2)], were synthesized by applying solvothermal techniques using ethanol as solvent. The crystal structures were solved by means of single-crystal X-ray diffraction (K-3[BP3O9(OH)(3)], monoclinic, C2/c (No. 15), a = 2454.6(8) pm, b = 736.3(2) pm, c = 1406.2(4) pm, beta = 118.35(2)degrees, Z = 8; Rb-3[B2P3O11(OH)(2)], monoclinic, P2(1)/c (No. 14), a = 781.6(2) pm, b:= 667.3(2) pm, c = 2424.8(5) pm, beta = 92.88(1)degrees, Z = 4). Both crystal structures comprise borophosphate chain anions. While for the rubidium compound a loop-branched chain motif is found as common for most of the chain anions in alkali metal borophosphates, the crystal structure of the potassium phase comprises the first open-branched chain with the highest phosphate content found so far in this group of compounds. Both chain anions are Closely related to known anhydrous or hydrated phases, and the structural relations are discussed in terms of how the presence of OH groups and hydrogen bonds as well as number, charge, and size of charge balancing cations influence the 3D structural arrangement. The anionic entities are classified in terms of general principles of structural systematics for borophosphates.

Large molecular motions are tolerated in crystals of diamine double salt of trans-chlorocinnamic acids with trans-1,2-diaminocyclohexane

Contrary to the general assumption that photoreactions in crystals may not proceed with large molecular motions, a pedal-like motion prompted by electronic excitation is believed to be involved during the β-dimer formation from the crystals of the diamine double salt of trans-2,4-dichlorocinnamic acid and trans-1,2-diaminocyclohexane.

Studies in oxasteroids—I : Synthesis of 3-cyano-3-methyl-7-methoxychroman-4-one and the structure of an “abnormal” product

A synthesis of 3-cyano-3-methyl-7-methoxychroman-4-one is reported. The structure of an “abnormal” product obtained during isomerization (III) with potassium t-butoxide in t-butanol, followed by alkylation with methyl iodide has been proved to be 3-t-butoxy-2-cyano- 2-mehthyl-2′,4′-dimethoxypropiophenone (IVa).

Exclusion of an Exotic Top Quark with -4/3 Electric Charge Using Soft Lepton Tagging

We present a measurement of the electric charge of the top quark using $\ppbar$ collisions corresponding to an integrated luminosity of 2.7~fb$^{-1}$ at the CDF II detector. We reconstruct $\ttbar$ events in the lepton+jets final state and use kinematic information to determine which $b$-jet is associated with the leptonically- or hadronically-decaying $t$-quark. Soft lepton taggers are used to determine the $b$-jet flavor. Along with the charge of the $W$ boson decay lepton, this information permits the reconstruction of the top quark's electric charge. Out of 45 reconstructed events with $2.4\pm0.8$ expected background events, 29 are reconstructed as $\ttbar$ with the standard model $+$2/3 charge, whereas 16 are reconstructed as $\ttbar$ with an exotic $-4/3$ charge. This is consistent with the standard model and excludes the exotic scenario at 95\% confidence level. This is the strongest exclusion of the exotic charge scenario and the first to use soft leptons for this purpose.

Transverse momentum and pseudorapidity distributions of charged hadrons in pp collisions at sqrt(s) = 0.9 and 2.36 TeV

Measurements of inclusive charged-hadron transverse-momentum and pseudorapidity distributions are presented for proton-proton collisions at sqrt(s) = 0.9 and 2.36 TeV. The data were collected with the CMS detector during the LHC commissioning in December 2009. For non-single-diffractive interactions, the average charged-hadron transverse momentum is measured to be 0.46 +/- 0.01 (stat.) +/- 0.01 (syst.) GeV/c at 0.9 TeV and 0.50 +/- 0.01 (stat.) +/- 0.01 (syst.) GeV/c at 2.36 TeV, for pseudorapidities between -2.4 and +2.4. At these energies, the measured pseudorapidity densities in the central region, dN(charged)/d(eta) for |eta|

Oxidation of catechol in plants : III. Purification and properties of the diphenylene dioxide 2,3-quinone-forming enzyme system from Tecoma leaves

In attempting to determine the nature of the enzyme system mediating the conversion of catechol to diphenylenedioxide 2,3-quinone, in Tecoma leaves, further purification of the enzyme was undertaken. The crude enzyme from Tecoma leaves was processed further by protamine sulfate precipitation, positive adsorption on tricalcium phosphate gel, and elution and chromatography on DEAE-Sephadex. This procedure yielded a 120-fold purified enzyme which stoichiometrically converted catechol to diphenylenedioxide 2,3-quinone. The purity of the enzyme system was assessed by polyacrylamide gel electrophoresis. The approximate molecular weight of the enzyme was assessed as 200,000 by gel filtration on Sephadex G-150. The enzyme functioned optimally at pH 7.1 and at 35 °C. The Km for catechol was determined as 4 × 10−4 Image . The enzyme did not oxidize o-dihydric phenols other than catechol and it did not exhibit any activity toward monohydric and trihydric phenols and flavonoids. Copper-chelating agents did not inhibit the enzyme activity. Copper could not be detected in the purified enzyme preparations. The purified enzyme was not affected by extensive dialysis against copper-complexing agents. It did not show any peroxidase activity and it was not inhibited by catalase. Hydrogen peroxide formation could not be detected during the catalytic reaction. The enzymatic conversion of catechol to diphenylenedioxide 2,3-quinone by the purified Tecoma leaf enzyme was suppressed by such reducing agents as GSH and cysteamine. The purified enzyme was not sensitive to carbon monoxide. It was not inhibited by thiol inhibitors. The Tecoma leaf was found to be localized in the soluble fraction of the cell. Treatment of the purified enzyme with acid, alkali, and urea led to the progressive denaturation of the enzyme.

Synthesis and identification of a new class of (S)-2,6-diamino-4,5,6,7-tetrahydrobenzo[d]thiazole derivatives as potent antileukemic agents

Benzothiazoles are multitarget agents with broad spectrum of biological activity. Among the antitumor agents discovered in recent years, the identification of various 2-(4-aminophenyl) benzothiazoles as potent and selective antitumor drugs against different cancer cell lines has stimulated remarkable interest. Some of the benzothiazoles are known to induce cell cycle arrest, activation of caspases and interaction with DNA molecule. Based on these interesting properties of benzothiazoles and to obtain new biologically active agents, a series of novel 4,5,6,7-tetrahydrobenzo[d]thiazole derivatives 5(a-i) were synthesized and evaluated for their efficacy as antileukemic agents in human leukemia cells (K562 and Reh). The chemical structures of the synthesized compounds were confirmed by H-1 NMR, LCMS and IR analysis. The cytotoxicity of these compounds were determined using trypan blue exclusion, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. Results showed that, these compounds mediate a significant cytotoxic response to cancer cell lines tested. We found that the compounds having electron withdrawing groups at different positions of the phenyl ring of the thiourea moiety displayed significant cytotoxic effect with IC50 value less than 60 mu M. To rationalize the role of electron withdrawing group in the induction of cytotoxicity, we have chosen molecule 5g (IC50 similar to 15 mu M) which is having chloro substitution at ortho and para positions. Flow cytometric analysis of annexin V-FITC/ propidium iodide (PI) double staining and DNA fragmentation suggest that 5g can induce apoptosis.

Structure-Reactivity Correlations of Benzoin Alkyl Ethers. Structures of 2-Methoxy-1,2-diphenylethanone (I) and 2-Isopropoxy-1,2-diphenylethanone (II)

(I): C15H1402, Mr---226.27, triclinic, Pi,a=8.441 (2), b= 10.276 (1), c= 15.342 (2)A, a=91.02 (2), ~ t= 79.26 (2), y= 105.88 (2) °, V=1256.8 (4)A 3, Z=4, D,,= 1.209 (flotation in KI),D x - 1.195 g cm -3, #(Mo, 2 = 0.7107/~) = 0.44 cm -~,F(000) = 480, T= 293 K, R -- 0.060 for 1793 significant reflections. (II): C~THlsO2, Mr= 254.83, orthorhombic, Pca21, a=8.476 (1); b= 16.098 (3), c=10.802(3)A, V=1473.9 (5) A s, Z=4, Dm=1.161 (flotation in KI), Dx= 1.148gem -3, /~(Mo, 2=0.7107 A) =0.41 cm -~, F(000) = 544, T= 293 K, R = 0.071 for 867 significant reflections. Both (I) and (II) crystallize in a cisoid conformation for the carbonyl group and alkoxy groups. Compounds (I) and (II) are photostable on irradiation in the solid state in spite of the favourable conformation of the functional groups for intramolecular H abstraction. Absence of photoreaction of (I)and (II) in the solid state is rationalized in the light of unfavourable intramolecular geometry.

Synthesis, Reactivity, Structural Aspects, and Solution Dynamics of Cyclopalladated Compounds of N,N `,N `'-Tris(2-anisyl)guanidine

N,N',N `'-Tris(2-anisyl)guanidine, (ArNH)(2)C=NAr (Ar = 2-(MeO)C6H4), was cyclopallaclated with Pd(OC(O)R)(2) (R = Me, CF3) in toluene at 70 degrees C to afford palladacycles Pd{kappa(2)(C,N)-C6H3-(OMe)-3(NHC(NHAr)(=NAr))-2}(mu-OC(O)R)](2)(R = Me (1a) and CF3 (1b)) in 87% and 95% yield, respectively. Palladacycle 1a was subjected to a metathetical reaction with LiBr in aqueous ethanol at 78 degrees C to afford palladacycle Pd{kappa(2)(C,N)-C6H3(OMe)-3(NHC(NHAr)(=NAr))-2}(mu-Br)](2) (2) in 90% yield. Palladacycle 2 was subjected to a bridge-splitting reaction with Lewis bases in CH2Cl2 to afford the monomeric palladacycles Pd{kappa(2)(C,N)-C6H3(OMe)-3(NHC(NHAr)(=NAr))-2}Br(L)] (L = 2,6-Me2C5H3N (3a), 2,4-Me2C5H3N (3b), 3,5-Me2C5H3N (3c), XyNC (Xy = 2,6-Me2C6H3; 4a), (BuNC)-Bu-t (4b), and PPh3 (5)) in 87-95% yield. Palladacycle 2 upon reaction with 2 equiv of XyNC in CH2Cl2 afforded an unanticipated palladacycle, Pd{kappa(2)(C,N)-C(=NXy)(C6H3(OMe)-4)-2(N=C-(NH Ar)(2))-3} Br(CNXy)] (6) in 93% yield, and the driving force for the formation of 6 was ascribed to a ring contraction followed by amine-imine tautomerization. Palladacycles 1 a,b revealed a dimeric transoid in-in conformation with ``open book'' framework in the solid state. In solution, 1 a exhibited a fluxional behavior ascribed to the six-membered ``(C,N)Pd'' ring inversion and partly dissociates to the pincer type and kappa(2)-O,O'-OAc monomeric palladacycles by an anchimerically assisted acetate cleavage process as studied by variable-temperature H-1 NMR data. Palladacycles 3a,b revealed a unique trans configuration around the palladium with lutidine being placed trans to the Pd-C bond, whereas cis stereochemistry was observed between the Pd-C bond and the Lewis base in 4a (as determined by X-ray diffraction data) and 5 (as determined by P-31 and C-13 NMR data). The aforementioned stereochemical difference was explained by invoking relative hardness/softness of the donor atoms around the palladium center. In solution, palladacycles 3a-c exist as a mixture of two interconverting boat conformers via a planar intermediate without any bond breaking due to the six-membered ``(C,N)Pd'' ring inversion, whereas palladacycles 4a,b and 5 exist as a single isomer, as deduced from detailed H-1 NMR studies.

Hydrogen-bond-directed self-assembly of D-(+)-dibenzoyltartaric acid and 4-aminopyridine: optical nonlinearities and stoichiometry-dependent novel structural features

Attempts to prepare hydrogen-bond-directed nonlinear optical materials from a 1:1 molar mixture Of D-(+)-dibenzoyltartaric acid (DBT, I) and 4-aminopyridine (4-AP, II) resulted in two salts of different stoichiometry. One of them crystallizes in an unusual 1.5:1 (acid:base) monohydrate salt form III while the other one crystallizes as 1:1 (acid:base) salt IV. Crystal structures of both of the salts were determined from single-crystal X-ray diffraction data. The salt III crystallizes in a monoclinic space group C2 with a = 30.339(l), b = 7.881(2), c = 14.355(1) angstrom, beta = 97.48(1)degrees, V = 3403.1(9) angstrom3, Z = 4, R(w) = 0.058, R(w)= 0.058. The salt IV also crystallizes in a monoclinic space group P2(1) with a = 7.500(1), b = 14.968(2), c = 10.370(1) angstrom, beta = 102.67(1)degrees, V = 1135.9(2) angstrom3, Z = 2, R = 0.043, R(w) = 0.043. Interestingly, two DBT molecules with distinctly different conformation are present in the same crystal lattice of salt III. Extensive hydrogen-bonding interactions are found in both of the salts, and both of them show SHG intensity 1.4-1.6 times that of urea.

Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity

2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4 A resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coil CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes. (C) 2010 Elsevier Inc. All rights reserved.