Interactions of Bovine Brain Tubulin with Pyridostigmine Bromide and N,N′-Diethyl-m-Toluamide

Pyridostigmine bromide (PB), an inhibitor of acetylcholinesterase, has been used as a prophylactic for nerve gas poisoning. N,N-diethyl-m-toluamide (DEET) is the active ingredient in most insect repellents and is thought to interact synergistically with PB. Since PB can inhibit the binding of organophosphates to tubulin and since organophosphates inhibit microtubule assembly, we decided to examine the effects of PB and DEET on microtubule assembly as well as their interactions with tubulin, the subunit protein of microtubules. We found that PB binds to tubulin with an apparent K _d of about 60 M. PB also inhibits microtubule assembly in vitro, although at higher concentrations PB induces formation of tubulin aggregates of high absorbance. Like PB, DEET is a weak inhibitor of microtubule assembly and also induces formation of tubulin aggregates. Many tubulin ligands stabilize the conformation of tubulin as measured by exposure of sulfhydryl groups and hydrophobic areas and stabilization of colchicine binding. PB appears to have very little effect on tubulin conformation, and DEET appears to have no effect. Neither compound interferes with colchicine binding to tubulin. Our results raise the possibility that PB and DEET may exert some of their effects in vivo by interfering with microtubule assembly or function, although high intracellular levels of these compounds would be required.     

Stereoselective chlorination of methyl β-lactoside with mesyl chloride in N,N-dimethylformamide

Treatment of methyl β-lactoside with mesyl chloride in N,N-dimethylformamide under a variety of conditions gave complex mixtures of chlorinated products, of which nine were isolated and characterised. Chlorination at a secondary position always occurred with inversion of configuration. When the reaction was conducted at 94° for 9 days, a mixture of the 3,3′,4′,6,6′-pentachloride, the 3,3′,6,6′- and 3,4′,6,6′-tetrachlorides, and the 3,6,6′- and 3′,6,6′-trichlorides was obtained together with the 3′,4′-epoxide of the 6,6′-dichloride, which was an artefact. Under milder conditions, the 6,6′-dichloride was encountered, together with methyl 6-chloro-6-deoxy-β-D-glucopyranoside which had arisen by hydrolysis of the interglycosidic bond. It is particularly noteworthy that displacement occurred at C-3′ of the lactoside, in spite of the vic-axial group at C-4′ which should hinder nucleophilic displacement at C-3′. The cause of this anomaly is discussed.     

Formation of the thioester,N,S-diacetylcysteine,from acetaldehyde and N,N′diacetylcystine in aqueous solution with ultraviolet light

Summary The thioester, N,S-diacetylcysteine, is formed during the illumination of phosphate buffered (pH 7.0) aqueous solutions of acetaldehyde and N,N-diacetylcystine with ultraviolet light. The yield of N,S-diacetylcysteine relative to N-acetylcysteine and unidentified products progressively increases as ultraviolet light below 239 nm, 253 nm and 281 nm is cut off with optical filters. When ultraviolet light below 320 nm is removed with an optical filter, there is no detectable reaction. Illumination of 0.025 M N,N-diacetylcystine with 0.5 M and 1.0 M acetaldehyde with filtered ultraviolet light gives, respectively, 20% and 80% yields of N,S-diacetylcysteine. In the reaction with 1.0 M acetaldehyde, N-acetylcysteine forms early in the reaction and later decreases with its conversion to N,S-diacetylcysteine. The prebiotic significance of these reactions is discussed.Abbreviations Ac-Cys N-acetylcysteine - Ac-Cys(Ac) N,S-diacetylcysteine - Ac-Cys N,N-diacetylcystine     

Liquid scintillation counting of polyacrylamide gels crosslinked with N,N′-methylene-bis-acrylamide and N,N′-diallyltartardiamide

Tritium (³H)-labeled material within polyacrylamide gel slices is commonly quantified by four general liquid scintillation counting methods: combustion, gel solubilization, selective solubilization of modified crosslinked gels, and elution. Of these four methods examined in this study, only combustion ensured complete recovery of ³H label; however, a less expensive and more convenient elution method yielding both a high recovery and cocktail counting efficiency was the use of Soluene-350 with 0.55% Permablend III in toluene. This particular solubilizer-cocktail system eliminates almost all chemiluminescence as does combustion.     

Urinary zinc excretion and zinc status of patients with Β-thalassemia major

In this study, zinc status and urinary zinc excretion with and without desferrioxamine (DFO) infusion and the relationship between urinary zinc excretion and renal tubular dysfunction in thalassemia major (TM) patients were investigated. Forty TM patients were given four DFO infusions on alternate days over a 1-wk period prior to the transfusion. On each day that DFO was given, a 24-h urine collection initiated. DFO was omitted for 1-wk before the following transfusion and during the period four 24-h urine collections were performed. Twenty healthy children provided 24-h urine collection as controls. Blood samples were taken on each of two consecutive transfusion days of the patients and from the controls. Urinary zinc excretion was measured and plasma and red blood cell (RBC) zinc analysis were performed by inductively coupled plasma-atomic emission spectrophotometry. UrinaryN-acetyl-Β-D-glucosaminidase (NAG) activity and creatinine were determined in morning urine specimens. The mean plasma zinc concentration was significantly lower in the patients not given DFO compared to the values of the patients given DFO and the control group. The mean RBC zinc concentration (Μmol/g Hb) in the patients (with and without DFO) and the control group were similar. Urinary zinc excretion was significantly higher in the patients receiving DFO compared to the control group, whereas urinary zinc excretion in the patients not given DFO was not different from the controls. Urinary NAG indices (U/g Cr) were significantly higher in the patients compared to controls. Urinary zinc excretion was correlated with the urinary NAG indices.     

Synthesis of optically active lipidicα-amino acids and lipidic 2-amino alcohols

Summary Lipidic-amino acids (LAAs) are a class of compounds combining structural features of amino acids with those of fatty acids. They are non-natural-amino acids with saturated or unsaturated long aliphatic side chains. Synthetic approaches to optically active LAAs and lipidic 2-amino alcohols (LAALs) are summarized in this review. A general approach to enantioselective synthesis of saturated LAAs is based on the oxidative cleavage of 3-amino -1,2-diols obtained by the regioselective opening of enantiomerically enriched long chain 2,3-epoxy alcohols. Unsaturated LAAs are prepared in their enantiomeric forms by Wittig reactionvia methyl (S)-2di-tert-butoxycarbonylamino-5-oxo-pentanoate. This key intermediate aldehyde is obtained by selective reduction of dimethyl N,N-di-Boc glutamate with DIBAL. (R) or (S) LAALs may be prepared starting from D-mannitol or L-serine. LAAs are converted into LAALs by chemoselective reduction of their fluorides using sodium borohydride with retention of optical purity. Replacement of the hydroxyl group of LAALs by the azido group, followed by selective reduction leads to unsaturated optically active lipidic 1,2-diamines.Abbreviations Bn benzyl - Boc tert-butoxycarbonyl - DDQ 2,3-dichloro-5,6-dicyano-1,4-benzoquinone - DET diethyl tartrate - DIBAL diisobutyl aluminum hydride - DMAP 4-dimethylaminopyridine - DMF N,N-dimethylformamide - DMSO dimethyl sulfoxide - EDC N-ethyl-N-(3-dimethylaminopropyl)carbodiimide - Et₃N triethylamine - HMPA hexamethylphosphoramide - HOBt 1-hydroxybenzotriazole - KN(TMS)₂ potassium bis(trimethylsilyl)-amide - LAA lipidic-amino acid - LAAAl lipidic 2-amino alcohol - LDA lipidic 1,2-diamine - LP lipidic peptide - MPM-Cl p-methoxybenzyl chloride - MsCl methanesulphonyl chloride - MTPA -methoxy--(trifluoromethyl)phenylaccitc - PLA₂ phospholipase A₂ - TBIIP tert-butyl hydroperoxide - THF tetrahydrofuran - TMSCl trimethylsilyl chloride - Tr trityl - Z benzyloxycarbonyl     

Synthesis and reactions of cyclic acetal derivatives of 6,6′-dichloro-6,6′-dideoxysucrose

Treatment of 6,6′-dichloro-6,6′-dideoxysucrose with a combination of 2,2-dimethoxypropane, N,N-dimethylformamide, and toluene-p-sulphonic acid (reagent A), followed by acetylation, gave the 1′,2:3,4-diacetal 1 (39%) and the 1′,2-acetal 2 (37%). A similar reaction of methyl 6-chloro-6-deoxy-α-D-glucopyranoside with reagent A yielded the corresponding 2,3- and 3,4-acetal derivatives in yields of 29% and 9%, respectively. The structures of 1 and 2 have been confirmed by ¹H-n.m.r. spectroscopy and by chemical transformations.     

Syntheses and antitumor activities of N′1,N′3-dialkyl-N′1,N′3-di-(alkylcarbonothioyl) malonohydrazide: The discovery of elesclomol

A series of N′¹,N′³-dialkyl-N′¹,N′³-di(alkylcarbonothioyl) malonohydrazides have been designed and synthesized as anticancer agents by targeting oxidative stress and Hsp70 induction. Structure–activity relationship (SAR) studies lead to the discovery of STA-4783 (elesclomol), a novel small molecule that has been evaluated in a number of clinical trials as an anticancer agent in combination with Taxol.     

A Convenient and Stereoselective Synthesis of 2′-Deoxy-β-L-Ribonucleosides

Abstract

2′-Deoxy-β-L-ribonucleosides containing usual bases which are useful as synthons for modified oligodeoxyribonucleotides, were conveniently synthesized by a stereoselective glycosylation procedure. The method is suitable for large-scale preparations.     

Synthesis and properties of optically active nanostructured polymers bearing amino acid moieties by direct polycondensation of 4,4′-thiobis(2-tert-butyl-5-methylphenol) with chiral diacids

Four derivatives of N-trimellitylimido-l-amino acid (4a–4d) were prepared by the reaction of trimellitic anhydride (1) with the l-amino acids (2a–2d) in acetic acid as diacid monomers and were used with the aim to obtain a new family of amino acid based poly(ester-imide)s (PEI)s. The polymerization was performed by direct polycondensation of chiral diacids (4a–4d) with 4,4′-thiobis(2-tert-butyl-5-methylphenol) (5) in the presence of tosyl chloride (TsCl), pyridine and N,N-dimethyl formamide (DMF). Step-growth polymerization was carried out by varying the time of heating and the molar ratio of TsCl/diacid and the optimum conditions were achieved. The synthesized polymers were characterized by means of specific rotation experiments, FT-IR, ¹H-NMR, X-ray diffraction techniques and elemental analysis. The surface morphology of the obtained polymers was studied by field emission scanning electron microscopy. The result showed nanostructure morphology of the resulting polymers. The obtained PEIs were soluble in polar aprotic solvents such as DMF, N,N-dimethyl acetamide, dimethyl sulfoxide, N-methyl-2-pyrrolidone and protic solvents such as sulfuric acid. Thermal stability and the weight-loss behavior of the PEIs were studied by thermal gravimetric analysis (TGA) techniques. TGA showed that the 10% weight loss temperature in a nitrogen atmosphere was more than 402°C, therefore they had useful levels of thermal stability associated with excellent solubility.     

Molecular design of N-linked tetravalent glycosides bearing N-acetylglucosamine,N,N′-diacetylchitobiose and N-acetyllactosamine: Analysis of cross-linking activities with WGA and ECA lectins

Two types of nonspacer- and spacer-N-linked tetravalent glycosides bearing N-acetylglucosamine (GlcNAc), N,N′-diacetylchitobiose [(GlcNAc)₂] and N-acetyllactosamine (LacNAc) were designed and prepared as glycomimetics. The interactions of wheat germ (Triticum vulgaris) agglutinin (WGA) and coral tree (Erythrina cristagalli) agglutinin (ECA) with a series of tetravalent glycosides and related compounds were studied using a hemagglutination inhibition assay, a precipitation assay, double-diffusion test, and an optical biosensor based on surface plasmon resonance (SPR). The tetravalent glycosides were found to be capable of binding and precipitating the lectins as tetravalent ligands. Strong interactions with WGA, due to a combination of multivalency effects and spacer effects, were observed for tetravalent glycosides bearing flexible tandem GlcNAc. The chelate effect leads to large rate enhancement for the tetravalent system with favorable orientation of ligands. Our simple strategy produced multivalent glycosides with strong cross-linking activity for lectin as a specific coagulant.     

Diastereo- and Enantioselective Synthesis of 1′-C-Branched N,O-Nucleosides

Abstract

A synthetic approach towards 1′-C-branched N, O-nucleosides is reported, based on 1,3-dipolar cycloaddition of ethoxycarbonylnitrone. The asymmetric version of the process exploits the presence of a chiral auxiliary at the carbon atom of nitrone and leads to β-D and β-L nucleosides in good yields.     

Nonenzymatic hydrolysis reactions of adenosine 5′-triphosphate and its related compounds: I. Hydrolysis reactions of ATP with some continuous-chain polyamines

The nonenzymatic, polyamine catalyzed hydrolysis of adenosine 5′-triphosphate has been investigated in order to shed light on ATPase function.It has been discovered that pentaethylenehexamine(pentaen) and tetraethylene-pentamine(tetraen) enhance the hydrolysis of ATP by considerable factors, whereas small amines such as ethylenediamine(en) show virtually no effect on the hydrolysis of ATP. It has also been found that the enhancing effect on the hydrolysis of ATP increases with the increasing number of the imino nitrogen atoms in the polyamines, although the spacing in the carbon chain of those polyamines cannot be ignored.In all cases the products of the hydrolysis reactions have been ADP and inorganic orthophosphate(P_i), unaccompanied by either AMP and pyrophosphate(PP_i). During the course of the hydrolysis reactions, the formations of one-to-one complexes of ATP and the polyamines have been considered as intermediates, in which several hydrogen (or electrostatic) bonds between the positively charged amino or imino nitrogen atoms of the polyamines and the adenine, ribose and triphosphate moieties of ATP are formed, activating the phosphate group so that the hydrolytic attack of water becomes easier.     

Regulation of renal gluconeogenesis by calcium ions, hormones and adenosine 3′:5′-cyclic monophosphate

1. The effect of Ca(2+), glucagon, adrenaline and adenosine 3':5'-cyclic monophosphate on gluconeogenesis by rat kidney-cortex slices was studied. 2. Glucose formation from a range of substrates, with the exception of glycerol, was increased by an increase in extracellular Ca(2+) concentration. 3. Hormones and adenosine 3':5'-cyclic monophosphate, at low Ca(2+) concentrations, stimulated glucose production from several substrates, but not from glycerol, fructose, malate or fumarate. 4. Hormonal stimulation was not detected in the absence of Ca(2+) or at 2.5mm-Ca(2+). 5. Ca(2+), hormones and adenosine 3':5'-cyclic monophosphate had no effect on phosphoenolpyruvate carboxylase activity. 6. It is proposed that Ca(2+) and adenosine 3':5'-cyclic monophosphate-mediated hormone action activate the same rate-limiting step in gluconeogenesis: this step is tentatively identified as the rate of transfer of substrates across the mitochondrial membrane.     

Synthesis and spectroscopic studies of platinum(II) complexes of N,N′-dicyclohexylethylenediamine

The platinum(II) complexes of the formula [Pt(DCHEDA)X₂], where DCHEDA is N,N′-dicyclohexylethylenediamine and X⁻ is CL⁻, Br⁻, I⁻, 0.5C₂O₄²⁻ (oxalate), 0.5C₃H₂O₄²⁻ (malonate), 0.5C₉H₄O₆²⁻ (4-carboxyphthalate), 0.5S₂O₃²⁻ or 0.5SO₄²⁻, have been synthesized and characterized by UVVis, IR, and ¹H NMR spectral techniques. All the above complexes are non-electrolytes in DMF/H₂O, except the sulphate complex which becomes a 1:1 electrolyte after incubation for 24 h at 28 °C. The halide complexes were also studied by X-ray photoelectron spectroscopy and these data suggest that there is π-bonding from platinum to halide in these complexes. The oxalate, malonate and sulphate bind in their complexes as bidentate ligands to platinum through two oxygen atoms whereas the thiosulphate in its complex binds as a bidentate ligand to platinum through one oxygen atom and one sulphur atom.     

Complexes of magnesium(II) and other divalent metal ions with adenosine 5′-triphosphate and 2,2′-dipyridylamine in aqueous solution

Binary and ternary systems involving adenosine 5′-triphosphate (ATP), 2,2′-dipyridylamine (DPA) and magnesium, calcium, strontium, manganese, cobalt, copper, and zinc(II) metal ions have been investigated in aqueous media by potentiometric titrations. The analysis of the titration curves shows the existence of M(ATP)²⁻, M(ATP)(H)⁻, and M(ATP)₂(H)₂⁴⁻ species for alkaline-earth metal ions, while no ternary complex can be detected. For transition metal ions both binary and ternary species are found. Binary M(ATP)₂(H)₂⁴⁻ complexes are present in solutions containing manganese and cobalt(II) metal ions but these species cannot be revealed in the case of copper and zinc(II). Ternary complexes as M(ATP)(DPA)²⁻ and M(ATP)(DPA)(H)⁻ are common to all transition metals. Binuclear and hydroxo complexes as M₂(ATP)(OH)⁻ and M(ATP)(OH)³⁻ are found only for copper and zinc(II). A hypothesis on the possible role of the species M-ATP in 1:2 ratio in the dephosphorylation mechanism is advanced on the basis of a comparison between the equilibrium data in the solution phase and the solid state structures of the magnesium, calcium, and manganese(II)- ATP-DPA systems.     

Inhibition of α-, β-, γ-, and δ-carbonic anhydrases from bacteria and diatoms with N′-aryl-N-hydroxy-ureas

The inhibition of α-, β-, γ-, and δ-class carbonic anhydrases (CAs, EC 4.2.1.1) from bacteria (Vibrio cholerae and Porphyromonas gingivalis) and diatoms (Thalassiosira weissflogii) with a panel of N’-aryl-N-hydroxy-ureas is reported. The α-/β-CAs from V. cholerae (VchCAα and VchCAβ) were effectively inhibited by some of these derivatives, with K_Is in the range of 97.5?nM – 7.26?µM and 52.5?nM – 1.81?µM, respectively, whereas the γ-class enzyme VchCAγ was less sensitive to inhibition (K_Is of 4.75 – 8.87?µM). The β-CA from the pathogenic bacterium Porphyromonas gingivalis (PgiCAβ) was not inhibited by these compounds (K_Is?>?10?µM) whereas the corresponding γ-class enzyme (PgiCAγ) was effectively inhibited (K_Is of 59.8?nM – 6.42?µM). The δ-CA from the diatom Thalassiosira weissflogii (TweCAδ) showed effective inhibition with these derivatives (K_Is of 33.3?nM – 8.74?µM). As most of these N-hydroxyureas are also ineffective as inhibitors of the human (h) widespread isoforms hCA I and II (K_Is?>?10?µM), this class of derivatives may lead to the development of CA inhibitors selective for bacterial/diatom enzymes over their human counterparts and thus to anti-infectives or agents with environmental applications.