Transglycosylation Catalyzed by Almond β-glucosidase and Cloned Pichia etchellsii β-glucosidase II using Glycosylasparagine Mimetics as Novel Acceptors

The stability of almond β-glucosidase in five different organic media was evaluated. After 1 hour of incubation at 30°C, the enzyme retained 95, 91, 81, 74 and 56% relative activity in aqueous solutions [30% (v/v)] of dioxane, DMSO, DMF, acetone and acetonitrile, respectively. Transglucosylation involving p-nitrophenyl β-D-glucopyranoside as donor and β-1-N-acetamido-D-glucopyranose, which is a glycosylasparagine mimic, as acceptor was explored under different reaction conditions using almond βglucosidase and cloned Pichia etchellsii β-glucosidase II. The yield of disaccharides obtained in both reactions turned out to be 3%. Both enzymes catalyzed the formation of (1→3)- as well as (1→6)- regioisomeric disaccharides, the former being the major product in cloned β-glucosidase II reaction while the latter predominated in the almond enzyme catalyzed reaction. Use of β-1-N-acetamido-D-mannopyranose and β-1-N-acetamido-2-acetamido-2-deoxy-D-glucopyranose as acceptors in almond β-glucosidase catalyzed reactions, however, did not afford any disaccharide products revealing the high acceptor specificity of this enzyme.     

Synthesis and anti-HIV activity of 3′-deoxy-3′-(N-hydroxyamino) analogues of nucleosides

3′-Deoxy analogues of thymidine and uridine bearing a 3′-N-alkyl-(or N-aralkyl) -N-hydroxyamino group either on the or the β face of the furanose ring have been prepared. One of these (13), exhibited a moderate anti-HIV activity.     

Piezoelectric properties of poly-β-hydroxybutyrate and copolymers of β-hydroxybutyrate and β-hydroxyvalerate

The shear piezoelectricity was observed in oriented films of poly-β-hydroxybutyrate (PHB) and copolymers of β-hydroxybutyrate (HB) and β-hydroxyvalerate (HV). The piezoelectric stress constant 3₁₄ = e′₁₄ − ie″₁₄ (polarization/strain), the piezoelectric strain constant d₁₄ = d′₁₄ − id″₁₄ (polarization/stress), the elastic constant c = c′ + ic″ and the dielectric constant = ′ − i″ were determined at a frequency of 10 Hz over a temperature range from −150° to +150°C. Piezoelectric relaxations as well as elastic and dielectric relaxations were clearly observed at the glass transition temperature of about 15°C. In order to evaluate the piezoelectric constants (e₂ and d₂) for the piezoelectric phase which consists of the crystalline region and the oriented non-crystalline region, a spherical dispersion two phase model was utilized. Assuming the appropriate fixed values for the elastic and dielectric constants in the piezoelectric phase, d₂ and d₂ were calculated as a function of temperature. For a PHB and a copolymer (17 HV/83 HB), e₂ and d₂ showed relaxations, leading to a conclusion that the instantaneous piezoelectric constant in the crystalline phase is constant independent of temperature but the piezoelectric constant in the oriented non-crystalline phase is relaxational and has the opposite sign. For a copolymer (25 HV/75 HB) and a chloroform treated copolymer (17 HV/83 HB), e₂ and d₂ were constant independent of temperature, indicating that the oriented non-crystalline phase has disappeared owing to the increased molecular flexibility due to copolymerization or annealing in chloroform vapour.     

Enzymatic alcoholysis of 3′,5′-di-O-acetyl-2′-deoxynucleosides

Candida antarctica-B (CAL-B) lipase-catalysed alcoholysis of a set of 3′,5′-di-O-acetyl-2′-deoxynucleosides (1a–e) gave the corresponding 3′-O-acetyl-2′-deoxy-nucleosides (2a–e) in yields ranging from 50 to 96%. The alcohol employed in the biotransformation affected the rate of the enzymatic reaction and the yield of the 3′-O-acetylated product, but in all cases only this regioisomer was formed. The obtained results are in agreement with the regioselectivity displayed by CAL-B lipase in previously reported biotransformations of nucleosides. CAL-B catalysed alcoholysis of 2′,3′,5′-tri-O-acetyl-cytidine and 4-N-acetyl-2′,3′,5′-tri-O-acetylcytidine was also studied, affording with the same regioselectivity the corresponding free 5′-hydroxyl nucleosides.     

Twitches in the presence of ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid

Single muscle fibers continue to twitch for up to 20 min when immersed in ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid (EGTA) solutions containing less than 10⁻⁸ M free calcium. Failure of the twitch results from reversible depolarization, which occurs after 15–20 min in EGTA. The results make it clear that external calcium or calcium in the transverse tubules play no essential part in action potential propagation or excitation-contraction coupling.     

A new method for selective N-acylation of aminoglycoside antibiotics

Per-N-formylation of aminoglycoside (aminocyclitol) antibiotics followed by mild hydrolysis with aqueous ammonia gave mono-N-deformylated derivatives. Each positional isomer of the mono-N-deformylated derivatives thus obtained was separated by column chromatography on Amberlite CG-50 (NH₄⁺ ). Acylation of mono-N-deformylated derivatives gave the corresponding mono-N-acylated derivatives. The N-formyl groups of the mono-N-acylates were removed by the treatment with dilute aqueous hydrazine acetate, whereas the newly introduced N-acyl group was stable under these conditions. The 1-N-formyl group of the deoxystreptamine moiety of per-N-formylated aminoglycoside antibiotics containing neamine (or 3′-deoxyneamine) is more readily deformylated than the 3-N-formyl group. In this report, isolation and structural-elucidation studies, including ¹³C-n.m.r. spectral assignments, of positional isomers of tri-N-formyl derivatives of xylostasin (1), 3′-deoxyxylostasin (2), kanamycin A (3), and neamine (4) are described. This selective N-acylation provides a useful method for the preparation of 1-N-modified derivatives, and the synthesis of 3′-deoxybutirosin A (2f) from 2 is described in detail as an example.     

Synthesis and antibacterial activity of novel neamine derivatives

Synthesis and activity of derivatives at the O5 or O6 positions of 1-N-((S)-4-amino-2-hydroxybutyryl)-3′,4′-dideoxyneamine, which is the neamine moiety of arbekacin, were reported. Among these results, the 5-O-aminoethylaminocarbonyl derivative showed effective activity against Staphylococcus aureus expressing a bifunctional aminoglycoside-modifying enzyme AAC(6′)-APH(2″).     

Changes in levels of mRNAs of transforming growth factor (TGF)-β1, -β2, -β3, TGF-β type II receptor and sulfated glycoprotein-2 during apoptosis of mouse uterine epithelium

To examine the roles played by transforming growth factors (TGF)-β1, -β2, -β3, and TGF-β type II receptors in the induction of apoptosis in the mouse uterine epithelium after estrogen deprivation, we investigated the expression of their mRNAs and the mRNA of sulfated glycoprotein-2 (SGP-2). Pellets containing 100 μg estradiol-17β (E₂) were implanted into ovariectomized mice and removed four days later. Apoptotic indices (percentage of apoptotic cells) of both luminal and glandular epithelia increased after E₂ pellets were removed, but administration of progesterone (P), 5-dihydrotestosterone (DHT), or continued implantation of E₂ pellets suppressed this increase. Levels of mRNAs of TGF-β1, -β2, and -β3, and SGP-2 did not increase after estrogen deprivation. However, estrogen deprivation caused a gradual increase in the level of TGF-β type II receptor mRNA, and its level increased about six-fold six days later. Moreover, E₂, P, and DHT markedly decreased the level of TGF-β type II receptor mRNA. In situ hybridization demonstrated that mRNAs of TGF-β1, -β2, -β3 and TGF-β type II receptor were localized to the epithelium. Exogenous administration of TGF-β1 into the uterine stroma induced apoptosis in the epithelium, a finding that suggests that signals produced by TGF-βs can induce apoptosis. Therefore, the present results suggest that increased sensitivity of uterine epithelial cells to TGF-βs, as demonstrated by an increase in TGF-β type II receptor mRNA, is involved in the induction of apoptosis after estrogen deprivation, although signals produced by TGF-βs do not appear sufficient to induce apoptosis.     

Phospholipid-nucleoside conjugates The aggregational characteristics and morphological aspects of selected 1-β-D-arabinofuranosylcytosine 5′-diphosphate-L-1,2-diacylglycerols

1-β-D-Arabinofuranosylcytosine 5′-diphosphate-1,2-diacylglycerols have previously been shown to be promising candidates as prodrugs of the clinically useful antileukemic agent 1-β-D-arabinofuranosylcytosine. Because of the amphipathic nature of these liponucleotides and the potential that their morphological state may mediate their biological activity, it was necessary to undertake detailed studies of their aggregational and morphological characteristics. When samples of 1-β-D-arabinofuranosylcytosine 5′-diphosphate-L-1,2-diacylglycerols containing either dimyristoyl, dipalmitoyl or distearoyl fatty acid side chains) were prepared in buffered saline solutions using sonication methods, the morphological nature of the resulting aggregate was shown to be related to temperature and the length of the side chain. When sonicated at low temperatures all the above-mentioned derivatives gave turbid solutions containing large bilayer sheets. As the temperature was raised, a transition temperature was reached at which a stable three-dimensional cross-linked network of small, interlocking bilayer stacks was formed. This turbidity transition temperature was directly related to the chain length of the fatty acid side chain. Sonication at temperatures close to this turbidity transition temperature produced small disc-shaped micellar structures. These micelles were shown to exist in another aggregational equilibrium consisting of a stacking-destacking process, the position within this equilibrium being dependent upon the concentration. In contrast, a sample of 1-β-d-arabinofuranosylcytosine 5′-diphosphate-l-1,2-dioleoylgycerol (which contains an unsaturated carbon-carbon bond in each of the fatty acid side chains) was shown to give a multilamellar liposome structure when sonicated in buffered saline at temperatures above its turbidity transition temperature.     

Laccase-induced cross-coupling of 4-aminobenzoic acid with para-dihydroxylated compounds 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide and 2,5-dihydroxybenzoic acid methyl ester

A fungal laccase from Trametes villosa (EC 1.10.3.2 p-phenoloxidase) was used to mediate the oxidation and cross-coupling of two para-dihydroxylated benzoic acid derivatives with 4-aminobenzoic acid. The incubation of 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide and 4-aminobenzoic acid with laccase under oxygen conditions resulted in the formation of 2-(4′-carboxy-anilino)-N-(2″-hydroxyethyl)-3,6-dioxo-1,4-cyclohexadien-1-carboxamide as the main product (yield > 85%). When 2,5-dihydroxybenzoic acid methyl ester was a co-substrate of 4-aminobenzoic acid, 2-(4′-carboxy-anilino)-N-(2″-hydroxyethyl)-3,6-dioxo-1,4-cyclohexadien-1-carboxy methyl ester was produced (yield > 75%). Both products were N–C coupling dimers consisting of para-quinone and benzoic acid moieties. The formation of quinone structures in the presence of T. villosa laccase may be useful in pharmaceutical synthesis. Because of high product yields and low amount of by-products laccase of T. villosa seems to be a suitable enzyme among laccases acting at pH 5 for the synthesis of heterologous dimers.     

Δ-β-Sitosten-3-one from β-sitosterol by leaf homogenates

β-Sitosterol-4-¹⁴C is metabolized to Δ⁴-β-sitosten-3-one by Cheiranthus cheiri leaf homogenates. Greater than 60% conversion occurs within 2 hr. Under identical conditions, leaf homogenates of Strophanthus kombé fail to metabolize β-sitosterol, while Digitalis purpurea leaf homogenates yield only very small amounts of the metabolite.     

A new method for the 3′-deoxygenation of butirosins A and B

An aziridine ring-formation involving the reaction of adjacent amino and alcohol groups with triphenylphosphine, carbon tetrachloride, and triethylamine was applied at the 2′ and 3′ positions of butirosin A (1a) and B (1b). The amino groups at the 2′ position of 1a and 1b were p-methoxybenzylated to increase the nucleophilicity of the nitrogen atom and to avoid the formation of a P-N linkage, and the N-p-methoxybenzyl derivatives were converted into the aziridine derivatives, which were then subjected to hydrogenolysis and removal of the protecting groups to give 3′-deoxybutirosin A (7a) and B (7b), respectively. This new method is compared with the conventional N, O-protecting method that involves several complex steps.     

Design, synthesis and preliminary evaluation of novel 3′-Substituted carboxycyclopropylglycines as antagonists at group 2 metabotropic glutamate receptors

Two novel 3′-substituted carboxycylopropylglycines, (2S,1′S,2′S,3′R)-2-(3′-xanthenylmethyl-2′-carboxycyclopropyl)glycine (8a) and (2S,1′S,2′S,3′R)-2-(3′-xanthenylethyl-2′-carboxycyclopropyl)glycine (8b), were synthesized and evaluated as mGluR ligands. Compound 8b showed to be a potent group II antagonist with submicromolar activity.     

Olefin versus sulfur coordination of benzo[b]thiophene (BT) in Cp(CO)2Re(η:η-μ2-BT)Cr(CO)3

Reactions of Cr(CO)₃(η⁶-BT), in which the Cr is π-coordinated to the benzene ring of benzo[b]thiophene (BT), with Cp′(CO)₂Re(THF), where Cp′ = η⁵-C₅H₅ or η⁵-C₅Me₅, give the products Cp′(CO)₂Re(η²:η⁶-μ₂-BT)Cr(CO)₃ in which the Cr remains coordinated to the benzene ring and Re is bound to the C(2)=C(3) double bond. An X-ray diffraction study of Cp(CO)₂Re(η²:η⁶-μ₂-BT)Cr(CO)₃ (3) provides details of the geometry. This structure contrasts with that of the Cp′(CO)₂Re(BT) complexes that exist as mixtures of isomers in which the BT is coordinated to the Re through either the double bond (2,3-η²) or the sulfur (η¹(S)). Thus, the electron-withdrawing Cr(CO)₃ group in 3 stabilizes the 2,3-η² mode of BT coordination to the Cp′(CO)₂Re fragment. Implications of these results for catalytic hydrodesulfurization of BT are discussed. Crystal data for 3: triclinic, space group

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Structure-activity relationship of truncated analogs of caprazamycins as potential anti-tuberculosis agents

Systematic structure–activity relationship studies of caprazamycin (CPZ) analogs, including the aminoribose-truncated 5 and the uridine-truncated 6, have been carried out. Both 5 and 6 were synthesized efficiently via diazepanone ring construction by intramolecular reductive alkylation of aminoaldehyde derivatives. The antibacterial activity of a range of analogs, including 5 and 6, against Mycobacteriumosis was evaluated, and it was found that the uridine, the aminoribose, and the fatty acyl side chains are crucial for antibacterial activity. This study would be a guide for designing novel anti-tuberculosis agents based on the 6′-N-alkyl-5′-β-O-aminoribosyl-glycyluridine class of antibiotics including the CPZs.     

Temporal Changes of Acid Phosphatase, Arylsulphatase, β-Galactosidase and β-N-ACETYL-d-Glucosaminidase Activities in Subcellular Fractions of Rat Liver

In this paper circadian changes in the liver enzyme activities of rat housed under highly standardized conditions with 12:12 hour light-dark cycle are shown. Activities of acid phosphatase, arylsulphatase, β-galactosidase and β-N-acetyl-d-glucosaminidase in microsomal and lysosomal fractions and crude homogenate were estimated every 4 hr during one 24-hr period. The enzyme activities were related to 1 mg of protein, 1 mg of DNA and 1 g fresh tissue. Daily changes of enzyme activities were found. In case of activity calculated per 1 mg DNA two maxima at 0500 and at 2100 hr were observed, while activity calculated per 1 mg protein showed one maximum at 0500 hr. Activity calculated per 1 g fresh tissue showed the maximum at 0500 hr for each enzyme only in microsomal fraction. As far as acrophase table is concerned for all enzymes and fractions the acrophase occurred during the night. The obtained results are discussed in relation to lysosomal enzymes synthesis process as well as different reference values.     

Prooxidant activity of β-carotene under 100% oxygen pressure in rat liver microsomes

The effects of the partial pressure of oxygen (pO₂ on antioxidant efficiency of β-carotene in inhibiting 2,2′-azobis(2-amidinopropane) (AAPH)-induced lipid peroxidation are investigated in rat liver microsomal membranes. The rate of peroxyl radicals generated by thermolysis of AAPH at 37°C is markedly higher at 150 than 760 mm Hg pO₂. At 150 mm Hg pO₂ β-carotene acts as an antioxidant, inhibiting 2,2′-azobis(2-amidinopropane) (AAPH)-induced Malondialdehyde (MDA) formation. At 760 mm Hg pO₂, it loses its antioxidant activity and shows a prooxidant effect, increasing lipid peroxidation products, -Tocopherol prevents the prooxidant effect of β-carotene in a dose-dependent manner. Our data provide the first evidence of a prooxidant effect of β-carotene under 100% oxygen pressure in a biological membrane model and point out the existence of cooperative interactions between β-carotene and -tocopherol.     

Reactions of tungsten phosphonium carbyne complexes with aryloxide nucleophiles to form aryloxycarbyne and η-ketenyl complexes

Tungsten phosphoranylideneketene complexes of the type Tp′(CO)(p-OC₆H₄R)W(η²-(C,C)---O=CC---PR′₂Ph) (R=NO₂, R′=Me (6a); R=NO₂, R′=Ph (6b); R=CN, R′=Me (7a); R=CN, R′=Ph (7b); R=Cl, R′=Ph (8b)) have been synthesized from phosphonium carbyne precursors in a reaction that reflects coupling of carbonyl and carbyne ligands. In addition to these products, aryloxycarbyne complexes Tp′(CO)₂WCO(p-C₆H₄NO₂) (9a), Tp′(CO)₂WCO(p-C₆H₄CN) (9b), and Tp′(CO)₂WCO(p-C₆H₄Cl) (9c)) have been prepared via substitution of the phosphonium carbyne phosphine with an aryloxide nucleophile. The product ratio of substitution at the carbyne carbon to carbonyl–carbyne coupling can be tuned by variation of the aryloxide para-substituent. Aryloxy carbyne complexes are the favored products with stronger nucleophiles, while weaker nucleophiles result in a mixture of aryloxy carbyne complexes and η²-ketenyl coupled complexes. Formation of η²-ketenyl complexes is favored for the least nucleophilic aryloxides. Ketenyl complexes 6a and 6b were methylated at the ketenyl oxygen to form cationic alkyne complexes [Tp′(CO)(p-OC₆H₄NO₂)W(η²-(C,C)---CH₃OCCPR₂Ph)][OTf] (R=Me (10a), R=Ph (10b)). The structures of η²-ketenyl complexes 6a and 7b and the structure of cationic alkyne complex 10a were determined by X-ray crystallography.     

C2-Symmetrical tetrahydroxyazepanes as inhibitors of glycosidases and HIV/FIV proteases

C₂-Symmetrical tetrahydroxyazepanes were synthesized as inhibitors for glycosidases. Tetrahydroxyazepane 1 is a non-specific inhibitor of various glycosidases, while compounds 2, 3 and 4 specifically inhibit β-N-acetylglucosaminidase, β-glucosidase, and a-fucosidase, respectively, with K_i in the micromolar range. Compound 1 is not an inhibitor of HIV/FIV proteases, but its 3,6-difluorobenzyl derivatives are moderate inhibitors of both enzymes.     

Alkene rotation in [Ru(η-C5H5) (L2) (η-alkene][CF3SO3] with L2 = iPr- or pTol-diazabutadiene. X-ray crystal structure of [Ru(η-C5H5(pTol-DAB) (η-ethene][CF3SO3]

Reaction of RuCl(η⁵-C₅H₅(pTol-DAB) with AgOTf (OTf = CF₃SO₃) in CH₂Cl₂ or THF and subsequent addition of L′ (L′ = ethene (a), dimethyl fumarate (b), fumaronitrile (c) or CO (d) led to the ionic complexes [Ru(η⁵-C₅H₅)(pTol-DAB)(L′)][OTf] 2a, 2b and 2d and [Ru(η⁵-C₅H₅)(pTol-DAB)(fumarontrile-N)][OTf] 5c. With the use of resonance Raman spectroscopy, the intense absorption bands of the complexes have been assigned to MLCT transitions to the iPr-DAB ligand. The X-ray structure determination of [Ru(η⁵-C₅H₅)(pTol-DAB)(η²-ethene)][CF₃SO₃] (2a) has been carried out. Crystal data for 2a: monoclinic, space group P2₁/n with A = 10.840(1), b = 16.639(1), C = 14.463(2) Å, β = 109.6(1)°, V = 2465.6(5) ų, Z = 4. Complex 2a has a piano stool structure, with the Cp ring η⁵-bonded, the pTol-DAB ligand σN, σN′ bonded (Ru-N distances 2.052(4) and 2.055(4) Å), and the ethene η²-bonded to the ruthenium center (Ru-C distances 2.217(9) and 2.206(8) Å). The C = C bond of the ethene is almost coplanar with the plane of the Cp ring, and the angle between the plane of the Cp ring and the double of the ethene is 1.8(0.2)°. The reaction of [RuCl(η⁵-C₅H₅)(PPh)₃ with AgOTf and ligands L′ = a and d led to [Ru(η⁵-C₅H₅)(PPh₃)₂(L′)]OTf] (3a) and (3d), respectively. By variable temperature NMR spectroscopy the rottional barrier of ethene (a), dimethyl fumarate (b and fumaronitrile (c) in complexes [Ru(η⁵-C₅H₅)(L₂)(η²-alkene][OTf] with L₂ = iPr-DAB (a, 1b, 1c), pTol-DAB (2a, 2b) and L = PPh₃ (3a) was determined. For 1a, 1b and 2b the barrier is 41.5±0.5, 62±1 and 59±1 kJ mol⁻¹, respectively. The intermediate exchange could not be reached for 1c, and the ΔG^# was estimated to be at least 61 kJ mol⁻. For 2a and 3a the slow exchange could not be reached. The rotational barrier for 2a was estimated to be 40 kJ mol⁻. The rotational barier for methyl propiolate (HC≡CC(O)OCH₃) (k) in complex [Ru(η⁵-C₅H₅)(iPr-DAB) η²-HC≡CC(O)OCH₃)][OTf] (1k) is 45.3±0.2 kJ mol⁻¹. The collected data show that the barrier of rotational of the alkene in complexes 1a, 2a, 1b, 2b and 1c does not correlate with the strength of the metal-alkene interaction in the ground state.