Synthesis and evaluation of N-alkyl-beta-D-glucosylamines on the growth of two wood fungi, Coriolus versicolor and Poria placenta

Various glucosylamines were synthesized from glucose and different alkyl amine compounds. These amino compounds are beta-d-glucopyranosylamine (GPA), N-ethyl-beta-d-glucopyranosylamine (EtGPA), N-butyl-beta-d-glucopyranosylamine (BuGPA), N-hexyl-beta-d-glucopyranosylamine (HeGPA), N-octyl-beta-d-glucopyranosylamine (OcGPA), N-dodecyl-beta-d-glucopyranosylamine (DoGPA), N-(2-hydroxyethyl)-beta-d-glucopyranosylamine (HEtGPA) and N,N-di(2-hydroxyethyl)-beta-d-glucopyranosylamine (DHEtGPA). They were tested for their antifungal activity against the growth of Coriolus versicolor and Poria placenta. An improvement of the antifungal activity with the increase of alkyl chain length was observed. DoGPA exhibited the best antifungal activity against both strains. It completely inhibited the fungal growth at 0.01x10(-3)molmL(-1) and 0.0075x10(-3)molmL(-1) for C. versicolor and P. placenta, respectively. For other glucosylamines higher concentrations were needed for complete inhibition of fungi.     

Metabolism of hexadecyltrimethylammonium chloride in Pseudomonas strain B1.

A bacterium (strain B1) utilizing hexadecyltrimethylammonium chloride as a carbon and energy source was isolated from activated sludge and tentatively identified as a Pseudomonas sp. This bacterium only grew on alkyltrimethylammonium salts (C12 to C22) and possible intermediates of hexadecyltrimethylammonium chloride breakdown such as hexadecanoate and acetate. Pseudomonas strain B1 did not grow on amines. Simultaneous adaptation studies suggested that the bacterium oxidized only the alkyl chain of hexadecyltrimethylammonium chloride. This was confirmed by the stoichiometric formation of trimethylamine from hexadecyltrimethylammonium chloride. The initial hexadecyltrimethylammonium chloride oxygenase activity, measured by its ability to form trimethylamine, was NAD(P)H and O2 dependent. Finally, assays of aldehyde dehydrogenase, hexadecanoyl-coenzyme A dehydrogenase, and isocitrate lyase in cell extracts revealed the potential of Pseudomonas strain B1 to metabolize the alkyl chain via beta-oxidation.     

Metabolism of hexadecyltrimethylammonium chloride in Pseudomonas strain B1.

A bacterium (strain B1) utilizing hexadecyltrimethylammonium chloride as a carbon and energy source was isolated from activated sludge and tentatively identified as a Pseudomonas sp. This bacterium only grew on alkyltrimethylammonium salts (C12 to C22) and possible intermediates of hexadecyltrimethylammonium chloride breakdown such as hexadecanoate and acetate. Pseudomonas strain B1 did not grow on amines. Simultaneous adaptation studies suggested that the bacterium oxidized only the alkyl chain of hexadecyltrimethylammonium chloride. This was confirmed by the stoichiometric formation of trimethylamine from hexadecyltrimethylammonium chloride. The initial hexadecyltrimethylammonium chloride oxygenase activity, measured by its ability to form trimethylamine, was NAD(P)H and O2 dependent. Finally, assays of aldehyde dehydrogenase, hexadecanoyl-coenzyme A dehydrogenase, and isocitrate lyase in cell extracts revealed the potential of Pseudomonas strain B1 to metabolize the alkyl chain via beta-oxidation.     

Block of the Kir2.1 Channel Pore by Alkylamine Analogues of Endogenous Polyamines

Inward rectification induced by mono- and diaminoalkane application to inside-out membrane patches was studied in Kir2.1 (IRK1) channels expressed in Xenopus oocytes. Both monoamines and diamines block Kir2.1 channels, with potency increasing as the alkyl chain length increases (from 2 to 12 methylene groups), indicating a strong hydrophobic interaction with the blocking site. For diamines, but not monoamines, increasing the alkyl chain also increases the steepness of the voltage dependence, at any concentration, from a limiting minimal value of ∼1.5 (n = 2 methylene groups) to ∼4 (n = 10 methylene groups). These observations lead us to hypothesize that monoamines and diamines block inward rectifier K⁺ channels by entering deeply into a long, narrow pore, displacing K⁺ ions to the outside of the membrane, with this displacement of K⁺ ions contributing to “extra” charge movement. All monoamines are proposed to lie with the “head” amine at a fixed position in the pore, determined by electrostatic interaction, so that zδ is independent of monoamine alkyl chain length. The head amine of diamines is proposed to lie progressively further into the pore as alkyl chain length increases, thus displacing more K⁺ ions to the outside, resulting in charge movement (zδ) increasing with the increase in alkyl chain length.     

Isolation and characterization of 3-N-trimethylamino-1-propanol-degrading Rhodococcus sp. strain A2

The aerobic degradation of 3- N -trimethylamino-1-propanol (homocholine) as a sole source of carbon and nitrogen has been found for a Rhodococcus sp. bacterium isolated from soil. The isolate was identified as Rhodococcus sp. strain A2 based on its phenotypic features, physiological and biochemical characteristics, and results of phylogenetic analysis. The washed cells of strain A2 completely degraded homocholine within 6 h, with concomitant formation of several metabolites. Analysis of the metabolites using capillary electrophoresis, fast atom bombardment–MS, and GC–MS showed that trimethylamine was the major metabolite, in addition to β-alanine betaine (β-AB) and trimethylaminopropionaldehyde. Therefore, the possible degradation pathway of homocholine in the isolated strain is through consequent oxidation of the alcohol group (-OH) to aldehyde (-CHO) and acid (-COOH). Thereafter, the cleavage of β-AB C–N bonds yielded trimethylamine and alkyl chain.     

Synthesis and characterization of a cationic poly(beta-hydroxyalkanoate)

Poly(beta-hydroxyalkanoates) (PHAs) are biodegradable polyesters produced by a wide range of bacteria. The structures of these polymers may be tuned by controlling the carbon source composition in the feed stock, but the range of functional groups accessible in this manner is limited to those that the organism is able to metabolize. Much effort has been made to chemically modify the side chains of these polymers to achieve new materials. Here, we report the synthesis of the first cationic PHA, poly(beta-hydroxy-octanoate)- co-(beta-hydroxy-11-(bis(2-hydroxyethyl)-amino)-10-hydroxyundecanoate) (PHON). Pseudomonas putida Gpo1 was used to produce poly(beta-hydroxy-octanoate)- co-(beta-hydroxy-10-undecenoate) (PHOU), whose vinyl-terminated side chains were first converted to terminal epoxides and then modified with diethanolamine. The modification of PHOU was examined using (1)H, COSY, and HSQC NMR and GPC and resulted in a loss of molecular weight due to aminolysis and also in the introduction of side chains terminated with tertiary amine groups, which are protonated at physiological pH. The polycationic PHA is soluble in polar solvents such as DMSO, DMF, and water. The new biodegradable cationic polymers are envisioned as nucleic acid delivery systems.     

Structural optimization and evaluation of butenolides as potent antifouling agents: modification of the side chain affects the biological activities of compounds

A recent global ban on the use of organotin compounds as antifouling agents has increased the need for safe and effective antifouling compounds. In this study, a series of new butenolide derivatives with various amine side chains was synthesized and evaluated for their anti-larval settlement activities in the barnacle, Balanus amphitrite. Side chain modification of butenolide resulted in butenolides 3c-3d, which possessed desirable physico-chemical properties and demonstrated highly effective non-toxic anti-larval settlement efficacy. A structure-activity relationship analysis revealed that varying the alkyl side chain had a notable effect on anti-larval settlement activity and that seven to eight carbon alkyl side chains with a tert-butyloxycarbonyl (Boc) substituent on an amine terminal were optimal in terms of bioactivity. Analysis of the physico-chemical profile of butenolide analogues indicated that lipophilicity is a very important physico-chemical parameter contributing to bioactivity.     

Substrate specificity of the bovine serum amine oxidase and in situ characterisation of aminoaldehydes by NMR spectroscopy

The oxidation of spermidine or homospermidine with bovine serum amine oxidase (BSAO) was monitored in situ, using proton nuclear magnetic resonance spectroscopy in water with 10% D(2)O. NMR assignments were performed by spin decoupling and COSY spectra or by comparison with data from synthetic aminoaldehydes. The results represent the first in situ characterisation of the highly reactive aminoaldehydes and showed oxidation at the N(1) amino group of spermidine and homospermidine. Comparison of homospermidine with a variety of substrates revealed that among straight chain di- and polyamines both an aminopropyl group and two primary amino groups separated by seven (norspermidine) or eight (spermidine) carbon atoms were required for optimal substrate ability. However, highest activity was seen with the substrate N-(4-aminobutyl)hexahydropyrimidine, showing that the substrate channel of BSAO has a dual substrate preference, with moderately bulky substituents at the distal end of a diamine contributing equally well as an alkyl amino group. Cytotoxic investigations of a variety of substrates for BSAO, confirmed previous results, that cytotoxicity is primarily linked to polyamines encompassing the aminopropyl moiety. No acrolein was observed at any time during the oxidation showing that it reacts very fast with available amino groups forming a variety of derivatives.     

Rational design of novel CYP2A6 inhibitors

Inhibition of CYP2A6-mediated nicotine metabolism can reduce cigarette smoking. We sought potent and selective CYP2A6 inhibitors to be used as leads for drugs useful in smoking reduction therapy, by evaluating CYP2A6 inhibitory effect of novel formyl, alkyl amine or carbonitrile substituted aromatic core structures. The most potent CYP2A6 inhibitors were thienopyridine-2-carbaldehyde, benzothienophene-3-ylmethanamine, benzofuran-5-carbaldehyde and indole-5-carbaldehyde, with IC₅₀ values below 0.5 μM for coumarin 7-hydroxylation. Nicotine oxidation was effectively inhibited in vitro by two alkyl amine compounds and benzofuran-5-carbonitrile. Some of these molecules could serve as potential lead molecules when designing CYP2A6 inhibitory drugs for smoking reduction therapy.     

Structural optimization and evaluation of butenolides as potent antifouling agents: modification of the side chain affects the biological activities of compounds

A recent global ban on the use of organotin compounds as antifouling agents has increased the need for safe and effective antifouling compounds. In this study, a series of new butenolide derivatives with various amine side chains was synthesized and evaluated for their anti-larval settlement activities in the barnacle, Balanus amphitrite. Side chain modification of butenolide resulted in butenolides 3c-3d, which possessed desirable physico-chemical properties and demonstrated highly effective non-toxic anti-larval settlement efficacy. A structure-activity relationship analysis revealed that varying the alkyl side chain had a notable effect on anti-larval settlement activity and that seven to eight carbon alkyl side chains with a tert-butyloxycarbonyl (Boc) substituent on an amine terminal were optimal in terms of bioactivity. Analysis of the physico-chemical profile of butenolide analogues indicated that lipophilicity is a very important physico-chemical parameter contributing to bioactivity.     

Biological activity of new N-oxides of tertiary amines

Potential biological properties of newly synthesized single and double alkyl chain N-oxides of tertiary amines (NTA) were studied. Individual compounds in each of the series had alkyl chains of different length. Various experiments were performed to determine a mechanism of the interaction between NTA and model and biological membranes. These were measurements of hemolytic efficiencies of NTA (pig erythrocytes), their influence on the transition temperatures (DPPC liposomes), on potassium leakage from cucumber, its growth and chlorophyll content (Cucumis sativus cv. Krak F1), and on the resting membrane potential in alga cells (Nitellopsis obtusa). Also, prevention of erythrocyte membrane lipid oxidation induced by UV irradiation was studied. Potential antioxidative properties of NTA were additionally tested in radical chromogen (ABTS) experiments in which antioxidative efficiencies of NTA were compared to that of the standard antioxidant Trolox. It was found that NTA readily interacted with erythrocyte membranes. Their hemolyzing efficiency increased with the alkyl chain length. Slightly more intensive interaction was found for double alkyl chain compounds. Similar results were obtained in DSC experiments, where incorporation of NTA into liposomal membranes shifted the main transition temperatures and caused a broadening of the main transition peaks depending on the alkyl chain length. Double alkyl chain compounds were also found more efficiently influencing the growth of cucumber. Influence of NTA on the resting membrane potential of algae cells was not quite following the alkyl chain length rule found in erythrocyte and liposome experiments. Also potassium leakage and chlorophyll content determined in physiological experiments were not following the increase of lipophilicity of compounds. Most efficiently influencing those parameters were NTA having shorter alkyl chains, and efficiencies of single alkyl chain compounds were evidently stronger. Both methods used to test the antioxidative properties of NTA showed that they depended on the alkyl chain lengths of compounds within each series, but double alkyl chain ones exhibited markedly greater efficiency.     

Purification and properties of the P2 primary alkylsulphohydrolase of the detergent-degrading bacterium pseudomonas C12B.

The P2 primary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (200-250-fold) by column chromatography on DEAE-cellulose, Sephadex G-100 and butyl-agarose. The intact protein is a dimer with a mol. wt. of 160 000. Activity towards primary alkyl sulphate esters was maximal at pH 8.3, varied little in the range pH 7.8-8.7, but decreased sharply at higher pH. For a homologous series of primary alkyl sulphate substrates (C6-C12), logKm decreased linearly with increasing chain length, corresponding to a contribution to the free energy of association between enzyme and substrate of -2.5kJ/mol for each additional CH2 group in the alkyl chain. logKi for the competitive inhibition by secondary alkyl 2-sulphate esters followed a similar pattern (-2.4kJ/mol for each additional CH2 group) except that only n-1 carbon atoms effectively participate in hydrophobic bonding, implying that the C-1 methyl group is not involved. logKi values for inhibition primary alkanesulphonates also depended linearly on chain length but with a diminished gradient, indicating a free-energy increment of -1.2kJ/mol per additional CH2 group. The collective results showed the presence of a hydrophobic site on the enzyme capable of accomodating an alkyl chain of considerable length. Cationic structures (in the form of arginine, lysine or histidine), whose presence might be expected for binding the anionic sulphate group, were not detectable at the active site.     

Multiple modes of catalysis-dependent inhibition and inactivation of aortic lysyl oxidase

Lysyl oxidase purified from bovine aorta can oxidize simple alkyl mono- and diamine substrates yielding the respective aldehyde, H2O2, and ammonia as products. The oxidation of such substrates is limited to approximately 100 catalytic turnovers per enzyme molecule since lysyl oxidase is syncatalytically and irreversibly inactivated in the course of oxidation of these amines. The present study reveals that addition of oxidant scavengers protects significantly against inactivation of lysyl oxidase and that the ammonia product is a reversible competitive inhibitor of amine oxidation. Further, the enzyme becomes covalently labeled by the amine substrate or its enzyme-processed derivative during catalysis. Thus, lysyl oxidase appears subject to multiple modes of catalysis-dependent inhibition or inactivation. Syncatalytic inactivation of lysyl oxidase might represent a means of restricting the activity of this enzyme toward its elastin and collagen substrates in vivo.     

Preparation of sulfonic-functionalized graphene oxide as ion-exchange material and its application into electrochemiluminescence analysis

Graphene oxide (GO) obtained from chemical oxidation of flake graphite was derivatized with sulfonic groups to form sulfonic-functionalized GO (GO-SO(3)(-)) through four sulfonation routes: through amide formation between the carboxylic group of GO and amine of sulfanilic acid (AA-GO-SO(3)(-)), aryl diazonium reaction of sulfanilic acid (AD-GO-SO(3)(-)), amide formation between the carboxylic group of GO and amine of cysteamine and oxidation by H(2)O(2) (CA-GO-SO(3)(-)), and alkyl diazonium reaction of cysteamine and oxidation by H(2)O(2) (CD-GO-SO(3)(-)). Results of Fourier transform infrared spectroscopy and X-ray photoelectrospectrocopy showed that -SO(3)(-) groups were attached onto GO. Thermo gravimetric analysis showed that derivatization with sulfonic groups improved thermo stability of GO. X-ray diffraction results indicated that GO-SO(3)(-) had more ordered π-π stacking structure than the original GO. GO-SO(3)(-) and cationic polyelectrote, poly (diallyldimethylammoniumchloride) (PDDA) were adsorbed at indium tin oxide (ITO) glass surface through layer-by-layer assembling to form (GO-SO(3)(-)/PDDA)(n)/ITO multilayers. After tris-(2,2'-bipyridyl) ruthenium (II) dichloride (Ru(bpy)(3)(2+)) was incorporated into the multilayers, the obtained Ru(bpy)(3)(2+)/(GO-SO(3)(-)/PDDA)(n)/ITO electrodes can be used as electrochemiluminescence sensors for detection of organic amine with high sensitivity (limit of detection of 1 nM) and stability.     

Specificity of guinea pig liver transglutaminase for amine substrates.

The amine specificity of guinea pig liver transglutaminase, a model enzyme for endo-gamma-glutamine:epsilon-lysin transferases, was explored with the aid of synthetic substrates of high apparent affinities. As exemplified by dansyl- (5-dimethylamino-1-naphthalenesulfonyl), (2,4-dinitrobenzenesulfonyl)-, and (2,4,6-triisopropylbenzenesulfonyl)-cadaverines--each of which showed affinities of approximately 4 x 10(7) M-1--the best amine substrates carried a large hydrophobic substituent attached to an alkylamine side chain of about 7.2 A in length. Altogether, our results point to the importance of a hydrophobic binding region in the enzyme from where the alkyl side chain reaches into a narrow crevice toward the active center and positions the primary amine of the substrate for attacking the carbonyl group of the acyl enzyme intermediate.     

Superoxide generated by glutathione reductase initiates a vanadate-dependent free radical chain oxidation of NADH.

Vanadate V(V) markedly stimulated the oxidation of NADPH by GSSG reductase and this oxidation was accompanied by the consumption of O2 and the accumulation of H2O2. Superoxide dismutases completely eliminated this effect of V(V), whereas catalase was without effect, as was exogenous H2O2 added to 0.1 mM. These effects could be seen equally well in phosphate- or in 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid-buffered solutions. Under anaerobic conditions there was no V(V)-stimulated oxidation of NADPH. Approximately 4% of the electrons flowing from NADPH to O2, through GSSG reductase, resulted in release of O2-. The average length of the free radical chains causing the oxidation of NADPH, initiated by O2- plus V(V), was calculated to be in the range 140-200 NADPH oxidized per O2- introduced. We conclude that GSSG reductase, and by extension other O2(-)-producing flavoprotein dehydrogenases such as lipoyl dehydrogenase and ferredoxin reductase, catalyze V(V)-stimulated oxidation of NAD(P)H because they release O2- and because O2- plus V(V) initiate a free radical chain oxidation of NAD(P)H. There is no reason to suppose that these enzymes can act as NAD(P)H:V(V) oxidoreductases.     

Antioxidant properties of gingerol related compounds from ginger

Ginger (Zingiber officinale Roscoe) shows an antioxidant activity, and we have been engaging to determine the structures of more than 50 antioxidants isolated from the rhizomes of ginger. The isolated antioxidants are divided into two groups; gingerol related compounds and diarylheptanoids. In this study, structure-activity relationship of gingerol related compounds was evaluated. Gingerol related compounds substituted with an alkyl group bearing 10-, 12- or 14-carbon chain length were isolated from the dichloromethane extract of rhizomes using repeated chromatographic techniques. The antioxidant activities of these compounds were evaluated by the following measurements; 1) 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity, 2) inhibitory effect on oxidation of methyl linoleate under aeration and heating by the Oil Stability Index (OSI) method, and 3) inhibitory effect on oxidation of liposome induced by 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH). These results suggested that the substituents on the alkyl chain might contribute to both radical scavenging effect and inhibitory effect of autoxidation of oils, while inhibitory effects against the AAPH-induced peroxidation of liposome was somewhat influenced by the alkyl chain length; the antioxidant activity might be due to not only radical scavenging activity of antioxidants but also their affinity of the antioxidants to the substrates.     

Application of 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole in analysis: Fluorescent dyes and unexpected reaction with tertiary amines

4-Chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is widely applied as a fluorescent tagging reagent in biochemistry, as a derivatization agent in analytical chemistry, and as a component for design of fluorescent nanoparticles. Four new 7-nitrobenzo-2-oxa-1,3-diazole (NBD)-tagged polyamines containing two to four amine moieties were synthesized and used as an effective tool for staining of siliceous frustules of the diatom algae and spicules of the siliceous sponges, including fossilized samples. An unexpected reaction between NBD-Cl and tertiary amine groups was found, giving rise to NBD-tagged amines with elimination of an alkyl group. The reaction proceeds through the Meisenheimer complex and quaternary salt, which transform to the product by Hofmann reaction (alkene elimination) or nucleophilic substitution (halogenated compound formation). In the case of polyamines, NBD-Cl causes chain scissoring, giving a set of NBD-tagged amines. The found NBD-Cl reaction with tertiary amines must be taken into account when using NBD-Cl and similar activated aromatic systems for amine derivatization in analytical and biochemistry applications. The reaction with polyamines opens the way to libraries of NBD-tagged compounds.     

Anti-Salmonella activity of (2E)-alkenals

AIMS: This study investigated the effect of a series of naturally occurring aliphatic (2E)-alkenals against Salmonella choleraesuis subsp. choleraesuis ATCC 35640 and evaluated their antibacterial action. METHOD AND RESULTS: A homologous series of aliphatic (2E)-alkenals from C5-C13 were tested for their antibacterial activity against Salm. Choleraesuis. The antibacterial action of (2E)-alkenals against Salm. choleraesuis increases with increasing carbon chain length. (2E)-Dodecenal (C12) was the most effective against this food-borne bacterium with minimum bactericidal concentration (MBC) of 6.25 microg ml-1 (34 micromol l-1), followed by (2E)-undecenal (C11) with an MBC of 12.5 microg ml-1 (77 micromol l-1). The activity was found to correlate with the hydrophobic alkyl chain length from the hydrophilic aldehyde group. The time-kill curve study showed that (2E)-dodecenal was bactericidal against Salm. choleraesuis at any growth stage. CONCLUSIONS: The antibacterial activity of (2E)-alkenals against Salm. choleraesuis was found to correlate with the hydrophobic alkyl chain length. The conjugated double bond is not essential in eliciting the activity but is associated with increasing it. SIGNIFICANCE AND IMPACT OF THE STUDY: Because of their easy availability and wide distribution in many edible plants, (2E)-alkenals can be used as anti-Salmonella agents.     

Dibasic non-imidazole histamine H3 receptor antagonists with a rigid biphenyl scaffold

A class of rigid, dibasic, non-imidazole H3 antagonists was developed, starting from a series of previously described flexible compounds. The original polymethylene chain between two tertiary amine groups was replaced by a rigid scaffold, composed by a phenyl ring or a biphenyl fragment. Modulation of the distance between the two amine groups, and of their alkyl substituents, was driven by superposition of molecular models and docking into a receptor model, resulting in the identification of 1,1'-[biphenyl-4,4'-diylbis(methylene)]bis-piperidine (5) as a subtype-selective H3 antagonist with high binding affinity (pKi=9.47) at human H3 histamine receptor.