Binding of isotryptamines and indenes at h5-HT6 serotonin receptors

To determine if the indolic nitrogen atom is required for the binding of N(1)-benzyltryptamines at h5-HT(6) serotonin receptors, several isotryptamines and indene analogs were examined. The affinity of 3-benzyl-N(1)-(N,N-dimethylaminoethyl)indole (5, K(i)=32nM) and 1-benzyl-3-(N,N-dimethylaminoethyl)indene (11, K(i)=3nM) indicates that the indolic nitrogen atom is not essential for binding.     

Modulation of phosphatidylcholine synthesis in vitro. Inhibition of diacylglycerol cholinephosphotransferase and lysophosphatidylcholine acyltransferase by centrophenoxine and neophenoxine

1,2-Diacyl-sn-glycerol : CDPcholine cholinephosphotransferase (EC 2.7.8.2) and acyl-CoA : 1-acyl-sn-glycero-3-phosphocholine acyltransferase (EC 2.3.1.23) activities of rat liver microsomes can be inhibited by centrophenoxine (N,N-dimethylaminoethyl p-chlorophenoxyacetate). This inhibition is brought about by the intact centrophenoxine molecule rather than by the products of hydrolysis. A nonhydrolyzable ether analog of centrophenoxine was synthesized (neophenoxine; N,N-dimethylaminoethyl p-chlorophenoxyethyl ether) and proved most effective in inhibiting the two routes of phosphatidylcholine biosynthesis. While 50% inhibition of the cholinephosphotransferase was attained at 5 mM neophenoxine, 50% inhibition of the acyltransferase required 0.6 mM neophenoxine levels only. Inhibition of the cholinephosphotransferase (Ki approximately 1.5 mM) and the acyltransferase (Ki approximately 1 mM) by neophenoxine was shown to be noncompetitive. Other membrane-bound enzymes, such as glucose-6-phosphatase, monoacylglycerol lipase, alkaline phosphatase or phospholipase A2 were not affected by the inhibitors. Because of this specificity, and because of the high affinity of the microsomal membrane for such agents, centrophenoxine and neophenoxine should prove useful for controlling phosphatidylcholine synthesis and for modulating the phosphatidylcholine deacylation-reacylation cycle.     

Immobilization of DNA onto a polymer support and its potentiality as immunoadsorbent

Immobilization of DNA to the surface of poly(ethylene terephthalate) (PET) microfibers with a high specific surface area of 0.83 m(2)/g was carried out to give the fiber surface an affinity for anti-DNA antibody. Following ozone oxidation, the microfibers were subjected to graft polymerization of monomers including acrylic acid, methacryloyloxyethyl phosphate, N,N-dimethylaminoethyl methacrylate, N-vinylformamide, and glycidyl methacrylate. Calf thymus DNA was immobilized to the grafted fiber surface through either covalent binding or polyion complexation with the grafted polymer chains. The highest surface density of DNA immobilized (0.6 mug/cm(2)) was obtained when DNA was immobilized through formation of phosphodiester linkage between the hydroxyl group of DNA and the phosphate group in grafted poly(methacryloyloxyethyl phosphate) using 1,1-carbonyldiimidazole, or through polyion complexation between the anionic DNA and the cationic grafted poly(N,N-dimethylaminoethyl methacrylate) chains. Batch adsorption of anti-DNA antibody to the grafted PET fibers with and without DNA immobilized on their surface was conducted with serum obtained from systemic lupus erythematosus model mice. The DNA-immobilized PET fibers exhibited a higher adsorption capacity and specificity than the others. In addition, the DNA-immobilized fibers effectively adsorbed human anti-DNA antibody.     

Arachidonoylcholine and N,N-Dimethylaminoethyl Arachidonate,New Cholinergic Compounds

Choline and N,N-dimethylaminoethyl esters of arachidonic and some other fatty acids were synthesized. Experiments on the embryos and larvae of sea urchins, sensitive to cholinergic compounds, showed that arachidonoylcholine exhibited cholinomimetic activity similar to that of nicotine while N,N-dimethylaminoethyl arachidonate acted as acetylcholine antagonist. The corresponding esters of docosahexaenoic acid were found to manifest similar biological properties.     

Molecular modeling study of intercalation complexes of tricyclic carboxamides with d(CCGGCGCCGG)₂ and d(CGCGAATTCGCG)₂

Tricyclic dyes with different mesoatoms such as xanthenes (fluorescein, eosin) anthracenes and acridines (proflavine) approved by the Food and Drug Administration (FDA) for use in foods, pharmaceuticals and cosmetic preparations interact with DNA, and some of them do so through intercalation. Hyperchem 7.5, Spartan 04, Yasara 10.5.14 program packages and molecular modeling, molecular mechanics and dynamics techniques with the oligonucleotides d(CCGGCGCCGG)2 and d(CGCGAATTCGCG)2 were utilized in order to examine the mode of binding to DNA of a range of tricyclic carboxamides bearing N,N-dimethylaminoethyl side chain, i.e., 9-amino-DACA, anthracene, acridine-1-carboxamide, acridine-4-carboxamide (DACA), azacridine, phenazine, pyridoquinoxaline, oxopyridoquinoxaline, phenoxazine and xanthenone or N,N-dimethylaminobutyl moiety, i.e., phenazine and acridine. The bicyclic quinoline-8-carboxamide was also examined for comparison reasons. On the basis of our data, prerequisite for the interaction between protonated N,N-dimethylaminoethyl moiety and guanine is the formation of only one internal hydrogen bond between carboxamide and peri NH + in the case of 9-amino-DACA or peri N in the cases of DACA, azacridine, phenazine and pyridoquinoxaline. The presence of an additional internal hydrogen bond between oxygen carboxamide and protonated N,N-dimethylamino group in the cases of tricyclic systems bearing peri NH (phenoxazine) or O (xanthenone) group, prevents the interaction between side chain and guanine. Also, the formation of one internal hydrogen bond between oxygen carboxamide and protonated N,N-dimethylamino group inhibits the interaction between side chain and guanine in the case of acridine-1-carboxamide. Our findings are in accordance with previously reported results obtained from the kinetic studies of the binding of acridine and related tricyclic carboxamides to DNA.     

Phorbol esters stimulate spermidine/spermine N1-acetyltransferase activity in mitogen-stimulated bovine lymphocytes

Phorbol 12-myristate-13-acetate (PMA) is shown to induce spermidine/spermine N1-acetyltransferase, a rate-limiting enzyme of polyamine biodegradation, in bovine lymphocytes. When PMA and phytohemagglutinin (PHA) were added simultaneously, the enzyme activity was stimulated synergistically. The ability of phorbol esters to stimulate the enzyme activity was consistent with their tumor-promoting ability. Phorbol, which is not a tumor promotor, was incapable of stimulating the enzyme activity. Phorbol diacetate weakly stimulated the activity of the acetylase. Phorbol dibutyrate had a similar stimulatory effect to PMA. These results suggest that the spermidine/spermine N1-acetyltransferase may play an important role in changes in polyamine levels in phorbol ester-treated cells and that the increase in the enzyme activity may have some relationship to the control of cell growth and differentiation by phorbol esters.     

Synthesis of novel cationic bolaamphiphiles from vernonia oil and their aggregated structures

The present study describes the synthesis of a novel class of vesicle-forming bolaamphiphiles with choline ester head groups. These bolaamphiphiles were derived from vernonia oil, whose main constituent is vernolic acid, a fatty acid with a unique combination of epoxy, carboxy and unsaturated double bonds. A series of bolaamphiphiles containing amido or ester groups within the hydrophobic domain were synthesized from N,N'-alkylenebis (vernolamides) and alpha,omega-alkylene divernolate ester in a two-stage synthesis comprising opening of the epoxy ring with chloroacetic acid, followed by quaternization with N,N-dimethylaminoethyl acetate to form choline ester head groups. The products were characterized by FT-IR, (1)H and (13)C NMR, and ESI-MS. Vesicles prepared from these bolaamphiphiles have the potential to serve as a targeted drug delivery systems with selective decapsulation in the presence of the enzyme acetylcholine esterase, resulting in site-specific release of the drug.     

Trypanocidal activity of (-)-cubebin derivatives against free amastigote forms of Trypanosoma cruzi

Five (-)-cubebin derivative compounds, (-)-O-acetyl cubebin (3), (-)-O-benzyl cubebin (4), (-)-O-(N,N-dimethylaminoethyl)-cubebin (5), (-)-hinokinin (6) and (-)-6,6'-dinitrohinokinin (7), previously synthesised by our research group, were evaluated on in vitro assay against free amastigote forms of Trypanosoma cruzi, the asogic agent of Chagas' disease. It was observed that 6 was the most active compound (IC(50)=0.7 microM), and that 4 and 5 displayed moderate activity against the parasite, giving IC(50) values of 5.7 and 4.7 microM, respectively. In contrast, it was observed that compound 3 was inactive and that 7 displayed low activity with IC(50) values of congruent with 1.5 x 10(4) and 95.3 microM, respectively.     

Direct capture of plasmid DNA from non-clarified bacterial lysate using polycation-grafted monoliths

Monolith columns from macroporous polyacrylamide gel were grafted with polycations, poly(N,N-dimethylaminoethyl methacrylate) (polyDMAEMA), (2-(methacryloyloxy)ethyl)-trimethyl ammonium chloride (polyMETA) and partially quaternized polyDMAEMA prepared via treating polyDMAEMA-grafted columns with propylbromide. The polymer grafting degrees varied between 34 and 110%. The polycation-grafted monolithic columns are able to capture plasmid DNA directly from alkaline lysate of Escherichia coli cells. Due to the large pore size in macroporous monoliths the particulate material present in non-clarified feeds did not block the columns. The captured plasmid DNA was eluted with 1M NaCl as particulate-free preparation with significantly reduced content of protein and RNA as compared to the applied lysate.     

Aminoacyl thiol esters and the origins of genetic specificity

Reasons for believing that primitive mechanisms of translation may have employed thiol esters of the amino acids rather than oxygen esters are summarized. It is suggested that coenzyme A (HSCoA), which fulfills the role of aminoacyl transfer in the synthesis of peptide antibiotics, is a primitive analogue of tRNA which performs a similar role in protein synthesis. HSCoA—an adenylic acid moiety containing phosphates esterified at the 3′ and 5′ positions and linked to a peptide-like structure terminating in a reactive thiol—possesses chemical features suggestive of both peptides and polynucleotides. Examination of the chemistry of HSCoA-like molecules shows that a rather similar compound can carry out a repeating intramolecular peptide synthesis in the absence of enzymes. Condensation of further nucleotides onto the adenylic acid moiety gives rise to parallel modes of peptide and oligonucleotide synthesis. A “self-improving” ability to select available amino acids is inherent in the proposed mechanism of peptide synthesis. The hypothesis plausibly explains the universal occurrence of a sulphur-containing amino acid at the N terminus of nascent proteins.     

Enzymatic production of enantiopure ketoprofen in a solvent-free two-phase system

Enzymatic hydrolysis conducted in a medium composed of solely substrate is considered to resolve racemic ketoprofen esters. In a system composed of two components, the pure liquid substrate (organic phase) and water (aqueous phase), hydrolysis products can be efficiently removed from the reaction mixtures. Accordingly, in this study we designed a solvent-free two-phase system for the enantioselective enzymatic hydrolysis of ketoprofen esters. In order to further optimize this system, the influences of various factors, such as the pH of the aqueous phase, temperature, enzyme content, and the alcohol chain length of esters, were examined on conversion and enantiomeric excess. 1N NaHCO₃ was identified as the most efficient aqueous phase for the extraction of ketoprofen. Changes in the amount of enzyme did not significantly affect the maximum conversion or the enantiomeric excess. On the other hand, ketoprofen esters with shorter alcohol chains displayed higher initial reaction rates and conversions in solventless media. In the case of ketoprofen propyl ester, for example, the productivity of the solvent-free two-phase system was about 10–100 times higher than that obtained to date for ketoprofen esterification with alcohols in organic solvents. The enantioselectivities obtained in solvent-free media were similar to those obtained for the enantioselective esterification of ketoprofen in organic solvents.     

An efficient and highly selective deprotection of N-Fmoc-alpha-amino acid and lipophilic N-Fmoc-dipeptide methyl esters with aluminium trichloride and N,N-dimethylaniline.

A novel procedure for the deprotection of the carboxyl group of amino acid methyl esters is presented. The process is carried out by the reagent system aluminium trichloride/N,N-dimethylaniline that can successfully be applied to unblock the carboxyl moiety either of N-Fmoc-protected amino acid methyl esters and N-Fmoc-protected short dipeptide methyl esters. The chiralities of the optically pure amino acid or peptide precursors are maintained totally unchanged.     

N-benzhydryl-glycolamide: the first protecting group in peptide synthesis with a strong conformational bias

We have synthesized and examined the preferred conformation of a set of N-benzhydryl-glycolamide esters from N(alpha)-protected (or N(alpha)-blocked) alpha-amino acids. Experiments were performed in CDCl(3) solution by Fourier transform infrared absorption and (1)H-NMR techniques, and in the crystalline state by x-ray diffraction. The results of our analysis strongly support the view that this type of N(alpha)-acylated alpha-aminoacyl esters has a marked tendency to fold into a beta-turn conformation, the nature of which is dictated by the structural propensity of the amino acid constituent at the i+1 position.     

Free mycolic acids as criteria in the classification of Nocardia and the 'rhodochrous' complex.

The methyl esters of free mycolic acids from representative strains of Nocardia asteroides, N. brasiliensis, N. caviae and the 'rhodochrous' complex were subjected to detailed mass spectral analysis. The anhydromycolic esters of the Nocardia strains consisted of homologous series containing from zero to three double bonds, with the main components of the parent mycolic acids centred on C52 to C54 (range C46 to C58). The anhydromycolates from one rhodochrous strain, Nocardia opaca, had a molecular weight range similar to the nocardiae (C46 to C57) but the remaining rhodochrous strains gave an homologous series of anhydromycolates containing from zero to two double bonds, with the main components of the parent mycolic acids centred on C38, C42, C44 or C46 (total range from C34 to C50). The mycolic acids from the rhodochrous strains with chain lengths centred around C40 form a group intermediate in size between corynomycolic acids (centred around C32) and nocardomycolic acids (centred around C50). These data weaken the case for retaining the 'rhodochrous' complex in the genus Mycobacterium, and also show that many rhodochrous strains can be distinguished from true nocardiae and corynebacteria. These results confirm the value of lipid characters in the classification of these organisms.     

Design and biophysical characterization of bioresponsive degradable poly(dimethylaminoethyl methacrylate) based polymers for in vitro DNA transfection

Water-soluble, degradable polymers based on poly(N,N-dimethylaminoethyl methacrylate) (PDMAEMA) with low cytotoxicity and good p-DNA transfection efficiency are highlighted in this article. To solve the nondegradability issue of PDMAEMA, new polymers based on DMAEMA and 5,6-benzo-2-methylene-1,3-dioxepane (BMDO) for gene transfection were synthesized. A poly(ethylene oxide) (PEO) azo-initiator was used as free-radical initiator. PEGylation was performed to improve water solubility and to reduce cytotoxicity of the polymers. The resulting polymers contain hydrolyzable ester linkages in the backbone and were soluble in water even with very high amounts of ester linkages. These degradable copolymers showed significantly less toxicity with a MTT assay using L929 cell lines and demonstrated promising DNA transfection efficiency when compared with the gold standard poly(ethyleneimine). Bioresponsive properties of the corresponding quaternized DMAEMA based degradable polymers were also studied. Although the quaternized DMAEMA copolymers showed enhanced water solubility, they were inferior in gene transfection and toxicity as compared to the unquaternized copolymers.     

Comparison of the kinetics and mechanism of the papain-catalyzed hydrolysis of esters and thiono esters

The kinetic constants for the papain-catalyzed hydrolysis of the methyl thiono esters of N-benzoylglycine and N-(beta-phenylpropionyl)glycine are compared with those for the corresponding methyl ester substrates. The k2/Ks values for the thiono esters are 2-3 times higher than those for the esters, and both show bell-shaped pH dependencies with similar pKa's (approximately 4 and 9). The k3 values for the thiono esters are 30-60 times less than those for the esters and do not exhibit a pH dependency. Solvent deuterium isotope effects on k2/Ks and k3 were measured for the ester and thiono ester substrates of both glycine derivatives. Each thiono ester substrate showed an isotope effect similar to that for the corresponding ester substrate. Moreover, use of the proton inventory technique indicated that, as for esters, one proton is transferred in the transition state for deacylation during reactions involving thiono esters and the degree of heavy atom reorganization in the transition state is very similar in both cases. The k3 values for the hydrolysis of a series of para-substituted N-benzoylglycine esters were found to correlate with the k3 values for the corresponding para-substituted thiono esters [Carey, P. R., Lee, H., Ozaki, Y., & Storer, A. C. (1984) J. Am. Chem. Soc. 106, 8258-8262], showing that the rate-determining step for the deacylation of both thiolacyl and dithioacyl enzymes probably involves the disruption of a contact between the substrate's glycinic nitrogen atom and the sulfur of cysteine-25. It is concluded that the hydrolysis of esters and thiono esters proceeds by essentially the same reaction pathway. Due to an oxygen-sulfur exchange process the product released in the case of the N-(beta-phenylpropionyl)glycine thiono ester substrate is the dioxygen acid; however, for the N-benzoylglycine thiono ester substrate, the thiol acid is the initial product. This thiol acid then acts as a substrate for papain and reacylates the enzyme to eventually give the dioxygen acid product. It is shown that thiol acids are excellent substrates for papain.     

Presence of covalently attached fatty acids in rat apolipoprotein B via thiolester linkages

Thiolester linked lipids in rat apolipoprotein B (ApoB) were examined by incubating reduced and carboxymethylated ApoB in 6 M urea buffer with [14C]methylamine at pH 8.5, 30 degrees C. It was observed that [14C]methylamine was covalently incorporated into ApoB, and there was a [14C]methylamine modified product which was lipid in nature. After extraction with organic solvents, the [14C]methylamine labeled product showed its Rf on TLC to be similar to that of the synthetic N-methyl fatty acyl amide. After purification on TLC and transesterification with 3 N methanolic HC1, methyl esters of C16:0, C18:0 and C18:1 fatty acids were identified by gas-liquid chromatography. These results suggest that rat ApoB, similar to human ApoB, contained covalently linked fatty acids through the high energy, labile thiolester bonds.     

Nitrogen deficiency in Arabidopsis affects galactolipid composition and gene expression and results in accumulation of fatty acid phytyl esters

Nitrogen is an essential nutrient for plants because it represents a major constituent of numerous cellular compounds, including proteins, amino acids, nucleic acids and lipids. While N deprivation is known to have severe consequences for primary carbon metabolism, the effect on chloroplast lipid metabolism has not been analysed in higher plants. Nitrogen limitation in Arabidopsis led to a decrease in the chloroplast galactolipid monogalactosyldiacylglycerol (MGDG) and a concomitant increase in digalactosyldiacylglycerol (DGDG), which correlated with an elevated expression of the DGDG synthase genes DGD1 and DGD2. The amounts of triacylglycerol and free fatty acids increased during N deprivation. Furthermore, phytyl esters accumulated containing medium-chain fatty acids (12:0, 14:0) and a large amount of hexadecatrienoic acid (16:3). Fatty acid phytyl esters were localized to chloroplasts, in particular to thylakoids and plastoglobules. Different polyunsaturated acyl groups were found in phytyl esters accumulating in Arabidopsis lipid mutants and in other plants, including 16:3 and 18:3 species. Therefore N deficiency in higher plants results in a co-ordinated breakdown of galactolipids and chlorophyll with deposition of specific fatty acid phytyl esters in thylakoids and plastoglobules of chloroplasts.     

9-Fluorenylmethyl esters

N alpha-Protected amino acid 9-fluorenylmethyl esters (Fm esters) were prepared by imidazole-catalyzed transesterification of active esters with 9-fluorenylmethanol (9-hydroxymethylfluorene). The new carboxyl protection is unaffected by acids, but is efficiently removed by beta-elimination under the influence of secondary and tertiary amines. Primary amines and ammonia can cause slight amide formation. Deblocking was achieved also by catalytic hydrogenation.     

Synthesis of acylamino acid esters of nucleoside 5''-phosphates and their investigation with PMR and CD spectra.

The acylamino acid esters of nucleoside 5'-phosphates are synthesized via condensation of N-(N'-acylaminoacyl) imidazoles with nucleoside 5'-phosphates. The PMR and CD spectra of the esters obtained are studied. The 3'-isomers of the substances under study are observed to have a shift in the conformational N in equilibrium S equilibrium of the carbohydrate moiety in favour of the S-form as compared to the initial nucleosides and their 2'-acyl esters.