Identification of aromatic dihydroxy acids in biological fluids

3,5-Dihydroxyphenylpropionic acid, 3,5-dihydroxycinnamic acid and 2,3-dihydroxycinnamic acid were detected for the first time to be components of human urine. In the course of this investigation all constitutional isomers of dihydroxy-benzoic, -phenylpropionic, -phenylacetic and -cinnamic acid were synthesized. Mass spectra and retention indices of methyl and trimethylsilyl (TMS) derivatives were determined. In contrast to many other substituted aromatic compounds the mass spectra of methyl and TMS derivatives of dihydroxy aromatic acids often allow a firm distinction to be made between constitutional isomers: TMS derivatives of aromatic acids containing two hydroxy groups located in the ortho position to each other can be recognized by ions resulting from a primary cleavage reaction mainly in the side chain or ester group, followed by loss of tetramethylsilane. In methyl derivatives of 1,2,3-trisubstituted isomers, methoxy groups are lost much more easily from the ions corresponding to the benzylic cleavage than in other isomers. Methyl derivatives of dihydroxycinnamic acids containing at least one methoxy group in the ortho position to the side chain are characterized by a fragmentation reaction, corresponding to the loss of dimethyl ether. TMS and methyl derivatives of 3,5-dihydroxy aromatic acids show unique structure-specific fragmentation reactions.     

Caffeoylquinic Acids: Separation Method,Antiradical Properties and Cytotoxicity

Twelve chlorogenic acid derivatives and two flavones were isolated from Moquiniastrum floribundum (Asteraceae, other name: Gochnatia floribunda). Compounds were evaluated in relation to their cytotoxicity and antiradical properties. Cytotoxicity was not observed for compounds, however, chlorogenic acid derivatives showed antiradical activity and were more active than the Trolox standard. Quinic acid esterified with caffeoyl group at C‐4 position showed higher antiradical activity compared to acylation at C‐3 or C‐5 positions. Additional caffeoyl groups esterified in quinic acid increase the antiradical activity observed for 4‐caffeoylquinic acid. Excepted to 3,4‐dicaffeoylquinic acid methyl ester, methyl ester derivatives show higher capacity of trapping radicals than their respective acids. Consequently, the presence of caffeoyl group at C‐4 position of quinic acid is suggested as fundamental to obtain the highest antiradical activity.     

Synthesis and pharmacological activity of amides and ozonolysis product of maleopimaric acid

The synthesis of a new group of maleopimaric acid amides containing fragments of the methyl esters of amino acids, aliphatic amines, imidazole and N-methylpiperazine was carried out. Ozonolysis of methyl maleopimarate flows through the cleavage of double bond C18(19) and the disclosure of anhydrous cycle with formation of secotriacid. As a result of screening of anti-inflammatory and antiulcer activity of maleopimaric acid derivatives new effective compounds such as methyl esters of maleopimaric acid and product of ozonolysis - diterpenic secotriacid, maleopimaric acid amide with L-leucine were revealed. An important advantage of the compounds studied is the low toxicity and the presence of bidirectional activity in the absence of adverse effects on the animal.     

High yielding synthesis of N-ethyl dehydroamino acids

Recently we reported the use of a sequence of alkylation and dehydration methodologies to obtain N-ethyl-α, β-dehydroamino acid derivatives. The application of this N-alkylation procedure to several methyl esters of β,β-dibromo and β-bromo, β-substituted dehydroamino acids protected with standard amine protecting groups was subsequently reported. The corresponding N-ethyl, β-bromo dehydroamino acid derivatives were obtained in fair to high yields and some were used as substrates in Suzuki cross-coupling reactions to give N-ethyl, β,β-disubstituted dehydroalanine derivatives. Herein, we further explore N-ethylation of β-halo dehydroamino acid derivatives using triethyloxonium tetrafluoroborate as alkylating agent, but substituting N,N-diisopropylethylamine for potassium tert-butoxide as auxiliary base. In these conditions, for all β-halo dehydroamino acid derivatives, reactions were complete and the N-ethylated derivative could be isolated in high yield. This method was also applied for N-ethylation of non-halogenated dehydroamino acids. Again, with all compounds the reactions were complete and the N-ethyl dehydroamino acid derivatives could be isolated in high yields. Some of these N-ethyl dehydroamino acid methyl ester derivatives were converted in high yields to their corresponding acids and coupled to an amino acid methyl ester to give N-ethyl dehydrodipeptide derivatives in good yields. Thus, this method constitutes a general procedure for high yielding synthesis of N-ethylated dehydroamino acids, which can be further applied in peptide synthesis.     

Synthesis of 9-O-acetyl- and 4,9-di-O-acetyl derivatives of the methyl ester of N-acetyl-beta-D-neuraminic acid methylglycoside. Their use as models in periodate oxidation studies.

Reaction of the methyl ester of N-acetyl-beta-D-neuraminic acid methyl glycoside with N-acetylimidazole yielded the corresponding 9-O-acetyl- and 4,9-di-O-acetyl derivatives. The structures of these compounds were confirmed by mass spectrometry and both 1H and 13C NMR spectroscopy. The compounds served as model substances in a comparative study of the rate of periodate oxidation of unsubstituted and of 9-O-acetylated-N-acetyl-neuraminic acids. This reaction was strongly hampered by the presence of the 9-O-acetyl group. The low molar absorbancy coefficient of N-acetyl-9-O-acetylneuraminic acid in the periodic acid/thiobarbituric acid assay can be explained by this retardation.     

Hemagglutinins from two influenza virus variants bind to sialic acid derivatives with millimolar dissociation constants: a 500-MHz proton nuclear magnetic resonance study

The equilibrium binding of influenza virus hemagglutinin to derivatives of its cell-surface ligand, sialic acid, was measured by nuclear magnetic resonance (NMR) spectroscopy. Binding was quantified by observing perturbations of sialic acid resonances in the presence of protein. The major perturbation observed was a chemical shift of the N-acetyl methyl resonance, presumably due to the proximity of the methyl group to tryptophan 153. X-31 hemagglutinin binds to the methyl alpha-glycoside of sialic acid with a dissociation constant of 2.8 mM and does not bind to the methyl beta-glycoside. Replacing the 4-hydroxyl group of sialic acid with an acetyl group has little effect, while replacing the 7-hydroxyl group with an acetyl prevents binding. Experiments with sialylated oligosaccharides confirm literature reports that mutations at amino acid 226 change the specificity of hemagglutinin for alpha(2,6) and alpha(2,3) glycosidic linkages. The NMR line broadening of sialyloligosaccharides suggests that sialic acid is the only component that contacts the protein. Saccharides containing two sialic acid residues appear to have two separate binding modes. Hemagglutinin that has undergone a low pH induced conformational change retains the ability to bind sialic acid.     

Synthesis of methyl pyranosides and furanosides of 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) by acid-catalysed solvolysis of the acetylated derivatives

Treatment of methyl 2,4,5,7,8-penta-O-acetyl-3-deoxy-alpha-D-manno-oct- 2-ulopyranosonic acid, or its methyl ester, with refluxing methanolic 0.1 M hydrogen chloride for 16 h gave 95% of methyl (methyl 3-deoxy-alpha-D-manno-oct-2-ulopyranosid)onate. Acetylation of the methyl ester of 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) gave mainly methyl 2,4,6,7,8-penta-O-acetyl-3-deoxy-alpha,beta-D-manno-oct-2-ulofuranoso nate. Treatment of this mixture with methanolic 0.02 M hydrogen chloride at room temperature gave methyl (methyl 3-deoxy-alpha, beta-D-manno-oct-2-ulofuranosid)onate and the corresponding 4-acetates which were isolated by reverse-phase column chromatography of their 7,8-O-isopropylidene derivatives. Confirmation of the position of the isopropylidene group was obtained by acetylation to give methyl (methyl 4,6-di-O-acetyl-3-deoxy-7,8-O-isopropylidene-alpha,beta-D-manno-oct-2-ul ofuranosid)onate. The furanose anomers were differentiated primarily by J3,4 values (alpha approximately 6.1 Hz, beta approximately 2.2 Hz). The anomeric configuration in the furanose series has been assigned on the basis of optical rotation.     

Syntheses of the methyl glycosides of the repeating units of chondroitin 4- and 6-sulfate

3,4,6-Tri-O-acetyl-D-galactal was transformed into methyl 6-O-acetyl-2-azido-4-O-benzyl-2-deoxy-beta-D-galactopyranoside and its 4-O-acetyl-6-O-benzyl analogue, each of which was glycosylated with activated, O-acetylated derivatives of methyl D-glucopyranosyluronate. The resulting beta-(1----3)-linked disaccharide derivatives were each reductively N-acetylated, hydrogenolysed, O-sulfated, and saponified to afford the disodium salts of methyl 2-acetamido-2-deoxy-3-O-(beta-D-glucopyranosyluronic acid)-4-O-sulfo-beta-D-galactopyranoside and the 6-O-sulfo analogue. D-Galactal was also transformed into activated derivatives of 2-azido-3,6-di-O-benzyl-2-deoxy-D-galactopyranose and their 3,4-di-O-benzyl analogues with various substituents at O-4 and O-6. These glycosyl donors were condensed with 6-O-protected derivatives of methyl 2,3-di-O-benzyl-beta-D-glucopyranoside to give the beta-(1----4)-linked disaccharide derivatives, which were selectively deprotected, then oxidised at C-6 of the gluco unit, reductively N-acetylated, selectively deprotected, O-sulfated at C-4 or C-6 of the galacto unit, and hydrogenolysed to give the disodium salts of methyl 4-O-(2-acetamido-2-deoxy-4-O-sulfo-beta-D-galactopyranosyl)-beta-D- glucopyranosiduronic acid and the 6-O-sulfo analogue.     

Occurrence of fatty acid chlorohydrins in jellyfish lipids.

Fatty acid chlorohydrins are characterized as lipid components of an edible jellyfish. The four isomers 9-chloro-10-hydroxypalmitic acid, 10-chloro-9-hydroxypalmitic acid, 9-chloro-10-hydroxystearic acid, and 10-chloro-9-hydroxystearic acid were identified by gas chromatography-mass spectrometry comparison of the methyl esters and their trimethylsilyl derivatives with known synthetic samples. Two additional isomers, 11-chloro-12-hydroxystearic acid and 12-chloro-11-hydroxystearic acid, were also found in the lipid by the identification of the expected mass spectral fragments of the trimethylsilyl (Me3Si) derivative of their methyl esters. These six isomeric compounds represented approximately 1.4% of the total extractable jellyfish lipid and were released from the lipid as methyl esters by boron trifluoride-methanol treatment. These isomers account for only about 30% of the organic chlorine in the lipid. Evidence is given that the remaining organic chlorine is also present as fatty acid chlorohydrins containing more than one hydroxyl group.     

The immunotropic activity of lupane and oleanane 2,3-<Emphasis Type="Italic">seco</Emphasis>-triterpenoids

The immunotropic effect of the 2,3-seco derivatives of allobetulon, betulonic acid, and the methyl ester of betulonic acid were studied. It was found that the highest activity is shown by compounds with an oleanane fragment. The presence of a free C28-carboxyl group enhances the activity of lupane 2,3-seco derivatives. A significant contribution to the development of the immune response is introduced by a functional group at the C3 atom in the A ring—the C3-carboxy derivatives intensify the processes of antibody production and alter the number and ratio of leukocyte forms. It is shown that 2,3-seco-1-cyano-19β,28-epoxy-18α-olean-3-oic acid stimulates humoral immunity with a positive influence on hematopoiesis.     

Synthesis of new iso-C-nucleoside analogues from 2-(methyl 2-O-benzyl-4,6-O-benzylidene-3-deoxy-alpha-D-altropyranosid-3-yl)ethanal

Treatment of 2-(methyl 2-O-benzyl-4,6-O-benzylidene-3-deoxy-alpha-D-altropyranosid-3-yl)ethanal with malononitrile, cyanoacetamide and 2-cyano-N-(4-methoxyphenyl)acetamide, respectively, in the presence of aluminium oxide yielded 2-cyano-4-(methyl 2-O-benzyl-4,6-O-benzylidene-3-deoxy-alpha-D-altropyranosid-3-yl)crotonic acid derivatives. Cyclization with sulfur and triethylamine was performed to synthesize the 2-amino-5-(methyl 2-O-benzyl-4,6-O-benzylidene-3-deoxy-alpha-D-altropyranosid-3-yl)thiophene-3-carbonic acid derivatives, which were treated with triethyl orthoformate/ammonia and triethyl orthoformate, respectively, to furnish 6-(methyl 2-O-benzyl-4,6-O-benzylidene-3-deoxy-alpha-D-altropyranosid-3-yl)thieno[2.3-d]pyrimidine derivatives. Deprotection in two steps afforded 2-amino-5-(1,6-anhydro-3-deoxy-beta-D-altropyranos-3-yl)thiophene-3-carbonitrile and 6-(1,6-anhydro-3-deoxy-beta-D-altropyranos-3-yl)thieno[2.3-d]pyrimidine derivatives, respectively.     

Modification of chitosan to improve its hypocholesterolemic capacity.

Cholestyramine is the most widely used bile acid sequestrant in the treatment of hypercholesterolemia. However, cholestyramine has unpleasant side effects as a consequence of its hydrophobic backbone. Therefore, high-capacity bile acid sequestering biopolymers with cationic chitosan derivatives were developed, because electrostatic interactions are important for binding with bile acid anions. Dialkylaminoalkylation and reductive amination of chitosan were done to add dialkylaminoalkyl and an additional free amino group at a hydroxyl site in the chitosan backbone respectively and the amino-derivatized chitosan derivatives were quaternized with methyl iodide to produce a cationic polyelectrolyte. The in vitro bile acid binding capacity of the chitosan derivatives in aqueous NaCl was measured by reversed-phase HPLC. The binding capacities of sodium glycocholate (a major bile acid) to chitosan, DEAE-chitosan, quaternized DEAE-chitosan, and cholestyramine were 1.42, 3.12, 4.06, and 2.78 mmol/g resin, respectively. With quaternized DEAE-chitosan, the bile acid binding capacity increased approximately 50% over that of cholestyramine. The bile acid binding capacity of dialkylaminoalkyl chitosan derivatives increased with the number of carbons in the alkyl groups, indicating that hydrophobic interaction is a secondary factor for the sequestration of bile acids.     

Synthesis and stereochemistry of 7β- and 7α-amino-, acetamido-, hemisuccinamido- and terephthalamido derivatives of testosterone

The reduction of 3-ethylenedioxy-7-oximino-5-androsten-17β-yl acetate and of its 17β-tetrahydropyranyl ether analog with sodium in ethanol, followed by thin-layer chromatography, allowed the isolation of the corresponding 17β-hydroxy- and 17β-tetrahydropyranyioxy-5-en-7β- and 7α-amines which were also characte-rized as 7-acetamides. The acylation of the two epimeric 17β-hydroxy-5-en-7-amines with succinic anhydride followed by selective saponification of the 17β-hemisuccinate group and diazomethane esterification, gave the corresponding 17β-hydroxy-5-en-7β- and 7α-hemisuccinamido methyl esters characterized also as 17β-acetates. On the other hand, the acylation of the two 17β-tetrahydropyranyl-oxy-5-en-7-amines with the acid chloride of terephthalic acid monomethyi ester led to the more rigid 7β- and 7α-terephthalamido methyl ester side-chains. The acidolysis of the 3-ethyleneketal protecting group of the preceding 5-en-7-N-acyl derivatives regenerated the 4-en-3-oxo function while the 17β-tetrahydropyranyl ether group was cleaved simultaneously into the 17β-alcohol. The four desired 7β- and 7α-hemisuccinamido- and terephthalamido carboxylic side-chain derivatives of 17β-hydroxy-4-androsten-3-one (testosterone) were finally obtained by saponification of the corresponding methyl esters.     

Synthesis and biological activity of some new 3,6-dinitro-1:8-naphthaloyl- and 3,6-diamino-1:8-naphthaloylamino acids and dipeptide derivatives

Synthesis of a series of 3,6-dinitro-1:8-naphthaloylamino acids (II-IX) and some of their corresponding methyl esters (X-XVI) and 3,6-diamino-1:8-naphthaloylamino acid derivatives (XXIX-XXXVI) is described. Coupling of 3,6-dinitro-1:8-naphthaloylamino acids with amino acid methyl ester hydrochlorides in dioxane-DMF-Et3N medium using DCC method furnishes the desired 3,6-dinitro-1:8-naphthaloyldipeptide methyl esters (XVII-XXVIII). Most of the synthesized 3,6-dinitro-1:8-naphthaloylamino acids, esters and dipeptide derivatives (compounds III-VI, XI-XV, XVII, XIX-XXI, XXIII and XXV) and 3,6-diamino-1:8-naphthaloylamino acid derivatives (XXIX-XXXV) were found to be active against a number of microorganisms.     

The Chlorinolysis of Methionine Derivatives

The chlorinolysis of l-methionine methyl ester hydrochloride with molecular chlorine was carried out under various conditions, resulting in methyl l-2-amino-4,4,4-trichlorobutanoate and methyl l-2-amino-3,4,4,4-tetrachlorobutanoate which were isolated as N-benzoyl and N-carbobenzoxy derivatives. The chlorinolysis of N-acylmethionine ester and methionine sulfoxide ester proceeded also without cleavage of the N-protecting group to give the same products as above. However, the reaction of methionine sulfone derivative with chlorine did not proceed in the same conditions.

It was proved that the resulting polychloroamino acid derivatives are optically pure. The possible chlorinolysis mechanism was also proposed.     

Characterization and in vivo production of three glycolipids from Candida Bogoriensis: 13-glucopyranosylglucopyranosyloxydocosanoic acid and its mono- and diacetylated derivatives

Three glycosides of 13-hydroxydocosanoic acid isolated from Candida bogoriensis were characterized by quantitating the amount of carbohydrate, acetate, and hydroxy acid in each, and by gas-liquid chromatography and mass spectrometry of their methyl ester, trimethylsilyl ether derivatives. One of the glycosides was the diacetylated derivative of 13-glucosylglucosyloxydocosanoic acid previously characterized by Tulloch, Spencer, and Deinema (Can. J. Chem., 46: 345 [1968]), in which the disaccharide had the beta(1 --> 2) sophorose linkage and the acetyl groups were attached to the 6' and 6" positions of the glucose residues. The other two glycosides were 13-glucosylglucosyloxydocosanoic acid and its monoacetylated derivative. A comparison of the mass spectra of derivatives indicates that the acetyl group of the monoacetyl lipid is on the internal glucose. Methyl 13-glucosyloxydocosanoate was produced by acid hydrolysis of the methyl ester of the unacetylated glycolipid and was characterized by the same techniques as the other glycolipids. Time course of production of the three glycolipids is consistent with the diacetylated derivative being the first extra-cellular product and the other two glycolipids being formed by deacetylation. 13-Hydroxy[13-(3)H]docosanoic acid, methyl 13-hydroxy[13-(3)H]docosanoate, and 9-hydroxy[11,12-(3)H]-stearic acid were each incorporated into the glycolipid fraction.     

Methionine methyl group metabolism in lemna

To provide information upon the ways in which Lemna paucicostata uses the methyl group of methionine, plants were grown for various periods (from 1 minute to 6.8 days) in the presence of a tracer dose of radioactive methyl-labeled methionine. Protein methionine accounted for approximately 19% of the accumulated methyl moieties; other methylated products, about 81%. The latter group included (percent of total methyl in parentheses): methylated ethanolamine derivatives (46%); methyl esters of the pellet (chiefly, or solely, pectin methyl esters) (15%); chlorophyll methyl esters (8%); unidentified neutral lipids (6%); nucleic acid derivatives (2-5%); methylated basic amino acids (2%). No other major methylated compounds were observed in any plant fraction. Available evidence suggests that little, if any, oxidation of the methyl group of methionine, directly or indirectly, occurs in Lemna. Our results indicate that S-methyl-methionine sulfonium is formed relatively rapidly, but does not accumulate at a commensurate rate, probably being reconverted to methionine. To our knowledge, this is the first time a reasonably complete accounting of the metabolic fate of methionine methyl has been obtained for any plant. The extent to which the results with Lemna may be representative of the situation for other higher plants is discussed.     

Resolution of 4-amino-cyclopentanecarboxylic acid methyl esters using hydrolytic enzymes.

A number of esterases (EC 3.1.1.1) and lipases (EC 3.1.1.3) of microbial and mammalian origin were screened for the ability to resolve racemic 4-amino-cyclopentanecarboxylic acid methyl ester derivatives as potential intermediates in the production of carbocyclic nucleosides. Surprisingly, functionalization of the remote amino group had a profound effect on both the rate and enantioselectivity of hydrolysis of the methyl ester. 4-(Benzoylamino)-2-cyclopentenecarboxylic acid, methyl ester (V) with pig liver esterase gave the highest enantioselectivity. The residual ester, which was of the correct absolute stereochemistry [(+) 1S, 4R] for carbocyclic nucleoside synthesis, could be obtained in high optical purity. Optimization of pH, solvent type, and concentration improved the enantioselectivity of the process by a further twofold.     

Substrate specificity of an α-amino acid ester hydrolase produced by Acetobacter turbidans A.T.C.C. 9325

A partially purified preparation of an alpha-amino acid ester hydrolase was obtained from Acetobacter turbidans A.T.C.C. 9325, which catalyses synthesis of 7-(d-alpha-amino-alpha-phenylacetamido)-3-cephem-3-methyl-4- carboxylic acid (cephalexin) from methyl d-alpha-aminophenylacetate and 7-amino-3-deacetoxycephalosporanic acid. The enzyme preparation catalysed both cephalosprin synthesis from 7-amino-3-deacetoxycephalosporanic acid and suitable amino acid esters (e.g. methyl d-alpha-aminophenylacetate, l-cysteine methyl ester, glycine ethyl ester, d-alanine methyl ester, methyl dl-alpha-aminoiso-butyrate, l-serine methyl ester, d-leucine methyl ester, l-methionine methyl ester) and the hydrolysis of such esters. The substrate specificity of the enzyme preparation for the hydrolysis closely paralleled the acyl-donor specificity for cephalosporin synthesis, even to the reaction rates. Only alpha-amino acid derivatives could act as acyl donors. The hydrogen atom on the alpha-carbon atom was not always required by acyl donors. The hydrolysis rate was markedly diminished by adding 7-amino-3-deacetoxycephalosporanic acid to reaction mixtures, but no effect on the total reaction rate (the hydrolysis rate plus synthesis rate) was observed with various concentrations of 7-amino-3-deacetoxycephalosporanic acid. Both the hydrolytic and the synthetic activities of the enzyme preparation were inhibited by high concentrations of some acyl donors (e.g. methyl d-alpha-aminophenylacetate, ethyl d-alpha-aminophenylacetate). The enzyme preparation hydrolysed alpha-amino acid esters much more easily than alpha-amino acid derivatives with an acid-amide bond.     

Synthesis of the isomeric trisaccharides, methyl O-alpha-L-fucopyranosyl- (1----3, 4, and 6)-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----3)-beta- D-galactopyranoside

Benzylation of methyl 3-O-(2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D- glucopyranosyl)-2,4,6-tri-O-benzyl-beta-D-galactopyranoside with benzyl bromide in N,N-dimethylformamide in the presence of sodium hydride afforded methyl 3-O- (2-acetamido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranosyl) -2,4,6- tri-O-benzyl-beta-D-galactopyranoside (3). Reductive ring-opening of the benzylidene group of 3 gave methyl 3-O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-beta-D- glucopyranosyl)- 2,4,6-tri-O-benzyl-beta-D-galactopyranoside (4). Cleavage of the 4,6-acetal group of 3 with hot, 80% aqueous acetic acid afforded the diol (5). Compounds 3, 4, and 5 were each subjected to halide ion-catalyzed glycosylation with 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl bromide to produce the corresponding trisaccharide derivatives, which, on catalytic hydrogenation, furnished the title trisaccharides, respectively.