Enantiodiscrimination of chiral alpha-aminophosphonic acids by mass spectrometry.

Diastereomeric clusters between first-group metal ions (M(+)) and chiral alpha-aminophosphonic acids (A and B) have been readily generated in the gas phase by electrospray ionization (ESI) and their fragmentation investigated by mass spectrometry. The complexes studied had the general formula [MA(S)B(2)](+) and [MA(R)B(2)](+), where M = H, Li, Na, or K, A(S) and A(R) are the two enantiomers of a given acid A, and B is a reference alpha-aminophosphonic acid of defined configuration. Collision-induced decomposition (CID) of [MA(S)B(2)](+) and [MA(R)B(2)](+) leads to fragmentation patterns characterized by [MAB](+)/[MB(2)] abundance ratios which depend on the configuration of ligand A. These different spectral features were correlated to the different stability of the diastereomeric [MA(S)B(2)](+) and [MA(R)B(2)](+) complexes in the gas phase. The results have been discussed in the light of MM2 Molecular Mechanics Force Field calculations.     

Facile syntheses for (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid through a common synthetic route

The delta 5,9 fatty acids (5Z,9Z)-5,9-hexadecadienoic acid, (5Z,9Z)-5,9-nonadecadienoic acid, and (5Z,9Z)-5,9-eicosadienoic acid were synthesized for the first time in four steps (9-12% overall yield) starting from commercially available 2-(2-bromoethyl)-1,3-dioxolane. The synthetic approach provided enough material to corroborate the structure and stereochemistry of (5Z,9Z)-5,9-nonadecadienoic acid which was recently identified in the flowers of Malvaviscus arboreus (Malvaceae). The novel phospholipids 1-hexadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn-glycer o-3-phosphocholine and 1-octadecanoyl-2-[(5Z,9Z)-5,9-eicosadienoyl]-sn- glycero-3-phosphocholine were also synthesized from commercially available L-alpha-phosphatidylcholine (egg yolk) and characterized by positive ion electrospray mass spectrometry. These are the first examples of unsymmetrical phospholipids with saturated fatty acids at the sn-1 position and delta 5,9 fatty acids at the sn-2 position.     

Omega-cyclohexyl fatty acids in acidophilic thermophilic bacteria. Studies on their presence, structure, and biosynthesis using precursors labeled with stable isotopes and radioisotopes.

Omega-Cyclohexyl undecanoic acid and omega-cyclohexyl tridecanoic acid were found in 10 strains of acido-thermophilic bacteria isolated from different Japanese hot springs. These unusual fatty acids were found in the esterified form in glyceride type complex lipids and constituted 74 to 93% of the total fatty acids in the bacteria. The fatty acids other than omega-cyclohexyl fatty acids found were 14-methyl hexadecanoic acid (3 to 15%) and 15-methyl hexadecanoic acid (1 to 6%), and trace amounts of straight chain and methyl-branched tetra- and penta-decanoic acids. Biosynthesis of omega-cyclohexyl fatty acids increased with increase in the concentration of glucose in the culture medium. The metabolism of omega-cyclohexyl fatty acids was studied using deuterium-labeled precursors by mass fragmentation analysis. The deuterium of [2-D]glucose was specifically incorporated into position 2 of the cyclohexyl ring of the fatty acids, indicating that the ring was synthesized from the glucose molecule. Radioactivity was efficiently incorporated into the omega-cyclohexyl fatty acids from labeled glucose, shikimate, and cyclohexyl carboxylate. These findings indicate that omega-cyclohexyl fatty acids are synthesized with glucose through shikimic acid and probably cyclohexyl carboxylyl-CoA derivative as the intermediates.     

Synthesis of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine analogues. n-Butyramide, 3-chloropropionamide, 3-aminopropionamide, and isovaleramide analogues

The syntheses of four analogues of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine are described. Activated carboxylic acids were reacted with 2-acetamido-2-deoxy-beta-D-glucopyranosylamine. n-Butyric anhydride gave N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-n-butyramide. 3-Chloropropionic anhydride was synthesized from 3-chloropropionic acid and gave N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-3-chloropropionamide. Equilibration of the latter with ammonium bicarbonate gave N1-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-3-aminopropionamide. Succinimidyl isovalerate was synthesized and gave N-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-isovaleramide.     

Synthesis and biological evaluation of novel naphthoquinone fused cyclic aminoalkylphosphonates and aminoalkylphosphonic monoester

A series of novel naphthoquinone fused cyclic alpha-aminophosphonates, 2-alkoxy-3,4-dihydro-2H-naphtho[2,3-e][1,4,2]oxazaphosphinane-5,10-dione 2-oxide 3-17 and naphthoquinone fused cyclic alpha-aminophosphonic monoester 18 were synthesized for the first time. These cyclic alpha-aminophosphonates were evaluated for antitumor activity on four human tumor cell lines, and three of them showed significant cytotoxicity (IC(50): 0.019-5.15 microM) comparable to that of the reference drug doxorubicin. Furthermore, inhibition assays for topoisomerase II-mediated relaxation of supercoiled DNA indicated that the naphthoquinone fused cyclic aminophosphonates were catalytic inhibitors of topoisomerase II.     

Synthesis of fatty acids from [1-14C]acetylcoenzyme A in subcellular particles of rat epididymal adipose tissue

1. Mitochondrial and microsomal fractions of rat epididymal adipose tissue incorporated [1-(14)C]acetyl-CoA equally well into various fatty acids by a chain-elongation mechanism. C(18) and C(20) fatty acids were the two major products, and comprised about 80% of the total fatty acids synthesized in both particles. 2. When incubated in air, mitochondria synthesized stearic acid, octadecenoic acid and eicosamonoenoic acid in almost equal amounts (about 20% each), whereas in microsomal fractions, the synthesis of octadecenoic acid was more than fivefold the stearic acid formation. In both fractions, major components of synthesized monoenoic fatty acids were the Delta(11:12) isomers. Hexadecenoic acid and octadecenoic acid from whole adipose tissue contained approx. 11 and 14% of the Delta(11:12) isomer respectively. 3. When mitochondria or microsomal fractions were incubated in nitrogen, there was increased synthesis of stearic acid and palmitic acid and less of C(16) and C(18) monoenoic acids; synthesis of C(20) acids remained predominantly of the monoenoic acids. Determination of the position of the double bond in the monoenoic acids supported the view that the synthesis of hexadecenoic acid and octadecenoic acid involves a desaturase activity, whereas eicosamonoenoic acid and eicosadienoic acid are formed only by elongation of endogenous fatty acids. 4. Most of the radioactivity was found in free fatty acids (63%) and the phospholipid (26%) fraction. In phospholipids, phosphatidylcholine and phosphatidylethanolamine were the two major components. 5. Most of the fatty acids synthesized, including those not normally found in particle lipids (arachidic acid, eicosamonoenoic acid and eicosadienoic acid) were distributed fairly evenly in the phospholipid and free fatty acid fractions. However, stearic acid was found predominantly in the phospholipid fraction.     

Interactions of insulin, corticosterone and prolactin in promoting milk-fat synthesis by mammary explants from pregnant rabbits

1. The rate of fatty acid synthesis by mammary explants from rabbits pregnant for 16 days or from rabbits pseudopregnant for 11 days was stimulated up to 15-fold by culturing for 2-4 days with prolactin. This treatment initiated the predominant synthesis of C(8:0) and C(10:0) fatty acids, which are characteristic of rabbit milk. 2. Inclusion of insulin in the culture medium increased the rate of synthesis of these medium-chain fatty acids. By contrast the inclusion of corticosterone led to the predominant synthesis of long-chain fatty acids. When explants were cultured for 2-4 days with insulin, corticosterone and prolactin, the rate of fatty acid synthesis increased up to 42-fold, but both medium- and long-chain fatty acids were synthesized. 3. These results show that the stimulus to mammary-gland lipogenesis and the initiation of synthesis of medium-chain fatty acids observed between days 16 and 23 of pregnancy in the rabbit can be simulated in vitro by prolactin alone. 4. When mammary explants from rabbits pregnant for 23 days were cultured for 2 days with insulin, corticosterone and prolactin, the rate of fatty acid synthesis increased fivefold, but there was a preferential synthesis of long-chain fatty acids. Culture with prolactin alone had little effect on the rate or pattern of fatty acids synthesized. 5. The results are compared with findings in vivo on the control of lipogenesis in the rabbit mammary gland, and are contrasted with the known effects of hormones in vitro on the mammary gland of the mid-pregnant mouse.     

alpha-Biphenylsulfonylamino 2-methylpropyl phosphonates: enantioselective synthesis and selective inhibition of MMPs

(R)-alpha-Biphenylsulfonylamino 2-methylpropyl phosphonates attain nM potency against several MMPs and are the most effective inhibitors based on phosphonate as zinc binding group. Since their preparation by direct N-acylation of expensive, enantiopure, alpha-aminophosphonic acids proceeds in low yields, we devised and evaluated a stereoselective and straightforward method of synthesis that avoids the unfavourable step of N-acylation. The key intermediate (R)-4-bromophenylsulfonylamino 2-methylpropyl phosphonate 9 was obtained by highly stereoselective addition of dibenzylphosphite to the enantiopure (S)-N-isobutylidene-p-bromobenzenesulfinamide 3, followed by oxidation with m-CPBA. Suzuki coupling of 9 with the desired arylboronic acids, gave the expected biphenylsulfonylamino derivatives in satisfactory yields. Liberation of the phosphonic group by hydrogenolysis led to the desired (R)-alpha-biphenylsulfonylamino 2-methylpropyl phosphonates 14a-i. Screening of the new compounds on MMP-1, -2, -3, -7, -8, -9, -13 and -14 showed IC(50) in the range of nM in most cases.     

Fatty acid biosynthesis in rabbit mammary gland during pregnancy and early lactation

The pattern of fatty acids synthesized by mammary-gland explants from rabbits during pregnancy and early lactation has been studied. From day 12 to day 18 of pregnancy, long-chain (C(14:0)-C(18:1)) fatty acids were the major products. From day 18 to day 21 of pregnancy there was an increase of up to 12-fold in the rate of fatty acid synthesis per unit wet weight of tissue that was almost exclusively caused by the synthesis of octanoic fatty acid and decanoic fatty acid, which are characteristic of rabbit milk. These medium-chain fatty acids were mainly incorporated into triglycerides. From day 22 to day 27 of pregnancy there was little change in the rate of fatty acid synthesis and the proportions of fatty acids synthesized were essentially the same as those synthesized by the lactating gland, i.e. 80-90% octanoic acid plus decanoic acid. About 2-4 days before parturition a second lipogenic stimulus occurred, although the pattern of fatty acids synthesized did not change.     

Synthesis,antioxidative and antiviral activity of hydroxycinnamic acid amides of thiazole containing amino acid

The synthesis and the biological (antioxidant and antiviral) activities of novel hydroxycinnamic acid amides of a thiazole containing TFA.valine-4-carboxylic acid ethyl ester are reported. The amides have been synthesized from p-coumaric, ferulic and sinapic acids with the corresponding TFA.valine-thiazole-4-carboxylic acid ethyl ester using the coupling reagent N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and 4-(dimethylamino) pyridine (DMAP) as a catalyst. The antioxidant properties of the newly synthesized amides have been studied for then antioxidative activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH)* test. The newly synthesized compounds have been tested against the replication in vitro of influenza virus A (H3N2) and human herpes virus 1 and 2 (HSV-1 and HSV-2).     

Synthesis of N3-fumaramoyl-L-2,3-diaminopropanoic acid analogues, the irreversible inhibitors of glucosamine synthetase

Several analogues of N3-fumaramoyl-L-2,3-diaminopropanoic acid were synthesized and evaluated for inhibition of glucosamine-6-phosphate synthetase activity. The syntheses were accomplished by acylation reaction of N2-tert.-butoxycarbonyl-L-2,3-diaminopropanoic acid (Boc-A2pr) or N2-tert.-butoxycarbonyl-L-2,4-diaminobutanoic acid (Boc-A2-bu) with the N-succinimidoyl esters of several derivatives of alpha, beta-unsaturated acids 2a-d followed by deprotection of the Boc groups. The obtained compounds were tested for inhibition of glucosamine synthetase isolated from Salmonella typhimurium and Saccharomyces cerevisiae. The results indicated that among the synthesized compounds, N3-4-methoxyfumaroyl-L-2,3-diaminopropanoic acid (FMDP) was the most powerful inhibitor of glucosamine synthetase.     

N5-(1-carboxyethyl)-ornithine, a new amino acid from the intracellular pool of Streptococcus lactis.

Intracellular concentrations of amino acids were determined in cells of Streptococcus lactis 133 during growth in complex, spent, and chemically defined media. Glutamic and aspartic acids represented the major constituents of the amino acid pool. However, organisms grown in spent medium or in defined medium supplemented with ornithine also contained unusually high levels of two additional amino acids. One of these amino acids was ornithine. The second compound exhibited properties of a neutral amino acid by coelution with valine from the amino acid analyzer. The compound did not, however, comigrate with valine or any other standard amino acid by two-dimensional thin-layer chromatography. The unknown amino acid was purified by paper and thin-layer chromatography, and its molecular structure was determined by 1H and 13C nuclear magnetic resonance spectroscopy. This new amino acid was shown to be N5-(1-carboxyethyl)-ornithine. The 14C-labeled compound was formed by cells of S. lactis 133 during growth in spent medium or defined medium containing [14C]ornithine. Formation of the derivative by resting cells required ornithine and the presence of a metabolizable sugar. N5-(1-Carboxyethyl)-ornithine was synthesized chemically from both poly-S-ornithine and (2S)-N2-carbobenzyloxy-ornithine as a 1:1 mixture of two diastereomers. The physical and chemical properties of the amino acid purified from S. lactis 133 were identical to those of one of the synthetic diastereomers. The bis-N-trifluoroacetyl-di-n-butyl esters of the natural and synthetic compounds generated identical gas chromatography-mass spectrometry spectra. A mechanism is suggested for the in vivo synthesis of N5-(1-carboxyethyl)-ornithine, and the possible functions of this new amino acid are discussed.     

Desiccation-induced Changes in Antioxidant Enzymes,Fatty Acids,and Amino Acids in the Cyanobacterium <Emphasis Type="Italic">Tolypothrix scytonemoides</Emphasis>

Tolypothrix scytonemoides subjected to desiccation exhibited increased antioxidant enzyme activities when compared to fresh cells. The activities of catalase (EC 1.11.1.6) and superoxide dismutase (EC 1.15.1.1) were enhanced in desiccated cells by 42.8% and 8.1%, respectively. The isoforms of catalase and superoxide dismutase were detected by activity staining of crude samples separated on native gels. The isoforms of superoxide dismutase were identified based on their sensitivity to hydrogen peroxide and cyanide. The changes in fatty acids and amino acids in fresh and desiccated cells were also investigated and it was found that the quantity of certain fatty acids and amino acids was greater in desiccated cells. Palmitic acid, palmitoleic acid, heptadecanoic acid, linoleic acid, and myristic acid were more in desiccated cells when compared to fresh cells. Desiccated cells synthesized myristoleic acid, eicosenoic acid and behenic acid, acids which were not synthesized by the fresh cells, whereas tricosanoic acid was synthesized by the fresh cells and not by desiccated cells. The levels of lysine, serine, glycine, proline and cysteine were also comparatively greater in the desiccated cells.     

Fatty acid biosynthesis by a particulate preparation from germinating pea

1. Fatty acid synthesis was studied in microsomal preparations from germinating pea (Pisum sativum). 2. The preparations synthesized a mixture of saturated fatty acids up to a chain length of C(24) from [(14)C]malonyl-CoA. 3. Whereas hexadecanoic acid was made de novo, octadecanoic acid and icosanoic acid were synthesized by elongation. 4. The products formed during [(14)C]malonyl-CoA incubation were analysed, and unesterified fatty acids and polar lipids were found to be major products. [(14)C]Palmitic acid represented a high percentage of the acyl-carrier protein esters, whereas (14)C-labelled very-long-chain fatty acids were mainly present as unesterified fatty acids. CoA esters were minor products. 5. The addition of exogenous lipids to the incubation system usually resulted in stimulation of [(14)C]malonyl-CoA incorporation into fatty acids. The greatest stimulation was obtained with dipalmitoyl phosphatidylcholine. Both exogenous palmitic acid and dipalmitoyl phosphatidylcholine increased the amount of [(14)C]-stearic acid synthesized, relative to [(14)C]palmitic acid. Addition of stearic acid increased the amount of [(14)C]icosanoic acid formed. 6. [(14)C]Stearic acid was elongated more effectively to icosanoic acid than [(14)C]stearoyl-CoA, and its conversion was not decreased by addition of unlabelled stearoyl-CoA. 7. Incorporation of [(14)C]malonyl-CoA into fatty acids was markedly decreased by iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid). Palmitate elongation was sensitive to arsenite addition, and stearate elongation to the presence of Triton X-100 or fluoride. The action of fluoride was not, apparently, due to chelation. 8. The microsomal preparations differed from soluble fractions from germinating pea in (a) synthesizing very-long-chain fatty acids, (b) not utilizing exogenous palmitate-acyl-carrier protein as a substrate for palmitate elongation and (c) having fatty acid synthesis stimulated by the addition of certain complex lipids.     

Synthesis and beta-lactamase inhibitory activity of new 6beta-cysteinesulfonamidopenicillanic acids

New 6beta-cysteinesulfonamidopenicillanic acids and their sulfoxides were synthesized by sulfonylation of 6beta-aminopenicillanic acid or its (S)-sulfoxide with (R)-N-benzyloxycarbonylcysteinesulfonyl chloride ethyl ester (2a, 1b) and (R)-N-benzyloxycarbonylcysteinesulfonyl chloride benzyl ester (2a, 2b). The corresponding 6beta-cysteinesulfonamidopenicillanic acids sulfones 1c and 2c were prepared by oxidation of the sulfoxides 1b and 2b with potassium permanganate in aqueous medium. When combined with ampicillin some of the compounds reduced the minimal inhibitory concentrations of ampicillin against beta-lactamase producing strains.     

Transacylation as a chain-termination mechanism in fatty acid synthesis by mammalian fatty acid synthetase. Synthesis of medium-chain-length (C8-C12) acyl-CoA esters by goat mammary-gland fatty acid synthetase.

1. Ruminant mammary-gland fatty acid synthetases can, in contrast with non-ruminant mammary enzymes, synthesize medium-chain fatty acids. 2. Medium-chain fatty acids are only synthesized in the presence of a fatty acid-removing system such as albumin, beta-lactoglobulin or methylated cyclodextrin. 3. The short- and medium-chain fatty acids synthesized were released as acyl-CoA esters from the fatty acid synthetase.     

1- and 2-substituted naphthalenes: a new class of potential hypotensive agents

A series of 2-substituted aminomethyloxy naphthalenes 1 and 4-(1-naphthoxy-2-substituted aminomethyl)-butanoic acids 2 were synthesized by Mannich reaction of 4-(2-naphthoxy)-butanoic acid 3 and 4-(1-naphthoxy)-butanoic acid 4 with appropriate secondary amines and para-formaldehyde. The newly synthesized compounds were tested for their hypotensive activity at 5 mg/kg i.v. dose in cats. The results indicated that the analogue 2-(N4-phenyl-N1-piperazino)-methyloxy naphthalene 1d (> N = N4-phenyl-N1-piperazino) was the most active analogue when its hypotensive activity was compared to the reference compound propranolol.     

Synthesis and stereochemical determination of diunsaturated valproic acid analogs including its major diunsaturated metabolite

Valproic acid, an antiepileptic drug, is transformed into diunsaturated metabolites in humans. Synthesis of the geometric isomers of 2-(1'-propenyl)-2-pentenoic acid and 2-(1'-propenyl)-3-pentenoic acid was attempted using known procedures. The final product, a mixture of isomers, was converted into tert-butyldimethylsilyl or ethyl derivatives. Capillary gas-liquid chromatography-mass spectrometry analysis of the derivatives showed at least three isomeric dienoic acids from synthesized products. Argentation thin-layer chromatography was effective in resolving the isomeric mixture into a single isomer or mixture of two isomers. Thin-layer chromatography and gas-liquid chromatography retention data, photochemical isomerization studies, and nuclear magnetic resonance spectrometry were used to characterize the dienoic acids. By comparison of the retention times of the diunsaturated metabolites with synthesized reference compounds, the structure assigned to the major diunsaturated metabolite is 2-[(E)-1'-propenyl](E)-2-pentenoic acid.     

Developmental changes in fatty acid biosynthesis and composition in the house cricket,Acheta domesticus

Incorporation of [1-¹⁴C] acetate into various phospholipid and triacylglycerol fatty acids showed cyclic fluctuations in fatty acid biosynthesis that were similar for all of the major fatty acids in both male and female house crickets, Acheta domesticus, during development. All three stadia showed low levels of biosynthesis near ecdysis followed by increased synthesis to a peak at midstadium. In the phospholipid fraction, the incorporation of newly synthesized saturated fatty acids, 16:0 and 18:0, predominated near ecdysis, while at midstadium linoleic acid was the most actively synthesized fatty acid. In the triacylglycerol fraction, 18:0 and 18:1 predominated throughout the entire stadium. In contrast to the large fluctuations in fatty acid biosynthesis, the fatty acid compositions of the phospholipid and triacylglycerol fractions did not change within a stadium. However, significant differences were demonstrated between the stages and were associated primarily with differences between nymphal and adult stadia. Males and females differed in the proportions of 16:0 and 18:2 incorporated into phospholipids with females showing a greater proportion of 18:2 and a corresponding smaller proportion of 16:0 than males. The greater proportion of linoleic acid in females and in adults in general compared to nymphs and the predominance of the incorporation of newly synthesized linoleic acid into the phospholipid fraction of all stadia are consistent with the importance of this fatty acid in a number of biological roles.     

Characterization of surface n-alkanes and fatty acids of the epiphytic lichen Xanthoria parietina, its photobiont a green alga Trebouxia sp., and its mycobiont, from the Jerusalem hills.

Surface alkanes and fatty acids from the thalli of the lichen Xanthoria parietina, its photobiont Trebouxia sp., and its mycobiont were analysed by GC-MS. The green alga Trebouxia sp. synthesized mainly unsaturated fatty acids such as (Z,Z,Z)-9,12,15-18 : 3 (Z,Z)-9,12-18 : 2 and (Z)-9-18 : 1, and light alkanes C8-C15 (up to 83% of total n-alkanes). However, the mycobiont contained mainly saturated fatty acids such as hexadecanoic (16 : 0) and octadecanoic acid (18 : 0), and also very long-chain n-alkanes C22-C34. Dehydroabietic acid was found in both lichen and mycobiont. The occurrence of different amounts of n-alkanes and fatty acids in the photobionts and mycobionts of X. parietina was shown for the first time. Lichens collected from different locations in the Jerusalem hills contained n-alkanes ranging in concentration from 187 to 211 mg x (g dry wt)-1; n-alkane concentrations in the photobiont and mycobiont were 17-24 and 215-262 mg x (g dry wt)-1, respectively.