A preparation of N-Fmoc-N-methyl-α-amino acids and N-nosyl-N-methyl-α-amino acids

A convenient route for the synthesis of lipophilic N-Fmoc-N-methyl-α-amino acids and N-nosyl-N-methyl-α-amino acids, interesting building blocks to be used for the preparation of N-methylated peptides, is presented. Both nosyl- and Fmoc-protected monomers are accessible, so these compounds can be used in solution as well as in solid phase peptide synthesis. The methodology is based on the use of benzhydryl group to protect temporarily the carboxyl function of N-nosyl-α-amino acids and on the subsequent methylation of the N-nosyl-α-amino acid benzhydryl esters with diazomethane. The benzhydryl esters offer several beneficial features such as simple preparation, stability to methylation and selective deprotection under mild conditions. The overall procedure is highly efficient in that the adopted conditions keep the chiral integrity of amino acid precursors and the process does not require chromatographic purification of the methylated products.     

Grouping of amino acids and recognition of protein structurally conserved regions by reduced alphabets of amino acids

Sequence alignment is a common method for finding protein structurally conserved/similar regions. However, sequence alignment is often not accurate if sequence identities between to-be-aligned se- quences are less than 30%. This is because that for these sequences, different residues may play similar structural roles and they are incorrectly aligned during the sequence alignment using substitu- tion matrix consisting of 20 types of residues. Based on the similarity of physicochemical features, residues can be clustered into a few groups. Using such simplified alphabets, the complexity of protein sequences is reduced and at the same time the key information encoded in the sequences remains. As a result, the accuracy of sequence alignment might be improved if the residues are properly clustered. Here, by using a database of aligned protein structures (DAPS), a new clustering method based on the substitution scores is proposed for the grouping of residues, and substitution matrices of residues at different levels of simplification are constructed. The validity of the reduced alphabets is confirmed by relative entropy analysis. The reduced alphabets are applied to recognition of protein structurally conserved/similar regions by sequence alignment. The results indicate that the accuracy or efficiency of sequence alignment can be improved with the optimal reduced alphabet with N around 9.     

Synthesis and reactions of α- and β-d-glucopyranosyluronic esters of amino acids

The synthesis of the fully benzylated α- and β-d-glucopyranosyluronic esters of 1-benzyl N-benzyloxycarbonyl-l-aspartic and -glutamic acids and N-(tert-butoxycarbonyl)-l-phenylalanine, followed by hydrogenolysis, afforded the respective anomers of the 1-O-acyl-d-glucopyranuronic acids 2, 7, and 12. Esterification of both anomers of the N-acetylated derivatives of 2 and 7 by diazomethane was accompanied by glycosyl-bond cleavage, and, in the case of the α anomers, with concomitant 1→2 acyl migration to give, after O-acetylation, the 2-O-acyl O-acetyl methyl ester derivatives 5 and 10, respectively. Similarly, 12α yielded methyl 1,3,4-tri-O-acetyl-2-O-[N-(tert-butoxycarbonyl)-l-phenylalanyl]-d-glucopyranuronate and an analogue having a furanurono-6,3-lactone structure. Esterification of the C-5 carboxyl group, in 1-O-acyl-α-d-glucopyranuronic acids by methanol in the presence of the BF_3^?-MeOH reagent (1–1.5 equiv.) proceeded without acyl migration. By using this procedure, followed by acetylation, the N-acetylated derivative of 7α afforded methyl 2,3,4-tri-O-acetyl-1-O-(1-methyl N-acetyl-l-glutam-5-oyl)-α-d-glucopyranuronate, and 12α gave methyl 2,3,4-tri-O-acetyl-1-O-(N-acetyl-l-phenylalanyl)-α-d-glucopyranuronate; the formation of the latter involved cleavage of the tert-butoxycarbonyl group by BF₃, followed by N-acetylation in the next step.     

Dehydration polycondensation of dicarboxylic acids and diols using sublimating strong brønsted acids

We investigated catalytic activities of strong br?nsted acids for dehydration polycondensations of dicarboxylic acids and diols, which were carried out at low temperature (<100 °C) under reduced pressure (0.3-3 mmHg). Strong Br?nsted acids, bis(perfluoroalkanesulfonyl)imide and perfluoroalkanesulfonic acid, showed higher activity than p-toluenesulfonic acid or rare-earth catalysts at 60 °C. In particular, bis(nonafluorobutanesulfonyl)imide (Nf(2)NH) showed the highest activity to synthesize not only aliphatic polyester (M(n) > 19000) but also aromatic polyester (M(n) > 7000). The used Nf(2)NH was sublimated from the reaction flask during polycondensation, and the sublimate, Nf(2)NH, was extra pure so that we can reuse the catalyst without loss of the activity in the dehydration polycondensations.     

Convenient syntheses of phosphinic analogues of γ-aminobutyric- and glutamic acids

Three-steps, one-pot synthesis of 2-amino-4-(hydroxyphosphinyl)butyric acid from dibutyl ester of vinylphosphinic acid was carried out with an overall yield of 66%. 3-Aminopropylphosphinic acid was prepared from allylamine in three steps with an overall yield of 56%. These improved protocols allowed to obtain these commercially unavailable phosphinic analogues of glutamic acid and GABA for testing on potential molecular targets.     

Synthesis and anticancer activity of quinopimaric and maleopimaric acids’ derivatives

A series of quinopimaric and maleopimaric acids’ derivatives modified in the E-ring, at the carbonyl- and carboxyl-groups were synthesized and their in vitro cytotoxic activity was evaluated at the National Cancer Institute, USA. Methyl esters of dihydroquinopimaric, 1a,4a-dehydroquinopimaric, 2,3-epoxyquinopimaric, 1-ethylenketal-dihydroquinopimaric, 1-ethylenketal-4-hydroxyiminodihydroquinopimaric acids displayed an activity on renal cancer, leukemia, colon cancer and breast cancer cell lines in concentration 10⁻⁵ M. Methyl 1,4-dihydroxyiminodihydroquinopimarate showed both a potent and broad spectrum of cytotoxic activity against NSC lung cancer, colon cancer, breast cancer, renal cancer and leukemia and revealed in vivo antineoplastic activity towards mouse solid transplantable mammary carcinoma Ca755 and colon adenocarcinoma AKATOL. The information about antineoplastic activity of the studied quinopimaric and maleopimaric acids’ derivatives will be used for hit to lead optimization in these chemical series.     

Transport of imino acids and non-α-amino acids across the brush-border membrane of the rabbit ileum

Summary The transport of -alanine and MeAIB and their effects as inhibitors of the transport of alanine, leucine and lysine across the brush-border membrane of the intact epithelium from the rabbit's distal ileum has been examined. Two separate transport systems have been characterized: 1) A sodium-dependent, -alanine-accepting system, which is a high-affinity transport system for -amino-monocarboxylic acids (neutral a.a.) and for cationic a.a., accepts non--amino acids as well as non--imino acids, is moderately stereospecific, and for which the affinity of a neutral a.a. is greatly reduced by N-methylation. 2) A sodium-dependent transport system for imino acids, which is inaccessible to cationic amino acids and non--amino acids but accepts cyclic, non--imino acids, is moderately stereospecific, and for which neutral a.a. have much lower affinities than their N-methylated derivatives. On the basis of the observations of this and the preceding paper five transport systems for amino acids are ascribed to the rabbit ileum. Some discrepancies between the present results and those obtained with brush-border membrane microvesicles from the rabbit small intestine are discussed.     

Production of aromatic d-amino acids from α-keto acids and ammonia by coupling of four enzyme reactions

A multi-enzyme system composed of glutamate racemase, thermostable d-amino acid aminotransferase, glutamate dehydrogenase and formate dehydrogenase was employed for the production of aromatic d-amino acids, d-phenylalanine and d-tyrosine, from the corresponding α-keto acids, phenylpyruvate and hydroxyphenylpyruvate, respectively. The optimal concentration of ammonium formate for the production of these d-amino acids was found in the range of 0.25–1.0 M. The optimal concentration of α-keto acid was determined to be 50 mM, above which the productivity greatly decreased. To keep the concentration of α-keto acid around this concentration, α-keto acid was intermittently fed into the multi-enzyme system during the production period. By running the multi-enzyme system for 35 h, 48 g l⁻¹ of d-phenylalanine and 60 g l⁻¹ of d-tyrosine were produced with 100% of optical purity from the equimolar amounts of phenylpyruvate and hydroxyphenylpyruvate, respectively. The production levels of both aromatic d-amino acids were demonstrated to be dependent on the stability of glutamate racemase.     

Amino acids and β-aminopropionitrile as inhibitors of seed germination and growth

The germination of lettuce fruits and legume seeds was affected by the imbibition of solutions of certain amino acids. Seedling growth was inhibited more markedly than germination by these compounds. Non-protein amino acids were, as a group, more effective inhibitors of germination and seedling growth than were protein amino acids, with the exception of lysine. Anomalous results were obtained with β-aminopropionitrile.     

Stability of Schiff bases of amino acids and pyridoxal-5′-phosphate

Summary Stability of Schiff bases from Pyridoxal-5-phosphate and- and non-amino acids and amines have been studied in a wide range of pH. Furthermore the transamination process for the PLP-serine Schiff base and the cyclization reaction of PLP-histidine Schiff base have also been studied.Results show that the-position on carboxyl group of amino acids plays an important role on the mechanism of hydrolysis of imine bond. Absence of ionic groups in the surroundings of that bond seems to be an important fact of stability.In the transamination reaction, the rate-determining step is the isomerization of the Schiff base to ketoimine, since the rate constants for disappearance of Schiff base coincide with the rate constants for PMP formation. This process is catalyzed by the OH^–/H₂O system and the monoprotonated amino acid.     

Synthesis,cytotoxicity and clastogenicity of novel α-aminophosphonic acids

Summary. α-Ethyl-N-(phosphonomethyl) glycine is synthesized and characterized by NMR and FAB spectroscopy. The cytotoxicity, clastogenic and antiproliferative effect of 3-ethyl-2-hydroxyl-2-oxo-1,4,2-oxazaphosphorinane, sodium salt of 3-ethyl-2-hydroxyl-2-oxo-1,4,2-oxazaphosphorinane, α-ethyl-α-N-(hydroxyethylamino) methylphosphonic acid, α-ethyl-N-(phosphonomethyl) glycine, α-ethyl-N-(phosphonomethyl) glycine isopropylammonium salt, glyphosate isopropylammonium salt are tested.     

Acyl derivatives of boswellic acids as inhibitors of NF-κB and STATs

Boswellic acid acylates including their epimers were synthesized and screened against a panel of human cancer cell lines. They exhibited a range of cytotoxicity against various human cancer cell lines thereby leading to the development of a possible SAR. One of the identified lead compounds was found to be an inhibitor of the NF-κB and STAT proteins, warranting further investigations to be developed into a potential anticancer lead.     

Synthesis of β-hydroxy fatty acids and β-amino fatty acids by the strains of Bacillus subtilis producing iturinic antibiotics

The iturinic antibiotics, which contain long chain β-amino acids, are produced by Bacillus subtilis. Screening these strains for the presence of a possible precursor of the iturinic antibiotics, we isolated a lipopeptide containing β-hydroxy fatty acids. The structure of this compound was studied and it appears to be identical or structurally very similar to surfactin. The carbon chain of its β-hydroxy fatty acids was n C₁₆, iso C₁₆, iso C₁₅ or anteiso C₁₅. The percentages of each β-hydroxy fatty acids varied according to the strain producing iturinic antibiotics and were influenced by addition of branched-chain α-amino acids to the culture medium. These results demonstrate for the first time that iso C₁₄ β-hydroxy fatty acid is a constituent present in such a surfactin like lipopeptide. Besides, the presence of radioactive β-hydroxy fatty acids in the phospholipids when the strains were grown in the presence of sodium [¹⁴C]acetate seems also characterize the different strains producing iturinic antibiotics.     

Chemical synthesis and biological evaluation of ω-hydroxy polyunsaturated fatty acids

ω-Hydroxy polyunsaturated fatty acids (PUFAs), natural metabolites from arachidonic acid (ARA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were prepared via convergent synthesis approach using two key steps: Cu-mediated CC bond formation to construct methylene skipped poly-ynes and a partial alkyne hydrogenation where the presence of excess 2-methyl-2-butene as an additive that is proven to be critical for the success of partial reduction of the poly-ynes to the corresponding cis-alkenes without over-hydrogenation. The potential biological function of ω-hydroxy PUFAs in pain was evaluated in naive rats. Following intraplantar injection, 20-hydroxyeicosatetraenoic acid (20-HETE, ω-hydroxy ARA) generated an acute decrease in paw withdrawal thresholds in a mechanical nociceptive assay indicating pain, but no change was observed from rats which received either 20-hydroxyeicosapentaenoic acid (20-HEPE, ω-hydroxy EPA) or 22-hydroxydocosahexaenoic acid (22-HDoHE, ω-hydroxy DHA). We also found that both 20-HEPE and 22-HDoHE are more potent than 20-HETE to activate murine transient receptor potential vanilloid receptor1 (mTRPV1).     

Peroxisomal β-oxidation of fatty acids in bovine and rat liver

Hepatic peroxisomal β-oxidation rates were compared in liver homogenates from cows and rats during different nutritional and physiological states. Peroxisomal oxidation in liver homogenates from cows represented 50% and 77% of the total capacity for the initial cycle of β-oxidation of palmitate and octanoate, respectively, but only 26% and 65% for rats. Lactation or food deprivation did not alter rates of hepatic peroxisomal β-oxidation of palmitate or octanoate in cows. Fasting and clofibrate treatment increased rates of total and peroxisomal β-oxidation of palmitate and octanoate in rat liver.     

A method for the separation of peptides and α-amino acids

1. Peptides and alpha-amino acids, occurring in mixtures from various sources, can be separated into one fraction containing the amino acids and several peptide fractions. This is achieved by chelation of the mixture with Cu(2+) ions and subsequent chromatography of these chelates over the acetate form of diethylaminoethylcellulose or triethylaminoethylcellulose. 2. The amino acid fraction is obtained by elution with 0.01m-collidine-acetate buffer, pH8.0. 3. Peptide fractions are eluted with 0.01m-collidine-acetate buffer, pH4.5, 0.17n-acetic acid and 0.1n-hydrochloric acid respectively. 4. With the exception of aspartic acid and glutamic acid, which are partly found in the acidic peptide fraction, the amino acids are completely separated from the peptides. 5. Contamination of the acidic peptide fraction with glutamic acid and aspartic acid can be largely avoided by previous addition of an excess of arginine. 6. Copper is removed from the eluates by extraction with 8-hydroxyquinoline in chloroform.     

Separation and identification of lipids and fatty acids of the marine algaFucus vesiculosus by TLC and GC—MS

Ten fractions of polar and nonpolar lipids (phospholipids, glyeolipids and neutral lipids) were isolated from the brown marine algaFucus vesiculosus by TLC. In each fraction the quality and quantity of fatty acids were determined by GC—MS.F. vesiculosus is another type of marine alga suitable as a source of polyene fatty acids.     

Absorption of circularly polarizedγ-radiation in L- and D-amino acids

No difference within the experimental error ( 10³) was observed in the absorption of right- and left-circularly polarized-radiation in L- and D-tryptophan and tyrosine. For the measurements the 14.4 keV transition of⁵⁷Fe was used and the analysis of the circularly polarized-radiation was performed by Mössbauer method.     

Oxidation of ω-hydroxylated fatty acids and steroids by SS-isoenzyme of liver alcohol dehydrogenase

The SS-isoenzyme of alcohol dehydrogenase from horse liver was found to be active towards ω1- and ω2-hydroxylated fatty acids, an ω-hydroxylated steroid, ethanol and a 3β-hydroxysteroid. The main part of all these activities disappeared after carboxymethylation of a cysteineresidue at the active site of LADH_SS. The ω-hydroxyfatty acid dehydrogenase activity of LADH_SS was of similar magnitude as that of LADH_EE whereas the ω-hydroxysteroid dehydrogenase activity of LADH_SS was considerably higher than that of LADH_EE.