Synthesis and applications of oligoribonucleotides with selected 2''-O-methylation using the 2''-O-[1-(2-fluorophenyl)-4-methoxypiperidin-4-yl] protecting group.

The synthesis of base protected 5'-O-dimethoxytrityl-2'-O-[1-(2- fluorophenyl)-4-methoxypiperidin-4-yl]-3'-O-(2-cyanoethyl N,N-diisopropylphosphoramidites) is described, using phenoxyacetyl protection for the exocyclic amino groups of guanosine and adenosine and acetyl protection of the amino group of cytidine. High yield assembly of these building blocks into oligoribonucleotides on aminopropyl controlled pore glass was achieved using 5-(4-nitrophenyl)-1H-tetrazole as activator. Mixed sequences containing selected 2'-O-methylation were also synthesised and their significance for the study of RNA biochemistry is discussed.     

Chemical synthesis and biological activities of partially inosine-substituted 2',5'-oligoadenylates: functional discrimination of three adenine amino groups on 2-5A for the binding to and activation of 2-5A-dependent endonuclease

Binding and activation efficacies to the 2-5A-dependent endonucease by chemically synthesized partially inosine-substituted 2-5A analogs, namely, pppI2'p5'A2'p5'A, pppA2'p5'I2'p5'A and pppA2'p5'A2'p5'I were compared with that of native 2-5A in mouse L cell and human lymphoblastoid cell extracts. The results obtained in this study indicated that the first adenine amino group from the 5' terminus of 2-5A molecule plays critical role in binding to the endonuclease, whereas the third adenine amino group has a function for the activation of this enzyme.     

Mediated Na(+)-independent transport of L-glutamate and L-cystine in 1- and 2-cell mouse conceptuses.

L-Glutamate and L-cystine appeared to compete for transport via a mediated Na(+)-independent transport process in 1- and 2-cell conceptuses. Not only did these substances competitively inhibit each others' uptake by conceptuses, but their Ki values for inhibition were about equal to their Km values for transport in 1-cell conceptuses. Moreover, the transport process interacted strongly with L-amino acids that had 3-6 atoms in a chain between their negatively charged groups, whereas it interacted weakly or not at all with amino acids that did not have these characteristics or that were N-methylated. Transport of anionic amino acids was not altered greatly by pH in the range 4.5-8.0, but transport of L-cystine was much faster at higher pH values. The slower cystine transport at lower pH values was due primarily to protonation of its second amino group. A small increase in the degree of deprotonation of cystine's carboxyl groups also probably contributed slightly to its faster transport at higher pH values. By all of these criteria, the transport process in conceptuses appears to be a form of amino acid transport system xc-. System xc- activity was several-fold higher in 1- than in 2-cell conceptuses. Similarly, L-glutamate uptake by unfertilized eggs was relatively rapid, whereas it was much slower in immature, fully-grown oocytes. System xc- activity in 1-cell conceptuses also appeared to increase in response to the oxidative stress of culture, whereas no such increase was observed for 2-cell conceptuses. We suggest that this transient increase in the activity of system xc- activity during development of 2-cell conceptuses from immature, fully-grown oocytes could help protect unfertilized and fertilized eggs from oxidative stresses in situ.     

Synthesis of tetracontapeptide--a hypothetical evolutionary precursor of calcium-binding proteins. I. Synthesis of (1-9), (10-14), (15-20), (21-26), (29-33), (34-40) fragments

Fragments (1-9), (10-14), (15-20), (21-26), (29-33) and (34-40) of a tetracontapeptide hypothetical ancestor of calcium-binding proteins were synthesised with the use of pentafluorophenyl esters. Formation of a succinimide derivative was detected during synthesis of fragment (15-20) containing Asp(OBzl)-Gly sequence. To avoid this side process, tert-butylprotecting group was used instead of benzyl group. alpha-Carboxyls of C-terminal amino acids were protected by phenacyl group.     

Synthesis of enantiopure non-natural alpha-amino acids using tert-butyl (2S)-2-[bis-(tert-butoxycarbonyl)amino]-5-oxopentanoate as key-intermediate:the first synthesis of(S)-2-amino-oleic acid.

A general method for the synthesis of enantiopure non-natural alpha-amino acids is described. The key intermediate tert-butyl (2S)-2-[bis(tert-butoxycarbonyl)amino]-5-oxopentanoate was obtained from l-glutamic acid after suitable protection and selective reduction of the gamma-methyl ester group by DIBALH. Wittig reaction of this chiral aldehyde with various ylides led to a variety of delta,epsilon-unsaturated alpha-amino acids. This methodology was applied to the synthesis of (S)-2-amino-oleic acid.     

2-(4-Chlorophenyl)sulfonylethoxycarbonyl (Cps) amino-protecting group for the synthesis of peptides in a solution. Synthesis of [Leu]enkephalin and its [D-ala2]analog

Solution syntheses of [Leu]enkephalin and its [D-Ala2]analogue were accomplished using a new 2-(4-chlorophenyl)sulfonylethoxycarbonyl (Cps) base-labile group for amino protection and a chromogenic acid-labile 4-(4-phenylazo)benzyloxybenzyl (Abz) group for carboxyl protection. The syntheses were performed by stepwise attachment of Cps-amino acids, which were introduced as pentachlorophenyl esters or as dicyclohexylammonium salts in the presence of tris(dimethylamino)chlorophosphonium perchlorate. To remove Cps-group, Abz-esters of Cps-peptides were treated with two molar equivalents of 1,8-diazabicyclo[5.4.0]undec-7-ene in dimethylformamide followed by neutralization of the base with an excess of 1-hydroxybenzotriazole; the deblocked amino components were then used without isolation. Fully deblocked pentapeptides were purified and characterized by HPLC, FAB mass spectra and amino acid composition.     

Stereospecific synthesis of (2S)-2-methyl-3-(2',6'-dimethyl-4'-hydroxyphenyl)-propionic acid (Mdp) and its incorporation into an opioid peptide

To examine the effect of replacing the N-terminal amino group in opioid peptides with a methyl group on biological activity, a stereospecific synthesis of the tyrosine analogue (2S)-2-methyl-3-(2',6'-dimethyl-4'-hydroxyphenyl)-propionic acid (Mdp) was performed. The enkephalin analogue (2S)-Mdp-D-Ala-Gly-Phe-Leu-NH2 turned out to be a quite potent delta opioid antagonist and a somewhat less potent mu antagonist, indicating that a positively charged N-terminal amino group is not a conditio sine qua non for the binding of opioid peptides to delta and mu receptors but may be required for signal transduction.     

A novel 3-0 sulfatase from human urine acting on methyl-2-deoxy-2-sulfamino-α-D-glucopyranoside 3-sulfate

Human urine contains a novel sulfatase which is specific for the 3-0 sulfate ester of sulfaminoglucopyranoside 3-sulfate. Of the three isomeric sulfamate derivatives, 3,4 and 6-0 sulfate esters, only the 3-0 ester is hydrolyzed. Enzymatic activity requires that the amino group be sulfated; sulfate is not released if the amino group is free or acetylated. The enzyme has been purified 70-fold. It has a pH optimum of 6.3 and is inhibited by inorganic sulfate and phosphate. The specificity of this enzyme suggests that a 3-0 sulfated glucosamine moiety may have a role in the physiological activity of heparin or heparan sulfate.     

Influence of 1-[(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl]pyrrolidine-2-carboxylic acid and diphenyliodonium chloride on ruminal protein metabolism and ruminal microorganisms.

The effects of 1-[(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl]pyrrolidine-2-carboxy lic acid (LY29) and diphenyliodonium chloride (DIC) on the degradation of protein to ammonia were determined in a mixed rumen microbial population taken from sheep on a grass hay-concentrate diet. Both compounds decreased NH3 production by inhibiting deamination of amino acids. LY29, but not DIC, inhibited growth of the high-activity ammonia-producing species, Clostridium aminophilum and Clostridium sticklandii.     

Iso-lactam and reduced amide analogues of the peptidomimetic dopamine receptor modulator 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide

An analogue of the highly potent gamma-lactam Pro-Leu-Gly-NH(2) peptidomimetic, 3(R)-[(2(S)-pyrrolidinylcarbonyl) amino]-2-oxo-1-pyrrolidineacetamide (2), 4(R)-[[2(S)-pyrrolidinylcarbonyl]amino]-2-oxo-1-pyrrolidineacetamide (3), in which the lactam carbonyl moiety has been placed in a different position with respect to the 3-amino group was synthesized. Also, a series of analogues of 2, compounds 4-6, were synthesized in which each of the amide bonds of 2 were systematically replaced with a reduced amide bond surrogate. The analogues were tested for their ability to enhance the binding of [3H]N-propylnorapomorphine to dopamine receptors in a functional in vitro assay utilizing bovine striatal membranes. Peptidomimetic 3 was shown to be more potent than 2, while 4 and 5 were significantly less effective than 2. Peptidomimetic 6 had a pharmacological profile similar to that of 2.     

Design and synthesis of new 2-substituted-5-(2-benzylthiophenyl)-1,3,4-oxadiazoles as benzodiazepine receptor agonists

A series of new 2-substituted-5-(2-benzylthiophenyl)-1,3,4-oxadiazoles was designed and synthesized as anticonvulsant agents. Conformational analysis and superimposition of energy minima conformers of the designed molecules on estazolam, a known benzodiazepine receptor agonist, revealed that the main proposed benzodiazepine pharmacophores were well matched. Electroshock and pentylenetetrazole-induced lethal convulsion tests showed that the introduction of an amino group in position 2 of 1,3,4-oxadiazole ring and a fluoro substituent at para position of benzylthio moiety had the best anticonvulsant activity. It seems this effect is mediated through benzodiazepine receptors mechanism.     

Cloning and expression of fructosyl-amino acid oxidase gene from Corynebacterium sp. 2-4-1 in Escherichia coli

The gene encoding the fructosyl-amino acid oxidase (fructosyl-alpha-L-amino acid: oxygen oxidoreductase (defructosylating); EC 1.5.3) of Corynebacterium sp. 2-4-1 was cloned and expressed in Escherichia coli. The gene consists of 1,116 nucleotides and encodes a protein of 372 amino acids with a predicted molecular mass of 39,042. The open reading frame was confirmed as the gene of the fructosyl-amino acid oxidase by comparison with the N-terminal amino acid sequence of the purified fructosyl-amino acid oxidase from Corynebacterium sp. 2-4-1. The sequence of the AMP-binding motif, GXGXXG, was found in the deduced N-terminal region. The amino acid sequence of the fructosyl-amino acid oxidase showed no similarity to that of fungal fructosyl-amino acid oxidases. In addition, substrate specificities of this fructosyl-amino acid oxidase were different from those of other fructosyl-amino acid oxidases. The fructosyl-amino acid oxidase of Corynebacterium sp. 2-4-1 is an enzyme that has unique substrate specificity and primary structure in comparison with fungal fructosyl-amino acid oxidases.     

X-ray diffraction study of the interaction between carboxypeptidase A and (S)-(+)-1-amino-2-phenylethyl phosphonic acid.

The structure of the carboxypeptidase A complex with the inhibitor (S)-(+)-1-amino-2-phenylethylphosphonic acid has been determined at 0.23 nm resolution. The delta F map shows electron-density peaks both in the S1 and S'1 sites, where the inhibitor molecule can be modeled in two different orientations with approximate 50% occupancy. In the proposed model, the phosphonate group binds to the zinc ion in a monodentate fashion. Other anchoring groups for the inhibitor molecule are Arg127 (hydrogen bonds with the phosphonate oxygen atoms) and Glu270 (hydrogen bond with the amino group in one of the two orientations). A recent spectroscopic investigation of the complex between cobalt(II) carboxypeptidase A and (S)-(+)-1-amino-2-phenylethylphosphonic acid is essentially in agreement with our results.     

Reprint of "effect of carbohydrate amino group modifications on the cytotoxicity of glycosylated 2-phenyl-benzo[b]thiophenes and 2-phenyl-benzo[b]furans" [Bioorg. Med. Chem. Lett. 21 (2011) 2591-2596

In previous studies, we have identified a family of benzo[b]furan and benzo[b]thiophene derivatives linked to amino sugars (1-6) that are cytotoxic to a range of cancer cell lines. We describe here an exploration of the effect of structural modification of the amino group on one of the carbohydrate residues (4-amino-2,3,4,6-tetradeoxy-α-l-threo-hexopyranoside) on in vitro cytotoxicity. It has been found that maintaining at least one basic functional group around the C-4 position in the carbohydrate moiety is crucial for cytotoxicity. Furthermore, it appears that modifications around the C-4 position are limited by suitable hydrophilic/hydrophobic and/or ionic interactions, as well as steric constraints.     

Discovery and pharmacological characterization of N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide hydrochloride (anagliptin hydrochloride salt) as a potent and selective DPP-IV inhibitor

In the course of our program for discovery of novel DPP-IV inhibitors, a series of pyrazolo[1,5-a]pyrimidines were found to be novel DPP-IV inhibitors. We identified N-[2-({2-[(2S)-2-cyanopyrrolidin-1-yl]-2-oxoethyl}amino)-2-methylpropyl]-2-methylpyrazolo[1,5-a]pyrimidine-6-carboxamide hydrochloride (4a) and described its pharmacological profiles.     

Synthesis of an aza analogue of 2-deoxy-D-ribofuranose and its homologues.

Azasugars were obtained in one-pot reactions by catalytic reduction reactions of amino group precursors in aldosugars followed by intramolecular reductive amino alkylation reactions. (3R,4S)-4-[(1S)-1,2-Dihydroxyethyl]pyrrolidin-3-ol was obtained from D-xylose by two different strategies through 3-C-cyano-3-deoxy-D-ribo-pentofuranose or 3-C-azidomethyl-3-deoxy-D-ribo-pentofuranose in 6 and 16% overall yields, respectively. The oxidative cleavage of the diol group in the corresponding Fmoc-azasugar followed by deprotection afforded (3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol. (3R,4S)-4-[(1S,2R)-1,2,3-Trihydroxypropyl]pyrrolidin-3-ol was synthesized from diacetone-D-glucose through 3-deoxy-3-C-nitromethyl-D-allose and the overall yield was 7%.     

2-(Aminomethyl)-2-decarboxyprostaglandin F2 alpha type analogs

The 2-(aminomethyl)-2-decarboxy analogs of prostaglandin F2 alpha (PGF2 alpha), (15S)-15-methyl-PGF2 alpha, 16-phenoxy-omega-tetranor-PGF2 alpha and 16,16-dimethyl-PGF2 alpha were synthesized. The amino analogs closely resemble the parent PGF2 alpha compounds as antifertility agents in the hamster.     

2-(Substituted phenyl)amino analogs of 1-methoxyspirobrassinol methyl ether: Synthesis and anticancer activity

New analogs of indole phytoalexin 1-methoxyspirobrassinol methyl ether have been designed by replacement of its 2-methoxy group with 2-(substituted phenyl)amino group. Synthesized by spirocyclization methodology, trans- and cis-diastereoisomers of target compounds were isolated and evaluated as potential anticancer and antimicrobial agents. Their molecular geometries were refined by ab initio minimizations. Pharmacophore modeling and QSAR studies were performed in order to correlate their molecular structure and biological activity.     

Synthesis of cystine peptides 21-25/70-73 and 35-39/56-59 of the beta-subunit of human choriogonadotropin.

Syntheses of two asymmetrical cystine peptides with the amino acid residues 21-25/70-73 and 35-39/56-59, based on the linear amino acid sequence and the disulfide bond assignment in the beta-subunit of human choriogonadotropin (hCG-beta), are described. S-trityl and S-acetamidomethyl peptide fragments of each cystine peptide were prepared in solution phase and were subjected to oxidation with I2/MeOH to form the disulfide bridge. The cystine peptides were characterized by their amino acid analyses and fast atom bombardment mass spectrometry. Immunological characterization by several homologous radioimmunoassay systems showed that peptide 21-25/70-73 had significant hCG, hCG-beta, and hLH activities while peptide 35-39/56-59 failed to reveal any immunoreactivity.     

Molecular cloning and expression of human neurochondrin-1 and -2.

Human neurochondrins have been cloned from a brain cDNA library. The human neurochondrin-1 and -2 predict leucine-rich (15.8 and 15.9%) proteins of 729 and 712 amino acid residues, with molecular weights of 78.9 and 77.2 kDa, respectively. The deduced amino acid sequence indicates 98% identity among human, mouse and rat species. Northern analysis indicates that about 4 kb human neurochondrin mRNAs are abundant in the fetal and the adult brain.