Abstract  Qualitative and quantitative analysis of dehydroepiandrosterone and its conjugates in biological matrices and establishment of their relationships with physiological functions is a very active field. This review article discusses methods of separation and quantification of dehydroepiandrosterone and its conjugates using high-performance liquid chromatographic techniques.

Abstract  The oligosaccharide beta-D-Galf-(1-->3)-alpha-D-Manp-(1-->2)-[beta-D-Galf- (1-->3)]-alpha-D-Manp-(1-->2)-alpha-D-Manp corresponds to the terminal end of the glycosylinositolphospholipid oligosaccharide of the protozoan Trypanosoma cruzi, the causative agent of Chagas' disease. Syntheses of methyl or ethylthio glycosides of the terminal disaccharide, trisaccharide, tetrasaccharide, and pentasaccharide corresponding to this structure are described. These syntheses employ the selective activation of a phenyl 1-selenogalactofuranoside or a phenyl 1-selenomannopyranoside donor over ethyl 1-thioglycoside acceptors with NIS-TfOH.

Abstract  A new approach for removing O-glycosidically linked carbohydrate side chains from glycoproteins is described. Periodate oxidation of the C3 and C4 carbons in peptide-linked N-acetylgalactosamine (GalNAc) residues generates a dialdehyde product which, under mild alkaline conditions, undergoes a beta-elimination which releases carbohydrate and leaves an intact peptide core. The pH and time dependence, and intermediates of the elimination, have been extensively followed by carbon-13 NMR spectroscopy and amino acid analysis using ovine submaxillary mucin (OSM) as the substrate. The deglycosylation of OSM is complete and provides apomucin in high yield with an amino acid composition identical to the starting material. Carboxymethylated OSM when deglycosylated by this method gives an apomucin with an apparent molecular weight of ca. 700 x 10(3). The molecular weight is the same as that calculated for the peptide core of the starting mucin, demonstrating the absence of peptide core cleavage. This contrasts with the use of trifluoromethanesulfonic acid (TFMSA), which generates apomucin products of lower molecular weights. Oligosaccharide side chains substituted at C3 of the peptide-linked GalNAc residue are resistant to the oxidation and elimination. Glycoproteins containing these more complex side chains can be deglycosylated by pretreatment with TFMSA under mild (0 degree C) conditions, which removes peripheral sugars (while leaving the peptide-linked GalNAc residue intact), followed by oxidation and beta-elimination. Studies on the deglycosylation of porcine submaxillary mucin and human tracheobronchial mucin indicate that this approach provides more efficient removal of carbohydrate and less peptide core degradation than a more vigorous (25 degrees C) treatment with TFMSA alone. 13C NMR spectroscopic studies and carbohydrate analysis of the deglycosylation intermediates of the human mucin indicate that certain sialic acid containing and N-acetylglucosamine-containing oligosaccharides have elevated resistance to TFMSA treatment at 0 degrees C. By the use of neuraminidase, repeated mild TFMSA treatments, and multiple oxidations and beta-eliminations, the human mucin can be nearly completely deglycosylated. It is expected that all mucins and most glycoproteins containing O-glycosidic linkages can be readily and nearly completely deglycosylated using this combined approach.

Abstract  A procedure to map N-glycosylation sites is presented here. It can be applied to purified proteins as well as to highly complex mixtures. The method exploits deglycosylation by PNGase F in a diagonal, reverse-phase chromatographic setup. When applied to 10 microL of mouse serum, affinity-depleted for its three most abundant components, 117 known or predicted sites were mapped in addition to 10 novel sites. Several sites were detected on soluble membrane or receptor components. Our method furthermore senses the nature of glycan structures and can detect differential glycosylation on a given site. These properties--high sensitivity and dependence on glycan imprinting--can be exploited for glycan-biomarker analysis.

Abstract  Phospholipase A2 inhibitor (PLI), purified from the blood plasma of the Habu snake (Trimeresurus flavoviridis), was separated into two distinct subunits, PLI-A and PLI-B. These subunits were shown to be glycoproteins with molecular weights of around 21,000-22,000. When they were deglycosylated chemically with trifluoromethanesulfonic acid, the molecular weights were found to be 17,000. Their amino acid sequences were determined by alignment of peptides obtained by lysyl endopeptidase digestion and Staphylococcus aureus V8 protease digestion. PLI-A and PLI-B were each composed of 147 amino acid residues with one residue, Asn103, being for N-linked glycosylation, and the molecular weights of their protein portions were calculated to be 16,368 and 16,408, respectively. Each subunit contained four cysteine residues, all of which exist in disulfide linkages (Cys64-Cys141 and Cys119-Cys133). The sequences of PLI-A and PLI-B showed 89.9% homology to each other. When the sequences were compared with those of lipocortins, no significant homologies were detected. But the sequences were significantly homologous to those of COOH-terminal carbohydrate recognition portions of pulmonary surfactant apoprotein and animal lectins.

Abstract  To study zona pellucida antigens involved in human fertilization, five monoclonal antibodies (MAbs)--2A1, 2G3, 4A2, 4E12, and 5H4--were produced to a glycoprotein family (ZP4) isolated from heat-solubilized porcine zonae pellucidae. Each MAb reacted not only with solubilized porcine zona glycoproteins but also with the glycoproteins deglycosylated by trifluoromethanesulfonic acid treatment. They also reacted with intact zonae pellucidae of porcine and human oocytes. Three (4A2, 4E12, and 5H4) of the five MAbs showed a significant blocking effect on human sperm binding and penetration of human zonae pellucidae. The 5H4 MAb showed a strong reaction with ZP4 and ZP1 glycoprotein families of porcine zonae pellucidae, and four other MAbs reacted more strongly with ZP3 than with ZP4. The reactivity of 5H4 with porcine zona glycoproteins was destroyed by chymotrypsin digestion, but the antigen epitope was resistant to proteolysis by trypsin and endoproteinase Lys-C. A peptide fragment reactive to 5H4 was isolated by reverse-phase HPLC from endoproteinase Lys-C-treated ZP4 glycoproteins, and its molecular mass was determined to be 7 kDa by SDS-PAGE. These results suggested that the antigen epitope corresponding to 5H4 is a good candidate for development of a contraceptive vaccine.

Abstract  4-(p-Chlorophenyl)tetralone (6) and 7-chloro-5-(p-chlorophenyl)tetralone (9) are key intermediates for the development of benzazepinone derivative haftens. These compounds could be synthesized from 4-phenyltetralone derivatives by triflic acid catalyzed Friedel-Crafts reaction. The reaction mechanism of Friedel-Crafts alkylation/acylation with lactones in triflic acid is presented. According to our tentative research, ring opening of protonated lactone (2) occurs in alkyl cleavage and the rate of the reaction is not dependent on concentration of triflic acid. So, alkylation of lactone in Friedel-Crafts reaction is presumed to be A(AL)1. In second step, intramolecular acylation of the intermediates 4 to 6, 4 can be transformed to a triflic acid-carboxylic anhydride and then the cyclization is undergone after leaving of the triflate anion.

Abstract  The iron-copper dinuclear active site in heme-copper oxidases (e.g., cyctochrome c oxidase) has spurred the development of the inorganic: chemistry of bridged heme-copper complexes, including species possessing (porphyrinate)Fe(III)-O(H)-Cu(II)-L moieties. We describe here the synthesis, by two routes, of [(F(8)TPP)Fe(III)-O-Cu(II)(MePY2)](+) (5) {F(8)TPP = tetrakis(2,6-difluorophenyl)porphyrinate; MePY2 = N,N-bis[2-(2-pyridyl)ethyl] methylamine). First, 5-(CF(3)SO(3)) was generated by reaction of [(MePY2)Cu(II)](CF(3)SO(3))(2) (3-(CF(3)SO(3))(2)) and [(F(8)TPP)Fe(III)-OH] (4) with triethylamine in THF or CH(3)CN in 65-70% yield. The complex was also prepared by reduction of O(2) by a 1:1 mixture of copper(I) and iron(II) complexes, [(MePY2)Cu(I)(CH(3)CN)](BArF) (1-(BArF)) (BArF = tetrakis(3,5-bis-trifluoromethylphenyl)borate) and (F(8)TPP)Fe(II) (2) in O(2)-saturated THF or acetone, at -80 degrees C with subsequent warming to room temperature. Preliminary stopped-flow kinetics on the O(2) reaction with the 1:1 mixture show the formation of at least two intermediates (i.e., a superoxo species (F(8)TPP)Fe-O(2) first, and then a presumed peroxo-bridged Fe-O(2)-Cu species) prior to the formation of the final mu-oxo complex [(F(8)TPP)Fe(III)-O-Cu(II)(MePY2)](+) (5-(BArF)). The (1)H NMR spectrum of 5-(CF(3)SO(3)) in CD(2)Cl(2) exhibits a pyrrole peak at 67.7 ppm (corroborated by (2)H NMR), while downfield (23.4 and 18.9 ppm) and dramatically upfield-shifted resonances (-87.7, -155.4 and -189.4) have been assigned to hydrogens of the MePY2 moiety, by specific deuteration. The mu-hydroxo complex [(F(8)TPP)Fe-(OH)-Cu(MePY2)](OTf)(2) (6-(CF(3)SO(3))(2)) was synthesized by addition of 3-(CF(3)SO(3))(2) to 4 in CH(3)CN, or by protonation of 5-(CF(3)SO(3)) with CF(3)SO(3)H. In a (1)H. NMR-spectroscopic protonation titration (CF(3)SO(3)H)(2) the pyrrole 67.7 ppm resonance for 5-(CF(3)SO(3)) progressively converts to 70.3 ppm, diagnostic of 6-(CF(3)SO(3))(2). The protonation is slow on the NMR time scale. The (1)H NMR spectral properties are consistent with antiferromagnetically coupled high-spin iron(III) and Cu(II) ions (S = 2 spin state), and the interaction is weaker in 6-(CF(3)SO(3))(2) (5-(CF3SO3), mu(eff) = 5.05 mu(B); 6-(CF(3)SO(3))(2), mu(eff) = 5.60 mu(B); Evans NMR method). By titration using a series of organic acids, the pK(a) for 6-(CF3S03)2 has been estimated to be 16.7 < pK(a) < 17.6 (CH(3)CN solvent), or 9.6 +/- 2 (aqueous). Plots of delta vs 1/T for both mu-oxo and mu-hydroxo complexes 5-(CF(3)SO(3)) and 6-(CF(3)SO(3))(2) have been obtained, showing linear Curie (for downfield resonances) or anti-Curie (for upfield peaks) behavior.

Abstract  Microtubules are a target for a broad spectrum of drugs used as chemotherapeutics to treat hematological malignancies and solid tumors. Most of these drugs have significant dose-limiting toxicities including peripheral neuropathies that can be debilitating and permanent. In an ongoing effort to develop safer and more effective drugs, benzimidazole-based compounds are being developed as replacement for vincristine and similar agents. In this report, we describe radiosyntheses of novel microtubule-targeting methyl N-[5-(3'-radiohalobenzoyl)-1H-benzimidazol-2-yl]carbamates 4 that are intended as potential imaging agents and molecular radiotherapeutics. 125 I- and 131 I-radiolabeled derivatives were prepared either by direct radioiodination of methyl N-(6-benzoyl-1H-benzimidazol-2-yl)carbamate 1 or radioiododestannylation of the corresponding stannane precursor 3. The direct radioiodination was conducted in a solution of 1 in triflic acid and produced after ~1 hour at elevated temperatures and HPLC purification on average 62% of the no-carrier added products 125 I-4 and 131 I-4. Radioiododestannylation of 3'-trimethylstannane 3 proceeded with ease at room temperature in the presence of H2 O2 as the oxidant and produced no-carrier-added 125 I-4 and 131 I-4 in high isolated yields, on average 85%. The radiohalodestannylation protocol is universal and can be applied to other radiohalides including 124 I to produce 124 I-4, a positron emission tomography agent, and 211 At to produce 211 At-4, an α-particle emitting radiotherapeutic.

Abstract  OBJECTIVE(S): Chalcones and their rigid analogues represent an important class of small molecules having anticancer activities. Therefore, in this study the synthesis and cytotoxic activity of new 3-benzylidenchroman-4-ones were described as rigid chalcone analogues.

MATERIALS AND METHODS: The reaction of resorcinol with 3-chloropropionic acid in the presence of CF3SO3H was afforded corresponding propiophenone. It was cyclized using 2M NaOH to give 7-hydroxy-4-chromanone. O-Alkylation of 7-hydroxy-4-chromanone with alkyl iodide in the presence of K2CO3 gave 7-alkoxychroman-4-one. Finally, condensation of chroman-4-one derivatives with different aldehydes afforded target compounds in good yields. The newly synthesized compounds were tested in vitro against different human cancer cell lines including K562 (human erythroleukemia), MDA-MB-231 (human breast cancer), and SK-N-MC (human neuroblastoma) cells. The cell viability was evaluated using MTT colorimetric assay.

RESULTS: Most of the compounds showed good inhibitory activity against cancer cells. Among them, compound 4a containing 7-hydroxy group on chromanone ring and 3-bromo-4-hydroxy-5-methoxy substitution pattern on benzylidene moiety was the most potent compound with IC50 values ≤ 3.86 µg/ml. It was 6-17 times more potent than etoposide against tested cell lines.

CONCLUSION: We described synthesis and cytotoxic activity of poly-functionalized 3-benzylidenechroman-4-ones as new chalcone-like agents. These compounds can be considered as conformationally constrained congeners of chalcones to tolerate the poly-functionalization on the core structures for further optimization.

Abstract  STUDY OBJECTIVE: To study carbohydrate natures in the antigen epitopes corresponding to sperm-immobilizing antibodies in infertile women.

DESIGN: Antibody absorption with human sperm and seminal plasma before and after treatments with trifluoromethanesulfonic acid or sodium metaperiodate.

PATIENTS: Thirty-three patients who showed a positive sperm immobilization test provided their sera for the experiment.

RESULTS: In 25 patients' sera whose sperm-immobilizing antibodies were absorbed with human seminal plasma, the antibody absorbing capabilities were completely abolished by deglycosylation treatment with trifluoromethanesulfonic acid. The sperm-immobilizing antibodies in 4 patients' sera were absorbed out with sperm membrane fraction before the treatment but not after the treatment with trifluoromethanesulfonic acid. In some patients' sera, the antibody-absorbing capabilities of ejaculated sperm were markedly reduced by sodium metaperiodate treatment.

CONCLUSION: The majority of sperm-immobilizing antibodies in infertile patients might be generated to carbohydrate structures of the sperm-coating antigens or sperm membrane antigens.

Abstract  Nonsymmetrical furanose-pyranose difructose dianhydrides (DFAs), a class of cyclic disaccharides present in foodstuffs, have been prepared in high yield by connecting the reacting monosaccharide moieties through a xylylene bridge prior to triflic acid-promoted bis-spiroketalization. The reaction can then proceed either intra- or intermolecularly, both the regio- and the stereoselectivity being strongly dependent on the spacer length. Noteworthy, the longer m- and p-xylylene positional isomers led to the thermodynamic alpha-D-fructofuranose beta-D-fructopyranose 1,2':2,1'-dyanhydride 1, the major DFA in commercial caramel, in a stereoselective manner. The shorter o-xylylene tether afforded preferentially the elusive contra-thermodynamic beta-D-fructofuranose alpha-D-fructopyranose diastereomer 2, a trace constituent of caramel. The results have been rationalized in terms of stereoelectronic and conformational properties and offer new perspectives for the preparation of pure DFA standards for analytical and nutritional studies. (C) 2008 Elsevier Ltd. All rights reserved.

Abstract  The disaccharides allyl beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta- and alpha-D-galactopyranoside 10a and 10b and the trisaccharides allyl 2-O-methyl-alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta- and alpha-D-galactopyranoside 18a and 18b have been prepared using stepwise assembly of the sugar units. The glycosidic linkages were formed employing the trichloroacetimidate procedure for the attachment of the galactopyranosyl residue and N-iodosuccinimide/triflic acid activation of an ethyl 1-thiofucopyranoside donor for fucosylation. Deprotection furnished the allyl glycosides which were converted into cysteamine-spacered ligands, activated with thiophosgene and subsequently linked to bovine serum albumin. The neoglycoproteins serve as immunoreagents to determine epitope specificities of monoclonal antibodies directed against highly immunogenic O-glycans located at the surface of Toxocara larvae.