Abstract  Maltase-glucoamylase (MGA) was immunoprecipitated from detergent extracts of brush border membranes of the human small intestinal mucosa. Electrophoretic analysis of the precipitates under denaturing conditions revealed a single polypeptide of Mr = 335,000 in the presence or absence of reducing agents. Cross-linking of brush border membranes with the homobifunctional reagent dithiobis(succinimidylpropionate) did not result in considerable changes in the electrophoretic pattern of MGA. In contrast, aminopeptidase N, used in these studies as a control glycoprotein of the brush border membrane revealed dimeric structures of its single subunit in the presence of dithiobis(succinimidylpropionate). These data suggest that MGA is expressed in the human small intestinal brush border as a monomeric polypeptide. The biosynthesis of MGA was studied by pulse-labeling of human intestinal biopsy specimens or mucosal explants in organ culture. Continuous labeling with [35S]methionine for 30 min revealed a single polypeptide high mannose precursor of Mr = 285,000 (MGAh) which matures after 4 h of labeling to the Mr = 335,000 as judged by the susceptibility of these two forms to endo-beta-N-acetylglucosaminidase H. Owing to the absence of pancreatic secretions in the culture medium and the isolation of an identical species from nonlabeled mucosa, this result indicates that the Mr = 335,000 does not undergo an in situ extracellular cleavage by intraluminal proteases. Further, biosynthetically labeled, intracellularly cleaved polypeptides corresponding to the high mannose precursor or mature forms of MGA were not detected. The mature form of MGA (MGAm) bears in addition to N-linked glycans also O-glycosidically linked oligosaccharides. In fact, endo-beta-N-acetylglucosaminidase F/glycopeptidase F treatment of MGAm followed by chemical deglycosylation with trifluoromethanesulfonic acid revealed approximately 35,000 daltons of O-linked sugars. Furthermore, MGAm as well as its N-linked sugars-depleted form bound to Helix pomatia lectin which has specificity toward Gal-GalNAc structures. In addition, the data were suggestive of a post-translational O-glycosylation of the molecule since (i) the high mannose precursor of MGA did not bind to H. pomatia lectin and (ii) its endo-beta-N-acetylglucosaminidase H or endo-beta-N-acetylglucosaminidase F/glycopeptidase F form displayed an apparent molecular weight similar to that obtained upon endo-beta-N-acetylglucosaminidase F/glycopeptidase F/trifluoromethanesulfonic acid deglycosylation. Finally, pulse-chase experiments revealed a relatively slow rate of post-translational processing of MGA in comparison to aminopeptidase N.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract  The sequence-specific O-linked core 1 ([R1, R2]-beta-Gal(1-3)-alpha-GalNAc-O-Ser/Thr) glycosylation pattern has been quantitatively determined for 30 of the 31 Ser/Thr residues in the 81-residue porcine submaxillary gland mucin tandem repeat. This was achieved by Edman amino acid sequencing of the isolated tandem repeat after selective removal of non-C3-substituted, peptide-linked GalNAc residues by periodate oxidation and subsequent trimming of the remaining oligosaccharides to peptide-linked GalNAc residues by mild trifluoromethanesulfonic acid/anisole treatment. The sequencing reveals 61% (range, 12-95%) of the peptide alpha-N-acetylgalactosamine (GalNAc) residues to be substituted by core 1 chains, a value in agreement with the carbon-13 NMR analysis of the native mucin. Residues with the lowest C3 substitution were typically clustered in regions of sequence with the highest densities of (glycosylated) serine or threonine. This suggests that the porcine beta3-Gal, core 1, transferase is sensitive to peptide sequence and/or neighboring core GalNAc glycosylation in vivo, in keeping with earlier in vitro enzymatic glycosylation studies (Granovsky, M., Blielfeldt, T., Peters, S., Paulsen, H., Meldal, M., Brockhausen, J., and Brockhausen, I. (1994) Eur. J. Biochem. 221, 1039-1046). These results demonstrate that the O-glycan structures in mucin domains are not necessarily uniformly distributed along the polypeptide core and that their lengths can be modulated by peptide sequence. The data further suggest that hydroxyamino acid spacing may contribute to the regulation of glycan length, thereby, providing a mechanism for maintaining an optimally expanded, protease resistant, mucin conformation.

Abstract  The extension of the pyrene ring from dimethyl 2,2'-(pyrene-1,6-diyl)dibenzoate derivatives by an intramolecular Friedel-Crafts acylation can be realized in an efficient and regioselective manner using triflic acid as proton source. Naphtho-tetracenone derivatives are obtained in high yields at room temperature while Bis-tetracene-diones are prepared upon heating. Both products display interesting fluorescence properties in the visible range with quantum yields varying from 50 to 60 %.

Abstract  Solvent-free per-O-acetylation of hexoses with a stoichiometric amount of acetic anhydride employing 0.03 mol % Cu(OTf)2 proceeded in high yields (90-99%) at room temperature to give exclusively pyranosyl products as an anomeric mixture, the alpha/beta ratio of which was dependent on the temperature and amount of catalyst used. Sequential anomeric substitution with p-thiocresol in the presence of BF3.etherate gave the thioglycosides, isolated exclusively or predominantly as one anomer in 66-75% yields.

Abstract  Treatment of N-acyloxazolidinones with hydroxylamines using samarium triflate as a Lewis acid provides the corresponding hydroxamic acids in 50-98% yields at room temperature. The conversion proceeds with high degree of chemoselectivity and without racemization of chiral centers alpha- to the acyl group. [reaction: see text]

Abstract  It is demonstrated that the beta-selectivity observed in the insoluble silver salt mediated couplings of 2,3-O-carbonate-protected rhamnosyl bromides is uniquely due to the heterogeneous nature of the reaction. In homogeneous solution these same donors are alpha-selective, a fact that is attributed to the half-chair conformation of these substances which reduces the energy barrier to oxacarbenium ion formation. It is suggested that the 2,3-O-carbonate group be dubbed torsionally arming in the manno- and rhamnopyranose series. When the carbonate group is removed to the 3,4-O-position a beta-selective system is formed, in both homogeneous and heterogeneous conditions, and it is demonstrated that this selectivity arises from the combination of the electron-withdrawing nature of the carbonate and its inability to take part in neighboring participation.

Abstract  This paper describes the superacid-catalyzed chemistry of olefinic amines and related compounds. A variety of olefinic amines are found to react with benzene in CF(3)SO(3)H (triflic acid) to give addition products in good yields (75-99%), including the pharmaceutical agents fenpiprane and prozapine. A general mechanism is proposed that invokes the formation of reactive, dicationic electrophiles and the direct observation of a diprotonated species is reported from low-temperature NMR experiments. This chemistry is also used to conveniently prepare functionalized polystyrene beads having pendant amine groups.

Abstract  Direct chlorination of 1-CH(3)-CB(11)H(11)(-) in glacial acetic acid gave the highly chlorinated carborane anion 1-CH(3)-CB(11)Cl(11)(-), and treatment of 1-CH(3)-CB(11)H(11)(-) with ICl in triflic acid afforded the highly iodinated carborane anion 1-CH(3)-CB(11)I(11)(-). Under similar or more vigorous reaction conditions, however, the reaction of 1-CH(3)-CB(11)H(11)(-) with Br(2) in triflic acid did not proceed to completion. The highly brominated carborane anion 1-CH(3)-CB(11)Br(11)(-) was achieved via a sealed-tube reaction. This new method has led to the isolation of 1-H-CB(11)X(11)(-) (X = Cl, Br, I) and 1-Br-CB(11)Br(11)(-) in high yield. The lithiation of 1-H-CB(11)X(11)(-) resembles that of its parent anion CB(11)H(12)(-). Treatment of these lithio species with methyl iodide gave the methylated carborane anions 1-CH(3)-CB(11)X(11)(-). These new weakly coordinating anions were fully characterized by (1)H, (13)C, and (11)B NMR, IR, and negative-ion MALDI MS spectroscopy. Some were further confirmed by single-crystal X-ray analysis.

Abstract  A general method for the N-arylation of amino acid esters with aryl triflates is described. Both α- and β-amino acid esters, including methyl, tert-butyl, and benzyl esters, are viable substrates. Reaction optimization was carried out by design of experiment (DOE) analysis using JMP software. The mild reaction conditions, which use t-BuBrettPhos Pd G3 or G4 precatalyst, result in minimal racemization of the amino acid ester. This method is the first synthetic application of the t-BuBrettPhos Pd G4 precatalyst. Mechanistic studies show that the observed erosion in enantiomeric excess is due to racemization of the amino acid ester starting material and not of the product.

Abstract  The carbohydrate-protein linkage of the allergen Ag-54 in the mould Cladosporium herbarum was studied after alkaline-borohydride treatment of the glycoprotein. By incorporating tritium into the peptide-linked monosaccharide through the alkaline scission of the linkage, it was demonstrated that the highly branched galactomannan moiety of Ag-54 was linked to the protein through mannose. Threonine was identified as the carbohydrate-linked amino acid. In addition, a few glucose units were peptide-linked, but the corresponding amino acids were not identified. When deglycosylated with trifluoromethanesulfonic acid, Ag-54 retained only 5% of its allergenic activity as compared to the native Ag-54 when tested by rocket immunoelectrophoresis and autoradiography. Immunological identity between native and deglycosylated Ag-54 was observed. Deglycosylation also resulted in reduced stability towards denaturation by heat or urea.

Abstract  Trimethylsilyl ethers of 1,5-diaryl-3-(trifluoromethyl)pent-1-en-4-yn-3-oles in superacid CF3SO3H (TfOH) give rise to the corresponding intermediate CF3-pentenynyl cations. These species react with benzene to afford conjugated CF3-pentenynes, which undergo subsequent cyclization, first, into CF3-cycloheptadienes and, finally, into unusual CF3-"helicopter"-like bicyclic structures.

Abstract  Coordination-driven self-assembly of organometallic η6-arene ruthenium(ii) supramolecular architectures (MA1-MA4) was carried out by employing dinuclear ruthenium acceptors [Ru2(μ-η4-C2O4)(CH3OH)2(η6-p-cymene)2](CF3SO3)2 (Rua), [Ru2(μ-η4-C6H2O4)(CH3OH)2(η6-p-cymene)2](CF3SO3)2 (Rub), [Ru2(dhnq)(H2O)2(η6-p-cymene)2](CF3SO3)2 (Ruc) and [Ru2(dhtq)(H2O)2(η6-p-cymene)2](CF3SO3)2 (Rud) separately with a new tetratopic donor (TD) in methanol at room temperature [TD = N,N,N',N'-tetra(pyridin-4-yl)-[1,1'-biphenyl]-4,4'-diamine]. All the coordination architectures were characterized by using spectroscopic techniques. The potency of these self-assembled architectures against human cervical cancer HeLa and human lung adenocarcinoma A549 cell lines is explored in vitro using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), annexin V-FITC/PI and 2',7'-dichlorofluorescein-diacetate assays.

Abstract  Stereoselective allylations of carbonyl compounds such as aldehydes and ketones are useful but challenging reactions in organic chemistry. The resulting chiral secondary and tertiary homoallylic alcohols or ethers are valuable building blocks in the synthesis of biologically active natural compounds and pharmaceuticals. Although researchers have developed several methods for the facially selective allylation of aldehydes, the stereoselective allylation of ketones still poses a severe problem. We have developed a highly diastereoselective domino multicomponent allylation reaction of a ketone and allyltrimethyl silane using the trimethylsilyl ether of a norpseudoephedrine or mandelic acid derivative as an auxiliary with a diastereoselectivity of up to 98:2. The reaction is performed at -78 degrees C in the presence of a catalytic amount of trifluoromethanesulfonic acid and leads to the corresponding tertiary ethers. The procedure can also be used for the allylation of aliphatic aldehydes with a diastereomeric ratio >99:1. Ketones give the 4,1'-syn product while the aldehydes give the reversed selectivity to yield a 4,1'-anti product. In addition, the reaction of gamma-substituted allyl silanes with ketones yields a product with two stereogenic centers and an anti diastereoselectivity of >99:1. The homoallylic ethers formed in the domino multicomponent process can be used in further synthetic transformations: the auxiliary can serve as a protecting group or can be cleaved reductively to give the corresponding homoallylic alcohols. Based on a number of both experimental and theoretical studies of the reaction mechanism, we conclude that an intermediate oxocarbenium ion is formed in the reaction of ketones. The oxocarbenium ion is attacked by the allyl silane during the stereogenic step. Using density functional theory methods, we could trace the observed stereoselectivity phenomena back to open transition states (TSs) where there is no interaction between the silane's trimethylsilyl group and the former carbonyl oxygen. On the contrary, the reaction with aldehydes forms an intermediate oxazolidinium salt, which explains the opposite selectivity. We have used the new allylation procedure in several total syntheses of natural products such as vitamin E, (+)-hydroxymyoporone, 5,6-dihydrocineromycin B, and polyoxygenated cembrenes.

Abstract  Analysis of extended X-ray absorption fine structure (EXAFS) data for the MnIV -oxo complexes [MnIV (O)(DMM N4py)]2+ , [MnIV (O)(2pyN2B)]2+ , and [MnIV (O)(2pyN2Q)]2+ (DMM N4py=N,N-bis(4-methoxy-3,5-dimethyl-2-pyridylmethyl)-N-bis(2-pyridyl)methylamine; 2pyN2B=(N-bis(1-methyl-2-benzimidazolyl)methyl-N-(bis-2-pyridylmethyl)amine, and 2pyN2Q=N,N-bis(2-pyridyl)-N,N-bis(2-quinolylmethyl)methanamine) afforded Mn=O and Mn-N bond lengths. The Mn=O distances for [MnIV (O)(DMM N4py)]2+ and [MnIV (O)(2pyN2B)]2+ are 1.72 and 1.70 Å, respectively. In contrast, the Mn=O distance for [MnIV (O)(2pyN2Q)]2+ was significantly longer (1.76 Å). We attribute this long distance to sample heterogeneity, which is reasonable given the reduced stability of [MnIV (O)(2pyN2Q)]2+ . The Mn=O distances for [MnIV (O)(DMM N4py)]2+ and [MnIV (O)(2pyN2B)]2+ could only be well-reproduced using DFT-derived models that included strong hydrogen-bonds between second-sphere solvent 2,2,2-trifluoroethanol molecules and the oxo ligand. These results suggest an important role for the 2,2,2-trifluoroethanol solvent in stabilizing MnIV -oxo adducts. The DFT methods were extended to investigate the structure of the putative [MnIV (O)(N4py)]2+ ⋅(HOTf)2 adduct. These computations suggest that a MnIV -hydroxo species is most consistent with the available experimental data.

Abstract  Epoxidation of styrene derivatives, sulfoxidation of thioanisole derivatives, and hydroxylation of toluene derivatives by a nonheme manganese(IV)-oxo complex binding triflic acid, [(N4Py)MnIV(O)]2+-(HOTf)2 [1-(H+)2], and scandium triflate, [(N4Py)MnIV(O)]2+-(Sc(OTf)3)2 [1-(Sc3+)2], occur via outer-sphere electron-transfer (OSET) pathways, exhibiting singly unified driving force dependence, enabling one to predict absolute values of the second-order rate constants of these three types of substrate oxidations by the manganese(IV)-oxo complex, using the Marcus theory of electron transfer. When [(N4Py)MnIV(O)]2+ (1) was replaced by [(N4Py)FeIV(O)]2+ (2), OSET pathways were changed to inner-sphere electron-transfer (ISET) pathways. The difference in the OSET versus ISET pathways is clarified based on the difference in the Lewis basicity of the oxo moieties in 1 and 2.

Abstract  The acid-base switching of complexes formed from anti-electrostatic anion-anion homodimers of organophosphates and cyanostar macrocycles was investigated for the first time. High-fidelity 2:2 complexes were selected by using suitably sized organo substituents. Reversible and direct switching occurs with triflic acid and hydroxide base. An unexpected acid⋅⋅⋅anion heterodimer was discovered with weaker picric acid, which helped reveal some of the elementary steps. Switching can also proceed in a cooperative (strong anion then weak acid) or stepwise manner (weak acid then strong anion).

Abstract  A sialic acid-specific lectin was purified from the hemolymph of Fenneropenaeus merguiensis by repetitive affinity fetuin-agarose column chromatography. The purified F. merguiensis lectin (called FmL) consisted of two distinct 30.9 and 32kDa subunits with identical N-terminal amino acid sequences of ten residues. FmL was also composed of sugar moieties; glucosamine, glucose, mannose and N-acetyl neuraminic acid but not N-glycolyl neuraminic acid. It was postulated to be a glycoprotein as it was positively stained by glycoprotein staining kit and detected by some bionylated plant lectins. Deglycosylation by either peptide N-glycosidase F or trifluoromethanesulfonic acid turned both types of FmL subunits to 28kDa peptides. The internal peptide sequence of FmL was similar to a fibrinogen-related domain of human ficolin and the horseshoe crab lectin. Determination of the lectin concentrations in the hemolymph was performed by ELISA while its hemaglutinating activity (HA) was tested by hemagglutination. Both specific lectin concentrations and HA increased as shrimp developed ovarian maturation stages 2 to 4. Their constitutive levels were found in pre-vitellogenic females and higher than those of males. Both specific lectin concentrations and HA of FmL were inducible to the highest levels at 12h after F. merguiensis was challenged by pathogenic Vibrio harveyi. The FmL-induced agglutination of V. harveyi was specifically abolished by sialic acid, fetuin and bacterial cell wall components. These findings might indicate the implication in an immune response of FmL to protect the shrimp themselves or their spawning eggs towards pathogenic bacteria in surrounding environment.

Abstract  We expressed human macrophage colony-stimulating factor (M-CSF) in Chinese hamster ovary (CHO) cells by introducing an expression plasmid coding for a 554-amino-acid M-CSF precursor and dihydrofolate reductase (DHFR) gene, and by amplifying the sequence. A cell line was obtained that secreted approximately 200,000 units/ml after 6 days in culture. The expressed recombinant human M-CSF (rhM-CSF) primarily consisted of two molecular species, a main 80-90 kD M-CSF as a homodimer and a molecular form higher than 150 kD. Purification of a main rhM-CSF gave an apparently homogeneous protein disulfide-bonded from 42-kD subunits, but one of the purified rhM-CSFs was composed of two subunit species with molecular masses of 44 and 42 kD. These purified rhM-CSFs had substantially the same specific activity (1 to 4 x 10(7) units/mg protein). Deglycosylation experiments with the latter rhM-CSF using chemical (trifluoromethanesulfonic acid) and enzymatic methods found a terminal neuraminic acid in addition to N- and O-glycosylation, but the two subunit species did not coalesce into a single molecule.

Abstract  The asymmetric synthesis of enantiomerically pure a-substituted tertiary homoallylic ethers 4a, 11 and 12a-c by the allylation of ethyl methyl ketone (la) with gamma-substituted allylsilanes 9a-h is described. The allylsilanes were obtained by a nickel-catalysed Grignard cross-coupling reaction of (E)- and (Z)-(3-iodoallyl)trimethylsilane with various Grignard reagents. The reaction of the allylsilanes with la in the presence of the trimethylsilyl ether of N-trifluoroacetylnorpseudoephedrine (3), and catalytic amounts of a mixture of trimethylsilyl triflate and trifluoromethanesulfonic acid led to the homoallylic ethers 4a, 11 and 12a-c with two new stereogenic centres, with a selectivity of 1:9 to >20:1 for the homoallylic and of 1:99 to >60:1 for the allylic centre. The facial selectivity does not depend on the configuration of the allylsilane, and in all reactions the anti product is preferentially formed. Interestingly, a pronounced switch of facial selectivity takes place with increasing length of the alkyl group of the allylsilane.

Abstract  The pyranose spiroketal natural products pollenopyrroside A and shensongine A (also known as xylapyrroside A, ent-capparisine B) have been synthesized by stereoselective spirocyclizations of a common C1-functionalized glycal precursor. In conjunction with our previously reported syntheses of the corresponding furanose isomers, this provides a versatile family-level synthesis of the pyrrolomorpholine spiroketal natural products and analogues. In rat mesangial cells, hyperglycemia-induced production of reactive oxygen species, which is implicated in diabetic nephropathy, was inhibited by pollenopyrroside A and shensongine A with mid-μM IC50 values, while unnatural C2-hydroxy analogues exhibited more potent, sub-μM activity.

Abstract  Fractions of leaf gel from Aloe barbadensis Mill. were prepared by gel permeation using DEAE Sephadex A-25, Sepharose 6B, and Sephadex G-50 columns. These were then tested by in vitro assays for proliferation of human normal dermal and baby hamster kidney cells. The glycoprotein fraction promoted cell growth, while the neutral polysaccharide fraction did not show any growth stimulation. Moreover, the polar-colored glycoprotein fraction strongly inhibited the in vitro assays. An active glycoprotein fraction (protein 82%, carbohydrate 11%) examined on polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE showed a single band. Its molecular weight was 29 kD on a Sephadex G-50 column and its isoelectric point was pH 6.8. Immunoblotting after SDS-PAGE showed that the glycoprotein was composed of two subunits (14 kD). Deglycosylation of glycoprotein (Pg21-2b fraction) by trifluoromethanesulphonic acid provided a protein band with a molecular weight of 13 kD on SDS-PAGE. The colored glycoprotein fraction was shown on SDS-PAGE to be a mixture with a molecular weight of 18 kD-15 kD. It was later hydrolyzed with 10% H2SO4 to produce phenolic substances.

Abstract  In this work we search for antigens of Trichinella spiralis in sera and stool of rats experimentally infected. The kinetic of antibodies to excretory and secretory (ES) antigens of muscle larvae (ML) was also determined. Wistar rats were infected with 15 ML per gram of body weight and blood samples were collected weekly for 10 weeks. Antibodies were studied using an indirect ELISA. For detection of circulating antigens and coproantigens, a sandwich ELISA was developed with the use of polyclonal rabbit antibodies obtained against the total extract of ML and an IgM monoclonal antibody (Mab) against ES antigens of ML. No reactivity was observed between Mab and the total worm antigens of Angiostrongylus cantonensis, Ascaris suum, Echinococcus granulosus, Fasciola hepatica, Strongyloides stercoralis, Taenia solium, Toxocara canis and Trichuris trichiura. The IgM Mab recognized antigens of 45, 49, and 55 kDa in ES antigens and was unable to bind ES antigens deglycosylated with trifluoromethanesulphonic acid (TFMS) indicating that a glycan structure is present in the epitope recognized by this Mab. The sensitivity of sandwich ELISA was 1 ng/mL. Circulating antigens were detected in all infected rats between 3 and 8 weeks post infection and coproantigens were found during the first two days post infection. Antibodies were detected since the third week post infection through the end of experiment. These results suggested that antigen detection by our sandwich ELISA could be a useful complementary laboratory test for antibody detection.