Metabolic plasticity drives development during mammalian embryogenesis

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Modification of galactose and N-acetylgalactosamine residues by oxidation of C-6 hydroxyls to the aldehydes followed by reductive amination: model systems and antifreeze glycoproteins

Amino acids and peptides have been attached to the C-6 hydroxyls of the galactose and the N-acetylgalactosamine by first oxidizing the C-6 hydroxyls to the aldehydes by galactose oxidase in the presence of small amounts of catalase, followed by reductive amination (-amino group) in the presence of cyanoborohydride. The activity of oxidized antifreeze glycoprotein was >70% of the original, and considerable activity has been retained with some substitutions on reductive amination using cyanoborohydride. The following were some activities retained (as compared with the oxidized antifreeze glycoprotein): Gly, 64; (Gly)₂, 88; (Gly)₃, 82; (Gly)₄, 70; Gly-Gly-NH₂, 44, Gly-Glu, 13; Gly-Leu, 40; Gly-Tyr, 57; Gly-Gly-Leu, 50; Gly-Gly-Phe, 30; and Gly-Gly-Val, 35. On amino acid analysis of acid hydrolysates, some release of the amino acid attached by amination occurred; e.g., Gly-Tyr gave 0.26 Gly and 0.49 Tyr per disaccharide.     

Preparation of polysaccharide-enzyme conjugates for competitive binding assays

A variety of bacterial O-polysaccharides were covalently linked to enzymes and it was demonstrated with three discrete monoclonal antibodies that enzyme-glycoconjugates function as convenient labelled antigens in direct enzyme immunoassays, particularly competitive assays that quantify bacterial O-antigens. Two strategies, each involving reductive amination, were used to couple O-polysaccharides to enzymes, while retaining high enzymic activity. Reduction of the Schiff base formed between, 1,3-diaminopropane and the terminal reducing ketodeoxyoctanoic acid (KDO) residue present in the majority of the lipopolysaccharide (LPS) core domains, following mild acid removal of Lipid A, offered the most direct route to mono-aminated polysaccharide. Alternatively, mild periodate oxidation of KDO and heptose residues generated multiple aldehyde targets for Schiff base formation, without affecting the O-antigenic determinant. Hetero- and homobifunctional coupling reagents, sulphosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate and discuccinimidyl suberate, activated polysaccharide for coupling to enzymes at amino and sulphydryl sites and produced conjugates that retained at least 95% of the original enzymic activity. The most suitable enzyme conjugates, especially for competitive inhibition EIA were those bearing one polysaccharide chain, and these were easily prepared from horse-radish peroxidase. Although the extent of conjugation of activated polysaccharide to -galactosidase and alkaline phosphatase could be controlled by reaction stoichiometry, the use of these enzymes was a less effective utilization of valuable antigen and enzyme.Issued as NRCC No. 31634     

Reductive dechlorination of 1,2-dichloroethane and chloroethane by cell suspensions of methanogenic bacteria

Concentrated cell suspensions of methanogenic bacteria reductively dechlorinated 1,2-dichloroethane via two reaction-mechanisms: a dihalo-elimination yielding ethylene and two hydrogenolysis reactions yielding chloroethane and ethane, consecutively. The transformation of chloroethane to ethane was inhibited by 1,2-dichloroethane. Stimulation of methanogenesis caused an increase in the amount of dechlorination products formed, whereas the opposite was found when methane formation was inhibited. Cells of Methanosarcina barkeri grown on H₂/CO₂ converted 1,2-dichloroethane and chloroethane at higher rates than acetate or methanol grown cells.Abbreviations BrES 2-bromoethanesulfonic acid - CA chloroethane - 1,2-DCA 1,2-dichloroethane - F₄₃₀ Ni(II)tetrahydro-(12, 13)-corphin with an uroporphinoid (III) ligand skeleton     

Reductive dechlorination of perchloroethylene and the role of methanogens

Abstract Perchloroethylene (PCE) was reductively dechlorinated to trichloroethylene in a 10% anaerobic sewage sludge. About 80% of the initially added PCE (300 nmol) was dechlorinated within three weeks. The calculated rates were 250 nM and 445 nM · day⁻¹ during the first and second weeks of incubation, respectively. The depletion of PCE varied in sludges obtained from different sources.
The role of methanogenesis in the dechlorination of PCE was evaluated by inhibiting the methanogens by addition of bromoethane sulfonic acid, a potent methanogenic inhibitor. Dechlorination of PCE was significantly inhibited in sludges amended with the inhibitor. Almost 41–48% less PCE was dechlorinated in sludges containing 5 mM BESA, indicating a relation between the two processes (methanogenesis and dechlorination). Direct proof that methanogens can transform chlorinated aliphatic compounds was obtained using axenic cultures of acetate-cleaving methanogens. Methanosarcina sp , originally isolated from a chlorophenol degrading consortium, showed significantly higher dechlorinating activity as compared to Ms. mazei . Based on these studies and other recently reported observations, it appears that methanogens/methanogenesis play an important role in the anaerobic dechlorination of chlorinated aliphatics such as PCE.     

Expedient syntheses of neoglycoproteins using phase transfer catalysis and reductive amination as key reactions

Starting from peracetylated chloro- or bromo-glycosyl donors ofN-acetylneurmainic acid,N-acetylglucosamine, glucose and lactose, the correspondingp-formylphenyl glycosides were synthesized stereospecifically under phase transfer catalysed conditions at room temperature in yields of 38–67%. After Zemplén de-O-acetylation, the formyl groups were directly and chemoselectively coupled to the lysine residues of bovine serum albumin by reductive amination using sodium cyanoborohydride. The conjugation reactions were followed as a function of time and under a series of different molar ratios of the reactants to provide glycoconjugates of varying degree of antigenicities. Thus, carbohydrate protein conjugates were made readily available using essentially two key reactions.Presented in part at the 15th International Carbohydrate Symposium, Yokohama, Japan, August 12–17, 1990.     

The Reductive Deglycosylation Of Adriamycin In Aqueous Medium: A Pulse Radiolysis Study

The free radical (II) produced by one-electron reduction of adriamycin (I) exists in aqueous solution at pH 7.0 in equilibrium with the parent and the two-electron reduced form (III). Over some hundreds of milliseconds deglycosylation takes place yielding an aglycone (IV) which subsequently rearranges to form a more stable aglycone. 7-deoxyadriamycinone (V). The changes in the optical absorption spectrum accompanying these processes are reported. The rate constant for III + IV is 1.1 s^-1 and for IV + V is 1.5 × 10^--² s.^-1. At pH 4.0 the two electron reduced form of adriamycin exists predominantly in a different tautomeric form (VII). It is suggested that this deglycosylates via a free radical mechanism involving the acidic form of the semiquinone free radical (VI)     

Isolation of an anaerobic bacterium which reductively dechlorinates tetrachloroethene and trichloroethene

Strain TEA, a strictly anaerobic, motile rod with one to four lateral flagella and a crystalline surface layer was isolated from a mixed culture that completely reduces chlorinated ethenes to ethene. The organism coupled reductive dehalogenation of tetrachloroethene or trichloroethene to cis-1,2-dichloroethene to growth, using molecular hydrogen as the electron donor. It was unable to grow fermentatively or in the presence of tri- or tetrachloroethene with glucose, pyruvate, lactate, acetate or formate. The 16S rDNA sequence of strain TEA was 99.7% identical to that of Dehalobacter restrictus. The two organisms thus are representatives of the same species or the same genus within the Bacillus/Clostridium subphylum of the gram-positive bacteria.     

Reductive methylation andpKa determination of the lysine side chains in calbindin D9k

The Lys residues in the 75-residue Ca²⁺-binding protein calbindin D_9k were reductively methylated with¹³C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca²⁺- and apo-forms of the protein, the¹³C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (¹H,¹³C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca²⁺-calbindin D_9k and apo-calbindin D_9k were obtained by combiningpH titration experiments and (¹H,¹³C)-HMQC NMR spectroscopy. Each Lys residue in the Ca²⁺- and apo-forms of calbindin D_9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D_9k has a very similar structure compared to Ca²⁺-calbindin D_9k, the removal of two Ca²⁺ ions from the protein leads to an increase of thepKa values of the Lys residues.     

A new chemical tool for characterization and partial depolymerization of red algal galactans

Complete acid hydrolysis of red algal galactans in the presence of borane - 4-methylmorpholine complex has been shown to prevent the acid degradation of 3,6-anhydrogalactose derivatives by their reduction to the corresponding 3,6-anhydro-galactitols, whereas all the other monosaccharides are liberated essentially in the non-reduced form; the reductive hydrolysis products may be determined quantitatively using gas-liquid chromatography (GLC). The method is recommended for preliminary characterization of the polysaccharide composition of red algal biomass. Partial acid hydrolysis of galactans in the presence of the same reducing agent gives rise to reduced oligosaccharides having terminal 3,6-anhydrogalactitol residues. Based on this reaction, the attribution of unknown galactans to the agar or carrageenan groups is possible by partial reductive hydrolysis of small samples of algal biomass with subsequent identification of agarobiitol or carrabiitol acetates by GLC. Sulfate groups are substantially retained under partial reductive hydrolysis conditions; the isolation by liquid chromatography and elucidation of structures of reduced sulfated oligosaccharides may be of great value for the structural analysis of complex red algal galactans.