Modification of galactose andN-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 (α-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.     

2‐Picoline‐borane: A non‐toxic reducing agent for oligosaccharide labeling by reductive amination

Analysis of N‐glycans is often performed by LC coupled to fluorescence detection. The N‐glycans are usually labeled by reductive amination with a fluorophore containing a primary amine to allow fluorescence detection. Moreover, many of the commonly applied labels also allow improved mass spectrometric detection of oligosaccharides. For reductive amination, the amine group of the label reacts with the reducing‐end aldehyde group of the oligosaccharide to form a Schiff base, which is reduced to a secondary amine. Here, we propose the use of 2‐picoline‐borane as the reducing agent, as a non‐toxic alternative to the extensively used, but toxic sodium cyanoborohydride. Using dextran oligosaccharides and plasma N‐glycans, we demonstrate similar labeling efficacies for 2‐picoline‐borane and sodium cyanoborohydride. Therefore, 2‐picoline‐borane is a non‐toxic alternative to sodium cyanoborohydride for the labeling of oligosaccharides.     

Resolution of the enantiomers of aldoses by liquid chromatography of diastereoisomeric 1-(N-acetyl-α-methylbenzylamino)-1-deoxyalditol acetates

Acyclic diastereoisomers, namely, 1-(N-acetyl-α-methylbenzylamino)-1-deoxyalditol acetates are readily obtained by reductive amination of aldoses with chiral α-methylbenzylamine (MBA) in the presence of sodium cyanoborohydride, and may be separated by reversed-phase 1.c. and, more effectively, by adsorption 1.c. According to this procedure, enantiomers of the common, neutral aldoses are resolved. In adsorption 1.c., l-l_* [defined as an adduct of an l-aldose and l-(-)-MBA] is eluted before d-l_* for erythrose, xylose, ribose, glucose, 4-O-methylglucose, galactose, and fucose, and the elution order is the reverse for arabinose, lyxose, mannose, rhamnose, and glyceraldehyde. This behavior is probably related to the configuration of C-2 of the aldoses.     

Immunochemical characterization of polylysine conjugates containing reductively aminated cellulose oligosaccharides

Neoglycoproteins prepared by direct reductive amination of cellobiose, cellotetraose and cellopentaose to polylysine, were found to be effective antigens in rabbits, and the antisera were found by quantitative inhibition techniques to be predominantly hapten specific. Several analogs incorporating various structural features of the carbohydrate hapten were synthesized and examined as inhibitors of the precipition reaction between the neoglycoproteins and homologous antisera in order to identify those structural features of the hapten important in antibody recognition. Antibodies to the reductively aminated cellobiose-polylysine conjugate were found to recognize the terminal -glucopyranosyl residue, theacyclic reduced glucose residue, and the secondary ammonium linkage and methylene arm of the lysyl residue of the hapten. Antibodies to the cellotetraose-polylysine conjugate, in contrast, displayed no recognition for the secondary ammonium linkage region; they were found to recognize the non-reducing terminal -glucopyranosyl residue, the two internal 1,4-linked glucopyranosyl residues, and the reducing end glucose residue in anacyclic orcyclic form. Inhibition studies with antisera to the cellopentaose-polylysine conjugate again established that there was no recognition of the secondary ammonium linkage region, and demonstrated that the upper limit to the size, of the antibody combining site was four 1,4-linked glucopyranosyl residues.Abbreviations BSA bovine serum albumin - Glc glucose - (Glc)₂ (Glc)₄, (Glc)₅ compounds derived by reductive amination of cellobiose, cellotetraose and cellopentaose     

Modification and introduction of various radioactive labels into the sialic acid moiety of sialoglycoconjugates

A method for modifying and isotopic labeling the sialyl moiety of sialoglycoproteins is described. The basis of the procedure is the reductive amination of the exocyclic aldehyde group, generated on sialic acid by mild periodate oxidation, with a variety of amino compounds and sodium cyanoborohydride. Optimal conditions were selected to obtain maximum modification of sialic acid and minimal non-specific incorporation of the amino compound (glycine). The glycine modified model glycoproteins (α₁-acid glycoprotein, fetuin) yielded single homogenous peaks upon gel filtration and on ion exchange chromatography. On gel electrophoresis a major band accounting for 92–98% of the modified glycoprotein and two minor bands consisting of dimers and trimers of the glycoprotein were observed. The modification did not alter the ability of the sialoglycoproteins to bind to wheat germ agglutinin-Sepharose or to interact with antibodies. The modified sialic acid was only partially released by mild acid hydrolysis suggesting that the introduction of an amino compound into the polyol chain of sialic acid has a stabilizing effect on the ketosidic linkage of the sugar. Interestingly, the modification rendered the sialic acid resistant to a variety of sialidases. The potential uses of this modification procedure include 1) the introduction of different isotopic labels (³H,¹⁴C,³⁵S,¹²⁵I) into the sialic acid moiety of glycoproteins; 2) the preparations of biologically active sialoglycoprotein (hormones, enzymes, co-factors) with increased circulating half-lives in animals; 3) preparation of substrates to search for endoglycosidases; 4) the direct comparison of sialoglycoprotein patterns obtained in small amounts from normal and pathological cells or tissues, and 5) the isolation and purification of cell surface sialoglycoproteins.     

Synthesis and characterization of tyramine-derivatized (1 å 4)-linked α-d-oligogalacturonides

The reducing end C-1 of (1 → 4)-linked α-d-oligogalacturonides (oligogalacturonides), with degrees of polymerization (dp) 3 and 13, was coupled to tyramine via reductive amination in the presence of sodium cyanoborohydride. These derivatives were purified in milligram quantities and structurally characterized. Tyramination of trigalacturonic acid proceeded to completion. The yield of apparently homogeneous tyraminated trigalacturonic acid after desalting was 35%. Derivatization of tridecagalacturonide with tyramine was incomplete. The tyraminated tridecagalacturonide was purified to apparent homogeneity using semipreparative high-performance anion-exchange chromatography (HPAEC) with a yield of 30%. The structures of the derivatized oligogalacturonides were established by ¹H NMR spectroscopy and electrospray mass spectrometry.     

Preparation of neuraminidase-resistant human alpha 1-protease inhibitor and its clearance in rat blood circulation

The sialic acid residues of human alpha 1-protease inhibitor were modified by periodate oxidation and subsequent reductive amination with ethanolamine and sodium cyanoborohydride. The modified inhibitor retained its original trypsin inhibitory activity and was not digested by neuraminidase from Clostridium perfringens. The modified inhibitor disappeared from rat blood circulation at the same rate as the native inhibitor.     

Preparation of a high-affinity photolabeling reagent for the Gal/GalNAc lectin of mammalian liver: demonstration of galactose-combining sites on each subunit of rabbit hepatic lectin

On the basis of the knowledge that the D-galactose/N-acetyl-D-galactosamine-specific lectin of rabbit liver can tolerate a large group on the C-6 hydroxyl group of a galactoside [Lee, R. T. (1982) Biochemistry 21, 1045-1050], we prepared a high-affinity photolabeling reagent for this lectin from a triantennary glycopeptide fraction of asialofetuin. The C-6 hydroxyl group of a D-galactopyranoside was converted, under mild conditions, into a primary amino group. The procedure involves conversion of the hydroxyl group to an oxo group with galactose oxidase, followed by reductive amination using benzylamine and sodium cyanoborohydride. Catalytic hydrogenolysis of the benzylamino derivative yielded the desired 6-amino-6-deoxy-D-galactoside. A 4-azidobenzoyl group was attached to the newly produced amino group to yield a photoactivatable affinity-labeling reagent. The reagent labeled the Triton-solubilized, purified hepatic lectins of rabbit and rat in a photo- and affinity-dependent manner. All the polypeptide subunits of the lectins were labeled, indicating that each subunit contains at least one D-galactose-combining site. In the case of the rabbit hepatic lectin, the minor subunit (46 kDa) was labeled more efficiently than the major one (40 kDa).     

Fluorescent detection of α-aminoadipic and γ-glutamic semialdehydes in oxidized proteins

The oxidative modification of proteins is believed to play a critical role in the etiology and/or progression of several diseases. α-Aminoadipic semialdehyde (AAS) and γ-glutamic semialdehyde (GGS) residues represent major oxidized amino acids generated in oxidized proteins. This paper describes a novel procedure for the specific and sensitive determination of AAS and GGS after their reductive amination with sodium cyanoborohydride and p-aminobenzoic acid, a fluorescence reagent, to their corresponding derivatives, followed by a high-performance liquid chromatography (HPLC) analysis. This fluorescent labeling of protein-associated aldehyde moieties is a simple and accurate technique that may be widely used to reveal increased levels of oxidatively modified proteins with reactive oxygen species during aging and disease.     

Labeling of oligosaccharides for quantitative mass spectrometry

Quantitative analysis of oligosaccharide mixtures and their derivatives by electrospray ionization mass spectrometry (ESI MS) is challenging, for example, due to different affinities of the analytes to alkali ions. To overcome this source of discrimination and to enhance signal intensity, labeling studies with cellobiose as model compound were performed with the goal to develop a rapid, easy, and robust method. Hydrazone formation with the permanently charged Girard’s T reagent as well as reductive amination with five different charge providing amines were studied under various conditions. In both reaction types, the removal of water turned out to be the critical step because only under these conditions are the reactions pushed to completion. By working with only a slight excess of reagents, no purification is necessary to achieve excellent signal/noise ratios, avoiding further sources of discrimination. Comparing various reducing agents with respect to their selectivity and stability in the acidic reaction medium, 2-picoline borane turned out to be superior to the commonly used sodium cyanoborohydride. Thus, by replacement of the toxic NaCNBH₃ by the more selective and stable, non-toxic 2-picoline borane, complete reductive amination with low amounts of reagents and without unlabeled alditol formation was achieved with o-aminobenzoic acid, a useful reagent for ESI MS in negative mode. For MS in positive mode Girard’s T derivatization was very suitable.     

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.     

Acetate and carbon dioxide assimilation by Desulfovibrio vulgaris (Marburg), growing on hydrogen and sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source and acetate plus CO₂ as the sole carbon sources. The incorporation of U-¹⁴C acetate into alanine, aspartate, glutamate, and ribose was studied. The labelling data show that alanine is synthesized from one acetate (C-2 + C-3) and one CO₂ (C-1), aspartate from one acetate (C-2 + C-3) and two CO₂ (C-1 + C-4), glutamate from two acetate (C-1–C-4) and one CO₂ (C-5), and ribose from 1.8 acetate and 1.4 CO₂. These findings indicate that in Desulfovibrio vulgaris (Marburg) pyruvate is formed via reductive carboxylation of acetyl-CoA, oxaloacetate via carboxylation of pyruvate or phosphoenol pyruvate, and -ketoglutarate from oxaloacetate plus acetyl-CoA via citrate and isocitrate. Since C-5 of glutamate is derived from CO₂, citrate must have been formed via a (R)-citrate synthase rather than a(S)-citrate synthase. The synthesis of ribose from 1.8 mol of acetate and 1.4 mol of CO₂ excludes the operation of the Calvin cycle in this chemolithotrophically growing bacterium.     

Induction of an osteocyte-like phenotype by fibroblast growth factor-2

Cycloheximide (CHX) is one of the most interesting protein synthesis inhibitors. For this reason, fluorescent derivatives of CHX could find useful applications in cell biology. We report the successful synthesis of a set of novel fluorescent derivatives of CHX. The effect of different functional groups on the biological activity of CHX was studied upon their modification through suitable strategies, i.e., acetylation of the hydroxyl group and reductive amination of the ketone group. The first route induced a complete loss of biological activity, while the second approach allowed a retained inhibition of protein synthesis, as demonstrated by in vitro translation assays. Various fluorescent dyes for reductive amination were tested (i.e., ANTS, APTS, and Rhodamine-123), and the success of the syntheses was demonstrated by diverse analytical techniques. Cycloheximide labeling with fluorescent dyes is a promising approach for developing fluorescence reporters for various applications, both in vitro (fluorescence spectroscopy) and in vivo (live imaging).     

The chemical mechanism of action of glucose oxidase from Aspergillus niger

Glucose oxidase from Aspergillus niger (EC 1.1.3.4) is able to catalyze the oxidation of -D-glucose with p-benzoquinone, methyl-1,4-benzoquinone, 1,2-naphthoquinone, 1,2-naphthoquinone-4-sulfonic acid, potassium ferricyanide, phenazine methosulfate, and 2,6-dichloroindophenol. In this work, the steady-state kinetic parameters, V ₁/K _B , for reactions of these substrates were collected from pH 2.5–8. Further, the molecular models of the enzyme's active site were constructed for the free enzyme in the oxidized state, the complex of -D-glucose with the oxidized enzyme, the complex of reduced enzyme with methyl-1,4-benzoquinone, the reduced enzyme plus 1,2-naphthoquinone-4-sulfonic acid, oxidized enzyme plus reduced 1,2-naphthoquinone-4-sulfonic acid (hydroquinone anion), and oxidized enzyme plus fully reduced 1,2-naphthoquinone-4-sulfonic acid.Combining the steady-state kinetic and structural data, it was concluded that Glu412 bound to His559, in the active site of enzyme, modulates powerfully its catalytic activity by affecting all the rate constants in the reductive and the oxidative half-reaction of the catalytic cycle. His516 is the catalytic base in the oxidative and the reductive part of the catalytic cycle. It was estimated that the pK _a of Glu412 (bound to His559) in the free reduced enzyme is 3.4, and the pK _a of His516 in the free reduced enzyme is 6.9.     

γ-Hydroxyethyl piperidine iminosugar and N-alkylated derivatives: A study of their activity as glycosidase inhibitors and as immunosuppressive agents

An efficient and practical strategy for the synthesis of (3R,4s,5S)-4-(2-hydroxyethyl) piperidine-3,4,5-triol and its N-alkyl derivatives 8a–f, starting from the d-glucose, is reported. The chiral pool methodology involves preparation of the C-3-allyl-α-d-ribofuranodialdose 10, which was converted to the C-5-amino derivative 11 by reductive amination. The presence of C-3-allyl group gives an easy access to the requisite hydroxyethyl substituted compound 13. Intramolecular reductive aminocyclization of C-5 amino group with C-1 aldehyde provided the γ-hydroxyethyl substituted piperidine iminosugar 8a that was N-alkylated to get N-alkyl derivatives 8b–f. Iminosugars 8a–f were screened against glycosidase enzymes. Amongst synthetic N-alkylated iminosugars, 8b and 8c were found to be α-galactosidase inhibitors while 8d and 8e were selective and moderate α-mannosidase inhibitors. In addition, immunomodulatory activity of compounds 8a–f was examined. These results were substantiated by molecular docking studies using AUTODOCK 4.2 programme.     

Efficient one‐pot synthesis of N‐ethyl amino acids

Mono‐N‐ethylated α‐amino acid esters are obtained in high yields using reductive amination procedures. Formation of imine is achieved by excess of acetaldehyde, followed by removal of acetaldehyde and reduction by NaBH(OAc)₃. The elaborated one‐pot synthesis allows for the efficient synthesis of side‐chain protected amino acid derivatives. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Proteins containing reductively aminated disaccharides: Synthesis and chemical characterization

Synthetic glycoproteins can be prepared by reductive amination of protein and reducing disaccharide in the presence of sodium cyanoborohydride. The reaction proceeds readily in aqueous solutions over a broad pH range to give high degrees of substitution. The degree of substitution can be determined by amino acid analysis, as the secondary amine linkage formed by reductive amination in stable to acid-catalyzed protein hydrolysis conditions. In order to demonstrate that coupling occurs to lysine residues, synthetic α-N-1-(1-deoxyglucitol)-lysine and ?-N-1-(1-deoxyglucitol)-lysine were prepared and compared with bovine serum albumin conjugates of maltose, cellobiose, lactose, and melibiose by amino acid analysis after acid hydrolysis. These studies demonstrate that the expected secondary amine linkages are formed with the ?-amino groups of lysine.     

Evidence for an incomplete reductive carboxylic acid cycle in Methanobacterium thermoautotrophicum

The involvement of reactions of the tricarboxylic acid cycle in autotrophic CO₂ fixation in Methanobacterium thermoautotrophicum was investigated. The incorporation of succinate into glutamate (=-ketoglutarate), aspartate (=oxaloacetate) and alanine (=pyruvate) was studied. The organism was grown on H₂ plus CO₂ at pH 6.5 in the presence of 1 mM [U-¹⁴C-]succinate. Significant amounts of the dicarboxylic acid were incorporated into cellular material under these conditions. Alanine, aspartate, and glutamate were isolated and their specific radioactivities were determined. Only glutamate was found to be labelled. Degradation of glutamate revealed that C-1 of glutamate was derived from CO₂ and C-2-C-5 from succinate indicating that in M. thermoautotrophicum -ketoglutarate is synthesized via reductive carboxylation of succinyl CoA. The finding that succinate was not incorporated into alanine and aspartate excludes that oxaloacetate and pyruvate are synthesized from -ketoglutarate via isocitrate or citrate. This is taken as evidence that a complete reductive carboxylic acid cycle is not involved here in autotrophic CO₂ fixation.     

A model for the effects of primary substrates on the kinetics of reductive dehalogenation

A kinetic model that describes substrate interactions during reductive dehalogenation reactions is developed. This model describes how the concentrations of primary electron-donor and -acceptor substrates affect the rates of reductive dehalogenation reactions. A basic model, which considers only exogenous electron-donor and -acceptor substrates, illustrates the fundamental interactions that affect reductive dehalogenation reaction kinetics. Because this basic model cannot accurately describe important phenomena, such as reductive dehalogenation that occurs in the absence of exogenous electron donors, it is expanded to include an endogenous electron donor and additional electron acceptor reactions. This general model more accurately reflects the behavior that has been observed for reductive dehalogenation reactions. Under most conditions, primary electron-donor substrates stimulate the reductive dehalogenation rate, while primary electron acceptors reduce the reaction rate. The effects of primary substrates are incorporated into the kinetic parameters for a Monod-like rate expression. The apparent maximum rate of reductive dehalogenation (q _{m, ap} ) and the apparent half-saturation concentration (K _ap ) increase as the electron donor concentration increases. The electron-acceptor concentration does not affect q _{m, ap} , but K _ap is directly proportional to its concentration.Definitions for model parameters RX halogenated aliphatic substrate - E-M ⁿ reduced dehalogenase - E-M ⁿ⁺² oxidized dehalogenase - [E-M ⁿ ] steady-state concentration of the reduced dehalogenase (moles of reduced dehalogenase per unit volume) - [E-M ⁿ⁺²] steady-state concentration of the oxidized dehalogenase (moles of reduced dehalogenase per unit volume) - DH₂ primary exogenous electron-donor substrate - A primary exogenous electron-acceptor substrate - A2 second primary exogenous electron-acceptor substrate - X biomass concentration (biomass per unit volume) - f fraction of biomass that is comprised of the dehalogenase (moles of dehalogenase per unit biomass) - stoichiometric coefficient for the reductive dehalogenation reaction (moles of dehalogenase oxidized per mole of halogenated substrate reduced) - stoichiometric coefficient for oxidation of the primary electron donor (moles of dehalogenase reduced per mole of donor oxidized) - stoichiometric coefficient for oxidation of the endogenous electron donor (moles of dehalogenase reduced per unit biomass oxidized) - stoichiometric coefficient for reduction of the primary electron acceptor (moles of dehalogenase oxidized per mole of acceptor reduced) - stoichiometric coefficient for reduction of the second electron acceptor (moles of dehalogenase oxidized per mole of acceptor reduced) - r _RX rate of the reductive dehalogenation reaction (moles of halogenated substrate reduced per unit volume per unit time) - r _d1 rate of oxidation of the primary exogenous electron donor (moles of donor oxidized per unit volume per unit time) - r _d2 rate of oxidation of the endogenous electron donor (biomass oxidized per unit volume per unit time) - r _a1 rate of reduction of the primary exogenous electron acceptor (moles of acceptor reduced per unit volume per unit time) - r _a2 rate of reduction of the second primary electron acceptor (moles of acceptor reduced per unit volume per unit time) - k _RX mixed second-order rate coefficient for the reductive dehalogenation reaction (volume per mole dehalogenase per unit time) - k _d1 mixed-second-order rate coefficient for oxidation of the primary electron donor (volume per mole dehalogenase per unit time) - k _d2 mixed-second-order rate coefficient for oxidation of the endogenous electron donor (volume per mole dehalogenase per unit time) - b first-order biomass decay coefficient (biomass oxidized per unit biomass per unit time) - k _a1 mixed-second-order rate coefficient for reduction of the primary electron acceptor (volume per mole dehalogenase per unit time) - k _a2 mixed-second-order rate coefficient for reduction of the second primary electron acceptor (volume per mole dehalogenase per unit time) - q _m,ap apparent maximum specific rate of reductive dehalogenation (moles of RX per unit biomass per unit time) - K _ap apparent half-saturation concentration for the halogenated aliphatic substrate (moles of RX per unit volume) - k _ap apparent pseudo-first-order rate coefficient for reductive dehalogenation (volume per unit biomass per unit time)     

Isolation of the major glycoprotein from fat cell plasma membranes

The major glycoprotein of rabbit fat cell plasma membranes has been solubilized by Brij 99 extraction and purified to homogeneity by preparative polyacrylamide gel electrophoresis and concanavalin A-Sepharose affinity chromatography. The isolation procedure yielded a glycoprotein with an apparent molecular weight of 79,000 which appeared as a single component by Coomassie blue and periodic acid-Schiff staining as well as by distribution of radioactivity after ¹²⁵I labeling. The lectin chromatography was effective in removing polypeptides and Schiff-nonreactive glycoproteins which migrated in close proximity to the major glycoprotein during electrophoresis but were not retained on the concanavalin A column. Determination of the amino acid and sugar composition of the purified glycoprotein indicated that it contained 18% carbohydrate by weight which occurred in the form of 30 mannose, 14 galactose, 23 glucosamine, 3 galactosamine, 6 N-acetylneuraminic acid, and 1 fucose residues per molecule. Approximately one-fifth of the total protein-bound saccharide of the adipocyte plasma membrane was accounted for by this glycoprotein and its composition suggested that it was the source of some of the previously identified (Y. Kawai, and R. G. Spiro, 1977, J. Biol. Chem.252, 6236–6244) asparagine- and serine (threonine)-linked carbohydrate units of the fat cell surface.