Synthesis of N-linked pentasaccharides with isomeric glycosidic linkage

As part of a program to explore the structural requirement of N-glycans in the carbohydrate-mediated biological interactions, N-linked pentasaccharide core structure was stereochemically modified in terms of glycosidic linkage. Three isomers, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, -D-Man-(13)-[-D-Man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, and -D-Man-(13)-[-D-man-(16)]--D-Man-(14)--D-GlcNAc-(14)--D-GlcNAc-L-Asn, were synthesized. Synthesis of the pentasaccharide with natural linkage is also described.     

Structures of the N-linked sugar chains in the PAS-6 glycoprotein from the bovine milk fat globule membrane

The structures of the N-linked sugar chains in the PAS-6 glycoprotein (PAS-6) from the bovine milk fat globule membrane were determined. The sugar chains were liberated from PAS-6 by hydrazinolysis, and the pyridylaminated sugar chains were separated into a neutral (6N) and two acidic chains (6M and 6D), the acidic sugar chains then being converted to neutral sugar chains (6MN and 6DN). 6N was separated into two neutral fractions (6N13 and 6N5.5), while 6MN and 6DN each gave a single fraction (6MN13 and 6DN13). The structure of 6N5.5, which was the major sugar chain in PAS-6, is proposed to be Man16 (Man13) Man14GlcNAc14GlcNAc-PA; 6N13, 6MN13 and 6DN13 are proposed to be Gal13Gal14GlcNAc12Man16 (Gal13Gal14GlcNAc12Man13) Man14GlcNAc14 (Fuc16)GlcNAc-PA;6M and 6D had 1 or 2 additional NeuAc residues at the non-reducing ends of 6MN13 and 6DN13, respectively. © 1998 Rapid Science Ltd     

Differential expression of enzyme activities initiating anoxic metabolism of various aromatic compounds via benzoyl-CoA

The regulation of the expression of enzyme activities catalyzing initial reactions in the anoxic metabolism of various aromatic compounds was studied at the whole cell level in the denitrifying Pseudomonas strain K 172. The specific enzyme activities were determined after growth on six different aromatic substrates (phenol, 4-hydroxybenzoate, benzoate, p-cresol, phenylacetate, 4-hydroxyphenylacetate) all being proposed to be metabolized anaerobically via benzoyl-CoA. As a control cells were grown on acetate, or aerobically on benzoate. The expression of the following enzyme activities was determined.Phenol carboxylase, as studied by the isotope exchange between ¹⁴CO₂ and the carboxyl group of 4-hydroxybenzoate; 4-hydroxybenzoyl-CoA reductase (dehydroxylating); p-cresol methylhydroxylase; 4-hydroxybenzyl alcohol dehydrogenase; 4-hydroxybenzaldehyde dehydrogenase; coenzymeA ligases for the aromatic acids benzoate, 4-hydroxybenzoate, phenylacetate, and 4-hydroxyphenylacetate; phenylglyoxylate: acceptor oxidoreductase and 4-hydroxyphenylglyoxylate: acceptor oxidoreductase; aromatic alcohol and aldehyde dehydrogenases.The formation of most active enzymes is strictly regulated; they were only induced when required, the basic activities being almost zero. The observed whole cell regulation pattern supports the postulate that the enzyme activities play a role in anoxic aromatic metabolism and that the compounds are degraded via the following intermediates: Phenol 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; benzoate benzoyl-CoA; p-cresol 4-hydroxybenzaldehyde 4-hydroxybenzoate 4-hydroxybenzoyl-CoA benzoyl-CoA; phenylacetate phenylacetyl-CoA phenylglyoxylate benzoyl-CoA plus CO₂; 4-hydroxyphenylacetate 4-hydroxyphenylacetyl-CoA 4-hydroxyphenylglyoxylate 4-hydroxybenzoyl-CoA plus CO₂ benzoyl-CoA.     

Expression sites and developmental regulation of genes encoding (1→3,1→4)-β-glucanases in germinated barley

Expression sites of genes encoding (13,14)--glucan 4-glucanohydrolase (EC 3.2.1.73) have been mapped in germinated barley grains (Hordeum vulgare L.) by hybridization histochemistry. A³²P-labelled cDNA (copy DNA) probe was hybridized to cryosections of intact barley grains to localize complementary mRNAs. No mRNA encoding (13,14)--glucanase is detected in ungerminated grain. Expression of (13,14)--glucanase genes is first detected in the scutellum after 1 d and is confined to the epithelial layer. At this stage, no expression is apparent in the aleurone. After 2 d, levels of (13,14)--glucanase mRNA decrease in the scutellar epithelium but increase in the aleurone. In the aleurone layer, induction of (13,14)--glucanase gene expression, as measured by mRNA accumulation, progresses from the proximal to distal end of the grain as a front moving away from, and parallel to, the face of the scutellum.Abbreviations cDNA copy DNA - RNase ribonuclease     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Synthesis of Gal-β-(1→4)-GlcNAc-β-(1→6)-[Gal-β- (1→3)]-GalNAc-α-OBn oligosaccharides bearing O-methyl or O-sulfo groups at C-3 of the Gal residue: specific acceptors for Gal: 3-O-sulfotransferases

Our recent studies have revealed the existence of two distinct Gal: 3-O-sulfotransferases capable of acting on the C-3 position of galactose in a Core 2 branched structure, e.g., Gal14GlcNAc16(Gal13)GalNac1OBenzyl as acceptor to give 3-O-sulfoGal14GlcNAc13(Gal13)GalNAc1OB 20 and Gal14GlcNAc16(3-O-sulfoGal13)GalNAc1OB 23. We herein report the synthesis of these two compounds and also that of other modified analogs that are highly specific acceptors for the two sulfotransferases. Appropriately protected 1-thio-glycosides 7, 8, and 10 were employed as glycosyl donors for the synthesis of our target compounds.     

Structure-function relationships of β- D-glucan endo- and exohydrolases from higher plants

(13),(14)--d-Glucans represent an important component of cell walls in the Poaceae family of higher plants. A number of glycoside endo- and exohydrolases is required for the depolymerization of (13),(14)--d-glucans in germinated grain or for the partial hydrolysis of the polysaccharide in elongating vegetative tissues. The enzymes include (13),(14)--d-glucan endohydrolases (EC 3.2.1.73), which are classified as family 17 glycoside hydrolases, (14)--d-glucan glucohydrolases (family 1) and -d-glucan exohydrolases (family 3). Kinetic analyses of hydrolytic reactions enable the definition of action patterns, the thermodynamics of substrate binding, and the construction of subsite maps. Mechanism-based inhibitors and substrate analogues have been used to study the spatial orientation of the substrate in the active sites of the enzymes, at the atomic level. The inhibitors and substrate analogues also allow us to define the catalytic mechanisms of the enzymes and to identify catalytic amino acid residues. Three-dimensional structures of (13),(14)--d-glucan endohydrolases, (14)--d-glucan glucohydrolases and -d-glucan exohydrolases are available or can be reliably modelled from the crystal structures of related enzymes. Substrate analogues have been diffused into crystals for solving of the three-dimensional structures of enzyme-substrate complexes. This information provides valuable insights into potential biological roles of the enzymes in the degradation of the barley (13),(14)--d-glucans during endosperm mobilization and in cell elongation.     

Occurrence of reducing terminal N-acetylglucosamine 3-sulfate and fucosylated outer chains in acidic N-glycans of porcine zona pellucida glycoproteins

Structures of acidic N-glycans released from porcine zona pellucida glycoproteins by hydrazinolysis were studied. The results indicated that the acidic glycans are of mono- to tetraantennary complex-type with and without N-acetyllactosamine repeating units. Sulfated residues are not only located at the C-6 position of GlcNAc included in the N-acetyllactosamine repeating units, but also at the C-6 position of GlcNAc in the non-repeated antennae and at the C-3 position of reducing terminal GlcNAc residue. Analysis of the oligosaccharide fragments released by endo--galactosidase digestion and by hydrazine/nitrous acid treatment also revealed that various sulfated and non-sulfated forms of fucosylated structures such as Fuc12Gal14(±SO–36)GlcNAc (type 2H), Gal14(Fuc13)(±SO–36)GlcNAc(Lex) and Fuc13 or 4(±SO–36)GlcNAc, are expressed in the repeated outer chain moieties.     

Monoclonal antibody specific for α2→8-linked oligo deaminated neuraminic acid (KDN) sequences in glycoproteins. Preparation and characterization of a monoclonal antibody and its application in immunohistochemistry

Two particular types of sialoglycoproteins have been detected in fish: polysialoglycoproteins containing 28-linked polysialic acid (8Neu5Gc2) _n present in unfertilized Salmonidae fish eggs, and glycoproteins bearing oligo/polymers of deaminated neuraminic acids (KDN) found in the vitelline envelope of the eggs and ovarian fluid. We report the preparation and characterization of a monoclonal antibody specifically recognizing oligo/polymers of KDN sequences in glycoproteins and its application in immunohistochemistry. Fusion of spleen cells from a BALB/c mouse immunized with a KDN-rich glycoprotein (KDN-gp) containing (8KDN2) _n 6(KDN23Gal13GlNAc13) GalNAc1 residues, with mouse myeloma cells yielded a hybrid cell line producing a monoclonal antibody that bound to KDN-gp, but not to KDN-gp depleted of KDN residues. The specificity of the monoclonal antibody, designated mAb.kdn8kdn, was determined by an enzyme-linked immunosorbent assay using KDN-gp samples that varied in KDN content. These antigens were prepared by the selective removal of KDN residues from the native KDN-gp. The mAb.kdn8kdn reacted most strongly with the intact KDN-gp and less strongly with KDN-gp samples containing decreased numbers of KDN residues. The mAb.kdn8kdn was shown specifically to recognize the 28-linked oligo/polyKDN sequences, (8KDN2) _n , and to be able to distinguish specifically (8KDN2) _n chains from (8Neu5Ac2) _n and (8Neu5Gc2) _n chains. The antibody was used successfully for the immunohistochemical detection of reactive KDN epitopes in sections of paraffin embedded rat pancreas. Several controls verified the specificity of the immunohistochemical staining, thus providing the first demonstration of (8KDN2) _n sequences in a mammalian tissue. The mAb.kdn8kdn can now be used to search further for glycoconjugates containing (8KDN2) _n chains and will facilitate studies on their biosynthesis, intracellular localization and function.     

The effect of fumonisin B1 on developing chick embryos: correlation between de novo sphingolipid biosynthesis and gross morphological changes

Fumonisins, mycotoxins produced byFusarium moniliforme and a number of other fungi, are potent inhibitors of the sphinganine-N-acyltransferase, a key enzyme of sphingolipid biosynthesis, and cause neuronal degeneration, liver and renal toxicity, cancer and other injury to animals.In this study we investigated the effect of fumonisin B₁ on the sphingolipids of developing chick embryos. After yolk sac injection of fumonisin B₁ a concentration and time dependent increase of the sphinganine-over-sphingosine ratio of the embryos could be demonstrated. Studies were done to evaluate the effect of fumonisin B₁ on the glycosphingolipid pattern of the chick embryos. In the presence of 72 µg fumonisin B₁ per egg the incorporation of [¹⁴C]galactose and of [¹⁴C]serine into embryonic glycosphingolipids was reduced by about 70%, although the mass of glycosphingolipids was not affected by the toxin. However, a reduction of the wet weight of the treated embryos was observed. Additionally, histological examinations of whole embryo sections of control and fumonisin B₁ treated embryos are presented. Fumonisin B₁ caused haemorrhages under the skin as well as in the liver of treated embyros. A close correlation between disruption of sphingoid metabolism and light microscopic detectable tissue lesions could be observed.Abbreviations Cer ceramide (N-acylsphingosine) - FB₁ fumonisin B₁ - GM3 NeuAc23Gal14Glc11Cer - GD3 NeuAc28NeuAc23Gal14Glc11Cer - GD1a NeuAc23Gal13GalNAc14(NeuAc23)Gal14Glc11Cer - GT1b NeuAc23Gal13GalNAc14(NeuAc28NeuAc23) Gal14Glc11Cer - HPLC high pressure liquid chromatography - PBS phosphate buffered saline - PDMP 1-phenyl-2-dodecanoylamino-3-morpholino-1-propanol - Sa sphinganine - So sphingosine - Sa/So sphinganine-over-sphingosine - TLC thin layer chromatography - Tris Tris(hydroxymethyl)aminomethan Dedicated to Dr Sen-itiroh Hakomori in celebration of his 65th birthday.     

Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Structural diversity of O-polysaccharides and serological classification of <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">garcae</Emphasis> and other strains of genomospecies 4

Novel O-serotypes were revealed among Pseudomonas syringae pv. garcae strains by using a set of mouse monoclonal antibodies specific to the lipopolysaccharide O-polysaccharide. Structural studies showed that the O-polysaccharide of P. syringae pv. garcae NCPPB 2708 is a hitherto unknown linear L-rhamnan lacking strict regularity and having two oligosaccharide repeating units I and II, which differ in the position of substitution in one of the rhamnose residues and have the following structures: I:3)--L Rha (12)-- L Rha (12)--L-Rha-(13)--L Rha (1;II: 2)--L-Rha-(13) -L-Rha-(12)--L-Rha-(13)--L Rha (1.The branched O-polysaccharides of P. syringae pv. garcae ICMP 8047 and NCPPB 588^T have the same L-rhamnan backbone with repeating units I and II and a lateral chain of 14)- or 13)-linked residues of 3-acetamido-3,6-dideoxy-D-galactose (D-Fuc3NAc). Several monoclonal antibody epitopes associated with the L-rhamnan backbone or the lateral -D-Fuc3NAc residues were characterized.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 777–789.Original Russian Text Copyright © 2004 by Ovod, Zdorovenko, Shashkov, Kocharova, Knirel.     

New structures of the O-specific polysaccharides of Proteus. 3. Polysaccharides containing non-carbohydrate organic acids

Four new Proteus O-specific polysaccharides were isolated by mild acid degradation from the lipopolysaccharides of P. penneri 28 (1), P. vulgaris O44 (2), P. mirabilis G1 (O3) (3), and P. myxofaciens (4), and their structures were elucidated using NMR spectroscopy and chemical methods. They were found to contain non-carbohydrate organic acids, including ether-linked lactic acid and amide-linked amino acids, and the following structures of the repeating units were established: 3)--L-QuipNAc-(13)--D-GlcpNAc-(16)--D-GlcpNAc-(1 (S)-Lac-(2–3) (1) 4)--D-GlcpA-(13)--D-GalpNAc-(14)--D-Glcp-(13)--D-Galp-(14)--D-GalpNAc-(1 L-Ala-(2–6) (2) 3)--D-GalpNAc-(16)--D-GalpNAc-(14)--D-GlcpA-(1 L-Lys-(2–6)--D-GalpA-(14) (3) 4)--D-GlcpA-(16)--D-GalpNAc-(16)--D-GlcpNAc-(13)--D-GlcpNAc-(1 (R)-aLys-(2–6) (4) where (S)-Lac and (R)-aLys stand for (S)-1-carboxyethyl (residue of lactic acid) and N-[(R)-1-carboxyethyl]-L-lysine (alaninolysine), respectively. The data obtained in this work and earlier serve as the chemical basis for classification of the bacteria Proteus.     

Conformational analysis of the Sda determinant-containing tetrasaccharide and two mimics in aqueous solution by using 1H NMR ROESY spectroscopy in combination with MD simulations

The conformational behaviour of the spacer-linked synthetic Sd^a tetrasaccharide -d-GalpNAc-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (1) and the two mimics -d-Galp-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (2) and -d-GlcpNAc-(14)-[-Neu5Ac-(23)]--d-Galp-(14)--d-GlcpNAc-(1O)(CH₂)₅NH₂ (3) were investigated by ¹H NMR spectroscopy in combination with molecular dynamics (MD) simulations in water. Experimental 2D ¹H ROESY cross-peak intensities (ROEs) of the tetrasaccharides were compared with calculated ROEs derived from MD trajectories using the CROSREL program. Analysis of these data indicated that the oligosaccharidic skeletons of the compounds 1–3 are rather rigid, especially the -d-Hex(NAc)-(14)-[-Neu5Ac-(23)]--d-Galp fragments. The - Neu5-Ac-(23)--d-Galp linkage occurred in two different energy minima in the three-dimensional structure of the compounds 1–3 in aqueous solution. Experimental data and dynamics simulations supported the finding that the higher energy rotamer (CHEAT forcefield) was abundant in compounds 1 and 3 due to the existence of a hydrogen bond between the carboxyl group of the sialic acid and the acetamido group of the terminal monosaccharide (GalNAc or GlcNAc) unit. The conformational similarity between 1 and 3 leads to the suggestion that also their activities will be alike.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

Characterization of a mouse monoclonal IgG3 antibody to the tumor-associated globo H structure produced by immunization with a synthetic glycoconjugate

Globo H (Fuc12Gal13GalNAc13Gal14Gal14Glc) is a carbohydrate structure that shows enhanced expression in many human carcinomas. From mice immunized with a globo H-KLH (keyhole limpet hemocyanin) synthetic conjugate an IgG3 monoclonal antibody (mAb VK-9) was derived that recognizes the globo H structure. Serological analysis showed that the minimal structure recognized by this mAb was the tetrasaccharide sequence Fuc12Gal13GalNAc13Gal. An isomeric structure with an internal GalNAc linkage was also recognized but less efficiently. mAb VK-9 did not react with many related structures, such as galactosylgloboside, globoside, H type 1, H type 2 blood group structures or fucosyl-gangliotetraosyl ceramide, but did react weakly with globo A ceramide. Not only did mAb VK-9 react with carbohydrate-protein conjugates but it could also recognize globo H-ceramide and human tumor cells expressing globo H. These results suggest that globo H-KLH could be explored as a vaccine in the treatment of carcinoma patients.     

Mutation spectra of N-ethyl-N''-nitro-N-nitrosoguanidine and 1-(2-hydroxyethyl)-1-nitrosourea in Escherichia coli

Summary DNA sequencing was used to determine the specific types of DNA base changes induced following in vivo exposure of Escherichia coli to the ethylating agent N-ethyl-N-nitro-N-nitrosoguanidine (ENNG) and the hydroxyethylating agent 1-(2-hydroxyethyl)-1-nitrosourea (HENU) using the xanthine guanine phosphoribosyltransferase (gpt) gene as the genetic target. We observed that 22/30 of the ENNG-induced mutations were GCAT transitions, 4/30 were ATGC transitions, 3/30 were ATTA transversions, and 1/30 was an ATCG transversion. We observed that 37/40 HENU-induced mutations were GCAT transitions and that the remaining 3/40 were ATGC transitions. A majority of the GCAT transitions induced by ENNG and HENU (68% and 73%, respectively) occurred at the second guanine of the sequence 5-GG(A or T)-3; this sequence specificity was similar to that previously seen with the alkylating agents N-methyl- and N-ethyl-N-nitrosourea (MNU and ENU) and N-methyl-N-nitro-N-nitrosoguanidine (MNNG). A DNA strand preference for the GA changes (antisense strand), previously noted for MNU, ENU, and MNNG, was observed following exposure to HENU and ENNG. The ATGC transitions induced by ENNG, HENU, and ENU also exhibit a sequence specificity with 13/13 mutations occurring at the T of the sequence 5-NTC-3. A strand preference was not apparent for these mutations.     

Cytochrome P-450-dependent catabolism of triethanolamine in Rhodotorula mucilaginosa

The yeast Rhodotorula mucilaginosa was able to grow in media containing triethanolamine or diethanolamine as the sole nitrogen source. During growth in the presence of triethanolamine, extracts of yeast cells contained increased levels of cytochrome P-450 dependent monooxygenase which catalyzed the oxidative N-dealkylation of aminoalcohols. Formation of diethanolamine, ethanolamine and glyoxylate from triethanolamine was demonstrated, and the identity of the products was verified by thin layer chromatography. These observations suggested the following scheme of triethanolamine catabolism: triethanolamine diethanolamine + glycolaldehyde, diethanolamine ethanolamine + glycolaldehyde, ethanolamine NH₃ + glycolaldehyde glycolate glyoxylate glycerate pathway.     

Characterization of four monosialo and a novel disialo Asn N-glycosides from the urine of a patient with aspartylglycosaminuria

We previously reported for the first time two Japanese patients with aspartylglycosaminuria (AGU). A novel disialo Asn N-glycoside (AG-5) has been isolated from the urine of one of the patients in addition to four known monosialo Asn N-glycosides (AG-1 to AG-4) by gel filtration and anion exchange chromatography in this study. Final purification of AG-5 was achieved by an electrochemical chromatographic method, high performance liquid chromatography with pulsed amperometric detector (HPLC-PAD). The yield of AG-5 was approximately 1 mg l^–1 urine. The chemical structures of AG-1 to AG-5 were characterized by gas-liquid chromatography, a permethylation study, fast atom bombardment-mass spectrometry (FAB-MS), and nuclear magnetic resonance (NMR). Based on the structural analysis, AG-5 had the following novel structure: NeuAc2 8NeuAc2 3Gal1 4GlCNAc1 Asn.     

One-pot enzymatic synthesis of the Galα1→3Galβ1→4GlcNAc sequence within situ UDP-Gal regeneration

The trisaccharide Gal13Gal14GlcNAc1O-(CH₂)₈COOCH₃ was enzymatically synthesized, within situ UDP-Gal regeneration. By combination in one pot of only four enzymes, namely, sucrose synthase, UDP-Glc 4-epimerase, UDP-Gal:GlcNAc 4-galactosyltransferase and UDP-Gal:Gal14GlcNAc 3-galactosyltransferase, Gal13Gal14GlcNAc1O-(CH₂)₈COOCH₃ was formed in a 2.2 µmol ml^–1 yield starting from the acceptor GlcNAc1O-(CH₂)₈COOCH₃. This is an efficient and convenient method for the synthesis of the Gal13Gal14GlcNAc epitope which plays an important role in various biological and immunological processes.