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.     

Effects of supplied cinnamic acids and biosynthetic intermediates on the anthocyanins accumulated by wild carrot suspension cultures

Anthocyanins isolated and characterized from the wild carrot suspension cultures used here were 3-O--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D<-galactopyranosylcyanidin (1), 3-O-[-D- xylopyranosyl-(12)--D-galactopyranosyl]cyanidin (2), 3-O-(6-O-sinapoyl)--D-glucopyranosyl-(16)-[-D- xylopyranosyl-(12)-]-D-galactopyranos ylcyanidin (3), 3-O-(6-O-feruoyl)--D-glucopyranosyl-(16)-[- D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (4), 3-O-(6-O-coumaroyl)--D-glucopyranosyl-(16)- [-D-xylopyranosyl-(12)-]-D-galactopyrano sylcyanidin (5), 3-O-[6-O-(3,4,5-trimethoxycinnamoyl)]-- D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (6), 3-O-[6-O-(3,4-dime- thoxycinnamoyl)]--D-glucopyranosyl-(16)-[-D-xylopyranosyl-(12)-]-D-galactopyranosylcyanidin (7), 3-O-[(6-O-sinapoyl)--D-glucopyranosyl-(16)--D-galactopyranosyl]cyanidin (8), and 3-O-(-D-galactopyranosyl)cyanidin (9). Except when cinnamic acids were provided in the culture medium, the major anthocyanin present in the two clones examined was 2. When the naturally occurring and some non-naturally occurring cinnamic acids were provided individually in the medium, 1 and 2 were minor components and the anthocyanin acylated with the supplied cinnamic acid, namely 3, 4, 5, 6, or 7 was the major anthocyanin present in the tissue. When caffeic acid was provided the major anthocyanin in the tissue was 4, thereby suggesting that the caffeic acid was methylated before its use in anthocyanin biosynthesis. Other cinnamic acids supplied had limited effects on the anthocyanins accumulated and appeared not to result in the accumulation of new anthocyanins by the tissue. Thus the tissue can use some but not all analogues of sinapic acid to acylate anthocyanins. Additional anthocyanins were detected in extracts of the wild carrot tissue cultures using mass spectrometry (both MS/MS and HPLC/MS). The additional compounds detected have also been found in cultures of black carrot, an Afghan cultivar of Daucus carota ssp. sativa and the flowers of wild carrot giving no evidence for qualitative differences in the anthocyanins synthesized by subspecies, cell cultures from subspecies, or clones from cell cultures. There are major differences in the amounts of individual anthocyanins found in cultures from different subspecies and in different clones from cell cultures. Here anthocyanins without acyl groups were usually found in the tissues and their accumulation is discussed. On the basis of the structures of the isolated anthocyanins, a likely pathway from cyanidin to the accumulated anthocyanins is proposed and discussed.Abbreviations Sin sinapoyl - Fer feruoyl - 4-Coum. 4-coumaroyl - 3,4-MeO₂Cin 3,4-dimethoxyeinnamoyl - 3,4,5-MeO₃Cin 3,4,5-trimethoxycinnamoyl - Cya cyanidin     

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.     

Unbalanced translocation between chromosomes 2 and 7 with de novo deletion of band 35 on the long arm of chromosme 2

Summary Clinical and cytogenetical findings are described in an infant with a de novo deletion of the long arm of chromosome 2. The boy's karyotype is 46,XY, rec(2)delq,t(2;7) (2pter2q34::7p217pter) (7qter7p21::2q362qter). He showed developmental retardation, low-set ears, micrognathia, short neck, abundant skin of the neck, tetralogy of Fallot, bipartite labialike scrotum, clitorislike penis, cryptorchism, and deformities of the hands and feet.     

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.     

Possible intrachromosomal duplication in a case of trisomy 9p

Summary A 5-year-old boy with multiple minor anomalies and mental retardation was found to have chromosomal condition of 46,XY,inv dup(9p)(pterp13::p21p24::p13qter). The clinical features of the propositus fit well with those of trisomy 9p which have been established to be a clinical entity.     

Regional mapping of catalase and Wilms tumor—aniridia,genitourinary abnormalities,and mental retardation triad loci to the chromosome segment 11p1305→p1306

Summary Gene dosage effects for catalase (CAT) were studied in two unrelated patients with an interstitial deletion involving 11p13 to determine precisely the sites of the genes for CAT and the Wilms tumor—aniridia, genitourinary abnormalities, and mental retardation triad (WAGR) in the 11p13 band. Case 1 had the aniridia-Wilms tumor association, and case 2 showed the AGR triad. The karyotypes identified by high resolution banding techniques were 46,XY,del(11)(pterp13::p11.11qter) for case 1 and 46,XY,t(2;17) (q23;q25), del(11) (pterp13::p11.2 qter) for case 2. In both cases, the distal breakpoints of the deleted chromosomes 11 appeared to have occurred on the middle portion of 11p13 (11p1305p1306). The level of erythrocyte CAT activities in case 1 was reduced (47% of normal), while that in case 2 was normal. The results suggested not only that both the CAT and WAGR should be mapped to chromosome region 11p1305p1306, but also that in this region the CAT locus is more distally placed than the WAGR locus. Because of the proximity of the two gene loci, assays of erythrocyte CAT may be useful to identify a submicroscopic deletion in some patients with sporadic aniridia and to predict a risk of developing Wilms tumor.     

Regional mapping of the locus for hexokinase-1 (HK1) to 10p11→q23 by gene dosage in human fibroblasts

Summary The hexokinase (HK) activity in human fibroblasts was close to that expected for a gene dosage effect in a mosaic cell line with about 86% trisomy 10 cells (64% greater than four control lines with normal karyotypes). There was no dosage effect for HK in the cell line that was trisomic for 10q24qter, nor in the cell line monosomic for 10pterp12. The data suggest an assignment of the HK1 locus (the only hexokinase in fibroblasts) to 10p11q23 by exclusion.     

Enhancement of epidermal growth factor receptor expression on glioma cells by recombinant tumor necrosis factor α

Summary Recombinant tumor necrosis factor (rTNF; optimal dose 1000 U/ml) significantly increased the density of epidermal growth factor receptor (EGF-R) in three of four glioma cell lines in culture as determined by binding analysis of anti-EGF-R monoclonal antibody (mAb) 425. Since enhancement of EGF-R expression by rTNF- was inhibited when cells were treated with the protein synthesis inhibitor cycloheximide, the effects of rTNF may be protein-synthesis-dependent. The dose of rTNF that was optimal for up-regulation of EGF-R on glioma cells did not inhibit the growth of these cells.¹²⁵I-labeled mAb 425 lysed glioma cells in culture following its internalization into the cells. After glioma cells had been treated with rTNF, the growth-inhibitory effects of the mAb were significantly enhanced, probably a reflection of the increase in EGF-R density on the tumor cell surfaces. The rTNF effects were specific to the EGF-R and did not affect unrelated glioma-associated antigens. In our previous clinical trials,¹²⁵I-labeled mAb 425 showed immunotherapeutic effects in glioma patients. The present study provides the basis for considerations of combined immunotherapy of glioma patients with¹²⁵I-labeled mAb 425 and rTNF.     

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.     

A model of associative memory in the brain

A model of associative memory for time varying spatial patterns is proposed and simulated on a digital computer. This is a network composed of many neuron-like elements, and shows an ability for associative memory similar to that of the brain.Suppose a number of sequences of spatial patterns are presented to this network, for example, 12345, ABC, and so on. Then, these patterns are memorized in the network. After that, if any part of one of these sequences, say 23, is presented to the circuit, the rest of the sequence, 45, is recalled following to it. It resembles to such a situation — if we hear a part of a melody which we have memorized in the past, the rest of the melody is recalled even after it is stopped half-way. Although the recalled patterns are not always 100% correct, they are not completely destroyed even if the presented patterns are imperfect.     

Synthesis of FimH receptor-active manno-oligosaccharides by reverse hydrolysis using α-mannosidases from Penicillium citrinum,Aspergillus phoenicis and almond

Recombinant Penicillium citrinum -1,2-mannosidase, expressed in Aspergillus oryzae, was employed to carry out regioselective synthesis of -d-mannopyranosyl-(12)-d-mannose. Yields (w/w) of 16.68% disaccharide, 3.07% trisaccharide and 0.48% tetrasaccharide were obtained, with 12 linkages present at 98.5% of the total linkages formed. Non-specific -mannosidase from almond was highly efficient in reverse hydrolysis and oligosaccharide yields of 45–50% were achieved. The products of the almond mannosidase were a mixture of disaccharides (30.75%, w/w), trisaccharides (12.26%, w/w) and tetrasaccharides (1.89%, w/w) with 12, 13 and 16 isomers. -1,2-linkage specific mannosidase from P. citrinum and -1,6-linkage-specific mannosidase from Aspergillus phoenicis were used in combination to hydrolyse the respective linkages from the mixture of isomers, resulting in -d-mannopyranosyl-(13)-d-mannose in 86.4% purity. The synthesised oligosaccharides can potentially inhibit the adhesion of pathogens by acting as "decoys" of receptors of type-1 fimbriae carried by enterobacteria.     

Synthesis of oligosaccharides using immobilised 1,2-a-mannosidase from Aspergillus phoenicis: Immobilisation-dependent modulation of product spectrum

A specific 1,2-a-mannosidase from A. phoenicis was immobilised on China clay, cellulose DE-52 and by entrapment in sodium alginate beads with respective yields of 19%, 16% and 18% in synthesis reactions consisting of 80% (w/w) mannose. Mana16man was the predominant product with China clay- and DE-52-immobilised enzymes with lesser amounts of mana12 and mana13. With the alginate bead-immobilised enzyme, mana12 was the predominant linkage formed.     

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.     

Identification of a familial 19/21 translocation by Q and G band patterns

Summary The analysis of a familial 19/21 translocation revealed the formula: 46,t(19;21) (p or q ter q 13 or p 13:; p ter q22::p 13 or q 13 p or q ter). Within this family, only members with a normal karyotype or a balanced translocation occurred, including a trisomy 21. These findings may be derived from a meiotic configuration constructed here which is assumed to prevail in the translocation carriers.

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Zusammenfassung Die Analyse einer familiären 19/21-Translokation mit Hilfe der Bändertechniken ergab den Befund: 46,t(19;21) (p or q ter q 13 or p 13:; p ter q 22 :: p 13 or q 13 p or q ter). In der Familie finden sich neben Personen mit normalem Karyotyp nur solche, bei denen die Translokation balanciert ist, darunter eine Trisomie 21. Diese Befunde lassen sich unter der Annahme erklären, daß eine hier abgeleitete Paarungskonfiguration in der Meiose der Translokationsträger vorherrscht.

     

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.     

The identification of two mis-sense mutations at the PAH gene locus in a Turkish patient with phenylketonuria

Summary DNA sequence analysis of the 13 exons and intron/exon boundaries of the phenylalanine hydroxylase (PAH) gene has detected two base transitions, resulting in mis-sense mutations, in the genomic DNA of a Turkish patient (E1) with phenylketonuria (PKU). The Leu⁴⁸Ser amino acid substitution was associated with the mutant haplotype 3 allele and the Glu²²¹Gly amino acid substitution with the mutant haplotype 4 allele of this family. Allele-specific oligonucleotide (ASO) dotblot analysis subsequently detected the Leu⁴⁸Ser mutation in the haplotype 4 PKU alleles of nine (18.8%) of the 48 unrelated Caucasian PKU families investigated. This mutation results in mild PKU in the homozygous state. The Glu²²¹Gly mutation has only been detected within patient E1 and his father.     

Further regional localization of the genes for human acid alpha glucosidase (GAA), peptidase D (PEPD), and alpha mannosidase B (MANB) by somatic cell hybridization

Summary We have further regionally localized the gene for human acid alpha glucosidase (GAA) to 17q21q23 by examinaiton of hybrid clones derived from a fusion between human fibroblasts carrying a 17/19 balanced translocation (17pter17q23::19p13.319pter; 19qterp13.3::17q2317qter) and a mouse line deficient in thymidine kinase. These hybrids were constantly maintained in HAT selective media in order to select for the presence of the human thymidine kinase gene on the intact chromosome 17 (17q21-q22) or the 17/19 (17pter17q23::19p13.319pter) translocation chromosome. We detected human GAA by rocket immunoelectrophoresis, using a human specific heterologous antibody raised against human acid alpha glucosidase (GAA) (Honig et al. 1984). Three secondary clones, which contained the 17/19 translocation and no intact chromosome 17 or 19, were still positive for GAA. Two of these secondary clones contained the distal portion of the 17/19 translocation chromosome, with a break in the band 17q21 (probably at 17q21.2), attached to a mouse chromosome. Combined with earlier results (Weil et al. 1979; Nickel et al. 1982; Honig et al. 1984), the gene for GAA can be assigned to 17q21.217q23. Additionally, these clones were negative for human peptidase D (PEPD), alpha mannosidase B (MANB), and phosphohexose isomerase (PHI). Combined with previous results (Ingram et al. 1977; Bruns et al. 1979), these results exclude the genes for PEPD and MANB from 19pter19p13.3 and confirm the exclusion of the gene for PHI from this segment of chromosome 19 (Wilson et al. 1984; Ingram et al. 1977).     

The coagulation factor VII regulator is located on 8p23.1

Summary Cytogenetic and coagulation studies have been performed on two patients with different abnormalities of chromosome 8, i.e. del(8p23.1pter) and dup(8q23.1qter). Results confirm the existence of a regulatory mechanism for clotting factor VII on chromosome 8 and define its location to the p23.1p23.2 region.     

Synthesis of Aminoethyl Glycosides of the Ganglioside GM1 and Asialo-GM1 Oligosaccharide Chains

4-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)--D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-1-thio-2-trichloroacetamido--D-galactopyranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-(2,3-di-O-benzyl-6-O-benzoyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, in 69% yield. The complete removal of O-protecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction of the aglycone azide group to amine led to the target aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of asialo-GM₁ ganglioside in 72% overall yield. Selective 3-O-glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,6-di-O-benzyl--D-galactopyranosyl)--D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero--D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid afforded 2-azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)oate]-(2 3)-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside, the selectively protected derivative of the oligosaccharide chain of GM₃ ganglioside, in 79% yield. Its 4-O-glycosylation with a disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl--D-galactopyranosyl) 1-thio-2-trichloroacetamido--D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl--D-galactopyranosyl)-(1 3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido--D-galactopyranosyl)-(1 4)-{[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosyl)onate]-(2 3)}-(2,6-di-O-benzyl--D-galactopyranosyl)-(1 4)-2,3,6-tri-O-benzyl--D-glucopyranoside in 85% yield. The resulting pentasaccharide was O-deprotected, its N-trichloroacetyl group was replaced by N-acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of -D-Gal-(1 3)--D-GalNAc-(1 4)-[-D-Neu5Ac-(2 3)]--D-Gal-(1 4)--D-Glc-OCH₂CH₂NH₂ containing the oligosaccharide chain of GM₁ ganglioside.