Synthesis Of N-acetyllactosamine Research Articles

Efficient synthesis of N-acetyllactosamine using immobilized β-galactosidase on a novel 3D polymer support

A novel polymer support was prepared by curing of epoxy resin in ethanol solution in the macropores of a melamine sponge. The produced polymer gel could uniformly deposit on the surface of melamine in either porous or nonporous morphology. The composite sponge with porous coating can be used as a large-sized and well-mass transferred support for the immobilization of β-galactosidase from Bacillus circulans through method of adsorption and crosslinking, and a column reactor was made for the preparation of N-acetyllactosamine in a sealed circulation way. The porosity and specific surface area of the support were 91.6 % and 46 m2/g, respectively. The loading amount and the specific activity of immobilized β-galactosidase under the optimal immobilization conditions were 41.2 mg/gsupport and 16.5 U/mgprotein, respectively. In the biosynthesis of N-acetyllactosamine lactose and N-acetylglucosamine were used as donor and acceptor, respectively. Under optimized conditions the N-acetyllactosamine yield reached 54 % within 150 min at 50 °C. After 10 cycles, the immobilized β-galactosidase retained 70 % of the original activity.

Improving β-Galactosidase-Catalyzed Transglycosylation Yields by Cross-Linked Layer-by-Layer Enzyme Immobilization

The biotransformation of lactose into gut-bioactive glycans catalyzed by β-galactosidase can give economic value to lactose-rich side streams generated in the food or the dairy industry. Herein, we study the immobilization of the commercially used β-galactosidase from Bacillus circulans onto silica particles using an enzyme immobilization technology involving a cross-linked layer-by-layer encapsulation method. The immobilized β-galactosidase was used for the synthesis of N-acetyllactosamine (LacNAc) as an important precursor for numerous bioactive compounds and a prebiotic in itself. Techniques including molecular analysis, enzyme activity determination, secondary structure analysis, thermodynamic characterization, and the determination of thermal and operational stability were conducted to characterize the immobilized enzyme. Changes in the activity of the enzyme after immobilization were attributed to possible changes in electrostatic, covalent, and protein–protein interactions. Immobilization significantly improved the enzymatic LacNAc yield compared to the free enzyme. In turn, this improved the economics and the sustainability of the process. The immobilized enzyme encapsulated in multilayer films was significantly more stable in the presence of divalent cations and its thermostability also substantially increased with the thermal denaturation activation energy increasing from 53 to 294 kJ mol–1. The immobilized enzyme was successfully reused in eight consecutive reaction cycles with no significant reduction in the LacNAc yield. The improved transgalactosylation yield and productivity, higher stability, and reusability obtained with this immobilization method provide new opportunities for industrial applications.

Synthesis of N-acetyllactosamine based branched hexasaccharide

The protected spacer-armed branched tri-N-acetyllactosamine has been synthesized as a building block for the synthesis of biantennary ABH blood group antigens. The key synthetic step is the simultaneous glycosylation of the 3′- and 6′-OH groups of an N-acetyllactosamine glycosyl acceptor by N-Troc-lactosamine trichloroacetimidate.

Chemoenzymatic Synthesis of Galectin Binding Glycopolymers.

Chemoenzymatic synthesis is an important strategy for the formation of glycopolymers. The use of a smaller number of traditional chemical steps and enzyme catalyzed reactions increases the yield of glycopolymer that can be produced by reducing the overall number of synthetic steps. In addition, chemoenzymatic routes are likely to be more accessible to those without a background in carbohydrate synthesis, making glycopolymers more available for studies across a broader range of scientists. Here, the enzymatic addition of galactose to N-acetylglucosamine functionalized glycodendrimers reduced the requisite number of synthetic steps for the full chemical synthesis of N-acetyl lactosamine (Lac NAc) functionalized dendrimers to four steps. Unpurified cell lysate was used in the enzyme catalyzed glycosylation, and product glycodendrimers were readily purified by dialysis after enzymatic degradation of all protein components of the lysate in the crude reaction mixture. Lac NAc functionalized dendrimers were used very effectively in homotypic cancer cellular aggregation assays and were found to either inhibit or enhance galectin-3 mediated cancer cellular aggregation, with differences in outcomes dependent on the generation of Lac NAc functionalized dendrimers that were used.

Enzymatic Synthesis of N-Acetyllactosamine (LacNAc) Type 1 Oligomers and Characterization as Multivalent Galectin Ligands

Repeats of the disaccharide unit N-acetyllactosamine (LacNAc) occur as type 1 (Galβ1, 3GlcNAc) and type 2 (Galβ1, 4GlcNAc) glycosylation motifs on glycoproteins and glycolipids. The LacNAc motif acts as binding ligand for lectins and is involved in many biological recognition events. To the best of our knowledge, we present, for the first time, the synthesis of LacNAc type 1 oligomers using recombinant β1,3-galactosyltransferase from Escherichia coli and β1,3-N-acetylglucosaminyltranferase from Helicobacter pylori. Tetrasaccharide glycans presenting LacNAc type 1 repeats or LacNAc type 1 at the reducing or non-reducing end, respectively, were conjugated to bovine serum albumin as a protein scaffold by squarate linker chemistry. The resulting multivalent LacNAc type 1 presenting neo-glycoproteins were further studied for specific binding of the tumor-associated human galectin 3 (Gal-3) and its truncated counterpart Gal-3∆ in an enzyme-linked lectin assay (ELLA). We observed a significantly increased affinity of Gal-3∆ towards the multivalent neo-glycoprotein presenting LacNAc type 1 repeating units. This is the first evidence for differences in glycan selectivity of Gal-3∆ and Gal-3 and may be further utilized for tracing Gal-3∆ during tumor progression and therapy.

Automated Glycan Assembly of Oligo-N-Acetyllactosamine and Keratan Sulfate Probes to Study Virus-Glycan Interactions

Summary Oligo- N -acetyllactosamine (LacNAc) and keratan sulfate (KS) glycans exert crucial functions in disease-relevant processes, including cancer formation, inflammation, and viral infection. To facilitate structure-activity studies with these glycans, we established a universal strategy to synthesize linear and branched LacNAc as well as differentially sulfated KS oligosaccharides by automated glycan assembly. We synthesized oligosaccharides as long as hexamers by combining four monosaccharide building blocks. Key to the strategy was installing three orthogonal protection groups, 9-fluorenylmethoxycarbonyl (Fmoc), levulinoyl (Lev) ester, and 2-naphthylmethyl (Nap) ether, which were selectively removed from a common oligosaccharide precursor for differential sulfation. Microarrays presenting the synthetic oligosaccharides revealed a specific interaction between a disulfated KS tetrasaccharide and the adeno-associated virus AAVrh10 gene-therapy vector, which was further corroborated by surface plasmon resonance studies. Thus, KS represents a novel receptor candidate for AAVrh10. These insights could have implications for cell-type-specific gene-delivery approaches.

Donor substrate promiscuity of bacterial β1–3-N-acetylglucosaminyltransferases and acceptor substrate flexibility of β1–4-galactosyltransferases

β1–3-N-Acetylglucosaminyltransferases (β3GlcNAcTs) and β1–4-galactosyltransferases (β4GalTs) have been broadly used in enzymatic synthesis of N-acetyllactosamine (LacNAc)-containing oligosaccharides and glycoconjugates including poly-LacNAc, and lacto-N-neotetraose (LNnT) found in the milk of human and other mammals. In order to explore oligosaccharides and derivatives that can be synthesized by the combination of β3GlcNAcTs and β4GalTs, donor substrate specificity studies of two bacterial β3GlcNAcTs from Helicobacter pylori (Hpβ3GlcNAcT) and Neisseria meningitidis (NmLgtA), respectively, using a library of 39 sugar nucleotides were carried out. The two β3GlcNAcTs have complementary donor substrate promiscuity and 13 different trisaccharides were produced. They were used to investigate the acceptor substrate specificities of three β4GalTs from Neisseria meningitidis (NmLgtB), Helicobacter pylori (Hpβ4GalT), and bovine (Bβ4GalT), respectively. Ten of the 13 trisaccharides were shown to be tolerable acceptors for at least one of these β4GalTs. The application of NmLgtA in one-pot multienzyme (OPME) synthesis of two trisaccharides including GalNAcβ1–3Galβ1–4GlcβProN3 and Galβ1–3Galβ1–4Glc was demonstrated. The study provides important information for using these glycosyltransferases as powerful catalysts in enzymatic and chemoenzymatic syntheses of oligosaccharides and derivatives which can be useful probes and reagents.

Sustainable synthesis of N-acetyllactosamine using an immobilized β-galactosidase on a tailor made porous polymer

An efficient enzymatic synthesis of N-acetyllactosamine has been developed in biosolvents, mediated by the action of an immobilized β-galactosidase on a tailor made porous polymer.

Synthesis of multivalent N-acetyl lactosamine modified quantum dots for the study of carbohydrate and galectin-3 interactions

Ternary core/shell CdSeS/ZnS-QDs coated with N-acetyl lactosamine was prepared as a fluorescent probe to study the interactions of N-acetyl lactosamine and galectin-3. The synthesis of N-acetyl lactosamine was achieved through the ‘azidoiodoglycosylation’ method. The amount of ligand coated on QDs was determined by 1H NMR and ICP-OES. The interactions between carbohydrates and galectin-3 were measured using SPR. The results revealed that the affinity of galectin-3 with di- and multivalent N-acetyl lactosamine increased 20 and 184-fold, respectively. The prepared glyco-QDs could be used as an efficient fluorescent probe to study carbohydrates and galectin-3 interactions.

Use of Ionic Liquids to Increase the Yield and Enzyme Stability in the β-Galactosidase Catalysed Synthesis of N-Acetyllactosamine

The use of ionic liquids as alternative solvents for enzyme catalysis was investigated. β-Galactosidase from Bacillus circulans catalyses the synthesis of N-acetyllactosamine starting from lactose and N-acetylglucosamine in a transglycoslyation reaction. The addition of 25% v/v of 1,3-di-methyl-imidazol-methyl sulfate [MMIM] [MeSO4] as a water-miscible ionic liquid suppresses the secondary hydrolysis of the formed product, resulting in doubling the yield to almost 60%. The enzyme can be reused several times after ultrafiltration of the reaction mixture without loss of activity. Results of different amounts of ionic liquids in the reaction medium on the thermostability of the galactosidase as well as on oxidoreductases are presented as well.

Occurrence of poly- N-acetyllactosamine synthesis in Sf-9 cells upon transfection of individual human β-1,4-galactosyltransferase I, II, III, IV, V and VI cDNAs

Lectin blot analysis of membrane glycoprotein samples from Sf-9 cells upon transfection of individual human β-1,4-galactosyltransferase (β-1,4-GalT) I, II, III, IV, V et VI cDNAs showed that the endogenous N-linked oligosaccharides are galactosylated (Guo et al., Glycobiology (2001), in press). Further analysis revealed that membrane glycoprotein samples from all the gene-transfected cells are also reactive to Lycopersicon esculentum agglutinin (LEA) et Datura stramonium agglutinin (DSA), both of which bind to oligosaccharides with poly- N-acetyllactosamine chains while no lectin reactive protein bands are detected when blots are pretreated with a mixture of diplococcal β-1,4-galactosidase et jack bean β- N-acetylhexosaminidase or N-glycanase. Analysis of endo-β-galactosidase-digestion products revealed the presence of the Gal1→GlcNAc1→Gal and/or GlcNAc1→Gal structures in the gene-transfected cells. When the homogenates of the gene-transfected cells were used as enzyme sources towards oligosaccharides with the GlcNAcβ1→(3Galβ1→4GlcNAc) 1-3 structures, human recombinant β-1,4-GalTs I et II galactosylated these oligosaccharides more effectively than other β-1,4-GalTs. These results indicate that β-1,4-GalTs I-VI can synthesize poly- N-acetyllactosamine chains with β-1,3- N-acetylglucosaminyltransferase.

A highly selective synthesis of N-acetyllactosamine catalyzed by immobilised β-galactosidase from Bacillus circulans

Immobilisation of the β-galactosidase of Bacillus circulans on Eupergit C gave a highly selective and stable biocatalyst. The immobilised enzyme catalysed the transfer of the β-galactosyl residue of p-nitrophenyl β- d-galactopyranoside to N-acetylglucosamine with high selectivity for transfer to the 4-position of the glycosyl acceptor.

The efficient enzymatic synthesis of N-acetyllactosamine in an organic co-solvent

In the presence of β-galactosidase from Bifidobacterium bifidum, N-acetyllactosamine was synthesized in significantly enhanced yield compared with earlier routes. Different proportions of the (1→4)- and (1→6)-linked forms were obtained depending on the choice of enzyme and reaction conditions, viz. the nature of added organic co-solvent (20–80% of 2-ethoxy ethyl ether, trimethyl phosphate, or acetone). The β-(1→4)-linked disaccharide was the major product and the β-(1→6)-linked disaccharide was the minor product. With β-galactosidases from P. multicolor, A. oryzae, B. longum the β-(1→6) linkage was exclusively synthesized. Procedures for optimising the yield of N-acetyllactosamine are discussed. An immobilized enzyme on a nylon powder column was used for the efficient recycling of enzyme and synthesizing the disaccharide.

On the Effect of the Aglycon Structure of Three Aryl β-D-Galactosides on the Yield and the Regioselectivity of the Transglycolytic Synthesis of N-Acetyllactosamine

ABSTRACT We examined the effect as donors of three aryl β-D-galactosides (i.e. p-nitrophenyl β-D-galactopyranoside, o-nitrophenyl β-D-galactopyranoside and phenyl β-D-galacto-pyranoside) on the regioselectivity and the yield of the synthesis of N-acetyllactosamine obtained from the transglycosylation reaction catalyzed by a crude preparation of β-D-galactosidase from Bacillus circulans at 25 °C, 37 °C and 55 °C, respectively. Using p-nitrophenyl β-D-galactopyranoside the reaction results were fully regiospecific at all the temperatures considered: the maximum molar yield (74%) was obtained at an incubation temperature of 55 °C. Using o-nitrophenyl β-D-galactopyranoside as the donor the reaction was still highly regioselective and the maximum molar yield (50%) was achieved at an incubation temperature also of 55 °C. Using phenyl β-D-galactopyranoside transglycolytic products appear only at an incubation temperature of 55 °C but at very low molar yield (about 14%) and lower regioselectivity.

Differences in N-acetyllactosamine synthesis between beta-1,4-galactosyltransferases I and V.

Unlike classical beta-1,4-galactosyltransferase (beta-1,4-GalT I), beta-1,4-GalT V (formerly IV**) has little activity towards 1 mM N-acetylglucosamine [Sato et al. (1998) Proc Natl Acad Sci USA 95:472-477]. The human beta-1,4-GalTs I and V were expressed individually in Sf-9 cells by transfection of the full coding sequences, and their N-acetyllactosamine synthetase activities were determined towards different N-acetylglucosamine concentrations. Kinetic studies using the cell homogenates as an enzyme source revealed that beta-1,4-GalTs I and V possess Km values of 0.6 mM and 33 mM towards N-acetylglucosamine, and of 48 microM and 41 microM towards UDP-Gal, respectively. No significant inhibition of N-acetyllactosamine synthesis with alpha-lactalbumin was observed for beta-1,4-GalT V but the significant inhibition with alpha-lactalbumin was observed for beta-1,4-GalT I.

Use of Escherichia coli polyphosphate kinase for oligosaccharide synthesis.

The Escherichia coli polyphosphate kinase (PPK) has been known to catalyze the reversible transfer of phosphate molecules between ATP and polyphosphate (poly(P)). It has also been found that the PPK catalyzes the kination of not only ADP but also other nucleoside diphosphates (NDPs) using poly(P) as a phosphate donor, yielding nucleotide triphosphates (NTPs). We used the PPK and poly(P) in place of pyruvate kinase and phosphoenol pyruvate for NTP regeneration followed by synthesis of sugar nucleotides in a cyclic synthesis system for oligosaccharides. It was confirmed that the PPK efficiently catalyzed the UTP regeneration in the cyclic system of N-acetyllactosamine synthesis. This novel activity of PPK enables us to perform the practical synthesis of oligosaccharides.

Chemical and Enzymatic Synthesis of Glycoconjugates 2. High Yielding Regioselective Synthesis of N-Acetyllactosamine by Use of Recombinant Thermophilic Glycosidases Library

β-Galactosidase activities from the recombinant thermophilic CLONEZYME™ glycosidase library were screened at 70 °C for catalysis of a transgalactosylation from o-nitrophenyl-β-galactopyranoside to N-acetylglucosamine. Three thermophilic glycosidases (Gly001-06, -07 and -09) were found to produce predominantly the β(1–4)-linked isomer, Gal β(1–4)GlcNAc with up to 61% yield and less than 10% of the hydrolysis side reaction product. Thus, commercial recombinant thermophilic enzyme libraries constitute a novel class of biocatalysts for preparative organic synthesis. © 1997 Elsevier Science Ltd.

High-Yield Synthesis ofN-Acetyllactosamine by Regioselective Transglycosylation

The synthesis ofN-acetyllactosamine (D-Galpβ1-4D-GlcpNAc) with very low contamination of its isomerN-acetylallolactosamine (D-Galpβ1-6D-GlcpNAc) was obtained by use of regioselective transglycosylation activity of β-galactosidase fromBacillus circulansusing lactose as the donor of D-Galpand D-GlcpNAc as the acceptor. The reaction was conducted at 15°C and at pH 5.0. The incubation time was considerably reduced and the yield improved 100% with respect to the best results so far described in the literature.

A Novel Three-Enzyme Reaction Cycle for the Synthesis of N-Acetyllactosamine with in Situ Regeneration of Uridine 5‘-Diphosphate Glucose and Uridine 5‘-Diphosphate Galactose

A new three-enzyme reaction cycle consisting of sucrose synthase, UDP glucose 4‘-epimerase, and human β-1,4-galactosyltransferase was established for the synthesis of N-acetyllactosamine (LacNAc) with in situ regeneration of UDP galactose. We found that UDP glucose 4‘-epimerase is reductively inactivated in the presence of UMP and acceptor substrates of β-1,4-galactosyltransferase. Reactivation of UDP glucose 4‘-epimerase by the transition state analogues dUDP or dTDP 6-deoxy-d-xylo-4-hexulose in combination with the repetitive batch technique enabled us to use the native enzymes for 11 days in this cycle. With 10 U of sucrose synthase, 5 U of UDP glucose 4‘-epimerase, and 1.25 U of β-1,4-galactosyltransferase, 594 mg of LacNAc could be synthesized. N-Acetyllactosamine was also subsequently converted to Neu5Acα2,6Galβl,4GlcNAc with α-2,6-sialyltransferase and CMP-Neu5Ac.

Use of transformed whole yeast cells expressing β-1,4-galactosyltransferase for the synthesis of N-acetyllactosamine

We report the use of transformed whole yeast cells for the synthesis of N-acetyllactosamine (LacNAc), which is the core-disaccharide of the anti-inflammatory agent Sialyl-Lewis X . Transformed whole yeast cells, which carry plasmids for the heterologous expression of a soluble human b-1,4-galactosyltransferase (GalT), proved to be a suitable catalyst for the synthesis of LacNAc by employing multi-enzyme-systems. This convenient use of the recombinant enzyme, taken together with the high availability of the transformed yeast through fermentation technology, will provide sufficient amounts of glycosyltransferase for chemoenzymatic synthesis of oligosaccharides.