Kuriko Yamada

High performance polymer supports for enzyme-assisted synthesis of glycoconjugates☆

Efficient and practical methodology for the construction of carbohydrates, including oligosaccharide derivatives and sphingoglycolipids, was established on the basis of a water-soluble polymer supports having unique linkers that can be cleaved by specific conditions. Novel glycomonomers for the construction of polymer supports were synthesized and copolymerized with acrylamide to give three types of water-soluble glycopolymers having primer sugars through the specific linkers containing (i) p-substituted benzyl group, (ii) L-phenylalanine residue, and (iii) ceramide-mimetic L-serine derivative, respectively. These glycopolymers were employed for sugar elongation reactions with glycosyl transferases such as GlcNAc beta 1,4-galactosyl transferase, beta Gall-->3/4GlcNAc alpha-2,6-sialyl transferase, and beta Gall-->3/4GlcNAc alpha-2,3-sialyl transferase in the presence of each sugar nucleotide as glycosyl donor to afford polymers having N-acetyllactosamine, sialyl alpha-(2-->6) N-acetyllactosamine, and sialyl alpha-(2-->3) lactose residues in excellent yield. Subsequent hydrogenolysis, hydrolysis with alpha-chymotrypsin, or transglycosylation to ceramide with ceramide glycanase proceeds smoothly to give N-acetyllactosamine, a versatile sialyl alpha-(2-->6) N-acetyllactosamine derivative having a terminal amino group, and ganglioside GM3 in high yield.

AN EFFICIENT SYNTHESIS OF SIALOGLYCOCONJUGATES ON A PEPTIDASE-SENSITIVE POLYMER SUPPORT

Abstract A novel method for the enzymatic synthesis of oligosaccharide derivatives on a α-chymotrypsin-sensitive polymer support is described. The primer polymer having N-acetyl-D-glucosamine (GlcNAc) residue through a phenylalanine-containing spacer moiety was successfully elongated with galactosyl and sialyltransferases to give a glycopolymer bearing sialyl α(2→6) N-acetyllactosamine branches in high yield. Subsequent hydrolysis with α-chymotrypsin proceeded smoothly and afforded a versatile sialotrisaccharide derivative having a terminal amino group which can be used for creating neoglycoconjugates.

Artificial Golgi Apparatus: Globular Protein-like Dendrimer Facilitates Fully Automated Enzymatic Glycan Synthesis

Despite the growing importance of synthetic glycans as tools for biological studies and drug discovery, a lack of common methods for the routine synthesis remains a major obstacle. We have developed a new method for automated glycan synthesis that employs the enzymatic approach and a dendrimer as an ideal support within the chemical process. Recovery tests using a hollow fiber ultrafiltration module have revealed that monodisperse G6 (MW = 58 kDa) and G7 (MW = 116 kDa) poly(amidoamine) dendrimers exhibit a similar profile to BSA (MW = 66 kDa). Characteristics of the globular protein-like G7 dendrimer with high solubility and low viscosity in water greatly enhanced throughput and efficiency in automated synthesis while random polyacrylamide-based supports entail significant loss during the repetitive reaction/separation step. The present protocol allowed for the fully automated enzymatic synthesis of sialyl Lewis X tetrasaccharide derivatives over a period of 4 days in 16% overall yield from a simple N-acetyl-d-glucosamine linked to an aminooxy-functionalized G7 dendrimer.

An Engineered Biocatalyst for the Synthesis of Glycoconjugates: Utilization of β1,3-N-Acetyl-D-glucosaminyltransferase from Streptococcus agalactiae Type Ia Expressed in Escherichia coli as a Fusion with Maltose-Binding Protein

A fusion protein composed of β1,3-N-acetyl-D-glucosaminyltransferase (β1,3-GlcNAcT) from Streptococcus agalactiae type Ia and maltose-binding protein (MBP) was produced in Escherichia coli as a soluble and highly active form. Although this fusion protein (MBP-β1,3-GlcNAcT) did not show any sugar-elongation activity to some simple low-molecular weight acceptor substrates such as galactose, Galβ(14)Glc (lactose), Galβ(14)GlcNAc (N-acetyllactosamine), Galβ(14)GlcNAcβ(13)Galβ(14)Glc (lacto-N-tetraose), and Galβ(14)GlcβCer (lactosylceramide, LacCer), the multivalent glycopolymer having LacCer-mimic branches (LacCer mimic polymer, LacCer primer) was found to be an excellent acceptor substrate for the introduction of a β-GlcNAc residue at the O-3 position of the non-reducing galactose moiety by this engineered enzyme. Subsequently, the polymer having GlcNAcβ(13)Galβ(14)Glc was subjected to further enzymatic modifications by using recombinant β1,4-D-galactosyltransferase (β1,4-GalT), α2,3-sialyltransferase (α2,3-SiaT), α1,3-L-fucosyltransferase (α1,3-FucT), and ceramide glycanase (CGase) to afford a biologically important ganglioside; Neu5Aα(23)Galβ(14)[Fucα(13)]GlcNAcβ(13)Galβ(14)GlcCerα(IV3Neu5Acα,III3Fucα-nLc4Cer) in 40% yield (4 steps). Interestingly, it was suggested that MBP-β1,3-GlcNAcT could also catalyze a glycosylation reaction of the LacCer mimic polymer with N-acetyl-D-galactosamine served from UDP-GalNAc to afford a polymer carrying trisaccharide branches, GalNAcβ(13)Galβ(14)Glc. The versatility of the MBP-β1,3-GlcNAcT in the practical synthesis was preliminarily demonstrated by applying this fusion protein as an immobilized biocatalyst displayed on the amylose resin which is known as a solid support showing potent binding-affinity with MBP.

Synthesis and anti-influenza virus activity of novel glycopolymers having triantennary oligosaccharide branches

An efficient method for the synthesis of novel glycopolymers with triantennary sialooligosaccharides showing potent anti-influenza virus activity is described. Polymerisable glycoside of triantennary N-acetyllactosamine [β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-D-glucopyranose, Galβ(1→4)GlcNAc] is synthesised from lactose and 4-(3-hydroxypropyl)-4-nitroheptane-1,7-diol as key starting materials, and converted into water-soluble glycopolymers by radical copolymerisation with acrylamide. Subsequent enzymic sialylation using cytidine 5′-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) with α-2,3-sialyltransferase from porcine liver or with α-2,6-sialyltransferase from rat liver gives novel glycoprotein mimics having potent inhibitory activity against influenza virus infection. It is demonstrated that the present triantennary glycoligands exhibit much higher biological activities than the effects by glycopolymers derived from the simple monovalent-type glycomonomers.

An efficient synthesis of a biantennary sialooligosaccharide analog using a 1,6-anhydro-β-lactose derivative as a key synthetic block

Abstract An efficient and versatile method for the synthesis of a biantennary octasaccharide derivative was established by combined chemical and enzymatic manipulations of 1,6-anhydro-β-lactose as a key starting material. A key 1,6-anhydro-β-lactose derivative having two unprotected hydroxyl groups at C-3′ and C-6′ positions was prepared and employed for the chemical coupling reaction with a known 3,4,6-tri- O -acetyl-2-deoxy-2-phthalimido-β- d -glucopyranosyl imidate to afford a tetrasaccharide derivative with two GlcNAc branches in 69% yield. Enzymatic galactosylation using UDP-Gal with a bovine milk β1,4-galactosyltransferase and subsequent sialylation with a recombinant α2,3-sialyltransferase in the presence of CMP-Neu5Ac proceeded smoothly and gave a desired model compound, a bivalent sialooctasaccharide ( 1 ), in 73% overall yield from the tetrasaccharide intermediate.

Highly efficient oligosaccharide synthesis on water-soluble polymeric primers by recombinant glycosyltransferases immobilised on solid supports

Recombinant beta-1,4-galactosyltranferase (beta 1,4-GalT) and alpha-2,6-sialytransferase (alpha 2,6-SiaT) immobilised covalently with activated Sepharose beads were employed for the practical synthesis of a trisaccharide derivative, Neu-5Ac alpha(2-->6)Gal beta(1-->4)GlcNAc beta-O-(CH2)6-NH2, on a water-soluble primer having GlcNAc residues through a alpha-chymotrypsin-sensitive linker.

Efficient synthesis of non-natural ganglioside (pseudo-GM3) and fluorescent labelled lysoGM3 on the basis of polymer-assisted enzymatic strategy

The versatility of polymer-assisted enzymatic synthesis of non-natural and biologically significant glycolipid derivatives was demonstrated by constructing pseudo-ganglioside GM3 1 having the trisaccharide sequence Neu5Acα- (2→6)Galβ(1→4)Glc and a fluorescent labelled lysoGM3 2.