Katsuji Haneda

A novel disaccharide substrate having 1,2-oxazoline moiety for detection of transglycosylating activity of endoglycosidases.

A disaccharide substrate of Manbeta1-4GlcNAc-oxazoline 2 was designed and synthesized as a novel probe for detection of the transglycosylating activity of endoglycosidases. A regio- and stereoselective transglycosylation reaction of 2 to GlcNAcbeta1-O-pNP or Dns-Asn(GlcNAc)-OH catalyzed by endo-beta-N-acetylglucosaminidase from Mucor hiemalis (Endo-M) and endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) has been demonstrated for the first time, resulting in the core trisaccharide derivative Manbeta1-4GlcNAcbeta1-4GlcNAcbeta1-O-pNP 8 (or -(Dns)Asn-OH). Interestingly, the transglycosylation proceeds irreversibly; the resulting trisaccharide 8 was not hydrolyzed by Endo-M and Endo-A. Based on these results, a new mechanism including an oxazolinium ion intermediate has been proposed for the endoglycosidase-catalyzed hydrolysis or transglycosylation.

Transglycosylation of intact sialo complex-type oligosaccharides to the N-acetylglucosamine moieties of glycopeptides by Mucor hiemalis endo-β-N-acetylglucosaminidase

The endo-beta-N-acetylglucosaminidase (endo-beta-GlcNAc-ase) of Mucor hiemalis, endo-M, was found to transfer the sialo complex-type oligosaccharides from transferrin glycopeptide to the N-acetylglucosamine (GlcNAc) moieties of peptidyl-GlcNAc. Disialo complex-type oligosaccharide of transferrin glycopeptide was transferred to 9-fluorenylmethyloxycarbonyl (Fmoc)-asparaginyl-N-acetylglucosaminide (Fmoc-Asn-GlcNAc) by endo-M in a high yield. The structure of the reaction product was confirmed to be Fmoc-Asn-(GlcNAc)2-Man-(Man-GlcNac-Gal-NeuAc)2 by mass spectrometry. Endo-M also transferred disialo complex-type oligosaccharide to the GlcNAc residue of chemically synthesized H-Ile-Asn(GlcNAc)-Ala-Thr-Leu-OH. Asn-linked asialo complex-type oligosaccharide and Asn-linked high-mannose type oligosaccharide were also effective as oligosaccharide donors. Transfer of disialo complex-type oligosaccharide to the GlcNAc-peptide was the most effective among the three types of oligosaccharides, although the disialo complex-type oligosaccharide attached to the peptide was the poorest substrate for the hydrolytic activity of endo-M.

Chemoenzymatic synthesis of a novel glycopeptide using a microbial endoglycosidase.

The chemoenzymatic synthesis of a glycopeptide by chemical synthesis of N-acetylglucosaminyl peptide and enzymatic transfer of an oligosaccharide is described. We synthesized glycosylated Peptide T which blocks infection of human T cells by human immunodeficiency virus. The first step of the chemoenzymatic method is the solid-phase chemical synthesis of N-acetylglucosaminyl Peptide T (Ala-Ser-Thr-Thr-Thr-Asn(GlcNAc)-Tyr-Thr) with an N-acetylglucosamine moiety bound to the asparaginyl residue by a solid-phase method. This product was prepared in high yield by the dimethylphosphinothioic mixed anhydride method without protecting the hydroxyl functions of the sugar moiety using Fmoc-N-acetylglucosaminyl asparagine instead of Fmoc-asparagine. The second step was transglycosylation of complex type oligosaccharide to N-acetylglucosaminyl Peptide T by a microbial endoglycosidase. The endo-beta-N-acetylglucosaminidase of Mucor hiemalis transfer the oligosaccharide of human transferrin glycopeptide to N-acetylglucosaminyl Peptide T. The transglycosylation product was confirmed to be the glycosylated Peptide T with a sialo biantennary complex type oligosaccharide by mass spectrometry. The glycosylated Peptide T was highly stable against proteolysis in comparison to native Peptide T and N-acetylglucosaminyl Peptide T.

Chemo-enzymatic synthesis of calcitonin derivatives containing N-linked oligosaccharides.

Eel calcitonin derivatives containing various N-linked oligosaccharides were chemo-enzymatically synthesized by the transglycosylation reaction of Mucor hiemalis endo-beta-N-acetylglucosaminidase (Endo-M) to a glycosylated calcitonin derivative [Asn(GlcNac)3]-CT in which N-acetyl-D-glycosamine (GlcNAc) is attached to the L-asparagine (Asn) residue of the peptide.

Chemo-enzymatic synthesis of a bioactive peptide containing a glutamine-linked oligosaccharide and its characterization

A bioactive peptide containing a glutamine-linked oligosaccharide was chemo-enzymatically synthesized by use of the solid-phase method of peptide synthesis and the transglycosylation activity of endo-beta-N-acetylglucosaminidase. Substance P, a neuropeptide, is an undecapeptide containing two L-glutamine residues. A substance P derivative with an N-acetyl-D-glucosamine residue attached to the fifth or sixth L-glutamine residue from the N-terminal region was chemically synthesized. A sialo complex-type oligosaccharide derived from a glycopeptide of hen egg yolk was added to the N-acetyl-D-glucosamine moiety of the substance P derivative using the transglycosylation activity of endo-beta-N-acetylglucosaminidase from Mucor hiemalis, and a substance P derivative with a sialo complex-type oligosaccharide attached to the L-glutamine residue was synthesized. This glycosylated substance P was biologically active, although the activity was rather low, and stable against peptidase digestion. The oligosaccharide moiety attached to the L-glutamine residue of the peptide was not liberated by peptide-N(4)-(N-acetyl-beta-D-glucosaminyl) asparagine amidase F.

A Synthesis of a Glycopeptide Analogue of Eel Calcitonin

Abstract The glycopeptide analogue of eel calcitonin, [Asn(GlcNAc)3]-CT (1), in which N-acetylglucosamine (GlcNAc) is attached to the asparagine residue of the peptide was synthesized using a thioester method to build the polypeptide segment and a dimethylphosphinothioic mixed anhyride (Mpt-MA) method for the incorporation of the glycopeptide moiety.

Chemo-enzymatic synthesis of a calcitonin derivative containing a high-mannose type oligosaccharide by endo-β-N-acetylglucosaminidase from Arthrobacter protophormiae

Chemo-enzymatic addition of a high-mannose type oligosaccharide to eel calcitonin (CT), a calcium-regulating hormone, was examined. The endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A) transglycosylated the Man(6)-GlcNAc moiety from an ovalbumin-derived high-mannose type glycosyl asparagine, Asn(Man(6)-GlcNAc(2))-OH, to a calcitonin derivative, [Asn(GlcNAc)(3)]-CT, in which the N-acetyl-d-glucosamine (GlcNAc) is attached to the third l-asparagine (Asn) residue of the peptide, and a calcitonin derivative containing a high-mannose type oligosaccharide, [Asn(Man(6)-GlcNAc(2))(3)]-CT, was synthesized. The optimal reaction conditions for the synthesis of [Asn(Man(6)-GlcNAc(2))(3)]-CT from Asn(Man(6)-GlcNAc(2))-OH and [Asn(GlcNAc)(3)]-CT catalyzed by Endo-A were examined. The transglycosylation yield relative to the concentration of the [Asn(GlcNAc)(3)]-CT added was 32.7%, and 4.42 mg of [Asn(Man(6)-GlcNAc(2))(3)]-CT was prepared.

Chemo-enzymatic synthesis of the glycosylated α-mating factor of Saccharomyces cerevisiae and analysis of its biological activity ☆

Abstract The effect of glycosylation on a bioactive peptide was studied using yeast Saccharomyces cerevisiae α-mating factor, which is composed of 13 amino acids. In this study, we prepared glycosylated α-mating factor by chemo-enzymatic synthesis. At first, N-acetylglucosaminyl α-mating factor (Trp–His–Trp–Leu–Gln(GlcNAc)–Leu–Lys–Pro–Gly–Gln–Pro–Met–Tyr) was chemically synthesized by the solid-phase method. Then, using the transglycosylation activity of Mucor hiemalis endo-β-N-acetylglucosaminidase, we synthesized glycosylated α-mating factor with a glutamine-linked sialo complex type oligosaccharide. The biological activity of α-mating factor derivatives was examined by means of a growth arrest assay using secreted-protease-defective a cells of S. cerevisiae. The results showed that the bioactivity of glycosylated α-mating factor was lower than that of native α-mating factor. However, when sialic acid was removed from the complex type sugar chain of glycosylated α-mating factor, its bioactivity was recovered. Glycosylated α-mating factor exhibited higher resistance against proteolysis than native α-mating factor. It was found that the bioactivity of N-acetylglucosaminyl α-mating factor was higher than that of α-mating factor. Circular dichroism studies indicated that a slight change in the structure of α-mating factor may influence its activity.

The chemo-enzymatic synthesis and evaluation of oligosaccharide-branched cyclodextrins

Abstract The syntheses of oligosaccharide-branched cyclodextrins, which showed potential binding to the saccharide-interacting protein, lectin, were investigated. The transglycosylations to Fmoc-Asn(GlcNAc)-NH-cyclodextrin by endo-β-N-acetylglucosaminidase of Mucor hiemalis (Endo-M) gave three kinds of natural oligosaccharide-branched cyclodextrins (7–9) in satisfactory yields. The association constant of the high-mannose type oligosaccharide-branched CD (7) with immobilized concanavalin A was found to be approximately 1.6 × 107 M−1 using an optical biosensor.

Acceptor specificity in the transglycosylation reaction using Endo-M

To determine the structural specificity of the glycosyl acceptor of the transglycosylation reaction using endo-β-N-acetylglucosaminidase (ENGase) (EC 3.2.1.96) from Mucor hiemalis (Endo-M), several acceptor derivatives were designed and synthesized. The narrow regions of the 1,3-diol structure from the 4- to 6-hydroxy functions of GlcNAc were found to be essential for the transglycosylation reaction using Endo-M. Furthermore, it was determined that Endo-M strictly recognizes a 1,3-diol structure consisting of primary and secondary hydroxyl groups.