Synthesis of sugar oxazolines derived from natural N-glycan as substrates for transglycosylation reaction by Endo-M

Authors：

Category

N-Glycans

Protocol Name

Authors

Umekawa, Midori
College of Life Science, Ritsumeikan University

Yamamoto, Kenji
Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University

KeyWords

Sugar oxazoline Endoglycosidase Glycosynthase-like mutant Transglycosylation N-linked glycan

Reagents

●	Sialoglycopeptide from egg yolk (Taiyo Kagaku Co., Ltd., Yokkaichi, Japan)
●	2-Chloro-1,3-dimethyl-2-imidazolinium chloride (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan)
●	Man₉GlcNAc₂Asn from soybean flour (Sigma-Aldrich, St. Louis, MO)
●	Pronase (Sigma-Aldrich)

Methods

Synthesis and purification of a sialo-complex-type sugar oxazoline (Fig. 1)

1)	Sialoglycopeptide (SGP) from egg yolk (500 mg, final conc. 5 mM) was subjected to hydrolysis by Mucor hiemalis endo-β-N-acetylglucosaminidase, Endo-M (50 μg), in 50 mM sodium phosphate buffer (pH6.5) at 30˚C overnight. The reaction was stopped by heating at 100˚C for 5 min.	Comment 1

2)	The reaction mixture was applied to a Sephadex G-25 gel filtration column and eluted with ionized water. The fractions containing a sialo-complex-type glycan with a single GlcNAc at the reducing end (SG-GlcNAc) were collected and freeze dried (150 mg, -80% yield).	Comment 1

3)	The SG-GlcNAc (150 mg, final conc. 50 mM, 1eq.) was dissolved in 1.5 mL water and reacted with 15 eq. of 2-chloro-1,3-dimethyl-2-imidazolinium chloride (DMC) in the presence of 45 eq. of triethylamine on ice for 0.5–1h. The GlcNAc residue at the reducing end of SG-GlcNAc was almost completely converted to the sugar oxazoline derivative.	Comment 0

The reaction mixture was applied to a Sephadex G-25 gel filtration column in 0.01% aqueous ammonia and the sialo-complex-type sugar oxazoline was obtained in 63% yield (freeze dried, 94.5 mg, the overall yield from SGP was 50% ).

Comment 1

Synthesis and purification of a high-mannose-type sugar oxazoline (Fig. 2)

1)	The Man₉GlcNAc₂Asn (75 mg) was treated with immobilized Arthrobactor protophormiae endo-β-N-acetylglucosaminidase (Endo-A) in acetate buffer (pH 6.0). Subsequent gel filtration on a column of Sephadex G-25 gave pure Man₉GlcNAc (45 mg).	Comment 1

2)	The oligosaccharide Man₉GlcNAc (45 mg, 26.8 μmol) was dissolved in a mixture of pyridine (3 mL) and acetic anhydride (3 mL), and the solution was stirred at room temperature for 20 h.	Comment 1

3)	The freeze dried mixture was subjected to column chromatography on silica gel followed by elution with dichloromethane/methanol (20:1) to afford the fully acetylated Man₉GlcNAc as a pale yellow syrup (56 mg).	Comment 0

4)	The acetylated Man₉GlcNAc (56 mg) was dissolved in anhydrous 1,2-dichloroethane (4 mL), and then trimethylsilyl bromide (40 μL, 0.33 mmol), boron trifluoride etherate (40 μL, 0.33 mmol), and 2,4,6-collidine (47 μL, 0.33 mmol) were added sequentially under an argon atmosphere. The mixture was stirred at room temperature overnight.	Comment 0

5)	The solution was diluted with chloroform (20 mL) and washed with saturated sodium bicarbonate solution and brine. The organic layer was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated.	Comment 0

6)	The residue was purified by column chromatography on silica gel and eluted with dichloromethane/methanol (20:1). The crude yellow solid was further purified by gel filtration (Sephadex LH-20, eluted with methanol) to afford the acetylated Man₉GlcNAc-oxazoline derivative as a pale yellow foam (40 mg).	Comment 1

7)	The acetylated Man₉GlcNAc-oxazoline (40 mg, 13.7 mmol) was treated with sodium methoxide in anhydrous methanol (4 mL) for 16 h, and the de-O-acetylation was monitored by ESI-MS. The methanol was removed by evaporation.	Comment 0

The residue was dissolved in water and subjected to gel filtration (Sephadex G-10, eluted with water containing 0.03% triethylamine). The fractions containing the product were collected and freeze dried. The Man₉GlcNAc-oxazoline was obtained as a pale yellow solid (24 mg).

Comment 1

Initial amount

Title1-1) 500 mg

Title2-1) 75 mg

Produced amount

Title1-4) 94.5 mg

Title1-7) 24 mg

Figure & Legends

Fig. 1. Synthesis of sialo-complex-type sugar oxazoline.

SG-GlcNAc, decasaccharide of sialo-complex-type GlcNAc; DMC, 2-chloro-1,3-dimethyl-2-imidazolinium chloride; Et₃N, triethylamine

This figure was originally published in Biochim Biophys Acta. Umekawa M. et al. "Efficient transfer of sialo-oligosaccharide onto proteins by combined use of a gycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-complex-type sugar oxazoline" 2010, 1800(11): 1203-9.

Fig. 2. Synthesis of high-mannose-type sugar oxazoline.

AcO, acetic anhydride; TMS-Br, trimethylsilyl bromide; Bf₃Et₂O, boron trifluoride etherate; MeONa, sodium methoxide; MeOH, methanol

This figure was originally published in J Biol Chem. Umekawa M. et al. "Mutants of Mucor hiemalis endo-beta-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activivites" 2008, 283(8): 4469-79.

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Date of registration：2014-05-28 09:26:28