Large scale preparation of oligosaccharides from chicken eggs

Authors：

Category

Large scale preparation of glycans, glycoproteins & glycolipids

Protocol Name

Authors

Kajihara, Yasuhiro
Department of Chemistry, Osaka University

KeyWords

Asparaginyl-oligosaccharides sialyloligosaccharides complex type oligosaccarides

Reagents

●	Actinase-E (Kaken Pharmaceutical Co. Ltd., Tokyo, Japan)
●	All chemical reagents (Merck Millipore, Billerica, MA or Sigma-Aldrich, St. Louis, MO)
●	Scheme 1. Reagents: a) Actinase-E, Tris-HCl buffer, NaN₃, pH 7.5 (86%) b) 9-fluorenylmethyl N-succinimidyl carbonate (Fmoc-OSu), NaHCO₃, Acetone, H₂O (68%) c) Cs₂CO₃, H₂O then BnBr, DMF (85%)

Methods

Glycopeptide 1

1)	Prepare egg yolks (12 pieces) after crash of eggshell.	Comment 0

2)	Add distilled water (380 mL).	Comment 0

3)	Add phenol solution (34.2 g phenol / 3.8 g water).	Comment 0

4)	Stir for 2 h at 4°C.	Comment 0

5)	Add water (480 mL).	Comment 0

6)	Centrifuge (6000 rpm) for 30 min at 4°C.	Comment 0

7)	Filter the supernatant and then concentrate at 35°C.	Comment 0

8)	Prepare a solution of the residue in water (8 mL).	Comment 0

9)	Filter the solution by filter paper.	Comment 0

10)	Purify by gel permeation column (Sephadex G50, 0.1 M NaCl, 2.5 cm × 100 cm).	Comment 0

11)	Collect the fractions containing sialyloligosacchary peptide 1 and then concentrate.	Comment 0

12)	Desalt by Sephadex G50 column (water, id 2.5 cm × 100 cm).	Comment 0

13)	Collect the fractions containing sialyloligosacchary peptide 1 and then concentrate.	Comment 0

14)	Purify by DEAE-650M column (water, id 2.5 cm × 15 cm).	Comment 0

15)	Collect the fractions containing sialyloligosacchary peptide 1 and then concentrate.	Comment 0

16)

Isolate sialyloligosaccharyl peptide 1 (113 mg)¹⁾.

Comment 0

Asparaginyl sialyloligosaccharide 2

1)	Prepare a solution containing crude sialylglycopeptide 1 (809 mg) and NaN₃ in a Tris-HCl buffer (50 mM, CaCl₂ 10 mM, pH 7.5, 32 mL).	Comment 0

2)	Add Actinase-E (263 mg) to a solution of sialylglycopeptide 1.	Comment 0

3)	Stir this solution for 60 h at 37°C (the pH was kept at 7.5).	Comment 0

4)	After 60 h, add Actinase-E (25 mg) again and then incubate for 55 h.	Comment 0

5)	Lyophilize this solution.	Comment 0

6)	Purify by gel permeation (Sephadex-G-25, 2.5 × 100 cm, H₂O).	Comment 0

Isolate sialyloligosacchary-asparagine 2 (301 mg).

Comment 0

Synthesis of 3

1)	Prepare a solution containing asparagine linked sialyloligosaccharide 2 (80 mg, 0.034 mmol) and NaHCO₃ (11.5 mg, 0.137 mmol) in H₂O-acetone (2.7 mL–4.1 mL).	Comment 0

2)	Add a solution (acetone 4.1 mL) containing 9-fluorenylmethyl-N-succimidylcarbonate (34.7 mg, 0.103 mmol).	Comment 0

3)	Stir this mixture at room temperature for 2 h.	Comment 0

4)	Evaporate this mixture to remove acetone.	Comment 0

5)	Purify by reverse phase HPLC system (ODS-column, 1.6 × 14 cm, H₂O to 20% MeOH).	Comment 0

Isolate Fmoc-Asn-disialyloligosaccharide 3 (60 mg).

Comment 0

Synthesis of 4

1)	Prepare a solution (cold H₂O, 2 mL, 4°C) containing Fmoc-Asn-disialyloligosaccharide 3（20 mg）.	Comment 0

2)	Desalt sodium salt from Fmoc-Asn-disialyloligosaccharide 3 by passing through a pasteur pipette column (0.5 cm × 5 cm) containing resin of Dowex-50W X8(H⁺) and then wash with water.	Comment 0

3)	Lyophilize the eluant.	Comment 0

4)	Prepare a solution (water, 2 mL) containing Fmoc-Asn-disialyloligosaccharide 3.	Comment 0

5)	Add a solution of aqCs₂CO₃ (2.5 mg/1 mL) and adjust pH to 6.	Comment 0

6)	Lyophilize this solution.	Comment 0

7)	Prepare a solution (dry DMF, 1.3 mL) containing Fmoc-Asn-disialyloligosaccharide 3.	Comment 0

8)	Add BnBr (5.1 μL) to the above DMF solution and then stir for 45 h at room temperature.	Comment 0

9)	Add diethyl ether (10 mL) and then collect precipitate generated.	Comment 0

10)	Purify by ODS-column (1.6 cm × 14 cm, H₂O to 40% MeOH).	Comment 0

11)

Isolate dibenzyl-sialyloligosaccharide 4 (18 mg).

Comment 0

Notes

Glycopeptide 1

This glycopeptide was prepared according to the reported conditions²⁾.

Asparaginyl sialyloligosaccharide 2

Key points in this protocol are adjustment and keep of pH during enzymatic reaction. This reaction was monitored by TLC (1M NH₄OAc:isopropanol = 1:1).

¹H-NMR (400 MHz, 30°C in D₂O, HOD = δ 4.81) δ 5.21 (s, 1H, Man4-H-1), 5.15 (d, 1H, J = 9.5 Hz, GlcNAc1-H-1), 4.03 (s, 1H, Man4’-H-1), 4.86 (s, 1H, Man3-H-1), 4.70 (m, 3H, GlcNAc2,5,5’-H-1), 4.53 (d, 2H, J = 8.0 Hz, Gal6,6’-H-1), 4.34 (bs, 1H, Man3-H-2), 4.28 (bd, 1H, Man4-H-2), 4.20 (bd, 1H, Man4’-H-2), 3.03 (dd, 1H, J = 4.4 Hz, 17.2 Hz, Asn-bCH₂), 2.95 (dd, 1H, J = 7.0 Hz, 17.2 Hz, Asn-bCH₂), 2.76 (bdd, 2H, J = 4.6 Hz, 12.4 Hz, NeuAc7,7’-H-3_eq), 2.16 (s, 3H, Ac), 2.15 (s, 6H, Ac×2), 2.28 (s, 6H, Ac×2), 2.10 (s, 3H, Ac), 1.80 (dd, 2H, J = 12.4 Hz, 12.4 Hz, NeuAc7,7’-H-3_ax)

Synthesis of 3

¹H-NMR (400 MHz, 30°C in D₂O, HOD = δ 4.81) δ 8.01 (d, 2H, J =7.5 Hz, Fmoc), 7.80(d, 2H, J = 7.5 Hz, Fmoc ), 7.60 (dd, 2H, J = 7.5 Hz,　Fmoc), 7.53 (dd, 2H, J = 7.5 Hz, Fmoc),　5.22 (s, 1H, Man4-H-1), 5.09 (d, 1H, J = 9.4 Hz, GlcNAc1-H-1), 5.03 (s, 1H, Man4’-H-1), 4.86 (s, 1H, Man3-H-1), 4.69 (m, GlcNAc2,5,5’-H-1), 4.53 (d, 2H, J = 7.8 Hz, Gal6, 6’-H-1), 4.44 (1H, Fmoc), 4.34 (bd, 1H, Man3-H-2), 4.29 (bd, 1H, Man4-H-2), 4.20 (bd, 1H, Man4’-H-2), 2.83-2.72 (m, 3H, Asn-bCH₂, NeuAc7,7’-H-3_eq), 2.61 (bdd, 1H, Asn-bCH₂), 2.15 (s, 9H, Ac×3), 2.12 (s, 6H, Ac×2), 1.98 (s, 3H, Ac), 1.80 (dd, 2H, J = 12.1 Hz, 12.1 Hz, NeuAc7,7’-H-3_ax); HRMS Calcd for C₁₀₃H₁₅₄N₈NaO₆₆[M+Na⁺] 2581.8838, found 2581.8821

Synthesis of 4

¹H-NMR (400 MHz, 30°C in D₂O, HOD = δ 4.81) δ 8.00 (d, 2H, Fmoc), 7.80 (d, 2H, Fmoc), 7.65-7.50 (m, 12H, Ph, Fmoc), 5.46 (d, 2H, J = 11.6 Hz, PhCH₂), 5.40 (d, 2H, J = 11.6 Hz, PhCH₂), 5.21 (s, 1H, Man4-H-1), 5.08 (d, 1H, J = 9.3 Hz, GlcNAc1-H-1), 5.02 (s, 1H, Man4’-H-1), 4.86 (s, 1H, Man3-H-1), 4.67 (m, 3H, GlcNAc2,5,5’-H-1), 4.41 (bd, 3H, Gal6, 6’- H-1, Fmoc), 4.33 (bd, 1H, Man3- H-2), 4.27 (bd, 1H, Man4’-H-2), 4.20 (d, 1H, Man4- H-2), 2.79 (bd, 3H, Asn-bCH₂, NeuAc7, 7’- H_3eq), 2.61 (bdd, 1H, Asn-bCH₂), 2.15 (s, 3H, Ac), 2.12 (s, 6H, Ac×2), 2.10 (s, 6H, Ac×2), 1.98 (s, 3H, Ac), 1.93 (2H, dd, J = 12.2, 12.2 Hz, NeuAc7,7’-H-3_ax);HRMS Calcd for C₁₁₇H₁₆₅N₈Na₂O₆₆[M+Na⁺] 2783.9597, found 2783.9501

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Date of registration：2016-10-07 10:02:52