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Regioselective removal of sulfate groups from glycans using non-destructive chemical reaction
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Regioselective removal of sulfate groups from glycans using non-destructive chemical reaction

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Introduction Protocol References Credit lines
Category
Isolation & structural analysis of glycans
Protocol Name

Regioselective removal of sulfate groups from glycans using non-destructive chemical reaction

Authors
Takano, Ryo
Faculty of Agriculture, Department of Bioscience and Biotechnology, University of the Ryukyus
KeyWords
Reagents

Amberlite IR-120B H+ form (Rohm and Haas/The Dow Chemical Company, Midland, MI) (Note 1)

Pyridine (Wako Pure Chemical Industries, Ltd., Osaka, Japan) (Note 2)

N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) (Merck, Darmstadt, Germany) (Note 3)

Methanol (Wako Pure Chemical Industries Ltd.)

Sodium hydroxide (Wako Pure Chemical Industries Ltd.)

Ammonium fluoride (Nacalai Tesque Inc., Kyoto, Japan)

Instruments

Glass column (2 × 5 cm)

Dialysis membrane UC36-32-100 (Viscase Companies, Inc., Darien, IL)

Glass filter funnel

Oil bath or heating mantle equipped with magnetic stirrer

Freeze-dry equipment

Methods
1.

Preparation of pyridinium salt of sulfated polysaccharide

1) 

 Dissolve a sulfated polysaccharide in water (200 mg/ 20 mL) and cool below 4ºC.

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2) 

 Apply to an Amberlite IR120 column (H+ form, 2 × 5 cm).

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3) 

 Elute the column with water (ca. 50 mL).

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4) 

 Neutralize the effluent with pyridine (≒ pH 6).

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5) 

 Freeze-dry the neutralized effluent.

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2.

Specific 6-O-desulfation of polysaccharide pyridinium salt

1) 

 Soak the pyridinium salt of polysaccharide (ca. 200 mg) in dry pyridine (20 mL).

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2) 

 Add MSTFA (4 mL).

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3) 

 Heat and stir at 110ºC for 2 h.

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4) 

 Pour the reaction mixture into crushed ice (20 g) and add methanol (5 mL).

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5) 

 Dialyze against water.

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6) 

 Add several drops of 1 M NaOH to ≒ pH 9.

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7) 

 Dialyze against water.

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8) 

 Freeze-dry the dialyzed solution.

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3.

Removal of residual trimethylsilyl group

This procedure is necessary if the desulfated material contains water-insoluble material due to incomplete desilylation during dialysis 11).

1) 

 Soak the freeze-dried material in 50 mL of 0.5 M methanolic ammonium fluoride.

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2) 

 Incubate the mixture at 50ºC for 3 h in a plastic tube equipped with open tube at the top.

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3) 

 Filter the mixture with glass filter funnel, and wash the precipitate with methanol three times.

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4) 

 Dissolve the precipitate in water and add several drops of 1 M NaOH to ≒ pH 9.

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5) 

 Dialyze against water.

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6) 

 Freeze-dry the dialyzed solution.

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Notes
  1. Other strong cation exchanger carrying sulfonate groups (such as Dowex 1x8 or AG 50W 1x8 etc.) is substitutable, while weak cation exchanger, e.g. that containing carboxymethyl or phosphate group, is not suitable.
  2. To remove moisture, add ca. 10 g of KOH / 500 mL directly to the freshly opened bottle, and keep for more than a day at room temperature before use.
  3. N,O-bis(trimethylsilyl)acetamide (BSA) is also effective for 6-O-specific desulfation, while this reagent may cause side reactions at high temperature (over 90ºC) 7).  Both MSTFA and BSA should be free from other silylating reagent such as TMCS, which may cause desulfation of other position 5).  On the other hand, MSTFA and BSA may lower the desulfation rate of TMCS or TPN.
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Date of registration:2014-07-29 16:54:46
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