Removal of sulfate groups (desulfation) is one of the important ways to analyze functions and structure of sulfated glycans. Among the chemical desulfation methods reported so far 1), the "solvolytic" reaction has been widely employed due to its convenience and mild conditions, where pyridinium salt of sulfated polysaccharide is heated in dimethylsulfoxide containing water or methanol 2). Specific N-desulfation of heparin/heparan sulfate can be achieved with this method at room temperature since the N-sulfate group is much more sensitive than O-sulfate groups 3). However, preferential 6-O-desulfation would be difficult because the solvolysis rate of 6-O-sulfate is close to that of 2-O-sulfate. In addition, as the solvolytic conditions may cause acid hydrolysis or methanolysis of the glycosyl bonds due to pyridinium ion and water or methanol in the system, depolymerization occasionally occur at elevated temperature when applied to acid-labile polysaccharides.
Several silylating reagents appeared to be effective for desulfation when added to the solvolytic reaction system using pyridine as the solvent 4). Some of them such as N,O-bis(trimethylsilyl)acetamide (BSA) and N-methyl-N-(trimethylsilyl)trifuluoroacetamide (MSTFA) strongly promote desulfation specifically at primary hydroxyl groups, i.e. 6-O-desulfation, while they almost completely suppress O-desulfation at other positions and N-desulfation 5-8). Using these reagents, sulfated groups linked to hydroxyl and amino groups of the carbohydrates are replaced with trimethylsilyl groups, which are then removed to regenerate free hydroxyl and amino groups in the subsequent dialysis step. The silylating reagent also reacts with water and removes residual moisture in the system to prevent hydrolytic cleavage of the polysaccharide chain. In addition, other reagents such as 4-trimethylsilyloxypent-3-en-2-one (TPN) and trimethychlorolsilane (TMCS) promote O-desulfation at any positions 4) as well as N-desulfation (unpublished results).
A protocol for specific 6-O-desulfation of heparin is introduced here as an example. Non-specific O-desulfation and N-desulfation is also possible when other silylating reagents such as TPN and TMCS are employed instead of MSTFA and BSA 9). The optimized conditions for the highest degree of desulfation and the lowest degradation using TMCS is established for an algal polysaccharide 10). |
Category | Isolation & structural analysis of glycans |
Protocol Name | Regioselective removal of sulfate groups from glycans using non-destructive chemical reaction |
Authors
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Takano, Ryo
Faculty of Agriculture, Department of Bioscience and Biotechnology, University of the Ryukyus
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KeyWords |
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Reagents
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Amberlite IR-120B H+ form (Rohm and Haas/The Dow Chemical Company, Midland, MI) (Note 1) |
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Pyridine (Wako Pure Chemical Industries, Ltd., Osaka, Japan) (Note 2) |
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N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) (Merck, Darmstadt, Germany) (Note 3) |
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Methanol (Wako Pure Chemical Industries Ltd.) |
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Sodium hydroxide (Wako Pure Chemical Industries Ltd.) |
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Ammonium fluoride (Nacalai Tesque Inc., Kyoto, Japan) |
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Instruments
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Dialysis membrane UC36-32-100 (Viscase Companies, Inc., Darien, IL) |
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Oil bath or heating mantle equipped with magnetic stirrer |
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Methods |
1. |
Preparation of pyridinium salt of sulfated polysaccharide
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1) |
Dissolve a sulfated polysaccharide in water (200 mg/ 20 mL) and cool below 4ºC. |
Comment 0
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Apply to an Amberlite IR120 column (H+ form, 2 × 5 cm). |
Comment 1
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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
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Soak the pyridinium salt of polysaccharide (ca. 200 mg) in dry pyridine (20 mL). |
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Pour the reaction mixture into crushed ice (20 g) and add methanol (5 mL). |
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Add several drops of 1 M NaOH to ≒ pH 9. |
Comment 1
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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).
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Soak the freeze-dried material in 50 mL of 0.5 M methanolic ammonium fluoride. |
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Incubate the mixture at 50ºC for 3 h in a plastic tube equipped with open tube at the top. |
Comment 1
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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. |
Comment 1
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Freeze-dry the dialyzed solution. |
Comment 0
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Notes |
- 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.
- 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.
- 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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Copyrights |
Attribution-Non-Commercial Share Alike
This work is released underCreative Commons licenses
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Date of registration:2014-07-29 16:54:46 |
- Usov, A. I. (2011) Polysaccharides of the red algae. Adv. Carbohyr. Chem. Biochem. 65, 115–217 [PMID : 21763512]
- Takano, R. (2002) Desulfation of sulfated carbohydrate. Trends Glycosci. Glycotechn. 14, 343–351 [PMID : not found]
- Nagasawa, K., Inoue Y., and Kamata, T. (1977) Solvolytic desulfation of glycosaminoglycuronan sulfates with dimethyl sulfoxide containing water or methanol. Carbohydr. Res. 58, 47–55 [PMID : 144018]
- Inoue Y., and Nagasawa K. (1976) Selective N-desulfation of heparin with dimethyl sulfoxide containing water or methanol. Carbohydr. Res. 46, 87–95 [PMID : 1248016]
- Takano, R., Kanda T., Hayashi K., Yoshida K., and Hara, S. (1995) Desulfation of sulfated carbohydrates mediated by silylating reagents. J. Carbohydr. Chem. 14, 885–888 [PMID : not found]
- Takano, R., Matsuo, M., Kamei-Hayashi, K., Hara, S., and Hirase, S. (1992) A novel regioselective desulfation method specific to carbohydrate 6-sulfate using silylating reagent. Biosci. Biotech. Biochem. 56, 1577–1580 [PMID : not found]
- Matsuo, M, Takano, R., Kamei-Hayashi, K., and Hara, S. (1993) A novel regioselective desulfation of polysaccharide sulfates: Specific 6-O-desulfation with N,O-bis(trimethylsilyl)acetamide. Carbohydr. Res. 241, 209–215 [PMID : 8472253]
- Takano, R, Ye, Z., Ta T.-V., Hayashi, Kariya, Y., and Hara, S. (1998) Specific 6-O-desulfation of heparin. Carbohydr. Lett. 3, 71–77 [PMID : not found]
- Thanh, T. T. T., Yuguchi, Y., Mimura, M., Yasunaga, H, Takano, R., Urakawa, H, and Kajiwara, K. (2002) Molecular characteristics and gelling properties of the carrageenan family, 1. Preparation of novel carrageenans and their dilute solution properties. Macromol. Chem. Phys. 203, 15–23 [PMID : not found]
- Kolender, A. A., Matulewicz, M. C. (2004) Desulfation of sulfated galactans with chlorotrimethylsilane. Characterization of beta-carrageenan by 1H NMR spectroscopy. Carbohydr. Res. 339, 1619–1629. [PMID : 15183736]
- Zhang, W., and Robbins, M. J. (1992) Removal of silyl protecting groups from hydroxyl functions with ammonium fluoride in methanol. Tetrahedron Lett. 33, 1177–1180 [PMID : not found]
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