Abstract
A facile method for activating hydroxyl groups of Sepharose gel using 2-fluoro-1-methylpyridinium toluene-4-sulfonate (FMP) in the presence of triethylamine is described. Nucleophilic affinity ligands, i.e., primary amino or sulfhydryl bearing compounds in slightly alkaline (pH 8-9) aqueous solutions or in organic solvents can be coupled directly to the activated gel. Such nucleophilic reactions result in displacing the pyridinium moiety from the activated gel as 1-methyl-2-pyridone and in forming stable, non-ionic secondary amine or thioether linkages between the gel and the ligands being immobilized. Because 1-methyl-2-pyridone has a molar extinction coefficient of 5,900 at 297 nm, the activation and coupling reactions can be followed conveniently by a simple spectrophotometric method.
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References
Porath, J. and Axen, R. 1976. Immobilization of Enzymes to Agar, Agarose, and Sephadex Supports, p. 19–45. In: Methods in Enzymology. vol. 44. Academic Press, New York.
Kohn, J. and Wilcheck, M. 1982. A new approach (cyano-transfer) for cyanogen bromide activation of Sepharose at neutral pH, which yields activated resins free of interfering nitrogen derivatives. Biochem. Biophys. Res. Comm. 107:878–884.
Kohn, J. and Wilcheck, M. 1983. New approaches for the use of cyanogen bromide and related cyanylating agents for the preparation of activated polysaccharide resins, p. 197–207. In: Affinity Chromatography and Biological Recognition, Chaiken, I. M., Wilcheck, M. and Parikh, I.) (eds.). Academic Press, New York.
Tesser, G.I., Fisch, H.U. and Schwyzer, R. 1974. Limitations of affinity chromatography: solvolytic detachment of ligands from polymeric supports. Helv. Chim. Acta. 57:1718–1730.
Nishikawa, A.H. and Boilon, P. 1975. Affinity purification methods. Improved procedures for cyanogen bromide reaction on agarose. Anal. Biochem. 64:268–275.
Nilsson, K. and Mosbach, K. 1980. p-Toluenesulfonyl chloride as an activating agent of agarose for the preparation of immobilized affinity ligands and proteins. Eur. J. Biochem. 112:397–402.
Nilsson, K. and Mosbach, K. 1981. Immobilization of enzymes and affinity ligands to various hydroxyl group carrying supports using highly reactive sulfonyl chlorides. Biochem. Biophys. Res. Comm. 102:449–457.
Hojo, K., Yoshino, H. and Mukaiyama, T. 1977. New synthetic reactions based on 1-methyl-2-fluuoropydinium salts. Stereospecific preparation of thioalcohols from alcohols. Chem. Letters 437–440.
Ellman, G.L. 1959. Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82:70–77.
Axen, R., Drevin, H. and Carlsson, J. 1975. Preparation of modified agarose gels containing thiol groups. Acta Chem. Scan. B29:471–474.
Mukaiyama, T., Ikeda, S. and Kobayashi, S. 1975. A novel method for the preparation of various 2-pyridyl sulfides from alcohols. Chem. Letters 1159–1162.
Bald, E., Saigo, K. and Mukaiyama, T. 1975. A facile synthesis of carboxamide by using 1-methyl-2-halopyridinium iodide as coupling reagents. Chem. Letters 1163–1166.
Mukaiyama, T. 1979. New synthetic reactions based on the onium salts of aza-arenes. Angew. Chem. Int. Ed. Engl. 18:707–808.
Carlsson, J., Olsson, I., Axen, R. and Drevin, H. 1976. A new method for the preparation of jack-bean urease involving covalent chromatography. Acta Chem. Scan. B30:180–182.
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Ngo, T. Facile Activation of Sepharose Hydroxyl Groups by 2-Fluoro-1 Methylpyridinium Toluene-4-Sulfonate: Preparation of Affinity and Covalent Chromatographic Matrices. Nat Biotechnol 4, 134–137 (1986). https://doi.org/10.1038/nbt0286-134
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DOI: https://doi.org/10.1038/nbt0286-134