Joachim Kohn

A new approach (cyano-transfer) for cyanogen bromide activation of Sepharose at neutral pH, which yields activated resins, free of interfering nitrogen derivatives.

Abstract A new approach for the reaction of Sepharose with cyanogen bromide is described, using triethylamine as a “cyano-transfer” reagent. An optimized procedure for activation at neutral pH was developed. This procedure requires only about 5% of the usual amount of cyanogen bromide. Activated resins are free of imidocarbonates and carbamates, containing only active cyanate esters. Extremely high coupling capacities (75 μmol ligand/g wet Sepharose 4B) can be obtained using this method.

A colorimetric method for monitoring activation of Sepharose by cyanogen bromide.

Abstract A colorimetric method has been developed for monitoring the activation of polysaccharides with cyanogen bromide. The method is based on the “Kőnig” reaction of cyanate esters with pyridine and barbituric acid to yield a redpurple colored complex with λ max , 575 nm and an e M value of 15,000 M −1 cm −1 . Besides providing a means for a quantitative measurement of activation of the gel with CNBr, the method also enables us to determine (a) the extent of washing the activated gel required for the removal of the unreacted reagent; and (b) the amount and the decay of the reactive groups remaining after the coupling of the ligand to the activated gel.

1‐Cyano‐4‐dimethylamino pyridinium tetrafluoroborate as a cyanylating agent for the covalent attachment of ligand to polysaccharide resins

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Mechanism of activation of Sepharose and Sephadex by cyanogen bromide

The mechanism of CNBr activation of polysaccharide resins like Sepharose and Sephadex has been elucidated using recently published analytical procedures for the determination of cyanate esters and imido carbonates. It was found that on agarose-based resins coupling of ligand occurs predominantly via cyanate esters, and not via imidocarbonates as in the case of Sephadex. This explains the different behaviours of Sepharose and Sephadex during CNBr activation.

A highly sensitive colorimetric method for the determination of carbodiimides

Abstract A highly sensitive colorimetric method has been developed for determination of carbodiimides in solution. The method is based on the “Konig” reaction of carbodiimides with pyridine and barbituric or dimethyl barbituric acid to yield a purple red product with a λ max at 595 nm and ϵ = 150,000. Due to the sensitivity of the method, trace amounts of carbodiimide (1 μ m solutions) can be quantified. The method is fast, reliable, and free of side reactions. Precautions to be taken when carbodiimides are used in pyridine solutions are also discussed.

p-Nitrophenylcyanate—An efficient, convenient, and Nonhazardous substitute for cyanogen bromide as an activating agent for sepharose

The reaction of aromatic cyanates with agarose-based resins was investigated. Phenylcyanate yielded aliphatic-aromatic imidocarbonates as the major product, whereasp-nitrophenylcyanate acted as a cyanylating agent, yielding mainly cyanate esters on the resin. Such cyanate esters were recently also shown to be the active group on cyanogen bromide-activated Sepharose; hence, the stable and nonvolatilep-nitrophenylcyanate was found to be a very convenient substitute for the highly hazardous cyanogen bromide. Activations withp-nitrophenylcyanate could be done safely outside a hood. Employing triethylamine instead of the commonly used inorganic bases, an optimized activation procedure was developed that is about 10 times more efficient than conventional cyanogen bromide activation. Since both cyanogen bromide andp-nitrophenylcyanate-activated resins contain cyanate esters as active groups, the coupling of ligands proceeded in an identical fashion in both cases.

New Methods for Activation of Polysaccharides for Protein Immobilization and Affinity Chromatography

With the increased use of immobilized biochemicals in science and industry, it has become clear that new and improved carriers and coupling methods are of great importance. The cyanogen bromide method that is widely used as an activating agent’.2 is very convenient except for the extremely hazardous nature of the reagent. After we determined the mechanism of activation of polysaccharides with CNBr,’ we were able to develop improved, milder, and nonhazardous activating agents that yielded active resins, identical, for all practical purposes, with the original CNBr-activated resins. The activation of Sepharose with CNBr in the presence of triethylamine required smaller amounts of CNBr, and the reaction could be performed a t neutral pH4 The reaction of CNBr with tertiary amines and phenols enabled the synthesis and isolation and a variety of cyanylating reagents, including N-cyanothriethylammonium tetrafluoroborate, I-cyano-4-dimethylaminopyridinium tetrafluoroborate.’ and p-nitrophenylcyanate.6 These cyano derivatives are stable and effective cyanylating agents and are also highly efficient activating agents for polysaccharides. Activation yields up to 50%, as compared to 1-2% by the regular CNBr method, were obtained. The resulting activated resins contained largely cyanate esters (Resin-0-C-N). Activation with these reagents could be performed safely without a hood. The product of the reaction of these activated cyanate esters with amines is the &-substituted isourea,’ therefore the activation of polysaccharides with these new cyanylating agents do not overcome the inherent problems (such as leakage and charge) that accompany the CNBr immobilization procedure. In order to eliminate these problems, we developed alternative and improved methods for activation and immobilization of ligands and proteins to polysaccharides that gave chemically stable resins. The reagents used were p-nitrophenyl-chloroformate, N-hydroxysuccinimide chloroformate, and trichlorophenylchloroformate (FIG. I ) . These reagents form reactive carbonates, which upon reaction with amines, give stable and uncharged carbamates (urethanes).’ The activation and coupling can be followed spectrophotometrically and the yield of coupling is very high. The mild reaction conditions used for activation of polysaccharides with aromatic cyanates would make this method very attractive if the imidocarbonate moieties would be converted to active carbonates. When agarose were reacted with p-(methylsulfide)phenylcyanate (MSPC), a resin was obtained containing p-(methylsu1fide)phenylimidocarbonate as the active moiety. The imidocarbonates could easily be hydrolyzed to the corresponding carbonate’ by brief exposure of the resin to ice-cold dilute sulfuric acid. The MSP moiety is a weak leaving group and therefore reacts only