Dextran hydrazide derivatives

Bis-hydrazide-containing molecules also can be used to activate soluble polymeric sub-stances-containing aldehyde groups. For instance, dextran may be periodate oxidized to create numerous formyl functionalities on each molecule. Subsequent reaction with a homobifunctional hydrazide in large excess results in a hydrazide-activated polymer having multivalent-binding capability toward aldehydes or ketones (Chapter 25, Section 2.2). Insoluble support matrices suitable for affinity chromatography have been activated in a similar fashion to create the hydrazide derivative (O Shannessy and Wilchek, 1990). 

Hydrazide derivatives also may be prepared from a periodate-oxidized dextran polymer or from a carboxyl-containing dextran derivative by reaction with te-hydrazidc compounds (Chapter 4, Section 8). A hydrazide terminal spacer provides reactivity toward aldehyde- or ketone-containing molecules. Thus, the hydrazide-dextran polymer can be used to conjugate specifically glycoproteins or other polysaccharide-containing molecules after they have been oxidized with periodate to form aldehydes (Chapter 1, Section 4.4). 

The following protocols may be used to create carboxyl-, amine-, or hydrazide-containing derivatives of dextran. 

To make an amine derivative of dextran, dissolve ethylene diamine (or another suitable diamine) in 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2, at a concentration of 3 M. Note Use of the hydrochloride form of ethylene diamine is more convenient, since it avoids having to adjust the pH of the highly alkaline free-base form of the molecule. Alternatively, to prepare a hydrazide-dextran derivative, dissolve adipic acid dihydrazide (Chapter 4, Section 8.1) in the coupling buffer at a concentration of 30 mg/ml (heating under a hot water tap may be necessary to completely dissolve the hydrazide compound). Adjust the pH to 7.2 with HC1 and cool to room temperature. 

The ethylene diamine-dextran derivative may be used for the coupling of carboxylate-contain-ing molecules by the carbodiimide reaction, for the coupling of amine-reactive probes, or to modify further using heterobifunctional crosslinkers. The hydrazide-dextran derivative may be used to crosslink aldehyde-containing molecules, such as oxidized carbohydrates or glycoproteins. 

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