Carboxylates with Bis-Hydrazide Compounds

Carboxylic acids may be covalently modified with adipic acid dihydrazide or car-bohydrazide to yield stable imide bonds with extending terminal hydrazide groups. Hydrazide functionalities do not spontaneously react with carboxylate groups the way they do with formyl groups (Section 4.5). In this case, the carboxylic add first must be activated with another compound that makes it reactive toward nucleophiles. In organic solutions, this may be accomphshed by using a water-insoluble carbodiimide (Chapter 3, Section 1.4) or by creating an intermediate active ester, such as an NHS ester (Chapter 2, Section 1.4). 

In aqueous solutions, the easiest method for forming this type of bond is by use of the water-soluble carbodiimide EDC (Chapter 3, Section 1.1). For proteins and other water-soluble macromolecules, EDC reacts with their available carboxylate groups to form an intermediate, highly reactive, O-acylisourea. This active ester species may further react with nucleophiles such as a hydrazide to yield a stable imide product (Fig. 97). 

Most proteins contain an abundance of carboxylic acid groups from C-terminal functional groups and aspartic and glutamic acid side chains, these groups are readily modified with his-hydrazide compounds to yield useful hydrazide-activated derivatives. Both carbohydrazide and adipic acid dihydrazide have been employed in forming these modifications using the carbodiimide reaction (Wilchek and Bayer, 1987). 

Dissolve 32 mg of adipic acid dihydrazide per milliliter of 0.1 M sodium phosphate, 0.15 M NaCl, pH 7.2. 

Dissolve 5 mg of the protein or other macromolecule to be modified per millihter of the above solution. 

Purify the modified protein by dialysis or gel filtration using a desalting resin. 

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