Hydrazide coupling using carbodiimides

Hydrazide-containing compounds also can be coupled to carboxylate groups using a carbodiimide-mediated reaction. Using bifunctional hydrazide reagents, carboxylates can be modified to possess terminal hydrazide groups able to conjugate with other carbonyl compounds (Chapter 4, Section 8). 

Biotin-hydrazide also may be used to couple with carboxylate-containing molecules. Hydrazidcs can be coupled with carboxylic acid groups by using the carbodiimide reaction (Chapter 3, Section 1.1). The carbodiimide activates a carboxylate to an o-acylisourea intermediate. Biotin-hydrazide can react with this intermediate via nucleophilic addition to form a stable covalent bond. 

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. 

Add the aldehyde-derivatized alkaline phosphatase to 8 nmol of a 5 -hydrazide oligonucleotide preparation made according to Section 2.1 (this chapter) using a carbodiimide coupling protocol. 

One method proposed to overcome this problem involves the use of a PEG-hydrazide instead of the usual amino PEG. This allows the activation of the protein COOH groups by water-soluble carbodiimide at acidic pH in which all protein amino groups are protonated and rendered unsuitable for coupling, while the hydrazide group, which has a low piCa, can still react to form the polymer-protein adduct [107]. 

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