Polysaccharide hydrazide

Polysaccharides, glycoproteins, and other glycoconjugates therefore may be specifically labeled on their carbohydrate portions by creating aldehyde functionalities and subsequently derivatizing them with another molecule containing an amine or a hydrazide group. This route of derivatization is probably the most common way of modifying carbohydrates. 

Amine functionalities also may be created on polysaccharides (Section 4.3, this chapter). The reducing ends of carbohydrate molecules (or generated aldehydes) may be reacted with small diamine compounds to yield short alkylamine spacers that can be used for subsequent conjugation reactions. Hydrazide groups may be similarly created using f z s-hydrazide compounds (Sections 4.5 and 4.6, this chapter). 

The carbonyl-reactive group on these crosslinkers is a hydrazide that can form hydrazone bonds with aldehyde residues. To utilize this functional group with carbohydrate-containing molecules, the sugars first must be mildly oxidized to contain aldehyde groups by treatment with sodium periodate. Oxidation with this compound will cleave adjacent carbon-carbon bonds which possess hydroxyl groups, as are abundant in polysaccharide molecules (Chapter 1, Sections 2 and 4.4). 

PDPH also may be used as a thiolation reagent to add sulfhydryl functional groups to carbohydrate molecules. The reagent can be used in this sense similar to the protocol described for AMBH (Chapter 1, Section 4.1). After modification of an oxidized polysaccharide with the hydrazide end of PDPH, the pyridyl group is removed by treatment with DTT, leaving the exposed sulfhydryl (Figure 5.15). 

The hydrazide derivative of AMCA can be used to modify aldehyde- or ketone-containing molecules, including cytosine residues using the bisulfite activation procedure described in Chapter 27, Section 2.1. AMCA-hydrazide reacts with these target groups to form hydrazone bonds (Figure 9.26). Carbohydrates and glycoconjugates can be labeled specifically at their polysaccharide portion if the required aldehydes are first formed by periodate oxidation or another such method (Chapter 1, Section 4.4). 

Figure 20.9 Polysaccharide groups on antibody molecules may be oxidized with periodate to create aldehydes. Modification with biotin-hydrazide results in hydrazone linkages. The sites of modification using this technique often are away from the antibody-antigen binding regions, thus preserving antibody activity.

Figure 23.9 Reaction of adipic acid dihydrazide with (strept)avidin produces a hydrazide derivative that is highly reactive toward periodate-oxidized polysaccharides.

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). 

On the other end of the cross-linker, the hydrazide functional group can react with periodate-oxidized carbohydrate molecules to form hydrazone linkages (Chapter 1, Sections 2 and 4.5). Thus, glycoproteins can be targeted specifically at their polysaccharide chains, avoiding cross-linking at active sites which can lead to activity losses (Fig. 167). 

BODIPY 530/550 C3 hydrazide is 4,4-difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl hydrazide, a derivative of the basic BODIPY structure, which contains two phenyl rings off the No. 5 and 7 carbon atoms and a propionic acid hydrazide group on the No. 3 carbon atom (Molecular Probes). The hydrazide functional group reacts with aldehyde- or ketone-containing molecules to form hydrazone linkages (Fig. 229). The compound may be used to label glycoproteins or other carbohydrate-containing molecules after oxidation of their polysaccharide portions with sodium periodate to yield aldehyde residues. 

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