Hydrazides biotin-hydrazide

These derivatives have been prepared according to equation 62 and their reactivity was investigated140. [2-18F]Fluoropropionic acid derivatives have been used for NCA fluo-roacetylation of a variety of amines, R3NH2, including homoveratrylamine, benzoyl hydrazide, biotin hydrazide, -butylamine, 2-butylamine and phenylalanine ethyl ester1. 

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. 

Biotin-hydrazide has been used to biotinylate antibodies at their oxidized carbohydrate residues (O Shanessy et al., 1984, 1987 O Shanessy and Quarles, 1985 Hoffman and O Shannessy, 1988), to modify the low-density lipoprotein (LDL) receptor (Wade et al., 1985), to biotinylate nerve growth factor (NGF) (Rosenberg et al., 1986), and to modify cytosine groups in oligonucleotides to produce probes suitable for hybridization assays (Reisfeld et al., 1987) (Chapter 27, Section 2.3). 

An analog of this biotinylation reagent with a longer spacer arm also exists. Biotin-LC-hydrazide contains a 6-aminocaproic acid extension off its valeric acid group (Thermo Fisher). 

Biotin-hydrazide can be used to label aldehyde-containing molecules, creating hydrazone bonds. 

The increased length of this spacer (24.7 A) provides more efficient interaction potential with avidin or streptavidin probes, possibly increasing the sensitivity of assay systems. The reactions of biotin-LC-hydrazide are identical to those of biotin-hydrazide. 

The following protocol describes the use of biotin-hydrazide to label glycosylated proteins at their carbohydrate residues. Control of the periodate oxidation level can result in specific labeling of sialic acid groups or general sugar residues (Chapter 1, Section 4.4). 

Add biotin-hydrazide or biotin-LC-hydrazide to a final concentration of 5 mM. 

The reactivity and use of biocytin-hydrazide is similar to that described for biotin-hydrazide in Section 3, this chapter. The following protocol for labeling glycoproteins at oxidized carbohydrate (galactose) sites is from Bayer and Wilchek (1992). 

In some cases, the ability to modify glycans at the reducing end without reduction preserves the carbohydrate s native structure sufficiently to allow interactions with proteins that would otherwise not interact if the bond were reduced. Therefore, depending on the ultimate use of the biotinylated carbohydrate, using a hydrazide mediated conjugation process can have advantages over the use of amine-biotin compounds. 

Figure 18.19 Biotin-PEG4-hydrazide is a hydrophilic biotinylation reagent that can be used to modify glycans or carbohydrates at their reducing end or after periodate oxidation to create aldehydes.

Figure 18.20 Biotin-PEG4-hydrazide reacts with aldehyde-containing molecules to form a hydrazone linkage.

The following protocol describes a method for the periodate oxidation of a glycoprotein followed by biotinylation of the resultant aldehydes using hydrazide-PEG4-biotin. Chapter 1, Section 4.6 describes an alternative protocol for the modification of glycans at their reducing ends with hydrazide compounds. 

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.

Prepare bisulfite modification solution consisting of 3 M concentration of a diamine (i.e., ethylenediamine), 1M sodium bisulfite, pH 6. The use of the dihydrochloride form of the diamine avoids having to adjust the pH down from the severe alkaline pH of the free-base form. Note The optimum pH for transaminating biotin-hydrazide to cytosine residues using bisulfite is 4.5 (see Section 2.3, this chapter). 

Biotin-Hydrazide Modification of Bisulfite-Activated Cytosine Groups... 

Dissolve biotin-hydrazide in water at a concentration of lOmg/ml. 

Figure 27.13 Biotin-hydrazide may be incorporated into cytosine bases using a bisulfite-catalyzed transamination reaction.

Bayer, E.A., Ben-Hur, H., and Wilchek, M. (1988) Biocytin hydrazide—a selective label for sialic acids, galactose, and other sugars in glycoconjugates using avidin-biotin technology. Anal. Biochem. 170, 271-281. 

Reisfeld, A., Rothenberg, J.M., Bayer, E.A., and Wilchek, M. (1987) Nonradioactive hybridization probes prepared by the reaction of biotin hydrazide with DNA. Biochem. Biophys. Res. Comm. 142, 519-526. 

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