Adipic acid dihydrazide

Ryure 148 Adipic add dihydrazide spontaneously reacts with aldehydes to form hydrazone linkages. 

Protocols for the use of adipic acid dihydrazide in the modification of aldehyde or carboxylate functional groups can be found in Chapter 1, Section 4.5, and Chapter 13, Section 5. 

These techniques have been used to target, detect, or assay glycoproteins in solution or on cell surfaces by using hydrazide-activated enzymes, avidin, or streptavidin (Chapter 23, Section 5) (Bayer and Wilchek, 1990 Bayer et al., 1987a, b, 1990) and to form conjugates with glycoproteins. 

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). 

Diazonium groups react with active hydrogens on aromatic rings to give covalent diazo bonds. Generation of a diazonium-reactive group usually is done from an aromatic amine by reaction 

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The following protocols make use of the compounds adipic acid dihydrazide and carbohy-drazide to derivatize molecules containing aldehydes, carboxylates, and alkylphosphates. The protocols are applicable for the modification of proteins, including enzymes, soluble polymers such as dextrans and poly-amino acids, and insoluble polymers used as micro-carriers or chromatographic supports. 

Aldehyde-containing macromolecules will react spontaneously with hydrazide compounds to form hydrazone linkages. The hydrazone bond is a form of Schiff base that is more stable than the Schiff base formed from the interaction of an aldehyde and an amine. The hydrazone, however, may be reduced and further stabilized by the same reductants utilized for reductive amination purposes (Chapter 3, Section 4.8). The addition of sodium cyanoborohydride to a hydrazide-aldehyde reaction drives the equilibrium toward formation of a stable covalent complex. Mallia (1992) found that adipic acid dihydrazide derivatization of periodate-oxidized dextran (containing multiple formyl functionalities) proceeds with much greater yield when sodium cyanoborohydride is present. 

Add a quantity of adipic acid dihydrazide or carbohydrazide (Aldrich) to the protein solution to obtain at least a 10-fold molar excess over the amount of aldehyde functionality present. High molar ratios are necessary to avoid protein conjugation during the reaction process. If the concentration of aldehydes is unknown, the addition of 32mg adipic acid dihydrazide per ml of the protein solution to be modified should work well. 

Figure 1.108 Glycoproteins that have been treated with sodium periodate to produce aldehyde groups can be further modified with adipic acid dihydrazide to result in a hydrazide derivative.

Figure 1.109 Carboxylate groups on proteins may be modified with adipic acid dihydrazide in the presence of a carbodiimide to produce hydrazide derivatives.

Carboxylic acids may be covalently modified with adipic acid dihydrazide or carbohydrazide to yield stable imide bonds with extending terminal hydrazide groups. Hydrazide functionalities don t spontaneously react with carboxylate groups the way they do with aldehyde groups (Section 4.5, this chapter). In this case, the carboxylic acid first must be activated with another compound that makes it reactive toward nucleophiles. In organic solutions, this may be accomplished 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). 

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

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

Alkylphosphate Group Adipic Acid Dihydrazide Phosphoramidate Linkage with... 

Add to the tube 7.5 pi of RNA or DNA containing a 5 -phosphate group. The concentration of the oligonucleotide should be 7.5-15 nmol or total of about 57-115.5 pg. Also, immediately add 5 pi of 0.25 M adipic acid dihydrazide or carbohydrazide dissolved in 0.1 M imidazole, pH 6.0. Because EDC is labile in aqueous solutions, the addition of the oligo and fezs-hydrazide/imidazole solutions should occur quickly. 

Dissolve 160 mg of adipic acid dihydrazide (Aldrich) in 5 ml of 0.1 M sodium phosphate, pH 6.0. Some heating of the tube under a hot-water tap may be required to help solubilize the compound. Cool to room temperature. 

Dissolve 50mg of (strept)avidin in the adipic acid dihydrazide solution. 

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

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. 

Hydrazide groups can react with carbonyl groups to form stable hydrazone linkages. Derivatives of proteins formed from the reaction of their carboxylate side chains with adipic acid dihydrazide (Chapter 4, Section 8.1) and the water-soluble carbodiimide EDC (Chapter 3, Section 1.1) create activated proteins that can covalently bind to formyl residues. Hydrazide-modified enzymes prepared in this manner can bind specifically to aldehyde groups formed by mild periodate oxidation of carbohydrates (Chapter 1, Section 4.4). These reagents can be used in assay systems to detect or measure glycoproteins in cells, tissue sections, or blots (Gershoni et al., 1985). 

The activation of enzymes using adipic acid dihydrazide and EDC is identical to the procedure outlined for the modification of (strept)avidin (Chapter 23, Section 5). Alternatively, hydrazide groups may be created on enzymes using the heterobifunctional chemoselective reagents described in Chapter 17, Section 2. 

The following protocol describes the modification of DNA or RNA probes at their 5 -phosphate ends with a bis-hydrazide compound, such as adipic acid dihydrazide or carbohydrazide. A similar procedure for coupling the diamine compound cystamine can be found in Section 2.2 (this chapter). 

The reaction of an excess of adipic acid dihydrazide with aldehyde groups present on proteins or other molecules will result in modified proteins containing alkyl-hydrazide groups (Fig. 96). Another bis-hydrazide compound, carbohydrazide, also may be employed with similar results, except that the spacer afforded through its use is considerably shorter. Target aldehydes may be created on macromolecules according... 

Add a quantity of adipic acid dihydrazide or carbohydrazide (Aldrich) to the... 

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