Hydrazide containing

Figure 1.45 Reaction of bisulfite with cytosine bases is an important route of derivatization. It can lead to uracil formation or, in the presence of an amine (or hydrazide) containing compound, transamination can occur, resulting in covalent modification.

Figure 1.70 AMBH is a hydrazide-containing compound that reacts with carbonyl groups to form hydrazone bonds. The free thiol can be used for subsequent conjugation reactions.

Figure 1.107 The N-terminal aldehyde group on a peptide formed from periodate oxidation of serine or threonine residues can be conjugated with a hydrazide-containing molecule to produce a hydrazone bond.

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). 

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). 

In addition, DNA and RNA may be modified with hydrazide-containing fluorophores by a transamination reaction of their cytosine residues using bisulfite as a catalyst (Chapter 27, Section 2.1) (Draper and Gold, 1980). 

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). 

Figure 17.4 Ketone derivatives of phenylalanine and mannose can be fed to cells to incorporate the monomers into proteins and glycans. The resultant modifications can be probed using hydrazide-containing reagents.

Hydrazide-containing PEG-biotinylation reagents provide reactivity with carbonyl groups (e.g., aldehydes) to label carbohydrates or glycoproteins via hydrazone bond formation (Figures 18.19 and 18.20). The hydrazide group also may be coupled with carboxylate-containing... 

For conjugation to hydrazide-containing proteins, dissolve the periodate-oxidized antibody at a concentration of lOmg/ml in 0.1M sodium phosphate, 0.15M NaCl, pH 6.0-7.2. For conjugation to amine-containing molecules and proteins, dissolve the oxidized antibody at lOmg/ml in 0.2M sodium carbonate, pH 9.6. 

The following protocols may be used to create carboxyl-, amine-, or hydrazide-containing derivatives of dextran. 

D.J.O. Shannessy and W.L. Hoffman, Site-directed immobilization of glycoproteins on hydrazide-containing solid supports. Biotechnol. Appl. Biochem. 9, 488—496 (1987). 

In dim red light, the weighed hydrazide contained in one of the 500 ml flasks (ca. 67 g 95% of theory) is washed into a 1000 ml beaker with 263 ml IN hydrochloric acid. 239 ml distilled water, 239 ml ethanol (95%), and 37 ml 2,4-pentanedione are added, and the well-mixed solution left to stand in the dark at room temperature until the reaction is complete, i.e., about 30 minutes. The reaction mixture is neutralized with the addition of 263 ml 1 N sodium hydroxide, and the beaker covered with parafilm and refrigerated to ensure complete precipitation. The pyrazole is filtered at the pump, the mother liquor being returned to the beaker and used to wash out the last few crystals, washed with cold water, and sucked dry under a stream of dry nitrogen. The product is dried in vacuo over barium oxide or phosphorus pentoxide for at least twelve hours before proceeding to the next step, wherein anhydrous conditions will increase yield. Hofmann calls for drying the pyrazole in vacuo at 60°, which indicated the product to be fairly stable. So all the hydrazide is converted prior to aminization. 

Phosphate-containing carbohydrates that are stable, such as the 5 -phosphate of the ribose derivatives of oligonucleotides, may be targeted for modification using a carbodiimide-facilitated reaction (Section 4.3). The water-soluble carbodiimide EDC can react with the phosphate groups to form highly reactive phospho-ester intermediates. These intermediates can react with amine- or hydrazide-containing molecules to form stable phosphoramidate bonds. 

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