Hydrazone bond

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.

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. 

Figure 1.110 The reaction of SANH with an amine-containing molecule results in an amide bond derivative that terminates in a protected hydrazine group. Reaction with an aldehyde-containing molecule results in release of the acetone-protecting group and formation of a stable hydrazone bond.

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). 

Figure 5.12 MPBH reacts with sulfhydryl-containing molecules through its maleimide end to produce thioether linkages. Its hydrazide group then can be used to conjugate with carbonyl-containing molecules (such as periodate-oxidized carbohydrates that contain aldehydes) to give hydrazone bonds.

Figure 5.14 PDPH reacts with thiol-containing compounds through its pyridyl disulfide end to form reversible disulfide linkages. Its hydrazide end then may be subsequently conjugated with an aldehyde-containing molecule to form hydrazone bonds. Glycoproteins may be crosslinked using this approach after periodate activation to...

Figure 5.15 PDPH may be used to add a sulfhydryl group to an aldehyde-containing molecule. After reacting its hydrazide end with the aldehyde to form a hydrazone bond, the pyridyl disulfide may be reduced with DTT to create a free thiol.

To reduce the hydrazone bonds to more stable linkages, cool the cell suspension to 0°C and add an equal volume of 30 mM sodium cyanoborohydride in PBS. Incubate for 40 minutes. Note If the presence of a reducing agent is detrimental to protein activity, eliminate the reduction step. In most cases, the hydrazone linkage is stable enough for fluorescent labeling experiments. 

Figure 9.20 Texas Red hydrazide reacts with aldehydes to create hydrazone bonds.

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 9.40 Cascade Blue hydrazide can be used to modify aldehyde-containing molecules to form hydrazone bonds.

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

Figure 11.21 BNAH contains a hydrazide group that can be used to label the reducing end of released glycans through the formation of a hydrazone bond.

Figure 14.19 Aldehyde-containing molecules, such as periodate-oxidized carbohydrates or glycoproteins, can be coupled to hydrazide-particles to form a hydrazone bond. This bond can be further stabilized by reduction...

The hydrazine-aldehyde reaction has been used intracellularly to deliver non-toxic drug components, which when linked to form a hydrazone bond in situ, become cytotoxic (Rideout, 1986, 1994 Rideout et al., 1990). This same approach has been used to generate enzyme inhibitors in vivo, wherein the hydrazine and aldehyde precursors are not active, but when coupled together within cells to form a hydrazone linkage, become active site binders (Rotenberg etal, 1991). 

Figure 17.6 The reaction of SANH with amine-containing proteins or other molecules results in amide bond modifications containing terminal hydrazine groups. The reaction of SFB with amine-containing proteins or other molecules results in amide bond modifications containing terminal aldehyde groups. Subsequently, the two modified molecules can be reacted together to create a conjugate via hydrazone bond formation.

The bis-aryl hydrazone bond formed by this reaction is stable in aqueous solution over a broad pH range (pH 2-11) and under elevated temperature conditions (up to 94°C) (Solulink web site). 

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