Hydrazone linkages

Hydrazone Linkage with Terminal Thiol Group... 

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. 

Ring opening with reduced hydrazone linkage to C1... 

Derivatives of hydrazine, especially the hydrazide compounds formed from carboxylate groups, can react specifically with aldehyde or ketone functional groups in target molecules. Reaction with either group creates a hydrazone linkage (Reaction 44)—a type of Schiff base. This bond is relatively stable if it is formed with a ketone, but somewhat labile if the reaction is with an aldehyde group. However, the reaction rate of hydrazine derivatives with aldehydes typically is faster than the rate with ketones. Hydrazone formation with aldehydes, however, results in much more stable bonds than the easily reversible Schiff base interaction of an amine with an aldehyde. To further stabilize the bond between a hydrazide and an aldehyde, the hydrazone may be reacted with sodium cyanoborohydride to reduce the double bond and form a secure covalent linkage. 

H Hydrazide Compound H Aldehyde Compound H Hydrazone Linkage ... 

Figure 4.29 ADH spontaneously reacts with aldehydes to form hydrazone linkages.

Figure 5.35 ABH reacts with aldehyde-containing compounds through its hydrazide end to form hydrazone linkages. Glycoconjugates may be labeled by this reaction after oxidation with sodium periodate to form aldehyde groups. Subsequent photoactivation with UV light causes transformation of the phenyl azide to a nitrene. The nitrene undergoes rapid ring expansion to a dehydroazepine that can couple to nucleophiles, such as amines.

Figure 9.10 Fluorescein-5-thiosemicarbazide reacts with aldehyde groups to produce hydrazone linkages.

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. 

Lissamine rhodamine B sulfonyl hydrazine can react with aldehyde groups to form hydrazone linkages. 

Hydrazide groups react with aldehyde and ketone groups to form hydrazone linkages (Chapter 2, Section 5.1). Three BODIPY derivatives are available that contain a hydrazine group modification of carboxylate side chains. Biomolecules such as proteins that don t normally possess aldehyde residues can be modified to contain them by a number of chemical means (Chapter 1, Section 4.4). 

BODIPY 530/550 C3 hydrazide is insoluble in aqueous solution, but may be dissolved in DMF or methanol as a concentrated stock solution prior to addition of a small aliquot to a reaction. Coupling to aldehyde-containing molecules occurs rapidly with the formation of a hydrazone linkage. The reaction may be done in buffered environments having a pH range of 5-10. However, modification of glycoproteins with this fluorophore may not yield satisfactory... 

Figure 9.32 Reaction of this BODIPY fluorophore with aldehyde groups creates hydrazone linkages.

Figure 9.48 A cyanine dye containing a hydrazide group can be used to label glycans at their reducing end or other reducing sugars, forming a hydrazone linkage. Glycoproteins also can be labeled after periodate oxidation to form aldehyde groups.

Another route to the formation of a hydrazide on a surface is to use an aldehyde-containing particle (such as HEMA/acrolein copolymers) and subsequently modify the aldehydes to form hydrazone linkages with bis-hydrazide compounds, which then can be stabilized by reduction with sodium cyanoborohydride (Chapter 2, Section 5). The resulting derivative contains terminal hydrazides for immobilization of carbonyl ligands (see Figure 14.18). 

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