Hydrazine-Aldehyde Reagent

When measured manometrically, the cytochrome oxidase activity with purified cytochromes a and c was strongly inhibited by aldehyde reagents such as hydrazine, phenylhydrazine, hydroxylamine, and sodium bisulfite. As can be seen in Table VIII, these reagents were effective inhibitors... 

If an unknown compound gives a positive test with the 2 4-dinitrophenyl-hydrazine reagent, it then becomes necessary to decide whether it is an aldehyde or a ketone. Although the dimedone reagent (Section 111,70,2) reacts only with aldehydes, it is hardly satisfactory for routine use In class reactions. It is much simpler to make use of three other reagents given below, the preparation and properties of which have already been described (Section 111,70). 

Unfortunately, there are no universal methods to detect all types of protein oxidation, because the products formed can be so diverse in nature. However, some forms of protein oxidation can be assayed using chemical modification (Davies et al., 1999 Shacter, 2000). In particular, the formation of carbonyl groups on proteins can be targeted using the reagent 2,4-dinitrophenyl-hydrazine (DNPH). This compound reacts with aldehydes to form 2,4-dinitrophenylhydrazone derivatives, which create chromogenic modifications that can be detected at high sensitivity in microplate assays or Western blot analysis (Buss et al., 1997 Winterbourn et al., 1999). 

Add a quantity of the crosslinker solution of choice (SANH or SFB) to the antibody solution to obtain the desired molar excess of reagent over the antibody. Typically, antibody modification procedures are done with 10- to 20-fold molar excess, but for dilute antibody concentrations, this may have to be doubled, depending on how many hydrazine or aldehyde groups are desired to be introduced on the modified antibody. 

Once a molecule is modified with a hydrazine reagent and another molecule is modified with the benzaldehyde compound, they may be combined to form the final conjugate, which will result in a hydrazone linkage between the two molecules. In addition, chemoselective ligation using aldehyde/hydrazine reactions may be done to immobilize biomolecules. In this regard, one modified component may be a surface and the other one an antibody, protein, or oligonucleotide destined for immobilization onto the surface. 

Baxendale IR, Ley SV Sneddon H (2002a) A clean conversion of aldehydes to nitriles using a solid-supported hydrazine. Synlett 775-777 Baxendale IR, Lee A-L, Ley SV (2002b) A concise synthesis of carpanone using solid-supported reagents and scavengers. J Chem Soc Perkin Trans 1 1850-1867... 

The migration of a C=C bond to form a C=N bond was also observed with hydro-xylamine [78, 79], hydrazine [80, 81] and primary amines [82]. The /f iminylphos-phine oxide formed in the reaction may serve as a Wittig reagent in the presence of a base to react with a ketone or an aldehyde leading to ,/fun saturated alkenyl-imines 153 (Scheme 10.74). The phosphorus group can be a phosphonium salt as well as a phosphonate. 

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