Cytosine hydrazide derivative

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). 

Fluorescein-5-thiosemicarbazide is a hydrazide derivative of fluorescein that can spontaneously react with aldehyde- or ketone-containing molecules to form a covalent, hydrazone linkage (Fig. 208) (Pierce). It also can be used to label cytosine residues in DNA or RNA by use of the bisulfite activation procedure (Chapter 17, Section 2.1). The resulting fluorescent derivative exhibits an excitation maximum at a wavelength of 492 nm and a maximal emission wavelength of 519 nm when dissolved in buffer at pH 8.6. In the same buffered environment, the compound has an extinction coefficient of approximately 78,000 M-1cm 1 at 492 nm. 

The hydrazide derivative can be used to modify aldehyde- or ketone-containing molecules, including cytosine residues using the bisulfite activation procedure de-... 

DNA and RNA may be modified with hydrazide-reactive probes by reacting their cytosine residues with bisulfite to form reactive sulfone intermediates. These derivatives undergo transamination to couple hydrazide- or amine-containing probes (Draper and Gold, 1980) (Chapter 27, Section 2.1). 

Addition of a nucleophile to the C-6 position of cytosine often results in fascile displacement reactions occurring at the N4 location. With hydroxylamine attack, nucleophilic displacement causes the formation of an N4-hydroxy derivative. A particularly important reaction for bioconjugate chemistry, however, is that of nucleophilic bisulfite addition to the C-6 position. Sulfonation of cytosine can lead to two distinct reaction products. At acid pH wherein the N-3 nitrogen is protonated, bisulfite reaction results in the 6-sulfonate product followed by spontaneous hydrolysis. Raising the pH to alkaline conditions causes effective formation of uracil. If bisulfite addition is done in the presence of a nucleophile, such as a primary amine or hydrazide compound, then transamination at the N4 position can take place instead of hydrolysis (Fig. 38). This is an important mechanism for adding spacer arm functionalities and other small molecules to cytosine-containing oligonucleotides (see Chapter 17, Section 2.1). 

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