Cytosine transamination

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.

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). 

Figure 27.3 The reaction of cytosine with bisulfite in the presence of an excess of an amine nucleophile (such as a diamine compound) leads to transamination at the N-4 position. This process is a route to adding an amine functional group to cytosine residues in oligonucleotides.

Since the site of modification on cytosine bases is at a hydrogen bonding position in double helix formation, the degree of bisulfite derivatization should be carefully controlled. Reaction conditions such as pH, diamine concentration, and incubation time and temperature affect the yield and type of products formed during the transamination process. At low concentrations of diamine, deamination and uracil formation dramatically exceed transamination. At high concentrations of diamine (3M), transamination can approach 100 percent yield (Draper and Gold, 1980). Ideally, only about 30-40 bases should be modified per 1,000 bases to assure hybridization ability after derivatization. 

Prepare bisulfite modification solution consisting of 3 M concentration of a diamine (i.e., ethylenediamine), 1M sodium bisulfite, pH 6. The use of the dihydrochloride form of the diamine avoids having to adjust the pH down from the severe alkaline pH of the free-base form. Note The optimum pH for transaminating biotin-hydrazide to cytosine residues using bisulfite is 4.5 (see Section 2.3, this chapter). 

Figure 27.13 Biotin-hydrazide may be incorporated into cytosine bases using a bisulfite-catalyzed transamination reaction.

Shapiro, R., and Weisgras, J.M. (1970) Bisulfite-catalyzed transamination of cytosine and cytidine. Biochem. Biophys. Res. Comm. 40, 839-843. 

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). 

Transamination simple introduction of reactive amino groups in cytosine on ss strands many primary or secondary labels may be reacted with this nucleophile useful for universal probes... 

Fig. 7.19. Transamination of unpaired cytosine with a bifunctional amine (ethylene diamine) is catalyzed by sodium bisulfite. The primary amino groups on the cytosine derivatives can then be coupled with, e.g., SCN- or NHS-ligands.

In the second step, the purified, transaminated DNA is brought to a higher pH (8.5-10) in order to decrease the protonation of the reactive amino group. Viscidi et al. (1986) labeled the N -substituted cytosine residue with biotin using the NHS-biotin ester, whereas, e.g., Hurskainen et al. (1991) labeled the same intermediate with a europium chelate (for time-resolved fluorescence). The Cq/qj increases about 1 log but this is compensated by the high probe concentration. This method yields probes with a similar detectability (10 molecules) as enzymatically labeled biotin probes, but the reagents are inexpensive and easily available. 

A comparative evaluation of methylamine, methylamine/anunonium hydroxide and ammonium hydroxide/ethanol for the deprotection of oligonucleotides has also been undertaken. In order to avoid the possible transamination of cytosine when benzoyl protection is employed for the amino group an acetyl protected monomer was used instead. Methylamine or methylamine/ammonium hydroxide mixtures were found to liberate the oligoribonucleotide at room temperature in 90 minutes and improve the yield of the desired product. 

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