Bisulfite reaction with cytosine

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.

As in the case of pyrimidine bases discussed previously, adenine and guanine are subject to nucleophilic displacement reactions at particular sites on their ring structures (Figure 1.50). Both compounds are reactive with nucleophiles at C-2, C-6, and C-8, with C-8 being the most common target for modification. However, the purines are much less reactive to nucleophiles than the pyrimidines. Hydrazine, hydroxylamine, and bisulfite—all important reactive species with cytosine, thymine, and uracil—are almost unreactive with guanine and adenine. 

DNA and RNA may be modified with hydrazide-reactive probes by reacting their cytosine residues with bisulfite to form reactive sulfone intermediates. These derivatives can undergo transamination reactions with hydrazide- or amine-containing probes to yield covalent bonds (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.

II. Reaction of cytosine and uracil with sodium bisulfite. J. Biol. Chem. 248, 4060-4064. 

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). 

Shapiro, R., Braverman, B., Louis, J. B., and Servis, R. E. (1973) Nucleic acid reactivity and conformation. II. Reaction of cytosine and uracil with sodium bisulfite. J. Biol. Chem. 248, 4060-4064. 

The 5,6-double bond in uracil, 5-fluorouracil, A-alkyluracils, thiouracils, and uridines is involved in adduct formation with sodium sulfite or bisulfite to give the corresponding 5,6-dihydro- 6-sulfonic acid salts. Bisulfite addition to cytosines and cytidine may be succeeded by a second reaction involving nucleophilic replacement of the amino group, for example, by water . 

It is possible to study promoter methylation by using the chemical modification of cytosine to uracil resulting from treatment with sodium bisulfite. In this reaction all cytosines are converted to uracil, but those that are methylated (5-methylcytosine) are resistant to modification (Wang et al., 1980). Following amplification and sequencing the converted bases (nonmethylated) will appear as thymine compared to cytosine in the native sequence (methylated). This therefore provides a mechanism with which... 

Sulfur dioxide (bisulfites) also reacts with thiamine yielding inactive compounds (see Section 5.6.6), forms adducts with riboflavin, nicotinamide, vitamin K, inhibits ascorbic acid oxidation (see Section 5.14.6.1.6) and reacts with ascorbic acid degradation products, reduces o-quinones produced in enzymatic browning reactions back to 1,2-diphenols (see Section 9.12.4), causes decolourisation of fruit anthocyanins (see Section 9.4.1.5.7) and reacts with synthetic azo dyes to form coloured or colourless products (see Section 11.4.1.3.2). Sulfur dioxide also reacts with pyrimidine bases in vitro, specifically with cytosine and 5-methylcytosine. Important reactions of sulfur dioxide are shown in Figure 11.4. 

Figure 27.2 Treatment of cytosine bases with bisulfite results in a multi-step deamination reaction, ultimately leading to uracil formation.

Adducts 119 are of relevance as reaction intermediates in the chemistry of several uracil and cytosine derivatives, which show a strong tendency to undergo covalent nucleophilic addition across the 5,6 double bond with such reagents as water, alcohols, hydroxylamine, and bisulfite ion. 

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