(Figure 8.3). Such procedures have frequently been adopted to make the 3-terminus of an oligonucleotide
inert to 3-exonuclease degradation.
8.4 Reactions with Nucleophiles
In general, nucleophiles can attack the pyrimidine residues of nucleic acids at C-6 or C-4, while reactions
at C-6 of adenine or C-2 of guanine are more difficult. -Effect nucleophiles, such as hydrazine, hydrox-
ylamine and bisulfite, are especially effective reagents for nucleophilic attack on pyrimidines.
Hydrazineadds to uracil and cytosine bases first at C-6 and then reacts again at C-4. The bases are con-
verted into pyrazol-2-one and 3-aminopyrazole, respectively, leaving an N-ribosylurea, which can react
further to form a sugar hydrazone. These reactions were used in the Maxam–Gilbertchemical method
of DNA sequence determination(now obsolete, Section 5.1), where subsequent treatment of the modified
ribose residues with piperidine causes -elimination of both 3- and 5-phosphates at the site of depyrimidi-
nation (Figure 8.4).^10
Cytosine andits nucleosides react with hydroxylamine, semicarbazide and methoxylamine under mild,
neutral conditions to give N^4 -substituted products. The mechanism of this process involves reaction with
the cytosine cation, as illustrated for hydroxylamine (Figure 8.5). The formation of N^4 -hydroxydeoxycyti-
dineis an important mutagenic event in DNA because this modified base exists to a significant extent in the
unusual imino-tautomeric form (Section 2.1.2) and thus is capable of base-mispairing with adenine.^11
A third addition reaction at C-6 of cytosine and uracil residues involves the bisulfiteanion. While this
adds reversibly, the intermediate non-aromatic heterocycles undergo a variety of chemical substitution
reactions of which the most important are: (i) transamination of cytosine at C-4 by various primary or
secondary amines, (ii) hydrogen isotope exchange at C-5, and (iii) deamination of cytosine to uracil.^12
The third process provides the basis for the mutagenicity and cytotoxicity of bisulfite (which is equivalent
to aqueous sulfur dioxide). Such mutations are best carried out at pH 5–6 to bring about the deamination
and then at pH 8–9 to eliminate bisulfite (Figure 8.6a, they are the likely basis of bottle-sterilisation by
Camden tablets in home-brewing). The in vitro incorporation of deuterium at C-5 into cytosine is another
substitution reaction that requires the addition of a cysteine-thiol to C-6.
This easy nucleophilic addition of sulfur to C-6 of the pyrimidine ring is a key feature of the biological
methylation of pyrimidines. Dan Santi has established that the mechanism of action of thymidylate syn-
thaseinvolves addition of a catalytic cysteine to C-6 of the deoxyuridylate in conjunction with elec-
trophilic addition of the methylene group of tetrahydrofolate to C-5.^13 It is this process that underpins the
activity of the anti-cancer drug, 5-fluorouracilin which 5-FU acts as a suicide substrate (Section 3.7.1).
In a similar fashion, Rich Roberts has shown that cytosine-specific DNA restriction methylases, such as
M. HhaI, add a catalytic thiol to C-6 of a deoxycytidine residue in conjunction with transfer of a methyl
group from S-adenosyl-L–methionine to C-5 (Figure 8.6b).^14
8.5 Reactions with Electrophiles
8.5.1 Halogenation of Nucleic Acid Residues
Uracil, adenine and guanine can be halogenated directlyby chlorine or bromine and so offer easy routes
to 5-chloro-(or bromo-)uridines and 8-chloro-(or bromo-)purines (the latter are readily converted into
298 Chapter 8
O BHO OHRO O BOROORO O BHO OHRO BNR'OHO OHiiiiiiFigure 8.3 Periodate cleavageof a 3-terminal nucleotide (RRNA) and its subsequent modification. Reagents: (i)
NaIO 4 , pH 4.5; (ii) NaBH 4 ; and (iii) (RNH 2 )