Nucleic Acids in Chemistry and Biology

8.5.3.2 Alkylating Agents. Twelve of the nitrogen and oxygen residues of the four nucleic acid

bases, in addition to the phosphate oxygen, can be alkylated in aqueous solution at neutral pH. ‘Soft’elec-
trophiles, such as dimethyl sulfate(DMS), methyl methanesulfonate(MMS) and alkyl halides (such as
methyl iodide) react in an SN2-like fashion and such alkylation takes place mainly at nitrogen sites with a
general selectivity G-N-7A-N-1A-N-3 T-N-3. A key measure of ‘softness’is a very high ratio of
methylation at G-N-7 compared to G-O-6 (typically 250:1).^19 In double-stranded DNA, the major alkyla-
tion site for DMS with adenines is at N-3 with lesser substitution at N-7.^20 ‘Hard’ electrophiles, such as
N-methyl-N-nitrosourea(MNU) and its ethyl homologue, ENU, are SN1-like alkylating agents. In nucleic
acids, MNU methylation of phosphate esterscan account for up to 50% of total alkylation and also gives
higher ratios for G-O-6:G-N-7 products, ranging from 0.08 in liver to 0.15 in brain DNA.^21 Other sites for
O-alkylation include T-O-2, T-O-4 and C-O-2. The O^2 -alkylation of ribonucleosides is important for pro-
duction of modified nucleotides (Section 3.1.4).
In contrast to the C-methylation of nucleic acids by various enzymes, such as thymidylate synthase
(Section 3.4), products arising from C-alkylation using electrophilic chemical agents have not been observed.
Many alkylating agents are known to be primary carcinogens(agents that act directly on nucleic acids
without metabolic activation). An extensive list includes DMS and MMS and their ethyl homologues,
-propiolactone, 2-methylaziridine, 1,3-propanesultone and ethylene oxide. The list of bifunctional car-
cinogenic agents includes bis-chloromethyl ether, bis-chloroethyl sulfideand epichlorohydrinalong with
such ‘first generation’anti-cancer agents as myleran, chlorambucil and cyclophosphamide (Section 3.7.1).
In general, ‘hard’alkylating agents have been found to be a greater carcinogenic hazard than ‘soft’ones
(Section 8.10).

8.5.3.3 Bis-(2-Chloroethyl) Sulfide. This is the mustard gasof World War I as well as of more

recent conflicts. It is a typical bifunctional alkylating agentin addition to being a proven carcinogen of the
respiratory tract. In the early 1960s, Brookes and Lawley showed that it cross-linkstwo bases either in the
same or in opposite strands of DNA. The typical products isolated (Figure 8.9) have a five-atom bridge
between N-7 of one guanine joined either to a second guanine or to adenine-N-1 or to cytosine-N-3.
Similar products are formed on alkylation of DNA with 2-methylaziridine. These reagents show little
sequence selectivity although nitrogen mustard, MeN(CH 2 CH 2 Cl) 2 , shows some preference for alkylation
of internal residues in a run of guanines.^22

8.5.3.4 Chloroacetaldehyde. This reagent combines the reactivity of formaldehyde and the alkyl

halides. It reacts with adenine and cytosine residues, converting them into etheno-derivatives, which have
an additional five-membered ring fused on to the pyrimidine ring (Figure 8.10).^23 These modified bases
are strongly fluorescent and have been used to probe the biochemical and physiological modes of action
of a range of adenine and cytosine species.^24

8.5.3.5 Dimethyl Sulfate. The methylation of deoxyguanosine was also a major feature of the (now

obsolete) Maxam–Gilbertchemical method for DNA sequence determination (Sections 5.1.1 and 8.4)
but is now employed for in vivo DNA footprinting (Section 5.8).^25 Following the formation of a

Covalent Interactions of Nucleic Acids with Small Molecules and Their Repair 301

N

NH

O

N

N

dR

NH 2

X OH

N

HN

O

N

N

dR

H 2 N

X

N

NH

O

N

N

dR

NH 2

Figure 8.9 Mono- and bi-functional products of dG with sulfur mustard(XS) and nitrogen mustard (XNH or
NMe) reagents