Synthesis of stable-isotope enriched 5-methylpyrimidines and their use as probes of base reactivity in DNA - PubMed (original) (raw)

Synthesis of stable-isotope enriched 5-methylpyrimidines and their use as probes of base reactivity in DNA

Artur Burdzy et al. Nucleic Acids Res. 2002.

Abstract

A specific and efficient method is presented for the conversion of 2'-deoxyuridine to thymidine via formation and reduction of the intermediate 5-hydroxymethyl derivative. The method has been used to generate both thymidine and 5-methyl-2'-deoxycytidine containing the stable isotopes 2H, 13C and 15N. Oligodeoxyribonucleotides have been constructed with these mass-tagged bases to investigate sequence-selectivity in hydroxyl radical reactions of pyrimidine methyl groups monitored by mass spectrometry. Studying the reactivity of 5-methylcytosine (5mC) is difficult as the reaction products can deaminate to the corresponding thymine derivatives, making the origin of the reaction products ambiguous. The method reported here can distinguish products derived from 5mC and thymine as well as investigate differences in reactivity for either base in different sequence contexts. The efficiency of formation of 5-hydroxymethyluracil from thymine is observed to be similar in magnitude in two different sequence contexts and when present in a mispair with guanine. The oxidation of 5mC proceeds slightly more efficiently than that of thymine and generates both 5-hydroxymethylcytosine and 5-formylcytosine but not the deaminated products. Thymine glycol is generated by both thymine and 5mC, although with reduced efficiency for 5mC. The method presented here should be widely applicable, enabling the examination of the reactivity of selected bases in DNA.

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Figures

Figure 1

Synthesis of 5-substituted pyrimidine derivatives labeled with 13C, 15N and 2H atoms.

Figure 2

Methyl pyrimidine reaction products obtained from direct methylation of 2′-dU and via formation and reduction of 5-hydroxymethyl-2′-dU. Products were hydrolyzed in formic acid, converted to the TMS derivative and analyzed by GC/MS. The selective ion profile (270 amu) for products of the Bergstrom method (A) and for our products (B) are shown.

Figure 3

Sequences of self-complementary ODNs used for oxidation by Fenton-type reagent, where M = 5mC and T = 15N2-thymine (enriched). (A) Sequence 1: ODN containing enriched thymine at the inner positions (T:A base pair) and unenriched thymine (in a T:A base pair) at the outer position. (B) Sequence 2: ODN containing enriched thymine (inner positions, T:A base pair) and unenriched thymine at the outer position (T:G mispair). (C) Sequence 3: ODN containing enriched thymine (inner positions, T:A base pair) and unenriched 5mC (outer position, 5mC:G base pair).

Figure 4

Reaction pathways for thymine and 5mC.

Figure 5

Formation of HmU in T:A base pairs. GC/MS trace at 360 amu (A) and 358 amu (B) of a silylated hydrolysate of duplex sequence 1 containing T:A base pair treated with Fe(III)NTA/H2O2/ascorbic acid.

Figure 6

Formation of HmU from a T:A base pair and formation of HmC and FoC from a 5mC:G base pair. GC/MS trace at 360 amu (A), 357 amu (B) and 268 amu (C) of a silylated hydrolysate of duplex sequence 3 (containing a 5mC:G base pair) treated with Fe(III)NTA/H2O2/ascorbic acid.

Figure 7

Formation of thymine glycol from T:A and 5mC:G base pairs. GC/MS trace at 435 amu (A) and 433 amu (B) of a silylated hydrolysate of duplex sequence 3 Fe(III)NTA/H2O2/ascorbic acid.

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Synthesis of stable-isotope enriched 5-methylpyrimidines and their use as probes of base reactivity in DNA - PubMed (original) (raw)