Chemical synthesis of oligodeoxyribonucleotides containing N3- and O4-carboxymethylthymidine and their formation in DNA

Abstract

Humans are exposed to N-nitroso compounds from both endogenous and exogenous sources. Many N-nitroso compounds can be metabolically activated to give diazoacetate, which can result in the carboxymethylation of DNA. The remarkable similarity in p53 mutations found in human gastrointestinal tumors and in shuttle vector studies, where the human p53 gene-containing vector was treated with diazoacetate and propagated in yeast cells, suggests that diazoacetate might be an important etiological agent for human gastrointestinal tumors. The O(6)-carboxymethyl-2'-deoxyguanosine was previously detected in isolated DNA upon exposure to diazoacetate and in blood samples of healthy human subjects. The corresponding modifications of thymidine and 2'-deoxyadenosine have not been assessed, though significant mutations at A:T base pairs were found in the p53 tumor suppressor gene in human gastrointestinal tumors and in shuttle vector studies. To understand the implications of the carboxymethylation chemistry of thymidine in the observed mutations at A:T base pairs, here we synthesized authentic N3-carboxymethylthymidine (N3-CMdT) and O(4)-carboxymethylthymidine (O(4)-CMdT), incorporated them into DNA, and demonstrated, for the first time, that they were the major carboxymethylated derivatives of thymidine formed in calf thymus DNA upon exposure to diazoacetate. The demonstration of the formation of N3-CMdT and O(4)-CMdT in isolated DNA upon treatment with diazoacetate, together with the preparation of authentic oligodeoxyribonucleotide substrates housing these two lesions, laid the foundation for investigating the replication and repair of these lesions and for understanding their implications in the mutations observed in human gastrointestinal tumors.

Figure 1.

LC-MS/MS for monitoring the formation of N3-CMdT and O⁴-CMdT in calf thymus DNA upon treatment with diazoacetate. Shown are the SICs for monitoring the m/z 301→185 transition, corresponding to the loss of a 2-deoxyribose, from the LC-MS/MS analyses with the injection of: (a) N3-CMdT standard; (b) O⁴-CMdT standard; (c) enzymatic digestion mixture of calf thymus (ct) DNA treated with 20 mM potassium diazoacetate (KDA, and the LC-MS/MS results from the reaction with 8 mM KDA are shown in Figure S7); (d) the sample in (c) with the addition of standard N3-CMdT; and (e) the sample in (c) with the addition of standard O⁴-CMdT.

Figure 2.

Tandem mass spectra supporting the formation of N3-CMdT and O⁴-CMdT in calf thymus DNA. Shown are the MS³ results, which monitors the further fragmentation of the protonated carboxymethylthymine (m/z 185), for: (a) standard N3-CMdT; (b) standard O⁴-CMdT; (c) the 27.8 min fraction shown in Figure 1c; and (d) the 29.2 min fraction shown in Figure 1c. Depicted in the insets are the corresponding MS/MS for the [M + H]⁺ ions of the modified nucleosides. The ion of m/z 185 found in MS/MS is attributed the elimination of a 2-deoxyribose.

Figure 3.

The product-ion spectrum of the ESI-produced [M – 3H]^3– ions of d(ATGGCGXGCTAT), where ‘X’ represents O⁴-CMdT. Illustrated in the insets is the negative-ion ESI-MS for the modified ODN.

Scheme 1.

The formation of diazoacetate and its modification of 2′-deoxyguanosine.

Scheme 2.

The chemical synthesis of N3-CMdT and O⁴-CMdT.

Scheme 3.

The incorporation of N3-CMdT into ODNs.

Scheme 4.

The incorporation of O⁴-CMdT into ODNs.