Synthesis and characterization of oligodeoxyribonucleotides containing a site-specifically incorporated N6-carboxymethyl-2'-deoxyadenosine or N4-carboxymethyl-2'-deoxycytidine

Abstract

Humans are exposed to both endogenous and exogenous N-nitroso compounds (NOCs), and many NOCs can be metabolically activated to generate a highly reactive species, diazoacetate, which is capable of inducing carboxymethylation of nucleobases in DNA. Here we report, for the first time, the chemical syntheses of authentic N(6)-carboxymethyl-2'-deoxyadenosine (N(6)-CMdA) and N(4)-carboxymethyl-2'-deoxycytidine (N(4)-CMdC), liquid chromatography-ESI tandem MS confirmation of their formation in calf thymus DNA upon diazoacetate exposure, and the preparation of oligodeoxyribonucleotides containing a site-specifically incorporated N(6)-CMdA or N(4)-CMdC. Additionally, thermodynamic studies showed that the substitutions of a dA with N(6)-CMdA and dC with N(4)-CMdC in a 12-mer duplex increased Gibbs free energy for duplex formation at 25°C by 5.3 and 6.8 kcal/mol, respectively. Moreover, primer extension assay revealed that N(4)-CMdC was a stronger blockade to Klenow fragment-mediated primer extension than N(6)-CMdA. The polymerase displayed substantial frequency of misincorporation of dAMP opposite N(6)-CMdA and, to a lesser extent, misinsertion of dAMP and dTMP opposite N(4)-CMdC. The formation and the mutagenic potential of N(6)-CMdA and N(4)-CMdC suggest that these lesions may bear important implications in the etiology of NOC-induced tumor development.

Scheme 1.

Syntheses of authentic N⁶-CMdA (a) and N⁴-CMdC (b).

Scheme 2.

Syntheses of phosphoramidite building blocks of ethyl ester derivative of the N⁶-CMdA (a) and methyl ester derivative of N⁴-CMdC (b).

Figure 1.

LC–MS/MS for monitoring the formation of N⁶-CMdA and N⁴-CMdC in KDA-treated calf thymus DNA. Shown are the SICs for monitoring the m/z 310.1→194.1 and 286.1→170.1 transition, corresponding to the loss of a 2-deoxyribose, from the LC–MS/MS analyses with the injection of: (a) N⁶-CMdA standard; (b) enzymatic digestion mixture of calf thymus DNA treated with 10 mM of KDA; (c) N⁴-CMdC standard; and (d) the same sample as in (b).

Figure 2.

Collision-induced dissociation (CID) mass spectra supporting the formation of N⁶-CMdA and N⁴-CMdC in calf thymus DNA. Shown are the MS/MS results that monitor the fragmentation of the protonated-ions of N⁶-CMdA (m/z 310.1) and N⁴-CMdC (m/z 286.1), for: (a) standard N⁶-CMdA; (b) the 30.5-min fraction shown in Figure 1b; (c) standard N⁴-CMdC; and (d) the 12.2-min fraction shown in Figure 1d.

Figure 3.

ESI-MS and MS/MS characterizations of d(ATGGCGXGCTAT), X = N⁶-CMdA: (a) negative-ion ESI-MS; (b) product-ion spectrum of the [M-3H]³⁻ ion (m/z 1246.3).

Figure 4.

In vitro replication studies of N⁶-CMdA- or N⁴-CMdC-bearing ODNs and the corresponding unmodified substrates with Kf⁻ of E. coli DNA polymerase I. A template, d(ATGGCGXGCTATGATCCTAG) (‘X’ represents N⁶-CMdA, N⁴-CMdC, dA or dC), and a 5′-[³²P]-labeled primer, d(p*GCTAGGATCATAGC), were employed. The concentration of the resulting duplex ODNs was 50 nM. The replication experiments were carried out: (a) in the presence of all four dNTPs at a concentration of 1.0 mM each for 30 min and the amounts of Kf⁻ are indicated in the figure; and (b) in the presence of dNTPs individually ([dNTP] = 1.0 mM) at 37°C for 10 min, and 0.1 U of Kf⁻ was used.