6-Nitrochrysene-Derived C8-2′-Deoxyadenosine Adduct: Synthesis of Site-Specific Oligodeoxynucleotides and Mutagenicity in Escherichia coli

Abstract

6-Nitrochrysene (6-NC), the most potent carcinogen evaluated by the newborn mouse assay, is metabolically activated by nitroreduction and a combination of ring oxidation and nitroreduction pathways. The nitroreduction pathway yields three major DNA adducts: at the C8 and N 2 positions of 2′deoxyguanosine (dG), N-(dG-8-yl)-6-AC and 5-(dG-N 2 -yl)-6-AC, and at the C8 position of 2′-deoxyadenosine (dA), N-(dA-8-yl)-6-AC. A nucleotide excision repair assay demonstrated that N-(dA-8-yl)-6-AC is repaired much more slowly than many other bulky DNA adducts, including the other DNA adducts formed by 6-NC. But neither the total synthesis nor evaluation of other biological activities of this dA adduct has ever been reported. Herein, we report a convenient synthesis of the 6-NC-derived dA adduct by employing the Buchwald−Hartwig coupling strategy, which provided a high yield of the protected N-(dA-8-yl)-6-AC. The deprotected nucleoside showed syn conformational preference by NMR spectroscopy. Following DMT protection of the 5′-hydroxyl, N-(dA-8-yl)-6-AC was converted to its 3′phosphoramidite, which was used to prepare oligonucleotides containing a single N-(dA-8-yl)-6-AC adduct. Circular dichroism spectra of the adducted duplex showed only a slight departure from the B-DNA helix profile of the control duplex. The 15-mer N-(dA-8-yl)-6-AC oligonucleotide was used to construct a single-stranded plasmid vector containing a single adduct, which was replicated in Escherichia coli. Viability of the adducted construct was ∼60% of the control, indicating slower translesion synthesis of the adduct, which increased to nearly 90% upon induction of the SOS functions. Without SOS, the mutation frequency (MF) of the adduct was 5.2%, including 2.9% targeted and 2.3% semi-targeted mutations. With SOS, the targeted MF increased 3-fold to 9.0%, whereas semi-targeted mutation increased only marginally to 3.2%. The major type of targeted mutation was A*→G in both uninduced and SOS-induced cells.

Translesion synthesis of 6-nitrochrysene-derived 2ʹ-deoxyadenosine adduct in human cells

“…It is remarkable that N-(dA-8-yl)-6-AC induces predominantly A*→G mutations in both uninduced and SOS-induced E. coli cells, and A*→T mutations occur at a much lower frequency [18]. But in human cells the trend is reversed with A*→T occurring at a much higher frequency than A*→G.…”

“…A 15-mer 5ʹ-GCCCTCAA*CAAGATG where A* denotes N-(dA-8-yl)-6-AC was synthesized as reported [18]. The DNA sequence of this oligonucleotide was selected from the TP53 gene codon 129-133, since codon 131 is a hotspot for A→T transversions in patients with urothelial cancers by exposure to another nitroaromatic carcinogen, aristolochic acid [27][28][29].…”

“…The DNA sequence of this oligonucleotide was selected from the TP53 gene codon 129-133, since codon 131 is a hotspot for A→T transversions in patients with urothelial cancers by exposure to another nitroaromatic carcinogen, aristolochic acid [27][28][29]. The 15-mer containing N-(dA-8-yl)-6-AC and a control were ligated to a gapped plasmid DNA to prepare site-specifically modified pMS2 plasmid constructs, according to a method reported earlier [18]. The lesion containing pMS2 construct and an unmodified plasmid DNA (containing a 20-mer sequence 5ʹ-AGGGTTTACCCAGTCACGTT-3ʹ at the ligation site different from the DNA sequence of either the lesion containing or control DNA) in 3:1 ratio were co-transfected into HEK 293T cells with or without deficiency in TLS polymerases.…”

“…We have recently developed a total synthesis method to prepare site-specifically incorporated N-(dA-8-yl)-6-AC in any desired sequence [18]. We have also shown that this adduct is mutagenic in Escherichia coli.…”

Site-Specific Incorporation of N-(2′-Deoxyguanosine-8-yl)-6-aminochrysene Adduct in DNA and Its Replication in Human Cells

“…16 There are no reports to synthesize the 6-NC-DNA adducts by a total synthetic approach, except we recently published an efficient method to prepare N-(dA-8-yl)-6-AC-containing oligonucleotide. 17 reacting N-OH-6-AC or 1,2-DHD-6-NHOH-C with oligonucleotides containing either a single dG or dA, as appropriate, to prepare small amount of the adducted oligonucleotides. 18 They also investigated repair of these adducts in human cells.…”