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Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis.

Gillet LC, Alzeer J, Schärer OD - Nucleic Acids Res. (2005)

Bottom Line: Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of beta-mercaptoethanol) designed to remove protecting groups of commercially available 'ultra-mild' phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF.We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction.Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Cancer Research, University of Zürich August Forel Strasse 7, 8008 Zürich, Switzerland.

ABSTRACT
Aromatic amino and nitro compounds are potent carcinogens found in the environment that exert their toxic effects by reacting with DNA following metabolic activation. One important adduct is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF), which has been extensively used in studies of the mechanisms of DNA repair and mutagenesis. Despite the importance of dG-AAF adducts in DNA, an efficient method for its incorporation into DNA using solid-phase synthesis is still missing. We report the development of a modified 'ultra-mild' DNA synthesis protocol that allows the incorporation of dG-AAF into oligonucleotides of any length accessible by solid-phase DNA synthesis with high efficiency and independent of sequence context. Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of beta-mercaptoethanol) designed to remove protecting groups of commercially available 'ultra-mild' phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF. We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction. Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.

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Site-specific incorporation of dG-AAF and dG-AF into oligonucleotides. Reaction conditions: (a) DMTr-Cl, pyridine (76%); (b) N-ethyldiisopropylamine, 2-cyanoethoxydiisopropylchloro-phosphoramidite, CH2Cl2 (86%); (c) modified ‘ultra-mild’ DNA synthesis as shown in Table 1; and (d) 1 M NaOH, 0.25 M β-mercaptoethanol.
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fig2: Site-specific incorporation of dG-AAF and dG-AF into oligonucleotides. Reaction conditions: (a) DMTr-Cl, pyridine (76%); (b) N-ethyldiisopropylamine, 2-cyanoethoxydiisopropylchloro-phosphoramidite, CH2Cl2 (86%); (c) modified ‘ultra-mild’ DNA synthesis as shown in Table 1; and (d) 1 M NaOH, 0.25 M β-mercaptoethanol.

Mentions: With the improved ultra-mild DNA synthesis and deprotection protocol in hand, we assessed its usefulness to incorporate dG-AAF residues into oligonucleotides. To be consistent with the ‘ultra-mild’ iPrPac protected dG, we had previously reported the preparation of N2-iPrPac protected dG-AAF (6) (39). 5′-Dimethoxytrityl protection of iPrPac-dG-AAF (6) and subsequent 3′-phosphitylation to (7) were performed according to the standard procedures (Figure 2). The iPrPac-dG-AAF phosphoramidite (7) was dissolved to 0.1 M in CH2Cl2, which provided improved stability and coupling efficiency over acetonitrile. We prepared then a 9mer (9-AAF) containing a single dG-AAF adduct using the conditions summarized in Table 1, and purified the oligonucleotide by HPLC. The HPLC trace revealed the homogeneity and the purity of the dG-AAF modified oligonucleotide (Figure 3A). Treatment of 9-AAF for 3 h at 37°C in a 1 M NaOH solution containing 0.25 M of β-mercaptoethanol (12) led to selective cleavage of the N8 acetyl and to the formation of corresponding dG-AF modified 9mer (9-AF). The 9-AAF and 9-AF oligomers eluted at two distinct retention times on HPLC (Figure 3), confirming the effective removal of the acetyl group from 9-AAF to yield the less polar 9-AF (12), and proving that the purified 9-AAF was really exempt of decomposition into the deacetylated 9-AF. Nano-ESI analyses confirmed the identity and purity of the 9-AAF and 9-AF (Table 2 and Supplementary Material).


Site-specific incorporation of N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF) into oligonucleotides using modified 'ultra-mild' DNA synthesis.

Gillet LC, Alzeer J, Schärer OD - Nucleic Acids Res. (2005)

Site-specific incorporation of dG-AAF and dG-AF into oligonucleotides. Reaction conditions: (a) DMTr-Cl, pyridine (76%); (b) N-ethyldiisopropylamine, 2-cyanoethoxydiisopropylchloro-phosphoramidite, CH2Cl2 (86%); (c) modified ‘ultra-mild’ DNA synthesis as shown in Table 1; and (d) 1 M NaOH, 0.25 M β-mercaptoethanol.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1074722&req=5

fig2: Site-specific incorporation of dG-AAF and dG-AF into oligonucleotides. Reaction conditions: (a) DMTr-Cl, pyridine (76%); (b) N-ethyldiisopropylamine, 2-cyanoethoxydiisopropylchloro-phosphoramidite, CH2Cl2 (86%); (c) modified ‘ultra-mild’ DNA synthesis as shown in Table 1; and (d) 1 M NaOH, 0.25 M β-mercaptoethanol.
Mentions: With the improved ultra-mild DNA synthesis and deprotection protocol in hand, we assessed its usefulness to incorporate dG-AAF residues into oligonucleotides. To be consistent with the ‘ultra-mild’ iPrPac protected dG, we had previously reported the preparation of N2-iPrPac protected dG-AAF (6) (39). 5′-Dimethoxytrityl protection of iPrPac-dG-AAF (6) and subsequent 3′-phosphitylation to (7) were performed according to the standard procedures (Figure 2). The iPrPac-dG-AAF phosphoramidite (7) was dissolved to 0.1 M in CH2Cl2, which provided improved stability and coupling efficiency over acetonitrile. We prepared then a 9mer (9-AAF) containing a single dG-AAF adduct using the conditions summarized in Table 1, and purified the oligonucleotide by HPLC. The HPLC trace revealed the homogeneity and the purity of the dG-AAF modified oligonucleotide (Figure 3A). Treatment of 9-AAF for 3 h at 37°C in a 1 M NaOH solution containing 0.25 M of β-mercaptoethanol (12) led to selective cleavage of the N8 acetyl and to the formation of corresponding dG-AF modified 9mer (9-AF). The 9-AAF and 9-AF oligomers eluted at two distinct retention times on HPLC (Figure 3), confirming the effective removal of the acetyl group from 9-AAF to yield the less polar 9-AF (12), and proving that the purified 9-AAF was really exempt of decomposition into the deacetylated 9-AF. Nano-ESI analyses confirmed the identity and purity of the 9-AAF and 9-AF (Table 2 and Supplementary Material).

Bottom Line: Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of beta-mercaptoethanol) designed to remove protecting groups of commercially available 'ultra-mild' phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF.We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction.Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.

View Article: PubMed Central - PubMed

Affiliation: Institute for Molecular Cancer Research, University of Zürich August Forel Strasse 7, 8008 Zürich, Switzerland.

ABSTRACT
Aromatic amino and nitro compounds are potent carcinogens found in the environment that exert their toxic effects by reacting with DNA following metabolic activation. One important adduct is N-(deoxyguanosin-8-yl)-2-acetylaminofluorene (dG-AAF), which has been extensively used in studies of the mechanisms of DNA repair and mutagenesis. Despite the importance of dG-AAF adducts in DNA, an efficient method for its incorporation into DNA using solid-phase synthesis is still missing. We report the development of a modified 'ultra-mild' DNA synthesis protocol that allows the incorporation of dG-AAF into oligonucleotides of any length accessible by solid-phase DNA synthesis with high efficiency and independent of sequence context. Key to this endeavor was the development of improved deprotection conditions (10% diisopropylamine in methanol supplemented with 0.25 M of beta-mercaptoethanol) designed to remove protecting groups of commercially available 'ultra-mild' phosphoramidite building blocks without compromising the integrity of the exquisitely base-labile acetyl group at N8 of dG-AAF. We demonstrate the suitability of these oligonucleotides in the nucleotide excision repair reaction. Our synthetic approach should facilitate comprehensive studies of the mechanisms of repair and mutagenesis induced by dG-AAF adducts in DNA and should be of general use for the incorporation of base-labile functionalities into DNA.

Show MeSH
Related in: MedlinePlus