NMR and Computational Characterization of the iV-(Deoxyguanosin-8-yl)aminofluorene Adduct [(AF)G] opposite Adenosine in DNA: (AF)G[syn]·A[anti] Pair Formation and Its pH Dependence

This paper reports on a combined two-dimensional NMR and energy minimization computational characterization of the conformation of the N-(deoxyguanosyl-8-yl)aminofluorene adduct [(AF)G] positioned across adenosine in a DNA oligomer duplex as a function of pH in aqueous solution. This study was undertaken on the d[C1-C2-A3-T4-C5-(AF)G6-C7-T8-A9-C10-C11]·[G12-G13-T14-A15-G16-A17-G18-A19-T20-G21-G22] complementary undecamer [(AF)G 11-mer duplex]. The modification of the single G6 on the pyrimidine-rich strand was accomplished by reaction of the oligonucleotide with N-acetoxy-2-(acetylamino)fluorene and subsequent deacetylation under alkaline conditions. The HPLC-purified modified strand was annealed with the unmodified purine-rich strand to generate the (AF)G 11-mer duplex. The exchangeable and nonexchangeable protons are well resolved and narrow in the NMR spectra of the (AF)G 11-mer duplex so that the base and the majority of sugar nucleic acid protons, as well as several aminofluorene ring protons, have been assigned following analysis of two-dimensional NOESY and COSY data sets at pH 6.9, 30 °C in H₂O and D₂O solution. The NOE distance constraints establish that the glycosidic torsion angle is syn at (AF)G6 and anti at A17, which results in the aminofluorene ring being positioned in the minor groove. A very large downfield shift is detected at the H2' sugar proton of (AF)G6 associated with the (AF)G6[syn]·A17[anti] alignment in the (AF)G 11-mer duplex. The NMR parameters demonstrate formation of Watson-Crick C5·G18 and C7·G16 base pairs on either side of the (AF)G6[syn]·A17[anti] modification site with the imino proton of G18 more stable to exchange than the imino proton of G16. Several nonexchangeable aminofluorene protons undergo large downfield shifts as do the imino and H8 protons of G16 on lowering of the pH from neutrality to acidic values for the (AF)G 11-mer duplex. Both the neutral and acidic pH conformations have been defined by assigning the NOE constraints in the [C5-(AF)G6-C7]·[G16-A17-G18] segment centered about the modification site and incorporating them in distance constrained minimized potential energy calculations in torsion angle space with the DUPLEX program. A series of NOEs between the aminofluorene protons and the DNA sugar protons in the neutral pH conformation establish that the aminofluorene ring spans the minor groove and is directed toward the G16-A17-G18 sugar-phosphate backbone on the partner strand. An unconstrained minimum energy neutral pH conformation for the (AF)G 11-mer duplex was generated from the minimization runs which satisfies the NMR distance constraints and is stabilized by hydrophobic interactions between the aminofluorene ring and the walls of the minor groove. The backbone of A17 adopts a B_II conformation (ϵ, ζ = g^-, t) and the sugar ring of G16 adopts a C3'-endo pucker which facilitates, in part, a small displacement of A17 toward the major groove and minimizes the total solvent exposure of aminofluorene and A17 at the modification site. There is poor stacking between bases in the C5-(AF)G6-C7 segment but strong stacking between bases in the G16-A17-G18 segment consistent with the large upfield shifts of the H8 protons of G16 and G18 in the (AF)G 11-mer duplex. The NOE constraints establish that the (AF)G6[syn]·A17[anti] alignment is retained on lowering of the pH but is accompanied by the loss of contacts between the aminofluorene ring of (AF)G6 and the G16-A17-G18 sugar-phosphate backbone on the partner strand. A minimum energy acidic pH conformation is proposed for the [C5-(AF)G6-C7]·[G16-A17-G18] segment involving protonated (AF)G6[syn]·A17[anti] pairing stabilized by one hydrogen bond between the N1 of protonated A17 and the O6 of (AF)G6. This alignment results in the displacement of the aminofluorene ring away from the helix axis in the minor groove. Our studies establish an interplay between hydrophobic and hydrogen-bonding contributions to the stabilization of the (AF)G6[syn]·A17[anti] modification site.