A ¹⁹F‐NMR study of 3‐fluoro‐4‐demethoxydaunomycin intercalation complexes with the hexanucleotide d(CTGCAG)₂

Abstract

Equilibrium systems containing intercalation complexes formed between the novel anthracycline drug, 3‐fluoro‐4‐demethoxydaunomycin (3FD), and the hexanucleotide duplex d(CTGCAG)2 have been studied by 19FNMR spectroscopy. Solutions containing a 1:1 molar ratio of 3FD/d(CTGCAG)2 gave four 19F signals which have been assigned to each of four possible intercalation isomers for the 1:1 3FD · d(CTGCAG)2 complex, which we denote by [d(CTGCAG)2][3FD]; these were where 3FD bound between the 5′‐CT‐3′,5′‐TG‐3′, 5′‐GC‐3′ or 5′‐CA‐3′ base sequences, with the drug sugar moiety lying in the minor groove and pointed in the 3′ direction in each case. Changes in temperature and NaCl concentration affecting the equilibrium distribution of these isomers were studied and indicated that no overriding binding site preference prevailed under standard biochemical conditions. Formation of some of the 2:1 3FD · d(CTGCAG)2 complex occurred when a solution of [d(CTGCAG)2][3FD] was exposed to excess 3FD; however, this complex was unstable to gel filtration and no co‐operative binding of the second 3FD molecule was observed.

A 19F NMR study of the interaction of 3‐fluoro‐4‐demethoxydaunomycin with the hexanucleotide d(TCCGGA)2

“…At higher NaCl concentrations, where the duplex is significantly more stable, the results of the 'H NMR study of 3FDd(CTGCAG), complex showed that the structure corresponding to isomer 1 in Fig. 3 exhibited the fastest 3FD dissociation rate [13]. This is consistent with other studies which have shown that the individual base pair melting temperature progressively increases in going from the terminal base pairs to the central residues of an oligonucleotide [14,15].…”

“…3. These conclusion are strongly supported by the results of our earlier study [6] of the binding of 3FD with the hexanucleotide d(CTGCAG),. In an analogous fashion, four 19F resonances were also observed and assigned to the four possible intercalation isomers; that is where the 3FD bound between the S-CT-3', S-TG-3', 5'-GC-3' and 5'-CA-3' with the drug sugar moiety lying in the minor groove and pointed in the 3' direction in each case.…”

“…3, tentative assignments of peaks A and B were made. Previous work suggests that at low NaCl concentrations the broadest signal is due to the intercalation isomer where the sugar moiety is located between the penultimate and terminal base pairs [6], i.e. structure 4 in Fig.…”

“…In an analogous fashion, four 19F resonances were also observed and assigned to the four possible intercalation isomers; that is where the 3FD bound between the S-CT-3', S-TG-3', 5'-GC-3' and 5'-CA-3' with the drug sugar moiety lying in the minor groove and pointed in the 3' direction in each case. This proposal was further supported by a 'H NMR study in which additional nucleotide proton resonances were observed upon addition of 3FD to the d(CTGCAG), solution that allowed identification of individual binding sites [13]. It was not possible to obtain similar 'H NMR results for the 3FD-d(TCCGGA), complex, presumably due to the faster nucleotide dissociation rate of 3FD which results in only one exchange averaged resonance being observed for each nucleotide proton.…”

“…We have previously demonstrated that by incorporating a 19F label into the anthracycline molecule, the relative binding affinity at specific two base sequences in an oligonucleotide can be determined by 19F NMR spectroscopy [6]. In this initial study it was shown that 4-demethoxydaunomycin shows no significant preference for any particular binding site in the hexanucleotide d(CTGCAG),, which contains both 5'-GC and 5'-CA sites.…”