Molecular Recognition between Oligopeptides and Nucleic Acids: Sequence-Specific Binding of the Naturally Occurring Antibiotic (4S)-(+)-Anthelvencin A and its (4R)-(-) Enantiomer to Deoxyribonucleic Acids Deduced from ¹H NMR, Footprinting, and Thermodynamic Datax

The DNA sequence specificity of both the natural antibiotic (4S)-(+)-anthelvencin A (1a) and its unnatural (4R)-(-) enantiomer 1b was determined by DNase I footprinting to be similar to that of netropsin. Structural and dynamic aspects of the binding of 1a and 1b to the double-helical decadeoxyribonucleotide d(CGCAATTGCG)₂ were studied by high-field ¹H NMR spectroscopy. The drug-induced chemical shifts and NOE measurements of the 1:1 complexes of (4S)-(+)-1a and (4R)-(-)-1b to the decadeoxyribonucleotide (complexes A and B, respectively) reveal that both enantiomeric forms of anthelvencin A bind in the minor groove along the sequence 5'-A₄A₅T₆T₇-3' of the DNA. NOE studies show that the (4S) enantiomer 1a is propeller twisted between the two pyrrole moieties to provide the structural flexibility for the drug to bind tightly in the minor groove along the 5'-AATT sequence on the DNA in complex A. In contrast, the unnatural (4R) enantiomer 1b adopts a coplanar conformation in complex B. From molecular modeling studies, it is shown that the (4S) hydrogen of 1a is directed out of the minor groove thereby positioning the positively charged 2-amino-1-pyrrolinium moiety for more favorable electrostatic interaction between the drug and DNA. Conversely, for (4R)-1b of complex B, the positively charged 2-amino-1-pyrrolinium group is directed out of the minor groove, and as a result, the interaction of this group with the negative potential of that section of the DNA is restricted. The natural (4S) enantiomer 1a is not bound centrally between the d[(A₄A₅T₆T₇)·(A₄A₅T₆T₇)] DNA duplex but is located diagonally across the 5'-AATT base pair sequence. In contrast, the unnatural (4R) enantiomer 1b behaves like achiral lexitropsins and binds centrally in the minor groove at the 5'-AATT base pair sequence. The rate of exchange of the chiral lexitropsins between equivalent sites is found to be dependent on the chirality of the oligopeptides. The exchange rates of (4S)-1a and (4R)-1b between the two equivalent 5'-AATT sites via the intramolecular flip-flop mechanism are ~36 s^-1 at 21 °C with ΔG^‡ of 68.3 ± 5 kJ mol ^-1 and ~77 s^-1 at 21 °C with ΔG^‡ of 62.5 ± 5 kJ mol^-1, respectively. Spectroscopic and calorimetric studies reveal that compounds (4S)-1a and (4R)-1b exhibit similar binding affinities for the poly[d(AT)]•poly[d(AT)] duplex, with ΔG° values of –33.5 and –33.0 kJ/mol, respectively, at 25 °C. Thus, the stereochemical difference between the enantiomers does not alter significantly the binding affinities of the two ligands for the poly[d(AT)]•poly[d(AT)] duplex. Furthermore, at 25 °C, the binding of both enantiomers to poly[d(AT)]•poly[d(AT)] is strongly entropy driven while the corresponding binding of the structurally similar ligand netropsin is strongly enthalpy driven. These results suggest that relatively small changes in drug structure can cause dramatic changes in the binding thermodynamics. The binding constants for both enantiomers exhibit very similar salt dependencies. Thus, the difference in chirality between the enantiomers does not alter the overall electrostatic contribution to the binding of the two ligands to the poly[d(AT)]•poly[d(AT)] duplex. Furthermore, the magnitude of the salt dependence of the binding constants for both enantiomers is characteristic of monocationic binding despite their formal dicationic structures. CD titration curves reveal that both anthelvencin enantiomers exhibit saturation binding site sizes equivalent to two base pairs. A model is proposed for the saturation binding of the two anthelvencins to the poly[d(AT)]•poly[d(AT)] duplex that is consistent with both the monocationic binding behavior and the binding site size. Collectively the data suggest the anthelvencins have one cationic terminus strongly bound to the minor groove site while the other terminus is less strongly bound.