A Non‐Intercalating Proflavine Derivative

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Abstract
A proflavine derivative with bulky substituents, 2,7‐di‐t‐butyl‐proflavine, binds to DNA but apparently not by intercalation. The methods used to study this problem include equilibrium dialysis, relaxation kinetics and hydroxdynamic studies on low‐molecular‐weight and closed‐circular DNA. These show that binding produces no detectable lengthening or unwinding of the double helix, and that the binding reaction is much faster than is the case with unsubstituted proflavine. We conclude that an outside‐bound complex is formed.We also measured equilibrium binding isotherms for DNA samples of varying G · C content, and for several synthetic polynucleotides. These results are consistent with a model in which a bound 2,7‐di‐t‐butyl‐proflavine molecule occupies three base pairs, and strong binding requires that two of these three be A · T pairs. Comparison of poly(dA) · poly(dT) with poly(A) · poly(U) shows a preference of at least 100‐fold for binding to the former over the latter. Also, we found that the thymine methyl groups are probably not responsible for the A · T specificity.The optical properties of the outside complex include a red shift of the absorbance maximum, with an increase in maximum absorbance instead of the more common decrease. The fluorescence of the dye is enhanced on binding, especially to an A · T‐rich‐DNA.The kinetic results show that two complex forms are present, differing in intrinsic affinity by about an order of magnitude. Both are formed with second‐order rate constants in excess of 108 M−1 s−1 and it is therefore very unlikely that either is an intercalated form. The absorbance spectrum of the bound dye changes as the degree of binding increases, due, at least in part, to the changing contribution of the two binding mechanisms. The conclusions concerning binding specificity refer to the dominant complex form; we have no firm information about the specificity of the weaker complex.