Abstract: In this work we have focused on the synthesis of new derivatives of naphthalene diimides that bind to DNA by a threading intercalation mode. The advantage of having a threading intercalator as a "carrier" part of the drug is that it brings additional recognition pattern into the system by directing substituents into the opposite grooves of DNA. This allows the possibility of selectively varying substituents to develop medicinal agents as well as probes for large-amplitude DNA dynamics. A systematic set of naphthalene diimides, with side chains of increasing size, have been synthesized. Their physical properties as well as DNA interaction kinetics were studied to probe the factors which determine the rates of association and dissociation of DNA binding. We have used intercalators with large and rigid substituents to probe the opening of an intercalation site with disruption of an adjacent base pair.