The preparation and examination of 2-22 constituting a systematic study of the chromophore of sandramycin (1) are detailed. Fluorescence quenching studies were used to establish binding constants for 1-24 within calf thymus DNA, within a single high affinity bis-intercalation binding site 5'-d(GCATGC)2, and to establish the preference for sandramycin binding to 5'-d(GCXXGC)2 where XX = AT, TA, GC, and CG. From the latter studies, sandramycin was found to exhibit a preference that follows the order: 5'-d(GCATGC)2 > 5'-d(GCGCGC)2, deltadeltaGo = 0.3kcal/mol > 5'-d(GCTAGC)2, 5'-d(GCCGGC)2, deltadeltaGo = 0.6 kcal/mol although it binds with high affinity to all four deoxyoligonucleotides. The two highest affinity sequences constitute repeating 5'-PuPy motifs with each intercalation event occurring at a 5'-PyPu step. The most effective sequence constitutes the less stable duplex, contains the sterically most accessible minor groove central to the bis-intercalation site, and the ability to accept two gly-NH/TC2 carbonyl H-bonds identified in prior NMR studies. Similarly, the contribution of the individual structural features of the chromophore were assessed with the high affinity duplex sequence 5'-d(GCATGC)2. To a first approximation, the cytotoxic properties were found to parallel trends established in the DNA binding affinities. The exception to this generalization was 4 which lacks the sandramycin chromophore phenol. Although typically 4-10x less potent than sandramycin against leukemia cell lines, it proved to be 1-10,000x more potent against melanomas, carcinomas, and adenocarcinomas exhibiting IC50 values of 1 pM-10 nM placing it among the most potent agents identified to date. Additionally, the first disclosure of the HIV-1 reverse transcriptase inhibitory activity of sandramycin (1) as well as that of its key analogs are described and define the chromophore structural features required for their exceptional potency. Two analogs, 18 and 3, roughly maintain the HIV-1 reverse transcriptase inhibitory potency of 1 but exhibit substantially diminished cytotoxic activity (10(2)-10(3)x).