Total synthesis of distamycin A and 2640 analogues: A solution-phase combinatorial approach to the discovery of new, bioactive DNA binding agents anddevelopment of a rapid, high-throughput screen for determining relative DNA binding affinity or DNA binding sequence selectivity

The development of a solution-phase synthesis of distamycin A and its exten sion to the preparation of 2630 analogues are described. Thus, solution-pha se synthesis techniques with reaction workup and purification employing aci d/base liquid-liquid extractions were used in the multistep preparation of distamycin A (8 steps, 40% overall yield) and a prototypical library of 263 0 analogues providing intermediates and final products that are greater tha n or equal to 95% pure on conventional reaction scales. The complementary d evelopment of a simple, rapid, and high-throughput screen for DNA binding a ffinity based on the loss of fluorescence derived from displacement of preb ound ethidium bromide is disclosed which is applicable for assessing relati ve or absolute binding affinity to DNA homopolymers or specific sequences ( hairpin oligonucleotides). Using hairpin oligonucleotides, this method perm its the screening of a library of compounds against a single predefined seq uence to identify high affinity binders, or the screening of a single compo und against a full library of individual hairpin oligionucleotides to defin e its sequence selectivity. The combination permits the establishment of th e complete DNA binding profile of each member of a library of compounds. Sc reening the prototypical library provided compounds that are 1000 times mor e potent than distamycin A in cytotoxic assays (67, IC50 = 29 nM, L1210), t hat bind to poly[dA]-poly[dT] with comparable affinity, and that exhibit an altered DNA binding sequence selectivity. Several candidates were identifi ed which bound the five-base-pair AT-rich site of the PSA-ARE-3 sequence, a nd one (128, K = 3.2 x 10(6) M-1) maintained the high affinity binding (K = 4.5 x 10(6) M-1) to the ARE-consensus sequence containing a CTC base-pair interrupted five-base-pair AT-rich site suitable for inhibition of gene tra nscription initiated by hormone insensitive androgen receptor dimerization and DNA binding characteristic of therapeutic resistant prostate cancer.