Web Release Date: June 20,
Total Synthesis of Distamycin A and 2640 Analogues: A Solution-Phase Combinatorial Approach to the Discovery of New, Bioactive DNA Binding Agents and Development of a Rapid, High-Throughput Screen for Determining Relative DNA Binding Affinity or DNA Binding Sequence Selectivity
Contribution from the Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037
Received November 30, 1999
Revised Manuscript Received March 2, 2000
Abstract:
The development of a solution-phase synthesis of distamycin A and its extension to the preparation
of 2640 analogues are described. Thus, solution-phase synthesis techniques with reaction workup and purification
employing acid/base liquid-liquid extractions were used in the multistep preparation of distamycin A (8 steps,
40% overall yield) and a prototypical library of 2640 analogues providing intermediates and final products
that are 
95% pure on conventional reaction scales. The complementary development of a simple, rapid, and
high-throughput screen for DNA binding affinity based on the loss of fluorescence derived from displacement
of prebound ethidium bromide is disclosed which is applicable for assessing relative or absolute binding affinity
to DNA homopolymers or specific sequences (hairpin oligonucleotides). Using hairpin oligonucleotides, this
method permits the screening of a library of compounds against a single predefined sequence to identify high
affinity binders, or the screening of a single compound against a full library of individual hairpin oligionucleotides
to define its sequence selectivity. The combination permits the establishment of the complete DNA binding
profile of each member of a library of compounds. Screening the prototypical library provided compounds
that are 1000 times more potent than distamycin A in cytotoxic assays (67, IC50 = 29 nM, L1210), that bind
to poly[dA]-poly[dT] with comparable affinity, and that exhibit an altered DNA binding sequence selectivity.
Several candidates were identified which bound the five-base-pair AT-rich site of the PSA-ARE-3 sequence,
and one (128, K = 3.2 × 106 M-1) maintained the high affinity binding (K = 4.5 × 106 M-1) to the ARE-consensus sequence containing a GC base-pair interrupted five-base-pair AT-rich site suitable for inhibition
of gene transcription initiated by hormone insensitive androgen receptor dimerization and DNA binding
characteristic of therapeutic resistant prostate cancer.
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