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

The regulation of gene expression is based on the sequence selective recognition of nucleic acids by a host of repressor, activator, and enhancer proteins. Selective external disruption or control of such processes has been a long standing goal of molecular biology, chemistry and medicine. Small molecules that are able to selectively bind DNA and activate (block a repressor) or inhibit (block an activator) gene expression hold great promise as therapeutics. However, the discovery of such agents has been slow due in part to the biophysical complexity associated with understanding small molecule-DNA interactions, the iterative process of designing and synthesizing individual molecules toward specific DNA sequences, and the technically demanding techniques involved in determination of their selectivity and binding affinity for a given sequence. Herein the author  report the combinatorial synthesis of a prototypical library of 2640 analogues of distamycin A and the development of a rapid, technically nondemanding screen for the determination of DNA binding affinity and sequence selectivity applicable to such libraries which should help accelerate the discovery process.

Solution-Phase Total Synthesis of Distamycin A.

As an initial demonstration of the approach, the author first conducted a total synthesis of distamycin A utilizing solution-phase synthesis techniques that require only acid/base liquid liquid extraction purification protocols. Previous total syntheses of distamycin A generally have relied on the coupling of N-methyl-4-nitropyrrole-2-carboxylic acid chlorides followed by nitro group reduction and further coupling steps. In developing a general set of reaction conditions suitable for the preparation of libraries, several requirements not intrinsic to the natural product synthesis needed to be addressed. In all cases, unreacted starting materials, coupling agents, and their reaction byproducts needed to be removed by simple acid/base extraction. Although acid chlorides could be used, the preparation and long-term storage of numerous heterocyclic acid chlorides would be difficult, requiring the implementation of coupling protocols that use the carboxylic acids directly. In addition, a nitro group reduction step introduces a reaction of variable generality (reaction time, catalyst poisoning), precludes the inclusion of subunits sensitive to reduction conditions, and requires the resultant free amines to be relatively stable. Consequently, we adopted the more direct use of BOC-protected amines. A general set of coupling conditions that gives high yields of coupled product was developed enlisting the amines and carboxylic acids directly and the water soluble 1-[3-(dimethylamino)propyl]-3-ethylcar-bodiimide hydrochloride (EDCI) with (dimethylamino)pyridine (DMAP) as an additive. The unreacted starting materials, reagents, and reaction and reagent byproducts all may be removed by acid/base extractions.

An approach to the rapid, parallel solution-phase synthesis of distamycin A analogues was developed enlisting a simple acid/base liquid liquid extraction for purification and isolation of each intermediate and final product (≥95% pure). Its utility was demonstrated with the preparation of distamycin A and a prototypical library of 2640 analogues assembled in a small mixture format of two libraries of 132 mixtures of 10 compounds providing each in multimilligram quantities sufficient for screening in multiple assays.



Dale L. Boger, J. Am. Chem. Soc. 2000, 122, 6382-6394