Synthesis and screening of small molecule libraries active in binding to DNA (diversityycombinatorial chemistryyureasygel-shift assayyDNA binding)

Five synthetic combinatorial libraries of 2,080 components each were screened as mixtures for inhibi- tion of DNA binding to two transcription factors. Rapid, solution-phase synthesis coupled to a gel-shift assay led to the identification of two compounds active at a 5- to 10-mM concentration level. The likely mode of inhibition is interca- lation between DNA base pairs. The efficient deconvolution through sublibrary synthesis augurs well for the use of large mixtures of small, nonpeptide molecules in biological screens. One of the most rapidly developing areas in the chemical sciences is concerned with molecular diversity. In organic and bioorganic chemistry the activity takes the form of synthetic combinatorial libraries, and current issues deal with solid phase vs. solution methods; massive parallel synthesis of single compounds vs. synthesis of mixtures; the use of rigid core structures vs. flexible linear sequences; devising deconvolution strategies vs. tagging techniques; and generating unbiased molecular landscapes for lead discovery vs. biased structures for lead development. These and other topics are addressed in a commendable review (1), but the issues that appear to be resolved are the need for automation and utility. Mere syn- thesis of molecular libraries is not enough; the synthesis must be connected to a selection process. We describe here our recent efforts in the latter context. Although the majority of chemical diversity studies employ insoluble supports, recent innovations make solution-phase approaches more attractive (2-6). In addition, methodology to synthesize (7) and analyze (8) tetraurea-based libraries was recently introduced and permits replacement of the secondary amide (peptide) bond with a more bioavailable functionality (9, 10). Elaborated herein is a simplified and more general solution-phase route to tetraurea libraries derived from the isolated tetraisocyanate of xanthene. Derivatives that are differentially protected have also been prepared, allowing for synthetic access to individual tetraureas. The general method for library synthesis is outlined in Fig. 1. An activated core molecule is condensed with a number of building blocks (11), resulting in a combinatorial library of covalently linked, core-building block ensembles. The shape and rigidity of the core determines the orientation of the building blocks in shape space. The libraries can be biased by changing the core, linkage, or building blocks to target a characterized biological structure (''focused libraries'') or syn- thesized with less structural bias using flexible cores. The latter was the case in the situation described herein, because small molecules that inhibit transcription factor-DNA binding are rare (12). Active components in these mixtures were identified through an iterative synthesisyscreening protocol known as deconvolution.