One of the first steps in HIV gene expression is the recruitment of Tat protein to the transcription machinery after its binding to the RNA response element TAR. Starting from a pool of 3.2 ؋ 10 6 individual chemical entities, we were able to select a hybrid peptoid͞peptide oligomer of 9 residues (CGP64222) that was able to block the formation of the Tat͞TAR RNA complex in vitro at nanomolar concentrations. NMR studies demonstrated that the compound binds similarly to polypeptides derived from the Tat protein and induces a conformational change in TAR RNA at the Tat

A tripeptoid library was synthesized using 69 different primary amines in initially 69 individual reactions by the mix and split approach. The resulting library consisted of 328,509 (69(3)) single compounds, divided in 69 subpools each containing 4,761 entities. The 69 subpools were tested in two binding assays, one for alpha-MSH (alpha-melanotropin) and one for GRP (gastrin-releasing peptide)/bombesin. The sublibraries with the highest affinity to the MSH receptor (i.e. melanocortin type 1 or MC1 receptor) and, respectively, the GRP-preferring bombesin receptor were identified by an iterative process. Individual tripeptoids with good binding activity were resynthesized, analyzed and their dissociation constants and biological activity determined. The KD of the most potent MC1 receptor ligand was 1.58 mumol/l and that of the GRP-preferring bombesin receptor 3.40 mumol/l. Extension of this latter tripeptoid structure whose KD value increased to 280 nmol/l. A similar increase in activity was not observed with the most potent MSH tripeptoid ligand when extended by one residue, but a compound suitable for radioiodination and lacking the N-terminal amino group had a slightly higher binding activity than the tripeptoids (KD approximately 850 nmol/l). These results demonstrate that testing a peptoid library containing 328,509 single compounds led to the successful identification of new ligands for both the MC1 receptor as well as the GRP-preferring bombesin receptor.

SummaryXanthines represent a new, versatile scaffold for combinatorial chemistry. A five-step solid-phase synthesis of xanthine derivatives is described which includes alkylations, a nucleophilic displacement reaction at a heterocycle and a ring closure reaction by condensation of a nitroso function with an activated methylene group. The selected reaction sequence allows the production of a highly diverse small-molecule combinatorial compound library.

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A strategy for high-throughput evaluation of combinatorial compound libraries is reported, which circumvents the necessity to test complex mixtures. The method is based on a new combination of protecting groups, solid-phase linker and tags. The bulk of the library first undergoes a binding assay with the components grafted on beads. A selection of beads carrying strong ligands is stripped from the labelled target and distributed into microvessels. The ligands are cleaved and rinsed into microeluates. Subsequently, a more detailed characterization with a functional assay in solution determines the best performers, which are identified through the peptidic tag left behind on the corresponding mother bead.