In the "one-bead one-compound" (OBOC) combinatorial library method, each compound bead displays only one compound entity. Hundreds of thousands to millions of compound beads can be synthesized rapidly and screened simultaneously. Positive compound beads are then isolated for structural analysis. To fully exploit the power of OBOC combinatorial small molecule libraries, a robust and high throughput encoding method is needed to decode the positive compound beads. In this paper, we report on the development of a novel encoding strategy that combines the concepts of ladder-synthesis and chemical encoding on bilayer beads. In these encoded libraries, small molecule compounds are displayed on the bead surface, and cleavable coding tags consisting of a series of truncated molecules reside in the bead interior. Such a library can be easily constructed using the biphasic approach (J. Am. Chem. Soc. 2002, 124, 7678) to topologically segregate the functionalities of the beads during library synthesis. The ladder members and coding tags are then released for MALDI-TOF-MS analysis. To simplify the interpretation of the mass spectra, we purposely add bromine into the cleavable linker so that the cleavage products generate a characteristic isotope fingerprint. The chemical structure of library compounds can be determined by analyzing the mass differences between adjacent peaks on the mass spectra. This encoding strategy also provides valuable information on the quality of the testing compound on the surface of the bead. To validate this methodology, a model OBOC small molecule library with 12,288 members was synthesized on TentaGel beads and screened against streptavidin. The chemical structures of the compound on each positive bead were unambiguously identified.
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