Development and applications of topologically segregated bilayer beads in one-bead one-compound combinatorial libraries

Using a "split-mix" synthesis approach, "One-Bead One-Compound" (OBOC) combinatorial libraries can be generated such that each bead displays only one chemical entity. Tens of thousands to millions of compound-beads can be screened concurrently using a variety of biochemical and cell-based screening methods. Positive beads are then physically isolated for structure determination. Peptide beads or peptoid beads consisting of α-amino acids and with a free N-terminus can be routinely sequenced by an automatic microsequencer using Edman chemistry. Libraries with N-terminally blocked peptides, peptides with unsequenceable building blocks, or small molecules require encoding. To fully exploit the OBOC combinatorial library methods, we have developed topologically segregated bilayer beads. Such bilayer beads allow us to prepare library compound on the outer layer of each bead and the coding tags in the bead interior. In addition, we can use these bilayer beads to prepare OBOC combinatorial libraries that are down-substituted on the bead surface but fully substituted in the bead interior. This configuration enables one to screen at a much higher stringency and yet have enough peptides or coding tags retained in the bead interior for structure determination.