On-bead cyclization in a combinatorial library of 15,625 octapeptides

Combinatorial peptide libraries prepared by split-and-mix synthesis on solid support can be decoded by amino acid analysis (AAA) using the TAGSFREE method, which assigns variable amino acids to ‘unique pair’ positions. The method was used here to investigate on-bead cyclization in a library of 15,625 octapeptides X8X7X6X5X4-Lys-X2-glu(β-Ala-β-Ala-TentaGel Macrobead)-OAllyl, anchored via the side-chain carboxylate of the d-glutamate. Cyclization was carried out by amide bond formation between the free N-terminus and the α-carboxyl group of d-glutamate after selective removal of the Fmoc and allyl protecting groups, and followed using the TNBS test for free amines. Fast-cyclizing sequences often contained a turn element, in particular Ala-(Asp/Thr)-Pro at X8-X7-X6, and phenylalanine at X2. Slow-cyclizing sequences contained predominantly basic and polar residues, in particular Arg-His-Ser at X7-X6-X5 and threonine at X8. Fast-cyclizing sequences gave higher preparative yields of cyclic peptides (22–26% purified yields) than slow-cyclizing sequences (6–8%), showing that fast reaction is associated with efficient cyclization. This experiment demonstrates the first use of a TAGSFREE library of cyclic peptides.

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