A Powerful Combinatorial Screen to Identify High‐Affinity Terbium(III)‐Binding Peptides

Lanthanide‐binding tags (LBTs) are protein fusion partners consisting of encoded amino acids that bind lanthanide ions with high affinity. Herein, we present a new screening methodology for the identification of new LBT sequences with high affinity for Tb3+ ions and intense luminescence properties. This methodology utilizes solid‐phase split‐and‐pool combinatorial peptide synthesis. Orthogonally cleavable linkers allow an efficient two‐step screening procedure. The initial screen avoids the interference caused by on‐bead screening by photochemically releasing a portion of the peptides into an agarose matrix for evaluation. The secondary screen further characterizes each winning sequence in a defined aqueous solution. Employment of this methodology on a series of focused combinatorial libraries yielded a linear peptide sequence of 17 encoded amino acids that demonstrated a 140‐fold increase in affinity (57 nM dissociation constant, KD) over previously reported lanthanide‐binding peptides. This linear sequence was macrocyclized by introducing a disulfide bond between flanking cysteine residues to produce a peptide with a 2‐nM apparent dissociation constant for Tb3+ ions.