The incorporation of unnatural chelating amino acids in short peptide sequences leads to lanthanide-binding peptides with a higher stability than sequences built exclusively from natural residues. In particular, the hexadentate peptide P-22, which incorporates two unnatural amino acids Ada(2) with aminodiacetate chelating arms, showed picomolar affinity for Tb3+. To design peptides with higher denticity, expected to show higher affinity for Ln(3+), we synthesized the novel unnatural amino acid Ed3a(2) which carries an ethylenediamine triacetate side-chain and affords a pentadentate coordination site. The synthesis of the derivative Fmoc-Ed3a(2)(tBu)(3)-OH, with appropriate protecting groups for direct use in the solid phase peptide synthesis (Fmoc strategy), is described. The two high denticity peptides P-HD2 (Ac-Trp-Ed3a(2)-Pro-Gly-Ada(2)-Gly-NH2) and P-HD5 (Ac-Trp-Ada(2)-Pro-Gly-Ed3a(2)-Gly-NH2) led to octadentate Tb3+ complexes with femtomolar stability in water. The position of the high denticity amino acid Ed3a(2) in the hexapeptide sequence appears to be critical for the control of the metal complex speciation. Whereas P-HD5 promotes the formation of polymetallic species in excess of Ln(3+), P-HD2 forms exclusively the mononuclear complex. The octadentate coordination of Tb3+ by both P-HD leads to total dehydration of the metal ion in the mononuclear complexes with long luminescence lifetimes (>2 ms). Hence, we demonstrated that unnatural amino acids carrying polyaminocarboxylate side-chains are interesting building blocks to design high affinity Ln-binding peptides. In particular the novel peptide P-HD2 forms a unique octadentate Tb3+ complex with femtomolar stability in water and an improvement of the luminescence properties with respect to the trisaquo TbP22 complex by a factor of 4.