(1) Eu-DOTA-Gly4, NHS (1.8 equivalents), EDC (4.5 equivalents), 2-(N-morpholino)ethanesulfonic acid, pH 6.5, 0°C, 1 h; (2) G5 (0.072 equivalent), phosphate-buffered saline, pH 7.4, room temperature, 24 h; 37% yield (42 Eu-DOTA-Gly4 units among 128 NH3+ polyamidoamine chain termini); (3) 2 (70 μmol), DyLight® 680 NHS ester (1 equivalent), phosphate-buffered saline, room temperature, 24 h, 93% yield. The average stoichiometry is listed for each ligand. The chemical exchange saturation transfer effect arises from the bound water of Eu-DOTA-Gly4.
EDC: 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; Eu-DOTA-Gly4: Eu-1,4,7,10-tetraazacclododecane-1,4,7,10-tetraacetic acid-Gly4; G5: G5PAMAM; G5PAMAM: Generation 5 polyamidoamine; NHS: N-hydroxysuccinimide.