Fluorescent chelates for monitoring metal binding with macromolecules

Metals and radionuclides are usually coupled with proteins together with suitable ligands for therapeutic, tumor-imaging, pharmaceuticals, and biocompatibility applications. Several ligands that can strongly coordinate a given nuclide in a specific valency are already known. However, the demand for bifunctionality has limited the applications of these ligands. We hereby report the molecular design of a receptor system based on the linkage of protein to monoazo ligands. By use of basic coordination chemistry, 4-(3-quinolinoazo) hydroxybenzoic acid (QABA) and derivatives were successfully conjugated to ovalbumin, bovine serum albumin, and alkaline phsophatase at a site that was distinct from the metal binding site. The presence of carboxylic acid linkage in the QABA served as a convenient bridge for protein conjugation and may allow the generic application of these ligands for bioconjugate synthesis while ensuring a high in vivo stability. The ligand-protein conjugates were characterized using UV-vis spectroscopy, Fourier transform infrared spectroscopy, thin layer charoatography, NMR, and surface-enhanced laser desorption ionization time-of-flight mass spectrometry. The conjugate was tested for the ability to recognize nonradioactive Ga³⁺ at a physiological pH, and a binding constant of 1 × 10²⁰ was recorded. Also, the in vitro testing results indicated that the fluorescent conjugates exhibited significant selectivity for gallium compared to Pb²⁺, Hg²⁺, Zn²⁺, Cu²⁺, Fe³⁺, and Co²⁺ while no responses were obtained for alkaline and alkaline earth metals. These attributes could allow these conjugates to be used as a model for imaging sensors and for metal detection.