Engineering of highly luminescent lanthanide tags suitable for protein labeling and time-resolved luminescence imaging.
The synthesis of a new ligand LH(4) based on a glutamic acid skeleton bis-functionalized on its nitrogen atom by 6-methylene-6'-carboxy-2,2'-bipyridine chromophoric units is described. UV-vis spectrophotometric titrations revealed the formation of 1:1 M:L complexes with lanthanide(III) cations, and complexation of LH(4) with equimolar amounts of hydrated LnCl(3) salts (Ln = Eu, Gd, and Tb) gave water-soluble and stable complexes of the general formula [LnL(H(2)O)]Na, which were characterized by elemental analysis, IR, UV-vis absorption spectroscopy, (1)H NMR (Ln = Eu), and mass spectrometry. The conditional stability constant for formation of the [EuL(H(2)O)]Na complex was determined by competitive complexation experiments to be log K = 16.5 +/- 0.6 in 0.01 M TRIS/HCl buffer (pH = 7.0). In water solution, the [EuL(H(2)O)]Na and [TbL(H(2)O)]Na complexes were highly luminescent with quantum yields of 8% and 31%, respectively, despite the presence of ca. one water molecule in the first coordination sphere of the metal ions. Activation of the appended carboxylate function of the glutamate moiety in the form of an N-hydroxysuccinimidyl ester allows for the covalent linking of the complexes to primary amino groups of biological compounds. Bovine serum albumin (BSA) was labeled with both Eu or Tb complexes, and the Ln-BSA conjugates were characterized by UV-vis absorption and emission spectroscopy and MALDI-TOF mass spectrometry. Labeling ratios (number of complex molecules per BSA) of ca. 8:1 and 7:1 were established for Eu-BSA and Tb-BSA, respectively. The suitability of the tagged compound for use in bioanalytical time-resolved luminescence microscopy was established by comparison with fluorescein-labeled probes.
Weibel
Nicolas
N
Laboratoire de Chimie Moléculaire, Ecole de Chimie, Polymères et Matériaux, ULP, 25 rue Becquerel, 67087 Strasbourg Cedex 02, France.
Charbonnière
Loïc J
LJ
Guardigli
Massimo
M
Roda
Aldo
A
Ziessel
Raymond
R
D005971:Glutamates / Q000138:chemical synthesis* / Q000737:chemistry; D028581:Lanthanoid Series Elements / Q000138:chemical synthesis* / Q000737:chemistry; D008164:Luminescent Proteins / Q000138:chemical synthesis / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D011725:Pyridines / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013055:Spectrophotometry, Infrared; D013056:Spectrophotometry, Ultraviolet
D005971:Glutamates; D028581:Lanthanoid Series Elements; D008164:Luminescent Proteins; D011725:Pyridines; D012710:Serum Albumin, Bovine