The synthesis, structure, and photophysical properties of several Tb(III) complexes with octadentate, macrotricyclic ligands that feature a bicapped topology and 2-hydroxyisopthalamide (IAM) chelating units are reported. These Tb(III) complexes exhibit highly efficient emission (Φtotal ≥ 50%), large extinction coefficients (εmax ≥ 20,000 M−1cm−1), and long luminescence lifetimes (τH2O ≥ 2.45 ms) at dilute concentrations in standard biological buffers. The structure of the methyl-protected ligand was determined by single-crystal X-ray diffraction, and confirms the macrotricyclic structure of the parent ligand; the amide groups of the methyl-protected cage compound generate an anion binding cavity that complexes a chloride anion. Once the ligand is deprotected a conformational change generates a similar cavity, formed by the phenolate and ortho amide oxygen groups that strongly bind lanthanide ions. The Tb(III) complexes thus formed display long term stability, with little if any change in their spectral properties (including lifetime, quantum yield, and emission spectrum) over time or in different chemical environments. Procedures to prepare functionalized derivatives with a terminal amine, carboxylate and N-hydroxysuccinimide groups suitable for derivatization and protein bioconjugation have also been developed. These bifunctional ligands have been covalently attached to a number of different proteins and the terbium complexes' exceptional photophysical properties are retained. These compounds establish a new aqueous stability and quantum yield standard for long-lifetime lanthanide reporters.

Isotope-coded affinity tags (ICAT) represent an important new tool for the analysis of complex mixtures of proteins in living systems [Aebersold, R., and Mann, M. (2003) Nature, 422, 198-207]. We envisage an alternative protein-labeling technique based on tagging with different element-coded metal chelates, which affords affinity chromatography, quantification, and identification of a tagged peptide from a complex mixture. As proof of concept, a synthetic peptide was modified at a cysteine side chain with either a carboxymethyl group or acetamidobenzyl-1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid (AcBD) chelates of terbium or yttrium. A mixture of the three modified peptides in a mole ratio of 100:1.0:0.83 carboxymethyl:AcBD-Tb:AcBD-Y was trypsinized, purified on a new affinity column that binds rare-earth DOTA chelates, and analyzed by LC-MS/MS. Chelate-tagged tryptic peptides eluted cleanly from the affinity column; the tagged peptides chromatographically coeluted during LC-MS analysis, were present in the expected ratio as indicated by MS ion intensity, and were sequence-identified by tandem mass spectrometry. DOTA-rare earth chelates have exceptional properties for use as affinity tags. They are highly polar and water-soluble. Many of the rare earth elements are naturally monoisotopic, providing a variety of simple choices for preparing mass tags. Further, the rare earths are heavy elements, whose mass defects give the masses of tagged peptides exact values not normally shared by molecules that contain only light elements.

An antibody that binds rare earth complexes selectively could be used as a docking station for a set of probe molecules, of particular interest for medical imaging and therapy. The rare earths are rich in probe properties, such as the paramagnetism of Gd, the luminescence of Tb and Eu, and the nuclear properties of Lu and Y. We find that antibody 2D12.5, initially developed to bind analogues of Y-DOTA (1,4,7,10-tetraazacyclododecane-N,N',N' ',N' ''-tetraacetic acid) for radiotherapy, binds not only Y-DOTA analogues but also analogous DOTA complexes of all of the lanthanides. Surprisingly, chelates of some metals such as Gd3+ bind more tightly than the original Y3+ complex. When the shape of the complex is perturbed by either increasing or decreasing the radius of the lanthanide ion, the thermodynamic stability of the protein-ligand complex changes in a regular fashion. The behavior of DeltaDeltaG as a function of ionic radius fits a parabola, as might be expected for a system that behaves in a thermodynamically elastic way. The broad specificity and high affinity of this antibody for all rare earth-DOTA complexes make it particularly interesting for applications that take advantage of the unique characteristics of lanthanides. For example, UV excitation of the Tb-DOTA-2D12.5 complex leads to energy transfer from aromatic side chains of the antibody to bound Tb-DOTA, enhancing green terbium luminescence >104 relative to unbound Tb-DOTA.

Engineering the permanent formation of a receptor-ligand complex has a number of potential applications in chemistry and biology, including targeted medical imaging and therapy. Starting from the crystal structure of the rare-earth-DOTA binding antibody 2D12.5 (Corneillie, T. M., Fisher, A. J., and Meares, C. F. (2003) J. Am. Chem. Soc. 125, 15039-15048), we used the site-directed incorporation of cysteine nucleophiles at the periphery of the antibody's binding site, paired with the chemical design of a weakly electrophilic ligand, to produce a receptor-ligand pair that associates efficiently and permanently. Protein residues proximal to the ligand's side chain were identified for engineering cysteine mutants. Fab fragments incorporating a cysteine at position 54, 55, or 56 of the heavy chain (complementarity determining region 2) were designed from the structure and then cloned, expressed in Drosophila S2 cells, and tested for reactivity with mildly electrophilic DOTA-yttrium ligands. All showed permanent binding activity, indicating that there is some tolerance for the location of the reactive mutant on the protein surface near the binding site. The G54C Fab mutant displayed the highest expression levels and permanent binding activity in initial experiments and was produced in high yield for further study. Upon examining the behavior of the G54C mutant with a small set of electrophilic ligands, differences in reactivity were observed which indicated that the substituents near the electrophilic atom can be important determinants of permanent binding. The G54C mutant permanently attaches to Y(3+) complexes of (S)-2-(4-acrylamidobenzyl)-DOTA with a half-time of approximately 13 min at 37 degrees C, making it potentially useful for in vivo pretargeting applications.

We report the crystal structures of antibody 2D12.5 Fab bound to an yttrium-DOTA analogue and separately to a gadolinium-DOTA analogue. The rare earth elements have many useful properties as probes, and 2D12.5 binds the DOTA (1,4,7,10-tetraazacyclododecane-N,N',N' ',N' "-tetraacetic acid) complexes of all of them (Corneillie et al. J. Am. Chem. Soc. 2003, 125, 3436-3437). The structures show that there are no direct protein-metal interactions: a bridging water acts as a link between the protein and metal, with the chelate present as the M isomer in each case. DOTA forms an amphipathic cylinder with the charged carboxylate groups toward the face of the chelate near the metal ion, while nonpolar methylene groups from the macrocycle and the carboxymethyl groups occupy the rear and sides of the molecule. The orientation of the metal-DOTA in the 2D12.5 complex places most of the methylene carbon atoms of DOTA in hydrophobic contact with aromatic protein side chains. Other binding interactions between the metal complex and the antibody include a bidentate salt bridge, four direct H-bonds, and four to five water-mediated H-bonds. We find that 2D12.5 exhibits enantiomeric binding generality, binding yttrium chelates in both Lambda(deltadeltadeltadelta) and Delta(lambdalambdalambdalambda) configurations with modestly different affinities. This develops from the symmetrical nature of the DOTA chelate, which places heteroatoms and hydrophobic atoms in approximately the same relative positions regardless of the helicity of DOTA.