Optimal His-Tag Design for Efficient [^99mTc(CO)₃]⁺ and [¹⁸⁸Re(CO)₃]⁺ Labeling of Proteins for Molecular Imaging and Radionuclide Therapy by Analysis of Peptide Arrays

Abstract

Hexahistidine tags (His-tags), incorporated into recombinant proteins to facilitate purification using metal-affinity chromatography, are useful binding sites for radiolabeling with [99mTc­(CO)3]+ and [188Re­(CO)3]+ for molecular imaging and radionuclide therapy. Labeling efficiencies vary unpredictably, and the method is therefore not universally useful. To overcome this, we have made quantitative comparisons of radiolabeling of a bespoke Celluspots array library of 382 His-tag-containing peptide sequences with [99mTc­(CO)3]+ and [188Re­(CO)3]+ to identify key features that enhance labeling. A selected sequence with 10-fold enhanced labeling efficiency compared to the most effective literature-reported sequences was incorporated into an exemplar protein and compared biologically with non-optimized analogues, in vitro and in vivo. Optimal labeling with either [99mTc­(CO)3]+ or [188Re­(CO)3]+ required six consecutive His residues in the protein sequence, surrounded by several positively charged residues (Arg or Lys), and the presence of phosphate in the buffer. Cys or Met residues in the sequence were beneficial, to a lesser extent. Negatively charged residues were deleterious to labeling. His-tags with adjacent positively charged residues could be labeled as much as 40 times more efficiently than those with adjacent negatively charged residues. 31P NMR of [Re­(CO)3(H2O)3]+ and electrophoresis of solutions of [99mTc­(CO)3(H2O)3]+ suggest that phosphate bridges form between cationic residues and the cationic metal synthon during labeling. The trial optimized protein, a scFv targeted to the PSMA antigen expressed in prostate cancer, was readily labeled in >95% radiochemical yield, without the need for subsequent purification. Labeling occurred more quickly and to higher specific activity than comparable non-optimized proteins, while retaining specific binding to PSMA and prostate cancer in vivo. Thus, optimized His-tags greatly simplify radiolabeling of recombinant proteins making them potentially more widely and economically available for imaging and treating patients.

In Vitro Biological Activity of α-Diimine Rhenium Dicarbonyl Complexes and Their Reactivity with Different Functional Groups

“…We suggest coordination of his via the imidazole ring, based on the collected evidence of this study and the fact that complexations with other amino acids, such as glycine, only gave analytical traces (observed by HPLC-MS) of possible interaction with 2b, and the fact that carboxylic acid and primary amines did not show affinity for the cis-[Re(CO) 2 ] + core. Overall the reactivity of the cis-[Re(CO) 2 ] + core (i.e., with aromatic nitrogen-based ligands) is comparable to that of the fac-[Re(CO) 3 ] + , with several studies supporting the interaction of the latter core with guanines [85][86][87][88][89] and histidine [90][91][92][93][94][95][96].…”