American Chemical Society
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MO Tripeptide Diastereomers (M = 99/99mTc, Re):  Models To Identify the Structure of 99mTc Peptide Targeted Radiopharmaceuticals

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posted on 2007-09-03, 00:00 authored by Melchor V. Cantorias, Robertha C. Howell, Louis Todaro, John E. Cyr, Dietmar Berndorff, Robin D. Rogers, Lynn C. Francesconi
Biologically active molecules, such as many peptides, serve as targeting vectors for radiopharmaceuticals based on 99mTc. Tripeptides can be suitable chelates and are easily and conveniently synthesized and linked to peptide targeting vectors through solid-phase peptide synthesis and form stable TcVO complexes. Upon complexation with [TcO]3+, two products form; these are syn and anti diastereomers, and they often have different biological behavior. This is the case with the approved radiopharmaceutical [99mTcO]depreotide ([99mTcO]P829, NeoTect) that is used to image lung cancer. [99mTcO]depreotide indeed exhibits two product peaks in its HPLC profile, but assignment of the product peaks to the diastereomers has proven to be difficult because the metal peptide complex is difficult to crystallize for structural analysis. In this study, we isolated diastereomers of [99TcO] and [ReO] complexes of several tripeptide ligands that model the metal chelator region of [99mTcO]depreotide. Using X-ray crystallography, we observed that the early eluting peak (A) corresponds to the anti diastereomer, where the TcO group is on the opposite side of the plane formed by the ligand backbone relative to the pendant groups of the tripeptide ligand, and the later eluting peak (B) corresponds to the syn diastereomer, where the TcO group is on the same side of the plane as the residues of the tripeptide. 1H NMR and circular dichroism (CD) spectroscopy report on the metal environment and prove to be diagnostic for syn or anti diastereomers, and we identified characteristic features from these techniques that can be used to assign the diastereomer profile in 99mTc peptide radiopharmaceuticals like [99mTcO]depreotide and in 188Re peptide radiotherapeutic agents. Crystallography, potentiometric titration, and NMR results presented insights into the chemistry occurring under physiological conditions. The tripeptide complexes where lysine is the second amino acid crystallized in a deprotonated metallo-amide form, possessing a short N1−M bond. The pKa measurements of the N1 amine (pKa ∼5.6) suggested that this amine is rendered more acidic by both metal complexation and the presence of the lysine residue. Furthermore, peptide chelators incorporating a lysine (like the chelator of [TcO]depreotide) likely exist in the deprotonated form in vivo, comprising a neutral metal center. Deprotonation possibly mediates the interconversion process between the syn and anti diastereomers. The N1 amine group on non-lysine-containing metallopeptides is not as acidic (pKa ∼6.8) and does not deprotonate and crystallize as do the metallo-amide species. Three of the tripeptide ligands (FGC, FSC, and FKC) were radiolabeled with 99mTc, and the individual syn and anti isomers were isolated for biodistribution studies in normal female nude mice. The main organs of uptake were the liver, intestines, and kidneys, with the FGC compounds exhibiting the highest liver uptake. In comparing the diastereomers, the syn compounds had substantially higher organ uptake and slower blood clearance than the anti compounds.