Fluorine-18 labeling of phosphopeptides: a potential approach for the evaluation of phosphopeptide metabolism in vivo.

Phosphopeptides are very useful reagents to study signal transduction pathways related with cellular protein phosphorylation/dephosphorylation. Phosphopeptides have also been identified as important drug candidates to modulate intracellular signaling mechanisms through targeting phosphotyrosine, phosphoserine, or phosphothreonine residue-binding protein domains. In this report, we describe the development of a convenient method for the mild and sufficient radiolabeling of phosphopeptides with the short-lived positron emitter fluorine-18 (¹⁸F) to allow radiopharmacological studies on phosphopeptide metabolism in vivo by means of positron emission tomography (PET). Radiolabeling was accomplished via conjugation of the N-terminus of polo-box domain (PBD)-binding phosphopeptide H-Met-Gln-Ser-pThr-Pro-Leu-OH with the bifunctional labeling agent N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) in reproducible isolated radiochemical yields of 25-28%. Radiopharmacological evaluation in vitro and in vivo of radiolabeled phosphopeptide [¹⁸F]FB-Met-Gln-Ser-pThr-Pro-Leu-OH [¹⁸F]-3 and its non-phosphorylated analog [¹⁸F]FB-Met-Gln-Ser-Thr-Pro-Leu-OH [¹⁸F]-4 involved metabolic stability, cell uptake studies, and small animal PET experiments. Radiolabeled phosphopeptide [¹⁸F]-3 showed a remarkable high metabolic stability in vivo compared to the corresponding non-phosphorylated peptide [¹⁸F]-4. The presented method indicates that radiolabeling of phosphopeptides with [¹⁸F]SFB is a promising approach for studying phosphopeptide metabolism in vivo.