Fluorine-18 Labeling of Phosphorpeptides: A convenient Approach for the Evaluation of Phosphorpeptide Metabolism in vivo

Objectives: Phosphopeptides are very useful reagents to study signal transduction pathways related with cellular protein phosphorylation/dephosphorylation. Phosphopeptides also have been identified as important drug candidates to both inhibit and stimulate intracellular signaling mechanisms through targeting phosphotyrosine, phosphoserine or phosphothreonine residue-binding protein domains. In this work we describe a convenient method for the mild and sufficient radiolabeling of phosphopeptides with the short-lived positron emitter fluorine-18 to allow radiopharmacological studies on phosphopeptide metabolism in vivo by means of positron emission tomography (PET).
Methods: Peptide syntheses were performed on peptide synthesizer (Syro I, MultiSynTech, Germany) using standard Fmoc chemistry. Radiolabeling was accomplished via conjugation of the N-terminus of Polo-box domain (PBD)-binding phosphopeptide H-Met-Gln-Ser-pThr-Pro-Leu-OH 1 and its unphosphorylated analog 2 with the bifunctional labeling agent N-succinimidyl-p-[¹⁸F]fluorobenzoate ([¹⁸F]SFB) (Fig. 1).
The radiolabeled phosphopeptide [¹⁸F]FB-Met-Gln-Ser-pThr-Pro-Leu-OH [¹⁸F]-1 and its unphosphorylated analog [¹⁸F]FB-Met-Gln-Ser-Thr-Pro-Leu-OH [¹⁸F]-2 were subjected to radiopharmacological evaluation involving investigation of metabolic stability in vitro and in vivo, cell uptake studies in human adenocarcinoma (HT-29) and squamous cell carcinoma (FaDu) cell lines, and small-animal PET studies in Wistar rats and NMRI nu/nu HT-29 tumor-bearing mice.
Results: Radiolabeling was achieved via ¹⁸F-fluorobenzoylation using the Bolton-Hunter-type reagent N-succinimidyl-p-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). The optimized radiosynthesis was conducted in a 0.05 M Na₂HPO₄ buffer solution (pH 9.0) at 40°C within 30 min using low amounts of peptide precursor (0.5 mg) to afford reproducible radiochemical yields (25-29%) and high radiochemical purity (>98%) within 95-109 min including HPLC purification. Cell uptake studies in HT-29 and FaDu tumor cells revealed only very little radiotracer uptake (less than 0.6% ID/mg protein). Radiolabeled phosphopeptide [¹⁸F]-1 showed remarkable high metabolic stability in vivo (65% intact peptide after 20 min) compared to the corresponding unphosphorylated peptide [18F]-2 (<5% intact peptide after 20 min). A detailed discussion on the radiopharmacological profile in vitro and in vivo of peptides [¹⁸F]-1 and [¹⁸F]-2 will be presented.
Conclusions: We developed a reproducible synthesis for ¹⁸F-labeled phosphopeptides, and the presented method is a promising approach for studying phosphopeptide metabolism and kinetics in vivo. Furthermore, cell penetrating peptides (CPP) are currently under investigation as potential mediators to enhance cell uptake of the desired ¹⁸F-labeled phosphopeptides.