F-18-Labeled phosphopeptide-cell-penetrating peptide dimers with enhanced cell uptake properties in human cancer cells

Introduction: Phosphopeptides represent interesting compounds to study and elucidate cellular protein phosphorylation/dephosphorylation processes underlying various signal transduction pathways. However, studies of phosphopeptide action in cells are severely constrained by the negatively charged phosphate moiety of the phosphopeptide resulting in poor transport through the cell membrane. The following study describes the synthesis and radiopharmacological evaluation of two F-18-labeled phosphopeptide-cell-penetrating peptide dimers. The polo-like kinase-1-binding hexaphosphopeptide H-Met-Gln-Ser-pThr-ProLeu-OH was coupled to cell-penetrating peptides (CPPs), either sC18, a cathelicidin-derived peptide, or the human calcitonin derivative hCT(18-32)-k7. Methods: Radiolabeling was accomplished with the prosthetic group N-succinimidyl 4-[F-18]fluorobenzoate ([F-18]SFB) using both, conventional and microfluidic-based bioconjugation of [F-18]SEB to N-terminal end of phosphopeptide part of the peptide dimers. Cellular uptake studies in human cancer cell lines HT-29 and FaDu cells at 4 T and 37 T and small animal PET in BALB/c mice were utilized for radiopharmacological characterization. Results: Isolated radiochemical yields ranged from 2% to 4% for conventional bioconjugation with [F-18]SFB. Significantly improved isolated radiochemical yields of up to 26% were achieved using microfluidic technology. Cellular uptake studies of radiolabeled phosphopeptide and phosphopeptide-CPP dimers indicate enhanced internalization of 50% ID/mg protein after 2 h for both phosphopeptide dimers compared to the phosphopeptide alone (