Fluorine‐18 labeling of phosphopeptides: A potential approach for the evaluation of phosphopeptide metabolism in vivo

Abstract

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.

RETRACTED: Peptide-Based Inhibitors of Plk1 Polo-box Domain Containing Mono-anionic Phosphothreonine Esters and Their Pivaloyloxymethyl Prodrugs

“…Typically, short phosphopeptides modeled on cognate recognition sequences provide starting points for transformation into more drug-like peptide mimetics. However, phosphopeptides present particular challenges to cellular studies, due to the poor membrane transport of phosphoryl di-anionic species (Allentoff et al, 1999; Richter et al, 2009). Overcoming the paradoxical importance of the phosphoryl pharmacophore in promoting high binding affinity while at the same time serving as a limiting factor in cell membrane transport, is among the primary obstacles faced in the development of phospho-dependent PPI inhibitors (Burke and Lee, 2003).…”

Synthesis of an 18F‐labeled cyclin‐dependent kinase‐2 inhibitor

“…Better results could be obtained using microwave activation for heating. Microwave activation increased radiochemical yields for [ 18 F]fluoride incorporation into nitro‐substituted benzamides to 42% compared with 8% radiochemical yield using conventional heating at 150 °C as exemplified for 18 F‐labeled 5‐HT 1A agonist Org 13063 …”

“…This finding is consistent with previously reported low radiochemical yields on direct incorporation of n.c.a. [ 18 F]fluoride into benzamides using conventional heating as exemplified for 5‐HT 1A receptor ligands p ‐[ 18 F]MPPF, p ‐[ 18 F]FBP, and 18 F‐labeled Org 13063 …”

Bioactive Casein Phosphopeptides in Dairy Products as Nutraceuticals for Functional Foods

“…One possible mode of CPP action on the transmembrane flux of calcium is that CPP might insert themselves into the plasma membrane and form their own calcium selective channels or act as calcium-carrier peptides rapidly internalized via endocytosis or other processes and eventually provide ionized calcium in the cytosol [53]. Cellular uptake studies of fluorine-18 labeled CPP in human colorectal adenocarcinoma cell line (HT-29) and human head and neck squamous cell carcinoma line (FaDu) cells at 37 °C and 48 °C showed a poor cell penetration because of the poor transport of the phosphopeptides through the cell membrane [54]. The results from in vivo studies are still too controversial, as there are many factors affecting Ca availability, such as the various co-present dietary compounds in the intestinal lumen [55].…”