3,4,5-Tri-
O-acetyl-2-[
18F]fluoro-2-deoxy-
D-glucopyranosyl 1-phenylthiosulfonate (Ac
3-[
18F]FGlc-PTS) was developed as a thiol-reactive labeling reagent for the site-specific
18F-glycosylation of peptides. Taking advantageof highly accessible 1,3,4,6-tetra-
O-acetyl-2-deoxy-2-[
18F]fluoroglucopyranose, a three-step radiochemical pathwaywas investigated and optimized, providing Ac
3-[
18F]FGlc-PTS in a radiochemical yield of about 33% in 90 min(decay-corrected and based on starting [
18F]fluoride). Ac
3-[
18F]FGlc-PTS was reacted with the model pentapeptideCAKAY, confirming chemoselectivity and excellent conjugation yields of >90% under mild reaction conditions.The optimized method was adopted to the
18F-glycosylation of the
v3-affine peptide c(RGDfC), achieving highconjugation yields (95%, decay-corrected). The
v3 binding affinity of the glycosylated c(RGDfC) remaineduninfluenced as determined by competition binding studies versus
125I-echistatin using both isolated
v3 andhuman umbilical vein endothelial cells (
Ki = 68 ± 10 nM (
v3) versus
Ki = 77 ± 4 nM (HUVEC)). The wholeradiosynthetic procedure, including the preparation of the
18F-glycosylating reagent Ac
3-[
18F]FGlc-PTS, peptideligation, and final HPLC purification, provided a decay-uncorrected radiochemical yield of 13% after a totalsynthesis time of 130 min. Ac
3-[
18F]FGlc-PTS represents a novel
18F-labeling reagent for the mild chemoselective
18F-glycosylation of peptides indicating its potential for the design and development of
18F-labeled bioactiveS-glycopeptides suitable to study their pharmacokinetics in vivo by positron emission tomography (PET).