National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health; Center for Neuroscience and Regenerative Medicine (CNRM) through Henry Jackson Foundation ; A new tracer, N-5-[F-18]fluoropentylmaleimide ([F-18]FPenM), for site-specific labeling of free thiol group in proteins and peptides was developed. The tracer was synthesized in three steps (F-18 displacement of the aliphatic tosylate, di-Boc removal by TFA to expose free amine, and incorporation of the free amine into a maleimide). The radiosynthesis was completed in 110 min with 11-17% radiochemical yield (uncorrected), and specific activity of 20-49 GBq/mu mol. [F-18]FPenM showed comparable labeling efficiency with N-[2-(4-[F-18]-fluorobenzamido)ethyl]maleimide ([F-18]FBEM). Its application was demonstrated by conjugation with glucagon-like peptide type 1 (GLP-1) analogue [cys(40)]-exendin-4. The cell uptake, binding affinity, imaging properties, biodistribution, and metabolic stability of the radiolabeled [F-18]FPenM-[cys(40)]-exendin-4 were studied using INS-1 tumor cells and INS-1 xenograft model. Positron emission tomography (PET) results showed that the new thiol-specific tracer, [F-18]FPenM-[cys(40)]-exendin-4, had high tumor uptake (20.32 +/- 4.36%ID/g at 60 min postinjection) and rapid liver and kidney clearance, which was comparable to the imaging results with [F-18]FBEM-[cys(40)]-exendin-4 reported by our group.