title: 1-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione: design, synthesis, and radiosynthesis of a new [18F]fluoropyridine-based maleimide reagent for the labeling of peptides and proteins.
abstract: FPyME (1-[3-(2-fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione) was designed as a [(18)F]fluoropyridine-based maleimide reagent for the prosthetic labeling of peptides and proteins via selective conjugation with a thiol (sulfhydryl) function. Its pyridinyl moiety carries the radioactive halogen (fluorine-18) which can be efficiently incorporated via a nucleophilic heteroaromatic substitution, and its maleimido function ensures the efficient alkylation of a free thiol function as borne by cysteine residues. [(18)F]FPyME (HPLC-purified) was prepared in 17-20% non-decay-corrected yield, based on starting [(18)F]fluoride, in 110 min using a three-step radiochemical pathway. The developed procedure involves (1) a high-yield nucleophilic heteroaromatic ortho-radiofluorination on [3-(3-tert-butoxycarbonylaminopropoxy)pyridin-2-yl]trimethylammonium trifluoromethanesulfonate as the fluorine-18 incorporation step, followed by (2) rapid and quantitative TFA-induced removal of the N-Boc-protective group and (3) optimized maleimide formation using N-methoxycarbonylmaleimide. Typically, 4.8-6.7 GBq (130-180 mCi) of radiochemically pure [(18)F]FPyME ([(18)F]-1) could be obtained after semipreparative HPLC in 110 min starting from a cyclotron production batch of 33.3 GBq (900 mCi) of [(18)F]fluoride (overall radiochemical yields, based on starting [(18)F]fluoride: 28-37% decay-corrected). [(18)F]FPyME ([(18)F]-1) was first conjugated with a small model hexapeptide ((N-Ac)KAAAAC), confirming the excellent chemoselectivity of the coupling reaction (CH(2)SH versus CH(2)NH(2)) and then conjugated with two 8-kDa proteins of interest, currently being developed as tumor imaging agents (c-AFIM-0 and c-STxB). Conjugation was achieved in high yields (60-70%, isolated and non-decay-corrected) and used optimized, short-time reaction conditions (a 1/9 (v/v) mixture of DMSO and 0.05 M aq Tris NaCl buffer (pH 7.4) or 0.1 M aq PBS (pH 8), at room temperature for 10 min) and purification conditions (a gel filtration using a Sephadex NAP-10 cartridge or a SuperDex Peptide HR 10/30 column), both compatible with the chemical stability of the proteins and the relatively short half-life of the radioisotope concerned. The whole radiosynthetic procedure, including the preparation of the fluorine-18-labeled reagent, the conjugation with the protein and the final purification took 130-140 min. [(18)F]FPyME ([(18)F]-1) represents a new, valuable, thiol-selective, fluorine-18-labeled reagent for the prosthetic labeling with fluorine-18 of peptides and proteins. Because of its excellent chemoselectivity, [(18)F]FPyME offers an interesting alternative to the use of the nonselective carboxylate and amine-reactive [(18)F]reagents and can therefore advantageously be used for the design and development of new peptide- and protein-based radiopharmaceuticals for PET.
authors.affiliation: Service Hospitalier Frédéric Joliot, Département de Recherche Médicale, CEA/DSV, 4 place du Général Leclerc, F-91401 Orsay, France.
authors.forename: Béatrice
authors.initials: B
authors.lastname: de Bruin
authors.forename: Bertrand
authors.initials: B
authors.lastname: Kuhnast
authors.forename: Françoise
authors.initials: F
authors.lastname: Hinnen
authors.forename: Loïc
authors.initials: L
authors.lastname: Yaouancq
authors.forename: Mohamed
authors.initials: M
authors.lastname: Amessou
authors.forename: Ludger
authors.initials: L
authors.lastname: Johannes
authors.forename: Alain
authors.initials: A
authors.lastname: Samson
authors.forename: Raphaël
authors.initials: R
authors.lastname: Boisgard
authors.forename: Bertrand
authors.initials: B
authors.lastname: Tavitian
authors.forename: Frédéric
authors.initials: F
authors.lastname: Dollé
mesh_terms: D005462:Fluorine Radioisotopes; D007553:Isotope Labeling / Q000379:methods*; D008301:Maleimides; D009369:Neoplasms / Q000175:diagnosis; D010455:Peptides / Q000737:chemistry; D049268:Positron-Emission Tomography; D011506:Proteins / Q000737:chemistry; D011725:Pyridines; D019275:Radiopharmaceuticals / Q000138:chemical synthesis*
chemical_list: D005462:Fluorine Radioisotopes; D008301:Maleimides; D010455:Peptides; D011506:Proteins; D011725:Pyridines; D019275:Radiopharmaceuticals; C023666:pyridine