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

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Abstract
FPyME (1-[3-(2-fluoropyridin-3-yloxy)propyl]pyrrole-2,5-dione) was designed as a [18F]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. [18F]FPyME (HPLC-purified) was prepared in 17−20% non-decay-corrected yield, based on starting [18F]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 [18F]FPyME ([18F]-1) could be obtained after semipreparative HPLC in 110 min starting from a cyclotron production batch of 33.3 GBq (900 mCi) of [18F]fluoride (overall radiochemical yields, based on starting [18F]fluoride: 28−37% decay-corrected). [18F]FPyME ([18F]-1) was first conjugated with a small model hexapeptide ((N-Ac)KAAAAC), confirming the excellent chemoselectivity of the coupling reaction (CH2SH versus CH2NH2) 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. [18F]FPyME ([18F]-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, [18F]FPyME offers an interesting alternative to the use of the nonselective carboxylate and amine-reactive [18F]reagents and can therefore advantageously be used for the design and development of new peptide- and protein-based radiopharmaceuticals for PET.

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