Design and Synthesis of a New [18F]Fluoropyridine-Based Haloacetamide Reagent for the Labeling of Oligonucleotides: 2-Bromo-N-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]acetamide

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Abstract
Based on the recently highlighted potential of nucleophilic heteroaromatic ortho-radiofluorinations in the preparation of fluorine-18-labeled radiotracers and radiopharmaceuticals for PET, a [18F]fluoropyridine-based bromoacetamide reagent has been prepared and used in prosthetic group introduction for the labeling of oligonucleotides. [18F]FPyBrA (2-bromo-N-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]acetamide) was designed as a radiochemically feasible reagent, its pyridinyl moiety both carrying the radioactive halogen (fluorine-18) and allowing its efficient incorporation via a nucleophilic heteroaromatic substitution, and its 2-bromoacetamide function, ensuring the efficient alkylation of a phosphorothioate monoester group born at the 3‘- or 5‘-end of single-stranded oligonucleotides. [18F]FPyBrA (HPLC-purified) was efficiently prepared in 18−20% non-decay-corrected yield (based on starting [18F]fluoride) using a three-step radiochemical pathway in 80−85 min. The developed procedure involves (1) a high-yield nucleophilic heteroaromatic ortho-radiofluorination as the fluorine-18 incorporation-step (70−85% radiochemical yield) and uses [3-(3-tert-butoxycarbonylaminopropoxy)pyridin-2-yl]trimethylammonium trifluoromethanesulfonate as precursor for labeling, followed by (2) rapid and quantitative TFA-removal of the N-Boc-protective group and (3) condensation with 2-bromoacetyl bromide (45−65% radiochemical yield). Typically, 3.3−3.7 GBq (90−100 mCi) of HPLC-purified [18F]FPyBrA could be obtained in 80−85 min, starting from 18.5 GBq (500 mCi) of a cyclotron production batch of [18F]fluoride. [18F]FPyBrA was regioselectively conjugated with 9-mer and 18-mer single-stranded oligonucleotides, provided with a phosphorothioate monoester group at their 3‘-end. Both natural phosphodiester DNAs and in vivo-stable 2‘-methoxy and -fluoro-modified RNAs were used. Conjugation uses optimized, short-time reaction conditions (MeOH/0.1 M PBS pH 7.4, 15 min, 120 °C), both compatible with the chemical stability of the oligonucleotides (ONs) and the half-life of fluorine-18. Conjugated [18F]ONs were finally purified by RP-HPLC and desalted using a Sephadex NAP-10 column. The whole radiosynthetic procedure, including the preparation of the fluorine-18-labeled reagent, the conjugation with the oligonucleotide, and the HPLC purification and formulation lasted 140−160 min. [18F]FPyBrA represents a valuable alternative to the already reported N-(4-[18F]fluorobenzyl)-2-bromoacetamide for the design and development of oligonucleotide-based radiopharmaceuticals for PET.

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