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.

[18F]FPyKYNE, a fluoropyridine‐based alkyne reagent designed for the fluorine‐18 labelling of macromolecules using click chemistry

“…Derivative 11 was then cleanly obtained in 97% yield by methylation of 10 using methyl trifluoromethanesulphonate (1.3 equiv.) in toluene at room temperature (RT) for 1 h. 21,26,46 Radiochemistry FPyKYNE (1) was labelled with fluorine-18 at its 2-fluoropyridinyl moiety using the one-step radiochemical process outlined in Scheme 2.…”

“…18 F]fluorobenzoate [8][9][10][11][12] ( Figure 1, compound A) and alkylation of a sulphhydryl function with bromoacetamide- [13][14][15][16][17][18][19][20][21][22] (Figure 1, compounds B and C) or maleimide [23][24][25][26] ( Figure 1, compounds D-G) reagents.…”

Radiosynthesis and bioconjugation of [18F]FPy5yne, a prosthetic group for the 18F labeling of bioactive peptides

“…23 Unfortunately, 2 was found to elute closely with reaction by-product diisopropyl hydrazine-1,2-dicarboxylate, and the two compounds could not be separated in our hands using silica gel chromatography. Presumably, alternative Mitsonubu reagents with water-soluble by-products such as di-tertbutyl azodicarboxylate (DBAD) 24 and di-2-methoxyethyl azodicarboxylate (DMEAD) 25 could be employed.…”

“…21,22 [ 18 F]FPyME was used to successfully label variety of thiol-containing peptides and proteins. 23 We sought to employ a 2-substituted pyridine [ 18 F]fluorination in the preparation of a general labeling agent for use with biomolecules of interest to PET. We reasoned that bifunctional precursor molecules containing 2-nitro (2) and 2-trimethylammonium triflate (3) leaving groups and robust terminal alkyne moieties could be used to prepare prosthetic label 2-…”

Realizing the Full Potential of PET for Measuring the Biodistribution of Novel Anticancer Agents

“…We write to draw your readers' attention to potential solutions: the development and availability of low-cost, low-radiation-emitting, simple-to-operate positron-emitting radioisotope generators and of inexpensive disposable, good-manufacturing-practice-compliant "card-based" radiosynthesis platforms that lighten regulatory overload (7). These systems should be used to implement generic radiolabeling procedures, such as those reported for peptides and proteins (8) and oligonucleotides (9). We hope the development of these new technologies is encouraged by others in the molecular imaging community.…”