Radiofluorination of an Anionic, Azide-Functionalized Teroligomer by Copper-Catalyzed Azide-Alkyne Cycloaddition

Abstract

This study describes the synthesis, radiofluorination and purification of an anionic amphiphilic teroligomer developed as a stabilizer for siRNA-loaded calcium phosphate nanoparticles (CaP-NPs). As the stabilizing amphiphile accumulates on nanoparticle surfaces, the fluorine-18-labeled polymer should enable to track the distribution of the CaP-NPs in brain tumors by positron emission tomography after application by convection-enhanced delivery. At first, an unmodified teroligomer was synthesized with a number average molecular weight of 4550 ± 20 Da by free radical polymerization of a defined composition of methoxy-PEG-monomethacrylate, tetradecyl acrylate and maleic anhydride. Subsequent derivatization of anhydrides with azido-TEG-amine provided an azido-functionalized polymer precursor (o14PEGMA-N₃) for radiofluorination. The ¹⁸F-labeling was accomplished through the copper-catalyzed cycloaddition of o14PEGMA-N₃ with diethylene glycol-alkyne-substituted heteroaromatic prosthetic group [¹⁸F]2, which was synthesized with a radiochemical yield (RCY) of about 38% within 60 min using a radiosynthesis module. The ¹⁸F-labeled polymer [¹⁸F]fluoro-o14PEGMA was obtained after a short reaction time of 2-3 min by using CuSO₄/sodium ascorbate at 90 °C. Purification was performed by solid-phase extraction on an anion-exchange cartridge followed by size-exclusion chromatography to obtain [¹⁸F]fluoro-o14PEGMA with a high radiochemical purity and an RCY of about 15%.

Keywords: 18F-polymer; CuAAC; PEG-[18F]FPyKYNE; click reaction; fluorine-18; teroligomer.

Scheme 2

Synthesis of (A) the pristine teroligomer o14PEGMA and (B) the azide-functionalized teroligomer o14PEGMA-N₃. Reagents and conditions: (a) free-radical polymerization with AIBN, 60 °C for 18 h under magnetic stirring; (b) (i) triethylamine in acetone, stirring at RT for 4 h, (ii) removal of acetone under vacuum; (iii) 1M aqueous ammonia, magnetic stirring at 40 °C for 12 h.

Scheme 3

Radiosynthesis of the teroligomer [¹⁸F]fluoro-o14PEGMA by copper-catalyzed azide-alkyne cycloaddition of o14PEGMA-N₃ and [¹⁸F]2. Reaction conditions: (a) [¹⁸F]F^-/K₂₂₂/K₂CO₃, DMSO, 120 °C, 8 min; (b) NaAsc/CuSO₄ 4:1, DMSO/H₂O 1:4, 90 °C, 2–3 min.

Scheme 1

Schematic illustration of the material concept investigated in this study. An amphiphilic anhydride group-containing oligomer (o14PEGMA) is synthesized from tetradecyl acrylate (orange), mPEG-MAc (blue) and maleic anhydride (red) [9]. The anhydride groups are derivatized, and unreacted anhydrides are hydrolyzed, yielding the azide-functionalized (green) teroligomer o14PEGMA-N₃ (see also Scheme 2B). The water-soluble oligomer is then conjugated with a suitable alkyne-bearing and radiolabeled group in a copper-catalyzed azide-alkyne cycloaddition, yielding [¹⁸F]fluoro-o14PEGMA (see also Scheme 3). The radiofluorinated oligomer would then be available as a stabilizer for the fabrication of siRNA-loaded CaP-NPs in analogy to our previous study [9].

Figure 1

Flow sheet of the synthesis module TRACERlab FX2 N for the radiosynthesis of [¹⁸F]2. (1) Sep-Pak Accell Plus QMA Carbonate Plus light cartridge, (2) K₂CO₃ (1.8 mg (13 µmol) in 400 µL water, 100 µL ACN), (3) K_2.2.2. (11 mg (29 mmol) in 1.5 mL of ACN), (4) precursor 1 (1.0–1.5 mg (2.3–3.5 µmol) in 800 µL DMSO), (5) 3.5 mL of water + 0.5 mL of ACN, (6) injection vial, (7) Reprosil-Pur C18-AQ, 250 × 10 mm (ACN/H₂O/TFA (35:75:0.05, v/v/v), flow 4.0 mL/min), (8) 50 mL of water + 40 µL of 1M aq. NaOH, (9) Sep-Pak^® C18 light, (10) 2 mL of water.

Figure 2

Spectra of o14PEGMA-N₃, o14PEGMA, the ammonium salt of o14PEGMA and azido-TEG-amine obtained by (A) FT-IR and (B) confocal Raman spectroscopy. Both methods complementarily indicate that azido-TEG-amine could be grafted on the o14PEGMA backbone by aminolysis. Additional peaks originating from azido-TEG-amine are visible in the reaction product o14PEGMA-N₃ when compared to the non-derivatized ammonium salt of o14PEGMA.

Figure 3

Exemplary radio-chromatogram of the semi-preparative SEC purification of the radiolabeled teroligomer [¹⁸F]fluoro-o14PEGMA (conditions: 2 HiTrap^TM desalting 5 mL columns in line, 3.75 mM aq. phosphate pH 7, flow 1.0 mL/min).

Figure 4

Exemplary radio-chromatograms of the analytical quality control of [¹⁸F]fluoro-o14PEGMA using (A) radio-SEC (HiTrap^TM desalting 5 mL column, 10% ethanol/25 mM aq. phosphate pH 7, flow 1.0) and (B) radio-RP-HPLC (Reprosil Pur C18 AQ 250 × 4.6 mm, gradient ACN/20 mM aq. NH₄OAc, flow 1.0).