Fully automated 18F-fluorination of N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB) for indirect labelling of nanobodies

Abstract

N-succinimidyl-4-[18F]fluorobenzoate ([18F]SFB), a widely used labeling agent to introduce the 4-[18F]fluorobenzoyl-prosthetic group, is normally obtained in three consecutive steps from [18F]fluoride ion. Here, we describe an efficient one-step labeling procedure of [18F]SFB starting from a tin precursor. This method circumvents volatile radioactive side-products and simplifies automatization. [18F]SFB was obtained after HPLC purification in a yield of 42 + 4% and a radiochemical purity (RCP) > 99% (n = 6). In addition, we investigate the automation of the coupling of [18F]SFB to a nanobody (cAbBcII10, targeting β-lactamase enzyme) and purification by size exclusion chromatography (PD-10 desalting column) to remove unconjugated reagent. Production and use of [18F]SFB were implemented on a radiosynthesis unit (Neptis®). The fully automated radiosynthesis process including purification and formulation required 160 min of synthesis time. [18F]SFB-labeled nanobody was obtained in a yield of 21 + 2% (activity yield 12 + 1% non-decay corrected) and a radiochemical purity (RCP) of > 95% (n = 3). This approach simplifies [18F]SFB synthesis to one-step, enhances the yield in comparison to the previous report and enables the production of radiolabeled nanobody on the same synthesis module.

Bioorthogonal Radiolabeling of Azide-Modified Bacteria Using [18F]FB-sulfo-DBCO

“…Based on initial concerns that a hydrophobic cyclooctyne-derived PET probe might result in increased nonspecific binding, we sought a water-soluble PET tracer compatible for SPAAC ligation with robust and reproducible radiosynthesis. Based on the commercial availability of the strained cyclooctyne-containing and sulfated amine Sulfo DBCO amine, we envisioned rapid amide formation via the readily available activated ester N -succinimidyl 4-[ 18 F]­fluorobenzoate ([ 18 F]­SFB) (Figure A), analogous to other reported fluorine-18 prosthetic groups. , The amine precursor was reacted with [ 18 F]­SFB for 20 min at 40 °C in the presence of Et 3 N. The reaction mixture was purified via semiprep HPLC using a Phenomenex Luna C18 column. The calculated radiochemical yield (RCY) for [ 18 F]­FB-sulfo-DBCO starting from [ 18 F]­fluoride was 50.8% ± 9.3 (decay corrected) with radiochemical purity of >99% and a calculated molar activity of 2.9 ± 0.6 GBq/μmol (Figure B).…”

“…[ 18 F]SFB was synthesized using a procedure reported by Nagachinta et al 39 To a 4 mL borosilicate vial containing [ 18 F]SFB (370–555 MBq) were added a PTFE stir bar, Et 3 N (25 μL), DMF (500 μL), and Sulfo DBCO amine precursor (5 mg). The mixture was stirred at 40 °C for 20 min, then diluted with H 2 O before purification via semiprep HPLC using a Phenomenex Luna C18 column, 10 mm (40% MeCN/60% H 2 O + 0.1% TFA).…”

Arginine-Selective Bioconjugation Reagent for Effective 18F-labeling of Native Proteins

“…For example, active esters, such as N -succinimidyl-4-[ 18 F]­fluorobenzoate ([ 18 F]­SFB) and 2,3,5,6-tetrafluorophenyl 6-[ 18 F]­fluoronicotinate ([ 18 F]­TFPFN), modify the proteins at their lysine residues or the N -terminus. , However, the standard radiosynthesis of [ 18 F]­SFB is a time-consuming three-step procedure . Despite several 18 F-labeling strategies enabling one-step radiosynthesis of [ 18 F]­SFB, − only two methods using either the spirocyclic iodonium ylide precursor or the highly toxic organotin precursor reported the decay-corrected isolated RCYs of [ 18 F]­SFB in 3–22 and 38–46%, respectively. , On the other hand, [ 18 F]­TFPFN requires very high protein loading to achieve effective radiolabeling . Alternatively, the proteins are chemically modified or engineered with a functional group, such as hydrazine, (±)-H 3 RESCA, or cysteine.…”

“… 5 Despite several 18 F-labeling strategies enabling one-step radiosynthesis of [ 18 F]SFB, 7 − 9 only two methods using either the spirocyclic iodonium ylide precursor or the highly toxic organotin precursor reported the decay-corrected isolated RCYs of [ 18 F]SFB in 3–22 and 38–46%, respectively. 10 , 11 On the other hand, [ 18 F]TFPFN requires very high protein loading to achieve effective radiolabeling. 6 Alternatively, the proteins are chemically modified or engineered with a functional group, such as hydrazine, (±)-H 3 RESCA, or cysteine.…”

18F‐Fluorination of a supported 2‐(aryl‐di‐tert‐butylsilyl)‐N‐methyl‐imidazole for indirect 18F‐labeling of a VHH single‐variable domain

“…18 In all cases, possible automation of a part or of the whole synthesis remains highly desirable to facilitate the potential transfer to pre-clinical trials. [19][20][21] In this context, radiosyntheses starting from precursors on a solid supports can be beneficial, especially when cleavage is triggered selectively by the radiolabeling reaction. Indeed, this strategy may allow to replace the timeconsuming HPLC purifications by simple filtrations and/or solid-phase extractions.…”