Automated radiosynthesis and purification of the thiol-reactive labeling agent [¹⁸F]FBAM

Objectives: The interest on ¹⁸F-radiolabeled peptides and proteins has increased over the last decade. Consequently, prosthetic groups as N-succinimidyl 4-[¹⁸Ffluorobenzoate ([¹⁸F]SFB) have been designed for labeling peptides at the amino functionality. Due to in some cases unsatisfactory or inconsistent radiolabeling results with [¹⁸F]SFB a strategy directed to a single functionality would circumvent these drawbacks. Therefore, prosthetic groups reacting exclusively with thiol groups were designed1, one of them N-[6-(4-[¹⁸F]fluorobenzylidene)aminooxyhexyl] maleimide [¹⁸F]FBAM² (Figure 1). To make [¹⁸F]FBAM more applicable for routine use we developed an automated radiosynthesis of this labeling agent including a solid phase purification³.
Methods:
The labeling precursor 4-N,N,N-trimethylaminobenzaldehyde triflate, the FBAM precursor N-(6-aminooxyhexyl)maleimide hydrochloride and the non-radioactive FBAM were synthesized according to the literature². [¹⁸F]Fluoride was produced on a CYCLONE 18/9 Cyclotron (IBA) via the ¹⁸O(p,n)¹⁸F reaction. A commercially available synthesis module TRACERlab FX_FDG (GE) was adapted in terms of program and hardware. Results: For the ¹⁸F-labeling 10 mg of 4-N,N,N-trimethylaminobenzaldehyde triflate were reacted with the [¹⁸F]KF-Kryptofix₂₂₂ complex in 1.0 mL of acetonitrile for 10 min at 90°C in reactor 1. After addition of 12 mL of water the solution was passed over a Strata C18U cartridge (Phenomenex) to trap the 4-[¹⁸F]fluorobenzaldehyde. The cartridge was eluted with 3.5 mL of methanol into reactor 2 and 7 mg of the aminooxy-precursor was added dissolved in 0.5 mL of methanol. The Schiff-base reaction was performed for 6 min at 40°C, 10 mL of water were added and the content of reactor 2 was passed over a LiChrolut RP18 cartridge (Merck). After washing with 6 mL of acetonitrile/water=1:1 the [¹⁸F]FBAM was finally eluted with 2mL of acetonitrile into a product vial. A typical radiosynthesis started with 3.1 GBq [^18F]fluoride and yielded about 580 MBq [¹⁸F]FBAM (29% yield d.c.) within a total synthesis time of 40 min, the radiochemical purity was 94-98% .
Conclusions:
An automated procedure for the routine radiosynthesis of [¹⁸F]FBAM will help to make the thiolreactive prosthetic group more broadly available for selective ¹⁸F radiolabeling of peptides and proteins as
molecular imaging probes.
Research Support:
References:
[1] Toyokuni et al., (2003), Bioconjug. Chem., 14, 1253-159, [2] Berndt et al., (2007), Nucl. Med. Biol., 34, 5-15[3] Kniess et al., (2011), Appl. Radiat. Isot., submitted