Enzymatic Radiofluorination of Biomolecules: Development and Automation of Second Generation Prosthetic on ELIXYS Radiosynthesizer

Objectives: Previously, we reported an enzymatic radiofluorination methodology for proteins (Drake et al., ACS Chem. Biol., 2016, 11, 1587-1594). [¹⁸F]Fluorooctanoic acid and lipoic acid ligase (LplA) were incubated with an Fab antibody engineered to contain a 13-amino acid tag (‘LAP-tag’), resulting in site-specific conjugation of the radiofluorinated small molecule prosthetic to the LAP-tagged protein. Fab labeling was rapid and high yielding (95 ± 7%, n=4) at neutral pH and 30 °C, and required only minimal amounts of protein (10 nmol). Despite these pleasing results, 2 major problems remained: preliminary in vivo studies revealed metabolic defluorination, presumably due to the alkyl fluoride prosthetic structure, and the synthesis required extensive manual intervention. We sought to address both issues by i) developing a second-generation aryl fluoride prosthetic and ii) automating the entire synthetic process on the ELIXYS automated radiosynthesizer platform (Sofie Biosciences, Culver City).

Methods: Based on prior literature (Cohen et al., ChemBioChem, 2012, 13, 888-894), we identified 7-(4-[¹⁸F]-fluorophenyl)-7-oxyheptanoic acid (FPOA) and ^W37ILplA as a suitable second generation prosthetic/enzyme pair. FPOA was synthesized in 2 steps, nucleophilic radiofluorination of the analogous aryl trimethylammonium triflate followed by methyl ester hydrolysis, and purified via HPLC. Subsequently, labeling of a model Fab-LAP construct with FPOA, catalyzed by ^W37ILplA, was investigated under various conditions. Finally, the radiofluorinated Fab-LAP was purified via nickel-affinity chromatography. All synthetic procedures, including HPLC purification and purification of the final radiofluorinated construct, were fully automated on ELIXYS.

Results: FPOA was generated via a 2-pot protocol in high chemical and radiochemical purity (>97%) and reasonable specific activity (>500 Ci/mmol). Decay-corrected yields were moderate (~25%), however the high purity of the prosthetic prompted us to proceed with Fab-LAP labeling. Conjugation of FPOA to a model Fab-LAP was comparable to results obtained previously using our first -generation prosthetic (>75% yields using < 25 nmol of protein). Critically, the 3-reactor set-up of ELIXYS enabled us to synthesize FPOA and then conjugate it to Fab-LAP without manual intervention. In addition, the platform’s flexibility facilitated purification of the final radiofluorinated Fab-LAP via nickel affinity chromatography.

Conclusion: FPOA/^W37ILplA was shown to be a suitable enzyme/prosthetic pair for radiofluorinating LAP-tagged proteins. The complete labeling protocol, including FPOA synthesis and purification, and protein labeling and purification, was fully automated on the ELIXYS platform, highlighting the benefits of its flexible 3-reactor design. In vitro and in vivo experiments to investigate the metabolic stability and imaging capabilities of the radiofluorinated construct are forthcoming. Research Support: Research reported in this abstract was supported by the National Institute Of Biomedical Imaging And Bioengineering of the National Institutes of Health under Award Number R43EB023782.

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