A new substrate for enzymatic radiofluorination

Objectives Recently, a novel, mild and high yielding F-18 labeling method utilizing an enzyme, called 5’-fluoroadenosine synthase (fluorinase), has been reported to yield [18F]-FDA from S-adenosyl-L-methionine (SAM). Fluorinase was shown to only accept SAM and its 2’-deoxy derivative. The goal is to diversify the application of enzymatic fluorination by designing novel substrates to produce a wider range of PET probes based on the extremely mild, specific and high yielding enzymatic catalysis approach. Herein, we investigate the activity of fluorinase using a new substrate possesing an azamethionine leaving group.

Methods The non-native substrate for fluorinase, N-adenosyl-N-methyl-L-azamethionine (MeAzaAdoMet), was modelled in silico. Computational studies on docking with fluorinase enzyme were performed and the molecule was synthesized. Fluorinase was applied in the reaction of MeAzaAdoMet fluorination to yield 5’-[18F]fluoro-5’deoxyadenosine ([18F]FDA).

Results In our preliminary study, MeAzaAdoMet was found to resemble the SAM molecule with 96% similarity in docking experiments. Based on the computed model, the compound was synthesized via a multistep reaction involving a series of protection, coupling, quaternization and deprotection steps starting from adenosine and L-N2-Boc-2,4-diaminobutyric acid. This compound was then subjected to the radiofluorination using fluorinase/L-amino acid oxidase/[18F]fluoride/[18O]H2O system. Based on an unoptimized radiofluorination methodology, the fluorination efficiency was found to be 30%± 3% (n=10)

Conclusions We have investigate the enzymatic radiofluorination using a SAM-like using the wild type fluorinase. This preliminary investigation confirmed that the substrate specificity of the enzyme can be widened using a L-methionine like leaving group, which could be synthesized chemically. Furthermore, this work could serve as the building block towards the investigation of new precursors suitable for enzymatic fluorination to yield a diverse range of fluorinated compounds.

Research Support Department of Energy (DOE DE-SC0001249)