Exploring fluorinase substrate tolerance at C-2 of SAM

The fluorinase enzyme (EC 2.5.1.63) utilises fluoride ion and S-adenosyl-L-methionine (SAM) as substrates for conversion to 5′-fluoro-5′-deoxy-adenosine (5′-FDA) and L-methionine (L-Met). The enzyme has a very strict substrate specificity, however it has been shown to tolerate acetylenes and NH2 replacements for H at C-2 of the adenine ring of SAM. This substrate tolerance is explored further here with −NHR, −N3, −OR and −SR substituents attached to C-2. New activities are demonstrated, for example with NH-methyl, NH-propyl,NH-butyl and O-butyl substrates at C-2, however azide and thioethers were not tolerated. Outcomes are supported by in silico analysis, revealing favourable H-bonding interactions involving NH and O substituents at the adenine C-2 position with N278 and the backbone amide of A279 at the active site respectively. The study informs on the selectivity of the fluorinase as a tool for radiolabelling candidate ligands with fluorine-18 for positron emission tomography programmes.