Directed Evolution of a Fluorinase for Improved Fluorination Efficiency with a Non‐native Substrate

Abstract

Fluorinases offer an environmentally friendly alternative for selective fluorination under mild conditions. However, their diversity is limited in nature and they have yet to be engineered through directed evolution. Herein, we report the directed evolution of the fluorinase FlA1 for improved conversion of the non-native substrate 5'-chloro-5'-deoxyadenosine (5'-ClDA) into 5'-fluoro-5'-deoxyadenosine (5'-FDA). The evolved variants, fah2081 (A279Y) and fah2114 (F213Y, A279L), were successfully applied in the radiosynthesis of 5'-[ F]FDA, with overall radiochemical conversion (RCC) more than 3-fold higher than wild-type FlA1. Kinetic studies of the two-step reaction revealed that the variants show a significantly improved k value in the conversion of 5'-ClDA into S-adenosyl-l-methionine (SAM) but a reduced k value in the conversion of SAM into 5'-FDA.

Enzymatic Halogenation and Dehalogenation Reactions: Pervasive and Mechanistically Diverse

“…439 Directed evolution of halogenases too is expanding the substrate scope of halogenating enzymes, pushing them as viable complementary catalysts in complex synthetic schemes. 440–442 …”

18F‐Labeling of Sensitive Biomolecules for Positron Emission Tomography

“…[14] A direct evolution of fluorinase for improved fluorination efficiency was also recently reported. [15] For instance, arginylglycylaspartic acid (RDG) peptide 14 bound to 5′-chloro-5′-deoxy-2-ethynyladenosine (ClDEA) through a tetraethylene glycol (TEG) linker could be labeled with fluorine-18 for late-stage modification (Figure 3). Specifically, an aqueous solution of [18 F]fluoride, directly from the cyclotron target, was reacted with RDG-TEG-CIDEA in the presence of L-SeMet at room temperature for 30 min to afford the radiolabeled product 15 in 12% RCY (non-decay corrected).…”

Probing the molecular determinants of fluorinase specificity

“…[13][14][15][16] There are only a handful of reports demonstrating limited success at enhancing substrate specificity through enyzme modification. 4,6,10,13,18 The challenge here is the lack of understanding of the fluorinasesubstrate interactions to enable a better engineered enzyme for fluorination.…”

“…15,16,[19][20][21][22][23][24] We have recently demonstrated that the catalytic efficiency of FlA1 fluorinase on a non-native substrate 5 0 -ClDA can be improved via directed evolution. 18 With the improved variants as a starting point, we probed their promiscuity against substrates that are modified at the C-2 (R 1 substitution) and the unexplored C-6 (R 2 substitution) positions of the adenine ring (Table 1).…”

“…To understand how the mutants impact the binding of substrates, structural models of the FlA1 mutants were generated using ROSETTA, 25 with the recently solved crystal structure of FlA1 (PDB code 5B6I) 18 as the template. Substrate coordinates were derived from the crystal structure of the Streptomyces cattleya fluorinase trimer complexed with substrate 2 (PDB code 4CQJ) 15 and docked into the FlA1 mutant active site using PyMOL.…”