Fig. 2. Reaction Optimization, Scale-up and Batch Synthesis in the Development of an Efficient Scaleable One-Pot Synthesis.

A Chosen Route C (from Fig. 1) was tested in a one-pot format [¹⁹F]FDT using pyrogen-free enzymes OtsA^Sf and OtsB^Sf. B Example reaction optimization with fixed donor sugar Glc-UDP [30 mM] at different acceptor substrate concentrations (9.08, 18.17, 36.34 and 72.68 mM) of [¹⁹F]FDG; reactions were monitored in real time by calibrated ¹⁹F NMR. Please also see Source Data File. C Direct reaction monitoring of the steps of one-pot, 3-step [¹⁹F]FDT synthesis from [¹⁹F]FDG by ¹⁹F-NMR. NMR spectra: (i) ¹⁹F NMR spectrum of purified [¹⁹F]FDT; (ii) Crude ¹⁹F NMR spectrum of [¹⁹F]FDT with small amount of deoxy-fluoro-G6P ([¹⁹F]-1) still present; (iii) Crude ¹⁹F NMR spectrum of intermediates [¹⁹F]-1 and [¹⁹F]-2; (iv) Crude ¹⁹F NMR spectrum of [¹⁹F]-1 from [¹⁹F]FDG conversion; and (v) reference sample of starting material [¹⁹F]FDG. D Representative, ¹⁹F-NMR spectra of crude reaction mixtures containing [¹⁹F]FDT from repeated batches using fresh enzyme (including from newly expressed preparations) and reactants synthesized by 3-enzymes-3-steps, one-pot syntheses, prior to purification.