4-Fluoro-Threonine: From Diastereoselective Synthesis to pH-Dependent Conformational Equilibrium in Aqueous Solution

4-Fluoro-threonine, the only fluoro amino acid of natural origin discovered so far, is an interesting target for both synthetic and theoretical investigations. In this work, we lay the foundation for spectroscopic characterization of 4-fluoro-threonine. First, we report a diastereoselective synthetic route, which is suitable to produce synthetic material for experimental characterization. The addition of the commercially available ethyl isocyanoacetate to benzyloxyacetaldehyde led to the corresponding benzyloxy-oxazoline, which was hydrolyzed and transformed into ethyl (4S*,5S*)-5-hydroxymethyl-2-oxo-4-oxazolidinecarboxylate in a few steps. Fluorination with diethylamino sulfur trifluoride (DAST) afforded ethyl (4S*,5S*)-5-fluoromethyl-2-oxo-4-oxazolidinecarboxylate, which was deprotected to give the desired diastereomerically pure 4-fluoro-threonine, in 8-10% overall yield. With the synthetic material in our hands, acid-base titrations have been carried out to determine acid dissociation constants and the isoelectric point, which is the testing ground for the theoretical analysis. We have used machine learning coupled with quantum chemistry at the state-of-the-art to analyze the conformational space of 4-fluoro-threonine, with the aim of gaining insights from the comparison of computational and experimental results. Indeed, we have demonstrated that our approach, which couples a last-generation double-hybrid density functional including empirical dispersion contributions with a model combining explicit first-shell molecules and a polarizable continuum for describing solvent effects, provides results and trends in remarkable agreement with experiments. Finally, the conformational analysis applied to fluoro amino acids represents an interesting study for the effect of fluorine on the stability and population of conformers.