Biochemistry of fluoroprolines: the prospect of making fluorine a bioelement

Abstract

Due to the heterocyclic structure and distinct conformational profile, proline is unique in the repertoire of the 20 amino acids coded into proteins. Here, we summarize the biochemical work on the replacement of proline with (4R)- and (4S)-fluoroproline as well as 4,4-difluoroproline in proteins done mainly in the last two decades. We first recapitulate the complex position and biochemical fate of proline in the biochemistry of a cell, discuss the physicochemical properties of fluoroprolines, and overview the attempts to use these amino acids as proline replacements in studies of protein production and folding. Fluorinated proline replacements are able to elevate the protein expression speed and yields and improve the thermodynamic and kinetic folding profiles of individual proteins. In this context, fluoroprolines can be viewed as useful tools in the biotechnological toolbox. As a prospect, we envision that proteome-wide proline-to-fluoroproline substitutions could be possible. We suggest a hypothetical scenario for the use of laboratory evolutionary methods with fluoroprolines as a suitable vehicle to introduce fluorine into living cells. This approach may enable creation of synthetic cells endowed with artificial biodiversity, containing fluorine as a bioelement.

Natural and Synthetic Halogenated Amino Acids—Structural and Bioactive Features in Antimicrobial Peptides and Peptidomimetics

“…The presence of fluorine on the C γ atom, with its electron-withdrawing properties, can bias the proline ring pucker preferences and “pre-organize” the protein main chain, thus conferring additional stabilization to the whole protein [ 29 , 30 ]. Thus, (2S, 4S)-4-fluoroproline ((4S)-FPro), prefers the C γ -endo ring pucker and stabilizes the cis peptide-bond conformation, while (2S, 4R)-4-fluoroproline, ((4R)-FPro) stabilizes C γ -exo ring pucker favoring the trans conformation [ 28 , 31 , 32 ] ( Figure 2 ).…”

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

“…The effect is likely produced by protein misfolding. The latter may originate from the C 4 -exo conformation promoted by R-Flp, which is unfavored by the parent protein structures 27 . With Dfp, the misfolding is The diversity of the outcomes produced by mere atomic mutations in the examined fluorescent proteins illustrates the potential of the SPI production method in altering target protein properties.…”

“…Numerous mutations have been performed on GFP, providing variants with elevated stability, fast and reliable folding, and variable fluorescence properties 17 . Nonetheless, in most cases, mutation of proline residues is considered a risky approach due to the fact that none of the remaining 19 canonical amino acids can properly restore the conformational profile of the proline residue 27 .…”

“…produced by the diminished velocity of the transto-cis peptide bond isomerization at the proline residue27 .The latter is known to be among the limiting steps in the kinetic profile of the protein folding that affects β-barrel formation and subsequent chromophore maturation. Indeed, for both amino acids, R-Flp and Dfp, the protein production resulted in an aggregated and insoluble protein.…”

Chemoselective N- and O-Difluoromethylation of 2-Pyridones, Isoquinolinones, and Quinolinones with TMSCF2Br

“…Fluorofunctionalization to enhance the physical and chemical properties of organic molecules is a successful strategy in pharmaceutical, − biochemical, and material science , applications. Particularly, difluoromethylation has emerged as an important reaction paradigm due to the lipophilicity and hydrogen bond donor ability of −CF 2 H, − enabling bioisosteric replacement of commonly encountered functional groups including −OH and −SH .…”