On the one hand, owing to its electronegativity, relatively small size, and notable leaving group ability from anionic intermediates, fluorine offers unique opportunities for mechanism-based enzyme inhibitor design. On the other, the “bio-orthogonal” and NMR-active 19-fluorine nucleus allows the bioorganic chemist to follow the mechanistic fate of fluorinated substrate analogues or inhibitors as they are enzymatically processed. This article takes an overview of the field, highlighting key developments along these lines. It begins by highlighting new screening methodologies for drug discovery that involve appropriate tagging of either substrate or the target protein itself with 19F-markers, that then report back on turnover and binding, respectively, via an the NMR screen. Taking this one step further, substrate-tagging with fluorine can be done is such a manner as to provide stereochemical information on enzyme mechanism. For example, substitution of one of the terminal hydrogens in phosphoenolpyruvate, provides insight into the, otherwise latent, facial selectivity of C-C bond formation in KDO synthase. Perhaps, most importantly, from the point of view of this discussion, appropriately tailored fluorinated functionality can be used to form to stabilized “transition state analogue” complexes with a target enzymes. Thus, 5-fluorinated pyrimidines, α-fluorinated ketones, and 2-fluoro-2-deoxysugars each lead to covalent adduction of catalytic active site residues in thymidylate synthase, serine protease and glycosidase enzymes, respectively. In all such cases, 19F NMR allows the bioorganic chemist to spectrally follow “transition state analogue” formation. Finally, the use of specific fluorinated functionality to engineer “suicide substrates” is highlighted in a discussion of the development of the α-(2′Z-fluoro)vinyl trigger for amino acid decarboxylase inactivation. Here 19F NMR allows the bioorganic chemist to glean useful partition ratio data directly out of the NMR tube.

To test the prediction that extensively fluorinated (fluorous) proteins should be more stable and exhibit novel self-segregating behavior, the properties of the de novo designed model 4-alpha-helix bundle protein, alpha 4F 6, in which the hydrophobic core is packed entirely with the extensively fluorinated amino acid l-5,5,5,5',5',5'-hexafluoroleucine, have been compared with its nonfluorinated counterpart, alpha 4H, in which the core is packed with leucine. alpha 4F 6 exhibits much greater resistance to proteolysis by either chymotrypsin or trypsin than alpha 4H and resists unfolding by organic solvents far better than alpha 4H. Whereas increasing concentrations of ethanol or 2-propanol cause the helices of the alpha 4H tetramer first to dissociate into monomeric helices and then to completely unfold, these solvents have little effect on the structure of alpha 4F 6. In contrast, increasing the concentrations of the fluorinated alcohol trifluoroethanol promotes dissociation of both alpha 4H and alpha 4F 6 to monomeric helices, whereas the secondary structure of both peptides remains intact. (19)F NMR experiments indicate that the two peptides can form mixed alpha-helical alpha 4F 6:alpha 4H bundles and thus do not exhibit the self-segregating behavior predicted by the fluorous effect. We conclude that the properties of alpha 4F 6 are best explained by the more hydrophobic nature of the hexafluoroleucine side chain, rather than the low solubility of fluorocarbons in hydrocarbon solvents that forms the basis of the fluorous effect.

The incorporation of extensively fluorinated, or fluorous, analogues of hydrophobic amino acids into proteins potentially provides the opportunity to modulate the physicochemical properties of proteins in a predictable manner. On the basis of the properties of small fluorocarbon molecules, extensively fluorinated proteins should be both more thermodynamically stable and self-segregate through "fluorous" interactions between fluorinated amino acids. We have examined the effects of introducing the fluorous leucine analogue l-5,5,5,5',5',5',-hexafluoroleucine (hFLeu) at various positions within the hydrophobic core of a de novo-designed four-alpha-helix bundle protein, alpha(4). The stabilizing effect of hFLeu is strongly dependent on the positions at which it is incorporated, with per-residue DeltaDeltaG(degrees)((fold)) ranging from -0.09 to -0.8 kcal mol(-1) residue(-1). In particular, incorporating hFLeu at all the "a" positions or all the "d" positions of the hydrophobic core, thereby creating an alternating packing arrangement of leucine and hFLeu, leads to very stably folded proteins that exhibit higher per-residue DeltaDeltaG(degrees)((fold)) values than proteins that are packed entirely with hFleu. We conclude that efficient packing of the fluorous amino acid within the hydrophobic core provides a more important contribution to enhancing protein stability than do fluorocarbon-fluorocarbon interactions between fluorinated protein side chains.

Developing specific chemical functionalities to deploy in biological environments for targeted enzyme inactivation lies at the heart of mechanism-based inhibitor (MBI) development, but also is central to other protein-tagging methods in modern chemical biology including activity-based protein profiling (ABPP) and proteolysis-targeting chimeras (PROTACS). We describe here a previously unknown class of potential PLP enzyme inactivators; namely, a family of quaternary, α-(1′fluoro)vinyl amino acids, bearing the side chains of the cognate amino acids. These are obtained by the capture of suitably protected amino acid enolates with β,β-difluorovinyl phenyl sulfone, a new 1′-fluorovinyl cation equivalent, and an electrophile that previously eluded synthesis, capture and characterization. A significant variety of biologically relevant AA-side chains are tolerated including those for alanine, valine, leucine, methionine, lysine, phenylalanine, tyrosine and tryptophan. Following addition/elimination, the resulting transoid α-(1′-fluoro)-β-(phenylsulfonyl)vinyl AA esters undergo smooth sulfone-stannane interchange to stereoselectively give the corresponding transoid α-(1′fluoro)-β-(tributylstannyl)vinyl AA esters. Protodestannylation and global deprotection then yields these sterically encumbered and densely functionalized, quaternary amino acids. The α-(1′fluoro)vinyl trigger, a potential allene-generating functionality originally proposed by Abeles, is now available in a quaternary AA context for the first time. In an initial test of this new inhibitor class, α-(1′-fluoro)vinyllysine is seen to act as a time dependent, irreversible inactivator of lysine decarboxylase from Hafnia alvei. The enantiomers of the inhibitor could be resolved and each is seen to give time dependent inactivation with this enzyme. Kitz-Wilson analysis reveals similar inactivation parameters for the two antipodes, L-α-(1′-fluoro)vinyllysine (Ki = 630 ± 20 μM; t1/2 = 2.8 min) and D-α-(1′-fluoro)vinyllysine (Ki = 470 ± 30 μM; t1/2 = 3.6 min). The stage is now set for exploration of the efficacy of this trigger in other PLP-enzyme active sites.

Protected alpha-formyl amino acids, themselves available from the corresponding alpha-vinyl amino acids, are stereoselectively transformed into the (Z)-configured alpha-(2'-fluoro)vinyl amino acids via a three-step sequence. The route employs McCarthy's reagent, diethyl alpha-fluoro-alpha-(phenylsulfonyl)methyl phosphonate, and proceeds via the intermediate (E)-alpha-fluorovinyl sulfones and (E)-alpha-fluorovinylstannanes. The latter may either be exploited as novel cross-coupling partners for fluorovinyl branch extension or be globally deprotected, to provide the title compounds. [structure: see text]