The post-translational modification of collagen by the enzyme prolyl 4-hydroxylase increases markedly the conformational stability of the collagen triple helix. For 25 years, the prevailing paradigm had been that this enhanced stability arises from water molecules that form bridges between the hydroxyl group of (2S,4R)-4-hydroxyproline (Hyp) residues and a main-chain oxygen. We recognized that the hydroxyl group could instead have stereoelectronic consequences that stabilize the desired conformation. To test this hypothesis, we synthesized collagen chains in which (2S,4R)-4-fluoroproline residues replace Hyp. These chains form triple helices of extraordinary stability, despite the inability of organic fluorine to form strong hydrogen bonds. In contrast, the diastereomer (2S,4S)-4-fluoroproline was highly destabilizing, and the disubstituted residue (2S)-4,4-difluoroproline was comparable to (2S)-proline. These data provided the first demonstration that a stereoelectronic effect can stabilize a protein structure. To promote the utility of 4-fluoroprolines, we have developed practical routes for the synthesis of the 2S,4R, 2S,4S, and 2R,4S diastereomers of 4-fluoroproline from HypOH, and effected those routes on a kilogram scale.

Modulation of collagen stability with fluoro groups

245th National Meeting (2013)

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