O-acylation of hydroxyproline residues: Effect on peptide-bond isomerization and collagen stability

Collagen is the most prevalent component of the extracellular matrix in animals.1,2 Collagen has a characteristic tertiary structure consisting of three left-handed polyproline II-type helices wound around a common axis to form a triple helix with a shallow right-handed superhelical pitch. The close packing of the polypeptide chains in the triple helix requires that every third residue be glycine. Each polypeptide chain in fibrillar collagen contains about 300 repeats of the sequence XaaYaaGly, in which Xaa is often (2S)-proline (Pro) and Yaa is often (2S,4R)-4-hydroxyproline (Hyp). The Hyp residues arise from a post-translational modification of Pro residues by the enzyme prolyl 4-hydroxylase. The thermal stability of collagen has a positive correlation with its Hyp content.3,4 The effects of Hyp stereochemistry and sequence on collagen stability have been explored with peptide mimics.5,6 In seminal work, Prockop and coworkers showed that [(ProHypGly)10]3 is more stable than [(ProProGly)10]3, whereas (HypProGly)10 does not exhibit triple helix formation.7,8 The diastereomer (2S,4S)-4-hydroxyproline (hyp) in the Xaa or Yaa position precludes triple helix formation.9 More recent studies have shown that Hyp can enhance triple helical stability in the Xaa position, but not if the Yaa residue is Pro.10,11 Previous reports from our laboratory have shown that replacing Hyp in (ProHypGly)n (n = 7 or 10) with (2S,4R)-4-fluoroproline (Flp) (but not its diastereomer (2S,4S)-4-fluoroproline (flp)) greatly enhances the stability of its triple helices (Table I).12–14 The increase in stability derives from the enhanced electron-withdrawing ability of the fluoro group. That inductive effect constrains the pucker of the pyrrolidine ring, and thereby preorganizes the peptide backbone dihedral angles of the individual strands into conformations that are favorable for triple helix formation.15,16 Table I Effect of Hyp and Flp on the conformational stability of collagen triple helices No known reagent can be used to replace the hydroxyl group of Hyp residues with a fluoro group in aqueous solution.17 The electron-withdrawing ability of a hydroxyl group can, however, be increased by its chemical modification. Here, we synthesize and characterize O-acyl derivatives of AcHypOMe and AchypOMe. We also determine the effects of O-acetylation on the conformational stability of a [(ProHypGly)10]3 triple helix. We find that the influence of the hydroxyl group remains largely intact after its acylation.