Modulating collagen triple-helix stability with 4-chloro, 4-fluoro, and 4-methylprolines.

Introduction Collagen is a fibrous protein comprising a right-handed, triple-helical bundle of three parallel, left-handed polyproline II-type helices. Each strand consists of approximately 300 repeats of the trimer (Xaa–Yaa–Gly), where Xaa is often (2S)proline (Pro) and Yaa is often (2S,4R)-4-hydroxyproline (Hyp) [1]. The most abundant protein in vertebrates, collagen is of fundamental importance to the threedimensional architecture of such animals. Understanding the chemical determinants of the structure and stability of collagen is essential for both curing collagen-related diseases and creating collagen-based biomaterials. We have previously demonstrated that pyrrolidine ring pucker and thus triplehelical stability can be manipulated by functionalizing the -position of the proline ring with moieties that control the ring pucker via either steric or stereoelectronic effects (Figure 1). The presence of Hyp in the Yaa position of natural collagen greatly increases the stability of the triple helix [2]. This enhanced stability is presumably due to a stereoelectronic effect that defines the pyrrolidine ring pucker of Hyp residues as the C -exo pucker. The C -exo ring pucker fixes the and angles of Hyp residues to those required for triple-helical assembly [3]. Substitution of non-natural proline derivatives that prefer the C -exo ring pucker, such as (2S,4R)-4-fluoroproline (Flp) [4] or (2S,4S)-4-methylproline (Mep) [5], for proline in the Yaa position also stabilizes triple helices. In contrast, the C -endo ring pucker is favorable for triple-helix stability in the Xaa position. Consequently, substitution of (2S,4S)-4-fluoroproline (flp) and (2S,4R)-4-methylproline (mep) for proline in the Xaa position stabilizes triple helices, because flp and mep prefer the C -endo ring pucker [5,6]. Here, we introduce additional proline derivatives that can be utilized to endow conformational stability and unique properties on collagen triple helices, namely the 4-chloroprolines. Additionally, we summarize the ability to modulate the thermal stability of collagen over a wide range of temperatures by modifying the -carbon of proline residues.