The design, synthesis and characterization of novel triple helical collagen mimetic structures

In our efforts to understand triple helical propensities of collagen-like structures, we focused on the thermodynamics of folding for single chain and template (scaffold)-assembled Gly-Pro-Hyp structures. The helical forming tendencies based on concentration or chain length were evaluated. The influence of N-terminal acetylation and template assembly of polypeptides favored triple helical formation over that of N-terminal free amines.; As an extension of our studies of N-isobutylglycine (Nleu) in collagen structures composed of Gly-Pro-Nleu and Gly-Nleu-Pro, we synthesized guest-host structures Ac-(Gly-Nleu-Pro)₃-(Gly-Nx-Pro) ₂-(Gly-Nleu-Pro)₃-NH₂ (Nx denotes N-substituted glycines) to investigate the effects of various N-alkyl and N-aralkyl substituents on triple helix formation. Biophysical studies by CD spectroscopy and thermal denaturation indicated that various N-substituted glycines participate in triple helical structures.; In addition, we synthesized a series of guest-host structures with substitutions in the Y position of the Gly-Xaa-Yaa trimeric unit. Triple helicity of Ac-(Gly-Nleu-Pro) ₃-(Gly-Pro-Y)₂-(Gly-Nleu-Pro)₃-NH₂ where Y is Pro, Hyp, trans-4(R)-fluoroproline (Flp) and Nleu. Guest-host structures containing Pro, Hyp and Flp were not triple helical in solution, suggesting the structural incompatibilities of the trimeric units Gly-Pro-Y and Gly-Nleu-Pro. The structure Ac-(Gly-Nleu-Pro)₃-(Gly-Nleu-Hyp) ₂-(Gly-Nleu-Pro)₃-NH₂ was shown not to be triple helical while Ac-(Gly-Nleu-Pro)₃-(Gly-Nleu-Flp)₂-(Gly-Nleu-Pro) ₃-NH₂ proved to be triple helical (T_m = 47°C).; The guest-host model was utilized to probe the biological activity of the sequence GEKGEP on phagocytosis. This sequence was incorporated into the triple helical guest-host structure Ac-(Gly-Nleu-Pro)₅-(GEKGEP)-(Gly-Nleu-Pro) ₅-NH₂ with a T_m at 56°C. This collagen mimetic is currently being assayed for phagocytotic activity.; A new scaffold, tris(2-aminoethyl)amine (TREN) was incorporated to assemble triple helices composed of Gly-Nleu-Pro and Gly-Pro-Nleu. Extensive biophysical studies demonstrated that TREN-[suc-(Gly-Nleu-Pro)_n-NH₂] ₃ (n = 5, 6; suc denotes a succinic acid spacer) are more stable than their KTA-assembled counterparts. The results suggest that the flexibility of the TREN scaffold allows the chains to adjust their register for tighter packing of collagen-like triple helices.; Significant progress has been made in the use of peptides to elicit specific cellular responses, particularly in areas of cell attachment and tissue integration. Our collaborators in Australia demonstrated our novel collagen mimetic structures, specifically H-(Gly-Pro-Nleu)₁₀-Gly-Pro-NH₂ have potential use as cell and tissue support matrices. We are employing these peptidomimetics as the basis for biomedical applications and the design of novel biomaterials.