Chemical Methods to Tailor the Stability of Collagen Triple Helices

Chemical methods for the manipulation of the conformational properties and thermal stability of synthetic collagen triple helices are important for the design of collagen-based materials for, e.g., wound healing. This thesis explores different chemical approaches to tune the conformational properties of functional collagen model peptides and investigates the underlying molecular factors. In the first part, the effect of pH on the conformational stability of collagen model peptides is investigated. The first study focuses on the effect of charged versus neutral Nand C-terminal functional groups on the thermal stability of collagen triple helices. Thermal denaturation experiments of collagen triple helices at different pH values revealed that positively charged ammonium groups at the N-terminus destabilize the triple helix more than negatively charged carboxylates at the C-terminus. Furthermore, neutral C-terminal carboxylic acids stabilize the triple helix more than C-terminal amides. In a second study, g-aminoproline (Amp) was used as a pH-responsive probe to tune the thermal stability of collagen triple helices in acidic and basic environments. The different steric and stereoelectronic properties of amino and ammonium groups led to a switch of the pyrrolidine ring upon changing the pH. The choice of the position of the residue within the collagen model peptide as well as the absolute configuration of the probe enables tuning the stability of Amp-containing triple helices. NMR spectroscopic studies with monomeric model compounds as well as comparative quantum chemical studies support the experimental findings. In a third study, g-azaproline (g-azPro) and alkylated derivatives, which lack a stereogenic center at the Ng atom, were investigated. Conformational analysis by NMR spectroscopy and DFT calculations revealed that the imidazolidine ring of the g-azPro derivatives are flexible and that the Ng atom can invert. The studies further uncovered that upon protonation, the main chain dihedral angles are displaced, which leads to disassembly of the collagen triple helix. g-Aminoproline and gazaproline initiate triple helix disassembly at different pH values: g-aminoproline at pH ~7, g-azaproline at pH ~3–4; and thus both are valuable complementary probes for the development of pH-responsive materials for, e.g. the targeted drug delivery to cancer cells. The second part of this dissertation explores new chemical approaches to increase the thermal stability of the collagen triple helix. The first approach addresses the trans/cis isomerization of the tertiary amide bonds. Both trans amide bonds and a fast cis–trans isomerization of Gly-Pro and Pro-Hyp bonds are crucial for the stability and folding rate of collagen. Our approach uses pendant hydrophobic moieties attached to the triple helix that generate a local hydrophobic environment around the triple helix. Thermal denaturation studies showed that the hydrophobic appendages provide hyperstable triple helices. Kinetic studies further demonstrated that folding is accelerated to an extent that thermally induced unfolding and folding of the triple helix take place at the same speed (with a heating rate of 1 °C/100 s). The findings suggest that the hydrophobic moieties generate a hydrophobic microenvironment that favors trans amide bond over the cis conformer and thus accelerate cis-to-trans isomerization. The second approach to stabilize triple helices takes advantage of the proximity of two neighboring gfunctionalized Pro derivatives in coplanar positions on adjacent strands. We equipped collagen model peptides with two complementary functional groups that can interact synergistically through H bonding or minimization of steric constraints. For example, (4S)-ammoniumproline interacts with its neighboring Hyp through H bonding, and thus stabilizes the collagen triple helix up to 13 °C. The last part of the dissertation explores the effect of four- and six-membered ring-size analogs on collagen triple helix stability. The study revealed that ring-size analogs of Pro differ in their trans/cis ratio of the tertiary amide bond and the geometry of the main chain dihedral angles, which are properties important for the stability of the collagen triple helix. This study allowed us to quantify relatively the contributions of these two factors towards the collagen triple helix, a study that is otherwise challenging or even impossible with Cg-substituted proline derivatives. The four-membered ring-size analog of Pro reduces the conformational stability of collagen significantly due to its lower trans/cis ratio, whereas the six-membered ring-size analog lowers the stability or even disrupts the triple helix due to distortion of the main chain dihedral angles. Thus, proper dihedral angles are more important for the stability of the collagen triple helix than high trans/cis amide bond ratios. We next performed NMR spectroscopic and computational studies with monomeric ring-size analogs that differ in their C-terminal functional group. The study showed that the conformation of proline derivatives is influenced by a complex interplay of multiple factors including steric constraints, intramolecular H bonding, and n®p* interactions. In contrast, the conformation of the proline ring-size analogs is either governed by H bonding or steric constraints. These studies with proline and its ring-size analogs highlight that the unique geometry of proline makes it an ideal building block for collagen or other proteins and peptides, since it is conformationally sensitive towards its chemical environment. This thesis provides chemical methods that enable the manipulation of the collagen triple helix such that it is pH-responsive or hyperstable. Control over both stimuli responsiveness and thermostability are important for the design of synthetic collagen based materials