Synthesis of poly(Pro–Hyp–Gly)_<i>n</i> by direct polycondensation of (Pro–Hyp–Gly)_<i>n</i>, where <i>n</i> = 1, 5, and 10, and stability of the triple‐helical structure

Abstract

Pro-Hyp-Gly is a characteristic amino acid sequence found in fibrous collagens, and (Pro-Hyp-Gly)(10), which has been widely used as a collagen-model peptide, forms a stable triple-helical structure. Here, we synthesized polypeptides consisting of the Pro-Hyp-Gly sequence by direct poly-condensation of (Pro-Hyp-Gly)(n), where n=1, 5, and 10, using 1-hydroxybenzotriazole and 1-ethyl-3-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride in both phosphate buffer (pH=7.4) and dimethylsulfoxide (DMSO) solutions for 48 h at 20 degrees C. The reaction of (Pro-Hyp-Gly)(5) and (Pro-Hyp-Gly)(10) in DMSO successfully gave polypeptides with molecular weights over 10,000, whereas low molecular weight products were obtained by reaction in phosphate buffer (pH=7.4). In contrast, Pro-Hyp-Gly at a concentration of 50 mg/mL in phosphate buffer (pH=7.4) gave polypeptides with molecular weights over 10,000. The Fourier transform infrared (FTIR) and (1)H nuclear magnetic resonance (NMR) spectra of poly(Pro-Hyp-Gly)(10) revealed that the polymerization of (Pro-Hyp-Gly)(10) described in this report had no side reactions. Each polypeptide obtained shows a collagen-like triple-helical structure, and the triple-helical structures of poly(Pro-Hyp-Gly) and poly(Pro-Hyp-Gly)(10) were stable up to T=80 degrees C, which suggests that the high molecular weight promotes stability of the triple-helical structure, in addition to the high Hyp content. Furthermore, transmission electron microscopy (TEM) observations show that poly(Pro-Hyp-Gly)(10) aggregates to form nanofiber-like structures about 10 nm in width, which suggests that a Pro-Hyp-Gly repeating sequence contains enough information for triple-helix formation, and for subsequent nanofiber-like structure formation.