Sequence-controlled synthesis of beta3-peptides: Solid-phase synthesis and DNA-templated synthesis

The discovery of β3-peptides as foldamers has attracted substantial interest in their synthesis, analysis and application. The advantages of using β3-peptides as structural and functional mimics of natural peptides derives from their resistance to proteolysis, metabolic stability and ability to fold into discrete, stable and defined structures at shorter chain lengths than their α-peptide counterparts. Since the interaction between β3-peptides and biomolecules depends on precise substituent sequence of β3-peptides, we decided to use two different strategies to achieve the sequence-controlled synthesis of β3-peptides: solid phase peptide synthesis (SPPS) and DNA-templated synthesis (DTS). Although the SPPS of β3-peptides has been developed, it was hampered by the difficulties of deprotection and sluggish reactivity of β3-amino acids. Based on the α-ketoacid-hydroxylamine (KAHA) ligation discovered by our group, we have developed a new iterative α-ketoacid-isoxazolidine approach for β3-peptide synthesis. A versatile synthesis of enantiopure isoxazolidine monomers has also been developed. Isoxazolidine monomers with side chains of diverse functionalities (proteinogenic and non-proteinogenic) and different configurations have been synthesized. With the availability of different isoxazolidine monomers, we have developed a three-step procedure to synthesize β3-peptides, including coupling, hydrolysis and neutralization, for the implementation of the KAHA ligation on solid phase. However, the procedure was restricted to 5-residue β3-peptides due to the instability of PL-PEGA resin. By optimizing the procedure, including changing resins, coupling a less hindered monomer in the first residue, and thermal hydrolysis of α-ketoesters, we have successfully synthesized a β3-octapeptide with acid and base functionality on the side chains. This novel SPPS of β3-peptides via KAHA ligation provides an alternative to the traditional SPPS of long β3-peptides. We also applied the KAHA ligation to DNA-templated polymerization of β 3-peptides. Combining the advantage of iterative KAHA ligation and DTS which increases the effective reactant molarity, sequence-controlled synthesis of β3-peptides can be constructed. We have successfully coupled the α-ketoacid and isoxazolidine to DNAs. However, no desired KAHA ligation product was observed in our early test due to the instability of isoxazolidine monomer on DNA. After modifying the DNA construct and making a more stable monomer, we successfully perform the first KAHA ligation on DNA. To avoid thermal destruction of monomer, we have to adopt isothermal strategies to implement the multistep synthesis of β3-peptides on DNA. We believe that these two synthetic methods of β3-peptide can help the development of β3-peptides in biological study and therapeutic application