Protease-catalyzed synthesis of N-acryloyl-oligopeptides and histidine-containing oligopeptides

Abstract

Synthetic peptides, peptides built via chemical methodology or through recombinant technology, have been designed for use in therapeutics. Current chemical methods to create synthetic peptides rely largely on solid-phase peptide synthesis (SPPS), which lacks important green preparative qualities and is economically unsustainable, limiting the application of peptides synthesized via SPPS to high cost, low volume applications such as personalized medicine. Applications including bulk adhesives and soft materials would benefit from a low cost, high yielding method of peptide synthesis. Therefore, an alternative method of chemical peptide synthesis is needed that minimizes the use of toxic solvents, hazards coupling reagents, tedious protection-deprotection chemistry, and substantial post-modification. In response to the lack of green characteristics of commonly used peptide synthesis strategies, protease-catalyzed peptide synthesis offers an alternative method for peptide construction using enzymes to catalyze amide bond formation between weakly activated amino acid ethyl esters (AA-OEt) in aqueous buffer (pH 8) under mild conditions (40 °C) with short reaction times. The drawback to PCPS is the loss in control over the length and sequence of the oligopeptide structure. However, applications where short peptides with random sequences might be utilized could benefit from using protease-catalysis as this method is low cost, environmentally sustainable, and high yielding. In addition to peptides alone, peptide-polymer conjugates are a highly versatile and useful molecular hybrid that has found extensive application in biomedical and materials technology. When coupled together, peptides and polymers exhibit synergistic interactions including improving peptide solubility and protecting peptides from degradation in vivo, whereas the peptide bestows the polymer structure self-assembly characteristics which enable to synthesis of supramacromolecular structures not available to either moiety individually. However, construction of the peptide segment is often completed using SPPS and other chemical methods, like N-carboxyanhydride ring opening polymerization. Furthermore, the process to couple peptides involve multiple steps to complete the conjugation. Methods to reduce the number of steps necessary, such as using one-pot systems, improves the green chemical characteristics of the peptide-polymer conjugation. In response to concerns about the lack of green chemical preparative methods for both peptides and peptide-polymer conjugates, this dissertation focuses on the utilizing PCPS for the synthesis of N-acryloyl terminated oligopeptides as precursors to prepare peptide-polymer conjugates. In chapter 2, evaluation of N-terminally modified N-acryloyl-glutamic acid diethyl ester, N-acryloyl-leucine ethyl ester, and N-acryloyl-alanine ethyl ester as grafters, and papain as the protease, was conducted for the direct addition of amino acid ethyl ester (AA-OEt) monomers via protease catalyzed peptide synthesis (PCPS), to yield N-acryloyl functionalized oligopeptides in a one-pot, aqueous scheme. It was hypothesized that, by building N-acryl grafters from AA-OEt monomers that are known to be good substrates for papain-catalyzed PCPS, the corresponding grafters would yield high grafter conversion, ratio of grafter-oligopeptide to free oligopeptide, and high overall yield. However, the work reported in chapter 2 demonstrates that the dominant factor in N-acryloyl-AA-OEt grafter efficiency is the comonomer used in co-oligomerizations. This work further advances our knowledge of factors that determine the efficiency of preparing N-acryloyl terminated oligopeptides by PCPS, providing practical routes to peptide macromers for conjugation to polymers and surfaces for a broad range of applications.

In the second portion of this dissertation, the synthesis of histidine-containing peptides (HCP) using PCPS is reported. Specifically, the co-oligomerization of histidine ethyl ester and model hydrophobic substrate, leucine ethyl ester, conducted to examine the random incorporation of leucine and histidine residues by protease catalysis, without chemical modification of the histidine monomer to increase histidine affinity for the protease. Proteases papain and α-chymotrypsin were evaluated as catalysts in this work to probe the difference in co-oligomerization of leucine and histidine via cystine or serine protease. Unlike previously reported co-oligomerizations of leucine with other amino acid ethyl ester substrates, papain and α-chymotrypsin catalyzed the formation of separate oligo(leucine) and oligo(histidine) peptides, with minimal co-oligomerization of the two monomers. To increase incorporation of both residues into a HCP, a dipeptide ethyl ester monomer composed of leucine-histidine (NH2-Leu-His-OEt·2HCl), was synthesized and utilized in papain and α-chymotrypsin peptide synthesis reactions. Using the dipeptide monomer resulted in formation of HCP composed of alternating sequences leucine and histidine residues with mixed chain lengths. The hydrophobicity of the resulting Oligo(Leu¬-alt-His) peptides limited the solubility to pH ≤ 3. Nonetheless, the synthesis of HCP using PCPS was achieved and evaluation between papain and α-chymotrypsin revealed that α-chymotrypsin is the superior catalysts to synthesis alternating oligopeptide sequences, albeit with the tradeoff of %-yield.