Protease-catalyzed synthesis of oligopeptides and glycan-terminated peptides

Abstract

Peptides and peptide conjugates are used for many applications, including as therapeutics and biomedical materials. At present, established synthetic routes to peptides and their conjugates include solid-phase peptide synthesis (SPPS), liquid-phase peptide synthesis (LPPS), N-carboxyanhydride ring opening polymerization (NCA-ROP) and recombinant peptide synthesis. While each of these methodologies can provide the target structures, their use is limited due to high product cost. SPPS, LPPS and NCA-ROP require toxic reagents, multiple protection-deprotection steps, and excessive solvent utilization. Protease-catalyzed peptide synthesis (PCPS) provides a green alternative method to prepare peptides and peptide conjugates. Furthermore, PCPS is conducted in an aqueous environment, using amino acid ethyl ester starting materials, requires short reaction times (i.e. a few minutes up to a couple hours), and product isolation from reaction mixture is normally simple. This thesis reveals a strategy by which PCPS was used to prepare glycan terminated peptide conjugates. ‘Grafters’ were synthesized that consist of a glycan conjugated directly, or through a short ethylene glycol ([CH2-CH2-O-]x)spacer, to the amine group of L-Phe-ethyl ester (Phe-OEt). Phe-OEt increases the grafter’s recognition at the protease (papain) catalytic active site. While glycan-PheOEt grafters that lacked an oligo(ethylene glycol) spacer resulted in low grafter efficiency (8.3 ± 2.0%), insertion of a short oligo(ethylene glycol) spacer (glycan-[CH2-CH2-O-]x -Phe-OEt, x ≥ 3) increased the grafter efficiency by 3-fold to 24.5 ± 1.8%. Spacers with one and two ethylene glycol units were too short to relieve detrimental papain-glycan interactions that resulted in low grafter efficiencies. Computational modeling performed with Rosetta software identified specific papain-glycan interactions that appear to destabilize the complex formed when the spacer has less than three ethylene glycol units. In addition, the polymerization of L-His-OEt by both papain and α-chymotrypsin was conducted, and the monomer conversion over time was monitored by NMR spectroscopy. The DPavg of oligo(His) was calculated by ESI-MS. Within 15 min, crude papain catalyzed the conversion of L-His-OEt to water-soluble oligo(His) with a DPavg of 9.3 ± 0.05. In addition, computational modeling performed by Rosetta Software compared the pocket energy for hydrolysis and synthesis of His oligomers catalyzed by crude papain and αchymotrypsin. Lastly, recombinant papain and its isozymes were heterologously expressed, purified, and evaluated individually for their peptide synthetic activity. The literature of papain biotransformations is dominated by the use of the natural powder isolated from papaya latex consisting of a mixture of proteases, of which papain is a minor component. Knowledge of whether papain is the only protease of the natural mixture with peptide synthesis activity, or whether peptide synthesis results from some combination of activities of its constituent proteases, is unknown. Herein, the peptide synthetic activity of recombinant papain, as well as its isozymes (chymopapain (I-IV), proteinase omega) was determined for Leu-OEt oligomerization using NMR in situ monitoring. Recombinant papain exhibited a 31% higher yield than commercial papain, using equivalent enzyme units measured by an azocasein assay. Furthermore, each isozyme was active to different extents for oligo(Leu) synthesis.