Protease catalyzed oligopeptide synthesis: Control of composition and end group structure

This thesis is focused on reverse equilibrium peptide synthesis from amino acid ester derivatives using proteases as the catalyst.;In the first study, one-pot biotransformations gave oligo(gamma-L-Et-Glu) decorated with selected amine-functionalized end-groups at the C-termini. Several proteases were used as catalysts for oligomerization of gamma-L-(Et)2-Glu.HCl in the presence of mono-functional amines. The series of amine nucleophiles (NH2-R, acyl acceptors) studied, mimic phenylalanine in that they possess aromatic rings linked to amine groups by one or more methylenes. Generally, addition of increased quantities of NH2-R from 0 to 30, 50 and 70 mol% with respect to gamma-L-(Et)2-Glu.HCl results in decreased %-yield but increased mol% of NH2-R end-capped oligo(gamma-L-Et-Glu)-NH-R (determined by NMR).;In the second study, composition of co-oligopeptides was varied by changing the feed ratio of L-cysteine-ethyl ester hydrochloride (L-Et-Cys.HCl) and L-glutamic acid di-ethyl ester hydrochloride [gamma-L-(Et)2-Glu.HCl]. Papain catalyzed co-oligomerization, produced oligo-Glu/Cys peptides that have a ∼ 1:1 glutamic acid and cysteine residue content for an average chain length of 9 for a monomer feed ratio of 4:6 (gamma-L-(Et)2-Glu.HCl : L-Et-Cys.HCl). The significance of this composition is highlighted in the sequence homology exhibited by phytochelatins (PC's). PC's are a group of peptides isolated from plant and animal sources responsible for binding to heavy metals thus allowing the plant to accomplish cellular metal homeostasis and detoxification. Characterization of the co-oligopeptide was successfully carried out by 1HNMR and 2D NMR to calculate the DPavg of the co-oligopeptides. MALDI-TOF of the co-oligopeptide further revealed that the distribution of cysteine residues ranged between 2 and 4. Metal binding properties of the co-oligopeptides containing 47% +/- 4% L-Cysteine was investigated by UV-visible spectroscopic analysis which further provided evidence of metal binding.;Thirdly, enzymatic synthesis of oligopeptides from L-phenylalanine ethyl ester hydrochloride (L-Et-Phe.HCl) and other L-form hydrophobic amino acid ester hydrochlorides in water miscible organic co-solvents was studied. The importance of the use of water miscible co-solvents in transforming reactions from heterogeneous to homogeneous conditions as a potent medium engineering tool for protease catalyzed oligopeptide synthesis is highlighted.