Protease Catalyzed In Situ C-Terminal Modification of Oligoglutamate

One-pot biotransformations gave oligo(γ-l-Et-Glu) decorated with selected amine-functionalized end-groups at C-termini. Motivations for this work were to (i) control the end group structure of peptides synthesized by protease-catalyzed peptide synthesis and (ii) incorporate end-groups that can be used directly or after further modification as polymerizable entities. Papain, bromelain, α-chymotrypsin, Multifect P-3000, and Purafect prime 4000 L were used as catalysts for oligomerization of γ-l-(Et)2-Glu in the presence of monofunctional 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 γ-l-(Et)2-Glu results in decreased % yield, but increased mol % of NH2-R end-capped oligo(γ-l-Et-Glu)-NH-R (determined by NMR). Irrespective of the protease used, 2-thiophene methyl amine (TPMA) gave the highest fraction of oligo(γ-l-Et-Glu)-NH-R chains. For example, using Multifect P-3000 and a feed ratio of TPMA-to γ-l-(Et)2-Glu of 7:3, >90 mol % of oligopeptides formed had TPMA C-terminal groups. With all five proteases studied herein, l-phenylalanine and l-histidine did not produce end-capped oligo(γ-l-Et-Glu). In contrast, l-phenylalanine analogs benzylamine (BzA) and l-phenylalaninol (F-OH), both of which lack the α-carboxyl group, gave substantial quantities of oligo(γ-l-Et-Glu)-F-OH or -BzA chains. Hence, the results of this study prove that the promiscuity of proteases used herein can be exploited to create a diverse family of desired end-functionalized oligopeptides. MALDI-TOF spectra recorded of oligo(γ-l-Et-Glu) with amine nucleophiles showed molecular ions that affirmed the formation of corresponding NH2-R functionalized oligo(γ-l-Et-Glu)

Protease-Catalyzed Oligomerization of Hydrophobic Amino Acid Ethyl Esters in Homogeneous Reaction Media Using l-Phenylalanine as a Model System

Enzymatic synthesis of oligopeptides from l-phenylalanine ethyl ester hydrochloride (l-Phe-Et·HCl) and other l-form hydrophobic amino acid ester hydrochlorides in water miscible organic cosolvents was studied. Different proteases, water miscible cosolvents, and effect of different ratios of water miscible cosolvents for protease-catalyzed oligo-phenylalanine [oligo(l-Phe)] were compared. The importance of the use of water miscible cosolvents in transforming reactions from heterogeneous to homogeneous conditions as a potent medium engineering tool for protease-catalyzed oligopeptide synthesis is highlighted. For example, at 0.125 M l-Phe-Et·HCl, 20% (v/v) methanol, 18.6 mg/mL bromelain, in phosphate buffer (0.25M, pH 8), 40 °C, for 3 h, oligo(l-Phe) precipitated from the solution to yield 45 ± 5%, in contrast, in the absence of cosolvent oligo(l-Phe) yield of 29 ± 5% was obtained. The following reaction conditions were optimized for bromelain catalyzed oligo(l-Phe) synthesis: pH, temperature, substrate, enzyme, and cosolvent concentrations. DPavg and chain length distribution in the product peptides were investigated by 1H NMR and MALDI-TOF

Chemo-enzymatic Routes to Lipopeptides and Their Colloidal Properties

A unique chemo-enzymatic route to lipopeptides was demonstrated herein that, relative to alternative methods such as solid-phase peptide synthesis (SPPS) and microbial synthesis, is simple, efficient, and scalable. Homo- and co-oligopeptides were synthesized from amino acid ethyl esters via protease catalysis in an aqueous media, followed by chemical coupling to fatty acids to generate a library of lipopeptides. Synthesized lipopeptides were built from hydrophobic moieties with chain lengths ranging from 8 to 18 and peptides consisting of oligo­(l-Glu) or oligo­(l-Glu-<i>co</i>-l-Leu) with an average of seven to eight repeating units. The chemical structures of the lipopeptides were characterized and confirmed by NMR and matrix-assisted laser desorption/ionization (MALDI). The colloidal and interfacial properties of these lipopeptides were characterized and compared in terms of the hydrophobic chain length, oligopeptide composition, and solution pH. The results showed correlation between the interfacial activity of the lipopeptides and the hydrophobicity of the fatty acid and oligopeptide headgroup, the effects of which have been semiquantitatively described in the manuscript. Results from these studies provide insights into design principles that can be further expanded in future work to access lipopeptides from protease-catalysis with improved control over sequence and exploring a wider range of peptide and lipid compositions to further tune lipopeptide biochemical and physical properties