Our research group is exploring the use of enzyme-catalyzed peptide synthesis from amino acid ester monomers as a versatile platform to prepare peptide oligomers with useful properties such as self-assembly, antimicrobial activity, metal chelation and more. Relative to alternative methods of peptide synthesis that generally involved tedious protection-deprotection steps, peptide synthesis by protease catalysis can be conducted using amino acid ethyl esters as monomers in aqueous media at nearly ambient temperatures. A strategy is described in this paper by which protease-catalyzed oligopeptides synthesis is used to prepare perfectly alternating oligopeptides sequences. Indeed, alternating oligopeptides motifs prepared by solid-state peptide synthesis have been shown to have a number of attractive physical and biological properties. The first target selected was silk inspired (AG)x sequences. In the first step, we prepared AG-OEt dipeptide 'legos' in nearly quantitative yield. In order to convert these AB dipeptide building blocks to perfectly alternating oligopeptides, initial studies showed the importance of identifying the appropriate reaction conditions so that propagation at the C-terminus of peptide chains dominates other pathways that would tend to randomize oligopeptide sequences. Based on this work, oligomerizations of AG-OEt were conducted using papain as catalyst at pH=8 and 40oC. In 30 seconds (AG)x was prepared in about 80% yield with an average chain length of 9.4 dimeric units. Thus, a simple and scalable route to silk-like �-sheet forming domains was demonstrated. Subsequently, the general utility of preparing dipeptide 'legos' that can be converted to alternating peptides was tested by studying the oligomerization of Lys-Leu ethyl ester (KL-OEt). After exploring a diverse range of protease-catalysts, a-chymotrypsin was found to be most effective for the conversion of KL-OEt to (KL)x oligomers retaining this alternating motif. Indeed, 10 s after addition of a-chymotrypsin to KL-OEt, (KL)x oligomers were formed that resulted in a sol�gel transition. The mixture of (KL)x chain lengths under alkaline conditions self-assembles into �-sheets. Thereafter, intermolecular backbone hydrogen bonding between peptides results in physically entangled nanofibrillar networks. This work is currently being extended to a broad range of interesting alternating peptide sequences.

Simplifying alternating peptide synthesis by protease-catalyzed dipeptide oligomerization

247th National Meeting (2014)

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