Cone Snails, Cyclic Peptides, and Fluorophores - A Gateway to Stable Traceable Peptides

Abstract

In-situ peptide degradation is a major hurdle to overcome in the development of traceable/visually detectable peptides. The reason being is that peptides quickly loose its conformation – by cleavage at various amino acid moieties – rendering them nonfunctional. Enhancing the structural stability of the peptide may therefore reduce degradation significantly, allowing it to retain its executive functionality longer. This project aims to stabilize peptides via the introduction of N- to C-terminal cyclization. In order to investigate stability, four structurally related peptides are synthesized: i) Tx2005 – 18 amino acid peptide found in Conus textile; ii) Tx2005-L – the introduction of a 7 amino acid linker to Tx2005; iii) Tx2005-CL – the cyclic version of Tx2005-L; and iv) Tx2005-FCL – with a flurophore bioconjugated to the linker sequence of Tx2005-CL. The peptides are synthesized via Solid Phase Peptide Synthesis (SPPS) and purified via Reverse Phase High Performance Liquid Chromatography (RP-HPLC). Backbone cyclization is carried out exploring both Fmoc and Boc chemistry. Fluorophore linkage is attained via azide ‘Click’ chemistry. All four peptides have successfully been synthesized and have been tested for biostability utilizing enzyme digestion assays. Data indicate that stability is increased in cyclic peptide variants. Cyclization is shown to mitigate degradation by the exopeptidase, carboxypeptidase, by a near complete retention of configuration, as demonstrated by RP-HPLC. Cyclization via Fmoc and bioconjugation utilizing ‘click’ chemistry is effectively shown to be promising for future research regarding enzymatic stability, drug delivery/orally bioactive peptide research, and peptide pathway mapping.