Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Pascal Heimer; Thomas Schmitz; Charlotte A. Bäuml; Diana Imhof

doi:10.3791/58368

A subscription to JoVE is required to view this content. Sign in or start your free trial.

Method Article

Synthesis and Structure Determination of µ-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

10.1K views

⸱

DOI:

10.3791/58368

⸱

October 2nd, 2018

Pascal Heimer^*¹ , Thomas Schmitz^*¹ , Charlotte A. Bäuml^*¹ , Diana Imhof¹

¹Pharmaceutical Biochemistry and Bioanalytics, Pharmaceutical Institute, University of Bonn

^* These authors contributed equally

Erratum Notice

Important: There has been an erratum issued for this article. View Erratum Notice

Summary

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

Abstract

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of µ-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Introduction

The use of bioactive peptides in pharmaceutical research and development is highly recognized, because they represent potent and highly selective compounds for specific biological targets¹. For their bioactivity, however, the three-dimensional structure is of great importance in order to perform structure-activity relationship studies²^,³^,⁴. Apart from the primary amino acid sequence that influences the overall conformation, disulfide bonds significantly stabilize the structure of cysteine-rich peptides⁵. Multiple disulfide-bridg....