Synthesis and Evaluation of Non-Hydrolyzable Phospho-Lysine Peptide Mimics.

Anett Hauser,Fabian Müller,Christian P R Hackenberger,Eleftheria Poulou,Peter Schmieder

doi:10.1002/chem.202003947

Abstract

The intrinsic lability of the phosphoramidate P−N bond in phosphorylated histidine (pHis), arginine (pHis) and lysine (pLys) residues is a significant challenge for the investigation of these post‐translational modifications (PTMs), which gained attention rather recently. While stable mimics of pHis and pArg have contributed to study protein substrate interactions or to generate antibodies for enrichment as well as detection, no such analogue has been reported yet for pLys. This work reports the synthesis and evaluation of two pLys mimics, a phosphonate and a phosphate derivative, which can easily be incorporated into peptides using standard fluorenyl‐methyloxycarbonyl‐ (Fmoc‐)based solid‐phase peptide synthesis (SPPS). In order to compare the biophysical properties of natural pLys with our synthetic mimics, the pK a values of pLys and analogues were determined in titration experiments applying nuclear magnetic resonance (NMR) spectroscopy in small model peptides. These results were used to compute electrostatic potential (ESP) surfaces obtained after molecular geometry optimization. These findings indicate the potential of the designed non‐hydrolyzable, phosphonate‐based mimic for pLys in various proteomic approaches.

Highlights

2.1 Post-Translational Modification of ProteinsCompared to other biological macromolecules, such as carbohydrates, lipids and nucleic acids, proteins exhibit the largest structural diversity and each cell contains several thousand different species
In order to prepare caged pLys peptides via this route, up to three purification steps were required, which would remarkably reduce the overall yield to approximately 3%
The Fmoc-protected solid-phase peptide synthesis (SPPS) building block 1 could be obtained in a straight forward synthesis in 39% yield over four steps starting from a non-expensive commercial compound

Summary

Introduction

Compared to other biological macromolecules, such as carbohydrates, lipids and nucleic acids, proteins exhibit the largest structural diversity and each cell contains several thousand different species. They make up approximately 50% of the dry weight of cells[2] and serve as the executors of biological tasks. Depending on the superior objectives, protein families have evolved to varying magnitude of versatility This means, for core functions as in transport, transcription and translation, proteins have diversified less than for regulatory functions as signaling, metabolic processes and phosphorylation[4] Strikingly, the information for this broad variety in structure and functionality is originated in the relatively small encoding genome, e.g. approximately 19-20,000 genes [5] code more than 9.3 million proteins [6] in humans.

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