Synthesis of molecular probes targeting ER flippases

Abstract

Most proteins that enter the secretory pathway become glycoproteins, i.e. they are modified by sugars, such as N-glycans. The absence of N-glycans is lethal, and alterations in glycosylation patterns may lead to devastating diseases, including cancer. The assembly of the canonical oligosaccharide donor for N-glycosylation requires the flipping of dolichol-containing glycolipids from the cytoplasmic to the luminal side of the endoplasmic reticulum (ER). This process implies the existence of proteins (flippases) that facilitate the otherwise very slow movement of polar glycolipids through the membrane (FIGURE 1). The goal of this study is the synthesis of molecular probes, which should serve as tools for the identification of the proposed ER flippases (FIGURE 1). FIGURE 1 (A) Illustration of some of the steps involved in the protein N-glycosylation pathway, located in the ER membrane. (b) Structure of the natural substrate (top) and the corresponding fluorescent analogue (bottom). We are synthesizing lipid analogs for the purpose of assaying, capturing and isolation of candidate flippase proteins. For this purpose, we are synthesizing fluorescent mimics of the natural substrates, with an ω-terminal fluorophore (NBD, see FIGURE 1). Additionally, we are synthesizing analogs with chemically reactive moieties for crosslinking to the target proteins are required. The chemical synthesis of these probes will be presented. This work is part of an interdisciplinary project supported by the Swiss National Science Foundation (SNSF) – Sinergia Project Molecular identification of lipid transporters for protein glycosylation.