Synthesis of Glycopeptides in the Inhibition of Insulin Resistance Associated with FoxO1-Thr231-OGlcNAc
Diabetes is one of the most widespread diseases in the United States, affecting more than 100 million adults1 and the amount of cases of diabetes is expected to continually rise over the coming years. Therefore, it is of vital importance to find a method of therapeutic treatment to insulin resistance. Further analysis of the framework and inner workings of the disease will help to determine the best method for the reversal of insulin resistance, particularly in cases of Type 2 diabetes mellitus (T2DM). The Forkhead Box O1 (FoxO1) regulatory protein is considered to play a key role in cellular sugar metabolism and insulin response. In particular, an amino acid site Threonine231 (Thr231) is hypothesized to be modified by a sugar group, O-linked N-acetylglucosamine (O-GlcNAc), in a manner that regulates metabolic signaling and insulin sensitivity. Using current knowledge of the insulin signaling pathway, it is proposed that a FoxoxO1 peptide mimic with an attached O-GlcNAc may lead to inhibition of the pathway if FoxO1-Thr231-O-GlcNAC is indeed the a stimulator of metabolic signaling. This project sought to synthesize sugar-peptide mimics of FoxO1-Thr231-O-GlcNAc in order to reverse the effects of T2DM. The sugar linked GlcNAc was synthesized over a succession of eight steps. The glycopeptide mimic was produced with the coupling of amino acids and an addition of a nuclear localization sequence as well as a fluorescent molecule, 5(6)-carboxyfluorescein (FAM), attached for visualization. Extensive optimization was done to the synthesis of the O-GlcNAc sugar as well as the glycopeptide mimic synthetic route to try and produce sufficient yield and accuracy in the product. The first step of the series of reactions was the primary focus of the optimization process, in order to improve the overall yield of the sugar-linked GlcNAc and purity of the final product. Optimization techniques included quadrupling the starting material and the addition of an argon line to create an inert atmosphere and using a sodium bicarbonate outflow line to neutralize the HCl gas. This eliminated a byproduct of the synthesis, HCl, improving the purity of the final product. However, the yield was still not sufficient to carry on to the final product. The production of peptide mimics of FoxO1-Thr231 was completed by coupling a variety of amino acids and monitored using the Kaiser test. These await sugar-coupling and testing in a suitable insulin resistance assay, work ongoing in the Fehl Laboratory. Due to complications with percent yield and optimization we were unable to determine the best method for effective reversal of insulin resistance. FurtherFuture analysis and manipulations of the insulin signaling pathways are will be key to determining the optimal technique for producing a treatment for both types of diabetes.
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