DEHYDROALANINE MEDIATED UBIQUITINATION: DEVELOPMENT AND INSIGHTS INTO THE STEREOCHEMISTRY OF THE ADDITION STEP

Site-specific modification of proteins is a powerful approach to label a substrate with various substitutes such as posttranslational modifications (PTMs) and offers an opportunity to study different aspects of modifications at atomic resolution. In the current presentation we will show our ongoing development of a novel strategy for site-specific ubiquitination using dehydroalanine chemistry for the preparation of ubiquitin conjugates with a linkage that is very similar to the native isopeptide bond. This approach relies on the selective elimination of the thiol side chain to afford dehydroalanine employing known methods. This dehydroalanine moiety then serves as an electrophilic center for the conjugation with hexapeptide derived from the last six amino acid of ubiquitin bearing a thiol nucleophile on its C terminus and protected Cys at the N-terminal. Subsequently, the resulted synthetic intermediate undergoes native chemical ligation with the complementary part of ubiquitin bearing thioester functionality at its C-terminus. During this process the formation of two protein diastereomers, as a result of addition step of hexapeptide-thiol on the protein-dehydroalanine, were observed. These diastereomers were further characterized and quantified using trypsin digestion and high-pressure liquid chromatography. Our results offer new opportunity in proteins conjugations, in particular those involving an isopeptide bond. Our study, for the first time, offers new insights into the stereoselectivity issue when dehydroalanine is involved in chemical protein modifications.