Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. This proposal is developing core synthesis ? enzymatic extension (CSEE) approach to produce large glycans and glycopeptides with most natural structural diversity. In CSEE approach, a core comprising a few sugars in the reducing end is synthesized first. Convergent core synthesis is cost-efficient with well documented methodologies. Then glycosyltransferases are used to elongate the core by following a variety of different biosynthesis pathways to generate complex and larger glycoconjugates with high diversity. Depending on high region- and stereo-selectivity of glycosyltransferases, CSEE is the most efficient approach to produce complex glycoconjugates with high fidelity. Our recent success of using 7 chemically synthesized N-glycan cores and 4 glycosyltransferases to produce 73 complex N-glycans clearly demonstrates that CSEE is an answer to the complexity and diversity of glycomes. In this program, 8 cores of O-GalNAc-Ser/Thr will be synthesized and extended with 16 glycosyltransferases to afford focused and diverse O-GalNAc-glycans/glycopeptides libraries; 3 cores of O-Man-Ser/Thr will be synthesized and extended with 9 glycosyltransferase to produce O-Man glycans/glycopeptides; O-Fuc, O-Glc and O-Xyl glycans and glycopeptides will be produced similarly. CSEE for synthesis of these O-glycan/O-glycopeptides will be fully validated in other labs. The technology advance of this proposal is to automate the enzymatic extension by using a conventional peptide synthesizer. We will fully investigate stability and optimal reaction conditions for the most synthetic useful glycosyltransferases and make the information accessible via a website. In summary, this program should make such a dream come true: scientists can have affordable access to any N- & O-glycans and glycopeptides as they have for regular peptides.
(By integrating the well documented chemical production of smaller core sugar sequences with enzymatic extension, the core synthesis ? enzymatic extension (CSEE) approach in this research program will produce a large number of diverse, natural and complex O-glycans and O- glycopeptides. To make the synthetic approach more accessible, affordable and adaptable, CSEE is automated on a conventional peptide synthesizer. This program is working on technologies which will make such a dream come true: scientists can order any N- & O-glycans and glycopeptides as they routinely order pieces of DNA or peptides. Revised Milestones 2015.7 to 2016.6 1 g scale chemical synthesis of all O-GalNAc cores 1 g scale chemical synthesis of O-Man cores m1, m2, m3 Optimize expression, storage conditions of currently available enzymes Develop UDP-Xyl synthesis system Clone and expression of B3GAT1 and HNK-1ST for O-man glycan synthesis Proof-of-concept experiment of Enzymatic Extension B Proof-of-concept experiment of Enzymatic Extension on solid phase by peptide synthesizer 2016.7 to 2017.6 20 g scale chemical synthesis of O-GalNAc cores 1 g scale chemical synthesis of GlcA?1,4Xyl for polymer synthesis Clone and expression of other human GTs, e.g., FUT2, POMK Proof-of-concept experiment of Enzymatic Extension A2 Synthesis of target O-GalNAc Core 2 Glycans Synthesis of Core 2 glycopeptides on solid phase by peptide synthesizer Synthesis of target O-Man glycan library 2017.7 to 2018.6 20 g scale chemical synthesis of all O-Man cores Synthesis of target O-GalNAc cores 1, 3-8 glycans Automated Synthesis of target O-GalNAc glycopeptides Automated Synthesis of target O-Man glycopeptides Synthesis of O-Man core m1 glycans 2018.7 to 2019.6 Website updating (summarize data and information, put on website) Summarize data for publication Generate standard protocol for each GTs, and reactions. Prepare training materials for cross-validation Send for cross-validation (Chemical synthesis and Enzymatic Extension) Apply CSEE approach in the synthesis of O-Fuc, O- Xyl, and O-Glc glycans and glycopeptides
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