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 glycosyltransferaes, 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 for investigating PSGL-1, podoplanin and microbial glycan interaction; 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 kinetics, substrate specificity, 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. 25
In life the best way normally is following the nature?s way. To produce complex carbohydrate sequences and their conjugates to peptides and proteins, the most cost-efficient approach is to follow the natural biosynthetic pathways and use the natural enzymes called glycosyltransferases to link and elongate the sugar chains. Thus 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 divers, 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. Two Nobel prices were given to chemists for their invention of synthesizer machines which can costumer produce oligonucleotides and peptides and help bring out great scientific advances in life science. 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.
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