Enabling Chemoenzymatic Strategies and Enzymes for Synthesizing Sialyl Glycans and Sialyl Glycoconjugates
Year: 2023
Type: article
Abstract: High Resolution Image Download MS PowerPoint Slide Conspectus Sialic acids are fascinating negatively charged nine-carbon monosaccharides. Sialic acid-containing glycans and glycoconjugates are structurally diverse, functionally important, and synthetically challenging molecules. We have developed highly efficient chemoenzymatic strategies that combine the power of chemical synthesis and enzyme catalysis to make sialic acids, sialyl glycans, sialyl glycoconjugates, and their derivatives more accessible, enabling the efforts to explore their functions and applications. The Account starts with a brief description of the structural diversity and the functional importance of naturally occurring sialic acids and sialosides. The development of one-pot multienzyme (OPME) chemoenzymatic sialylation strategies is then introduced, highlighting its advantages in synthesizing structurally diverse sialosides with a sialyltransferase donor substrate engineering tactic. With the strategy, systematic access to sialosides containing different sialic acid forms with modifications at C3/4/5/7/8/9, various internal glycans, and diverse sialyl linkages is now possible. Also briefly described is the combination of the OPME sialylation strategy with bacterial sialidases for synthesizing sialidase inhibitors. With the goal of simplifying the product purification process for enzymatic glycosylation reactions, glycosphingolipids that contain a naturally existing hydrophobic tag are attractive targets for chemoenzymatic total synthesis. A user-friendly highly efficient chemoenzymatic strategy is developed which involves three main processes, including chemical synthesis of lactosyl sphingosine as a water-soluble hydrophobic tag-containing intermediate, OPME enzymatic extension of its glycan component with a single C18-cartridge purification of the product, followed by a facile chemical acylation reaction. The strategy allows the introduction of different sialic acid forms and diverse fatty acyl chains into the products. Gram-scale synthesis has been demonstrated. OPME sialylation has also been demonstrated for the chemoenzymatic synthesis of sialyl glycopeptides and in vitro enzymatic N - glycan processing for the formation of glycoproteins with disialylated biantennary complex-type N-glycans. For synthesizing human milk oligosaccharides (HMOs) which are glycans with a free reducing end, acceptor substrate engineering and process engineering strategies are developed, which involve the design of a hydrophobic tag that can be easily installed into the acceptor substrate to allow facile purification of the product from enzymatic reactions and can be conveniently removed in the final step to produce target molecules. The process engineering involves heat-inactivation of enzymes in the intermediate steps in multistep OPME reactions for the production of long-chain sialoside targets in a single reaction pot and with a single C18-cartridge purification process. In addition, a chemoenzymatic synthon strategy has been developed. It involves the design of a derivative of the sialyltransferase donor substrate precursor, which is tolerated by enzymes in OPME reactions, introduced to enzymatic products, and then chemically converted to the desired target structures in the final step. The chemoenzymatic synthon approach has been used together with the acceptor substrate engineering method in the synthesis of complex bacterial glycans containing sialic acids, legionaminic acids, and derivatives. The biocatalysts characterized and their engineered mutants developed by the Chen group are described, with highlights on synthetically useful enzymes. We anticipate further development of chemoenzymatic strategies and biocatalysts to enable exploration of the sialic acid space.
Source: Accounts of Chemical Research
Author: Xi Chen
Institution: University of California, Davis
Language: English
Subfield: Organic Chemistry
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $5,000
Awards: Developing chemoenzymatic strategies, enzymes, and kits for accessible and affordable gangliosides, Engineered probes for sialoglycan detection, Chemoenzymatic synthesis of bacterial nonulosonic acids and glycans, Affordable sialoglycans and associated reagents for expanded chemoenzymatic production, G786782417, TITLE: DISTRIBUTED MANUFACTURING OF ANTIGEN FOR SEROLOGICAL TESTING & COUNTERMEASURES? PURPOSE: THE PURPOSE OF THIS PROJECT IS TO DEVELOP TECHNOLOGIES CURRENTLY AT PROOF-OF-CONCEPT STAGE FOR AGILE AND DISTRIBUTED MANUFACTURING OF CORONAVIRUS FAMILY SPIKE PROTEIN VARIANTS BY DEPLOYING EASILY SCALABLE PROTEIN EXPRESSION SYSTEMS. ACTIVITIES TO BE PERFORMED: THE PROJECT TEAM WILL DEVELOP METHODS TO RAPIDLY SCREEN SARS-COV-2 ANTIGENS THAT BIND TO PATIENT SERA AND/OR ANTIBODIES THAT TARGET DIFFERENT VIRAL PROTEINS; IDENTIFY PRODUCTION PLATFORMS THAT CAN MOST EFFICIENTLY PRODUCE THE TARGET ANTIGENS THAT MEET QUALITY SPECIFICATIONS AT A SPEED, SCALE, AND COST THAT IS NOT ACHIEVABLE WITH CURRENT ANTIGEN PRODUCTION PROCESSES; CONDUCT RESEARCH TO UNDERSTAND HOW THE CHOICE OF HOST-CELL PRODUCTION PLATFORM IMPACTS FUNCTIONALITY OF THE ANTIGENS PRODUCED; AND DEVELOP CAPABILITIES FOR SCALEUP AND POST-PRODUCTION MODIFICATION OF GLYCOFORMS ON PURIFIED ANTIGENS; AND PROVIDE A TECHNO-ECONOMIC ASSESSMENT OF PROMISING PRODUCTION PLATFORMS. EXPECTED OUTCOMES: THIS RESEARCH WILL ENABLE AGILE DOMESTIC BIOMANUFACTURING OF REAGENTS FOR CORONAVIRUS RESEARCH AND DIAGNOSTICS, DEVELOP NOVEL PRODUCTION TECHNOLOGIES THAT ENHANCE U.S. BIOINDUSTRIAL COMPETITIVENESS, DE-RISK INVESTMENT IN ANTIGEN PRODUCTION INFRASTRUCTURE, AND EXPAND THE BIOMANUFACTURING WORKFORCE. BY ESTABLISHING METHODS FOR RAPID, DISTRIBUTED ANTIGEN PRODUCTION, THIS PROJECT WILL SIGNIFICANTLY SPEED DISTRIBUTED PRODUCTION AND DEPLOYMENT OF REAGENTS FOR DIAGNOSTICS AND COUNTERMEASURES IN RESPONSE TO ANY NEW DISEASE OUTBREAK. INTENDED BENEFICIARIES: AS NEW CORONAVIRUSES AND VARIANTS ARISE, HEALTHCARE RESEARCHERS WILL BENEFIT BY HAVING THE ABILITY TO QUICKLY DESIGN AND PRODUCE ANTIGENS NEEDED TO ACCELERATE DIAGNOSTICS AND COUNTERMEASURE DEVELOPMENT. THE PUBLIC WILL BENEFIT THROUGH FASTER RESPONSES TO FUTURE PANDEMICS, AND THE NATION WILL BENEFIT MORE BROADLY BY THE INCREASED CAPABILITIES OF THE U.S. BIOINDUSTRIAL SECTOR. SUBRECIPIENT ACTIVITIES: THE RECIPIENT PLANS TO SUBAWARD FUNDS TO ACADEMIC PARTNERS TO CONDUCT RESEARCH FOR ANTIGEN DISCOVERY, STANDARDIZED CLONING, PRODUCTION OF ANTIGENS IN DIFFERENT HOST CELLS, AND CHARACTERIZATION AND ?GLYCO-TAILORING? OF THE TARGET ANTIGENS PRODUCED. THE RECIPIENT WILL ALSO SUBAWARD FUNDS FOR AND TECHNO-ECONOMIC ANALYSIS FOR THIS PROJECT., Microbial production of fucosylated human milk oligosaccharides, Glycolipid biointerface to decipher disease-implicated ganglioside-protein interactions, Chemoenzymatic construction of synthetic human milk oligosaccharide (HMO) glycome
OpenAlex ID: W4390062786