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Hierarchical and programmable one-pot synthesis of oligosaccharides.

ABSTRACT: The programmable one-pot oligosaccharide synthesis method was designed to enable the rapid synthesis of a large number of oligosaccharides, using the software Optimer to search Building BLocks (BBLs) with defined relative reactivity values (RRVs) to be used sequentially in the one-pot reaction. However, there were only about 50 BBLs with measured RRVs in the original library and the method could only synthesize small oligosaccharides due to the RRV ordering requirement. Here, we increase the library to include 154 validated BBLs and more than 50,000 virtual BBLs with predicted RRVs by machine learning. We also develop the software Auto-CHO to accommodate more data handling and support hierarchical one-pot synthesis using fragments as BBLs generated by the one-pot synthesis. This advanced programmable one-pot method provides potential synthetic solutions for complex glycans with four successful examples demonstrated in this work.

SUBMITTER: Cheng CW

PROVIDER: S-EPMC6283847 | biostudies-other | 2018 Dec

REPOSITORIES: biostudies-other

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Hierarchical and programmable one-pot synthesis of oligosaccharides.

Cheng Cheng-Wei CW Zhou Yixuan Y Pan Wen-Harn WH Dey Supriya S Wu Chung-Yi CY Hsu Wen-Lian WL Wong Chi-Huey CH

Nature communications 20181206 1

The programmable one-pot oligosaccharide synthesis method was designed to enable the rapid synthesis of a large number of oligosaccharides, using the software Optimer to search Building BLocks (BBLs) with defined relative reactivity values (RRVs) to be used sequentially in the one-pot reaction. However, there were only about 50 BBLs with measured RRVs in the original library and the method could only synthesize small oligosaccharides due to the RRV ordering requirement. Here, we increase the lib ...[more]

PMID: 30523255

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Project description:Glycosylation of proteins is a key function of the biosynthetic-secretory pathway in the endoplasmic reticulum (ER) and Golgi apparatus. Glycosylated proteins play a crucial role in cell trafficking and signaling, cell-cell adhesion, blood-group antigenicity, and immune response. In addition, the glycosylation of proteins is an important parameter in the optimization of many glycoprotein-based drugs such as monoclonal antibodies. In vitro glycoengineering of proteins requires glycosyltransferases as well as expensive nucleotide sugars. Here, we present a designed pathway consisting of five enzymes, glucokinase (Glk), phosphomannomutase (ManB), mannose-1-phosphate-guanyltransferase (ManC), inorganic pyrophosphatase (PmPpA), and 1-domain polyphosphate kinase 2 (1D-Ppk2) expressed in E. coli for the cell-free production and regeneration of GDP-mannose from mannose and polyphosphate with catalytic amounts of GDP and ADP. It was shown that GDP-mannose is produced at various conditions, that is pH 7-8, temperature 25-35°C and co-factor concentrations of 5-20?mM MgCl2 . The maximum reaction rate of GDP-mannose achieved was 2.7??M/min at 30°C and 10?mM MgCl2 producing 566?nmol GDP-mannose after a reaction time of 240?min. With respect to the initial GDP concentration (0.8?mM) this is equivalent to a yield of 71%. Additionally, the cascade was coupled to purified, transmembrane-deleted Alg1 (ALG1?TM), the first mannosyltransferase in the ER-associated lipid-linked oligosaccharide (LLO) assembly. Thereby, in a one-pot reaction, phytanyl-PP-(GlcNAc)2 -Man1 was produced with efficient nucleotide sugar regeneration for the first time. Phytanyl-PP-(GlcNAc)2 -Man1 can serve as a substrate for the synthesis of LLO for the cell-free in vitro glycosylation of proteins. A high-performance anion exchange chromatography method with UV and conductivity detection (HPAEC-UV/CD) assay was optimized and validated to determine the enzyme kinetics. The established kinetic model enabled the optimization of the GDP-mannose regenerating cascade and can further be used to study coupling of the GDP-mannose cascade with glycosyltransferases. Overall, the study envisages a first step towards the development of a platform for the cell-free production of LLOs as precursors for in vitro glycoengineering of proteins.

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Hierarchical and programmable one-pot synthesis of oligosaccharides.

Publications

Hierarchical and programmable one-pot synthesis of oligosaccharides.

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