Modification and application of glycosidases to create homogeneous glycoconjugates
<p>In the post-genomic era, recognition of the importance of sugars is increasing in biological research. For the precise analysis of their functions, homogeneous materials are required. Chemical synthesis is a powerful tool for preparation of homogeneous oligosaccharides and glycoconjugates....
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| Médium: | Diplomová práce |
| Jazyk: | English |
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2013
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| Témata: |
| _version_ | 1826273929992339456 |
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| author | Yamamoto, K Yamamoto, K |
| author2 | Davis, BG |
| author_facet | Davis, BG Yamamoto, K Yamamoto, K |
| author_sort | Yamamoto, K |
| collection | OXFORD |
| description | <p>In the post-genomic era, recognition of the importance of sugars is increasing in biological research. For the precise analysis of their functions, homogeneous materials are required. Chemical synthesis is a powerful tool for preparation of homogeneous oligosaccharides and glycoconjugates. Glycosidases are potent catalysts for this purpose because they realize high stereo- and regio- selectivities under conditions benign to biomolecules without repetitive protection/deprotection procedures.</p> <p>A glycosynthase is an aritificial enzyme which is derived from a glycosidase and is devised for glycosylation reaction. To suppress the mechanistically inherent oligomerization side reaction of this class of biocatalysts, a glycosidase with plastic substrate recognition was engineered to afford the first α-mannosynthase. This novel biocatalyst showed low occurrence of oligomerized products as designed and was applied to prepare a wide range of oligosaccharides.</p> <p>Glycosidases are also valuable tools for glycan engineering of glycoconjugates, which is a pivotal issue in the development of pharmaceutical agents, including immunoglobulin G (IgG)-based drugs. EndoS, an endo-β-N-acetylglucosaminidase from Streptococcus pyogenes, natively cleaves N-glycans on IgG specifically. When the latent glycosylation activity of this enzyme was applied, the N-glycan remodelling of full-length IgG was successfully achieved for the first time and a highly pure glycoform was obtained using the chemically synthesized oxazoline tetrasaccharide as glycosyl donor. This biocatalytic reaction allows development of a novel type of antibody-drug conjugates (ADCs) in which drug molecules are linked to N-glycans site-specifically. For this purpose, glycans with bioorthogonal reaction handles were synthesized and conjugated to IgG. A model reaction using a dye compound as reaction partner worked successfully and the synthetic method for this newly designed ADC was validated.</p> <p>Glycan trimming of glycoproteins expressed from Pichia pastoris was performed using exoglycosidases to derive homogeneous glycoform. Jack Bean α-mannosidase (JBM) trimmed native N-glycans down to the core trisaccharide structure but some of the glycoforms were discovered to be resistant to the JBM activity. Enzymatic analyses using exoglycosidases suggested that the JBM-resistant factor was likely to be β-mannoside. In summary, this work advanced application of modified glycosidases for preparation of oligosaccharides and also demonstrated biocatalytic utility of glycosidases to produce biologically relevant glycoconjugates with homogeneous glycoforms.</p> |
| first_indexed | 2024-03-06T22:35:42Z |
| format | Thesis |
| id | oxford-uuid:59d1917c-345d-4fe3-ace4-67dd3c8bc017 |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-06T22:35:42Z |
| publishDate | 2013 |
| record_format | dspace |
| spelling | oxford-uuid:59d1917c-345d-4fe3-ace4-67dd3c8bc0172022-03-26T17:11:59ZModification and application of glycosidases to create homogeneous glycoconjugatesThesishttp://purl.org/coar/resource_type/c_db06uuid:59d1917c-345d-4fe3-ace4-67dd3c8bc017Organic chemistryGlycobiologyChemical biologyBiomimetic synthesisProtein chemistryEnzymesEnglishOxford University Research Archive - Valet2013Yamamoto, KYamamoto, KDavis, BG<p>In the post-genomic era, recognition of the importance of sugars is increasing in biological research. For the precise analysis of their functions, homogeneous materials are required. Chemical synthesis is a powerful tool for preparation of homogeneous oligosaccharides and glycoconjugates. Glycosidases are potent catalysts for this purpose because they realize high stereo- and regio- selectivities under conditions benign to biomolecules without repetitive protection/deprotection procedures.</p> <p>A glycosynthase is an aritificial enzyme which is derived from a glycosidase and is devised for glycosylation reaction. To suppress the mechanistically inherent oligomerization side reaction of this class of biocatalysts, a glycosidase with plastic substrate recognition was engineered to afford the first α-mannosynthase. This novel biocatalyst showed low occurrence of oligomerized products as designed and was applied to prepare a wide range of oligosaccharides.</p> <p>Glycosidases are also valuable tools for glycan engineering of glycoconjugates, which is a pivotal issue in the development of pharmaceutical agents, including immunoglobulin G (IgG)-based drugs. EndoS, an endo-β-N-acetylglucosaminidase from Streptococcus pyogenes, natively cleaves N-glycans on IgG specifically. When the latent glycosylation activity of this enzyme was applied, the N-glycan remodelling of full-length IgG was successfully achieved for the first time and a highly pure glycoform was obtained using the chemically synthesized oxazoline tetrasaccharide as glycosyl donor. This biocatalytic reaction allows development of a novel type of antibody-drug conjugates (ADCs) in which drug molecules are linked to N-glycans site-specifically. For this purpose, glycans with bioorthogonal reaction handles were synthesized and conjugated to IgG. A model reaction using a dye compound as reaction partner worked successfully and the synthetic method for this newly designed ADC was validated.</p> <p>Glycan trimming of glycoproteins expressed from Pichia pastoris was performed using exoglycosidases to derive homogeneous glycoform. Jack Bean α-mannosidase (JBM) trimmed native N-glycans down to the core trisaccharide structure but some of the glycoforms were discovered to be resistant to the JBM activity. Enzymatic analyses using exoglycosidases suggested that the JBM-resistant factor was likely to be β-mannoside. In summary, this work advanced application of modified glycosidases for preparation of oligosaccharides and also demonstrated biocatalytic utility of glycosidases to produce biologically relevant glycoconjugates with homogeneous glycoforms.</p> |
| spellingShingle | Organic chemistry Glycobiology Chemical biology Biomimetic synthesis Protein chemistry Enzymes Yamamoto, K Yamamoto, K Modification and application of glycosidases to create homogeneous glycoconjugates |
| title | Modification and application of glycosidases to create homogeneous glycoconjugates |
| title_full | Modification and application of glycosidases to create homogeneous glycoconjugates |
| title_fullStr | Modification and application of glycosidases to create homogeneous glycoconjugates |
| title_full_unstemmed | Modification and application of glycosidases to create homogeneous glycoconjugates |
| title_short | Modification and application of glycosidases to create homogeneous glycoconjugates |
| title_sort | modification and application of glycosidases to create homogeneous glycoconjugates |
| topic | Organic chemistry Glycobiology Chemical biology Biomimetic synthesis Protein chemistry Enzymes |
| work_keys_str_mv | AT yamamotok modificationandapplicationofglycosidasestocreatehomogeneousglycoconjugates AT yamamotok modificationandapplicationofglycosidasestocreatehomogeneousglycoconjugates |