Glycoproteins play a pivotal role in bacterial and viral infections as well as cancer metastasis. The synthesis of glycoconjugates is hampered by the complexity of the chemical strategies required for the preparation of the saccharide moiety, and consequently the development of new drug candidates is strongly limited. Due to the structural complexity and the arduous extraction from natural sources, the synthesis of pure oligosaccharides is a paramount challenge in glycobiology. In this thesis, an efficient and scalable method based on an integrated approach between chemistry and biotechnology for the synthesis of oligosaccharides has been developed. In particular, by means of a chemo-biocatalytic approach, different mannose-based glycans activated with 2-iminomethoxyethyl reactive group were synthesized in good yields. With a convergent synthetic strategy, mono- disaccharides were used in coupling reactions with ε-amino groups of lysine residues in highly immunogenic proteins over-expressed by Mycobacterium tuberculosis (Mtb). The different neoglycoproteins prepared have been assayed through ex vivo tests as potential vaccines active against Mtb. The glycosylation of immunogenic proteins results either in a co-adjuvant effect, or in an additional stimulation of humoral response over the T-cell mediated one.
Le glicoproteine ricoprono un ruolo chiave nelle infezioni batteriche e virali, così come nelle infezioni tumorali. La sintesi e lo sviluppo di nuovi farmaci a struttura complessa, come gli oligosaccaridi e i loro derivati glicoconiugati, è fortemente condizionata dalla mancanza di strategie chimiche semplici ed efficienti. Ciò si traduce nella scarsa disponibilità di nuovi candidati farmaci. Vista la complessità strutturale e le difficoltà di estrazione da fonti naturali, la sintesi di oligosaccaridi risulta la principale sfida in glicobiologia. In questa tesi, è stato sviluppato un metodo di sintesi efficiente e facilmente “scalabile” basato su un approccio integrato tra chimica e biotecnologie. In particolare, mediante un approccio chemoenzimatico, sono stati sintetizzati con buone rese differenti glicani derivati del mannosio attivati con il gruppo 2-imminometossietile. Mono- e disaccaridi precedentemente preparati sono stati usati nella reazione di coupling con i gruppi ε-amminici di lisine di proteine immunogeniche espresse dal Mycobacterium tuberculosis. I diversi glicoconiugati sono stati saggiati come potenziali vaccini antitubercolari mediante test ex vivo. La glicosilazione di proteine immunogeniche co-adiuva e rafforza la risposta immunitaria umorale mediata da cellule T.
SINTESI CHEMOENZIMATICA DI NEOGLICOPROTEINE CON PROPRIETA’ ANTIGENICA CONTRO LA TUBERCOLOSI
LEGNAZZI, FEDERICA
2013/2014
Abstract
Glycoproteins play a pivotal role in bacterial and viral infections as well as cancer metastasis. The synthesis of glycoconjugates is hampered by the complexity of the chemical strategies required for the preparation of the saccharide moiety, and consequently the development of new drug candidates is strongly limited. Due to the structural complexity and the arduous extraction from natural sources, the synthesis of pure oligosaccharides is a paramount challenge in glycobiology. In this thesis, an efficient and scalable method based on an integrated approach between chemistry and biotechnology for the synthesis of oligosaccharides has been developed. In particular, by means of a chemo-biocatalytic approach, different mannose-based glycans activated with 2-iminomethoxyethyl reactive group were synthesized in good yields. With a convergent synthetic strategy, mono- disaccharides were used in coupling reactions with ε-amino groups of lysine residues in highly immunogenic proteins over-expressed by Mycobacterium tuberculosis (Mtb). The different neoglycoproteins prepared have been assayed through ex vivo tests as potential vaccines active against Mtb. The glycosylation of immunogenic proteins results either in a co-adjuvant effect, or in an additional stimulation of humoral response over the T-cell mediated one.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/21429