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Herein, different conjugation methods were investigated with the aim of controlling glycosylation site and glycosylation density on the carrier protein. <jats:italic>S.<\/jats:italic> Typhimurium lipopolysaccharide O-Antigen and CRM<jats:sub>197<\/jats:sub> carrier protein were used as models. In particular, thiol and click chemistry were examined, both involving the linkage of the terminal reducing sugar unit of the O-Antigen chain to different amino acids on the carrier protein. Thiol chemistry allowed O-Antigen conjugation only when the carrier protein was activated on the lysines and with a relative high number of linkers, while click chemistry allowed conjugate generation even when just one position on the protein was activated and to both lysine and tyrosine sites. The study highlights click chemistry as a leading approach for the synthesis of well-defined glycoconjugates, useful to investigate the relationship between conjugate design and immune response.<\/jats:p>","DOI":"10.1007\/s10719-020-09930-2","type":"journal-article","created":{"date-parts":[[2020,6,13]],"date-time":"2020-06-13T07:02:35Z","timestamp":1592031755000},"page":"611-622","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Click chemistry compared to thiol chemistry for the synthesis of site-selective glycoconjugate vaccines using CRM197 as carrier protein"],"prefix":"10.1007","volume":"37","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-2796-4817","authenticated-orcid":false,"given":"G.","family":"Stefanetti","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"M.","family":"Allan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A.","family":"Usera","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"F.","family":"Micoli","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,6,13]]},"reference":[{"key":"9930_CR1","doi-asserted-by":"publisher","first-page":"107","DOI":"10.1146\/annurev-immunol-030409-101159","volume":"28","author":"FY Avci","year":"2010","unstructured":"Avci, F.Y., Kasper, D.L.: How bacterial carbohydrates influence the adaptive immune system. 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