title: Tyrosine-directed conjugation of large glycans to proteins via copper-free click chemistry.
abstract: We have demonstrated that the insertion of alkyne-containing bifunctional linkers into the tyrosine residues of the carrier protein, followed by the copper mediated azide-alkyne [3 + 2] cycloaddition of carbohydrates, is a robust approach for the preparation of glycoconjugates with defined glycans, carrier, and connectivity. Conjugation of Group B Streptococcus (GBS) capsular polysaccharides to streptococcal pilus protein could extend the vaccine coverage to a variety of strains. Application of our protocol to these large charged polysaccharides occurred at low yields. Herein we developed a tyrosine-directed conjugation approach based on the copper-free click chemistry of sugars modified with cyclooctynes, which enables efficient condensation of synthetic carbohydrates. Most importantly, this strategy was demonstrated to be more effective than the corresponding copper catalyzed reaction for the insertion of GBS onto the tyrosine residues of GBS pilus proteins, previously selected as vaccine antigens through the so-called reverse vaccinology. Integrity of protein epitopes in the modified proteins was ascertained by competitive ELISA, and conjugation of polysaccharide to protein was confirmed by SDS page electrophoresis and immunoblot assays. The amount of conjugated polysaccharide was estimated by high-performance anion-exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD). The described technology is particularly suitable for proteins used with the dual role of vaccine antigen and carrier for the carbohydrate haptens.
authors.affiliation: Novartis Vaccines & Diagnostics , Via Fiorentina 1, 53100 Siena, Italy.
authors.forename: Alberto
authors.initials: A
authors.lastname: Nilo
authors.forename: Martin
authors.initials: M
authors.lastname: Allan
authors.forename: Barbara
authors.initials: B
authors.lastname: Brogioni
authors.forename: Daniela
authors.initials: D
authors.lastname: Proietti
authors.forename: Vittorio
authors.initials: V
authors.lastname: Cattaneo
authors.forename: Stefano
authors.initials: S
authors.lastname: Crotti
authors.forename: Samantha
authors.initials: S
authors.lastname: Sokup
authors.forename: Huili
authors.initials: H
authors.lastname: Zhai
authors.forename: Immaculada
authors.initials: I
authors.lastname: Margarit
authors.forename: Francesco
authors.initials: F
authors.lastname: Berti
authors.forename: Qi-Ying
authors.initials: QY
authors.lastname: Hu
authors.forename: Roberto
authors.initials: R
authors.lastname: Adamo
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry; D002240:Carbohydrate Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods; D057930:Click Chemistry / Q000379:methods*; D003300:Copper / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000737:chemistry / Q000276:immunology; D006001:Glycoconjugates / Q000138:chemical synthesis*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry* / Q000276:immunology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013292:Streptococcus agalactiae / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D000939:Epitopes; D006001:Glycoconjugates; D011134:Polysaccharides; D011506:Proteins; D014443:Tyrosine; D003300:Copper