and A. which three or more reactants are combined in one pot to render a product that incorporates atoms from all starting materials.1 Owing to their high chemical efficiency, diversity and complexity-generating character,2 these processes have been widely exploited in drug discovery3 and natural product synthesis. 4 Amazing applications of MCRs are also found in heterocyclic, 5 cyclic peptide6 and carbohydrate chemistry.7 However, MCRs have been rarely exploited in protein bioconjugation8,9 and, to our knowledge, they have never been employed in the development of glycoconjugate vaccines or in immunological applications. Among the MCRs, the Ugi four-component reaction stands out as one of the most powerful approaches to produce biologically relevant molecules.10 This procedure, which comprises the efficient condensation of a primary amine, a carboxylic acid, an oxo-compound and an isocyanide, has been previously used for the conjugation of oligo- and polysaccharides to other molecules.11 This reaction has been seldom employed in the covalent modification of proteins, such as bovine serum albumin (BSA), horseradish peroxidase (HRP) and bovine pancreatic trypsin,9 but not in such a way that several saccharidic components are simultaneously conjugated to a protein. As part of our effort to develop synthetic methods toward antibacterial conjugate vaccines,12 herein we report an innovative application of MCRs in the fields of glycobiology and bioconjugation. The strategy encompasses the utilization of the Ugi reaction as an effective method for the conjugation of either one or two bacterial capsular polysaccharides (CPs) to carrier proteins, thus leading to uni- or bivalent glycoconjugates. The scope of this multicomponent bioconjugation is usually assessed by employing oxo- and carboxylic acid-functionalized CPs of different bacteria or serotypes as well as tetanus toxoid (TT) and diphtheria toxoid (DT) as the amino components of the process, along with commercial isocyanides. VAL-083 This work represents the VAL-083 first endeavor toward the development of glycoconjugate vaccine candidates by means of MCRs. The immunogenic role of bacterial CPs and their importance in the development of prophylactic vaccines have been known since the beginning of the last century.13 Although the introduction of antibiotics discouraged the application of antibacterial carbohydrate-based vaccines, the emergence of resistance against antibiotics has led to a renaissance of this strategy.14 Owing to their T-cell independent response, carbohydrate-based vaccines provide only short term protection for most bacterial infections, being of limited value in high risk groups such as infants and immuno-compromised people.15 Proteins, on the other hand, are T-cell dependent antigens with a resulting long lasting immunization. The conjugation of CPs to carrier proteins is known to enhance the immunogenicity when compared with the natural polysaccharide.16 Several methods have been established for the synthesis of glycoproteins and glycoconjugate vaccines, including classical reductive amination, peptide coupling and maleimide-mediated conjugation,17 as well as new site-specific methods such as alkyneCazide dipolar cycloaddition18 and thioether formation,19 among others.20 Results and discussion An advantage of using the Ugi reaction in the conjugation step is that many of the Ugi-reactive functional groups are either already present in the protein (serotypes of global incidence, the CPs of serotypes 14 (CPs14), 9V (CPs9V) and 7F (CPs7F) were chosen at first and subjected to a controlled periodic oxidation,24 aimed at generating the carbonyl groups (1 carbonyl every 6 or 7 repetitive models) VAL-083 required for the Ugi reaction without altering their antigenic determinants (see the ESI?). TT and DT were selected as carrier proteins, as they have shown great efficacy in vaccination strategies worldwide.25 These proteins were produced in-house and then detoxified according to production procedures recommended by the World Health Organization (WHO).26 Both proteins were next subjected to hydrazide activation by the controlled reaction of hydrazine with aspartic and glutamic acid side chains (see the ESI?), aimed at introducing more reactive amino groups. As oxo-functionalized CPs have VAL-083 never been conjugated to such large proteins by means of isocyanide-MCRs, we initially focused on assessing the scope of the multicomponent conjugation protocol and the immunogenicity of the Ugi-derived glycoconjugates. The conjugation of the oxo-functionalized CPs Rabbit Polyclonal to STAT5A/B of the three serotypes (Scheme 1A, shown as putative generic structures) was first carried out for non-activated DT and TT. Acetic acid and to (A) non-activated DT and TT and (B) hydrazide-activated DT and TT. (C) Repetitive units of the CPs of serotypes 14, 7F and 9V,.