Quantitative NMR for the structural analysis of novel bivalent glycoconjugates as vaccine candidates.

Novel unimolecular bivalent glycoconjugates were assembled combining several functionalized capsular polysaccharides of Streptococcus pneumoniae and Neisseria meningitidis to a carrier protein by using an effective strategy based on the Ugi 4-component reaction. The development of multivalent glycoconjugates opens new opportunities in the field of vaccine design, but their high structural complexity involves new analytical challenges. Nuclear Magnetic Resonance has found wide applications in the characterization and impurity profiling of carbohydrate-based vaccines. Eight bivalent conjugates were studied by quantitative NMR analyzing the structural identity, the content of each capsular polysaccharide, the ratios between polysaccharides, the polysaccharide to protein ratios and undesirable contaminants. The qNMR technique involves experiments with several modified parameters for obtaining spectra with quantifiable signals. In addition, the achieved NMR results were combined with the results of colorimetric assay and Size Exclusion HPLC for assessing the protein content and free protein percentage, respectively. The application of quantitative NMR showed to be efficient to clear up the new structural complexities while allowing the quantitative assessment of the components.

Isonitriles: Versatile Handles for the Bioorthogonal Functionalization of Proteins.

The property of the isonitrile group to enable the simultaneous α-addition of a strong electrophile and a nucleophile has always attracted the attention of organic chemists. Its versatility is augmented when recognizing that its high structural compactness, the inertia to most of the naturally occurring functional groups, and relatively prolonged physiological and metabolical stability, convert it into the smallest bioorthogonal group. The discovery and optimization of the isonitrile-tetrazine [4+1] cycloaddition as an alternative tool for the development of ligation and decaging strategies and the recently reported reaction of isonitriles with chlorooximes bring new opportunities for the utilization of this functional group in biological systems. Although several approaches have been reported for the synthesis of isonitrile-modified carbohydrates and polysaccharides, its incorporation in proteins has been barely explored. Besides compiling the reported methods for the assembly of isonitrile-modified proteins, this Mini-Review aims at calling attention to the real potential of this modification for protein ligation, decaging, immobilization, imaging, and many other applications at a low structural and functional cost.

Expanding the Scope of Ugi Multicomponent Bioconjugation to Produce Pneumococcal Multivalent Glycoconjugates as Vaccine Candidates.

Conjugate vaccines against encapsulated pathogens like Streptococcus pneumoniae face many challenges, including the existence of multiple serotypes with a diverse global distribution that constantly requires new formulations and higher coverage. Multivalency is usually achieved by combining capsular polysaccharide-protein conjugates from invasive serotypes, and for S. pneumoniae, this has evolved from 7- up to 20-valent vaccines. These glycoconjugate formulations often contain high concentrations of carrier proteins, which may negatively affect glycoconjugate immune response. This work broadens the scope of an efficient multicomponent strategy, leading to multivalent pneumococcal glycoconjugates assembled in a single synthetic operation. The bioconjugation method, based on the Ugi four-component reaction, enables the one-pot incorporation of two different polysaccharide antigens to a tetanus toxoid carrier, thus representing the fastest approach to achieve multivalency. The reported glycoconjugates incorporate three combinations of capsular polysaccharides 1, 6B, 14, and 18C from S. pneumoniae. The glycoconjugates were able to elicit functional specific antibodies against pneumococcal strains comparable to those shown by mixtures of the two monovalent glycoconjugates.

Multicomponent polysaccharide-protein bioconjugation in the development of antibacterial glycoconjugate vaccine candidates.

A new synthetic strategy for the development of multivalent antibacterial glycoconjugate vaccines is described. The approach comprises the utilization of an isocyanide-based multicomponent process for the conjugation of functionalized capsular polysaccharides of S. pneumoniae and S. Typhi to carrier proteins such as diphtheria and tetanus toxoids. For the first time, oxo- and carboxylic acid-functionalized polysaccharides could be either independently or simultaneously conjugated to immunogenic proteins by means of the Ugi-multicomponent reaction, thus leading to mono- or multivalent unimolecular glycoconjugates as vaccine candidates. Despite the high molecular weight of the two or three reacting biomolecules, the multicomponent bioconjugation proved highly efficient and reproducible. The Ugi-derived glycoconjugates showed notable antigenicity and elicited good titers of functional specific antibodies. To our knowledge, this is the only bioconjugation method that enables the incorporation of two different polysaccharidic antigens to a carrier protein in a single step. Applications in the field of self-adjuvanting, eventually anticancer, multicomponent vaccines are foreseeable.

Multicomponent Reactions in Ligation and Bioconjugation Chemistry.

Multicomponent reactions (MCRs) encompass an exciting class of chemical transformations that have proven success in almost all fields of synthetic organic chemistry. These convergent procedures incorporate three or more reactants into a final product in one pot, thus combining high levels of complexity and diversity generation with low synthetic cost. Striking applications of these processes are found in heterocycle, peptidomimetic, and natural product syntheses. However, their potential in the preparation of large macro- and biomolecular constructs has been realized just recently. This Account describes the most relevant results of our group in the utilization of MCRs for ligation/conjugation of biomolecules along with significant contributions from other laboratories that validate the utility of this special class of bioconjugation process. Thus, MCRs have proven to be efficient in the ligation of lipids to peptides and oligosaccharides as well as the ligation of steroids, carbohydrates, and fluorescent and affinity tags to peptides and proteins. In the field of glycolipids, we highlight the power of isocyanide-based MCRs with the one-pot double lipidation of glycan fragments functionalized as either the carboxylic acid or amine. In peptide chemistry, the versatility of the multicomponent ligation strategy is demonstrated in both solution-phase lipidation protocols and solid-phase procedures enabling the simultaneous lipidation and biotinylation of peptides. In addition, we show that MCRs are powerful methods for synchronized lipidation/labeling and macrocyclization of peptides, thus accomplishing in one step what usually requires long sequences. In the realm of protein bioconjugation, MCRs have also proven to be effective in labeling, site-selective modification, immobilization, and glycoconjugation processes. For example, we illustrate a successful application of multicomponent polysaccharide-protein conjugation with the preparation of multivalent glycoconjugate vaccine candidates by the ligation of two antigenic capsular polysaccharides of a pathogenic bacterium to carrier proteins. By highlighting the ability to join several biomolecules in only one synthetic operation, we hope to encourage the biomolecular chemistry community to apply this powerful chemistry to novel biomedicinal challenges.