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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.

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.

Peptide stapling is traditionally used to lock peptide conformations into α-helical structures using a variety of macrocyclization chemistries. In an endeavor to add a diversity-generating tool to this repertoire, we introduce a multicomponent stapling approach enabling the simultaneous stabilization of helical secondary structures and the exocyclic N-functionalization of the side chain-tethering lactam bridge. This is accomplished by means of a novel solid-phase methodology comprising, for the first time, the on-resin Ugi reaction-based macrocyclization of peptide side chains bearing amino and carboxylic acid groups. The exocyclic diversity elements arise from the isocyanide component used in the Ugi multicomponent stapling protocol, which allows for the incorporation of relevant fragments such as lipids, sugars, polyethylene glycol, fluorescent labels, and reactive handles. We prove the utility of such exocyclic reactive groups in the bioconjugation of a maleimide-armed lactam-bridged peptide to a carrier protein. The on-resin multicomponent stapling proved efficient for the installation of not only one, but also two consecutive lactam bridges having either identical or dissimilar N-functionalities. The easy access to helical peptides with a diverse set of exocyclic functionalities shows prospect for applications in peptide drug discovery and chemical biology.

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.

Plasmodium falciparum neutral metallo-aminopeptidase (PfAM1), a member of the M1 family of metallo proteases, is a promising target for malaria, a devastating human parasitic disease. We report the high-level expression of PfAM1 in Escherichia coli BL21. An optimized gene, with a codon adaptation index and an average G/C content higher than the native gene, was synthesized and cloned in the pTrcHis2B vector. Optimal expression was achieved by induction with 1 mM IPTG at 37 °C for 18 h. This allowed obtaining 100 mg of recombinant PfAM1 (rPfAM1) per L of culture medium; 19% of the E. coli soluble protein mass was from rPFAM1. rPfAM1, fused to an amino-terminal 6×His tag, was purified in a single step by immobilized metal ion affinity chromatography. The protein showed only limited signs of proteolytic degradation, and this step increased purity 27-fold. The kinetic characteristics of rPfAM1, such as a neutral optimal pH, a preference for substrates with basic or hydrophobic amino acids at the P1 position, an inhibition profile typical of metallo-aminopeptidases, and inhibition from Zn2+ excess, were similar to those of the native PfAM1. We have thus optimized an expression system that should be useful for identifying new PfAM1 inhibitors.

Malaria is a global human parasitic disease mainly caused by the protozoon Plasmodium falciparum. Increased parasite resistance to current drugs determines the relevance of finding new treatments against new targets. A novel target is the M1 alanyl-aminopeptidase from P. falciparum (PfA-M1), which is essential for parasite development in human erythrocytes and is inhibited by the pseudo-peptide bestatin. In this work, we used a combinatorial multicomponent approach to produce a library of peptidomimetics and screened it for the inhibition of recombinant PfA-M1 (rPfA-M1) and the in vitro growth of P. falciparum erythrocytic stages (3D7 and FcB1 strains). Dose-response studies with selected compounds allowed identifying the bestatin-based peptidomimetic KBE009 as a submicromolar rPfA-M1 inhibitor (Ki=0.4μM) and an in vitro antimalarial compound as potent as bestatin (IC50=18μM; without promoting erythrocyte lysis). At therapeutic-relevant concentrations, KBE009 is selective for rPfA-M1 over porcine APN (a model of these enzymes from mammals), and is not cytotoxic against HUVEC cells. Docking simulations indicate that this compound binds PfA-M1 without Zn2+ coordination, establishing mainly hydrophobic interactions and showing a remarkable shape complementarity with the active site of the enzyme. Moreover, KBE009 inhibits the M1-type aminopeptidase activity (Ala-7-amido-4-methylcoumarin substrate) in isolated live parasites with a potency similar to that of the antimalarial activity (IC50=82μM), strongly suggesting that the antimalarial effect is directly related to the inhibition of the endogenous PfA-M1. These results support the value of this multicomponent strategy to identify PfA-M1 inhibitors, and make KBE009 a promising hit for drug development against malaria.

The development of selective inhibitors of microbial metallo-aminopeptidases is an important goal in the pursuit of antimicrobials for therapeutic applications. Herein, we disclose a combinatorial approach relying on two Ugi reactions for the generation of peptidomimetics inspired by natural metallo-aminopeptidase inhibitors. The library was screened for inhibitory activity against the neutral metallo-aminopeptidase of Escherichia coli (ePepN) and the porcine kidney cortex metallo-aminopeptidase (pAPN), which was used as a model of the M1-aminopeptidases of mammals. Six compounds showed typical dose-response inhibition profiles toward recombinant ePepN, with two of them being very potent and highly selective for ePepN over pAPN. Another compound showed moderate ePepN inhibition but total selectivity for this bacterial enzyme over its mammalian orthologue at concentrations of physiological relevance. This strategy proved to be useful for the identification of lead compounds for further optimization and development.