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We have developed a means of presenting relatively small glycans in a context to make them T cell-dependent antigens. This approach requires synthesis of glycans that remain close to carrier proteins upon conjugation, allowing T cell recognition and generation of B cells that produce high-affinity antibodies and memory toward target pathogens. In this work, we describe the syntheses of three disaccharides of the capsular polysaccharides from serotypes 4, 7F and 9V Streptococcus pneumoniae (Sp) as propargyl glycosides for use in this vaccine strategy. While variations of these disaccharides have previously been synthesized, none have been synthesized with an alkyne as the linker. Incorporation of the alkyne led to redesign of the synthesis of each disaccharide. All three disaccharides were synthesized without use of benzyl protective groups, one of the most used carbohydrate protective groups due to its stability and ease of removal via hydrogenation. We report challenges that arose while synthesizing the targets with less common protective groups, such as naphthyl ethers, and the methods used to successfully overcome these challenges. [ABSTRACT FROM AUTHOR]

Vaccines targeting glycan structures at the surface of pathogenic microbes must overcome the inherent T cell-independent nature of immune responses against glycans. Carbohydrate conjugate vaccines achieve this by coupling bacterial polysaccharides to a carrier protein that recruits heterologous CD4 T cells to help B cell maturation. Yet they most often produce low- to medium-affinity immune responses of limited duration in immunologically fit individuals and disappointing results in the elderly and immunocompromised patients. Here, we hypothesized that these limitations result from suboptimal T cell help. To produce the next generation of more efficacious conjugate vaccines, we have explored a synthetic design aimed at focusing both B cell and T cell recognition to a single short glycan displayed at the surface of a virus-like particle. We tested and established the proof of concept of this approach for 2 serotypes of Streptococcus pneumoniae. In both cases, these vaccines elicited serotype-specific, protective, and long-lasting IgG antibodies of nanomolar affinity against the target glycans in mice. We further identified a requirement for CD4 T cells in the anti-glycan antibody response. Our findings establish the design principles for improved glycan conjugate vaccines. We surmise that the same approach can be used for any microbial glycan of interest., Introduction Over the last 150 years, efficient control of the most dangerous infectious diseases has been achieved through a combination of higher hygiene and the widespread use of vaccines. At [...]

Antibody 2G12 uniquely neutralizes a broad range of HIV-1 isolates by binding the high-mannose glycans on the HIV-1 surface glycoprotein, gp120. Antigens that resemble these natural epitopes of 2G12 would be highly desirable components for an HIV-1 vaccine. However, host-produced (self)-carbohydrate motifs have been unsuccessful so far at eliciting 2G12-like antibodies that cross-react with gp120. Based on the surprising observation that 2G12 binds nonproteinaceous monosaccharide D-fructose with higher affinity than D-mannose, we show here that a designed set of nonself, synthetic monosaccharides are potent antigens. When introduced to the terminus of the D1 arm of protein glycans recognized by 2G12, their antigenicity is significantly enhanced. Logical variation of these unnatural sugars pinpointed key modifications, and the molecular basis of this increased antigenicity was elucidated using high-resolution crystallographic analyses. Virus-like particle protein conjugates containing such nonself glycans are bound more tightly by 2G12. As immunogens they elicit higher titers of antibodies than those immunogenic conjugates containing the self D1 glycan motif. These antibodies generated from nonself immunogens also crossreact with this self motif, which is found in the glycan shield, when it is presented in a range of different conjugates and glycans. However, these antibodies did not bind this glycan motif when present on gp120. 2G12 | gp120 | oligosaccharide | glycoconjugate | glycomimetic doi/ 10.1073/pnas.1002717107

Carbohydrate--protein binding is important to many areas of biochemistry. Here, backscattering interferometry (BSI) has been shown to be a convenient and sensitive method for obtaining quantitative information about the strengths and selectivities of such interactions. The surfaces of glass microfluidic channels were covalently modified with extravidin, to which biotinylated lectins were subsequently attached by incubation and washing. The binding of unmodified carbohydrates to the resulting avidin-immobilized lectins was monitored by BSI. Dose--response curves that were generated within several minutes and were highly reproducible in multiple wash/measure cycles provided adsorption coefficients that showed mannose to bind to concanavalin A (conA) with 3.7 times greater affinity than glucose consistent with literature values. Galactose was observed to bind selectively and with similar affinity to the lectin BS-1. The avidities of polyvalent sugar-coated virus particles for immobilized conA were much higher than monovalent glycans, with increases of 60-200 fold per glycan when arrayed on the exterior surface of cowpea mosaic virus or bacteriophage O[beta]. Sugar-functionalized PAMAM dendrimers showed size-dependent adsorption, which was consistent with the expected density of lectins on the surface. The sensitivity of BSI matches or exceeds that of surface plasmon resonance and quartz crystal microbalance techniques, and is sensitive to the number of binding events, rather than changes in mass. The operational simplicity and generality of BSI, along with the near-native conditions under which the target binding proteins are immobilized, make BSI an attractive method for the quantitative characterization of the binding functions of lectins and other proteins.

The plasma clearance and tissue distribution in mice of the versatile capsid of bacteriophage Q[beta] are determined by decorating the particles with gadolinium complexes using the [Cu.sup.I]-mediated azide-alkyne cycloaddition reaction. The results have shown that nanoparticle trafficking is designed in predictable ways by chemical and genetic modifications that modulate surface charge.

Heteroaryldihydropyrimidines (HAPs) are a new class of antivirals inhibiting production of hepatitis B virus (HBV) virions in tissue culture. Here, we examine the effect of a representative HAP molecule, methyl 4-(2-chloro-4-fluorophenyl)-6-methyl-2-(pyridin-2-yl)-l, 4-dihydropyrimidine-5-carboxylate (HAP-1), on the in vitro assembly of HBV capsid protein (Cp). HAP-1 enhances the rate and extent of Cp assembly over a broad concentration range. Aberrant particles, dominated by hexagonal arrays of Cp, were observed from assembly reactions with high HAP-1 concentrations. HAP-1 also led to dissociation of metastable HBV capsids, overcoming a kinetic barrier to dissociation by scavenging Cp and redirecting its assembly into hexamer-rich structures. Thus, HAP drugs act as allosteric effectors that induce an assembly-active state and, at high concentration, preferentially stabilize noncapsid polymers of Cp. HAP compounds may have multiple effects in vivo stemming from inappropriate assembly of Cp. These results show that activating and deregulating virus assembly may be a powerful general approach for antiviral therapeutics. antiviral | virus assembly | protein polymerization

Sulfur mustards react with Grignard reagents with neighboring group participation of the sulfur atom along with proper activation of the leaving group. 2,6-Dichloro-9-thiabicyclo[3.3.1]nonane provides clean reactivity with organomagnesium nucleophiles on a topologically constrained scaffold.

Silylation chemistry on porous silicon provides for ultrahigh sensitivity and analyte specificity with desorption/ ionization on silicon mass spectrometry (DIOS-MS) analysis. Here, we report that the silylation of oxidized porous silicon offers a DIOS platform that is resistant to air oxidation and acid/base hydrolysis. Furthermore, surface modification with appropriate hydrophobic silanes allows analytes to absorb to the surface via hydrophobic interactions for direct analyte extraction from complex matrixes containing salts and other nonvolatile interferences present in the sample matrix. This enables rapid cleanup by simply spotting the sample onto the modified DIOS target and removing the liquid phase containing the interferences. This approach is demonstrated in the analysis of protein digests and metabolites in biofluids, as well as for the characterizing of inhibitors from their enzyme complex. An unprecedented detection limit of 480 molecules (800 ymol) for des-[Arg.sup.9]-bradykinin is reported on a pentafluorophenyl-functionalized DIOS chip.

Synthesis and reactivity of 2,6-dichloro-9-thiabicyclo[3.3.1]nonane are discussed. Data reveal that the compound is highly reactive and possesses stereochemical control mediated by the sulfur center.

Titanium and zirconium Et2C2B4H4 metal phosphine compounds were synthesized and characterized, and their ethylene polymerization activity was investigated.

The one-pot conversion of aromatic aldehydes to allenes in the presence of titanium-substituted ylides involved three reaction processes. The formation of allenes was characterized by the elimination of titanium oxides to form vinylphosphonium salts which produced allenic phosphorane via aldehyde-induced deprotonation and condensation. The ylide species involved in the one-pot synthetic process were metal complexes with different titanium ligand substitutions.

The reaction of titanium-substituted ylide species with carbony compounds led to the formation of vinylphosphonium salts. The stepwise coupling procedure involved a double olefination and a single reaction using boron-substituted phosphorus ylide. The resulting vinylphosphonium salts undergo deprotonation and condensation reactions with an aldehyde to produce 1,3-disubstituted allenes. Furthermore, irradiation of the vinylphosphonium salts induced reversible isomerization forming Z-vinylphosphonium isomers.

Titanium(IV)-substituted ylide reagent and bis(trimethylsilyl)amide bases were utilized for the one-pot double deoxygenation of simple alkyl- and polyether-tethered aromatic dialdehydes. The double olefination methodology involved the conversion of dialdehyde to vinylphosphonium salt. An allenic phosphorane unit of (Me2N)3P=C(H)TiCl(OiPr)2 was also formed by the deprotonation of vinylphosphonium with an excess base. Furthermore, the success of the cyclization process was dependent on the slow addition of phosphorus ylide to intramolecular aldehyde in dilute solution.

The results of kinetic trapping studies on pentaammineosmium(II) in water were reported. It was demonstrated that an active source of (NH3)5Os(super II) other than the aquo complex is formed in aqueous solution and decomposes by a first-order process with a half-life of hours at room temperature. The rate of the decomposition process increases at lower pH and is greater in H2O than in D2O.