Optimization of labile esters for esterase-assisted accumulation of nitroxides into cells: a model for in vivo EPR imaging.

Nitroxide-based electron paramagnetic resonance (EPR) imaging agents are useful quantitative probes of O2 concentration in vivo in real time. Lipophilic, labile alkanoyloxymethyl esters of nitroxides can cross the blood-brain barrier, and after hydrolysis, the corresponding anionic nitroxide is intracellularly entrapped at levels sufficient to permit O2 measurements. The utility of nitroxides as EPR imaging agents depends critically on their ability to accumulate in the brain to high levels. In this study, we systematically investigated the relationship between the structure of the alkanoyl moiety and the ability of the corresponding labile ester to deliver nitroxide intracellularly. We demonstrate, in a cultured cell model, that for nitroxide labile esters with unbranched alkanoyl chains, increasing the chain length improves intracellular loading. Moreover, by studying an isomeric series of labile esters, we conclude that branching of the alkanoyl chain drastically reduces intracellular loading. These structural insights improve our general ability to use labile esters to deliver carboxylates intracellularly, and suggest a strategy for enhancing delivery of nitroxide imaging agents across the blood-brain barrier in a living animal.

Toward a carbohydrate-based HIV-1 vaccine: synthesis and immunological studies of oligomannose-containing glycoconjugates.

Human antibody 2G12 is a broadly neutralizing antibody that exerts its anti-HIV activity by targeting a novel oligomannose cluster on HIV-1 gp120. It was previously demonstrated that synthetic oligomannose clusters could mimic the carbohydrate epitope of 2G12 and showed enhanced antigenicity (Wang L. X. et al. (2004) Chem.Biol. 11, 127). This paper describes the synthesis of oligomannose-containing glycoconjugates that include either a carrier protein or a universal T-helper epitope peptide to provide an effective immunogen. It was shown that the synthetic neoglycoconjugates containing oligomannose clusters could be recognized by the human antibody 2G12. Rabbit immunization studies revealed that only a small fraction of antibodies raised by the glycoconjugates was directed to the carbohydrate antigens, with the majority of the IgG type antibodies being directed to the linkers in the conjugates. The anti-sera showed weak cross-reactivity to HIV-1 gp120.

Chemoenzymatic synthesis of HIV-1 gp41 glycopeptides: effects of glycosylation on the anti-HIV activity and alpha-helix bundle-forming ability of peptide C34.

C34 is a 34-mer peptide derived from the C-terminal ectodomain of HIV-1 envelope glycoprotein, gp41. The C34 region in native gp41 carries a conserved N-glycan at Asn637 and the sequence is directly involved in the virus-host membrane fusion, an essential step for HIV-1 infection. This paper describes the synthesis of glycoforms of C34 which carry a monosaccharide, a disaccharide, and a native oligosaccharide moiety. The synthesis of the glycopeptide which carries a native high-mannose type N-glycan was achieved by a chemoenzymatic approach by using an endoglycosidase-catalyzed oligosaccharide transfer as the key step. The effects of glycosylation on the inhibitory activity and the helix-bundle forming ability of C34 were investigated. It was found that glycosylation moderately decreases the anti-HIV activity of C34 and, in comparison with C34, glyco-C34 forms less compact six-helix bundles with the corresponding N-terminal peptide, N36. This study suggests that conserved glycosylation modulates the anti-HIV activity and conformations of the gp41 C-peptide, C34.

Synthesis, conformation, and immunogenicity of monosaccharide-centered multivalent HIV-1 gp41 peptides containing the sequence of DP178.

Several monosaccharide-centered multivalent HIV-1 gp41 peptides containing the sequence of DP178 were synthesized. Conformational studies showed that multivalent assembly enhanced the alpha-helical content of the peptide. Therefore, 2-, 3-, or 4-alpha-helix bundles of peptide DP178 could be obtained by assembling the peptide on a suitable bi-, tri-, or tetravalent template. Immunization studies indicated that while peptide DP178 alone was poorly immunogenic, the tetravalent peptide MVP-1 raised high titers of antibodies in mice that recognize not only peptide DP178 but also the native HIV-1 glycoprotein gp41, even in the absence of a carrier protein or adjuvant. The study suggests that carbohydrate-centered multivalent peptides provide not only a model for mimicking protein alpha-helix-bundle structure, but also an effective immunogen for raising high-titer antibodies against HIV-1 envelope glycoprotein gp41.

Binding of high-mannose-type oligosaccharides and synthetic oligomannose clusters to human antibody 2G12: implications for HIV-1 vaccine design.

Human antibody 2G12 broadly neutralizes human immunodeficiency virus type 1 (HIV-1) isolates and shows protective activity against viral challenge in animal models. Previous mutational analysis suggested that 2G12 recognized a novel cluster of high-mannose type oligosaccharides on HIV-1 gp120. To explore the carbohydrate antigen for HIV-1 vaccine design, we have studied the binding of 2G12 to an array of HIV-1 high-mannose type oligosaccharides by competitive ELISAs and found that Man9GlcNAc is 210- and 74-fold more effective than Man5GlcNAc and Man6GlcNAc in binding to 2G12. The results establish that the larger high-mannose oligosaccharide on HIV-1 is the favorable subunit for 2G12 recognition. To mimic the putative epitope of 2G12, we have created scaffold-based multivalent Man9 clusters and found that the galactose-scaffolded bi-, tri-, and tetra-valent Man9 clusters are 7-, 22-, and 73-fold more effective in binding to 2G12 than the monomeric Man9GlcNAc2Asn. The experimental data shed light on further structural optimization of epitope mimics for developing a carbohydrate-based HIV-1 vaccine.

Synthesis and anti-HIV-1 activity of 4-[4-(4,6-bisphenylamino-triazin-2-ylamino)-5-methoxy-2-methylphenylazo]-5-hydroxynaphthalene-2,7-disulfonic acid and its derivatives.

A structure-based design approach has been used to optimize a lead HIV-1 entry inhibitor targeted to the envelope glycoprotein gp41. The docking study on this lead compound revealed important structural requirements that need to be preserved as well as structural non-requirements that could be eliminated to substantially reduce the molecular size of the lead compound. Based on the results from docking study, a limited number of analogues were designed and synthesized. This approach yielded a new analogue (compound 4) that retained the anti-HIV-1 activity with reduced molecular size approaching towards more drug-like character.

Chemoenzymatic synthesis of high-mannose type HIV-1 gp120 glycopeptides.

A chemoenzymatic approach to the synthesis of glycoforms of HIV-1 gp120 glycopeptides is described. Thus, the high-mannose type glycopeptides [gp120 (336-342)] containing Man(9), Man(6) and Man(5) moieties, respectively, were synthesized in satisfactory yields via transglycosylation to the acetylglucosaminyl peptide, using the recombinant Arthrobacter Endo-beta-N-acetyl-glucosaminidase (Endo-A) as the key enzyme.

Synthesis of maleimide-activated carbohydrates as chemoselective tags for site-specific glycosylation of peptides and proteins.

An array of maleimide-activated mono- and oligosaccharides were synthesized to permit site-specific glycosylation of cysteine-containing peptides and proteins. Maleimide-activated monosaccharides, in which the native alpha- or beta-O-glycosidic linkages found for nonreducing terminal sugars of native glycoproteins are preserved, were prepared using 2'-aminoethyl glycosides as the key intermediates. In addition, a native high-mannose type oligosaccharide, Man(9)GlcNAc(2)Asn, was converted into its maleimide-activated form by taking advantage of the existing amino group in the Asn portion. The application of these maleimide-activated carbohydrates was exemplified by the site-specific glycosylation of a 36-mer HIV-1 gp41 peptide, T20, which is a potent inhibitor against HIV infection. The chemoselective ligation was found to be rapid, highly efficient, and essentially quantitative. Tagging the biologically active peptide with a mannose and/or oligomannose moiety will be useful for targeting the drug to macrophage and dendritic cells, which are primary targets for HIV-1 infection and are expressing mannose- and oligomanose-specific receptors on their surface. In combination with site-specific mutagenesis, the maleimide-activated carbohydrates can serve as generally applicable tags for site-specific glycosylation of proteins via the highly efficient maleimide-thiol ligation reaction.

Carbohydrate-centered maleimide cluster as a new type of templates for multivalent peptide assembling. synthesis of multivalent HIV-1 gp41 peptides.

This paper describes a facile synthesis of carbohydrate-centered maleimide clusters and their application as a new type of templates for multivalent peptide assembling. Simultaneous introduction of multiple maleimide functionalities onto a carbohydrate core was achieved through the reaction of carbohydrate-based polyamines with methoxycarbonylmaleimide or with the N-hydroxylsuccinimide ester of 6-maleimidohexanoic acid. The clustered maleimides placed on the carbohydrate core allow rapid and highly chemoselective ligation with multiple copies of cysteine-containing peptides under virtually neutral conditions at room temperature. This mild and highly efficient ligation method is extremely valuable for synthesizing large and complex multivalent peptides that may not be easily obtained by conventional ligation methods. The usefulness of the maleimide clusters as a new type of templates for multivalent peptide synthesis was exemplified by the synthesis of two tetravalent gp41 peptides incorporating the sequence of the potent HIV inhibitor, T20. The synthetic multivalent gp41 peptides are useful as novel immunogens to raise specific antibodies for HIV studies. They are also useful probes for studying HIV membrane fusion mechanisms.

Improved preparation of perallylated cyclodextrins: facile synthesis of cyclodextrin-based polycationic and polyanionic compounds.

An improved procedure for the perallylation of cyclodextrins allowed the preparation of O-perallylated alpha-, beta-, and gamma-cyclodextrins in 89, 91, and 88% yields, respectively. These were converted into two cyclodextrin-based functionalized compounds, the polycationic heptakis[2,3,6-tri-O-(6-amino-3-thiahexyl)]-beta-cyclodextrin hydrochloride (3), and the polyanionic heptakis[2,3,6-tri-O-(sodium 5-carboxyl-3-thiapentyl)]-beta-cyclodextrin (4), a potential inhibitor of HIV-1 replication.

&figsp;A Novel Sequential Transformations of Phosphonate. Highly Stereoselective Synthesis of Perfluoroalkylated alpha-Fluoro-alpha,beta unsaturated Esters.

The phosphoryl-stabilized carbanion 2, generated from diethyl (1-fluoro-1-carbethoxymethyl) phosphonate (1) and n-butyllithium in tetrahydrofuran (THF), was acylated by the addition of perfluoroalkanoic anhydride to give perfluoroacylated phosphonates (3). The latter were reacted in-situ with suitable organolithium derivatives. Elimination of phosphonic acid anion afforded perfluoroalkylated alpha-fluoro-alpha,beta-unsaturated esters in 58-87% yields with the E-isomer produced exclusively or predominately. Thus the sequential transformations of phosphonate provides a convenient synthesis of the title compounds. The effect of base and solvent as well as reaction temperature has been investigated in more detail. A possible mechanism for the explanation of stereochemical results is proposed.