Design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines.

The development of vaccines against specific types of cancers will offer new modalities for therapeutic intervention. Here, we describe the synthesis of a novel vaccine construction prepared from spherical gold nanoparticles of 3-5 nm core diameters. The particles were coated with both the tumor-associated glycopeptides antigens containing the cell-surface mucin MUC4 with Thomsen Friedenreich (TF) antigen attached at different sites and a 28-residue peptide from the complement derived protein C3d to act as a B-cell activating "molecular adjuvant". The synthesis entailed solid-phase glycopeptide synthesis, design of appropriate linkers, and attachment chemistry of the various molecules to the particles. Attachment to the gold surface was mediated by a novel thiol-containing 33 atom linker which was further modified to be included as a third "spacer" component in the synthesis of several three-component vaccine platforms. Groups of mice were vaccinated either with one of the nanoplatform constructs or with control particles without antigen coating. Evaluation of sera from the immunized animals in enzyme immunoassays (EIA) against each glycopeptide antigen showed a small but statistically significant immune response with production of both IgM and IgG isotypes. Vaccines with one carbohydrate antigen (B, C, and E) gave more robust responses than the one with two contiguous disaccharides (D), and vaccine E with a TF antigen attached to threonine at the 10th position of the peptide was selected for IgG over IgM suggesting isotype switching. The data suggested that this platform may be a viable delivery system for tumor-associated glycopeptide antigens.

Synthesis of 6-PEtN-α-D-GalpNAc-(1->6)-ß-D-Galp-(1->4)-ß-D-GlcpNAc-(1->3)-ß-D-Galp-(1->4)-ß-D-Glcp, a Haemophilus influenzae lipopolysacharide structure, and biotin and protein conjugates thereof.

BACKGROUND: In bacteria with truncated lipopolysaccharide structures, i.e., lacking the O-antigen polysaccharide part, core structures are exposed to the immune system upon infection and thus their use as carbohydrate surface antigens in glycoconjugate vaccines can be considered and investigated. One such suggested structure from Haemophilus influenzae LPS is the phosphorylated pentasaccharide 6-PEtN-α-D-GalpNAc-(1→6)-ß-D-Galp-(1→4)-ß-D-GlcpNAc-(1→3)-ß-D-Galp-(1→4)-ß-D-Glcp. RESULTS: Starting from a spacer-containing lactose derivative a suitably protected lacto-N-neotetraose tetrasaccharide structure was constructed through subsequential couplings with two thioglycoside donors, a glucosamine residue followed by a galactose derivative, using NIS/AgOTf as promoter. Removal of a silyl protecting group at the primary position of the non-reducing end residue afforded an acceptor to which the terminal α-galactosamine moiety was introduced using a 2-azido bromo sugar and halide assisted coupling conditions. Global deprotection afforded the non-phosphorylated target pentasaccharide, whereas removal of a silyl group from the primary position of the non-reducing end residue produced a free hydroxy group which was phosphorylated using H-phosphonate chemistry to yield the phosphoethanolamine-containing protected pentasaccharide. Partial deprotection afforded the phosphorylated target pentasaccharide with a free spacer amino group but with a protected phosphoethanolamino group. Conjugation of the spacer amino group to biotin or dimethyl squarate followed by deprotection of the phosphoethanolamino group and, in the case of the squarate derivative, further reaction with a protein then afforded the title conjugates. CONCLUSION: An effective synthesis of a biologically interesting pentasaccharide structure has been accomplished. The target pentasaccharide, an α-GalNAc substituted lacto-N-neotetraose structure, comprises a phosphoethanolamine motif and a spacer aglycon. Through the spacer, biotin and protein conjugates of the title compound have been constructed to allow further use in biological experiments.

Identification of the smallest structure capable of evoking opsonophagocytic antibodies against Streptococcus pneumoniae type 14.

Synthetic overlapping oligosaccharide fragments of Streptococcus pneumoniae serotype 14 capsular polysaccharide (Pn14PS), [6)-[beta-D-Galp-(1-->4)-]beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->](n), were conjugated to CRM(197) protein and injected into mice to determine the smallest immunogenic structure. The resulting antibodies were then tested for Pn14PS specificity and for their capacity to promote the phagocytosis of S. pneumoniae type 14 bacteria. Earlier studies have reported that the oligosaccharide corresponding to one structural repeating unit of Pn14PS, i.e., Gal-Glc-(Gal-)GlcNAc, induces a specific antibody response to Pn14PS. The broader study described here, which evaluated 16 oligosaccharides, showed that the branched trisaccharide element Glc-(Gal-)GlcNAc is essential in inducing Pn14PS-specific antibodies and that the neighboring galactose unit at the nonreducing end contributes clearly to the immunogenicity of the epitope. Only the oligosaccharide conjugates that produce antibodies recognizing Pn14PS were capable of promoting the phagocytosis of S. pneumoniae type 14. In conclusion, the branched tetrasaccharide Gal-Glc-(Gal-)GlcNAc may be a serious candidate for a synthetic oligosaccharide conjugate vaccine against infections caused by S. pneumoniae type 14.

Synthesis of 2'-([1,2,3]triazol-1-yl)-2'-deoxyadenosines.

A reliable and efficient protocol for the synthesis of 2 '-([1,2,3]triazol-1-yl)-2 '-deoxyadenosine derivatives from vidarabine is presented. Vidarabine was converted to 2'-azido-2'-deoxy-3',5-O-(tetraisopropyldisiloxane-1,3-diyl)-adenosine. This azide was used as the starting material for the Cu(I)-catalyzed parallel synthesis of 1,2,3-triazoles using a variety of alkynes. The reactions proceeded in good yield and gave almost exclusively the 1,4-disubstituted 1,2,3-triazoles.

Varied presentation of the Thomsen-Friedenreich disaccharide tumor-associated carbohydrate antigen on gold nanoparticles.

Three-dimensional self-assembled monolayers of gold coated with the Thomsen-Friedenreich antigen (TF(ag)) disaccharide (beta-Galp-(1-->3)-GalpNAc) in a variety of presentations have been prepared and characterized. Anomalies in the size distribution of our originally synthesized TF(ag)-bearing nanoparticles as shown in dynamic light scattering experiments prompted us to explore the effect of antigen density on the uniformity of the particles. Gold nanoparticles containing a range of densities 'diluted' with copies of the PEG-thiol spacer unit showed that lower antigen density affords more uniform particles. We also wanted to study the constitution of the actual antigen by synthesizing nanoparticles not only with the linker-extended disaccharide, but also within the context of the surrounding peptide sequence where it may be presented in vivo. The synthesis of TF(ag)-containing glycopeptide thiols based on a mucin peptide repeating unit were prepared, assembled into gold nanoparticles and their physical properties evaluated. These novel multivalent tools should prove extremely useful in exploring the binding properties and immune response to this important carbohydrate antigen.

Synthesis of oligosaccharides corresponding to Vibrio cholerae O139 polysaccharide structures containing dideoxy sugars and a cyclic phosphate.

A spacer-equipped tetrasaccharide, p-aminocyclohexylethyl alpha-l-Colp-(1-->2)-beta-d-Galp-(1-->3)-[alpha-l-Colp-(1-->4)]-beta-D-GlcpNAc, containing a 4,6-cyclic phosphate in the galactose residue, has been synthesised. The structure corresponds to a part of the repeating unit of the capsular (and lipo-) polysaccharide of the endemic bacteria Vibrio cholerae type O139 synonym Bengal. The synthetic strategy allows continuous syntheses of the complete O139 hexasaccharide repeating unit as well as of the structurally related repeating unit of serotype O22. Starting from ethyl 2-azido-4,6-O-benzylidene-2-deoxy-1-thio-beta-D-glucopyranoside, a thioglycoside tetrasaccharide donor block was constructed through two orthogonal glycosylations with glycosyl bromide donors. First, a properly protected galactose moiety was introduced using silver triflate as promoter and subsequently the two colitose residues, carrying electron-withdrawing protecting groups for stability reasons, under halide-assisted conditions. The tetrasaccharide block was then linked to the spacer in a NIS-TMSOTf-promoted coupling. Transformation of the azido group into an acetamido group using H2S followed by removal of temporary protecting acetyl groups gave a 4',6'-diol, which was next phosphorylated with methyl dichlorophosphate and deprotected to yield the 4,6-cyclic phosphate tetrasaccharide target structure.

SmI2/water/amine mediates cleavage of allyl ether protected alcohols: application in carbohydrate synthesis and mechanistic considerations.

[reaction: see text]. SmI2/H2O/amine provides selective cleavage of unsubstituted allyl ethers in good to excellent yields. This method is therefore useful in deprotection of alcohols and carbohydrates.