A highly efficient chemoenzymatic synthesis of N-glycopeptides was achieved. It was found that using synthetic oligosaccharide oxazolines, the mimics of the presumed oxazolinium ion intermediate formed in a retaining mechanism of substrate-assisted catalysis, as the donor substrates and GlcNAc-peptides as the acceptors, the endo-beta-N-acetylglucosaminidase (ENGase)-catalyzed transglycosylation gave a high yield (73-82%) of the corresponding glycopeptides in a regio- and stereospecific manner, regardless of the size of the peptide portions. The use of the oligosaccharide oxazolines as donor substrates not only expanded the substrate availability but also led to a substantial enhancement of the synthetic efficiency, compared to the use of natural N-glycans.

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.

[reaction: see text] A highly efficient endoglycosidase-catalyzed synthesis of homogeneous glycoproteins is described. By using ribonuclease B as a model system, it was demonstrated that Endo-A could efficiently attach a preassembled oligosaccharide to a GlcNAc-containing protein in a regio- and stereospecific manner, when the corresponding sugar oxazoline was used as the donor substrate. The method allows the synthesis of both natural and tailor-made N-linked glycoproteins in excellent yield.

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[structure: see text] A highly efficient chemoenzymatic synthesis of HIV-1 V3 domain glycopeptides carrying two N-linked core tri- and pentasaccharides was achieved. The synthesis consisted of two key steps: a solid-phase synthesis of the cyclic, 47-mer V3 domain peptide containing two GlcNAc residues and a novel endoglycosidase-catalyzed transglycosylation that simultaneously added two N-glycan moieties to the peptide precursor from the oligosaccharide oxazoline donor substrates. The availability of the synthetic glycopeptides allowed the probing of the effects of glycosylation on the HIV-1 V3 domain. It was demonstrated that glycosylation influenced the global conformations of the V3 domain and provided protection of the V3 domain against protease digestion.

Bone marrow-derived mesenchymal stem cells (BM-MSCs) have properties that make them promising for the treatment of chronic nonhealing wounds. The major challenge is ensuring an efficient, safe, and painless delivery of BMMSCs. Tissue-engineered skin substitutes have considerable benefits in skin damage resulting from chronic nonhealing wounds. Here, we have constructed a three-dimensional biomimetic scaffold known as collagen-chitosan sponge scaffolds (CCSS) using the cross-linking and freeze-drying method. Scanning electron microscopy images showed that CCSS had an interconnected network pore configuration about 100 lm and exhibited a suitable swelling ratio for maintaining morphological stability and appropriate biodegradability to improve biostability using swelling and degradation assays. Furthermore, BM-MSCs were seeded in CCSS using the two-step seeding method to construct tissueengineered skin substitutes. In addition, in this three-dimensional biomimetic CCSS, BM-MSCs secreted their own collagen and maintain favorable survival ability and viability. Importantly, BM-MSCs exhibited a significant upregulated expression of proangiogenesis factors, including HIF-1a, VEGF, and PDGF following hypoxia pretreatment. In vivo, hypoxia pretreatment of the skin substitute observably accelerated wound closure via the reduction of inflammation and enhanced angiogenesis in diabetic rats with hindlimb ischemia. Thus, hypoxia pretreatment of the skin substitutes can serve as ideal bioengineering skin substitutes to promote optimal diabetic skin wound healing.Diabetes mellitus (DM) is reaching epidemic proportions worldwide. The number of DM patients is approximately 350 million at the moment and is expected to more than double by 2030 without intervention. In addition, 15% of all diabetic patients suffer from foot ulcerations, which often become chronic nonhealing wounds.

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.

An efficient chemo-enzymatic synthesis of alpha Gal-conjugated peptide T20 as novel HIV-immuno-targeting agent is described. The synthesis involves chemo-enzymatic preparation of maleimide-functionalized alpha Gal epitope and its chemoselective ligation with the peptide T20. The title compound contains two functional domains: the trisaccharide alpha Gal epitope that binds to human natural anti-Gal antibodies and the 36-amino acid gp41 peptide (T20) that recognizes the gp41 N-terminal ectodomain of the HIV envelope. Biological assays demonstrated that the synthetic conjugate could readily bind to natural anti-Gal antibodies (both IgG and IgM type) in normal human serum and exhibited potent anti-HIV activity even in the absence of human antibodies and complement system. The experimental data suggest that the synthetic alpha Gal-T20 might be valuable for in vivo HIV-immuno-targeting via antibody-mediated cytotoxicity and/or antibody-dependent, complement-mediated lysis of HIV particles and HIV-infected cells, thus providing an additional dimension of HIV intervention.