Synthesis of carbohydrate antigens of the human immunodeficiency virus-1

Abstract

N-glycans play an important role in HIV-1 pathogenesis and immune evasion, and are also important in antibody recognition of the virus. The glycoprotein gp 120, which displays a large number of N-linked oligosaccharides, has been a target for vaccine design as it is responsible for viral entry and infection. The neuralizing antibody 2G12 recognizes a conserved set of high mannose glycans on the gp120. Many vaccine candidates have tried to replicate an immune response similar to 2G12, an antibody that recognizes conserved mannans on the surface of gp120 but have met with limited success. We have developed a synthetic carbohydrate antigen that mimics the D1 arm of the high mannose oligosaccharide. The tetrasaccharide construct was synthesized using a linear approach that installed three mannose residues sequentially to the 5-Aminopentyl-linked mannose sugar. Though we were successful at the synthesis of the tetrasaccharide antigen, the conjugation of this oligosaccharide with amino acid was extremely challenging. Recently, several potent broadly neutralizing antibodies have been isolated from seronegative patients that are able to penetrate the HIV-1 ‘glycan shield’. Screening of sera from asymptomatic patients against a library of HIV-1 N-glycans could thus lead to the identification of new target for development of a fully synthetic carbohydrate-based anti-HIV-1 vaccine. Here we report a convergent approach for the combinatorial synthesis of a library of high mannose-type N-glycans through the regioselective manipulation of an orthogonally protected N-glycan core pentasaccharide with a small library of sugars to rapidly prepare asymmetrically substituted oligosaccharides. We have demonstrated that the levulinic ester (Lev), 9-fluorenylmethyloxycarbonate (Fmoc) and allyloxycarbonate (Alloc) used to protect key branching points on the oligosaccharide are orthogonal to each other. They can be regioselectively removed allowing selective extension of each arm with different donors to prepare asymmetrically branched high mannose oligosaccharides. Using this methodology, we synthesized Man5GlcNAc2 oligosaccharide that has recently been reported to be the most abundant and conserved glycan on the surface of native HIV-1 virions.