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The Development of a Bifunctional Toolkit to study Carbohydrate Interactions in Biology

Reports of research work funded by grants prior to 2015

Victoria University of Wellington

The Development of a Bifunctional Toolkit to study Carbohydrate Interactions in Biology

MSM Timmer, B Stocker
School of Chemical and Physical Sciences

The specific objectives of this project were the development of a series of bifunctional tools that can be used to study the effect of carbohydrates in biological systems.  To this end, we have extend our recently development oxyamine-linker methodology and

  1. Explored the attachment of glycans to a variety of biological molecules and assay systems.
  2. Prepared specific glycan conjugates that can be evaluated in subsequent biological assays.  Specifically, have prepared glycosylated proteins to study the role of N-glycans and have started the evaluation of oxyamine linked glycans in ELISAs for the detection of anti-carbohydrate antibodies in serum.

The synthesis of a number of N-glycosyl-N-alkyl-methoxyamine bifunctional linkers were accomplished.  The linkers contain an N-methoxyamine functional group for conjugation to carbohydrates and a terminal group, such as an amine, azide, thiol, or carboxylic acid, for conjugation to the probe of choice.  The strategy for the linker synthesis is rapid (3–4 steps) and efficient (51–96% overall yield), and many of the linkers were synthesized using a three-step one-pot strategy.  Moreover, the linkers can be conjugated to glycans in excellent yield and they show excellent stability toward hydrolytic cleavage.  Effective conjugation to biologically relevant target structures was accomplished, as illustrated by the conjugation of the blood group carbohydrate antigen Lex (CD15) to a fluorescent dendrimer and the antigen N-acetylglucosamine to biotin.  The biotin conjugated carbohydrate antigens are now being incorporated into ELISA protocols.

The fluorescent Lex functionalised dendrimer was subsequently used in the detection of DC-SIGN on macrophages.  To this end, THP-1 cells were differentiated into M1-like macrophages using PMA (phorbol myristate acetate), followed by treatment with LPS.  Subsequently analysis by flow cytometry using both the synthetic Lex functionalised dendrimers and a DC-SIGN specific antibody as positive control, showed that all DC-SIGN-positive cells were co-stained with Lex.  However, an additional DC-SIGN negative population of macrophages was observed that stained with Lex, indicating that perhaps an additional receptor is involved with the immune recognition of Lex.  Studies to identify this additional receptor are currently under investigation.

The Development of a Bifunctional Toolkit to study Carbohydrate Interactions in Biology

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