In our research group, newly-developed methodologies are used for the preparation of compounds of biological importance. These projects are often performed in collaboration with biochemists or pharmaceutical companies.

We are involved in several programs that are directed towards the development of vaccines against life-threatening bacterial infections. One of our long-term aims is to make fully synthetic carbohydrate-based vacines. For example, we report a new synthetic methodology that makes it possible to construct fully synthetic glycolipopeptides that contain a carbohydrate-based B-epitope, a peptide T-epitope and a lipopeptide. Such a conjugate constitutes the minimal structural features required for an effective immunological response. We have synthesized a compound (Figure 1), which contains a L-glycero-D-manno-heptose sugar that will act as a B-epitope, the peptide sequence YAFKYARHANVGRNAFELFL that has been identified as a MHC class II restricted recognition site for human T-cells and is derived from an outer-membrane protein of Neisseria meningitidis and the lipopeptide S-2-3[dipalmitoyloxy]-(R/S)-propyl-N-palmitoyl-R-Cysteine (Pam3Cys). The lipopeptide Pam3Cys is a highly potent B-cell and macrophage activator derived from the immunologically active

N-terminal sequence of the principal lipoprotein of Escherichia coli and has been used in synthetic peptide-based vaccine and cancer vaccine development. Such a project involves a combination of carbohydrate-, peptide and lipid chemistry and is very challenging because these areas of chemistry have been developed independently and are often not

compatible with each other.

Tumor-associated antigens are another group of our synthetic targets. Human cancer cells are often characterized by the presence of tumor-associated glycosphingolipids (GSL). Several GSL antigens have been identified as adhesion molecules, which may promote tumor cell metastases. It has been shown that active immunization with GSL can induce or enhance antibody titters and several studies indicate that these antibodies can suppress metastases. In general, carbohydrates and glycolipids are

poor immunogens. However, a much-improved immunological reaction can be obtained by conjugating saccharide epitopes to a carrier protein or synthetic lipopetide. We are involved in the synthesis of several tumor-associated oligosaccharides, which will be equipped with an artificial aminopropyl spacer (Figure 2). The latter moiety allows clean

and selectiveconjugation to a carrier protein or a lipopeptide.The heparin sulfates are complex linear sulphated polysaccharides and the initial biosynthetic product is extensively modified by amino (N) and O-sulphation and uronate epimerisation. The degree and distribution of these modifications varies between different cell-types and this may be very important in cellular recognition and growth factor regulation. There is a growing body of literature indicating important neurobiological roles for heparan sulphate proteoglycans, including neuroepithelial growth and differentiation, neurite outgrowth, nerve regeneration axonal guidance and branching, deposition of amyloidotic plaques in Alzheimer's disease and astrocyte proliferation.

In particular both aFGF and bFGF are abundant expressed both during development and in adult neural tissues with different spatial and temporal distributions. They are believed to play important roles in regulating growth and differentiation and contribute to the process of neural repair in the adult CNS. We are employing a latent-active glycosylation strategy for the to

preparation of libraries of disaccharide building blocks, which in a parallel fashion, can be assembled to heparine sulfates. Thesecompounds will be used to elucidate ligands for both aFGF and bFGF.

In collaboration with the research group of Dr Michael Pearce, who is also a faculty member of the Complex Carbohydrate Research Center (CCRC), we are studying a new class of animal and fungal lectins, known as X-lectins. A lectin has been found in the cortical granules of the Xenopus oocyte. Upon fertilization, the cortical granules fuse with the plasma membrane and release their content, 70% of which is lectin, into the egg jelly. A vigorous cross-linking reaction occurs, as the lectin is multimeric(probably octameric). This reaction is postulated to cause the formation of a fertilization membrane, which most likely prevents polyspermy. The saccharides of the jelly glycoproteins are an ill-defined heterogeneous mixture. Therefore it will be difficult to determine the structure of the endogenous ligands for the protein. A search of the human EST data base revealed a 251 bp fragment obtained from a human heart cDNA which showed over 98% identity with the nucleotide sequence of the Xenopus oocyte lectin. This new human lectin is cloned and over-expressed. We are developing new procedures for the synthesis and screening of oligosaccharide libraries to elucidate the endogenous ligands of these new lectins.