Chemical and Enzymatic Synthesis of N-Glycan Libraries and Glycoarrays

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The importance of carbohydrates in biological systems has stimulated the recent development of new screening technologies. With the advent of glycomics, the characterization of entire glycomes of individual cell types, organs and organisms can be attempted. The variety of natural oligosaccharides is typically determined by mass spectrometry but leaves bioscientists with many unresolved questions. Glycan structures can be determined from minute quantities, however elucidation of the biological properties requires much larger quantities of the compounds. In order to detect the biological recognition of glycans by lectins or antibodies miniaturized methods were developed. Robust nanoscale chemical immobilization of glycans on surfaces by micro contact printing (glycan arrays) allows the analysis of the specificity of carbohydrate binding proteins in a high throughput manner. These arrays are based on the availability of many defined carbohydrate structures, which need to immobilized after chemical derivatization. Despite worldwide efforts the number of glycans printed on current arrays is still low and represents glycomes only very incompletely. The main reason is the difficult isolation of pure oligosaccharides from natural sources and thus the required structures need to provided by synthesis. Access to oligosaccharides is quite demanding and requires specialized labs for each class of compounds. Thus current arrays are focusing on a limited number of structures whereas the systematic variation of the most important structures shows many gaps. Despite several shortcomings the carbohydrate binding specificity of an unknown protein can be determined using glycan arrays in most of the cases. This project aims at:1) developing further the methods for modular synthesis of complex N-glycans and their selective deprotection to universally applicable N-glycan azides.2) the enzymatic elongation of all mammalian N-glycan core structures by glycosyltransferases in solution.3) generating stable conjugates of N-glycans for arrays and NMR-measurements.4) use of N-glycanarrays for the systematic elucidation of specificity profiles of lectins followed by detailed structure determination using NMR and crystallography.5) establishing a rapid semisynthesis of all mammalian N-glycan core structures of the complex type using recombinant glycosyltransferases and an N-glycan isolated from egg yolk.