<p>Glycoconjugates are biologically important molecules with diverse functions. They consist of carbohydrates of varying size and complexity, attached to a non-sugar moiety as a lipid or a protein. Glycoconjugate structures are often very complex and their intricate biosynthetic pathways makes overexpression difficult. This renders the isolation of pure, structurally defined compounds from natural sources cumbersome. Therefore, to better address questions in glycobiology, synthetic glycoconjugates are an appealing alternative. In addition, synthetic methods allow for the preparation of non-natural glycoconjugates that can enhance the understanding of the influence of structural features on the biological responses.</p><p>In this thesis, synthetic methods for the preparation of glycoconjugates, especially glycolipid analogues, have been developed. These methods make use of solid-phase chemistry and are amenable to library synthesis of series of similar compounds. Solid-phase synthesis is a technique where the starting material of the reaction is attached to small plastic beads through a linker. This allows large excess of reagents to speed up the reactions and the sometimes difficult purifications of intermediate products are reduced to simple washings of the beads.</p><p>One problem with solid-phase synthesis is the difficulties to monitor the reactions and characterize the intermediate products. Gel-phase 19 F-NMR spectroscopy, using fluorinated linkers and protecting groups, is an excellent tool to overcome this problem and to monitor solid-phase synthesis of e.g. glycoconjugates. Two novel fluorinated linkers for the attachment of carboxylic acids have been developed and are presented in the thesis. These linkers can be cleaved with both acids of varying strengths and nucleophiles like hydroxide ions, and they are stable to glycosylation conditions. In addition, a novel filter reactor for solid-phase synthesis was designed. The reactor fits into an ordinary NMR spectrometer to facilitate the reaction monitoring with gel-phase 19 F-NMR spectroscopy.</p><p>The biological applications of the synthesized glycolipids were demonstrated in two different settings. The CD1d restricted binding of glycolipids carrying the monosaccharide α-GalNAc as carbohydrate could be detected on viable cells of mouse origin. CD1d is one of several antigen presenting molecules (the CD1 proteins) that presents lipids and glycolipids to circulating T-cells that in turn can initiate an immune response. The CD1 molecules are relatively sparsely investigated, and the method to measure glycolipid binding on viable cells, as described in the thesis, has the possibility to greatly enhance the knowledge of the structural requirements for CD1-binding.</p><p>Serine-based neoglycolipids with terminal carboxylic acids were used to prepare glycoconjugate arrays with covalent bonds to secondary amines on microtiter plates. Carbohydrate arrays have great possibilities to simplify the study of interactions between carbohydrates and e.g. proteins and microbes. The usefulness of the glycolipid arrays constructed in the thesis was illustrated with two lectins, RCA120 from Ricinus communis and BS-1 from Bandeiraea simplicifolia. Both lectins bound to the array of neoglycolipids in agreement with their respective specificity for galactosides.</p><p>Glycobiology is a large area of great interest and the methods described in this thesis can be used to answer a variety of glycoconjugaterelated biological questions.</p>

Glycoconjugates are biologically important molecules with diverse functions. They consist of carbohydrates of varying size and complexity, attached to a non-sugar moiety as a lipid or a protein. Glycoconjugate structures are often very complex and their intricate biosynthetic pathways makes overexpression difficult. This renders the isolation of pure, structurally defined compounds from natural sources cumbersome. Therefore, to better address questions in glycobiology, synthetic glycoconjugates are an appealing alternative. In addition, synthetic methods allow for the preparation of non-natural glycoconjugates that can enhance the understanding of the influence of structural features on the biological responses. In this thesis, synthetic methods for the preparation of glycoconjugates, especially glycolipid analogues, have been developed. These methods make use of solid-phase chemistry and are amenable to library synthesis of series of similar compounds. Solid-phase synthesis is a technique where the starting material of the reaction is attached to small plastic beads through a linker. This allows large excess of reagents to speed up the reactions and the sometimes difficult purifications of intermediate products are reduced to simple washings of the beads. One problem with solid-phase synthesis is the difficulties to monitor the reactions and characterize the intermediate products. Gel-phase 19 F-NMR spectroscopy, using fluorinated linkers and protecting groups, is an excellent tool to overcome this problem and to monitor solid-phase synthesis of e.g. glycoconjugates. Two novel fluorinated linkers for the attachment of carboxylic acids have been developed and are presented in the thesis. These linkers can be cleaved with both acids of varying strengths and nucleophiles like hydroxide ions, and they are stable to glycosylation conditions. In addition, a novel filter reactor for solid-phase synthesis was designed. The reactor fits into an ordinary NMR spectrometer to facilitate the reaction monitoring with gel-phase 19 F-NMR spectroscopy. The biological applications of the synthesized glycolipids were demonstrated in two different settings. The CD1d restricted binding of glycolipids carrying the monosaccharide α-GalNAc as carbohydrate could be detected on viable cells of mouse origin. CD1d is one of several antigen presenting molecules (the CD1 proteins) that presents lipids and glycolipids to circulating T-cells that in turn can initiate an immune response. The CD1 molecules are relatively sparsely investigated, and the method to measure glycolipid binding on viable cells, as described in the thesis, has the possibility to greatly enhance the knowledge of the structural requirements for CD1-binding. Serine-based neoglycolipids with terminal carboxylic acids were used to prepare glycoconjugate arrays with covalent bonds to secondary amines on microtiter plates. Carbohydrate arrays have great possibilities to simplify the study of interactions between carbohydrates and e.g. proteins and microbes. The usefulness of the glycolipid arrays constructed in the thesis was illustrated with two lectins, RCA120 from Ricinus communis and BS-1 from Bandeiraea simplicifolia. Both lectins bound to the array of neoglycolipids in agreement with their respective specificity for galactosides. Glycobiology is a large area of great interest and the methods described in this thesis can be used to answer a variety of glycoconjugaterelated biological questions.

Mucin is a term used to describe a large family of heavily glycosylated proteins which are present on the surfaces of secretory epithelial cells and are overexpressed by many carcinomas. Membrane-bound mucin MUC1 is of special interest. Its backbone consists of repeating units of twenty amino acids with five potential glycosylation sites. These sites are expanded to structures like the T (Galβ(1-&gt;3)GalNAcα-Ser/Thr) and Tn (GalNAcα-Ser/Thr) antigens by the action of various glycosyltransferases. In different types of carcinomas these epitopes are being terminated by sialic acid residues to form among others: 2,3-sialyl-T and sialyl-Tn structures due to the elevated levels of different sialyltransferases. Solid-phase synthesis of the selected antigens derived from the mucin MUC1 has been developed and optimized. A chemoenzymatic approach has been used to effectively prepare 2,3-sialyl-T and 2,6-sialyl-Tn glycopeptides. The formation of intramolecular sialic acid lactones in presence of acetic acid was investigated. The stability of lactones formed from 2,3-sialyl-T towards water was studied using NMR spectroscopy and it appeared that 1''-&gt;2' lactone displayed remarkable strength to hydrolysis and it was suggested as a candidate for cancer vaccine. Gel-phase 19F NMR spectroscopy is known to be a very good tool to characterize resin-bound products using fluorinated protecting groups and linker molecules. The hydrophobic peptide LLLLTVLTV, which is a fragment from the MUC1 signal sequence, was prepared using solid-phase synthesis according to a modified Fmoc protocol with more active coupling reagent, stronger base, and the isopropylidene dipeptide Fmoc-Leu-Thr-(ΨMe,Mepro)-OH. Gel-phase 19F NMR spectroscopy was used to evaluate peptide chain aggregation and coupling and deprotection efficiency. A carbamate linker strategy proved to be effective in solid-phase synthesis of serine-based neoglycolipids with terminal amino functionality. Neoglycolipids were covalently bound to secondary amines in microtiter plates using squaric acid ester methodology. These arrays have potential to study the interactions between carbohydrates and e.g. proteins and microbes. The new fluorinated α-amino protective group [1-(4-(4-fluorophenyl)-2,6-dioxocyclohexylidene)ethyl] Fde was developed. This group is cleaved with hydrazine in DMF solution. By using amino acids protected with this group, it was possible to quantify the efficiency of peptide coupling using gel-phase 19F NMR spectroscopy.

To study the functions of glycoconjugates in biological systems reliable and efficient protocols for glycoconjugate synthesis are needed. To reach this goal we have developed methods for solid-phase synthesis of glycoconjugates that can be monitored with gel-phase 19F spectroscopy using fluorinated linkers, building blocks, and protecting groups. We have developed a new fluorine containing linker suitable for solid-phase synthesis of glycoconjugates. The linker was more acid-labile than similar linkers in order to enable cleavage under mild conditions of the target compound from the linker resin.  A carbamate-based strategy has been applied to attach a spacer carrying an amino group to a fluorinated Wang linker for synthesis of amino-functionalized glycoconjugates using thioglycoside donors with fluorinated protective groups. Cleavage from the solid support was performed with trifluoroacetic acid and subsequent protecting group removal gave the target compound. The terminal amine was conjugated with didecyl squarate and this derivative can be attached to various proteins and solid surfaces carrying primary or secondary amines. To evaluate this methodology we have immobilized glycoconjugates in amino-functionalized microtiter plates and successfully probed them with lectin. In addition, a novel fluorine containing protecting group has been designed, synthesized and evaluated. The protecting group was used for protection of the unreactive 4-OH in a galactose building block that was applied in the synthesis of 6-aminohexyl galabioside and was removed with TBAF in THF. Adenovirus serotype 8 (Ad8), Ad19, and Ad37 cause the severe ocular infection, epidemic keratoconjunctivities (EKC). During infection, the adenoviruses interact with sialic acid containing glycoconjugates on the epithelial cells via fiber structures extending from the viral particles. The virus particle most likely binds to the host cell in a multivalent way by simultaneously using multiple fiber proteins and binding sites. Multivalent sialic acid containing conjugates could efficiently inhibit Ad37 cell attachment and subsequent infection of human corneal epithelial (HCE) cells. Three compact tri- and tetravalent sialic acid conjugates were prepared and evaluated as inhibitors of adenoviral host cell attachment and subsequent infection and all conjugates were potent as anti-adenoviral agents. The conjugates can readily be synthesized from accessible starting materials. A crystal structure of the Ad37 fiber knob protein and the trivalent sialic acid conjugate showed that the three binding sites were all occupied by one sialic acid residue each.