Synthesis of C-linked Disaccharides and Analogues as Biomimetics of the TF Epitope

Glycoproteins and glycolipids are major components of the outer surface of mammalian cells. Their carbohydrate moieties, which are directed into the outer environment, serve as ligands for receptor proteins, such as lectins, pathogen proteins and antibodies. Recognition processes between the receptor proteins and the carbohydrates have a significant impact on different aspects of cell biology as they turn structure into biological response in a selective way. Cellular carbohydrate structures are found to change dramatically during embryonic development or under pathological conditions including malignant transformation. One example is the Thomsen-Friedenreich (TF) epitope, a disaccharide consisting of galactose-β-(1→3)-N-acetyl-galactosamine α-linked to a serine or threonine of a carrier protein. The TF epitope is considered as a cancer-specific carbohydrate antigen that is found on various epithelial carcinomas of human adults and can be recognized by the anti-TF antibody. It has been demonstrated that synthetic glycoconjugates of the TF epitope can act as anti-cancer vaccines by stimulating the immune system against tumor growth and proliferation. Native carbohydrates are liable to chemical and enzymatic hydrolysis of their glycosidic bonds and therefore short-lived in the blood. Their stable C-analogues in which the glycosidic oxygens have been replaced by methylene or substituted methylene units are particularly interesting as therapeutic agents. We report herein our efforts to synthesize non-hydrolysable methylene and fluorinated methylene linked disaccharides analogues of the TF epitope. Our synthesis follows a methodology previously developed in our laboratory for the preparation of C(1→3)-disaccharides. The key step is an Oshima-Nozaki coupling between a β-C-galactosyl carbaldehyde and an enone, isolevoglucosenone. The hydroxyl methylene linker that is created by this coupling can be deoxygenated or can be substituted by one fluorine atom (or two fluorine atoms via the corresponding ketone). 1,4-Addition of an amine to the enone derived from the Oshima-Nozaki coupling, ketone reduction and some protecting group manipulations gave an hydroxyl methylene linked disaccharide that could smoothly undergo Barton-McCombie deoxygenation. On the contrary, the attempts of nucleophilic fluorination of this substrate failed to give the desired outcome probably due to an intramolecular rearrangement. With the deoxygenated product in hand and after some protecting group transformations, we attempted C-allylation of the anomeric centre and at the same time cleavage of the 1,6-anhydro bridge. Some preliminary results are presented. An allyl-C-disaccharide can lead to a C-glycosyl amino acid according to the literature and therefore it can be used for preparing a C,C-analogue of the TF epitope. In our search for biomimetics of the TF epitope with less synthetic requirements, we attempted to replace the second saccharidic unit by a phenyl ring. Simple phenyl CH2- and CF2-galactosides were prepared starting from α- and β-C-galactosyl carbaldehydes. NMR studies in combination with theoretical calculations revealed that the conformational behaviour of these galactosides in solution depends on the substitution at pseudoanomeric center. Similarities and differences to their O-analogues are discussed. Additionally, NMR spectroscopy was used to detect binding to a lectin protein. The β-galactosyl carbaldehyde –common starting material for the synthesis of C-linked disaccharides and phenyl C-galactosides mentioned above – was also used to prepare a small library of CH2-galactosyl aryl ethers bearing different aromatic amides. The affinities of these derivatives towards some galactoside-specific lectins, the galectins, were measured. None of the synthesized C-galactosyl analogues appeared to be high-affinity ligand of galectins. However, such studies are useful for the future design of sugar analogues with the minimum of functionalities that assure strong binding to carbohydrate recognition proteins.

Synthesis of a C-linked disaccharide analogue of the Thomsen Friedenreich (TF)-epitope a-O-conjugated to L-serine and formation of a cluster as potential anticancer vaccine

Loay Awad

Cell surface glycoproteins and glycolipids are responsible for cellular recognition processes. Vaccination is the procedure whereby the immune system is induced to create antibodies against a foreign molecule involved in disease or viral infection. In many disease states, the oligosaccharide chains presented on cell surface glycoprotein are altered. In some tumors, the glycan chains of glycoproteins are attenuated to only a few sugar residues. In the case of the TF-antigen, the polysaccharide chains have been shortened to a galactose-β-(1–>3)-N-acetyl-galactosamine disaccharide structure α-linked to a serine or threonine. Immunogenicity of this epitope in synthetic vaccines has been demonstrated. However, this disaccharide conjugate is relatively short-lived in the blood stream because of its hydrolysis catalysed by ubiquitous glycosidases in vivo. C-disaccharides are sugar mimetics whose interglycosidic linkage is non-hydrolysable as required for a disaccharide-based vaccine. In the first part of the work, we report the first synthesis of TF-antigen analogues applying the methodology developed by our group for the synthesis of C(1–>3)-disaccharides. Conjugate addition of diethylaluminium iodide (Et2AlI) to isolevoglucosenone leads to an aluminium enolate, which reacts with the sugar derived carbaldehyde, 2,6-anhydro-3,4,5,7-tetrakis-O-[(tert-butyldimethyl)silyl]-D-glycero-L-manno-heptose, to give an aldol. The convergent and stereoselective synthesis of this adduct allows us to obtain C(1–>3)-disaccharides. Reduction of the moiety derived from isolevoglucosenone with lithium borohydride, followed by cleavage of the 1,6-anhydro bridge produces C-disaccharides with D-galacto configuration. Königs-Knorr glycosidation of N-Fmoc-serine tert-butylester, followed by reduction of azide moiety to the corresponding acetamido group allows us to obtain TF-antigen analogues linked either by hydroxymethano (-CH(OH)-) or methano (-CH2-) group. In a second part we report the synthesis of a fully deprotected thio-glycotripeptide based on the TFantigen -C-analogues linked by hydroxymethano - N-acetyl-O-{α-D-glyco}-D-seryl-O-{α-D-glyco}-D-seryl-rac-N-(3-[(acetylthio)amino]propyl)-O-{α-D-glyco}-D-serinamide, which was covalently conjugated to the KLH protein carrier via Michael addition reaction. Biological trials with the TF (analogues)-KLH are in progress. We report also the synthesis of fully deprotected TF-antigen -C-analogues linked by hydroxymethano (-CH(OH) and methano (-CH2-) group. their conformational analysis is currently in progress.

EPFL2005

Synthesis of aza-C-disaccharides

Eliazar Rodriguez Garcia

Cell surface oligosaccharides control cell adhesion, fertilization, inflammation, the immune response and metastasis. Their biosynthesis implies sequences of reactions catalysed by glycosidases and glycosyltranferases. Inhibitors of these enzymes are therefore potential antiviral, antibacterial, antimetastatic, antidiabetes, antihyperglycemic, antiadhesive or immunostimulatory agents. Carbohydrate mimetics are among the most powerful inhibitors of these enzymes. They are more stable towards endogenous degradative enzymes, they have improved bioavailability and reduced clearances rates, and they have a higher affinity and selectivity for their cognate receptors by taking advantages of interactions that the natural saccharide does not. Besides the polyhydroxylated piperidines, pyrrolidines, indolizidines, pyrrolizidines and nortropanes, important classes of glycomimetics are C-glycosides and aza-C-disaccharides. Aza C-disaccharides are an emergent class of glycomimetics that combine the features of azasugars and C-glycosyl compounds. The first part of this work was devoted to the synthesis of new aza-C(1—>2)-disaccharides. The type of reaction applied to generate the C-C linkage-bond to join the two monosaccharide fragments was an aldol reaction. Reaction of 4,6-O-benzylidene-2-deoxy-α-D-erythro-hexopyranosid-3-ulose and a N-protected pyrrolidine-derived aldehyde generated the corresponding aldol adduct. First assays of aldol coupling gave poor yields. The reaction was improved by realizing the instability of the aldehyde partner, increasing the concentration, decreasing the temperature and by addition of HMPA as stabilizer of the enolate intermediate. From the aldol adduct a first aza-C-disaccharide with a hydroxy group on the methano linker and with D-allose configuration for the pyranose ring was prepared. In order to synthesize branched aza-C-disaccharides without heteroatom on the methano linker, dehydration of the β-hydroxyketone (aldol adduct) was done. Best results for this transformation were obtained using DAST as dehydration agent, in order to avoid secondary reactions. Dehydrated E and Z isomers were thus obtained. Further transformations from these isomers generated two other aza-C(1—>2)-disaccharides with D-allose and D-glucose configuration for the pyranose ring, respectively. In the second part of this work a new and efficient combinatorial methodology has been developed for the design and screening of new inhibitors of α-mannosidases. A family of iminederivatives was formed by mixing amines and aldehydes in a 96-wells microplate. The inhibitory activities against two different mannosidases were measured in situ. On the other hand, isolated imines showed analogous activities to the mixtures prepared in the microplates. The rapid biological screening makes the method a valuable tool. Moreover minutes amounts of products are required. This method can be applied for the discovery of new lead compounds of all classes of enzymes and biopolymers.

EPFL2005

Synthesis of Glycomimetics as Potential Anti-Cancer Agents

Annabelle Gillig

Cancer cells exhibit different properties that make them dangerous to the host, such as the ability to invade other tissues and the abnormal response to the control mechanisms that regulate the division of normal cells, which allow them to divide in a relatively uncontrolled fashion. Moreover, cancer cells require fewer protein growth factors than normal cells and can go on dividing indefinitely, in contrast with normal. Thus, malignant tumors cells defy not only the normal controls on their proliferation, but also the normal controls on their position. The more widely such cells spread in the body, the harder they become to eradicate surgically. Therefore, cancer research has the difficult task of analyzing malignant transformation and metastatic diffusion in order to develop new therapies. In this context, we report in the present work the development of new chemical tools related to cancer research. The first part relies on the salvage pathway of nicotinamide adenine dinucleotide NAD+ by the enzyme nicotinamide phosphoribosyl transferase. The latter enzyme is over-expressed in cancer cells and a synthetic inhibitor, called FK866, shows efficient anti-cancer activity while having little toxicity to normal cells. However, FK866 has a poor bioavailability, which highlights the need to develop new drugs. In order to optimize the bioavailability and the interactions in the active site, we report in the present work the strategy for the design of a new class of potential nicotinamide phosphoribosyl transferase inhibitors. An efficient synthetic pathway involving the preparation of an iminoribitol, its substitution on the position 1 through a three stepone pot reaction, a cross-metathesis and an ester-amide exchange has been set up. A library of new glycomimetics analogous to FK866 has been prepared and their encouraging biological evaluation on cancer cells led to the selection of eight candidates for further biological studies. The second part of the project is based on the development of cancer vaccines. Carcinomas express a multitude of different carbohydrate antigens, such as the Thomsen-Friedenreich antigen, on plasma membrane. Interestingly, the changes of the glycopatterns during the malignant transformation generally result in shorter carbohydrate chains. In usual tissue, the Thomsen-Friedenreich antigen is masked and inaccessible to the immune system, but in various tumors, it is exposed and immunoreactive. A cluster of Thomsen-Friedenreich antigen administered as vaccines has proven to be a target for the immune system. Nevertheless, due to hydrolysis catalysed by glycosidases in vivo, these disaccharides conjugates are relatively short-lived in the blood stream. Our strategy to avoid hydrolytic degradation is the use of C-linked- glycosides. Thus, we report the synthesis of non-hydrolysable clusters of C-linked disaccharides analogous to the TF-antigen through the coupling of two glycosides, the construction of disaccharides analogous to the Thomsen-Friedenreich epitope and their clustering through a peptide synthesis strategy. Conjugation of these clusters to a carrier protein such as KLH will provide the expected cancer vaccines. The last part refers to an outstanding strategy in human therapy, which use oligonucleotide analogues to inhibit the genes expression at the level of translation. This strategy, called the antisense approach, is based on the introduction of an antisense oligonucleotide, complementary to the mRNA of interest, into the cell. Base-pairing with the targeted mRNA thus inhibits its translation into a protein. RNAs tend to fold into a variety of secondary structure such as hairpin loops. Therefore, the antisense oligonucleotide should have a restricted conformation in order to bind efficiently, and with high specificity, to the targeted nucleic acid structure. Thus, we report the synthesis of a conformationally restricted dinucleoside through the application of cross-metathesis in nucleic acid chemistry.

EPFL2010

Synthesis of aza-C-disaccharides

Eliazar Rodriguez Garcia

EPFL2005

Synthesis of a C-linked disaccharide analogue of the Thomsen Friedenreich (TF)-epitope a-O-conjugated to L-serine and formation of a cluster as potential anticancer vaccine

Loay Awad

EPFL2005

Synthesis of Glycomimetics as Potential Anti-Cancer Agents

Annabelle Gillig

EPFL2010