Synthesis and unexpected binding of monofluorinated N,N'-diacetylchitobiose and LacdiNAc to wheat germ agglutinin.

Fluorination of carbohydrate ligands of lectins is a useful approach to examine their binding profile, improve their metabolic stability and lipophilicity, and convert them into 19F NMR-active probes. However, monofluorination of monovalent carbohydrate ligands often leads to a decreased or completely lost affinity. By chemical glycosylation, we synthesized the full series of methyl ß-glycosides of N,N'-diacetylchitobiose (GlcNAcß(1-4)GlcNAcß1-OMe) and LacdiNAc (GalNAcß(1-4)GlcNAcß1-OMe) systematically monofluorinated at all hydroxyl positions. A competitive enzyme-linked lectin assay revealed that the fluorination at the 6'-position of chitobioside resulted in an unprecedented increase in affinity to wheat germ agglutinin (WGA) by one order of magnitude. For the first time, we have characterized the binding profile of a previously underexplored WGA ligand LacdiNAc. Surprisingly, 4'-fluoro-LacdiNAc bound WGA even stronger than unmodified LacdiNAc. These observations were interpreted using molecular dynamic calculations along with STD and transferred NOESY NMR techniques, which gave evidence for the strengthening of CH/π interactions after deoxyfluorination of the side chain of the non-reducing GlcNAc. These results highlight the potential of fluorinated glycomimetics as high-affinity ligands of lectins and 19F NMR-active probes.

Exploring long-range fluorine-carbon J-coupling for conformational analysis of deoxyfluorinated disaccharides: A combined computational and NMR study.

In this study, we investigated the potential of long-range fluorine-carbon J-coupling for determining the structures of deoxyfluorinated disaccharides. Three disaccharides, previously synthesized as potential galectin inhibitors, exhibited through-space fluorine-carbon J-couplings. In our independent conformational analysis of these disaccharide derivatives, we employed a combination of density functional theory (DFT) calculations and nuclear magnetic resonance (NMR) experiments. By comparing the calculated nuclear shieldings with the experimental carbon chemical shifts, we were able to identify the most probable conformers for each compound. A model comprising fluoromethane and methane molecules was used to study the relationship between molecular arrangements and intermolecular through-space J-coupling. Our study demonstrates the important effect of internuclear distance and molecular orientation on the magnitude of fluorine-carbon coupling. The experimental values for the fluorine-carbon through-space couplings (TSCs) of the disaccharides corresponded with values calculated for the most probable conformers identified by the conformational analysis. These results unlock the broader application of fluorine-carbon TSCs as powerful tools for conformational analysis of flexible molecules, offering valuable insights for future structural investigations.

Selectively Deoxyfluorinated N-Acetyllactosamine Analogues as 19 F NMR Probes to Study Carbohydrate-Galectin Interactions.

Galectins are widely expressed galactose-binding lectins implied, for example, in immune regulation, metastatic spreading, and pathogen recognition. N-Acetyllactosamine (Galß1-4GlcNAc, LacNAc) and its oligomeric or glycosylated forms are natural ligands of galectins. To probe substrate specificity and binding mode of galectins, we synthesized a complete series of six mono-deoxyfluorinated analogues of LacNAc, in which each hydroxyl has been selectively replaced by fluorine while the anomeric position has been protected as methyl ß-glycoside. Initial evaluation of their binding to human galectin-1 and -3 by ELISA and 19 F NMR T2 -filter revealed that deoxyfluorination at C3, C4' and C6' completely abolished binding to galectin-1 but very weak binding to galectin-3 was still detectable. Moreover, deoxyfluorination of C2' caused an approximately 8-fold increase in the binding affinity towards galectin-1, whereas binding to galectin-3 was essentially not affected. Lipophilicity measurement revealed that deoxyfluorination at the Gal moiety affects log P very differently compared to deoxyfluorination at the GlcNAc moiety.

Synthesis of multiply fluorinated N-acetyl-D-glucosamine and D-galactosamine analogs via the corresponding deoxyfluorinated glucosazide and galactosazide phenyl thioglycosides.

Multiple fluorination of glycostructures has emerged as an attractive way of modulating their protein affinity, metabolic stability, and lipophilicity. Here we described the synthesis of a series of mono-, di- and trifluorinated N-acetyl-ᴅ-glucosamine and ᴅ-galactosamine analogs. The key intermediates are the corresponding multiply fluorinated glucosazide and galactosazide thioglycosides prepared from deoxyfluorinated 1,6-anhydro-2-azido-ß-ᴅ-hexopyranose precursors by ring-opening reaction with phenyl trimethylsilyl sulfide. Nucleophilic deoxyfluorination at C4 and C6 by reaction with DAST, thioglycoside hydrolysis and azide/acetamide transformation completed the synthesis.

The effect of deoxyfluorination and O-acylation on the cytotoxicity of N-acetyl-D-gluco- and D-galactosamine hemiacetals.

Fully acetylated deoxyfluorinated hexosamine analogues and non-fluorinated 3,4,6-tri-O-acylated N-acetyl-hexosamine hemiacetals have previously been shown to display moderate anti-proliferative activity. We prepared a set of deoxyfluorinated GlcNAc and GalNAc hemiacetals that comprised both features: O-acylation at the non-anomeric positions with an acetyl, propionyl and butanoyl group, and deoxyfluorination at selected positions. Determination of the in vitro cytotoxicity towards the MDA-MB-231 breast cancer and HEK-293 cell lines showed that deoxyfluorination enhanced cytotoxicity in most analogues. Increasing the ester alkyl chain length had a variable effect on the cytotoxicity of fluoro analogues, which contrasted with non-fluorinated hemiacetals where butanoyl derivatives had always higher cytotoxicity than acetates. Reaction with 2-phenylethanethiol indicated that the recently described S-glyco-modification is an unlikely cause of cytotoxicity.

Use of remote acyl groups for stereoselective 1,2-cis-glycosylation with fluorinated glucosazide thiodonors.

Fluorinated glycans are valuable probes for studying carbohydrate-protein interactions at the atomic level. Glucosamine is a ubiquitous component of glycans, and the stereoselective synthesis of α-linked fluorinated glucosamine is a challenge associated with the chemical synthesis of fluorinated glycans. We found that introducing a 6-O-acyl protecting group onto 3-fluoro and 4-fluoro glucosazide thiodonors endowed them with moderate α-selectivity in the glycosylation of carbohydrate acceptors, which was further improved by adjusting the acceptor reactivity via O-benzoylation. Excellent stereoselectivity was achieved for 3,6-di-O-acyl-4-fluoro analogues. The glycosylation of threonine-derived acceptors enabled the stereoselective synthesis of the protected fluorinated analogue of α-GlcNAc-O-Thr, a moiety abundant in cell-surface O-glycans of the protozoan parasite Trypanosoma cruzi. DFT calculations supported the involvement of transient cationic species which resulted from the stabilization of the oxocarbenium ion through O-6 acyl group participation.

ESI-TOF mass spectrometry of cationic carbosilane dendrimers: A potent tool for characterization of structural defects.

Macromolecular polyelectrolytes are gaining considerable attention for the application in medicine that implies their detailed characterization. We have successfully applied electrospray ionization mass spectrometry (ESI MS) to the analysis of defects in the structure of three generations of polycationic carbosilane dendrimers bearing series of quarternary phosphonium groups at their periphery. Besides expected defects caused by incomplete conversion of particular reaction steps during the synthesis of dendritic scaffold and subsequent peripheral functionalization, also, several products of side reactions were observed together with defects created in the course of measurement (particularly ion exchange products). Defective molecules can be to some extent separated by means of gel permeation chromatography that proves that they are not products of in source fragmentation processes. Within the reaction sequence used for the synthesis of dendrimers under study, hydrosilylation was the source of most defects; the effectivity of quarternization depends on the type of phosphine. Results confirm high sensitivity of ESI MS towards defects, stability of the carbosilane skeleton towards fragmentation under the conditions of ESI ionization, and capability to detect both lower- and higher-molecular weight impurities arising from the synthetic sequence in the same m/z range as the target dendrimer, thus providing valuable view of the polydispersity.

Synthesis and in vitro cytotoxicity of acetylated 3-fluoro, 4-fluoro and 3,4-difluoro analogs of D-glucosamine and D-galactosamine.

BACKGROUND: Derivatives of D-glucosamine and D-galactosamine represent an important family of the cell surface glycan components and their fluorinated analogs found use as metabolic inhibitors of complex glycan biosynthesis, or as probes for the study of protein-carbohydrate interactions. This work is focused on the synthesis of acetylated 3-deoxy-3-fluoro, 4-deoxy-4-fluoro and 3,4-dideoxy-3,4-difluoro analogs of D-glucosamine and D-galactosamine via 1,6-anhydrohexopyranose chemistry. Moreover, the cytotoxicity of the target compounds towards selected cancer cells is determined. RESULTS: Introduction of fluorine at C-3 was achieved by the reaction of 1,6-anhydro-2-azido-2-deoxy-4-O-benzyl-ß-D-glucopyranose or its 4-fluoro analog with DAST. The retention of configuration in this reaction is discussed. Fluorine at C-4 was installed by the reaction of 1,6:2,3-dianhydro-ß-D-talopyranose with DAST, or by fluoridolysis of 1,6:3,4-dianhydro-2-azido-ß-D-galactopyranose with KHF2. The amino group was introduced and masked as an azide in the synthesis. The 1-O-deacetylated 3-fluoro and 4-fluoro analogs of acetylated D-galactosamine inhibited proliferation of the human prostate cancer cell line PC-3 more than cisplatin and 5-fluorouracil (IC50 28 ± 3 µM and 54 ± 5 µM, respectively). CONCLUSION: A complete series of acetylated 3-fluoro, 4-fluoro and 3,4-difluoro analogs of D-glucosamine and D-galactosamine is now accessible by 1,6-anhydrohexopyranose chemistry. Intermediate fluorinated 1,6-anhydro-2-azido-hexopyranoses have potential as synthons in oligosaccharide assembly.