Influence of Selective Deoxyfluorination on the Molecular Structure of Type-2 N-Acetyllactosamine.

N-Acetyllactosamine is a common saccharide motif found in various biologically active glycans. This motif usually works as a backbone for additional modifications and thus significantly influences glycan conformational behavior and biological activity. In this work, we have investigated the type-2 N-acetyllactosamine scaffold using the complete series of its monodeoxyfluorinated analogs. These glycomimetics have been studied by molecular mechanics, quantum mechanics, X-ray crystallography, and various NMR techniques, which have provided a comprehensive and complete insight into the role of individual hydroxyl groups in the conformational behavior and lipophilicity of N-acetyllactosamine.

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.

The Effect of Deoxyfluorination on Intermolecular Interactions in the Crystal Structures of 1,6-Anhydro-2,3-epimino-hexopyranoses.

The effect of substitution on intermolecular interactions was investigated in a series of 1,6-anhydro-2,3-epimino-hexopyranoses. The study focused on the qualitative evaluation of intermolecular interactions using DFT calculations and the comparison of molecular arrangements in the crystal lattice. Altogether, ten crystal structures were compared, including two structures of C4-deoxygenated, four C4-deoxyfluorinated and four parent epimino pyranoses. It was found that the substitution of the original hydroxy group by hydrogen or fluorine leads to a weakening of the intermolecular interaction by approximately 4 kcal/mol. The strength of the intermolecular interactions was found to be in the following descending order: hydrogen bonding of hydroxy groups, hydrogen bonding of the amino group, interactions with fluorine and weak electrostatic interactions. The intermolecular interactions that involved fluorine atom were rather weak; however, they were often supported by other weak interactions. The fluorine atom was not able to substitute the role of the hydroxy group in molecular packing and the fluorine atoms interacted only weakly with the hydrogen atoms located at electropositive regions of the carbohydrate molecules. However, the fluorine interaction was not restricted to a single molecule but was spread over at least three other molecules. This feature is a base for similar molecule arrangements in the structures of related compounds, as we found for the C4-Fax and C4-Feq epimines presented here.

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.

Development of α-Selective Glycosylation for the Synthesis of Deoxyfluorinated TN Antigen Analogues.

The Tn antigen (GalNAcα1-Thr/Ser) is abundantly expressed in many tumors but rarely found in healthy tissues, which makes it an attractive epitope for antitumor immunotherapy. The use of the Tn antigen in the development of therapeutic antitumor vaccines is hampered by its low immunogenicity, which may be enhanced by deoxyfluorination of the GalNAc moiety. Here, we report the synthesis of protected 3- and 4-fluoro analogues of the threonine-containing Tn antigen. As the stereoselective synthesis of α-linked fluorinated GalNAc is difficult, we prepared a panel of C3 and C4 deoxyfluorinated galactosazide thiodonors and evaluated their stereoselectivity in the glycosylation of carbohydrate acceptors and threonine derivatives. Glycosylation of threonine derivatives with O-benzylated C4 fluoro donors gave only modest but usable α-selectivity of α/ß = 2.5-3/1. The use of acyl and silyl protection at the 3- and 6-positions of the C4 fluoro donors did not enhance the selectivity. Installing a 4,6-di-tert-butylsilylene-protecting group in C3 fluoro donors resulted in exclusive α-selectivity and reaffirmed the strong α-directing effect of this protective group in glycosylation with galacto-configured glycosyl donors.

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.

Synthesis of a Helical Phosphine and a Catalytic Study of Its Palladium Complex.

In this study, 9-(diphenylphosphanyl)[7]helicene was prepared as a suitable ligand for the subsequent synthesis of palladium complexes. The corresponding PdL2Cl2 complex was then successfully obtained in both racemic and enantiopure forms. The PdL2Cl2 complex emerges exclusively in the trans arrangement showing dynamic interconversion between its homo- and heterochiral forms as evidenced by 31P NMR. The trans arrangement was ultimately confirmed by X-ray crystallography using single crystals of the homochiral complex. Additionally, the PdL2Cl2 complex was subjected to screening of its catalytic activity in a Suzuki-type reaction of aryl bromides with aryl boronic acids showing fair yields of the resulting biaryls. However, the final asymmetric reactions catalyzed by the optically pure PdL2Cl2 complex provided targeted binaphtyls only in negligible enantiomeric excess.

Ferrocenes as new anticancer drug candidates: Determination of the mechanism of action.

Chemotherapy plays an essential role in the management of cancer worldwide. However, it is a non-specific treatment limited by major drawbacks, thus identification and testing of new promising molecular structures representing potential drug candidates are urgently needed. In this work, ferrocene complexes as potential antitumor drugs that display cytotoxicity in low micromolar concentrations against ovarian cancer cells A2780 and SK-OV-3 were investigated to identify their mode of action. Their mechanism of cellular accumulation was studied using differential pulse voltammetry and inductively coupled plasma - mass spectrometry. Their mode of cell death induction was determined by changes in the mitochondrial membrane potential, production of reactive oxygen species and by Annexin V staining. Transferrin receptors were identified as key mediators of intracellular accumulation of ferrocenes and the extent of cellular uptake reflected the anticancer activity of individual compounds. Functional analysis revealed activation of intrinsic apoptosis as a dominant mechanism leading to regulated cell death induced in ovarian cancer cells by ferrocenes. Ferrocenes represent a group of promising sandwich organometallic complexes exerting cytotoxic activity. We suggest their application not only as standalone chemotherapeutics but also as modifying substituents of known drugs to improve their antitumor effects.

Stereoselectivity in Glycosylation with Deoxofluorinated Glucosazide and Galactosazide Thiodonors.

Control of anomeric stereoselectivity in glycosylation with deoxofluorinated glycosyl donors is critical for assembly of fluorinated oligosaccharides. Here, we report the synthesis of benzylated 3-fluoro and 4-fluoro analogues of phenyl 1-thioglucosazide and galactosazide donors and evaluation of their stereoselectivity in glycosylation of a series of model carbohydrate acceptors using the Tf2O/Ph2SO promoter system. Low-temperature NMR revealed formation of covalent α-triflate and both anomers of oxosulfonium triflates under selected glycosylation conditions. This study demonstrates how the stereoselectivity depends on acceptor reactivity and glycosyl donor configuration. Reactive acceptors favor formation of 1,2- trans-ß-glycosides with both d- gluco and d- galacto donors, whereas poorly reactive acceptors favor formation of 1,2- cis-α-glycosides with d- galacto donors but are unselective with d- gluco donors.

A comprehensive LC/MS analysis of novel cyclopentenedione library.

Cyclopentenediones (CPDs) are compounds with a variety of applications ranging from the preparation of functional polymers to the development of antimicrobial agents, suggesting the potential use of CPDs as novel bioactive compounds or drugs. For this reason, a detailed characterization of CPDs and the development of robust analytical methods for their trace analysis are being sought. Here we focused on the design and synthesis of a library of novelized benzylidene CPD derivatives that were consequently characterized by ultra-high performance liquid chromatography (UHPLC) on-line connected with tandem mass spectrometry (MS/MS). The library design was based on a 2-benzylidene-4-cyclopentene-1,3-dione skeleton substituted with a variety of hydroxy, methoxy, halogen, linear aliphatic, heterocyclic and saccharide moieties, primarily modulating the skeleton's hydrophobicity. The prepared CPDs were effectively ionized by positive/negative atmospheric pressure photoionization (APPI) and atmospheric pressure chemical ionization (APCI). After careful optimization of the dopant composition and flow rate, positive-mode APPI proved to be more sensitive than APCI. In negative mode, both ionization techniques gave similar results. Further, a detailed MS fragmentation study was performed, confirming the structure of the compounds and enabling positional isomers of CPDs to be differentiated on the basis of their collision spectra analysis. Finally, an optimization of the composition of the mobile phase and reversed-phased separation mode were done, followed by a selection of the most suitable UHPLC stationary phases, i.e. C18, C8 and phenyl. The applicability of the method was evaluated by the inclusion of the other two substances in the study, i.e. monomeric and dimeric bioactive CPDs, compound TX-1123 and nostotrebin 6 with cytostatic and antimicrobial activities, respectively. The results presented here could be used in further investigations of the chromatographic retention and MS behavior of CPDs, which could be utilized for their isolation, detailed characterization and analysis in biological systems.

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.

Evaluation of cytotoxic activity of titanocene difluorides and determination of their mechanism of action in ovarian cancer cells.

BACKGROUND: Ovarian cancer is the seventh-most common cancer amongst women and the most deadly gynecologic cancer. Cisplatin based drugs are used in first line therapy, but resistance represents a major obstacle for successful treatment. In this study, we investigated the anticancer effects and mechanism of action of three titanocene difluorides, two bearing a pendant carbohydrate moiety (α-D-ribofuranos-5-yl) on their periphery and one without any substitution. RESULTS: The efficacy of these compounds on ovarian cancer cell lines was evaluated in relation to their particular chemical structure and compared with cisplatin as the most common treatment modality for this type of cancer. The typical mechanism of cisplatin action involves DNA damage, activation of p53 protein and induction of cell death, as previously described for titanium ions. Nevertheless, our data indicate that the effect of titanocene difluoride derivatives is mediated via the endoplasmic reticulum stress pathway and autophagy. CONCLUSION: We anticipate that the presence of substituents on cyclopentadienyl ring(s) might play an important role in modulation of the activity of particular compounds. Titanocene difluorides exert comparable cytotoxic activity as cisplatin and are more efficient in cisplatin-resistant cell lines. Our results suggest potential utilization of these compounds especially in the treatment of cisplatin-resistant tumor cells.

The effect of transient operations on the levels and congener profiles of PCBz, PCPh and PCDD/F in raw flue gases of MSWI plant.

The concentrations and congener profiles of polychlorinated benzenes, phenols, dibenzo-p-dioxin and dibenzofurans, measured in raw flue gases of a real MSWI plant, are discussed in this paper. The data covers steady-state and transient operation conditions, including the memory effect period close to two shutdowns. The highest levels of concentrations of the pollutants were observed the first day after the shutdown with subsequent gradual decrease towards steady-state values. The conditions of the steady-state period prior to shutdown are decisive for the increase of start-up values, memory effect values and memory effect length. We found that the above-mentioned pollutants had a different length of the memory effect period. It can be concluded that under plant transient operation the formation of pollutants by the de novo synthesis is higher than under steady-state conditions.

LC-NMR Technique in the Analysis of Phytosterols in Natural Extracts.

The ability of LC-NMR to detect simultaneously free and conjugated phytosterols in natural extracts was tested. The advantages and disadvantages of a gradient HPLC-NMR method were compared to the fast composition screening using SEC-NMR method. Fractions of free and conjugated phytosterols were isolated and analyzed by isocratic HPLC-NMR methods. The results of qualitative and quantitative analyses were in a good agreement with the literature data.

Skeletal rearrangements resulting from reactions of 1,6:2,3- and 1,6:3,4-dianhydro-ß-D-hexopyranoses with diethylaminosulphur trifluoride.

A complete series of eight 1,6:2,3- and 1,6:3,4-dianhydro-ß-D-hexopyranoses were subjected to fluorination with DAST. The 1,6:3,4-dianhydropyranoses yielded solely products of skeletal rearrangement resulting from migration of the tetrahydropyran oxygen (educts of D-altro and D-talo configuration) or of the 1,6-anhydro bridge oxygen (D-allo, D-galacto). The major products yielded by the 1,6:2,3-dianhydropyranoses were compounds arising from nucleophilic substitution, with configuration at C4 either retained (D-talo, D-gulo) or inverted (D-manno), or from C6 migration (D-allo). The minor products in the 1,6:2,3-series resulted from migration of the tetrahydropyran oxygen (D-gulo) or the oxirane oxygen (D-manno), or from nucleophilic substitution with retention of configuration (D-manno). The structure of most of the rearranged products was verified by X-ray crystallography.

Synthesis of all configurational isomers of 1,6-anhydro-2,3,4-trideoxy-2,3-epimino-4-fluoro-beta-d-hexopyranoses.

We have prepared a full series of 1,6-anhydro-2,3,4-trideoxy-4-fluoro-2,3-epimino-beta-d-hexopyranoses. The key step was the reaction of azido sulfonates possessing a free C-4 hydroxyl with DAST and subsequent LiAlH(4) reduction. Nucleophilic displacement of the hydroxyl activated by DAST proceeded without rearrangement and with moderate to good yields. A convenient synthesis of d-mannoepimine from a readily available 3-benzylamino derivative was also developed.

Synthesis of 2-aza[6]helicene and attempts to synthesize 2,14-diaza[6]helicene utilizing metal-catalyzed cycloisomerization.

A modular synthetic approach leading to 2-aza[6]helicene is reported. It involves assembly of key hetero biphenylylnaphthalenes from functionalized building blocks and study of their metal catalyzed cycloisomerization.