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Antibiotic resistance is a major worldwide concern, and new drugs with mechanistically novel modes of action are urgently needed. Here, we report the structure-based drug design, synthesis, and evaluation in vitro and in cellular systems of sialic acid derivatives able to inhibit the bacterial sialic acid symporter SiaT. We designed and synthesized 21 sialic acid derivatives and screened their affinity for SiaT by a thermal shift assay and elucidated the inhibitory mechanism through binding thermodynamics, computational methods, and inhibitory kinetic studies. The most potent compounds, which have a 180-fold higher affinity compared to the natural substrate, were tested in bacterial growth assays and indicate bacterial growth delay in methicillin-resistant

Supplementary Figures 1-3, Methods from Attenuation of Tumor Growth by Formation of Antiproliferative Glycosaminoglycans Correlates with Low Acetylation of Histone H3

In search for novel antibacterial compounds, bacterial sialic acid uptake inhibition represents a promising strategy. Sialic acid plays a critical role for growth and colonisation of several pathogenic bacteria, and its uptake inhibition in bacteria was recently demonstrated to be a viable strategy by targeting the SiaT sodium solute symporters from Proteus mirabilis and Staphylococcus aureus. Here we report the design, synthesis and evaluation of potential sialic acid uptake inhibitors bearing 4-N-piperidine and piperazine moieties. The 4-N-derivatives were obtained via 4-N-functionalization with piperidine and piperazine nucleophiles in an efficient direct substitution of the 4-O-acetate of Neu5Ac. Evaluation for binding to bacterial transport proteins with nanoDSF and ITC revealed compounds possessing nanomolar affinity for the P. mirabilis SiaT symporter. Computational analyses indicate the engagement of a previously untargeted portion of the binding site.

We present a xylosylated naphthoxyloside carrying a terminal azide functionality that can be used for conjugation using click chemistry. We show that this naphthoxyloside serves as a substrate for β4GalT7 and induces the formation of soluble glycosaminoglycan (GAG) chains with physiologically relevant lengths and sulfation patterns. Finally, we demonstrate its usefulness by conjugation to the Alexa Fluor 647 and TAMRA fluorophores and coupling to a surface plasmon resonance chip for interaction studies with the hepatocyte growth factor known to interact with the GAG heparan sulfate.

Although glycosaminoglycans (GAGs) are known to be involved in a variety of physiological and pathological processes, knowledge about their expression by cells or tissues, the GAGome, is limited. Xylosides can be used to induce the formation of GAGs without the presence of a proteoglycan core protein. The administration of xylosides to living cells tends to result in a considerable amplification in GAG production, and the xylosides can, therefore, be used as analytical tools to study the GAG produced by a certain cell type. One of the most common ways to analyze the GAGs structurally is by disaccharide analysis, which involves depolymerization of the GAGs into disaccharides, fluorescent labeling of the disaccharides with 2-aminoacridone, and quantification using high-pressure liquid chromatography (HPLC). Here, we describe the procedure of producing xyloside-primed GAGs and how to study them structurally by disaccharide analysis.

β-1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme in the synthesis of two classes of glycosaminoglycans (GAG), i.e., heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS). GAG chains are linear polysaccharides of alternating hexuronic acid and N-acetylhexosamine residues, commonly linked to core proteins to form proteoglycans with important roles in the regulation of a range of biological processes. The biosynthesis of GAGs is initiated by xylosylation of a serine residue of the core protein followed by galactosylation, catalyzed by β4GalT7. The biosynthesis can also be initiated by xylosides carrying hydrophobic aglycons, such as 2-naphthyl β-D-xylopyranoside. We have cloned and expressed β4GalT7, and designed a cell-free assay to measure the activity of this enzyme. The assay employs a 96-well plate format for high throughput. In this chapter, we describe the cloning, expression, and purification of β4GalT7, as well as assays proposed for development of substrates for GAG priming and for investigating inhibitors of β4GalT7.

Although glycosaminoglycans (GAGs) are known to be involved in a variety of physiological and pathological processes, knowledge about their expression by cells or tissues, the GAGome, is limited. Xylosides can be used to induce the formation of GAGs without the presence of a proteoglycan core protein. The administration of xylosides to living cells tends to result in a considerable amplification in GAG production, and the xylosides can, therefore, be used as analytical tools to study the GAG produced by a certain cell type. One of the most common ways to analyze the GAGs structurally is by disaccharide analysis, which involves depolymerization of the GAGs into disaccharides, fluorescent labeling of the disaccharides with 2-aminoacridone, and quantification using high-pressure liquid chromatography (HPLC). Here, we describe the procedure of producing xyloside-primed GAGs and how to study them structurally by disaccharide analysis.

β-1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme in the synthesis of two classes of glycosaminoglycans (GAG), i.e., heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS). GAG chains are linear polysaccharides of alternating hexuronic acid and N-acetylhexosamine residues, commonly linked to core proteins to form proteoglycans with important roles in the regulation of a range of biological processes. The biosynthesis of GAGs is initiated by xylosylation of a serine residue of the core protein followed by galactosylation, catalyzed by β4GalT7. The biosynthesis can also be initiated by xylosides carrying hydrophobic aglycons, such as 2-naphthyl β-D-xylopyranoside. We have cloned and expressed β4GalT7, and designed a cell-free assay to measure the activity of this enzyme. The assay employs a 96-well plate format for high throughput. In this chapter, we describe the cloning, expression, and purification of β4GalT7, as well as assays proposed for development of substrates for GAG priming and for investigating inhibitors of β4GalT7.

The glycosaminoglycan dermatan sulfate (DS) is a well-known activator of heparin cofactor II-dependent inactivation of thrombin. In contrast to heparin, dermatan sulfate has never been prepared recombinantly from material of non-animal origin. Here we report on the enzymatic synthesis of structurally well-defined DS with high anticoagulant activity. Using a microbial K4 polysaccharide and the recombinant enzymes DS-epimerase 1, dermatan 4-O-sulfotransferase 1, uronyl 2-O-sulfotransferase and N-acetylgalactosamine 4-sulfate 6-O-sulfotransferase, several new glycostructures have been prepared, such as a homogenously sulfated IdoA-GalNAc-4S polymer and its 2-O-, 6-O- and 2,6-O-sulfated derivatives. Importantly, the recombinant highly 2,4-O-sulfated DS inhibits thrombin via heparin cofactor II, approximately 20 times better than heparin, enabling manipulation of vascular and extravascular coagulation. The potential of this method can be extended to preparation of specific structures that are of importance for binding and activation of cytokines, and control of inflammation and metastasis, involving extravasation and migration.

Five novel xylosides tagged with the fluorescent probe Pacific Blue™ were synthesized and found to act as substrates for β4GalT7, a bottleneck enzyme in the biosynthetic pathways leading to glycosaminoglycans. By confocal microscopy of A549 cells, we showed that the xylosides were taken up by the cells, but did not enter the Golgi apparatus where most of the glycosaminoglycan biosynthesis occurs. Instead, after a possible double galactosylation by β4GalT7 and β3GalT6, the biosynthesis was terminated. We hypothesize this is due to the charge of the fluorescent probe, which is required for fluorescent ability and stability under physiological conditions.

Proteoglycans have important biological activities. To improve the overall synthetic efficiency, a new chemoenzymatic route has been established for the proteoglycan linkage region bearing a galactose-xylose disaccharide. The xylosylated glycopeptides were synthesized via solid phase synthesis, which was followed by the addition of the galactose unit by the galactosyl transferase β4GalT7. This work leads to a better understanding of the acceptor preference of β4GalT7 and opens the door for expeditious synthesis of the proteoglycan linkage region.

Structural studies of human DS-epi1 suggests a new catalytic isomerization mechanism and reveals remarkable similarities to bacterial proteins., Dermatan sulfate epimerase 1 (DS-epi1, EC 5.1.3.19) catalyzes the conversion of d-glucuronic acid to l-iduronic acid on the polymer level, a key step in the biosynthesis of the glycosaminoglycan dermatan sulfate. Here, we present the first crystal structure of the catalytic domains of DS-epi1, solved at 2.4 Å resolution, as well as a model of the full-length luminal protein obtained by a combination of macromolecular crystallography and targeted cross-linking mass spectrometry. Based on docking studies and molecular dynamics simulations of the protein structure and a chondroitin substrate, we suggest a novel mechanism of DS-epi1, involving a His/double-Tyr motif. Our work uncovers detailed information about the domain architecture, active site, metal-coordinating center and pattern of N-glycosylation of the protein. Additionally, the structure of DS-epi1 reveals a high structural similarity to proteins from several families of bacterial polysaccharide lyases. DS-epi1 is of great importance in a range of diseases, and the structure provides a necessary starting point for design of active site inhibitors.

The vaginal microbiome of healthy women is a diverse and dynamic system of various microorganisms. Any sudden change in microbe composition can increase the vaginal pH and thus lead to vaginal infections, conditions that affect a large percentage of women each year. The most common fungal strains involved in infections belong to the yeast species Candida albicans. The main virulence factor of C. albicans is the ability to transform from planktonic yeast-form cells into a filamentous form (hyphae or pseudohyphae), with the subsequent formation of biofilm. The hyphal form, constituted by filamentous cells, has the ability to invade tissue and induce inflammation. Our hypothesis is that certain polyhydroxylated carboxylic acids, that may serve as an alternative carbohydrate source and at the same time lower the pH, function as an indicator of a nutrient-rich environment for C. albicans, which favors planktonic cells over hyphae, and thus diminish the formation of biofilm. We have shown that the biofilm formation in C. albicans and other Candida species can be significantly reduced by the addition of glucono-δ-lactone (GDL).

Structural characterization of glycosaminoglycans remains a challenge but is essential for determining structure-function relationships between glycosaminoglycans and the biomolecules with which they interact and for gaining insight into the biosynthesis of glycosaminoglycans. We have recently reported that xyloside-primed chondroitin/dermatan sulfate derived from a human breast carcinoma cell line, HCC70, has cytotoxic effects and shown that it differs in disaccharide composition from nontoxic chondroitin/dermatan sulfate derived from a human breast fibroblast cell line, CCD-1095Sk. To further investigate the structural requirements for the cytotoxic effect, we developed a novel LC-MS/MS approach based on reversed-phase dibutylamine ion-pairing chromatography and negative-mode higher-energy collision dissociation and used it in combination with cell growth studies and disaccharide fingerprinting. This strategy enabled detailed structural characterization of linkage regions, internal oligosaccharides, and nonreducing ends, revealing not only differences between xyloside-primed chondroitin/dermatan sulfate from HCC70 cells and CCD-1095Sk cells, but also sialylation of the linkage region and previously undescribed methylation and sulfation of the nonreducing ends. Although the xyloside-primed chondroitin/dermatan sulfate from HCC70 cells was less complex in terms of presence and distribution of iduronic acid than that from CCD-1095Sk cells, both glucuronic acid and iduronic acid appeared to be essential for the cytotoxic effect. Our data have moved us one step closer to understanding the structure of the cytotoxic chondroitin/dermatan sulfate from HCC70 cells primed on xylosides and demonstrate the suitability of the LC-MS/MS approach for structural characterization of glycosaminoglycans.

β-1,4-Galactosyltransferase 7 (β4GalT7) is a key enzyme in the biosynthesis of glycosaminoglycan (GAG) chains. Natural and synthetic xylosides can be used to both inhibit and prime GAG synthesis by acting as inhibitors or substrates for β4GalT7. In this report, we exploit hydroxylated oxanes as deoxygenated xyloside analogs to clarify the minimum protein-ligand interactions required for galactosylation and/or inhibition. Enantiomerically pure substances were synthesized using a chiral pool approach whereas the corresponding racemates were obtained from simple starting materials. The results of a β4GalT7 assay show that a single hydroxyl group on an oxane ring is insufficient to induce galactosylation or inhibition, which implies that at least two substituents, one of which being 3-OH, needs to be present.

The opportunistic human fungal pathogen Candida albicans rely on cell morphological transitions to develop biofilm and invade the host. In the current study, we developed new regulatory molecules, which inhibit the morphological transition of C. albicans from yeast-form cells to cells forming hyphae. These compounds, benzyl α-L-fucopyranoside and benzyl β-D-xylopyranoside, inhibit the morphological switching and adhesion of C. albicans to a polystyrene surface, resulting in a reduced biofilm formation. The addition of cAMP to cells treated with α-L-fucopyranoside restored the yeast-hyphae switch and the biofilm level to that of the untreated control. In the β-D-xylopyranoside treated cells, the biofilm level was only partially restored by the addition of cAMP, and these cells remained mainly as yeast-form cells.

In an era characterized by a move towards a “knowledge society”, universities are central in fostering “knowledgeability”, that is the reflexive understanding of knowledge in knowledge societies. The objective of “knowledgeability” can be met through creating a stronger link between education and research. Furthermore, overall student performance, for example in critical thinking and problem solving, can be improved if research-related activities are incorporated into the curriculum. The aim of this paper is to use inter-national examples to discuss the research- education nexus from four different perspectives, namely context, policy, im- plementation and quality, with case studies from higher education institutions in Singapore and Sweden. We suggest that different integrative technologies can be used to enhance the links, but it will be essential to consider the inputs of training, service and support in using new technology. Interestingly, the act of evaluating the link between edu- cation and research will increase awareness of this linkage by stakeholders involved in both education and research. In turn the link can be strengthened, contributing to increased quality in both education and research.

The vaginal microbiome of healthy women is a diverse and dynamic system of various microorganisms. Any sudden change in microbe composition can increase the vaginal pH and thus lead to vaginal infections, conditions that affect a large percentage of women each year. The most common fungal strains involved in infections belong to the yeast species

During the biosynthesis of chondroitin/dermatan sulfate (CS/DS), a variable fraction of glucuronic acid is converted to iduronic acid through the activities of two epimerases, dermatan sulfate epimerases 1 (DS-epi1) and 2 (DS-epi2). Previous in vitro studies indicated that without association with other enzymes, DS-epi1 activity produces structures that have only a few adjacent iduronic acid units. In vivo, concomitant with epimerization, dermatan 4-O-sulfotransferase 1 (D4ST1) sulfates the GalNAc adjacent to iduronic acid. This sulfation facilitates DS-epi1 activity and enables the formation of long blocks of sulfated iduronic acid–containing domains, which can be major components of CS/DS. In this report, we used recombinant enzymes to confirm the concerted action of DS-epi1 and D4ST1. Confocal microscopy revealed that these two enzymes colocalize to the Golgi, and FRET experiments indicated that they physically interact. Furthermore, FRET, immunoprecipitation, and cross-linking experiments also revealed that DS-epi1, DS-epi2, and D4ST1 form homomers and are all part of a hetero-oligomeric complex where D4ST1 directly interacts with DS-epi1, but not with DS-epi2. The cooperation of DS-epi1 with D4ST1 may therefore explain the processive mode of the formation of iduronic acid blocks. In conclusion, the iduronic acid–forming enzymes operate in complexes, similar to other enzymes active in glycosaminoglycan biosynthesis. This knowledge shed light on regulatory mechanisms controlling the biosynthesis of the structurally diverse CS/DS molecule.

Xylosides can induce the formation and secretion of xyloside-primed glycosaminoglycans when administered to living cells; however, their impact on the detailed glycosaminoglycan structure remains unknown. Here, we have systematically investigated how the xyloside concentration and the type of xyloside, as well as the cell type, influenced the structure of xyloside-primed glycosaminoglycans in terms of the heparan sulfate and chondroitin/dermatan sulfate proportion and disaccharide composition. We found that although greatest influence was exerted by the cell type, both the xyloside concentration and type of xyloside impacted the proportion of heparan sulfate and the complexity of chondroitin/dermatan sulfate. The disaccharide composition of the chondroitin/dermatan sulfate was influenced by the xyloside concentration and type of xyloside to a higher extent than that of the heparan sulfate; the proportion of 4S-sulfated disaccharides in the chondroitin/dermatan sulfate decreased and the proportions of 6S-sulfated and/or nonsulfated disaccharides increased both with increasing concentrations of xyloside and with increasing xyloside hydrophobicity, whereas the proportion of nonsulfated disaccharides was primarily altered in the heparan sulfate with increasing concentrations of xyloside. Our results indicate that it is feasible to not only produce large amounts of glycosaminoglycans in living cells but also to fine-tune their structures by using xylosides of different types and at different concentrations.

During the biosynthesis of chondroitin/dermatan sulfate (CS/DS), a variable fraction of glucuronic acid is converted to iduronic acid through the activities of two epimerases, dermatan sulfate epimerases 1 (DS-epi1) and 2 (DS-epi2). Previous in vitro studies indicated that without association with other enzymes, DS-epi1 activity produces structures that have only a few adjacent iduronic acid units. In vivo, concomitant with epimerization, dermatan 4-O-sulfotransferase 1 (D4ST1) sulfates the GalNAc adjacent to iduronic acid. This sulfation facilitates DS-epi1 activity and enables the formation of long blocks of sulfated iduronic acid-containing domains, which can be major components of CS/DS. In this report, we used recombinant enzymes to confirm the concerted action of DS-epi1 and D4ST1. Confocal microscopy revealed that these two enzymes colocalize to the Golgi, and FRET experiments indicated that they physically interact. Furthermore, FRET, immunoprecipitation, and cross-linking experiments also revealed that DS-epi1, DS-epi2, and D4ST1 form homomers and are all part of a hetero-oligomeric complex where D4ST1 directly interacts with DS-epi1, but not with DS-epi2. The cooperation of DS-epi1 with D4ST1 may therefore explain the processive mode of the formation of iduronic acid blocks. In conclusion, the iduronic acid-forming enzymes operate in complexes, similar to other enzymes active in glycosaminoglycan biosynthesis. This knowledge shed light on regulatory mechanisms controlling the biosynthesis of the structurally diverse CS/DS molecule.

Monosubstituted naphthoxylosides have been shown to function as substrates for, and inhibitors of, the enzyme β4GalT7, a key enzyme in the biosynthetic pathway leading to glycosaminoglycans and proteoglycans. In this article, we explore the synthesis of 16 xyloside analogues, modified at two different positions, as well as their function as inhibitors of and/or substrates for the enzyme. Seemingly simple compounds turned out to require complex synthetic pathways. A meta-analysis of the synthetic work shows that, regardless of the abundance of methods available for carbohydrate synthesis, even simple modifications can turn out to be problematic, and double modifications present additional challenges due to conformational, steric, and stereoelectronic effects.

International audience; Xyloside analogues with substitution of the endocyclic oxygen atom by sulfur or carbon were investigated as substrates for β-1,4-galactosyltransferase 7 (β4GalT7), a key enzyme in the biosynthesis of glycosaminoglycan chains. The analogues with an endocyclic sulfur atom proved to be excellent substrates for β4GalT7, and were galactosylated approximately fifteen times more efficiently than the corresponding xyloside. The 5a-carba-β-xylopyranoside in the d-configuration proved to be a good substrate for β4GalT7, whereas the enantiomer in the l-configuration showed no activity. Further investigations by X-ray crystallography, NMR spectroscopy, and molecular modeling provided a rationale for the pronounced activity of the sulfur analogues. Favorable π-π interactions between the 2-naphthyl moiety and a tyrosine side chain of the enzyme were observed for the thio analogues, which open up for the design of efficient GAG primers and inhibitors.

Glycosaminoglycans contain a beta-D-xylopyranose residue at its reducing end, which links the polysaccharide to the protein in proteoglycans. 2-Naphthyl beta-D-xylopyranosides have shown inhibition of tumor growth and we herein investigate conformation and dynamics of compounds structurally and stereochemically modified at the C3 position as well as the influence of solvent. The 3-deoxygenated compound, the 3-C-methyl-substituted beta-D-xylopyranoside, beta-D-ribopyranoside, the 3-C-methyl-substituted beta-D-ribopyranoside as well as 2-naphthyl beta-D-xylopyranoside were analyzed by NMR spectroscopy. Conformational equilibria were dependent on the solvent of choice, either methanol-d(4) or chloroform-d, with mainly C-4(1) and C-1(4) conformations present but also skew conformations to some extent. Intramolecular hydrogen bonding was concluded to be important for the 3-C-methyl-substituted beta-D-xylopyranosides in the non-polar solvent. Dynamic NMR (DNMR) spectroscopy was carried out for the 3-deoxygenated compound, which at 25 degrees C in methanol-d(4) exists with equally populated states of the C-4(1) and the C-1(4) conformations, but at -100 degrees C only a few percent is present of the latter. Using C-13 NMR detection for DNMR, resonance lines were shown to broaden at -40 degrees C and to sharpen again below -90 degrees C, without the emergence of a second set of NMR resonances, a typical behavior for an unequally populated equilibrium. The enthalpy and entropy activation barriers were calculated and resulted in Delta H-double dagger = 47.3 kJ mol(-1) and Delta S-double dagger = 54 J mol(-1) K-1. AuthorCount:4

We previously reported that the xyloside 2-(6-hydroxynaphthyl) β-d-xylopyranoside (XylNapOH), in contrast to 2-naphthyl β-d-xylopyranoside (XylNap), specifically reduces tumor growth both in vitro and in vivo. Although there are indications that this could be mediated by the xyloside-primed glycosaminoglycans (GAGs) and that these differ in composition depending on xyloside and cell type, detailed knowledge regarding a structure-function relationship is lacking. In this study we isolated XylNapOH- and XylNap-primed GAGs from a breast carcinoma cell line, HCC70, and a breast fibroblast cell line, CCD-1095Sk, and demonstrated that both XylNapOH- and XylNap-primed chondroitin sulfate/dermatan sulfate GAGs derived from HCC70 cells had a cytotoxic effect on HCC70 cells and CCD-1095Sk cells. The cytotoxic effect appeared to be mediated by induction of apoptosis and was inhibited in a concentration-dependent manner by the XylNap-primed heparan sulfate GAGs. In contrast, neither the chondroitin sulfate/dermatan sulfate nor the heparan sulfate derived from CCD-1095Sk cells primed on XylNapOH or XylNap had any effect on the growth of HCC70 cells or CCD-105Sk cells. These observations were related to the disaccharide composition of the XylNapOH- and XylNap-primed GAGs, which differed between the two cell lines but was similar when the GAGs were derived from the same cell line. To our knowledge this is the first report on cytotoxic effects mediated by chondroitin sulfate/dermatan sulfate.

The use of benzylidene acetals as protecting groups in carbohydrate chemistry is utterly important. The main advantage of benzylidene acetal is the ability for regioselective openings. 4,6-benzylidene acetal can be opened selectively under reductive conditions to yield either free 4-OH or 6-OH. There are a plethora of methods available for regioselective openings, but only a few of these are widely used. In recent years, the mechanism has been investigated for borane mediated openings and it seems likely that the regioselectivity is determined by borane, rather than Lewis acid. When borane is activated by Lewis acids, borane is the most electrophilic species that consequently coordinates to the most nucleophilic oxygen of the acetals, usually O-6. This results in the formation of 6-O-benzyl ethers. If borane is not activated, Lewis acid is the most electrophilic species that thus adds to O-6 and hence generates the 4-O-benzyl ether.

Despite the importance of regioselective reductive openings of cyclic acetals, mechanistic details are scarce. In this study 4,6-O-benzylidene acetals were used as model compounds for deciphering the mechanism of regioselective openings using a variety of reducing agents. Competitive isotopic studies aiming at primary and secondary isotope effects, as well as an electron-deficient substrate, were used to evaluate stereo- and regioselectivity. We show that there are three distinctly different mechanistic pathways. In nonpolar solvents, such as toluene, the acetal is activated by the very reactive naked Lewis acid to give a fully developed oxocarbenium ion that is then reduced by the borane, with low stereoselectivity. In THF the reactivity of the Lewis acid is moderated by complex formation with the solvent. These reactions are thus much slower and proceed through an intimate ion pair and thereby show high stereoselectivities. The regioselectivity in these reactions is directed by the interaction between the Lewis acid and the most nucleophilic oxygen of the acetal, thus yielding a free 6-hydroxyl group. Finally, boranes such as BH(3)·NMe(3) are activated by Lewis acid, which results in the borane being the most electrophilic species, and consequently the reaction shows inversed regioselectivity to give a free 4-hydroxyl group. These reactions proceed through an oxocarbenium ion and thus show low stereoselectivity.

Glycosaminoglycan (GAG) chains anchored to core proteins form proteoglycans, widely distributed cell-surface macromolecules with multiple functions, such as regulation of growth factor and cytokine signaling, cell-cell interactions, and uptake of biomolecules. The biosynthesis of GAG can be manipulated by xylosides attached to various hydrophobic groups, and we have earlier reported that a naphthoxyloside, 2-(6-hydroxynaphthyl) β-d-xylopyranoside (XylNapOH), which serves as a primer for GAG synthesis, reduces tumor load up to 97% in vivo, despite lower efficiency in vitro. Here we show, using radiolabeled xylosides and coculture experiments, that XylNapOH-treated bladder and breast carcinoma cells secrete antiproliferative GAG chains that are taken up by both normal and cancer cells and transported to the cell nuclei where they induce an antiproliferative effect, accompanied by apoptosis. We also show that XylNapOH treatment lowers the level of histone H3 acetylation selectively in bladder and breast carcinoma cells without affecting expression of histone H3. However, XylNapOH-primed GAG chains from normal cells are not internalized and do not cause growth retardation. Using in vitro and in vivo C6 glioma cell and tumor models, we show that XylNapOH is much more effective in vivo than in vitro. We propose that, in vivo, the antiproliferative XylNapOH-primed GAG chains produced by tumor cells inhibit tumor growth in an autocrine fashion by formation of antiproliferative GAG chains on the xyloside prodrug, whereas no antiproliferative GAG chains are produced by surrounding normal cells. This is a novel mechanism for targeting tumor cells, making these xylosides promising drug candidates for antitumor therapy. Cancer Res; 70(9); 3771–9. ©2010 AACR.

Three different series of xylosylated naphthoic acid-amino acid conjugates containing one or two amino acid residues were synthesized for the investigation of glycosaminoglycan priming and potential use as anti-tumor drugs. All xylosylated naphthoic acid-conjugates inhibited the growth of normal lung fibroblasts to some extent, whereas the growth of tumor derived T24 carcinoma cells was not affected. There was no correlation between amino acid conjugation, retention time and the antiproliferative activity. Only one compound initiated the priming of glycosaminoglycans. Modification of the naphthalene ring with one or two amino acid residues did not have any effect on proteoglycan biosynthesis or glycosaminoglycan priming in T24 carcinoma cells.

The mechanisms of regioselective reductive openings of acetals were investigated in several model systems by a combination of Hammett plots, kinetic experiments, density functional calculations, and (11)B NMR. The regioselectivity of borane reductions of cyclic acetals can be controlled by the choice of borane. Lewis acid activation of BH3 x NMe3 increases the reaction rate and renders the borane the most electrophilic species, which associates to the more electron-rich oxygen of the acetal. In contrary, without activation, the regioselectivity is instead directed by the Lewis acid, as exemplified by the reaction with BH3 x THF.

The mechanism for the acid-mediated substitution of a phenolic hydroxyl group with a sulfur nucleophile has been investigated by a combination of experimental and theoretical methods. We conclude that the mechanism is distinctively different in nonpolar solvents (i.e., toluene) compared with polar solvents. The cationic mechanism, proposed for the reaction in polar solvents, is not feasible and the reaction instead proceeds through a multistep mechanism in which the acid (pTsOH) mediates the proton shuffling. From DFT calculations, we found a rate-determining transition state with protonation of the hydroxyl group to generate free water and a tight ion pair between a cationic protonated naphthalene species and a tosylate anion. Kinetic experiments support this mechanism and show that, at moderate concentrations, the reaction is first order with respect to 2-naphthol, n-propanethiol, and p-toluenesulfonic acid (pTsOH). Experimentally determined activation parameters are similar to the calculated values (DeltaH(exp) ( not equal)=105+/-9, DeltaH(calcd) ( not equal)=118 kJ mol(-1); DeltaG(exp) ( not equal)=112+/-18, DeltaG(calcd) ( not equal)=142 kJ mol(-1)).

Distinct from template-directed biosynthesis of nucleic acids and proteins, the enzymatic synthesis of heterogeneous polysaccharides is a complex process that is difficult to study using common analytical tools. Therefore, the mode of action and processivity of those enzymes are largely unknown. Dermatan sulfate epimerase 1 (DS-epi1) is the predominant enzyme during the formation of iduronic acid residues in the glycosaminoglycan dermatan sulfate. Using recombinant DS-epi1 as a model enzyme, we describe a tandem mass spectrometry-based method to study the mode of action of polysaccharide processing enzymes. The enzyme action on the substrate was monitored by hydrogen–deuterium exchange mass spectrometry and the sequence information was then fed into mathematical models with two different assumptions of the mode of action for the enzyme: processive reducing end to non-reducing end, and processive non-reducing end to reducing end. Model data was scored by correlation to experimental data and it was found that DS-epi1 attacks its substrate on a random position, followed by a processive mode of modification towards the non-reducing end and that the substrate affinity of the enzyme is negatively affected by each additional epimerization event. It could also be shown that the smallest active substrate was the reducing end uronic acid in a tetrasaccharide and that octasaccharides and longer oligosaccharides were optimal substrates. The method of using tandem mass spectrometry to generate sequence information of the complex enzymatic products in combination with in silico modeling can be potentially applied to study the mode of action of other enzymes involved in polysaccharide biosynthesis.

Thin layer chromatography (TLC) using staining reagents is a superior method for analyzing organic compounds without chromophores. It is fast, versatile and sometimes the only viable method. We have investigated quantitative TLC using staining reagents, in combination with modern image analysis software. Our results show that it is possible to get reliable measurements, suitable for high-throughput screening or physical organic investigations. The range of detection and the errors for the different parts of the process are illustrated. We show that the errors are largely due to the staining process and can be diminished by measuring ratios of compounds.

Three series of sulfur-containing analogs to the selectively antiproliferative 2-(6-hydroxynaphthyl) beta-D-xylopyranoside were synthesized and their biological properties investigated. A short, general route to hydroxynaphthyl disulfides from dihydroxynaphthalenes was developed to utilize the disulfide bond as a sulfur-selective protecting group to enable the orthogonal protection of hydroxyls and thiols. The results indicate that hydrophobic, uncharged oxygen-sulfur substituted naphthoxylosides are taken up by cells and initiate priming of GAG chains to a greater extent compared to the oxygen analogs. No correlation between priming ability and antiproliferative activity was observed.

Xylosides are a group of compounds that can induce glycosaminoglycan (GAG) chain synthesis independently of a proteoglycan core protein. We have previously shown that the xyloside 2-(6-hydroxynaphthyl)β-D-xylopyranoside has a tumor-selective growth inhibitory effect both in vitro and in vivo, and that the effect in vitro was correlated to a reduction in histone H3 acetylation. In addition, GAG chains have previously been reported to inhibit histone acetyltransferases (HAT). To investigate if xylosides, or the corresponding xyloside-primed GAG chains, can be used as HAT inhibitors, we have synthesized a series of naphthoxylosides carrying structural motifs similar to the aromatic moieties of the known HAT inhibitors garcinol and curcumin, and studied their biological activities. Here, we show that the disubstituted naphthoxylosides induced GAG chain synthesis, and that the ones with at least one free phenolic group exhibited moderate HAT inhibition in vitro, without affecting histone H3 acetylation in cell culture. The xyloside-primed GAG chains, on the other hand, had no effect on HAT activity, possibly explaining why the effect of the xylosides on histone H3 acetylation was absent in cell culture as the xylosides were recruited for GAG chain synthesis. Further investigations are required to find xylosides that are effective HAT inhibitors or xylosides producing GAG chains with HAT inhibitory effects.

Two formally C-xylosylated analogs to 2-naphthyl beta-D-xylopyranoside, which is known to initiate priming of glucosaminoglycan chains, were synthesized by a position inversion of glucose (i.e., position 1 becomes position 5). The D-C-xyloside showed priming, while the L-C-xyloside did not initiate priming.

We here study the interactions of a polyamide with large DNA, and compare to those of minor groove binder distamycin (DST), including high ligand/DNA binding ratios. Specific as well as nonspecific binding is probed using polarized-light spectroscopy combined with singular value decomposition analysis. Circular and linear dichroism data confirm binding geometries consistent with minor groove binding for both of the ligands. Interestingly, at high and intermediate ligand/DNA ratios the polyamide exhibits no significant sequence discrimination between mixed-sequence (calf thymus) and AT DNA as compared to DST. Each ligand is concluded to exhibit two different binding modes depending upon ligand/DNA ratio and nucleo-base sequence. At high binding ratios, distinct differences between the ligands are observed: circular dichroism spectra exciton effects provide evidence of bimolecular interactions of the polyamide when bound to AT-DNA, whereas no effects are seen with DST or mixed-sequence DNA. Also linear dichroism indicates that a change in binding geometry occurs at high polyamide/AT ratios, and that the effect occurs only with polyamide in contrast to DST. Since the effect is insignificant with DST, or with calf thymus DNA, it is concluded that it relates to the sizes of the ligands and the minor grooves, becoming critical in the limit of crowding.

Aryl iodides are important synthetic intermediates that can be transformed into tritium labelled compounds by metal-mediated hydrodehalogenation and also react in a number of important synthetic transformations. We present ICI/ln(OTf)(3) as a new reagent combination for mild iodination, suitable for acid-sensitive substrates such as carbohydrates. (c) 2005 Elsevier Ltd. All rights reserved.

2-Bromoethyl glycosides can easily and in high yields be transformed into sulfones by treatment with a suitable thiol followed by oxidation with mCPBA. The observation that the so formed sulfones were cleaved by treatment with NaOMe/MeOH was used to design a new safety catch linker for synthesis of glycoconjugates on solid support. (C) 2005 Elsevier Ltd. All rights reserved.

Proteoglycans (PGs) are macromolecules that consist of long linear polysaccharides, glycosaminoglycan (GAG) chains, covalently attached to a core protein by the carbohydrate xylose. The biosynthesis of GAG chains is initiated by xylosylation of the core protein followed by galactosylation by the galactosyltransferase beta 4GalT7. Some beta-D-xylosides, such as 2-naphthyl beta-D-xylopyranoside, can induce GAG synthesis by serving as acceptor substrates for beta 4GalT7 and by that also compete with the GAG synthesis on core proteins. Here we present structure-activity relationships for beta 4GalT7 and xylosides with modifications of the aromatic aglycon, using enzymatic assays, cell studies, and molecular docking simulations. The results show that the aglycons reside on the outside of the active site of the enzyme and that quite bulky aglycons are accepted. By separating the aromatic aglycon from the xylose moiety by linkers, a trend towards increased galactosylation with increased linker length is observed. The galactosylation is influenced by the identity and position of substituents in the aromatic framework, and generally, only xylosides with beta-glycosidic linkages function as good substrates for beta 4GalT7. We also show that the galactosylation ability of a xyloside is increased by replacing the anomeric oxygen with sulfur, but decreased by replacing it with carbon. Finally, we propose that reaction kinetics of galactosylation by beta 4GalT7 is dependent on subtle differences in orientation of the xylose moiety. AuthorCount:11

Xylose is one of the few monosaccharidic building blocks that are used by mammalian cells. In comparison with other monosaccharides, xylose is rather unusual and, so far, only found in two different mammalian structures, i.e. in the Notch receptor and as the linker between protein and glycosaminoglycan (GAG) chains in proteoglycans. Interestingly, simple soluble xylopyranosides can not only initiate the biosynthesis of soluble GAG chains but also function as inhibitors of important enzymes in the biosynthesis of proteoglycans. Furthermore, xylose is a major constituent of hemicellulosic xylans and thus one of the most abundant carbohydrates on Earth. Altogether, this has spurred a strong interest in xylose chemistry. The scope of this review is to describe synthesis of xylopyranosyl donors, as well as protective group chemistry, modifications, and conformational analysis of xylose.

We have studied interhalogen/silver trifluoromethanesulfonate (IX/AgOTf) promoted glycosylations and found differences in the sensitivity of the formed oxocarbenium ions (e.g. from compounds with or without participating groups) toward halide nucleophiles. These differences can be explained using the HSAB theory. By applying this theory on sialylations, we increased the yield for a model reaction from a highly unpredictable 35-46% using ICl to 74% using IBr. We have also showed that the most prominent role of the silver ions is lowering the concentration of the halide nucleophile rather than activating the interhalogen compound and, by increasing the amount of AgOTf from 1 to 1.5 equiv (with respect to IBr), the yield in the model reaction improved from 74% to 89%. A comparison of two different anomeric leaving groups showed that glycal formation can be minimized using a thiophenyl donor instead of xanthate. By combining these observations, we were able to increase the yield of the model reaction to 97%.

In order to investigate the selective antiproliferative effects shown by 2-(6-hydroxynaphthyl)-beta-D-xylopyranoside the 14 possible beta-D-xylopyranosidic compounds were synthesized on solid support. An aminomethylated polystyrene resin was converted into an acid chloride resin and then esterified using dihydroxynaphthalene. The free hydroxy group was then xylosylated Under BF3.OEt2 mediated conditions. The xyloside was deprotected and simultaneously cleaved off the resin Using NaOMe/MeOH. Final purification using reverse phase HPLC gave the pure xylosides in 6-42% yield with Virtually no formation of alpha-xylosides. (C) 2002 Elsevier Science Ltd. All rights reserved.

In order to expand the recognition code by hairpin polyamides to include DNA sequences of the type 5'-CWWC-3' two polyamides, PyPyPyPy-(R)(H2N)gamma-ImPyPyIm-beta-Dp (1) and PyPyPyPy-(R)(H2N)gamma-ImPy-beta-Im-beta-Dp (2) were synthesized which have in common an Py/Im pair in the terminal position for targeting C x G but differ with respect to internal placement of a beta-alanine residue. The equilibrium association constants (Ka) were determined at four DNA sites which differ at a single common position, 5'-TNTACA-3' (N = T, A, G, C). Quantitative DNase I footprint titration experiments reveal that the eight-ring hairpin PyPyPyPy-(R)(H2N)gamma-ImPyPyIm-beta-Dp (1) binds the four binding sites with similar affinities, Ka = 1.3-1.9 x 10(10) M(-1) indicating that there is no preference for the position N. In contrast, a redesigned polyamide PyPyPyPy-(R)(H2N)gamma-ImPy-beta-Im-beta-Dp (2) that places an internal flexible aliphatic beta-alanine to the 5'-side of a key imidazole group bound the match site 5'-TCTACA-3' with high affinity and good sequence discrimination (Ka(match) = 4.9 x 10(10) M(-1) and the single base pair mismatch sites with 5- to 25-fold lower affinity). These results expand the repertoire of sequences targetable by hairpins and emphasize the importance of beta-alanine as a key element for minor groove recognition.

An improved, chemoselective hydrogenolysis method has been developed. By employing a solvent-acid combination (i.e., DMF-aq HCl) we were able to favor debenzylation rather than aromatic hydrogenation and acid-mediated bond cleavage which are the two main drawbacks of these reactions. The generality of the method, which was primarily developed as a solution to a carbohydrate problem, is shown by the successful hydrogenolysis of 1,8-naphthalide, a previously unsolved problem. (C) 2010 Elsevier Ltd. All rights reserved.

Virtually complete regioselective galactosylation of the diol acceptor p-methoxyphenyl 6-O-benzyl-2-deoxy-2-tetrachlorophthalimido-β-d-glucopyranoside (8) with the donor phenyl 2,3,4-tri-O-acetyl-6...

Virtually complete regioselective galactosylation of the diol acceptor p-methoxyphenyl 6-O-benzyl-2-deoxy-2-tetrachlorophthalimido-β-d-glucopyranoside (15) with the donors ethyl 3,4-di-O-acetyl-6-O-benzyl-2-deoxy-2-[[(2,2,2-trichloroethoxy)carbonyl]amino]-1-thio-β-d-galactopyranoside (14), 4methylphenyl 2,3-di-O-acetyl-4-azido-6-O-benzyl-4-deoxy-1-thio-β-d-galactopyranoside (30), and 4-methylphenyl 2-O-acetyl-4-azido-6-O-benzyl-4-deoxy-3-O-(methoxyethanoyl)-1-thio-β-d-galactopyranoside (44) gave the lactose-diamine derivatives 16, 33, and 45, respectively. Fucosylation of the NHAc derivatives of 16 and 33 (17 and 34) with the donor 18 gave, after deprotection and N-acetylation, the 2-NHAc-Lex and 4-NHAc-Lex trisaccharides 3 and 5, respectively. Removal of the Troc group from the tetrasaccharide intermediate 23, followed by N-acetylation (→ 24), gave the NHAc-SLex tetrasaccharide 2. Regioselective sialylation of the partially protected trisaccharide diols 21 and 37 with the sialyl donors 22 and 38 gave, af...

Boron trifluoride etherate-induced glucosylation of methanol, 1-propanol, 2-propanol, 2-bromoethanol, and 3-bromo-2-(bromomethyl)propan-1-ol, using 1,2,3,4,6-penta-O-acetyl-β-D-glucopyranose as donor, gave the corresponding β-glucopyranosides. The α-glucosides and 1,2,3,4,6-penta-O-acetyl-α-D-glucopyranose were formed as byproducts in varying amounts, according to GLC analysis. The propensity of the different glucopyranosides to anomerize was determined in separate experiments. 1. Dedicated to the memory of Professor Akira Hasegawa.