Synthesis and cytotoxicity of the conjugates of diterpenoid isosteviol and N-acetyl-D-glucosamine.

A series of conjugates of diterpenoid isosteviol (16-oxo-ent-beyeran-19-oic acid) and N-acetyl-D-glucosamine was synthesised and their cytotoxicity against several human cancer cell lines (M-Hela, MCF-7, Hep G2, Panc-1, PC-3), as well as normal human cell lines (WI-38, Chang liver) was assayed. Most of the conjugates were found to be cytotoxic against the mentioned cancer cell lines in the range of IC50 values 13-89 µM. Two lead compounds 14a and 14b showed selective cytotoxicity against M-Hela (IC50 13 and 14 µM) that was two times as high as the cytotoxicity of the anti-cancer drug Tamoxifen in control (IC50 28 µM). It was found that cytotoxic activity of the lead compounds against M-Hela cells is due to induction of apoptosis.

Large scale production of lacto-N-biose I, a building block of type I human milk oligosaccharides, using sugar phosphorylases.

Human milk oligosaccharides (HMOs) have drawn attention for their contribution to the explosive bifidobacterial growth in the intestines of neonates. We found that bifidobacteria can efficiently metabolize lacto-N-biose I (LNB), the major building blocks of HMOs, and we have developed a method to synthesize LNB by applying this system. We produced LNB on a kilogram scale by the method. This proved that, among the enterobacteria, only bifidobacteria can assimilate LNB, and provided the data that supported the explosive growth of bifidobacteria in neonates. Furthermore, we were also able to reveal the structure of LNB crystal and the low stability for heating at neutral pH, which has not been clarified so far. In this paper, using bifidobacteria and LNB as examples, I describe the research on oligosaccharide synthesis that was conducted by utilizing a sugar metabolism.Abbreviations: LNB: lacto-N-biose I; GNB: galacto-N-biose; HMOs: human milk oligosaccharides; GLNBP: GNB/LNB phosphorylase; NahK: N-acetylhexosamine 1-kinase; GalT: UDP-glucose-hexose-1-phosphate uridylyltransferase; GalE: UDP-glucose 4-epimerase; SP: sucrose phosphorylase.

Synthesis of defined mono-de-N-acetylated ß-(1→6)-N-acetyl-d-glucosamine oligosaccharides to characterize PgaB hydrolase activity.

Many clinically-relevant biofilm-forming bacterial strains produce partially de-N-acetylated poly-ß-(1→6)-N-acetyl-d-glucosamine (dPNAG) as an exopolysaccharide. In Gram-negative bacteria, the periplasmic protein PgaB is responsible for partial de-N-acetylation of PNAG prior to its export to the extracellular space. In addition to de-N-acetylase activity found in the N-terminal domain, PgaB contains a C-terminal hydrolase domain that can disrupt dPNAG-dependent biofilms and hydrolyzes dPNAG but not fully acetylated PNAG. The role of this C-terminal domain in biofilm formation has yet to be determined in vivo. Further characterization of the enzyme's hydrolase activity has been hampered by a lack of specific dPNAG oligosaccharides. Here, we report the synthesis of a defined mono de-N-acetylated dPNAG penta- and hepta-saccharide. Using mass spectrometry analysis and a fluorescence-based thin-layer chromatography (TLC) assay, we found that our defined dPNAG oligosaccharides are hydrolase substrates. In addition to the expected cleavage site, two residues to the reducing side of the de-N-acetylated residue, minor cleavage products on the non-reducing side of the de-N-acetylation site were observed. These findings provide quantitative data to support how PNAG is processed in Gram-negative bacteria.

Multivalent assembly of a pyrene functionalized thio-N-acetylglucosamine: Synthesis, spectroscopic and WGA binding studies.

We introduce here a new fluorescent derivative of 1-thio-ß-N-acetylglucosamine linked to a pyrene system through a triazolylpentyl spacer, designed to self-assemble into a multivalent glycocluster. The synthesis was achieved by efficient CuAAC click reaction between a pyrene functionalized with an azide group and a suitable alkynyl thiomonosaccharide. Spectroscopic studies by fluorometry indicated that the self-assembly in aqueous medium is modulated by concentration and pH changes, the latter due to the presence of the amino group close to the π system. Circular dichroism experiments revealed a moderate positive signal, suggesting that the pyrene-thioGlcNAc conjugate can aggregate into a chiral supramolecular assembly. The sugar moiety showed to specifically and reversibly interact with the wheat germ agglutinin, a fact that was demonstrated by turbidity assay. SEM microscopy of a lyophilized solution at pH 10 revealed a fibrillar morphology compatible with the presence of tubular micelles, whereas crystalline and amorphous solids are formed at lower pHs.

Enzymatic hydrolysis of ionic liquid-extracted chitin.

Chitin, one of Nature's most abundant biopolymers, can be obtained by either traditional chemical pulping or by extraction using the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate. The IL extraction and coagulation process provides access to a unique chitin, with an open hydrated gel-like structure. Here, enzymatic hydrolysis of this chitin hydrogel, dried shrimp shell, chitin extracted from shrimp shells using IL and then dried, and commercial chitin was carried out using chitinase from Streptomyces griseus. The enzymatic hydrolysis of shrimp shells resulted only in the monomer N-acetylglucosamine, while much higher amounts of the dimer (N,N'-diacetylchitobiose) compared to the monomer were detected when using all forms of 'pure' chitin. Interestingly, small amounts of the trimer (N,N',N''-triacetylchitotriose) were also detected when the IL-chitin hydrogel was used as substrate. Altogether, our findings indicate that the product distribution and yield are highly dependent on the substrate selected for the reaction and its hydrated state.

A constraint scaffold enhances affinity of a bivalent N-acetylglucosamine ligand against wheat germ agglutinin.

Bivalent glycoconjugates have a minimal valence with avidity potential on protein-carbohydrate interactions as well as simplicity of chemical structures enabling simple synthesis with low cost. Understanding the way to maximize the affinities of bivalent glycoconjugates is important for the development of cost-effective tools for therapeutic and diagnostic research. However, there has been little discussion about the effects of constraints imposed from ligand scaffolds on the binding abilities. We synthesized three kinds of biantennary N-acetylglucosamine glycosides with different scaffolds using isobutenyl bis(propargyl)ether as a common scaffold precursor. Decoration of the scaffold branches with GlcNAc moieties through copper-catalyzed azide-alkyne cycloaddition and grafting of the alkenyl focal point to another bivalent biotin dendron through thiol-ene and nucleophilic substitution reactions were successfully carried out in an orthogonal manner. The association constants of the ligands against wheat germ agglutinin were determined by a fluorometric titration assay. A bivalent biotin counterpart provided higher affinity than an isobutyl scaffold, whereas an isobutenyl scaffold yielded more enhancement than a bivalent biotin counterpart. The present work suggested that the constraint and steric bulk of ligand scaffolds are possible factors for improving binding properties of glycoconjugates against lectins or proteins.

Intracellular Imaging of Protein-Specific Glycosylation.

Posttranslational protein glycosylation is conserved in all kingdoms of life and implicated in the regulation of protein structure, function, and localization. The visualization of glycosylation states of designated proteins within living cells is of great importance for unraveling the biological roles of intracellular protein glycosylation. Our generally applicable approach is based on the incorporation of a glucosamine analog, Ac4GlcNCyoc, into the cellular glycome via metabolic engineering. Ac4GlcNCyoc can be labeled in a second step via inverse-electron-demand Diels-Alder chemistry with fluorophores inside living cells. Additionally, target proteins can be expressed as enhanced green fluorescent protein (EGFP)-fusion proteins. To assess the proximity of the donor EGFP and the glycan-anchored acceptor fluorophore, Förster resonance energy transfer (FRET) is employed and read out with high contrast by fluorescence lifetime imaging (FLIM) microscopy. In this chapter, we present a detailed description of methods required to perform protein-specific imaging of glycosylation inside living cells. These include the complete synthesis of Ac4GlcNCyoc, immunoprecipitation of EGFP-fusion proteins to examine the Ac4GlcNCyoc modification state, and a complete section on basics, performance, as well as data analysis for FLIM-FRET microscopy. We also provide useful notes necessary for reproducibility and point out strengths and limitations of the approach.

Enzymatic production of N-acetyl-d-glucosamine from crayfish shell wastes pretreated via high pressure homogenization.

This study presents an efficient pretreatment of crayfish shell using high pressure homogenization that enables N-acetyl-d-glucosamine (GlcNAc) production by chitinase. Firstly, the chitinase from Serratia proteamaculans NJ303 was screened for its ability to degrade crayfish shell and produce GlcNAc as the sole product. Secondly, high pressure homogenization, which caused the crayfish shell to adopt a fluffy netted structure that was characterized by Scanning electron microscope (SEM), Fourier transform infrared spectrometer (FT-IR), X-ray diffraction (XRD), was evaluated as the best pretreatment method. In addition, the optimal conditions of high pressure homogenization of crayfish shell were determined to be five cycles at a pressure of 400bar, which achieved a yield of 3.9g/L of GlcNAc from 25g/L of crayfish shell in a batch enzymatic reaction over 1.5h. The results showed high pressure homogenization might be an efficient method for direct utilization of crayfish shell for enzymatic production of GlcNAc.

Sugar-Based Arylsulfonamide Carboxylates as Selective and Water-Soluble Matrix Metalloproteinase-12 Inhibitors.

Matrix metalloproteinase-12 (MMP-12) can be considered an attractive target to study selective inhibitors useful in the development of new therapies for lung and cardiovascular diseases. In this study, a new series of arylsulfonamide carboxylates, with increased hydrophilicity resulting from conjugation with a ß-N-acetyl-d-glucosamine moiety, were designed and synthesized as MMP-12 selective inhibitors. Their inhibitory activity was evaluated on human MMPs by using the fluorimetric assay, and a crystallographic analysis was performed to characterize their binding mode. Among these glycoconjugates, a nanomolar MMP-12 inhibitor with improved water solubility, compound 3 [(R)-2-(N-(2-(3-(2-acetamido-2-deoxy-ß-d-glucopyranosyl)thioureido)ethyl)biphenyl-4-ylsulfonamido)-3-methylbutanoic acid], was identified.

Synthesis of NAG-thiazoline-derived inhibitors for ß-N-acetyl-d-hexosaminidases.

ß-N-Acetyl-d-hexosaminidases are responsible for the metabolism of glycoconjugates in diverse physiological processes that are important targets for medicine and pesticide development. Fourteen new NAG-thiazoline derivatives were synthesized by cyclization and click reaction using d-glucosamine hydrochloride as the starting material. All the compounds created were characterized by NMR and HRMS spectra. A preliminary bioassay, using four enzymes from two ß-N-acetyl-d-hexosaminidase families, showed that most of the compounds synthesized exhibit selective inhibition of GH84 ß-N-acetyl-d-hexosaminidase. Among the compounds tested, compounds 5a (IC50=12.6 µM, hOGA) and 5e (IC50=12.5 µM, OfOGA) proved to be a highly selective and potent inhibitor.

Glycosylation with 2-Acetamido-2-deoxyglycosyl Donors at a Low Temperature: Scope of the Non-Oxazoline Method.

A direct construction of 1,2-trans-ß-linked 2-acetamido-2-deoxyglycosides was investigated. The 3,4,6-tri-O-benzyl- and 3,4,6-tri-O-acetyl-protected glycosyl diethyl phosphites and 4,6-O-benzylidene-protected galactosyl diethyl phosphite each reacted with a variety of acceptor alcohols in the presence of a stoichiometric amount of Tf2NH in CH2Cl2 at -78 °C to afford the corresponding ß-glycosides in good to high yields with complete stereoselectivity. Some experiments provided strong evidence that the corresponding oxazolinium ions are not responsible for the reaction. We demonstrated that glycosylations with the corresponding glycosyl imidate and thioglycoside also proceeded at a low temperature, indicating the possibility of these donors being attractive alternatives to the phosphite. A plausible reaction mechanism, which features glycosyl triflimide and contact ion pair as reactive intermediates, is proposed on the basis of the results obtained with 2-acetamido-2-deoxymannosyl donors.

Bi- to tetravalent glycoclusters presenting GlcNAc/GalNAc as inhibitors: from plant agglutinins to human macrophage galactose-type lectin (CD301) and galectins.

Emerging insights into the functional spectrum of tissue lectins leads to identification of new targets for the custom-made design of potent inhibitors, providing a challenge for synthetic chemistry. The affinity and selectivity of a carbohydrate ligand for a lectin may immensely be increased by a number of approaches, which includes varying geometrical or topological features. This perspective leads to the design and synthesis of glycoclusters and their testing using assays of physiological relevance. Herein, hydroquinone, resorcinol, benzene-1,3,5-triol and tetra(4-hydroxyphenyl)ethene have been employed as scaffolds and propargyl derivatives obtained. The triazole-containing linker to the α/ß-O/S-glycosides of GlcNAc/GalNAc presented on these scaffolds was generated by copper-catalysed azide-alkyne cycloaddition. This strategy was used to give a panel of nine glycoclusters with bi-, tri- and tetravalency. Maintained activity for lectin binding after conjugation was ascertained for both sugars in solid-phase assays with the plant agglutinins WGA (GlcNAc) and DBA (GalNAc). Absence of cross-reactivity excluded any carbohydrate-independent reactivity of the bivalent compounds, allowing us to proceed to further testing with a biomedically relevant lectin specific for GalNAc. Macrophage galactose(-binding C)-type lectin, involved in immune defence by dendritic cells and in virus uptake, was produced as a soluble protein without/with its α-helical coiled-coil stalk region. Binding to ligands presented on a matrix and on cell surfaces was highly susceptible to the presence of the tetravalent inhibitor derived from the tetraphenylethene-containing scaffold, and presentation of GalNAc with an α-thioglycosidic linkage proved favorable. Cross-reactivity of this glycocluster to human galectins-3 and -4, which interact with Tn-antigen-presenting mucins, was rather small. Evidently, the valency and spatial display of α-GalNAc residues is a key factor to design potent and selective inhibitors for this lectin.

High-efficient synthesis and biological activities of allosamidins.

The pseudo-trisaccharide allosamidin 1 is a potent inhibitor of all family-18 chitinases, and it is confirmed to have insecticidal and antifungal activities. But the synthesis of allosamidins is very difficult, and it is a challengeable subject. Allosamidins were synthesized in solid-liquid phase, total solid-phase and total liquid-phase, respectively. Solid-liquid phase method realizes the partial solid-phase synthesis of allosamidins. Total solid-phase method greatly simplifies the purification process. Total liquid-phase method shortens the synthetic steps of allosamidins. The insecticidal and antifungal activities of allosamidins were also reported herein.

Inhibition of microbial ß-N-acetylhexosaminidases by 4-deoxy- and galacto-analogues of NAG-thiazoline.

NAG-thiazoline is a well-established competitive inhibitor of two physiologically relevant glycosidase families-ß-N-acetylhexosaminidases (GH20) and ß-N-acetylglucosaminidases (GH84). Based on the different substrate flexibilities of these enzyme groups, we designed and synthesized the 4-deoxy derivative of NAG-thiazoline aiming at the selective inhibition of GH20 ß-N-acetylhexosaminidases. One GH84 and two GH20 microbial glycosidases were employed as model enzymes for the inhibition assays. Surprisingly, the new compound 4-deoxy-thiazoline exhibited no activity inhibition with either of the enzyme families of interest. Unlike with the substrates, the 4-hydroxyl group of the inhibitor's sugar ring seems to be crucial for binding the inhibitor to the active sites of these enzymes.

Inhibition of GlcNAc-processing glycosidases by C-6-azido-NAG-thiazoline and its derivatives.

NAG-thiazoline is a strong competitive inhibitor of GH20 ß-N-acetyl- hexosaminidases and GH84 ß-N-acetylglucosaminidases. Here, we focused on the design, synthesis and inhibition potency of a series of new derivatives of NAG-thiazoline modified at the C-6 position. Dimerization of NAG-thiazoline via C-6 attached triazole linkers prepared by click chemistry was employed to make use of multivalency in the inhibition. Novel compounds were tested as potential inhibitors of ß-N-acetylhexosaminidases from Talaromyces flavus, Streptomyces plicatus (both GH20) and ß-N-acetylglucosaminidases from Bacteroides thetaiotaomicron and humans (both GH84). From the set of newly prepared NAG-thiazoline derivatives, only C-6-azido-NAG-thiazoline displayed inhibition activity towards these enzymes; C-6 triazole-substituted NAG-thiazolines lacked inhibition activity against the enzymes used. Docking of C-6-azido-NAG-thiazoline into the active site of the tested enzymes was performed. Moreover, a stability study with GlcNAc-thiazoline confirmed its decomposition at pH < 6 yielding 2-acetamido-2-deoxy-1-thio-α/ß-D-glucopyranoses, which presumably dimerize oxidatively into S-S linked dimers; decomposition products of NAG-thiazoline are void of inhibitory activity.

Synthesis and anti-cancer activity of a glycosyl library of N-acetylglucosamine-bearing oleanolic acid.

N-Acetylglucosamine-bearing triterpenoid saponins (GNTS) were reported to be a unique type of saponins with potent anti-tumor activity. In order to study the structure-activity relationship of GNTS, 24 oleanolic acid saponins with (1 --> 3)-linked, (1 --> 4)-linked, (1 --> 6)-linked N-acetylglucosamine oligosaccharide residues were synthesized in a combinatorial and concise method. The cytotoxicity of these compounds toward the leukemia cell line HL-60 and the colorectal cancer cell line HT-29 could not be improved. Half maximal inhibition below 10 µM was achieved in one single case. The study revealed that the activity decreased following the order of 3' > 4' > 6' glycosyl modifications. GNTS that incorporated (D/L)-xylose and L-arabinose at positions 3' and 4' were more potent than those bearing other sugars.

Syntheses of sulfated glycopolymers and analyses of their BACE-1 inhibitory activity.

The glycopolymers for glycosaminoglycan mimic were synthesized, and the inhibitory effects of Alzheimer's ß-secretase (BACE-1) were examined. The regio-selective sulfation was conducted on N-acetyl glucosamine (GlcNAc), and the acrylamide derivatives were synthesized with the consequent sulfated GlcNAc. The glycopolymers were synthesized with acrylamide using radical initiator. The glycopolymer with sulfated GlcNAc showed the strong inhibitory effect on BACE-1, and the inhibitory effects were dependent on the sulfation positions. Especially, glycopolymers carrying 3,4,6-O-sulfo-GlcNAc showed the strong inhibitory effect. The docking simulation suggested that glycopolymers bind to the active site of BACE-1.

Discovery and validation of a new class of small molecule Toll-like receptor 4 (TLR4) inhibitors.

Many inflammatory diseases may be linked to pathologically elevated signaling via the receptor for lipopolysaccharide (LPS), toll-like receptor 4 (TLR4). There has thus been great interest in the discovery of TLR4 inhibitors as potential anti-inflammatory agents. Recently, the structure of TLR4 bound to the inhibitor E5564 was solved, raising the possibility that novel TLR4 inhibitors that target the E5564-binding domain could be designed. We utilized a similarity search algorithm in conjunction with a limited screening approach of small molecule libraries to identify compounds that bind to the E5564 site and inhibit TLR4. Our lead compound, C34, is a 2-acetamidopyranoside (MW 389) with the formula C17H27NO9, which inhibited TLR4 in enterocytes and macrophages in vitro, and reduced systemic inflammation in mouse models of endotoxemia and necrotizing enterocolitis. Molecular docking of C34 to the hydrophobic internal pocket of the TLR4 co-receptor MD-2 demonstrated a tight fit, embedding the pyran ring deep inside the pocket. Strikingly, C34 inhibited LPS signaling ex-vivo in human ileum that was resected from infants with necrotizing enterocolitis. These findings identify C34 and the ß-anomeric cyclohexyl analog C35 as novel leads for small molecule TLR4 inhibitors that have potential therapeutic benefit for TLR4-mediated inflammatory diseases.

Synthesis of ß-C-GlcNAc Ser from ß-C-Glc Ser.

The glycosylation of proteins, specifically installation of O-GlcNAc on Ser/Thr residues, is a dynamic control element for transcription repression, protein degradation, and nutrient sensing. To provide homogeneous and stable structures with this motif, the synthesis of a C-linked mimic, C-GlcNAc Ser, has been prepared from the C-Glc Ser by a double inversion strategy using azide to insert the C-2 nitrogen functionality. The C-Glc Ser was available by a ring-closing metathesis and hydroalkoxylation route.