Design, Synthesis, and Biological Evaluation of Thioglucoside Analogues of Gliflozin as Potent New Gliflozin Drugs.

In this study, we have investigated the potential of two classes of thioglucoside analogues of gliflozins as antidiabetic drugs, one with substitutions of S-atoms in meta-positions (similar to C-glucoside SGLT2 inhibitors, TAGs A, B, and C) and the other with substitutions of S-atoms in ortho-positions (similar to O-glucoside SGLT2 inhibitors, TAGs D, E, F, and G). These TAGs were confirmed to show good stability against ß-glucosidase and to have no acute toxicity to cultured cells. Most importantly, TAGs D, E, F, and G all showed high inhibitory activity against SGLT2 (IC50: 2.0-5.9 nM) and thus have great potential to be developed as new gliflozin drugs. Compared with the synthesis of C-glucoside gliflozins, the synthesis of TAGs is simple, efficient, and associated with low costs, high yields, and very mild reaction conditions.

Tetracyclic 1,4-Naphthoquinone Thioglucoside Conjugate U-556 Blocks the Purinergic P2X7 Receptor in Macrophages and Exhibits Anti-Inflammatory Activity In Vivo.

P2X7 receptors (P2X7Rs) are ligand-gated ion channels that play a significant role in inflammation and are considered a potential therapeutic target for some inflammatory diseases. We have previously shown that a number of synthetic 1,4-naphthoquinones are capable of blocking P2X7Rs in neuronal and macrophage cells. In the present investigation, we have demonstrated the ability of the tetracyclic quinone-thioglucoside conjugate U-556, derived from 1,4-naphthoquinone thioglucoside, to inhibit ATP-induced Ca2+ influx and YO-PRO-1 dye uptake, which indicates blocking P2X7R in RAW 264.7 macrophages. This process was accompanied by the inhibition of ATP-induced reactive oxygen species production in macrophages, as well as the macrophage survival strengthening under ATP toxic effects. Nevertheless, U-556 had no noticeable antioxidant capacity. Naphthoquinone-thioglucoside conjugate U-556 binding to the extracellular part of the P2X7R was confirmed by SPR analysis, and the kinetic characteristics of this complex formation were established. Computer modeling predicted that U-556 binds the P2X7R allosteric binding site, topographically similar to that of the specific A438079 blocker. The study of biological activity in in vivo experiments shows that tetracylic conjugate significantly reduces inflammation provoked by carrageenan. The data obtained points out that the observed physiological effects of U-556 may be due to its ability to block the functioning of the P2X7R.

Two new flavonoid thioglucosides from the seeds of Lepidium apetalum.

Two previously undescribed flavonoid thioglucosides lepidiumflavonosides A and B (1-2) and two known megastigmane compounds (7E,9S)-9-hydroxy-5,7-megastigmadien-4-one 9-O-ß-D-glucopyranoside (3) and (9S)-4-oxo-ß-inol ß-D-glucopyranoside (4) were isolated from the water extract of the seeds of Lepidium apetalum Willd. The structural elucidation of isolated compounds was unambiguously determined based on extensive 1D and 2D NMR spectroscopic analyses. All compounds were evaluated for their estrogen-like effects on MCF-7 cells in vitro. The results showed that compounds 1-4 significantly promoted the proliferation of MCF-7 cells, and the proliferation was antagonized by the specific ER antagonist ICI182,780, suggesting that compounds 1-4 might have the estrogen-like effect in vitro potentially.

4'-Nitrobiphenyl thioglucoside as the Smallest, fluorescent photosensitizer with cancer targeting ligand.

We have previously developed a glucose-linked biphenyl photosensitizer that can pass through glucose transporters, aiming for cancer-selective photodynamic therapy (PDT). Its small size (MW: 435) will allow oral administration and a fast clearance avoiding photosensitivity. However, its fluorescence efficiency was quite low, causing difficulty in monitoring cellular uptake. We thus synthesized a series of monosaccharide-linked biphenyl derivatives with a sulfur atom replacing an oxygen atom, in search of a photosensitizer with a brighter fluorescence. Among them, 4'-nitrobiphenyl thioglucoside showed a fluorescence emission extending to near infra-red region with a strength three times greater than that of the previous compound. This compound was found to have a higher 1O2-producing efficiency (ΦΔ: 0.75) than the previous compound (ΦΔ: 0.65). The thioglucoside indicated a significant photodamaging effect (IC50: 250 µM) against cancer cells. Although the galactose and mannose analogs exerted similar photodamaging effects, they were moderately toxic in the dark at a concentration of 300 µM. The thioglucoside and thiomannoside were at least partially uptaken through glucose transporters as demonstrated by inhibition with cytochalasin B, whereas no inhibition was observed for the galactoside. The behavior of d-glucose toward the cellular uptakes of these photosensitizers was bipolar: inhibitory at a low concentration and recovery or acceleratory at a higher concentration. These results indicate that 4'-nitrobiphenyl thioglucoside is the smallest (MW: 393) cancer-targeting photosensitizer with a trackable fluorescence property.

Structural Insights into the Substrate Transport Mechanisms in GTR Transporters through Ensemble Docking.

Glucosinolate transporters (GTRs) are part of the nitrate/peptide transporter (NPF) family, members of which also transport specialized secondary metabolites as substrates. Glucosinolates are defense compounds derived from amino acids. We selected 4-methylthiobutyl (4MTB) and indol-3-ylmethyl (I3M) glucosinolates to study how GTR1 from Arabidopsis thaliana transports these substrates in computational simulation approaches. The designed pipeline reported here includes massive docking of 4MTB and I3M in an ensemble of GTR1 conformations (in both inward and outward conformations) extracted from molecular dynamics simulations, followed by clustered and substrate-protein interactions profiling. The identified key residues were mutated, and their role in substrate transport was tested. We were able to identify key residues that integrate a major binding site of these substrates, which is critical for transport activity. In silico approaches employed here represent a breakthrough in the plant transportomics field, as the identification of key residues usually takes a long time if performed from a purely wet-lab experimental perspective. The inclusion of structural bioinformatics in the analyses of plant transporters significantly speeds up the knowledge-gaining process and optimizes valuable time and resources.

Lepidium graminifolium L.: Glucosinolate Profile and Antiproliferative Potential of Volatile Isolates.

Glucosinolates (GSLs) from Lepidium graminifolium L. were analyzed qualitatively and quantitatively by their desulfo-counterparts using UHPLC-DAD-MS/MS technique and by their volatile breakdown products-isothiocyanates (ITCs) using GC-MS analysis. Thirteen GSLs were identified with arylaliphatic as the major ones in the following order: 3-hydroxybenzyl GSL (glucolepigramin, 7), benzyl GSL (glucotropaeolin, 9), 3,4,5-trimethoxybenzyl GSL (11), 3-methoxybenzyl GSL (glucolimnanthin, 12), 4-hydroxy-3,5-dimethoxybenzyl GSL (3,5-dimethoxysinalbin, 8), 4-hydroxybenzyl GSL (glucosinalbin, 6), 3,4-dimethoxybenzyl GSL (10) and 2-phenylethyl GSL (gluconasturtiin, 13). GSL breakdown products obtained by hydrodistillation (HD) and CH2Cl2 extraction after hydrolysis by myrosinase for 24 h (EXT) as well as benzyl ITC were tested for their cytotoxic activity using MTT assay. Generally, EXT showed noticeable antiproliferative activity against human bladder cancer cell line UM-UC-3 and human glioblastoma cell line LN229, and can be considered as moderately active, while IC50 of benzyl ITC was 12.3 µg/mL, which can be considered as highly active.

Bioengineering potato plants to produce benzylglucosinolate for improved broad-spectrum pest and disease resistance.

In traditional, small-scale agriculture in the Andes, potatoes are frequently co-cultivated with the Andean edible tuber Tropaeolum tuberosum, commonly known as mashua, which is believed to exert a pest and disease protective role due to its content of the phenylalanine-derived benzylglucosinolate (BGLS). We bioengineered the production of BGLS in potato by consecutive generation of stable transgenic events with two polycistronic constructs encoding for expression of six BGLS biosynthetic genes from Arabidopsis thaliana. First, we integrated a polycistronic construct coding for the last three genes of the pathway (SUR1, UGT74B1 and SOT16) into potato driven by the cauliflower mosaic virus 35S promoter. After identifying the single-insertion transgenic event with the highest transgene expression, we stacked a second polycistronic construct coding for the first three genes in the pathway (CYP79A2, CYP83B1 and GGP1) driven by the leaf-specific promoter of the rubisco small subunit from chrysanthemum. We obtained transgenic events producing as high as 5.18 pmol BGLS/mg fresh weight compared to the non-transgenic potato plant producing undetectable levels of BGLS. Preliminary bioassays suggest a possible activity against Phytophthora infestans, causing the late blight disease and Premnotrypes suturicallus, referred to as the Andean potato weevil. However, we observed altered leaf morphology, abnormally thick and curlier leaves, reduced growth and tuber production in five out of ten selected transgenic events, which indicates that the expression of BGLS biosynthetic genes has an undesirable impact on the potato. Optimization of the expression of the BGLS biosynthetic pathway in potato is required to avoid alterations of plant development.

Synthesis and Evaluation of Antimicrobial and Cytotoxic Activity of Oxathiine-Fused Quinone-Thioglucoside Conjugates of Substituted 1,4-Naphthoquinones.

A series of new tetracyclic oxathiine-fused quinone-thioglycoside conjugates based on biologically active 1,4-naphthoquinones and 1-mercapto derivatives of per-O-acetyl d-glucose, d-galactose, d-xylose, and l-arabinose have been synthesized, characterized, and evaluated for their cytotoxic and antimicrobial activities. Six tetracyclic conjugates bearing a hydroxyl group in naphthoquinone core showed high cytotoxic activity with EC50 values in the range of 0.3 to 0.9 µM for various types of cancer and normal cells and no hemolytic activity up to 25 µM. The antimicrobial activity of conjugates was screened against Gram-positive bacteria (Staphylococcus aureus, Bacillus cereus), Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli), and fungus Candida albicans by the agar diffusion method. The most effective juglone conjugates with d-xylose or l-arabinose moiety and hydroxyl group at C-7 position of naphthoquinone core at concentration 10 µg/well showed antimicrobial activity comparable with antibiotics vancomicin and gentamicin against Gram-positive bacteria strains. In liquid media, juglone-arabinosidic tetracycles showed highest activity with MIC 6.25 µM. Thus, a positive effect of heterocyclization with mercaptosugars on cytotoxic and antimicrobial activity for group of 1,4-naphthoquinones was shown.

Promoting crystallization of intrinsic membrane proteins with conjugated micelles.

A new technique for promoting nucleation and growth of membrane protein (MP) crystals from micellar environments is reported. It relies on the conjugation of micelles that sequester MPs in protein detergent complexes (PDCs). Conjugation via amphiphilic [metal:chelator] complexes presumably takes place at the micelle/water interface, thereby bringing the PDCs into proximity, promoting crystal nucleation and growth. We have successfully applied this approach to two light-driven proton pumps: bacteriorhodopsin (bR) and the recently discovered King Sejong 1-2 (KS1-2), using the amphiphilic 4,4'-dinonyl-2,2'-dipyridyl (Dinonyl) (0.7 mM) chelator in combination with Zn2+, Fe2+, or Ni2+ (0.1 mM). Crystal growth in the presence of the [metal-chelator] complexes leads to purple, hexagonal crystals (50-75 µm in size) of bR or pink, rectangular/square crystals (5-15 µm) of KS1-2. The effects of divalent cation identity and concentration, chelator structure and concentration, ionic strength and pH on crystal size, morphology and process kinetics, are described.

1,2-cis-Selective glucosylation enabled by halogenated benzyl protecting groups.

We report on our initial results from a systematic effort to implement electron-withdrawing protecting groups and Lewis basic solvents/additives as an approach to 1,2-cis(α)-selective O-glucosylation. 1,2-cis-Selective O-glucosylations are reported with thioglucosides and glucosyl trichloroacetimidates and a range of acceptors. A correlation between electron-withdrawing effects and 1,2-cis selectivity has been established. This phenomenon may prove to be broadly applicable in the area of chemical O-glycosylation.

Structural, spectral characterization and molecular docking analyses of mer-ruthenium (II) complexes containing the bidentate chelating ligands.

In this study, we analyzed some monofunctional Ru (II) complexes containing chlorine, bromine and fluorine atoms around the central atom. The best calculation level among HF, B3LYP and M062X methods for [Ru (Cl-Ph-tpy)(NN)X]+ (X = F, Cl, Br) was determined in the light of Benchmark analysis and according to this analysis results, the best level is shown as B3LYP-LANL2DZ/6-31G(d). In addition to this, the spectroscopic data (IR, NMR and UV-Vis) were also obtained in agreement with experimental results. The tendency of anticancer activity and structural activity relationship (SAR) parameters are predicted with some quantum chemical methods. Surface and contour diagrams, as well as electron densities on mentioned complexes were interpreted through theoretically obtained results. Finally, the anticancer activity tendency of the relevant complexes on the human cervical carcinoma cell line (ID: 1 M17) is supported by molecular docking calculations.

Structural transition dynamics of biologically active flavins in alkylglucoside surfactants aggregates.

The photophysics and structural transition dynamics of a bio-active flavin lumichrome (LM) entrapped in two sugars based neutral surfactants were reported. Sugar-based surfactants, which were used for research purpose are potential environmentally friendly, green alternative amphiphilic surface active substance compared to other kinds of common surfactants. Here, two alkyl glucoside surfactants n-octyl-ß-D-glucopyranoside (OBG) and n-octyl-ß-D-thioglucopyranoside (OBTG) were used. This work is carried out by using steady-state absorption and fluorescence emission spectroscopy along with time-resolved fluorescence emission techniques. Photophysics of LM was modulated several folds in these two sugar-based neutral micelles. LM exhibits excitation and emission wavelength dependent fluorescence properties in these two sugars based neutral micelles. LM confined in the micellar environments exhibited structural transition dynamism, i.e. different kinds of conformers are equilibrated. Herein, different conformers of LM are identified and explained with the help of spectroscopic methods. From the fluorescence anisotropy measurement, it was found that the rotational relaxation time of LM in OBG micelle was more compared to that in OBTG micelle, which indicates that the LM molecule faced much more constrained environment in OBG micellar media.

Membrane matters: The impact of a nanodisc-bilayer or a detergent microenvironment on the properties of two eubacterial rhodopsins.

Multi-spanning membrane proteins usually require solubilization to allow proper purification and characterization, which generally impairs their structural and functional integrity. We have tested the efficacy of several commonly used detergents and membrane-mimicking nanodiscs with respect to solubilization, spectral properties, thermal stability and oligomeric profile of two membrane proteins from the eubacterial rhodopsin family, green proteorhodopsin (PR) and Gloeobacter violaceus rhodopsin (GR). Good solubilization was observed for the detergents TritonX-100 and dodecylphosphocholine (DPC), but DPC in particular strongly affected the thermal stability of PR and especially GR. The least deleterious effects were obtained with n-dodecyl-ß-D-maltopyranoside (DDM) and octyl glucose neopentyl glycol (OGNG), which adequately stabilized the native oligomeric and monomeric state of PR and GR, respectively. The transition from the oligomeric to the monomeric state is accompanied by a small red-shift. Both GR and PR were rather unstable in SMA-nanodiscs, but the highest thermal stability was realized by the MSP-nanodisc environment. The size of the MSP-nanodisc was too small to fit the PR hexamer, but large enough to contain the PR monomer and GR trimer. This permitted the comparison of the photocycle of trimeric GR in a membrane-mimicking (MSP-nanodisc) and a detergent (DDM) environment. The ultrarapid early phase of the photocycle (femto- to picosecond lifetimes) showed very similar kinetics in either environment, but the slower part, initiated with proton transfer and generation of the M intermediate, proceeded faster in the nanodisc environment. The implications of our results for the biophysical characterization of PR and GR are discussed.

One-pot synthesis of cyclic oligosaccharides by the polyglycosylation of monothioglycosides.

Cyclic oligosaccharides such as cyclodextrins (CyDs) have been known as excellent host molecules for the inclusion of various organic guest molecules. The development of new synthetic methods for preparing cyclic oligosaccharides from simple and readily available glycosyl donors would be highly desirable, since the current traditional synthetic routes include multiple reaction steps (glycosylation reactions and deprotections). We herein report on the synthesis of cyclic oligosaccharides by polyglycosylation of monothioglycosides, typically, 2,3,4-protected 1-thioglycosides. A series of promoters and solvents were tested for the glycosylation of thiogalactosides that contain a hydroxy group at the 6-position, and glycosylation using a N-iodosuccinimide (NIS)/trimethylsilyl triflate (TMSOTf) promoter system in dichloromethane afforded cyclic oligosaccharides which consist of tri~penta galactosides containing repeating ß-(1→6) glycosidic linkage as major products, as evidenced by a single crystal X-ray structure analysis of the cyclic tetragalactoside. The effect of reaction temperature and reactant concentrations on the glycosylation products was also investigated. The cyclic glucosides were obtained by the glycosylation of the thioglucosides. Moreover, protecting groups of the synthesized cyclic tetragalactoside were removed to obtain deprotected cyclic tetragalactoside.

GH3 Auxin-Amido Synthetases Alter the Ratio of Indole-3-Acetic Acid and Phenylacetic Acid in Arabidopsis.

Auxin is the first discovered plant hormone and is essential for many aspects of plant growth and development. Indole-3-acetic acid (IAA) is the main auxin and plays pivotal roles in intercellular communication through polar auxin transport. Phenylacetic acid (PAA) is another natural auxin that does not show polar movement. Although a wide range of species have been shown to produce PAA, its biosynthesis, inactivation and physiological significance in plants are largely unknown. In this study, we demonstrate that overexpression of the CYP79A2 gene, which is involved in benzylglucosinolate synthesis, remarkably increased the levels of PAA and enhanced lateral root formation in Arabidopsis. This coincided with a significant reduction in the levels of IAA. The results from auxin metabolite quantification suggest that the PAA-dependent induction of GRETCHEN HAGEN 3 (GH3) genes, which encode auxin-amido synthetases, promote the inactivation of IAA. Similarly, an increase in IAA synthesis, via the indole-3-acetaldoxime pathway, significantly reduced the levels of PAA. The same adjustment of IAA and PAA levels was also observed by applying each auxin to wild-type plants. These results show that GH3 auxin-amido synthetases can alter the ratio of IAA and PAA in plant growth and development.

Bailcalin Protects against Diabetic Cardiomyopathy through Keap1/Nrf2/AMPK-Mediated Antioxidative and Lipid-Lowering Effects.

Previous studies demonstrated that Bailcalin (BAI) prevented cardiac injuries under different disease models. Whether BAI protected against type 2 diabetes mellitus- (T2DM-) associated cardiomyopathy was investigated in this study. T2DM was established by the combination of streptozotocin injection and high-fat diet in mice. BAI was administered daily for 6 months. After evaluating cardiac functions, mice hearts were removed and processed for morphological, biochemical, and molecular mechanism analyses. Neonatal rat cardiomyocytes (NRCM) were isolated and treated with high glucose and palmitate (HG/Pal) for in vitro investigation. BAI significantly ameliorated T2DM-induced cardiomyocyte hypertrophy, interstitial fibrosis, and lipid accumulation accompanied by markedly improved cardiac functions in diabetic mice. Mechanically, BAI restored decreased phosphorylation of AMPK and enhanced expression and nuclei translocation of Nrf2. In in vitro experiments, BAI also prevented NRCM from HG/Pal-induced apoptosis and oxidative stress injuries by increasing p-AMPK and Nrf2 accumulation. The means by which BAI restored p-AMPK seemed to be related to the antioxidative effects of Nrf2 after silencing AMPK or Nrf2 in NRCM. Furthermore, BAI regulated Nrf2 by inhibiting Nrf2 ubiquitination and consequent degradation mediated by Keap1. This study showed that BAI alleviated diabetes-associated cardiac dysfunction and cardiomyocyte injuries in vivo and in vitro via Keap1/Nrf2/AMPK-mediated antioxidation and lipid-lowering effects. BAI might be a potential adjuvant drug for diabetes cardiomyopathy treatment.

Gold-catalyzed synthesis of α-D-glucosides using an o-ethynylphenyl ß-D-1-thioglucoside donor.

A gold-catalyzed glucosylation method using an o-ethynylphenyl ß-D-1-thioglucoside as donor is described. The reaction proceeds in a mostly SN2 pathway. A series of α-D-glucosides are obtained in good yields and with up to 19:1 α-selectivity.

Antitumor effect of a liposome-encapsulated ß1,4-galactosyltransferase inhibitor.

Galactosyltransferases are a family of enzymes responsible for the synthesis of glycan chains which are involved in cell proliferation, adhesion and apoptosis. A recently synthesized galactosyltransferase inhibitor, 2-naphthyl 2-butanamido-2-deoxy-1-thio-ß-D-glucopyranoside (612), has been found to selectively inhibit ß1,4-galactosyltransferase over ß1,3-galactosyltransferase and, therefore, has potential to suppress the synthesis of cancer associated epitopes. However, the application of this inhibitory activity in biological systems remains unknown. In this study, 612 was introduced into a cationic liposome (LP) delivery system, and the anti-proliferative effects of both free and the LP-incorporated 612 (612-LP) were investigated in A549 lung cancer cells, which actively express anionic sialic acid moieties on the surfaces of cells. The anti-proliferative effects were evaluated via MTT assays. The results revealed that free 612 and empty LP impose neither anti-proliferative nor apoptotic effects on cancer cells at low doses, whereas the 612-LP system inhibited cancer cell growth at a concentration as low as 0.1 µg/mL after 3 days of incubation, suggesting that this formulation enabled efficient delivery of 612 into cells and promoted the anti-proliferative activity of 612 against cancer cells. Therefore, this highly specific inhibitor 612 has the potential for development as an effective anti-cancer agent and merits further investigation.

Endotoxin reduction in protein solutions using octyl ß-D-1-thioglucopyranoside wash on chromatography media.

Endotoxins are complex molecules and one of the most challenging impurities requiring separation in biopharmaceutical protein purification processes. Usually these contaminants are cleared during the downstream process, but if endotoxin interacts with the target protein it becomes difficult to remove. In the present study we identified a detergent, octyl-ß-D-1-thioglucopyranoside (OTG), that disrupted endotoxin-protein interactions. The integration of this detergent into washes on several chromatography media was demonstrated to provide a separation tool for decreasing endotoxin from target proteins. This study also examined the mechanism of OTG endotoxin-protein disruption through phase modification incubation and chromatographic studies. The non-ionic OTG wash was shown to break both hydrophobic and electrostatic interactions between the endotoxin and protein. This mechanism contrasts with the breaking of hydrophobic interactions by washing with known endotoxin decreasing Triton X-100 detergent. The difference in mechanisms likely results in the ability of OTG to decrease endotoxin to levels less than those resulting from a detergent wash such as Triton X-100. Finally, we show the impact of the OTG endotoxin removal tool on the biopharmaceutical industry. While maintaining monomer purity and activity levels, endotoxin removal from a fusion protein allowed for decreased background levels in a T cell functional assay. The lowered baseline of T cell responses allowed for more effective detection of molecular interaction with the cells. The detergent wash can be used to both decrease the overall level of endotoxin in a purified protein solution and to enable more effective screening of lead candidate molecules.

Diverse Excretion Pathways of Benzyl Glucosinolate in Humans after Consumption of Nasturtium (Tropaeolum majus L.)-A Pilot Study.

SCOPE: Different metabolic and excretion pathways of the benzyl glucosinolate breakdown products benzyl isothiocyanate and benzyl cyanide are investigated to obtain information about their multiple fate after ingestion. Detailed focus is on the so far underestimated transformation/excretion pathways-protein conjugation and exhalation. METHODS AND RESULTS: Metabolites, protein conjugates, and non-conjugated isothiocyanates are determined in plasma, urine, and breath of seven volunteers after consuming freeze-dried nasturtium or bread enriched with nasturtium. Samples are collected up to 48 h at selected time points. The metabolites of the mercapturic acid pathway are detectable in plasma up to 24 h after consumption. Additionally, mercapturic acid is the main metabolite in urine, but non-conjugated benzyl isothiocyanate is detectable as well. Protein conjugates show high amounts in plasma even 48 h after consumption. In breath, benzyl isothiocyanate and benzyl cyanide are detectable up to 48 h after consumption. CONCLUSION: Isothiocyanates are not only metabolized via the mercapturic acid pathway, but also form protein conjugates in blood and are exhaled. To balance intake and excretion, it is necessary to investigate all potential metabolites and excretion routes. This has important implications for the understanding of physiological and pharmacological effects of isothiocyanate-containing products.