Synthesis, pharmacological evaluation and mechanistic study of scutellarin methyl ester -4'-dipeptide conjugates for the treatment of hypoxic-ischemic encephalopathy (HIE) in rat pups.

A series of novel scutellarin methyl ester-4'-dipeptide conjugates exhibiting active transport characteristics and protection against pathological damage caused by hypoxic-ischemic encephalopathy (HIE) were successfully designed and synthesized. The physiochemical properties of the obtained compounds, as well as the Caco-2 cell-based permeability and uptake into hPepT1-MDCK cells were evaluated using various analytical methods. Scutellarin methyl ester-4'-Val-homo-Leu dipeptide (5k) was determined as the optimal candidate with a high apparent permeability coefficient (Papp A to B) of 1.95 ± 0.24 × 10-6 cm/s, low ER (Papp BL to AP/Papp AP to BL) of 0.52 in Caco-2 cells, and high uptake of 25.47 µmol/mg/min in hPepT1-MDCK cells. Comprehensive mechanistic studies demonstrated that pre-treatment of PC12 cells with 5k resulted in more potent anti-oxidative activity, which was manifested by a significant decrease in the malondialdehyde (MDA) and reactive oxygen species (ROS) levels, attenuation of the H2O2-induced apoptotic cell accumulation in the sub-G1 peak, and improvement in the expression of the relevant apoptotic proteins (Bcl-2, Bax, and cleave-caspase-3). Moreover, evaluation of in vivo neuroprotective characteristics in hypoxic-ischemic rat pups revealed that 5k significantly reduced infarction and alleviated the related pathomorphological damage. The compound was also shown to ameliorate the neurological deficit at 48 h as well as to decrease the brain tissue loss at 4 weeks. Conjugate 5k was demonstrated to reduce the amyloid precursor protein (APP) and ß-site APP-converting enzyme-1 (BACE-1) expression. Pharmacokinetic characterization of 5k indicated favorable druggability and pharmacokinetic properties. The conducted docking studies revealed optimal binding of 5k to PepT1. Hydrogen bonding as well as cation-π interactions with the corresponding amino acid residues in the target active site were clearly observed. The obtained results suggest 5k as a potential candidate for anti-HIE therapy, which merits further investigation.

Demystifying a hexuronic acid ligand that recognizes Toxoplasma gondii and blocks its invasion into host cells.

Toxoplasma gondii is a ubiquitous eukaryotic pathogen responsible for toxoplasmosis in humans and animals. This parasite is an obligate intracellular pathogen and actively invades susceptible host cells, a process which is mediated by specific receptor-ligand interactions. Here, we have identified an unnatural 2,4-disulfated d-glucuronic acid (Di-S-GlcA), a hexuronic acid composed of heparin/heparan sulfate, as a potential carbohydrate ligand that can selectively bind to T. gondii parasites. More importantly, the gelatin conjugated Di-S-GlcA multivalent probe displayed strong inhibition of parasite entry into host cells. These results open perspective for the future use of Di-S-GlcA epitopes in biomedical applications against toxoplasmosis.

Highly Specific near-Infrared Fluorescent Probe for the Real-Time Detection of ß-Glucuronidase in Various Living Cells and Animals.

ß-Glucuronidase (GLU) is an important biomarker for primary cancers and intestinal metabolism of drugs or endogenous substances; however, an effective optical probe for near-infrared (NIR) monitoring in vivo is still lacking. Herein, we design an enzyme-activated off-on NIR fluorescent probe, HC-glu, based on a hemicyanine keleton, which is conjugated with a d-glucuronic acid residue via a glycosidic bond, for the fluorescent quantification and trapping of endogenous GLU activity in vitro and in vivo. The newly developed NIR probe exhibited prominent features including prominent selectivity, high sensitivity, and ultrahigh imaging resolution. It has been successfully used to detect and image endogenous GLU in various hepatoma carcinoma cells, tumor tissues, and tumor-bearing mouse models, for cancer diagnosis and therapy. Moreover, it could detect the in vivo activity of GLU in the intestinal tracts of animals including mice and zebrafish, where GLU performs a vital biological function and is mainly distributed. It could also evaluate real intestinal distribution and real-time variations of GLU in development and growth, all of which are very helpful to guide rational drug use in the clinic. Our results fully demonstrated that HC-glu may serve as a promising tool for evaluating the biological function and process of GLU in living systems.

Synthesis of Scutellarein Derivatives with a Long Aliphatic Chain and Their Biological Evaluation against Human Cancer Cells.

Scutellarin is the major active flavonoid extracted from the traditional Chinese herbal medicine Erigeron breviscapus (Vant.) Hand-Mazz., which is widely used in China. Recently, accumulating evidence has highlighted the potential role of scutellarin and its main metabolite scutellarein in the treatment of cancer. To explore novel anticancer agents with high efficiency, a series of new scutellarein derivatives with a long aliphatic chain were synthesized, and the antiproliferative activities against Jurkat, HCT-116 and MDA-MB-231 cancer cell lines were assessed. Among them, compound 6a exhibited the strongest antiproliferative effects on Jurkat (IC50 = 1.80 µM), HCT-116 (IC50 = 11.50 µM) and MDA-MB-231 (IC50 = 53.91 µM). In particular, 6a even showed stronger antiproliferative effects than the positive control NaAsO2 on Jurkat and HCT-116 cell lines. The results showed that a proper long aliphatic chain enhanced the antiproliferative activity of scutellarein.

Exploitation of new structurally diverse d-glucuronamide-containing N-glycosyl compounds: synthesis and anticancer potential.

The synthesis and anticancer evaluation of novel N-glycosyl derivatives containing N-substituted glucuronamide moieties, as nucleoside analogs or as prospective mimetics of glycosyl phosphates or of nucleotides, is reported. These compounds comprise N-anomerically-linked nucleobases or motifs that are surrogates of a phosphate group, such as sulfonamide or phosphoramidate moieties. 1-Sulfonamido glucuronamides containing N-benzyl, N-propargyl or N-dodecyl carboxamide units were synthesized through glycosylation of methanesulfonamide with tetra-O-acetyl glucuronamides. 1-Azido glucuronamides were accessed by microwave-assisted reactions of tetra-O-acetyl glucuronamides with TMSN3 and were further converted into N-glycosylphosphoramidates by treatment with trimethyl phosphite. Potential glucuronamide-based nucleotide mimetics comprising both an anomeric sulfonamide/phosphoramidate group and a benzyltriazolylmethyl amide system at C-5, as nucleobase mimetics, were synthesized via 'click' cycloaddition of N-propargyl glucuronamide derivatives with benzyl azide. N-Dodecyl tetra-O-acetyl glucuronamides were converted into uracil and purine nucleosides via N-glycosylation of the corresponding silylated nucleobases. Biological screening revealed significant antiproliferative activities of the N-dodecyl glucuronamide-containing sulfonamide, phosphoramidate and nucleosides in K562 and MCF-7 cells. The highest effect was exhibited by the N9-linked purine nucleoside in the breast cancer cell MCF-7 with a GI50 value similar to that of clinically used 5-fluorouracil. Immunoblotting and cell cycle analysis of K562 cells treated with the most active compound as well as evaluation of the effect of this nucleoside on the activities of caspases 3 and 7 showed induction of apoptosis as the mechanism of cell death.

An Efficient Chemical Synthesis of Scutellarein: An in Vivo Metabolite of Scutellarin.

Scutellarein (2), which is an important in vivo metabolite of scutellarin (1), was synthesized from 3,4,5-trimethoxyphenol (3) in high yield in four steps. This strategy relies on acetylation, aldolization, cyclization and hydrolysis reactions, respectively.

Design, synthesis and DNA interaction study of new potential DNA bis-intercalators based on glucuronic acid.

A series of novel potential DNA bis-intercalators were designed and synthesized, in which two glucuronic acids were linked by ethylenediamine, and the glucuronic acid was coupled with various chromophores, including quinoline, acridine, indole and purine, at the C-1 position. The preliminary binding properties of these compounds to calf thymus DNA (CT-DNA) have been investigated by UV-absorption and fluorescence spectroscopy. The results indicated that all the target compounds can interact with CT-DNA, and the acridine derivative, 3b, showed the highest key selection vector (KSV) value, which suggested that compound 3b binds most strongly to CT-DNA.

A pH-responsive carboxylic ß-1,3-glucan polysaccharide for complexation with polymeric guests.

The helix-forming nature of ß-1,3-glucan polysaccharides is a characteristic that has potential for producing gene carriers, bio-nanomaterials and other chiral nanowires. Herein, carboxylic curdlan (CurCOOH) bearing the ß-1,3-polyglucuronic acid structure was successfully prepared from ß-1,3-glucan polysaccharide curdlan (Cur) by one-step oxidation using a 4-acetamido-TEMPO/NaClO/NaClO(2) system as the oxidant. The resulting high-molecular-weight CurCOOH was proved to bear the 6-COOH group in 100% purity. The optical rotatory dispersion (ORD) spectra indicated that the obtained CurCOOH behaves as a water-soluble single-strand in various pH aqueous media. This advantage has allowed us to use CurCOOH as a polymeric host to form various macromolecular complexes. For example, complexation of CurCOOH with single-walled carbon nanotubes (SWNTs) resulted in a water-soluble one-dimensional architecture, which formed a dispersion in aqueous solution that was stable for several months, and much more stable than SWNTs complexes of the similar negatively-charged polyacrylic acid (PAA) and polymethacrylic acid (PMAA). It was shown that in the complex, SWNTs are effectively wrapped by a small amount of CurCOOH, enabling them to avoid electrostatic repulsion. This pH-responsive CurCOOH formed a very stable complex with cationic water-soluble polythiophenes (PT-1), which was stabilized not only by the hydrophobic interaction but also by the electrostatic attraction between trimethylammonium cations in PT-1 and dissociated anionic COO(-) groups in CurCOOH. The included PT-1 became CD-active only in the neutral to basic pH region, and the positive Cotton effect suggested that the conjugated main chain is twisted in the right-handed direction. We also found that CurCOOH can interact with polycytidylic acid (poly(C)) only under high NaCl concentrations, the binding and release of which could be controlled by a change in the salt concentration. We believe, therefore, that CurCOOH bearing a dissociable COOH group can act as a new potential polymeric host to construct novel polymeric complexes applicable for gene carriers, biosensors, chiral polymer assemblies, etc.

Synthesis of some novel D-glucuronic acid acetylated derivatives as potential anti-tumor agents.

A structurally diverse series of Δ(4,5) -uronamide derivatives have been chemically synthesized starting from D-glucuronic acid itself by means of acetylation, activation, amide bond formation and base-catalyzed elimination protocols. Structure elucidation for all products along with optimization of the synthetic steps is described. The synthesized compounds were evaluated for their in-vitro anti-tumor activity against MCF-7, TK-10 and UACC-62 cell lines. The compounds 5, 11, 13, 15 and 16 were the most active against TK-10 cell line. On the other hand, the most active compounds against the MCF-7 cell line were 11 and 15. However, compounds 5, 7, 11, 13, 15 and 16 were the most active against the UACC-62 cell line.

Xylooligosaccharides production from a sugarcane biomass mixture: Effects of commercial enzyme combinations on bagasse/straw hydrolysis pretreated using different strategies.

Xylooligosaccharides (XOS) are non-digestible food ingredients with prebiotic properties for selectively promoting the growth of probiotics, which provide many health benefits and several applications in the food and pharmaceutical industry. The objective of this study was to optimize the concentration of commercial hemicellulases for the production of XOS, with a 2-6 polymerization degree, using a mixture of sugarcane bagasse and straw pretreated with ionic liquid or diluted sulfuric acid. The concentrations of enzymes endo-1,4-xylanase (NS50030, Novozyme®) and α-L-arabinofuranosidase (GH51) (Megazyme®) were optimized using a central composite rotatable design (CCRD). The xylooligosaccharides (XOS) released by hydrolysis were analyzed via capillary electrophoresis and quantified with HPAEC-PAD. The XOS profile obtained from the hydrolisis of the pretreated sugarcane biomass mixture (MPSA) was similar to that obtained with the hydrolisis of MBX, which provided higher xylobiose (X2) concentration. Our results also demonstrated that pretreatment with an ionic liquid favored the requirement of lower enzyme concentration in enzymatic hydrolysis for having provided a biomass with lower lignin content than the pretreatment with dilute sulfuric acid. It required up to 20% less of the optimum concentration of the endo-1,4-xylanase mixture to achieve similar values to those obtained with the biomass pretreated with dilute sulfuric acid, representing a possible alternative to reduce enzymatic cost.

Total synthesis of scutellarin and apigenin 7-O-ß-d-glucuronide.

A general protocol for direct glucuronic linkages formation featuring Au(I)-catalyzed appropriately protected glucuronyl o-alkynylbenzoate-involved glycosylation reaction, as well as a concise approach for easy access of scutellarein prominent for the mild and efficient hydroxyl group installation via borylation-oxidation sequence from flavanone derivative, has been established, based on which a novel route for scutellarin derivatives preparation has been devised. The developed strategies, among which the stepwise deprotection process was also included, guarantee the high whole synthetic efficiency, and definitely will find broad application in diversity-oriented synthesis of bioactive flavonoid glycosides.

Production of High Commercial Value Xylooligosaccharides from Meranti Wood Sawdust Using Immobilised Xylanase.

The present study explores the utilisation of a new raw material from lignocellulose biomass, Meranti wood sawdust (MWS) for high commercial value xylooligosaccharides (XOS) production using immobilised xylanase. The xylanase was immobilised by a combination of entrapment and covalent binding techniques. The hemicellulosic xylan from MWS was extracted using a standard chlorite delignification method. The production of total and derivatives of XOS from the degradation of the hemicellulosic xylan of MWS were compared to the production from the commercial xylan from Beechwood. The utilisation of the extracted xylan from MWS yielded 0.36 mg/mL of total XOS after 60 h of hydrolysis. During the hydrolysis reaction, the immobilised xylanase released a lower degree of polymerisation (DP) of XOS, mainly X2 and X3, which were the major products of xylan degradation by xylanase enzymes. The production of XOS with a lower DP from MWS demonstrated the biotechnological potential of the MWS in the future. The XOS production retained about 70% of its initial XOS production during the second cycle. This is also the first report on the utilisation of MWS wastes in enzymatic hydrolysis using immobilised xylanase for XOS production.

Anti-Dengue, Cytotoxicity, Antifungal, and In Silico Study of the Newly Synthesized 3-O-Phospo-α-D-Glucopyranuronic Acid Compound.

The aim of the current study was to synthesize new bioactive compounds and evaluate their therapeutic relevance. The chemical structure of compound 7 (methyl 3-O-phospo-α-D-glucopyranuronic acid was elucidated by physical and advance spectral technique. Also, this compound was assessed for various in vitro biological screening. The results showed that compound 7 has promising antifungal activity against selected fungal strains. Computational study was also carried out to find antimalarial efficacy of the synthesized compounds. Compounds (2-7) were tested for cytotoxicity by MTT assay, and no considerable cytotoxicity was observed. Molecular docking study was performed to predict the binding modes of new compound (7). The docking results revealed that the compound has strong attraction towards the target protein, as characterized by good bonding networks. On the basis of the acquired results, it can be predicted that compound (7) might show good inhibitory activity against dengue envelope protein.

Lithocholate glucuronide is a cholestatic agent.

Lithocholic acid and its taurine, glycine, and sulfate derivatives are potent cholestatic agents. Lithocholate glucuronide is present in the plasma and urine of patients with cholestatic syndromes, but little is known of its metabolism, excretion, and cholestatic potential. [3 beta-3H]lithocholate 3-O-beta-D-glucuronide was synthesized, and chemical and radiochemical purity were established. The aqueous solubility of lithocholate glucuronide was determined and found to be greater than that of lithocholic acid or several of its derivatives. In the range of concentrations examined, calcium ions precipitated lithocholate glucuronide stoichiometrically. The material was administered to rats prepared with an external biliary fistula. When 17-25 micrograms quantities were administered, 89.1 +/- 4.5% (mean +/- SEM) of the radiolabel was secreted in bile within the first 20 h after administration, the major fraction being secreted in less than 20 min. Four-fifths of the radiolabeled material in bile was the administered unaltered parent compound, while a minor fraction consisted of a more polar derivative(s). We showed that increasing biliary concentrations of more polar derivatives were observed with milligram doses of [3H]lithocholate glucuronide, and with time after the administration of these loading doses. Milligram doses of [3H]lithocholate glucuronide resulted in partial or complete cholestasis. When induced cholestasis was partial, secretion in bile remained the primary excretory route (82.5-105.6% recovery in bile), while, when complete cholestasis was induced, wide tissue distribution of radiolabel was observed. Cholestasis developed rapidly during infusion of [3H]lithocholate glucuronide. Bile flow was diminished within 10-20 min of the start of an infusion of 0.05 mumol, 100 g-1 body weight, minute-1, administered concomitantly with an equimolar infusion of taurocholate. The results establish that lithocholate glucuronide exerts cholestatic effects comparable to those exerted by unconjugated lithocholic acid.

Production of xylooligosaccharides by microwave-induced, organic acid-catalyzed hydrolysis of different xylan-type hemicelluloses: Optimization by response surface methodology.

A feasible approach to produce xylooligosaccharides (XOS) using organic acids as catalysts by microwave-induced hydrolysis of different hemicelluloses was developed. The effects of different acids (oxalic acid, maleic acid, citric acid and sulfuric acid), acid concentration, reaction temperature and reaction time on the hemicelluloses hydrolysis were investigated. Results demonstrated that organic acid was more beneficial to the XOS production than the conventional sulfuric acid. Higher acid concentration, higher reaction temperature and longer reaction time accelerated the further depolymerization of XOS to form monosaccharide. Response surface methodology was employed to optimize the reaction conditions (temperature and time) for the production of XOS from beechwood xylan (BX), corncob hemicelluloses (CH) and recovered hemicelluloses from the industrial waste liquor of dissolving pulp (RH), respectively. The predicted highest XOS yields were achieved to 39.31% (126.54°C-7.95min), 27.29% (120.00°C-0min), 30.32% (122.63°C-15.85min), respectively, being close to the experimental value (39.42%, 27.46% and 30.89%) from BX, CH and RH, indicating the fitted models of XOS yield were in good agreement with the experimental results.

Co-production of functional xylooligosaccharides and fermentable sugars from corncob with effective acetic acid prehydrolysis.

A novel and green approach for the coproduction of xylooligosaccharides (XOS), in terms of a series of oligosaccharide components from xylobiose to xylohexose, and fermentable sugars was developed using the prehydrolysis of acetic acid that was fully recyclable and environmentally friendly, followed by enzymatic hydrolysis. Compared to hydrochloric acid and sulfuric acid, acetic acid hydrolysis provided the highest XOS yield of 45.91% and the highest enzymatic hydrolysis yield. More than 91% conversion of cellulose was achieved in a batch-hydrolysis using only a cellulase loading of 20FPU/g cellulose and even a high solid loading of 20% without any special strategies. The acetic acid pretreated corncob should be washed adequately before saccharification to achieve complete hydrolysis. Consequently, a mass balance analysis showed that 139.8g XOS, 328.1g glucose, 25.1g cellobiose, and 147.8g xylose were produced from 1000g oven dried raw corncob.

Chemical input reduction in the arabinoxylan and lignocellulose alkaline extraction and xylooligosaccharides production.

Lignocellulosic material breakdown by hydrolysis is an important step to open new perspectives for bioenergy and special foods production like prebiotic xylooligosaccharides. Improvement of lignocellulose and arabinoxylan alkaline extraction from sugarcane bagasse and enzymatic hydrolysis were performed. Treatments 1 (10% KOH at 70°C), 3 (5% KOH at 121°C) and ZD method (24% KOH at 35°C) showed solid lignocellulose recovery of respectively 75.2%, 74.2% and 73%. A range of 24.8-27% extracted material with high arabinoxylan content (72.1-76.3%) was obtained with these treatments. Treatment 1 and 3 exhibited great KOH reduction in the method reaction, 54.1% and 76.2%, respectively. Likewise, in treatment 3 there was a decrease in ethanol consumption (40.9%) when compared to ZD method. The extracted arabinoxylan showed susceptibility to enzymatic hydrolysis with high solid loading (7%) since Trichoderma reesei xylanases were advantageous for xylose production (54.9%), while Aspergillus fumigatus xylanases achieved better XOS production (27.1%).

Scutellarin derivatives as apoptosis inducers: Design, synthesis and biological evaluation.

To explore novel antitumor agents with high efficiency and low toxicity, a series of NO-donating scutellarin derivatives (14-17) were synthesized and the antiproliferative activities against MCF-7, HCT-116, PC-3 and HepG2 cancer cell lines were assessed. Among them, compound 14b was the strongest with IC50 values of 2.96 µM, 7.25 µM, 0.09 µM and 0.50 µM, respectively, and displayed low toxicity against normal human liver L-O2 cells with an IC50 of 47.96 µM, showing good selectivity between normal and malignant liver cells. Moreover, NO releasing ability of the derivatives has been studied. Mechanism studies of the most promising compounds 14b and 15a were carried out. The results indicated that 14b and 15a could induce apoptosis, cell cycle arrest at the S phase and led to mitochondrial dysfunction in the HepG2 and PC-3 cell lines, respectively. Furthermore, Human Apoptosis Protein Array kit assay demonstrated that 14b could induce apoptosis through down-regulating the levels of procaspase-3 and inhibiting the expression of survivin, c-IAP1, HSP27, HSP60, HSP70, HO-1/HMOX1/HSP32 and HO-2/HMOX2 in HepG2 cell line. These results guaranteed compound 14b to be a drug candidate against liver cancer for further investigation.

Photoinduced electron-transfer alpha-deoxygenation of aldonolactones. Efficient synthesis of 2-deoxy-D-arabino-hexono-1,4-lactone.

A photoinduced electron-transfer (PET) reaction was used for the deoxygenation at C-2 of aldonolactones derivatized as 2-O-[3-(trifluoromethyl)benzoyl] or benzoyl esters. By irradiation of different D-galactono- and D-glucono-1,4-derivatives, with a 450W lamp, using 9-methylcarbazole as photosensitizer, the corresponding 2-deoxy-D-lyxo- and 2-deoxy-D-arabino-hexono-1,4-lactones were efficiently obtained.

Synthesis, characterization and in situ intestinal absorption of different molecular weight scutellarin-PEG conjugates.

Highly water soluble esters of scutellarin with different molecular weight polyethylene glycol (PEG) were synthesized. The physicochemical properties, the stabilities under different conditions and the in situ intestinal absorption of the conjugates in rats were investigated. By PEG modification, greatly increased water solubility and a desirable partition coefficient were obtained. These compounds act as prodrugs i.e. breakdown occurrs in a predictable fashion: in vitro, the t1/2 of them in PBS buffer at pH 7.4 was above 12 h (37 degrees C), while in plasma a more rapid breakdown was observed (t1/2 1.5-3 h). PEGylation could enhance the absorption of scutellarin in rat intestine, and scutellarin, its PEG conjugates are absorbed through intestine mainly via passive transport. When the molecular weight of PEG increased from 200 to 1000 Da, the absorption of the conjugates decreased accordingly. The range of PEG molecular weight used for the PEGylation of scutellarin was about 400-1000 Da based on considerations of the yield, the stability and the absorption.