A kinetic study of almond-β-glucosidase catalysed synthesis of hexyl-glycosides in low aqueous media: Influence of glycosyl donor and water activity

A variety of alkyl and aryl glycosides were investigated as substrates for almond β-glucosidase catalysed synthesis of hexyl-β- -glycosides in low aqueous hexanol media. The rate-limiting step in the organic media was determined to be the glycosylation of the enzyme. The kinetic constants V_max, K_m (glycosyl donor) and V_max/K_m were all influenced by the water activity and they all increased in value with increasing water activity. The increase in V_max/K_m was mainly determined by the increase in V_max and a plot of log(V_max/K_m) versus water activity resulted in a straight line with similar slopes for all glycosides but with different absolute values and thus the most reactive substrate p-nitrophenyl glucoside was the best one in the entire water activity range studied (0.53–0.96). The preference for the two competing acceptors, hexanol and water, was not affected by the aglycon part of the glucoside. Surprisingly, the ratio between trans glycosylation and hydrolysis increased with increasing water activity. A decrease in water activity caused an increase in equilibrium yield of hexyl glycoside, as expected, but was not beneficial for the kinetically controlled yield.     

GM3, GM2 and GM1 mimics designed for biosensing: chemoenzymatic synthesis, target affinities and 900 MHz NMR analysis

Undec-10-enyl, undec-10-ynyl and 11-azidoundecyl glycoside analogues corresponding to the oligosaccharides of human gangliosides GM3, GM2 and GM1 were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. Due to poor water solubility of the substrates, the reactions were carried out in methanol-water media, which for the first time were shown to be compatible with the C. jejuni α-(2→3)-sialyltransferase (CST-06) and β-(1→4)-N-acetylgalactosaminyltransferase (CJL-30). Bioequivalence of our synthetic analogues and natural gangliosides was examined by binding to Vibrio cholerae toxin and to the B subunit of Escherichia coli heat-labile enterotoxin. This bioequivalence was confirmed by binding mouse and human monoclonal antibodies to GM1 and acute phase sera containing IgM and IgG antibodies to GM1 from patients with the immune-mediated polyneuropathy Guillain-Barré syndrome. The synthesized compounds were analyzed by 1D and 2D 900 MHz NMR spectroscopy. TOCSY and DQF-COSY experiments in combination with ¹³C-¹H correlation measurements (HSQC, HMBC) were carried out for primary structural characterization, and a complete assignment of all ¹H and ¹³C chemical shifts is presented.     

Isoetin and its derivatives: Analytics,chemosystematics, and bioactivities

Isoetin, 5,7,2′,4′,5′-pentahydroxyflavone, is a rare, structurally simple natural product belonging to the flavone sub-group of flavonoids. The first reports on naturally occurring isoetin derivatives were published in the 1970s though methoxy-derivatives with the same substitution pattern had already been synthesized a decade earlier. A glucoside of isoetin was first discovered in the genus Isoetes (Lycopodiopsida). In the forty years following the discovery of the new naturally occurring flavonoid aglycone, only a limited number of reports on isoetin and its derivatives have been published. Simple, i.e. non-methyl-ether derivatives of isoetin have been found in the Isoetaceae, Asteraceae, Ranunculaceae, Rosaceae, and Rubiaceae families; while methyl ethers and their derivatives have been found in the Lycopodiaceae, Asteraceae, Cucurbitaceae, Fabaceae, and Pedaliaceae. A total of 14 non-methyl-ether-derivatives (including isoetin) and the same number of methyl ether derivatives have been described, some methyl derivatives only as synthetic compounds, others even only as virtual compounds generated for in silico studies. The published NMR data of isoetin and its derivatives as well as chemosystematic studies using isoetin derivatives as markers are compiled and critically assessed. Moreover, the papers dealing with bioactivities of isoetin and its derivatives are summarized.     

The metabolism of [3−3H]oleanolic acid-3-O-mono-[14C]glucoside in isolated cells from Calendula officinalis leaves

The radioactive precursor, [3?³H]oleanolic acid-3-O-mono-[¹⁴C]glucoside was administrated to isolated cells obtained from the leaves of Calendula officinalis. The radioactivity of the precursor was incorporated into fractions containing free oleanolic acid, individual glucosides, glucuronide F and other glucuronides. The ratio of ³H: ¹⁴C radioactivity in these fractions indicated that glucosides were formed in a process involving direct glycosylation of the precursor, whereas the glucuronides were formed from oleanolic acid released by hydrolysis of the precursor. Dynamics curves showed that glucoside II formed by direct glycosylation of the precursor was intensively transformed to other derivatives.     

Identification of a β-glucosidase hydrolyzing tuberonic acid glucoside in rice (Oryza sativa L.)

Tuberonic acid (TA) and its glucoside (TAG) have been isolated from potato (Solanum tuberosum L.) leaflets and shown to exhibit tuber-inducing properties. These compounds were reported to be biosynthesized from jasmonic acid (JA) by hydroxylation and subsequent glycosylation, and to be contained in various plant species. Here we describe the in vivo hydrolytic activity of TAG in rice. In this study, the TA resulting from TAG was not converted into JA. Tuberonic acid glucoside (TAG)-hydrolyzing β-glucosidase, designated OsTAGG1, was purified from rice by six purification steps with an ～4300-fold purification. The purified enzyme migrated as a single band on native PAGE, but as two bands with molecular masses of 42 and 26 kDa on SDS–PAGE. Results from N-terminal sequencing and peptide mass fingerprinting of both polypeptides suggested that both bands were derived from a single polypeptide, which is a member of the glycosyl hydrolase family 1. In the native enzyme, the K_m and V_max values of TAG were 31.7 μM and 0.25 μkatal/mg protein, OsTAGG1 preferentially hydrolyzed TAG and methyl TAG. Here we report that OsTAGG1 is a specific β-glucosidase hydrolyzing TAG, which releases the physiologically active TA.     

A new neolignan glucoside from hairy roots of Cichorium intybus

A new neolignan glucoside was isolated from hairy roots of Cichorium intybus. Its structure was determined as (7S, 8R)-3′-demethyl-dehydrodiconiferyl alcohol-3′-O-β-glucopyranoside based on a combination of 1D and 2D NMR techniques and CD data. In addition, further water soluble constituents, including four known phenolic compounds and one known sesquiterpene lactone glucoside, were isolated from the same source.     

13C-N.M.R. Spectroscopy of α- and β-anomeric series of alkyl l-arabinopyranosides

Anomeric pairs of l-arabinopyranosides of a variety of aliphatic alcohols were prepared, and their n.m.r. spectroscopy, especially the glycosylation shift of their ¹³C signals, was investigated in comparison with those of d-glucopyranosides, d-mannopyranosides, and l-rhamnopyranosides reported previously. It was found that the glycosylation shift of the l-arabinopyranosides in the present study is almost the same as that of d-glucopyranosides, and the conformational equilibrium of each of these l-arabinopyranosides is very similar to that of the corresponding anomer of methyl l-arabinopyranoside, namely, a preponderance of the ⁴C₁, form, regardless of the structure of the aglycon alcohol. The present results are also useful for structural study of naturally occurring arabinopyranosides.     

Chemical preparation of sialyl Lewis x using an enzymatically synthesized sialoside building block

The sialyl Lewis x tetrasaccharide with a propylamine aglycon was assembled by chemoselective glycosylation from a p-tolyl thioglycosyl donor obtained from an enzymatically synthesized sialodisaccharide. Combining the advantages of highly efficient enzymatic synthesis of sialoside building blocks, and diverse chemical glycosylation, this chemoenzymatic approach is practical for obtaining complex sialosides and their analogues.     

Enhanced chemical and biological activities of a newly biosynthesized eugenol glycoconjugate, eugenol α-d-glucopyranoside

Eugenol, the essential component (over 90 %) of clove oil from Eugenia caryophyllata Thunb. (Myrtaceae), is a phenolic compound well known for its versatile pharmacological actions, including analgesic, local anesthetic, anti-inflammatory, antimicrobial, antitumor, and hair-growing effects. However, the application of eugenol is greatly limited mainly because of its unwanted physicochemical properties, such as low solubility, liability to sublimation, and pungent odor. Since glycosylation has been suggested to improve the physicochemical and biological properties of the parental compound, we have previously developed a novel and efficient way to biosynthesize highly purified eugenol α-D-glucopyranoside (α-EG). In light of the widely acknowledged importance of pure eugenol and the potential superiority of the glycosylation, it is crucial to further explore and compare the physicochemical and biological properties of these two phenolic compounds. In this study, we demonstrate that glucosylation is a promising method for modification of phenolic compound, and that α-EG is superior over its parent eugenol, in all of the tested aspects, including physicochemical properties, antioxidation activity, and antimicrobial and antitumor activities. These results strongly suggest that α-EG, as a novel prodrug, may serve as a useful probe and potential therapeutic drug in both fundamental research and clinical application in the coming future.     

Biosynthesis of dehydrodiconiferyl alcohol glucosides: implications for the control of tobacco cell growth

The dehydrodiconiferyl alcohol glucosides A and B are factors isolated from transformed Vinca rosea tumor cells that can replace the cytokinin requirement for growth of tobacco (Nicotiana tabacum) pith and callus cells in culture. These factors, present in tobacco pith cells, have their concentrations elevated approximately 2 orders of magnitude after cytokinin exposure. Biosynthesis experiments showed that these compounds are not cell wall fragments, as previously suggested, but are produced directly from coniferyl alcohol. Their synthesis is probably associated with the existing pathway for cell wall biosynthesis in both Vinca tumors and tobacco pith explants. The pathway requires only two steps, the dimerization of coniferyl alcohol by a soluble intracellular peroxidase and subsequent glycosylation. Biosynthetic experiments suggested that dehydrodiconiferyl alcohol glucoside breakdown was very slow and control of its concentration was exerted through restricted availability of coniferyl alcohol.     

Grayanotoxin-XVIII and grayanoside B,a new a-nor-b-homo-ent-kaurene and its glucoside from Leucothoe grayana

From Leucothoe grayana a new grayanoid (diterpene) and its glucoside have been isolated. The structures of these new natural compounds have been established by chemical and spectroscopic means and by correlation with a known compound.     

New sesterterpenoids and other constituents from Salvia dominica growing wild in Jordan

A new nor-oleanane triterpene 24-nor-4(23)-12-oleanadien-2α,3α,28-triol (1) and two new sesterterpenes salvidominicolide A (2), salvidominicolide B (3) together with fifteen known compounds were isolated from Salvia dominica L. growing wild in Jordan. The known compounds comprised six flavones, three triterpenes, one diterpene, one sesterterpene, two oxygenated monoterpenes, one sterol glucoside and one flavone glucoside. The isolated compounds were elucidated by extensive spectroscopic methods including NMR (1D and 2D), UV, IR and MS (HRMS).     

Synthesis and antibacterial activity of novel modified 5-O-desosamine ketolides

A series of novel modified 5-O-desosamine-ketolides were synthesized. The 5-O-desosamine fragment was removed from ketolide by an efficient and mild manipulation. 4-O-substituted desosamine was introduced into the ketolide aglycon and various coupling methods were essayed for the glycosylation. Three novel ketolides were tested for in vitro antibacterial activity against a panel of susceptible and resistant pathogens. Compound 26 showed potent activity against all the methicillin-sensitivity and resistant pathogens.     

New acyclic sesquiterpenoid derivatives and a monoterpene disaccharide from Dichondra repens Forst.

A new highly oxygenated acyclic sesquiterpenoid (2E, 6E)-8,10,11-trihydroxyl-7,11-dimethyl-3-hydroxymethyl-2,6-dodecadienoic acid (1) and its glucoside (2), together with a new pinane monoterpene disaccharide glucoside 6,6-dimethyl-2-methlenebicyclo [3.1.1]hept-3-O-(6-O-apiofuranosyl)-β-d-glucopyranoside (3) were isolated from hydrophilic extract of Dichondra repens. Their structures were elucidated on the basis of spectroscopic analyses and chemical methods. The three compounds did not show any cytotoxic activity (IC50 > 20 μM) against two human lung cancer cell lines (NCI-H661 and A549).     

Chlorine-containing iridoid and iridoid glucoside, and other glucosides from leaves of Myoporum bontioides

Sixteen compounds were isolated from the MeOH extract of leaves of Myoporum bontioides. The five compounds hitherto unknown, were elucidated to be a chlorine-containing iridoid, named myopochlorin, and an iridoid glucoside, an acylated iridoid glucoside, a linear acetogenin glucoside, and an acyclic monoterpene glucoside, named myobontiosides A-D, respectively, by means of spectroscopic analyses.     

Highly hydroxylated steroids of the starfish Archaster typicus from the Vietnamese waters

Five new steroidal compounds, including an unusual glucoside, along with several known steroids were isolated from the starfish Archaster typicus collected in shallow waters of Quang Ninh province (Vietnam). Three new compounds are 27-nor-cholestane derivatives and the other two are 24,26-dihydroxycholestane derivatives. A biogenesis pathway for the unusual side chain of 27-nor-cholestane derivatives is proposed. Isolated compounds presented moderate toxic effects in the sperm- and 8-blastomere tests on embryonal development of the sea urchin Strongylocentrotusintermedius.     

In vitro stability and ex vivo absorption of thymol monoglucosides in the porcine gut

Thymol α-D-glucopyranoside (TαG) and thymol β-D-glucopyranoside (TβG) are believed to have different kinetic behaviours in the porcine gut than its parent aglycon thymol. However, recently, it was shown that concentrations of both glucosides decreased rapidly in the stomach and proximal small intestine following oral supplementation to piglets as did thymol. Yet, the stability of thymol glucosides in gut contents and their absorption route remains obscure. Therefore, a series of in vitro incubations were performed, simulating the impact of pH, digestive enzymes, bacterial activity and mucosal extracts on stability of these glucosides. Their absorption mechanisms were investigated using the Ussing chamber model in the presence or the absence of inhibitors of sodium-dependent glucose linked transporter 1 and lactase phlorizin hydrolase. Both glucosides remained intact at physiological pH levels in the presence of digestive enzymes. Recoveries from TαG and TβG were below 90% when incubated with small intestinal homogenates from the distal jejunum or from all sampled sites, respectively. However, no aglycon could be detected in these samples. Bacterial inoculum of the small intestine, on the other hand, hydrolysed TβG quickly with up to 44% of free aglycon appearing. TαG proved more resistant to porcine gastro-intestinal bacterial glucosidases with only trace amounts (<1%) of free thymol at the end of the incubations. Electrophysiological measurements in Ussing chambers did not suggest active transport of the glucosides. Mucosal TαG and TβG concentrations were unchanged between start and end of the absorption measurements. Additionally, no TαG and only a very limited amount of TβG were retrieved from the serosal side. Tissue associated concentrations, although marginal (<1% of luminal concentration), were mainly as intact glucoside or as aglycon for TαG and TβG, respectively. Addition of both inhibitors significantly increased the amount of intact glucosides retrieved from the mucosal tissues as compared to controls. In conclusion, bacterial hydrolysis was identified as the most important source of TβG loss, whereas TαG seemed less prone to degradation or absorption in these in vitro and ex vivo models.     

Formation Mechanism of 2,6-Dimethyl-2,6-Octadienes from Thermal Decomposition of Linalyl β-D-Glucopyranoside

Thermally decomposed products of (±)-linalyl β-D-glucoside were analyzed by GC and GC/MS. 2,6-Dimethyl-2,6-octadienes produced by mild pyrolysis of linalyl β-D-glucopyranoside under a vacuum were detected and characterized by MS and NMR spectroscopy. This suggests that 2,6-dimethyl-2,6-octadienes are produced during thermal decomposition of the glucoside via proton transfer from the anomeric position to C-6 in the aglycon moiety. A stable isotope labeling experiment directly indicated the new reaction mechanism.     

Cytotoxic properties of the alkaloid rutaecarpine and its oligocyclic derivatives and chemical modifications to enhance water-solubility

The alkaloid rutaecarpine and its derivatives have been described as cytotoxic and hold potential as antitumor agents. Nevertheless, their synthesis is demanding and compounds display poor water solubility. Herein, we describe the synthesis of two sets of rutaecarpine derivatives with amine functions to improve solubility. Using a classic shake-flask experiment and a potentiometric titration platform, the water solubility of the compounds was determined. Solubility improved significantly with the amine functions connected over the indole-N atom. Reduction of metabolic activity and cell viability on HeLa cells was in the same range or better for these derivatives compared to the chemically unaltered parent compounds prepared in a new synthetic procedure established in our group.     

Regioselective glycosylation of hydroquinone to α-arbutin by cyclodextrin glucanotransferase from Thermoanaerobacter sp.

Hydroquinone glycosides were produced by transglycosylation reactions catalyzed by cyclodextrin glucanotransferase (CGTase) from Thermoanaerobacter sp. (Toruzyme^® 3.0L). The reactions were carried out in an aqueous system containing hydroquinone (HQ) and maltodextrin as acceptor and donor substrate molecules respectively. The conditions for the synthesis of hydroquinone glucoside (α-arbutin) were 9 mM hydroquinone, maltodextrin (5%, w/v) in 20 mM citrate phosphate buffer, pH 5.5 and 0.025 mg/ml toruzyme at 40 °C for 24 h. The transfer efficiency of hydroquinone glycosylation was 31.8% and 29.2% respectively, when α-cyclodextrin and maltodextrin were employed as donor substrates. The major glycoside product was identified as hydroquinone-1-O-α-d-glucopyranoside (α-arbutin) on the basis of mass spectrometric, nuclear magnetic resonance analysis and component analysis of its enzymatic hydrolysates. The highest molar yield of α-arbutin (21.2%) was obtained when α-cyclodextrin was used as the donor substrate. A two step enzymatic reaction system comprising of CGTase and amyloglucosidase helped to attain a molar yield of 30% for α-arbutin. At room temperature the solubility of α-arbutin in water was determined to be 12.8 g/100 ml which is approximately 1.8 fold higher than that of hydroquinone.