Unravelling the Specificity of Laminaribiose Phosphorylase from Paenibacillus sp. YM-1 towards Donor Substrates Glucose/Mannose 1-Phosphate by Using X-ray Crystallography and Saturation Transfer Difference NMR Spectroscopy

Glycoside phosphorylases (GPs) carry out a reversible phosphorolysis of carbohydrates into oligosaccharide acceptors and the corresponding sugar 1-phosphates. The reversibility of the reaction enables the use of GPs as biocatalysts for carbohydrate synthesis. Glycosyl hydrolase family 94 (GH94), which only comprises GPs, is one of the most studied GP families that have been used as biocatalysts for carbohydrate synthesis, in academic research and in industrial production. Understanding the mechanism of GH94 enzymes is a crucial step towards enzyme engineering to improve and expand the applications of these enzymes in synthesis. In this work with a GH94 laminaribiose phosphorylase from Paenibacillus sp. YM-1 (PsLBP), we have demonstrated an enzymatic synthesis of disaccharide 1 (β-d -mannopyranosyl-(1→3)-d -glucopyranose) by using a natural acceptor glucose and noncognate donor substrate α-mannose 1-phosphate (Man1P). To investigate how the enzyme recognises different sugar 1-phosphates, the X-ray crystal structures of PsLBP in complex with Glc1P and Man1P have been solved, providing the first molecular detail of the recognition of a noncognate donor substrate by GPs, which revealed the importance of hydrogen bonding between the active site residues and hydroxy groups at C2, C4, and C6 of sugar 1-phosphates. Furthermore, we used saturation transfer difference NMR spectroscopy to support crystallographic studies on the sugar 1-phosphates, as well as to provide further insights into the PsLBP recognition of the acceptors and disaccharide products.     

Engineering of a Thermostable Biocatalyst for the Synthesis of 2-O-Glucosylglycerol

2-O-Glucosylglycerol is accumulated by various bacteria and plants in response to environmental stress. It is widely applied as a bioactive moisturising ingredient in skin care products, for which it is manufactured via enzymatic glucosylation of glycerol by the sucrose phosphorylase from Leuconostoc mesenteroides. This industrial process is operated at room temperature due to the mediocre stability of the biocatalyst, often leading to microbial contamination. The highly thermostable sucrose phosphorylase from Bifidobacterium adolescentis could be a better alternative in that regard, but this enzyme is not fit for production of 2-O-glucosylglycerol due to its low regioselectivity and poor affinity for glycerol. In this work, the thermostable phosphorylase was engineered to alleviate these problems. Several engineering approaches were explored, ranging from site-directed mutagenesis to conventional, binary, iterative or combinatorial randomisation of the active site, resulting in the screening of ∼3,900 variants. Variant P134Q displayed a 21-fold increase in catalytic efficiency for glycerol, as well as a threefold improvement in regioselectivity towards the 2-position of the substrate, while retaining its activity for several days at elevated temperatures.     

Trehalose Analogues: Latest Insights in Properties and Biocatalytic Production

Trehalose (α-d-glucopyranosyl α-d-glucopyranoside) is a non-reducing sugar with unique stabilizing properties due to its symmetrical, low energy structure consisting of two 1,1-anomerically bound glucose moieties. Many applications of this beneficial sugar have been reported in the novel food (nutricals), medical, pharmaceutical and cosmetic industries. Trehalose analogues, like lactotrehalose (α-d-glucopyranosyl α-d-galactopyranoside) or galactotrehalose (α-d-galactopyranosyl α-d-galactopyranoside), offer similar benefits as trehalose, but show additional features such as prebiotic or low-calorie sweetener due to their resistance against hydrolysis during digestion. Unfortunately, large-scale chemical production processes for trehalose analogues are not readily available at the moment due to the lack of efficient synthesis methods. Most of the procedures reported in literature suffer from low yields, elevated costs and are far from environmentally friendly. “Greener” alternatives found in the biocatalysis field, including galactosidases, trehalose phosphorylases and TreT-type trehalose synthases are suggested as primary candidates for trehalose analogue production instead. Significant progress has been made in the last decade to turn these into highly efficient biocatalysts and to broaden the variety of useful donor and acceptor sugars. In this review, we aim to provide an overview of the latest insights and future perspectives in trehalose analogue chemistry, applications and production pathways with emphasis on biocatalysis.     

2-氯-4-硝基苯基-β-D-吡喃岩藻糖苷的合成

2-氯-4-硝基苯基糖苷是一类重要的糖基酶底物试剂,在相关酶抑制剂制备、酶生物活性研究中有着广泛的应用.以D-半乳糖为原料,通过4步反应得到中间体D-岩藻糖;再经乙酰化制备1,2,3,4-三-O-乙酰基-α,β-D-吡喃岩藻糖,溴代反应获得1-溴-2,3,4-三-O-乙酰基-α-D-吡喃岩藻糖,通过溴代糖与2-氯4-硝基苯酚偶联,立体选择性获得2-氯-4-硝基苯-2,3,4-三-O-乙酰基-β-D-吡喃岩藻糖基糖苷,最后脱除乙酰基保护得标题化合物.该化合物可以作为一种新型的β-D-岩藻糖苷酶底物试剂,用于酶活性测试,其合成方法未见文献报道.     

Non-Chlorine Bleaching of Kraft Pulp II. Ozonation of Methyl 4-O-Ethyl-β-D-glucopyranoside (1) Preparation of Authentic Carbonyl Sugars and Their Analysis by Gas Chromatography and Mass Spectrometry

Methyl 4-O-ethyl-β-D-glucopyranoside (1) was prepared as a model compound for cellulose to investigate the reactions of ozone with polysaccharides during ozone bleaching of kraft pulp. The model compound was converted into authentic carbonyl sugars, methyl 3,6-di-O-acetyl-4-O-ethyl-β-D-arabino-hexopyranosidulose (2), methyl 2,6-di-O-acetyl-4-O-ethyl-β-D-ribo-hexopyranoside-3-ulose (3), methyl 2,3-di-O-acetyl-4-O-ethyl-β-D-gluco-hexodialdo-1,5-pyranoside (4). These carbonyl sugars were converted into O-methyloximes and analyzed by gas chromatography and mass spectrometry.     

Application of high-speed counter-current chromatography coupled with high performance liquid chromatography for the separation and purification of Quercetin-3-O-sambubioside from the leaves of Nelumbo nucifera

Quercetin-3-O-sambubioside [Quercetin-3-O-β-D-xylopyranosyl (1→2)-β-D-glucopyranoside] was separated and purified by semi-preparative high-speed counter-current chromatography (HSCCC) with a two-phase-solvent system composed of ethyl acetate-n-butanol-water (4:1:5, v/v) from the leaves of Nelumbo nucifera (Lotus). A total of 5.0 mg of the targeted compound with a purity of 98.6% as determined by high performance liquid chromatography (HPLC) was obtained from 100 mg of the crude extract cleaned up by AB-8 macroporous resin in a one-step separation. Quercetin-3-O-sambubioside was a novel flavonoid glycoside from the leaves of Nelumbo nucifera, and its chemical structure was identified by means of ESI-MS, 1D NMR and 2D NMR.     

Effect of Counter Cation on Alkaline Reaction of β-O-4-Type Substructure in Lignin

This study aimed to clarify the effects of counter cations on the alkaline-induced β-O-4 bond cleavage and further reactions of β-O-4-type substructures in lignin. For this purpose, a non-phenolic β-O-4-type lignin model compound, the erythro isomer of 2-(2-methoxyphenoxy)-1-(3,4-dimethoxyphenyl)propane-1,3-diol (veratrylglycerol-β-guaiacyl ether), was treated in a 100% water solution or an aqueous methanol, ethanol, or 1,4-dioxane solution containing LiOH, NaOH, or CsOH as an alkaline source at 150?°C. The rates of β-O-4 bond cleavage were in the order of CsOH?>?NaOH?>?LiOH in all solvents. This order can rationally be attributed to the strength of the interactions between HO^– and the counter cations. Because Cs⁺ has the lowest positive charge density among the counter cations and hence interacts with HO^– most weakly, HO^– can exert its reactivity most actively in the reactions using CsOH. We also discuss how the counter cations affect the profile of reaction products.     

Further studies on sphingolipids in wheat grain

The principal molecular species of sphingolipids in wheat grain were confirmed to beN-2′-hydroxylignoceroyl-4-hydroxysphinganine for ceramide, and 1-O-β-glucosyl-, 1-O-[β-mannosyl(1→4)-O-β-glucosyl]-, 1-O-[β-mannosyl(1→4)-O-β-mannosyl(1→4)-O-β-glucosyl]-and 1-O[β-mannosyl(1→4)-O-β-mannosyl(1→4)-O-β-mannosyl(1→4)-O-β-glucosyl]-N-2′-hydroxypalmitoyl (or hydroxyarachidoyl)-cis-8-sphingenine for mono-, di-, tri- and tetraglycosylceramide, respectively. A novel glycolipid, cellobiosylceramide, was found as the minor diglycosylceramide; the major species was characterized to be 1-O-[β-glucosyl(1→4)-O-β-glucosyl]-N-2′-hydroxypalmitoyl (or hydroxyarachidoyl)-cis-8-sphingenine. It was observed in these sphingolipids that the dihydroxy bases were combined mainly with C₁₆ and C₂₀ acids, whereas the trihydroxy bases combined mostly with acids of chain length of 20 or more.     

Activity-Guided Isolation and Purification of Three Flavonoid Glycosides from Neo-Taraxacum siphonanthum by High-Speed Counter-Current Chromatography

DPPH (1,1-diphenyl-2-picryhydrazyl) radical scavenging assay was used to screen different fractions of Neo-Taraxacum siphonanthum ethanol extracts. The potent active fraction was isolated and purified by preparative high-speed counter-current chromatography (HSCCC) with a solvent system composed of n-hexane-n-butanol-water (3:4:7, v/v/v). The flow rate was 1.5 mL/min and resolution speed was 800 rpm. Three flavonoid glycosides with the purity over 99% were obtained and identified as luteolin- 3′-O-β-D-glucopyranoside (I), luteolin-7-O-β-D-glucopyranoside (II), and luteolin-4′-O-β-D-glucopyranoside (III) by ESI-MS, ¹H NMR and ¹³C NMR analysis. Antioxidant activity of three flavonoid glycosides was assessed by DPPH assay, all of which showed potent activity.     

Identification of Genes Essential for Sulfamate and Fluorine Incorporation During Nucleocidin Biosynthesis

Nucleocidin is an adenosine derivative containing 4’-fluoro and 5’-O-sulfamoyl substituents. In this study, nucleocidin biosynthesis is examined in two newly discovered producers, Streptomyces virens B-24331 and Streptomyces aureorectus B-24301, which produce nucleocidin and related derivatives at titers 30-fold greater than S. calvus. This enabled the identification of two new O-acetylated nucleocidin derivatives, and a potential glycosyl-O-acetyltransferase. Disruption of nucJ, nucG, and nucI, within S. virens B-24331, specifying a radical SAM/Fe−S dependent enzyme, sulfatase, and arylsulfatase, respectively, led to loss of 5’-O-sulfamoyl biosynthesis, but not fluoronucleoside production. Disruption of nucN, nucK, and nucO specifying an amidinotransferase, and two sulfotransferases respectively, led to loss of fluoronucleoside production. Identification of S. virens B-24331 as a genetically tractable and high producing strain sets the stage for understanding nucleocidin biosynthesis and highlights the utility of using 16S-RNA sequences to identify alternative producers of valuable compounds in the absence of genome sequence data.     

Glycosynthase activity of Geobacillus stearothermophilus GH52 beta-xylosidase: efficient synthesis of xylooligosaccharides from alpha-D-xylopyranosyl fluoride through a conjugated reaction

Glycosynthases are mutant glycosidases in which the acidic nucleophile is replaced by a small inert residue. In the presence of glycosyl fluorides of the opposite anomeric configuration (to that of their natural substrates), these enzymes can catalyze glycosidic bond formation with various acceptors. In this study we demonstrate that XynB2E335G, a nucleophile-deficient mutant of a glycoside hydrolase family 52 beta-xylosidase from G. stearothermophilus, can function as an efficient glycosynthase, using alpha-D-xylopyranosyl fluoride as a donor and various aryl sugars as acceptors. The mutant enzyme can also catalyze the self-condensation reaction of alpha-D-xylopyranosyl fluoride, providing mainly alpha-D-xylobiosyl fluoride. The self-condensation kinetics exhibited apparent classical Michaelis-Menten behavior, with kinetic constants of 1.3 s(-1) and 2.2 mM for k(cat) and K(M(acceptor)), respectively, and a k(cat)/K(M(acceptor)) value of 0.59 s(-1) mM(-1). When the beta-xylosidase E335G mutant was combined with a glycoside hydrolase family 10 glycosynthase, high-molecular-weight xylooligomers were readily obtained from the affordable alpha-D-xylopyranosyl fluoride as the sole substrate.     

Iron‐Catalyzed Direct Alkylamination of Phenols with O‐Benzoyl‐N‐alkylhydroxylamines under Mild Conditions

A novel iron‐catalyzed direct alkylamination reaction of phenols has been achieved with O‐benzoyl‐N‐alkylhydroxylamines as aminating agents. This protocol provides a facile access to N‐alkyl‐substituted aminophenols though a radical reaction from phenols. The catalytic direct alkylamination operates at room temperature without the need of any ligands and additives to afford the desired products with excellent regioselectivity and functional group tolerance.

     

An Advanced Apralog with Increased in vitro and in vivo Activity toward Gram-negative Pathogens and Reduced ex vivo Cochleotoxicity

We describe the convergent synthesis of a 5-O-β-D-ribofuranosyl-based apramycin derivative (apralog) that displays significantly improved antibacterial activity over the parent apramycin against wild-type ESKAPE pathogens. In addition, the new apralog retains excellent antibacterial activity in the presence of the only aminoglycoside modifying enzyme (AAC(3)-IV) acting on the parent, without incurring susceptibility to the APH(3’) mechanism that disables other 5-O-β-D-ribofuranosyl 2-deoxystreptamine type aminoglycosides by phosphorylation at the ribose 5-position. Consistent with this antibacterial activity, the new apralog has excellent 30 nM activity (IC₅₀) for the inhibition of protein synthesis by the bacterial ribosome in a cell-free translation assay, while retaining the excellent across-the-board selectivity of the parent for inhibition of bacterial over eukaryotic ribosomes. Overall, these characteristics translate into excellent in vivo efficacy against E. coli in a mouse thigh infection model and reduced ototoxicity vis à vis the parent in mouse cochlear explants.     

Structure of the O-Antigen and the Lipid A from the Lipopolysaccharide of Fusobacterium nucleatum ATCC 51191

Fusobacterium nucleatum is a common member of the oral microbiota. However, this symbiont has been found to play an active role in disease development. As a Gram-negative bacterium, F. nucleatum has a protective outer membrane layer whose external leaflet is mainly composed of lipopolysaccharides (LPSs). LPSs play a crucial role in the interaction between bacteria and the host immune system. Here, we characterised the structure of the O-antigen and lipid A from F. nucleatum ssp. animalis ATCC 51191 by using a combination of GC-MS, MALDI and NMR techniques. The results revealed a novel repeat of the O-antigen structure of the LPS, [→4)-β-d -GlcpNAcA-(1→4)-β-d -GlcpNAc3NAlaA-(1→3)-α-d -FucpNAc4NR-(1→], (R=acetylated 60 %), and a bis-phosphorylated hexa-acylated lipid A moiety. Taken together these data showed that F. nucleatum ATCC 51191 has a distinct LPS which might differentially influence recognition by immune cells.     

Contribution of the γ-hydroxy group to the β-O-4 bond cleavage of lignin model compounds in a basic system using tert-butoxide

Abstract

The most common non-phenolic β-O-4-type lignin model compounds with or without the γ-hydroxymethyl group (C₆-C₃- or C₆-C₂-type, respectively) were treated in a 0.5?mol/L potassium tert-butoxide in DMSO solution at 30?°C to examine the effects of presence of the group. The β-O-4 bond of the C₆-C₃-type cleaved more rapidly than the C₆-C₂-type, indicating that the γ-hydroxy group contributes to the cleavage, in contrast to their reactions in alkaline pulping processes. Furthermore, the β-O-4 bond of the threo isomer of the C₆-C₃-type cleaved more rapidly than that of the erythro isomer. This result can be attributed to the fact that the erythro isomer has the hydrogen bond between a generated alkoxide and the other hydroxy group at its α- and γ-positions in its most-preferential conformer, interfering with the β-O-4 bond cleavage. It was also suggested in treatments of their methyl-etherified derivatives at the α- or γ-hydroxy group that the contribution of the γ-hydroxy group of the threo isomer is greater than that of the erythro isomer. Detailed examination of the distribution profile of reaction products supported this greater contribution of the γ-hydroxy group of the threo isomer.     

Effect of Ionic Liquids on Preparative Separation of Flavonoid Compounds in the Extract from Brassica napus L. Pollen using High-Performance Counter-Current Chromatography

To investigate the effect of ionic liquids (ILs) as additives to a two-phase solvent system using high-performance counter-current chromatography, the two structurally similar flavonoids, Kaempferol-3, 4′-di-O-β-D-glucoside and Kaempferol 3-O-β-D-(2-O-β-D-glucosyl)-glucopyranoside, were chosen as models. The effects of the length of the alkyl groups on the imidazolium ring and its counterion, and the concentrations of ILs on the K values were investigated. The results showed that the K values of the target compounds could be improved when ILs were used as additives in the two-phase solvent system. The mechanism of ILs as additives to the solvent system was discussed for the first time.     

Nonhydrolyzable Heptose Bis- and Monophosphate Analogues Modulate Pro-inflammatory TIFA-NF-κB Signaling

d -Glycero-d -manno-heptose-1β,7-bisphosphate (HBP) and d -glycero-d -manno-heptose-1β-phosphate (H1P) are bacterial metabolites that were recently shown to stimulate inflammatory responses in host cells through the activation of the TIFA-dependent NF-κB pathway. To better understand structure-based activity in relation to this process, a family of nonhydrolyzable phosphonate analogues of HBP and H1P was synthesized. The inflammation modulation by which these molecules induce the TIFA-NF-κB signal axis was evaluated in vivo at a low-nanomolar concentration (6 nM) and compared to that of the natural metabolites. Our data showed that three phosphonate analogues had similar stimulatory activity to HBP, whereas two phosphonates antagonized HBP-induced TIFA-NF-κB signaling. These results open new horizons for the design of pro-inflammatory and innate immune modulators that could be used as vaccine adjuvant.     

Investigation on the Hydrogen Abstraction from Methyl Glucoside by Active Oxygen Species under Oxygen Delignification Conditions. Part 2: Study on the C-2 Position

Abstract

It was investigated whether active oxygen species (AOS) generated in situ by reacting a phenolic compound, 2,4,6-trimethylphenol, with O₂ abstract the C-2 hydrogen from a carbohydrate model compound, methyl β-d-glucopyranoside (MGPβ), under conditions simulating oxygen delignification (0.5 mol/l NaOH, 0.36 mmol/l FeCl₃, 1.1 MPa O₂, 95°C). MGPβ was degraded less than methyl β-d-mannopyranoside (MMPβ) when they were reacted together with AOS. It was suggested that the configurational difference at their C-2 positions results in their different reactivity toward AOS. When MGPβ and methyl β-d-(2-²H)glucopyranoside (MGPβ-2D) were reacted together with AOS, MGPβ was degraded slightly but more than MGPβ-2D, which clearly indicates that the C-2 hydrogen of MGPβ is abstracted by AOS. MGPβ and MGPβ-2D were also subjected together to alkaline H₂O₂ treatment (0.5 mol/l NaOH, 0.36 mmol/l FeCl₃, 58.8 mmol/l H₂O₂, 95°C) in which oxyl anion radical (O^-?) is likely the only AOS to degrade these compounds. At least some AOS other than O^-? abstract the C-2 hydrogen from MGPβ in the aforementioned oxygen-alkali treatments.     

Selective Monovalent Galectin-8 Ligands Based on 3-Lactoylgalactoside

Galectin-8 has gained attention as a potential new pharmacological target for the treatment of various diseases, including cancer, inflammation, and disorders associated with bone mass reduction. To that end, new molecular probes are needed in order to better understand its role and its functions. Herein we aimed to improve the affinity and target selectivity of a recently published galectin-8 ligand, 3-O-[1-carboxyethyl]-β-d -galactopyranoside, by introducing modifications at positions 1 and 3 of the galactose. Affinity data measured by fluorescence polarization show that the most potent compound reached a K_D of 12 μM. Furthermore, reasonable selectivity versus other galectins was achieved, making the highlighted compound a promising lead for the development of new selective and potent ligands for galectin-8 as molecular probes to examine the protein's role in cell-based and in vivo studies.     

An efficient and novel method for the synthesis of cardiolipin and its analogs

A novel synthetic method has been developed for cardiolipin and its analog via a chlorophosphoramidite coupling reaction followed by oxidation. The reagent, N,N-diisopropylmethylphosphoramidic chloride, couples effectively with 1,2-O-dimyristoyl-sn-glycerol in the presence of an amidite activator to form a phosphoamidite intermediate, which then reacts with 2-O-benzylglycerol in the presence of a basic catalyst followed by in situ oxidation to give the corresponding protected cardiolipin. Deprotection of the protecting groups provides tetramyristoyl cardiolipin in good overall yield of 60%. The synthetic method is applicable to large-scale synthesis of cardiolipin and various analogs with or without unsaturation for liposomal drug delivery. Presented at the 94th AOCS Annual Meeting & Expo, Kansas City, Missouri, May 4–7, 2003.