Synthesis and ab initio conformational investigation of a series of model sulfated α-L-iduronopyranosides.

Due to the all-axial orientation of the OH-groups in the 1C4 chair conformation considered standard for L-hexapyranosides, including l-iduronopyranoside - a component of many biologically and medically significant sulfated glycans, these monosaccharides can be anticipated to display unusual conformations upon the introduction of bulky and charged substituents. Herein we describe the synthesis of a series of iduronopyranoside derivatives with varying sulfation patterns, which were studied computationally using the DLPNO-MP2 approach and by means of analyzing their chemical shifts to ascertain the effects sulfation has on the conformation of the iduronopyranoside ring.

Structural studies of the O polysaccharides from the lipopolysaccharides of Azospirillum thiophilum BV-ST and Azospirillum griseum L-25-5w-1T.

Diazotrophic bacteria of the genus Azospirillum are known widely, because they are ubiquitous in the rhizosphere and can promote the growth and performance of nonlegume plants. Recently, more Azospirillum species have been isolated from sources other than plants or soil. We report the structures of the O polysaccharides (OPSs) from the lipopolysaccharides of the type strains A. thiophilum BV-ST (1) and A. griseum L-25-5w-1T (2), isolated from aquatic environments. Both structures have a common tetrarhamnan in the repeating-unit, which is decorated with a side xylose in the OPS of A. thiophilum BV-ST.

Structure, Physicochemical Properties and Biological Activity of Lipopolysaccharide from the Rhizospheric Bacterium Ochrobactrum quorumnocens T1Kr02, Containing d-Fucose Residues.

Lipopolysaccharides (LPSs) are major components of the outer membranes of Gram-negative bacteria. In this work, the structure of the O-polysaccharide of Ochrobactrum quorumnocens T1Kr02 was identified by nuclear magnetic resonance (NMR), and the physical-chemical properties and biological activity of LPS were also investigated. The NMR analysis showed that the O-polysaccharide has the following structure: →2)-ß-d-Fucf-(1→3)-ß-d-Fucp-(1→. The structure of the periplasmic glucan coextracted with LPS was established by NMR spectroscopy and chemical methods: →2)-ß-d-Glcp-(1→. Non-stoichiometric modifications were identified in both polysaccharides: 50% of d-fucofuranose residues at position 3 were O-acetylated, and 15% of d-Glcp residues at position 6 were linked with succinate. This is the first report of a polysaccharide containing both d-fucopyranose and d-fucofuranose residues. The fatty acid analysis of the LPS showed the prevalence of 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, lactobacillic, and 27-hydroxyoctacosanoic acids. The dynamic light scattering demonstrated that LPS (in an aqueous solution) formed supramolecular particles with a size of 72.2 nm and a zeta-potential of -21.5 mV. The LPS solution (10 mkg/mL) promoted the growth of potato microplants under in vitro conditions. Thus, LPS of O. quorumnocens T1Kr02 can be recommended as a promoter for plants and as a source of biotechnological production of d-fucose.

Fucosylated Chondroitin Sulfates with Rare Disaccharide Branches from the Sea Cucumbers Psolus peronii and Holothuria nobilis: Structures and Influence on Hematopoiesis.

Two fucosylated chondroitin sulfates were isolated from the sea cucumbers Psolus peronii and Holothuria nobilis using a conventional extraction procedure in the presence of papain, followed by anion-exchange chromatography on DEAE-Sephacel. Their composition was characterized in terms of quantitative monosaccharide and sulfate content, and structures were mainly elucidated using 1D- and 2D-NMR spectroscopy. As revealed by the data of the NMR spectra, both polysaccharides along with the usual fucosyl branches contained rare disaccharide branches α-D-GalNAc4S6R-(1→2)-α-L-Fuc3S4R → attached to O-3 of the GlcA of the backbone (R = H or SO3-). The polysaccharides were studied as stimulators of hematopoiesis in vitro using mice bone marrow cells as the model. The studied polysaccharides were shown to be able to directly stimulate the proliferation of various progenitors of myelocytes and megakaryocytes as well as lymphocytes and mesenchymal cells in vitro. Therefore, the new fucosylated chondroitin sulfates can be regarded as prototype structures for the further design of GMP-compatible synthetic analogs for the development of new-generation hematopoiesis stimulators.

The Acinetobacter baumannii K70 and K9 capsular polysaccharides consist of related K-units linked by the same Wzy polymerase and cleaved by the same phage depolymerases.

IMPORTANCE: Bacteriophage show promise for the treatment of Acinetobacter baumannii infections that resist all therapeutically suitable antibiotics. Many tail-spike depolymerases encoded by phage that are able to degrade A. baumannii capsular polysaccharide (CPS) exhibit specificity for the linkage present between K-units that make up CPS polymers. This linkage is formed by a specific Wzy polymerase, and the ability to predict this linkage using sequence-based methods that identify the Wzy at the K locus could assist with the selection of phage for therapy. However, little is known about the specificity of Wzy polymerase enzymes. Here, we describe a Wzy polymerase that can accommodate two different but similar sugars as one of the residues it links and phage depolymerases that can cleave both types of bond that Wzy forms.

Stereocontrolled Synthesis and Conformational Analysis of a Series of Disaccharides α,ß-d-GlcA-(1→3)-α-L-Fuc.

D-Glucuronic acid is a fundamental building block of many biologically important polysaccharides, either in its non-substituted form or bearing a variety of substituents, among them sulfates. We have previously performed a study of the effects of exhaustive sulfation on the conformational behavior of ß-gluronopyranosides. Herein, we report an investigation comparing α- and ß-derivatives of this monosaccharide within the title disaccharides using NMR and quantum chemistry approaches. It was found that for α-linked disaccharides, the introduction of sulfates did not greatly affect their conformational behavior. However, for ß-derivatives, considerable conformational changes were observed. In general, they resemble those that took place for the monosaccharides, except that NOESY experiments and calculations of intra-ring spin-spin coupling constants suggest the presence of a 1S5 conformer along with 3S1 in the fully sulfated disaccharide. During the synthesis of model compounds, hydrogen bond-mediated aglycone delivery was used as an α-directing stereocontrol approach in the glucuronidation reaction.

Enthalpy of formation of 6-phenyl-1,5-diazabicyclo[3.1.0]hexane by combustion calorimetry and theoretical approach for efficient prediction of thermochemistry of diaziridines.

The combustion energy and standard molar enthalpy of formation of crystalline 6-phenyl-1,5-diazabicyclo[3.1.0]hexane (PDABH) were determined using an isoperibolic calorimeter with a static bomb. PDABH is the first diaziridine for which the experimental value of the enthalpy of formation was obtained. This value was validated by the theoretical values of gas phase enthalpy of formation and enthalpy of sublimation. The gas phase enthalpy of formation was calculated using the DLPNO-CCSD(T1)/CBS method in conjunction with isodesmic-type reactions. This method was chosen in comparison to another high quality evaluative method (G4), which has been shown to provide unreliable results for cyclic nitrogen containing compounds. The descriptors of the molecular electrostatic potential (MEP) were used to estimate the enthalpy of sublimation of PDABH. The proposed MEP model is based on experimental enthalpies of sublimation for 75 compounds structurally similar to PDABH. The high-level ab initio calculations of gas phase enthalpies of formation combined with enthalpies of sublimations estimated using descriptors of MEP allow predicting the enthalpies of formation of diaziridines in the solid phase.

A highly branched novel galactofuranan in the cell wall of Clavibacter tesselarius VKM Ac-1406T.

The structures of two cell wall glycopolymers were studied in the plant pathogenic bacterium Clavibacter tesselarius VKM Ac-1406T (family Microbacteriaceae, order Micrococcales, class Actinomycetes). The predominant polymer was a novel (1 â†’ 6)-linked ß-d-galactofuranan with a highly branched repeating unit, α-L-Rhap-(1 â†’ 3)-α-D-Galp-(1 â†’ 2)-[α-L-Rhap-(1 â†’ 3)]-α-D-Fucp-(1 →, at O-2 on every second galactofuranose residue. The second polymer present in small amounts was acidic with the repeating unit, →3)-α-D-Galp-(1 â†’ 3)-α-D-[4,6-S-Pyr]-Manp-(1 â†’ 3)-α-D-Manp-[2OAc]0.2-(1→, and was reported in all Clavibacter species investigated to date. The presented results expand our knowledges of structural diversity of phosphate-free cell wall glycopolymers and provide evidence in support of their taxonomic specificity for bacterial species and genera.

Glycosaminoglycans from the Starfish Lethasterias fusca: Structures and Influence on Hematopoiesis.

Crude anionic polysaccharides extracted from the Pacific starfish Lethasterias fusca were purified by anion-exchange chromatography. The main fraction LF, having MW 14.5 kDa and dispersity 1.28 (data of gel-permeation chromatography), was solvolytically desulfated and giving rise to preparation LF-deS with a structure of dermatan core [→3)-ß-d-GalNAc-(1→4)-α-l-IdoA-(1→]n, which was identified according to NMR spectroscopy data. Analysis of the NMR spectra of the parent fraction LF led to identification of the main component as dermatan sulfate LF-Derm →3)-ß-d-GalNAc4R-(1→4)-α-l-IdoA2R3S-(1→ (where R was SO3 or H), bearing sulfate groups at O-3 or both at O-2 and O-3 of α-l-iduronic acid, as well as at O-4 of some N-acetyl-d-galactosamine residues. The minor signals in NMR spectra of LF were assigned as resonances of heparinoid LF-Hep composed of the fragments →4)-α-d-GlcNS3S6S-(1→4)-α-l-IdoA2S3S-(1→. The 3-O-sulfated and 2,3-di-O-sulfated iduronic acid residues are very unusual for natural glycosaminoglycans, and further studies are needed to elucidate their possible specific influence on the biological activity of the corresponding polysaccharides. To confirm the presence of these units in LF-Derm and LF-Hep, a series of variously sulfated model 3-aminopropyl iduronosides were synthesized and their NMR spectra were compared with those of the polysaccharides. Preparations LF and LF-deS were studied as stimulators of hematopoiesis in vitro. Surprisingly, it was found that both preparations were active in these tests, and hence, the high level of sulfation is not necessary for hematopoiesis stimulation in this particular case.

Structure of the O-specific polysaccharide of Ochrobactrum endophyticum KCTC 42485T containing 3-(3-hydroxy-2,3-dimethyl-5-oxoprolyl)amino-3,6-dideoxy-d-galactose.

Ochrobactrum endophyticum (syn. Brucella endophytica) is an aerobic species of Alphaproteobacteria isolated from healthy roots of Glycyrrhiza uralensis. Here we report the structure of the O-specific polysaccharide obtained by mild acid hydrolysis of the lipopolysaccharide of the type strain KCTC 42485:→3)-α-l-FucpNAc-(1→3)-ß-d-QuipNAc-(1→2)-ß-d-Fucp3NAcyl-(1→ where Acyl is 3-hydroxy-2,3-dimethyl-5-oxoprolyl. The structure was elucidated using chemical analyses along with 1H and 13C NMR spectroscopy (including 1H,1H COSY, TOCSY, ROESY and 1H,13C HSQC, HMBC, HSQC-TOCSY and HSQC-NOESY experiments). To our knowledge the OPS structure is novel and has not been previously published.

A novel cell wall galactofuranan in Clavibacter phaseoli VKM Ac-2641T.

A glycopolymer of novel structure was found in the cell wall of plant pathogen Clavibacter phaseoli VKM Ac-2641T (family Microbacteriaceae, class Actinomycetes). The glycopolymer was (1 â†’ 6)-linked ß-d-galactofuranan with side branched trisaccharide, α-D-Manp-(1 â†’ 2)-[α-D-Manp-(1 â†’ 3)]-α-D-Ribf-(1→ at O-2 on every second galactofuranose residue. The galactofuranan structure was established by chemical and NMR spectroscopic methods using one- and two-dimensional techniques 1H,1H COSY, TOCSY, ROESY and 1H,13C HSQC, HMBC. The results of this study provide new data on diversity of bacterial glycopolymers, may prove useful for bacterial taxonomy and contribute to the understanding of the host plant-microbiota interaction mechanisms.

Overclocking Nitronyl Nitroxide Gold Derivatives in Cross-Coupling Reactions.

Nitronyl nitroxides are functional building blocks in cutting-edge research fields, such as the design of molecular magnets, the development of redox and photoswitchable molecular systems and the creation of redox-active components for organic and hybrid batteries. The key importance of the nitronyl nitroxide function is to translate molecular-level-optimized structures into nano-scale devices and new technologies. In spite of great importance, efficient and versatile synthetic approaches to these compounds still represent a challenge. Particularly, methods for the direct introduction of a nitronyl nitroxide moiety into aromatic systems possess many limitations. Here, we report gold derivatives of nitronyl nitroxide that can enter Pd(0)-catalysed cross-coupling reactions with various aryl bromides, affording the corresponding functionalized nitronyl nitroxides. Based on the high thermal stability and enhanced reactivity in catalytic transformation, a new reagent is suggested for the synthesis of radical systems via a universal cross-coupling approach.

Polysaccharides of Salsola passerina: Extraction, Structural Characterization and Antioxidant Activity.

The above-ground part of the Salsola passerine was found to contain ~13% (w/w) of polysaccharides extractable with water and aqueous solutions of ammonium oxalate and sodium carbonate. The fractions extracted with aqueous sodium carbonate solutions had the highest yield. The polysaccharides of majority fractions are characterized by similar monosaccharide composition; namely, galacturonic acid and arabinose residues are the principal components of their carbohydrate chains. The present study focused on the determination of antioxidant activity of the extracted polysaccharide fractions and elucidation of the structure of polysaccharides using nuclear magnetic resonance (NMR) spectroscopy. Homogalacturonan (HG), consisting of 1,4-linked residues of α-D-galactopyranosyluronic acid (GalpA), rhamnogalacturonan-I (RG-I), which contains a diglycosyl repeating unit with a strictly alternating sequence of 1,4-linked D-GalpA and 1,2-linked L-rhamnopyranose (Rhap) residues in the backbone, and arabinan, were identified as the structural units of the obtained polysaccharides. HMBC spectra showed that arabinan consisted of alternating regions formed by 3,5-substituted and 1,5-linked arabinofuranose residues, but there was no alternation of these residues in the arabinan structure. Polysaccharide fractions scavenged the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical at 0.2-1.8 mg/mL. The correlation analysis showed that the DPPH scavenging activity of polysaccharide fractions was associated with the content of phenolic compounds (PCs).

Epinephrine extensively changes the biofilm matrix composition in Micrococcus luteus C01 isolated from human skin.

The importance of the impact of human hormones on commensal microbiota and microbial biofilms is established in lots of studies. In the present investigation, we continued and extended the research of epinephrine effects on the skin commensal Micrococcus luteus C01 and its biofilms, and also the matrix changes during the biofilm growth. Epinephrine in concentration 4.9 × 10-9 M which is close to normal blood plasma level increased the amount of polysaccharides and extracellular DNA in the matrix, changed extensively its protein, lipid and polysaccharide composition. The Ef-Tu factor was one of the most abundant proteins in the matrix and its amount increased in the presence of the hormone. One of the glucose-mannose polysaccharide was absent in the matrix in presence of epinephrine after 24 h of incubation. The matrix phospholipids were also eradicated by the addition of the hormone. Hence, epinephrine has a great impact on the M. luteus biofilms and their matrix composition, and this fact opens wide perspectives for the future research.

Fucose-Rich Sulfated Polysaccharides from Two Vietnamese Sea Cucumbers Bohadschia argus and Holothuria (Theelothuria) spinifera: Structures and Anticoagulant Activity.

Fucosylated chondroitin sulfates (FCSs) FCS-BA and FCS-HS, as well as fucan sulfates (FSs) FS-BA-AT and FS-HS-AT were isolated from the sea cucumbers Bohadschia argus and Holothuria (Theelothuria) spinifera, respectively. Purification of the polysaccharides was carried out by anion-exchange chromatography on DEAE-Sephacel column. Structural characterization of polysaccharides was performed in terms of monosaccharide and sulfate content, as well as using a series of non-destructive NMR spectroscopic methods. Both FCSs were shown to contain a chondroitin core [→3)-ß-d-GalNAc-(1→4)-ß-d-GlcA-(1→]n bearing sulfated fucosyl branches at O-3 of every GlcA residue in the chain. These fucosyl residues were different in pattern of sulfation: FCS-BA contained Fuc2S4S, Fuc3S4S and Fuc4S at a ratio of 1:8:2, while FCS-HS contained these residues at a ratio of 2:2:1. Polysaccharides differed also in content of GalNAc4S6S and GalNAc4S units, the ratios being 14:1 for FCS-BA and 4:1 for FCS-HS. Both FCSs demonstrated significant anticoagulant activity in clotting time assay and potentiated inhibition of thrombin, but not of factor Xa. FS-BA-AT was shown to be a regular linear polymer of 4-linked α-L-fucopyranose 3-sulfate, the structure being confirmed by NMR spectra of desulfated polysaccharide. In spite of considerable sulfate content, FS-BA-AT was practically devoid of anticoagulant activity. FS-HS-AT cannot be purified completely from contamination of some FCS. Its structure was tentatively represented as a mixture of chains identical with FS-BA-AT and other chains built up of randomly sulfated alternating 4- and 3-linked α-L-fucopyranose residues.

Synthesis and evaluation of avermectin-imidazo[1,2-a]pyridine hybrids as potent GABAA receptor modulators.

The γ-aminobutyric acid type A (GABAA) receptors are pentameric transmembrane protein complexes. They have attracted extensive attention from the scientific community due to their significant pharmacological potential. Here we report the first synthesis of avermectin-imidazo[1,2-a]pyridine hybrids promising as GABAA receptor positive allosteric modulators (PAMs). An efficient multi-step protocol was elaborated for the installation of the 6-methyl-2-(p-tolyl)imidazo[1,2-a]pyridine pendant to the Avermectin B1a and Ivermectin skeletons through a linker. A variety of linkers were used in order to study the effect of disturbances in the hybrid structure on the GABAA receptor affinity. In vitro experiments showed that the lead compounds exhibited high potency (IC50 = 207 and 359 nM) for binding at the benzodiazepine site of GABAA receptors. In silico studies suggest that the hybrids are able to bind at the Ivermectin binding site of the GABAA receptor. The functional properties of the highest-affinity hybrid (compound 15e) as GABAAR PAM were evaluated by patch-clamp electrophysiological recordings of GABA-mediated currents in rat cerebellar Purkinje neurons. The results obtained suggest that the potentiating effect of hybrid compound 15e is due to its interaction both with benzodiazepine- and Ivermectin-binding sites of GABAARs. Drug-induced behavioral responses in adult zebrafish for hybrids correlate with an alternative mode of action of avermectin and imidazo[1,2-a]pyridine pharmacophores. The investigation of avermectin-imidazo[1,2-a]pyridine hybrid molecules with activity as GABAA receptor modulators is important for the discovery of safe and effective drugs for the treatment of neurological disorders and pest control agents.

Pectin from leaves of birch (Betula pendula Roth.): Results of NMR experiments and hypothesis of the RG-I structure.

We report that in birch leaf pectin, rhamnogalacturonan-I (RG-I) and galacturonan (HG) were found as separate polymers rather than domains of a complex macromolecule. RG-I and HG were separated by anion-exchange and size-exclusion chromatography and studied by using NMR spectroscopy. NMR spectra showed that methyl-esterified D-galactosyluronic acid residues were located only in HG. Oligosaccharides of similar structure to the backbone, but without terminal reducing residues in the NMR spectra, were found in RG-I. We hypothesize, these oligosaccharides and RG-I backbone can be covalently bound due to its co-eluted of from DEAE-cellulose and Sepharose CL-4B. This result differs from the classical RG-I model, which assumes that all Rhap and GalpA residues are located only in the RG-I backbone. In the heteronuclear multiple bond correlation (HMBC) and rotating frame Overhauser effect spectroscopy (ROESY) spectra, the correlation peaks confirming the substitution of 2,4-rhamnose residues at O-4 by only single D-galactose residues were identified.

Depolymerization of a fucosylated chondroitin sulfate from Cucumaria japonica: Structure and activity of the product.

Fucosylated chondroitin sulfate CJ from the body wall of sea cucumber Cucumaria japonica was depolymerized by the treatment with H2O2 in the presence of Cu(OAc)2. The molecular weight of the polysaccharide was decreased from 32 kDa to 5 kDa. The product CJ-DP was shown to contain l-Fuc, d-GalNAc, d-GlcA, and sulfate in molar proportions of 0.96:0.90:1.00:5.4, which were quite similar to those of the parent polysaccharide CJ. The NMR analysis revealed that CJ-DP, like the parent polysaccharide CJ, consisted of both branched →4)-[3-O-α-l-Fuc]-ß-d-GlcA-(1 â†’ 3)-ß-d-GalNAc-(1→ and linear →4)-ß-d-GlcA-(1 â†’ 3)-ß-d-GalNAc-(1→ repeating blocks. Sulfate groups occupy O-4 and the majority of O-6 of GalNAc, as well as O-3 of GlcA, in linear blocks and different positions in Fuc branches. This result indicates that depolymerization practically does not diminish the amount of branches and sulfate groups in the product. Both polysaccharides CJ and CJ-DP demonstrated anticoagulant and hematopoiesis-stimulatory activities in vitro.

Cell wall galactofuranan and pyruvate-containing galactomannan in the cell walls of Clavibacter strains.

The cell wall glycopolymer structures of plant-associated strains Clavibacter sp. VKM Ac-1371, Clavibacter sp. VKM Ac-1372 and Clavibacter sp. VKM Ac-1374, members of three putative new species (family Microbacteriaceae, class Actinobacteria) were studied. Each strain was found to contain two glycopolymers, neutral and acidic ones. The main chain of neutral polymer, identical in all three strains, is (1 â†’ 6)-linked ß-d-galactofuranan with every second galactofuranose residue substituted at position 2 by side disaccharide, α-d-Manp-(1 â†’ 2)-α-d-Ribf-(1 â†’ . The second, acidic polymer, is pyruvate-containing galactomannan with the repeating unit, →3)-α-d-Galp-(1 â†’ 3)-α-d-[4,6-S-Pyr]-Manp-(1 â†’ 3)-α-d-Manp-(1 â†’ . Reducing mannopyranose residues of the acidic polysaccharides repeating unit from strains VKM Ac-1372 and VKM Ac-1374 bear O-acetyl residues additionally. The cell wall glycopolymer structures were established by chemical and NMR spectroscopic methods with using one- and two-dimensional techniques 1H,1H COSY, TOCSY, ROESY and 1H,13C HSQC, HMBC. The results obtained provide new data on diversity of the bacterial cell wall glycopolymers and may prove valuable for microbial taxonomy and insight into the molecular mechanisms of interactions between bacteria and plants and also of bacterial adaptation to survival in desert systems.

Novel Acinetobacter baumannii Bacteriophage Aristophanes Encoding Structural Polysaccharide Deacetylase.

Acinetobacter baumannii appears to be one of the most crucial nosocomial pathogens. A possible component of antimicrobial therapy for infections caused by extremely drug-resistant A. baumannii strains may be specific lytic bacteriophages or phage-derived enzymes. In the present study, we observe the biological features, genomic organization, and phage-host interaction strategy of novel virulent bacteriophage Aristophanes isolated on A. baumannii strain having K26 capsular polysaccharide structure. According to phylogenetic analysis phage Aristophanes can be classified as a representative of a new distinct genus of the subfamily Beijerinckvirinae of the family Autographiviridae. This is the first reported A. baumannii phage carrying tailspike deacetylase, which caused O-acetylation of one of the K26 sugar residues.