Almond shell xylo-oligosaccharides exhibiting immunostimulatory activity

Partially O-acetylated xylo-oligosaccharides (DXO) isolated from almond shells by autohydrolysis as well as their de-acetylated form (DeXO) were subjected to chemical, molecular, and structural analyses. They represent a mixture of neutral and acidic oligomers and low-molecular weight polymers related to (4-O-methyl-D-glucurono)-D-xylan. DXO and DeXO showed direct mitogenic activity and enhancement of the T-mitogen-induced proliferation of rat thymocytes, indicating the immunostimulatory potential of the almond shell xylo-oligosaccharides.     

NMR characterization of endogenously O-acetylated oligosaccharides isolated from tomato (Lycopersicon esculentum) xyloglucan

Eight oligosaccharide subunits, generated by endoglucanase treatment of the plant polysaccharide xyloglucan isolated from the culture filtrate of suspension-cultured tomato (Lycopersicon esculentum) cells, were structurally characterized by NMR spectroscopy. These oligosaccharides, which contain up to three endogenous O-acetyl substituents, consist of a cellotetraose core with alpha-D-Xylp residues at O-6 of the two beta-D-Glcp residues at the non-reducing end of the core. Some of the alpha-D-Xylp residues themselves bear either an alpha-L-Arap or a beta-D-Galp residue at O-2. O-Acetyl substituents are located at O-6 of the unbranched (internal) beta-D-Glcp residue, O-6 of the terminal beta-D-Galp residue, and/or at O-5 of the terminal alpha-L-Arap residue. Structural assignments were facilitated by long-range scalar coupling interactions observed in the high-resolution gCOSY spectra of the oligosaccharides. The presence of five-bond scalar coupling constants in the gCOSY spectra provides a direct method of assigning O-acetylation sites, which may prove generally useful in the analysis of O-acylated glycans. Spectral assignment of these endogenously O-acetylated oligosaccharides makes it possible to deduce correlations between their structural features and the chemical shifts of diagnostic resonances in their NMR spectra.     

Nod factors and chitooligomers elicit an increase in cytosolic calcium in aequorin-expressing soybean cells

Rhizobial Nod factors (NFs) function as nodulation signals that trigger symbiotic responses of leguminous host plants. NFs consist of a chitin oligomer backbone carrying a fatty acid at the non-reducing end. Depending on the rhizobial strain, NFs carry additional substituents, which may determine host specificity. Transgenic suspension-cultured soybean (Glycine max [L.] Merr.) cells expressing aequorin have been used to record cytosolic [Ca(2+)] changes upon treatment with purified NFs and chitin fragments. Both compounds elicited an increase of cytosolic [Ca(2+)] at nanomolar concentrations. The shape and amplitude of cytosolic [Ca(2+)] changes was similar to the response elicited by un-derivatized chitin oligomers. Cells challenged first with NFs did not respond to a subsequent treatment with chitin oligomers and vice versa. Dose-response experiments showed that un-derivatized chitin oligomers were more active compared with NFs. The capacity of NFs to elicit the calcium response depended on their structure. The presence of reducing end substituents in methylfucosylated NFs from Rhizobium sp. NGR234 and the O-acetyl group at the non-reducing end in NFs from Sinorhizobium meliloti attenuated the activity to cause the calcium changes. The sulfate group in NFs from Rhizobium tropici did not affect the elicitor activity. Pentameric S. meliloti NFs were more active than tetrameric molecules, whereas trimeric or dimeric degradation products were inactive. Substituents in NFs may have the function to avoid stimulation of defense reactions mediated by the perception system for chitin oligomers.     

Structural studies of the capsular polysaccharide of Klebsiella type 59.

The structure of the capsular polysaccharide from Klebsiella type 59 has been investigated by methylation analysis, a modified Smith-degradation procedure, and uronic acid degradation followed by oxidation and elimination of the substituents of the oxidized residue. The oligomeric fragments produced by these degradative procedures were isolated and characterized. O-Acetyl groups were identified and their position determined. The polysaccharide consists of the following pentasaccharide repeating-unit (dotted lines indicate that only some of the residues carry the O-acetyl substituent). See article.     

Probing the proposed phenyl-A region of the sigma-1 receptor

The proposed phenyl-A region of sigma (sigma) receptors accommodates several structural features. In this study we explored the possibility that appropriate structural features located at the phenyl-A region of sigma receptor sites could lead to more potent and selective agents for the sigma receptor subtypes. By keeping the phenyl-B substituent as the optimum omega-phenylpentyl moiety, and varying substituents in the phenyl-A region, we have observed changes in binding potency and selectivity at the sigma receptor subtypes. SAR for the binding of these compounds at sigma-2 sites was also examined.     

Synthesis and pharmacological evaluation of substituted naphth[1,2,3-de]isoquinolines (dinapsoline analogues) as D1 and D2 dopamine receptor ligands

Dinapsoline ((2); (+/-)-dihydroxy-2,3,7,11b-tetrahydro-1H-naphth[1,2,3-de]isoquinoline) is a full D(1) dopamine agonist that also has significant D(2) receptor affinity. Based on a similar pharmacophore, dinapsoline has pharmacological similarities to dihydrexidine ((1); (+/-)-trans-10,11-dihydroxy-5,6,6a,7,8,12b-hexahydrobenzo[a]phenanthridine), the first high affinity full D(1) agonist. Small alkyl substitutions on the dihydrexidine backbone are known to alter markedly the D(1):D(2) selectivity of dihydrexidine, and it was of interest to determine whether similar SAR exists within the dinapsoline series. This report describes the synthesis and pharmacological evaluation of six analogues of dinapsoline: N-allyl-(3);N-n-propyl- (4); 6-methyl- (5); 4-methyl- (6); 4-methyl-N-allyl- (7); and 4-methyl-N-n-propyl-dinapsoline (8). As expected from earlier studies with the dihydrexidine backbone, N-allyl (3) or N-n-propyl (4) analogues had markedly decreased D(1) affinity. Unexpectedly, and unlike the dihydrexidine series, these same substituents did not markedly increase D(2) affinity. The addition of a methyl group to position 6 (5) increased D(1):D(2) selectivity, but less markedly than did the analogous 2-methyl substituent added to 1. Unlike the analogous 4-methyl substituent of 1, the addition of a 4-methyl-group (6) actually decreased D(1) affinity without affecting D(2) affinity. These data demonstrate that the dinapsoline (2) backbone can be modified to produce dopamine agonists with novel properties. Moreover, as rigid ligands in which small substituents can cause significant changes in selectivity, they are important tools for deriving 'differential' SARs of the dopamine receptor isoforms.     

Structural studies of the O-antigenic polysaccharides from the enteroaggregative Escherichia coli strain 87/D2 and international type strains from E. coli O128

The O-antigen of the lipopolysaccharide (LPS) from the enteroaggregative Escherichia coli strain 87/D2 has been determined by component analysis together with NMR spectroscopy. The polysaccharide has pentasaccharide repeating units in which all the residues have the galacto-configuration. The repeating unit of the O-antigen, elucidated using the O-deacylated LPS, is branched with the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately 0.7 per repeating unit) distributed over two positions. Subsequent analysis showed that the galactose residue carries acetyl groups at either O-3 or O-4 in a ratio of approximately 2:1. The international reference strain from E. coli O128ab was investigated and the repeating unit of the O-antigens has the following structure: Analysis of the 1H NMR spectrum of the LPS revealed O-acetyl groups (approximately one per repeating unit) distributed over two positions. The integrals of the resonances for the O-acetyl groups indicated similarities between the O-antigen from E. coli O128ab and that of E. coli strain 87/D2, whereas the O-acetyl substitution pattern in the E. coli O128ac O-antigen differed slightly. Enzyme immunoassay using specific anti-E. coli O128ab and anti-E. coli O128ac rabbit sera confirmed the results.     

Isolation and structural identification of diarabinosyl 8-O-4-dehydrodiferulate from maize bran insoluble fibre

The first saccharide ester of a dehydrodiferulic acid (DFA) other than 5-5-DFA has been isolated from maize bran insoluble fibre after acidic hydrolysis and fractionation by gel chromatography and semi-preparative RP-HPLC. HPLC-MS along with 1D, 2D and 3D NMR spectra provided the requisite structural evidence that it is the di-5-O-l-arabinosyl ester of 8-O-4-DFA. Although a range of DFAs have been well authenticated as components released from the cell walls of grasses, the only structural evidence for a DFA attached to polysaccharides had been from 5-5-DFA. The isolation of the 8-O-4-ester demonstrates that polysaccharides in maize cell walls, and presumably in all grasses, are cross-linked through dehydrodiferulates other than 5-5-dehydrodiferulate.     

Growth-Related Substituent Changes in Exopolysaccharides of Fast-Growing Rhizobia

Pyruvic acid and O-acetyl groups are the major noncarbohydrate substituents in exopolysaccharides (EPS) produced by fast-growing species of Rhizobium. EPS substituent variations were observed among strains of the same species. The amounts of these substituents also varied with culture age; pyruvic acid increased in the EPS of all four species, whereas O-acetyl increased in Rhizobium trifolii and R. leguminosarum EPS, decreased in R. meliloti EPS, and remained constant in R. phaseoli EPS. The use of glycerol as a substrate for R. meliloti significantly increased EPS yields, whereas mannitol increased those of the other three Rhizobium species.     

Distribution analyses of chain substituents of lipoteichoic acids by chemical degradation

The lipoteichoic acid from Lactococcus lactis Kiel 48337 was analyzed. It had 61% of its glycerophosphate residues substituted with alpha-D-galactopyranosyl residues. Non-substituted glycerophosphate residues were split off by two alkaline hydrolyses and an intermediate enzymatic phosphomonoester cleavage. The resulting (GalGroP)nGroGal and (GalGroP)nGlc2Gro oligomers were separated by chromatography on DEAE-Sephadex into 10 pairs of molecular species with n from 1 to 10. The relative frequencies of GalGro and these oligomers were close to the values calculated by computer simulation for a random distribution of chain substituents. A similar series of oligomers was obtained in one step by hydrolysis of the lipoteichoic acid with 98% (by vol.) acetic acid. Due to side reactions, the picture was less precise but nevertheless indicative of the same distribution pattern. The data provide indirect evidence that the alanine ester substituents of the native lipoteichoic acid (Ala/P = 0.38) occupy the free positions between the galactosylated oligomers and are therefore themselves distributed randomly.     

ADMET rules of thumb II: A comparison of the effects of common substituents on a range of ADMET parameters

A systematic analysis of data generated in key in vitro assays within GSK has been undertaken to identify what impact a range of common substituents have on a range of ADMET parameters. These include; P450 1A2, 2C9, 2C19, 2D6 and 3A4 inhibition, hERG inhibition, phosphate buffer solubility and artificial membrane permeability. We do this by identifying all matched molecular pairs, differing by the replacement of a hydrogen atom with a list of predefined substituents.For each substituent we calculate the mean difference in the ADMET parameter for all the matched molecular pairs identified, making a statistical assessment of the difference, as well as assessing the diversity for each example to ensure that the results can be generalized. We also relate the change in activity observed for each substituent to differences in their molecular properties in an effort to identify any structural alerts.     

Rapid analysis of mono-saccharides and oligo-saccharides in hydrolysates of lignocellulosic biomass by HPLC

HPLC using pre-column derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) was used to analyse mono-saccharides and oligo-saccharides in hydrolysates of lignocellulosic biomass. PMP derivatives, including those of mannose, rhamnose, cellobiose, glucose, xylose and arabinose, were separated within 14 min with detection at 254 nm. The method was also suitable for xylo-oligosaccharides (XOS): PMP derivatives of xylohexaose, xylopentaose, xylotetraose, xylotriose and xylobiose were well separated under the same conditions. The method was used to determine the mono-saccharide composition of Miscanthus and evaluate the production of XOS from enzymatic hydrolysis of crude xylan.     

Synthesis of GN8 derivatives and evaluation of their antiprion activity in TSE-infected cells

A series of GN8 derivatives were synthesized from various diamines, carboxylic acid derivatives, and nitrogen nucleophiles, and their antiprion activity was tested in TSE-infected mouse neuronal cells. We found that two ethylenediamine units, hydrophobic substituents on the nitrogen atoms, and the diphenylmethane scaffold were essential structural features responsible for the activity. Seven derivatives bearing substituents at the benzylic position exhibited an improved antiprion activity with the IC₅₀ values of 0.51-0.83 μM. Conformational analysis of model compounds suggested that the introduction of the substituent at the benzylic position restricted the conformational variability of the diphenylmethane unit.     

First report of a lyase for cepacian, the polysaccharide produced by Burkholderia cepacia complex bacteria

Bacteria belonging to the Burkholderia cepacia complex (Bcc) are interesting for their involvement in pulmonary infections in patients affected by cystic fibrosis (CF) or chronic granulomatous disease. Many Bcc strains isolated from CF patients produce high amounts of exopolysaccharides (EPS). Although different strains sometimes biosynthesise different EPS, the majority of Bcc bacteria produce only one type of polysaccharide, which is called cepacian. The polymer has a unique heptasaccharidic repeating unit, containing three side chains, and up to three O-acetyl substituents.. We here report for the first time the isolation and characterisation of a lyase active towards cepacian produced by a Bacillus sp., which was isolated in our laboratory. The enzyme molecular mass, evaluated by size-exclusion chromatography, is 32,700+/-1500Da. The enzyme catalyses a beta-elimination reaction of the disaccharide side chain beta-d-Galp-(1-->2)-alpha-d-Rhap-(1--> from the C-4 of the glucuronic acid residue present in the polymer backbone. Although active on both native and de-acetylated cepacian, the enzyme showed higher activity on the latter polymer.     

Endoglucanase sensitivity for substituents in methyl cellulose hydrolysis studied using MALDI-TOFMS for oligosaccharide analysis and structural analysis of enzyme active sites

The properties of modified cellulose polymers, such as methylcellulose, are significantly influenced by the distribution of substituents along the polymer backbone. This distribution is difficult to determine due to the lack of suitable analytical methods. One approach is to use cellulose-degrading enzymes to gain information from the capability of the enzymes to cleave the bonds between glucose units. Endoglucanases are cellulase enzymes that can break internal glycosidic linkages and degrade low substituted regions of modified cellulose where the substituents do not interfere with the enzyme active site. In this work methyl cellulose was degraded using five endoglucanases from glycosyl hydrolase families 5 and 7 from three different species. The products were analyzed with reducing end analysis, chromatography (SEC-MALS-RI), and MALDI-TOFMS. The results were correlated with available determined enzyme structures and using structural alignment for unknown enzyme structures. This was performed in order to elucidate the relationship between active site structures and sensitivity for substituents on derivatized cellulose. The evaluation of endoglucanase hydrolysis of methyl cellulose showed that differences in sensitivity could be related to differences in steric hindrance of substituents in the active site, which could explain differences within family 5 and 7 enzymes, as well as the generally higher substituent tolerance for family 5 enzymes. This information is important for use of endoglucanases as tools for characterization of substituent distribution. The results are also valuable since soluble cellulose derivatives are generally used as substrates during enzyme characterization and in endoglucanase activity assays.     

Hygrophorones A-G: fungicidal cyclopentenones from Hygrophorus species (Basidiomycetes)

Twenty new 5-(hydroxyalkyl)-2-cyclopentenone derivatives (hygrophorones) could be isolated from Hygrophorus latitabundus, H. olivaceoalbus, H. persoonii, and H. pustulatus. Their fungicidal activity was exemplarily tested. The hygrophorones have structural similarities to the antibiotic pentenomycin. Chemically, hygrophorones are 2-cyclopentenones with hydroxy or acetoxy substituents at C-4 and/or C-5. An odd-numbered 1' oxidized alkyl chain (C(11), C(13), C(15), or C(17)) is attached at C-5. In addition, from H. persoonii the new gamma-butyrolactone derivative [5-(E)-2-hydroxytetradexylidene-5H-furan-2-one] could be isolated. Some hygrophorones are responsible for the color reaction of the stipes of these fungi upon treatment with potassium hydroxide solution. Structural elucidations are based on 1D ((1)H, (13)C) and 2D (COSY, NOESY, HSQC, HMBC) NMR spectroscopic analyses as well as HR-FT-ICR-MS investigations.     

Structural and serological studies of the O-antigen of Proteus mirabilis O-9

The following structure of the O-polysaccharide (O-antigen) of the lipopolysaccharide of Proteus mirabilis O-9 was determined by NMR spectroscopy, including 2D 1H,(1)H COSY, TOCSY, ROESY, and 1H,(13)C HMQC experiments, along with chemical methods: [chemical structure: see text] where the degree of O-acetylation is approximately 70%. Immunochemical studies using rabbit polyclonal anti-Proteus mirabilis O-9 serum showed the importance of the O-acetyl groups in manifesting the serological specificity of the O-9 antigen. Anti-P. mirabilis O-9 cross-reacted with the lipopolysaccharides (LPS) of P. vulgaris O-25 and Proteus penneri 14, which could be accounted for by a structural similarity of their O-polysaccharides.     

Proton transfer from H2O to p-substituted anilide anion: can the size of water cluster influence the N-???H–OH→N–H???OH- switching

The effects of the H(2)O cluster size and the substituent (X=H, Me, OMe, CHO, NO and NO(2)) in the para position of anilide ion on the HN?···H-OH→HN-H···OH? switching were investigated by means of B3LYP and MP2 methods. The changes in the H-bond strength due to variation of the substituent and H(2)O cluster size were well monitored by changes in the interaction energy, structural parameter, electron density topology, natural charge, charge transfer and percentage of p-character of N atom in the C-N bond. The HN(-)[Symbol: see text][Symbol: see text][Symbol: see text]H-OH→HN-H[Symbol: see text][Symbol: see text][Symbol: see text]OH(-) switching was enhanced by an increase in the extent of solvation and the insertion of the electron-donating substituents in the para position. The results demonstrate that the strength of the HN(-)???HO H-bond increases and that of NH???OH(-) H-bond decreases by electron-donating substituents. The reverse situations were found for electron-accepting substituents.     

Chemical and structural analysis of the capsular polysaccharide from Escherichia coli O9:K28(A):H- (K28 antigen)

The structure of the capsular polysaccharide from Escherichia coli O9:K28(A):H- (K28 antigen) has been determined by using the techniques of methylation, periodate oxidation, and partial hydrolysis. N.m.r. spectroscopy (1H and 13C) was used to establish the nature of the anomeric linkages. O-Acetyl groups were determined spectrophotometrically and were located using methyl vinyl ether as a protective reagent. The polysaccharide is comprised of repeating units of the tetrasaccharide shown (three-plus-one type) with 70% of the fucosyl residues carrying an O-acetyl substituent. (formula; see text) This structure resembles that of E. coli K27 and has the structural pattern of Klebsiella K54 polysaccharide.     

Structural dynamics of archaeal small heat shock proteins

Small heat shock proteins (sHsps) are a widespread and diverse class of molecular chaperones. In vivo, sHsps contribute to thermotolerance. Recent evidence suggests that their function in the cellular chaperone network is to maintain protein homeostasis by complexing a variety of non-native proteins. One of the most characteristic features of sHsps is their organization into large, sphere-like structures commonly consisting of 12 or 24 subunits. Here, we investigated the functional and structural properties of Hsp20.2, an sHsp from Archaeoglobus fulgidus, in comparison to its relative, Hsp16.5 from Methanocaldococcus jannaschii. Hsp20.2 is active in suppressing the aggregation of different model substrates at physiological and heat-stress temperatures. Electron microscopy showed that Hsp20.2 forms two distinct types of octahedral oligomers of slightly different sizes, indicating certain structural flexibility of the oligomeric assembly. By three-dimensional analysis of electron microscopic images of negatively stained specimens, we were able to reconstitute 3D models of the assemblies at a resolution of 19 Å. Under conditions of heat stress, the distribution of the structurally different Hsp20.2 assemblies changed, and this change was correlated with an increased chaperone activity. In analogy to Hsp20.2, Hsp16.5 oligomers displayed structural dynamics and exhibited increased chaperone activity under conditions of heat stress. Thus, temperature-induced conformational regulation of the activity of sHsps may be a general phenomenon in thermophilic archaea.