betadex and maltoheptaose

Entirely oligosaccharide-based supramolecular amphiphiles were constructed via host-guest interactions between ferrocene-terminated acetylated-maltoheptaose (Fc-AcMH) and β-cyclodextrin-terminated four-arm star maltoheptaose (MH

Topics: Antibiotics, Antineoplastic; beta-Cyclodextrins; Cell Line, Tumor; Cell Proliferation; Cell Survival; Doxorubicin; Drug Delivery Systems; Ferrous Compounds; Fluorescent Dyes; Glucans; Humans; Hydrogen-Ion Concentration; Hydrophobic and Hydrophilic Interactions; Macromolecular Substances; Metallocenes; Micelles; Nanoparticles; Oligosaccharides; Optical Imaging; Particle Size

MALDI in-source decay (ISD) technique described to date has proven to be a convenient and rapid method for sequencing purified peptides and proteins. However, the general ISD still can not produce adequate fragments for the detailed structural elucidation of oligosaccharides. In this study, an efficient and practical method termed the laser-enhanced ISD (LEISD) technique of MALDI-FTICR MS allows highly reliable and abundant fragmentation of the neutral oligosaccharides, which was attributed to the ultrahigh irradiation laser of mJ level. The yield of ISD fragmentation was evaluated under different laser powers for 7 neutral oligosaccharides using DHB as matrix. Better quality ISD spectra including fragment ions in low-mass region were obtained at higher laser power. Results from the LEISD of oligosaccharides demonstrated that a significantly better signal-to-noise ratio (S/N) and more structural information could be obtained in comparison to the conventional CID. It was also suggested that the valuable A ions derived from cross-ring cleavage of the linear oligosaccharides allowed the distinction among α(1→4)-, α(1→6)-, β(1→4)-, and β(1→3)-linked isobaric structures according to fragment types and intensities. In addition, ideal fragmentation ions observed by LEISD method facilitated the determination of the sequences and branched points of complex oligosaccharides from human milk.

Topics: beta-Cyclodextrins; Dextrans; Fourier Analysis; Glucans; Humans; Milk, Human; Oligosaccharides; Reproducibility of Results; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

We report new approaches using alkali-hydroxide-doped matrices to facilitate structural characterization of neutral underivatized oligosaccharides by matrix-assisted laser desorption/ionization (MALDI) time-of-flight (TOF) MS. The approaches involved pretreatment of the analytes with NaOH or LiOH in aqueous solution, followed by mixing them with MALDI matrices prior to MS analysis. It was found that for open-ended neutral underivatized oligosaccharides partial alkaline degradation occurred upon laser desorption and ionization of the hydroxide-pretreated analytes in 2,5-dihydroxybenzoic acid (DHBA). The effect intensified when nonacidic compounds such as 2,4,6-trihydroxyacetophenone (THAP) and 5-amino-2-mercapto-1,3,4-thiadiazole (AMT) were used as matrix. The degradation allowed facile identification of the reducing end residue of the analyte and facilitated its structural characterization by postsource decay TOF-MS. Applying the same technique using matrices composed of LiOH and THAP or AMT led to the production of singly as well as multiple lithiated ions of oligosaccharides containing hexoses with free 3-OH groups. Extensive lithiation through multiple hydrogen-lithium exchanges up to 6 Li atoms was observed for maltoheptaose, beta-cyclodextrin, and dextran 1500. Such a 'lithium tagging' technique makes it possible to differentiate positional isomers of milk-neutral oligosaccharides, lacto-N-difucohexaose I and II (LNDFH-I and LNDFH-II), without the need of chemical derivatization or tandem MS analysis.

Topics: Acetophenones; beta-Cyclodextrins; Catechols; Dextrans; Glucans; Hydroxybenzoates; Lithium Compounds; Oligosaccharides; Sodium Hydroxide; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Thiadiazoles

The human gut microbiota performs functions that are not encoded in our Homo sapiens genome, including the processing of otherwise undigestible dietary polysaccharides. Defining the structures of proteins involved in the import and degradation of specific glycans by saccharolytic bacteria complements genomic analysis of the nutrient-processing capabilities of gut communities. Here, we describe the atomic structure of one such protein, SusD, required for starch binding and utilization by Bacteroides thetaiotaomicron, a prominent adaptive forager of glycans in the distal human gut microbiota. The binding pocket of this unique alpha-helical protein contains an arc of aromatic residues that complements the natural helical structure of starch and imposes this conformation on bound maltoheptaose. Furthermore, SusD binds cyclic oligosaccharides with higher affinity than linear forms. The structures of several SusD/oligosaccharide complexes reveal an inherent ligand recognition plasticity dominated by the three-dimensional conformation of the oligosaccharides rather than specific interactions with the composite sugars.

Topics: Amylose; Bacterial Outer Membrane Proteins; Bacteroides; beta-Cyclodextrins; Binding Sites; Calorimetry; Carbohydrate Conformation; Gastrointestinal Tract; Glucans; Humans; Models, Molecular; Oligosaccharides; Protein Binding; Starch; Trisaccharides

Heptadeoxy-6(I-VII)-halo, -azido, and hepta-6(I-VII)S-hepta(N-Boc-2-amino)ethyl-6(I-VII)-heptathiomaltoheptaose derivatives were prepared by acetolysis of the corresponding per-C-6-modified beta-cyclodextrin derivatives. The rapid and convenient structural characterisation of all of the modified oligosaccharides by ESIMS is described.

Topics: beta-Cyclodextrins; Carbohydrate Sequence; Cyclization; Glucans; Magnetic Resonance Spectroscopy; Spectrometry, Mass, Electrospray Ionization

AMP-activated protein kinase (AMPK) coordinates cellular metabolism in response to energy demand as well as to a variety of stimuli. The AMPK beta subunit acts as a scaffold for the alpha catalytic and gamma regulatory subunits and targets the AMPK heterotrimer to glycogen. We have determined the structure of the AMPK beta glycogen binding domain in complex with beta-cyclodextrin. The structure reveals a carbohydrate binding pocket that consolidates all known aspects of carbohydrate binding observed in starch binding domains into one site, with extensive contact between several residues and five glucose units. beta-cyclodextrin is held in a pincer-like grasp with two tryptophan residues cradling two beta-cyclodextrin glucose units and a leucine residue piercing the beta-cyclodextrin ring. Mutation of key beta-cyclodextrin binding residues either partially or completely prevents the glycogen binding domain from binding glycogen. Modeling suggests that this binding pocket enables AMPK to interact with glycogen anywhere across the carbohydrate's helical surface.

Topics: Amino Acid Sequence; AMP-Activated Protein Kinases; Animals; beta-Cyclodextrins; Binding Sites; Binding, Competitive; Carbohydrate Conformation; Catalytic Domain; Crystallography, X-Ray; Glucans; Glucose; Glycogen; Leucine; Liver; Models, Molecular; Molecular Sequence Data; Multienzyme Complexes; Mutagenesis, Site-Directed; Mutation; Oligosaccharides; Protein Binding; Protein Serine-Threonine Kinases; Protein Structure, Tertiary; Protein Subunits; Rats; Sequence Homology, Amino Acid; Spectrum Analysis, Raman; Tryptophan; Water

A number of regulatory binding sites of glycogen phosphorylase (GP), such as the catalytic, the inhibitor, and the new allosteric sites are currently under investigation as targets for inhibition of hepatic glycogenolysis under high glucose concentrations; in some cases specific inhibitors are under evaluation in human clinical trials for therapeutic intervention in type 2 diabetes. In an attempt to investigate whether the storage site can be exploited as target for modulating hepatic glucose production, alpha-, beta-, and gamma-cyclodextrins were identified as moderate mixed-type competitive inhibitors of GPb (with respect to glycogen) with K(i) values of 47.1, 14.1, and 7.4 mM, respectively. To elucidate the structural basis of inhibition, we determined the structure of GPb complexed with beta- and gamma-cyclodextrins at 1.94 A and 2.3 A resolution, respectively. The structures of the two complexes reveal that the inhibitors can be accommodated in the glycogen storage site of T-state GPb with very little change of the tertiary structure and provide a basis for understanding their potency and subsite specificity. Structural comparisons of the two complexes with GPb in complex with either maltopentaose (G5) or maltoheptaose (G7) show that beta- and gamma-cyclodextrins bind in a mode analogous to the G5 and G7 binding with only some differences imposed by their cyclic conformations. It appears that the binding energy for stabilization of enzyme complexes derives from hydrogen bonding and van der Waals contacts to protein residues. The binding of alpha-cyclodextrin and octakis (2,3,6-tri-O-methyl)-gamma-cyclodextrin was also investigated, but none of them was bound in the crystal; moreover, the latter did not inhibit the phosphorylase reaction.

Topics: alpha-Cyclodextrins; Animals; beta-Cyclodextrins; Binding Sites; Circular Dichroism; Crystallography, X-Ray; Cyclodextrins; gamma-Cyclodextrins; Glucans; Glucose; Glycogen; Glycogen Phosphorylase, Muscle Form; Hydrogen Bonding; Kinetics; Models, Chemical; Oligosaccharides; Phosphorylation; Protein Binding; Protein Structure, Tertiary; Rabbits

A new maltoheptaose derivative was prepared as a useful substrate for continual assay of alpha-amylase. The maltoheptaoside has thionaphtyl group as a fluorescent energy donor at the reducing end and dansyl group as an acceptor group at the non-reducing end. Excitation of the thionaphthyl group at 290 nm results in emission at 523 nm from the dansyl group, while the emission from the thionaphthyl group is quenched by the dansyl group. This fluorescence energy transfer is reduced by the hydrolytic action with alpha-amylase and a significant decrease in the dansyl emission concomitant with an increase in the thionaphthyl emission was observed. Usefulness of this substrate was demonstrated for sensitive and continuous assay of alpha-amylase from Aspergillus oryzae.

Topics: alpha-Amylases; Aspergillus oryzae; beta-Cyclodextrins; Carbohydrate Sequence; Cyclodextrins; Dansyl Compounds; Fluorescent Dyes; Glucans; Kinetics; Molecular Sequence Data; Molecular Structure; Spectrometry, Fluorescence; Substrate Specificity

Hydrothermolysis of D-glucose, cellobiose, and beta-cyclodextrin leads to the formation of small amounts of 3-deoxyhexonic acids. These acids are known to be produced by the alkaline degradation of carbohydrates. The formation under neutral hydrothermal conditions of the 3-deoxyhexonic acids provides evidence for the formation of 3-deoxy-D-erythro-hex-2-ulose, a compound that has been reported to play a role in both alkaline and acidic conversion of carbohydrates. Hydrothermolysis of 2- and 3-deoxy-D-arabino-hexonic acid does not lead to significant decarboxylation, and therefore these compounds cannot be considered to play a major role in the initial hydrothermal decarboxylation of biomass.

Topics: beta-Cyclodextrins; Biomass; Carbohydrate Conformation; Carbohydrate Metabolism; Carbohydrate Sequence; Cellobiose; Chromatography, High Pressure Liquid; Cyclodextrins; Decarboxylation; Glucans; Gluconates; Glucose; Ketoses; Molecular Sequence Data; Sugar Acids; Temperature

The 3D structure of 6-deoxy-6-L-tyrosinylamidocyclomaltoheptaose, a self-complexing beta-cyclodextrin derivative, was determined by NMR and molecular modelling. The aminoacyl side-chain is included in the cavity and induces chemical-shift variations in the CD proton signals, allowing their complete assignment. Dipolar interactions between protons of the tyrosine ring and internal protons of the cyclodextrin were used to obtain distance constraints. Then 42 structures were calculated from 32 distance constraints--21 shorter than 4 A involve the host-guest interactions--using a simulated annealing procedure. Starting from one of the resulting structures, a 250-ps molecular dynamics simulation was carried out in a waterbox without constraint. The simulation data are in agreement with NMR data such as nOe and ring-current effects. The cyclodextrin part takes an elliptical shape, which tightly fits the aromatic moiety. As a consequence, the respective motion of the host and the guest moieties have the same amplitude and time scale: the self-inclusion complex shows only little flexibility.

Topics: beta-Cyclodextrins; Carbohydrate Conformation; Carbohydrate Sequence; Computer Graphics; Cyclodextrins; Glucans; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Sequence Data; Molecular Structure; Protons; Tyrosine

The characterization is reported of two peptide fragments (SBD106 and SBD122) containing the starch-binding domain (SBD) of Aspergillus sp. glucoamylase I. The starch-binding peptides were produced in Escherichia coli as fusion proteins of the maltose-binding protein (MBP). SBD106 (11.9 kDa) and SBD122 (13.8 kDa) were purified from the factor Xa digest of MBP fusion proteins. The amino acid compositions were similar to those deduced from their amino acid sequences. The interactions of beta-cyclodextrin and maltoheptaose with purified SBD peptides were investigated by UV difference spectroscopy. SBD106 and SBD122 bound specifically beta-cyclodextrin with a dissociation constant (Kd) of 34 microM and 23.5 microM, respectively. Maltoheptaose binding to SBD106 and SBD122 was weaker than that of beta-cyclodextrin; dissociation constants were 0.57 and 0.50 mM, respectively. The results indicate that the intramolecular disulfide bonding is not required for the domain functioning and that O-glycosylation is not critical for the functioning of the starch-binding domain, but may affect its conformation and dynamics.

Topics: Amino Acid Sequence; Amino Acids; Aspergillus; beta-Cyclodextrins; Binding Sites; Carbohydrate Conformation; Cyclodextrins; Genes, Fungal; Genetic Engineering; Glucan 1,4-alpha-Glucosidase; Glucans; Kinetics; Molecular Sequence Data; Peptide Fragments; Recombinant Fusion Proteins; Starch

A serum-free medium has been developed which is able to support primary antibody responses by cultured murine lymphocytes. This medium is based on RPMI 1640 that is supplemented with beta-cyclodextrin, insulin, transferrin, albumin, low density lipoprotein, putrescine and L-alanine as substitutes for fetal calf serum. Omission of anyone of these components resulted in a marked decrease of antibody responses. By employing the serum-free culture conditions, it was clearly demonstrated that polyamines such as putrescine, spermidine and spermine had positive effects on the development of antibody-forming cells. This serum-free medium supported the antibody response to sheep erythrocytes, trinitrophenyl (TNP)-Ficoll or TNP-lipopolysaccharide as efficiently as 10% fetal calf serum-containing medium. In addition, murine lymphocytes proliferated in response to an antigen or a mitogen equally well in these two types of medium.

Topics: Animals; Antibody Formation; Antibody-Producing Cells; beta-Cyclodextrins; Culture Media; Cyclodextrins; Dextrins; Erythrocytes; Female; Glucans; In Vitro Techniques; Lymphocyte Activation; Mice; Mice, Inbred BALB C; Polyamines; Putrescine; Starch; Time Factors