Beta-1,3-glucan polysaccharides as novel one-dimensional hosts for DNA/RNA, conjugated polymers and nanoparticles

Beta-1,3-glucan polysaccharides have triple-stranded helical structures whose sense and pitch are comparable to those of polynucleotides. We recently revealed that the beta-1,3-glucans could interact with certain polynucleotides to form triple-stranded and helical macromolecular complexes consisting of two polysaccharide-strands and one polynucleotide-strand. This unique property of the beta-1,3-glucans has made it possible to utilize these polysaccharides as potential carriers for various functional polynucleotides. In particular, cell-uptake efficiency of the resultant polysaccharide/polynucleotide complexes was remarkably enhanced when functional groups recognized in a biological system were introduced as pendent groups. The beta-1,3-glucans can also interact with various one-dimensional architectures, such as single-walled carbon nanotubes, to produce unique nanocomposites, in which the single-walled carbon nanotubes are entrapped within the helical superstructure of beta-1,3-glucans. Various conductive polymers and gold nanoparticles are also entrapped within the helical superstructure in a similar manner. In addition, diacetylene monomers entrapped within the helical superstructure can be photo-polymerized to afford the corresponding poly(diacetylene)-nanofibers with a uniform diameter. These findings indicate that the beta-1,3-glucans are very attractive and useful materials not only in biotechnology but also in nanotechnology. These unique properties of the beta-1,3-glucans undoubtedly originate from their inherent, very strong helix-forming character which has never been observed for other polysaccharides.     

Lactose-appended schizophyllan is a potential candidate as a hepatocyte-targeted antisense carrier

A schizophyllan (beta-1,3-glucan) derivative carrying lactose-appendages prepared by reductive amination can form stable macromolecular complexes with polynucleotides, shows excellent affinity with a lactose-binding lectin, and effectively mediates gene transfection into hepatocytes.     

A molecular template designed by the modification of a helix-forming β-1,3-glucan polysaccharide to fabricate one-dimensional nanostructures

We report the development of a new templating molecule designed by the modification of a helix-forming β-1,3-glucan polysaccharide to the cationic semiartificial one and its application to the fabrication of one-dimensional (1D) gold nanostructures by simple photoirradiation. Transmission electron microscopy observation showed that Au(III) ions are primarily reduced to gold nanoparticles self-assembling into the 1D array with the aid of the cationic β-1,3-glucan polysaccharide, which gradually fuse into the 1D gold nanostructure with the tapelike structure. The gold nanotape structure could not be created by neutral β-1,3-glucan polysaccharides or random coil synthetic cationic polymers. These findings consistently support the view that Au(III) ions are reduced by unmodified OH groups to gold nanoparticles under the photoirradiation, which are wrapped in the helical structure of the cationic β-1,3-glucan polysaccharide and eventually fuse into gold nanotapes. One may regard, therefore, that this cationic β-1,3-glucan polysaccharide can act as an "all-in-one" template playing three roles of reduction, 1D arrangement, and fusion of gold nanoparticles. In addition, we found an interesting phenomenon that the obtained gold nanotapes coated with cationic β-1,3-glucan polysaccharides show unique surface-enhanced Raman scattering for anionic porphyrines organized on the surface of gold nanotapes through the electrostatic interaction.     

A pH-responsive carboxylic β-1,3-glucan polysaccharide for complexation with polymeric guests

The helix-forming nature of β-1,3-glucan polysaccharides is a characteristic that has potential for producing gene carriers, bio-nanomaterials and other chiral nanowires. Herein, carboxylic curdlan (CurCOOH) bearing the β-1,3-polyglucuronic acid structure was successfully prepared from β-1,3-glucan polysaccharide curdlan (Cur) by one-step oxidation using a 4-acetamido-TEMPO/NaClO/NaClO(2) system as the oxidant. The resulting high-molecular-weight CurCOOH was proved to bear the 6-COOH group in 100% purity. The optical rotatory dispersion (ORD) spectra indicated that the obtained CurCOOH behaves as a water-soluble single-strand in various pH aqueous media. This advantage has allowed us to use CurCOOH as a polymeric host to form various macromolecular complexes. For example, complexation of CurCOOH with single-walled carbon nanotubes (SWNTs) resulted in a water-soluble one-dimensional architecture, which formed a dispersion in aqueous solution that was stable for several months, and much more stable than SWNTs complexes of the similar negatively-charged polyacrylic acid (PAA) and polymethacrylic acid (PMAA). It was shown that in the complex, SWNTs are effectively wrapped by a small amount of CurCOOH, enabling them to avoid electrostatic repulsion. This pH-responsive CurCOOH formed a very stable complex with cationic water-soluble polythiophenes (PT-1), which was stabilized not only by the hydrophobic interaction but also by the electrostatic attraction between trimethylammonium cations in PT-1 and dissociated anionic COO(-) groups in CurCOOH. The included PT-1 became CD-active only in the neutral to basic pH region, and the positive Cotton effect suggested that the conjugated main chain is twisted in the right-handed direction. We also found that CurCOOH can interact with polycytidylic acid (poly(C)) only under high NaCl concentrations, the binding and release of which could be controlled by a change in the salt concentration. We believe, therefore, that CurCOOH bearing a dissociable COOH group can act as a new potential polymeric host to construct novel polymeric complexes applicable for gene carriers, biosensors, chiral polymer assemblies, etc.     

Carbohydrate-appended curdlans as a new family of glycoclusters with binding properties both for a polynucleotide and lectins

Beta-1,3-glucans having carbohydrate-appendages (alpha-D-mannoside, N-acetyl-beta-D-glucosaminide and beta-lactoside) at the C6-position of every repeating unit can be readily prepared from curdlan (a linear beta-1,3-glucan) through regioselective bromination/azidation to afford 6-azido-6-deoxycurdlan followed by chemo-selective Cu(i)-catalyzed [3 + 2]-cycloaddition with various carbohydrate modules having a terminal alkyne. The resultant carbohydrate-appended curdlans can interact with polycytosine to form stable macromolecular complexes consistent with two polysaccharide strands and one polycytosine strand. Furthermore, these macromolecular complexes show strong and specific affinity toward carbohydrate-binding proteins (lectins). Therefore, one can utilize these carbohydrate-appended curdlans as a new family of glycoclusters.     

Curdlan as a Polymeric Starting Material to Access C6-Modified Glucose Derivatives

We evaluated the potential of a linear β-1,3-glucan (curdlan) as a starting material to access C6-modified glucose derivatives and found that 6-bromo-6-deoxyglucose, 6-azide-6-deoxyglucose, and 6-acetamido-6-deoxyglucose could be readily prepared from curdlan through its C6-selective and quantitative modifications and subsequent acid-catalyzed hydrolysis.     

Amylose‐grafted Curdlan: A New Class of Semi‐artificial Branched Polysaccharides for Hierarchical Polymeric Superstructures Created by the Action of “Orthogonal” Binding Sites

The semi‐artificial branched‐polysaccharides, amylose‐grafted curdlans, were synthesized utilizing an enzymatic polymerization. Both a curdlan main chain and amylose side chains on the polysaccharides maintain the original helical structure as well as the molecular binding ability. Thanks to the difference in their molecular recognition properties between β‐1,3‐glucan chain and α‐1,4‐glucan chain, the amylose‐grafted curdlans can provide two different orthogonal binding sites within one polymeric system. When a water‐soluble polythiophene was mixed with the amylose‐grafted curdlan, the polythiophene was twisted in two different modes and therein, fluorescence energy of the polythiophene wrapped by the amylose side chains was successfully transferred to the polythiophene wrapped by the curdlan main chain. We thus concluded that in the dendritic superstructure of this polysaccharide, a self‐organized “Janus‐type FRET system” was successfully constructed.     

Instantaneous inclusion of a polynucleotide and hydrophobic guest molecules into a helical core of cationic beta-1,3-glucan polysaccharide

We succeeded in the quantitative and selective introduction of an ammonium cationic group into the C6 position of Curdlan (CUR) by "Click Chemistry", and the obtained cationic Curdlan (CUR-N+) showed good solubility in water. ORD studies suggested that CUR-N+ adopts a single-stranded structure, different from a right-handed, triple-stranded helical structure of beta-1,3-glucan polysaccharides in water. It has been revealed that the polymeric complexes of CUR-N+ with polymeric guest molecules, such as polycytidylic acid (poly(C)), permethyldecasilane (PMDS), and single-walled carbon nanotubes (SWNTs), can be easily obtained by just mixing them in water with sonication. The characterization of the resultant CUR-N+-poly(C) complexes by UV-vis, CD spectroscopic measurements, and AFM and TEM observations revealed that they have stoichiometric, nanosized fibrous structures. From these experimental results as well as our precedent studies (e.g., refs 6 and 23), we propose that the complexation would be driven by the cooperative action of (1) the hydrogen-bonding interaction between the OH group at the C2 position and hydrogen-bonding sites of the cytosine ring (ref 6d), (2) the electrostatic interaction between the ammonium cation and the phosphate anion (ref 23), as well as (3) the background hydrophobic interaction. In addition, the complexed polynucleotide chain showed a strong resistance against enzymatic hydrolysis. Likewise, the dispersion of PMDS and SWNTs in water by CUR-N+ and the fibrous structures of the complexes were confirmed by spectroscopic measurements as well as microscopic observations. These binding properties of CUR-N+, which can proceed spontaneously in water, clearly differ from those of schizophyllan (SPG), which inevitably require a denature-renature process corresponding to a conversion of a triple strand to single strands induced by DMSO or base for inclusion of polymeric guest molecules.     

Controlled Stability of the Triple‐Stranded Helical Structure of a β‐1,3‐Glucan with a Chromophoric Aromatic Moiety at a Peripheral Position

We synthesized a semiartificial β‐1,3‐glucan, curdlan with dialkylaniline groups (CUR‐DA), that bears chromophoric aromatic groups at its peripheral positions. Spectroscopic studies as well as microscopic observations indicate that CUR‐DA adopts a triple‐stranded helical structure in water‐ or methanol‐rich solutions of dimethyl sulfoxide (DMSO). This triple‐stranded helical structure exhibits high thermal stability and resistance to base, attributes that are similar to those of the triple‐stranded helical structure of native β‐1,3‐glucans such as schizophyllan. Moreover, we found that the stability of the triple‐stranded helical structure can be easily modulated by solvent composition and metal‐ion (Zn²⁺) binding. As β‐1,3‐glucan polysaccharides are known to serve as “polymeric” hosts, including certain DNA molecules, carbon nanotubes, and conjugated polymers, and complexation occurs only with the single‐stranded structure, this information is very useful for the creation of these attractive polymeric composites, the controlled release of DNA, and so on.     

Synthesis of thermoplastic curdlan alkyl carbamates having hydrogen bonding ability

Rigid and little moldable curdlan, a linear β-1,3-glucan having intra- and interchain hydrogen bonds, was reacted with several alkyl isocyanates, which gave thermoplastic curdlan alkyl carbamates (CrdC) with degree of substitution about 2. The alkyl carbamation at hydroxy groups in the glucan skeleton effectively broke the interchain hydrogen bonds of curdlan and increased flexibility of CrdC, while the newly formed carbamate moieties could moderately keep the hydrogen bonding ability in CrdC. Elongating the alkyl groups in the carbamate side chains increased solubility in organic solvents and thermoplasticity of CrdC, which enabled to make homogeneous and free-standing films by both methods of solution-casting and hot-pressing.     

Solution self-assembly, spontaneous deprotonation, and crystal structures of bipyrazolate-bridged metallomacrocycles with dimetal centers

A series of nanosized cavity-containing bipyrazolate-bridged metallomacrocycles with dimetal centers, namely, {[(bpy)M]8L4}(NO3)8 [L=3,3',5,5'-tetramethyl-4,4'-bipyrazolyl, Pd, Pt; 1,4-bis-4'-(3',5'-dimethyl)-pyrazolylbenzene), Pd; and 1,4-bis-4'-(3',5'-dimethyl)-pyrazolylbiphenyl, Pd], {[(phen)M]8L4}(NO3)8 [L=3,3',5,5'-tetramethyl-4,4'-bipyrazolyl, Pd, Pt; 1,4-bis-4'-(3',5'-dimethyl)-pyrazolylbenzene, Pd; and 1,4-bis-4'-(3',5'-dimethyl)-pyrazolylbiphenyl, Pd], {[(bpy)Pd]6L3}(NO3)6 [L=1,4-bis-4'-(3',5'-dimethyl)-pyrazolylbenzene,], {[(phen)Pd]6L3}(NO3)6 [L=1,4-bis-4'-(3',5'-dimethyl)-pyrazolylbenzene,], {[(bpy)Pd]4L2}(NO3)4 [L=1,3-bis-4'-(3',5'-dimethyl)-pyrazolylbenzene, and 1,2-bis-4'-(3',5'-dimethyl)-pyrazolylbenzene,], and {[(phen)Pd]4L2}(NO3)4 [L=1,3-bis-4'-(3',5'-dimethyl)-pyrazolylbenzene, and 1,2-bis-4'-(3',5'-dimethyl)-pyrazolylbenzene,] (where bpy=2,2'-bipyridine and phen=1,10-phenanthroline) have been synthesized through a directed self-assembly approach that involves spontaneous deprotonation of the 1H-bipyrazolyl ligands in aqueous solution. These complexes, with weak Pd(II)...Pd(II) or Pt(II)...Pt(II) interactions, have been characterized by elemental analysis, 1H and 13C NMR, cold-spray ionization or electrospray ionization mass spectrometry, UV-visible spectroscopy, and luminescence spectroscopy. Complexes and have also been characterized by single-crystal X-ray diffraction analysis.     

Theoretical study of β and γ-cyclodextrins inclusion complexes with nineteen atropisomeric polychlorobiphenyls

Inclusion complexes of β and γ-cyclodextrins (CDs), chirally selective macrocycles, with 19 atropisomeric polychlorinated biphenyls (PCBs), stable at physiological temperatures, were studied by molecular mechanics optimized potential for liquid simulations and semi-empirical quantum AM1 methods. Bimodal manual docking, geometry optimization and single point calculations were done. PCB:CD complex formation was studied considering two ways of entry in the large base of CD cavity: with biphenyl C1–C1′ bond axis of PCBs oriented parallel and anti-parallel with the CD trunk height axis. A distance dependent dielectric constant was used to account for the solvent effect. The values of complexation and binding energies were calculated, confirming the existence of a specific van der Waals (vdW) type interaction. β and/or γ-CD chiral recognition of the 19 atropisomeric PCBs, is described by means of the complexation and binding energy values. The binding energy is a better discriminator of PCBs enantiomers than complexation energy considering the average energy differences between (+) and (?) PCB:β/γ-CD complexes. The chromatographic elution order of several PCB enantiomers from literature was correlated with the complexation and binding energies. The molecular modeling of inclusion complexes is recommended to be used as enantiomer identification tool in correlation with chromatographic data.     

A novel pyrrolidinyl PNA showing high sequence specificity and preferential binding to DNA over RNA

A novel conformationally constrained pyrrolidinyl peptide nucleic acid (PNA) carrying an D-aminopyrrolidine carboxylic acid (D-Apc) spacer was synthesized, and its interactions with complementary oligo- and polynucleotides were studied by UV and CD spectroscopy. The decathymine PNA formed very stable PNA-DNA complexes with poly(dA) and (dA)(10) by a sequence-specific A-T pairing. The interaction with poly(rA) gave the corresponding PNA-RNA complex with much lower stability.     

TiO2纳米颗粒对钌(II)三联吡啶配合物光损伤小牛胸腺DNA能力的影响

由于极短的激发态寿命, 钌(II)三联吡啶配合物对脱氧核糖核酸(DNA)的光损伤能力低下. 设计合成了三个钌(II)三联吡啶配合物[Ru(ttp)(tpy)]^2＋ (1), [Ru(ttp-COOH)(tpy)]^2＋ (2)和[Ru(ttp-COOH)(tpy-pyr)]^2＋ (3), 其中tpy为2,2':6',2"-三联吡啶, ttp为4′-(4-甲苯基)-2,2':6',2"-三联吡啶, ttp-COOH为4′-(4-羧基苯基)-2,2':6',2"-三联吡啶, tpy-pyr为4'-(1-芘基)-2,2':6',2"-三联吡啶. 比较了TiO₂纳米颗粒对它们光损伤小牛胸腺DNA的影响. 发现TiO₂纳米颗粒在空气和氩气条件下均可显著提高配合物3光损伤DNA的能力. TiO₂纳米颗粒和配合物3间的光诱导电子转移作用及其该作用生成的钌(III)物种可能是促进配合物3对DNA光损伤的主要原因.     

Bioreactor design considerations in the production of high-quality microbial exopolysaccharide

An examination into the effect of bioreactor design on the production of β,1,3-glucan exopolysaccharide (“curdlan”) by selected patent cultures ofAlcaligenes faecalis andAgrobacterium radiobacter revealed that low shear mixing achieved through the replacement of the radial-flow flat-blade impellers that are commonly supplied in “standard” commercial bioreactors, by low shear (high-pumping) axialflow impellers, leads to an increase in thequality of the exopolymer recovered during the stationary-phase of batch fermentations. Whereas “Rushton turbine” impellers were effective in providing high rates of oxygen transfer necessary for high cell density fermentations, the high shear-to-flow ratio characteristic of this design produced a product of inferior quality, but with characteristics very similar to that of the commercially available “curdlan standard.” Curdlan is water insoluble, and consequently, the fermentation broth is of a relative low viscosity compared to other soluble microbial polysaccharides. Whereas curdlan does not constrain mass transfer from gas to liquid, it nevertheless offers a resistance to oxygen transfer from the liquid to the cell by virtue of the layer of insoluble exopolymer surrounding the cell mass, thereby necessitating an unexpectedly high dissolved oxygen concentration for maximal productivity. The requirement for high volumetric oxygen transfer can be met by low shear designs with axial-flow impellers, providing gas dispersion is assisted by the use of sparging devices consisting of microporous materials.

     

Rapid proton-coupled electron-transfer of hydroquinone through phenylenevinylene bridges

We describe the synthesis of two oligo(phenylene vinylene)s (OPVs) with a hydroquinone moiety and a thiol anchor group: 4-(2',5'-dihydroxystyryl)benzyl thioacetate and 4-[4'-(2' ',5' '-dihydroxystyryl)styryl]benzyl thioacetate. Monolayers on gold of these molecules were examined by electrochemical techniques to determine the electron transfer kinetics of the hydroquinone functionality (H2Q) through these delocalized tethers ("molecular wires") as a function of pH. Between pH 4 and 9, rate constants were ca. 100-fold faster than for the same H2Q functionality confined to the surface via alkane tethers. Also, in this same pH range rate constants were independent of the length of the OPV bridge. These new electroactive molecules in which the hydroquinone functionality is wired to the gold surface by means of OPV tethers should be useful platforms for constructing bioelectronic devices such as biosensors, biofuel cells, and biophotovoltaic cells with a fast response time.     

Self-assembled monolayers of Ru/Os dinuclear complexes: probing monolayer structure and interaction energies by electrochemical means

Monolayers of [Ru(bpy)2(micro-1)M2][PF6]4 salts (M = Os, Ru; bpy = 2,2'-bipyridine, 1 = 4'-(2,2'-bipyridin-4-yl)-2,2':6',2' '-terpyridine, tpy = 2,2':6',2' '-terpyridine, and 2 = 4'-(4-pyridyl)-2,2':6',2' '-terpyridine) were self-assembled on platinum and investigated by fast-scan electrochemistry. The electrochemistry of the complexes in solution and confined to the surface in self-assembled monolayers (SAMs) exhibited an almost ideal behavior. Scan-rate-dependent measurements of the peak current density (jp) were used to determine interaction energies within the monolayer. It is shown that the tpy coordination sites of the dinuclear complexes interact more strongly within the SAM than the bipyridine-coordinated fragments. This result was supported by peak potential shifts, which are due to interaction forces in SAMs. The alignment of the rodlike complexes relative to the surface is discussed, and the results of molecular mechanics calculations indicate that the species adopt a tilted orientation.     

Hybrid Curdlan Poly(γ ‐Glutamic Acid) Nanoassembly for Immune Modulation in Macrophage

A nanoformulation composed of curdlan, a linear polysaccharide of 1,3‐β‐linked d ‐glucose units, hydrogen bonded to poly(γ ‐glutamic acid) (PGA), was developed to stimulate macrophage. Curdlan/PGA nanoparticles (C‐NP) are formulated by physically blending curdlan (0.2 mg mL^?1 in 0.4 m NaOH) with PGA (0.8 mg mL^?1). Forster resonance energy transfer (FRET) analysis demonstrates a heterospecies interpolymer complex formed between curdlan and PGA. The ¹H‐NMR spectra display significant peak broadening as well as downfield chemical shifts of the hydroxyl proton resonances of curdlan, indicating potential intermolecular hydrogen bonding interactions. In addition, the cross peaks in ¹H‐¹H 2D‐NOESY suggest intermolecular associations between the OH‐2/OH‐4 hydroxyl groups of curdlan and the carboxylic‐/amide‐groups of PGA via hydrogen bonding. Intracellular uptake of C‐NP occurs over time in human monocyte‐derived macrophage (MDM). Furthermore, C‐NP nanoparticles dose‐dependently increase gene expression for TNF‐α, IL‐6, and IL‐8 at 24 h in MDM. C‐NP nanoparticles also stimulate the release of IL‐lβ, MCP‐1, TNF‐α, IL‐8, IL‐12p70, IL‐17, IL‐18, and IL‐23 from MDM. Overall, this is the first demonstration of a simplistic nanoformulation formed by hydrogen bonding between curdlan and PGA that modulates cytokine gene expression and release of cytokines from MDM.     

Template-directed synthesis using the heterogeneous templates produced by montmorillonite catalysis. A possible bridge between the prebiotic and RNA worlds

The synthesis of oligoguanylates [oligo(G)s] is catalyzed by a template of oligocytidylates [oligo(C)s] containing 2',5'- and 3',5'-linked phosphodiester bonds with and without incorporated C5'ppC groupings. An oligo(C) template containing exclusively 2',5'-phosphodiester bonds also serves as a template for the synthesis of complementary oligo(G)s. The oligo(C) template was prepared by the condensation of the 5'-phosphorimidazolide of cytidine on montmorillonite clay. These studies establish that RNA oligomers prepared by mineral catalysis, or other routes on the primitive earth, did not have to be exclusively 3',5'-linked to catalyze template-directed synthesis, since oligo(C)s containing a variety of linkage isomers serve as templates for the formation of complementary oligo(G)s. These findings support the postulate that origin of the RNA world was initiated by the RNA oligomers produced by polymerization of activated monomers formed by prebiotic processes.     

A new strategy for a DNA assay based on a target-triggered isothermal exponential degradation reaction

An ultra-sensitive, fast, simple and easily operated method for sequence-specific detection of polynucleotides is proposed herein, which is based on a novel target-triggered isothermal exponential degradation reaction (TT-isoTexpDR) and the color change of probe-functionalized gold nanoparticles.