Preparation of ß-1,3-glucan mimics via modification of polymer backbone, and evaluation of cytokine production using the polymer library in immune activation.

ß-1,3-Glucans possess therapeutic potential owing to their ability to exhibit immunostimulating activity. ß-1,3-Glucans, isolated from various organisms, differ in their chemical structures, molecular weight, and branching degree, potentially forming particulate, helix, or random coil conformations in water. Therefore, this study used synthesized ß-1,3-glucan mimic polymers to investigate the difference in binding affinity for dectin-1 and induced cytokine productions based on polymer structures. The ß-1,3-glucan mimic polymers were synthesized using ß-1,3-glucan tetrasaccharyl monomer, with subsequent modifications to the polymer backbones through the introduction of hydrogen or a hydroxy group. Polymers with different structures in both ligands and polymer backbones were utilized to comprehensively investigate their binding affinity to dectin-1 and cytokine-inducing in macrophages. Hydroxylated polymers exhibited a high binding affinity for dectin-1, similar to that of schizophyllan, whereas the polymer composed of only saccharyl monomers did not bind to dectin-1. Further, when administered to macrophage RAW264 cells, polymers with branched and hydrophobic polymer backbones exhibited strong cytokine-inducing activities. Moreover, the results revealed that the essential factors for cytokine induction include the branches of ß-1,3-glucans, high (tens of thousands) molecular weights, and hydrophobicity. The results suggests that artificial polymers comprising these factors exhibit immunostimulating activity and could be developed as therapeutic agents.

Identification of a tomato UDP-arabinosyltransferase for airborne volatile reception.

Volatiles from herbivore-infested plants function as a chemical warning of future herbivory for neighboring plants. (Z)-3-Hexenol emitted from tomato plants infested by common cutworms is taken up by uninfested plants and converted to (Z)-3-hexenyl ß-vicianoside (HexVic). Here we show that a wild tomato species (Solanum pennellii) shows limited HexVic accumulation compared to a domesticated tomato species (Solanum lycopersicum) after (Z)-3-hexenol exposure. Common cutworms grow better on an introgression line containing an S. pennellii chromosome 11 segment that impairs HexVic accumulation, suggesting that (Z)-3-hexenol diglycosylation is involved in the defense of tomato against herbivory. We finally reveal that HexVic accumulation is genetically associated with a uridine diphosphate-glycosyltransferase (UGT) gene cluster that harbors UGT91R1 on chromosome 11. Biochemical and transgenic analyses of UGT91R1 show that it preferentially catalyzes (Z)-3-hexenyl ß-D-glucopyranoside arabinosylation to produce HexVic in planta.

UDP-glucose, cereblon-dependent proinsulin degrader.

Insulin secretion is regulated in multiple steps, and one of the main steps is in the endoplasmic reticulum (ER). Here, we show that UDP-glucose induces proinsulin ubiquitination by cereblon, and uridine binds and competes for proinsulin degradation and behaves as sustainable insulin secretagogue. Using insulin mutagenesis of neonatal diabetes variant-C43G and maturity-onset diabetes of the young 10 (MODY10) variant-R46Q, UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT1) protects cereblon-dependent proinsulin ubiquitination in the ER. Cereblon is a ligand-inducible E3 ubiquitin ligase, and we found that UDP-glucose is the first identified endogenous proinsulin protein degrader. Uridine-containing compounds, such as uridine, UMP, UTP, and UDP-galactose, inhibit cereblon-dependent proinsulin degradation and stimulate insulin secretion from 3 to 24 h after administration in ß-cell lines as well as mice. This late and long-term insulin secretion stimulation is designated a day sustainable insulin secretion stimulation. Uridine-containing compounds are designated as proinsulin degradation regulators.

UDP-Glucose: A Cereblon-Dependent Glucokinase Protein Degrader.

We previously reported that glucokinase is ubiquitinated and degraded by cereblon with an unknown endogenous glucokinase protein degrader. Here, we show that UDP-glucose is a glucokinase protein degrader. We identified that both glucose and UDP-glucose bind to glucokinase and that both uridine and UDP-glucose bind to cereblon in a similar way to thalidomide. From these results, UDP-glucose was identified as a molecular glue between cereblon and glucokinase. Glucokinase produces glucose-6-phosphate in the pancreas and liver. Especially in ß-cells, glucokinase is the main target of glucose for glucose-induced insulin secretion. UDP-glucose administration ubiquitinated and degraded glucokinase, lowered glucose-6-phosphate production, and then reduced insulin secretion in ß-cell lines and mice. Maturity-onset diabetes of the young type 2 (MODY2) glucokinaseE256K mutant protein was resistant to UDP-glucose induced ubiquitination and degradation. Taken together, glucokinase ubiquitination and degradation signaling might be impaired in MODY2 patients.

Antimicrobial activities of amphiphilic cationic polymers and their efficacy of combination with novobiocin.

Today, drug-resistant bacteria represent a significant problem worldwide. In fact, bacteria are becoming resistant even to newly developed antibiotics. Therefore, there is an urgent need to develop antibiotics to which bacteria cannot become resistant. In this study, antimicrobial polymers to which bacteria cannot develop resistance were prepared from 6-aminohexyl methacrylamide and N-isopropyl acrylamide. The polymers with molecular weights of the order of 105 showed little antimicrobial activity against Staphylococcus aureus and Escherichia coli as well as low toxicity. On the other hand, polymers with lower molecular weights (of the order of 104) did show antimicrobial activity against S. aureus and E. coli. These polymers were combined with novobiocin to investigate the combined usage effects against E. coli. The combined usage of novobiocin and the low-molecular-weight polymers reduced the minimum inhibitory concentration, which was less than 0.0625 µg/mL against E. coli. This result indicates that the combination is useful for increasing the efficacy of antibiotics and broadening their antimicrobial spectrum. Furthermore, the results showed the possibility that the antimicrobial polymers serve not only as antibiotics to which bacteria have not developed resistance but also as adjuvants for other antibiotics.

Antibacterial Activity of Membrane-Permeabilizing Bactericidal Cyclodextrin Derivatives.

Antimicrobial peptides that act by disrupting bacterial membranes are attractive agents for treating drug-resistant bacteria. This study investigates a membrane-disrupting peptide mimic made of a cyclic oligosaccharide cyclodextrin scaffold that can be chemically polyfunctionalized. An antibacterial functional group on the peptide was simplified to an alkylamino group that combines cationic and hydrophobic moieties, the former to interact with the anionic bacterial membrane and the latter with the membrane interior. The cyclodextrins equipped with eight alkylamino groups on the molecules using a poly-click reaction exhibited antibacterial activity against Gram-positive and Gram-negative bacteria, including drug-resistant pathogens such as carbapenem-resistant Enterobacteriaceae. Several lines of evidence showed that these agents disrupt bacterial membranes, leading to rapid bacterial cell death. The resulting membrane perturbation was directly visualized using high-speed atomic force microscopy imaging. In Gram-negative bacteria, the membrane-permeabilizing action of these derivatives allowed the entry of co-treated traditional antibiotics, which were then active against these bacteria.

One-Step Synthesis of Sugar Nucleotides.

The chemical synthesis of sugar nucleotides requires a multistep procedure to ensure a selective reaction. Herein, sugar nucleotides were synthesized in one step using 2-chloro-1,3-dimethylimidazolinium chloride as the condensation reagent. The products were obtained in yields of 12-30%, and the yields were increased to 35-47% by the addition of a tuning reagent. NMR identification of the sugar nucleotides showed that mainly 1,2-trans-glycosides were present. The reported method represents a one-step route to sugar nucleotides from commercially available materials.

Synthesis of ß-1,3-glucan mimics by ß-1,3-glucan trisaccharyl monomer polymerization.

ß-1,3-Glucans are important as immunostimulating agents in living organisms. The multivalent binding of ß-1,3-glucans to dectin-1, a cell surface receptor, activates immunological defenses. To study artificial immunostimulating agents, glycopolymers carrying ß-1,3-glucan trisaccharides as artificial ligands were synthesized. The ß-1,3-glucan trisaccharide, defined as an active unit of ß-1,3-glucan, was constructed from D-glucose by glycosylation. A norbornene group was introduced as a polymerizable group into the trisaccharide derivative at the aglycone. The prepared endo/exo norbornene stereoisomers of the monomers were separated by silica gel chromatography and identified by NMR spectroscopy and mass spectrometry. The synthesized glycosyl monomers were polymerized and copolymerized with norbornene using 2nd generation Hoveyda-Grubbs catalyst, deprotected, and purified by gel filtration to prepare water-soluble glycopolymers of varied compositions and high molecular weights. These polymers will have the potential for multivalent binding to dectin-1 to activate immune response and facilitate studies to understand the binding mechanisms of ß-1,3-glucans with dectin-1.

Gramicidin S-inspired antimicrobial cyclodextrin to disrupt gram-negative and gram-positive bacterial membranes.

A membrane-active antimicrobial peptide gramicidin S-like amphiphilic structure was prepared from cyclodextrin. The mimic was a cyclic oligomer composed of 6-amino-modified glucose 2,3-di-O-propanoates and it exhibited antimicrobial activity against Gram-positive and Gram-negative bacteria, together with no resistance development and low haemolytic activity against red blood cells.

Development of New Antimicrobial Agents from Cationic PG-Surfactants Containing Oligo-Lys Peptides.

Peptide gemini-surfactant (PG-surfactant), a kind of lipopeptide, is composed of a short linker peptide (X) between two alkyl-chain-modified Cys residues and peripheral peptides at the N-terminal (Y) and the C-terminal (Z) sides, respectively, of the alkylated Cys residues. In this study, we developed and examined a series of PG-surfactants containing two C12 saturated alkanes and oligo-Lys, arranged at the X-, Y-, or Z-positions. To arrange oligo-Lys at the Y- or Z-positions, a repeat sequence of -Asp-Lys-Asp-Lys- was used at the X-position. All of the PG-surfactants exhibited high antimicrobial activity against both Gram-positive and -negative bacteria. In addition to high antimicrobial activity, a low hemolysis activity is prerequisite for efficient intravenous administration. Among the synthesized PG-surfactants, those having -(Lys)3- at the Y- or Z-positions, i.e. K3-DKDKC12 and DKDKC12K3, showed reasonably low hemolytic activities. This combination of high antimicrobial activity along with low hemolytic activity is an essential and unique property and has not been previously reported for the synthesized lipopeptides. Further, using scanning electron microscopy (SEM) and N-phenyl-1-naphthylamine (NPN) uptake assay we showed that the antimicrobial activity of these PG-surfactants may be attributed to membrane disruptive mechanisms. Although the PG-surfactants with low hemolytic activity could interact and localize onto red blood cell surfaces and cause slight expansion of cell morphologies, no subsequent penetration occurred. In summary, we describe here the successful development of PG-surfactants having high antibacterial and low hemolytic activity, thus providing a significant molecular platform to develop novel antimicrobial agents.

Membrane-active antimicrobial poly(amino-modified alkyl) ß-cyclodextrins synthesized via click reactions.

The emergence of drug-resistant bacteria has led to the high demand for new antibiotics. In this report, we investigated membrane-active antimicrobial ß-cyclodextrins. These contain seven amino-modified alkyl groups on a molecule, which act as functional moieties to permeabilize bacterial cell membranes. The polyfunctionalization of cyclodextrins was achieved through a click reaction assisted by microwave irradiation. A survey using derivatives with systematically varied functionalities clarified the unique correlation of the antimicrobial activity of these compounds with their molecular structure and hydrophobicity/hydrophilicity balances. The optimum hydrophobicity for the compounds being membrane-active was specific to bacterial strains and animal cells; this led to specific compounds having selective toxicity against bacteria including multidrug-resistant pathogens. The results demonstrate that cyclodextrin is a versatile molecular scaffold for rationally designed structures and can be used for the development of new antibiotics.

Transglycosylation Activity of Catalytic Domain Mutant of Endo-1,3-ß-glucanase from Cellulosimicrobium cellulans.

BACKGROUND: Oligosaccharides are of great value in drug discovery programs which address a wide range of therapeutic strategies in medical specialties. However, owing to difficulties in oligosaccharide synthesis by conventional methods, oligosaccharide assembly using enzymes has been explored. The transglycosylases have been demonstrated to be effective for the oligosaccharide synthesis. Further studies are required to improve the specificity and activity of transglycosylases. There is an additional approach to use mutated glycosidase which transforms into glycosyltransferase with a decreased hydrolytic activity. The substitution of catalytic residue in glycosidase results in the loss of hydrolytic activity. During the reaction with glucanase, reaction of water with the substrate - enzyme intermediate results in the production of a hydrolyzed sugar. When the water molecule is replaced by a competing sugar, a new glycoside linkage is formed as a result of transglycosylation. OBJECTIVE: In this article, we evaluated the transglycosylation activity of endo-1,3-ß-glucanase mutant, E119G, toward laminarioligosaccharides under various pH and temperature conditions, in comparison with those of the wild-type enzyme. We also analyzed the effect of glucose and laminaribiose on the transglycosylation activity. METHOD: In this article, we generated the E119G mutant of endo-1,3-ß-glucanase from Cellulosimicrobium cellulans DK-1. The residue, Glu119, would act as a nucleophile in the reaction and affect the balance between hydrolysis and transglycosylation. The enzymatic activities of wild-type and E119G were estimated by detecting the products obtained from laminarioligosaccharides as substrates. We also analyzed the effect of reaction conditions such as temperature and pH on the enzymatic activity of E119G toward laminaritriose. We further analyzed the enzymatic activity of E119G toward laminaritriose in the presence of glucose or laminaribiose to investigate whether these additional molecules could accelerate the transglycosylation activity. RESULTS: The purified E119G mutant of endo-1,3-ß-glucanase was properly folded, and exhibited the secondary structure, similar to that of wild-type. The E119G mutant exhibited enhanced transglycosylation activity and decreased hydrolytic activity, relative to the wild-type. The hydrolytic as well as transglycosylation activities of E119G decreased with the decrease in temperature, however, the ratio of transglycosylation products increased. The temperature-dependent degree of reduction in hydrolytic activity was higher than that in the transglycosylation activity. The enzymatic activities were similar within the range of pH 4.0 - 7.4, while those at pH 8.0 and 8.5 were slightly decreased. The enzymatic activity of E119G toward laminaritriose in the presence of glucose was ineffective, while the addition of laminaribiose evidently increased the transglycosylation products such as laminaritetraose and laminaripentaose. CONCLUSION: A mutation of catalytic residue, Glu119 to Gly, in endo-1,3-ß-glucanase from Cellulosimicrobium cellulans exhibited transglycosylation activity on laminarioligosaccharides. The combination of laminaribiose and laminaritriose as a substrate enhanced the transglycosylation activity. According to the structural information previously reported, laminaritriose mainly binds to the enzyme at the subsites from -1 to -3 and forms a link with laminaribiose, which transiently binds to the subsites +1 and +2. To increase the amount of transglycosylation product, the reaction was found to be effective at low temperature.

Syntheses and structure-membrane active antimicrobial activity relationship of alkylamino-modified glucose, maltooligosaccharide, and amylose.

Antimicrobial alkylamine-modified sugars were prepared. Microwave-assisted click reaction efficiently achieved poly-functionalization of oligo- and polysaccharides. The sugars exhibited a unique relationship of their bacterial membrane permeabilization and antimicrobial activity with the number of functional groups and the structure of the molecular scaffold. It shows that the assembly of the functional groups is necessary for being antimicrobial. The amylose derivatives also exhibited synergy to minimize the necessary dose of conventional antibiotics and increase their antimicrobial potency.

Chemical Modification of Cyclodextrin and Amylose by Click Reaction and Its Application to the Synthesis of Poly-alkylamine-Modified Antibacterial Sugars.

Cyclodextrin (CD) can be chemically modified into desired and sophisticated functional molecules. However, poly-modification often produces complicated mixtures, resulting in a low yield of the desired product. As the most promising procedure to solve such problems and to achieve poly-modification of the CD molecule, we present here the Huisgen 1,3-dipolar cycloaddition, known as a click reaction. This review will describe the results of our microwave-assisted click reaction for the poly-modification of CD and amylose molecules, and its application to the study of synthetic membrane active antibacterial derivatives.

Chemical approach for the syntheses of GM4 isomers with sialic acid to non-natural linkage positions on galactose.

Cell-surface glycans containing sialic acid are involved in various biological phenomena. However, the syntheses of GM4 derivatives with (2 → 2) and (2 → 4) linkages have not been investigated to date. In this study, sialylation of all of the hydroxyl groups on galactose were investigated for the syntheses of GM4 isomers. Regioselective sialylation was achieved via protection of galactosyl acceptors using electron-rich benzyl groups. These synthetic sialylated glycans will prove to be useful tools for studying unidentified carbohydrate-mediated biological roles.

Synthesis of antimicrobial cyclodextrins bearing polyarylamino and polyalkylamino groups via click chemistry for bacterial membrane disruption.

Cyclodextrin derivatives are synthesized as membrane-disrupting agents via a microwave-assisted Huisgen reaction. Their ability to permeabilize bacterial membranes depends on the amino substituents and an appropriate balance of hydrophobicity and hydrophilicity, thus enabling the preparation of derivatives with selective toxicity against bacteria.

Mimicking an antimicrobial peptide polymyxin B by use of cyclodextrin.

Cyclodextrin derivatives prepared to mimic a membrane active antibacterial peptide polymyxin B strongly permeabilized bacterial membrane and inhibited bacterial proliferation.

Supramolecular complexation of N-alkyl- and N,N'-dialkylpiperazines with cucurbit[6]uril in aqueous solution and in the solid state.

Complex stoichiometry/composition and degree of oligomerization (oligomeric supramolecular complex formation) of cucurbit[6]uril (CB[6]) with N-alkyl- and N,N'-dialkylpiperazine were investigated in aqueous solutions by means of isothermal titration calorimetry (ITC), ESI-MS, NMR and light scattering measurements. It was found that the complex stability and the degree of oligomerization increase with elongating the alkyl chain attached to the piperazine core. X-ray crystallographic studies revealed a clear correlation between the structure of CB[6]-alkylpiperazine crystals obtained from aqueous solutions and the molecular weight/properties of host-guest oligomers existed in the solution as supramolecular "seeds" of crystal formation.

Sequence recognition and self-sorting of a dipeptide by cucurbit[6]uril and cucurbit[7]uril.

Cucurbit[7]uril forms very strong complex with zwitterionic dipeptide Phe-Gly with affinity exceeding 10(7) M(-1) and effectively recognizes peptide sequence of Phe-Gly over Gly-Phe as well as Tyr-Gly over Gly-Tyr and Trp-Gly over Gly-Trp with relative affinities of 23 000, 18 000 and 2000, respectively.