A novel dextrin produced by the enzymatic reaction of 6-α-glucosyltransferase. I. The effect of nonreducing ends of glucose with by α-1,6 bonds on the retrogradation inhibition of high molecular weight dextrin.

We prepared a high-molecular-weight modified dextrin (MWS-1000) from a partial hydrolysate of waxy corn starch with a weight average molecular weight of 1 × 106 (WS-1000) using Paenibacillus alginolyticus PP710 α-glucosyltransferase. The gel permeation chromatography showed that the weight average molecular weight of MWS-1000 was almost the same as that of WS-1000. The side chain lengths of WS-1000 and MWS-1000 after isomaltodextranase digestion were also shown to be similar to each other by high-performance anion exchange chromatography with pulsed amperometric detection. Since MWS-1000 confirmed the presence of α-1,6 bonds by enzyme digestibility, methylation, and 1H-NMR analyses, it was presumed that the structure of MWS-1000 was based on the introduction of α-1,6 glucosyl residues at the nonreducing ends of the partial hydrolysate of waxy corn starch. Furthermore, the MWS-1000 solution was not retrograded even during refrigerated storage or after repeated freeze-thaw cycles.

Synthesis of Randomly Substituted Anionic Cyclodextrins in Ball Milling.

A number of influencing factors mean that the random substitution of cyclodextrins (CD) in solution is difficult to reproduce. Reaction assembly in mechanochemistry reduces the number of these factors. However, lack of water can improve the reaction outcomes by minimizing the reagent's hydrolysis. High-energy ball milling is an efficient, green and simple method for one-step reactions and usually reduces degradation and byproduct formation. Anionic CD derivatives have successfully been synthesized in the solid state, using a planetary ball mill. Comparison with solution reactions, the solvent-free conditions strongly reduced the reagent hydrolysis and resulted in products of higher degree of substitution (DS) with more homogeneous DS distribution. The synthesis of anionic CD derivatives can be effectively performed under mechanochemical activation without significant changes to the substitution pattern but the DS distributions were considerably different from the products of solution syntheses.

Synthesis, characterization and biocompatibility of cadmium sulfide nanoparticles capped with dextrin for in vivo and in vitro imaging application.

BACKGROUND: The safe use in biomedicine of semiconductor nanoparticles, also known as quantum dots (QDs), requires a detailed understanding of the biocompatibility and toxicity of QDs in human beings. The biological characteristics and physicochemical properties of QDs entail new challenges regarding the management of potential adverse health effects following exposure. At certain concentrations, the synthesis of semiconductor nanoparticles of CdS using dextrin as capping agent, at certain concentration, to reduce their toxicity and improves their biocompatibility. RESULTS: This study successfully synthesized and characterized biocompatible dextrin-coated cadmium sulfide nanoparticles (CdS-Dx/QDs). The results show that CdS-Dx/QDs are cytotoxic at high concentrations (>2 µg/mL) in HepG2 and HEK293 cells. At low concentrations (<1 µg/mL), CdS-Dx/QDs were not toxic to HepG2 or HeLa cells. CdS-Dx nanoparticles only induced cell death by apoptosis in HEK293 cells at 1 µg/mL concentrations. The in vitro results showed that the cells efficiently took up the CdS-Dx/QDs and this resulted in strong fluorescence. The subcellular localization of CdS-Dx/QDs were usually small and apparently unique in the cytoplasm in HeLa cells but, in the case of HEK293 cells it were more abundant and found in cytoplasm and the nucleus. Animals treated with 100 µg/kg of CdS-Dx/QDs and sacrificed at 3, 7 and 18 h showed a differential distribution in their organs. Intense fluorescence was detected in lung and kidney, with moderate fluorescence detected in liver, spleen and brain. The biocompatibility and toxicity of CdS-Dx/QDs in animals treated daily with 100 µg/kg for 1 week showed the highest level of fluorescence in kidney, liver and brain. Less fluorescence was detected in lung and spleen. There was also evident presence of fluorescence in testis. The histopathological and biochemical analyses showed that CdS-Dx/QDs were non-toxic for rodents. CONCLUSIONS: The in vitro and in vivo studies confirmed the effective cellular uptake and even distribution pattern of CdS-Dx/QDs in tissues. CdS-Dx/QDs were biocompatible with tissues from rodents. The CdS-Dx/QDs used in this study can be potentially used in bio-imaging applications.

Dextrin-phospholipase A2: synthesis and evaluation as a bioresponsive anticancer conjugate.

There is still an urgent need for improved treatments for metastatic cancer. Although the phospholipase A(2) (PLA(2)) crotoxin, an antitumor protein that appears to act by interaction with epidermal growth factor receptors (EGFR), has recently shown activity in breast cancer in phase I clinical trials, it also displayed nonspecific neurotoxicity. Therefore, the aim of this study was to apply a novel concept called polymer-masked-unmasked-protein therapy (PUMPT) to give a bioresponsive dextrin-PLA(2) conjugate that would reduce PLA(2) systemic toxicity but retain antitumor activity following alpha-amylase triggered degradation of dextrin in the tumor interstitium. Dextrin (M(w) approximately 60000 g/mol; approximately 22 mol % succinoylation) and PLA(2) (from honey bee venom) were chosen as models for these initial studies, and the conjugates synthesized contained 6.1 wt % PLA(2), with <1% free enzyme. The conjugate showed decreased ( approximately 36%) enzyme activity compared to native PLA(2), but activity was restored to approximately 100% following incubation with alpha-amylase. Whereas dextrin conjugation caused a marked reduction in PLA(2)'s hemolytic activity, the conjugate was cytotoxic toward MCF-7, HT29, and B16F10 cells at a level that was comparable to, or greater than, that seen for free PLA(2). In these cell lines, cytotoxicity showed partial correlation with the level of EGFR expression. The reduced toxicity and alpha-amylase triggered activity of the dextrin-PLA(2) conjugate confirmed the potential of this approach for further development as a novel anticancer treatment.

A novel anti-HIV dextrin-zidovudine conjugate improving the pharmacokinetics of zidovudine in rats.

The aim of this study was to investigate a newly synthesized dextrin-zidovudine (AZT) conjugate designed as a sustained release prodrug of AZT for parenteral administration. AZT was first reacted with succinic anhydride to form a succinoylated AZT which was subsequently coupled with dextrin to yield the dextrin-AZT conjugate. The structure of the conjugate was characterized by FT-IR and (1)H-NMR spectroscopy. The drug content of the conjugate was 18.9 wt.%. The release in vitro of free AZT and succinoylated AZT was investigated in buffer solutions at pH 5.5 and 7.4 and in human plasma. AZT and succinoylated AZT release from the conjugate was 1.4% (pH 5.5), 41.7% (pH 7.4) and 78.4% in human plasma after 24 h. Release was complete in human plasma after 48 h. A pharmacokinetic study in rats following intravenous administration of the conjugate showed prolonged plasma levels of AZT compared to free AZT. The use of the conjugate extended the plasma half-life of AZT from 1.3 to 19.3 h and the mean residence time from 0.4 to 23.6 h. Furthermore, the conjugate provided a significant greater area under the plasma concentration-time curve and reduced the systemic clearance of AZT. This study suggested the potential of this novel dextrin-AZT conjugate as a new intravenous preparation of AZT.

Infection by HIV-1 blocked by binding of dextrin 2-sulphate to the cell surface of activated human peripheral blood mononuclear cells and cultured T-cells.

1. Structural analogues of a sulphated polysaccharide, dextrin sulphate, were synthesized and tested for their ability to block infection by HIV-1. Using the T-cell lines, C8166 and HPB-ALL, and the laboratory adapted strains of HIV-1.MN, HIV-1.IIIb and HIV-1.RF, dextrin 2-sulphate (D2S) combined the best combination of high anti-HIV-1 activity (95% inhibitory concentration (IC95) = 230 nM) and low anticoagulant activity. It also blocked infection of activated peripheral blood mononuclear (PBMN) cells by five primary viral isolates at an IC95 of 230-3700 nM depending upon the primary viral isolate tested. 2. In saturation binding studies, [3H]-D2S bound to a cell surface protein on HPB-ALL cells in a specific and saturable manner with a Kd of 82 +/- 14 nM and a Bmax of 4.8 +/- 0.3 pmol/10(6) cells. It bound to other human T-cell lines in a similar manner. 3. There was very little binding of [3H]-D2S to freshly isolated PBMN cells (Bmax 0.18 +/- 0.03 pmol/10(6) cells) and these cells could not be infected by HIV-1. Culture of PBMN cells in lymphocyte growth medium (LGM) containing IL-2 did not significantly change the Bmax of [3H]-D2S. In contrast, PBMN cells which had been cultured with phytohaemagglutinin (PHA; 5 micrograms ml-1) for 72 h had a Bmax of [3H]-D2S binding of 7.2 +/- 0.1 pmol/10(6) cells and these cells could be infected by HIV-1. Removal of the PHA and further culture of the PBMN cells in LGM containing IL-2 resulted in a fall in the Bmax to 2.0 +/- 0.1 pmol/10(6) cells. The Kd of binding did not change significantly during the course of these experiments.4. [3H]-D2S did not bind to freshly isolated erythrocytes or to erythrocytes which had been cultured in PHA for 72 h.5. These results suggest that there is a relationship between the expression of the [3H]-D2S binding protein on the plasma membrane of PBMN cells and the susceptibility of these cells to infection by HIV- 1.

Synthesis of branched cyclomalto-oligosaccharides using Pseudomonas isoamylase.

Branched cyclomalto-oligosaccharides (cyclodextrins) were synthesised from cyclomalto-oligosaccharides and maltose or maltotriose through the reverse action of Pseudomonas isoamylase. The reaction rate was greater with maltotriose than with maltose, and with increasing size of the cyclomalto-oligosaccharide (cG6 less than cG7 less than cG8). Maltotriose is effective as both a side-chain donor and acceptor, and three isomers of 6-O-alpha-maltotriosylmaltotriose (branched G6) were formed through mutual condensation, but maltose was effective only as a side-chain donor. Each branched cyclomalto-oligosaccharide and G6 was purified by liquid chromatography, and their structures were determined by chemical, enzymic, and 13C-n.m.r. spectroscopic analyses.

Porcine-pancreatic alpha amylase hydrolysis of substrates containing 6-deoxy-D-glucose and 6-deoxy-6-fluoro-D-glucose and the specificity of subsite binding.

Hydrolysis of 6-deoxyamylose and mono-6-deoxy-6-fluorocyclomaltoheptaose by porcine-pancreatic alpha amylase produces low-molecular-weight modified products, which have been analyzed by chemical and chromatographic techniques. Results for both substrates show that modified D-glucose and two isomers of modified maltoses are produced in the enzyme reaction. In addition, the formation of maltoses modified in the nonreducing residue is more favored than the formation of maltoses modified in the reducing residue. These results indicate that productive binding of 6-fluoro- and 6-deoxy-D-glucose residues is permitted at subsites 1 through 4 of the amylase-active site but that binding of these modified residues may be less favorable at subsite 3, the subsite at which catalytic attack occurs.

Structure studies of amylose-V complexes and retrograded amylose by action of alpha amylases, and a new method for preparing amylodextrins.

Human-salivary, porcine-pancreatic, and Bacillus subtilis alpha amylases were used to study the structure of amylose-V complexes with butyl alcohol, tert-butyl alcohol, 1,1,2,2-tetrachloroethane, and 1-naphthol, and of retrograded amylose. Alpha amylase hydrolyzes the amorphous, folding areas on the surfaces of the lamella of packed helices, with the formation of resistant, amylodextrin fragments. Their degree of polymerization (d.p.) corresponds to the diameter of the helices and the folding length of the chain. The resistant fragments were fractionated on a column of Bio-Gel A-0.5m. Gel filtration of human-salivary and porcine-pancreatic alpha amylase hydrolyzates gave resistant fragments whose peak fractions, i.e., the three pooled fractions from the gel-filtration column with the highest amount of carbohydrate, had a d.p. of 75 +/- 4 for the amylose complex with butyl alcohol, 90 +/- 3 for those with tert-butyl alcohol and tetrachloroethane, and 123 +/- 2 for that with 1-naphthol. These d.p. values correspond to helices of six residues per turn with a folding length of 10 nm, seven residues per turn with a folding length of 10 nm, and eight residues per turn with a folding length of 12 nm (or nine residues per turn with a folding length of 10 nm), respectively. Acid hydrolysis of retrograded amylose gave a resistant fragment having an average d.p. of 32, human-salivary and porcine-pancreatic alpha amylases gave a resistant fragment of d.p. 43, and Bacillus subtilis alpha amylase gave a resistant fragment of d.p. 50. A structure for retrograded amylose is proposed in which there are crystalline, double-helical regions that are 10 nm long, interspersed with amorphous regions. The amorphous regions are hydrolyzed by acid and by alpha amylases, leaving the crystalline regions intact. The differences in the sizes of the resistant amylodextrins depend on the differences in the specificities of the hydrolyzing agents: acid hydrolyzes right up to the edge of the crystalline region, whereas the alpha amylases hydrolyze up to some point several D-glucosyl residues away from the crystalline region, leaving "stubs" on the ends of the amylodextrins whose sizes are dependent on the sizes of the binding sites of the individual alpha amylases.(ABSTRACT TRUNCATED AT 400 WORDS)

The synthesis of an active derivative of cyclomaltoheptaose for the hydrolysis of esters and the formation of amide bonds.

The synthesis is described of a derivative of cyclomaltoheptaose (beta-cyclodextrin) to which the tripeptide Ser-His-Asp, the catalytic triad found in chymotrypsin, has been coupled. The derivative enhanced the rates of hydrolysis of activated esters, as measured by the release of p-nitrophenol, and the formation of amine bonds.

Synthesis and characterization of dextrin monosuccinate.

Reaction conditions, including reaction solvents, reaction time, reaction temperature, and molar ratio of succinic anhydride (SA) to anhydroglucose units (AGU) in dextrin, were investigated for preparing dextrin monosuccinate with high degree of substitution (DS). The results showed the optimum conditions as follows: Solvent, dimethyl sulfoxide; reaction temperature, 50°C; reaction time, 16 h; and molar ratio of SA to AGU in dextrin, 6:1. Under these conditions, the maximum DS reached 2.64. The chemical structure of dextrin monosuccinate was identified using FT-IR and (13)C NMR. The FT-IR data indicated the absorption bands of esters and carbonyl acids at 1726 and 1,574 cm(-1). Signals at 173.13, 171.81, 28.79, and 28.61 ppm in (13)C NMR spectrum were ascribed to carbons in ester, carbonyl acid, and methylene. These data suggest that the prepared dextrin succinate was monoester with functional carbonyl acid groups and could be used in polymer therapy as drug carriers.

Self-assembled nanogel of hydrophobized dendritic dextrin for protein delivery.

Highly branched cyclic dextrin derivatives (CH-CDex) that are partly substituted with cholesterol groups have been synthesized. The CH-CDex forms monodisperse and stable nanogels with a hydrodynamic radii of approximately 10 nm by the self-assembly of 4-6 CH-CDex macromolecules in water. The CH-CDex nanogels spontaneously trap 10-16 molecules of fluorescein isothiocyanate-labeled insulin (FITC-Ins). The complex shows high colloidal stability: no dissociation of trapped insulin is observed after at least 1 month in phosphate buffer (0.1 M, pH 8.0). In the presence of bovine serum albumin (BSA, 50 mg . mL(-1)), which is a model blood system, the FITC-Ins trapped in the nanogels is continuously released ( approximately 20% at 12 h) without burst release. The high-density nanogel structure derived from the highly branched CDex significantly affects the stability of the nanogel-protein complex.

Design of iodine-lithium-alpha-dextrin liquid crystal with potent antimicrobial and anti-inflammatory properties.

An ideal antimicrobial should be not toxic and possess board spectrum antiviral, antibacterial, antifungal activity, excluding resistance and should affect pathogen-mediated damage of host physiology including immune, nervous and endocrine systems. With the purpose of a combination of nonspecific antimicrobial action of molecular and ionized iodine with systemic immune-modulating property of the negatively charged polysaccharides a complex drug of iodine and lithium on a template of a alpha-dextrin liquid crystal was designed. The physicochemical model of iodine-lithium-alpha-dextrin (ILalphaD) is based on the human blood and the stereochemistry of moving equilibred systems of dynamically balanced organic polymers conformation complexed with the iodine and lithium molecules. Here we reviewed the antibacterial, antiviral, immune-modulating and anti-inflammatory mechanisms in vivo and in vitro as well as pharmacokinetics, metabolism, chronic toxicity, cumulative properties, embryo toxicity and carcinogenicity of ILalphaD. Clinical efficacy, tolerability and safety of ILalphaD monotherapy have been evaluated in HIV-infected patients, administered intravenously for a total of 12 infusions in 4 cycles. ILalphaD therapy contributes to anti-HIV and anti-inflammatory effects, resolution of dermatological and neurological pathology and dramatically improves the quality of life reflecting on enhanced treatment adherence. ILalphaD appears to be safe and perspective for an adjuvant therapy of bacterial and viral infections, including HIV/AIDS, hypothyroid, autoimmune and inflammatory diseases for controlling pathogen production from infected cells, immune response, inflammation and metabolism.

Synthesis and NMR characterization of beta-alanine-bridged hemispherodextrin, a very efficient chiral selector in EKC.

A capped derivative of beta-CD (THALAH) was synthesized and characterized by NMR spectroscopy at different pH values. A trehalose moiety, bonded through beta-alanine bridges to the CD cavity, is included in the capping unit, giving peculiar properties to this molecule. The hemispherodextrin thus obtained was tested as a chiral selector in EKC. At neutral pH, the monocationic species of THALAH behaves as a very efficient selector separating successfully all the 11 tested enantiomeric pairs of dansyl-derivatives of amino acids, some of them even at concentrations as low as 0.15 mM. The differences observed in the migration order among the different systems give suggestions about the mechanism of molecular recognition between the selector and the analytes.

Fluorogenic substrates of glycogen debranching enzyme for assaying debranching activity.

Glycogen debranching enzyme (GDE) degrades glycogen in concert with glycogen phosphorylase. GDE has two distinct active sites for maltooligosaccharide transferase and amylo-1,6-glucosidase activities. Phosphorylase limit dextrin from glycogen is debranched by cooperation of the two activities. Fluorogenic branched dextrins were prepared as substrates of GDE from pyridylaminated maltooctaose (PA-maltooctaose) and maltotetraose, taking advantage of the synthetic action of Klebsiella pneumoniae pullulanase. Their structures were as follows: Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4GlcPA (B3), Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B4), Glcalpha1-4Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B5), Glcalpha1-4Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B6), Glcalpha1-4(Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6)Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B7), and Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-6Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4Glcalpha1-4GlcPA (B8). These dextrins were incubated with porcine skeletal muscle GDE. No fluorogenic product was found in the digest of B8. The fluorogenic products from B3, B4, and B5 were PA-maltooctaose only. PA-maltooctaose, PA-maltoundecaose, and 6(7)-O-alpha-glucosyl-PA-maltooctaose were from B7. PA-maltooctaose and 6(6)-O-alpha-glucosyl-PA-maltooctaose were from B6. These results indicate that the maltooligosaccharide transferase removed the maltotriosyl residues from the maltotetraosyl branches by hydrolysis or intramolecular transglycosylation to expose 6-O-alpha-glucosyl residues, and then the amylo-1,6-glucosidase hydrolyzed the alpha-1,6-glycosidic linkages of the products rapidly. Probably, 6-O-alpha-glucosyl-PA-maltooctaoses from B7 and B6 were less susceptible to the amylo-1,6-glucosidase than were those from B3, B4, and B5. Taking this into account, B3, B4, and B5 are suitable substrates for GDE assay.

Pharmaceutical usefulness of hydroxypropylcyclodextrins: "e pluribus unum" is an essential feature.

A series of hydroxypropyl-beta-cyclodextrins was prepared by a method that leads to a preferential substitution on the secondary hydroxyls, mainly O-2, of beta-cyclodextrin with (S)-2-hydroxypropyl groups. The series consisted of mixtures of compounds with average degrees of substitution of 8, 3, and 1.6 and of a specially isolated monosubstituted compound; thus, the number of components progressively decreased in this series. The crystallinity in the series increased progressively, the first member being fully amorphous and the last one fully crystalline. All members of the series formed clear aqueous solutions at concentrations of greater than 50.0, 2.0, 0.6, and 0.3%, respectively. Therefore, pharmaceutically useful hydroxypropylcyclodextrin preparations are those containing a large number of chemically individual compounds--a feature resulting in an amorphous state and high water solubility.

Photoinduced electron transfer in a supramolecular species building of mono-6-p-nitrobenzoyl-beta-cyclodextrin with naphthalene derivatives.

Photoinduced electron transfer was observed in the supramolecular complexes of p-nitrobenzoyl-beta-cyclodextrin (NBCD) with a number of naphthalene derivatives, which were stabilized clearly via hydrophobic interactions in aqueous solution. Both steady-state and time-resolved fluorescence measurements were conducted, which revealed that there were two routes of electron transfer, i.e., electron transfer between the free donor and free acceptor in solution and electron transfer between the donor and acceptor bound in a supramolecular assembly. The evidence collected demonstrates that the latter route was very efficient. As a result, the rate and quantum yield of the fluorescence quenching in the present supramolecular system were appreciably large.