Toru Mimura

Synthesis of curdlan sulfates having inhibitory effects in vitro against AIDS viruses HIV-1 and HIV-2

Nucleoside analogues such as azidothymidine (AZT), dideoxyinosine (ddI), and dideoxycytidine (ddC) [1] inhibit the viral reverse transcriptase (RT) of human immunodeficiency virus (HIV) and terminate DNA chain synthesis from viral RNA inside the infected cells. However, these nucleosides manifest serious side-effects such as bonemarrow toxicity and the appearance of AZT-resistant viruses upon long-term treatment [2]. It is difficult to make an effective vaccine owing to the multiplicity of AIDS viruses. Therefore, anti-AIDS compounds having a different blocking mechanism are of interest. We have synthesized sulfated polysaccharides demonstrating high anti-AIDS virus activity by sulfation of synthetic and natural-occurring polysaccharides [3-6]. The mechanism of action of sulfated polysaccharides is assumed to be different from that of the nucleosides, that is, they bind to HIV virions and prevent them from penetrating into target cells [7-9]. However, sulfated polysaccharides such as dextran sulfate have high blood anticoagulant activity, leading to undesirable side-effects when used as anti-AIDS drugs [10].

Synthesis, structure and antiviral activity of sulfates of cellulose and its branched derivatives

Abstract Cellulose and branched cellulose having d -glucopyranose or d -galacto-pyranose as side chains were sulfated with piperidine N-sulfonic acid or SO3-dimethylformamide complex. The structures of the sulfated polysaccharides were analysed by elemental analysis and 13[C]-NMR spectroscopy. The 13[C]-NMR spectra of cellulose sulfate were assigned and degree of substitution values of each hydroxyl group were obtained. The reactivity was in the order C-6 ⪢ C-2> C-3. Sulfated polysaccharides with high degree of substitution values showed high anti-human-immuno-deficiency-virus activity.

Synthesis of polymethacrylate derivatives having sulfated maltoheptaose side chains with anti‐HIV activities

Anti-HIV (human immunodeficiency virus) active polymethacrylates having pendant sulfated oligosaccharides were synthesized, and the relationship between structures and biological activities of the polymethacrylates was examined. Acetylated 1-O-methacryloyl maltoheptaoside (MA-AcM7) was polymerized with AIBN as an initiator to give polymethacrylates having a pendant acetylated maltoheptaose in every repeating unit, poly(MA-AcM7)s. After hydroxyl groups were recovered by deacetylation, the polymethacrylates having maltoheptaose units, poly(MA-M7)s, were sulfated to give polymethacrylates having sulfated maltoheptaose side-chains, poly(MA-SM7)s, with degrees of sulfation of 1.1 to 2.7 (maximum, 3.0). These polymethacrylates including sulfated oligosaccharides exhibited low anti-HIV activities represented by the 50% protecting concentration (EC 50 ) in the range of 15-62 μg/mL and low blood anticoagulant activities around 10 unit/mg (standard dextran sulfate, 22.7 unit/mg). The anti-HIV activity increased with increasing degree of sulfation to reach EC 50 of 15-16 μg/mL. In addition, copolymerization of MA-AcM7 with methyl methacrylate (MMA) and subsequent sulfation gave polymethacrylates consisting of various proportions of highly sulfated maltoheptaose and MMA units. It was revealed that the anti-HIV activity increased with decreasing proportion of the sulfated oligosaccharide moiety and that a copolymethacrylate having 22 mol % of sulfated maltoheptaose units (DS = 3.0) had a high anti-HIV activity in the EC 50 of 0.3 μg/mL. The blood anticoagulant activity increased slightly from 9 to 18 unit/mg with decreasing proportion of the sulfated maltoheptaose units. These results suggested that the biological activities were influenced strongly by the spatial distance between sulfated oligosaccharide substituents in the polymethacrylate main chain. Distinction and conformation of the oligosaccharide side chains also played an important role.

SYNTHESIS OF SULFONATED AMINO-POLYSACCHARIDES HAVING ANTI-HIV AND BLOOD ANTICOAGULANT ACTIVITIES

Sulfonated 3-amino-3-deoxy-(1-->6)-alpha-D-allopyranans were synthesized to elucidate the relationship between structure and such specific biological activities as anti-HIV and blood anticoagulant activities. Ring-opening copolymerization of 1,6-anhydro-3-azido-2,4-di-O-benzyl-3-deoxy-beta-D-allopyranose 1 with 1,6-anhydro- 2,3,4-tri-O-benzyl-beta-D-allopyranose 2 with PF5 catalyst gave copolymers having various proportions of the 1 unit. 13C NMR spectroscopy showed the resulting copolymers to have alpha-(1-->6)-stereoregularity, and the monomer reactivity ratios were calculated to be r1 = 0.92 and r2 = 1.11 by the Kelen-Tüdös method. Reduction of the azido groups and subsequent debenzylation of the copolymers afforded new amino-polysaccharides consisting of 3-amino-3-deoxy-allose and allose units. Sulfonation of 3-amino-3-deoxy-(1-->6)-alpha-D-allopyranan and copolysaccharides consisting of 3-amino-allose and allose or glucose units was performed with piperidine-N-sulfonic acid to give polysaccharides containing sulfoamido and sulfonate groups in good yields. The sulfoamido-copolysaccharides containing of 3-amino-3-deoxy-allose and glucose units exhibited high anti-HIV activities manifested by the 50% protecting concentration (EC50) of 0.2-0.5 mg/mL and low cytotoxicity shown by a 50% cytotoxic concentration (CC50) of > 1000 micrograms/mL. The sulfoamido-copolysaccharides containing 3-amino-allose and allose units had somewhat lower anti-HIV activities (EC50 = 0.8-0.9 microgram/mL) and slightly higher cytotoxicities (CC50 = 740-797 micrograms/mL). In addition, it was found that the blood anti-coagulant activity increased, with increasing proportion of the amino-allose unit, from 30 to 58 unit/mg (standard dextran sulfate, 22.7 unit/mg). These results suggest that the sulfonated 3-amino-3-deoxyallose unit plays important roles on both anti-HIV and blood anticoagulant activities.

Biosynthesis of curdlan from culture media containing 13C-labeled glucose as the carbon source

13C-Labeled curdlans were biosynthesized by Agrobacterium sp. (ATCC 31749) from culture media containing D-(1-13C)glucose, D-(6-13C)glucose, or D-(2-13C)glucose as the carbon source, and their structures were analyzed by 13C NMR spectroscopy. The labeling was mainly found in the original position, that is, C-1, C-6, or C-2, indicating direct polymerization of introduced glucose. In addition, C-3 in curdlan obtained from D-(1-13C)glucose, C-1 in curdlan obtained from D-(6-13C)glucose, and C-1 and C-3 in curdlan obtained from D-(2-13)glucose were labeled. From analysis of this labeling, the biosynthesis of curdlan was interpreted as involving five routes: (1) direct synthesis from glucose; (2) rearrangement (1-13C-->3-13C); and (3) isomerization (6-13C-->1-13C) of cleaved trioses by the Embden-Meyerhof pathway, followed by neogenesis of glucose and formation of curdlan; (4) from fructose 6-phosphate formed in the pentose cycle (2-13C-->1-13C, 3-13C); and (5) neogenesis of glucose from fragments produced in various pathways of glycolysis. The 13C-labeling at C-6 and C-2 in the starting glucoses is well preserved in the C-6 carbon and the C-1 to C-3 carbons, respectively, in the curdlan produced.

Dietary aspartame with protein on plasma and brain amino acids, brain monoamines and behavior in rats

Aspartame (APM; L-aspartyl-L-phenylalanine methyl ester), was investigated for its ability to alter levels of the large neutral amino acids and monoamines in overnight fasted rats allowed to consume meals with or without protein for two hours. Additionally, the possible long term behavioral consequences of APM in 25% casein diets with or without 10% sucrose were determined. Acute APM ingestion increased both plasma and brain phenylalanine and tyrosine levels, but brain tryptophan levels were not altered regardless of dietary protein. Brain norepinephrine and dopamine levels were unaltered by any of the diet while serotonin levels were slightly increased when a protein-free diet was consumed. But APM and/or protein ingestion minimized this increase of brain serotonin levels as much as controls. Chronic APM ingestion failed to influence diurnal feeding patterns, meal size distributions, or diurnal patterns of spontaneous motor activity. The chronic ingestion of abuse doses of APM produced no significant chemical changes in brain capable of altering behavioral parameters believed to be controlled by monoamines in rats.

A strong inhibition of HIV-induced cytopathic effects by synthetic (1→6)-α-d-mannopyranan sulfate☆

Abstract The anti-HIV effects of mannopyranan sulfate ( 1 ) were investigated by using MT-4 cells, namely, an HTLV-I-carrying CD-4 positive cell-line, in vitro . Stereoregular (1→6)-α- d -mannopyranan, which had been synthesized by ring-opening polymerization of a 1,6-anhydromannose derivative, was sulfated with piperidine- N -sulfonic acid to provide 1 . N.m.r. analysis of 1 indicated that the reactivity of hydroxyl groups was in the order, 3-OH > 2-OH > > 4-OH. Mannopyranan sulfate having degree of substitution (d.s.) of 1.19–1.83 effectively inhibited HIV-induced cytopathic effects at a concentration of >3.3 μg/mL. The anticoagulant activity and the adsorption on concanavalin A of 1 indicated the possibility of selective binding of 1 having d.s. of 1.19–1.83 to HIV-protein.

Synthesis of sulfated octadecyl ribo-oligosaccharides with potent anti-AIDS virus activity by ring-opening polymerization of a 1,4-anhydroribose derivative

The synthesis and anti-AIDS virus activity of sulfated octadecyl ribofuranans with medium-range molecular weights have been investigated. Selective ring-opening polymerization of 1,4-anhydro-2,3-di-O-benzyl-alpha-D-ribopyranose with 10-20 mol% of boron triflouride etherate as a catalyst in a large amount of dichloromethane gave 2,3-di-O-benzyl-(1-->5)-alpha-D-ribofuranan in good yield. The molecular weight of the benzylated ribofuranan was in the range of 9 x 10(3) to 10 x 10(3). Debenzylation of the polymer followed by acetylation gave peracetylated (1-->5)-alpha-D-ribofuranans. The peracetylated ribofuranans were treated with octadecyl alcohol and a stannic chloride catalyst to afford acetylated ribofuranans having octadecyl groups at the reducing terminal. The molecular weights of the resulting acetylated octadecyl ribofuranans were below 9 x 10(3). Sulfation of the deacetylated octadecyl ribofuranans by piperidine-N-sulfonic acid in dry Me2SO gave sulfated octadecyl ribofuranans with molecular weights of 3 x 10(3) to 9 x 10(3) and sulfur contents of 13.0-16.2%. The sulfated octadecyl ribofuranans had potent anti-AIDS virus activity, EC50 = 0.6-2.5 micrograms/mL (a standard curdlan sulfate showed EC50 = 0.43 micrograms/mL), and low anticoagulant activity, 4-17 units/mg (a standard dextran sulfate, 22.7 unit/mg). Structural analysis of the ribofuranans was performed by NMR at 400 and 600 MHz.

Effect of Degree of Sulfation on Anti-Hiv Activity of Synthetic (1→5)-α-D-Ribofuranan Sulfate

ABSTRACT Stereoregular (1→5)-α-D-ribofuranan prepared by selective ring-opening polymerization of anhydro-ribose derivatives was sulfated with piperidine N-sulfonic acid to give ribofuranan sulfates (RFS) with various degrees of sulfation (DS). NMR analysis of RFS indicated a similar reactivity of two hydroxyl groups (2-OH and 3-OH). Anti-HIV effects of RFS were investigated by using MT-4 cells, i.e., an HTLV-I-carrying CD-4 positive cell line in vitro. RFS with higher DS showed remarkably higher anti-HIV activity. Anticoagulant activity of RFS was also investigated. RFS with a high DS interacted more selectively with virus protein rather than other proteins in the blood than RFS with a low DS.

Effect of mealing on plasma and brain amino acid, and brain monoamine in rats after oral aspartame.

Aspartame (APM; L-aspartyl-L-phenylalanine methyl ester) was investigated for its ability to alter brain amino acids and monoamines in overnight fasted rats allowed to consume commercial diets for 60 minutes. In addition, the effects of mealing on the changes in plasma and brain amino acids and brain monoamines induced by glucose and/or insulin, and known pharmacologically active compounds, were studied. The consumption of the commercial chow largely prevented changes in blood glucose and amino acids, and brain amino acids and the monoamines dopamine, norepinephrine and serotonin that might be expected to occur following glucose with or without insulin. Feeding failed to prevent changes in the above parameters when 5-hydroxy-tryptophan, p-chlorophenylalanine and reserpine were administered. The oral administration of up to 250 mg/kg BW APM with water or glucose followed by free feeding failed to alter brain monoamines. These studies demonstrate the potent ability of food to normalize biochemical parameters in blood and brain that otherwise might occur, and clearly show the lack of effect on brain monoamine levels of abuse doses of APM when administered with food.