Taku Chiba

Application of a radical reaction to the synthesis of L-iduronic acid derivatives from D-glucuronic acid analogues

Abstract Commercially available d -glucofuranurono-6,3-lactone was transformed into the known, crystalline methyl (5R)-1,2,3,4-tetra-O-acetyl-5-C-bromo-β- d -glucopyranuronate (3) in three steps. Reduction with tributyltin hydride gave crystalline methyl 1,2,3,4-tetra-O-acetyl-α- l -idopyranuronate (4) in ∼30% yield, together with the crystalline methyl 1,2,3,4-tetra-O-acetyl-β- d -glucopyranuronate (1, 63.5%) which may be separated and converted back by Ferriers photobromination into 3. This procedure provides the first practical and expeditious conversion of d -glucuronic acid into d -iduronic acid by epimerization. Acetate 4 was converted in quantitative yield into methyl (2,3,4-tri-O-acetyl-α- l -idopyranosyl bromide)-uronate (22), and then into methyl 3,4-di-O-acetyl-β- l -idopyranuronate 1,2-(methyl orthoester) (23), which are useful compounds for glycosylation reactions. Various β- d -glucuronic acid derivatives have been epimerized to α- l -iduronic acid analogues by this novel procedure.

Multiple mechanisms involved in the inhibition of proinflammatory cytokine production from human monocytes by N-(p-coumaroyl)serotonin and its derivatives.

We have reported that N-(p-coumaroyl)serotonin(CS) isolated from safflower oil cake (Carthamus tinctorius L.) inhibits the production of proinflammatory cytokines by endotoxin (LPS)- stimulated human monocytes. In this study, the effects of CS and its three derivatives, N-(trans-cinnamoyl)serotonin (Cin.S), N-(trans-cinnamoyl)tryptamine (Cin.T), and N-(p-coumaroyl)tryptamine (CT) on the production of proinflammatory cytokines were compared. Cin.S possessed radical scavenging activity at a comparable level to CS, while CT and Cin.T exhibited lower activity, suggesting that hydroxyl group in serotonin is essential for the antioxidative activity. CS and CT strongly inhibited the production of proinflammatory cytokines (IL-1alpha, IL-1beta, IL-6, IL-8, and TNF-alpha) from LPS-stimulated human monocytes. However, Cin.S inhibited the production of only IL-1alpha and IL-1beta, and Cin.T inhibited none of these cytokines production. CS and CT markedly inhibited the protein synthesis in monocytes, the inhibitory effect of Cin.S was moderate, and that of Cin.T was quite weak. These results indicate that CS and its derivatives inhibit the production of proinflammatory cytokines through multiple mechanisms.

Chemical synthesis of l-iduronic acid-containing disaccharidic fragments of heparin ☆ ☆☆

Abstract Condensation of methyl 3- O -benzyl-2-benzyloxycarbonylamino-6- O -chloro-acetyl-2-deoxy-α- d -glucopyranoside with methyl (2,3,4-tri- O -acetyl-α- l -idopyranosyl bromide)uronate in 1,2-dichloroethane, in the presence of silver triflate and molecular sieves, provided 83% of crystalline methyl 3- O -benzyl-2-benzyloxy-carbonylamino-6- O -chloroacetyl-2-deoxy-4- O -(methyl 2,3,4-tri- O -acetyl-α- l -idopyranosyluronate)-α- d -glucopyranoside. Dechloroacetylation followed successively by O -sulfation with the sulfur trioxide-trimethylamine complex, acetylation, and saponification gave the disodium salt of methyl 2-acetamido-2-deoxy-4- O -(α- l -idopyranosyluronic acid)-6- O -sulfo-α- d -glucopyranoside. The N -sulfated analogue of this disaccharide was also synthesized, the acetylation being replaced by a selective N -sulfation. Condensation of methyl (methyl 2,3-di- O -benzyl-β- l -idopyranosid)uronate with 6- O -acetyl-2-azido-3,4-di- O -benzyl-2-deoxy-α- d -glucopyranosyl bromide in dichloromethane, in the presence of silver triflate and 2,4,6-trimethylpyridine, gave methyl [methyl 4- O -(6- O -acetyl-2-azido-3,4-di- O -benzyl-2-deoxy-α- d -glucopyranosyl)-2,3-di- O -benzyl-β- l -idopyranosid]uronate. Saponification followed successively by esterification, O -sulfation, saponification, catalytic hydrogenolysis, and selective N -sulfation gave the trisodium salt of methyl 4- O -(2-deoxy-6- O -sulfo-2-sulfoamino-α- d -glucopyranosyl)-β- l -idopyranosiduronic acid.

Differences in interleukin 1 (IL-1), IL-6, tumor necrosis factor and IL-1 receptor antagonist production by human monocytes stimulated with muramyl dipeptide (MDP) and its stearoyl derivative, romurtide

The immunostimulatory reagents muramyl dipeptide (MDP) and its stearoyl derivative romurtide [MDP-Lys(L18)] were assessed for cytokine inducing activity in human monocytes. Both MDP and romurtide stimulated the production of interleukin-1 (IL-1), IL-6, tumor necrosis factor (TNF) and IL-1 receptor antagonist (IL-1Ra). Kinetics study indicated that IL-1, TNF and IL-1Ra were induced after 4 h stimulation but IL-6 was produced at a later phase. Romurtide induced these cytokines for longer period that MDP. Dose-response study indicated that romurtide was far more potent than MDP in induction of IL-1, IL-6 and TNF. Although the magnitude of the IL-1 and IL-6 induction was almost the same, that of TNF induction was greater in romurtide-stimulated monocytes than in MDP-stimulated cells. Among IL-1, IL-1 beta appeared to be a major product. In contrast to other cytokines, IL-1Ra was induced by MDP and romurtide in a similar dose and time dependent manner with similar magnitude of response. These studies indicate that MDP and romurtide, especially romurtide, are very potent inducers of both immunostimulatory and immunosuppressive cytokines by human monocytes but with different efficacy and kinetics.

Synthesis of new sugar derivatives and evaluation of their antibacterial activities against Mycobacterium tuberculosis.

A series of sugar derivatives (1-13) were synthesized and evaluated for antibacterial activity against Mycobacteriumtuberculosis (MTB), especially multi-drug resistant (MDR) MTB, and the structure-activity relationships of these compounds were studied. The results showed that the compound OCT313 (2-acetamido-2deoxy-beta-D-glucopyranosyl N,N-dimethyldithiocarbamate) (4) exhibited significant in vitro bactericidal activity, and that the dithiocarbamate group at C-1 position of the glucopyranoside ring was requisite for the antibacterial activity.

Simple Fibroblast-Based Assay for Screening of New Antimicrobial Drugs against Mycobacterium tuberculosis

ABSTRACT In this study, we propose a simple and reproducible host-cell-based assay for the screening of antimycobacterial drugs that is suitable for drug discovery. The method evaluates both antimycobacterial activity of the drugs and their cytotoxicity to host cells. The basis of this simple fibroblast-based assay (SFA) is that cells of human lung fibroblast cell line MRC-5, which are highly sensitive to mycobacterial cytotoxicity, are killed by virulent Mycobacterium tuberculosis strain H37Rv bacilli in response to the viability of bacilli. Clinically used antimycobacterial drugs inhibited the mycobacterial cytotoxicity to MRC-5 cells in a dose-dependent manner. MICs of isoniazid, streptomycin, rifampin, and ethambutol determined by this SFA (0.428, 1.816, 0.013, and 3.465 μg/ml, respectively) were within 1 log of MICs determined by the broth dilution test (BDT) using Middlebrook 7H9 medium. The MIC of pyrazinamide, which exhibits bactericidal activity only at a high dose by BDT (1,231 μg/ml at pH 6.6 and 492 μg/ml at pH 5.8), was 3.847 μg/ml in the modified method of SFA. On the other hand, sodium azide, a toxic agent for both mammalian cells and bacteria, exhibited cytotoxicity to fibroblasts at a dose lower than that required to inhibit mycobacterial growth. Thus, this fibroblast-based method enabled us to evaluate both antibacterial activity of drugs and their cytotoxicity to human cells within a short period of time.

Synthesis and evaluation of anti-tubercular activity of new dithiocarbamate sugar derivatives.

The present study was undertaken to optimize the anti-tubercular activity of 2-acetamido-2-deoxy-β-D-glucopyranosyl N,N-dimethyldithiocarbamate (OCT313, Glc-NAc-DMDC), a lead compound previously reported by us. Structural modifications of OCT313 included the replacements of the DMDC group at C-1 by pyrrolidine dithiocarbamate (PDTC) and the acetyl group at C-2 by either propyl, butyl, benzyl or oleic acid groups. The antimycobacterial activities of these derivatives were evaluated against Mycobacterium tuberculosis (MTB). Glc-NAc-pyrrolidine dithiocarbamate (OCT313HK, Glc-NAc-PDTC) exhibited the most potent anti-tubercular activity with the minimal inhibitory concentration (MIC) of 6.25-12.5 μg/ml. The antibacterial activity of OCT313HK was highly specific to MTB and Mycobacterium bovis BCG, but not against Mycobacterium avium, Mycobacterium smegmatis, Staphylococcus aureus or Escherichia coli. Importantly, OCT313HK was also effective against MTB clinical isolates, including multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains. Interestingly, OCT313HK was exerted the primary bactericidal activity, and it was also exhibited the bacteriolytic activity at high concentrations. We next investigated whether the mycobacterial monooxygenase EthA, a common activator of thiocarbamide-containing anti-tubercular drugs, also activated OCT313HK. Contrary to our expectations, the anti-tubercular activity of dithiocarbamate sugar derivatives and dithiocarbamates were not dependent on ethA expression, in contrast to thiocarbamide-containing drugs. Overall, this study presents OCT313HK as a novel and potent compound against MTB, particularly promising to overcome drug resistance.

CHOP, a basic leucine zipper transcriptional factor, contributes to the antiproliferative effect of IL-1 on A375 human melanoma cells through augmenting transcription of IL-6.

Interleukin-1 (IL-1) inhibits the proliferation of A375 human melanoma cells. We have demonstrated previously that p38 mitrogen-activated protein kinase (MAPK) mediated the antiproliferative effect of IL-1 partially through the downregulation of activity and protein level of ornithine decarboxylase (ODC). In this study, we investigated the role of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), one of the p38 MAPK target transcriptional factors. The mRNA level of CHOP was not affected by IL-1 treatment in A375-6 cells. Unexpectedly, CHOP was constitutively phosphorylated, and IL-1 or p38 MAPK inhibitor, SB203580, did not affect the phosphorylation level. However, A375-6 cells exhibited enhanced sensitivity to IL-1 by transfecting CHOP expression plasmid and reduced sensitivity to IL-1 by antisense CHOP mRNA expression plasmid. Furthermore, CHOP appeared to regulate positively IL-6 production at the transcriptional level. The experiments using CHOP muteins revealed that dimerization ability - but not p38 MAPK-dependent phosphorylation or DNA binding activity - is important for the IL-6 inducing activity of CHOP. These results indicate that CHOP contributes to the IL-1 growth-inhibitory signal through augmenting IL-6 production.

Liquid chromatographic-atmospheric pressure chemical ionization mass spectrometric analysis of glycine conjugates and urinary isovalerylglycine in isovaleric acidemia

n-Acetylglycine, n-propionylglycine, n-butyrylglycine, isobutyrylglycine, n-valerylglycine, isovalerylglycine, heptanoylglycine, phenylacetylglycine and isovalerylglucuronide were identified based on their liquid chromatographic-atmospheric pressure chemical ionization mass spectra (LC-APCI-MS). We were able to detect the presence of urinary isovalerylglycine in two cases of isovaleric acidemia using LC-APCI-MS. Membrane-filtered urine samples were injected into the LC-APCI-MS system in the negative-ion mode without any further pretreatment, and large amounts of isovalerylglycine were detected as the [M-H]- ion. The urinary excretion of isovalerylglycine appeared to increase after L-carnitine therapy. This analytical method is quick and easy and it may be a useful tool in understanding dysfunctional conditions in isovaleric acidemia.

Studies on the reactivities of the secondary hydroxyl groups in 1,6-anhydro-4′,6′-o-benzylidene-β-lactose by selective benzoylation☆

Abstract Selective benzoylation of 1,6-anhydro-4′,6′- O -benzylidene-β-lactose ( 1 ), using 2.1 molar equivalents of benzoyl chloride in pyridine at — 20°, yielded five benzoates which were designated 2 to 6 in order of decreasing R F value on t.l.c. After column chromatography on silica gel, compounds 2 – 6 were separated as the 2,2′,3,3′-tetra-benzoate ( 2 , 3%), 2,3,3′-tribenzoate ( 3 , 11%), 2,2′,3′-tribenzoate ( 4 , 5%), 2,3′-dibenzoate ( 5 , 30%), and 3′-benzoate ( 6 , 22%), respectively. Selective benzoylation of 5 , using 1.1 molar equivalents of benzoyl chloride, afforded 2 , 3 , and 4 in yields of 15, 56, and 8%, respectively, together with 5% of 5 . Thus, the order of reactivities of the secondary hydroxyl groups in 1 is 3′>3>2′. Compounds 3 – 6 have potential value in the chemical modification of lactose or the synthesis of lactose-containing oligosaccharides.