d-Glucose- and d-mannose-based antimetabolites. Part 2. Facile synthesis of 2-deoxy-2-halo-d-glucoses and -d-mannoses

Modified d-glucose and d-mannose analogs are potentially clinically useful metabolic inhibitors. Biological evaluation of 2-deoxy-2-halo analogs has been impaired by limited availability and lack of efficient methods for their preparation. We have developed practical synthetic approaches to 2-deoxy-2-fluoro-, 2-chloro-2-deoxy-, 2-bromo-2-deoxy-, and 2-deoxy-2-iodo derivatives of d-glucose and d-mannose that exploit electrophilic addition reactions to a commercially available 3,4,6-tri-O-acetyl-d-glucal.     

Using S-adenosyl-l-homocysteine capture compounds to characterize S-adenosyl-l-methionine and S-adenosyl-l-homocysteine binding proteins

S-Adenosyl-l-methionine (SAM) is recognized as an important cofactor in a variety of biochemical reactions. As more proteins and pathways that require SAM are discovered, it is important to establish a method to quickly identify and characterize SAM binding proteins. The affinity of S-adenosyl-l-homocysteine (SAH) for SAM binding proteins was used to design two SAH-derived capture compounds (CCs). We demonstrate interactions of the proteins COMT and SAHH with SAH–CC with biotin used in conjunction with streptavidin–horseradish peroxidase. After demonstrating SAH-dependent photo-crosslinking of the CC to these proteins, we used a CC labeled with a fluorescein tag to measure binding affinity via fluorescence anisotropy. We then used this approach to show and characterize binding of SAM to the PR domain of PRDM2, a lysine methyltransferase with putative tumor suppressor activity. We calculated the K_d values for COMT, SAHH, and PRDM2 (24.1 ± 2.2 μM, 6.0 ± 2.9 μM, and 10.06 ± 2.87 μM, respectively) and found them to be close to previously established K_d values of other SAM binding proteins. Here, we present new methods to discover and characterize SAM and SAH binding proteins using fluorescent CCs.     

d-Polyglutamine Amyloid Recruits l-Polyglutamine Monomers and Kills Cells

Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases such as Huntington's disease. Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either l- or d-polyQ peptides and found that d-fibrils are as cytotoxic as l-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced l-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized d- and l-polyQ in vitro. We found that, as expected, d-polyQ monomers are not recognized by proteins that recognize l-polyQ monomers. However, amyloid fibrils prepared from d-polyQ peptides can efficiently seed the aggregation of l-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a “rippled β-sheet” interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins.     

Electric field-induced orientation of l- and dl-phosphatidylcholine bilayers

Membrane orientation induced by an alternating electric field has been examined for the l-enantiomer and racemic dipalmitoylphosphatidylcholine (DPPC) bilayers. The orientation effect was measured by bending curvature of hairpin-like deformation of the multilamellar cylindrical tubes with varying field-strength, frequency and tube size. It has been observed that both l- and dl-DPPC tubes are similar in the profiles of field-strength dependence and frequency dependence on the curvature deformation, but different in the deformed curvatures. dl-DPPC tubes deform largely as compared with l-DPPC tubes. The square of the deformed curvature of dl-DPPC tubes is larger than that of l-DPPC by about 37% on average. The result indicates that the racemic membrane is responsive to the electric field as compared with the l-enantiomer membrane. This suggests that a hybrid arrangement of head groups of the racemic lipid leads an effective response of the membrane due to the head group orientation.     

Steady state kinetics of soluble and membrane-bound mitochondrial ATPase

Steady state kinetic measurements of the rate of hydrolysis of ATP to ADP and inorganic phosphate by beef heart mitochondrial ATPase have been performed with both the solubilized enzyme and with the enzyme attached to a mitochondrial membrane fraction at 25° in 0.1 M NaCl with Mg²⁺ as the metal ion activator. These studies indicate the ATP Michaelis constants are somewhat larger for the soluble enzyme and the turnover numbers are considerably larger. In addition, the steady state parameters are essentially independent of pH over the range 7–9 for the membrane-bound enzyme, while the turnover number for the soluble enzyme varies considerably with pH. The product, ADP, is a competitive inhibitor of ATP and inhibits the soluble enzyme much more strongly than the membrane-bound enzyme. Oligomycin inhibits the membrane-bound enzyme very strongly, but has no effect on the activity of the soluble enzyme. The oligomycin inhibition is noncompetitive in nature.     

Single-step bioconversion for the preparation of l-gulose and l-galactose

Both carbohydrate monomers l-gulose and l-galactose are rarely found in nature, but are of great importance in pharmacy R&D and manufacturing. A method for the production of l-gulose and l-galactose is described that utilizes recombinant Escherichia coli harboring a unique mannitol dehydrogenase. The recombinant E. coli system was optimized by genetic manipulation and directed evolution of the recombinant protein to improve conversion. The resulting production process requires a single step, represents the first readily scalable system for the production of these sugars, is environmentally friendly, and utilizes inexpensive reagents, while producing l-galactose at 4.6 g L⁻¹ d⁻¹ and l-gulose at 0.90 g L⁻¹ d⁻¹.     

Triton x-100 inhibits agonist-induced currents and suppresses benzodiazepine modulation of GABAA receptors in Xenopus oocytes

Changes in lipid bilayer elastic properties have been proposed to underlie the modulation of voltage-gated Na⁺ and L-type Ca²⁺ channels and GABA_A receptors by amphiphiles. The amphiphile Triton X-100 increases the elasticity of lipid bilayers at micromolar concentrations, assessed from its effects on gramicidin channel A appearance rate and lifetime in artificial lipid bilayers. In the present study, the pharmacological action of Triton-X 100 on GABA_A receptors expressed in Xenopus laevis oocytes was examined. Triton-X 100 inhibited GABA_A α₁β₃γ_2S receptor currents in a noncompetitive, time- and voltage-dependent manner and increased the apparent rate and extent of desensitization at 10 μM, which is 30 fold below the critical micelle concentration. In addition, Triton X-100 induced picrotoxin-sensitive GABA_A receptor currents and suppressed allosteric modulation by flunitrazepam at α₁β₃γ_2S receptors. All effects were independent of the presence of a γ_2S subunit in the GABA_A receptor complex. The present study suggests that Triton X-100 may stabilize open and desensitized states of the GABA_A receptor through changes in lipid bilayer elasticity.     

Metabolism of 2-deoxy-d-glucose by baker's yeast : IV. Incorporation of 2-deoxy-d-glucose into cell wall mannan

1.

1. Saccharomyces cerevisiae grown in the presence of 2-deoxy-d-glucose incorporate this glucose and mannose analogue into cell wall polysaccharides. Fractionation of cell walls to mannan- and glucan-containing fractions followed by analysis for glucose, mannose and deoxyglucose showed that deoxyglucose was incorported mainly, if not exclusively, into cell wall mannan.     

Synthesis of 4-O-glycosylated 1,5-anhydro-d-fructose and of 1,5-anhydro-d-tagatose from a common intermediate 2,3-O-isopropylidene-d-fructose

Four novel disaccharides of glycosylated 1,5-anhydro-d-ketoses have been prepared: 1,5-anhydro-4-O-β-d-glucopyranosyl-d-fructose, 1,5-anhydro-4-O-β-d-galactopyranosyl-d-fructose, 1,5-anhydro-4-O-β-d-glucopyranosyl-d-tagatose, and 1,5-anhydro-4-O-β-d-galactopyranosyl-d-tagatose. The common intermediate, 1,5-anhydro-2,3-O-isopropylidene-β-d-fructopyranose, was prepared from d-fructose and was converted into the d-tagatose derivative by oxidation followed by stereoselective reduction to the 4-epimer. The anhydroketoses thus prepared were glycosylated and deprotected to give the disaccharides.     

Permeation and inhibition of polycystin-l channel by monovalent organic cations

Polycystin-L (PCL), homologous to polycystin-2 (71% similarity in protein sequence), is the third member of the polycystin family of proteins. Polycystin-1 and -2 are mutated in autosomal dominant polycystic kidney disease, but the physiological role of PCL has not been determined. PCL acts as a Ca-regulated non-selective cation channel permeable to mono- and divalent cations. To further understand the biophysical and pharmacological properties of PCL, we examined a series of organic cations for permeation and inhibition, using single-channel patch clamp and whole-cell two-microelectrode voltage clamp techniques in conjunction with Xenopus oocyte expression. We found that PCL is permeable to organic amines, methlyamine (MA, 3.8 Å), dimethylamine (DMA, 4.6 Å) and triethylamine (TriEA, 6 Å), and to tetra-alkylammonium cation (TAA) tetra-methylammonium (TMA, 5.5-6.4 Å). TAA compounds tetra-ethylammonium (TEA, 6.1-8.2 Å) and tetra-propylammonium (TPA, 9.8 Å) were impermeable through PCL and exhibited weak inhibition on PCL (IC₅₀ values>13 mM). Larger TAA cations tetra-butylammonium (TBA, 11.6 Å) and tetra-pentylammonium (TPeA, 13.2 Å) were impermeable through PCL as well and showed strong inhibition (IC₅₀ values of 2.7 mM and 1.3 μM, respectively). Inhibition by TBA was on decreasing the single-channel current amplitude and exhibited no effect on open probability (NP_o) or mean open time (MOT), suggesting that it blocks the PCL permeation pathway. In contract, TEA, TPA and TPeA reduced NP_o and MOT values but had no effect on the amplitude, suggesting their binding to a different site in PCL, which affects the channel gating. Taken together, our studies revealed that PCL is permeable to organic amines and TAA cation TMA, and that inhibition of PCL by large TAA cations exhibits two different mechanisms, presumably through binding either to the pore pathway to reduce permeant flux or to another site to regulate the channel gating. These data allow to estimate a channel pore size of ∼7 Å for PCL.     

Fluorinated carbohydrates : Part XIII. 2-deoxy-2-fluoro-D-galactose

Reaction of trifluoro(fluoroxy)methane at ca. −80° with 3,4,6-tri-O-acetyl-D-galactal affords trifluoromethyl 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-α-D-galactopyranoside (2, 39%), 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-α-D-galactopyranosyl fluoride (3, 37%), trifluoromethyl 3,4,6-tri-O-acetyl-2-deoxy-2-fluoro-α-D-talopyranoside (4, 3%), and 3,4,6-tri-O-acetyl-2-deoxy-2-fluor-α-D-talopyranosyl fluoride (5, 2%). The structures of compounds 2–5 have been established by n.m.r. spectroscopy. Acid hydrolysis of 2 or 3 allords 2-deoxy-2-fluoro-D-galactose.     

Oral supplementation with a combination of l-citrulline and l-arginine rapidly increases plasma l-arginine concentration and enhances NO bioavailability

Background

Chronic supplementation with l-citrulline plus l-arginine has been shown to exhibit anti-atherosclerotic effects. However, the short-term action of this combination on the nitric oxide (NO)–cGMP pathway remains to be elucidated. The objective of the present study was to investigate the acute effects of a combination of oral l-citrulline and l-arginine on plasma l-arginine and NO levels, as well as on blood circulation.

Methods

Rats or New Zealand white rabbits were treated orally with l-citrulline, or l-arginine, or a combination of each at half dosage. Following supplementation, plasma levels of l-arginine, NO_x, cGMP and changes in blood circulation were determined sequentially.

Results

l-Citrulline plus l-arginine supplementation caused a more rapid increase in plasma l-arginine levels and marked enhancement of NO bioavailability, including plasma cGMP concentrations, than with dosage with the single amino acids. Blood flow in the central ear artery in rabbits was also significantly increased by l-citrulline plus l-arginine administration as compared with the control.

Conclusion

Our data show for the first time that a combination of oral l-citrulline and l-arginine effectively and rapidly augments NO-dependent responses at the acute stage. This approach may have clinical utility for the regulation of cardiovascular function in humans.     

Formation of l-quebrachitol from d-bornesitol in leaves of Acer pseudoplatanus

d-Bornesitol and l-quebrachitol have been found in the leaves of Acer pseudoplatanus L. The results of incorporation studies using labeled myo-inositol-¹⁴C, l-inositol-¹⁴C and d-bornesitol-¹⁴C indicate that l-quebrachitol is produced by epimerization of d-bornesitol. In Artemisia vulgaris, however, the precursor of l-quebrachitol is l-inositol.     

Structural Basis for Catalysis by the Mono- and Dimetalated Forms of the dapE-Encoded N-succinyl-l,l-Diaminopimelic Acid Desuccinylase

Biosynthesis of lysine and meso-diaminopimelic acid in bacteria provides essential components for protein synthesis and construction of the bacterial peptidoglycan cell wall. The dapE operon enzymes synthesize both meso-diaminopimelic acid and lysine and, therefore, represent potential targets for novel antibacterials. The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase functions in a late step of the pathway and converts N-succinyl-l,l-diaminopimelic acid to l,l-diaminopimelic acid and succinate. Deletion of the dapE gene is lethal to Helicobacter pylori and Mycobacterium smegmatis, indicating that DapE's are essential for cell growth and proliferation. Since there are no similar pathways in humans, inhibitors that target DapE may have selective toxicity against only bacteria. A major limitation in developing antimicrobial agents that target DapE has been the lack of structural information. Herein, we report the high-resolution X-ray crystal structures of the DapE from Haemophilus influenzae with one and two zinc ions bound in the active site, respectively. These two forms show different activity. Based on these newly determined structures, we propose a revised catalytic mechanism of peptide bond cleavage by DapE enzymes. These structures provide important insight into catalytic mechanism of DapE enzymes as well as a structural foundation that is critical for the rational design of DapE inhibitors.     

Synthesis and antitumor activity of new d-seco and d-homo androstane derivatives

Starting from 3β-hydroxy-17-oxo-16,17-secoandrost-5-ene-16-nitrile (1), the new 16,17-secoandrostane derivatives 4-9 were synthesized. On the other hand, 3β-hydroxy-17-oxa-d-homoandrost-5-ene-16-one (10) yielded the new d-homo derivatives 12, 13 and 15. In vitro antiproliferative activity of selected compounds against three tumor cell lines (human breast adenocarcinoma ER+, MCF-7, human breast adenocarcinoma ER−, MDA-MB-231, prostate cancer AR−, PC-3, and normal fetal lung fibroblasts, MRC-5) was evaluated. Compounds 3 and 12 showed strong antiproliferative activity against PC-3 cells, the IC₅₀ values being 2 μM and 0.55 μM, respectively. Compounds 6 (10 μM) and 14 (9 μM) showed moderate activity against MDA-MB-231 cells. The synthesized compounds 1-3, 5-8, 10 and 12-15 were not toxic to normal fetal lung fibroblasts cells, MRC-5.     

The oxidation of D-glucose and D-fructose with oxygen in aqueous,alkaline solutions : Part II. The overall kinetics

Based on a previously reported, integral reaction-scheme, a very simple, kinetic model for the homogeneous oxidation of D-glucose and D-fructose in aqueous, alkaline solutions has been developed. It involves the influence of the type and concentration of the hexose, the hydroxyl-ion concentration, the oxygen concentration in the liquid phase, and the temperature on the rate of formation of the acidic reaction products. No attention is paid to the product distribution.