An improved synthesis of 5-thio-D-ribose from D-ribono-1,4-lactone

5-Thio-D-ribopyranose was synthesized from D-ribono-1,4-lactone (1) by two approaches: (i) 5-bromo-5-deoxy-D-ribono-1,4-lactone (2) was successively transformed into 5-bromo-5-deoxy, 5-S-acetyl-5-thio or 5-thiocyanato-D-ribofuranose derivatives; appropriate treatment then lead to 5-thio-D-ribopyranose (7) in 46-48% overall yield and; (ii) 2 was transformed into the 5-S-acetyl-5-thio-D-ribono-1,4-lactone derivative (11). Reduction and deprotection of 11 afforded 5-thio-D-ribopyranose (7) in 57% overall yield.     

Evaluation of 4-hydroxy-6-methyl-3-pyridinecarboxylic acid and 2,6-dimethyl-4-hydroxy-3-pyridinecarboxylic acid as chelating agents for iron and aluminium

4-Hydroxy-6-methyl-3-pyridinecarboxylic acid (DQ6) and the new compound 2,6-dimethyl-4-hydroxy-3-pyridinecarboxylic acid (DQ726) were evaluated for possible application for iron (Fe) and aluminium (Al) chelation therapy. Metal/ligand solution chemistry, cytotoxicity, octanol/water partitioning (D_o/w), and chelation efficiency were studied. The solution chemistry of the two ligands with Fe(III) and Al(III) was investigated in aqueous 0.6 m (Na)Cl at 25 °C by means of potentiometric titrations, UV-Vis spectrophotometry, and ¹H NMR spectroscopy. DQ6 exhibited a high coordination efficiency towards Al(III). Fe(III)/DQ6, Al(III)/DQ726, and Fe(III)/DQ726 complexes were less stable. These results were confirmed by chelation efficiency measurements performed in an octanol/aqueous solution. Accordingly, the effects of the substitution at various ring positions of 4-hydroxy-3-pyridinecarboxylic acid were rationalised. Partitioning experiments at pH 7.4 showed both DQ6 and DQ726, and their Fe(III) and Al(III) complexes, to be hydrophilic. The toxicity of DQ6 and of DQ726 was investigated with human cancer cell lines and normal human primary cells: no cytotoxic effects were observed up to 0.1 mM, following a 3 days exposure. According to our results, DQ6 has the favourable properties to be a chelating agent for Al.     

Ascorbic acid synthesis due to L-gulono-1,4-lactone oxidase expression enhances NO production in endothelial cells

As a primary antioxidant, ascorbic acid (AA) provides beneficial effects for vascular health mitigating oxidative stress and endothelial dysfunction. However, the association of intracellular AA with NO production occurring inside the endothelial cells remains unclear. In the present study, we addressed this issue by increasing intracellular AA directly through de novo synthesis. To restore AA synthesis pathway, bovine aortic endothelial cells were transfected with the plasmid vector encoding L-gulono-1,4-lactone oxidase (GULO, EC 1.1.3.8), the missing enzyme converting L-gulono-1,4-lactone (GUL) to AA. Functional expression of GULO was verified by Western blotting and in vitro enzyme activity assay. GULO expression alone did not lead to AA synthesis but the supply of GUL resulted in a marked increase of intracellular AA. When the cells were stimulated with calcium ionophore, A23187, NO production was more active in the GULO-expressing cells supplied with GUL, in comparison with the cells without GULO expression or without GUL supply, indicating that intracellular AA regulated NO production. Enhancement of NO production by intracellular AA was further verified in aortic endothelial cells obtained from eNOS knockout mice that were cotransfected with eNOS and GULO constructs. GULO-dependent AA synthesis also elevated intracellular tetrahydrobiopterin content, implicating that this essential cofactor of endothelial nitric oxide synthase (eNOS) might mediate the AA effect. The present study strongly suggests that intracellular AA plays critical roles in vascular physiology through enhancing endothelial NO production.     

Photoinduced electron-transfer alpha-deoxygenation of aldonolactones. Efficient synthesis of 2-deoxy-D-arabino-hexono-1,4-lactone

A photoinduced electron-transfer (PET) reaction was used for the deoxygenation at C-2 of aldonolactones derivatized as 2-O-[3-(trifluoromethyl)benzoyl] or benzoyl esters. By irradiation of different D-galactono- and D-glucono-1,4-derivatives, with a 450W lamp, using 9-methylcarbazole as photosensitizer, the corresponding 2-deoxy-D-lyxo- and 2-deoxy-D-arabino-hexono-1,4-lactones were efficiently obtained.     

Conformation of 4-thio- -lyxono-1,4-lactone in solution and in the crystalline state

The conformation in ²H₂O of 4-thio- -lyxono-1,4-lactone (1) was studied by nuclear magnetic resonance spectroscopy, by means of homonuclear (J_1H,1H) and heteronuclear (J_1H,13C) coupling constants. The couplings were directly measured by a two-dimensional heteronucleus-coupled ω₁ hetero-half-filtered proton-proton correlation (HETLOC) experiment, which does not require ¹³C isotopic enrichment. In solution, the thiolactone ring of 1 adopts preferentially the E₃ conformation, and its hydroxymethyl group populates mainly the gt rotamer. The X-ray diffraction data of a single crystal of 1 indicates that also in the solid state the thiolactone ring adopts an E₃ conformation, with a puckering somewhat larger than that observed for aldono-1,4-lactones and furanose rings. The molecules are linked by hydrogen bonds, which form chains. Particularly, O-5 is fully engaged as donor and acceptor in hydrogen bonding and the rotameric conformation of the hydroxymethyl group of 1 is fixed in the tg form.     

Facile synthesis and rearrangement of L-threo-2,3-hexodiulosono-1,4-lactone 2-(2-arylhydrazones)

Controlled reaction of L-threo-2,3-hexodiulosono-1,4-lactone with substituted phenylhydrazines gave the 2-(monoarylhydrazones) (2), which underwent dehydrative acetylation to 4-(2-acetoxyethylidene)-4-hydroxy-2,3-dioxohutyro-1,4-lactone 2-(2-arylhydrazones) (3). The latter reacted with methylhydrazine to give 1-methyl-3-(1-methylpyrazolin-3-yl)-4,5-pyrazoledione 4-(2-arylhydrazones) (4). Reaction of the monoarythydrazones (2) with phenylhydrazine gave the mixed bishydrazones (5), which were rearranged by alkali and acidification to the pyrazolediones (6). Compounds 6 gave triacetyl (7) and tribenzoyl derivatives (8), and, on periodite oxidation, the aldehydes (9), which afforded the monohydrazones (10). The i.r.. n.m.r.. and mass-spectral data of some of the compounds were investigated.     

Vanillic acid, a metabolite of 4-hydroxy-3-methoxyphenylglycol and 4-hydroxy-3-methoxymandelic acid in man

Abstract: 4-Hydroxy-3-methoxyphenylglycol (HMPG) labelled with ¹⁴C was used to study the metabolic fate of HMPG in six healthy volunteers. Besides conjugation and oxidation to 4-hydroxy-3-methoxymandelic acid (HMMA, VMA) a minor portion, 8.4 ± 1.1% (mean ± SEM) was excreted as ¹⁴C-labelled vantllic acid (VA). To study if VA was formed from HMPG or HMMA (VMA), deuterium-labelled HMPG ([²H₃]HMPG) and HMMA ([²H₆]HMMA) were simultaneously injected intravenously to seven healthy volunteers. The recovery of [²H₃]VA from [²H₃]HMPG was 8.3 ± 2.1% and the recovery of [²H₆]VA from [²H₆]HMMA was 9.0 ± 2.1%. The ²H-labelled VAs were probably formed by a decar boxylation reaction, in the case of HMPG after previous oxidation to HMMA.     

Tautomerism of 4-hydroxy-2,5-dimethyl-3(2H)-furanone: evidence for its enantioselective biosynthesis

Chiral natural flavor compounds exhibit characteristic enantiomeric excesses due to stereoselective, enzymatically catalyzed reactions during biogenesis. Although the enzymatic formation of the strawberry key flavor compound 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF; Furaneol(R)) is anticipated, the naturally occurring compound is racemic. As racemization due to keto-enol-tautomerism of HDMF could account for this observation, HDMF was investigated by (1)H-NMR spectroscopy tracing the exchange of the proton bound to the furanone-ring at C2 with deuteron from the medium (D(2)O). In addition, the racemization rate of HDMF was directly determined by cyclodextrin-modified capillary electrophoresis of enantiomerically enriched HDMF stored at different pH values. Tautomerism and the racemization rate of HDMF was lowest at pH values between 4 and 5. However, tautomerism and thus racemization was catalyzed under stronger acidic conditions (pH 2) and especially at pH values greater than 7, the value published for plant cell cytosol. Approximately 50% of the protons at C2 were exchanged with deuteron within 1 h at pH 7.2. Therefore, in order to demonstrate the enzymatic formation of HDMF, incubation experiments with Zygosaccharomyces rouxii as well as strawberry protein extract were carried out under slightly acidic conditions (pH 5), the most suitable pH value for studies on the enantiomeric ratio of HDMF. In both experiments the formation of enantiomerically enriched HDMF could be demonstrated for the first time, whereas incubation experiments under neutral conditions resulted in the detection of racemic HDMF.     

A novel type of oxygenolytic ring cleavage: 2,4-oxygenation and decarbonylation of 1H-3-hydroxy-4-oxoquinaldine and 1H-3-hydroxy-4-oxoquinoline

Abstract The utilization of quinaldine (2-methylquinoline) by Arthrobacter sp. Rü61a proceeds via 1 H -4-oxoquinaldine, 1 H -3-hydroxy-4-oxoquinaldine, and N -acetyl-anthranilic acid. By analogy, 1 H -4-oxoquinoline is degraded by Pseudomonas putida 33/1 via 1 H -3-hydroxy-4-oxoquinoline and N -formylanthranilic acid. Using the purified enzymes from both organisms, the mode of N -heterocyclic ring cleavage was investigated. The conversions of 1 H -3-hydroxy-4-oxoquinaldine and 1 H -3-hydroxy-4-oxoquinoline to N -acetyl- and N -formylanthranilic acid, respectively, were both accompanied by the release of carbon monoxide. The enzyme-catalysed transformations were performed in an [¹⁸O]O₂ atmosphere and resulted in the incorporation of two oxygen atoms into the respective products, N -acetyl- and N -formylanthranilic acid, indicating an oxygenolytic attack at C-2 and C-4 of both 1 H -3-hydroxy-4-oxoquinaldine and 1 H -3-hydroxy-4-oxoquinolone.     

Safe and easy route for the synthesis of 1,3-dimethyl-1,2,3-triazolium salt and investigation of its anticancer activities

We have developed a new safe and easy route for the synthesis of 1,3-dimethyl-1,2,3-triazolium derivatives. We have reported the synthesis of 4,9-dioxo-1,3-dimethylnaphtho[2,3-d][1,2,3]triazol-3-ium chloride from methylation of 1-methyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione. The synthesis of 1-methyl-1H-naphtho[2,3-d][1,2,3]triazole-4,9-dione is inefficient as a significant amount of by-product is formed that is difficult to separate and also unsafe as it requires the use of hazardous methylazide as a starting material. It is, however, important to develop an improved method for the synthesis of 4,9-dioxo-1,3-dimethylnaphtho[2,3-d][1,2,3]triazol-3-ium salt due to its significant anticancer activities. Herein, we report a safe and convenient route for the synthesis of this compound, which lead to more detailed exploration of its profound anticancer activities. The improved method can be applicable for the synthesis of other 1,3-dimethyl-1,2,3-triazolium salts of interest without the use of potentially explosive methylazide. The compound synthesized in this new method shows significant anticancer activities against melanoma, colon cancer, non-small cell lung cancer and central nervous system (CNS) cancer with GI50 values ranging from low μM to nM.     

Mycobacterium tuberculosis possesses a functional enzyme for the synthesis of vitamin C, L-gulono-1,4-lactone dehydrogenase

The last step of the biosynthesis of L-ascorbic acid (vitamin C) in plants and animals is catalyzed by L-gulono-1,4-lactone oxidoreductases, which use both L-gulono-1,4-lactone and L-galactono-1,4-lactone as substrates. L-gulono-1,4-lactone oxidase is missing in scurvy-prone, vitamin C-deficient animals, such as humans and guinea pigs, which are also highly susceptible to tuberculosis. A blast search using the rat L-gulono-1,4-lactone oxidase sequence revealed the presence of closely related orthologs in a limited number of bacterial species, including several pathogens of human lungs, such as Mycobacterium tuberculosis, Pseudomonas aeruginosa, Burkholderia cepacia and Bacillus anthracis. The genome of M. tuberculosis, the etiologic agent of tuberculosis, encodes a protein (Rv1771) that shows 32% identity with the rat L-gulono-1,4-lactone oxidase protein. The Rv1771 gene was cloned and expressed in Escherichia coli, and the corresponding protein was affinity-purified and characterized. The FAD-binding motif-containing Rv1771 protein is a metalloenzyme that oxidizes L-gulono-1,4-lactone (Km 5.5 mm) but not L-galactono-1,4-lactone. The enzyme has a dehydrogenase activity and can use both cytochrome c (Km 4.7 microm) and phenazine methosulfate as exogenous electron acceptors. Molecular oxygen does not serve as a substrate for the Rv1771 protein. Dehydrogenase activity was measured in cellular extracts of a Mycobacterium bovis BCG strain. In conclusion, M. tuberculosis produces a novel, highly specific L-gulono-1,4-lactone dehydrogenase (Rv1771) and has the capacity to synthesize vitamin C.     

A new synthesis of L-threo-hex-2-enaro-1,4-lactone (“saccharoascorbic” acid): A method for the protection of the enediol of ascorbic acid

A new synthesis of l-threo-hex-2-enaro-1,4-lactone (4) (“saccharoascorbic acid”) is presented, whose unique feature involves oxidation of the side chain of ascorbic acid. Ascorbate 2-sulfate (1) was selectively oxidized in water at pH 8–8.5 with platinum-on-carbon catalyst to yield the 2-sulfate (3) of 4. Hydrolysis of 3 in 15% trifluoroacetic acid for 90 min at 70° yielded 4. The procedure affords a useful preparation of 4, and demonstrates the excellence of sulfation for protection of the enediol of ascorbic acid during synthetic manipulations of the side chain. The sulfated ring is stable to oxidizing agents and to base, yet sulfate is readily removed by acid hydrolysis. The properties of a new compound (3) of biological significance, and those of the previously uncharacterized 4, are reported.