Biotransformation of raspberry ketone and zingerone by cultured cells of Phytolacca americana

The biotransformation of raspberry ketone and zingerone were individually investigated using cultured cells of Phytolacca americana. In addition to (2S)-4-(4-hydroxyphenyl)-2-butanol (2%), (2S)-4-(3,4-dihydroxyphenyl)-2-butanol (5%), 4-[4-(beta-d-glucopyranosyloxy)phenyl]-2-butanone (19%), 4-[(3S)-3-hydroxybutyl]phenyl-beta-d-glucopyranoside (23%), and (2S)-4-(4-hydroxyphenyl)but-2-yl-beta-d-glucopyranoside (20%), two biotransformation products, i.e., 2-hydroxy-4-[(3S)-3-hydroxybutyl]phenyl-beta-d-glucopyranoside (12%) and 2-hydroxy-5-[(3S)-3-hydroxybutyl]phenyl-beta-d-glucopyranoside (11%), were isolated from suspension cells after incubation with raspberry ketone for three days. On the other hand, two compounds, i.e., (2S)-4-(4-hydroxy-3-methoxyphenyl)but-2-yl-beta-d-glucopyranoside (17%) and (2S)-2-(beta-d-glucopyranosyloxy)-4-[4-(beta-d-glucopyranosyloxy)-3-methoxyphenyl]butane (16%), together with (2S)-4-(4-hydroxy-3-methoxyphenyl)-2-butanol (15%), 4-[4-(beta-d-glucopyranosyloxy)-3-methoxyphenyl]-2-butanone (21%), and 4-[(3S)-3-hydroxybutyl]-2-methoxyphenyl-beta-d-glucopyranoside (24%) were obtained upon addition of zingerone. Cultured cells of P. americana can reduce, and regioselectively hydroxylate and glucosylate, these food ingredients to their beta-glycosides.     

Synthesis of methyl glycoside derivatives of tri- and penta-saccharides related to the antithrombin III-binding sequence of heparin, employing cellobiose as a key starting-material

Two key synthons for the title pentasaccharide derivative, methyl O-(methyl 2-O-benzoyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-acetyl- 2-azido - 3-O- benzyl-2-deoxy-beta-D-glucopyranoside and O-(methyl 2,3-di-O-benzyl-4-O- chloroacetyl-beta-D-glucopyranosyluronate)-(1----4)-3,6-di-O-acetyl-2-az ido-2- deoxy-alpha-D- glucopyranosyl bromide, were prepared from a common starting material, cellobiose. They were coupled to give a tetrasaccharide derivative that underwent O-dechloroacetylation to the corresponding glycosyl acceptor. Its condensation with the known 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl bromide afforded a 77% yield of suitably protected pentasaccharide, methyl O-(6-O- acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)- O- (methyl 2,3- di-O-benzyl-beta-D-glucopyranosyluronate)-(1----4)-O-(3,6-di-O-acetyl-2- azido-2 - deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-benzoyl-3-O-benzyl-alpha-L- idopyranosyluronate)- (1----4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranoside. Sequential deprotection and sulfation gave the decasodium salt of methyl O-(2- deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl)-(1----4)-O-(be ta-D- glucopyranosyl-uronic acid)-(1----4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-gluco pyranosyl)- (1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1----4)-2-deoxy-2- sulfamido-6-O- sulfo-beta-D-glucopyranoside (3). In a similar way, the trisaccharide derivative, the hexasodium salt of methyl O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D- glucopyranosyl)- (1----4)-O-(beta-D-glucopyranosyluronic acid)-(1----4)-2-deoxy-2-sulfamido-3,6- di-O- sulfo-alpha-D-glucopyranoside (4) was synthesized from methyl O-(6-O-acetyl-2- azido- 3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2,3-di-O- benzyl-beta- D-glucopyranosyluronate)-3,6-di-O-acetyl-2-azido-2-deoxy-alpha-D- glucopyranoside. The pentasaccharide 3 binds strongly to antithrombin III with an association constant almost equivalent to that of high-affinity heparin, but the trisaccharide 4 appears not to bind.     

Biosynthetic studies on the alpha-glucosidase inhibitor acarbose: the chemical synthesis of isotopically labeled 2-epi-5-epi-valiolone analogs

2-epi-5-epi-valiolone is a cyclization product of the C(7) sugar phosphate, sedoheptulose 7-phosphate, involved in the biosynthesis of the aminocyclitol moieties of acarbose, validamycin, and pyralomicin. As part of our investigation into the pathway from 2-epi-5-epi-valiolone to the valienamine moiety of acarbose, we prepared 1-epi-5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-6], 5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-17], 1-epi-2-epi-5-epi-(6-(2)H(2))valiolol [(6-(2)H(2))-12] and 2-epi-5-epi-(6-(2)H(2))valiolamine [(6-(2)H(2))-11]. Compounds (6-(2)H(2))-6 and (6-(2)H(2))-17 were synthesized from 2,3,4,6-tetra-O-benzyl-D-glucopyranose in 10 and seven steps, respectively, whereas (6-(2)H(2))-12 and (6-(2)H(2))-11 were synthesized from 2,3,4,6-tetra-O-benzyl-D-mannopyranose in eight and 10 steps, respectively.     

Metabolism of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate in the neuronal and glial compartments of the adult rat brain as detected by [(13)C, (2)H] NMR spectroscopy

Ex vivo ?(13)C, (2)H? NMR spectroscopy allowed to estimate the relative sizes of neuronal and glial glutamate pools and the relative contributions of (1-(13)C) glucose and (2-(13)C, 2-(2)H(3)) acetate to the neuronal and glial tricarboxylic acid cycles of the adult rat brain. Rats were infused during 60 min in the right jugular vein with solutions containing (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose or (2-(13)C, 2-(2)H(3)) acetate only. At the end of the infusion the brains were frozen in situ and perchloric acid extracts were prepared and analyzed by high resolution (13)C NMR spectroscopy (90.5 MHz). The relative sizes of the neuronal and glial glutamate pools and the contributions of acetyl-CoA molecules derived from (2-(13)C, (2)H(3)) acetate or (1-(13)C) glucose entering the tricarboxylic acid cycles of both compartments, could be determined by the analysis of (2)H-(13)C multiplets and (2)H induced isotopic shifts observed in the C4 carbon resonances of glutamate and glutamine. During the infusions with (2-(13)C, 2-(2)H(3)) acetate and (1-(13)C) glucose, the glial glutamate pool contributed 9% of total cerebral glutamate being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (4%), (2-(13)C) acetyl-CoA (3%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (2%). The neuronal glutamate pool accounted for 91% of the total cerebral glutamate being mainly originated from (2-(13)C) acetyl-CoA (86%) and (2-(13)C, 2-(2)H) acetyl-CoA (5%). During the infusions of (2-(13)C, 2-(2)H(3)) acetate only, the glial glutamate pool contributed 73% of the cerebral glutamate, being derived from (2-(13)C, 2-(2)H(3)) acetyl-CoA (36%), (2-(13)C, 2-(2)H) acetyl-CoA (27%) and (2-(13)C) acetyl-CoA (10%). The neuronal pool contributed 27% of cerebral glutamate being formed from (2-(13)C) acetyl-CoA (11%) and recycled (2-(13)C, 2-(2)H) acetyl-CoA (16%). These results illustrate the potential of ?(13)C, (2)H? NMR spectroscopy as a novel approach to investigate substrate selection and metabolic compartmentation in the adult mammalian brain.     

Primary structure of the glycan chains of normal C 1 esterase inhibitor (C 1-INH) after NMR analysis at 400 MHz

The primary structural analysis of O- and N-linked carbohydrate chains of the C-1-esterase inhibitor purified from normal serum was carried out by 400-MHz 1H-NMR spectroscopy. C-1-esterase inhibitor protein of a molecular weight of 116,000 daltons contains 24 O-glycans: NeuAc (alpha 2-3) Gal (beta 1-3) GalNAc, 4 N-glycans: NeuAc (alpha 2-6) Gal (beta 1-4) (GlcNAc (beta 1-2) Man (alpha 1-3) [NeuAc (alpha 2-6) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-6)] Man (beta 1-4) GlcNAc (beta 1-4) GlcNAc and 2 N-glycans: NeuAc (alpha 2-3) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-3) [NeuAc (alpha 2-3) Gal (beta 1-4) GlcNAc (beta 1-2) Man (alpha 1-6)] Man (beta 1-4) GlcNAc (beta 1-4) GlcNAc. 30% of the N-glycans are fucosylated.     

Versatile precursor to ruthenium-bis(phosphine) hydrogenation catalysts

The known complex trans-RuCl2(NBD)Py2 (1, NBD is norbornadiene, Py is pyridine) reacts with either (R)-BINAP ((R)-2, 2'-bis(diphenylphosphino)-1,1'-binaphthyl), (S;S)-Chiraphos ((2S;3S-(-)-2,3-bis(diphenylphosphino)butane), (S;S)-Skewphos ((2S;4S)-(-)-2,4-bis(diphenylphosphino)pentane), (R)-(S)-Josiphos ((R)-(-)-1-[(S)-2-(diphenylphosphino)ferrocenyl]ethyl-dicyclohexylpho sphine), (R;R)-Norphos ((2R;3R)-(-)-2, 3-bis(diphenylphosphino)bicyclo[2.2.1]hept-5-ene), or (R;R)-Me-DUPHOS ((-)-1,2-bis((2R;5R)-2, 5-dimethylphospholano)benzene) to generate in high yields the crystalline complexes trans-RuCl2(P-P*)Py2 (P-P* is the corresponding chiral bis(phosphine)). The complexes trans-RuCl2(P-P*)Py2 are active enantioselective hydrogenation catalysts for ketoesters and noncarboxylic olefins in the presence of small amounts of HBF4 (aq.). They are active for hydrogenation of carboxylic substrates in the presence of Et3N. Reaction of trans-RuCl2(P-P*)Py2 with (rac)-1,2-diphenylethylene-diamine (N-N*, either enantiomer) forms in good yields the corresponding compounds trans-RuCl2(P-P*)(N-N*). Representative hydrogenations with these catalysts are presented.     

Synthesis of an octasaccharide fragment of high-mannose-type glycans of glycoproteins.

O-(alpha-D-Mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----3)- O- [(alpha-D-mannopyranosyl)-(1----2)-O-(alpha-D-mannopyranosyl)-(1----6)]- O- (alpha-D-mannopyranosyl)-(1----6)-O-(beta-D-mannopyranosyl)-(1----4)-O-( 2- acetamido-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-2-deoxy- glucopyranose, an octasaccharide fragment of high-mannose type glycan of glycoproteins, was synthesized. Crucial glycosylation of trisaccharide intermediate, benzyl O-(2,4-di-O-benzyl-beta-D-mannopyranosyl)-(1----4)-O-(2-acetamido-3,6-di -O- benzyl-2-deoxy-beta-D-glucopyranosyl)-(1----4)-2-acetamido-3,6-di-O-benz yl-2- deoxy-beta-D-glucopyranoside, was successful only with a di-O-acetyltetradeca-O-benzyl-D-mannopentaosyl chloride. The use of the corresponding hexadeca-O-acetyl-D-mannopentaosyl bromide did not give the desired product.     

Synthesis and biological evaluation of 1,3-oxathiolane 5-azapyrimidine, 6-azapyrimidine, and fluorosubstituted 3-deazapyrimidine nucleosides

(2R,5S)-5-Amino-2-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]- 1,2,4-triazine-3(2H)-one (8) and (2R,5R)-5-amino-2-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,2,4-tr iazine-3(2H)-one (9) have been synthesized via a multi-step procedure from 6-azauridine. (2R,5S)-4-Amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]-1,3, 5-triazine-2(1H)-one (11) and (2R,5R)-4-amino-1-[2-(hydroxymethyl)-1,3-oxathiolan-5-yl]- 1,3,5-triazine-2(1H)-one (12), and the fluorosubstituted 3-deazanucleosides (19-24) have been synthesized by the transglycosylation of (2R,5S)-1-[2-[[(tert-butyldiphenylsilyl) oxy]methyl]-1,3-oxathiolan-5-yl] cytosine (2) with silylated 5-azacytosine and the corresponding silylated fluorosubstituted 3-deazacytosines, respectively, in the presence of trimethylsilyl trifluoromethanesulfonate as the catalyst in anhydrous dichloroethane, followed by deprotection of the blocking groups. These compounds were tested in vitro for cytotoxicity against L1210, B16F10, and CCRF-CEM tumor cell lines and for antiviral activity against HIV-1 and HBV.     

Stereochemical aspects of the formation of double bonds in abscisic acid

The stereochemistry of the hydrogen elimination that occurs during the formation of the Delta(4)- and Delta(2)'-double bonds of abscisic acid has been determined from the (14)C/(3)H ratios in abscisic acid biosynthesized by avocado fruit from [2-(14)C,(2R)-2-(3)H(1)]-, [2-(14)C,(2S)-2-(3)H(1)]- and [2-(14)C,(5S)-5-(3)H(1)]-mevalonate. Setting the (14)C/(3)H ratio at 3:3 for [2-(14)C,(2R)-2-(3)H(1)]mevalonate, the corresponding ratio in derived methyl abscisate was 3:2.28; the analogous ratio for methyl abscisate from [2-(14)C,(2S)-2-(3)H(1)]mevalonate was 3:1.63. Removal of the 3'-hydrogen atom of abscisic acid by base-catalysed exchange altered the ratios to 3:1.55 and 3:1.44 respectively. It was concluded that this 3'-hydrogen atom is derived from the pro-2R-hydrogen atom of mevalonate. Removal of the 4-hydrogen atom from methyl abscisate by formation of a derivative, a lactone, lacking this hydrogen atom changed the ratio to 3:1.04 for material derived from [2-(14)C,(2R)-2-(3)H(1)]-mevalonate and to 3:1.05 for [2-(14)C,(2S)-2-(3)H(1)]mevalonate, showing that this hydrogen atom also is derived from the pro-2R-hydrogen atom of mevalonate. These ratios of the lactones are consistent with their retaining one (3)H atom at the 6'-methyl position of abscisic acid from the [(2R)-2-(3)H(1)]- and [(2S)-2-(3)H(1)]-mevalonate. The presence of some label at positions 3' and 4 when [(2S)-2-(3)H(1)]mevalonate was the precursor is attributed to the action of isopentenyl pyrophosphate isomerase. The hydrogen atom at C-5 of abscisic acid is derived from the pro-5S-hydrogen atom of mevalonate.     

Total synthesis of sialosylcerebroside, GM4

Described are total syntheses of O-[sodium (5-acetamido-3,5-dideoxy-D -glycero-alpha-D-galacto-2-nonulopyranosyl)onate]-(2----3)-O -beta-D -galactopyranosyl-(1----1)-(2R,3S,4E)-2-N-tetracosanoylsphingen ine,O-[sodium (5-acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosyl+ ++)onate] -(2----3)-O-alpha-D-galactopyranosyl-(1----1)-(2R,3S,4E)-2-N -tetracosanoylsphingenine, O-[sodium (5-acetamido-3,5-dideoxy-D-glycero-beta -D-galacto-2-nonulopyranosyl)onate]-(2----3)-O-beta-D-gal act opyranosyl -(1----1)-(2R,3S,4E)-2-N-tetracosanoylsphingenine, and O-[sodium (5-acetamido-3,5-dideoxy-D-glycero-beta-D-galacto-2-nonulopyranosyl++ +)onate] -(2----3)-O-alpha-D-galactopyranosyl-(1----1)-(2R,3S,4E)-2-N -tetracosanoylsphingenine by using O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D -galacto-2-nonulopyranosyl)onate]-(2----3)-2,3,4,6-tetra-O-a cetyl-D -galactopyrano-syl trichloroacetimidate and O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-beta -D-galacto-2-nonulopyranosyl)onate]-(2----3)-2,4,6-tri-O-ace tyl-D-galactopyranosyl trichloroacetimidate as key glycosyl donors, and (2S,3R,4E)-3 -O-benzoyl-2-N-tetracosanoylsphingenine as a key glycosyl acceptor.     

Ventilatory responses to acute and chronic hypoxia in mice: effects of dopamine D(2) receptors.

We used genetically engineered D(2) receptor-deficient [D(2)-(-/-)] and wild-type [D(2)-(+/+)] mice to test the hypothesis that dopamine D(2) receptors modulate the ventilatory response to acute hypoxia [hypoxic ventilatory response (HVR)] and hypercapnia [hypercapnic ventilatory response (HCVR)] and time-dependent changes in ventilation during chronic hypoxia. HVR was independent of gender in D(2)-(+/+) mice and significantly greater in D(2)-(-/-) than in D(2)-(+/+) female mice. HCVR was significantly greater in female D(2)-(+/+) mice than in male D(2)-(+/+) and was greater in D(2)-(-/-) male mice than in D(2)-(+/+) male mice. Exposure to hypoxia for 2-8 days was studied in male mice only. D(2)-(+/+) mice showed time-dependent increases in "baseline" ventilation (inspired PO(2) = 214 Torr) and hypoxic stimulated ventilation (inspired PO(2) = 70 Torr) after 8 days of acclimatization to hypoxia, but D(2)-(-/-) mice did not. Hence, dopamine D(2) receptors modulate the acute HVR and HCVR in mice in a gender-specific manner and contribute to time-dependent changes in ventilation and the acute HVR during acclimatization to hypoxia.     

An Efficient reproductive method for Irs2-/- mice with C57BL/6JJcl genetic background

Efficient reproduction using natural mating and reproduction technology [in vitro fertilization (IVF) and embryo transfer (ET)] was investigated in IRS2 deficient mice with C57BL/6JJcl genetic background (Irs2(-/-) mice) as a typical type 2 diabetes model. From the results using various combinations of Irs2(-/-) and Irs2(-/+) mice, the combination of female Irs2(-/+) x male Irs2(-/-) was found to be more efficient than other combinations. In applications of reproduction technology using IVF and ET, the combination of female Irs2(-/+) x male Irs2(-/-) involves the possibility of Irs2(-/-) production by repeats using female Irs2(-/+) mice. However, reproductive continuity using this combination is difficult because of dependence on human technique and the cost of ET. Therefore, we concluded that Irs2(-/-) mice should be produced by embryo transfer using Irs2(-/-) mice from a colony consisting of female Irs2(-/+) x male Irs2(-/-).     

Investigation of the mechanism of the methylmalonyl-CoA mutase reaction with the substrate analogue: ethylmalonyl-CoA.

1. Ethylmalonyl-CoA was found to be a substrate for methylmalonyl-CoA mutase from Propionibacterium shermanii, the product being mainly (2R)-methylsuccinyl-CoA along with some (2S)-diastereoisomer. 2. The relevant 1H-nuclear magnetic resonance signals of methylsuccinic acid and of its dimethyl ester were assigned to the diastereotopic methylene hydrogens using sterospecifically dideuterated specimens of known configuration. 3. [2(-2)H1]Ethylmalonyl-CoA was converted by methylmalonyl-CoA mutase in 2H2O mainly to (2R, 3S)-[3(-2)H1]methylsuccinyl-CoA. No dideuterated product was observed. 4. Starting from (1R)-[1(-2)H1]-ethathanol, (1S)-[1(-2)H1]ethanol and [2H6] ethanol the following deuterated specimens of ethylmalonic acid were synthesised and characterised: (3S)-[3(-2)H1], (3R)-[3(-2)H1] and [3(-2)H2, 4(-2)H3], respectively. 5. Conversion of (3S)-[3(-2)H1]-ethylmalonyl-CoA (70% 2H1 and 2% 2H2 species) on the mutase in water afforded mainly (2R)-[2(-2)H1]methylsuccinyl-CoA along with some (2S)-diastereoisomer. No deuterium loss was observed. 6. Methylmalonyl-CoA mutase converted (3R)-[3(-2)H1]ethylmalonyl-CoA (81% 2H1 and 2% 2H2 species) to the following methylsuccinyl-CoA species: 33% [3(-2)H1], the deuterium being in the threo position with respect to the methyl group; 21% [2(-2)H1]; 46% unlabelled. The ratio of the species with (2R) and (2S) configuration was about 60:40. 7. Reaction of [3(-2)H2, 4(-2)H3]ethylmalonyl-CoA (94.5% [2H5] species) with the mutase gave the following labelled methylsuccinyl-CoA species:53.4% [methyl-2H3, 2(-2)H1, 3(-2)H1], the 3-deuterium being in the threo position with respect to the methyl group; 37.6% [methyl-2H3, 2(-2)H1]; 5% [methyl(-2)H3, 2(-2)H1, 2(-2)H1, 3(-2)H1] the 3-deuterium being in erythro position with respect to the methyl group; 4% [methyl(-2)H3, 3(-2)H1]. The ratio of the species with (2R) and (2S) configuration was about 70:30. 8. Implications of these findings for the mechanism of the rearrangements catalysed by coenzyme B12 are discussed.     

Chiral synthesis of (2S,3S)-2-(2-morpholin-2-yl-2-phenylmethoxy)phenol

Resolution of (2RS,3RS)-2-[alpha-(2-methoxymethoxyphenoxy)phenylmethyl]morpholine, 11, with (+) mandelic acid led to the formation of (+)-(2S,3S)-2-[alpha-(2-methoxymethoxyphenoxy)phenyl methyl] morpholine (11a). Compound 11 was synthesized in seven steps from (2RS,3RS)-cinnamyl alcohol-2,3-epoxide (4), with an overall yield of 17%. Cleavage of the methoxymethyl group of the Fmoc derivative 12 with catalytic amounts of p-toluenesulfonic acid in methanol afforded (+)-(2S,3S)-2-(2-morpholin-2-yl-2-phenylmethoxy)phenol 2. The synthetic utility as well as the configuration of compound 2 has been demonstrated by converting (S,S)-2-(2-morpholin-2-yl-2-phenylmethoxy)phenol 2 to (2S,3S)-2-[alpha-(2-ethoxyphenoxy)phenylmethyl]morpholine (1) and (2S,3S)-2-(2-methoxyphenoxy) benzyl)morpholine (16), two potential norepinephrine reuptake inhibitors under clinical evaluation.     

New antioxidative phenolic glycosides isolated from Kokuto non-centrifuged cane sugar

Nine compounds, 3-hydroxy-4,5-dimethoxyphenyl-beta-D-glucopyranoside (1), beta-D-fructfuranosyl-alpha-D-(6-vanilloyl)-glucopyranoside (2), beta-D-fructfuranosyl-alpha-D-(6-syringyl)-glucopyranoside (3), 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxy-1-(E)-propenyl)-2-methoxyphenoxy]propyl-beta-D-glucopyranoside (4), 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)-2-[4-(3-hydroxy-1-(E)-propenyl)-2,6-dimethoxyphenoxy] propyl-beta-D-glucopyranoside (5), dehydrodiconiferyl alcohol-9'-beta-D-glucopyranoside (6), 4-[ethane-2-[3-(4-hydroxy-3-methoxyphenyl)-2-propen]oxy]-2,6-dimethoxyphenyl-beta-D-glucopyranoside (7), 4-[ethane-2-[3-(4-hydroxy-3-methoxyphenyl)-2-propen]oxy]-2-methoxyphenyl-beta-D-glucopyranoside (8), and 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)-2-[4-(3-hydroxy-1-(E)-propenyl)-2,6-dimethoxyphenoxy]propyl-beta-D-glucopyranoside (9), were isolated from Kokuto non-centrifuged cane sugar. Their structures were elucidated by spectroscopic evidence, mainly based on the NMR technique. Among them, seven new glycosides were identified. The 2-deoxyribose oxidation method was used to measure their antioxidative activity. All of these compounds showed antioxidative activities.     

Enzymatic- and renal-dependent catabolism of the intestinotropic hormone glucagon-like peptide-2 in rats

The intestinotropic hormone glucagon-like peptide (GLP)-2-(1-33) is cleaved in vitro to GLP-2-(3-33) by dipeptidyl peptidase IV (DP IV). To determine the importance of DP IV versus renal clearance in the regulation of circulating GLP-2-(1-33) levels in vivo, GLP-2-(1-33) or the DP IV-resistant analog [Gly(2)]GLP-2 was injected in normal or DP IV-negative rats and assayed by HPLC and RIA. Normal rats showed a steady degradation of GLP-2-(1-33) to GLP-2-(3-33) over time, whereas little or no conversion was detected for GLP-2-(1-33) in DP IV-negative rats and for [Gly(2)]GLP-2 in normal rats. To determine the role of the kidney in clearance of GLP-2-(1-33) from the circulation, normal rats were bilaterally nephrectomized, and plasma immunoreactive GLP-2 levels were measured. The slope of the disappearance curves for both GLP-2-(1-33) and [Gly(2)]GLP-2 were significantly reduced in nephrectomized compared with non-nephrectomized rats (P < 0.01). In contrast to both GLP-2-(1-33) and [Gly(2)]GLP-2, GLP-2-(3-33) did not stimulate intestinal growth in a murine assay in vivo. Thus the intestinotropic actions of GLP-2-(1-33) are determined both by the actions of DP IV and by the kidney in vivo in the rat.     

Acylated anthocyanins from red radish (Raphanus sativus L.)

Twelve acylated anthocyanins were isolated from the red radish (Raphanus sativus L.) and their structures were determined by spectroscopic analyses. Six of these were identified as pelargonidin 3-O-[6-O-(E)-feruloyl-2-O-beta-D-glucopyranosyl]-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), pelargonidin 3-O-[6-O-(E)-caffeoyl-2-O-(6-(E)-feruloyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), pelargonidin 3-O-[6-O-(E)-p-coumaroyl-2-O-(6-(E)-caffeoyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), pelargonidin 3-O-[6-O-(E)-feruloyl-2-O-(6-(E)-caffeoyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), pelargonidin 3-O-[6-O-(E)-p-coumaroyl-2-O-(6-(E)-feruloyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside), and pelargonidin 3-O-[6-O-(E)-feruloyl-2-O-(2-(E)-feruloyl-beta-D-glucopyranosyl)-(1-->2)-beta-D-glucopyranoside]-5-O-(beta-D-glucopyranoside).     

Expression and distribution of pituitary adenylate cyclase-activating peptide in human prostate and prostate cancer tissues

The therapeutic potential of the intestinotrophic mediator glucagon-like peptide-2 (1-33) [GLP-2 (1-33)] has increased interest in the pharmacokinetics of the peptide. This study was undertaken to investigate whether the primary degradation product GLP-2 (3-33) interacts with the GLP-2 receptor. Functional (cAMP) and binding in vitro studies were carried out in cells expressing the transfected human GLP-2 receptor. Furthermore, a biologic response of GLP-2 (3-33) was tested in vivo. Mice were allocated to groups treated for 10 days (twice daily) with: (1) 5 microg GLP-2 (1-33), (2) 25 microg GLP-2 (3-33), (3) 5 microg GLP-2 (1-33)+100 microg GLP-2 (3-33), or (4) 5 microg GLP-2 (1-33)+500 microg GLP-2 (3-33). The intestine was investigated for growth changes. GLP-2 (3-33) bound to the GLP-2 receptor with a binding affinity of 7.5% of that of GLP-2 (1-33). cAMP accumulation was stimulated with an efficacy of 15% and a potency more than two orders of magnitude lower than that of GLP-2 (1-33). Increasing doses of GLP-2 (3-33) (10(-7)-10(-5) M) caused a shift to the right in the dose-response curve of GLP-2 (1-33). Treatment of mice with either GLP-2 (1-33) or (3-33) induced significant growth responses in both the small and large intestines, but the response induced by GLP-2 (3-33) was much smaller. Co-administration of 500 microg of GLP-2 (3-33) and 5 microg GLP-2 (1-33) resulted in a growth response that was smaller than that of 5 microg GLP-2 (1-33) alone. Consistent with the observed in vivo activities, our functional studies and binding data indicate that GLP-2 (3-33) acts as a partial agonist with potential competitive antagonistic properties on the GLP-2 receptor.     

Stereospecific synthesis and bio-activity of novel beta(3)-adrenoceptor agonists and inverse agonists

Since it is widely distributed into the body, beta(3)-adrenoceptor is becoming an attractive target for the treatment of several pathologies such as obesity, type 2 diabetes, metabolic syndrome, cachexia, overactive bladder, ulcero-inflammatory disorder of the gut, preterm labour, anxiety and depressive disorders, and heart failure. New compounds belonging to the class of arylethanolamines bearing one or two stereogenic centres were prepared in good yields as racemates and optically active forms. They were, then, evaluated for their intrinsic activity towards beta(3)-adrenoceptor and their affinity for beta(1)- and beta(2)-adrenergic receptors. Stereochemical features were found to play a crucial role in determining the behaviour of such compounds. In particular, alpha-racemic, (alphaR)- and (alphaS)-2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}-2- methylpropanoic acid, (alpha-rac, beta-rac)-, (alphaR, betaS)- and (alphaR, betaR)- 2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}propanoic acid were found to be endowed with beta(3)-adrenoceptor agonistic activity. Whereas, (alphaS, betaS)- and (alphaS, betaR)-2-{4-[2-(2-hydroxy-2-phenylethylamino)ethyl]phenoxy}propanoic acid behaved as beta(3)-adrenoceptor inverse agonists. Such compounds showed no affinity for beta(1)- and beta(2)-adrenergic receptor, respectively. Thus, resulting highly selective beta(3)-adrenoceptor ligands.     

New bisabolane sesquiterpenes and coumarin from Leontopodium longifolium

From the roots of Leontopotium longifolium, three new bisabolane sesquiterpenes, rel-(1S,4R,5S,6R)-4,5-diacetoxy-6-[(R)-1,5-dimethylhexa-3,5-dienyl]-3-methylcyclohex-2-enyl (Z)-2-methylbut-2-enoate (1), rel-(1S,4R,5S,6R)-4,5-diacetoxy-6-[(R)-5-hydroxy-1,5-dimethylhex-3-enyl]-3-methylcyclohex-2-enyl (Z)-2-methylbut-2-enoate (2), rel-(1R,2S,4R,5S)-4-acetoxy-2-[(R)-5-hydroxy-1,5-dimethylhex-3-enyl]-5-methylcyclohexyl (Z)-2-methylbut-2-enoate (3), and a new coumarin, 2,3-dihydro-5-hydroxy-2-(1-methylethenyl)-7H-pyrano[2,3-g][1,4]benzodioxin-7-one (4) together with nine known compounds have been isolated. The structures of these compounds were established by spectroscopic methods. Compounds 1 and 2 exhibited moderate cytotoxic activities against human promyelocytic leukemia (HL-60) cells.