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.     

Characterisation and X-ray crystallography of products from the Bucherer-Bergs reaction of methyl 2,3-O-isopropylidene-alpha-D-lyxo-pentodialdo-1,4-furanoside

Methyl 2,3-O-isopropylidene-alpha-D-mannofuranosidurononitrile [alternative name: methyl (5R)-5-C-cyano-2,3-O-isopropylidene-alpha-D-lyxofuranoside] (2), methyl 2,3-O-isopropylidene-alpha-D-mannofuranosiduronamide [methyl (5S)-5-C-carbamoyl-2,3-O-isopropylidene-alpha-D-lyxofuranoside; methyl (5S)-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosiduronamide] (3), methyl 2,3-O-isopropylidene-alpha-D-mannofuranosiduronic acid [methyl (5S)-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosiduronic acid] (4), methyl 5-deoxy-2,3-O-isopropylidene-5-ureido-beta-L-gulofuranosiduronamide [methyl (5R)-5-deoxy-2,3-O-isopropylidene-5-ureido-alpha-D-lyxo-hexofuranosiduronamide (5), and (4S,5S,6R)-5,6-dihydro-6-hydroxy-4,5-isopropylidenedioxy-4H-pyrido[2,1-e]imidazolidine-2',4'-dione [IUPAC name: (3aS,4R,8aS)-4-hydroxy-2,2-dimethyl-3a,8a-dihydro-4H-1,3-dioxa-4a,6-diaza-s-indacene-5,7-dione] (6), instead of the expected hydantoin derivative, were obtained from the Bucherer-Bergs reaction of methyl 2,3-O-isopropylidene-alpha-D-lyxo-pentodialdo-1,4-furanoside (1). The structure of 6 was deduced from NMR and mass spectral data and confirmed by X-ray crystallography. The configuration at C-5 in 2-5 was confirmed by establishing the 5S configuration of 3 by X-ray crystallography. Conformations of the six- and five-membered rings in 3 and 6 are also discussed.     

攀援孔药花化学成分研究

从攀援孔药花全草95%乙醇提取物中首次分离得到19个化合物,通过波谱数据或与已知物对照,它们分别鉴定为:(2S,3S,4R)-2-[(2R)-2-羟基-二十一酰胺基]-二十一烷-1,3,4-三醇(1)、(2S,3S,4R)-2-二十四酰胺基-十八烷-1,3,4-三醇(2)、胡萝卜甙(3)、β-谷甾醇(4)、(20S,22E,24R)-5α,8α-表二氧-麦角甾-6,22-二烯-3β-醇(5)、6β-羟基-豆甾-4-烯-3-酮(6)、十六烷酸-1-甘油酯(7)、桦木酸(8)、大黄素(9)、二十二烷酸-1-甘油酯(10)、对羟基苯甲醛(11)、十七烷酸-1-甘油酯(12)、金色酰胺醇乙酸酯(13)、十九烷酸-1-甘油酯(14)、棕榈酸(15)(、E)-p-香豆酸(16)、(22E,24S)-24-甲基-5α-胆甾-7,22-二烯-3β,5α,6β-三醇(17)、2-去氧-β-蜕皮激素(18)和auranamide(19)。     

Systematic synthesis of four epicatechin series procyanidin trimers and their inhibitory activity on the Maillard reaction and antioxidant activity

A systematic synthesis of four natural epicatechin series procyanidin trimers [[4,8:4",8"]-2,3-cis-3,4-trans: 2",3"-cis-3",4"-trans: 2,3-trans-(-)-epi-catechin-(-)-epicatechin-(+)-catechin, [4,8:4",8"]-2,3-cis-3,4-trans: 2",3"-cis-3",4"-trans: 2,3-cis-tri-(-)-epicatechin: procyanidin C1, [4,8:4",8"]-2,3-cis-3,4-trans: 2",3"-trans-3",4"-trans: 2,3-trans-(-)-epicatechin-(+)-catechin-(+)-catechin: procyanidin C4, and [4,8:4",8"]-2,3-cis-3,4-trans: 2",3"-trans-3",4"-trans: 2,3-cis-(-)-epicatechin-(+)-catechin-(-)-epicatechin] is described. Condensation of (2R,3R,4S)-5,7,3'4'-tetra-O-benzyl-4-(2"-ethoxyethyloxy)flavan derived from (-)-epicatechin as an electrophile with the dimeric nucleophiles in the presence of TMSOTf followed by deprotection yielded trimers. Inhibitory activities on the Maillard reaction and antioxidant activity on lipid peroxide of the synthesized oligomers were also investigated.     

The stereochemistry of beta-lactam formation in penicillin biosynthesis.

1. (2R,3S)-[U-14C,3-3H1]- and (2R,3R)-[U-14C,2,3-3H2] Cysteine hydrochlorides have been separately synthesised. The latter compound has been shown to have uniform distributions of tritium between C-2 and C-3. 2. The abvoe cysteines and (2R)-[U-14C,3,3,3',3'-3H4]cystine have been converted to samples of penicillin G by Penicillium chrysogenum. 3. Incorporation results indicate that all but 14% of the tritium is lost from the (2R,3S)-[3-3H1]isomer; that 42% of tritium is retained by the non-stereospecifically C-3 tritiated cystine; and that 58% of tritium is retained by the (2R,3R)-[2,3-3H2]isomer on conversion to penicillin G. 4. Degradation of the penicillin G derived from (2R,3R)-[U-14C,2,3-3H2]cysteine hydrochloride has indicated that in fact about 87% of the original C-3 tritium of cysteine is retained at C-5 of penicillin G. 5. The results indicate stereospecificity in the cyclisation giving rise to the beta-lactam ring in penicillin G in nature with loss of the 3-pro-S-hydrogen and rentention of the 3-pro-R-hydrogen of cysteine. Thus there is net retention of stereochemistry in the cyclisation.     

Assay of physiological levels of 2,3-butanediol diastereomers in blood and urine by gas chromatography-mass spectrometry

We present an assay for 2,3-butanediol by gas chromatography-mass spectrometry of its trimethylsilyl ethers. 2R,3R- and/or 2S,3S-2,3-butanediol and meso-2,3-butanediol are quantitated with corresponding internal standards of [2,3-2H2]butanediol. Limits of detection are 1 and 0.1 microM for split and splitless injections, respectively. Blood concentrations of 2,3-butanediol in nonalcoholics are 0.5 +/- 0.3 (SD) microM for 2R,3R- and/or 2S,3S-2,3-butanediol and 0.8 +/- 0.4 microM for meso-2,3-butanediol (n = 9). Two hours after alcohol ingestion, blood levels had risen in eight of nine subjects to 1.2 +/- 0.7 microM for 2R,3R-/2S,3S-2,3-butanediol and to 1.2 +/- 0.6 microM for meso-2,3-butanediol. Baseline urinary excretion of 2,3-butanediol is 0.4 +/- 0.2 mumol/mmol creatinine for 2R,3R-/2S,3S-2,3-butanediol and 0.9 +/- 0.5 mumol/mmol creatinine for meso-2,3-butanediol.     

Phytotoxic naphthopyranone derivatives from the coprophilous fungus Guanomyces polythrix.

Reinvestigation of the fermentation broth and mycelium of the coprophilous fungus Guanomyces polythrix, grown in static conditions, led to the isolation of several phytotoxic compounds, including two new naphthopyranone derivatives, namely (2S, 3R)-5-hydroxy-6,8-dimethoxy-2,3-dimethyl-2,3-dihydro-4H-naphtho[2,3-b]-pyran-4-one and (2S, 3R)-5-hydroxy-6,8,10-trimethoxy-2,3-dimethyl-2,3-dihydro-4H-naphtho[2,3-b]-pyran-4-one. The structures of the new compounds were established by spectral and chiroptical methods. In addition, the structure of 8-hydroxy-6-methyl-9-oxo-9H-xanthene-1-carboxylic acid methyl ester was unambiguously determined by X-ray analysis. The isolates caused significant inhibition of radicle growth of two weed seedlings (Amaranthus hypochondriacus and Echinochloa crusgalli) and interacted with both spinach and bovine brain calmodulins.     

New synthesis of the (3Z,6Z,9S,10R)-isomers of 9,10-epoxy-3,6-henicosadiene and 9,10-epoxy-1,3,6-henicosatriene, pheromone components of the female fall webworm moth, Hyphantria cunea

(3Z,6Z,9S,10R)-9,10-Epoxy-3,6-henicosadiene (1) and (3Z,6Z,9S,10R)-9,10-epoxy-1,3,6-henicosatriene (5), pheromone components of the female fall webworm moth (Hyphantria cunea Drury), were synthesized by starting from (2S,3R)-2,3-epoxy-4-t-butyldimethylsilyloxy-1-butanol (8). Epoxide 8 was prepared by employing lipase-catalyzed asymmetric acetylation of (+/-)-8 as the key optical resolution step.     

Stereochemical course of two arene-cis-diol dehydrogenases specifically induced in Pseudomonas putida.

Catabolism of nonphenolic arenes is frequently initiated by dioxygenases, yielding single isomer products with two adjacent hydroxylated asymmetric centers. The next enzymic reaction dehydrogenates these cyclic cis-diols, with aromatization yielding catechols for ring cleavage. There are two stereochemical questions to answer. (i) To which face of NAD is hydride transferred giving NADH? (ii) Which hydrogen of the arene-cis-diols is donated to NAD? We report the results of 1H nuclear magnetic resonance [1H NMR] experiments for two diol dehydrogenases induced during growth of Pseudomonas putida PaW1(TOL) and JT105 with p-xylene and p-toluate, respectively. per-[2H5]benzoate-1,2-dihydrodiol and per-[2H7]- and specifically [2H]p-toluate-2,3-dihydrodiols were the substrates used to examine this by 1H NMR, as the two protons of the prochiral center (C-4 of the nicotinamide ring) are easily distinguished in the region of 2.6 to 2.7 ppm. We found that with the partially purified dehydrogenases (i) 2H from the (2R) center of per-(1S,2R)-benzoate-1,2-dihydrodiol was donated to the Si-face of NAD to give (4S)-NAD2H; (ii) p-toluate-2,3-diol dehydrogenase also provided exclusively (4S)-NAD2H, but the 2H was transferred from both the 2- and 3-C atoms of (2S,3R)-p-toluate-2,3-dihydrodiol with specifically deuterated species in approximately equal amounts; and (iii) the unexpected lack of stereo- and regioselectivity of p-toluate-2,3-diol dehydrogenase was supported by kinetic isotope effect studies.     

钮子瓜化学成分研究

从民间药物钮子瓜全草95%乙醇提取物中首次分离得到14个化合物,应用波谱方法及与已知品对照的手段鉴定它们为(2S,3S,4R,10E)2[(2R)2-羟基二十四烷酰氨基]10十八烷-1,3,4-三醇(1)、(2S,3S,4R)2-二十四烷酰胺基十八烷-1,3,4-三醇(2)、胡萝卜苷(3)、swertish(4)、苯甲酸(5)、水杨酸(6)、loliolide(7)、胸腺嘧啶(8)、尿嘧啶(9)、(23Z)-9,19-环阿尔廷-23-烯-3β,25-二醇(10)、(20S,22E,24R)5α,8α-表二氧麦角甾6,22二烯3β醇(11)、十六烷酸1甘油酯(12)、大豆脑苷Ⅰ(13)、(22E,24S)24甲基5α胆甾7,22二烯3β,5α,6β三醇(14)。     

Stereoselective biosynthesis of chloroarylpropane diols by the basidiomycete Bjerkandera adusta: exploring the roles of amino acids, pyruvate, glycerol and phenyl acetyl carbinol

Bjerkandera adusta produces many chlorometabolites including chlorinated anisyl metabolites (CAMs) and 1-arylpropane-1,2-diols (1, 2, 3, 4) as idiophasic metabolic products of L-phenylalanine. These diols are stereoselectively biosynthesized from a C7-unit (benzylic, from L-phenylalanine) and a C2-unit, of unknown origin, as predominantly erythro (1R,2S) enantiomers. Of the labeled amino acids tested as possible C2-units, at the 4-10 mM level, none were found to efficiently label the 2,3-propane carbons of the diols. However, glycine (2-13C), L-serine (2,3,3-d3) and L-methionine (methyl-d3) entered the biomethylation pathway. Neither pyruvate (2,3-13C2), acetate (1,2-13C2), acetaldehyde (d4) nor ethanol (ethyl-d5) labeled the 2,3-propane carbons of the diols at the 4-10 mM level. Pyruvate (2,3-13C2) and L-serine (2,3,3-d3) (which also entered the biomethylation pathway) did, however, effectively label the 2,3-propane carbons of the alpha-ketols and diols at the 40 mM level as evidenced by mass spectrometry. Glycerol (1,1,2,3,3-d5) also appeared to label one of the 2,3-propane carbons (ca. 5% as 2H2 in the C3 side chain) as suggested by mass spectrometric data and also entered the biomethylation pathway, likely via amino acid synthesis. Glycerol (through pyruvate), therefore, likely supplies C2 and C3 of the propane side chain with arylpropane diol biosynthesis. Incubation of B. adusta with synthetic [2-2H1, 2-18O]-glycerol showed that neither 2H nor 18O were incorporated in the alpha-ketols or diols. The oxygen atom on the C2 of the ketols/diols, therefore, does not appear to come from the oxygen atom on the C2 of glycerol. Glycerol, however, can readily form L-serine (which can then form pyruvate via PLP/serine dehydratase and involve transamination washing out the 18O label and providing the oxygen from water), and can then go on to label the C2-unit. Labeled alpha-ketol, phenyl acetyl carbinol (5) (PAC; ring-d(5), 2,3-13C2 propane) cultured with B. adusta leads to stereospecific reduction to the (1R,2S)-diol (6) (ring-d5 and 2,3-13C2); in all other metabolites produced, the 2,3-13C2) label is washed out. Incubation of the fungus with 4-fluorobenzaldehyde (13) produces a pooling of predominantly erythro (1R,2S) 1-(4'-fluorophenyl)-1,2-propane diol (18 as diacetate) (through the corresponding alpha-ketols 16, 17). Blocking the para-position with fluorine thus appears to prevent ring oxygenation and also chlorination, forcing the conclusion that para-ring oxygenation precedes meta-chlorination.     

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 and KCNQ2 opener activity of N-(1-benzo[1,3]dioxol-5-yl-ethyl, N-[1-(2,3-dihydro-benzofuran-5-yl)-ethyl, and N-[1-(2,3-dihydro-1H-indol-5-yl)-ethyl acrylamides

Bioisosteric replacement studies led to the identification of N-(1-benzo[1,3]dioxol-5-yl-ethyl)-3-(2-chloro-phenyl)-acrylamide ((S)-3) as a highly potent KCNQ2 opener, and 3-(2,6-difluoro-phenyl)-N-[1-(2,3-dihydro-benzofuran-5-yl)-ethyl]-acrylamide ((S)-4), and N-[1-(2,3-dihydro-1H-indol-5-yl)-ethyl]-3-(2-fluoro-phenyl)-acrylamide ((S)-5) as highly efficacious KCNQ2 openers. In contrast, their respective R enantiomers showed significantly less or no appreciable KCNQ2 opener activity even at the highest concentration tested (10 microM). Because of its high potency and moderate efficacy as well as its convenient synthesis, (+/-)-3 was selected as a reference compound for analyzing efficacies of KCNQ openers in electrophysiology studies. Compounds (S)-4 and (S)-5 demonstrated significant activity in reducing neuronal hyperexcitability in rat hippocampal slices. The synthesis and the KCNQ2 opener activity of these acrylamides are described.     

Synthesis of stereospecifically deuterated phenylalamines and determination of their configuration.

1. Starting from trans-cinnamic acid a chiral (-)3-phenyl-[2,3-2H]propionic acid has been synthesized using Clostridium kiuyveri cells as catalyst. 2. The chiral dideuterated acid has been converted by chemical methods to a mixture of (2R) and (2S)-phenyl[2,3-2H]-alanine. 3. By means of 1H nuclear magnetic resonance spectroscopy and the action of D and L-amino acid oxidase the configuration of the phenylalamine has been shown to be (2R, 3S) and (2S, 3S), respectively. The labelled phenylalanine is thus sterically and isotopically homogenous at position 3 but heterogenous at position 2.     

Stereospecific oxidation of (R)- and (S)-1-indanol by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

A recombinant Escherichia coli strain which expresses naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized (S)-1-indanol to trans-(1S,3S)-indan-1,3-diol (95.5%) and (R)-3-hydroxy-1-indanone (4.5%). The same cells oxidized (R)-1-indanol to cis-1,3-indandiol (71%), (R)-3-hydroxy-1-indanone (18.2%), and cis-1,2,3-indantriol (10.8%). Purified NDO oxidized (S)-1-indenol to both syn- and anti-2,3-dihydroxy-1-indanol.     

5'-Fluoro-5'-deoxyaristeromycin

5'-Fluoro-5'-deoxyaristeromycin (2) has been prepared via a Mitsunobu coupling of (1S,2S,3R,4S)-2,3-(cyclopentylidenedioxy)-4-fluoromethylcyclopentan-1-ol with N6-bis-boc protected adenine. This procedure is adaptable to preparing a number of 5'-fluoro-5'-deoxycarbocyclic nucleoside analogs with diversity in the heterocyclic base. Antiviral analysis found promising activity for 2 toward measles but no other viruses. No cytotoxicity was observed for 2.     

Biosynthesis of 1-alkenes in higher plants: stereochemical implications. A model study with Carthamus tinctorius (Asteraceae)

Odd numbered 1-alkenes, such as 1-pentadecene or 1,8,11,14-heptadecatetraene are formed from palmitic or linolenic acid by fragmentative decarboxylation. Incubation studies with germinating safflower (Carthamus tinctorius) and (2R,3R)-12-phenyl[2,3-2H2]dodecanoic acid, (2S,3S)-12-phenyl[2,3-2H2]dodecanoic acid, (2R)-12-phenyl[2-2H]dodecanoic acid and (2S)-12-phenyl[2-2H]dodecanoic acid instead of the natural alpha-linolenic acid precursor revealed the fragmentation to be an overall anti elimination of the 3-pro(S) hydrogen and the carboxyl group (anti-periplanar transition state geometry). Externally offered 3-hydroxy acids are not fragmented to 1-alkenes. The most probable mechanistic alternatives are in agreement with abstraction of the 3-pro(S) hydrogen as a radical followed by electron transfer and fragmentation, or transient insertion of oxygen into the 3-pro(S) C-H bond and subsequent fragmentation into an 1-alkene and CO2 after appropriate activation. The mechanism seems to be of general importance for the biosynthesis of vinylic substructures of natural products from oxygenated precursors.     

Novel (2R,3R)-2,3-butanediol dehydrogenase from potential industrial strain Paenibacillus polymyxa ATCC 12321

A (2R,3R)-2,3-butanediol dehydrogenase (BDH99::67) from Paenibacillus polymyxa ATCC 12321 was functionally characterized. The genetic characteristics of BDH99::67 are completely different from those of meso- and (2S,3S)-2,3-butanediol dehydrogenases. The results showed that BDH99::67 belongs to the medium-chain dehydrogenase/reductase superfamily and not to the short-chain dehydrogenase/reductase superfamily, to which meso- and (2S,3S)-2,3-butanediol dehydrogenases belong.     

Synthesis and structure determination of some sugar amino acids related to alanine and 6-deoxymannojirimycin.

(5'R)-5'-Methyl-5'-[methyl (4S)-2,3-O-isopropylidene-beta-L-erythrofuranosid-4-C-yl]-imidazolidin-2',4'-dione was synthesised starting from methyl 6-deoxy-2,3-O-isopropylidene-alpha-D-lyxo-hexofuranosid-5-ulose applying the Bucherer-Bergs reaction. Its 5'-R configuration was confirmed by X-ray crystallography. Corresponding alpha-amino acid-methyl (5R)-5-amino-5-C-carboxy-5,6-dideoxy-alpha-D-lyxo-hexofuranoside (alternative name: 2-[methyl (4S)-2,3-O-isopropylidene-beta-L-erythrofuranosid-4-C-yl]-D-alanine) was obtained from the above hydantoin by acid hydrolysis of the isopropylidene group followed by basic hydrolysis of the hydantoin ring. Total deprotection afforded 5-C-carboxy-6-deoxymannojirimycin. Analogously, methyl (5S)-5-amino-5-C-carboxy-5,6-dideoxy-alpha-L-lyxo-hexofuranoside and 5-C-carboxy-6-deoxy-L-mannojirimycin were prepared from the corresponding (5'S)-5'-methyl-5'-[methyl (4R)-2,3-O-isopropylidene-beta-D-erythrofuranosid-4-C-yl]-imidazolidin-2',4'-dione starting from methyl 6-deoxy-2,3-O-isopropylidene-alpha-L-lyxo-hexofuranosid-5-ulose.     

On the steric course of the adenosylcobalamin-dependent 2-methyleneglutarate mutase reaction in Clostridium barkeri

The enzymatically active enantiomer of 3-methylitaconate in Clostridium barkeri has (R)-configuration. This was checked by fermentation of the racemate and reisolation of the (S)-enantiomer. In addition (R)-3-methylitaconate was synthesized by enzymatic isomerisation of 2,3-dimethylmaleate which was protonated at the Si-face. 2-Methylene[2-2H1]glutarate was synthesized via (R)-3-methyl[3-2H1]itaconate by brief incubation of 2,3-dimethylmaleate with a cell-free extract of Clostridium barkeri in 2H2O. The predominantly monodeuterated compound was oxidized to (S)-[2-2H1]succinate as analysed by circular dichroism. The results demonstrate that 2-methyleneglutarate mutase catalyses the reversible migration of an acryloyl residue from the alpha-carbon to the beta-carbon of propionate with inversion of configuration at the alpha-carbon.