Morphinan alkaloids in Papaver bracteatum: Biosynthesis and fate

The known metabolic pathway for hydrophenanthrene alkaloids in Papaver somniferum has been examined for occurrence in P. bracteatum, a species reported to contain thebaine but no codeine or morphine. 1,2-Dehydro-reticulinium-[3-¹⁴C] chloride and (±)-reticuline-[3-¹⁴C] were fed to P. bracteatum plants and both were incorporated, the former into reticuline and thebaine and the latter into thebaine, suggesting that thebaine biosynthesis is the same in the two species. Studies of the natural abundance of morphinan alkaloids in P. bracteatum and the results from feeding codeinone-[16-³H] and codeine-[16-³H] indicate that this species can reduce codeinone to codeine but can not perform either of the demethylations to produce codeinone or morphine. Fed thebaine-[16-³H] was substantially metabolized but not by pathways that involved demethylations to either oripavine or northebaine.     

Alkaloid biosynthesis in Croton flavens

The biosynthesis of the morphinandienone alkaloids norsinoacutine, sinoacutine and flavinantine has been studied using 1-³ H-sinoacutine, 1-³H-norsinoacutine, 1-³H-norsinoacutinols, l-[S-methyl-¹⁴C]-methionine, glycine-2-¹⁴C, 1-³H-8,14-dihydronorsalutaridine, 1-³ H-8,14-dihydrosalutaridine, 1-³H-sinomenine, 1-³H-isosinomenine, (±)-[2-¹⁴C]phenylalanine, (±)-[N-methyl-¹⁴C]orientaline and (±)-[N-methyl-¹⁴C]reticuline.     

Reticuline: A dopamine receptor blocker

Reticuline, a benzylisoquinoline alkaloid, inhibited specific [³H] dopamine binding to dopamine receptors in tissue homogenates from rat corpora striata. The alkaloid blocked amphetamine-induced circling behavior in mice with unilateral (chemically-induced) degeneration of dopaminergic neurons in the corpus striatum. Blockade of apomorphine-induced climbing behavior by reticuline was observed in mice. Reticuline did not produce catalepsy at doses which blocked circling behavior. These results show that reticuline is a dopamine receptor blocking agent in the central nervous system.     

Purification and characterization of the berberine bridge enzyme from berberis beaniana cell cultures

The berberine bridge-forming enzyme (BBE) has been found in 66 samples taken from differentiated plants and from cell suspension cultures. It was purified 450-fold from Berberis beaniana cell cultures by gel-filtration, DEAE and phenyl-Sepharose chromatography, electrophoresis and isoclectric focusing. The enzyme was shown to be homogeneous by gel electrophoresis (M_r = 52 kD ± 4). The enzyme, which requires the presence of oxygen, catalyses the conversion of the (S)-enantiomers of reticuline, protosinomenine and laudanosoline to the corresponding (S)-tetrahydroprotoberberines and released stoichiometric amounts of H₂O₂. Within the cells the enzyme is located in a particle with the density p = 1.14 g/ml.     

Aporphine alkaloids from Phoebe pittieri

The wood and bark of Phoebe pittieri afforded 1,2,3-trimethoxy-9,10-methylenedioxynoraporphine and 1,2,9-trimethoxy-10-hydroxynoraporphine (noraporphine), which are new alkaloids, and norpurpureine and reticuline, which have not been reported from this source before.     

Azafluorenones from Oxandra cf. major and biogenetic considerations

The stem bark of Oxandra cf. major, which contains large amounts of 7,7′-bisdehydroaporphine alkaloids, has also yielded three commonplace steroids, reticuline, four known aporphinoids and the new azafluorenones darienine, macondine and ursuline. Three biogenetic hypotheses are discussed in the light of the structures of the azafluorenones found to date in the Annonaceae.     

Peroxidases of Papaver somniferum

Extracts of Papaver somniferum that had peroxidase activity were ineffective in catalysing oxidation of reticuline. Two peroxidases were purified from young seedlings and their properties examined. Only one of them was active toward indole-3-acetic acid (IAA).     

Biotransformation of reticuline into coreximine,scoulerine, pallidine,and isoboldine with rat liver enzyme

(±)-Reticuline (1) was biotransformed into the protoberberine alkaloids, coreximine (12) and scoulerine (10), the morphinandienone alkaloid, pallidine (14), and the aporphine alkaloid, isoboldine (16). The transformation was stimulated by O₂ and the cofactor NAD, NADP, or NADPH, NADPH being more effective than the other cofactors. The N-methyl group of (±)-reticuline was not incorporated intact into protoberberines.     

Knockdown of berberine bridge enzyme by RNAi accumulates (<Emphasis Type="Italic">S</Emphasis>)-reticuline and activates a silent pathway in cultured California poppy cells

Reticuline is a key compound in the biosynthetic pathway for isoquinoline alkaloids in plants, which include morphine, codeine and berberine. We established cultured California poppy (Eschscholzia californica) cells, in which berberine bridge enzyme (BBE) was knocked down by RNA interference, to accumulate the important key intermediate reticuline. Both BBE mRNA accumulation and enzyme activity were effectively suppressed in transgenic cells. In these transgenic cells, end-products of isoquinoline alkaloid biosynthesis, such as sanguinarine, were considerably reduced and reticuline was accumulated at a maximum level of 310 μg/g-fresh weight. In addition, 1 g-fresh weight of these cells secreted significant amounts of reticuline into the medium, with a maximum level of 6 mg/20 mL culture medium. These cells also produced a methylated derivative of reticuline, laudanine, which could scarcely be detected in control cells. We discuss the potential application of RNAi technology in metabolic modification and the flexibility of plant secondary metabolism.     

大花地不容块根生物碱成分的研究

为研究大花地不容块根的生物碱成分,该研究采用酸提碱沉法从大花地不容块根中提取出总生物碱,通过硅胶柱色谱法和制备液相色谱技术对大花地不容块根的总生物碱提取物进行分离纯化,并利用波谱学手段对化合物进行结构鉴定。结果表明:(1)从总生物碱提取物中分离得到11个化合物,分别鉴定为青藤碱(1)、青风藤碱(2)、斯帝酚灵碱(3)、瑞枯灵(4)、异紫堇定(5)、紫堇单酚碱(6)、巴婆碱(7)、sukhodianine(8)、荷包牡丹碱(9)、7-氧代克班宁(10)和巴马汀(11)。(2)体外细胞毒性测试显示,大花地不容总生物碱和其主成分青藤碱对人类肺癌细胞A549的IC₅₀值分别为7.5×10^-4 g·mL^-1和6.59×10^-9 g·mL^-1。化合物2、3、4、7、8、9和10系首次从大花地不容中分离得到。大花地不容块根中含有吗啡烷、原阿朴啡、阿朴啡、苄基四氢异喹啉和原小檗碱5种类型的生物碱。     

Chemical constituents from the bark of Annona salzmannii (Annonaceae)

1. Download : Download full-size image

Highlights? Bark of Annona salzmannii yielded sesquiterpenes, steroids, and isoquinoline alkaloids. ? Isoquinoline alkaloids are the chemotaxonomic marker of the genus Annona. ? The NMR data for reticuline (12) were revised. ? Selin-11-en-4α-ol (3) is reported for the first time in the genus Annona.     

Catecholamine receptors and cyclic AMP formation in the central nervous system: effects of tetrahydroisoquinoline derivatives.

The ability of a series of tetrahydroisoquinoline (THIQ) alkaloids to inhibit the binding of radioligands to catecholamine receptors in the CNS has been examined. $\text{(+)}$ THP was the most potent inhibitor of [³H] dihydroalprenolol binding to β-adrenergic receptors and of [³H] haloperidol to dopaminergic receptors and was the least potent inhibitor of [³H] WB-4101 binding to α-adrenergic receptors. Other THIQ alkaloids examined such as salsoline, salsolinol, and reticuline were less potent than $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic and dopaminergic receptors, and more potent than $\text{(+)}$ THP in inhibiting radioligand to α-adrenergic receptors. The marked potency of $\text{(+)}$ THP in inhibiting radioligand binding to β-adrenergic receptors (IC₅₀ ～ 10^?7 M) was confirmed by the potency of this compound in inhibiting (?) isoproternol elicited accumulations of cyclic AMP in brain slice preparations. These data indicate that, if formed $\text{in}$$\text{vivo}$ during alcohol consumption, THIQ derivatives such as THP may affect catecholamine neurons in the CNS.     

The preparation and properties of a stable mercury derivative of transfer RNAs from Escherichia coli

Further investigations into the properties of the mercury derivative formed by the reaction of 4-thiouridine-containing tRNAs and pentafluorophenylmercury chloride have been carried out. tRNA^fMet (which contains only one 4-thiouridine residue) has been isolated by a one-step column Chromatographic procedure from unfractionated Escherichia coli tRNA and has been shown to react with the mercury compound to give a derivative which has similar properties to those previously reported for the corresponding mercury derivative of tRNA^Tyr which contains two adjacent 4-thiouridine residues. The mercury derivative of tRNA^Tyr appears to be a competitive inhibitor of tRNA^Tyr in the aminoacylation reaction (tRNA^TyrK_m = 0.42 μM, mercury derivative of tRNA^TyrK_i = 0.11 μM). The mercury derivative of Tyr-tRNA^Tyr can be made, but only by the reaction of the mercury compound with the aminoacylated tRNA.     

Intramolecular participation of the amide group in the hydrolysis of p-nitrophenyl N-(bromoacetyl) anthranilate

The rate of hydrolysis of p-nitrophenyl N-(bromoacetyl) anthranilate (Ib) has been measured in aqueous solution between pH 1 and 6.5 has been found to increase linearly with pH at pH higher than 3. An abnormally large apparent alkaline rate constant of 3.8 × 10⁶M^?1 sec^?1 has been determined. Intramolecular nucleophilic displacement by the amide group at the carbonyl carbon of the ester occurred and a cyclic intermediate was formed. This intermediate has been detected by direct isolation and by measurements of the proton release accompanying the reaction. The rates of hydrolysis of analogous derivatives (IIb-IIIb-IV), for which this intramolecular assistance was not possible, were slower by a factor of about 5 × 10⁵. Such an example of intramolecular catalysis may be useful for a better understanding of the enzymatic catalysis.     

Purification and properties of a D-fructose 1,6-bisphosphatase from Saccharomyces cerevisiae.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) from Saccharomyces cerevisiae has been purified to homogeneity. A molecular weight of 115,000 has been obtained by gel filtration. The enzyme appears to be a dimer with identical subunits. The apparent K_m for fructose bisphosphatase varies with the Mg²⁺ concentration of the enzyme, being 1 × 10^?6m at 10 mm Mg²⁺ and 1 × 10^?5m at 2 mm Mg²⁺. Other phosphorylated compounds are not significantly hydrolyzed by the enzyme. An optimum pH of 8.0 is exhibited by the enzyme. This optimum is not changed by addition of EDTA. AMP inhibits the enzyme with a K_i of 8.0 × 10^?5m at 25 °C. The inhibition is temperature dependent, the value of K_i increasing with raising temperature. 2-Deoxy-AMP is also inhibitory with a K_i value at 25 °C of 1.6 × 10^?4m. An ordered uni-bi mechanism has been deduced for the reaction with phosphate leaving the enzyme as the first product and the fructose 6-phosphate as the second one.     

Cytochrome P450 3A Enzymes Catalyze the O6-Demethylation of Thebaine,a Key Step in Endogenous Mammalian Morphine Biosynthesis

Morphine, first characterized in opium from the poppy Papaver somniferum, is one of the strongest known analgesics. Endogenous morphine has been identified in several mammalian cells and tissues. The synthetic pathway of morphine in the opium poppy has been elucidated. The presence of common intermediates in plants and mammals suggests that biosynthesis occurs through similar pathways (beginning with the amino acid l-tyrosine), and the pathway has been completely delineated in plants. Some of the enzymes in the mammalian pathway have been identified and characterized. Two of the latter steps in the morphine biosynthesis pathway are demethylation of thebaine at the O³- and the O⁶-positions, the latter of which has been difficult to demonstrate. The plant enzymes responsible for both the O³-demethylation and the O⁶-demethylation are members of the Fe^II/α-ketoglutarate-dependent dioxygenase family. Previous studies showed that human cytochrome P450 (P450) 2D6 can catalyze thebaine O³-demethylation. We report that demethylation of thebaine at the O⁶-position is selectively catalyzed by human P450s 3A4 and 3A5, with the latter being more efficient, and rat P450 3A2. Our results do not support O⁶-demethylation of thebaine by an Fe^II/α-ketoglutarate-dependent dioxygenase. In rat brain microsomes, O⁶-demethylation was inhibited by ketoconazole, but not sulfaphenazole, suggesting that P450 3A enzymes are responsible for this activity in the brain. An alternate pathway to morphine, oripavine O⁶-demethylation, was not detected. The major enzymatic steps in mammalian morphine synthesis have now been identified.     

Reversion from Suppression to Nonsuppression in SUQ5 [psi+] Strains of Yeast: The Classification of Mutations

Reversion from the suppressed to nonsuppressed phenotype in strains of genotype SUQ5 [psi⁺] ade2-1 his5-2 lys1-1 can1-100 ura3-1 has been induced by treatment with ethyl methanesulphonate, nitrosoguanidine or UV (254 nm) light. Spontaneously occurring revertants have also been selected by two different methods. Reversion has been shown to occur through a variety of nuclear mutations and through mutation of [psi⁺] to [psi^-]. Nuclear mutations included back-mutation of SUQ5, antisuppressor mutations that were recessive, semi-dominant or dominant, and dominant or recessive mutations of genes required for the maintenance of the [psi⁺] factor. Complementation tests by which the various kinds of mutations could be distinguished from one another were designed. The spectra of spontaneously occurring and induced mutations have been described.     

A new regioselective method of macrobicyclic Schiff bases synthesis

A new regioselective method of di-gem-thio-substituted PNP-crown derivatives synthesis is presented. The geminalmercaptoethanolanetricyclophosphaza-PNP-lariat ether structure has been determined by X-ray crystallography and characterised by ab initio calculations. The 16-membered PNP-crown ether ring exists in unique conformation: ac⁻ap ac⁻sc⁺sc⁺ap sc⁺ap ap sc⁻ap sc⁻sc⁻ac⁺ap ac⁺. All the ether oxygen atoms are directed into the interior of the ring. All endocyclic P-N bond lengths are equal within experimental error with the mean value 1.578(2) Å. The P-S bond properties have been characterised in terms of natural bond orbital (NBO) analysis, and its interactions with other NBO have been described. The spirocyclisation mechanism at cyclophosphazene phosphorus atom has been proposed.     

In vitro iodination of plant ribonucleic acids

The optimum conditions for in vitro iodination of RNAs have been established which yield specific radioactivities ranging from 10 × 10⁴ to 10 × 10⁶ cpm/μg. A nomogram has been constructed by correlating specific radioactivities of RNA with concentration of KI, RNA, and ¹²⁵I. This nomogram can be used to determine the conditions for the desired specific radioactivities for any unknown RNA. The in vitro iodinated RNA has been compared with in vivo labeled RNA for hybridization characteristics. Competition hybridization between ¹²⁵I-labeled chloroplast-rRNA and unlabeled pea (Pisum sativum) chloroplast-rRNA was identical to that found using [³²P]chloroplast-rRNA. Thermal stability of DNA-¹²⁵I-rRNA hybrids was similar to the thermal stability of DNA-[³²P]rRNA hybrids. The iodinated RNA was not found to have undergone any changes in its hydrogen-bonding properties.