Aliphatic Halogenase Enables Late‐Stage C−H Functionalization: Selective Synthesis of a Brominated Fischerindole Alkaloid with Enhanced Antibacterial Activity

The anion promiscuity of a newly discovered standalone aliphatic halogenase WelO5 was probed and enabled the selective synthesis of 13R‐bromo‐12‐epi‐fischerindole U via late‐stage enzymatic functionalization of an unactivated sp³ C?H bond. Pre‐saturating the WelO5 active site with a non‐native bromide anion was found to be critical to the highly selective in vitro transfer of bromine, instead of chlorine, to the target carbon center and also allowed the relative binding affinity of bromide and chloride towards the WelO5 enzyme to be assessed. This study further revealed the critical importance of halogen substitution on modulating the antibiotic activity of fischerindole alkaloids and highlights the promise of WelO5‐type aliphatic halogenases as enzymatic tools to fine‐tune the bioactivity of complex natural products.     

Direct Evidence for Membrane Transport of Host-Plant-Derived Pyrrolizidine Alkaloid N-Oxides in Two Leaf Beetle Genera

The chrysomelid leaf beetles Longitarsus jacobaeae, Oreina cacaliae, and O. speciosissima sequester pyrrolizidine alkaloids from their asteracean host plants and store them as nontoxic N-oxides. Previous analyses showed that Longitarsus is able to N-oxidize protoxic tertiary PAs, but did not resolve in which form N-oxides are taken up. For Oreina, beetles seem able to directly transmit the polar PA N-oxides from the gut into the hemolymph and prevent any reduction of them in the gut yielding protoxic free bases. Here, we confirm the predicted direct uptake of PAs as N-oxides by Oreina, and elucidate the situation for Longitarsus by applying double-labeled [14C]senecionine [18O]N-oxide as tracer. The beetles were fed with the tracer and subsequently senecionine N-oxide was recovered from the defensive secretions (Oreina) and beetle extracts (Longitarsus), purified by HPLC, and submitted to ESI-MS, GC-MS, and analysis of the specific radioactivity. The 18O-label is retained without any loss in the labeled senecionine N-oxide recovered from the two Oreina species. Analysis of the Longitarsus experiment was complicated by a contamination of the HPLC-purified senecionine N-oxide with a second compound, identified as a dihydrosenecionine N-oxide by high-resolution CID analysis. The dihydrosenecionine N-oxide, probably the 15,20-dihydro derivative, constitutes a major idiosyncratic senecionine metabolite present in the beetle. The recovered senecionine N-oxide retained 74% 18O-label. The remaining 25% is mostly due to loss of 18O by reduction and subsequent re-N-oxidation. The experiments confirm for both beetle genera a direct uptake of the polar nontoxic PA N-oxides, which requires specific membrane carriers. Accumulation of detrimental free base PA is prevented by glucosylation (Oreina) or N-oxidation (Longitarsus).     

Winter Cherry Bugs Feed on Plant Tropane Alkaloids and De-epoxidize Scopolamine to Atropine

The winter cherry bug colonizes the Duboisia leichhardtii tree, which is a rich source of scopolamine. It consumes the tropane alkaloids atropine and scopolamine. Quantitative analysis revealed that the ratio of scopolamine to atropine in the winter cherry bug (0.46) was far from that found in the leaves of the host plant (7.20). To elucidate whether the winter cherry bugs selectively excrete or decompose scopolamine, they were fed scopolamine and/or atropine together with sucrose. They took up scopolamine as well as atropine, and converted scopolamine into atropine.     

Quinolizidine alkaloids in Ormosia arborea seeds inhibit predation but not hoarding by agoutis (Dasyprocta leporina)

Quinolizidine alkaloids (QAs) are secondary compounds found in seeds of many species of plants, possibly protecting them against pathogens and seed predators. QAs were isolated from Ormosia arborea seeds and bioassayed against red-rumped agoutis (Dasyprocta leporina, Rodentia: Caviomorpha) to verify if they inhibit seed predation and food hoarding (seed dispersal). Three treatments were used: (1) seeds of O. arborea, (2) palatable seeds of Mimusops coriacea (Sapotaceae) treated with MeOH, and (3) seeds of M. coriacea treated with QAs dissolved in MeOH in similar concentration to that present in O. arborea. Palatable seeds were significantly more preyed upon than seeds treated with QAs and Ormosia seeds, but QAs did not influence hoarding behavior. QAs in O. arborea may have a strong effect in avoiding seed predation by rodents, without reducing dispersal.     

Do Naïve Ruminants Degrade Alkaloids in the Rumen?

Three different methods for the culture of rumen microorganisms (Hungates technique, the Hohenheim in vitro gas production method, and the semicontinuous rumen simulation technique) were employed to study the influence of various alkaloids (sparteine, lupanine, cytisine, atropine, quinidine, lobeline, harmaline, arecoline, nicotine, caffeine, pilocarpine, gramine, senecionine, and monocrotaline) on rumen microorganisms. Rumen microorganisms from naive ruminants (sheep, cattle) that had not been exposed to the alkaloids before were generally not able to degrade most of the alkaloids. Only the alkaloids pilocarpine, gramine, and monocrotaline appeared to be degradable. Rumen microorganisms from a sheep preconditioned to lupin alkaloids tolerated lupanine much better than nonadapted microorganisms, but no degradation occurred. The findings indicate that the main site of detoxification in naive ruminants is not the rumen but more likely the liver and kidneys as in nonruminants.     

Bovine neuronal vesicular glutamate transporter activity is inhibited by ergovaline and other ergopeptines

l-Glutamate (Glu) is a major excitatory neurotransmitter responsible for neurotransmission in the vertebrate central nervous system. Vesicular Glu transporters VGLUT1 and VGLUT2 concentrate (50 mM) Glu [Michaelis constant (measuring affinity), or K_m, = 1 to 4 mM] into synaptic vesicles (SV) for subsequent release into the synaptic cleft of glutamatergic neurons. Vesicular Glu transporter activity is dependent on vacuolar H⁺-ATPase function. Previous research has shown that ergopeptines contained in endophyte-infected tall fescue interact with dopaminergic and serotoninergic receptors, thereby affecting physiology regulated by these neuron types. To test the hypothesis that ergopeptine alkaloids inhibit VGLUT activity of bovine cerebral SV, SV were isolated from cerebral tissue of Angus-cross steers that were naive to ergot alkaloids. Immunoblot analysis validated the enrichment of VGLUT1, VGLUT2, synaptophysin 1, and vacuolar H⁺-ATPase in purified SV. Glutamate uptake assays demonstrated the dependence of SV VGLUT-like activity on the presence of ATP, H⁺-gradients, and H⁺-ATPase function. The effect of ergopeptines on VGLUT activity was evaluated by ANOVA. Inhibitory competition (IC₅₀) experiments revealed that VGLUT-mediated Glu uptake (n = 9) was inhibited by ergopeptine alkaloids: bromocriptine (2.83 ± 0.59 μM) < ergotamine (20.5 ± 2.77 μM) < ergocornine (114 ± 23.1 μM) < ergovaline (137 ± 6.55 μM). Subsequent ergovaline kinetic inhibition analysis (n = 9; Glu = 0.05, 0.10, 0.50, 1, 2, 4, 5 mM) demonstrated no change in apparent K_m. However, the maximum reaction rate (V_max) of Glu uptake was decreased when evaluated in the presence of 50, 100, and 200 μM ergovaline, suggesting that ergovaline inhibited SV VGLUT activity through a noncompetitive mechanism. The findings of this study suggest cattle with fescue toxicosis may have a decreased glutamatergic neurotransmission capacity due to consumption of ergopeptine alkaloids.     

Chemical basis of egg cannibalism in a caterpillar (Utetheisa ornatrix)

Larvae of the mothUtetheisa ornatrix are shown to cannibalize eggs in the laboratory. They proved most cannibalistic if they were systemically deficient in pyrrolizidine alkaloid (PA), the defensive agent that protectsUtetheisa at all stages of development against predation, and whichUtetheisa acquire as larvae from their food plant. In exercising cannibalistic choice,Utetheisa larvae feed preferentially on eggs that are PA-Iaden rather than PA-free. Egg cannibalism can therefore provideUtetheisa with a supplemental means of PA procurement. Moreover, presence of PA in the egg, while providing the egg with defense against predation, can increase its vulnerability to cannibalism. Although evidence is presented thatUtetheisa larvae cannibalize eggs in nature, it is argued that such feeding may occur only opportunistically in the wild, rather than as a matter of course.Paper No. 104 of the series Defense Mechanisms of Arthropods, No. 103 in Eisner and Eisner, 1991.Psyche 98:111–118.     

Effects of Root Isoquinoline Alkaloids from <Emphasis Type="Italic">Hydrastis canadensis</Emphasis> on <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> Isolated from <Emphasis Type="Italic">Hydrastis</Emphasis> Root Tissue

Goldenseal (Hydrastis canadensis L.) is a popular medicinal plant distributed widely in North America. The rhizome, rootlets, and root hairs produce medicinally active alkaloids. Berberine, one of the Hydrastis alkaloids, has shown antifungal activity. The influence of a combination of the major Hydrastis alkaloids on the plant rhizosphere fungal ecology has not been investigated. A bioassay was developed to study the effect of goldenseal isoquinoline alkaloids on three Fusarium isolates, including the two species isolated from Hydrastis rhizosphere. The findings suggest that the Hydrastis root extract influences macroconidia germination, but that only the combined alkaloids—berberine, canadine, and hydrastine—appear to synergistically stimulate production of the mycotoxin zearalenone in the Fusarium oxysporum isolate. The Hydrastis root rhizosphere effect provided a selective advantage to the Fusarium isolates closely associated with the root tissue in comparison with the Fusarium isolate that had never been exposed to Hydrastis.     

响应面法优化水提白附片中的总生物碱

以白附片为原料,利用酸水法提取,在单因素实验基础上根据Box-Behnken实验设计原理,采用四因素三水平的响应面分析法对总生物碱的提取工艺进行了优化.通过响应面实验,建立了白附片中总生物碱提取率与四个因素变化的二次回归方程.同时依据回归模型进行了计算机模拟实验及绘制曲面图,了解白附片中总生物碱提取率随主要因素水平的变化方向及优化点.结果表明:白附片中总生物碱提取的最佳工艺条件为pH 3.4,液料比11 mL/g,温度30℃,时间4.6 h,在此条件下,白附片中总生物碱的提取率为48.5％±1.7％.     

芋艿皮渣生物碱提取工艺研究

生物碱是一类具有生理活性的物质,是许多药用植物的有效成分,具有多种生物活性,在植物体内还具有良好的分布和较强的输导能力,是高效、低毒、无污染、对人畜安全的天然产物,在医药和农药领域有广阔的应用前景。本文研究了芋艿总生物碱的提取工艺技术。以盐酸小檗碱为标准物质,采用酸性染料比色法测定生物碱含量。考察乙醇浓度,料液比,提取时间,提取温度四个因素对总生物碱提取效果的影响。实验结果表明:影响芋艿总生物碱提取效果的因素主次顺序为乙醇浓度,并通过正交实验确定最佳提取工艺条件为乙醇70%,料液比1∶15,提取时间2.5h,提取温度60℃,此条件生物碱提取率为0.212%。