Substrate specificity of NovM: implications for novobiocin biosynthesis and glycorandomization

[reaction: see text] In an effort to expand the scope of natural product in vitro glycorandomization (IVG), the substrate specificity of NovM was investigated. A test of four aglycon analogues and over 40 nucleotide sugars revealed NovM has a surprisingly stringent substrate specificity and provided only three new "unnatural" natural products. On the basis of the determined substrate specificity, an alternative to the sugar nucleotide biosynthetic dogma and a cautionary note for the general applicability of IVG are introduced.     

Does activation of the anti proton, rather than concertedness, determine the stereochemistry of base-catalyzed 1,2-elimination reactions? Anti stereospecificity in E1cB eliminations of β-3-trifluoromethylphenoxy esters, thioesters, and ketones

As part of a comprehensive investigation on the stereochemical aspects of base-catalyzed 1,2-elimination reactions, we have studied a set of acyclic carbonyl substrates that react by an irreversible E1cB mechanism with largely anti stereospecificity. (2)H NMR data show that these reactions using KOH in EtOH/H(2)O under non-ion-pairing conditions produce a minimum of 85-89% anti elimination on stereospecifically labeled tert-butyl (2R*,3R*)- and (2R*,3S*)-3-(3-trifluoromethylphenoxy)-2,3-(2)H(2)-butanoate, S-tert-butyl (2R*,3R*)- and (2R*,3S*)-3-(3-trifluoromethylphenoxy)-2,3-(2)H(2)-butanethioate, and the related ketones, (4R*,5R*)- and (4R*,5S*)-5-(3-trifluoromethylphenoxy)-4,5-(2)H(2)-3-hexanone. With both diastereomers of each substrate available, the KIEs can be calculated and the innate stereoselectivities determined. The elimination reactions of the β-3-trifluoromethylphenoxy substrates occur by E1cB mechanisms with diffusionally equilibrated enolate-anion intermediates. Thus, it is clear that anti elimination does not depend solely upon concerted E2 mechanisms. Negative hyperconjugation provides a satisfactory explanation for the anti stereospecificity exhibited by our carbonyl substrates, where the leaving group activates the anti proton, leading to the enolate intermediate. The activation of the anti proton by negative hyperconjugation may also play a role in the concerted pathways of E2 mechanisms. We have also measured the rates of the hydroxide-catalyzed elimination reactions of butanoate, thiobutanoate, and ketone substrates in EtOH/H(2)O, with β-tosyloxy, acetoxy, and 3-trifluoromethylphenoxy nucleofuges.     

Effects of Pulsed Electric Field on Secondary Metabolism of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. Gamay Fréaux Suspension Culture and Exudates

Plant cell cultures provide a large potential for the production of secondary metabolites. Through the application of different physical and chemical cell stress factors, we investigated the production of the secondary metabolites in plant cell cultures. The effects of pulsed electric field (PEF) and ethephon on growth and secondary metabolism, particularly anthocyanins and phenolic acids synthesis, were investigated by using suspension culture of Vitis vinifera L. cv. Gamay Fréaux as a model system. Anthocyanins were measured by spectrophotometer and extracellular phenolic acids were determined by high-performance liquid chromatography. The compounds were identified by liquid chromatography–mass spectrometry and nuclear magnetic resonance. After the treatments with PEF and ethephon, the concentrations of anthocyanins and phenolic acids in cell culture were higher than in the control, without loss of biomass. The combination of PEF treatment and ethephon improved secondary metabolites formation. Production levels of extracellular phenolic acids, 3-O-glucosyl-resveratrol were increased by PEF and ethephon treatments. The results show that PEF induced a defense response of plant cells and may have altered the cell/membrane’s dielectric properties. PEF, an external stimulus or stress, is proposed as a promising new abiotic elicitor for stimulating secondary metabolites biosynthesis in plant cell cultures.     

Organic matter flow in the food web at a temperate heath under multifactorial climate change

The rising atmospheric CO(2) concentration, increasing temperature and changed patterns of precipitation currently expose terrestrial ecosystems to altered environmental conditions. This may affect belowground nutrient cycling through its intimate relationship with the belowground decomposers. Three climate change factors (elevated CO(2), increased temperature and drought) were investigated in a full factorial field experiment at a temperate heathland location. The combined effect of biotic and abiotic factors on nitrogen and carbon flows was traced in plant root → litter → microbe → detritivore/omnivore → predator food-web for one year after amendment with (15)N(13)C(2)-glycine. Isotope ratio mass spectrometry (IRMS) measurement of (15)N/(14)N and (13)C/(12)C in soil extracts and functional ecosystem compartments revealed that the recovery of (15)N sometimes decreased through the chain of consumption, with the largest amount of bioactive (15)N label pool accumulated in the microbial biomass. The elevated CO(2) concentration at the site for 2 years increased the biomass, the (15)N enrichment and the (15)N recovery in detritivores. This suggests that detritivore consumption was controlled by both the availability of the microbial biomass, a likely major food source, and the climatic factors. Furthermore, the natural abundance δ(13)C of enchytraeids was significantly altered in CO(2)-fumigated plots, showing that even small changes in δ(13)C-CO(2) can be used to detect transfer of carbon from primary producers to detritivores. We conclude that, in the short term, the climate change treatments affected soil organism activity, possibly with labile carbohydrate production controlling the microbial and detritivore biomass, with potential consequences for the decomposition of detritus and nutrient cycling. Hence, there appears to be a strong coupling of responses in carbon and nitrogen cycling at this temperate heath.     

Vibrationally mediated photodissociation of ammonia: the influence of N-H stretching vibrations on passage through conical intersections

Velocity map ion imaging of the H atoms formed in the photodissociation of vibrationally excited ammonia molecules measures the extent of adiabatic and nonadiabatic dissociation for different vibrations in the electronically excited state. Decomposition of molecules with an excited symmetric N-H stretch produces primarily ground state NH(2) along with a H atom. The kinetic energy release distribution is qualitatively similar to the ones from dissociation of ammonia excited to the electronic origin or to several different levels of the bending vibration and umbrella vibration. The situation is very different for electronically excited molecules containing a quantum of antisymmetric N-H stretch. Decomposition from that state produces almost solely electronically excited NH(2)*, avoiding the conical intersection between the excited state and ground state surfaces. These rotationally resolved measurements agree with our previous inferences from lower resolution Doppler profile measurements. The production of NH(2)* suggests that the antisymmetric stretching excitation in the electronically excited molecule carries it away from the conical intersection that other vibrational states access.     

Modeling of mass and charge transfer in an inverted rotating disk electrode (IRDE) reactor

In this work, the validation of a newly constructed inverted rotating disk electrode (IRDE) reactor is reported. Compared to the rotating disk electrode (RDE) reactor, the working electrode is changed in position from the top to the bottom of the electrochemical cell. The IRDE reactor is designed to facilitate the actual study of gas evolution reactions. It is studied whether the first-order analytical expression for the velocity field in an RDE reactor is also acceptable for an IRDE configuration. To that purpose, the kinetic parameters of the well-known ferri/ferro cyanide redox system are determined in both configurations and compared. This is done qualitatively by comparing the polarization curves obtained in the inverted and the conventional RDE configuration. Additionally, a statistically founded fitting algorithm is used to quantitatively determine the model parameters of the oxidation and reduction reaction. Not only the diffusion coefficients of Fe²⁺ and Fe³⁺ are calculated, but also the rate constants (k_ox and k_red) and the transfer coefficients (α_ox and α_red) are quantified and compared together with their respective standard deviation. It is found that the parameters of mass and charge transfer in both configurations agree well. So it is concluded that the same analytical equations of mass and charge transfer can be used in both the RDE and the IRDE reactor.