Biosynthesis of Indole Diterpene Lolitrems: Radical-Induced Cyclization of an Epoxyalcohol Affording a Characteristic Lolitremane Skeleton

Lolitrems are tremorgenic indole diterpenes that exhibit a unique 5/6 bicyclic system of the indole moiety. Although genetic analysis has indicated that the prenyltransferase LtmE and the cytochrome P450 LtmJ are involved in the construction of this unique structure, the detailed mechanism remains to be elucidated. Herein, we report the reconstitution of the biosynthetic pathway for lolitrems employing a recently established genome-editing technique for the expression host Aspergillus oryzae. Heterologous expression and bioconversion of the various intermediates revealed that LtmJ catalyzes multistep oxidation to furnish the lolitrem core. We also isolated the key reaction intermediate with an epoxyalcohol moiety. This observation allowed us to establish the mechanism of radical-induced cyclization, which was firmly supported by density functional theory calculations and a model experiment with a synthetic analogue.     

Analysis of a Cahn–Hilliard–Brinkman model for tumour growth with chemotaxis

Phase field models recently gained a lot of interest in the context of tumour growth models. Typically Darcy-type flow models are coupled to Cahn–Hilliard equations. However, often Stokes or Brinkman flows are more appropriate flow models. We introduce and mathematically analyse a new Cahn–Hilliard–Brinkman model for tumour growth allowing for chemotaxis. Outflow boundary conditions are considered in order not to influence tumour growth by artificial boundary conditions. Existence of global-in-time weak solutions is shown in a very general setting.     

A probabilistic approach to spectral analysis of growth-fragmentation equations

The growth-fragmentation equation describes a system of growing and dividing particles, and arises in models of cell division, protein polymerisation and even telecommunications protocols. Several important questions about the equation concern the asymptotic behaviour of solutions at large times: at what rate do they converge to zero or infinity, and what does the asymptotic profile of the solutions look like? Does the rescaled solution converge to its asymptotic profile at an exponential speed? These questions have traditionally been studied using analytic techniques such as entropy methods or splitting of operators. In this work, we present a probabilistic approach: we use a Feynman–Kac formula to relate the solution of the growth-fragmentation equation to the semigroup of a Markov process, and characterise the rate of decay or growth in terms of this process. We then identify the Malthus exponent and the asymptotic profile in terms of a related Markov process, and give a spectral interpretation in terms of the growth-fragmentation operator and its dual.     

Solid‐state 31P NMR investigation on the status of guanine nucleotides in paclitaxel‐stabilized microtubules

Microtubule dynamics is a target for many chemotherapeutic drugs. In order to understand the biochemical effects of paclitaxel on the GTPase activity of tubulin, the status of guanine nucleotides in microtubules was investigated by ³¹P cross‐polarization magic angle spinning (CPMAS) NMR. Microtubules were freshly prepared in vitro in the presence of paclitaxel and then lyophilized in sucrose buffer for solid‐state NMR experiments. A ³¹P CPMAS NMR spectrum with the SNR of 25 was successfully acquired from the lyophilized microtubule sample. The broadness of the ³¹P spectral lines in the spectrum indicates that the molecular environments around the guanine nucleotides inside tubulin may not be as crystalline as reported by many diffraction studies. Deconvolution of the spectrum into four spectral components was carried out in comparison with the ³¹P NMR spectra obtained from five control samples. The spectral analysis suggested that about 13% of the nucleotides were present as GTP and 37% as GDP in the β‐tubulin (E‐site) of the microtubules. It was found that most of the GDPs were present as GDP‐P_i complex in the microtubules, which seems to be one of the effects of paclitaxel binding. Copyright © 2015 John Wiley & Sons, Ltd.     

Studies on metabolites and metabolic pathways of bulleyaconitine A in rat liver microsomes using LC‐MSn combined with specific inhibitors

Bulleyaconitine A (BLA) from Aconitum bulleyanum plants is usually used as anti‐inflammatory drug in some Asian countries. It has a variety of bioactivities, and at the same time some toxicities. Since the bioactivities and toxicities of BLA are closely related to its metabolism, the metabolites and the metabolic pathways of BLA in rat liver microsomes were investigated by HPLC–MSⁿ. In this research, the 12 metabolites of BLA were identified according to the results of HPLC‐MSⁿ data and the relevant literature. The results showed that there are multiple metabolites of BLA in rat liver microsomes, including demethylation, deacetylation, dehydrogenation deacetylation and hydroxylation. The major metabolic pathways of BLA in rat liver microsomes were clarified by HPLC‐MS combined with specific inhibitors of CYP450 isoforms. As a result, CYP3A and 2C were found to be the principal CYP isoforms contributing to the metabolism of BLA. Moreover, CYP2D6 and 2E1 are also more important CYP isoforms for the metabolism of BLA. While CYP1A2 only affected the formation rate of M11, its effect on the metabolism of BLA is very small. Copyright © 2014 John Wiley & Sons, Ltd.     

Local atomic and electronic structure in the LiVPO4(F,O) tavorite-type materials from solid-state NMR combined with DFT calculations

⁷Li, ³¹P, and ¹⁹F solid-state nuclear magnetic resonance (NMR) spectroscopy was used to investigate the local arrangement of oxygen and fluorine in LiVPO₄F_1-yO_y materials, interesting as positive electrode materials for Li-ion batteries. From the evolution of the 1D spectra versus y, 2D ⁷Li radiofrequency-driven recoupling (RFDR) experiments combined, and a tentative signal assignment based on density functional theory (DFT) calculations, it appears that F and O are not randomly dispersed on the bridging X position between two X–VO₄–X octahedra (X = O or F) but tend to segregate at a local scale. Using DFT calculations, we analyzed the impact of the different local environments on the local electronic structure. Depending on the nature of the VO₄X₂ environments, vanadium ions are either in the +III or in the +IV oxidation state and can exhibit different distributions of their unpaired electron(s) on the d orbitals. Based on those different local electronic structures and on the computed Fermi contact shifts, we discuss the impact on the spin transfer mechanism on adjacent nuclei and propose tentative signal assignments. The O/F clustering tendency is discussed in relation with the formation of short V^IVO vanadyl bonds with a very specific electronic structure and possible cooperative effect along the chain.     

Synthesis and characterization of modular polyphosphonate homopolymers and copolymers

Linear polyphosphonates with the generic formula –[P(Ph)(X)OR′O]_n– (X = S or Se) have been synthesized by polycondensations of P(Ph)(NEt₂)₂ and a diol (HOR′OH = 1,4-cyclohexanedimethanol, 1,4-benzenedimethanol, tetraethylene glycol, or 1,12-dodecanediol) followed by reaction with a chalcogen. Random copolymers have been synthesized by polycondensations of P(Ph)(NEt₂)₂ and mixture of two of the diols in a 2:1:1 mol ratio followed by reaction with a chalcogen. Block copolymers with the generic formula –[P(Ph)(X)OR′O]_{(x + 2)} –[P(Ph)(X)OR′O]_{(x + 3)}– (X = S or Se) have been synthesized by the polycondensations of Et₂N[P(Ph)(X)OR′O]_{(x + 2)}P(Ph)NEt₂ oligomers with HOR′O[P(Ph)(X)OR′O]_{(x + 3)}H oligomers followed by reaction with a chalcogen. The Et₂N[P(Ph)(X)OR′O]_{(x + 2)}P(Ph)NEt₂ oligomers are prepared by the reaction of an excess of P(Ph)(NEt₂)₂ with a diol while the HOR′O[P(Ph)(X)OR′O]_{(x + 3)}H oligomers are prepared by the reaction of P(Ph)(NEt₂)₂ with an excess of the diol. In each case the excess, x is the same and determines the average block sizes. All of the polymers were characterized using ¹H, ¹³C{¹H}, and ³¹P{¹H} NMR spectroscopy, TGA, DSC, and SEC. ³¹P{¹H} NMR spectroscopy demonstrates that the random and block copolymers have the expected arrangements of monomers and, in the case of block copolymers, verifies the block sizes. All polymers are thermally stable up to ~300°C, and the arrangements of monomers in the copolymers (block vs. random) affect their degradation temperatures and T_g profiles. The polymers have weight average MWs of up to 3.8 × 10⁴ Da.     

Second-Coordination Sphere Effects on Selectivity and Specificity of Heme and Nonheme Iron Enzymes

Mononuclear iron-containing enzymes are highly versatile oxidants that often react stereospecifically and/or regioselectively with substrates. Combined experimental and computational studies on heme monooxygenases, nonheme iron dioxygenases and halogenases have revealed the intricate details of the second-coordination sphere, which determine this specificity and selectivity. These second-coordination sphere effects originate from the positioning of the substrate and oxidant, which involve the binding of the co-factors and substrate into the active site of the protein. In addition, some enzymes affect the selectivity and reactivity through charge-stabilization from nearby bound cations/anions, an induced electric field or through the positioning of salt bridges and hydrogen-bonding interactions to first-coordination sphere iron ligands and/or the substrate. Examples of all of these second-coordination sphere effects in iron-containing enzymes and how these influence structure and reactivity are given.