Heterologous expression of the avirulence gene ACE1 from the fungal rice pathogen Magnaporthe oryzae

The ACE1 and RAP1 genes from the avirulence signalling gene cluster of the rice blast fungus Magnaporthe oryzae were expressed in Aspergillus oryzae and M. oryzae itself. Expression of ACE1 alone produced a polyenyl pyrone (magnaporthepyrone), which is regioselectively epoxidised and hydrolysed to give different diols, 6 and 7, in the two host organisms. Analysis of the three introns present in ACE1 determined that A. oryzae does not process intron 2 correctly, while M. oryzae processes all introns correctly in both appressoria and mycelia. Co-expression of ACE1 and RAP1 in A. oryzae produced an amide 8 which is similar to the PKS-NRPS derived backbone of the cytochalasans. Biological testing on rice leaves showed that neither the diols 6 and 7, nor amide 8 was responsible for the observed ACE1 mediated avirulence, however, gene cluster analysis suggests that the true avirulence signalling compound may be a tyrosine-derived cytochalasan compound.     

Hyperpolarization of amino acid derivatives in water for biological applications

We report on the successful synthesis and hyperpolarization of N-unprotected α-amino acid ethyl propionate esters and extensively, on an alanine derivative hyperpolarized by PHIP (4.4 ± 1.0% ¹³C-polarization), meeting required levels for in vivo detection. Using water as solvent increases biocompatibility and the absence of N-protection is expected to maintain biological activity.     

Doping-induced memory effect in Li-ion batteries: the case of Al-doped Li4Ti5O12

In Li-ion batteries (LIBs), a memory effect has been revealed in two-phase electrode materials such as olivine LiFePO₄ and anatase TiO₂, which complicates the two-phase transition and influences the estimation of the state of charge. Practical electrode materials are usually optimized by the element doping strategy, however, its impact on the memory effect has not been reported yet. Here we firstly present the doping-induced memory effect in LIBs. Pristine Li₄Ti₅O₁₂ is free from the memory effect, while a distinct memory effect could be induced by Al-doping. After being discharged to a lower cutoff potential, Al-doped Li₄Ti₅O₁₂ exhibits poorer electrochemical kinetics, delivering a larger overpotential during the charging process. This dependence of the overpotential on the discharging cutoff leads to the memory effect in Al-doped Li₄Ti₅O₁₂. Our discovery emphasizes the impact of element doping on the memory effect of electrode materials, and thus has implications for battery design.     

Calcium ions tune the zinc-sequestering properties and antimicrobial activity of human S100A12

Human S100A12 is a host-defense protein expressed and released by neutrophils that contributes to innate immunity. Apo S100A12 is a 21 kDa antiparallel homodimer that harbors two Ca(ii)-binding EF-hand domains per subunit and exhibits two His₃Asp motifs for chelating transition metal ions at the homodimer interface. In this work, we present results from metal-binding studies and microbiology assays designed to ascertain whether Ca(ii) ions modulate the Zn(ii)-binding properties of S100A12 and further evaluate the antimicrobial properties of this protein. Our metal-depletion studies reveal that Ca(ii) ions enhance the ability of S100A12 to sequester Zn(ii) from microbial growth media. We report that human S100A12 has antifungal activity against Candida albicans, C. krusei, C. glabrata and C. tropicalis, all of which cause human disease. This antifungal activity is Ca(ii)-dependent and requires the His₃Asp metal-binding sites. We expand upon prior studies of the antibacterial activity of S100A12 and report Ca(ii)-dependent and strain-selective behavior. S100A12 exhibits in vitro growth inhibitory activity against Listeria monocytogenes. In contrast, S100A12 has negligible effect on the growth of Escherichia coli K-12 and Pseudomonas aeruginosa PAO1. Loss of functional ZnuABC, a high-affinity Zn(ii) import system, increases the susceptibility of E. coli and P. aeruginosa to S100A12, indicating that S100A12 deprives these mutant strains of Zn(ii). To evaluate the Zn(ii)-binding sites of S100A12 in solution, we present studies using Co(ii) as a spectroscopic probe and chromophoric small-molecule chelators in Zn(ii) competition titrations. We confirm that S100A12 binds Zn(ii) with a 2 : 1 stoichiometry, and our data indicate sub-nanomolar affinity binding. Taken together, these data support a model whereby S100A12 uses Ca(ii) ions to tune its Zn(ii)-chelating properties and antimicrobial activity.     

Quantification of chondroitin sulfate and dermatan sulfate in danaparoid sodium by <Superscript>1</Superscript>H NMR spectroscopy and PLS regression

Danaparoid sodium (the active pharmaceutical ingredient in Orgaran; Merck Sharp and Dohme) is a biopolymeric non-heparin drug used as anticoagulant and antithrombotic agent approved for the prophylaxis of post-operative deep-vein thrombosis, which may lead to pulmonary embolism in patients undergoing, e.g., elective hip replacement surgery. It consists of a mixture of three glycosaminoglycans (GAGs): heparan sulfate (HS), dermatan sulfate (DS), and chondroitin sulfate (CS). Currently, the CS and DS content are quantified by means of a time-consuming enzymatic method. In this paper the use of ¹H NMR in combination with multivariate regression (partial least-squares, PLS) is proposed as a new method. In order to evaluate the proposed method, a series of danaparoid sodium samples were analyzed and the results were compared with those obtained by the enzymatic method (reference method). The results showed that the proposed ¹H NMR method is a good alternative for analysis of CS and DS in danaparoid sodium. Accuracy of ±0.7% (w/w) and ±1.1% (w/w) for CS and DS was obtained by the ¹H NMR method and accuracy of ±1.0% (w/w) and ±1.3% (w/w) by the enzymatic method. Furthermore, the use of ¹H NMR in combination with PLS results in a fast quantification. The analysis time is reduced to 35 min per sample instead of 60 h for a maximum of 16 samples.     

A comparative study of the coordination behavior of cyclo-P5 and cyclo-As5 ligand complexes towards the trinuclear Lewis acid complex (perfluoro-ortho-phenylene)mercury

Reactions of the cyclo-E₅ sandwich complexes [Cp*Fe(η⁵-P₅)] (1) and [Cp*Fe(η⁵-As₅)] (2) with the planar Lewis acid trimeric (perfluoro-ortho-phenylene)mercury [(o-C₆F₄Hg)₃] (3) afford compounds that show distinctly different assemblies in the solid state. The phosphorus containing ligand 1 forms dimeric coordination units with two molecules of 3, with one P atom of each cyclo-P₅ ligand positioned in close proximity to the center of a molecule of 3. In contrast to the coordination behavior of 1, the arsenic analog 2 shows simultaneous interaction of three As atoms with the Hg atoms of 3. A DFT study and subsequent AIM analyses of the products suggest that electrostatic forces are prevalent over donor–acceptor interactions in these adducts, and may play a role in the differences in the observed coordination behavior. Subsequently, a series of [Cp^RFe(η⁵-P₅)] (Cp^R = C₅H_5–n tBu_n, n = 1–3, 6a–c) sandwich complexes was prepared and also reacted with [(o-C₆F₄Hg)₃]. In the solid state the obtained products 7a–c with increasing steric demand of the Cp^R ligands show no significant change in their assembly compared to the Cp* analog 4. All of the products were characterized by single crystal X-ray structure analysis, mass spectrometry and elemental analysis as well as NMR spectroscopy and IR spectrometry.     

The importance of local chemical structure for chemical metabolism by human uridine 5'-diphosphate-glucuronosyltransferase

The uridine 5'-diphosphate- (UDP-)glucuronosyltransferase (UGT) family of enzymes catalyzes the conjugation of chemicals containing a suitable nucleophilic atom with glucuronic acid. Despite the importance of glucuronidation as an elimination and detoxification mechanism for drugs, environmental chemicals, and endogenous compounds, the structural features of substrates that confer isoform selectivity are poorly understood. The relationship between the local molecular structure of nucleophilic atoms of chemicals and the ability of UGT isoforms to glucuronidate the nucleophilic atoms was investigated here. The proximity of an aromatic ring to the nucleophilic atom was highly associated with a greater likelihood of glucuronidation by most UGT isoforms. Similarly, most UGT isoforms were found to have a statistically significant preference for oxygen over nitrogen as the nucleophilic atom. The converse was established only for UGT1A4. Na?ve Bayes models were trained to predict the site of glucuronidation for eight UGT isoforms on the basis of the partial charge and Fukui function of the nucleophilic atom and whether an aromatic ring was attached to the nucleophilic atom. On average, the cross-validated sensitivity and specificity of the models were approximately 75-80%. For all but UGT2B7, the area under the receiver operating characteristics curve of the model was greater than 0.8, indicating strong predictive ability. A chemical diversity analysis of the currently available data indicates bias toward chemicals containing phenolic groups, and it is likely that the availability of chemical data sets with greater diversity will facilitate further insights into the structural features of substrates that confer enzyme selectivity.     

Photoanodic and photocathodic behaviour of La5Ti2CuS5O7 electrodes in the water splitting reaction

The particulate semiconductor La₅Ti₂CuS₅O₇ (LTC) with a band gap energy of 1.9 eV functioned as either a photocathode or a photoanode when embedded onto Au or Ti metal layers, respectively. By applying an LTC/Au photocathode and LTC/Ti photoanode to, respectively, photoelectrochemical (PEC) water reduction and oxidation concurrently, zero-bias overall water splitting was accomplished under visible light irradiation. The band structures of LTC/Au and LTC/Ti calculated using a semiconductor device simulator (AFORS-HET) confirmed the critical role of the solid/solid junction of the metal back contact in the charge separation and PEC properties of LTC photoelectrodes. The prominently long lifetime of photoexcited charge carriers in LTC, confirmed by transient absorption spectroscopy, allowed the utilization of both photoexcited electrons and holes depending on the band structure at the solid/solid junction.     

Two more pieces of the colibactin genotoxin puzzle from Escherichia coli show incorporation of an unusual 1-aminocyclopropanecarboxylic acid moiety

Colibactin represents a structurally undefined class of bacterial genotoxin inducing DNA damage and genomic instability in mammalian cells, thus promoting tumour development and exacerbating lymphopenia in animal models. The colibactin biosynthetic gene cluster (clb) has been known for ten years and it encodes a hybrid nonribosomal peptide synthetase (NRPS)/polyketide synthase (PKS) assembly line. Nevertheless, the final chemical product(s) remain unknown. Previously, we and others reported several colibactin pathway-related metabolites including N-myristoyl-d-asparagine (1) as part of a prodrug precursor that is cleaved from the putative precolibactin to form active colibactin by the peptidase ClbP. Herein, we report two new colibactin pathway-related metabolites (2 and 3) isolated from a clbP mutant of the probiotic E. coli Nissle 1917 strain. Their structures were established by HRMS and NMR. Compound 2 shows an additional 4-aminopenatanoic acid moiety with respect to 1, while 3 is characterized by the presence of an unusual 7-methyl-4-azaspiro[2.4]hept-6-en-5-one residue. Moreover, we propose the biosynthetic pathway towards both intermediates on the basis of extensive gene inactivation and feeding experiments. The identification of 2 and 3 provides further insight into colibactin biosynthesis including the involvement and formation of a rare 1-aminocyclopropanecarboxylic acid unit. Thus, our work establishes additional steps of the pathway forming the bacterial genotoxin colibactin.     

Absolute structure determination of compounds with axial and planar chirality using the crystalline sponge method

The absolute stereochemistry of compounds with axial and planar chirality is successfully determined by the crystalline sponge method without crystallization or derivatization of the compounds. This method is applied to absolute structure determination in the asymmetric synthesis of unique compounds with axial and planar chirality.     

An efficient synthesis of certain 5-substituted-2'-deoxyuridine 3',5'-cyclic monophosphate p-o-alkyl(aralkyl) esters. The crystal and molecular structure of 5-IODO-2'-deoxyuridine 3',5'-cyclic monophosphate p-o-methyl ester with axial methoxy group

Some 5-alkyl- and 5-halogeno-2'-deoxyuridine 3',5'-cyclic mono- phosphate P-O-alkyl(aralkyl) esters have been prepared from the silver salts of the parent cyclic monophosphates and alkyl(aralkyl) iodides. The diastereomeric phosphotriesters have been characterized by spectroscopic methods. The crystal and molecular structure of 5-iodo-2'-deoxyuridine 3',5'-cyclic monophosphate P-O-methyl ester with axial methoxy group has been determined. We failed to obtain the desired triesters from the reactions of the cAMP silver salt with alkyl iodides, contrary to previous claims in the literature.     

The crystal structure of 10-acetonylphenoxatellurine nitrate,C15H13NO5Te

The crystal structure of C₁₅ H₁₃NO₅Te was determined from X-ray diffractometer data. The unit cell is triclinic (Bī): a = 17.118(5), b = 7.402(2), c = 12.225(2) Å, α = 87.96(1), β = 93.31 (1), γ = 92.13(2)° at 22°C, Z = 4/cell, and the conventional R = 0.025 for 2497 independent reflections. The molecule is folded along the Te-ring oxygen axis (135° ). The average Te-ring carbon distance is 2.101 Å, the Te-acetonyl carbon distance is 2.129 Å and the average C-ring oxygen distance is 1.388 Å. The acetonyl group and phenyl rings have normal distances and angles, and the nitrate group is nearly regular, with Te…ONO₂ = 2.775 Å. The coordination around Te is that of an extremely distorted trigonal bipyramid, with apical positions occupied by one ring carbon and the ONO₂ group (167.4°), and two axial positions occupied by the acetonyl carbon and the other ring carbon (94.6°). Coordinates of all hydrogen atoms were determined.     

The addition of benzocyclobutenylidene to benzene : The facile rearrangement of spiro[benzocyclobutene-1,7'-cyclohepta-1',3',5'-triene] to 9a,10-dihydrobenz[α]azulene

Thermal decomposition of the sodium salts of benzocyclobutenone tosylhydrazone and 2-methylbenzocyclobutenone tosylhydrazone in benzene affords 9a,10-dihydrobenz[α]azulene 4 and trans-10-methyl-9a, 10-dihydrobenz[α]azulene 3, respectively. A mechanism involving initially the addition of the carbene benzocyclobutenylidene, or its 2-Me derivative, to the benzene ring is postulated. A proposed intermediate in the reaction, spiro [benzocyclobutene 1,7' cyclohepta-1',3',5'-triene] 12 has been synthesised, and shown to give rise to 4 under the reaction conditions. The rate of rearrangement of 12 → 4 has been measured, and the activation energy determined: E_a = 125.9 ± O.8 KJmol^?1 and A = 1.38 × lO¹⁴sec^?1. The mechanism for the rearrangement must involve ring opening of the benzocyclobutene moiety of 12 to give an o- xylylene intermediate which is postulated to possess considerable diradical character. At 71.8 °, this ring opening is 2.7 × 10⁶ times faster than the ring opening of the parent benzocyclobutene molecule. The decomposition of the sodium salt of 2-(7' -cyclohepta-1',3',5' trienyl)benzaldehyde tosylhydrazone has also been investigated and is shown to yield 4a,10-dihydrobenz[α]azulene, 9,10-dihydrobenz[α]azulene and 8,9-benzotricyclo [5.3.0.0^2.10]deca-3,5,8-triene. A mechanism involving intramolecular 1,3-dipolar addition of a diazo grouping to a cycloheptatriene Π-bond, followed by decomposition of the resulting pyrazoline intermediate, is proposed.     

Reactive species involved in the regioselective photooxidation of heptamethine cyanines

Heptamethine cyanines are important near-IR fluorophores used in many fluorescence applications. Despite this utility, these molecules are susceptible to light-promoted reactions (photobleaching) involving photochemically generated reactive oxygen species (ROS). Here, we have sought to define key chemical aspects of this nearly inescapable process. Near-IR photolysis of a model heptamethine cyanine leads to the regioselective oxidative cleavage of the characteristic polyene. We report the first quantitative analysis of the major reaction pathway following either photolysis or exposure to candidate ROS. These studies clearly indicate that only singlet oxygen (¹O₂), and not other feasible ROS, recapitulates the direct photolysis pathway. Computational studies were employed to investigate the regioselectivity of the oxidative cleavage process, and the theoretical ratio is comparable to observed experimental values. These results provide a more complete picture of heptamethine cyanine photooxidation, and provide insight for the design of improved compounds for future applications.     

Investigation of the structure and 13C NMR spectrum of 5-phenylbarbituric acid

Crystals of 5-phenylbarbituric acid, 2 , are monoclinic, space group C2/c. There are 8 molecules in the unit cell of dimensions a = 20.218(3), b = 7.477(1), c, = 12.443(2), β = 111.54° and V = 1749.6(4) ų. Full-matrix least-squares refinement has reached R = 0.039. This is the first barbiturate reported to exist in the enol form in the solid state. There is extensive hydrogen bonding between adjacent molecules which gives rise to very dense crystals. The ¹³C-nmr spectroscopy shows that 2 exists in both enol and keto forms in DMSO solution.