2-[2-(Trifluoro­meth­yl)phen­yl]-2H-1-benzopyran-4(3H)-one

In the title compound, C16H11F3O2, the γ-pyran­one ring adopts an envelope conformation with the chiral C atom standing out of the ring plane. In the crystal, molecules are linked by C—H⋯O and C—H⋯F inter­actions

Importance of N-terminal proline for the promiscuous activity of 4-oxalocrotonate tautomerase (4-OT)

Michael addition of aldehydes to nitro-olefins is an effective method to obtain useful chiral gamma-nitroaldehydes. gamma-Nitroaldehydes are precursors for chiral gamma-aminobutyric acid analogues, which have numerous pharmacological activities and are used for the treatment of neurological disorders. A whole-cell system based on recombinantly expressed 4-oxalocrotonate tautomerase (4-OT) was developed and shown to be an effective biocatalyst for the Michael addition of branched aldehydes to beta-nitrostyrenes. The aim of this study was to investigate the influence of the substitution of the N-terminal proline with lysine and arginine, both containing a reactive epsilon-amino group, on the Michael addition catalyzed by 4-OT. First, the effects of these mutations were examined by in silico analysis, followed by the generation of three terminal lysine mutants. The generated mutants, 4-OT_K, 4-OT_PK and 4-OT_KK were tested for their ability to utilise beta-nitrostyrene (1), (E)-1-nitro-2-(2-thienyl)ethene (2) and trans-p-chloro-beta-nitrostyrene (3) as Michael acceptors with isobutanal (2-methylpropanal) as the donor. For comparison, the lithium salt of lysine was used in the same organocatalytic reactions. In general, the introduction of lysine had a negative effect on Michael additions based on overall product yields. However, additional lysine residues at the N-terminus of the protein resulted in structural changes that enhanced the activity towards 2 and 3. Therefore, the N-terminal proline is important for 4-OT-catalysed Michael-additions, but it is not essential

Struktur des MjRelBE Proteinkomplexes aus Methanococcus jannaschii

In the last decades a number of plasmid stabilization systems were discovered. Although difficult to study because they exhibit high toxicity to host cells, their function became clear after the toxin – antitoxin interplay was elucidated. They play an important part in stable maintenance and inheritance of plasmids and, as their function was now clear, only new members of similar traits were expected to be discovered. As the number of sequenced genomes grew, the number of putative chromosomally encoded TA systems rouse quickly. These new members of the TA superfamilies shared the common features of the already known TA systems. They were comprised of a toxin that is counteracted by tight binding of an upstream encoded antitoxin. These two proteins form a stable non-toxic toxin – antitoxin complex but the antitoxin is susceptible to protease degradation. If the antitoxin pool is not replenished the toxin is free to act upon its target, poisoning the cell and leading to cell stasis or even cell death. The delay in antitoxin production may be caused due to plasmid loss, in cases when the TA system is plasmid borne or inhibition of protein synthesis in cases of chromosomally encoded TA systems. The discovery of these chromosomally encoded TA systems was surprising as it contradicted the idea of the TA systems being the failsafe of plasmid inheritance. What was their purpose if they are not coded on the plasmid but on the chromosome? The current opinion is that these systems represent an advantage to the cell harbouring the system as they function like an emergency brake that holds the cell metabolism and redirects it to a new pathway. The advantage lies in a swift transfer to minimal protein production targeted for cell survival in stress conditions. The known TA systems share little sequence identity, even within a toxin superfamily. Although bioinformatic methods have advanced and new members of the TA system toxins are identified only on their primary sequence, the real connections between the proteins can be realized only through the determinations of their three-dimensional structures. The structure determination of the M. jannaschii MjRelBE protein complex was successful. This structure represents the bridge between the RelE superfamily members PhRelE and YoeB because it possesses the characteristics of both. It did raise the question again whether the RelE toxin really needs another component, like the ribosome, in order to be active. Comparing the endoribonucleases and the TA toxins some conclusions can be drawn. The cleft shape, the arrangement of secondary structure elements and the positioning of positively charged residues are the essential ingredients for RNA binding. This common fold between the TA systems and other endoribonucleases is associated with the substrate (RNA) and therefore wide spread. The positioning of the residues responsible for RNA cleavage differs regardless of the common fold.Die Toxin - Antitoxin (TA) Systeme werden von Chromosomen oder Plasmiden verschiedener Bakterienstämme kodiert. Die Rolle der Toxin - Antitoxin Systeme in Apoptose- ähnlichen Prozessen bei Bakterien, sowie der Zusammenhang mit der Stressantwort auf Nährstoffmangel, ist Gegenstand aktueller Forschung. So wurden während Untersuchungen an Toxin - Antitoxin Systemen in Bakterien auch die Proteine RelE und RelB entdeckt. Das relB Gen zog zuerst die Aufmerksamkeit auf sich, da Zellen, deren relB Gen im E. coli Chromosom Fehler aufwies, ein eigentümliches Verhalten nach Nährstoff- Entzug aufwiesen. Diese Zellen verlangsamten nach einem Aminosäurenentzug ihre Proteinbiosynthese, wie es für Wild Typ Zellen zu erwarten wäre, setzten aber nach etwa 10 Minuten die Biosynthese fort. Der Proteinkomplex, der von den aufeinander folgenden Genen relB und relE kodiert wird, wurde charakterisiert und gefunden, dass das Protein RelE als Toxin und RelB als sein spezifisches Antitoxin wirken. Das RelBE System ähnelt den schon bekannten Toxin-Antitoxin Systemen (TA Systeme). Sie alle haben ähnliche Charakteristiken: a) sie bestehen aus zwei aufeinander folgenden Genen, b) das Gen-Produkt des einen ist ein langlebiges toxisches Protein und das des anderen ein kurzlebiges (Proteolyse- empfindliches) antitoxisches Protein, c) das Gen des Antitoxins liegt vor dem Gen des Toxins, d) beide Proteine werden gleichzeitig exprimiert (produziert), e) das Antitoxin wird im Überschuss zum Toxin produziert, f) das Antitoxin wird von Proteasen verdaut, h) das Operon wird entweder vom Antitoxin allein oder vom Antitoxin – Toxin Komplex autoreguliert. In dieser Arbeit wurde die Struktur des M. jannaschii MjRelBE Protein Komplexes ermittelt. Diese wies zu einem Zusammenhang mit den Endoribonucleasen, da sie eine sehr eng verwandte Faltung haben. Aus der Struktur konnte auch hergeleitet werden, weshalb die spontan entstandene Mutante MjRelE(R62S) nicht toxisch für die E. coli Zellen ist. Diese Aminosäure nimmt eine Schlüsselrolle bei der Spaltung von mRNA an und hat die gleiche Rolle in den strukturverwandten Endoribonucleasen YoeB (gehört zur RelE toxin superfamilie) und RNase Sa

Electronic features and hydrogen bonding capacity of the sulfur acceptor in thioureido-based compounds. Part 2. Further insight by theoretical charge density study

Theoretical charge density analysis of the thioureido based compound 4-methyl-3-thiosemicarbazide (MeTSC) is used in this study with the aim to provide additional insight into electronic features of the thioureido sulfur acceptor and the corresponding D-H...S hydrogen bonding (D=N, C). The present work is motivated by our earlier experimental charge density study on the same compound that pointed out to a great flexibility of the thioureido S acceptor and its ability to adjust the lone pair electron density to the donor groups in simultaneous hydrogen bonding. Through the additional theoretical approach we were able to single out different fragments of MeTSC crystal and to carefully follow the changes in electron density properties of the S acceptors belonging to: isolated MeTSC monomers (i.e. two crystallographically independent molecules), eight D-H...S bonded dimers and MeTSC molecules placed in full crystal environment, where each S atom engages in four hydrogen bonds. Deformation density of the sulfur's lone electron pair, topological properties of D-H...S interactions and electrostatic potential are here examined in order to comprehend the mutual influence and potential cooperative effects of the four simultaneously formed interactions to each of the S acceptors. The hydrogen bonding involving thioureido S acceptor is also investigated in terms of the energetic properties of the eight real MeTSC dimers existing in the crystal structure, and additional systems MeTSC/MeOH and acetone/MeOH. The latter systems are designed with the purpose of direct comparison and ranking of interactions involving thioureido S to those involving more conventional, carbonyl O acceptor. Energetic features were thoroughly studied through electrostatic interactions energies (XD2006), cohesive energies (CRYSTAL09) and ab initio approach employing the coupled-cluster single S and doubles augmented by a perturbational correction for connected triple excitations (CCSD(T)) method

Synthesis, crystal structure and local anti-inflammatory activity of the L-phenylalanine methyl ester derivative of dexamethasone-derived cortienic acid

L-phenylalanine methyl ester derivative of dexamethasone - derived cortienic acid (DF) was synthesized and its crystal structure was characterized by X-ray diffraction method. The crystal system is orthorhombic with space group P212121 and cell constants a = 8.2969 (3) Å, b = 18.9358 (8) Å, c = 20.0904 (6) Å, V = 3156.4 (2) Å3 and Z = 4. Ring A of the steroid nucleus and phenyl ring in the 17β-side chain are almost planar. Rings B and C have a slightly distorted chair conformation, whereas ring D has an envelope conformation. The packing of DF is characterized by a network of intermolecular hydrogen bonds involving the O4 atom from one side of the steroid nucleus and O1 and F1 atoms from the other side as hydrogen bond acceptors. Apart from the intermolecular hydrogen bonds in the crystal packing, there are also numerous intramolecular hydrogen bonds of the N-H...O, C-H...O and C-H...F type. Local anti-inflammatory activity of DF was evaluated by use of croton oil-induced ear edema test. This derivative achieved maximal inhibition of ear edema at significantly lower concentration in comparison with dexamethasone. [Projekat Ministarstva nauke Republike Srbije, br. 172041, 172014 i 172035

Charge Density Analysis of 2‑Pyridineformamide N(4)-Methylthiosemicarbazone (<i>Z</i>′ = 4): Role of an Enhanced N–H···S Thioureido Dimer

Charge density distribution in 2-pyridineformamide N(4)-methylthiosemicarbazone (TSC4) has been determined using high-resolution X-ray diffraction data (100 K) and Hansen–Coppens multipole formalism. The results are interpreted in terms of Bader’s quantum theory of atoms in molecules (QTAIM), electrostatic potential analysis, and theoretical calculations (CRYSTAL09). The study highlights the molecular dissimilarity at the electronic level. The N–H···S interactions have a dominant role in stabilization of the TSC4 crystal structure. As each of the four S acceptors simultaneously engages in several interactions, the focus of study is on the lone pairs’ electron density of these acceptors which is particularly able to adjust to the various spatial distributions of the interacting donor groups. The main structural feature of TSC4 is the formation of specific N–H···S bonded dimers between two pairs of independent molecules. These dimers are the main building block in the crystal structure, and with the two additional N–H···S contacts they represent a significant enhancement of a typical R²₂(8) dimer synthon which dominates among the thioureido-based crystal structures. Two TSC4 dimers are structurally very similar and exhibit considerable cohesive energy (−20.8 and −21.4 kcal/mol). This type of dimer is the only structural motif that is common for S acceptors of all four independent molecules

Oxidative Stress and Polymorphism of Xenobiotic-Metabolizing Enzymes in Two Patients with Severe Alpha-1-Antitrypsin Deficiency

Alpha-1-antitrypsin deficiency (AATD) and tobacco smoke play a key role in the pathogenesis of early-onset emphysema. Differences in AATD-related chronic obstructive pulmonary disease stages imply the existence of modifying factors associated with disease severity. We present two male patients with emphysema caused by severe AATD (PiZZ genotype). Both are former smokers and have epoxide hydrolase low-activity phenotype. Extremely high level of oxidative stress (high urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine), increased inflammation (high serum CRP), and GSTP1 105Val mutation were found in patient with a worse lung function and prognosis. These data provide more evidence that oxidative stress-related gene variants and inflammation are associated with worse symptoms of AATD-related emphysema. Therefore, prevention against severe stage of AATD-related emphysema would include early identification of the risk gene variants, cessation or never smoking, and treatment with anti-inflammatory and anti-oxidant drugs. Additionally, urinary 8-oxodG could be a candidate for predictive biomarker for routine assessment of the oxidative stress level in AATD patients

Gender-related reference intervals of urinary 8-oxo-7,8-dihydro-2 '-deoxyguanosine determined by liquid chromatography-tandem mass spectrometry in Serbian population

Objectives: Although there are many nucleobase modifications, 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) is one of the dominant form of oxidative modifications of DNA. Urinary 8-oxodG is potentially the best non-invasive biomarker of oxidative stress. Defining reference interval for urinary 8-oxodG is a prerequisite for its clinical use as biomarker. Design and methods: Reference population included 229 healthy Serbian adults (130 males and 99 females). The spot urinary 8-oxodG was determined using high performance liquid chromatography and tandem mass spectrometry (HPLC-MS/MS). Urinary creatinine was measured by the kinetic Jaffe method. Results: Analytical performances of the HPLC-MS/MS: CVs within and between-run variations were 5.6% and 2.6%; LOD and LOQ were 1.65 nmol/L and 330 nmol/L; mean recovery and relative accuracy were 96% and 97%. Creatinine level was higher in males than in females, but no gender difference in 8-oxodG level was observed. Upon the adjustment of 8-oxodG to creatinine (8-oxodG/creatinine), higher values were obtained in females (1.38 +/- 0.65 nmol/mmol) than in males (1.05 +/- 0.48 nmol/mmol). Distribution of 8-oxodG/creatinine in spot urine sample was log-normal and gender-related reference intervals (estimated as the 2.5th-97.5th percentiles) were 0.45-2.22 nmol/mmol for males, and 0.54-3.11 nmol/mmol for females. Body mass index (BMI) affects excretion of the 8-oxodG in males, independently of urinary creatinine, while in females it does not. Therefore, BMI might contribute to the gender-related differences of 8-oxodG/creatinine in spot urine samples. Conclusions: This is the first established gender-related reference intervals of spot urinary 8-oxodG/creatinine. Our results contribute to the full validation of 8-oxodG as biomarker of oxidative stress

Oxidative Stress and Genetic Variants of Xenobiotic-Metabolising Enzymes Associated with COPD Development and Severity in Serbian Adults

The genetic and non-genetic factors that contribute to the development of chronic obstructive pulmonary disease (COPD) are still poorly understood. We investigated the potential role of genetic variants of xenobiotic-metabolising enzymes (glutathione-S-transferase M1, GSTM1; glutathione-S-transferase T1, GSTT1; microsomal epoxide hydrolase, mEH), oxidative stress (assessed by urinary 8-oxo-7,8-dihydro-2'-deoxyguanosine, 8-oxodG/creatinine), sex, ageing and smoking habits on susceptibility to development of COPD and its severity in Serbian population. The investigated population consisted of 153 healthy subjects (85 males and 68 females) and 71 patients with COPD (33 males and 38 females). Detection of GSTM1*null, GSTT1*null, mEH Tyr113His and mEH His139Arg gene variants was performed by PCR/RFLP method. Urinary 8-oxodG was determined using HPLC-MS/MS, and expressed as 8-oxodG/creatinine. We revealed that increased urinary 8-oxodG/creatinine and leucocytosis are the strongest independent predictors for COPD development. Increased level of oxidative stress increased the risk for COPD in males [odds ratio (OR), 95% confidence interval (CI): 8.42, 2.26-31.28], more than in females (OR, 95% CI: 3.60, 1.37-9.45). Additionally, independent predictors for COPD were ageing in males (OR, 95% CI: 1.29, 1.12-1.48), while in females they were at least one GSTM1 or GSTT1 gene deletion in combination (OR, 95% CI: 23.67, 2.62-213.46), and increased cumulative cigarette consumption (OR, 95% CI: 1.09, 1.01-1.16). Severity of COPD was associated with the combined effect of low mEH activity phenotype, high level of oxidative stress and heavy smoking. In conclusion, early identification of GSTM1*null or GSTT1*null genotypes in females, low mEH activity phenotype in heavy smokers and monitoring of oxidative stress level can be useful diagnostic and prognostic biomarkers

The Role of Oxidative Stress in the Clinical Manifestations of Childhood Asthma

Introduction: The significance of oxidative stress in pathogenesis of childhood asthma was recognized, but its role in the clinical manifestations of disease is still unclear. Materials and Methods: The study was conducted in 96 asthmatic children. The urinary biomarker of oxidative stress, 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG/creatinine) was determined by using HPLC-MS/MS. ELISA was performed to measure myeloperoxidase (MPO) and Cu,Zn-superoxide dismutase (Cu, Zn-SOD) in serum. Results: Logistic regression analysis revealed that female gender, tobacco smoke exposure, and increased 8-oxodG/creatinine were associated with risk for intermittent asthma, while the positive allergy test and increased Cu, Zn-SOD were associated with eczema in asthmatic children. Higher MPO (p = 0.033), and percent of granulocytes (p = 0.030) were found in severe persistent asthma in comparison to intermittent or mild persistent asthma. Conclusion: The main findings that TSE-induced oxidative stress is a risk for intermittent asthma and eczema may be clinically significant for the disease prevention and therapeutic improvements