Assessing the rigidity of cubanes and bicyclo(1.1.1)pentanes as benzene bioisosteres.

Aromatic rings are critical core substructures in the majority of pharmaceutical compounds. There is much recent interest in replacing aromatic structures with saturated bioisosteres of benzene, which are generally fused or bridged ring systems. These bioisosteres often show improved solubility properties compared to benzene, and may also undergo fewer unwanted metabolic processes. One key reason why aromatic rings have proven so successful in drug design is their rigidity. This paper uses molecular dynamics simulations supported by crystallographic data to assess the rigidity of bicyclopentane and cubane ring systems as two of the most common benzene bioisosteres and compares this to benzene. Whilst a benzene ring is shown to be more flexible than these two bioisosteres in terms of its dihedral ring flexibility, substituents around the ring tend to behave in a much more similar way in both benzene and the bioisosteric systems.

Carbon monoxide release from ultrasound-sensitive microbubbles improves endothelial cell growth.

Carbon monoxide is a gasotransmitter that may be beneficial for vascular tissue engineering and regenerative medicine strategies because it can promote endothelial cell (EC) proliferation and migration by binding to heme-containing compounds within cells. For example, CO may be beneficial for vascular cognitive impairment and dementia because many patients' disrupted blood-brain barriers do not heal naturally. However, control of the CO dose is critical, and new controlled delivery methods need to be developed. This study developed ultrasound-sensitive microbubbles with a carefully controlled precipitation technique, loaded them with CO, and assessed their ability to promote EC proliferation and function. Microbubbles fabricated with perfluoropentane exhibited good stability at room temperature, but they could still be ruptured and release CO in culture with application of ultrasound. Microbubbles synthesized from the higher boiling point compound, perfluorohexane, were too stable at physiological temperature. The lower-boiling point perfluoropentane microbubbles had good biocompatibility and appeared to improve VE-cadherin expression when CO was loaded in the bubbles. Finally, tissue phantoms were used to show that an imaging ultrasound probe can efficiently rupture the microbubbles and that the CO-loaded microbubbles can improve EC spreading and proliferation compared to control conditions without microbubbles as well as microbubbles without application of ultrasound. Overall, this study demonstrated the potential for use of these ultrasound-sensitive microbubbles for improving blood vessel endothelialization.

The isoprene-responsive phosphoproteome provides new insights into the putative signalling pathways and novel roles of isoprene.

Many plants, especially trees, emit isoprene in a highly light- and temperature-dependent manner. The advantages for plants that emit, if any, have been difficult to determine. Direct effects on membranes have been disproven. New insights have been obtained by RNA sequencing, proteomic and metabolomic studies. We determined the responses of the phosphoproteome to exposure of Arabidopsis leaves to isoprene in the gas phase for either 1 or 5 h. Isoprene effects that were not apparent from RNA sequencing and other methods but were apparent in the phosphoproteome include effects on chloroplast movement proteins and membrane remodelling proteins. Several receptor kinases were found to have altered phosphorylation levels. To test whether potential isoprene receptors could be identified, we used molecular dynamics simulations to test for proteins that might have strong binding to isoprene and, therefore might act as receptors. Although many Arabidopsis proteins were found to have slightly higher binding affinities than a reference set of Homo sapiens proteins, no specific receptor kinase was found to have a very high binding affinity. The changes in chloroplast movement, photosynthesis capacity and so forth, found in this work, are consistent with isoprene responses being especially useful in the upper canopy of trees.

The 3-(3-oxoisoindolin-1-yl)pentane-2,4-dione (ISOAC1) as a new molecule able to inhibit Amyloid ß aggregation and neurotoxicity.

Amyloid ß 1-42 (Aß1-42) protein aggregation is considered one of the main triggers of Alzheimer's disease (AD). In this study, we examined the in vitro anti-amyloidogenic activity of the isoindolinone derivative 3-(3-oxoisoindolin-1-yl)pentane-2,4-dione (ISOAC1) and its neuroprotective potential against the Aß1-42 toxicity. By performing the Thioflavin T fluorescence assay, Western blotting analyses, and Circular Dichroism experiments, we found that ISOAC1 was able to reduce the Aß1-42 aggregation and conformational transition towards ß-sheet structures. Interestingly, in silico studies revealed that ISOAC1 was able to bind to both the monomer and a pentameric protofibril of Aß1-42, establishing a hydrophobic interaction with the PHE19 residue of the Aß1-42 KLVFF motif. In vitro analyses on primary cortical neurons showed that ISOAC1 counteracted the increase of intracellular Ca2+ levels and decreased the Aß1-42-induced toxicity, in terms of mitochondrial activity reduction and increase of reactive oxygen species production. In addition, confocal microscopy analyses showed that ISOAC1 was able to reduce the Aß1-42 intraneuronal accumulation. Collectively, our results clearly show that ISOAC1 exerts a neuroprotective effect by reducing the Aß1-42 aggregation and toxicity, hence emerging as a promising compound for the development of new Aß-targeting therapeutic strategies for AD treatment.

Hydroxymethylbutenyl diphosphate accumulation reveals MEP pathway regulation for high CO2-induced suppression of isoprene emission.

Isoprene is emitted by some plants and is the most abundant biogenic hydrocarbon entering the atmosphere. Multiple studies have elucidated protective roles of isoprene against several environmental stresses, including high temperature, excessive ozone, and herbivory attack. However, isoprene emission adversely affects atmospheric chemistry by contributing to ozone production and aerosol formation. Thus, understanding the regulation of isoprene emission in response to varying environmental conditions, for example, elevated CO2, is critical to comprehend how plants will respond to climate change. Isoprene emission decreases with increasing CO2 concentration; however, the underlying mechanism of this response is currently unknown. We demonstrated that high-CO2-mediated suppression of isoprene emission is independent of photosynthesis and light intensity, but it is reduced with increasing temperature. Furthermore, we measured methylerythritol 4-phosphate (MEP) pathway metabolites in poplar leaves harvested at ambient and high CO2 to identify why isoprene emission is reduced under high CO2. We found that hydroxymethylbutenyl diphosphate (HMBDP) was increased and dimethylallyl diphosphate (DMADP) decreased at high CO2. This implies that high CO2 impeded the conversion of HMBDP to DMADP, possibly through the inhibition of HMBDP reductase activity, resulting in reduced isoprene emission. We further demonstrated that although this phenomenon appears similar to abscisic acid (ABA)-dependent stomatal regulation, it is unrelated as ABA treatment did not alter the effect of elevated CO2 on the suppression of isoprene emission. Thus, this study provides a comprehensive understanding of the regulation of the MEP pathway and isoprene emission in the face of increasing CO2.

Abatement of photochemical smog precursors through complete hydrocarbon oxidation over commercial Pd catalysts under fuel-lean conditions with NO promoting effect.

Currently, severe environmental issues have led to a great transition in the automotive industry from internal combustion engine vehicles to electric vehicles, but this transition will take time more than 10 years, which still requires the use of internal combustion engine vehicles. However, these vehicles emit a significant amount of hydrocarbons, in addition to nitrogen oxides (NOx), due to incomplete fuel combustion. They contribute to the formation of photochemical smog when they react with NOx in the presence of sunlight. To effectively remove these hydrocarbons from the exhaust gas of turbo-gasoline engines or diesel engines, we investigated the abatement of propane and iso-pentane, two typical hydrocarbons. In particular, we studied commercial Pd catalysts and revealed how the Pd loading and aging process simulating 4k and 100k mileage affected hydrocarbon abatement abilities, and their phases were identified using characterization technique, including CO chemisorption, X-ray diffraction (XRD), and high-resolution transmission electron microscopy (HR-TEM). We also suggested the reaction pathway for the complete oxidation of propane over Pd catalyst based on the reaction orders of propane and oxygen: Propane adsorbs on O atoms of PdO, and the kinetically relevant C-H bond cleavage step occurs by the interaction with abundant neighboring O atoms of PdO. Finally, the propane and iso-pentane abatement ability of the Pd catalyst aged for 100k mileage were evaluated under realistic exhaust gas conditions, and the effect of each gas component in the realistic exhaust gas was identified; water inhibits the catalytic reaction of hydrocarbons by occupying the active sites, whereas NO catalyzes the hydrocarbon oxidation reaction by either changing the reaction pathway or active sites under fuel-lean conditions. These findings enable us to effectively reduce environmental pollution and facilitate a smoother transition from internal combustion engine vehicles to electric vehicles.

Relationship between seasonal variation in isoprene emission and plant hormone profiles in the tropical plant Ficus septica.

In Ficus septica, the short-term control of isoprene production and, therefore, isoprene emission has been linked to the hormone balance between auxin (IAA) and jasmonic acid (JA). However, the relationship between long-term changes in isoprene emission and that of plant hormones remains unknown. This study tracked isoprene emissions from F. septica leaves, plant hormone concentrations and signalling gene expression, MEP pathway metabolite concentrations, and related enzyme gene expression for 1 year in the field to better understand the role of plant hormones and their long-term control. Seasonality of isoprenes was mainly driven by temperature- and light-dependent variations in substrate availability through the MEP route, as well as transcriptional and post-transcriptional control of isoprene synthase (IspS). Isoprene emissions are seasonally correlated with plant hormone levels. This was especially evident in the cytokinin profiles, which decreased in summer and increased in winter. Only 4-hydroxy-3-methylbut-2-butenyl-4-diphosphate (HMBDP) exhibited a positive connection with cytokinins among the MEP metabolites examined, suggesting that HMBDP and its biosynthetic enzyme, HMBDP synthase (HDS), play a role in channelling of MEP pathway metabolites to cytokinin production. Thus, it is probable that cytokinins have potential feed-forward regulation of isoprene production. Under long-term natural conditions, the hormonal balance of IAA/JA-Ile was not associated with IspS transcripts or isoprene emissions. This study builds on prior work by revealing differences between short- and long-term hormonal modulation of isoprene emissions in the tropical tree F. septica.

Automatization of metabolite extraction for high-throughput metabolomics: case study on transgenic isoprene-emitting birch.

Metabolomics studies are becoming increasingly common for understanding how plant metabolism responds to changes in environmental conditions, genetic manipulations and treatments. Despite the recent advances in metabolomics workflow, the sample preparation process still limits the high-throughput analysis in large-scale studies. Here, we present a highly flexible robotic system that integrates liquid handling, sonication, centrifugation, solvent evaporation and sample transfer processed in 96-well plates to automatize the metabolite extraction from leaf samples. We transferred an established manual extraction protocol performed to a robotic system, and with this, we show the optimization steps required to improve reproducibility and obtain comparable results in terms of extraction efficiency and accuracy. We then tested the robotic system to analyze the metabolomes of wild-type and four transgenic silver birch (Betula pendula Roth) lines under unstressed conditions. Birch trees were engineered to overexpress the poplar (Populus × canescens) isoprene synthase and to emit various amounts of isoprene. By fitting the different isoprene emission capacities of the transgenic trees with their leaf metabolomes, we observed an isoprene-dependent upregulation of some flavonoids and other secondary metabolites as well as carbohydrates, amino acid and lipid metabolites. By contrast, the disaccharide sucrose was found to be strongly negatively correlated to isoprene emission. The presented study illustrates the power of integrating robotics to increase the sample throughput, reduce human errors and labor time, and to ensure a fully controlled, monitored and standardized sample preparation procedure. Due to its modular and flexible structure, the robotic system can be easily adapted to other extraction protocols for the analysis of various tissues or plant species to achieve high-throughput metabolomics in plant research.

Phytochemical Composition and Antioxidant Activity of Various Extracts of Fibre Hemp (Cannabis sativa L.) Cultivated in Lithuania.

The phytochemistry of fibre hemp (Cannabis sativa L., cv. Futura 75 and Felina 32) cultivated in Lithuania was investigated. The soil characteristics (conductivity, pH and major elements) of the cultivation field were determined. The chemical composition of hemp extracts and essential oils (EOs) from different plant parts was determined by the HPLC/DAD/TOF and GC/MS techniques. Among the major constituents, ß-caryophyllene (≤46.64%) and its oxide (≤14.53%), α-pinene (≤20.25%) or α-humulene (≤11.48) were determined in EOs. Cannabidiol (CBD) was a predominant compound (≤64.56%) among the volatile constituents of the methanolic extracts of hemp leaves and inflorescences. Appreciable quantities of 2-monolinolein (11.31%), methyl eicosatetraenoate (9.70%) and γ-sitosterol (8.99%) were detected in hemp seed extracts. The octadecenyl ester of hexadecenoic acid (≤31.27%), friedelan-3-one (≤21.49%), dihydrobenzofuran (≤17.07%) and γ-sitosterol (14.03%) were major constituents of the methanolic extracts of hemp roots, collected during various growth stages. The CBD quantity was the highest in hemp flower extracts in pentane (32.73%). The amounts of cannabidiolic acid (CBDA) were up to 24.21% in hemp leaf extracts. The total content of tetrahydrocannabinol (THC) isomers was the highest in hemp flower pentane extracts (≤22.43%). The total phenolic content (TPC) varied from 187.9 to 924.7 (average means, mg/L of gallic acid equivalent (GAE)) in aqueous unshelled hemp seed and flower extracts, respectively. The TPC was determined to be up to 321.0 (mg/L GAE) in root extracts. The antioxidant activity (AA) of hemp extracts and Eos was tested by the spectrophotometric DPPH● scavenging activity method. The highest AA was recorded for hemp leaf EOs (from 15.034 to 35.036 mmol/L, TROLOX equivalent). In the case of roots, the highest AA (1.556 mmol/L, TROLOX) was found in the extracts of roots collected at the seed maturation stage. The electrochemical (cyclic and square wave voltammetry) assays correlated with the TPC. The hydrogen-peroxide-scavenging activity of extracts was independent of the TPC.

Preparation of bicyclo[1.1.1]pentane-derived nucleoside analogues.

Nucleoside analogues are prevalent in drug design and call for more diversified structures. Bicyclo[1.1.1]pentane (BCP) structure has recently found wide applications in drug discovery. However, incorporation of BCP fragment into nucleoside analogues is hitherto unknown. Thus, from readily available BCP-containing building blocks, six new compounds, including pyrimidine nucleoside analogues, purine nucleoside analogues, and C-nucleoside analogues were prepared in 1-4 steps, generally with good yields.

Photocatalytic Three-Component Synthesis of 3-Heteroarylbicyclo[1.1.1]pentane-1-acetates.

Herein, we report a visible-light-induced three-component reaction involving [1.1.1]propellane, diazoates, and various heterocycles for the synthesis of 3-heteroarylbicyclo[1.1.1]pentane-1-acetates. Throughout this reaction, the radicals generated from diazoate species react with [1.1.1]propellane in an addition reaction to form bicyclo[1.1.1]pentane (BCP) radicals that subsequently react with heterocycles, leading to the formation of 1,3-disubstituted BCP acetates. Notably, this methodology exhibits excellent functional group compatibility, high atom economy, and mild reaction conditions, thus facilitating suitable synthetic access to 1,3-disubstituted BCP acetates.

Conformationally Controlled sp3 -Hydrocarbon-Based α-Helix Mimetics.

With over 60 % of protein-protein interfaces featuring an α-helix, the use of α-helix mimetics as inhibitors of these interactions is a prevalent therapeutic strategy. However, methods to control the conformation of mimetics, thus enabling maximum efficacy, can be restrictive. Alternatively, conformation can be controlled through the introduction of destabilizing syn-pentane interactions. This tactic, which is often adopted by Nature, is not a common feature of lead optimization owing to the significant synthetic effort required. Through assembly-line synthesis with NMR and computational analysis, we have shown that alternating syn-anti configured contiguously substituted hydrocarbons, by avoiding syn-pentane interactions, adopt well-defined conformations that present functional groups in an arrangement that mimics the α-helix. The design of a p53 mimetic that binds to Mdm2 with moderate to good affinity, demonstrates the therapeutic promise of these scaffolds.

Bicyclo[1.1.1]pentane Embedded in Porphyrinoids.

We report a two-step approach to obtain synthetically versatile bicyclo[1.1.1]pentane (BCP) derivatives using Grignard reagents. This method allows the incorporation of BCP units in tetrapyrrolic macrocycles and the synthesis of a new class of calix[4]pyrrole analogues by replacing two bridging methylene groups with two BCP units. In addition, a doubly N-confused system was also formed in the presence of electron-withdrawing substituents at the BCP bridgeheads. The pyrrole rings in BCP containing macrocycles exist in 1,3-alternate or αßαß conformations, as observed from single-crystal X-ray diffraction analyses and 2D NMR spectroscopy.

A review on isoprene in human breath.

We summarize the history and review the literature on isoprene in exhaled breath and discuss the current evidence and models that describe its endogenous origin and consequence for understanding isoprene levels and their variations in exhaled breath.

Peering down the sink: A review of isoprene metabolism by bacteria.

Isoprene (2-methyl-1,3-butadiene) is emitted to the atmosphere each year in sufficient quantities to rival methane (>500 Tg C yr-1 ), primarily due to emission by trees and other plants. Chemical reactions of isoprene with other atmospheric compounds, such as hydroxyl radicals and inorganic nitrogen species (NOx ), have implications for global warming and local air quality, respectively. For many years, it has been estimated that soil-dwelling bacteria consume a significant amount of isoprene (~20 Tg C yr-1 ), but the mechanisms underlying the biological sink for isoprene have been poorly understood. Studies have indicated or confirmed the ability of diverse bacterial genera to degrade isoprene, whether by the canonical iso-type isoprene degradation pathway or through other less well-characterized mechanisms. Here, we review current knowledge of isoprene metabolism and highlight key areas for further research. In particular, examples of isoprene-degraders that do not utilize the isoprene monooxygenase have been identified in recent years. This has fascinating implications both for the mechanism of isoprene uptake by bacteria, and also for the ecology of isoprene-degraders in the environments.

Alternating [1.1.1]Propellane-(Meth)Acrylate Copolymers: A New Class of Dielectrics with High Energy Density for Film Capacitors.

Polymer dielectrics with high energy density are of urgent demand in electric and electronic devices, but the tradeoff between dielectric constant and breakdown strength is still unsolved. Herein, the synthesis and molar mass control of three alternating [1.1.1]propellane-(meth)acrylate copolymers, denoted as P-MA, P-MMA, and P-EA, respectively, are reported. These copolymers exhibit high thermal stability and are semi-crystalline with varied glass transition temperatures and melting temperatures. The rigid bicyclo[1.1.1]pentane units in the polymer backbone promote the orientational polarization of the polar ester groups, thus enhancing the dielectric constants of these polymers, which are 4.50 for P-EA, 4.55 for P-MA, and 5.11 for P-MMA at 10 Hz and room temperature, respectively. Moreover, the high breakdown strength is ensured by the non-conjugated nature of bicyclo[1.1.1]pentane unit. As a result, these copolymers show extraordinary energy storage performance; P-MA exhibits a discharge energy density of 9.73 J cm-3 at 750 MV m-1 and ambient temperature. This work provides a new type of promising candidates as polymer dielectrics for film capacitors, and offers an efficient strategy to improve the dielectric and energy storage properties by introducing rigid non-conjugated bicyclo[1.1.1]pentane unit into the polymer backbone.

Origin, evolution, and future of isoprene and nitric oxide interactions within leaves.

Photolytic generation of nitric oxide (NO), isoprene, and reactive oxygen species (ROS) pre-dated life on Earth (~4 billion years ago). However, isoprene-ROS-NO interactions became relevant to climate chemistry ~50 million years ago, after aquatic and terrestrial ecosystems became dominated by isoprene-emitting diatoms and angiosperms. Today, NO and NO2 (together referred to as NOx) are dangerous biogenic gaseous atmospheric pollutants. In plants, NO, with its multiple sources and sinks, acts as a secondary messenger that regulates development at low doses and induces cell death at high doses. Likewise, biogenic isoprene is a putative antioxidant and hormone 'enabler' that hastens plant (and leaf) growth and reproduction, and improves plant tolerance to transient abiotic stresses. Using examples from controlled-chamber simulation and field studies of isoprene oxidation, we discuss the likely nature and extent of isoprene oxidation within leaves. We argue that isoprene-NO interactions vary greatly among plant species, driven by differences in isoprene emission rate and nitrate assimilation capacity (i.e. NO sink strength), ROS availability, and the within-leaf ratio between free-NO and isoprene. In a warmer and CO2-fertilized future climate, antagonism between isoprene and NO within leaves will probably occur in a NO-rich (relative to present) environment, yielding a greater proportion of isoprene oxidation products, and inducing major changes in NO-mediated growth and stress responses.

Molecular characteristics of isoprene synthase and its control effects on isoprene emissions from tropical trees.

The isoprene emission rate from plants is simulated by a function of light intensity and leaf temperature, and the G-93 formula is the most extensively applied algorithm for this purpose. Isoprene is biosynthesized by the enzyme isoprene synthase (IspS), and instantly emitted from the leaf. Enzyme kinetics of IspS and substrate availability are important factors involved in the short-term leaf-level control of isoprene emissions. It is thus assumed that the parameters of G-93 may correlate with the kinetics of IspSs, however, at present there is no data available on the relationship between these two parameters. In this investigation, six IspS genes from tropical trees were cloned, their properties characterized, and the relationship between the enzyme kinetics of IspSs and the parameters of G-93 examined. There was a negative correlation between the enzyme kinetics of IspS Km and parameter CT1 of G93, which is used to define the temperature dependency of isoprene emissions. However, performance constant of IspS (kcat/Km) only showed slight positive correlation with CT1.suggesting that the enzyme kinetics of IspS has limited significance in controlling the temperature response of isoprene emissions. The molecular structure of IspS was further elucidated using a molecular dynamics simulation with a focus on the active site in the 6 α-helices bundle. The simulation of the enzyme-substrate complex of IspS from B. variegata predicted a new metal binding domain in helix F (E383) and catalytic motif FXRDRLXE in the A-C loop that could involve the deprotonation of dimethylallyl diphosphate (DMADP) to form a carbocation. Notably, after the binding of a metal ion and DMADP, the active-site closure mechanism was found to involve conformational alterations in the helix H-α1 and transition from a loose to tight enclosure of the 6 α-helices bundles to tune the active pocket size. The characteristics identified for the IspSs from tropical trees could help to explain regional isoprene emissions in tropical areas.

Rapid Freezing of Skeletal and Cardiac Muscles Using Isopentane Cooled with Liquid Nitrogen and Tragacanth Gum for Histological, Genetic, and Protein Expression Studies.

Histological and molecular genetic evaluation of skeletal and cardiac muscles is an indispensable part of understanding muscle biology and the pathology of muscle disorders. Proper processing of the muscle tissue is a prerequisite for optimal evaluation. However, the processing of skeletal muscle samples often comes with many challenges. One of the commonly used methods of frozen tissue preparation involves optimal cutting temperature compound (OCT compound) embedding. This method is considered optimal for the processing of most of the routinely studied tissue samples. However, the processing of skeletal muscle samples using this method is often unsuitable as it causes artifacts and low DNA, RNA, and protein yield and quality due to the slow freezing of skeletal muscle tissues that allows ice crystals to form. One of the most suitable methods for skeletal muscle tissue processing for histological, genetic, and molecular studies is rapid freezing of freshly collected tissue samples using isopentane cooled with liquid nitrogen and tragacanth gum, which provides distinct advantages in consuming less time, preserving the cell morphology, and helping higher nucleic acids and protein yields. This chapter describes a protocol for rapid freezing of freshly collected skeletal muscle tissues using isopentane pre-chilled with liquid nitrogen and tragacanth gum. Skeletal muscle tissue samples processed using this protocol can be used for histological and immunological staining investigations and studies requiring DNA, RNA, and proteins from these tissues.