Applications of the Vilsmeier reaction. 13. Vilsmeier approach to polycyclic aromatic hydrocarbons

The Synthesis of three typical polycyclic hydrocarbons (PAH) has been described, wherein the Vilsmeier reaction plays a major role. Vilsmeier reaction of the tetraloll gives the dihydronaphthaldehyde 2 which on cyclodehydration gives the dihydroarene 3. Ita dehydrogenation affords 3-methoxybenz[a]anthracene (4). Vilsmeier reaction on the dimethoxydihydronaphthalene 5 gives the versatile dimethoxydihydronaphthaldehyde 6 which has been converted to the dimethoxybenzo[c]fluorene 7 by direct cyclodehydration and the fulvene 10 by cyclodehydration of allylic alcohol 8 derived from 6 followed by dehydrogenation. The saturated alcohol 12 corresponding to 8 undergoes cyclodehydration to give the dimethoxyhexahydrobenzo[c]phenanthrene (13). Some of the advantages of the Vilsmeier approach to PAH have been pointed out.

The ozone transfer between atmosphere and vegetation. A study on Scots pine in the field

Ozone (O3) is a reactive gas present in the troposphere in the range of parts per billion (ppb), i.e. molecules of O3 in 109 molecules of air. Its strong oxidative capacity makes it a key element in tropospheric chemistry and a threat to the integrity of materials, including living organisms. Knowledge and control of O3 levels are an issue in relation to indoor air quality, building material endurance, respiratory human disorders, and plant performance. Ozone is also a greenhouse gas and its abundance is relevant to global warming. The interaction of the lower troposphere with vegetated landscapes results in O3 being removed from the atmosphere by reactions that lead to the oxidation of plant-related components. Details on the rate and pattern of removal on different landscapes as well as the ultimate mechanisms by which this occurs are not fully resolved. This thesis analysed the controlling processes of the transfer of ozone at the air-plant interface. Improvement in the knowledge of these processes benefits the prediction of both atmospheric removal of O3 and its impact on vegetation. This study was based on the measurement and analysis of multi-year field measurements of O3 flux to Scots pine (Pinus sylvestris L.) foliage with a shoot-scale gas-exchange enclosure system. In addition, the analyses made use of simultaneous CO2 and H2O exchange, canopy-scale O3, CO2 and H2O exchange, foliage surface wetness, and environmental variables. All data was gathered at the SMEAR measuring station (southern Finland). Enclosure gas-exchange techniques such as those commonly used for the measure of CO2 and water vapour can be applied to the measure of ozone gas-exchange in the field. Through analysis of the system dynamics the occurring disturbances and noise can be identified. In the system used in this study, the possible artefacts arising from the ozone reactivity towards the system materials in combination with low background concentrations need to be taken into account. The main artefact was the loss of ozone towards the chamber walls, which was found to be very variable. The level of wall-loss was obtained from simultaneous and continuous measurements, and was included in the formulation of the mass balance of O3 concentration inside the chamber. The analysis of the field measurements in this study show that the flux of ozone to the Scots pine foliage is generated in about equal proportions by stomatal and non-stomatal controlled processes. Deposition towards foliage and forest is sustained also during night and winter when stomatal gas-exchange is low or absent. The non-stomatal portion of the flux was analysed further. The pattern of flux in time was found to be an overlap of the patterns of biological activity and presence of wetness in the environment. This was seen to occur both at the shoot and canopy scale. The presence of wetness enhanced the flux not only in the presence of liquid droplets but also during existence of a moisture film on the plant surfaces. The existence of these films and their relation to the ozone sinks was determined by simultaneous measurements of leaf surface wetness and ozone flux. The results seem to suggest ozone would be reacting at the foliage surface and the reaction rate would be mediated by the presence of surface wetness. Alternative mechanisms were discussed, including nocturnal stomatal aperture and emission of reactive volatile compounds. The prediction of the total flux could thus be based on a combination of a model of stomatal behaviour and a model of water absorption on the foliage surfaces. The concepts behind the division of stomatal and non-stomatal sinks were reconsidered. This study showed that it is theoretically possible that a sink located before or near the stomatal aperture prevents or diminishes the diffusion of ozone towards the intercellular air space of the mesophyll. This obstacle to stomatal diffusion happens only under certain conditions, which include a very low presence of reaction sites in the mesophyll, an extremely strong sink located on the outer surfaces or stomatal pore. The relevance, or existence, of this process in natural conditions would need to be assessed further. Potentially strong reactions were considered, including dissolved sulphate, volatile organic compounds, and apoplastic ascorbic acid. Information on the location and the relative abundance of these compounds would be valuable. The highest total flux towards the foliage and forest happens when both the plant activity and ambient moisture are high. The highest uptake into the interior of the foliage happens at large stomatal apertures, provided that scavenging reactions located near the stomatal pore are weak or non-existent. The discussion covers the methodological developments of this study, the relevance of the different controlling factors of ozone flux, the partition amongst its component, and the possible mechanisms of non-stomatal uptake.

Studying the diurnal and seasonal acclimation of photosystem Ii using chlorophyll-a fluorescence

A small fraction of the energy absorbed in the light reactions of photosynthesis is re-emitted as chlorophyll-a fluorescence. Chlorophyll-a fluorescence and photochemistry compete for excitation energy in photosystem II (PSII). Therefore, changes in the photochemical capacity can be detected through analysis of chlorophyll fluorescence. Chlorophyll fluorescence techniques have been widely used to follow the diurnal (fast), and the seasonal (slow) acclimation in the energy partitioning between photochemical and non-photochemical processes in PSII. Energy partitioning in PSII estimated through chlorophyll fluorescence can be used as a proxy of the plant physiological status, and measured at different spatial and temporal scales. However, a number of technical and theoretical limitations still limit the use of chlorophyll fluorescence data for the study of the acclimation of PSII. The aim of this Thesis was to study the diurnal and seasonal acclimation of PSII in field conditions through the development and testing of new chlorophyll fluorescence-based tools, overcoming these limitations. A new model capable of following the fast acclimation of PSII to rapid fluctuations in light intensity was developed. The model was used to study the rapid acclimation in the electron transport rate under fluctuating light. Additionally, new chlorophyll fluorescence parameters were developed for estimating the seasonal acclimation in the sustained rate constant of thermal energy dissipation and photochemistry. The parameters were used to quantitatively evaluate the effect of light and temperature on the seasonal acclimation of PSII. The results indicated that light environment not only affected the degree but also the kinetics of response of the acclimation to temperature, which was attributed to differences in the structural organization of PSII during seasonal acclimation. Furthermore, zeaxanthin-facilitated thermal dissipation appeared to be the main mechanisms modulating the fraction of absorbed energy being dissipated thermally during winter in field Scots pine. Finally, the integration between diurnal and seasonal acclimation mechanisms was studied using a recently developed instrument MONI-PAM (Walz GmbH, Germany) capable of continuously monitoring the energy partitioning in PSII.

Zinc, cadmium and lead resistance mechanisms in bacteria and their contribution to biosensing

In bacteria resistance to heavy metals is mainly achieved through active efflux, but also sequestration with proteins or as insoluble compounds is used. Although numerous studies have dealt with zinc, cadmium and lead resistance mechanisms in bacteria, it has still remained unclear how different transporters are integrated into an effective homeostasis/resistance network and whether specific mechanisms for lead sequestration exist. Furthermore, since metals are toxic not only to bacteria but to higher organisms as well, it is important to be able to estimate possible biological effects of heavy metals in the environment. This could be done by determining the bioavailable amount of the metals in the environment with bacterial bioreporters. That is, one can employ bacteria that respond to metal contamination by a measurable signal to assess the property of metals to cross biological membranes and to cause harmful effects in a possibly polluted environment. In this thesis a new lead resistance mechanism is described, interplay between CBA transporters and P-type ATPases in zinc and cadmium resistance is presented and finally the acquired knowledge is used to construct bacterial bioreporters for heavy metals with increased sensitivity and specificity. The new lead resistance model employs a P-type ATPase that removes Pb2+ ions from the cytoplasm and a phosphatase that produces inorganic phosphate for lead sequestration in the periplasm. This was the first study where the molecular mechanism of lead sequestration has been described. Characterization of two P-type ATPases and two CBA transporters showed that resistance mechanisms for Zn2+ and Cd2+ are somewhat different than for Pb2+ as these metals cannot be sequestered as insoluble compounds as easily. Resistance to Zn2+ was conferred merely by the CBA transporter that could export both cytoplasmic and periplasmic ions; whereas, full resistance to Cd2+ required interplay of a P-type ATPase that exported cytoplasmic ions to periplasm and a CBA transporter that further exported periplasmic ions to the outside. The knowledge on functionality of the transporters and metal-inducible promoters was exploited in bioreporter technology. A transporter-deficient bioreporter strain that lacked exporters for Zn2+/Cd2+/Pb2+ could detect up to 45-fold lower metal concentrations than its wild type counterpart due to the accumulation of metals in the cell. The broad specificity issue of bioreporters was overcome by using Zn-specific promoter as a sensor element, thus achieving Zn-specific bioreporter.

Thermal decomposition of doped calcium hydroxide for chemical energy storage

Thermal decomposition of Ca(OH)2 with and without additives has been experimentally investigated for its application as a thermochemical energy storage system. The homogeneous reaction model gives a satisfactory fit for the kinetic data on pure and Ni(OH)2---, Zn(OH)2--- and Al(OH)3---doped Ca(OH)2 and the order of reaction is 0.76 in all cases except for the Al(OH)3-doped sample for which the decomposition is zero order. These additives are shown not only to enhance the reaction rate but also to reduce the decomposition temperature significantly. Some models for solid decomposition reactions, and possible mechanisms in the decomposition of solids containing additives, are also discussed.

Photolysis of the dithiolactone 4-isopropylidene-3,3-dimethyl-1-thietan-2-thione; a Norrish type I reaction

The dithiolactone (1) upon excitation gives the dithione (2) in cyclohexane and other aprotic solvents and a 1 : 1 adduct in hydroxylic solvents from an n* excited singlet state via an -cleavage process.

Mechanisms of transmembrane cation transport studied by nuclear magnetic resonance spectroscopy

Several molecules like ionophores, vitamins, ion-binding cyclic peptides, acidic phospholipids, surfactants are known to expose the inner side of vesicles, to the externally added cations. Whereas ionophores and certain other systems bring about these changes by a selective transport (influx) of the cation by specialized mechanisms known as the carrier and channel mechanism, other systems cause lysis and vesicle fusion. These systems have been successfully studied using1H,31 P and13C nuclear magnetic resonance spectroscopy after the demonstration, fifteen years ago, of the ability of paramagnetic lanthanide ions to distinguish the inside of the vesicle from the outside. The results of these ’nuclear magnetic resonance kinetics’ experiments are reviewed.

HPLC analysis of plant sterol oxidation products

Increased interest in the cholesterol-lowering effect of plant sterols has led to development of plant sterol-enriched foods. When products are enriched, the safety of the added components must be evaluated. In the case of plant sterols, oxidation is the reaction of main concern. In vitro studies have indicated that cholesterol oxides may have harmful effects. Due their structural similarity, plant sterol oxidation products may have similar health implications. This study concentrated on developing high-performance liquid chromatography (HPLC) methods that enable the investigation of formation of both primary and secondary oxidation products and thus can be used for oxidation mechanism studies of plant sterols. The applicability of the methods for following the oxidation reactions of plant sterols was evaluated by using oxidized stigmasterol and sterol mixture as model samples. An HPLC method with ultraviolet and fluorescence detection (HPLC-UV-FL) was developed. It allowed the specific detection of hydroperoxides with FL detection after post-column reagent addition. The formation of primary and secondary oxidation products and amount of unoxidized sterol could be followed by using UV detection. With the HPLC-UV-FL method, separation between oxides was essential and oxides of only one plant sterol could be quantified in one run. Quantification with UV can lead to inaccuracy of the results since the number of double bonds had effect on the UV absorbance. In the case of liquid chromatography-mass spectrometry (LC-MS), separation of oxides with different functionalities was important because some oxides of the same sterol have similar molecular weight and moreover epimers have similar fragmentation behaviour. On the other hand, coelution of different plant sterol oxides with the same functional group was acceptable since they differ in molecular weights. Results revealed that all studied plant sterols and cholesterol seem to have similar fragmentation behaviour, with only relative ion abundances being slightly different. The major advantage of MS detection coupled with LC separation is the capability to analyse totally or partly coeluting analytes if these have different molecular weights. The HPLC-UV-FL and LC-MS methods were demonstrated to be suitable for studying the photo-oxidation and thermo-oxidation reactions of plant sterols. The HPLC-UV-FL method was able to show different formation rates of hydroperoxides during photo-oxidation. The method also confirmed that plant sterols have similar photo-oxidation behaviour to cholesterol. When thermo-oxidation of plant sterols was investigated by HPLC-UV-FL and LC-MS, the results revealed that the formation and decomposition of individual hydroperoxides and secondary oxidation products could be studied. The methods used revealed that all of the plant sterols had similar thermo-oxidation behaviour when compared with each other, and the predominant reactions and oxidation rates were temperature dependent. Overall, these findings showed that with these LC methods the oxidation mechanisms of plant sterols can be examined in detail, including the formation and degradation of individual hydroperoxides and secondary oxidation products, with less sample pretreatment and without derivatization.

Lost in Translation : Translation Mechanisms in Production of Cocksfoot Mottle Virus Proteins

The present study focuses on the translational strategies of Cocksfoot mottle virus (CfMV, genus Sobemovirus), which infects monocotyledonous plants. CfMV RNA lacks the 5'cap and the 3'poly(A) tail that ensure efficient translation of cellular messenger RNAs (mRNAs). Instead, CfMV RNA is covalently linked to a viral protein VPg (viral protein, genome-linked). This indicates that the viral untranslated regions (UTRs) must functionally compensate for the lack of the cap and poly(A) tail. We examined the efficacy of translation initiation in CfMV by comparing it to well-studied viral translational enhancers. Although insertion of the CfMV 5'UTR (CfMVe) into plant expression vectors improved gene expression in barley more than the other translational enhancers examined, studies at the RNA level showed that CfMVe alone or in combination with the CfMV 3'UTR did not provide the RNAs translational advantage. Mutation analysis revealed that translation initiation from CfMVe involved scanning. Interestingly, CfMVe also promoted translation initiation from an intercistronic position of dicistronic mRNAs in vitro. Furthermore, internal initiation occurred with similar efficacy in translation lysates that had reduced concentrations of eukaryotic initiation factor (eIF) 4E, suggesting that initiation was independent of the eIF4E. In contrast, reduced translation in the eIF4G-depleted lysates indicated that translation from internally positioned CfMVe was eIF4G-dependent. After successful translation initiation, leaky scanning brings the ribosomes to the second open reading frame (ORF). The CfMV polyprotein is produced from this and the following overlapping ORF via programmed -1 ribosomal frameshift (-1 PRF). Two signals in the mRNA at the beginning of the overlap program approximately every fifth ribosome to slip one nucleotide backwards and continue translation in the new -1 frame. This leads to the production of C-terminally extended polyprotein, which encodes the viral RNA-dependent RNA polymerase (RdRp). The -1 PRF event in CfMV was very efficient, even though it was programmed by a simple stem-loop structure instead of a pseudoknot, which is usually required for high -1 PRF frequencies. Interestingly, regions surrounding the -1 PRF signals improved the -1 PRF frequencies. Viral protein P27 inhibited the -1 PRF event in vivo, putatively by binding to the -1 PRF site. This suggested that P27 could regulate the occurrence of -1 PRF. Initiation of viral replication requires that viral proteins are released from the polyprotein. This is catalyzed by viral serine protease, which is also encoded from the polyprotein. N-terminal amino acid sequencing of CfMV VPg revealed that the junction of the protease and VPg was cleaved between glutamate (E) and asparagine (N) residues. This suggested that the processing sites used in CfMV differ from the glutamate and serine (S) or threonine (T) sites utilized in other sobemoviruses. However, further analysis revealed that the E/S and E/T sites may be used to cleave out some of the CfMV proteins.

Food and Indoor Air Isolated Bacillus Non-Protein Toxins: : Structures, Physico-Chemical Properties and Mechanisms of Effects on Eukaryotic Cells

We report here the structures and properties of heat-stable, non-protein, and mammalian cell-toxic compounds produced by spore-forming bacilli isolated from indoor air of buildings and from food. Little information is available on the effects and occurrence of heat-stable non-protein toxins produced by bacilli in moisture-damaged buildings. Bacilli emit spores that move in the air and can serve as the carriers of toxins, in a manner similar to that of the spores of toxic fungi found in contaminated indoor air. Bacillus spores in food cause problems because they tolerate the temperatures applied in food manufacture and the spores later initiate growth when food storage conditions are more favorable. Detection of the toxic compounds in Bacillus is based on using the change in mobility of boar spermatozoa as an indicator of toxic exposure. GC, LC, MS, and nuclear magnetic resonance NMR spectroscopy were used for purification, detection, quantitation, and analysis of the properties and structures of the compounds. Toxicity and the mechanisms of toxicity of the compounds were studied using boar spermatozoa, feline lung cells, human neural cells, and mitochondria isolated from rat liver. The ionophoric properties were studied using the BLM (black-lipid membrane) method. One novel toxin, forming ion channels permeant to K+ > Na+ > Ca2+, was found and named amylosin. It is produced by B. amyloliquefaciens isolated from indoor air of moisture-damaged buildings. Amylosin was purified with an RP-HPLC and a monoisotopic mass of 1197 Da was determined with ESI-IT-MS. Furthermore, acid hydrolysis of amylosin followed by analysis of the amino acids with the GS-MS showed that it was a peptide. The presence of a chromophoric polyene group was found using a NMR spectroscopy. The quantification method developed for amylosin based on RP-HPLC-UV, using the macrolactone polyene, amphotericin B (MW 924), as a reference compound. The B. licheniformis strains isolated from a food poisoning case produced a lipopeptide, lichenysin A, that ruptured mammalian cell membranes and was purified with a LC. Lichenysin A was identified by its protonated molecules and sodium- and potassium- cationized molecules with MALDI-TOF-MS. Its protonated forms were observed at m/z 1007, 1021 and 1035. The amino acids of lichenysin A were analyzed with ESI-TQ-MS/MS and, after acid hydrolysis, the stereoisomeric forms of the amino acids with RP-HPLC. The indoor air isolates of the strain of B. amyloliquefaciens produced not only amylosin but also lipopeptides: the cell membrane-damaging surfactin and the fungicidal fengycin. They were identified with ESI-IT-MS observing their protonated molecules, the sodium- and potassium-cationized molecules and analysing the MS/MS spectra. The protonated molecules of surfactin and fengycin showed m/z values of 1009, 1023, and 1037 and 1450, 1463, 1493, and 1506, respectively. Cereulide (MW 1152) was purified with RP-HPLC from a food poisoning strain of B. cereus. Cereulide was identified with ESI-TQ-MS according to the protonated molecule observed at m/z 1154 and the ammonium-, sodium- and potassium-cationized molecules observed at m/z 1171, 1176, and 1192, respectively. The fragment ions of the MS/MS spectrum obtained from the protonated molecule of cereulide at m/z 1154 were also interpreted. We developed a quantification method for cereulide, using RP-HPLC-UV and valinomycin (MW 1110, which structurally resembles cereulide) as the reference compound. Furthermore, we showed empirically, using the BLM method, that the emetic toxin cereulide is a specific and effective potassium ionophore of whose toxicity target is especially the mitochondria.

Effect of surface resistance on gas absorption accompanied by a chemical reaction in a gas - liquid contactor

An analysis of gas absorption accompanied by chemical reaction in the presence of interfacial resistance is presented. The analysis indicates that the effect of interfacial resistance on interphase mass transfer is significantly higher in presence of a reaction compared to the pure absorption case. For fixed values of surface resistance and contact time, the difference between the amount of gas transferred across the interface with and without surface resistance increases as the value of reaction velocity increases. For ranges of contact time and surface resistance of practical relevance, the influence of surface resistance is too high to be neglected while designing gas-liquid contactors.

Photochemical Reactions in Oriented Systems

Modification of chemical reactions through the use of constrained and/or organized media has attracted a great deal of attention recently. Results from our laboratory in this direction which include a study of photochemical reactions in solid state and in cyclodextrins are presented here. A study of solid state photochemical behavior of coumarins has provided information regarding subtler aspects of topochemical postulates of photodimerization. Results pertaining to geometrical criteria for photodimerization and "chloro" as a crystal engineering group are discussed. As a part of an attempt to correlate chemical reactivity with molecular packing in the solid state, photooxidation of diarylthioketones in the solid state has been investigated. The observed differences in the reactivity of these crystals are rationalized in terms of crystal packing. Though cyclodextrins have been extensively studied, very few photochemical reactions involving molecules complexed to cyclodextrins have been examined. In this connection, the utility of cyclodextrins in bringing about selectivity in photochemical reactions through the study of excited state behavior of olefins and aryl alkyl ketones has been demonstrated in our laboratory.