Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    High-resolution mass spectrometry for the analysis of interfacial kinetics of organic surface reactions
    Sen, Rickdeb - \ 2017
    Wageningen University. Promotor(en): H. Zuilhof. - Wageningen : Wageningen University - ISBN 9789463436243 - 308
    surface chemistry - unimolecular films - chemical reactions - analytical methods - mass spectrometry - oppervlaktechemie - unimoleculaire films - chemische reacties - analytische methoden - massaspectrometrie

    In this thesis, XPS and DART–HRMS have been used in close conjugation to supplement each other, since the latter is a relatively new addition to surface chemist’s repertoire that – after development – needed a firm comparison to build up a reputation of its own. The strength of our approach has been underlined by the high correlation between these two independent analytical techniques. Central to our approach has been the formation of mixed monolayers in case of aluminum oxide substrates. As presented in Chapters 2, 3 and 4, we have succeeded in the rapid formation of range stable, covalently bound mixed monolayers. The subsequent development of a general and fast analytical technique to determine the interfacial reaction kinetics, including the activation parameters DH‡ and DS‡, provided unparalleled insights. We have developed a “MS–ionizable tag” technique, which has been applied for the analysis of surface–bound organic reactions, to the best of our knowledge, for the first time.

    The Strain–Promoted Alkyne–Azide Cycloaddition (SPAAC) reaction was chosen as a model reaction given the fact that its kinetics had been well–studied in solution. As shown in Chapter 2, the microenvironment around the reactive surface group was carefully controlled by the length of the inert alkyl chains surrounding it. We observed a few interesting trends which could be of great interest to future surface chemists. First, the SPAAC reaction – which is a click reaction in solution – does not retain this nature on the surface (It does not proceed to full conversion and converges sluggishly to around 37% yield after significant temporal passage). A partially accessible microenvironment, where the motion of reactive groups is slightly restricted, was found to provide a high rate with the highest surface yield. In contrast, a freely accessible reactive moiety afforded a lower surface yield albeit with the highest overall rate. Finally, a buried microenvironment led to the highest overall rate albeit with a lower surface yield. As a corollary, for the surface–bound SPAAC reaction we can compare the partially accessible microenvironment to a marathon runner who is able to run further but at a pace slower than a sprinter (free microenvironment). This provides the surface chemist with a handle for tuning the monolayer as per her/his reaction goals.

    Harnessing the valuable insights gained from the SPAAC reaction, our concept of ionizable MS tag coupled with DART–HRMS was further extended to a more novel and yet unstudied interfacial reaction in Chapter 3. The Strain–Promoted Oxidation–Controlled cycloalkyne–1,2–Quinone (SPOCQ) cycloaddition was applied for the first time on a surface and afforded a quantitative yield for a free microenvironment in under 4 h. It is to be noted here, that for the first time a 100% (quantitative) metal–free click reaction was observed at a surface. This proved that our approach of engineering the microenvironment around the reactive site provides a distinct edge needed to attain quantitative yields. Quinones are hard to synthesize/store/use in solution given their high propensity to polymerize. However, we demonstrated that on the surface, quinones can be easily generated and stored over–extended period of time by a facile periodate oxidation. Auto–polymerization of surface–bound quinones is precluded by their tether and enforced distal separation by surrounding inert alkyl chains (3:1 ratio). The wider application of this interesting mixture has been further rigorously demonstrated in later chapters too. The bioorthogonality of the SPOCQ reaction coupled with its higher speed and its quantitative yields on the surface are definitely its most salient features.

    After studying strain–promoted click reactions on the surface (culminating for SPOCQ in quantitative conversion within 4 h), the question arose if DART–HRMS could also be used to reproducibly and precisely determine a different class of cycloadditions, for which we selected the interfacial inverse electron demand Diels–Alder (IEDDA) reaction as this reaction was reported to be really fast –at least for click reactions– in solution. This was studied in Chapter 4 extensively and we surpassed our previous kinetic record (SPOCQ) by obtaining a quantitative yield in a mere 15 min. The other interesting observation of this study was that reversing the reaction counterparts on the surface produced a discernible reaction rate difference. We found that one of the reactants when tethered in a particular stereochemistry (exo– form) gave the highest surface coverage (100%) within the shortest amount of time. This was also the first time that the effect of diastereomerism on interfacial reaction rates was studied.

    In Chapter 5, covalent modification of native non–activated mica has been carried out utilizing catechol linkers. Previous studies for mica modification produced poorly defined polymeric structures on the surface or required extensive and tedious organic synthesis. We have addressed both these issues head–on in this thesis. Well–defined and characterized ultrathin layers were constructed on mica using a catechol–based molecule involving a two–step synthesis. Mica being atomically flat provides an ideal surface upon which to study various phenomena by AFM and other forms of microscopy. However, most research until now was restricted to simply drop–casting the pre–fabricated moieties followed by studying their final structures. Our method now allows for the step–wise formation and characterization of these very interesting structures. Along with it, we also performed several click attachment chemistries on these ultrathin layers which can be harnessed by surface chemists to put various functional and structurally complex moieties on the surface. This opens the pathway for the attachment of more complex architectures on the surface with higher functionality along with the ability to study their formation in a step–wise controlled fashion.

    Overall, this thesis wishes to understand organic surface chemistry and several of its intricate mysteries. It clearly outlines several modification techniques and unravels interfacial kinetics of several interesting “metal–free click reactions”. It strives to rationalize the activation parameters in conjunction with classical organic chemistry and gives details on how surrounding “inert” alkyl chains can play a profound role in reaction rates. Lastly, we have striven to and achieved rapid and quantitative reactions on the surface by virtue of optimization of this microenvironment. Personally I believe, we have treaded on a road seldom traveled and unraveled a new understanding about molecular interactions on the ever–interesting and an infinitely–complex surface.

    Chemie - Van suiker naar mobiel : Kennisclip Bogo-project e-learning
    Baltissen, A.H.M.C. - \ 2016
    Groen Kennisnet
    biopolymeren - suiker - biobased economy - biochemie - chemische reacties - polymelkzuur - melkzuur - bioplastics - tuinbouw - lesmaterialen - biopolymers - sugar - biobased economy - biochemistry - chemical reactions - polylactic acid - lactic acid - bioplastics - horticulture - teaching materials
    Deze kennisclip maakt onderdeel uit van de lesmodule Biobased Economy van het CIV T&U.
    New insight into enzymatic cross-linking of globular proteins: from nanostructure to functionality
    Sariçay, Y. - \ 2014
    Wageningen University. Promotor(en): Martien Cohen Stuart, co-promotor(en): Renko de Vries; Peter Wierenga. - Wageningen : Wageningen University - ISBN 9789462571211 - 217
    enzymatische cross-linking - eiwitten - chemische reacties - alfa-lactalbumine - lactalbumine - nanotechnologie - enzymatic cross-linking - proteins - chemical reactions - alpha-lactalbumin - lactalbumin - nanotechnology


    In last two decades, enzymatic cross-linking of proteins has a growing interest in food technology for better tailoring protein functionality. However, the relation between physical and functional properties of enzymatically cross-linked proteins has been hardly addressed so far. The aim of this thesis was to elucidate the effect of enzymatic protein cross- linking on the physical and functional properties of protein nanoparticles at multiple lengthscale. In the first part of this thesis, as a model system, the enzymatic cross-linking of globular whey protein apo-α-lactalbumin (α-LA) by horseradish peroxidase (HRP) was discussed in details. In comparison with HRP, in the second part of the thesis, we also addressed to what extent both laccase (LC) (from trametes versicolor) and tyrosinase (TYR) (from agaricus bisporus) differ in catalyzing oxidative cross-linking of α-LA. Both HRP and LC were capable of creating self-similar large α-LA nanoparticles that have an open architecture at similar lengthscales whereas TYR led to the formation of α-LA oligomers only. All HRP-, LC-nanoparticles and TYR-oligomers exhibited a high extent of secondary structure content preserved whereas their almost all tertiary structure was lost upon enzymatic cross-linking. HRP-catalyzed cross-linking of α-LA resulted in more hydrophilic nanoparticles than LC-cross-linked α-LA nanoparticles. Whereas both HRP- and LC-nanoparticles exhibited very high colloidal and thermal stability against protein aggregation at pH 5.8 and 7.0, HRP- nanoparticles were more stable than LC-nanoparticles upon heating and in the presence of dithiothreitol (DTT). This suggests that, unlike HRP- nanoparticles, not only the dityrosine bonds but also disulfide cross-linking stabilizes LC-nanoparticles. Dilute dispersions of HRP-nanoparticles exhibited a high viscosity and a hydrophilic nature. As these dispersions were concentrated, they jammed above their critical overlapping concentration and thus created physical transparent protein hydrogels at relatively low protein concentration (4% w/v). These properties of HRP- nanoparticle dispersions offer high thickening properties that are comparable with polysaccharide in food applications as protein-based thickeners.

    Multi-response modeling of acrylamide formation in biscuits
    Capuano, E. ; Fels, H.J. van der; Atac-Mogol, B. ; Kocadagli, T. ; Göncüoglu, N. ; Hamzalioglu, B.A. ; Gokmen, V. - \ 2013
    biscuits - acrylamiden - bakken (in de oven) - kinetica - modellen - chemische reacties - biscuits - acrylamides - baking - kinetics - models - chemical reactions
    The aim of this study was to model acrylamide formation during baking of biscuits, using multi-response kinetic modelling.
    Reaction and separation opportunities with microfluidic devices
    Kolfschoten, R.C. - \ 2011
    Wageningen University. Promotor(en): Remko Boom, co-promotor(en): Anja Janssen. - [S.l.] : S.n. - ISBN 9789085858645 - 153
    microfluidics - chemische reacties - scheiding - kunstmatige membranen - enzymen - diffusie - microfluidics - chemical reactions - separation - artificial membranes - enzymes - diffusion

    Microfluidic devices make precisely controlled processing of substances possible on a microliter level. The advantage is that, due to the small sizes, the driving forces for mass and heat transfer are high. The surface to volume ratios are also high, which can benefit many surface oriented processes. In addition, because of their small volumes, microfluidic devices reduce reagent consumption and risk of failure compared to larger counterparts. Furthermore, the parallelization of such devices can increase productivity while maintaining their characteristics. Overall, these advantageous properties give many opportunities for reaction and separation processes.

    Although researchers have intensively studied microfluidics for analytical and sensory applications, microfluidics for preparative processes is still in its infancy. This thesis research involved exploring these processes for biocatalysis and bio-separations with microfluidic devices. The purpose of this thesis was to yield a better understanding of microfluidics for the preparative processes and larger-scale production. We therefore addressed subjects including microfluidic parallelization, membrane separation, biocatalysis, and design. The presented research is useful for further developing innovative process intensification by means of microfluidic devices.

    Parallelization of microfluidic devices can facilitate the generation of more data or product in less time. In Chapter 2, we present a proof of concept of such a parallelization for obtaining information on reaction and separation kinetics. We assembled different microfluidic contactors into a single device in order to perform distinct experiments simultaneously. The concept of the parallelization was based on the decoupling of pressure drop from residence time. We demonstrated this by microfluidic membrane separations and determination of membrane properties. The reported device enabled a three times higher throughput compared to devices with a single separation region.

    Processes such as chromatographic separation and nanofiltration can remove low molecular weight sugars from liquid mixtures of oligosaccharides. In Chapter 3, we present a novel separation process based on the concept of mass diffusion. Differences between diffusivities of the components drive such a separation, while membranes, in particular nanofiltration membranes, can enhance the separation. We demonstrate this by the use of a membrane microfluidic device for the separation of small molecular weight components. Our results show that mass diffusion separation in liquids is a feasible concept. With optimized microchannel and membrane dimensions, the presented separation process might compete with currently available separation technologies.

    For diffusion-based processes, such as mass diffusion separation shown in Chapter 3, small diffusion distances – and thus thinner membranes – can reduce diffusion times significantly. In Chapter 4, we used a microfluidic contactor to contact liquid streams via such extremely thin membranes. We show that the presented concept can be useful for diffusion-based pre-concentration or downstream processes such as fractionation and enrichment. Our results indicate that also this method can yield a feasible process. Moreover, the technology is generally applicable to any diffusing component – regardless of its absolute diffusivity or concentration.

    Fast mass transfer and low reagent consumption have made enzyme microreactors popular research tools. In Chapter 5, we used such a microreactor to study the effect of diffusion on enzyme activity. We found that the Michaelis-Menten kinetic parameters were similar at the microscale and bench scale. Our results show that with residence times below a few seconds, diffusion effects limited the reaction rate and therefore reduced the conversion per volume of enzyme microreactor. The critical residence time where this limitation occurred increased quadratically with channel width, increased with enzyme concentration, and decreased with substrate concentration. We concluded that in order to use an enzyme microreactor efficiently, such effects should be taken into account.

    Many parameters such as the enzyme properties, operating conditions, and dimensions of the microreactor determine to what extent mass transfer restrictions affect the reaction rate and the productivity. The use of microchannels can indeed shorten the characteristic mass transfer time, as shown in Chapter 5, but may also affect the productivity of the microreactor. Chapter 6 provides the correlations between these parameters for coflow enzyme microreactors obeying Michaelis-Menten kinetics. These correlations outline the design space based on reduced mass transfer restrictions and maximum productivity respectively. The methodology that yields the design space provides a generic hands-on approach to optimally design coflow enzyme microreactors.

    Microfluidics involves the exploitation of the phenomena that manifest themselves on microscale. This thesis shows that microscale applications can indeed offer unprecedented benefits. The discussion in Chapter 7 summarizes and reflects on the previous parts of this thesis. We conclude that it is important to explore and exploit other characteristics of continuous production in microfluidic devices beyond mass transfer effects in order to develop novel processes. In addition, we stress the importance of adoption of microfluidics, and show which determinants are involved in this. Knowledge of these determinants is of utmost importance to reduce skepticism towards and stimulate the adoption of microfluidics by industry.

    New approach to analyse spin probe and spin trap ESR
    Makarova, K. - \ 2011
    Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): Henk van As. - [S.l. : S.n. - ISBN 9789085858409 - 145
    paramagnetische elektronenresonantiespectroscopie - chemische reacties - vrije radicalen - biofysica - electron paramagnetic resonance spectroscopy - chemical reactions - free radicals - biophysics
    The goal of this thesis is to develop new comprehensive methods for the analysis of ESR spectra and interpretation of magnetic parameters. A new approach for the analysis of fast isotropic spectra is proposed. It is based on a combination of an experimental approach (multifrequency ESR) and accurate spectra simulation using an improved model, that will be further introduced below. The determined magnetic parameters of the spin probe are directly interpreted in terms of structural information about the spin probe surroundings (lipid bilayer). The obtained magnetic parameters of various spin traps are interpreted by artificial neural networks (ANN) in order to obtain information about the identities of trapped radicals. Then, Density Functional Theory (DFT) calculations are applied to study the mechanism of reactions involving free radicals detected by spin trapping ESR and to calculate magnetic parameters of the radical adducts.

    Kinetic modeling of reactions in Foods
    Boekel, M.A.J.S. van - \ 2008
    Boca Raton : CRC Press - ISBN 9781574446142 - 400
    wiskundige modellen - kinetica - chemische reacties - voedselkwaliteit - voedingsmiddelen - houdbaarheid (kwaliteit) - opslagkwaliteit - fysische eigenschappen - fysicochemische eigenschappen - modelleren - mathematical models - kinetics - chemical reactions - food quality - foods - keeping quality - storage quality - physical properties - physicochemical properties - modeling
    The level of quality that food maintains as it travels down the production-to-consumption path is largely determined by the chemical, biochemical, physical, and microbiological changes that take place during its processing and storage. Kinetic Modeling of Reactions in Foods demonstrates how to effectively capture these changes in an integrative fashion using mathematical models. Thus, kinetic modeling of food changes creates the possibility to control and predict food quality from a technological point of view.
    High-Rate Sulfate Reduction at High Salinity (up to 90 in Mesophilic UASB Reactors
    Vallero, M.V.G. ; Sipma, J. ; Lettinga, G. ; Lens, P.N.L. - \ 2004
    Biotechnology and Bioengineering 86 (2004)2. - ISSN 0006-3592 - p. 226 - 235.
    sulfaat - reductie - chemische reacties - zoutgehalte - geactiveerd slib - biodegradatie - acetaten - propionaten - ethanol - waterzuivering - sulfate - reduction - chemical reactions - salinity - activated sludge - biodegradation - acetates - propionates - ethanol - water treatment - desulfitobacterium-frappieri pcp-1 - anaerobic granular sludge - long-term competition - waste-water - methanogenic bacteria - biological treatment - reducing reactors - bed reactor - wastewaters - ammonia
    Sulfate reduction in salt-rich wastewaters using unadapted granular sludge was investigated in 0.9 L UASB reactors (pH 7.0 ± 0.2; hydraulic retention time from 8-14 h) fed with acetate, propionate, or ethanol at organic loading rates up to 10 and in excess sulfate (COD/SO of 0.5). High-rate sulfate reduction rates (up to 3.7 were achieved at salinities exceeding 50 gNaCl.L-1 and 1 gMgCl2.L-1. Sulfate reduction proceeded at a salinity of up to 70 gNaCl.L-1 and 1 gMgCl2.L-1 (corresponding to a conductivity of about 85-90, although at lower rates compared to a conductivity of 60-70 Ethanol as well as propionate were suitable substrates for sulfate reduction, with acetate and sulfide as the end products. The successful high-rate treatment was due to the proliferation of a halotolerant incomplete oxidizing SRB population present in the unadapted inoculum sludge. Bioaugmentation of this sludge with the acetate oxidizing halotolerant SRB Desulfobacter halotolerans was unsuccessful, as the strain washed out from the UASB reactor without colonizing the UASB granules. © 2004 Wiley Periodicals, Inc.
    Vetoxidatie en het voorspellen van houdbaarheid
    Dekker, M. - \ 2002
    Voedingsmiddelentechnologie 35 (2002)1-2. - ISSN 0042-7934 - p. 37 - 39.
    vetten - lipiden - geoxideerde vetten - oxidatie - chemische reacties - houdbaarheid (kwaliteit) - kwaliteit - bewaartijd - opslagkwaliteit - voedingsmiddelen - conferenties - fats - lipids - oxidized fats - oxidation - chemical reactions - keeping quality - quality - storage life - storage quality - foods - conferences
    Verslag van een symposium als afsluiting van een onderzoeksproject
    Wheat bran glucuronoarabinoxylans : biochemical and physical aspects
    Schooneveld - Bergmans, M.E.F. - \ 1997
    Agricultural University. Promotor(en): A.G.J. Voragen; G. Beldman. - S.l. : Schooneveld-Bergmans - ISBN 9789054857167 - 125
    graansoorten - maling - Triticum aestivum - tarwe - hexaploïdie - voedsel - voedingsmiddelen - koolhydraten - zetmeel - vezel - polysacchariden - structuur - chemische reacties - cereals - milling - Triticum aestivum - wheat - hexaploidy - food - foods - carbohydrates - starch - fibre - polysaccharides - structure - chemical reactions

    Arabinoxylans are present in cereal cell walls and in vitro they have interesting physicochemical properties, such as viscosity and gelation. Although many studies on these properties were reported for wheat flour arabinoxylan, not much research has been directed towards exploitation of these polysaccharides as food gum. For that purpose glucuronoarabinoxylans of wheat bran, a cheap by-product of the cereal industry, were studied with regard to their extractability, their structural and physicochemical properties.

    Approximately 50% of the glucuronoarabinoxylans of wheat bran cell wall material were recovered in high purity by barium hydroxide extraction at 70 to 95°C. Delignification or other treatments to open up the cell wall structure were not effective in increasing the yield. The extracted glucuronoarabinoxylans were very diverse in chemical structure and physicochemical properties. About 30% of them had a low degree of substitution, were easily degradable by xylanolytic enzymes and hardly influenced the viscosity of the solvent as a result of extensive aggregation. Over 50% of them had a high degree of substitution, were supposed to contain dimeric branches of arabinose and xylose, were scarcely degradable by xylanolytic enzymes, gave moderate viscosity to solutions and were very effective in stabilizing emulsions. The structure of these glucuronoarabinoxylans could only be speculated upon and it could not be enzymatically modified as a consequence of its complexity and the lack of appropriate enzymes. The remaining glucuronoarabinoxylans either had an intermediate or very high degree of substitution, of which the latter was presumed to be connected to lignin-fragments.

    Gel-forming glucuronoarabinoxylans were recovered only in low yield by dilute alkali extraction and subsequent purification was necessary. These feruloylated glucuronoarabinoxylans gelled upon addition of oxidative agents, of which peroxide - peroxidase, glucose - glucoseoxidase - peroxidase and ammonium persulphate were investigated. In comparison with wheat flour arabinoxylans, those of wheat bran appeared to give less flexible networks at high concentration, which was ascribed to their high degree of substitution and high ferulic acid content. Of the dimers formed upon cross-linking, the generally known diferulic acid, being a 5-5 coupled dimer, was only present in relatively low amounts. Dimers, in which the 8-position of the ferulic acid residue is involved were preponderant. The distribution of the dimers was not affected by the type of cross- linking agent or the type of arabinoxylan. However, the presence of lignin fragments in the bran extract was presumed to cause a low ferulic acid recovery upon cross-linking.

    Growth and death of animal cells in bioreactors
    Martens, D.E. - \ 1996
    Agricultural University. Promotor(en): J. Tramper; C.D. de Gooijer. - S.l. : Martens - ISBN 9789054855934 - 222
    weefselkweek - celkweek - meristemen - zoölogie - chemische reacties - uitrusting - biotechnologie - chemische industrie - biochemie - tissue culture - cell culture - meristems - zoology - chemical reactions - equipment - biotechnology - chemical industry - biochemistry - cum laude

    Animal-cell cultivation is becoming increasingly important especially for the area of hunian- health products. The products range from vaccines to therapeutic proteins and the cells themselves. The therapeutic application of proteins puts high demands upon their quality with respect to purity and structure. For example, a correct folding and glycosylation is of importance for the activity, the in vivo clearance rate and the possible immunogenicity of the protein, and can often only be obtained by production in animal cells. An important class of proteins produced by animal cells is formed by monoclonal antibodies. Monoclonal antibodies are produced by hybridoma cells and have the capacity to bind very specifically to a particular molecular structure (epitope), a quality that makes them suitable for application in in vivo and in vitro diagnostics, in separation technology and for the in vivo targeting of drugs.

    The occurrence of substantial cell death and the presence of cell debris is a major problem in animal-cell cultivation. It interferes with the attainment of high volumetric productivities and with a proper functioning of the process. In addition, it may affect the quality of the product and cause problems in down-stream processing. Cell death may follow two different pathways, being apoptosis and necrosis, which have very distinct physiological and morphological features. Necrosis is a passive process generally caused by sudden high levels of environmental stress, whereas apoptosis is an active, genetically controlled process induced by mild stress conditions or specific signals from the environment.

    After the introduction in Chapter 1, the application of a general framework for the construction of segregated models is discussed in Chapter 2 with respect to the behaviour of animal-cell populations. For the construction of segregated models, the physiological state of an animal cell must be specified, which is discussed in this chapter with special attention for the experimental verification of the models. Finally, a number of age-structured, segregated models, which are of importance for animal-cell cultivation are reviewed in this chapter.

    The required amounts of animal-cell products are expected to be in the order of kilograms or even tonnes on a yearly basis. In order to produce these amounts, scale-up is necessary, which is most easily done in conventional reactor systems like the stirred-tank, bubble-column, and air-lift reactor. A main problem in the scale-up of these reactors is the supply of sufficient oxygen to the culture, which often requires sparging. Hydrodynamic forces associated with sparging cause cell death. In Chapters 3, 4, and 5 the specific death rate of hybridoma cells in bubble-column and air-lift reactors is studied with the hypothetical-killing-volume theory as a central theme. The hypothetical killing volume is a hypothetical volume associated with an air bubble during its lifetime in the reactor in which all cells are killed. The first-order death-rate constant in bubble- column and air-lift reactors can then be derived to be the product of this hypothetical killing volume and the number of bubbles introduced into the reactor per unit time and per unit reactor volume. The specific death rate of the hybridoma cells in the bubble-column and air-lift reactors is shown to be proportional to the gas flow rate and the reciprocal reactor height. Furthermore, in bubble columns the specific death rate is shown to be proportional to the square of the reciprocal reactor diameter. These results are in accordance with the hypothetical-killing-volume theory. The main cause of cell death is found to be bubble breakup at the surface, although detrimental effects at the sparger cannot be excluded. In Chapter 6 the specific death rate of Vero cells immobilized on microcarriers is shown to be proportional to the gas flow rate. Since the height of the reactor is not varied, it cannot be excluded that in this case also the rising of the bubbles or the associated liquid flow cause cell damage.

    A common method to reduce the detrimental effects of air bubbles is the use of protective additives. In this thesis it is shown that the addition of two such protectants, Pluronic F68 (Chapter 3) and serum (Chapter 4), respectively, reduces the amount of cell death as a consequence of sparging. Furthermore, as demonstrated in Chapter 4, the protective effect of serum has a fast-acting, physical, and a slow-acting, physiological component. In Chapter 5 the effect of the specific growth rate on the specific death rate of cells due to sparging is studied in air-lift loop reactors. Cells with varying specific growth rates are obtained from steady-state continuous cultures run at different dilution rates. Remarkably, the specific death rate of the cells due to sparging decreased as their specific growth rate decreased. Furthermore, in Chapter 6 it is shown that the specific death rate of Vero cells is reduced by immobilisation of the cells inside porous carriers.

    Below a critical dilution rate in continuous culture as well as towards the end of batch cultures, the specific death rate of hybridoma cells increases rapidly. In this case, the cells mainly die through apoptosis as a consequence of substrate depletion and the accumulation of toxic products. In Chapter 7 an age-structured model is developed to describe the rate of apoptosis as a function of the dilution rate in continuous culture. In this model a critical specific growth rate is introduced below which the cells start becoming apoptotic. In addition to the specific deathand growth rate, the average cell volume of the viable cells and the specific consumption and production rates for glucose, glutamine, lactate and ammonia are calculated. The model can reasonably well describe a set of literature data, with respect to the specific growth- and death rate and the concentrations of viable cells, dead cells, glucose, glutamine, lactate, and ammonia. In Chapter 8 the model is extended with equations concerning two hypotheses for the production of monoclonal antibody being:
    (1) Passive release of antibody from dead cells.
    (2) Increased productivity by the apoptotic cells.
    Both hypotheses can describe the increase in productivity at decreasing dilution rates as observed in Chapter 5. Furthermore, the distribution of cells over the different phases of the cell cycle is calculated and the equations for the average cell volume are rewritten in terms of forward scatter as measured by flow cytometry. Model predictions concerning these variables are compared to results obtained in Chapter 5. The G 1 - and G 2 /M-phase fractions are not predicted correctly, which may be caused by a cell-cycle-phase specificity of apoptosis. In addition, a good prediction of all cell-cycle fractions can be obtained if it is assumed that the duration of the G 2 /M phase is not constant, but increases as the specific growth rate decreases from the maximum to its critical value. Furthermore, the calculated fraction of apoptotic cells is shown to be proportional to the forward scatter.

    Cell death associated with sparging may be minimised by:
    (1) Maximizing the amount of oxygen transferred per bubble introduced in the reactor.
    (2) The addition of a shear-protective agent like Pluronic F68 or serum.
    (3) The immobilisation of cells inside porous microcarriers.
    Point one is most easily done by increasing the reactor height and the oxygen tension in the air bubbles. With respect to the second point, it should be mentioned that the additive must be removed from the final product and can cause problems in down-stream processing. This may lead to an increase in the product cost. Finally, in the case of immobilisation of cells inside carriers, transport limitations may occur, which may in turn induce apoptosis. Cell death through apoptosis as caused by low levels of shear, substrate limitation and the presence of toxic products may be reduced through a careful process design and genetic manipulation of the cells.

    Glucuronoarabinoxylans from sorghum grain
    Verbruggen, M.A. - \ 1996
    Agricultural University. Promotor(en): A.G.J. Voragen; G. Beldman. - S.l. : Verbruggen - ISBN 9789054855026 - 131
    sorghum bicolor - polysacchariden - structuur - chemische reacties - sorghum - sorghum bicolor - polysaccharides - structure - chemical reactions - sorghum

    Water-unextractable cell wall materials (WUS) were prepared from raw, polished, and malted sorghum ( Sorghum vulgare cv. Fara Fara). Except for the amounts, hardly any difference could be observed between the WUS of these three raw materials. This means that cell wall materials of the endosperm cell walls are basically the same as those of the outer endosperm and pericarp layers, and that the cell walls largely persist, during malting. These preparations were further fractionated by a sequential extraction procedure using aqueous solutions of saturated Ba(OH) 2 , 1M KOH and 4M KOH. The WUS preparations were composed of glucuronoarabinoxylans (GAX), (1→3),(1→4)-β-D-glucans, cellulose, and some protein. GAX was primarily extracted by Ba(OH) 2 solutions. All GAX fractions were composed of a highly substituted (1→4)-β-D-xylan backbone, substituted by arabinose and uronic acid. It was concluded that sorghum GAX populations were characterized by a reasonable homogeneity, since they could not be separated further by several chromatographic and precipitation techniques.

    Degradation studies using purified xylanases, arabinofuranosidases and a glucuronidase alone or in combination, showed that the GAX populations were hardly broken down. Some oligomers were formed by digesting Ba(OH) 2 extracted GAX with a combination of endoxylanase I and (1→4)-β-D-arabinoxylan arabinofuranohydrolase, both purified from Aspergillus awamori . These oligomers were found to have a main chain of three or four xylose units, and to contain α-glucuronic acid linked to O -2 of the non-reducing terminal xylose unit. Two oligomers were found to have a dimeric (1→2)-linked arabinose side, chain linked at O -3 of an internal xylose unit. Also single arabinose substitution occured at O -3 of an internal xylose unit. There are strong indications that these side groups can also be linked at O -2 of an internal xylose residue. The reducing xylose units were unsubstituted. A model for the GAX populations from sorghum was proposed combining the results of the degradation studies, the identification of the oligomers, and knowledge about the mode of action of the enzymes used.

    Finally, the developed techniques to investigate GAX in particular, were used to study the behaviour of GAX in the brewing process. Worts and spent grains of mashes, supplemented with commercial enzyme preparations containing xylanases among others were studied. Except for the amount of solubilized GAX, the GAX hardly changed with respect to the sugar composition and molecular weight distribution. A direct relationship between GAX, xylanases, and filtration behaviour of worts prepared from malted sorghum, could therefore not be established.

    Modelling nutrient removal in a sequencing batch reactor with respirometry
    Bernardes, R.S. - \ 1996
    Agricultural University. Promotor(en): W.H. Rulkens; A. Klapwijk. - S.l. : Bernardes - ISBN 9789054854715 - 173
    waterzuivering - verwijdering - chemische reacties - uitrusting - afvalwaterbehandeling - rioolafvalwaterverwijdering - zuiveringsinstallaties - water treatment - removal - chemical reactions - equipment - waste water treatment - sewage effluent disposal - purification plants

    The main objectives of the present thesis can be summarized as: i) the development and validation of simplified mathematical models for activated sludge processes in an SBR treating real domestic wastewater; ii) the application of these simplified models for analysing the respirometric response and for obtaining information about the oxygen uptake for the different processes; iii) the application of the monitored respirometric values for model calibration and determination of parameter values, which are used to predict the processes in the next cycle; iv) the use of models as theoretical background for the development of control strategies for plug-flow systems and for SBR; v) relating the basic time scale for the models to the short term.

    The starting points for the model development and simplification were: i) the Activated Sludge Model No. 1, for carbon oxidation, nitrification and denitrification; and ii) the Activated Sludge Model No. 2, for biological phosphorus removal .

    In this study an SBR pilot plant was used and seen as a model for a plug-flow system. During the two and a half years of operation, the plant underwent three different technological phases. The first phase began with the removal of organics and nitrification. Denitrification was incorporated in the second phase. The last phase included biological phosphorus removal.

    In the first phase, two simplified mathematical activated sludge models are presented. The first model gives the response of the respiration rate in an SBR with nitrification, the oxidation of readily biodegradable matter, and endogenous respiration during one cycle. This model is used to predict the respiration rate during a complete SBR cycle. For this, it uses parameter values calibrated during the previous cycle, some default values and information about the ammonia concentration in the influent. The endogenous respiration rate is described with an exponential equation. The second model is used to predict the changes in nitrification capacity after a change in the loading rate and/or sludge wastage rate. For model calibration and validation, an SBR pilot plant receiving domestic wastewater was operated for nine weeks.

    In the second phase, a mathematical model is presented for the behaviour of the respiration rate and nitrate removal in an SBR with nitrification, denitrification and carbon oxidation. This model is based on the response of the respiration rate measured during nitrification and carbon oxidation and the nitrate removal rate during the post-denitrification period. For model calibration and validation, an SBR pilot plant receiving domestic wastewater was operated for three months. The respiration rate was used to calibrate several parameters of the model.

    In the third phase, a mathematical model for an activated sludge SBR with nitrification, denitrification, carbon oxidation and phosphate removal is presented. This model is based on the response of the respiration rate measured during nitrification, carbon oxidation and phosphate removal, together with the behaviour of phosphate and acetate as proposed in the Activated Sludge Model No. 2. For model calibration and validation, an SBR pilot plant receiving settled domestic wastewater plus acetic acid solution was operated for five months.

    In all the three phases the model for the respiration rate ( r ) in an SBR during one cycle, including nitrification, oxidation of readily biodegradable matter, endogenous respiration and a fraction for the respiration rate for phosphorus uptake, gives a good simulation of the measured respiration rate. A good prediction of the total oxygen consumption and distribution during one cycle is found from a simulation, using parameters calculated from the previous cycle together with the variables from the influent. Therefor this model can be used in control strategies as long as it is used for a short time- scale. During long-term operation, parameter variation is significant and too complex to be predicted. In the particular case of nitrification capacity variation in an SBR during long-term operation, the model can explain the variation trend but cannot explain the abrupt changes.

    Simplified mathematical models for the activated sludge process on the bases of the respiration rate are validated. On short-time scale, the models give a good response prediction of the activated sludge process feed with wastewater. The models are good tools for control strategies, however periodical parameter calibration is needed.

    Transannular cyclisation reactions and the germacrane system mediated by enzymes from Cichorium intybus
    Piet, D.P. - \ 1996
    Agricultural University. Promotor(en): Æ. de Groot; M.C.R. Franssen. - S.l. : Piet - ISBN 9789054855880 - 155
    enzymen - biofysica - cichorium intybus - chemische reacties - cyclische verbindingen - enzymes - biophysics - cichorium intybus - chemical reactions - cyclic compounds

    Chicory ( Cichorium intybus L.), one of the many species of the Compositae family, has been cultivated for the production of the leaves since 300 BC as a food supplement and since the 16th century as a substitute for coffee. The sprouts of the chicory are appreciated for their bitter taste. This bitter taste is associated with the presence of sesquiterpene lactones. The majority of these sesquiterpene lactones possess a guaiane framework, a small number possesses a eudesmane- or a germacrane framework. The abundance of these sesquiterpene lactones is not limited to the leaves of the plant. Considerable amounts of are also present in the root, currently an agricultural waste product. Not only is the root a rich source of sesquiterpene lactones, it also contains a large amount of inulin ( 1 ), a storage carbohydrate which is based on fructose instead of glucose. Fructose, an interesting sweetener, is a versatile building block in the synthesis of several polymers and natural products. The bitter principles in the chicory may find their application as a bitter tasting additive in consumer goods.

    The biosynthesis of the sesquiterpene lactones in the chicory is believed to start from a head to tail cyclisation of farnesyl pyrophosphate ( 23 ) into a germacrane, followed by cyclisation into eudesmanes and guaianes. This thesis deals with the cyclisation of germacrane synthons and natural germacranes, induced by a root homogenate of fresh chicory. The goal is to determine the substrate specificity of the germacrane cyclase of chicory and to obtain more insight in the biosynthesis of the sesquiterpene bitter principles in C.intybus.

    A general introduction on the history, use and contents of the chicory is given in chapter 1. In chapter 2, an overview of the literature on the (bio)synthesis of germacrane sesquiterpenes and their possible biotransformation into a variety of cyclised products, is presented.

    In chapter 3, the synthesis of two (E,E)-cyclodeca-1,5-dienols possessing the germacrane framework ( 100 and 101 ), is described. The cyclisation behaviour of these compounds and the natural germacrane (+)-hedycaryol ( 39 ) towards a chicory root homogenate is discussed. The cyclising enzymes in this homogenate transform the 10-membered ring compounds into products with a eudesmane skeleton by protonation of the C l -C 10 double bond followed by transannular cyclisation and subsequent stereoselective incorporation of a water molecule at C 4 . The flexibility of the 10-membered ring system was demonstrated by the formation of the epimeric diols 117-120 from 100 and 101 . The relatively small hydroxyl function at C 7 permitted inversion of the germacrane framework, enabling cyclisation through two different syn- conformations. The large isopropanol group of 39 prohibits this inversion to such an extent that cyclisation takes place only through one conformation to give cryptomeridiol ( 125 ).

    In chapter 4, the synthesis of the three (E,E)-cyclodeca-1,6-dienols 131-133 and their cyclisation by a chicory root homogenate is described. Two kinds of hydroazulene alcohols were obtained in these reactions arising from 1,5- and 1,7-cyclisation. The 1,5-cyclisation products ( 134-136 ) are formed through an internal nucleophilic displacement of the allylic alcohol moiety by the Cj-CjO double bond, while in the formation of the 1,7-cyclisation products ( 137-139 ), an allylic isomerisation reaction of the (E,E)-cyclodeca-1,6-dienol skeleton into an allylic (E,E)-cyclodeca-1,5- dienol skeleton preceded the internal nucleophilic displacement reaction. Hydroazulenes possessing a C 6 -C 7 double bond like 134 resemble natural products like alismol ( 43) .

    Recently, the structure of a trinor-guaiane, dictamnol ( 140 ), similar to alismol, was published. The ring fusion of dictamnol ( 140 ) was postulated as cis. Since 140 was already synthesised at our laboratory and major discrepancies were found between our NMR spectral data of 140 and those reported in the literature, serious doubt about the stereochemistry and the ring junction arose. Therefore, natural dictamnol was isolated, its stereochemistry was reinvestigated and a structural revision into a transfused hydroazulene ( 152 ) is proposed.

    In chapter 5, the biotransformation of derivatives of germacrone, a readily available sesquiterpene germacrane, is described. In a number of cases, enzyme mediated cyclisation of the chemically epoxidised germacrone derivatives, had to compete with spontaneous cyclisation reactions. However, some selectivity was observed, especially in the biotransformation of germacrone-4,5-epoxide ( 48 ) into neoprocurcumenol ( 161 ). Compound 161 is the only product obtained through an enzyme-mediated cyclisation of 48 . The C l -C 10 double bond in 161 is characteristic for guaiane bitter principles in the chicory. In boiled root samples, the only conversion that was observed was a homofragmentation reaction of 48 into curcumenone ( 160 ).

    The synthesis of isogermacrone ( 166 ) paved the way for studying the influence of the position and the stereochemistry of the double bond on the ring fusion of the cyclisation products. The 4,5-epoxides of isogermacrone ( 167 ) and isogermacrene B ( 176 ) were transformed by a chicory root homogenate into a cis-fused eudesmane and two tricyclo[]sesquiterpenes.

    Chapter 6 deals with the synthesis of (E,Z)-cyclodeca-1,5-dienone 184 and the biotransformation of 184 and structurally related compounds. Transannular cyclisation reactions of (E,Z)-cyclodeca- 1,5-dienes appear to proceed in a different way as compared to the (E,E)-cyclodeca-1,5-dienes in chapter 3-5. Instead of a carbon-carbon bond formation between both double bonds of the germacrane skeleton, ring substituents are involved in the cyclisation process to relieve ring strain. If no additional ring substituents are present, e.g. in E-epoxide 201 , a cis-fused hydroazulene diol ( 202 ) is obtained. The Z-epoxides 211 and 214 were not or not unambiguously transformed by a chicory root homogenate.

    In chapter 7, the biotransformation of farnesyl pyrophosphate ( 23 ) by a partially purified chicory root homogenate is described. Radio-GC and GC-MS analysis of the incubation products obtained from [1- 3H]-farnesyl pyrophosphate revealed that 23 was initially transformed into germacrene A ( 36 ). However, the Cope rearrangement product, (β-elemene, 222) and two cyclisation products of 36 , α- and β-selinene ( 223 and 224 ) were the only products that were detected in the assay, since 36 is sensitive towards acid and elevated temperatures.

    In conclusion, the substrate specificity of the germacrane cyclase is discussed and an active site model for the germacrane cyclase in proposed together with two tentative biosyntheses of the sesquiterpene lactones in chicory. The most likely biosynthesis starts with cyclisation of farnesyl pyrophosphate ( 23 ) in germacrene A ( 36 ) followed by several oxidation steps to give intermediate 227 . Enzyme mediated cyclisation of 227 would start with the protonation and subsequent dehydration of the C 3 -hydroxyl group giving allylic cation 228 . This cation then would give 229 after a 1,5-cyclisation, followed by a selective deprotonation towards the bridgehead carbon atom. Further oxidation of 229 would give the guaianolides 12-17 .

    Glucosidation of the C 3 -hydroxyl function of 227 gives sonchuside A ( 20 ) and cichorioside C ( 21 ) which may be cyclised by germacrane cyclasing enzymes into the corresponding eudesmanolides, e.g. 18 . Presumably, glucosidation of the C 3 -hydroxyl group prevents the 1,5-cyclisation process towards the guaianolides.

    Two-stage baculovirus production in insect-cell bioreactors
    Lier, F. van - \ 1995
    Agricultural University. Promotor(en): J. Tramper; J.M. Vlak. - S.l. : Van Lier - ISBN 9789054853978 - 184
    baculovirus - kernpolyedervirussen - experimenten - weefselkweek - celkweek - chemische reacties - uitrusting - biologische bestrijding - virussen - organismen ingezet bij biologische bestrijding - baculovirus - nuclear polyhedrosis viruses - experiments - tissue culture - cell culture - chemical reactions - equipment - biological control - viruses - biological control agents

    Baculoviruses are insect-pathogenic viruses with a narrow host range. The viruses can be an alternative to chemical insecticides. From research aimed at improving the efficacy of the viruses in insect control another application evolved: the use of the baculovirus to express foreign proteins in insect cells. To produce large amounts of baculovirus or baculovirus- expressed proteins, large-scale culture and subsequent infection of insect cells is necessary. Chapter 1 of this thesis reviews the research on factors influencing insect-cell culture and infection and the status of production with insect-cell cultures.

    One of the production strategies reviewed in chapter 1 is a continuous two-stage bioreactor configuration. In the first reactor cells are cultured and the effluent is led to second reactor where the cells are infected with the baculovirus. The first results reported with this system showed two disadvantages. The number of cells infected was relatively small and production in the infection reactor was limited to about one month.

    To increase the number of infected cells the residence time distribution in the infection reactor was altered (chapter 2). The infection reactor was replaced by two infection reactors in series, each containing half the volume of the original infection reactor. Therefore, the mixing characteristics of the infection part of the production system became more plug-flow like. This resulted in a higher number of cells which produced polyhedra (encapsulated virus). However, the time during which the cells in the infection reactors produced polyhedra was diminished to about two weeks.

    To gain more insight in the decrease of production a recombinant virus was used in a study described in chapter 3. In this recombinant virus the polyhedrin gene (coding for the major protein in the virus matrix) was replaced by the lacZ gene of Escherichia coli. Production of β- galactosidase in a continuous two-reactor configuration gave analogous results to production with the wild-type virus. Production was maintained for about four weeks then it rapidly decreased. Upon restriction enzyme analysis it was shown that the decrease of β-galactosidase resulted from the disappearance of the gene from the virus population rather than inactivation of the gene.

    Research at the department of Virology of the Agricultural University of Wageningen revealed that the reduction of production resulted from the occurrence of defective mutants of the baculovirus. These defective virus lack the polyhedrin-promoter driven gene and became predominant in the bioreactor due to interference with the replication of non-mutated virus.

    On the defective viruses another highly expressed gene, the p10 gene, was found to be still present. A recombinant virus containing the lacZ from E. coli under control of the p10 promoter was used in the study described in chapter 4. The recombinant virus produced both polyhedra and β-galactosidase in a continuous bioreactor system. Again production lasted for about four weeks both for polyhedra and β-galactosidase This indicates that the presence of the p10-promoter driven gene per se is not enough for recombinant protein production.

    In chapter 5 a model is presented which describes the kinetics of virus infection. Besides the infectious virus particles and the defective mutants, a third virus type is taken into account. This so-called abortive virus is capable of blocking an entry site of a cell but is not leading to production of new virus particles. By assuming a limited amount of entry sites on an insect cell it was possible to calculate when production of infectious virus decreased. The model indicates that the build-up of defective viruses can be postponed by keeping the number of infectious viruses per cell low. A way of accomplishing this is the use of repeated batch infections.

    In chapter 6 experiments with repeated (fed-)batch infections are presented. These experiments confirmed calculations with the model. A production involving a series of batch infections resulted in prolongation of production time to 60 days.

    The application of the baculovirus expression vector in the light of the findings described in the thesis is discussed in chapter 7.

    Design, characterization and application of the multiple air-lift loop bioreactor
    Bakker, W.A.M. - \ 1995
    Agricultural University. Promotor(en): J. Tramper; C.D. de Gooijer; H.H. Beeftink. - S.l. : Bakker - ISBN 9789054854791 - 172
    chemische reacties - uitrusting - chemische eigenschappen - automatische regeling - instrumentatie - systemen - zuurstof - monoclonale antilichamen - hybridoma's - chemical reactions - equipment - chemical properties - automatic control - instrumentation - systems - oxygen - monoclonal antibodies - hybridomas

    A new bioreactor is introduced: the Multiple Air-lift Loop reactor (MAL). The MAL consists of a series of air-lift loop reactors within one vessel. With the MAL, a new type of geometry for air-lift reactors with an internal loop is introduced. This new geometry was characterized with respect to hydrodynamics, mixing and oxygen transfer. The hydrodynamics were described by an existing model. Hydrodynamics, mixing and oxygen transfer in the new reactor configuration were comparable to that in conventional air-lifts with an internal loop.

    The design and use of the MAL as a reactor cascade, to approximate plug-flow behaviour, were studied. Biological model systems were used to compare the reactor series to a single vessel. These model systems included immobilized invertase and nitrifying bacteria. With the immobilized invertase it was shown that a threecompartment MAL gives an improved substrate conversion when compared to a single vessel of the same overall volume. This could be described with a previously developed model. Also for the immobilized nitrifying bacteria improved substrate conversion was shown in the comparison between a series and a single vessel. Free suspended hybridomas were used for monoclonal antibody (MAb) production. It was shown that reactor series can be useful research tools for kinetic studies. In the second vessel in the series conditions were obtained that can hardly be reached in a single vessel. Not only growth, but also death could be studied under stable conditions. A model was derived that describes hybridoma growth and their MAb production.

    Vessels in a series can be of equal volume, but very often unequal volumes can be more advantageous. Therefore, choosing the appropriate reactor volumes is an important design step, which is discussed for different applications. Finally, a general procedure for choosing the optimal bioreactor cascade configuration for any application is given.

    Chemical consequences of long-range orbital interactions in Perhydronaphtalene-1,4-diol monosulfonate esters
    Orru, R.V.A. - \ 1994
    Agricultural University. Promotor(en): Æ. de Groot; J.B.P.A. Wijnberg. - S.l. : Orru - ISBN 9789054852995 - 145
    diterpenoïden - sesquiterpenoïden - terpenen - etherische oliën - sesquiterpenen - chemische structuur - chemische reacties - sulfonaten (esters) - diterpenoids - sesquiterpenoids - terpenoids - essential oils - sesquiterpenes - chemical structure - chemical reactions - sulfonates (esters)

    In this thesis the base-induced reactions of perhydronaphthalene-1,4-diol monosulfonate esters are described. These compounds undergo smoothly, typical carbocationic processes upon treatment with sodium tert -amylate in refluxing benzene. The product outcome, product ratio, and (relative) rate of these reactions is satisfactorily explained when through-bond orbital interactions (TBI) over four abonds are invoked. In order to gather more detailed information about the basic stereochemical and stereoelectronic principles underlying these processes, synthetical organic, computational, and kinetic investigations were undertaken.

    Most experimental studies on TBI have focussed on its spectroscopic manifestations and are reviewed repeatedly. On the other hand, there are relatively few reviews on the chemical consequences of TBI over three or more σ-bonds. A number of illustrative examples of chemical reactions in which long-range orbital interactions are believed to play an essential role are discussed in chapter 1. In the same chapter also attention is drawn to the synthetic utility of some of these reactions.

    In chapter 2 the syntheses of the mesylates 39 , 40 , and 58-67 are described. The compounds 39 , 40 , 58 , and 59 are prepared in order to investigate how the orientation of the sulfonate ester group in combination with the orientation of the tertiary hydroxyl group determines the outcome and rate of their reactions with sodium tert -amylate. The results of these investigations are described in chapter 3. It was found that an equatorial sulfonate ester group favors homofragmentation leading to the cyclopropane derivative 105 . In case of an axial sulfonate ester group β-elimination, which strongly depends on the stereochemistry of the tertiary deprotonated hydroxyl group, is the main reaction path. In the chapter 3 the synthesis of the O-silylated mesylates 106 and 107 is also described. These compounds show no reaction at all upon treatment with strong base. On the other hand, fast reactions are observed when 106 and 107 are treated with TBAF. Generation of an alcoholate is crucial for the observed reactions. Homofragmentation and an internal return reaction with inversion of configuration of the mesylate group in the axial mesylates 39 and 58 is explained by assuming a 1,3-bridged intermediate carbocation.

    The mesylates 60-65 are prepared (Chapter 2) to determine the influence of the geometry of the relaying σ-bonds on the reactions with sodium tert -amylate. In chapter 4 the results of these studies are described in detail. An alcoholate function intramolecularly induces heterolysis of the sulfonate ester group in an apolar solvent via orbital interactions through three intervening C-C single bonds. It is shown that the reactivity of the compounds 60-65 is only affected by the relative position of the hydroxyl function to the sulfonate ester group and not by the orientation of the hydroxyl group. The two chief pathways by which these compounds react are rearrangement ( 60 , 62 , and 63 ) and homofragmentation ( 64 and 65 ). Stereoelectronic effects play a dominant role here, except in compound 61 where steric factors primarily determine the reactivity and product outcome (ether formation). Homofragmentation is much faster than rearrangement and is only possible when a 1,3-bridged through-space interaction accompanies TBI. The extent of TBI as well as the product composition is strongly determined by the σ-relay of the four σ-bonds between the electron donor (alcoholate) and the electron acceptor (sulfonate ester bond). These results are consistent with the " trans rule", which is in line with predictions from theoretical models regarding TBI.

    The product outcome, product ratio, and relative rate of the base-induced reactions of perhydronaphthalene-1,4-diol monosulfonate esters described in chapter 4 are satisfactorily explained with the concept of TBI. However, the conclusions are all based on empirical results. In chapter 5 the results of semi-empirical calculations, using the MNDO method, performed on model systems are presented. In this way a more detailed understanding of the stereoelectronic features underlying the homofragmentation and rearrangement reaction is obtained. The trends in the results of the MNDO simulations are the same as those found in the reactions of the compounds 60-65 . Whether rearrangement or homofragmentation takes place depends on the geometry of the σ-relay and the inductivity of the system. Cyclopropanoid bridged structures seem to be involved in the rearrangement process as well as in the homofragmentation process.

    In order to explore the effects of the order of substitution of the carbon atom that borders the carbon atom to which the mesylate group is attached the compounds 40 , 66 , and 67 were synthesized. This subject is discussed in chapter 6. The product formation is strongly dependent on the steric consequences of alkyl substituents at βcarbon atoms. Homofragmentation is highly favored when the repulsive steric interactions do not prevent a homohyperconjugatively stabilized transition state. This is only possible in an ideal "W" arrangement of theσ-relay ( 40 ). Due to the repulsive 1,3- peri -effect in 66 , and a combination of the 1,3- peri -effect and the 1,3- diaxial -effect in 67 the σ-relay diverges from the ideal "all trans " geometry as a result of which other reaction pathways (elimination, 1,3-H, and 1,2-Me shifts) are favored over homofragmentation. Introduction of inductively electron-donating substituents leads to an increase in reaction rate, despite the (slight) deviation of the "W" arrangement. It is concluded that although bridged ions are important intermediates in the observed reaction paths, they are not decisive for the reactivity of these compounds.

    The O-silylated mesylates 106 and 107 react fast upon treatment with TBAF in refluxing benzene (Chapter 3). At room temperature only desilylation takes place. To investigate the influence of a remote nucleofugal mesylate group on the rate of desilylation, apart from 106 and 107 , also the O-silylated compounds 127-131 are synthesized and treated with TBAF as is described in chapter 7. The rates of desilylation are determined conveniently by HPLC monitoring of the disappearance of the starting material. The desilylation rate of compounds with a mesylate group is much higher than the desilylation rate of corresponding compounds with a hydroxyl group instead ( 130 and 131 ). Furthermore, compounds having a "W" arrangement ( 107 and 129 ) of the relaying σ-bonds react considerably faster than their "sickle relay" analogs ( 106 , 127 , and 128 ). The results presented in this chapter show nicely that longrange electronic effects of distant substituents can exert a substantial influence on the reactivity of certain functional groups in general.

    In conclusion, the concept of TBI offers a good explanation for the reactivity of the compounds studied throughout this thesis. The stereochemical and stereoelectronic requirements for the base-induced reactions of perhydronaphthalene-1,4-diol monosulfonate esters are now well established. The general utilitly of the concept of σ-delocalization and TBI in everyday chemistry is demonstrated

    Gasvormige stikstofverliezen bij de teelt van sla op een zandbed onder glas.
    Postma, R. ; Oenema, O. ; Bussink, D.W. ; Heinen, M. - \ 1994
    Meststoffen : Dutch/English annual on fertilizers and fertilization (1994). - ISSN 0169-2267 - p. 28 - 34.
    ammoniak - watervrije ammoniak - lactuca sativa - slasoorten - hydrocultuur - chemische reacties - mineralen - boekhouding - stikstofdioxide - glastuinbouw - ammonia - anhydrous ammonia - lactuca sativa - lettuces - hydroponics - chemical reactions - minerals - accounting - nitrogen dioxide - greenhouse horticulture
    Uit mineralenbalansen van teelten op kunstmatige substraten in de glastuinbouw is gebleken dat de aanvoer van stikstof (N) via recirculerende voedingsoplossingen wat hoger is dan de afvoer van N via oogstprodukten en gewasresten. In deze studie zijn de ammoniak-vervluchtiging, denitrificatie en lachgas-emissie bij de teelt van sla gekwantificeerd
    Oligosacchariden als bifidogene factoren.
    Hartemink, R. ; Nout, M.J.R. ; Rombouts, F.M. - \ 1994
    Voedingsmiddelentechnologie 27 (1994)20. - ISSN 0042-7934 - p. 27 - 29.
    bifidobacterium - koolhydraten - chemische reacties - microbiële afbraak - polysacchariden - structuur - bifidobacterium - carbohydrates - chemical reactions - microbial degradation - polysaccharides - structure
    Levende bacterien die een gunstige invloed hebben op de darmflora. Ze worden probiotica genoemd en ze worden toegepast in zowel de humane voeding als de veevoeding
    Tailor-made produktie van oligosacchariden.
    Laere, K.M.J. van; Schols, H.A. ; Voragen, A.G.J. - \ 1994
    Voedingsmiddelentechnologie 27 (1994)20. - ISSN 0042-7934 - p. 33 - 35.
    koolhydraten - chemische reacties - chemische structuur - chemicaliën - voedselindustrie - voedseltechnologie - polysacchariden - eigenschappen - structuur - invloeden - carbohydrates - chemical reactions - chemical structure - chemicals - food industry - food technology - polysaccharides - properties - structure - influences
    De gewenste oligosacchariden kunnen met behulp van specifieke enzymen en chromatografische technieken 'op maat' worden gemaakt
    Orienterend onderzoek naar de integrale inpassing van aanzuren van varkensmengmest = Pre-investigation on the integrated implementation of pig slurry acidification
    Derikx, P.J.L. ; Vijn, T.K. ; Willers, H.C. - \ 1993
    Wageningen : IMAG-DLO (Rapport / Instituut voor Mechanisatie, Arbeid en Gebouwen 93-34) - 41
    luchtverontreiniging - ammoniak - chemische reacties - emissie - stikstof - vervluchtiging - air pollution - ammonia - chemical reactions - emission - nitrogen - volatilization
    Enzymatic synthesis of polyol seters in aqueous - organic two-phase systems
    Janssen, A. - \ 1993
    Agricultural University. Promotor(en): K. van 't Riet. - S.l. : Janssen - ISBN 9789054851271 - 181
    emulgeermiddelen - chemische reacties - synthese - carboxyl ester hydrolasen - tannase - choline esterase - triacylglycerol lipase - koolhydraten - vetzuren - carbonzuren - emulsifiers - chemical reactions - synthesis - carboxylic ester hydrolases - tannase - cholinesterase - triacylglycerol lipase - carbohydrates - fatty acids - carboxylic acids

    The last decade increasingly attention is paid to lipases as catalysts for synthesis of components, such as fatty acid-based surfactants, flavors, edible oil equivalents, monomers and polymers, and amides. In this thesis, the lipase-catalyzed esterification of polyols and fatty acids is described. These esters consist of a nonpolar part (fatty acid) and a polar part (polyol). Therefore, polyol esters have surface-active properties and are used as emulsifier in food, pharmaceutics; and cosmetics. One of the aims of this thesis is to develop a reaction system for the esterification of polyols (carbohydrates) and fatty acids, without any modification of the substrates. Also, high reaction rates are desired.

    Enzymatic esterification is often performed in the presence of organic solvents. Besides activity and stability of the enzymes, the solvents will affect the equilibrium position of reactions. In literature, models were described for the prediction of the equilibrium position in dilute two-phase systems. However, for industrial applications, high product concentrations are desired, which implicate the use of nondilute reaction systems. Another aim of this thesis is to gain a better insight in factors that affect the equilibrium position of a reaction and to predict the product concentrations at equilibrium in non-dilute two-phase systems.

    In chapter 2 and 3, the lipase-catalyzed esterification of sorbitol and fatty acid is studied in two different two-phase reaction systems. In chapter 2, 2-pyrrolidone is used as a cosolvent for sorbitol. In this study, the lipase from Chromobacterium viscosum is used and the initial esterification rate is high as compared to literature data. The water activity is found to be important for the ester concentrations at equilibrium. High concentrations of the cosolvent 2-pyrrolidone should be avoided, because these will inactivate the lipase. In the reaction system that is described in chapter 3, water is used to dissolve sorbitol. Candida rugosa lipase is used in this study and initial esterification rates are slightly higher than in chapter 2. The water activity is dependent on the sorbitol mole fraction in the aqueous phase and lowering of the water activity is limited by the solubility of sorbitol. A two-phase membrane reactor is a suitable type of reactor, since the water activity of the aqueous phase can be kept constant during the experiment and lipase possesses a good stability. In both reaction systems, besides sorbitol also glucose and fructose can be used as a substrate, while disaccharides, such as sucrose, are not reactive at all.

    In chapter 4, the lipase-catalyzed esterification of glycerol and decanoic acid has been studied in aqueous-organic two-phase systems. The addition of an organic solvent is found to influence the ester mole fractions at equilibrium. For the synthesis of polar products (monoesters), a polar solvent (low log P) is favorable, while for the synthesis of nonpolar products (triesters), it is better to choose a nonpolar solvent (high log P). The computer program 'Two-phase Reaction Equilibrium Prediction' (TREP) has been developed for the prediction of the ester concentrations in nondilute two-phase systems, in case both the reaction equilibrium as well as the phase equilibrium are achieved. This program is based on mass balances and the UNIFAC group contribution method. Deviations in the prediction with TREP are generally less then a factor of 2 and are due to inaccuracies of the UNIFAC group contribution method.

    The lipase-catalyzed acylglycerol synthesis with fatty acids of different chain length is studied in chapter 5. For predictions with TREP, one set of equilibrium constants is used for monoester, diester, and triester synthesis. It is shown that with this set the equilibrium position of the reaction between glycerol and all saturated fatty acids with a chain length from 6 to 18 and oleic acid can be calculated within some margins. For fatty acids with different chain length, the ester mole fractions at equilibrium are clearly different. With the short-chain hexanoic acid, the monoester mole fraction is highest, while for the long-chain oleic acid, the diester mole fraction is the highest one. Besides the equilibrium position, also the reaction rates are affected by the solvent that is added. In polar solvents, the monoester production rate is enhanced. This is caused by the shift in the equilibrium mole fractions.

    In chapter 6, the effect of solvents on the esterification of decanoic acid and several alcohols, such as 1-dodecanol, 1-butanol, 1,3-propanediol, and sorbitol is studied. In agreement with the previous results, the ester mole fractions at the reaction equilibrium are dependent on the solvability of the ester in the organic phase. This effect is most striking for the polar sorbitol esters. Almost no esters are present at equilibrium in systems with nonpolar solvents, while reasonable high ester mole fractions can be obtained in systems with polar solvents. In contrast with the results of chapter 5, the equilibrium constants are clearly affected by the type of alcohol that is chosen as a substrate. Calculations with TREP showed that the calculated ester mole fractions did not deviate more than a factor of 1.5 from the measured ones. However, it appears that the calculated water mole fractions deviate systematically in the downwards direction.

    Chapter 7 shows a comparison between models in literature for the prediction of the equilibrium position in dilute two-phase reaction systems and calculations with TREP. It is shown that the models from literature are limited to reaction systems in which partition coefficients are constant. The program TREP can be used for nondilute as well as dilute reaction systems.

    Furthermore, this chapter shows that the ester mole fractions at equilibrium can be increased with increasing temperature. This is due to the increase of the solubility of sorbitol with increasing temperature. Most pronounced is the effect of temperature on the reaction rate, which is increased enormously. However, for long-term processes at high temperatures it is important that heat-stable lipases will be used.

    Enzymatic acylglycerol synthesis in membrane reactor systems
    Padt, A. van der - \ 1993
    Agricultural University. Promotor(en): K. van 't Riet. - S.l. : Van der Padt - ISBN 9789054851264 - 151
    derivaten - alcoholen - glycerol - acylglycerolen - diacylglycerolen - triacylglycerolen - chemische reacties - membranen - omgekeerde osmose - ultrafiltratie - fermentatie - voedselbiotechnologie - vetzuren - carbonzuren - derivatives - alcohols - glycerol - acylglycerols - diacylglycerols - triacylglycerols - chemical reactions - membranes - reverse osmosis - ultrafiltration - fermentation - food biotechnology - fatty acids - carboxylic acids

    Up till twenty years ago, only chemical modifications of agricultural oils for novel uses were studied. Because of the instability of various fatty acids, enzymatic biomodifications can have advantages above the chemical route. Nowadays, enzymatic catalysis can be used for the modification of oils and fats. One way of biomodification is the enzymatic esterification of glycerol with fatty acid for the synthesis of mono- and triacylglycerols. Monoesters (monoacylglycerols) are used as emulsifiers in food and in cosmetics, tailor made triesters (triacylglycerols) are used to adjust the melting range of foods and cosmetics. This thesis describes a number of membrane reactor systems for the enzymatic esterification of glycerol with decanoic acid in hexadecane as solvent. Description and modelling of the kinetics and thermodynamic equilibrium have resulted in reactor concepts to reach the objective of mono- and triester synthesis.

    The basic reactor studied is a two-phase immobilized enzyme membrane reactor. In the membrane reactor, lipase from Candida ragosa is immobilized at the inner fibre side of a hydrophilic hollow fibre module. Decanoic acid in n-hexadecane is circulated at the same side, meanwhile a water-glycerol phase is circulated at the shell side. The glycerol diffuses through the membrane matrix allowing the synthesis to take place at the interface. The water produced diffuses backwards.

    Chapter 2 describes the enzymatic esterification of decanoic acid with glycerol for an emulsion system and for a hydrophilic membrane system. In a two-phase system, the enzyme activity is related to the oil-phase volume, the interface area and the enzyme load. The rate per unit interface area of the membrane system approximates the rate measured in an emulsion system. This implies that the cellulose membrane does not affect the esterification. Another consequence is that the activity per oil-phase volume is only specific surface area related, therefore a hollow fibre device is desirable. The optimum enzyme load in the membrane system is half of that in the emulsion system.

    The enzyme stability in glycerol-water mixtures is described in chapter 3. The activity of lipase from Candida rugosa with time can be described with a two-step model, assuming the native lipase reversibly altering its conformation to a form having no activity. The reversibility is experimentally verified. Both, the native and inactive form do inactivate irreversible at the same time to a completely inactive form. The inactivation is a function of the glycerol concentration. The activity of immobilized enzyme is reduced to the same level of activity as is found for free lipase.

    Not only activity and stability of the enzymatic system are of importance, also the equilibrium ester concentrations must be known in the non-ideal two-phase system. Chapter 4 presents the program TREP (Two-phase Reaction Equilibrium Prediction). With the use of measured thermodynamic activity based equilibrium constants, mass balances and the UNIFAC group contribution method, TREP predicts the equilibrium product and substrate concentrations for given initial amounts. Equilibrium predictions show that an excess of triesters can be obtained only at low water activity conditions, in this case an one-phase system is predicted. Predictions show that pure monoesters cannot be obtained in a two-phase system of decanoic acid-hexadecane phase and a glycerol-water phase, even with a high glycerol to fatty acid ratio. This is experimentally verified.

    From the knowledge gathered in these chapters, two membrane reactor systems are designed, one membrane reactor for the triester production and a second membrane reactor system equipped with an in-line adsorption column for the synthesis of monoesters.

    Chapter 5 describes a pervaporation system in which an excess of triesters can be synthesized at low water activity conditions. Lipase is immobilized onto the lumen side of a cellulose membrane where the organic phase is present. At the shell side, air circulates and the water activity is controlled with the use of a condenser. The lipase catalyzed esterification of decanoic acid with partial glycerides is studied in this reactor. In agreement with the predictions made in chapter 4, an excess of triacylglycerols, is obtained at low water activity conditions only.

    A second membrane reactor concept is described in chapter 6, the organic-phase is led over an adsorption column in order to adsorb the monoglycerides onto the adsorbate. When the column is saturated with monoesters, the column can be desorbed off-line in a continuous membrane/repeated batch column process. If a 5 % ethanol in hexane solution is used as desorption solvent, monoesters are desorbed selectively leading to a 90 % purity.

    Finally, in chapter 7, the potentials and limitations of the enzymatic esterification are discussed. To predict the steady-state concentration of a continuous reactor, the enzyme kinetics must be described. The membrane reactor is reaction limited, this could be overcome by placing a column packed with immobilized enzyme in the organic phase recirculation loop. Not only esterification can be performed in the pervaporation system, this system could also be suitable for interesterification or transesterification. Then the program TREP should be extended for reactions with different types of fatty acids.

    Engineering aspects of nitrification with immobilized cells
    Hunik, J.H. - \ 1993
    Agricultural University. Promotor(en): J. Tramper. - Wageningen : Hunik - 167
    chemische reacties - biotechnologie - chemische industrie - biochemie - cellen - immobilisatie - bradyrhizobiaceae - geïmmobiliseerde cellen - chemical reactions - biotechnology - chemical industry - biochemistry - cells - immobilization - bradyrhizobiaceae - immobilized cells

    Several aspects of a nitrification process with artificially immobilized cells in an airlift loop reactor have been investigated and are described in this thesis. In chapter 1 an overview of immobilization methods, suitable reactors, modelling, small-scale
    applications and scale-up strategy is given. The subjects of chapter 1 provide the starting point of the following chapters. Application of immobilized cells is beneficial for the nitrification process at high product and substrate concentrations and with a process temperature far below the optimal temperature of 30-35°C. In chapter 2 and 3 the kinetics of, respectively, Nitrosomonas europaea and Nitrobacter agilis cells at high product and substrate concentrations is presented. The results show a severe product inhibition of Nitrobacter agilis by nitrite, while Nitrosomonas europaea seems to be more sensitive
    for a high osmotic pressure. In chapter 4 a theoretical background of the immobilization method and further scale-up is presented. For the immobilization method the theory for the break-up of liquid jets, with Newtonian behaviour, is evaluated and a method to apply this theory for non-Newtonian liquids like a K-carrageenan solution is presented. In chapter 6 a dynamic model for the nitrification with immobilized Nitrosomonas europaea and Nitrobacter agilis cells is presented. The model includes mass-transfer rates, kinetic behaviour of the microorganisms, and reactor and gel bead properties. Predictions
    of reactor bulk concentrations of NH 4+, NO 2-and NO 3-(N-compounds) are given by the model together with concentration profiles of N-compounds, oxygen and biomass in the gel beads. A sensitivity analysis of the model parameters shows that the diffusion coefficient of oxygen in the gel beads and the radius of the gel beads are the most important parameters influencing the model output. The model is experimentally validated by means of reactor bulk concentrations and biomass profiles of Nitrosomonas europaea and Nitrobacter agilis in the gel beads. Predicted and measured values agree very well and the assumptions and equations used in the model seem to be valid. The biomass profiles of the two microorganisms co-immobilized in the gel beads are determined with immunofluorescence and a stereological method (chapter 5). The immunofluorescence technique was used to separate the Nitrosomonas europaea and Nitrobacter agilis colonies in the beads. From the position and diameter of the colonies it is possible to determine the spatial distribution of the two microorganisms in the gel beads. In chapter 7 the model is used as a tool to develop a strategy to scale-up the nitrification process with immobilized cells. A design for a large-scale application should be optimized with respect to the transport of oxygen to the immobilized cells in the gel beads. This is an advantage over the nitrification process with suspended cells, which is limited by the growth rate of the nitrifying bacteria. The growth of nitrifying bacteria is a slower process than the transport of oxygen to the cells. Some interesting aspects, which were not treated elsewhere in this thesis, are discussed in the general discussion in chapter 8.

    De beweeglijke balans tussen chemische bindingsprocessen en biologische opnameprocessen.
    Riemsdijk, W.H. van - \ 1993
    Bodem 3 (1993). - ISSN 0925-1650 - p. 173 - 175.
    chemische speciatie - chemische reacties - anorganische verbindingen - mineralen - voedingsstoffen - biologische beschikbaarheid - plantenvoeding - bodemchemie - bodemfumigatie - bodemeigenschappen - grondsterilisatie - binding (scheikundig) - chemical speciation - chemical reactions - inorganic compounds - minerals - nutrients - bioavailability - plant nutrition - soil chemistry - soil fumigation - soil properties - soil sterilization - bonding
    In het kader van bodembeschermingsbeleid wordt aandacht geschonken aan de problematiek van de biologische beschikbaarheid van (anorganische) stoffen in de bodem
    Physical aspects of liquid-impelled loop reactors
    Sonsbeek, H. van - \ 1992
    Agricultural University. Promotor(en): J. Tramper. - S.l. : [s.n.] - 135
    chemische reacties - uitrusting - massaoverdracht - vloeistofmechanica - vloeistoffen (liquids) - vloeistoffen (fluids) - vermenging - dichtheid - chemical reactions - equipment - mass transfer - fluid mechanics - liquids - fluids - mixing - density

    The liquid-impelled loop reactor (LLR) is a reactor that consists of two parts : the main tube and the circulation tube. Both parts are in open connection at the bottom and at the top. The reactor is filled with a liquid phase: the continuous phase. Another liquid phase is injected in the main tube by means of pumping. This liquid phase is immiscible with the continuous phase and its density is significantly different. If the density is higher than the density of the continuous phase, injection takes places at the top of the main tube. For a lower density injection takes place at the bottom. Due to the density difference the dispersed-phase droplets that are formed will fall or rise, respectively, and coalesce at the other end of the tube. The coalesced liquid is discharged from the reactor. Due to the presence of dispersed phase in the main tube a pressure difference exists which causes circulation of the continuous phase in the reactor. This results in good mixing without the use of an impeller. For biotechnological purposes it is most likely that the continuous phase is an aqueous phase that contains the biocatalyst, possibly immobilized. The work on the liquid-impelled loop reactor originates from two previous research studies. First, the physical characterisation and modelling of air-lift loop reactors for application in cultivating shear-sensitive biocatalysts. Second, the research focused on application of organic solvents in biological processes, which is a promising area for many years already.

    In a review the current state of the art is given with respect to biocatalysis in media consisting of two liquid phases that are not miscible. This research area has shown much progress in recent years, however, industrial applications seem still not very numerous. To carry out two-liquid-phase experiments on a scale bigger than shake flasks, in most cases existing bioreactors are used. Adjustments are made to the reactor and the processing to make them suitable for use with two liquid phases. The liquid-impelled loop reactor can be seen as a special case, where an air-lift loop reactor is adjusted for use with two liquid phases instead of a liquid and a gas phase.

    The research was started with characterisation of important physical aspects such as drop size, dispersed-phase concentration (holdup) and continuous-phase velocity as function of the dispersed-phase flow rate. Description of drop sizes that are formed in the liquid-impelled loop reactor at the liquid sparger, show good agreement with theoretical predictions. The hydrodynamic model that was used in the air-lift loop study is applied. It shows to be a good method to describe holdup and liquid velocity. The best results with this model are obtained when it is assumed that the continuous phase flows fastest in the centre of the tube and that the dispersed phase concentrates in the centre of the tube.

    On the topic of hydrodynamic models for air-lift loop reactors many articles are published. Because this still continues, this literature is analysed and the basic principles of the several models are described and compared. It appears that in all models the relative velocity between dispersed-phase bubbles and continuous phase plays an essential role. This quantity is difficult to determine and shows a wide spread due to the distribution in bubble size. Furthermore, velocity profiles or turbulence can have much influence but are not taken into account in the described models. Comparison of the models by means of using literature data did not yield a clear preference for one of the models nor for a particular basic principle.

    To describe mixing in the continuous phase, the one-dimensional axial dispersion model is used, which is in general suitable for flow in tubular devices. The mixing parameter is determined per reactor section. For the main tube a correlation between mixing parameter and energy dissipation is given. The mixing parameter can be used to describe the flow of the continuous phase as a plug flow with axial disturbances. Furthermore, dimensionless mixing times can be estimated. The dimensionless mixing time is the number of circulations that is necessary to achieve complete mixing of the continuous phase, where the criterium must be defined by the user.

    Mass transfer is investigated in an FC40 water system. For this purpose a new method is developed based on the principle of a steady-state measurement, in stead of the most widely used dynamic measurement. Compared to a gas/liquid system at equal dispersed-phase flow rates, the mass-transfer rate in the liquid/liquid system is favorable. This is due to the larger exchange area, because the drops are smaller than the bubbles and the drop velocity relative to the continuous phase is lower than the relative velocity of the bubbles. The mass-transfer coefficient for the liquid/liquid system, derived from experimental results, however, is lower than literature values for gas/liquid systems. This is probably caused by the lower diffusion coefficient of oxygen in liquid than of oxygen in gas. The transfer capacity can often be the highest for gas/liquid systems because the maximum dispersed-phase flow rate in liquid/liquid systems is limited with respect to drop formation and coalescence. Further physical reseach must be focussed on this limitation.

    Basic bioreactor design.
    Riet, K. van 't; Tramper, J. - \ 1991
    New York : CRC Press - ISBN 9780824784461 - 480
    biochemie - biotechnologie - chemische industrie - chemische reacties - uitrusting - fermentatie - voedselbiotechnologie - studieboeken - biochemistry - biotechnology - chemical industry - chemical reactions - equipment - fermentation - food biotechnology - textbooks
    Based on a graduate course in biochemical engineering, provides the basic knowledge needed for the efficient design of bioreactors and the relevant principles and data for practical process engineering, with an emphasis on enzyme reactors and aerated reactors for microorganisms. Includes exercises.
    Modelling and characterization of an airlift-loop bioreactor
    Verlaan, P. - \ 1987
    Agricultural University. Promotor(en): K. van 't Riet, co-promotor(en): K.C.A.M. Luyben. - S.l. : Verlaan - 129
    chemische reacties - uitrusting - biotechnologie - chemische industrie - biochemie - chemical reactions - equipment - biotechnology - chemical industry - biochemistry

    An airlift-loop reactor is a bioreactor for aerobic biotechnological processes. The special feature of the ALR is the recirculation of the liquid through a downcomer connecting the top and the bottom of the main bubbling section. Due to the high circulation-flow rate, efficient mixing and oxygen transfer is combined with a controlled liquid flow in the absence of mechanical agitators.

    Liquid velocities and gas hold-ups in an external-loop airlift reactor (ALR) on different scales were modelled on the basis of a simple pressure balance. The model is adapted for non-isobaric conditions and takes into account nonuniform flow profiles and gas hold-up distributions across the duct. The friction coefficient together with the reactor dimensions are input parameters. It has been shown that the friction coefficient can be obtained from simple one-phase flow calculations based on known data of the seperate reactor parts. The model predicts liquid velocities and local gas hold-ups in an ALR to within 10% and can be applied easily to an internal loop reactor.

    Mixing in the individual sections of the ALR is determined by a newly developed parameter estimation procedure which has proven to be reliable for the estimation of axial dispersion coefficients in the individual sections of the ALR. From the results it can be concluded, that in an ALR the liquid flow behaves like plug-flow with superimposed dispersion except for the topsection for which it is not reasonable to assume plug-flow. The mixing results simplified the modelling of oxygen transfer in the ALR as it appeared not to be necessary to incorporate the dispersion contribution Into the oxygen model.

    The non-isobaric plug-flow model, presented in this thesis, predicts dynamic and stationary dissolved oxygen concentration (DOC) profiles in large-scale ALRs and has been applied also to estimate the volumetric oxygen transfer coefficient, k 1 a, in the pertinent ALR. Comparison with the results on the basis of a simple isobaric stirred-tank-reactor model demonstrates, that such a model yields conservative values though for the present situation the underestimation did not exceed a value of 10% relative to the plug-flow model. Therefore, due to its simplicity, it is recommended to use the stirred tank model for a rapid characterization of the overall aeration capacity of laboratory scale and pilot-scale ALRs. Oxygen depletion of the gas phase, even during a fermentation, appeared to be very limited and was fairly well predicted by the plug-flow model. For this reason an ALR is a very suitable reactor for aerobic processes having a high oxygen demand. If necessary, the aeration capacity of the ALR can be enhanced by injection of a small amount of gas at the entrance of the downflow region. This phenomenom is also accurately predicted by the plug-flow model. In the present ALR the aeration capacity of the air-sparger region did not significantly differ from the main aeration process in the upflow region due to its special geometry.

    The intermediate flow region between the ALR and the bubble-column (BC) flow regime was investigated by gradually closing a butterfly valve at the bottom of the downcomer. When the valve is further shut and thus the friction is enhanced, the liquid velocity will be reduced thereby enlarging the gas hold-up. The maximum value for the gas hold-up is obtained when the ALR is operated as a BC. In the transition flow regime between ALR and BC flow, the liquid velocity was found to be a simple power law function of the gas flow rate. The coefficients of the power law depend on the flow characteristics in the reactor. In the transition flow regime the hydrodynamic calculations based on the plug-flow behaviour of an ALR are only valid up to a certain defined value of the total gas-liquid flow rate. For greater values, the ALR type of flow will change Into a BC type of flow. A simple criterium qualifies the distinction between both flow patterns, determined by the superficial liquid velocity and the liquid circulation velocity.

    The transition of ALR to BC flow coincides with the decrease of the Bodenstein number which also indicates a less established plug flow. As the dispersion coefficient at a constant gas-flow rate, remained constant for as well the ALR, the BC and the transition flow, the decreased Bodenstein number in the BC-type of flow is mainly attributed to the decreased convective transport as the liquid circulation is impeded. The number of circulations required to achieve complete mixing diminshes when the liquid circulation is impeded and appeared to be proportional to the Bodenstein number.

    In the transition flow regime, the volumetric oxygen transfer coefficient was estimated by both the stirred-tank model and the plug-flow model. The stirred-tank model yielded reliable results for the entire range of operation while the plug-flow model only appeared to be appropiate for the ALR operation mode. The volumetric oxygen transfer coefficient was found to increase for the BC operation mode and appeared to be a power law function of the ratio of the superficial liquid and gas velocity and the Bodenstein number.

    Addition of immobilized biocatalysts to the ALR, in our case simulated by neutral buoyant particles with diameters ranging from 2.4-2.7 am, significantly reduces the liquid velocity and the gas hold-up in an ALR. The decrease in liquid velocity is attributed to the decrease in gas hold-up and an increased friction in the ALR. The gas hold-up is reduced mainly because the presence of the particles increases the collision frequency of the air bubbles thereby increasing coalescence due to the diminished flowed area available for the air-water mixture. In comparison to a gas-liquid flow, axial dispersion in the three-phase flow is reduced as the presence of the particles damps the small eddies which are, apart from other mechanisms, responsible for the axial dispersion. Moreover. the increased coalescence also contributes to a decrease in axial dispersion. The presence of the particles negatively influences aeration due to a reduction in the gas-liquid interfacial area as a result of the increased coalescence. The effect of the increase in apparent viscosity in the ALR was not supposed to contribute to the decrease in the aeration process.

    Design of an organic-liquid-phase / immobilized-cell reactor for the microbial epoxidation of propene
    Brink, L.E.S. - \ 1986
    Landbouwhogeschool Wageningen. Promotor(en): K. van 't Riet; K.C.A.M. Luyben; J. Tramper. - Wageningen : Brink - 114
    biochemie - biotechnologie - chemische industrie - chemische reacties - constructie - ontwerp - ontwikkeling - uitrusting - biochemistry - biotechnology - chemical industry - chemical reactions - construction - design - development - equipment
    Replacement of a considerable part of the traditional, aqueous reaction medium in biotechnology by an organic medium is a promising technique to broaden the scope and range of biotechnological processes. This seems especially to be true for the conversion of non-polar substances. The high capacity of solvents for sparingly water-soluble substrates and products could reduce the required volume of the reaction mixture significantly, and may also lead to less substrate and/or product inhibition in the aqueous biocatalyst phase, when these mechanisms are involved. Furthermore, the use of an organic solvent could shift reaction equilibria favourably and facilitate down-stream processing. In chapter 1 a general review is presented of non-aqueous solvent systems in biocatalytic processes. Special attention is paid to two-liquid-phase systems, involving water-immiscible solvents. Several facets of these biphasic systems have been studied in this thesis using the epoxidation of propene by gel-entrapped Mycobacterium cells as a model.

    After the description of the throughout this work employed techniques of gas-analysis automatization and of substrate-level control (chapter 2), the far-reaching consequences of the solvent choice are treated in chapter 3. Many solvents cause rapid inactivation of the free, propene-epoxidizing cells. This appears also to be the case if the cells are immobilized in calcium alginate. However, the support material prevents direct cell-organic solvent contact and the associated aggregation and clotting of cells, mostly accompanied with loss of activity. High activity retentions of the immobilized cells relate to low polarities and high molecular weights of the used solvents. The polarity, as expressed by the Hildebrand solubility parameter, is also useful for describing the solvent capacity for one of the two substrates, oxygen, and for the product, propene oxide. The capacity for propene is less well described by the Hildebrand solubility parameter, but also less relevant, as the capacity of the solvents for propene is always about two orders of magnitude higher than that of water, and thus limitation of the rate by unsufficient supply of propene is less likely to occur. It is stressed that optimization of the solvent polarity is necessary, as the requirement of a high activity retention conflicts with the need for a high solvent capacity for the polar propene oxide. Optimization of the polarity will also be likely in case of other types of two-liquid-phase bioconversions.

    External and internal-diffusion limitations, which are to be expected when using cells entrapped in a hydrophilic: gel, are quantified in chapters 4 and 5. With negligible product inhibition, satisfactory predictions of the mass-transfer effects on the intrinsic Michaelis-Menten kinetics of the immobilized cells are obtained by using a simple pore-diffusion model (chapter 4). Internal diffusion is found to severely limit the epoxidation rate. A more complex model for the intrinsic epoxidation kinetics has been derived for modelling of mass-transfer rates in case of product inhibition (chapter 5).

    The microkinetic model defined in chapter 4 is integrated in a macrokinetic model to describe the behaviour of a packed-bed immobilized-cell reactor (chapter 6). Depletion of the limiting substrate, oxygen, along the length of the bioreactor can be prevented by using an organic solvent, n-hexadecane, as the transport medium. It is argued that this finding may eliminate the need for a separate gas phase in the fixed-bed reactor. Model predictions of the oxygen conversion in the bioreactor at various degrees of external and internal-diffusion limitation, at various liquid space times and with water or n-hexadecane as the continuous phase are in good agreement with experimentally obtained values. In chapter 7 some other, main limitations of the epoxide production in the packed-bed organic-liquid-phase/immobilized-cell reactor are quantified. Product inhibition is reduced by absorption of the inhibitory epoxide in a cold di-n-octyl phthalate phase. The stability of the immobilized cells is increased by supplying the cells alternately with propene and a co-substrate (ethene). About 50 g dry weight of cells in a 1.7 dm 3packed-bed reactor were used, which produced ~ 1.5 g chiral propene oxide; two third of the epoxide was absorbed in the octyl phthalate phase.

    Finally, in the last chapter of this thesis a general discussion is presented. The significance of optimization of the solvent polarity and of the interphase polarity, i.e. the polarity of the phase between biocatalyst and organic solvent is underlined. In case of entrapment in prepolymers, the hydrophobicity/hydrophilicity balance of the gel can be optimized with respect to polarities of substrates and products. Several features of hydrophilic and hydrophobic gels are compared. A quantitative illustration is given concerning the design on a technical scale of a fixed-bed organic-liquid-phase/immobilized-cell reactor. The advantages of using solvents with a high substrate capacity (often oxygen in case of aerobic processes) are demonstrated.

    Biocatalysts in organic syntheses : proceedings of an International symposium organized under auspices of the Working Party on Immobilized Biocatalysts of the European Federation of Biotechnology : Noordwijkerhout, The Netherlands, 14-17 April 1985
    Tramper, J. ; Plas, H.C. van der; Linko, P. - \ 1985
    Amsterdam : Elsevier (Studies in organic chemistry 22) - ISBN 9780444425416 - 259
    biochemie - biotechnologie - katalysatoren - katalytische activiteit - chemische industrie - chemische reacties - enzymen - fermentatie - organische verbindingen - synthese - biochemistry - biotechnology - catalysts - catalytic activity - chemical industry - chemical reactions - enzymes - fermentation - organic compounds - synthesis
    The Chichibabin amination of diazines geometrical isomerism in anions of aromatic amines
    Breuker, J. - \ 1982
    Landbouwhogeschool Wageningen. Promotor(en): H.C. van der Plas. - Wageningen : Breuker - 101
    azinen - pyridazinen - pyrimidines - purinen - chemische reacties - aminen - reactiemechanisme - azines - pyridazines - pyrimidines - purines - chemical reactions - amines - reaction mechanism
    The first part of this thesis describes investigations into the mechanistic aspects of the Chichibabin amination of some diazines in liquid ammonia containing potassium amide.

    The nucleophilic attack of the amide ion on 4-phenylpyrimidine readily takes place at C-2, due to its low electron density, and at C-6 because of the thermodynamic stability of the resulting σ-adduct. The former kinetically determined C-2 adduct isomerizes into the latter as shown by NMR spectros copy. Both adducts, but no analogous isomerization are observed in 4- t -butyl-pyrimidine. In 5-phenylpyrimidine an adduct on C-2 is not formed.

    Phenylpyrazine initially undergoes nucleophilic addition in KNH 2 /NH 3 at all three unsubstituted pyrazine carbon atoms. The C-5 adduct is thermodynamically. the most stable one.

    Amination of 4-phenylpyrimidine in 15N-labeled KNH 2 /NH 3 clearly shows that a ring opening-ring closure sequence (the S N (ANRORC) mechanism) must be in volved in the formation of the main product 2-amino-4- phenylpyrimidine. Quenching of the reaction with ammonium salt is an essential requirement for this mechanism. The conclusion is that the intermediate 6-amino-1,6-dihydro-4-phenylpyrimidine undergoes the ring opening. In the amination of 5-phenylpyrimidine the product 2-amino-5-phenylpyrimidine is also formed via an acyclic intermediate. In contrast, 4- t -butylpyrimidine, pyrazine and phenyl-pyrazine do not follow this S N (ANRORC) mechanism.

    The second part of this thesis deals with the occurrence of geometrical isomerism in the anions of aromatic amino compounds. NMR spectroscopy reveals the presence of two isomers of azaaromatic amines in liquid ammonia containing potassium amide, and even of anilines, in which the rotational barrier is lower. Coalescence is observed on increasing the temperature.

    The 1H and 13C NMR spectra are assigned to the syn - and anti -isomers. In all anions the ortho -hydrogen atom in the syn position relative to the lone pair of the exocyclic nitrogen atom resonates at lower field than in the anti position.

    In contrast, the ortho13C atoms do not show such a straightforward rela tionship in the anions of amino- as well as (methylamino)pyridines. In the former ions the signal of the ortho -carbon in the syn position relative to the nitrogen lone pair is found at higher field than in the anti position, whereas in the (methylamino)pyridine anions this signal is observed at lower field.

    With these data it is shown that the presence of a methyl substituent ortho to the amino group in aminopyridine anions causes a preference for the iso mer in which the amino hydrogen and the methyl group are directed towards each other. The conclusion is that the effective size of the lone pair is larger than that of an amino hydrogen, probably due to solvation. Stabilization of the preferred isomer by other effects, however, cannot be excluded.

    The reactivity of substituted purines in strongly basic medium : the occurrence of geometrical isomerism in the anions of aromatic amino compounds
    Kos, N.J. - \ 1981
    Landbouwhogeschool Wageningen. Promotor(en): H.C. van der Plas. - Wageningen : Kos - 107
    aminen - azinen - chemische reacties - chemie - kinetica - purinen - pyridazinen - pyrimidines - stereochemie - isomeren - heterocyclische verbindingen - amines - azines - chemical reactions - chemistry - kinetics - purines - pyridazines - pyrimidines - stereochemistry - isomers - heterocyclic compounds
    In this thesis two subjects are described: a. the amination of substituted purines by potassium amide in liquid ammonia and b. the occurrence of geometri cal isomerism in the anions of aromatic amino compounds.

    It is shown that the first step in the amination of purines, being present as anions under these strongly basic conditions, is the formation of a σ-adduct as position 6 to give a 6-amino-1,6-dihydropurinide. If position 6 is occupied by a blocking group an attack at position 2 or 8 does not occur. The further reaction course depends on the nature of the substituents and their position in the purine ring. i. If a leaving group (Cl,SCH 3 ) is present at the same position where the amide ion has attacked, this substituent is expelled (S N (AE) mechanism). In case no leaving group is present a Chichibabin amination occurs due to expulsion of a hydride ion from position 6 (this reaction is described in Chapter 2).

    The Chichibabin amination can also occur at position 6 when a leaving group (Cl,SCH 3 ) is present at position 8. ii. In the last-mentioned system a tele substitution is possible besides the S N (AE) reaction. This reaction is exemplified in the conversion of 8-chloropurine into adenine (formed besides 8-chloro adenine). The σ-adduct at position 6 is protonated at position 8, after which dehydrohalogenation occurs (S N (AE) tele , see Chapter 3). iii. If a leaving group is present at position 2 (Cl,F,SCH 3 ) the σ-adduct at position 6 undergoes ring opening of the pyrimidine ring with expulsion of the leaving group. The resulting imidazole derivative undergoes ring closure to give a 2-aminopurine. This type of reaction is referred to as an S N (ANRORC) mechanism and is described in Chapter 4.

    It has been established that in an S N (AE) mechanism the second step, involving the expulsion of the leaving group, is fast; the intermediary a-adduct cannot be observed. However, in the Chichibabin amination, tele amination and reaction according to the S N (ANRORC) mechanism, the second step is slow and therefore the σ-adduct can be observed by low temperature NMR spectroscopy.

    In Chapter 5 a new method is presented for the reductive removal of amino and alkylamino groups from position 6 of 9-substituted purines with sodium in liquid ammonia. The reaction involves reduction of the N(1) - C(6) bond, followed by elimination. This reaction is of special interest since the alternative method for the removal of amino groups i.e. the diazotization cannot be used with alkylamino groups. Therefore this new method is especially useful for the deamination of 6-(alkylamino)-9-substituted purines.

    In the last part of this thesis the occurrence of geometrical isomerism in the anions of aromatic amino compounds in liquid ammonia containing potassium amide is described. It is shown that this phenomenon occurs even in anilines, where the rotational barrier will be lower than in azaaromatic systems. This is confirmed by the occurrence of coalescence with increasing temperature (Chapter 6). The 1H and 13C NMR spectra of the anions of aminopyridines, aminopyrimidines and N-methylaminopyridines are assigned to the syn - and anti isomers. It has been revealed that in all these anions the ortho hydrogen atom in the s yn position relative to the lone pair resonates at a lower field than the hydrogen atom in the anti position. For the 13C NMR shifts of the ortho carbon atoms it was found that in the anions of N-methylaminopyridines the ortho carbon atom in the syn position relative to the lone pair resonates at lower field than the ortho carbon atom in the anti position. In the anions of aminopyridines and aminopyrimidines this phenomenon is reversed. We have also shown that the presence of a methyl group ortho to the anionic amino group causes a preference for the isomer, in which the proton of the NH group is in a syn position relative to the methyl group. This is explained in terms of the electron pair being "larger" than a proton, but it is possible that the preferred isomer is also stabilized by a better solvation and by an electronical effect.

    Synthesis and amination of naphthyridines
    Haak, H.J.W. van den - \ 1981
    Landbouwhogeschool Wageningen. Promotor(en): H.C. van der Plas. - S.l. : S.n. - 88
    pyridines - chemische reacties - aminen - synthese - organische verbindingen - pyridines - chemical reactions - amines - synthesis - organic compounds
    In the introduction of this thesis (chapter 1) the reactions of nephthyridines with potassium amide which were known at the start of our research are reviewed. It is shown in chapter 2, that in the amination of 1,X-naphthyridines with potassium amide in liquid ammonia at about -35° to -45°C the initial adduct formation is charge controlled. Thus, at these temperatures the site with the lowest electron density is most susceptible for amide attack (C-2 in 1,5 naphthyridine, C-2 in 1,6-naphthyridine, C-2 and C-8 in 1,7-naphthyridine, C-2 in 1,8-naphthyridine), as proved by NMR spectroscopy. On raising the temperature to about 10°C the site of addition has been found to change for 1,5- and 1,7-naphthyridine (NMR spectroscopy):from C-2 to C-4 in 1,5-naphthyridine and from C-2 and C-8 to C-8 only in 1,7-naphthyridine. Thus, at about 10°C the amination is thermodynamically controlled. The several factors which contribute to the stability of these addition products have been discussed. It has been found that the anionic a adducts (2(4,8)-aminodihydro-l,X-naphthyridinides) can easily be oxidized with potassium permanganate into their corresponding 2(4,8)-amino-1,X-naphthyridines.
    In chapter 3 a facile synthesis of 2,6-naphthyridine is described. Both 2,6 and 2,7-naphthyridine undergo with potassium amide under kinetically and thermodynamically controlled conditions a adduct formation at position 1. Chichibabin amination of 2,6-naphthyridine yields 1-amino-2,6-naphthyridine in 54% yield. The conversion of 1-halogeno-2,6-naphthyridines into 1-amino-2,6-naphthyridine is shown in chapter 4 to proceed via an even telesubstitution process [S N (AE) tele process]. The amination of 2-bromo-1,5-naphthyridine into 2-amino-1,5-naphthyridine is shown to proceed via an S N (AE) ipso substitution mechanism.
    Chapter 5 deals with the reaction of 1-halogeno-2,7-naphthyridines with KNH 2 /NH 3 yielding 1-amino-2,7-naphthyridine. Experiments with deuterated compounds show that these reactions proceed via an S N (AE) ipso process and not via an S N (AE) tele process, even though a adduct formation at C-8 takes place, as is shown by NMR spectroscopy.
    In chapter 6 the occurrence of an open-chain intermediate in the amination of 8-bromo-1,7-phenanthroline is shown by NMR spectroscopy. The reaction of 3-bromo-2-ethoxy-1,5-naphthyridine with KNH 2 /NH 3 is described in chapter 7. The procedure in the literature for its preparation does not lead to this compound but to the isomeric 3-bromo-1-ethyl-1,5-naphthyridin-2(1H)-one. Reaction of this compound with KNH 2 /NH 3 yields 3- and 4-amino-1-ethyl-1,b- naphthyridin-2(1H)one, the latter being the main product. 3-Bromo-2-ethoxy-1,5-naplithyridine was prepared on reacting 2,3-dibromo-1,5-naphthyridine with sodium ethoxide. A mixture of 3- and 4-amino-2-ethoxy- 1,5-naphthyridine was obtained on amination of 3-bromo-2-ethoxy-1,5-naphthyridine. In both cases the intermediacy of the respective 3,4,-dihydro compounds was proposed.

    Homoaromatics as intermediates in the substitution reactions of 1,2,4,5-tetrazines with ammonia and hydrazine
    Counotte-Potman, A. - \ 1981
    Landbouwhogeschool Wageningen. Promotor(en): H.C. van der Plas. - S.l. : - 113
    chemische reacties - hydrogenering - oxidatie - reductie - substitutie - heterocyclische verbindingen - chemical reactions - hydrogenation - oxidation - reduction - substitution - heterocyclic compounds
    This thesis describes some nucleophilic substitution reactions between the red 1,2,4,5-tetrazines and hydrazine-hydrate or ammonia. Special attention was paid to the occurrence of the S N (ANRORC) mechanism in these substitution reactions. This mechanism comprises a sequence of reactions, involving the A ddition of a N ucleophile to a heteroaromatic species, followed by a R ing- O pening and R ing C losure reaction to the substitution product.

    σ-Adducts, namely 6-hydrazino- and 6-amino-3-aryl(alkyl)-1,6-dihydro-1,2,4,5-tetrazines, are formed upon addition of hydrazine or ammonia to 3-aryl(alkyl)-1,2,4,5-tetrazines. This is accompanied by a change in colour from red to yellow. These adducts can be observed by NMR spectroscopy. ln heteroaromatics in liquid ammonia, an upfield shift (Δδ) of 4-5 ppm is usually measured for the hydrogen atom, attached to the carbon atom to which addition takes place. An extra ordinary large upfield shift is observed however upon addition to 1,2,4,5-tetrazines; Δδ= ~ 8.5 ppm in hydrazine and Δδ= ~ 8.7 ppm in liquid ammonia (at 230 K, chapters 4 and 6).

    The fact that 3-aryl(alkyl)-1,2,4,5-tetrazines are converted into the 6-amino compounds by oxidation of the intermediate in liquid ammonia (chapter 2), indicates that an intermediary 1,6-dihydro-6-amino structure must exist. 1H NMR measurements at various temperatures of 1,6-dihydro-1,2,4,5-tetrazines as model compounds for these σ-adducts gave an explanation for the large up field shift (Δδ). 1,6-Dihydro-1,2,4,5-tetrazines and their conjugate acids and bases were found to be homoaromatic and they are present in the monohomotetrazole conformation. The hydrogens at the sp 3carbon atom have a different orientation towards the tetrazole ring. One (H A) is oriented above the aromatic ring, in the shielding regio; H Bis in the exo position, in the deshielding regio; thus resulting in a large difference in chemical shift. The homoaromatic species show a ring inversion. The kinetic parameters (ΔH, ΔS and ΔG) were determined by dynamic NMR measurements (chapter 3). Since a large substituent at C 6 of the homotetrazole (e.g. methyl or ethyl) is found exclusively in the exo position, the hydrogen of the above mentioned a-adducts is oriented above the ring current of the tetrazole ring, resulting in a chemical shift at high field.

    The charge of the tetrazole ring exerts an influence through space on H A, H Bis hardly influenced. This became obvious from δH Ain 1H NMR and JCH Ain 13C NMR (chapters 3 and 4).

    The homoaromatic σ-adducts in liquid ammonia and even in hydrazine- hydrate/ methanol are anionic species, as was primarily proven by a 13C NMR study (chapters 4 and 6). The driving force for the deprotonation is probably the larger resonance stabilization of the homoaromatic anion with respect to the neutral homoaromatic species.
    3-Alkyl(aryl)-1,2,4,5-tetrazines were found to undergo a Chichibabin hydrazination into 6-hydrazino-3-alkyl(aryl)-1,2,4,5-tetrazines on treatment with hydrazine-hydrate. The first step in this reaction sequence was the formation of a homoaromatic σ-adduct. Subsequently an open-chain intermediate was observed by NMR, on raising the temperature. Finally the hydrazino compound is formed by ring closure. This reaction sequence can be considered as an S N (ANRORC) process. With 15N-labelled hydrazine, only part of the label was found to be built in the 1,2,4,5-tetrazine ring of the 6-hydrazino compounds. This is the first example of a reaction in which both the hydrazino compound with the 15N-label in the ring and with the 15N-label in the exocyclic hydrazino group are formed according to the S N (ANRORC) mechanism (chapter 6).

    During the hydrazino-deamination and hydrazino-dehalogenation of 6-amino- and 6-halogeno-1,2,4,5-tetrazines only a part of the molecules was found to react according to the S N (ANRORC) process. The other part followed the alternative S N (AE), A ddition- E limination, pathway (chapters 5 and 6).

    The crystal structure of 6-ethyl-3-phenyl-1,6-dihydro-1,2,4,5-tetrazine was elucidated by X-ray structural analysis very recently. This analysis revealed that the molecule is in a boat-conformation. C 6 points upwards with a dihedral angle of 49.3° and C 3 with an angle of 26.7°. N 1 was found to be sp 2hybridized and the N(1)-N(2), N(2)-N(3), C(3)-N(4) and N(4)-N(5) bond distances were found to be between single- en double bond length, in agreement with the expected electron delocalization. Therefore we came to the conclusion that the crystal structure agrees with the homoaromatic character of the compound (chapter 7).

    Thermal and photochemical reactions of dihydrodiazines
    Stoel, R.E. van der - \ 1979
    Landbouwhogeschool Wageningen. Promotor(en): H.C. van der Plas. - Wageningen : van der Stoel - 103
    azinen - derivaten - chemische reacties - fotochemie - azines - derivatives - chemical reactions - photochemistry
    This thesis describes the results of an investigation into the thermal and photochemical reactivity of dihydrodiazines.

    In order to prepare the title compounds the diazines and some phenyldiazines are treated with phenyllithium in ether, yielding adducts resulting from attack of phenyllithium on the various positions of the heteroaromatic ring. With pyrimidine addition takes place mainly at C(4), with pyridazine at C(3). By using TMEDA, addition at C(2) in 4-phenylpyrimidine and at C(4) in pyridazine is strongly promoted. The structure of the adducts is studied by n.m.r. spectroscopy. The charge distribution pattern in the C(4)-adduct of pyrimidine and in both the C(3)-adduct and the C(4)-adduct of pyridazine is determined by comparing the carbon chemical shifts of these compounds with those of the corresponding dihydrodiazines obtained by hydrolysis of the adducts. C(5) in the phenyllithium-pyrimidine adduct carries little negative charge, C(4) in the phenyllithium-pyridazine adduct has a considerable amount of charge while the charge density at C(6) in the 3-adduct and both C(3) and C(5) in the 4-adduct of pyridazine is moderate.

    Some organolithium-diazine adducts and some dihydropyrimidines are treated with electrophilic reagents. Both 4,6-diphenyl-1(3)-lithio-1,4(3,4)-dihydropyrimidine and 4,6-diphenyl-1,4(3.4)-dihydropyrimidine are attacked by the electrophilic reagent (methyliodide, methyl chloroformate) at N(3), yielding 4,6-dipheny]-3-methyl(methoxycarbonyl)-3,4-dihydropyrimidine. 4,4,6-Triphenyl-1,4(3,4)-dihydropyrimidine gives upon treatment with methyliodide mainly 3-methyl-4,4,6-triphenyl-3,4-dihydropyrimidine. The 3,4-dihydro structure of the products is established both spectroscopically and chemically. Reaction of 2-lithio-3-methyl-2,3-dihydropyridazine with methyliodide (methyl chloroformate, tosylchloride) gives the corresponding 2,3-dimethyl-(2-methoxycarbonyl-3-methyl-, 2-tosyl-3-methyl-)2,3-dihydropyridazine. 1-Lithio-2-phenyl-1,2-dihydropyrazine yields upon treatment with methyliodide 5-methyl-2-phenylpyrazine. Reaction with carbonyl compounds only yields high molecular material.

    Photolysis of 4-R-1,4(3,4)-dihydropyrimidines causes rearrangement to 5-R-1,2(2,3)-dihydropyrimidines, provided that the substituent R contains a π-bond in αposition to the heterocyclic ring (R=phenyl,isobutenyl,phenylethynyl). 4-Methyl-1,4(3,4)-dihydropyrimidine does not show this rearrangement. Chemical evidence is presented that the rearrangement occurs via the di-π-methane mechanism leading to 6-R-2,4-diazabicyclo [3.1.0] hex-2(3)-ene. This latter intermediate undergoes a thermal homo [1,5] hydrogen shift into 5-R-2,5-dihydropyrimidine which on tautomerization gives the final product. The reaction can be sensitized by acetone. 4,5-Diphenyl-, 5-methyl-4-phenyl and 5-bromo-4-phenyl-1,4(3,4)-dihydropyrimidine do not rearrange under photochemical conditions.

    Several 4-R-1,4(3,4)-dihydropyrimidines (R=2- or 3-thienyl,2-furyl, 1-methyl-2-pyrrolyl and 3-pyridyl) containing heteroaryl vinyl methane moieties undergo photochemical rearrangement into 5-R-1,2(2,3)-dihydropyrimidines. Oxidation of these compounds yield 5-heteroarylpyrimidines. The chemical yields are strongly dependent of the nature of the heteroaryl group.

    The existence of a 6-R-2,4-diazabicyclo [3.1.0] hex-2(3)-ene as an intermediate in the photoisomerization of 4-R-1,4(3,4)- dihydropyrimidines into 5-R-1,2(2,3)-dihydropyrimidines is confirmed spectroscopically in case R= p -trifluoromethylphenyl. It is established that the p -trifluoromethylphenyl group is in exo position in the bicyclic compound. 6- Exo -( p -trifluoromethylphenyl)-2,4-diazabicyclo [3.1.0] hex- 2(3)-ene immediately gives 5-( p -trifluoromethylphenyl)-1,2(2,3)- dihydropyrimidine upon addition of potassium hydroxide in methanol.

    Photolysis of 4-R-1,4(3,4)-dihydropyrimidines causes ring contraction into imidazoles, provided that the substituent R is sufficiently capable of stabilizing an anionic centre (R=2-thiazolyl and 2- or 4-pyridyl). Chemical evidence is presented that the ring contraction of 6-phenyl-4-(2-pyridyl)-1,4(3,4)-dihydropyrimidine occurs via heterolytic fission of the C(1)-C(6) bond of intermediate 1-phenyl-6-(2-pyridyl)-2,4-diazabicyclo [3.1.0] hex-2(3)-ene. The anion stabilizing effect of R is correlated with the acid strength (pKa) of R-CH 3 . A pKa value around 30 determines the border-line between ring contraction into an imidazole and formation of an isomeric 5-R-1,2(2,3)-dihydropyrimidine.

    [Sigma]-Adducts of pteridines and 3-deazapteridines, structure and reactivity
    Nagel, A. - \ 1978
    Landbouwhogeschool Wageningen. Promotor(en): H.C. van der Plas. - Wageningen : [s.n.] - 92
    azinen - purinen - pyridazinen - pyrimidines - chemische structuur - chemische reacties - structuuractiviteitsrelaties - azines - purines - pyridazines - pyrimidines - chemical structure - chemical reactions - structure activity relationships
    In the introduction of this thesis the reactions of pteridines and pyrido[2,3- b ]-pyrazines with nucleophiles are reviewed. In the following chapters the results of an NMR investigation on the formation of σ-adducts between these azaaromatic ring systems and nitrogen nucleophiles, especially KNH 2 /NH 3 , are described. In order to establish the structures of these - not isolable - σ-adducts, the 1H and 13C NMR spectra of pteridine, pyrido[2,3- b ]pyrazine and a number of derivatives of both these heterocyclic systems, containing one or more OCH 3 , SCH 3 , CH 3 , t-C 4 H 9 , OH, NH 2 , NHNH 2 , F, Cl, Br and C 6 H 5 substituents, were extensively analyzed. All resonance signals in the NMR spectra were unequivocally assigned.

    By means of 1H and 13C NMR, pteridines are shown to form in principle two different σ-adducts with NH 3 : at -60°C one molecule of NH 3 adds to C-4, yielding 4-amino-3,4-dihydro-2-R-pteridines (R=H, Cl), or alternatively, at temperatures up to +25°C, the addition of two molecules of NH 3 to C-7 and C-6 takes place, causing the formation of 6,7-diamino-4-R-2-X-5,6,7,8-tetrahydropteridines (R=X=H, R=H, X=Cl, OCH 3 , SCH 3 , C 6 H 5 , R=CH 3 , X=Cl. R=C 6 H 5 , X=Cl,H). This detailed NMR spectral information allowed straightforward interpretation of the 13C NMR spectra of the covalent hydrates 3,4-dihydro-4-hydroxypteridine, 6,7-dihydroxy-5,6,7,8-tetrahydroxypteridine and their cationic species.

    Due to the rapid decomposition of pteridine in KNH 2 /NH 3 , no σ-adduct could ever be detected. In sharp contrast, three σ-adducts between KNH 2 and pyrido[2,3- b ]-pyrazines are described i.e. the 3-amino-3,4-dihydropyrido[2,3- b ]pyrazinide ion, the 3-amino-2-t-butyl-3,4-dihydro-6-chloropyrido[2,3- b ]pyrazinide ion and the 2-amino-1,2-dihydro-3-phenylpyrido[2,3- b ]pyrazinide ion.

    The results are subsequently presented concerning the investigation of the reaction of KNH 2 /NH 3 with 2-X-4,6,7-triphenylpteridines (X=SCH 3 , Cl, F, H). Two reactions are found to take place : aminolysis at C-2, yielding 2-amino-4,6,7-triphenylpteridine (X=SCH 3 , Cl, F) and ring contraction, giving rise to the formation of 2-X-6,8-diphenylpurines (X=SCH 3 , H). By studying the aminolysis with both 15N labelled pteridines and with K 15NH 2 / 15NH 3 it is proved that the displacement at C-2 in the case of X=SCH 3 , occurs via a ring-opening and ring closing sequence [S N (ANRORC)]mechanism to the extent of 50-85% (depending on [KNH 2 ]); in the case of X=F this amounts to 40% and in the case of X=Cl to 100%.

    It is further proved that in the ring contraction of 2-methylthio-4,6,7-triphenylpteridine 85% of C-7 is expelled and 10% of C-6, both processes being preceded by addition of amide ion to C-7 and C-6 respectively.

    The possible elimination of C-7 and C-6 is clearly demonstrated by the fact that both 4,6- and 4,7-diphenyl-2-methylthiopteridines undergo ring contraction to the same product i.e. 6,8-diphenyl-2-methylthiopurine. As a consequence in the former isomer only C-7 is eliminated, while in the latter exclusively C-6 is expelled.

    In the next chapter the reactions of 6-chloro-2-R 1 , 3-R 2 -pyrido[2,3- b ]pyrazines [R 1 =H, R 2 =C 6 H 5 , t -C 4 H 9 , R= t -C 4 H 9 , R 2 =H, R 1 =R 2 =H, CH 3 , C 6 H 5 , phenanthro(9,10)] with KNH 2 /NH 3 are described.

    These compounds undergo ring contraction into 2-R-1H-imidazo[4,5- b ]pyridines (R=H, C 6 H 5 , t -C 4 H 9 ), besides reductive dechlorination. It is found that ring contraction of 2,3-diphenyl-6-X-pyrido[2,3- b ]pyrazines takes place exclusively if X=Cl; in the case of X=F only aminolysis is found, and in the case of X=Br reductive debromination occurs exclusively.

    The investigation on the mechanism of the ring contraction of 6-chloropyrido-[2,3- b ]pyrazine into 1H-imidazo[4,5- b ]pyridine is performed by using both is 15N-4 and 13C-2 labelled compounds and K 15NH 2 / 15NH 3 . The results can be explained by the initial formation of a σ-adduct of amide ion at C-2 - unfortunately not detectable by spectroscopic methods - in which σ-adduct, by an intramolecular rearrangement, the chlorine atom and C-2 are expelled simultaneously.

    Ring transformations in reactions of pyrimidine and N-alkylpyrimidinium salts with nucleophile
    Oostveen, E.A. - \ 1977
    Landbouwhogeschool Wageningen. Promotor(en): H.C. van der Plas. - Wageningen : Pudoc - 58
    pyrimidines - chemische reacties - pyrimidines - chemical reactions

    Paper I

    On treatment with liquid ammonia at -33°C, the quaternary pyrimidinium salts, i.e. 1-methylpyrimidinium methyl sulfate, 1,2-dimethylpyrimidinium iodide, 1,4,6-trimethyl-pyrimidinium iodide and 1,2,4,6-tetramethylpyrimidinium iodide demethylate yielding pyrimidine. 2-methyl-, 4,6-dimethyl- and 2,4.6-trimethylpyrimidine, respectively. It was observed that under these conditions 1-methyl-[1,3- 15N]-pyriniidiniuni methyl sulfate yields [1- 15N]-pyrimidine. By measuring the PMR spectra of above- mentioned pyrimidinium salts in liquid ammonia it is shown that these salts undergo covalent amination on the 1,6-azomethin bond. These results indicate that the demethylation reaction occurs via an Addition-NucleophileRing-Opening-Ring Closure mechanism.

    Paper II

    On treatment with active methylene compounds in basic media the quaternary pyrimidinium salts, i.e. methyl 1-methylpyrimidinium sulfate, 1-methyl-4-phenylpyrimidinium iodide and 1-methyl-5- phenylpyrimidinium iodide are converted into pyridine derivatives. The mechanism of the reaction is discussed.

    Paper III

    On treatment of the quaternary pyrimidinium salts i.e. 1-methyl-4-phenylpyrimidinium iodide and 1-methyl-5-phenylpyrimidinium iodide with cyanamide, O -methylisouronium chloride or bis[S-methylisothiouronium] sulfate in basic media, 2-amino-4-phenylpyrimidine and 2-amino-5-phenylpyrimidine are formed respectively. A ring transformation is involved in which the two-atom fragment N(1)-C(2) of the pyrimidine ring is replaced by an N-C fragment of the reagent. On reacting 1-methylpyrimidinium iodide with benzamidinium chloride or pivalamidinium chloride in a solution of sodium ethoxide in ethanol, 2-phenylpyrimidine and 2- tert -butylpyrimidine are formed respectively.

    It is proved by 15N-labelling that this nucleophilic substitution occurs via a ring transformation in which the N(1)- C(2)-N(3) fragment of the pyrimidine is replaced by the N-C-N fragment of the amidine. These reactions are new examples of a nucleophilic substitution occurring according to an S N (ANRORC) mechanism.

    Paper IV

    Reaction of 4-alkoxy- or 4,6-dialkoxypyrimidines with 1 equivalent of triethyloxonium tetrafluoroborate yields 4-alkoxy-N-ethyl or 4,6-dialkoxy-N-ethylpyrimidinium salts, respectively. With two or more equivalents of this reagent, rearrangement of N-ethyl-alkoxypyrimidinium salts into 1-ethyl-3-alkyl-1,4(3,4)-dihydro-4-oxopyrimidinium salts takes place. These rearrangements can also be performed by heating. The mechanism of these rearrangement reactions is discussed.

    Paper V

    The crystal and molecular structures of two isomeric compounds, 1-ethyl-4,6-diethoxypyrimidinium tetrafluoroborate and 1,3-diethyl-1,4(3,4)-dihydro-6-ethoxy-4-oxopyrimidinium tetrafluoroborate, reaction products of 4,6-diethoxypyrimidine with Meerwein reagent [O(C 2 H 5 )3+BF4-] , have been determined by means of X-ray diffraction.
    1-Ethyl-4,6-diethoxypyrimidinium tetrafluoroborate is monoclinic a=10.794, b=13.361,c=10.892 Å, β =112.6°, space group P2 1 /n, four molecules per unit cell.
    1,3-Diethyl-1,4(3,4)-dihydro-6-ethoxy-4-oxopyrimidinium tetrafluoroborate is monoclinic, a=17.637, b=14.054, c=11.501 Å, β =101.7°, space group C2/c, eight molecules per unit cell.
    In both structures the fluoroborate ions are disordered. The bond distances in the π-electron systems are reasonably well described in terms of a small number of resonance structures.

    Paper VI

    Treatment of 1,3-diethyl-1,4(3,4)-dihydro-4-oxopyrimidinium tetrafluoroborate and its 2-phenyl, 6-phenyl, 6-methyl and 6-ethoxy derivatives with aqueous ammonia resulted in the formation of a mixture of open-chain compounds i.e. N -formyl(acetyl,benzoyl)- N -ethyl-3-(ethylamino)acrylamides and N -ethyl-3-[formyl(acetyl,benzoyl)ethylamino]-acrylamides. They are formed by cleavage of the pyrimidine ring between the N(1)-C(2) and N(3)-C(2) bond, respectively. In liquid ammonia the same ring cleavage generally occurs; however, in the case where a 6-ethoxy group is present, recyclisation can take place, leading to 6-(ethylamino)pyrimidine derivatives. This degenerate ring transformation has been observed also with the 2-methyl and 2-phenyl derivative of 1,3-diethyl-1,4(3,4)-dihydro-6-ethoxy-4-oxopyrimidinium tetrafluoroborate. Evidence is presented by means of 1H-NMR and 13C-NMR spectroscopy that all these reactions are iniated by attack of NH 3 at the C(2)-position. Some of the above-mentioned open-chain compounds underwent a ring closure to the initially used 1,3-diethyl-1,4(3,4)-dihydro-4-oxopyrimidinium tetrafluoroborates on treating them with hydrofluoroboric acid in absolute ethanol.

    Paper VII

    On treatment with liquid ammonia at -33° the quaternary pyrimidinium salts i.e. 4-ethoxy-1-ethyl- and 4,6-diethoxy-1-ethylpyrimidinium tetrafluoroborate undergo amino-de-ethoxylation, yielding 1,4-dihydro-1-ethyl-4-iminopyrimidine hydrogen tetrafluoroborate and a mixture of 1,4-dihydro-6-ethoxy-1-ethyl-4-imino- and 1,6-dihydro-4-ethoxy-1-ethyl-6-iminopyrimidine hydrogen tetrafluoroberate, respectively. 1H-NMR and 13C-NMR spectroscopic evidence is presented for the fact that compounds 1 and 3 easily give σ-adducts at position 2. Using 15N-labelled ammonia it was shown that in these amino-de-ethoxylation reactions the substitution at C(4) or C(6) does not involve ring opening but probably occurs via an S N (AE n ) process. Reaction of 4-ethoxy-1-ethyl-2-phenyl-, 6-ethoxy-1-ethyl-4-phenyl-, 4,6-dimethoxy-1-ethyl-2-phenyl- and 4,6-dimethoxy-1-ethyl-2-methylpyrimidinium tetrafluoroborate with liquid ammonia gives besides the amino-de-ethoxylation product degenerate ring transformations leading to the N-deethylated products 14-16 and 4(6)-ethylaminopyrimidines 17-19. The salt 11 and 1,6-dihydro-1-ethyl-6-imino-4-phenylpyrimidine hydrogen tetrafluoroborate undergo, with potassium hydroxide, a Dimroth rearrangement to pyrimidines 20 and 17, respectively.

    Paper VIII

    The mechanism of the conversion of pyrimidine into 5-ethyl-2-methylpyridine has been investigated. It has been proved, using the labelled compounds [1,3- 15N]pyrimidine, [4,6- 14C]pyrimidine and [5- 14C]pyrimidine, that this reaction proceeds via a mechanism, in which the pyrimidine ring is fragmentated into two molecules of HCN and one molecule of N -methylacetaldimine. Four molecules of this imine undergo an aldol type condensation leading to 5-ethyl-2-methylpyridine.

    Reactions of pyridazines and pyridazine 1-oxides with nitrogen-containing nucleophiles
    Klinge, D.E. - \ 1976
    Landbouwhogeschool Wageningen. Promotor(en): H.C. van der Plas. - Wageningen : [s.n.] - 40
    pyridazinen - chemische reacties - pyridazines - chemical reactions
    In dit proefschrift is een orienterend onderzoek beschreven naar het chemisch gedrag van halogeen-pyridazinen en halogeen-pyridazine-N-oxiden met kaliumamide in vloeibare ammoniak, met methanolische ammoniak en met vloeibare ammoniak. Dit onderzoek hangt nauw samen met uitvoerige studies over de reactiviteit van pyridinen, pyrimidinen en pyrazinen, die de afgelopen jaren in het laboratorium voor organische chemie te Wageningen zijn verricht. De resultaten van het door ons uitgevoerde onderzoek zijn in een vijftal publikaties verwerkt en laten zich als volgt samenvatten:

    Publikatie I:
    4-X-3-(methoxymethyl)-6-methylpyridazine (X=Cl, Br, I) geeft na aminering een mengsel van 4- and 5-amino-3-(methoxymethyl)6-methylpyridazine (verhouding 1 : 5); het is gebleken dat deze verhouding onafhankelijk is van de aard van het halogeen atoom. Daarom wordt als intermediair in deze reactie het 3-(methoxy-methyl)-6-methyl-4,5-didehydropyridazine aangenomen.

    Publikatie II:
    De reactie van 4-chloor-3,6-difenylpyridazine met kaliumamide in vloeibare ammoniak levert twee produkten op, namelijk 4-amino-3,6-difenylpyridazine en imino-4,4'-bis(3,6-difenylpyridazine). Uitgaande van 4-chloor-3,6-difenylpyridazine- [5- 13C] werd, door middel van quantitatieve 13C-NMR spectrometrie, van beide aminoverbindingen aangetoond dat zij gevormd zijn via het symmetrisch intermediair 3,6-difenyl-4,5-didehydropyridazine.

    De resultaten vermeld in I en II geven voor het eerst zeer gefundeerde aanwijzingen voor het bestaan van een intermediair 3,6-digesubstitueerd 4,5-didehydropyridazine in reacties van 3,6-digesubstitueerde 4-halogeenpyridazinen met kaliumamide in vloeibare ammoniak.

    Publikatie III:
    In de reactie van 6-chloor-3-methoxy-4-nitropyridazine 1-oxide met methanolische ammoniak en met vloeibare ammoniak treedt amino-demethoxylering op. Met behulp van 1H-NMR metingen kon worden aangetoond dat deze substitutie op een zeer ongewone wijze verloopt, n.l. in het op C(5) gevormde Meisenheimer complex.

    Publikatie IV:
    Van verschillende 3-mono- en 3,6-digesubstitueerde pyridazinen, 3-, 4- en 6-mono- en 3,6-digesubstitueerde pyridazine 1-oxiden en 4-nitro-3,6-digesubstitueerde pyridazine 1-oxiden zijn de 13C-absorpties bepaald. Hieruit werden de 13C-substituent effecten van substituenten in de 3-, 4- en 6-positie van de pyridazine ring en die van de N-oxide functie berekend. Door toepassing van deze substituent effecten konden de 13C-NMR spectra van de σ-adducten van 4-nitro-3,6-digesubstitueerde pyridazine 1-oxiden met vloeibare ammoniak worden geinterpreteerd.

    Uit de resultaten van de 1H-NMR en 13C-NMR metingen vermeld in III en IV kon zeer duidelijk worden vastgesteld dat 3-methoxy-4-nitropyridazine 1-oxiden een nucleofiele substitutie op CM kunnen ondergaan volgens een mechanisme, dat tot nu toe niet eerder in de literatuur is beschreven.

    Publikatie V:
    Bij de behandeling van 4-amino-3-X-pyridazine (X=Cl, Br) met kaliumamide in vloeibare ammoniak wordt 4-cyaanpyrazool gevormd. Onder dezelfde reactie omstandigheden ondergaat 4-amino-3,6-di-X-pyridazine (X=Cl, Br) een ringcontractie tot 3-(cyaanmethyl)-1,2,4-triazool.

    De in V beschreven ringcontractie tot 4-cyaanpyrazool kan worden beschouwd als een nieuw voorbeeld van een reactie, die reeds in andere ringsystemen beschreven is. De in V beschreven ringcontractie tot 3-(cyaanmethyl)-1,2,4-triazool is echter het eerste voorbeeld van een door kaliumamide gekatalyseerde ringcontractie, waarbij het gevormde vijfringsysteem meer stikstofatomen bevat dan het oorspronkelijke zesringsysteem.

    Inwerking van basen op N-oxiden van pyridine en pyrimidine
    Peereboom, R. - \ 1975
    Landbouwhogeschool Wageningen. Promotor(en): H.J. den Hertog, co-promotor(en): H.C. van der Plas. - Wageningen : [s.n.] - 70
    piperidinen - pyridines - pyrimidines - stikstofoxiden - chemische reacties - basen - alkaliteit - piperidines - pyridines - pyrimidines - nitrogen oxides - chemical reactions - bases - alkalinity
    A survey is given of investigations on reactions of halogeno- azahetarenes in basic media as described to date in the literature, chiefly of those on the behaviour of N-oxides of halogeno-azahetarenes and in some cases those of otherwise N-quaternised azahetarenes towards liquid ammonia and towards potassium amide in liquid ammonia. The reactions were found to proceed according to S N (AE)-, S N (EA)- and S N (AE a )-mechanisms and/or ring openings. The open-chain compounds formed in the latter process close either to the same (S N (ANRORC)- mechanism) or a new ring system (ring transformation) (Chapter

    In this connection we studied reactions of 3-bromopyridine 1-oxides with potassium amide in liquid ammonia and reactions of 4-X-6-methyl-(phenyl)pyrimidine 1-oxides with liquid ammonia and potassium amide in liquid ammonia.

    It was found that the reaction of 3-bromopyridine 1-oxide with potassium amide in liquid ammonia in the presence of isopropylamine affords 3-amino- and 3-(isopropylamino)pyridine 1-oxide whereas 3-bromopyridine 1-oxide remains unchanged in a mixture of liquid ammonia and isopro pylamine alone. These results affirm the previous hypothesis, that the amination of 3-bromopyridine 1-oxide with potassium amide in liquid ammonia proceeds by the S N (EA)-mechanism via 2,3-didehydropyridine 1-oxide (Chapter 2).

    3-Bromo-6-methylpyridine 1-oxide was converted into 4-amino- (main product), 3-amino- and 2-amino-6-methylpyridine 1-oxide by potassium amide in liquid ammonia, whereas 3-bromo-6-ethoxypyridine 1-oxide when treated with the same reagent only yielded 3-amino-6-ethoxypyridine 1-oxide Thus the methyl group occupying the 6-position changes the pathway of the amination of 3-bromopyridine 1-oxide, whereas the ethoxy group at the C(6)-atom does not. This difference can be explained by assuming that the methyl derivative is partly deprotonated yielding an anionic group which has a strong mesomeric interaction with the N-oxide group leading to a change of the charge distribution in the ring of the substrate. This causes the occurrence of a second reaction pathway, an S N (EA)-mechanism via 3,4-didehydro-6-methylpyridine 1-oxide (Chapter 2).

    The reaction of 3-bromoquinoline 1-oxide with potassium amide in liquid ammonia affords 3-hydroxy-4-[3-amino-2-quinolyl]quinoline 1-oxide together with 3- and 4-aminoquinoline 1-oxide. These products must be formed via 3,4-didehydroquinoline 1-oxide as an intermediate. That 3-bromoquinoline 1-oxide reacts differently from 3-bromopyridine 1-oxide is caused by the fused benzogroup which changes the charge distribution in the substrate considerably and enhances the stability of the intermediary 3,4-didehydroquinoline 1-oxide compared to that of 2,3-didehydroquinoline 1-oxide (Chapter 2).

    The oxidation of 4-X-6-methyl(phenyl)pyrimidines (X=Cl, Br and OC 6 H 5 can theoretically give two isomeric N-oxides (N(1)- and N(3)-oxide). The structure determination of the formed 4-X-6- methyl(phenyl)pyrimidine N-oxides was based on the structure of 4- chloro-6-methyl(phenyl)pyrimidine 1-oxide which was established by means of PMR spectroscopy on the dechlorinated compound (Chapter 3).

    The reactions of 4-chloro-6-methyl(phenyl)pyrimidine 1-oxide with liquid ammonia and with potassium amide in liquid ammonia yield a compound formed by amino-dechlorination, 4-amino-6-methyl(phenyl)pyrimidine 1-oxide and a ringtransformation product, 5-amino-3-methyl(phenyl)isoxazole.

    The results of the reactions of 4-chloro-6-methyl- and 4-chloro-6-phenyl-[l(3)- 15N]pyrimidine 1-oxide with liquid ammonia and with pot assium amide in liquid ammonia indicate that an S N (ANRORC)-mechanism is not operative in the conversion to the 4-amino-6-methyl- and 4-amino-6-phenylpyrimidine 1-oxide respectively. Furthermore it could be established that the dechlorination in liquid ammonia does not take place via a 4,5-didehydropyrimidine 1-oxide as intermediate, but according to an S N (AE)-process. This is based on the results of the reaction of 4-chloro-5-deutero-6-phenylpyrimidine 1-oxide The amino-dechlorination of the same substrate with potassium amide in liquid ammonia presumably takes place via the same pathway. This result is supported by studying the reactions of 4-chloro-5,6-diphenylpyrimidine 1-oxide in both media. 4-Amino-5,6-diphenylpyrimidine 1-oxide as well as 5-amino-3,4-diphenyl-isoxazole are formed. Since an S N (EA)-mechanism is prohibited because of the presence of a phenylgroup on position 5, this result is good evidence for the occurrence of an S N (AE)-mechanism in the nucleophilic displacement of the halogen atom in the 4-halogenopyrimidine 1-oxides (Chapter 4).

    The results of the reactions of 4-chloro-6-methyl-[l(3)- 15N]pyrimidine 1-oxide with liquid ammonia and with pot de in liquid ammonia have established two concurrent pathways for the formation of 5-amino-3-methylisoxazole. The first pathway involves addition of the nucleophile to the C(2)-atom, resulting in an isoxazole compound with the same 15N-enrichment as present in the substrate. In the second route addition to the C(4)-atom occurs leading to amino-dechlorination, as well as ring transformation to 5-amino-3-methylisoxazole with 15N enrichment at the ring nitrogen only . 6-Methyl-4-phenoxypyrimidine 1-oxide forms 5-amino-3-methylisoxazole but no 4-amino-6-methylpyrimidine 1-oxide in the reaction with potassium amide in liquid ammonia. In good accordance with these data it has been found that in this reaction the isoxazole formation only takes place via an addition of the amide ion to the C(2)-atom (Chapter 5).

    Variation of the substituent X of the 4-X-6-methyl(phenyl)pyrimidine 1-oxides influences the competition between the amination and the ring contraction. The substrates, in which X=Cl, Br and I, yield 4- aminopyrimidine 1-oxides, as well as 5-aminoisoxazoles, in both media. The addition of the amide ion or ammonia to the C(4)-atom is the principal reaction pathway. In the cases where X=OC 6 H 5 and X=SC 6 H 5 the activation by the substituent at the 4-position must be low, because these substrates only react with potassium amide in liquid ammonia at -33°C. The addition of the amide ion at the C(2)-atom is strongly favoured to the addition at the C(4)-atom. 6-Methyl-4-(trimethylammonio)pyrimidine 1-oxide affords 4-amino-6-methylpyrimidine 1-oxide as sole product in the reaction with liquid ammonia. The trimethylammonio group, a good leaving group, strongly activates the 4- position for the direct nucleophilic substitution and prohibits the addition of ammonia at the C(4)-atom (Chapter 6).

    Inwerking van stikstofhoudende nucleofielen op enige 15N-gemerkte pyrimidine- en chinazolinederivaten
    Kroon, A.P. - \ 1974
    Landbouwhogeschool Wageningen. Promotor(en): H.C. van der Plas. - S.l. : S.n. - 71
    chemische reacties - stikstof - pyrimidines - derivaten - chemical reactions - nitrogen - pyrimidines - derivatives
    In this thesis an investigation is described on the mechanism of aminations of pyrimidine- and quinazoline derivatives with nitrogen containing bases.

    In the introduction a survey is given of investigations, reported in the literature, concerning σ-complex formation on azahetarenes and their derivatives. The complex forming ability of different carbon atoms in these heterocyclic substrates with nucleophiles is very important for the explanation of the results of many reactions (Chapter I).

    On amination of 2-X-4-phenylpyrimidines (X=F, Cl, Br, J) with potassium amide in liquid ammonia at -75 °C, 2-amino-4-phenylpyrimidine is obtained in good yield. A second product, 3-amino-3-phenylacrylonitrile, is isolated in low yield.

    The formation of the 2-amino compound occurs to a large extent via a series of reactions, involving an initial Addition of the Nucleophile, to the C 6 atom, Ring-opening and Ring-Closure [S N (ANRORC)- mechanism] (X=F, Cl, Br, J; resp. 82, 88, 88, 73%). Proof for this mechanism is based on studies with 2-halogeno-4-phenyl-[1,3- 15N]-pyrimidines. In the case of the 2-chloro- and 2-bromo compound the open- chain intermediate postulated in the S N (ANRORC)-mechanism can be isolated. This intermediate cyclized slowly at room temperature to the 2- amino compound. Reaction with potassium amide or sodium hydroxide gave the same result. Surprisingly the 3-amino-3-phenylacrylonitrile obtained from the 15N-labelled compounds, contains no excess of 15N. Apparently both N- atoms present in this compound must come from the amide ion. It is proposed that the nitrile is formed by an initial attack of the amide ion on C 4 . Due to steric hindrance this addition is difficult and thus must be only a minor pathway. After ring opening of the C 4 adduct it is assumed that a second amide ion adds across the azomethine bond in this open-chain product. Loss of cyanamide and a subsequent reduction-oxidation process can then give rise to the formation of acrylonitrile. This second S N (ANRORC)- mechanism, via the C 4 adduct, cannot be ruled out but it is considered to be of less importance (Chapter II).

    Since there is very little information on the effect of leaving group mobility on the S N (ANRORC)-mechanism the influence of different groups on the occurrence of this mechanism was studied in the reaction of the 2-X- 4-phenylpyrimidines (X=SCH 3 , SO 2 CH 3 , SC 6 H 5 , SO 2 C 6 H 5 , SCN, CN and +N(CH 3 ) 3 ) with potassium amide. Using the [1,3- 15N]-labelled substrates, the corresponding 2-amino compounds are isolated and investigated by mass spectrometry. From the results the conclusion can be drawn that the methylthio-, the thiocyanato- and the methylsulfonyl group show a behaviour nearly identical to the 2-halogeno compounds [%-S N (ANRORC)mechanism resp. 91, 90 and 73] . The trimethylammonio- and the cyanogroup undergo almost exclusively a S N (AE) displacement process (90% and 95% resp.), while the phenylsulfonyl group has no special preference 34% S N (AE). It is very striking that the methylsulfonyl group - in contrast to the phenylsulfonyl group - mainly undergoes a S N (ANRORC)-amination. Deprotonation of the methyl group possibly is the cause of this difference; formation of the C 2 -NH 2 adduct, the first step in the addition-elimination, is then less favourable. 1H-NMR spectrometric measurements of the methylsulfonyl substrate in the amination medium showed the disappearance of the CH 3 signal. The C 6 -NH 2 adduct, postulated in the S N (ANRORC)-amination of the 2-substituted 4-phenylpyrimidines, is proven by measuring 5-deuterio-2-methylthio-4-phenylpyrimidine in its reaction medium with 1H-NMR (Chapter III).

    Investigations were carried out as to how the course of the reaction is influenced by the presence of a substituent in position 6, choosing for that purpose the phenyl group. This voluminous group can possibly prevent (or retard) the addition of the amide ion at position 6(4), making the competitive reaction pathway via an addition-elimination reaction more favourable. The results, obtained when aminating 2-X-4- phenyl- [1,3- 15N] -pyrimidine (X=F, Cl, Br) with potassium amide, give evidence that in the amination of the fluoro compound no ring opening occurs, but that 2-chloro- and 2-bromo-4-phenylpyrimidine react for a considerable part (~ 70%) via ring opening into the 2-amino compound. Since in the reaction of the 2-chloro- and 2-bromo-4-phenylpyrimidine about 90% reacts by a S N (ANRORC)-mechanism one has to conclude that the phenyl group in position 4 or 6 does actually influence the addition of the amide ion on that position. The reaction intermediate, postulated for the S N (ANRORC)-mechanism in the 2-halogeno-4,6- diphenylpyrimidines, is isolated for the 2-bromo- and 2-chloro compound in low yield, using short reaction times. (Chapter IV).

    In extension of the work on the amination of 2-halogeno-4,6-difenylpyrimidines, the possibility that a ring opening is also involved in the conversion of 2-chloro-4-phenylquinazoline into 2-amino-4- phenylquinazoline with potassium amide was investigated. Mass spectrometric determinations of this product indicate that about 70% of the substrate reacts via ring opening into the 2-amino compound. In this quinazoline substrate attack of amide ion at only one carbon atom can give rise to a S N (ANRORC)-mechanism, so we can conclude that this quinazoline is more vulnerable to the ring opening reaction than the 2-chloro-4,6-diphenyl-pyrimidine. This can be explained with the well-known high reactivity of the 3,4-bond in quinazoline. Experiments with other 2-substituted 4-phenyl-[3- 15N]-pyrimidines showed the same trend in occurrence of the S N (ANRORC)-mechanism as found for the 2-substituted 4- phenylpyrimidines: the 2-fluoro compound was aminated for 55% and the 2-cyano compound for 15% via a ring opening. investigation of (-chloro-4-phenylquinazoline in the amination reaction with ethanolic ammonia, using 15N labelled substrate showed as unexpected result that in the amination the S N (ANRORC)mechanism is operative for 34%. This percentage was found to be dependent on the concentration of the ammonia. The occurrence of the same ring opening reaction was shown in the amination of 2-chloro- and 4-chloroquinazoline with ethanolic ammonia (Chapter V).

    It is reported ill the literature that on heating 4-quinazolinone with phenyl phosphorodiamidate 4-aminoquinazoline is formed. It is now shown, using [3- 15N]-4-quinazolinon that the formed 4-amino compound contains a certain amount of 15N-label in the amino group. It is determined that in this reaction a ring opening is partly operative in the amination of the oxo compound. The amino compound however, also undergoes an exchange reaction with PPDA involving a ring opening (Chapter VI).

    Cinesubstitutie en broomverplaatsing bij inwerking van kaliumamide op 4-gesubstitueerde halogeenpyridinen
    Vrijhof, P. - \ 1974
    Landbouwhogeschool Wageningen. Promotor(en): H.J. den Hertog. - Wageningen : Veenman - 90
    chemische reacties - halogenen - pyridines - chemical reactions - halogens - pyridines

    A survey is given of investigations on the reactions of halogeno-azahetarenes in strongly basic media, especially towards potassium amide and lithium piperidide, which were found to comprise direct and cine substitutions, ring openings and ring transformations, halogen migrations and several other processes. In this connection we studied reactions of 4-substituted halogenopyridines with potassium amide, involving cine substitutions and halogen migrations (chapter 1).

    Reaction procedures and methods of analysis applied are described in chapter 2.

    First we investigated the amination of 3-bromo-4-ethoxypyridine, a substrate previously supposed to be converted at -33° into 2-amino-4-ethoxypyridine, 4-ethoxypyridine and 2-amino-5-bromo-4-ethoxypyridine via a mechanism involving 4-ethoxy-2,3-didehydropyridine as an intermediate and a bromine migration (PIETERSE and DEN HERTOG, 1962). By varying reaction conditions and applying 2H-labelling it was established that 3-bromo-4-ethoxypyridine is transformed into 4-ethoxypyridine and 3,5-dibromo-4-ethoxypyridine by an intermolecular trans-bromination, that 4-ethoxypyridine is formed at the same time by potassium-bromine exchange and that 2(=6)-amino-4-ethoxypyridine does not originate from a didehydropyridine derivative as an intermediate but results from an abnormal-addition-elimination reaction (AE a ) starting with an attack on the nucleus at C-6 by the amide ion. In a base-catalyzed reaction the 3,5-dibromo derivative is converted into 2,5-dibromo-4-ethoxypyridine, which at -33° is aminated to yield 2-amino-5- bromo-4-ethoxypyridine.

    Action of potassium amide on 3-bromo-2,4-diethoxypyridine results in a bromine shift to C-5 and subsequent formation of 2,4-diethoxypyridine, 6-amino-2,4- diethoxypyridine and 6-amino-3-bromo-2,4-diethoxypyridine (chapter 3).

    Variation of the substituent at C-4 and the halogen atom at C-3 gave the following results.

    3-Chloro-4-ethoxypyridine is converted into 4-amino-3-chloropyridine (AE-mechanism). The 3-iodo derivative yields 4-ethoxypyridine and 3-amino-4-ethoxypyridine at -33° whereas at -75° only deiodination takes place.

    3-Chloro-, 3-bromo- and 3-iodo-4-piperidinopyridine give the 3-amino derivative according to the S N Ar 2 - or AE-mechanism, this strongly temperature dependent process being favoured by the voluminosity of the substituent. Evidence for a nucleophilic substitution via radical anions as a side-reaction is obtained in the conversion of 3-iodo- into 3-amino-4-piperidinopyridine. Only 3-bromo-4-piperidinopyridine yields the 2-amino compound through the AE a -pathway with initial attack of the amide ion at C-2 and C-6. Dehalogenation was found to occur in the aminations of 3-bromo- and 3-iodo-4-piperidino pyridine (potassium-halogen exchange).

    3-Bromo-4-(4'-pyridyl)pyridine containing a -I, -M substituent is rapidly transformed into 2-amino- and 3-amino-4-(4'-pyridyl)pyridine and into debrominated material. In a mixture of liquid ammonia and ether some 2-bromo-4-(4'- pyridyl)pyridine is formed together with 4-(4'-pyridyl)pyridine; a result indicating that possibly in both media bromine migration takes place, in liquid ammonia followed by a rapid amination.

    3-Bromo-4-cyanopyridine, the second compound with a -I, -M group studied, yields 4-aminopyridine, 3-bromo-4-aminopyridine, 3,5-dibromo-4-aminopyridine and 2-bromo-4-aminopyridine. Thus in this case bromine migrations occur and subsequently replacements of the cyano by the amino group (chapter 4).

    In connection with the results described above the reactivities of dibromo- and tribromo-4-ethoxypyridines in liquid ammonia and in a mixture of liquid ammonia and ether were studied.

    In liquid ammonia 2,3-dibromo-4-ethoxypyridine yields 2,5-dibromo-4-ethoxypyridine and 2-amino-5-bromo-4-ethoxypyridine; 2,5-dibromo-4-ethoxypyridine also gives 2-amino-5-bromo-4-ethoxypyridine, while 3,5-dibromo-4-ethoxypyridine is converted into the 2,5-dibromo- and 2-amino-5-bromo derivative as well, together with substances resulting from side and subsequent reactions. It is supposed that all the reactions start with the abstraction of the most acidic proton. The formed carbanion then abstracts a bromonium ion from a molecule of the starting material yielding 2,3,5-tribromo-4-ethoxypyridine and the anion of the 2- or 5-bromo derivative. From these products by a second migration of a bromonium ion 2,5-dibromo-4-ethoxypyridyl-3-anion and the starting substance are obtained.

    In the mixture of liquid ammonia and ether the same disproportionation products are formed. In this medium however, they react independently according to different routes. Thus 2,3-dibromo- and 2,5-dibromo-4-ethoxypyridine give mixtures of 2-amino-4-ethoxypyridine, 2,6-dibromo-4-ethoxypyridine and 2,3,6-tribromo-4-ethoxypyridine of the same composition, whereas 3,5-dibromo-4-ethoxypyridine is converted into 3-bromo-4-ethoxypyridine as main product, together with 2,3,6-tribromo-4-ethoxypyridine.

    2,3,5-Tribromo-4-ethoxypyridine is transformed in both media into 2,3,6-tribromo-4-ethoxypyridine and in liquid ammonia also debrominated to 2,5-dibromo-4-ethoxypyridine.

    In order to check the validity of the mechanisms proposed several experiments were carried out, viz. the action of potassium amide on mixtures of two substrates. The results are in good agreement with the schemes given and corroborate the introduced reaction mechanisms. Although in nearly all the reactions studied the bromine migration is an intermolecular process, there is an indication of an intramolecular transformation in the isomerization of 3,5-dibromo-6-deutero-4-ethoxypyridine into 2,5-dibromo-6-deutero-4-ethoxypyridine of nearly the same deuterium content.

    The above was an incentive to investigate the base-catalyzed bromine shift in bromo-chloro-4-ethoxypyridines in both media. The reaction of 3-bromo-2-chloro-4-ethoxypyridine proceeds analogously to that of 2,3-dibromo-4-ethoxypyridine. 2-Bromo-3-chloro-4-ethoxypyridine was found to be the only example of a substrate from which a bromonium ion is abstracted from C-2 in the pyridine nucleus, producing 3-chloro-4-ethoxypyridine and 2,6-dibromo-3-chloro-4-ethoxypyridine. From 3-bromo-5-chloro-4-ethoxypyridine together with the last-mentioned products 2-bromo-5-chloro-4-ethoxypyridine is formed.

    Finally it is emphasized that several isomerizations described may be used for the synthesis of compounds otherwise not easily accessible (chapter 5).

    Inwerking van kaliumamide op enige 15N-gemerkte 4-halogeenpyrimidinederivaten
    Valk, J. de - \ 1973
    Wageningen University. Promotor(en): H.C. van der Plas. - Wageningen : Landbouwhogeschool - 44
    ammoniak - isotopen - stikstof - kalium - pyrimidines - derivaten - chemische reacties - ammonia - isotopes - nitrogen - potassium - pyrimidines - derivatives - chemical reactions
    Aan de hand van de in de inleiding gegeven voorbeelden is het duidelijk dat tal van gesubstitueerde halogeenpyrimidinen, in reactie gebracht met een geschikt nucleofiel reagens, aanleiding kunnen geven tot het optreden van ringopeningsreacties. De daarbij gevormde, vaak instabiele open-ketenverbindingen kunnen hetzij verder reageren door een ringsluiting dan wel overgaan in meer stabiele open-ketenverbindingen.

    De vorming van (1.31) in de reactie van (1.29) met lithiumpiperidide in piperidine deed ons besluiten de aminering van (1.29) met kaliumamide in vloeibare ammoniak opnieuw aan een onderzoek te onderwerpen. De overweging die hierbij een rol speelt, was de volgende. Het amide-ion zou, op analoge wijze als het lithiumpiperidide in de reactie met (1.29) op de 2-plaats van het pyrimidinemolecuul kunnen aanvallen. Een daaropvolgende verbreking van de C 2 - N 3 band levert dan een open-ketenverbinding (1.33) op, die echter hierin wezenlijk verschilt van de piperidinoverbinding (1.31) dat door additie van de aminogroep in (1.33) over de drievoudige band van de nitrilfunctie ringsluiting kan optreden, waarbij 4-amino-6-fenylpyrimidine (1.34) ontstaat. Daar bij een aminering volgens het zojuist beschreven mechanisme een van de ringstikstofatomen van (1.29) zich na de reactie in de aminogroep buiten de ring zal bevinden, is het mogelijk d.m.v. is N-gemerkte moleculen na te gaan of de aminering van (1.29) via dit mechanisme (geheel dan wel gedeeltelijk) verloopt dan wel via een mechanisme, waarbij de ring niet wordt geopend.

    Het mechanisme, verlopend via een intermediaire open-ketenverbinding, blijkt zowel in de aminering van (1.29) als in de aminering van diverse andere gesubstitueerde 4-halogeenpyrimidinen een belangrijke rol te spelen. Voorgesteld is dit nieuwe substitutie-mechanisme het A(additie)N(nucleofiel)R(ring) O(opening)R(ring)S(sluiting)-mechanisme te noemen (ANRORS- mechanisme).

    In dit proefschrift wordt het onderzoek beschreven naar het voorkomen van het ANRORS-mechanisme in de aminering van diverse gesubstitueerde 4-halogeenpyrimidinen met kaliumamide in vloeibare ammoniak; tevens wordt ook aandacht geschonken aan het optreden van dit mechanisme in de aminering van een halogeen bevattend bicyclisch systeem, n.l. 4-chloorchinazoline.

    De resultaten van de aminering en het voorkomen van het ANRORS-mechanisme in de aminering van de vier 4-halogeen-6-fenylpyrimidinen zal worden beschreven in hoofdstuk II, terwijl in hoofdstuk III de invloed van substituenten op dit ANRORS-mechanisme aan de orde zal komen . In hoofdstuk IV zijn resultaten bijeengebracht van een 14C-onderzoek dat als doel had meer informatie te verkrijgen over de structuur van de in dit basisch milieu niet te isoleren open-ketenverbindingen. Het onderzoek naar het voorkomen van het ANRORS-mechanisme in de aminering van 4-chloorchinazoline staat beschreven in hoofdstuk V. In hoofdstuk VI zullen de in dit proefschrift beschreven resultaten worden besproken en vergeleken worden met analoge, in de literatuur vermelde reactietypen.

    Metasubstituties bij inwerking van sterke basen op gesubstitueerde 2-halogeenpyridinen
    Boer, H. - \ 1973
    Landbouwhogeschool Wageningen. Promotor(en): H.J. den Hertog. - Wageningen : Veenman - 89
    aminen - chemische reacties - gehalogeneerde koolwaterstoffen - organische halogeenverbindingen - pyridines - amines - chemical reactions - halogenated hydrocarbons - organic halogen compounds - pyridines

    In this thesis an investigation is described on the mechanisms of aminations of derivatives of 2-halogenopyridines with potassium amide in which part of the reaction products contain the amino group occupying the position meta to the leaving halogen atom. Special attention is paid to the possibility that 2,4-didehydropyridine derivatives occur as reaction intermediates.

    In the introduction a survey is given of all types of reactions recorded in the literature, occurring when halogenopyridines are treated with strong bases. From stability calculations of the various didehydropyridines the conclusion can be drawn that in some reactions 2,4-didehydropyridines might act as intermediates (Chapter 1).

    Subsequently the various procedures for carrying out reactions and the analytical methods used are described (Chapter 2).

    First results are given of experiments on the amination of some monohalogenopyridines with potassium amide in liquid ammonia, occurring via 3,4-didehydropyridine as an intermediate, in the presence of other nucleophiles in order to test whether these substances might act as 'didehydro scavengers' (Chapter 3).

    Thereupon results of reactions of a series of 2-halogeno-6-ethoxypyridines with potassium amide in liquid ammonia with or without didehydro scavengers are reported. 2-Chloro-6-ethoxypyridine reacts very slowly with potassium amide producing about equal amounts of 2-amino-6-ethoxypyridine and 4-ethoxy-2- methylpyrimidine. This pyrimidine derivative is obviously formed analogous to 4-phenoxy-2-methylpyrimidine from 2-bromo-6-phenoxypyridine with potassium amide: the amide ion attacks the substrate at carbon atom 4, then the ring is opened between carbon atoms 3 and 4 to an intermediate which by ring closure turns into a pyrimidine derivative. From 2-bromo- and 2-iodo-6-ethoxypyridine 2-amino- and 4-amino-6-ethoxypyridine were obtained as main products. The aminations of the bromo and the iodo compounds mentioned above were also carried out in different reaction conditions i.e. various concentrations of potassium amide and in the presence of several didehydro scavengers. From the results obtained, the probability of a route via 6-ethoxy-2,4-didehydropyridine as intermediate is discussed (Chapter 4).

    Amination reactions of several 3-substituted 2-bromo-6-ethoxypyridines are described in the next chapter. In some experiments didehydro scavengers were used.

    When reacting 2-bromo-3,6-diethoxypyridine and 2-bromo-3-N,N-dimethyl-amino-6-ethoxypyridine with potassium amide in liquid ammonia the product with the amino group on carbon atom 4 appeared to be formed together with the 2-amino compound. In the presence of a didehydro scavenger products containing the substituent derived from this scavenger at the 2- and the 4-position are formed from these substrates together with the amino derivatives. It is likely that in these aminations 2,4-didehydropyridines are involved as intermediates. Likewise, on reacting 2-bromo-6-ethoxy-3-fluoropyridine with potassium amide 2- and 4-amino-6-ethoxy-3-fluoropyridine are formed. In the presence of the thiophenoxide ion as a didehydro scavenger no other products are found. Thus, here a combination of addition-elimination processes is supposed to occur (Chapter 5).

    In investigations on the aminations of some 5-substituted 2-bromo-6-ethoxypyridines it was established that the 2-amino derivatives and never the 4-amino compounds are formed. From the 5-ethoxy, 5-N,N-dimethylamino and 5-amino derivative in each reaction the 2-amino derivative is obtained as sole product. In the presence of the 3-oxo-2-pentyl anion the following results were obtained:

    In 2-bromo-5,6-diethoxypyridine the 3-oxo-2-pentyl group is introduced into the 2-position. Furthermore 1,3-dicyano-1-ethoxy-4-ethyl-1,3-hexadiene is formed as a by-product.

    From 2-bromo-5-N,N-dimethylamino-6-ethoxypyridine only 5-N,N-dimethylamino-6-ethoxy-2-(3-oxo-2-pentyl)pyridine results.

    5-Amino-2-bromo-6-ethoxypyridine gives in the presence of 3-pentanone no other products than those formed when the ketone is not added to the reaction mixture.

    Thus, whereas didehydro reaction intermediates may be involved to a certain extent in the aminations of 2-bromo-5,6-diethoxypyridine and 2-bromo-S-N,N- dimethylamino-6-ethoxypyridine, the reaction of 5-amino-2-bromo-6-ethoxypyridine probably proceeds by an addition-elimination mechanism only.

    2-Bromo-6-ethoxy-5-fluoropyridine gives a totally different reaction mixture on amination. The main product in this case is 4-ethoxy-5-fluoro-2-methylpyrimidine, formed according to the mechanism playing a role in the amination of 2-chloro-6-ethoxypyridine (Chapter 6).

    The next chapter (7) gives a survey of the aminations of some 3-ethoxy-2-halogenopyridines. Information has been obtained that the reactions of both 2-bromo-3-ethoxy- and 3-ethoxy-2-iodopyridine might occur via 3-ethoxy-2,4-didehydropyridine as an intermediate.

    Finally the results mentioned in the preceding chapters are listed and some general conclusions are drawn (Chapter 8).

    Inwerking van kaliumamide op enige chloorpyrazinen
    Lont, P.J. - \ 1973
    Landbouwhogeschool Wageningen. Promotor(en): H.C. van der Plas. - Wageningen : [s.n.] - 59
    amiden - chemische reacties - organische chloorverbindingen - pyrazinen - cum laude - amides - chemical reactions - organochlorine compounds - pyrazines
    Sinds 1960 wordt in het Laboratorium voor Organische Chemie te Wageningen het gedrag van halogeenmonoaza- en halogeen-1,3-diaza-aromaten t.o.v. sterke nucleofiele reagentia, met name kaliumamide in vloeibare ammoniak en lithiumpiperidide in piperidine, bestudeerd. Uit dit onderzoek is gebleken dat afhankelijk van het soort reagens, de aard van het halogeenatoom en de plaats van het halogeen in de heterocyclische kern, een groot aantal verschillende reactietypen kunnen optreden: normale substituties, cine-substituties, koppelingsreacties, halogeenmigraties, ringopeningen al of niet gevolgd door ringsluitingen.

    In verband met het door ons uitgevoerde onderzoek naar het gedrag van chloorpyrazine en enige derivaten van deze verbinding met kaliumamide in vloeibare ammoniak, zal in deze inleiding aan de hand van een aantal literatuurvoorbeelden de belangrijkste aspecten van de tot nu toe bij halogeenmonoaza- en halogeendiazaaromaten gevonden reactietypen worden besproken.

    Het blijkt dat de vele soorten reacties, die door inwerking van het kaliumamide op halogeenpyridinen en halogeenpyrimidinen kunnen plaatsvinden, ingedeeld kunnen worden in twee groepen:

    a reacties, waarbij het kaliumamide door zijn sterk basische eigenschappen in staat is een proton aan het halogeenhoudende substraat te onttrekken (§ 1.1.) en

    b reacties, waarbij het kaliumamide vanwege zijn nucleofiel karakter aan het heterocyclische substraat addeert, al of niet op plaatsen waar zich het halogeenatoom bevindt (§ 1.2.).

    Aminering van hydroxyderivaten van halogeenazahetarenen
    Roelfsema, W.A. - \ 1972
    Landbouwhogeschool Wageningen. Promotor(en): H.J. den Hertog. - Wageningen : Veenman - 57
    pyridines - piperidinen - derivaten - chinolinen - chinoline - indolen - chemische reacties - aminen - pyrrool - pyridines - piperidines - derivatives - quinolines - quinoline - indoles - chemical reactions - amines - pyrrole

    In this thesis an introductory investigation is described on the reactivity of hydroxy derivatives of halogenopyridines and a bromohydroxyquinoline towards strong bases.

    It is a sequel to earlier work on the effect of substituents present in the nucleus of halogenopyridines on the course of aminations of these substances (chapter 1).

    Halogenohydroxy compounds were reacted with potassium amide or lithium piperidide, whereupon products were isolated, identified and determined quantitatively. Mechanisms were suggested interpreting the results obtained. Details about amination procedures and analyses of reaction mixtures are given in chapter 2.

    When various halogenohydroxypyridines are reacted with potassium amide in liquid ammonia at -33°C, substitutions, cine-substitutions and ring contractions occur.

    It was shown that part of the bromohydroxypyridines are aminated according to the elimination-addition (EA)mechanism via hydroxy-3,4-didehydropyridines as intermediates. The addition of ammonia to the triple bond in the intermediate is directed by the hydroxy group. Depending on this effect one or two aminohydroxy compounds are formed. Thus, cine-substitution takes place exclusively in the transformation of 3-bromo-5-hydroxypyridine via 5-hydroxy-3,4-didehydropyridine into 4-amino-5-hydroxypyridine. The amination of 4-bromo-2-hydroxypyridine follows a more complicated pattern, presumably involving two isomeric hydroxydidehydropyridines. It leads to the formation of a mixture of 3- and 4- amino-2-hydroxypyridine (chapter 3).

    From both 2- and 3-bromo-4-hydroxypyridine, 2-amino-4-hydroxypyridine is formed. These reactions can be explained, assuming divergent AE-processes (chapter 4).

    The amination of 2-bromo-6-hydroxypyridine takes place without rearrangement. Continued investigation is required to establish whether this process follows the AE pathway.

    Remarkable results were met when studying the amination of 2-chloro-3-hydroxypyridine, 2-bromo-3-hydroxypyridine and three derivatives of the latter. These substances change into pyrrole derivatives. In contrast to the reaction of 3-amino-2-bromopyridine with potassium amide yielding 3-cyanopyrrole, the halogeno-hydroxypyridines are converted into pyrrole carbonamides, in which the substituent occupies the 2-position. A 3-substituted pyrrole derivative was obtained from our substrates however, when lithium piperidide was used as a reagent. Together with this product (i.e. pyrrole-3-carbopiperidide), 3-hydroxy-2-piperidinopyridine is obtained. The mechanism of the reactions of 2-halogeno-3- hydroxypyridines and those of the corresponding amino and methyl compounds are discussed (chapter 5).

    Ring contraction takes place too in the amination with potassium amide, of 4-bromo-3-hydroxyquinoline i.e. a derivative of 4-bromo-3-hydroxypyridine from which no hydrogen bromide can be abstracted leading to the formation of a 4,5-didehydro compound. In this reaction oxindole is the product. Thus, along with the ring-fission a fragment of the chain is split off containing one carbon atom, yielding cyanide.

    By the action of lithium piperidide or of piperidine at elevated temperature on 3-bromo-4-hydroxyquinoline the ring system remains unaltered. In the former case 3-hydroxy-4-piperidinoquinoline is formed as a substitution product, in the latter a cine-substitution results in the formation of 3-hydroxy-2-piperidinoquinoline.

    Mechanisms were proposed for the reactions of 4-bromo-3-hydroxyquinoline; they were compared again with those, suggested for the aminations of the corresponding amino compound (chapter 6).

    nieuw apparaat voor de bepaling van ruwe celstof; Bereiding van het uranylreagens volgens Kahane door verwerking van uraanresten
    Kampen, G.B. van; Westenberg, L. - \ 1936
    's-Gravenhage : [s.n.] (Verslagen van landbouwkundige onderzoekingen no. 42,10) - 8
    analyse - koolhydraten - celmembranen - celwanden - cellulose - chemische reacties - chemie - samenstelling - uitrusting - voer - verwerking - uranium - analytische scheikunde - chemische proceskunde - chemische processen - analysis - carbohydrates - cell membranes - cell walls - cellulose - chemical reactions - chemistry - composition - equipment - feeds - processing - uranium - analytical chemistry - chemical engineering - chemical processes
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