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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.

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    Adducten van nitrofuranen: metabolisme, uitscheidingskinetiek en analytiek
    Zuidema, T. - \ 2003
    Wageningen : RIKILT (Rapport / RIKILT 2003.022) - 56
    nitrofuranen - derivaten - vleeskuikens - blootstelling - metabolisme - furazolidon - in vitro - heterocyclische verbindingen - analytische scheikunde - nitrofurans - derivatives - broilers - exposure - metabolism - furazolidone - in vitro - heterocyclic compounds - analytical chemistry
    Synthesis and oxidation by xanthine oxidase from arthrobacter M-4 of 6-aryl-4(3H)-pteridinones and related compounds
    Meester, J.W.G. De - \ 1987
    Agricultural University. Promotor(en): H.C. van der Plas. - S.l. : De Meester - 126
    organische verbindingen - oxidoreductasen - synthese - heterocyclische verbindingen - organic compounds - oxidoreductases - synthesis - heterocyclic compounds

    In this thesis xanthine oxidase from Arthrobacter M-4 in the form of a cell-free extract or as immobilized cells has been studied with regard to its application In preparative organic chemistry. The enzyme has a broad substrate specificity towards azaheterocycles as purines and pteridines.

    The unequivocal preparation of 6-aryl-4(3H)-pteridinones and 7-aryl-4(3H)- pteridinones with different substituents at the para position of the phenyl group is described. The oxidation of these compounds by (immobilized) xanthine oxidase from Arthrobacter M-4 usually goes fast for all the studied 6-aryl-4(3H)-pteridinones as well as for 7-(pX-phenyl)-4(3H)-pteridinones (X= H and F). All the compounds of the last mentioned series with a substituent larger than hydrogen or fluoro are slowly oxidized. Oxidation only takes place at C-2 of the pteridine nucleus. No oxidation at the pyrazine site, is established. Small laboratory-scale oxidations have been carried out with cells entrapped in gelatine and crosslinked with glutaraldehyde. Based on spectral data the products of the oxidation reactions are 6- and 7-aryllumazines (chapter 2).

    By comparison of the kinetic parameters of 6- and 7-phenyl-4(3H)-pteridinones with unsubstituted 4(3H)-pteridinone the existence of a hydrophobic pocket in the vicinity of the active site has been suggested. The rate-limiting step in the oxidation of 6-aryl-4(3H)-pteridinones by the bacterial xanthine oxidase is not significantly affected by the nature of the aryl substituent. The inhibition of the oxidation of 1-methylxanthine into 1-methyluric acid by 7-aryl-4(3H)-pteridinones is sensitive to electronic factors. A positive σ-value of 0.73 has been calculated for the inhibition indicating that a more electron-donating aryl substituent increases the affinity of this compound towards the enzyme (chapter 3).

    Only 1-methyl-, 3-methyl-6-phenyl-4(3H)-pteridinone and 3-methyl-6-phenyl4-(3H)-pteridinone-8-oxide are found to be substrates althought their reactivity is still very low. The site of oxidation is not changed. All the 3-alkyl derivatives are less tightly bound to the enzyme than 6-phenyl-4(3H)-pteridinone. Introduction of the N-oxide at N-8 considerably lowers the binding of the substrates. Inhibition studies have revealed that 3-methyl-6-phenyl-4(3H)-pteridinone is a non-competitive inhibitor (Ki= 47 μM) whereas the 3-ethyl derivative is an uncompetitive one (Ki= 19.6 μM) (chapter 4).

    The purified bacterial enzyme from Arthrobacter M-4 proves to be monomeric. This is quite remarkable since the flavoproteins to which xanthine oxidase belongs are usually dimeric. The inhibitory effects of 5,6-diaminouracil and bisalloxazine on the oxidation of xanthine into uric acid by bovine milk xanthine oxidase and xanthine oxidase from Arthrobacter M-4 have been examined. 5,6-Diaminouracil is about nine times more potent as inhibitor for xanthine oxidase from Arthrobacter M-4 than allopurinol. Inhibition constants are 8.4 μM for allopurinol and 0.98 μM for 5,6-diaminouracil. Bisalloxazine has a negligible inhibitory effect on the activity of both xanthine oxidases (chapter 5).

    7-Phenyl-, 7-p-methoxyphenyl-, 7-methyl-, 7-t-butyl-, 6,7- diphenyl-, 6,7-dimethyl- and 2-phenylpteridine are converted in good yields into their respective 4-amino compounds when they are dissolved in liquid ammonia (-40°) and potassium permanganate has been added to the solution. Increase of the temperature of the amino-oxidation does not change the position of substitution, however the yields are lower. The intermediary of 4-aminodihydropteri dines in these reactions has been proven by 1H nmr spectroscopy (chapter 6).

    By comparison of the substrate specificity between bovine milk xanthine oxidase and xanthine oxidase from Arthrobacter M-4 it is concluded that the hydrophobic site of the active site of both enzymes must be different. From the kinetic data it is clear that the bacterial enzyme has a less hydrophobic site (chapter 5) than the milk enzyme and that the bacterial enzyme only can perform oxidation at the pyrimidine site (chapter 7).

    Despite the interesting complementary behaviour of the bacterial and the bovine milk enzyme towards both 6-and 7-aryl-4(3H)-pteridinones the only serious drawback for use of xanthine oxidase from Arthrobacter M-4 as an immobilized biocatalyst in heterocyclic chemistry is the low specific acitivity obtained after growing of these cells.

    Bio-organic heterocycles. Synthesis, mechanisms and bioactivity : proceedings of the 4th FECHEM Conference on Heterocycles in Bio-organic Chemistry, Houthalen, Belgium, 25-28 May 1986
    Plas, H.C. van der; Simonyi, M. ; Alderweireldt, F.C. ; Lepoivre, J.A. - \ 1986
    Amsterdam : Elsevier (Studies in organic chemistry 27) - ISBN 9780444427113 - 325
    heterocyclische verbindingen - organische scheikunde - heterocyclic compounds - organic chemistry
    On the electronic structure of free and protein-bound isoalloxazines
    Eweg, J.K. - \ 1982
    Landbouwhogeschool Wageningen. Promotor(en): F. Mueller, co-promotor(en): C. Veeger. - S.l. : S.n. - 164
    enzymen - spectroscopie - spectraalanalyse - ionisatie - heterocyclische verbindingen - foto-elektronenspectroscopie - enzymes - spectroscopy - spectral analysis - ionization - heterocyclic compounds - photoelectron spectroscopy
    The subject of this thesis, as apparent from its title, is a detailed study of the isoalloxazine molecule by a variety of techniques belonging to the field of optical spectroscopy. After a brief introduction to the subject in general terms, together with some historical background and citation of relevant review papers which appeared before 1979 (Chapter 1), a general outline of experimental techniques and theoretical methods used is given (Chapter 2). More detailed information about specific experiments or calculations is provided in the experimental sections of the individual chapters.

    Chapters 3 and 4 describe the continuous wave and time-resolved spectral properties of (iso)alloxazines determined under experimental conditions, in which external perturbation of the molecules of interest is minimized as much as possible. This could be achieved by measurements in apolar solvents (Chapter 3) and in the vapour phase (Chapter 4), a state of aggregation in which the molecule could be brought with some experimental skill. Thus, a large temperature range (77 to 520 K) could be employed. In solution at 77 and 300 K (Chapter 3), the spectra revealed a 1250 cm -1vibrational progression in all isoalloxazines investigated (probably a stretching mode); actual lifetimes of 5 - 10 ns for fluorescence and ~ 300 ms for phosphorescence (77 K, no phosphorescence could be detected in fluid solution) were observed; the ratio of the actual and radiative lifetimes of the electronically first excited singlet state agreed very well with an independent quantum yield determination and solvent interactions were found to affect primarily the Franck-Condon envelopes of the spectra and not the electronic transition energies. The non-radiative decay of isoalloxazine, on the other hand, is strongly dependent on the molecule's environment. An anomalously large Stokes-loss in fluid and glassy solution is indicative of a conformational change of the molecule occurring upon electronic excitation. In alkane solution at 77 K, isoalloxazines form clusters exhibiting P-type (triplet-triplet annihilation process) delayed fluorescence. Vapour phase spectra (Chapter 4) , although less structured than solution spectra owing to sequence congestion, provided primary information on isolated (iso)alloxazines when comparison with spectra obtained in condensed media was made. Fluorescence lifetimes range from < 0.5 ns for alloxazine to 1-18 ns for isoalloxazine vapour. The data indicate possible intramolecular complex formation between the isoalloxazine ring system and its own aliphatic side-chain carrying a (polar) hydroxyl group. Direct photodissociation of isoalloxazine in the electronically first excited singlet state is not a probable process in the vapour phase.

    Chapter 5 describes the continuation of (iso)alloxazine vapour phase experiments by the application of ultraviolet photoelectron spectroscopy. Both He(I) and He(II) excitation was used. The spectra are interpreted using various methyl substituted isoalloxazines and by comparison with the results obtained from CNDO/S calculations and photoionization cross- sections derived therefrom. The dependence of the electronic properties of isoalloxazines on the redox state and the degree of substitution is analyzed. A critical review of the data obtained from semiempirical MO calculations by various other authors and the CNDO/S results shows that a few methods give a fairly good prediction of the πorbital energies only. Without exception, the calculated σorbital energies contain considerable error. Particularly, all theoretical methods fail to predict a threefold degeneracy in the orbital level scheme at ~-9.6 eV, in which both πand σorbitals are involved. Possible reasons for the failures are discussed. Analysis of the experimental and theoretical results reveals a planar molecular conformation to be the most probable me for an isolated reduced isoalloxazine molecule in the vapour phase, contrary to the bent conformation which is encountered in solution or the solid state. Bending is, therefore, most likely caused by interaction of the molecule with its environment. Such interactions, leading to changes in orbital energies, may in part be responsible for the ability of the protein-bound flavocoenzyme to be involved in a broad diversity of biological reactions.

    In chapter 6, all foregoing data are put together in a combined analysis to establish relationships between the photoelectron and ordinary optical spectra. First, additional photoelectron spectra of 10-hydroxyalkyl-isoalloxazines confirmed the existence of the intramolecular complex proposed on the basis of vapour phase fluorescence lifetimes (vide supra). The hydrogen-bond between the side-chain OH group and the N 1 imine-like nitrogen atom (cf. Scheme 1.1, p. 1) leads to a destabilization of the lone pair on the latter atom, contrary to common experience. This anomalous behaviour can be ascribed to rearrangement of existing through- bond interactions in the free isoalloxazine molecule. Results from CNDO/S and INDO/S calculations agree reasonably with the observations. Secondly these findings have considerable impact on the interpretation of existing and in this thesis presented optical spectra. The new photoelectron spectroscopic data lead, in combination with optical spectra, to the assignment of the isoalloxazine S 2 to a mixed state containing (anomalous) ny* character arising from the anomalous behaviour of the N 1 lone pair (cf. Scheme 1.1, p.1). The oscillator strength of the
    S 0 ->S 2 π-> π* component is very sensitive to external perturbation. Finally, the possible implications for biochemical catalysis by flavins are discussed.

    Chapter 7 describes a thorough investigation of Old Yellow Enzyme (OYE), a (flavo)protein containing the isoalloxazine derivative flavin mononucleotide (M). It is attempted to interpret the spectral properties of this protein in terms of intermolecular interactions between its constituents, using the knowledge and experience acquired in the preceeding research. The results demonstrate the inadequacy of the existing explanation of the phenomena occurring upon the addition of phenols to OYE , based on the formation of a simple phenolate -FMN donor- acceptor charge transfer complex. Instead,it was found that the phenolate anion interferes strongly with an existing tight complex between FMN and the apoprotein, probably a H-bonded structure in which FMN is tautomerized and interacts with a L-chiral center. This is concluded from a separate electronic transition with an origin at 496 m, thus far not recognized as such, and the circular dichroism observed. The emission of OYE is dominated by that of free FMN, although protein-bound FMN seems also to become luminescent in glassy solution at 143 K. A second fluorescence/phosphorescence emission appears upon UV-excitation of both native and complexed OYE . This emission is quenched by the addition of phenol to OYE , shows a large (3000 cm -1) blue shift on going to a low temperature glass and is tentatively assigned to excimers of nucleic acids. Long-wavelength excitation with a synchronously pumped, mode-locked Rhodamine 6G dye laser revealed a third, extremely weak, emission in both native OYE and its complexes. It decays with ~3 ns lifetime at 143 K. ESR spectra revealed the presence of a low amount of an unpaired spin in OYE. Owing to an unusual relaxational behaviour it could only be observed below 15 K and the signal was measured in both the free enzyme and its complexes. Possible assignment and consequences of this observation are discussed. In frozen aqueous solutions of the OYE-phenolate complex, a phase transition was discovered at which the colour reverted to that of the native enzyme. Subsequent melting restored the original colour. The observed phenomena and existing literature data lead to the conclusion that the only model from which no apparent inconsistencies emerge, is that of a very complicated network of hydrogen-bonded structures in the protein. These involve several, partly unknown, chromophores. Phenols interfere with this network, leading to the formation of the long- wavelength absorption band in OYE.

    Finally, a brief postscript (Chapter 8) is given, containing an overview of recent spectroscopic literature on isoalloxazines, a discussion in response to a polemic in papers by other scientists and the ESCA (XPS) spectrum of 3,7,8,10-tetramethyl-isoalloxazine (3-methyllumiflavin).

    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.

    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).

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