Oxidation of N-alkyl and N-aryl azaheterocycles by free and immobilized rabbit liver aldehyde oxidase

S.A.G.F. Angelino

Research output: Thesisinternal PhD, WU

Abstract

Aldehyde oxidase isolated from rabbit liver is studied in this thesis with regard to its application in organic synthesis. The enzyme has a broad substrate specificity towards azaheterocycles and therefore offers great potential for profitable use.<p/>The oxidation of <em>1-alkyl(aryl)-3-aminocarbonylpyridiniwn chlorides</em> by aldehyde oxidase shows that reaction can occur in principle at two different positions in the pyridinium ring. Only the 1-alkyl-1,6-dihydro-6-oxo-3-pyridinecarboxamides are obtained with the 1-methyl, 1-ethyl and 1- <em>n</em> -propyl derivatives. The corres ponding 4-oxo compound is found as sole product with the 1-Oxidation of N-alkyl and N-aryl azaheterocycles by free and immobilized rabbit liver aldehyde oxidase-butyl analogue, while the 1- <em>i</em> -propyl derivative yields a mixture of 4- and 6-oxo compounds. Evidence for the presence of a hydrophobic region in the vicinity of the enzymic active site has been acquired from the kinetic data of the oxidation reactions (chapter 2). Oxidation of the 1-aryl analogues results predominantly in 1-aryl-1,6-dihydro-6-oxo-3-pyridinecarboxamides, together with the corresponding 4-oxo compounds as minor products. In general the site of oxidation is determined by steric factors, although the maximum rate of oxidation by aldehyde oxidase is very sensitive to electronic effects. A more electron-withdrawing aryl substi tuent increases the reaction rate for oxidation at C-6. Consequently a positive ρ-value of about 3.6 is calculated for free aldehyde oxidase, indicating that a nucleophilic attack is the rate-limiting step in the oxidation mechanism of these azinium compounds. The oxidation of the 1-aryl derivatives by bovine milk xanthine oxidase yields mainly 4-oxo products. The maximum rate of oxidation with xanthine oxidase is only slightly affected by the nature of the aryl substi tuent (chapter 3). <em></em><p/><em>1-Alkyl(aryl)quinolinium chloridess</em> are also oxidized by aldehyde oxidase essen tially at two positions, C-2 and C-4. The site and the maximum rate of oxidation are dependent on the size and the steric conformation of the N-substituent. The presence of an aminocarbonyl group at C-3 directs the oxidation completely to C-4, irrespective of the size of the N-substituent (chapter 5).<p/>A comparison is made between the site(s) of oxidation of several azinium compounds by aldehyde oxidase and the covalent amination pattern in liquid ammonia. It is shown that covalent amination is particularly valuable as a model reaction with those substrates in which the enzyme reaction is predominantly controlled by steric factors. Thus the oxidation positions of the 1-alkyl-3-aminocarbonylpyridinium chlorides are predicted quite accurately by the model reaction (chapters 4 and 5).<p/>N-Methyl and N-benzyl derivatives of <em>2(1H)- and 4(3H)-pyrimidinone</em> (at N-1 or N-3) are oxidized at the same site by aldehyde oxidase. Despite the difference in steric size only one type of product has been obtained from all substrates studied, viz. the corresponding N-1 or N-3 substituted uracil. The maximum rates of oxidation with this enzyme exhibit an optimum in the pH range 6.5-7.8, which is little affected by the site and the size of the N-substituent (chapter 6).<p/>A study of the immobilization of aldehyde oxidase to several supports by various methods in order to make continuous operation feasible and to improve the enzyme stability, was undertaken. This showed that of the various matrices and coupling methods tested, the activity of aldehyde oxidase is best retained upon adsorp tion to modified Sepharose matrices. It is established that the storage stabili ty of enzyme adsorbed to n-alkylamine-substituted Sepharose 4B or diethylamino ethyl Sepharose 6B is significantly lower in comparison to free enzyme. The operational stability of the immobilized enzyme preparation, however, has improv ed substantially compared to soluble enzyme, although the corresponding pro ductivity is still very poor. The inactivation of aldehyde oxidase during turnover is dependent on the rate of oxidation. Coimmobilization of catalase and/or superoxide dismutase provides a further increase of the operational stability and productivity. A positive effect on both parameters is also found for aldehyde oxidase/n-alkylamine Sepharose 4B preparations by increasing the amount of enzyme adsorbed per unit weight of support (chapter 7). The aldehyde oxidase/Sepharose preparations were used throughout this study to perform small scale syntheses (chapters 2, 3, 5 and 6). Despite the interesting oxidative capabilities, it is concluded that the appli cation of aldehyde oxidase as an immobilized biocatalyst in organic synthesis is still unattractive at present and awaits further research on the stabilization of this enzyme.
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • van der Plas, H.C., Promotor
  • Mueller, F., Co-promotor, External person
Award date3 Feb 1984
Place of PublicationWageningen
Publisher
Publication statusPublished - 1984

Keywords

  • oxidoreductases
  • aldehyde oxidoreductases

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