Synthesis and oxidation by xanthine oxidase from arthrobacter M-4 of 6-aryl-4(3H)-pteridinones and related 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.
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
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Landbouwuniversiteit Wageningen
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| Subjects: | heterocyclic compounds, organic compounds, oxidoreductases, synthesis, heterocyclische verbindingen, organische verbindingen, oxidoreductasen, synthese, |
| Online Access: | https://research.wur.nl/en/publications/synthesis-and-oxidation-by-xanthine-oxidase-from-arthrobacter-m-4 |
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