Diamine Oxidase: Kinetic Studies and Use in Organic Synthesis

Equi, Angela Mary (1991) Diamine Oxidase: Kinetic Studies and Use in Organic Synthesis. PhD thesis, University of Glasgow.

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The subject of this thesis is the enzyme diamine oxidase and four main topics are discussed: (1) oxidation of diamines using diamine oxidase; (2) inhibition of diamine oxidase; (3) stereochemistry and regiochemistry of the reactions catalysed by diamine oxidase; and (4) applications of diamine oxidase. (1) Oxidation of diamines using diamine oxidase. Diamine oxidase catalyses the oxidative deamination of diamines to their corresponding aminoaldehydes (Scheme A). H2N(CH2)nNH2 + H2O + O2 →Diamine Oxidase → H2N(CH2)n-1CHO + H2O2 + NH3 Scheme A n = 4 (i) n = 5 (ii) Putrescine (i) and cadaverine (ii) are the best substrates of diamine oxidase. A-Alkylputrescines and C-alkylcadaverines were synthesised and tested as substrates of diamine oxidase using an improved spectrophotometric assay. The assay involves the measurement of the hydrogen peroxide produced as a by-product of the enzymic reaction. From this assay Km and Vmax values were obtained for the oxidation of these substrates using diamine oxidase. The KM is a measure of the strength of the enzyme-substrate complex and determines the binding efficiency of the substrate to the enzyme. The Vmax is the maximal rate and is related to the turnover number of an enzyme. Analysis of these results provided information on the steric constraints of the active site. Also studied were a,w-diamines with chain lengths varying from two to twelve. Analysis of these results showed that the best binding (lowest KM value) was observed with diamines with chain lengths from five to seven and the highest Vmax value was obtained with cadaverine (ii) as the substrate (chain length five). These results suggest that formation of a cyclic diamine intermediate with the enzyme is essential for recognition and catalysis. (2) Inhibition of diamine oxidase. The oxidative deamination of diamines by diamine oxidase is a key step in polyamine metabolism. Polyamines are known to be essential for cell growth and replication. Inhibitors of the reaction catalysed by diamine oxidase should have a considerable effect on the polyamine metabolism and therefore on cell growth. Substrate analogues [eg 3,3-dimethylcadaverine (iii)] were examined as inhibitors of diamine oxidase. Compounds resembling substrates although with no primary amine groups present [eg 1,6-bis(A-piperidyl)hexane (iv)] were also tested as inhibitors. These tests were carried out using the same spectrophotometric assay as before. Most of the compounds did inhibit the diamine oxidase catalysed reaction and were shown to be competitive inhibitors. Ki values were obtained for the compounds tested as inhibitors. (3) Stereochemistry and regiochemistry of the reaction catalysed by diamine oxidase. The stereochemistry and regiochemistry of most enzymic reactions are controlled. As not a great deal is known about the stereochemistry or regiochemistry of the oxidative deamination of diamines catalysed by diamine oxidase studies were carried out to find out more. Analysis of the products of the reaction with C- alkylcadaverines was performed to determine any selectivity from the reactions. Isolation of the products was achieved by trapping the imines from the enzymic reaction using 3,4-dimethoxybenzoylacetic acid (v). The compounds examined for selectivity in the diamine oxidase catalysed oxidation were (a) 3-methylcadaverine; (b) 2-methylcadaverine; and (c) 3-phenylcadaverine. With the first two selectivity does occur although it is not yet clear if this is due to the enzyme catalysed reaction or the second reaction involving the coupling with the B-keto acid (v). Surprisingly, no product was obtained with 3-phenylcadaverine. (4) Applications of diamine oxidase. Enzymes can often be used to catalyse reactions which are difficult to carry out by other methods. The main advantages of using enzymes in synthesis are the mildness of the reaction conditions and also the possible control of the stereochemistry and regiochemistry. Diamine oxidase catalyses the oxidation of diamines to their corresponding aminoaldehydes for which there are no chemically convenient methods. Therefore the use of this enzyme in synthesis could be very favourable. The alkaloid, cryptopleurine (vi), has been synthesised using diamine oxidase in a key step. Cryptopleurine (vi) has known anti-cancer activity. Oxidation of cadaverine using diamine oxidase and subsequent coupling to 3,4-dimethoxybenzoylacetic acid (v) formed an intermediate in the synthesis of cryptopleurine (vi). Using C-alkylcadaverines and following the same procedure, a number of intermediate analogues of cryptopleurine were formed. These alkaloid analogues when made may also possess interesting biological activity. The pyrrolizidine alkaloid, trachelanthamidine (viii), was also synthesised using diamine oxidase. Oxidative deamination of homospermidine (vii) and subsequent reduction of the likely product, 1-formylpyrrolizidine produced the alkaloid (viii).

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Organic chemistry
Date of Award: 1991
Depositing User: Enlighten Team
Unique ID: glathesis:1991-78343
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 28 Feb 2020 12:09
Last Modified: 28 Feb 2020 12:09
URI: https://theses.gla.ac.uk/id/eprint/78343

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