Studies on the use of haloperoxidases in organic synthesis

Research output: Thesisinternal PhD, WU

Abstract

The subject of this thesis is the use of haloperoxidases in synthetic organic chemistry. Haloperoxidases are enzymes capable of halogenating a variety of organic compounds. They require hydrogen peroxide and halide ions as cosubstrates. The enzymes operate under mild conditions, compared to conventional halogenating agents, resulting in increased yields and purity of products. This thesis presents some new substrates for a chloroperoxidase from the mold Caldariomyces fumago (CPO) and a bromoperoxidase from the brown alga Ascophyllum nodosum (BPO). The reaction mechanisms of both enzymes and their natural function are discussed. The immobilization of CPO is studied.

CPO catalyzes the smooth halogenation of various heterocyclic compounds. Barbituric acid and some of its derivatives give the corresponding 5-chloro or 5,5-dichloro compounds in very high yields (Ch. 2). 5-Monochlorobarbituric acid is obtained in high yield when the CPO-mediated chlorination reaction is combined with an electrochemical process. The 5,5-dichloro compound is reduced to its 5-monochloro analogue at an electrode, which is simultaneously used for the production of the cosubstrate hydrogen peroxide. The system is very efficient, as shown by the high turnover obtained for CPO (10 6-10 7) (Ch.3). Pyrazoles are also good substrates for the enzyme, giving their 4-chloro derivatives in high yields. CPO further converts 2-aminopyridine into 2-amino-3-chloropyridine and 8-hydroxyquinoline into the corresponding 5,7-dibromo compound (Ch. 4). Kinetic studies indicate that there are two possible reaction routes for CPO: I) the organic substrate binds to an oxidized enzyme intermediate and direct transfer of halogen takes place; II) the enzyme produces hypohalous acid as the active halogenating agent. The available data do not as yet permit a definitive choice of reaction mechanism. (Ch. 2).

BPO smoothly brominates barbituric acid and some of its derivatives giving the corresponding 5-bromo or 5,5-dibromo compounds in good yields. Kinetic measurements show that BPO produces free hypobromous acid which either brominates the organic halogen acceptor or reacts with hydrogen peroxide giving singlet oxygen (Ch. 5).

Relatively apolar substrates like monochlorodimedon and resorcinol are efficiently halogenated by CPO entrapped in reversed micelles. The system is composed of cetyltrimethyl ammonium halide (CTAX, X=Br or Cl), pentanol, octane and a small amount of aqueous buffer containing the enzyme and hydrogen peroxide. The CTAX serves a dual function: i) as a surfactant, it stabilizes the reversed micelle, and ii) as a supplier of the halide substrate. The reaction rates obtained are twice as high as in water (Ch. 6).

CPO can be immobilized on various solid supports, but the low stability of the enzyme-support bond and the support itself at the optimal pH of the enzyme is a serious problem. The best way to immobilize CPO seems to be the crosslinking of a mixture of the enzyme and a water-soluble acrylamide- N -acryloxysuccinimide copolymer with an α-ω-diamine. In this way CPO is bound by means of both entrapment and covalent bonds, in 40% yield (Ch.7).

Six new halometabolites have been detected by GC/MS analysis of the dichloromethane extract of the culture medium of Caldariomyces fumago. The compounds are chlorinated derivatives of ethanol, propanol-2 and 2-(4-hydroxyphenyl)ethanol. Their possible biosynthetic routes and the implications for the reaction mechanism of CPO are discussed (Ch.8).

In short, the two haloperoxidases studied here are biocatalysts which are potentially useful in synthetic organic methodology, because they perform their halogenation reactions in a very smooth and mild way. However, haloperoxidases with greater specificity in their reactions would certainly be of more advantage to the organic chemist.

Original languageDutch
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • van der Plas, H.C., Promotor
  • Laane, C., Promotor, External person
Award date9 Oct 1987
Place of PublicationS.l.
Publisher
Publication statusPublished - 1987

Keywords

  • organic compounds
  • oxygenases
  • synthesis

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