|M.Sc Thesis||Department of Chemistry|
|Supervisor:||Prof. Keinan Ehud|
An active site nucleophilic lysine residue with a highly perturbed pKa is an essential element of the catalytic machinery that is available to both the natural, type I aldolase enzymes and the aldolase antibodies that were elicited by reactive immunization with 1,3-diketones. These, chemically programmed antibodies were shown to catalyze aldol reactions with broad substrate scope, enantioselectivity and high reaction rates, as well as retroaldol, decarboxylation, alkylation, b-elinmination and deuteration reactions.
The catalytic antibodies provide us an opportunity to learn the details of their catalytic machinery, particularly in comparison with their naturally occurring counterparts. The aldolase antibodies, which accept a wide scope of substrates allow for mechanistic studies that could not be preformed with the natural aldolases. Identification of enzyme-substrate intermediates could be achieved by using various reactions as mechanistic probes.
In order to study the initial steps of the antibody-catalyzed aldol mechanism we used the benzoin oxidation reaction to the corresponding benzils by potassium ferricyanide. Antibody 24H6, which accepts large substrate molecules, catalyzes oxidation reactions that are not catalyzed by other aldolase antibodies. These properties allowed for a Hammett correlation study of this reaction using a series of substituted benzoin substrates.
In this work we show that the aldolase antibody, 24H6, effectively catalyzes the oxidation reaction of the substituted benzoins to the corresponding benzils. Linear free energy relationship studies suggests that the rate-determining step of the catalyzed reaction is the Schiff-base to enamine tautomerization, while in the uncatalyzed reaction the rate-limiting step is the formation of the enol intermediate. The dependence of the binding constants on the Hammett sigma values support the assumption that the substrate-antibody binding involves covalent bond formation.