Design and Synthesis of Photoreleasable Ubiquinol and its Biologically Active Analogues

Author: Wang, Guangyang

Year: 1999

Degree: Dissertation (Ph.D.)

Advisor: Hoffmann, Michael R.

Committee Members: Okumura, Mitchio; Rees, Douglas C.; Anson, Fred C.; Hoffmann, Michael R.

Option: Chemistry

DOI: 10.7907/str8-et07

Abstract

Every organism contains a respiratory chain that converts energy from food molecules into adenosine triphosphate (ATP) which drives a multitude of biochemical reactions. A respiratory chain achieves this via a series of integral membrane protein complexes that produce a transmembrane proton gradient as a result of sequential electron transfers through these complexes. Ubiquinol and ubiquinol oxidase enzymes are important components of the respiratory chains in most aerobic organisms. The energy from the oxidation of ubiquinol is used to promote the generation of an electrochemical gradient across the cytoplasmic or mitochondrial membrane for ATP synthesis.

Study of the catalytic mechanisms of ubiquinol oxidase enzymes is very difficult due to the structural complexity of these proteins. Since electron input from ubiquinol plays a very important role in enzyme function, to understand the detailed enzymatic mechanism of these enzymes, it is critical to understand the kinetics of individual electron transfer events in enzymatic turnover. On account of the rapidity of electron transfer in these proteins, traditional stopped-flow methods for following the kinetic course of electron transfer reactions are limited, due to the millisecond order of mixing dead time.

Photochemical initiation of ubiquinol release, a method of circumventing the mixing limitation, is investigated. The method is based on the photolysis of small organic protecting groups, or "cage" compounds. These cage compounds rendered inactive quinol-cage complex prior to photolysis by linking to the key functional groups in ubiquinol or by generating a large steric hindrance that blocks ubiquinol binding. Photolysis of the cage-quinol complex rapidly releases the two-electron donor and triggers on the enzymatic electron transfer. Such a complex can be used not only in enzymatic electron transfer study, but also in time-resolved protein conformation change study.

The cage compounds that have been studied for this purpose are based on 3',5'- dimethoxybenzoin (DMB). Esters of DMB are known to photolyze rapidly (on the sub-nanosecond time scale), generating the parent acid and the inert photoproduct 5,7- dimethoxy-2-phenylbenzofuran. A water-soluble derivative of 3', 5'-dimethoxybenzoin (DMB),3',5'-bis (carboxylmethoxy)benzoin (BCMB) was linked to the hydroxy group in ubiquinol via a carbonate linkage. Such a complex has been measured to yield a photorelease rate of 990s-1 in detergent solution. Another cage compound, N-hydroxypyridine- 2-thione, was also used to "cage" ubiquinol via a carbonate linkage. In the presence of efficient hydrogen donor, such as mercaptoethanol, such a complex has the potential to photolytically generate ubiquinol in sub-millisecond time scale.

A series of ubiquinol analogues were synthesized with a carboxylic acid functionality attached to benzoquinone at different positions. These analogues can be "caged" by BCMB via an ester bond with the potential to trigger electron transfer in sub-microsecond.

In the mean time, efforts have been made to elucidate the controversial photolysis mechanisms of benzoin compounds by synthetic substitution study and quantum chemistry calculation. Very efficient synthetic schemes for the preparation of large-size combinatorial libraries of benzoins were developed.

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