Bench, Bennie John (2009-12). Investigations of the Biological Roles of Substituted Cyclohexadienes. Doctoral Dissertation. Thesis uri icon

abstract

  • In recent years there have been two cycloterpenals, molecules consisting of a cyclohexadienal core, isolated from nature. Cyclocitral, the condensation product of citral, has been isolated from the North Sea bryozoans Flustra foliacea. In the human eyes, cycloretinal has been isolated and is a toxic by product of the vision cycle. This retinal dimer is believed to contribute to age related macular degeneration, the leading cause of blindness in the elderly. In 1992, it was discovered that if [beta]-ionylideneacetaldehyde was incubated with beta-lactoglobulin ([beta]-LG), the principal whey protein in dairy milk, that it would mediate the formation of cyclo-[beta]-ional. No follow up studies were performed on this protein mediated reaction or what biological activities these cycloterpenals may possess. This dissertation investigates the biological roles of substituted cyclohexadienes including cycloterpenals and cyclohexadiene enaminonitriles. To mimic the protein mediated reaction, we developed a synthetic procedure to produce a wide array of cycloterpenal by utilizing L-proline. Over 100 cycloterpenals were synthesized and screened for their biological activities against an array of cell based screens. The phenotypic effects of these cycloterpenals were screened against a PC12 assay where dramatic effects were observed on neurite outgrowth. During the synthesis of starting materials for the production of our cycloterpenal library, it was discovered that if excess base was added to the Horner-Wadsworth-Emmons reaction between a methyl-ketone and diethyl-(cyanomethyl)-phosphonate, conversion of the [alpha]-[beta]-unsaturated nitrile into an enaminonitrile was observed. This new synthetic procedure was optimized to generate a library of enaminonitriles as well as their quinazoline derivatives. The work within also includes the investigation of the [beta]-LG mediated reaction formation of cycloterpenals with natural and unnatural [beta]-methyl aldehydes. We were able to demonstrate that [beta]-LG could mediate the conversion of [alpha],[beta]-unsaturated aldehydes into their corresponding cycloterpenal. In vitro analysis was also performed with store bought milks and the [beta]-LG present was able to mediate the formation of cyclocitral. An in vivo experiment was also performed by utilizing New Zealand White rabbits to demonstrate the formation of cycloretinal within the blood stream by feeding a source of [beta]-LG with retinal. Interestingly, in human blood, [beta]-LG is present at concentrations of 0.7-1.2 g/dL. The protein has been identified within drusen pigments and lipofuscin granules that accumulate in the retina of macular degeneration patients. As humans do not produce beta-lactoglobulin, the source of this protein is from milk and milk products. With these experiments, we clearly demonstrate that under the appropriate conditions, cycloretinal can be produced with [beta]-LG. We have clearly established a direct link between [beta]-LG chemistry and age-related macular degeneration.
  • In recent years there have been two cycloterpenals, molecules consisting of a

    cyclohexadienal core, isolated from nature. Cyclocitral, the condensation product of

    citral, has been isolated from the North Sea bryozoans Flustra foliacea. In the human

    eyes, cycloretinal has been isolated and is a toxic by product of the vision cycle. This

    retinal dimer is believed to contribute to age related macular degeneration, the leading

    cause of blindness in the elderly. In 1992, it was discovered that if [beta]-ionylideneacetaldehyde was incubated with beta-lactoglobulin ([beta]-LG), the principal

    whey protein in dairy milk, that it would mediate the formation of cyclo-[beta]-ional. No

    follow up studies were performed on this protein mediated reaction or what biological

    activities these cycloterpenals may possess.

    This dissertation investigates the biological roles of substituted

    cyclohexadienes including cycloterpenals and cyclohexadiene enaminonitriles. To

    mimic the protein mediated reaction, we developed a synthetic procedure to produce a

    wide array of cycloterpenal by utilizing L-proline. Over 100 cycloterpenals were

    synthesized and screened for their biological activities against an array of cell based

    screens. The phenotypic effects of these cycloterpenals were screened against a PC12 assay where dramatic effects were observed on neurite outgrowth. During the

    synthesis of starting materials for the production of our cycloterpenal library, it was

    discovered that if excess base was added to the Horner-Wadsworth-Emmons reaction

    between a methyl-ketone and diethyl-(cyanomethyl)-phosphonate, conversion of the

    [alpha]-[beta]-unsaturated nitrile into an enaminonitrile was observed. This new synthetic

    procedure was optimized to generate a library of enaminonitriles as well as their

    quinazoline derivatives.

    The work within also includes the investigation of the [beta]-LG mediated reaction

    formation of cycloterpenals with natural and unnatural [beta]-methyl aldehydes. We were

    able to demonstrate that [beta]-LG could mediate the conversion of [alpha],[beta]-unsaturated

    aldehydes into their corresponding cycloterpenal. In vitro analysis was also

    performed with store bought milks and the [beta]-LG present was able to mediate the

    formation of cyclocitral. An in vivo experiment was also performed by utilizing New Zealand White rabbits to demonstrate the formation of cycloretinal within the blood

    stream by feeding a source of [beta]-LG with retinal.

    Interestingly, in human blood, [beta]-LG is present at concentrations of 0.7-1.2

    g/dL. The protein has been identified within drusen pigments and lipofuscin granules

    that accumulate in the retina of macular degeneration patients. As humans do not produce beta-lactoglobulin, the source of this protein is from milk and milk products.

    With these experiments, we clearly demonstrate that under the appropriate conditions,

    cycloretinal can be produced with [beta]-LG. We have clearly established a direct link

    between [beta]-LG chemistry and age-related macular degeneration.

publication date

  • December 2009