Progress 09/01/05 to 02/28/08
Outputs
OUTPUTS: Broadly, the experiments conducted in the synthesis of the fungal metabolite oosporein fall into two large categories - first, addition of functional groups to toluene with dimerization intended as a later step and, second, addition of functional groups to an already dimerized quinone. As many experiments had already been attempted with aromatic precursors, the majority of experiments conducted center on using compounds that were dimerized early in the synthesis, usually in the first step, as in the case of 2,5-dimethoxytoluene/ceric ammonium nitrate (CAN). Early dimerization and oxidation proved successful. The current method of producing oosporein is a five-step synthesis with an overall yield of approximately 30% percent. Addition of functional groups to aromatic precursors. Early experiments attempted to synthesize 2,3,5,6-tetramethoxytoluene and 3,6-dimethoxy-2-methyl-1,4-benzoquinone (1B), which would then be dimerized to form precursors to oosporein.
2,6-dimethoxytoluene was reacted with 1 equivalent of bromine to produce 3-bromo-2,6-dimethoxytoluene (Fig 1C). In an effort to form a 6-bromo-3-methoxy-1,4-benzoquinone , 3-bromo-2,6-dimethoxytoluene was unsuccessfully combined with CrO3, yielding a complex mixture. 3-bromo-2,6-dimethoxytoluene was allowed to react with H2O2 in acetic acid in an attempt to add OH groups to open sites, but the reaction yielded a complex mixture.
PARTICIPANTS: Dr. Subbiah is the PI, Founder/President, and Chief Scientific Officer of PhytoMyco Research Corp., Greenville, NC; Founder/Chairman, of PhytoMyco Res. Pvt. Ltd. Mysore, India, a natural product processing and product development company, holds Adjunct Professor positions at Department of Biology, East Carolina University, Greenville, NC. and North Carolina State University, Raleigh, NC. Dr. Subbiah was Post-doctoral Research Associate at N.C. State University, Raleigh, NC, USA. and as a Matsumae International Foundation Post-doctoral Fellow at Tottori University, Japan. Dr. Subbiah received his Ph. D., M. Phil., M. Sc and B. Sc from University of Mysore, Mysore. Lori Forrest, project assistant, MS, chemistry Jeff Bonner, Project assistant, MS, Chemistry Brian Love, Ph. D. Consultant
TARGET AUDIENCES: Rust diseases represent a significant market globally for plant disease control products, particularly with the emergence of Asian soybean rust in South America and recently in the southern U.S. The cereal rusts (wheat, barley, oat, rice, corn and millets) alone is a major global issue, and the cereal rust market is well over $10 billion. Rust diseases have occurred for centuries and affect several economically important crops such as cereal grains, soybean, peanut, coffee and ornamental plants in greenhouses. And more recently have been a problem in the US with the recent reports of the tropical Asian rust pathogen of soybean. Because the fungi that cause rust diseases are transported great distances via wind, epidemics of rust disease occur frequently on local and regional scales. but PhytoMyco is poised to offer solutions that primarily address rust disease of cereals (barley, oat, and wheat) and soybean, which are major food crops in the world.
PROJECT MODIFICATIONS: No modifications, except more emphasis has been given to commercialization as well as identifying another potential opportunity for rapid commercialization: This new research can use new gene cloning technologies, combination of amplification of cDNA library by degenerate primers and enzyme purification, to clone and characterize genes encoding orcinol hydroxylase and 2,3,5-trihydroxytoluene 1,2-oxygenase, which are important enzymes involved in the oosporein biosynthesis. Oosporein is the metabolic chemical product that confers resistant to single and multiple fungal diseases in plant. Production of oosporein in cells is controlled by several enzymes.
Impacts
Five-Step Synthesis of Oosporein PURPOSE The following procedure provides a five-step synthesis for 10 grams of the fungal metabolite oosporein (3,6,3',6'-Tetrahydroxy-4,4'-dimethyl-bicyclohexyl-3,6,3',6'-tetraene -2,5,2',5'-tetraone) beginning with an aromatic monomer. SCOPE The reactions rely heavily on isolation of products as precipitates and evaporation of solvents to provide high yields of very clean products. The overall reaction yield for all five steps of this synthesis is 27.6 per cent. The following reaction procedure was developed specifically for synthesis of a 10-gram batch of oosporein. The feasibility of large scale-up has not been evaluated. Step 5 of the reaction is still in active development and is subject to change as the procedure is refined based on new experimental data. PROCEDURE 1. Synthesis of Ditoluquinone (4,4'-Dimethyl-bicyclohexyl-3,6,3',6'-tetraene-2,5,2',5'-tetraone) 1.1 Dissolve 227 g of ceric ammonium nitrate (3.5 equivalents) in 0.3
L of water. Three equivalents of CAN are required, but 3.5 equivalents are used to speed the rate of reaction. Two equivalents of 2,5-dimethoxytoluene are necessary to make one equivalent of dimerized compound. 1.2 Dissolve 36.02 g of 2,5-dimethoxytoluene in 0.3 L of acetonitrile. 1.3 Add the 2,5-dimethoxytoluene solution to the CAN solution dropwise via addition funnel. The 2,5-dimethoxytoluene solution will turn very dark purple upon entry but will then become orange/yellow. As the reaction progresses, the bright yellow product will crash out of the solution and be visible as a yellow precipitate. Stir for 5 days at room temperature in a loosely-stoppered flask. 1.4 Once the reaction is complete, pour the reaction solution into 1.5 L of rapidly-stirring water (5 times the volume of water used in step 1.1). Stir the aqueous solution for one hour at room temperature. A yellow powdery precipitate will be visible in the aqueous solution. 1.5 Subject the aqueous solution/mixture to
vacuum filtration to isolate the yellow solid. Rinse the solid with three 0.15 L portions of water to remove any excess CAN and acetonitrile. The yield for this reaction is 92-93% (26.37 g product from this example). Dry the product thoroughly before using it in step 2, as acetic anhydride reacts with water.
Publications
- Subbiah, Ven. 2007. United States Patent Application, 20070110726 Rust disease control by Aphanocladium album and/or Beauveria brongniartii Abstract There is disclosed a method and composition for controlling rust disease in plants. Metabolites produced by Aphanocladium album mycoparasites are recovered and applied in an effective amount to plants at risk for acquiring rust disease More specifically, the application of the metabolite converts infective urediniospores that cause rust disease into non-infective teliospores. In a more specific aspect, the metabolite is reacted with another substance or under a specific reaction to result in a different compound which is also effective against rust disease.
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