Cercosporin-Photocatalyzed [4+1]- and [4+2]-Annulations of Azoalkenes Under Mild Conditions

This protocol can be used for the biosynthesis of cercosporins through microbial fermentation, which in turn can be used to synthesize nitrogen-containing heterocycles in the green and mild conditions. An advantage of this technique is that it can be used as a bridge between the microbial fermentation methods and the organic synthesized processes. Nitrogen-containing heterocycles are not only important skeleton for many natural product with bioactivities, but they are synthetic precursors for many agrochemicals and drugs.

Demonstrating the procedure with Philippe Icyishaka will be Yan Zhang, a technician from my laboratory. For alpha-Halo-N-acyl-hydrazone preparation, weigh out 10 millimolar of ketone and 10 millimolar of benzoyl hydrazine into a flask. Add 20 milliliters of methanol to the flask, and equip the flask with a stir bar and a rubber stopper.

Slowly inject 250 microliters of hydrochloric acid into the mixture, and incubate the flask in air at room temperature. After one hour, collect the precipitate after reaction by filtration, and wash the product with acetone. Then dry the precipitate by vacuum, and identify the product by NMR.

To prepare cercosporin, charge a three-liter shake flask with one liter of S-7 medium, and inoculate the cercosporin-producing strain into the shake flask. Culture the mixture under light conditions at 135 revolutions per minute and 25 degrees Celsius for two weeks. At the end of the incubation, use a vacuum pump to subject the fermentation broth to vacuum filtration, and extract the pellet and the supernatant separately with three 50-milliliter volumes of dichloromethane.

Collect the pellet for drying in a freezer dryer, and combine the organic phases. Wash the organic phases with water two to three times, and concentrate the solution under vacuum. Redissolve the residue with analytical methanol, and filter the product through a 18-micrometer, organic microfiltration membrane.

Then purify the cercosporin with a Sephadex LH-20 column, and identify the product by HPLC according to standard protocols. To prepare 1, 2, 3-thiadiazole, add the entire volume of the alpha-Halo-N-acyl-hydrazone, one milligram of cercosporin, 27 milligrams of potassium tert-butoxide, and 39 milligrams of potassium thiocyanate into a 10-millimeter Schlenk tube equipped with a rubber stopper and a stirring bar. Purge the Schlenk tube with oxygen three times before injecting two milliliters of dry acetonitrile into the tube.

Subject the tube to a five-watt, blue LED from the bottom for 16 hours before washing four times with 15 milliliters of saturated sodium chloride solution. After the last wash, combine the aqueous phase, and re-extract the aqueous phase with four 15-milliliter volumes of ethyl acetate. Combine the organic phase, and dry with anhydrous sodium sulfate.

Remove the solvent with a vacuum evaporator. To purify the product with thin-layer chromatography, draw a straight line on the silica plate, and add the dissolved product to the line. Put the silica plate in the chromatography cylinder with 10 to one PE to EA.After, dissolve the silica in DCM.

Remove the silica from the product by filtration, and dry the DCM with a rotary evaporator. Then identify the compound by NMR according to standard protocol. To prepare 1, 4, 5, 6-tetrahydropyridazine, add the entire volume of the alpha-Halo-N-acyl-hydrazone, 2.7 milligrams of cercosporin, and 195 milligrams of cesium carbonate into a 10-milliliter Schlenk tube equipped with a rubber stopper and a stirring bar.

Purge the Schlenk tube three times with nitrogen, and inject two milliliters of acetonitrile in water into the tube. Subject the Schlenk tube to a five-watt, blue LED from the bottom for 16 hours followed by four washes with 15 milliliters of saturated sodium chloride per wash. Combine the aqueous phase, and re-extract the aqueous phase with four 15-milliliter volumes of ethyl acetate.

Combine the organic phase, and dry with anhydrous sodium sulfate. Remove the solvent with a vacuum evaporator. To purify the product with thin-layer chromatography, draw a straight line on the silica plate, and add the dissolved product to the line.

Put the silica plate in the chromatography cylinder with 10 to one PE to EA.After, dissolve the silica in DCM. Remove the silica from the product with filtration, and dry the DCM with a rotary evaporator. Then identify the compound by NMR according to standard protocol.

These representative results demonstrate how 4-aryl-1, 2, 3-thiadiazoles and 1, 4, 5, 6-tetrahydropyridazines can be conveniently synthesized by cercosporin-catalyzed photocatalytic reactions from alpha-Halo-N-acyl-hydrazone. The 4-aryl-1, 2, 3-thiadiazoles acquisition procedure is suitable for substrates bearing both electron-donating and electron-accepting groups in the phenyl ring, providing the desired products with moderate to good yields. Always remember to use nitrogen and oxygen for two different categories of the reactions.