Cercosporin-Photocatalyzed [4+1]- and [4+2]-Annulations of Azoalkenes Under Mild Conditions
July 17th, 2020
New routes for the synthesis of nitrogen-containing heterocycles utilizing cercosporin as a metal-free photocatalyst were developed.
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.
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Overview
This study presents a method for the biosynthesis of cercosporins through microbial fermentation, which can then be utilized to synthesize nitrogen-containing heterocycles. This approach offers a green and mild alternative for organic synthesis, bridging microbial fermentation and synthetic processes.
Key Study Components
Area of Science
- Organic Chemistry
- Microbial Fermentation
- Photocatalysis
Background
- Nitrogen-containing heterocycles are crucial for many bioactive natural products.
- They serve as synthetic precursors for agrochemicals and pharmaceuticals.
- Cercosporin acts as a metal-free photocatalyst in this synthesis.
- The method emphasizes environmentally friendly practices in organic synthesis.
Purpose of Study
- To develop new synthetic routes for nitrogen-containing heterocycles.
- To demonstrate the use of cercosporin in a sustainable synthesis process.
- To connect microbial fermentation with organic synthesis techniques.
Methods Used
- Microbial fermentation for cercosporin biosynthesis.
- Preparation of alpha-Halo-N-acyl-hydrazone as a synthetic step.
- Utilization of methanol as a solvent in the synthesis process.
- Demonstration of procedures by laboratory technician Yan Zhang.
Main Results
- Successful biosynthesis of cercosporins through microbial fermentation.
- Demonstrated the feasibility of synthesizing nitrogen-containing heterocycles.
- Highlighted the advantages of using green and mild conditions.
- Established a method that bridges fermentation and organic synthesis.
Conclusions
- The developed method provides a sustainable approach to organic synthesis.
- Cercosporin serves as an effective photocatalyst in the synthesis process.
- This research contributes to the field of organic chemistry and sustainable practices.
Frequently Asked Questions
Executive Industry Relevance
This method bridges microbial fermentation and organic synthesis to produce nitrogen-containing heterocycles under mild, sustainable conditions. It enables early-stage target validation by providing access to bioactive scaffolds relevant to drug and agrochemical discovery. The approach supports predictive confidence through reproducible, metal-free photocatalytic pathways.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Enables interrogation of therapeutic hypotheses via synthesis of nitrogen-containing heterocycles as bioactive scaffolds.
- Operational Value: Uses metal-free cercosporin photocatalyst derived from fermentation, reducing reliance on precious metals.
- Predictive Value: Supports functional target validation through accessible heterocyclic libraries for screening.
Screening & Assay Development
- Scientific Value: Generates quantifiable heterocyclic products (e.g., 1,2,3-thiadiazoles, tetrahydropyridazines) for structure-activity relationship analysis.
- Operational Value: Delivers reproducible yields under mild LED irradiation, supporting assay standardization.
- Scalability: Fermentation-derived cercosporin allows batch-to-batch consistency for screening campaigns.
Translational & Preclinical Research
- Translational Value: Produces heterocycles that serve as precursors for agrochemicals and drugs, enabling continuity to preclinical evaluation.
- Mechanistic De-risking: Clarifies structure-function relationships in nitrogen-rich scaffolds under physiologically compatible conditions.
- Predictive Confidence: Facilitates lead identification by providing diverse, synthetically accessible heterocyclic cores.
Pipeline & Workflow Integration
The method integrates fermentation biology with downstream synthesis, positioning it from early discovery through lead identification stages.
- Discovery Biology: Supports hypothesis testing by generating diverse nitrogen heterocycles for target engagement studies.
- Screening: Enables assay-ready compound preparation with consistent purity and yield under mild conditions.
- Analytics: Provides NMR- and HPLC-verifiable outputs for quantitative comparison across reaction conditions.
- Translational Research: Connects biosynthetic heterocycles to drug-like molecules for preclinical progression.
- Enterprise Reuse: Cercosporin production via fermentation creates a reusable, sustainable photocatalytic platform.
Operational & Enterprise Impact
- Scientific Value: Reduces mechanistic ambiguity in heterocycle synthesis through defined photocatalytic pathways.
- Operational Value: Ensures reproducibility via standardized fermentation and LED-driven photoprocesses.
- Strategic Value: Improves capital efficiency by lowering catalyst cost and enabling milder process conditions.
- Portfolio Impact: Supports risk-adjusted prioritization of heterocyclic scaffolds in early discovery.
Implementation Considerations
- Requires expertise in microbial fermentation and photochemical reaction setup.
- Needs LED irradiation equipment and anaerobic/oxygen-controlled Schlenk tube handling.
- Demands cross-team standardization between fermentation and synthetic chemistry teams.
- Involves adaptation considerations for substrate scope across azoalkene derivatives.
- Includes practical limitations such as substrate-dependent yields and purification via TLC and evaporation.
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