So our protocol allows us to prepare enantiopure epoxy fatty acids, and this method might help advance research on the mechanisms by which these metabolites act in the body. Historically, pure epoxy fatty acids are very difficult to obtain. So our protocol is simple, rapid, cost-effective, and it allows us to prepare significant quantities of these metabolites.
Yes, we developed a protocol for DHA, but it also works very well for EPA and arachidonic acid. Work quickly, as some of the reagents are air-sensitive, specifically DHA and NADPH, and be sure to pair everything in advance so that setup is simple. Also, be aware of the hazards.
Our protocol uses techniques in both enzymology and organic chemistry. Visualization is critical to aid readers and viewers who aren't familiar with the specialized techniques in these fields. To inoculate, use a sterile loop or pipette dipped in glycerol stock of pBS-BM3 transfected DH5-alpha E.coli into five milliliters of sterile LB broth supplemented with 0.5 milligrams of ampicillin.
Incubate the cell culture in a shaker at 37 degrees Celsius for 24 hours at 200 rpm. Then, add the five-milliliter starter culture and 100 milligrams of ampicillin to one liter of sterile LB broth in an Erlenmeyer flask. Shake at 37 degrees Celsius for six hours at 200 rpm, then at 30 degrees Celsius for 18 hours at 200 rpm.
After that, collect the cell culture into centrifuge tubes, and centrifuge the cell culture at four degrees Celsius for 10 minutes at 1, 000 times g. Pour off the supernatant into another flask. Decontaminate with bleach or using the autoclave, and store the cell pellet at minus 78 degrees Celsius until enzyme purification.
To perform cell lysis, first thaw the cell pellet on ice, and resuspend in 40 milliliters of ice-cold solubilization buffer containing Tris, PMSF, and EDTA. While on ice, sonicate the cells for one minute with an ultrasonic homogenizer, with an output power setting of 10 and duty of 100, followed by a one-minute break on ice in order to lyse the cells. Repeat this procedure six times.
Centrifuge the cell lysate at four degrees Celsius for 30 minutes at 11, 000 times g to pellet the cell debris. To perform affinity chromatography, in a cold room or refrigerator with a sliding door, at four degrees Celsius, first prepare a strong anion exchange chromatography column by washing the resin with five column volumes of buffer A of 10-millimolar Tris at pH 7.8. Next, add the supernatant of the cell lysates to the equilibrated column, and wash the column with three column volumes of cold buffer A.Then, elute the BM3 by washing the column with six column volumes of cold buffer B of 10-millimolar Tris and 600-millimolar sodium chloride.
Collect the reddish-brown eluent fraction. If the protein is not being used immediately, mix it with an equal volume of glycerol, and flash-freeze with liquid nitrogen. Store the frozen solution at minus 78 degrees Celsius.
To perform epoxidation of DHA, first determine the concentration of the thawed BM3 enzyme, according to the carbon monoxide dithionite spectral assay method. Then, in a beaker containing two liters of reaction buffer being stirred on a magnetic stir plate, add 0.94 millimoles of DHA diluted in 18.8 milliliters of DMSO and the BM3 enzyme diluted to 20 nanomolar. While the solution is stirring, begin the epoxidation reaction by adding 8.7 milliliters of reaction buffer containing 0.94 millimoles of NADPH.
Stir the reaction for 30 minutes while bubbling air into the reaction mixture with an air-filled balloon attached to a syringe and needle taped to the inside of the beaker. After that, draw up one microliter of the reaction mixture, and put it into a NanoDrop spectrophotometer to measure the absorbance at 340 nanometers. Use water as the blank.
If there is no remaining absorbance, which indicates NADPH has been consumed, the reaction is complete. Quench the reaction mixture by slowly adding one-molar oxalic acid dropwise, until the pH of the solution reaches approximately four. Now, extract the quenched buffer solution with two liters of anhydrous, peroxide-free diethyl ether.
Use a separatory funnel to shake and separate the layers, and collect the ether into an Erlenmeyer flask. Repeat two additional times, and add approximately 50 grams of anhydrous magnesium sulfate to dry the ether. On a vacuum filter funnel, pour the dried ether to filter the magnesium sulfate, and transfer the filtered ether layer to a round-bottom flask of the rotary evaporator.
Adjust the rotation speed to medium, and start the vacuum to concentrate the liquid to yield crude EDP residue. Load the residue into a 40-gram silica cartridge to perform flash column chromatography using an automated flash purification machine. Start at 10%ethyl acetate in hexanes, and ramp up to 60%ethyl acetate in hexanes over 22 minutes.
The fractions are automatically collected in tubes. Then, combine the fractions from each peak into a round-bottom flask, put on the rotary evaporator, and start the evaporation. Three fractions are obtained, DHA, EDP isomers, and diepoxide.
To esterify the obtained 0.151 grams of epoxide, in a round-bottom flask or small vial, add two milliliters of anhydrous methanol and three milliliters of anhydrous toluene, and add a stir bar. Connect the flask with a balloon filled with argon through a rubber septum and a needle, and add 0.26 milliliters of two-molar TMS-diazomethane through a syringe while stirring. Wait 10 minutes, and add an additional 05 milliliters of TMS-diazomethane until a pale yellow color appears.
After 30 minutes, carefully concentrate the mixture using the rotary evaporator, and use a 40-gram silica gel column cartridge on the flash chromatography machine to purify the residue. Elute with an isocratic concentration of 4%ethyl acetate in hexanes over 22 minutes. Collect the fractions containing the purified 19S, 20R-EDP methyl ester eluates first, followed by 16S, 17R-EDP methyl ester.
Now, assess the fractions by thin-layer chromatography in an eight-to-one hexane-to-ethyl-acetate mixture. Stain with potassium permanganate to check for any mixed fractions containing both isomers. Concentrate the fractions corresponding to each peak on a rotary evaporator, and then weigh on a balance to determine the amount of EDP esters obtained.
If mixed fractions remain, re-chromatograph them with the same solvent system as previously used. Concentrate each fraction containing the individual regioisomer on a rotary evaporator. To convert individual EDP methyl ester regioisomers to their acid forms, dilute the EDP ester in a small vial with approximately 0.7 milliliters of THF per 0.1 millimoles of ester, along with 0.175 milliliters of water.
Cool the vial to zero degrees Celsius in an ice bath, add three equivalents of lithium hydroxide solution, and stir on a stir plate under argon overnight while warming to room temperature. In the morning, quench the reaction by slowly adding formic acid, until the pH of the mixture reaches three to four. Then, add water and two milliliters of ethyl acetate to separate the layers.
Extract the water layer with 10 milliliters of ethyl acetate three times, wash with 20 milliliters of saturated brine solution, and add several grams of anhydrous sodium sulfate in the solution to dry the ethyl acetate layer. Concentrate the ethyl acetate solution using the rotary evaporator, add 10 milliliters of hexanes, and concentrate again. Repeat twice to azeotropically remove residual formic acid.
Purify the residue by flash column chromatography, eluting with 10 to 30%ethyl acetate over 15 minutes. Concentrate the desired fractions in the rotary evaporator, and dry in vacuo to afford the purified acid. The flash column chromatogram obtained upon purification of the crude mixture from enzymatic epoxidation is shown here.
Three major peaks were obtained, eluting in the order of, first, unreacted DHA, second, mixture of EDP isomers, and, third, diepoxide, which are normal over-oxidation products. Following esterification and separation of the regioisomers, the proton NMR spectra indicate that high-purity 16S, 17R-EDP and 19S, 20R-EDP methyl esters were obtained. Chiral HPLC confirmed enantiomeric purity of the acid forms of the EDPs.
Be sure to keep your broth sterile while handling and inoculating to avoid contamination. The epoxidation can be used for other fatty acids. So we also tried it with arachidonic and eicosapentaenoic acids, and we obtained good yields of those enantiopure epoxides, so 14, 15-EET and 17, 18-EEQ.
So a protocol that allows researchers to access epoxy fatty acids might aid in research on the biological functions of these compounds, which could lead to their use as drug leads. The hazardous chemicals are PMSF, which is hazardous by contact, and TMS-diazomethane, which is very toxic by contact and inhalation. So the latter should be used in a fume hood and with proper personal protective equipment.
