The overall goal of this procedure is to prepare and purify an end toal I oto iodine. This is accomplished by first generating di ito methyl lithium by deprotonation of di ito methane at low temperature and reacting with an immune. The second step is to rapidly warm the reaction mixture to promote cyclization of the di iodide intermediate to the desired I oto uridine product.
Next, the stability of the I oto Uridine product to various stationary phases used for chromatography is assessed to determine the most appropriate for purification. The final step is to purify the I oto a iodine and perform proton NMR analysis. Ultimately, this screen of stationary phases for chromatography is used to show that basic Illumina modified to activity four is most appropriate for the purification of the IDO odine.
We first had the idea of this method when we found that the new IDO iodine products were unstable to silica gel chromatography and we required an alternative purification. From here, we designed a procedure to assess the stability of the IO odine to arrange of stationary phases for chromatography, which can also be applied to other potentially sensitive compounds. Demonstrating this procedure will be Tom and Dom are both PhD students in my research group.
First flame dry, a 100 milliliter round bottom flask containing a stir bar and fitted with a septum under a stream of argonne. After allowing the flask to cool her room temperature, add 5.7 milliliters of anhydrous tetrahedran and 2.7 milliliters of anhydrous ethyl ether via syringe and 315 microliters of freshly distilled hexa ethyl dilane via a micro syringe. Place the flask in a suitably sized doer that will allow it to be well submerged.
After stirring is initiated, add dry ice and acetone to the doer to cool the solution to minus 78 degrees Celsius. Then cover the doer with aluminum foil to minimize exposure of the reaction vessel. To light following this add 0.6 milliliters of 2.5 molar and butyl lithium in hexanes dropwise via syringe over two to three minutes.
After allowing the mixture to stir at minus 78 degrees Celsius for 30 minutes. To form the lithium hexa ethyl dilane solution, add one milliliter of anhydrous terah hydro ferran to a flame dried 10 milliliter round bottom flask via syringe. Then add 135 microliters of di ITOM methane via a micro syringe and gently swirl the flask to ensure the solution is well mixed.
Next, add the di ITOM methane solution dropwise over two minutes to the solution of lithium hexa ethyl dilane at minus 78 degrees Celsius while allowing the solution to stir for 20 minutes. Weigh out 135 milligrams of the amin into another flame dried 10 milliliter round bottom flask and dissolve it in two milliliters of anhydrous tetra hydro ferran. After the 20 minutes of deprotonation time, add the immune solution dropwise to the di ito methyl lithium solution Over five minutes at minus 78 degrees Celsius.
Immediately after the dropwise edition is complete, lift the reaction vessel out of the dry ice bath. Recover the flask with aluminum foil and transfer it to an ice water bath at zero degrees Celsius. Stir the solution for 15 minutes at zero degrees Celsius.
Following this, quench the reaction by adding 30 milliliters of saturated aqueous sodium bicarbonate solution. Transfer the mixture to a separating funnel and add 30 milliliters of di chloro methane. Shake the mixture, vent the funnel and remove the lower organic layer.
After repeating the extraction two times, add sodium sulfate to the combined organic layers to remove any water present in the solution. Then filter off the sodium sulfate under vacuum filtration using a glass funnel with cotton plug, collecting the filtrate in a 250 milliliter round bottom flask, remove the solvent under reduced pressure on a rotary evaporator. To afford an impure sample of the desired ITO uridine product.
Add 1 3 5 TRIMETH oxy benzene as an internal standard to the crude dine and dissolve the crude dine sample in di chloro methane. Ensuring this is fully dissolved. Take an aliquot from this mixture, then remove the solvent under reduced pressure.
When finished, analyze the sample by proton NMR spectroscopy. Following NMR analysis, open the recorded proton NMR spectrum using standard NMR processing software such as mast nova. Right click on the spectrum and choose integration.
Then choose manual to provide the integration tool. Click and drag to cover the width of the peaks at 6.08 PP m and 4.87 PP M to integrate the signals of the internal standard and the aine CHI signal respectively. Right click on the integral for the peak at 6.08 PP m select edit integral and change the normalized value to 3.0.
Next, use the updated integral value for the iodine CHI signal to determine the yield of the I OTO iodine. Once the NMR analysis is complete, mix 25 grams of the following stationary phases with 50 milliliters of 5%ethyl acetate hexane in six separate 250 milliliter conical flasks containing stir bars in conical flask. Prepare a 50 milliliter solution, a 5%ethyl acetate heane to be used as a control.
Then stir the slurries on a magnetic stir plate. Add two milliliter aliquots of the I OTO Azarin internal standard solution to each of the conical flask set room temperature. Then stir the slurry mixtures for 30 minutes.
After 30 minutes, filter each of the slurry mixtures under vacuum using a centered funnel, collecting each filtrate in a 250 milliliter round bottom flask. Wash the residue in the centered funnel twice with 30 milliliters of di chloro methane. Remove the solvent from the resulting samples using a rotary evaporator.
Following this, analyze the samples by proton NMR spectroscopy to calculate the amount of iota odine recovered in each case. As described previously, record the yields in the laboratory notebook. Compare the yields of I OTO Aine obtained from each stationary phase tested with that obtained previously to determine the sample giving the highest yield.
Next, repeat the previously described synthesis to generate the crude IO iodine mixture to generate basic Illumina activity. Four, add 100 grams of basic Illumina activity, one to a 500 milliliter round bottom flask, and then add 10 milliliters of water to the flask. After fitting the flask with a glass stopper, shake it vigorously until no lumps are visible, indicating even spreading of water throughout the Illumina.
Once the Illumina has cooled to room temperature, purify the crude IO iodine by column chromatography using the basic Illumina activity four. As the stationary phase, elute with hexane grading to 5%YL acetate taxane. Combine the product containing fractions in a round bottom flask and remove the solvent using a rotary evaporator to obtain the pure I oto iodine.
Prior to purification, a 59%yield of I oto a iodine was calculated by proton NMR. However, this I oto a iodine was challenging to purify and underwent significant decomposition on silica purification on basic Illumina. Activity four afforded the product in 48%yield.
Proton NMR analysis gave a series of yields for the stationary phases with respect to the internal standard basic Illumina activity four afforded the highest yield of 53%which is closest to the NMR yield. Therefore, basic Illumina activity four was chosen for the end. Toal iota was IR redeemed purification.
A wide selection of ITO aines can be accessed in high yield. Both al alcohol and aromatic and toal amines are compatible with the reaction, including hysterically demanding turt and ortho. To examples, the reaction is proposed to occur by deprotonation, followed by Nucleophilic edition.
To give the amino gem di iodide intermediate subsequent warming induces a highly DIA stereo selective cyclization favoring the cis iodine. Due to subtle steric interactions in the cyclization transition state during reaction optimization, temperature control and timing were essential to the reaction outcome. Quenching the reaction without warming afforded the end toal IO iodine and the amge di iodide.
However, the products undergo degradation under the reaction conditions, which is avoided by warming the reaction and reducing the reaction times. After watching this video, you should have a good understanding of how to synthesize the IO iridium product and assess its stability to a wide range of stationary phases for purification by chromatography. Don't forget that working with mbu lithium can be extremely hazardous and precautions such as working under a nut atmosphere and safely quenching access.
Reagent should always be taken while performing this procedure.
