ペプチド第三級アミドライブラリのスプリット·アンド·プールの合成とキャラクタリゼーション

The overall goal of this procedure is to demonstrate how to synthesize combinatorial libraries containing peptide tertiary amid or PTA units. This is accomplished by first preparing a PTA sub monomer from natural amino acids. The second step is to synthesize a peptide linker region on a solid support.

Next, a combinatorial library containing PTA and peptide subunits is synthesized. The final step is to characterize the synthesized library to ensure the quality of the synthesis. Ultimately, a combinatorial library containing confirmational restricted PTA subunits is synthesized and such a library can be used for high throughput screening to provide protein ligands and lead to new drug discovery.

So the chemistry that is the subject of this article was developed in in an attempt to try to find molecules that were much stiffer than some of the peptides and other things we'd worked with previously. The idea is that if one identifies libraries of stiffer molecules and they'll bind to protein targets with much higher affinities, and we thought this was very, very important in trying to develop drugs moving forward or, or even interesting probe molecules. Visual demonstration of this PTA suborder synthesis is really critical as the synthesis is hard to learn because the temperature and the timing control are really critical to a successful synthesis.

First, add 8.9 grams of dine and 11.9 grams of potassium bromide to a 500 milliliter three neck round bottom flask with a magnetic stir bar. Then add 100 milliliters of a previously prepared 30%hydrogen bromide solution to the flask. After placing the flask in a negative 10 degrees Celsius ethylene glycol, dry ice bath bubble argon through a long needle from the bottom of the flask for 10 minutes.

Stirred the solution with the magnetic stir bar at 300 RPM. Following this, add a previously prepared sodium nitrite solution to a pressure equalizing dropping funnel attached to the three neck, round bottom flask and seal it with a septum. Slowly open the valve of the dropping funnel, allowing the sodium nitrite solution to drip into the flask.

Control the valve to adjust the dripping rate to approximately two drops per second. After the sodium nitrite addition is complete, keep stirring the solution for three more hours, allowing the temperature to warm up from negative 10 degrees Celsius to room temperature. If the color of the solution is too dark, apply a vacuum to remove the excess nitrogen oxides and possible bromine generated during the reaction.

Once the solution has been transferred to an extracting funnel, extract the product three times with 35 milliliters of dathyl ether. After combining the organic phase and washing with saturated brine, collect it into a flask and dry over sodium sulfate for six hours. Following filtration of the sodium sulfate, evaporate the solvent under vacuum to yield a clear to pale yellow oil.

Purify the crude product by silica gel column chromatography with three to one hexane ethyl acetate for isotopic labeling dissolve 300 milligrams of L alanine with 10 milliliters of deuterated water in a 50 milliliter polyethylene tube. After adding 10 milligrams of alpha ketoglutarate as a cos substrate, warm up the tube to 37 degrees Celsius. When finished, adjust the pH to 8.5 to 8.7 using a one molar sodium dute oxide solution and pH test strips.

Next, add 0.1 milligrams of alanine transaminase to the solution. Place the tube in a 37 degrees Celsius incubator and incubated overnight with mild shaking at 10 to 30 RPM. At this point, swell one gram of 90 micron, 10 to gel beads with Ram linker in 10 milliliters of dimethylformamide for three hours in a 12 milliliter syringe reactor with mild shaking, drain the dimethylformamide from the reactor and add 10 milliliters of 20%pi Perine dimethylformamide solution to de protect the FOC group from the rink amid linker.

After shaking the beads with the 20%Pepperdine solution for 30 minutes, wash them five times with dimethyl forma meat to remove all of the Pepperdine. Next, remove a few beads from the syringe and test them with a chloral test. Add two milliliters of a two molar bromo acetic acid dimethylformamide solution to the beads and shake gently.

Then add two milliliters of a two molar diop propyl carbo diamine dimethylformamide solution to the beads. After sealing the syringe with a plunger, shake the beads on a shaker for 10 minutes. Once the beads have been washed thoroughly with dimethylformamide, add two milliliters of a previously prepared one molar methoxy ethyl dimethylformamide solution to them.

After shaking the beads and washing them five times with dimethylformamide, check a few of the beads with a chloral test. Following this, add 10 milliliters of a one-to-one di chloral methane dimethylformamide solution to the previously used syringe. Split all one gram beads evenly into three five milliliters syringe reactors using a 1000 microliter pipette with a truncated pipette tip.

After washing the three syringes three times with chloro methane wash the r and s labeled syringes three times with anhydrous tet hydro furin, and the B labeled syringe three times with dimethylformamide. For tristine coupling of bromo propan NOIC acid, add approximately 200 milligrams of tristine to a vial in a fume hood. Once the vial has been capped, weigh the amount of tristine in the vial.

Then add the appropriate amount of anhydrous tetra hydro ferran to the vial to make a 20 milligram per milliliter Tri phosgene tetra hydro ferran solution. To prepare the bromo acids tri phosgene mixture, add 89 microliters of R two Bromo propan NOIC acid and S two Bromo propan NOIC acid D four in two small vials separately to each vial, add five milliliters of the tristine tetra hydro furan solution. After sealing the vials, place them in a negative 20 degrees Celsius freezer for 20 minutes.

In the meantime, add 1, 125 microliters of a two to one tetra hydro furan di isopropyl amine solution to syringe r and s. Separately, add 356 microliters of 2 4 6 trimethyl purine to each of the vials containing the cooled bromo acids phosgene mixtures yielding white precipitates. Immediately apply the corresponding suspension to directly to the ified beads, and then place them on a shaker to shake under 120 RPM for two hours.

For bromo acetic acid coupling with di isopropyl carbo diamine, add five milliliters of a two molar bromo acetic acid dimethylformamide solution to syringe B.After shaking, add five milliliters of a two molar di propyl carbo diamine dimethylformamide solution to the same syringe and shake gently following this place syringe B on the same shaker as syringe r and s. Then shake the syringes for two hours after thoroughly washing the syringes with di chloro methane and dimethylformamide. Pool all the beads from syringes R, s and B into one 12 milliliter syringe reactor.

Once the beads have been washed five times with dimethylformamide, add 10 milliliters of a one-to-one di chloro methane dimethylformamide solution to the syringe. Split all the beads evenly into seven individual two milliliter syringes using a 1000 microliter pipette with a truncated pipette tip for emanation, add five milliliters of seven previously prepared two molar amine solutions to the corresponding syringe. Incubate all seven syringes in a 60 degree Celsius incubator with shaking overnight Following incubation.

Wash the beads that need to be cleaved five times with di chloral methane. After shaking the beads 15 minutes and washing them again with di chloro methane, drain the di chloro methane from the syringe. Then transfer each individual bead into a 96 well plate.

Using a light microscope and a pipette with a truncated tip cover the 96 well plate with a cover slip and place it in the negative 20 degrees Celsius freezer for 15 minutes. Once the plate has been removed from the freezer, add 20 microliters of a previously prepared cooled one-to-one TFA DI chloro methane solution to each of the wells that contains a bead. Replace the cover slip and place the 96 wall plate on a shaker back in the negative 20 degrees Celsius freezer.

After shaking for 20 minutes, remove the 96 wall plate from the freezer and peel off the cover slip. Blow dry the di chloro methane from each well by blowing air over it. When cleaved under room temperature using a 50%TFA DI CHLORO methane solution, significant degradation is observed.

Peaks 593 and 484 correspond to the linker and PTA trimer respectively showing that the whole molecule was successfully synthesized on bead but degraded during cleavage. When cleaved under low temperature conditions as described above, the amount of TFA induced degradation is greatly suppressed. The cleavage mechanism has been described in previous literature and it is believed to go through an olaine intermediate PTA molecules can be sequenced by Ms.MS, and the fragmentation pattern is similar to peptides and peptides.

PTA molecules synthesized with S two bromo propole acid D four generally give broader peaks in MS and msm. Ms.Spectra due to the presence of incomplete duration products such as S two bromo propan, NOIC acid D three, this could be used as an indication of the presence of the R Chiral center during the sequencing procedure, PTA molecules also tend to form more ated adduct than peptide peptide. Therefore, low sodium water and plastic apparatus are preferred.

Another potential byproduct is the acrylamide formed from the bromine elimination during amination. Once the acrylamide is formed, the sequence is terminated. This can be solved by lowering the primary amine concentration to one molar in order to reduce the solution.

Basicity Once mastered a good size library, can be prepared in two days if it's performed properly. Now that this chemistry has been fully developed and and the synthesis is quite effective, I hope that other researchers will adopt this powerful technology to make conformationally constrained peptide mimetics for drug development and probe development.