The overall goal of the following experiment is to synthesize a functionalized BSP peptide using solid phase techniques. This is achieved by loading a protected BIS amino acid onto hydroxyethyl benzoic acid resin. Then deep protection coupling cycles are repeated to attach functionalized bis amino acids, a process that elongates the BIS peptide and displays desired functionality.
Finally, the first bis amino acid is modified with an alpha amino acid in order to cleave the BIS peptide from the resin by DI Keto pyrazine formation. Results are obtained that show the successful synthesis of a functionalized BSP peptide in good crude purity and consistent isolated yields of approximately 10%based on liquid chromatography mass spectrometry analysis. The implications of this technique extend towards parallel and library synthesis of bis peptides because the BIS peptides are assembled on a solid resin, which is more easily manipulated than reactions in solution and byproducts are easily removed by filtration.
Visual demonstration of this method is critical as many of the steps are difficult to learn because there are many important details necessary to ensure that all of the reactions approach completion as closely as possible. With solid phase synthesis, there is no way to purify intermediates. So in order to obtain a clean final product, every reaction must be driven as far as it can.
We recommend creating a checklist of experimental procedures to avoid confusion since the steps are repetitive in nature. This protocol begins with loading of the first BSP peptide, the protection of the first BSP peptide and simultaneous resin capping. To start load the first BSP peptide by weighing 114 milligrams of H-M-B-A-A resin into an eight milliliter reaction vessel and add a magnetic stir bar.
Cap the top of the vessel with the rubber septum and purge the tube with Argonne for at least five minutes. In the meantime, prepare the activated BIS amino acid solution as described in the written protocol. Accompanying this video, transfer the solution to the reaction vessel via syringe and stir under Argonne overnight the following day, remove the septum and drain the reaction mixture.
Wash the resin by adding approximately two milliliters of chloro methane to the resin. Stir for 30 seconds to a minute and then drain. Repeat this process four times with DCM and then wash the resin five times with dimethylformamide.
To perform deep protection of the first bis amino acid and simultaneous resin capping slowly add two milliliters of a solution of 33%hydrogen bromide in acetic acid and DCM to the reaction vessel. Allow to stir for 15 minutes after draining and washing the resin five times in DCM. Repeat the D protection sequence once more.
Next, wash the resin five times in DCM followed by five times in DMF, neutralize the resin by washing twice with the 5%volume, volume solution of D-I-P-E-A in DMF. Then wash five times with DCM and five times with DMF once more. Now coupling of the protected functionalized bis amino acid can be performed.
First, reintroduce an inert atmosphere to the resin containing reaction vessel by washing three times with anhydrous DCM. Then attach a septum and argonne line purge and wash the vessel by adding one to two milliliters of anhydrous DCM and stirring for 30 seconds. Then drain the vessel until the argonne line bubbler begins to rise.
Repeat this process at least once more. Next, prepare a solution of functionalized bis amino acid in a flame dried test tube under Argonne atmosphere. Add 47 microliters of DIC and stir for 90 minutes.
Then add 35 microliters of D-I-P-E-A in 666 microliters and hydrus DMF to the resin and stir for an additional five minutes. Transfer the pre activated BIS amino acid solution to the vessel via syringe and stir overnight the following day. Drain the reaction mixture and wash twice with anhydrous DCM while under argon.
A critical step in this procedure is the biz amino acid couplings. To ensure di keto piperazines ring closures, we employ longer reaction times with additional activating agents. To promote the closure of the di keto pyrazine, add an HOAT and DIC solution stir under argon for one hour.
Next, remove the septum and drain the reaction mixture. Wash the resin five times with DCM and five times with DMF. This section covers the deep protection of the functionalized BIS amino acid, the deep protection of F moc and the acylation of the first BIS amino acid.
To perform deep protection of the protected functionalized bis amino acid, slowly add two milliliters of a solution of TFA and TIPS to the reaction vessel and stir for one hour after draining, wash the resin with DCM for about 30 seconds and then drain repeat DCM wash five times. Then repeat the entire deep protection resin washing sequence following A-D-C-M-D-M-F wash. Neutralize the resin by washing twice with a 5%volume volume solution of D-I-P-E-A in DMF.
Then perform another D-C-M-D-M-F wash. From here, the BIS peptide can be elongated with other functionalized bis amino acids or functionalized on the leading nitrogen carboxylic acid or both. To de protect the FM group, add a two milliliter solution of 20%pipe paridine in DMF and mix the reaction for 20 minutes.
Drain and wash the resin five times in DMF. Repeat this process once more following an additional wash of the resin. A isolate the first bis amino acid by adding the prepared amino acid solution to the reaction vessel and stir for six hours as a final step.
Wash the resin five times with DCM and five times with DMF. The final portion of this protocol includes removal of the Bach group from the resin bound amino acid and cleavage from the resin. First, add two milliliters of a one-to-one T-F-A-D-C-M solution to the reaction vessel and stir for 30 minutes.
Drain and wash the resin five times in DCM. Then repeat this process once more. Wash and drain the resin for 30 seconds, five times with DCM and five times with DMF.
Next, add two milliliters of a solution of 10%D-I-P-E-A in anhydrous DMF and stir for 24 to 48 hours. Finally, collect the reaction mixture into a pre weighed round bottom flask. Transfer 30 microliters of this solution to 450 microliters of Tetra Hydro FU in an lc MS file and submit it for analysis.
Wash the resin with additional aliquots of DMF and collect into the round bottom flask. The following methods may be used to monitor on resin chemical transformations throughout the synthesis. To detect free hydroxyl groups, perform the methyl red test by first removing approximately one milligram of dry resin via disposable pipette.
Rinse into a four milliliter reaction vessel. Add a methyl red solution prepared as described in the text and stir for five to 10 minutes. Drain and wash the resin five times with DCM orange or red resin beads indicate the presence of free hydroxyl groups.
This test can be used to assess the loading of the first bis amino acid and resin capping steps. To detect secondary means, perform the chloral test by transferring approximately one milligram of dry resin into a small vial via disposable pipette. Add three drops of both 0.8 millimolar chloral in DMF solution and 2%acid aldehyde in DMF solution.
And let's sit at room temperature for five to 10 minutes. In this case, blue or purple resin beads are an indication that free secondary means are present on the resin bound compound. To confirm activation efficiencies perform the activation trap test activated compound during the synthesis can be assessed by transferring five to 10 microliters of the activated solution to a liquid chromatography mass spectrometry vial containing 50 microliters of olaine mixed by hand.
For a few seconds, the solution should become yellow, then dilute with 450 microliters of tetra hydro uran and submit for lc MS analysis. The final product and activated intermediates can be assessed using an LCM S system equipped with a C 18 reverse-phase column and a solvent system of water acetyl nitrile with 0.1%formic acid. This L-C-M-S-U-V trace provides an example of the good purity of the crude product.
The major peak in the spectrum found at 21.3 minutes is found to have a mask consistent with the expected product mass. This can be visualized in the mass spectrum of the crude product, peak revealing major peaks corresponding to the mass, plus the mass of the sodium ion, as well as the mass minus the mass of the IV DDE protecting group. Shown here is the LCMS UV trace of the purified spectra revealing a very pure product at 21.3 minutes and the mass spectrum confirming the identity of this purified trier peak.
After watching this video, you should have a good understanding of how to use solid face techniques to synthesize functionalized bis peptides after its development. This technique paved the way for researchers in the field of biz peptides to explore applications in protein-protein interactions and catalysis Once mastered. This technique can lead to the synthesis of a functionalized biz peptide trimer in four to five days if it is performed properly.
When attempting this procedure, it's important to remember to keep the beads immersed in the reaction and washing solutions Following this procedure. Other methods like parallel or library synthesis can be performed in order to answer additional questions like the effect of stereochemistry or alternate functionalities on protein binding or catalytic activity. Don't forget that working with organic reagents can be extremely hazardous and precautions such as the use of proper personal safety equipment and a fume hood should always be taken when performing this procedure.
