自己組織化ペプチドのポリアミンによる両親媒性物質 (Ppa の) と関連生体材料の合成のための安易なプロトコル

This method can answer key questions on how to decorate peptide amphiphiles to polyamines and on the use of octagonal protecting groups The main advantage of this technique is that you can generate hybrid peptide amphiphiles with unprotected chemical building blocks containing multiple reactive sides. Demonstrating the procedure will be Mehdi, Nathalia and Krishnaiah, laboratory students from the Conda Sheridan Laboratory. First, carefully weigh out 2-Chlorotrityl chloride resin place the resin in a fritted, medium porocity, synthesis vessel with 50 milliliter capacity.

Next, affix the synethesis vessel to a variable speed mechanical shaker and turn the vessel to a 45 degree angle to maximize agitation. Then add 15 milliliters of DCM to the resin. After allowing the resin beads to swell for 15 minutes, add eight equivalents of the desired polyamine to the resin, and allow it to react for five hours.

After five hours, perform a kaiser test to confirm a successful reaction of the polyamine to the resin. Following this, protect the primary amine group by adding four equivalents of DDE in anhydrous methanol and shaking the reaction mixture overnight. On the following day, perform a kaiser test to confirm successful protection by the absence of blue color from the resin bead.

Then, drain the DCM and wash the resin twice with a two to one mixture of DCM and DMF. Now, add 20 equivalents of Boc Anhydride in DCM to the resin and allow the reaction to proceed for three hours. After performing a chloranil test to confirm protection of the secondary amine, drain the solvent mixture and wash the resin twice with a two to one mixture of DCM and DMF.

Following this, add 10 milliliters of a 2%solution of hydrazine in DMF to the resin. After shaking for one hour, perform a kaiser test to confirm successful deprotection of the primary amine. Next, mix four equivalents of the Fmoc protected amino acid, 3.95 equivalents of HBTU, and 15 equivalents of DIPEA.

Add a one to one mixture of DCM and DMF and sonicate the cocktail until complete dissolution. Make sure the amino acid, coupling agent, and DIPEA are truly mixed and activated before adding the mixture to the resin. After waiting three to five minutes to ensure carboxylic acid activation, add the reaction mixture to the vessel containing the resin and allow the reaction to proceed for two to four hours at ambient temperature.

Perform a kaiser or chloranil test at every step to confirm successful coupling. After performing a kaiser test to confirm successful coupling, deprotect the Fmoc group from the amino acid by adding 10 milliliters of a 20%solution of 4-Methylpyridine in DMF. Once the reaction is finished, perform a kaiser test to confirm successful deprotection of the amino acid.

Then, wash the resin twice with 10 milliliters of DMF each wash lasting for 5 minutes, and finally with 10 milliliters of DCM for 10 minutes. After coupling all the required amino acids, conjugate the hydrophobic tail to the last amino acid by adding 10 equivalents of the desired carboxylic acid functionality to 9.5 equivalents of HBTU, and 12 equivalents of DIPEA. Sonicate the cocktail until complete dissolution, then add the cocktail to the vessel.

Carry out the reaction for at least five hours, although it is advisable to carry it out overnight for the highest yield. Wash the resin with eight milliliters of DMF for two minutes, and twice with eight milliliters of DCM for five minutes. Before each addition, drain the solvent from the vessel.

Once the last wash is performed, dry the resin under vacuum for 15 minutes. To prepare 15 milliliters of cleavage cocktail, add 14 milliliters of TFA to 0.5 milliliters of water, and 0.5 milliliters of Triisopropylsilane. Add this cleavage cocktail to the resin, and shake for two to four hours at room temperature.

After the cleavage reaction is complete, collect the solution in a 50 milliliter round bottom flask. Then, concentrate the TFA in vacuo to one to two milliliters using a rotary evaporator at reduced pressure, while heating the mixture at 40 degrees Celsius After evaporation, add the obtained TFA solution, dropwise, to a round bottom flask containing 15 milliliters of anhydrous cold ether to precipitate the PPA. Next, add 5 milliliters anhydrous cold ether to the original flask containing the concentrated TFA solution.

Sonicate to recover additional solids. Then, combine with the ether solution from the previous step. Cover the flask and place it inside the refrigerator overnight to maximize precipitation.

On the following day, collect the precipitated material by vacuum filtration using a fine or medium pour size center disk filter funnel. Finally, wash the precipitate twice with five to ten milliliters of cold ether to remove any residual organics. The HPLC trace and MALDI Spectrum confirm the presence of the PPA product which should have a purity of greater than 95%for material characterization or biological evaluation.

A single sharp peak is observed in the UV based HPLC trace and the MALDI Spectrum corresponds to that of the calculated molecular weight of the PPA within plus or minus one dalton. The self assembly of the PPAs can be visualized and analyzed by transmission electron microscopy, atomic force microscopy, small angle X-Ray scattering, scanning electron microscopy, and dynamic light scattering. Successful self assembly will result in well defined nanostructures, in both transmission electron microscopy and atomic force microscopy.

Once mastered, this protocol can be done in three days if performed properly. While attempting this procedure, remember to purify the products and assess their purity before any subsequent studies. Following this procedure, other molecules, such as peptide amphiphiles, peptides, and peptide-polyamine hybrids can be prepared.

After its development, this technique helps research in the field of self assembly peptide amphiphiles to explore the influence of polyamine nano structures in areas such as drug delivery, imaging, or catalysis After watching this video, you should have a good understanding of how to carry the synthesis of polyamine-based peptide amphiphiles and related peptide amphiphiles. Trifluoroacetic acid and 4-Methylpyridine can be hazardous, and precautions such as wearing gloves, lab coats and working in the fume hood should be taken while performing this procedure.