The overall goal of this procedure is to describe a simple and efficient method for solid phase peptide synthesis and the aqueous self-assembly of peptides into highly ordered nano sheets. Structurally similar to polypeptides peptides are in substituted glycine polymers where the side chains are attached to the nitrogen rather than the alpha carbon. The process begins by synthesizing a specific peptide sequence via other sub monomer method.
The sub monomer method consists of two simple chemical steps that introduce each monomer into the chain sequentially. First, an amine attached to a solid support is bromo acetylated. Second, the bromide is displaced with a primary amine.
These steps are repeated until the desired chain length is achieved After chain elongation, the peptide is cleaved from the solid support and the resulting crude peptide oil is purified by HPLC and characterized to initiate the self-assembly of peptides into nano sheets. A dilute aqueous buffer solution of the peptide is prepared in a glass vial and gently mixed. As a final step.
The nano sheets are examined under the microscope. The results demonstrate that an iterative cycle of two simple chemical steps can yield a sequence specific polymer that spontaneously self assembles into nano sheets. Peptides are novel class of bio-inspired polymer with properties in between those of proteins and traditional polymers.
They're robust and synthetically versatile like traditional polymers, but are also highly tuneable and sequence specific like proteins and DNA peptides are emerging as an advanced material to address key problems in biomedicine and material science, such as the identification of bioactive drugs or the design of artificial proteins. The major advantages of peptides include precise sequence control, the availability of incredibly diverse building blocks, efficient synthesis and protease stability. The general peptide manual synthesis method involving basic equipment and commercially available reagents is outlined enabling peptides to be easily synthesized in most Laboratories.
Visual demonstration of the synthetic steps is critical and serves as a valuable reference for individuals entering the field of solid phase synthesis. For the first time here, the purification and characterization of an amphiphilic peptide 36 mers described as well as its self-assembly into highly ordered nano sheets. Begin this protocol with setup of the experimental apparatus as described in the written protocol.
The following steps will be performed in a glass reaction vessel instead of a disposable polypropylene fritted cartridge for clarity. Following setup at 100 milligrams of R amide resin to a fritted reaction vessel swell the resin by adding two milliliters of dimethyl form amide or DMF agitate by bubbling for 10 minutes. Then drain the solution by vacuum to isolate the swelled resin.
Next, add one milliliter of 20%full methyl pyridine in DMF to de protect the FM O group. Agitate for two minutes and drain. Repeat this process for 12 minutes, then rinse the resin by adding two milliliters of DMF agitating for 15 seconds and draining.
Begin growing the peptide chain with the first sub monomer cycle, which includes a bromo acetylation and a displacement step. For bromo acetylation, add one milliliter of 0.6 molar bromo acetic acid in DMF and 86 microliters of di isopropyl carbo mite. After agitating for 30 minutes, drain and rinse with two milliliters of DMF.
Next, perform displacement by adding one milliliter of one to two molar amine in end methyl pyro genone incubate for 30 to 120 minutes following the incubation, drain and rinse with two milliliters of DMF. Again, continue to grow the peptide chain by repeating the sub monomer cycle. After the final displacement is done, rinse with two milliliters of DMF.
Follow this with a diora methane wash cap and store the reaction vessel until cleavage. To begin cleavage, transfer all of the dried resin to a 20 milliliter scintillation glass vial working inside a hood and using proper personal protective equipment, add four milliliters of tri fluoro acetic acid, or TFA cleavage cocktail to the scintillation glass vial and cap. Tightly stir for 10 minutes to two hours of room temperature.
Alternatively, use a rotary shaker to mix the solution. Collect the TFA cleavage solution by filtering the resin through a disposable polypropylene fritted cartridge into a new pre weighed 20 milliliter scintillation glass vial. Next, add one milliliter of fresh cleavage cocktail to rinse the resin and collect any residual peptide.
Repeat this, rinse twice. Evaporate the TFA by blowing a gentle stream of nitrogen or by using a biot charge. V 10 evaporator red dissolve the crude oil in six milliliters of one to one aceto nitrile and water.
Then freeze and lyophilize the sample. This process should be repeated once more following the second lyophilization. Record the weight of the crude product store as a dry powder at minus 20 degrees Celsius through a combination of moldy analytical HPLC and or electro spray LCMS.
The purity of the crude product and whether the desired molecular weight is present can be determined by following the procedures outlined in the text. The crude peptide mixture can then be purified with reverse phase prep HPLC according to the written procedure. This section describes the protocol to form sheets from a single chain sequence specific amphiphilic 36 me peptide.
After the peptide strand is synthesized, purified and lyophilized, the resulting white powder is dissolved in DMSO to make a two millimolar stock solution. Next, obtain a one Dr.Glass file to prepare a 20 micromolar peptide solution. First, add 445 microliters of milli Q water and 50 microliters of 10 x sheet formation buffer.
Vortex the vial to mix. Then add five microliters of a two millimolar peptide stock solution and gently swell. The resulting solution sheets are formed by the gentle agitation of the dilute aqueous peptide solution.
Slowly tilting the capped glass vial from the horizontal position to the upright position. Results in sheets gentle shaking, also yields sheets. However, the sheets tend to be smaller and with fewer straight edges for many high quality sheets.
Rotate the glass vials about the horizontal axis slowly for one day using a tube rotator or a customized rocker for fluorescent staining of nano sheets, add one microliter of 100 micromolar Nile red to 100 microliters of the nano sheet solution to obtain a final concentration of one micromolar Nile. Red is an environmentally sensitive dye whose fluorescence intensity increases when it is localized in hydrophobic environments. Next, make a 1%aros solution in hot water and pour into a plastic Petri dish.
Ensure that the aros solution is approximately one eighth of an inch thick and allow the solution to cool undisturbed on a flat surface. After the aros sets, use a spatula to cut one centimeter by one centimeter squares. Then transfer the cut squares onto a glass slide to collect the sheets in the same plane spot one microliter of sheet solution onto the piece of aros.
After two minutes, the aros should absorb the buffer leaving the sheets at the surface. Image the sheet within 15 minutes. Alternatively, to image sheets and solution load 15 microliters inside a 20 millimeter diameter 0.12 millimeter gasket on a glass slide.
Cover the sample with a cover slip and image within a few days to perform SEM of nano sheets. First plasma etch silicon chips to aid in the absorption of the nano sheets. Then drop 20 microliters of peptide sheet solution on a plasma treated silicon substrate and allow the solution to sit after three minutes, remove excess solution with the tip of a Kim wipe pipette 20 microliters of water onto the surface and again wick away the excess solution to remove buffer and salts.
Alternatively, the peptid sheet solutions are dialyzed against water to remove buffer and salt. In this case, 20 microliters of the dialyzed sheet solution can be dropped onto the plasma treated silicon substrates and allowed to air dry. After the sheet solution has dried on the plasma treated silicon substrates, the sheet can be imaged with SEM using an in lens detector at beam energies between one kilovolt and five kilovolts.
The synthesis characterization and purification of a sequence specific 36 me block charge peptide that folds into a highly ordered two dimensional nano sheet was performed. The amines using the synthesis of the 36 me block charge peptide will bok ethylene diamine to phenethyl, amine and t butyl beta alanine. After the synthesis, the crude peptide was cleaved from the resin with 95%aqueous TFA.
After the TFA solution was collected and evaporated. The crude 36 me block charge peptide was purified with reverse phase HPLC. The mass of the purified block charge peptide was then confirmed by moldy.
The observed mass 4981.2 matches closely to the calculated mass of 4981.74. The lyophilized powder was dissolved in DMSO to make a two millimolar stock solution and stored at four degrees Celsius. Sheets were prepared by the aforementioned protocol and image with fluorescence optical microscopy.
A variety of shapes with feature sizes ranging up to 300 microns are observed, and notably straight edges are prominent. SEM was also used to image the sheets similarly revealing prominent straight edges. This video describes a straightforward and efficient protocol for the solid face synthesis of peptides and the aqueous self-assembly of peptides into nano sheets.
Since literally hundreds of am mean building blocks are readily available and can be used in this protocol, the design space accessible with polypeptides is practically limitless. Peptides are promising materials for biomedicine and nanoscience research because of their synthetic flexibility, robustness, and ordering. At the atomic level in particular, the nano sheets serve as a potential platform for two dimensional display scaffolds, membrane mimetics, biological sensors, protein mimetics, And device fabrication.
Working with corrosive reagents such as trior, acetic acid, and bromo acetic acid is extremely hazardous to prevent injury, perform all reactions in a fume hood and wear proper personal protective equipment.
