A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.
As structures to increase the functionality of supermolecular structures grows, this method of incorporating 1, 2-dithiolane groups with self-assembling peptides can help answer key questions about reactivity on supermolecular surfaces. The main advantage of this technique is that the coupling and deprotection of the 1, 2-dithiolane precursor molecule occurs on resin with only a single purification step of the final modified peptide. As the time it takes the assemblies to mature into their final supermolecular structures varies, visual demonstration of the characterization methods and results for the supermolecular assemblies is critical.
To synthesize the dithiolane precursor, first dissolve one gram of 3-bromo-2-proprionic acid in approximately four milliliters of one-molar sodium hydroxide in a 50-milliliter round-bottomed reaction flask at 55 degrees Celsius with stirring. When all of the reagent has been suspended in solution, seal the reaction flask with a septa and place the flask under nitrogen atmosphere. Next, add 1.49 grams of potassium thioacetate and three milliliters of two-molar sulfuric acid to four milliliters of deionized water to create thioacetic acid in situ.
Aspirate the thioacetic acid solution into a plastic, disposable 10-milliliter syringe and equip the syringe with an 18-gauge needle, then pierce the septa with the needle to add the mixture dropwise to the reaction flask and continue the reaction overnight at 55 degrees Celsius. The next morning, monitor the reaction by thin-layer chromatography on silica gel 60 F254 plates using a mixture of methanol and dichloromethane and visualizing the reaction progress by bromocresol green stain. When the completed reaction has cooled to room temperature, acidify the mixture with two-molar sulfuric acid to a pH of one.
A yellow oil should separate out of solution, then extract the product with three 40-milliliter additions of cold chloroform and combine the organic layers for drying over magnesium sulfate, removing the chloroform under reduced pressure. The product should be a yellow oil with an overall yield of 83%and can be used without further purification. To couple the dithiolane precursor to the N-terminus of the on-resin peptide, add four equivalents of dithiolane precursor to five milliliters of dimethylformamide, four equivalents of HBTU, and 10 equivalents of DIPEA.
Allow the coupling mixture to preactivate for 10 minutes at room temperature before adding the sample to the N-terminus-resin-containing fritted syringe. Shake the coupling reaction for two hours, followed by three five-milliliter washes in fresh dimethylformamide and repeat the coupling reaction and overnight shaking. After the second coupling, wash the resin with three five-milliliter dimethylformamide washes followed by three five-milliliter dichloromethane resin washes and transfer the dried resin to a 10-milliliter microwave reaction tube.
Then, to deprotect the thioacetate group from the N-terminus dithiolane precursor, add two milliliters of dimethylformamide to the tube. Allow the resin to swell, add a small magnetic stir bar to the vessel, and resuspend the resin with low-speed magnetic stirring. After 15 minutes, add two milliliters of concentrated ammonium hydroxide to the tube, cap the reaction vessel with a silicone septa, and place the vessel in a microwave reactor for 45 minutes at 75 degrees Celsius with stirring.
When the microwave reaction is complete, transfer the resin into a clean, disposable, fritted syringe and wash the resin with two five-milliliter dimethylformamide and two five-milliliter methanol washes. After the last wash, add five milliliters of cleavage cocktail to the resin-containing syringe for a 1 1/2 hour incubation with gentle shaking at room temperature. To prepare one milliliter of crude peptide for high-performance liquid chromatography or HPLC purification, add 400 microliters of concentrated peptide stock in acetonitrile to 600 microliters of water supplemented with 0.1%of trifluoroacetic acid and filter the solution through a 22-micrometer syringe filter into an HPLC vial.
To purify the peptide, use a C18 semi-preparative column at a three-milliliters-per-minute flow rate over a linear gradient of 15 to 55%acetonitrile in 20 minutes with the UV-detectors set to 222 nanometers and 330 nanometers. Then collect and combine the peaks of interest. For peptide product confirmation by matrix-assisted laser desorption/ionization time of flight or MALDI-TOF mass spectrometry, mix 0.5 microliters of the collected peak on a matrix-assisted laser desorption/ionization plate containing 0.5 microliters of 2, 5-dihydroxybenzoic acid matrix.
To prepare a self-assembly solution, dissolve one milligram of the lyophilized peptide powder in a mixture of 20%acetonitrile and 10-millimolar HEPES in a 1.5-milliliter microcentrifuge tube, then vortex the assembly solution and leave the sample to assemble at room temperature. When the peptide assembly reaches maturation, dry eight to 10 microliters of the assembly solution as a thin film on the attenuated total-reflection diamond crystal and monitor the disappearance of a large and broad water peak from 1640 to 1631 inverse centimeters as the dry film forms. Acquire background and infrared spectra from 1500 to 1800 inverse centimeters averaging 50 scans with a two inverse centimeter resolution, subtracting the background scans prior to each sample scan.
The infrared signature for beta sheet assembly should be observed as a sharp peak between 1625 and 1635 inverse centimeters. When the peptide samples have matured into beta-sheet-rich supermolecular structures, load 10 microliters of the peptide assembly solution onto the surface of a transmission electron microscope carbon grid and allow the assemblies to adsorb onto the grid's surface for one to two minutes. Touch filter paper to the edge of the grid to remove the excess sample, and add 10 microliters of freshly-prepared 2%uranyl acetate stain to the grid surface for a two to three-minute incubation, then remove excess stain with filter paper and place the grid in a vacuum desiccator overnight before next-day imaging by transmission electron microscopy.
Aside from the initial one-step synthesis of the dithiolane precursor molecule, the rest of the 1, 2-dithiolane-modified peptide synthesis occurs on solid support. The conversion of 3-bromo-2-proprionic acid to 3-2-propanoic acid, the dithiolane precursor, is confirmed by proton and Carbon-13 nuclear magnetic resonance before it is coupled to the free N-terminus amine of a peptide still on resin. After deprotection, the crude peptide is purified by reverse-phase HPLC and the product's mass is confirmed by MALDI-TOF mass spectrometry.
Fourier-transform infrared and circular dichroism spectroscopy can be used to monitor the self-assembly of the purified 1, 2-dithiolane peptide into mature amyloid fibers to characterize their extended beta sheet conformation. The fibers can then be imaged by transmission electron microscopy. While these methods can be used to modify the N-terminus of any peptide sequence still on resin, it's important to remember that the peptide-self-assembly conditions should be optimized for each peptide.
These techniques, which add 1, 2-dithiolane groups to self-assembling peptides, will allow researchers in the field of biomaterials to explore supermolecular surface reactivity.
