Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

1. Synthesis and Purification of 1,2-Dithiolane Modified Peptide

1. Synthesis of dithiolane precursor, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid¹⁹.
   1. Add 1 g of 3-bromo-2-(bromomethyl)propionic acid (1 equiv.) dissolved in minimal amount of 1 M NaOH (approximately 4 mL) to a 25-mL round bottom reaction flask with stirring at 55 °C. Seal the reaction flask with a septa and place under nitrogen atmosphere.
   2. Prepare a solution containing 1.49 g of potassium thioacetate (3.2 equiv.) in 4 mL of deionized water and 3 mL of 2 M sulfuric acid (H₂SO₄) to create thioacetic acid in situ.
   3. Pull the thioacetic acid solution into a plastic disposable 10 mL syringe and place a needle on the syringe. Add the mixture dropwise to the reaction flask by piercing through the septa with the needle. Continue the reaction overnight at 55 °C.
   4. Monitor the reaction by thin layer chromatography (TLC) on silica gel 60 F₂₅₄ plates using a mixture of methanol and dichloromethane (1:9). Visualize the reaction progress by bromocresol green stain. The product has an R_f = 0.57.
   5. After the reaction is complete and cooled to room temperature, acidify the mixture to pH 1 with 2 M H₂SO₄. A yellow oil separates out of solution.
   6. Extract the product with cold chloroform (40 mL x 3). Combine the organic layers and dry over magnesium sulfate. Remove the chloroform under reduced pressure.
   7. Confirm the identity of the isolated product, 1, by nuclear magnetic resonance (NMR) spectroscopy as shown in Figures 1B and C. Expect the following results: ¹H NMR, CDCl₃, 300 MHz: d = 10.1 (b, 1 H), 3.2 (m, 4 H), 2.9 (m, 1 H), 2.4 (s, 6 H); ¹³C NMR, CDCl₃, 75 MHz: d = 195.1 (CH₃COS-), 177.6 (-COOH), 45.1 (-CH₂S-), 30.5 (CH), 29.2 (-SCOCH₃).
      Note: The product is a yellow oil and has an overall yield of 83%. Use the product without further purification.
2. SPPS and On-Resin Coupling of Dithiolane Precursor
   Note: The solid-phase peptide synthesis described below was conducted on an automated peptide synthesizer, following the recommended manufacturer’s protocols. Settings and reagents may be adapted for other commercial instruments or when using specialized amino acids.
   1. Weigh out 0.156 g of Rink amide 4-methylbenzhydrylamine (MBHA) resin (0.1 mmol) and place in a reaction vessel. Swell the resin in dimethylformamide (DMF) for at least 15 min prior to the start of the synthesis.
   2. Weigh out 4 equivalents of each fluorenylmethyloxycarbonyl (Fmoc) protected amino acid (0.4 mmol) in the sequence and 0.152 g of N,N,N’,N’-tetramethyl-O-(1H-benotriazol-1-yl)uranium hexafluorophosphate (0.4 mmol, HBTU) for each amino acid in the sequence. Each cartridge contains both the Fmoc-protected amino acid and HBTU.
   3. After running all pre-synthesis checks on the synthesizer (refill reagents, flushed reagent lines and pressurized all reagent bottles), place the amino acid cartridges into the carousel in the C’ to N’ terminus direction. Place an empty cartridge after the final amino acid position for the final N-terminal Fmoc deprotection step.
   4. Synthesize the peptide using the standard recommended settings.
      1. Deprotect the Fmoc group from the resin with 5 mL of 20% piperidine in DMF (5 min x 2).
      2. Wash the resin with DMF (6 x 5 mL) before the coupling step.
      3. For a single coupling step, add 4 mL of 0.4 M N-methylmorpholine in DMF to the Fmoc-protected amino acid and HBTU. Activate the Fmoc-protected amino acid solution for 30 s before transferring the solution to the reaction vessel.
         Note: The automated peptide synthesizer mixes the resin and the solution by bubbling N₂ gas every 30 s for 20 min while the coupling reaction takes places. For manual peptide synthesis, place the reaction vessel on an orbital shaker at low speed for the duration of the coupling step.
      4. Drain the solution and wash the resin with DMF (3 x 5 mL).
      5. Repeat steps 1.2.4.1 through 1.2.4.4 for each Fmoc-protected amino acid in the C’ to N’-terminus direction to synthesize the peptide of interest.
   5. After the final N-terminal deprotection step, transfer the resin into a disposable fritted syringe. Wash the resin with DMF (3 x 5 mL) and dichloromethane (DCM, 3 x 5 mL).
      Note: The resin may be stored after DCM washing in a vacuum desiccator. If the resin was stored previous to coupling, ensure to swell the resin in DMF before the coupling reaction.
   6. Couple the dithiolane precursor (1) to the N-terminus of the on-resin peptide by adding 4 equivalents of 1, 5 mL of DMF, 4 equivalents of HBTU and 10 equivalents of N,N-diisopropylethylamine (DIPEA). Pre-activate the coupling mixture for 10 min before adding to the resin containing fritted syringe.
   7. Shake the coupling reaction for 2 h. After 2 h, wash the resin with DMF (3 x 5 mL) and repeat the coupling reaction with shaking overnight.
   8. After the second coupling, wash the resin with DMF (3 x 5 mL) and DCM (3 x 5 mL).
      Note: The resin can be stored at this point under vacuum until cleavage.
3. Thioacetate Deprotection and Peptide Cleavage from Resin
   1. To deprotect the thioacetate group from the N-terminus dithiolane precursor, transfer the dried resin to a 10 mL microwave reaction tube and add 2 mL of DMF. Allow the resin to swell, add a small magnetic stir bar to the vessel, and re-suspend with a low speed of magnetic stirring for 15 min.
   2. Add 2 mL of concentrated ammonium hydroxide, cap the reaction vessel with the silicone septa, and place the reaction vessel into a microwave reactor using the microwave settings of 75 °C for 45 min with stirring.
   3. After the microwave reaction is complete, transfer the resin into a clean disposable fritted syringe. Wash with DMF (2 x 5 mL) and methanol (MeOH, 2 x 5 mL).
   4. Add a solution of concentrated ammonium hydroxide in methanol (1:4), for a total volume of 5 mL. Leave to shake overnight to increase intramolecular oxidation of disulfide bond in dithiolane ring.
   5. Wash the resin with MeOH (2 x 5 mL) and DCM (3 x 5 mL).
      Note: The dried resin may be stored in a vacuum desiccator at this point.
   6. Add the cleavage cocktail to the resin containing syringe with gently shaking for 1.5 h. The cleavage cocktail used is 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS), and 2.5% water at a total volume of 5 mL.
      CAUTION: Work under chemical fume hood only. TFA is volatile and corrosive.
      Note: For the majority of peptide sequences and amino acid sidechain protecting groups, the above cleavage cocktail solution is sufficient; however, alternative cleavage cocktails may be needed for certain amino acid side chain protecting groups (in particular peptides containing Cys, Met, Trp and Arg) or other resin chemistry²⁰.
   7. Precipitate the crude peptide into 25 mL of cold diethyl ether in a 50 mL conical tube by dropwise addition from the fritted syringe. The peptide precipitates as a white solid. Pellet the peptide by centrifugation at 1300 x g for 10 min. Carefully decant the diethyl ether into a separate container for waste collection.
   8. Add another 25 mL of diethyl ether to the conical tube and re-suspend the precipitate by vortexing. Repeat the centrifugation at 1300 x g for 10 min and decant the diethyl ether again. Dry the pellet under vacuum.
4. Purification of 1,2-Dithiolane Modified Peptide
   Note: Purify the crude peptide by reverse phase-HPLC. Collect and combine the peptide peaks and confirm the mass by MALDI-TOF mass spectrometry.
   1. Dissolve the crude peptide pellet in minimal amount of acetonitrile with 0.1% TFA. Due to the peptide’s hydrophobicity and aggregation propensity, gently heat the sample at 40 °C to aid in solubility.
      Note: Avoid higher temperatures and sonication of the peptide in order to prevent potential disulfide exchange reactions^21,22,23.
   2. To prepare 1 mL of crude peptide for HPLC purification, add 400 μL of concentrated peptide stock in acetonitrile to 600 μL of H₂O with 0.1 % TFA and filter through a 22 μm syringe filter into a HPLC vial. An additional 5% isopropanol can be added to prevent peptide aggregation and precipitation.
   3. Purify the peptide using C-18 semi-preparative column with a flow rate of 3 mL/min over a linear gradient of 15-55% acetonitrile in 20 min. Set the UV detectors to 222 nm (amide backbone) and 330 nm (disulfide bond). Collect and combine the peaks of interest (Figure 2A).
   4. Confirm the peptide product mass by MALDI-TOF mass spectrometer in the reflectron mode (Figure 2B). For analysis, mix 0.5 μL of collected peak on the MALDI plate with 0.5 μL of 2,5-dihydroxybenzoic acid (DHB) matrix (10 mg/mL DHB in 50% acetonitrile, 0.1% TFA).
      Note: Common adducts in MALDI-TOF mass spectrometry include the sodium and potassium salt adduct ([M+Na]⁺ and [M+K]⁺) peaks. Desalting the sample prior to the analysis is recommended if the salt adduct peaks suppress the signal of the main [M+H]⁺ peak. Additionally, an oxidized peak of [M+O]⁺ is also detected in the 1,2-dithiolane modified peptide. A report on laser induced oxidation from the MALDI ionization using DHB matrix suggests that factors including sample concentration, solvent, and laser intensity can be modified to limit the MALDI induced oxidation artifact²⁴.
   5. After MALDI-TOF confirmation of the correct mass, lyophilize the peptide after flash freezing. Keep the lyophilized peptide powder under vacuum until assembly.

2. Characterization of Supramolecular Self-Assembly Structures

1. Formation of Amyloid Fibers
   1. To prepare self-assembly solution, weigh out 1 mg of the peptide powder using an analytical balance. Dissolve into a mixture (pH 7.5) of 20% acetonitrile and 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in a 1.5 mL microcentrifuge tube, to a final concentration of 1 mg/mL peptide assembly mixture. Vortex the assembly solution and leave to assemble at room temperature.
2. Spectroscopic Characterization of Amyloid Fibers
   1. Follow the peptide assembly process via Fourier-transform infrared (FTIR) spectroscopy every few days. A broad peak centered around 1670 cm^-1 is the IR signature arising from unassembled peptides in the sample¹⁷. The peptide assembly samples usually take one to two weeks for the broad unassembled peak to disappear and reach maturation.
      1. Dry an aliquot of 8-10 μL of the assembly solution as a thin film on the ATR diamond crystal. Monitor the disappearance of a large and broad water peak from 1640 to 1630 cm^-1 as the dry film forms.
      2. Acquire IR spectra from 1500-1800 cm^-1 averaging 50 scans with a 2 cm^-1 resolution. Acquire and subtract the background scans prior to each sample scan. The IR signature for β-sheet assembly is a sharp peak between 1625 and 1635 cm^-1 (Figure 3A)^25,26.
   2. Characterize the peptide assembly into β-sheet rich supramolecular structures by circular dichroism (CD). Record the spectra using a CD spectropolarimeter with a Peltier temperature control system.
      1. Pipette 30 μL of the assembly solution in a 0.1 mm path length microcuvette.
         Note: A cell holder is needed to clamp and position the short path length cell in the instrument.
      2. For each spectrum, set the CD instrument to the following parameters: scanning wavelengths of 300 nm to 180 nm, scanning rate of 100/min, band width of 1 nm, 25 °C, average of three scans.
      3. Collect a spectrum of the buffer (20% acetonitrile/10 mM HEPES, pH 7.5) and subtract from each sample scan as a control. The CD signature for β-sheets is an ellipticity minimum centered around 220 nm (Figure 3B)²⁷.
3. Microscopy of Amyloid Fibers
   1. Allow two to three weeks for the peptide samples to mature into β-sheet rich supramolecular structures.
      Note: The assemblies can be imaged using transmission electron microscopy (TEM) at earlier stages of the assembly process as well.
      1. Pipette 10 μL of the peptide assembly solution onto the surface of TEM carbon grid.
         Note: Take care not to touch the pipette tip to the grid surface. High precision, self-closing tweezers are used to hold the TEM grid during preparation.
      2. Wait 1-2 min to allow the assemblies to adsorb onto the grid surface. Remove excess sample by touching filter paper to the edge of the grid.
      3. Prepare a 2% uranyl acetate stain by adding 100 μL of deionized water to commercially available 4% uranyl acetate solution. Pipette 10 μL of the 2% uranyl acetate stain onto the grid surface and incubate for 2-3 min. After the incubation, remove excess stain by touching filter paper to the edge of the grid.
      4. Place the TEM grids in a vacuum desiccator overnight. Store under vacuum until imaging.
      5. Image the prepared samples with TEM (Figure 3C). Typical parameters for microscopy are as follows: images at magnifications ranging from 9,300X to 23,000X, tungsten filament with an accelerating voltage of 120 kV.
         Note: ImageJ can be used to measure average fiber width of the supramolecular structures of acquired TEM images²⁸.
         CAUTION: Please consult all relevant safety data sheets (SDS) before use. Several chemicals used in the synthesis, purification, and characterization of the described 1,2-dithiolane modified self-assembling peptides are corrosive or toxic and should only be used under a chemical fume hood. Always use appropriate personal protective equipment (including safety glasses, lab coat, full length pants, closed-toe shoes) when working in the laboratory.