Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles (PPAs) and Related Biomaterials

1. General Protocol for Synthesis of PPAs

1. Calculate the scale of synthesis (usually mmol). This scale is based on the mass of the target PPA amount. Keep in mind that the reaction efficiency of SPPS gradually decreases with an increase of amino acid sequence. Therefore, the exact reaction efficiency is difficult to calculate.
2. Calculate the weight of the resin to be used according to the loading of the resin. The loading is found on the container or the analysis protocol of the resin and expressed in mmol/g. The following formula can be used for calculating the weight of the resin:
3. Carefully weigh out 2-chlorotrityl chloride resin (in our case the loading is ~0.85 mmol)
4. Place the resin in a fritted synthesis vessel with the following specifications: Capacity – 50 mL, Fritted Disc – 25 mm, Porosity – Medium.
5. Add 15 mL of dichloromethane (DCM) to the resin.
6. Affix the synthesis vessel to a variable speed mechanical shaker (flask shaker/stirrer), and turn the vessels to a 45° angle (or horizontally) to maximize agitation.
7. Allow the resin beads to swell for 15 min. Swelling enhances the yield of reaction because it facilitates molecular diffusion and accessibility to the active site, enhancing coupling efficiency.
8. Add 8 equivalents (2 mmol for a 0.25 mmol scale) of the desired polyamine (Spermine, Spermidine, Diethyelenetriamine, 1,3-diaminopropane, etc.) to the resin and allow it to react for 5 h.
   Note: A lesser reaction period would reduce yield.
9. Do a Kaiser test (see Table of Materials) to confirm successful coupling of the polyamine to the resin. Successful coupling of the primary amines yields a violet/blue color, which corresponds to the free amine.
10. Protect the primary amine group using 4 equivalents of 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), dissolved in anhydrous methanol (15 mL), by shaking the reaction mixture overnight¹¹.
    Note: A lesser reaction time reduces yield.
11. Perform a Kaiser test. An absence of blue color from the resin bead confirms successful protection. In case of unsuccessful protection of the primary amine, repeat the previous step.
12. Drain the DCM and wash the resins twice with a mixture of DCM and DMF (2:1, 15 mL).
13. Dissolve 20 equivalents (5 mmol) of di-tert butyl di-carbonate (Boc) in DCM (20 mL) and allow the reaction to proceed for 3 h.
14. Do a chloranil test (see Table of Materials) to confirm complete protection of the secondary amine. A positive test (protection) yields a colorless or light yellow color. Free secondary amines give a dark green/blue color, while primary amines give a red color.
15. Drain the solvent mixture and wash twice with a mixture of DCM and DMF (2:1, 15 mL).
16. Add a 2% solution of hydrazine in DMF (10 mL) and shake for 1 h.
17. Do a Kaiser test to confirm successful deprotection of the primary amine.
18. Add the desired amino acids in the reverse sequence in which you would write/draw them. Peptides are drawn from the N termini to the C termini but are synthesized in the opposite direction, C to N. For example, for a PPA requiring the following peptide core -GLFD-, add the Aspartic acid (D), followed by Phenylalanine (F), Leucine (L), and finally Glycine (G).
    NOTE: For most amino acids, the following coupling cocktail is appropriate for attachment to the polyamine loaded resins.
    1. Mix 4 equivalents (1 mmol) of the Fmoc-protected amino acid, 3.95 equivalents of 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), and 15 equivalents of diisopropylethyl amine (DIPEA).
    2. Dissolve them in a mixture of DCM and DMF (1:1, 15 mL) and sonicate the cocktail for 2 min (until complete dissolution).
    3. Wait an additional 3–5 min to ensure activation of the carboxylic acid on the amino acid.
    4. Add the reaction mixture to the vessel. Carry out the reaction for 2-4 h at ambient temperature.
       NOTE: We have found the following as efficient alternatives to HBTU (usually requiring lower amounts of the amino acids and the coupling agent): COMU, PyBOP, HATU, DIC/Oxima.
    5. Do a Kaiser test to confirm successful coupling.
    6. De-protect the Fmoc-group from the amino acid by adding a 20% solution of 4-methyl piperidine (10 mL) in DMF. Do it twice, each time shaking the reaction mixture for 15 min.
       NOTE: An efficient alternative for Fmoc removal is piperazine/DBU¹².
       1. Perform a DCM (15 mL) wash between additions.
    7. Do a Kaiser test to confirm successful de-protection of the amino acid.
    8. Wash the resin thoroughly to remove all traces of 4-methyl. Wash the resin twice with DMF (10 mL), each wash lasting for 5 min, and finally with DCM (10 mL) for 10 min.
    9. Add all the remaining amino acids by successively repeating the coupling and de-protection steps.
19. Finally, after coupling all the required amino acids, conjugate the hydrophobic tail to the last amino acid of the polyamine-peptide construct:
    1. Add 10 equivalents of the desired carboxylic acid functionality to 9.5 equivalents of HBTU and 12 equivalents of DIPEA dissolved in DCM and DMF mixture (1:1, 20 mL).
       Note: Bear in mind than some tails may need a different proportion of solvent (usually a larger% of DCM) or the addition or another solvent such as N-methylpyrrolidone (NMP).
    2. Sonicate the cocktail for 5–10 min until dissolution (we have seen it takes longer for the tail to completely dissolve) and add to the vessel.
       Note: For tails containing multiple reactive sites, they will need to be protected before coupling them.
    3. Carry out this reaction (coupling the hydrophobic tail) for at least 5 h, although it is advisable to carry it out overnight for the highest yield.

2. PPA Cleavage from Solid Support

The purpose of this step is the cleavage of the PPA from the resin, and to remove the Boc protecting groups from the amino acids and polyamine residues.

1. Wash the resin with DMF (8 mL for 2 min) and twice with DCM (8 mL, each time for 5 min). Before each addition, drain the solvent from the vessel. Once the last wash is performed, dry the resin under vacuum for 15 min.
2. Prepare the cleavage solution using the following mixture ratio: 28:1:1 trifluoroacetic acid (TFA): H₂O : Triisopropyl silane (TIPS). To make 15 mL of the cleavage cocktail add 14 mL of TFA, to 0.5 mL of H₂O and 0.5 mL of TIPS.
3. Add this cleavage solution to the resin and shake for 2–4 h at room temperature.
4. Collect the solution in a 25 or 50 mL round bottom flask.
5. Concentrate the TFA in vacuo to 1–2 mL using a rotary evaporator at reduced pressure while heating the mixture at 40 °C (do not surpass 55 °C to avoid decomposition of the PPA).
6. After evaporation, add the obtained TFA solution (dropwise) to a round bottom flask containing anhydrous cold ether (15 mL). This will immediately precipitate the PPA.
7. In addition, add anhydrous cold ether (5 mL) to the original flask where the TFA was concentrated.
8. Sonicate this flask to recover additional solids and combine with the ether solution from step 2.6.
   Note: The transferring pipette can be washed with a small volume of DCM. Avoid introducing DMF during the process because it tends to form a gel-like substance.
9. Place the flask (covered) inside the refrigerator and let it stand overnight to maximize precipitation.
10. Collect the precipitated material by vacuum filtration using a sintered disc filter funnel. The ideal pore sizes are fine or medium.
11. Wash the precipitate twice with cold ether (5–10 mL) to remove any residual organics.

3. Identification of the Crude Product Using the MALDI Dried-drop Method

1. Matrix preparation for analysis in positive mode:
   1. To start molecular weight analysis using Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry, add 10 to 20 mg of α-cyano-4-hydroxycinnamic acid to a microcentrifuge tube.
   2. Add a solution of water/acetonitrile (1:1, 1.0 mL) with 0.1% Trifluoroacetic acid (TFA). Mix thoroughly by vortexing.
      Note: This matrix should be used for positively charged peptides. MALDI negative mode is similar but requires a matrix containing 9-aminoacridine with 0.1% ammonia as solvent additive.
   3. Add 1–2 µL of the sample to the stainless steel target plate for MALDI and dry the sample in air. Add 1–2 µL of the matrix and dry it again.
      Note: The plates have circular markings gridded along the target plate to help locate the particular spot.
2. Analyze the samples in MALDI instrument to verify their identity. Electrospray ionization (ESI) is a valid alternative to confirm the identity of the PPAs.

4. Purification of PPAs Using Preparative Reverse-phase High-performance Liquid Chromatography (HPLC) Purification

1. Dissolve the PPA precipitate in a minimum amount of acetonitrile and water.
   1. As a rule of thumb, dissolve 100 mg of dry crude PPA in less than 5 mL of acetonitrile and water. More hydrophobic PPAs might require a greater percentage of acetonitrile to dissolve and might also require a larger volume of solvent in general, depending on the net charge of the PPAs (Table 1).
   2. If complete dissolution does not take place, add 1% TFA (ACN or H₂O). It is also possible to add trace amounts of other compatible solvents including dimethylsulfoxide, methanol, or isopropanol (limit their content to 5%).

Table 1: Solvent Systems. Proposed solvent system for positively and negatively charged PPAs.

2. Sonicate the PPA using a horn sonicator for 20 min at 10 s pulses with Amp1 of 48% or for 2-3 h in an ultrasonic bath.
3. Then, filter using a 0.45 μm syringe filter followed by filtration using a 0.20 μm Polytetrafluoroethylene (PTFE) syringe filter. The PPA solution should be clear and free of all particulate materials.
   1. If filtration is too difficult, sonicate for an extended period of time. It is strongly advised that the PPA solution is purified immediately after syringe filtration, as prolonged storage might cause it to aggregate or gelate, which will necessitate re-sonication and, perhaps, re-filtration.
4. During and after purification, use either HPLC grade solvents or ones that are filtered through a 0.25 μm syringe filter/membrane.
   NOTE: The most common solvent conditions to purify PPAs consist in a gradient of H₂O and ACN.
5. Use a standard reverse-phase HPLC instrument for this protocol. It should include an elution gradient programmer, a binary solvent delivery system, a UV detector capable of detecting at 220 and 254 nm, and a programmable fraction collector. The maximum flow rate should be 50 mL/min.
6. Use a C-18 reversed phase column for separation. Columns of the following dimensions can be used depending on the mass of solid being purified and the net charge of the PPA (Table 2).

Table 2: Suggested columns: Column dimensions, particle sizes and the maximum load capacity per injection for C18 reverse phase HPLC columns

7. Inject PPA with a volume determined by both the capacity of the column and by the volume of the injection loop of the HPLC. Run the HPLC gradient method according to the settings seen in Table 3 (elution time of 40 min).

Table 3: Suggested gradient: Suggested reverse phase gradient showing the relative composition of water vs acetonitrile over a period of time. The flow rate will depend on the column specifications.

8. Analyze the fractions collected on the various test tubes using MALDI and determine which tube (or tubes) contains the PPA. This is assessed by studying the molecular weight found in each tube.
   1. Check purity of the fractions on an analytical HPLC. Use an HPLC-gradient system equipped with a UV detector set at 220 nm.
   2. If there are impurities, do an additional cycle of purification by HPLC. The above gradient used for preparative HPLC can be scaled down to use with the analytical HPLC using the column converter online software. Each fraction much be ≥95% pure for physical characterization or biological evaluation.
9. Collect the fractions in a large round-bottomed flask or evaporation flask and remove all the acetonitrile and most of the water. The final PPA solution should be no more than 10 mL.
10. Transfer this concentrate to a 50 mL centrifuge tube. Be advised that PPAs are detergent-like substances; thus, bubbles will be generated during evaporation of the solvent. We have found that addition of ethyl alcohol diminishes bubble formation.
11. Vacuum concentrate the concentrate. During the vacuum evaporation, a large amount of the PPA might adhere to the walls of the container. Recover this by repeatedly rinsing the flask wall with HPLC water. The combined volume of the concentrated PPA and the rinsate should be no more than 30 mL.
12. Place the aqueous solution inside of a 50 mL tube.
13. Flash freeze this PPA solution in liquid nitrogen:
    NOTE: Flash freezing results in smaller ice-crystals which will sublime faster in the lyophilizer. Moreover, slower freezing may allow the formation of self-assembled supramolecular structures. Another alternative (but less desirable) is to gradually freeze in a -80 °C freezer or to freeze using a dry ice + acetone mixture
    1. Before placing the centrifuge tube in the lyophilizer, perforate or loosen the cap of the tube to allow moisture to escape the sample and get collected in the refrigeration unit. Set the lyophilization unit set to -54 °C and 0.016 mbar.
       NOTE: Another option is to remove the cap and place a wipe (with a rubber band) on top of the opening. Also, we have found that freezing samples at an angle or breaking the ice into small portions allows for a faster and better lyophilization due to an increase in surface area.
    2. If the solid starts to melt, this may indicate traces of an organic solvent (usually acetonitrile) are present. Repeat the freezing step and assess the condition of lyophilization.
    3. If the melting persists, there are two potential solutions:
       1. Split the sample in two portions (to be placed in tubes), dilute with water, and freeze again. Do not use this solution often because large amounts of organic solvents may damage the equipment.
       2. Alternatively, place the sample in a flask and further evaporate the organic solvent under vacuum. Lyophilizing a sample that is in the liquid form usually leads to bumping and loss of the PPA compound.

5. Storage of PPAs

1. Store the PPAs in a -20 °C with the caps tightened and sealed with Parafilm on a tube with a proper label.
2. Before use, bring the PPA back to ambient temperature in a vacuum chamber to avoid condensation of water vapor on the PPAs.