This project was funded by the University of Nebraska Medical Center (Start-up funds, MC-S); NIH-COBRE, 5P20GM103480 (T. Bronich) and the American Chemical Society, PRF# 57434-DNI7(MC-S).

1. General Protocol for Synthesis of PPAs
<ol>
	<li>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.</li>
	<li>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:</li>
	<li>Carefully weigh out 2-chlorotrityl chloride resin (in our case the loading is ~0.85 mmol)</li>
	<li>Place the resin in a fritted synthesis vessel with the following specifications: Capacity &#8211; 50 mL, Fritted Disc &#8211; 25 mm, Porosity &#8211; Medium.</li>
	<li>Add 15 mL of dichloromethane (DCM) to the resin.</li>
	<li>Affix the synthesis vessel to a variable speed mechanical shaker (flask shaker/stirrer), and turn the vessels to a 45&#176; angle (or horizontally) to maximize agitation.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Drain the DCM and wash the resins twice with a mixture of DCM and DMF (2:1, 15 mL).</li>
	<li>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.</li>
	<li>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.</li>
	<li>Drain the solvent mixture and wash twice with a mixture of DCM and DMF (2:1, 15 mL).</li>
	<li>Add a 2% solution of hydrazine in DMF (10 mL) and shake for 1 h.</li>
	<li>Do a Kaiser test to confirm successful deprotection of the primary amine.</li>
	<li>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.
	<ol>
		<li>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 hexa&#64258;uorophosphate (HBTU), and 15 equivalents of diisopropylethyl amine (DIPEA).</li>
		<li>Dissolve them in a mixture of DCM and DMF (1:1, 15 mL) and sonicate the cocktail for 2 min (until complete dissolution).</li>
		<li>Wait an additional 3&#8211;5 min to ensure activation of the carboxylic acid on the amino acid.</li>
		<li>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.</li>
		<li>Do a Kaiser test to confirm successful coupling.</li>
		<li>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.
		<ol>
			<li>Perform a DCM (15 mL) wash between additions.</li>
		</ol>
		</li>
		<li>Do a Kaiser test to confirm successful de-protection of the amino acid.</li>
		<li>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.</li>
		<li>Add all the remaining amino acids by successively repeating the coupling and de-protection steps.</li>
	</ol>
	</li>
	<li>Finally, after coupling all the required amino acids, conjugate the hydrophobic tail to the last amino acid of the polyamine-peptide construct:
	<ol>
		<li>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).</li>
		<li>Sonicate the cocktail for 5&#8211;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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Add this cleavage solution to the resin and shake for 2&#8211;4 h at room temperature.</li>
	<li>Collect the solution in a 25 or 50 mL round bottom flask.</li>
	<li>Concentrate the TFA in vacuo to 1&#8211;2 mL using a rotary evaporator at reduced pressure while heating the mixture at 40 &#176;C (do not surpass 55 &#176;C to avoid decomposition of the PPA).</li>
	<li>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.</li>
	<li>In addition, add anhydrous cold ether (5 mL) to the original flask where the TFA was concentrated.</li>
	<li>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.</li>
	<li>Place the flask (covered) inside the refrigerator and let it stand overnight to maximize precipitation.</li>
	<li>Collect the precipitated material by vacuum filtration using a sintered disc filter funnel. The ideal pore sizes are fine or medium.</li>
	<li>Wash the precipitate twice with cold ether (5&#8211;10 mL) to remove any residual organics.</li>
</ol>
3. Identification of the Crude Product Using the MALDI Dried-drop Method
<ol>
	<li>Matrix preparation for analysis in positive mode:
	<ol>
		<li>To start molecular weight analysis using Matrix Assisted Laser Desorption/Ionization (MALDI) mass spectrometry, add 10 to 20 mg of &#945;-cyano-4-hydroxycinnamic acid to a microcentrifuge tube.</li>
		<li>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.</li>
		<li>Add 1&#8211;2 &#181;L of the sample to the stainless steel target plate for MALDI and dry the sample in air. Add 1&#8211;2 &#181;L of the matrix and dry it again. 
		Note: The plates have circular markings gridded along the target plate to help locate the particular spot.</li>
	</ol>
	</li>
	<li>Analyze the samples in MALDI instrument to verify their identity. Electrospray ionization (ESI) is a valid alternative to confirm the identity of the PPAs.</li>
</ol>
4. Purification of PPAs Using Preparative Reverse-phase High-performance Liquid Chromatography (HPLC) Purification
<ol>
	<li>Dissolve the PPA precipitate in a minimum amount of acetonitrile and water.
	<ol>
		<li>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).</li>
		<li>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%).</li>
	</ol>
	</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Solvent</td>
			<td colspan="2">Positively Charged PPAs</td>
			<td colspan="2">Negatively Charged PPAs</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">0.1% TFA in Water</td>
			<td colspan="2">0.1% NH3 in Water</td>
		</tr>
		<tr>
			<td></td>
			<td colspan="2">0.1% TFA in ACN</td>
			<td colspan="2">0.1% NH3 in ACN</td>
		</tr>
	</tbody>
</table>
Table 1: Solvent Systems. Proposed solvent system for positively and negatively charged PPAs.
<ol start="2">
	<li>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.</li>
	<li>Then, filter using a 0.45 &#956;m syringe filter followed by filtration using a 0.20 &#956;m Polytetrafluoroethylene (PTFE) syringe filter. The PPA solution should be clear and free of all particulate materials.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>During and after purification, use either HPLC grade solvents or ones that are filtered through a 0.25 &#956;m syringe filter/membrane. 
	NOTE: The most common solvent conditions to purify PPAs consist in a gradient of H2O and ACN.</li>
	<li>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.</li>
	<li>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).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>PPA Charge</td>
			<td>Particle Size</td>
			<td>Column Size</td>
			<td>Mass of crude PPA</td>
		</tr>
		<tr>
			<td>+ ve Charged</td>
			<td>5 &#956;m</td>
			<td>150 x 30 mm</td>
			<td>170 mg</td>
		</tr>
		<tr>
			<td>-&#160; ve Charged</td>
			<td>5 &#956;m</td>
			<td>150 x 30 mm</td>
			<td>170 mg</td>
		</tr>
		<tr>
			<td>+ ve Charged</td>
			<td>5 &#956;m</td>
			<td>150 x 21.2 mm</td>
			<td>90 mg</td>
		</tr>
		<tr>
			<td>-&#160; ve Charged</td>
			<td>5 &#956;m</td>
			<td>150 x 21.2 mm</td>
			<td>90 mg</td>
		</tr>
	</tbody>
</table>
Table 2: Suggested columns: Column dimensions, particle sizes and the maximum load capacity per injection for C18 reverse phase HPLC columns
<ol start="7">
	<li>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).</li>
</ol>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Time</td>
			<td>Solvent A (Acetonitrile)</td>
			<td>Solvent B (Water)</td>
			<td>Flow Rate (mL/min)</td>
		</tr>
		<tr>
			<td>0</td>
			<td>5%</td>
			<td>95%</td>
			<td rowspan="5">Flow rate depends on the column packing and its size.</td>
		</tr>
		<tr>
			<td>2</td>
			<td>5%</td>
			<td>95%</td>
		</tr>
		<tr>
			<td>35</td>
			<td>95%</td>
			<td>5%</td>
		</tr>
		<tr>
			<td>38</td>
			<td>100%</td>
			<td>0%</td>
		</tr>
		<tr>
			<td>40</td>
			<td>5%</td>
			<td>95%</td>
		</tr>
	</tbody>
</table>
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.
<ol start="8">
	<li>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.
	<ol>
		<li>Check purity of the fractions on an analytical HPLC. Use an HPLC-gradient system equipped with a UV detector set at 220 nm.</li>
		<li>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 &#8805;95% pure for physical characterization or biological evaluation.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Place the aqueous solution inside of a 50 mL tube.</li>
	<li>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 &#176;C freezer or to freeze using a dry ice + acetone mixture
	<ol>
		<li>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 &#176;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.</li>
		<li>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.</li>
		<li>If the melting persists, there are two potential solutions:
		<ol>
			<li>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.</li>
			<li>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.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
5. Storage of PPAs
<ol>
	<li>Store the PPAs in a -20 &#176;C with the caps tightened and sealed with Parafilm on a tube with a proper label.</li>
	<li>Before use, bring the PPA back to ambient temperature in a vacuum chamber to avoid condensation of water vapor on the PPAs.</li>
</ol>

<ol>
	<li>Cui, H., Pashuck, E. T., Velichko, Y. S., Weigand, S. J., Cheetham, A. G., Newcomb, C. J., Stupp, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+and+x-ray-triggered+crystallization+at+long+range+in+self-assembling+filament+networks.">Spontaneous and x-ray-triggered crystallization at long range in self-assembling filament networks.</a> Science. 327, 555-559 (2010).</li><li>Pashuck, E. T., Cui, H., Stupp, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tuning+supramolecular+rigidity+of+peptide+fibers+through+molecular+structure.">Tuning supramolecular rigidity of peptide fibers through molecular structure.</a> Journal of the American Chemical Society. 132, 6041-6046 (2010).</li><li>Stupp, S. I., Zha, R. H., Palmer, L. C., Cui, H., Bitton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-assembly+of+biomolecular+soft+matter.">Self-assembly of biomolecular soft matter.</a> Faraday Discussions. 166, 9-30 (2013).</li><li>Conda-Sheridan, M., Lee, S. S., Preslar, A. T., Stupp, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Esterase-activated+release+of+naproxen+from+supramolecular+nanofibres.">Esterase-activated release of naproxen from supramolecular nanofibres.</a> Chemical Communications. 50, 13757-13760 (2014).</li><li>Mata, A., Palmer, L., Tejeda-Montes, E., Stupp, S. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+of+biomolecules+for+nanoengineered+biomaterials+for+regenerative+medicine.">Design of biomolecules for nanoengineered biomaterials for regenerative medicine.</a> Nanotechnology in Regenerative Medicine. , 39-49 (2012).</li><li>Samad, M. B., Chhonker, Y. S., Contreras, J. I., McCarthy, A., McClanahan, M. M., Murry, D. J., Conda-Sheridan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developing+Polyamine-Based+Peptide+Amphiphiles+with+Tunable+Morphology+and+Physicochemical+Properties.">Developing Polyamine-Based Peptide Amphiphiles with Tunable Morphology and Physicochemical Properties.</a> Macromolecular bioscience. 17, (2017).</li><li>Nel, A. E., M&#228;dler, L., Velegol, D., Xia, T., Hoek, E. M., Somasundaran, P., Klaessig, F., Castranova, V., Thompson, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+biophysicochemical+interactions+at+the+nano-bio+interface.">Understanding biophysicochemical interactions at the nano-bio interface.</a> Nature Materials. 8, 543 (2009).</li><li>Gujrati, M., Malamas, A., Shin, T., Jin, E., Sun, Y., Lu, Z. -. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+cationic+lipid-based+nanoparticles+facilitate+endosomal+escape+and+reduction-triggered+cytosolic+siRNA+release.">Multifunctional cationic lipid-based nanoparticles facilitate endosomal escape and reduction-triggered cytosolic siRNA release.</a> Molecular Pharmaceutics. 11, 2734-2744 (2014).</li><li>Zhu, Y., Li, J., Kanvinde, S., Lin, Z., Hazeldine, S., Singh, R. K., Oupicky&#769;, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-immolative+polycations+as+gene+delivery+vectors+and+prodrugs+targeting+polyamine+metabolism+in+cancer.">Self-immolative polycations as gene delivery vectors and prodrugs targeting polyamine metabolism in cancer.</a> Molecular Pharmaceutics. 12, 332-341 (2014).</li><li>Planas-Portell, J., Gallart, M., Tiburcio, A. F., Altabella, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-containing+amine+oxidases+contribute+to+terminal+polyamine+oxidation+in+peroxisomes+and+apoplast+of+Arabidopsis+thaliana.">Copper-containing amine oxidases contribute to terminal polyamine oxidation in peroxisomes and apoplast of Arabidopsis thaliana.</a> BMC Plant Biology. 13, 109 (2013).</li><li>Nash, I. A., Bycroft, B. W., Chan, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dde+-+A+selective+primary+amine+protecting+group:+A+facile+solid+phase+synthetic+approach+to+polyamine+conjugates.">Dde - A selective primary amine protecting group: A facile solid phase synthetic approach to polyamine conjugates.</a> Tetrahedron Letters. 37, 2625-2628 (1996).</li><li>Ralhan, K., KrishnaKumar, V. G., Gupta, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Piperazine+and+DBU:+a+safer+alternative+for+rapid+and+efficient+Fmoc+deprotection+in+solid+phase+peptide+synthesis.">Piperazine and DBU: a safer alternative for rapid and efficient Fmoc deprotection in solid phase peptide synthesis.</a> RSC Advances. 5, 104417-104425 (2015).</li><li>Casero, R. A., Marton, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+polyamine+metabolism+and+function+in+cancer+and+other+hyperproliferative+diseases.">Targeting polyamine metabolism and function in cancer and other hyperproliferative diseases.</a> Nature Reviews Drug Discovery. 6, 373 (2007).</li><li>Wuts, P. G. M., Greene, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Protection for the Amino Group. In Greene's Protective Groups in Organic Synthesis. , 696-926 (2006).</li><li>Palasek, S. A., Cox, Z. J., Collins, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limiting+racemization+and+aspartimide+formation+in+microwave-enhanced+Fmoc+solid+phase+peptide+synthesis.">Limiting racemization and aspartimide formation in microwave-enhanced Fmoc solid phase peptide synthesis.</a> Journal of Peptide Science. 13, 143-148 (2007).</li></ol>

The authors have no conflict of interest to declare

The protocols described here can be used to synthesize PPAs as wells as PAs and related peptide-based molecules (such as hybrid PA-peptoids). Although the synthesis of peptides using SPPS is a straightforward procedure, the synthesis of peptides containing biological homing molecules can be particularly challenging. Polyamines like spermine, spermidine, diethyelenetriamine, etc., can function as homing molecules for targeting cancer cells13. The PPAs can self-assemble into nanostructures ...

Self-assembling peptide amphiphiles (PAs) are a class of biomaterials typically comprised of the following segments: (a) hydrophilic head, (b) linker region, and (c) hydrophobic tail. Most PAs described in the literature possess a hydrophilic head comprised of charged or polar amino acid residues1,2,3,4. PAs have found a wide range of applications in biomedicine including drug delivery, disease diagnostics, regenerative medicine, etc.5. Based on their amino acid sequence, PAs can form a wide variety of nanostructures including spherical micelles, and nano-filaments. We have recently reported a class of hybrid polyamine-based peptide amphiphiles, termed PPAs6. The morphologies, self-assembling kinetics, and metabolic degradation, of these biomaterials, were found to be related to their solubilizing head group. Moreover, the PPA nanostructures did not show toxicity towards mammalian cells (MiaPaCa2 and HeLa cell lines) at the tested concentrations. PPA-based nanocarriers are attractive drug-delivery vehicles because: (1) polyamine uptake and metabolism has been shown to be increased in cancerous cells, (2) cationic nanostructures can achieve endosomal escape7,8, which leads to higher circulation and residence within a cell, and (3) they should have a distinct metabolic profile when compared with PA; for example, they will be more stable towards proteases found in the human body (although they maybe sensitive to other enzymes, such as amine oxidases)9,10. Also, PPAs have been found to have diverse morphologies, physicochemical properties, nanoparticle stiffness, and assembly kinetics depending on the length and charge of individual PPA molecule6. Herein, we describe a detailed protocol for the synthesis, identification, and purification of PPAs that can also be applied to the preparation of PAs or similar hybrid peptide molecules.
Because polyamines are not commonly commercially available in their protected forms, and because protecting the primary and secondary amines of polyamines is of utmost importance for conjugating them with amino acids and other molecules, we have outlined the synthetic steps to achieve their protection. The overall goal of this protocol is to provide a simple method for conjugating polyamines to amino acids. Polyamines lack a carboxylic group; thus, they cannot be coupled to Rink Amide or Wang resins. Instead, resins such as 2-chlorotrityl chloride are recommended for the synthetic protocol. The main challenge for PPA synthesis is the presence of primary and secondary amine functional groups. For our purposes, we protected all the secondary amines in the polyamine while keeping the primary amino group on the polyamine free to allow the coupling reaction. The reaction was done on a solid support following the principles of solid phase peptide synthesis (SPPS) to facilitate the work-up after each coupling and deprotection step. The following protocol is for both the manual and automated synthesis of PPAs (although the verification of some steps will be challenging in an automated system). The synthesis of these molecules can also be carried out on an automated synthesizer or with the aid of a microwave reactor (automated or semi-automated). The reaction scheme has been summarized in Figure 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57908/57908fig1.jpg" /> 
Figure 1: (A) A general reaction scheme for the synthesis of PPAs. (B) Representative polyamines that can be used to synthesized PPAs described here. <a href="/files/ftp_upload/57908/57908fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table><tbody><tr><td>2-Chlorotrityl chloride resin&amp;nbsp;</td><td>AappTec</td><td>RTZ001</td><td></td></tr><tr><td>SynthwareTM synthesis vessel&amp;nbsp;</td><td>Aldrich</td><td>SYNP120050M</td><td></td></tr><tr><td>Dichloromethane</td><td>Acros</td><td>AC406920250</td><td>Fisher Sci. Catalogue #</td></tr><tr><td>Wrist Shaker</td><td>Boekel Scientific</td><td>401000-2</td><td></td></tr><tr><td>Kaiser test kit</td><td>Sigma-Aldrich</td><td>60017</td><td></td></tr><tr><td>2-[(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl-amino]-ethanol</td><td>Sigma-Aldrich</td><td>CDS004772</td><td></td></tr><tr><td>Anhydrous Methanol</td><td>Acros</td><td>AC610981000</td><td>Fisher Sci. Catalogue #</td></tr><tr><td>Chloranil test kit</td><td>TCI</td><td>TCC1771-KIT</td><td>VWR Catalogue #</td></tr><tr><td>Di-tert butyl di-carbonate&amp;nbsp;</td><td>Acros</td><td>AC194670250</td><td>Fisher Sci. Catalogue #</td></tr><tr><td>Dimethylformamide</td><td>Fisher Scientific</td><td>BP1160-4</td><td></td></tr><tr><td>Hydrazine</td><td>Acros</td><td>AC296815000</td><td>FIsher Sci. Catalogue #</td></tr><tr><td>(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)</td><td>p3biosystems</td><td>31001</td><td></td></tr><tr><td>4-methyl piperidine&amp;nbsp;</td><td>Acros</td><td>AC127515000</td><td>FIsher Sci. Catalogue #</td></tr><tr><td>Trifluoroacetic Acid</td><td>AappTec</td><td>CXZ035</td><td></td></tr><tr><td>Triisopropyl Silane</td><td>Sigma-Aldrich</td><td>233781</td><td></td></tr><tr><td>Ether</td><td>Fisher Scientific</td><td>E138-1</td><td></td></tr><tr><td>&amp;alpha;-Cyano-4-hydroxycinnamic acid</td><td>Sigma-Aldrich</td><td>C8982</td><td></td></tr><tr><td>9-Aminoacridine</td><td>Sigma-Aldrich</td><td>92817</td><td></td></tr><tr><td>Fisherbrand&amp;nbsp;Syringe Filters: PTFE Membrane</td><td>Fisher Scientific</td><td>09-730-21</td><td></td></tr></tbody></table>

facile protocol for the synthesis of self-assembling polyamine-based peptide amphiphiles (ppas) and related biomaterials

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). Traditional PAs possess charged amino acids as solubilizing groups (lysine, arginine), which are directly connected to a lipid segment or can contain a linker region made of neutral amino acids. Tuning the peptide sequence of PAs can yield diverse morphologies. Similarly, PPAs possess a hydrophobic segment and neutral amino acids, but also contain polyamine molecules as water solubilizing (hydrophilic) groups. As is the case with PAs, PPAs can also self-assemble into diverse morphologies, including small rods, twisted nano-ribbons, and fused nano-sheets, when dissolved in water. However, the presence of both primary and secondary amines on a single polyamine molecule poses a significant challenge when synthesizing PPAs. In this paper, we show a simple protocol, based on literature precedents, to achieve a facile synthesis of PPAs using solid phase peptide synthesis (SPPS). This protocol can be extended to the synthesis of PAs and other similar systems. We also illustrate the steps that are needed for cleavage from the resin, identification, and purification.

After the synthesis and purification and before physicochemical or biological evaluation, it is recommended the masses of the PPAs are re-checked and the purity ascertained using analytical HPLC. For material characterization or biological evaluation, PPAs need to have a purity of &#62;95%. Figure 2 shows the HPLC trace (Top) and MALDI spectra (Bottom) confirming the presence of the product. HPLC analytical systems will integrate the area under the curve (AUC...

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

The synthesis of polyamine-based peptide amphiphiles (PPAs) is a significant challenge due to the presence of multiple amine nitrogens, which requires judicious use of protecting groups to mask these reactive functionalities. In this paper, we describe a facile method for the preparation of these new class of self-assembling molecules.

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

Polyamine-based Peptide Amphiphiles (PPAs) are a new class of self-assembling amphiphilic biomaterials-related to the peptide amphiphiles (PAs). ...

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Research

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Chemistry

8.0K Views.  University of Nebraska Medical Center. De synthese van peptide polyamine gebaseerde amfifielen (PPA) is een grote uitdaging wegens de aanwezigheid van meerdere amine stikstofatomen, waarvoor oordeelkundig gebruik om groepen te maskeren deze reactieve functionaliteiten te beschermen. In deze paper beschrijven we een facile methode voor de bereiding van deze nieuwe klasse van zelfassemblerende moleculen.

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

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

Biochemistry

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Split-and-pool Synthesis and Characterization of Peptide Tertiary Amide Library

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid phase by using combinatorial chemistry. One of the most well studied classes of peptidomimetics is peptoids. Peptoids are easy to synthesize and have been shown to be proteolysis-resistant and cell-permeable. Over the past decade, many useful protein ligands have been identified through screening of peptoid libraries. However, most of the ligands identified from peptoid libraries do not display high affinity, with rare exceptions. This may be due, in part, to the lack of chiral centers and conformational constraints in peptoid molecules. Recently, we described a new synthetic route to access peptide tertiary amides (PTAs). PTAs are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. With side chains on both &#945;-carbon and main chain nitrogen atoms, the conformation of these molecules are greatly constrained by sterical hindrance and allylic 1,3 strain. (Figure 1) Our study suggests that these PTA molecules are highly structured in solution and can be used to identify protein ligands. We believe that these molecules can be a future source of high-affinity protein ligands. Here we describe the synthetic method combining the power of both split-and-pool and sub-monomer strategies to synthesize a sample one-bead one-compound (OBOC) library of PTAs.

Peptide tertiary amides (PTAs) are a superfamily of peptidomimetics that include but are not limited to peptides, peptoids and N-methylated peptides. Here we describe a synthetic method which combines&#160;both split-and-pool and sub-monomer strategies to synthesize a one-bead one-compound library of PTAs.

Peptidomimetics are great sources of protein ligands. The oligomeric nature of these compounds enables us to access large synthetic libraries on solid ...

Solid-phase Submonomer Synthesis of Peptoid Polymers and their Self-Assembly into Highly-Ordered Nanosheets

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and fold into higher order nanostructures. Structurally similar to peptides, peptoids are poly N-substituted glycines, where the side chains are attached to the nitrogen rather than the alpha-carbon. Their ease of synthesis and structural diversity allows testing of basic design principles to drive de novo design and engineering of new biologically-active and nanostructured materials.
Here, a simple manual peptoid synthesis protocol is presented that allows the synthesis of long chain polypeptoids ( up to 50mers) in excellent yields. Only basic equipment, simple techniques (e.g. liquid transfer, filtration), and commercially available reagents are required, making peptoids an accessible addition to many researchers' toolkits. The peptoid backbone is grown one monomer at a time via the submonomer method which consists of a two-step monomer addition cycle: acylation and displacement. First, bromoacetic acid activated in situ with N,N'-diisopropylcarbodiimide acylates a resin-bound secondary amine. Second, nucleophilic displacement of the bromide by a primary amine follows to introduce the side chain. The two-step cycle is iterated until the desired chain length is reached. The coupling efficiency of this two-step cycle routinely exceeds 98% and enables the synthesis of peptoids as long as 50 residues. Highly tunable, precise and chemically diverse sequences are achievable with the submonomer method as hundreds of readily available primary amines can be directly incorporated.
 
Peptoids are emerging as a versatile biomimetic material for nanobioscience research because of their synthetic flexibility, robustness, and ordering at the atomic level. The folding of a single-chain, amphiphilic, information-rich polypeptoid into a highly-ordered nanosheet was recently demonstrated. This peptoid is a 36-mer that consists of only three different commercially available monomers: hydrophobic, cationic and anionic. The hydrophobic phenylethyl side chains are buried in the nanosheet core whereas the ionic amine and carboxyl side chains align on the hydrophilic faces. The peptoid nanosheets serve as a potential platform for membrane mimetics, protein mimetics, device fabrication, and sensors. Methods for peptoid synthesis, sheet formation, and microscopy imaging are described and provide a simple method to enable future peptoid nanosheet designs.

A simple and general manual peptoid synthesis method involving basic equipment and commercially available reagents is outlined, enabling peptoids to be easily synthesized in most laboratories. The synthesis, purification and characterization of an amphiphilic peptoid 36mer is described, as well as its self-assembly into highly-ordered nanosheets.

Peptoids are a novel class of biomimetic, non-natural, sequence-specific heteropolymers that resist proteolysis, exhibit potent biological activity, and ...

Bioengineering

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

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

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.

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as ...

 Peptide Hydrogels: Methods for Environmentally Responsive Gelation of ECF-5

Preparation of Mechanically Stable Self-Assembled Peptides Hydrogels

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking networks. Their morphology closely resembles that of the extracellular matrix, allowing them to exhibit both the biological functions of peptides and responsive gelation properties. These unique characteristics have led to their extensive application in tissue engineering, three-dimensional cell culture, cancer therapy, regenerative medicine, and other biomedical fields. This article describes three methods for preparing ECF-5 peptide hydrogels using self-assembling peptides with environmentally responsive gelation processes: (1) pH-responsive gelation: varying pH levels induce the protonation or deprotonation of amino acid residues, altering electrostatic interactions between peptide molecules and promoting their self-assembly into hydrogels; (2) Metal ion addition: polyvalent metal ions chelate with negatively charged amino acid residues, acting as bridges between peptides to form a network hydrogel; (3) Solvent exchange: hydrophobic peptides are initially dissolved in non-polar organic solvents and subsequently induce self-assembly into hydrogels upon transitioning to a polar aqueous environment. These methods utilize conventional experimental procedures to facilitate peptide self-assembly into hydrogels. By designing peptide sequences to align with specific gelation-inducing conditions, it is possible to achieve finely tuned micro/nanostructures and biological functions, highlighting the significant potential of peptide hydrogels in the biomedical domain.

Author Spotlight: Improving the Production of Self-Assembling Fibers and Peptide Hydrogels for Superior Biocompatibility

This protocol presents three fast and simple preparation methods that use environmental conditions to trigger the self-assembly of peptides into hydrogels. Additionally, the characterization of peptide hydrogels is described, demonstrating that mechanically stable peptide hydrogels can be formed under these straightforward conditions.

Peptide hydrogels are highly hydrophilic, three-dimensional network gels formed by the self-assembly of nanofibers or polymers, creating water-locking ...

A Tripeptide-Stabilized Nanoemulsion of Oleic Acid

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The KYF-Pt-NE encapsulates Pt(II) at 10 wt. %, has a diameter of 107 &#177; 27 nm and a negative surface charge. The KYF-Pt-NE is stable in water and in serum, and is biologically active. The conjugation of a fluorophore to KYF allows the synthesis of a fluorescent nanoemulsion that is suitable for biological imaging. The synthesis of the nanoemulsion is performed in an aqueous environment, and the KYF-Pt-NE forms via self-assembly of a short KYF peptide and an oleic acids-platinum(II) conjugate. The self-assembly process depends on the temperature of the solution, the molar ratio of the substrates, and the flow rate of the substrate addition. Crucial steps include maintaining the optimal stirring rate during the synthesis, permitting sufficient time for self-assembly, and pre-concentrating the nanoemulsion gradually in a centrifugal concentrator.

This protocol describes an efficient method to synthesize a nanoemulsion of an oleic acids-platinum(II) conjugate stabilized with a lysine-tyrosine-phenylalanine (KYF) tripeptide. The nanoemulsion forms under mild synthetic conditions via self-assembly of the KYF and the conjugate.

We describe a method to produce a nanoemulsion composed of an oleic acids-Pt(II) core and a lysine-tyrosine-phenylalanine (KYF) coating (KYF-Pt-NE). The ...

Self-Assembly of Gamma-Modified Peptide Nucleic Acids into Complex Nanostructures in Organic Solvent Mixtures

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated media. In this article, we describe detailed protocols that enable the construction of nanofiber architectures in organic solvent mixtures through the self-assembly of uniquely addressable, single-stranded, gamma-modified peptide nucleic acid (&#947;PNA) tiles. Each single-stranded tile (SST) is a 12-base &#947;PNA oligomer composed of two concatenated modular domains of 6 bases each. Each domain can bind to a mutually complimentary domain present on neighboring strands using programmed complementarity to form nanofibers that can grow to microns in length. The SST motif is made of 9 total oligomers to enable the formation of 3-helix nanofibers. In contrast with analogous DNA nanostructures, which form diameter-monodisperse structures, these &#947;PNA systems form nanofibers that bundle along their widths during self-assembly in organic solvent mixtures. Self-assembly protocols described here therefore also include a conventional surfactant, Sodium Dodecyl Sulfate (SDS), to reduce bundling effects.

This article provides protocols for the design and self-assembly of nanostructures from gamma-modified peptide nucleic acid oligomers in organic solvent mixtures.

Current strategies in DNA and RNA nanotechnology enable the self-assembly of a variety of nucleic acid nanostructures in aqueous or substantially hydrated ...

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

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