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...

<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&#160;</td>
			<td>AappTec</td>
			<td>RTZ001</td>
			<td></td>
		</tr>
		<tr>
			<td>SynthwareTM synthesis vessel&#160;</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&#160;</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&#160;</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>&#945;-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&#160;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...

Watch this Scientific Journal Video about Facile Protocol for the Synthesis of Self-assembling Polyamine-based Peptide Amphiphiles PPAs and Related Biomaterials at JoVE.com

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). ...

This method can answer key questions on how to decorate peptide amphiphiles to polyamines and on the use of octagonal protecting groups The main advantage of this technique is that you can generate hybrid peptide amphiphiles with unprotected chemical building blocks containing multiple reactive sides. Demonstrating the procedure will be Mehdi, Nathalia and Krishnaiah, laboratory students from the Conda Sheridan Laboratory. First, carefully weigh out 2-Chlorotrityl chloride resin place the resin in a fritted, medium porocity, synthesis vessel with 50 milliliter capacity.Next, affix the synethesis vessel to a variable speed mechanical shaker and turn the vessel to a 45 degree angle to maximize agitation. Then add 15 milliliters of DCM to the resin. After allowing the resin beads to swell for 15 minutes, add eight equivalents of the desired polyamine to the resin, and allow it to react for five hours.After five hours, perform a kaiser test to confirm a successful reaction of the polyamine to the resin. Following this, protect the primary amine group by adding four equivalents of DDE in anhydrous methanol and shaking the reaction mixture overnight. On the following day, perform a kaiser test to confirm successful protection by the absence of blue color from the resin bead.Then, drain the DCM and wash the resin twice with a two to one mixture of DCM and DMF. Now, add 20 equivalents of Boc Anhydride in DCM to the resin and allow the reaction to proceed for three hours. After performing a chloranil test to confirm protection of the secondary amine, drain the solvent mixture and wash the resin twice with a two to one mixture of DCM and DMF.Following this, add 10 milliliters of a 2%solution of hydrazine in DMF to the resin. After shaking for one hour, perform a kaiser test to confirm successful deprotection of the primary amine. Next, mix four equivalents of the Fmoc protected amino acid, 3.95 equivalents of HBTU, and 15 equivalents of DIPEA.Add a one to one mixture of DCM and DMF and sonicate the cocktail until complete dissolution. Make sure the amino acid, coupling agent, and DIPEA are truly mixed and activated before adding the mixture to the resin. After waiting three to five minutes to ensure carboxylic acid activation, add the reaction mixture to the vessel containing the resin and allow the reaction to proceed for two to four hours at ambient temperature.Perform a kaiser or chloranil test at every step to confirm successful coupling. After performing a kaiser test to confirm successful coupling, deprotect the Fmoc group from the amino acid by adding 10 milliliters of a 20%solution of 4-Methylpyridine in DMF. Once the reaction is finished, perform a kaiser test to confirm successful deprotection of the amino acid.Then, wash the resin twice with 10 milliliters of DMF each wash lasting for 5 minutes, and finally with 10 milliliters of DCM for 10 minutes. After coupling all the required amino acids, conjugate the hydrophobic tail to the last amino acid by adding 10 equivalents of the desired carboxylic acid functionality to 9.5 equivalents of HBTU, and 12 equivalents of DIPEA. Sonicate the cocktail until complete dissolution, then add the cocktail to the vessel.Carry out the reaction for at least five hours, although it is advisable to carry it out overnight for the highest yield. Wash the resin with eight milliliters of DMF for two minutes, and twice with eight milliliters of DCM for five minutes. Before each addition, drain the solvent from the vessel.Once the last wash is performed, dry the resin under vacuum for 15 minutes. To prepare 15 milliliters of cleavage cocktail, add 14 milliliters of TFA to 0.5 milliliters of water, and 0.5 milliliters of Triisopropylsilane. Add this cleavage cocktail to the resin, and shake for two to four hours at room temperature.After the cleavage reaction is complete, collect the solution in a 50 milliliter round bottom flask. Then, concentrate the TFA in vacuo to one to two milliliters using a rotary evaporator at reduced pressure, while heating the mixture at 40 degrees Celsius After evaporation, add the obtained TFA solution, dropwise, to a round bottom flask containing 15 milliliters of anhydrous cold ether to precipitate the PPA. Next, add 5 milliliters anhydrous cold ether to the original flask containing the concentrated TFA solution.Sonicate to recover additional solids. Then, combine with the ether solution from the previous step. Cover the flask and place it inside the refrigerator overnight to maximize precipitation.On the following day, collect the precipitated material by vacuum filtration using a fine or medium pour size center disk filter funnel. Finally, wash the precipitate twice with five to ten milliliters of cold ether to remove any residual organics. The HPLC trace and MALDI Spectrum confirm the presence of the PPA product which should have a purity of greater than 95%for material characterization or biological evaluation.A single sharp peak is observed in the UV based HPLC trace and the MALDI Spectrum corresponds to that of the calculated molecular weight of the PPA within plus or minus one dalton. The self assembly of the PPAs can be visualized and analyzed by transmission electron microscopy, atomic force microscopy, small angle X-Ray scattering, scanning electron microscopy, and dynamic light scattering. Successful self assembly will result in well defined nanostructures, in both transmission electron microscopy and atomic force microscopy.Once mastered, this protocol can be done in three days if performed properly. While attempting this procedure, remember to purify the products and assess their purity before any subsequent studies. Following this procedure, other molecules, such as peptide amphiphiles, peptides, and peptide-polyamine hybrids can be prepared.After its development, this technique helps research in the field of self assembly peptide amphiphiles to explore the influence of polyamine nano structures in areas such as drug delivery, imaging, or catalysis After watching this video, you should have a good understanding of how to carry the synthesis of polyamine-based peptide amphiphiles and related peptide amphiphiles. Trifluoroacetic acid and 4-Methylpyridine can be hazardous, and precautions such as wearing gloves, lab coats and working in the fume hood should be taken while performing this procedure.

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7.9K vistas.  University of Nebraska Medical Center. This method can answer key questions on how to decorate peptide amphiphiles to polyamines and on the use of octagonal protecting groups The main advantage of this technique is that you can generate hybrid peptide amphiphiles with unprotected chemical building blocks containing multiple reactive sides. Demonstrating the procedure will be Mehdi, Nathalia and Krishnaiah, laboratory students from the Conda Sheridan Laboratory. First, carefully weigh out 2-Chlorotrityl chloride resin place the res...