Name: synthesis and characterization of 1,2-dithiolane modified self-assembling peptides
Uploaded: 2018-08-20T13:00:00
Description: Watch this Scientific Journal Video about Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides at JoVE.com

The authors would like to thank Dr. B. Ellen Scanley for her technical training and help using the TEM at the Connecticut State Colleges and University (CSCU) Center for Nanotechnology and Dr. Ishita Mukerji at Wesleyan University for access to her CD spectrophotometer. The work reported was in part supported by the Science Institute at Fairfield University, the NASA Connecticut Space Grant Consortium, and by the National Science Foundation under Grant Number CHE-1624774.

1. Synthesis and Purification of 1,2-Dithiolane Modified Peptide
<ol>
	<li>Synthesis of dithiolane precursor, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid19.
	<ol>
		<li>Add 1 g of 3-bromo-2-(bromomethyl)propionic acid (1 equiv.) dissolved in minimal amount of 1 M NaOH (approximately 4 mL) to a 25-mL round bottom reaction flask with stirring at 55 &#176;C. Seal the reaction flask with a septa and place under nitrogen atmosphere.</li>
		<li>Prepare a solution containing 1.49...

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disulfide+exchange:+Exposing+supramolecular+reactivity+through+dynamic+covalent+chemistry.">Disulfide exchange: Exposing supramolecular reactivity through dynamic covalent chemistry.</a> Chemical Society Reviews. 43, 1861-1872 (2014).</li><li>Vendetti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dihydroasparagusic+acid:+Antioxidant+and+tyrosinase+inhibitory+activities+and+improved+synthesis.">Dihydroasparagusic acid: Antioxidant and tyrosinase inhibitory activities and improved synthesis.</a> Journal of Agricultural and Food Chemistry. 61, 6848-6855 (2013).</li><li>Stawikowski, M., Fields, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+to+peptide+synthesis.">Introduction to peptide synthesis.</a> Current Protocols in Protein Science. 26, (2002).</li><li>Canadell, J., Goossens, H., Klumperman, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-healing+materials+based+on+disulfide+links.">Self-healing materials based on disulfide links.</a> Macromolecules. 44, 2536-2541 (2011).</li><li>Lafont, U., van Zeijl, H., van der Zwaag, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+cross-linkers+on+the+cohesive+and+adhesive+self-healing+ability+of+polydisulfide-based+thermosets.">Influence of cross-linkers on the cohesive and adhesive self-healing ability of polydisulfide-based thermosets.</a> ACS Applied Materials and Interfaces. 4, 6280-6288 (2012).</li><li>Komaromy, D., Stuart, M. C. A., Santiago, G. M., Tezcan, M., Krasnikov, V. V., Otto, S. <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+can+direct+dynamic+covalent+bond+formation+toward+diversity+or+specificity.">Self-assembly can direct dynamic covalent bond formation toward diversity or specificity.</a> Journal of the American Chemical Society. 139, 6234-6241 (2017).</li><li>McAvery, K. M., Guan, B., Fortier, C. A., Tarr, M. A., Cole, R. 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This article discusses the details of both the synthesis and the purification of an N-terminal 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting supramolecular structures. The synthesis of the 1,2-dithiolane peptide reported here has the advantages, including a one-step synthesis to produce the dithiolane precursor, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the on-resin microwave deprotection reaction of the precursor thioacetate protecting group to yield the oxidized ...

The robust peptide bond forming chemistry involved in solid-phase peptide synthesis and the ability to control sequence length and composition make the peptides that self-assemble into supramolecular structures a heavily researched field. The factors that control and stabilize peptide self-assembled structures, including side chain steric and electrostatic interactions, hydrogen bonding, and hydrophobic effects1, serve as a set of design rules. As the research into these fundamental design rules continues to progress, the logical next step in peptide self-assembly involves expanding the diversity of peptide-based structures and functions. While self-assembling peptides are a versatile biomaterial that have been used for many biomedical applications by tuning the peptide sequence or assembly conditions2,3,4, the development of strategies for post-assembly modifications to peptide nanofibers5,6,7,8,9 remains a relatively unexplored area.
Dynamic disulfide exchange and thiol chemistry at the surface of supramolecular structures is one area that has the potential to yield new and functional biomaterials. The incorporation of 1,2-dithiolane moieties (commonly a derivative of lipoic acid (la) or asparagusic acid (aa)) have been reported in liposome systems10,11, block copolymers12,13, and as organizing anchors at surfaces14,15. Herein, we report the synthesis and characterization of a self-assembling peptide derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease that is modified at the N-terminus with a 1,2-dithiolane functional group16,17. The resulting supramolecular fibers now serve as an experimental platform to study the disulfide-exchange and thiol reactivity at the supramolecular surface of amyloid fibers18.

<table>
	<tbody>
		<tr>
			<td>Rink amide MBHA resin, high load</td>
			<td>Gyros Protein Technologies</td>
			<td>RAM-5-HL</td>
			<td>Avoid contact with skin and eyes; do not inhale</td>
		</tr>
		<tr>
			<td>N,N-Dimethylformamide</td>
			<td>Fisher Scientific</td>
			<td>D119-4</td>
			<td>Flammable liquid and vapor; irritating to eyes and skin; Use personal protective equipment; keep away from open flame</td>
		</tr>
		<tr>
			<td>Fmoc-L-Val-OH</td>
			<td>Gyros Protein Technologies</td>
			<td>FLA-25-V</td>
			<td>Wear personal protective equipment; do not inhale</td>
		</tr>
		<tr>
			<td>Fmoc-L-Leu-OH</td>
			<td>Gyros Protein Technologies</td>
			<td>FLA-25-L</td>
			<td>Wear personal protective equipment; do not inhale</td>
		</tr>
		<tr>
			<td>Fmoc-L-Lys(Boc)-OH</td>
			<td>Gyros Protein Technologies</td>
			<td>FLA-25-KBC</td>
			<td>Wear personal protective equipment; do not inhale</td>
		</tr>
		<tr>
			<td>Fmoc-L-Phe-OH</td>
			<td>Gyros Protein Technologies</td>
			<td>FLA-25-F</td>
			<td>Wear personal protective equipment; do not inhale</td>
		</tr>
		<tr>
			<td>Fmoc-L-Ala-OH</td>
			<td>Gyros Protein Technologies</td>
			<td>FLA-25-A</td>
			<td>Wear personal protective equipment; do not inhale</td>
		</tr>
		<tr>
			<td>Fmoc-L-Gln(Trt)-OH</td>
			<td>Gyros Protein Technologies</td>
			<td>FLA-25-QT</td>
			<td>Wear personal protective equipment; do not inhale</td>
		</tr>
		<tr>
			<td>N,N,N&#8242;,N&#8242;-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate</td>
			<td>Gyros Protein Technologies</td>
			<td>26432</td>
			<td>Causes skin, eye and respiratory irritation; do not inhale; use under hood or in well ventilated area</td>
		</tr>
		<tr>
			<td>0.4 M N-methylmorpholine in DMF</td>
			<td>Gyros Protein Technologies</td>
			<td>PS3-MM-L</td>
			<td>highly flammable; wear personal protective equipment; keep away from heat and keep container tightly closed; do not inhale or swallow; wash skin thoroughly after handling</td>
		</tr>
		<tr>
			<td>20% piperidine in DMF</td>
			<td>Gyros Protein Technologies</td>
			<td>PS3-PPR-L</td>
			<td>Causes severe eye and skin burns; Flammable Liquid and vapor; Do not inhale</td>
		</tr>
		<tr>
			<td>dichloromethane</td>
			<td>Fisher Scientific</td>
			<td>D37-4</td>
			<td>May cause cancer; Do not inhale; Wear personal protective equipment; use under hood only; if contacted rise with water for at least 15 minutes and obtain medical attention</td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Fisher Scientific</td>
			<td>A998-4</td>
			<td>Flammable; irritating to eyes; Use personal protective equipment; Use only under a fume hood; keep away from open flame or hot surface; if contacted rinse wiith water for at least 15 minutes and obtain medical attention</td>
		</tr>
		<tr>
			<td>trifluoroacetic acid</td>
			<td>Fisher Scientific</td>
			<td>A116-50</td>
			<td>Causes severe burns; do not inhale; harmful to aquatic life; use personal protective equipment; use only under fume hood; if contacted rinse with water for at least 15 minutes and obain immediate medical attention</td>
		</tr>
		<tr>
			<td>4% uranyl acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400-4</td>
			<td>Do not inhale; harmful to aquatic life</td>
		</tr>
		<tr>
			<td>4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid</td>
			<td>Acros Organics</td>
			<td>AC172571000</td>
			<td>Do not inhale; use outdoors or in well-ventilated area</td>
		</tr>
		<tr>
			<td>nitrogen Gas</td>
			<td>TechAir</td>
			<td></td>
			<td>Contents under pressure, may explode if heated</td>
		</tr>
		<tr>
			<td>3-bromo-2-(bromomethyl)propionic acid</td>
			<td>Alfa Aesar</td>
			<td>AAA1963014</td>
			<td>Do not inhale; causes irritation to skin and eyes; corrosive</td>
		</tr>
		<tr>
			<td>sodium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>S318-100</td>
			<td>Use personal protective equipment; use only under fume hood; if contact rinse area for at least 15 minutes and obtain medical attention</td>
		</tr>
		<tr>
			<td>potassium thioacetate</td>
			<td>Acros Organics</td>
			<td>AC221300250</td>
			<td>Causes skin and eye irritation; do not inhale; use personal protective equipment</td>
		</tr>
		<tr>
			<td>sulfuric acid</td>
			<td>Fisher Scientific</td>
			<td>SA213</td>
			<td>Causes burns; keep away from water; keep away from combustible material; do not inhale; use personal protective equipment; if contact rinse area for at least 15 minutes and obtain medical attention</td>
		</tr>
		<tr>
			<td>chloroform-d</td>
			<td>Acros Organics</td>
			<td>AC320690075</td>
			<td>Possible cancer hazard; irritating to skin and eyes; do not inhale; Use personal protective equipment; use only under fume hood; If contact rinse area for at least 15 minutes and obtain medical attention</td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td>Fisher Scientific</td>
			<td>C298-4</td>
			<td>Possible cancer hazard; irritating to skin and eyes; do not inhale; Use personal protective equipment; use only under fume hood; If contact rinse area for at least 15 minutes and obtain medical attention</td>
		</tr>
		<tr>
			<td>N,N-diisopropylethylamine</td>
			<td>Acros Organics</td>
			<td>AC367841000</td>
			<td>Highly flammable; harmful to aquatic life; wear personal protective equipment; do not swallow</td>
		</tr>
		<tr>
			<td>ammonium hydroxide</td>
			<td>Fisher Scientific</td>
			<td>A669S-500</td>
			<td>Corrosive; do not inhale</td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Fisher Scientific</td>
			<td>A452-4</td>
			<td>Flammable liquid and vapor; use personal protective equipment; do not inhale; If contact rinse area for at least 15 minutes and obtain medical attention</td>
		</tr>
		<tr>
			<td>triisopropylsilane</td>
			<td>Sigma Aldrich</td>
			<td>233781</td>
			<td>Flammable; use personal proctective safety equipment; keep container tightly closed</td>
		</tr>
		<tr>
			<td>diethyl ether</td>
			<td>Fisher Scientific</td>
			<td>E138-1</td>
			<td>Extremely flammable; Irritating to skin and eyes; Use personal protective equipment</td>
		</tr>
		<tr>
			<td>2,5-dihydroxybenzoic acid</td>
			<td>Sigma Aldrich</td>
			<td>39319-10x10MG-F</td>
			<td>do not inhale; irritating to skin and eyes</td>
		</tr>
		<tr>
			<td>alpha-cyano-4-hydroxycinnamic acid</td>
			<td>Alfa Aesar</td>
			<td>AAJ67635EXK</td>
			<td></td>
		</tr>
		<tr>
			<td>c18 zip-tip</td>
			<td>Millipore</td>
			<td>ZTC18S096</td>
			<td></td>
		</tr>
		<tr>
			<td>tris(2-carboxyethyl) phospine hydrochloride</td>
			<td>Thermo Scientific</td>
			<td>PI20490</td>
			<td></td>
		</tr>
		<tr>
			<td>silica gel 60 F254 coated aluminum-backed TLC sheets</td>
			<td>EMD Millipore</td>
			<td>1.05549.0001</td>
			<td></td>
		</tr>
		<tr>
			<td>Thin walled Precision NMR tubes</td>
			<td>Bel-Art</td>
			<td>663000585</td>
			<td>5mm O.D.</td>
		</tr>
		<tr>
			<td>All-plastic Norm-Ject syringes</td>
			<td>Air Tite</td>
			<td>AL10</td>
			<td></td>
		</tr>
		<tr>
			<td>single-use needle</td>
			<td>BD PrecisionGlide</td>
			<td>BD 305185</td>
			<td>used needles get disposed on in sharps waste container</td>
		</tr>
		<tr>
			<td>disposable fritted syringe</td>
			<td>Torviq</td>
			<td>SF1000LL</td>
			<td>10mL fritted syringes were used in the report, but larger syringes are avaibale if needed for larger scale synthesis.</td>
		</tr>
		<tr>
			<td>carbon grid</td>
			<td>Ted Pella, Inc.</td>
			<td>CF200-CU</td>
			<td>Make sure to prepare samples and staining on the carbon grid side, not the shiny copper side of grid</td>
		</tr>
		<tr>
			<td>self-closing tweezers</td>
			<td>Electron Microscopy Sciences</td>
			<td>78318-3X</td>
			<td>very sharp tips, length: 120 mm</td>
		</tr>
		<tr>
			<td>0.1 mm short path length cell</td>
			<td>Starna Cells, Inc.</td>
			<td>20/C-Q-0.1</td>
			<td>Fragile</td>
		</tr>
		<tr>
			<td>10mL Vessel Caps</td>
			<td>CEM</td>
			<td>909210</td>
			<td></td>
		</tr>
		<tr>
			<td>10mL Pressure Vessels</td>
			<td>CEM</td>
			<td>908035</td>
			<td></td>
		</tr>
		<tr>
			<td>Aeris Semi-Prep HPLC column</td>
			<td>Phenomenex</td>
			<td>00F-4632-N0</td>
			<td>150 x 10mm</td>
		</tr>
		<tr>
			<td>cell holder</td>
			<td>Starna Cells, Inc.</td>
			<td>CH-2049</td>
			<td>Needed when using short pathlength cells</td>
		</tr>
		<tr>
			<td>PS3 peptide synthesizer</td>
			<td>Gyros Protein Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DiscoverSP Microwave Reactor</td>
			<td>CEM</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>centrifuge</td>
			<td>HERMLE</td>
			<td>Z 206 A</td>
			<td>used a fixed 6x50 mL rotor</td>
		</tr>
		<tr>
			<td>HPLC</td>
			<td>Shimadzu</td>
			<td></td>
			<td>UV Detector</td>
		</tr>
		<tr>
			<td>nuclear magnetic resonance spectrometer</td>
			<td>Avance, Bruker</td>
			<td></td>
			<td>300 MHz</td>
		</tr>
		<tr>
			<td>MALDI-TOF mass spectrometer</td>
			<td>Axima Confidence, Shimadzu</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>lyophilizer</td>
			<td>Millrock Technology</td>
			<td>BT85A</td>
			<td></td>
		</tr>
		<tr>
			<td>Fourier-Transform Infrared Spectrometer</td>
			<td>Alpha Tensor, Bruker</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission Electron Microscope</td>
			<td>Tecnai Spirit, FEI</td>
			<td></td>
			<td>Used with Gatan Orius Fiberoptic CCD digital camera. Accessed at CSCU Center for Nanotechnology</td>
		</tr>
		<tr>
			<td>Circular Dichroism Spectropolarimeter</td>
			<td>J-810, JASCO</td>
			<td></td>
			<td>Used with a six-cell Peltier temperature controller. Accessed at Wesleyan University.</td>
		</tr>
	</tbody>
</table>

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.

Aside from the initial one-step synthesis of the dithiolane precursor molecule, the rest of the 1,2-dithiolane modified peptide synthesis occurs on solid support (Figure 1A). The conversion of 3-bromo-2-(bromomethyl)propionic acid to 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, the dithiolane precursor, is confirmed by 1H and 13C NMR (Figure 1B and C) before it is coupled to the free N...

Watch this Scientific Journal Video about Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides at JoVE.com

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

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 ...

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