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 g of potassium thioacetate (3.2 equiv.) in 4 mL of deionized water and 3 mL of 2 M sulfuric acid (H2SO4) to create thioacetic acid in situ.</li>
		<li>Pull the thioacetic acid solution into a plastic disposable 10 mL syringe and place a needle on the syringe. Add the mixture dropwise to the reaction flask by piercing through the septa with the needle. Continue the reaction overnight at 55 &#176;C.</li>
		<li>Monitor the reaction by thin layer chromatography (TLC) on silica gel 60 F254 plates using a mixture of methanol and dichloromethane (1:9). Visualize the reaction progress by bromocresol green stain. The product has an Rf = 0.57.</li>
		<li>After the reaction is complete and cooled to room temperature, acidify the mixture to pH 1 with 2 M H2SO4. A yellow oil separates out of solution.</li>
		<li>Extract the product with cold chloroform (40 mL x 3). Combine the organic layers and dry over magnesium sulfate. Remove the chloroform under reduced pressure.</li>
		<li>Confirm the identity of the isolated product, 1, by nuclear magnetic resonance (NMR) spectroscopy as shown in Figures 1B and C. Expect the following results: 1H NMR, CDCl3, 300 MHz: d = 10.1 (b, 1 H), 3.2 (m, 4 H), 2.9 (m, 1 H), 2.4 (s, 6 H); 13C NMR, CDCl3, 75 MHz: d = 195.1 (CH3COS-), 177.6 (-COOH), 45.1 (-CH2S-), 30.5 (CH), 29.2 (-SCOCH3). 
		Note: The product is a yellow oil and has an overall yield of 83%. Use the product without further purification.</li>
	</ol>
	</li>
	<li>SPPS and On-Resin Coupling of Dithiolane Precursor 
	Note: The solid-phase peptide synthesis described below was conducted on an automated peptide synthesizer, following the recommended manufacturer&#8217;s protocols. Settings and reagents may be adapted for other commercial instruments or when using specialized amino acids.
	<ol>
		<li>Weigh out 0.156 g of Rink amide 4-methylbenzhydrylamine (MBHA) resin (0.1 mmol) and place in a reaction vessel. Swell the resin in dimethylformamide (DMF) for at least 15 min prior to the start of the synthesis.</li>
		<li>Weigh out 4 equivalents of each fluorenylmethyloxycarbonyl (Fmoc) protected amino acid (0.4 mmol) in the sequence and 0.152 g of N,N,N&#8217;,N&#8217;-tetramethyl-O-(1H-benotriazol-1-yl)uranium hexafluorophosphate (0.4 mmol, HBTU) for each amino acid in the sequence. Each cartridge contains both the Fmoc-protected amino acid and HBTU.</li>
		<li>After running all pre-synthesis checks on the synthesizer (refill reagents, flushed reagent lines and pressurized all reagent bottles), place the amino acid cartridges into the carousel in the C&#8217; to N&#8217; terminus direction. Place an empty cartridge after the final amino acid position for the final N-terminal Fmoc deprotection step.</li>
		<li>Synthesize the peptide using the standard recommended settings.
		<ol>
			<li>Deprotect the Fmoc group from the resin with 5 mL of 20% piperidine in DMF (5 min x 2).</li>
			<li>Wash the resin with DMF (6 x 5 mL) before the coupling step.</li>
			<li>For a single coupling step, add 4 mL of 0.4 M N-methylmorpholine in DMF to the Fmoc-protected amino acid and HBTU. Activate the Fmoc-protected amino acid solution for 30 s before transferring the solution to the reaction vessel. 
			Note: The automated peptide synthesizer mixes the resin and the solution by bubbling N2 gas every 30 s for 20 min while the coupling reaction takes places. For manual peptide synthesis, place the reaction vessel on an orbital shaker at low speed for the duration of the coupling step.</li>
			<li>Drain the solution and wash the resin with DMF (3 x 5 mL).</li>
			<li>Repeat steps 1.2.4.1 through 1.2.4.4 for each Fmoc-protected amino acid in the C&#8217; to N&#8217;-terminus direction to synthesize the peptide of interest.</li>
		</ol>
		</li>
		<li>After the final N-terminal deprotection step, transfer the resin into a disposable fritted syringe. Wash the resin with DMF (3 x 5 mL) and dichloromethane (DCM, 3 x 5 mL). 
		Note: The resin may be stored after DCM washing in a vacuum desiccator. If the resin was stored previous to coupling, ensure to swell the resin in DMF before the coupling reaction.</li>
		<li>Couple the dithiolane precursor (1) to the N-terminus of the on-resin peptide by adding 4 equivalents of 1, 5 mL of DMF, 4 equivalents of HBTU and 10 equivalents of N,N-diisopropylethylamine (DIPEA). Pre-activate the coupling mixture for 10 min before adding to the resin containing fritted syringe.</li>
		<li>Shake the coupling reaction for 2 h. After 2 h, wash the resin with DMF (3 x 5 mL) and repeat the coupling reaction with shaking overnight.</li>
		<li>After the second coupling, wash the resin with DMF (3 x 5 mL) and DCM (3 x 5 mL). 
		Note: The resin can be stored at this point under vacuum until cleavage.</li>
	</ol>
	</li>
	<li>Thioacetate Deprotection and Peptide Cleavage from Resin
	<ol>
		<li>To deprotect the thioacetate group from the N-terminus dithiolane precursor, transfer the dried resin to a 10 mL microwave reaction tube and add 2 mL of DMF. Allow the resin to swell, add a small magnetic stir bar to the vessel, and re-suspend with a low speed of magnetic stirring for 15 min.</li>
		<li>Add 2 mL of concentrated ammonium hydroxide, cap the reaction vessel with the silicone septa, and place the reaction vessel into a microwave reactor using the microwave settings of 75 &#176;C for 45 min with stirring.</li>
		<li>After the microwave reaction is complete, transfer the resin into a clean disposable fritted syringe. Wash with DMF (2 x 5 mL) and methanol (MeOH, 2 x 5 mL).</li>
		<li>Add a solution of concentrated ammonium hydroxide in methanol (1:4), for a total volume of 5 mL. Leave to shake overnight to increase intramolecular oxidation of disulfide bond in dithiolane ring.</li>
		<li>Wash the resin with MeOH (2 x 5 mL) and DCM (3 x 5 mL). 
		Note: The dried resin may be stored in a vacuum desiccator at this point.</li>
		<li>Add the cleavage cocktail to the resin containing syringe with gently shaking for 1.5 h. The cleavage cocktail used is 95% trifluoroacetic acid (TFA), 2.5% triisopropylsilane (TIPS), and 2.5% water at a total volume of 5 mL. 
		CAUTION: Work under chemical fume hood only. TFA is volatile and corrosive. 
		Note: For the majority of peptide sequences and amino acid sidechain protecting groups, the above cleavage cocktail solution is sufficient; however, alternative cleavage cocktails may be needed for certain amino acid side chain protecting groups (in particular peptides containing Cys, Met, Trp and Arg) or other resin chemistry20.</li>
		<li>Precipitate the crude peptide into 25 mL of cold diethyl ether in a 50 mL conical tube by dropwise addition from the fritted syringe. The peptide precipitates as a white solid. Pellet the peptide by centrifugation at 1300 x g for 10 min. Carefully decant the diethyl ether into a separate container for waste collection.</li>
		<li>Add another 25 mL of diethyl ether to the conical tube and re-suspend the precipitate by vortexing. Repeat the centrifugation at 1300 x g for 10 min and decant the diethyl ether again. Dry the pellet under vacuum.</li>
	</ol>
	</li>
	<li>Purification of 1,2-Dithiolane Modified Peptide 
	Note: Purify the crude peptide by reverse phase-HPLC. Collect and combine the peptide peaks and confirm the mass by MALDI-TOF mass spectrometry.
	<ol>
		<li>Dissolve the crude peptide pellet in minimal amount of acetonitrile with 0.1% TFA. Due to the peptide&#8217;s hydrophobicity and aggregation propensity, gently heat the sample at 40 &#176;C to aid in solubility. 
		Note: Avoid higher temperatures and sonication of the peptide in order to prevent potential disulfide exchange reactions21,22,23.</li>
		<li>To prepare 1 mL of crude peptide for HPLC purification, add 400 &#956;L of concentrated peptide stock in acetonitrile to 600 &#956;L of H2O with 0.1 % TFA and filter through a 22 &#956;m syringe filter into a HPLC vial. An additional 5% isopropanol can be added to prevent peptide aggregation and precipitation.</li>
		<li>Purify the peptide using C-18 semi-preparative column with a flow rate of 3 mL/min over a linear gradient of 15-55% acetonitrile in 20 min. Set the UV detectors to 222 nm (amide backbone) and 330 nm (disulfide bond). Collect and combine the peaks of interest (Figure 2A).</li>
		<li>Confirm the peptide product mass by MALDI-TOF mass spectrometer in the reflectron mode (Figure 2B). For analysis, mix 0.5 &#956;L of collected peak on the MALDI plate with 0.5 &#956;L of 2,5-dihydroxybenzoic acid (DHB) matrix (10 mg/mL DHB in 50% acetonitrile, 0.1% TFA). 
		Note: Common adducts in MALDI-TOF mass spectrometry include the sodium and potassium salt adduct ([M+Na]+ and [M+K]+) peaks. Desalting the sample prior to the analysis is recommended if the salt adduct peaks suppress the signal of the main [M+H]+ peak. Additionally, an oxidized peak of [M+O]+ is also detected in the 1,2-dithiolane modified peptide. A report on laser induced oxidation from the MALDI ionization using DHB matrix suggests that factors including sample concentration, solvent, and laser intensity can be modified to limit the MALDI induced oxidation artifact24.</li>
		<li>After MALDI-TOF confirmation of the correct mass, lyophilize the peptide after flash freezing. Keep the lyophilized peptide powder under vacuum until assembly.</li>
	</ol>
	</li>
</ol>
2. Characterization of Supramolecular Self-Assembly Structures
<ol>
	<li>Formation of Amyloid Fibers
	<ol>
		<li>To prepare self-assembly solution, weigh out 1 mg of the peptide powder using an analytical balance. Dissolve into a mixture (pH 7.5) of 20% acetonitrile and 10 mM (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) in a 1.5 mL microcentrifuge tube, to a final concentration of 1 mg/mL peptide assembly mixture. Vortex the assembly solution and leave to assemble at room temperature.</li>
	</ol>
	</li>
	<li>Spectroscopic Characterization of Amyloid Fibers
	<ol>
		<li>Follow the peptide assembly process via Fourier-transform infrared (FTIR) spectroscopy every few days. A broad peak centered around 1670 cm-1 is the IR signature arising from unassembled peptides in the sample17. The peptide assembly samples usually take one to two weeks for the broad unassembled peak to disappear and reach maturation.
		<ol>
			<li>Dry an aliquot of 8-10 &#956;L of the assembly solution as a thin film on the ATR diamond crystal. Monitor the disappearance of a large and broad water peak from 1640 to 1630 cm-1 as the dry film forms.</li>
			<li>Acquire IR spectra from 1500-1800 cm-1 averaging 50 scans with a 2 cm-1 resolution. Acquire and subtract the background scans prior to each sample scan. The IR signature for &#946;-sheet assembly is a sharp peak between 1625 and 1635 cm-1 (Figure 3A)25,26.</li>
		</ol>
		</li>
		<li>Characterize the peptide assembly into &#946;-sheet rich supramolecular structures by circular dichroism (CD). Record the spectra using a CD spectropolarimeter with a Peltier temperature control system.
		<ol>
			<li>Pipette 30 &#956;L of the assembly solution in a 0.1 mm path length microcuvette. 
			Note: A cell holder is needed to clamp and position the short path length cell in the instrument.</li>
			<li>For each spectrum, set the CD instrument to the following parameters: scanning wavelengths of 300 nm to 180 nm, scanning rate of 100/min, band width of 1 nm, 25 &#176;C, average of three scans.</li>
			<li>Collect a spectrum of the buffer (20% acetonitrile/10 mM HEPES, pH 7.5) and subtract from each sample scan as a control. The CD signature for &#946;-sheets is an ellipticity minimum centered around 220 nm (Figure 3B)27.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Microscopy of Amyloid Fibers 
	<ol>
		<li>Allow two to three weeks for the peptide samples to mature into &#946;-sheet rich supramolecular structures. 
		Note: The assemblies can be imaged using transmission electron microscopy (TEM) at earlier stages of the assembly process as well.
		<ol>
			<li>Pipette 10 &#956;L of the peptide assembly solution onto the surface of TEM carbon grid. 
			Note: Take care not to touch the pipette tip to the grid surface. High precision, self-closing tweezers are used to hold the TEM grid during preparation.</li>
			<li>Wait 1-2 min to allow the assemblies to adsorb onto the grid surface. Remove excess sample by touching filter paper to the edge of the grid.</li>
			<li>Prepare a 2% uranyl acetate stain by adding 100 &#956;L of deionized water to commercially available 4% uranyl acetate solution. Pipette 10 &#956;L of the 2% uranyl acetate stain onto the grid surface and incubate for 2-3 min. After the incubation, remove excess stain by touching filter paper to the edge of the grid.</li>
			<li>Place the TEM grids in a vacuum desiccator overnight. Store under vacuum until imaging.</li>
			<li>Image the prepared samples with TEM (Figure 3C). Typical parameters for microscopy are as follows: images at magnifications ranging from 9,300X to 23,000X, tungsten filament with an accelerating voltage of 120 kV. 
			Note: ImageJ can be used to measure average fiber width of the supramolecular structures of acquired TEM images28. 
			CAUTION: Please consult all relevant safety data sheets (SDS) before use. Several chemicals used in the synthesis, purification, and characterization of the described 1,2-dithiolane modified self-assembling peptides are corrosive or toxic and should only be used under a chemical fume hood. Always use appropriate personal protective equipment (including safety glasses, lab coat, full length pants, closed-toe shoes) when working in the laboratory.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+[FeFe]-hydrogenase:+On+resin+modification+of+an+amino+acid+to+anchor+a+hexacarbonyldiiron+cluster+in+a+peptide+framework.">Artificial [FeFe]-hydrogenase: On resin modification of an amino acid to anchor a hexacarbonyldiiron cluster in a peptide framework.</a> European Journal of Inorganic Chemistry. 2011, 1050-1055 (2011).</li><li>Van Duinen, S. G., Castano, E. M., Prelli, F., Bots, G. T. A. B., Luyendijk, W., Frangione, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hereditary+cerebral+hemorrhage+with+amyloidosis+in+patients+of+Dutch+origin+is+related+to+Alzheimer+disease.">Hereditary cerebral hemorrhage with amyloidosis in patients of Dutch origin is related to Alzheimer disease.</a> Proceedings of the National Academy of Sciences of the United States of America. 84, 5991-5994 (1987).</li><li>Barth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+infrared+absorption+of+amino+acid+sidechains.">The infrared absorption of amino acid sidechains.</a> Progress in Biophysics and Molecular Biology. 74, 141-173 (2000).</li><li>Jayaraman, M., 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=Slow+amyloid+nucleation+via+&#945;-helix-rich+oligomeric+intermediates+in+short+polyglutamine-containing+Huntingtin+fragments.">Slow amyloid nucleation via &#945;-helix-rich oligomeric intermediates in short polyglutamine-containing Huntingtin fragments.</a> Journal of Molecular Biology. 415, 881-899 (2012).</li></ol>

The authors have nothing to disclose.

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 1,2-dithiolane moiety utilizing ammonium hydroxide as a safer alternative to the toxic hydrazine deprotection reported previously29. The overall solid-phase peptide synthesis of the 1,2-dithiolane peptide (Figure 1A) can be easily modified by changing the sequence length and composition, including using Fmoc-protected unnatural amino acids, and C-terminal resin chemistry to suit many different research applications.
In order to prevent unwanted peptide by-products when the amino acids with nucleophilic side chains or the amino acids with protecting groups have reactive cleavage products, additional scavengers should be added to the cleavage cocktail20. A test cleavage of a small portion (less than 10% of total resin) may be performed before the on-resin microwave deprotection step to ensure a high yield of coupling reaction. Using the column and HPLC conditions described in the report, the thioacetate protected 1,2-dithiolane precursor peptide peak elutes 5 min after the uncoupled free amine peptide. If a significant amount of free amine peptide remains, another coupling step with 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid is recommended. The intramolecular 1,2-dithiolane disulfide bond is detected on the HPLC by monitoring the weak disulfide bond absorbance at 330 nm. The HPLC peak corresponding to the oxidized 1,2-dithiolane is confirmed by adding 100 L of 100 mM tris(2-carboxyethyl)phosphine(TCEP) to a peptide sample in a separate HPLC run. The TCEP reduced thiol containing peptide will have a different retention time than the oxidized disulfide peptide. The thiol containing peptide elutes approximately 1 min later than the disulfide peptide using the column and conditions described in the report. A suitable alternative MALDI matrix for peptides is &#945;-cyano-4-hydroxycinnamic acid (CHCA) and is prepared by adding 1 mL of 50% acetonitrile with 0.1% TFA to 5 mg of CHCA. The samples can be desalted by using a C18 zip-tip prior to MALDI-TOF mass analysis if the salt adduct (Na+ and K+) peaks significantly suppress the [M+H]+ peak.
The peptide sequence chosen for these studies is derived from Dutch mutant of the A&#946;-peptide associated with Alzheimer&#39;s disease30, and has previously been shown to self-assemble into amyloid fibers of parallel &#946;-sheets16,17. As shown in the representative results, Figure 3A-C, the 1,2-dithiolane-KLVFFAQ-NH2 peptide also assembles into amyloid fibers. The FTIR amide I stretch centered at 1627cm-1 and CD ellipticity minimum at 218 nm are spectroscopic signatures of &#946;-sheet assemblies25,26,27, and the carbonyl CO stretch at 1676 cm-1 corresponding to ordered glutamine side-chain interactions suggest that the peptides are organized in parallel &#946;-strands16,31,32.
Transmission electron microscopy images, using 2% uranyl acetate negative stain, show extended and twisting supramolecular fibers that are approximately 10 nm in width. The fibers of the N-terminal acetylated peptide, Ac-KLVFFAQ-NH2, are smooth and straight, with a slightly larger width of almost 12 nm16. Since preparing TEM grids and acquiring images is a time intensive process, it is best to prepare several TEM grids from the same assembly sample at once of varying peptide concentrations. Prepare a diluted sample for TEM (1:50 or 1:100) alongside the original assembly solution sample. The amyloid fibers at high concentrations may adhere to the grid in clumps or patches and acquiring images of individual fibers may be difficult. It is important to see the edges of individual fibers for width measurements and the visualization of fiber helicity is often obscured by overcrowded samples.
As more applications using supramolecular biomaterials are being explored, there is a need to integrate potentially reactive functional groups that are orthogonal to the self-assembly process into the self-assembling monomers. The methods described in this report highlight an on-resin approach to the synthesis of a 1,2-dithiolane modified self-assembling peptide and spectroscopic characterization of the supramolecular structures.

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&amp;prime;,N&amp;prime;-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-terminus amine of a peptide still on-resin. The deprotection of thioacetate to thiols with ammonium hydroxide is performed using a microwave reactor and the 1,2-dithiolane is oxidized overnight in methanol before the 1,2-dithiolane modified peptide is cleaved from the resin. The crude peptide is purified by reverse phase HPLC (Figure 2A) and the product&#39;s mass is confirmed by MALDI-TOF mass spectrometry (Figure 2B).
The purified 1,2-dithiolane peptide self-assembles into mature amyloid fibers over a 2-3 week period. FT-IR (Figure 3A) and CD spectroscopy (Figure 3B) are used to follow the assembly process and to characterize the extended &#946;-sheet conformation. The fibers are imaged by TEM (Figure 3C).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58135/58135fig1.jpg" /> 
Figure 1. Synthetic scheme for the characterization of 1,2-dithiolane precursor molecule. (A) Synthetic scheme of the final 1,2-dithiolane modified peptide, 1,2-dithiolane-KLVFFAQ-NH2. (B) 1H-NMR of 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid in CDCl3 at 300 MHz. (C)13C-NMR of 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid in CDCl3 at 75 MHz. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58135/58135fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58135/58135fig2.jpg" /> 
Figure 2. Synthesis of 1,2-dithiolane modified peptide. (A) HPLC chromatogram from the purification of 1,2-dithiolane -KLVFFAQ-NH2. (B) MALDI-TOF mass spectrum of the main peak from HPLC purification (retention time of ~17.5 min) in reflectron mode using DHB matrix confirms the calculated mass of 1,2-dithiolane-KLVFFAQ-NH2. Common adducts are also identified. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58135/58135fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58135/58135fig3.jpg" /> 
Figure 3. Supramolecular characterization of 1,2-dithiolane modified peptide. (A) FT-IR of 1 mg/mL 1,2-dithiolane-KLVFFAQ-NH2 fibers assembled in 10mM HEPES, pH 7.5 in 20% CH3CN. The peak at 1627 cm-1 is consistent with the peptides assembled in a &#946;-sheet conformation. (B) CD of 1 mg/mL 1,2-dithiolane-KLVFFAQ-NH2 fibers assembled in 10 mM HEPES, pH 7.5 in 20% CH3CN. The ellipticity minimum at 218 nm is consistent with the peptides assembled in a &#946;-sheet conformation. (C) Image of the 1,2-dithiolane-KLVFFAQ-NH2 amyloid fiber (negative stain of 2% uranyl acetate) by TEM. Scale bar is 100 nm. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58135/58135fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

synthesis-characterization-12-dithiolane-modified-self-assembling

Nanyang Technological University

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Chemistry

7.1K Views.  Fairfield University. 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.

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

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 ...

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

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

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

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 ...

Biochemistry

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 ...

Evaluating Biofunctional Self-Assembling Peptides for Cell Adhesion and Morphology

Fabrication and Characterization of Colorectal Cancer Organoids from SW1222 Cell Line in Ultrashort Self&#45;Assembling Peptide Matrix

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of hydrogels that are biocompatible, biodegradable, and non-immunogenic. We have previously proven that SAPs, when biofunctionalized with protein-derived motifs, can mimic the extracellular matrix characteristics that support colorectal organoid formation. These biofunctional peptide hydrogels retain the original parent peptide's mechanical properties, tunability, and printability while incorporating cues that allow cell-matrix interactions to increase cell adhesion. This paper presents the protocols needed to evaluate and characterize the effects of various biofunctional peptide hydrogels on cell adhesion and lumen formation using an adenocarcinoma cancer cell line able to form colorectal cancer organoids cost-effectively. These protocols will help evaluate biofunctional peptide hydrogel effects on cell adhesion and luminal formation using immunostaining and fluorescence image analysis. The cell line used in this study has been previously utilized for generating organoids in animal-derived matrices.

Author Spotlight: Optimization of Ultrashort Peptide Matrices for Colorectal Cancer Organoids

This protocol aims to evaluate biofunctional self-assembling peptides for cell adhesion, organoid morphology, and gene expression by immunostaining. We will use a colorectal cancer cell line to provide a cost-effective way of obtaining organoids for intensive testing.

Ultrashort self-assembling peptides (SAPs) can spontaneously form nanofibers that resemble the extracellular matrix. These fibers allow the formation of ...