The authors acknowledge financial support from the Natural Science Foundation of China Grants (No. 21372023, 21778009 and 81701818); the Ministry of Science and Technology of the People&#39;s Republic of China (No. 2015DFA31590); the Shenzhen Science and Technology Innovation Committee (No. JCYJ20170412150719814, JCYJ20170412150609690, JCYJ20150403101146313, JCYJ20160301111338144, JCYJ20160331115853521, JSGG20160301095829250, and GJHS20170310093122365); and the China Postdoctoral Science Foundation (No. 2017M610704).

1. Equipment Preparation
<ol>
	<li>For the manual peptide-synthesis apparatus, place a vacuum manifold (Table of Materials) in an efficient fume hood. Next, place three-way stopcocks onto the vacuum manifold and connect them to a nitrogen or argon gas line. Cap all unused inlets using rubber septa.</li>
	<li>Connect resin-filled columns (0.8 x 4 cm, 10-mL reservoir, see Table of Materials) to the manifold using the three-way stopcocks (Figure 1). Use a pump connected to a vacuum system as vacuum filtration or a rubber pipette bulb by extruding to remove the solvent in the columns.</li>
	<li>Use a photoreactor (Figure 2), equipped with ten 350 nm lamps (Table of Materials) for UV irradiation. Connect these to an argon gas tank via the photoreactor air inlet to ensure that the photoreactor is filled with argon gas prior to and during the photoreactions.</li>
	<li>Before switching on the UV lamp of the photoreactor, ensure that the photoreactor door is closed in case there is irradiation from the UV light.</li>
</ol>
2. Resin Preparation
NOTE: In general, the construction of peptide substrates is carried out using Fmoc-based solid-phase peptide synthesis protocols. These are carried out using the rink amide resin which leaves a C-terminal amide remaining following peptide cleavage. This protocol is used throughout the paper.
CAUTION: N,N-dimethylformamide (DMF), dichloromethane (DCM), 4-methylmorpholine (NMM), and N,N-diisoproylethylamine (DIPEA) are toxic and harmful by inhalation, ingestion, or skin contact. Diethyl ether is highly flammable. Trifluoroacetic acid (TFA) is corrosive. 1,2-ethanedithiol (EDT) is highly malodorous. Therefore, all organic solvents and chemicals should be handled with appropriate personal protective equipment (nitrile gloves, lab coat, and protective eyeglasses) and handled inside a chemical fume hood.
<ol>
	<li>Calculate the amount of resin required using the following formula: 
	scale (mmol) / (resin loading capacity (mmol/g) 1,000 (mg/g)) = mass of resin (mg) 
	E.g., the amount of rink amide MBHA resin (0.5 mmol/g) for 25 &#181;mol = 0.025 mmol / (0.5 mmol/g 1,000 mg/g) = 50 mg. Next, weigh 50 mg of resin in a column and set it up on the vacuum manifold using three-way stopcocks.</li>
	<li>Add 1 - 2 mL of DCM to the resin in a column (0.8 x 4 cm, 10-mL reservoir). To swell the resin, gently agitate it using a nitrogen or argon stream for 10 min. Next, remove the solvent using vacuum filtration.</li>
</ol>
3. N-terminal Fmoc Deprotection and Washing
<ol>
	<li>Prepare the N-terminal Fmoc deprotecting solution: prepare an adequate volume (200 mL) of 50% (v/v) morpholine in DMF in a glass bottle.</li>
	<li>Add 1 - 2 mL of the deprotection solution to the resin, gently agitate it for 10 min and then drain the solution using a vacuum. Using DMF (1 - 2 mL) and DCM (1 - 2 mL) in that order, wash the resin and the reaction vessel thoroughly for a total of 3x. Next, repeat the deprotection and wash procedures 1x.</li>
</ol>
4. Fmoc-protected Amino Acid Coupling
<ol>
	<li>Using the coupling of alanine residue as an example, in the case of a 25 &#181;mol-scale manual synthesis, weigh Fmoc-Ala-OH (5 equiv., 41.4 mg), 2-(6-chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU; 4.9 equiv., 50.5 mg) in a polypropylene container and dissolve it in DMF (0.5 mL).</li>
	<li>Add DIPEA (10 equiv., 43.5 &#181;l) in order to generate a 0.25-M activated amino acid solution ( Table 1). Following an approximate 1 min pre-activation, add the solution to the resin, and then bubble it with N2 for approximately 1 - 2 h.</li>
	<li>From this step on, incorporate each amino acid in the peptide chain as a sequence of steps: first the deprotection of the N-terminal Fmoc-group, and then the washing, followed by the coupling of the amino acid via activation using HCTU. 
	NOTE: A longer coupling time (e.g., 2 h) is recommended if coupling following a sterically hindered amino acid residue [e.g., Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-His(Trt)-OH, Fmoc-Arg(Pbf)-OH]. The alkene/alkyne bearing un-natural amino acids are used in 3 equivalents instead of 5 and are left to react for 3 h.</li>
	<li>Monitor the peptide synthesis progress using Kaiser or chloranil tests as described by Arora et al.36 These tests provide qualitative assessments of the presence or absence of free primary and secondary amines. Alternatively, approximately 2 - 3 mg of the peptide can be cleaved from the resin and analyzed by LC-MS.</li>
</ol>
5. Thiol-ene Hydrothiolation and Thiol-yne Cyclization
<ol>
	<li>Construct thioether linker through on-resin cyclization (Figure 3).
	<ol>
		<li>Prepare approximately 50 mL of the Trt deprotection solution (TFA/TIS/DCM 0.03/0.06/1.0). Treat the Cys- and mS5-bearing resin [NH2-R-mS5-A-A-A-Cys(Trt)-R&#39;-resin, 50 mg] with 1 - 2 mL of the Trt deprotection solution in a 10-mL column. Gently agitate the solution for 10 min using N2. Finally, wash it with DCM (1 -2 mL) for a total of 3x. 
		NOTE: MS5 represents the alkylene-bearing building block (see the structure depicted in Figure 6) used for peptide coupling and thio-ene cyclization38.</li>
		<li>Repeat the procedure described above for a total of approximately 6x in order to remove the trityl protection group of cysteine, until the solution color is no longer yellow.</li>
		<li>Bubbled with N2, wash the R-mS5-A-A-A-Cys(-SH)-R&#39;-resin [cysteine with free thio (-SH)] with DMF (1 - 2 mL) and DCM (1 - 2 mL) in that order. Shrink the resin using methanol (1 - 2 mL) for 2 min and then remove the solvent using filtration. Next, sequentially dry the resin under a steam of N2 gas for approximately 5 min in the column.</li>
		<li>Prepare degassed DMF beforehand, within a mouth flask, by bubbling nitrogen gas for approximately 1 h through a long needle that was stretched into the solvent.</li>
		<li>Transfer the resin into a 10-mL round-bottomed flask through weigh paper. Suspend the resin in 2 mL of the degassed DMF followed by the addition of the photoinitiator 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP; 1 equiv., 5.6 mg), 4-methoxyacetophenone (MAP; 1 equiv., 3.8 mg).</li>
		<li>Add a stir bar (0.3 cm) into the flask and cap the flask with a suitable rubber plug, then displace the air in the flask with nitrogen gas using an oil pump. 
		NOTE: The inert atmosphere is not strictly required for the effective thio-ene photoreaction on a solid phase. However, it is very necessary for the thio-yne photoreaction in solution phase in Figure 5. Otherwise, sulfur will oxidize during the UV irradiation.</li>
		<li>Set the reaction flask into the photoreactor and stir the resin for 1 h under UV irradiation at room temperature (Figure 2). 
		CAUTION: Prior to switching the photoreactor UV lamp on, ensure that the photoreactor door is closed in case there is harmful irradiation from the UV light. 
		NOTE: Frequently sampling the reaction mixture during the photoreactions is recommended for new sequences, as the linear peptide precursor has the identical molecular weight of the product. In general, linear and cyclic peptides display significantly different hydrophilicity. This could be easily distinguished using HPLC. Alternatively, the 5,5&#39;-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent could also be used to study the presence of free thiol37.</li>
		<li>Transfer the resin from the flask into the column and remove the solvent using vacuum filtration. Wash and dry the resin as described in step 5.1.3.</li>
		<li>Prepare approximately 10 mL of the cleavage cocktail (TFA/TIPS/EDT/H2O 94/1/2.5/2.5) in the fume hood.</li>
		<li>Transfer the resin into a 2-mL polypropylene container, add 1 mL of cleavage cocktail (TFA/TIPS/EDT/H2O 94/1/2.5/2.5) to the container, and seal the container tightly using a screw cap. Then, gently agitate the container on an orbital shaker at a rate of 60 rpm in the fume hood for 1.5 - 2 h. 
		CAUTION: TFA is highly corrosive. Wear protective clothing and work in an efficient fume hood. EDT is a highly malodorous substance and must be handled in an efficient fume hood.</li>
		<li>Remove the TFA cocktail by evaporation under an N2 gas stream in the fume hood. Next, precipitate the residue using cold diethyl ether (1 mL) for 30 s and isolate it via centrifugation at 12,000 x g for 2 min. Following the centrifugation, gently pour the ether component out. Repeat the precipitate and centrifugation steps for 2x. Evaporate the residue to dryness.</li>
		<li>Finally, dissolve the residue in 1 mL H2O/acetonitrile (2:1) and purify by HPLC using a C18 analytic column (4.6 x 250 mm, flow rate 1.0 mL/min). Use H2O (containing 0.1% TFA) and pure acetonitrile as solvents in a 2%/min linear gradient from 20% to 70% acetonitrile over 25 min. Monitor HPLC spectra using UV 280 nm and 220 nm wavelengths ( Table 2).</li>
	</ol>
	</li>
	<li>Construct the thioether linker through intermolecular thio-ene reaction, and then cyclize the peptide by macrolactamization (Figure 4).
	<ol>
		<li>Synthesize the alkylene residue bearing the linear peptide H2N-A-A-A-mS5(2-R&#39;&#39;)-R&#39;-resin (50 mg) using standard Fmoc-based solid-phase peptide synthesis (SPPS) as described in steps 2 - 4. Next, wash and dry the resin as described in step 5.1.3.</li>
		<li>Suspend the resin in a 10-mL round-bottomed flask containing 2 mL of the degassed DMF as described in step 5.1.4.</li>
		<li>Add the photoinitiator MMP and MAP (MMP: 1 equiv., 5.6 mg; MAP: 1 equiv., 3.8 mg), Fmoc-Cys-OH (3 equiv., 25.7 mg), and a stir bar (0.3 cm) into the flask. Cap the flask using a suitable rubber plug and then use the oil pump to replace the air in the flask with nitrogen.</li>
		<li>Set the reaction flask into the photoreactor. Stir for 1 - 2 h under UV irradiation at room temperature (Figure 2).</li>
		<li>Monitor the reaction under an LC-MS analysis: cleave 2 - 3 mg of the resin using the cleavage cocktail. Then precipitate the residue with cold diethyl ether (300 &#181;L), isolate the residue by centrifugation, and evaporate the residue to dryness as described in step 5.1.11. After that, dissolve the residue in 100 &#181;L of H2O/acetonitrile (2:1). Filtrate the peptide solution using a 0.22-&#181;m microporous film and analyze it using LC-MS with the compound ionized in the electrospray ionization (ESI) and operated in positive mode.</li>
		<li>If necessary, repeat steps 5.2.2 - 5.2.4 to ensure the reaction is carried out to completion.</li>
		<li>Following the completion of the photo-reaction, transfer the resin from the flask into the column, and remove the solvent using vacuum filtration. Wash and dry the resin as described in step 5.1.3.</li>
		<li>Add the DMF solution of benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBob; 2.4 equiv., 31.2 mg), 1-hydroxybenzotriazole (HoBt; 2.4 equiv., 8.1 mg), and NMM (4 equiv., 11 &#181;L) to the resin in the column for the macrolactamization. Bubble this solution with N2 for 2 h.
		<ol>
			<li>In addition, monitor this coupling reaction using LC-MS as described in step 5.2.3. If necessary, repeat this step to ensure the reaction is carried out to completion.</li>
		</ol>
		</li>
		<li>Elongate the peptide using standard Fmoc-based SPPS as described in steps 3 and 4.</li>
		<li>Upon assembly of all the amino acid residues, cleave the peptide from the resin as described in steps 5.1.10 and 5.1.11 and purify it as described in step 5.1.12.</li>
	</ol>
	</li>
	<li>Construct vinyl sulfide linker in solution phase (Figure 5).
	<ol>
		<li>Synthesize the alkyne residue bearing linear peptide using standard Fmoc-based SPPS as described in steps 2 - 4. Synthesize the alkyne bearing amino acid according to a well-established protocol as described in a previous study20.</li>
		<li>Cleave the peptide from the resin and precipitate it using cold diethyl ether as described in steps 5.1.9 - 5.1.11. Following the cleavage and precipitation of the resin, collect the peptide using centrifugation at 12,000 x g for 2 min.</li>
		<li>Dry the resulting residue in a vacuum. Dissolve the residue in the degassed DMF (50 mL) in a 100-mL round-bottomed flask in order to reach a final concentration of 0.5 mM (based on the loading of the resin, 0.025 mmol (1000 mL/L / 0.5 mmol/L)= 50 mL).
		<ol>
			<li>Add the photoinitiator DMPA (0.5 equiv., 3.2 mg) and then degas the reaction solution for 10 min using N2 through a long needle stretched into the solution. Next, irradiate the sample under UV light at room temperature for 0.5 - 1 h without agitation.</li>
		</ol>
		</li>
		<li>Remove the DMF under a high vacuum and precipitate the crude residue by adding diethyl ether in order to dissolve its organic byproducts. Then, isolate the residue using centrifugation at 12,000 x g for 2 min. Following the centrifugation, gently pour the ether component out. Evaporate the residue to dryness. Finally, dissolve the residue in 1 mL of H2O/acetonitrile (2:1) and purify it using HPLC as described in step 5.1.7.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

In the on-resin intramolecular thio-ene cyclization described in Figure 3, the removal of the trityl group of a cysteine residue was found to be a critical step for the subsequent photoreaction. In addition, the peptide molecular weight prior to and following the reaction was found to be identical as depicted in Figure 6B. Therefore, the use of an HPLC identification or a DTNB assay is required in order to monitor the reaction. In the case of the intermolecular ...

The development of ligands to modulate protein-protein interactions (PPIs) provides an attractive approach for modern drug discovery. Thus, a great deal of effort has been invested into studying novel chemical modalities that could efficiently modulate PPIs1,2,3. PPIs generally consist of shallow, large, and/or discontinued interacting surfaces, and small molecules are typically considered to be unsuitable ligands for the modulation of PPIs4,5. With a suitable exposed interacting surface area, short peptides that mimic the structural features of protein interfaces represent ideal candidates to address this problem6,7. However, short peptides are typically unstructured in an aqueous solution. This is due to the fact that water molecules which compete with the intramolecular hydrogen bonding network of the peptide backbone and well-defined conformations are entropically unfavorable in water8. In addition, the peptides&#39; inherently low stability and cell permeability properties largely limit their use in biological applications9,10. According to the protein data bank (PDB) analysis, &#62;50% of PPIs involve short &#945;-helix interactions11. Thus, different chemical methods have been developed in regard to helix stabilization. These include disulfide/thioether bond formation12,13,14, ring-closing metathesis15, lactam ring formation16, &#34;click&#34; chemistry17, addition of perfluoroarenes18,19, and vinyl-sulfide formation20.
Stabilized helical peptides are widely utilized for various intracellular targets, including p53, estrogen receptors, Ras, BCL-2 family proteins, and others21,22,23,24. ALRN-6924, an all-hydrocarbon stapled peptide dual inhibitor of MDM2 and MDMX, is currently being used for clinical investigation25. In the past few years, our group has focused on the development of novel peptide stabilization methods using thiol-ene and thiol-yne reactions26,27,28. In general, we have demonstrated that these photo-initiated reactions are efficient under mild conditions when naturally abundant cysteine is used. In addition, we have shown that these reactions have an excellent functional group tolerance, are bio-orthogonal, and have been proven to be applicable for peptide and protein modifications29. The resulting thioether/vinyl sulfide tethered peptides largely improve the chemical space of constraint peptides, provide a labile on-tether modification center, and is proven to be applicable for uses in numerous biological applications30,31,32. To date, only limited reports have been described regarding thiol-ene/thiol-yne peptide cyclization. In a study published by Anseth et al. in 2009, an on-resin intramolecular thiol-ene reaction for peptide cyclization between activated alkenes with cysteine was demonstrated33. In 2015, Chou et al. described a two-component radical initiated thiol-ene reaction for peptide stapling34 and a subsequent, sequential thiol-yne/ene coupling reaction35. Recently, we described a series of work based on thioether/vinyl sulfide tethered peptides20,26,27. This protocol describes a detailed synthesis of the above-mentioned thioether/vinyl sulfide tethered peptides in hope that it will be helpful for the broader research community.

<table><tbody><tr><td>Rink Amide MBHA resin(0.53 mmol/g)</td><td>HECHENG</td><td>GRM50407</td><td></td></tr><tr><td>Standard Fmoc-protected amino acids</td><td>GL Biochem (Shanghai) Ltd.</td><td></td><td></td></tr><tr><td>N-Methyl-2-pyrrolidinone</td><td>Shenzhen endi Biotechnology Co.Ltd.</td><td>3230</td><td>skin harmful</td></tr><tr><td>N,N-Dimethyl formamide</td><td>Energy</td><td>B020051</td><td>skin harmful</td></tr><tr><td>Dichloromethane</td><td>Energy</td><td>W330229</td><td>skin harmful</td></tr><tr><td>N,N-Diisoproylethylamine</td><td>Aldrich</td><td>9578</td><td>irritant</td></tr><tr><td>Trifluoroacetic acid</td><td>J&amp;amp;K</td><td>101398</td><td>corrosive</td></tr><tr><td>Triisopropylsilane</td><td>J&amp;amp;K</td><td>973821</td><td></td></tr><tr><td>1,2-Ethanedithiol</td><td>J&amp;amp;K</td><td>248897</td><td>Stench</td></tr><tr><td>2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate&amp;nbsp;</td><td>GL Biochem (Shanghai) Ltd.</td><td>851012</td><td></td></tr><tr><td>Morpholine</td><td>Aldrich</td><td>M109062</td><td>irritant</td></tr><tr><td>Diethyl ether</td><td>Aldrich</td><td>673811</td><td>flammable</td></tr><tr><td>Acetonitrile</td><td>Aldrich</td><td>9758</td><td>toxicity</td></tr><tr><td>Methanol</td><td>Aldrich</td><td>9758</td><td>toxicity</td></tr><tr><td>2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone</td><td>Energy</td><td>A050035</td><td></td></tr><tr><td>4-methoxyacetophenone</td><td>Energy</td><td>A050098</td><td></td></tr><tr><td>2,2-dimethoxy-2-phenylacetophenone</td><td>Energy</td><td>D070132</td><td></td></tr><tr><td>5,5&#039;-Dithiobis-(2-nitrobenzoic acid)</td><td>J&amp;amp;K</td><td>281281</td><td></td></tr><tr><td>Benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate</td><td>Energy</td><td>E020172</td><td></td></tr><tr><td>1-Hydroxybenzotriazole</td><td>Energy</td><td>D050256</td><td></td></tr><tr><td>4-Methylmorpholine</td><td>Energy</td><td>W320038</td><td></td></tr><tr><td>High Performance Liquid Chromatography</td><td>SHIMADZU</td><td>LC-30AD</td><td></td></tr><tr><td>Electrospray Ionization Mass</td><td>SHIMADZU</td><td>LCMS-8030</td><td></td></tr><tr><td>Lyophilizer</td><td>Labconco</td><td>FreeZone</td><td></td></tr><tr><td>SpeedVac concentration system</td><td>Thermo</td><td>Savant</td><td></td></tr><tr><td>vacuum manifold</td><td>promega</td><td>A7231</td><td></td></tr><tr><td>three-way stopcocks</td><td>Bio-Rad</td><td>7328107</td><td></td></tr><tr><td>poly-prep chromatography columns&amp;nbsp;</td><td>Bio-Rad</td><td>7311550</td><td></td></tr></tbody></table>

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.

The HPLC and MS spectra of the peptide Ac-YmS5AAAC-NH2 and its cyclized product Ac-Y-(cyclo-1,5)-[mS5AAAC]-NH2 that were generated using the on-resin intramolecular thiol-ene photoreaction are depicted in Figure 6B. The cyclic peptide was found to have an identical molecular weight relative to its linear precursor. However, its HPLC retention time was observed to be approximately 2 min earlier than that of its precu...

Watch this Scientific Journal Video about Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation at JoVE.com

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

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 ...

constructing-thioethervinyl-sulfide-tethered-helical-peptides-via

Research

JoVE Journal

Chemistry

10.7K Views.  Peking University Shenzhen Graduate School. We presenteren een protocol voor de bouw van thioether/vinyl sulfide-aangebonden spiraalvormige peptiden met behulp van foto-geïnduceerde thiol-Ono/thiol-yne hydrothiolation.

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

A Facile and Efficient Approach for the Production of Reversible Disulfide Cross-linked Micelles

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. Improving drug delivery to maximize therapeutic outcomes and to reduce drug-associated side effects are some of the cornerstones of present-day nanomedicine. Nanoparticles in particular have found a wide application in cancer treatment. Nanoparticles that offer a high degree of flexibility in design, application, and production based on the tumor microenvironment are projected to be more effective with rapid translation into clinical practice. The polymeric micellar nano-carrier is a popular choice for drug delivery applications.
In this article, we describe a simple and effective protocol for synthesizing drug-loaded, disulfide cross-linked micelles based on the self-assembly of a well-defined amphiphilic linear-dendritic copolymer (telodendrimer, TD). TD is composed of polyethylene glycol (PEG) as the hydrophilic segment and a thiolated cholic acid cluster as the core-forming hydrophobic moiety attached stepwise to an amine-terminated PEG using solution-based peptide chemistry. Chemotherapy drugs, such as paclitaxel (PTX), can be loaded using a standard solvent evaporation method. The O2-mediated oxidation was previously utilized to form intra-micellar disulfide cross-links from free thiol groups on the TDs. However, the reaction was slow and not feasible for large-scale production. Recently, an H2O2-mediated oxidation method was explored as a more feasible and efficient approach, and it was 96 times faster than the previously reported method. Using this approach, 50 g of PTX-loaded, disulfide cross-linked nanoparticles have been successfully produced with narrow particle size distribution and high drug loading efficiency. The stability of the resulting micelle solution is analyzed using disrupting conditions such as co-incubation with a detergent, sodium dodecyl sulfate, with or without a reducing agent. The drug-loaded, disulfide cross-linked micelles demonstrated less hemolytic activity when compared to their non-cross-linked counterparts.

To deliver cancer drugs to tumor sites with high specificity and reduced side effects, new methods based on nanoparticles are required. Here, we describe disulfide cross-linked micelles that can be easily prepared by hydrogen peroxide-mediated oxidation and are able to dissociate efficiently under a reducing tumor environment to release payloads.

Nanomedicine is an emerging form of therapy that harnesses the unique properties of particles that are nanometers in scale for biomedical application. ...

Biochemistry

Synthesis of Programmable Main-chain Liquid-crystalline Elastomers Using a Two-stage Thiol-acrylate Reaction

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline elastomers (LCEs) with facile control over network structure and programming of an aligned monodomain. Tailored LCE networks were synthesized using routine mixing of commercially available starting materials and pouring monomer solutions into molds to cure. An initial polydomain LCE network is formed via a self-limiting thiol-acrylate Michael-addition reaction. Strain-to-failure and glass transition behavior were investigated as a function of crosslinking monomer, pentaerythritol tetrakis(3-mercaptopropionate) (PETMP). An example non-stoichiometric system of 15 mol% PETMP thiol groups and an excess of 15 mol% acrylate groups was used to demonstrate the robust nature of the material. The LCE formed an aligned and transparent monodomain when stretched, with a maximum failure strain over 600%. Stretched LCE samples were able to demonstrate both stress-driven thermal actuation when held under a constant bias stress or the shape-memory effect when stretched and unloaded. A permanently programmed monodomain was achieved via a second-stage photopolymerization reaction of the excess acrylate groups when the sample was in the stretched state. LCE samples were photo-cured and programmed at 100%, 200%, 300%, and 400% strain, with all samples demonstrating over 90% shape fixity when unloaded. The magnitude of total stress-free actuation increased from 35% to 115% with increased programming strain. Overall, the two-stage TAMAP methodology is presented as a powerful tool to prepare main-chain LCE systems and explore structure-property-performance relationships in these fascinating stimuli-sensitive materials.

A novel methodology is presented to synthesize and program main-chain liquid-crystalline elastomers using commercially available starting monomers. A wide range of thermomechanical properties was tailored by adjusting the amount of crosslinker, while the actuation performance was dependent on the amount of applied strain during programming.

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline ...

Generation and Recovery of &beta;-cell Spheroids From Step-growth PEG-peptide Hydrogels

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional microenvironment with tissue-like elasticity and high permeability for culturing therapeutically relevant cells or tissues. Hydrogels prepared from poly(ethylene glycol) (PEG) derivatives are increasingly used for a variety of tissue engineering applications, in part due to their tunable and cytocompatible properties. In this protocol, we utilized thiol-ene step-growth photopolymerizations to fabricate PEG-peptide hydrogels for encapsulating pancreatic MIN6 b-cells. The gels were formed by 4-arm PEG-norbornene (PEG4NB) macromer and a chymotrypsin-sensitive peptide crosslinker (CGGYC). The hydrophilic and non-fouling nature of PEG offers a cytocompatible microenvironment for cell survival and proliferation in 3D, while the use of chymotrypsin-sensitive peptide sequence (CGGY&darr;C, arrow indicates enzyme cleavage site, while terminal cysteine residues were added for thiol-ene crosslinking) permits rapid recovery of cell constructs forming within the hydrogel. The following protocol elaborates techniques for: (1) Encapsulation of MIN6 &beta;-cells in thiol-ene hydrogels; (2) Qualitative and quantitative cell viability assays to determine cell survival and proliferation; (3) Recovery of cell spheroids using chymotrypsin-mediated gel erosion; and (4) Structural and functional analysis of the recovered spheroids.

Generation and Recovery of &#946;-cell Spheroids From Step-growth PEG-peptide Hydrogels

The following protocol provides techniques for encapsulating pancreatic &#x03B2;-cells in step-growth PEG-peptide hydrogels formed by thiol-ene photo-click reactions. This material platform not only offers a cytocompatible microenvironment for cell encapsulation, but also permits user-controlled rapid recovery of cell structures formed within the hydrogels.

Hydrogels are hydrophilic crosslinked polymers that provide a three-dimensional  microenvironment with tissue-like elasticity and high permeability for  ...

Bioengineering

Self-assembling Morphologies Obtained from Helical Polycarbodiimide Copolymers and Their Triazole Derivatives

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous networks, ribbons, worm-like aggregates, toroidal structures, and spherical particles) is described. These aggregates are morphologically influenced by extensive hydrophobic side chain-side chain interactions of the singular polycarbodiimide strands, as inferred by atomic force microscopy (AFM) and scanning electron microscopy (SEM) techniques. Polycarbodiimide-g-polystyrene copolymers (PS-PCDs) were prepared by a combination of synthetic methods, including coordination-insertion polymerization, copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) &#34;click&#34; chemistry, and atom transfer radical polymerization (ATRP). PS-PCDs were found to form specific toroidal architectures at low concentrations in CHCl3. To determine the influence of a more polar solvent medium (i.e., THF and THF/EtOH) on polymer aggregation behavior, a number of representative PS-PCD composites have been tested to show discrete concentration-dependent spherical particles. These fundamental studies are of practical interest to the development of experimental procedures for desirable architectures by directed self-assembly in thin film. These architectures may be exploited as drug carriers, whereas other morphological findings represent certain interest in the area of novel functional materials.

Here, we present a protocol to prepare and visualize secondary structures (e.g., fibers, toroidal architectures, and nano-spheres) derived from helical polycarbodiimides. The morphology characterized by both atomic force microscopy (AFM) and scanning electron microscopy (SEM) was shown to depend on molecular structure, concentration, and the solvent of choice.

A facile method for the preparation of polycarbodiimide-based secondary structures (e.g., nano-rings, &#34;craters,&#34; fibers, looped fibers, fibrous ...

Synthesis of a Thiol Building Block for the Crystallization of a Semiconducting Gyroidal Metal-sulfur Framework

We present a method for preparing thioester molecules as the masked form of the thiol linkers and their utilization for accessing a semiconducting and porous metal-dithiolene network in the highly ordered single crystalline state. Unlike the highly reactive free-standing thiols, which tend to decompose and complicate the crystallization of metal-thiolate open frameworks, the thioester reacts in situ to provide the thiol species, serving to mitigate the reaction between the mercaptan units and the metal centers, and to improve crystallization consequently. Specifically, the thioester was synthesized in a one-pot procedure: an aromatic bromide (hexabromotriphenylene) reacted with excess sodium thiomethoxide under vigorous conditions to first form the thioether intermediate product. The thioether was then demethylated by the excess thiomethoxide to provide the thiolate anion that was acylated to form the thioester product. The thioester was conveniently purified by standard column chromatography, and then used directly in the framework synthesis, wherein NaOH and ethylenediamine serve to revert in situ the thioester to the thiol linker for assembling the single-crystalline Pb(II)-dithiolene network. Compared with other methods for thiol synthesis (e.g., by cleaving alkyl thioether using sodium metal and liquid ammonia), the thioester synthesis here uses simple conditions and economical reagents. Moreover, the thioester product is stable and can be conveniently handled and stored. More importantly, in contrast to the generic difficulty in accessing crystalline metal-thiolate open frameworks, we demonstrate that using the thioester for in situ formation of the thiol linker greatly improves the crystallinity of the solid-state product. We intend to encourage broader research efforts on the technologically important metal-sulfur frameworks by disclosing the synthetic protocol for the thioester as well as the crystalline framework solid.

Here, we present a one-pot, transition-metal-free synthesis of thiols and thioesters from aromatic halides and sodium thiomethoxide, followed by the preparation of single crystals of a metal-dithiolene network using thiol species generated in situ from the more stable and tractable thioester.

We present a method for preparing thioester molecules as the masked form of the thiol linkers and their utilization for accessing a semiconducting and ...

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

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

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

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

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

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 ...

In Vesiculo Synthesis of Peptide Membrane Precursors for Autonomous Vesicle Growth

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an attractive alternative to liposomes or fatty acid-based vesicles. Externally or within the vesicles, peptides can be easily expressed and simplify the synthesis of membrane precursors. Provided here is a protocol for the creation of vesicles with diameters of ~200 nm based on the amphiphilic elastin-like polypeptides (ELP) utilizing dehydration-rehydration from glass beads. Also presented are protocols for bacterial ELP expression and purification via inverse temperature cycling, as well as their covalent functionalization with fluorescent dyes. Furthermore, this report describes a protocol to enable the transcription of RNA aptamer dBroccoli inside ELP vesicles as a less complex example for a biochemical reaction. Finally, a protocol is provided, which allows in vesiculo expression of fluorescent proteins and the membrane peptide, whereas synthesis of the latter results in vesicle growth.

Presented here are protocols for the creation of peptide-based small unilamellar vesicles capable of growth. To facilitate in vesiculo production of the membrane peptide, these vesicles are equipped with a transcription-translation system and the peptide-encoding plasmid.

Compartmentalization of biochemical reactions is a central aspect of synthetic cells. For this purpose, peptide-based reaction compartments serve as an ...

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

Samenvatting

Meer video's verkennen