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 border="0" cellpadding="0" cellspacing="0" width="644">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Rink Amide MBHA resin(0.53 mmol/g)</td>
			<td style="width:143px;">HECHENG</td>
			<td style="width:119px;">GRM50407</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard Fmoc-protected amino acids</td>
			<td style="width:143px;">GL Biochem (Shanghai) Ltd.</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">N-Methyl-2-pyrrolidinone</td>
			<td style="width:143px;">Shenzhen endi Biotechnology Co.Ltd.</td>
			<td style="width:119px;">3230</td>
			<td style="width:167px;">skin harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N,N-Dimethyl formamide</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">B020051</td>
			<td style="width:167px;">skin harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dichloromethane</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">W330229</td>
			<td style="width:167px;">skin harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N,N-Diisoproylethylamine</td>
			<td style="width:143px;">Aldrich</td>
			<td style="width:119px;">9578</td>
			<td style="width:167px;">irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trifluoroacetic acid</td>
			<td style="width:143px;">J&#38;K</td>
			<td style="width:119px;">101398</td>
			<td style="width:167px;">corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triisopropylsilane</td>
			<td style="width:143px;">J&#38;K</td>
			<td style="width:119px;">973821</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1,2-Ethanedithiol</td>
			<td style="width:143px;">J&#38;K</td>
			<td style="width:119px;">248897</td>
			<td style="width:167px;">Stench</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate&#160;</td>
			<td style="width:143px;">GL Biochem (Shanghai) Ltd.</td>
			<td style="width:119px;">851012</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Morpholine</td>
			<td style="width:143px;">Aldrich</td>
			<td>M109062</td>
			<td style="width:167px;">irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diethyl ether</td>
			<td style="width:143px;">Aldrich</td>
			<td style="width:119px;">673811</td>
			<td style="width:167px;">flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetonitrile</td>
			<td style="width:143px;">Aldrich</td>
			<td style="width:119px;">9758</td>
			<td style="width:167px;">toxicity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Methanol</td>
			<td style="width:143px;">Aldrich</td>
			<td style="width:119px;">9758</td>
			<td style="width:167px;">toxicity</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">A050035</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-methoxyacetophenone</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">A050098</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2,2-dimethoxy-2-phenylacetophenone</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">D070132</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5,5&#39;-Dithiobis-(2-nitrobenzoic acid)</td>
			<td style="width:143px;">J&#38;K</td>
			<td style="width:119px;">281281</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">E020172</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1-Hydroxybenzotriazole</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">D050256</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">4-Methylmorpholine</td>
			<td style="width:143px;">Energy</td>
			<td style="width:119px;">W320038</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">High Performance Liquid Chromatography</td>
			<td style="width:143px;">SHIMADZU</td>
			<td style="width:119px;">LC-30AD</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Electrospray Ionization Mass</td>
			<td style="width:143px;">SHIMADZU</td>
			<td style="width:119px;">LCMS-8030</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Lyophilizer</td>
			<td style="width:143px;">Labconco</td>
			<td style="width:119px;">FreeZone</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SpeedVac concentration system</td>
			<td style="width:143px;">Thermo</td>
			<td style="width:119px;">Savant</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">vacuum manifold</td>
			<td style="width:143px;">promega</td>
			<td style="width:119px;">A7231</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">three-way stopcocks</td>
			<td style="width:143px;">Bio-Rad</td>
			<td style="width:119px;">7328107</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">poly-prep chromatography columns&#160;</td>
			<td style="width:143px;">Bio-Rad</td>
			<td style="width:119px;">7311550</td>
			<td style="width:167px;"></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 ...

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JoVE Journal

Chemistry

10.6K Views.  Peking University Shenzhen Graduate School. We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

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

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

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 ...

Preparing Silica Aerogel Monoliths via a Rapid Supercritical Extraction Method

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The procedure requires 15-20 min of preparation time, during which a liquid precursor mixture is prepared and poured into wells of a metal mold that is placed between the platens of a hydraulic hot press, followed by several hours of processing within the hot press. The precursor solution consists of a 1.0:12.0:3.6:3.5 x 10-3 molar ratio of tetramethylorthosilicate (TMOS):methanol:water:ammonia. In each well of the mold, a porous silica sol-gel matrix forms. As the temperature of the mold and its contents is increased, the pressure within the mold rises. After the temperature/pressure conditions surpass the supercritical point for the solvent within the pores of the matrix (in this case, a methanol/water mixture), the supercritical fluid is released, and monolithic aerogel remains within the wells of the mold. With the mold used in this procedure, cylindrical monoliths of 2.2 cm diameter and 1.9 cm height are produced. Aerogels formed by this rapid method have comparable properties (low bulk and skeletal density, high surface area, mesoporous morphology) to those prepared by other methods that involve either additional reaction steps or solvent extractions (lengthier processes that generate more chemical waste).The rapid supercritical extraction method can also be applied to the fabrication of aerogels based on other precursor recipes.

This article describes a rapid supercritical extraction method for fabricating silica aerogels. By utilizing a confined mold and hydraulic hot press, monolithic aerogels can be made in eight hours or less.

A procedure for the fabrication of monolithic silica aerogels in eight hours or less via a rapid supercritical extraction process is described. The ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Epitaxial Growth of Perovskite Strontium Titanate on Germanium via Atomic Layer Deposition

Atomic layer deposition (ALD) is a commercially utilized deposition method for electronic materials. ALD growth of thin films offers thickness control and conformality by taking advantage of self-limiting reactions between vapor-phase precursors and the growing film. Perovskite oxides present potential for next-generation electronic materials, but to-date have mostly been deposited by physical methods. This work outlines a method for depositing SrTiO3 (STO) on germanium using ALD. Germanium has higher carrier mobilities than silicon and therefore offers an alternative semiconductor material with faster device operation. This method takes advantage of the instability of germanium's native oxide by using thermal deoxidation to clean and reconstruct the Ge (001) surface to the 2&#215;1 structure. 2-nm thick, amorphous STO is then deposited by ALD. The STO film is annealed under ultra-high vacuum and crystallizes on the reconstructed Ge surface. Reflection high-energy electron diffraction (RHEED) is used during this annealing step to monitor the STO crystallization. The thin, crystalline layer of STO acts as a template for subsequent growth of STO that is crystalline as-grown, as confirmed by RHEED. In situ X-ray photoelectron spectroscopy is used to verify film stoichiometry before and after the annealing step, as well as after subsequent STO growth. This procedure provides framework for additional perovskite oxides to be deposited on semiconductors via chemical methods in addition to the integration of more sophisticated heterostructures already achievable by physical methods.

This work details the procedures for the growth and characterization of crystalline SrTiO3 directly on germanium substrates by atomic layer deposition. The procedure illustrates the ability of an all-chemical growth method to integrate oxides monolithically onto semiconductors for metal-oxide semiconductor devices.

Atomic layer deposition (ALD) is a commercially utilized deposition method for electronic materials. ALD growth of thin films offers thickness control and ...

Insights into the Interactions of Amino Acids and Peptides with Inorganic Materials Using Single-Molecule Force Spectroscopy

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals leads to the formation of composite materials with unique properties. In addition, the undesirable process of biofouling is initiated by the adsorption of biomolecules, mainly proteins, on surfaces. This organic layer is an adhesion layer for bacteria and allows them to interact with the surface. Understanding the fundamental forces that govern the interactions at the organic-inorganic interface is therefore important for many areas of research and could lead to the design of new materials for optical, mechanical and biomedical applications. This paper demonstrates a single-molecule force spectroscopy technique that utilizes an AFM to measure the adhesion force between either peptides or amino acids and well-defined inorganic surfaces. This technique involves a protocol for attaching the biomolecule to the AFM tip through a covalent flexible linker and single-molecule force spectroscopy measurements by atomic force microscope. In addition, an analysis of these measurements is included.

Here we present a protocol to measure the force of interactions between a well-defined inorganic surface and either peptides or amino acids by single-molecule force spectroscopy measurements using an atomic force microscope (AFM). The information obtained from the measurement is important to better understand the peptide-inorganic material interphase.

The interactions between proteins or peptides and inorganic materials lead to several interesting processes. For example, combining proteins with minerals ...

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 ...

Synthesizing Sodium Tungstate and Sodium Molybdate Microcapsules via Bacterial Mineral Excretion

We present a method, the bacterial mineral excretion (BME), for synthesizing two kinds of microcapsules, sodium tungstate and sodium molybdate, and the two metal oxides&#39; corresponding nanoparticles&#8212;the former being as small as 22 nm and the latter 15 nm. We fed two strains of bacteria, Shewanella algae and Pandoraea sp., with various concentrations of tungstate or molybdate ions. The concentrations of tungstate and molybdate were adjusted to make microcapsules of different length-to-diameter ratios. We found that the higher the concentration the smaller the nanoparticles were. The nanoparticles came in with three length-to-diameter ratios: 10:1, 3:1 and 1:1, which were achieved by feeding the bacteria respectively with a low concentration, a medium concentration, and a high concentration. The images of the hollow microcapsules were taken via the scanning electron microsphere (SEM). Their crystal structures were verified by X-ray diffraction (XRD)&#8212;the crystal structure of molybdate microcapsules is Na2MoO4 and that of tungstate microcapsules is Na2WO4 with Na2W2O7. These syntheses all were accomplished under a near ambient condition.

This work presents a protocol for manufacture sodium tungstate and sodium molybdate microcapsules via bacteria and their corresponding nanoparticles.

We present a method, the bacterial mineral excretion (BME), for synthesizing two kinds of microcapsules, sodium tungstate and sodium molybdate, and the ...

Fabrication and Testing of Catalytic Aerogels Prepared Via Rapid Supercritical Extraction

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three preparation methods are described: (a) the incorporation of metal salts into silica or alumina wet gels using an impregnation method; (b) the incorporation of metal salts into alumina wet gels using a co-precursor method; and (c) the addition of metal nanoparticles directly into a silica aerogel precursor mixture. The methods utilize a hydraulic hot press, which allows for rapid (&#60;6 h) supercritical extraction and results in aerogels of low density (0.10 g/mL) and high surface area (200-800 m2/g). While the work presented here focuses on the use of copper salts and copper nanoparticles, the approach can be implemented using other metal salts and nanoparticles. A protocol for testing the three-way catalytic ability of these aerogels for automotive pollution mitigation is also presented. This technique uses custom-built equipment, the Union Catalytic Testbed (UCAT), in which a simulated exhaust mixture is passed over an aerogel sample at a controlled temperature and flow rate. The system is capable of measuring the ability of the catalytic aerogels, under both oxidizing and reducing conditions, to convert CO, NO and unburned hydrocarbons (HCs) to less harmful species (CO2, H2O and N2). Example catalytic results are presented for the aerogels described.

Here we present protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms. Methods for preparing materials using copper salts and copper-containing nanoparticles are featured. Catalytic testing protocols demonstrate the effectiveness of these aerogels for three-way catalysis applications.

Protocols for preparing and testing catalytic aerogels by incorporating metal species into silica and alumina aerogel platforms are presented. Three ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.