Name: constructing thioether/vinyl sulfide-tethered helical peptides via photo-induced thiol-ene/yne hydrothiolation
Uploaded: 2018-08-01T13:30:00
Description: Watch this Scientific Journal Video about Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation at JoVE.com

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 pum...

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">
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		<tr height="42">
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			<td height="21" style="height:21px;width:216px;">1,2-Ethanedithiol</td>
			<td style="width:143px;">J&#38;K</td>
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			<td style="width:167px;">Stench</td>
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			<td>M109062</td>
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			<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>
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			<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>
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			<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>
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		<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>
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		<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>
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		<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>
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			<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>
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		<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>
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	</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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