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

Summary

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

Abstract

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.

Introduction

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 PPIs^1,2,3. 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 PPIs^4,5. With a suitable exposed interacting surface area, short peptides that mimi....

Protocol

1. Equipment Preparation

1. 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.
2. 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.
3. Use a photor....

Results

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.......

Discussion

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 .......

Materials

List of materials used in this article

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