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 mimic the structural features of protein interfaces represent ideal candidates to address this problem^6,7. 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' inherently low stability and cell permeability properties largely limit their use in biological applications^9,10. According to the protein data bank (PDB) analysis, >50% of PPIs involve short α-helix interactions¹¹. Thus, different chemical methods have been developed in regard to helix stabilization. These include disulfide/thioether bond formation^12,13,14, ring-closing metathesis¹⁵, lactam ring formation¹⁶, "click" chemistry¹⁷, addition of perfluoroarenes^18,19, and vinyl-sulfide formation²⁰.

Stabilized helical peptides are widely utilized for various intracellular targets, including p53, estrogen receptors, Ras, BCL-2 family proteins, and others^21,22,23,24. 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 reactions^26,27,28. 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 applications^30,31,32. 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 peptides^20,26,27. 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.

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 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.
4. 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.

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.

1. 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 µmol = 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.
2. 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.

3. N-terminal Fmoc Deprotection and Washing

1. Prepare the N-terminal Fmoc deprotecting solution: prepare an adequate volume (200 mL) of 50% (v/v) morpholine in DMF in a glass bottle.
2. 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.

4. Fmoc-protected Amino Acid Coupling

1. Using the coupling of alanine residue as an example, in the case of a 25 µmol-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).
2. Add DIPEA (10 equiv., 43.5 µl) 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.
3. 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.
4. 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.

5. Thiol-ene Hydrothiolation and Thiol-yne Cyclization

1. Construct thioether linker through on-resin cyclization (Figure 3).
   1. 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'-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.
   2. 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.
   3. Bubbled with N₂, wash the R-mS₅-A-A-A-Cys(-SH)-R'-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.
   4. 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.
   5. 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).
   6. 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.
   7. 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'-dithiobis-(2-nitrobenzoic acid) (DTNB) reagent could also be used to study the presence of free thiol³⁷.
   8. 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.
   9. Prepare approximately 10 mL of the cleavage cocktail (TFA/TIPS/EDT/H₂O 94/1/2.5/2.5) in the fume hood.
   10. 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.
   11. 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.
   12. 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).
2. Construct the thioether linker through intermolecular thio-ene reaction, and then cyclize the peptide by macrolactamization (Figure 4).
   1. Synthesize the alkylene residue bearing the linear peptide H₂N-A-A-A-mS₅(2-R'')-R'-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.
   2. Suspend the resin in a 10-mL round-bottomed flask containing 2 mL of the degassed DMF as described in step 5.1.4.
   3. 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.
   4. Set the reaction flask into the photoreactor. Stir for 1 - 2 h under UV irradiation at room temperature (Figure 2).
   5. 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 µL), 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 µL of H₂O/acetonitrile (2:1). Filtrate the peptide solution using a 0.22-µm microporous film and analyze it using LC-MS with the compound ionized in the electrospray ionization (ESI) and operated in positive mode.
   6. If necessary, repeat steps 5.2.2 - 5.2.4 to ensure the reaction is carried out to completion.
   7. 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.
   8. 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 µL) to the resin in the column for the macrolactamization. Bubble this solution with N₂ for 2 h.
      1. 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.
   9. Elongate the peptide using standard Fmoc-based SPPS as described in steps 3 and 4.
   10. 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.
3. Construct vinyl sulfide linker in solution phase (Figure 5).
   1. 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.
   2. 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.
   3. 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).
      1. 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.
   4. 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.

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