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JoVE Journal Chemistry

Chemistry

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

Published: September 28, 2022 doi: 10.3791/64289
Automatic Translation
*1, *1,2, *1, 3, 1,2, 1, 3, 1,2, 1,2
1Pingshan Translational Medicine Center, Shenzhen Bay Laboratory, 2Key Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School, 3Department of Radiation Oncology, the First Affiliated Hospital, Anhui Medical University
* These authors contributed equally

Summary

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.

Abstract

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.

Introduction

Protein-protein interactions (PPIs)1 play a pivotal role in drug research and development. Constructing stabilized peptides with a fixed conformation by chemical means is one of the most important methods for developing mimetic motifs of PPIs2. To date, several cyclic peptides that target PPIs have been developed for clinical use3. Most peptides are constrained to an α-helix conformation to decrease the conformational entropy and improve the metabolic stability, target-binding affinity, and cell permeability4,5. In the past 2 decades, the side chains of Cys6,7, lysine8,9, tryptophan10, arginine11, and Met12,13 have been inserted into unnatural amino acids to fix the peptide into a cyclic conformation. Such cyclic peptides can target a unique chemical space or special sites, thereby triggering a covalent reaction to form protein-peptide covalent binding14,15,16,17. In a recent report by Yu et al., a chloroacetamide was anchored onto the domain of peptide ligands, ensuring a covalent conjugation reaction with excellent protein specificity18. Moreover, electrophilic warheads, such as acrylamide and aryl sulfonyl fluoride (ArSO2F), were further incorporated into peptides by Walensky et al.19 to form stabilized peptide covalent inhibitors and improve the anti-tumor effect of peptide inhibitors. Therefore, it is very important to introduce an additional functional group in order to covalently modify protein-peptide ligands20. These groups not only react with proteins on the side chain but also stabilize the secondary structure of the peptide21. However, the application of covalently modified proteins induced by peptide ligands is limited due to the complicated synthetic route and the non-specific binding of the chemical groups22,23. Effective strategies for the synthesis of cyclic peptides are, therefore, urgently required.

Inspired by the multifarious strategies of cyclic peptides2,24,25,26, this protocol attempts to develop a simple and efficient method for stabilizing peptides. In addition, we noted that the side chain group of a stable peptide could react covalently with a target protein when it was spatially close to the peptide ligands. The lack of chemically modified Met was filled by the Deming group in 2013 by developing a novel method for producing selectively modified peptide methionine27. Based on this background, the Shi et al. focused on the development of the ring closure of side chains to form a sulfonium salt center. When the peptide ligand combines with the target protein, the sulfonium salt group reacts covalently with the spatially close Cys protein. In recent years, the Shi et al. have designed a new method for stabilizing cyclic peptide28. The sulfonium salt on the cyclic peptide was reduced by a reducing agent with a sulfhydryl group that was reversibly reduced to Met. However, the reaction had low efficiency, which was harmful to subsequent biological application studies. In the current study, a Met-Cys and propargyl bromide-Cys ring-closure reaction was designed, with a single sulfonium salt remaining on the side chain of the cyclic peptide. The sulfonium salt acted as a new warhead that reacted covalently with the protein Cys under spatial proximity. Briefly, a Cys and Met mutated peptide was cyclized by intramolecular alkylation, resulting in the generation of an on-tether sulfonium center. In this process, the formation of a side chain bridge was critical for cyclic peptides. Overall, this protocol describes a detailed sulfonium-based peptide cyclization that is achieved using simple reaction conditions and operations. The aim is to develop a potential method for further broad biological applications.

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Protocol

1. Equipment preparation

CAUTION: Morpholine, N,N-dimethylformamide (DMF), dichloromethane (DCM), N,N-diisopropylethylamine (DIPEA), TFA, morpholine, piperidine, diethyl ether, and methanol are toxic, volatile, and corrosive. These reagents can harm the human body through inhalation, ingestion, or skin contact. For all chemical experiments, use protective equipment, including disposable gloves, experimental coats, and protective eyeglasses.

  1. Construct all peptide substrates on Rink-amide 4-methylbenzhydrylamine (MBHA) resin by standard manual Fmoc-based solid-phase peptide synthesis (SPPS)29, as shown in Figure 1.
  2. Use either of the two options for constructing linear peptides: one, synthesize the peptide utilizing condensing agent 2-(7-azabenzotriazol-1-yl)-N,N, N',N'-tetramethyluronium hexafluorophosphate (HATU) and a reagent of DIPEA; or two, synthesize utilizing 1-hydroxybenzotriazole (HOBT) and N,N'-diisopropylcarbodiimide (DIC) as the amide condensation reagent. Select an appropriate protocol for peptide synthesis according to the sequence of the peptide.
  3. To establish a manual peptide-synthesis device, set up modified vacuum solid phase extraction equipment in a fume hood and connect it with a three-way stopcock. Next, place a polypropylene filter cartridge or glass reactor on the equipment while connected to nitrogen (N2) gas.
    NOTE: For modifying the vacuum solid phase extraction equipment, remove the extraction tube and keep the vacuum-sealed system.
  4. Load MBHA resin into the resin-filled columns and dissolve it in DMF. Adjust the switch of the three-way stopcocks for N2 bubbling, and then remove the solvent in the column using a working pump connected to a vacuum system. Calculate the amount of amino acid or condensing agent required using the following formula:
    Amino acid (g) and condensing agent (g) = resin scale (g) × resin loading capacity (mM/g) × equivalent (5 eq) × molecular weight (g/M)
    ​NOTE: Choose the amount of loaded resin according to the length of the coupling peptide. Multiple equivalents (eq) of amino acids are used to react more fully. Liquid solvents need to be converted to volume by density. The 5 eq shows that the input amount of the calculated compound is amplified by a factor of five.

2. Resin preparation

NOTE: Choose the amount of loaded resin according to the length of the coupling peptide.

  1. Weigh 302 mg of Rink-amide MBHA resin (0.331 mM/g loaded) into a column (20 mL reservoir). Add 5-10 mL of DCM or DMF into the column to swell the resin under N2 bubbling for 30 min.
  2. Next, close the N2 switch, and then turn on the vacuum pump suction switch to remove the solvent. Then, wash the resins sequentially with DCM (5-10 mL) and DMF (5-10 mL) 5x.

3. N-terminal Fmoc deprotection

NOTE: Deprotection by morpholine requires 30 min, and deprotection by piperidine takes 5 min.

  1. Prepare the N-terminal 9-fluorenylmethyloxycarbonyl (Fmoc) deprotection solution: prepare a sufficient volume (500 mL) of 20% (v/v) piperidine or 50% (v/v) morpholine in DMF in a glass container for Fmoc group deprotection.
  2. Add 10 mL of the deprotection solution to the column, bubble N2 into the solution for 30 min or 5 min 2x, and repeat the deprotection procedures 2x.
  3. Drain the solution by a vacuum pump and wash the resin sequentially with DCM (5-10 mL) and DMF (5-10 mL) 5x.
  4. Detect the resin as a dark yellow color solution by 5% ninhydrin (Kaiser test) after every deprotection to confirm the absence of the Fmoc group before the coupling step. In detail, add 1 mL of DMF to a small amount of resin and add 200 µL of 5% ninhydrin to a glass tube heated at 130 °C for 3 min to assess changes in the resin color.
  5. Drain the solution by a vacuum pump and wash the resin sequentially with DCM (5-10 mL) and DMF (5-10 mL) 5x.

4. Coupling the linear peptide (Figure 2)

NOTE: When the synthetic peptide sequences contain two or more repeating units, the coupling procedure can be directly carried out by selecting the amino acid type, such as Fmoc-AA-OH or Fmoc-AAA-OH, and so on. Some special amino acids with steric hindrance and peptides with longer amino acid sequences are required to properly extend the reaction time for coupling.

  1. For a single coupling step, take the coupling of Cys residue as an example (synthesize 300 mg resin scales). Prepare a mixture solution including Fmoc-Cys(Trt)-OH (5 eq, 292 mg) and HATU (5 eq, 206 mg) or Fmoc-Cys(Trt)-OH (5 eq, 292 mg) and HOBT (5 eq, 106 mg) in a polypropylene tube and dissolve it in 3 mL of DMF.
  2. Add 173 µL of DIPEA (10 eq) or 154 µL of DIC (10 eq) to the solution of Cys to activate the amino acid. Let the mixture pre-activate for 1 min. Add the mixture to the column with resin, and bubble it with N2 for 2 h.
    NOTE: The specific reaction time required should be standardized to detect 5% ninhydrin.
  3. After coupling, add 1 mL of DMF to a small amount of resin and add 200 µL of 5% ninhydrin to a glass tube heated at 130 °C for 3 min. Observe the resin change to colorless to confirm the absence of a free amino group before the deprotection step.
  4. Drain the solution using a vacuum pump and wash the resin sequentially with DCM (5-10 mL) and DMF (5-10 mL) 5x.
  5. Add 10 mL of the deprotection solution to the column, bubble with N2 for 30 min or 5 min 2x, add fresh solution, and repeat the deprotection procedures 2x.
  6. Repeat the following steps: deprotection of the Fmoc group; detecting resin deprotection; washing the resin; coupling the amino acid; detecting the coupling reaction. Repeat the coupling step until all the peptides are synthesized.
    ​NOTE: Use the Kaiser test to monitor whether every step of deprotection is complete or whether each step of the amino acid coupling is thorough. Alternatively, a small amount of peptide can be cleaved from the resin and checked by LC-MS for successful coupling.

5. Bisalkylation between Met and Cys (Figure 3)

  1. Construct a linear peptide with Met and Cys by repeating steps 4.1-4.6. Add 10 mL of anhydrous methanol to the column with resin for dehydrating and dry with N2 for the next use (repeat 2x) after linear peptide coupling.
  2. Weigh 100 mg of the resin obtained in the previous step into a column (20 mL reservoir) and wash the resin with DCM (5-10 mL) and DMF (5-10 mL) before the coupling step.
  3. Prepare a solution for removing the Cys trt(trityl) protection group. Prepare a sufficient volume (100 mL) of TFA/TIS/DCM (3:5:92) mixture in a glass container for removing the protectiove group.
  4. Add 5-10 mL of the solution of TFA/TIS/DCM (3:5:92) to the column to remove the protection group for 10 min. Repeat six times with N2 bubbling until the yellow color completely disappears.
  5. Drain the solution by a vacuum pump and wash the resin sequentially with DCM (5-10 mL) and DMF (5-10 mL) 5x.
  6. Prepare a solution for reacting with deprotected Cys. Prepare a sufficient volume (50 mL) of mixture solution (DMF) including di-halogenated linker (2 eq) and DIPEA (4 eq) for reacting with deprotected Cys.
  7. Add 5-10 mL of the reaction solution to the column to react with deprotected Cys for at least 3 h with N2 bubbling. Dehydrate with anhydrous methanol and dry with N2 for the next use.
  8. Drain the solution by a vacuum pump and wash the resin sequentially with DCM (5-10 mL) and DMF (5-10 mL) 5x.
  9. Prepare a solution for peptide cyclization: prepare a sufficient volume (20 mL) of TFA mixture (TFA:TIS:H2O = 95:2.5:2.5) in a glass container in the fume hood for peptide cyclization.
  10. Add 5-10 mL of the TFA mixture solution to the polypropylene tube and cleave the resin under the TFA cocktail for 3 h.
    CAUTION: TFA is highly corrosive and irritating; the peptide cleavage process should be performed in a fume hood.
  11. Perform steps 7.1-7.5 to obtain a linear peptide solution by reverse-phase liquid phase purification (HPLC). Freeze-dry the solution for the next use.

6. Propargyl sulfonium salt cyclization (Figure 4)

  1. Construct a linear peptide with Met and Cys by repeating steps 4.1-4.6. Add 10 mL of anhydrous methanol to the column with the resin for dehydrating and dry with N2 for the next use (repeat twice) after linear peptide coupling.
  2. Weigh 100 mg of the resin obtained in the previous step into a column (20 mL reservoir) and wash the resin with DCM (5-10 mL) and DMF (5-10 mL) before the coupling step.
  3. Perform steps 7.1-7.5 to obtain a linear peptide solution by HPLC, and freeze-dry the sample, as mentioned, for next use.
  4. Prepare a 1% HCOOH aqueous solution (in volume) and 1.0 mM propargyl bromide (5 eq) and add to the Met peptide solution (0.2 mM, 1 eq; 0.2 mL of MeCN/H2O [1:1, v/v]).
  5. Next, shake the coupling reaction of Met and propargyl bromide at room temperature for 12 h.
  6. After the reaction, dissolve the product in a polypropylene tube in acetonitrile and filter it through a 0.22 µmfilter membrane. Then, purify the solution by reverse-phase HPLC immediately and freeze-dry it into powder for the next use.
  7. In the last step, add the peptide with propargyl bromide to a polypropylene tube, maintain the reaction solution pH at 8.0 by adding (NH4)2CO3 solution, and shake the reaction mixture at 37 °C for 12 h. This step obtains the cyclic peptide of propargyl sulfonium salt.
  8. Collect the last reaction mixture: dissolve it in a polypropylene tube in acetonitrile and filter it through a 0.22 µm filter membrane. Then, purify the solution by reverse-phase HPLC immediately and freeze-dry it into powder for the next use.

7. Purification of cyclic peptides

  1. Construct linear peptide substrates on Rink-amide MBHA resin by repeating steps 4.1-4.6. Add 10 mL of anhydrous methanol to the column with resin for dehydrating and dry with N2 for the next use (repeat twice) after linear peptide coupling.
  2. Prepare a sufficient volume of cleavage cocktail (TFA/H2O/TIS, v/v/v, 95:2.5:2.5) in the fume hood. Add 1-5 mL of TFA mixture solution to the polypropylene tube and cleave the resin under the TFA cocktail for 3 h.
  3. Next, sequentially dry the resin under a steam of N2 in the column. Alternatively, use a filter device to filter the resin and collect the peptide solution. Then, add 20 mL of ether to the peptide solution to precipitate the peptide, centrifuge at 3,500 x g for 5 min, and repeat twice. Collect the crude peptide precipitate and dry it under a stream of N2 for the next step.
  4. Dissolve 200 mg of crude peptide in a polypropylene tube in 4 mL of acetonitrile-water solution and filter it through a 0.22 µm filter. Transfer the peptide into an HPLC vial insert. Place the insert into the autosampler of a semi-preparative reverse-phase HPLC system equipped with a C18 5 µm, 4.6 mm x 250 mm column, and a 1 mL injection loop.
  5. Purify and isolate the peptide using a gradient program of 5%-95% acetonitrile in water with 0.1% TFA over 30 min while monitoring the HPLC spectra using UV at 254 nm. Confirm the peptide molecular weight by LC-MS, collect the solution of the peptide, and freeze-dry it into powder for the next use.

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Representative Results

All the linear peptides were synthesized on Rink-amide MBHA resin by standard manual Fmoc solid-phase synthesis. A model cyclic hexapeptide (Ac (cyclo-I)-WMAAAC-NH2) was constructed as described in Figure 5A. Notably, a new on-tether chiral center was generated by Met alkylation, with the two epimers of cyclic peptide (Ia, Ib) confirmed by reverse-phase HPLC. Further, the conversion and ratio of epimers were determined using the integration of reverse-phase HPLC. Cyclic Ac-(cyclo-I)-WMAAAC-NH2 peptides 1-Ia and 1-Ib, generated from hexapeptide Ac-WMAAAC-NH2, exhibited distinct retention times and identical molecular weights (Figure 5B). Next, the tolerance of the cyclic peptide to different functional groups was further tested, as shown in Figure 5C. The loop closure efficiency of the 10 linear peptides was evaluated using a di-halogenated linker, with the results showing that all the peptides efficiently generated the corresponding cyclic peptides. Compared with the other cyclic peptides, a model hexacyclic cyclic peptide with high conversion rates produced a differential ratio of 1:1 of the epimers and could be separated by HPLC. However, in some cases, the peptide epimers could not be separated under HPLC conditions, likely due to the sulfonium chiral center of the epimer not being very stable and being slowly racemized into epimer mixtures. The reaction efficiency of the epimers (1-Ia) with pyridinrthiols (PyS) (10 mM) was then examined by HPLC. Figure 5D shows the HPLC traces of the time-dependent conversion between the cyclic peptide (1 mM) and its conjugated product. The traces clearly show the time-dependent reduction of peptide 1-Ia with PyS in PBS (pH 7.4).

Another strategy for peptide ring closure was that, firstly, a propargyl group was attached to Met, and then the formed propargyl sulfonium center triggered a thiol-yne reaction to generate a cyclic peptide. In this strategy, the ring size and peptide sequence did not affect the cyclization reaction, with the peptide containing two or three amino acids just used as a model to close the loop. The synthetic route of intramolecular peptide cyclization is described in Figure 6A. In addition, a simplified propargylated Met model peptide was constructed as described in Figure 6B. The results showed that the model peptide MC's yield could be up to 80% when the reaction solution pH was set to 8.0 (MC refers to a model cyclic peptide synthesized by this route; Figure 6A). Moreover, the model peptide MC was isolated and purified by HPLC (Figure 6D), and its molecular weight was confirmed by LC-MS (Figure 6C). As shown in Figure 6E, the chemical shift was further characterized by 1H NMR and heteronuclear single quantum coherence (HSQC). In addition, the dithiothreitol (DTT) was added to attempt to open the sulfonium ring to explore the stability of MC. The results showed no addition or ring-opening products after 24 h (Figure 6F).

Figure 1
Figure 1: Diagram of the experimental setup for a Fmoc-based solid phase peptide for synthesizing peptides. The peptide column was placed on the solid phase reactor via three-way stopcocks with nitrogen or argon bubbling through the column during peptide synthesis. Please click here to view a larger version of this figure.

Figure 2
Figure 2: On-resin intermolecular synthetic linear CM peptides. All the linear peptides were synthesized on Rink-amide MBHA resin by standard manual Fmoc solid-phase synthesis. Please click here to view a larger version of this figure.

Figure 3
Figure 3: Synthetic routes for peptide cyclization via Cys and Met bisalkylation. The linear peptides containing Met and Cys were constructed as stabilized cyclic peptides. First, the trt-protected Cys was deprotected with 3% TFA in DCM. Then, a di-halogenated linker (2 eq) and DIPEA (4 eq) were added to react with Cys for 3 h. Finally, cyclization between Cys and Met was completed when the resin was cleaved in a TFA cleavage solution. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Synthetic routes for peptide cyclization via Cys and Met thiol-yne. First, the linear peptides were purified and characterized by HPLC and LC-MS. The reaction occurred under the following conditions: a solution including the compound (0.2 mM, 1.0 eq) in 0.2 mL of MeCN/H2O (1:1, v/v), 1% HCOOH aqueous solution (in volume), and propargyl bromide (1.0 mM, 5.0 eq) was shaken at room temperature for 12 h at a pH of 8.0. Please click here to view a larger version of this figure.

Figure 5
Figure 5: Synthesis scheme of cyclic peptides using bisalkylation between Cys and Met. (A) Schematic illustration of peptide cyclization by Cys and Met. (B) The HPLC and LC-MS spectrum of the epimers (1-Ia and 1-Ib). (C) The functional residue tolerance of the different peptides was tested. (D) HPLC traces of the time-dependent conversion between epimers (1-Ia; 1 mM) and their conjugated products. This figure is a modification of that reported by Wang et al.30. Please click here to view a larger version of this figure.

Figure 6
Figure 6: Synthesis scheme of cyclic peptides using a thiol-yne type reaction. (A) Facile construction of peptide cyclization by the thiol-yne reaction. MC refers to a model cyclic peptide synthesized by the route. (B) Intramolecular peptide cyclization of different linear peptides. a = 1 mL of 1 M (NH4)2CO3 aqueous solution. b = 1% Et3N in 1 mL of MeCN/H2O (1:1). Abbreviations: Ahx = 6-aminocaproic acid. The yield was calculated as the percentage of the product determined by weighing. Conversion represented the amount of starting material that reacted by HPLC. (C) The LC-MS spectrum of the MC cyclization peptide. (D) The HPLC spectrum of the MC cyclization peptide. (E) 1H NMR and HSQC spectra characterization of the MC cyclic peptides. (F) 1H NMR spectra of the time-dependent conversion between MC cyclic peptide and DTT in D2O for 3 h, 6 h, 12 h, and 24 h. This figure is a modification of that reported by Hou et al.31. Please click here to view a larger version of this figure.

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Discussion

The synthetic approach described in this paper provides a method for synthesizing cyclic peptides using Cys and Met in the peptide sequence, in which the basic linear peptides are constructed by common solid-phase peptide synthesis techniques. For the bisalkylation of cyclic peptides between Cys and Met, 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 cleavage solution. Notably, the removal of the protective group of Cys was found to be a critical step for the subsequent ring-closure reaction. Therefore, trt-Cys was deprotected, and this was done until the solution had no apparent yellow color. Further studies showed that cyclic peptides developed by the bisalkylation between Cys and Met methodology could be extended to loop closure in a variety of peptides, with the condition being that the peptide contains the amino acids Cys and Met. In addition, the peptide sequence and linker could also be further adjusted according to the experimental design (Figure 5). It was, therefore, necessary to monitor the synthesis by LC-MS.

For cyclic peptides in the thiol-yne type reaction, firstly, linear peptides were also constructed by common solid-phase peptide synthesis techniques on resin, with the following reactions carried out in the liquid phase solution. Crude peptides were then cleaved from the resin and purified by reverse-phase HPLC. A solution of peptides and propargyl bromide was added to the reaction for 12 h, along with 0.2 mL of MeCN/H2O (1:1, v/v) and 1% HCOOH aqueous solution, and the products were then purified immediately by reverse-phase HPLC. Surprisingly, the ring-closure reaction occurred when the pH of the reaction solution was increased to 8.0. This study has developed a method that constrains the peptide to a cyclic conformation structure with good stability via a thiol-yne type reaction. Moreover, it was necessary to adjust the solvent for the peptide loop closure due to the hydrophilicity and hydrophobicity of the peptides being different. For example, the pH of the hydrophilic peptide was adjusted by (NH4)2CO3, and the hydrophobic peptide was then adjusted by a mixed solvent (1% Et3N solution in 50% MeCN/H2O; Figure 6).

In recent years, various ligation technologies have been developed for synthesizing cyclic peptides, with these technologies generating natural peptide bonds and unnatural backbone linkages32. However, challenges remain in the synthetic cyclic peptide field. Firstly, the amino acid sequence of the peptide has a steric effect, and the efficiency of cyclization may be affected by the residues close to the cyclization site. Secondly, the spatial structure of peptides is diverse, and it may be necessary to carefully choose the connection site during ring closure at the same site. Finally, peptide sequences contain hydrophilic or hydrophobic amino acids, and, therefore, the solubility of the reaction solvent needs to be considered. Therefore, more effort should be put into identifying cyclization at the site, solubility, and isomers to further develop cyclic peptide applications in the pharmaceutical industry.

In this research, a series of facile macrocyclization protocols using bisalkylation between Cys and Met or via a thiol-yne type reaction were designed to resolve the challenges of synthesizing cyclic peptides. Fortunately, these reactions were facile, highly efficient, and metal catalyst-free. The methods developed have been demonstrated to perform both intermolecularly and intramolecularly and have satisfying functional group tolerance. Furthermore, these methods were developed to introduce constraint cyclization, ensuring the peptide chain is more conformationally stabilized, thereby improving target protein binding affinity and reducing nonspecific protein binding. In addition, by using reasonable designs, the reaction site selectivity could also be enhanced by forming a conformationally stabilized cyclization peptide. Overall, peptide chain cyclization generated biologically active compounds, indicating that cyclic peptides are promising drug candidates.

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Disclosures

The authors have nothing to disclose.

Acknowledgments

We acknowledge financial support from the National Key R&D Program of China (2021YFC2103900); the Natural Science Foundation of China grants (21778009, and 21977010); the Natural Science Foundation of Guangdong Province (2022A1515010996 and 2020A1515010521): the Shenzhen Science and Technology Innovation Committee, (RCJC20200714114433053, JCYJ201805081522131455, and JCYJ20200109140406047); and the Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions grant (2019SHIBS0004). The authors acknowledge journal support from Chemical Science, The Royal Society of Chemistry for reference 30 and The Journal of Organic Chemistry, American Chemical Society, for reference 31.

Materials

Name Company Catalog Number Comments
1,3-bis(bromomethyl)-benzen Energy D0215
1,3-Dimethylbarbituric acid Energy A46873
1H NMR and HSQC Bruker  AVANCE-III 400
1-Hydroxybenzotriazole hydrate Energy E020543
2-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) Energy A1797
2-mercaptopyridine Energy Y31130
6-Aminocaproic acid Energy A010678
Acetic anhydride Energy A01021454
Acetonitrile Aldrich 9758
Ammonium carbonate Energy 12980
Dichloromethane (DCM) Energy W330229
Digital Heating Cooling Drybath  Thermo Scientific 88880029
Diisopropylethylamine (DIPEA) Energy W320014
Dimethyl formamide (DMF) Energy B020051
Dithiothreitol Energy A10027
Electrospray Ionization Mass SHIMADZU2020  LC-MS2020
Fmoc-Ala-OH Nanjing Peptide Biotech Ltd R30101
Fmoc-Arg(Pbf)-OH Nanjing Peptide Biotech Ltd R30201
Fmoc-Cys(Trt)-OH Nanjing Peptide Biotech Ltd R30501
Fmoc-Gln(Trt)-OH Nanjing Peptide Biotech Ltd R30601
Fmoc-Glu(OtBu)-OH Nanjing Peptide Biotech Ltd R30701
Fmoc-His(Boc)-OH Nanjing Peptide Biotech Ltd R30902
Fmoc-Ile-OH Nanjing Peptide Biotech Ltd R31001
Fmoc-Lys(Boc)-OH Nanjing Peptide Biotech Ltd R31201
Fmoc-Met-OH Nanjing Peptide Biotech Ltd R31301
Fmoc-Pro-OH Nanjing Peptide Biotech Ltd R31501
Fmoc-Ser(tBu)-OH Nanjing Peptide Biotech Ltd R31601
Fmoc-Thr(tBu)-OH Nanjing Peptide Biotech Ltd R31701
Fmoc-Trp(Boc)-OH Nanjing Peptide Biotech Ltd R31801
Fmoc-Tyr(tBu)-OH Nanjing Peptide Biotech Ltd R31901
Fmoc-Val-OH Nanjing Peptide Biotech Ltd R32001
Formic acid Energy W810042
High Performance Liquid
Chromatography		 SHIMADZU LC-2030
Methanol Aldrich 9758
Morpholine Aldrich M109062
N,N'-Diisopropylcarbodiimide Energy B010023
Ninhydrin Reagent Energy N7285
Propargyl bromide Energy W320293
Rink Amide MBHA resin Nanjing Peptide Biotech Ltd.
Solid Phase Extraction (SPE) Sample Collection Plates  Thermo Scientific 60300-403
Tetrakis(triphenylphosphine) palladium Energy T1350
Three-way stopcocks Bio-Rad 7328107
Triethylamine Energy B010737
Trifluoroacetic acid (TFA) J&K 101398
Triisopropylsilane (TIS) Energy T1533

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Cyclic Peptides On-tether Sulfonium Center Sulfonium Salt Fast Reaction Efficient Method Peptide Stabilization N-terminal Deprotection Solution Piperidine Morpholine DMF Vacuum Pump Dichloromethane N,N-dimethylformamide Resin Color Detection Coupling Of Peptides Polypropylene Tube N,N-diisopropylethylamine N,N'diisopropylcarbodiimide
Constructing Cyclic Peptides Using an On-Tether Sulfonium Center
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Song, C., Hou, Z., Jiao, Z., Liu,… More

Song, C., Hou, Z., Jiao, Z., Liu, Z., Lian, C., Zhang, M., Liang, W., Yin, F., Li, Z. Constructing Cyclic Peptides Using an On-Tether Sulfonium Center. J. Vis. Exp. (187), e64289, doi:10.3791/64289 (2022).

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