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.
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.
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.
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.
2. Resin preparation
NOTE: Choose the amount of loaded resin according to the length of the coupling peptide.
3. N-terminal Fmoc deprotection
NOTE: Deprotection by morpholine requires 30 min, and deprotection by piperidine takes 5 min.
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.
5. Bisalkylation between Met and Cys (Figure 3)
6. Propargyl sulfonium salt cyclization (Figure 4)
7. Purification of cyclic peptides
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: 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: 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: 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: 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: 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: 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.
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.
The authors have nothing to disclose.
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.
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 |