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* These authors contributed equally
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,....
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.
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-.......
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. .......
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 Chemistr....
Name | Company | Catalog Number | Comments |
1,3-bis(bromomethyl)-benzen | Energy | D0215 | |
1,3-Dimethylbarbituric acid | Energy | A46873 | |
1H NMR and HSQC | Bruker | Â 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 | Â Thermo Scientific | 88880029 | |
Diisopropylethylamine (DIPEA) | Energy | W320014 | |
Dimethyl formamide (DMF) | Energy | B020051 | |
Dithiothreitol | Energy | A10027 | |
Electrospray Ionization Mass | SHIMADZU2020 | Â 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 | Â 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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