Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

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 permeability^4,5. In the past 2 decades,....

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

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-.......

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. .......

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