An amphiphilic D / L cyclic peptide capable of self-assembly to form a nanohelix and a synthesis and assembly method thereof

Abstract

The present invention relates to an amphiphilic D / L cyclic peptide capable of self-assembly to form a nanohelix and a synthesis and assembly method thereof. The synthesis method of the amphiphilic D / L cyclic peptide comprises the following steps: (a) sequentially connecting glycine and threonine on a solid phase resin to prepare a resin for linear peptide synthesis; (b) generating a linear peptide by solid phase peptide synthesis on the resin prepared in step (a); (c) using a cutting liquid to cut the linear peptide to obtain a linear peptide; (d) under heating conditions of 50-70°C and a pH of 7-8, the linear peptide obtained in step (c) is cyclized to form a cyclic peptide. The cyclic peptide synthesis method of the present application has the following advantages: cyclization can be performed under mild conditions, no protection of side chain groups and addition of additional activation reagents are required, the method does not depend on the chirality of the C-terminal amino acid, and a consistent main product can be obtained after cyclization of either D-type or L-type amino acids or a mixture of the two.

Description

Technical Field

[0001] The present invention belongs to the technical field of cyclic peptide synthesis, and in particular, relates to an amphiphilic D / L cyclic peptide composed of alternating L- and D-amino acids and a synthesis and assembly method thereof. Background Art

[0002] Compared with linear peptides, the synthesis of cyclic peptides is challenging. First, racemic products are easily produced in the cyclization reaction, which requires the precursor linear peptide to have a high purity. Therefore, linear peptides synthesized by solid phase often require a relatively fine separation gradient to remove a small amount or part of the isomers present in the system, which will result in a relatively low separation efficiency. In addition, the cyclization of most cyclic peptides needs to be carried out in an organic solvent such as DMF, requires a coupling reagent, and requires a side chain protecting group. In the present invention, the synthesis of cyclic peptides can be carried out in aqueous solution without the need for a side chain protecting group.

[0003] D / L cyclopeptides are a type of cyclopeptides that can be assembled into one-dimensional nanotubes, showing great application prospects in ion channels, sensors, nanomedicine and drug delivery. Currently reported cyclopeptides of this type are all symmetrical in the backbone, while the assembly of cyclopeptides with asymmetric backbones remains to be explored.

[0004] In addition, the traditional process of controlling the assembly and disassembly of small molecule peptides relies on the additional addition of coupling chemicals as energy to drive. Therefore, from the perspective of environmental friendliness, it is very important to be able to control the assembly and disassembly without the need for additional coupling chemicals. Therefore, there is still a need for an effective method to synthesize and separate cyclic peptides, and there is still a need for a method to achieve the self-assembly and disassembly process of cyclic peptides without the need for chemical reagents to drive. Summary of the invention

[0005] Technical Purpose

[0006] A technical purpose of the present invention is to provide a novel method for synthesizing cyclic peptides. The cyclization method can be carried out in aqueous solution, does not require activation reagents and side chain protecting groups, and has mild reaction conditions. It first synthesizes D / L linear peptides through solid phase, and a small amount of racemate does not affect the yield and separation of the cyclization product. In addition, the chirality of the aldehyde derivative of the C-terminal amino acid does not affect the final cyclization product. That is to say, if the C-terminal linear peptide aldehyde group is L-type, the final cyclization product will also be D-type.

[0007] Another technical purpose of the present invention is to provide an amphiphilic D / L cyclic peptide synthesized by the above method.

[0008] Another technical purpose of the present invention is to provide a nanohelical structure formed by self-assembly of the above-mentioned amphiphilic D / L-cyclic peptide.

[0009] Another technical purpose of the present invention is to provide uses of the above nanohelical structure.

[0010] Technical content

[0011] In one aspect, the present invention provides a method for synthesizing an amphiphilic D / L-cyclic peptide, comprising the following steps:

[0012] (a) Resin preparation:

[0013] The resin for linear peptide synthesis is prepared by sequentially connecting glycine and threonine to the solid phase resin;

[0014] (b) Synthetic linear peptides:

[0015] Generate a linear peptide by solid phase peptide synthesis (SPPS) on the resin prepared in step (a), wherein the N-terminus of the linear peptide is an L-amino acid with a side chain nucleophilic group, and the C-terminus is a D-amino acid or an aldehyde derivative of glycine, preferably, the C-terminal amino acid is an aldehyde derivative of a hydrophobic amino acid, wherein the aldehyde derivative of the amino acid specifically refers to a product in which the carboxyl group of the amino acid is reduced to an aldehyde group, for example, in the present application, AA-OH is used to represent a certain amino acid, and AA-H can be used to represent its aldehyde derivative;

[0016] (c) Peptide cleavage

[0017] Using a TFA / TIS / water system as a cutting solution to cut a linear peptide to obtain a linear peptide, wherein in the system, the volume fraction of TIS in the cutting solution is 0.5-1%;

[0018] (d) Cyclization

[0019] Under heating conditions at 50-70°C and at a pH of 7-8, the linear peptide obtained in step (c) is cyclized to form a cyclic peptide. Specifically, the N-terminal amino group and the C-terminal aldehyde group of the linear peptide are condensed to form an imine, and then the side chain group of the N-terminal amino acid attacks the imine to form a cyclic peptide.

[0020] In the following, each of the above steps is explained in detail.

[0021] Step (a)

[0022] In a specific embodiment, in step (a), the solid phase resin is an amino resin, preferably Rink Amide Resin; optionally, its amino acid substitution degree (resin substitution value) can be 0.3 to 1.0 mmol / g, for example, 0.94 mmol / g.

[0023] In a specific embodiment, in step (a), the amount of the coupled amino acid is 3 times the molar amount compared to the resin substitution value (eg, 0.94 mmol / g), and the reaction time is 2 hours.

[0024] Step (b)

[0025] In a specific embodiment, step (b) may include the following steps:

[0026] (b1) Connecting the first amino acid: connecting the first amino acid to the resin obtained in step (a), wherein the first amino acid is an aldehyde derivative of an amino acid, Fmoc-D-AA-H (D means that the amino acid is D-type) or Fmoc-Gly-H before connecting to the solid phase resin;

[0027] (b2) Accession of subsequent amino acids: Based on step (b1), seven other amino acids in alternating L and D forms are sequentially connected, that is, the second amino acid and the N-terminal amino acid are L-form, and finally the Fmoc protecting group is removed.

[0028] For the synthetic linear peptide, preferably, the C-terminal is mainly distributed with hydrophobic amino acids, the N-terminal is mainly distributed with hydrophilic amino acids, and the N-terminal amino acid is an amino acid with a nucleophilic group, for example, the N-terminal amino acid can be selected from serine, cysteine, threonine, tryptophan and tyrosine. The arrangement of the C-terminal hydrophobic amino acid and the N-terminal hydrophilic amino acid makes the synthetic cyclic peptide have good amphiphilicity.

[0029] In a specific embodiment, the first amino acid of step (b1) is Fmoc-D-Leu-H or Fmoc-Gly-H.

[0030] In a specific embodiment, the amino acid aldehyde derivative Fmoc-D-AA-H or Fmoc-Gly-H used in step (b1) can be prepared by the following method:

[0031] The carboxyl group of the amino acid Fmoc-D-AA-OH or Fmoc-Gly-OH is first subjected to a one-step coupling reaction to obtain an amide intermediate, which is then reduced to an aldehyde group through a one-step reduction reaction to obtain an aldehyde-modified amino acid derivative Fmoc-D-AA-H or Fmoc-Gly-H.

[0032] Specifically, the aldehyde derivative Fmoc-D-AA-H can be prepared by the following route:

[0033]

[0034] As shown in the above reaction formula, the C-terminal amino acid Fmoc-D-AA-OH of the cyclic peptide to be synthesized is reacted with DIC and HOBt in DMF to generate an amide intermediate, and the amide intermediate is reduced with LiAH4 in THF to generate an aldehyded amino acid derivative Fmoc-D-AA-H. The R group in the above reaction formula represents a side chain group of a common amino acid. For example, when the first amino acid is Fmoc-D-Leu-H or Fmoc-Gly-H, R is isobutyl and H, respectively.

[0035] In a specific embodiment, in step (b1), the first amino acid (e.g., Fmoc-D-Leu-H) is used in a 4-fold molar amount compared to the resin substitution value, is dissolved in a protic mixed solvent, and then added to the resin, reacted under an inert gas environment, and then the secondary amine generated by the reaction is protected using di-tert-butyl dicarbonate (Boc2O).

[0036] Specifically, in step (b1), first, the resin with threonine at the N-terminus prepared in step (a) is suspended in a container, and then Fmoc-AA-H is dissolved in a mixed solution of methanol (MeOH), dichloromethane (DCM), dimethylformamide (DMF) and acetic acid (AcOH), and the air in the system is replaced with N2, and gently shaken at room temperature, and then the filtered resin is thoroughly washed with DMF and DCM, and tetrahydrofuran (THF) containing Boc2O and N,N-diisopropylethylamine (DIPEA) is added thereto to react, and the resin is collected by filtration and thoroughly washed with DMF, THF and DCM.

[0037] In a specific embodiment, the amino acid sequences (from N-terminus to C-terminus) sequentially inserted in step (b2) are selected from: NH2-SeQeAlW (SEQ ID No: 8), NH2-TeQeAlW (SEQ ID No: 9), NH2-CeQeAlW (SEQ ID No: 10), NH2-NeQeAlW (SEQ ID No: 11), NH2-WeQeAlW (SEQ ID No: 12) and NH2-SeQeClW (SEQ ID No: 13).

[0038] Step (c)

[0039] In a specific embodiment, in step (c), TFA / TIS / water are mixed in a certain volume ratio (for example, a volume ratio of 95:0.5:4.5) as a cutting solution, added to the resin, and after shaking at room temperature, the cut solution is filtered, and the resin is washed with TFA and DCM, and this part of the solution is also filtered together, and the combined lysate is concentrated under vacuum, and the resulting residue is treated with cold ether to precipitate the crude peptide, and then the supernatant is discarded by centrifugation, and the precipitate is subsequently purified by reverse phase HPLC. Specifically, the amount of TIS used is 0.1 mL, and the room temperature shaking time is less than 1.5 h. In step (c), preferably, the amount of TIS should not be too much, otherwise the aldehyde group will be reduced to alcohol, because alcohol cannot undergo cyclization, which will reduce the cyclization yield.

[0040] Specifically, the following reaction formula shows a representative embodiment of the above steps (a)-(c):

[0041]

[0042] Step (d)

[0043] In a specific embodiment, in step (d), the concentration of the linear peptide is less than 5 mM.

[0044] In particular, step (d) is performed as follows:

[0045] The linear peptide obtained in the above step (c) is dissolved in water or phosphate buffer and reacted at a pH value of 7.4-8.0 to generate a cyclic peptide.

[0046] In the above pH range, the N-terminal amino group and C-terminal aldehyde group of the linear peptide condense to form an imine, and then the side chain group of the N-terminal amino acid attacks the imine to form a cyclic peptide.

[0047] In a specific embodiment, in step (d), the linear peptide obtained in step (c) is reacted at 60-70° C. for 24-48 hours at a pH of 7.4-8.0 to generate a cyclic peptide, and the progress of the reaction can be monitored by HPLC.

[0048] In a specific embodiment, step (d) further includes the step of purifying the cyclic peptide: purifying the generated cyclic peptide using a reverse phase high pressure liquid chromatography column. For example, A (water / acetonitrile = 95%: 5%) and B (acetonitrile) containing 0.1% TFA are used as mobile phases, the target peak is collected, and then the acetonitrile is removed by rotary evaporation, and the target cyclic peptide is obtained by concentration and freeze drying. It has been identified that the main product in the obtained product maintains the C-terminal D-type. In the present application, it is found that the chirality of the C-terminal aldehyde amino acid does not affect the configuration of the final cyclization product. That is to say, whether it is an L-type aldehyde group or a D-type aldehyde group, the residue in the final generated cyclization product is D-type.

[0049] In a specific embodiment, the method further comprises the step of introducing a fluorescent group into a suitable side chain of the synthesized cyclic peptide. Preferably, the fluorescent group is derived from Cy5.5.

[0050] As mentioned above, the amphiphilic D / L cyclic peptide of the present application is condensed by the N-terminal amino group and the C-terminal aldehyde group through the head-to-tail cyclization method, and the imine formed by the attack of the side chain nucleophilic group promotes the cyclization reaction. The reaction can be carried out in an aqueous solution, without the need to add an organic solvent, only mild heating conditions are required, and it is insensitive to air. It is important that the N-terminal amino acid must be an amino acid with a side chain nucleophilic group (such as Ser, Thr, Cys, Asn, Trp); in addition, a small amount or part of the racemate appears in the C-terminal aldehyde group of the linear peptide, and there is no need to deliberately separate and remove it.

[0051] In another aspect, the present invention provides an amphiphilic D / L cyclic peptide synthesized by the above method.

[0052] In a specific embodiment, the cyclic peptide is selected from the following structures:

[0053]

[0054] In another aspect, the present invention provides a method for self-assembling the above-mentioned amphiphilic D / L-cyclic peptide to form a nanohelical structure, the method comprising:

[0055] The amphiphilic D / L cyclic peptide is added to a phosphate buffer to prepare an amphiphilic D / L cyclic peptide solution, the pH is adjusted to 7.4-8.0, the solution is allowed to stand at 50-80° C. for 12-24 hours, and then cooled to room temperature to obtain a nanohelical structure.

[0056] In a specific embodiment, the concentration of the phosphate buffer may be 10 mM, and the concentration of the prepared amphiphilic D / L-cyclic peptide solution is 1-2 mM.

[0057] In another aspect, the present invention provides a nanohelical structure, which is formed by an amphiphilic D / L-cyclic peptide. In particular, the nanohelical structure is formed by the above method.

[0058] In another aspect, the present invention provides use of the nanohelical structure in preparing ion channel drugs, sensors or drug delivery systems.

[0059] The beneficial effects of the present invention are as follows:

[0060] (1) The present application aims to solve the racemization problem often encountered in cyclization, that is, it is necessary to separate a linear peptide with relatively high purity and then use it for cyclization, and provides a novel method for synthesizing amphiphilic D / L cyclic peptides, which is independent of the chirality of the C-terminal amino acid. Whether it is D-type or L-type or a mixture of the two, a consistent main product, i.e., D-type, can be obtained after cyclization.

[0061] (2) In the preparation method of the amphiphilic D / L cyclic peptide of the present application, the cyclization process can be carried out under neutral or weakly alkaline conditions (7.4-8.0) under mild heating conditions in an aqueous solution, without the need for organic solvents and activating agents. In addition, the amphiphilic D / L cyclic peptide of the present application can also be reversibly restored to a linear structure under acidic conditions. The existence form of the cyclic peptide can be adjusted by pH, which can provide a reference for the design of stimulus-responsive drug carriers in the future.

[0062] (3) The asymmetric amphiphilic D / L cyclic peptide synthesized in the present application can self-assemble into a braided nanohelix after a heating and cooling process, which is not a traditional nanotube. The structure of these "braided" nanohelices is mainly attributed to the five-membered heterocycles produced in the cyclization reaction. If the main chain skeleton of the cyclic peptide is symmetrical, it will maintain a planar structure, thereby forming a one-dimensional nanotube. The introduction of the five-membered heterocycle destroys the planar structure of the cyclic peptide skeleton, which is more conducive to the formation of a spiral assembly.

[0063] (4) The assemblies formed by the self-assembly of the cyclic peptides of the present invention maintain low cytotoxicity to human bone marrow stromal cells (HS-5).

[0064] In summary, the method of the present invention is suitable for the cyclization of linear peptides with an aldehyde group at the C-terminus, and the cyclization can be carried out under mild conditions without the need for protection of side chain groups and the addition of additional activation reagents. The amphiphilic D / L cyclic peptide synthesized by the dynamic covalent bond can regulate the opening and closing of the ring and the changes in the assembly morphology caused by regulating the pH of the solution. The synthesis of the asymmetric amphiphilic D / L cyclic peptide proposed in the present invention solves the problem of the difficulty in cyclization and purification of linear peptides with partial racemization at the C-terminus, and proposes that the chiral inversion mechanism is a prerequisite for the synthesis of the main cyclized product. The cyclization reaction involved only requires a rough purification of the linear peptide, and does not require the removal of the racemic isomers contained in the linear peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 : Chemical structural formulas of linear peptides 1a and 1b.

[0066] Figure 2 : Congo red staining experiment of compound 2.

[0067] Figure 3: NMR (DMSO-d6) spectra of the amide bond portion of linear peptide 1 (containing 80% 1a and 20% 1b) and linear peptide 1' (containing 11% 1a and 89% 1b).

[0068] Figure 4 : Comparison of the NMR spectra (DMSO-d6) of the main products 2 and 2' isolated from linear peptide 1 and linear peptide 1' under the same cyclization conditions.

[0069] Figure 5 : NMR spectrum of compound 2 in H2O:D2O=9:1.

[0070] Figure 6 : NMR spectrum of compound 3 in DMSO-d6.

[0071] Figure 7 : NMR spectrum of compound 4 in DMSO-d6.

[0072] Figure 8 : NMR spectrum of compound 5 in DMSO-d6.

[0073] Fig. 9 : NMR spectrum of compound 6 in DMSO-d6.

[0074] Fig.10 : NMR spectrum of compound 7 in DMSO-d6.

[0075] Fig.11 : Time-dependent assembly morphology changes of compound 2.

[0076] Fig.12 : Micrograph of compound 8 assembled at 1 mM concentration at room temperature for 24 hours.

[0077] Fig.13 : Assembly morphology of compound 2 at different concentrations (500μm, 1mM, 2mM, 3mM, 6mM).

[0078] Fig.14 : Cytotoxicity of assemblies of some compounds (2, 3, 5). DETAILED DESCRIPTION

[0079] In order to more clearly demonstrate the implementation purpose / technical solution and advantages of the present invention, the technical solution in the implementation mode of the present invention will be clearly and completely described below with reference to specific examples.

[0080] the term

[0081] Unless otherwise specifically defined or otherwise interpreted according to the context, the "amino acid" in the present application refers to common amino acids, for example, common amino acids may include glycine, alanine, valine, leucine, isoleucine, methionine (methionine), proline, tryptophan, serine, tyrosine, cysteine, phenylalanine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine and histidine.

[0082] In the present application, AA in "Fmoc-D-AA-OH" represents a specific amino acid with the OH portion removed. For example, "Leu" in "Fmoc-D-Leu-OH" represents the -OH portion of leucine with the -OH portion removed.

[0083] In the present application, "D / L cyclic peptide" refers to a cyclic peptide in which D-amino acids and L-amino acids are arranged alternately.

[0084] In the present application, unless otherwise specified, the compound refers to the cyclic peptide of the present application, for example, compound 2 and cyclic peptide 2 are synonymous.

[0085] In this application, "room temperature" refers to 25±2°C.

[0086] Materials and reagents

[0087] The chemicals used in the present invention were purchased from companies or prepared by the authors. Rink Amide resin (0.94mmol / g), 2-chloro-trityl resin (0.42mmol / g) and Fmoc-amino acid were purchased from Gill Biochemical (Shanghai, China). Other chemical reagents and solvents were purchased from Aladdin Industrial Co., Ltd. (Shanghai, China). Cy5.5-maleimide was purchased from McLean (Shanghai, China). Nuclear magnetic resonance spectra (NMR) were obtained using 500 and 600MHz (Cryo) AVANCE NEO spectrometers (Bruker, USA). Transmission electron microscopy (TEM) images were obtained on a Talos L120CTEM (Thermo Fisher, USA).

[0088] Abbreviations: Boc2O: tert-butyl dicarbonate;

[0089] DCM: dichloromethane;

[0090] DIPEA: diisopropylethylamine;

[0091] DMF: N,N-dimethylformamide;

[0092] DMSO: dimethyl sulfoxide;

[0093] Fmoc: 9-fluorenylmethoxycarbonyl;

[0094] HBTU: benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate;

[0095] PyAOP: 2-(7-azabenzotriazole)-tetramethyluronium hexafluorophosphate;

[0096] TCEP: Tris(2-carboxyethyl)phosphine hydrochloride

[0097] MeOH: methanol;

[0098] THF: tetrahydrofuran;

[0099] TFA: trifluoroacetic acid;

[0100] ThT: Thioflavin T;

[0101] TIS: triisopropylsilane;

[0102] AcOH: acetic acid;

[0103] Trt: trityl;

[0104] Gly: glycine;

[0105] Thr: threonine;

[0106] Leu: leucine;

[0107] Trp: tryptophan;

[0108] Asn: Asparagine.

[0109] ROESY: Rotating Frame Overhauser Effect Spectroscopy.

[0110] The 10 mM phosphate buffer used in the following examples was prepared as follows: 57.2 mg of disodium hydrogen phosphate (0 hydrate) and 11.6 mg of sodium dihydrogen phosphate (0 hydrate) were taken and the volume was made up to 50 mL with dd water.

[0111] Preparation Example 1: Synthesis of Compound 2

[0112] (1) Select Rink Amide resin with a substitution degree of 0.94 mmol / g, weigh 0.85 g (0.8 mmol) of the resin into a solid phase reaction tube, add DMF (20 mL), and swell for 30 minutes. Then squeeze out the solution with an ear bulb, add 15 mL of 20% piperidine solution to submerge the resin (5 min x 2 times) for deprotection. After a while, remove the above solution, and wash the resin alternately with DCM and DMF for 5 times, each time for one minute.

[0113] (2) In a 20 mL glass weighing bottle, weigh Fmoc-Gly-OH (2.4 mmol, 713 mg), HBTU (2.4 mmol, 912 mg), then add 15 mL of dry DMF, shake thoroughly to dissolve the powder, and finally add DIPEA (700 μL). Add the above solution to the solid phase synthesis tube of step (1), shake gently at room temperature for 1.5-2 hours, remove the reaction solution, wash thoroughly with DCM and DMF, and then carry out the deprotection and washing steps in step (1).

[0114] (3) In a 20 mL glass weighing bottle, weigh Fmoc-Thr-OH (2.4 mmol, 819 mg), HBTU (2.4 mmol, 912 mg), then add 15 mL of dry DMF, shake thoroughly to dissolve the powder, and finally add DIPEA (700 μL). Add the above solution to the solid phase synthesis tube of step (2), shake gently at room temperature for 1.5-2 hours, remove the reaction solution, wash thoroughly with DCM and DMF, and then carry out the deprotection and washing steps as in step (1).

[0115] (4) The resin with threonine at the N-terminus prepared above was transferred to a Schlenk tube, and then Fmoc-D-Leu-H (i.e., aldehyde-protected Fmoc-protected D-Leu) (4 equivalents, 1.08 g) was dissolved in a mixed solution of MeOH, DCM, DMF and AcOH (30:3:2:0.35, v / v / v / v; 16 mL), and the air in the system was removed by bubbling with N2, and the mixture was gently shaken on a shaker at room temperature for 7 h. The filtered resin was then thoroughly washed with DMF and DCM. The resin was transferred to a Schlenk tube, and THF (3 mL) containing Boc2O (5 equivalents, 860 mg) and DIPEA (5.0 equivalents, 700 μL) was added thereto. The reactants were heated at 50°C for 5 h (no shaking was required), the resin was collected by filtration, and was thoroughly washed with DMF, THF and DCM to obtain a resin with the first amino acid (Leu) attached.

[0116] (5) The resin treated in step (4) was transferred back to the synthesis tube, and Fmoc was removed by the deprotection method of step (1). Then, a reaction solution containing Fmoc-Trp(Boc)-OH (2.4 mmol, 1.26 g), HBTU (2.4 mmol, 912 mg) and DIPEA (4.0 mmol, 700 μL) was added. After two hours, the washing / deprotection / washing operation was repeated.

[0117] (6) Similar to the operation in step (5), Fmoc-D-Leu-OH, Fmoc-Ala-OH, Fmoc-D-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, and Fmoc-D-Glu(OtBu)-OH are sequentially connected, and deprotection / washing operations are performed.

[0118] (7) Take 0.2 mmol of the deprotected resin in step (6) and add a reaction solution containing Fmoc-Ser-OH (0.6 mmol, 200 mg), HBTU (0.6 mmol, 228 mg), and DIPEA (1 mmol, 175 μL). After two hours, remove the reaction solution, wash to remove Fmoc, and then wash thoroughly with DMF and DCM.

[0119] (8) TFA / TIS / water (95:0.5:4.5 v / v / v, 20 mL) were mixed according to the aforementioned volume ratio and then added to the resin obtained in step (7). After shaking at room temperature for 1.5 hours, the cleavage solution was carefully filtered into a 100 mL round-bottom flask. The resin was then washed with TFA (2 mL) and DCM (10 mL), and this portion of the solution was also filtered into the bottle. The combined lysate was concentrated under vacuum, and the resulting residue was treated with cold Et2O to precipitate the crude peptide, which was then centrifuged and the supernatant was discarded. The precipitate was then dissolved in water / acetonitrile (1 / 1, v / v) containing 0.1% TFA, filtered, and purified by reverse phase HPLC. The purified product was freeze-dried to obtain linear peptide 1 (SEQ ID No: 1) (this linear peptide contains 80% 1a and 20% 1b, wherein the structures of linear peptides 1a and 1b refer to Figure 1 , the NMR spectrum of linear peptide 1 can be found in Figure 3 ). Note: To avoid the reduction of the C-terminal aldehyde group, only TIS (0.1 mL) was used and the reaction time was shortened to 1.5 h. In the initial exploration, it was found that when the amount of TIS was increased to 2.5%, part of the aldehyde group was reduced to alcohol, which was undesirable.

[0120] (9) The linear peptide freeze-dried in step (8) was dissolved in dd water to a concentration of 1 mM, and the pH of the solution was adjusted to 7.4. The reaction solution was placed in a 68° C. water bath, heated for 48 h, and ultrasonicated for 3 min. A pair of diastereoisomers of cyclized products were obtained by reverse phase HPLC separation (the main product compound 2 accounted for 80%, and the other diastereoisomer accounted for 20%, wherein the stereochemistry of compound 2 at the cyclization site was determined by two-dimensional nuclear magnetic resonance ROESY, and the stereochemistry of compounds 3, 6, and 7 described below was also determined by two-dimensional nuclear magnetic resonance ROESY). A small amount of uncyclized linear peptide can be recovered.

[0121] (10) The isolated compound 2 was subjected to rotary evaporation to remove the organic solvent, and then placed at -80°C for 1 hour and placed in a freeze dryer until a powdered compound 2 (the amino acid sequence of the corresponding linear peptide is SEQ ID No: 1) was obtained. Its structure and NMR spectrum in H2O:D2O=9:1 are shown in Figure 5 .

[0122] In addition, in order to explore whether there is a chiral inversion mechanism at the C-terminal aldehyde amino acid (leucine) during the cyclization of the linear peptide 1, in addition to using Fmoc-L-Leu-H (i.e., aldehyde-protected Fmoc-protected L-Leu) to replace the above-mentioned Fmoc-D-Leu-H, the aldehyde-based linear peptide 1' (11% of 1a and 89% of 1b, whose NMR spectrum is shown in Figure 2) with the C-terminal L-type as the main product was synthesized by the same solid-phase peptide synthesis method as above. Figure 3 ). Under the same cyclization conditions as above, the linear peptide 1' was cyclized to generate the main product 2', and its NMR spectrum was verified to be the same as that of compound 2 (that is, 2' and 2 are the same compound, such as Figure 4 This indicates that the C-terminal partially racemized amino acid can generate a major cyclic peptide product with consistent structure.

[0123] Preparation Example 2: Synthesis of Compound 3

[0124] The synthesis of compound 3 was carried out according to the synthesis steps of compound 2, except that Fmoc-Thr-OH was used instead of Fmoc-Ser-OH in step (7) to obtain powdered compound 3 (the amino acid sequence of the corresponding linear peptide is SEQ ID No: 2), the structure and NMR spectrum of which in H2O:D2O=9:1 are shown in Figure 6 .

[0125] Preparation Example 3: Synthesis of Compound 4

[0126] The synthesis of compound 4 was carried out according to the synthesis steps of compound 2, except that Fmoc-Cys(Trt)-OH was used instead of Fmoc-Ser-OH in step (7) to obtain powdered compound 4 (the amino acid sequence of the corresponding linear peptide is SEQ ID No: 3), the structure and NMR spectrum of which in H2O:D2O=9:1 are shown in Figure 7 The two-dimensional NMR spectrum (not shown here) shows two groups of signals, so compound 4 is a mixture of a pair of diastereomers.

[0127] Preparation Example 4: Synthesis of Compound 5

[0128] The synthesis of compound 5 was carried out according to the synthesis steps of compound 2, except that in step (4), the aldehyde amino acid derivative used was Fmoc-Gly-H instead of Fmoc-D-Leu-H, to obtain compound 5 (the amino acid sequence of the corresponding linear peptide is SEQ ID No: 4), the structure and NMR spectrum of which in H2O:D2O=9:1 are shown in Figure 8 The two-dimensional NMR spectrum (not shown here) showed two groups of signals, so compound 5 was a mixture of a pair of diastereomers.

[0129] Preparation Example 5: Synthesis of Compound 6

[0130] The synthesis of compound 6 was carried out according to the synthesis steps of compound 2, except that Fmoc-Asn(Trt)-OH was used instead of Fmoc-Ser-OH in step (7) to obtain powdered compound 6 (the amino acid sequence of the corresponding linear peptide is SEQ ID No: 5), the structure and NMR spectrum of which in H2O:D2O=9:1 are shown in Fig. 9 .

[0131] Preparation Example 6: Synthesis of Compound 7

[0132] The synthesis of compound 7 was carried out according to the synthesis steps of compound 2, except that Fmoc-Trp(Boc)-OH was used instead of Fmoc-Ser-OH in step (7) to obtain powdered compound 7 (the amino acid sequence of the corresponding linear peptide is SEQ ID No: 6), the structure and NMR spectrum of which in H2O:D2O=9:1 are shown in Fig.10 .

[0133] Preparation Example 7: Synthesis of Compound 8

[0134]

[0135] The synthesis of compound 9 was carried out according to the synthesis steps of compound 2, except that Fmoc-Ala-OH was replaced with Fmoc-Cys(StBu)-OH in step (6) to obtain powdered compound 9.

[0136] Compound 8 was synthesized in the following manner: Compound 9 (5.4 mg, 1 mM) was reduced by TCEP (10 mM, pH 7.4) for 5 hours to obtain compound 10 (the amino acid sequence of the corresponding linear peptide is SEQ ID No: 7) (3 mg, 65%). Then 1.1 mg of compound 10 was dissolved in 10 mM phosphate buffer, and then 2 times the amount of TCEP was added to adjust the pH value of the solution to 7.4, and a DMSO solution (200 μL) containing Cy5.5-maleimide was added. After reacting for 5 hours, compound 8 (0.9 mg) was separated and purified by reversed-phase high performance liquid chromatography.

[0137] Example 1: Assembly of cyclic peptides

[0138] The compound 2 prepared in Preparation Example 1 (about 1 mg) was weighed in a 1.5 mL centrifuge tube, and then a 10 mM phosphate buffer was added to prepare a concentration of 1 mM. The centrifuge tube with a lid was then placed in a 68°C water bath. After 24 hours, it was taken out and placed at room temperature, and the changes in the assembly morphology were observed using a transmission electron microscope at different time points.

[0139] Results Fig.11 . Fig.11 It shows that after heating and cooling, the assembly of compound 2 can be observed after 5 minutes. The assembly morphology is a short spindle-shaped nanoassembly, showing a state of continued extension. After two hours of observation, it appears as a multi-stranded helical nanoassembly, and gradually becomes thicker and longer (24-hour morphology). This shows that the assembly of this type of cyclic peptide is induced by heating and cooling, and there is a time-dependent assembly morphology change.

[0140] Example 2: Congo red test

[0141] In this experiment, the excitation wavelength of Congo red was 490 nm.

[0142] A) Compound 2 (1 mM) and phosphate buffer (pH 7.4) containing Congo red (0.1 mM) were heated in a water bath at 68°C for 25 hours, then cooled to room temperature and the emission spectrum peaks of Congo red were detected at different time points. The results are shown in Figure 2 A, wherein the emission spectrum peak detected at 1 minute is represented by line a; the emission spectrum peak detected at 10 minutes is represented by line b; and the emission spectrum peak detected at 24 hours is represented by line c.

[0143] B) After incubating compound 2 (1 mM) and phosphate buffer (pH 7.4) containing Congo red (0.1 mM) at room temperature for 25 hours, the fluorescence changes were detected at different time points. Figure 2 In B, the fluorescence spectra at three time points basically overlap.

[0144] Figure 2 A in the middle shows that when compound 2 and the small molecule fluorescent dye Congo red are co-incubated, the maximum emission spectrum of the dye moves toward the low spectrum direction. After a heating process, the maximum emission peak of Congo red blue shifts after a short period of time, which indicates that the assembly of Congo red and cyclic peptide has a certain effect.

[0145] Congo red, as a commonly used indicator of β-amyloid protein, is used in scientific research and clinical practice to characterize whether there is abnormal protein aggregation and to study whether there are amyloid lesions in different organs.

[0146] As confirmed by the above experiment, after a heating and cooling process of Congo red (0.1 mM) and compound 2 (1 mM), the maximum emission peak of Congo red showed a certain blue shift in the assembly of compound 2, from 640 nm to 580 nm ( Figure 2 In contrast, if there is no heating process, the above phenomenon will not occur ( Figure 2 (B) These phenomena indicate that the assembly of the present invention has potential application value in drug carrier and spectral detection.

[0147] Example 3: Fluorescence staining experiment

[0148] Cy5.5-labeled cyclic peptide 8 was dissolved in 10 mM phosphate buffer at pH 7.4 and incubated at room temperature for 24 h for assembly. Afterwards, 10 μL of the solution was dropped on a glass slide and covered with a cover glass for confocal imaging (instrument: Zeiss 900).

[0149] Results Fig.12 Due to the presence of fluorescent groups, Fig.12 Peptide assemblies with lengths of several micrometers can be clearly observed, accompanied by loosely arranged areas. This phenomenon also indicates the dynamic assembly process of this type of cyclic peptide.

[0150] Example 4: Assembly experiments at different concentrations

[0151] Compound 2 was prepared in 10mM phosphate buffer (pH 7.4) to a starting concentration of 6mM, and then diluted proportionally to prepare different concentrations (3mM, 2mM, 1mM, 500μM). The above solutions of different concentrations were incubated in a water bath at 68°C for 24h and then cooled to room temperature. After 24 hours, TEM samples were prepared for observation. 10μL of the sample was placed on a carbon-coated copper grid for 1 minute and completely absorbed with filter paper. The sample was negatively stained with 3% uranyl acetate for one minute, and the dye was then absorbed with filter paper. After the sample was dry, the specimen was observed using a Talos L120C TEM instrument with an operating voltage of 120kV.

[0152] Observation results such as Fig.13 shown.

[0153] Fig.13 This shows that compound 2 exhibits extremely strong assembly ability at concentrations of 0.5-6mM, and assembles into very similar nanohelical structures.

[0154] Example 5: Cytotoxicity Experiment

[0155] In order to evaluate the properties of cyclic peptide assemblies as drug delivery carriers, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay was used to evaluate the toxicity of several cyclic peptide assemblies to normal cells.

[0156] The cytotoxicity of three different cyclic peptides (cyclic peptides 2, 3, and 5) at different concentrations on bone marrow stromal cells HS-5 was detected by standard MTT assay. Bone marrow stromal cells HS-5 were cultured in DMEM medium containing 10% FBS and 1% double antibiotics (penicillin and streptomycin). When the cell density reached 70-80%, the medium was discarded, the cells were washed with cold PBS, and then digested with trypsin. After counting, 96-well plates (cell density was 104 cells / well) were plated and cultured overnight in a 37°C, 5% CO2 incubator. Different concentrations of cyclic peptide assembly (prepared as described in Example 1) solution were added and incubated for 24 hours. 10 μL of PBS solution containing MTT was added to each well. After further incubation for 4 hours, 100 μL of SDS·HCl was used to dissolve the crystals. At room temperature, the absorbance of each well at 595 nm was measured using an enzyme reader.

[0157] The experimental results are as follows Fig.14 shown.

[0158] from Fig.14 It can be seen that the assemblies of these compounds showed less cytotoxicity even at a concentration of 100 μM, thus meeting the low toxicity requirements of drug carriers.

Claims

1. An amphiphilic D / L cyclic peptide selected from the following structures:

2. A method for synthesizing the amphiphilic D / L-cyclic peptide described in claim 1, comprising the following steps: (a) Resin preparation: The resin for linear peptide synthesis is prepared by sequentially connecting glycine and threonine to the solid phase resin; (b) Synthetic linear peptides: Generate a linear peptide by solid phase peptide synthesis (SPPS) on the resin prepared in step (a), wherein the N-terminus of the linear peptide is an L-amino acid with a side chain nucleophilic group, and the C-terminus is an aldehyde derivative of D-leucine or glycine; in, Step (b) comprises the following sub-steps: (b1) introduction of the first amino acid: introducing the first amino acid to the resin obtained in step (a), wherein the first amino acid is an aldehyde derivative of the amino acid before introduction to the solid phase resin; (b2) Insertion of subsequent amino acids: on the basis of step (b1), seven other amino acids in alternating L and D forms are sequentially inserted, i.e., the second amino acid and the N-terminal amino acid are L-form, and finally the Fmoc protecting group is removed, wherein the amino acid sequences sequentially inserted in step (b2) are selected from the group consisting of NH2-SeQeAlW (SEQ ID No: 8), NH2-TeQeAlW (SEQ ID No: 9), NH2-CeQeAlW (SEQ ID No: 10), NH2-NeQeAlW (SEQ ID No: 11), NH2-WeQeAlW (SEQ ID No: 12) and NH2-SeQeClW (SEQ ID No: 13); (c) Peptide cleavage Using trifluoroacetic acid (TFA) / triisopropylsilane (TIS) / water system as cutting liquid to cut linear peptide to obtain linear peptide, wherein in the system, the volume fraction of TIS in the cutting liquid is 0.5-1%; (d) Cyclization The linear peptide obtained in step (c) is cyclized to form a cyclic peptide under heating conditions at 50-70°C and pH 7-8.

3. The method according to claim 2: wherein: In step (a), the solid phase resin is an amino resin.

4. The method according to claim 2: wherein: In step (c), a mixture of TFA / TIS / water in a volume ratio of 95:0.5:4.5 is added to the resin as a cutting solution, and after shaking at room temperature, the cutting solution is filtered, and the resin is washed with TFA and DCM, filtered, and the combined cleavage solution is concentrated under vacuum. The resulting residue is treated with cold ether to precipitate the crude peptide, and then centrifuged to discard the supernatant, and the precipitate is subsequently purified by reverse phase HPLC.

5. The method according to claim 2: wherein: In step (d), the linear peptide obtained in step (c) is dissolved in water or phosphate buffer and reacted at pH 7.4-8.0 and 60-70° C. for 24-48 hours to generate a cyclic peptide.

6. A method for self-assembling a nanohelical structure from the amphiphilic D / L-cyclic peptide of claim 1, the method comprising: The amphiphilic D / L cyclic peptide as claimed in claim 1 is added to a phosphate buffer to prepare an amphiphilic D / L cyclic peptide solution, the pH is adjusted to 7.4-8.0, the solution is allowed to stand at 50-80° C. for 12-24 hours, and then cooled to room temperature to obtain a nanohelical structure. 7 . A nanohelical structure, formed by the amphiphilic D / L-cyclic peptide according to claim 1 . The nanohelix structure according to claim 7 , which is formed by the method according to claim 6 .

9. Use of the nanohelical structure as claimed in claim 7 in the preparation of ion channel drugs, sensors or drug delivery systems.

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