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CN118530294B - A kind of aromatic diamine immobilized linker, its precursor, preparation method and application - Google Patents

A kind of aromatic diamine immobilized linker, its precursor, preparation method and application Download PDF

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CN118530294B
CN118530294B CN202410573195.2A CN202410573195A CN118530294B CN 118530294 B CN118530294 B CN 118530294B CN 202410573195 A CN202410573195 A CN 202410573195A CN 118530294 B CN118530294 B CN 118530294B
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hydroxyl
halogen
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CN118530294A (en
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李承喜
谢佩
于怡
万枫
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Zhejiang University ZJU
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    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D213/78—Carbon atoms having three bonds to hetero atoms, with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07C237/20—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atoms of the carboxamide groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton containing six-membered aromatic rings
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    • C07C237/30—Carboxylic acid amides, the carbon skeleton of the acid part being further substituted by amino groups having the carbon atom of at least one of the carboxamide groups bound to a carbon atom of a non-condensed six-membered aromatic ring of the carbon skeleton having the nitrogen atom of the carboxamide group bound to hydrogen atoms or to acyclic carbon atoms
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    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D241/08—Heterocyclic compounds containing 1,4-diazine or hydrogenated 1,4-diazine rings not condensed with other rings having one or two double bonds between ring members or between ring members and non-ring members with oxygen atoms directly attached to ring carbon atoms
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    • C07D333/26—Heterocyclic compounds containing five-membered rings having one sulfur atom as the only ring hetero atom not condensed with other rings not substituted on the ring sulphur atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
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    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
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    • C07K5/12—Cyclic peptides with only normal peptide bonds in the ring
    • C07K5/126—Tetrapeptides

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Abstract

本发明公开了一种芳基二胺固载连接子、其前体以及制备方法和在制备环肽中的应用。芳基二胺固载连接子包括固相承载材料，固相承载材料通过其上的活性基团直接或经中间连接单元间接连接芳基二胺，芳基二胺具有式(1)～(6)任一所示结构：式(1)～(6)中：X为C或N；R1、R2分别独立为H或者氨基酸与氨基发生缩合反应脱去一分子水的羰基化合物结构单元，且R1、R2中至少有一个为H。

The present invention discloses an aryl diamine solid-supported linker, a precursor thereof, a preparation method thereof, and an application thereof in the preparation of a cyclopeptide. The aryl diamine solid-supported linker comprises a solid-phase support material, the solid-phase support material is directly connected to the aryl diamine through an active group thereon or indirectly connected via an intermediate connection unit, and the aryl diamine has a structure shown in any one of formulas (1) to (6): In formulae (1) to (6), X is C or N; R 1 and R 2 are independently H or carbonyl compound structural units formed by condensation reaction between amino acid and amino group to remove one molecule of water, and at least one of R 1 and R 2 is H.

Description

Aryl diamine immobilized linker, precursor thereof, preparation method and application
Technical Field
The invention relates to the technical field of polypeptide synthesis, in particular to an aryl diamine immobilized linker, a precursor thereof, a preparation method and application.
Background
The solid-phase flow synthesis is an effective method for preparing the cyclopeptide molecule, and has the advantages of simple and convenient operation, no need of separation and purification of intermediates, easy automation and the like. Related prior art discloses a method for synthesizing cyclic peptide by an enzyme method on a solid carrier, wherein the method comprises the steps of synthesizing linear peptide on the solid carrier, and cyclizing the linear peptide on the carrier to form cyclic peptide by enzyme catalysis, and the related prior art discloses a method for preparing polypeptide cyclic peptide by using azide-alkyne cycloaddition reaction under specific conditions, so as to prepare a series of histamine head-tail cyclic peptides with triazole rings.
Currently, there are four main methods for preparing cyclic peptides, namely "head-tail", "side chain-side chain", "head-side chain" and "side chain-tail" connection. Among them, the latter three methods all require the incorporation of amino acids of specific functional groups or unnatural amino acids, thereby limiting structural diversity and all altering the structure of natural amino acids. The natural amino acid structure of the cyclic peptide can be retained to the greatest extent by utilizing a head-tail connection method.
In order to promote intramolecular acylation and inhibit intermolecular polymerization, a resin mode with low concentration and medium loading is often adopted, but the problems of long reaction time, low efficiency, difficult purification and the like in the preparation process cannot be solved.
Disclosure of Invention
Aiming at the technical problems and the defects in the field, the invention provides an aryl diamine immobilized linker, a precursor thereof, a preparation method and application.
The invention combines the solid phase flow synthesis method of polypeptide, adopts the mode of 'head-tail' connection, develops 'aryl diamine' as a connector main body, fixes the connector main body on a solid phase bearing material such as resin and the like, forms an aryl triazole structure with nitrous acid compounds such as 'activator' nitrite and the like, and then carries out intramolecular cyclization to obtain a cyclized product. The invention is successfully applied to the preparation of the cyclic peptide by designing the structure and the preparation process of the novel 'linker' and the precursor thereof, and the process flow is also applicable to the high-temperature flow automatic synthesis of the cyclic peptide.
The invention adopts aryl diamine immobilized linker to prepare the cyclopeptide, has mild condition of the whole process flow, simple operation and no need of multiple separation and purification, can effectively solve the technical problems of lower synthesis efficiency, poor selectivity and the like in the reaction process, realizes high-temperature flow automatic cyclopeptide synthesis, effectively shortens the reaction time, and can meet the high-throughput screening of cyclopeptide drugs.
In the present invention, the solid phase support material is not particularly limited, and may include any one or more of Wang resin, MBHA resin, trityl resin, 2-CTC resin, RINK AMIDE resin, sieber resin, DHP resin, merrifield resin, NO 2 -Merr. Resin, REM resin, ONb resin, HMNb resin, HMBA resin, HMPA resin, HMPB resin, PAM resin, sasrin resin, HAL resin, CBHO resin, PAL resin, AMNb resin, ANP resin, BHA resin, knorr resin, XAL resin, moBHB resin, pore-controllable glass, magnetic beads, etc., as is conventional in the art.
In a first aspect, the invention provides an aryl diamine immobilization connector, comprising a solid phase bearing material, wherein the solid phase bearing material is directly connected with aryl diamine through active groups on the solid phase bearing material or indirectly connected with aryl diamine through an intermediate connecting unit, and the aryl diamine has a structure shown in any one of formulas (1) - (6):
in the formulas (1) - (6):
X is C or N, preferably N, and the N-containing heterocycle is favorable for improving the selectivity in the preparation process of the cyclopeptide compared with the benzene ring structure, and the selectivity is more than 95:5;
r 1、R2 is H or carbonyl compound structural unit of which one molecule of water is removed by condensation reaction of amino acid and amino, and at least one of R 1、R2 is H. The source amino acid of the carbonyl compound structural unit may comprise any one or more of glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile), methionine (Met), phenylalanine (Phe), tryptophan (Trp), proline (Pro), serine (Ser), threonine (Thr), cysteine (Cys), tyrosine (Tyr), asparagine (Asn), glutamine (Gln), aspartic acid (Asp), glutamic acid (Glu), lysine (Lys), arginine (Arg), histidine (His), selenocysteine (Sec), and pyrrolysine (Pyl).
The reactive group may include at least one of an amino group, a halogen atom (e.g., cl, etc.), a hydroxyl group.
The intermediate connection unit may comprise any one or more of the following combinations:
One or more linked amino acid fragments;
n 1 is any integer between 1 and 15;
R 2 is O, -N (R 3) -, S, -C (=O) -or-S (=O) 2-,R3 is H or C 1～C4 alkyl;
n 2 is any integer between 1 and 15;
R 3 is H, substituted or unsubstituted C 1～C4 alkyl, C 1～C4 alkyl substituted or unsubstituted phenyl, C 1～C4 alkyl substituted or unsubstituted thienyl, C 1～C4 alkyl substituted or unsubstituted naphthyl, C 1～C4 alkyl substituted or unsubstituted furyl, C 1～C4 alkyl substituted or unsubstituted indolyl, mercapto, C 2～C4 amide, C 2～C4 carboxyl or C 1～C4 alkylamino, wherein the substituent on the C 1～C4 alkyl is selected from one or more of mercapto, C 1～C4 alkylthio, hydroxy and halogen;
r 4 is one or more of H, halogen, alkynyl, substituted or unsubstituted C 1～C4 alkyl, C 1～C4 alkyl substituted or unsubstituted phenyl, C 1～C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C 1～C4 alkoxy, ester group, hydroxyl, C 2～C4 acyl and cyano, wherein the substituents on the C 1～C4 alkyl and the C 1～C4 alkoxy are respectively and independently selected from one or more of mercapto, C 1～C4 alkylthio, hydroxyl and halogen;
R 5 is one or more of H, halogen, alkynyl, substituted or unsubstituted C 1～C4 alkyl, C 1～C4 alkyl substituted or unsubstituted phenyl, C 1～C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C 1～C4 alkoxy, ester group, hydroxyl, C 2～C4 acyl and cyano, wherein the substituents on the C 1～C4 alkyl and the C 1～C4 alkoxy are respectively and independently selected from one or more of mercapto, C 1～C4 alkylthio, hydroxyl and halogen;
r 6 is one or more of H, halogen, alkynyl, substituted or unsubstituted C 1～C4 alkyl, C 1～C4 alkyl substituted or unsubstituted phenyl, C 1～C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C 1～C4 alkoxy, ester group, hydroxyl, C 2～C4 acyl and cyano, wherein the substituents on the C 1～C4 alkyl and the C 1～C4 alkoxy are respectively and independently selected from one or more of mercapto, C 1～C4 alkylthio, hydroxyl and halogen;
R 7 is one or more of H, halogen, alkynyl, substituted or unsubstituted C 1～C4 alkyl, C 1～C4 alkyl substituted or unsubstituted phenyl, C 1～C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C 1～C4 alkoxy, ester group, hydroxyl, C 2～C4 acyl and cyano, wherein the substituents on the C 1～C4 alkyl and the C 1～C4 alkoxy are respectively and independently selected from one or more of mercapto, C 1～C4 alkylthio, hydroxyl and halogen;
R 8 is -(CH2)n-、-CH2(CH3)-、-CH2(CH2CH3)-、-CH2(CH2Ph)-、-CH2OCH2CH2- or-CH 2(CH2CH2SCH3) -, n=1-20, or R 8 is any one structure of formulas (a) - (h):
n 3、n4、n5 is independently 1 or 2, r 10、R11 is independently C 1～C4 alkyl or C 1～C4 alkoxy;
R 9 is -(CH2)m-、-CH2(CH3)-、-CH2(CH2CH3)-、-CH2(CH2Ph)-、-CH2OCH2CH2- or-CH 2(CH2CH2SCH3) -, m=1-20, or R 9 is any one structure of formulas (a) - (h).
In a second aspect, the present invention provides a method for preparing an aryl diamine immobilization linker according to the first aspect, comprising the steps of:
1) The solid phase bearing material is directly connected with any one of the structures shown in (7) - (12) through active groups on the solid phase bearing material to obtain a precursor of the immobilized linker, or
The solid-phase bearing material is firstly connected with the intermediate connection unit through active groups on the solid-phase bearing material, and then any one of structures (7) - (12) is connected with the intermediate connection unit to obtain a precursor of the immobilized connector;
in the formulas (7) - (12):
X is C or N, preferably N, and the N-containing heterocycle is favorable for improving the selectivity in the preparation process of the cyclopeptide compared with the benzene ring structure, and the selectivity is more than 95:5;
R 4、R5 is respectively and independently nitro, -NH 2, -NHFmoc or-NHR 6,R6 is an amino unprotected or Fmoc protected amino acid and amino condensed to remove a molecule of water carbonyl compound structural unit, and R 4、R5 is not simultaneously selected from-NH 2 and amino unprotected amino acid and amino condensed to remove a molecule of water carbonyl compound structural unit;
2) And (3) carrying out reductive amination and/or at least one Fmoc protection removal on the nitro group on the structure shown in any one of formulas (7) to (12) of the immobilized linker precursor to obtain the aryl diamine immobilized linker.
The reaction system used in step 2) of the nitroreductive amination may include sodium borohydride and weak acid system, sodium cyanoborohydride and weak acid system, or sodium triacetoxyborohydride and weak acid system, etc. Further, the weak acid may include dilute hydrochloric acid, glacial acetic acid. Further, the concentration of the dilute hydrochloric acid can be 1-2 mmol/L.
The reducing agent used in step 2) nitroreductive amination may include tetrahydroxydiboron and 4,4' -bipyridine systems, pd/C hydrogenation systems, raney Ni hydrogenation systems, iron powder and acid systems, zinc powder and acid systems, snCl 2 and acid systems, tiCl 3 and acid systems, lithium aluminum hydride systems, sodium borohydride-Lewis acid systems, sodium dithionite and basic systems, hydrazine hydrate systems.
In a third aspect, the present invention provides an immobilized linker precursor, comprising a solid phase support material, wherein the solid phase support material is directly or indirectly connected to a precursor structure through an intermediate connection unit by an active group thereon, and the precursor structure has any one of structures represented by formulas (7) to (12):
in the formulas (7) - (12):
X is C or N, preferably N, and the N-containing heterocycle is favorable for improving the selectivity in the preparation process of the cyclopeptide compared with the benzene ring structure, and the selectivity is more than 95:5;
R 4、R5 is respectively and independently nitro, -NH 2, -NHFmoc or-NHR 6,R6 is carbonyl compound structural unit of which amino is unprotected or amino acid protected by Fmoc and amino undergo condensation reaction to remove one molecule of water, and R 4、R5 is not simultaneously selected from-NH 2 and carbonyl compound structural unit of which amino is unprotected and amino undergoes condensation reaction and removes one molecule of water. The optional range of source amino acids for the carbonyl compound building block may be referred to in the description of the source amino acid for the carbonyl compound building block in the first aspect.
In a third aspect, the optional scope of the reactive group and the intermediate linking unit may be as described in relation to the first aspect.
In a fourth aspect, the present invention provides the use of an aryl diamine immobilization linker according to the first aspect or an immobilization linker precursor according to the third aspect for the preparation of a cyclic peptide.
In a fifth aspect, the present invention provides a method for preparing a cyclic peptide, which adopts the aryl diamine immobilization linker according to the first aspect, and specifically comprises the steps of:
s1, synthesizing a polypeptide chain on the aryl diamine immobilization linker through an amino acid condensation reaction;
s2, treating the product of the step S1 with nitrous acid compounds to form an aryl triazole structure;
S3, cyclizing the product of the step S2 in an alkaline environment to generate cyclic peptide.
In step S1, the condensing agent used in the amino acid condensation reaction may include at least one of a carbodiimide condensing agent, a phosphorus positive ion condensing agent, and a urea positive ion condensing agent. Further:
the carbodiimide type condensing agent may include at least one of N, N '-Dicyclohexylcarbodiimide (DCC), N' -Diisopropylcarbodiimide (DIC), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (edc·hcl);
The phospho-cation condensing agent may include at least one of 1H-benzotriazol-1-yloxytripyrrolidinyl hexafluorophosphate (PyBOP), (7-azabenzotriazol-1-oxy) tripyrrolidinyl phosphohexafluorophosphate (PyAOP), bis (2-oxo-3-oxazolidinyl) phosphinic chloride (BOP-Cl), chlorotrimyrrolidinyl hexafluorophosphate (PyCloP), tripyrrolidinyl phosphonium bromide hexafluorophosphate (PyBrop);
The urea positive ion condensing agent may include benzotriazole-N, N, N ', N' -tetramethylurea Hexafluorophosphate (HBTU), N, N, N ', N' -tetramethyl-O- (7-azabenzotriazol-1-yl) hexafluoro phosphate urea (HATU), 2- (7-azabenzotriazol) -N, N, N ', at least one of N' -tetramethyluronium tetrafluoroborate (TATU), O- [ (ethoxycarbonyl) cyanomethylamine ] -N, N, N ', N' -tetramethylthiourea tetrafluoroborate (TOTU), O- (benzotriazol-1-yl) -N, N, N ', N' -dipyrromethene hexafluorophosphate (HBPyU).
In step S1, the condensation activator used in the amino acid condensation reaction may include at least one of 1-hydroxy-7-azobenzotriazole (HOAt), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-1H-1, 2, 3-triazole-4-carboxylic acid ethyl ester (HOCT), 3-hydroxy-3, 4-dihydro-4-oxo-1, 2, 3-benzotriazine (HOOBt), 2-hydroxypyridine-N-oxide (HOPO), 4-Dimethylaminopyridine (DMAP), N-hydroxysuccinimide (HOSu), p-nitrophenol, N-Diisopropylethylamine (DIEA), oxyma, and derivatives thereof.
In step S1 to step S3, the solvent of each reaction process may independently include at least one of N, N-Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), dichloromethane (DCM), ethyl Acetate (EA), tetrahydrofuran (THF), toluene, acetone, diethyl ether, acetonitrile, methanol, water, respectively.
In the step S1 to the step S3, the reaction temperature in each reaction process can be 0-100 ℃ respectively and independently.
In step S2, the nitrous compound may include at least one of sodium nitrite, nitrosylsulfuric acid, methyl nitrite, ethyl nitrite, isopropyl nitrite, butyl nitrite, tert-butyl nitrite, amyl nitrite, isoamyl nitrite, and hexyl nitrite.
Compared with the prior art, the invention has the beneficial effects that:
1. the 'linker' synthesis method can adopt a solid phase synthesis technology, strong reducing agents such as palladium carbon, metallic reagent zinc powder, iron powder, tin dichloride and the like with explosion risk are not needed in the process, and the method has the advantages of mild reaction conditions, simple and convenient operation, high safety and high biological activity.
2. The N, O, S heterocyclic linker effectively solves the chemical selectivity problem in the synthesis process, avoids the generation of a large number of bimolecular acylation products, improves the reaction efficiency, and has the characteristics that the selectivity of monoacylation to bisacylation is not less than 90:10, wherein the N heterocyclic linker is optimal, and the selectivity of monoacylation to bisacylation is more than 95:5, so that the reaction efficiency can be effectively improved.
3. The linker of the invention can not only manually synthesize the cyclic peptide, but also realize high-temperature flow automatic synthesis of the cyclic peptide.
4. The invention does not need to reserve a special functional group amino acid as a connection point in the chain peptide skeleton for the synthesis of the cyclic peptide, breaks through the limit of the structural design of the cyclic peptide medicine, and realizes the preparation of the cyclic peptide by 'traceless' cyclization.
5. The invention can be used for the synthesis of cyclic peptides with different orders, including but not limited to the preparation of cyclic peptides with milligram, gram, kilogram and the like, and can be industrially applied.
Drawings
FIG. 1 is a validated selectivity chromatogram of a heteroaryl diamine resin.
FIG. 2 is a graph of a phenyl diamine resin validated selectivity chromatogram.
Detailed Description
The invention will be further elucidated with reference to the drawings and to specific embodiments. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The methods of operation, under which specific conditions are not noted in the examples below, are generally in accordance with conventional conditions, or in accordance with the conditions recommended by the manufacturer.
Examples of different linker synthesis methods and cyclic peptide synthesis routes are described below.
A) "Azacyclic" linkers
The method utilizes polypeptide solid-phase synthesis technology, firstly obtains corresponding 5-nitro-6-amino nicotinamide compound through the condensation reaction of RINK AMIDE resin and 5-nitro-6-amino nicotinic acid, and then carries out nitro reduction on the 5-nitro-6-amino nicotinamide compound under the action of tetrahydroxy diboron and 4,4' -bipyridine.
The technical scheme route is schematically as follows:
Wherein, RINK AMIDE resins are shown.
B) "benzene ring" linker
The method utilizes polypeptide solid-phase synthesis technology, firstly obtains corresponding 4-aminomethyl-2-nitroaniline compound through the reductive amination reaction of RINK AMIDE resin and 4-nitro-3-aminobenzaldehyde, and then carries out nitroreduction on the 4-aminomethyl-2-nitroaniline compound under the action of tetrahydroxy diboron and 4,4' -bipyridine.
The technical scheme route is schematically as follows:
Wherein, RINK AMIDE resins are shown.
C) Synthesis of natural cyclic peptides
The method utilizes polypeptide solid-phase synthesis technology to synthesize long-chain peptide, under the action of isoamyl nitrite, 2, 3-diaminopyridine generates a pyridotriazole analogue with higher dissociation capability, then intramolecular cyclization reaction is carried out under alkaline condition to obtain cyclic peptide with side chain protection, and finally protecting group is removed by acidic condition to obtain natural cyclic peptide.
The technical scheme route is schematically as follows:
wherein Trt represents a triphenylmethyl protecting group.
Example 1
(1) Prepared using a polypeptide synthesizer, 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) was placed in a 10mL syringe with a sieve plate, followed by 5mL DMF to swell for 30min, solvent was removed, 5-nitro-6-aminonicotinic acid or 3-nitro-4-aminobenzoic acid (0.41 mmol), HATU (156 mg,0.41 mmol), DIEA (110. Mu.L, 0.62 mmol) was dissolved in 2mL DMF, added to the resin and allowed to stand for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
(2) Tetrahydroxydiboron (28 mg,0.31 mmol) and 4,4' -bipyridine (1 mg,0.005 mmol) were dissolved in 2.5mL DMF and then added to the resin from (1) and reacted on a shaker for 30min. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
1A cleavage of the resin from the lysate (TFA: TIPS: EDT: H 2 O=94:1:2.5:2.5), collecting the cleavage filtrate, volatilizing the solution by air flow, adding diethyl ether to the resulting solid, settling, and resolving the centrifuged solid by LCMS: MS (ESI) exact mass calcd.for C 6H9N4O[M+H]+:153.08; found:153.16.
2A cleavage of the resin from the lysate (TFA: TIPS: EDT: H 2 O=94:1:2.5:2.5), collecting the cleavage filtrate, volatilizing the solution by air flow, adding diethyl ether to the resulting solid, settling, and resolving the centrifuged solid by LCMS: MS (ESI) exact mass calcd.for C 6H9N4O[M+H]+:152.08; found:152.22.
Example 2
(1) Prepared using a polypeptide synthesizer, 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) was placed in a 10mL syringe with a sieve plate, followed by 5mL DMF to swell for 30min, solvent was removed, fmoc-Gly-OH (92 mg,0.31 mmol), HATU (118 mg,0.31 mmol), DIEA (110. Mu.L, 0.62 mmol) was dissolved in 2mL DMF, followed by addition to the resin, standing for 30min, and after the reaction was completed, three washes with DCM and DMF, respectively. Finally 20% piperidine was added and Fmoc removal was performed.
(2) 5-Nitro-6-aminonicotinic acid or 3-nitro-4-aminobenzoic acid (0.41 mmol), HATU (156 mg,0.41 mmol), DIEA (110. Mu.L, 0.62 mmol) were dissolved in 2mL DMF, followed by addition of the resin from (1), followed by a 1h reaction, and after completion of the reaction, three washes each with DCM and DMF.
(3) Tetrahydroxydiboron (28 mg,0.31 mmol) and 4,4' -bipyridine (1 mg,0.005 mmol) were dissolved in 2.5mL DMF and then added to the resin from (2) and reacted on a shaker for 30min. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
1B cleavage of the resin from the lysate (TFA: TIPS: EDT: H 2 O=94:1:2.5:2.5), collecting the cleavage filtrate, volatilizing the solution with an air stream, adding diethyl ether to the obtained solid, settling, centrifuging the obtained solid, and resolving the solid by LCMS ：MS(ESI)exact mass calcd.for C8H12N5O2[M+H]+:210.10;Found:210.21.
2B cleavage of the resin from the lysate (TFA: TIPS: EDT: H 2 O=94:1:2.5:2.5), collecting the cleavage filtrate, volatilizing the solution with an air stream, adding diethyl ether to the obtained solid, settling, centrifuging the obtained solid, and resolving the solid by LCMS ：MS(ESI)exact mass calcd.for C9H13N4O2[M+H]+:209.10;Found:209.22.
Example 3
(1) Preparation was performed using a polypeptide synthesizer by placing 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) in a 10mL syringe with a sieve plate, followed by swelling with 5mL DMF for 30min, removing the solvent, dissolving 4-amino-3-nitrophenylacetic acid (81 mg,0.41 mmol), HATU (156 mg,0.41 mmol), DIEA (110. Mu.L, 0.62 mmol) in 2mL DMF, adding to the resin, standing for 1h, and washing three times with DCM and DMF, respectively, after the reaction.
(2) Tetrahydroxydiboron (28 mg,0.31 mmol) and 4,4' -bipyridine (1 mg,0.005 mmol) were dissolved in 2.5mL DMF and then added to the resin from (1) and reacted on a shaker for 30min. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
2C resin was cleaved from the lysate (TFA: TIPS: EDT: H 2 O=94:1:2.5:2.5), the cleavage filtrate was collected, the solution was evaporated using an air stream, the resulting solid was then added with diethyl ether, settled, and the centrifuged resulting solid was resolved by LCMS: MS (ESI) exact mass calcd.for C 8H12N3O[M+H]+:166.10; found:166.24.
Example 4
(1) Preparation was performed using a polypeptide synthesizer by placing 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) in a 10mL syringe with a sieve plate, followed by 5mL DMF for swelling for 30min, removing the solvent, dissolving 6-amino-5-nitropyridine-3-carbaldehyde or 4-amino-3-nitrobenzaldehyde (0.51 mmol) in 2mL DMF:MeOH:AcOH in the mixed solution, followed by adding to the resin, adding sodium cyanoborohydride (64 mg,0.51 mmol) to the reaction system, reacting on a shaker for 48H, and washing three times with DCM, H 2 O and DMF, respectively, after the reaction.
(2) Tetrahydroxydiboron (28 mg,0.31 mmol) and 4,4' -bipyridine (1 mg,0.005 mmol) were dissolved in 2.5mL DMF and then added to the resin from (1) and reacted on a shaker for 30min. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
(3) Verification of 1d aryl diamine resin preparation of chain peptide, first condensation HATU (159.2 mg,0.41 mmol), fmoc-Gly-OH (124.7 mg,0.41 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF and then added to the resin from (2) and allowed to stand for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence. Second condensation HATU (159.2 mg,0..41 mmol), fmoc-Gln-OH (192.4 mg,0.31 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF followed by addition of resin and reaction was allowed to stand for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence.
Cyclization of peptide chain 1.2mL of isoamyl nitrite was dissolved in 2mL of DMF and added to the resin obtained in (3), reacted on a shaker for 1.5h, and after the reaction was completed, washed three times with DCM and DMF, respectively. A further 5% DIEA in DMF was added and reacted on a shaker for 1.5h after the reaction was completed. The analysis result of LCMS of the cyclopeptide product is as follows ：MS(ESI)exact mass calcd.for C26H25N3O3Na[M+Na]+:450.18;Found:450.22.
(4) Verification of 2d aryl diamine resin preparation of chain peptide, first condensation HBTU (159.2 mg,0.41 mmol), fmoc-Gly-OH (124.7 mg,0.41 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF and then added to the resin from (2) and allowed to stand for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence. Second condensation HBTU (159.2 mg,0..41 mmol), fmoc-Gln-OH (192.4 mg,0.31 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF followed by addition of resin and reaction for 1h by standing. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence.
Cyclization of peptide chain 1.2mL of isoamyl nitrite was dissolved in 2mL of DMF and added to the resin obtained in (4), reacted on a shaker for 1.5h, and after the reaction was completed, washed three times with DCM and DMF, respectively. A further 5% DIEA in DMF was added and reacted on a shaker for 1.5h after the reaction was completed. The analysis result of LCMS of the cyclopeptide product is as follows ：MS(ESI)exact mass calcd.for C26H25N3O3Na[M+Na]+:450.18;Found:450.22.
Example 5
(1) Prepared using a polypeptide synthesizer, 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) was placed in a 10mL syringe with a sieve plate, followed by 5mL DMF to swell for 30min, solvent was removed, 4-hydroxymethylbenzoic acid (92 mg,0.31 mmol), HATU (118 mg,0.31 mmol), DIEA (110. Mu.L, 0.62 mmol) were dissolved in 2mL DMF, followed by addition to the resin, standing for 1h, and after the reaction was completed, three washes with DCM and DMF, respectively.
(2) 5-Nitro-6-aminonicotinic acid (0.41 mmol), HATU (156 mg,0.41 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in 2mL DMF, followed by addition of the resin from (1), standing for 1h, and after completion of the reaction, washing three times with DCM and DMF, respectively.
(3) Tetrahydroxydiboron (28 mg,0.31 mmol) and 4,4' -bipyridine (1 mg,0.005 mmol) were dissolved in 2.5mL DMF and then added to the resin from (2) and reacted on a shaker for 30min. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
1E resin was cut from lysate (TFA: TIPS: EDT: H 2 O=94:1:2.5:2.5), the lysate was collected, the solution was evaporated with air flow, the resulting solid was again added with diethyl ether, settled, and the centrifuged solid was resolved by LCMS ：MS(ESI)exact mass calcd.for C14H15N4O3[M+H]+:287.11;Found:287.25.
Example 6
(1) Prepared using a polypeptide synthesizer, 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) was placed in a 10mL syringe with a sieve plate, followed by 5mL DMF to swell for 30min, solvent was removed, 4-hydroxymethylbenzoic acid (92 mg,0.31 mmol), HATU (118 mg,0.31 mmol), DIEA (110. Mu.L, 0.62 mmol) were dissolved in 2mL DMF, followed by addition to the resin, standing for 1h, and after the reaction was completed, three washes with DCM and DMF, respectively.
(2) 3- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -4-aminobenzoic acid (154 mg,0.41 mmol), HATU (156 mg,0.41 mmol), DIEA (110. Mu.L, 0.62 mmol) was dissolved in 2mL DMF, followed by addition to the resin obtained in (1), standing for 1H, and after completion of the reaction, washing three times with DCM and DMF, respectively. Finally, 20% piperidine solution was added for Fmoc removal.
Verification of 2e aryl diamine resin HBTU (159.2 mg,0.41 mmol), fmoc-Gly-OH (124.7 mg,0.41 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF and then added to the resin from (2) and allowed to stand for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence.
The resin was cleaved from the lysate (TFA: TIPS: EDT: H 2 o=94:1:2.5:2.5), the cleavage filtrate was collected, the solution was evaporated with air flow, the resulting solid was evaporated, diethyl ether was added, sedimentation was performed, and the centrifuged solid was resolved by LCMS ：MS(ESI)exact mass calcd.for C17H19N4O4[M+H]+:343.14;Found:343.18.
Example 7
(1) Prepared using a polypeptide synthesizer, 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) was placed in a 10mL syringe with a sieve plate, followed by 5mL DMF to swell for 30min, solvent was removed, 4-chloromethylbenzoic acid (91 mg,0.31 mmol), HATU (118 mg,0.31 mmol), DIEA (110. Mu.L, 0.62 mmol) were dissolved in 2mL DMF, followed by addition to the resin, standing for 1h, and after the reaction was completed, three washes with DCM and DMF, respectively.
(2) 4- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -3-nitrophenol (313 mg,0.9 mmol) and NaH (60%, 40mg,1 mmol) were reacted in 5mL THF at 65℃for 3 hours. Then, to the resin obtained in (1) was added 5mL of DMF and the reaction was continued at 65℃for 2h. After the reaction was completed, the reaction mixture was washed three times with water, DCM and DMF, respectively.
(3) Tetrahydroxydiboron (28 mg,0.31 mmol) and 4,4' -bipyridine (1 mg,0.005 mmol) were dissolved in 2.5mL DMF and then added to the resin from (2) and reacted on a shaker for 30min. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
2F' resin verification, resin was cut from lysate (TFA: TIPS: EDT: H 2 O=94:1:2.5:2.5), the lysate was collected, the solution was evaporated with air flow, the resulting solid was again added with diethyl ether, settled, and the centrifuged solid was resolved by LCMS ：MS(ESI)exact mass calcd.for C14H14N3O4[M+H]+:288.10;Found:288.09.
Example 8
(1) Preparation was performed using a polypeptide synthesizer by placing 1.58mmol/g 2-CTC resin (460 mg,0.72 mmol) in a 10mL syringe with a sieve plate, followed by 5mL DMF for 30min for swelling, removing the solvent, dissolving 3-nitro-4-aminophenol (554.4 mg,3.6 mmol), DMAP (284 mg,2.4 mmol), TEA (488. Mu.L, 3.6 mmol) in 6mL DCM, followed by addition to the resin, reacting overnight on a shaker, and after the reaction was completed, washing three times with DCM, water, DMF, respectively.
(2) Ethyl viologen diiodide (337 mg,0.72 mmol), na 2S2O4 (335 mg,1.8 mmol) and K 2CO3 (199mg, 1.4 mmol) were dissolved in 4mL of a mixed solvent of water and DMF (ratio 1:1), followed by addition to the resin obtained in (1) and reaction on a shaker for 48h. After the reaction was completed, the reaction mixture was washed three times with DCM, water and DMF.
(3) Verification of 2g aryl diamine resin preparation of chain peptide, first condensation HATU (159.2 mg,0.41 mmol), fmoc-Gly-OH (124.7 mg,0.41 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF and then added to the resin from (2) and allowed to stand for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence. Second condensation HATU (159.2 mg,0..41 mmol), fmoc-Gln-OH (192.4 mg,0.31 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF followed by addition of resin and reaction was allowed to stand for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence.
Cyclization of peptide chain 1.2mL of isoamyl nitrite was dissolved in 2mL of DMF and added to the resin obtained in (3), reacted on a shaker for 1.5h, and after the reaction was completed, washed three times with DCM and DMF, respectively. A further 5% DIEA in DMF was added and reacted on a shaker for 1.5h after the reaction was completed. The corresponding cyclic peptide product was detected by LCMS to verify the linker preparation was successful.
Example 9
(1) Preparation was performed using a polypeptide synthesizer by placing 1.10mmol/g Wang resin (300 mg,0.3 mmol) in a 10mL syringe with a sieve plate, followed by swelling with 5mL DCM for 30min, removing the solvent, dissolving 4-amino-3-nitrobenzenesulfonyl chloride (313 mg,0.9 mmol), TEA (125.1. Mu.L, 0.9 mmol) in 3mL DCM, followed by adding to the resin, heating to 60℃and washing three times with DCM, DMF, respectively, after 2h reaction.
(2) Tetrahydroxydiboron (28 mg,0.31 mmol) and 4,4' -bipyridine (1 mg,0.005 mmol) were dissolved in 2.5mL DMF and then added to the resin from (1) and reacted on a shaker for 30min. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
(3) Verification of the aryl diamine resin for 2h preparation of chain peptide, first condensation HBTU (159.2 mg,0.41 mmol), fmoc-Gly-OH (124.7 mg,0.41 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF and then added to the resin from (2) and allowed to stand for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence. Second condensation HBTU (159.2 mg,0..41 mmol), fmoc-Gln-OH (192.4 mg,0.31 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF followed by addition of resin and reaction for 1h by standing. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence.
Cyclization of peptide chain 1.2mL of isoamyl nitrite was dissolved in 2mL of DMF and added to the resin obtained in (3), reacted on a shaker for 1.5h, and after the reaction was completed, washed three times with DCM and DMF, respectively. A further 5% DIEA in DMF was added and reacted on a shaker for 1.5h after the reaction was completed. The corresponding cyclic peptide product was detected by LCMS to verify the linker preparation was successful.
Example 10
(1) Prepared using a polypeptide synthesizer, 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) was placed in a 10mL syringe with a sieve plate, followed by 5mL DMF to swell for 30min, solvent was removed, 4-hydroxymethylbenzoic acid (92 mg,0.31 mmol), HATU (118 mg,0.31 mmol), DIEA (110. Mu.L, 0.62 mmol) were dissolved in 2mL DMF, followed by addition to the resin, standing for 1h, and after the reaction was completed, three washes with DCM and DMF, respectively.
(2) 4-Amino-3-nitrobenzenesulfonyl chloride (313 mg,0.9 mmol) and TEA (125.1. Mu.L, 0.9 mmol) were dissolved in 3mL DCM and then added to the resin obtained in (1), the temperature was raised to 60℃and after 2h the reaction was completed, each was washed three times with DCM and DMF, respectively. Finally, nitro reduction was performed, tetrahydroxydiboron (28 mg,0.31 mmol) and 4,4' -bipyridine (1 mg,0.005 mmol) were dissolved in 2.5mL DMF and then added to the resulting resin and reacted on a shaker for 30min. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
2I resin verification, resin was cut from lysate (TFA: TIPS: EDT: H 2 o=94:1:2.5:2.5), the lysate was collected, the solution was evaporated with air flow, the resulting solid was again added with diethyl ether, settled, and the centrifuged resulting solid was resolved by LCMS ：MS(ESI)exact mass calcd.for C14H14N3O4S[M+H]+:322.08;Found:322.22.
Example 11 comparison of Selectivity of heteroaryl diamine resins and phenyl diamine resins
(1) The preparation was carried out using a polypeptide synthesizer, and 1a and 2a aryl diamine resins (300 mg) were placed in 10mL syringe with sieve plate, followed by 5mL DMF for swelling for 30min.
(2) HATU (159.2 mg,0.41 mmol), fmoc-Gly-OH (124.7 mg,0.41 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF and then added to a syringe of the 1a and 2a aryl diamine resin from (1) and allowed to stand for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence.
(3) 1A and 2a aryl diamine resins were cut from the lysate (TFA: TIPS: EDT: H 2 o=94:1:2.5:2.5), the lysate was collected, the solution was evaporated using an air stream, the resulting solid was again added with diethyl ether, settled, and the resulting solid was centrifuged and analyzed by liquid chromatography. The results are shown in figures 1 and 2.
Conclusion that 1a aryl diamine resin was monoacylated to bisacylated product ratio was 96:4,2a aryl diamine resin was monoacylated to bisacylated product ratio was 85:15, and comparing the two resin liquid phases results showed that heteroaryl diamine resin selectivity was much higher than phenyl diamine resin.
Example 12
(1) Prepared using a polypeptide synthesizer, 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) was placed in a 10mL syringe with a sieve plate, followed by 5mL DMF for swelling for 30min, solvent removed, 7- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) -6-amino-2-naphthoic acid (131 mg,0.31 mmol), HATU (118 mg,0.31 mmol), DIEA (110 μl,0.62 mmol) was dissolved in 2mL DMF, followed by addition to the resin, standing for 1H, and washing three times with DCM and DMF, respectively, after the reaction was completed.
(2) Fmoc removal was performed by adding 20% piperidine solution to the aryl resin of (1) for a period of 5min, washing three times with DCM and DMF, respectively, and repeating the reaction step once.
(3) 1J verification of aryl diamine resin preparation of chain peptide, first condensation HBTU (159.2 mg,0.41 mmol), fmoc-Gly-OH (124.7 mg,0.41 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF and then added to the resin from (2) and allowed to stand for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence. Second condensation HBTU (159.2 mg,0..41 mmol), fmoc-Gln-OH (192.4 mg,0.31 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF followed by addition of resin and reaction for 1h by standing. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence.
Cyclization of peptide chain 1.2mL of isoamyl nitrite was dissolved in 2mL of DMF and added to the resin obtained in (3), reacted on a shaker for 1.5h, and after the reaction was completed, washed three times with DCM and DMF, respectively. A further 5% DIEA in DMF was added and reacted on a shaker for 1.5h after the reaction was completed. The corresponding cyclic peptide product was detected by LCMS to verify the linker preparation was successful.
Selectivity tests were performed as in example 11, with 1j aryl diamine resins monoacylated to bisacylated products selectivity greater than 80:20, selectivity approaching that of phenyl diamine resins.
Example 13
(1) Prepared using a polypeptide synthesizer, 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) was placed in a 10mL syringe with a sieve plate, followed by 5mL DMF for swelling for 30min, solvent was removed, 4-bromo-5-nitrothiophene-3-carboxylic acid (100 mg,0.40 mmol), HATU (152 mg,0.4 mmol), DIEA (110 μl,0.62 mmol) were dissolved in 2mL DMF, followed by addition to the resin, allowed to stand for reaction for 1h, and after the reaction was completed, washed three times with DCM and DMF, respectively.
(2) Transferring the reaction resin into a sealable reaction tube, then adding 2mL of ethanol and 0.5mL of NH 3·H2 O (30%), and stirring for 24h at 70 ℃, wherein the stirring speed is required to be noted, the resin is prevented from being broken, and the rotating speed is 100-200 revolutions. After the reaction was completed, filtration was performed using a syringe with a sieve plate, and washing was performed three times with water, DMF and DCM, respectively.
(3) Tetrahydroxydiboron (28 mg,0.31 mmol) and 4,4' -bipyridine (1 mg,0.005 mmol) were dissolved in 2.5mL DMF and then added to the resulting resin and reacted on a shaker for 30min. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
(4) Verification of the aryl diamine resin 4a preparation of chain peptide was performed, first condensation by dissolving HBTU (159.2 mg,0.41 mmol), fmoc-Gly-OH (124.7 mg,0.41 mmol) and DIEA (110. Mu.L, 0.62 mmol) in DMF followed by addition to the resulting resin of (3) and standing for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence. Second condensation HBTU (159.2 mg,0..41 mmol), fmoc-Gln-OH (192.4 mg,0.31 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF followed by addition of resin and reaction for 1h by standing. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence.
Cyclization of peptide chain 1.2mL of isoamyl nitrite was dissolved in 2mL of DMF and added to the resin obtained in (4), reacted on a shaker for 1.5h, and after the reaction was completed, washed three times with DCM and DMF, respectively. A further 5% DIEA in DMF was added and reacted on a shaker for 1.5h after the reaction was completed. The corresponding cyclic peptide product was detected by LCMS to verify the linker preparation was successful.
Selectivity test was performed as in example 11, with 4a aryl diamine resins monoacylated to bisacylated product selectivities of not less than 90:10, superior to phenyl diamine resins.
Example 14
(1) Prepared using a polypeptide synthesizer, 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) was placed in a 10mL syringe with a sieve plate, followed by 5mL DMF for swelling for 30min, solvent was removed, 3-bromo-4-nitrothiophene-2-carboxylic acid (100 mg,0.40 mmol), HATU (152 mg,0.4 mmol), DIEA (110 μl,0.62 mmol) were dissolved in 2mL DMF, followed by addition to the resin, allowed to stand for reaction for 1h, and after the reaction was completed, washed three times with DCM and DMF, respectively.
(2) Transferring the reaction resin into a sealable reaction tube, then adding 2mL of ethanol and 0.5mL of NH 3·H2 O (30%), and stirring for 24h at 70 ℃, wherein the stirring speed is required to be noted, the resin is prevented from being broken, and the rotating speed is 100-200 revolutions. After the reaction was completed, filtration was performed using a syringe with a sieve plate, and washing was performed three times with water, DMF and DCM, respectively.
(3) Tetrahydroxydiboron (28 mg,0.31 mmol) and 4,4' -bipyridine (1 mg,0.005 mmol) were dissolved in 2.5mL DMF and then added to the resulting resin and reacted on a shaker for 30min. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
(4) Verification of the aryl diamine resin 4a preparation of chain peptide was performed, first condensation by dissolving HBTU (159.2 mg,0.41 mmol), fmoc-Gly-OH (124.7 mg,0.41 mmol) and DIEA (110. Mu.L, 0.62 mmol) in DMF followed by addition to the resulting resin of (3) and standing for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence. Second condensation HBTU (159.2 mg,0..41 mmol), fmoc-Gln-OH (192.4 mg,0.31 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF followed by addition of resin and reaction for 1h by standing. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence.
Cyclization of peptide chain 1.2mL of isoamyl nitrite was dissolved in 2mL of DMF and added to the resin obtained in (4), reacted on a shaker for 1.5h, and after the reaction was completed, washed three times with DCM and DMF, respectively. A further 5% DIEA in DMF was added and reacted on a shaker for 1.5h after the reaction was completed. The corresponding cyclic peptide product was detected by LCMS to verify the linker preparation was successful.
Selectivity test was performed as in example 11, with 4a aryl diamine resins monoacylated to bisacylated product selectivities of not less than 90:10, superior to phenyl diamine resins.
Example 15
(1) Preparation was performed using a polypeptide synthesizer by placing 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) in a 10mL syringe with a sieve plate followed by 5mL DMF for 30min swelling, removing the solvent, dissolving 4-amino-5-nitrofuran-3-carboxylic acid (69 mg,0.40 mmol), HATU (152 mg,0.4 mmol), DIEA (110. Mu.L, 0.62 mmol) in 2mL DMF followed by adding to the resin, standing for 1h, washing three times with DCM and DMF, respectively, after the reaction was completed.
(2) Tetrahydroxydiboron (28 mg,0.31 mmol) and 4,4' -bipyridine (1 mg,0.005 mmol) were dissolved in 2.5mL DMF and then added to the resulting resin and reacted on a shaker for 30min. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
(3) Verification of the aryl diamine resin 5a preparation of chain peptide was performed, first condensation by dissolving HBTU (159.2 mg,0.41 mmol), fmoc-Gly-OH (124.7 mg,0.41 mmol) and DIEA (110. Mu.L, 0.62 mmol) in DMF followed by addition to the resulting resin of (3) and standing for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence. Second condensation HBTU (159.2 mg,0..41 mmol), fmoc-Gln-OH (192.4 mg,0.31 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF followed by addition of resin and reaction for 1h by standing. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence.
Cyclization of peptide chain 1.2mL of isoamyl nitrite was dissolved in 2mL of DMF and added to the resin obtained in (3), reacted on a shaker for 1.5h, and after the reaction was completed, washed three times with DCM and DMF, respectively. A further 5% DIEA in DMF was added and reacted on a shaker for 1.5h after the reaction was completed. The corresponding cyclic peptide product was detected by LCMS to verify the linker preparation was successful.
Selectivity test was performed as in example 11, with 5a aryl diamine resins monoacylated to bisacylated product selectivities of not less than 90:10, superior to phenyl diamine resins.
Example 16
(1) Prepared using a polypeptide synthesizer, 0.34mmol/G RINK AMIDE resin (300 mg,0.102 mmol) was placed in a 10mL syringe with a sieve plate, followed by 5mL DMF for swelling for 30min, solvent was removed, 3-amino-4-nitrofuran-2-carboxylic acid (69 mg,0.40 mmol), HATU (152 mg,0.4 mmol), DIEA (110 μl,0.62 mmol) were dissolved in 2mL DMF, followed by addition to the resin, allowed to stand for reaction for 1h, and after the reaction was completed, washed three times with DCM and DMF, respectively.
(2) Tetrahydroxydiboron (28 mg,0.31 mmol) and 4,4' -bipyridine (1 mg,0.005 mmol) were dissolved in 2.5mL DMF and then added to the resulting resin and reacted on a shaker for 30min. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively.
(3) Verification of the aryl diamine resin 6a preparation of chain peptide was performed, first condensation by dissolving HBTU (159.2 mg,0.41 mmol), fmoc-Gly-OH (124.7 mg,0.41 mmol) and DIEA (110. Mu.L, 0.62 mmol) in DMF followed by addition to the resulting resin of (3) and standing for 1h. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence. Second condensation HBTU (159.2 mg,0..41 mmol), fmoc-Gln-OH (192.4 mg,0.31 mmol) and DIEA (110. Mu.L, 0.62 mmol) were dissolved in DMF followed by addition of resin and reaction for 1h by standing. After the reaction was completed, the reaction mixture was washed three times with DCM and DMF, respectively. Fmoc removal was performed by adding 20% piperidine solution for 5min, and washing with DCM and DMF was performed three times in sequence.
Cyclization of peptide chain 1.2mL of isoamyl nitrite was dissolved in 2mL of DMF and added to the resin obtained in (3), reacted on a shaker for 1.5h, and after the reaction was completed, washed three times with DCM and DMF, respectively. A further 5% DIEA in DMF was added and reacted on a shaker for 1.5h after the reaction was completed. The corresponding cyclic peptide product was detected by LCMS to verify the linker preparation was successful.
Selectivity test was performed as in example 11, with the 6a aryl diamine resin monoacylated to bisacylated product selectivity not less than 90:10, being superior to the phenyl diamine resin.
EXAMPLE 17 Synthesis of Cyclo-GQ
(1) Preparation was performed using a polypeptide synthesizer and 1a aryl diamine resin (300 mg) was placed in a 10mL syringe with a sieve plate, followed by swelling with 5mL DMF for 30min.
(2) The linear peptides were prepared according to the following table conditions
(3) Cyclization 1.2mL of isoamyl nitrite is dissolved in 2mL of DMF, then added into the swollen chain peptide resin, reacted for 1.5h on a shaking table, and after the reaction is finished, the solvent is removed, and DMF is washed. Subsequently, DIEA (100. Mu.L) in 2mL of DMF was added, the reaction was performed for 1.5h, concentrated under reduced pressure, and the solvent was evaporated to give the crude product, which was purified by HPLC. The analysis result of LCMS of the cyclopeptide product is as follows ：MS(ESI)exact mass calcd.for C26H25N3O3Na[M+Na]+:450.18;Found:450.22.
The cyclic peptide can also be automatically synthesized by high-temperature flow, and the reaction steps are as follows:
(1) Using a self-grinding automated synthesis apparatus, 1a aryl diamine resin (20 mg) was placed in the reaction module.
(2) The reaction conditions are shown in the following table.
The table illustrates:
a) The preparation method of the polypeptide long chain comprises the following steps:
pump 1. Delivering a solution of 0.4M amino acid monomer in DMF and DMF;
Pump 2 delivery of DMF solution of 0.38M HATU and DMF;
pump 3 delivers DIEA, 99.9% purity and DMF;
b) Deprotection step:
Pump 2. Delivering 20% (volume ratio) piperidine and 0.5% (volume ratio) HCOOH in DMF;
c) And (3) an activation step:
pump 3. Delivering a 50% by volume solution of isoamyl nitrite in DMF;
d) Cyclization step:
Pump 3. Delivering 1% (volume ratio) DIEA in DMF;
the volumes presented in the table all refer to the volume of a single pump, and if three pumps are used simultaneously, the total volume is three times the table.
In addition, the aryl diamine resins of examples 1-10 and examples 12-16 were used to successfully prepare Cyclo-GQ, and the procedure was the same as in example 17, and is not repeated here. The aryl diamine immobilization linker has universality for preparing cyclic peptides, can be applied to preparing cyclic dipeptide and even cyclic heptapeptide, and is applicable to the synthesis scheme of larger cyclic peptide compounds. In addition, the aryl diamine immobilization linker of the invention can be used for high-temperature flow automatic preparation of cyclic peptides.
EXAMPLE 18 Synthesis of Cyclo-KT
(1) Preparation was performed using a polypeptide synthesizer and 1a aryl diamine resin (300 mg) was placed in a 10mL syringe with a sieve plate, followed by swelling with 5mL DMF for 30min.
(2) The linear peptides were prepared according to the following table conditions
(3) Cyclization 1.2mL of isoamyl nitrite is dissolved in 2mL of DMF, then added into the swollen chain peptide resin, reacted for 1.5h on a shaking table, and after the reaction is finished, the solvent is removed, and DMF is washed. Subsequently, DIEA (100. Mu.L) in 2mL of DMF was added, the reaction was performed for 1.5h, concentrated under reduced pressure, and the solvent was evaporated to give the crude product, which was purified by HPLC. The analysis result of LCMS of the cyclopeptide product is as follows ：MS(ESI)exact mass calcd.for C19H36N3O5[M+H]+:386.26;Found:386.24.
High temperature flow automated synthesis can also be successful in preparing the cyclic peptide, and reference is made to example 17 for specific embodiments.
EXAMPLE 19 Synthesis of Cyclo-GDT
(1) Preparation was performed using a polypeptide synthesizer and 1a aryl diamine resin (300 mg) was placed in a 10mL syringe with a sieve plate, followed by swelling with 5mL DMF for 30min.
(2) The linear peptides were prepared according to the following table conditions
(3) Cyclization 1.2mL of isoamyl nitrite is dissolved in 2mL of DMF, then added into the swollen chain peptide resin, reacted for 1.5h on a shaking table, and after the reaction is finished, the solvent is removed, and DMF is washed. Subsequently, DIEA (100. Mu.L) in 2mL of DMF was added, the reaction was performed for 1.5h, concentrated under reduced pressure, and the solvent was evaporated to give the crude product, which was purified by HPLC. The analysis result of LCMS of the cyclopeptide product is as follows ：MS(ESI)exact mass calcd.for C36H62N6O12Na[2M+Na]+:793.43;Found:793.32.
High temperature flow automated synthesis can also be successful in preparing the cyclic peptide, and reference is made to example 17 for specific embodiments.
EXAMPLE 20 Synthesis of Cyclo-GKYA
(1) Preparation was performed using a polypeptide synthesizer and 1a aryl diamine resin (300 mg) was placed in a 10mL syringe with a sieve plate, followed by swelling with 5mL DMF for 30min.
(2) The linear peptides were prepared according to the following table conditions
(3) Cyclization 1.2mL of isoamyl nitrite is dissolved in 2mL of DMF, then added into the swollen chain peptide resin, reacted for 1.5h on a shaking table, and after the reaction is finished, the solvent is removed, and DMF is washed. Subsequently, DIEA (100. Mu.L) in 2mL of DMF was added, the reaction was performed for 1.5h, concentrated under reduced pressure, and the solvent was evaporated to give the crude product, which was purified by HPLC. The analysis result of LCMS of the cyclopeptide product is as follows ：MS(ESI)exact mass calcd.for C58H90N10O14Na[2M+Na]+:1173.65;Found:1173.39.
High temperature flow automated synthesis can also be successful in preparing the cyclic peptide, and reference is made to example 17 for specific embodiments.
EXAMPLE 21 Synthesis of Cyclo-FSNMG
(1) Preparation was performed using a polypeptide synthesizer and 1a aryl diamine resin (300 mg) was placed in a 10mL syringe with a sieve plate, followed by swelling with 5mL DMF for 30min.
(2) The linear peptides were prepared according to the following table conditions
(3) Cyclization 1.2mL of isoamyl nitrite is dissolved in 2mL of DMF, then added into the swollen chain peptide resin, reacted for 1.5h on a shaking table, and after the reaction is finished, the solvent is removed, and DMF is washed. Subsequently, DIEA (100. Mu.L) in 2mL of DMF was added, the reaction was performed for 1.5h, concentrated under reduced pressure, and the solvent was evaporated to give the crude product, which was purified by HPLC. The analysis result of LCMS of the cyclopeptide product is as follows ：MS(ESI)exact mass calcd.for C46H54N6O7Na[M+Na]+:857.37;Found:857.23.
High temperature flow automated synthesis can also be successful in preparing the cyclic peptide, and reference is made to example 17 for specific embodiments.
Synthesis of example 22Yunnanin C
(1) Preparation was performed using a polypeptide synthesizer and 1a aryl diamine resin (300 mg) was placed in a 10mL syringe with a sieve plate, followed by swelling with 5mL DMF for 30min.
(2) The linear peptides were prepared according to the following table conditions
(3) Cyclization 1.2mL of isoamyl nitrite is dissolved in 2mL of DMF, then added into the swollen chain peptide resin, reacted for 1.5h on a shaking table, and after the reaction is finished, the solvent is removed, and DMF is washed. DIEA (100. Mu.L) in 2mL DMF was then added and reacted for 1.5h. The crude product was obtained and purified by HPLC.
(4) Deprotection, namely obtaining a crude product, dissolving the crude product in 1mL TFA:TIPS:EDT:H 2 O (the ratio is 94:1:2.5:2.5) solution, reacting for 2 hours on a shaking table to obtain a deprotected natural cyclic peptide, volatilizing the reaction solution by using air flow to obtain a solid, adding diethyl ether, settling, centrifuging, and purifying the obtained solid by HPLC. The analysis result of LCMS of the cyclopeptide product is as follows ：MS(ESI)exact mass calcd.for C36H47N7O9Na[M+Na]+:744.23;Found:744.34.
High temperature flow automated synthesis can also be successful in preparing the cyclic peptide, and reference is made to example 17 for specific embodiments.
Further, it is to be understood that various changes and modifications of the present application may be made by those skilled in the art after reading the above description of the application, and that such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims (11)

1.一种芳基二胺固载连接子，包括固相承载材料，所述固相承载材料通过其上的活性基团直接或经中间连接单元间接连接芳基二胺，其特征在于，所述芳基二胺具有式(1)所示结构：1. An aromatic diamine solid-supported linker, comprising a solid-phase supporting material, wherein the solid-phase supporting material is directly connected to an aromatic diamine through an active group thereon or indirectly connected via an intermediate connecting unit, wherein the aromatic diamine has a structure shown in formula (1): 式(1)中：In formula (1): X为N；X is N; R1、R2分别独立为H或者氨基酸与氨基发生缩合反应脱去一分子水的羰基化合物结构单元，且R1、R2中至少有一个为H。R 1 and R 2 are independently H or a carbonyl compound structural unit obtained by condensation reaction between an amino acid and an amino group to remove a molecule of water, and at least one of R 1 and R 2 is H. 2.根据权利要求1所述的芳基二胺固载连接子，其特征在于，所述活性基团包括氨基、卤素原子、羟基中的至少一种；2. The aromatic diamine immobilized linker according to claim 1, characterized in that the active group comprises at least one of an amino group, a halogen atom, and a hydroxyl group; 所述中间连接单元包括以下任意一种或多种组合结构：The intermediate connection unit includes any one or more of the following combined structures: 一个或多个连接的氨基酸片段；one or more linked amino acid segments; n1为1至15之间任一整数； n 1 is any integer between 1 and 15; R2为O、-N(R3)-、S、-C(＝O)-或-S(＝O)2-，R3为H或C1～C4烷基； R 2 is O, -N(R 3 )-, S, -C(=O)- or -S(=O) 2 -, R 3 is H or C 1 ～C 4 alkyl; n2为1至15之间任一整数； n 2 is any integer between 1 and 15; R3为H、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的噻吩基、C1～C4烷基取代或未取代的萘基、C1～C4烷基取代或未取代的呋喃基、C1～C4烷基取代或未取代的吲哚基、巯基、C2～C4的酰胺基、C2～C4羧基或C1～C4烷胺基，所述C1～C4烷基上的取代基选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R3 is H, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted thienyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, C1 - C4 alkyl substituted or unsubstituted furyl, C1-C4 alkyl substituted or unsubstituted indolyl, thiol, C2 - C4 amide, C2 - C4 carboxyl or C1 - C4 alkylamino, and the substituent on the C1 - C4 alkyl is selected from one or more of thiol, C1 - C4 alkylthio, hydroxyl and halogen; R4为H、卤素、炔基、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的萘基、取代或未取代的C1～C4烷氧基、酯基、羟基、C2～C4酰基、氰基中的一种或多种，所述C1～C4烷基、所述C1～C4烷氧基上的取代基分别独立选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R4 is one or more of H, halogen, alkynyl, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1 - C4 alkoxy, ester, hydroxyl, C2 - C4 acyl, and cyano, and the substituents on the C1 - C4 alkyl and the C1 - C4 alkoxy are independently selected from one or more of mercapto, C1 - C4 alkylthio, hydroxyl, and halogen; R5为H、卤素、炔基、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的萘基、取代或未取代的C1～C4烷氧基、酯基、羟基、C2～C4酰基、氰基中的一种或多种，所述C1～C4烷基、所述C1～C4烷氧基上的取代基分别独立选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R5 is one or more of H, halogen, alkynyl, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1 - C4 alkoxy, ester, hydroxyl, C2 - C4 acyl, and cyano, and the substituents on the C1 - C4 alkyl and the C1 - C4 alkoxy are independently selected from one or more of mercapto, C1 - C4 alkylthio, hydroxyl, and halogen; R6为H、卤素、炔基、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的萘基、取代或未取代的C1～C4烷氧基、酯基、羟基、C2～C4酰基、氰基中的一种或多种，所述C1～C4烷基、所述C1～C4烷氧基上的取代基分别独立选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R6 is one or more of H, halogen, alkynyl, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1 - C4 alkoxy, ester, hydroxyl, C2 - C4 acyl, and cyano, and the substituents on the C1 - C4 alkyl and the C1 - C4 alkoxy are independently selected from one or more of mercapto, C1 - C4 alkylthio, hydroxyl, and halogen; R7为H、卤素、炔基、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的萘基、取代或未取代的C1～C4烷氧基、酯基、羟基、C2～C4酰基、氰基中的一种或多种，所述C1～C4烷基、所述C1～C4烷氧基上的取代基分别独立选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R7 is one or more of H, halogen, alkynyl, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1 - C4 alkoxy, ester, hydroxyl, C2 - C4 acyl, and cyano, and the substituents on the C1 - C4 alkyl and the C1 - C4 alkoxy are independently selected from one or more of mercapto, C1 - C4 alkylthio, hydroxyl, and halogen; R8为-(CH2)n-、-CH2(CH3)-、-CH2(CH2CH3)-、-CH2(CH2Ph)-、-CH2OCH2CH2-或-CH2(CH2CH2SCH3)-，n＝1～20，或者R8为式(a)～(h)中任一结构： R 8 is -(CH 2 ) n -, -CH 2 (CH 3 )-, -CH 2 (CH 2 CH 3 )-, -CH 2 (CH 2 Ph)-, -CH 2 OCH 2 CH 2 - or -CH 2 (CH 2 CH 2 SCH 3 )-, n=1-20, or R 8 is any one of the structures of formulae (a) to (h): n3、n4、n5分别独立为1或2，R10、R11分别独立为C1～C4烷基或C1～C4烷氧基；n 3 , n 4 , n 5 are each independently 1 or 2, R 10 , R 11 are each independently C 1 ～C 4 alkyl or C 1 ～C 4 alkoxy; R9为-(CH2)m-、-CH2(CH3)-、-CH2(CH2CH3)-、-CH2(CH2Ph)-、-CH2OCH2CH2-或-CH2(CH2CH2SCH3)-，m＝1～20，或者R9为式(a)～(h)中任一结构。 R 9 is -(CH 2 ) m -, -CH 2 (CH 3 ) -, -CH 2 (CH 2 CH 3 ) -, -CH 2 (CH 2 Ph) -, -CH 2 OCH 2 CH 2 - or -CH 2 (CH 2 CH 2 SCH 3 ) -, m = 1 to 20, or R 9 is any one of the structures of formulae (a) to (h). 3.根据权利要求1或2所述的芳基二胺固载连接子的制备方法，其特征在于，包括步骤：3. The method for preparing the aryl diamine immobilized linker according to claim 1 or 2, characterized in that it comprises the steps of: 1)所述固相承载材料通过其上的活性基团直接连接式(7)所示结构，得到固载连接子前体；或者，1) The solid-phase support material is directly connected to the structure shown in formula (7) through the active groups on it to obtain a solid-phase support linker precursor; or, 所述固相承载材料通过其上的活性基团先连接所述中间连接单元，然后在所述中间连接单元上连接式(7)所示结构，得到固载连接子前体；The solid-phase support material is first connected to the intermediate connection unit through the active groups on it, and then the structure shown in formula (7) is connected to the intermediate connection unit to obtain a solid-phase support linker precursor; 式(7)中：In formula (7): X为N；X is N; R4、R5分别独立为硝基、-NH2、-NHFmoc或-NHR6，R6为氨基无保护或被Fmoc保护的氨基酸与氨基发生缩合反应脱去一分子水的羰基化合物结构单元，且R4、R5不同时选自-NH2和氨基无保护的氨基酸与氨基发生缩合反应脱去一分子水的羰基化合物结构单元；R 4 and R 5 are independently nitro, -NH 2 , -NHFmoc or -NHR 6 , R 6 is a carbonyl compound structural unit obtained by condensation reaction between an amino acid without protected amino group or protected by Fmoc and an amino group to remove a molecule of water, and R 4 and R 5 are not simultaneously selected from -NH 2 and a carbonyl compound structural unit obtained by condensation reaction between an amino acid without protected amino group and an amino group to remove a molecule of water; 2)将所述固载连接子前体的式(7)所示结构上的硝基还原胺化和/或脱至少一个Fmoc保护，得到所述芳基二胺固载连接子。2) reductively aminate the nitro group on the structure of formula (7) of the solid-supported linker precursor and/or remove at least one Fmoc protection to obtain the aryl diamine solid-supported linker. 4.一种固载连接子前体，包括固相承载材料，所述固相承载材料通过其上的活性基团直接或经中间连接单元间接连接前体结构，其特征在于，所述前体结构具有式(7)所示结构：4. A solid-phase linker precursor, comprising a solid-phase support material, wherein the solid-phase support material is directly connected to a precursor structure through an active group thereon or indirectly connected via an intermediate connecting unit, wherein the precursor structure has a structure shown in formula (7): 式(7)中：In formula (7): X为N；X is N; R4、R5分别独立为硝基、-NH2、-NHFmoc或-NHR6，R6为氨基无保护或被Fmoc保护的氨基酸与氨基发生缩合反应脱去一分子水的羰基化合物结构单元，且R4、R5不同时选自-NH2和氨基无保护的氨基酸与氨基发生缩合反应脱去一分子水的羰基化合物结构单元。R 4 and R 5 are independently nitro, -NH 2 , -NHFmoc or -NHR 6 , R 6 is a carbonyl compound structural unit obtained by condensation reaction between an amino acid without protected amino group or protected by Fmoc and an amino group to remove a molecule of water, and R 4 and R 5 are not simultaneously selected from -NH 2 and a carbonyl compound structural unit obtained by condensation reaction between an amino acid without protected amino group and an amino group to remove a molecule of water. 5.根据权利要求4所述的固载连接子前体，其特征在于，所述活性基团包括氨基、卤素原子、羟基中的至少一种；5. The immobilized linker precursor according to claim 4, characterized in that the active group comprises at least one of an amino group, a halogen atom, and a hydroxyl group; 所述中间连接单元包括以下任意一种或多种组合结构：The intermediate connection unit includes any one or more of the following combined structures: 一个或多个连接的氨基酸片段；one or more linked amino acid segments; n1为1至15之间任一整数； n 1 is any integer between 1 and 15; R2为O、-N(R3)-、S、-C(＝O)-或-S(＝O)2-，R3为H或C1～C4烷基； R 2 is O, -N(R 3 )-, S, -C(=O)- or -S(=O) 2 -, R 3 is H or C 1 ～C 4 alkyl; n2为1至15之间任一整数； n 2 is any integer between 1 and 15; R3为取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的噻吩基、C1～C4烷基取代或未取代的萘基、C1～C4烷基取代或未取代的呋喃基、C1～C4烷基取代或未取代的吲哚基、巯基、C2～C4的酰胺基、C2～C4羧基或C1～C4烷胺基，所述C1～C4烷基上的取代基选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R3 is substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted thienyl, C1 -C4 alkyl substituted or unsubstituted naphthyl, C1 - C4 alkyl substituted or unsubstituted furyl, C1 - C4 alkyl substituted or unsubstituted indolyl, thiol, C2 - C4 amide, C2 - C4 carboxyl or C1 - C4 alkylamino, and the substituent on the C1 - C4 alkyl is selected from one or more of thiol, C1 - C4 alkylthio, hydroxyl and halogen; R4为H、卤素、炔基、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的萘基、取代或未取代的C1～C4烷氧基、酯基、羟基、C2～C4酰基、氰基中的一种或多种，所述C1～C4烷基、所述C1～C4烷氧基上的取代基分别独立选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R4 is one or more of H, halogen, alkynyl, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1 - C4 alkoxy, ester, hydroxyl, C2 - C4 acyl, and cyano, and the substituents on the C1 - C4 alkyl and the C1 - C4 alkoxy are independently selected from one or more of mercapto, C1 - C4 alkylthio, hydroxyl, and halogen; R5为H、卤素、炔基、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的萘基、取代或未取代的C1～C4烷氧基、酯基、羟基、C2～C4酰基、氰基中的一种或多种，所述C1～C4烷基、所述C1～C4烷氧基上的取代基分别独立选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R5 is one or more of H, halogen, alkynyl, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1 - C4 alkoxy, ester, hydroxyl, C2 - C4 acyl, and cyano, and the substituents on the C1 - C4 alkyl and the C1 - C4 alkoxy are independently selected from one or more of mercapto, C1 - C4 alkylthio, hydroxyl, and halogen; R6为H、卤素、炔基、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的萘基、取代或未取代的C1～C4烷氧基、酯基、羟基、C2～C4酰基、氰基中的一种或多种，所述C1～C4烷基、所述C1～C4烷氧基上的取代基分别独立选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R6 is one or more of H, halogen, alkynyl, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1 - C4 alkoxy, ester, hydroxyl, C2 - C4 acyl, and cyano, and the substituents on the C1 - C4 alkyl and the C1 - C4 alkoxy are independently selected from one or more of mercapto, C1 - C4 alkylthio, hydroxyl, and halogen; R7为H、卤素、炔基、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的萘基、取代或未取代的C1～C4烷氧基、酯基、羟基、C2～C4酰基、氰基中的一种或多种，所述C1～C4烷基、所述C1～C4烷氧基上的取代基分别独立选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R7 is one or more of H, halogen, alkynyl, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1 - C4 alkoxy, ester, hydroxyl, C2 - C4 acyl, and cyano, and the substituents on the C1 - C4 alkyl and the C1 - C4 alkoxy are independently selected from one or more of mercapto, C1 - C4 alkylthio, hydroxyl, and halogen; R8为-(CH2)n-、-CH2(CH3)-、-CH2(CH2CH3)-、-CH2(CH2Ph)-、-CH2OCH2CH2-或-CH2(CH2CH2SCH3)-，n＝1～20，或者R8为式(a)～(h)中任一结构： R 8 is -(CH 2 ) n -, -CH 2 (CH 3 )-, -CH 2 (CH 2 CH 3 )-, -CH 2 (CH 2 Ph)-, -CH 2 OCH 2 CH 2 - or -CH 2 (CH 2 CH 2 SCH 3 )-, n=1-20, or R 8 is any one of the structures of formulae (a) to (h): n3、n4、n5分别独立为1或2，R10、R11分别独立为C1～C4烷基或C1～C4烷氧基；n 3 , n 4 , n 5 are each independently 1 or 2, R 10 , R 11 are each independently C 1 ～C 4 alkyl or C 1 ～C 4 alkoxy; R9为-(CH2)m-、-CH2(CH3)-、-CH2(CH2CH3)-、-CH2(CH2Ph)-、-CH2OCH2CH2-或-CH2(CH2CH2SCH3)-，m＝1～20，或者R9为式(a)～(h)中任一结构。 R 9 is -(CH 2 ) m -, -CH 2 (CH 3 ) -, -CH 2 (CH 2 CH 3 ) -, -CH 2 (CH 2 Ph) -, -CH 2 OCH 2 CH 2 - or -CH 2 (CH 2 CH 2 SCH 3 ) -, m = 1 to 20, or R 9 is any one of the structures of formulae (a) to (h). 6.根据权利要求1或2所述的芳基二胺固载连接子或者根据权利要求4或5所述的固载连接子前体在制备环肽中的应用。6. Use of the aryl diamine immobilized linker according to claim 1 or 2 or the immobilized linker precursor according to claim 4 or 5 in the preparation of a cyclic peptide. 7.一种环肽制备方法，其特征在于，采用芳基二胺固载连接子，所述芳基二胺固载连接子包括固相承载材料，所述固相承载材料通过其上的活性基团直接或经中间连接单元间接连接芳基二胺，所述芳基二胺具有式(1)所示结构：7. A method for preparing a cyclic peptide, characterized in that an aromatic diamine solid-supported linker is used, wherein the aromatic diamine solid-supported linker comprises a solid-phase supporting material, wherein the solid-phase supporting material is directly connected to the aromatic diamine through an active group thereon or indirectly connected via an intermediate connecting unit, and the aromatic diamine has a structure shown in formula (1): 式(1)中：In formula (1): X为C或N；X is C or N; R1、R2分别独立为H或者氨基酸与氨基发生缩合反应脱去一分子水的羰基化合物结构单元，且R1、R2中至少有一个为H；R 1 and R 2 are independently H or a carbonyl compound structural unit obtained by condensation reaction between an amino acid and an amino group to remove a molecule of water, and at least one of R 1 and R 2 is H; 所述环肽制备方法具体包括步骤：The cyclic peptide preparation method specifically comprises the steps of: S1，在所述芳基二胺固载连接子上通过氨基酸缩合反应合成多肽链；S1, synthesizing a polypeptide chain on the aromatic diamine immobilized linker through an amino acid condensation reaction; S2，用亚硝酸类化合物处理步骤S1的产物形成芳基三氮唑结构；S2, treating the product of step S1 with a nitrous acid compound to form an aryl triazole structure; S3，步骤S2的产物在碱性环境下环化生成环肽。S3, the product of step S2 is cyclized under alkaline conditions to generate a cyclopeptide. 8.根据权利要求7所述的环肽制备方法，其特征在于，所述活性基团包括氨基、卤素原子、羟基中的至少一种；8. The method for preparing a cyclic peptide according to claim 7, characterized in that the active group comprises at least one of an amino group, a halogen atom, and a hydroxyl group; 所述中间连接单元包括以下任意一种或多种组合结构：The intermediate connection unit includes any one or more of the following combined structures: 一个或多个连接的氨基酸片段；one or more linked amino acid segments; n1为1至15之间任一整数； n 1 is any integer between 1 and 15; R2为O、-N(R3)-、S、-C(＝O)-或-S(＝O)2-，R3为H或C1～C4烷基； R 2 is O, -N(R 3 )-, S, -C(=O)- or -S(=O) 2 -, R 3 is H or C 1 ～C 4 alkyl; n2为1至15之间任一整数； n 2 is any integer between 1 and 15; R3为H、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的噻吩基、C1～C4烷基取代或未取代的萘基、C1～C4烷基取代或未取代的呋喃基、C1～C4烷基取代或未取代的吲哚基、巯基、C2～C4的酰胺基、C2～C4羧基或C1～C4烷胺基，所述C1～C4烷基上的取代基选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R3 is H, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted thienyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, C1 - C4 alkyl substituted or unsubstituted furyl, C1-C4 alkyl substituted or unsubstituted indolyl, thiol, C2 - C4 amide, C2 - C4 carboxyl or C1 - C4 alkylamino, and the substituent on the C1 - C4 alkyl is selected from one or more of thiol, C1 - C4 alkylthio, hydroxyl and halogen; R4为H、卤素、炔基、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的萘基、取代或未取代的C1～C4烷氧基、酯基、羟基、C2～C4酰基、氰基中的一种或多种，所述C1～C4烷基、所述C1～C4烷氧基上的取代基分别独立选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R4 is one or more of H, halogen, alkynyl, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1 - C4 alkoxy, ester, hydroxyl, C2 - C4 acyl, and cyano, and the substituents on the C1 - C4 alkyl and the C1 - C4 alkoxy are independently selected from one or more of mercapto, C1 - C4 alkylthio, hydroxyl, and halogen; R5为H、卤素、炔基、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的萘基、取代或未取代的C1～C4烷氧基、酯基、羟基、C2～C4酰基、氰基中的一种或多种，所述C1～C4烷基、所述C1～C4烷氧基上的取代基分别独立选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R5 is one or more of H, halogen, alkynyl, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1 - C4 alkoxy, ester, hydroxyl, C2 - C4 acyl, and cyano, and the substituents on the C1 - C4 alkyl and the C1 - C4 alkoxy are independently selected from one or more of mercapto, C1 - C4 alkylthio, hydroxyl, and halogen; R6为H、卤素、炔基、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的萘基、取代或未取代的C1～C4烷氧基、酯基、羟基、C2～C4酰基、氰基中的一种或多种，所述C1～C4烷基、所述C1～C4烷氧基上的取代基分别独立选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R6 is one or more of H, halogen, alkynyl, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1 - C4 alkoxy, ester, hydroxyl, C2 - C4 acyl, and cyano, and the substituents on the C1 - C4 alkyl and the C1 - C4 alkoxy are independently selected from one or more of mercapto, C1 - C4 alkylthio, hydroxyl, and halogen; R7为H、卤素、炔基、取代或未取代的C1～C4烷基、C1～C4烷基取代或未取代的苯基、C1～C4烷基取代或未取代的萘基、取代或未取代的C1～C4烷氧基、酯基、羟基、C2～C4酰基、氰基中的一种或多种，所述C1～C4烷基、所述C1～C4烷氧基上的取代基分别独立选自巯基、C1～C4烷硫基、羟基、卤素中的一种或多种； R7 is one or more of H, halogen, alkynyl, substituted or unsubstituted C1 - C4 alkyl, C1 - C4 alkyl substituted or unsubstituted phenyl, C1 - C4 alkyl substituted or unsubstituted naphthyl, substituted or unsubstituted C1 - C4 alkoxy, ester, hydroxyl, C2 - C4 acyl, and cyano, and the substituents on the C1 - C4 alkyl and the C1 - C4 alkoxy are independently selected from one or more of mercapto, C1 - C4 alkylthio, hydroxyl, and halogen; R8为-(CH2)n-、-CH2(CH3)-、-CH2(CH2CH3)-、-CH2(CH2Ph)-、-CH2OCH2CH2-或-CH2(CH2CH2SCH3)-，n＝1～20，或者R8为式(a)～(h)中任一结构： R 8 is -(CH 2 ) n -, -CH 2 (CH 3 )-, -CH 2 (CH 2 CH 3 )-, -CH 2 (CH 2 Ph)-, -CH 2 OCH 2 CH 2 - or -CH 2 (CH 2 CH 2 SCH 3 )-, n=1-20, or R 8 is any one of the structures of formulae (a) to (h): n3、n4、n5分别独立为1或2，R10、R11分别独立为C1～C4烷基或C1～C4烷氧基；n 3 , n 4 , n 5 are each independently 1 or 2, R 10 , R 11 are each independently C 1 ～C 4 alkyl or C 1 ～C 4 alkoxy; R9为-(CH2)m-、-CH2(CH3)-、-CH2(CH2CH3)-、-CH2(CH2Ph)-、-CH2OCH2CH2-或-CH2(CH2CH2SCH3)-，m＝1～20，或者R9为式(a)～(h)中任一结构。 R 9 is -(CH 2 ) m -, -CH 2 (CH 3 ) -, -CH 2 (CH 2 CH 3 ) -, -CH 2 (CH 2 Ph) -, -CH 2 OCH 2 CH 2 - or -CH 2 (CH 2 CH 2 SCH 3 ) -, m = 1 to 20, or R 9 is any one of the structures of formulae (a) to (h). 9.根据权利要求7或8所述的环肽制备方法，其特征在于，步骤S1中：9. The method for preparing a cyclic peptide according to claim 7 or 8, characterized in that in step S1: 所述氨基酸缩合反应所采用的缩合剂包括碳二亚胺型缩合剂、磷正离子型缩合剂、脲正离子型缩合剂中的至少一种；The condensing agent used in the amino acid condensation reaction includes at least one of a carbodiimide condensing agent, a phosphorus cation condensing agent, and a urea cation condensing agent; 所述碳二亚胺型缩合剂包括N,N'-二环己基碳二亚胺、N,N'-二异丙基碳二亚胺、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺盐酸盐中的至少一种；The carbodiimide type condensing agent includes at least one of N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; 所述磷正离子型缩合剂包括1H-苯并三唑-1-基氧三吡咯烷基六氟磷酸盐、(7-氮杂苯并三唑-1-氧)三吡咯磷六氟磷酸盐、双(2-氧代-3-恶唑烷基)次磷酰氯、氯代三吡咯烷基六氟磷酸盐、三吡咯烷基溴化鏻六氟磷酸盐中的至少一种；The phosphorus cation type condensing agent includes at least one of 1H-benzotriazole-1-yloxytripyrrolidino hexafluorophosphate, (7-azabenzotriazole-1-oxy)tripyrrolidinophosphine hexafluorophosphate, bis(2-oxo-3-oxazolidinyl)phosphinyl chloride, chlorotripyrrolidinophosphine hexafluorophosphate, and tripyrrolidinophosphonium bromide hexafluorophosphate; 所述脲正离子型缩合剂包括苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸盐、N,N,N',N'-四甲基-O-(7-氮杂苯并三唑-1-基)六氟磷酸脲、2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲四氟硼酸盐、O-[(乙氧基羰基)氰基甲胺]-N,N,N',N'-四甲基硫脲四氟硼酸盐、O-(苯并三唑-1-基)-N,N,N',N'-二吡咯基脲六氟磷酸酯中的至少一种；The urea cationic condensing agent includes at least one of benzotriazole-N,N,N',N'-tetramethyl uronium hexafluorophosphate, N,N,N',N'-tetramethyl-O-(7-azabenzotriazole-1-yl) uronium hexafluorophosphate, 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyl uronium tetrafluoroborate, O-[(ethoxycarbonyl) cyanomethylamine]-N,N,N',N'-tetramethyl thiourea tetrafluoroborate, and O-(benzotriazole-1-yl)-N,N,N',N'-dipyrrolyl uronium hexafluorophosphate; 所述氨基酸缩合反应所采用的缩合活化剂包括1-羟基-7-偶氮苯并三氮唑、1-羟基苯并三唑、1-羟基-1H-1,2,3-三唑-4-羧酸乙酯、3-羟基-3,4-二氢-4-氧代-1,2,3-苯并三嗪、2-羟基吡啶-N-氧化物、4-二甲氨基吡啶、N-羟基丁二酰亚胺、对硝基苯酚、N,N-二异丙基乙胺、Oxyma及其衍生物中的至少一种。The condensation activator used in the amino acid condensation reaction includes at least one of 1-hydroxy-7-azobenzotriazole, 1-hydroxybenzotriazole, 1-hydroxy-1H-1,2,3-triazole-4-carboxylic acid ethyl ester, 3-hydroxy-3,4-dihydro-4-oxo-1,2,3-benzotriazine, 2-hydroxypyridine-N-oxide, 4-dimethylaminopyridine, N-hydroxysuccinimide, p-nitrophenol, N,N-diisopropylethylamine, Oxyma and its derivatives. 10.根据权利要求7或8所述的环肽制备方法，其特征在于，步骤S1至步骤S3中：10. The method for preparing a cyclic peptide according to claim 7 or 8, characterized in that in step S1 to step S3: 各反应过程的溶剂分别独立包括N,N-二甲基甲酰胺、二甲基亚砜、二氯甲烷、乙酸乙酯、四氢呋喃、甲苯、丙酮、乙醚、乙腈、甲醇、水中的至少一种；The solvent of each reaction process independently includes at least one of N,N-dimethylformamide, dimethyl sulfoxide, dichloromethane, ethyl acetate, tetrahydrofuran, toluene, acetone, ether, acetonitrile, methanol and water; 各反应过程的反应温度分别独立为0～100℃。The reaction temperature of each reaction process is independently 0 to 100°C. 11.根据权利要求7或8所述的环肽制备方法，其特征在于，步骤S2中，所述亚硝酸类化合物包括亚硝酸钠、亚硝酰硫酸、亚硝酸甲酯、亚硝酸乙酯、亚硝酸异丙酯、亚硝酸丁酯、亚硝酸叔丁酯、亚硝酸戊酯、亚硝酸异戊酯、亚硝酸己酯中的至少一种。11. The method for preparing a cyclic peptide according to claim 7 or 8, characterized in that in step S2, the nitrite compound includes at least one of sodium nitrite, nitrosyl sulfuric acid, methyl nitrite, ethyl nitrite, isopropyl nitrite, butyl nitrite, tert-butyl nitrite, amyl nitrite, isoamyl nitrite, and hexyl nitrite.
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