Polypeptide cyclization method

Abstract

The invention discloses a polypeptide cyclization method which is characterized in that amino acid with alkyne amide and a peptide chain containing cysteine are used as precursors, cyclization labeling is rapidly and efficiently carried out in three modes of amino acid side chain-Cys, amino acid C terminal-Cys and amino acid N terminal-Cys under mild conditions, and formed alkenyl thioether is stable in structure, specific in configuration and high in stability. And subsequent modification can be carried out through condensation of end-to-end amides in a peptide chain, so that a more complex topological structure can be obtained. Besides, the cyclopeptide compound prepared by the method is stable in property under different pH values, can tolerate strong acid and strong alkali, and has relatively good storage stability.

Description

Technical Field

[0001] The invention belongs to the field of chemical engineering, and particularly relates to a polypeptide cyclization method. Background Art

[0002] Peptide drugs are highly favored for their good drug properties such as good target selectivity, strong affinity, low toxicity, clear biological functions and good metabolic stability. Compared with linear peptides, cyclic peptides have strong thermal and protease hydrolysis stability, cell membrane permeability and bioavailability due to their unique structure. They have gradually become an emerging direction for peptide drug research and show great prospects. At present, many strategies for constructing cyclic peptides have been developed, such as the method of synthesizing cyclic peptides by connecting amide bonds head to tail, using Cu-catalyzed alkynyl [3+2] cycloaddition, Ru-catalyzed olefin metathesis, Pd-catalyzed arylation of cysteine ​​Cys-SH and lysine Lys-NH2, metal-free catalyzed disulfide bond formation, oxadiazole formation and Petasis-borono-Manich reaction to construct cyclic peptides. The disadvantages of these strategies are: (1) the introduction of non-natural amino acids is required; (2) transition metal catalysis is required; (3) non-water solubility is required. Therefore, it is urgent to develop a method for cyclizing unprotected peptide chains to form cyclic peptides under mild conditions without metal catalysts. Summary of the invention

[0003] In order to overcome the problems existing in the prior art, one of the objectives of the present invention is to provide a method for polypeptide cyclization.

[0004] The second object of the present invention is to provide a cyclic peptide compound prepared by the above method.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] The first aspect of the present invention is to provide a method for polypeptide cyclization, which adopts method one or method two;

[0007] Method 1: Prepared by intramolecular cyclization reaction of formula S1A, formula S1B, formula S2 or formula S3;

[0008] Method 2: From formula S4 and Prepared by cyclization reaction;

[0009] The formulas S1A, S1B, S2, S3 and S4 are:

[0010]

[0011] Wherein, in the formula S1A and the formula S1B, AA x The corresponding amino acid is selected from one of natural amino acids or synthetic amino acids;

[0012] In formula S2, AA x The corresponding amino acid is selected from amino acids having active functional groups in the side chains, wherein the active functional groups are amino, carboxyl or hydroxyl groups;

[0013] AA1, AA y or The corresponding amino acid is selected from at least one of natural amino acids or synthetic amino acids;

[0014] The EWG is selected from alkanesulfonyl, arylsulfonyl, alkanoyl, aroyl, nitrile or nitro;

[0015] AA y Where y is an integer from 0 to 100;

[0016] n is an integer from 1 to 100.

[0017] The natural amino acids include tryptophan, lysine, phenylalanine, methionine, threonine, valine, leucine, isoleucine, cysteine, serine, glycine, tyrosine, aspartic acid, asparagine, glutamic acid, glutamine, alanine, arginine, histidine, and proline.

[0018] In Formula S1A and Formula S1B, AA x The corresponding amino acid can be any amino acid, which is formed by introducing an acetylamide group into the amino acid, and then the acetylamide group and the cysteine ​​sulfhydryl group or the cystine sulfhydryl group on the peptide chain undergo an intramolecular β-sulfhydration reaction to form a peptide ring. x The corresponding amino acid does not need to be limited to a specific amino acid.

[0019] In formula S2, AA x As long as the side chain of the corresponding amino acid contains any one of a carboxyl group, an amino group or a hydroxyl group, an alkynamide group can be introduced on the carboxyl group, the amino group or the hydroxyl group, and then a subsequent ring-closing reaction can occur.

[0020] Preferably, in the formula S1A and S1B, AA x The corresponding amino acid is selected from lysine and cystine.

[0021] Preferably, in the formula S2, AA x The corresponding amino acids are selected from glutamine, asparagine and tyrosine.

[0022] Preferably, AA1, AA y or The corresponding amino acid is selected from at least one of glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, tyrosine, asparagine, glutamine, lysine, methionine, serine, threonine, proline and histidine.

[0023] In the formulas S1A, S1B, S2, S3 and S4, AA1, AA y , AA x A linear peptide chain is formed, and the total number of amino acids in the linear peptide chain is an integer between 2 and 100.

[0024] The cyclization reaction in the method one and the method two of the present invention refers to the peptide ring formed by the intramolecular β-sulfhydration reaction of the alkyne amide group with the cysteine ​​sulfhydryl group or the cystine sulfhydryl group that breaks the disulfide bond on the peptide chain. The present invention forms a cyclic peptide compound by introducing an alkyne amide structure on the polypeptide, and then performing an intramolecular β-sulfhydration reaction with the cysteine ​​sulfhydryl group or the cysteine ​​sulfhydryl group that breaks the disulfide bond on the peptide chain. The advantage of this method is that alkyne amide can be introduced at multiple positions (carbon end, nitrogen end and side chain) of the peptide chain, thereby realizing various types of cyclization methods, with great flexibility and universality; the method has simple reaction conditions, clean system and stable product structure, which can provide very favorable support for the construction of subsequent cyclic peptide molecular libraries and the screening of high-throughput drugs. In addition, the method in the present invention does not need to pre-prepare the cyclization handle in advance, the reaction is fast and highly selective, the conditions are mild and the product is stable. In order to provide guarantee for the screening and modification of subsequent polypeptide and protein drugs, a method of polypeptide cyclization with high universality and great significance has been developed.

[0025] In addition, the cyclization reaction in the present invention is highly selective. First, from the group perspective, the alkyne amide group only reacts with the sulfhydryl group in the peptide chain, and does not react with the amino group, carboxyl group, and hydroxyl group in the peptide chain, which has a high functional group selectivity; secondly, from the configuration analysis, the cyclization reaction products are all cis structures, which shows that the configuration is highly selective. In addition, the cyclization reaction in the present invention has a high yield and few side reactions. During the cyclization reaction in method one, very few intermolecular reactions occur.

[0026] Preferably, the AA y Where y represents the total number of amino acids contained in the entire peptide chain, and y is preferably 1-100; more preferably, y is 2-20; and even more preferably, y is 2-10.

[0027] Preferably, n is an integer of 1-100; more preferably, n is 1-10.

[0028] Preferably, the EWG is trifluoromethanesulfonyl (CF3SO2-).

[0029] Preferably, the method further comprises the step of adding Bu3P.

[0030] Preferably, the concentrations of Formula S1A, Formula S1B, Formula S2, Formula S3, and Formula S4 are 1 mmol / L to 10 mmol / L.

[0031] Preferably, the solvent for the intramolecular cyclization reaction is selected from CH3CN, DMF, and a mixed solution of PB buffer / CH3CN.

[0032] Preferably, the temperature of the intramolecular cyclization reaction is -20 to 100°C; and the reaction time is 5 min to 48 h.

[0033] Preferably, the amino acid sequence in Formula S1A, Formula S1B, Formula S2, Formula S3, and Formula S4 is selected from any one of the following sequences: (Cbz-Cys-OH)2, (Cbz-Cys-Pro-OH)2, (Cbz-Cys-Gly-OH)2, (Cbz-Cys-Gly-Glu(O t Bu)-OH)2, (Cbz-Cys-Gly-Ala-OH)2, (Cbz-Cys-Gly-Ala-Phe-OH)2, (H-Cys-OH)2, (Boc-Lys-Cys-OEt)2 , (Boc-Lys-Ala-Cys-OEt)2, (Boc-Lys-Val-Cys-OEt)2, (Boc-Lys-Thr-Cys-OEt)2, Boc-Lys-Ala-Cys(SS t Bu)-OMe, Ac-Gln-Ala-Cys(SS t Bu)-OMe, Cbz-Gln-Cys(SS t Bu)-OMe, Cbz-Gln-Ala-Cys(SS t Bu)-OMe, Cbz-Gln-His-Cys(SS t Bu)-OMe, Cbz-Gln-Met-Cys(SS t Bu)-OMe, Cbz-Gln-Thr-Cys(SS t Bu)-OMe, Cbz-Gln-Ser-Cys(SS t Bu)-OMe, Cbz-Gln-Trp-Cys(SS t Bu)-OMe, Cbz-Gln-Ile-Cys(SS t Bu)-OMe, Cbz-Gln-Pro-Cys(SS t Bu)-OMe, Cbz-Tyr-Ala-Cys(SS t Bu)-OMe, Cbz-Asn-Ala-Cys(SS tBu)-OMe、Cbz-Gln-Phe-Ala-Cys(SS t Bu)-OMe、Cbz-Gln-Phe-Ala-Cys(SS t Bu)-OH、Cbz-Gln-Gly-Phe-Ala-Cys(SS t Bu)-OH、Cbz-Gln-Phe-Met-Cys(SS t Bu)-Gly-OH, Cbz-Gln-Ala-Pro-Leu-Met-Cys(SS t Bu)-OH、Cbz-Gln-Met-Leu-Ala-Trp-Gly-Cys(SS t Bu)-OH、Cbz-Gln-Ala-Met-Phe-Ala-Cys(SS t Bu)-Gly-OH, Cbz-Gln-Met-Ala-Pro-Phe-Ala-Cys-Ala-OH, Cbz-Gln-Gly-Leu-Trp-Ala-Cys(SS t Bu)-Gly-Ala-OH, Cbz-Gln-Ala-Leu-Phe-Met-Trp-Ala-Cys(SS t Bu)-Met-OH, Cbz-Gln-Tyr-Ile-Gln-Asn-Cys(SS t Bu)-Pro-Leu-Gly-OH, NH2-Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2 or Fmoc-Gln-Pro-Cys(SS t Bu)-Ser(O t Bu)-Ala-OH.

[0034] Preferably, the method adopts solid phase synthesis or liquid phase synthesis.

[0035] Preferably, the solid phase synthesis method specifically comprises: performing a cyclization reaction on a solid phase carrier to obtain a cyclic peptide connected to a resin; and cleaving the cyclic peptide with a cleavage solution to obtain a cyclic peptide compound.

[0036] Preferably, the solid phase carrier is: Rink amide resin, Rink Amide AM resin, Rink Amide MBHA resin, CTC resin.

[0037] Preferably, the lysate is: a mixed solution of acetic acid, trifluoroethanol and dichloromethane; or a mixed solution of trifluoroacetic acid, triisopropylsilane and water.

[0038] Preferably, the method further comprises the step of condensing the head and tail amides of formula S2 / formula S3 / formula S4 to form a peptide ring. Specifically, the method further comprises the step of subjecting the amino group at the head end and the carboxyl group at the tail end of formula S2 (formula S3 or formula S4) to an amide condensation reaction to form a peptide ring; this step may be before the cyclization step in method one or method two, or after the cyclization step in method one or method two. Through the head and tail amide condensation reaction and cyclization reaction, a bicyclic peptide can be formed, and a more complex cyclic peptide structure can be constructed, which provides a new idea for the structural design of cyclic peptides.

[0039] Preferably, in the AA x The structure after the introduction of the alkyne amide group is selected from:

[0040] The second aspect of the present invention is to provide a cyclic peptide compound prepared by the method provided in the first aspect of the present invention, wherein the cyclic peptide compound has a general structural formula as shown in any one of Formulas C1 to C4:

[0041]

[0042] Among them, AA1, AA x AA y , n. EWG is as defined above.

[0043] Preferably, the cyclic peptide compound is selected from any one of the following compounds:

[0044]

[0045]

[0046] The invention has the following beneficial effects: the polypeptide cyclization method of the invention has a fast reaction speed, the cyclization reaction is completed in 5 minutes at the fastest, and the reaction is completed in no more than 48 hours at the slowest, the yield is high, the reaction adaptability is high, and amino acids can be introduced into various amino acids such as lysine, cystine, tyrosine, aspartic acid, etc.; it can be synthesized and applied on a solid phase, and a large cyclic peptide containing multiple amino acids can be prepared, and after a single cyclic peptide is formed by the head-to-tail condensation of amides, a subsequent alkyne amide β-sulfhydration reaction can form a bicyclic peptide. The invention uses an amino acid with alkyne amide and a peptide chain containing cysteine ​​or cystine as precursors, and under mild conditions, quickly and efficiently performs cyclization labeling in three ways: amino acid side chain-Cys, amino acid C-terminal-Cys, and amino acid N-terminal-Cys. The formed alkenyl thioether structure is stable and has a specific configuration, and can also be subsequently modified by the head-to-tail amide condensation in the peptide chain to obtain a more complex topological structure.

[0047] The cyclic peptide compound prepared in the present invention has an alkenyl sulfide structure obtained by the addition of alkyne amide and thiol, which is very stable, not sensitive to light, oxygen and acidity and alkalinity; can tolerate strong acid and alkali, and has good storage stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 For cyclic peptide compound 5 1 H-NMR spectrum.

[0049] Figure 2 For cyclic peptide compound 5 13 C-NMR spectrum.

[0050] Figure 3 For cyclic peptide compound 7 1 H-NMR spectrum.

[0051] Figure 4 For cyclic peptide compound 7 13 C-NMR spectrum.

[0052] Figure 5 For cyclic peptide compound 9 1 H-NMR spectrum.

[0053] Figure 6 For cyclic peptide compound 9 13 C-NMR spectrum.

[0054] Figure 7 For cyclic peptide compound 13 1 H-NMR spectrum.

[0055] Figure 8 For cyclic peptide compound 13 13 C-NMR spectrum. DETAILED DESCRIPTION

[0056] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples, but the implementation and protection of the present invention are not limited thereto. It should be noted that if there are processes that are not particularly described in detail below, they can be implemented or understood by those skilled in the art with reference to the prior art. If the manufacturer of the reagents or instruments used is not indicated, they are deemed to be conventional products that can be purchased commercially.

[0057] The preparation route of the cyclic peptide compound ring-closing precursor linear peptide in the present invention is shown in the following formula:

[0058]

[0059]

[0060] In the above reaction formula, i) Fmoc protecting group removal; ii) amino acid condensation; iii) resin cleavage; IV) amino acid condensation; V) ester condensation; VI) amino acid condensation, wherein i) Fmoc protecting group removal, ii) amino acid condensation and iii) resin cleavage are solid phase reactions on CTC resin or Rink Amide resin; IV) amino acid condensation, V) ester condensation and VI) amino acid condensation are liquid phase reactions, and the specific reaction conditions are as follows:

[0061] i) Removal of Fmoc protecting group: Add 50% morpholine / DMF into the solid phase synthesis tube and shake for 15 minutes. Then remove the reaction solvent and wash twice with DMF and DCM respectively; repeat the above operation once to complete the removal of Fmoc protecting group.

[0062] ii) Amino acid condensation: Fmoc-AA y -OH includes (Fmoc-Cys(SS t Bu)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Trp-OH, Fmoc-Met-O H, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Tyr(Boc)-OH, Cbz-Gln(R)-O H) (3.0 equiv), ethyl 2-oximecyanoacetate (3.0 equiv) was dissolved in N,N-dimethylformamide (DMF) and dichloromethane (DCM) to prepare a clear solution, and then N,N-diisopropylcarbodiimide (DIC) (3.0 equiv) was added thereto and reacted in an ice-water bath for 5 min. The reaction solution was then added to a solid phase synthesis tube and reacted at room temperature for 2 hours. The reaction solvent was then drained and washed twice with DMF and DCM, respectively, for the next step of deprotection group reaction.

[0063] iii) Resin cleavage: Acetic acid, trifluoroethanol, and dichloromethane are prepared in a volume ratio of 1:2:7 (or trifluoroacetic acid, triisopropylsilane and water in a ratio of 95:2.5 2.5) to prepare a cleavage solution, which is then added to a solid phase synthesis tube and cleaved at room temperature for 1 hour. The cleavage solution is then collected, the solvent is removed, and cold ether is added to the residue to precipitate the polypeptide, which is then centrifuged to obtain a crude polypeptide of the compound of formula I with a carboxylic acid or amide at the C-terminus.

[0064] IV) Amino acid condensation: cystine-containing polypeptides are condensed with N-terminally exposed polypeptides (including NH2-Pro-OH, NH2-Gly-OH, NH2-Gly-Ala-OH, NH2-Gly-Glu (Ot Bu)-OH and other condensation.

[0065] V) Ester condensation: The polypeptide containing cystine is condensed with hydroxyethylenediamine alkynamide via 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) ester.

[0066] IV) Amino acid condensation: The N-terminally exposed cysteine ​​or cystine-containing polypeptide is condensed with an amino acid activated ester or an amino acid alkyne amide activated ester.

[0067] Among them, the method of introducing an alkyne amide group at the N-terminus of an amino acid is as follows:

[0068] 1) dissolving an amino acid having a general structural formula (II) and triethylamine in a first solvent, and then adding trifluoromethanesulfonyl chloride to react. After the reaction is completed, a trifluoromethanesulfonamide compound having a general structural formula (III) is obtained:

[0069]

[0070] 2) dissolving a trifluoromethanesulfonamide compound having a general structural formula (III) and a base in a second solvent to obtain a solution A; then dissolving compound (IV) in a first solvent to obtain a solution B; and finally adding solution B to solution A to react, and after the reaction is completed, obtaining a trifluoromethanesulfonyl alkynamide compound having a general structural formula (I):

[0071]

[0072] Wherein, R is an amino acid.

[0073] The method for introducing an alkynamide group at the C-terminus of an amino acid is:

[0074] 1) Dissolving hydroxyethylamine and triethylamine in a first solvent, and then adding trifluoromethanesulfonyl chloride to react. After the reaction is completed, a hydroxyethyl trifluoromethanesulfonamide compound having a general structural formula (V) is obtained:

[0075]

[0076] 2) dissolving a hydroxyethyl trifluoromethanesulfonamide compound having a general structural formula (V) and a base in a second solvent to obtain a solution A; then dissolving a compound (IV) in a first solvent to obtain a solution B; finally, adding the solution B to the solution A for reaction, and after the reaction is completed, obtaining a hydroxyethyl trifluoromethanesulfonyl alkynamide compound having a general structural formula (VI), then dissolving an amino acid having a general structural formula (VII) and (VI) in the first solvent, adding 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) for ester condensation to obtain a compound (VIII) having an alkynamide at the C-terminus of the amino acid:

[0077]

[0078] Wherein, R is an amino acid.

[0079] The method for introducing an alkynamide group into the side chain of an amino acid can be obtained by referring to the following method, specifically:

[0080] A trifluoromethanesulfonyl ethylenediamine compound (IX), an amino acid with a side chain (X) and a condensing agent (EDCI) are dissolved in a first solvent for reaction to obtain a compound with a general structural formula (XI); a trifluoromethanesulfonyl alkynamide compound with a general structural formula (XI) and a base are dissolved in a second solvent to obtain a solution A; then a compound (IV) is dissolved in the first solvent to obtain a solution B; finally, solution B is added to solution A for reaction, and after the reaction is completed, a trifluoromethanesulfonyl alkynamide compound with a general structural formula (XII) is obtained.

[0081]

[0082] During the reaction, in order to prevent the amino group or carboxyl group from affecting the reaction, the amino group (for example, the amino group can be protected by a Boc or Cbz functional group) or the carboxyl group (for example, the carboxyl group can be protected by an esterification reaction) can be protected by using a commonly used group protecting functional group, and the protecting group can be removed after the target compound is synthesized.

[0083] The invention introduces an alkyne amide group into a specific position of a target amino acid first, and then introduces the target amino acid into a peptide chain through a condensation reaction.

[0084] The raw material information used in the embodiment of the present invention is as follows:

[0085] The CAS number is 73724-43-3 and it can be purchased.

[0086] The CAS number is 51499-82-2, which can be purchased or synthesized according to patent: WO2013100132A1. The CAS number is 6968-11-2 and can be purchased;

[0087] The CAS number is 32854-09-4 and can be purchased;

[0088] Example 1

[0089] The structural formula of the cyclic peptide compound 1 in this example is:

[0090]

[0091] The reaction scheme and preparation method of cyclopeptide compound 1 are as follows:

[0092] The specific reaction route is as follows:

[0093]

[0094] Compound S1a (0.1 mmol, 83.4 mg) was weighed and placed in a reaction flask, acetonitrile (CH3CN) (20 mL) was added to completely dissolve S1a, and then tributylphosphine ( n Bu3P)(4 equiv, 0.4 mmol, 80.9 mg), stirred for 10 min, and then separated by column chromatography to obtain 91% of the intramolecular cyclic peptide compound 1. Due to the low reaction concentration, the intermolecular cyclization product 1* was very small.

[0095] 1 H NMR (400MHz, CDCl3) δ6.42(d,J=5.9Hz,1H),6.25(d,J=6.1Hz,1H),5.27(d,J=7.4Hz,1H),4.72(d,J=9.2Hz,1H),4.54(d,J=5.9Hz,1H),4. 26(d,J=12.0Hz,1H), 4.04(d,J=14.2Hz,1H), 3.85–3.65(m,1H), 3.27(dd,J=13.9,5.5Hz,1H), 2.57(dd,J=13.7,8.8Hz,1H), 1.44(s,9H).

[0096] The mass spectrum peak of 419.09 was found by MS detection.

[0097] Effect of Reaction Concentration and Reducing Amount on Reaction Time in Example 1

[0098] Four portions of the same mass of compound S1a (0.1 mmol, 83.4 mg) were placed in four reaction bottles, and then acetonitrile (CH3CN) (2 mL, 4 mL, 10 mL, 20 mL) was added to the four reaction bottles to completely dissolve S1a. Then, tributylphosphine ( n Bu3P) (2 equiv, 0.2 mmol, 40.5 mg), and monitored by TLC spot plate. It was found that the higher the concentration of compound S1a, the faster the reaction. However, the increase in concentration would aggravate the production of dimers.

[0099] The amount of reducing agent accelerates the reaction. When the concentration is 10 mmol / L and the reducing agent is 4 times the amount, the cyclopeptide compound 1 can be obtained with a yield of 91% in just 10 minutes. Therefore, the reaction can be accelerated by increasing the amount of reducing agent, and the formation of dimers can be avoided by adjusting the concentration of the compound S1a reaction solution.

[0100] Embodiment 2-6

[0101] Examples 2-6 are used to synthesize cyclic peptide compounds 2-5, respectively. The structural formulas of cyclic peptide compounds 2-5 are as follows:

[0102]

[0103] The preparation method of cyclic peptide compound 5 is as follows:

[0104]

[0105] The linear peptide S5a (0.1 mmol, 116.3 mg) was weighed and placed in a reaction flask, acetonitrile (CH3CN) (20 mL) was added to completely dissolve S5a, and then tributylphosphine ( n Bu3P) (4 equiv, 0.4 mmol, 80.9 mg), TLC monitoring, after the reaction of compound S5a is complete, CH3CN is removed by vacuum distillation, then H2O and DCM are added, the aqueous layer is extracted three times with DCM (10 mL×3), then dried with anhydrous Na2SO4, the organic solvent is removed by vacuum distillation, and the cyclopeptide compound 5 (103.5 mg, 89%) is separated by silica gel column chromatography (eluent PE / EtOAc=1:2 (volume ratio)). Cyclic peptide compound 5 1 H-NMR, 13 C-NMR Figure 1 and Figure 2 shown.

[0106] 1H NMR (400MHz, DMSO-d6) δ8.41(t,J=5.5Hz,1H),7.98(d,J=7.2Hz,1H),7.83(d,J=8.0Hz,1H),7. 46–7.29(m,5H),6.56(d,J=6.8Hz,1H),6.02(d,J=6.7Hz,1H),5.04(d,J=9.3Hz,2H),4.43–4.3 3(m,1H),4.31–4.19(m,2H),4.13–4.02(m,1H),3.83(dt,J=10.5,5.7Hz,3H),3.66(dd,J=15.9 ,4.9Hz,1H),3.25(dd,J=13.4,3.6Hz,1H),3.09(dd,J=13.1,8.7Hz,1H),1.31(d,J=7.1Hz,3H).

[0107] 13 C NMR (101MHz, DMSO) δ171.8,170.1,168.6,155.9,137.1,133.9,130.1,128.8,128 .4,119.9(q,J=323.0Hz),119.4,66.3,62.9,55.0,49.0,48.7,43.3,35.2,17.6.

[0108] The preparation methods of Examples 2-4 and Example 6 can be prepared by referring to the preparation method of Example 5.

[0109] The reaction conditions and concentrations of Examples 1-6 were consistent, and the yields of the target cyclopeptide compounds obtained were shown in Table 1.

[0110] Table 1 Yields of cyclic peptide compounds of Examples 1-6

[0111]

[0112] Example 7

[0113] The structural formula of cyclic peptide compound 7 is as follows:

[0114]

[0115] The preparation method of cyclic peptide compound 7 is as follows:

[0116]

[0117] The N-terminus-to-side-chain ring closure was achieved by introducing alkynamide at the N-terminus of cystine. We successfully introduced the alkynamide group at the N-terminus of (H-Cys-OEt)2 through sulfonylation and alkynyl introduction, thus synthesizing S7a (cystine alkynamide). n The cyclic peptide compound 7 was obtained with a yield of 82% under the action of Bu3P (4 equiv, 0.4 mmol, 80.9 mg). 1 H-NMR and 13 C-NMR Figure 3 and Figure 4 shown.

[0118] 1 H NMR (400MHz, CDCl3) δ6.71 (d, J=8.6Hz, 1H), 5.58 (dd, J=8.7, 2.1Hz, 1H), 5.18 (s, 1H), 4.2 9(q,J=7.2Hz,2H),3.50–3.42(m,1H),3.12(dd,J=13.3,3.2Hz,1H),1.31(t,J=7.1Hz,3H).

[0119] 13 C NMR (101MHz, CDCl3) δ 166.1, 119.4 (q, J = 311.0Hz), 118.2, 104.2, 62.8, 55.7, 26.8, 14.0.

[0120] Examples 8-12

[0121] Examples 8 to 12 are used to synthesize cyclic peptide compounds 8 to 12, respectively. The structural formulas of cyclic peptide compounds 8 to 12 are as follows:

[0122]

[0123] The preparation method of cyclopeptide compound 9 is as follows:

[0124]

[0125] The linear peptide S9a (lysine side chain N-terminal introduced with alkyne amide) (0.1 mmol, 125.2 mg) was weighed and placed in a reaction bottle, acetonitrile (CH3CN) (20 mL) was added to completely dissolve S9a, and then tributylphosphine ( nBu3P) (4 equiv, 0.4 mmol, 80.9 mg), TLC monitoring After the reaction of compound S9a is complete, CH3CN is removed by vacuum distillation, and then H2O and DCM are added, and the aqueous layer is extracted with DCM (10 mL×3). Then, it is dried over anhydrous Na2SO4, and the organic solvent is removed by vacuum distillation. The cyclopeptide compound 9 (107.7 mg, 86%) is separated by silica gel column chromatography (eluent: PE / EtOAc=1:2 (volume ratio)). Cyclic peptide compound 9 1 H-NMR, 13 C-NMR Figure 5 and Figure 6 shown.

[0126] 1 H NMR (400MHz, DMSO-d6) δ8.25(d,J=8.6Hz,1H),8.07(d,J=8.3Hz,1H),6.52(dd ,J=17.2,7.2Hz,2H),5.87(d,J=6.9Hz,1H),4.37(dd,J=15.7,8.1Hz,2H),4.0 3(dq,J=12.1,6.8Hz,3H),3.38(d,J=19.7Hz,2H),3.24(d,J=3.5Hz,1H),2.93 (dd,J=14.3,10.1Hz,1H),1.64–1.35(m,4H),1.30(s,9H),1.21–1.11(m,8H).

[0127] 13 C NMR (101MHz, DMSO) δ172.8,171.8,170.4,155.3,130.1,119.9,119.7(q,J=323.0 Hz),78.4,61.5,53.4,52.3,50.7,48.0,34.9,32.1,28.6,27.7,21.6,18.4,14.4.

[0128] The reaction conditions and concentrations of Examples 8 and 10 to 12 are consistent with those of Example 9. The yields of Examples 8 to 12 are shown in Table 2.

[0129] Table 2 Yields of Examples 8 to 12

[0130] Example No. Example 8 Example 9 Example 10 Embodiment 11 Example 12 Cyclic peptide number 8 9 10 11 12 Isolation yield% 47 86 91 70 89

[0131] Examples 13-24

[0132] Examples 13-24 are used to synthesize cyclic peptide compounds 13-24, respectively. The structural formulas of cyclic peptide compounds 13-24 are as follows:

[0133]

[0134] Wherein, the preparation method of cyclic peptide compound 13 is as follows:

[0135]

[0136] The linear peptide S13a (0.1 mmol, 75.5 mg) was weighed and placed in a reaction flask, acetonitrile (CH3CN) (10 mL) was added to completely dissolve S13a, and then tributylphosphine ( n Bu3P) (4 equiv, 0.4 mmol, 80.9 mg), TLC monitoring After the reaction of compound S13a is complete, CH3CN is removed by vacuum distillation, and then H2O and DCM are added, and the aqueous layer is extracted with DCM (10 mL×3). Then, it is dried over anhydrous Na2SO4, and the organic solvent is removed by vacuum distillation. The cyclopeptide compound 13 (57 mg, 86%) is separated by silica gel column chromatography (eluent: PE / EtOAc=1:2 (volume ratio)). Cyclic peptide compound 13 1 H-NMR, 13 C-NMR Figure 7 and Figure 8 shown.

[0137] The reaction conditions and reaction concentrations of Examples 13 to 24 are the same. It can be seen from Examples 13 to 24 that the acetylamide group is highly selective for the sulfhydryl group on cysteine ​​during the cyclization process, that is, the acetylamide group does not react with the amino, hydroxyl, and carboxyl groups in the peptide chain, but only reacts with the sulfhydryl group, which is highly selective.

[0138] The yields of the cyclic peptide compounds in Examples 13 to 24 are shown in Table 3.

[0139] Table 3 Yields of Examples 13 to 24

[0140]

[0141]

[0142] Examples 25-34

[0143] Examples 25 to 34 are used to synthesize cyclic peptide compounds 25 to 34, respectively. The structural formulas of cyclic peptide compounds 25 to 34 are as follows:

[0144]

[0145]

[0146] Wherein, the preparation method of cyclic peptide compound 30 is as follows:

[0147]

[0148]

[0149] I) Preparation of ring-closing precursor: First, dichloromethane (DCM) was used to swell the resin, and Fmoc-AA-OH (Fmoc-Cys (S t Bu)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH, Fmoc-Phe-OH, Fmoc-Met-OH, Fmoc-Gln(Trt)-OH, Cbz-Gln(R 1 )-OH(R 1 For the connection of alkyne amide) (3.0 equiv), ethyl 2-oxime cyanoacetate (3.0 equiv) was dissolved in N, N-dimethylformamide (DMF) and dichloromethane (DCM) to prepare a clear solution, and then N, N-diisopropylcarbodiimide (DIC) (3.0 equiv) was added thereto and reacted in an ice-water bath for 5 min, and then the reaction solution was added to a solid phase synthesis tube, reacted at room temperature for 2 hours, and then the reaction solvent was drained, and washed twice with DMF and DCM, respectively, for the next step of deprotection reaction. 50% morpholine / DMF was added to the solid phase synthesis tube and shaken for 15 minutes, and then the reaction solvent was removed, and washed twice with DMF and DCM, respectively; the above operation was repeated once to complete the removal of the Fmoc protecting group. The above operation cycle completes the preparation of the linear peptide (30a-2). Finally, the amino acid copper-activated ester containing alkyne amide (30a) is used to react with the peptide chain, an equivalent amount of 1-hydroxybenzotriazole (HOAt) is added, and appropriate amounts of DMF and DCM are added for dissolution. The reaction is carried out at room temperature for 2 hours, and then the reaction solvent is drained and washed three times with DMF and DCM respectively to complete the preparation of the ring-closing precursor (i.e., S30a).

[0150] II) Cyclization on resin: DMF was selected as the ring closure solvent, and tributylphosphine ( n Bu3P)(4equiv, 0.4mmol, 80.9mg), the ring-closing concentration was 10mmol / L, and the reaction time was 3h.

[0151] III) Resin cleavage: A cleavage solution was prepared by mixing acetic acid, trifluoroethanol and dichloromethane in a volume ratio of 1:2:7 (or trifluoroacetic acid, dichloromethane, triisopropylsilane and water in a volume ratio of 50:45:2.5:2.5), and then added to a solid phase synthesis tube and cleaved at room temperature for 1 hour. The cleavage solution was then collected, the solvent was removed, and cold ether was added to the residue to precipitate the polypeptide, which was then centrifuged to obtain the ring-closed product, i.e., cyclopeptide compound 30.

[0152] It is worth noting that, since the alkynamide group has a certain tolerance to acid, the cyclization process can also be carried out by separating the ring-closing precursor and then performing the ring-closing in the liquid phase. The solid phase cyclization method of the present invention is compatible with both alkynamide and cysteine, avoiding side reactions between alkynamide and cysteine.

[0153] The preparation steps of the cyclic peptide compounds in Examples 25 to 29 and Examples 31 to 34 are the same as those in Example 30, and the reaction raw materials used include Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Tyr(Boc)-OH. The reaction yields and MS characterization data of Examples 25 to 30 are recorded in Tables 4 and 5, respectively.

[0154] Table 4 Reaction yields of Examples 25 to 30

[0155]

[0156] The reaction conditions B in Table 4 are n Bu3P (4equiv), DMF, concentration is 1mmol / L~10mmol / L.

[0157] Table 5 MS characterization data of Examples 25 to 30

[0158] Cyclic peptide compound number Cal.MS(ESI) Obtained MS(ESI) Separation purity % 25 <![CDATA[[M+H] + =801.22]]> 800.80 84 26 <![CDATA[[M+Na] + =940.23]]> 940.19 77 27 <![CDATA[[M+Na] + =880.22]]> 880.14 73 28 <![CDATA[[M] - =993.31]]> 993.79 64 29 <![CDATA[[M] - =1139.36]]> 1139.99 87 30 <![CDATA[[M+K] + =1098.27]]> 1099.29 88 31 <![CDATA[[M+Na] + =1193.37]]> 1193.26 74 32 <![CDATA[[M+H] + =1138.39]]> 1138.12. 85 33 <![CDATA[[M] - =1431.49]]> 1432.17 75 34 <![CDATA[[M+Na] + =1390.50]]> 1390.08 63

[0159] Embodiment 35

[0160] The structural formula of the cyclic peptide compound 35 in this example is as follows:

[0161]

[0162] The preparation method of cyclic peptide compound 35 is as follows:

[0163]

[0164] The synthesized diacetylene amide (3.0 equiv) and the linear polypeptide oxytocin containing two Cys were reacted in a buffer solution of pH = 8.0 with a concentration of 1 mmol / L. The reaction process was monitored by HPLC and MS. The reaction yield and MS characterization data of the cyclic peptide compound 35 are shown in Table 6.

[0165] Table 6 Reaction yield and MS characterization data of cyclic peptide compound 35

[0166] Cyclic peptide number Cal.MS(ESI) Obtained MS(ESI) Separation purity % 35 <![CDATA[[M+H] + =1395.44]]> 1394.87 73

[0167] Embodiment 36

[0168] The structural formula of the cyclic peptide compound 36 in this example is as follows:

[0169]

[0170] The preparation method of cyclic peptide compound 36 is as follows:

[0171]

[0172] After solid-phase peptide synthesis of (36-1a) containing alkyne amide, the peptide chain protecting group is removed to obtain (36-2a), which is cleaved from the resin to obtain (36-3a), and a single cyclic peptide is constructed by head-to-tail amide condensation, and then a second cyclic peptide is constructed by alkyne amide and thiol β-sulfhydration reaction to obtain a bicyclic peptide (36). This example uses the synthesis of cyclic peptide compound 36 as an example to illustrate the preparation method of bicyclic peptides. This type of synthesis method can be applied to the synthesis of more large cyclic peptides.

[0173] Performance Testing:

[0174] Stability test:

[0175] In order to test the stability of the prepared cyclic peptide compounds at different pH values, some cyclic peptide compounds, namely cyclic peptide compounds 1, 4, 14, 16 and 19, were randomly selected from the cyclization products of amino acid C-terminal-Cys and side chain N-terminal-Cys, and were treated at room temperature for 24 h under PBS (pH=7.3), 0.01 mol / L NaOH (pH=12.0) and 0.1 mol / L HCl (pH=1.0), and left open at room temperature for 30 days. The test results are recorded in Table 7.

[0176] Table 7 Stability test results of cyclic peptide compounds 1, 4, 14, 16, and 19

[0177]

[0178] It can be seen from Table 7 above that the cyclic peptide compound prepared in the present invention is stable at different pH values, can withstand strong acids and bases, and has good storage stability.

[0179] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for polypeptide cyclization, characterized in that: This method adopts method one or method two; Method 1: Prepared by intramolecular cyclization reaction of formula S1A, formula S1B, formula S2 or formula S3; Method 2: From formula S4 and Prepared by cyclization reaction; The formulas S1A, S1B, S2, S3 and S4 are: Wherein, in the formula S1A and the formula S1B, AA x The corresponding amino acid is selected from one of natural amino acids or synthetic amino acids; In formula S2, AA x The corresponding amino acid is selected from amino acids having active functional groups in the side chains, wherein the active functional groups are amino, carboxyl or hydroxyl groups; AA1, AA y or The corresponding amino acid is selected from at least one of natural amino acids or synthetic amino acids; The EWG is selected from alkanesulfonyl, arylsulfonyl, alkanoyl, aroyl, nitrile or nitro; AA y Where y is an integer from 0 to 100; n is an integer from 1 to 100.

2. The polypeptide cyclization method according to claim 1, characterized in that: The method further comprises the step of adding Bu3P.

3. The polypeptide cyclization method according to claim 1, characterized in that: The concentrations of Formula S1A, Formula S1B, Formula S2, Formula S3, and Formula S4 are 1 mmol / L to 10 mmol / L.

4. The method for polypeptide cyclization according to claim 1, characterized in that: The solvent for the intramolecular cyclization reaction is selected from a mixed solution of CH3CN, DMF, and PB buffer / CH3CN.

5. The method for polypeptide cyclization according to claim 1, characterized in that: The temperature of the intramolecular cyclization reaction is -20 to 100° C.; the reaction time is 5 min to 48 h.

6. The method for polypeptide cyclization according to claim 1, characterized in that: The method adopts a solid phase synthesis method or a liquid phase synthesis method; the solid phase synthesis method specifically comprises: performing a cyclization reaction on a solid phase carrier to obtain a cyclic peptide connected to a resin; The product was cleaved with a cleavage solution to obtain a cyclic peptide compound.

7. The method for polypeptide cyclization according to claim 1, characterized in that: The method further comprises the step of forming a peptide ring by amide condensation of the head and tail of Formula S2 / Formula S3 / Formula S4.

8. The method for polypeptide cyclization according to claim 1, characterized in that: In the AA x The structure after the introduction of the alkyne amide group is selected from:

9. The cyclic peptide compound prepared by the method according to any one of claims 1 to 8, characterized in that: It has the general structural formula of any one of formulas C1 to C4: Among them, AA1, AA x AA y , n. EWG as defined in any one of claims 1 to 8.

10. The cyclic peptide compound according to claim 9, characterized in that: The cyclic peptide compound is selected from any one of the following compounds:

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