Interchain disulfide bonds (Cys) A24 -Cys B23 H2 Relaxin derivatives with thioether bonds as substitutes

Abstract

This invention discloses an interchain disulfide bond Cys A24 -Cys B23 H2Relaxin derivatives with thioether bonds as alternatives and their synthetic methods, wherein the structural formula of the H2Relaxin derivatives is shown below: This invention first utilizes diaminodioic acid molecules to synthesize interchain disulfide bonds (Cys) in a single solid-phase process using an N-fluorenemethyloxycarbonyl (Fmoc) solid-phase polypeptide synthesis method. A24 -Cys B23 The folded refolding precursor of H2Relaxin derivatives with thioether bonds as substitutes was then efficiently obtained via one-step oxidative folding and refolding to yield interchain disulfide bonds (Cys). A24 -Cys B23 The H2Relaxin derivative is replaced by a thioether bond.

Description

Technical Field

[0001] This invention relates to an interchain disulfide bond Cys A24 -Cys B23 The H2 Relaxin derivative with thioether bonds as substitutes and its synthetic method belong to the field of peptide drug synthesis technology. Background Technology

[0002] Polypeptides possess characteristics such as high activity, high selectivity, and low toxicity, enabling them to play important regulatory roles as hormones, neurotransmitters, growth factors, and ion channel ligands, thus exhibiting significant clinical medicinal value. Examples include insulin for treating diabetes, oxytocin for promoting labor, and hepcidin for treating iron metabolism disorders.

[0003] H2 Relaxin is a member of the insulin superfamily and is a heterodimeric polypeptide containing 53 amino acids with two pairs of interchain disulfide bonds (Cys). A24 -Cys B23 Cys A11 -Cys B11 ) and a pair of intrachain disulfide bonds (Cys A10 -Cys A15 H2 Relaxin participates in regulating the physiological functions of many organs, exhibiting effects such as promoting vasodilation, angiogenesis, and cardioprotection. H2 Relaxin has entered phase III clinical trials as a treatment for acute heart failure. However, H2 Relaxin exhibits cross-activity with RXFP2, which is outside the drug target RXFP1 (relaxin family peptide receptor 1), and has a short in vivo half-life (10-20 minutes), making its drug modification urgent. Chemical synthesis is the preferred method for early-stage medicinal chemistry exploration and structure-function studies in most cases due to its faster derivative preparation speed and the ability to precisely control the structure of each amino acid at the atomic level. However, the difficult folding and refolding (disulfide bond pairing) of H2 Relaxin presents certain challenges for its chemical synthesis and drug modification.

[0004] The following are existing chemical synthesis methods for H2 Relaxin and its derivatives:

[0005] The article (J. Biol. Chem. 1991, 266: 10754-10761.) reports a strategy for sequentially constructing disulfide bonds based on orthogonal thiol protection. First, chains A and B, each containing two pairs of orthogonal thiol protecting groups, are synthesized. Folding and refolding are achieved through successive disulfide bond oxidation and orthogonal thiol deprotection, ultimately yielding the H2 Relaxin derivative. This method requires multiple chemical reactions and repeated separation and purification steps, making it complex, time-consuming, and inefficient.

[0006] The article (J. Pept. Sci. 2017, 23, 455-465) reports a synthetic strategy that mimics natural single chains. First, N-terminal aldehyde functional groups were synthesized. A-chain linker 1 and carbon-terminated hydroxylamine functional groups B-chain linker 2 Then, via oxime connection A-chain linker 1 The nitrogen end is covalently connected to B-chain linker 2 The carbon terminus forms a single-chain intermediate ( Chain A linker 1 -linker 2 -B chain After disulfide bond pairing is completed, the excess linker is removed through a chemical reaction. 1 -linker 2 The linker is removed, ultimately yielding full-length H2 Relaxin. This method requires the construction of a linker through multiple chemical reactions. 1 -linker 2 Furthermore, the removal of linkers is time-consuming and, under harsh conditions, can cause side reactions.

[0007] The article (Chem. Sci., 2016, 7, 3805-3819) reports the chemical synthesis of B-chain single-chain derivatives of H2 Relaxin. B7-33 can be obtained simply and efficiently via Fmoc solid-phase synthesis. However, due to significant sequence and structural differences from full-length H2 Relaxin, B7-33 exhibits poor activity. Summary of the Invention

[0008] To address the shortcomings of the existing technology, this invention provides an interchain disulfide bond (Cys). A24 -Cys B23 H2 Relaxin derivatives with thioether bonds as alternatives and their synthetic methods. This invention utilizes diaminodiacid molecules to link the AB chain of H2Relaxin via interchain disulfide bonds (Cys). A24 -Cys B23 The thioether bond analogues were pre-bound, thus enabling a single solid-phase synthesis of the H2 Relaxin derivative. Furthermore, disulfide bond pairing could be achieved through a single-step oxidative folding without the need for orthogonal thiol protection or linker assistance.

[0009] The interchain disulfide bond Cys of this invention A24 -Cys B23 The H2 Relaxin derivative with the thioether bond as a substitute, abbreviated as H2-1, has the following structural formula:

[0010]

[0011] The interchain disulfide bond Cys of this invention A24 -Cys B23The synthetic method for H2 Relaxin derivatives with thioether bonds as substitutes first involves synthesizing a C-terminal 6-peptide of the H2 Relaxin B chain using an N-fluorenemethyloxycarbonyl (Fmoc) solid-phase peptide synthesis method. Then, a diamino diacid molecule is condensed onto the 6-peptide, and the remaining 22 amino acids of the H2 Relaxin B chain are synthesized using the Fmoc solid-phase peptide synthesis method. After removing the allyloxycarbonyl protecting group from the diamino diacid, the remaining 23 amino acids of the H2 Relaxin A chain are synthesized using the Fmoc solid-phase peptide synthesis method. Finally, after TFA cleavage and purification, unfolded-H2-1 is obtained, and H2-1 is efficiently obtained through one-step folding and refolding. The specific steps include the following:

[0012] Step 1: Synthesis of Compound I

[0013] 1a. Take 0.1 mmol of Rink Amide AM resin (degree of substitution 0.32 mmol / g), add 5 mL of a mixed solution of dichloromethane (DCM) / N,N-dimethylformamide (DMF) (volume ratio 1:1), and allow the resin to swell for 30 minutes. Use a diaphragm pump as the power source to drain the mixed solution to obtain the swollen resin.

[0014] 1b. Amino acid condensation was performed using the standard Fmoc solid-phase polypeptide synthesis method (5 equivalents of Fmoc amino acids, 5 equivalents of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), N-hydroxy-7-azabenzotriazole (HOAt) and 10 equivalents of N,N-diisopropylethylamine (DIEA; the equivalents of other raw materials added in the following preparation process are all relative to the molar amount of the resin) and Fmoc-Ser(tBu)-OH and Fmoc-Trp(Boc)-OH were sequentially linked to the 1a resin to obtain compound I;

[0015] Step 2: Synthesis of Compound II

[0016] 2a. Add 2 mL of DMF solution containing 20% ​​piperidine to resin 1b and react for 5 minutes. Then rinse three times with DMF, DCM and DMF respectively. Repeat the above steps (react for 10 minutes) to complete the removal of Fmoc.

[0017] 2b. Add 1 mL of DMF solution containing twice the amount of pseudo-dipeptide Fmoc-Ser(tBu)-Thr(Psi(Me, Me)Pro)-OH and condensation reagent to resin 2a to allow the exposed carboxyl groups of the pseudo-dipeptide to undergo a condensation reaction with the exposed amino groups of resin 2a. Place the mixture in a shaker at room temperature and shake for 12 hours to obtain compound II.

[0018] In step 2b, the condensation reagents are (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tri-1-pyrrolidinyl hexafluorophosphate (PyAop), 1-hydroxy-7-azobenzotriazole (HOAt), and N-methylmorpholine (NMM), with the following reagent ratios: 2 molar amounts of PyAop, 2 molar amounts of HOAt, and 4 molar amounts of NMM.

[0019] Step 3: Synthesis of Compound III

[0020] Amino acid condensation was performed using the standard Fmoc solid-phase peptide synthesis method, and Fmoc-Met-OH and Fmoc-Gly-OH were sequentially linked onto 2b resin to obtain compound III;

[0021] Step 4: Synthesis of Compound IV

[0022] 4a. Add 2 mL of DMF solution containing 20% ​​piperidine to the resin in step 3 and react for 5 minutes. Then rinse three times with DMF, DCM and DMF respectively. Repeat the above steps (react for 10 minutes) to complete the removal of Fmoc and expose the amino group.

[0023] 4b. Add 1 mL of DMF solution containing 2 equivalents of diamino diacid molecules and condensation reagent to the resin of 4a, so that the exposed carboxyl groups of the pre-synthesized diamino diacid molecules and the exposed amino groups of the resin of 4a undergo a condensation reaction. Place the mixture in a shaker at room temperature and shake for 12 hours to obtain compound IV.

[0024] In step 4b, the condensation reagents are (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tri-1-pyrrolidinyl hexafluorophosphate (PyAop), 1-hydroxy-7-azobenzotriazole (HOAt), and N-methylmorpholine (NMM), with the following reagent ratios: 2 molar amounts of PyAop, 2 molar amounts of HOAt, and 4 molar amounts of NMM.

[0025] Step 5: Synthesis of Compound V

[0026] Amino acid condensation was performed using the standard Fmoc solid-phase peptide synthesis method, and Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, and Fmoc-Val-OH were sequentially linked onto 4b resin. Fmoc-Glu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Boc-Asp(OtBu)-OH were used to obtain compound V;

[0027] Step 6: Synthesis of Compound VI

[0028] 6a. Add 3 mL of DCM solution of 1 equivalent of Pd(PPh3)4 and 10 equivalent of PhSiH3 to the resin in step 5 to remove the Alloc protecting group of diamino diacid. Shake at room temperature for 3 hours, and then rinse with DMF, DCM and DMF in sequence.

[0029] 6b. Add 2 mL of DMF solution containing sodium diethyldithiocarbamate to the resin from 6a, shake at room temperature for 5 min, and then rinse sequentially with DMF, DCM, and DMF. Repeat the above steps several times until the black color on the resin disappears, obtaining compound VI;

[0030] Step 7: Obtaining compound unfolded-H2-1

[0031] 7a. Amino acid condensation was performed using the standard Fmoc solid-phase peptide synthesis method, and Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc- Gly-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Boc-Pyr-OH;

[0032] 7b. Add DMF and DCM sequentially to resin 7a and rinse three times each. Use a diaphragm pump as a power source to drain the washed product to obtain dry resin;

[0033] 7c. Add 6 mL of cleavage reagent to the resin in 7b and react at room temperature for 3 hours. Then transfer the cleavage solution to a centrifuge tube and concentrate it using nitrogen bubbling. Finally, when the cleavage solution is concentrated to less than 3 mL, add 40 mL of ice-cold ether, centrifuge (4500 rpm) to remove the supernatant, add another 40 mL of ice-cold ether, remove the supernatant, and air dry to obtain solid crude peptide.

[0034] In step 7c, the cutting reagent is a mixture of trifluoroacetic acid, phenol, water, anisole sulfide and ethylenedithiol, with a volume ratio of: trifluoroacetic acid: water: phenol: anisole sulfide: ethylenedithiol = 82.5:5:5:5:2.5.

[0035] 7d. Dissolve a small amount of the crude peptide solid obtained in step 7c in a pure aqueous solution containing 20% ​​acetonitrile. After filtration through a membrane, analyze using reversed-phase high-performance liquid chromatography (HPLC). The analyte concentration gradient is 20%-90% acetonitrile, and the time is 30 min. After chromatographic analysis, identify the main peak by ESI-MS to verify the correctness of unfolded-H2-1. After verification, purify the crude peptide solid obtained in step 7c using a C4 semi-preparative column (semi-preparative gradient of 20%-90% acetonitrile concentration, time 30 min). Collect the purified solutions, combine them, and freeze-dry to obtain a white solid unfolded-H2-1.

[0036] Step 8: Refold to obtain H2-1

[0037] 8a. Dissolve 6 mg of unfolded-H2-1 obtained in 7d in 100 mL of pure water containing 6.2 mg of oxidized glutathione (GSSH) and 31 mg of reduced glutathione (GSH). After refolding at room temperature for 10 min, purify the refolded solution using a C4 semi-preparative column (semi-preparative gradient of 1%-90% acetonitrile concentration, time 30 min). Collect the purified solution and verify the correctness of H2-1 by ESI-MS identification. After freeze-drying, obtain H2-1.

[0038] The synthetic route of this invention is shown below:

[0039]

[0040] The interchain disulfide bond Cys of this invention A24 -Cys B23 The activity (dose-response) assay of H2-1, an H2 Relaxin derivative with a thioether bond, against receptors RXFP1 and RXFP2 was conducted by the University of Science and Technology of China. The results showed that H2-1 had a potency (half-maximal effective concentration, EC50) against the drug target receptor RXFP1. 50 The titer of H2-1 against the non-drug target receptor RXFP2 was 1.3 ± 0.2 nM, consistent with that of natural H2 Relaxin (1.1 ± 0.1 nM); the titer of H2-1 against the non-drug target receptor RXFP2 was 53.9 ± 6.5 nM, lower than that of natural H2 Relaxin (20.1 ± 0.9 nM).

[0041] The beneficial effects of this invention are reflected in:

[0042] This invention designs an interchain disulfide bond Cys A24 -Cys B23 A full-length H2 Relaxin derivative (H2-1) with a thioether bond as a substitute and its synthetic method are presented. First, by loading a diamino diacid molecule, full-length unfolded-H2-1 can be obtained through a single Fmoc solid-phase peptide synthesis, followed by a one-step folding and refolding process to obtain H2-1. The entire synthetic process is carried out on a solid-phase resin, and only one folding and refolding step is required to complete the disulfide bond pairing, resulting in fewer steps, shorter processing time, simpler operation, and higher yield. Activity tests show that compared with natural H2 Relaxin, H2-1 retains the activity of the drug target receptor RXFP1 but has reduced activity against the non-drug target receptor RXFP1. Attached Figure Description

[0043] Figure 1 This is the high-performance liquid chromatogram of compound unfolded-H2-1.

[0044] Figure 2 This is the mass spectrum of compound unfolded-H2-1.

[0045] Figure 3 This is the high-performance liquid chromatogram of compound H2-1.

[0046] Figure 4 This is the mass spectrum of compound H2-1.

[0047] Figure 5 This is the dose-response curve of compound H2-1 to receptor RXFP1.

[0048] Figure 6 This is the dose-response curve of compound H2-1 to receptor RXFP2. Detailed Implementation

[0049] To facilitate understanding of the present invention, the implementation process of the present invention will be further described below with reference to specific embodiments. These descriptions are only for further illustrating the features and advantages of the present invention, and are not intended to limit the scope of the claims.

[0050] Example 1:

[0051] 315 mg (0.1 mmol) of Rink Amide AM resin (degree of substitution 0.32 mmol / g) was added to a solid-phase synthesis tube. 5 mL of a mixed solution of dichloromethane (DCM) / N,N-dimethylformamide (DMF) with a volume ratio of 1:1 was added to the synthesis tube to allow the resin to swell for 30 minutes. The mixed solution was then drained using a diaphragm pump to obtain the swollen resin.

[0052] Add 2 mL of DMF solution containing 20% ​​piperidine (volume fraction) to the swollen resin, and place it in a shaker at room temperature for 5 minutes. Then rinse three times each with DMF, DCM, and DMF. Add 2 mL of DMF solution containing 20% ​​piperidine to the resin again, and place it in a shaker at room temperature for 10 minutes. Then rinse three times each with DMF, DCM, and DMF to complete the removal of Fmoc. The first amino acid requiring condensation, Fmoc-Ser(tBu)-OH (191.72 mg, 5 eq, 0.5 mmol), and 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU, 206.845 mg, 5 eq, 0.5 mmol) were dissolved in 2 mL of DMF. N,N-diisopropylethylamine (DIEA, 165 μL, 10 eq, 1 mmol) was added, and the mixture was activated by shaking for 30 s. The mixture was then added to a solid-phase synthesis tube and reacted at room temperature for 1 hour. After the reaction, the mixture was washed three times each with DMF, DCM, and DMF. Then, Fmoc-Ser(tBu)-OH (191.72 mg, 5 eq, 0.5 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU, 190.12 mg, 5 eq, 0.5 mmol), were added to the solid-phase synthesis tube. 0.5 mmol. of N-hydroxy-7-azabenzotriazole (HOAt, 68.0 mg, 5 eq, 0.5 mmol.) was dissolved in DMF. After activation with DIEA (165 μL, 10 eq, 1 mmol.) for 30 seconds, the solution was added to the resin, and the mixture was placed in a shaker and reacted at room temperature for 1 hour. After the reaction, the solution was washed three times each with DMF, DCM, and DMF. Subsequent amino acid condensation was repeated using the above steps and reagent equivalents (the amino acid shaking condensation time was changed to 30 minutes).

[0053] Fmoc-Trp(Boc)-OH was further linked to the resin using the Fmoc solid-phase peptide synthesis method.

[0054] Add 2 mL of DMF solution containing 20% ​​piperidine to the swollen resin, place it in a shaker at room temperature and shake for 5 minutes, then wash three times with DMF, DCM and DMF respectively. Add 2 mL of DMF solution containing 20% ​​piperidine to the resin again, place it in a shaker at room temperature and shake for 10 minutes, then wash three times with DMF, DCM and DMF respectively to complete the removal of Fmoc. Dissolve the pseudo-dipeptide Fmoc-Ser(tBu)-Thr(Psi(Me,Me)Pro)-OH (104.92 mg, 2 eq, 0.2 mmol), (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tri-1-pyrrolidinyl hexafluorophosphate (PyAop, 104.278 mg, 2 eq, 0.2 mmol) and HOAt (27.22 mg, 2 eq, 0.2 mmol) in 1 mL of DMF. After activation with N-methylmorpholine (NMM, 45 μL, 4 eq, 0.4 mmol) for 30 s, the solution was added to the resin and reacted in a shaker at room temperature for 12 hours. After the reaction was completed, the solution was washed three times sequentially with DMF, DCM, and DMF.

[0055] Fmoc-Met-OH and Fmoc-Gly-OH were further linked to the resin using the Fmoc solid-phase polypeptide synthesis method.

[0056] Add 2 mL of DMF solution containing 20% ​​piperidine to the swollen resin, and shake in a shaker at room temperature for 5 minutes. Then wash three times each with DMF, DCM, and DMF. Add another 2 mL of DMF solution containing 20% ​​piperidine to the resin, and shake in a shaker at room temperature for 10 minutes. Then wash three times each with DMF, DCM, and DMF to complete the removal of Fmoc. Dissolve diamino diacid molecules (105.5 mg, 2 eq, 0.2 mmol), PyAop (104.2 mg, 2 eq, 0.2 mmol), and HOAt (27.22 mg, 2 eq, 0.2 mmol) in 1 mL of DMF. Activate with NMM (45 μL, 4 eq, 0.4 mmol) for 30 seconds, then add to the resin and shake in a shaker at room temperature for 12 hours. After the reaction, wash three times each with DMF, DCM, and DMF.

[0057] The following peptides were sequentially linked to resin using the Fmoc solid-phase peptide synthesis method: Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-Glu(OtBu)-OH, and Fmoc-Glu(OtBu)-OH. Fmoc-Met-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Boc-Asp(OtBu)-OH.

[0058] Tetra(triphenylphosphine)palladium (Pd(PPh3)4, 115.5 mg, 1 eq, 0.1 mmol) and phenylsilane (PhSiH3, 123.39 μL, 10 eq, 1 mmol) were dissolved in 3 mL of DCM solution and added to the synthesis tube. The reaction was carried out at room temperature with shaking for 3 hours, followed by washing three times each with DMF, DCM, and DMF, respectively.

[0059] Dissolve 400 mg of sodium diethyldithiocarbamate in 10 mL of DMF solution. Add 2 mL of DMF solution to the synthesis tube and react with the solution in a shaker at room temperature for 5 min. Then rinse the tube three times each with DMF, DCM, and DMF, respectively. Repeat the above steps several times until the black palladium reagent residue on the resin is removed.

[0060] The following peptides were sequentially linked to resin using the Fmoc solid-phase peptide synthesis method: Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Trt)-OH, and Fmoc-Ala-OH. Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, and Boc-Pyr-OH were used to complete the one-time solid-phase synthesis of H2 Relaxin derivatives on resins, in which the interchain disulfide bonds CysA24-CysB23 were replaced by thioether bonds.

[0061] Example 2:

[0062] DMF, DCM, DMF, and DCM were added sequentially to the resin obtained in Example 1, and the mixture was rinsed three times each. The washed resin was then dried using a diaphragm pump to obtain a dry resin.

[0063] 321 mg of phenol was dissolved in a mixed solution of 300 μL water, 300 μL anisole, and 150 μL ethylenedithiol. The mixture was then diluted to 6 mL with trifluoroacetic acid (TFA) to prepare the cutting solution. 6 mL of the cutting solution was added to a solid-phase synthesis tube containing dried resin, and the mixture was shaken in a shaker at room temperature for 3 hours. The cutting solution was transferred to a centrifuge tube, and the TFA was purged using nitrogen bubbling to concentrate the solution to less than 3 mL. 40 mL of ice-cold ether was added to the centrifuge tube to precipitate the crude peptide. The solution was centrifuged at 4500 rpm to allow the solid crude peptide to settle at the bottom. After removing the supernatant, another 40 mL of ice-cold ether was added, and the settled crude peptide was resuspended by sonication. After centrifugation again, the supernatant was removed, and the solid precipitate was air-dried to obtain unfolded-H2-1.

[0064] 20 mg of crude peptide was dissolved in 3 mL of pure aqueous solution containing 20% ​​acetonitrile (volume fraction) by sonication. After passing through a membrane, the solid crude peptide was separated and purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C4 semi-preparative column (semi-preparative gradient of 20%-90% acetonitrile concentration, time 30 min). The main peak solution was collected. The correctness of unfolded-H2-1 was verified by ESI-MS identification of the main peak.

[0065] Example 3:

[0066] 6.2 mg of oxidized glutathione (GSSH) and 31 mg of reduced glutathione (GSH) were dissolved in 100 mL of pure water to prepare the refolding solution. 6 mg of unfolded-H2-1 was dissolved in 100 mL of the refolding solution and mixed well. After adjusting the pH to 7.5-8, the solution was refolded at room temperature for 10 min. The refolding solution was then purified using a C4 semi-preparative process (a semi-preparative gradient of 1%-90% acetonitrile concentration for 30 min). The main peak was collected and identified by ESI-MS to verify the correctness of H2-1. The purified H2-1 solution was collected and lyophilized to obtain a white solid, H2-1.

[0067] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto, nor does the order of the various embodiments limit the present invention in any way. Any changes or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in the present invention should be included within the scope of protection of the present invention.

Claims

1. An interchain disulfide bond (Cys) A24 -Cys B23 The H2 Relaxin derivative with a thioether bond as a substitute, abbreviated as H2-1, is characterized by... Its structural formula is shown below: 。 2. A Cys interchain disulfide bond as described in claim 1 A24 -Cys B23 A method for synthesizing H2 Relaxin derivatives with thioether bonds as substitutes, characterized in that: First, the C-terminal 6-peptide of the H2 Relaxin B chain was synthesized using the N-fluorenemethyloxycarbonyl Fmoc solid-phase peptide synthesis method. Then, the diamino diacid molecule was condensed onto the 6-peptide, and the remaining 22 amino acids of the H2 Relaxin B chain were synthesized using the Fmoc solid-phase peptide synthesis method. After removing the allyloxycarbonyl protecting group from the diamino diacid, the remaining 23 amino acids of the H2 Relaxin A chain were synthesized using the Fmoc solid-phase peptide synthesis method, thus completing the single solid-phase synthesis of unfolded-H2-1. Finally, unfolded-H2-1 was obtained by TFA cleavage and purification, and then subjected to one-step oxidative folding refolding to efficiently obtain H2-1. Specifically, the steps include the following: Step 1: Synthesis of Compound I 1a. Add a 1:1 volume ratio of DCM and DMF mixed solution to Rink Amide AM resin to swell the resin. Use a diaphragm pump as a power source to remove the mixed solvent and obtain the swollen resin. 1b. Amino acid condensation was performed using the standard Fmoc solid-phase polypeptide synthesis method. Fmoc-Ser(tBu)-OH and Fmoc-Trp(Boc)-OH were sequentially linked to resin 1a to obtain compound I. Step 2: Synthesis of Compound II 2a. Add a DMF solution containing 20% ​​piperidine to resin 1b and react for 5 minutes. Then rinse with DMF, DCM and DMF in sequence. Repeat the above steps to complete the removal of Fmoc. 2b. Add a DMF solution containing a pseudo-dipeptide (Fmoc-Ser(tBu)-Thr(Psi(Me, Me)Pro)-OH) and a condensation reagent to resin 2a, so that the exposed carboxyl group of the pseudo-dipeptide and the exposed amino group of resin 2a undergo a condensation reaction. Place the mixture in a shaker at room temperature and shake for 12 hours to obtain compound II. Step 3: Synthesis of Compound III Amino acid condensation was performed using the standard Fmoc solid-phase peptide synthesis method, and Fmoc-Met-OH and Fmoc-Gly-OH were sequentially linked onto 2b resin to obtain compound III; Step 4: Synthesis of Compound IV 4a. Add a DMF solution containing 20% ​​piperidine to resin 3a and react for 5 minutes. Then rinse with DMF, DCM and DMF in sequence. Repeat the above steps to complete the removal of Fmoc. 4b. Add a DMF solution containing diamino diacid molecules and a condensation reagent to resin 4a to allow the exposed carboxyl groups of the diamino diacid molecules to undergo a condensation reaction with the exposed amino groups of resin 4a. Place the mixture in a shaker at room temperature and shake for 12 hours to obtain compound IV. Step 5: Synthesis of Compound V Amino acid condensation was performed using the standard Fmoc solid-phase peptide synthesis method, and Fmoc-Ile-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ala-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc- Arg(Pbf)-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ile-OH, Fmoc-Val-OH, Fmoc-G lu(OtBu)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Met-OH, Fmoc-Trp(Boc)-OH, Fmoc-Ser(tBu)-OH, Boc-Asp(OtBu)-OH, to obtain compound V; Step 6: Synthesis of Compound VI 6a. Add a DCM solution of tetra(triphenylphosphine)palladium Pd(PPh3)4 and benzenesilane PhSiH3 to resin 5a to remove the amino protecting group of diaminodioic acid. Shake at room temperature for 3 hours, and then rinse with DMF, DCM and DMF in sequence. 6b. Add a DMF solution containing sodium diethyldithiocarbamate to the resin in 6a, shake at room temperature for 5 min, and then rinse with DMF, DCM and DMF in sequence; repeat the above steps several times until the black color of the resin fades to obtain compound VI; Step 7: Obtaining compound unfolded-H2-1 7a. Amino acid condensation was performed using the standard Fmoc solid-phase peptide synthesis method, and Fmoc-Phe-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Cys(Trt)-OH, and Fmoc- Gly-OH, Fmoc-Val-OH, Fmoc-His(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asn(Tr t)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Leu-OH, Boc-Pyr-OH; 7b. Add DMF, DCM, DMF and DCM sequentially to the resin in 7a and rinse. Use a diaphragm pump as a power source to dry the washed product to obtain a dry resin. 7c. Add the cleavage reagent to the resin in 7b and react at room temperature for 3 hours. Then transfer the cleavage solution to a centrifuge tube and concentrate the cleavage solution using nitrogen bubbling. Finally, add ice-cold ether, centrifuge to remove the supernatant, add ice-cold ether again, centrifuge again to remove the supernatant, and air dry to obtain solid crude peptide. After separation and purification, obtain unfolded-H2-1. Step 8: Refold to obtain H2-1 The unfolded-H2-1 obtained in 7 days was dissolved in the folding and refolding solution, refolded at room temperature for 10 min, and then separated and purified to obtain H2-1; In step 2b, the condensation reagents are (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tri-1-pyrrolidinyl hexafluorophosphate PyAop, 1-hydroxy-7-azobenzotriazole HOAt, and N-methylmorpholine NMM, with the following reagent ratios: 2 molar amounts of PyAop, 2 molar amounts of HOAt, and 4 molar amounts of NMM relative to the resin. In step 4b, the condensation reagents are (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tri-1-pyrrolidinyl hexafluorophosphate PyAop, 1-hydroxy-7-azobenzotriazole HOAt, and N-methylmorpholine NMM, with the following reagent ratios: 2 molar amounts of PyAop, 2 molar amounts of HOAt, and 4 molar amounts of NMM relative to the resin. In step 8, the refolding solution is a pure aqueous solution containing oxidized glutathione (GSSG) and reduced glutathione (GSH), with the reagent ratio as follows: 10 equivalents of GSSG and 100 equivalents of GSH.

3. The synthesis method according to claim 2, characterized in that: In step 6a, the amino protecting group of the diaminodica acid is allyloxycarbonyl (Alloc).

4. The synthesis method according to claim 2, characterized in that: In step 7c, the cutting reagent is a mixture of trifluoroacetic acid, phenol, water, anisole sulfide and ethylenedithiol, with a volume ratio of: trifluoroacetic acid: water: phenol: anisole sulfide: ethylenedithiol = 82.5:5:5:5:2.5.

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