CN111303272A - Disulfide analogue of pramlintide and preparation method thereof - Google Patents
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Abstract
The invention discloses a disulfide analogue of pramlintide and a preparation method thereof, and the disulfide analogue of pramlintide which is stable under a reducing condition is obtained by replacing a disulfide bond of pramlintide which is a polypeptide drug. The target polypeptide is synthesized by Fmoc solid phase synthesis, and the crude product is purified and lyophilized to obtain the disulfide analogue of pramlintide. Reduction stability experiments show that the disulfide analogue of pramlintide has good reduction stability.
Description
Technical Field
The invention relates to a disulfide analogue of pramlintide and a preparation method thereof.
Background
Diabetes is a common endocrine disease, and more than 1 million people suffer from diabetes in China. Diabetes is a chronic disease characterized by hyperglycemia and has painful daily symptoms including fatigue, blurred vision, thirst, frequent urination, and the like. If hyperglycemia is not controlled and treated in a timely manner, diabetic ketoacidosis and hypertonic hyperglycemia will result, which are the 2 most serious acute metabolic complications of diabetes.
Pramlintide, an amylin analog, is an effective therapeutic approach to regulate blood glucose balance in patients in conjunction with insulin. Pramlintide contains 37 amino acids and a pair of disulfide bonds, and has a half-life of about 38 minutes. Research shows that the disulfide bond plays an important role in maintaining the three-dimensional space structure of pramlintide. However, disulfide bonds are unstable under in vivo reducing conditions and are readily reduced by thiols, resulting in structural rearrangement of the polypeptide and loss of biological activity. Therefore, it is important to find a stable alternative bond to the disulfide bond.
Disclosure of Invention
Aiming at the potential defect of application of disulfide bond of pramlintide in-vivo reduction environment, the invention aims to provide a disulfide analogue of pramlintide and a preparation method thereof. The invention utilizes diamino diacid Fmoc-A2(all/Alloy) -OH replaces the labile disulfide bond of pramlintide to the reducing system with a stable thioether bond. Reduction stability experiments show that the disulfide analogue of pramlintide has good reduction stability.
The disulfide analog of pramlintide is obtained by replacing pramlintide disulfide bond with diamino diacid.
The diamino diacid is Fmoc-A2(all/Alloy) -OH, having the formula:
the disulfide analogue of pramlintide has the following structure:
the preparation method of the disulfide analogue of pramlintide comprises the following steps:
step 1: synthesis of hydrazide resins
37.5mg of 2-CI-Trt resin (loading 0.40mmol/g, 15. mu. mol) was swollen with 375. mu.L of DMF and 50. mu.L of DIEA (300. mu. mol), 15. mu.L of N were added dropwise with stirring in a ice-salt bath2H4·H2And reacting the mixture of O (300umol) and 150uL DMF for 20 minutes, then placing the reaction system at room temperature for further reaction for 70 minutes, adding 7.5uL MeOH (185umol) for reaction for 20 minutes, washing the obtained resin with DMF, MeOH and diethyl ether for 5 times in sequence, and drying the resin under vacuum at 55 ℃ for 1 hour to obtain the hydrazide resin for synthesizing the polypeptide hydrazide.
Step 2: synthesis of hydrazide polypeptide by Fmoc solid phase synthesis method
2a, transferring the hydrazide resin obtained in step 1 into a solid phase synthesis tube, swelling with DMF for 15 minutes, adding a DMF mixture of DIC, Oxyma, and the 1 st amino acid at the C-terminal of the hydrazide polypeptide (4.5 times equivalent DIC:4.5 times equivalent Oxyma: 4.5 times equivalent protected 1 st amino acid at the C-terminal of the target peptide, 500uL DMF), reacting at 55 ℃ for 40 minutes, washing the resin with DMF, subsequently soaking the resin with a blocking reagent (acetic anhydride: 2, 6-lutidine: DMF 5:6:89, V%) for 2 minutes, and washing the resin with DMF again. The resin was soaked for 8 min and 6 min with the addition of 20% piperidine in DMF, respectively. After washing with DMF in bulk, the amino acids Fmoc-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ala-OH, Fmoc-A were condensed in sequence by Fmoc solid phase synthesis2(aly/Alloy) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ala-OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (Trt) -OH and Fmoc-Lys (Boc) -OH. Fmoc-A for a particular amino acid2(Ally/Alloy) -OH condensation, preparation of a mixture (2 times equivalent PyBOP: 2 times equivalent Oxyma: 500uL DMF:1.5 times equivalent Fmoc-A)2(all/Alloy) -OH) was added to the resin and reacted overnight at ambient temperature. Pd (PPh) is prepared after the completion of Fmoc-Asn (Trt) -OH condensation of 10 th amino acid at C-terminal of hydrazide polypeptide3)4THF mixture of N-methylaniline (1-fold equivalent of Pd (PPh)3)428 times equivalent of N-methylaniline: 500uL THF), adding into resin, keeping out of the sun, and reacting for 2 hours at normal temperature; the obtained resin is sequentially prepared with DMF and 0.5% NaS prepared from DMF2CN(C2H5)2The solution was washed with DCM and DMF. Adding 20% piperidine DMF solution to soak the resin for 8 min and 6 min respectivelyDMF washes the resin. DMF mixture of PyAOP, HOAT and NMM (5 times equivalent PyAOP: 5 times equivalent HOAT: 10 times equivalent NMM:500uL DMF) was prepared, added to the resin and reacted for 4 hours, and after the reaction was completed, the obtained resin was washed with a large amount of DMF. The amino acid Fmoc-Lys (Boc) -OH is condensed sequentially by adopting Fmoc solid phase synthesis method.
After the final amino acid Fmoc-Lys (Boc) -OH condensation of the hydrazide polypeptide was completed, the resin was washed with DMF and DCM in bulk and dried naturally, followed by treatment with additional cleavage reagent (TFA: phenol: water: TIPS ═ 88:5: 2, V%) 0.5mL-0.8mL for 2 hours. The cleavage reagent was collected, precipitated by adding 15 volume equivalents of glacial ethyl ether, powdered nascent peptide was centrifuged, and hydrazide polypeptide was purified by semi-preparative HPLC. The product was confirmed by analytical HPLC and mass spectrometry (FIGS. 3a, 3b), and lyophilized in vacuo to give the purified hydrazide polypeptide H-Lys-Cys-Asn-Thr-Ala-Thr-Gln-Arg-Leu-NHNH2(Cys2-Ala7, thioether bond).
And step 3: fmoc solid phase synthesis method for synthesizing cysteine polypeptide
3a, 45.5mg Rink Amide-AM resin (loading 0.33mmol/g, 15. mu. mol) was transferred to a solid phase synthesis tube and swollen with DMF for 15 minutes, the resin was soaked with 20% piperidine in DMF for 8 minutes and 6 minutes, and the resin was washed with bulk DMF. A DMF mixture of DIC, Oxyma and the first amino acid at the C-terminus of the cysteine polypeptide was prepared (4.5 Xeq DIC:4.5 Xyma: 500uL DMF:4.5 Xeq protected first amino acid at the terminus of the target peptide), added to the resin and reacted at 55 ℃ for 40 min, and the resin was washed with copious amounts of DMF. The blocking reagent (acetic anhydride: 2, 6-lutidine: DMF ═ 5:6:89, V%) soaked the resin for 2 minutes, and the resin was washed again with copious amounts of DMF. Adding 20% piperidine in DMF solution, soaking the resin for 8 min and 6 min, washing the resin with DMF, condensing amino acids Fmoc-Thr (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Ser (tBu) -OH, Fmoc-His (Trt) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Cys (Trt) -OH.
3b, after the condensation of the last amino acid Fmoc-Cys (Trt) -OH of the cysteine polypeptide is finished, washing the resin with a large amount of DMF and DCM, naturally drying, and then adding a cutting reagent (TFA: phenol: water: TIPS ═ 88:5: 2, V%) for treating for 2 hours in a range of 0.5mL to 0.8 mL; collecting a cutting reagent, adding 15 times of ice ether with equivalent volume for precipitation, and centrifuging powdery primary peptide; semi-preparative HPLC purified the target peptide. Analyzing HPLC and mass spectrum to confirm the product (FIG. 4b,4c), vacuum freeze drying to obtain purified cysteine polypeptide H-Cys-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH2。
And 4, step 4: natural splicing of hydrazide and cysteine polypeptides
Weighing 1.6mg of the hydrazide polypeptide H-Lys-Cys-Asn-Thr-Ala-Thr-Ala-Ala-Thr-Gln-Arg-Leu-NHNH obtained in step 22(Cys2-Ala7, thioether bond) was dissolved in 0.5mL of PBS (6.0M guanidine hydrochloride, 0.2M phosphate, pH3.0), magnetons were added, and 120uL of NaNO was added dropwise with stirring in an ice salt bath (about-10 ℃ C.)2(0.1M) aqueous solution, and reacting at low temperature for 20 minutes; after the reaction was completed, 200uL of MESNa guanidine hydrochloride solution (0.6M MESNa,6.0M guanidine hydrochloride, 0.2M phosphate, pH7.0) was added dropwise to the system, and 3.8mg of the cysteine polypeptide H-Cys-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH-obtained in step 3 was added2The ice salt bath was removed, the pH of the reaction solution was carefully adjusted to neutral by NaOH (2.0M), after 2 days of reaction, HPLC detection was performed to isolate a new peak, and the ligation product was determined by analytical HPLC and mass spectrometry (FIGS. 6a,6 b).
And 5: desulfurization of natural splice products
Weighing 1mg of the natural splicing peptide H-Lys-Cys-Asn-Thr-Ala-Thr-Ala-Ala-Thr-Gln-Arg-Leu-Cys-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH obtained in the step 42(Cys2-Ala7, thioether bond) was dissolved in 1mL of PBS (6.0M guanidine hydrochloride, 0.2M phosphate, pH7.0), magnetons were added, 143mg of TCEP. HCl was added under stirring at 37 ℃ to adjust the pH of the reaction solution to 6.9, followed by 50uL of 2-methyl-2-propanethiol and 500uL of a radical initiator VA044(0.1M aqueous solution), adjusting the pH of the reaction solution to 6.9; after 6 hours of reaction, HPLC detection separated a new peak and analytical HPLC and mass spectrometry identified the desulfurization product, i.e., pramlintide disulfide analog (fig. 7a,7 b).
Compared with the existing pramlintide, the pramlintide disulfide analog improves the reduction stability through reduction stability experiments, has only one atom difference in structure, and avoids larger interference in structure.
Drawings
FIG. 1 is a schematic representation of a structural comparison of pramlintide and pramlintide disulfide analogues.
FIG. 2 is a schematic diagram of the synthesis of the hydrazide polypeptides of the invention.
FIG. 3 is an HPLC chart (a) and a mass spectrum chart (b) of the hydrazide polypeptide of the invention.
FIG. 4 shows the structure (a), HPLC (b) and mass spectrum (c) of the cysteine polypeptide of the present invention.
FIG. 5 is a schematic diagram of pramlintide disulfide analog synthesis.
FIG. 6 is an HPLC (a) and mass spectrum (b) of the naturally spliced product of a hydrazide polypeptide and a cysteine polypeptide.
FIG. 7 is an HPLC (a) and mass spectrum (b) of pramlintide disulfide analog.
FIG. 8 is a pramlintide and pramlintide disulfide analog reduction stability test.
Detailed description of the invention
The following is further illustrated with reference to the examples:
the meanings of the abbreviations used in the present invention are listed in the following table:
example 1: synthesis of hydrazide resins
37.5mg of 2-CI-Trt resin (loading 0.40mmol/g, 15. mu. mol) were swollen with 375uL DMF, washed with ice salt and stirredUnder the condition (2), 50uL DIEA (300umol) and 15uL N were added dropwise2H4·H2And reacting the mixture of O (300umol) and 150uL DMF for 20 minutes, then placing the reaction system at room temperature for further reaction for 70 minutes, adding 7.5uL MeOH (185umol) for reaction for 20 minutes, washing the obtained resin with DMF, MeOH and diethyl ether for 5 times in sequence, and drying the resin under vacuum at 55 ℃ for 1 hour to obtain the hydrazide resin for synthesizing the polypeptide hydrazide.
Example 2: synthesis of hydrazide polypeptide by Fmoc solid phase synthesis method
2a, the hydrazide resin obtained in example 1 was transferred to a solid phase synthesis tube, swollen with DMF for 15 min, added with a DMF mixture of DIC, Oxyma, the 1 st amino acid at the C-terminus of the hydrazide polypeptide (4.5 fold equivalent DIC:4.5 fold equivalent Oxyma: 4.5 fold equivalent protected 1 st amino acid at the C-terminus of the target peptide, 500uL DMF), reacted at 55 ℃ for 40 min, the resin was washed with DMF, followed by soaking the resin with a blocking reagent (acetic anhydride: 2, 6-lutidine: DMF 5:6:89, V%) for 2 min, and again washing the resin with DMF. The resin was soaked for 8 min and 6 min with the addition of 20% piperidine in DMF, respectively. After washing with DMF in bulk, the amino acids Fmoc-Arg (Pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ala-OH, Fmoc-A were condensed in sequence by Fmoc solid phase synthesis2(all/Alloy) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Ala-OH, Fmoc-Thr (tBu) -OH, Fmoc-Asn (Trt) -OH. Fmoc-A for a particular amino acid2(Ally/Alloy) -OH condensation, preparation of a mixture (2 times equivalent PyBOP: 2 times equivalent Oxyma: 500uL DMF:1.5 times equivalent Fmoc-A)2(all/Alloy) -OH) was added to the resin and reacted overnight at ambient temperature. Pd (PPh) is prepared after the completion of Fmoc-Asn (Trt) -OH condensation of 10 th amino acid at C-terminal of hydrazide polypeptide3)4THF mixture of N-methylaniline (1-fold equivalent of Pd (PPh)3)428 times equivalent of N-methylaniline: 500uL THF), adding into resin, keeping out of the sun, and reacting for 2 hours at normal temperature; the obtained resin is sequentially prepared with DMF and 0.5% NaS prepared from DMF2CN(C2H5)2The solution was washed with DCM and DMF. The resin was soaked separately for 8 min and 6 min with the addition of 20% piperidine in DMF and the resin was washed with DMF. DMF mixture of PyAOP, HOAT and NMM was prepared (5 times equivalent PyAOP: 5 times equivalent HOAT: 10 times equivalent NMM:500 uL)DMF) was added to the resin and reacted for 4 hours, and after the reaction was completed, the resulting resin was washed with a large amount of DMF. The amino acid Fmoc-Lys (Boc) -OH is condensed sequentially by adopting Fmoc solid phase synthesis method.
After completion of Fmoc-Lys (Boc) -OFmoc solid phase condensation of the last amino acid of the hydrazide polypeptide, the resin was washed with a large amount of DMF and DCM, and after drying naturally, treated with additional cleavage reagent (TFA: phenol: water: TIPS ═ 88:5:5:2, V%) for 2 hours in a range of 0.5mL to 0.8 mL. The cleavage reagent was collected, precipitated by adding 15 volume equivalents of glacial ethyl ether, powdered nascent peptide was centrifuged, and hydrazide polypeptide was purified by semi-preparative HPLC. The product was confirmed by analytical HPLC and mass spectrometry (FIGS. 3a, 3b), and lyophilized in vacuo to give the purified hydrazide polypeptide H-Lys-Cys-Asn-Thr-Ala-Thr-Gln-Arg-Leu-NHNH2(Cys2-Ala7, thioether bond).
Example 3: fmoc solid phase synthesis method for synthesizing cysteine polypeptide
3a, 45.5mg Rink Amide-AM resin (loading 0.33mmol/g, 15. mu. mol) was transferred to a solid phase synthesis tube and swollen with DMF for 15 minutes, the resin was soaked with 20% piperidine in DMF for 8 minutes and 6 minutes, and the resin was washed with bulk DMF. A DMF mixture of DIC, Oxyma and the first amino acid at the C-terminus of the cysteine polypeptide was prepared (4.5 Xeq DIC:4.5 Xyma: 500uL DMF:4.5 Xeq first amino acid at the C-terminus of the cysteine polypeptide), added to the resin and reacted at 55 ℃ for 40 min, and the resin was washed with copious amounts of DMF. The blocking reagent (acetic anhydride: 2, 6-lutidine: DMF ═ 5:6:89, V%) soaked the resin for 2 minutes, and the resin was washed again with copious amounts of DMF. Adding 20% piperidine in DMF solution, soaking the resin for 8 min and 6 min, washing the resin with DMF, condensing amino acids Fmoc-Thr (tBu) -OH, Fmoc-Asn (Trt) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-Pro-OH, Fmoc-Pro-OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Ser (tBu) -OH, Fmoc-His (Trt) -OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Cys (Trt) -OH.
3b, last amino acid F of polypeptide to be cysteineAfter the condensation with moc-Cys (Trt) -OH was completed, the resin was washed with a large amount of DMF and DCM, and after drying naturally, 0.5mL-0.8mL of a cleavage reagent (TFA: phenol: water: TIPS ═ 88:5: 2: V%) was added for treatment for 2 hours. The cleavage reagent was collected, precipitated with 15 volume equivalents of glacial ethyl ether, and the powdered primary peptide was centrifuged and semi-preparative HPLC purified to the target peptide. Analyzing HPLC and mass spectrum to confirm the product (FIG. 4b,4c), vacuum freeze drying to obtain purified cysteine polypeptide H-Cys-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH2。
Example 4: natural splicing of hydrazide and cysteine polypeptides
1. 1.6mg of the hydrazide polypeptide H-Lys-Cys-Asn-Thr-Ala-Thr-Ala-Ala-Thr-Gln-Arg-Leu-NHNH obtained in example 2 was weighed2(Cys2-Ala7, thioether bond) was dissolved in 0.5mL PBS (6.0M guanidinium hydrochloride, 0.2M phosphate, pH 3.0).
2. Adding magneton, and adding 120uL NaNO dropwise into ice salt bath (about-10 deg.C) under stirring2(0.1M) aqueous solution, and reacting at low temperature for about 20 minutes.
3. After the oxidation was completed, 200uL of MESNa guanidine hydrochloride solution (0.6M MESNa,6.0M guanidine hydrochloride, 0.2M phosphate, pH7.0) was added dropwise, and 3.8mg of the cysteine polypeptide H-Cys-Asn-Phe-Leu-Val-His-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH obtained in example 3 was added2The ice salt bath was removed, and the pH of the reaction solution was carefully adjusted to neutral with NaOH (2.0M).
4. After 2 days of reaction, HPLC detection separated a new peak, analytical HPLC and mass spectrometry confirmed the ligation product (FIGS. 6a,6 b).
Example 5: desulfurization of natural splice products
1. Weighing 1mg of the natural splicing peptide H-Lys-Cys-Asn-Thr-Ala-Thr-Ala-Ala-Thr-Gln-Arg-Leu-Cys-Asn-Phe-Leu-Val-His-Ser-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH obtained in example 42(Cys2-Ala7, thioether bond) was dissolved in 1mL PBS (6.0M guanidinium hydrochloride, 0.2M phosphate, pH 7.0).
2. Magnetitum was added, and 143mg of TCEP & HCl was added thereto in an oil bath (37 ℃ C.) with stirring to adjust the pH of the reaction mixture to 6.9.
3. 50uL of 2-methyl-2-propanethiol and 500uL of a radical initiator VA-044(0.1M aqueous solution) were added to adjust the pH of the reaction mixture to 6.9.
4. After 6 hours of reaction, HPLC analysis separated a new peak, analytical HPLC and mass spectrometry determined the desulfurization product H-Lys-Cys-Asn-Thr-Ala-Thr-Gln-Arg-Leu-Ala-Asn-Phe-Leu-Val-His-Ser-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH2(Cys2-Ala7, thioether bond), pramlintide disulfide analog (FIGS. 7a,7 b).
Example 6: pramlintide and pramlintide disulfide analogue reduction stability comparison test
Pramlintide and pramlintide disulfide analogs 100ug each was weighed and dissolved in 100uL of DTT buffer (20mM Tris-HCl, pH 7.4,150mM NaCl,1mM CaCl)2,1mM MgCl20.1% Triton X-100,3mM DTT). Changes in the percentage content of pramlintide and pramlintide disulfide analogues were monitored by HPLC at 0,0.5,1,2,4, and 8 hours, respectively, under incubation conditions at 25 ℃ (fig. 8). From figure 8 we can see that pramlintide disulfide analogues remain substantially stable while most of the pramlintide is reduced after 8 hours in a buffered solution of DTT. Indicating that the pramlintide disulfide analog is more stable than pramlintide in a reducing environment.
Claims (3)
3. a process for the preparation of a dithio analog of pramlintide according to claim 1 or 2, characterized by comprising the steps of:
step 1: synthesis of hydrazide resins
Swelling 2-CI-Trt resin with DMF, and dropwise adding DIEA and N in a salt bath under the condition of stirring2H4·H2Reacting the mixed solution of O and DMF, then placing the reaction system at room temperature for continuous reaction, and then adding MeOH for reaction to obtain hydrazide resin for synthesizing hydrazide polypeptide;
step 2: synthesis of hydrazide polypeptide by Fmoc solid phase synthesis method
Transferring the hydrazide resin obtained in the step (1) into a solid phase synthesis tube, sequentially carrying out condensation connection on amino acids by adopting an Fmoc solid phase synthesis method after DMF is swelled, removing resin peptide Alloc and aly protecting groups after the 10 th amino acid Fmoc-Asn (Trt) -OH condensation at the C end of the hydrazide polypeptide is finished, carrying out intramolecular cyclization after the Fmoc protecting groups, and continuously condensing the amino acids by adopting the Fmoc solid phase synthesis method according to the peptide sequence; after the solid phase synthesis is finished, a cutting reagent is added for reaction for 2 hours, and after the glacial ethyl ether precipitation and purification, the hydrazide polypeptide H-Lys-Cys-Asn-Thr-Ala-Thr-Gln-Arg-Leu-NHNH is obtained2(Cys2-Ala7, thioether bond);
and step 3: fmoc solid phase synthesis method for synthesizing cysteine polypeptide
Transferring Rink Amide-AM resin into a solid-phase synthesis tube, performing condensation connection of amino acids according to peptide sequence by adopting an Fmoc solid-phase synthesis method after DMF swelling, adding a cutting reagent for reaction for 2 hours after the Fmoc solid-phase synthesis of the last amino acid of the cysteine polypeptide is finished, and obtaining the cysteine polypeptide H-Cys-Asn-Phe-Leu-Val-His-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH after precipitation and purification of ethyl acetate2;
And 4, step 4: natural splicing of hydrazide and cysteine polypeptides
Step 2Dissolving the obtained hydrazide polypeptide in PBS solution, adding NaNO2Carrying out reaction on the aqueous solution; after the reaction is finished, adding MESNa guanidine hydrochloride solution and the cysteine polypeptide obtained in the step 3 into the system, adjusting the PH of the reaction solution to be neutral, and obtaining a connecting product-natural splicing peptide H-Lys-Cys-Asn-Thr-Ala-Thr-Gln-Arg-Leu-Cys-Asn-Phe-Leu-Val-His-Ser-Asn-Asn-Phe-Gly-Pro-Ile-Leu-Pro-Thr-Asn-Val-Gly-Ser-Asn-Thr-Tyr-NH2;
And 5: desulfurization of natural splice products
And (3) dissolving the connecting product obtained in the step (4) in a PBS solution, adding TCEP & HCl, adjusting the pH of the reaction solution to 6.9, then adding 2-methyl-2-propyl mercaptan and a free radical initiator VA-044, adjusting the pH of the reaction solution to 6.9, and reacting to obtain the target product.
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Publication number | Priority date | Publication date | Assignee | Title |
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CN113527517A (en) * | 2021-07-16 | 2021-10-22 | 苏州大学 | Method for synthesizing U. hensis probe |
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Application publication date: 20200619 |