Synthesis method of salmon calcitonin acetylation impurity

Abstract

The invention provides a synthesis method of salmon calcitonin acetylation impurities. The method comprises the following steps: 1) coupling amino resin with corresponding amino acid to obtain linear peptide resin; 2) performing solid-phase oxidation on the peptide resin to form disulfide bonds; 3) acetylation modification of peptide resin; and 4) cracking the peptide resin to obtain the corresponding salmon calcitonin impurity crude peptide. Firstly, according to the method, a solid-phase synthesis method is adopted, corresponding impurity crude peptide can be directly obtained after cracking, and the cost is reduced; secondly, the method is simple to operate, and a series of acetylated impurities can be prepared at the same time; and finally, the crude peptide synthesized by the method is relatively high in purity, and the purification difficulty is reduced.

Description

Technical Field

[0001] The invention relates to the technical field of drug synthesis, and in particular to a method for synthesizing salmon calcitonin acetylated impurities. Background Art

[0002] Salmon calcitonin, also known as salmon calcitonin, is a synthetic polypeptide preparation derived from salmon. It is the most active calcitonin currently, and its calcium-lowering effect is 10 to 40 times greater than that of human calcitonin. Salmon calcitonin can significantly reduce bone calcium loss in high-turnover bone diseases, such as osteoporosis, deforming bone disease (Paget's disease), painful neuropathy (Sudeck's disease) and malignant osteolysis. It has a more significant effect on the trunk bones of postmenopausal osteoporosis than on the limb bones, and on high-turnover bone diseases than low-turnover bone diseases. Salmon calcitonin can inhibit osteoclast activity and stimulate osteoblast formation and activity. Calcitonin can also increase urinary calcium, phosphorus and sodium excretion by inhibiting osteolysis and reducing tubular reabsorption, but serum calcium will not drop below the normal range. This drug also inhibits the secretory activity of the stomach and pancreas, but does not affect gastrointestinal motility. There is evidence that salmon calcitonin has an analgesic effect on patients with certain painful bone diseases. All calcitonins are similar in structure, with a single chain and 32 amino acids arranged differently, the order of the amino acids depends on the species, and their effects are basically the same. Salmon calcitonin and human calcitonin have 16 different amino acids out of the 32, but their effects and applications for Paget's disease of bone are the same. This drug has a high affinity for its receptor binding sites present in certain areas of the central nervous system, and the duration of action is longer than that of synthetic mammalian calcitonin. The structural formula is as follows:

[0003]

[0004] Acetylated impurities are common impurities in the production of peptide drugs and are often used as internal control impurities (N-acetyl-cysteinyl 1 -Salmon calcitonin is controlled as a related substance), but there is no report on the acetylated impurities of salmon calcitonin. In view of the above problems existing in the prior art, it is necessary to invent a method for synthesizing the acetylated impurities of salmon calcitonin. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for synthesizing salmon calcitonin acetylated impurities.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The invention provides a method for synthesizing salmon calcitonin acetylated impurities, which comprises the following steps: 1) coupling an amino resin with corresponding amino acids to obtain a linear peptide resin; 2) solid phase oxidation of the peptide resin to form a disulfide bond; 3) acetylation modification of the peptide resin; and 4) cleavage of the peptide resin to obtain a corresponding salmon calcitonin impurity crude peptide.

[0008] Furthermore, the step 1) specifically includes: washing the amino resin with a solvent and performing a swelling treatment with the solvent; then, adding a de-Fmoc reagent to perform a de-Fmoc reaction; weighing the amino acid raw material and the activation reagent, dissolving them with a solvent, and cooling to below 10° C. to perform an activation reaction; repeating the above operations to couple one by one to obtain a linear peptide resin.

[0009] Furthermore, in the step 1), the Fmoc strategy coupling method is used to sequentially couple the following amino acid raw materials: Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc- oc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Boc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ser-OH, Fmoc-Thr-OH, Fmoc-Lys(Alloc)-OH, the coupling time is 1-3 hours, preferably 2 hours.

[0010] Furthermore, in the step 1), the amino resin includes RinkAmide Resin, RinkAmideAM Resin, Sieber Resin and RinkAmide MBHAResin, and the amino resin has a substitution degree of 0.2 to 0.4 mmol / g.

[0011] Further, the solvent used in the step 1) is one or a combination of DMF, DMAc, DCM, NMP, and DMSO, preferably DMF; the de-Fmoc reagent in the step 1) is any one of 10-30% PIP / DMF, 10-30% PIP / DCM, 10-30% PIP / NMP, and 10-30% PIP / DMAc, preferably 20% PIP / DMF; the synthesis method in the step 1) is: the activation reagent is any one of HOBT / DIC, Oxyma Pure / DIC, HOAT / DIC, PyBOP / DIPEA, HBTU / DIC, HATU / DIC, HOBT / DIPEA, HOAT / DIPEA, HBTU / DIPEA, HATU / DIPEA, TBTU / DIPEA, TBTU / DIC, and PyAOP / DIPEA, preferably DIC / HOBt.

[0012] Furthermore, in the step 2), the oxidation system used for solid phase oxidation of the peptide resin to form disulfide bonds is I2 / DMF.

[0013] Furthermore, the step 3) specifically includes: weighing the reagent for the acetylation reaction, dissolving it in a reaction solvent, and then adding it to the peptide resin obtained in the step 2) to react.

[0014] Further, in the step 3), the reagent for the acetylation reaction is one or a combination of two of Ac2O, Ac2O / Py, CH3COOH / DIC, CH3COOH / DIC / DMAP, CH3COCl / TEA, and CH3COCl / DIPEA; in the step 3), the reaction solvent is any one or more of DCM, DMF, NMP, and DMSO, preferably DMF and DCM.

[0015] Furthermore, the step 4) specifically includes: collecting the peptide resin obtained in the step 3), cleaving it with a pre-cooled cleavage solution, filtering the resin, adding a pre-cooled sedimentation solution for sedimentation, centrifuging, washing the obtained solid, centrifuging and drying, and obtaining a crude salmon calcitonin acetylated impurity product, that is, the crude salmon calcitonin impurity peptide.

[0016] Furthermore, in the step 4), the cleavage solution used for cleavage is a mixture of more than 80% TFA and any one or more of H2O, EDT, TIS, thioanisole, phenol, and DODT, preferably TFA:Tis:H2O=90:5:5; in the step 4), the sedimentation liquid used for cleavage is any one or more of petroleum ether, diethyl ether, methyl tert-butyl ether, isopropyl ether, dipropyl ether, and ethylene glycol dimethyl ether, preferably tert-methyl ether and diethyl ether.

[0017] Compared with the prior art, the technical solution provided by the present invention has at least the following advantages:

[0018] The present invention provides a method for synthesizing salmon calcitonin acetylated impurities. Firstly, the method uses a solid phase synthesis method, and can directly obtain the corresponding impurity crude peptide after cleavage, thereby reducing the cost; secondly, the method is simple to operate, and is conducive to the simultaneous preparation of a series of acetylated impurities; finally, the crude peptide synthesized by the method has a high purity, thereby reducing the difficulty of purification.

[0019] In summary, the method for synthesizing salmon calcitonin acetylated impurities provided by the present invention shortens the synthesis time, reduces the cost, and improves the purity of the crude peptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a synthetic route diagram of a method for synthesizing salmon calcitonin acetylated impurities provided by the present invention;

[0021] Figure 2 This is the chromatogram of the acetylated impurity on the side chain of Ser at position 2; Figure 3 This is the mass spectrum of the acetylated impurity on the Ser side chain at position 2. DETAILED DESCRIPTION

[0022] The present invention is described in detail below in conjunction with specific implementation modes.

[0023] The present invention provides a method for synthesizing salmon calcitonin acetylated impurities, the synthesis route is as follows Figure 1 As shown, the following steps are included:

[0026] 1. The amino resin is coupled with the corresponding amino acid to obtain a linear peptide resin;

[0027] 2. Solid phase oxidation of peptide resin to form disulfide bonds;

[0028] 3. Acetylation modification of peptide resin;

[0029] 4. The peptide resin is cleaved to obtain the corresponding salmon calcitonin impurity crude peptide.

[0030] In the step 1), the following amino acid raw materials are coupled in sequence using the classic Fmoc strategy coupling method: Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc -Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Boc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ser-OH, Fmoc-Thr-OH, Fmoc-Lys(Alloc)-OH, the coupling time is 1-3 hours, preferably 2 hours.

[0031] In the step 1), the amino resin includes RinkAmide Resin, RinkAmideAM Resin, SieberResin and RinkAmide MBHAResin, and the amino resin substitution degree is 0.2-0.4 mmol / g;

[0032] The solvent used in step 1) is one or a combination of DMF, DMAc, DCM, NMP, and DMSO, preferably DMF;

[0033] In the step 1), the de-Fmoc reagent is any one of 10-30% PIP / DMF, 10-30% PIP / DCM, 10-30% PIP / NMP, and 10-30% PIP / DMAc, preferably 20% PIP / DMF;

[0034] The synthesis method in step 1) is as follows: the activating reagent is any one of HOBT / DIC, Oxyma Pure / DIC, HOAT / DIC, PyBOP / DIPEA, HBTU / DIC, HATU / DIC, HOBT / DIPEA, HOAT / DIPEA, HBTU / DIPEA, HATU / DIPEA, TBTU / DIPEA, TBTU / DIC, and PyAOP / DIPEA, preferably DIC / HOBt;

[0035] In the step 2), the oxidation system is I2 / DMF;

[0036] In the step 3), the reagent for the acetylation reaction is one or a combination of two of Ac2O, Ac2O / Py, CH3COOH / DIC, CH3COOH / DIC / DMAP, CH3COCl / TEA, and CH3COCl / DIPEA;

[0037] In the step 3), the reaction solvent is any one or more of DCM, DMF, NMP, and DMSO, preferably DMF and DCM;

[0038] In the step 4), the lysis solution used for lysis is a mixture of 80% or more TFA and any one or more of H2O, EDT, TIS, thioanisole, phenol, and DODT, preferably TFA:Tis:H2O=90:5:5;

[0039] In the step 4), the sedimentation liquid used for cracking is any one or more of petroleum ether, diethyl ether, methyl tert-butyl ether, isopropyl ether, dipropyl ether, and ethylene glycol dimethyl ether, preferably tert-methyl ether and diethyl ether;

[0040] Examples

[0041] Salmon calcitonin Ser side chain acetylated impurities:

[0042] Example 1 (acetylation of the Ser side chain at position 2):

[0043] (1) Amino resin is coupled with amino acid to obtain linear peptide resin:

[0044] 4 g of RinkAmide Resin with a substitution degree of 0.25 mmol / g was transferred to the reactor, washed with DMF twice, and swelled with DMF for more than 30 min. 20% PIP / DMF was added, and the reaction was carried out with aeration for 10 min. After drying, 20% PIP / DMF was added again, and the reaction was carried out with aeration for 15 min. After drying, the resin was washed with DMF for 6 times, and the resin was colored by Kaiser detection.

[0045] Weigh 1.01 g (3 mmol) of Fmoc-Pro-OH and 0.48 g (3.15 mmol) of HOBT, dissolve in DMF, cool to below 10°C, add 0.6 g (3.15 mmol) of DIC, activate for 5 min, pour into the reactor, react at 25°C for 2 h, and Kaiser test shows colorless.

[0046] Repeat the above steps to couple Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, one by one). Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt )-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH , Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser-OH, Boc-Cys(Trt)-OH;

[0047] (2) Solid phase oxidation of peptide resin to form disulfide bonds:

[0048] Weigh 1.55 g I2 (6 mmol), dissolve it in 30 mL DMF, pour it into the reactor, react for 1 h, drain and wash it with DMF 3 times, 5% PIP / DMF 3 times, and DMF 6 times.

[0049] (3) Peptide resin acetylation modification:

[0050] Weigh 2.1 g (20 mmol) of acetic anhydride and 1.6 g (20 mmol) of pyridine, dissolve them in DCM, pour them into a reactor, and react at 25° C. for 4 h.

[0051] (4) Peptide resin cleavage to obtain the corresponding salmon calcitonin impurity crude peptide:

[0052] The resin was washed with DMF 3 times, DCM 3 times, and shrunk with methanol 3 times for 5+5+10 min respectively, and then dried after being drained.

[0053] Prepare a cleavage solution of TFA:TIS:H2O=90:5:5, precool, collect the resin and cleave it with the precooled cleavage solution (1g peptide resin corresponds to 8mL cleavage solution), cleave at 25°C for 2h, filter the resin, add precooled ether (1g resin corresponds to 80mL ether) to precipitate, centrifuge, wash the solid with ether twice, centrifuge and dry, and obtain 4.14g of crude salmon calcitonin acetylated impurity as a white solid with a purity of 66.34% (see chromatogram) Figure 2 , mass spectrum see Figure 3 ).

[0054] Example 2 (acetylation of the Ser side chain at position 5):

[0055] Referring to Example 1, the order of coupling amino acids in step (1) is: Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc- Leu-OH, Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Boc-Cys(Trt)-OH;

[0056] 4.21 g of the corresponding impurity crude product as a white solid was obtained with a purity of 63.21%.

[0057] Example 3 (acetylation of the Ser side chain at position 13):

[0058] Referring to Example 1, the order of coupling amino acids in step (1) is: Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-O H, Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Boc-Cys(Trt)-OH;

[0059] 4.01 g of the corresponding impurity crude product was obtained as a white solid with a purity of 61.13%.

[0060] Example 4 (acetylation of the Ser side chain at position 29):

[0061] Referring to Example 1, the order of coupling amino acids in step (1) is: Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc- His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-O H, Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Boc-Cys(Trt)-OH;

[0062] 3.96 g of the corresponding impurity crude product was obtained as a white solid with a purity of 63.51%.

[0063] Salmon calcitonin Thr side chain acetylated impurities:

[0064] Example 5 (acetylation of Thr side chain at position 6):

[0065] (1) Amino resin is coupled with amino acid to obtain linear peptide resin:

[0066] 4 g of RinkAmide Resin with a substitution degree of 0.25 mmol / g was transferred to the reactor, washed with DMF twice, and swelled with DMF for more than 30 min. 20% PIP / DMF was added, and the reaction was carried out with aeration for 10 min. After drying, 20% PIP / DMF was added again, and the reaction was carried out with aeration for 15 min. After drying, the resin was washed with DMF for 6 times, and the resin was colored by Kaiser detection.

[0067] Weigh 1.01 g (3 mmol) of Fmoc-Pro-OH and 0.48 g (3.15 mmol) of HOBT, dissolve in DMF, cool to below 10°C, add 0.6 g (3.15 mmol) of DIC, activate for 5 min, pour into the reactor, react at 25°C for 2 h, and Kaiser test shows colorless.

[0068] Repeat the above steps to couple Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, one by one). Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt )-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH , Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Boc-Cys(Trt)-OH;

[0069] (2) Peptide resin oxidation:

[0070] Weigh 1.55 g I2 (6 mmol), dissolve it in 30 mL DMF, pour it into the reactor, react for 1 h, drain and wash it with DMF 3 times, 5% PIP / DMF 3 times, and DMF 6 times.

[0071] (3) Peptide resin acetylation:

[0072] Weigh 2.1 g (20 mmol) of acetic anhydride and 1.6 g (20 mmol) of pyridine, dissolve them in DCM, pour them into a reactor, and react at 25° C. for 4 h.

[0073] (4) Resin cracking:

[0074] The resin was washed with DMF 3 times, DCM 3 times, and shrunk with methanol 3 times for 5+5+10 min respectively, and then dried after being drained.

[0075] Prepare a lysis solution of TFA:TIS:H2O=90:5:5, precool it, collect the resin and lyse it with the precooled lysis solution (1g peptide resin corresponds to 8mL lysis solution), lyse it at 25℃ for 2h, filter the resin, add precooled ether (1g resin corresponds to 80mL ether) to precipitate, centrifuge, wash the obtained solid with ether twice, centrifuge and dry to obtain 4.07g of crude white solid of salmon calcitonin acetylated impurity with a purity of 58.66%.

[0076] Example 6 (acetylation of Thr side chain at position 21):

[0077] Referring to Example 5, the order of coupling amino acids in step (1) is: Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc- His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-O H, Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Boc-Cys(Trt)-OH;

[0078] 4.11 g of the corresponding impurity crude product was obtained as a white solid with a purity of 59.47%.

[0079] Example 7 (acetylation of Thr side chain at position 25):

[0080] Referring to Example 5, the order of coupling amino acids in step (1) is: Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc- His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-O H, Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Boc-Cys(Trt)-OH;

[0081] 3.94 g of the corresponding impurity crude product was obtained as a white solid with a purity of 58.96%.

[0082] Example 8 (acetylation of Thr side chain at position 27):

[0083] Referring to Example 5, the order of coupling amino acids in step (1) is: Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc- His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-O H, Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Boc-Cys(Trt)-OH;

[0084] 3.99 g of the corresponding impurity crude product was obtained as a white solid with a purity of 59.03%.

[0085] Example 9 (acetylation of Thr side chain at position 31):

[0086] Referring to Example 5, the order of coupling amino acids in step (1) is: Fmoc-Pro-OH, Fmoc-Thr-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc- His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-O H, Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Boc-Cys(Trt)-OH;

[0087] 3.89 g of the corresponding impurity crude product as a white solid was obtained with a purity of 60.22%.

[0088] Salmon calcitonin Lys side chain acetylated impurities:

[0089] Example 10 (Acetylation of the Lys side chain at position 11)

[0090] (1) Amino resin is coupled with amino acid to obtain linear peptide resin:

[0091] 4 g of RinkAmide Resin with a substitution degree of 0.25 mmol / g was transferred to the reactor, washed with DMF twice, and swelled with DMF for more than 30 min. 20% PIP / DMF was added, and the reaction was carried out with aeration for 10 min. After drying, 20% PIP / DMF was added again, and the reaction was carried out with aeration for 15 min. After drying, the resin was washed with DMF for 6 times, and the resin was colored by Kaiser detection.

[0092] Weigh 1.01 g (3 mmol) of Fmoc-Pro-OH and 0.48 g (3.15 mmol) of HOBT, dissolve in DMF, cool to below 10°C, add 0.6 g (3.15 mmol) of DIC, activate for 5 min, pour into the reactor, react at 25°C for 2 h, and Kaiser test shows colorless.

[0093] Repeat the above steps to couple Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fm one by one). oc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH , Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Alloc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fm oc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Boc-Cys(Trt)-OH;

[0094] (2) Peptide resin oxidation:

[0095] Weigh 1.55 g I2 (6 mmol), dissolve it in 30 mL DMF, pour it into the reactor, react for 1 h, drain and wash it with DMF 3 times, 5% PIP / DMF 3 times, and DMF 6 times.

[0096] (3) Peptide resin acetylation:

[0097] Weigh 40.3 g (0.25 mmol) of Pd(PPh3) and 0.9 g (10 mmol) of morpholine, dissolve them in DCM, pour them into the reactor, and react at 25°C for 1 h. Drain and wash with DCM 3 times, 5% PIP / DCM 3 times, and DCM 6 times.

[0098] Weigh 2.1 g (20 mmol) of acetic anhydride and 1.6 g (20 mmol) of pyridine, dissolve them in DCM, pour them into a reactor, and react at 25° C. for 4 h.

[0099] (4) Resin cracking:

[0100] The resin was washed with DMF 3 times, DCM 3 times, and shrunk with methanol 3 times for 5+5+10 min respectively, and then dried after being drained.

[0101] Prepare a lysis solution of TFA:TIS:H2O=90:5:5, precool it, collect the resin and lyse it with the precooled lysis solution (1g peptide resin corresponds to 8mL lysis solution), lyse it at 25℃ for 2h, filter the resin, add precooled ether (1g resin corresponds to 80mL ether) to precipitate, centrifuge, wash the obtained solid with ether twice, centrifuge and dry to obtain 4.36g of crude white solid of salmon calcitonin acetylated impurity with a purity of 64.18%.

[0102] Example 11 (Acetylation of the Lys side chain at position 18)

[0103] Referring to Example 10, the order of coupling amino acids in step (1) is: Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Alloc)-OH, Fmoc- moc-His(Trt)-OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu -OH, Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Boc-Cys(Trt)-OH;

[0104] 4.29 g of the corresponding impurity crude product was obtained as a white solid with a purity of 62.93%.

[0105] Terminal Cys acetylation:

[0106] Embodiment 12:

[0107] (1) Amino resin is coupled with amino acid to obtain linear peptide resin:

[0108] 4 g of RinkAmide Resin with a substitution degree of 0.25 mmol / g was transferred to the reactor, washed with DMF twice, and swelled with DMF for more than 30 min. 20% PIP / DMF was added, and the reaction was carried out with aeration for 10 min. After drying, 20% PIP / DMF was added again, and the reaction was carried out with aeration for 15 min. After drying, the resin was washed with DMF for 6 times, and the resin was colored by Kaiser detection.

[0109] Weigh 1.01 g (3 mmol) of Fmoc-Pro-OH and 0.48 g (3.15 mmol) of HOBT, dissolve in DMF, cool to below 10°C, add 0.6 g (3.15 mmol) of DIC, activate for 5 min, pour into the reactor, react at 25°C for 2 h, and Kaiser test shows colorless.

[0110] Repeat the above steps to couple Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Gly-OH, Fmoc-Thr(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, F moc-Arg(Pbf)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt) -OH, Fmoc-Leu-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Gly-OH, Fmoc-Leu-OH, Fmoc-Val-OH, Fmoc-Cys(Trt)-OH, Fmoc-Thr-OH, Fmoc-Ser(tBu)-OH, Fmoc-Leu-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Cys(Trt)-OH;

[0111] (2) Peptide resin oxidation:

[0112] Weigh 1.55 g I2 (6 mmol), dissolve it in 30 mL DMF, pour it into the reactor, react for 1 h, drain and wash it with DMF 3 times, 5% PIP / DMF 3 times, and DMF 6 times.

[0113] (3) Peptide resin acetylation:

[0114] Weigh 2.1 g (20 mmol) of acetic anhydride and 1.6 g (20 mmol) of pyridine, dissolve them in DCM, pour them into a reactor, and react at 25° C. for 4 h.

[0115] (4) Resin cracking:

[0116] The resin was washed with DMF 3 times, DCM 3 times, and shrunk with methanol 3 times for 5+5+10 min respectively, and then dried after being drained.

[0117] Prepare a lysis solution of TFA:TIS:H2O=90:5:5, precool it, collect the resin and lyse it with the precooled lysis solution (1g peptide resin corresponds to 8mL lysis solution), lyse it at 25℃ for 2h, filter the resin, add precooled ether (1g resin corresponds to 80mL ether) to precipitate, centrifuge, wash the obtained solid with ether twice, centrifuge and dry to obtain 4.24g of crude white solid of salmon calcitonin acetylated impurity with a purity of 61.35%.

[0118] Abbreviations and English meanings

[0119]

[0120]

[0121] Those skilled in the art can understand that the above-mentioned embodiments are specific examples for implementing the present application, and in practical applications, various changes can be made to them in form and detail without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application, so the scope of protection of the present application shall be based on the scope defined in the claims.

Claims

1. A method for synthesizing salmon calcitonin acetylated impurities, characterized in that: The method comprises the following steps: 1) amino resin is coupled with corresponding amino acid to obtain linear peptide resin; 2) Solid phase oxidation of peptide resin to form disulfide bonds; 3) Peptide resin acetylation modification; 4) The peptide resin is cleaved to obtain the corresponding salmon calcitonin impurity crude peptide.

2. The method for synthesizing salmon calcitonin acetylated impurities according to claim 1, characterized in that: The step 1) specifically comprises: washing the amino resin with a solvent and performing swelling treatment with the solvent; then, adding a de-Fmoc reagent to perform a de-Fmoc reaction; weighing the amino acid raw material and the activation reagent, dissolving them with a solvent, and cooling to below 10° C. to perform an activation reaction; repeating the above operations to couple one by one to obtain a linear peptide resin.

3. The method for synthesizing the acetylated impurity of salmon calcitonin according to claim 2, characterized in that: In the step 1), the Fmoc strategy coupling method is used to sequentially couple the following amino acid raw materials: Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc- Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Glu(OtBu)-OH, Fmoc-Val-OH, Boc-Cys(Trt)-OH, Fmoc-Cys(Trt)-OH, Fmoc-Ser-OH, Fmoc-Thr-OH, Fmoc-Lys(Alloc)-OH, the coupling time is 1-3 hours, preferably 2 hours.

4. The method for synthesizing salmon calcitonin acetylated impurities according to claim 2, characterized in that: In the step 1), the amino resin includes RinkAmide Resin, RinkAmide AM Resin, Sieber Resin and RinkAmideMBHAResin, and the substitution degree of the amino resin is 0.2-0.4 mmol / g.

5. The method for synthesizing salmon calcitonin acetylated impurities according to claim 2, characterized in that: The solvent used in step 1) is one or a combination of DMF, DMAc, DCM, NMP, and DMSO, preferably DMF; In the step 1), the de-Fmoc reagent is any one of 10-30% PIP / DMF, 10-30% PIP / DCM, 10-30% PIP / NMP, and 10-30% PIP / DMAc, preferably 20% PIP / DMF; The synthesis method in the step 1) is: the activating reagent is any one of HOBT / DIC, Oxyma Pure / DIC, HOAT / DIC, PyBOP / DIPEA, HBTU / DIC, HATU / DIC, HOBT / DIPEA, HOAT / DIPEA, HBTU / DIPEA, HATU / DIPEA, TBTU / DIPEA, TBTU / DIC, and PyAOP / DIPEA, preferably DIC / HOBt.

6. The method for synthesizing salmon calcitonin acetylated impurities according to claim 1, characterized in that: In the step 2), the oxidation system used for solid phase oxidation of the peptide resin to form disulfide bonds is I2 / DMF.

7. The method for synthesizing salmon calcitonin acetylated impurities according to claim 1, characterized in that: The step 3) specifically includes: weighing the reagent for the acetylation reaction, dissolving it in a reaction solvent, and then adding it to the peptide resin obtained in the step 2) to react.

8. The method for synthesizing salmon calcitonin acetylated impurities according to claim 7, characterized in that: In the step 3), the reagent for the acetylation reaction is one or a combination of two of Ac2O, Ac2O / Py, CH3COOH / DIC, CH3COOH / DIC / DMAP, CH3COCl / TEA, and CH3COCl / DIPEA; In the step 3), the reaction solvent is any one or more of DCM, DMF, NMP, and DMSO, preferably DMF and DCM.

9. The method for synthesizing salmon calcitonin acetylated impurities according to claim 1, characterized in that: The step 4) specifically comprises: collecting the peptide resin obtained in the step 3), cleaving it with a pre-cooled cleavage solution, filtering the resin, adding a pre-cooled sedimentation solution for sedimentation, centrifuging, washing the obtained solid, centrifuging and drying, and obtaining a crude salmon calcitonin acetylated impurity product, that is, the crude salmon calcitonin impurity peptide.

10. The method for synthesizing salmon calcitonin acetylated impurities according to claim 9, characterized in that: In the step 4), the lysis solution used for lysis is a mixture of 80% or more TFA and any one or more of H2O, EDT, TIS, thioanisole, phenol, and DODT, preferably TFA:Tis:H2O=90:5:5; In the step 4), the sedimentation liquid used for cracking is any one or more of petroleum ether, diethyl ether, methyl tert-butyl ether, isopropyl ether, dipropyl ether, and ethylene glycol dimethyl ether, preferably tert-methyl ether and diethyl ether.

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