Method for manufacturing oxytocin by liquid-phase polypeptide synthesis
20200002382 ยท 2020-01-02
Assignee
Inventors
Cpc classification
Y02P20/55
GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
C07K5/0606
CHEMISTRY; METALLURGY
International classification
Abstract
The present disclosure discloses a liquid-phase synthesis method of oxytocin under mild conditions for the first time, which is characterized in that three oxytocin fragments which include Boc-Cys(Acm)-Tyr(tBu)-OH, H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe and H-Leu-Gly-NH.sub.2 are synthesized for the first time, the fragments are assembled to synthesize an all-protected oxytocin amino acid sequence, iodine is used to remove Acm while cyclization is performed to form a disulfide bond to obtain protected cyclic oxytocin, trifluoroacetic acid is used to remove residual protecting groups to obtain crude oxytocin, ethyl acetate is used to perform recrystallization, and reversed-phase chromatography purification is performed to reach high-purity (crude product purity reaches 95%) and high biological value of oxytocin (588 IU/mg). In the present invention, a Boc-polypeptide synthesis and Fmoc-polypeptide synthesis combined method is provided, wherein, all the reactions are carried out under mild conditions without using the ammonia-sodium-method decapping reaction which is reported by literature referring to oxytocin liquid-phase synthesis domestically and abroad. Furthermore, the liquid-phase synthesis method of oxytocin is first performed without highly toxic reagents and unsafe reaction conditions, thus greatly reducing the cost of oxytocin synthesis and providing access to industrial production of oxytocin.
Claims
1-9. (canceled)
10. A method for manufacturing oxytocin by liquid-phase polypeptide synthesis, which comprises the following steps: (1) synthesizing fragment 1: Boc-Cys(Acm)-Tyr(tBu)-OH; (2) synthesizing fragment 2: H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe; (3) synthesizing fragment 3: H-Leu-Gly-NH.sub.2; (4) synthesizing compound 4: Boc-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe; (5) synthesizing compound 5: Boc-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OH; (6) synthesizing compound 6: Boc-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-Leu-Gly-NH.sub.2; (7) synthesizing compound 7: Boc-Cys-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys-Pro-Leu-Gly-NH.sub.2 (8) decapping compound 7 with trifluoroacetic acid and concentrating the reaction mixture to obtain a crude oxytocin solution; (9) purifying the crude oxytocin by C18 reversed-phase silica gel chromatography to obtain pure oxytocin.
11. The method for manufacturing oxytocin by liquid-phase polypeptide synthesis according to claim 10, wherein, the fragment 1 in the step (1) is: Boc-Cys(Acm)-Tyr(tBu)-OH; amino acid units for synthesizing the fragment 1 are Boc-Cys(Acm)-OH and H-Tyr(tBu)-OH; DIC or DCC is employed as a condensing agent; HOSu or HOBt is employed as an activator; THF and water are employed as a solvent; and the ratio of the THF to the water is from 1:1 to 5:1, wherein the solvent may also be DMF or DMSO.
12. The method for manufacturing oxytocin by liquid-phase polypeptide synthesis according to claim 10, wherein, the fragment 2 in the step (2) is: H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe; and synthesizing the fragment 2 comprises the following steps: (1) synthesizing compound 2.1: Fmoc-Cys(Acm)-Pro-OMe: a ratio of an amount of Fmoc-Cys(Acm)-OH to an amount of H-Pro-OMe.HCl is from 1:1.05 to 1:1.5; DIC or DCC is employed as a condensing agent; a ratio of an amount of the condensing agent to the amount of H-Pro-OMe.HCl is 1:1; HOSu or HOBt or HOOBt is employed as an activator; a ratio of an amount of the activator to the amount of H-Pro-OMe.HCl is 1:1; triethylamine (TEA) diisopropylethylamine (DIPEA) or N-methylmorpholine (NMM) is employed as a base; a ratio of an amount of the base to the amount of H-Pro-OMe.HCl is 1:1; and DMF, DMSO, DCM or THF is employed as a solvent; (2) synthesizing compound 2.2: H-Cys(Acm)-Pro-OMe: decapping the compound 2.1 with diethylamine (DEA) or 5% piperazine/DCM as a decapping reagent, concentrating after completion of the decapping reaction, using petroleum ether to separate a product, filtrating and vacuum-drying to give compound 2.2; (3) synthesizing compound 2.3: Fmoc-Asn(Trt)-Cys(Acm)-Pro-OMe: a ratio of an amount of the compound 2.2 to an amount of Fmoc-Asn(Trt)-OH is from 1:1.05 to 1:1.5; DIC or DCC is employed as a condensing agent, a ratio of an amount of the condensing agent to the amount of Fmoc-Asn(Trt)-OH is 1:1; HOSu, HOOBt or HOBt is employed as an activator; a ratio of an amount of the activator to the amount of Fmoc-Asn(Trt)-OH is 1:1; and DMF, DMSO, DCM, or THF is employed as a solvent; (4) synthesizing compound 2.4: H-Asn(Trt)-Cys(Acm)-Pro-OMe: decapping the compound 2.3 with diethylamine (DEA) or 5% piperazine/DCM as a decapping reagent; concentrating after completion of the decapping reaction, using saturated NaHCO.sub.3 solution to separate the product, filtrating, washing with water to neutral and drying in vacuum, and then washing a crude product with petroleum ether while stirring, filtrating and drying in vacuum to give compound 2.4; (5) synthesizing compound 2.5: Fmoc-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe: a ratio of an amount of the compound 2.4 to an amount of Fmoc-Gln(Trt)-OH is from 1:1.05 to 1:1.5; DIC or DCC is employed as a condensing agent; a ratio of an amount of the condensing agent to the amount of Fmoc-Gln(Trt)-OH is 1:1; HOSu, HOOBt or HOBt is employed as an activator, a ratio of an amount of the activator to the amount of Fmoc-Gln(Trt)-OH is 1:1; and DMF, DMSO, DCM or THF is employed as a solvent; (6) synthesizing compound 2.6: H-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe: decapping the compound 2.5 with diethylamine (DEA) or 5% piperazine/DCM as a decapping reagent; concentrating after completion of the decapping reaction, using saturated NaHCO.sub.3 solution to separate a product, filtrating, washing with water to neutral and drying in vacuum, and then washing a crude product with petroleum ether while stirring, filtrating and drying in vacuum to give compound 2.6; (7) synthesizing compound 2.7: Fmoc-Ile-Gln (Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe: a ratio of an amount of compound 2.6 to an amount of Fmoc-Ile-OH is 1:1.05-1:1.5; DIC or DCC is employed as a condensing agent; a ratio of an amount of the condensing agent to an amount of Fmoc-Ile-OH is 1:1; HOSu, HOOBt or HOBt is employed as an activator; a ratio of an amount of the activator to the amount of Fmoc-Ile-OH is 1:1; and DMF, DMSO, DCM or THF is employed as a solvent; (8) synthesizing fragment 2: H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe: decapping the compound 2.7 with diethylamine (DEA) or 5% piperazine/DCM as a decapping reagent; concentrating after completion of the decapping reaction, using saturated NaHCO.sub.3 solution to separate a product, filtrating, washing with water to neutral and drying in vacuum, and then washing a crude product with petroleum ether while stirring, filtrating and drying in vacuum to give the fragment 2.
13. The method for manufacturing oxytocin by liquid-phase polypeptide synthesis according to claim 10, wherein the fragment 3 in the step (3) is: H-Leu-Gly-NH.sub.2; and the fragment 3 may be synthesized through solution 1 or solution 2 as follows: solution 1: Boc-Leu-OH and H-Gly-NH.sub.2.HCl are employed as amino acid units; BOP, HBTU, TBTU, DIC or DCC/HOBt or HOSu is employed as a condensing agent; TEA, NMM or DIPEA is employed as a base; DMF, DMSO, THF or DCM is employed as a solvent; synthesizing Boc-Leu-Gly-NH.sub.2 and then removing the boc protecting group of it with TFA to obtain H-Leu-Gly-NH.sub.2; solution 2: Fmoc-Leu-OH, H-Gly-NH.sub.2.HCl are employed as amino acid units; BOP, HBTU, TBTU, DIC or DCC/HOBt or HOSu is employed as a condensing agent; TEA, NMM or DIPEA is employed as a base; DMF, DMSO, THF or DCM is employed as a solvent; after synthesizing Fmoc-Leu-Gly-NH.sub.2, removing the Fmoc protecting group of it to obtain H-Leu-Gly-NH.sub.2; and a decapping agent is diethylamine (DEA) or 5% piperazine/DCM.
14. The method for manufacturing oxytocin by liquid-phase polypeptide synthesis according to claim 10, wherein the compound 4 in the step (4) is: Boc-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe and the synthesis of compound 4 comprises the following step: a ratio of an amount of the fragment 1 to an amount of the fragment 2 is 1.05-1.5:1; BOP, DCC, DIC or an azide reagent is employed as a condensing agent; a ratio of an amount of the condensing agent to an amount of the fragment 1 is 1:1; HOSu or HOBt is employed as an activator; a ratio of an amount of the activator to the fragment 1 is 1:1; and DCM, THF, DMF or DMSO is employed as a solvent.
15. The method for manufacturing oxytocin by liquid-phase polypeptide synthesis according to claim 10, wherein the compound 5 in the step (5) is: Boc-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OH and the synthesis of compound 5 comprises the following steps: LiOH or NaOH is employed as a saponification reagent, an amount of which is 5-15 equivalents of the compound 4; a concentration is 0.05-0.2 M; and THF, DMF, methanol, acetonitrile or DMSO is employed as a solvent.
16. The method for manufacturing oxytocin by liquid-phase polypeptide synthesis according to claim 10, wherein the compound 6 in the step (6) is: Boc-Cys(Acm)-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-Leu-Gly-NH.sub.2; and the synthesis of compound 6 comprises the following step: a ratio of an amount of the compound 5 to an amount of the fragment 3 is 1:1:1.05-2; DCC, DIC, BOP or an azide reagent is employed as a condensing agent; HOSu, HOOBt or HOBt is employed as an activator; and THF, DMF or DMSO is employed as a solvent.
17. The method for manufacturing oxytocin by liquid-phase polypeptide synthesis according to claim 10, wherein in the step (7), synthesizing compound 7 using iodine as a reagent, an amount of the iodine is 5-10 equivalents of the compound 6 and a concentration of the iodine is 0.1-0.5M, and DMF is employed as a solvent; after completion of the reaction, using 0.1% sodium thiosulfate solution to separate a solid, washing the solid with water and drying in vacuum to give the compound 7.
18. The method for manufacturing oxytocin by liquid-phase polypeptide synthesis according to claim 10, wherein an amino acid sequence of the compound 7 is: Boc-Cys-Tyr(tBu)-Ile-Gln(Trt)-Asn(Trt)-Cys-Pro-Leu-Gly-NH.sub.2.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0055]
[0056]
[0057]
[0058]
[0059]
[0060]
[0061]
[0062]
[0063]
[0064]
[0065]
[0066]
[0067]
[0068]
[0069]
[0070]
[0071]
[0072]
[0073]
[0074]
[0075]
DETAILED DESCRIPTION
[0076] Full names corresponding to the abbreviation of the substance appearing in the claims and description of the embodiments of the present invention are shown in Table 1.
TABLE-US-00001 TABLE 1 Abbreviation Full name Fmoc 9-fluorene methoxy carbonyl Trt trityl HOBt I-hydroxybenzotriazole Boc t-Butyloxy carbonyl tBu tertiary butyl TFA trifluoroacetic acid TIS Triisopropylsilane DIPEA n,n-diisopropylethylamine IPA isopropanol DMF N,N-dimethylformamide Gly glycine Leu leucine Pro proline Asn L-Asparagine Monohydrate Gln glutamine Cys cysteine Ile isoleucine Tyr Tyrosine THF Tetrahydrofuran DCC Dicyclohexylcarbodiimide HOOBt 3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine TEA triethylamine DEA diethylamine HOSu 1-hydroxy-5-pyrrolidinedione
Example 1: Synthesis of Fragment 1: Boc-Cys(Acm)-Tyr(tBu)-OH
[0077] Boc-Cys(Acm)-OH (100 mol, 29.2 g, 1 eq.) and HOSu (110 mmol, 12.6 g, 1.1 eq.) were dissolved in 200 mL THF, and then cooled to 10 C. in an ice bath for 10 min. To the cool solution was added DCC (110 mmol, 22.7 g, 1.1 eq.) dissolved in 20 mL THF. After reaction for 20 minutes, H-Tyr(tBu)-OH (110 mmol, 26.1 g, 1.1 eq.) and NaHCO.sub.3 (110 mmol, 9.2 g, 1.1 eq.) were dissolve in 150 mL water and then added to the above solution. The reaction was kept at 15-25 C., monitored by HPLC, until it was completed. The mixture solution was regulated to neutral by addition of 0.5M HCl, filtrated with removal of insoluble matter, concentrated to remove THF and subsequently regulated to pH=2 by addition of 0.5M HCl. The obtained white solid was then collected by filtration, washed with water until neutral, and then dried in vacuum to give 47.1 g fragment 1 with 99.1% purity and 92% yield. Fragment 1: Boc-Cys(Acm)-Tyr(tBu)-OH. Its RP-HPLC image is shown in
Example 2 Synthesis of Fragment 2: H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe
[0078] (1) Synthesis of Compound 2.1: Fmoc-Cys(Acm)-Pro-OMe:
[0079] Fmoc-Cys(Acm)-OH (100 mmol, 41.5 g, 1 eq.) and HOBt (105 mmol, 14.1 g, 1.05 eq.) were dissolved in the mixture solvent of 200 mL THF and 30 mL DMF, and then cooled to 10 C. in an ice bath for 10 min. To the cool solution was added DCC (105 mmol, 21.6 g, 1.05 eq.) dissolved in 20 mL THF. After reaction for 20 minutes, H-Pro-OMe.HCl (105 mmol, 17.4 g, 1.05 eq.), dissolved in 100 mL THF and well-mixed with TEA (105 mmol, 14.5 ml, 1.05 eq.), was added to the above reaction solution. The reaction was kept at 15-25 C., monitored by HPLC, until it was completed. The mixture solution was filtrated with removal of insoluble matter, concentrated and subsequently separated solid out by addition of 0.1M HCl. The solid was then collected by filtration, washed to neutral by water, and then dried in vacuum to give 50.9 g compound 2.1 with 95.3% purity and 97% yield (the yield is calculated in mmol, the same as below). The HPLC image is shown in
[0080] (2) Synthesis of Compound 2.2: H-Cys(Acm)-Pro-OMe:
[0081] The dried compound 2.1 was dissolved in 200 mL diethylamine, concentrated to a minimum amount, precipitated by addition of petroleum ether, filtered, washed with petroleum ether, and dried in vacuum to give 28.8 g compound 2.2 with 98.0% purity and 95% yield. The HPLC image is shown in
[0082] (3) Synthesis of Compound 2.3: Fmoc-Asn(Trt)-Cys(Acm)-Pro-OMe:
[0083] Fmoc-Asn(Trt)-OH (1.1 eq., 104.4 mmol, 62.3 g) and HOBt (1.1 eq., 104.4 mmol, 14.1 g) were dissolved in the mixture solvent of 200 mL DCM and 20 mL DMF, and then cooled to 10 C. for 10 min. To the cool solution was added DCC (1.1 eq., 104.4 mmol, 21.5 g) dissolved in 20 mL DCM. After reaction for 20 minutes, compound 2.2 (1 eq., 94.9 mmol, 28.8 g), dissolved in 50 mL DMF, was added to the above reaction solution. The reaction was kept at 15-25 C., monitored by HPLC, until it was complete. The mixture solution was filtrated with removal of insoluble matter, concentrated and subsequently separated solid out by addition of 0.1M HCl. The solid was then collected by filtration, washed to neutral by water, and then dried in vacuum to give 75 g compound 2.3 with 99% purity and 85% yield. The HPLC image is shown in
[0084] (4) Synthesis of Compound 2.4: H-Asn(Trt)-Cys(Acm)-Pro-OMe:
[0085] The dried compound 2.3 was dissolved with 200 mL diethylamine, concentrated to a minimum amount, precipitated by addition of saturated NaHCO.sub.3, filtered, washed to neutral with water, dried in vacuum, washed by stirring in petroleum ether three times, filtered, and dried in vacuum to give 55.7 g compound 2.4 with 96.3% purity and 84.4% yield. The HPLC image is shown in
[0086] (5) Synthesis of Compound 2.5: Fmoc-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe:
[0087] Fmoc-Gln(Trt)-OH (1.1 eq., 92.8 mmol, 56.7 g) and HOBt (1.1 eq., 92.8 mmol, 12.5 g) were dissolved in the mixture solvent of 150 mL THF and 50 mL DMF, and then cooled to 10 C. for 10 min. To the cool solution was added DCC (1.1 eq., 92.8 mmol, 19.1 g) dissolved in 20 mL THF. After reaction for 20 minutes, compound 2.4 (1 eq., 84.4 mmol, 55.7 g), dissolved in 150 mL THF, was added to the above reaction solution. The reaction was kept at 15-25 C., monitored by HPLC, until it was complete. The mixture solution was filtrated with removal of insoluble matter, concentrated and subsequently separated solid out by addition of 0.1M HCl. The solid was then collected by filtration, washed to neutral by water, and then dried in vacuum to give 106.4 g compound 2.5 with 85% yield.
[0088] (6) Synthesis of Compound 2.6: H-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe:
[0089] The dried compound 2.5 was dissolved in 150 mL diethylamine, concentrated to a minimum amount, precipitated by addition of saturated NaHCO.sub.3, filtered, washed to neutral with water, dried in vacuum, washed by stirring in petroleum ether three times, filtered, and dried in vacuum to give 86.5 g compound 2.6 with 98.3. % purity and 84% yield. The HPLC image is shown in
[0090] (7) Synthesis of Compound 2.7: Fmoc-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe:
[0091] Fmoc-Ile-OH (1.1 eq., 92.4 mmol, 32.6 g) and HOBt (1.1 eq., 92.4 mmol, 12.5 g) were dissolved in the mixture solvent of 150 mL THF and 50 mL DMF, and then cooled to 10 C. for 10 min. To the cool solution was added DCC (1.1 eq., 92.4 mmol, 19 g) dissolved in 20 mLTHF. After reaction for 20 minutes, compound 2.6 (1 eq., 84 mmol, 86.5 g), dissolved in 150 mL THF, was added to the above reaction solution. The reaction was kept at 15-25 C., monitored by HPLC, until it was complete. The mixture solution was filtrated with removal of insoluble matter, concentrated and subsequently separated solid out by addition of 0.1M HCl. The solid was then collected by filtration, washed to neutral by water, and then dried in vacuum to give 114.4 g compound 2.7 with 90.3% purity and 83.8% yield.
[0092] (8) Synthesis of Fragment 2:
[0093] H-Ile-Gln(Trt)-Asn(Trt)-Cys(Acm)-Pro-OMe: he dried compound 2.7 was dissolved in 150 mL diethylamine, concentrated to a minimum amount, precipitated by addition of saturated NaHCO.sub.3, filtered, washed to neutral with water, dried in vacuum, washed by stirring in petroleum ether three times, filtered, and dried in vacuum to give 91.5 g fragment 2 with 80.1% yield. The HPLC image is shown in
Example 3
[0094] Synthesis of Fragment 3 According to Solution 2: H-Leu-Gly-NH.sub.2:
[0095] Fmoc-Leu-OH (1 eq., 100 mmol, 35.3 g) and BOP (1.01 eq., 101 mmol, 44.7 g) were dissolved in 200 mL DMF and added with DIPEA (1.2 eq., 120 mmol, 21 ml). After 5 minutes, H-Gly-NH.sub.2. HCl (1.1 eq., 110 mmol, 12.2 g), dissolved in 100 mL DMF, was added with TEA (1.1 eq., 110 mmol, 15 ml), and poured into the above reaction solution. After the TLC (DCM:MeOH:AcOH=100:6:1) monitored reaction was completed, the solid product was precipitated by addition of 0.1M HCl, washed with water until neutral, and then dried in vacuum to give 43.7 g product Fmoc-Ile-Gly-NH.sub.2. HCl with 97% purity and 98% yield.
[0096] The dried Fmoc-Ile-Gly-NH.sub.2.HCl was dissolved in 400 mL diethylamine. After the reaction was completed, indicated by TLC (DCM:MeOH:AcOH=100:6:1), the reaction mixture was concentrated, precipitated by petroleum ether, filtered, washed with petroleum ether and dried in vacuum to give 17.9 g fragment 3 with 98.4% purity and 95.6% yield. The HPLC image is shown in
Example 4
[0097] Synthesis of Compound 4:
[0098] Fragment 1 (1.1 eq., 88 mmol, 45 g) and HOBt ((1.1 eq., 88 mmol, 11.9 mg) were dissolved in the mixture solvent of 150 mL THF and 60 mL DMF, and then cooled to 10 C. in an ice bath for 10 min. To the cool solution was added DCC (1.1 eq., 88 mmol, 18.1 mg) dissolved in 20 mL THF. After reaction for 20 minutes, fragment 2 (1 eq., 80 mmol, 91.5 g), dissolved in 100 mL THF, was added to the above reaction solution. The reaction was kept at 15-25 C., monitored by HPLC, until it was completed. The mixture solution was filtrated with removal of insoluble matter, concentrated and precipitated by addition of saturated NaHCO.sub.3. The solid was then collected by filtration, washed with water until neutral, and then dried in vacuum to give 115.5 g compound 4 with 86% purity and 70.6% yield. The RP-HPLC image is shown in
Example 5
[0099] Synthesis of Compound 5:
[0100] Compound 4 (1 eq., 115.5 g) was dissolve in 800 mL THF, cooled to 10 C. in an ice bath for 10 min and the reaction solution was maintained in the ice bath while 1.5M NaOH (5 eq., 353 mmol, 235 mL) was added. After the reaction was completed, indicated by HPLC, the mixture solution was regulated to neutral by addition of 0.5M HCl, concentrated to precipitate a white solid which is then filtered, washed with water and dried in vacuum to give 106.2 g compound 5 with 93% purity and 65.5% yield. The RP-HPLC image is shown in
Example 6
[0101] Synthesis of Compound 6:
[0102] Compound 5 (1 eq., 65.5 mmol, 106.2 g) and BOP (1.05 eq., 68.8 mmol, 30.4 g) were dissolve in 150 mL DMF and added with DIPEA (1.1 eq., 72 mmol, 12.5 ml). After 5 min, fragment 3 (1.1 eq., 72 mmol, 12.5 ml), dissolved in 100 mL DMF, was added to the above reaction solution. After the reaction was completed, indicated by HPLC, a white solid was precipitated by addition of 0.1M HCl, filtered, washed with water until neutral and dried in vacuum to give 107.5 g compound 6 with 88.6% purity and 60% yield. The RP-HPLC image is shown in
Example 7
[0103] Synthesis of Compound 7:
[0104] Iodine (10 eq., 600 mmol, 152 g), dissolve in 120 mL DMF, was slowly added to compound 6 (1 eq., 60 mmol, 107.5 g). After the reaction was completed, indicated by HPLC, a white solid is precipitated by addition of 0.1% sodium thiosulfate solution, filtered, washed with water and dried in vacuum to give 96.5 g compound 7 with 95% purity and 58.6% yield. The RP-HPLC image is shown in
Example 8
[0105] Synthesis of Crude Oxytocin:
[0106] Compound 7 was dissolved in 200 mL trifluoroacetic acid hydrolysis reagent (TFA:TIS:H.sub.2O=95:5:5), concentrated to a minimum amount after 10 min, added with DCM and concentrated to a minimum amount (three times), and then added with ethyl acetate, refrigerated for 10 h, filtered, and dried in vacuum to obtain 26.6 g crude oxytocin with 94.5% purity and 26.4% yield. The RP-HPLC image is shown in
Example 9
[0107] Pretreatment of the crude peptide: 0.5 g crude peptide was dissolved in 10 ml NaH.sub.2PO.sub.4 solution (0.1 M), followed by addition of acetonitrile to make the concentration of acetonitrile reach 15% in the solution, and then filtered through a 0.45 m microporous membrane.
[0108] Purification Conditions and Gradients:
[0109] Preparation column: DAC HB-50, Fuji silica gel 100 A-10 m-C18
[0110] Detection wavelength: 220 nm, flow rate: 50 ml/min
[0111] Mobile phase: A 0.1M NaH.sub.2PO.sub.4 solution, B 50% acetonitrile/A, 0-60 min: 30% B-50% B
[0112] Liquid contained oxytocin with purity above 99% was collected, concentrated at 25 C., desalted by PS polymer, added with acetic acid, concentrated, and freeze-dried to obtain 10.6 g oxytocin (the HPLC image is shown in
Example 10
[0113] Synthesis of Fragment 3 According to Solution 1:
[0114] Boc-Leu-OH (1 eq., 100 mmol, 23.1 g) and BOP (1.01 eq., 101 mmol, 44.7 g) were dissolved in 200 ml DCM and 20 ml DMF, and added with DIPEA (1.2 eq., 120 mmol, 21 ml). After 5 min, H-Gly-NH.sub.2. HCl (1.1 eq., 110 mmol, 12.2 g), dissolved in 100 ml DMF and added with TEA (1.1 eq., 110 mmol, 15 ml), was poured to the above reaction mixture, traced by TLC analysis (DCM:MeOH:AcOH=100:20:1). After the reaction was completed, the white thick material was precipitated with 0.5 M HCl solution, dissolved in EA, extracted, washed with water three times, washed with saturated NaCl once, dried by anhydrous MgSO.sub.4 for 30 min, filtered, and concentrated to obtain dry product with 99.1% purity. The HPLC image is shown in
[0115] The obtained product was added with 200 ml of 30% TFA/DCM (trifluoroacetic acid/dichloromethane) solution, after 30 minutes reaction, concentrated to dryness, and dried in vacuum to obtain 25.6 g product H-Leu-Gly-NH.sub.2 with 96% purity, and 85% yield. The HPLC image is shown in