阅读说明：本技术 一种生长抑素的制备方法 (Preparation method of somatostatin ) 是由 张启鹏 刘慧敏 刘志国 于 2021-06-09 设计创作，主要内容包括：本申请公开了一种生长抑素的制备方法,所述制备方法包括以下步骤：(S1)将R～(1)-Cys(SH)-OR～(I)通过其侧链巯基官能团装载于树脂Resin上,得到氨基酸树脂R～(1)-Cys(S-Resin)-OR～(I)；(S2)在所述氨基酸树脂R～(1)-Cys(S-Resin)-OR～(I)依次偶联其它保护氨基酸和/或保护肽片段,得到还原型的全保护线性肽树脂；(S3)将所述还原型的全保护线性肽树脂闭环氧化,得到全保护环肽；(S4)将所述全保护环肽脱除侧链保护基团,得到所述生长抑素。所述制备方法以半胱氨酸的巯基与树脂位点结合来代替常规的羧基结合,制备方法更简单、环保、节能。(The application discloses a preparation method of somatostatin, which comprises the following steps: (S1) adding R 1 ‑Cys(SH)‑OR I Loading the side chain mercapto functional group on Resin to obtain amino acid Resin R 1 ‑Cys(S‑Resin)‑OR I (ii) a (S2) in the amino acid resin R 1 ‑Cys(S‑Resin)‑OR I Sequentially coupling other protected amino acids and/or protected peptide fragments to obtain a reduced fully-protected linear peptide resin; (S3) ring-closing and oxidizing the reduced fully-protected linear peptide resin to obtain a fully-protected cyclic peptide; (S4) removing side chain protecting groups from the full-protection cyclic peptide to obtain the somatostatin. The preparation method has the advantages that the sulfydryl of the cysteine is combined with the resin sites to replace the conventional carboxyl combination, and the preparation method is simpler, environment-friendly and energy-saving.)

1. A preparation method of somatostatin, which is characterized by comprising the following steps:

(S1) adding R¹-Cys(SH)-OR^ICoupling the side chain mercapto functional group on Resin to obtain amino acid Resin R¹-Cys(S-Resin)-OR^I；

(S2) in the amino acid resin R¹-Cys(S-Resin)-OR^IOther protected amino acid and/or protected peptide fragment are coupled in sequence to obtain the reduced fully-protected linear peptide resin R¹⁴-Ala-Gly-Cys(R^H)-Lys(R^G)-Asn(R^F)-Phe-Phe-Trp(R^E)-Lys(R^D)-Thr(R^C)-Phe-Thr(R^B)-Ser(R^A)-Cys(S-Resin)-OR^I；

(S3) ring-closing and oxidizing the reduced fully-protected linear peptide resin to obtain a fully-protected cyclic peptide;

(S4) removing side chain protecting groups from the fully-protected cyclic peptide to obtain the somatostatin;

R¹、R¹⁴is a main chain amino protecting group of amino acid, and is any one selected from amino protecting groups;

R^Ia main chain carboxyl protecting group which is Cys and is any one selected from carboxyl protecting groups;

R^A、R^B、R^C、R^D、R^E、R^F、R^G、R^His a side chain protecting group of an amino acid;

wherein R is^AA side chain protecting group which is Ser, selected from any one of Ser side chain protecting groups;

R^B、R^Ca side chain protecting group which is Thr, independently selected from any one of Thr side chain protecting groups;

R^D、R^Gis a side chain protecting group of Lys, independently selected from any one of Lys side chain protecting groups;

R^Ea side chain protecting group which is Trp, selected from any one of Trp side chain protecting groups;

R^Fany one of side chain protecting groups selected from Asn side chain protecting groups, which are Asn;

R^His a side chain protecting group of Cys, and is any one of Cys side chain protecting groups;

the resin contains active sites that react with thiol groups.

2. The method for preparing the peptide of claim 1, wherein the amino protecting group is selected from any one of H, Fmoc, Boc, Dde, ivDde, Mtt, MMt and Alloc;

the carboxyl protecting group is selected from^tBu, OAll, OBzl;

the Ser side chain protecting group is selected from^tAny one of Bu, Bzl, Z;

the Thr side chain protecting group is selected from^tAny one of Bu, Bzl, Z;

the Lys side chain protecting group is selected from any one of Boc, Dde, ivDde, Mtt, MMt and Alloc;

the Trp side chain protecting group is selected from any one of Boc, For and Z;

the Asn side chain protecting group is selected from any one of Trt, Xan and Dmb;

the Cys side chain protecting group is selected from Meb, Mob, Trt, Acm, Tacm,^tAny one of Bu, H, Mtt, MMt;

the active site reacting with sulfhydryl group is selected from any one of halogen groups;

preferably, the halogen group is selected from any one of-Cl, -Br, -I;

preferably, the resin is selected from any one of Trt chloride resins;

preferably, the Trt chloride resin is selected from any one of trityl chloride resin, 2-chlorotrityl chloride resin, methyltrityl chloride resin, and methoxytrityl chloride resin;

preferably, the degree of substitution of the resin is 0.3 to 1.5 mmol/g.

3. The method of claim 1, wherein said (S1) includes the steps of:

in the presence of a resin, R¹-Cys(SH)-OR^IAdding an alkaline reagent into a raw material I of a solvent I, reacting I, and adding an end-capping reagent to react II; obtaining the amino acid resin;

preferably, the alkaline agent comprises NMM, (Et)₃N, DIEA, pyridine, sodium acetate, and/or collidineSeed growing;

the solvent I comprises at least one of DCM, NMP, DCE, TCM and DMF;

the end capping reagent comprises at least one of methanol, ethanol, n-propanol, isopropanol and tert-butanol;

preferably, the resin, R¹-Cys(SH)-OR^IThe proportion of the solvent I, the alkaline reagent and the end-capping reagent is 1 mmol: 1-3 mmol: 10-30 ml: 2-6 mmol: 0.5-3.5 ml;

preferably, the conditions of reaction I include: under the condition of inactive gas, the time is 4-5 h, and the temperature is 20-30 ℃;

preferably, the reaction II is carried out for 20-40 min at 20-30 ℃.

4. The preparation method according to claim 3, characterized in that the raw material I further contains an activating agent, and the raw material I is subjected to activating treatment;

preferably, the activator comprises at least one of potassium carbonate, sodium hydroxide, potassium hydroxide;

the activator and R¹-Cys(SH)-OR^IIn a molar ratio of 0.5 to 1: 1;

preferably, the conditions of the activation treatment include: the time is 40-60 min.

5. The process according to claim 1, wherein said sequential coupling of further protected amino acids and/or protected peptide fragments in (S2) is carried out in particular from said amino acid resin R¹-Cys(S-Resin)-OR^IThe following protected amino acids and/or protected peptide fragments are coupled in sequence:

R²-Ser(R^A)-OH、R³-Thr(R^B)-OH、R⁴-Phe-OH、R⁵-Thr(R^C)-OH、R⁶-Lys(R^D)-OH、R⁷-Trp(R^E)-OH、R⁸-Phe-OH、R⁹-Phe-OH、R¹⁰-Asn(R^F)-OH、R¹¹-Lys(R^G)-OH、R¹²-Cys(R^H)-OH、R¹³-Gly-OH、R¹⁴-Ala-OH；

or

R²-Ser(R^A)-OH、R³-Thr(R^B)-OH、R⁴-Phe-OH、R⁵-Thr(R^C)-OH、R⁶-Lys(R^D)-OH、R⁷-Trp(R^E)-OH、R⁸-Phe-OH、R⁹-Phe-OH、R¹⁰-Asn(R^F)-OH、R¹¹-Lys(R^G)-OH、R¹²-Cys(R^H)-OH、R¹⁴-Ala-Gly-OH；

Wherein R is²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵Is a main chain amino protecting group, and is any one selected from amino protecting groups;

preferably, the amino protecting group is selected from any one of H, Fmoc, Boc, Dde, ivDde, Mtt, MMt, Alloc.

6. The method according to claim 1, wherein the coupling step (S2) comprises the steps of:

removing the main chain amino protection from the amino acid resin or the peptide resin obtained by the previous coupling to obtain an intermediate;

pre-activating the solution containing the protected amino acid or protected peptide fragment to be coupled, condensing agent and solvent II, adding the intermediate to perform coupling reaction, and obtaining the reduced fully-protected linear peptide resin after coupling of all the protected amino acid and/or protected peptide fragment.

7. The production method according to claim 1, wherein the ring-closure oxidation described in (S3) comprises the steps of:

carrying out ring-closing oxidation reaction on raw materials containing reduced full-protection linear peptide resin and oxidant solution to obtain full-protection cyclic peptide;

preferably, the oxidizing agent in the oxidizing agent solution is selected from I₂、TI(TFA)₃、(SCN)₂At least one of;

the solvent in the oxidant solution is selected from methanol, ethanol, DMF, DCM, DCE, NMP, TCE, H₂At least one of O;

the concentration of the oxidant solution is 0.01-2 mol/L;

the ratio of the reduced fully-protected linear peptide resin to the oxidant solution was 1.88 g: 30-40 ml;

preferably, the conditions of the ring-closing oxidation reaction include: the reaction time is 0.5-2 h under the condition of inactive gas.

8. The method according to claim 7, further comprising a step of removing the resin after the ring-closure oxidation reaction is completed:

carrying out suction filtration on the solution after the ring-closing oxidation reaction to obtain filtrate and resin, washing the resin by using a solvent III and carrying out suction filtration to obtain filtrate, and combining the filtrates to obtain a fully-protected cyclopeptide solution;

preferably, the solvent III is selected from methanol, ethanol, DMF, DCM, DCE, NMP, TCE, H₂At least one of O.

9. The method according to claim 7, further comprising the step of removing excess oxidizing agent after obtaining the fully protected cyclic peptide solution:

adding a reducing agent solution into the fully-protected cyclic peptide solution until the solution is colorless;

preferably, the reducing agent in the reducing agent solution is selected from at least one of vitamin C and sodium thiosulfate;

the solvent in the reducing agent solution is selected from methanol, ethanol, DMF, DCM, DCE, NMP, TCE, H₂At least one of O;

the concentration of the reducing agent solution is 0.01-2 mol/L.

10. The process according to claim 1, wherein the removal of the side chain protecting group in (S3) comprises the steps of:

carrying out cracking reaction on a raw material containing the fully-protected cyclic peptide and a cracking solution;

preferably, the lysate contains TFA and a scavenger;

preferably, the scavenger is selected from Tis, H₂O, EDT, phenol, anisole;

preferably, the lysis solution is TFA, Tis and H₂O＝85～95ml：5～10ml：5～10ml；

Preferably, the ratio of the fully protected cyclic peptide to the lysis solution is 1 g: 0.5-2 ml;

preferably, the conditions of the cleavage reaction include: the cracking time is 1.0-3.0 h.

Technical Field

The application relates to a preparation method of somatostatin, belonging to the technical field of polypeptide synthesis.

Background

Somatostatin, English name Somatostatin, molecular formula C₇₆H₁₀₄N₁₈O₁₉S₂Structural formula and structural formula:

somatostatin, a growth hormone release inhibitor, is an endogenous regulatory hormone present in the human body and can inhibit the secretion of growth hormone, thyroid stimulating hormone, insulin and glucagon, inhibit the secretion of gastric acid stimulated by 5-peptide gastrin, inhibit the release of pepsin and gastrin, obviously reduce the visceral blood flow, reduce the pressure of portal vein, reduce the blood flow and pressure of collateral circulation, reduce the blood flow of liver, reduce the internal and external secretion of pancreas and the secretion of small intestine and gall bladder, reduce the activity of enzyme, protect pancreatic cells, inhibit the secretion of glucagon and influence the absorption of gastrointestinal tract and the nutritional function.

Is mainly suitable for upper gastrointestinal bleeding caused by esophageal variceal bleeding, peptic ulcer, stress ulcer and erosive gastritis caused by liver cirrhosis portal hypertension clinically, and can be used for preventing and treating acute pancreatitis and its complications, and adjuvant treatment of pancreatic, biliary and intestinal fistulas. Other functions and applications are continuously discovered along with basic research and clinical application, so that the application prospect of the somatostatin is wide, and the market demand is increasing year by year.

The liquid phase synthesis methods adopted in the reports of U.S. Pat. Nos. 4,4337194, 3862925 and 3917578 have the disadvantages of multiple reaction steps, long route, difficult purification and separation of intermediates, high material source and preparation difficulty, high cost and difficulty in meeting the requirements of the existing industrial production.

Compared with liquid phase coupling, the solid phase synthesis method of the polypeptide has the advantages of low cost, convenient operation, short route period, environment-friendly and easily obtained solvent reagent, simple post-treatment and the like. The solid phase synthesis method patents (CN1923851A, CN1508152A, CN1552728A, CN102952175A and the like) which are published at present use carrier resin to gradually or fragment couple, then use cracking reagent to crack, firstly obtain reduced somatostatin linear peptide, then obtain oxidized somatostatin cyclic peptide through liquid phase cyclization, and finally obtain the product through reversed phase preparation chromatography purification, salt conversion and freeze drying.

However, all the above patents of solid phase synthesis methods are to obtain reduced somatostatin and then obtain oxidized somatostatin by means of liquid phase cyclization. However, when the preparation is performed by the liquid phase cyclization method, in order to reduce intermolecular disulfide bond byproducts formed in the oxidation process, the sample concentration needs to be controlled not to be too high, and usually only to reach 1mg/ml or even lower, so that the reaction volume during the liquid phase cyclization reaction is very large, the scale of industrial production is restricted, and a large amount of waste liquid is generated. In addition, the oxidation reaction speed of liquid phase cyclization is generally slow, and many active sites in a polypeptide sequence also generate more byproducts during oxidation, thereby affecting the purity and the yield.

Disclosure of Invention

According to one aspect of the application, a preparation method of somatostatin is provided, wherein the thiol group of cysteine is combined with a resin site to replace the conventional carboxyl group combination, so that the reduced fully-protected linear peptide resin can be subjected to closed-loop oxidation and simultaneously falls off from the resin, and the problem that the conventional method requires that the linear peptide is firstly cracked from the resin and then subjected to liquid-phase cyclization to obtain an oxidized cyclic peptide product is avoided.

A preparation method of somatostatin, which comprises the following steps:

(S1) adding R¹-Cys(SH)-OR^ICoupling the side chain mercapto functional group on Resin to obtain amino acid Resin R¹-Cys(S-Resin)-OR^I；

(S2) in the amino acid resin R¹-Cys(S-Resin)-OR^IOther protected amino acid and/or protected peptide fragment are coupled in sequence to obtain the reduced fully-protected linear peptide resin R¹⁴-Ala-Gly-Cys(R^H)-Lys(R^G)-Asn(R^F)-Phe-Phe-Trp(R^E)-Lys(R^D)-Thr(R^C)-Phe-Thr(R^B)-Ser(R^A)-Cys(S-Resin)-OR^I；

(S3) ring-closing and oxidizing the reduced fully-protected linear peptide resin to obtain a fully-protected cyclic peptide;

(S4) removing side chain protecting groups from the fully-protected cyclic peptide to obtain the somatostatin;

R¹、R¹⁴being main-chain amino protecting groups of amino acidsAny one selected from amino protecting groups;

R^Ia main chain carboxyl protecting group which is Cys and is any one selected from carboxyl protecting groups;

R^A、R^B、R^C、R^D、R^E、R^F、R^G、R^His a side chain protecting group of an amino acid;

wherein R is^AA side chain protecting group which is Ser, selected from any one of Ser side chain protecting groups;

R^B、R^Ca side chain protecting group which is Thr, independently selected from any one of Thr side chain protecting groups;

R^D、R^Gis a side chain protecting group of Lys, independently selected from any one of Lys side chain protecting groups;

R^Ea side chain protecting group which is Trp, selected from any one of Trp side chain protecting groups;

R^Fany one of side chain protecting groups selected from Asn side chain protecting groups, which are Asn;

R^His a side chain protecting group of Cys, and is any one of Cys side chain protecting groups;

the resin contains active sites that react with thiol groups.

Optionally, the amino protecting group is selected from any one of H, Fmoc, Boc, Dde, ivDde, Mtt, MMt, Alloc;

the carboxyl protecting group is selected from^tBu, OAll, OBzl;

the Ser side chain protecting group is selected from^tAny one of Bu, Bzl, Z;

the Thr side chain protecting group is selected from^tAny one of Bu, Bzl, Z;

the Lys side chain protecting group is selected from any one of Boc, Dde, ivDde, Mtt, MMt and Alloc;

the Trp side chain protecting group is selected from any one of Boc, For and Z;

the Asn side chain protecting group is selected from any one of Trt, Xan and Dmb;

the Cys side chain protecting group is selected from Meb, Mob, Trt, Acm, Tacm,^tAny one of Bu, H, Mtt, MMt;

the active site reacting with sulfhydryl group is selected from any one of halogen groups;

optionally, the halogen group is selected from any one of-Cl, -Br, -I;

optionally, the resin is selected from any one of Trt chloride resins;

optionally, the Trt chloride resin is selected from any one of trityl chloride resin, 2-chlorotrityl chloride resin, methyltrityl chloride resin, and methoxytrityl chloride resin;

optionally, the degree of substitution of the resin is 0.3 to 1.5 mmol/g.

Optionally, the (S1) includes the steps of:

in the presence of a resin, R¹-Cys(SH)-OR^IAdding an alkaline reagent into a raw material I of a solvent I, reacting I, and adding an end-capping reagent to react II; obtaining the amino acid resin;

alternatively, the alkaline reagent comprises NMM, (Et)₃N, DIEA, pyridine, sodium acetate, and/or collidine;

the solvent I comprises at least one of DCM, NMP, DCE, TCM and DMF;

the end capping reagent comprises at least one of methanol, ethanol, n-propanol, isopropanol and tert-butanol;

optionally, the resin, R¹-Cys(SH)-OR^IThe proportion of the solvent I, the alkaline reagent and the end-capping reagent is 1 mmol: 1-3 mmol: 10-30 ml: 2-6 mmol: 0.5-3.5 ml;

optionally, the conditions of reaction I include: under the condition of inactive gas, the time is 4-5 h, and the temperature is 20-30 ℃;

optionally, the reaction II is carried out for 20-40 min at 20-30 ℃;

optionally, the raw material I also contains an activating agent, and the raw material I is subjected to activation treatment;

optionally, the activator comprises at least one of potassium carbonate, sodium hydroxide, potassium hydroxide;

optionally, the activator is reacted with R¹-Cys(SH)-OR^IIn a molar ratio of 0.5 to 1: 1;

optionally, the conditions of the activation treatment include: the time is 40-60 min.

Alternatively, said sequential coupling of further protected amino acids and/or protected peptide fragments in (S2) is specifically from said amino acid resin R¹-Cys(S-Resin)-OR^IThe following protected amino acids and/or protected peptide fragments are coupled in sequence:

R²-Ser(R^A)-OH、R³-Thr(R^B)-OH、R⁴-Phe-OH、R⁵-Thr(R^C)-OH、R⁶-Lys(R^D)-OH、R⁷-Trp(R^E)-OH、R⁸-Phe-OH、R⁹-Phe-OH、R¹⁰-Asn(R^F)-OH、R¹¹-Lys(R^G)-OH、R¹²-Cys(R^H)-OH、R¹³-Gly-OH、R¹⁴-Ala-OH；

or

R²-Ser(R^A)-OH、R³-Thr(R^B)-OH、R⁴-Phe-OH、R⁵-Thr(R^C)-OH、R⁶-Lys(R^D)-OH、R⁷-Trp(R^E)-OH、R⁸-Phe-OH、R⁹-Phe-OH、R¹⁰-Asn(R^F)-OH、R¹¹-Lys(R^G)-OH、R¹²-Cys(R^H)-OH、R¹⁴-Ala-Gly-OH；

Wherein R is²、R³、R⁴、R⁵、R⁶、R⁷、R⁸、R⁹、R¹⁰、R¹¹、R¹²、R¹³、R¹⁴、R¹⁵Is a main chain amino protecting group, and is any one selected from amino protecting groups;

optionally, the amino protecting group is selected from any one of H, Fmoc, Boc, Dde, ivDde, Mtt, MMt, Alloc.

Alternatively, the coupling described in (S2) comprises the steps of:

removing the main chain amino protection from the amino acid resin or the peptide resin obtained by the previous coupling to obtain an intermediate;

pre-activating the solution containing the protected amino acid or protected peptide fragment to be coupled, condensing agent and solvent II, adding the intermediate to perform coupling reaction, and obtaining the reduced fully-protected linear peptide resin after coupling of all the protected amino acid and/or protected peptide fragment.

Optionally, the backbone amino group removal protection specifically comprises:

carrying out deprotection reaction on raw materials containing amino acid resin or peptide resin obtained by previous coupling and deprotection agent solution;

optionally, the deprotecting agent in the deprotecting agent solution is selected from Pip, DBU, NH₂NH₂Any one of (a);

the solvent in the deprotection agent solution is selected from one or the combination of DMF, NMP, DCM, TCE and DCE;

optionally, the deprotection agent solution is selected from 5-30% of Pip, 0.5-2% of DBU and 1-3% of NH₂NH₂Any one of the above.

Optionally, the volume ratio of the amino acid resin or peptide resin obtained by the previous coupling to the deprotection agent solution is 1: 2-3;

optionally, the deprotection reaction conditions include: under the condition of inactive gas, the time is 5-40 min;

optionally, the condensing agent is selected from any one of the following combinations of condensing agents: DIC/HOBt, DIC/HOOBt, DIC/HOAt, HBTU/NMM, PyBOP/DIEA;

the solvent II comprises DCM and/or DMF;

optionally, the ratio of the amino acid resin or peptide resin from the previous coupling, the protected amino acid and/or protected peptide fragment to be coupled, the condensing agent, solvent II is 1 mmol: 1.5-3 mmol: 3-6 mmol: 10-50 ml;

optionally, the pre-activation conditions include: the preactivation time is 5-15 min, and the preactivation temperature is 0-10 ℃;

optionally, the coupling reaction conditions are: under the condition of inactive gas, the coupling reaction time is 1-2 h, and the coupling reaction temperature is 25-35 ℃.

Alternatively, the closed-loop oxidation described in (S3) includes the steps of:

carrying out ring-closing oxidation reaction on raw materials containing reduced full-protection linear peptide resin and oxidant solution to obtain full-protection cyclic peptide;

optionally, the oxidant in the oxidant solution is selected from I₂、TI(TFA)₃、(SCN)₂At least one of;

the solvent in the oxidant solution is selected from methanol, ethanol, DMF, DCM, DCE, NMP, TCE, H₂At least one of O;

the concentration of the oxidant solution is 0.01-2 mol/L;

the ratio of the reduced fully-protected linear peptide resin to the oxidant solution was 1.88 g: 30-40 ml;

optionally, the conditions of the ring-closing oxidation reaction include: the reaction time is 0.5-2 h under the condition of inactive gas.

Optionally, after the ring-closing oxidation reaction is finished, the method further comprises the step of removing the resin: carrying out suction filtration on the solution after the ring-closing oxidation reaction to obtain filtrate and resin, washing the resin by using a solvent III and carrying out suction filtration to obtain filtrate, and combining the filtrates to obtain a fully-protected cyclopeptide solution;

alternatively, the solvent III is selected from methanol, ethanol, DMF, DCM, DCE, NMP, TCE, H₂At least one of O;

optionally, the number of washes is 2.

Optionally, after obtaining the fully protected cyclic peptide solution, the method further comprises the step of removing the excess oxidizing agent:

adding a reducing agent solution into the fully-protected cyclic peptide solution until the solution is colorless;

optionally, the reducing agent in the reducing agent solution is selected from at least one of vitamin C and sodium thiosulfate;

the solvent in the reducing agent solution is selected from methanol, ethanol, DMF, DCM, DCE, NMP, TCE, H₂At least one of O;

the concentration of the reducing agent solution is 0.01-2 mol/L.

Alternatively, the removal of the side chain protecting group described in (S3) comprises the steps of:

carrying out cracking reaction on a raw material containing the fully-protected cyclic peptide and a cracking solution;

optionally, the lysate contains TFA and a scavenger;

optionally, the scavenger is selected from Tis, H₂O, EDT, phenol, anisole;

optionally, the lysing solution is TFA, Tis, H₂O＝85～95ml：5～10ml：5～10ml；

Optionally, the ratio of the fully protected cyclic peptide to the lysis solution is 1 g: 0.5-2 ml;

optionally, the conditions of the cleavage reaction include: the cracking time is 1.0-3.0 h.

As an embodiment, the present application provides a method for preparing somatostatin, comprising the steps of:

step 1, adding a resin carrier into a reactor, swelling and then extracting a solvent;

step 2, Fmoc-Cys (SH) -O^tBu is activated and then added into a reactor, a certain equivalent of alkali is added under stirring, nitrogen is introduced for reaction for 4-5 hours, then methanol is added for reaction for 30min, suction filtration is carried out, and the Resin is washed and dried to finally obtain Fmoc-Cys (S-Resin) -O^tBu, where the thiol group of cysteine is bound to a resin site instead of the conventional carboxyl binding;

step 3, coupling each protected amino acid in a somatostatin peptide sequence in sequence by adopting a general Fmoc strategy of polypeptide solid phase synthesis to obtain fully-protected linear peptide resin;

step 4, swelling the obtained linear peptide resin by using a solvent, adding an oxidant under stirring to perform disulfide bond cyclization on the resin, transferring a product from the resin to a solution while performing cyclization reaction, filtering the resin, and collecting filtrate to obtain an oxidized fully-protected cyclic peptide crude peptide solution;

step 5, adding a reducing agent to remove redundant oxidizing agent;

step 6, extracting, washing and rotary steaming to obtain a fully-protected crude cyclic peptide solid;

step 7, removing side chain protecting groups from the fully-protected crude cyclic peptide solid by using a lysate, adding a settling solvent ether for settling, and centrifuging to obtain the crude somatostatin cyclic peptide;

and 8, performing reverse phase preparation and purification, salt conversion and freeze drying on the cyclic peptide crude peptide through a C18 bonded silica gel column to obtain a final product.

The preparation method described above, wherein the step 1 is swelling a resin carrier selected from the group consisting of: trityl chloride resin, 2-chlorotrityl chloride resin, methyltrityl chloride resin, methoxytrityl chloride resin and other Trt series resins, and in some embodiments, the resin support is more preferably: the substitution degree of the 2-chlorotrityl chloride resin is 0.3 to 1.5mmol/g, and the more preferable substitution degree is 0.5 to 1.0 mmol/g.

The preparation method as described above, wherein the step 2 is to subject the starting material Fmoc-Cys (SH) -O^tBu is activated and then assembled with a resin carrier in a reaction solvent. Wherein the activating reagent is selected from: potassium carbonate, sodium hydroxide, potassium hydroxide, and in some embodiments, potassium carbonate is preferred; the reaction solvent is selected from: DCM, NMP, DCE, TCM, DMF, etc., and in some embodiments, the reaction solvent is more preferably: DCM, DMF or a mixed DMF/DCM solvent; the added base is selected from: NMM, (Et)₃N, DIEA, pyridine, sodium acetate, collidine, etc., and in some embodiments, the reaction reagent is more preferably: DIEA, pyridine, NMM, (Et)₃N; the reaction temperature is 20-30 ℃.

The preparation method as described above, wherein the step 3 is a step of coupling each protected amino acid Fmoc-Ser (in the peptide sequence) in sequence^tBu)-OH、Fmoc-Thr(^tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(^tBu)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Trp(Boc)-OH、Fmoc-Phe-OH、Fmoc-Phe-OH、Fmoc-Asn(Trt)-OH、Fmoc-Lys(Boc)-OH、Fmoc-Cys(R^H) -OH, Fmoc-Gly-OH, Fmoc/Boc-Ala-OH; wherein R is^HSelected from Meb, Mob, Trt, Acm, Tacm,^tBu, H, Mtt, MMt, etc., and in some embodiments, R^HPreferably Trt, Acm, Mtt, MMt; the condensing agent is selected from: DIC/HOBt, DIC/HOAt, HBTU/NMM, PyBOP/DIEA, etc., preferably DIC/HOBt.

The protected amino acids in the above coupling peptide sequence can be coupled or fragment-linked one by one in different protection modes (such as Fmoc/Boc-Ala-OH, Fmoc/Boc-Ala-Gly-OH and the like).

The production method described above, wherein the step 4 is to subject the linear peptide resin to on-resin cyclization. The cyclization reaction solvent is selected from: DMF, NMP, DCM, tetrahydrofuran, DCE, TCM, ACN, ethanol, methanol, H₂O, and the like, singly or in combination, and in some embodiments, preferably DCM; the oxidant is selected from: i is₂、TI(TFA)₃、(SCN)₂In some embodiments, it is preferably I₂。

The preparation method described above, wherein the step 5 is to add a reducing agent to the fully protected crude cyclic peptide solution to remove excess oxidizing agent, wherein the reducing agent is selected from: vitamin C, sodium thiosulfate, and the like.

The preparation method described above, wherein the step 6 is extracting, washing, rotary steaming to obtain the fully protected crude cyclic peptide solid, and in some embodiments, the fully protected crude cyclic peptide solid can also be obtained by adjusting pH, filtering and drying.

In the preparation method, in the step 7, the side chain protecting group is removed by using a lysate, and the crude somatostatin cyclic peptide is obtained by adding ether for sedimentation and centrifugation. The lysis solution contains TFA and scavenger selected from TIS and H₂O, EDT, phenol, anisole, etc., preferably the lysate combination is TFA/TIS/H₂O, in some embodiments the volume ratio of the lysing solution is: TFA TIS H₂O＝90:5:5。

The existing patent routes are that the carboxyl terminal of cysteine at the tail end of a peptide sequence is combined with a resin site, amino acids are coupled one by one to obtain reduced type full-protection peptide resin, then a large amount of lysis solution is needed to be used for carrying out pyrolysis on the resin, the obtained pyrolysis combined solution needs to be subjected to sedimentation, washing and centrifugation by using a large amount of ether, and the obtained reduced type product needs to be dissolved by using a large amount of solvent and then subjected to liquid phase cyclization to obtain an oxidized cyclic peptide product.

The invention has the advantages that: the process comprises the steps of combining sulfydryl of cysteine with resin sites to replace conventional carboxyl, coupling amino acids one by one to obtain reduced type full-protection peptide resin, firstly using a small amount of solvent to complete cyclization on the resin, automatically dropping oxidized cyclopeptide from the resin, collecting filtrate, carrying out rotary evaporation to obtain the full-protection cyclopeptide crude peptide, then using a small amount of cracking solution to crack and remove side chain protecting groups, adding a small amount of ether to carry out sedimentation, washing and centrifugation to obtain the cyclopeptide crude peptide.

The cyclic peptide obtained by the process can automatically fall off from the resin, the peptide resin cracking is not needed, the operation is simple and convenient, the production amount of the solvent/reagent and the waste liquid is greatly reduced, the byproducts generated in the peptide resin cracking process can be avoided, and the purity of the crude peptide is improved. Meanwhile, the oxidation type full-protection cyclic peptide is directly cyclized on the solid-phase resin in one step without liquid-phase cyclization operation, and due to the false dilution effect of active sites on the solid-phase resin, the generation of mistaken impurities such as intermolecular dimerization, multimerization and the like can be avoided, so that the oxidation reaction for cyclizing on the resin can be carried out at a higher concentration, the use of solvents is reduced, and the industrial expanded production is facilitated.

The english/abbreviation of the present application is described below:

the beneficial effects that this application can produce include:

(1) according to the preparation method of the somatostatin, the sulfydryl of cysteine is combined with the resin site to replace the conventional carboxyl combination, the subsequent oxidation and cyclization processes of the linear peptide can be simultaneously carried out on the resin by the aid of the first amino acid fixing strategy, the linear peptide can fall off from the resin after cyclization is completed, and the problem that the linear peptide is firstly cracked from the resin and then subjected to liquid phase cyclization to obtain an oxidized cyclic peptide product in the conventional method is avoided.

(2) According to the preparation method of the somatostatin, the obtained linear peptide resin is subjected to disulfide bond cyclization on the resin under the action of the oxidant, the using amount of the solvent is small, the steps are simple, and compared with the scheme that in the traditional process, a large amount of lysate is used for cracking the resin, a large amount of ether is used for settling, washing and centrifuging the obtained cracking combined solution, and the oxidized cyclic peptide product can be obtained only by performing liquid phase cyclization after the obtained reduced product is dissolved by using a large amount of solvent, the preparation method is more environment-friendly and energy-saving.

Drawings

FIG. 1 is a schematic diagram of a process for preparing somatostatin according to the present application.

Detailed Description

The present application will be described in detail with reference to examples, but the present application is not limited to these examples.

The raw materials in the examples of the present application were all purchased commercially, unless otherwise specified.

The preparation process route of the somatostatin is schematically shown in figure 1.

Example 1: preparation of somatostatin

(1)Fmoc-Cys(S-CTC Resin)-O^tPreparation of Bu resin

2.3g (2mmol) of 2-CTC Resin (the degree of substitution is 0.88mmol/g) is weighed into a glass sand core reactor, 30ml of DCM is added, the mixture is stirred and swelled for 1h at room temperature, and the solvent is removed for standby.

1.60g (4mmol) of Fmoc-Cys (SH) -O were weighed^tBu、2.76g(2mmol)K₂CO₃Reaction in 20ml DCM for 1h, filtration, addition of filtrate to the above reactor, slow addition of 2.06ml DIEA (12mmol) with stirring, N addition₂Controlling the temperature to be 25 ℃, stirring and reacting for 5h, adding 2.3ml of methanol, continuing to react for 30min, performing suction filtration after the reaction is finished, washing the resin for 3 times (30 ml each time) by using DMF (dimethyl formamide), washing for 2 times (30 ml each time) by using DCM (DCM) and washing for 2 times (20 ml each time) by using methyl tert-butyl ether in sequence, and drying under reduced pressure (40 ℃ and-0.09 Mpa) to obtain Fmoc-Cys (S)-CTC Resin)-O^t2.9g of Bu resin, and the detected resin substitution degree is 0.55 mmol/g.

(2)Boc-Ala-Gly-Cys(Trt)-Lys(Boc)-Asn(Trt)-Phe-Phe-Trp(Boc)-Lys(Boc)-Thr(^tBu)-Phe-Thr(^tBu)-Ser(^tBu)-Cys(S-CTC Resin)-O^tPreparation of Bu peptide resin

Swelling: weighing Fmoc-Cys (S-CTC Resin) -O^t2.9g of Bu (1.6mmol, the substitution degree is 0.55mmol/g) is added into a sand core reactor, 20ml of DMF is added for swelling for 1 hour, and the solvent is pumped out;

deprotection: to the reactor was added 3 resin volumes of 20% Pip/DMF and N was added₂Carrying out bubbling reaction for 30min, washing for 6 times by using DMF (dimethyl formamide) with the volume 3 times that of the resin after deprotection for later use;

pre-activation: weighing 1.22g of Fmoc-Ser (A)^tBu) -OH (3.2mmol) and 0.43g HOBt (3.2mmol) are added into a beaker, dissolved in 20ml of DMF, and then 0.50ml (3.2mmol) of DIC is added to activate for 10min at 0-10 ℃;

coupling: adding the activating solution into the reactor after deprotection, and introducing N₂Bubbling reaction, intermediate sampling, detecting with ninhydrin to determine reaction end point, and washing resin with DMF 3 times the volume of the resin for 4 times after coupling to obtain Fmoc-Ser (ii)^tBu)-Cys(S-CTC Resin)-O^tBu resin.

Repeating the above deprotection, preactivation and coupling procedures to couple Fmoc-Thr (F-Fmoc-Thr^tBu)-OH、Fmoc-Phe-OH、Fmoc-Thr(^tBu) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Phe-OH, Fmoc-Asn (Trt) -OH, Fmoc-Lys (Boc) -OH, Fmoc-Cys (Trt) -OH, Boc-Ala-Gly-OH, washing the resin 4 times with DMF after coupling is finished, washing 2 times with DCM, washing 2 times with MTBE, drying under reduced pressure (40 ℃, -0.09MPa) to obtain the dry peptide resin Boc-Ala-Gly-Cys (Trt) -Lys (Boc) -Asn (Trt) -Phe-Trp (Boc) -Lys (Boc) -Thr (Boc) (Trt)^tBu)-Phe-Thr(^tBu)-Ser(^tBu)-Cys(S-CTC Resin)-O^tBu 7.52g (yield 98.4%).

(3) Preparation of somatostatin crude peptide solid

3.76g of the above dried peptide resin was added to the reactor and re-swollen with DCM and the solution was removed. Adding into a reactor80ml of 0.05mol/L I₂Per DCM solution, N₂Reacting for 2h, performing suction filtration to obtain filtrate containing the full-protection somatostatin cyclopeptide which falls off from the resin, washing the resin with DCM for 2 times, and repeating the operation for 2 times. All filtrates from 3 treatments were combined to give 240ml of fully protected cyclic peptide solution.

Slowly adding 0.05mol/L vitamin C/methanol solution into 240ml of the all-protected somatostatin cyclic peptide solution until the solution becomes colorless, and removing the solvent by rotary evaporation to obtain 2.58g of the all-protected somatostatin cyclic peptide solid (the yield is 99.3%). The solid was treated with 2ml of a lysis solution (TFA: Tis: H)₂O-90: 5:5, v: v: v) cracking for 1.5h, adding 10ml of methyl tert-butyl ether for settling, centrifuging the obtained suspension, pulping and washing the lower layer of solid for 2 times by using the methyl tert-butyl ether, drying the wet product under reduced pressure (35-45 ℃ and-0.06-1.0 Mpa) to obtain 1.55g of somatostatin cyclic peptide crude peptide (the yield is 94.9 percent), and the HPLC purity is 77.74 percent. The detection profile data are shown in table 1.

TABLE 1 detection profile data for crude somatostatin

Retention time	Peak area	Peak area%
			13.535	37.466	0.1368
15.269	329.6971	1.2036
			16.403	2.12953e4	77.7392
18.680	430.40137	1.5712
			20.086	112.30130	0.4100
22.782	1526.44116	5.5723
			33.466	413.69553	1.5102

(4) Preparation of somatostatin

1.55g of the crude cyclic peptide was weighed and dissolved in 100ml of purified water, followed by filtration through a 0.45um membrane.

Adopting high performance liquid chromatography, selecting 50mm C18 filler to prepare a column, loading a crude peptide solution, taking 1% triethylamine phosphate (pH2.0) as a mobile phase system A and acetonitrile as a mobile phase B, performing gradient elution for 60min according to 10% -40% of the phase B, and collecting qualified fractions.

Desalting and trans-acetate treating qualified fraction, re-sampling the qualified fraction, eluting with 0.5% acetic acid/water as mobile phase A and acetonitrile as mobile phase B for 60min according to gradient of 20-40% of phase B, and collecting qualified fraction.

The above-mentioned qualified fraction is filtered by using 0.22um filter membrane, and freeze-dried so as to obtain 0.65g of somatostatin finished product (yield is 39.7%) whose purity is 99.84%. The detection profile data are shown in table 2.

TABLE 2 somatostatin product detection profile data

Example 2: preparation of somatostatin

The procedure for the preparation of example 2 is the same as in example 1, except that step (1) is different.

Step (1) of example 2 is as follows:

(1)Fmoc-Cys(S-CTC Resin)-O^tbu resin preparation

2.3g (2mmol) of 2-CTC Resin (the degree of substitution is 0.88mmol/g) is weighed into a glass sand core reactor, 30ml of DCM is added, the mixture is stirred and swelled for 1h at room temperature, and the solvent is removed for standby.

1.60g (4mmol) of Fmoc-Cys (SH) -O were weighed^tBu was added to the above reactor, 20ml DCM was added, 2.06ml DIEA (12mmol) was added slowly with stirring, N was added₂Controlling the temperature to be 25 ℃, stirring and reacting for 5h, adding 2.3ml of methanol, continuing to react for 30min, performing suction filtration after the reaction is finished, washing the Resin for 3 times by using DMF (dimethyl formamide), washing for 2 times by DCM (DCM), washing for 2 times by using methyl tert-butyl ether, and drying under reduced pressure (40 ℃ and-0.09 Mpa) to obtain Fmoc-Cys (S-CTC Resin) -O^tBu resin 2.58g, through the detection of resin substitution degree of 0.35 mmol/g.

2.58g of Fmoc-Cys (S-CTC Resin) -O prepared in step (1) of example 2 was used^tBu Resin was used in place of 2.9g of Fmoc-Cys (S-CTC Resin) -O prepared in step (1) of example 1^tBu resin, the same procedures and conditions as in example 1 were used for the subsequent experiments.

Example 3: preparation of somatostatin

The procedure for the preparation of example 3 is the same as in example 1, except that step (3) is different.

Step (3) of example 3 is as follows:

(3) preparation of somatostatin crude peptide solid

1.88g of the above dried peptide resin was taken and added to the reactor and re-swollen with 20ml of DCM, and the solvent was removed. 40ml of 0.05mol/L I are added to the reactor₂Methanol solution, N₂Reacting for 2h, performing suction filtration to obtain a filtrate containing the full-protection somatostatin cyclic peptide which falls off from the resin, washing the resin with methanol, and performing suction filtration for 2 times. All filtrates from 3 treatments were combined to give 180ml of fully protected somatostatin cyclopeptide solution.

To 180ml of the all-protected somatostatin cyclic peptide solution, 0.05mol/L of vitamin C/methanol solution was slowly added until the solution became colorless, and 2.37g of the all-protected somatostatin cyclic peptide solid was obtained after the solvent was removed by rotary evaporation (yield 95.9%). The solid was treated with 2ml of a lysis solution (TFA: Tis: H)₂O-90: 5:5, v: v: v) cracking for 1.5h, adding 10ml of methyl tert-butyl ether for sedimentation, centrifuging the obtained suspension, pulping and washing the lower layer of solid for 2 times by using the methyl tert-butyl ether, and drying the wet product under reduced pressure (40 ℃ and-0.09 Mpa) to obtain 0.66g of somatostatin cyclopeptide crude peptide (the yield is 80.4%).

The subsequent experiment was carried out by the same procedure and conditions as in example 1, using 0.66g of the crude somatostatin cyclic peptide prepared in step (3) of example 3 instead of 1.55g of the crude somatostatin cyclic peptide prepared in step (3) of example 1.

Although the present application has been described with reference to a few embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the application as defined by the appended claims.