CN113801197B

CN113801197B - Preparation method of procalcitonin

Info

Publication number: CN113801197B
Application number: CN202110886927.XA
Authority: CN
Inventors: 刘宝生; 张巍; 李湘; 徐琪
Original assignee: Chinese Peptide Co
Current assignee: Chinese Peptide Co
Priority date: 2021-08-03
Filing date: 2021-08-03
Publication date: 2023-11-03
Anticipated expiration: 2041-08-03
Also published as: CN113801197A

Abstract

The invention discloses a solid-phase fragment synthesis method of procalcitonin, which belongs to the technical field of polypeptide synthesis, wherein 4-16 fragments are connected one by using a solid-phase synthesis method, then 1-3 fragments are connected to a 4-16 fragment solid-phase resin to obtain a full peptide resin, and acidolysis is carried out synchronously to remove side chain protecting groups and cleave peptides, so as to obtain a crude product of procalcitonin, and purification and freeze-drying are carried out to obtain a pure product of procalcitonin. The invention adopts the protected amino acid fragment Fmoc-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -OH as the raw material, reduces the generation of impurities, shortens the synthesis period, and has high purity of the obtained crude product, easy purification and better application prospect.

Description

Preparation method of procalcitonin

Technical field: the invention relates to the field of polypeptide medicine preparation, in particular to a method for preparing procalcitonin by a solid phase method

The background technology is as follows: the platanide is an analogue of Uroguanylin (UGN), has the effect of promoting guanylate cyclase receptor agonist of natriuresis, can regulate acid-base ions in gastrointestinal tract, induce liquid transport into gastrointestinal tract, and increase gastrointestinal peristalsis, and can be used for treating chronic idiopathic constipation (chronic idiopathic constipation, CIC) and constipation-predominant irritable bowel syndrome (IBS-C) of adults.

The procalcitonin is a 16 peptide which is difficult to prepare, contains double disulfide bonds in the sequence and has a unique space winding structure, and is extremely easy to generate polycondensation in the solid phase connection process, so that the synthesis is difficult. Patent CN105764916a reports the synthesis of procalcitonin by solid-liquid phase combination, first solid phase synthesis of 1-6 fragment 1, solid phase synthesis of 7-14 fragment 2, liquid phase synthesis of 15-16 fragment 3, then sequentially ligating fragments 2,3 in liquid phase, and then ligating fragment 1 to obtain procalcitonin linear peptide. The method has the advantages of complex process, more intermediates, complex operation and high production cost. Patent CN104628827B reports a stepwise synthesis of procalcitonin, wherein Cys is protected with StBu, mmt, acm and oxidized on a solid phase resin. The method has higher difficulty in removing the Cys protecting group. And the oxidation is carried out on the resin, the reaction control difficulty is high, and the complete oxidation cannot be ensured. Patent CN108864254a reports solid phase synthetic peptide resins, with Cys at positions 4,12 protected with StBu; the amino acid at positions 7 and 15 replaces Cys amino acid with 3-Cl-Ala. In the method, in the step of converting 3-Cl-Ala into Cys, the risk of incomplete conversion exists, which is unfavorable for large-scale production. Patent CN107383170a reports a method for solid phase synthesis of procalcitonin, which uses air natural oxidation method to construct two pairs of disulfide bonds. Because the procalcitonin has two pairs of disulfide bonds, natural oxidation can generate two mispronounced peptides, topoisomerase and multimer, which is unfavorable for mass production. Patent CN1080003222a reports a method for solid phase synthesis of procalcitonin, first liquid phase stepwise synthesis of fully protected 1-6 fragments, then solid phase synthesis of 7-16 fragments, and then enzymatic ligation of the 6 peptide fragments to the peptide resin. The method has great difficulty in synthesizing 1-6 by a one-step method, and is not suitable for industrial production.

The procalcitonin is an oral polypeptide raw material medicine with the market demand reaching the level of hundred kilograms, and the patent has a certain technical novelty, but has large-scale production and great difficulty. In the research of the procalcitonin, the specificity of the procalcitonin sequence is found to cause the polycondensation phenomenon to be easy, and the procalcitonin belongs to difficult peptide, and the coupling is difficult when the procalcitonin is connected by a conventional method, so that amino acid with a large steric hindrance protecting group is required to be added, the secondary structure of the procalcitonin is changed, and the polycondensation phenomenon is relieved. In addition, when the last three amino acids are linked in a solid phase, it is found that impurities such as β -Asp, assu, D-Asp, D-Asn, and missing peptides are easily generated, and the original developer is analyzed to find that these impurities are controlled at an extremely low level, and an appropriate method is required to solve the problem of the high content of the impurities.

The invention comprises the following steps:

in order to solve the above problems, it is necessary to develop a method for preparing procalcitonin which is easy to synthesize and has a low impurity content. In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

a method for preparing procalcitonin, comprising:

step 1) Fmoc-Leu-OH was attached to the solid phase resin.

Step 2) connecting 4-16 fragments one by one according to the sequence by utilizing a solid phase synthesis method to obtain H-Cys (Trt) -Glu (OtBu) -Leu-Cys (Acm) -Val-Asn (Trt) -Val-Ala-Cys (Trt) -Thr (tBu) - (Hmb) Gly-Cys (Acm) -Leu-Wang resin.

The tripeptide fragment Fmoc-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -OH was ligated to the 4-16 fragment peptide resin described above to give, after removal of Fmoc protection, a H-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -Cys (Trt) -Glu (OtBu-Leu-Cys (Acm) -Val-Asn (Trt) -Val-Ala-Cys (Trt) -Thr (tBu) - (Hmb) Gly-Cys (Acm) -Leu-Wang resin.

Or connecting a tripeptide fragment Boc-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -OH to the 4-16 fragment peptide resin to obtain Boc-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -Cys (Trt) -Glu (OtBu) -Leu-Cys (Acm) -Val-Asn (Trt) -Val-Ala-Cys (Trt) -Thr (tBu) - (Hmb) Gly-Cys (Acm) -Leu-Wang resin.

And 3, cracking the resin, and simultaneously removing side chain protecting groups to obtain a crude product of the procalcitonin.

And 4, oxidizing to form two pairs of disulfide bridges respectively, and purifying to obtain pure product of the procalcitonin.

Preferably, the resin used in step 1) is Wang resin or CTC resin, and the degree of substitution of the peptide resin Fmoc-Leu-resin is 0.2 to 0.6mmol/g.

Preferably, step 2) uses Hmb protected Gly to effectively prevent condensation of peptide chains.

Preferably, fmoc-Cys (Trt) -OH is used in position 4,12 of step 2). Fmoc-Cys (Acm) -OH was used at positions 7, 15.

Preferably, step 2) uses Fmoc-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -OH or Boc-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -OH fragments in 1.2-4 fold

Preferably, the coupling reagent used in step 2) is a mixture of HOBt and DIC, or HBTU with an organic base. Wherein the organic base is selected from one of DIPEA and Et3N, NMM. More preferably a combination of HOBt and DIC.

Preferably, the cleavage reagent used in step 3) comprises a combination of TFA, EDT, TIS, phenol, anisole sulfide, phenyl sulfide, and water. More preferably the TFA to EDT to TIS to H2O ratio is 86.5:4.5:4.5:4.5

Preferably, the oxidizing agent used in the step 4) comprises hydrogen peroxide and iodine, the oxidation concentration is 0.5-2g/L, and the oxidation end point is judged by HPLC (high performance liquid chromatography) central control. The purification adopts reversed phase high performance liquid preparation chromatographic column separation and purification.

The invention adopts the amino acid Fmoc- (Hmb) Gly-OH, fmoc-Cys (Acm) -OH and fragment R-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -OH with special protection, and has the following advantages:

1. can prevent the condensation polymerization of peptide chains and reduce the coupling difficulty

Cys adopts an orthogonal protecting group strategy, and can directionally construct two pairs of disulfide bonds

3. The fragment R-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -OH is adopted to control the sources of racemized impurities, degraded impurities, missing peptides and other impurities in the finished product on raw materials, so that the quality of the product is ensured to be superior to that of an original grinding preparation.

The special amino acid or fragment adopted by the invention is purchased or self-made, establishes the quality standard matched with the quality standard of the finished product, has short production period, small production difficulty and stable quality of the finished product, and can meet the industrial scale production.

Detailed Description

The embodiment of the invention discloses a preparation method of procalcitonin. Modifications of process parameters including segmentation methods and condensation conditions can be made by those skilled in the art in light of the present disclosure. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included within the present invention. While the method of the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the method described herein without departing from the spirit and scope of the invention.

The invention is further illustrated by the following examples.

The meanings of the abbreviations used in the specification and claims are listed in the following table:

english abbreviations	Chinese meaning
		DIC	Diisopropylcarbodiimide
HOBt	1-hydroxybenzotriazoles
		HBTU	O-benzotriazol-tetramethylurea hexafluorophosphate
DIPEA	N, N-diisopropylethylamine
		NMM	N-methylmorpholine
Et ₃ N	Triethylamine
		DMF	N, N-dimethylformamide
DCM	Dichloromethane (dichloromethane)
		Trt	Trityl radical
tBu	Tert-butyl group
		OtBu	Tert-butoxy radical
Boc	Boc-group

Example 1:

weighing blank Wang resin with the substitution degree of 0.75mmol/g, adding Fmoc-Leu-OH with the substitution degree of 2 times, mixing, adding DCM for dissolving, adding pyridine with the substitution degree of 4 times of amino acid, slowly adding 2, 6-dichlorobenzoyl chloride with the substitution degree of 8 times, stirring for 3 hours, transferring to a reaction column, pumping out the reaction liquid, washing with DMF for 3 times, washing with methanol for 1 time, washing with DMF for 3 times, washing with methanol for 3 times, pumping out the solvent, and vacuum drying to obtain Fmoc-Leu-Wang resin, wherein the detection substitution degree is 0.43mmol/g.

Fmoc-Leu-Wang resin with the substitution degree of 0.43mmol/g is accurately weighed, 47.6g is put into a solid phase reactor, DMF with the volume of 2-3 times of the resin is added to swell for 2 hours, 20% PIP/DMF solution is added to stir for 30 minutes after DMF is pumped out to remove Fmoc protecting groups, then the resin is washed by DMF for 6 times, each time for 2 minutes, after ninhydrin detection is positive, activated amino acid solution (Fmoc-Cys (Acm) -OH24.9g and HOBt8.1g are added to a beaker to dissolve in a small amount of DMF, DIC9.3mL is added to activate for 15 minutes under ice bath) and then the reaction solution is pumped out after ninhydrin detection is negative, and the reaction solution is washed by DMF for 3 times and pumped out.

The above operations are then performed sequentially: deprotection, washing, coupling, washing operations. Wherein the coupled Fmoc protected amino acid is Fmoc- (Hmb) Gly-OH, fmoc-Thr (tBu) -OH, fmoc-Cys (Trt) -OH, fmoc-Ala-OH, fmoc-Val-OH, fmoc-Asn (Trt) -OH, fmoc-Val-OH, fmoc-Cys (Acm) -OH, fmoc-Leu-OH, fmoc-Glu (OtBu) -OH, fmoc-Cys (Trt) -OH in sequence. The coupling temperature was 25℃and the end point of each amino acid reaction was detected by Kaiser test to give an H-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -Cys (Trt) -Glu (OtBu) -Leu-Cys (Acm) -Val-Asn (Trt) -Val-Ala-Cys (Trt) -Thr (tBu) - (Hmb) Gly-Cys (Acm) -Leu-Wang resin.

Activated amino acid solution (Fmoc-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -OH57.2g and HOBt8.1g were added in a beaker to dissolve, DIC9.3mL was added under ice bath to activate for 15 min) and reacted at 25℃for 1.5 hours, 20% PIP/DMF solution was added and stirred for 30 min, after the reaction was completed DMF was washed 6 times, ninhydrin detection was negative, the reaction solution was drained, DMF was washed 4 times, methanol was washed 2 times, DCM was washed 1 time, meOH was washed 3 times and drained. The resulting resin was dried under vacuum to give 99g of H-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -Cys (Trt) -Glu (OtBu) -Leu-Cys (Acm) -Val-Asn (Trt) -Val-Ala-Cys (Trt) -Thr (tBu) - (Hmb) Gly-Cys (Acm) -Leu-Wang resin.

The TFA/H2O/EDT/TIS was prepared as a cutting fluid 1000mL in a volume ratio of 86.5:4.5:4.5:4.5, and stored at-5℃for further use. The cut-off (8-10 mL/g crude) was added to the reactor and the stirrer was started. And adding the procalcitonin peptide resin into the cutting fluid, controlling the temperature of the mixture in the reactor to be about 25 ℃ after the addition, starting to record the reaction time, reacting for 3 hours, filtering, and collecting the filtrate.

10L of glacial ethyl ether with the volume 10 times of that of the cutting fluid is measured and added into a precipitator, filtrate is slowly added into the precipitator, and a large amount of solid precipitate is formed by stirring. Transferring the mixture in the precipitator to a filter, filtering to remove supernatant, and washing filter residues with diethyl ether; the filtration and washing were repeated three times. The obtained product is dried in vacuum until the weight is constant, 36.5g of the linear peptide of the procalcitonin is obtained, the purity of the crude product is 81.1, and the yield of the crude product is 100%.

Weighing 30g of the procalcitonin linear peptide, adding the solution into 15L of water with the concentration of 2g/L, adding hydrogen peroxide for oxidation, reacting for 3 hours, sampling, and performing HPLC central control monitoring until the reaction is complete. Adding iodine solution, reacting for 3 hours, sampling, performing HPLC central control monitoring until the reaction is complete, and obtaining the crude solution of the procalcitonin.

The crude solution of procalcitonin was diluted and purified by loading onto a C18 reverse phase chromatography column. Collecting sample peaks and qualified samples, desalting, freeze-drying to obtain the procalcitonin, wherein the refined peptide yield in the purification step is 39.9%, the purity is 98.7%, and compared with the original developer, the new simple substances are less than 0.1%.

The conditions and reaction steps of the following examples are the same as in example 1, except for the following table:

Claims

1. a method of synthesizing procalcitonin, said method comprising the steps of:

step 1, fmoc-Leu-OH is connected to solid phase resin,

step 2, sequentially connecting the rest of protective amino acids or peptide fragments under the action of a coupling reagent according to the sequence from the C end to the N end of the amino acid sequence of the procalcitonin to obtain the complete protective peptide resin of the procalcitonin, wherein the complete protective peptide resin comprises the following specific steps:

the 4-16 fragments are connected one by one according to the sequence by utilizing a solid phase synthesis method to obtain H-Cys (Trt) -Glu (OtBu) -Leu-Cys (Acm) -Val-Asn (Trt) -Val-Ala-Cys (Trt) -Thr (tBu) - (Hmb) Gly-Cys (Acm) -Leu-Wang resin;

the tripeptide fragment Fmoc-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -OH was ligated to the 4-16 fragment peptide resin described above to give, after removal of Fmoc protection, a H-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -Cys (Trt) -Glu (OtBu) -Leu-Cys (Acm) -Va l-Asn (Trt) -Val-Ala-Cys (Trt) -Thr (tBu) - (Hmb) Gly-Cys (Acm) -Leu-Wang resin,

wherein specifically protected amino acids or fragments are employed: fmoc- (Hmb) Gly-OH at position 14, R-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -OH at positions 1-3, fmoc-Cys (Trt) -OH at positions 4,12, fmoc-Cys (Acm) -OH at positions 7,15,

step 3, cracking the resin, simultaneously removing side chain protecting groups to obtain a crude product of the procalcitonin,

step 4, oxidizing to form two pairs of disulfide bridges respectively, and purifying to obtain pure product of the procalcitonin;

the oxidation described in step 4 includes two steps, first completing the first pair of disulfide bond formation using hydrogen peroxide, and then completing the second pair of disulfide bond formation using iodine.

2. The method of claim 1, wherein the solid phase resin is Wang or CTC resin.

3. The method of claim 1, wherein the solid phase resin substitution range is 0.2-0.6mmol/g.

4. The process according to claim 1, wherein the coupling reagent is a mixture of HOBt and DIC, or HBTU and an organic base selected from DIPEA, et ₃ N, NMM.

5. The method according to claim 1, wherein the R group in R-Asn (Trt) -Asp (OtBu) -Glu (OtBu) -OH is Fmoc or Boc.

6. The method of claim 1, wherein the lysate component of the lysate resin is a combination of TFA, EDT, TIS, phenol, anisole, dimethyl sulfide, and water.

7. The method of claim 1, wherein the endpoint of oxidation is determined from HPLC control results.

8. The method of claim 1, wherein the purifying is performed by reverse phase high performance liquid chromatography.