CN112279894A - Method for synthesizing alarelin by polypeptide solid-liquid combination - Google Patents

Abstract

The invention provides a method for synthesizing alanorelin by polypeptide solid-liquid combination, which comprises the following steps: 1) and synthesizing compound 1 by a polypeptide solid phase synthesis method: Boc-Pglu-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Ala-Leu-OH; 2) and synthesizing a compound 2 by a polypeptide liquid phase synthesis method: H-Arg (pbf) -Pro-NHEt; 3) synthesis of compound 3 in liquid phase: Boc-Pglu-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Ala-Leu-Arg (pbf) -Pro-NHEt; 4) and synthesizing a crude product of alanorelin: Pglu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt, the invention takes piperazine as a CTC resin synthetic segment for a decapping reagent, combines a liquid phase method to synthesize alanrelin, the piperazine is not an easy-to-detoxify reagent, the cost is lower than that of the easy-to-detoxify reagent piperidine used in a classical synthesis method, and the transport and storage are more convenient; the CTC resin synthetic segment can be conveniently and automatically produced; the CTC resin can be recycled; is suitable for large-scale industrial production, and has no violent chemical reaction; the production cost is reduced.

Description

Method for synthesizing alarelin by polypeptide solid-liquid combination

Technical Field

The invention relates to the technical field of medicines, in particular to a method for synthesizing alanyl-relin by polypeptide solid-liquid combination.

Background

The alanrelin is a synthetic nonapeptide analogue of gonadotropin releasing hormone (GnRH), and can stimulate pituitary to release Luteinizing Hormone (LH) and Follicle Stimulating Hormone (FSH) at the initial stage of administration to cause the transient increase of the ovarian steroid hormone; repeated administration can inhibit the pituitary from releasing LH and FSH, reduce the level of estradiol in blood, and achieve the effect of removing ovary, and the inhibition effect can be used for treating hormone-dependent diseases such as endometriosis.

In the prior art, application No. CN201710851059.5 provides a solid-phase preparation method of alarelin, which comprises the following steps:

(1) activating Boc-Pro: respectively dissolving Boc-Pro and Cs2CO3, and then mixing to prepare an activated Boc-Pro product Boc-Pro-Cs;

(2) Boc-Pro curing: dissolving Boc-Pro-Cs and chloromethyl resin, and curing Boc-Pro to prepare Boc-Pro resin;

(3) and peptide grafting: the Boc-Pro resin is sequentially connected with Boc-Arg, Boc-Leu, Boc-D-Ala, Boc-Tyr, Boc-Ser- (But), Boc-Trp, Boc-His- (Tos) and pGlu in a one-by-one coupling mode to synthesize alanrelin-resin;

(4) aminolysis resin is carried out on the alarelin-resin, and acetate is converted to obtain an alarelin crude product;

(5) and purifying the crude product of the alarelin by HPLC and freeze-drying to obtain the finished product of the alarelin.

The preparation method disclosed by the prior art can obtain the alanrelin with high yield and purity, and adopts a Boc strategy, so that the deprotection cost is higher, ammonolysis is required, the reaction conditions are difficult to control, side reactions are more, and a production process with lower cost and more convenience for industrialization is required.

Disclosure of Invention

Based on the above, there is a need to provide a method for synthesizing alanorelin by solid-liquid combination of polypeptides, which addresses the problems in the background art.

The technical scheme of the invention is to construct a method for synthesizing alanyl-relin by polypeptide solid-liquid combination, which comprises the following steps:

1) and synthesizing compound 1 by a polypeptide solid phase synthesis method:

Boc-Pglu-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OH；

2) and synthesizing a compound 2 by a polypeptide liquid phase synthesis method: H-Arg (pbf) -Pro-NHEt;

3) synthesis of compound 3 in liquid phase:

Boc-Pglu-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg(pbf)-Pro-NHEt；

4) and synthesizing a crude product of alanorelin: Pglu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt.

Further: in step 1), compound 1 can be synthesized sequentially from C to N terminus by a solid phase method;

the method comprises the following steps:

1): CTC resin is taken as a solid phase carrier;

Boc-Pglu-OH, Fmoc-His (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-D-Ala-OH, Fmoc-Leu-OH are amino acid monomers;

the condensing agent is one of HBTU, HATU, TATU, TBTU, PyBOP, BOP, DIC and DCC;

the uncapping reagent is a piperazine solution, the solvent is a dipolar aprotic solvent, and the mass concentration of the piperazine solution is 2-5%;

the organic base is DIEA or NMM;

sequentially synthesized from C to N terminal:

Boc-Pglu-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Ala-Leu-CTC resin;

2): cleavage with trifluoroacetic acid/dichloromethane solution, concentration and precipitation with water gave compound 1:

Boc-Pglu-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OH。

further: in step 2), compound 2 can be synthesized by a liquid phase method:

Fmoc-Arg (pbf) -OH, H-Pro-NHEt HCl are taken as amino acid monomers, a condensing agent is one of HBTU, HATU, TATU, TBTU, PyBOP, BOP, DIC and DCC, an activating agent is HOSu or HOBt, an organic base is mainly DIEA or TEA or NMM, and the synthesis is carried out in sequence:

1)、Fmoc-Arg(pbf)-Pro-NHEt；

2)、H-Arg(pbf)-Pro-NHEt。

further: in step 3), compound 3 can be synthesized by a liquid phase method: taking a compound 1 and a compound 2 as synthesis fragments, a condensing agent as one of HBTU, HATU, TATU, TBTU, PyBOP, BOP, DIC and DCC, an activating agent as HOSu or HOBt, an organic base mainly as DIEA or TEA or NMM, and synthesizing a compound 3:

Boc-Pglu-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg(pbf)-Pro-NHEt；

the molar ratio of compound 1 to compound 2 is 1: 1.01-1: 1.5, the molar ratio of compound 2 to the condensing agent, activator and organic base is 1: 1: 1: 1;

further, the purity of the crude alanrelin can reach more than 90 percent, the crude alanrelin can be obtained by dissolving the compound 3 in a TFA solution for reaction, after the reaction is finished, the reaction product is concentrated, ether reagents are added for precipitation, and the crude alanrelin is obtained by filtration.

In one embodiment, the post-use resin recovery steps are as follows:

1) washing the resin with DMF;

2) washing with methanol, removing filtrate, and vacuum drying;

3) and (3) preparing a DCM solution of thionyl chloride, adding the solution into a solid-phase synthesis reactor filled with resin, reacting for 2 hours, then pumping out the reaction solution, and washing the DCM.

In one example, the procedure for obtaining crude product from compound 3 is as follows:

1) a flask was taken, weighed as Compound 3, and charged with a peptide cleaving reagent (TFA: TIS: H)₂O95: 2.5:2.5) to obtain a TFA solution containing crude alanrelin;

2) the TFA solution was concentrated to a small amount and diethyl ether was added;

3) centrifuging and drying in vacuum to obtain crude alanyl-relin trifluoroacetate.

In one example, compound 3 was synthesized as follows:

1) dissolving the compound 1 and a condensing agent in DMF, adding an activating agent and organic alkali for dissolving, and carrying out cold bath at the temperature of-10-5 ℃ for 5-15 min;

2) and dissolving the compound 2 in THF, reacting for 1.5-2.5 h, and detecting by HPLC to complete the reaction. Concentrating, adding hydrochloric acid solution to precipitate off white solid, filtering, and washing with water to neutrality;

3) vacuum drying to obtain compound 3.

The invention has the following advantages and beneficial effects:

1) piperazine is used as a CTC resin synthesis segment for a decapping reagent, and is combined with a liquid phase method to synthesize alanelin, wherein piperazine is not an easy-to-detoxify reagent, the cost is lower than that of piperidine which is an easy-to-detoxify reagent used in a classical synthesis method, and the piperazine is more convenient to transport and store;

2) the CTC resin synthetic segment can be conveniently and automatically produced;

3) the CTC resin can be recycled;

4) is suitable for large-scale industrial production, and has no violent chemical reaction;

5) the production cost is reduced.

Drawings

FIG. 1 is an HPLC chart of Compound 1 of the present invention.

FIG. 2 is a MS plot of an unprotected sample cleaved by Compound 1 of the present invention.

FIG. 3 is an RP-HPLC plot of Fmoc-Arg (Pbf) -Pro-NHEt.HCl of the present invention.

FIG. 4 is an RP-HPLC chart of H-Arg (Pbf) -Pro-NHEt according to the present invention.

FIG. 5 is an MS map of H-Arg (Pbf) -Pro-NHEt according to the present invention.

FIG. 6 is an HPLC chart of Compound 3 (alarelin precursor) of the present invention.

FIG. 7 is an HPLC chart of crude alanrelin of the present invention.

FIG. 8 is a MS plot of alarelin of the invention.

FIG. 9 is an HPLC plot of Compound 1 of another embodiment of the present invention.

Detailed Description

To facilitate an understanding of the invention, the invention will now be described more fully with reference to the accompanying drawings. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Example 1

The Chinese names corresponding to the English abbreviations of the substances appearing in the claims and the specification of the present invention are shown in Table 1.

TABLE 1 Chinese names corresponding to English abbreviations for appearing substances in the present invention

A method for synthesizing alanorelin by polypeptide solid-liquid combination comprises the following steps:

1) and synthesizing compound 1 by a polypeptide solid phase synthesis method:

Boc-Pglu-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OH；

2) and synthesizing a compound 2 by a polypeptide liquid phase synthesis method: H-Arg (pbf) -Pro-NHEt;

3) synthesis of compound 3 in liquid phase:

Boc-Pglu-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg(pbf)-Pro-NHEt；

4) and synthesizing a crude product of alanorelin: Pglu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt.

Further: in step 1), compound 1 can be synthesized sequentially from C to N terminus by a solid phase method;

the method comprises the following steps:

1): CTC resin is taken as a solid phase carrier;

Boc-Pglu-OH, Fmoc-His (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-D-Ala-OH, Fmoc-Leu-OH are amino acid monomers;

the condensing agent is one of HBTU, HATU, TATU, TBTU, PyBOP, BOP, DIC and DCC;

the uncapping reagent is a piperazine solution with the mass concentration of 2-5%, and the solvent is a dipolar aprotic solvent;

still more preferably, the solvent of the uncapping reagent is DMF, and the mass concentration of piperazine is 3%;

the organic base is DIEA or NMM;

sequentially synthesized from C to N terminal:

Boc-Pglu-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Ala-Leu-CTC resin;

2): cleavage with trifluoroacetic acid/dichloromethane solution, concentration and precipitation with water gave compound 1:

Boc-Pglu-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OH。

specifically, the synthesis process of the compound 1 is as follows:

the feeding table is as follows:

name (R)	Molecular weight	Density of	Volume μ l	Mass mg
					CTCresin(25g)	(degree of substitution 1.0mmol/g)			\
Fmoc-Leu-OH	353.4	\	\	8835
					DIEA	129.24	0.782	41200	32310
Fmoc-D-Ala-OH	311.34	\	\	15567
					HBTU	379.24	\	\	18962
DIEA	129.24	0.782	9916.11	7754.4
					Fmoc-Tyr(tBu)-OH	459.5	\	\	22975
HBTU	379.24	\	\	18962
					DIEA	129.24	0.782	9916.11	7754.4
Fmoc-Ser(tBu)-OH	383.4	\	\	19170
					HBTU	379.24	\	\	18962
DIEA	129.24	0.782	9916.11	7754.4
					Fmoc-Trp(Boc)-OH	526.6	\	\	26330
HBTU	379.24	\	\	18962
					DIEA	129.24	0.782	9916.11	7754.4
Fmoc-His(Boc)-OH	477.51	\	\	23875.5
					HBTU	379.24	\	\	18962
DIEA	129.24	0.782	9916.11	7754.4
					Boc-Pglu-OH	229.2	\	\	11460
HBTU	379.24	\	\	18962
					DIEA	129.24	0.782	9916.11	7754.4

Firstly, synthesizing Leu-resin;

1) weighing 25g of CTC resin in a 250ml solid phase synthesis reactor, taking Fmoc-Leu-OH in a 250ml triangular flask, measuring DCM120ml in a 100ml graduated cylinder, shaking up, adding DIEA41ml, shaking up to dissolve, pouring into the reactor, and starting to react.

2) When the reaction is carried out for 1h, a suction filtration device is used for pumping out the reaction liquid, and DMF is used for washing for 4 times;

3) weighing 200ml of piperazine/DMF solution with the mass concentration of 3% as a uncapping reagent, pouring the uncapping reagent into a reactor, reacting for 20min, then pumping out reaction liquid, and washing with DMF for 4 times.

4) Ninhydrin detection: placing the resin in a glass tube, adding isopropanol, vortex5 s, pouring off the isopropanol after the resin sinks at the bottom of the glass tube, repeating the washing three times, checking ninhydrin, dropping ninhydrin solution in a small test tube, heating in a dry heater at 110 deg.C for 3min, and obtaining positive result.

Secondly, synthesizing D-Ala-Leu-resin;

1) after Leu-resin is obtained, adding a second amino acid protective monomer Fmoc-D-Ala-OH, a condensing agent HBTU and alkali DIEA into a reaction system, wherein a solvent is DMF, the reaction time is 2 hours, and after the reaction is finished, washing the reaction system for 4 times by using DMF;

2) detection of reaction completion: placing the resin in a glass tube, adding isopropanol, vortex5 s, pouring off the isopropanol after the resin sinks to the bottom of the glass tube, repeating the washing three times, checking ninhydrin, dropping ninhydrin solution in a small test tube, heating in a dry heater at 110 deg.C for 3min, and obtaining negative.

3) After Fmoc-D-Ala-Leu-resin was obtained, 200ml of uncapped reagent piperazine solution was weighed and poured into a reactor, reacted for 20min, the reaction solution was withdrawn and washed 4 times with DMF.

4) Ninhydrin detection: placing the resin in a glass tube, adding isopropanol, vortex5 s, pouring off the isopropanol after the resin sinks at the bottom of the glass tube, repeating the washing three times, checking ninhydrin, dropping ninhydrin solution in a small test tube, heating in a dry heater at 110 deg.C for 3min, and obtaining positive result.

Thirdly, after obtaining the D-Ala-Leu-resin, connecting amino acid protective monomers on the D-Ala-Leu-resin in sequence according to the condensation reaction method:

Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Boc) -OH and Boc-Pglu-OH:

Boc-Pglu-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Ala-Leu-resin.

Fourthly, the protection polypeptide is cut from the resin by the following method:

adding 150ml of 1% TFA/DCM solution, carrying out swing reaction for 10min, collecting the reaction solution, repeating for 6 times, concentrating the reaction solution, adding a citric acid solution with the mass concentration of 10%, precipitating a white solid, filtering, washing with water to be neutral, and drying in vacuum. To give compound 1:

Boc-Pglu-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Ala-Leu-OH, 29.8g, 91.9% purity, 91% yield. (RP-HPLC is shown in FIG. 1; MS is shown in FIG. 2 for the cleaved unprotected sample)

Referring to fig. 1, compound 1 was analyzed as follows:

agilent model 1260 HPLC, Krosmail100-5C18column4.6 x 250 mm;

a: 5% acetonitrile/H2O solution, 0.1% TFA; b: acetonitrile, 0.1% tfa (hplc);

gradient elution: 0-30 min; 70% of B to 75% of B;

λ 210 nm; the flow rate was 1.0 ml/min.

Further: in step 2), compound 2 can be synthesized by a liquid phase method:

Fmoc-Arg (pbf) -OH, H-Pro-NHEt HCl are taken as amino acid monomers, a condensing agent is one of HBTU, HATU, TATU, TBTU, PyBOP, BOP, DIC, DCC and the like, an activating agent is HOSu or HOBt, an organic base is mainly DIEA or TEA or NMM, and the synthesis is carried out in sequence:

1)、Fmoc-Arg(pbf)-Pro-NHEt；

2)、H-Arg(pbf)-Pro-NHEt。

specifically, the synthesis process of the compound 2 is as follows:

synthesis of H-Arg (Pbf) -Pro-NHEt

1) Synthesis of Fmoc-arg (pbf) -Pro-nhet.hcl: Fmoc-Arg (Pbf) -OH, 100mmol, 65g, BOP110mmol, 49g were dissolved in 500ml DMF, cooled for 10min, TEA110 mmol, 12g were added.

2) Then, H-Pro-NHEt.HCl, 110mmol, 20g was weighed, dissolved in 100ml DMF +12g TEA, poured into the above reaction solution, reacted for 2H, HPLC showed complete reaction, 0.5M hydrochloric acid was added to precipitate, filtered to obtain white solid product, which was dried under vacuum to obtain 87.4g product with yield of 108% and purity of 96.8%. (RP-HPLC chart is shown in FIG. 3).

Referring to fig. 3, the analysis method is as follows:

agilent model 1260 HPLC, Krosmail100-5C18column4.6 x 250 mm;

a: 5% acetonitrile/H₂O solution, 0.1% TFA; b: acetonitrile/H₂O，0.1％TFA(HPLC)；

Gradient elution: 0-30 min; 55% of B to 65% of B;

λ＝210nm；

the flow rate was 1.0 ml/min.

Thirdly, synthesizing H-Arg (Pbf) -Pro-NHEt

1) Dissolve the product with 500ml diethylamine, stir for 30min and HPLC shows reaction complete. 2) Concentrating to remove diethylamine, adding petroleum ether to precipitate white solid H-Arg (Pbf) -Pro-NHEt, and vacuum drying to obtain 51g product with yield of 92.6% and purity of 97.5%. (the RP-HPLC chart is shown in FIG. 4, and the MS chart is shown in FIG. 5).

Referring to fig. 4, the analysis method is as follows:

agilent model 1260 HPLC, Krosmail100-5C18column4.6 x 250mm

A: 5% acetonitrile/H₂O solution, 0.1% TFA; b: acetonitrile/H₂O，0.1％TFA(HPLC)

Gradient elution: 0-30 min; 0% B to 10% B

λ＝210nm；

The flow rate was 1.0 ml/min.

Further: in step 3), compound 3 can be synthesized by a liquid phase method: taking a compound 1 and a compound 2 as synthesis fragments, a condensing agent as one of HBTU, HATU, TATU, TBTU, PyBOP, BOP, DIC and DCC, an activating agent as HOSu or HOBt, an organic base mainly as DIEA or TEA or NMM, and synthesizing a compound 3:

Boc-Pglu-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg(pbf)-Pro-NHEt；

the molar ratio of compound 1 to compound 2 is 1: 1.01-1: 1.5, the molar ratio of compound 2 to the condensing agent, activator and organic base is 1: 1: 1: 1.

specifically, the synthesis process of the compound 3 is as follows:

1) alanorelin precursor (compound 3) synthesis:

taking a compound 1: 10mmol, 13g and BOP11mmol, 4.9g are dissolved in 20ml DMF, and the solution is dissolved by adding activating agent HOBt and organic base DIEA, and the solution is cooled and bathed at-5 ℃ for 10min, and then the compound 2: 12mmol, 6.6g, dissolved in 20ml THF, reacted for 2h, and the reaction was complete as detected by HPLC.

Filtering to remove white solid, concentrating, adding 0.5M hydrochloric acid solution to precipitate off-white solid, filtering, and washing with water to neutrality. Vacuum drying to obtain the product alanyl-relin precursor (compound 3)15.8g, with 86.3% yield and 91% purity. (RP-HPLC FIG. 6).

Referring to fig. 6, the analysis method is as follows:

agilent model 1260 HPLC, Krosmail100-5C18column4.6 x 250 mm;

a: 5% acetonitrile/H₂O solution, 0.1% TFA; b: acetonitrile/H₂O，0.1％TFA(HPLC)；

Gradient elution: 0-30 min; 70% of B to 75% of B;

λ 210 nm; the flow rate was 1.0 ml/min.

2) Crude alanorelin synthesis

A100 ml round-bottomed flask was taken, and 5mmol (9.2g) of the alanorelin precursor (compound 3) was charged, and a peptide-cleaving reagent (TFA: TIS: H) was added₂O95: 2.5:2.5) to obtain a TFA solution containing the crude alanrelin.

The TFA solution was concentrated to a small amount and diethyl ether was added. Centrifuging, and vacuum drying to obtain 5.3g of crude alanyl-relin with purity of 91% and yield of 83%. (RP-HPLC is shown in FIG. 7, and MS is shown in FIG. 8)

Referring to fig. 7, the analysis method is as follows:

agilent model 1260 HPLC, Krosmail100-5C18column4.6 x 250 mm;

A：0.1％TFA/H₂o solution B: h₂O, 0.1% TFA/acetonitrile (HPLC);

gradient elution: 0-30 min; 20% of B to 30% of B;

λ 210 nm; the flow rate was 1.0 ml/min.

According to the preparation method of the alarelin, the solid-phase synthesis method is adopted to synthesize the compound 1, the liquid-phase synthesis method is adopted to synthesize the compound 2, then the two intermediates are condensed to obtain the compound 3, and the alarelin is obtained after deprotection, so that the preparation steps of the alarelin are simplified, and the preparation cost of the alarelin is reduced; the purity of the alanorelin prepared by the method is up to more than 90%.

Example 2

The difference from the above example is that compound 1:

firstly, recovering resin;

1) the resin from example 1 was washed with DMF, washed with methanol, the filtrate was withdrawn and dried in vacuo.

2) A DCM solution (10 ml of thionyl chloride, 200ml of DCM) was prepared, added to a 250ml solid phase synthesis reactor containing resin, reacted for 2h, the reaction solution was removed and washed with DCM.

The feeding table is as follows:

secondly, synthesizing Leu-resin;

1) weighing 25g of CTC resin in a 250ml solid phase synthesis reactor, taking Fmoc-Leu-OH in a 250ml triangular flask, measuring DCM120ml in a 100ml graduated cylinder, shaking up, adding DIEA41ml, shaking up to dissolve, pouring into the reactor and starting reaction.

2) When the reaction is carried out for 1h, a suction filtration device is used for pumping out the reaction liquid, and DMF is used for washing for 4 times;

3) 200ml of the uncapped reagent piperazine solution is measured and poured into a reactor for reaction for 20min, and then the reaction solution is pumped out and washed 4 times by DMF.

4) Ninhydrin detection: placing the resin in a glass tube, adding isopropanol, vortex5 s, pouring off the isopropanol after the resin sinks at the bottom of the glass tube, repeating the washing three times, checking ninhydrin, dropping ninhydrin solution in a small test tube, heating in a dry heater at 110 deg.C for 3min, and obtaining positive result.

Thirdly, synthesizing D-Ala-Leu-resin;

1) after Leu-resin is obtained, adding a second amino acid protective monomer Fmoc-D-Ala-OH, a condensing agent HBTU and alkali DIEA into a reaction system, wherein a solvent is DMF, the reaction time is 2 hours, and after the reaction is finished, washing the reaction system for 4 times by using DMF;

2) detection of reaction completion: placing the resin in a glass tube, adding isopropanol, vortex5 s, pouring off the isopropanol after the resin sinks to the bottom of the glass tube, repeating the washing three times, checking ninhydrin, dropping ninhydrin solution in a small test tube, heating in a dry heater at 110 deg.C for 3min, and obtaining negative.

3) After Fmoc-D-Ala-Leu-resin was obtained, 200ml of the decapping reagent piperazine solution was measured and poured into the reactor for 20min, and then the reaction solution was extracted and washed 4 times with DMF.

4) Ninhydrin detection: placing the resin in a glass tube, adding isopropanol, vortex5 s, pouring off the isopropanol after the resin sinks at the bottom of the glass tube, repeating the washing three times, checking ninhydrin, dropping ninhydrin solution in a small test tube, heating in a dry heater at 110 deg.C for 3min, and obtaining positive result.

After obtaining the D-Ala-Leu-resin, connecting amino acid protective monomers on the D-Ala-Leu-resin according to the condensation reaction method:

Fmoc-Tyr (tBu) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Trp (Boc) -OH, Fmoc-His (Boc) -OH and Boc-Pglu-OH;

obtaining Boc-Pglu-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Ala-Leu-resin. The protected polypeptide is then cleaved from the resin by the following method:

adding 1% TFA/DCM solution 150ml, rocking reaction for 10min, collecting reaction solution, repeating 6 times, concentrating reaction solution, adding 10% citric acid solution, precipitating white solid, filtering, washing with water to neutrality, and vacuum drying. To give compound 1: Boc-Pglu-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Ala-Leu-OH, 27.3g, purity 88.5%, yield 84%. (RP-HPLC chart is shown in FIG. 9)

Referring to fig. 9, the analysis method is as follows:

agilent model 1260 HPLC, Krosmail100-5C18column4.6 x 250 mm;

a: 5% acetonitrile/H₂O solution, 0.1% TFA; b: acetonitrile, 0.1% tfa (hplc);

gradient elution: 0-30 min; 70% of B to 90% of B;

λ 210 nm; the flow rate was 1.0 ml/min.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (8)

1. A method for synthesizing alanorelin by polypeptide solid-liquid combination is characterized by comprising the following steps:

1) and synthesizing compound 1 by a polypeptide solid phase synthesis method:

Boc-Pglu-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OH；

2) and synthesizing a compound 2 by a polypeptide liquid phase synthesis method: H-Arg (pbf) -Pro-NHEt;

3) synthesis of compound 3 in liquid phase:

Boc-Pglu-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg(pbf)-Pro-NHEt；

4) and synthesizing a crude product of alanorelin: Pglu-His-Trp-Ser-Tyr-D-Ala-Leu-Arg-Pro-NHEt.

2. The method for synthesizing alanorelin by polypeptide solid-liquid combination according to claim 1, characterized in that: in step 1), compound 1 can be synthesized sequentially from C to N terminus by a solid phase method comprising the steps of:

1): CTC resin is taken as a solid phase carrier;

Boc-Pglu-OH, Fmoc-His (Boc) -OH, Fmoc-Trp (Boc) -OH, Fmoc-Ser (tBu) -OH, Fmoc-Tyr (tBu) -OH, Fmoc-D-Ala-OH, Fmoc-Leu-OH are amino acid monomers;

the condensing agent is one of HBTU, HATU, TATU, TBTU, PyBOP, BOP, DIC and DCC;

the uncapping reagent is a piperazine solution, the solvent is a dipolar aprotic solvent, and the mass concentration of the piperazine solution is 2-5%;

the organic base is DIEA or NMM;

sequentially synthesized from C to N terminal:

Boc-Pglu-His (Boc) -Trp (Boc) -Ser (tBu) -Tyr (tBu) -D-Ala-Leu-CTC resin;

2): cleavage with trifluoroacetic acid/dichloromethane solution, concentration and precipitation with water gave compound 1:

Boc-Pglu-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-OH。

3. the method for synthesizing alanorelin by polypeptide solid-liquid combination according to claim 1, characterized in that: in step 2), compound 2 can be synthesized by a liquid phase method:

Fmoc-Arg (pbf) -OH, H-Pro-NHEt HCl are taken as amino acid monomers, a condensing agent is one of HBTU, HATU, TATU, TBTU, PyBOP, BOP, DIC and DCC, an activating agent is HOSu or HOBt, an organic base is mainly DIEA or TEA or NMM, and the synthesis is carried out in sequence:

1)、Fmoc-Arg(pbf)-Pro-NHEt；

2)、H-Arg(pbf)-Pro-NHEt。

4. the method for synthesizing alanorelin by polypeptide solid-liquid combination according to claim 1, characterized in that: in step 3), compound 3 can be synthesized by a liquid phase method: taking a compound 1 and a compound 2 as synthesis fragments, taking a condensing agent as one of HBTU, HATU, TATU, TBTU, PyBOP, BOP, DIC, DCC and the like, taking an activating agent as HOSu or HOBt, taking an organic base as main DIEA or TEA or NMM, and synthesizing a compound 3:

Boc-Pglu-His(Boc)-Trp(Boc)-Ser(tBu)-Tyr(tBu)-D-Ala-Leu-Arg(pbf)-Pro-NHEt；

the molar ratio of compound 1 to compound 2 is 1: 1.01-1: 1.5, the molar ratio of compound 2 to the condensing agent, activator and organic base is 1: 1: 1: 1.

5. the method for synthesizing alanorelin according to the solid-liquid polypeptide fragment of claim 4, wherein the resin recovery steps after use are as follows:

1) washing the resin with DMF;

2) washing with methanol, removing filtrate, and vacuum drying;

3) and (3) preparing a DCM solution of thionyl chloride, adding the solution into a solid-phase synthesis reactor filled with resin, reacting for 2 hours, then pumping out the reaction solution, and washing the DCM.

6. The method for synthesizing alarelin by polypeptide solid-liquid combination according to claim 1, characterized in that the purity of the crude product of alarelin can reach more than 90%, and the crude product of alarelin can be obtained by dissolving compound 3 in TFA solution for reaction, concentrating after the reaction is finished, adding ether reagent for precipitation, and filtering to obtain the crude product of alarelin.

7. The method for synthesizing alarelin by polypeptide solid-liquid fragments as claimed in claim 6, characterized in that the crude product obtained from compound 3 is obtained by the following steps:

1) a flask was taken, weighed as Compound 3, and charged with a peptide cleaving reagent (TFA: TIS: H)₂O95: 2.5:2.5) to obtain a TFA solution containing crude alanrelin;

2) the TFA solution was concentrated to a small amount and diethyl ether was added;

3) centrifuging and drying in vacuum to obtain crude alanyl-relin trifluoroacetate.

8. The method for synthesizing alanorelin by the polypeptide solid-liquid fragment according to any one of claims 1 to 6, wherein the synthesis step of compound 3 is as follows:

1) dissolving the compound 1 and a condensing agent in DMF, adding an activating agent and organic alkali for dissolving, and carrying out cold bath at the temperature of-10-5 ℃ for 5-15 min;

2) and dissolving the compound 2 in THF, reacting for 1.5-2.5 h, and detecting by HPLC to complete the reaction. Concentrating, adding hydrochloric acid solution to precipitate off white solid, filtering, and washing with water to neutrality;

3) vacuum drying to obtain compound 3.

Images