阅读说明：本技术 一种pt141合成方法 (PT141 synthesis method ) 是由 郑春旭 孔亚 顾维炜 黄保胜 周黎 王良友 于 2019-12-02 设计创作，主要内容包括：本发明公开了一种PT141合成方法,属于多肽合成技术领域。本发明的合成方法包括如下步骤(1)以Rink HMBA-AM Resin和Fmoc-Lys(Boc)-OH为原料,制备Fmoc-Lys(Boc)-HMBA-Am Resin；(2)在步骤(1)制得的Fmoc-Lys(Boc)-HMBA-Am Resin上通过Fmoc固相合成法依次偶联保护基氨基酸,得到线性PT141全保护肽树脂；(3)将步骤(2)制得的线性PT141全保护肽树脂用裂解液裂解脱除氨基酸保护基,对所得产物进行环化处理,使得肽链在2位的Asp处与7位Lys处成环；(4)对步骤(3)所得环化产物进行甲酯化脱除HMBA-Am Resin残基,而后将产物水解,经过纯化,冷冻干燥得PT141。本发明的方法相对其他固相方法收率更高,相对酶制剂方法成本较低,且易于操作,工艺较为简单。(The invention discloses a PT141 synthesis method, and belongs to the technical field of polypeptide synthesis. The synthesis method comprises the following steps of (1) preparing Fmoc-Lys (Boc) -HMBA-AM Resin by taking Rink HMBA-AM Resin and Fmoc-Lys (Boc) -OH as raw materials; (2) sequentially coupling protecting group amino acids on Fmoc-Lys (Boc) -HMBA-Am Resin prepared in the step (1) by an Fmoc solid-phase synthesis method to obtain linear PT141 fully-protected peptide Resin; (3) cracking the linear PT141 full-protection peptide resin prepared in the step (2) by using a cracking solution to remove an amino acid protecting group, and carrying out cyclization treatment on the obtained product to ensure that a peptide chain forms a ring at an Asp position at a 2-position and a Lys position at a 7-position; (4) and (4) performing methyl esterification on the cyclized product obtained in the step (3) to remove HMBA-Am Resin residues, hydrolyzing the product, purifying, and performing freeze drying to obtain PT 141. Compared with other solid phase methods, the method has the advantages of higher yield, lower cost compared with an enzyme preparation method, easy operation and simpler process.)

1. A synthetic method of PT141 is characterized by comprising the following steps:

(1) using Rink HMBA-Am Resin and Fmoc-Lys (Boc) -OH as raw materials to prepare Fmoc-Lys (Boc) -HMBA-Amresin;

(2) sequentially coupling protecting group amino acids on Fmoc-Lys (Boc) -HMBA-Am Resin prepared in the step (1) by an Fmoc solid-phase synthesis method to obtain linear PT141 fully-protected peptide Resin;

(3) cracking the linear PT141 full-protection peptide resin prepared in the step (2) by using a cracking solution to remove an amino acid protecting group, and carrying out cyclization treatment on the obtained product to ensure that a peptide chain forms a ring at an Asp position at a 2-position and a Lys position at a 7-position;

(4) and (4) performing methyl esterification on the cyclized product obtained in the step (3) to remove HMBA-Am Resin residues, hydrolyzing the product, purifying, and performing freeze drying to obtain PT 141.

2. The method of claim 1, wherein in step (2), the Fmoc removal reagent is 15-25% piperidine/DMF.

3. The method of claim 1, wherein in step (3), the lysis solution comprises 85-95% trifluoroacetic acid, 4-6% thioanisole, 1-3% anisole, and 2-4% ethanedithiol.

4. The method according to claim 3, wherein the amount of the lysis buffer is 6-10ml/g of the linear PT141 fully-protected peptide resin and the lysis time is 1-3 h.

5. The method of claim 1, wherein in step (3), the step of washing with DCM for 2-4 times and DMF for 5-7 times is further included after the cleavage.

6. The process according to claim 1, wherein in step (3), the cyclization is carried out on a support resin and the condensing agent used is HOBt, PyBoP or DIEA.

7. The process of claim 1, wherein in step (4), the reagents used for methyl esterification are DIEA, MeOH and DMF in a ratio of 1: 1-3: 4-6 volume ratio.

8. The method of claim 1, wherein in the step (4), the hydrolysis is performed by adjusting the solution to be alkaline with 30-200mg/L NaOH to remove methyl ester.

9. The method according to claim 1, wherein in the step (4), the purification is performed by high performance liquid chromatography, provided that: and (3) purification: mobile phase: phase A: 0.5-1.5% per mill TFA; phase B: acetonitrile; salt conversion: mobile phase: phase A: 1)40-60mmol/L NH₄Ac aqueous solution, 2)0.3-0.5 per mill acetic acid; phase B: and (3) acetonitrile.

Technical Field

The invention relates to a PT141 synthesis method, belonging to the technical field of polypeptide synthesis.

Background

PT141, Brelemanolide (Bremelanotide), is an alpha-melanocyte stimulating hormone analog. Developed by Palatin, USA, as a cyclic peptide drug for treating sexual dysfunction. PT141 is an artificially synthesized nitrogen-terminal acetylated cyclic heptapeptide compound with the peptide sequence of

Ac-Nle-[Asp-His-D-Phe-Arg-Trp-Lys]-OH

The chemical structure of the method has two carboxyl groups, the side chain carboxyl of aspartic acid and the side chain amino of lysine form amide bond cyclization, and the direct liquid phase cyclization product is not unique due to the existence of C-terminal carboxyl, so that the method has a challenge to the synthesis process. At present, the method for synthesizing PT141 is mainly a solid-phase synthesis method, and Fmoc-Asp (OAllyl) -OH and Fmoc-Lys (alloc) -OH are used as raw materials for synthesis, but the problems that protected amino acid and deprotection reagent are expensive, the requirement on equipment is high by adopting an HF cracking mode, the environmental pollution is serious, and the product yield is low are existed. Chinese patent 200710048824.6 discloses a solid phase synthesis method using Fmoc (fluorenylmethyloxycarbonyl) protection with a yield of 17%. The solid phase synthesis method of PT-141 disclosed in Chinese patent 200610086841.4 has a yield of 40% or less.

Chinese patent 201510667256.2 discloses a method for synthesizing Ac-Nle-Asp (COOH) -otBu and Boc-His (Trt) -D-Phe-Arg (pbf) -TrP (Boc) -Lys (IVDde) -HMBA-P respectively, which needs to synthesize a large amount of fragments Ac-Nle-Asp (COOH) -otBu, and needs to use hydrazine hydrate and other dangerous compounds when removing Lys protecting groups.

The Chinese patent 201710619602.9 uses an enzyme preparation for cyclization, which has strict requirements on the control of reaction environmental conditions and is expensive.

Disclosure of Invention

In order to solve the technical problems, the invention provides a PT141 synthesis method, which has higher yield compared with other solid phase methods, lower cost compared with an enzyme preparation method, easy operation and simpler process.

The invention aims to provide a PT141 synthesis method, which is characterized by comprising the following steps:

(1) using Rink HMBA-Am Resin and Fmoc-Lys (Boc) -OH as raw materials to prepare Fmoc-Lys (Boc) -HMBA-Am Resin;

(2) sequentially coupling protecting group amino acids on Fmoc-Lys (Boc) -HMBA-Am Resin prepared in the step (1) by an Fmoc solid-phase synthesis method to obtain linear PT141 fully-protected peptide Resin;

(3) cracking the linear PT141 full-protection peptide resin prepared in the step (2) by using a cracking solution to remove an amino acid protecting group, and carrying out cyclization treatment on the obtained product to ensure that a peptide chain forms a ring at an Asp position at a 2-position and a Lys position at a 7-position;

(4) and (4) performing methyl esterification on the cyclized product obtained in the step (3) to remove HMBA-Am Resin residues, hydrolyzing the product, purifying, and performing freeze drying to obtain PT 141.

Further, in step (2), the reagent used for Fmoc removal is 15-25% piperidine/DMF.

Further, in the step (3), the lysis solution comprises 85-95% of trifluoroacetic acid, 4-6% of thioanisole, 1-3% of anisole and 2-4% of 1, 2-ethanedithiol.

Furthermore, the addition amount of the lysis solution is 6-10ml/g of linear PT141 full-protection peptide resin, and the lysis time is 1-3 h.

Further, in step (3), after the cleavage, the steps of washing with DCM for 2-4 times and DMF for 5-7 times are included.

Further, in step (3), the cyclization process is carried out on a carrier resin using a condensing agent of HOBt, PyBoP or DIEA.

Further, in step (4), the reagents used for methyl esterification are DIEA, MeOH and DMF in accordance with a 1: 1-3: 4-6 volume ratio.

Further, in the step (4), the hydrolysis is carried out by adjusting the solution to be alkaline with 30-200mg/L NaOH to remove methyl ester.

Further, in the step (4), the purification is performed by using high performance liquid chromatography, and the conditions are as follows: and (3) purification: mobile phase: phase A: 0.5-1.5% per mill TFA; phase B: acetonitrile; salt conversion: mobile phase: phase A: 1)40-60mmol/L NH₄Ac aqueous solution, 2)0.3-0.5 per mill acetic acid; phase B: and (3) acetonitrile.

The invention has the beneficial effects that: the direct coupling of peptide sequences avoids fragment synthesis, and conventional amino acids Fmoc-Asp (OtBu) -OH and Fmoc-Lys (Boc) -OH are adopted as raw materials to replace expensive raw materials such as Fmoc-Asp (OAllyl) -OH, Fmoc-Lys (alloc) -OH or Fmoc-Lys (IVDde) -OH and the like, thereby avoiding the use of dangerous compounds such as hydrogenation process or hydrazine hydrate and the like. Solid phase cyclization, and amido bond cyclization structure is unique. The method of the invention is easy to operate, the process is simple, the product purity can reach 99.3%, the total yield is 55.0%, and the method is suitable for industrial production.

Drawings

FIG. 1 is an HPLC chart of PT141 product;

FIG. 2 is the MS diagram of PT141 product.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

In this process, "degree of substitution" refers to the amount of the resin-supported substance per unit amount, in units of "mmol/g". Abbreviations appearing herein have the following meanings:

TABLE 1 explanation of the terms related to the present invention

Abbreviations	Means of
		Fmoc	9-fluorenylmethoxycarbonyl group
Boc	Tert-butyloxycarbonyl radical
		Trt	Trityl radical
HOBt	1-hydroxybenzotriazoles
		Pbf
	2,2,4,6, 7-pentamethyldihydrobenzofuran-5-sulfonyl
		OtBu	Tert-butoxy radical
DMF	N, N-dimethylformamide
		DIC	N, N' -diisopropylcarbodiimide
TFA	Trifluoroacetic acid
		DBLK
	20% piperidine/DMF solution
		HPLC	High performance liquid chromatography
Ac	Acetate salt
		DIEA	N, N-diisopropylethylamine
EDT
		1, 2-ethanedithiol