阅读说明：本技术 一种阿巴帕肽的固相合成方法 (Solid-phase synthesis method of abamectin ) 是由 陶志强 李国弢 姚志军 宓鹏程 陶安进 袁建成 于 2018-09-06 设计创作，主要内容包括：本发明涉及一种阿巴帕肽的固相合成方法,具体公开了一种通过使用本发明的Asp上的Fmoc保护基脱除剂脱除Fmoc保护基,所述的脱除剂为AlCl<Sub>3</Sub>的NMP溶液及吲哚；本发明的方法有效地降低杂质含量,提高产率。(The invention relates to a solid-phase synthesis method of abamectin, and particularly discloses a method for removing Fmoc protective groups by using an AlCl removing agent on Asp (Asp) 3 NMP solution and indole; the method of the invention effectively reduces the impurity content and improves the yield.)

1. A preparation method of abamectin comprises the following steps:

step 1: Fmoc-Asp (OtBu) -Leu-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -Lys (Boc) -Leu-Leu-Aib-Lys (Boc) -Leu-His (Trt) -Thr (tBu) -Ala-solid phase synthetic resin;

step 2: adding AlCl under the protection of inert gas₃Removing the Fmoc protecting group on Asp from the NMP solution and the indole;

and step 3: continuing to sequentially couple and synthesize Fmoc-Asp (OtBu) -Lys (Boc) -Gly-Lys (Boc) -Ser (tBu) -Ile-Gln (Trt) -Asp (OtBu) -Leu-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -Lys (Boc) -Leu-Leu-Aib-Lys (Boc) -Leu-His (Trt) -Thr (tBu) -Ala-solid-phase synthetic resin according to the Fmoc protection strategy;

and 4, step 4: adding AlCl under the protection of inert gas₃Removing the Fmoc protecting group on Asp from the NMP solution and the indole;

and 5: sequentially coupling according to Fmoc protection strategy to obtain fully-protected Abapatide-solid phase synthetic resin;

step 6: adding a cracking agent, and cracking the solid-phase synthetic resin and the side chain protecting group to obtain the abapa peptide;

optionally, step 7: and (5) preparing and purifying by reverse phase chromatography to obtain the refined peptide.

2. The method of claim 1, wherein said AlCl is included in the reagent for removing Fmoc protecting group at Asp in step 2 and step 4₃The dosage is 3 times of molar equivalent of the reaction substrate, and the dosage of the indole is 1 time of molar equivalent of the reaction substrate.

3. The method according to claim 1, wherein the Fmoc protecting group at Asp is removed in steps 2 and 4, followed by washing with an organic solvent and then DMF; the organic solvent for washing is capable of dissolving AlCl₃The solvent of (1).

4. The method of claim 1, wherein the Fmoc protection strategy of steps 1, 3 or 5 is: coupling Fmoc-protected amino acid on solid-phase synthetic resin or polypeptide-solid-phase synthetic resin; the terminal Fmoc protecting group is removed and the next Fmoc protected amino acid is coupled until completion.

5. The process according to claim 4, wherein the removing agent for removing the Fmoc protecting group at the non-Asp amino acid terminal is a 20% piperidine/DMF solution.

6. The process according to claim 4, wherein the Fmoc-protected amino acid is coupled to the solid-phase synthetic resin or the polypeptide-solid-phase synthetic resin by activating the amino acid to be coupled to the solid-phase synthetic resin or the polypeptide-solid-phase synthetic resin with a coupling agent until the reaction is completed.

7. The process according to claim 6, wherein the coupling agent is a combination of DIPCDI and Compound A or a combination of DIPEA and Compound A and Compound B, wherein Compound A is HOAt or HOBt and Compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, preferably a combination of DIPCDI and Compound A, more preferably the ratio of the components in the coupling agent is DIPCDI: A ═ 1.2:1.1 and DIPEA: B ═ 2.0:1.1:1.0 in terms of molar ratio.

8. The method according to claim 1, wherein the lysis solution used in step 6 is a mixture of TFA, H2O, EDT, anisole, thioanisole and phenol in different ratios, preferably R cleavage reagent (TFA: thioanisole: anisole: EDT: 90:5:3: 2).

9. The method of claim 1, wherein the purification step of step 7 is performed by reversed-phase high-pressure liquid chromatography, preferably the reversed-phase high-pressure liquid chromatography comprises: using reverse octadecylsilane as stationary phase and 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting target peak fraction, concentrating, and lyophilizing.

Technical Field

The invention relates to the field of medicinal chemistry, in particular to a solid-phase synthesis method of abamectin

Background

Abapatide (Abaloperatide) is a novel parathyroid hormone-related peptide developed by Radius Health corporation for the treatment of postmenopausal osteoporosis with a high risk of fracture. Marketed in the united states on 28 th 4 th 2017 under the name TYMLOS. Teriparatide and abapa peptide are parathyroid hormone-related peptide (PTHrP), but abapa peptide is better able to reduce the rate of bone fracture and incidence of hypercalcemia.

The Abapatide consists of 34 amino acids and has a molecular formula of C₁₇₄H₃₀₀N₅₆O₄₉The molecular weight is 3961. The peptide sequence is shown as follows:

H-Ala¹-Val-Ser-Glu-His-Gln-Leu-Leu-His-Asp-Lys¹¹-Gly-Lys-Ser-Ile-Gln-Asp-Leu-Arg-Arg-Arg²¹-Glu-Leu-Leu-Glu-Lys-Leu-Leu-Aib-Lys-Leu³¹-His-Thr-Ala-NH₂

the original patent US5969095 mainly adopts Boc strategy solid phase stepwise coupling method for synthesis, needs strong acid HF cracking, and has high risk coefficient. The synthesis of fully protected polypeptide fragments in patent CN201510136475 requires a large amount of expensive 2-CTC resin, and the production cost is high. CN108047329 preferentially synthesizes Fmoc-Thr (OtBu) -Ala-OH, Fmoc-Lys (Boc) -Gly-OH, Fmoc-Ala-Val-OH, Fmoc-Arg (Pbf) -Glu (OtBu) -OH and other small fragments, and then solid phase coupling is carried out, although the generation of Gly, Ala and Arg deficiency impurities can be reduced, the preferential synthesis of small fragment impurities increases the process complexity. Furthermore, the abapa peptide sequence has two aspartic acids at positions 10 and 17, and after the solid phase is coupled with the aspartic acids sequentially in the polypeptide synthesis, the deprotection with 20% piperidine DMF solution is usually prone to generate asparagine impurities and piperidine impurities, and the reaction formula is shown as follows:

as shown in the reaction formula, when piperidine is used as a base for removing the N-terminal Fmoc protecting group, an asparagine imide side reaction is easy to occur. The literature reports that the addition of 1% HOBt to a 20% piperidine DMF solution is effective in reducing the occurrence of this side reaction (org. lett., vol.14, No.20,2012), but the production of this impurity is still unavoidable.

Disclosure of Invention

Aiming at the defects of the prior art, a new synthesis method is needed to avoid the generation of impurities.

One aspect of the invention provides a specific preparation method of abamectin, which comprises the following steps:

step 1: Fmoc-Asp (OtBu) -Leu-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -Lys (Boc) -Leu-Leu-Aib-Lys (Boc) -Leu-His (Trt) -Thr (tBu) -Ala-solid phase synthetic resin;

step 2: adding AlCl under the protection of inert gas₃Removing the Fmoc protecting group on Asp from the NMP solution and the indole;

and step 3: continuing to sequentially couple and synthesize Fmoc-Asp (OtBu) -Lys (Boc) -Gly-Lys (Boc) -Ser (tBu) -Ile-Gln (Trt) -Asp (OtBu) -Leu-Arg (Pbf) -Glu (OtBu) -Leu-Leu-Glu (OtBu) -Lys (Boc) -Leu-Leu-Aib-Lys (Boc) -Leu-His (Trt) -Thr (tBu) -Ala-solid-phase synthetic resin according to the Fmoc protection strategy;

and 4, step 4: adding AlCl under the protection of inert gas₃Removing the Fmoc protecting group on Asp from the NMP solution and the indole;

and 5: sequentially coupling according to Fmoc protection strategy to obtain fully-protected Abapatide-solid phase synthetic resin;

step 6: adding a cracking agent, and cracking the solid-phase synthetic resin and the side chain protecting group to obtain the abapa peptide;

optionally, step 7: and (5) preparing and purifying by reverse phase chromatography to obtain the refined peptide.

The Fmoc protection strategy in the steps 1, 3 and 5 is as follows: coupling Fmoc-protected amino acid on solid-phase synthetic resin or polypeptide-solid-phase synthetic resin; the terminal Fmoc protecting group is removed and the next Fmoc protected amino acid is coupled until completion.

In the technical scheme of the invention, the solid phase synthetic resin is swelled by a solvent before the solid phase synthetic resin is used, and the selected solvent comprises DMF, NMP, dichloromethane and the like, preferably DMF.

In the technical scheme of the invention, the remover for removing the Fmoc protecting group at the end of non-Asp amino acid is 20% piperidine/DMF solution.

In the technical scheme of the invention, the method for coupling the Fmoc-protected amino acid on the solid-phase synthetic resin or the polypeptide-solid-phase synthetic resin is to activate the amino acid to be coupled and the solid-phase synthetic resin or the polypeptide-solid-phase synthetic resin by using a coupling agent until the reaction is finished.

In the technical scheme of the invention, the coupling agent is a composition of DIPCDI and a compound A or a composition of DIPEA and a compound A and a compound B, wherein the compound A is HOAt or HOBt, the compound B is PyAOP, PyBOP, HATU, HBTU or TBTU, and the coupling agent is preferably a composition of DIPCDI and the compound A. Further, the ratio of each component in the coupling agent is, in terms of molar ratio, DIPCDI: a: 1.2:1.1, DIPEA: a: B: 2.0:1.1: 1.0.

In the technical scheme of the invention, the time for carrying out the coupling reaction of each amino acid is generally 1.5 to 4 hours, preferably 2 to 3 hours; the temperature is preferably room temperature (i.e., 20. + -. 5 ℃ C.), and may be suitably elevated or reduced.

In the technical scheme of the invention, the solid-phase synthetic Resin is amino solid-phase synthetic Resin, preferably RinkAmide AM Resin, Rink Amide MBHA Resin or Rink Amide Resin.

In the technical scheme of the invention, the substitution degree of the solid phase synthetic resin is 0.1-1.0mmol/g, preferably 0.2-0.8mmol/g, and more preferably 0.2-0.4 mmol/g.

In the technical scheme of the invention, the removing reagent for removing the Fmoc protecting group on Asp in the step 2 and the step 4 is AlCl₃And AlCl₃Indole is added into the organic solution of (1), and the AlCl is added₃The dosage is 3 times of molar equivalent of reaction substrate, and the dosage of indole is 1 time of molar equivalent of reaction substrate. The organic solvent used in the organic solution was NMP.

In the technical scheme of the invention, in the step 2 and the step 4, removing the Fmoc protecting group on Asp, washing with an organic solvent, and washing with DMF; the solvent is capable of dissolving AlCl₃The solvent of (4) is preferably toluene, water, ethanol, diethyl ether and carbon tetrachloride. The deprotection time is usually 2 to 4 hours, preferably 3 hours. Temperature preferenceAt room temperature (i.e., 20. + -. 5 ℃ C.), and also at an appropriately elevated or reduced temperature.

In the technical scheme of the invention, the lysis solution used in the step 6 is TFA or H₂O, EDT, anisole, thioanisole, phenol, preferably R cleavage reagent (TFA: thioanisole: EDT: 90:5:3: 2).

In the technical scheme of the invention, the purification step in the step 7 adopts reversed-phase high-pressure liquid chromatography. Further, the reversed-phase high-pressure liquid chromatography comprises: using reverse octadecylsilane as stationary phase and 0.1% trifluoroacetic acid water solution/acetonitrile as mobile phase, collecting target peak fraction, concentrating, and lyophilizing.

The invention uses AlCl after coupling two Fmoc-Asp (OtBu) -OH in peptide sequence₃The NMP solution replaces the DBLK solution to carry out deprotection reaction. With AlCl₃The method has the advantages that organic base piperidine is replaced for deprotection, and the side reaction of the aspartimide under the action of alkali can be well reduced, so that the purity of crude peptide is improved, the purification difficulty is reduced, and the product yield is improved. The addition of indole can capture fluorenene, thereby promoting complete Fmoc protecting group removal and reducing separate use of AlCl₃Defective peptide impurities resulting from incomplete removal of the Fmoc protecting group in toluene solution.

Without wishing to be bound by theory, it is shown by the protocol of the present invention and by experiments in the comparative group that the technical protocol of the present invention achieves unexpected technical effects. The inventor believes that the AlCl is possibly caused₃The toluene solution of (1) can effectively remove the N-terminal Fmoc protecting group (Eur.J.org.chem.2000(4),573-575), while the invention adopts AlCl₃Applied to removing Fmoc protecting group of Asp in Abapatide sequence by using AlCl₃The method has the advantages that organic base piperidine is replaced for deprotection, and the side reaction of the aspartimide under the action of alkali can be well avoided, so that the purity of the crude peptide is improved, the purification difficulty is reduced, and the product yield is improved. AlCl in the literature₃The conversion rate of the toluene solution in the liquid phase reaction is only 86-95%, and when the protecting group is removed in the solid phase reaction, the deprotection effect is poorer due to the heterogeneous reaction, so that the method cannot be applied to solid phase polypeptide synthesis. The invention uses N-methyl pyrrole with better solubilityThe preparation method comprises the steps of using an alkyl ketone (NMP) as a solvent, adding a small amount of indole as a trapping agent, and trapping fluorene positive ions by double actions of Friedel-crafts alkylation reaction and lone pair electrons on indole ring nitrogen, so that AlCl is improved₃The solid phase deprotection reaction efficiency ensures that the Fmoc protecting group is completely removed and avoids the occurrence of the side reaction of the aspartimide.

Advantageous effects

The method greatly reduces the impurity content, and obviously improves the yield and the purity; the preparation method is simple and the cost of raw materials is low.

Drawings

FIG. 1 shows the mass spectrum of the protamine.

FIG. 2 is a mass spectrum of an impurity from asparagine.

Detailed Description